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TITLE Pectin Biosynthesis is Critical for Cell Wall Integrity and Immunity in Arabidopsis 1

thaliana 2

3

RUNNING TITLE GAE1 and GAE6 in Arabidopsis immunity 4

5

AUTHORS Gerit Bethke1 Amanda Thao1 Guangyan Xiong2 Baohua Li4 Nicole E 6

Soltis4Noriyuki Hatsugai1 Rachel A Hillmer15 Fumiaki Katagiri1 Daniel J 7

Kliebenstein4 Markus Pauly26 and Jane Glazebrook1 8

9 1 Department of Plant Biology University of Minnesota 1500 Gortner Ave St Paul MN 10

55108 USA 11 2 Energy Biosciences Institute University of California 2151 Berkeley Way Berkeley 12

CA 94720 USA 13 4 Department of Plant Sciences University of California One Shields Avenue Davis 14

CA 95616 USA 15 5 Plant Biological Sciences Graduate Program 1445 Gortner Ave St Paul MN 55108 16

USA 17 6 Department of Plant and Microbial Biology University of California Berkeley CA 18

94720 USA 19

20

Present address University of Louisville 500 S Preston Street Louisville KY4020221

USA 22

23

CORRESPONDING AUTHOR Gerit Bethke gbethkeumnedu 24

25

FOOTNOTE The author responsible for distribution of materials integral to the findings 26

presented in this article in accordance with the policy described in the Instructions for 27

Authors (wwwplantcellorg) is Jane Glazebrook (jglazebrumnedu) 28

29

Plant Cell Advance Publication Published on January 26 2016 doi101105tpc1500404

copy2016 American Society of Plant Biologists All Rights Reserved

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Loss of UDP-glucuronate 4-epimerases GAE1 and GAE6 in Arabidopsis thaliana 31

causes a dramatic reduction of pectin in the cell wall and compromised immunity to 32

Pseudomonas syringae and Botrytis cinerea 33

34

35

SYNOPSIS 30

ABSTRACT 36

37

Plant cell walls are important barriers against microbial pathogens Cell walls of 38

Arabidopsis thaliana leaves contain three major types of polysaccharides cellulose 39

various hemicelluloses and pectins UDP-D-galacturonic acid the key building block of 40

pectins is produced from the precursor UDP-D-glucuronic acid by the action of 41

glucuronate 4-epimerases (GAEs) Pseudomonas syringae pv maculicola ES4326 42

(Pma ES4326) repressed expression of GAE1 and GAE6 in Arabidopsis and immunity 43

to Pma ES4326 was compromised in gae6 and gae1 gae6 mutant plants These plants 44

had brittle leaves and cell walls of leaves had less galacturonic acid (GalA) Resistance 45

to specific Botrytis cinerea isolates was also compromised in gae1 gae6 double mutant 46

plants Although oligogalacturonide (OG)-induced immune signaling was unaltered in 47

gae1 gae6 mutant plants immune signaling induced by a commercial pectinase 48

macerozyme was reduced Macerozyme treatment or infection with B cinerea released 49

less soluble uronic acid likely reflecting fewer OGs from gae1 gae6 cell walls than from 50

wild-type Col-0 Although both OGs and macerozyme induced immunity to B cinerea in 51

Col-0 only OGs also induced immunity in gae1 gae6 Pectin is thus an important 52

contributor to plant immunity and this is due at least in part to the induction of immune 53

responses by soluble pectin likely OGs that are released during plant-pathogen 54

interactions 55

56

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INTRODUCTION 57

The cell walls in Arabidopsis thaliana leaves are mainly primary cell walls 58

consisting of three major types of polysaccharidesmdashcellulose various hemicelluloses 59

and various pectic polysaccharidesmdashas well as some structural proteins (Liepman et 60

al 2010) Arabidopsis leaf walls contain approximately 14 cellulose a homopolymer 61

of (1 4)-β linked D-glucose subunits (Zablackis et al 1995 Carpita 2010) 62

Hemicelluloses are a diverse class of polysaccharides that includes xylans xyloglucans 63

mannans glucomannans and mixed-linkage β-glucans (Scheller and Ulvskov 2010) In 64

Arabidopsis leaves the major hemicellulose is xyloglucan which constitutes about 20 65

of the wall polysaccharides Xyloglucan contains a (1 4)-β-linked glucan backbone 66

substituted with (1 6)-α-linked xylosyl residues or side chains of xylosyl galactosyl and 67

fucosyl residues (Zablackis et al 1995 Liepman et al 2010) Glucuronoarabinoxylan 68

(4 of the wall) is also found in Arabidopsis leaves (Zablackis et al 1995) Primary 69

walls of dicotyledonous plants generally also contain 3-5 of the hemicelluloses 70

mannan and glucomannan (Scheller and Ulvskov 2010) Hence hemicelluloses in 71

primary walls of dicotyledonous plants are mainly composed of glucose (Glc) xylose 72

(Xyl) arabinose (Ara) galactose (Gal) and mannose (Man) 73

Pectins are a diverse group of polysaccharides that all contain galacturonic acid 74

(GalA) and make up about 50 of Arabidopsis leaf walls (Zablackis et al 1995 Harholt 75

et al 2010) Homogalacturonan (HG) is a linear homopolymer of (14)-α-linked 76

galacturonic acid (GalA) residues and it makes up about 65 of all pectin in 77

Arabidopsis leaf walls (Zablackis et al 1995 Mohnen 2008) A linear (14)-α-linked 78

GalA backbone substituted with single xylose residues is called xylogalacturonan 79

(XGA) and a polymer with complex side-chains containing borate ions and sugars such 80

as Ara rhamnose (Rha) Gal Xyl or fucose (Fuc) is referred to as rhamnogalacturonan 81

II (RGII) (Mohnen 2008 Harholt et al 2010) XGA and RGII make up less than 10 of 82

leaf cell wall pectin (Zandleven et al 2007 Mohnen 2008) In contrast 83

rhamnogalacturonan I (RGI) backbones consist of a repeating α-14-D-GalA-α-12-L-Rha 84

disaccharide and are substituted with β-(14)-galactan branched arabinan or 85

arabinogalactan side chains (Mohnen 2008 Harholt et al 2010) RGI constitutes about 86

20 ndash 25 of pectin in primary walls (Mohnen 2008) Hence pectin in Arabidopsis leaf 87

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cell walls consists mostly of GalA Rha and smaller amounts of other sugars including 88

Ara Gal Xyl and Fuc 89

In general carbohydrate biosynthesis requires nucleotide sugars provided by 90

nucleotide sugar interconversion pathways (Seifert 2004) Most nucleotide sugars are 91

synthesized from uridine diphosphate-Glc (UDP-Glc) UDP-glucuronic acid (UDP-GlcA) 92

is made from UDP-Glc by UDP-glucose dehydrogenase activity or via an alternative 93

pathway requiring inositol oxygenase activity (Tenhaken and Thulke 1996 Loewus and 94

Murthy 2000) UDP-D-Glucuronate 4-epimerases (GAEs) interconvert UDP-D-GlcA and 95

UDP-D-GalA the monomeric precursor of pectin There are six GAE genes encoded by 96

the Arabidopsis genome When heterologously expressed in Escherichia coli or Pichia 97

pastori GAE1 GAE4 and GAE6 showed activity in interconverting UDP-D-GlcA and 98

UDP-D-GalA (Gu and Bar-Peled 2004 Molhoj et al 2004 Usadel et al 2004) GAE1 99

and GAE6 were found to be Golgi-localized and strongly expressed in various plant 100

tissues (Molhoj et al 2004 Usadel et al 2004 Parsons et al 2012) GAE1 and GAE6 101

have been hypothesized to be evolutionarily older than the other GAE family members 102

(Usadel et al 2004) and might have overlapping functions that are distinct from the 103

other family members 104

Plant cell wall composition and architecture affects wall strength and flexibility 105

and cell walls present a physical barrier to potential plant pathogens Besides preformed 106

physical barriers such as a cell wall plants have a sophisticated immune system to 107

defend themselves against harmful microbial pathogens (Chisholm et al 2006 Jones 108

and Dangl 2006) Immune signaling involves changes in phytohormone levels the most 109

important for plant immunity being salicylic acid (SA) jasmonic acid (JA) and ethylene 110

(ET) (Grant and Jones 2009 Pieterse et al 2012) Other major regulators of plant 111

immunity include PHYTOALEXIN DEFICIENT 4 (PAD4) and ENHANCED DISEASE 112

SUSCEPTIBILITY 1 (EDS1) (Wiermer et al 2005) which are predicted to encode 113

lipases Immune signaling generally involves changes in the transcriptome that in turn 114

lead to altered cellular responses (Tao et al 2003) For instance induced expression of 115

the cytochrome P450 PAD3 is thought to increase production of the anti-microbial 116

secondary metabolite camalexin (Zhou et al 1999) 117

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Because plant cell walls are important barriers against pathogenic microbes 118

alterations in wall structural properties can lead to changes in plant immunity To date 119

only a few examples of cell wall related mutants with altered pathogen phenotypes have 120

been studied in detail Mutants impaired in callose biosynthesis powdery mildew 121

resistant 4 (pmr4) display enhanced resistance to the powdery mildew pathogens 122

Golovinomyces orontii and G cichoracearum (Nishimura et al 2003) These plants 123

have constitutively high SA levels and SA signaling is required for the resistance 124

phenotype (Nishimura et al 2003) Other plants with enhanced resistance to these 125

powdery mildew pathogens include pmr5 a mutant enriched in pectin and pmr6 a 126

mutant with a defect in a pectate lyase-like gene (Vogel et al 2002 Vogel et al 2004) 127

Plants with mutations in cellulose synthases required for primary and secondary wall 128

formation also show increased resistance to pathogens (Ellis and Turner 2001 129

Hernandez-Blanco et al 2007) Enhanced resistance to powdery mildew and 130

Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) in constitutive 131

expression of VSP 1 (cev1) which is mutated in the primary wall cellulose synthase 132

CesA3 was linked to constitutive high JA and ET levels in these plants (Ellis and 133

Turner 2001 Ellis et al 2002a) Interestingly irregular xylem1 (irx1) irx3 and irx5 134

plants which carry mutations in the secondary wall-associated cellulose synthases 135

CesA8 CesA7 and CesA4 showed enhanced resistance to the necrotrophic pathogens 136

Plectosphaerella cucumerina Ralstonia solanacearum and Botrytis cinerea as well as 137

the biotrophic powdery mildew G cichoracearum whereas growth of P cucumerina and 138

R solanacearum pathogens was unaltered in pmr5 pmr6 and cev1 plants (Hernandez-139

Blanco et al 2007) This enhanced resistance of the irx mutants was independent of 140

SA JA or ET signaling but instead involved abscisic acid signaling (Hernandez-Blanco 141

et al 2007) Changes in wall structure are likely monitored by the plants and effects 142

may be counteracted through initiation of defense responses that can lead to altered 143

phytohormone levels 144

Although plant cell walls serve as barriers against pathogen entry many 145

pathogens secrete cell wall-degrading enzymes (CWDEs) and thus are able to 146

successfully infect their host plants (Albersheim et al 1969) For example the B 147

cinerea polygalacturonase Bc-PG1 is required for full virulence of this pathogen and the 148

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gene encoding it shows evidence for diversifying selection as would be expected for a 149

virulence gene (ten Have et al 1998 Rowe and Kliebenstein 2007) Secretion of 150

CWDEs can trigger the release of cell wall fragments which can act as damage-151

associated molecular patterns (DAMPs) that activate plant immune responses (Hahn et 152

al 1981 Ferrari et al 2013) Similarly pectin fragments known as oligogalacturonides 153

(OGs) can trigger plant immune responses (Cote and Hahn 1994 Ferrari et al 2013) 154

Such responses include the production of reactive oxygen species (ROS) activation of 155

mitogen-activated protein kinase (MAPK) activation and enhanced expression of 156

defense-related genes such as PAD3 (Ferrari et al 2007 Denoux et al 2008 Galletti 157

et al 2008 Galletti et al 2011) Some of these responses require SA JA and ET 158

whereas others are independent of these phytohormones (Ferrari et al 2007) 159

Here we describe two GAE family members GAE1 and GAE6 whose 160

expression is repressed upon pathogen challenge A gae1 gae6 double mutant has 161

brittle leaves and its walls are reduced in pectin specifically HG and likely RGI 162

Resistance to the bacterial pathogen Pma ES4326 and the fungal necrotroph B cinerea 163

is compromised in gae1 gae6 Callose deposition is reduced in gae1 gae6 plants after 164

treatment with B cinerea isolate Gallo 1 Furthermore these plants show altered 165

activation of immune signaling in response to treatment with macerozyme a 166

polygalacturonase that degrades pectin and they also are hyperresponsive to JA 167

signaling Macerozyme treatment released less soluble likely low molecular weight 168

pectin from isolated gae1 gae6 cell walls than from Col-0 cell walls B cinerea-induced 169

accumulation of soluble likely low molecular weight pectin was also decreased in gae1 170

gae6 plants Additionally gae1 gae6 plants are impaired in macerozyme-induced 171

immunity to B cinerea possibly due to decreased release of OGs from their cell walls 172

173

174

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RESULTS 175

176

GAE1 and GAE6 Expression Is Repressed by Pma ES4326-Induced Immune 177

Signaling 178

Our previous research suggested that the status of pectin specifically pectin 179

modification by methylesterification is important for plant immunity against Pma 180

ES4326 (Bethke et al 2014) Here we investigate whether pectin abundance in 181

addition to its specific structure is also important for plant immunity by taking a closer 182

look at the formation of the pectin precursor UDP-GalA by GAEs 183

184 Microarray experiments showed that expression of GAE1 and GAE6 was

185 repressed in A thaliana (Arabidopsis) plants treated with Pma ES4326 (Wang et al

186 2008) Repression of GAE1 and GAE6 in wild-type Arabidopsis Col-0 plants treated with

187 Pma ES4326 was confirmed by quantitative real time (qRT)-PCR Figure 1) To test if

188 this repression is mediated by immune signaling qRT-PCR was also performed in

189 mutants with blocked immune signaling (Figure 1 and Supplemental Figure 1) 190 Generally immunity to Pma ES4326 requires intact SA signaling Repression of both

191 GAE1 and GAE6 required the presence of the major immune regulators EDS1 and

192 PAD4 required for SA signaling as well as other immune responses (Jirage et al 1999

193 Wiermer et al 2005 Bartsch et al 2006 Wang et al 2008) at both early and late time

194 points The presence of AVRPPHB SUSCEPTIBLE 3 (PBS3) a regulator of many

195 immune responses including SA signaling (Nobuta et al 2007) was also required for

196 repression of GAE1 and GAE6 at an early time point (Figure 1) CALMODULIN-

197 BINDING PROTEIN 60-LIKE a (CBP60a) (a negative regulator of SA synthesis and

198 immunity (Truman et al 2013)) CBP60g and SYSTEMIC ACQUIRED RESISTANCE

199 DEFICIENT 1 (SARD1) (both promote SA biosynthesis and regulation of other immune

200 responses (Wang et al 2011)) SALICYLIC ACID INDUCTION DEFICIENT 2 SID2

201 (encodes a biosynthetic enzyme for SA (Wildermuth et al 2001)) and

202 NONEXPRESSER OF PR GENES 1 NPR1 (a regulator of the majority of SA-induced

203 immune signaling (Cao et al 1997 Yan and Dong 2014)) were not required for this

204 repression (Figure 1 and Supplemental Figure1) Thus repression of GAE1 and GAE6

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205 requires a regulatory function that is dependent on EDS1 and PAD4 partially

206 dependent on PBS3 but independent of SA

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There are six GAE family members in Arabidopsis GAE2 expression was 207

induced by Pma ES4326 in the absence of PAD4 or EDS1 GAE3 expression was 208

unaltered GAE4 was repressed early and GAE5 expression was induced in all 209

genotypes (Supplemental Figure 2) The similarity of the immune-regulated expression 210

of GAE1 and GAE6 suggested a redundant role of these genes in Arabidopsis immunity 211

to Pma ES4326 212

213

Resistance to Pma ES4326 Is Compromised in gae1 gae6 and gae6 Plants 214

To study the roles of GAE1 and GAE6 in plant immunity in more detail plants 215

with T-DNA insertion mutations in GAE1 and GAE6 were obtained from the SALK 216

collection as described in the Accession Numbers section of Methods We identified two 217

GAE6 knock-out alleles gae6-1 and gae6-2 and a T-DNA insertion in GAE1 gae1-1 218

that caused reduced expression of this gene (Supplemental Figure 3) A double mutant 219

was created by crossing gae1-1 and gae6-1 Both gae6 and gae1 gae6 plants were 220

more susceptible to Pma ES4326 than wild-type Col-0 and gae1 plants supporting a 221

role of GAE6 in plant immunity to Pma ES4326 (Figure 2) 222

223

gae6 and gae1 gae6 Plants Have Brittle Leaves 224

While inoculating leaves of gae1 gae6 plants we noticed that the petioles and 225

midribs of these plants tended to break easily when leaves were bent against the 226

proximal-distal axis of the leaves (Figure 3 see Supplemental Figure 4D to F) They 227

tended to break less often when twisted around the proximal-distal axis To quantify 228

this phenotype leaves were bent by 180deg (Figure 3A) and breaking of midribs or 229

petioles was recorded Most gae1 gae6 leaves broke whereas fewer leaves of gae6 230

plants and very few leaves of wild-type Col-0 and gae1 plants did (Figure 3B and 231

Supplemental Figure 4C) Although gae1 gae6 plants had slightly shorter leaves and 232

petioles than wild type plants (Supplemental Figure 4A and B) no correlation between 233

leaf size or petiole length and breaking frequency was detected We also investigated 234

tissue integrity by monitoring ion leakage Interestingly when leaves were inoculated 235

with deionized water leaf discs of gae1 gae6 leaked more ions than wild-type Col-0 236

discs (Figure 3C) suggesting that the increased brittleness is correlated with a 237

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_____________________________________________________________________________

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Cell Walls of gae6 and gae1 gae6 Plants Have Lower Levels of Pectin 243

GalA is a major component of pectin We analyzed wall monosaccharide 244

composition in gae mutants to investigate whether the increased brittleness was 245

associated with an altered wall composition of gae leaves To approximate pectin 246

content of gae mutant walls the total uronic acid concentration was measured using a 247

simple colorimetric assay (Filisetti-Cozzi and Carpita 1991 van den Hoogen et al 248

1998) Total uronic acid was reduced in gae6-2 strongly reduced in gae1 gae6 plants 249

but was indistinguishable from wild-type Col-0 in gae6-1 and gae1 (Supplemental 250

Figure 5) This suggested that gae1 gae6 and possibly gae6 plants have reduced 251

pectin 252

To investigate the cell wall composition in leaves of gae plants in more detail we 253

measured the levels of seven neutral monosaccharides the acidic sugars GalA (the 254

monomeric subunit of the pectin backbone (Harholt et al 2010)) and GlcA (Figure 4) 255

We also estimated the crystalline cellulose content (Updegraff 1969) Cell walls of both 256

gae6 mutants and gae1 gae6 were reduced in GalA (Figure 4A) In gae1 gae6 GalA 257

content in cell walls was reduced by more than 40 compared to the wild-type level 258

GlcA levels were indistinguishable between mutants and wild-type (Figure 4B) 259

Cellulose content was higher in gae6-2 and gae1 gae6 walls likely due to the loss of 260

pectins (Figure 4C) Cell walls of gae1 gae6 plants were also enriched in all neutral 261

sugars measured presumably due to the substantial proportional loss of GalA (Figure 262

4D) The levels of most wall monosaccharides were even higher in the gae1 gae6 263

double mutant with the exception of Rha which did not exhibit a significant increase 264

compared to the gae6 single mutants The absence of a compensational increase in 265

Rha suggested that RGI the dominant Rha contributor to the wall may be considered 266

reduced in gae1 gae6 mutants compared to wild-type albeit not to such an extent as 267

HG 268

238 structural defect that promotes increased ion leakage Taken together these results

239 showed that loss of GAE6 causes brittle leaves and additional losss of GAE1 strongly

240 enhances this phenotype We conclude that GAE1 and GAE6 contribute to normal leaf

241 flexibility 242

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gae6 cell walls are reduced in HG Using the same samples we detected no difference 274

from wild type in binding of LM5 which recognizes a tetramer of (1-4)-β-D-galactans 275

found in RGI (Jones et al 1997) LM6 which recognizes a linear pentasaccharide in (1-276

To further investigate which pectic components were altered in gae1 gae6 dot 269

blot experiments using pectin isolated from the various genotypes were performed Both 270

LM19 and LM20 antibodies recognize HG (Verhertbruggen et al 2009a) As expected 271

LM19 binding was reduced in gae6-1 and gae1 gae6 (Figure 5A and B) and LM20 272

binding was reduced in gae1 gae6 (Figure 5C and D) This result confirmed that gae1 273

277 5)-α-L-arabinans found in RGI (Willats et al 1998 Verhertbruggen et al 2009b) LM8

278 which recognizes xylogalacturonan (Willats et al 2004) or CCRC-M7 which

279 recognizes an arabinosylated (1-6)-β-D-galactan epitope occurring on RGI (Puhlmann et

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al 1994 Steffan et al 1995 Pattathil et al 2010) (Supplemental Figure 6) 280

Collectively these cell wall composition data show that gae1 gae6 plants are reduced in 281

HG Due to the strong proportional reduction of HG in the walls levels of all other 282

components should be higher However Rha indicative of RGI was not higher in gae1 283

gae6 suggesting that RGI may also be reduced in gae1 gae6 walls 284

285

GAE1 and GAE6 Are Required for Immunity to Specific B cinerea Isolates 286

We hypothesized that plant immunity to B cinerea might be affected by pectin 287

content in the host cell wall because this pathogen requires polygalacturonases and 288

pectin methylesterases for full virulence (ten Have et al 1998 Valette-Collet et al 289

2003 Kars et al 2005b Espino et al 2010) Because Bcinerea is genetically diverse 290

and has a high diversity of polygalacturonases (Rowe and Kliebenstein 2007) we 291

measured relative fungal growth at both 2 and 3 d post-inoculation (dpi) using ten 292

different B cinerea isolates (Figure 6) The B cinerea isolates Fresa 525 and Gallo 1 293

showed enhanced virulence on gae1 gae6 plants at both time points whereas KT and 294

UK Razz showed enhanced virulence in gae1 gae6 plants at 3 but not 2 d dpi (Figure 295

6A) We did not detect any significant changes in relative fungal growth between wild-296

type Col-0 and gae1 gae6 plants when using Acacia Apple 517 DN Grape Pepper or 297

Rasp isolates of B cinerea Therefore we continued our study using Gallo 1 and Fresa 298

525 We also included Pepper for comparison as this isolate has been used in a large-299

scale analysis of plant gene expression in response to B cinerea infection (Windram et 300

al 2012) 301

We next investigated whether expression of GAE1 and GAE6 was affected by 302

challenge with B cinerea Inoculation of wild-type Col-0 plants with any isolate 303

repressed GAE1 and GAE6 expression (Figure 6B and C) and this repression was 304

generally independent of PAD4 (Figure 6B and C) Expression of GAE2 and GAE3 was 305

unaltered by B cinerea treatment GAE4 was repressed only by Fresa 525 treatment 306

while GAE5 expression was enhanced with all three isolates tested (Supplemental 307

Figure 7) As seen for infection with Pma ES4326 the expression of GAE1 and GAE6 308

was repressed upon B cinerea treatment This indicates a role for GAE1 and GAE6 in 309

the immune response to B cinerea 310

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Because the amount of pectin substrate for pathogen polygalacturonases is 313

reduced we hypothesized that immunity phenotypes of gae1 gae6 plants may result 314

from reduced generation of OGs by these enzymes In turn the reduced OGs could 315

lead to reduced immune responses To test this we used macerozyme a commercial 316

pectinase as a proxy for pathogen pectinases We isolated cell walls as a subcellular 317

fraction known as alcohol insoluble residue (AIR Gille et al 2009) This was treated 318

with macerozyme and tested for release of soluble likely low molecular weight pectin 319

fragments (Figure 7) Treatment with 025 macerozyme released significantly more 320

uronic acid from wild-type Col-0 AIR relative to gae1 gae6 AIR (Figure 7B) To test 321

activity of these pectin-containing solutions we infiltrated them into wild-type Col-0 322

plants and measured PAD3 expression PAD3 is required for synthesis of the 323

phytoalexin camalexin (Zhou et al 1999) and its expression is known to be induced by 324

OG treatment (Ferrari et al 2007) Fractions from macerozyme treated Col-0 walls 325

induced significantly more PAD3 expression than fractions derived from gae1 gae6 326

walls suggesting that macerozyme treatment did indeed release more active soluble 327

uronic acid possibly OGs from wild-type Col-0 walls than from gae1 gae6 walls (Figure 328

7C) 329

Next we studied the effect of macerozyme treatment on expression of PAD3 in 330

intact plants (Figure 8) This treatment did not lead to any visible leaf damage 331

(Supplemental Figure 8) In wild-type Col-0 PAD3 expression was induced three h after 332

treatment with 001 macerozyme (Figure 8A) By contrast no macerozyme-induced 333

induction of PAD3 expression was detected in gae1 gae6 (Figure 8A) However OG 334

treatment induced the expression of PAD3 in both genotypes indicating that gae1 gae6 335

plants do respond to OGs (Figure 8B) Macerozyme treatment also induced expression 336

of the SA marker genes PATHOGENESIS RELATED 1 (PR1) and SID2 in Col-0 but 337

not in gae1 gae6 (Figure 9 panel A and B) PR1 was not significantly induced by 338

purified OGs (Figure 9C) at the time and concentration tested but SID2 was induced by 339

OG treatment in both genotypes (Figure 9D) However the induction was stronger in 340

wild-type Col-0 In addition the JA markers JASMONATE-ZIM-DOMAIN PROTEIN 5 341

(JAZ5) and JAZ10 were induced by macerozyme and OG treatment in both Col-0 and 342

gae1 gae6 (Figure 10A and Supplemental Figure 11) Interestingly expression of JAZ5 343

312 Pectinase-Induced Immune Signaling Is Altered in gae1 gae6 Plants

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344

345

346

and JAZ10 was already strongly induced in gae1 gae6 after mock inoculation (Figure 10

and Supplemental Figure 11) We observed no hyperresponsive induction by mock

treatment of PLANT DEFENSIN 12 (PDF12) a necrotrophy and late JA signaling

347 marker gene (Supplemental Figure 12A-D) PDF12 was also not induced by

348 macerozyme or OG treatments (Supplemental Figure 12A-D) This suggests that early

349 JA signaling is induced by mock-treatment in gae1 gae6 Because it is known that JA

350 and SA signaling act antagonistically (Pieterse et al 2012) the strong up-regulation of

351 JA-induced immune signaling in gae1 gae6 might explain the lower OG-induced SID2

352 expression in this genotype

353 To test whether other immune responses are altered in gae1 gae6 plants we

354 analyzed Mitogen-activated protein kinase (MAPK) activity following macerozyme and

355 OG treatments OGs are known to activate the MAPKs MPK3 and MPK6 in Arabidopsis

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(Galletti et al 2011) Macerozyme treatment activated MPK3 and MPK6 in wild-type 356

Col-0 whereas MAPK activation in gae1 gae6 was slightly reduced in four out of five 357

experiments performed (Supplemental Figure 13) By contrast OG-induced MAPK 358

activity was similar or increased in gae1 gae6 in four out of five experiments 359

(Supplemental Figure 13) Additionally mock treatment induced MAPK activity in gae1 360

gae6 more strongly than it did in wild-type Col-0 in four out of five experiments 361

performed (Fig S13) Collectively these results illustrate that activation of several 362

immune signaling componentsmdashcamalexin synthesis SA and JA signaling and 363

possibly MAPK activationmdashby macerozyme is substantially altered in gae1 gae6 This 364

suggests that release of soluble pectin fragments possibly OGs by pectinolytic 365

enzymes might be reduced in gae1 gae6 plants while recognition of OGs is intact 366

367

Following B cinerea Infection Production of Soluble Pectin and Deposition of 368

Callose Are Reduced in gae1 gae6 Plants 369

Although pad3 plants were more susceptible to the B cinerea isolates Fresa 525 370

and Gallo 1 which also grew better on gae1 gae6 growth of the isolate Pepper was 371

similar on Col-0 and pad3 (Supplemental Figure 9A) However we did not detect any 372

significant changes in camalexin production after treatment with B cinerea isolate Gallo 373

1 and Fresa 525 in gae1 gae6 (Figure 8D) Neither did we detect changes in PAD3 374

expression after treatment with any B cinerea isolate in gae1 gae6 (Figure 8E) 375

Additionally SID2 and PR1 were similarly induced by all B cinerea isolates in both Col-376

0 and gae1 gae6 (Figure 9E and F) and sid2 plants which are deficient for pathogen-377

induced SA biosynthesis did not show any differences in relative B cinerea growth 378

(Supplemental Figure 9B) Expression of JAZ10 was induced more strongly by Gallo 1 379

treatment in gae1 gae6 than in wild-type Col-0 but not by Fresa 525 or Pepper (Fig 380

10C) while dde2 plants deficient for JA biosynthesis showed enhanced susceptibility to 381

all B cinerea isolates tested (Supplemental Figure 9B) Curiously exogenous 382

application of MeJA did not lead to altered relative fungal growth of B cinerea 383

(Supplemental Figure 9C) and cev1 isoxaben resistant 1 (ixr1) plants which have 384

constitutively high JA levels (Ellis and Turner 2001) did not show any changes in 385

susceptibility to the B cinerea isolates tested (Supplemental Figure 9D) Hence neither 386

Page 22 of 54

differences in B cinerea isolate susceptibility to camalexin nor altered SA or JA 387

signaling by themselves explained the enhanced susceptibility of gae1 gae6 to these B 388

cinerea isolates 389

Hydrogen peroxide accumulation in response to B cinerea isolates Fresa 525 390

Gallo 1 and Pepper was not obviously altered in gae1 gae6 plants (Supplemental Figure 391

14) However callose deposition upon treatment with B cinerea isolate Gallo 1 was 392

strongly reduced in gae1 gae6 (Figure 11) It was previously shown that OG treatment 393

induces callose deposition (Denoux et al 2008) suggesting that the reduction in 394

callose deposition might be due to reduced OG release in gae1 gae6 It is possible that 395

this reduction in callose deposition contributes to the reduced immunity of gae1 gae6 396

plants to B cinerea isolate Gallo 1 397

Because macerozyme could be used to release soluble likely low molecular 398

weight pectin from isolated cell walls we next investigated whether infection with B 399

cinerea was accompanied by accumulation of soluble pectin as well We prepared cell 400

walls (AIR fraction) from leaves infected with B cinerea incubated these cell walls with 401

water and measured uronic acid content of the extracts B cinerea treatment did in fact 402

lead to increased accumulation of soluble pectin in wild-type Col-0 plants when 403

compared to mock-treated samples (Figure 12) No such increased accumulation of 404

water-soluble pectin fractions was observed in gae1 gae6 (Figure 12) 405

406

Macerozyme-Induced Pattern-Triggered Immunity against B cinerea Is Abolished 407

in gae1 gae6 408

It was previously shown that OG treatment can induce patternndashtriggered 409

immunity (PTI) to B cinerea (Ferrari et al 2007) We tested macerozyme treatment to 410

determine whether it could also induce PTI to the B cinerea isolate Gallo 1 (Figure 13) 411

Indeed pretreatment with 001 macerozyme for 24 h led to reduced B cinerea growth 412

in wild-type Col-0 but not gae1 gae6 plants (Figure 13A) Interestingly mock and 413

macerozyme pretreated gae1 gae6 plants exhibited similar growth of B cinerea Gallo 1 414

as did macerozyme-treated Col-0 plants This result is likely due to activation of JA 415

signaling in gae1 gae6 during the pretreatment procedure (Figure 13C and D) OG-416

induced PTI was observed in both genotypes when using a high dose of OGs (Figure 417

Page 23 of 54

13B) whereas a lower dose of OGs only induced PTI in Col-0 (Supplemental Figure 418

15) This suggests that OG perception in gae1 gae6 at least above a certain threshold 419

is unaltered while release of immunity-activating pectin fragments possibly OGs is 420

421 reduced We conclude that immune responses induced by macerozyme treatment

422 which do not occur in gae1 gae6 plants are important for plant immunity to the B

423 cinerea isolate Gallo 1

Page 24 of 54

____________________________________________________________________________

Page 25 of 54

DISCUSSION 426

Here we showed that loss of GAE1 and GAE6 leads to a dramatic reduction in 427

pectin content in the cell wall (Figure 4) likely causing the brittleness of leaves that we 428

observed (Figure 3) Petioles and midribs of gae1 gae6 plants broke easily when bent 429

and inoculation of leaves with deionized water resulted in increased ion leakage 430

possibly due either to microscopic fractures of the cell wall induced by inoculation stress 431

or to a reduced capability of these walls to withstand increased turgor pressure The 432

increased brittleness was associated with altered cell wall composition In our analysis 433

the GalA content of gae1 gae6 leaf walls was reduced by more than 40 and the total 434

uronic acids by over 60 (Figure 4 and Supplemental Figure 5) This is a major change 435

in the overall cell wall composition considering that pectin makes up about 50 of the 436

plant cell walls found in Arabidopsis leaves (Zablackis et al 1995 Nobuta et al 2007 437

Harholt et al 2010) Interestingly quasimodo1 (qua1) plants which have a mutation in 438

a glycosyltransferase family 8 gene are reduced in GalA by 25 and show reduced 439

binding of HG specific antibodies (Bouton et al 2002) 440

Furthermore qua2 plants which have a mutation in a putative methyltransferase 441

have about 13 less total GalA and lack 50 of cell wall HG (Mouille et al 2007) 442

Likewise cgr2 cgr3 plants carrying mutations in another class of Golgi-localized 443

putative methyl-transferases have leaf cell walls that are reduced by about 40 in 444

uronic acid (Kim et al 2015) All three mutants are severely dwarfed (Bouton et al 445

2002 Mouille et al 2007 Kim et al 2015) Additionally qua1 and qua2 mutants show 446

a distorted overall shape likely due to reduced cell cohesion and are extremely fragile 447

(Bouton et al 2002 Mouille et al 2007) By contrast gae1 gae6 plants with a similar 448

strong reduction in GalA and total uronic acids have an overall normal shape and are 449

only slightly smaller than wild-type Col-0 (Supplemental Figure 4) In the wall analysis 450

performed here we did not detect changes in other wall polysaccharides or structures 451

that might compensate for the reduction of pectin in gae1 gae6 It is possible the severe 452

morphological alterations in qua2 and cgr2 cgr3 plants are due to reduction in pectin 453

methylesterification which is unaffected in gae1 gae6 It was speculated that quality 454

control mechanisms in the Golgi prevent secretion of inefficiently esterified pectin in 455

cgr2 cgr3 (Kim et al 2015) suggesting that the Golgi of these plants might over-456

Page 26 of 54

accumulate pectin and that this may contribute to the severe dwarf phenotype of these 457

plants The gae1 gae6 plants described here provide an opportunity to study the effects 458

of reduction of pectin in plants that do not show severe morphological phenotypes 459

We found that gae1 gae6 plants are also compromised in resistance to B 460

cinerea and Pma ES4326 indicating that loss of pectin reduces immunity to these 461

pathogens (Figure 2 and 6) We found that gae1 gae6 plants were slightly more 462

susceptible to Pma ES4326 but showed a strong increase in susceptibility to specific B 463

cinerea isolates This was not unexpected because B cinerea is known to interact with 464

pectin as part of its infection strategy It secretes various pectin-degrading enzymes 465

early during infection and requires some of these enzymes including PG1 PG2 and 466

PME1 for full virulence (ten Have et al 1998 Valette-Collet et al 2003 Kars et al 467

2005b Espino et al 2010) Additionally Arabidopsis plants expressing an antisense 468

construct of the polygalacturonase inhibitor PGIP1 are significantly more susceptible to 469

B cinerea (Ferrari et al 2006) On the other hand Pma ES4326 is not known to470

require cell wall degrading enzymes for full virulence even though it encodes two 471

pectate lyase genes in its genome (PMA4326_07094 and PMA4326_04621 472

wwwbacteriaensemblorg) B cinerea isolates are also known to be genetically quite 473

diverse in their polygalacturonase (PG) loci and genetic variability in PG2 is associated 474

with varying growth on pectin containing media (Rowe and Kliebenstein 2007) 475

Furthermore the contribution of PG1 and PG2 to B cinerea virulence seems to depend 476

on the specific B cinerea isolate and the host species (Kars et al 2005a Zhang and 477

Van Kan 2013) B cinerea isolates are also known to be differently sensitive to the anti-478

microbial secondary metabolite camalexin (Kliebenstein et al 2005) In line with these 479

observations the susceptibility of gae1 gae6 plants differed depending on the specific 480

B cinerea isolate Whereas Fresa 525 Gallo 1 UK Razz and KT grew better in gae1481

gae6 we detected no difference in Acacia Apple 517 DN Grape Pepper or Rasp 482

growth (Figure 6) 483

B cinerea was shown to metabolize pectin and to possibly utilize GalA as a484

carbon source during in planta growth (Zhang et al 2011 Zhang and Van Kan 2013) 485

Our data show that gae1 gae6 plants which are reduced in GalA content did allow 486

enhanced growth of specific B cinerea isolates but no reduction in growth of any B 487

Page 27 of 54

cinerea isolates tested was found This suggests that utilization of pectin as a carbon 488

source was not a contributing factor to alterations in B cinerea susceptibility It is 489

possible that changes in the cell wall composition affect the structural integrity of the 490

wall and affect hyphal penetration of specific B cinerea isolates However OGs (small 491

HG fragments) are likely released during the infection process (Ferrari et al 2013) 492

OGs function as DAMPs and initiate immune responses in Arabidopsis (Ferrari et al 493

2013) Treatment of Arabidopsis with OGs having a degree of polymerization (DP) of 494

10-15 sugar subunits was previously shown to enhance resistance to B cinerea (Ferrari495

et al 2007) We showed that pretreatment with OGs or macerozyme induced enhanced 496

immunity to B cinerea isolate Gallo 1 in wild-type Col-0 but only OGs and not 497

macerozyme induced immunity in gae1 gae6 (Figure 13) Our data suggest gae1 gae6 498

plants which have reduced pectin content may release less OGs and that this 499

reduction leads to the increased susceptibility to B cinerea 500

Consistently we found that macerozyme treatment released less soluble pectin 501

from gae1 gae6 cell walls than from wild-type walls Fractions containing soluble pectin 502

released from gae1 gae6 were also less active in inducing PAD3 gene expression than 503

the fractions released from wild-type walls (Figure 7) We also showed that less soluble 504

pectin accumulated in gae1 gae6 than in wild-type plants 48 h after treatment with B 505

cinerea (Figure 12) 506

We observed that loss of pectin leads to alterations in A thaliana immune 507

signaling Macerozyme treatment induced expression of PAD3 and the SA marker 508

genes PR1 and SID2 in wild-type Col-0 but not in gae1 gae6 whereas OG treatment 509

led to increased PAD3 and SID2 expression in both wild-type and gae1 gae6 (Figure 8 510

and 9) This suggests that although release of active soluble pectin in these plants 511

might be altered OG recognition is intact In fact cell walls isolated from qua1 another 512

mutant known to be reduced in HG released less OGs (DP1-6) than did wild-type cell 513

walls upon endopolygalacturonase treatment (Bouton et al 2002) This was also true 514

for gae1 gae6 cell walls as macerozyme treatment of isolated gae1 gae6 cell walls 515

released less soluble pectin than treatment of Col-0 cell walls Because the fraction 516

from gae1 gae6 plants was also less active in inducing PAD3 expression (Figure 7) it 517

seems possible that biologically active OGs were the active component inducing PAD3 518

Page 28 of 54

expression Interestingly expression of the JA marker genes JAZ5 and JAZ10 was 519

higher in gae1 gae6 than in wild-type mock-inoculated plants but was comparable to 520

wild-type in untreated plants suggesting that JA signaling in gae1 gae6 is 521

hyperresponsive (Figure 10 and Supplemental Figure 11) This induction of JA signaling 522

might be due to wounding stress incurred during the inoculation process JA and SA 523

signaling generally act antagonistically (Pieterse et al 2012) Increased JA signaling 524

activity might hence explain why mock- and macerozyme-treated gae1 gae6 plants 525

exhibited reduced expression of the SA marker PR1 and why OG treatment induced 526

expression of SID2 another SA marker to a lower extent in gae1 gae6 than in wild-type 527

plants (Figure 9) 528

No single immune signaling component was found to be the sole contributor to 529

the compromised immunity of gae1 gae6 to specific B cinerea isolates such as Gallo 1 530

and Fresa 525 These isolates grew better on pad3 while growth of Pepper another B 531

cinerea isolate that did not show altered virulence on gae1 gae6 was unaltered 532

(Supplemental Figure 9) This suggested that differences in camalexin sensitivity of 533

these B cinerea isolates might account for the differences in gae1 gae6 susceptibility 534

However camalexin accumulation and PAD3 expression was the same for wild-type 535

Col-0 and gae1 gae6 for the B cinerea isolates Fresa 525 and Gallo 1 (Figure 8) which 536

showed enhanced growth on gae1 gae6 plants It should be noted that B cinerea 537

virulence is often estimated by comparing lesion sizes Lesion size and measurements 538

of relative fungal growth by qPCR do not always show the same results For example 539

although Denby et al (2004) showed that Pepper causes larger lesions on pad3 than on 540

wild-type this study showed that relative fungal growth of Pepper on pad3 was 541

unaltered 542

We observed that callose deposition was strongly reduced in gae1 gae6 plants 543

72 h after treatment with B cinerea isolate Gallo 1 (Figure 11) possibly due to reduced 544

release of active soluble pectin Interestingly OG-induced PTI to an unspecified isolate 545

of B cinerea was intact in the pmr4 mutant which does not produce callose (Galletti et 546

al 2008) Due to the high SA levels in pmr4 plants (Nishimura et al 2003) and the fact 547

that different B cinerea isolates vary both in induction of and reaction to defense 548

responses (Kliebenstein et al 2005 Rowe et al 2010) we could not determine 549

Page 29 of 54

whether reduced callose deposition causes the increased susceptibility of gae1 gae6 to 550

B cinerea isolate Gallo 1551

We did not observe any changes in B cinerea growth either on cev1 ixr1 plants 552

which are known to have constitutively high JA and OPDA levels (Ellis et al 2002b) or 553

on plants exogenously treated with MeJA (Supplemental Figure 9) Because plants 554

were kept uncovered for 3 h after MeJA treatment it is unclear how much MeJA was 555

bioavailable in the plant However JAZ10 expression was induced 3 h after spraying 556

with MeJA (Supplemental Figure 10) Because pathogen growth generally depends on 557

the combination of a multitude of immune signaling events we conclude that multiple 558

changes to the immune signaling in gae1 gae6 together account for the altered B 559

cinerea growth phenotypes detected 560

We showed that expression of GAE1 and GAE6 is repressed by the bacterial 561

hemibiotroph Pma ES4326 This repression did not occur in an eds1 or pad4 562

background or in pbs3 at the 24-h time point (Figure 1 and Supplemental Figure 1) 563

During the Arabidopsis - Pma ES4326 interaction the expression of many defense 564

related genes is altered in a PAD4EDS1 dependent fashion (Wang et al 2008) Some 565

of these expression changes require SA CBP60g and SARD1 are thought to act 566

downstream of PAD4 and EDS1 but upstream of SA-dependent responses (Wang et 567

al 2011) PBS3 is also thought to act downstream of PAD4 and EDS1 and upstream of 568

SA but the set of genes whose expression it affects is only partially overlapping with 569

those affected by CBP60gSARD1 (Wang et al 2008 Wang et al 2011) Thus GAE1 570

and GAE6 repression is regulated by the part of the immune signaling network that 571

requires PAD4 EDS1 and PBS3 but is independent of CBP60gSARD1 and SA 572

Because EDS1 PAD4 and PBS3 are important components of the plant immune 573

signaling network (Wiermer et al 2005 Nobuta et al 2007) one might conclude that 574

repression of GAE1 and GAE6 may be part of the plant immune response to Pma 575

ES4326 B cinerea treatment also reduced GAE1 and GAE6 expression but this was 576

mostly independent of PAD4 (Figure 6) while both Pma ES4326 and B cinerea repress 577

GAE1 and GAE6 expression the host signaling mechanisms responsible likely differ 578

In summary our data show that Arabidopsis GAE1 and GAE6 are required for 579

pectin biosynthesis leaf flexibility and immunity to Pma ES4326 and to specific B 580

Page 30 of 54

cinerea isolates We used macerozyme a commercial pectinase to mimic the effect of 581

B cinerea pectinases Macerozyme treatment released less pectin from gae1 gae6 cell582

walls and induced less immune signaling in gae1 gae6 plants Less water soluble pectin 583

also accumulated in B cinerea infected gae1 gae6 plants and macerozyme-induced 584

immunity to B cinerea isolate Gallo 1 was reduced in these plants We conclude that 585

the reduced pectin content in these mutants may lead to reduced release of soluble 586

pectin including active OGs during pathogen attack and that this compromises 587

immunity 588

589

590

Page 31 of 54

METHODS 591

Plant Materials and Growth Conditions 592

Wild-type Columbia (Col-0) and all mutant plants (in Col-0 background) were 593

grown on sterilized BM2 germinating mix (Berger Inc Quebec Canada) in a controlled 594

environment chamber (Conviron Pembina ND USA) with a 12-h photoperiod under 595

100 microM m-2 s-1 fluorescent illumination at 22degC and 75 relative humidity Germplasm 596

used is described in the Accession Numbers section at the end of Methods 597

598

Data Analysis and Replicates 599

For biological replicates samples were obtained from separate plant tissues and 600

in many cases from plants grown in different flats In some experiments multiple 601

technical replicates were measured per biological replicate For statistical analysis all 602

technical replicates were averaged and the averages were considered one biological 603

replicate Independent experiments were performed at different times and often 604

consisted of multiple biological replicates per experiment 605

Data analysis was performed using mixed linear models in the R programming 606

environment (Bates et al 2015) unless otherwise indicated Generally genotypes 607

treatments and time points were used as fixed effects and replicate-specific effects like 608

different pots flats etc were considered random effects We reported p-values 609

obtained from the mixed linear model when less than 10 comparisons were performed 610

and q-values for more than 10 comparisons to correct for multiple comparison testing 611

Q-values were calculated using the Benjamini-Hochberg procedure (Benjamini and612

Hochberg 1995) unless specified otherwise 613

614

Pathogen Strains Growth Conditions and Pathogen Growth Assays 615

Growth of Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) and 616

bacterial growth assays were performed as previously described (Bethke et al 2014) 617

For qRT-PCR assays four week-old plants were inoculated with bacteria (OD600 = 618

0002) or mock (5 mM MgSO4) At least four leaves from two plants were collected at 619

the indicated time points per biological replicate 620

Page 32 of 54

Prior to each experiment Botrytis cinerea isolates were grown on 1x PDA 621

medium (Difco) for 10 d at room temperature All isolates used have been described 622

previously (Rowe and Kliebenstein 2007) The spores were washed from the surface of 623

the plate using an inoculation medium (1x Gamborgrsquos B-5 basal salt mixture (Sigma-624

Aldrich G5768) 2 (wv) glucose 10 mM phosphate buffer pH 64) and fungal hyphae 625

were removed from the suspension by filtering through four layers of cheesecloth The 626

concentration of spores was determined using a hemocytometer and adjusted to 25 x 627

105 spores mL-1 Four week-old Arabidopsis plants were inoculated by placing one 10-628

microL droplet of the B cinerea spore solution or inoculation medium (mock) on the adaxial 629

leaf surface of fully expanded leaves Inoculated plants were kept at 100 relative 630

humidity Infection sites were collected at various time points using a cork borer For 631

qRT-PCR assays at least four leaf discs from one plant were combined for each 632

biological sample For B cinerea growth assays at least 12 leaf discs from three to six 633

plants were pooled per biological sample Relative fungal growth was determined by 634

measuring the abundance of a fungal gene relative to a plant gene as described below 635

In brief DNA from infected tissue was extracted and equal amounts of total DNA were 636

used to perform qPCR reactions using the B cinerea cutinase A and the Arabidopsis 637

SK11 gene respectively Primers were described in (Gachon and Saindrenan 2004) 638

and are listed in Supplemental Table 1 Two technical replicates were combined for 639

each biological replicate B cinerea is a necrotrophic fungal pathogen and may destroy 640

plant DNA as disease progresses However decreases in the amount of plant DNA 641

over the course of our experiments were only observed at late time points with very 642

susceptible plants so the qPCR assay is generally a reasonable estimate of fungal 643

biomass (Supplemental Figure 16) 644

645

Treatment with Macerozyme or OGs 646

For PTI assays four week-old plants either were inoculated with 001 (wv) 647

macerozyme R-10 (Yakult) a Rhizopus polygalacturonase (EC 32115) that possesses 648

high pectinase and hemicellulase activity or were mock inoculated using heat-649

inactivated macerozyme Other similar plants were either inoculated with 250 or 500 microg 650

mL-1 OGs or mock inoculated with water One d later plants were infected with B 651

Page 33 of 54

cinerea and samples were collected immediately (0 dpi) and two or three d later To 652

investigate the role of MeJA on B cinerea growth plants were sprayed with 1mM MeJA 653

in water with 001 (vv) Tween 20 Covers were removed so that leaves could dry off 654

before plants were inoculated with B cinerea three h later Samples were harvested at 655

the indicated time points For expression analysis and MAPK activation assays plants 656

were either inoculated with 100 microg mL-1 OGs or mock inoculated with water OGs used 657

for experiments described in Figures 8 to 10 and Supplemental Figure 12 were a kind 658

gift from Simone Ferrari OGs used in experiments described in Figure 13 and 659

Supplemental Figures 11 13 and 15 were prepared as previously described (Kohorn et 660

al 2014) 661

662

Expression Analysis 663

Leaf tissue from inoculated plants was harvested at the indicated time points 664

flash frozen pulverized and RNA extracted using Trizol (Invitrogen) Quantitative RT-665

PCR was performed using the SuperScript III Platinum SYBR Green One-Step 666

quantitative RT-PCR kit (Invitrogen) and a Lightcycler 480 Real-Time PCR system 667

(Roche) as previously described (Truman and Glazebrook 2012) In brief equal 668

amounts of total RNA and a gene specific primer were used for each reaction The 669

crossing point (Cp) was calculated using the 2nd derivative max method provided with 670

the Lightcycler software for each amplification curve Each reaction was run with two 671

technical replicates and the Cp values for these replicates were averaged ACTIN2 was 672

used as a stably expressed reference gene At least three biological replicates each 673

were performed and analyzed using a mixed-linear model RT-PCR was performed to 674

verify T-DNA insertion lines using the One-Step RT-PCR kit (Qiagen) according to the 675

manufacturerrsquos instructions The primers used can be found in Supplemental Table 1 676

677

Electrolyte Leakage Assay 678

Fully expanded leaves of four week-old plants were inoculated with water (Milli-Q 679

grade) using a needleless syringe Leaf discs were collected using a cork borer and 680

three leaf discs from one leaf were placed with the adaxial surface down onto 2 mL 681

water in a 12-well cell culture plate Conductivity was measured using a Horiba B-173 682

Page 34 of 54

conductivity meter after 30 min and then the water was replaced Conductivity was 683

measured again 30 min later (1 h total) as indicated in Figure 3C Six biological 684

replicates each were performed in three independent experiments and means and SEs 685

were calculated Two-sided t-tests were performed for each time point 686

687

DAB and Aniline Blue Staining 688

Leaves from four week-old plants were either mock inoculated with B cinerea 689

inoculation medium or inoculated with 10 microL droplets of a B cinerea spore solution (25 690

x 105 spores mL-1) of the indicated isolates Samples were collected 72 h later For 33prime-691

diaminobenzidine (DAB) staining two to five inoculated leaves were vacuum infiltrated 692

with 33prime-Diaminobenzidine (1 mg mL-1 in water pH 38) solution and stained overnight 693

in the dark Leaves were destained using 100 ethanol at 65degC and then transferred to 694

50 (vv) glycerol and photographed 695

Callose was visualized by staining leaves with aniline blue as previously 696

described (Adam and Somerville 1996) Briefly leaves were vacuum infiltrated with 697

alcoholic lactophenol (one volume of phenolglycerollactic acidwater (1111) mixed 698

with two volumes of ethanol) and destained at 65degC Next leaves were moved through 699

an alcohol gradient (50 20 and 10 (vv) ethanol) into water The destained leaves 700

were stained for 30 min in 150 mM K2HPO4 (pH 95) with 001 (wv) aniline blue 701

Samples were mounted in water and examined using a Nikon Eclipse Ni-U microscope 702

at 10x magnification using a DAPI filter (excitation at 325-375 nm emission at 435-485 703

nm) For callose quantification six 200 microm x 200 microm squares were counted for each 704

sample type Callose deposits per mm2 were calculated and data analyzed using a t-705

test Note that there is an inherent uncertainty associated with such counts because 706

callose deposits may not all be in focus at the same time 707

708

MAPK Activity Assay 709

Fully expanded leaves of four week-old plants were inoculated with 001 710

macerozyme 100 microg mL-1 OGs 100 nM flg22 or water (mock) flg22 a peptide from 711

bacterial flagella that induces immune responses was used as a positive control for 712

Page 35 of 54

MAPK activation (Asai et al 2002) and was purchased from EZBiolab Inc (Westfield 713

IN USA) Samples were harvested 10 min later and immediately frozen in liquid 714

nitrogen Protein was extracted using an extraction buffer containing 25 mM Tris pH 78 715

75 mM NaCl 1 mM DTT 1 mM NaF 05mM Na3VO4 01 (vv) Tween 20 one 716

cOmplete Mini protease inhibitor cocktail tablet (Roche) and one PhosSTOP 717

phosphatase inhibitor cocktail tablet (Roche) per 10 mL buffer 15 microg of total protein 718

were electrophoresed on a 10 SDS-polyacrylamide gel and then blotted onto a PVDF 719

membrane (Bio-Rad 162-0177) Activated MAPKs were detected using p4442 MAPK 720

(Erk12) antibody (9102S Cell Signal 12500 in TBST (20 mM Tris pH 75 150 mM 721

NaCl 01 (vv) Tween 20)) Anti-Rabbit IgG-HRP (115000 in TBST A6154 Sigma) 722

and ECL Plus substrate (Pierce 32132) MPK3 and MPK6 were detected with an anti-723

MPK3 antibody (M8318 Sigma 12000 in TBST and 3 (wv) milk) or an anti-MPK6 724

antibody (A7104 Sigma 16000 in TBST and 3 (wv) milk) respectively The 725

secondary antibody and detection were the same as for the p4442 antibody Five 726

independent experiments were performed 727

728

Preparation of Alcohol Insoluble Residue (AIR) Pectin Extraction and Uronic Acid 729

Measurements 730

Fully expanded leaves from four week-old plants that were incubated for 48 h in 731

the dark to reduce starch were harvested flash frozen and pulverized AIR was 732

extracted by washing ground material twice in 70 (vv) ethanol three times in a 733

mixture of chloroform and methanol (11 vv) and once in acetone (Gille et al 2009) 734

and was then air dried Cell wall pectin was extracted from AIR with cell wall extraction 735

buffer (50 mM Trizma 50 mM CDTA pH 72) at 95degC for 15 min Samples were 736

homogenized using a paint shaker (Harbil 5G-HD) and glass beads (3 mm diameter) 737

For dot blot experiments 500 microL of cell wall extraction buffer were used per 10 mg of 738

AIR Debris was precipitated by centrifugation for 10 min at 10000 g 739

Total uronic acid content of pectin extracted as described above using 500 microL of 740

cell wall extraction buffer per 1 mg of AIR was measured as described previously 741

(Filisetti-Cozzi and Carpita 1991 van den Hoogen et al 1998) First 36 microL of pectin 742

extract were mixed with 4 microL of 4 M sulfamic acid Then 200 microL of sulfuric acid 743

Page 36 of 54

containing 120 mM sodium tetraborate was added and samples were incubated at 80degC 744

for 1 h Following cooling on ice the optical density of samples was measured at 490 745

nm Next 40 microL of m-hydroxydiphenyl reagent (100 microL of m-hydroxydiphenyl in dimethyl 746

sulfoxide at 100 mgmL mixed with 49 mL 80 (vv) sulfuric acid just before use) was 747

added and the samples were mixed The optical density at 490 nm was measured 748

again The optical density before m-hydroxydiphenyl reagent addition was subtracted 749

from the optical density measured after addition of the dye The concentration of uronic 750

acid was calculated using known amounts of GalA as a standard 751

752

Release of Pectin from Arabidopsis Cell Walls Using Macerozyme 753

AIR preparations from leaves of four week-old plants were treated with 025 754

(wv) macerozyme R-10 (Yakult) or water (mock) at a concentration of 100 microL per mg 755

AIR Samples were incubated for 3 h at room temperature on a vortex shaker and 756

centrifuged for 10 min at 12000 g Total uronic acid concentration of 5-fold diluted 757

supernatants was determined for two technical replicates each The concentration of 758

uronic acid was calculated using known amounts of GalA as a standard For 759

subsequent qRT-PCR experiments supernatants from multiple extractions were 760

combined boiled for 20 min to inactivate macerozyme diluted 10-fold with water and 761

inoculated into four week-old Col-0 plants Heat-inactivated 10-fold diluted macerozyme 762

solutions were used as mock treatment Samples were collected 3 h later and PAD3 763

expression was determined 764

765

Release of Pectin from Arabidopsis Leaves Infected with B cinerea 766

Fully expanded leaves of four week-old plants were either inoculated with up to 767

five 10 microL droplets of B cinerea spore solution (25 x 105 spores mL-1) or mock 768

inoculated with B cinerea inoculation medium For each biological replicate leaves with 769

a combined count of at least 20 infection sites were collected 48 h later AIR was 770

extracted as described above but plants were not dark-treated or freeze-dried and the 771

first ethanol step was performed using 85 (vv) ethanol To extract soluble pectin 772

fragments AIR samples were mixed with water at a concentration of 20 microL per infection 773

site incubated for 7 h at room temperature on a vortex shaker and centrifuged for 10 774

Page 37 of 54

min at 12000 g Total uronic acid concentration of supernatants was determined for two 775

technical replicates each The concentration of uronic acid was calculated using known 776

amounts of GalA as a standard 777

778

Measurement of Uronic Acids Neutral Monosaccharides and Cellulose Content in 779

Cell Walls 780

Neutral sugars and uronic acids from non-cellulosic polysaccharides in cell walls 781

(AIR) were released by trifluoroacetic acid (TFA) hydrolysis Approximately 1 mg of AIR 782

was hydrolyzed with 2 M TFA at 121degC for 90 min The TFA-soluble material was split 783

into two equal parts one for uronic acid and one for neutral sugar determination The 784

uronic acid content was determined by analyzing the hydrolyzate using a CarboPac 785

PA200 anion-exchange column with an ICS-3000 Dionex chromatography system 786

(Dionex Sunnyvale CA USA) The elution profile consisted of a linear gradient of 50 to 787

200 mM sodium acetate in 01 M NaOH in 10 min at 04 mL per min Alditol acetate 788

derivatives of neutral sugars were produced as described (York et al 1985) In brief 789

monosaccharides in the hydrolyzate were reduced with NaBH4 and peracetylated with 790

acetic anhydride and pyridine at 121degC for 20 min The generated alditol acetates were 791

analyzed by GC-MS Crystalline cellulose was measured according to Updegraff (1969) 792

After hydrolysis of noncellulosic polysaccharides from AIR with the Updegraff reagent 793

(acetic acid nitric acids water 812 (vv) ) the remaining pellet was hydrolyzed in 794

72 sulfuric acid The resulting glucose quantity was determined by the anthrone 795

method (Scott and Melvin 1953) 796

797

Dot Blot Experiments 798

Pectin solutions extracted using 50 microL of cell wall extraction buffer per mg AIR 799

were serially diluted and nitrocellulose membranes were spotted with 1 microL of the diluted 800

pectin solutions Membranes were dried overnight blocked with 5 milk (wv) in 1x 801

PBS (8 g L-1 NaCl 02 g L-1 KCl 144 g L-1 Na2HPO4 024 g L-1 KH2PO4 pH 74) and 802

probed with LM5 (Jones et al 1997) LM6 (Willats et al 1998) LM8 (Willats et al 803

2004) LM19 and LM20 (Verhertbruggen et al 2009a) or CCRC-M7 (Steffan et al 804

Page 38 of 54

1995) antibodies LM series antibodies were diluted 1250 and CCRC-M7 was diluted 805

1500 in 5 milk powder (Nestle Carnation) in 1 x PBS For LM series antibodies a 806

goat anti-Rat HRP conjugated antibody (Bethyl A110-105P 15000 diluted) and for 807

CCRC-M7 an anti-Mouse IgG HRP conjugate (Promega W402B 12500 diluted) was 808

used as secondary antibody in 5 milk powder in 1x PBS LM series antibodies were 809

obtained from plant probes (wwwplantprobesnet) and CCRC-M7 from CarboSource 810

(wwwcarbosourcenet) Membranes were washed after incubation with the primary and 811

secondary antibodies with 1x PBS Dot blots were developed using the ECL system 812

(GE Healthcare Amersham ECL prime) For quantification of signals dot blot results on 813

x-ray films were photographed using a CCD camera and intensities were measured814

using Image Jrsquos Integrated Density function Locally adjusted background was 815

subtracted As the dynamic range of the signal on an x-ray film was narrow non-816

linearity in the measured values was corrected for each genotype in each biological 817

replicate using the values from different dilutions in two technical replicates We noticed 818

that the residuals were not normally distributed and performed Box-Cox power 819

transformation (Box and Cox 1964) to obtain normally distributed values for the 820

subsequent statistical analysis of the data For LM19 Box-Cox selected power 821

transformation was done to the power of 038th and for LM20 to the power 075th After 822

correction of the transformed measured values with the effect of the biological 823

replicates the values were fit to a linear model with the Arabidopsis genotype as the 824

fixed effect This linear model was used both to generate the plots in Figures 5B and D 825

and to test significant differences 826

827

Camalexin Measurements 828

Four week-old plants were infected with B cinerea and samples (three leaf disks 829

from one plant each) were harvested at the indicated time points flash frozen and 830

pulverized Each sample was extracted with 300 microL of 90 methanol (vv) 50 microL of the 831

extract was run on an Agilent Lichrocart 250-4 RP18e 5-microm column using an Agilent 832

1100 series HPLC Camalexin was detected using a DiodeArray (DAD) at 330 nm and 833

with a fluorescence detector at emission 318 nmexcitation 385 nm (Agilent) Separation 834

was achieved using the following program with aqueous acetonitrile 5 min gradient 835

Page 39 of 54

from 63 to 69 acetonitrile 30 s gradient from 69 to 99 acetonitrile 2 min at 99 836

acetonitrile and a post-run equilibration of 35 min at 63 acetonitrile (Denby et al 837

2004 Kliebenstein et al 2005) Purified camalexin was used to produce a standard 838

curve to identify and quantitate camalexin 839

840

Accession Numbers 841

Microarray data used can be accessed at NCBI Gene Expression Omnnibus 842

(httpwwwncbinlmnihgovgeo) GSE18978 Sequence information for all Arabidopsis 843

genes described in this article can be found in TAIR (wwwarabidopsisorg) using the 844

following accession numbers PAD3 (CYP71B15 At3g26830) SID2 (ICS1 At1g74710) 845

GAE1 (At4g30440) GAE2 (At1g02000) GAE3 (At4g00110) GAE4 (At2g45310) GAE5 846

(At4g12250) GAE6 (At3g23820) PR1 (At2g14610) JAZ5 (At1g17380) JAZ10 847

(At5g13220) ACTIN2 (At3g18780) PDF12a (At5g44420) PDF12b (At2g26020) 848

AtSK11 (At5g26751) The sequence of the B cinerea cutinase Bc-CutA (Z69264) can 849

be found in the DNA Data Bank of Japan (httpgetentryddbjnigacjp) Germplasm 850

used included dde2-2 (At5g42650 (von Malek et al 2002)) pad3-1 (At3g26830 (Zhou 851

et al 1999)) pad4-1 (At3g52430 (Jirage et al 1999)) sid2-2 (At1g74710 (Wildermuth 852

et al 2001)) pbs3-2 (At5g13320 SALK_018225 (Nobuta et al 2007)) cbp60a-1 853

(At5g62570 SALK_124410 (Truman et al 2013)) cbp60g-1 sard1-2 (At5g26920 854

SALK_023199 At1g73805 SALK_052422 (Wang et al 2011) cbp60a-1 cbp60g-1 855

sard1-2 (Truman et al 2013) gae1-1 (At4g30440 SALK_085554) gae6-1 (At3g23280 856

SALK_104454) gae6-2 (At3g23280 SALK_017191) ixr1-1 cev1 (At5g05170 (Ellis 857

and Turner 2001 Scheible et al 2001)) eds1 plants were derived by introgression of 858

the Ler eds1-2 allele into Col-0 that contains two copies of EDS1 (At3g48090 859

At3g48080 (Bartsch et al 2006) mpk6-2 (At2g43790 SALK_073907 (Liu and Zhang 860

2004)) mpk3-DG (At3g45640 a fast neutron deletion mutant (Miles et al 2005)) and 861

npr1-1 (At1g64280 (Cao et al 1997)) T-DNA insertion lines were part of the SALK 862

collection (Alonso et al 2003) and were obtained from the Arabidopsis Biological 863

Resource Center 864

865

Supplemental Data 866

Page 40 of 54

Supplemental Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 867

Expression Does Not Require CBP60s or SID2 868

Supplemental Figure 2 Expression of GAE Family Members uponTreatment with Pma 869

ES4326 870

Supplemental Figure 3 Characterization of T-DNA Insertions in GAE1 and GAE6 871

Supplemental Figure 4 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves and 872

Are Slightly Smaller than Wild-Type Col-0 Plants 873

Supplemental Figure 5 Cell Walls of gae1 gae6 Plants Contain Less Uronic Acid than 874

Wild-Type Col-0 Cell Walls 875

Supplemental Figure 6 Antibodies Raised Against the RGI or XG Components of 876

Pectin Show No Difference in Binding to gae Mutant and Col-0 Cell Walls 877

Supplemental Figure 7 The Effect of B cinerea Treatment on the Expression of GAE 878

Family Genes 879

Supplemental Figure 8 Treatment with 001 Macerozyme Does Not Cause Any 880

Visible Damage to Col-0 Plants 881

Supplemental Figure 9 B cinerea Growth on Plants with Defects in Immune 882

Signaling 883

Supplemental Figure 10 MeJA-Induced Expression of the JA marker gene JAZ10 884

Supplemental Figure 11 The Expression of the JA Marker Gene JAZ5 Is Highly 885

Responsive in gae1 gae6 Plants 886

Supplemental Figure 12 The Expression of PDF12 Is Unaltered in gae1 gae6 Plants 887

Supplemental Figure 13 Macerozyme Treatment Activates MPK3 and MPK6 888

Supplemental Figure 14 B cinerea-Induced ROS Production in Wild-Type Col-0 and 889

gae1 gae6 Plants 890

Supplemental Figure 15 OG-Induced PTI Using 250 microg mL-1 OGs 891

Supplemental Figure 16 Abundance of Plant DNA in B cinerea Treated Plants 892

Supplemental Table 1 Primers used in this study 893

894

ACKNOWLEDGEMENTS 895

We thank William Truman for helpful discussions Simone Ferrari (Sapienza 896

Universita di Roma) for the generous gift of OGs Bruna Bucciarelli (USDA-ARS) for 897

Page 41 of 54

help with fluorescence microscopy the Arabidopsis Biological Resource Center for T-898

DNA insertion lines Paul Knoxrsquos lab (University of Leeds) for LM5 LM6 LM8 LM19 899

and LM20 antibodies and the Complex Carbohydrate Resource Center (University of 900

Georgia) for the CCRC-M7 antibody Most of this work was funded by the Division of 901

Chemical Sciences Geosciences and Biosciences Office of Basic Energy Sciences of 902

the US Department of Energy through Grant DE-FG02-05ER15670 to JG This work 903

was also supported by NSF awards IOS 1339125 and IOS 1021861 to DJK NSF 904

award IOS 1121425 to F K and the USDA National Institute of Food and Agriculture 905

Hatch project number CA-D-PLS-7033-H to DJK 906

907

AUTHOR CONTRIBUTIONS 908

GB and JG designed the research GB GX AT BL NES and NH 909

performed research DJK provided tools GB GX MP AD BL RAH and FK 910

analyzed data GB and JG wrote the paper with input from all authors 911

912

FIGURE LEGENDS 913

Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 Expression 914

Requires PAD4 EDS1 and PBS3 915

(A) Expression of GAE1 in leaves of wild-type Col-0 and mutant plants Expression was916

measured 24 or 32 h after either inoculation with Pma ES4326 (OD600 = 0002) or mock 917

inoculation with 5 mM MgSO4 and was then normalized to the level of ACTIN2 Data 918

from three biological replicates were combined using a mixed linear model Bars 919

represent mean log2-ratios to ACTIN2 plusmn SE Letters indicate significantly different 920

groups with q lt 005 where 005 represents the maximum false discovery rate at which 921

the test may be called significant 922

(B) Expression of GAE6 measured as described in (A)923

924

Figure 2 Susceptibility of gae6 and gae1 gae6 Plants to Pma ES4326 Is Increased 925

Relative to Wild-Type Col-0 926

Leaves of plants of the indicated genotypes were inoculated with Pma ES4326 (OD600 = 927

00002) as described in Methods Bacterial titers in leaves were determined either 928

Page 42 of 54

immediately (0 dpi) or after three days (3 dpi) Bars represent the mean plusmn SE of four 929

independent experiments each with 4 or 12 biological replicates at 0 and 3 dpi 930

respectively Data were combined using a mixed linear model Asterisks indicate 931

significant differences from Col-0 wild-type (q lt 005) Susceptible pad4 plants were 932

included as a positive control 933

934

Figure 3 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves 935

(A) Depiction of the assay for brittleness of gae mutant leaves Fully expanded leaves of936

four to five week-old plants were bent by 180ordm so that the adaxial surface of the leaf was 937

parallel with the soil Broken leaf petioles or midribs were considered brittle 938

(B) Quantification of broken midribs or petioles For each genotype 66 to 82 leaves 939

were analyzed for brittleness as described in (A) Bars represent the ratio of broken to 940

total number of leaves tested Results were analyzed using Fisherrsquos exact test Letters 941

indicate significantly different groups at p lt 005 942

(C) Conductivity measurements Leaves of four week-old plants were inoculated with943

deionized water and then leaf discs were cut out and placed on deionized water 944

Conductivity was measured at the indicated time points After 30 min the water was 945

replaced with fresh water (30 min f w) to account for wounding-related ion leakage from 946

the edges of the leaf discs Three independent experiments with six biological replicates 947

each were performed and data were analyzed using a t-test for each time point P-948

values were corrected using the Bonferroni method Data show means plusmn SE asterisks 949

indicate significant differences from wild-type Col-0 at q lt 001 950

951

Figure 4 Cell Walls of gae6 and gae1 gae6 Plants Are Reduced in GalA 952

(A) to (D) Levels of GalA (A) GlcA (B) cellulose (C) and neutral sugars (D) in alcohol953

insoluble residue (AIR) extracted from leaves of four week-old plants of the indicated 954

genotypes Bars represent means plusmn SE of four biological replicates combined using a 955

mixed linear model Letters indicate significantly different groups (p lt 005) 956

957

Figure 5 Cell Walls of gae1 gae6 Plants Are Reduced in Homogalacturonan 958

Page 43 of 54

Dot blot analyses with anti-LM19 and anti-LM20 antibodies Pectin was extracted from 959

AIR prepared from the indicated genotypes using 1 microL of extraction buffer per 10 microg of 960

AIR Pectin was serially diluted and one microL of undiluted 13 diluted and 19 diluted 961

samples were spotted on a nitrocellulose membrane Three biological replicates were 962

were spotted twice to obtain two technical replicates each All replicates produced 963

similar results 964

(A) Representative dot blot using anti-LM19 antibody965

(B) Relative signal intensities of the spots in the dot blot in (A)966

(C) Representative dot blot using anti-LM20 antibody967

(D) Relative signal intensities of the LM20 dot blot shown in (C)968

Relative signal intensities were measured using Image J software Measurements from 969

the three biological replicates were combined using a linear model after Box-Cox power 970

transformation as described in Methods Bars represent means plusmn SE Asterisks indicate 971

a difference from wild-type Col-0 at p lt 005 972

973

Figure 6 Growth of B cinerea on gae1 gae6 Plants and the Effect of B cinerea on 974

Expression of GAE1 and GAE6 975

(A) Growth of B cinerea isolates Leaves of Col-0 and gae1 gae6 plants were976

inoculated with 25 x 105 spores mL-1 of the B cinerea isolates indicated in the figure 977

Samples were collected two (2 dpi) and three (3 dpi) days later Bars represent means plusmn 978

SE of six biological replicates for each fungus combined using a mixed linear model 979

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 980

(B) and (C) Expression of GAE1 (B) and GAE6 (C) 48 h after inoculation with the B981

cinerea isolates indicated Expression levels were measured by qRT-PCR Bars 982

represent mean log2-ratios to ACTIN2 plusmn SE of three biological replicates combined 983

using a mixed linear model Letters indicate significantly different groups (q lt 005) 984

985

Figure 7 Levels of Soluble Pectin and Expression of PAD3 in Fractions Produced 986

Following Macerozyme Treatment of gae1 gae6 Cell Walls 987

(A) Schematic overview of the experimental procedure for (B) and (C)988

Page 44 of 54

(B) Uronic acid measurements Alcohol-insoluble residue (AIR) from leave tissue of four989

week-old wild-type Col-0 or gae1 gae6 plants was treated with water or 025 990

macerozyme and assayed for uronic acid levels Data from 11 biological replicates were 991

combined using a mixed linear model Bars represent means plusmn SE Letters indicate 992

significant differences (p lt 005) 993

(C) Expression of PAD3 in Col-0 plants treated with the fractions from (B) Extracts from994

(B) were heat inactivated pooled and diluted 5-fold with water Pooled extracts were995

inoculated into four week-old Col-0 plants PAD3 expression was measured 3 h after 996

inoculation and normalized to the level of ACTIN2 expression Data from four biological 997

replicates were combined using a mixed linear model Bars represent mean log2-ratios 998

to ACTIN2 plusmn SE Letters indicate significant differences (p lt 005) 999

1000

Figure 8 Macerozyme-Induced PAD3 Expression Is Absent in gae1 gae6 1001

(A) Expression of PAD3 after macerozyme treatment Four week-old plants were either1002

inoculated with 001 macerozyme or mock inoculated with water and PAD3 1003

expression was measured 3 h later by qRT-PCR Data from four biological replicates 1004

were combined using a mixed linear model Mean log2-ratios to ACTIN2 plusmn SE were 1005

plotted Letters indicate significantly different groups (p lt 005) 1006

(B) Expression of PAD3 after OG treatment PAD3 expression was measured as1007

described in (A) 3 h after inoculation with 100 microg mL-1 OGs or mock inoculation with 1008

water Data are from six biological replicates 1009

(C) PAD3 expression in untreated plants Three biological replicates were performed1010

and data analyzed as described in (A) 1011

(D) Camalexin accumulation after B cinerea treatment Camalexin accumulation was1012

measured in four week-old plants inoculated with the B cinerea strains shown in the 1013

figure Eight biological replicates each were combined using a mixed linear model 1014

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 1015

(E) PAD3 expression 28 h after treatment with the indicated B cinerea strains Data1016

from six biological replicates were collected and analyzed as described in (A) Data in 1017

all figure parts represent means plusmn SE Letters indicate significantly different groups (q lt 1018

005) 1019

Page 45 of 54

1020

Figure 9 Macerozyme-Induced Expression of the SA Marker Genes SID2 and PR1 1021

Is Abolished in gae1 gae6 Plants 1022

(A) to (H) Expression of PR1 (A) (C) (E) and (G) or SID2 (B) (D) (F) and (H) in four1023

week-old Col-0 and gae1 gae6 plants Data were combined using a mixed linear model 1024

Bars represent mean log2-ratios of expression vs ACTIN2 plusmn SE for all figure parts 1025

Letters indicate significantly different groups at p lt 005 for panels (A) to (D) (G) and 1026

(H) and qlt005 for panels (E) to (F) 1027

(A) and (B) Plants were treated with 001 macerozyme or mock treated with water1028

and samples were collected 3 h later Data from four biological replicates are shown 1029

(C) and (D) Plants were inoculated with 100 microg mL-1 OGs or mock treated with water1030

and samples collected 3 h later Data from six biological replicates are shown 1031

(E) and (F) Plants were inoculated with the indicated B cinerea isolates or mock1032

inoculated with B cinerea inoculation medium and samples collected 48 h later Data 1033

from eight biological replicates are shown 1034

(G) and (H) Expression in untreated plants Three biological replicates were performed1035

1036

Figure 10 The Expression of the JA Marker Gene JAZ10 is Highly Responsive in 1037

gae1 gae6 Plants 1038

(A) to (D) JAZ10 expression in four week-old plants Data were combined using a mixed1039

linear model Mean log2-ratios to ACTIN2 plusmn SE are shown Letters indicate significantly 1040

different groups at p lt 005 for panels (A) (B) and (D) and q lt 005 for panel (C) 1041

(A) Plants were inoculated with 001 macerozyme or mock inoculated using water and1042

samples were collected 3 h later Data from four biological replicates are shown 1043

(B) Plants were inoculated with 100 microg mL-1 OGs or mock inoculated using water and1044

samples collected 3 h later Data are from six biological replicates 1045

(C) JAZ10 expression 48 h after treatment with the indicated B cinerea strains Data1046

are from six biological replicates 1047

(D) JAZ10 expression in untreated plants Data are from three biological replicates1048

1049

Figure 11 Gallo 1-Induced Callose Deposition Is Reduced in gae1 gae6 Plants 1050

Page 46 of 54

(A) Callose deposition in four week-old Col-0 and gae1 gae6 plants Plants were1051

inoculated with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) or mock inoculated 1052

with B cinerea inoculation medium and samples were collected after 72 h Callose was 1053

stained with aniline blue and visualized using a Nikon Eclipse Ni-U microscope with a 1054

DAPI filter Three leaves were stained per treatment Scale bars are 100 microm Asterisks 1055

indicate fungal hyphae arrows indicate representative callose spots 1056

(B) Quantification of callose deposition Callose deposits were counted in 200 microm x 2001057

microm squares laid out across the images shown here Bars represent means plusmn SE from 1058

six squares each Letters indicate significantly different groups (p lt 005) according to a 1059

t-test analysis1060

1061

Figure 12 Less Soluble Pectin Accumulates in B cinerea-Infected gae1 gae6 1062

Leaves 1063

Four week-old Col-0 and gae1 gae6 plants were inoculated with 10 microL droplets of the 1064

indicated B cinerea isolate (25 x 105 spores mL-1) or mock inoculated with B cinerea 1065

inoculation medium Leaves were collected 48 h later AIR was extracted from leaf 1066

samples and water added (20 microL infection site) to extract soluble pectin fragments 1067

Samples were vortexed for 7 h centrifuged at 12000 g and the uronic acid 1068

concentration of the supernatant was measured Data from nine biological replicates 1069

were combined using a mixed linear model Means plusmn SE are shown Letters indicate 1070

significant differences (q lt 005) 1071

1072

Figure 13 Macerozyme-Induced PTI Is Lost in gae1 gae6 While OG-Induced PTI Is 1073

Unaltered 1074

(A) Macerozyme-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1075

inoculated with 001 macerozyme or mock inoculated with boiled macerozyme and 1076

were infected with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) 24 h later Samples 1077

were collected immediately (0 dpi) and after two (2 dpi) and three (3 dpi) days Data 1078

show mean plusmn SE of six to 14 biological replicates each combined using a linear model 1079

Letters indicate significantly different groups (q lt 005) 1080

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

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Page 2 of 54

Loss of UDP-glucuronate 4-epimerases GAE1 and GAE6 in Arabidopsis thaliana 31

causes a dramatic reduction of pectin in the cell wall and compromised immunity to 32

Pseudomonas syringae and Botrytis cinerea 33

34

35

SYNOPSIS 30

ABSTRACT 36

37

Plant cell walls are important barriers against microbial pathogens Cell walls of 38

Arabidopsis thaliana leaves contain three major types of polysaccharides cellulose 39

various hemicelluloses and pectins UDP-D-galacturonic acid the key building block of 40

pectins is produced from the precursor UDP-D-glucuronic acid by the action of 41

glucuronate 4-epimerases (GAEs) Pseudomonas syringae pv maculicola ES4326 42

(Pma ES4326) repressed expression of GAE1 and GAE6 in Arabidopsis and immunity 43

to Pma ES4326 was compromised in gae6 and gae1 gae6 mutant plants These plants 44

had brittle leaves and cell walls of leaves had less galacturonic acid (GalA) Resistance 45

to specific Botrytis cinerea isolates was also compromised in gae1 gae6 double mutant 46

plants Although oligogalacturonide (OG)-induced immune signaling was unaltered in 47

gae1 gae6 mutant plants immune signaling induced by a commercial pectinase 48

macerozyme was reduced Macerozyme treatment or infection with B cinerea released 49

less soluble uronic acid likely reflecting fewer OGs from gae1 gae6 cell walls than from 50

wild-type Col-0 Although both OGs and macerozyme induced immunity to B cinerea in 51

Col-0 only OGs also induced immunity in gae1 gae6 Pectin is thus an important 52

contributor to plant immunity and this is due at least in part to the induction of immune 53

responses by soluble pectin likely OGs that are released during plant-pathogen 54

interactions 55

56

Page 3 of 54

INTRODUCTION 57

The cell walls in Arabidopsis thaliana leaves are mainly primary cell walls 58

consisting of three major types of polysaccharidesmdashcellulose various hemicelluloses 59

and various pectic polysaccharidesmdashas well as some structural proteins (Liepman et 60

al 2010) Arabidopsis leaf walls contain approximately 14 cellulose a homopolymer 61

of (1 4)-β linked D-glucose subunits (Zablackis et al 1995 Carpita 2010) 62

Hemicelluloses are a diverse class of polysaccharides that includes xylans xyloglucans 63

mannans glucomannans and mixed-linkage β-glucans (Scheller and Ulvskov 2010) In 64

Arabidopsis leaves the major hemicellulose is xyloglucan which constitutes about 20 65

of the wall polysaccharides Xyloglucan contains a (1 4)-β-linked glucan backbone 66

substituted with (1 6)-α-linked xylosyl residues or side chains of xylosyl galactosyl and 67

fucosyl residues (Zablackis et al 1995 Liepman et al 2010) Glucuronoarabinoxylan 68

(4 of the wall) is also found in Arabidopsis leaves (Zablackis et al 1995) Primary 69

walls of dicotyledonous plants generally also contain 3-5 of the hemicelluloses 70

mannan and glucomannan (Scheller and Ulvskov 2010) Hence hemicelluloses in 71

primary walls of dicotyledonous plants are mainly composed of glucose (Glc) xylose 72

(Xyl) arabinose (Ara) galactose (Gal) and mannose (Man) 73

Pectins are a diverse group of polysaccharides that all contain galacturonic acid 74

(GalA) and make up about 50 of Arabidopsis leaf walls (Zablackis et al 1995 Harholt 75

et al 2010) Homogalacturonan (HG) is a linear homopolymer of (14)-α-linked 76

galacturonic acid (GalA) residues and it makes up about 65 of all pectin in 77

Arabidopsis leaf walls (Zablackis et al 1995 Mohnen 2008) A linear (14)-α-linked 78

GalA backbone substituted with single xylose residues is called xylogalacturonan 79

(XGA) and a polymer with complex side-chains containing borate ions and sugars such 80

as Ara rhamnose (Rha) Gal Xyl or fucose (Fuc) is referred to as rhamnogalacturonan 81

II (RGII) (Mohnen 2008 Harholt et al 2010) XGA and RGII make up less than 10 of 82

leaf cell wall pectin (Zandleven et al 2007 Mohnen 2008) In contrast 83

rhamnogalacturonan I (RGI) backbones consist of a repeating α-14-D-GalA-α-12-L-Rha 84

disaccharide and are substituted with β-(14)-galactan branched arabinan or 85

arabinogalactan side chains (Mohnen 2008 Harholt et al 2010) RGI constitutes about 86

20 ndash 25 of pectin in primary walls (Mohnen 2008) Hence pectin in Arabidopsis leaf 87

Page 4 of 54

cell walls consists mostly of GalA Rha and smaller amounts of other sugars including 88

Ara Gal Xyl and Fuc 89

In general carbohydrate biosynthesis requires nucleotide sugars provided by 90

nucleotide sugar interconversion pathways (Seifert 2004) Most nucleotide sugars are 91

synthesized from uridine diphosphate-Glc (UDP-Glc) UDP-glucuronic acid (UDP-GlcA) 92

is made from UDP-Glc by UDP-glucose dehydrogenase activity or via an alternative 93

pathway requiring inositol oxygenase activity (Tenhaken and Thulke 1996 Loewus and 94

Murthy 2000) UDP-D-Glucuronate 4-epimerases (GAEs) interconvert UDP-D-GlcA and 95

UDP-D-GalA the monomeric precursor of pectin There are six GAE genes encoded by 96

the Arabidopsis genome When heterologously expressed in Escherichia coli or Pichia 97

pastori GAE1 GAE4 and GAE6 showed activity in interconverting UDP-D-GlcA and 98

UDP-D-GalA (Gu and Bar-Peled 2004 Molhoj et al 2004 Usadel et al 2004) GAE1 99

and GAE6 were found to be Golgi-localized and strongly expressed in various plant 100

tissues (Molhoj et al 2004 Usadel et al 2004 Parsons et al 2012) GAE1 and GAE6 101

have been hypothesized to be evolutionarily older than the other GAE family members 102

(Usadel et al 2004) and might have overlapping functions that are distinct from the 103

other family members 104

Plant cell wall composition and architecture affects wall strength and flexibility 105

and cell walls present a physical barrier to potential plant pathogens Besides preformed 106

physical barriers such as a cell wall plants have a sophisticated immune system to 107

defend themselves against harmful microbial pathogens (Chisholm et al 2006 Jones 108

and Dangl 2006) Immune signaling involves changes in phytohormone levels the most 109

important for plant immunity being salicylic acid (SA) jasmonic acid (JA) and ethylene 110

(ET) (Grant and Jones 2009 Pieterse et al 2012) Other major regulators of plant 111

immunity include PHYTOALEXIN DEFICIENT 4 (PAD4) and ENHANCED DISEASE 112

SUSCEPTIBILITY 1 (EDS1) (Wiermer et al 2005) which are predicted to encode 113

lipases Immune signaling generally involves changes in the transcriptome that in turn 114

lead to altered cellular responses (Tao et al 2003) For instance induced expression of 115

the cytochrome P450 PAD3 is thought to increase production of the anti-microbial 116

secondary metabolite camalexin (Zhou et al 1999) 117

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Because plant cell walls are important barriers against pathogenic microbes 118

alterations in wall structural properties can lead to changes in plant immunity To date 119

only a few examples of cell wall related mutants with altered pathogen phenotypes have 120

been studied in detail Mutants impaired in callose biosynthesis powdery mildew 121

resistant 4 (pmr4) display enhanced resistance to the powdery mildew pathogens 122

Golovinomyces orontii and G cichoracearum (Nishimura et al 2003) These plants 123

have constitutively high SA levels and SA signaling is required for the resistance 124

phenotype (Nishimura et al 2003) Other plants with enhanced resistance to these 125

powdery mildew pathogens include pmr5 a mutant enriched in pectin and pmr6 a 126

mutant with a defect in a pectate lyase-like gene (Vogel et al 2002 Vogel et al 2004) 127

Plants with mutations in cellulose synthases required for primary and secondary wall 128

formation also show increased resistance to pathogens (Ellis and Turner 2001 129

Hernandez-Blanco et al 2007) Enhanced resistance to powdery mildew and 130

Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) in constitutive 131

expression of VSP 1 (cev1) which is mutated in the primary wall cellulose synthase 132

CesA3 was linked to constitutive high JA and ET levels in these plants (Ellis and 133

Turner 2001 Ellis et al 2002a) Interestingly irregular xylem1 (irx1) irx3 and irx5 134

plants which carry mutations in the secondary wall-associated cellulose synthases 135

CesA8 CesA7 and CesA4 showed enhanced resistance to the necrotrophic pathogens 136

Plectosphaerella cucumerina Ralstonia solanacearum and Botrytis cinerea as well as 137

the biotrophic powdery mildew G cichoracearum whereas growth of P cucumerina and 138

R solanacearum pathogens was unaltered in pmr5 pmr6 and cev1 plants (Hernandez-139

Blanco et al 2007) This enhanced resistance of the irx mutants was independent of 140

SA JA or ET signaling but instead involved abscisic acid signaling (Hernandez-Blanco 141

et al 2007) Changes in wall structure are likely monitored by the plants and effects 142

may be counteracted through initiation of defense responses that can lead to altered 143

phytohormone levels 144

Although plant cell walls serve as barriers against pathogen entry many 145

pathogens secrete cell wall-degrading enzymes (CWDEs) and thus are able to 146

successfully infect their host plants (Albersheim et al 1969) For example the B 147

cinerea polygalacturonase Bc-PG1 is required for full virulence of this pathogen and the 148

Page 6 of 54

gene encoding it shows evidence for diversifying selection as would be expected for a 149

virulence gene (ten Have et al 1998 Rowe and Kliebenstein 2007) Secretion of 150

CWDEs can trigger the release of cell wall fragments which can act as damage-151

associated molecular patterns (DAMPs) that activate plant immune responses (Hahn et 152

al 1981 Ferrari et al 2013) Similarly pectin fragments known as oligogalacturonides 153

(OGs) can trigger plant immune responses (Cote and Hahn 1994 Ferrari et al 2013) 154

Such responses include the production of reactive oxygen species (ROS) activation of 155

mitogen-activated protein kinase (MAPK) activation and enhanced expression of 156

defense-related genes such as PAD3 (Ferrari et al 2007 Denoux et al 2008 Galletti 157

et al 2008 Galletti et al 2011) Some of these responses require SA JA and ET 158

whereas others are independent of these phytohormones (Ferrari et al 2007) 159

Here we describe two GAE family members GAE1 and GAE6 whose 160

expression is repressed upon pathogen challenge A gae1 gae6 double mutant has 161

brittle leaves and its walls are reduced in pectin specifically HG and likely RGI 162

Resistance to the bacterial pathogen Pma ES4326 and the fungal necrotroph B cinerea 163

is compromised in gae1 gae6 Callose deposition is reduced in gae1 gae6 plants after 164

treatment with B cinerea isolate Gallo 1 Furthermore these plants show altered 165

activation of immune signaling in response to treatment with macerozyme a 166

polygalacturonase that degrades pectin and they also are hyperresponsive to JA 167

signaling Macerozyme treatment released less soluble likely low molecular weight 168

pectin from isolated gae1 gae6 cell walls than from Col-0 cell walls B cinerea-induced 169

accumulation of soluble likely low molecular weight pectin was also decreased in gae1 170

gae6 plants Additionally gae1 gae6 plants are impaired in macerozyme-induced 171

immunity to B cinerea possibly due to decreased release of OGs from their cell walls 172

173

174

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RESULTS 175

176

GAE1 and GAE6 Expression Is Repressed by Pma ES4326-Induced Immune 177

Signaling 178

Our previous research suggested that the status of pectin specifically pectin 179

modification by methylesterification is important for plant immunity against Pma 180

ES4326 (Bethke et al 2014) Here we investigate whether pectin abundance in 181

addition to its specific structure is also important for plant immunity by taking a closer 182

look at the formation of the pectin precursor UDP-GalA by GAEs 183

184 Microarray experiments showed that expression of GAE1 and GAE6 was

185 repressed in A thaliana (Arabidopsis) plants treated with Pma ES4326 (Wang et al

186 2008) Repression of GAE1 and GAE6 in wild-type Arabidopsis Col-0 plants treated with

187 Pma ES4326 was confirmed by quantitative real time (qRT)-PCR Figure 1) To test if

188 this repression is mediated by immune signaling qRT-PCR was also performed in

189 mutants with blocked immune signaling (Figure 1 and Supplemental Figure 1) 190 Generally immunity to Pma ES4326 requires intact SA signaling Repression of both

191 GAE1 and GAE6 required the presence of the major immune regulators EDS1 and

192 PAD4 required for SA signaling as well as other immune responses (Jirage et al 1999

193 Wiermer et al 2005 Bartsch et al 2006 Wang et al 2008) at both early and late time

194 points The presence of AVRPPHB SUSCEPTIBLE 3 (PBS3) a regulator of many

195 immune responses including SA signaling (Nobuta et al 2007) was also required for

196 repression of GAE1 and GAE6 at an early time point (Figure 1) CALMODULIN-

197 BINDING PROTEIN 60-LIKE a (CBP60a) (a negative regulator of SA synthesis and

198 immunity (Truman et al 2013)) CBP60g and SYSTEMIC ACQUIRED RESISTANCE

199 DEFICIENT 1 (SARD1) (both promote SA biosynthesis and regulation of other immune

200 responses (Wang et al 2011)) SALICYLIC ACID INDUCTION DEFICIENT 2 SID2

201 (encodes a biosynthetic enzyme for SA (Wildermuth et al 2001)) and

202 NONEXPRESSER OF PR GENES 1 NPR1 (a regulator of the majority of SA-induced

203 immune signaling (Cao et al 1997 Yan and Dong 2014)) were not required for this

204 repression (Figure 1 and Supplemental Figure1) Thus repression of GAE1 and GAE6

Page 8 of 54

205 requires a regulatory function that is dependent on EDS1 and PAD4 partially

206 dependent on PBS3 but independent of SA

Page 9 of 54

There are six GAE family members in Arabidopsis GAE2 expression was 207

induced by Pma ES4326 in the absence of PAD4 or EDS1 GAE3 expression was 208

unaltered GAE4 was repressed early and GAE5 expression was induced in all 209

genotypes (Supplemental Figure 2) The similarity of the immune-regulated expression 210

of GAE1 and GAE6 suggested a redundant role of these genes in Arabidopsis immunity 211

to Pma ES4326 212

213

Resistance to Pma ES4326 Is Compromised in gae1 gae6 and gae6 Plants 214

To study the roles of GAE1 and GAE6 in plant immunity in more detail plants 215

with T-DNA insertion mutations in GAE1 and GAE6 were obtained from the SALK 216

collection as described in the Accession Numbers section of Methods We identified two 217

GAE6 knock-out alleles gae6-1 and gae6-2 and a T-DNA insertion in GAE1 gae1-1 218

that caused reduced expression of this gene (Supplemental Figure 3) A double mutant 219

was created by crossing gae1-1 and gae6-1 Both gae6 and gae1 gae6 plants were 220

more susceptible to Pma ES4326 than wild-type Col-0 and gae1 plants supporting a 221

role of GAE6 in plant immunity to Pma ES4326 (Figure 2) 222

223

gae6 and gae1 gae6 Plants Have Brittle Leaves 224

While inoculating leaves of gae1 gae6 plants we noticed that the petioles and 225

midribs of these plants tended to break easily when leaves were bent against the 226

proximal-distal axis of the leaves (Figure 3 see Supplemental Figure 4D to F) They 227

tended to break less often when twisted around the proximal-distal axis To quantify 228

this phenotype leaves were bent by 180deg (Figure 3A) and breaking of midribs or 229

petioles was recorded Most gae1 gae6 leaves broke whereas fewer leaves of gae6 230

plants and very few leaves of wild-type Col-0 and gae1 plants did (Figure 3B and 231

Supplemental Figure 4C) Although gae1 gae6 plants had slightly shorter leaves and 232

petioles than wild type plants (Supplemental Figure 4A and B) no correlation between 233

leaf size or petiole length and breaking frequency was detected We also investigated 234

tissue integrity by monitoring ion leakage Interestingly when leaves were inoculated 235

with deionized water leaf discs of gae1 gae6 leaked more ions than wild-type Col-0 236

discs (Figure 3C) suggesting that the increased brittleness is correlated with a 237

Page 10 of 54

_____________________________________________________________________________

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Cell Walls of gae6 and gae1 gae6 Plants Have Lower Levels of Pectin 243

GalA is a major component of pectin We analyzed wall monosaccharide 244

composition in gae mutants to investigate whether the increased brittleness was 245

associated with an altered wall composition of gae leaves To approximate pectin 246

content of gae mutant walls the total uronic acid concentration was measured using a 247

simple colorimetric assay (Filisetti-Cozzi and Carpita 1991 van den Hoogen et al 248

1998) Total uronic acid was reduced in gae6-2 strongly reduced in gae1 gae6 plants 249

but was indistinguishable from wild-type Col-0 in gae6-1 and gae1 (Supplemental 250

Figure 5) This suggested that gae1 gae6 and possibly gae6 plants have reduced 251

pectin 252

To investigate the cell wall composition in leaves of gae plants in more detail we 253

measured the levels of seven neutral monosaccharides the acidic sugars GalA (the 254

monomeric subunit of the pectin backbone (Harholt et al 2010)) and GlcA (Figure 4) 255

We also estimated the crystalline cellulose content (Updegraff 1969) Cell walls of both 256

gae6 mutants and gae1 gae6 were reduced in GalA (Figure 4A) In gae1 gae6 GalA 257

content in cell walls was reduced by more than 40 compared to the wild-type level 258

GlcA levels were indistinguishable between mutants and wild-type (Figure 4B) 259

Cellulose content was higher in gae6-2 and gae1 gae6 walls likely due to the loss of 260

pectins (Figure 4C) Cell walls of gae1 gae6 plants were also enriched in all neutral 261

sugars measured presumably due to the substantial proportional loss of GalA (Figure 262

4D) The levels of most wall monosaccharides were even higher in the gae1 gae6 263

double mutant with the exception of Rha which did not exhibit a significant increase 264

compared to the gae6 single mutants The absence of a compensational increase in 265

Rha suggested that RGI the dominant Rha contributor to the wall may be considered 266

reduced in gae1 gae6 mutants compared to wild-type albeit not to such an extent as 267

HG 268

238 structural defect that promotes increased ion leakage Taken together these results

239 showed that loss of GAE6 causes brittle leaves and additional losss of GAE1 strongly

240 enhances this phenotype We conclude that GAE1 and GAE6 contribute to normal leaf

241 flexibility 242

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gae6 cell walls are reduced in HG Using the same samples we detected no difference 274

from wild type in binding of LM5 which recognizes a tetramer of (1-4)-β-D-galactans 275

found in RGI (Jones et al 1997) LM6 which recognizes a linear pentasaccharide in (1-276

To further investigate which pectic components were altered in gae1 gae6 dot 269

blot experiments using pectin isolated from the various genotypes were performed Both 270

LM19 and LM20 antibodies recognize HG (Verhertbruggen et al 2009a) As expected 271

LM19 binding was reduced in gae6-1 and gae1 gae6 (Figure 5A and B) and LM20 272

binding was reduced in gae1 gae6 (Figure 5C and D) This result confirmed that gae1 273

277 5)-α-L-arabinans found in RGI (Willats et al 1998 Verhertbruggen et al 2009b) LM8

278 which recognizes xylogalacturonan (Willats et al 2004) or CCRC-M7 which

279 recognizes an arabinosylated (1-6)-β-D-galactan epitope occurring on RGI (Puhlmann et

Page 13 of 54

Page 14 of 54

al 1994 Steffan et al 1995 Pattathil et al 2010) (Supplemental Figure 6) 280

Collectively these cell wall composition data show that gae1 gae6 plants are reduced in 281

HG Due to the strong proportional reduction of HG in the walls levels of all other 282

components should be higher However Rha indicative of RGI was not higher in gae1 283

gae6 suggesting that RGI may also be reduced in gae1 gae6 walls 284

285

GAE1 and GAE6 Are Required for Immunity to Specific B cinerea Isolates 286

We hypothesized that plant immunity to B cinerea might be affected by pectin 287

content in the host cell wall because this pathogen requires polygalacturonases and 288

pectin methylesterases for full virulence (ten Have et al 1998 Valette-Collet et al 289

2003 Kars et al 2005b Espino et al 2010) Because Bcinerea is genetically diverse 290

and has a high diversity of polygalacturonases (Rowe and Kliebenstein 2007) we 291

measured relative fungal growth at both 2 and 3 d post-inoculation (dpi) using ten 292

different B cinerea isolates (Figure 6) The B cinerea isolates Fresa 525 and Gallo 1 293

showed enhanced virulence on gae1 gae6 plants at both time points whereas KT and 294

UK Razz showed enhanced virulence in gae1 gae6 plants at 3 but not 2 d dpi (Figure 295

6A) We did not detect any significant changes in relative fungal growth between wild-296

type Col-0 and gae1 gae6 plants when using Acacia Apple 517 DN Grape Pepper or 297

Rasp isolates of B cinerea Therefore we continued our study using Gallo 1 and Fresa 298

525 We also included Pepper for comparison as this isolate has been used in a large-299

scale analysis of plant gene expression in response to B cinerea infection (Windram et 300

al 2012) 301

We next investigated whether expression of GAE1 and GAE6 was affected by 302

challenge with B cinerea Inoculation of wild-type Col-0 plants with any isolate 303

repressed GAE1 and GAE6 expression (Figure 6B and C) and this repression was 304

generally independent of PAD4 (Figure 6B and C) Expression of GAE2 and GAE3 was 305

unaltered by B cinerea treatment GAE4 was repressed only by Fresa 525 treatment 306

while GAE5 expression was enhanced with all three isolates tested (Supplemental 307

Figure 7) As seen for infection with Pma ES4326 the expression of GAE1 and GAE6 308

was repressed upon B cinerea treatment This indicates a role for GAE1 and GAE6 in 309

the immune response to B cinerea 310

Page 15 of 54

Page 16 of 54

Because the amount of pectin substrate for pathogen polygalacturonases is 313

reduced we hypothesized that immunity phenotypes of gae1 gae6 plants may result 314

from reduced generation of OGs by these enzymes In turn the reduced OGs could 315

lead to reduced immune responses To test this we used macerozyme a commercial 316

pectinase as a proxy for pathogen pectinases We isolated cell walls as a subcellular 317

fraction known as alcohol insoluble residue (AIR Gille et al 2009) This was treated 318

with macerozyme and tested for release of soluble likely low molecular weight pectin 319

fragments (Figure 7) Treatment with 025 macerozyme released significantly more 320

uronic acid from wild-type Col-0 AIR relative to gae1 gae6 AIR (Figure 7B) To test 321

activity of these pectin-containing solutions we infiltrated them into wild-type Col-0 322

plants and measured PAD3 expression PAD3 is required for synthesis of the 323

phytoalexin camalexin (Zhou et al 1999) and its expression is known to be induced by 324

OG treatment (Ferrari et al 2007) Fractions from macerozyme treated Col-0 walls 325

induced significantly more PAD3 expression than fractions derived from gae1 gae6 326

walls suggesting that macerozyme treatment did indeed release more active soluble 327

uronic acid possibly OGs from wild-type Col-0 walls than from gae1 gae6 walls (Figure 328

7C) 329

Next we studied the effect of macerozyme treatment on expression of PAD3 in 330

intact plants (Figure 8) This treatment did not lead to any visible leaf damage 331

(Supplemental Figure 8) In wild-type Col-0 PAD3 expression was induced three h after 332

treatment with 001 macerozyme (Figure 8A) By contrast no macerozyme-induced 333

induction of PAD3 expression was detected in gae1 gae6 (Figure 8A) However OG 334

treatment induced the expression of PAD3 in both genotypes indicating that gae1 gae6 335

plants do respond to OGs (Figure 8B) Macerozyme treatment also induced expression 336

of the SA marker genes PATHOGENESIS RELATED 1 (PR1) and SID2 in Col-0 but 337

not in gae1 gae6 (Figure 9 panel A and B) PR1 was not significantly induced by 338

purified OGs (Figure 9C) at the time and concentration tested but SID2 was induced by 339

OG treatment in both genotypes (Figure 9D) However the induction was stronger in 340

wild-type Col-0 In addition the JA markers JASMONATE-ZIM-DOMAIN PROTEIN 5 341

(JAZ5) and JAZ10 were induced by macerozyme and OG treatment in both Col-0 and 342

gae1 gae6 (Figure 10A and Supplemental Figure 11) Interestingly expression of JAZ5 343

312 Pectinase-Induced Immune Signaling Is Altered in gae1 gae6 Plants

Page 17 of 54

Page 18 of 54

Page 19 of 54

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344

345

346

and JAZ10 was already strongly induced in gae1 gae6 after mock inoculation (Figure 10

and Supplemental Figure 11) We observed no hyperresponsive induction by mock

treatment of PLANT DEFENSIN 12 (PDF12) a necrotrophy and late JA signaling

347 marker gene (Supplemental Figure 12A-D) PDF12 was also not induced by

348 macerozyme or OG treatments (Supplemental Figure 12A-D) This suggests that early

349 JA signaling is induced by mock-treatment in gae1 gae6 Because it is known that JA

350 and SA signaling act antagonistically (Pieterse et al 2012) the strong up-regulation of

351 JA-induced immune signaling in gae1 gae6 might explain the lower OG-induced SID2

352 expression in this genotype

353 To test whether other immune responses are altered in gae1 gae6 plants we

354 analyzed Mitogen-activated protein kinase (MAPK) activity following macerozyme and

355 OG treatments OGs are known to activate the MAPKs MPK3 and MPK6 in Arabidopsis

Page 21 of 54

(Galletti et al 2011) Macerozyme treatment activated MPK3 and MPK6 in wild-type 356

Col-0 whereas MAPK activation in gae1 gae6 was slightly reduced in four out of five 357

experiments performed (Supplemental Figure 13) By contrast OG-induced MAPK 358

activity was similar or increased in gae1 gae6 in four out of five experiments 359

(Supplemental Figure 13) Additionally mock treatment induced MAPK activity in gae1 360

gae6 more strongly than it did in wild-type Col-0 in four out of five experiments 361

performed (Fig S13) Collectively these results illustrate that activation of several 362

immune signaling componentsmdashcamalexin synthesis SA and JA signaling and 363

possibly MAPK activationmdashby macerozyme is substantially altered in gae1 gae6 This 364

suggests that release of soluble pectin fragments possibly OGs by pectinolytic 365

enzymes might be reduced in gae1 gae6 plants while recognition of OGs is intact 366

367

Following B cinerea Infection Production of Soluble Pectin and Deposition of 368

Callose Are Reduced in gae1 gae6 Plants 369

Although pad3 plants were more susceptible to the B cinerea isolates Fresa 525 370

and Gallo 1 which also grew better on gae1 gae6 growth of the isolate Pepper was 371

similar on Col-0 and pad3 (Supplemental Figure 9A) However we did not detect any 372

significant changes in camalexin production after treatment with B cinerea isolate Gallo 373

1 and Fresa 525 in gae1 gae6 (Figure 8D) Neither did we detect changes in PAD3 374

expression after treatment with any B cinerea isolate in gae1 gae6 (Figure 8E) 375

Additionally SID2 and PR1 were similarly induced by all B cinerea isolates in both Col-376

0 and gae1 gae6 (Figure 9E and F) and sid2 plants which are deficient for pathogen-377

induced SA biosynthesis did not show any differences in relative B cinerea growth 378

(Supplemental Figure 9B) Expression of JAZ10 was induced more strongly by Gallo 1 379

treatment in gae1 gae6 than in wild-type Col-0 but not by Fresa 525 or Pepper (Fig 380

10C) while dde2 plants deficient for JA biosynthesis showed enhanced susceptibility to 381

all B cinerea isolates tested (Supplemental Figure 9B) Curiously exogenous 382

application of MeJA did not lead to altered relative fungal growth of B cinerea 383

(Supplemental Figure 9C) and cev1 isoxaben resistant 1 (ixr1) plants which have 384

constitutively high JA levels (Ellis and Turner 2001) did not show any changes in 385

susceptibility to the B cinerea isolates tested (Supplemental Figure 9D) Hence neither 386

Page 22 of 54

differences in B cinerea isolate susceptibility to camalexin nor altered SA or JA 387

signaling by themselves explained the enhanced susceptibility of gae1 gae6 to these B 388

cinerea isolates 389

Hydrogen peroxide accumulation in response to B cinerea isolates Fresa 525 390

Gallo 1 and Pepper was not obviously altered in gae1 gae6 plants (Supplemental Figure 391

14) However callose deposition upon treatment with B cinerea isolate Gallo 1 was 392

strongly reduced in gae1 gae6 (Figure 11) It was previously shown that OG treatment 393

induces callose deposition (Denoux et al 2008) suggesting that the reduction in 394

callose deposition might be due to reduced OG release in gae1 gae6 It is possible that 395

this reduction in callose deposition contributes to the reduced immunity of gae1 gae6 396

plants to B cinerea isolate Gallo 1 397

Because macerozyme could be used to release soluble likely low molecular 398

weight pectin from isolated cell walls we next investigated whether infection with B 399

cinerea was accompanied by accumulation of soluble pectin as well We prepared cell 400

walls (AIR fraction) from leaves infected with B cinerea incubated these cell walls with 401

water and measured uronic acid content of the extracts B cinerea treatment did in fact 402

lead to increased accumulation of soluble pectin in wild-type Col-0 plants when 403

compared to mock-treated samples (Figure 12) No such increased accumulation of 404

water-soluble pectin fractions was observed in gae1 gae6 (Figure 12) 405

406

Macerozyme-Induced Pattern-Triggered Immunity against B cinerea Is Abolished 407

in gae1 gae6 408

It was previously shown that OG treatment can induce patternndashtriggered 409

immunity (PTI) to B cinerea (Ferrari et al 2007) We tested macerozyme treatment to 410

determine whether it could also induce PTI to the B cinerea isolate Gallo 1 (Figure 13) 411

Indeed pretreatment with 001 macerozyme for 24 h led to reduced B cinerea growth 412

in wild-type Col-0 but not gae1 gae6 plants (Figure 13A) Interestingly mock and 413

macerozyme pretreated gae1 gae6 plants exhibited similar growth of B cinerea Gallo 1 414

as did macerozyme-treated Col-0 plants This result is likely due to activation of JA 415

signaling in gae1 gae6 during the pretreatment procedure (Figure 13C and D) OG-416

induced PTI was observed in both genotypes when using a high dose of OGs (Figure 417

Page 23 of 54

13B) whereas a lower dose of OGs only induced PTI in Col-0 (Supplemental Figure 418

15) This suggests that OG perception in gae1 gae6 at least above a certain threshold 419

is unaltered while release of immunity-activating pectin fragments possibly OGs is 420

421 reduced We conclude that immune responses induced by macerozyme treatment

422 which do not occur in gae1 gae6 plants are important for plant immunity to the B

423 cinerea isolate Gallo 1

Page 24 of 54

____________________________________________________________________________

Page 25 of 54

DISCUSSION 426

Here we showed that loss of GAE1 and GAE6 leads to a dramatic reduction in 427

pectin content in the cell wall (Figure 4) likely causing the brittleness of leaves that we 428

observed (Figure 3) Petioles and midribs of gae1 gae6 plants broke easily when bent 429

and inoculation of leaves with deionized water resulted in increased ion leakage 430

possibly due either to microscopic fractures of the cell wall induced by inoculation stress 431

or to a reduced capability of these walls to withstand increased turgor pressure The 432

increased brittleness was associated with altered cell wall composition In our analysis 433

the GalA content of gae1 gae6 leaf walls was reduced by more than 40 and the total 434

uronic acids by over 60 (Figure 4 and Supplemental Figure 5) This is a major change 435

in the overall cell wall composition considering that pectin makes up about 50 of the 436

plant cell walls found in Arabidopsis leaves (Zablackis et al 1995 Nobuta et al 2007 437

Harholt et al 2010) Interestingly quasimodo1 (qua1) plants which have a mutation in 438

a glycosyltransferase family 8 gene are reduced in GalA by 25 and show reduced 439

binding of HG specific antibodies (Bouton et al 2002) 440

Furthermore qua2 plants which have a mutation in a putative methyltransferase 441

have about 13 less total GalA and lack 50 of cell wall HG (Mouille et al 2007) 442

Likewise cgr2 cgr3 plants carrying mutations in another class of Golgi-localized 443

putative methyl-transferases have leaf cell walls that are reduced by about 40 in 444

uronic acid (Kim et al 2015) All three mutants are severely dwarfed (Bouton et al 445

2002 Mouille et al 2007 Kim et al 2015) Additionally qua1 and qua2 mutants show 446

a distorted overall shape likely due to reduced cell cohesion and are extremely fragile 447

(Bouton et al 2002 Mouille et al 2007) By contrast gae1 gae6 plants with a similar 448

strong reduction in GalA and total uronic acids have an overall normal shape and are 449

only slightly smaller than wild-type Col-0 (Supplemental Figure 4) In the wall analysis 450

performed here we did not detect changes in other wall polysaccharides or structures 451

that might compensate for the reduction of pectin in gae1 gae6 It is possible the severe 452

morphological alterations in qua2 and cgr2 cgr3 plants are due to reduction in pectin 453

methylesterification which is unaffected in gae1 gae6 It was speculated that quality 454

control mechanisms in the Golgi prevent secretion of inefficiently esterified pectin in 455

cgr2 cgr3 (Kim et al 2015) suggesting that the Golgi of these plants might over-456

Page 26 of 54

accumulate pectin and that this may contribute to the severe dwarf phenotype of these 457

plants The gae1 gae6 plants described here provide an opportunity to study the effects 458

of reduction of pectin in plants that do not show severe morphological phenotypes 459

We found that gae1 gae6 plants are also compromised in resistance to B 460

cinerea and Pma ES4326 indicating that loss of pectin reduces immunity to these 461

pathogens (Figure 2 and 6) We found that gae1 gae6 plants were slightly more 462

susceptible to Pma ES4326 but showed a strong increase in susceptibility to specific B 463

cinerea isolates This was not unexpected because B cinerea is known to interact with 464

pectin as part of its infection strategy It secretes various pectin-degrading enzymes 465

early during infection and requires some of these enzymes including PG1 PG2 and 466

PME1 for full virulence (ten Have et al 1998 Valette-Collet et al 2003 Kars et al 467

2005b Espino et al 2010) Additionally Arabidopsis plants expressing an antisense 468

construct of the polygalacturonase inhibitor PGIP1 are significantly more susceptible to 469

B cinerea (Ferrari et al 2006) On the other hand Pma ES4326 is not known to470

require cell wall degrading enzymes for full virulence even though it encodes two 471

pectate lyase genes in its genome (PMA4326_07094 and PMA4326_04621 472

wwwbacteriaensemblorg) B cinerea isolates are also known to be genetically quite 473

diverse in their polygalacturonase (PG) loci and genetic variability in PG2 is associated 474

with varying growth on pectin containing media (Rowe and Kliebenstein 2007) 475

Furthermore the contribution of PG1 and PG2 to B cinerea virulence seems to depend 476

on the specific B cinerea isolate and the host species (Kars et al 2005a Zhang and 477

Van Kan 2013) B cinerea isolates are also known to be differently sensitive to the anti-478

microbial secondary metabolite camalexin (Kliebenstein et al 2005) In line with these 479

observations the susceptibility of gae1 gae6 plants differed depending on the specific 480

B cinerea isolate Whereas Fresa 525 Gallo 1 UK Razz and KT grew better in gae1481

gae6 we detected no difference in Acacia Apple 517 DN Grape Pepper or Rasp 482

growth (Figure 6) 483

B cinerea was shown to metabolize pectin and to possibly utilize GalA as a484

carbon source during in planta growth (Zhang et al 2011 Zhang and Van Kan 2013) 485

Our data show that gae1 gae6 plants which are reduced in GalA content did allow 486

enhanced growth of specific B cinerea isolates but no reduction in growth of any B 487

Page 27 of 54

cinerea isolates tested was found This suggests that utilization of pectin as a carbon 488

source was not a contributing factor to alterations in B cinerea susceptibility It is 489

possible that changes in the cell wall composition affect the structural integrity of the 490

wall and affect hyphal penetration of specific B cinerea isolates However OGs (small 491

HG fragments) are likely released during the infection process (Ferrari et al 2013) 492

OGs function as DAMPs and initiate immune responses in Arabidopsis (Ferrari et al 493

2013) Treatment of Arabidopsis with OGs having a degree of polymerization (DP) of 494

10-15 sugar subunits was previously shown to enhance resistance to B cinerea (Ferrari495

et al 2007) We showed that pretreatment with OGs or macerozyme induced enhanced 496

immunity to B cinerea isolate Gallo 1 in wild-type Col-0 but only OGs and not 497

macerozyme induced immunity in gae1 gae6 (Figure 13) Our data suggest gae1 gae6 498

plants which have reduced pectin content may release less OGs and that this 499

reduction leads to the increased susceptibility to B cinerea 500

Consistently we found that macerozyme treatment released less soluble pectin 501

from gae1 gae6 cell walls than from wild-type walls Fractions containing soluble pectin 502

released from gae1 gae6 were also less active in inducing PAD3 gene expression than 503

the fractions released from wild-type walls (Figure 7) We also showed that less soluble 504

pectin accumulated in gae1 gae6 than in wild-type plants 48 h after treatment with B 505

cinerea (Figure 12) 506

We observed that loss of pectin leads to alterations in A thaliana immune 507

signaling Macerozyme treatment induced expression of PAD3 and the SA marker 508

genes PR1 and SID2 in wild-type Col-0 but not in gae1 gae6 whereas OG treatment 509

led to increased PAD3 and SID2 expression in both wild-type and gae1 gae6 (Figure 8 510

and 9) This suggests that although release of active soluble pectin in these plants 511

might be altered OG recognition is intact In fact cell walls isolated from qua1 another 512

mutant known to be reduced in HG released less OGs (DP1-6) than did wild-type cell 513

walls upon endopolygalacturonase treatment (Bouton et al 2002) This was also true 514

for gae1 gae6 cell walls as macerozyme treatment of isolated gae1 gae6 cell walls 515

released less soluble pectin than treatment of Col-0 cell walls Because the fraction 516

from gae1 gae6 plants was also less active in inducing PAD3 expression (Figure 7) it 517

seems possible that biologically active OGs were the active component inducing PAD3 518

Page 28 of 54

expression Interestingly expression of the JA marker genes JAZ5 and JAZ10 was 519

higher in gae1 gae6 than in wild-type mock-inoculated plants but was comparable to 520

wild-type in untreated plants suggesting that JA signaling in gae1 gae6 is 521

hyperresponsive (Figure 10 and Supplemental Figure 11) This induction of JA signaling 522

might be due to wounding stress incurred during the inoculation process JA and SA 523

signaling generally act antagonistically (Pieterse et al 2012) Increased JA signaling 524

activity might hence explain why mock- and macerozyme-treated gae1 gae6 plants 525

exhibited reduced expression of the SA marker PR1 and why OG treatment induced 526

expression of SID2 another SA marker to a lower extent in gae1 gae6 than in wild-type 527

plants (Figure 9) 528

No single immune signaling component was found to be the sole contributor to 529

the compromised immunity of gae1 gae6 to specific B cinerea isolates such as Gallo 1 530

and Fresa 525 These isolates grew better on pad3 while growth of Pepper another B 531

cinerea isolate that did not show altered virulence on gae1 gae6 was unaltered 532

(Supplemental Figure 9) This suggested that differences in camalexin sensitivity of 533

these B cinerea isolates might account for the differences in gae1 gae6 susceptibility 534

However camalexin accumulation and PAD3 expression was the same for wild-type 535

Col-0 and gae1 gae6 for the B cinerea isolates Fresa 525 and Gallo 1 (Figure 8) which 536

showed enhanced growth on gae1 gae6 plants It should be noted that B cinerea 537

virulence is often estimated by comparing lesion sizes Lesion size and measurements 538

of relative fungal growth by qPCR do not always show the same results For example 539

although Denby et al (2004) showed that Pepper causes larger lesions on pad3 than on 540

wild-type this study showed that relative fungal growth of Pepper on pad3 was 541

unaltered 542

We observed that callose deposition was strongly reduced in gae1 gae6 plants 543

72 h after treatment with B cinerea isolate Gallo 1 (Figure 11) possibly due to reduced 544

release of active soluble pectin Interestingly OG-induced PTI to an unspecified isolate 545

of B cinerea was intact in the pmr4 mutant which does not produce callose (Galletti et 546

al 2008) Due to the high SA levels in pmr4 plants (Nishimura et al 2003) and the fact 547

that different B cinerea isolates vary both in induction of and reaction to defense 548

responses (Kliebenstein et al 2005 Rowe et al 2010) we could not determine 549

Page 29 of 54

whether reduced callose deposition causes the increased susceptibility of gae1 gae6 to 550

B cinerea isolate Gallo 1551

We did not observe any changes in B cinerea growth either on cev1 ixr1 plants 552

which are known to have constitutively high JA and OPDA levels (Ellis et al 2002b) or 553

on plants exogenously treated with MeJA (Supplemental Figure 9) Because plants 554

were kept uncovered for 3 h after MeJA treatment it is unclear how much MeJA was 555

bioavailable in the plant However JAZ10 expression was induced 3 h after spraying 556

with MeJA (Supplemental Figure 10) Because pathogen growth generally depends on 557

the combination of a multitude of immune signaling events we conclude that multiple 558

changes to the immune signaling in gae1 gae6 together account for the altered B 559

cinerea growth phenotypes detected 560

We showed that expression of GAE1 and GAE6 is repressed by the bacterial 561

hemibiotroph Pma ES4326 This repression did not occur in an eds1 or pad4 562

background or in pbs3 at the 24-h time point (Figure 1 and Supplemental Figure 1) 563

During the Arabidopsis - Pma ES4326 interaction the expression of many defense 564

related genes is altered in a PAD4EDS1 dependent fashion (Wang et al 2008) Some 565

of these expression changes require SA CBP60g and SARD1 are thought to act 566

downstream of PAD4 and EDS1 but upstream of SA-dependent responses (Wang et 567

al 2011) PBS3 is also thought to act downstream of PAD4 and EDS1 and upstream of 568

SA but the set of genes whose expression it affects is only partially overlapping with 569

those affected by CBP60gSARD1 (Wang et al 2008 Wang et al 2011) Thus GAE1 570

and GAE6 repression is regulated by the part of the immune signaling network that 571

requires PAD4 EDS1 and PBS3 but is independent of CBP60gSARD1 and SA 572

Because EDS1 PAD4 and PBS3 are important components of the plant immune 573

signaling network (Wiermer et al 2005 Nobuta et al 2007) one might conclude that 574

repression of GAE1 and GAE6 may be part of the plant immune response to Pma 575

ES4326 B cinerea treatment also reduced GAE1 and GAE6 expression but this was 576

mostly independent of PAD4 (Figure 6) while both Pma ES4326 and B cinerea repress 577

GAE1 and GAE6 expression the host signaling mechanisms responsible likely differ 578

In summary our data show that Arabidopsis GAE1 and GAE6 are required for 579

pectin biosynthesis leaf flexibility and immunity to Pma ES4326 and to specific B 580

Page 30 of 54

cinerea isolates We used macerozyme a commercial pectinase to mimic the effect of 581

B cinerea pectinases Macerozyme treatment released less pectin from gae1 gae6 cell582

walls and induced less immune signaling in gae1 gae6 plants Less water soluble pectin 583

also accumulated in B cinerea infected gae1 gae6 plants and macerozyme-induced 584

immunity to B cinerea isolate Gallo 1 was reduced in these plants We conclude that 585

the reduced pectin content in these mutants may lead to reduced release of soluble 586

pectin including active OGs during pathogen attack and that this compromises 587

immunity 588

589

590

Page 31 of 54

METHODS 591

Plant Materials and Growth Conditions 592

Wild-type Columbia (Col-0) and all mutant plants (in Col-0 background) were 593

grown on sterilized BM2 germinating mix (Berger Inc Quebec Canada) in a controlled 594

environment chamber (Conviron Pembina ND USA) with a 12-h photoperiod under 595

100 microM m-2 s-1 fluorescent illumination at 22degC and 75 relative humidity Germplasm 596

used is described in the Accession Numbers section at the end of Methods 597

598

Data Analysis and Replicates 599

For biological replicates samples were obtained from separate plant tissues and 600

in many cases from plants grown in different flats In some experiments multiple 601

technical replicates were measured per biological replicate For statistical analysis all 602

technical replicates were averaged and the averages were considered one biological 603

replicate Independent experiments were performed at different times and often 604

consisted of multiple biological replicates per experiment 605

Data analysis was performed using mixed linear models in the R programming 606

environment (Bates et al 2015) unless otherwise indicated Generally genotypes 607

treatments and time points were used as fixed effects and replicate-specific effects like 608

different pots flats etc were considered random effects We reported p-values 609

obtained from the mixed linear model when less than 10 comparisons were performed 610

and q-values for more than 10 comparisons to correct for multiple comparison testing 611

Q-values were calculated using the Benjamini-Hochberg procedure (Benjamini and612

Hochberg 1995) unless specified otherwise 613

614

Pathogen Strains Growth Conditions and Pathogen Growth Assays 615

Growth of Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) and 616

bacterial growth assays were performed as previously described (Bethke et al 2014) 617

For qRT-PCR assays four week-old plants were inoculated with bacteria (OD600 = 618

0002) or mock (5 mM MgSO4) At least four leaves from two plants were collected at 619

the indicated time points per biological replicate 620

Page 32 of 54

Prior to each experiment Botrytis cinerea isolates were grown on 1x PDA 621

medium (Difco) for 10 d at room temperature All isolates used have been described 622

previously (Rowe and Kliebenstein 2007) The spores were washed from the surface of 623

the plate using an inoculation medium (1x Gamborgrsquos B-5 basal salt mixture (Sigma-624

Aldrich G5768) 2 (wv) glucose 10 mM phosphate buffer pH 64) and fungal hyphae 625

were removed from the suspension by filtering through four layers of cheesecloth The 626

concentration of spores was determined using a hemocytometer and adjusted to 25 x 627

105 spores mL-1 Four week-old Arabidopsis plants were inoculated by placing one 10-628

microL droplet of the B cinerea spore solution or inoculation medium (mock) on the adaxial 629

leaf surface of fully expanded leaves Inoculated plants were kept at 100 relative 630

humidity Infection sites were collected at various time points using a cork borer For 631

qRT-PCR assays at least four leaf discs from one plant were combined for each 632

biological sample For B cinerea growth assays at least 12 leaf discs from three to six 633

plants were pooled per biological sample Relative fungal growth was determined by 634

measuring the abundance of a fungal gene relative to a plant gene as described below 635

In brief DNA from infected tissue was extracted and equal amounts of total DNA were 636

used to perform qPCR reactions using the B cinerea cutinase A and the Arabidopsis 637

SK11 gene respectively Primers were described in (Gachon and Saindrenan 2004) 638

and are listed in Supplemental Table 1 Two technical replicates were combined for 639

each biological replicate B cinerea is a necrotrophic fungal pathogen and may destroy 640

plant DNA as disease progresses However decreases in the amount of plant DNA 641

over the course of our experiments were only observed at late time points with very 642

susceptible plants so the qPCR assay is generally a reasonable estimate of fungal 643

biomass (Supplemental Figure 16) 644

645

Treatment with Macerozyme or OGs 646

For PTI assays four week-old plants either were inoculated with 001 (wv) 647

macerozyme R-10 (Yakult) a Rhizopus polygalacturonase (EC 32115) that possesses 648

high pectinase and hemicellulase activity or were mock inoculated using heat-649

inactivated macerozyme Other similar plants were either inoculated with 250 or 500 microg 650

mL-1 OGs or mock inoculated with water One d later plants were infected with B 651

Page 33 of 54

cinerea and samples were collected immediately (0 dpi) and two or three d later To 652

investigate the role of MeJA on B cinerea growth plants were sprayed with 1mM MeJA 653

in water with 001 (vv) Tween 20 Covers were removed so that leaves could dry off 654

before plants were inoculated with B cinerea three h later Samples were harvested at 655

the indicated time points For expression analysis and MAPK activation assays plants 656

were either inoculated with 100 microg mL-1 OGs or mock inoculated with water OGs used 657

for experiments described in Figures 8 to 10 and Supplemental Figure 12 were a kind 658

gift from Simone Ferrari OGs used in experiments described in Figure 13 and 659

Supplemental Figures 11 13 and 15 were prepared as previously described (Kohorn et 660

al 2014) 661

662

Expression Analysis 663

Leaf tissue from inoculated plants was harvested at the indicated time points 664

flash frozen pulverized and RNA extracted using Trizol (Invitrogen) Quantitative RT-665

PCR was performed using the SuperScript III Platinum SYBR Green One-Step 666

quantitative RT-PCR kit (Invitrogen) and a Lightcycler 480 Real-Time PCR system 667

(Roche) as previously described (Truman and Glazebrook 2012) In brief equal 668

amounts of total RNA and a gene specific primer were used for each reaction The 669

crossing point (Cp) was calculated using the 2nd derivative max method provided with 670

the Lightcycler software for each amplification curve Each reaction was run with two 671

technical replicates and the Cp values for these replicates were averaged ACTIN2 was 672

used as a stably expressed reference gene At least three biological replicates each 673

were performed and analyzed using a mixed-linear model RT-PCR was performed to 674

verify T-DNA insertion lines using the One-Step RT-PCR kit (Qiagen) according to the 675

manufacturerrsquos instructions The primers used can be found in Supplemental Table 1 676

677

Electrolyte Leakage Assay 678

Fully expanded leaves of four week-old plants were inoculated with water (Milli-Q 679

grade) using a needleless syringe Leaf discs were collected using a cork borer and 680

three leaf discs from one leaf were placed with the adaxial surface down onto 2 mL 681

water in a 12-well cell culture plate Conductivity was measured using a Horiba B-173 682

Page 34 of 54

conductivity meter after 30 min and then the water was replaced Conductivity was 683

measured again 30 min later (1 h total) as indicated in Figure 3C Six biological 684

replicates each were performed in three independent experiments and means and SEs 685

were calculated Two-sided t-tests were performed for each time point 686

687

DAB and Aniline Blue Staining 688

Leaves from four week-old plants were either mock inoculated with B cinerea 689

inoculation medium or inoculated with 10 microL droplets of a B cinerea spore solution (25 690

x 105 spores mL-1) of the indicated isolates Samples were collected 72 h later For 33prime-691

diaminobenzidine (DAB) staining two to five inoculated leaves were vacuum infiltrated 692

with 33prime-Diaminobenzidine (1 mg mL-1 in water pH 38) solution and stained overnight 693

in the dark Leaves were destained using 100 ethanol at 65degC and then transferred to 694

50 (vv) glycerol and photographed 695

Callose was visualized by staining leaves with aniline blue as previously 696

described (Adam and Somerville 1996) Briefly leaves were vacuum infiltrated with 697

alcoholic lactophenol (one volume of phenolglycerollactic acidwater (1111) mixed 698

with two volumes of ethanol) and destained at 65degC Next leaves were moved through 699

an alcohol gradient (50 20 and 10 (vv) ethanol) into water The destained leaves 700

were stained for 30 min in 150 mM K2HPO4 (pH 95) with 001 (wv) aniline blue 701

Samples were mounted in water and examined using a Nikon Eclipse Ni-U microscope 702

at 10x magnification using a DAPI filter (excitation at 325-375 nm emission at 435-485 703

nm) For callose quantification six 200 microm x 200 microm squares were counted for each 704

sample type Callose deposits per mm2 were calculated and data analyzed using a t-705

test Note that there is an inherent uncertainty associated with such counts because 706

callose deposits may not all be in focus at the same time 707

708

MAPK Activity Assay 709

Fully expanded leaves of four week-old plants were inoculated with 001 710

macerozyme 100 microg mL-1 OGs 100 nM flg22 or water (mock) flg22 a peptide from 711

bacterial flagella that induces immune responses was used as a positive control for 712

Page 35 of 54

MAPK activation (Asai et al 2002) and was purchased from EZBiolab Inc (Westfield 713

IN USA) Samples were harvested 10 min later and immediately frozen in liquid 714

nitrogen Protein was extracted using an extraction buffer containing 25 mM Tris pH 78 715

75 mM NaCl 1 mM DTT 1 mM NaF 05mM Na3VO4 01 (vv) Tween 20 one 716

cOmplete Mini protease inhibitor cocktail tablet (Roche) and one PhosSTOP 717

phosphatase inhibitor cocktail tablet (Roche) per 10 mL buffer 15 microg of total protein 718

were electrophoresed on a 10 SDS-polyacrylamide gel and then blotted onto a PVDF 719

membrane (Bio-Rad 162-0177) Activated MAPKs were detected using p4442 MAPK 720

(Erk12) antibody (9102S Cell Signal 12500 in TBST (20 mM Tris pH 75 150 mM 721

NaCl 01 (vv) Tween 20)) Anti-Rabbit IgG-HRP (115000 in TBST A6154 Sigma) 722

and ECL Plus substrate (Pierce 32132) MPK3 and MPK6 were detected with an anti-723

MPK3 antibody (M8318 Sigma 12000 in TBST and 3 (wv) milk) or an anti-MPK6 724

antibody (A7104 Sigma 16000 in TBST and 3 (wv) milk) respectively The 725

secondary antibody and detection were the same as for the p4442 antibody Five 726

independent experiments were performed 727

728

Preparation of Alcohol Insoluble Residue (AIR) Pectin Extraction and Uronic Acid 729

Measurements 730

Fully expanded leaves from four week-old plants that were incubated for 48 h in 731

the dark to reduce starch were harvested flash frozen and pulverized AIR was 732

extracted by washing ground material twice in 70 (vv) ethanol three times in a 733

mixture of chloroform and methanol (11 vv) and once in acetone (Gille et al 2009) 734

and was then air dried Cell wall pectin was extracted from AIR with cell wall extraction 735

buffer (50 mM Trizma 50 mM CDTA pH 72) at 95degC for 15 min Samples were 736

homogenized using a paint shaker (Harbil 5G-HD) and glass beads (3 mm diameter) 737

For dot blot experiments 500 microL of cell wall extraction buffer were used per 10 mg of 738

AIR Debris was precipitated by centrifugation for 10 min at 10000 g 739

Total uronic acid content of pectin extracted as described above using 500 microL of 740

cell wall extraction buffer per 1 mg of AIR was measured as described previously 741

(Filisetti-Cozzi and Carpita 1991 van den Hoogen et al 1998) First 36 microL of pectin 742

extract were mixed with 4 microL of 4 M sulfamic acid Then 200 microL of sulfuric acid 743

Page 36 of 54

containing 120 mM sodium tetraborate was added and samples were incubated at 80degC 744

for 1 h Following cooling on ice the optical density of samples was measured at 490 745

nm Next 40 microL of m-hydroxydiphenyl reagent (100 microL of m-hydroxydiphenyl in dimethyl 746

sulfoxide at 100 mgmL mixed with 49 mL 80 (vv) sulfuric acid just before use) was 747

added and the samples were mixed The optical density at 490 nm was measured 748

again The optical density before m-hydroxydiphenyl reagent addition was subtracted 749

from the optical density measured after addition of the dye The concentration of uronic 750

acid was calculated using known amounts of GalA as a standard 751

752

Release of Pectin from Arabidopsis Cell Walls Using Macerozyme 753

AIR preparations from leaves of four week-old plants were treated with 025 754

(wv) macerozyme R-10 (Yakult) or water (mock) at a concentration of 100 microL per mg 755

AIR Samples were incubated for 3 h at room temperature on a vortex shaker and 756

centrifuged for 10 min at 12000 g Total uronic acid concentration of 5-fold diluted 757

supernatants was determined for two technical replicates each The concentration of 758

uronic acid was calculated using known amounts of GalA as a standard For 759

subsequent qRT-PCR experiments supernatants from multiple extractions were 760

combined boiled for 20 min to inactivate macerozyme diluted 10-fold with water and 761

inoculated into four week-old Col-0 plants Heat-inactivated 10-fold diluted macerozyme 762

solutions were used as mock treatment Samples were collected 3 h later and PAD3 763

expression was determined 764

765

Release of Pectin from Arabidopsis Leaves Infected with B cinerea 766

Fully expanded leaves of four week-old plants were either inoculated with up to 767

five 10 microL droplets of B cinerea spore solution (25 x 105 spores mL-1) or mock 768

inoculated with B cinerea inoculation medium For each biological replicate leaves with 769

a combined count of at least 20 infection sites were collected 48 h later AIR was 770

extracted as described above but plants were not dark-treated or freeze-dried and the 771

first ethanol step was performed using 85 (vv) ethanol To extract soluble pectin 772

fragments AIR samples were mixed with water at a concentration of 20 microL per infection 773

site incubated for 7 h at room temperature on a vortex shaker and centrifuged for 10 774

Page 37 of 54

min at 12000 g Total uronic acid concentration of supernatants was determined for two 775

technical replicates each The concentration of uronic acid was calculated using known 776

amounts of GalA as a standard 777

778

Measurement of Uronic Acids Neutral Monosaccharides and Cellulose Content in 779

Cell Walls 780

Neutral sugars and uronic acids from non-cellulosic polysaccharides in cell walls 781

(AIR) were released by trifluoroacetic acid (TFA) hydrolysis Approximately 1 mg of AIR 782

was hydrolyzed with 2 M TFA at 121degC for 90 min The TFA-soluble material was split 783

into two equal parts one for uronic acid and one for neutral sugar determination The 784

uronic acid content was determined by analyzing the hydrolyzate using a CarboPac 785

PA200 anion-exchange column with an ICS-3000 Dionex chromatography system 786

(Dionex Sunnyvale CA USA) The elution profile consisted of a linear gradient of 50 to 787

200 mM sodium acetate in 01 M NaOH in 10 min at 04 mL per min Alditol acetate 788

derivatives of neutral sugars were produced as described (York et al 1985) In brief 789

monosaccharides in the hydrolyzate were reduced with NaBH4 and peracetylated with 790

acetic anhydride and pyridine at 121degC for 20 min The generated alditol acetates were 791

analyzed by GC-MS Crystalline cellulose was measured according to Updegraff (1969) 792

After hydrolysis of noncellulosic polysaccharides from AIR with the Updegraff reagent 793

(acetic acid nitric acids water 812 (vv) ) the remaining pellet was hydrolyzed in 794

72 sulfuric acid The resulting glucose quantity was determined by the anthrone 795

method (Scott and Melvin 1953) 796

797

Dot Blot Experiments 798

Pectin solutions extracted using 50 microL of cell wall extraction buffer per mg AIR 799

were serially diluted and nitrocellulose membranes were spotted with 1 microL of the diluted 800

pectin solutions Membranes were dried overnight blocked with 5 milk (wv) in 1x 801

PBS (8 g L-1 NaCl 02 g L-1 KCl 144 g L-1 Na2HPO4 024 g L-1 KH2PO4 pH 74) and 802

probed with LM5 (Jones et al 1997) LM6 (Willats et al 1998) LM8 (Willats et al 803

2004) LM19 and LM20 (Verhertbruggen et al 2009a) or CCRC-M7 (Steffan et al 804

Page 38 of 54

1995) antibodies LM series antibodies were diluted 1250 and CCRC-M7 was diluted 805

1500 in 5 milk powder (Nestle Carnation) in 1 x PBS For LM series antibodies a 806

goat anti-Rat HRP conjugated antibody (Bethyl A110-105P 15000 diluted) and for 807

CCRC-M7 an anti-Mouse IgG HRP conjugate (Promega W402B 12500 diluted) was 808

used as secondary antibody in 5 milk powder in 1x PBS LM series antibodies were 809

obtained from plant probes (wwwplantprobesnet) and CCRC-M7 from CarboSource 810

(wwwcarbosourcenet) Membranes were washed after incubation with the primary and 811

secondary antibodies with 1x PBS Dot blots were developed using the ECL system 812

(GE Healthcare Amersham ECL prime) For quantification of signals dot blot results on 813

x-ray films were photographed using a CCD camera and intensities were measured814

using Image Jrsquos Integrated Density function Locally adjusted background was 815

subtracted As the dynamic range of the signal on an x-ray film was narrow non-816

linearity in the measured values was corrected for each genotype in each biological 817

replicate using the values from different dilutions in two technical replicates We noticed 818

that the residuals were not normally distributed and performed Box-Cox power 819

transformation (Box and Cox 1964) to obtain normally distributed values for the 820

subsequent statistical analysis of the data For LM19 Box-Cox selected power 821

transformation was done to the power of 038th and for LM20 to the power 075th After 822

correction of the transformed measured values with the effect of the biological 823

replicates the values were fit to a linear model with the Arabidopsis genotype as the 824

fixed effect This linear model was used both to generate the plots in Figures 5B and D 825

and to test significant differences 826

827

Camalexin Measurements 828

Four week-old plants were infected with B cinerea and samples (three leaf disks 829

from one plant each) were harvested at the indicated time points flash frozen and 830

pulverized Each sample was extracted with 300 microL of 90 methanol (vv) 50 microL of the 831

extract was run on an Agilent Lichrocart 250-4 RP18e 5-microm column using an Agilent 832

1100 series HPLC Camalexin was detected using a DiodeArray (DAD) at 330 nm and 833

with a fluorescence detector at emission 318 nmexcitation 385 nm (Agilent) Separation 834

was achieved using the following program with aqueous acetonitrile 5 min gradient 835

Page 39 of 54

from 63 to 69 acetonitrile 30 s gradient from 69 to 99 acetonitrile 2 min at 99 836

acetonitrile and a post-run equilibration of 35 min at 63 acetonitrile (Denby et al 837

2004 Kliebenstein et al 2005) Purified camalexin was used to produce a standard 838

curve to identify and quantitate camalexin 839

840

Accession Numbers 841

Microarray data used can be accessed at NCBI Gene Expression Omnnibus 842

(httpwwwncbinlmnihgovgeo) GSE18978 Sequence information for all Arabidopsis 843

genes described in this article can be found in TAIR (wwwarabidopsisorg) using the 844

following accession numbers PAD3 (CYP71B15 At3g26830) SID2 (ICS1 At1g74710) 845

GAE1 (At4g30440) GAE2 (At1g02000) GAE3 (At4g00110) GAE4 (At2g45310) GAE5 846

(At4g12250) GAE6 (At3g23820) PR1 (At2g14610) JAZ5 (At1g17380) JAZ10 847

(At5g13220) ACTIN2 (At3g18780) PDF12a (At5g44420) PDF12b (At2g26020) 848

AtSK11 (At5g26751) The sequence of the B cinerea cutinase Bc-CutA (Z69264) can 849

be found in the DNA Data Bank of Japan (httpgetentryddbjnigacjp) Germplasm 850

used included dde2-2 (At5g42650 (von Malek et al 2002)) pad3-1 (At3g26830 (Zhou 851

et al 1999)) pad4-1 (At3g52430 (Jirage et al 1999)) sid2-2 (At1g74710 (Wildermuth 852

et al 2001)) pbs3-2 (At5g13320 SALK_018225 (Nobuta et al 2007)) cbp60a-1 853

(At5g62570 SALK_124410 (Truman et al 2013)) cbp60g-1 sard1-2 (At5g26920 854

SALK_023199 At1g73805 SALK_052422 (Wang et al 2011) cbp60a-1 cbp60g-1 855

sard1-2 (Truman et al 2013) gae1-1 (At4g30440 SALK_085554) gae6-1 (At3g23280 856

SALK_104454) gae6-2 (At3g23280 SALK_017191) ixr1-1 cev1 (At5g05170 (Ellis 857

and Turner 2001 Scheible et al 2001)) eds1 plants were derived by introgression of 858

the Ler eds1-2 allele into Col-0 that contains two copies of EDS1 (At3g48090 859

At3g48080 (Bartsch et al 2006) mpk6-2 (At2g43790 SALK_073907 (Liu and Zhang 860

2004)) mpk3-DG (At3g45640 a fast neutron deletion mutant (Miles et al 2005)) and 861

npr1-1 (At1g64280 (Cao et al 1997)) T-DNA insertion lines were part of the SALK 862

collection (Alonso et al 2003) and were obtained from the Arabidopsis Biological 863

Resource Center 864

865

Supplemental Data 866

Page 40 of 54

Supplemental Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 867

Expression Does Not Require CBP60s or SID2 868

Supplemental Figure 2 Expression of GAE Family Members uponTreatment with Pma 869

ES4326 870

Supplemental Figure 3 Characterization of T-DNA Insertions in GAE1 and GAE6 871

Supplemental Figure 4 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves and 872

Are Slightly Smaller than Wild-Type Col-0 Plants 873

Supplemental Figure 5 Cell Walls of gae1 gae6 Plants Contain Less Uronic Acid than 874

Wild-Type Col-0 Cell Walls 875

Supplemental Figure 6 Antibodies Raised Against the RGI or XG Components of 876

Pectin Show No Difference in Binding to gae Mutant and Col-0 Cell Walls 877

Supplemental Figure 7 The Effect of B cinerea Treatment on the Expression of GAE 878

Family Genes 879

Supplemental Figure 8 Treatment with 001 Macerozyme Does Not Cause Any 880

Visible Damage to Col-0 Plants 881

Supplemental Figure 9 B cinerea Growth on Plants with Defects in Immune 882

Signaling 883

Supplemental Figure 10 MeJA-Induced Expression of the JA marker gene JAZ10 884

Supplemental Figure 11 The Expression of the JA Marker Gene JAZ5 Is Highly 885

Responsive in gae1 gae6 Plants 886

Supplemental Figure 12 The Expression of PDF12 Is Unaltered in gae1 gae6 Plants 887

Supplemental Figure 13 Macerozyme Treatment Activates MPK3 and MPK6 888

Supplemental Figure 14 B cinerea-Induced ROS Production in Wild-Type Col-0 and 889

gae1 gae6 Plants 890

Supplemental Figure 15 OG-Induced PTI Using 250 microg mL-1 OGs 891

Supplemental Figure 16 Abundance of Plant DNA in B cinerea Treated Plants 892

Supplemental Table 1 Primers used in this study 893

894

ACKNOWLEDGEMENTS 895

We thank William Truman for helpful discussions Simone Ferrari (Sapienza 896

Universita di Roma) for the generous gift of OGs Bruna Bucciarelli (USDA-ARS) for 897

Page 41 of 54

help with fluorescence microscopy the Arabidopsis Biological Resource Center for T-898

DNA insertion lines Paul Knoxrsquos lab (University of Leeds) for LM5 LM6 LM8 LM19 899

and LM20 antibodies and the Complex Carbohydrate Resource Center (University of 900

Georgia) for the CCRC-M7 antibody Most of this work was funded by the Division of 901

Chemical Sciences Geosciences and Biosciences Office of Basic Energy Sciences of 902

the US Department of Energy through Grant DE-FG02-05ER15670 to JG This work 903

was also supported by NSF awards IOS 1339125 and IOS 1021861 to DJK NSF 904

award IOS 1121425 to F K and the USDA National Institute of Food and Agriculture 905

Hatch project number CA-D-PLS-7033-H to DJK 906

907

AUTHOR CONTRIBUTIONS 908

GB and JG designed the research GB GX AT BL NES and NH 909

performed research DJK provided tools GB GX MP AD BL RAH and FK 910

analyzed data GB and JG wrote the paper with input from all authors 911

912

FIGURE LEGENDS 913

Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 Expression 914

Requires PAD4 EDS1 and PBS3 915

(A) Expression of GAE1 in leaves of wild-type Col-0 and mutant plants Expression was916

measured 24 or 32 h after either inoculation with Pma ES4326 (OD600 = 0002) or mock 917

inoculation with 5 mM MgSO4 and was then normalized to the level of ACTIN2 Data 918

from three biological replicates were combined using a mixed linear model Bars 919

represent mean log2-ratios to ACTIN2 plusmn SE Letters indicate significantly different 920

groups with q lt 005 where 005 represents the maximum false discovery rate at which 921

the test may be called significant 922

(B) Expression of GAE6 measured as described in (A)923

924

Figure 2 Susceptibility of gae6 and gae1 gae6 Plants to Pma ES4326 Is Increased 925

Relative to Wild-Type Col-0 926

Leaves of plants of the indicated genotypes were inoculated with Pma ES4326 (OD600 = 927

00002) as described in Methods Bacterial titers in leaves were determined either 928

Page 42 of 54

immediately (0 dpi) or after three days (3 dpi) Bars represent the mean plusmn SE of four 929

independent experiments each with 4 or 12 biological replicates at 0 and 3 dpi 930

respectively Data were combined using a mixed linear model Asterisks indicate 931

significant differences from Col-0 wild-type (q lt 005) Susceptible pad4 plants were 932

included as a positive control 933

934

Figure 3 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves 935

(A) Depiction of the assay for brittleness of gae mutant leaves Fully expanded leaves of936

four to five week-old plants were bent by 180ordm so that the adaxial surface of the leaf was 937

parallel with the soil Broken leaf petioles or midribs were considered brittle 938

(B) Quantification of broken midribs or petioles For each genotype 66 to 82 leaves 939

were analyzed for brittleness as described in (A) Bars represent the ratio of broken to 940

total number of leaves tested Results were analyzed using Fisherrsquos exact test Letters 941

indicate significantly different groups at p lt 005 942

(C) Conductivity measurements Leaves of four week-old plants were inoculated with943

deionized water and then leaf discs were cut out and placed on deionized water 944

Conductivity was measured at the indicated time points After 30 min the water was 945

replaced with fresh water (30 min f w) to account for wounding-related ion leakage from 946

the edges of the leaf discs Three independent experiments with six biological replicates 947

each were performed and data were analyzed using a t-test for each time point P-948

values were corrected using the Bonferroni method Data show means plusmn SE asterisks 949

indicate significant differences from wild-type Col-0 at q lt 001 950

951

Figure 4 Cell Walls of gae6 and gae1 gae6 Plants Are Reduced in GalA 952

(A) to (D) Levels of GalA (A) GlcA (B) cellulose (C) and neutral sugars (D) in alcohol953

insoluble residue (AIR) extracted from leaves of four week-old plants of the indicated 954

genotypes Bars represent means plusmn SE of four biological replicates combined using a 955

mixed linear model Letters indicate significantly different groups (p lt 005) 956

957

Figure 5 Cell Walls of gae1 gae6 Plants Are Reduced in Homogalacturonan 958

Page 43 of 54

Dot blot analyses with anti-LM19 and anti-LM20 antibodies Pectin was extracted from 959

AIR prepared from the indicated genotypes using 1 microL of extraction buffer per 10 microg of 960

AIR Pectin was serially diluted and one microL of undiluted 13 diluted and 19 diluted 961

samples were spotted on a nitrocellulose membrane Three biological replicates were 962

were spotted twice to obtain two technical replicates each All replicates produced 963

similar results 964

(A) Representative dot blot using anti-LM19 antibody965

(B) Relative signal intensities of the spots in the dot blot in (A)966

(C) Representative dot blot using anti-LM20 antibody967

(D) Relative signal intensities of the LM20 dot blot shown in (C)968

Relative signal intensities were measured using Image J software Measurements from 969

the three biological replicates were combined using a linear model after Box-Cox power 970

transformation as described in Methods Bars represent means plusmn SE Asterisks indicate 971

a difference from wild-type Col-0 at p lt 005 972

973

Figure 6 Growth of B cinerea on gae1 gae6 Plants and the Effect of B cinerea on 974

Expression of GAE1 and GAE6 975

(A) Growth of B cinerea isolates Leaves of Col-0 and gae1 gae6 plants were976

inoculated with 25 x 105 spores mL-1 of the B cinerea isolates indicated in the figure 977

Samples were collected two (2 dpi) and three (3 dpi) days later Bars represent means plusmn 978

SE of six biological replicates for each fungus combined using a mixed linear model 979

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 980

(B) and (C) Expression of GAE1 (B) and GAE6 (C) 48 h after inoculation with the B981

cinerea isolates indicated Expression levels were measured by qRT-PCR Bars 982

represent mean log2-ratios to ACTIN2 plusmn SE of three biological replicates combined 983

using a mixed linear model Letters indicate significantly different groups (q lt 005) 984

985

Figure 7 Levels of Soluble Pectin and Expression of PAD3 in Fractions Produced 986

Following Macerozyme Treatment of gae1 gae6 Cell Walls 987

(A) Schematic overview of the experimental procedure for (B) and (C)988

Page 44 of 54

(B) Uronic acid measurements Alcohol-insoluble residue (AIR) from leave tissue of four989

week-old wild-type Col-0 or gae1 gae6 plants was treated with water or 025 990

macerozyme and assayed for uronic acid levels Data from 11 biological replicates were 991

combined using a mixed linear model Bars represent means plusmn SE Letters indicate 992

significant differences (p lt 005) 993

(C) Expression of PAD3 in Col-0 plants treated with the fractions from (B) Extracts from994

(B) were heat inactivated pooled and diluted 5-fold with water Pooled extracts were995

inoculated into four week-old Col-0 plants PAD3 expression was measured 3 h after 996

inoculation and normalized to the level of ACTIN2 expression Data from four biological 997

replicates were combined using a mixed linear model Bars represent mean log2-ratios 998

to ACTIN2 plusmn SE Letters indicate significant differences (p lt 005) 999

1000

Figure 8 Macerozyme-Induced PAD3 Expression Is Absent in gae1 gae6 1001

(A) Expression of PAD3 after macerozyme treatment Four week-old plants were either1002

inoculated with 001 macerozyme or mock inoculated with water and PAD3 1003

expression was measured 3 h later by qRT-PCR Data from four biological replicates 1004

were combined using a mixed linear model Mean log2-ratios to ACTIN2 plusmn SE were 1005

plotted Letters indicate significantly different groups (p lt 005) 1006

(B) Expression of PAD3 after OG treatment PAD3 expression was measured as1007

described in (A) 3 h after inoculation with 100 microg mL-1 OGs or mock inoculation with 1008

water Data are from six biological replicates 1009

(C) PAD3 expression in untreated plants Three biological replicates were performed1010

and data analyzed as described in (A) 1011

(D) Camalexin accumulation after B cinerea treatment Camalexin accumulation was1012

measured in four week-old plants inoculated with the B cinerea strains shown in the 1013

figure Eight biological replicates each were combined using a mixed linear model 1014

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 1015

(E) PAD3 expression 28 h after treatment with the indicated B cinerea strains Data1016

from six biological replicates were collected and analyzed as described in (A) Data in 1017

all figure parts represent means plusmn SE Letters indicate significantly different groups (q lt 1018

005) 1019

Page 45 of 54

1020

Figure 9 Macerozyme-Induced Expression of the SA Marker Genes SID2 and PR1 1021

Is Abolished in gae1 gae6 Plants 1022

(A) to (H) Expression of PR1 (A) (C) (E) and (G) or SID2 (B) (D) (F) and (H) in four1023

week-old Col-0 and gae1 gae6 plants Data were combined using a mixed linear model 1024

Bars represent mean log2-ratios of expression vs ACTIN2 plusmn SE for all figure parts 1025

Letters indicate significantly different groups at p lt 005 for panels (A) to (D) (G) and 1026

(H) and qlt005 for panels (E) to (F) 1027

(A) and (B) Plants were treated with 001 macerozyme or mock treated with water1028

and samples were collected 3 h later Data from four biological replicates are shown 1029

(C) and (D) Plants were inoculated with 100 microg mL-1 OGs or mock treated with water1030

and samples collected 3 h later Data from six biological replicates are shown 1031

(E) and (F) Plants were inoculated with the indicated B cinerea isolates or mock1032

inoculated with B cinerea inoculation medium and samples collected 48 h later Data 1033

from eight biological replicates are shown 1034

(G) and (H) Expression in untreated plants Three biological replicates were performed1035

1036

Figure 10 The Expression of the JA Marker Gene JAZ10 is Highly Responsive in 1037

gae1 gae6 Plants 1038

(A) to (D) JAZ10 expression in four week-old plants Data were combined using a mixed1039

linear model Mean log2-ratios to ACTIN2 plusmn SE are shown Letters indicate significantly 1040

different groups at p lt 005 for panels (A) (B) and (D) and q lt 005 for panel (C) 1041

(A) Plants were inoculated with 001 macerozyme or mock inoculated using water and1042

samples were collected 3 h later Data from four biological replicates are shown 1043

(B) Plants were inoculated with 100 microg mL-1 OGs or mock inoculated using water and1044

samples collected 3 h later Data are from six biological replicates 1045

(C) JAZ10 expression 48 h after treatment with the indicated B cinerea strains Data1046

are from six biological replicates 1047

(D) JAZ10 expression in untreated plants Data are from three biological replicates1048

1049

Figure 11 Gallo 1-Induced Callose Deposition Is Reduced in gae1 gae6 Plants 1050

Page 46 of 54

(A) Callose deposition in four week-old Col-0 and gae1 gae6 plants Plants were1051

inoculated with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) or mock inoculated 1052

with B cinerea inoculation medium and samples were collected after 72 h Callose was 1053

stained with aniline blue and visualized using a Nikon Eclipse Ni-U microscope with a 1054

DAPI filter Three leaves were stained per treatment Scale bars are 100 microm Asterisks 1055

indicate fungal hyphae arrows indicate representative callose spots 1056

(B) Quantification of callose deposition Callose deposits were counted in 200 microm x 2001057

microm squares laid out across the images shown here Bars represent means plusmn SE from 1058

six squares each Letters indicate significantly different groups (p lt 005) according to a 1059

t-test analysis1060

1061

Figure 12 Less Soluble Pectin Accumulates in B cinerea-Infected gae1 gae6 1062

Leaves 1063

Four week-old Col-0 and gae1 gae6 plants were inoculated with 10 microL droplets of the 1064

indicated B cinerea isolate (25 x 105 spores mL-1) or mock inoculated with B cinerea 1065

inoculation medium Leaves were collected 48 h later AIR was extracted from leaf 1066

samples and water added (20 microL infection site) to extract soluble pectin fragments 1067

Samples were vortexed for 7 h centrifuged at 12000 g and the uronic acid 1068

concentration of the supernatant was measured Data from nine biological replicates 1069

were combined using a mixed linear model Means plusmn SE are shown Letters indicate 1070

significant differences (q lt 005) 1071

1072

Figure 13 Macerozyme-Induced PTI Is Lost in gae1 gae6 While OG-Induced PTI Is 1073

Unaltered 1074

(A) Macerozyme-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1075

inoculated with 001 macerozyme or mock inoculated with boiled macerozyme and 1076

were infected with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) 24 h later Samples 1077

were collected immediately (0 dpi) and after two (2 dpi) and three (3 dpi) days Data 1078

show mean plusmn SE of six to 14 biological replicates each combined using a linear model 1079

Letters indicate significantly different groups (q lt 005) 1080

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

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Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk RGadrinab C Heller C Jeske A Koesema E Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt MHazari N Hom E Karnes M Mulholland C Ndubaku R Schmidt I Guzman P Aguilar-Henonin L Schmid M Weigel DCarter DE Marchand T Risseeuw E Brogden D Zeko A Crosby WL Berry CC and Ecker JR (2003) Genome-wideinsertional mutagenesis of Arabidopsis thaliana Science 301 653-657

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Asai T Tena G Plotnikova J Willmann MR Chiu WL Gomez-Gomez L Boller T Ausubel FM and Sheen J (2002) MAPkinase signalling cascade in Arabidopsis innate immunity Nature 415 977-983

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Bartsch M Gobbato E Bednarek P Debey S Schultze JL Bautor J and Parker JE (2006) Salicylic acid-independentENHANCED DISEASE SUSCEPTIBILITY1 signaling in Arabidopsis immunity and cell death is regulated by the monooxygenaseFMO1 and the nudix hydrolase NUDT7 Plant Cell 18 1038-1051

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Bates D Machler M Bolker BM and Walker SC (2015) Fitting Linear Mixed-Effects Models Using lme4 J Stat Softw 67 1-48Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Benjamini Y and Hochberg Y (1995) Controlling the False Discovery Rate - a Practical and Powerful Approach to MultipleTesting J Roy Stat Soc B Met 57 289-300

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Bethke G Grundman RE Sreekanta S Truman W Katagiri F and Glazebrook J (2014) Arabidopsis PECTINMETHYLESTERASEs Contribute to Immunity against Pseudomonas syringae Plant Physiol 164 1093-1107

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Bouton S Leboeuf E Mouille G Leydecker MT Talbotec J Granier F Lahaye M Hofte H and Truong HN (2002)Quasimodo1 encodes a putative membrane-bound glycosyltransferase required for normal pectin synthesis and cell adhesion inArabidopsis Plant Cell 14 2577-2590

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Box GEP and Cox DR (1964) An analysis of transformations Journal of the Royal Statistical Society Series B 26 211-252Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Cao H Glazebrook J Clarke JD Volko S and Dong XN (1997) The Arabidopsis NPR1 gene that controls systemic acquiredresistance encodes a novel protein containing ankyrin repeats Cell 88 57-63

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Carpita N (2010) Update on Mechanisms of Plant Cell Wall Biosynthesis How plants make cellulose and other (1-gt4)-beta-D-glycans Plant Physiol

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Chisholm ST Coaker G Day B and Staskawicz BJ (2006) Host-microbe interactions Shaping the evolution of the plantimmune response Cell 124 803-814

Page 3 of 54

INTRODUCTION 57

The cell walls in Arabidopsis thaliana leaves are mainly primary cell walls 58

consisting of three major types of polysaccharidesmdashcellulose various hemicelluloses 59

and various pectic polysaccharidesmdashas well as some structural proteins (Liepman et 60

al 2010) Arabidopsis leaf walls contain approximately 14 cellulose a homopolymer 61

of (1 4)-β linked D-glucose subunits (Zablackis et al 1995 Carpita 2010) 62

Hemicelluloses are a diverse class of polysaccharides that includes xylans xyloglucans 63

mannans glucomannans and mixed-linkage β-glucans (Scheller and Ulvskov 2010) In 64

Arabidopsis leaves the major hemicellulose is xyloglucan which constitutes about 20 65

of the wall polysaccharides Xyloglucan contains a (1 4)-β-linked glucan backbone 66

substituted with (1 6)-α-linked xylosyl residues or side chains of xylosyl galactosyl and 67

fucosyl residues (Zablackis et al 1995 Liepman et al 2010) Glucuronoarabinoxylan 68

(4 of the wall) is also found in Arabidopsis leaves (Zablackis et al 1995) Primary 69

walls of dicotyledonous plants generally also contain 3-5 of the hemicelluloses 70

mannan and glucomannan (Scheller and Ulvskov 2010) Hence hemicelluloses in 71

primary walls of dicotyledonous plants are mainly composed of glucose (Glc) xylose 72

(Xyl) arabinose (Ara) galactose (Gal) and mannose (Man) 73

Pectins are a diverse group of polysaccharides that all contain galacturonic acid 74

(GalA) and make up about 50 of Arabidopsis leaf walls (Zablackis et al 1995 Harholt 75

et al 2010) Homogalacturonan (HG) is a linear homopolymer of (14)-α-linked 76

galacturonic acid (GalA) residues and it makes up about 65 of all pectin in 77

Arabidopsis leaf walls (Zablackis et al 1995 Mohnen 2008) A linear (14)-α-linked 78

GalA backbone substituted with single xylose residues is called xylogalacturonan 79

(XGA) and a polymer with complex side-chains containing borate ions and sugars such 80

as Ara rhamnose (Rha) Gal Xyl or fucose (Fuc) is referred to as rhamnogalacturonan 81

II (RGII) (Mohnen 2008 Harholt et al 2010) XGA and RGII make up less than 10 of 82

leaf cell wall pectin (Zandleven et al 2007 Mohnen 2008) In contrast 83

rhamnogalacturonan I (RGI) backbones consist of a repeating α-14-D-GalA-α-12-L-Rha 84

disaccharide and are substituted with β-(14)-galactan branched arabinan or 85

arabinogalactan side chains (Mohnen 2008 Harholt et al 2010) RGI constitutes about 86

20 ndash 25 of pectin in primary walls (Mohnen 2008) Hence pectin in Arabidopsis leaf 87

Page 4 of 54

cell walls consists mostly of GalA Rha and smaller amounts of other sugars including 88

Ara Gal Xyl and Fuc 89

In general carbohydrate biosynthesis requires nucleotide sugars provided by 90

nucleotide sugar interconversion pathways (Seifert 2004) Most nucleotide sugars are 91

synthesized from uridine diphosphate-Glc (UDP-Glc) UDP-glucuronic acid (UDP-GlcA) 92

is made from UDP-Glc by UDP-glucose dehydrogenase activity or via an alternative 93

pathway requiring inositol oxygenase activity (Tenhaken and Thulke 1996 Loewus and 94

Murthy 2000) UDP-D-Glucuronate 4-epimerases (GAEs) interconvert UDP-D-GlcA and 95

UDP-D-GalA the monomeric precursor of pectin There are six GAE genes encoded by 96

the Arabidopsis genome When heterologously expressed in Escherichia coli or Pichia 97

pastori GAE1 GAE4 and GAE6 showed activity in interconverting UDP-D-GlcA and 98

UDP-D-GalA (Gu and Bar-Peled 2004 Molhoj et al 2004 Usadel et al 2004) GAE1 99

and GAE6 were found to be Golgi-localized and strongly expressed in various plant 100

tissues (Molhoj et al 2004 Usadel et al 2004 Parsons et al 2012) GAE1 and GAE6 101

have been hypothesized to be evolutionarily older than the other GAE family members 102

(Usadel et al 2004) and might have overlapping functions that are distinct from the 103

other family members 104

Plant cell wall composition and architecture affects wall strength and flexibility 105

and cell walls present a physical barrier to potential plant pathogens Besides preformed 106

physical barriers such as a cell wall plants have a sophisticated immune system to 107

defend themselves against harmful microbial pathogens (Chisholm et al 2006 Jones 108

and Dangl 2006) Immune signaling involves changes in phytohormone levels the most 109

important for plant immunity being salicylic acid (SA) jasmonic acid (JA) and ethylene 110

(ET) (Grant and Jones 2009 Pieterse et al 2012) Other major regulators of plant 111

immunity include PHYTOALEXIN DEFICIENT 4 (PAD4) and ENHANCED DISEASE 112

SUSCEPTIBILITY 1 (EDS1) (Wiermer et al 2005) which are predicted to encode 113

lipases Immune signaling generally involves changes in the transcriptome that in turn 114

lead to altered cellular responses (Tao et al 2003) For instance induced expression of 115

the cytochrome P450 PAD3 is thought to increase production of the anti-microbial 116

secondary metabolite camalexin (Zhou et al 1999) 117

Page 5 of 54

Because plant cell walls are important barriers against pathogenic microbes 118

alterations in wall structural properties can lead to changes in plant immunity To date 119

only a few examples of cell wall related mutants with altered pathogen phenotypes have 120

been studied in detail Mutants impaired in callose biosynthesis powdery mildew 121

resistant 4 (pmr4) display enhanced resistance to the powdery mildew pathogens 122

Golovinomyces orontii and G cichoracearum (Nishimura et al 2003) These plants 123

have constitutively high SA levels and SA signaling is required for the resistance 124

phenotype (Nishimura et al 2003) Other plants with enhanced resistance to these 125

powdery mildew pathogens include pmr5 a mutant enriched in pectin and pmr6 a 126

mutant with a defect in a pectate lyase-like gene (Vogel et al 2002 Vogel et al 2004) 127

Plants with mutations in cellulose synthases required for primary and secondary wall 128

formation also show increased resistance to pathogens (Ellis and Turner 2001 129

Hernandez-Blanco et al 2007) Enhanced resistance to powdery mildew and 130

Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) in constitutive 131

expression of VSP 1 (cev1) which is mutated in the primary wall cellulose synthase 132

CesA3 was linked to constitutive high JA and ET levels in these plants (Ellis and 133

Turner 2001 Ellis et al 2002a) Interestingly irregular xylem1 (irx1) irx3 and irx5 134

plants which carry mutations in the secondary wall-associated cellulose synthases 135

CesA8 CesA7 and CesA4 showed enhanced resistance to the necrotrophic pathogens 136

Plectosphaerella cucumerina Ralstonia solanacearum and Botrytis cinerea as well as 137

the biotrophic powdery mildew G cichoracearum whereas growth of P cucumerina and 138

R solanacearum pathogens was unaltered in pmr5 pmr6 and cev1 plants (Hernandez-139

Blanco et al 2007) This enhanced resistance of the irx mutants was independent of 140

SA JA or ET signaling but instead involved abscisic acid signaling (Hernandez-Blanco 141

et al 2007) Changes in wall structure are likely monitored by the plants and effects 142

may be counteracted through initiation of defense responses that can lead to altered 143

phytohormone levels 144

Although plant cell walls serve as barriers against pathogen entry many 145

pathogens secrete cell wall-degrading enzymes (CWDEs) and thus are able to 146

successfully infect their host plants (Albersheim et al 1969) For example the B 147

cinerea polygalacturonase Bc-PG1 is required for full virulence of this pathogen and the 148

Page 6 of 54

gene encoding it shows evidence for diversifying selection as would be expected for a 149

virulence gene (ten Have et al 1998 Rowe and Kliebenstein 2007) Secretion of 150

CWDEs can trigger the release of cell wall fragments which can act as damage-151

associated molecular patterns (DAMPs) that activate plant immune responses (Hahn et 152

al 1981 Ferrari et al 2013) Similarly pectin fragments known as oligogalacturonides 153

(OGs) can trigger plant immune responses (Cote and Hahn 1994 Ferrari et al 2013) 154

Such responses include the production of reactive oxygen species (ROS) activation of 155

mitogen-activated protein kinase (MAPK) activation and enhanced expression of 156

defense-related genes such as PAD3 (Ferrari et al 2007 Denoux et al 2008 Galletti 157

et al 2008 Galletti et al 2011) Some of these responses require SA JA and ET 158

whereas others are independent of these phytohormones (Ferrari et al 2007) 159

Here we describe two GAE family members GAE1 and GAE6 whose 160

expression is repressed upon pathogen challenge A gae1 gae6 double mutant has 161

brittle leaves and its walls are reduced in pectin specifically HG and likely RGI 162

Resistance to the bacterial pathogen Pma ES4326 and the fungal necrotroph B cinerea 163

is compromised in gae1 gae6 Callose deposition is reduced in gae1 gae6 plants after 164

treatment with B cinerea isolate Gallo 1 Furthermore these plants show altered 165

activation of immune signaling in response to treatment with macerozyme a 166

polygalacturonase that degrades pectin and they also are hyperresponsive to JA 167

signaling Macerozyme treatment released less soluble likely low molecular weight 168

pectin from isolated gae1 gae6 cell walls than from Col-0 cell walls B cinerea-induced 169

accumulation of soluble likely low molecular weight pectin was also decreased in gae1 170

gae6 plants Additionally gae1 gae6 plants are impaired in macerozyme-induced 171

immunity to B cinerea possibly due to decreased release of OGs from their cell walls 172

173

174

Page 7 of 54

RESULTS 175

176

GAE1 and GAE6 Expression Is Repressed by Pma ES4326-Induced Immune 177

Signaling 178

Our previous research suggested that the status of pectin specifically pectin 179

modification by methylesterification is important for plant immunity against Pma 180

ES4326 (Bethke et al 2014) Here we investigate whether pectin abundance in 181

addition to its specific structure is also important for plant immunity by taking a closer 182

look at the formation of the pectin precursor UDP-GalA by GAEs 183

184 Microarray experiments showed that expression of GAE1 and GAE6 was

185 repressed in A thaliana (Arabidopsis) plants treated with Pma ES4326 (Wang et al

186 2008) Repression of GAE1 and GAE6 in wild-type Arabidopsis Col-0 plants treated with

187 Pma ES4326 was confirmed by quantitative real time (qRT)-PCR Figure 1) To test if

188 this repression is mediated by immune signaling qRT-PCR was also performed in

189 mutants with blocked immune signaling (Figure 1 and Supplemental Figure 1) 190 Generally immunity to Pma ES4326 requires intact SA signaling Repression of both

191 GAE1 and GAE6 required the presence of the major immune regulators EDS1 and

192 PAD4 required for SA signaling as well as other immune responses (Jirage et al 1999

193 Wiermer et al 2005 Bartsch et al 2006 Wang et al 2008) at both early and late time

194 points The presence of AVRPPHB SUSCEPTIBLE 3 (PBS3) a regulator of many

195 immune responses including SA signaling (Nobuta et al 2007) was also required for

196 repression of GAE1 and GAE6 at an early time point (Figure 1) CALMODULIN-

197 BINDING PROTEIN 60-LIKE a (CBP60a) (a negative regulator of SA synthesis and

198 immunity (Truman et al 2013)) CBP60g and SYSTEMIC ACQUIRED RESISTANCE

199 DEFICIENT 1 (SARD1) (both promote SA biosynthesis and regulation of other immune

200 responses (Wang et al 2011)) SALICYLIC ACID INDUCTION DEFICIENT 2 SID2

201 (encodes a biosynthetic enzyme for SA (Wildermuth et al 2001)) and

202 NONEXPRESSER OF PR GENES 1 NPR1 (a regulator of the majority of SA-induced

203 immune signaling (Cao et al 1997 Yan and Dong 2014)) were not required for this

204 repression (Figure 1 and Supplemental Figure1) Thus repression of GAE1 and GAE6

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205 requires a regulatory function that is dependent on EDS1 and PAD4 partially

206 dependent on PBS3 but independent of SA

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There are six GAE family members in Arabidopsis GAE2 expression was 207

induced by Pma ES4326 in the absence of PAD4 or EDS1 GAE3 expression was 208

unaltered GAE4 was repressed early and GAE5 expression was induced in all 209

genotypes (Supplemental Figure 2) The similarity of the immune-regulated expression 210

of GAE1 and GAE6 suggested a redundant role of these genes in Arabidopsis immunity 211

to Pma ES4326 212

213

Resistance to Pma ES4326 Is Compromised in gae1 gae6 and gae6 Plants 214

To study the roles of GAE1 and GAE6 in plant immunity in more detail plants 215

with T-DNA insertion mutations in GAE1 and GAE6 were obtained from the SALK 216

collection as described in the Accession Numbers section of Methods We identified two 217

GAE6 knock-out alleles gae6-1 and gae6-2 and a T-DNA insertion in GAE1 gae1-1 218

that caused reduced expression of this gene (Supplemental Figure 3) A double mutant 219

was created by crossing gae1-1 and gae6-1 Both gae6 and gae1 gae6 plants were 220

more susceptible to Pma ES4326 than wild-type Col-0 and gae1 plants supporting a 221

role of GAE6 in plant immunity to Pma ES4326 (Figure 2) 222

223

gae6 and gae1 gae6 Plants Have Brittle Leaves 224

While inoculating leaves of gae1 gae6 plants we noticed that the petioles and 225

midribs of these plants tended to break easily when leaves were bent against the 226

proximal-distal axis of the leaves (Figure 3 see Supplemental Figure 4D to F) They 227

tended to break less often when twisted around the proximal-distal axis To quantify 228

this phenotype leaves were bent by 180deg (Figure 3A) and breaking of midribs or 229

petioles was recorded Most gae1 gae6 leaves broke whereas fewer leaves of gae6 230

plants and very few leaves of wild-type Col-0 and gae1 plants did (Figure 3B and 231

Supplemental Figure 4C) Although gae1 gae6 plants had slightly shorter leaves and 232

petioles than wild type plants (Supplemental Figure 4A and B) no correlation between 233

leaf size or petiole length and breaking frequency was detected We also investigated 234

tissue integrity by monitoring ion leakage Interestingly when leaves were inoculated 235

with deionized water leaf discs of gae1 gae6 leaked more ions than wild-type Col-0 236

discs (Figure 3C) suggesting that the increased brittleness is correlated with a 237

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Cell Walls of gae6 and gae1 gae6 Plants Have Lower Levels of Pectin 243

GalA is a major component of pectin We analyzed wall monosaccharide 244

composition in gae mutants to investigate whether the increased brittleness was 245

associated with an altered wall composition of gae leaves To approximate pectin 246

content of gae mutant walls the total uronic acid concentration was measured using a 247

simple colorimetric assay (Filisetti-Cozzi and Carpita 1991 van den Hoogen et al 248

1998) Total uronic acid was reduced in gae6-2 strongly reduced in gae1 gae6 plants 249

but was indistinguishable from wild-type Col-0 in gae6-1 and gae1 (Supplemental 250

Figure 5) This suggested that gae1 gae6 and possibly gae6 plants have reduced 251

pectin 252

To investigate the cell wall composition in leaves of gae plants in more detail we 253

measured the levels of seven neutral monosaccharides the acidic sugars GalA (the 254

monomeric subunit of the pectin backbone (Harholt et al 2010)) and GlcA (Figure 4) 255

We also estimated the crystalline cellulose content (Updegraff 1969) Cell walls of both 256

gae6 mutants and gae1 gae6 were reduced in GalA (Figure 4A) In gae1 gae6 GalA 257

content in cell walls was reduced by more than 40 compared to the wild-type level 258

GlcA levels were indistinguishable between mutants and wild-type (Figure 4B) 259

Cellulose content was higher in gae6-2 and gae1 gae6 walls likely due to the loss of 260

pectins (Figure 4C) Cell walls of gae1 gae6 plants were also enriched in all neutral 261

sugars measured presumably due to the substantial proportional loss of GalA (Figure 262

4D) The levels of most wall monosaccharides were even higher in the gae1 gae6 263

double mutant with the exception of Rha which did not exhibit a significant increase 264

compared to the gae6 single mutants The absence of a compensational increase in 265

Rha suggested that RGI the dominant Rha contributor to the wall may be considered 266

reduced in gae1 gae6 mutants compared to wild-type albeit not to such an extent as 267

HG 268

238 structural defect that promotes increased ion leakage Taken together these results

239 showed that loss of GAE6 causes brittle leaves and additional losss of GAE1 strongly

240 enhances this phenotype We conclude that GAE1 and GAE6 contribute to normal leaf

241 flexibility 242

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gae6 cell walls are reduced in HG Using the same samples we detected no difference 274

from wild type in binding of LM5 which recognizes a tetramer of (1-4)-β-D-galactans 275

found in RGI (Jones et al 1997) LM6 which recognizes a linear pentasaccharide in (1-276

To further investigate which pectic components were altered in gae1 gae6 dot 269

blot experiments using pectin isolated from the various genotypes were performed Both 270

LM19 and LM20 antibodies recognize HG (Verhertbruggen et al 2009a) As expected 271

LM19 binding was reduced in gae6-1 and gae1 gae6 (Figure 5A and B) and LM20 272

binding was reduced in gae1 gae6 (Figure 5C and D) This result confirmed that gae1 273

277 5)-α-L-arabinans found in RGI (Willats et al 1998 Verhertbruggen et al 2009b) LM8

278 which recognizes xylogalacturonan (Willats et al 2004) or CCRC-M7 which

279 recognizes an arabinosylated (1-6)-β-D-galactan epitope occurring on RGI (Puhlmann et

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al 1994 Steffan et al 1995 Pattathil et al 2010) (Supplemental Figure 6) 280

Collectively these cell wall composition data show that gae1 gae6 plants are reduced in 281

HG Due to the strong proportional reduction of HG in the walls levels of all other 282

components should be higher However Rha indicative of RGI was not higher in gae1 283

gae6 suggesting that RGI may also be reduced in gae1 gae6 walls 284

285

GAE1 and GAE6 Are Required for Immunity to Specific B cinerea Isolates 286

We hypothesized that plant immunity to B cinerea might be affected by pectin 287

content in the host cell wall because this pathogen requires polygalacturonases and 288

pectin methylesterases for full virulence (ten Have et al 1998 Valette-Collet et al 289

2003 Kars et al 2005b Espino et al 2010) Because Bcinerea is genetically diverse 290

and has a high diversity of polygalacturonases (Rowe and Kliebenstein 2007) we 291

measured relative fungal growth at both 2 and 3 d post-inoculation (dpi) using ten 292

different B cinerea isolates (Figure 6) The B cinerea isolates Fresa 525 and Gallo 1 293

showed enhanced virulence on gae1 gae6 plants at both time points whereas KT and 294

UK Razz showed enhanced virulence in gae1 gae6 plants at 3 but not 2 d dpi (Figure 295

6A) We did not detect any significant changes in relative fungal growth between wild-296

type Col-0 and gae1 gae6 plants when using Acacia Apple 517 DN Grape Pepper or 297

Rasp isolates of B cinerea Therefore we continued our study using Gallo 1 and Fresa 298

525 We also included Pepper for comparison as this isolate has been used in a large-299

scale analysis of plant gene expression in response to B cinerea infection (Windram et 300

al 2012) 301

We next investigated whether expression of GAE1 and GAE6 was affected by 302

challenge with B cinerea Inoculation of wild-type Col-0 plants with any isolate 303

repressed GAE1 and GAE6 expression (Figure 6B and C) and this repression was 304

generally independent of PAD4 (Figure 6B and C) Expression of GAE2 and GAE3 was 305

unaltered by B cinerea treatment GAE4 was repressed only by Fresa 525 treatment 306

while GAE5 expression was enhanced with all three isolates tested (Supplemental 307

Figure 7) As seen for infection with Pma ES4326 the expression of GAE1 and GAE6 308

was repressed upon B cinerea treatment This indicates a role for GAE1 and GAE6 in 309

the immune response to B cinerea 310

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Because the amount of pectin substrate for pathogen polygalacturonases is 313

reduced we hypothesized that immunity phenotypes of gae1 gae6 plants may result 314

from reduced generation of OGs by these enzymes In turn the reduced OGs could 315

lead to reduced immune responses To test this we used macerozyme a commercial 316

pectinase as a proxy for pathogen pectinases We isolated cell walls as a subcellular 317

fraction known as alcohol insoluble residue (AIR Gille et al 2009) This was treated 318

with macerozyme and tested for release of soluble likely low molecular weight pectin 319

fragments (Figure 7) Treatment with 025 macerozyme released significantly more 320

uronic acid from wild-type Col-0 AIR relative to gae1 gae6 AIR (Figure 7B) To test 321

activity of these pectin-containing solutions we infiltrated them into wild-type Col-0 322

plants and measured PAD3 expression PAD3 is required for synthesis of the 323

phytoalexin camalexin (Zhou et al 1999) and its expression is known to be induced by 324

OG treatment (Ferrari et al 2007) Fractions from macerozyme treated Col-0 walls 325

induced significantly more PAD3 expression than fractions derived from gae1 gae6 326

walls suggesting that macerozyme treatment did indeed release more active soluble 327

uronic acid possibly OGs from wild-type Col-0 walls than from gae1 gae6 walls (Figure 328

7C) 329

Next we studied the effect of macerozyme treatment on expression of PAD3 in 330

intact plants (Figure 8) This treatment did not lead to any visible leaf damage 331

(Supplemental Figure 8) In wild-type Col-0 PAD3 expression was induced three h after 332

treatment with 001 macerozyme (Figure 8A) By contrast no macerozyme-induced 333

induction of PAD3 expression was detected in gae1 gae6 (Figure 8A) However OG 334

treatment induced the expression of PAD3 in both genotypes indicating that gae1 gae6 335

plants do respond to OGs (Figure 8B) Macerozyme treatment also induced expression 336

of the SA marker genes PATHOGENESIS RELATED 1 (PR1) and SID2 in Col-0 but 337

not in gae1 gae6 (Figure 9 panel A and B) PR1 was not significantly induced by 338

purified OGs (Figure 9C) at the time and concentration tested but SID2 was induced by 339

OG treatment in both genotypes (Figure 9D) However the induction was stronger in 340

wild-type Col-0 In addition the JA markers JASMONATE-ZIM-DOMAIN PROTEIN 5 341

(JAZ5) and JAZ10 were induced by macerozyme and OG treatment in both Col-0 and 342

gae1 gae6 (Figure 10A and Supplemental Figure 11) Interestingly expression of JAZ5 343

312 Pectinase-Induced Immune Signaling Is Altered in gae1 gae6 Plants

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344

345

346

and JAZ10 was already strongly induced in gae1 gae6 after mock inoculation (Figure 10

and Supplemental Figure 11) We observed no hyperresponsive induction by mock

treatment of PLANT DEFENSIN 12 (PDF12) a necrotrophy and late JA signaling

347 marker gene (Supplemental Figure 12A-D) PDF12 was also not induced by

348 macerozyme or OG treatments (Supplemental Figure 12A-D) This suggests that early

349 JA signaling is induced by mock-treatment in gae1 gae6 Because it is known that JA

350 and SA signaling act antagonistically (Pieterse et al 2012) the strong up-regulation of

351 JA-induced immune signaling in gae1 gae6 might explain the lower OG-induced SID2

352 expression in this genotype

353 To test whether other immune responses are altered in gae1 gae6 plants we

354 analyzed Mitogen-activated protein kinase (MAPK) activity following macerozyme and

355 OG treatments OGs are known to activate the MAPKs MPK3 and MPK6 in Arabidopsis

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(Galletti et al 2011) Macerozyme treatment activated MPK3 and MPK6 in wild-type 356

Col-0 whereas MAPK activation in gae1 gae6 was slightly reduced in four out of five 357

experiments performed (Supplemental Figure 13) By contrast OG-induced MAPK 358

activity was similar or increased in gae1 gae6 in four out of five experiments 359

(Supplemental Figure 13) Additionally mock treatment induced MAPK activity in gae1 360

gae6 more strongly than it did in wild-type Col-0 in four out of five experiments 361

performed (Fig S13) Collectively these results illustrate that activation of several 362

immune signaling componentsmdashcamalexin synthesis SA and JA signaling and 363

possibly MAPK activationmdashby macerozyme is substantially altered in gae1 gae6 This 364

suggests that release of soluble pectin fragments possibly OGs by pectinolytic 365

enzymes might be reduced in gae1 gae6 plants while recognition of OGs is intact 366

367

Following B cinerea Infection Production of Soluble Pectin and Deposition of 368

Callose Are Reduced in gae1 gae6 Plants 369

Although pad3 plants were more susceptible to the B cinerea isolates Fresa 525 370

and Gallo 1 which also grew better on gae1 gae6 growth of the isolate Pepper was 371

similar on Col-0 and pad3 (Supplemental Figure 9A) However we did not detect any 372

significant changes in camalexin production after treatment with B cinerea isolate Gallo 373

1 and Fresa 525 in gae1 gae6 (Figure 8D) Neither did we detect changes in PAD3 374

expression after treatment with any B cinerea isolate in gae1 gae6 (Figure 8E) 375

Additionally SID2 and PR1 were similarly induced by all B cinerea isolates in both Col-376

0 and gae1 gae6 (Figure 9E and F) and sid2 plants which are deficient for pathogen-377

induced SA biosynthesis did not show any differences in relative B cinerea growth 378

(Supplemental Figure 9B) Expression of JAZ10 was induced more strongly by Gallo 1 379

treatment in gae1 gae6 than in wild-type Col-0 but not by Fresa 525 or Pepper (Fig 380

10C) while dde2 plants deficient for JA biosynthesis showed enhanced susceptibility to 381

all B cinerea isolates tested (Supplemental Figure 9B) Curiously exogenous 382

application of MeJA did not lead to altered relative fungal growth of B cinerea 383

(Supplemental Figure 9C) and cev1 isoxaben resistant 1 (ixr1) plants which have 384

constitutively high JA levels (Ellis and Turner 2001) did not show any changes in 385

susceptibility to the B cinerea isolates tested (Supplemental Figure 9D) Hence neither 386

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differences in B cinerea isolate susceptibility to camalexin nor altered SA or JA 387

signaling by themselves explained the enhanced susceptibility of gae1 gae6 to these B 388

cinerea isolates 389

Hydrogen peroxide accumulation in response to B cinerea isolates Fresa 525 390

Gallo 1 and Pepper was not obviously altered in gae1 gae6 plants (Supplemental Figure 391

14) However callose deposition upon treatment with B cinerea isolate Gallo 1 was 392

strongly reduced in gae1 gae6 (Figure 11) It was previously shown that OG treatment 393

induces callose deposition (Denoux et al 2008) suggesting that the reduction in 394

callose deposition might be due to reduced OG release in gae1 gae6 It is possible that 395

this reduction in callose deposition contributes to the reduced immunity of gae1 gae6 396

plants to B cinerea isolate Gallo 1 397

Because macerozyme could be used to release soluble likely low molecular 398

weight pectin from isolated cell walls we next investigated whether infection with B 399

cinerea was accompanied by accumulation of soluble pectin as well We prepared cell 400

walls (AIR fraction) from leaves infected with B cinerea incubated these cell walls with 401

water and measured uronic acid content of the extracts B cinerea treatment did in fact 402

lead to increased accumulation of soluble pectin in wild-type Col-0 plants when 403

compared to mock-treated samples (Figure 12) No such increased accumulation of 404

water-soluble pectin fractions was observed in gae1 gae6 (Figure 12) 405

406

Macerozyme-Induced Pattern-Triggered Immunity against B cinerea Is Abolished 407

in gae1 gae6 408

It was previously shown that OG treatment can induce patternndashtriggered 409

immunity (PTI) to B cinerea (Ferrari et al 2007) We tested macerozyme treatment to 410

determine whether it could also induce PTI to the B cinerea isolate Gallo 1 (Figure 13) 411

Indeed pretreatment with 001 macerozyme for 24 h led to reduced B cinerea growth 412

in wild-type Col-0 but not gae1 gae6 plants (Figure 13A) Interestingly mock and 413

macerozyme pretreated gae1 gae6 plants exhibited similar growth of B cinerea Gallo 1 414

as did macerozyme-treated Col-0 plants This result is likely due to activation of JA 415

signaling in gae1 gae6 during the pretreatment procedure (Figure 13C and D) OG-416

induced PTI was observed in both genotypes when using a high dose of OGs (Figure 417

Page 23 of 54

13B) whereas a lower dose of OGs only induced PTI in Col-0 (Supplemental Figure 418

15) This suggests that OG perception in gae1 gae6 at least above a certain threshold 419

is unaltered while release of immunity-activating pectin fragments possibly OGs is 420

421 reduced We conclude that immune responses induced by macerozyme treatment

422 which do not occur in gae1 gae6 plants are important for plant immunity to the B

423 cinerea isolate Gallo 1

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DISCUSSION 426

Here we showed that loss of GAE1 and GAE6 leads to a dramatic reduction in 427

pectin content in the cell wall (Figure 4) likely causing the brittleness of leaves that we 428

observed (Figure 3) Petioles and midribs of gae1 gae6 plants broke easily when bent 429

and inoculation of leaves with deionized water resulted in increased ion leakage 430

possibly due either to microscopic fractures of the cell wall induced by inoculation stress 431

or to a reduced capability of these walls to withstand increased turgor pressure The 432

increased brittleness was associated with altered cell wall composition In our analysis 433

the GalA content of gae1 gae6 leaf walls was reduced by more than 40 and the total 434

uronic acids by over 60 (Figure 4 and Supplemental Figure 5) This is a major change 435

in the overall cell wall composition considering that pectin makes up about 50 of the 436

plant cell walls found in Arabidopsis leaves (Zablackis et al 1995 Nobuta et al 2007 437

Harholt et al 2010) Interestingly quasimodo1 (qua1) plants which have a mutation in 438

a glycosyltransferase family 8 gene are reduced in GalA by 25 and show reduced 439

binding of HG specific antibodies (Bouton et al 2002) 440

Furthermore qua2 plants which have a mutation in a putative methyltransferase 441

have about 13 less total GalA and lack 50 of cell wall HG (Mouille et al 2007) 442

Likewise cgr2 cgr3 plants carrying mutations in another class of Golgi-localized 443

putative methyl-transferases have leaf cell walls that are reduced by about 40 in 444

uronic acid (Kim et al 2015) All three mutants are severely dwarfed (Bouton et al 445

2002 Mouille et al 2007 Kim et al 2015) Additionally qua1 and qua2 mutants show 446

a distorted overall shape likely due to reduced cell cohesion and are extremely fragile 447

(Bouton et al 2002 Mouille et al 2007) By contrast gae1 gae6 plants with a similar 448

strong reduction in GalA and total uronic acids have an overall normal shape and are 449

only slightly smaller than wild-type Col-0 (Supplemental Figure 4) In the wall analysis 450

performed here we did not detect changes in other wall polysaccharides or structures 451

that might compensate for the reduction of pectin in gae1 gae6 It is possible the severe 452

morphological alterations in qua2 and cgr2 cgr3 plants are due to reduction in pectin 453

methylesterification which is unaffected in gae1 gae6 It was speculated that quality 454

control mechanisms in the Golgi prevent secretion of inefficiently esterified pectin in 455

cgr2 cgr3 (Kim et al 2015) suggesting that the Golgi of these plants might over-456

Page 26 of 54

accumulate pectin and that this may contribute to the severe dwarf phenotype of these 457

plants The gae1 gae6 plants described here provide an opportunity to study the effects 458

of reduction of pectin in plants that do not show severe morphological phenotypes 459

We found that gae1 gae6 plants are also compromised in resistance to B 460

cinerea and Pma ES4326 indicating that loss of pectin reduces immunity to these 461

pathogens (Figure 2 and 6) We found that gae1 gae6 plants were slightly more 462

susceptible to Pma ES4326 but showed a strong increase in susceptibility to specific B 463

cinerea isolates This was not unexpected because B cinerea is known to interact with 464

pectin as part of its infection strategy It secretes various pectin-degrading enzymes 465

early during infection and requires some of these enzymes including PG1 PG2 and 466

PME1 for full virulence (ten Have et al 1998 Valette-Collet et al 2003 Kars et al 467

2005b Espino et al 2010) Additionally Arabidopsis plants expressing an antisense 468

construct of the polygalacturonase inhibitor PGIP1 are significantly more susceptible to 469

B cinerea (Ferrari et al 2006) On the other hand Pma ES4326 is not known to470

require cell wall degrading enzymes for full virulence even though it encodes two 471

pectate lyase genes in its genome (PMA4326_07094 and PMA4326_04621 472

wwwbacteriaensemblorg) B cinerea isolates are also known to be genetically quite 473

diverse in their polygalacturonase (PG) loci and genetic variability in PG2 is associated 474

with varying growth on pectin containing media (Rowe and Kliebenstein 2007) 475

Furthermore the contribution of PG1 and PG2 to B cinerea virulence seems to depend 476

on the specific B cinerea isolate and the host species (Kars et al 2005a Zhang and 477

Van Kan 2013) B cinerea isolates are also known to be differently sensitive to the anti-478

microbial secondary metabolite camalexin (Kliebenstein et al 2005) In line with these 479

observations the susceptibility of gae1 gae6 plants differed depending on the specific 480

B cinerea isolate Whereas Fresa 525 Gallo 1 UK Razz and KT grew better in gae1481

gae6 we detected no difference in Acacia Apple 517 DN Grape Pepper or Rasp 482

growth (Figure 6) 483

B cinerea was shown to metabolize pectin and to possibly utilize GalA as a484

carbon source during in planta growth (Zhang et al 2011 Zhang and Van Kan 2013) 485

Our data show that gae1 gae6 plants which are reduced in GalA content did allow 486

enhanced growth of specific B cinerea isolates but no reduction in growth of any B 487

Page 27 of 54

cinerea isolates tested was found This suggests that utilization of pectin as a carbon 488

source was not a contributing factor to alterations in B cinerea susceptibility It is 489

possible that changes in the cell wall composition affect the structural integrity of the 490

wall and affect hyphal penetration of specific B cinerea isolates However OGs (small 491

HG fragments) are likely released during the infection process (Ferrari et al 2013) 492

OGs function as DAMPs and initiate immune responses in Arabidopsis (Ferrari et al 493

2013) Treatment of Arabidopsis with OGs having a degree of polymerization (DP) of 494

10-15 sugar subunits was previously shown to enhance resistance to B cinerea (Ferrari495

et al 2007) We showed that pretreatment with OGs or macerozyme induced enhanced 496

immunity to B cinerea isolate Gallo 1 in wild-type Col-0 but only OGs and not 497

macerozyme induced immunity in gae1 gae6 (Figure 13) Our data suggest gae1 gae6 498

plants which have reduced pectin content may release less OGs and that this 499

reduction leads to the increased susceptibility to B cinerea 500

Consistently we found that macerozyme treatment released less soluble pectin 501

from gae1 gae6 cell walls than from wild-type walls Fractions containing soluble pectin 502

released from gae1 gae6 were also less active in inducing PAD3 gene expression than 503

the fractions released from wild-type walls (Figure 7) We also showed that less soluble 504

pectin accumulated in gae1 gae6 than in wild-type plants 48 h after treatment with B 505

cinerea (Figure 12) 506

We observed that loss of pectin leads to alterations in A thaliana immune 507

signaling Macerozyme treatment induced expression of PAD3 and the SA marker 508

genes PR1 and SID2 in wild-type Col-0 but not in gae1 gae6 whereas OG treatment 509

led to increased PAD3 and SID2 expression in both wild-type and gae1 gae6 (Figure 8 510

and 9) This suggests that although release of active soluble pectin in these plants 511

might be altered OG recognition is intact In fact cell walls isolated from qua1 another 512

mutant known to be reduced in HG released less OGs (DP1-6) than did wild-type cell 513

walls upon endopolygalacturonase treatment (Bouton et al 2002) This was also true 514

for gae1 gae6 cell walls as macerozyme treatment of isolated gae1 gae6 cell walls 515

released less soluble pectin than treatment of Col-0 cell walls Because the fraction 516

from gae1 gae6 plants was also less active in inducing PAD3 expression (Figure 7) it 517

seems possible that biologically active OGs were the active component inducing PAD3 518

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expression Interestingly expression of the JA marker genes JAZ5 and JAZ10 was 519

higher in gae1 gae6 than in wild-type mock-inoculated plants but was comparable to 520

wild-type in untreated plants suggesting that JA signaling in gae1 gae6 is 521

hyperresponsive (Figure 10 and Supplemental Figure 11) This induction of JA signaling 522

might be due to wounding stress incurred during the inoculation process JA and SA 523

signaling generally act antagonistically (Pieterse et al 2012) Increased JA signaling 524

activity might hence explain why mock- and macerozyme-treated gae1 gae6 plants 525

exhibited reduced expression of the SA marker PR1 and why OG treatment induced 526

expression of SID2 another SA marker to a lower extent in gae1 gae6 than in wild-type 527

plants (Figure 9) 528

No single immune signaling component was found to be the sole contributor to 529

the compromised immunity of gae1 gae6 to specific B cinerea isolates such as Gallo 1 530

and Fresa 525 These isolates grew better on pad3 while growth of Pepper another B 531

cinerea isolate that did not show altered virulence on gae1 gae6 was unaltered 532

(Supplemental Figure 9) This suggested that differences in camalexin sensitivity of 533

these B cinerea isolates might account for the differences in gae1 gae6 susceptibility 534

However camalexin accumulation and PAD3 expression was the same for wild-type 535

Col-0 and gae1 gae6 for the B cinerea isolates Fresa 525 and Gallo 1 (Figure 8) which 536

showed enhanced growth on gae1 gae6 plants It should be noted that B cinerea 537

virulence is often estimated by comparing lesion sizes Lesion size and measurements 538

of relative fungal growth by qPCR do not always show the same results For example 539

although Denby et al (2004) showed that Pepper causes larger lesions on pad3 than on 540

wild-type this study showed that relative fungal growth of Pepper on pad3 was 541

unaltered 542

We observed that callose deposition was strongly reduced in gae1 gae6 plants 543

72 h after treatment with B cinerea isolate Gallo 1 (Figure 11) possibly due to reduced 544

release of active soluble pectin Interestingly OG-induced PTI to an unspecified isolate 545

of B cinerea was intact in the pmr4 mutant which does not produce callose (Galletti et 546

al 2008) Due to the high SA levels in pmr4 plants (Nishimura et al 2003) and the fact 547

that different B cinerea isolates vary both in induction of and reaction to defense 548

responses (Kliebenstein et al 2005 Rowe et al 2010) we could not determine 549

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whether reduced callose deposition causes the increased susceptibility of gae1 gae6 to 550

B cinerea isolate Gallo 1551

We did not observe any changes in B cinerea growth either on cev1 ixr1 plants 552

which are known to have constitutively high JA and OPDA levels (Ellis et al 2002b) or 553

on plants exogenously treated with MeJA (Supplemental Figure 9) Because plants 554

were kept uncovered for 3 h after MeJA treatment it is unclear how much MeJA was 555

bioavailable in the plant However JAZ10 expression was induced 3 h after spraying 556

with MeJA (Supplemental Figure 10) Because pathogen growth generally depends on 557

the combination of a multitude of immune signaling events we conclude that multiple 558

changes to the immune signaling in gae1 gae6 together account for the altered B 559

cinerea growth phenotypes detected 560

We showed that expression of GAE1 and GAE6 is repressed by the bacterial 561

hemibiotroph Pma ES4326 This repression did not occur in an eds1 or pad4 562

background or in pbs3 at the 24-h time point (Figure 1 and Supplemental Figure 1) 563

During the Arabidopsis - Pma ES4326 interaction the expression of many defense 564

related genes is altered in a PAD4EDS1 dependent fashion (Wang et al 2008) Some 565

of these expression changes require SA CBP60g and SARD1 are thought to act 566

downstream of PAD4 and EDS1 but upstream of SA-dependent responses (Wang et 567

al 2011) PBS3 is also thought to act downstream of PAD4 and EDS1 and upstream of 568

SA but the set of genes whose expression it affects is only partially overlapping with 569

those affected by CBP60gSARD1 (Wang et al 2008 Wang et al 2011) Thus GAE1 570

and GAE6 repression is regulated by the part of the immune signaling network that 571

requires PAD4 EDS1 and PBS3 but is independent of CBP60gSARD1 and SA 572

Because EDS1 PAD4 and PBS3 are important components of the plant immune 573

signaling network (Wiermer et al 2005 Nobuta et al 2007) one might conclude that 574

repression of GAE1 and GAE6 may be part of the plant immune response to Pma 575

ES4326 B cinerea treatment also reduced GAE1 and GAE6 expression but this was 576

mostly independent of PAD4 (Figure 6) while both Pma ES4326 and B cinerea repress 577

GAE1 and GAE6 expression the host signaling mechanisms responsible likely differ 578

In summary our data show that Arabidopsis GAE1 and GAE6 are required for 579

pectin biosynthesis leaf flexibility and immunity to Pma ES4326 and to specific B 580

Page 30 of 54

cinerea isolates We used macerozyme a commercial pectinase to mimic the effect of 581

B cinerea pectinases Macerozyme treatment released less pectin from gae1 gae6 cell582

walls and induced less immune signaling in gae1 gae6 plants Less water soluble pectin 583

also accumulated in B cinerea infected gae1 gae6 plants and macerozyme-induced 584

immunity to B cinerea isolate Gallo 1 was reduced in these plants We conclude that 585

the reduced pectin content in these mutants may lead to reduced release of soluble 586

pectin including active OGs during pathogen attack and that this compromises 587

immunity 588

589

590

Page 31 of 54

METHODS 591

Plant Materials and Growth Conditions 592

Wild-type Columbia (Col-0) and all mutant plants (in Col-0 background) were 593

grown on sterilized BM2 germinating mix (Berger Inc Quebec Canada) in a controlled 594

environment chamber (Conviron Pembina ND USA) with a 12-h photoperiod under 595

100 microM m-2 s-1 fluorescent illumination at 22degC and 75 relative humidity Germplasm 596

used is described in the Accession Numbers section at the end of Methods 597

598

Data Analysis and Replicates 599

For biological replicates samples were obtained from separate plant tissues and 600

in many cases from plants grown in different flats In some experiments multiple 601

technical replicates were measured per biological replicate For statistical analysis all 602

technical replicates were averaged and the averages were considered one biological 603

replicate Independent experiments were performed at different times and often 604

consisted of multiple biological replicates per experiment 605

Data analysis was performed using mixed linear models in the R programming 606

environment (Bates et al 2015) unless otherwise indicated Generally genotypes 607

treatments and time points were used as fixed effects and replicate-specific effects like 608

different pots flats etc were considered random effects We reported p-values 609

obtained from the mixed linear model when less than 10 comparisons were performed 610

and q-values for more than 10 comparisons to correct for multiple comparison testing 611

Q-values were calculated using the Benjamini-Hochberg procedure (Benjamini and612

Hochberg 1995) unless specified otherwise 613

614

Pathogen Strains Growth Conditions and Pathogen Growth Assays 615

Growth of Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) and 616

bacterial growth assays were performed as previously described (Bethke et al 2014) 617

For qRT-PCR assays four week-old plants were inoculated with bacteria (OD600 = 618

0002) or mock (5 mM MgSO4) At least four leaves from two plants were collected at 619

the indicated time points per biological replicate 620

Page 32 of 54

Prior to each experiment Botrytis cinerea isolates were grown on 1x PDA 621

medium (Difco) for 10 d at room temperature All isolates used have been described 622

previously (Rowe and Kliebenstein 2007) The spores were washed from the surface of 623

the plate using an inoculation medium (1x Gamborgrsquos B-5 basal salt mixture (Sigma-624

Aldrich G5768) 2 (wv) glucose 10 mM phosphate buffer pH 64) and fungal hyphae 625

were removed from the suspension by filtering through four layers of cheesecloth The 626

concentration of spores was determined using a hemocytometer and adjusted to 25 x 627

105 spores mL-1 Four week-old Arabidopsis plants were inoculated by placing one 10-628

microL droplet of the B cinerea spore solution or inoculation medium (mock) on the adaxial 629

leaf surface of fully expanded leaves Inoculated plants were kept at 100 relative 630

humidity Infection sites were collected at various time points using a cork borer For 631

qRT-PCR assays at least four leaf discs from one plant were combined for each 632

biological sample For B cinerea growth assays at least 12 leaf discs from three to six 633

plants were pooled per biological sample Relative fungal growth was determined by 634

measuring the abundance of a fungal gene relative to a plant gene as described below 635

In brief DNA from infected tissue was extracted and equal amounts of total DNA were 636

used to perform qPCR reactions using the B cinerea cutinase A and the Arabidopsis 637

SK11 gene respectively Primers were described in (Gachon and Saindrenan 2004) 638

and are listed in Supplemental Table 1 Two technical replicates were combined for 639

each biological replicate B cinerea is a necrotrophic fungal pathogen and may destroy 640

plant DNA as disease progresses However decreases in the amount of plant DNA 641

over the course of our experiments were only observed at late time points with very 642

susceptible plants so the qPCR assay is generally a reasonable estimate of fungal 643

biomass (Supplemental Figure 16) 644

645

Treatment with Macerozyme or OGs 646

For PTI assays four week-old plants either were inoculated with 001 (wv) 647

macerozyme R-10 (Yakult) a Rhizopus polygalacturonase (EC 32115) that possesses 648

high pectinase and hemicellulase activity or were mock inoculated using heat-649

inactivated macerozyme Other similar plants were either inoculated with 250 or 500 microg 650

mL-1 OGs or mock inoculated with water One d later plants were infected with B 651

Page 33 of 54

cinerea and samples were collected immediately (0 dpi) and two or three d later To 652

investigate the role of MeJA on B cinerea growth plants were sprayed with 1mM MeJA 653

in water with 001 (vv) Tween 20 Covers were removed so that leaves could dry off 654

before plants were inoculated with B cinerea three h later Samples were harvested at 655

the indicated time points For expression analysis and MAPK activation assays plants 656

were either inoculated with 100 microg mL-1 OGs or mock inoculated with water OGs used 657

for experiments described in Figures 8 to 10 and Supplemental Figure 12 were a kind 658

gift from Simone Ferrari OGs used in experiments described in Figure 13 and 659

Supplemental Figures 11 13 and 15 were prepared as previously described (Kohorn et 660

al 2014) 661

662

Expression Analysis 663

Leaf tissue from inoculated plants was harvested at the indicated time points 664

flash frozen pulverized and RNA extracted using Trizol (Invitrogen) Quantitative RT-665

PCR was performed using the SuperScript III Platinum SYBR Green One-Step 666

quantitative RT-PCR kit (Invitrogen) and a Lightcycler 480 Real-Time PCR system 667

(Roche) as previously described (Truman and Glazebrook 2012) In brief equal 668

amounts of total RNA and a gene specific primer were used for each reaction The 669

crossing point (Cp) was calculated using the 2nd derivative max method provided with 670

the Lightcycler software for each amplification curve Each reaction was run with two 671

technical replicates and the Cp values for these replicates were averaged ACTIN2 was 672

used as a stably expressed reference gene At least three biological replicates each 673

were performed and analyzed using a mixed-linear model RT-PCR was performed to 674

verify T-DNA insertion lines using the One-Step RT-PCR kit (Qiagen) according to the 675

manufacturerrsquos instructions The primers used can be found in Supplemental Table 1 676

677

Electrolyte Leakage Assay 678

Fully expanded leaves of four week-old plants were inoculated with water (Milli-Q 679

grade) using a needleless syringe Leaf discs were collected using a cork borer and 680

three leaf discs from one leaf were placed with the adaxial surface down onto 2 mL 681

water in a 12-well cell culture plate Conductivity was measured using a Horiba B-173 682

Page 34 of 54

conductivity meter after 30 min and then the water was replaced Conductivity was 683

measured again 30 min later (1 h total) as indicated in Figure 3C Six biological 684

replicates each were performed in three independent experiments and means and SEs 685

were calculated Two-sided t-tests were performed for each time point 686

687

DAB and Aniline Blue Staining 688

Leaves from four week-old plants were either mock inoculated with B cinerea 689

inoculation medium or inoculated with 10 microL droplets of a B cinerea spore solution (25 690

x 105 spores mL-1) of the indicated isolates Samples were collected 72 h later For 33prime-691

diaminobenzidine (DAB) staining two to five inoculated leaves were vacuum infiltrated 692

with 33prime-Diaminobenzidine (1 mg mL-1 in water pH 38) solution and stained overnight 693

in the dark Leaves were destained using 100 ethanol at 65degC and then transferred to 694

50 (vv) glycerol and photographed 695

Callose was visualized by staining leaves with aniline blue as previously 696

described (Adam and Somerville 1996) Briefly leaves were vacuum infiltrated with 697

alcoholic lactophenol (one volume of phenolglycerollactic acidwater (1111) mixed 698

with two volumes of ethanol) and destained at 65degC Next leaves were moved through 699

an alcohol gradient (50 20 and 10 (vv) ethanol) into water The destained leaves 700

were stained for 30 min in 150 mM K2HPO4 (pH 95) with 001 (wv) aniline blue 701

Samples were mounted in water and examined using a Nikon Eclipse Ni-U microscope 702

at 10x magnification using a DAPI filter (excitation at 325-375 nm emission at 435-485 703

nm) For callose quantification six 200 microm x 200 microm squares were counted for each 704

sample type Callose deposits per mm2 were calculated and data analyzed using a t-705

test Note that there is an inherent uncertainty associated with such counts because 706

callose deposits may not all be in focus at the same time 707

708

MAPK Activity Assay 709

Fully expanded leaves of four week-old plants were inoculated with 001 710

macerozyme 100 microg mL-1 OGs 100 nM flg22 or water (mock) flg22 a peptide from 711

bacterial flagella that induces immune responses was used as a positive control for 712

Page 35 of 54

MAPK activation (Asai et al 2002) and was purchased from EZBiolab Inc (Westfield 713

IN USA) Samples were harvested 10 min later and immediately frozen in liquid 714

nitrogen Protein was extracted using an extraction buffer containing 25 mM Tris pH 78 715

75 mM NaCl 1 mM DTT 1 mM NaF 05mM Na3VO4 01 (vv) Tween 20 one 716

cOmplete Mini protease inhibitor cocktail tablet (Roche) and one PhosSTOP 717

phosphatase inhibitor cocktail tablet (Roche) per 10 mL buffer 15 microg of total protein 718

were electrophoresed on a 10 SDS-polyacrylamide gel and then blotted onto a PVDF 719

membrane (Bio-Rad 162-0177) Activated MAPKs were detected using p4442 MAPK 720

(Erk12) antibody (9102S Cell Signal 12500 in TBST (20 mM Tris pH 75 150 mM 721

NaCl 01 (vv) Tween 20)) Anti-Rabbit IgG-HRP (115000 in TBST A6154 Sigma) 722

and ECL Plus substrate (Pierce 32132) MPK3 and MPK6 were detected with an anti-723

MPK3 antibody (M8318 Sigma 12000 in TBST and 3 (wv) milk) or an anti-MPK6 724

antibody (A7104 Sigma 16000 in TBST and 3 (wv) milk) respectively The 725

secondary antibody and detection were the same as for the p4442 antibody Five 726

independent experiments were performed 727

728

Preparation of Alcohol Insoluble Residue (AIR) Pectin Extraction and Uronic Acid 729

Measurements 730

Fully expanded leaves from four week-old plants that were incubated for 48 h in 731

the dark to reduce starch were harvested flash frozen and pulverized AIR was 732

extracted by washing ground material twice in 70 (vv) ethanol three times in a 733

mixture of chloroform and methanol (11 vv) and once in acetone (Gille et al 2009) 734

and was then air dried Cell wall pectin was extracted from AIR with cell wall extraction 735

buffer (50 mM Trizma 50 mM CDTA pH 72) at 95degC for 15 min Samples were 736

homogenized using a paint shaker (Harbil 5G-HD) and glass beads (3 mm diameter) 737

For dot blot experiments 500 microL of cell wall extraction buffer were used per 10 mg of 738

AIR Debris was precipitated by centrifugation for 10 min at 10000 g 739

Total uronic acid content of pectin extracted as described above using 500 microL of 740

cell wall extraction buffer per 1 mg of AIR was measured as described previously 741

(Filisetti-Cozzi and Carpita 1991 van den Hoogen et al 1998) First 36 microL of pectin 742

extract were mixed with 4 microL of 4 M sulfamic acid Then 200 microL of sulfuric acid 743

Page 36 of 54

containing 120 mM sodium tetraborate was added and samples were incubated at 80degC 744

for 1 h Following cooling on ice the optical density of samples was measured at 490 745

nm Next 40 microL of m-hydroxydiphenyl reagent (100 microL of m-hydroxydiphenyl in dimethyl 746

sulfoxide at 100 mgmL mixed with 49 mL 80 (vv) sulfuric acid just before use) was 747

added and the samples were mixed The optical density at 490 nm was measured 748

again The optical density before m-hydroxydiphenyl reagent addition was subtracted 749

from the optical density measured after addition of the dye The concentration of uronic 750

acid was calculated using known amounts of GalA as a standard 751

752

Release of Pectin from Arabidopsis Cell Walls Using Macerozyme 753

AIR preparations from leaves of four week-old plants were treated with 025 754

(wv) macerozyme R-10 (Yakult) or water (mock) at a concentration of 100 microL per mg 755

AIR Samples were incubated for 3 h at room temperature on a vortex shaker and 756

centrifuged for 10 min at 12000 g Total uronic acid concentration of 5-fold diluted 757

supernatants was determined for two technical replicates each The concentration of 758

uronic acid was calculated using known amounts of GalA as a standard For 759

subsequent qRT-PCR experiments supernatants from multiple extractions were 760

combined boiled for 20 min to inactivate macerozyme diluted 10-fold with water and 761

inoculated into four week-old Col-0 plants Heat-inactivated 10-fold diluted macerozyme 762

solutions were used as mock treatment Samples were collected 3 h later and PAD3 763

expression was determined 764

765

Release of Pectin from Arabidopsis Leaves Infected with B cinerea 766

Fully expanded leaves of four week-old plants were either inoculated with up to 767

five 10 microL droplets of B cinerea spore solution (25 x 105 spores mL-1) or mock 768

inoculated with B cinerea inoculation medium For each biological replicate leaves with 769

a combined count of at least 20 infection sites were collected 48 h later AIR was 770

extracted as described above but plants were not dark-treated or freeze-dried and the 771

first ethanol step was performed using 85 (vv) ethanol To extract soluble pectin 772

fragments AIR samples were mixed with water at a concentration of 20 microL per infection 773

site incubated for 7 h at room temperature on a vortex shaker and centrifuged for 10 774

Page 37 of 54

min at 12000 g Total uronic acid concentration of supernatants was determined for two 775

technical replicates each The concentration of uronic acid was calculated using known 776

amounts of GalA as a standard 777

778

Measurement of Uronic Acids Neutral Monosaccharides and Cellulose Content in 779

Cell Walls 780

Neutral sugars and uronic acids from non-cellulosic polysaccharides in cell walls 781

(AIR) were released by trifluoroacetic acid (TFA) hydrolysis Approximately 1 mg of AIR 782

was hydrolyzed with 2 M TFA at 121degC for 90 min The TFA-soluble material was split 783

into two equal parts one for uronic acid and one for neutral sugar determination The 784

uronic acid content was determined by analyzing the hydrolyzate using a CarboPac 785

PA200 anion-exchange column with an ICS-3000 Dionex chromatography system 786

(Dionex Sunnyvale CA USA) The elution profile consisted of a linear gradient of 50 to 787

200 mM sodium acetate in 01 M NaOH in 10 min at 04 mL per min Alditol acetate 788

derivatives of neutral sugars were produced as described (York et al 1985) In brief 789

monosaccharides in the hydrolyzate were reduced with NaBH4 and peracetylated with 790

acetic anhydride and pyridine at 121degC for 20 min The generated alditol acetates were 791

analyzed by GC-MS Crystalline cellulose was measured according to Updegraff (1969) 792

After hydrolysis of noncellulosic polysaccharides from AIR with the Updegraff reagent 793

(acetic acid nitric acids water 812 (vv) ) the remaining pellet was hydrolyzed in 794

72 sulfuric acid The resulting glucose quantity was determined by the anthrone 795

method (Scott and Melvin 1953) 796

797

Dot Blot Experiments 798

Pectin solutions extracted using 50 microL of cell wall extraction buffer per mg AIR 799

were serially diluted and nitrocellulose membranes were spotted with 1 microL of the diluted 800

pectin solutions Membranes were dried overnight blocked with 5 milk (wv) in 1x 801

PBS (8 g L-1 NaCl 02 g L-1 KCl 144 g L-1 Na2HPO4 024 g L-1 KH2PO4 pH 74) and 802

probed with LM5 (Jones et al 1997) LM6 (Willats et al 1998) LM8 (Willats et al 803

2004) LM19 and LM20 (Verhertbruggen et al 2009a) or CCRC-M7 (Steffan et al 804

Page 38 of 54

1995) antibodies LM series antibodies were diluted 1250 and CCRC-M7 was diluted 805

1500 in 5 milk powder (Nestle Carnation) in 1 x PBS For LM series antibodies a 806

goat anti-Rat HRP conjugated antibody (Bethyl A110-105P 15000 diluted) and for 807

CCRC-M7 an anti-Mouse IgG HRP conjugate (Promega W402B 12500 diluted) was 808

used as secondary antibody in 5 milk powder in 1x PBS LM series antibodies were 809

obtained from plant probes (wwwplantprobesnet) and CCRC-M7 from CarboSource 810

(wwwcarbosourcenet) Membranes were washed after incubation with the primary and 811

secondary antibodies with 1x PBS Dot blots were developed using the ECL system 812

(GE Healthcare Amersham ECL prime) For quantification of signals dot blot results on 813

x-ray films were photographed using a CCD camera and intensities were measured814

using Image Jrsquos Integrated Density function Locally adjusted background was 815

subtracted As the dynamic range of the signal on an x-ray film was narrow non-816

linearity in the measured values was corrected for each genotype in each biological 817

replicate using the values from different dilutions in two technical replicates We noticed 818

that the residuals were not normally distributed and performed Box-Cox power 819

transformation (Box and Cox 1964) to obtain normally distributed values for the 820

subsequent statistical analysis of the data For LM19 Box-Cox selected power 821

transformation was done to the power of 038th and for LM20 to the power 075th After 822

correction of the transformed measured values with the effect of the biological 823

replicates the values were fit to a linear model with the Arabidopsis genotype as the 824

fixed effect This linear model was used both to generate the plots in Figures 5B and D 825

and to test significant differences 826

827

Camalexin Measurements 828

Four week-old plants were infected with B cinerea and samples (three leaf disks 829

from one plant each) were harvested at the indicated time points flash frozen and 830

pulverized Each sample was extracted with 300 microL of 90 methanol (vv) 50 microL of the 831

extract was run on an Agilent Lichrocart 250-4 RP18e 5-microm column using an Agilent 832

1100 series HPLC Camalexin was detected using a DiodeArray (DAD) at 330 nm and 833

with a fluorescence detector at emission 318 nmexcitation 385 nm (Agilent) Separation 834

was achieved using the following program with aqueous acetonitrile 5 min gradient 835

Page 39 of 54

from 63 to 69 acetonitrile 30 s gradient from 69 to 99 acetonitrile 2 min at 99 836

acetonitrile and a post-run equilibration of 35 min at 63 acetonitrile (Denby et al 837

2004 Kliebenstein et al 2005) Purified camalexin was used to produce a standard 838

curve to identify and quantitate camalexin 839

840

Accession Numbers 841

Microarray data used can be accessed at NCBI Gene Expression Omnnibus 842

(httpwwwncbinlmnihgovgeo) GSE18978 Sequence information for all Arabidopsis 843

genes described in this article can be found in TAIR (wwwarabidopsisorg) using the 844

following accession numbers PAD3 (CYP71B15 At3g26830) SID2 (ICS1 At1g74710) 845

GAE1 (At4g30440) GAE2 (At1g02000) GAE3 (At4g00110) GAE4 (At2g45310) GAE5 846

(At4g12250) GAE6 (At3g23820) PR1 (At2g14610) JAZ5 (At1g17380) JAZ10 847

(At5g13220) ACTIN2 (At3g18780) PDF12a (At5g44420) PDF12b (At2g26020) 848

AtSK11 (At5g26751) The sequence of the B cinerea cutinase Bc-CutA (Z69264) can 849

be found in the DNA Data Bank of Japan (httpgetentryddbjnigacjp) Germplasm 850

used included dde2-2 (At5g42650 (von Malek et al 2002)) pad3-1 (At3g26830 (Zhou 851

et al 1999)) pad4-1 (At3g52430 (Jirage et al 1999)) sid2-2 (At1g74710 (Wildermuth 852

et al 2001)) pbs3-2 (At5g13320 SALK_018225 (Nobuta et al 2007)) cbp60a-1 853

(At5g62570 SALK_124410 (Truman et al 2013)) cbp60g-1 sard1-2 (At5g26920 854

SALK_023199 At1g73805 SALK_052422 (Wang et al 2011) cbp60a-1 cbp60g-1 855

sard1-2 (Truman et al 2013) gae1-1 (At4g30440 SALK_085554) gae6-1 (At3g23280 856

SALK_104454) gae6-2 (At3g23280 SALK_017191) ixr1-1 cev1 (At5g05170 (Ellis 857

and Turner 2001 Scheible et al 2001)) eds1 plants were derived by introgression of 858

the Ler eds1-2 allele into Col-0 that contains two copies of EDS1 (At3g48090 859

At3g48080 (Bartsch et al 2006) mpk6-2 (At2g43790 SALK_073907 (Liu and Zhang 860

2004)) mpk3-DG (At3g45640 a fast neutron deletion mutant (Miles et al 2005)) and 861

npr1-1 (At1g64280 (Cao et al 1997)) T-DNA insertion lines were part of the SALK 862

collection (Alonso et al 2003) and were obtained from the Arabidopsis Biological 863

Resource Center 864

865

Supplemental Data 866

Page 40 of 54

Supplemental Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 867

Expression Does Not Require CBP60s or SID2 868

Supplemental Figure 2 Expression of GAE Family Members uponTreatment with Pma 869

ES4326 870

Supplemental Figure 3 Characterization of T-DNA Insertions in GAE1 and GAE6 871

Supplemental Figure 4 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves and 872

Are Slightly Smaller than Wild-Type Col-0 Plants 873

Supplemental Figure 5 Cell Walls of gae1 gae6 Plants Contain Less Uronic Acid than 874

Wild-Type Col-0 Cell Walls 875

Supplemental Figure 6 Antibodies Raised Against the RGI or XG Components of 876

Pectin Show No Difference in Binding to gae Mutant and Col-0 Cell Walls 877

Supplemental Figure 7 The Effect of B cinerea Treatment on the Expression of GAE 878

Family Genes 879

Supplemental Figure 8 Treatment with 001 Macerozyme Does Not Cause Any 880

Visible Damage to Col-0 Plants 881

Supplemental Figure 9 B cinerea Growth on Plants with Defects in Immune 882

Signaling 883

Supplemental Figure 10 MeJA-Induced Expression of the JA marker gene JAZ10 884

Supplemental Figure 11 The Expression of the JA Marker Gene JAZ5 Is Highly 885

Responsive in gae1 gae6 Plants 886

Supplemental Figure 12 The Expression of PDF12 Is Unaltered in gae1 gae6 Plants 887

Supplemental Figure 13 Macerozyme Treatment Activates MPK3 and MPK6 888

Supplemental Figure 14 B cinerea-Induced ROS Production in Wild-Type Col-0 and 889

gae1 gae6 Plants 890

Supplemental Figure 15 OG-Induced PTI Using 250 microg mL-1 OGs 891

Supplemental Figure 16 Abundance of Plant DNA in B cinerea Treated Plants 892

Supplemental Table 1 Primers used in this study 893

894

ACKNOWLEDGEMENTS 895

We thank William Truman for helpful discussions Simone Ferrari (Sapienza 896

Universita di Roma) for the generous gift of OGs Bruna Bucciarelli (USDA-ARS) for 897

Page 41 of 54

help with fluorescence microscopy the Arabidopsis Biological Resource Center for T-898

DNA insertion lines Paul Knoxrsquos lab (University of Leeds) for LM5 LM6 LM8 LM19 899

and LM20 antibodies and the Complex Carbohydrate Resource Center (University of 900

Georgia) for the CCRC-M7 antibody Most of this work was funded by the Division of 901

Chemical Sciences Geosciences and Biosciences Office of Basic Energy Sciences of 902

the US Department of Energy through Grant DE-FG02-05ER15670 to JG This work 903

was also supported by NSF awards IOS 1339125 and IOS 1021861 to DJK NSF 904

award IOS 1121425 to F K and the USDA National Institute of Food and Agriculture 905

Hatch project number CA-D-PLS-7033-H to DJK 906

907

AUTHOR CONTRIBUTIONS 908

GB and JG designed the research GB GX AT BL NES and NH 909

performed research DJK provided tools GB GX MP AD BL RAH and FK 910

analyzed data GB and JG wrote the paper with input from all authors 911

912

FIGURE LEGENDS 913

Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 Expression 914

Requires PAD4 EDS1 and PBS3 915

(A) Expression of GAE1 in leaves of wild-type Col-0 and mutant plants Expression was916

measured 24 or 32 h after either inoculation with Pma ES4326 (OD600 = 0002) or mock 917

inoculation with 5 mM MgSO4 and was then normalized to the level of ACTIN2 Data 918

from three biological replicates were combined using a mixed linear model Bars 919

represent mean log2-ratios to ACTIN2 plusmn SE Letters indicate significantly different 920

groups with q lt 005 where 005 represents the maximum false discovery rate at which 921

the test may be called significant 922

(B) Expression of GAE6 measured as described in (A)923

924

Figure 2 Susceptibility of gae6 and gae1 gae6 Plants to Pma ES4326 Is Increased 925

Relative to Wild-Type Col-0 926

Leaves of plants of the indicated genotypes were inoculated with Pma ES4326 (OD600 = 927

00002) as described in Methods Bacterial titers in leaves were determined either 928

Page 42 of 54

immediately (0 dpi) or after three days (3 dpi) Bars represent the mean plusmn SE of four 929

independent experiments each with 4 or 12 biological replicates at 0 and 3 dpi 930

respectively Data were combined using a mixed linear model Asterisks indicate 931

significant differences from Col-0 wild-type (q lt 005) Susceptible pad4 plants were 932

included as a positive control 933

934

Figure 3 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves 935

(A) Depiction of the assay for brittleness of gae mutant leaves Fully expanded leaves of936

four to five week-old plants were bent by 180ordm so that the adaxial surface of the leaf was 937

parallel with the soil Broken leaf petioles or midribs were considered brittle 938

(B) Quantification of broken midribs or petioles For each genotype 66 to 82 leaves 939

were analyzed for brittleness as described in (A) Bars represent the ratio of broken to 940

total number of leaves tested Results were analyzed using Fisherrsquos exact test Letters 941

indicate significantly different groups at p lt 005 942

(C) Conductivity measurements Leaves of four week-old plants were inoculated with943

deionized water and then leaf discs were cut out and placed on deionized water 944

Conductivity was measured at the indicated time points After 30 min the water was 945

replaced with fresh water (30 min f w) to account for wounding-related ion leakage from 946

the edges of the leaf discs Three independent experiments with six biological replicates 947

each were performed and data were analyzed using a t-test for each time point P-948

values were corrected using the Bonferroni method Data show means plusmn SE asterisks 949

indicate significant differences from wild-type Col-0 at q lt 001 950

951

Figure 4 Cell Walls of gae6 and gae1 gae6 Plants Are Reduced in GalA 952

(A) to (D) Levels of GalA (A) GlcA (B) cellulose (C) and neutral sugars (D) in alcohol953

insoluble residue (AIR) extracted from leaves of four week-old plants of the indicated 954

genotypes Bars represent means plusmn SE of four biological replicates combined using a 955

mixed linear model Letters indicate significantly different groups (p lt 005) 956

957

Figure 5 Cell Walls of gae1 gae6 Plants Are Reduced in Homogalacturonan 958

Page 43 of 54

Dot blot analyses with anti-LM19 and anti-LM20 antibodies Pectin was extracted from 959

AIR prepared from the indicated genotypes using 1 microL of extraction buffer per 10 microg of 960

AIR Pectin was serially diluted and one microL of undiluted 13 diluted and 19 diluted 961

samples were spotted on a nitrocellulose membrane Three biological replicates were 962

were spotted twice to obtain two technical replicates each All replicates produced 963

similar results 964

(A) Representative dot blot using anti-LM19 antibody965

(B) Relative signal intensities of the spots in the dot blot in (A)966

(C) Representative dot blot using anti-LM20 antibody967

(D) Relative signal intensities of the LM20 dot blot shown in (C)968

Relative signal intensities were measured using Image J software Measurements from 969

the three biological replicates were combined using a linear model after Box-Cox power 970

transformation as described in Methods Bars represent means plusmn SE Asterisks indicate 971

a difference from wild-type Col-0 at p lt 005 972

973

Figure 6 Growth of B cinerea on gae1 gae6 Plants and the Effect of B cinerea on 974

Expression of GAE1 and GAE6 975

(A) Growth of B cinerea isolates Leaves of Col-0 and gae1 gae6 plants were976

inoculated with 25 x 105 spores mL-1 of the B cinerea isolates indicated in the figure 977

Samples were collected two (2 dpi) and three (3 dpi) days later Bars represent means plusmn 978

SE of six biological replicates for each fungus combined using a mixed linear model 979

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 980

(B) and (C) Expression of GAE1 (B) and GAE6 (C) 48 h after inoculation with the B981

cinerea isolates indicated Expression levels were measured by qRT-PCR Bars 982

represent mean log2-ratios to ACTIN2 plusmn SE of three biological replicates combined 983

using a mixed linear model Letters indicate significantly different groups (q lt 005) 984

985

Figure 7 Levels of Soluble Pectin and Expression of PAD3 in Fractions Produced 986

Following Macerozyme Treatment of gae1 gae6 Cell Walls 987

(A) Schematic overview of the experimental procedure for (B) and (C)988

Page 44 of 54

(B) Uronic acid measurements Alcohol-insoluble residue (AIR) from leave tissue of four989

week-old wild-type Col-0 or gae1 gae6 plants was treated with water or 025 990

macerozyme and assayed for uronic acid levels Data from 11 biological replicates were 991

combined using a mixed linear model Bars represent means plusmn SE Letters indicate 992

significant differences (p lt 005) 993

(C) Expression of PAD3 in Col-0 plants treated with the fractions from (B) Extracts from994

(B) were heat inactivated pooled and diluted 5-fold with water Pooled extracts were995

inoculated into four week-old Col-0 plants PAD3 expression was measured 3 h after 996

inoculation and normalized to the level of ACTIN2 expression Data from four biological 997

replicates were combined using a mixed linear model Bars represent mean log2-ratios 998

to ACTIN2 plusmn SE Letters indicate significant differences (p lt 005) 999

1000

Figure 8 Macerozyme-Induced PAD3 Expression Is Absent in gae1 gae6 1001

(A) Expression of PAD3 after macerozyme treatment Four week-old plants were either1002

inoculated with 001 macerozyme or mock inoculated with water and PAD3 1003

expression was measured 3 h later by qRT-PCR Data from four biological replicates 1004

were combined using a mixed linear model Mean log2-ratios to ACTIN2 plusmn SE were 1005

plotted Letters indicate significantly different groups (p lt 005) 1006

(B) Expression of PAD3 after OG treatment PAD3 expression was measured as1007

described in (A) 3 h after inoculation with 100 microg mL-1 OGs or mock inoculation with 1008

water Data are from six biological replicates 1009

(C) PAD3 expression in untreated plants Three biological replicates were performed1010

and data analyzed as described in (A) 1011

(D) Camalexin accumulation after B cinerea treatment Camalexin accumulation was1012

measured in four week-old plants inoculated with the B cinerea strains shown in the 1013

figure Eight biological replicates each were combined using a mixed linear model 1014

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 1015

(E) PAD3 expression 28 h after treatment with the indicated B cinerea strains Data1016

from six biological replicates were collected and analyzed as described in (A) Data in 1017

all figure parts represent means plusmn SE Letters indicate significantly different groups (q lt 1018

005) 1019

Page 45 of 54

1020

Figure 9 Macerozyme-Induced Expression of the SA Marker Genes SID2 and PR1 1021

Is Abolished in gae1 gae6 Plants 1022

(A) to (H) Expression of PR1 (A) (C) (E) and (G) or SID2 (B) (D) (F) and (H) in four1023

week-old Col-0 and gae1 gae6 plants Data were combined using a mixed linear model 1024

Bars represent mean log2-ratios of expression vs ACTIN2 plusmn SE for all figure parts 1025

Letters indicate significantly different groups at p lt 005 for panels (A) to (D) (G) and 1026

(H) and qlt005 for panels (E) to (F) 1027

(A) and (B) Plants were treated with 001 macerozyme or mock treated with water1028

and samples were collected 3 h later Data from four biological replicates are shown 1029

(C) and (D) Plants were inoculated with 100 microg mL-1 OGs or mock treated with water1030

and samples collected 3 h later Data from six biological replicates are shown 1031

(E) and (F) Plants were inoculated with the indicated B cinerea isolates or mock1032

inoculated with B cinerea inoculation medium and samples collected 48 h later Data 1033

from eight biological replicates are shown 1034

(G) and (H) Expression in untreated plants Three biological replicates were performed1035

1036

Figure 10 The Expression of the JA Marker Gene JAZ10 is Highly Responsive in 1037

gae1 gae6 Plants 1038

(A) to (D) JAZ10 expression in four week-old plants Data were combined using a mixed1039

linear model Mean log2-ratios to ACTIN2 plusmn SE are shown Letters indicate significantly 1040

different groups at p lt 005 for panels (A) (B) and (D) and q lt 005 for panel (C) 1041

(A) Plants were inoculated with 001 macerozyme or mock inoculated using water and1042

samples were collected 3 h later Data from four biological replicates are shown 1043

(B) Plants were inoculated with 100 microg mL-1 OGs or mock inoculated using water and1044

samples collected 3 h later Data are from six biological replicates 1045

(C) JAZ10 expression 48 h after treatment with the indicated B cinerea strains Data1046

are from six biological replicates 1047

(D) JAZ10 expression in untreated plants Data are from three biological replicates1048

1049

Figure 11 Gallo 1-Induced Callose Deposition Is Reduced in gae1 gae6 Plants 1050

Page 46 of 54

(A) Callose deposition in four week-old Col-0 and gae1 gae6 plants Plants were1051

inoculated with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) or mock inoculated 1052

with B cinerea inoculation medium and samples were collected after 72 h Callose was 1053

stained with aniline blue and visualized using a Nikon Eclipse Ni-U microscope with a 1054

DAPI filter Three leaves were stained per treatment Scale bars are 100 microm Asterisks 1055

indicate fungal hyphae arrows indicate representative callose spots 1056

(B) Quantification of callose deposition Callose deposits were counted in 200 microm x 2001057

microm squares laid out across the images shown here Bars represent means plusmn SE from 1058

six squares each Letters indicate significantly different groups (p lt 005) according to a 1059

t-test analysis1060

1061

Figure 12 Less Soluble Pectin Accumulates in B cinerea-Infected gae1 gae6 1062

Leaves 1063

Four week-old Col-0 and gae1 gae6 plants were inoculated with 10 microL droplets of the 1064

indicated B cinerea isolate (25 x 105 spores mL-1) or mock inoculated with B cinerea 1065

inoculation medium Leaves were collected 48 h later AIR was extracted from leaf 1066

samples and water added (20 microL infection site) to extract soluble pectin fragments 1067

Samples were vortexed for 7 h centrifuged at 12000 g and the uronic acid 1068

concentration of the supernatant was measured Data from nine biological replicates 1069

were combined using a mixed linear model Means plusmn SE are shown Letters indicate 1070

significant differences (q lt 005) 1071

1072

Figure 13 Macerozyme-Induced PTI Is Lost in gae1 gae6 While OG-Induced PTI Is 1073

Unaltered 1074

(A) Macerozyme-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1075

inoculated with 001 macerozyme or mock inoculated with boiled macerozyme and 1076

were infected with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) 24 h later Samples 1077

were collected immediately (0 dpi) and after two (2 dpi) and three (3 dpi) days Data 1078

show mean plusmn SE of six to 14 biological replicates each combined using a linear model 1079

Letters indicate significantly different groups (q lt 005) 1080

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

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Page 4 of 54

cell walls consists mostly of GalA Rha and smaller amounts of other sugars including 88

Ara Gal Xyl and Fuc 89

In general carbohydrate biosynthesis requires nucleotide sugars provided by 90

nucleotide sugar interconversion pathways (Seifert 2004) Most nucleotide sugars are 91

synthesized from uridine diphosphate-Glc (UDP-Glc) UDP-glucuronic acid (UDP-GlcA) 92

is made from UDP-Glc by UDP-glucose dehydrogenase activity or via an alternative 93

pathway requiring inositol oxygenase activity (Tenhaken and Thulke 1996 Loewus and 94

Murthy 2000) UDP-D-Glucuronate 4-epimerases (GAEs) interconvert UDP-D-GlcA and 95

UDP-D-GalA the monomeric precursor of pectin There are six GAE genes encoded by 96

the Arabidopsis genome When heterologously expressed in Escherichia coli or Pichia 97

pastori GAE1 GAE4 and GAE6 showed activity in interconverting UDP-D-GlcA and 98

UDP-D-GalA (Gu and Bar-Peled 2004 Molhoj et al 2004 Usadel et al 2004) GAE1 99

and GAE6 were found to be Golgi-localized and strongly expressed in various plant 100

tissues (Molhoj et al 2004 Usadel et al 2004 Parsons et al 2012) GAE1 and GAE6 101

have been hypothesized to be evolutionarily older than the other GAE family members 102

(Usadel et al 2004) and might have overlapping functions that are distinct from the 103

other family members 104

Plant cell wall composition and architecture affects wall strength and flexibility 105

and cell walls present a physical barrier to potential plant pathogens Besides preformed 106

physical barriers such as a cell wall plants have a sophisticated immune system to 107

defend themselves against harmful microbial pathogens (Chisholm et al 2006 Jones 108

and Dangl 2006) Immune signaling involves changes in phytohormone levels the most 109

important for plant immunity being salicylic acid (SA) jasmonic acid (JA) and ethylene 110

(ET) (Grant and Jones 2009 Pieterse et al 2012) Other major regulators of plant 111

immunity include PHYTOALEXIN DEFICIENT 4 (PAD4) and ENHANCED DISEASE 112

SUSCEPTIBILITY 1 (EDS1) (Wiermer et al 2005) which are predicted to encode 113

lipases Immune signaling generally involves changes in the transcriptome that in turn 114

lead to altered cellular responses (Tao et al 2003) For instance induced expression of 115

the cytochrome P450 PAD3 is thought to increase production of the anti-microbial 116

secondary metabolite camalexin (Zhou et al 1999) 117

Page 5 of 54

Because plant cell walls are important barriers against pathogenic microbes 118

alterations in wall structural properties can lead to changes in plant immunity To date 119

only a few examples of cell wall related mutants with altered pathogen phenotypes have 120

been studied in detail Mutants impaired in callose biosynthesis powdery mildew 121

resistant 4 (pmr4) display enhanced resistance to the powdery mildew pathogens 122

Golovinomyces orontii and G cichoracearum (Nishimura et al 2003) These plants 123

have constitutively high SA levels and SA signaling is required for the resistance 124

phenotype (Nishimura et al 2003) Other plants with enhanced resistance to these 125

powdery mildew pathogens include pmr5 a mutant enriched in pectin and pmr6 a 126

mutant with a defect in a pectate lyase-like gene (Vogel et al 2002 Vogel et al 2004) 127

Plants with mutations in cellulose synthases required for primary and secondary wall 128

formation also show increased resistance to pathogens (Ellis and Turner 2001 129

Hernandez-Blanco et al 2007) Enhanced resistance to powdery mildew and 130

Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) in constitutive 131

expression of VSP 1 (cev1) which is mutated in the primary wall cellulose synthase 132

CesA3 was linked to constitutive high JA and ET levels in these plants (Ellis and 133

Turner 2001 Ellis et al 2002a) Interestingly irregular xylem1 (irx1) irx3 and irx5 134

plants which carry mutations in the secondary wall-associated cellulose synthases 135

CesA8 CesA7 and CesA4 showed enhanced resistance to the necrotrophic pathogens 136

Plectosphaerella cucumerina Ralstonia solanacearum and Botrytis cinerea as well as 137

the biotrophic powdery mildew G cichoracearum whereas growth of P cucumerina and 138

R solanacearum pathogens was unaltered in pmr5 pmr6 and cev1 plants (Hernandez-139

Blanco et al 2007) This enhanced resistance of the irx mutants was independent of 140

SA JA or ET signaling but instead involved abscisic acid signaling (Hernandez-Blanco 141

et al 2007) Changes in wall structure are likely monitored by the plants and effects 142

may be counteracted through initiation of defense responses that can lead to altered 143

phytohormone levels 144

Although plant cell walls serve as barriers against pathogen entry many 145

pathogens secrete cell wall-degrading enzymes (CWDEs) and thus are able to 146

successfully infect their host plants (Albersheim et al 1969) For example the B 147

cinerea polygalacturonase Bc-PG1 is required for full virulence of this pathogen and the 148

Page 6 of 54

gene encoding it shows evidence for diversifying selection as would be expected for a 149

virulence gene (ten Have et al 1998 Rowe and Kliebenstein 2007) Secretion of 150

CWDEs can trigger the release of cell wall fragments which can act as damage-151

associated molecular patterns (DAMPs) that activate plant immune responses (Hahn et 152

al 1981 Ferrari et al 2013) Similarly pectin fragments known as oligogalacturonides 153

(OGs) can trigger plant immune responses (Cote and Hahn 1994 Ferrari et al 2013) 154

Such responses include the production of reactive oxygen species (ROS) activation of 155

mitogen-activated protein kinase (MAPK) activation and enhanced expression of 156

defense-related genes such as PAD3 (Ferrari et al 2007 Denoux et al 2008 Galletti 157

et al 2008 Galletti et al 2011) Some of these responses require SA JA and ET 158

whereas others are independent of these phytohormones (Ferrari et al 2007) 159

Here we describe two GAE family members GAE1 and GAE6 whose 160

expression is repressed upon pathogen challenge A gae1 gae6 double mutant has 161

brittle leaves and its walls are reduced in pectin specifically HG and likely RGI 162

Resistance to the bacterial pathogen Pma ES4326 and the fungal necrotroph B cinerea 163

is compromised in gae1 gae6 Callose deposition is reduced in gae1 gae6 plants after 164

treatment with B cinerea isolate Gallo 1 Furthermore these plants show altered 165

activation of immune signaling in response to treatment with macerozyme a 166

polygalacturonase that degrades pectin and they also are hyperresponsive to JA 167

signaling Macerozyme treatment released less soluble likely low molecular weight 168

pectin from isolated gae1 gae6 cell walls than from Col-0 cell walls B cinerea-induced 169

accumulation of soluble likely low molecular weight pectin was also decreased in gae1 170

gae6 plants Additionally gae1 gae6 plants are impaired in macerozyme-induced 171

immunity to B cinerea possibly due to decreased release of OGs from their cell walls 172

173

174

Page 7 of 54

RESULTS 175

176

GAE1 and GAE6 Expression Is Repressed by Pma ES4326-Induced Immune 177

Signaling 178

Our previous research suggested that the status of pectin specifically pectin 179

modification by methylesterification is important for plant immunity against Pma 180

ES4326 (Bethke et al 2014) Here we investigate whether pectin abundance in 181

addition to its specific structure is also important for plant immunity by taking a closer 182

look at the formation of the pectin precursor UDP-GalA by GAEs 183

184 Microarray experiments showed that expression of GAE1 and GAE6 was

185 repressed in A thaliana (Arabidopsis) plants treated with Pma ES4326 (Wang et al

186 2008) Repression of GAE1 and GAE6 in wild-type Arabidopsis Col-0 plants treated with

187 Pma ES4326 was confirmed by quantitative real time (qRT)-PCR Figure 1) To test if

188 this repression is mediated by immune signaling qRT-PCR was also performed in

189 mutants with blocked immune signaling (Figure 1 and Supplemental Figure 1) 190 Generally immunity to Pma ES4326 requires intact SA signaling Repression of both

191 GAE1 and GAE6 required the presence of the major immune regulators EDS1 and

192 PAD4 required for SA signaling as well as other immune responses (Jirage et al 1999

193 Wiermer et al 2005 Bartsch et al 2006 Wang et al 2008) at both early and late time

194 points The presence of AVRPPHB SUSCEPTIBLE 3 (PBS3) a regulator of many

195 immune responses including SA signaling (Nobuta et al 2007) was also required for

196 repression of GAE1 and GAE6 at an early time point (Figure 1) CALMODULIN-

197 BINDING PROTEIN 60-LIKE a (CBP60a) (a negative regulator of SA synthesis and

198 immunity (Truman et al 2013)) CBP60g and SYSTEMIC ACQUIRED RESISTANCE

199 DEFICIENT 1 (SARD1) (both promote SA biosynthesis and regulation of other immune

200 responses (Wang et al 2011)) SALICYLIC ACID INDUCTION DEFICIENT 2 SID2

201 (encodes a biosynthetic enzyme for SA (Wildermuth et al 2001)) and

202 NONEXPRESSER OF PR GENES 1 NPR1 (a regulator of the majority of SA-induced

203 immune signaling (Cao et al 1997 Yan and Dong 2014)) were not required for this

204 repression (Figure 1 and Supplemental Figure1) Thus repression of GAE1 and GAE6

Page 8 of 54

205 requires a regulatory function that is dependent on EDS1 and PAD4 partially

206 dependent on PBS3 but independent of SA

Page 9 of 54

There are six GAE family members in Arabidopsis GAE2 expression was 207

induced by Pma ES4326 in the absence of PAD4 or EDS1 GAE3 expression was 208

unaltered GAE4 was repressed early and GAE5 expression was induced in all 209

genotypes (Supplemental Figure 2) The similarity of the immune-regulated expression 210

of GAE1 and GAE6 suggested a redundant role of these genes in Arabidopsis immunity 211

to Pma ES4326 212

213

Resistance to Pma ES4326 Is Compromised in gae1 gae6 and gae6 Plants 214

To study the roles of GAE1 and GAE6 in plant immunity in more detail plants 215

with T-DNA insertion mutations in GAE1 and GAE6 were obtained from the SALK 216

collection as described in the Accession Numbers section of Methods We identified two 217

GAE6 knock-out alleles gae6-1 and gae6-2 and a T-DNA insertion in GAE1 gae1-1 218

that caused reduced expression of this gene (Supplemental Figure 3) A double mutant 219

was created by crossing gae1-1 and gae6-1 Both gae6 and gae1 gae6 plants were 220

more susceptible to Pma ES4326 than wild-type Col-0 and gae1 plants supporting a 221

role of GAE6 in plant immunity to Pma ES4326 (Figure 2) 222

223

gae6 and gae1 gae6 Plants Have Brittle Leaves 224

While inoculating leaves of gae1 gae6 plants we noticed that the petioles and 225

midribs of these plants tended to break easily when leaves were bent against the 226

proximal-distal axis of the leaves (Figure 3 see Supplemental Figure 4D to F) They 227

tended to break less often when twisted around the proximal-distal axis To quantify 228

this phenotype leaves were bent by 180deg (Figure 3A) and breaking of midribs or 229

petioles was recorded Most gae1 gae6 leaves broke whereas fewer leaves of gae6 230

plants and very few leaves of wild-type Col-0 and gae1 plants did (Figure 3B and 231

Supplemental Figure 4C) Although gae1 gae6 plants had slightly shorter leaves and 232

petioles than wild type plants (Supplemental Figure 4A and B) no correlation between 233

leaf size or petiole length and breaking frequency was detected We also investigated 234

tissue integrity by monitoring ion leakage Interestingly when leaves were inoculated 235

with deionized water leaf discs of gae1 gae6 leaked more ions than wild-type Col-0 236

discs (Figure 3C) suggesting that the increased brittleness is correlated with a 237

Page 10 of 54

_____________________________________________________________________________

Page 11 of 54

Cell Walls of gae6 and gae1 gae6 Plants Have Lower Levels of Pectin 243

GalA is a major component of pectin We analyzed wall monosaccharide 244

composition in gae mutants to investigate whether the increased brittleness was 245

associated with an altered wall composition of gae leaves To approximate pectin 246

content of gae mutant walls the total uronic acid concentration was measured using a 247

simple colorimetric assay (Filisetti-Cozzi and Carpita 1991 van den Hoogen et al 248

1998) Total uronic acid was reduced in gae6-2 strongly reduced in gae1 gae6 plants 249

but was indistinguishable from wild-type Col-0 in gae6-1 and gae1 (Supplemental 250

Figure 5) This suggested that gae1 gae6 and possibly gae6 plants have reduced 251

pectin 252

To investigate the cell wall composition in leaves of gae plants in more detail we 253

measured the levels of seven neutral monosaccharides the acidic sugars GalA (the 254

monomeric subunit of the pectin backbone (Harholt et al 2010)) and GlcA (Figure 4) 255

We also estimated the crystalline cellulose content (Updegraff 1969) Cell walls of both 256

gae6 mutants and gae1 gae6 were reduced in GalA (Figure 4A) In gae1 gae6 GalA 257

content in cell walls was reduced by more than 40 compared to the wild-type level 258

GlcA levels were indistinguishable between mutants and wild-type (Figure 4B) 259

Cellulose content was higher in gae6-2 and gae1 gae6 walls likely due to the loss of 260

pectins (Figure 4C) Cell walls of gae1 gae6 plants were also enriched in all neutral 261

sugars measured presumably due to the substantial proportional loss of GalA (Figure 262

4D) The levels of most wall monosaccharides were even higher in the gae1 gae6 263

double mutant with the exception of Rha which did not exhibit a significant increase 264

compared to the gae6 single mutants The absence of a compensational increase in 265

Rha suggested that RGI the dominant Rha contributor to the wall may be considered 266

reduced in gae1 gae6 mutants compared to wild-type albeit not to such an extent as 267

HG 268

238 structural defect that promotes increased ion leakage Taken together these results

239 showed that loss of GAE6 causes brittle leaves and additional losss of GAE1 strongly

240 enhances this phenotype We conclude that GAE1 and GAE6 contribute to normal leaf

241 flexibility 242

Page 12 of 54

gae6 cell walls are reduced in HG Using the same samples we detected no difference 274

from wild type in binding of LM5 which recognizes a tetramer of (1-4)-β-D-galactans 275

found in RGI (Jones et al 1997) LM6 which recognizes a linear pentasaccharide in (1-276

To further investigate which pectic components were altered in gae1 gae6 dot 269

blot experiments using pectin isolated from the various genotypes were performed Both 270

LM19 and LM20 antibodies recognize HG (Verhertbruggen et al 2009a) As expected 271

LM19 binding was reduced in gae6-1 and gae1 gae6 (Figure 5A and B) and LM20 272

binding was reduced in gae1 gae6 (Figure 5C and D) This result confirmed that gae1 273

277 5)-α-L-arabinans found in RGI (Willats et al 1998 Verhertbruggen et al 2009b) LM8

278 which recognizes xylogalacturonan (Willats et al 2004) or CCRC-M7 which

279 recognizes an arabinosylated (1-6)-β-D-galactan epitope occurring on RGI (Puhlmann et

Page 13 of 54

Page 14 of 54

al 1994 Steffan et al 1995 Pattathil et al 2010) (Supplemental Figure 6) 280

Collectively these cell wall composition data show that gae1 gae6 plants are reduced in 281

HG Due to the strong proportional reduction of HG in the walls levels of all other 282

components should be higher However Rha indicative of RGI was not higher in gae1 283

gae6 suggesting that RGI may also be reduced in gae1 gae6 walls 284

285

GAE1 and GAE6 Are Required for Immunity to Specific B cinerea Isolates 286

We hypothesized that plant immunity to B cinerea might be affected by pectin 287

content in the host cell wall because this pathogen requires polygalacturonases and 288

pectin methylesterases for full virulence (ten Have et al 1998 Valette-Collet et al 289

2003 Kars et al 2005b Espino et al 2010) Because Bcinerea is genetically diverse 290

and has a high diversity of polygalacturonases (Rowe and Kliebenstein 2007) we 291

measured relative fungal growth at both 2 and 3 d post-inoculation (dpi) using ten 292

different B cinerea isolates (Figure 6) The B cinerea isolates Fresa 525 and Gallo 1 293

showed enhanced virulence on gae1 gae6 plants at both time points whereas KT and 294

UK Razz showed enhanced virulence in gae1 gae6 plants at 3 but not 2 d dpi (Figure 295

6A) We did not detect any significant changes in relative fungal growth between wild-296

type Col-0 and gae1 gae6 plants when using Acacia Apple 517 DN Grape Pepper or 297

Rasp isolates of B cinerea Therefore we continued our study using Gallo 1 and Fresa 298

525 We also included Pepper for comparison as this isolate has been used in a large-299

scale analysis of plant gene expression in response to B cinerea infection (Windram et 300

al 2012) 301

We next investigated whether expression of GAE1 and GAE6 was affected by 302

challenge with B cinerea Inoculation of wild-type Col-0 plants with any isolate 303

repressed GAE1 and GAE6 expression (Figure 6B and C) and this repression was 304

generally independent of PAD4 (Figure 6B and C) Expression of GAE2 and GAE3 was 305

unaltered by B cinerea treatment GAE4 was repressed only by Fresa 525 treatment 306

while GAE5 expression was enhanced with all three isolates tested (Supplemental 307

Figure 7) As seen for infection with Pma ES4326 the expression of GAE1 and GAE6 308

was repressed upon B cinerea treatment This indicates a role for GAE1 and GAE6 in 309

the immune response to B cinerea 310

Page 15 of 54

Page 16 of 54

Because the amount of pectin substrate for pathogen polygalacturonases is 313

reduced we hypothesized that immunity phenotypes of gae1 gae6 plants may result 314

from reduced generation of OGs by these enzymes In turn the reduced OGs could 315

lead to reduced immune responses To test this we used macerozyme a commercial 316

pectinase as a proxy for pathogen pectinases We isolated cell walls as a subcellular 317

fraction known as alcohol insoluble residue (AIR Gille et al 2009) This was treated 318

with macerozyme and tested for release of soluble likely low molecular weight pectin 319

fragments (Figure 7) Treatment with 025 macerozyme released significantly more 320

uronic acid from wild-type Col-0 AIR relative to gae1 gae6 AIR (Figure 7B) To test 321

activity of these pectin-containing solutions we infiltrated them into wild-type Col-0 322

plants and measured PAD3 expression PAD3 is required for synthesis of the 323

phytoalexin camalexin (Zhou et al 1999) and its expression is known to be induced by 324

OG treatment (Ferrari et al 2007) Fractions from macerozyme treated Col-0 walls 325

induced significantly more PAD3 expression than fractions derived from gae1 gae6 326

walls suggesting that macerozyme treatment did indeed release more active soluble 327

uronic acid possibly OGs from wild-type Col-0 walls than from gae1 gae6 walls (Figure 328

7C) 329

Next we studied the effect of macerozyme treatment on expression of PAD3 in 330

intact plants (Figure 8) This treatment did not lead to any visible leaf damage 331

(Supplemental Figure 8) In wild-type Col-0 PAD3 expression was induced three h after 332

treatment with 001 macerozyme (Figure 8A) By contrast no macerozyme-induced 333

induction of PAD3 expression was detected in gae1 gae6 (Figure 8A) However OG 334

treatment induced the expression of PAD3 in both genotypes indicating that gae1 gae6 335

plants do respond to OGs (Figure 8B) Macerozyme treatment also induced expression 336

of the SA marker genes PATHOGENESIS RELATED 1 (PR1) and SID2 in Col-0 but 337

not in gae1 gae6 (Figure 9 panel A and B) PR1 was not significantly induced by 338

purified OGs (Figure 9C) at the time and concentration tested but SID2 was induced by 339

OG treatment in both genotypes (Figure 9D) However the induction was stronger in 340

wild-type Col-0 In addition the JA markers JASMONATE-ZIM-DOMAIN PROTEIN 5 341

(JAZ5) and JAZ10 were induced by macerozyme and OG treatment in both Col-0 and 342

gae1 gae6 (Figure 10A and Supplemental Figure 11) Interestingly expression of JAZ5 343

312 Pectinase-Induced Immune Signaling Is Altered in gae1 gae6 Plants

Page 17 of 54

Page 18 of 54

Page 19 of 54

Page 20 of 54

344

345

346

and JAZ10 was already strongly induced in gae1 gae6 after mock inoculation (Figure 10

and Supplemental Figure 11) We observed no hyperresponsive induction by mock

treatment of PLANT DEFENSIN 12 (PDF12) a necrotrophy and late JA signaling

347 marker gene (Supplemental Figure 12A-D) PDF12 was also not induced by

348 macerozyme or OG treatments (Supplemental Figure 12A-D) This suggests that early

349 JA signaling is induced by mock-treatment in gae1 gae6 Because it is known that JA

350 and SA signaling act antagonistically (Pieterse et al 2012) the strong up-regulation of

351 JA-induced immune signaling in gae1 gae6 might explain the lower OG-induced SID2

352 expression in this genotype

353 To test whether other immune responses are altered in gae1 gae6 plants we

354 analyzed Mitogen-activated protein kinase (MAPK) activity following macerozyme and

355 OG treatments OGs are known to activate the MAPKs MPK3 and MPK6 in Arabidopsis

Page 21 of 54

(Galletti et al 2011) Macerozyme treatment activated MPK3 and MPK6 in wild-type 356

Col-0 whereas MAPK activation in gae1 gae6 was slightly reduced in four out of five 357

experiments performed (Supplemental Figure 13) By contrast OG-induced MAPK 358

activity was similar or increased in gae1 gae6 in four out of five experiments 359

(Supplemental Figure 13) Additionally mock treatment induced MAPK activity in gae1 360

gae6 more strongly than it did in wild-type Col-0 in four out of five experiments 361

performed (Fig S13) Collectively these results illustrate that activation of several 362

immune signaling componentsmdashcamalexin synthesis SA and JA signaling and 363

possibly MAPK activationmdashby macerozyme is substantially altered in gae1 gae6 This 364

suggests that release of soluble pectin fragments possibly OGs by pectinolytic 365

enzymes might be reduced in gae1 gae6 plants while recognition of OGs is intact 366

367

Following B cinerea Infection Production of Soluble Pectin and Deposition of 368

Callose Are Reduced in gae1 gae6 Plants 369

Although pad3 plants were more susceptible to the B cinerea isolates Fresa 525 370

and Gallo 1 which also grew better on gae1 gae6 growth of the isolate Pepper was 371

similar on Col-0 and pad3 (Supplemental Figure 9A) However we did not detect any 372

significant changes in camalexin production after treatment with B cinerea isolate Gallo 373

1 and Fresa 525 in gae1 gae6 (Figure 8D) Neither did we detect changes in PAD3 374

expression after treatment with any B cinerea isolate in gae1 gae6 (Figure 8E) 375

Additionally SID2 and PR1 were similarly induced by all B cinerea isolates in both Col-376

0 and gae1 gae6 (Figure 9E and F) and sid2 plants which are deficient for pathogen-377

induced SA biosynthesis did not show any differences in relative B cinerea growth 378

(Supplemental Figure 9B) Expression of JAZ10 was induced more strongly by Gallo 1 379

treatment in gae1 gae6 than in wild-type Col-0 but not by Fresa 525 or Pepper (Fig 380

10C) while dde2 plants deficient for JA biosynthesis showed enhanced susceptibility to 381

all B cinerea isolates tested (Supplemental Figure 9B) Curiously exogenous 382

application of MeJA did not lead to altered relative fungal growth of B cinerea 383

(Supplemental Figure 9C) and cev1 isoxaben resistant 1 (ixr1) plants which have 384

constitutively high JA levels (Ellis and Turner 2001) did not show any changes in 385

susceptibility to the B cinerea isolates tested (Supplemental Figure 9D) Hence neither 386

Page 22 of 54

differences in B cinerea isolate susceptibility to camalexin nor altered SA or JA 387

signaling by themselves explained the enhanced susceptibility of gae1 gae6 to these B 388

cinerea isolates 389

Hydrogen peroxide accumulation in response to B cinerea isolates Fresa 525 390

Gallo 1 and Pepper was not obviously altered in gae1 gae6 plants (Supplemental Figure 391

14) However callose deposition upon treatment with B cinerea isolate Gallo 1 was 392

strongly reduced in gae1 gae6 (Figure 11) It was previously shown that OG treatment 393

induces callose deposition (Denoux et al 2008) suggesting that the reduction in 394

callose deposition might be due to reduced OG release in gae1 gae6 It is possible that 395

this reduction in callose deposition contributes to the reduced immunity of gae1 gae6 396

plants to B cinerea isolate Gallo 1 397

Because macerozyme could be used to release soluble likely low molecular 398

weight pectin from isolated cell walls we next investigated whether infection with B 399

cinerea was accompanied by accumulation of soluble pectin as well We prepared cell 400

walls (AIR fraction) from leaves infected with B cinerea incubated these cell walls with 401

water and measured uronic acid content of the extracts B cinerea treatment did in fact 402

lead to increased accumulation of soluble pectin in wild-type Col-0 plants when 403

compared to mock-treated samples (Figure 12) No such increased accumulation of 404

water-soluble pectin fractions was observed in gae1 gae6 (Figure 12) 405

406

Macerozyme-Induced Pattern-Triggered Immunity against B cinerea Is Abolished 407

in gae1 gae6 408

It was previously shown that OG treatment can induce patternndashtriggered 409

immunity (PTI) to B cinerea (Ferrari et al 2007) We tested macerozyme treatment to 410

determine whether it could also induce PTI to the B cinerea isolate Gallo 1 (Figure 13) 411

Indeed pretreatment with 001 macerozyme for 24 h led to reduced B cinerea growth 412

in wild-type Col-0 but not gae1 gae6 plants (Figure 13A) Interestingly mock and 413

macerozyme pretreated gae1 gae6 plants exhibited similar growth of B cinerea Gallo 1 414

as did macerozyme-treated Col-0 plants This result is likely due to activation of JA 415

signaling in gae1 gae6 during the pretreatment procedure (Figure 13C and D) OG-416

induced PTI was observed in both genotypes when using a high dose of OGs (Figure 417

Page 23 of 54

13B) whereas a lower dose of OGs only induced PTI in Col-0 (Supplemental Figure 418

15) This suggests that OG perception in gae1 gae6 at least above a certain threshold 419

is unaltered while release of immunity-activating pectin fragments possibly OGs is 420

421 reduced We conclude that immune responses induced by macerozyme treatment

422 which do not occur in gae1 gae6 plants are important for plant immunity to the B

423 cinerea isolate Gallo 1

Page 24 of 54

____________________________________________________________________________

Page 25 of 54

DISCUSSION 426

Here we showed that loss of GAE1 and GAE6 leads to a dramatic reduction in 427

pectin content in the cell wall (Figure 4) likely causing the brittleness of leaves that we 428

observed (Figure 3) Petioles and midribs of gae1 gae6 plants broke easily when bent 429

and inoculation of leaves with deionized water resulted in increased ion leakage 430

possibly due either to microscopic fractures of the cell wall induced by inoculation stress 431

or to a reduced capability of these walls to withstand increased turgor pressure The 432

increased brittleness was associated with altered cell wall composition In our analysis 433

the GalA content of gae1 gae6 leaf walls was reduced by more than 40 and the total 434

uronic acids by over 60 (Figure 4 and Supplemental Figure 5) This is a major change 435

in the overall cell wall composition considering that pectin makes up about 50 of the 436

plant cell walls found in Arabidopsis leaves (Zablackis et al 1995 Nobuta et al 2007 437

Harholt et al 2010) Interestingly quasimodo1 (qua1) plants which have a mutation in 438

a glycosyltransferase family 8 gene are reduced in GalA by 25 and show reduced 439

binding of HG specific antibodies (Bouton et al 2002) 440

Furthermore qua2 plants which have a mutation in a putative methyltransferase 441

have about 13 less total GalA and lack 50 of cell wall HG (Mouille et al 2007) 442

Likewise cgr2 cgr3 plants carrying mutations in another class of Golgi-localized 443

putative methyl-transferases have leaf cell walls that are reduced by about 40 in 444

uronic acid (Kim et al 2015) All three mutants are severely dwarfed (Bouton et al 445

2002 Mouille et al 2007 Kim et al 2015) Additionally qua1 and qua2 mutants show 446

a distorted overall shape likely due to reduced cell cohesion and are extremely fragile 447

(Bouton et al 2002 Mouille et al 2007) By contrast gae1 gae6 plants with a similar 448

strong reduction in GalA and total uronic acids have an overall normal shape and are 449

only slightly smaller than wild-type Col-0 (Supplemental Figure 4) In the wall analysis 450

performed here we did not detect changes in other wall polysaccharides or structures 451

that might compensate for the reduction of pectin in gae1 gae6 It is possible the severe 452

morphological alterations in qua2 and cgr2 cgr3 plants are due to reduction in pectin 453

methylesterification which is unaffected in gae1 gae6 It was speculated that quality 454

control mechanisms in the Golgi prevent secretion of inefficiently esterified pectin in 455

cgr2 cgr3 (Kim et al 2015) suggesting that the Golgi of these plants might over-456

Page 26 of 54

accumulate pectin and that this may contribute to the severe dwarf phenotype of these 457

plants The gae1 gae6 plants described here provide an opportunity to study the effects 458

of reduction of pectin in plants that do not show severe morphological phenotypes 459

We found that gae1 gae6 plants are also compromised in resistance to B 460

cinerea and Pma ES4326 indicating that loss of pectin reduces immunity to these 461

pathogens (Figure 2 and 6) We found that gae1 gae6 plants were slightly more 462

susceptible to Pma ES4326 but showed a strong increase in susceptibility to specific B 463

cinerea isolates This was not unexpected because B cinerea is known to interact with 464

pectin as part of its infection strategy It secretes various pectin-degrading enzymes 465

early during infection and requires some of these enzymes including PG1 PG2 and 466

PME1 for full virulence (ten Have et al 1998 Valette-Collet et al 2003 Kars et al 467

2005b Espino et al 2010) Additionally Arabidopsis plants expressing an antisense 468

construct of the polygalacturonase inhibitor PGIP1 are significantly more susceptible to 469

B cinerea (Ferrari et al 2006) On the other hand Pma ES4326 is not known to470

require cell wall degrading enzymes for full virulence even though it encodes two 471

pectate lyase genes in its genome (PMA4326_07094 and PMA4326_04621 472

wwwbacteriaensemblorg) B cinerea isolates are also known to be genetically quite 473

diverse in their polygalacturonase (PG) loci and genetic variability in PG2 is associated 474

with varying growth on pectin containing media (Rowe and Kliebenstein 2007) 475

Furthermore the contribution of PG1 and PG2 to B cinerea virulence seems to depend 476

on the specific B cinerea isolate and the host species (Kars et al 2005a Zhang and 477

Van Kan 2013) B cinerea isolates are also known to be differently sensitive to the anti-478

microbial secondary metabolite camalexin (Kliebenstein et al 2005) In line with these 479

observations the susceptibility of gae1 gae6 plants differed depending on the specific 480

B cinerea isolate Whereas Fresa 525 Gallo 1 UK Razz and KT grew better in gae1481

gae6 we detected no difference in Acacia Apple 517 DN Grape Pepper or Rasp 482

growth (Figure 6) 483

B cinerea was shown to metabolize pectin and to possibly utilize GalA as a484

carbon source during in planta growth (Zhang et al 2011 Zhang and Van Kan 2013) 485

Our data show that gae1 gae6 plants which are reduced in GalA content did allow 486

enhanced growth of specific B cinerea isolates but no reduction in growth of any B 487

Page 27 of 54

cinerea isolates tested was found This suggests that utilization of pectin as a carbon 488

source was not a contributing factor to alterations in B cinerea susceptibility It is 489

possible that changes in the cell wall composition affect the structural integrity of the 490

wall and affect hyphal penetration of specific B cinerea isolates However OGs (small 491

HG fragments) are likely released during the infection process (Ferrari et al 2013) 492

OGs function as DAMPs and initiate immune responses in Arabidopsis (Ferrari et al 493

2013) Treatment of Arabidopsis with OGs having a degree of polymerization (DP) of 494

10-15 sugar subunits was previously shown to enhance resistance to B cinerea (Ferrari495

et al 2007) We showed that pretreatment with OGs or macerozyme induced enhanced 496

immunity to B cinerea isolate Gallo 1 in wild-type Col-0 but only OGs and not 497

macerozyme induced immunity in gae1 gae6 (Figure 13) Our data suggest gae1 gae6 498

plants which have reduced pectin content may release less OGs and that this 499

reduction leads to the increased susceptibility to B cinerea 500

Consistently we found that macerozyme treatment released less soluble pectin 501

from gae1 gae6 cell walls than from wild-type walls Fractions containing soluble pectin 502

released from gae1 gae6 were also less active in inducing PAD3 gene expression than 503

the fractions released from wild-type walls (Figure 7) We also showed that less soluble 504

pectin accumulated in gae1 gae6 than in wild-type plants 48 h after treatment with B 505

cinerea (Figure 12) 506

We observed that loss of pectin leads to alterations in A thaliana immune 507

signaling Macerozyme treatment induced expression of PAD3 and the SA marker 508

genes PR1 and SID2 in wild-type Col-0 but not in gae1 gae6 whereas OG treatment 509

led to increased PAD3 and SID2 expression in both wild-type and gae1 gae6 (Figure 8 510

and 9) This suggests that although release of active soluble pectin in these plants 511

might be altered OG recognition is intact In fact cell walls isolated from qua1 another 512

mutant known to be reduced in HG released less OGs (DP1-6) than did wild-type cell 513

walls upon endopolygalacturonase treatment (Bouton et al 2002) This was also true 514

for gae1 gae6 cell walls as macerozyme treatment of isolated gae1 gae6 cell walls 515

released less soluble pectin than treatment of Col-0 cell walls Because the fraction 516

from gae1 gae6 plants was also less active in inducing PAD3 expression (Figure 7) it 517

seems possible that biologically active OGs were the active component inducing PAD3 518

Page 28 of 54

expression Interestingly expression of the JA marker genes JAZ5 and JAZ10 was 519

higher in gae1 gae6 than in wild-type mock-inoculated plants but was comparable to 520

wild-type in untreated plants suggesting that JA signaling in gae1 gae6 is 521

hyperresponsive (Figure 10 and Supplemental Figure 11) This induction of JA signaling 522

might be due to wounding stress incurred during the inoculation process JA and SA 523

signaling generally act antagonistically (Pieterse et al 2012) Increased JA signaling 524

activity might hence explain why mock- and macerozyme-treated gae1 gae6 plants 525

exhibited reduced expression of the SA marker PR1 and why OG treatment induced 526

expression of SID2 another SA marker to a lower extent in gae1 gae6 than in wild-type 527

plants (Figure 9) 528

No single immune signaling component was found to be the sole contributor to 529

the compromised immunity of gae1 gae6 to specific B cinerea isolates such as Gallo 1 530

and Fresa 525 These isolates grew better on pad3 while growth of Pepper another B 531

cinerea isolate that did not show altered virulence on gae1 gae6 was unaltered 532

(Supplemental Figure 9) This suggested that differences in camalexin sensitivity of 533

these B cinerea isolates might account for the differences in gae1 gae6 susceptibility 534

However camalexin accumulation and PAD3 expression was the same for wild-type 535

Col-0 and gae1 gae6 for the B cinerea isolates Fresa 525 and Gallo 1 (Figure 8) which 536

showed enhanced growth on gae1 gae6 plants It should be noted that B cinerea 537

virulence is often estimated by comparing lesion sizes Lesion size and measurements 538

of relative fungal growth by qPCR do not always show the same results For example 539

although Denby et al (2004) showed that Pepper causes larger lesions on pad3 than on 540

wild-type this study showed that relative fungal growth of Pepper on pad3 was 541

unaltered 542

We observed that callose deposition was strongly reduced in gae1 gae6 plants 543

72 h after treatment with B cinerea isolate Gallo 1 (Figure 11) possibly due to reduced 544

release of active soluble pectin Interestingly OG-induced PTI to an unspecified isolate 545

of B cinerea was intact in the pmr4 mutant which does not produce callose (Galletti et 546

al 2008) Due to the high SA levels in pmr4 plants (Nishimura et al 2003) and the fact 547

that different B cinerea isolates vary both in induction of and reaction to defense 548

responses (Kliebenstein et al 2005 Rowe et al 2010) we could not determine 549

Page 29 of 54

whether reduced callose deposition causes the increased susceptibility of gae1 gae6 to 550

B cinerea isolate Gallo 1551

We did not observe any changes in B cinerea growth either on cev1 ixr1 plants 552

which are known to have constitutively high JA and OPDA levels (Ellis et al 2002b) or 553

on plants exogenously treated with MeJA (Supplemental Figure 9) Because plants 554

were kept uncovered for 3 h after MeJA treatment it is unclear how much MeJA was 555

bioavailable in the plant However JAZ10 expression was induced 3 h after spraying 556

with MeJA (Supplemental Figure 10) Because pathogen growth generally depends on 557

the combination of a multitude of immune signaling events we conclude that multiple 558

changes to the immune signaling in gae1 gae6 together account for the altered B 559

cinerea growth phenotypes detected 560

We showed that expression of GAE1 and GAE6 is repressed by the bacterial 561

hemibiotroph Pma ES4326 This repression did not occur in an eds1 or pad4 562

background or in pbs3 at the 24-h time point (Figure 1 and Supplemental Figure 1) 563

During the Arabidopsis - Pma ES4326 interaction the expression of many defense 564

related genes is altered in a PAD4EDS1 dependent fashion (Wang et al 2008) Some 565

of these expression changes require SA CBP60g and SARD1 are thought to act 566

downstream of PAD4 and EDS1 but upstream of SA-dependent responses (Wang et 567

al 2011) PBS3 is also thought to act downstream of PAD4 and EDS1 and upstream of 568

SA but the set of genes whose expression it affects is only partially overlapping with 569

those affected by CBP60gSARD1 (Wang et al 2008 Wang et al 2011) Thus GAE1 570

and GAE6 repression is regulated by the part of the immune signaling network that 571

requires PAD4 EDS1 and PBS3 but is independent of CBP60gSARD1 and SA 572

Because EDS1 PAD4 and PBS3 are important components of the plant immune 573

signaling network (Wiermer et al 2005 Nobuta et al 2007) one might conclude that 574

repression of GAE1 and GAE6 may be part of the plant immune response to Pma 575

ES4326 B cinerea treatment also reduced GAE1 and GAE6 expression but this was 576

mostly independent of PAD4 (Figure 6) while both Pma ES4326 and B cinerea repress 577

GAE1 and GAE6 expression the host signaling mechanisms responsible likely differ 578

In summary our data show that Arabidopsis GAE1 and GAE6 are required for 579

pectin biosynthesis leaf flexibility and immunity to Pma ES4326 and to specific B 580

Page 30 of 54

cinerea isolates We used macerozyme a commercial pectinase to mimic the effect of 581

B cinerea pectinases Macerozyme treatment released less pectin from gae1 gae6 cell582

walls and induced less immune signaling in gae1 gae6 plants Less water soluble pectin 583

also accumulated in B cinerea infected gae1 gae6 plants and macerozyme-induced 584

immunity to B cinerea isolate Gallo 1 was reduced in these plants We conclude that 585

the reduced pectin content in these mutants may lead to reduced release of soluble 586

pectin including active OGs during pathogen attack and that this compromises 587

immunity 588

589

590

Page 31 of 54

METHODS 591

Plant Materials and Growth Conditions 592

Wild-type Columbia (Col-0) and all mutant plants (in Col-0 background) were 593

grown on sterilized BM2 germinating mix (Berger Inc Quebec Canada) in a controlled 594

environment chamber (Conviron Pembina ND USA) with a 12-h photoperiod under 595

100 microM m-2 s-1 fluorescent illumination at 22degC and 75 relative humidity Germplasm 596

used is described in the Accession Numbers section at the end of Methods 597

598

Data Analysis and Replicates 599

For biological replicates samples were obtained from separate plant tissues and 600

in many cases from plants grown in different flats In some experiments multiple 601

technical replicates were measured per biological replicate For statistical analysis all 602

technical replicates were averaged and the averages were considered one biological 603

replicate Independent experiments were performed at different times and often 604

consisted of multiple biological replicates per experiment 605

Data analysis was performed using mixed linear models in the R programming 606

environment (Bates et al 2015) unless otherwise indicated Generally genotypes 607

treatments and time points were used as fixed effects and replicate-specific effects like 608

different pots flats etc were considered random effects We reported p-values 609

obtained from the mixed linear model when less than 10 comparisons were performed 610

and q-values for more than 10 comparisons to correct for multiple comparison testing 611

Q-values were calculated using the Benjamini-Hochberg procedure (Benjamini and612

Hochberg 1995) unless specified otherwise 613

614

Pathogen Strains Growth Conditions and Pathogen Growth Assays 615

Growth of Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) and 616

bacterial growth assays were performed as previously described (Bethke et al 2014) 617

For qRT-PCR assays four week-old plants were inoculated with bacteria (OD600 = 618

0002) or mock (5 mM MgSO4) At least four leaves from two plants were collected at 619

the indicated time points per biological replicate 620

Page 32 of 54

Prior to each experiment Botrytis cinerea isolates were grown on 1x PDA 621

medium (Difco) for 10 d at room temperature All isolates used have been described 622

previously (Rowe and Kliebenstein 2007) The spores were washed from the surface of 623

the plate using an inoculation medium (1x Gamborgrsquos B-5 basal salt mixture (Sigma-624

Aldrich G5768) 2 (wv) glucose 10 mM phosphate buffer pH 64) and fungal hyphae 625

were removed from the suspension by filtering through four layers of cheesecloth The 626

concentration of spores was determined using a hemocytometer and adjusted to 25 x 627

105 spores mL-1 Four week-old Arabidopsis plants were inoculated by placing one 10-628

microL droplet of the B cinerea spore solution or inoculation medium (mock) on the adaxial 629

leaf surface of fully expanded leaves Inoculated plants were kept at 100 relative 630

humidity Infection sites were collected at various time points using a cork borer For 631

qRT-PCR assays at least four leaf discs from one plant were combined for each 632

biological sample For B cinerea growth assays at least 12 leaf discs from three to six 633

plants were pooled per biological sample Relative fungal growth was determined by 634

measuring the abundance of a fungal gene relative to a plant gene as described below 635

In brief DNA from infected tissue was extracted and equal amounts of total DNA were 636

used to perform qPCR reactions using the B cinerea cutinase A and the Arabidopsis 637

SK11 gene respectively Primers were described in (Gachon and Saindrenan 2004) 638

and are listed in Supplemental Table 1 Two technical replicates were combined for 639

each biological replicate B cinerea is a necrotrophic fungal pathogen and may destroy 640

plant DNA as disease progresses However decreases in the amount of plant DNA 641

over the course of our experiments were only observed at late time points with very 642

susceptible plants so the qPCR assay is generally a reasonable estimate of fungal 643

biomass (Supplemental Figure 16) 644

645

Treatment with Macerozyme or OGs 646

For PTI assays four week-old plants either were inoculated with 001 (wv) 647

macerozyme R-10 (Yakult) a Rhizopus polygalacturonase (EC 32115) that possesses 648

high pectinase and hemicellulase activity or were mock inoculated using heat-649

inactivated macerozyme Other similar plants were either inoculated with 250 or 500 microg 650

mL-1 OGs or mock inoculated with water One d later plants were infected with B 651

Page 33 of 54

cinerea and samples were collected immediately (0 dpi) and two or three d later To 652

investigate the role of MeJA on B cinerea growth plants were sprayed with 1mM MeJA 653

in water with 001 (vv) Tween 20 Covers were removed so that leaves could dry off 654

before plants were inoculated with B cinerea three h later Samples were harvested at 655

the indicated time points For expression analysis and MAPK activation assays plants 656

were either inoculated with 100 microg mL-1 OGs or mock inoculated with water OGs used 657

for experiments described in Figures 8 to 10 and Supplemental Figure 12 were a kind 658

gift from Simone Ferrari OGs used in experiments described in Figure 13 and 659

Supplemental Figures 11 13 and 15 were prepared as previously described (Kohorn et 660

al 2014) 661

662

Expression Analysis 663

Leaf tissue from inoculated plants was harvested at the indicated time points 664

flash frozen pulverized and RNA extracted using Trizol (Invitrogen) Quantitative RT-665

PCR was performed using the SuperScript III Platinum SYBR Green One-Step 666

quantitative RT-PCR kit (Invitrogen) and a Lightcycler 480 Real-Time PCR system 667

(Roche) as previously described (Truman and Glazebrook 2012) In brief equal 668

amounts of total RNA and a gene specific primer were used for each reaction The 669

crossing point (Cp) was calculated using the 2nd derivative max method provided with 670

the Lightcycler software for each amplification curve Each reaction was run with two 671

technical replicates and the Cp values for these replicates were averaged ACTIN2 was 672

used as a stably expressed reference gene At least three biological replicates each 673

were performed and analyzed using a mixed-linear model RT-PCR was performed to 674

verify T-DNA insertion lines using the One-Step RT-PCR kit (Qiagen) according to the 675

manufacturerrsquos instructions The primers used can be found in Supplemental Table 1 676

677

Electrolyte Leakage Assay 678

Fully expanded leaves of four week-old plants were inoculated with water (Milli-Q 679

grade) using a needleless syringe Leaf discs were collected using a cork borer and 680

three leaf discs from one leaf were placed with the adaxial surface down onto 2 mL 681

water in a 12-well cell culture plate Conductivity was measured using a Horiba B-173 682

Page 34 of 54

conductivity meter after 30 min and then the water was replaced Conductivity was 683

measured again 30 min later (1 h total) as indicated in Figure 3C Six biological 684

replicates each were performed in three independent experiments and means and SEs 685

were calculated Two-sided t-tests were performed for each time point 686

687

DAB and Aniline Blue Staining 688

Leaves from four week-old plants were either mock inoculated with B cinerea 689

inoculation medium or inoculated with 10 microL droplets of a B cinerea spore solution (25 690

x 105 spores mL-1) of the indicated isolates Samples were collected 72 h later For 33prime-691

diaminobenzidine (DAB) staining two to five inoculated leaves were vacuum infiltrated 692

with 33prime-Diaminobenzidine (1 mg mL-1 in water pH 38) solution and stained overnight 693

in the dark Leaves were destained using 100 ethanol at 65degC and then transferred to 694

50 (vv) glycerol and photographed 695

Callose was visualized by staining leaves with aniline blue as previously 696

described (Adam and Somerville 1996) Briefly leaves were vacuum infiltrated with 697

alcoholic lactophenol (one volume of phenolglycerollactic acidwater (1111) mixed 698

with two volumes of ethanol) and destained at 65degC Next leaves were moved through 699

an alcohol gradient (50 20 and 10 (vv) ethanol) into water The destained leaves 700

were stained for 30 min in 150 mM K2HPO4 (pH 95) with 001 (wv) aniline blue 701

Samples were mounted in water and examined using a Nikon Eclipse Ni-U microscope 702

at 10x magnification using a DAPI filter (excitation at 325-375 nm emission at 435-485 703

nm) For callose quantification six 200 microm x 200 microm squares were counted for each 704

sample type Callose deposits per mm2 were calculated and data analyzed using a t-705

test Note that there is an inherent uncertainty associated with such counts because 706

callose deposits may not all be in focus at the same time 707

708

MAPK Activity Assay 709

Fully expanded leaves of four week-old plants were inoculated with 001 710

macerozyme 100 microg mL-1 OGs 100 nM flg22 or water (mock) flg22 a peptide from 711

bacterial flagella that induces immune responses was used as a positive control for 712

Page 35 of 54

MAPK activation (Asai et al 2002) and was purchased from EZBiolab Inc (Westfield 713

IN USA) Samples were harvested 10 min later and immediately frozen in liquid 714

nitrogen Protein was extracted using an extraction buffer containing 25 mM Tris pH 78 715

75 mM NaCl 1 mM DTT 1 mM NaF 05mM Na3VO4 01 (vv) Tween 20 one 716

cOmplete Mini protease inhibitor cocktail tablet (Roche) and one PhosSTOP 717

phosphatase inhibitor cocktail tablet (Roche) per 10 mL buffer 15 microg of total protein 718

were electrophoresed on a 10 SDS-polyacrylamide gel and then blotted onto a PVDF 719

membrane (Bio-Rad 162-0177) Activated MAPKs were detected using p4442 MAPK 720

(Erk12) antibody (9102S Cell Signal 12500 in TBST (20 mM Tris pH 75 150 mM 721

NaCl 01 (vv) Tween 20)) Anti-Rabbit IgG-HRP (115000 in TBST A6154 Sigma) 722

and ECL Plus substrate (Pierce 32132) MPK3 and MPK6 were detected with an anti-723

MPK3 antibody (M8318 Sigma 12000 in TBST and 3 (wv) milk) or an anti-MPK6 724

antibody (A7104 Sigma 16000 in TBST and 3 (wv) milk) respectively The 725

secondary antibody and detection were the same as for the p4442 antibody Five 726

independent experiments were performed 727

728

Preparation of Alcohol Insoluble Residue (AIR) Pectin Extraction and Uronic Acid 729

Measurements 730

Fully expanded leaves from four week-old plants that were incubated for 48 h in 731

the dark to reduce starch were harvested flash frozen and pulverized AIR was 732

extracted by washing ground material twice in 70 (vv) ethanol three times in a 733

mixture of chloroform and methanol (11 vv) and once in acetone (Gille et al 2009) 734

and was then air dried Cell wall pectin was extracted from AIR with cell wall extraction 735

buffer (50 mM Trizma 50 mM CDTA pH 72) at 95degC for 15 min Samples were 736

homogenized using a paint shaker (Harbil 5G-HD) and glass beads (3 mm diameter) 737

For dot blot experiments 500 microL of cell wall extraction buffer were used per 10 mg of 738

AIR Debris was precipitated by centrifugation for 10 min at 10000 g 739

Total uronic acid content of pectin extracted as described above using 500 microL of 740

cell wall extraction buffer per 1 mg of AIR was measured as described previously 741

(Filisetti-Cozzi and Carpita 1991 van den Hoogen et al 1998) First 36 microL of pectin 742

extract were mixed with 4 microL of 4 M sulfamic acid Then 200 microL of sulfuric acid 743

Page 36 of 54

containing 120 mM sodium tetraborate was added and samples were incubated at 80degC 744

for 1 h Following cooling on ice the optical density of samples was measured at 490 745

nm Next 40 microL of m-hydroxydiphenyl reagent (100 microL of m-hydroxydiphenyl in dimethyl 746

sulfoxide at 100 mgmL mixed with 49 mL 80 (vv) sulfuric acid just before use) was 747

added and the samples were mixed The optical density at 490 nm was measured 748

again The optical density before m-hydroxydiphenyl reagent addition was subtracted 749

from the optical density measured after addition of the dye The concentration of uronic 750

acid was calculated using known amounts of GalA as a standard 751

752

Release of Pectin from Arabidopsis Cell Walls Using Macerozyme 753

AIR preparations from leaves of four week-old plants were treated with 025 754

(wv) macerozyme R-10 (Yakult) or water (mock) at a concentration of 100 microL per mg 755

AIR Samples were incubated for 3 h at room temperature on a vortex shaker and 756

centrifuged for 10 min at 12000 g Total uronic acid concentration of 5-fold diluted 757

supernatants was determined for two technical replicates each The concentration of 758

uronic acid was calculated using known amounts of GalA as a standard For 759

subsequent qRT-PCR experiments supernatants from multiple extractions were 760

combined boiled for 20 min to inactivate macerozyme diluted 10-fold with water and 761

inoculated into four week-old Col-0 plants Heat-inactivated 10-fold diluted macerozyme 762

solutions were used as mock treatment Samples were collected 3 h later and PAD3 763

expression was determined 764

765

Release of Pectin from Arabidopsis Leaves Infected with B cinerea 766

Fully expanded leaves of four week-old plants were either inoculated with up to 767

five 10 microL droplets of B cinerea spore solution (25 x 105 spores mL-1) or mock 768

inoculated with B cinerea inoculation medium For each biological replicate leaves with 769

a combined count of at least 20 infection sites were collected 48 h later AIR was 770

extracted as described above but plants were not dark-treated or freeze-dried and the 771

first ethanol step was performed using 85 (vv) ethanol To extract soluble pectin 772

fragments AIR samples were mixed with water at a concentration of 20 microL per infection 773

site incubated for 7 h at room temperature on a vortex shaker and centrifuged for 10 774

Page 37 of 54

min at 12000 g Total uronic acid concentration of supernatants was determined for two 775

technical replicates each The concentration of uronic acid was calculated using known 776

amounts of GalA as a standard 777

778

Measurement of Uronic Acids Neutral Monosaccharides and Cellulose Content in 779

Cell Walls 780

Neutral sugars and uronic acids from non-cellulosic polysaccharides in cell walls 781

(AIR) were released by trifluoroacetic acid (TFA) hydrolysis Approximately 1 mg of AIR 782

was hydrolyzed with 2 M TFA at 121degC for 90 min The TFA-soluble material was split 783

into two equal parts one for uronic acid and one for neutral sugar determination The 784

uronic acid content was determined by analyzing the hydrolyzate using a CarboPac 785

PA200 anion-exchange column with an ICS-3000 Dionex chromatography system 786

(Dionex Sunnyvale CA USA) The elution profile consisted of a linear gradient of 50 to 787

200 mM sodium acetate in 01 M NaOH in 10 min at 04 mL per min Alditol acetate 788

derivatives of neutral sugars were produced as described (York et al 1985) In brief 789

monosaccharides in the hydrolyzate were reduced with NaBH4 and peracetylated with 790

acetic anhydride and pyridine at 121degC for 20 min The generated alditol acetates were 791

analyzed by GC-MS Crystalline cellulose was measured according to Updegraff (1969) 792

After hydrolysis of noncellulosic polysaccharides from AIR with the Updegraff reagent 793

(acetic acid nitric acids water 812 (vv) ) the remaining pellet was hydrolyzed in 794

72 sulfuric acid The resulting glucose quantity was determined by the anthrone 795

method (Scott and Melvin 1953) 796

797

Dot Blot Experiments 798

Pectin solutions extracted using 50 microL of cell wall extraction buffer per mg AIR 799

were serially diluted and nitrocellulose membranes were spotted with 1 microL of the diluted 800

pectin solutions Membranes were dried overnight blocked with 5 milk (wv) in 1x 801

PBS (8 g L-1 NaCl 02 g L-1 KCl 144 g L-1 Na2HPO4 024 g L-1 KH2PO4 pH 74) and 802

probed with LM5 (Jones et al 1997) LM6 (Willats et al 1998) LM8 (Willats et al 803

2004) LM19 and LM20 (Verhertbruggen et al 2009a) or CCRC-M7 (Steffan et al 804

Page 38 of 54

1995) antibodies LM series antibodies were diluted 1250 and CCRC-M7 was diluted 805

1500 in 5 milk powder (Nestle Carnation) in 1 x PBS For LM series antibodies a 806

goat anti-Rat HRP conjugated antibody (Bethyl A110-105P 15000 diluted) and for 807

CCRC-M7 an anti-Mouse IgG HRP conjugate (Promega W402B 12500 diluted) was 808

used as secondary antibody in 5 milk powder in 1x PBS LM series antibodies were 809

obtained from plant probes (wwwplantprobesnet) and CCRC-M7 from CarboSource 810

(wwwcarbosourcenet) Membranes were washed after incubation with the primary and 811

secondary antibodies with 1x PBS Dot blots were developed using the ECL system 812

(GE Healthcare Amersham ECL prime) For quantification of signals dot blot results on 813

x-ray films were photographed using a CCD camera and intensities were measured814

using Image Jrsquos Integrated Density function Locally adjusted background was 815

subtracted As the dynamic range of the signal on an x-ray film was narrow non-816

linearity in the measured values was corrected for each genotype in each biological 817

replicate using the values from different dilutions in two technical replicates We noticed 818

that the residuals were not normally distributed and performed Box-Cox power 819

transformation (Box and Cox 1964) to obtain normally distributed values for the 820

subsequent statistical analysis of the data For LM19 Box-Cox selected power 821

transformation was done to the power of 038th and for LM20 to the power 075th After 822

correction of the transformed measured values with the effect of the biological 823

replicates the values were fit to a linear model with the Arabidopsis genotype as the 824

fixed effect This linear model was used both to generate the plots in Figures 5B and D 825

and to test significant differences 826

827

Camalexin Measurements 828

Four week-old plants were infected with B cinerea and samples (three leaf disks 829

from one plant each) were harvested at the indicated time points flash frozen and 830

pulverized Each sample was extracted with 300 microL of 90 methanol (vv) 50 microL of the 831

extract was run on an Agilent Lichrocart 250-4 RP18e 5-microm column using an Agilent 832

1100 series HPLC Camalexin was detected using a DiodeArray (DAD) at 330 nm and 833

with a fluorescence detector at emission 318 nmexcitation 385 nm (Agilent) Separation 834

was achieved using the following program with aqueous acetonitrile 5 min gradient 835

Page 39 of 54

from 63 to 69 acetonitrile 30 s gradient from 69 to 99 acetonitrile 2 min at 99 836

acetonitrile and a post-run equilibration of 35 min at 63 acetonitrile (Denby et al 837

2004 Kliebenstein et al 2005) Purified camalexin was used to produce a standard 838

curve to identify and quantitate camalexin 839

840

Accession Numbers 841

Microarray data used can be accessed at NCBI Gene Expression Omnnibus 842

(httpwwwncbinlmnihgovgeo) GSE18978 Sequence information for all Arabidopsis 843

genes described in this article can be found in TAIR (wwwarabidopsisorg) using the 844

following accession numbers PAD3 (CYP71B15 At3g26830) SID2 (ICS1 At1g74710) 845

GAE1 (At4g30440) GAE2 (At1g02000) GAE3 (At4g00110) GAE4 (At2g45310) GAE5 846

(At4g12250) GAE6 (At3g23820) PR1 (At2g14610) JAZ5 (At1g17380) JAZ10 847

(At5g13220) ACTIN2 (At3g18780) PDF12a (At5g44420) PDF12b (At2g26020) 848

AtSK11 (At5g26751) The sequence of the B cinerea cutinase Bc-CutA (Z69264) can 849

be found in the DNA Data Bank of Japan (httpgetentryddbjnigacjp) Germplasm 850

used included dde2-2 (At5g42650 (von Malek et al 2002)) pad3-1 (At3g26830 (Zhou 851

et al 1999)) pad4-1 (At3g52430 (Jirage et al 1999)) sid2-2 (At1g74710 (Wildermuth 852

et al 2001)) pbs3-2 (At5g13320 SALK_018225 (Nobuta et al 2007)) cbp60a-1 853

(At5g62570 SALK_124410 (Truman et al 2013)) cbp60g-1 sard1-2 (At5g26920 854

SALK_023199 At1g73805 SALK_052422 (Wang et al 2011) cbp60a-1 cbp60g-1 855

sard1-2 (Truman et al 2013) gae1-1 (At4g30440 SALK_085554) gae6-1 (At3g23280 856

SALK_104454) gae6-2 (At3g23280 SALK_017191) ixr1-1 cev1 (At5g05170 (Ellis 857

and Turner 2001 Scheible et al 2001)) eds1 plants were derived by introgression of 858

the Ler eds1-2 allele into Col-0 that contains two copies of EDS1 (At3g48090 859

At3g48080 (Bartsch et al 2006) mpk6-2 (At2g43790 SALK_073907 (Liu and Zhang 860

2004)) mpk3-DG (At3g45640 a fast neutron deletion mutant (Miles et al 2005)) and 861

npr1-1 (At1g64280 (Cao et al 1997)) T-DNA insertion lines were part of the SALK 862

collection (Alonso et al 2003) and were obtained from the Arabidopsis Biological 863

Resource Center 864

865

Supplemental Data 866

Page 40 of 54

Supplemental Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 867

Expression Does Not Require CBP60s or SID2 868

Supplemental Figure 2 Expression of GAE Family Members uponTreatment with Pma 869

ES4326 870

Supplemental Figure 3 Characterization of T-DNA Insertions in GAE1 and GAE6 871

Supplemental Figure 4 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves and 872

Are Slightly Smaller than Wild-Type Col-0 Plants 873

Supplemental Figure 5 Cell Walls of gae1 gae6 Plants Contain Less Uronic Acid than 874

Wild-Type Col-0 Cell Walls 875

Supplemental Figure 6 Antibodies Raised Against the RGI or XG Components of 876

Pectin Show No Difference in Binding to gae Mutant and Col-0 Cell Walls 877

Supplemental Figure 7 The Effect of B cinerea Treatment on the Expression of GAE 878

Family Genes 879

Supplemental Figure 8 Treatment with 001 Macerozyme Does Not Cause Any 880

Visible Damage to Col-0 Plants 881

Supplemental Figure 9 B cinerea Growth on Plants with Defects in Immune 882

Signaling 883

Supplemental Figure 10 MeJA-Induced Expression of the JA marker gene JAZ10 884

Supplemental Figure 11 The Expression of the JA Marker Gene JAZ5 Is Highly 885

Responsive in gae1 gae6 Plants 886

Supplemental Figure 12 The Expression of PDF12 Is Unaltered in gae1 gae6 Plants 887

Supplemental Figure 13 Macerozyme Treatment Activates MPK3 and MPK6 888

Supplemental Figure 14 B cinerea-Induced ROS Production in Wild-Type Col-0 and 889

gae1 gae6 Plants 890

Supplemental Figure 15 OG-Induced PTI Using 250 microg mL-1 OGs 891

Supplemental Figure 16 Abundance of Plant DNA in B cinerea Treated Plants 892

Supplemental Table 1 Primers used in this study 893

894

ACKNOWLEDGEMENTS 895

We thank William Truman for helpful discussions Simone Ferrari (Sapienza 896

Universita di Roma) for the generous gift of OGs Bruna Bucciarelli (USDA-ARS) for 897

Page 41 of 54

help with fluorescence microscopy the Arabidopsis Biological Resource Center for T-898

DNA insertion lines Paul Knoxrsquos lab (University of Leeds) for LM5 LM6 LM8 LM19 899

and LM20 antibodies and the Complex Carbohydrate Resource Center (University of 900

Georgia) for the CCRC-M7 antibody Most of this work was funded by the Division of 901

Chemical Sciences Geosciences and Biosciences Office of Basic Energy Sciences of 902

the US Department of Energy through Grant DE-FG02-05ER15670 to JG This work 903

was also supported by NSF awards IOS 1339125 and IOS 1021861 to DJK NSF 904

award IOS 1121425 to F K and the USDA National Institute of Food and Agriculture 905

Hatch project number CA-D-PLS-7033-H to DJK 906

907

AUTHOR CONTRIBUTIONS 908

GB and JG designed the research GB GX AT BL NES and NH 909

performed research DJK provided tools GB GX MP AD BL RAH and FK 910

analyzed data GB and JG wrote the paper with input from all authors 911

912

FIGURE LEGENDS 913

Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 Expression 914

Requires PAD4 EDS1 and PBS3 915

(A) Expression of GAE1 in leaves of wild-type Col-0 and mutant plants Expression was916

measured 24 or 32 h after either inoculation with Pma ES4326 (OD600 = 0002) or mock 917

inoculation with 5 mM MgSO4 and was then normalized to the level of ACTIN2 Data 918

from three biological replicates were combined using a mixed linear model Bars 919

represent mean log2-ratios to ACTIN2 plusmn SE Letters indicate significantly different 920

groups with q lt 005 where 005 represents the maximum false discovery rate at which 921

the test may be called significant 922

(B) Expression of GAE6 measured as described in (A)923

924

Figure 2 Susceptibility of gae6 and gae1 gae6 Plants to Pma ES4326 Is Increased 925

Relative to Wild-Type Col-0 926

Leaves of plants of the indicated genotypes were inoculated with Pma ES4326 (OD600 = 927

00002) as described in Methods Bacterial titers in leaves were determined either 928

Page 42 of 54

immediately (0 dpi) or after three days (3 dpi) Bars represent the mean plusmn SE of four 929

independent experiments each with 4 or 12 biological replicates at 0 and 3 dpi 930

respectively Data were combined using a mixed linear model Asterisks indicate 931

significant differences from Col-0 wild-type (q lt 005) Susceptible pad4 plants were 932

included as a positive control 933

934

Figure 3 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves 935

(A) Depiction of the assay for brittleness of gae mutant leaves Fully expanded leaves of936

four to five week-old plants were bent by 180ordm so that the adaxial surface of the leaf was 937

parallel with the soil Broken leaf petioles or midribs were considered brittle 938

(B) Quantification of broken midribs or petioles For each genotype 66 to 82 leaves 939

were analyzed for brittleness as described in (A) Bars represent the ratio of broken to 940

total number of leaves tested Results were analyzed using Fisherrsquos exact test Letters 941

indicate significantly different groups at p lt 005 942

(C) Conductivity measurements Leaves of four week-old plants were inoculated with943

deionized water and then leaf discs were cut out and placed on deionized water 944

Conductivity was measured at the indicated time points After 30 min the water was 945

replaced with fresh water (30 min f w) to account for wounding-related ion leakage from 946

the edges of the leaf discs Three independent experiments with six biological replicates 947

each were performed and data were analyzed using a t-test for each time point P-948

values were corrected using the Bonferroni method Data show means plusmn SE asterisks 949

indicate significant differences from wild-type Col-0 at q lt 001 950

951

Figure 4 Cell Walls of gae6 and gae1 gae6 Plants Are Reduced in GalA 952

(A) to (D) Levels of GalA (A) GlcA (B) cellulose (C) and neutral sugars (D) in alcohol953

insoluble residue (AIR) extracted from leaves of four week-old plants of the indicated 954

genotypes Bars represent means plusmn SE of four biological replicates combined using a 955

mixed linear model Letters indicate significantly different groups (p lt 005) 956

957

Figure 5 Cell Walls of gae1 gae6 Plants Are Reduced in Homogalacturonan 958

Page 43 of 54

Dot blot analyses with anti-LM19 and anti-LM20 antibodies Pectin was extracted from 959

AIR prepared from the indicated genotypes using 1 microL of extraction buffer per 10 microg of 960

AIR Pectin was serially diluted and one microL of undiluted 13 diluted and 19 diluted 961

samples were spotted on a nitrocellulose membrane Three biological replicates were 962

were spotted twice to obtain two technical replicates each All replicates produced 963

similar results 964

(A) Representative dot blot using anti-LM19 antibody965

(B) Relative signal intensities of the spots in the dot blot in (A)966

(C) Representative dot blot using anti-LM20 antibody967

(D) Relative signal intensities of the LM20 dot blot shown in (C)968

Relative signal intensities were measured using Image J software Measurements from 969

the three biological replicates were combined using a linear model after Box-Cox power 970

transformation as described in Methods Bars represent means plusmn SE Asterisks indicate 971

a difference from wild-type Col-0 at p lt 005 972

973

Figure 6 Growth of B cinerea on gae1 gae6 Plants and the Effect of B cinerea on 974

Expression of GAE1 and GAE6 975

(A) Growth of B cinerea isolates Leaves of Col-0 and gae1 gae6 plants were976

inoculated with 25 x 105 spores mL-1 of the B cinerea isolates indicated in the figure 977

Samples were collected two (2 dpi) and three (3 dpi) days later Bars represent means plusmn 978

SE of six biological replicates for each fungus combined using a mixed linear model 979

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 980

(B) and (C) Expression of GAE1 (B) and GAE6 (C) 48 h after inoculation with the B981

cinerea isolates indicated Expression levels were measured by qRT-PCR Bars 982

represent mean log2-ratios to ACTIN2 plusmn SE of three biological replicates combined 983

using a mixed linear model Letters indicate significantly different groups (q lt 005) 984

985

Figure 7 Levels of Soluble Pectin and Expression of PAD3 in Fractions Produced 986

Following Macerozyme Treatment of gae1 gae6 Cell Walls 987

(A) Schematic overview of the experimental procedure for (B) and (C)988

Page 44 of 54

(B) Uronic acid measurements Alcohol-insoluble residue (AIR) from leave tissue of four989

week-old wild-type Col-0 or gae1 gae6 plants was treated with water or 025 990

macerozyme and assayed for uronic acid levels Data from 11 biological replicates were 991

combined using a mixed linear model Bars represent means plusmn SE Letters indicate 992

significant differences (p lt 005) 993

(C) Expression of PAD3 in Col-0 plants treated with the fractions from (B) Extracts from994

(B) were heat inactivated pooled and diluted 5-fold with water Pooled extracts were995

inoculated into four week-old Col-0 plants PAD3 expression was measured 3 h after 996

inoculation and normalized to the level of ACTIN2 expression Data from four biological 997

replicates were combined using a mixed linear model Bars represent mean log2-ratios 998

to ACTIN2 plusmn SE Letters indicate significant differences (p lt 005) 999

1000

Figure 8 Macerozyme-Induced PAD3 Expression Is Absent in gae1 gae6 1001

(A) Expression of PAD3 after macerozyme treatment Four week-old plants were either1002

inoculated with 001 macerozyme or mock inoculated with water and PAD3 1003

expression was measured 3 h later by qRT-PCR Data from four biological replicates 1004

were combined using a mixed linear model Mean log2-ratios to ACTIN2 plusmn SE were 1005

plotted Letters indicate significantly different groups (p lt 005) 1006

(B) Expression of PAD3 after OG treatment PAD3 expression was measured as1007

described in (A) 3 h after inoculation with 100 microg mL-1 OGs or mock inoculation with 1008

water Data are from six biological replicates 1009

(C) PAD3 expression in untreated plants Three biological replicates were performed1010

and data analyzed as described in (A) 1011

(D) Camalexin accumulation after B cinerea treatment Camalexin accumulation was1012

measured in four week-old plants inoculated with the B cinerea strains shown in the 1013

figure Eight biological replicates each were combined using a mixed linear model 1014

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 1015

(E) PAD3 expression 28 h after treatment with the indicated B cinerea strains Data1016

from six biological replicates were collected and analyzed as described in (A) Data in 1017

all figure parts represent means plusmn SE Letters indicate significantly different groups (q lt 1018

005) 1019

Page 45 of 54

1020

Figure 9 Macerozyme-Induced Expression of the SA Marker Genes SID2 and PR1 1021

Is Abolished in gae1 gae6 Plants 1022

(A) to (H) Expression of PR1 (A) (C) (E) and (G) or SID2 (B) (D) (F) and (H) in four1023

week-old Col-0 and gae1 gae6 plants Data were combined using a mixed linear model 1024

Bars represent mean log2-ratios of expression vs ACTIN2 plusmn SE for all figure parts 1025

Letters indicate significantly different groups at p lt 005 for panels (A) to (D) (G) and 1026

(H) and qlt005 for panels (E) to (F) 1027

(A) and (B) Plants were treated with 001 macerozyme or mock treated with water1028

and samples were collected 3 h later Data from four biological replicates are shown 1029

(C) and (D) Plants were inoculated with 100 microg mL-1 OGs or mock treated with water1030

and samples collected 3 h later Data from six biological replicates are shown 1031

(E) and (F) Plants were inoculated with the indicated B cinerea isolates or mock1032

inoculated with B cinerea inoculation medium and samples collected 48 h later Data 1033

from eight biological replicates are shown 1034

(G) and (H) Expression in untreated plants Three biological replicates were performed1035

1036

Figure 10 The Expression of the JA Marker Gene JAZ10 is Highly Responsive in 1037

gae1 gae6 Plants 1038

(A) to (D) JAZ10 expression in four week-old plants Data were combined using a mixed1039

linear model Mean log2-ratios to ACTIN2 plusmn SE are shown Letters indicate significantly 1040

different groups at p lt 005 for panels (A) (B) and (D) and q lt 005 for panel (C) 1041

(A) Plants were inoculated with 001 macerozyme or mock inoculated using water and1042

samples were collected 3 h later Data from four biological replicates are shown 1043

(B) Plants were inoculated with 100 microg mL-1 OGs or mock inoculated using water and1044

samples collected 3 h later Data are from six biological replicates 1045

(C) JAZ10 expression 48 h after treatment with the indicated B cinerea strains Data1046

are from six biological replicates 1047

(D) JAZ10 expression in untreated plants Data are from three biological replicates1048

1049

Figure 11 Gallo 1-Induced Callose Deposition Is Reduced in gae1 gae6 Plants 1050

Page 46 of 54

(A) Callose deposition in four week-old Col-0 and gae1 gae6 plants Plants were1051

inoculated with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) or mock inoculated 1052

with B cinerea inoculation medium and samples were collected after 72 h Callose was 1053

stained with aniline blue and visualized using a Nikon Eclipse Ni-U microscope with a 1054

DAPI filter Three leaves were stained per treatment Scale bars are 100 microm Asterisks 1055

indicate fungal hyphae arrows indicate representative callose spots 1056

(B) Quantification of callose deposition Callose deposits were counted in 200 microm x 2001057

microm squares laid out across the images shown here Bars represent means plusmn SE from 1058

six squares each Letters indicate significantly different groups (p lt 005) according to a 1059

t-test analysis1060

1061

Figure 12 Less Soluble Pectin Accumulates in B cinerea-Infected gae1 gae6 1062

Leaves 1063

Four week-old Col-0 and gae1 gae6 plants were inoculated with 10 microL droplets of the 1064

indicated B cinerea isolate (25 x 105 spores mL-1) or mock inoculated with B cinerea 1065

inoculation medium Leaves were collected 48 h later AIR was extracted from leaf 1066

samples and water added (20 microL infection site) to extract soluble pectin fragments 1067

Samples were vortexed for 7 h centrifuged at 12000 g and the uronic acid 1068

concentration of the supernatant was measured Data from nine biological replicates 1069

were combined using a mixed linear model Means plusmn SE are shown Letters indicate 1070

significant differences (q lt 005) 1071

1072

Figure 13 Macerozyme-Induced PTI Is Lost in gae1 gae6 While OG-Induced PTI Is 1073

Unaltered 1074

(A) Macerozyme-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1075

inoculated with 001 macerozyme or mock inoculated with boiled macerozyme and 1076

were infected with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) 24 h later Samples 1077

were collected immediately (0 dpi) and after two (2 dpi) and three (3 dpi) days Data 1078

show mean plusmn SE of six to 14 biological replicates each combined using a linear model 1079

Letters indicate significantly different groups (q lt 005) 1080

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

Parsed CitationsAdam L and Somerville SC (1996) Genetic characterization of five powdery mildew disease resistance loci in Arabidopsisthaliana Plant J 9 341-356

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

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Because plant cell walls are important barriers against pathogenic microbes 118

alterations in wall structural properties can lead to changes in plant immunity To date 119

only a few examples of cell wall related mutants with altered pathogen phenotypes have 120

been studied in detail Mutants impaired in callose biosynthesis powdery mildew 121

resistant 4 (pmr4) display enhanced resistance to the powdery mildew pathogens 122

Golovinomyces orontii and G cichoracearum (Nishimura et al 2003) These plants 123

have constitutively high SA levels and SA signaling is required for the resistance 124

phenotype (Nishimura et al 2003) Other plants with enhanced resistance to these 125

powdery mildew pathogens include pmr5 a mutant enriched in pectin and pmr6 a 126

mutant with a defect in a pectate lyase-like gene (Vogel et al 2002 Vogel et al 2004) 127

Plants with mutations in cellulose synthases required for primary and secondary wall 128

formation also show increased resistance to pathogens (Ellis and Turner 2001 129

Hernandez-Blanco et al 2007) Enhanced resistance to powdery mildew and 130

Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) in constitutive 131

expression of VSP 1 (cev1) which is mutated in the primary wall cellulose synthase 132

CesA3 was linked to constitutive high JA and ET levels in these plants (Ellis and 133

Turner 2001 Ellis et al 2002a) Interestingly irregular xylem1 (irx1) irx3 and irx5 134

plants which carry mutations in the secondary wall-associated cellulose synthases 135

CesA8 CesA7 and CesA4 showed enhanced resistance to the necrotrophic pathogens 136

Plectosphaerella cucumerina Ralstonia solanacearum and Botrytis cinerea as well as 137

the biotrophic powdery mildew G cichoracearum whereas growth of P cucumerina and 138

R solanacearum pathogens was unaltered in pmr5 pmr6 and cev1 plants (Hernandez-139

Blanco et al 2007) This enhanced resistance of the irx mutants was independent of 140

SA JA or ET signaling but instead involved abscisic acid signaling (Hernandez-Blanco 141

et al 2007) Changes in wall structure are likely monitored by the plants and effects 142

may be counteracted through initiation of defense responses that can lead to altered 143

phytohormone levels 144

Although plant cell walls serve as barriers against pathogen entry many 145

pathogens secrete cell wall-degrading enzymes (CWDEs) and thus are able to 146

successfully infect their host plants (Albersheim et al 1969) For example the B 147

cinerea polygalacturonase Bc-PG1 is required for full virulence of this pathogen and the 148

Page 6 of 54

gene encoding it shows evidence for diversifying selection as would be expected for a 149

virulence gene (ten Have et al 1998 Rowe and Kliebenstein 2007) Secretion of 150

CWDEs can trigger the release of cell wall fragments which can act as damage-151

associated molecular patterns (DAMPs) that activate plant immune responses (Hahn et 152

al 1981 Ferrari et al 2013) Similarly pectin fragments known as oligogalacturonides 153

(OGs) can trigger plant immune responses (Cote and Hahn 1994 Ferrari et al 2013) 154

Such responses include the production of reactive oxygen species (ROS) activation of 155

mitogen-activated protein kinase (MAPK) activation and enhanced expression of 156

defense-related genes such as PAD3 (Ferrari et al 2007 Denoux et al 2008 Galletti 157

et al 2008 Galletti et al 2011) Some of these responses require SA JA and ET 158

whereas others are independent of these phytohormones (Ferrari et al 2007) 159

Here we describe two GAE family members GAE1 and GAE6 whose 160

expression is repressed upon pathogen challenge A gae1 gae6 double mutant has 161

brittle leaves and its walls are reduced in pectin specifically HG and likely RGI 162

Resistance to the bacterial pathogen Pma ES4326 and the fungal necrotroph B cinerea 163

is compromised in gae1 gae6 Callose deposition is reduced in gae1 gae6 plants after 164

treatment with B cinerea isolate Gallo 1 Furthermore these plants show altered 165

activation of immune signaling in response to treatment with macerozyme a 166

polygalacturonase that degrades pectin and they also are hyperresponsive to JA 167

signaling Macerozyme treatment released less soluble likely low molecular weight 168

pectin from isolated gae1 gae6 cell walls than from Col-0 cell walls B cinerea-induced 169

accumulation of soluble likely low molecular weight pectin was also decreased in gae1 170

gae6 plants Additionally gae1 gae6 plants are impaired in macerozyme-induced 171

immunity to B cinerea possibly due to decreased release of OGs from their cell walls 172

173

174

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RESULTS 175

176

GAE1 and GAE6 Expression Is Repressed by Pma ES4326-Induced Immune 177

Signaling 178

Our previous research suggested that the status of pectin specifically pectin 179

modification by methylesterification is important for plant immunity against Pma 180

ES4326 (Bethke et al 2014) Here we investigate whether pectin abundance in 181

addition to its specific structure is also important for plant immunity by taking a closer 182

look at the formation of the pectin precursor UDP-GalA by GAEs 183

184 Microarray experiments showed that expression of GAE1 and GAE6 was

185 repressed in A thaliana (Arabidopsis) plants treated with Pma ES4326 (Wang et al

186 2008) Repression of GAE1 and GAE6 in wild-type Arabidopsis Col-0 plants treated with

187 Pma ES4326 was confirmed by quantitative real time (qRT)-PCR Figure 1) To test if

188 this repression is mediated by immune signaling qRT-PCR was also performed in

189 mutants with blocked immune signaling (Figure 1 and Supplemental Figure 1) 190 Generally immunity to Pma ES4326 requires intact SA signaling Repression of both

191 GAE1 and GAE6 required the presence of the major immune regulators EDS1 and

192 PAD4 required for SA signaling as well as other immune responses (Jirage et al 1999

193 Wiermer et al 2005 Bartsch et al 2006 Wang et al 2008) at both early and late time

194 points The presence of AVRPPHB SUSCEPTIBLE 3 (PBS3) a regulator of many

195 immune responses including SA signaling (Nobuta et al 2007) was also required for

196 repression of GAE1 and GAE6 at an early time point (Figure 1) CALMODULIN-

197 BINDING PROTEIN 60-LIKE a (CBP60a) (a negative regulator of SA synthesis and

198 immunity (Truman et al 2013)) CBP60g and SYSTEMIC ACQUIRED RESISTANCE

199 DEFICIENT 1 (SARD1) (both promote SA biosynthesis and regulation of other immune

200 responses (Wang et al 2011)) SALICYLIC ACID INDUCTION DEFICIENT 2 SID2

201 (encodes a biosynthetic enzyme for SA (Wildermuth et al 2001)) and

202 NONEXPRESSER OF PR GENES 1 NPR1 (a regulator of the majority of SA-induced

203 immune signaling (Cao et al 1997 Yan and Dong 2014)) were not required for this

204 repression (Figure 1 and Supplemental Figure1) Thus repression of GAE1 and GAE6

Page 8 of 54

205 requires a regulatory function that is dependent on EDS1 and PAD4 partially

206 dependent on PBS3 but independent of SA

Page 9 of 54

There are six GAE family members in Arabidopsis GAE2 expression was 207

induced by Pma ES4326 in the absence of PAD4 or EDS1 GAE3 expression was 208

unaltered GAE4 was repressed early and GAE5 expression was induced in all 209

genotypes (Supplemental Figure 2) The similarity of the immune-regulated expression 210

of GAE1 and GAE6 suggested a redundant role of these genes in Arabidopsis immunity 211

to Pma ES4326 212

213

Resistance to Pma ES4326 Is Compromised in gae1 gae6 and gae6 Plants 214

To study the roles of GAE1 and GAE6 in plant immunity in more detail plants 215

with T-DNA insertion mutations in GAE1 and GAE6 were obtained from the SALK 216

collection as described in the Accession Numbers section of Methods We identified two 217

GAE6 knock-out alleles gae6-1 and gae6-2 and a T-DNA insertion in GAE1 gae1-1 218

that caused reduced expression of this gene (Supplemental Figure 3) A double mutant 219

was created by crossing gae1-1 and gae6-1 Both gae6 and gae1 gae6 plants were 220

more susceptible to Pma ES4326 than wild-type Col-0 and gae1 plants supporting a 221

role of GAE6 in plant immunity to Pma ES4326 (Figure 2) 222

223

gae6 and gae1 gae6 Plants Have Brittle Leaves 224

While inoculating leaves of gae1 gae6 plants we noticed that the petioles and 225

midribs of these plants tended to break easily when leaves were bent against the 226

proximal-distal axis of the leaves (Figure 3 see Supplemental Figure 4D to F) They 227

tended to break less often when twisted around the proximal-distal axis To quantify 228

this phenotype leaves were bent by 180deg (Figure 3A) and breaking of midribs or 229

petioles was recorded Most gae1 gae6 leaves broke whereas fewer leaves of gae6 230

plants and very few leaves of wild-type Col-0 and gae1 plants did (Figure 3B and 231

Supplemental Figure 4C) Although gae1 gae6 plants had slightly shorter leaves and 232

petioles than wild type plants (Supplemental Figure 4A and B) no correlation between 233

leaf size or petiole length and breaking frequency was detected We also investigated 234

tissue integrity by monitoring ion leakage Interestingly when leaves were inoculated 235

with deionized water leaf discs of gae1 gae6 leaked more ions than wild-type Col-0 236

discs (Figure 3C) suggesting that the increased brittleness is correlated with a 237

Page 10 of 54

_____________________________________________________________________________

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Cell Walls of gae6 and gae1 gae6 Plants Have Lower Levels of Pectin 243

GalA is a major component of pectin We analyzed wall monosaccharide 244

composition in gae mutants to investigate whether the increased brittleness was 245

associated with an altered wall composition of gae leaves To approximate pectin 246

content of gae mutant walls the total uronic acid concentration was measured using a 247

simple colorimetric assay (Filisetti-Cozzi and Carpita 1991 van den Hoogen et al 248

1998) Total uronic acid was reduced in gae6-2 strongly reduced in gae1 gae6 plants 249

but was indistinguishable from wild-type Col-0 in gae6-1 and gae1 (Supplemental 250

Figure 5) This suggested that gae1 gae6 and possibly gae6 plants have reduced 251

pectin 252

To investigate the cell wall composition in leaves of gae plants in more detail we 253

measured the levels of seven neutral monosaccharides the acidic sugars GalA (the 254

monomeric subunit of the pectin backbone (Harholt et al 2010)) and GlcA (Figure 4) 255

We also estimated the crystalline cellulose content (Updegraff 1969) Cell walls of both 256

gae6 mutants and gae1 gae6 were reduced in GalA (Figure 4A) In gae1 gae6 GalA 257

content in cell walls was reduced by more than 40 compared to the wild-type level 258

GlcA levels were indistinguishable between mutants and wild-type (Figure 4B) 259

Cellulose content was higher in gae6-2 and gae1 gae6 walls likely due to the loss of 260

pectins (Figure 4C) Cell walls of gae1 gae6 plants were also enriched in all neutral 261

sugars measured presumably due to the substantial proportional loss of GalA (Figure 262

4D) The levels of most wall monosaccharides were even higher in the gae1 gae6 263

double mutant with the exception of Rha which did not exhibit a significant increase 264

compared to the gae6 single mutants The absence of a compensational increase in 265

Rha suggested that RGI the dominant Rha contributor to the wall may be considered 266

reduced in gae1 gae6 mutants compared to wild-type albeit not to such an extent as 267

HG 268

238 structural defect that promotes increased ion leakage Taken together these results

239 showed that loss of GAE6 causes brittle leaves and additional losss of GAE1 strongly

240 enhances this phenotype We conclude that GAE1 and GAE6 contribute to normal leaf

241 flexibility 242

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gae6 cell walls are reduced in HG Using the same samples we detected no difference 274

from wild type in binding of LM5 which recognizes a tetramer of (1-4)-β-D-galactans 275

found in RGI (Jones et al 1997) LM6 which recognizes a linear pentasaccharide in (1-276

To further investigate which pectic components were altered in gae1 gae6 dot 269

blot experiments using pectin isolated from the various genotypes were performed Both 270

LM19 and LM20 antibodies recognize HG (Verhertbruggen et al 2009a) As expected 271

LM19 binding was reduced in gae6-1 and gae1 gae6 (Figure 5A and B) and LM20 272

binding was reduced in gae1 gae6 (Figure 5C and D) This result confirmed that gae1 273

277 5)-α-L-arabinans found in RGI (Willats et al 1998 Verhertbruggen et al 2009b) LM8

278 which recognizes xylogalacturonan (Willats et al 2004) or CCRC-M7 which

279 recognizes an arabinosylated (1-6)-β-D-galactan epitope occurring on RGI (Puhlmann et

Page 13 of 54

Page 14 of 54

al 1994 Steffan et al 1995 Pattathil et al 2010) (Supplemental Figure 6) 280

Collectively these cell wall composition data show that gae1 gae6 plants are reduced in 281

HG Due to the strong proportional reduction of HG in the walls levels of all other 282

components should be higher However Rha indicative of RGI was not higher in gae1 283

gae6 suggesting that RGI may also be reduced in gae1 gae6 walls 284

285

GAE1 and GAE6 Are Required for Immunity to Specific B cinerea Isolates 286

We hypothesized that plant immunity to B cinerea might be affected by pectin 287

content in the host cell wall because this pathogen requires polygalacturonases and 288

pectin methylesterases for full virulence (ten Have et al 1998 Valette-Collet et al 289

2003 Kars et al 2005b Espino et al 2010) Because Bcinerea is genetically diverse 290

and has a high diversity of polygalacturonases (Rowe and Kliebenstein 2007) we 291

measured relative fungal growth at both 2 and 3 d post-inoculation (dpi) using ten 292

different B cinerea isolates (Figure 6) The B cinerea isolates Fresa 525 and Gallo 1 293

showed enhanced virulence on gae1 gae6 plants at both time points whereas KT and 294

UK Razz showed enhanced virulence in gae1 gae6 plants at 3 but not 2 d dpi (Figure 295

6A) We did not detect any significant changes in relative fungal growth between wild-296

type Col-0 and gae1 gae6 plants when using Acacia Apple 517 DN Grape Pepper or 297

Rasp isolates of B cinerea Therefore we continued our study using Gallo 1 and Fresa 298

525 We also included Pepper for comparison as this isolate has been used in a large-299

scale analysis of plant gene expression in response to B cinerea infection (Windram et 300

al 2012) 301

We next investigated whether expression of GAE1 and GAE6 was affected by 302

challenge with B cinerea Inoculation of wild-type Col-0 plants with any isolate 303

repressed GAE1 and GAE6 expression (Figure 6B and C) and this repression was 304

generally independent of PAD4 (Figure 6B and C) Expression of GAE2 and GAE3 was 305

unaltered by B cinerea treatment GAE4 was repressed only by Fresa 525 treatment 306

while GAE5 expression was enhanced with all three isolates tested (Supplemental 307

Figure 7) As seen for infection with Pma ES4326 the expression of GAE1 and GAE6 308

was repressed upon B cinerea treatment This indicates a role for GAE1 and GAE6 in 309

the immune response to B cinerea 310

Page 15 of 54

Page 16 of 54

Because the amount of pectin substrate for pathogen polygalacturonases is 313

reduced we hypothesized that immunity phenotypes of gae1 gae6 plants may result 314

from reduced generation of OGs by these enzymes In turn the reduced OGs could 315

lead to reduced immune responses To test this we used macerozyme a commercial 316

pectinase as a proxy for pathogen pectinases We isolated cell walls as a subcellular 317

fraction known as alcohol insoluble residue (AIR Gille et al 2009) This was treated 318

with macerozyme and tested for release of soluble likely low molecular weight pectin 319

fragments (Figure 7) Treatment with 025 macerozyme released significantly more 320

uronic acid from wild-type Col-0 AIR relative to gae1 gae6 AIR (Figure 7B) To test 321

activity of these pectin-containing solutions we infiltrated them into wild-type Col-0 322

plants and measured PAD3 expression PAD3 is required for synthesis of the 323

phytoalexin camalexin (Zhou et al 1999) and its expression is known to be induced by 324

OG treatment (Ferrari et al 2007) Fractions from macerozyme treated Col-0 walls 325

induced significantly more PAD3 expression than fractions derived from gae1 gae6 326

walls suggesting that macerozyme treatment did indeed release more active soluble 327

uronic acid possibly OGs from wild-type Col-0 walls than from gae1 gae6 walls (Figure 328

7C) 329

Next we studied the effect of macerozyme treatment on expression of PAD3 in 330

intact plants (Figure 8) This treatment did not lead to any visible leaf damage 331

(Supplemental Figure 8) In wild-type Col-0 PAD3 expression was induced three h after 332

treatment with 001 macerozyme (Figure 8A) By contrast no macerozyme-induced 333

induction of PAD3 expression was detected in gae1 gae6 (Figure 8A) However OG 334

treatment induced the expression of PAD3 in both genotypes indicating that gae1 gae6 335

plants do respond to OGs (Figure 8B) Macerozyme treatment also induced expression 336

of the SA marker genes PATHOGENESIS RELATED 1 (PR1) and SID2 in Col-0 but 337

not in gae1 gae6 (Figure 9 panel A and B) PR1 was not significantly induced by 338

purified OGs (Figure 9C) at the time and concentration tested but SID2 was induced by 339

OG treatment in both genotypes (Figure 9D) However the induction was stronger in 340

wild-type Col-0 In addition the JA markers JASMONATE-ZIM-DOMAIN PROTEIN 5 341

(JAZ5) and JAZ10 were induced by macerozyme and OG treatment in both Col-0 and 342

gae1 gae6 (Figure 10A and Supplemental Figure 11) Interestingly expression of JAZ5 343

312 Pectinase-Induced Immune Signaling Is Altered in gae1 gae6 Plants

Page 17 of 54

Page 18 of 54

Page 19 of 54

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344

345

346

and JAZ10 was already strongly induced in gae1 gae6 after mock inoculation (Figure 10

and Supplemental Figure 11) We observed no hyperresponsive induction by mock

treatment of PLANT DEFENSIN 12 (PDF12) a necrotrophy and late JA signaling

347 marker gene (Supplemental Figure 12A-D) PDF12 was also not induced by

348 macerozyme or OG treatments (Supplemental Figure 12A-D) This suggests that early

349 JA signaling is induced by mock-treatment in gae1 gae6 Because it is known that JA

350 and SA signaling act antagonistically (Pieterse et al 2012) the strong up-regulation of

351 JA-induced immune signaling in gae1 gae6 might explain the lower OG-induced SID2

352 expression in this genotype

353 To test whether other immune responses are altered in gae1 gae6 plants we

354 analyzed Mitogen-activated protein kinase (MAPK) activity following macerozyme and

355 OG treatments OGs are known to activate the MAPKs MPK3 and MPK6 in Arabidopsis

Page 21 of 54

(Galletti et al 2011) Macerozyme treatment activated MPK3 and MPK6 in wild-type 356

Col-0 whereas MAPK activation in gae1 gae6 was slightly reduced in four out of five 357

experiments performed (Supplemental Figure 13) By contrast OG-induced MAPK 358

activity was similar or increased in gae1 gae6 in four out of five experiments 359

(Supplemental Figure 13) Additionally mock treatment induced MAPK activity in gae1 360

gae6 more strongly than it did in wild-type Col-0 in four out of five experiments 361

performed (Fig S13) Collectively these results illustrate that activation of several 362

immune signaling componentsmdashcamalexin synthesis SA and JA signaling and 363

possibly MAPK activationmdashby macerozyme is substantially altered in gae1 gae6 This 364

suggests that release of soluble pectin fragments possibly OGs by pectinolytic 365

enzymes might be reduced in gae1 gae6 plants while recognition of OGs is intact 366

367

Following B cinerea Infection Production of Soluble Pectin and Deposition of 368

Callose Are Reduced in gae1 gae6 Plants 369

Although pad3 plants were more susceptible to the B cinerea isolates Fresa 525 370

and Gallo 1 which also grew better on gae1 gae6 growth of the isolate Pepper was 371

similar on Col-0 and pad3 (Supplemental Figure 9A) However we did not detect any 372

significant changes in camalexin production after treatment with B cinerea isolate Gallo 373

1 and Fresa 525 in gae1 gae6 (Figure 8D) Neither did we detect changes in PAD3 374

expression after treatment with any B cinerea isolate in gae1 gae6 (Figure 8E) 375

Additionally SID2 and PR1 were similarly induced by all B cinerea isolates in both Col-376

0 and gae1 gae6 (Figure 9E and F) and sid2 plants which are deficient for pathogen-377

induced SA biosynthesis did not show any differences in relative B cinerea growth 378

(Supplemental Figure 9B) Expression of JAZ10 was induced more strongly by Gallo 1 379

treatment in gae1 gae6 than in wild-type Col-0 but not by Fresa 525 or Pepper (Fig 380

10C) while dde2 plants deficient for JA biosynthesis showed enhanced susceptibility to 381

all B cinerea isolates tested (Supplemental Figure 9B) Curiously exogenous 382

application of MeJA did not lead to altered relative fungal growth of B cinerea 383

(Supplemental Figure 9C) and cev1 isoxaben resistant 1 (ixr1) plants which have 384

constitutively high JA levels (Ellis and Turner 2001) did not show any changes in 385

susceptibility to the B cinerea isolates tested (Supplemental Figure 9D) Hence neither 386

Page 22 of 54

differences in B cinerea isolate susceptibility to camalexin nor altered SA or JA 387

signaling by themselves explained the enhanced susceptibility of gae1 gae6 to these B 388

cinerea isolates 389

Hydrogen peroxide accumulation in response to B cinerea isolates Fresa 525 390

Gallo 1 and Pepper was not obviously altered in gae1 gae6 plants (Supplemental Figure 391

14) However callose deposition upon treatment with B cinerea isolate Gallo 1 was 392

strongly reduced in gae1 gae6 (Figure 11) It was previously shown that OG treatment 393

induces callose deposition (Denoux et al 2008) suggesting that the reduction in 394

callose deposition might be due to reduced OG release in gae1 gae6 It is possible that 395

this reduction in callose deposition contributes to the reduced immunity of gae1 gae6 396

plants to B cinerea isolate Gallo 1 397

Because macerozyme could be used to release soluble likely low molecular 398

weight pectin from isolated cell walls we next investigated whether infection with B 399

cinerea was accompanied by accumulation of soluble pectin as well We prepared cell 400

walls (AIR fraction) from leaves infected with B cinerea incubated these cell walls with 401

water and measured uronic acid content of the extracts B cinerea treatment did in fact 402

lead to increased accumulation of soluble pectin in wild-type Col-0 plants when 403

compared to mock-treated samples (Figure 12) No such increased accumulation of 404

water-soluble pectin fractions was observed in gae1 gae6 (Figure 12) 405

406

Macerozyme-Induced Pattern-Triggered Immunity against B cinerea Is Abolished 407

in gae1 gae6 408

It was previously shown that OG treatment can induce patternndashtriggered 409

immunity (PTI) to B cinerea (Ferrari et al 2007) We tested macerozyme treatment to 410

determine whether it could also induce PTI to the B cinerea isolate Gallo 1 (Figure 13) 411

Indeed pretreatment with 001 macerozyme for 24 h led to reduced B cinerea growth 412

in wild-type Col-0 but not gae1 gae6 plants (Figure 13A) Interestingly mock and 413

macerozyme pretreated gae1 gae6 plants exhibited similar growth of B cinerea Gallo 1 414

as did macerozyme-treated Col-0 plants This result is likely due to activation of JA 415

signaling in gae1 gae6 during the pretreatment procedure (Figure 13C and D) OG-416

induced PTI was observed in both genotypes when using a high dose of OGs (Figure 417

Page 23 of 54

13B) whereas a lower dose of OGs only induced PTI in Col-0 (Supplemental Figure 418

15) This suggests that OG perception in gae1 gae6 at least above a certain threshold 419

is unaltered while release of immunity-activating pectin fragments possibly OGs is 420

421 reduced We conclude that immune responses induced by macerozyme treatment

422 which do not occur in gae1 gae6 plants are important for plant immunity to the B

423 cinerea isolate Gallo 1

Page 24 of 54

____________________________________________________________________________

Page 25 of 54

DISCUSSION 426

Here we showed that loss of GAE1 and GAE6 leads to a dramatic reduction in 427

pectin content in the cell wall (Figure 4) likely causing the brittleness of leaves that we 428

observed (Figure 3) Petioles and midribs of gae1 gae6 plants broke easily when bent 429

and inoculation of leaves with deionized water resulted in increased ion leakage 430

possibly due either to microscopic fractures of the cell wall induced by inoculation stress 431

or to a reduced capability of these walls to withstand increased turgor pressure The 432

increased brittleness was associated with altered cell wall composition In our analysis 433

the GalA content of gae1 gae6 leaf walls was reduced by more than 40 and the total 434

uronic acids by over 60 (Figure 4 and Supplemental Figure 5) This is a major change 435

in the overall cell wall composition considering that pectin makes up about 50 of the 436

plant cell walls found in Arabidopsis leaves (Zablackis et al 1995 Nobuta et al 2007 437

Harholt et al 2010) Interestingly quasimodo1 (qua1) plants which have a mutation in 438

a glycosyltransferase family 8 gene are reduced in GalA by 25 and show reduced 439

binding of HG specific antibodies (Bouton et al 2002) 440

Furthermore qua2 plants which have a mutation in a putative methyltransferase 441

have about 13 less total GalA and lack 50 of cell wall HG (Mouille et al 2007) 442

Likewise cgr2 cgr3 plants carrying mutations in another class of Golgi-localized 443

putative methyl-transferases have leaf cell walls that are reduced by about 40 in 444

uronic acid (Kim et al 2015) All three mutants are severely dwarfed (Bouton et al 445

2002 Mouille et al 2007 Kim et al 2015) Additionally qua1 and qua2 mutants show 446

a distorted overall shape likely due to reduced cell cohesion and are extremely fragile 447

(Bouton et al 2002 Mouille et al 2007) By contrast gae1 gae6 plants with a similar 448

strong reduction in GalA and total uronic acids have an overall normal shape and are 449

only slightly smaller than wild-type Col-0 (Supplemental Figure 4) In the wall analysis 450

performed here we did not detect changes in other wall polysaccharides or structures 451

that might compensate for the reduction of pectin in gae1 gae6 It is possible the severe 452

morphological alterations in qua2 and cgr2 cgr3 plants are due to reduction in pectin 453

methylesterification which is unaffected in gae1 gae6 It was speculated that quality 454

control mechanisms in the Golgi prevent secretion of inefficiently esterified pectin in 455

cgr2 cgr3 (Kim et al 2015) suggesting that the Golgi of these plants might over-456

Page 26 of 54

accumulate pectin and that this may contribute to the severe dwarf phenotype of these 457

plants The gae1 gae6 plants described here provide an opportunity to study the effects 458

of reduction of pectin in plants that do not show severe morphological phenotypes 459

We found that gae1 gae6 plants are also compromised in resistance to B 460

cinerea and Pma ES4326 indicating that loss of pectin reduces immunity to these 461

pathogens (Figure 2 and 6) We found that gae1 gae6 plants were slightly more 462

susceptible to Pma ES4326 but showed a strong increase in susceptibility to specific B 463

cinerea isolates This was not unexpected because B cinerea is known to interact with 464

pectin as part of its infection strategy It secretes various pectin-degrading enzymes 465

early during infection and requires some of these enzymes including PG1 PG2 and 466

PME1 for full virulence (ten Have et al 1998 Valette-Collet et al 2003 Kars et al 467

2005b Espino et al 2010) Additionally Arabidopsis plants expressing an antisense 468

construct of the polygalacturonase inhibitor PGIP1 are significantly more susceptible to 469

B cinerea (Ferrari et al 2006) On the other hand Pma ES4326 is not known to470

require cell wall degrading enzymes for full virulence even though it encodes two 471

pectate lyase genes in its genome (PMA4326_07094 and PMA4326_04621 472

wwwbacteriaensemblorg) B cinerea isolates are also known to be genetically quite 473

diverse in their polygalacturonase (PG) loci and genetic variability in PG2 is associated 474

with varying growth on pectin containing media (Rowe and Kliebenstein 2007) 475

Furthermore the contribution of PG1 and PG2 to B cinerea virulence seems to depend 476

on the specific B cinerea isolate and the host species (Kars et al 2005a Zhang and 477

Van Kan 2013) B cinerea isolates are also known to be differently sensitive to the anti-478

microbial secondary metabolite camalexin (Kliebenstein et al 2005) In line with these 479

observations the susceptibility of gae1 gae6 plants differed depending on the specific 480

B cinerea isolate Whereas Fresa 525 Gallo 1 UK Razz and KT grew better in gae1481

gae6 we detected no difference in Acacia Apple 517 DN Grape Pepper or Rasp 482

growth (Figure 6) 483

B cinerea was shown to metabolize pectin and to possibly utilize GalA as a484

carbon source during in planta growth (Zhang et al 2011 Zhang and Van Kan 2013) 485

Our data show that gae1 gae6 plants which are reduced in GalA content did allow 486

enhanced growth of specific B cinerea isolates but no reduction in growth of any B 487

Page 27 of 54

cinerea isolates tested was found This suggests that utilization of pectin as a carbon 488

source was not a contributing factor to alterations in B cinerea susceptibility It is 489

possible that changes in the cell wall composition affect the structural integrity of the 490

wall and affect hyphal penetration of specific B cinerea isolates However OGs (small 491

HG fragments) are likely released during the infection process (Ferrari et al 2013) 492

OGs function as DAMPs and initiate immune responses in Arabidopsis (Ferrari et al 493

2013) Treatment of Arabidopsis with OGs having a degree of polymerization (DP) of 494

10-15 sugar subunits was previously shown to enhance resistance to B cinerea (Ferrari495

et al 2007) We showed that pretreatment with OGs or macerozyme induced enhanced 496

immunity to B cinerea isolate Gallo 1 in wild-type Col-0 but only OGs and not 497

macerozyme induced immunity in gae1 gae6 (Figure 13) Our data suggest gae1 gae6 498

plants which have reduced pectin content may release less OGs and that this 499

reduction leads to the increased susceptibility to B cinerea 500

Consistently we found that macerozyme treatment released less soluble pectin 501

from gae1 gae6 cell walls than from wild-type walls Fractions containing soluble pectin 502

released from gae1 gae6 were also less active in inducing PAD3 gene expression than 503

the fractions released from wild-type walls (Figure 7) We also showed that less soluble 504

pectin accumulated in gae1 gae6 than in wild-type plants 48 h after treatment with B 505

cinerea (Figure 12) 506

We observed that loss of pectin leads to alterations in A thaliana immune 507

signaling Macerozyme treatment induced expression of PAD3 and the SA marker 508

genes PR1 and SID2 in wild-type Col-0 but not in gae1 gae6 whereas OG treatment 509

led to increased PAD3 and SID2 expression in both wild-type and gae1 gae6 (Figure 8 510

and 9) This suggests that although release of active soluble pectin in these plants 511

might be altered OG recognition is intact In fact cell walls isolated from qua1 another 512

mutant known to be reduced in HG released less OGs (DP1-6) than did wild-type cell 513

walls upon endopolygalacturonase treatment (Bouton et al 2002) This was also true 514

for gae1 gae6 cell walls as macerozyme treatment of isolated gae1 gae6 cell walls 515

released less soluble pectin than treatment of Col-0 cell walls Because the fraction 516

from gae1 gae6 plants was also less active in inducing PAD3 expression (Figure 7) it 517

seems possible that biologically active OGs were the active component inducing PAD3 518

Page 28 of 54

expression Interestingly expression of the JA marker genes JAZ5 and JAZ10 was 519

higher in gae1 gae6 than in wild-type mock-inoculated plants but was comparable to 520

wild-type in untreated plants suggesting that JA signaling in gae1 gae6 is 521

hyperresponsive (Figure 10 and Supplemental Figure 11) This induction of JA signaling 522

might be due to wounding stress incurred during the inoculation process JA and SA 523

signaling generally act antagonistically (Pieterse et al 2012) Increased JA signaling 524

activity might hence explain why mock- and macerozyme-treated gae1 gae6 plants 525

exhibited reduced expression of the SA marker PR1 and why OG treatment induced 526

expression of SID2 another SA marker to a lower extent in gae1 gae6 than in wild-type 527

plants (Figure 9) 528

No single immune signaling component was found to be the sole contributor to 529

the compromised immunity of gae1 gae6 to specific B cinerea isolates such as Gallo 1 530

and Fresa 525 These isolates grew better on pad3 while growth of Pepper another B 531

cinerea isolate that did not show altered virulence on gae1 gae6 was unaltered 532

(Supplemental Figure 9) This suggested that differences in camalexin sensitivity of 533

these B cinerea isolates might account for the differences in gae1 gae6 susceptibility 534

However camalexin accumulation and PAD3 expression was the same for wild-type 535

Col-0 and gae1 gae6 for the B cinerea isolates Fresa 525 and Gallo 1 (Figure 8) which 536

showed enhanced growth on gae1 gae6 plants It should be noted that B cinerea 537

virulence is often estimated by comparing lesion sizes Lesion size and measurements 538

of relative fungal growth by qPCR do not always show the same results For example 539

although Denby et al (2004) showed that Pepper causes larger lesions on pad3 than on 540

wild-type this study showed that relative fungal growth of Pepper on pad3 was 541

unaltered 542

We observed that callose deposition was strongly reduced in gae1 gae6 plants 543

72 h after treatment with B cinerea isolate Gallo 1 (Figure 11) possibly due to reduced 544

release of active soluble pectin Interestingly OG-induced PTI to an unspecified isolate 545

of B cinerea was intact in the pmr4 mutant which does not produce callose (Galletti et 546

al 2008) Due to the high SA levels in pmr4 plants (Nishimura et al 2003) and the fact 547

that different B cinerea isolates vary both in induction of and reaction to defense 548

responses (Kliebenstein et al 2005 Rowe et al 2010) we could not determine 549

Page 29 of 54

whether reduced callose deposition causes the increased susceptibility of gae1 gae6 to 550

B cinerea isolate Gallo 1551

We did not observe any changes in B cinerea growth either on cev1 ixr1 plants 552

which are known to have constitutively high JA and OPDA levels (Ellis et al 2002b) or 553

on plants exogenously treated with MeJA (Supplemental Figure 9) Because plants 554

were kept uncovered for 3 h after MeJA treatment it is unclear how much MeJA was 555

bioavailable in the plant However JAZ10 expression was induced 3 h after spraying 556

with MeJA (Supplemental Figure 10) Because pathogen growth generally depends on 557

the combination of a multitude of immune signaling events we conclude that multiple 558

changes to the immune signaling in gae1 gae6 together account for the altered B 559

cinerea growth phenotypes detected 560

We showed that expression of GAE1 and GAE6 is repressed by the bacterial 561

hemibiotroph Pma ES4326 This repression did not occur in an eds1 or pad4 562

background or in pbs3 at the 24-h time point (Figure 1 and Supplemental Figure 1) 563

During the Arabidopsis - Pma ES4326 interaction the expression of many defense 564

related genes is altered in a PAD4EDS1 dependent fashion (Wang et al 2008) Some 565

of these expression changes require SA CBP60g and SARD1 are thought to act 566

downstream of PAD4 and EDS1 but upstream of SA-dependent responses (Wang et 567

al 2011) PBS3 is also thought to act downstream of PAD4 and EDS1 and upstream of 568

SA but the set of genes whose expression it affects is only partially overlapping with 569

those affected by CBP60gSARD1 (Wang et al 2008 Wang et al 2011) Thus GAE1 570

and GAE6 repression is regulated by the part of the immune signaling network that 571

requires PAD4 EDS1 and PBS3 but is independent of CBP60gSARD1 and SA 572

Because EDS1 PAD4 and PBS3 are important components of the plant immune 573

signaling network (Wiermer et al 2005 Nobuta et al 2007) one might conclude that 574

repression of GAE1 and GAE6 may be part of the plant immune response to Pma 575

ES4326 B cinerea treatment also reduced GAE1 and GAE6 expression but this was 576

mostly independent of PAD4 (Figure 6) while both Pma ES4326 and B cinerea repress 577

GAE1 and GAE6 expression the host signaling mechanisms responsible likely differ 578

In summary our data show that Arabidopsis GAE1 and GAE6 are required for 579

pectin biosynthesis leaf flexibility and immunity to Pma ES4326 and to specific B 580

Page 30 of 54

cinerea isolates We used macerozyme a commercial pectinase to mimic the effect of 581

B cinerea pectinases Macerozyme treatment released less pectin from gae1 gae6 cell582

walls and induced less immune signaling in gae1 gae6 plants Less water soluble pectin 583

also accumulated in B cinerea infected gae1 gae6 plants and macerozyme-induced 584

immunity to B cinerea isolate Gallo 1 was reduced in these plants We conclude that 585

the reduced pectin content in these mutants may lead to reduced release of soluble 586

pectin including active OGs during pathogen attack and that this compromises 587

immunity 588

589

590

Page 31 of 54

METHODS 591

Plant Materials and Growth Conditions 592

Wild-type Columbia (Col-0) and all mutant plants (in Col-0 background) were 593

grown on sterilized BM2 germinating mix (Berger Inc Quebec Canada) in a controlled 594

environment chamber (Conviron Pembina ND USA) with a 12-h photoperiod under 595

100 microM m-2 s-1 fluorescent illumination at 22degC and 75 relative humidity Germplasm 596

used is described in the Accession Numbers section at the end of Methods 597

598

Data Analysis and Replicates 599

For biological replicates samples were obtained from separate plant tissues and 600

in many cases from plants grown in different flats In some experiments multiple 601

technical replicates were measured per biological replicate For statistical analysis all 602

technical replicates were averaged and the averages were considered one biological 603

replicate Independent experiments were performed at different times and often 604

consisted of multiple biological replicates per experiment 605

Data analysis was performed using mixed linear models in the R programming 606

environment (Bates et al 2015) unless otherwise indicated Generally genotypes 607

treatments and time points were used as fixed effects and replicate-specific effects like 608

different pots flats etc were considered random effects We reported p-values 609

obtained from the mixed linear model when less than 10 comparisons were performed 610

and q-values for more than 10 comparisons to correct for multiple comparison testing 611

Q-values were calculated using the Benjamini-Hochberg procedure (Benjamini and612

Hochberg 1995) unless specified otherwise 613

614

Pathogen Strains Growth Conditions and Pathogen Growth Assays 615

Growth of Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) and 616

bacterial growth assays were performed as previously described (Bethke et al 2014) 617

For qRT-PCR assays four week-old plants were inoculated with bacteria (OD600 = 618

0002) or mock (5 mM MgSO4) At least four leaves from two plants were collected at 619

the indicated time points per biological replicate 620

Page 32 of 54

Prior to each experiment Botrytis cinerea isolates were grown on 1x PDA 621

medium (Difco) for 10 d at room temperature All isolates used have been described 622

previously (Rowe and Kliebenstein 2007) The spores were washed from the surface of 623

the plate using an inoculation medium (1x Gamborgrsquos B-5 basal salt mixture (Sigma-624

Aldrich G5768) 2 (wv) glucose 10 mM phosphate buffer pH 64) and fungal hyphae 625

were removed from the suspension by filtering through four layers of cheesecloth The 626

concentration of spores was determined using a hemocytometer and adjusted to 25 x 627

105 spores mL-1 Four week-old Arabidopsis plants were inoculated by placing one 10-628

microL droplet of the B cinerea spore solution or inoculation medium (mock) on the adaxial 629

leaf surface of fully expanded leaves Inoculated plants were kept at 100 relative 630

humidity Infection sites were collected at various time points using a cork borer For 631

qRT-PCR assays at least four leaf discs from one plant were combined for each 632

biological sample For B cinerea growth assays at least 12 leaf discs from three to six 633

plants were pooled per biological sample Relative fungal growth was determined by 634

measuring the abundance of a fungal gene relative to a plant gene as described below 635

In brief DNA from infected tissue was extracted and equal amounts of total DNA were 636

used to perform qPCR reactions using the B cinerea cutinase A and the Arabidopsis 637

SK11 gene respectively Primers were described in (Gachon and Saindrenan 2004) 638

and are listed in Supplemental Table 1 Two technical replicates were combined for 639

each biological replicate B cinerea is a necrotrophic fungal pathogen and may destroy 640

plant DNA as disease progresses However decreases in the amount of plant DNA 641

over the course of our experiments were only observed at late time points with very 642

susceptible plants so the qPCR assay is generally a reasonable estimate of fungal 643

biomass (Supplemental Figure 16) 644

645

Treatment with Macerozyme or OGs 646

For PTI assays four week-old plants either were inoculated with 001 (wv) 647

macerozyme R-10 (Yakult) a Rhizopus polygalacturonase (EC 32115) that possesses 648

high pectinase and hemicellulase activity or were mock inoculated using heat-649

inactivated macerozyme Other similar plants were either inoculated with 250 or 500 microg 650

mL-1 OGs or mock inoculated with water One d later plants were infected with B 651

Page 33 of 54

cinerea and samples were collected immediately (0 dpi) and two or three d later To 652

investigate the role of MeJA on B cinerea growth plants were sprayed with 1mM MeJA 653

in water with 001 (vv) Tween 20 Covers were removed so that leaves could dry off 654

before plants were inoculated with B cinerea three h later Samples were harvested at 655

the indicated time points For expression analysis and MAPK activation assays plants 656

were either inoculated with 100 microg mL-1 OGs or mock inoculated with water OGs used 657

for experiments described in Figures 8 to 10 and Supplemental Figure 12 were a kind 658

gift from Simone Ferrari OGs used in experiments described in Figure 13 and 659

Supplemental Figures 11 13 and 15 were prepared as previously described (Kohorn et 660

al 2014) 661

662

Expression Analysis 663

Leaf tissue from inoculated plants was harvested at the indicated time points 664

flash frozen pulverized and RNA extracted using Trizol (Invitrogen) Quantitative RT-665

PCR was performed using the SuperScript III Platinum SYBR Green One-Step 666

quantitative RT-PCR kit (Invitrogen) and a Lightcycler 480 Real-Time PCR system 667

(Roche) as previously described (Truman and Glazebrook 2012) In brief equal 668

amounts of total RNA and a gene specific primer were used for each reaction The 669

crossing point (Cp) was calculated using the 2nd derivative max method provided with 670

the Lightcycler software for each amplification curve Each reaction was run with two 671

technical replicates and the Cp values for these replicates were averaged ACTIN2 was 672

used as a stably expressed reference gene At least three biological replicates each 673

were performed and analyzed using a mixed-linear model RT-PCR was performed to 674

verify T-DNA insertion lines using the One-Step RT-PCR kit (Qiagen) according to the 675

manufacturerrsquos instructions The primers used can be found in Supplemental Table 1 676

677

Electrolyte Leakage Assay 678

Fully expanded leaves of four week-old plants were inoculated with water (Milli-Q 679

grade) using a needleless syringe Leaf discs were collected using a cork borer and 680

three leaf discs from one leaf were placed with the adaxial surface down onto 2 mL 681

water in a 12-well cell culture plate Conductivity was measured using a Horiba B-173 682

Page 34 of 54

conductivity meter after 30 min and then the water was replaced Conductivity was 683

measured again 30 min later (1 h total) as indicated in Figure 3C Six biological 684

replicates each were performed in three independent experiments and means and SEs 685

were calculated Two-sided t-tests were performed for each time point 686

687

DAB and Aniline Blue Staining 688

Leaves from four week-old plants were either mock inoculated with B cinerea 689

inoculation medium or inoculated with 10 microL droplets of a B cinerea spore solution (25 690

x 105 spores mL-1) of the indicated isolates Samples were collected 72 h later For 33prime-691

diaminobenzidine (DAB) staining two to five inoculated leaves were vacuum infiltrated 692

with 33prime-Diaminobenzidine (1 mg mL-1 in water pH 38) solution and stained overnight 693

in the dark Leaves were destained using 100 ethanol at 65degC and then transferred to 694

50 (vv) glycerol and photographed 695

Callose was visualized by staining leaves with aniline blue as previously 696

described (Adam and Somerville 1996) Briefly leaves were vacuum infiltrated with 697

alcoholic lactophenol (one volume of phenolglycerollactic acidwater (1111) mixed 698

with two volumes of ethanol) and destained at 65degC Next leaves were moved through 699

an alcohol gradient (50 20 and 10 (vv) ethanol) into water The destained leaves 700

were stained for 30 min in 150 mM K2HPO4 (pH 95) with 001 (wv) aniline blue 701

Samples were mounted in water and examined using a Nikon Eclipse Ni-U microscope 702

at 10x magnification using a DAPI filter (excitation at 325-375 nm emission at 435-485 703

nm) For callose quantification six 200 microm x 200 microm squares were counted for each 704

sample type Callose deposits per mm2 were calculated and data analyzed using a t-705

test Note that there is an inherent uncertainty associated with such counts because 706

callose deposits may not all be in focus at the same time 707

708

MAPK Activity Assay 709

Fully expanded leaves of four week-old plants were inoculated with 001 710

macerozyme 100 microg mL-1 OGs 100 nM flg22 or water (mock) flg22 a peptide from 711

bacterial flagella that induces immune responses was used as a positive control for 712

Page 35 of 54

MAPK activation (Asai et al 2002) and was purchased from EZBiolab Inc (Westfield 713

IN USA) Samples were harvested 10 min later and immediately frozen in liquid 714

nitrogen Protein was extracted using an extraction buffer containing 25 mM Tris pH 78 715

75 mM NaCl 1 mM DTT 1 mM NaF 05mM Na3VO4 01 (vv) Tween 20 one 716

cOmplete Mini protease inhibitor cocktail tablet (Roche) and one PhosSTOP 717

phosphatase inhibitor cocktail tablet (Roche) per 10 mL buffer 15 microg of total protein 718

were electrophoresed on a 10 SDS-polyacrylamide gel and then blotted onto a PVDF 719

membrane (Bio-Rad 162-0177) Activated MAPKs were detected using p4442 MAPK 720

(Erk12) antibody (9102S Cell Signal 12500 in TBST (20 mM Tris pH 75 150 mM 721

NaCl 01 (vv) Tween 20)) Anti-Rabbit IgG-HRP (115000 in TBST A6154 Sigma) 722

and ECL Plus substrate (Pierce 32132) MPK3 and MPK6 were detected with an anti-723

MPK3 antibody (M8318 Sigma 12000 in TBST and 3 (wv) milk) or an anti-MPK6 724

antibody (A7104 Sigma 16000 in TBST and 3 (wv) milk) respectively The 725

secondary antibody and detection were the same as for the p4442 antibody Five 726

independent experiments were performed 727

728

Preparation of Alcohol Insoluble Residue (AIR) Pectin Extraction and Uronic Acid 729

Measurements 730

Fully expanded leaves from four week-old plants that were incubated for 48 h in 731

the dark to reduce starch were harvested flash frozen and pulverized AIR was 732

extracted by washing ground material twice in 70 (vv) ethanol three times in a 733

mixture of chloroform and methanol (11 vv) and once in acetone (Gille et al 2009) 734

and was then air dried Cell wall pectin was extracted from AIR with cell wall extraction 735

buffer (50 mM Trizma 50 mM CDTA pH 72) at 95degC for 15 min Samples were 736

homogenized using a paint shaker (Harbil 5G-HD) and glass beads (3 mm diameter) 737

For dot blot experiments 500 microL of cell wall extraction buffer were used per 10 mg of 738

AIR Debris was precipitated by centrifugation for 10 min at 10000 g 739

Total uronic acid content of pectin extracted as described above using 500 microL of 740

cell wall extraction buffer per 1 mg of AIR was measured as described previously 741

(Filisetti-Cozzi and Carpita 1991 van den Hoogen et al 1998) First 36 microL of pectin 742

extract were mixed with 4 microL of 4 M sulfamic acid Then 200 microL of sulfuric acid 743

Page 36 of 54

containing 120 mM sodium tetraborate was added and samples were incubated at 80degC 744

for 1 h Following cooling on ice the optical density of samples was measured at 490 745

nm Next 40 microL of m-hydroxydiphenyl reagent (100 microL of m-hydroxydiphenyl in dimethyl 746

sulfoxide at 100 mgmL mixed with 49 mL 80 (vv) sulfuric acid just before use) was 747

added and the samples were mixed The optical density at 490 nm was measured 748

again The optical density before m-hydroxydiphenyl reagent addition was subtracted 749

from the optical density measured after addition of the dye The concentration of uronic 750

acid was calculated using known amounts of GalA as a standard 751

752

Release of Pectin from Arabidopsis Cell Walls Using Macerozyme 753

AIR preparations from leaves of four week-old plants were treated with 025 754

(wv) macerozyme R-10 (Yakult) or water (mock) at a concentration of 100 microL per mg 755

AIR Samples were incubated for 3 h at room temperature on a vortex shaker and 756

centrifuged for 10 min at 12000 g Total uronic acid concentration of 5-fold diluted 757

supernatants was determined for two technical replicates each The concentration of 758

uronic acid was calculated using known amounts of GalA as a standard For 759

subsequent qRT-PCR experiments supernatants from multiple extractions were 760

combined boiled for 20 min to inactivate macerozyme diluted 10-fold with water and 761

inoculated into four week-old Col-0 plants Heat-inactivated 10-fold diluted macerozyme 762

solutions were used as mock treatment Samples were collected 3 h later and PAD3 763

expression was determined 764

765

Release of Pectin from Arabidopsis Leaves Infected with B cinerea 766

Fully expanded leaves of four week-old plants were either inoculated with up to 767

five 10 microL droplets of B cinerea spore solution (25 x 105 spores mL-1) or mock 768

inoculated with B cinerea inoculation medium For each biological replicate leaves with 769

a combined count of at least 20 infection sites were collected 48 h later AIR was 770

extracted as described above but plants were not dark-treated or freeze-dried and the 771

first ethanol step was performed using 85 (vv) ethanol To extract soluble pectin 772

fragments AIR samples were mixed with water at a concentration of 20 microL per infection 773

site incubated for 7 h at room temperature on a vortex shaker and centrifuged for 10 774

Page 37 of 54

min at 12000 g Total uronic acid concentration of supernatants was determined for two 775

technical replicates each The concentration of uronic acid was calculated using known 776

amounts of GalA as a standard 777

778

Measurement of Uronic Acids Neutral Monosaccharides and Cellulose Content in 779

Cell Walls 780

Neutral sugars and uronic acids from non-cellulosic polysaccharides in cell walls 781

(AIR) were released by trifluoroacetic acid (TFA) hydrolysis Approximately 1 mg of AIR 782

was hydrolyzed with 2 M TFA at 121degC for 90 min The TFA-soluble material was split 783

into two equal parts one for uronic acid and one for neutral sugar determination The 784

uronic acid content was determined by analyzing the hydrolyzate using a CarboPac 785

PA200 anion-exchange column with an ICS-3000 Dionex chromatography system 786

(Dionex Sunnyvale CA USA) The elution profile consisted of a linear gradient of 50 to 787

200 mM sodium acetate in 01 M NaOH in 10 min at 04 mL per min Alditol acetate 788

derivatives of neutral sugars were produced as described (York et al 1985) In brief 789

monosaccharides in the hydrolyzate were reduced with NaBH4 and peracetylated with 790

acetic anhydride and pyridine at 121degC for 20 min The generated alditol acetates were 791

analyzed by GC-MS Crystalline cellulose was measured according to Updegraff (1969) 792

After hydrolysis of noncellulosic polysaccharides from AIR with the Updegraff reagent 793

(acetic acid nitric acids water 812 (vv) ) the remaining pellet was hydrolyzed in 794

72 sulfuric acid The resulting glucose quantity was determined by the anthrone 795

method (Scott and Melvin 1953) 796

797

Dot Blot Experiments 798

Pectin solutions extracted using 50 microL of cell wall extraction buffer per mg AIR 799

were serially diluted and nitrocellulose membranes were spotted with 1 microL of the diluted 800

pectin solutions Membranes were dried overnight blocked with 5 milk (wv) in 1x 801

PBS (8 g L-1 NaCl 02 g L-1 KCl 144 g L-1 Na2HPO4 024 g L-1 KH2PO4 pH 74) and 802

probed with LM5 (Jones et al 1997) LM6 (Willats et al 1998) LM8 (Willats et al 803

2004) LM19 and LM20 (Verhertbruggen et al 2009a) or CCRC-M7 (Steffan et al 804

Page 38 of 54

1995) antibodies LM series antibodies were diluted 1250 and CCRC-M7 was diluted 805

1500 in 5 milk powder (Nestle Carnation) in 1 x PBS For LM series antibodies a 806

goat anti-Rat HRP conjugated antibody (Bethyl A110-105P 15000 diluted) and for 807

CCRC-M7 an anti-Mouse IgG HRP conjugate (Promega W402B 12500 diluted) was 808

used as secondary antibody in 5 milk powder in 1x PBS LM series antibodies were 809

obtained from plant probes (wwwplantprobesnet) and CCRC-M7 from CarboSource 810

(wwwcarbosourcenet) Membranes were washed after incubation with the primary and 811

secondary antibodies with 1x PBS Dot blots were developed using the ECL system 812

(GE Healthcare Amersham ECL prime) For quantification of signals dot blot results on 813

x-ray films were photographed using a CCD camera and intensities were measured814

using Image Jrsquos Integrated Density function Locally adjusted background was 815

subtracted As the dynamic range of the signal on an x-ray film was narrow non-816

linearity in the measured values was corrected for each genotype in each biological 817

replicate using the values from different dilutions in two technical replicates We noticed 818

that the residuals were not normally distributed and performed Box-Cox power 819

transformation (Box and Cox 1964) to obtain normally distributed values for the 820

subsequent statistical analysis of the data For LM19 Box-Cox selected power 821

transformation was done to the power of 038th and for LM20 to the power 075th After 822

correction of the transformed measured values with the effect of the biological 823

replicates the values were fit to a linear model with the Arabidopsis genotype as the 824

fixed effect This linear model was used both to generate the plots in Figures 5B and D 825

and to test significant differences 826

827

Camalexin Measurements 828

Four week-old plants were infected with B cinerea and samples (three leaf disks 829

from one plant each) were harvested at the indicated time points flash frozen and 830

pulverized Each sample was extracted with 300 microL of 90 methanol (vv) 50 microL of the 831

extract was run on an Agilent Lichrocart 250-4 RP18e 5-microm column using an Agilent 832

1100 series HPLC Camalexin was detected using a DiodeArray (DAD) at 330 nm and 833

with a fluorescence detector at emission 318 nmexcitation 385 nm (Agilent) Separation 834

was achieved using the following program with aqueous acetonitrile 5 min gradient 835

Page 39 of 54

from 63 to 69 acetonitrile 30 s gradient from 69 to 99 acetonitrile 2 min at 99 836

acetonitrile and a post-run equilibration of 35 min at 63 acetonitrile (Denby et al 837

2004 Kliebenstein et al 2005) Purified camalexin was used to produce a standard 838

curve to identify and quantitate camalexin 839

840

Accession Numbers 841

Microarray data used can be accessed at NCBI Gene Expression Omnnibus 842

(httpwwwncbinlmnihgovgeo) GSE18978 Sequence information for all Arabidopsis 843

genes described in this article can be found in TAIR (wwwarabidopsisorg) using the 844

following accession numbers PAD3 (CYP71B15 At3g26830) SID2 (ICS1 At1g74710) 845

GAE1 (At4g30440) GAE2 (At1g02000) GAE3 (At4g00110) GAE4 (At2g45310) GAE5 846

(At4g12250) GAE6 (At3g23820) PR1 (At2g14610) JAZ5 (At1g17380) JAZ10 847

(At5g13220) ACTIN2 (At3g18780) PDF12a (At5g44420) PDF12b (At2g26020) 848

AtSK11 (At5g26751) The sequence of the B cinerea cutinase Bc-CutA (Z69264) can 849

be found in the DNA Data Bank of Japan (httpgetentryddbjnigacjp) Germplasm 850

used included dde2-2 (At5g42650 (von Malek et al 2002)) pad3-1 (At3g26830 (Zhou 851

et al 1999)) pad4-1 (At3g52430 (Jirage et al 1999)) sid2-2 (At1g74710 (Wildermuth 852

et al 2001)) pbs3-2 (At5g13320 SALK_018225 (Nobuta et al 2007)) cbp60a-1 853

(At5g62570 SALK_124410 (Truman et al 2013)) cbp60g-1 sard1-2 (At5g26920 854

SALK_023199 At1g73805 SALK_052422 (Wang et al 2011) cbp60a-1 cbp60g-1 855

sard1-2 (Truman et al 2013) gae1-1 (At4g30440 SALK_085554) gae6-1 (At3g23280 856

SALK_104454) gae6-2 (At3g23280 SALK_017191) ixr1-1 cev1 (At5g05170 (Ellis 857

and Turner 2001 Scheible et al 2001)) eds1 plants were derived by introgression of 858

the Ler eds1-2 allele into Col-0 that contains two copies of EDS1 (At3g48090 859

At3g48080 (Bartsch et al 2006) mpk6-2 (At2g43790 SALK_073907 (Liu and Zhang 860

2004)) mpk3-DG (At3g45640 a fast neutron deletion mutant (Miles et al 2005)) and 861

npr1-1 (At1g64280 (Cao et al 1997)) T-DNA insertion lines were part of the SALK 862

collection (Alonso et al 2003) and were obtained from the Arabidopsis Biological 863

Resource Center 864

865

Supplemental Data 866

Page 40 of 54

Supplemental Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 867

Expression Does Not Require CBP60s or SID2 868

Supplemental Figure 2 Expression of GAE Family Members uponTreatment with Pma 869

ES4326 870

Supplemental Figure 3 Characterization of T-DNA Insertions in GAE1 and GAE6 871

Supplemental Figure 4 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves and 872

Are Slightly Smaller than Wild-Type Col-0 Plants 873

Supplemental Figure 5 Cell Walls of gae1 gae6 Plants Contain Less Uronic Acid than 874

Wild-Type Col-0 Cell Walls 875

Supplemental Figure 6 Antibodies Raised Against the RGI or XG Components of 876

Pectin Show No Difference in Binding to gae Mutant and Col-0 Cell Walls 877

Supplemental Figure 7 The Effect of B cinerea Treatment on the Expression of GAE 878

Family Genes 879

Supplemental Figure 8 Treatment with 001 Macerozyme Does Not Cause Any 880

Visible Damage to Col-0 Plants 881

Supplemental Figure 9 B cinerea Growth on Plants with Defects in Immune 882

Signaling 883

Supplemental Figure 10 MeJA-Induced Expression of the JA marker gene JAZ10 884

Supplemental Figure 11 The Expression of the JA Marker Gene JAZ5 Is Highly 885

Responsive in gae1 gae6 Plants 886

Supplemental Figure 12 The Expression of PDF12 Is Unaltered in gae1 gae6 Plants 887

Supplemental Figure 13 Macerozyme Treatment Activates MPK3 and MPK6 888

Supplemental Figure 14 B cinerea-Induced ROS Production in Wild-Type Col-0 and 889

gae1 gae6 Plants 890

Supplemental Figure 15 OG-Induced PTI Using 250 microg mL-1 OGs 891

Supplemental Figure 16 Abundance of Plant DNA in B cinerea Treated Plants 892

Supplemental Table 1 Primers used in this study 893

894

ACKNOWLEDGEMENTS 895

We thank William Truman for helpful discussions Simone Ferrari (Sapienza 896

Universita di Roma) for the generous gift of OGs Bruna Bucciarelli (USDA-ARS) for 897

Page 41 of 54

help with fluorescence microscopy the Arabidopsis Biological Resource Center for T-898

DNA insertion lines Paul Knoxrsquos lab (University of Leeds) for LM5 LM6 LM8 LM19 899

and LM20 antibodies and the Complex Carbohydrate Resource Center (University of 900

Georgia) for the CCRC-M7 antibody Most of this work was funded by the Division of 901

Chemical Sciences Geosciences and Biosciences Office of Basic Energy Sciences of 902

the US Department of Energy through Grant DE-FG02-05ER15670 to JG This work 903

was also supported by NSF awards IOS 1339125 and IOS 1021861 to DJK NSF 904

award IOS 1121425 to F K and the USDA National Institute of Food and Agriculture 905

Hatch project number CA-D-PLS-7033-H to DJK 906

907

AUTHOR CONTRIBUTIONS 908

GB and JG designed the research GB GX AT BL NES and NH 909

performed research DJK provided tools GB GX MP AD BL RAH and FK 910

analyzed data GB and JG wrote the paper with input from all authors 911

912

FIGURE LEGENDS 913

Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 Expression 914

Requires PAD4 EDS1 and PBS3 915

(A) Expression of GAE1 in leaves of wild-type Col-0 and mutant plants Expression was916

measured 24 or 32 h after either inoculation with Pma ES4326 (OD600 = 0002) or mock 917

inoculation with 5 mM MgSO4 and was then normalized to the level of ACTIN2 Data 918

from three biological replicates were combined using a mixed linear model Bars 919

represent mean log2-ratios to ACTIN2 plusmn SE Letters indicate significantly different 920

groups with q lt 005 where 005 represents the maximum false discovery rate at which 921

the test may be called significant 922

(B) Expression of GAE6 measured as described in (A)923

924

Figure 2 Susceptibility of gae6 and gae1 gae6 Plants to Pma ES4326 Is Increased 925

Relative to Wild-Type Col-0 926

Leaves of plants of the indicated genotypes were inoculated with Pma ES4326 (OD600 = 927

00002) as described in Methods Bacterial titers in leaves were determined either 928

Page 42 of 54

immediately (0 dpi) or after three days (3 dpi) Bars represent the mean plusmn SE of four 929

independent experiments each with 4 or 12 biological replicates at 0 and 3 dpi 930

respectively Data were combined using a mixed linear model Asterisks indicate 931

significant differences from Col-0 wild-type (q lt 005) Susceptible pad4 plants were 932

included as a positive control 933

934

Figure 3 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves 935

(A) Depiction of the assay for brittleness of gae mutant leaves Fully expanded leaves of936

four to five week-old plants were bent by 180ordm so that the adaxial surface of the leaf was 937

parallel with the soil Broken leaf petioles or midribs were considered brittle 938

(B) Quantification of broken midribs or petioles For each genotype 66 to 82 leaves 939

were analyzed for brittleness as described in (A) Bars represent the ratio of broken to 940

total number of leaves tested Results were analyzed using Fisherrsquos exact test Letters 941

indicate significantly different groups at p lt 005 942

(C) Conductivity measurements Leaves of four week-old plants were inoculated with943

deionized water and then leaf discs were cut out and placed on deionized water 944

Conductivity was measured at the indicated time points After 30 min the water was 945

replaced with fresh water (30 min f w) to account for wounding-related ion leakage from 946

the edges of the leaf discs Three independent experiments with six biological replicates 947

each were performed and data were analyzed using a t-test for each time point P-948

values were corrected using the Bonferroni method Data show means plusmn SE asterisks 949

indicate significant differences from wild-type Col-0 at q lt 001 950

951

Figure 4 Cell Walls of gae6 and gae1 gae6 Plants Are Reduced in GalA 952

(A) to (D) Levels of GalA (A) GlcA (B) cellulose (C) and neutral sugars (D) in alcohol953

insoluble residue (AIR) extracted from leaves of four week-old plants of the indicated 954

genotypes Bars represent means plusmn SE of four biological replicates combined using a 955

mixed linear model Letters indicate significantly different groups (p lt 005) 956

957

Figure 5 Cell Walls of gae1 gae6 Plants Are Reduced in Homogalacturonan 958

Page 43 of 54

Dot blot analyses with anti-LM19 and anti-LM20 antibodies Pectin was extracted from 959

AIR prepared from the indicated genotypes using 1 microL of extraction buffer per 10 microg of 960

AIR Pectin was serially diluted and one microL of undiluted 13 diluted and 19 diluted 961

samples were spotted on a nitrocellulose membrane Three biological replicates were 962

were spotted twice to obtain two technical replicates each All replicates produced 963

similar results 964

(A) Representative dot blot using anti-LM19 antibody965

(B) Relative signal intensities of the spots in the dot blot in (A)966

(C) Representative dot blot using anti-LM20 antibody967

(D) Relative signal intensities of the LM20 dot blot shown in (C)968

Relative signal intensities were measured using Image J software Measurements from 969

the three biological replicates were combined using a linear model after Box-Cox power 970

transformation as described in Methods Bars represent means plusmn SE Asterisks indicate 971

a difference from wild-type Col-0 at p lt 005 972

973

Figure 6 Growth of B cinerea on gae1 gae6 Plants and the Effect of B cinerea on 974

Expression of GAE1 and GAE6 975

(A) Growth of B cinerea isolates Leaves of Col-0 and gae1 gae6 plants were976

inoculated with 25 x 105 spores mL-1 of the B cinerea isolates indicated in the figure 977

Samples were collected two (2 dpi) and three (3 dpi) days later Bars represent means plusmn 978

SE of six biological replicates for each fungus combined using a mixed linear model 979

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 980

(B) and (C) Expression of GAE1 (B) and GAE6 (C) 48 h after inoculation with the B981

cinerea isolates indicated Expression levels were measured by qRT-PCR Bars 982

represent mean log2-ratios to ACTIN2 plusmn SE of three biological replicates combined 983

using a mixed linear model Letters indicate significantly different groups (q lt 005) 984

985

Figure 7 Levels of Soluble Pectin and Expression of PAD3 in Fractions Produced 986

Following Macerozyme Treatment of gae1 gae6 Cell Walls 987

(A) Schematic overview of the experimental procedure for (B) and (C)988

Page 44 of 54

(B) Uronic acid measurements Alcohol-insoluble residue (AIR) from leave tissue of four989

week-old wild-type Col-0 or gae1 gae6 plants was treated with water or 025 990

macerozyme and assayed for uronic acid levels Data from 11 biological replicates were 991

combined using a mixed linear model Bars represent means plusmn SE Letters indicate 992

significant differences (p lt 005) 993

(C) Expression of PAD3 in Col-0 plants treated with the fractions from (B) Extracts from994

(B) were heat inactivated pooled and diluted 5-fold with water Pooled extracts were995

inoculated into four week-old Col-0 plants PAD3 expression was measured 3 h after 996

inoculation and normalized to the level of ACTIN2 expression Data from four biological 997

replicates were combined using a mixed linear model Bars represent mean log2-ratios 998

to ACTIN2 plusmn SE Letters indicate significant differences (p lt 005) 999

1000

Figure 8 Macerozyme-Induced PAD3 Expression Is Absent in gae1 gae6 1001

(A) Expression of PAD3 after macerozyme treatment Four week-old plants were either1002

inoculated with 001 macerozyme or mock inoculated with water and PAD3 1003

expression was measured 3 h later by qRT-PCR Data from four biological replicates 1004

were combined using a mixed linear model Mean log2-ratios to ACTIN2 plusmn SE were 1005

plotted Letters indicate significantly different groups (p lt 005) 1006

(B) Expression of PAD3 after OG treatment PAD3 expression was measured as1007

described in (A) 3 h after inoculation with 100 microg mL-1 OGs or mock inoculation with 1008

water Data are from six biological replicates 1009

(C) PAD3 expression in untreated plants Three biological replicates were performed1010

and data analyzed as described in (A) 1011

(D) Camalexin accumulation after B cinerea treatment Camalexin accumulation was1012

measured in four week-old plants inoculated with the B cinerea strains shown in the 1013

figure Eight biological replicates each were combined using a mixed linear model 1014

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 1015

(E) PAD3 expression 28 h after treatment with the indicated B cinerea strains Data1016

from six biological replicates were collected and analyzed as described in (A) Data in 1017

all figure parts represent means plusmn SE Letters indicate significantly different groups (q lt 1018

005) 1019

Page 45 of 54

1020

Figure 9 Macerozyme-Induced Expression of the SA Marker Genes SID2 and PR1 1021

Is Abolished in gae1 gae6 Plants 1022

(A) to (H) Expression of PR1 (A) (C) (E) and (G) or SID2 (B) (D) (F) and (H) in four1023

week-old Col-0 and gae1 gae6 plants Data were combined using a mixed linear model 1024

Bars represent mean log2-ratios of expression vs ACTIN2 plusmn SE for all figure parts 1025

Letters indicate significantly different groups at p lt 005 for panels (A) to (D) (G) and 1026

(H) and qlt005 for panels (E) to (F) 1027

(A) and (B) Plants were treated with 001 macerozyme or mock treated with water1028

and samples were collected 3 h later Data from four biological replicates are shown 1029

(C) and (D) Plants were inoculated with 100 microg mL-1 OGs or mock treated with water1030

and samples collected 3 h later Data from six biological replicates are shown 1031

(E) and (F) Plants were inoculated with the indicated B cinerea isolates or mock1032

inoculated with B cinerea inoculation medium and samples collected 48 h later Data 1033

from eight biological replicates are shown 1034

(G) and (H) Expression in untreated plants Three biological replicates were performed1035

1036

Figure 10 The Expression of the JA Marker Gene JAZ10 is Highly Responsive in 1037

gae1 gae6 Plants 1038

(A) to (D) JAZ10 expression in four week-old plants Data were combined using a mixed1039

linear model Mean log2-ratios to ACTIN2 plusmn SE are shown Letters indicate significantly 1040

different groups at p lt 005 for panels (A) (B) and (D) and q lt 005 for panel (C) 1041

(A) Plants were inoculated with 001 macerozyme or mock inoculated using water and1042

samples were collected 3 h later Data from four biological replicates are shown 1043

(B) Plants were inoculated with 100 microg mL-1 OGs or mock inoculated using water and1044

samples collected 3 h later Data are from six biological replicates 1045

(C) JAZ10 expression 48 h after treatment with the indicated B cinerea strains Data1046

are from six biological replicates 1047

(D) JAZ10 expression in untreated plants Data are from three biological replicates1048

1049

Figure 11 Gallo 1-Induced Callose Deposition Is Reduced in gae1 gae6 Plants 1050

Page 46 of 54

(A) Callose deposition in four week-old Col-0 and gae1 gae6 plants Plants were1051

inoculated with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) or mock inoculated 1052

with B cinerea inoculation medium and samples were collected after 72 h Callose was 1053

stained with aniline blue and visualized using a Nikon Eclipse Ni-U microscope with a 1054

DAPI filter Three leaves were stained per treatment Scale bars are 100 microm Asterisks 1055

indicate fungal hyphae arrows indicate representative callose spots 1056

(B) Quantification of callose deposition Callose deposits were counted in 200 microm x 2001057

microm squares laid out across the images shown here Bars represent means plusmn SE from 1058

six squares each Letters indicate significantly different groups (p lt 005) according to a 1059

t-test analysis1060

1061

Figure 12 Less Soluble Pectin Accumulates in B cinerea-Infected gae1 gae6 1062

Leaves 1063

Four week-old Col-0 and gae1 gae6 plants were inoculated with 10 microL droplets of the 1064

indicated B cinerea isolate (25 x 105 spores mL-1) or mock inoculated with B cinerea 1065

inoculation medium Leaves were collected 48 h later AIR was extracted from leaf 1066

samples and water added (20 microL infection site) to extract soluble pectin fragments 1067

Samples were vortexed for 7 h centrifuged at 12000 g and the uronic acid 1068

concentration of the supernatant was measured Data from nine biological replicates 1069

were combined using a mixed linear model Means plusmn SE are shown Letters indicate 1070

significant differences (q lt 005) 1071

1072

Figure 13 Macerozyme-Induced PTI Is Lost in gae1 gae6 While OG-Induced PTI Is 1073

Unaltered 1074

(A) Macerozyme-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1075

inoculated with 001 macerozyme or mock inoculated with boiled macerozyme and 1076

were infected with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) 24 h later Samples 1077

were collected immediately (0 dpi) and after two (2 dpi) and three (3 dpi) days Data 1078

show mean plusmn SE of six to 14 biological replicates each combined using a linear model 1079

Letters indicate significantly different groups (q lt 005) 1080

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

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Page 6 of 54

gene encoding it shows evidence for diversifying selection as would be expected for a 149

virulence gene (ten Have et al 1998 Rowe and Kliebenstein 2007) Secretion of 150

CWDEs can trigger the release of cell wall fragments which can act as damage-151

associated molecular patterns (DAMPs) that activate plant immune responses (Hahn et 152

al 1981 Ferrari et al 2013) Similarly pectin fragments known as oligogalacturonides 153

(OGs) can trigger plant immune responses (Cote and Hahn 1994 Ferrari et al 2013) 154

Such responses include the production of reactive oxygen species (ROS) activation of 155

mitogen-activated protein kinase (MAPK) activation and enhanced expression of 156

defense-related genes such as PAD3 (Ferrari et al 2007 Denoux et al 2008 Galletti 157

et al 2008 Galletti et al 2011) Some of these responses require SA JA and ET 158

whereas others are independent of these phytohormones (Ferrari et al 2007) 159

Here we describe two GAE family members GAE1 and GAE6 whose 160

expression is repressed upon pathogen challenge A gae1 gae6 double mutant has 161

brittle leaves and its walls are reduced in pectin specifically HG and likely RGI 162

Resistance to the bacterial pathogen Pma ES4326 and the fungal necrotroph B cinerea 163

is compromised in gae1 gae6 Callose deposition is reduced in gae1 gae6 plants after 164

treatment with B cinerea isolate Gallo 1 Furthermore these plants show altered 165

activation of immune signaling in response to treatment with macerozyme a 166

polygalacturonase that degrades pectin and they also are hyperresponsive to JA 167

signaling Macerozyme treatment released less soluble likely low molecular weight 168

pectin from isolated gae1 gae6 cell walls than from Col-0 cell walls B cinerea-induced 169

accumulation of soluble likely low molecular weight pectin was also decreased in gae1 170

gae6 plants Additionally gae1 gae6 plants are impaired in macerozyme-induced 171

immunity to B cinerea possibly due to decreased release of OGs from their cell walls 172

173

174

Page 7 of 54

RESULTS 175

176

GAE1 and GAE6 Expression Is Repressed by Pma ES4326-Induced Immune 177

Signaling 178

Our previous research suggested that the status of pectin specifically pectin 179

modification by methylesterification is important for plant immunity against Pma 180

ES4326 (Bethke et al 2014) Here we investigate whether pectin abundance in 181

addition to its specific structure is also important for plant immunity by taking a closer 182

look at the formation of the pectin precursor UDP-GalA by GAEs 183

184 Microarray experiments showed that expression of GAE1 and GAE6 was

185 repressed in A thaliana (Arabidopsis) plants treated with Pma ES4326 (Wang et al

186 2008) Repression of GAE1 and GAE6 in wild-type Arabidopsis Col-0 plants treated with

187 Pma ES4326 was confirmed by quantitative real time (qRT)-PCR Figure 1) To test if

188 this repression is mediated by immune signaling qRT-PCR was also performed in

189 mutants with blocked immune signaling (Figure 1 and Supplemental Figure 1) 190 Generally immunity to Pma ES4326 requires intact SA signaling Repression of both

191 GAE1 and GAE6 required the presence of the major immune regulators EDS1 and

192 PAD4 required for SA signaling as well as other immune responses (Jirage et al 1999

193 Wiermer et al 2005 Bartsch et al 2006 Wang et al 2008) at both early and late time

194 points The presence of AVRPPHB SUSCEPTIBLE 3 (PBS3) a regulator of many

195 immune responses including SA signaling (Nobuta et al 2007) was also required for

196 repression of GAE1 and GAE6 at an early time point (Figure 1) CALMODULIN-

197 BINDING PROTEIN 60-LIKE a (CBP60a) (a negative regulator of SA synthesis and

198 immunity (Truman et al 2013)) CBP60g and SYSTEMIC ACQUIRED RESISTANCE

199 DEFICIENT 1 (SARD1) (both promote SA biosynthesis and regulation of other immune

200 responses (Wang et al 2011)) SALICYLIC ACID INDUCTION DEFICIENT 2 SID2

201 (encodes a biosynthetic enzyme for SA (Wildermuth et al 2001)) and

202 NONEXPRESSER OF PR GENES 1 NPR1 (a regulator of the majority of SA-induced

203 immune signaling (Cao et al 1997 Yan and Dong 2014)) were not required for this

204 repression (Figure 1 and Supplemental Figure1) Thus repression of GAE1 and GAE6

Page 8 of 54

205 requires a regulatory function that is dependent on EDS1 and PAD4 partially

206 dependent on PBS3 but independent of SA

Page 9 of 54

There are six GAE family members in Arabidopsis GAE2 expression was 207

induced by Pma ES4326 in the absence of PAD4 or EDS1 GAE3 expression was 208

unaltered GAE4 was repressed early and GAE5 expression was induced in all 209

genotypes (Supplemental Figure 2) The similarity of the immune-regulated expression 210

of GAE1 and GAE6 suggested a redundant role of these genes in Arabidopsis immunity 211

to Pma ES4326 212

213

Resistance to Pma ES4326 Is Compromised in gae1 gae6 and gae6 Plants 214

To study the roles of GAE1 and GAE6 in plant immunity in more detail plants 215

with T-DNA insertion mutations in GAE1 and GAE6 were obtained from the SALK 216

collection as described in the Accession Numbers section of Methods We identified two 217

GAE6 knock-out alleles gae6-1 and gae6-2 and a T-DNA insertion in GAE1 gae1-1 218

that caused reduced expression of this gene (Supplemental Figure 3) A double mutant 219

was created by crossing gae1-1 and gae6-1 Both gae6 and gae1 gae6 plants were 220

more susceptible to Pma ES4326 than wild-type Col-0 and gae1 plants supporting a 221

role of GAE6 in plant immunity to Pma ES4326 (Figure 2) 222

223

gae6 and gae1 gae6 Plants Have Brittle Leaves 224

While inoculating leaves of gae1 gae6 plants we noticed that the petioles and 225

midribs of these plants tended to break easily when leaves were bent against the 226

proximal-distal axis of the leaves (Figure 3 see Supplemental Figure 4D to F) They 227

tended to break less often when twisted around the proximal-distal axis To quantify 228

this phenotype leaves were bent by 180deg (Figure 3A) and breaking of midribs or 229

petioles was recorded Most gae1 gae6 leaves broke whereas fewer leaves of gae6 230

plants and very few leaves of wild-type Col-0 and gae1 plants did (Figure 3B and 231

Supplemental Figure 4C) Although gae1 gae6 plants had slightly shorter leaves and 232

petioles than wild type plants (Supplemental Figure 4A and B) no correlation between 233

leaf size or petiole length and breaking frequency was detected We also investigated 234

tissue integrity by monitoring ion leakage Interestingly when leaves were inoculated 235

with deionized water leaf discs of gae1 gae6 leaked more ions than wild-type Col-0 236

discs (Figure 3C) suggesting that the increased brittleness is correlated with a 237

Page 10 of 54

_____________________________________________________________________________

Page 11 of 54

Cell Walls of gae6 and gae1 gae6 Plants Have Lower Levels of Pectin 243

GalA is a major component of pectin We analyzed wall monosaccharide 244

composition in gae mutants to investigate whether the increased brittleness was 245

associated with an altered wall composition of gae leaves To approximate pectin 246

content of gae mutant walls the total uronic acid concentration was measured using a 247

simple colorimetric assay (Filisetti-Cozzi and Carpita 1991 van den Hoogen et al 248

1998) Total uronic acid was reduced in gae6-2 strongly reduced in gae1 gae6 plants 249

but was indistinguishable from wild-type Col-0 in gae6-1 and gae1 (Supplemental 250

Figure 5) This suggested that gae1 gae6 and possibly gae6 plants have reduced 251

pectin 252

To investigate the cell wall composition in leaves of gae plants in more detail we 253

measured the levels of seven neutral monosaccharides the acidic sugars GalA (the 254

monomeric subunit of the pectin backbone (Harholt et al 2010)) and GlcA (Figure 4) 255

We also estimated the crystalline cellulose content (Updegraff 1969) Cell walls of both 256

gae6 mutants and gae1 gae6 were reduced in GalA (Figure 4A) In gae1 gae6 GalA 257

content in cell walls was reduced by more than 40 compared to the wild-type level 258

GlcA levels were indistinguishable between mutants and wild-type (Figure 4B) 259

Cellulose content was higher in gae6-2 and gae1 gae6 walls likely due to the loss of 260

pectins (Figure 4C) Cell walls of gae1 gae6 plants were also enriched in all neutral 261

sugars measured presumably due to the substantial proportional loss of GalA (Figure 262

4D) The levels of most wall monosaccharides were even higher in the gae1 gae6 263

double mutant with the exception of Rha which did not exhibit a significant increase 264

compared to the gae6 single mutants The absence of a compensational increase in 265

Rha suggested that RGI the dominant Rha contributor to the wall may be considered 266

reduced in gae1 gae6 mutants compared to wild-type albeit not to such an extent as 267

HG 268

238 structural defect that promotes increased ion leakage Taken together these results

239 showed that loss of GAE6 causes brittle leaves and additional losss of GAE1 strongly

240 enhances this phenotype We conclude that GAE1 and GAE6 contribute to normal leaf

241 flexibility 242

Page 12 of 54

gae6 cell walls are reduced in HG Using the same samples we detected no difference 274

from wild type in binding of LM5 which recognizes a tetramer of (1-4)-β-D-galactans 275

found in RGI (Jones et al 1997) LM6 which recognizes a linear pentasaccharide in (1-276

To further investigate which pectic components were altered in gae1 gae6 dot 269

blot experiments using pectin isolated from the various genotypes were performed Both 270

LM19 and LM20 antibodies recognize HG (Verhertbruggen et al 2009a) As expected 271

LM19 binding was reduced in gae6-1 and gae1 gae6 (Figure 5A and B) and LM20 272

binding was reduced in gae1 gae6 (Figure 5C and D) This result confirmed that gae1 273

277 5)-α-L-arabinans found in RGI (Willats et al 1998 Verhertbruggen et al 2009b) LM8

278 which recognizes xylogalacturonan (Willats et al 2004) or CCRC-M7 which

279 recognizes an arabinosylated (1-6)-β-D-galactan epitope occurring on RGI (Puhlmann et

Page 13 of 54

Page 14 of 54

al 1994 Steffan et al 1995 Pattathil et al 2010) (Supplemental Figure 6) 280

Collectively these cell wall composition data show that gae1 gae6 plants are reduced in 281

HG Due to the strong proportional reduction of HG in the walls levels of all other 282

components should be higher However Rha indicative of RGI was not higher in gae1 283

gae6 suggesting that RGI may also be reduced in gae1 gae6 walls 284

285

GAE1 and GAE6 Are Required for Immunity to Specific B cinerea Isolates 286

We hypothesized that plant immunity to B cinerea might be affected by pectin 287

content in the host cell wall because this pathogen requires polygalacturonases and 288

pectin methylesterases for full virulence (ten Have et al 1998 Valette-Collet et al 289

2003 Kars et al 2005b Espino et al 2010) Because Bcinerea is genetically diverse 290

and has a high diversity of polygalacturonases (Rowe and Kliebenstein 2007) we 291

measured relative fungal growth at both 2 and 3 d post-inoculation (dpi) using ten 292

different B cinerea isolates (Figure 6) The B cinerea isolates Fresa 525 and Gallo 1 293

showed enhanced virulence on gae1 gae6 plants at both time points whereas KT and 294

UK Razz showed enhanced virulence in gae1 gae6 plants at 3 but not 2 d dpi (Figure 295

6A) We did not detect any significant changes in relative fungal growth between wild-296

type Col-0 and gae1 gae6 plants when using Acacia Apple 517 DN Grape Pepper or 297

Rasp isolates of B cinerea Therefore we continued our study using Gallo 1 and Fresa 298

525 We also included Pepper for comparison as this isolate has been used in a large-299

scale analysis of plant gene expression in response to B cinerea infection (Windram et 300

al 2012) 301

We next investigated whether expression of GAE1 and GAE6 was affected by 302

challenge with B cinerea Inoculation of wild-type Col-0 plants with any isolate 303

repressed GAE1 and GAE6 expression (Figure 6B and C) and this repression was 304

generally independent of PAD4 (Figure 6B and C) Expression of GAE2 and GAE3 was 305

unaltered by B cinerea treatment GAE4 was repressed only by Fresa 525 treatment 306

while GAE5 expression was enhanced with all three isolates tested (Supplemental 307

Figure 7) As seen for infection with Pma ES4326 the expression of GAE1 and GAE6 308

was repressed upon B cinerea treatment This indicates a role for GAE1 and GAE6 in 309

the immune response to B cinerea 310

Page 15 of 54

Page 16 of 54

Because the amount of pectin substrate for pathogen polygalacturonases is 313

reduced we hypothesized that immunity phenotypes of gae1 gae6 plants may result 314

from reduced generation of OGs by these enzymes In turn the reduced OGs could 315

lead to reduced immune responses To test this we used macerozyme a commercial 316

pectinase as a proxy for pathogen pectinases We isolated cell walls as a subcellular 317

fraction known as alcohol insoluble residue (AIR Gille et al 2009) This was treated 318

with macerozyme and tested for release of soluble likely low molecular weight pectin 319

fragments (Figure 7) Treatment with 025 macerozyme released significantly more 320

uronic acid from wild-type Col-0 AIR relative to gae1 gae6 AIR (Figure 7B) To test 321

activity of these pectin-containing solutions we infiltrated them into wild-type Col-0 322

plants and measured PAD3 expression PAD3 is required for synthesis of the 323

phytoalexin camalexin (Zhou et al 1999) and its expression is known to be induced by 324

OG treatment (Ferrari et al 2007) Fractions from macerozyme treated Col-0 walls 325

induced significantly more PAD3 expression than fractions derived from gae1 gae6 326

walls suggesting that macerozyme treatment did indeed release more active soluble 327

uronic acid possibly OGs from wild-type Col-0 walls than from gae1 gae6 walls (Figure 328

7C) 329

Next we studied the effect of macerozyme treatment on expression of PAD3 in 330

intact plants (Figure 8) This treatment did not lead to any visible leaf damage 331

(Supplemental Figure 8) In wild-type Col-0 PAD3 expression was induced three h after 332

treatment with 001 macerozyme (Figure 8A) By contrast no macerozyme-induced 333

induction of PAD3 expression was detected in gae1 gae6 (Figure 8A) However OG 334

treatment induced the expression of PAD3 in both genotypes indicating that gae1 gae6 335

plants do respond to OGs (Figure 8B) Macerozyme treatment also induced expression 336

of the SA marker genes PATHOGENESIS RELATED 1 (PR1) and SID2 in Col-0 but 337

not in gae1 gae6 (Figure 9 panel A and B) PR1 was not significantly induced by 338

purified OGs (Figure 9C) at the time and concentration tested but SID2 was induced by 339

OG treatment in both genotypes (Figure 9D) However the induction was stronger in 340

wild-type Col-0 In addition the JA markers JASMONATE-ZIM-DOMAIN PROTEIN 5 341

(JAZ5) and JAZ10 were induced by macerozyme and OG treatment in both Col-0 and 342

gae1 gae6 (Figure 10A and Supplemental Figure 11) Interestingly expression of JAZ5 343

312 Pectinase-Induced Immune Signaling Is Altered in gae1 gae6 Plants

Page 17 of 54

Page 18 of 54

Page 19 of 54

Page 20 of 54

344

345

346

and JAZ10 was already strongly induced in gae1 gae6 after mock inoculation (Figure 10

and Supplemental Figure 11) We observed no hyperresponsive induction by mock

treatment of PLANT DEFENSIN 12 (PDF12) a necrotrophy and late JA signaling

347 marker gene (Supplemental Figure 12A-D) PDF12 was also not induced by

348 macerozyme or OG treatments (Supplemental Figure 12A-D) This suggests that early

349 JA signaling is induced by mock-treatment in gae1 gae6 Because it is known that JA

350 and SA signaling act antagonistically (Pieterse et al 2012) the strong up-regulation of

351 JA-induced immune signaling in gae1 gae6 might explain the lower OG-induced SID2

352 expression in this genotype

353 To test whether other immune responses are altered in gae1 gae6 plants we

354 analyzed Mitogen-activated protein kinase (MAPK) activity following macerozyme and

355 OG treatments OGs are known to activate the MAPKs MPK3 and MPK6 in Arabidopsis

Page 21 of 54

(Galletti et al 2011) Macerozyme treatment activated MPK3 and MPK6 in wild-type 356

Col-0 whereas MAPK activation in gae1 gae6 was slightly reduced in four out of five 357

experiments performed (Supplemental Figure 13) By contrast OG-induced MAPK 358

activity was similar or increased in gae1 gae6 in four out of five experiments 359

(Supplemental Figure 13) Additionally mock treatment induced MAPK activity in gae1 360

gae6 more strongly than it did in wild-type Col-0 in four out of five experiments 361

performed (Fig S13) Collectively these results illustrate that activation of several 362

immune signaling componentsmdashcamalexin synthesis SA and JA signaling and 363

possibly MAPK activationmdashby macerozyme is substantially altered in gae1 gae6 This 364

suggests that release of soluble pectin fragments possibly OGs by pectinolytic 365

enzymes might be reduced in gae1 gae6 plants while recognition of OGs is intact 366

367

Following B cinerea Infection Production of Soluble Pectin and Deposition of 368

Callose Are Reduced in gae1 gae6 Plants 369

Although pad3 plants were more susceptible to the B cinerea isolates Fresa 525 370

and Gallo 1 which also grew better on gae1 gae6 growth of the isolate Pepper was 371

similar on Col-0 and pad3 (Supplemental Figure 9A) However we did not detect any 372

significant changes in camalexin production after treatment with B cinerea isolate Gallo 373

1 and Fresa 525 in gae1 gae6 (Figure 8D) Neither did we detect changes in PAD3 374

expression after treatment with any B cinerea isolate in gae1 gae6 (Figure 8E) 375

Additionally SID2 and PR1 were similarly induced by all B cinerea isolates in both Col-376

0 and gae1 gae6 (Figure 9E and F) and sid2 plants which are deficient for pathogen-377

induced SA biosynthesis did not show any differences in relative B cinerea growth 378

(Supplemental Figure 9B) Expression of JAZ10 was induced more strongly by Gallo 1 379

treatment in gae1 gae6 than in wild-type Col-0 but not by Fresa 525 or Pepper (Fig 380

10C) while dde2 plants deficient for JA biosynthesis showed enhanced susceptibility to 381

all B cinerea isolates tested (Supplemental Figure 9B) Curiously exogenous 382

application of MeJA did not lead to altered relative fungal growth of B cinerea 383

(Supplemental Figure 9C) and cev1 isoxaben resistant 1 (ixr1) plants which have 384

constitutively high JA levels (Ellis and Turner 2001) did not show any changes in 385

susceptibility to the B cinerea isolates tested (Supplemental Figure 9D) Hence neither 386

Page 22 of 54

differences in B cinerea isolate susceptibility to camalexin nor altered SA or JA 387

signaling by themselves explained the enhanced susceptibility of gae1 gae6 to these B 388

cinerea isolates 389

Hydrogen peroxide accumulation in response to B cinerea isolates Fresa 525 390

Gallo 1 and Pepper was not obviously altered in gae1 gae6 plants (Supplemental Figure 391

14) However callose deposition upon treatment with B cinerea isolate Gallo 1 was 392

strongly reduced in gae1 gae6 (Figure 11) It was previously shown that OG treatment 393

induces callose deposition (Denoux et al 2008) suggesting that the reduction in 394

callose deposition might be due to reduced OG release in gae1 gae6 It is possible that 395

this reduction in callose deposition contributes to the reduced immunity of gae1 gae6 396

plants to B cinerea isolate Gallo 1 397

Because macerozyme could be used to release soluble likely low molecular 398

weight pectin from isolated cell walls we next investigated whether infection with B 399

cinerea was accompanied by accumulation of soluble pectin as well We prepared cell 400

walls (AIR fraction) from leaves infected with B cinerea incubated these cell walls with 401

water and measured uronic acid content of the extracts B cinerea treatment did in fact 402

lead to increased accumulation of soluble pectin in wild-type Col-0 plants when 403

compared to mock-treated samples (Figure 12) No such increased accumulation of 404

water-soluble pectin fractions was observed in gae1 gae6 (Figure 12) 405

406

Macerozyme-Induced Pattern-Triggered Immunity against B cinerea Is Abolished 407

in gae1 gae6 408

It was previously shown that OG treatment can induce patternndashtriggered 409

immunity (PTI) to B cinerea (Ferrari et al 2007) We tested macerozyme treatment to 410

determine whether it could also induce PTI to the B cinerea isolate Gallo 1 (Figure 13) 411

Indeed pretreatment with 001 macerozyme for 24 h led to reduced B cinerea growth 412

in wild-type Col-0 but not gae1 gae6 plants (Figure 13A) Interestingly mock and 413

macerozyme pretreated gae1 gae6 plants exhibited similar growth of B cinerea Gallo 1 414

as did macerozyme-treated Col-0 plants This result is likely due to activation of JA 415

signaling in gae1 gae6 during the pretreatment procedure (Figure 13C and D) OG-416

induced PTI was observed in both genotypes when using a high dose of OGs (Figure 417

Page 23 of 54

13B) whereas a lower dose of OGs only induced PTI in Col-0 (Supplemental Figure 418

15) This suggests that OG perception in gae1 gae6 at least above a certain threshold 419

is unaltered while release of immunity-activating pectin fragments possibly OGs is 420

421 reduced We conclude that immune responses induced by macerozyme treatment

422 which do not occur in gae1 gae6 plants are important for plant immunity to the B

423 cinerea isolate Gallo 1

Page 24 of 54

____________________________________________________________________________

Page 25 of 54

DISCUSSION 426

Here we showed that loss of GAE1 and GAE6 leads to a dramatic reduction in 427

pectin content in the cell wall (Figure 4) likely causing the brittleness of leaves that we 428

observed (Figure 3) Petioles and midribs of gae1 gae6 plants broke easily when bent 429

and inoculation of leaves with deionized water resulted in increased ion leakage 430

possibly due either to microscopic fractures of the cell wall induced by inoculation stress 431

or to a reduced capability of these walls to withstand increased turgor pressure The 432

increased brittleness was associated with altered cell wall composition In our analysis 433

the GalA content of gae1 gae6 leaf walls was reduced by more than 40 and the total 434

uronic acids by over 60 (Figure 4 and Supplemental Figure 5) This is a major change 435

in the overall cell wall composition considering that pectin makes up about 50 of the 436

plant cell walls found in Arabidopsis leaves (Zablackis et al 1995 Nobuta et al 2007 437

Harholt et al 2010) Interestingly quasimodo1 (qua1) plants which have a mutation in 438

a glycosyltransferase family 8 gene are reduced in GalA by 25 and show reduced 439

binding of HG specific antibodies (Bouton et al 2002) 440

Furthermore qua2 plants which have a mutation in a putative methyltransferase 441

have about 13 less total GalA and lack 50 of cell wall HG (Mouille et al 2007) 442

Likewise cgr2 cgr3 plants carrying mutations in another class of Golgi-localized 443

putative methyl-transferases have leaf cell walls that are reduced by about 40 in 444

uronic acid (Kim et al 2015) All three mutants are severely dwarfed (Bouton et al 445

2002 Mouille et al 2007 Kim et al 2015) Additionally qua1 and qua2 mutants show 446

a distorted overall shape likely due to reduced cell cohesion and are extremely fragile 447

(Bouton et al 2002 Mouille et al 2007) By contrast gae1 gae6 plants with a similar 448

strong reduction in GalA and total uronic acids have an overall normal shape and are 449

only slightly smaller than wild-type Col-0 (Supplemental Figure 4) In the wall analysis 450

performed here we did not detect changes in other wall polysaccharides or structures 451

that might compensate for the reduction of pectin in gae1 gae6 It is possible the severe 452

morphological alterations in qua2 and cgr2 cgr3 plants are due to reduction in pectin 453

methylesterification which is unaffected in gae1 gae6 It was speculated that quality 454

control mechanisms in the Golgi prevent secretion of inefficiently esterified pectin in 455

cgr2 cgr3 (Kim et al 2015) suggesting that the Golgi of these plants might over-456

Page 26 of 54

accumulate pectin and that this may contribute to the severe dwarf phenotype of these 457

plants The gae1 gae6 plants described here provide an opportunity to study the effects 458

of reduction of pectin in plants that do not show severe morphological phenotypes 459

We found that gae1 gae6 plants are also compromised in resistance to B 460

cinerea and Pma ES4326 indicating that loss of pectin reduces immunity to these 461

pathogens (Figure 2 and 6) We found that gae1 gae6 plants were slightly more 462

susceptible to Pma ES4326 but showed a strong increase in susceptibility to specific B 463

cinerea isolates This was not unexpected because B cinerea is known to interact with 464

pectin as part of its infection strategy It secretes various pectin-degrading enzymes 465

early during infection and requires some of these enzymes including PG1 PG2 and 466

PME1 for full virulence (ten Have et al 1998 Valette-Collet et al 2003 Kars et al 467

2005b Espino et al 2010) Additionally Arabidopsis plants expressing an antisense 468

construct of the polygalacturonase inhibitor PGIP1 are significantly more susceptible to 469

B cinerea (Ferrari et al 2006) On the other hand Pma ES4326 is not known to470

require cell wall degrading enzymes for full virulence even though it encodes two 471

pectate lyase genes in its genome (PMA4326_07094 and PMA4326_04621 472

wwwbacteriaensemblorg) B cinerea isolates are also known to be genetically quite 473

diverse in their polygalacturonase (PG) loci and genetic variability in PG2 is associated 474

with varying growth on pectin containing media (Rowe and Kliebenstein 2007) 475

Furthermore the contribution of PG1 and PG2 to B cinerea virulence seems to depend 476

on the specific B cinerea isolate and the host species (Kars et al 2005a Zhang and 477

Van Kan 2013) B cinerea isolates are also known to be differently sensitive to the anti-478

microbial secondary metabolite camalexin (Kliebenstein et al 2005) In line with these 479

observations the susceptibility of gae1 gae6 plants differed depending on the specific 480

B cinerea isolate Whereas Fresa 525 Gallo 1 UK Razz and KT grew better in gae1481

gae6 we detected no difference in Acacia Apple 517 DN Grape Pepper or Rasp 482

growth (Figure 6) 483

B cinerea was shown to metabolize pectin and to possibly utilize GalA as a484

carbon source during in planta growth (Zhang et al 2011 Zhang and Van Kan 2013) 485

Our data show that gae1 gae6 plants which are reduced in GalA content did allow 486

enhanced growth of specific B cinerea isolates but no reduction in growth of any B 487

Page 27 of 54

cinerea isolates tested was found This suggests that utilization of pectin as a carbon 488

source was not a contributing factor to alterations in B cinerea susceptibility It is 489

possible that changes in the cell wall composition affect the structural integrity of the 490

wall and affect hyphal penetration of specific B cinerea isolates However OGs (small 491

HG fragments) are likely released during the infection process (Ferrari et al 2013) 492

OGs function as DAMPs and initiate immune responses in Arabidopsis (Ferrari et al 493

2013) Treatment of Arabidopsis with OGs having a degree of polymerization (DP) of 494

10-15 sugar subunits was previously shown to enhance resistance to B cinerea (Ferrari495

et al 2007) We showed that pretreatment with OGs or macerozyme induced enhanced 496

immunity to B cinerea isolate Gallo 1 in wild-type Col-0 but only OGs and not 497

macerozyme induced immunity in gae1 gae6 (Figure 13) Our data suggest gae1 gae6 498

plants which have reduced pectin content may release less OGs and that this 499

reduction leads to the increased susceptibility to B cinerea 500

Consistently we found that macerozyme treatment released less soluble pectin 501

from gae1 gae6 cell walls than from wild-type walls Fractions containing soluble pectin 502

released from gae1 gae6 were also less active in inducing PAD3 gene expression than 503

the fractions released from wild-type walls (Figure 7) We also showed that less soluble 504

pectin accumulated in gae1 gae6 than in wild-type plants 48 h after treatment with B 505

cinerea (Figure 12) 506

We observed that loss of pectin leads to alterations in A thaliana immune 507

signaling Macerozyme treatment induced expression of PAD3 and the SA marker 508

genes PR1 and SID2 in wild-type Col-0 but not in gae1 gae6 whereas OG treatment 509

led to increased PAD3 and SID2 expression in both wild-type and gae1 gae6 (Figure 8 510

and 9) This suggests that although release of active soluble pectin in these plants 511

might be altered OG recognition is intact In fact cell walls isolated from qua1 another 512

mutant known to be reduced in HG released less OGs (DP1-6) than did wild-type cell 513

walls upon endopolygalacturonase treatment (Bouton et al 2002) This was also true 514

for gae1 gae6 cell walls as macerozyme treatment of isolated gae1 gae6 cell walls 515

released less soluble pectin than treatment of Col-0 cell walls Because the fraction 516

from gae1 gae6 plants was also less active in inducing PAD3 expression (Figure 7) it 517

seems possible that biologically active OGs were the active component inducing PAD3 518

Page 28 of 54

expression Interestingly expression of the JA marker genes JAZ5 and JAZ10 was 519

higher in gae1 gae6 than in wild-type mock-inoculated plants but was comparable to 520

wild-type in untreated plants suggesting that JA signaling in gae1 gae6 is 521

hyperresponsive (Figure 10 and Supplemental Figure 11) This induction of JA signaling 522

might be due to wounding stress incurred during the inoculation process JA and SA 523

signaling generally act antagonistically (Pieterse et al 2012) Increased JA signaling 524

activity might hence explain why mock- and macerozyme-treated gae1 gae6 plants 525

exhibited reduced expression of the SA marker PR1 and why OG treatment induced 526

expression of SID2 another SA marker to a lower extent in gae1 gae6 than in wild-type 527

plants (Figure 9) 528

No single immune signaling component was found to be the sole contributor to 529

the compromised immunity of gae1 gae6 to specific B cinerea isolates such as Gallo 1 530

and Fresa 525 These isolates grew better on pad3 while growth of Pepper another B 531

cinerea isolate that did not show altered virulence on gae1 gae6 was unaltered 532

(Supplemental Figure 9) This suggested that differences in camalexin sensitivity of 533

these B cinerea isolates might account for the differences in gae1 gae6 susceptibility 534

However camalexin accumulation and PAD3 expression was the same for wild-type 535

Col-0 and gae1 gae6 for the B cinerea isolates Fresa 525 and Gallo 1 (Figure 8) which 536

showed enhanced growth on gae1 gae6 plants It should be noted that B cinerea 537

virulence is often estimated by comparing lesion sizes Lesion size and measurements 538

of relative fungal growth by qPCR do not always show the same results For example 539

although Denby et al (2004) showed that Pepper causes larger lesions on pad3 than on 540

wild-type this study showed that relative fungal growth of Pepper on pad3 was 541

unaltered 542

We observed that callose deposition was strongly reduced in gae1 gae6 plants 543

72 h after treatment with B cinerea isolate Gallo 1 (Figure 11) possibly due to reduced 544

release of active soluble pectin Interestingly OG-induced PTI to an unspecified isolate 545

of B cinerea was intact in the pmr4 mutant which does not produce callose (Galletti et 546

al 2008) Due to the high SA levels in pmr4 plants (Nishimura et al 2003) and the fact 547

that different B cinerea isolates vary both in induction of and reaction to defense 548

responses (Kliebenstein et al 2005 Rowe et al 2010) we could not determine 549

Page 29 of 54

whether reduced callose deposition causes the increased susceptibility of gae1 gae6 to 550

B cinerea isolate Gallo 1551

We did not observe any changes in B cinerea growth either on cev1 ixr1 plants 552

which are known to have constitutively high JA and OPDA levels (Ellis et al 2002b) or 553

on plants exogenously treated with MeJA (Supplemental Figure 9) Because plants 554

were kept uncovered for 3 h after MeJA treatment it is unclear how much MeJA was 555

bioavailable in the plant However JAZ10 expression was induced 3 h after spraying 556

with MeJA (Supplemental Figure 10) Because pathogen growth generally depends on 557

the combination of a multitude of immune signaling events we conclude that multiple 558

changes to the immune signaling in gae1 gae6 together account for the altered B 559

cinerea growth phenotypes detected 560

We showed that expression of GAE1 and GAE6 is repressed by the bacterial 561

hemibiotroph Pma ES4326 This repression did not occur in an eds1 or pad4 562

background or in pbs3 at the 24-h time point (Figure 1 and Supplemental Figure 1) 563

During the Arabidopsis - Pma ES4326 interaction the expression of many defense 564

related genes is altered in a PAD4EDS1 dependent fashion (Wang et al 2008) Some 565

of these expression changes require SA CBP60g and SARD1 are thought to act 566

downstream of PAD4 and EDS1 but upstream of SA-dependent responses (Wang et 567

al 2011) PBS3 is also thought to act downstream of PAD4 and EDS1 and upstream of 568

SA but the set of genes whose expression it affects is only partially overlapping with 569

those affected by CBP60gSARD1 (Wang et al 2008 Wang et al 2011) Thus GAE1 570

and GAE6 repression is regulated by the part of the immune signaling network that 571

requires PAD4 EDS1 and PBS3 but is independent of CBP60gSARD1 and SA 572

Because EDS1 PAD4 and PBS3 are important components of the plant immune 573

signaling network (Wiermer et al 2005 Nobuta et al 2007) one might conclude that 574

repression of GAE1 and GAE6 may be part of the plant immune response to Pma 575

ES4326 B cinerea treatment also reduced GAE1 and GAE6 expression but this was 576

mostly independent of PAD4 (Figure 6) while both Pma ES4326 and B cinerea repress 577

GAE1 and GAE6 expression the host signaling mechanisms responsible likely differ 578

In summary our data show that Arabidopsis GAE1 and GAE6 are required for 579

pectin biosynthesis leaf flexibility and immunity to Pma ES4326 and to specific B 580

Page 30 of 54

cinerea isolates We used macerozyme a commercial pectinase to mimic the effect of 581

B cinerea pectinases Macerozyme treatment released less pectin from gae1 gae6 cell582

walls and induced less immune signaling in gae1 gae6 plants Less water soluble pectin 583

also accumulated in B cinerea infected gae1 gae6 plants and macerozyme-induced 584

immunity to B cinerea isolate Gallo 1 was reduced in these plants We conclude that 585

the reduced pectin content in these mutants may lead to reduced release of soluble 586

pectin including active OGs during pathogen attack and that this compromises 587

immunity 588

589

590

Page 31 of 54

METHODS 591

Plant Materials and Growth Conditions 592

Wild-type Columbia (Col-0) and all mutant plants (in Col-0 background) were 593

grown on sterilized BM2 germinating mix (Berger Inc Quebec Canada) in a controlled 594

environment chamber (Conviron Pembina ND USA) with a 12-h photoperiod under 595

100 microM m-2 s-1 fluorescent illumination at 22degC and 75 relative humidity Germplasm 596

used is described in the Accession Numbers section at the end of Methods 597

598

Data Analysis and Replicates 599

For biological replicates samples were obtained from separate plant tissues and 600

in many cases from plants grown in different flats In some experiments multiple 601

technical replicates were measured per biological replicate For statistical analysis all 602

technical replicates were averaged and the averages were considered one biological 603

replicate Independent experiments were performed at different times and often 604

consisted of multiple biological replicates per experiment 605

Data analysis was performed using mixed linear models in the R programming 606

environment (Bates et al 2015) unless otherwise indicated Generally genotypes 607

treatments and time points were used as fixed effects and replicate-specific effects like 608

different pots flats etc were considered random effects We reported p-values 609

obtained from the mixed linear model when less than 10 comparisons were performed 610

and q-values for more than 10 comparisons to correct for multiple comparison testing 611

Q-values were calculated using the Benjamini-Hochberg procedure (Benjamini and612

Hochberg 1995) unless specified otherwise 613

614

Pathogen Strains Growth Conditions and Pathogen Growth Assays 615

Growth of Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) and 616

bacterial growth assays were performed as previously described (Bethke et al 2014) 617

For qRT-PCR assays four week-old plants were inoculated with bacteria (OD600 = 618

0002) or mock (5 mM MgSO4) At least four leaves from two plants were collected at 619

the indicated time points per biological replicate 620

Page 32 of 54

Prior to each experiment Botrytis cinerea isolates were grown on 1x PDA 621

medium (Difco) for 10 d at room temperature All isolates used have been described 622

previously (Rowe and Kliebenstein 2007) The spores were washed from the surface of 623

the plate using an inoculation medium (1x Gamborgrsquos B-5 basal salt mixture (Sigma-624

Aldrich G5768) 2 (wv) glucose 10 mM phosphate buffer pH 64) and fungal hyphae 625

were removed from the suspension by filtering through four layers of cheesecloth The 626

concentration of spores was determined using a hemocytometer and adjusted to 25 x 627

105 spores mL-1 Four week-old Arabidopsis plants were inoculated by placing one 10-628

microL droplet of the B cinerea spore solution or inoculation medium (mock) on the adaxial 629

leaf surface of fully expanded leaves Inoculated plants were kept at 100 relative 630

humidity Infection sites were collected at various time points using a cork borer For 631

qRT-PCR assays at least four leaf discs from one plant were combined for each 632

biological sample For B cinerea growth assays at least 12 leaf discs from three to six 633

plants were pooled per biological sample Relative fungal growth was determined by 634

measuring the abundance of a fungal gene relative to a plant gene as described below 635

In brief DNA from infected tissue was extracted and equal amounts of total DNA were 636

used to perform qPCR reactions using the B cinerea cutinase A and the Arabidopsis 637

SK11 gene respectively Primers were described in (Gachon and Saindrenan 2004) 638

and are listed in Supplemental Table 1 Two technical replicates were combined for 639

each biological replicate B cinerea is a necrotrophic fungal pathogen and may destroy 640

plant DNA as disease progresses However decreases in the amount of plant DNA 641

over the course of our experiments were only observed at late time points with very 642

susceptible plants so the qPCR assay is generally a reasonable estimate of fungal 643

biomass (Supplemental Figure 16) 644

645

Treatment with Macerozyme or OGs 646

For PTI assays four week-old plants either were inoculated with 001 (wv) 647

macerozyme R-10 (Yakult) a Rhizopus polygalacturonase (EC 32115) that possesses 648

high pectinase and hemicellulase activity or were mock inoculated using heat-649

inactivated macerozyme Other similar plants were either inoculated with 250 or 500 microg 650

mL-1 OGs or mock inoculated with water One d later plants were infected with B 651

Page 33 of 54

cinerea and samples were collected immediately (0 dpi) and two or three d later To 652

investigate the role of MeJA on B cinerea growth plants were sprayed with 1mM MeJA 653

in water with 001 (vv) Tween 20 Covers were removed so that leaves could dry off 654

before plants were inoculated with B cinerea three h later Samples were harvested at 655

the indicated time points For expression analysis and MAPK activation assays plants 656

were either inoculated with 100 microg mL-1 OGs or mock inoculated with water OGs used 657

for experiments described in Figures 8 to 10 and Supplemental Figure 12 were a kind 658

gift from Simone Ferrari OGs used in experiments described in Figure 13 and 659

Supplemental Figures 11 13 and 15 were prepared as previously described (Kohorn et 660

al 2014) 661

662

Expression Analysis 663

Leaf tissue from inoculated plants was harvested at the indicated time points 664

flash frozen pulverized and RNA extracted using Trizol (Invitrogen) Quantitative RT-665

PCR was performed using the SuperScript III Platinum SYBR Green One-Step 666

quantitative RT-PCR kit (Invitrogen) and a Lightcycler 480 Real-Time PCR system 667

(Roche) as previously described (Truman and Glazebrook 2012) In brief equal 668

amounts of total RNA and a gene specific primer were used for each reaction The 669

crossing point (Cp) was calculated using the 2nd derivative max method provided with 670

the Lightcycler software for each amplification curve Each reaction was run with two 671

technical replicates and the Cp values for these replicates were averaged ACTIN2 was 672

used as a stably expressed reference gene At least three biological replicates each 673

were performed and analyzed using a mixed-linear model RT-PCR was performed to 674

verify T-DNA insertion lines using the One-Step RT-PCR kit (Qiagen) according to the 675

manufacturerrsquos instructions The primers used can be found in Supplemental Table 1 676

677

Electrolyte Leakage Assay 678

Fully expanded leaves of four week-old plants were inoculated with water (Milli-Q 679

grade) using a needleless syringe Leaf discs were collected using a cork borer and 680

three leaf discs from one leaf were placed with the adaxial surface down onto 2 mL 681

water in a 12-well cell culture plate Conductivity was measured using a Horiba B-173 682

Page 34 of 54

conductivity meter after 30 min and then the water was replaced Conductivity was 683

measured again 30 min later (1 h total) as indicated in Figure 3C Six biological 684

replicates each were performed in three independent experiments and means and SEs 685

were calculated Two-sided t-tests were performed for each time point 686

687

DAB and Aniline Blue Staining 688

Leaves from four week-old plants were either mock inoculated with B cinerea 689

inoculation medium or inoculated with 10 microL droplets of a B cinerea spore solution (25 690

x 105 spores mL-1) of the indicated isolates Samples were collected 72 h later For 33prime-691

diaminobenzidine (DAB) staining two to five inoculated leaves were vacuum infiltrated 692

with 33prime-Diaminobenzidine (1 mg mL-1 in water pH 38) solution and stained overnight 693

in the dark Leaves were destained using 100 ethanol at 65degC and then transferred to 694

50 (vv) glycerol and photographed 695

Callose was visualized by staining leaves with aniline blue as previously 696

described (Adam and Somerville 1996) Briefly leaves were vacuum infiltrated with 697

alcoholic lactophenol (one volume of phenolglycerollactic acidwater (1111) mixed 698

with two volumes of ethanol) and destained at 65degC Next leaves were moved through 699

an alcohol gradient (50 20 and 10 (vv) ethanol) into water The destained leaves 700

were stained for 30 min in 150 mM K2HPO4 (pH 95) with 001 (wv) aniline blue 701

Samples were mounted in water and examined using a Nikon Eclipse Ni-U microscope 702

at 10x magnification using a DAPI filter (excitation at 325-375 nm emission at 435-485 703

nm) For callose quantification six 200 microm x 200 microm squares were counted for each 704

sample type Callose deposits per mm2 were calculated and data analyzed using a t-705

test Note that there is an inherent uncertainty associated with such counts because 706

callose deposits may not all be in focus at the same time 707

708

MAPK Activity Assay 709

Fully expanded leaves of four week-old plants were inoculated with 001 710

macerozyme 100 microg mL-1 OGs 100 nM flg22 or water (mock) flg22 a peptide from 711

bacterial flagella that induces immune responses was used as a positive control for 712

Page 35 of 54

MAPK activation (Asai et al 2002) and was purchased from EZBiolab Inc (Westfield 713

IN USA) Samples were harvested 10 min later and immediately frozen in liquid 714

nitrogen Protein was extracted using an extraction buffer containing 25 mM Tris pH 78 715

75 mM NaCl 1 mM DTT 1 mM NaF 05mM Na3VO4 01 (vv) Tween 20 one 716

cOmplete Mini protease inhibitor cocktail tablet (Roche) and one PhosSTOP 717

phosphatase inhibitor cocktail tablet (Roche) per 10 mL buffer 15 microg of total protein 718

were electrophoresed on a 10 SDS-polyacrylamide gel and then blotted onto a PVDF 719

membrane (Bio-Rad 162-0177) Activated MAPKs were detected using p4442 MAPK 720

(Erk12) antibody (9102S Cell Signal 12500 in TBST (20 mM Tris pH 75 150 mM 721

NaCl 01 (vv) Tween 20)) Anti-Rabbit IgG-HRP (115000 in TBST A6154 Sigma) 722

and ECL Plus substrate (Pierce 32132) MPK3 and MPK6 were detected with an anti-723

MPK3 antibody (M8318 Sigma 12000 in TBST and 3 (wv) milk) or an anti-MPK6 724

antibody (A7104 Sigma 16000 in TBST and 3 (wv) milk) respectively The 725

secondary antibody and detection were the same as for the p4442 antibody Five 726

independent experiments were performed 727

728

Preparation of Alcohol Insoluble Residue (AIR) Pectin Extraction and Uronic Acid 729

Measurements 730

Fully expanded leaves from four week-old plants that were incubated for 48 h in 731

the dark to reduce starch were harvested flash frozen and pulverized AIR was 732

extracted by washing ground material twice in 70 (vv) ethanol three times in a 733

mixture of chloroform and methanol (11 vv) and once in acetone (Gille et al 2009) 734

and was then air dried Cell wall pectin was extracted from AIR with cell wall extraction 735

buffer (50 mM Trizma 50 mM CDTA pH 72) at 95degC for 15 min Samples were 736

homogenized using a paint shaker (Harbil 5G-HD) and glass beads (3 mm diameter) 737

For dot blot experiments 500 microL of cell wall extraction buffer were used per 10 mg of 738

AIR Debris was precipitated by centrifugation for 10 min at 10000 g 739

Total uronic acid content of pectin extracted as described above using 500 microL of 740

cell wall extraction buffer per 1 mg of AIR was measured as described previously 741

(Filisetti-Cozzi and Carpita 1991 van den Hoogen et al 1998) First 36 microL of pectin 742

extract were mixed with 4 microL of 4 M sulfamic acid Then 200 microL of sulfuric acid 743

Page 36 of 54

containing 120 mM sodium tetraborate was added and samples were incubated at 80degC 744

for 1 h Following cooling on ice the optical density of samples was measured at 490 745

nm Next 40 microL of m-hydroxydiphenyl reagent (100 microL of m-hydroxydiphenyl in dimethyl 746

sulfoxide at 100 mgmL mixed with 49 mL 80 (vv) sulfuric acid just before use) was 747

added and the samples were mixed The optical density at 490 nm was measured 748

again The optical density before m-hydroxydiphenyl reagent addition was subtracted 749

from the optical density measured after addition of the dye The concentration of uronic 750

acid was calculated using known amounts of GalA as a standard 751

752

Release of Pectin from Arabidopsis Cell Walls Using Macerozyme 753

AIR preparations from leaves of four week-old plants were treated with 025 754

(wv) macerozyme R-10 (Yakult) or water (mock) at a concentration of 100 microL per mg 755

AIR Samples were incubated for 3 h at room temperature on a vortex shaker and 756

centrifuged for 10 min at 12000 g Total uronic acid concentration of 5-fold diluted 757

supernatants was determined for two technical replicates each The concentration of 758

uronic acid was calculated using known amounts of GalA as a standard For 759

subsequent qRT-PCR experiments supernatants from multiple extractions were 760

combined boiled for 20 min to inactivate macerozyme diluted 10-fold with water and 761

inoculated into four week-old Col-0 plants Heat-inactivated 10-fold diluted macerozyme 762

solutions were used as mock treatment Samples were collected 3 h later and PAD3 763

expression was determined 764

765

Release of Pectin from Arabidopsis Leaves Infected with B cinerea 766

Fully expanded leaves of four week-old plants were either inoculated with up to 767

five 10 microL droplets of B cinerea spore solution (25 x 105 spores mL-1) or mock 768

inoculated with B cinerea inoculation medium For each biological replicate leaves with 769

a combined count of at least 20 infection sites were collected 48 h later AIR was 770

extracted as described above but plants were not dark-treated or freeze-dried and the 771

first ethanol step was performed using 85 (vv) ethanol To extract soluble pectin 772

fragments AIR samples were mixed with water at a concentration of 20 microL per infection 773

site incubated for 7 h at room temperature on a vortex shaker and centrifuged for 10 774

Page 37 of 54

min at 12000 g Total uronic acid concentration of supernatants was determined for two 775

technical replicates each The concentration of uronic acid was calculated using known 776

amounts of GalA as a standard 777

778

Measurement of Uronic Acids Neutral Monosaccharides and Cellulose Content in 779

Cell Walls 780

Neutral sugars and uronic acids from non-cellulosic polysaccharides in cell walls 781

(AIR) were released by trifluoroacetic acid (TFA) hydrolysis Approximately 1 mg of AIR 782

was hydrolyzed with 2 M TFA at 121degC for 90 min The TFA-soluble material was split 783

into two equal parts one for uronic acid and one for neutral sugar determination The 784

uronic acid content was determined by analyzing the hydrolyzate using a CarboPac 785

PA200 anion-exchange column with an ICS-3000 Dionex chromatography system 786

(Dionex Sunnyvale CA USA) The elution profile consisted of a linear gradient of 50 to 787

200 mM sodium acetate in 01 M NaOH in 10 min at 04 mL per min Alditol acetate 788

derivatives of neutral sugars were produced as described (York et al 1985) In brief 789

monosaccharides in the hydrolyzate were reduced with NaBH4 and peracetylated with 790

acetic anhydride and pyridine at 121degC for 20 min The generated alditol acetates were 791

analyzed by GC-MS Crystalline cellulose was measured according to Updegraff (1969) 792

After hydrolysis of noncellulosic polysaccharides from AIR with the Updegraff reagent 793

(acetic acid nitric acids water 812 (vv) ) the remaining pellet was hydrolyzed in 794

72 sulfuric acid The resulting glucose quantity was determined by the anthrone 795

method (Scott and Melvin 1953) 796

797

Dot Blot Experiments 798

Pectin solutions extracted using 50 microL of cell wall extraction buffer per mg AIR 799

were serially diluted and nitrocellulose membranes were spotted with 1 microL of the diluted 800

pectin solutions Membranes were dried overnight blocked with 5 milk (wv) in 1x 801

PBS (8 g L-1 NaCl 02 g L-1 KCl 144 g L-1 Na2HPO4 024 g L-1 KH2PO4 pH 74) and 802

probed with LM5 (Jones et al 1997) LM6 (Willats et al 1998) LM8 (Willats et al 803

2004) LM19 and LM20 (Verhertbruggen et al 2009a) or CCRC-M7 (Steffan et al 804

Page 38 of 54

1995) antibodies LM series antibodies were diluted 1250 and CCRC-M7 was diluted 805

1500 in 5 milk powder (Nestle Carnation) in 1 x PBS For LM series antibodies a 806

goat anti-Rat HRP conjugated antibody (Bethyl A110-105P 15000 diluted) and for 807

CCRC-M7 an anti-Mouse IgG HRP conjugate (Promega W402B 12500 diluted) was 808

used as secondary antibody in 5 milk powder in 1x PBS LM series antibodies were 809

obtained from plant probes (wwwplantprobesnet) and CCRC-M7 from CarboSource 810

(wwwcarbosourcenet) Membranes were washed after incubation with the primary and 811

secondary antibodies with 1x PBS Dot blots were developed using the ECL system 812

(GE Healthcare Amersham ECL prime) For quantification of signals dot blot results on 813

x-ray films were photographed using a CCD camera and intensities were measured814

using Image Jrsquos Integrated Density function Locally adjusted background was 815

subtracted As the dynamic range of the signal on an x-ray film was narrow non-816

linearity in the measured values was corrected for each genotype in each biological 817

replicate using the values from different dilutions in two technical replicates We noticed 818

that the residuals were not normally distributed and performed Box-Cox power 819

transformation (Box and Cox 1964) to obtain normally distributed values for the 820

subsequent statistical analysis of the data For LM19 Box-Cox selected power 821

transformation was done to the power of 038th and for LM20 to the power 075th After 822

correction of the transformed measured values with the effect of the biological 823

replicates the values were fit to a linear model with the Arabidopsis genotype as the 824

fixed effect This linear model was used both to generate the plots in Figures 5B and D 825

and to test significant differences 826

827

Camalexin Measurements 828

Four week-old plants were infected with B cinerea and samples (three leaf disks 829

from one plant each) were harvested at the indicated time points flash frozen and 830

pulverized Each sample was extracted with 300 microL of 90 methanol (vv) 50 microL of the 831

extract was run on an Agilent Lichrocart 250-4 RP18e 5-microm column using an Agilent 832

1100 series HPLC Camalexin was detected using a DiodeArray (DAD) at 330 nm and 833

with a fluorescence detector at emission 318 nmexcitation 385 nm (Agilent) Separation 834

was achieved using the following program with aqueous acetonitrile 5 min gradient 835

Page 39 of 54

from 63 to 69 acetonitrile 30 s gradient from 69 to 99 acetonitrile 2 min at 99 836

acetonitrile and a post-run equilibration of 35 min at 63 acetonitrile (Denby et al 837

2004 Kliebenstein et al 2005) Purified camalexin was used to produce a standard 838

curve to identify and quantitate camalexin 839

840

Accession Numbers 841

Microarray data used can be accessed at NCBI Gene Expression Omnnibus 842

(httpwwwncbinlmnihgovgeo) GSE18978 Sequence information for all Arabidopsis 843

genes described in this article can be found in TAIR (wwwarabidopsisorg) using the 844

following accession numbers PAD3 (CYP71B15 At3g26830) SID2 (ICS1 At1g74710) 845

GAE1 (At4g30440) GAE2 (At1g02000) GAE3 (At4g00110) GAE4 (At2g45310) GAE5 846

(At4g12250) GAE6 (At3g23820) PR1 (At2g14610) JAZ5 (At1g17380) JAZ10 847

(At5g13220) ACTIN2 (At3g18780) PDF12a (At5g44420) PDF12b (At2g26020) 848

AtSK11 (At5g26751) The sequence of the B cinerea cutinase Bc-CutA (Z69264) can 849

be found in the DNA Data Bank of Japan (httpgetentryddbjnigacjp) Germplasm 850

used included dde2-2 (At5g42650 (von Malek et al 2002)) pad3-1 (At3g26830 (Zhou 851

et al 1999)) pad4-1 (At3g52430 (Jirage et al 1999)) sid2-2 (At1g74710 (Wildermuth 852

et al 2001)) pbs3-2 (At5g13320 SALK_018225 (Nobuta et al 2007)) cbp60a-1 853

(At5g62570 SALK_124410 (Truman et al 2013)) cbp60g-1 sard1-2 (At5g26920 854

SALK_023199 At1g73805 SALK_052422 (Wang et al 2011) cbp60a-1 cbp60g-1 855

sard1-2 (Truman et al 2013) gae1-1 (At4g30440 SALK_085554) gae6-1 (At3g23280 856

SALK_104454) gae6-2 (At3g23280 SALK_017191) ixr1-1 cev1 (At5g05170 (Ellis 857

and Turner 2001 Scheible et al 2001)) eds1 plants were derived by introgression of 858

the Ler eds1-2 allele into Col-0 that contains two copies of EDS1 (At3g48090 859

At3g48080 (Bartsch et al 2006) mpk6-2 (At2g43790 SALK_073907 (Liu and Zhang 860

2004)) mpk3-DG (At3g45640 a fast neutron deletion mutant (Miles et al 2005)) and 861

npr1-1 (At1g64280 (Cao et al 1997)) T-DNA insertion lines were part of the SALK 862

collection (Alonso et al 2003) and were obtained from the Arabidopsis Biological 863

Resource Center 864

865

Supplemental Data 866

Page 40 of 54

Supplemental Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 867

Expression Does Not Require CBP60s or SID2 868

Supplemental Figure 2 Expression of GAE Family Members uponTreatment with Pma 869

ES4326 870

Supplemental Figure 3 Characterization of T-DNA Insertions in GAE1 and GAE6 871

Supplemental Figure 4 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves and 872

Are Slightly Smaller than Wild-Type Col-0 Plants 873

Supplemental Figure 5 Cell Walls of gae1 gae6 Plants Contain Less Uronic Acid than 874

Wild-Type Col-0 Cell Walls 875

Supplemental Figure 6 Antibodies Raised Against the RGI or XG Components of 876

Pectin Show No Difference in Binding to gae Mutant and Col-0 Cell Walls 877

Supplemental Figure 7 The Effect of B cinerea Treatment on the Expression of GAE 878

Family Genes 879

Supplemental Figure 8 Treatment with 001 Macerozyme Does Not Cause Any 880

Visible Damage to Col-0 Plants 881

Supplemental Figure 9 B cinerea Growth on Plants with Defects in Immune 882

Signaling 883

Supplemental Figure 10 MeJA-Induced Expression of the JA marker gene JAZ10 884

Supplemental Figure 11 The Expression of the JA Marker Gene JAZ5 Is Highly 885

Responsive in gae1 gae6 Plants 886

Supplemental Figure 12 The Expression of PDF12 Is Unaltered in gae1 gae6 Plants 887

Supplemental Figure 13 Macerozyme Treatment Activates MPK3 and MPK6 888

Supplemental Figure 14 B cinerea-Induced ROS Production in Wild-Type Col-0 and 889

gae1 gae6 Plants 890

Supplemental Figure 15 OG-Induced PTI Using 250 microg mL-1 OGs 891

Supplemental Figure 16 Abundance of Plant DNA in B cinerea Treated Plants 892

Supplemental Table 1 Primers used in this study 893

894

ACKNOWLEDGEMENTS 895

We thank William Truman for helpful discussions Simone Ferrari (Sapienza 896

Universita di Roma) for the generous gift of OGs Bruna Bucciarelli (USDA-ARS) for 897

Page 41 of 54

help with fluorescence microscopy the Arabidopsis Biological Resource Center for T-898

DNA insertion lines Paul Knoxrsquos lab (University of Leeds) for LM5 LM6 LM8 LM19 899

and LM20 antibodies and the Complex Carbohydrate Resource Center (University of 900

Georgia) for the CCRC-M7 antibody Most of this work was funded by the Division of 901

Chemical Sciences Geosciences and Biosciences Office of Basic Energy Sciences of 902

the US Department of Energy through Grant DE-FG02-05ER15670 to JG This work 903

was also supported by NSF awards IOS 1339125 and IOS 1021861 to DJK NSF 904

award IOS 1121425 to F K and the USDA National Institute of Food and Agriculture 905

Hatch project number CA-D-PLS-7033-H to DJK 906

907

AUTHOR CONTRIBUTIONS 908

GB and JG designed the research GB GX AT BL NES and NH 909

performed research DJK provided tools GB GX MP AD BL RAH and FK 910

analyzed data GB and JG wrote the paper with input from all authors 911

912

FIGURE LEGENDS 913

Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 Expression 914

Requires PAD4 EDS1 and PBS3 915

(A) Expression of GAE1 in leaves of wild-type Col-0 and mutant plants Expression was916

measured 24 or 32 h after either inoculation with Pma ES4326 (OD600 = 0002) or mock 917

inoculation with 5 mM MgSO4 and was then normalized to the level of ACTIN2 Data 918

from three biological replicates were combined using a mixed linear model Bars 919

represent mean log2-ratios to ACTIN2 plusmn SE Letters indicate significantly different 920

groups with q lt 005 where 005 represents the maximum false discovery rate at which 921

the test may be called significant 922

(B) Expression of GAE6 measured as described in (A)923

924

Figure 2 Susceptibility of gae6 and gae1 gae6 Plants to Pma ES4326 Is Increased 925

Relative to Wild-Type Col-0 926

Leaves of plants of the indicated genotypes were inoculated with Pma ES4326 (OD600 = 927

00002) as described in Methods Bacterial titers in leaves were determined either 928

Page 42 of 54

immediately (0 dpi) or after three days (3 dpi) Bars represent the mean plusmn SE of four 929

independent experiments each with 4 or 12 biological replicates at 0 and 3 dpi 930

respectively Data were combined using a mixed linear model Asterisks indicate 931

significant differences from Col-0 wild-type (q lt 005) Susceptible pad4 plants were 932

included as a positive control 933

934

Figure 3 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves 935

(A) Depiction of the assay for brittleness of gae mutant leaves Fully expanded leaves of936

four to five week-old plants were bent by 180ordm so that the adaxial surface of the leaf was 937

parallel with the soil Broken leaf petioles or midribs were considered brittle 938

(B) Quantification of broken midribs or petioles For each genotype 66 to 82 leaves 939

were analyzed for brittleness as described in (A) Bars represent the ratio of broken to 940

total number of leaves tested Results were analyzed using Fisherrsquos exact test Letters 941

indicate significantly different groups at p lt 005 942

(C) Conductivity measurements Leaves of four week-old plants were inoculated with943

deionized water and then leaf discs were cut out and placed on deionized water 944

Conductivity was measured at the indicated time points After 30 min the water was 945

replaced with fresh water (30 min f w) to account for wounding-related ion leakage from 946

the edges of the leaf discs Three independent experiments with six biological replicates 947

each were performed and data were analyzed using a t-test for each time point P-948

values were corrected using the Bonferroni method Data show means plusmn SE asterisks 949

indicate significant differences from wild-type Col-0 at q lt 001 950

951

Figure 4 Cell Walls of gae6 and gae1 gae6 Plants Are Reduced in GalA 952

(A) to (D) Levels of GalA (A) GlcA (B) cellulose (C) and neutral sugars (D) in alcohol953

insoluble residue (AIR) extracted from leaves of four week-old plants of the indicated 954

genotypes Bars represent means plusmn SE of four biological replicates combined using a 955

mixed linear model Letters indicate significantly different groups (p lt 005) 956

957

Figure 5 Cell Walls of gae1 gae6 Plants Are Reduced in Homogalacturonan 958

Page 43 of 54

Dot blot analyses with anti-LM19 and anti-LM20 antibodies Pectin was extracted from 959

AIR prepared from the indicated genotypes using 1 microL of extraction buffer per 10 microg of 960

AIR Pectin was serially diluted and one microL of undiluted 13 diluted and 19 diluted 961

samples were spotted on a nitrocellulose membrane Three biological replicates were 962

were spotted twice to obtain two technical replicates each All replicates produced 963

similar results 964

(A) Representative dot blot using anti-LM19 antibody965

(B) Relative signal intensities of the spots in the dot blot in (A)966

(C) Representative dot blot using anti-LM20 antibody967

(D) Relative signal intensities of the LM20 dot blot shown in (C)968

Relative signal intensities were measured using Image J software Measurements from 969

the three biological replicates were combined using a linear model after Box-Cox power 970

transformation as described in Methods Bars represent means plusmn SE Asterisks indicate 971

a difference from wild-type Col-0 at p lt 005 972

973

Figure 6 Growth of B cinerea on gae1 gae6 Plants and the Effect of B cinerea on 974

Expression of GAE1 and GAE6 975

(A) Growth of B cinerea isolates Leaves of Col-0 and gae1 gae6 plants were976

inoculated with 25 x 105 spores mL-1 of the B cinerea isolates indicated in the figure 977

Samples were collected two (2 dpi) and three (3 dpi) days later Bars represent means plusmn 978

SE of six biological replicates for each fungus combined using a mixed linear model 979

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 980

(B) and (C) Expression of GAE1 (B) and GAE6 (C) 48 h after inoculation with the B981

cinerea isolates indicated Expression levels were measured by qRT-PCR Bars 982

represent mean log2-ratios to ACTIN2 plusmn SE of three biological replicates combined 983

using a mixed linear model Letters indicate significantly different groups (q lt 005) 984

985

Figure 7 Levels of Soluble Pectin and Expression of PAD3 in Fractions Produced 986

Following Macerozyme Treatment of gae1 gae6 Cell Walls 987

(A) Schematic overview of the experimental procedure for (B) and (C)988

Page 44 of 54

(B) Uronic acid measurements Alcohol-insoluble residue (AIR) from leave tissue of four989

week-old wild-type Col-0 or gae1 gae6 plants was treated with water or 025 990

macerozyme and assayed for uronic acid levels Data from 11 biological replicates were 991

combined using a mixed linear model Bars represent means plusmn SE Letters indicate 992

significant differences (p lt 005) 993

(C) Expression of PAD3 in Col-0 plants treated with the fractions from (B) Extracts from994

(B) were heat inactivated pooled and diluted 5-fold with water Pooled extracts were995

inoculated into four week-old Col-0 plants PAD3 expression was measured 3 h after 996

inoculation and normalized to the level of ACTIN2 expression Data from four biological 997

replicates were combined using a mixed linear model Bars represent mean log2-ratios 998

to ACTIN2 plusmn SE Letters indicate significant differences (p lt 005) 999

1000

Figure 8 Macerozyme-Induced PAD3 Expression Is Absent in gae1 gae6 1001

(A) Expression of PAD3 after macerozyme treatment Four week-old plants were either1002

inoculated with 001 macerozyme or mock inoculated with water and PAD3 1003

expression was measured 3 h later by qRT-PCR Data from four biological replicates 1004

were combined using a mixed linear model Mean log2-ratios to ACTIN2 plusmn SE were 1005

plotted Letters indicate significantly different groups (p lt 005) 1006

(B) Expression of PAD3 after OG treatment PAD3 expression was measured as1007

described in (A) 3 h after inoculation with 100 microg mL-1 OGs or mock inoculation with 1008

water Data are from six biological replicates 1009

(C) PAD3 expression in untreated plants Three biological replicates were performed1010

and data analyzed as described in (A) 1011

(D) Camalexin accumulation after B cinerea treatment Camalexin accumulation was1012

measured in four week-old plants inoculated with the B cinerea strains shown in the 1013

figure Eight biological replicates each were combined using a mixed linear model 1014

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 1015

(E) PAD3 expression 28 h after treatment with the indicated B cinerea strains Data1016

from six biological replicates were collected and analyzed as described in (A) Data in 1017

all figure parts represent means plusmn SE Letters indicate significantly different groups (q lt 1018

005) 1019

Page 45 of 54

1020

Figure 9 Macerozyme-Induced Expression of the SA Marker Genes SID2 and PR1 1021

Is Abolished in gae1 gae6 Plants 1022

(A) to (H) Expression of PR1 (A) (C) (E) and (G) or SID2 (B) (D) (F) and (H) in four1023

week-old Col-0 and gae1 gae6 plants Data were combined using a mixed linear model 1024

Bars represent mean log2-ratios of expression vs ACTIN2 plusmn SE for all figure parts 1025

Letters indicate significantly different groups at p lt 005 for panels (A) to (D) (G) and 1026

(H) and qlt005 for panels (E) to (F) 1027

(A) and (B) Plants were treated with 001 macerozyme or mock treated with water1028

and samples were collected 3 h later Data from four biological replicates are shown 1029

(C) and (D) Plants were inoculated with 100 microg mL-1 OGs or mock treated with water1030

and samples collected 3 h later Data from six biological replicates are shown 1031

(E) and (F) Plants were inoculated with the indicated B cinerea isolates or mock1032

inoculated with B cinerea inoculation medium and samples collected 48 h later Data 1033

from eight biological replicates are shown 1034

(G) and (H) Expression in untreated plants Three biological replicates were performed1035

1036

Figure 10 The Expression of the JA Marker Gene JAZ10 is Highly Responsive in 1037

gae1 gae6 Plants 1038

(A) to (D) JAZ10 expression in four week-old plants Data were combined using a mixed1039

linear model Mean log2-ratios to ACTIN2 plusmn SE are shown Letters indicate significantly 1040

different groups at p lt 005 for panels (A) (B) and (D) and q lt 005 for panel (C) 1041

(A) Plants were inoculated with 001 macerozyme or mock inoculated using water and1042

samples were collected 3 h later Data from four biological replicates are shown 1043

(B) Plants were inoculated with 100 microg mL-1 OGs or mock inoculated using water and1044

samples collected 3 h later Data are from six biological replicates 1045

(C) JAZ10 expression 48 h after treatment with the indicated B cinerea strains Data1046

are from six biological replicates 1047

(D) JAZ10 expression in untreated plants Data are from three biological replicates1048

1049

Figure 11 Gallo 1-Induced Callose Deposition Is Reduced in gae1 gae6 Plants 1050

Page 46 of 54

(A) Callose deposition in four week-old Col-0 and gae1 gae6 plants Plants were1051

inoculated with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) or mock inoculated 1052

with B cinerea inoculation medium and samples were collected after 72 h Callose was 1053

stained with aniline blue and visualized using a Nikon Eclipse Ni-U microscope with a 1054

DAPI filter Three leaves were stained per treatment Scale bars are 100 microm Asterisks 1055

indicate fungal hyphae arrows indicate representative callose spots 1056

(B) Quantification of callose deposition Callose deposits were counted in 200 microm x 2001057

microm squares laid out across the images shown here Bars represent means plusmn SE from 1058

six squares each Letters indicate significantly different groups (p lt 005) according to a 1059

t-test analysis1060

1061

Figure 12 Less Soluble Pectin Accumulates in B cinerea-Infected gae1 gae6 1062

Leaves 1063

Four week-old Col-0 and gae1 gae6 plants were inoculated with 10 microL droplets of the 1064

indicated B cinerea isolate (25 x 105 spores mL-1) or mock inoculated with B cinerea 1065

inoculation medium Leaves were collected 48 h later AIR was extracted from leaf 1066

samples and water added (20 microL infection site) to extract soluble pectin fragments 1067

Samples were vortexed for 7 h centrifuged at 12000 g and the uronic acid 1068

concentration of the supernatant was measured Data from nine biological replicates 1069

were combined using a mixed linear model Means plusmn SE are shown Letters indicate 1070

significant differences (q lt 005) 1071

1072

Figure 13 Macerozyme-Induced PTI Is Lost in gae1 gae6 While OG-Induced PTI Is 1073

Unaltered 1074

(A) Macerozyme-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1075

inoculated with 001 macerozyme or mock inoculated with boiled macerozyme and 1076

were infected with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) 24 h later Samples 1077

were collected immediately (0 dpi) and after two (2 dpi) and three (3 dpi) days Data 1078

show mean plusmn SE of six to 14 biological replicates each combined using a linear model 1079

Letters indicate significantly different groups (q lt 005) 1080

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

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Page 7 of 54

RESULTS 175

176

GAE1 and GAE6 Expression Is Repressed by Pma ES4326-Induced Immune 177

Signaling 178

Our previous research suggested that the status of pectin specifically pectin 179

modification by methylesterification is important for plant immunity against Pma 180

ES4326 (Bethke et al 2014) Here we investigate whether pectin abundance in 181

addition to its specific structure is also important for plant immunity by taking a closer 182

look at the formation of the pectin precursor UDP-GalA by GAEs 183

184 Microarray experiments showed that expression of GAE1 and GAE6 was

185 repressed in A thaliana (Arabidopsis) plants treated with Pma ES4326 (Wang et al

186 2008) Repression of GAE1 and GAE6 in wild-type Arabidopsis Col-0 plants treated with

187 Pma ES4326 was confirmed by quantitative real time (qRT)-PCR Figure 1) To test if

188 this repression is mediated by immune signaling qRT-PCR was also performed in

189 mutants with blocked immune signaling (Figure 1 and Supplemental Figure 1) 190 Generally immunity to Pma ES4326 requires intact SA signaling Repression of both

191 GAE1 and GAE6 required the presence of the major immune regulators EDS1 and

192 PAD4 required for SA signaling as well as other immune responses (Jirage et al 1999

193 Wiermer et al 2005 Bartsch et al 2006 Wang et al 2008) at both early and late time

194 points The presence of AVRPPHB SUSCEPTIBLE 3 (PBS3) a regulator of many

195 immune responses including SA signaling (Nobuta et al 2007) was also required for

196 repression of GAE1 and GAE6 at an early time point (Figure 1) CALMODULIN-

197 BINDING PROTEIN 60-LIKE a (CBP60a) (a negative regulator of SA synthesis and

198 immunity (Truman et al 2013)) CBP60g and SYSTEMIC ACQUIRED RESISTANCE

199 DEFICIENT 1 (SARD1) (both promote SA biosynthesis and regulation of other immune

200 responses (Wang et al 2011)) SALICYLIC ACID INDUCTION DEFICIENT 2 SID2

201 (encodes a biosynthetic enzyme for SA (Wildermuth et al 2001)) and

202 NONEXPRESSER OF PR GENES 1 NPR1 (a regulator of the majority of SA-induced

203 immune signaling (Cao et al 1997 Yan and Dong 2014)) were not required for this

204 repression (Figure 1 and Supplemental Figure1) Thus repression of GAE1 and GAE6

Page 8 of 54

205 requires a regulatory function that is dependent on EDS1 and PAD4 partially

206 dependent on PBS3 but independent of SA

Page 9 of 54

There are six GAE family members in Arabidopsis GAE2 expression was 207

induced by Pma ES4326 in the absence of PAD4 or EDS1 GAE3 expression was 208

unaltered GAE4 was repressed early and GAE5 expression was induced in all 209

genotypes (Supplemental Figure 2) The similarity of the immune-regulated expression 210

of GAE1 and GAE6 suggested a redundant role of these genes in Arabidopsis immunity 211

to Pma ES4326 212

213

Resistance to Pma ES4326 Is Compromised in gae1 gae6 and gae6 Plants 214

To study the roles of GAE1 and GAE6 in plant immunity in more detail plants 215

with T-DNA insertion mutations in GAE1 and GAE6 were obtained from the SALK 216

collection as described in the Accession Numbers section of Methods We identified two 217

GAE6 knock-out alleles gae6-1 and gae6-2 and a T-DNA insertion in GAE1 gae1-1 218

that caused reduced expression of this gene (Supplemental Figure 3) A double mutant 219

was created by crossing gae1-1 and gae6-1 Both gae6 and gae1 gae6 plants were 220

more susceptible to Pma ES4326 than wild-type Col-0 and gae1 plants supporting a 221

role of GAE6 in plant immunity to Pma ES4326 (Figure 2) 222

223

gae6 and gae1 gae6 Plants Have Brittle Leaves 224

While inoculating leaves of gae1 gae6 plants we noticed that the petioles and 225

midribs of these plants tended to break easily when leaves were bent against the 226

proximal-distal axis of the leaves (Figure 3 see Supplemental Figure 4D to F) They 227

tended to break less often when twisted around the proximal-distal axis To quantify 228

this phenotype leaves were bent by 180deg (Figure 3A) and breaking of midribs or 229

petioles was recorded Most gae1 gae6 leaves broke whereas fewer leaves of gae6 230

plants and very few leaves of wild-type Col-0 and gae1 plants did (Figure 3B and 231

Supplemental Figure 4C) Although gae1 gae6 plants had slightly shorter leaves and 232

petioles than wild type plants (Supplemental Figure 4A and B) no correlation between 233

leaf size or petiole length and breaking frequency was detected We also investigated 234

tissue integrity by monitoring ion leakage Interestingly when leaves were inoculated 235

with deionized water leaf discs of gae1 gae6 leaked more ions than wild-type Col-0 236

discs (Figure 3C) suggesting that the increased brittleness is correlated with a 237

Page 10 of 54

_____________________________________________________________________________

Page 11 of 54

Cell Walls of gae6 and gae1 gae6 Plants Have Lower Levels of Pectin 243

GalA is a major component of pectin We analyzed wall monosaccharide 244

composition in gae mutants to investigate whether the increased brittleness was 245

associated with an altered wall composition of gae leaves To approximate pectin 246

content of gae mutant walls the total uronic acid concentration was measured using a 247

simple colorimetric assay (Filisetti-Cozzi and Carpita 1991 van den Hoogen et al 248

1998) Total uronic acid was reduced in gae6-2 strongly reduced in gae1 gae6 plants 249

but was indistinguishable from wild-type Col-0 in gae6-1 and gae1 (Supplemental 250

Figure 5) This suggested that gae1 gae6 and possibly gae6 plants have reduced 251

pectin 252

To investigate the cell wall composition in leaves of gae plants in more detail we 253

measured the levels of seven neutral monosaccharides the acidic sugars GalA (the 254

monomeric subunit of the pectin backbone (Harholt et al 2010)) and GlcA (Figure 4) 255

We also estimated the crystalline cellulose content (Updegraff 1969) Cell walls of both 256

gae6 mutants and gae1 gae6 were reduced in GalA (Figure 4A) In gae1 gae6 GalA 257

content in cell walls was reduced by more than 40 compared to the wild-type level 258

GlcA levels were indistinguishable between mutants and wild-type (Figure 4B) 259

Cellulose content was higher in gae6-2 and gae1 gae6 walls likely due to the loss of 260

pectins (Figure 4C) Cell walls of gae1 gae6 plants were also enriched in all neutral 261

sugars measured presumably due to the substantial proportional loss of GalA (Figure 262

4D) The levels of most wall monosaccharides were even higher in the gae1 gae6 263

double mutant with the exception of Rha which did not exhibit a significant increase 264

compared to the gae6 single mutants The absence of a compensational increase in 265

Rha suggested that RGI the dominant Rha contributor to the wall may be considered 266

reduced in gae1 gae6 mutants compared to wild-type albeit not to such an extent as 267

HG 268

238 structural defect that promotes increased ion leakage Taken together these results

239 showed that loss of GAE6 causes brittle leaves and additional losss of GAE1 strongly

240 enhances this phenotype We conclude that GAE1 and GAE6 contribute to normal leaf

241 flexibility 242

Page 12 of 54

gae6 cell walls are reduced in HG Using the same samples we detected no difference 274

from wild type in binding of LM5 which recognizes a tetramer of (1-4)-β-D-galactans 275

found in RGI (Jones et al 1997) LM6 which recognizes a linear pentasaccharide in (1-276

To further investigate which pectic components were altered in gae1 gae6 dot 269

blot experiments using pectin isolated from the various genotypes were performed Both 270

LM19 and LM20 antibodies recognize HG (Verhertbruggen et al 2009a) As expected 271

LM19 binding was reduced in gae6-1 and gae1 gae6 (Figure 5A and B) and LM20 272

binding was reduced in gae1 gae6 (Figure 5C and D) This result confirmed that gae1 273

277 5)-α-L-arabinans found in RGI (Willats et al 1998 Verhertbruggen et al 2009b) LM8

278 which recognizes xylogalacturonan (Willats et al 2004) or CCRC-M7 which

279 recognizes an arabinosylated (1-6)-β-D-galactan epitope occurring on RGI (Puhlmann et

Page 13 of 54

Page 14 of 54

al 1994 Steffan et al 1995 Pattathil et al 2010) (Supplemental Figure 6) 280

Collectively these cell wall composition data show that gae1 gae6 plants are reduced in 281

HG Due to the strong proportional reduction of HG in the walls levels of all other 282

components should be higher However Rha indicative of RGI was not higher in gae1 283

gae6 suggesting that RGI may also be reduced in gae1 gae6 walls 284

285

GAE1 and GAE6 Are Required for Immunity to Specific B cinerea Isolates 286

We hypothesized that plant immunity to B cinerea might be affected by pectin 287

content in the host cell wall because this pathogen requires polygalacturonases and 288

pectin methylesterases for full virulence (ten Have et al 1998 Valette-Collet et al 289

2003 Kars et al 2005b Espino et al 2010) Because Bcinerea is genetically diverse 290

and has a high diversity of polygalacturonases (Rowe and Kliebenstein 2007) we 291

measured relative fungal growth at both 2 and 3 d post-inoculation (dpi) using ten 292

different B cinerea isolates (Figure 6) The B cinerea isolates Fresa 525 and Gallo 1 293

showed enhanced virulence on gae1 gae6 plants at both time points whereas KT and 294

UK Razz showed enhanced virulence in gae1 gae6 plants at 3 but not 2 d dpi (Figure 295

6A) We did not detect any significant changes in relative fungal growth between wild-296

type Col-0 and gae1 gae6 plants when using Acacia Apple 517 DN Grape Pepper or 297

Rasp isolates of B cinerea Therefore we continued our study using Gallo 1 and Fresa 298

525 We also included Pepper for comparison as this isolate has been used in a large-299

scale analysis of plant gene expression in response to B cinerea infection (Windram et 300

al 2012) 301

We next investigated whether expression of GAE1 and GAE6 was affected by 302

challenge with B cinerea Inoculation of wild-type Col-0 plants with any isolate 303

repressed GAE1 and GAE6 expression (Figure 6B and C) and this repression was 304

generally independent of PAD4 (Figure 6B and C) Expression of GAE2 and GAE3 was 305

unaltered by B cinerea treatment GAE4 was repressed only by Fresa 525 treatment 306

while GAE5 expression was enhanced with all three isolates tested (Supplemental 307

Figure 7) As seen for infection with Pma ES4326 the expression of GAE1 and GAE6 308

was repressed upon B cinerea treatment This indicates a role for GAE1 and GAE6 in 309

the immune response to B cinerea 310

Page 15 of 54

Page 16 of 54

Because the amount of pectin substrate for pathogen polygalacturonases is 313

reduced we hypothesized that immunity phenotypes of gae1 gae6 plants may result 314

from reduced generation of OGs by these enzymes In turn the reduced OGs could 315

lead to reduced immune responses To test this we used macerozyme a commercial 316

pectinase as a proxy for pathogen pectinases We isolated cell walls as a subcellular 317

fraction known as alcohol insoluble residue (AIR Gille et al 2009) This was treated 318

with macerozyme and tested for release of soluble likely low molecular weight pectin 319

fragments (Figure 7) Treatment with 025 macerozyme released significantly more 320

uronic acid from wild-type Col-0 AIR relative to gae1 gae6 AIR (Figure 7B) To test 321

activity of these pectin-containing solutions we infiltrated them into wild-type Col-0 322

plants and measured PAD3 expression PAD3 is required for synthesis of the 323

phytoalexin camalexin (Zhou et al 1999) and its expression is known to be induced by 324

OG treatment (Ferrari et al 2007) Fractions from macerozyme treated Col-0 walls 325

induced significantly more PAD3 expression than fractions derived from gae1 gae6 326

walls suggesting that macerozyme treatment did indeed release more active soluble 327

uronic acid possibly OGs from wild-type Col-0 walls than from gae1 gae6 walls (Figure 328

7C) 329

Next we studied the effect of macerozyme treatment on expression of PAD3 in 330

intact plants (Figure 8) This treatment did not lead to any visible leaf damage 331

(Supplemental Figure 8) In wild-type Col-0 PAD3 expression was induced three h after 332

treatment with 001 macerozyme (Figure 8A) By contrast no macerozyme-induced 333

induction of PAD3 expression was detected in gae1 gae6 (Figure 8A) However OG 334

treatment induced the expression of PAD3 in both genotypes indicating that gae1 gae6 335

plants do respond to OGs (Figure 8B) Macerozyme treatment also induced expression 336

of the SA marker genes PATHOGENESIS RELATED 1 (PR1) and SID2 in Col-0 but 337

not in gae1 gae6 (Figure 9 panel A and B) PR1 was not significantly induced by 338

purified OGs (Figure 9C) at the time and concentration tested but SID2 was induced by 339

OG treatment in both genotypes (Figure 9D) However the induction was stronger in 340

wild-type Col-0 In addition the JA markers JASMONATE-ZIM-DOMAIN PROTEIN 5 341

(JAZ5) and JAZ10 were induced by macerozyme and OG treatment in both Col-0 and 342

gae1 gae6 (Figure 10A and Supplemental Figure 11) Interestingly expression of JAZ5 343

312 Pectinase-Induced Immune Signaling Is Altered in gae1 gae6 Plants

Page 17 of 54

Page 18 of 54

Page 19 of 54

Page 20 of 54

344

345

346

and JAZ10 was already strongly induced in gae1 gae6 after mock inoculation (Figure 10

and Supplemental Figure 11) We observed no hyperresponsive induction by mock

treatment of PLANT DEFENSIN 12 (PDF12) a necrotrophy and late JA signaling

347 marker gene (Supplemental Figure 12A-D) PDF12 was also not induced by

348 macerozyme or OG treatments (Supplemental Figure 12A-D) This suggests that early

349 JA signaling is induced by mock-treatment in gae1 gae6 Because it is known that JA

350 and SA signaling act antagonistically (Pieterse et al 2012) the strong up-regulation of

351 JA-induced immune signaling in gae1 gae6 might explain the lower OG-induced SID2

352 expression in this genotype

353 To test whether other immune responses are altered in gae1 gae6 plants we

354 analyzed Mitogen-activated protein kinase (MAPK) activity following macerozyme and

355 OG treatments OGs are known to activate the MAPKs MPK3 and MPK6 in Arabidopsis

Page 21 of 54

(Galletti et al 2011) Macerozyme treatment activated MPK3 and MPK6 in wild-type 356

Col-0 whereas MAPK activation in gae1 gae6 was slightly reduced in four out of five 357

experiments performed (Supplemental Figure 13) By contrast OG-induced MAPK 358

activity was similar or increased in gae1 gae6 in four out of five experiments 359

(Supplemental Figure 13) Additionally mock treatment induced MAPK activity in gae1 360

gae6 more strongly than it did in wild-type Col-0 in four out of five experiments 361

performed (Fig S13) Collectively these results illustrate that activation of several 362

immune signaling componentsmdashcamalexin synthesis SA and JA signaling and 363

possibly MAPK activationmdashby macerozyme is substantially altered in gae1 gae6 This 364

suggests that release of soluble pectin fragments possibly OGs by pectinolytic 365

enzymes might be reduced in gae1 gae6 plants while recognition of OGs is intact 366

367

Following B cinerea Infection Production of Soluble Pectin and Deposition of 368

Callose Are Reduced in gae1 gae6 Plants 369

Although pad3 plants were more susceptible to the B cinerea isolates Fresa 525 370

and Gallo 1 which also grew better on gae1 gae6 growth of the isolate Pepper was 371

similar on Col-0 and pad3 (Supplemental Figure 9A) However we did not detect any 372

significant changes in camalexin production after treatment with B cinerea isolate Gallo 373

1 and Fresa 525 in gae1 gae6 (Figure 8D) Neither did we detect changes in PAD3 374

expression after treatment with any B cinerea isolate in gae1 gae6 (Figure 8E) 375

Additionally SID2 and PR1 were similarly induced by all B cinerea isolates in both Col-376

0 and gae1 gae6 (Figure 9E and F) and sid2 plants which are deficient for pathogen-377

induced SA biosynthesis did not show any differences in relative B cinerea growth 378

(Supplemental Figure 9B) Expression of JAZ10 was induced more strongly by Gallo 1 379

treatment in gae1 gae6 than in wild-type Col-0 but not by Fresa 525 or Pepper (Fig 380

10C) while dde2 plants deficient for JA biosynthesis showed enhanced susceptibility to 381

all B cinerea isolates tested (Supplemental Figure 9B) Curiously exogenous 382

application of MeJA did not lead to altered relative fungal growth of B cinerea 383

(Supplemental Figure 9C) and cev1 isoxaben resistant 1 (ixr1) plants which have 384

constitutively high JA levels (Ellis and Turner 2001) did not show any changes in 385

susceptibility to the B cinerea isolates tested (Supplemental Figure 9D) Hence neither 386

Page 22 of 54

differences in B cinerea isolate susceptibility to camalexin nor altered SA or JA 387

signaling by themselves explained the enhanced susceptibility of gae1 gae6 to these B 388

cinerea isolates 389

Hydrogen peroxide accumulation in response to B cinerea isolates Fresa 525 390

Gallo 1 and Pepper was not obviously altered in gae1 gae6 plants (Supplemental Figure 391

14) However callose deposition upon treatment with B cinerea isolate Gallo 1 was 392

strongly reduced in gae1 gae6 (Figure 11) It was previously shown that OG treatment 393

induces callose deposition (Denoux et al 2008) suggesting that the reduction in 394

callose deposition might be due to reduced OG release in gae1 gae6 It is possible that 395

this reduction in callose deposition contributes to the reduced immunity of gae1 gae6 396

plants to B cinerea isolate Gallo 1 397

Because macerozyme could be used to release soluble likely low molecular 398

weight pectin from isolated cell walls we next investigated whether infection with B 399

cinerea was accompanied by accumulation of soluble pectin as well We prepared cell 400

walls (AIR fraction) from leaves infected with B cinerea incubated these cell walls with 401

water and measured uronic acid content of the extracts B cinerea treatment did in fact 402

lead to increased accumulation of soluble pectin in wild-type Col-0 plants when 403

compared to mock-treated samples (Figure 12) No such increased accumulation of 404

water-soluble pectin fractions was observed in gae1 gae6 (Figure 12) 405

406

Macerozyme-Induced Pattern-Triggered Immunity against B cinerea Is Abolished 407

in gae1 gae6 408

It was previously shown that OG treatment can induce patternndashtriggered 409

immunity (PTI) to B cinerea (Ferrari et al 2007) We tested macerozyme treatment to 410

determine whether it could also induce PTI to the B cinerea isolate Gallo 1 (Figure 13) 411

Indeed pretreatment with 001 macerozyme for 24 h led to reduced B cinerea growth 412

in wild-type Col-0 but not gae1 gae6 plants (Figure 13A) Interestingly mock and 413

macerozyme pretreated gae1 gae6 plants exhibited similar growth of B cinerea Gallo 1 414

as did macerozyme-treated Col-0 plants This result is likely due to activation of JA 415

signaling in gae1 gae6 during the pretreatment procedure (Figure 13C and D) OG-416

induced PTI was observed in both genotypes when using a high dose of OGs (Figure 417

Page 23 of 54

13B) whereas a lower dose of OGs only induced PTI in Col-0 (Supplemental Figure 418

15) This suggests that OG perception in gae1 gae6 at least above a certain threshold 419

is unaltered while release of immunity-activating pectin fragments possibly OGs is 420

421 reduced We conclude that immune responses induced by macerozyme treatment

422 which do not occur in gae1 gae6 plants are important for plant immunity to the B

423 cinerea isolate Gallo 1

Page 24 of 54

____________________________________________________________________________

Page 25 of 54

DISCUSSION 426

Here we showed that loss of GAE1 and GAE6 leads to a dramatic reduction in 427

pectin content in the cell wall (Figure 4) likely causing the brittleness of leaves that we 428

observed (Figure 3) Petioles and midribs of gae1 gae6 plants broke easily when bent 429

and inoculation of leaves with deionized water resulted in increased ion leakage 430

possibly due either to microscopic fractures of the cell wall induced by inoculation stress 431

or to a reduced capability of these walls to withstand increased turgor pressure The 432

increased brittleness was associated with altered cell wall composition In our analysis 433

the GalA content of gae1 gae6 leaf walls was reduced by more than 40 and the total 434

uronic acids by over 60 (Figure 4 and Supplemental Figure 5) This is a major change 435

in the overall cell wall composition considering that pectin makes up about 50 of the 436

plant cell walls found in Arabidopsis leaves (Zablackis et al 1995 Nobuta et al 2007 437

Harholt et al 2010) Interestingly quasimodo1 (qua1) plants which have a mutation in 438

a glycosyltransferase family 8 gene are reduced in GalA by 25 and show reduced 439

binding of HG specific antibodies (Bouton et al 2002) 440

Furthermore qua2 plants which have a mutation in a putative methyltransferase 441

have about 13 less total GalA and lack 50 of cell wall HG (Mouille et al 2007) 442

Likewise cgr2 cgr3 plants carrying mutations in another class of Golgi-localized 443

putative methyl-transferases have leaf cell walls that are reduced by about 40 in 444

uronic acid (Kim et al 2015) All three mutants are severely dwarfed (Bouton et al 445

2002 Mouille et al 2007 Kim et al 2015) Additionally qua1 and qua2 mutants show 446

a distorted overall shape likely due to reduced cell cohesion and are extremely fragile 447

(Bouton et al 2002 Mouille et al 2007) By contrast gae1 gae6 plants with a similar 448

strong reduction in GalA and total uronic acids have an overall normal shape and are 449

only slightly smaller than wild-type Col-0 (Supplemental Figure 4) In the wall analysis 450

performed here we did not detect changes in other wall polysaccharides or structures 451

that might compensate for the reduction of pectin in gae1 gae6 It is possible the severe 452

morphological alterations in qua2 and cgr2 cgr3 plants are due to reduction in pectin 453

methylesterification which is unaffected in gae1 gae6 It was speculated that quality 454

control mechanisms in the Golgi prevent secretion of inefficiently esterified pectin in 455

cgr2 cgr3 (Kim et al 2015) suggesting that the Golgi of these plants might over-456

Page 26 of 54

accumulate pectin and that this may contribute to the severe dwarf phenotype of these 457

plants The gae1 gae6 plants described here provide an opportunity to study the effects 458

of reduction of pectin in plants that do not show severe morphological phenotypes 459

We found that gae1 gae6 plants are also compromised in resistance to B 460

cinerea and Pma ES4326 indicating that loss of pectin reduces immunity to these 461

pathogens (Figure 2 and 6) We found that gae1 gae6 plants were slightly more 462

susceptible to Pma ES4326 but showed a strong increase in susceptibility to specific B 463

cinerea isolates This was not unexpected because B cinerea is known to interact with 464

pectin as part of its infection strategy It secretes various pectin-degrading enzymes 465

early during infection and requires some of these enzymes including PG1 PG2 and 466

PME1 for full virulence (ten Have et al 1998 Valette-Collet et al 2003 Kars et al 467

2005b Espino et al 2010) Additionally Arabidopsis plants expressing an antisense 468

construct of the polygalacturonase inhibitor PGIP1 are significantly more susceptible to 469

B cinerea (Ferrari et al 2006) On the other hand Pma ES4326 is not known to470

require cell wall degrading enzymes for full virulence even though it encodes two 471

pectate lyase genes in its genome (PMA4326_07094 and PMA4326_04621 472

wwwbacteriaensemblorg) B cinerea isolates are also known to be genetically quite 473

diverse in their polygalacturonase (PG) loci and genetic variability in PG2 is associated 474

with varying growth on pectin containing media (Rowe and Kliebenstein 2007) 475

Furthermore the contribution of PG1 and PG2 to B cinerea virulence seems to depend 476

on the specific B cinerea isolate and the host species (Kars et al 2005a Zhang and 477

Van Kan 2013) B cinerea isolates are also known to be differently sensitive to the anti-478

microbial secondary metabolite camalexin (Kliebenstein et al 2005) In line with these 479

observations the susceptibility of gae1 gae6 plants differed depending on the specific 480

B cinerea isolate Whereas Fresa 525 Gallo 1 UK Razz and KT grew better in gae1481

gae6 we detected no difference in Acacia Apple 517 DN Grape Pepper or Rasp 482

growth (Figure 6) 483

B cinerea was shown to metabolize pectin and to possibly utilize GalA as a484

carbon source during in planta growth (Zhang et al 2011 Zhang and Van Kan 2013) 485

Our data show that gae1 gae6 plants which are reduced in GalA content did allow 486

enhanced growth of specific B cinerea isolates but no reduction in growth of any B 487

Page 27 of 54

cinerea isolates tested was found This suggests that utilization of pectin as a carbon 488

source was not a contributing factor to alterations in B cinerea susceptibility It is 489

possible that changes in the cell wall composition affect the structural integrity of the 490

wall and affect hyphal penetration of specific B cinerea isolates However OGs (small 491

HG fragments) are likely released during the infection process (Ferrari et al 2013) 492

OGs function as DAMPs and initiate immune responses in Arabidopsis (Ferrari et al 493

2013) Treatment of Arabidopsis with OGs having a degree of polymerization (DP) of 494

10-15 sugar subunits was previously shown to enhance resistance to B cinerea (Ferrari495

et al 2007) We showed that pretreatment with OGs or macerozyme induced enhanced 496

immunity to B cinerea isolate Gallo 1 in wild-type Col-0 but only OGs and not 497

macerozyme induced immunity in gae1 gae6 (Figure 13) Our data suggest gae1 gae6 498

plants which have reduced pectin content may release less OGs and that this 499

reduction leads to the increased susceptibility to B cinerea 500

Consistently we found that macerozyme treatment released less soluble pectin 501

from gae1 gae6 cell walls than from wild-type walls Fractions containing soluble pectin 502

released from gae1 gae6 were also less active in inducing PAD3 gene expression than 503

the fractions released from wild-type walls (Figure 7) We also showed that less soluble 504

pectin accumulated in gae1 gae6 than in wild-type plants 48 h after treatment with B 505

cinerea (Figure 12) 506

We observed that loss of pectin leads to alterations in A thaliana immune 507

signaling Macerozyme treatment induced expression of PAD3 and the SA marker 508

genes PR1 and SID2 in wild-type Col-0 but not in gae1 gae6 whereas OG treatment 509

led to increased PAD3 and SID2 expression in both wild-type and gae1 gae6 (Figure 8 510

and 9) This suggests that although release of active soluble pectin in these plants 511

might be altered OG recognition is intact In fact cell walls isolated from qua1 another 512

mutant known to be reduced in HG released less OGs (DP1-6) than did wild-type cell 513

walls upon endopolygalacturonase treatment (Bouton et al 2002) This was also true 514

for gae1 gae6 cell walls as macerozyme treatment of isolated gae1 gae6 cell walls 515

released less soluble pectin than treatment of Col-0 cell walls Because the fraction 516

from gae1 gae6 plants was also less active in inducing PAD3 expression (Figure 7) it 517

seems possible that biologically active OGs were the active component inducing PAD3 518

Page 28 of 54

expression Interestingly expression of the JA marker genes JAZ5 and JAZ10 was 519

higher in gae1 gae6 than in wild-type mock-inoculated plants but was comparable to 520

wild-type in untreated plants suggesting that JA signaling in gae1 gae6 is 521

hyperresponsive (Figure 10 and Supplemental Figure 11) This induction of JA signaling 522

might be due to wounding stress incurred during the inoculation process JA and SA 523

signaling generally act antagonistically (Pieterse et al 2012) Increased JA signaling 524

activity might hence explain why mock- and macerozyme-treated gae1 gae6 plants 525

exhibited reduced expression of the SA marker PR1 and why OG treatment induced 526

expression of SID2 another SA marker to a lower extent in gae1 gae6 than in wild-type 527

plants (Figure 9) 528

No single immune signaling component was found to be the sole contributor to 529

the compromised immunity of gae1 gae6 to specific B cinerea isolates such as Gallo 1 530

and Fresa 525 These isolates grew better on pad3 while growth of Pepper another B 531

cinerea isolate that did not show altered virulence on gae1 gae6 was unaltered 532

(Supplemental Figure 9) This suggested that differences in camalexin sensitivity of 533

these B cinerea isolates might account for the differences in gae1 gae6 susceptibility 534

However camalexin accumulation and PAD3 expression was the same for wild-type 535

Col-0 and gae1 gae6 for the B cinerea isolates Fresa 525 and Gallo 1 (Figure 8) which 536

showed enhanced growth on gae1 gae6 plants It should be noted that B cinerea 537

virulence is often estimated by comparing lesion sizes Lesion size and measurements 538

of relative fungal growth by qPCR do not always show the same results For example 539

although Denby et al (2004) showed that Pepper causes larger lesions on pad3 than on 540

wild-type this study showed that relative fungal growth of Pepper on pad3 was 541

unaltered 542

We observed that callose deposition was strongly reduced in gae1 gae6 plants 543

72 h after treatment with B cinerea isolate Gallo 1 (Figure 11) possibly due to reduced 544

release of active soluble pectin Interestingly OG-induced PTI to an unspecified isolate 545

of B cinerea was intact in the pmr4 mutant which does not produce callose (Galletti et 546

al 2008) Due to the high SA levels in pmr4 plants (Nishimura et al 2003) and the fact 547

that different B cinerea isolates vary both in induction of and reaction to defense 548

responses (Kliebenstein et al 2005 Rowe et al 2010) we could not determine 549

Page 29 of 54

whether reduced callose deposition causes the increased susceptibility of gae1 gae6 to 550

B cinerea isolate Gallo 1551

We did not observe any changes in B cinerea growth either on cev1 ixr1 plants 552

which are known to have constitutively high JA and OPDA levels (Ellis et al 2002b) or 553

on plants exogenously treated with MeJA (Supplemental Figure 9) Because plants 554

were kept uncovered for 3 h after MeJA treatment it is unclear how much MeJA was 555

bioavailable in the plant However JAZ10 expression was induced 3 h after spraying 556

with MeJA (Supplemental Figure 10) Because pathogen growth generally depends on 557

the combination of a multitude of immune signaling events we conclude that multiple 558

changes to the immune signaling in gae1 gae6 together account for the altered B 559

cinerea growth phenotypes detected 560

We showed that expression of GAE1 and GAE6 is repressed by the bacterial 561

hemibiotroph Pma ES4326 This repression did not occur in an eds1 or pad4 562

background or in pbs3 at the 24-h time point (Figure 1 and Supplemental Figure 1) 563

During the Arabidopsis - Pma ES4326 interaction the expression of many defense 564

related genes is altered in a PAD4EDS1 dependent fashion (Wang et al 2008) Some 565

of these expression changes require SA CBP60g and SARD1 are thought to act 566

downstream of PAD4 and EDS1 but upstream of SA-dependent responses (Wang et 567

al 2011) PBS3 is also thought to act downstream of PAD4 and EDS1 and upstream of 568

SA but the set of genes whose expression it affects is only partially overlapping with 569

those affected by CBP60gSARD1 (Wang et al 2008 Wang et al 2011) Thus GAE1 570

and GAE6 repression is regulated by the part of the immune signaling network that 571

requires PAD4 EDS1 and PBS3 but is independent of CBP60gSARD1 and SA 572

Because EDS1 PAD4 and PBS3 are important components of the plant immune 573

signaling network (Wiermer et al 2005 Nobuta et al 2007) one might conclude that 574

repression of GAE1 and GAE6 may be part of the plant immune response to Pma 575

ES4326 B cinerea treatment also reduced GAE1 and GAE6 expression but this was 576

mostly independent of PAD4 (Figure 6) while both Pma ES4326 and B cinerea repress 577

GAE1 and GAE6 expression the host signaling mechanisms responsible likely differ 578

In summary our data show that Arabidopsis GAE1 and GAE6 are required for 579

pectin biosynthesis leaf flexibility and immunity to Pma ES4326 and to specific B 580

Page 30 of 54

cinerea isolates We used macerozyme a commercial pectinase to mimic the effect of 581

B cinerea pectinases Macerozyme treatment released less pectin from gae1 gae6 cell582

walls and induced less immune signaling in gae1 gae6 plants Less water soluble pectin 583

also accumulated in B cinerea infected gae1 gae6 plants and macerozyme-induced 584

immunity to B cinerea isolate Gallo 1 was reduced in these plants We conclude that 585

the reduced pectin content in these mutants may lead to reduced release of soluble 586

pectin including active OGs during pathogen attack and that this compromises 587

immunity 588

589

590

Page 31 of 54

METHODS 591

Plant Materials and Growth Conditions 592

Wild-type Columbia (Col-0) and all mutant plants (in Col-0 background) were 593

grown on sterilized BM2 germinating mix (Berger Inc Quebec Canada) in a controlled 594

environment chamber (Conviron Pembina ND USA) with a 12-h photoperiod under 595

100 microM m-2 s-1 fluorescent illumination at 22degC and 75 relative humidity Germplasm 596

used is described in the Accession Numbers section at the end of Methods 597

598

Data Analysis and Replicates 599

For biological replicates samples were obtained from separate plant tissues and 600

in many cases from plants grown in different flats In some experiments multiple 601

technical replicates were measured per biological replicate For statistical analysis all 602

technical replicates were averaged and the averages were considered one biological 603

replicate Independent experiments were performed at different times and often 604

consisted of multiple biological replicates per experiment 605

Data analysis was performed using mixed linear models in the R programming 606

environment (Bates et al 2015) unless otherwise indicated Generally genotypes 607

treatments and time points were used as fixed effects and replicate-specific effects like 608

different pots flats etc were considered random effects We reported p-values 609

obtained from the mixed linear model when less than 10 comparisons were performed 610

and q-values for more than 10 comparisons to correct for multiple comparison testing 611

Q-values were calculated using the Benjamini-Hochberg procedure (Benjamini and612

Hochberg 1995) unless specified otherwise 613

614

Pathogen Strains Growth Conditions and Pathogen Growth Assays 615

Growth of Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) and 616

bacterial growth assays were performed as previously described (Bethke et al 2014) 617

For qRT-PCR assays four week-old plants were inoculated with bacteria (OD600 = 618

0002) or mock (5 mM MgSO4) At least four leaves from two plants were collected at 619

the indicated time points per biological replicate 620

Page 32 of 54

Prior to each experiment Botrytis cinerea isolates were grown on 1x PDA 621

medium (Difco) for 10 d at room temperature All isolates used have been described 622

previously (Rowe and Kliebenstein 2007) The spores were washed from the surface of 623

the plate using an inoculation medium (1x Gamborgrsquos B-5 basal salt mixture (Sigma-624

Aldrich G5768) 2 (wv) glucose 10 mM phosphate buffer pH 64) and fungal hyphae 625

were removed from the suspension by filtering through four layers of cheesecloth The 626

concentration of spores was determined using a hemocytometer and adjusted to 25 x 627

105 spores mL-1 Four week-old Arabidopsis plants were inoculated by placing one 10-628

microL droplet of the B cinerea spore solution or inoculation medium (mock) on the adaxial 629

leaf surface of fully expanded leaves Inoculated plants were kept at 100 relative 630

humidity Infection sites were collected at various time points using a cork borer For 631

qRT-PCR assays at least four leaf discs from one plant were combined for each 632

biological sample For B cinerea growth assays at least 12 leaf discs from three to six 633

plants were pooled per biological sample Relative fungal growth was determined by 634

measuring the abundance of a fungal gene relative to a plant gene as described below 635

In brief DNA from infected tissue was extracted and equal amounts of total DNA were 636

used to perform qPCR reactions using the B cinerea cutinase A and the Arabidopsis 637

SK11 gene respectively Primers were described in (Gachon and Saindrenan 2004) 638

and are listed in Supplemental Table 1 Two technical replicates were combined for 639

each biological replicate B cinerea is a necrotrophic fungal pathogen and may destroy 640

plant DNA as disease progresses However decreases in the amount of plant DNA 641

over the course of our experiments were only observed at late time points with very 642

susceptible plants so the qPCR assay is generally a reasonable estimate of fungal 643

biomass (Supplemental Figure 16) 644

645

Treatment with Macerozyme or OGs 646

For PTI assays four week-old plants either were inoculated with 001 (wv) 647

macerozyme R-10 (Yakult) a Rhizopus polygalacturonase (EC 32115) that possesses 648

high pectinase and hemicellulase activity or were mock inoculated using heat-649

inactivated macerozyme Other similar plants were either inoculated with 250 or 500 microg 650

mL-1 OGs or mock inoculated with water One d later plants were infected with B 651

Page 33 of 54

cinerea and samples were collected immediately (0 dpi) and two or three d later To 652

investigate the role of MeJA on B cinerea growth plants were sprayed with 1mM MeJA 653

in water with 001 (vv) Tween 20 Covers were removed so that leaves could dry off 654

before plants were inoculated with B cinerea three h later Samples were harvested at 655

the indicated time points For expression analysis and MAPK activation assays plants 656

were either inoculated with 100 microg mL-1 OGs or mock inoculated with water OGs used 657

for experiments described in Figures 8 to 10 and Supplemental Figure 12 were a kind 658

gift from Simone Ferrari OGs used in experiments described in Figure 13 and 659

Supplemental Figures 11 13 and 15 were prepared as previously described (Kohorn et 660

al 2014) 661

662

Expression Analysis 663

Leaf tissue from inoculated plants was harvested at the indicated time points 664

flash frozen pulverized and RNA extracted using Trizol (Invitrogen) Quantitative RT-665

PCR was performed using the SuperScript III Platinum SYBR Green One-Step 666

quantitative RT-PCR kit (Invitrogen) and a Lightcycler 480 Real-Time PCR system 667

(Roche) as previously described (Truman and Glazebrook 2012) In brief equal 668

amounts of total RNA and a gene specific primer were used for each reaction The 669

crossing point (Cp) was calculated using the 2nd derivative max method provided with 670

the Lightcycler software for each amplification curve Each reaction was run with two 671

technical replicates and the Cp values for these replicates were averaged ACTIN2 was 672

used as a stably expressed reference gene At least three biological replicates each 673

were performed and analyzed using a mixed-linear model RT-PCR was performed to 674

verify T-DNA insertion lines using the One-Step RT-PCR kit (Qiagen) according to the 675

manufacturerrsquos instructions The primers used can be found in Supplemental Table 1 676

677

Electrolyte Leakage Assay 678

Fully expanded leaves of four week-old plants were inoculated with water (Milli-Q 679

grade) using a needleless syringe Leaf discs were collected using a cork borer and 680

three leaf discs from one leaf were placed with the adaxial surface down onto 2 mL 681

water in a 12-well cell culture plate Conductivity was measured using a Horiba B-173 682

Page 34 of 54

conductivity meter after 30 min and then the water was replaced Conductivity was 683

measured again 30 min later (1 h total) as indicated in Figure 3C Six biological 684

replicates each were performed in three independent experiments and means and SEs 685

were calculated Two-sided t-tests were performed for each time point 686

687

DAB and Aniline Blue Staining 688

Leaves from four week-old plants were either mock inoculated with B cinerea 689

inoculation medium or inoculated with 10 microL droplets of a B cinerea spore solution (25 690

x 105 spores mL-1) of the indicated isolates Samples were collected 72 h later For 33prime-691

diaminobenzidine (DAB) staining two to five inoculated leaves were vacuum infiltrated 692

with 33prime-Diaminobenzidine (1 mg mL-1 in water pH 38) solution and stained overnight 693

in the dark Leaves were destained using 100 ethanol at 65degC and then transferred to 694

50 (vv) glycerol and photographed 695

Callose was visualized by staining leaves with aniline blue as previously 696

described (Adam and Somerville 1996) Briefly leaves were vacuum infiltrated with 697

alcoholic lactophenol (one volume of phenolglycerollactic acidwater (1111) mixed 698

with two volumes of ethanol) and destained at 65degC Next leaves were moved through 699

an alcohol gradient (50 20 and 10 (vv) ethanol) into water The destained leaves 700

were stained for 30 min in 150 mM K2HPO4 (pH 95) with 001 (wv) aniline blue 701

Samples were mounted in water and examined using a Nikon Eclipse Ni-U microscope 702

at 10x magnification using a DAPI filter (excitation at 325-375 nm emission at 435-485 703

nm) For callose quantification six 200 microm x 200 microm squares were counted for each 704

sample type Callose deposits per mm2 were calculated and data analyzed using a t-705

test Note that there is an inherent uncertainty associated with such counts because 706

callose deposits may not all be in focus at the same time 707

708

MAPK Activity Assay 709

Fully expanded leaves of four week-old plants were inoculated with 001 710

macerozyme 100 microg mL-1 OGs 100 nM flg22 or water (mock) flg22 a peptide from 711

bacterial flagella that induces immune responses was used as a positive control for 712

Page 35 of 54

MAPK activation (Asai et al 2002) and was purchased from EZBiolab Inc (Westfield 713

IN USA) Samples were harvested 10 min later and immediately frozen in liquid 714

nitrogen Protein was extracted using an extraction buffer containing 25 mM Tris pH 78 715

75 mM NaCl 1 mM DTT 1 mM NaF 05mM Na3VO4 01 (vv) Tween 20 one 716

cOmplete Mini protease inhibitor cocktail tablet (Roche) and one PhosSTOP 717

phosphatase inhibitor cocktail tablet (Roche) per 10 mL buffer 15 microg of total protein 718

were electrophoresed on a 10 SDS-polyacrylamide gel and then blotted onto a PVDF 719

membrane (Bio-Rad 162-0177) Activated MAPKs were detected using p4442 MAPK 720

(Erk12) antibody (9102S Cell Signal 12500 in TBST (20 mM Tris pH 75 150 mM 721

NaCl 01 (vv) Tween 20)) Anti-Rabbit IgG-HRP (115000 in TBST A6154 Sigma) 722

and ECL Plus substrate (Pierce 32132) MPK3 and MPK6 were detected with an anti-723

MPK3 antibody (M8318 Sigma 12000 in TBST and 3 (wv) milk) or an anti-MPK6 724

antibody (A7104 Sigma 16000 in TBST and 3 (wv) milk) respectively The 725

secondary antibody and detection were the same as for the p4442 antibody Five 726

independent experiments were performed 727

728

Preparation of Alcohol Insoluble Residue (AIR) Pectin Extraction and Uronic Acid 729

Measurements 730

Fully expanded leaves from four week-old plants that were incubated for 48 h in 731

the dark to reduce starch were harvested flash frozen and pulverized AIR was 732

extracted by washing ground material twice in 70 (vv) ethanol three times in a 733

mixture of chloroform and methanol (11 vv) and once in acetone (Gille et al 2009) 734

and was then air dried Cell wall pectin was extracted from AIR with cell wall extraction 735

buffer (50 mM Trizma 50 mM CDTA pH 72) at 95degC for 15 min Samples were 736

homogenized using a paint shaker (Harbil 5G-HD) and glass beads (3 mm diameter) 737

For dot blot experiments 500 microL of cell wall extraction buffer were used per 10 mg of 738

AIR Debris was precipitated by centrifugation for 10 min at 10000 g 739

Total uronic acid content of pectin extracted as described above using 500 microL of 740

cell wall extraction buffer per 1 mg of AIR was measured as described previously 741

(Filisetti-Cozzi and Carpita 1991 van den Hoogen et al 1998) First 36 microL of pectin 742

extract were mixed with 4 microL of 4 M sulfamic acid Then 200 microL of sulfuric acid 743

Page 36 of 54

containing 120 mM sodium tetraborate was added and samples were incubated at 80degC 744

for 1 h Following cooling on ice the optical density of samples was measured at 490 745

nm Next 40 microL of m-hydroxydiphenyl reagent (100 microL of m-hydroxydiphenyl in dimethyl 746

sulfoxide at 100 mgmL mixed with 49 mL 80 (vv) sulfuric acid just before use) was 747

added and the samples were mixed The optical density at 490 nm was measured 748

again The optical density before m-hydroxydiphenyl reagent addition was subtracted 749

from the optical density measured after addition of the dye The concentration of uronic 750

acid was calculated using known amounts of GalA as a standard 751

752

Release of Pectin from Arabidopsis Cell Walls Using Macerozyme 753

AIR preparations from leaves of four week-old plants were treated with 025 754

(wv) macerozyme R-10 (Yakult) or water (mock) at a concentration of 100 microL per mg 755

AIR Samples were incubated for 3 h at room temperature on a vortex shaker and 756

centrifuged for 10 min at 12000 g Total uronic acid concentration of 5-fold diluted 757

supernatants was determined for two technical replicates each The concentration of 758

uronic acid was calculated using known amounts of GalA as a standard For 759

subsequent qRT-PCR experiments supernatants from multiple extractions were 760

combined boiled for 20 min to inactivate macerozyme diluted 10-fold with water and 761

inoculated into four week-old Col-0 plants Heat-inactivated 10-fold diluted macerozyme 762

solutions were used as mock treatment Samples were collected 3 h later and PAD3 763

expression was determined 764

765

Release of Pectin from Arabidopsis Leaves Infected with B cinerea 766

Fully expanded leaves of four week-old plants were either inoculated with up to 767

five 10 microL droplets of B cinerea spore solution (25 x 105 spores mL-1) or mock 768

inoculated with B cinerea inoculation medium For each biological replicate leaves with 769

a combined count of at least 20 infection sites were collected 48 h later AIR was 770

extracted as described above but plants were not dark-treated or freeze-dried and the 771

first ethanol step was performed using 85 (vv) ethanol To extract soluble pectin 772

fragments AIR samples were mixed with water at a concentration of 20 microL per infection 773

site incubated for 7 h at room temperature on a vortex shaker and centrifuged for 10 774

Page 37 of 54

min at 12000 g Total uronic acid concentration of supernatants was determined for two 775

technical replicates each The concentration of uronic acid was calculated using known 776

amounts of GalA as a standard 777

778

Measurement of Uronic Acids Neutral Monosaccharides and Cellulose Content in 779

Cell Walls 780

Neutral sugars and uronic acids from non-cellulosic polysaccharides in cell walls 781

(AIR) were released by trifluoroacetic acid (TFA) hydrolysis Approximately 1 mg of AIR 782

was hydrolyzed with 2 M TFA at 121degC for 90 min The TFA-soluble material was split 783

into two equal parts one for uronic acid and one for neutral sugar determination The 784

uronic acid content was determined by analyzing the hydrolyzate using a CarboPac 785

PA200 anion-exchange column with an ICS-3000 Dionex chromatography system 786

(Dionex Sunnyvale CA USA) The elution profile consisted of a linear gradient of 50 to 787

200 mM sodium acetate in 01 M NaOH in 10 min at 04 mL per min Alditol acetate 788

derivatives of neutral sugars were produced as described (York et al 1985) In brief 789

monosaccharides in the hydrolyzate were reduced with NaBH4 and peracetylated with 790

acetic anhydride and pyridine at 121degC for 20 min The generated alditol acetates were 791

analyzed by GC-MS Crystalline cellulose was measured according to Updegraff (1969) 792

After hydrolysis of noncellulosic polysaccharides from AIR with the Updegraff reagent 793

(acetic acid nitric acids water 812 (vv) ) the remaining pellet was hydrolyzed in 794

72 sulfuric acid The resulting glucose quantity was determined by the anthrone 795

method (Scott and Melvin 1953) 796

797

Dot Blot Experiments 798

Pectin solutions extracted using 50 microL of cell wall extraction buffer per mg AIR 799

were serially diluted and nitrocellulose membranes were spotted with 1 microL of the diluted 800

pectin solutions Membranes were dried overnight blocked with 5 milk (wv) in 1x 801

PBS (8 g L-1 NaCl 02 g L-1 KCl 144 g L-1 Na2HPO4 024 g L-1 KH2PO4 pH 74) and 802

probed with LM5 (Jones et al 1997) LM6 (Willats et al 1998) LM8 (Willats et al 803

2004) LM19 and LM20 (Verhertbruggen et al 2009a) or CCRC-M7 (Steffan et al 804

Page 38 of 54

1995) antibodies LM series antibodies were diluted 1250 and CCRC-M7 was diluted 805

1500 in 5 milk powder (Nestle Carnation) in 1 x PBS For LM series antibodies a 806

goat anti-Rat HRP conjugated antibody (Bethyl A110-105P 15000 diluted) and for 807

CCRC-M7 an anti-Mouse IgG HRP conjugate (Promega W402B 12500 diluted) was 808

used as secondary antibody in 5 milk powder in 1x PBS LM series antibodies were 809

obtained from plant probes (wwwplantprobesnet) and CCRC-M7 from CarboSource 810

(wwwcarbosourcenet) Membranes were washed after incubation with the primary and 811

secondary antibodies with 1x PBS Dot blots were developed using the ECL system 812

(GE Healthcare Amersham ECL prime) For quantification of signals dot blot results on 813

x-ray films were photographed using a CCD camera and intensities were measured814

using Image Jrsquos Integrated Density function Locally adjusted background was 815

subtracted As the dynamic range of the signal on an x-ray film was narrow non-816

linearity in the measured values was corrected for each genotype in each biological 817

replicate using the values from different dilutions in two technical replicates We noticed 818

that the residuals were not normally distributed and performed Box-Cox power 819

transformation (Box and Cox 1964) to obtain normally distributed values for the 820

subsequent statistical analysis of the data For LM19 Box-Cox selected power 821

transformation was done to the power of 038th and for LM20 to the power 075th After 822

correction of the transformed measured values with the effect of the biological 823

replicates the values were fit to a linear model with the Arabidopsis genotype as the 824

fixed effect This linear model was used both to generate the plots in Figures 5B and D 825

and to test significant differences 826

827

Camalexin Measurements 828

Four week-old plants were infected with B cinerea and samples (three leaf disks 829

from one plant each) were harvested at the indicated time points flash frozen and 830

pulverized Each sample was extracted with 300 microL of 90 methanol (vv) 50 microL of the 831

extract was run on an Agilent Lichrocart 250-4 RP18e 5-microm column using an Agilent 832

1100 series HPLC Camalexin was detected using a DiodeArray (DAD) at 330 nm and 833

with a fluorescence detector at emission 318 nmexcitation 385 nm (Agilent) Separation 834

was achieved using the following program with aqueous acetonitrile 5 min gradient 835

Page 39 of 54

from 63 to 69 acetonitrile 30 s gradient from 69 to 99 acetonitrile 2 min at 99 836

acetonitrile and a post-run equilibration of 35 min at 63 acetonitrile (Denby et al 837

2004 Kliebenstein et al 2005) Purified camalexin was used to produce a standard 838

curve to identify and quantitate camalexin 839

840

Accession Numbers 841

Microarray data used can be accessed at NCBI Gene Expression Omnnibus 842

(httpwwwncbinlmnihgovgeo) GSE18978 Sequence information for all Arabidopsis 843

genes described in this article can be found in TAIR (wwwarabidopsisorg) using the 844

following accession numbers PAD3 (CYP71B15 At3g26830) SID2 (ICS1 At1g74710) 845

GAE1 (At4g30440) GAE2 (At1g02000) GAE3 (At4g00110) GAE4 (At2g45310) GAE5 846

(At4g12250) GAE6 (At3g23820) PR1 (At2g14610) JAZ5 (At1g17380) JAZ10 847

(At5g13220) ACTIN2 (At3g18780) PDF12a (At5g44420) PDF12b (At2g26020) 848

AtSK11 (At5g26751) The sequence of the B cinerea cutinase Bc-CutA (Z69264) can 849

be found in the DNA Data Bank of Japan (httpgetentryddbjnigacjp) Germplasm 850

used included dde2-2 (At5g42650 (von Malek et al 2002)) pad3-1 (At3g26830 (Zhou 851

et al 1999)) pad4-1 (At3g52430 (Jirage et al 1999)) sid2-2 (At1g74710 (Wildermuth 852

et al 2001)) pbs3-2 (At5g13320 SALK_018225 (Nobuta et al 2007)) cbp60a-1 853

(At5g62570 SALK_124410 (Truman et al 2013)) cbp60g-1 sard1-2 (At5g26920 854

SALK_023199 At1g73805 SALK_052422 (Wang et al 2011) cbp60a-1 cbp60g-1 855

sard1-2 (Truman et al 2013) gae1-1 (At4g30440 SALK_085554) gae6-1 (At3g23280 856

SALK_104454) gae6-2 (At3g23280 SALK_017191) ixr1-1 cev1 (At5g05170 (Ellis 857

and Turner 2001 Scheible et al 2001)) eds1 plants were derived by introgression of 858

the Ler eds1-2 allele into Col-0 that contains two copies of EDS1 (At3g48090 859

At3g48080 (Bartsch et al 2006) mpk6-2 (At2g43790 SALK_073907 (Liu and Zhang 860

2004)) mpk3-DG (At3g45640 a fast neutron deletion mutant (Miles et al 2005)) and 861

npr1-1 (At1g64280 (Cao et al 1997)) T-DNA insertion lines were part of the SALK 862

collection (Alonso et al 2003) and were obtained from the Arabidopsis Biological 863

Resource Center 864

865

Supplemental Data 866

Page 40 of 54

Supplemental Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 867

Expression Does Not Require CBP60s or SID2 868

Supplemental Figure 2 Expression of GAE Family Members uponTreatment with Pma 869

ES4326 870

Supplemental Figure 3 Characterization of T-DNA Insertions in GAE1 and GAE6 871

Supplemental Figure 4 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves and 872

Are Slightly Smaller than Wild-Type Col-0 Plants 873

Supplemental Figure 5 Cell Walls of gae1 gae6 Plants Contain Less Uronic Acid than 874

Wild-Type Col-0 Cell Walls 875

Supplemental Figure 6 Antibodies Raised Against the RGI or XG Components of 876

Pectin Show No Difference in Binding to gae Mutant and Col-0 Cell Walls 877

Supplemental Figure 7 The Effect of B cinerea Treatment on the Expression of GAE 878

Family Genes 879

Supplemental Figure 8 Treatment with 001 Macerozyme Does Not Cause Any 880

Visible Damage to Col-0 Plants 881

Supplemental Figure 9 B cinerea Growth on Plants with Defects in Immune 882

Signaling 883

Supplemental Figure 10 MeJA-Induced Expression of the JA marker gene JAZ10 884

Supplemental Figure 11 The Expression of the JA Marker Gene JAZ5 Is Highly 885

Responsive in gae1 gae6 Plants 886

Supplemental Figure 12 The Expression of PDF12 Is Unaltered in gae1 gae6 Plants 887

Supplemental Figure 13 Macerozyme Treatment Activates MPK3 and MPK6 888

Supplemental Figure 14 B cinerea-Induced ROS Production in Wild-Type Col-0 and 889

gae1 gae6 Plants 890

Supplemental Figure 15 OG-Induced PTI Using 250 microg mL-1 OGs 891

Supplemental Figure 16 Abundance of Plant DNA in B cinerea Treated Plants 892

Supplemental Table 1 Primers used in this study 893

894

ACKNOWLEDGEMENTS 895

We thank William Truman for helpful discussions Simone Ferrari (Sapienza 896

Universita di Roma) for the generous gift of OGs Bruna Bucciarelli (USDA-ARS) for 897

Page 41 of 54

help with fluorescence microscopy the Arabidopsis Biological Resource Center for T-898

DNA insertion lines Paul Knoxrsquos lab (University of Leeds) for LM5 LM6 LM8 LM19 899

and LM20 antibodies and the Complex Carbohydrate Resource Center (University of 900

Georgia) for the CCRC-M7 antibody Most of this work was funded by the Division of 901

Chemical Sciences Geosciences and Biosciences Office of Basic Energy Sciences of 902

the US Department of Energy through Grant DE-FG02-05ER15670 to JG This work 903

was also supported by NSF awards IOS 1339125 and IOS 1021861 to DJK NSF 904

award IOS 1121425 to F K and the USDA National Institute of Food and Agriculture 905

Hatch project number CA-D-PLS-7033-H to DJK 906

907

AUTHOR CONTRIBUTIONS 908

GB and JG designed the research GB GX AT BL NES and NH 909

performed research DJK provided tools GB GX MP AD BL RAH and FK 910

analyzed data GB and JG wrote the paper with input from all authors 911

912

FIGURE LEGENDS 913

Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 Expression 914

Requires PAD4 EDS1 and PBS3 915

(A) Expression of GAE1 in leaves of wild-type Col-0 and mutant plants Expression was916

measured 24 or 32 h after either inoculation with Pma ES4326 (OD600 = 0002) or mock 917

inoculation with 5 mM MgSO4 and was then normalized to the level of ACTIN2 Data 918

from three biological replicates were combined using a mixed linear model Bars 919

represent mean log2-ratios to ACTIN2 plusmn SE Letters indicate significantly different 920

groups with q lt 005 where 005 represents the maximum false discovery rate at which 921

the test may be called significant 922

(B) Expression of GAE6 measured as described in (A)923

924

Figure 2 Susceptibility of gae6 and gae1 gae6 Plants to Pma ES4326 Is Increased 925

Relative to Wild-Type Col-0 926

Leaves of plants of the indicated genotypes were inoculated with Pma ES4326 (OD600 = 927

00002) as described in Methods Bacterial titers in leaves were determined either 928

Page 42 of 54

immediately (0 dpi) or after three days (3 dpi) Bars represent the mean plusmn SE of four 929

independent experiments each with 4 or 12 biological replicates at 0 and 3 dpi 930

respectively Data were combined using a mixed linear model Asterisks indicate 931

significant differences from Col-0 wild-type (q lt 005) Susceptible pad4 plants were 932

included as a positive control 933

934

Figure 3 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves 935

(A) Depiction of the assay for brittleness of gae mutant leaves Fully expanded leaves of936

four to five week-old plants were bent by 180ordm so that the adaxial surface of the leaf was 937

parallel with the soil Broken leaf petioles or midribs were considered brittle 938

(B) Quantification of broken midribs or petioles For each genotype 66 to 82 leaves 939

were analyzed for brittleness as described in (A) Bars represent the ratio of broken to 940

total number of leaves tested Results were analyzed using Fisherrsquos exact test Letters 941

indicate significantly different groups at p lt 005 942

(C) Conductivity measurements Leaves of four week-old plants were inoculated with943

deionized water and then leaf discs were cut out and placed on deionized water 944

Conductivity was measured at the indicated time points After 30 min the water was 945

replaced with fresh water (30 min f w) to account for wounding-related ion leakage from 946

the edges of the leaf discs Three independent experiments with six biological replicates 947

each were performed and data were analyzed using a t-test for each time point P-948

values were corrected using the Bonferroni method Data show means plusmn SE asterisks 949

indicate significant differences from wild-type Col-0 at q lt 001 950

951

Figure 4 Cell Walls of gae6 and gae1 gae6 Plants Are Reduced in GalA 952

(A) to (D) Levels of GalA (A) GlcA (B) cellulose (C) and neutral sugars (D) in alcohol953

insoluble residue (AIR) extracted from leaves of four week-old plants of the indicated 954

genotypes Bars represent means plusmn SE of four biological replicates combined using a 955

mixed linear model Letters indicate significantly different groups (p lt 005) 956

957

Figure 5 Cell Walls of gae1 gae6 Plants Are Reduced in Homogalacturonan 958

Page 43 of 54

Dot blot analyses with anti-LM19 and anti-LM20 antibodies Pectin was extracted from 959

AIR prepared from the indicated genotypes using 1 microL of extraction buffer per 10 microg of 960

AIR Pectin was serially diluted and one microL of undiluted 13 diluted and 19 diluted 961

samples were spotted on a nitrocellulose membrane Three biological replicates were 962

were spotted twice to obtain two technical replicates each All replicates produced 963

similar results 964

(A) Representative dot blot using anti-LM19 antibody965

(B) Relative signal intensities of the spots in the dot blot in (A)966

(C) Representative dot blot using anti-LM20 antibody967

(D) Relative signal intensities of the LM20 dot blot shown in (C)968

Relative signal intensities were measured using Image J software Measurements from 969

the three biological replicates were combined using a linear model after Box-Cox power 970

transformation as described in Methods Bars represent means plusmn SE Asterisks indicate 971

a difference from wild-type Col-0 at p lt 005 972

973

Figure 6 Growth of B cinerea on gae1 gae6 Plants and the Effect of B cinerea on 974

Expression of GAE1 and GAE6 975

(A) Growth of B cinerea isolates Leaves of Col-0 and gae1 gae6 plants were976

inoculated with 25 x 105 spores mL-1 of the B cinerea isolates indicated in the figure 977

Samples were collected two (2 dpi) and three (3 dpi) days later Bars represent means plusmn 978

SE of six biological replicates for each fungus combined using a mixed linear model 979

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 980

(B) and (C) Expression of GAE1 (B) and GAE6 (C) 48 h after inoculation with the B981

cinerea isolates indicated Expression levels were measured by qRT-PCR Bars 982

represent mean log2-ratios to ACTIN2 plusmn SE of three biological replicates combined 983

using a mixed linear model Letters indicate significantly different groups (q lt 005) 984

985

Figure 7 Levels of Soluble Pectin and Expression of PAD3 in Fractions Produced 986

Following Macerozyme Treatment of gae1 gae6 Cell Walls 987

(A) Schematic overview of the experimental procedure for (B) and (C)988

Page 44 of 54

(B) Uronic acid measurements Alcohol-insoluble residue (AIR) from leave tissue of four989

week-old wild-type Col-0 or gae1 gae6 plants was treated with water or 025 990

macerozyme and assayed for uronic acid levels Data from 11 biological replicates were 991

combined using a mixed linear model Bars represent means plusmn SE Letters indicate 992

significant differences (p lt 005) 993

(C) Expression of PAD3 in Col-0 plants treated with the fractions from (B) Extracts from994

(B) were heat inactivated pooled and diluted 5-fold with water Pooled extracts were995

inoculated into four week-old Col-0 plants PAD3 expression was measured 3 h after 996

inoculation and normalized to the level of ACTIN2 expression Data from four biological 997

replicates were combined using a mixed linear model Bars represent mean log2-ratios 998

to ACTIN2 plusmn SE Letters indicate significant differences (p lt 005) 999

1000

Figure 8 Macerozyme-Induced PAD3 Expression Is Absent in gae1 gae6 1001

(A) Expression of PAD3 after macerozyme treatment Four week-old plants were either1002

inoculated with 001 macerozyme or mock inoculated with water and PAD3 1003

expression was measured 3 h later by qRT-PCR Data from four biological replicates 1004

were combined using a mixed linear model Mean log2-ratios to ACTIN2 plusmn SE were 1005

plotted Letters indicate significantly different groups (p lt 005) 1006

(B) Expression of PAD3 after OG treatment PAD3 expression was measured as1007

described in (A) 3 h after inoculation with 100 microg mL-1 OGs or mock inoculation with 1008

water Data are from six biological replicates 1009

(C) PAD3 expression in untreated plants Three biological replicates were performed1010

and data analyzed as described in (A) 1011

(D) Camalexin accumulation after B cinerea treatment Camalexin accumulation was1012

measured in four week-old plants inoculated with the B cinerea strains shown in the 1013

figure Eight biological replicates each were combined using a mixed linear model 1014

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 1015

(E) PAD3 expression 28 h after treatment with the indicated B cinerea strains Data1016

from six biological replicates were collected and analyzed as described in (A) Data in 1017

all figure parts represent means plusmn SE Letters indicate significantly different groups (q lt 1018

005) 1019

Page 45 of 54

1020

Figure 9 Macerozyme-Induced Expression of the SA Marker Genes SID2 and PR1 1021

Is Abolished in gae1 gae6 Plants 1022

(A) to (H) Expression of PR1 (A) (C) (E) and (G) or SID2 (B) (D) (F) and (H) in four1023

week-old Col-0 and gae1 gae6 plants Data were combined using a mixed linear model 1024

Bars represent mean log2-ratios of expression vs ACTIN2 plusmn SE for all figure parts 1025

Letters indicate significantly different groups at p lt 005 for panels (A) to (D) (G) and 1026

(H) and qlt005 for panels (E) to (F) 1027

(A) and (B) Plants were treated with 001 macerozyme or mock treated with water1028

and samples were collected 3 h later Data from four biological replicates are shown 1029

(C) and (D) Plants were inoculated with 100 microg mL-1 OGs or mock treated with water1030

and samples collected 3 h later Data from six biological replicates are shown 1031

(E) and (F) Plants were inoculated with the indicated B cinerea isolates or mock1032

inoculated with B cinerea inoculation medium and samples collected 48 h later Data 1033

from eight biological replicates are shown 1034

(G) and (H) Expression in untreated plants Three biological replicates were performed1035

1036

Figure 10 The Expression of the JA Marker Gene JAZ10 is Highly Responsive in 1037

gae1 gae6 Plants 1038

(A) to (D) JAZ10 expression in four week-old plants Data were combined using a mixed1039

linear model Mean log2-ratios to ACTIN2 plusmn SE are shown Letters indicate significantly 1040

different groups at p lt 005 for panels (A) (B) and (D) and q lt 005 for panel (C) 1041

(A) Plants were inoculated with 001 macerozyme or mock inoculated using water and1042

samples were collected 3 h later Data from four biological replicates are shown 1043

(B) Plants were inoculated with 100 microg mL-1 OGs or mock inoculated using water and1044

samples collected 3 h later Data are from six biological replicates 1045

(C) JAZ10 expression 48 h after treatment with the indicated B cinerea strains Data1046

are from six biological replicates 1047

(D) JAZ10 expression in untreated plants Data are from three biological replicates1048

1049

Figure 11 Gallo 1-Induced Callose Deposition Is Reduced in gae1 gae6 Plants 1050

Page 46 of 54

(A) Callose deposition in four week-old Col-0 and gae1 gae6 plants Plants were1051

inoculated with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) or mock inoculated 1052

with B cinerea inoculation medium and samples were collected after 72 h Callose was 1053

stained with aniline blue and visualized using a Nikon Eclipse Ni-U microscope with a 1054

DAPI filter Three leaves were stained per treatment Scale bars are 100 microm Asterisks 1055

indicate fungal hyphae arrows indicate representative callose spots 1056

(B) Quantification of callose deposition Callose deposits were counted in 200 microm x 2001057

microm squares laid out across the images shown here Bars represent means plusmn SE from 1058

six squares each Letters indicate significantly different groups (p lt 005) according to a 1059

t-test analysis1060

1061

Figure 12 Less Soluble Pectin Accumulates in B cinerea-Infected gae1 gae6 1062

Leaves 1063

Four week-old Col-0 and gae1 gae6 plants were inoculated with 10 microL droplets of the 1064

indicated B cinerea isolate (25 x 105 spores mL-1) or mock inoculated with B cinerea 1065

inoculation medium Leaves were collected 48 h later AIR was extracted from leaf 1066

samples and water added (20 microL infection site) to extract soluble pectin fragments 1067

Samples were vortexed for 7 h centrifuged at 12000 g and the uronic acid 1068

concentration of the supernatant was measured Data from nine biological replicates 1069

were combined using a mixed linear model Means plusmn SE are shown Letters indicate 1070

significant differences (q lt 005) 1071

1072

Figure 13 Macerozyme-Induced PTI Is Lost in gae1 gae6 While OG-Induced PTI Is 1073

Unaltered 1074

(A) Macerozyme-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1075

inoculated with 001 macerozyme or mock inoculated with boiled macerozyme and 1076

were infected with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) 24 h later Samples 1077

were collected immediately (0 dpi) and after two (2 dpi) and three (3 dpi) days Data 1078

show mean plusmn SE of six to 14 biological replicates each combined using a linear model 1079

Letters indicate significantly different groups (q lt 005) 1080

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

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Page 8 of 54

205 requires a regulatory function that is dependent on EDS1 and PAD4 partially

206 dependent on PBS3 but independent of SA

Page 9 of 54

There are six GAE family members in Arabidopsis GAE2 expression was 207

induced by Pma ES4326 in the absence of PAD4 or EDS1 GAE3 expression was 208

unaltered GAE4 was repressed early and GAE5 expression was induced in all 209

genotypes (Supplemental Figure 2) The similarity of the immune-regulated expression 210

of GAE1 and GAE6 suggested a redundant role of these genes in Arabidopsis immunity 211

to Pma ES4326 212

213

Resistance to Pma ES4326 Is Compromised in gae1 gae6 and gae6 Plants 214

To study the roles of GAE1 and GAE6 in plant immunity in more detail plants 215

with T-DNA insertion mutations in GAE1 and GAE6 were obtained from the SALK 216

collection as described in the Accession Numbers section of Methods We identified two 217

GAE6 knock-out alleles gae6-1 and gae6-2 and a T-DNA insertion in GAE1 gae1-1 218

that caused reduced expression of this gene (Supplemental Figure 3) A double mutant 219

was created by crossing gae1-1 and gae6-1 Both gae6 and gae1 gae6 plants were 220

more susceptible to Pma ES4326 than wild-type Col-0 and gae1 plants supporting a 221

role of GAE6 in plant immunity to Pma ES4326 (Figure 2) 222

223

gae6 and gae1 gae6 Plants Have Brittle Leaves 224

While inoculating leaves of gae1 gae6 plants we noticed that the petioles and 225

midribs of these plants tended to break easily when leaves were bent against the 226

proximal-distal axis of the leaves (Figure 3 see Supplemental Figure 4D to F) They 227

tended to break less often when twisted around the proximal-distal axis To quantify 228

this phenotype leaves were bent by 180deg (Figure 3A) and breaking of midribs or 229

petioles was recorded Most gae1 gae6 leaves broke whereas fewer leaves of gae6 230

plants and very few leaves of wild-type Col-0 and gae1 plants did (Figure 3B and 231

Supplemental Figure 4C) Although gae1 gae6 plants had slightly shorter leaves and 232

petioles than wild type plants (Supplemental Figure 4A and B) no correlation between 233

leaf size or petiole length and breaking frequency was detected We also investigated 234

tissue integrity by monitoring ion leakage Interestingly when leaves were inoculated 235

with deionized water leaf discs of gae1 gae6 leaked more ions than wild-type Col-0 236

discs (Figure 3C) suggesting that the increased brittleness is correlated with a 237

Page 10 of 54

_____________________________________________________________________________

Page 11 of 54

Cell Walls of gae6 and gae1 gae6 Plants Have Lower Levels of Pectin 243

GalA is a major component of pectin We analyzed wall monosaccharide 244

composition in gae mutants to investigate whether the increased brittleness was 245

associated with an altered wall composition of gae leaves To approximate pectin 246

content of gae mutant walls the total uronic acid concentration was measured using a 247

simple colorimetric assay (Filisetti-Cozzi and Carpita 1991 van den Hoogen et al 248

1998) Total uronic acid was reduced in gae6-2 strongly reduced in gae1 gae6 plants 249

but was indistinguishable from wild-type Col-0 in gae6-1 and gae1 (Supplemental 250

Figure 5) This suggested that gae1 gae6 and possibly gae6 plants have reduced 251

pectin 252

To investigate the cell wall composition in leaves of gae plants in more detail we 253

measured the levels of seven neutral monosaccharides the acidic sugars GalA (the 254

monomeric subunit of the pectin backbone (Harholt et al 2010)) and GlcA (Figure 4) 255

We also estimated the crystalline cellulose content (Updegraff 1969) Cell walls of both 256

gae6 mutants and gae1 gae6 were reduced in GalA (Figure 4A) In gae1 gae6 GalA 257

content in cell walls was reduced by more than 40 compared to the wild-type level 258

GlcA levels were indistinguishable between mutants and wild-type (Figure 4B) 259

Cellulose content was higher in gae6-2 and gae1 gae6 walls likely due to the loss of 260

pectins (Figure 4C) Cell walls of gae1 gae6 plants were also enriched in all neutral 261

sugars measured presumably due to the substantial proportional loss of GalA (Figure 262

4D) The levels of most wall monosaccharides were even higher in the gae1 gae6 263

double mutant with the exception of Rha which did not exhibit a significant increase 264

compared to the gae6 single mutants The absence of a compensational increase in 265

Rha suggested that RGI the dominant Rha contributor to the wall may be considered 266

reduced in gae1 gae6 mutants compared to wild-type albeit not to such an extent as 267

HG 268

238 structural defect that promotes increased ion leakage Taken together these results

239 showed that loss of GAE6 causes brittle leaves and additional losss of GAE1 strongly

240 enhances this phenotype We conclude that GAE1 and GAE6 contribute to normal leaf

241 flexibility 242

Page 12 of 54

gae6 cell walls are reduced in HG Using the same samples we detected no difference 274

from wild type in binding of LM5 which recognizes a tetramer of (1-4)-β-D-galactans 275

found in RGI (Jones et al 1997) LM6 which recognizes a linear pentasaccharide in (1-276

To further investigate which pectic components were altered in gae1 gae6 dot 269

blot experiments using pectin isolated from the various genotypes were performed Both 270

LM19 and LM20 antibodies recognize HG (Verhertbruggen et al 2009a) As expected 271

LM19 binding was reduced in gae6-1 and gae1 gae6 (Figure 5A and B) and LM20 272

binding was reduced in gae1 gae6 (Figure 5C and D) This result confirmed that gae1 273

277 5)-α-L-arabinans found in RGI (Willats et al 1998 Verhertbruggen et al 2009b) LM8

278 which recognizes xylogalacturonan (Willats et al 2004) or CCRC-M7 which

279 recognizes an arabinosylated (1-6)-β-D-galactan epitope occurring on RGI (Puhlmann et

Page 13 of 54

Page 14 of 54

al 1994 Steffan et al 1995 Pattathil et al 2010) (Supplemental Figure 6) 280

Collectively these cell wall composition data show that gae1 gae6 plants are reduced in 281

HG Due to the strong proportional reduction of HG in the walls levels of all other 282

components should be higher However Rha indicative of RGI was not higher in gae1 283

gae6 suggesting that RGI may also be reduced in gae1 gae6 walls 284

285

GAE1 and GAE6 Are Required for Immunity to Specific B cinerea Isolates 286

We hypothesized that plant immunity to B cinerea might be affected by pectin 287

content in the host cell wall because this pathogen requires polygalacturonases and 288

pectin methylesterases for full virulence (ten Have et al 1998 Valette-Collet et al 289

2003 Kars et al 2005b Espino et al 2010) Because Bcinerea is genetically diverse 290

and has a high diversity of polygalacturonases (Rowe and Kliebenstein 2007) we 291

measured relative fungal growth at both 2 and 3 d post-inoculation (dpi) using ten 292

different B cinerea isolates (Figure 6) The B cinerea isolates Fresa 525 and Gallo 1 293

showed enhanced virulence on gae1 gae6 plants at both time points whereas KT and 294

UK Razz showed enhanced virulence in gae1 gae6 plants at 3 but not 2 d dpi (Figure 295

6A) We did not detect any significant changes in relative fungal growth between wild-296

type Col-0 and gae1 gae6 plants when using Acacia Apple 517 DN Grape Pepper or 297

Rasp isolates of B cinerea Therefore we continued our study using Gallo 1 and Fresa 298

525 We also included Pepper for comparison as this isolate has been used in a large-299

scale analysis of plant gene expression in response to B cinerea infection (Windram et 300

al 2012) 301

We next investigated whether expression of GAE1 and GAE6 was affected by 302

challenge with B cinerea Inoculation of wild-type Col-0 plants with any isolate 303

repressed GAE1 and GAE6 expression (Figure 6B and C) and this repression was 304

generally independent of PAD4 (Figure 6B and C) Expression of GAE2 and GAE3 was 305

unaltered by B cinerea treatment GAE4 was repressed only by Fresa 525 treatment 306

while GAE5 expression was enhanced with all three isolates tested (Supplemental 307

Figure 7) As seen for infection with Pma ES4326 the expression of GAE1 and GAE6 308

was repressed upon B cinerea treatment This indicates a role for GAE1 and GAE6 in 309

the immune response to B cinerea 310

Page 15 of 54

Page 16 of 54

Because the amount of pectin substrate for pathogen polygalacturonases is 313

reduced we hypothesized that immunity phenotypes of gae1 gae6 plants may result 314

from reduced generation of OGs by these enzymes In turn the reduced OGs could 315

lead to reduced immune responses To test this we used macerozyme a commercial 316

pectinase as a proxy for pathogen pectinases We isolated cell walls as a subcellular 317

fraction known as alcohol insoluble residue (AIR Gille et al 2009) This was treated 318

with macerozyme and tested for release of soluble likely low molecular weight pectin 319

fragments (Figure 7) Treatment with 025 macerozyme released significantly more 320

uronic acid from wild-type Col-0 AIR relative to gae1 gae6 AIR (Figure 7B) To test 321

activity of these pectin-containing solutions we infiltrated them into wild-type Col-0 322

plants and measured PAD3 expression PAD3 is required for synthesis of the 323

phytoalexin camalexin (Zhou et al 1999) and its expression is known to be induced by 324

OG treatment (Ferrari et al 2007) Fractions from macerozyme treated Col-0 walls 325

induced significantly more PAD3 expression than fractions derived from gae1 gae6 326

walls suggesting that macerozyme treatment did indeed release more active soluble 327

uronic acid possibly OGs from wild-type Col-0 walls than from gae1 gae6 walls (Figure 328

7C) 329

Next we studied the effect of macerozyme treatment on expression of PAD3 in 330

intact plants (Figure 8) This treatment did not lead to any visible leaf damage 331

(Supplemental Figure 8) In wild-type Col-0 PAD3 expression was induced three h after 332

treatment with 001 macerozyme (Figure 8A) By contrast no macerozyme-induced 333

induction of PAD3 expression was detected in gae1 gae6 (Figure 8A) However OG 334

treatment induced the expression of PAD3 in both genotypes indicating that gae1 gae6 335

plants do respond to OGs (Figure 8B) Macerozyme treatment also induced expression 336

of the SA marker genes PATHOGENESIS RELATED 1 (PR1) and SID2 in Col-0 but 337

not in gae1 gae6 (Figure 9 panel A and B) PR1 was not significantly induced by 338

purified OGs (Figure 9C) at the time and concentration tested but SID2 was induced by 339

OG treatment in both genotypes (Figure 9D) However the induction was stronger in 340

wild-type Col-0 In addition the JA markers JASMONATE-ZIM-DOMAIN PROTEIN 5 341

(JAZ5) and JAZ10 were induced by macerozyme and OG treatment in both Col-0 and 342

gae1 gae6 (Figure 10A and Supplemental Figure 11) Interestingly expression of JAZ5 343

312 Pectinase-Induced Immune Signaling Is Altered in gae1 gae6 Plants

Page 17 of 54

Page 18 of 54

Page 19 of 54

Page 20 of 54

344

345

346

and JAZ10 was already strongly induced in gae1 gae6 after mock inoculation (Figure 10

and Supplemental Figure 11) We observed no hyperresponsive induction by mock

treatment of PLANT DEFENSIN 12 (PDF12) a necrotrophy and late JA signaling

347 marker gene (Supplemental Figure 12A-D) PDF12 was also not induced by

348 macerozyme or OG treatments (Supplemental Figure 12A-D) This suggests that early

349 JA signaling is induced by mock-treatment in gae1 gae6 Because it is known that JA

350 and SA signaling act antagonistically (Pieterse et al 2012) the strong up-regulation of

351 JA-induced immune signaling in gae1 gae6 might explain the lower OG-induced SID2

352 expression in this genotype

353 To test whether other immune responses are altered in gae1 gae6 plants we

354 analyzed Mitogen-activated protein kinase (MAPK) activity following macerozyme and

355 OG treatments OGs are known to activate the MAPKs MPK3 and MPK6 in Arabidopsis

Page 21 of 54

(Galletti et al 2011) Macerozyme treatment activated MPK3 and MPK6 in wild-type 356

Col-0 whereas MAPK activation in gae1 gae6 was slightly reduced in four out of five 357

experiments performed (Supplemental Figure 13) By contrast OG-induced MAPK 358

activity was similar or increased in gae1 gae6 in four out of five experiments 359

(Supplemental Figure 13) Additionally mock treatment induced MAPK activity in gae1 360

gae6 more strongly than it did in wild-type Col-0 in four out of five experiments 361

performed (Fig S13) Collectively these results illustrate that activation of several 362

immune signaling componentsmdashcamalexin synthesis SA and JA signaling and 363

possibly MAPK activationmdashby macerozyme is substantially altered in gae1 gae6 This 364

suggests that release of soluble pectin fragments possibly OGs by pectinolytic 365

enzymes might be reduced in gae1 gae6 plants while recognition of OGs is intact 366

367

Following B cinerea Infection Production of Soluble Pectin and Deposition of 368

Callose Are Reduced in gae1 gae6 Plants 369

Although pad3 plants were more susceptible to the B cinerea isolates Fresa 525 370

and Gallo 1 which also grew better on gae1 gae6 growth of the isolate Pepper was 371

similar on Col-0 and pad3 (Supplemental Figure 9A) However we did not detect any 372

significant changes in camalexin production after treatment with B cinerea isolate Gallo 373

1 and Fresa 525 in gae1 gae6 (Figure 8D) Neither did we detect changes in PAD3 374

expression after treatment with any B cinerea isolate in gae1 gae6 (Figure 8E) 375

Additionally SID2 and PR1 were similarly induced by all B cinerea isolates in both Col-376

0 and gae1 gae6 (Figure 9E and F) and sid2 plants which are deficient for pathogen-377

induced SA biosynthesis did not show any differences in relative B cinerea growth 378

(Supplemental Figure 9B) Expression of JAZ10 was induced more strongly by Gallo 1 379

treatment in gae1 gae6 than in wild-type Col-0 but not by Fresa 525 or Pepper (Fig 380

10C) while dde2 plants deficient for JA biosynthesis showed enhanced susceptibility to 381

all B cinerea isolates tested (Supplemental Figure 9B) Curiously exogenous 382

application of MeJA did not lead to altered relative fungal growth of B cinerea 383

(Supplemental Figure 9C) and cev1 isoxaben resistant 1 (ixr1) plants which have 384

constitutively high JA levels (Ellis and Turner 2001) did not show any changes in 385

susceptibility to the B cinerea isolates tested (Supplemental Figure 9D) Hence neither 386

Page 22 of 54

differences in B cinerea isolate susceptibility to camalexin nor altered SA or JA 387

signaling by themselves explained the enhanced susceptibility of gae1 gae6 to these B 388

cinerea isolates 389

Hydrogen peroxide accumulation in response to B cinerea isolates Fresa 525 390

Gallo 1 and Pepper was not obviously altered in gae1 gae6 plants (Supplemental Figure 391

14) However callose deposition upon treatment with B cinerea isolate Gallo 1 was 392

strongly reduced in gae1 gae6 (Figure 11) It was previously shown that OG treatment 393

induces callose deposition (Denoux et al 2008) suggesting that the reduction in 394

callose deposition might be due to reduced OG release in gae1 gae6 It is possible that 395

this reduction in callose deposition contributes to the reduced immunity of gae1 gae6 396

plants to B cinerea isolate Gallo 1 397

Because macerozyme could be used to release soluble likely low molecular 398

weight pectin from isolated cell walls we next investigated whether infection with B 399

cinerea was accompanied by accumulation of soluble pectin as well We prepared cell 400

walls (AIR fraction) from leaves infected with B cinerea incubated these cell walls with 401

water and measured uronic acid content of the extracts B cinerea treatment did in fact 402

lead to increased accumulation of soluble pectin in wild-type Col-0 plants when 403

compared to mock-treated samples (Figure 12) No such increased accumulation of 404

water-soluble pectin fractions was observed in gae1 gae6 (Figure 12) 405

406

Macerozyme-Induced Pattern-Triggered Immunity against B cinerea Is Abolished 407

in gae1 gae6 408

It was previously shown that OG treatment can induce patternndashtriggered 409

immunity (PTI) to B cinerea (Ferrari et al 2007) We tested macerozyme treatment to 410

determine whether it could also induce PTI to the B cinerea isolate Gallo 1 (Figure 13) 411

Indeed pretreatment with 001 macerozyme for 24 h led to reduced B cinerea growth 412

in wild-type Col-0 but not gae1 gae6 plants (Figure 13A) Interestingly mock and 413

macerozyme pretreated gae1 gae6 plants exhibited similar growth of B cinerea Gallo 1 414

as did macerozyme-treated Col-0 plants This result is likely due to activation of JA 415

signaling in gae1 gae6 during the pretreatment procedure (Figure 13C and D) OG-416

induced PTI was observed in both genotypes when using a high dose of OGs (Figure 417

Page 23 of 54

13B) whereas a lower dose of OGs only induced PTI in Col-0 (Supplemental Figure 418

15) This suggests that OG perception in gae1 gae6 at least above a certain threshold 419

is unaltered while release of immunity-activating pectin fragments possibly OGs is 420

421 reduced We conclude that immune responses induced by macerozyme treatment

422 which do not occur in gae1 gae6 plants are important for plant immunity to the B

423 cinerea isolate Gallo 1

Page 24 of 54

____________________________________________________________________________

Page 25 of 54

DISCUSSION 426

Here we showed that loss of GAE1 and GAE6 leads to a dramatic reduction in 427

pectin content in the cell wall (Figure 4) likely causing the brittleness of leaves that we 428

observed (Figure 3) Petioles and midribs of gae1 gae6 plants broke easily when bent 429

and inoculation of leaves with deionized water resulted in increased ion leakage 430

possibly due either to microscopic fractures of the cell wall induced by inoculation stress 431

or to a reduced capability of these walls to withstand increased turgor pressure The 432

increased brittleness was associated with altered cell wall composition In our analysis 433

the GalA content of gae1 gae6 leaf walls was reduced by more than 40 and the total 434

uronic acids by over 60 (Figure 4 and Supplemental Figure 5) This is a major change 435

in the overall cell wall composition considering that pectin makes up about 50 of the 436

plant cell walls found in Arabidopsis leaves (Zablackis et al 1995 Nobuta et al 2007 437

Harholt et al 2010) Interestingly quasimodo1 (qua1) plants which have a mutation in 438

a glycosyltransferase family 8 gene are reduced in GalA by 25 and show reduced 439

binding of HG specific antibodies (Bouton et al 2002) 440

Furthermore qua2 plants which have a mutation in a putative methyltransferase 441

have about 13 less total GalA and lack 50 of cell wall HG (Mouille et al 2007) 442

Likewise cgr2 cgr3 plants carrying mutations in another class of Golgi-localized 443

putative methyl-transferases have leaf cell walls that are reduced by about 40 in 444

uronic acid (Kim et al 2015) All three mutants are severely dwarfed (Bouton et al 445

2002 Mouille et al 2007 Kim et al 2015) Additionally qua1 and qua2 mutants show 446

a distorted overall shape likely due to reduced cell cohesion and are extremely fragile 447

(Bouton et al 2002 Mouille et al 2007) By contrast gae1 gae6 plants with a similar 448

strong reduction in GalA and total uronic acids have an overall normal shape and are 449

only slightly smaller than wild-type Col-0 (Supplemental Figure 4) In the wall analysis 450

performed here we did not detect changes in other wall polysaccharides or structures 451

that might compensate for the reduction of pectin in gae1 gae6 It is possible the severe 452

morphological alterations in qua2 and cgr2 cgr3 plants are due to reduction in pectin 453

methylesterification which is unaffected in gae1 gae6 It was speculated that quality 454

control mechanisms in the Golgi prevent secretion of inefficiently esterified pectin in 455

cgr2 cgr3 (Kim et al 2015) suggesting that the Golgi of these plants might over-456

Page 26 of 54

accumulate pectin and that this may contribute to the severe dwarf phenotype of these 457

plants The gae1 gae6 plants described here provide an opportunity to study the effects 458

of reduction of pectin in plants that do not show severe morphological phenotypes 459

We found that gae1 gae6 plants are also compromised in resistance to B 460

cinerea and Pma ES4326 indicating that loss of pectin reduces immunity to these 461

pathogens (Figure 2 and 6) We found that gae1 gae6 plants were slightly more 462

susceptible to Pma ES4326 but showed a strong increase in susceptibility to specific B 463

cinerea isolates This was not unexpected because B cinerea is known to interact with 464

pectin as part of its infection strategy It secretes various pectin-degrading enzymes 465

early during infection and requires some of these enzymes including PG1 PG2 and 466

PME1 for full virulence (ten Have et al 1998 Valette-Collet et al 2003 Kars et al 467

2005b Espino et al 2010) Additionally Arabidopsis plants expressing an antisense 468

construct of the polygalacturonase inhibitor PGIP1 are significantly more susceptible to 469

B cinerea (Ferrari et al 2006) On the other hand Pma ES4326 is not known to470

require cell wall degrading enzymes for full virulence even though it encodes two 471

pectate lyase genes in its genome (PMA4326_07094 and PMA4326_04621 472

wwwbacteriaensemblorg) B cinerea isolates are also known to be genetically quite 473

diverse in their polygalacturonase (PG) loci and genetic variability in PG2 is associated 474

with varying growth on pectin containing media (Rowe and Kliebenstein 2007) 475

Furthermore the contribution of PG1 and PG2 to B cinerea virulence seems to depend 476

on the specific B cinerea isolate and the host species (Kars et al 2005a Zhang and 477

Van Kan 2013) B cinerea isolates are also known to be differently sensitive to the anti-478

microbial secondary metabolite camalexin (Kliebenstein et al 2005) In line with these 479

observations the susceptibility of gae1 gae6 plants differed depending on the specific 480

B cinerea isolate Whereas Fresa 525 Gallo 1 UK Razz and KT grew better in gae1481

gae6 we detected no difference in Acacia Apple 517 DN Grape Pepper or Rasp 482

growth (Figure 6) 483

B cinerea was shown to metabolize pectin and to possibly utilize GalA as a484

carbon source during in planta growth (Zhang et al 2011 Zhang and Van Kan 2013) 485

Our data show that gae1 gae6 plants which are reduced in GalA content did allow 486

enhanced growth of specific B cinerea isolates but no reduction in growth of any B 487

Page 27 of 54

cinerea isolates tested was found This suggests that utilization of pectin as a carbon 488

source was not a contributing factor to alterations in B cinerea susceptibility It is 489

possible that changes in the cell wall composition affect the structural integrity of the 490

wall and affect hyphal penetration of specific B cinerea isolates However OGs (small 491

HG fragments) are likely released during the infection process (Ferrari et al 2013) 492

OGs function as DAMPs and initiate immune responses in Arabidopsis (Ferrari et al 493

2013) Treatment of Arabidopsis with OGs having a degree of polymerization (DP) of 494

10-15 sugar subunits was previously shown to enhance resistance to B cinerea (Ferrari495

et al 2007) We showed that pretreatment with OGs or macerozyme induced enhanced 496

immunity to B cinerea isolate Gallo 1 in wild-type Col-0 but only OGs and not 497

macerozyme induced immunity in gae1 gae6 (Figure 13) Our data suggest gae1 gae6 498

plants which have reduced pectin content may release less OGs and that this 499

reduction leads to the increased susceptibility to B cinerea 500

Consistently we found that macerozyme treatment released less soluble pectin 501

from gae1 gae6 cell walls than from wild-type walls Fractions containing soluble pectin 502

released from gae1 gae6 were also less active in inducing PAD3 gene expression than 503

the fractions released from wild-type walls (Figure 7) We also showed that less soluble 504

pectin accumulated in gae1 gae6 than in wild-type plants 48 h after treatment with B 505

cinerea (Figure 12) 506

We observed that loss of pectin leads to alterations in A thaliana immune 507

signaling Macerozyme treatment induced expression of PAD3 and the SA marker 508

genes PR1 and SID2 in wild-type Col-0 but not in gae1 gae6 whereas OG treatment 509

led to increased PAD3 and SID2 expression in both wild-type and gae1 gae6 (Figure 8 510

and 9) This suggests that although release of active soluble pectin in these plants 511

might be altered OG recognition is intact In fact cell walls isolated from qua1 another 512

mutant known to be reduced in HG released less OGs (DP1-6) than did wild-type cell 513

walls upon endopolygalacturonase treatment (Bouton et al 2002) This was also true 514

for gae1 gae6 cell walls as macerozyme treatment of isolated gae1 gae6 cell walls 515

released less soluble pectin than treatment of Col-0 cell walls Because the fraction 516

from gae1 gae6 plants was also less active in inducing PAD3 expression (Figure 7) it 517

seems possible that biologically active OGs were the active component inducing PAD3 518

Page 28 of 54

expression Interestingly expression of the JA marker genes JAZ5 and JAZ10 was 519

higher in gae1 gae6 than in wild-type mock-inoculated plants but was comparable to 520

wild-type in untreated plants suggesting that JA signaling in gae1 gae6 is 521

hyperresponsive (Figure 10 and Supplemental Figure 11) This induction of JA signaling 522

might be due to wounding stress incurred during the inoculation process JA and SA 523

signaling generally act antagonistically (Pieterse et al 2012) Increased JA signaling 524

activity might hence explain why mock- and macerozyme-treated gae1 gae6 plants 525

exhibited reduced expression of the SA marker PR1 and why OG treatment induced 526

expression of SID2 another SA marker to a lower extent in gae1 gae6 than in wild-type 527

plants (Figure 9) 528

No single immune signaling component was found to be the sole contributor to 529

the compromised immunity of gae1 gae6 to specific B cinerea isolates such as Gallo 1 530

and Fresa 525 These isolates grew better on pad3 while growth of Pepper another B 531

cinerea isolate that did not show altered virulence on gae1 gae6 was unaltered 532

(Supplemental Figure 9) This suggested that differences in camalexin sensitivity of 533

these B cinerea isolates might account for the differences in gae1 gae6 susceptibility 534

However camalexin accumulation and PAD3 expression was the same for wild-type 535

Col-0 and gae1 gae6 for the B cinerea isolates Fresa 525 and Gallo 1 (Figure 8) which 536

showed enhanced growth on gae1 gae6 plants It should be noted that B cinerea 537

virulence is often estimated by comparing lesion sizes Lesion size and measurements 538

of relative fungal growth by qPCR do not always show the same results For example 539

although Denby et al (2004) showed that Pepper causes larger lesions on pad3 than on 540

wild-type this study showed that relative fungal growth of Pepper on pad3 was 541

unaltered 542

We observed that callose deposition was strongly reduced in gae1 gae6 plants 543

72 h after treatment with B cinerea isolate Gallo 1 (Figure 11) possibly due to reduced 544

release of active soluble pectin Interestingly OG-induced PTI to an unspecified isolate 545

of B cinerea was intact in the pmr4 mutant which does not produce callose (Galletti et 546

al 2008) Due to the high SA levels in pmr4 plants (Nishimura et al 2003) and the fact 547

that different B cinerea isolates vary both in induction of and reaction to defense 548

responses (Kliebenstein et al 2005 Rowe et al 2010) we could not determine 549

Page 29 of 54

whether reduced callose deposition causes the increased susceptibility of gae1 gae6 to 550

B cinerea isolate Gallo 1551

We did not observe any changes in B cinerea growth either on cev1 ixr1 plants 552

which are known to have constitutively high JA and OPDA levels (Ellis et al 2002b) or 553

on plants exogenously treated with MeJA (Supplemental Figure 9) Because plants 554

were kept uncovered for 3 h after MeJA treatment it is unclear how much MeJA was 555

bioavailable in the plant However JAZ10 expression was induced 3 h after spraying 556

with MeJA (Supplemental Figure 10) Because pathogen growth generally depends on 557

the combination of a multitude of immune signaling events we conclude that multiple 558

changes to the immune signaling in gae1 gae6 together account for the altered B 559

cinerea growth phenotypes detected 560

We showed that expression of GAE1 and GAE6 is repressed by the bacterial 561

hemibiotroph Pma ES4326 This repression did not occur in an eds1 or pad4 562

background or in pbs3 at the 24-h time point (Figure 1 and Supplemental Figure 1) 563

During the Arabidopsis - Pma ES4326 interaction the expression of many defense 564

related genes is altered in a PAD4EDS1 dependent fashion (Wang et al 2008) Some 565

of these expression changes require SA CBP60g and SARD1 are thought to act 566

downstream of PAD4 and EDS1 but upstream of SA-dependent responses (Wang et 567

al 2011) PBS3 is also thought to act downstream of PAD4 and EDS1 and upstream of 568

SA but the set of genes whose expression it affects is only partially overlapping with 569

those affected by CBP60gSARD1 (Wang et al 2008 Wang et al 2011) Thus GAE1 570

and GAE6 repression is regulated by the part of the immune signaling network that 571

requires PAD4 EDS1 and PBS3 but is independent of CBP60gSARD1 and SA 572

Because EDS1 PAD4 and PBS3 are important components of the plant immune 573

signaling network (Wiermer et al 2005 Nobuta et al 2007) one might conclude that 574

repression of GAE1 and GAE6 may be part of the plant immune response to Pma 575

ES4326 B cinerea treatment also reduced GAE1 and GAE6 expression but this was 576

mostly independent of PAD4 (Figure 6) while both Pma ES4326 and B cinerea repress 577

GAE1 and GAE6 expression the host signaling mechanisms responsible likely differ 578

In summary our data show that Arabidopsis GAE1 and GAE6 are required for 579

pectin biosynthesis leaf flexibility and immunity to Pma ES4326 and to specific B 580

Page 30 of 54

cinerea isolates We used macerozyme a commercial pectinase to mimic the effect of 581

B cinerea pectinases Macerozyme treatment released less pectin from gae1 gae6 cell582

walls and induced less immune signaling in gae1 gae6 plants Less water soluble pectin 583

also accumulated in B cinerea infected gae1 gae6 plants and macerozyme-induced 584

immunity to B cinerea isolate Gallo 1 was reduced in these plants We conclude that 585

the reduced pectin content in these mutants may lead to reduced release of soluble 586

pectin including active OGs during pathogen attack and that this compromises 587

immunity 588

589

590

Page 31 of 54

METHODS 591

Plant Materials and Growth Conditions 592

Wild-type Columbia (Col-0) and all mutant plants (in Col-0 background) were 593

grown on sterilized BM2 germinating mix (Berger Inc Quebec Canada) in a controlled 594

environment chamber (Conviron Pembina ND USA) with a 12-h photoperiod under 595

100 microM m-2 s-1 fluorescent illumination at 22degC and 75 relative humidity Germplasm 596

used is described in the Accession Numbers section at the end of Methods 597

598

Data Analysis and Replicates 599

For biological replicates samples were obtained from separate plant tissues and 600

in many cases from plants grown in different flats In some experiments multiple 601

technical replicates were measured per biological replicate For statistical analysis all 602

technical replicates were averaged and the averages were considered one biological 603

replicate Independent experiments were performed at different times and often 604

consisted of multiple biological replicates per experiment 605

Data analysis was performed using mixed linear models in the R programming 606

environment (Bates et al 2015) unless otherwise indicated Generally genotypes 607

treatments and time points were used as fixed effects and replicate-specific effects like 608

different pots flats etc were considered random effects We reported p-values 609

obtained from the mixed linear model when less than 10 comparisons were performed 610

and q-values for more than 10 comparisons to correct for multiple comparison testing 611

Q-values were calculated using the Benjamini-Hochberg procedure (Benjamini and612

Hochberg 1995) unless specified otherwise 613

614

Pathogen Strains Growth Conditions and Pathogen Growth Assays 615

Growth of Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) and 616

bacterial growth assays were performed as previously described (Bethke et al 2014) 617

For qRT-PCR assays four week-old plants were inoculated with bacteria (OD600 = 618

0002) or mock (5 mM MgSO4) At least four leaves from two plants were collected at 619

the indicated time points per biological replicate 620

Page 32 of 54

Prior to each experiment Botrytis cinerea isolates were grown on 1x PDA 621

medium (Difco) for 10 d at room temperature All isolates used have been described 622

previously (Rowe and Kliebenstein 2007) The spores were washed from the surface of 623

the plate using an inoculation medium (1x Gamborgrsquos B-5 basal salt mixture (Sigma-624

Aldrich G5768) 2 (wv) glucose 10 mM phosphate buffer pH 64) and fungal hyphae 625

were removed from the suspension by filtering through four layers of cheesecloth The 626

concentration of spores was determined using a hemocytometer and adjusted to 25 x 627

105 spores mL-1 Four week-old Arabidopsis plants were inoculated by placing one 10-628

microL droplet of the B cinerea spore solution or inoculation medium (mock) on the adaxial 629

leaf surface of fully expanded leaves Inoculated plants were kept at 100 relative 630

humidity Infection sites were collected at various time points using a cork borer For 631

qRT-PCR assays at least four leaf discs from one plant were combined for each 632

biological sample For B cinerea growth assays at least 12 leaf discs from three to six 633

plants were pooled per biological sample Relative fungal growth was determined by 634

measuring the abundance of a fungal gene relative to a plant gene as described below 635

In brief DNA from infected tissue was extracted and equal amounts of total DNA were 636

used to perform qPCR reactions using the B cinerea cutinase A and the Arabidopsis 637

SK11 gene respectively Primers were described in (Gachon and Saindrenan 2004) 638

and are listed in Supplemental Table 1 Two technical replicates were combined for 639

each biological replicate B cinerea is a necrotrophic fungal pathogen and may destroy 640

plant DNA as disease progresses However decreases in the amount of plant DNA 641

over the course of our experiments were only observed at late time points with very 642

susceptible plants so the qPCR assay is generally a reasonable estimate of fungal 643

biomass (Supplemental Figure 16) 644

645

Treatment with Macerozyme or OGs 646

For PTI assays four week-old plants either were inoculated with 001 (wv) 647

macerozyme R-10 (Yakult) a Rhizopus polygalacturonase (EC 32115) that possesses 648

high pectinase and hemicellulase activity or were mock inoculated using heat-649

inactivated macerozyme Other similar plants were either inoculated with 250 or 500 microg 650

mL-1 OGs or mock inoculated with water One d later plants were infected with B 651

Page 33 of 54

cinerea and samples were collected immediately (0 dpi) and two or three d later To 652

investigate the role of MeJA on B cinerea growth plants were sprayed with 1mM MeJA 653

in water with 001 (vv) Tween 20 Covers were removed so that leaves could dry off 654

before plants were inoculated with B cinerea three h later Samples were harvested at 655

the indicated time points For expression analysis and MAPK activation assays plants 656

were either inoculated with 100 microg mL-1 OGs or mock inoculated with water OGs used 657

for experiments described in Figures 8 to 10 and Supplemental Figure 12 were a kind 658

gift from Simone Ferrari OGs used in experiments described in Figure 13 and 659

Supplemental Figures 11 13 and 15 were prepared as previously described (Kohorn et 660

al 2014) 661

662

Expression Analysis 663

Leaf tissue from inoculated plants was harvested at the indicated time points 664

flash frozen pulverized and RNA extracted using Trizol (Invitrogen) Quantitative RT-665

PCR was performed using the SuperScript III Platinum SYBR Green One-Step 666

quantitative RT-PCR kit (Invitrogen) and a Lightcycler 480 Real-Time PCR system 667

(Roche) as previously described (Truman and Glazebrook 2012) In brief equal 668

amounts of total RNA and a gene specific primer were used for each reaction The 669

crossing point (Cp) was calculated using the 2nd derivative max method provided with 670

the Lightcycler software for each amplification curve Each reaction was run with two 671

technical replicates and the Cp values for these replicates were averaged ACTIN2 was 672

used as a stably expressed reference gene At least three biological replicates each 673

were performed and analyzed using a mixed-linear model RT-PCR was performed to 674

verify T-DNA insertion lines using the One-Step RT-PCR kit (Qiagen) according to the 675

manufacturerrsquos instructions The primers used can be found in Supplemental Table 1 676

677

Electrolyte Leakage Assay 678

Fully expanded leaves of four week-old plants were inoculated with water (Milli-Q 679

grade) using a needleless syringe Leaf discs were collected using a cork borer and 680

three leaf discs from one leaf were placed with the adaxial surface down onto 2 mL 681

water in a 12-well cell culture plate Conductivity was measured using a Horiba B-173 682

Page 34 of 54

conductivity meter after 30 min and then the water was replaced Conductivity was 683

measured again 30 min later (1 h total) as indicated in Figure 3C Six biological 684

replicates each were performed in three independent experiments and means and SEs 685

were calculated Two-sided t-tests were performed for each time point 686

687

DAB and Aniline Blue Staining 688

Leaves from four week-old plants were either mock inoculated with B cinerea 689

inoculation medium or inoculated with 10 microL droplets of a B cinerea spore solution (25 690

x 105 spores mL-1) of the indicated isolates Samples were collected 72 h later For 33prime-691

diaminobenzidine (DAB) staining two to five inoculated leaves were vacuum infiltrated 692

with 33prime-Diaminobenzidine (1 mg mL-1 in water pH 38) solution and stained overnight 693

in the dark Leaves were destained using 100 ethanol at 65degC and then transferred to 694

50 (vv) glycerol and photographed 695

Callose was visualized by staining leaves with aniline blue as previously 696

described (Adam and Somerville 1996) Briefly leaves were vacuum infiltrated with 697

alcoholic lactophenol (one volume of phenolglycerollactic acidwater (1111) mixed 698

with two volumes of ethanol) and destained at 65degC Next leaves were moved through 699

an alcohol gradient (50 20 and 10 (vv) ethanol) into water The destained leaves 700

were stained for 30 min in 150 mM K2HPO4 (pH 95) with 001 (wv) aniline blue 701

Samples were mounted in water and examined using a Nikon Eclipse Ni-U microscope 702

at 10x magnification using a DAPI filter (excitation at 325-375 nm emission at 435-485 703

nm) For callose quantification six 200 microm x 200 microm squares were counted for each 704

sample type Callose deposits per mm2 were calculated and data analyzed using a t-705

test Note that there is an inherent uncertainty associated with such counts because 706

callose deposits may not all be in focus at the same time 707

708

MAPK Activity Assay 709

Fully expanded leaves of four week-old plants were inoculated with 001 710

macerozyme 100 microg mL-1 OGs 100 nM flg22 or water (mock) flg22 a peptide from 711

bacterial flagella that induces immune responses was used as a positive control for 712

Page 35 of 54

MAPK activation (Asai et al 2002) and was purchased from EZBiolab Inc (Westfield 713

IN USA) Samples were harvested 10 min later and immediately frozen in liquid 714

nitrogen Protein was extracted using an extraction buffer containing 25 mM Tris pH 78 715

75 mM NaCl 1 mM DTT 1 mM NaF 05mM Na3VO4 01 (vv) Tween 20 one 716

cOmplete Mini protease inhibitor cocktail tablet (Roche) and one PhosSTOP 717

phosphatase inhibitor cocktail tablet (Roche) per 10 mL buffer 15 microg of total protein 718

were electrophoresed on a 10 SDS-polyacrylamide gel and then blotted onto a PVDF 719

membrane (Bio-Rad 162-0177) Activated MAPKs were detected using p4442 MAPK 720

(Erk12) antibody (9102S Cell Signal 12500 in TBST (20 mM Tris pH 75 150 mM 721

NaCl 01 (vv) Tween 20)) Anti-Rabbit IgG-HRP (115000 in TBST A6154 Sigma) 722

and ECL Plus substrate (Pierce 32132) MPK3 and MPK6 were detected with an anti-723

MPK3 antibody (M8318 Sigma 12000 in TBST and 3 (wv) milk) or an anti-MPK6 724

antibody (A7104 Sigma 16000 in TBST and 3 (wv) milk) respectively The 725

secondary antibody and detection were the same as for the p4442 antibody Five 726

independent experiments were performed 727

728

Preparation of Alcohol Insoluble Residue (AIR) Pectin Extraction and Uronic Acid 729

Measurements 730

Fully expanded leaves from four week-old plants that were incubated for 48 h in 731

the dark to reduce starch were harvested flash frozen and pulverized AIR was 732

extracted by washing ground material twice in 70 (vv) ethanol three times in a 733

mixture of chloroform and methanol (11 vv) and once in acetone (Gille et al 2009) 734

and was then air dried Cell wall pectin was extracted from AIR with cell wall extraction 735

buffer (50 mM Trizma 50 mM CDTA pH 72) at 95degC for 15 min Samples were 736

homogenized using a paint shaker (Harbil 5G-HD) and glass beads (3 mm diameter) 737

For dot blot experiments 500 microL of cell wall extraction buffer were used per 10 mg of 738

AIR Debris was precipitated by centrifugation for 10 min at 10000 g 739

Total uronic acid content of pectin extracted as described above using 500 microL of 740

cell wall extraction buffer per 1 mg of AIR was measured as described previously 741

(Filisetti-Cozzi and Carpita 1991 van den Hoogen et al 1998) First 36 microL of pectin 742

extract were mixed with 4 microL of 4 M sulfamic acid Then 200 microL of sulfuric acid 743

Page 36 of 54

containing 120 mM sodium tetraborate was added and samples were incubated at 80degC 744

for 1 h Following cooling on ice the optical density of samples was measured at 490 745

nm Next 40 microL of m-hydroxydiphenyl reagent (100 microL of m-hydroxydiphenyl in dimethyl 746

sulfoxide at 100 mgmL mixed with 49 mL 80 (vv) sulfuric acid just before use) was 747

added and the samples were mixed The optical density at 490 nm was measured 748

again The optical density before m-hydroxydiphenyl reagent addition was subtracted 749

from the optical density measured after addition of the dye The concentration of uronic 750

acid was calculated using known amounts of GalA as a standard 751

752

Release of Pectin from Arabidopsis Cell Walls Using Macerozyme 753

AIR preparations from leaves of four week-old plants were treated with 025 754

(wv) macerozyme R-10 (Yakult) or water (mock) at a concentration of 100 microL per mg 755

AIR Samples were incubated for 3 h at room temperature on a vortex shaker and 756

centrifuged for 10 min at 12000 g Total uronic acid concentration of 5-fold diluted 757

supernatants was determined for two technical replicates each The concentration of 758

uronic acid was calculated using known amounts of GalA as a standard For 759

subsequent qRT-PCR experiments supernatants from multiple extractions were 760

combined boiled for 20 min to inactivate macerozyme diluted 10-fold with water and 761

inoculated into four week-old Col-0 plants Heat-inactivated 10-fold diluted macerozyme 762

solutions were used as mock treatment Samples were collected 3 h later and PAD3 763

expression was determined 764

765

Release of Pectin from Arabidopsis Leaves Infected with B cinerea 766

Fully expanded leaves of four week-old plants were either inoculated with up to 767

five 10 microL droplets of B cinerea spore solution (25 x 105 spores mL-1) or mock 768

inoculated with B cinerea inoculation medium For each biological replicate leaves with 769

a combined count of at least 20 infection sites were collected 48 h later AIR was 770

extracted as described above but plants were not dark-treated or freeze-dried and the 771

first ethanol step was performed using 85 (vv) ethanol To extract soluble pectin 772

fragments AIR samples were mixed with water at a concentration of 20 microL per infection 773

site incubated for 7 h at room temperature on a vortex shaker and centrifuged for 10 774

Page 37 of 54

min at 12000 g Total uronic acid concentration of supernatants was determined for two 775

technical replicates each The concentration of uronic acid was calculated using known 776

amounts of GalA as a standard 777

778

Measurement of Uronic Acids Neutral Monosaccharides and Cellulose Content in 779

Cell Walls 780

Neutral sugars and uronic acids from non-cellulosic polysaccharides in cell walls 781

(AIR) were released by trifluoroacetic acid (TFA) hydrolysis Approximately 1 mg of AIR 782

was hydrolyzed with 2 M TFA at 121degC for 90 min The TFA-soluble material was split 783

into two equal parts one for uronic acid and one for neutral sugar determination The 784

uronic acid content was determined by analyzing the hydrolyzate using a CarboPac 785

PA200 anion-exchange column with an ICS-3000 Dionex chromatography system 786

(Dionex Sunnyvale CA USA) The elution profile consisted of a linear gradient of 50 to 787

200 mM sodium acetate in 01 M NaOH in 10 min at 04 mL per min Alditol acetate 788

derivatives of neutral sugars were produced as described (York et al 1985) In brief 789

monosaccharides in the hydrolyzate were reduced with NaBH4 and peracetylated with 790

acetic anhydride and pyridine at 121degC for 20 min The generated alditol acetates were 791

analyzed by GC-MS Crystalline cellulose was measured according to Updegraff (1969) 792

After hydrolysis of noncellulosic polysaccharides from AIR with the Updegraff reagent 793

(acetic acid nitric acids water 812 (vv) ) the remaining pellet was hydrolyzed in 794

72 sulfuric acid The resulting glucose quantity was determined by the anthrone 795

method (Scott and Melvin 1953) 796

797

Dot Blot Experiments 798

Pectin solutions extracted using 50 microL of cell wall extraction buffer per mg AIR 799

were serially diluted and nitrocellulose membranes were spotted with 1 microL of the diluted 800

pectin solutions Membranes were dried overnight blocked with 5 milk (wv) in 1x 801

PBS (8 g L-1 NaCl 02 g L-1 KCl 144 g L-1 Na2HPO4 024 g L-1 KH2PO4 pH 74) and 802

probed with LM5 (Jones et al 1997) LM6 (Willats et al 1998) LM8 (Willats et al 803

2004) LM19 and LM20 (Verhertbruggen et al 2009a) or CCRC-M7 (Steffan et al 804

Page 38 of 54

1995) antibodies LM series antibodies were diluted 1250 and CCRC-M7 was diluted 805

1500 in 5 milk powder (Nestle Carnation) in 1 x PBS For LM series antibodies a 806

goat anti-Rat HRP conjugated antibody (Bethyl A110-105P 15000 diluted) and for 807

CCRC-M7 an anti-Mouse IgG HRP conjugate (Promega W402B 12500 diluted) was 808

used as secondary antibody in 5 milk powder in 1x PBS LM series antibodies were 809

obtained from plant probes (wwwplantprobesnet) and CCRC-M7 from CarboSource 810

(wwwcarbosourcenet) Membranes were washed after incubation with the primary and 811

secondary antibodies with 1x PBS Dot blots were developed using the ECL system 812

(GE Healthcare Amersham ECL prime) For quantification of signals dot blot results on 813

x-ray films were photographed using a CCD camera and intensities were measured814

using Image Jrsquos Integrated Density function Locally adjusted background was 815

subtracted As the dynamic range of the signal on an x-ray film was narrow non-816

linearity in the measured values was corrected for each genotype in each biological 817

replicate using the values from different dilutions in two technical replicates We noticed 818

that the residuals were not normally distributed and performed Box-Cox power 819

transformation (Box and Cox 1964) to obtain normally distributed values for the 820

subsequent statistical analysis of the data For LM19 Box-Cox selected power 821

transformation was done to the power of 038th and for LM20 to the power 075th After 822

correction of the transformed measured values with the effect of the biological 823

replicates the values were fit to a linear model with the Arabidopsis genotype as the 824

fixed effect This linear model was used both to generate the plots in Figures 5B and D 825

and to test significant differences 826

827

Camalexin Measurements 828

Four week-old plants were infected with B cinerea and samples (three leaf disks 829

from one plant each) were harvested at the indicated time points flash frozen and 830

pulverized Each sample was extracted with 300 microL of 90 methanol (vv) 50 microL of the 831

extract was run on an Agilent Lichrocart 250-4 RP18e 5-microm column using an Agilent 832

1100 series HPLC Camalexin was detected using a DiodeArray (DAD) at 330 nm and 833

with a fluorescence detector at emission 318 nmexcitation 385 nm (Agilent) Separation 834

was achieved using the following program with aqueous acetonitrile 5 min gradient 835

Page 39 of 54

from 63 to 69 acetonitrile 30 s gradient from 69 to 99 acetonitrile 2 min at 99 836

acetonitrile and a post-run equilibration of 35 min at 63 acetonitrile (Denby et al 837

2004 Kliebenstein et al 2005) Purified camalexin was used to produce a standard 838

curve to identify and quantitate camalexin 839

840

Accession Numbers 841

Microarray data used can be accessed at NCBI Gene Expression Omnnibus 842

(httpwwwncbinlmnihgovgeo) GSE18978 Sequence information for all Arabidopsis 843

genes described in this article can be found in TAIR (wwwarabidopsisorg) using the 844

following accession numbers PAD3 (CYP71B15 At3g26830) SID2 (ICS1 At1g74710) 845

GAE1 (At4g30440) GAE2 (At1g02000) GAE3 (At4g00110) GAE4 (At2g45310) GAE5 846

(At4g12250) GAE6 (At3g23820) PR1 (At2g14610) JAZ5 (At1g17380) JAZ10 847

(At5g13220) ACTIN2 (At3g18780) PDF12a (At5g44420) PDF12b (At2g26020) 848

AtSK11 (At5g26751) The sequence of the B cinerea cutinase Bc-CutA (Z69264) can 849

be found in the DNA Data Bank of Japan (httpgetentryddbjnigacjp) Germplasm 850

used included dde2-2 (At5g42650 (von Malek et al 2002)) pad3-1 (At3g26830 (Zhou 851

et al 1999)) pad4-1 (At3g52430 (Jirage et al 1999)) sid2-2 (At1g74710 (Wildermuth 852

et al 2001)) pbs3-2 (At5g13320 SALK_018225 (Nobuta et al 2007)) cbp60a-1 853

(At5g62570 SALK_124410 (Truman et al 2013)) cbp60g-1 sard1-2 (At5g26920 854

SALK_023199 At1g73805 SALK_052422 (Wang et al 2011) cbp60a-1 cbp60g-1 855

sard1-2 (Truman et al 2013) gae1-1 (At4g30440 SALK_085554) gae6-1 (At3g23280 856

SALK_104454) gae6-2 (At3g23280 SALK_017191) ixr1-1 cev1 (At5g05170 (Ellis 857

and Turner 2001 Scheible et al 2001)) eds1 plants were derived by introgression of 858

the Ler eds1-2 allele into Col-0 that contains two copies of EDS1 (At3g48090 859

At3g48080 (Bartsch et al 2006) mpk6-2 (At2g43790 SALK_073907 (Liu and Zhang 860

2004)) mpk3-DG (At3g45640 a fast neutron deletion mutant (Miles et al 2005)) and 861

npr1-1 (At1g64280 (Cao et al 1997)) T-DNA insertion lines were part of the SALK 862

collection (Alonso et al 2003) and were obtained from the Arabidopsis Biological 863

Resource Center 864

865

Supplemental Data 866

Page 40 of 54

Supplemental Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 867

Expression Does Not Require CBP60s or SID2 868

Supplemental Figure 2 Expression of GAE Family Members uponTreatment with Pma 869

ES4326 870

Supplemental Figure 3 Characterization of T-DNA Insertions in GAE1 and GAE6 871

Supplemental Figure 4 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves and 872

Are Slightly Smaller than Wild-Type Col-0 Plants 873

Supplemental Figure 5 Cell Walls of gae1 gae6 Plants Contain Less Uronic Acid than 874

Wild-Type Col-0 Cell Walls 875

Supplemental Figure 6 Antibodies Raised Against the RGI or XG Components of 876

Pectin Show No Difference in Binding to gae Mutant and Col-0 Cell Walls 877

Supplemental Figure 7 The Effect of B cinerea Treatment on the Expression of GAE 878

Family Genes 879

Supplemental Figure 8 Treatment with 001 Macerozyme Does Not Cause Any 880

Visible Damage to Col-0 Plants 881

Supplemental Figure 9 B cinerea Growth on Plants with Defects in Immune 882

Signaling 883

Supplemental Figure 10 MeJA-Induced Expression of the JA marker gene JAZ10 884

Supplemental Figure 11 The Expression of the JA Marker Gene JAZ5 Is Highly 885

Responsive in gae1 gae6 Plants 886

Supplemental Figure 12 The Expression of PDF12 Is Unaltered in gae1 gae6 Plants 887

Supplemental Figure 13 Macerozyme Treatment Activates MPK3 and MPK6 888

Supplemental Figure 14 B cinerea-Induced ROS Production in Wild-Type Col-0 and 889

gae1 gae6 Plants 890

Supplemental Figure 15 OG-Induced PTI Using 250 microg mL-1 OGs 891

Supplemental Figure 16 Abundance of Plant DNA in B cinerea Treated Plants 892

Supplemental Table 1 Primers used in this study 893

894

ACKNOWLEDGEMENTS 895

We thank William Truman for helpful discussions Simone Ferrari (Sapienza 896

Universita di Roma) for the generous gift of OGs Bruna Bucciarelli (USDA-ARS) for 897

Page 41 of 54

help with fluorescence microscopy the Arabidopsis Biological Resource Center for T-898

DNA insertion lines Paul Knoxrsquos lab (University of Leeds) for LM5 LM6 LM8 LM19 899

and LM20 antibodies and the Complex Carbohydrate Resource Center (University of 900

Georgia) for the CCRC-M7 antibody Most of this work was funded by the Division of 901

Chemical Sciences Geosciences and Biosciences Office of Basic Energy Sciences of 902

the US Department of Energy through Grant DE-FG02-05ER15670 to JG This work 903

was also supported by NSF awards IOS 1339125 and IOS 1021861 to DJK NSF 904

award IOS 1121425 to F K and the USDA National Institute of Food and Agriculture 905

Hatch project number CA-D-PLS-7033-H to DJK 906

907

AUTHOR CONTRIBUTIONS 908

GB and JG designed the research GB GX AT BL NES and NH 909

performed research DJK provided tools GB GX MP AD BL RAH and FK 910

analyzed data GB and JG wrote the paper with input from all authors 911

912

FIGURE LEGENDS 913

Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 Expression 914

Requires PAD4 EDS1 and PBS3 915

(A) Expression of GAE1 in leaves of wild-type Col-0 and mutant plants Expression was916

measured 24 or 32 h after either inoculation with Pma ES4326 (OD600 = 0002) or mock 917

inoculation with 5 mM MgSO4 and was then normalized to the level of ACTIN2 Data 918

from three biological replicates were combined using a mixed linear model Bars 919

represent mean log2-ratios to ACTIN2 plusmn SE Letters indicate significantly different 920

groups with q lt 005 where 005 represents the maximum false discovery rate at which 921

the test may be called significant 922

(B) Expression of GAE6 measured as described in (A)923

924

Figure 2 Susceptibility of gae6 and gae1 gae6 Plants to Pma ES4326 Is Increased 925

Relative to Wild-Type Col-0 926

Leaves of plants of the indicated genotypes were inoculated with Pma ES4326 (OD600 = 927

00002) as described in Methods Bacterial titers in leaves were determined either 928

Page 42 of 54

immediately (0 dpi) or after three days (3 dpi) Bars represent the mean plusmn SE of four 929

independent experiments each with 4 or 12 biological replicates at 0 and 3 dpi 930

respectively Data were combined using a mixed linear model Asterisks indicate 931

significant differences from Col-0 wild-type (q lt 005) Susceptible pad4 plants were 932

included as a positive control 933

934

Figure 3 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves 935

(A) Depiction of the assay for brittleness of gae mutant leaves Fully expanded leaves of936

four to five week-old plants were bent by 180ordm so that the adaxial surface of the leaf was 937

parallel with the soil Broken leaf petioles or midribs were considered brittle 938

(B) Quantification of broken midribs or petioles For each genotype 66 to 82 leaves 939

were analyzed for brittleness as described in (A) Bars represent the ratio of broken to 940

total number of leaves tested Results were analyzed using Fisherrsquos exact test Letters 941

indicate significantly different groups at p lt 005 942

(C) Conductivity measurements Leaves of four week-old plants were inoculated with943

deionized water and then leaf discs were cut out and placed on deionized water 944

Conductivity was measured at the indicated time points After 30 min the water was 945

replaced with fresh water (30 min f w) to account for wounding-related ion leakage from 946

the edges of the leaf discs Three independent experiments with six biological replicates 947

each were performed and data were analyzed using a t-test for each time point P-948

values were corrected using the Bonferroni method Data show means plusmn SE asterisks 949

indicate significant differences from wild-type Col-0 at q lt 001 950

951

Figure 4 Cell Walls of gae6 and gae1 gae6 Plants Are Reduced in GalA 952

(A) to (D) Levels of GalA (A) GlcA (B) cellulose (C) and neutral sugars (D) in alcohol953

insoluble residue (AIR) extracted from leaves of four week-old plants of the indicated 954

genotypes Bars represent means plusmn SE of four biological replicates combined using a 955

mixed linear model Letters indicate significantly different groups (p lt 005) 956

957

Figure 5 Cell Walls of gae1 gae6 Plants Are Reduced in Homogalacturonan 958

Page 43 of 54

Dot blot analyses with anti-LM19 and anti-LM20 antibodies Pectin was extracted from 959

AIR prepared from the indicated genotypes using 1 microL of extraction buffer per 10 microg of 960

AIR Pectin was serially diluted and one microL of undiluted 13 diluted and 19 diluted 961

samples were spotted on a nitrocellulose membrane Three biological replicates were 962

were spotted twice to obtain two technical replicates each All replicates produced 963

similar results 964

(A) Representative dot blot using anti-LM19 antibody965

(B) Relative signal intensities of the spots in the dot blot in (A)966

(C) Representative dot blot using anti-LM20 antibody967

(D) Relative signal intensities of the LM20 dot blot shown in (C)968

Relative signal intensities were measured using Image J software Measurements from 969

the three biological replicates were combined using a linear model after Box-Cox power 970

transformation as described in Methods Bars represent means plusmn SE Asterisks indicate 971

a difference from wild-type Col-0 at p lt 005 972

973

Figure 6 Growth of B cinerea on gae1 gae6 Plants and the Effect of B cinerea on 974

Expression of GAE1 and GAE6 975

(A) Growth of B cinerea isolates Leaves of Col-0 and gae1 gae6 plants were976

inoculated with 25 x 105 spores mL-1 of the B cinerea isolates indicated in the figure 977

Samples were collected two (2 dpi) and three (3 dpi) days later Bars represent means plusmn 978

SE of six biological replicates for each fungus combined using a mixed linear model 979

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 980

(B) and (C) Expression of GAE1 (B) and GAE6 (C) 48 h after inoculation with the B981

cinerea isolates indicated Expression levels were measured by qRT-PCR Bars 982

represent mean log2-ratios to ACTIN2 plusmn SE of three biological replicates combined 983

using a mixed linear model Letters indicate significantly different groups (q lt 005) 984

985

Figure 7 Levels of Soluble Pectin and Expression of PAD3 in Fractions Produced 986

Following Macerozyme Treatment of gae1 gae6 Cell Walls 987

(A) Schematic overview of the experimental procedure for (B) and (C)988

Page 44 of 54

(B) Uronic acid measurements Alcohol-insoluble residue (AIR) from leave tissue of four989

week-old wild-type Col-0 or gae1 gae6 plants was treated with water or 025 990

macerozyme and assayed for uronic acid levels Data from 11 biological replicates were 991

combined using a mixed linear model Bars represent means plusmn SE Letters indicate 992

significant differences (p lt 005) 993

(C) Expression of PAD3 in Col-0 plants treated with the fractions from (B) Extracts from994

(B) were heat inactivated pooled and diluted 5-fold with water Pooled extracts were995

inoculated into four week-old Col-0 plants PAD3 expression was measured 3 h after 996

inoculation and normalized to the level of ACTIN2 expression Data from four biological 997

replicates were combined using a mixed linear model Bars represent mean log2-ratios 998

to ACTIN2 plusmn SE Letters indicate significant differences (p lt 005) 999

1000

Figure 8 Macerozyme-Induced PAD3 Expression Is Absent in gae1 gae6 1001

(A) Expression of PAD3 after macerozyme treatment Four week-old plants were either1002

inoculated with 001 macerozyme or mock inoculated with water and PAD3 1003

expression was measured 3 h later by qRT-PCR Data from four biological replicates 1004

were combined using a mixed linear model Mean log2-ratios to ACTIN2 plusmn SE were 1005

plotted Letters indicate significantly different groups (p lt 005) 1006

(B) Expression of PAD3 after OG treatment PAD3 expression was measured as1007

described in (A) 3 h after inoculation with 100 microg mL-1 OGs or mock inoculation with 1008

water Data are from six biological replicates 1009

(C) PAD3 expression in untreated plants Three biological replicates were performed1010

and data analyzed as described in (A) 1011

(D) Camalexin accumulation after B cinerea treatment Camalexin accumulation was1012

measured in four week-old plants inoculated with the B cinerea strains shown in the 1013

figure Eight biological replicates each were combined using a mixed linear model 1014

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 1015

(E) PAD3 expression 28 h after treatment with the indicated B cinerea strains Data1016

from six biological replicates were collected and analyzed as described in (A) Data in 1017

all figure parts represent means plusmn SE Letters indicate significantly different groups (q lt 1018

005) 1019

Page 45 of 54

1020

Figure 9 Macerozyme-Induced Expression of the SA Marker Genes SID2 and PR1 1021

Is Abolished in gae1 gae6 Plants 1022

(A) to (H) Expression of PR1 (A) (C) (E) and (G) or SID2 (B) (D) (F) and (H) in four1023

week-old Col-0 and gae1 gae6 plants Data were combined using a mixed linear model 1024

Bars represent mean log2-ratios of expression vs ACTIN2 plusmn SE for all figure parts 1025

Letters indicate significantly different groups at p lt 005 for panels (A) to (D) (G) and 1026

(H) and qlt005 for panels (E) to (F) 1027

(A) and (B) Plants were treated with 001 macerozyme or mock treated with water1028

and samples were collected 3 h later Data from four biological replicates are shown 1029

(C) and (D) Plants were inoculated with 100 microg mL-1 OGs or mock treated with water1030

and samples collected 3 h later Data from six biological replicates are shown 1031

(E) and (F) Plants were inoculated with the indicated B cinerea isolates or mock1032

inoculated with B cinerea inoculation medium and samples collected 48 h later Data 1033

from eight biological replicates are shown 1034

(G) and (H) Expression in untreated plants Three biological replicates were performed1035

1036

Figure 10 The Expression of the JA Marker Gene JAZ10 is Highly Responsive in 1037

gae1 gae6 Plants 1038

(A) to (D) JAZ10 expression in four week-old plants Data were combined using a mixed1039

linear model Mean log2-ratios to ACTIN2 plusmn SE are shown Letters indicate significantly 1040

different groups at p lt 005 for panels (A) (B) and (D) and q lt 005 for panel (C) 1041

(A) Plants were inoculated with 001 macerozyme or mock inoculated using water and1042

samples were collected 3 h later Data from four biological replicates are shown 1043

(B) Plants were inoculated with 100 microg mL-1 OGs or mock inoculated using water and1044

samples collected 3 h later Data are from six biological replicates 1045

(C) JAZ10 expression 48 h after treatment with the indicated B cinerea strains Data1046

are from six biological replicates 1047

(D) JAZ10 expression in untreated plants Data are from three biological replicates1048

1049

Figure 11 Gallo 1-Induced Callose Deposition Is Reduced in gae1 gae6 Plants 1050

Page 46 of 54

(A) Callose deposition in four week-old Col-0 and gae1 gae6 plants Plants were1051

inoculated with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) or mock inoculated 1052

with B cinerea inoculation medium and samples were collected after 72 h Callose was 1053

stained with aniline blue and visualized using a Nikon Eclipse Ni-U microscope with a 1054

DAPI filter Three leaves were stained per treatment Scale bars are 100 microm Asterisks 1055

indicate fungal hyphae arrows indicate representative callose spots 1056

(B) Quantification of callose deposition Callose deposits were counted in 200 microm x 2001057

microm squares laid out across the images shown here Bars represent means plusmn SE from 1058

six squares each Letters indicate significantly different groups (p lt 005) according to a 1059

t-test analysis1060

1061

Figure 12 Less Soluble Pectin Accumulates in B cinerea-Infected gae1 gae6 1062

Leaves 1063

Four week-old Col-0 and gae1 gae6 plants were inoculated with 10 microL droplets of the 1064

indicated B cinerea isolate (25 x 105 spores mL-1) or mock inoculated with B cinerea 1065

inoculation medium Leaves were collected 48 h later AIR was extracted from leaf 1066

samples and water added (20 microL infection site) to extract soluble pectin fragments 1067

Samples were vortexed for 7 h centrifuged at 12000 g and the uronic acid 1068

concentration of the supernatant was measured Data from nine biological replicates 1069

were combined using a mixed linear model Means plusmn SE are shown Letters indicate 1070

significant differences (q lt 005) 1071

1072

Figure 13 Macerozyme-Induced PTI Is Lost in gae1 gae6 While OG-Induced PTI Is 1073

Unaltered 1074

(A) Macerozyme-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1075

inoculated with 001 macerozyme or mock inoculated with boiled macerozyme and 1076

were infected with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) 24 h later Samples 1077

were collected immediately (0 dpi) and after two (2 dpi) and three (3 dpi) days Data 1078

show mean plusmn SE of six to 14 biological replicates each combined using a linear model 1079

Letters indicate significantly different groups (q lt 005) 1080

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

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Page 9 of 54

There are six GAE family members in Arabidopsis GAE2 expression was 207

induced by Pma ES4326 in the absence of PAD4 or EDS1 GAE3 expression was 208

unaltered GAE4 was repressed early and GAE5 expression was induced in all 209

genotypes (Supplemental Figure 2) The similarity of the immune-regulated expression 210

of GAE1 and GAE6 suggested a redundant role of these genes in Arabidopsis immunity 211

to Pma ES4326 212

213

Resistance to Pma ES4326 Is Compromised in gae1 gae6 and gae6 Plants 214

To study the roles of GAE1 and GAE6 in plant immunity in more detail plants 215

with T-DNA insertion mutations in GAE1 and GAE6 were obtained from the SALK 216

collection as described in the Accession Numbers section of Methods We identified two 217

GAE6 knock-out alleles gae6-1 and gae6-2 and a T-DNA insertion in GAE1 gae1-1 218

that caused reduced expression of this gene (Supplemental Figure 3) A double mutant 219

was created by crossing gae1-1 and gae6-1 Both gae6 and gae1 gae6 plants were 220

more susceptible to Pma ES4326 than wild-type Col-0 and gae1 plants supporting a 221

role of GAE6 in plant immunity to Pma ES4326 (Figure 2) 222

223

gae6 and gae1 gae6 Plants Have Brittle Leaves 224

While inoculating leaves of gae1 gae6 plants we noticed that the petioles and 225

midribs of these plants tended to break easily when leaves were bent against the 226

proximal-distal axis of the leaves (Figure 3 see Supplemental Figure 4D to F) They 227

tended to break less often when twisted around the proximal-distal axis To quantify 228

this phenotype leaves were bent by 180deg (Figure 3A) and breaking of midribs or 229

petioles was recorded Most gae1 gae6 leaves broke whereas fewer leaves of gae6 230

plants and very few leaves of wild-type Col-0 and gae1 plants did (Figure 3B and 231

Supplemental Figure 4C) Although gae1 gae6 plants had slightly shorter leaves and 232

petioles than wild type plants (Supplemental Figure 4A and B) no correlation between 233

leaf size or petiole length and breaking frequency was detected We also investigated 234

tissue integrity by monitoring ion leakage Interestingly when leaves were inoculated 235

with deionized water leaf discs of gae1 gae6 leaked more ions than wild-type Col-0 236

discs (Figure 3C) suggesting that the increased brittleness is correlated with a 237

Page 10 of 54

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Page 11 of 54

Cell Walls of gae6 and gae1 gae6 Plants Have Lower Levels of Pectin 243

GalA is a major component of pectin We analyzed wall monosaccharide 244

composition in gae mutants to investigate whether the increased brittleness was 245

associated with an altered wall composition of gae leaves To approximate pectin 246

content of gae mutant walls the total uronic acid concentration was measured using a 247

simple colorimetric assay (Filisetti-Cozzi and Carpita 1991 van den Hoogen et al 248

1998) Total uronic acid was reduced in gae6-2 strongly reduced in gae1 gae6 plants 249

but was indistinguishable from wild-type Col-0 in gae6-1 and gae1 (Supplemental 250

Figure 5) This suggested that gae1 gae6 and possibly gae6 plants have reduced 251

pectin 252

To investigate the cell wall composition in leaves of gae plants in more detail we 253

measured the levels of seven neutral monosaccharides the acidic sugars GalA (the 254

monomeric subunit of the pectin backbone (Harholt et al 2010)) and GlcA (Figure 4) 255

We also estimated the crystalline cellulose content (Updegraff 1969) Cell walls of both 256

gae6 mutants and gae1 gae6 were reduced in GalA (Figure 4A) In gae1 gae6 GalA 257

content in cell walls was reduced by more than 40 compared to the wild-type level 258

GlcA levels were indistinguishable between mutants and wild-type (Figure 4B) 259

Cellulose content was higher in gae6-2 and gae1 gae6 walls likely due to the loss of 260

pectins (Figure 4C) Cell walls of gae1 gae6 plants were also enriched in all neutral 261

sugars measured presumably due to the substantial proportional loss of GalA (Figure 262

4D) The levels of most wall monosaccharides were even higher in the gae1 gae6 263

double mutant with the exception of Rha which did not exhibit a significant increase 264

compared to the gae6 single mutants The absence of a compensational increase in 265

Rha suggested that RGI the dominant Rha contributor to the wall may be considered 266

reduced in gae1 gae6 mutants compared to wild-type albeit not to such an extent as 267

HG 268

238 structural defect that promotes increased ion leakage Taken together these results

239 showed that loss of GAE6 causes brittle leaves and additional losss of GAE1 strongly

240 enhances this phenotype We conclude that GAE1 and GAE6 contribute to normal leaf

241 flexibility 242

Page 12 of 54

gae6 cell walls are reduced in HG Using the same samples we detected no difference 274

from wild type in binding of LM5 which recognizes a tetramer of (1-4)-β-D-galactans 275

found in RGI (Jones et al 1997) LM6 which recognizes a linear pentasaccharide in (1-276

To further investigate which pectic components were altered in gae1 gae6 dot 269

blot experiments using pectin isolated from the various genotypes were performed Both 270

LM19 and LM20 antibodies recognize HG (Verhertbruggen et al 2009a) As expected 271

LM19 binding was reduced in gae6-1 and gae1 gae6 (Figure 5A and B) and LM20 272

binding was reduced in gae1 gae6 (Figure 5C and D) This result confirmed that gae1 273

277 5)-α-L-arabinans found in RGI (Willats et al 1998 Verhertbruggen et al 2009b) LM8

278 which recognizes xylogalacturonan (Willats et al 2004) or CCRC-M7 which

279 recognizes an arabinosylated (1-6)-β-D-galactan epitope occurring on RGI (Puhlmann et

Page 13 of 54

Page 14 of 54

al 1994 Steffan et al 1995 Pattathil et al 2010) (Supplemental Figure 6) 280

Collectively these cell wall composition data show that gae1 gae6 plants are reduced in 281

HG Due to the strong proportional reduction of HG in the walls levels of all other 282

components should be higher However Rha indicative of RGI was not higher in gae1 283

gae6 suggesting that RGI may also be reduced in gae1 gae6 walls 284

285

GAE1 and GAE6 Are Required for Immunity to Specific B cinerea Isolates 286

We hypothesized that plant immunity to B cinerea might be affected by pectin 287

content in the host cell wall because this pathogen requires polygalacturonases and 288

pectin methylesterases for full virulence (ten Have et al 1998 Valette-Collet et al 289

2003 Kars et al 2005b Espino et al 2010) Because Bcinerea is genetically diverse 290

and has a high diversity of polygalacturonases (Rowe and Kliebenstein 2007) we 291

measured relative fungal growth at both 2 and 3 d post-inoculation (dpi) using ten 292

different B cinerea isolates (Figure 6) The B cinerea isolates Fresa 525 and Gallo 1 293

showed enhanced virulence on gae1 gae6 plants at both time points whereas KT and 294

UK Razz showed enhanced virulence in gae1 gae6 plants at 3 but not 2 d dpi (Figure 295

6A) We did not detect any significant changes in relative fungal growth between wild-296

type Col-0 and gae1 gae6 plants when using Acacia Apple 517 DN Grape Pepper or 297

Rasp isolates of B cinerea Therefore we continued our study using Gallo 1 and Fresa 298

525 We also included Pepper for comparison as this isolate has been used in a large-299

scale analysis of plant gene expression in response to B cinerea infection (Windram et 300

al 2012) 301

We next investigated whether expression of GAE1 and GAE6 was affected by 302

challenge with B cinerea Inoculation of wild-type Col-0 plants with any isolate 303

repressed GAE1 and GAE6 expression (Figure 6B and C) and this repression was 304

generally independent of PAD4 (Figure 6B and C) Expression of GAE2 and GAE3 was 305

unaltered by B cinerea treatment GAE4 was repressed only by Fresa 525 treatment 306

while GAE5 expression was enhanced with all three isolates tested (Supplemental 307

Figure 7) As seen for infection with Pma ES4326 the expression of GAE1 and GAE6 308

was repressed upon B cinerea treatment This indicates a role for GAE1 and GAE6 in 309

the immune response to B cinerea 310

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Page 16 of 54

Because the amount of pectin substrate for pathogen polygalacturonases is 313

reduced we hypothesized that immunity phenotypes of gae1 gae6 plants may result 314

from reduced generation of OGs by these enzymes In turn the reduced OGs could 315

lead to reduced immune responses To test this we used macerozyme a commercial 316

pectinase as a proxy for pathogen pectinases We isolated cell walls as a subcellular 317

fraction known as alcohol insoluble residue (AIR Gille et al 2009) This was treated 318

with macerozyme and tested for release of soluble likely low molecular weight pectin 319

fragments (Figure 7) Treatment with 025 macerozyme released significantly more 320

uronic acid from wild-type Col-0 AIR relative to gae1 gae6 AIR (Figure 7B) To test 321

activity of these pectin-containing solutions we infiltrated them into wild-type Col-0 322

plants and measured PAD3 expression PAD3 is required for synthesis of the 323

phytoalexin camalexin (Zhou et al 1999) and its expression is known to be induced by 324

OG treatment (Ferrari et al 2007) Fractions from macerozyme treated Col-0 walls 325

induced significantly more PAD3 expression than fractions derived from gae1 gae6 326

walls suggesting that macerozyme treatment did indeed release more active soluble 327

uronic acid possibly OGs from wild-type Col-0 walls than from gae1 gae6 walls (Figure 328

7C) 329

Next we studied the effect of macerozyme treatment on expression of PAD3 in 330

intact plants (Figure 8) This treatment did not lead to any visible leaf damage 331

(Supplemental Figure 8) In wild-type Col-0 PAD3 expression was induced three h after 332

treatment with 001 macerozyme (Figure 8A) By contrast no macerozyme-induced 333

induction of PAD3 expression was detected in gae1 gae6 (Figure 8A) However OG 334

treatment induced the expression of PAD3 in both genotypes indicating that gae1 gae6 335

plants do respond to OGs (Figure 8B) Macerozyme treatment also induced expression 336

of the SA marker genes PATHOGENESIS RELATED 1 (PR1) and SID2 in Col-0 but 337

not in gae1 gae6 (Figure 9 panel A and B) PR1 was not significantly induced by 338

purified OGs (Figure 9C) at the time and concentration tested but SID2 was induced by 339

OG treatment in both genotypes (Figure 9D) However the induction was stronger in 340

wild-type Col-0 In addition the JA markers JASMONATE-ZIM-DOMAIN PROTEIN 5 341

(JAZ5) and JAZ10 were induced by macerozyme and OG treatment in both Col-0 and 342

gae1 gae6 (Figure 10A and Supplemental Figure 11) Interestingly expression of JAZ5 343

312 Pectinase-Induced Immune Signaling Is Altered in gae1 gae6 Plants

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Page 18 of 54

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344

345

346

and JAZ10 was already strongly induced in gae1 gae6 after mock inoculation (Figure 10

and Supplemental Figure 11) We observed no hyperresponsive induction by mock

treatment of PLANT DEFENSIN 12 (PDF12) a necrotrophy and late JA signaling

347 marker gene (Supplemental Figure 12A-D) PDF12 was also not induced by

348 macerozyme or OG treatments (Supplemental Figure 12A-D) This suggests that early

349 JA signaling is induced by mock-treatment in gae1 gae6 Because it is known that JA

350 and SA signaling act antagonistically (Pieterse et al 2012) the strong up-regulation of

351 JA-induced immune signaling in gae1 gae6 might explain the lower OG-induced SID2

352 expression in this genotype

353 To test whether other immune responses are altered in gae1 gae6 plants we

354 analyzed Mitogen-activated protein kinase (MAPK) activity following macerozyme and

355 OG treatments OGs are known to activate the MAPKs MPK3 and MPK6 in Arabidopsis

Page 21 of 54

(Galletti et al 2011) Macerozyme treatment activated MPK3 and MPK6 in wild-type 356

Col-0 whereas MAPK activation in gae1 gae6 was slightly reduced in four out of five 357

experiments performed (Supplemental Figure 13) By contrast OG-induced MAPK 358

activity was similar or increased in gae1 gae6 in four out of five experiments 359

(Supplemental Figure 13) Additionally mock treatment induced MAPK activity in gae1 360

gae6 more strongly than it did in wild-type Col-0 in four out of five experiments 361

performed (Fig S13) Collectively these results illustrate that activation of several 362

immune signaling componentsmdashcamalexin synthesis SA and JA signaling and 363

possibly MAPK activationmdashby macerozyme is substantially altered in gae1 gae6 This 364

suggests that release of soluble pectin fragments possibly OGs by pectinolytic 365

enzymes might be reduced in gae1 gae6 plants while recognition of OGs is intact 366

367

Following B cinerea Infection Production of Soluble Pectin and Deposition of 368

Callose Are Reduced in gae1 gae6 Plants 369

Although pad3 plants were more susceptible to the B cinerea isolates Fresa 525 370

and Gallo 1 which also grew better on gae1 gae6 growth of the isolate Pepper was 371

similar on Col-0 and pad3 (Supplemental Figure 9A) However we did not detect any 372

significant changes in camalexin production after treatment with B cinerea isolate Gallo 373

1 and Fresa 525 in gae1 gae6 (Figure 8D) Neither did we detect changes in PAD3 374

expression after treatment with any B cinerea isolate in gae1 gae6 (Figure 8E) 375

Additionally SID2 and PR1 were similarly induced by all B cinerea isolates in both Col-376

0 and gae1 gae6 (Figure 9E and F) and sid2 plants which are deficient for pathogen-377

induced SA biosynthesis did not show any differences in relative B cinerea growth 378

(Supplemental Figure 9B) Expression of JAZ10 was induced more strongly by Gallo 1 379

treatment in gae1 gae6 than in wild-type Col-0 but not by Fresa 525 or Pepper (Fig 380

10C) while dde2 plants deficient for JA biosynthesis showed enhanced susceptibility to 381

all B cinerea isolates tested (Supplemental Figure 9B) Curiously exogenous 382

application of MeJA did not lead to altered relative fungal growth of B cinerea 383

(Supplemental Figure 9C) and cev1 isoxaben resistant 1 (ixr1) plants which have 384

constitutively high JA levels (Ellis and Turner 2001) did not show any changes in 385

susceptibility to the B cinerea isolates tested (Supplemental Figure 9D) Hence neither 386

Page 22 of 54

differences in B cinerea isolate susceptibility to camalexin nor altered SA or JA 387

signaling by themselves explained the enhanced susceptibility of gae1 gae6 to these B 388

cinerea isolates 389

Hydrogen peroxide accumulation in response to B cinerea isolates Fresa 525 390

Gallo 1 and Pepper was not obviously altered in gae1 gae6 plants (Supplemental Figure 391

14) However callose deposition upon treatment with B cinerea isolate Gallo 1 was 392

strongly reduced in gae1 gae6 (Figure 11) It was previously shown that OG treatment 393

induces callose deposition (Denoux et al 2008) suggesting that the reduction in 394

callose deposition might be due to reduced OG release in gae1 gae6 It is possible that 395

this reduction in callose deposition contributes to the reduced immunity of gae1 gae6 396

plants to B cinerea isolate Gallo 1 397

Because macerozyme could be used to release soluble likely low molecular 398

weight pectin from isolated cell walls we next investigated whether infection with B 399

cinerea was accompanied by accumulation of soluble pectin as well We prepared cell 400

walls (AIR fraction) from leaves infected with B cinerea incubated these cell walls with 401

water and measured uronic acid content of the extracts B cinerea treatment did in fact 402

lead to increased accumulation of soluble pectin in wild-type Col-0 plants when 403

compared to mock-treated samples (Figure 12) No such increased accumulation of 404

water-soluble pectin fractions was observed in gae1 gae6 (Figure 12) 405

406

Macerozyme-Induced Pattern-Triggered Immunity against B cinerea Is Abolished 407

in gae1 gae6 408

It was previously shown that OG treatment can induce patternndashtriggered 409

immunity (PTI) to B cinerea (Ferrari et al 2007) We tested macerozyme treatment to 410

determine whether it could also induce PTI to the B cinerea isolate Gallo 1 (Figure 13) 411

Indeed pretreatment with 001 macerozyme for 24 h led to reduced B cinerea growth 412

in wild-type Col-0 but not gae1 gae6 plants (Figure 13A) Interestingly mock and 413

macerozyme pretreated gae1 gae6 plants exhibited similar growth of B cinerea Gallo 1 414

as did macerozyme-treated Col-0 plants This result is likely due to activation of JA 415

signaling in gae1 gae6 during the pretreatment procedure (Figure 13C and D) OG-416

induced PTI was observed in both genotypes when using a high dose of OGs (Figure 417

Page 23 of 54

13B) whereas a lower dose of OGs only induced PTI in Col-0 (Supplemental Figure 418

15) This suggests that OG perception in gae1 gae6 at least above a certain threshold 419

is unaltered while release of immunity-activating pectin fragments possibly OGs is 420

421 reduced We conclude that immune responses induced by macerozyme treatment

422 which do not occur in gae1 gae6 plants are important for plant immunity to the B

423 cinerea isolate Gallo 1

Page 24 of 54

____________________________________________________________________________

Page 25 of 54

DISCUSSION 426

Here we showed that loss of GAE1 and GAE6 leads to a dramatic reduction in 427

pectin content in the cell wall (Figure 4) likely causing the brittleness of leaves that we 428

observed (Figure 3) Petioles and midribs of gae1 gae6 plants broke easily when bent 429

and inoculation of leaves with deionized water resulted in increased ion leakage 430

possibly due either to microscopic fractures of the cell wall induced by inoculation stress 431

or to a reduced capability of these walls to withstand increased turgor pressure The 432

increased brittleness was associated with altered cell wall composition In our analysis 433

the GalA content of gae1 gae6 leaf walls was reduced by more than 40 and the total 434

uronic acids by over 60 (Figure 4 and Supplemental Figure 5) This is a major change 435

in the overall cell wall composition considering that pectin makes up about 50 of the 436

plant cell walls found in Arabidopsis leaves (Zablackis et al 1995 Nobuta et al 2007 437

Harholt et al 2010) Interestingly quasimodo1 (qua1) plants which have a mutation in 438

a glycosyltransferase family 8 gene are reduced in GalA by 25 and show reduced 439

binding of HG specific antibodies (Bouton et al 2002) 440

Furthermore qua2 plants which have a mutation in a putative methyltransferase 441

have about 13 less total GalA and lack 50 of cell wall HG (Mouille et al 2007) 442

Likewise cgr2 cgr3 plants carrying mutations in another class of Golgi-localized 443

putative methyl-transferases have leaf cell walls that are reduced by about 40 in 444

uronic acid (Kim et al 2015) All three mutants are severely dwarfed (Bouton et al 445

2002 Mouille et al 2007 Kim et al 2015) Additionally qua1 and qua2 mutants show 446

a distorted overall shape likely due to reduced cell cohesion and are extremely fragile 447

(Bouton et al 2002 Mouille et al 2007) By contrast gae1 gae6 plants with a similar 448

strong reduction in GalA and total uronic acids have an overall normal shape and are 449

only slightly smaller than wild-type Col-0 (Supplemental Figure 4) In the wall analysis 450

performed here we did not detect changes in other wall polysaccharides or structures 451

that might compensate for the reduction of pectin in gae1 gae6 It is possible the severe 452

morphological alterations in qua2 and cgr2 cgr3 plants are due to reduction in pectin 453

methylesterification which is unaffected in gae1 gae6 It was speculated that quality 454

control mechanisms in the Golgi prevent secretion of inefficiently esterified pectin in 455

cgr2 cgr3 (Kim et al 2015) suggesting that the Golgi of these plants might over-456

Page 26 of 54

accumulate pectin and that this may contribute to the severe dwarf phenotype of these 457

plants The gae1 gae6 plants described here provide an opportunity to study the effects 458

of reduction of pectin in plants that do not show severe morphological phenotypes 459

We found that gae1 gae6 plants are also compromised in resistance to B 460

cinerea and Pma ES4326 indicating that loss of pectin reduces immunity to these 461

pathogens (Figure 2 and 6) We found that gae1 gae6 plants were slightly more 462

susceptible to Pma ES4326 but showed a strong increase in susceptibility to specific B 463

cinerea isolates This was not unexpected because B cinerea is known to interact with 464

pectin as part of its infection strategy It secretes various pectin-degrading enzymes 465

early during infection and requires some of these enzymes including PG1 PG2 and 466

PME1 for full virulence (ten Have et al 1998 Valette-Collet et al 2003 Kars et al 467

2005b Espino et al 2010) Additionally Arabidopsis plants expressing an antisense 468

construct of the polygalacturonase inhibitor PGIP1 are significantly more susceptible to 469

B cinerea (Ferrari et al 2006) On the other hand Pma ES4326 is not known to470

require cell wall degrading enzymes for full virulence even though it encodes two 471

pectate lyase genes in its genome (PMA4326_07094 and PMA4326_04621 472

wwwbacteriaensemblorg) B cinerea isolates are also known to be genetically quite 473

diverse in their polygalacturonase (PG) loci and genetic variability in PG2 is associated 474

with varying growth on pectin containing media (Rowe and Kliebenstein 2007) 475

Furthermore the contribution of PG1 and PG2 to B cinerea virulence seems to depend 476

on the specific B cinerea isolate and the host species (Kars et al 2005a Zhang and 477

Van Kan 2013) B cinerea isolates are also known to be differently sensitive to the anti-478

microbial secondary metabolite camalexin (Kliebenstein et al 2005) In line with these 479

observations the susceptibility of gae1 gae6 plants differed depending on the specific 480

B cinerea isolate Whereas Fresa 525 Gallo 1 UK Razz and KT grew better in gae1481

gae6 we detected no difference in Acacia Apple 517 DN Grape Pepper or Rasp 482

growth (Figure 6) 483

B cinerea was shown to metabolize pectin and to possibly utilize GalA as a484

carbon source during in planta growth (Zhang et al 2011 Zhang and Van Kan 2013) 485

Our data show that gae1 gae6 plants which are reduced in GalA content did allow 486

enhanced growth of specific B cinerea isolates but no reduction in growth of any B 487

Page 27 of 54

cinerea isolates tested was found This suggests that utilization of pectin as a carbon 488

source was not a contributing factor to alterations in B cinerea susceptibility It is 489

possible that changes in the cell wall composition affect the structural integrity of the 490

wall and affect hyphal penetration of specific B cinerea isolates However OGs (small 491

HG fragments) are likely released during the infection process (Ferrari et al 2013) 492

OGs function as DAMPs and initiate immune responses in Arabidopsis (Ferrari et al 493

2013) Treatment of Arabidopsis with OGs having a degree of polymerization (DP) of 494

10-15 sugar subunits was previously shown to enhance resistance to B cinerea (Ferrari495

et al 2007) We showed that pretreatment with OGs or macerozyme induced enhanced 496

immunity to B cinerea isolate Gallo 1 in wild-type Col-0 but only OGs and not 497

macerozyme induced immunity in gae1 gae6 (Figure 13) Our data suggest gae1 gae6 498

plants which have reduced pectin content may release less OGs and that this 499

reduction leads to the increased susceptibility to B cinerea 500

Consistently we found that macerozyme treatment released less soluble pectin 501

from gae1 gae6 cell walls than from wild-type walls Fractions containing soluble pectin 502

released from gae1 gae6 were also less active in inducing PAD3 gene expression than 503

the fractions released from wild-type walls (Figure 7) We also showed that less soluble 504

pectin accumulated in gae1 gae6 than in wild-type plants 48 h after treatment with B 505

cinerea (Figure 12) 506

We observed that loss of pectin leads to alterations in A thaliana immune 507

signaling Macerozyme treatment induced expression of PAD3 and the SA marker 508

genes PR1 and SID2 in wild-type Col-0 but not in gae1 gae6 whereas OG treatment 509

led to increased PAD3 and SID2 expression in both wild-type and gae1 gae6 (Figure 8 510

and 9) This suggests that although release of active soluble pectin in these plants 511

might be altered OG recognition is intact In fact cell walls isolated from qua1 another 512

mutant known to be reduced in HG released less OGs (DP1-6) than did wild-type cell 513

walls upon endopolygalacturonase treatment (Bouton et al 2002) This was also true 514

for gae1 gae6 cell walls as macerozyme treatment of isolated gae1 gae6 cell walls 515

released less soluble pectin than treatment of Col-0 cell walls Because the fraction 516

from gae1 gae6 plants was also less active in inducing PAD3 expression (Figure 7) it 517

seems possible that biologically active OGs were the active component inducing PAD3 518

Page 28 of 54

expression Interestingly expression of the JA marker genes JAZ5 and JAZ10 was 519

higher in gae1 gae6 than in wild-type mock-inoculated plants but was comparable to 520

wild-type in untreated plants suggesting that JA signaling in gae1 gae6 is 521

hyperresponsive (Figure 10 and Supplemental Figure 11) This induction of JA signaling 522

might be due to wounding stress incurred during the inoculation process JA and SA 523

signaling generally act antagonistically (Pieterse et al 2012) Increased JA signaling 524

activity might hence explain why mock- and macerozyme-treated gae1 gae6 plants 525

exhibited reduced expression of the SA marker PR1 and why OG treatment induced 526

expression of SID2 another SA marker to a lower extent in gae1 gae6 than in wild-type 527

plants (Figure 9) 528

No single immune signaling component was found to be the sole contributor to 529

the compromised immunity of gae1 gae6 to specific B cinerea isolates such as Gallo 1 530

and Fresa 525 These isolates grew better on pad3 while growth of Pepper another B 531

cinerea isolate that did not show altered virulence on gae1 gae6 was unaltered 532

(Supplemental Figure 9) This suggested that differences in camalexin sensitivity of 533

these B cinerea isolates might account for the differences in gae1 gae6 susceptibility 534

However camalexin accumulation and PAD3 expression was the same for wild-type 535

Col-0 and gae1 gae6 for the B cinerea isolates Fresa 525 and Gallo 1 (Figure 8) which 536

showed enhanced growth on gae1 gae6 plants It should be noted that B cinerea 537

virulence is often estimated by comparing lesion sizes Lesion size and measurements 538

of relative fungal growth by qPCR do not always show the same results For example 539

although Denby et al (2004) showed that Pepper causes larger lesions on pad3 than on 540

wild-type this study showed that relative fungal growth of Pepper on pad3 was 541

unaltered 542

We observed that callose deposition was strongly reduced in gae1 gae6 plants 543

72 h after treatment with B cinerea isolate Gallo 1 (Figure 11) possibly due to reduced 544

release of active soluble pectin Interestingly OG-induced PTI to an unspecified isolate 545

of B cinerea was intact in the pmr4 mutant which does not produce callose (Galletti et 546

al 2008) Due to the high SA levels in pmr4 plants (Nishimura et al 2003) and the fact 547

that different B cinerea isolates vary both in induction of and reaction to defense 548

responses (Kliebenstein et al 2005 Rowe et al 2010) we could not determine 549

Page 29 of 54

whether reduced callose deposition causes the increased susceptibility of gae1 gae6 to 550

B cinerea isolate Gallo 1551

We did not observe any changes in B cinerea growth either on cev1 ixr1 plants 552

which are known to have constitutively high JA and OPDA levels (Ellis et al 2002b) or 553

on plants exogenously treated with MeJA (Supplemental Figure 9) Because plants 554

were kept uncovered for 3 h after MeJA treatment it is unclear how much MeJA was 555

bioavailable in the plant However JAZ10 expression was induced 3 h after spraying 556

with MeJA (Supplemental Figure 10) Because pathogen growth generally depends on 557

the combination of a multitude of immune signaling events we conclude that multiple 558

changes to the immune signaling in gae1 gae6 together account for the altered B 559

cinerea growth phenotypes detected 560

We showed that expression of GAE1 and GAE6 is repressed by the bacterial 561

hemibiotroph Pma ES4326 This repression did not occur in an eds1 or pad4 562

background or in pbs3 at the 24-h time point (Figure 1 and Supplemental Figure 1) 563

During the Arabidopsis - Pma ES4326 interaction the expression of many defense 564

related genes is altered in a PAD4EDS1 dependent fashion (Wang et al 2008) Some 565

of these expression changes require SA CBP60g and SARD1 are thought to act 566

downstream of PAD4 and EDS1 but upstream of SA-dependent responses (Wang et 567

al 2011) PBS3 is also thought to act downstream of PAD4 and EDS1 and upstream of 568

SA but the set of genes whose expression it affects is only partially overlapping with 569

those affected by CBP60gSARD1 (Wang et al 2008 Wang et al 2011) Thus GAE1 570

and GAE6 repression is regulated by the part of the immune signaling network that 571

requires PAD4 EDS1 and PBS3 but is independent of CBP60gSARD1 and SA 572

Because EDS1 PAD4 and PBS3 are important components of the plant immune 573

signaling network (Wiermer et al 2005 Nobuta et al 2007) one might conclude that 574

repression of GAE1 and GAE6 may be part of the plant immune response to Pma 575

ES4326 B cinerea treatment also reduced GAE1 and GAE6 expression but this was 576

mostly independent of PAD4 (Figure 6) while both Pma ES4326 and B cinerea repress 577

GAE1 and GAE6 expression the host signaling mechanisms responsible likely differ 578

In summary our data show that Arabidopsis GAE1 and GAE6 are required for 579

pectin biosynthesis leaf flexibility and immunity to Pma ES4326 and to specific B 580

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cinerea isolates We used macerozyme a commercial pectinase to mimic the effect of 581

B cinerea pectinases Macerozyme treatment released less pectin from gae1 gae6 cell582

walls and induced less immune signaling in gae1 gae6 plants Less water soluble pectin 583

also accumulated in B cinerea infected gae1 gae6 plants and macerozyme-induced 584

immunity to B cinerea isolate Gallo 1 was reduced in these plants We conclude that 585

the reduced pectin content in these mutants may lead to reduced release of soluble 586

pectin including active OGs during pathogen attack and that this compromises 587

immunity 588

589

590

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METHODS 591

Plant Materials and Growth Conditions 592

Wild-type Columbia (Col-0) and all mutant plants (in Col-0 background) were 593

grown on sterilized BM2 germinating mix (Berger Inc Quebec Canada) in a controlled 594

environment chamber (Conviron Pembina ND USA) with a 12-h photoperiod under 595

100 microM m-2 s-1 fluorescent illumination at 22degC and 75 relative humidity Germplasm 596

used is described in the Accession Numbers section at the end of Methods 597

598

Data Analysis and Replicates 599

For biological replicates samples were obtained from separate plant tissues and 600

in many cases from plants grown in different flats In some experiments multiple 601

technical replicates were measured per biological replicate For statistical analysis all 602

technical replicates were averaged and the averages were considered one biological 603

replicate Independent experiments were performed at different times and often 604

consisted of multiple biological replicates per experiment 605

Data analysis was performed using mixed linear models in the R programming 606

environment (Bates et al 2015) unless otherwise indicated Generally genotypes 607

treatments and time points were used as fixed effects and replicate-specific effects like 608

different pots flats etc were considered random effects We reported p-values 609

obtained from the mixed linear model when less than 10 comparisons were performed 610

and q-values for more than 10 comparisons to correct for multiple comparison testing 611

Q-values were calculated using the Benjamini-Hochberg procedure (Benjamini and612

Hochberg 1995) unless specified otherwise 613

614

Pathogen Strains Growth Conditions and Pathogen Growth Assays 615

Growth of Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) and 616

bacterial growth assays were performed as previously described (Bethke et al 2014) 617

For qRT-PCR assays four week-old plants were inoculated with bacteria (OD600 = 618

0002) or mock (5 mM MgSO4) At least four leaves from two plants were collected at 619

the indicated time points per biological replicate 620

Page 32 of 54

Prior to each experiment Botrytis cinerea isolates were grown on 1x PDA 621

medium (Difco) for 10 d at room temperature All isolates used have been described 622

previously (Rowe and Kliebenstein 2007) The spores were washed from the surface of 623

the plate using an inoculation medium (1x Gamborgrsquos B-5 basal salt mixture (Sigma-624

Aldrich G5768) 2 (wv) glucose 10 mM phosphate buffer pH 64) and fungal hyphae 625

were removed from the suspension by filtering through four layers of cheesecloth The 626

concentration of spores was determined using a hemocytometer and adjusted to 25 x 627

105 spores mL-1 Four week-old Arabidopsis plants were inoculated by placing one 10-628

microL droplet of the B cinerea spore solution or inoculation medium (mock) on the adaxial 629

leaf surface of fully expanded leaves Inoculated plants were kept at 100 relative 630

humidity Infection sites were collected at various time points using a cork borer For 631

qRT-PCR assays at least four leaf discs from one plant were combined for each 632

biological sample For B cinerea growth assays at least 12 leaf discs from three to six 633

plants were pooled per biological sample Relative fungal growth was determined by 634

measuring the abundance of a fungal gene relative to a plant gene as described below 635

In brief DNA from infected tissue was extracted and equal amounts of total DNA were 636

used to perform qPCR reactions using the B cinerea cutinase A and the Arabidopsis 637

SK11 gene respectively Primers were described in (Gachon and Saindrenan 2004) 638

and are listed in Supplemental Table 1 Two technical replicates were combined for 639

each biological replicate B cinerea is a necrotrophic fungal pathogen and may destroy 640

plant DNA as disease progresses However decreases in the amount of plant DNA 641

over the course of our experiments were only observed at late time points with very 642

susceptible plants so the qPCR assay is generally a reasonable estimate of fungal 643

biomass (Supplemental Figure 16) 644

645

Treatment with Macerozyme or OGs 646

For PTI assays four week-old plants either were inoculated with 001 (wv) 647

macerozyme R-10 (Yakult) a Rhizopus polygalacturonase (EC 32115) that possesses 648

high pectinase and hemicellulase activity or were mock inoculated using heat-649

inactivated macerozyme Other similar plants were either inoculated with 250 or 500 microg 650

mL-1 OGs or mock inoculated with water One d later plants were infected with B 651

Page 33 of 54

cinerea and samples were collected immediately (0 dpi) and two or three d later To 652

investigate the role of MeJA on B cinerea growth plants were sprayed with 1mM MeJA 653

in water with 001 (vv) Tween 20 Covers were removed so that leaves could dry off 654

before plants were inoculated with B cinerea three h later Samples were harvested at 655

the indicated time points For expression analysis and MAPK activation assays plants 656

were either inoculated with 100 microg mL-1 OGs or mock inoculated with water OGs used 657

for experiments described in Figures 8 to 10 and Supplemental Figure 12 were a kind 658

gift from Simone Ferrari OGs used in experiments described in Figure 13 and 659

Supplemental Figures 11 13 and 15 were prepared as previously described (Kohorn et 660

al 2014) 661

662

Expression Analysis 663

Leaf tissue from inoculated plants was harvested at the indicated time points 664

flash frozen pulverized and RNA extracted using Trizol (Invitrogen) Quantitative RT-665

PCR was performed using the SuperScript III Platinum SYBR Green One-Step 666

quantitative RT-PCR kit (Invitrogen) and a Lightcycler 480 Real-Time PCR system 667

(Roche) as previously described (Truman and Glazebrook 2012) In brief equal 668

amounts of total RNA and a gene specific primer were used for each reaction The 669

crossing point (Cp) was calculated using the 2nd derivative max method provided with 670

the Lightcycler software for each amplification curve Each reaction was run with two 671

technical replicates and the Cp values for these replicates were averaged ACTIN2 was 672

used as a stably expressed reference gene At least three biological replicates each 673

were performed and analyzed using a mixed-linear model RT-PCR was performed to 674

verify T-DNA insertion lines using the One-Step RT-PCR kit (Qiagen) according to the 675

manufacturerrsquos instructions The primers used can be found in Supplemental Table 1 676

677

Electrolyte Leakage Assay 678

Fully expanded leaves of four week-old plants were inoculated with water (Milli-Q 679

grade) using a needleless syringe Leaf discs were collected using a cork borer and 680

three leaf discs from one leaf were placed with the adaxial surface down onto 2 mL 681

water in a 12-well cell culture plate Conductivity was measured using a Horiba B-173 682

Page 34 of 54

conductivity meter after 30 min and then the water was replaced Conductivity was 683

measured again 30 min later (1 h total) as indicated in Figure 3C Six biological 684

replicates each were performed in three independent experiments and means and SEs 685

were calculated Two-sided t-tests were performed for each time point 686

687

DAB and Aniline Blue Staining 688

Leaves from four week-old plants were either mock inoculated with B cinerea 689

inoculation medium or inoculated with 10 microL droplets of a B cinerea spore solution (25 690

x 105 spores mL-1) of the indicated isolates Samples were collected 72 h later For 33prime-691

diaminobenzidine (DAB) staining two to five inoculated leaves were vacuum infiltrated 692

with 33prime-Diaminobenzidine (1 mg mL-1 in water pH 38) solution and stained overnight 693

in the dark Leaves were destained using 100 ethanol at 65degC and then transferred to 694

50 (vv) glycerol and photographed 695

Callose was visualized by staining leaves with aniline blue as previously 696

described (Adam and Somerville 1996) Briefly leaves were vacuum infiltrated with 697

alcoholic lactophenol (one volume of phenolglycerollactic acidwater (1111) mixed 698

with two volumes of ethanol) and destained at 65degC Next leaves were moved through 699

an alcohol gradient (50 20 and 10 (vv) ethanol) into water The destained leaves 700

were stained for 30 min in 150 mM K2HPO4 (pH 95) with 001 (wv) aniline blue 701

Samples were mounted in water and examined using a Nikon Eclipse Ni-U microscope 702

at 10x magnification using a DAPI filter (excitation at 325-375 nm emission at 435-485 703

nm) For callose quantification six 200 microm x 200 microm squares were counted for each 704

sample type Callose deposits per mm2 were calculated and data analyzed using a t-705

test Note that there is an inherent uncertainty associated with such counts because 706

callose deposits may not all be in focus at the same time 707

708

MAPK Activity Assay 709

Fully expanded leaves of four week-old plants were inoculated with 001 710

macerozyme 100 microg mL-1 OGs 100 nM flg22 or water (mock) flg22 a peptide from 711

bacterial flagella that induces immune responses was used as a positive control for 712

Page 35 of 54

MAPK activation (Asai et al 2002) and was purchased from EZBiolab Inc (Westfield 713

IN USA) Samples were harvested 10 min later and immediately frozen in liquid 714

nitrogen Protein was extracted using an extraction buffer containing 25 mM Tris pH 78 715

75 mM NaCl 1 mM DTT 1 mM NaF 05mM Na3VO4 01 (vv) Tween 20 one 716

cOmplete Mini protease inhibitor cocktail tablet (Roche) and one PhosSTOP 717

phosphatase inhibitor cocktail tablet (Roche) per 10 mL buffer 15 microg of total protein 718

were electrophoresed on a 10 SDS-polyacrylamide gel and then blotted onto a PVDF 719

membrane (Bio-Rad 162-0177) Activated MAPKs were detected using p4442 MAPK 720

(Erk12) antibody (9102S Cell Signal 12500 in TBST (20 mM Tris pH 75 150 mM 721

NaCl 01 (vv) Tween 20)) Anti-Rabbit IgG-HRP (115000 in TBST A6154 Sigma) 722

and ECL Plus substrate (Pierce 32132) MPK3 and MPK6 were detected with an anti-723

MPK3 antibody (M8318 Sigma 12000 in TBST and 3 (wv) milk) or an anti-MPK6 724

antibody (A7104 Sigma 16000 in TBST and 3 (wv) milk) respectively The 725

secondary antibody and detection were the same as for the p4442 antibody Five 726

independent experiments were performed 727

728

Preparation of Alcohol Insoluble Residue (AIR) Pectin Extraction and Uronic Acid 729

Measurements 730

Fully expanded leaves from four week-old plants that were incubated for 48 h in 731

the dark to reduce starch were harvested flash frozen and pulverized AIR was 732

extracted by washing ground material twice in 70 (vv) ethanol three times in a 733

mixture of chloroform and methanol (11 vv) and once in acetone (Gille et al 2009) 734

and was then air dried Cell wall pectin was extracted from AIR with cell wall extraction 735

buffer (50 mM Trizma 50 mM CDTA pH 72) at 95degC for 15 min Samples were 736

homogenized using a paint shaker (Harbil 5G-HD) and glass beads (3 mm diameter) 737

For dot blot experiments 500 microL of cell wall extraction buffer were used per 10 mg of 738

AIR Debris was precipitated by centrifugation for 10 min at 10000 g 739

Total uronic acid content of pectin extracted as described above using 500 microL of 740

cell wall extraction buffer per 1 mg of AIR was measured as described previously 741

(Filisetti-Cozzi and Carpita 1991 van den Hoogen et al 1998) First 36 microL of pectin 742

extract were mixed with 4 microL of 4 M sulfamic acid Then 200 microL of sulfuric acid 743

Page 36 of 54

containing 120 mM sodium tetraborate was added and samples were incubated at 80degC 744

for 1 h Following cooling on ice the optical density of samples was measured at 490 745

nm Next 40 microL of m-hydroxydiphenyl reagent (100 microL of m-hydroxydiphenyl in dimethyl 746

sulfoxide at 100 mgmL mixed with 49 mL 80 (vv) sulfuric acid just before use) was 747

added and the samples were mixed The optical density at 490 nm was measured 748

again The optical density before m-hydroxydiphenyl reagent addition was subtracted 749

from the optical density measured after addition of the dye The concentration of uronic 750

acid was calculated using known amounts of GalA as a standard 751

752

Release of Pectin from Arabidopsis Cell Walls Using Macerozyme 753

AIR preparations from leaves of four week-old plants were treated with 025 754

(wv) macerozyme R-10 (Yakult) or water (mock) at a concentration of 100 microL per mg 755

AIR Samples were incubated for 3 h at room temperature on a vortex shaker and 756

centrifuged for 10 min at 12000 g Total uronic acid concentration of 5-fold diluted 757

supernatants was determined for two technical replicates each The concentration of 758

uronic acid was calculated using known amounts of GalA as a standard For 759

subsequent qRT-PCR experiments supernatants from multiple extractions were 760

combined boiled for 20 min to inactivate macerozyme diluted 10-fold with water and 761

inoculated into four week-old Col-0 plants Heat-inactivated 10-fold diluted macerozyme 762

solutions were used as mock treatment Samples were collected 3 h later and PAD3 763

expression was determined 764

765

Release of Pectin from Arabidopsis Leaves Infected with B cinerea 766

Fully expanded leaves of four week-old plants were either inoculated with up to 767

five 10 microL droplets of B cinerea spore solution (25 x 105 spores mL-1) or mock 768

inoculated with B cinerea inoculation medium For each biological replicate leaves with 769

a combined count of at least 20 infection sites were collected 48 h later AIR was 770

extracted as described above but plants were not dark-treated or freeze-dried and the 771

first ethanol step was performed using 85 (vv) ethanol To extract soluble pectin 772

fragments AIR samples were mixed with water at a concentration of 20 microL per infection 773

site incubated for 7 h at room temperature on a vortex shaker and centrifuged for 10 774

Page 37 of 54

min at 12000 g Total uronic acid concentration of supernatants was determined for two 775

technical replicates each The concentration of uronic acid was calculated using known 776

amounts of GalA as a standard 777

778

Measurement of Uronic Acids Neutral Monosaccharides and Cellulose Content in 779

Cell Walls 780

Neutral sugars and uronic acids from non-cellulosic polysaccharides in cell walls 781

(AIR) were released by trifluoroacetic acid (TFA) hydrolysis Approximately 1 mg of AIR 782

was hydrolyzed with 2 M TFA at 121degC for 90 min The TFA-soluble material was split 783

into two equal parts one for uronic acid and one for neutral sugar determination The 784

uronic acid content was determined by analyzing the hydrolyzate using a CarboPac 785

PA200 anion-exchange column with an ICS-3000 Dionex chromatography system 786

(Dionex Sunnyvale CA USA) The elution profile consisted of a linear gradient of 50 to 787

200 mM sodium acetate in 01 M NaOH in 10 min at 04 mL per min Alditol acetate 788

derivatives of neutral sugars were produced as described (York et al 1985) In brief 789

monosaccharides in the hydrolyzate were reduced with NaBH4 and peracetylated with 790

acetic anhydride and pyridine at 121degC for 20 min The generated alditol acetates were 791

analyzed by GC-MS Crystalline cellulose was measured according to Updegraff (1969) 792

After hydrolysis of noncellulosic polysaccharides from AIR with the Updegraff reagent 793

(acetic acid nitric acids water 812 (vv) ) the remaining pellet was hydrolyzed in 794

72 sulfuric acid The resulting glucose quantity was determined by the anthrone 795

method (Scott and Melvin 1953) 796

797

Dot Blot Experiments 798

Pectin solutions extracted using 50 microL of cell wall extraction buffer per mg AIR 799

were serially diluted and nitrocellulose membranes were spotted with 1 microL of the diluted 800

pectin solutions Membranes were dried overnight blocked with 5 milk (wv) in 1x 801

PBS (8 g L-1 NaCl 02 g L-1 KCl 144 g L-1 Na2HPO4 024 g L-1 KH2PO4 pH 74) and 802

probed with LM5 (Jones et al 1997) LM6 (Willats et al 1998) LM8 (Willats et al 803

2004) LM19 and LM20 (Verhertbruggen et al 2009a) or CCRC-M7 (Steffan et al 804

Page 38 of 54

1995) antibodies LM series antibodies were diluted 1250 and CCRC-M7 was diluted 805

1500 in 5 milk powder (Nestle Carnation) in 1 x PBS For LM series antibodies a 806

goat anti-Rat HRP conjugated antibody (Bethyl A110-105P 15000 diluted) and for 807

CCRC-M7 an anti-Mouse IgG HRP conjugate (Promega W402B 12500 diluted) was 808

used as secondary antibody in 5 milk powder in 1x PBS LM series antibodies were 809

obtained from plant probes (wwwplantprobesnet) and CCRC-M7 from CarboSource 810

(wwwcarbosourcenet) Membranes were washed after incubation with the primary and 811

secondary antibodies with 1x PBS Dot blots were developed using the ECL system 812

(GE Healthcare Amersham ECL prime) For quantification of signals dot blot results on 813

x-ray films were photographed using a CCD camera and intensities were measured814

using Image Jrsquos Integrated Density function Locally adjusted background was 815

subtracted As the dynamic range of the signal on an x-ray film was narrow non-816

linearity in the measured values was corrected for each genotype in each biological 817

replicate using the values from different dilutions in two technical replicates We noticed 818

that the residuals were not normally distributed and performed Box-Cox power 819

transformation (Box and Cox 1964) to obtain normally distributed values for the 820

subsequent statistical analysis of the data For LM19 Box-Cox selected power 821

transformation was done to the power of 038th and for LM20 to the power 075th After 822

correction of the transformed measured values with the effect of the biological 823

replicates the values were fit to a linear model with the Arabidopsis genotype as the 824

fixed effect This linear model was used both to generate the plots in Figures 5B and D 825

and to test significant differences 826

827

Camalexin Measurements 828

Four week-old plants were infected with B cinerea and samples (three leaf disks 829

from one plant each) were harvested at the indicated time points flash frozen and 830

pulverized Each sample was extracted with 300 microL of 90 methanol (vv) 50 microL of the 831

extract was run on an Agilent Lichrocart 250-4 RP18e 5-microm column using an Agilent 832

1100 series HPLC Camalexin was detected using a DiodeArray (DAD) at 330 nm and 833

with a fluorescence detector at emission 318 nmexcitation 385 nm (Agilent) Separation 834

was achieved using the following program with aqueous acetonitrile 5 min gradient 835

Page 39 of 54

from 63 to 69 acetonitrile 30 s gradient from 69 to 99 acetonitrile 2 min at 99 836

acetonitrile and a post-run equilibration of 35 min at 63 acetonitrile (Denby et al 837

2004 Kliebenstein et al 2005) Purified camalexin was used to produce a standard 838

curve to identify and quantitate camalexin 839

840

Accession Numbers 841

Microarray data used can be accessed at NCBI Gene Expression Omnnibus 842

(httpwwwncbinlmnihgovgeo) GSE18978 Sequence information for all Arabidopsis 843

genes described in this article can be found in TAIR (wwwarabidopsisorg) using the 844

following accession numbers PAD3 (CYP71B15 At3g26830) SID2 (ICS1 At1g74710) 845

GAE1 (At4g30440) GAE2 (At1g02000) GAE3 (At4g00110) GAE4 (At2g45310) GAE5 846

(At4g12250) GAE6 (At3g23820) PR1 (At2g14610) JAZ5 (At1g17380) JAZ10 847

(At5g13220) ACTIN2 (At3g18780) PDF12a (At5g44420) PDF12b (At2g26020) 848

AtSK11 (At5g26751) The sequence of the B cinerea cutinase Bc-CutA (Z69264) can 849

be found in the DNA Data Bank of Japan (httpgetentryddbjnigacjp) Germplasm 850

used included dde2-2 (At5g42650 (von Malek et al 2002)) pad3-1 (At3g26830 (Zhou 851

et al 1999)) pad4-1 (At3g52430 (Jirage et al 1999)) sid2-2 (At1g74710 (Wildermuth 852

et al 2001)) pbs3-2 (At5g13320 SALK_018225 (Nobuta et al 2007)) cbp60a-1 853

(At5g62570 SALK_124410 (Truman et al 2013)) cbp60g-1 sard1-2 (At5g26920 854

SALK_023199 At1g73805 SALK_052422 (Wang et al 2011) cbp60a-1 cbp60g-1 855

sard1-2 (Truman et al 2013) gae1-1 (At4g30440 SALK_085554) gae6-1 (At3g23280 856

SALK_104454) gae6-2 (At3g23280 SALK_017191) ixr1-1 cev1 (At5g05170 (Ellis 857

and Turner 2001 Scheible et al 2001)) eds1 plants were derived by introgression of 858

the Ler eds1-2 allele into Col-0 that contains two copies of EDS1 (At3g48090 859

At3g48080 (Bartsch et al 2006) mpk6-2 (At2g43790 SALK_073907 (Liu and Zhang 860

2004)) mpk3-DG (At3g45640 a fast neutron deletion mutant (Miles et al 2005)) and 861

npr1-1 (At1g64280 (Cao et al 1997)) T-DNA insertion lines were part of the SALK 862

collection (Alonso et al 2003) and were obtained from the Arabidopsis Biological 863

Resource Center 864

865

Supplemental Data 866

Page 40 of 54

Supplemental Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 867

Expression Does Not Require CBP60s or SID2 868

Supplemental Figure 2 Expression of GAE Family Members uponTreatment with Pma 869

ES4326 870

Supplemental Figure 3 Characterization of T-DNA Insertions in GAE1 and GAE6 871

Supplemental Figure 4 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves and 872

Are Slightly Smaller than Wild-Type Col-0 Plants 873

Supplemental Figure 5 Cell Walls of gae1 gae6 Plants Contain Less Uronic Acid than 874

Wild-Type Col-0 Cell Walls 875

Supplemental Figure 6 Antibodies Raised Against the RGI or XG Components of 876

Pectin Show No Difference in Binding to gae Mutant and Col-0 Cell Walls 877

Supplemental Figure 7 The Effect of B cinerea Treatment on the Expression of GAE 878

Family Genes 879

Supplemental Figure 8 Treatment with 001 Macerozyme Does Not Cause Any 880

Visible Damage to Col-0 Plants 881

Supplemental Figure 9 B cinerea Growth on Plants with Defects in Immune 882

Signaling 883

Supplemental Figure 10 MeJA-Induced Expression of the JA marker gene JAZ10 884

Supplemental Figure 11 The Expression of the JA Marker Gene JAZ5 Is Highly 885

Responsive in gae1 gae6 Plants 886

Supplemental Figure 12 The Expression of PDF12 Is Unaltered in gae1 gae6 Plants 887

Supplemental Figure 13 Macerozyme Treatment Activates MPK3 and MPK6 888

Supplemental Figure 14 B cinerea-Induced ROS Production in Wild-Type Col-0 and 889

gae1 gae6 Plants 890

Supplemental Figure 15 OG-Induced PTI Using 250 microg mL-1 OGs 891

Supplemental Figure 16 Abundance of Plant DNA in B cinerea Treated Plants 892

Supplemental Table 1 Primers used in this study 893

894

ACKNOWLEDGEMENTS 895

We thank William Truman for helpful discussions Simone Ferrari (Sapienza 896

Universita di Roma) for the generous gift of OGs Bruna Bucciarelli (USDA-ARS) for 897

Page 41 of 54

help with fluorescence microscopy the Arabidopsis Biological Resource Center for T-898

DNA insertion lines Paul Knoxrsquos lab (University of Leeds) for LM5 LM6 LM8 LM19 899

and LM20 antibodies and the Complex Carbohydrate Resource Center (University of 900

Georgia) for the CCRC-M7 antibody Most of this work was funded by the Division of 901

Chemical Sciences Geosciences and Biosciences Office of Basic Energy Sciences of 902

the US Department of Energy through Grant DE-FG02-05ER15670 to JG This work 903

was also supported by NSF awards IOS 1339125 and IOS 1021861 to DJK NSF 904

award IOS 1121425 to F K and the USDA National Institute of Food and Agriculture 905

Hatch project number CA-D-PLS-7033-H to DJK 906

907

AUTHOR CONTRIBUTIONS 908

GB and JG designed the research GB GX AT BL NES and NH 909

performed research DJK provided tools GB GX MP AD BL RAH and FK 910

analyzed data GB and JG wrote the paper with input from all authors 911

912

FIGURE LEGENDS 913

Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 Expression 914

Requires PAD4 EDS1 and PBS3 915

(A) Expression of GAE1 in leaves of wild-type Col-0 and mutant plants Expression was916

measured 24 or 32 h after either inoculation with Pma ES4326 (OD600 = 0002) or mock 917

inoculation with 5 mM MgSO4 and was then normalized to the level of ACTIN2 Data 918

from three biological replicates were combined using a mixed linear model Bars 919

represent mean log2-ratios to ACTIN2 plusmn SE Letters indicate significantly different 920

groups with q lt 005 where 005 represents the maximum false discovery rate at which 921

the test may be called significant 922

(B) Expression of GAE6 measured as described in (A)923

924

Figure 2 Susceptibility of gae6 and gae1 gae6 Plants to Pma ES4326 Is Increased 925

Relative to Wild-Type Col-0 926

Leaves of plants of the indicated genotypes were inoculated with Pma ES4326 (OD600 = 927

00002) as described in Methods Bacterial titers in leaves were determined either 928

Page 42 of 54

immediately (0 dpi) or after three days (3 dpi) Bars represent the mean plusmn SE of four 929

independent experiments each with 4 or 12 biological replicates at 0 and 3 dpi 930

respectively Data were combined using a mixed linear model Asterisks indicate 931

significant differences from Col-0 wild-type (q lt 005) Susceptible pad4 plants were 932

included as a positive control 933

934

Figure 3 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves 935

(A) Depiction of the assay for brittleness of gae mutant leaves Fully expanded leaves of936

four to five week-old plants were bent by 180ordm so that the adaxial surface of the leaf was 937

parallel with the soil Broken leaf petioles or midribs were considered brittle 938

(B) Quantification of broken midribs or petioles For each genotype 66 to 82 leaves 939

were analyzed for brittleness as described in (A) Bars represent the ratio of broken to 940

total number of leaves tested Results were analyzed using Fisherrsquos exact test Letters 941

indicate significantly different groups at p lt 005 942

(C) Conductivity measurements Leaves of four week-old plants were inoculated with943

deionized water and then leaf discs were cut out and placed on deionized water 944

Conductivity was measured at the indicated time points After 30 min the water was 945

replaced with fresh water (30 min f w) to account for wounding-related ion leakage from 946

the edges of the leaf discs Three independent experiments with six biological replicates 947

each were performed and data were analyzed using a t-test for each time point P-948

values were corrected using the Bonferroni method Data show means plusmn SE asterisks 949

indicate significant differences from wild-type Col-0 at q lt 001 950

951

Figure 4 Cell Walls of gae6 and gae1 gae6 Plants Are Reduced in GalA 952

(A) to (D) Levels of GalA (A) GlcA (B) cellulose (C) and neutral sugars (D) in alcohol953

insoluble residue (AIR) extracted from leaves of four week-old plants of the indicated 954

genotypes Bars represent means plusmn SE of four biological replicates combined using a 955

mixed linear model Letters indicate significantly different groups (p lt 005) 956

957

Figure 5 Cell Walls of gae1 gae6 Plants Are Reduced in Homogalacturonan 958

Page 43 of 54

Dot blot analyses with anti-LM19 and anti-LM20 antibodies Pectin was extracted from 959

AIR prepared from the indicated genotypes using 1 microL of extraction buffer per 10 microg of 960

AIR Pectin was serially diluted and one microL of undiluted 13 diluted and 19 diluted 961

samples were spotted on a nitrocellulose membrane Three biological replicates were 962

were spotted twice to obtain two technical replicates each All replicates produced 963

similar results 964

(A) Representative dot blot using anti-LM19 antibody965

(B) Relative signal intensities of the spots in the dot blot in (A)966

(C) Representative dot blot using anti-LM20 antibody967

(D) Relative signal intensities of the LM20 dot blot shown in (C)968

Relative signal intensities were measured using Image J software Measurements from 969

the three biological replicates were combined using a linear model after Box-Cox power 970

transformation as described in Methods Bars represent means plusmn SE Asterisks indicate 971

a difference from wild-type Col-0 at p lt 005 972

973

Figure 6 Growth of B cinerea on gae1 gae6 Plants and the Effect of B cinerea on 974

Expression of GAE1 and GAE6 975

(A) Growth of B cinerea isolates Leaves of Col-0 and gae1 gae6 plants were976

inoculated with 25 x 105 spores mL-1 of the B cinerea isolates indicated in the figure 977

Samples were collected two (2 dpi) and three (3 dpi) days later Bars represent means plusmn 978

SE of six biological replicates for each fungus combined using a mixed linear model 979

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 980

(B) and (C) Expression of GAE1 (B) and GAE6 (C) 48 h after inoculation with the B981

cinerea isolates indicated Expression levels were measured by qRT-PCR Bars 982

represent mean log2-ratios to ACTIN2 plusmn SE of three biological replicates combined 983

using a mixed linear model Letters indicate significantly different groups (q lt 005) 984

985

Figure 7 Levels of Soluble Pectin and Expression of PAD3 in Fractions Produced 986

Following Macerozyme Treatment of gae1 gae6 Cell Walls 987

(A) Schematic overview of the experimental procedure for (B) and (C)988

Page 44 of 54

(B) Uronic acid measurements Alcohol-insoluble residue (AIR) from leave tissue of four989

week-old wild-type Col-0 or gae1 gae6 plants was treated with water or 025 990

macerozyme and assayed for uronic acid levels Data from 11 biological replicates were 991

combined using a mixed linear model Bars represent means plusmn SE Letters indicate 992

significant differences (p lt 005) 993

(C) Expression of PAD3 in Col-0 plants treated with the fractions from (B) Extracts from994

(B) were heat inactivated pooled and diluted 5-fold with water Pooled extracts were995

inoculated into four week-old Col-0 plants PAD3 expression was measured 3 h after 996

inoculation and normalized to the level of ACTIN2 expression Data from four biological 997

replicates were combined using a mixed linear model Bars represent mean log2-ratios 998

to ACTIN2 plusmn SE Letters indicate significant differences (p lt 005) 999

1000

Figure 8 Macerozyme-Induced PAD3 Expression Is Absent in gae1 gae6 1001

(A) Expression of PAD3 after macerozyme treatment Four week-old plants were either1002

inoculated with 001 macerozyme or mock inoculated with water and PAD3 1003

expression was measured 3 h later by qRT-PCR Data from four biological replicates 1004

were combined using a mixed linear model Mean log2-ratios to ACTIN2 plusmn SE were 1005

plotted Letters indicate significantly different groups (p lt 005) 1006

(B) Expression of PAD3 after OG treatment PAD3 expression was measured as1007

described in (A) 3 h after inoculation with 100 microg mL-1 OGs or mock inoculation with 1008

water Data are from six biological replicates 1009

(C) PAD3 expression in untreated plants Three biological replicates were performed1010

and data analyzed as described in (A) 1011

(D) Camalexin accumulation after B cinerea treatment Camalexin accumulation was1012

measured in four week-old plants inoculated with the B cinerea strains shown in the 1013

figure Eight biological replicates each were combined using a mixed linear model 1014

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 1015

(E) PAD3 expression 28 h after treatment with the indicated B cinerea strains Data1016

from six biological replicates were collected and analyzed as described in (A) Data in 1017

all figure parts represent means plusmn SE Letters indicate significantly different groups (q lt 1018

005) 1019

Page 45 of 54

1020

Figure 9 Macerozyme-Induced Expression of the SA Marker Genes SID2 and PR1 1021

Is Abolished in gae1 gae6 Plants 1022

(A) to (H) Expression of PR1 (A) (C) (E) and (G) or SID2 (B) (D) (F) and (H) in four1023

week-old Col-0 and gae1 gae6 plants Data were combined using a mixed linear model 1024

Bars represent mean log2-ratios of expression vs ACTIN2 plusmn SE for all figure parts 1025

Letters indicate significantly different groups at p lt 005 for panels (A) to (D) (G) and 1026

(H) and qlt005 for panels (E) to (F) 1027

(A) and (B) Plants were treated with 001 macerozyme or mock treated with water1028

and samples were collected 3 h later Data from four biological replicates are shown 1029

(C) and (D) Plants were inoculated with 100 microg mL-1 OGs or mock treated with water1030

and samples collected 3 h later Data from six biological replicates are shown 1031

(E) and (F) Plants were inoculated with the indicated B cinerea isolates or mock1032

inoculated with B cinerea inoculation medium and samples collected 48 h later Data 1033

from eight biological replicates are shown 1034

(G) and (H) Expression in untreated plants Three biological replicates were performed1035

1036

Figure 10 The Expression of the JA Marker Gene JAZ10 is Highly Responsive in 1037

gae1 gae6 Plants 1038

(A) to (D) JAZ10 expression in four week-old plants Data were combined using a mixed1039

linear model Mean log2-ratios to ACTIN2 plusmn SE are shown Letters indicate significantly 1040

different groups at p lt 005 for panels (A) (B) and (D) and q lt 005 for panel (C) 1041

(A) Plants were inoculated with 001 macerozyme or mock inoculated using water and1042

samples were collected 3 h later Data from four biological replicates are shown 1043

(B) Plants were inoculated with 100 microg mL-1 OGs or mock inoculated using water and1044

samples collected 3 h later Data are from six biological replicates 1045

(C) JAZ10 expression 48 h after treatment with the indicated B cinerea strains Data1046

are from six biological replicates 1047

(D) JAZ10 expression in untreated plants Data are from three biological replicates1048

1049

Figure 11 Gallo 1-Induced Callose Deposition Is Reduced in gae1 gae6 Plants 1050

Page 46 of 54

(A) Callose deposition in four week-old Col-0 and gae1 gae6 plants Plants were1051

inoculated with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) or mock inoculated 1052

with B cinerea inoculation medium and samples were collected after 72 h Callose was 1053

stained with aniline blue and visualized using a Nikon Eclipse Ni-U microscope with a 1054

DAPI filter Three leaves were stained per treatment Scale bars are 100 microm Asterisks 1055

indicate fungal hyphae arrows indicate representative callose spots 1056

(B) Quantification of callose deposition Callose deposits were counted in 200 microm x 2001057

microm squares laid out across the images shown here Bars represent means plusmn SE from 1058

six squares each Letters indicate significantly different groups (p lt 005) according to a 1059

t-test analysis1060

1061

Figure 12 Less Soluble Pectin Accumulates in B cinerea-Infected gae1 gae6 1062

Leaves 1063

Four week-old Col-0 and gae1 gae6 plants were inoculated with 10 microL droplets of the 1064

indicated B cinerea isolate (25 x 105 spores mL-1) or mock inoculated with B cinerea 1065

inoculation medium Leaves were collected 48 h later AIR was extracted from leaf 1066

samples and water added (20 microL infection site) to extract soluble pectin fragments 1067

Samples were vortexed for 7 h centrifuged at 12000 g and the uronic acid 1068

concentration of the supernatant was measured Data from nine biological replicates 1069

were combined using a mixed linear model Means plusmn SE are shown Letters indicate 1070

significant differences (q lt 005) 1071

1072

Figure 13 Macerozyme-Induced PTI Is Lost in gae1 gae6 While OG-Induced PTI Is 1073

Unaltered 1074

(A) Macerozyme-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1075

inoculated with 001 macerozyme or mock inoculated with boiled macerozyme and 1076

were infected with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) 24 h later Samples 1077

were collected immediately (0 dpi) and after two (2 dpi) and three (3 dpi) days Data 1078

show mean plusmn SE of six to 14 biological replicates each combined using a linear model 1079

Letters indicate significantly different groups (q lt 005) 1080

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

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Page 10 of 54

_____________________________________________________________________________

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Cell Walls of gae6 and gae1 gae6 Plants Have Lower Levels of Pectin 243

GalA is a major component of pectin We analyzed wall monosaccharide 244

composition in gae mutants to investigate whether the increased brittleness was 245

associated with an altered wall composition of gae leaves To approximate pectin 246

content of gae mutant walls the total uronic acid concentration was measured using a 247

simple colorimetric assay (Filisetti-Cozzi and Carpita 1991 van den Hoogen et al 248

1998) Total uronic acid was reduced in gae6-2 strongly reduced in gae1 gae6 plants 249

but was indistinguishable from wild-type Col-0 in gae6-1 and gae1 (Supplemental 250

Figure 5) This suggested that gae1 gae6 and possibly gae6 plants have reduced 251

pectin 252

To investigate the cell wall composition in leaves of gae plants in more detail we 253

measured the levels of seven neutral monosaccharides the acidic sugars GalA (the 254

monomeric subunit of the pectin backbone (Harholt et al 2010)) and GlcA (Figure 4) 255

We also estimated the crystalline cellulose content (Updegraff 1969) Cell walls of both 256

gae6 mutants and gae1 gae6 were reduced in GalA (Figure 4A) In gae1 gae6 GalA 257

content in cell walls was reduced by more than 40 compared to the wild-type level 258

GlcA levels were indistinguishable between mutants and wild-type (Figure 4B) 259

Cellulose content was higher in gae6-2 and gae1 gae6 walls likely due to the loss of 260

pectins (Figure 4C) Cell walls of gae1 gae6 plants were also enriched in all neutral 261

sugars measured presumably due to the substantial proportional loss of GalA (Figure 262

4D) The levels of most wall monosaccharides were even higher in the gae1 gae6 263

double mutant with the exception of Rha which did not exhibit a significant increase 264

compared to the gae6 single mutants The absence of a compensational increase in 265

Rha suggested that RGI the dominant Rha contributor to the wall may be considered 266

reduced in gae1 gae6 mutants compared to wild-type albeit not to such an extent as 267

HG 268

238 structural defect that promotes increased ion leakage Taken together these results

239 showed that loss of GAE6 causes brittle leaves and additional losss of GAE1 strongly

240 enhances this phenotype We conclude that GAE1 and GAE6 contribute to normal leaf

241 flexibility 242

Page 12 of 54

gae6 cell walls are reduced in HG Using the same samples we detected no difference 274

from wild type in binding of LM5 which recognizes a tetramer of (1-4)-β-D-galactans 275

found in RGI (Jones et al 1997) LM6 which recognizes a linear pentasaccharide in (1-276

To further investigate which pectic components were altered in gae1 gae6 dot 269

blot experiments using pectin isolated from the various genotypes were performed Both 270

LM19 and LM20 antibodies recognize HG (Verhertbruggen et al 2009a) As expected 271

LM19 binding was reduced in gae6-1 and gae1 gae6 (Figure 5A and B) and LM20 272

binding was reduced in gae1 gae6 (Figure 5C and D) This result confirmed that gae1 273

277 5)-α-L-arabinans found in RGI (Willats et al 1998 Verhertbruggen et al 2009b) LM8

278 which recognizes xylogalacturonan (Willats et al 2004) or CCRC-M7 which

279 recognizes an arabinosylated (1-6)-β-D-galactan epitope occurring on RGI (Puhlmann et

Page 13 of 54

Page 14 of 54

al 1994 Steffan et al 1995 Pattathil et al 2010) (Supplemental Figure 6) 280

Collectively these cell wall composition data show that gae1 gae6 plants are reduced in 281

HG Due to the strong proportional reduction of HG in the walls levels of all other 282

components should be higher However Rha indicative of RGI was not higher in gae1 283

gae6 suggesting that RGI may also be reduced in gae1 gae6 walls 284

285

GAE1 and GAE6 Are Required for Immunity to Specific B cinerea Isolates 286

We hypothesized that plant immunity to B cinerea might be affected by pectin 287

content in the host cell wall because this pathogen requires polygalacturonases and 288

pectin methylesterases for full virulence (ten Have et al 1998 Valette-Collet et al 289

2003 Kars et al 2005b Espino et al 2010) Because Bcinerea is genetically diverse 290

and has a high diversity of polygalacturonases (Rowe and Kliebenstein 2007) we 291

measured relative fungal growth at both 2 and 3 d post-inoculation (dpi) using ten 292

different B cinerea isolates (Figure 6) The B cinerea isolates Fresa 525 and Gallo 1 293

showed enhanced virulence on gae1 gae6 plants at both time points whereas KT and 294

UK Razz showed enhanced virulence in gae1 gae6 plants at 3 but not 2 d dpi (Figure 295

6A) We did not detect any significant changes in relative fungal growth between wild-296

type Col-0 and gae1 gae6 plants when using Acacia Apple 517 DN Grape Pepper or 297

Rasp isolates of B cinerea Therefore we continued our study using Gallo 1 and Fresa 298

525 We also included Pepper for comparison as this isolate has been used in a large-299

scale analysis of plant gene expression in response to B cinerea infection (Windram et 300

al 2012) 301

We next investigated whether expression of GAE1 and GAE6 was affected by 302

challenge with B cinerea Inoculation of wild-type Col-0 plants with any isolate 303

repressed GAE1 and GAE6 expression (Figure 6B and C) and this repression was 304

generally independent of PAD4 (Figure 6B and C) Expression of GAE2 and GAE3 was 305

unaltered by B cinerea treatment GAE4 was repressed only by Fresa 525 treatment 306

while GAE5 expression was enhanced with all three isolates tested (Supplemental 307

Figure 7) As seen for infection with Pma ES4326 the expression of GAE1 and GAE6 308

was repressed upon B cinerea treatment This indicates a role for GAE1 and GAE6 in 309

the immune response to B cinerea 310

Page 15 of 54

Page 16 of 54

Because the amount of pectin substrate for pathogen polygalacturonases is 313

reduced we hypothesized that immunity phenotypes of gae1 gae6 plants may result 314

from reduced generation of OGs by these enzymes In turn the reduced OGs could 315

lead to reduced immune responses To test this we used macerozyme a commercial 316

pectinase as a proxy for pathogen pectinases We isolated cell walls as a subcellular 317

fraction known as alcohol insoluble residue (AIR Gille et al 2009) This was treated 318

with macerozyme and tested for release of soluble likely low molecular weight pectin 319

fragments (Figure 7) Treatment with 025 macerozyme released significantly more 320

uronic acid from wild-type Col-0 AIR relative to gae1 gae6 AIR (Figure 7B) To test 321

activity of these pectin-containing solutions we infiltrated them into wild-type Col-0 322

plants and measured PAD3 expression PAD3 is required for synthesis of the 323

phytoalexin camalexin (Zhou et al 1999) and its expression is known to be induced by 324

OG treatment (Ferrari et al 2007) Fractions from macerozyme treated Col-0 walls 325

induced significantly more PAD3 expression than fractions derived from gae1 gae6 326

walls suggesting that macerozyme treatment did indeed release more active soluble 327

uronic acid possibly OGs from wild-type Col-0 walls than from gae1 gae6 walls (Figure 328

7C) 329

Next we studied the effect of macerozyme treatment on expression of PAD3 in 330

intact plants (Figure 8) This treatment did not lead to any visible leaf damage 331

(Supplemental Figure 8) In wild-type Col-0 PAD3 expression was induced three h after 332

treatment with 001 macerozyme (Figure 8A) By contrast no macerozyme-induced 333

induction of PAD3 expression was detected in gae1 gae6 (Figure 8A) However OG 334

treatment induced the expression of PAD3 in both genotypes indicating that gae1 gae6 335

plants do respond to OGs (Figure 8B) Macerozyme treatment also induced expression 336

of the SA marker genes PATHOGENESIS RELATED 1 (PR1) and SID2 in Col-0 but 337

not in gae1 gae6 (Figure 9 panel A and B) PR1 was not significantly induced by 338

purified OGs (Figure 9C) at the time and concentration tested but SID2 was induced by 339

OG treatment in both genotypes (Figure 9D) However the induction was stronger in 340

wild-type Col-0 In addition the JA markers JASMONATE-ZIM-DOMAIN PROTEIN 5 341

(JAZ5) and JAZ10 were induced by macerozyme and OG treatment in both Col-0 and 342

gae1 gae6 (Figure 10A and Supplemental Figure 11) Interestingly expression of JAZ5 343

312 Pectinase-Induced Immune Signaling Is Altered in gae1 gae6 Plants

Page 17 of 54

Page 18 of 54

Page 19 of 54

Page 20 of 54

344

345

346

and JAZ10 was already strongly induced in gae1 gae6 after mock inoculation (Figure 10

and Supplemental Figure 11) We observed no hyperresponsive induction by mock

treatment of PLANT DEFENSIN 12 (PDF12) a necrotrophy and late JA signaling

347 marker gene (Supplemental Figure 12A-D) PDF12 was also not induced by

348 macerozyme or OG treatments (Supplemental Figure 12A-D) This suggests that early

349 JA signaling is induced by mock-treatment in gae1 gae6 Because it is known that JA

350 and SA signaling act antagonistically (Pieterse et al 2012) the strong up-regulation of

351 JA-induced immune signaling in gae1 gae6 might explain the lower OG-induced SID2

352 expression in this genotype

353 To test whether other immune responses are altered in gae1 gae6 plants we

354 analyzed Mitogen-activated protein kinase (MAPK) activity following macerozyme and

355 OG treatments OGs are known to activate the MAPKs MPK3 and MPK6 in Arabidopsis

Page 21 of 54

(Galletti et al 2011) Macerozyme treatment activated MPK3 and MPK6 in wild-type 356

Col-0 whereas MAPK activation in gae1 gae6 was slightly reduced in four out of five 357

experiments performed (Supplemental Figure 13) By contrast OG-induced MAPK 358

activity was similar or increased in gae1 gae6 in four out of five experiments 359

(Supplemental Figure 13) Additionally mock treatment induced MAPK activity in gae1 360

gae6 more strongly than it did in wild-type Col-0 in four out of five experiments 361

performed (Fig S13) Collectively these results illustrate that activation of several 362

immune signaling componentsmdashcamalexin synthesis SA and JA signaling and 363

possibly MAPK activationmdashby macerozyme is substantially altered in gae1 gae6 This 364

suggests that release of soluble pectin fragments possibly OGs by pectinolytic 365

enzymes might be reduced in gae1 gae6 plants while recognition of OGs is intact 366

367

Following B cinerea Infection Production of Soluble Pectin and Deposition of 368

Callose Are Reduced in gae1 gae6 Plants 369

Although pad3 plants were more susceptible to the B cinerea isolates Fresa 525 370

and Gallo 1 which also grew better on gae1 gae6 growth of the isolate Pepper was 371

similar on Col-0 and pad3 (Supplemental Figure 9A) However we did not detect any 372

significant changes in camalexin production after treatment with B cinerea isolate Gallo 373

1 and Fresa 525 in gae1 gae6 (Figure 8D) Neither did we detect changes in PAD3 374

expression after treatment with any B cinerea isolate in gae1 gae6 (Figure 8E) 375

Additionally SID2 and PR1 were similarly induced by all B cinerea isolates in both Col-376

0 and gae1 gae6 (Figure 9E and F) and sid2 plants which are deficient for pathogen-377

induced SA biosynthesis did not show any differences in relative B cinerea growth 378

(Supplemental Figure 9B) Expression of JAZ10 was induced more strongly by Gallo 1 379

treatment in gae1 gae6 than in wild-type Col-0 but not by Fresa 525 or Pepper (Fig 380

10C) while dde2 plants deficient for JA biosynthesis showed enhanced susceptibility to 381

all B cinerea isolates tested (Supplemental Figure 9B) Curiously exogenous 382

application of MeJA did not lead to altered relative fungal growth of B cinerea 383

(Supplemental Figure 9C) and cev1 isoxaben resistant 1 (ixr1) plants which have 384

constitutively high JA levels (Ellis and Turner 2001) did not show any changes in 385

susceptibility to the B cinerea isolates tested (Supplemental Figure 9D) Hence neither 386

Page 22 of 54

differences in B cinerea isolate susceptibility to camalexin nor altered SA or JA 387

signaling by themselves explained the enhanced susceptibility of gae1 gae6 to these B 388

cinerea isolates 389

Hydrogen peroxide accumulation in response to B cinerea isolates Fresa 525 390

Gallo 1 and Pepper was not obviously altered in gae1 gae6 plants (Supplemental Figure 391

14) However callose deposition upon treatment with B cinerea isolate Gallo 1 was 392

strongly reduced in gae1 gae6 (Figure 11) It was previously shown that OG treatment 393

induces callose deposition (Denoux et al 2008) suggesting that the reduction in 394

callose deposition might be due to reduced OG release in gae1 gae6 It is possible that 395

this reduction in callose deposition contributes to the reduced immunity of gae1 gae6 396

plants to B cinerea isolate Gallo 1 397

Because macerozyme could be used to release soluble likely low molecular 398

weight pectin from isolated cell walls we next investigated whether infection with B 399

cinerea was accompanied by accumulation of soluble pectin as well We prepared cell 400

walls (AIR fraction) from leaves infected with B cinerea incubated these cell walls with 401

water and measured uronic acid content of the extracts B cinerea treatment did in fact 402

lead to increased accumulation of soluble pectin in wild-type Col-0 plants when 403

compared to mock-treated samples (Figure 12) No such increased accumulation of 404

water-soluble pectin fractions was observed in gae1 gae6 (Figure 12) 405

406

Macerozyme-Induced Pattern-Triggered Immunity against B cinerea Is Abolished 407

in gae1 gae6 408

It was previously shown that OG treatment can induce patternndashtriggered 409

immunity (PTI) to B cinerea (Ferrari et al 2007) We tested macerozyme treatment to 410

determine whether it could also induce PTI to the B cinerea isolate Gallo 1 (Figure 13) 411

Indeed pretreatment with 001 macerozyme for 24 h led to reduced B cinerea growth 412

in wild-type Col-0 but not gae1 gae6 plants (Figure 13A) Interestingly mock and 413

macerozyme pretreated gae1 gae6 plants exhibited similar growth of B cinerea Gallo 1 414

as did macerozyme-treated Col-0 plants This result is likely due to activation of JA 415

signaling in gae1 gae6 during the pretreatment procedure (Figure 13C and D) OG-416

induced PTI was observed in both genotypes when using a high dose of OGs (Figure 417

Page 23 of 54

13B) whereas a lower dose of OGs only induced PTI in Col-0 (Supplemental Figure 418

15) This suggests that OG perception in gae1 gae6 at least above a certain threshold 419

is unaltered while release of immunity-activating pectin fragments possibly OGs is 420

421 reduced We conclude that immune responses induced by macerozyme treatment

422 which do not occur in gae1 gae6 plants are important for plant immunity to the B

423 cinerea isolate Gallo 1

Page 24 of 54

____________________________________________________________________________

Page 25 of 54

DISCUSSION 426

Here we showed that loss of GAE1 and GAE6 leads to a dramatic reduction in 427

pectin content in the cell wall (Figure 4) likely causing the brittleness of leaves that we 428

observed (Figure 3) Petioles and midribs of gae1 gae6 plants broke easily when bent 429

and inoculation of leaves with deionized water resulted in increased ion leakage 430

possibly due either to microscopic fractures of the cell wall induced by inoculation stress 431

or to a reduced capability of these walls to withstand increased turgor pressure The 432

increased brittleness was associated with altered cell wall composition In our analysis 433

the GalA content of gae1 gae6 leaf walls was reduced by more than 40 and the total 434

uronic acids by over 60 (Figure 4 and Supplemental Figure 5) This is a major change 435

in the overall cell wall composition considering that pectin makes up about 50 of the 436

plant cell walls found in Arabidopsis leaves (Zablackis et al 1995 Nobuta et al 2007 437

Harholt et al 2010) Interestingly quasimodo1 (qua1) plants which have a mutation in 438

a glycosyltransferase family 8 gene are reduced in GalA by 25 and show reduced 439

binding of HG specific antibodies (Bouton et al 2002) 440

Furthermore qua2 plants which have a mutation in a putative methyltransferase 441

have about 13 less total GalA and lack 50 of cell wall HG (Mouille et al 2007) 442

Likewise cgr2 cgr3 plants carrying mutations in another class of Golgi-localized 443

putative methyl-transferases have leaf cell walls that are reduced by about 40 in 444

uronic acid (Kim et al 2015) All three mutants are severely dwarfed (Bouton et al 445

2002 Mouille et al 2007 Kim et al 2015) Additionally qua1 and qua2 mutants show 446

a distorted overall shape likely due to reduced cell cohesion and are extremely fragile 447

(Bouton et al 2002 Mouille et al 2007) By contrast gae1 gae6 plants with a similar 448

strong reduction in GalA and total uronic acids have an overall normal shape and are 449

only slightly smaller than wild-type Col-0 (Supplemental Figure 4) In the wall analysis 450

performed here we did not detect changes in other wall polysaccharides or structures 451

that might compensate for the reduction of pectin in gae1 gae6 It is possible the severe 452

morphological alterations in qua2 and cgr2 cgr3 plants are due to reduction in pectin 453

methylesterification which is unaffected in gae1 gae6 It was speculated that quality 454

control mechanisms in the Golgi prevent secretion of inefficiently esterified pectin in 455

cgr2 cgr3 (Kim et al 2015) suggesting that the Golgi of these plants might over-456

Page 26 of 54

accumulate pectin and that this may contribute to the severe dwarf phenotype of these 457

plants The gae1 gae6 plants described here provide an opportunity to study the effects 458

of reduction of pectin in plants that do not show severe morphological phenotypes 459

We found that gae1 gae6 plants are also compromised in resistance to B 460

cinerea and Pma ES4326 indicating that loss of pectin reduces immunity to these 461

pathogens (Figure 2 and 6) We found that gae1 gae6 plants were slightly more 462

susceptible to Pma ES4326 but showed a strong increase in susceptibility to specific B 463

cinerea isolates This was not unexpected because B cinerea is known to interact with 464

pectin as part of its infection strategy It secretes various pectin-degrading enzymes 465

early during infection and requires some of these enzymes including PG1 PG2 and 466

PME1 for full virulence (ten Have et al 1998 Valette-Collet et al 2003 Kars et al 467

2005b Espino et al 2010) Additionally Arabidopsis plants expressing an antisense 468

construct of the polygalacturonase inhibitor PGIP1 are significantly more susceptible to 469

B cinerea (Ferrari et al 2006) On the other hand Pma ES4326 is not known to470

require cell wall degrading enzymes for full virulence even though it encodes two 471

pectate lyase genes in its genome (PMA4326_07094 and PMA4326_04621 472

wwwbacteriaensemblorg) B cinerea isolates are also known to be genetically quite 473

diverse in their polygalacturonase (PG) loci and genetic variability in PG2 is associated 474

with varying growth on pectin containing media (Rowe and Kliebenstein 2007) 475

Furthermore the contribution of PG1 and PG2 to B cinerea virulence seems to depend 476

on the specific B cinerea isolate and the host species (Kars et al 2005a Zhang and 477

Van Kan 2013) B cinerea isolates are also known to be differently sensitive to the anti-478

microbial secondary metabolite camalexin (Kliebenstein et al 2005) In line with these 479

observations the susceptibility of gae1 gae6 plants differed depending on the specific 480

B cinerea isolate Whereas Fresa 525 Gallo 1 UK Razz and KT grew better in gae1481

gae6 we detected no difference in Acacia Apple 517 DN Grape Pepper or Rasp 482

growth (Figure 6) 483

B cinerea was shown to metabolize pectin and to possibly utilize GalA as a484

carbon source during in planta growth (Zhang et al 2011 Zhang and Van Kan 2013) 485

Our data show that gae1 gae6 plants which are reduced in GalA content did allow 486

enhanced growth of specific B cinerea isolates but no reduction in growth of any B 487

Page 27 of 54

cinerea isolates tested was found This suggests that utilization of pectin as a carbon 488

source was not a contributing factor to alterations in B cinerea susceptibility It is 489

possible that changes in the cell wall composition affect the structural integrity of the 490

wall and affect hyphal penetration of specific B cinerea isolates However OGs (small 491

HG fragments) are likely released during the infection process (Ferrari et al 2013) 492

OGs function as DAMPs and initiate immune responses in Arabidopsis (Ferrari et al 493

2013) Treatment of Arabidopsis with OGs having a degree of polymerization (DP) of 494

10-15 sugar subunits was previously shown to enhance resistance to B cinerea (Ferrari495

et al 2007) We showed that pretreatment with OGs or macerozyme induced enhanced 496

immunity to B cinerea isolate Gallo 1 in wild-type Col-0 but only OGs and not 497

macerozyme induced immunity in gae1 gae6 (Figure 13) Our data suggest gae1 gae6 498

plants which have reduced pectin content may release less OGs and that this 499

reduction leads to the increased susceptibility to B cinerea 500

Consistently we found that macerozyme treatment released less soluble pectin 501

from gae1 gae6 cell walls than from wild-type walls Fractions containing soluble pectin 502

released from gae1 gae6 were also less active in inducing PAD3 gene expression than 503

the fractions released from wild-type walls (Figure 7) We also showed that less soluble 504

pectin accumulated in gae1 gae6 than in wild-type plants 48 h after treatment with B 505

cinerea (Figure 12) 506

We observed that loss of pectin leads to alterations in A thaliana immune 507

signaling Macerozyme treatment induced expression of PAD3 and the SA marker 508

genes PR1 and SID2 in wild-type Col-0 but not in gae1 gae6 whereas OG treatment 509

led to increased PAD3 and SID2 expression in both wild-type and gae1 gae6 (Figure 8 510

and 9) This suggests that although release of active soluble pectin in these plants 511

might be altered OG recognition is intact In fact cell walls isolated from qua1 another 512

mutant known to be reduced in HG released less OGs (DP1-6) than did wild-type cell 513

walls upon endopolygalacturonase treatment (Bouton et al 2002) This was also true 514

for gae1 gae6 cell walls as macerozyme treatment of isolated gae1 gae6 cell walls 515

released less soluble pectin than treatment of Col-0 cell walls Because the fraction 516

from gae1 gae6 plants was also less active in inducing PAD3 expression (Figure 7) it 517

seems possible that biologically active OGs were the active component inducing PAD3 518

Page 28 of 54

expression Interestingly expression of the JA marker genes JAZ5 and JAZ10 was 519

higher in gae1 gae6 than in wild-type mock-inoculated plants but was comparable to 520

wild-type in untreated plants suggesting that JA signaling in gae1 gae6 is 521

hyperresponsive (Figure 10 and Supplemental Figure 11) This induction of JA signaling 522

might be due to wounding stress incurred during the inoculation process JA and SA 523

signaling generally act antagonistically (Pieterse et al 2012) Increased JA signaling 524

activity might hence explain why mock- and macerozyme-treated gae1 gae6 plants 525

exhibited reduced expression of the SA marker PR1 and why OG treatment induced 526

expression of SID2 another SA marker to a lower extent in gae1 gae6 than in wild-type 527

plants (Figure 9) 528

No single immune signaling component was found to be the sole contributor to 529

the compromised immunity of gae1 gae6 to specific B cinerea isolates such as Gallo 1 530

and Fresa 525 These isolates grew better on pad3 while growth of Pepper another B 531

cinerea isolate that did not show altered virulence on gae1 gae6 was unaltered 532

(Supplemental Figure 9) This suggested that differences in camalexin sensitivity of 533

these B cinerea isolates might account for the differences in gae1 gae6 susceptibility 534

However camalexin accumulation and PAD3 expression was the same for wild-type 535

Col-0 and gae1 gae6 for the B cinerea isolates Fresa 525 and Gallo 1 (Figure 8) which 536

showed enhanced growth on gae1 gae6 plants It should be noted that B cinerea 537

virulence is often estimated by comparing lesion sizes Lesion size and measurements 538

of relative fungal growth by qPCR do not always show the same results For example 539

although Denby et al (2004) showed that Pepper causes larger lesions on pad3 than on 540

wild-type this study showed that relative fungal growth of Pepper on pad3 was 541

unaltered 542

We observed that callose deposition was strongly reduced in gae1 gae6 plants 543

72 h after treatment with B cinerea isolate Gallo 1 (Figure 11) possibly due to reduced 544

release of active soluble pectin Interestingly OG-induced PTI to an unspecified isolate 545

of B cinerea was intact in the pmr4 mutant which does not produce callose (Galletti et 546

al 2008) Due to the high SA levels in pmr4 plants (Nishimura et al 2003) and the fact 547

that different B cinerea isolates vary both in induction of and reaction to defense 548

responses (Kliebenstein et al 2005 Rowe et al 2010) we could not determine 549

Page 29 of 54

whether reduced callose deposition causes the increased susceptibility of gae1 gae6 to 550

B cinerea isolate Gallo 1551

We did not observe any changes in B cinerea growth either on cev1 ixr1 plants 552

which are known to have constitutively high JA and OPDA levels (Ellis et al 2002b) or 553

on plants exogenously treated with MeJA (Supplemental Figure 9) Because plants 554

were kept uncovered for 3 h after MeJA treatment it is unclear how much MeJA was 555

bioavailable in the plant However JAZ10 expression was induced 3 h after spraying 556

with MeJA (Supplemental Figure 10) Because pathogen growth generally depends on 557

the combination of a multitude of immune signaling events we conclude that multiple 558

changes to the immune signaling in gae1 gae6 together account for the altered B 559

cinerea growth phenotypes detected 560

We showed that expression of GAE1 and GAE6 is repressed by the bacterial 561

hemibiotroph Pma ES4326 This repression did not occur in an eds1 or pad4 562

background or in pbs3 at the 24-h time point (Figure 1 and Supplemental Figure 1) 563

During the Arabidopsis - Pma ES4326 interaction the expression of many defense 564

related genes is altered in a PAD4EDS1 dependent fashion (Wang et al 2008) Some 565

of these expression changes require SA CBP60g and SARD1 are thought to act 566

downstream of PAD4 and EDS1 but upstream of SA-dependent responses (Wang et 567

al 2011) PBS3 is also thought to act downstream of PAD4 and EDS1 and upstream of 568

SA but the set of genes whose expression it affects is only partially overlapping with 569

those affected by CBP60gSARD1 (Wang et al 2008 Wang et al 2011) Thus GAE1 570

and GAE6 repression is regulated by the part of the immune signaling network that 571

requires PAD4 EDS1 and PBS3 but is independent of CBP60gSARD1 and SA 572

Because EDS1 PAD4 and PBS3 are important components of the plant immune 573

signaling network (Wiermer et al 2005 Nobuta et al 2007) one might conclude that 574

repression of GAE1 and GAE6 may be part of the plant immune response to Pma 575

ES4326 B cinerea treatment also reduced GAE1 and GAE6 expression but this was 576

mostly independent of PAD4 (Figure 6) while both Pma ES4326 and B cinerea repress 577

GAE1 and GAE6 expression the host signaling mechanisms responsible likely differ 578

In summary our data show that Arabidopsis GAE1 and GAE6 are required for 579

pectin biosynthesis leaf flexibility and immunity to Pma ES4326 and to specific B 580

Page 30 of 54

cinerea isolates We used macerozyme a commercial pectinase to mimic the effect of 581

B cinerea pectinases Macerozyme treatment released less pectin from gae1 gae6 cell582

walls and induced less immune signaling in gae1 gae6 plants Less water soluble pectin 583

also accumulated in B cinerea infected gae1 gae6 plants and macerozyme-induced 584

immunity to B cinerea isolate Gallo 1 was reduced in these plants We conclude that 585

the reduced pectin content in these mutants may lead to reduced release of soluble 586

pectin including active OGs during pathogen attack and that this compromises 587

immunity 588

589

590

Page 31 of 54

METHODS 591

Plant Materials and Growth Conditions 592

Wild-type Columbia (Col-0) and all mutant plants (in Col-0 background) were 593

grown on sterilized BM2 germinating mix (Berger Inc Quebec Canada) in a controlled 594

environment chamber (Conviron Pembina ND USA) with a 12-h photoperiod under 595

100 microM m-2 s-1 fluorescent illumination at 22degC and 75 relative humidity Germplasm 596

used is described in the Accession Numbers section at the end of Methods 597

598

Data Analysis and Replicates 599

For biological replicates samples were obtained from separate plant tissues and 600

in many cases from plants grown in different flats In some experiments multiple 601

technical replicates were measured per biological replicate For statistical analysis all 602

technical replicates were averaged and the averages were considered one biological 603

replicate Independent experiments were performed at different times and often 604

consisted of multiple biological replicates per experiment 605

Data analysis was performed using mixed linear models in the R programming 606

environment (Bates et al 2015) unless otherwise indicated Generally genotypes 607

treatments and time points were used as fixed effects and replicate-specific effects like 608

different pots flats etc were considered random effects We reported p-values 609

obtained from the mixed linear model when less than 10 comparisons were performed 610

and q-values for more than 10 comparisons to correct for multiple comparison testing 611

Q-values were calculated using the Benjamini-Hochberg procedure (Benjamini and612

Hochberg 1995) unless specified otherwise 613

614

Pathogen Strains Growth Conditions and Pathogen Growth Assays 615

Growth of Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) and 616

bacterial growth assays were performed as previously described (Bethke et al 2014) 617

For qRT-PCR assays four week-old plants were inoculated with bacteria (OD600 = 618

0002) or mock (5 mM MgSO4) At least four leaves from two plants were collected at 619

the indicated time points per biological replicate 620

Page 32 of 54

Prior to each experiment Botrytis cinerea isolates were grown on 1x PDA 621

medium (Difco) for 10 d at room temperature All isolates used have been described 622

previously (Rowe and Kliebenstein 2007) The spores were washed from the surface of 623

the plate using an inoculation medium (1x Gamborgrsquos B-5 basal salt mixture (Sigma-624

Aldrich G5768) 2 (wv) glucose 10 mM phosphate buffer pH 64) and fungal hyphae 625

were removed from the suspension by filtering through four layers of cheesecloth The 626

concentration of spores was determined using a hemocytometer and adjusted to 25 x 627

105 spores mL-1 Four week-old Arabidopsis plants were inoculated by placing one 10-628

microL droplet of the B cinerea spore solution or inoculation medium (mock) on the adaxial 629

leaf surface of fully expanded leaves Inoculated plants were kept at 100 relative 630

humidity Infection sites were collected at various time points using a cork borer For 631

qRT-PCR assays at least four leaf discs from one plant were combined for each 632

biological sample For B cinerea growth assays at least 12 leaf discs from three to six 633

plants were pooled per biological sample Relative fungal growth was determined by 634

measuring the abundance of a fungal gene relative to a plant gene as described below 635

In brief DNA from infected tissue was extracted and equal amounts of total DNA were 636

used to perform qPCR reactions using the B cinerea cutinase A and the Arabidopsis 637

SK11 gene respectively Primers were described in (Gachon and Saindrenan 2004) 638

and are listed in Supplemental Table 1 Two technical replicates were combined for 639

each biological replicate B cinerea is a necrotrophic fungal pathogen and may destroy 640

plant DNA as disease progresses However decreases in the amount of plant DNA 641

over the course of our experiments were only observed at late time points with very 642

susceptible plants so the qPCR assay is generally a reasonable estimate of fungal 643

biomass (Supplemental Figure 16) 644

645

Treatment with Macerozyme or OGs 646

For PTI assays four week-old plants either were inoculated with 001 (wv) 647

macerozyme R-10 (Yakult) a Rhizopus polygalacturonase (EC 32115) that possesses 648

high pectinase and hemicellulase activity or were mock inoculated using heat-649

inactivated macerozyme Other similar plants were either inoculated with 250 or 500 microg 650

mL-1 OGs or mock inoculated with water One d later plants were infected with B 651

Page 33 of 54

cinerea and samples were collected immediately (0 dpi) and two or three d later To 652

investigate the role of MeJA on B cinerea growth plants were sprayed with 1mM MeJA 653

in water with 001 (vv) Tween 20 Covers were removed so that leaves could dry off 654

before plants were inoculated with B cinerea three h later Samples were harvested at 655

the indicated time points For expression analysis and MAPK activation assays plants 656

were either inoculated with 100 microg mL-1 OGs or mock inoculated with water OGs used 657

for experiments described in Figures 8 to 10 and Supplemental Figure 12 were a kind 658

gift from Simone Ferrari OGs used in experiments described in Figure 13 and 659

Supplemental Figures 11 13 and 15 were prepared as previously described (Kohorn et 660

al 2014) 661

662

Expression Analysis 663

Leaf tissue from inoculated plants was harvested at the indicated time points 664

flash frozen pulverized and RNA extracted using Trizol (Invitrogen) Quantitative RT-665

PCR was performed using the SuperScript III Platinum SYBR Green One-Step 666

quantitative RT-PCR kit (Invitrogen) and a Lightcycler 480 Real-Time PCR system 667

(Roche) as previously described (Truman and Glazebrook 2012) In brief equal 668

amounts of total RNA and a gene specific primer were used for each reaction The 669

crossing point (Cp) was calculated using the 2nd derivative max method provided with 670

the Lightcycler software for each amplification curve Each reaction was run with two 671

technical replicates and the Cp values for these replicates were averaged ACTIN2 was 672

used as a stably expressed reference gene At least three biological replicates each 673

were performed and analyzed using a mixed-linear model RT-PCR was performed to 674

verify T-DNA insertion lines using the One-Step RT-PCR kit (Qiagen) according to the 675

manufacturerrsquos instructions The primers used can be found in Supplemental Table 1 676

677

Electrolyte Leakage Assay 678

Fully expanded leaves of four week-old plants were inoculated with water (Milli-Q 679

grade) using a needleless syringe Leaf discs were collected using a cork borer and 680

three leaf discs from one leaf were placed with the adaxial surface down onto 2 mL 681

water in a 12-well cell culture plate Conductivity was measured using a Horiba B-173 682

Page 34 of 54

conductivity meter after 30 min and then the water was replaced Conductivity was 683

measured again 30 min later (1 h total) as indicated in Figure 3C Six biological 684

replicates each were performed in three independent experiments and means and SEs 685

were calculated Two-sided t-tests were performed for each time point 686

687

DAB and Aniline Blue Staining 688

Leaves from four week-old plants were either mock inoculated with B cinerea 689

inoculation medium or inoculated with 10 microL droplets of a B cinerea spore solution (25 690

x 105 spores mL-1) of the indicated isolates Samples were collected 72 h later For 33prime-691

diaminobenzidine (DAB) staining two to five inoculated leaves were vacuum infiltrated 692

with 33prime-Diaminobenzidine (1 mg mL-1 in water pH 38) solution and stained overnight 693

in the dark Leaves were destained using 100 ethanol at 65degC and then transferred to 694

50 (vv) glycerol and photographed 695

Callose was visualized by staining leaves with aniline blue as previously 696

described (Adam and Somerville 1996) Briefly leaves were vacuum infiltrated with 697

alcoholic lactophenol (one volume of phenolglycerollactic acidwater (1111) mixed 698

with two volumes of ethanol) and destained at 65degC Next leaves were moved through 699

an alcohol gradient (50 20 and 10 (vv) ethanol) into water The destained leaves 700

were stained for 30 min in 150 mM K2HPO4 (pH 95) with 001 (wv) aniline blue 701

Samples were mounted in water and examined using a Nikon Eclipse Ni-U microscope 702

at 10x magnification using a DAPI filter (excitation at 325-375 nm emission at 435-485 703

nm) For callose quantification six 200 microm x 200 microm squares were counted for each 704

sample type Callose deposits per mm2 were calculated and data analyzed using a t-705

test Note that there is an inherent uncertainty associated with such counts because 706

callose deposits may not all be in focus at the same time 707

708

MAPK Activity Assay 709

Fully expanded leaves of four week-old plants were inoculated with 001 710

macerozyme 100 microg mL-1 OGs 100 nM flg22 or water (mock) flg22 a peptide from 711

bacterial flagella that induces immune responses was used as a positive control for 712

Page 35 of 54

MAPK activation (Asai et al 2002) and was purchased from EZBiolab Inc (Westfield 713

IN USA) Samples were harvested 10 min later and immediately frozen in liquid 714

nitrogen Protein was extracted using an extraction buffer containing 25 mM Tris pH 78 715

75 mM NaCl 1 mM DTT 1 mM NaF 05mM Na3VO4 01 (vv) Tween 20 one 716

cOmplete Mini protease inhibitor cocktail tablet (Roche) and one PhosSTOP 717

phosphatase inhibitor cocktail tablet (Roche) per 10 mL buffer 15 microg of total protein 718

were electrophoresed on a 10 SDS-polyacrylamide gel and then blotted onto a PVDF 719

membrane (Bio-Rad 162-0177) Activated MAPKs were detected using p4442 MAPK 720

(Erk12) antibody (9102S Cell Signal 12500 in TBST (20 mM Tris pH 75 150 mM 721

NaCl 01 (vv) Tween 20)) Anti-Rabbit IgG-HRP (115000 in TBST A6154 Sigma) 722

and ECL Plus substrate (Pierce 32132) MPK3 and MPK6 were detected with an anti-723

MPK3 antibody (M8318 Sigma 12000 in TBST and 3 (wv) milk) or an anti-MPK6 724

antibody (A7104 Sigma 16000 in TBST and 3 (wv) milk) respectively The 725

secondary antibody and detection were the same as for the p4442 antibody Five 726

independent experiments were performed 727

728

Preparation of Alcohol Insoluble Residue (AIR) Pectin Extraction and Uronic Acid 729

Measurements 730

Fully expanded leaves from four week-old plants that were incubated for 48 h in 731

the dark to reduce starch were harvested flash frozen and pulverized AIR was 732

extracted by washing ground material twice in 70 (vv) ethanol three times in a 733

mixture of chloroform and methanol (11 vv) and once in acetone (Gille et al 2009) 734

and was then air dried Cell wall pectin was extracted from AIR with cell wall extraction 735

buffer (50 mM Trizma 50 mM CDTA pH 72) at 95degC for 15 min Samples were 736

homogenized using a paint shaker (Harbil 5G-HD) and glass beads (3 mm diameter) 737

For dot blot experiments 500 microL of cell wall extraction buffer were used per 10 mg of 738

AIR Debris was precipitated by centrifugation for 10 min at 10000 g 739

Total uronic acid content of pectin extracted as described above using 500 microL of 740

cell wall extraction buffer per 1 mg of AIR was measured as described previously 741

(Filisetti-Cozzi and Carpita 1991 van den Hoogen et al 1998) First 36 microL of pectin 742

extract were mixed with 4 microL of 4 M sulfamic acid Then 200 microL of sulfuric acid 743

Page 36 of 54

containing 120 mM sodium tetraborate was added and samples were incubated at 80degC 744

for 1 h Following cooling on ice the optical density of samples was measured at 490 745

nm Next 40 microL of m-hydroxydiphenyl reagent (100 microL of m-hydroxydiphenyl in dimethyl 746

sulfoxide at 100 mgmL mixed with 49 mL 80 (vv) sulfuric acid just before use) was 747

added and the samples were mixed The optical density at 490 nm was measured 748

again The optical density before m-hydroxydiphenyl reagent addition was subtracted 749

from the optical density measured after addition of the dye The concentration of uronic 750

acid was calculated using known amounts of GalA as a standard 751

752

Release of Pectin from Arabidopsis Cell Walls Using Macerozyme 753

AIR preparations from leaves of four week-old plants were treated with 025 754

(wv) macerozyme R-10 (Yakult) or water (mock) at a concentration of 100 microL per mg 755

AIR Samples were incubated for 3 h at room temperature on a vortex shaker and 756

centrifuged for 10 min at 12000 g Total uronic acid concentration of 5-fold diluted 757

supernatants was determined for two technical replicates each The concentration of 758

uronic acid was calculated using known amounts of GalA as a standard For 759

subsequent qRT-PCR experiments supernatants from multiple extractions were 760

combined boiled for 20 min to inactivate macerozyme diluted 10-fold with water and 761

inoculated into four week-old Col-0 plants Heat-inactivated 10-fold diluted macerozyme 762

solutions were used as mock treatment Samples were collected 3 h later and PAD3 763

expression was determined 764

765

Release of Pectin from Arabidopsis Leaves Infected with B cinerea 766

Fully expanded leaves of four week-old plants were either inoculated with up to 767

five 10 microL droplets of B cinerea spore solution (25 x 105 spores mL-1) or mock 768

inoculated with B cinerea inoculation medium For each biological replicate leaves with 769

a combined count of at least 20 infection sites were collected 48 h later AIR was 770

extracted as described above but plants were not dark-treated or freeze-dried and the 771

first ethanol step was performed using 85 (vv) ethanol To extract soluble pectin 772

fragments AIR samples were mixed with water at a concentration of 20 microL per infection 773

site incubated for 7 h at room temperature on a vortex shaker and centrifuged for 10 774

Page 37 of 54

min at 12000 g Total uronic acid concentration of supernatants was determined for two 775

technical replicates each The concentration of uronic acid was calculated using known 776

amounts of GalA as a standard 777

778

Measurement of Uronic Acids Neutral Monosaccharides and Cellulose Content in 779

Cell Walls 780

Neutral sugars and uronic acids from non-cellulosic polysaccharides in cell walls 781

(AIR) were released by trifluoroacetic acid (TFA) hydrolysis Approximately 1 mg of AIR 782

was hydrolyzed with 2 M TFA at 121degC for 90 min The TFA-soluble material was split 783

into two equal parts one for uronic acid and one for neutral sugar determination The 784

uronic acid content was determined by analyzing the hydrolyzate using a CarboPac 785

PA200 anion-exchange column with an ICS-3000 Dionex chromatography system 786

(Dionex Sunnyvale CA USA) The elution profile consisted of a linear gradient of 50 to 787

200 mM sodium acetate in 01 M NaOH in 10 min at 04 mL per min Alditol acetate 788

derivatives of neutral sugars were produced as described (York et al 1985) In brief 789

monosaccharides in the hydrolyzate were reduced with NaBH4 and peracetylated with 790

acetic anhydride and pyridine at 121degC for 20 min The generated alditol acetates were 791

analyzed by GC-MS Crystalline cellulose was measured according to Updegraff (1969) 792

After hydrolysis of noncellulosic polysaccharides from AIR with the Updegraff reagent 793

(acetic acid nitric acids water 812 (vv) ) the remaining pellet was hydrolyzed in 794

72 sulfuric acid The resulting glucose quantity was determined by the anthrone 795

method (Scott and Melvin 1953) 796

797

Dot Blot Experiments 798

Pectin solutions extracted using 50 microL of cell wall extraction buffer per mg AIR 799

were serially diluted and nitrocellulose membranes were spotted with 1 microL of the diluted 800

pectin solutions Membranes were dried overnight blocked with 5 milk (wv) in 1x 801

PBS (8 g L-1 NaCl 02 g L-1 KCl 144 g L-1 Na2HPO4 024 g L-1 KH2PO4 pH 74) and 802

probed with LM5 (Jones et al 1997) LM6 (Willats et al 1998) LM8 (Willats et al 803

2004) LM19 and LM20 (Verhertbruggen et al 2009a) or CCRC-M7 (Steffan et al 804

Page 38 of 54

1995) antibodies LM series antibodies were diluted 1250 and CCRC-M7 was diluted 805

1500 in 5 milk powder (Nestle Carnation) in 1 x PBS For LM series antibodies a 806

goat anti-Rat HRP conjugated antibody (Bethyl A110-105P 15000 diluted) and for 807

CCRC-M7 an anti-Mouse IgG HRP conjugate (Promega W402B 12500 diluted) was 808

used as secondary antibody in 5 milk powder in 1x PBS LM series antibodies were 809

obtained from plant probes (wwwplantprobesnet) and CCRC-M7 from CarboSource 810

(wwwcarbosourcenet) Membranes were washed after incubation with the primary and 811

secondary antibodies with 1x PBS Dot blots were developed using the ECL system 812

(GE Healthcare Amersham ECL prime) For quantification of signals dot blot results on 813

x-ray films were photographed using a CCD camera and intensities were measured814

using Image Jrsquos Integrated Density function Locally adjusted background was 815

subtracted As the dynamic range of the signal on an x-ray film was narrow non-816

linearity in the measured values was corrected for each genotype in each biological 817

replicate using the values from different dilutions in two technical replicates We noticed 818

that the residuals were not normally distributed and performed Box-Cox power 819

transformation (Box and Cox 1964) to obtain normally distributed values for the 820

subsequent statistical analysis of the data For LM19 Box-Cox selected power 821

transformation was done to the power of 038th and for LM20 to the power 075th After 822

correction of the transformed measured values with the effect of the biological 823

replicates the values were fit to a linear model with the Arabidopsis genotype as the 824

fixed effect This linear model was used both to generate the plots in Figures 5B and D 825

and to test significant differences 826

827

Camalexin Measurements 828

Four week-old plants were infected with B cinerea and samples (three leaf disks 829

from one plant each) were harvested at the indicated time points flash frozen and 830

pulverized Each sample was extracted with 300 microL of 90 methanol (vv) 50 microL of the 831

extract was run on an Agilent Lichrocart 250-4 RP18e 5-microm column using an Agilent 832

1100 series HPLC Camalexin was detected using a DiodeArray (DAD) at 330 nm and 833

with a fluorescence detector at emission 318 nmexcitation 385 nm (Agilent) Separation 834

was achieved using the following program with aqueous acetonitrile 5 min gradient 835

Page 39 of 54

from 63 to 69 acetonitrile 30 s gradient from 69 to 99 acetonitrile 2 min at 99 836

acetonitrile and a post-run equilibration of 35 min at 63 acetonitrile (Denby et al 837

2004 Kliebenstein et al 2005) Purified camalexin was used to produce a standard 838

curve to identify and quantitate camalexin 839

840

Accession Numbers 841

Microarray data used can be accessed at NCBI Gene Expression Omnnibus 842

(httpwwwncbinlmnihgovgeo) GSE18978 Sequence information for all Arabidopsis 843

genes described in this article can be found in TAIR (wwwarabidopsisorg) using the 844

following accession numbers PAD3 (CYP71B15 At3g26830) SID2 (ICS1 At1g74710) 845

GAE1 (At4g30440) GAE2 (At1g02000) GAE3 (At4g00110) GAE4 (At2g45310) GAE5 846

(At4g12250) GAE6 (At3g23820) PR1 (At2g14610) JAZ5 (At1g17380) JAZ10 847

(At5g13220) ACTIN2 (At3g18780) PDF12a (At5g44420) PDF12b (At2g26020) 848

AtSK11 (At5g26751) The sequence of the B cinerea cutinase Bc-CutA (Z69264) can 849

be found in the DNA Data Bank of Japan (httpgetentryddbjnigacjp) Germplasm 850

used included dde2-2 (At5g42650 (von Malek et al 2002)) pad3-1 (At3g26830 (Zhou 851

et al 1999)) pad4-1 (At3g52430 (Jirage et al 1999)) sid2-2 (At1g74710 (Wildermuth 852

et al 2001)) pbs3-2 (At5g13320 SALK_018225 (Nobuta et al 2007)) cbp60a-1 853

(At5g62570 SALK_124410 (Truman et al 2013)) cbp60g-1 sard1-2 (At5g26920 854

SALK_023199 At1g73805 SALK_052422 (Wang et al 2011) cbp60a-1 cbp60g-1 855

sard1-2 (Truman et al 2013) gae1-1 (At4g30440 SALK_085554) gae6-1 (At3g23280 856

SALK_104454) gae6-2 (At3g23280 SALK_017191) ixr1-1 cev1 (At5g05170 (Ellis 857

and Turner 2001 Scheible et al 2001)) eds1 plants were derived by introgression of 858

the Ler eds1-2 allele into Col-0 that contains two copies of EDS1 (At3g48090 859

At3g48080 (Bartsch et al 2006) mpk6-2 (At2g43790 SALK_073907 (Liu and Zhang 860

2004)) mpk3-DG (At3g45640 a fast neutron deletion mutant (Miles et al 2005)) and 861

npr1-1 (At1g64280 (Cao et al 1997)) T-DNA insertion lines were part of the SALK 862

collection (Alonso et al 2003) and were obtained from the Arabidopsis Biological 863

Resource Center 864

865

Supplemental Data 866

Page 40 of 54

Supplemental Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 867

Expression Does Not Require CBP60s or SID2 868

Supplemental Figure 2 Expression of GAE Family Members uponTreatment with Pma 869

ES4326 870

Supplemental Figure 3 Characterization of T-DNA Insertions in GAE1 and GAE6 871

Supplemental Figure 4 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves and 872

Are Slightly Smaller than Wild-Type Col-0 Plants 873

Supplemental Figure 5 Cell Walls of gae1 gae6 Plants Contain Less Uronic Acid than 874

Wild-Type Col-0 Cell Walls 875

Supplemental Figure 6 Antibodies Raised Against the RGI or XG Components of 876

Pectin Show No Difference in Binding to gae Mutant and Col-0 Cell Walls 877

Supplemental Figure 7 The Effect of B cinerea Treatment on the Expression of GAE 878

Family Genes 879

Supplemental Figure 8 Treatment with 001 Macerozyme Does Not Cause Any 880

Visible Damage to Col-0 Plants 881

Supplemental Figure 9 B cinerea Growth on Plants with Defects in Immune 882

Signaling 883

Supplemental Figure 10 MeJA-Induced Expression of the JA marker gene JAZ10 884

Supplemental Figure 11 The Expression of the JA Marker Gene JAZ5 Is Highly 885

Responsive in gae1 gae6 Plants 886

Supplemental Figure 12 The Expression of PDF12 Is Unaltered in gae1 gae6 Plants 887

Supplemental Figure 13 Macerozyme Treatment Activates MPK3 and MPK6 888

Supplemental Figure 14 B cinerea-Induced ROS Production in Wild-Type Col-0 and 889

gae1 gae6 Plants 890

Supplemental Figure 15 OG-Induced PTI Using 250 microg mL-1 OGs 891

Supplemental Figure 16 Abundance of Plant DNA in B cinerea Treated Plants 892

Supplemental Table 1 Primers used in this study 893

894

ACKNOWLEDGEMENTS 895

We thank William Truman for helpful discussions Simone Ferrari (Sapienza 896

Universita di Roma) for the generous gift of OGs Bruna Bucciarelli (USDA-ARS) for 897

Page 41 of 54

help with fluorescence microscopy the Arabidopsis Biological Resource Center for T-898

DNA insertion lines Paul Knoxrsquos lab (University of Leeds) for LM5 LM6 LM8 LM19 899

and LM20 antibodies and the Complex Carbohydrate Resource Center (University of 900

Georgia) for the CCRC-M7 antibody Most of this work was funded by the Division of 901

Chemical Sciences Geosciences and Biosciences Office of Basic Energy Sciences of 902

the US Department of Energy through Grant DE-FG02-05ER15670 to JG This work 903

was also supported by NSF awards IOS 1339125 and IOS 1021861 to DJK NSF 904

award IOS 1121425 to F K and the USDA National Institute of Food and Agriculture 905

Hatch project number CA-D-PLS-7033-H to DJK 906

907

AUTHOR CONTRIBUTIONS 908

GB and JG designed the research GB GX AT BL NES and NH 909

performed research DJK provided tools GB GX MP AD BL RAH and FK 910

analyzed data GB and JG wrote the paper with input from all authors 911

912

FIGURE LEGENDS 913

Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 Expression 914

Requires PAD4 EDS1 and PBS3 915

(A) Expression of GAE1 in leaves of wild-type Col-0 and mutant plants Expression was916

measured 24 or 32 h after either inoculation with Pma ES4326 (OD600 = 0002) or mock 917

inoculation with 5 mM MgSO4 and was then normalized to the level of ACTIN2 Data 918

from three biological replicates were combined using a mixed linear model Bars 919

represent mean log2-ratios to ACTIN2 plusmn SE Letters indicate significantly different 920

groups with q lt 005 where 005 represents the maximum false discovery rate at which 921

the test may be called significant 922

(B) Expression of GAE6 measured as described in (A)923

924

Figure 2 Susceptibility of gae6 and gae1 gae6 Plants to Pma ES4326 Is Increased 925

Relative to Wild-Type Col-0 926

Leaves of plants of the indicated genotypes were inoculated with Pma ES4326 (OD600 = 927

00002) as described in Methods Bacterial titers in leaves were determined either 928

Page 42 of 54

immediately (0 dpi) or after three days (3 dpi) Bars represent the mean plusmn SE of four 929

independent experiments each with 4 or 12 biological replicates at 0 and 3 dpi 930

respectively Data were combined using a mixed linear model Asterisks indicate 931

significant differences from Col-0 wild-type (q lt 005) Susceptible pad4 plants were 932

included as a positive control 933

934

Figure 3 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves 935

(A) Depiction of the assay for brittleness of gae mutant leaves Fully expanded leaves of936

four to five week-old plants were bent by 180ordm so that the adaxial surface of the leaf was 937

parallel with the soil Broken leaf petioles or midribs were considered brittle 938

(B) Quantification of broken midribs or petioles For each genotype 66 to 82 leaves 939

were analyzed for brittleness as described in (A) Bars represent the ratio of broken to 940

total number of leaves tested Results were analyzed using Fisherrsquos exact test Letters 941

indicate significantly different groups at p lt 005 942

(C) Conductivity measurements Leaves of four week-old plants were inoculated with943

deionized water and then leaf discs were cut out and placed on deionized water 944

Conductivity was measured at the indicated time points After 30 min the water was 945

replaced with fresh water (30 min f w) to account for wounding-related ion leakage from 946

the edges of the leaf discs Three independent experiments with six biological replicates 947

each were performed and data were analyzed using a t-test for each time point P-948

values were corrected using the Bonferroni method Data show means plusmn SE asterisks 949

indicate significant differences from wild-type Col-0 at q lt 001 950

951

Figure 4 Cell Walls of gae6 and gae1 gae6 Plants Are Reduced in GalA 952

(A) to (D) Levels of GalA (A) GlcA (B) cellulose (C) and neutral sugars (D) in alcohol953

insoluble residue (AIR) extracted from leaves of four week-old plants of the indicated 954

genotypes Bars represent means plusmn SE of four biological replicates combined using a 955

mixed linear model Letters indicate significantly different groups (p lt 005) 956

957

Figure 5 Cell Walls of gae1 gae6 Plants Are Reduced in Homogalacturonan 958

Page 43 of 54

Dot blot analyses with anti-LM19 and anti-LM20 antibodies Pectin was extracted from 959

AIR prepared from the indicated genotypes using 1 microL of extraction buffer per 10 microg of 960

AIR Pectin was serially diluted and one microL of undiluted 13 diluted and 19 diluted 961

samples were spotted on a nitrocellulose membrane Three biological replicates were 962

were spotted twice to obtain two technical replicates each All replicates produced 963

similar results 964

(A) Representative dot blot using anti-LM19 antibody965

(B) Relative signal intensities of the spots in the dot blot in (A)966

(C) Representative dot blot using anti-LM20 antibody967

(D) Relative signal intensities of the LM20 dot blot shown in (C)968

Relative signal intensities were measured using Image J software Measurements from 969

the three biological replicates were combined using a linear model after Box-Cox power 970

transformation as described in Methods Bars represent means plusmn SE Asterisks indicate 971

a difference from wild-type Col-0 at p lt 005 972

973

Figure 6 Growth of B cinerea on gae1 gae6 Plants and the Effect of B cinerea on 974

Expression of GAE1 and GAE6 975

(A) Growth of B cinerea isolates Leaves of Col-0 and gae1 gae6 plants were976

inoculated with 25 x 105 spores mL-1 of the B cinerea isolates indicated in the figure 977

Samples were collected two (2 dpi) and three (3 dpi) days later Bars represent means plusmn 978

SE of six biological replicates for each fungus combined using a mixed linear model 979

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 980

(B) and (C) Expression of GAE1 (B) and GAE6 (C) 48 h after inoculation with the B981

cinerea isolates indicated Expression levels were measured by qRT-PCR Bars 982

represent mean log2-ratios to ACTIN2 plusmn SE of three biological replicates combined 983

using a mixed linear model Letters indicate significantly different groups (q lt 005) 984

985

Figure 7 Levels of Soluble Pectin and Expression of PAD3 in Fractions Produced 986

Following Macerozyme Treatment of gae1 gae6 Cell Walls 987

(A) Schematic overview of the experimental procedure for (B) and (C)988

Page 44 of 54

(B) Uronic acid measurements Alcohol-insoluble residue (AIR) from leave tissue of four989

week-old wild-type Col-0 or gae1 gae6 plants was treated with water or 025 990

macerozyme and assayed for uronic acid levels Data from 11 biological replicates were 991

combined using a mixed linear model Bars represent means plusmn SE Letters indicate 992

significant differences (p lt 005) 993

(C) Expression of PAD3 in Col-0 plants treated with the fractions from (B) Extracts from994

(B) were heat inactivated pooled and diluted 5-fold with water Pooled extracts were995

inoculated into four week-old Col-0 plants PAD3 expression was measured 3 h after 996

inoculation and normalized to the level of ACTIN2 expression Data from four biological 997

replicates were combined using a mixed linear model Bars represent mean log2-ratios 998

to ACTIN2 plusmn SE Letters indicate significant differences (p lt 005) 999

1000

Figure 8 Macerozyme-Induced PAD3 Expression Is Absent in gae1 gae6 1001

(A) Expression of PAD3 after macerozyme treatment Four week-old plants were either1002

inoculated with 001 macerozyme or mock inoculated with water and PAD3 1003

expression was measured 3 h later by qRT-PCR Data from four biological replicates 1004

were combined using a mixed linear model Mean log2-ratios to ACTIN2 plusmn SE were 1005

plotted Letters indicate significantly different groups (p lt 005) 1006

(B) Expression of PAD3 after OG treatment PAD3 expression was measured as1007

described in (A) 3 h after inoculation with 100 microg mL-1 OGs or mock inoculation with 1008

water Data are from six biological replicates 1009

(C) PAD3 expression in untreated plants Three biological replicates were performed1010

and data analyzed as described in (A) 1011

(D) Camalexin accumulation after B cinerea treatment Camalexin accumulation was1012

measured in four week-old plants inoculated with the B cinerea strains shown in the 1013

figure Eight biological replicates each were combined using a mixed linear model 1014

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 1015

(E) PAD3 expression 28 h after treatment with the indicated B cinerea strains Data1016

from six biological replicates were collected and analyzed as described in (A) Data in 1017

all figure parts represent means plusmn SE Letters indicate significantly different groups (q lt 1018

005) 1019

Page 45 of 54

1020

Figure 9 Macerozyme-Induced Expression of the SA Marker Genes SID2 and PR1 1021

Is Abolished in gae1 gae6 Plants 1022

(A) to (H) Expression of PR1 (A) (C) (E) and (G) or SID2 (B) (D) (F) and (H) in four1023

week-old Col-0 and gae1 gae6 plants Data were combined using a mixed linear model 1024

Bars represent mean log2-ratios of expression vs ACTIN2 plusmn SE for all figure parts 1025

Letters indicate significantly different groups at p lt 005 for panels (A) to (D) (G) and 1026

(H) and qlt005 for panels (E) to (F) 1027

(A) and (B) Plants were treated with 001 macerozyme or mock treated with water1028

and samples were collected 3 h later Data from four biological replicates are shown 1029

(C) and (D) Plants were inoculated with 100 microg mL-1 OGs or mock treated with water1030

and samples collected 3 h later Data from six biological replicates are shown 1031

(E) and (F) Plants were inoculated with the indicated B cinerea isolates or mock1032

inoculated with B cinerea inoculation medium and samples collected 48 h later Data 1033

from eight biological replicates are shown 1034

(G) and (H) Expression in untreated plants Three biological replicates were performed1035

1036

Figure 10 The Expression of the JA Marker Gene JAZ10 is Highly Responsive in 1037

gae1 gae6 Plants 1038

(A) to (D) JAZ10 expression in four week-old plants Data were combined using a mixed1039

linear model Mean log2-ratios to ACTIN2 plusmn SE are shown Letters indicate significantly 1040

different groups at p lt 005 for panels (A) (B) and (D) and q lt 005 for panel (C) 1041

(A) Plants were inoculated with 001 macerozyme or mock inoculated using water and1042

samples were collected 3 h later Data from four biological replicates are shown 1043

(B) Plants were inoculated with 100 microg mL-1 OGs or mock inoculated using water and1044

samples collected 3 h later Data are from six biological replicates 1045

(C) JAZ10 expression 48 h after treatment with the indicated B cinerea strains Data1046

are from six biological replicates 1047

(D) JAZ10 expression in untreated plants Data are from three biological replicates1048

1049

Figure 11 Gallo 1-Induced Callose Deposition Is Reduced in gae1 gae6 Plants 1050

Page 46 of 54

(A) Callose deposition in four week-old Col-0 and gae1 gae6 plants Plants were1051

inoculated with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) or mock inoculated 1052

with B cinerea inoculation medium and samples were collected after 72 h Callose was 1053

stained with aniline blue and visualized using a Nikon Eclipse Ni-U microscope with a 1054

DAPI filter Three leaves were stained per treatment Scale bars are 100 microm Asterisks 1055

indicate fungal hyphae arrows indicate representative callose spots 1056

(B) Quantification of callose deposition Callose deposits were counted in 200 microm x 2001057

microm squares laid out across the images shown here Bars represent means plusmn SE from 1058

six squares each Letters indicate significantly different groups (p lt 005) according to a 1059

t-test analysis1060

1061

Figure 12 Less Soluble Pectin Accumulates in B cinerea-Infected gae1 gae6 1062

Leaves 1063

Four week-old Col-0 and gae1 gae6 plants were inoculated with 10 microL droplets of the 1064

indicated B cinerea isolate (25 x 105 spores mL-1) or mock inoculated with B cinerea 1065

inoculation medium Leaves were collected 48 h later AIR was extracted from leaf 1066

samples and water added (20 microL infection site) to extract soluble pectin fragments 1067

Samples were vortexed for 7 h centrifuged at 12000 g and the uronic acid 1068

concentration of the supernatant was measured Data from nine biological replicates 1069

were combined using a mixed linear model Means plusmn SE are shown Letters indicate 1070

significant differences (q lt 005) 1071

1072

Figure 13 Macerozyme-Induced PTI Is Lost in gae1 gae6 While OG-Induced PTI Is 1073

Unaltered 1074

(A) Macerozyme-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1075

inoculated with 001 macerozyme or mock inoculated with boiled macerozyme and 1076

were infected with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) 24 h later Samples 1077

were collected immediately (0 dpi) and after two (2 dpi) and three (3 dpi) days Data 1078

show mean plusmn SE of six to 14 biological replicates each combined using a linear model 1079

Letters indicate significantly different groups (q lt 005) 1080

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

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Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk RGadrinab C Heller C Jeske A Koesema E Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt MHazari N Hom E Karnes M Mulholland C Ndubaku R Schmidt I Guzman P Aguilar-Henonin L Schmid M Weigel DCarter DE Marchand T Risseeuw E Brogden D Zeko A Crosby WL Berry CC and Ecker JR (2003) Genome-wideinsertional mutagenesis of Arabidopsis thaliana Science 301 653-657

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Asai T Tena G Plotnikova J Willmann MR Chiu WL Gomez-Gomez L Boller T Ausubel FM and Sheen J (2002) MAPkinase signalling cascade in Arabidopsis innate immunity Nature 415 977-983

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Bartsch M Gobbato E Bednarek P Debey S Schultze JL Bautor J and Parker JE (2006) Salicylic acid-independentENHANCED DISEASE SUSCEPTIBILITY1 signaling in Arabidopsis immunity and cell death is regulated by the monooxygenaseFMO1 and the nudix hydrolase NUDT7 Plant Cell 18 1038-1051

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Bates D Machler M Bolker BM and Walker SC (2015) Fitting Linear Mixed-Effects Models Using lme4 J Stat Softw 67 1-48Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Benjamini Y and Hochberg Y (1995) Controlling the False Discovery Rate - a Practical and Powerful Approach to MultipleTesting J Roy Stat Soc B Met 57 289-300

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Bethke G Grundman RE Sreekanta S Truman W Katagiri F and Glazebrook J (2014) Arabidopsis PECTINMETHYLESTERASEs Contribute to Immunity against Pseudomonas syringae Plant Physiol 164 1093-1107

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Bouton S Leboeuf E Mouille G Leydecker MT Talbotec J Granier F Lahaye M Hofte H and Truong HN (2002)Quasimodo1 encodes a putative membrane-bound glycosyltransferase required for normal pectin synthesis and cell adhesion inArabidopsis Plant Cell 14 2577-2590

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Box GEP and Cox DR (1964) An analysis of transformations Journal of the Royal Statistical Society Series B 26 211-252Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Cao H Glazebrook J Clarke JD Volko S and Dong XN (1997) The Arabidopsis NPR1 gene that controls systemic acquiredresistance encodes a novel protein containing ankyrin repeats Cell 88 57-63

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Carpita N (2010) Update on Mechanisms of Plant Cell Wall Biosynthesis How plants make cellulose and other (1-gt4)-beta-D-glycans Plant Physiol

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Chisholm ST Coaker G Day B and Staskawicz BJ (2006) Host-microbe interactions Shaping the evolution of the plantimmune response Cell 124 803-814

Page 11 of 54

Cell Walls of gae6 and gae1 gae6 Plants Have Lower Levels of Pectin 243

GalA is a major component of pectin We analyzed wall monosaccharide 244

composition in gae mutants to investigate whether the increased brittleness was 245

associated with an altered wall composition of gae leaves To approximate pectin 246

content of gae mutant walls the total uronic acid concentration was measured using a 247

simple colorimetric assay (Filisetti-Cozzi and Carpita 1991 van den Hoogen et al 248

1998) Total uronic acid was reduced in gae6-2 strongly reduced in gae1 gae6 plants 249

but was indistinguishable from wild-type Col-0 in gae6-1 and gae1 (Supplemental 250

Figure 5) This suggested that gae1 gae6 and possibly gae6 plants have reduced 251

pectin 252

To investigate the cell wall composition in leaves of gae plants in more detail we 253

measured the levels of seven neutral monosaccharides the acidic sugars GalA (the 254

monomeric subunit of the pectin backbone (Harholt et al 2010)) and GlcA (Figure 4) 255

We also estimated the crystalline cellulose content (Updegraff 1969) Cell walls of both 256

gae6 mutants and gae1 gae6 were reduced in GalA (Figure 4A) In gae1 gae6 GalA 257

content in cell walls was reduced by more than 40 compared to the wild-type level 258

GlcA levels were indistinguishable between mutants and wild-type (Figure 4B) 259

Cellulose content was higher in gae6-2 and gae1 gae6 walls likely due to the loss of 260

pectins (Figure 4C) Cell walls of gae1 gae6 plants were also enriched in all neutral 261

sugars measured presumably due to the substantial proportional loss of GalA (Figure 262

4D) The levels of most wall monosaccharides were even higher in the gae1 gae6 263

double mutant with the exception of Rha which did not exhibit a significant increase 264

compared to the gae6 single mutants The absence of a compensational increase in 265

Rha suggested that RGI the dominant Rha contributor to the wall may be considered 266

reduced in gae1 gae6 mutants compared to wild-type albeit not to such an extent as 267

HG 268

238 structural defect that promotes increased ion leakage Taken together these results

239 showed that loss of GAE6 causes brittle leaves and additional losss of GAE1 strongly

240 enhances this phenotype We conclude that GAE1 and GAE6 contribute to normal leaf

241 flexibility 242

Page 12 of 54

gae6 cell walls are reduced in HG Using the same samples we detected no difference 274

from wild type in binding of LM5 which recognizes a tetramer of (1-4)-β-D-galactans 275

found in RGI (Jones et al 1997) LM6 which recognizes a linear pentasaccharide in (1-276

To further investigate which pectic components were altered in gae1 gae6 dot 269

blot experiments using pectin isolated from the various genotypes were performed Both 270

LM19 and LM20 antibodies recognize HG (Verhertbruggen et al 2009a) As expected 271

LM19 binding was reduced in gae6-1 and gae1 gae6 (Figure 5A and B) and LM20 272

binding was reduced in gae1 gae6 (Figure 5C and D) This result confirmed that gae1 273

277 5)-α-L-arabinans found in RGI (Willats et al 1998 Verhertbruggen et al 2009b) LM8

278 which recognizes xylogalacturonan (Willats et al 2004) or CCRC-M7 which

279 recognizes an arabinosylated (1-6)-β-D-galactan epitope occurring on RGI (Puhlmann et

Page 13 of 54

Page 14 of 54

al 1994 Steffan et al 1995 Pattathil et al 2010) (Supplemental Figure 6) 280

Collectively these cell wall composition data show that gae1 gae6 plants are reduced in 281

HG Due to the strong proportional reduction of HG in the walls levels of all other 282

components should be higher However Rha indicative of RGI was not higher in gae1 283

gae6 suggesting that RGI may also be reduced in gae1 gae6 walls 284

285

GAE1 and GAE6 Are Required for Immunity to Specific B cinerea Isolates 286

We hypothesized that plant immunity to B cinerea might be affected by pectin 287

content in the host cell wall because this pathogen requires polygalacturonases and 288

pectin methylesterases for full virulence (ten Have et al 1998 Valette-Collet et al 289

2003 Kars et al 2005b Espino et al 2010) Because Bcinerea is genetically diverse 290

and has a high diversity of polygalacturonases (Rowe and Kliebenstein 2007) we 291

measured relative fungal growth at both 2 and 3 d post-inoculation (dpi) using ten 292

different B cinerea isolates (Figure 6) The B cinerea isolates Fresa 525 and Gallo 1 293

showed enhanced virulence on gae1 gae6 plants at both time points whereas KT and 294

UK Razz showed enhanced virulence in gae1 gae6 plants at 3 but not 2 d dpi (Figure 295

6A) We did not detect any significant changes in relative fungal growth between wild-296

type Col-0 and gae1 gae6 plants when using Acacia Apple 517 DN Grape Pepper or 297

Rasp isolates of B cinerea Therefore we continued our study using Gallo 1 and Fresa 298

525 We also included Pepper for comparison as this isolate has been used in a large-299

scale analysis of plant gene expression in response to B cinerea infection (Windram et 300

al 2012) 301

We next investigated whether expression of GAE1 and GAE6 was affected by 302

challenge with B cinerea Inoculation of wild-type Col-0 plants with any isolate 303

repressed GAE1 and GAE6 expression (Figure 6B and C) and this repression was 304

generally independent of PAD4 (Figure 6B and C) Expression of GAE2 and GAE3 was 305

unaltered by B cinerea treatment GAE4 was repressed only by Fresa 525 treatment 306

while GAE5 expression was enhanced with all three isolates tested (Supplemental 307

Figure 7) As seen for infection with Pma ES4326 the expression of GAE1 and GAE6 308

was repressed upon B cinerea treatment This indicates a role for GAE1 and GAE6 in 309

the immune response to B cinerea 310

Page 15 of 54

Page 16 of 54

Because the amount of pectin substrate for pathogen polygalacturonases is 313

reduced we hypothesized that immunity phenotypes of gae1 gae6 plants may result 314

from reduced generation of OGs by these enzymes In turn the reduced OGs could 315

lead to reduced immune responses To test this we used macerozyme a commercial 316

pectinase as a proxy for pathogen pectinases We isolated cell walls as a subcellular 317

fraction known as alcohol insoluble residue (AIR Gille et al 2009) This was treated 318

with macerozyme and tested for release of soluble likely low molecular weight pectin 319

fragments (Figure 7) Treatment with 025 macerozyme released significantly more 320

uronic acid from wild-type Col-0 AIR relative to gae1 gae6 AIR (Figure 7B) To test 321

activity of these pectin-containing solutions we infiltrated them into wild-type Col-0 322

plants and measured PAD3 expression PAD3 is required for synthesis of the 323

phytoalexin camalexin (Zhou et al 1999) and its expression is known to be induced by 324

OG treatment (Ferrari et al 2007) Fractions from macerozyme treated Col-0 walls 325

induced significantly more PAD3 expression than fractions derived from gae1 gae6 326

walls suggesting that macerozyme treatment did indeed release more active soluble 327

uronic acid possibly OGs from wild-type Col-0 walls than from gae1 gae6 walls (Figure 328

7C) 329

Next we studied the effect of macerozyme treatment on expression of PAD3 in 330

intact plants (Figure 8) This treatment did not lead to any visible leaf damage 331

(Supplemental Figure 8) In wild-type Col-0 PAD3 expression was induced three h after 332

treatment with 001 macerozyme (Figure 8A) By contrast no macerozyme-induced 333

induction of PAD3 expression was detected in gae1 gae6 (Figure 8A) However OG 334

treatment induced the expression of PAD3 in both genotypes indicating that gae1 gae6 335

plants do respond to OGs (Figure 8B) Macerozyme treatment also induced expression 336

of the SA marker genes PATHOGENESIS RELATED 1 (PR1) and SID2 in Col-0 but 337

not in gae1 gae6 (Figure 9 panel A and B) PR1 was not significantly induced by 338

purified OGs (Figure 9C) at the time and concentration tested but SID2 was induced by 339

OG treatment in both genotypes (Figure 9D) However the induction was stronger in 340

wild-type Col-0 In addition the JA markers JASMONATE-ZIM-DOMAIN PROTEIN 5 341

(JAZ5) and JAZ10 were induced by macerozyme and OG treatment in both Col-0 and 342

gae1 gae6 (Figure 10A and Supplemental Figure 11) Interestingly expression of JAZ5 343

312 Pectinase-Induced Immune Signaling Is Altered in gae1 gae6 Plants

Page 17 of 54

Page 18 of 54

Page 19 of 54

Page 20 of 54

344

345

346

and JAZ10 was already strongly induced in gae1 gae6 after mock inoculation (Figure 10

and Supplemental Figure 11) We observed no hyperresponsive induction by mock

treatment of PLANT DEFENSIN 12 (PDF12) a necrotrophy and late JA signaling

347 marker gene (Supplemental Figure 12A-D) PDF12 was also not induced by

348 macerozyme or OG treatments (Supplemental Figure 12A-D) This suggests that early

349 JA signaling is induced by mock-treatment in gae1 gae6 Because it is known that JA

350 and SA signaling act antagonistically (Pieterse et al 2012) the strong up-regulation of

351 JA-induced immune signaling in gae1 gae6 might explain the lower OG-induced SID2

352 expression in this genotype

353 To test whether other immune responses are altered in gae1 gae6 plants we

354 analyzed Mitogen-activated protein kinase (MAPK) activity following macerozyme and

355 OG treatments OGs are known to activate the MAPKs MPK3 and MPK6 in Arabidopsis

Page 21 of 54

(Galletti et al 2011) Macerozyme treatment activated MPK3 and MPK6 in wild-type 356

Col-0 whereas MAPK activation in gae1 gae6 was slightly reduced in four out of five 357

experiments performed (Supplemental Figure 13) By contrast OG-induced MAPK 358

activity was similar or increased in gae1 gae6 in four out of five experiments 359

(Supplemental Figure 13) Additionally mock treatment induced MAPK activity in gae1 360

gae6 more strongly than it did in wild-type Col-0 in four out of five experiments 361

performed (Fig S13) Collectively these results illustrate that activation of several 362

immune signaling componentsmdashcamalexin synthesis SA and JA signaling and 363

possibly MAPK activationmdashby macerozyme is substantially altered in gae1 gae6 This 364

suggests that release of soluble pectin fragments possibly OGs by pectinolytic 365

enzymes might be reduced in gae1 gae6 plants while recognition of OGs is intact 366

367

Following B cinerea Infection Production of Soluble Pectin and Deposition of 368

Callose Are Reduced in gae1 gae6 Plants 369

Although pad3 plants were more susceptible to the B cinerea isolates Fresa 525 370

and Gallo 1 which also grew better on gae1 gae6 growth of the isolate Pepper was 371

similar on Col-0 and pad3 (Supplemental Figure 9A) However we did not detect any 372

significant changes in camalexin production after treatment with B cinerea isolate Gallo 373

1 and Fresa 525 in gae1 gae6 (Figure 8D) Neither did we detect changes in PAD3 374

expression after treatment with any B cinerea isolate in gae1 gae6 (Figure 8E) 375

Additionally SID2 and PR1 were similarly induced by all B cinerea isolates in both Col-376

0 and gae1 gae6 (Figure 9E and F) and sid2 plants which are deficient for pathogen-377

induced SA biosynthesis did not show any differences in relative B cinerea growth 378

(Supplemental Figure 9B) Expression of JAZ10 was induced more strongly by Gallo 1 379

treatment in gae1 gae6 than in wild-type Col-0 but not by Fresa 525 or Pepper (Fig 380

10C) while dde2 plants deficient for JA biosynthesis showed enhanced susceptibility to 381

all B cinerea isolates tested (Supplemental Figure 9B) Curiously exogenous 382

application of MeJA did not lead to altered relative fungal growth of B cinerea 383

(Supplemental Figure 9C) and cev1 isoxaben resistant 1 (ixr1) plants which have 384

constitutively high JA levels (Ellis and Turner 2001) did not show any changes in 385

susceptibility to the B cinerea isolates tested (Supplemental Figure 9D) Hence neither 386

Page 22 of 54

differences in B cinerea isolate susceptibility to camalexin nor altered SA or JA 387

signaling by themselves explained the enhanced susceptibility of gae1 gae6 to these B 388

cinerea isolates 389

Hydrogen peroxide accumulation in response to B cinerea isolates Fresa 525 390

Gallo 1 and Pepper was not obviously altered in gae1 gae6 plants (Supplemental Figure 391

14) However callose deposition upon treatment with B cinerea isolate Gallo 1 was 392

strongly reduced in gae1 gae6 (Figure 11) It was previously shown that OG treatment 393

induces callose deposition (Denoux et al 2008) suggesting that the reduction in 394

callose deposition might be due to reduced OG release in gae1 gae6 It is possible that 395

this reduction in callose deposition contributes to the reduced immunity of gae1 gae6 396

plants to B cinerea isolate Gallo 1 397

Because macerozyme could be used to release soluble likely low molecular 398

weight pectin from isolated cell walls we next investigated whether infection with B 399

cinerea was accompanied by accumulation of soluble pectin as well We prepared cell 400

walls (AIR fraction) from leaves infected with B cinerea incubated these cell walls with 401

water and measured uronic acid content of the extracts B cinerea treatment did in fact 402

lead to increased accumulation of soluble pectin in wild-type Col-0 plants when 403

compared to mock-treated samples (Figure 12) No such increased accumulation of 404

water-soluble pectin fractions was observed in gae1 gae6 (Figure 12) 405

406

Macerozyme-Induced Pattern-Triggered Immunity against B cinerea Is Abolished 407

in gae1 gae6 408

It was previously shown that OG treatment can induce patternndashtriggered 409

immunity (PTI) to B cinerea (Ferrari et al 2007) We tested macerozyme treatment to 410

determine whether it could also induce PTI to the B cinerea isolate Gallo 1 (Figure 13) 411

Indeed pretreatment with 001 macerozyme for 24 h led to reduced B cinerea growth 412

in wild-type Col-0 but not gae1 gae6 plants (Figure 13A) Interestingly mock and 413

macerozyme pretreated gae1 gae6 plants exhibited similar growth of B cinerea Gallo 1 414

as did macerozyme-treated Col-0 plants This result is likely due to activation of JA 415

signaling in gae1 gae6 during the pretreatment procedure (Figure 13C and D) OG-416

induced PTI was observed in both genotypes when using a high dose of OGs (Figure 417

Page 23 of 54

13B) whereas a lower dose of OGs only induced PTI in Col-0 (Supplemental Figure 418

15) This suggests that OG perception in gae1 gae6 at least above a certain threshold 419

is unaltered while release of immunity-activating pectin fragments possibly OGs is 420

421 reduced We conclude that immune responses induced by macerozyme treatment

422 which do not occur in gae1 gae6 plants are important for plant immunity to the B

423 cinerea isolate Gallo 1

Page 24 of 54

____________________________________________________________________________

Page 25 of 54

DISCUSSION 426

Here we showed that loss of GAE1 and GAE6 leads to a dramatic reduction in 427

pectin content in the cell wall (Figure 4) likely causing the brittleness of leaves that we 428

observed (Figure 3) Petioles and midribs of gae1 gae6 plants broke easily when bent 429

and inoculation of leaves with deionized water resulted in increased ion leakage 430

possibly due either to microscopic fractures of the cell wall induced by inoculation stress 431

or to a reduced capability of these walls to withstand increased turgor pressure The 432

increased brittleness was associated with altered cell wall composition In our analysis 433

the GalA content of gae1 gae6 leaf walls was reduced by more than 40 and the total 434

uronic acids by over 60 (Figure 4 and Supplemental Figure 5) This is a major change 435

in the overall cell wall composition considering that pectin makes up about 50 of the 436

plant cell walls found in Arabidopsis leaves (Zablackis et al 1995 Nobuta et al 2007 437

Harholt et al 2010) Interestingly quasimodo1 (qua1) plants which have a mutation in 438

a glycosyltransferase family 8 gene are reduced in GalA by 25 and show reduced 439

binding of HG specific antibodies (Bouton et al 2002) 440

Furthermore qua2 plants which have a mutation in a putative methyltransferase 441

have about 13 less total GalA and lack 50 of cell wall HG (Mouille et al 2007) 442

Likewise cgr2 cgr3 plants carrying mutations in another class of Golgi-localized 443

putative methyl-transferases have leaf cell walls that are reduced by about 40 in 444

uronic acid (Kim et al 2015) All three mutants are severely dwarfed (Bouton et al 445

2002 Mouille et al 2007 Kim et al 2015) Additionally qua1 and qua2 mutants show 446

a distorted overall shape likely due to reduced cell cohesion and are extremely fragile 447

(Bouton et al 2002 Mouille et al 2007) By contrast gae1 gae6 plants with a similar 448

strong reduction in GalA and total uronic acids have an overall normal shape and are 449

only slightly smaller than wild-type Col-0 (Supplemental Figure 4) In the wall analysis 450

performed here we did not detect changes in other wall polysaccharides or structures 451

that might compensate for the reduction of pectin in gae1 gae6 It is possible the severe 452

morphological alterations in qua2 and cgr2 cgr3 plants are due to reduction in pectin 453

methylesterification which is unaffected in gae1 gae6 It was speculated that quality 454

control mechanisms in the Golgi prevent secretion of inefficiently esterified pectin in 455

cgr2 cgr3 (Kim et al 2015) suggesting that the Golgi of these plants might over-456

Page 26 of 54

accumulate pectin and that this may contribute to the severe dwarf phenotype of these 457

plants The gae1 gae6 plants described here provide an opportunity to study the effects 458

of reduction of pectin in plants that do not show severe morphological phenotypes 459

We found that gae1 gae6 plants are also compromised in resistance to B 460

cinerea and Pma ES4326 indicating that loss of pectin reduces immunity to these 461

pathogens (Figure 2 and 6) We found that gae1 gae6 plants were slightly more 462

susceptible to Pma ES4326 but showed a strong increase in susceptibility to specific B 463

cinerea isolates This was not unexpected because B cinerea is known to interact with 464

pectin as part of its infection strategy It secretes various pectin-degrading enzymes 465

early during infection and requires some of these enzymes including PG1 PG2 and 466

PME1 for full virulence (ten Have et al 1998 Valette-Collet et al 2003 Kars et al 467

2005b Espino et al 2010) Additionally Arabidopsis plants expressing an antisense 468

construct of the polygalacturonase inhibitor PGIP1 are significantly more susceptible to 469

B cinerea (Ferrari et al 2006) On the other hand Pma ES4326 is not known to470

require cell wall degrading enzymes for full virulence even though it encodes two 471

pectate lyase genes in its genome (PMA4326_07094 and PMA4326_04621 472

wwwbacteriaensemblorg) B cinerea isolates are also known to be genetically quite 473

diverse in their polygalacturonase (PG) loci and genetic variability in PG2 is associated 474

with varying growth on pectin containing media (Rowe and Kliebenstein 2007) 475

Furthermore the contribution of PG1 and PG2 to B cinerea virulence seems to depend 476

on the specific B cinerea isolate and the host species (Kars et al 2005a Zhang and 477

Van Kan 2013) B cinerea isolates are also known to be differently sensitive to the anti-478

microbial secondary metabolite camalexin (Kliebenstein et al 2005) In line with these 479

observations the susceptibility of gae1 gae6 plants differed depending on the specific 480

B cinerea isolate Whereas Fresa 525 Gallo 1 UK Razz and KT grew better in gae1481

gae6 we detected no difference in Acacia Apple 517 DN Grape Pepper or Rasp 482

growth (Figure 6) 483

B cinerea was shown to metabolize pectin and to possibly utilize GalA as a484

carbon source during in planta growth (Zhang et al 2011 Zhang and Van Kan 2013) 485

Our data show that gae1 gae6 plants which are reduced in GalA content did allow 486

enhanced growth of specific B cinerea isolates but no reduction in growth of any B 487

Page 27 of 54

cinerea isolates tested was found This suggests that utilization of pectin as a carbon 488

source was not a contributing factor to alterations in B cinerea susceptibility It is 489

possible that changes in the cell wall composition affect the structural integrity of the 490

wall and affect hyphal penetration of specific B cinerea isolates However OGs (small 491

HG fragments) are likely released during the infection process (Ferrari et al 2013) 492

OGs function as DAMPs and initiate immune responses in Arabidopsis (Ferrari et al 493

2013) Treatment of Arabidopsis with OGs having a degree of polymerization (DP) of 494

10-15 sugar subunits was previously shown to enhance resistance to B cinerea (Ferrari495

et al 2007) We showed that pretreatment with OGs or macerozyme induced enhanced 496

immunity to B cinerea isolate Gallo 1 in wild-type Col-0 but only OGs and not 497

macerozyme induced immunity in gae1 gae6 (Figure 13) Our data suggest gae1 gae6 498

plants which have reduced pectin content may release less OGs and that this 499

reduction leads to the increased susceptibility to B cinerea 500

Consistently we found that macerozyme treatment released less soluble pectin 501

from gae1 gae6 cell walls than from wild-type walls Fractions containing soluble pectin 502

released from gae1 gae6 were also less active in inducing PAD3 gene expression than 503

the fractions released from wild-type walls (Figure 7) We also showed that less soluble 504

pectin accumulated in gae1 gae6 than in wild-type plants 48 h after treatment with B 505

cinerea (Figure 12) 506

We observed that loss of pectin leads to alterations in A thaliana immune 507

signaling Macerozyme treatment induced expression of PAD3 and the SA marker 508

genes PR1 and SID2 in wild-type Col-0 but not in gae1 gae6 whereas OG treatment 509

led to increased PAD3 and SID2 expression in both wild-type and gae1 gae6 (Figure 8 510

and 9) This suggests that although release of active soluble pectin in these plants 511

might be altered OG recognition is intact In fact cell walls isolated from qua1 another 512

mutant known to be reduced in HG released less OGs (DP1-6) than did wild-type cell 513

walls upon endopolygalacturonase treatment (Bouton et al 2002) This was also true 514

for gae1 gae6 cell walls as macerozyme treatment of isolated gae1 gae6 cell walls 515

released less soluble pectin than treatment of Col-0 cell walls Because the fraction 516

from gae1 gae6 plants was also less active in inducing PAD3 expression (Figure 7) it 517

seems possible that biologically active OGs were the active component inducing PAD3 518

Page 28 of 54

expression Interestingly expression of the JA marker genes JAZ5 and JAZ10 was 519

higher in gae1 gae6 than in wild-type mock-inoculated plants but was comparable to 520

wild-type in untreated plants suggesting that JA signaling in gae1 gae6 is 521

hyperresponsive (Figure 10 and Supplemental Figure 11) This induction of JA signaling 522

might be due to wounding stress incurred during the inoculation process JA and SA 523

signaling generally act antagonistically (Pieterse et al 2012) Increased JA signaling 524

activity might hence explain why mock- and macerozyme-treated gae1 gae6 plants 525

exhibited reduced expression of the SA marker PR1 and why OG treatment induced 526

expression of SID2 another SA marker to a lower extent in gae1 gae6 than in wild-type 527

plants (Figure 9) 528

No single immune signaling component was found to be the sole contributor to 529

the compromised immunity of gae1 gae6 to specific B cinerea isolates such as Gallo 1 530

and Fresa 525 These isolates grew better on pad3 while growth of Pepper another B 531

cinerea isolate that did not show altered virulence on gae1 gae6 was unaltered 532

(Supplemental Figure 9) This suggested that differences in camalexin sensitivity of 533

these B cinerea isolates might account for the differences in gae1 gae6 susceptibility 534

However camalexin accumulation and PAD3 expression was the same for wild-type 535

Col-0 and gae1 gae6 for the B cinerea isolates Fresa 525 and Gallo 1 (Figure 8) which 536

showed enhanced growth on gae1 gae6 plants It should be noted that B cinerea 537

virulence is often estimated by comparing lesion sizes Lesion size and measurements 538

of relative fungal growth by qPCR do not always show the same results For example 539

although Denby et al (2004) showed that Pepper causes larger lesions on pad3 than on 540

wild-type this study showed that relative fungal growth of Pepper on pad3 was 541

unaltered 542

We observed that callose deposition was strongly reduced in gae1 gae6 plants 543

72 h after treatment with B cinerea isolate Gallo 1 (Figure 11) possibly due to reduced 544

release of active soluble pectin Interestingly OG-induced PTI to an unspecified isolate 545

of B cinerea was intact in the pmr4 mutant which does not produce callose (Galletti et 546

al 2008) Due to the high SA levels in pmr4 plants (Nishimura et al 2003) and the fact 547

that different B cinerea isolates vary both in induction of and reaction to defense 548

responses (Kliebenstein et al 2005 Rowe et al 2010) we could not determine 549

Page 29 of 54

whether reduced callose deposition causes the increased susceptibility of gae1 gae6 to 550

B cinerea isolate Gallo 1551

We did not observe any changes in B cinerea growth either on cev1 ixr1 plants 552

which are known to have constitutively high JA and OPDA levels (Ellis et al 2002b) or 553

on plants exogenously treated with MeJA (Supplemental Figure 9) Because plants 554

were kept uncovered for 3 h after MeJA treatment it is unclear how much MeJA was 555

bioavailable in the plant However JAZ10 expression was induced 3 h after spraying 556

with MeJA (Supplemental Figure 10) Because pathogen growth generally depends on 557

the combination of a multitude of immune signaling events we conclude that multiple 558

changes to the immune signaling in gae1 gae6 together account for the altered B 559

cinerea growth phenotypes detected 560

We showed that expression of GAE1 and GAE6 is repressed by the bacterial 561

hemibiotroph Pma ES4326 This repression did not occur in an eds1 or pad4 562

background or in pbs3 at the 24-h time point (Figure 1 and Supplemental Figure 1) 563

During the Arabidopsis - Pma ES4326 interaction the expression of many defense 564

related genes is altered in a PAD4EDS1 dependent fashion (Wang et al 2008) Some 565

of these expression changes require SA CBP60g and SARD1 are thought to act 566

downstream of PAD4 and EDS1 but upstream of SA-dependent responses (Wang et 567

al 2011) PBS3 is also thought to act downstream of PAD4 and EDS1 and upstream of 568

SA but the set of genes whose expression it affects is only partially overlapping with 569

those affected by CBP60gSARD1 (Wang et al 2008 Wang et al 2011) Thus GAE1 570

and GAE6 repression is regulated by the part of the immune signaling network that 571

requires PAD4 EDS1 and PBS3 but is independent of CBP60gSARD1 and SA 572

Because EDS1 PAD4 and PBS3 are important components of the plant immune 573

signaling network (Wiermer et al 2005 Nobuta et al 2007) one might conclude that 574

repression of GAE1 and GAE6 may be part of the plant immune response to Pma 575

ES4326 B cinerea treatment also reduced GAE1 and GAE6 expression but this was 576

mostly independent of PAD4 (Figure 6) while both Pma ES4326 and B cinerea repress 577

GAE1 and GAE6 expression the host signaling mechanisms responsible likely differ 578

In summary our data show that Arabidopsis GAE1 and GAE6 are required for 579

pectin biosynthesis leaf flexibility and immunity to Pma ES4326 and to specific B 580

Page 30 of 54

cinerea isolates We used macerozyme a commercial pectinase to mimic the effect of 581

B cinerea pectinases Macerozyme treatment released less pectin from gae1 gae6 cell582

walls and induced less immune signaling in gae1 gae6 plants Less water soluble pectin 583

also accumulated in B cinerea infected gae1 gae6 plants and macerozyme-induced 584

immunity to B cinerea isolate Gallo 1 was reduced in these plants We conclude that 585

the reduced pectin content in these mutants may lead to reduced release of soluble 586

pectin including active OGs during pathogen attack and that this compromises 587

immunity 588

589

590

Page 31 of 54

METHODS 591

Plant Materials and Growth Conditions 592

Wild-type Columbia (Col-0) and all mutant plants (in Col-0 background) were 593

grown on sterilized BM2 germinating mix (Berger Inc Quebec Canada) in a controlled 594

environment chamber (Conviron Pembina ND USA) with a 12-h photoperiod under 595

100 microM m-2 s-1 fluorescent illumination at 22degC and 75 relative humidity Germplasm 596

used is described in the Accession Numbers section at the end of Methods 597

598

Data Analysis and Replicates 599

For biological replicates samples were obtained from separate plant tissues and 600

in many cases from plants grown in different flats In some experiments multiple 601

technical replicates were measured per biological replicate For statistical analysis all 602

technical replicates were averaged and the averages were considered one biological 603

replicate Independent experiments were performed at different times and often 604

consisted of multiple biological replicates per experiment 605

Data analysis was performed using mixed linear models in the R programming 606

environment (Bates et al 2015) unless otherwise indicated Generally genotypes 607

treatments and time points were used as fixed effects and replicate-specific effects like 608

different pots flats etc were considered random effects We reported p-values 609

obtained from the mixed linear model when less than 10 comparisons were performed 610

and q-values for more than 10 comparisons to correct for multiple comparison testing 611

Q-values were calculated using the Benjamini-Hochberg procedure (Benjamini and612

Hochberg 1995) unless specified otherwise 613

614

Pathogen Strains Growth Conditions and Pathogen Growth Assays 615

Growth of Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) and 616

bacterial growth assays were performed as previously described (Bethke et al 2014) 617

For qRT-PCR assays four week-old plants were inoculated with bacteria (OD600 = 618

0002) or mock (5 mM MgSO4) At least four leaves from two plants were collected at 619

the indicated time points per biological replicate 620

Page 32 of 54

Prior to each experiment Botrytis cinerea isolates were grown on 1x PDA 621

medium (Difco) for 10 d at room temperature All isolates used have been described 622

previously (Rowe and Kliebenstein 2007) The spores were washed from the surface of 623

the plate using an inoculation medium (1x Gamborgrsquos B-5 basal salt mixture (Sigma-624

Aldrich G5768) 2 (wv) glucose 10 mM phosphate buffer pH 64) and fungal hyphae 625

were removed from the suspension by filtering through four layers of cheesecloth The 626

concentration of spores was determined using a hemocytometer and adjusted to 25 x 627

105 spores mL-1 Four week-old Arabidopsis plants were inoculated by placing one 10-628

microL droplet of the B cinerea spore solution or inoculation medium (mock) on the adaxial 629

leaf surface of fully expanded leaves Inoculated plants were kept at 100 relative 630

humidity Infection sites were collected at various time points using a cork borer For 631

qRT-PCR assays at least four leaf discs from one plant were combined for each 632

biological sample For B cinerea growth assays at least 12 leaf discs from three to six 633

plants were pooled per biological sample Relative fungal growth was determined by 634

measuring the abundance of a fungal gene relative to a plant gene as described below 635

In brief DNA from infected tissue was extracted and equal amounts of total DNA were 636

used to perform qPCR reactions using the B cinerea cutinase A and the Arabidopsis 637

SK11 gene respectively Primers were described in (Gachon and Saindrenan 2004) 638

and are listed in Supplemental Table 1 Two technical replicates were combined for 639

each biological replicate B cinerea is a necrotrophic fungal pathogen and may destroy 640

plant DNA as disease progresses However decreases in the amount of plant DNA 641

over the course of our experiments were only observed at late time points with very 642

susceptible plants so the qPCR assay is generally a reasonable estimate of fungal 643

biomass (Supplemental Figure 16) 644

645

Treatment with Macerozyme or OGs 646

For PTI assays four week-old plants either were inoculated with 001 (wv) 647

macerozyme R-10 (Yakult) a Rhizopus polygalacturonase (EC 32115) that possesses 648

high pectinase and hemicellulase activity or were mock inoculated using heat-649

inactivated macerozyme Other similar plants were either inoculated with 250 or 500 microg 650

mL-1 OGs or mock inoculated with water One d later plants were infected with B 651

Page 33 of 54

cinerea and samples were collected immediately (0 dpi) and two or three d later To 652

investigate the role of MeJA on B cinerea growth plants were sprayed with 1mM MeJA 653

in water with 001 (vv) Tween 20 Covers were removed so that leaves could dry off 654

before plants were inoculated with B cinerea three h later Samples were harvested at 655

the indicated time points For expression analysis and MAPK activation assays plants 656

were either inoculated with 100 microg mL-1 OGs or mock inoculated with water OGs used 657

for experiments described in Figures 8 to 10 and Supplemental Figure 12 were a kind 658

gift from Simone Ferrari OGs used in experiments described in Figure 13 and 659

Supplemental Figures 11 13 and 15 were prepared as previously described (Kohorn et 660

al 2014) 661

662

Expression Analysis 663

Leaf tissue from inoculated plants was harvested at the indicated time points 664

flash frozen pulverized and RNA extracted using Trizol (Invitrogen) Quantitative RT-665

PCR was performed using the SuperScript III Platinum SYBR Green One-Step 666

quantitative RT-PCR kit (Invitrogen) and a Lightcycler 480 Real-Time PCR system 667

(Roche) as previously described (Truman and Glazebrook 2012) In brief equal 668

amounts of total RNA and a gene specific primer were used for each reaction The 669

crossing point (Cp) was calculated using the 2nd derivative max method provided with 670

the Lightcycler software for each amplification curve Each reaction was run with two 671

technical replicates and the Cp values for these replicates were averaged ACTIN2 was 672

used as a stably expressed reference gene At least three biological replicates each 673

were performed and analyzed using a mixed-linear model RT-PCR was performed to 674

verify T-DNA insertion lines using the One-Step RT-PCR kit (Qiagen) according to the 675

manufacturerrsquos instructions The primers used can be found in Supplemental Table 1 676

677

Electrolyte Leakage Assay 678

Fully expanded leaves of four week-old plants were inoculated with water (Milli-Q 679

grade) using a needleless syringe Leaf discs were collected using a cork borer and 680

three leaf discs from one leaf were placed with the adaxial surface down onto 2 mL 681

water in a 12-well cell culture plate Conductivity was measured using a Horiba B-173 682

Page 34 of 54

conductivity meter after 30 min and then the water was replaced Conductivity was 683

measured again 30 min later (1 h total) as indicated in Figure 3C Six biological 684

replicates each were performed in three independent experiments and means and SEs 685

were calculated Two-sided t-tests were performed for each time point 686

687

DAB and Aniline Blue Staining 688

Leaves from four week-old plants were either mock inoculated with B cinerea 689

inoculation medium or inoculated with 10 microL droplets of a B cinerea spore solution (25 690

x 105 spores mL-1) of the indicated isolates Samples were collected 72 h later For 33prime-691

diaminobenzidine (DAB) staining two to five inoculated leaves were vacuum infiltrated 692

with 33prime-Diaminobenzidine (1 mg mL-1 in water pH 38) solution and stained overnight 693

in the dark Leaves were destained using 100 ethanol at 65degC and then transferred to 694

50 (vv) glycerol and photographed 695

Callose was visualized by staining leaves with aniline blue as previously 696

described (Adam and Somerville 1996) Briefly leaves were vacuum infiltrated with 697

alcoholic lactophenol (one volume of phenolglycerollactic acidwater (1111) mixed 698

with two volumes of ethanol) and destained at 65degC Next leaves were moved through 699

an alcohol gradient (50 20 and 10 (vv) ethanol) into water The destained leaves 700

were stained for 30 min in 150 mM K2HPO4 (pH 95) with 001 (wv) aniline blue 701

Samples were mounted in water and examined using a Nikon Eclipse Ni-U microscope 702

at 10x magnification using a DAPI filter (excitation at 325-375 nm emission at 435-485 703

nm) For callose quantification six 200 microm x 200 microm squares were counted for each 704

sample type Callose deposits per mm2 were calculated and data analyzed using a t-705

test Note that there is an inherent uncertainty associated with such counts because 706

callose deposits may not all be in focus at the same time 707

708

MAPK Activity Assay 709

Fully expanded leaves of four week-old plants were inoculated with 001 710

macerozyme 100 microg mL-1 OGs 100 nM flg22 or water (mock) flg22 a peptide from 711

bacterial flagella that induces immune responses was used as a positive control for 712

Page 35 of 54

MAPK activation (Asai et al 2002) and was purchased from EZBiolab Inc (Westfield 713

IN USA) Samples were harvested 10 min later and immediately frozen in liquid 714

nitrogen Protein was extracted using an extraction buffer containing 25 mM Tris pH 78 715

75 mM NaCl 1 mM DTT 1 mM NaF 05mM Na3VO4 01 (vv) Tween 20 one 716

cOmplete Mini protease inhibitor cocktail tablet (Roche) and one PhosSTOP 717

phosphatase inhibitor cocktail tablet (Roche) per 10 mL buffer 15 microg of total protein 718

were electrophoresed on a 10 SDS-polyacrylamide gel and then blotted onto a PVDF 719

membrane (Bio-Rad 162-0177) Activated MAPKs were detected using p4442 MAPK 720

(Erk12) antibody (9102S Cell Signal 12500 in TBST (20 mM Tris pH 75 150 mM 721

NaCl 01 (vv) Tween 20)) Anti-Rabbit IgG-HRP (115000 in TBST A6154 Sigma) 722

and ECL Plus substrate (Pierce 32132) MPK3 and MPK6 were detected with an anti-723

MPK3 antibody (M8318 Sigma 12000 in TBST and 3 (wv) milk) or an anti-MPK6 724

antibody (A7104 Sigma 16000 in TBST and 3 (wv) milk) respectively The 725

secondary antibody and detection were the same as for the p4442 antibody Five 726

independent experiments were performed 727

728

Preparation of Alcohol Insoluble Residue (AIR) Pectin Extraction and Uronic Acid 729

Measurements 730

Fully expanded leaves from four week-old plants that were incubated for 48 h in 731

the dark to reduce starch were harvested flash frozen and pulverized AIR was 732

extracted by washing ground material twice in 70 (vv) ethanol three times in a 733

mixture of chloroform and methanol (11 vv) and once in acetone (Gille et al 2009) 734

and was then air dried Cell wall pectin was extracted from AIR with cell wall extraction 735

buffer (50 mM Trizma 50 mM CDTA pH 72) at 95degC for 15 min Samples were 736

homogenized using a paint shaker (Harbil 5G-HD) and glass beads (3 mm diameter) 737

For dot blot experiments 500 microL of cell wall extraction buffer were used per 10 mg of 738

AIR Debris was precipitated by centrifugation for 10 min at 10000 g 739

Total uronic acid content of pectin extracted as described above using 500 microL of 740

cell wall extraction buffer per 1 mg of AIR was measured as described previously 741

(Filisetti-Cozzi and Carpita 1991 van den Hoogen et al 1998) First 36 microL of pectin 742

extract were mixed with 4 microL of 4 M sulfamic acid Then 200 microL of sulfuric acid 743

Page 36 of 54

containing 120 mM sodium tetraborate was added and samples were incubated at 80degC 744

for 1 h Following cooling on ice the optical density of samples was measured at 490 745

nm Next 40 microL of m-hydroxydiphenyl reagent (100 microL of m-hydroxydiphenyl in dimethyl 746

sulfoxide at 100 mgmL mixed with 49 mL 80 (vv) sulfuric acid just before use) was 747

added and the samples were mixed The optical density at 490 nm was measured 748

again The optical density before m-hydroxydiphenyl reagent addition was subtracted 749

from the optical density measured after addition of the dye The concentration of uronic 750

acid was calculated using known amounts of GalA as a standard 751

752

Release of Pectin from Arabidopsis Cell Walls Using Macerozyme 753

AIR preparations from leaves of four week-old plants were treated with 025 754

(wv) macerozyme R-10 (Yakult) or water (mock) at a concentration of 100 microL per mg 755

AIR Samples were incubated for 3 h at room temperature on a vortex shaker and 756

centrifuged for 10 min at 12000 g Total uronic acid concentration of 5-fold diluted 757

supernatants was determined for two technical replicates each The concentration of 758

uronic acid was calculated using known amounts of GalA as a standard For 759

subsequent qRT-PCR experiments supernatants from multiple extractions were 760

combined boiled for 20 min to inactivate macerozyme diluted 10-fold with water and 761

inoculated into four week-old Col-0 plants Heat-inactivated 10-fold diluted macerozyme 762

solutions were used as mock treatment Samples were collected 3 h later and PAD3 763

expression was determined 764

765

Release of Pectin from Arabidopsis Leaves Infected with B cinerea 766

Fully expanded leaves of four week-old plants were either inoculated with up to 767

five 10 microL droplets of B cinerea spore solution (25 x 105 spores mL-1) or mock 768

inoculated with B cinerea inoculation medium For each biological replicate leaves with 769

a combined count of at least 20 infection sites were collected 48 h later AIR was 770

extracted as described above but plants were not dark-treated or freeze-dried and the 771

first ethanol step was performed using 85 (vv) ethanol To extract soluble pectin 772

fragments AIR samples were mixed with water at a concentration of 20 microL per infection 773

site incubated for 7 h at room temperature on a vortex shaker and centrifuged for 10 774

Page 37 of 54

min at 12000 g Total uronic acid concentration of supernatants was determined for two 775

technical replicates each The concentration of uronic acid was calculated using known 776

amounts of GalA as a standard 777

778

Measurement of Uronic Acids Neutral Monosaccharides and Cellulose Content in 779

Cell Walls 780

Neutral sugars and uronic acids from non-cellulosic polysaccharides in cell walls 781

(AIR) were released by trifluoroacetic acid (TFA) hydrolysis Approximately 1 mg of AIR 782

was hydrolyzed with 2 M TFA at 121degC for 90 min The TFA-soluble material was split 783

into two equal parts one for uronic acid and one for neutral sugar determination The 784

uronic acid content was determined by analyzing the hydrolyzate using a CarboPac 785

PA200 anion-exchange column with an ICS-3000 Dionex chromatography system 786

(Dionex Sunnyvale CA USA) The elution profile consisted of a linear gradient of 50 to 787

200 mM sodium acetate in 01 M NaOH in 10 min at 04 mL per min Alditol acetate 788

derivatives of neutral sugars were produced as described (York et al 1985) In brief 789

monosaccharides in the hydrolyzate were reduced with NaBH4 and peracetylated with 790

acetic anhydride and pyridine at 121degC for 20 min The generated alditol acetates were 791

analyzed by GC-MS Crystalline cellulose was measured according to Updegraff (1969) 792

After hydrolysis of noncellulosic polysaccharides from AIR with the Updegraff reagent 793

(acetic acid nitric acids water 812 (vv) ) the remaining pellet was hydrolyzed in 794

72 sulfuric acid The resulting glucose quantity was determined by the anthrone 795

method (Scott and Melvin 1953) 796

797

Dot Blot Experiments 798

Pectin solutions extracted using 50 microL of cell wall extraction buffer per mg AIR 799

were serially diluted and nitrocellulose membranes were spotted with 1 microL of the diluted 800

pectin solutions Membranes were dried overnight blocked with 5 milk (wv) in 1x 801

PBS (8 g L-1 NaCl 02 g L-1 KCl 144 g L-1 Na2HPO4 024 g L-1 KH2PO4 pH 74) and 802

probed with LM5 (Jones et al 1997) LM6 (Willats et al 1998) LM8 (Willats et al 803

2004) LM19 and LM20 (Verhertbruggen et al 2009a) or CCRC-M7 (Steffan et al 804

Page 38 of 54

1995) antibodies LM series antibodies were diluted 1250 and CCRC-M7 was diluted 805

1500 in 5 milk powder (Nestle Carnation) in 1 x PBS For LM series antibodies a 806

goat anti-Rat HRP conjugated antibody (Bethyl A110-105P 15000 diluted) and for 807

CCRC-M7 an anti-Mouse IgG HRP conjugate (Promega W402B 12500 diluted) was 808

used as secondary antibody in 5 milk powder in 1x PBS LM series antibodies were 809

obtained from plant probes (wwwplantprobesnet) and CCRC-M7 from CarboSource 810

(wwwcarbosourcenet) Membranes were washed after incubation with the primary and 811

secondary antibodies with 1x PBS Dot blots were developed using the ECL system 812

(GE Healthcare Amersham ECL prime) For quantification of signals dot blot results on 813

x-ray films were photographed using a CCD camera and intensities were measured814

using Image Jrsquos Integrated Density function Locally adjusted background was 815

subtracted As the dynamic range of the signal on an x-ray film was narrow non-816

linearity in the measured values was corrected for each genotype in each biological 817

replicate using the values from different dilutions in two technical replicates We noticed 818

that the residuals were not normally distributed and performed Box-Cox power 819

transformation (Box and Cox 1964) to obtain normally distributed values for the 820

subsequent statistical analysis of the data For LM19 Box-Cox selected power 821

transformation was done to the power of 038th and for LM20 to the power 075th After 822

correction of the transformed measured values with the effect of the biological 823

replicates the values were fit to a linear model with the Arabidopsis genotype as the 824

fixed effect This linear model was used both to generate the plots in Figures 5B and D 825

and to test significant differences 826

827

Camalexin Measurements 828

Four week-old plants were infected with B cinerea and samples (three leaf disks 829

from one plant each) were harvested at the indicated time points flash frozen and 830

pulverized Each sample was extracted with 300 microL of 90 methanol (vv) 50 microL of the 831

extract was run on an Agilent Lichrocart 250-4 RP18e 5-microm column using an Agilent 832

1100 series HPLC Camalexin was detected using a DiodeArray (DAD) at 330 nm and 833

with a fluorescence detector at emission 318 nmexcitation 385 nm (Agilent) Separation 834

was achieved using the following program with aqueous acetonitrile 5 min gradient 835

Page 39 of 54

from 63 to 69 acetonitrile 30 s gradient from 69 to 99 acetonitrile 2 min at 99 836

acetonitrile and a post-run equilibration of 35 min at 63 acetonitrile (Denby et al 837

2004 Kliebenstein et al 2005) Purified camalexin was used to produce a standard 838

curve to identify and quantitate camalexin 839

840

Accession Numbers 841

Microarray data used can be accessed at NCBI Gene Expression Omnnibus 842

(httpwwwncbinlmnihgovgeo) GSE18978 Sequence information for all Arabidopsis 843

genes described in this article can be found in TAIR (wwwarabidopsisorg) using the 844

following accession numbers PAD3 (CYP71B15 At3g26830) SID2 (ICS1 At1g74710) 845

GAE1 (At4g30440) GAE2 (At1g02000) GAE3 (At4g00110) GAE4 (At2g45310) GAE5 846

(At4g12250) GAE6 (At3g23820) PR1 (At2g14610) JAZ5 (At1g17380) JAZ10 847

(At5g13220) ACTIN2 (At3g18780) PDF12a (At5g44420) PDF12b (At2g26020) 848

AtSK11 (At5g26751) The sequence of the B cinerea cutinase Bc-CutA (Z69264) can 849

be found in the DNA Data Bank of Japan (httpgetentryddbjnigacjp) Germplasm 850

used included dde2-2 (At5g42650 (von Malek et al 2002)) pad3-1 (At3g26830 (Zhou 851

et al 1999)) pad4-1 (At3g52430 (Jirage et al 1999)) sid2-2 (At1g74710 (Wildermuth 852

et al 2001)) pbs3-2 (At5g13320 SALK_018225 (Nobuta et al 2007)) cbp60a-1 853

(At5g62570 SALK_124410 (Truman et al 2013)) cbp60g-1 sard1-2 (At5g26920 854

SALK_023199 At1g73805 SALK_052422 (Wang et al 2011) cbp60a-1 cbp60g-1 855

sard1-2 (Truman et al 2013) gae1-1 (At4g30440 SALK_085554) gae6-1 (At3g23280 856

SALK_104454) gae6-2 (At3g23280 SALK_017191) ixr1-1 cev1 (At5g05170 (Ellis 857

and Turner 2001 Scheible et al 2001)) eds1 plants were derived by introgression of 858

the Ler eds1-2 allele into Col-0 that contains two copies of EDS1 (At3g48090 859

At3g48080 (Bartsch et al 2006) mpk6-2 (At2g43790 SALK_073907 (Liu and Zhang 860

2004)) mpk3-DG (At3g45640 a fast neutron deletion mutant (Miles et al 2005)) and 861

npr1-1 (At1g64280 (Cao et al 1997)) T-DNA insertion lines were part of the SALK 862

collection (Alonso et al 2003) and were obtained from the Arabidopsis Biological 863

Resource Center 864

865

Supplemental Data 866

Page 40 of 54

Supplemental Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 867

Expression Does Not Require CBP60s or SID2 868

Supplemental Figure 2 Expression of GAE Family Members uponTreatment with Pma 869

ES4326 870

Supplemental Figure 3 Characterization of T-DNA Insertions in GAE1 and GAE6 871

Supplemental Figure 4 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves and 872

Are Slightly Smaller than Wild-Type Col-0 Plants 873

Supplemental Figure 5 Cell Walls of gae1 gae6 Plants Contain Less Uronic Acid than 874

Wild-Type Col-0 Cell Walls 875

Supplemental Figure 6 Antibodies Raised Against the RGI or XG Components of 876

Pectin Show No Difference in Binding to gae Mutant and Col-0 Cell Walls 877

Supplemental Figure 7 The Effect of B cinerea Treatment on the Expression of GAE 878

Family Genes 879

Supplemental Figure 8 Treatment with 001 Macerozyme Does Not Cause Any 880

Visible Damage to Col-0 Plants 881

Supplemental Figure 9 B cinerea Growth on Plants with Defects in Immune 882

Signaling 883

Supplemental Figure 10 MeJA-Induced Expression of the JA marker gene JAZ10 884

Supplemental Figure 11 The Expression of the JA Marker Gene JAZ5 Is Highly 885

Responsive in gae1 gae6 Plants 886

Supplemental Figure 12 The Expression of PDF12 Is Unaltered in gae1 gae6 Plants 887

Supplemental Figure 13 Macerozyme Treatment Activates MPK3 and MPK6 888

Supplemental Figure 14 B cinerea-Induced ROS Production in Wild-Type Col-0 and 889

gae1 gae6 Plants 890

Supplemental Figure 15 OG-Induced PTI Using 250 microg mL-1 OGs 891

Supplemental Figure 16 Abundance of Plant DNA in B cinerea Treated Plants 892

Supplemental Table 1 Primers used in this study 893

894

ACKNOWLEDGEMENTS 895

We thank William Truman for helpful discussions Simone Ferrari (Sapienza 896

Universita di Roma) for the generous gift of OGs Bruna Bucciarelli (USDA-ARS) for 897

Page 41 of 54

help with fluorescence microscopy the Arabidopsis Biological Resource Center for T-898

DNA insertion lines Paul Knoxrsquos lab (University of Leeds) for LM5 LM6 LM8 LM19 899

and LM20 antibodies and the Complex Carbohydrate Resource Center (University of 900

Georgia) for the CCRC-M7 antibody Most of this work was funded by the Division of 901

Chemical Sciences Geosciences and Biosciences Office of Basic Energy Sciences of 902

the US Department of Energy through Grant DE-FG02-05ER15670 to JG This work 903

was also supported by NSF awards IOS 1339125 and IOS 1021861 to DJK NSF 904

award IOS 1121425 to F K and the USDA National Institute of Food and Agriculture 905

Hatch project number CA-D-PLS-7033-H to DJK 906

907

AUTHOR CONTRIBUTIONS 908

GB and JG designed the research GB GX AT BL NES and NH 909

performed research DJK provided tools GB GX MP AD BL RAH and FK 910

analyzed data GB and JG wrote the paper with input from all authors 911

912

FIGURE LEGENDS 913

Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 Expression 914

Requires PAD4 EDS1 and PBS3 915

(A) Expression of GAE1 in leaves of wild-type Col-0 and mutant plants Expression was916

measured 24 or 32 h after either inoculation with Pma ES4326 (OD600 = 0002) or mock 917

inoculation with 5 mM MgSO4 and was then normalized to the level of ACTIN2 Data 918

from three biological replicates were combined using a mixed linear model Bars 919

represent mean log2-ratios to ACTIN2 plusmn SE Letters indicate significantly different 920

groups with q lt 005 where 005 represents the maximum false discovery rate at which 921

the test may be called significant 922

(B) Expression of GAE6 measured as described in (A)923

924

Figure 2 Susceptibility of gae6 and gae1 gae6 Plants to Pma ES4326 Is Increased 925

Relative to Wild-Type Col-0 926

Leaves of plants of the indicated genotypes were inoculated with Pma ES4326 (OD600 = 927

00002) as described in Methods Bacterial titers in leaves were determined either 928

Page 42 of 54

immediately (0 dpi) or after three days (3 dpi) Bars represent the mean plusmn SE of four 929

independent experiments each with 4 or 12 biological replicates at 0 and 3 dpi 930

respectively Data were combined using a mixed linear model Asterisks indicate 931

significant differences from Col-0 wild-type (q lt 005) Susceptible pad4 plants were 932

included as a positive control 933

934

Figure 3 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves 935

(A) Depiction of the assay for brittleness of gae mutant leaves Fully expanded leaves of936

four to five week-old plants were bent by 180ordm so that the adaxial surface of the leaf was 937

parallel with the soil Broken leaf petioles or midribs were considered brittle 938

(B) Quantification of broken midribs or petioles For each genotype 66 to 82 leaves 939

were analyzed for brittleness as described in (A) Bars represent the ratio of broken to 940

total number of leaves tested Results were analyzed using Fisherrsquos exact test Letters 941

indicate significantly different groups at p lt 005 942

(C) Conductivity measurements Leaves of four week-old plants were inoculated with943

deionized water and then leaf discs were cut out and placed on deionized water 944

Conductivity was measured at the indicated time points After 30 min the water was 945

replaced with fresh water (30 min f w) to account for wounding-related ion leakage from 946

the edges of the leaf discs Three independent experiments with six biological replicates 947

each were performed and data were analyzed using a t-test for each time point P-948

values were corrected using the Bonferroni method Data show means plusmn SE asterisks 949

indicate significant differences from wild-type Col-0 at q lt 001 950

951

Figure 4 Cell Walls of gae6 and gae1 gae6 Plants Are Reduced in GalA 952

(A) to (D) Levels of GalA (A) GlcA (B) cellulose (C) and neutral sugars (D) in alcohol953

insoluble residue (AIR) extracted from leaves of four week-old plants of the indicated 954

genotypes Bars represent means plusmn SE of four biological replicates combined using a 955

mixed linear model Letters indicate significantly different groups (p lt 005) 956

957

Figure 5 Cell Walls of gae1 gae6 Plants Are Reduced in Homogalacturonan 958

Page 43 of 54

Dot blot analyses with anti-LM19 and anti-LM20 antibodies Pectin was extracted from 959

AIR prepared from the indicated genotypes using 1 microL of extraction buffer per 10 microg of 960

AIR Pectin was serially diluted and one microL of undiluted 13 diluted and 19 diluted 961

samples were spotted on a nitrocellulose membrane Three biological replicates were 962

were spotted twice to obtain two technical replicates each All replicates produced 963

similar results 964

(A) Representative dot blot using anti-LM19 antibody965

(B) Relative signal intensities of the spots in the dot blot in (A)966

(C) Representative dot blot using anti-LM20 antibody967

(D) Relative signal intensities of the LM20 dot blot shown in (C)968

Relative signal intensities were measured using Image J software Measurements from 969

the three biological replicates were combined using a linear model after Box-Cox power 970

transformation as described in Methods Bars represent means plusmn SE Asterisks indicate 971

a difference from wild-type Col-0 at p lt 005 972

973

Figure 6 Growth of B cinerea on gae1 gae6 Plants and the Effect of B cinerea on 974

Expression of GAE1 and GAE6 975

(A) Growth of B cinerea isolates Leaves of Col-0 and gae1 gae6 plants were976

inoculated with 25 x 105 spores mL-1 of the B cinerea isolates indicated in the figure 977

Samples were collected two (2 dpi) and three (3 dpi) days later Bars represent means plusmn 978

SE of six biological replicates for each fungus combined using a mixed linear model 979

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 980

(B) and (C) Expression of GAE1 (B) and GAE6 (C) 48 h after inoculation with the B981

cinerea isolates indicated Expression levels were measured by qRT-PCR Bars 982

represent mean log2-ratios to ACTIN2 plusmn SE of three biological replicates combined 983

using a mixed linear model Letters indicate significantly different groups (q lt 005) 984

985

Figure 7 Levels of Soluble Pectin and Expression of PAD3 in Fractions Produced 986

Following Macerozyme Treatment of gae1 gae6 Cell Walls 987

(A) Schematic overview of the experimental procedure for (B) and (C)988

Page 44 of 54

(B) Uronic acid measurements Alcohol-insoluble residue (AIR) from leave tissue of four989

week-old wild-type Col-0 or gae1 gae6 plants was treated with water or 025 990

macerozyme and assayed for uronic acid levels Data from 11 biological replicates were 991

combined using a mixed linear model Bars represent means plusmn SE Letters indicate 992

significant differences (p lt 005) 993

(C) Expression of PAD3 in Col-0 plants treated with the fractions from (B) Extracts from994

(B) were heat inactivated pooled and diluted 5-fold with water Pooled extracts were995

inoculated into four week-old Col-0 plants PAD3 expression was measured 3 h after 996

inoculation and normalized to the level of ACTIN2 expression Data from four biological 997

replicates were combined using a mixed linear model Bars represent mean log2-ratios 998

to ACTIN2 plusmn SE Letters indicate significant differences (p lt 005) 999

1000

Figure 8 Macerozyme-Induced PAD3 Expression Is Absent in gae1 gae6 1001

(A) Expression of PAD3 after macerozyme treatment Four week-old plants were either1002

inoculated with 001 macerozyme or mock inoculated with water and PAD3 1003

expression was measured 3 h later by qRT-PCR Data from four biological replicates 1004

were combined using a mixed linear model Mean log2-ratios to ACTIN2 plusmn SE were 1005

plotted Letters indicate significantly different groups (p lt 005) 1006

(B) Expression of PAD3 after OG treatment PAD3 expression was measured as1007

described in (A) 3 h after inoculation with 100 microg mL-1 OGs or mock inoculation with 1008

water Data are from six biological replicates 1009

(C) PAD3 expression in untreated plants Three biological replicates were performed1010

and data analyzed as described in (A) 1011

(D) Camalexin accumulation after B cinerea treatment Camalexin accumulation was1012

measured in four week-old plants inoculated with the B cinerea strains shown in the 1013

figure Eight biological replicates each were combined using a mixed linear model 1014

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 1015

(E) PAD3 expression 28 h after treatment with the indicated B cinerea strains Data1016

from six biological replicates were collected and analyzed as described in (A) Data in 1017

all figure parts represent means plusmn SE Letters indicate significantly different groups (q lt 1018

005) 1019

Page 45 of 54

1020

Figure 9 Macerozyme-Induced Expression of the SA Marker Genes SID2 and PR1 1021

Is Abolished in gae1 gae6 Plants 1022

(A) to (H) Expression of PR1 (A) (C) (E) and (G) or SID2 (B) (D) (F) and (H) in four1023

week-old Col-0 and gae1 gae6 plants Data were combined using a mixed linear model 1024

Bars represent mean log2-ratios of expression vs ACTIN2 plusmn SE for all figure parts 1025

Letters indicate significantly different groups at p lt 005 for panels (A) to (D) (G) and 1026

(H) and qlt005 for panels (E) to (F) 1027

(A) and (B) Plants were treated with 001 macerozyme or mock treated with water1028

and samples were collected 3 h later Data from four biological replicates are shown 1029

(C) and (D) Plants were inoculated with 100 microg mL-1 OGs or mock treated with water1030

and samples collected 3 h later Data from six biological replicates are shown 1031

(E) and (F) Plants were inoculated with the indicated B cinerea isolates or mock1032

inoculated with B cinerea inoculation medium and samples collected 48 h later Data 1033

from eight biological replicates are shown 1034

(G) and (H) Expression in untreated plants Three biological replicates were performed1035

1036

Figure 10 The Expression of the JA Marker Gene JAZ10 is Highly Responsive in 1037

gae1 gae6 Plants 1038

(A) to (D) JAZ10 expression in four week-old plants Data were combined using a mixed1039

linear model Mean log2-ratios to ACTIN2 plusmn SE are shown Letters indicate significantly 1040

different groups at p lt 005 for panels (A) (B) and (D) and q lt 005 for panel (C) 1041

(A) Plants were inoculated with 001 macerozyme or mock inoculated using water and1042

samples were collected 3 h later Data from four biological replicates are shown 1043

(B) Plants were inoculated with 100 microg mL-1 OGs or mock inoculated using water and1044

samples collected 3 h later Data are from six biological replicates 1045

(C) JAZ10 expression 48 h after treatment with the indicated B cinerea strains Data1046

are from six biological replicates 1047

(D) JAZ10 expression in untreated plants Data are from three biological replicates1048

1049

Figure 11 Gallo 1-Induced Callose Deposition Is Reduced in gae1 gae6 Plants 1050

Page 46 of 54

(A) Callose deposition in four week-old Col-0 and gae1 gae6 plants Plants were1051

inoculated with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) or mock inoculated 1052

with B cinerea inoculation medium and samples were collected after 72 h Callose was 1053

stained with aniline blue and visualized using a Nikon Eclipse Ni-U microscope with a 1054

DAPI filter Three leaves were stained per treatment Scale bars are 100 microm Asterisks 1055

indicate fungal hyphae arrows indicate representative callose spots 1056

(B) Quantification of callose deposition Callose deposits were counted in 200 microm x 2001057

microm squares laid out across the images shown here Bars represent means plusmn SE from 1058

six squares each Letters indicate significantly different groups (p lt 005) according to a 1059

t-test analysis1060

1061

Figure 12 Less Soluble Pectin Accumulates in B cinerea-Infected gae1 gae6 1062

Leaves 1063

Four week-old Col-0 and gae1 gae6 plants were inoculated with 10 microL droplets of the 1064

indicated B cinerea isolate (25 x 105 spores mL-1) or mock inoculated with B cinerea 1065

inoculation medium Leaves were collected 48 h later AIR was extracted from leaf 1066

samples and water added (20 microL infection site) to extract soluble pectin fragments 1067

Samples were vortexed for 7 h centrifuged at 12000 g and the uronic acid 1068

concentration of the supernatant was measured Data from nine biological replicates 1069

were combined using a mixed linear model Means plusmn SE are shown Letters indicate 1070

significant differences (q lt 005) 1071

1072

Figure 13 Macerozyme-Induced PTI Is Lost in gae1 gae6 While OG-Induced PTI Is 1073

Unaltered 1074

(A) Macerozyme-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1075

inoculated with 001 macerozyme or mock inoculated with boiled macerozyme and 1076

were infected with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) 24 h later Samples 1077

were collected immediately (0 dpi) and after two (2 dpi) and three (3 dpi) days Data 1078

show mean plusmn SE of six to 14 biological replicates each combined using a linear model 1079

Letters indicate significantly different groups (q lt 005) 1080

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

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Page 12 of 54

gae6 cell walls are reduced in HG Using the same samples we detected no difference 274

from wild type in binding of LM5 which recognizes a tetramer of (1-4)-β-D-galactans 275

found in RGI (Jones et al 1997) LM6 which recognizes a linear pentasaccharide in (1-276

To further investigate which pectic components were altered in gae1 gae6 dot 269

blot experiments using pectin isolated from the various genotypes were performed Both 270

LM19 and LM20 antibodies recognize HG (Verhertbruggen et al 2009a) As expected 271

LM19 binding was reduced in gae6-1 and gae1 gae6 (Figure 5A and B) and LM20 272

binding was reduced in gae1 gae6 (Figure 5C and D) This result confirmed that gae1 273

277 5)-α-L-arabinans found in RGI (Willats et al 1998 Verhertbruggen et al 2009b) LM8

278 which recognizes xylogalacturonan (Willats et al 2004) or CCRC-M7 which

279 recognizes an arabinosylated (1-6)-β-D-galactan epitope occurring on RGI (Puhlmann et

Page 13 of 54

Page 14 of 54

al 1994 Steffan et al 1995 Pattathil et al 2010) (Supplemental Figure 6) 280

Collectively these cell wall composition data show that gae1 gae6 plants are reduced in 281

HG Due to the strong proportional reduction of HG in the walls levels of all other 282

components should be higher However Rha indicative of RGI was not higher in gae1 283

gae6 suggesting that RGI may also be reduced in gae1 gae6 walls 284

285

GAE1 and GAE6 Are Required for Immunity to Specific B cinerea Isolates 286

We hypothesized that plant immunity to B cinerea might be affected by pectin 287

content in the host cell wall because this pathogen requires polygalacturonases and 288

pectin methylesterases for full virulence (ten Have et al 1998 Valette-Collet et al 289

2003 Kars et al 2005b Espino et al 2010) Because Bcinerea is genetically diverse 290

and has a high diversity of polygalacturonases (Rowe and Kliebenstein 2007) we 291

measured relative fungal growth at both 2 and 3 d post-inoculation (dpi) using ten 292

different B cinerea isolates (Figure 6) The B cinerea isolates Fresa 525 and Gallo 1 293

showed enhanced virulence on gae1 gae6 plants at both time points whereas KT and 294

UK Razz showed enhanced virulence in gae1 gae6 plants at 3 but not 2 d dpi (Figure 295

6A) We did not detect any significant changes in relative fungal growth between wild-296

type Col-0 and gae1 gae6 plants when using Acacia Apple 517 DN Grape Pepper or 297

Rasp isolates of B cinerea Therefore we continued our study using Gallo 1 and Fresa 298

525 We also included Pepper for comparison as this isolate has been used in a large-299

scale analysis of plant gene expression in response to B cinerea infection (Windram et 300

al 2012) 301

We next investigated whether expression of GAE1 and GAE6 was affected by 302

challenge with B cinerea Inoculation of wild-type Col-0 plants with any isolate 303

repressed GAE1 and GAE6 expression (Figure 6B and C) and this repression was 304

generally independent of PAD4 (Figure 6B and C) Expression of GAE2 and GAE3 was 305

unaltered by B cinerea treatment GAE4 was repressed only by Fresa 525 treatment 306

while GAE5 expression was enhanced with all three isolates tested (Supplemental 307

Figure 7) As seen for infection with Pma ES4326 the expression of GAE1 and GAE6 308

was repressed upon B cinerea treatment This indicates a role for GAE1 and GAE6 in 309

the immune response to B cinerea 310

Page 15 of 54

Page 16 of 54

Because the amount of pectin substrate for pathogen polygalacturonases is 313

reduced we hypothesized that immunity phenotypes of gae1 gae6 plants may result 314

from reduced generation of OGs by these enzymes In turn the reduced OGs could 315

lead to reduced immune responses To test this we used macerozyme a commercial 316

pectinase as a proxy for pathogen pectinases We isolated cell walls as a subcellular 317

fraction known as alcohol insoluble residue (AIR Gille et al 2009) This was treated 318

with macerozyme and tested for release of soluble likely low molecular weight pectin 319

fragments (Figure 7) Treatment with 025 macerozyme released significantly more 320

uronic acid from wild-type Col-0 AIR relative to gae1 gae6 AIR (Figure 7B) To test 321

activity of these pectin-containing solutions we infiltrated them into wild-type Col-0 322

plants and measured PAD3 expression PAD3 is required for synthesis of the 323

phytoalexin camalexin (Zhou et al 1999) and its expression is known to be induced by 324

OG treatment (Ferrari et al 2007) Fractions from macerozyme treated Col-0 walls 325

induced significantly more PAD3 expression than fractions derived from gae1 gae6 326

walls suggesting that macerozyme treatment did indeed release more active soluble 327

uronic acid possibly OGs from wild-type Col-0 walls than from gae1 gae6 walls (Figure 328

7C) 329

Next we studied the effect of macerozyme treatment on expression of PAD3 in 330

intact plants (Figure 8) This treatment did not lead to any visible leaf damage 331

(Supplemental Figure 8) In wild-type Col-0 PAD3 expression was induced three h after 332

treatment with 001 macerozyme (Figure 8A) By contrast no macerozyme-induced 333

induction of PAD3 expression was detected in gae1 gae6 (Figure 8A) However OG 334

treatment induced the expression of PAD3 in both genotypes indicating that gae1 gae6 335

plants do respond to OGs (Figure 8B) Macerozyme treatment also induced expression 336

of the SA marker genes PATHOGENESIS RELATED 1 (PR1) and SID2 in Col-0 but 337

not in gae1 gae6 (Figure 9 panel A and B) PR1 was not significantly induced by 338

purified OGs (Figure 9C) at the time and concentration tested but SID2 was induced by 339

OG treatment in both genotypes (Figure 9D) However the induction was stronger in 340

wild-type Col-0 In addition the JA markers JASMONATE-ZIM-DOMAIN PROTEIN 5 341

(JAZ5) and JAZ10 were induced by macerozyme and OG treatment in both Col-0 and 342

gae1 gae6 (Figure 10A and Supplemental Figure 11) Interestingly expression of JAZ5 343

312 Pectinase-Induced Immune Signaling Is Altered in gae1 gae6 Plants

Page 17 of 54

Page 18 of 54

Page 19 of 54

Page 20 of 54

344

345

346

and JAZ10 was already strongly induced in gae1 gae6 after mock inoculation (Figure 10

and Supplemental Figure 11) We observed no hyperresponsive induction by mock

treatment of PLANT DEFENSIN 12 (PDF12) a necrotrophy and late JA signaling

347 marker gene (Supplemental Figure 12A-D) PDF12 was also not induced by

348 macerozyme or OG treatments (Supplemental Figure 12A-D) This suggests that early

349 JA signaling is induced by mock-treatment in gae1 gae6 Because it is known that JA

350 and SA signaling act antagonistically (Pieterse et al 2012) the strong up-regulation of

351 JA-induced immune signaling in gae1 gae6 might explain the lower OG-induced SID2

352 expression in this genotype

353 To test whether other immune responses are altered in gae1 gae6 plants we

354 analyzed Mitogen-activated protein kinase (MAPK) activity following macerozyme and

355 OG treatments OGs are known to activate the MAPKs MPK3 and MPK6 in Arabidopsis

Page 21 of 54

(Galletti et al 2011) Macerozyme treatment activated MPK3 and MPK6 in wild-type 356

Col-0 whereas MAPK activation in gae1 gae6 was slightly reduced in four out of five 357

experiments performed (Supplemental Figure 13) By contrast OG-induced MAPK 358

activity was similar or increased in gae1 gae6 in four out of five experiments 359

(Supplemental Figure 13) Additionally mock treatment induced MAPK activity in gae1 360

gae6 more strongly than it did in wild-type Col-0 in four out of five experiments 361

performed (Fig S13) Collectively these results illustrate that activation of several 362

immune signaling componentsmdashcamalexin synthesis SA and JA signaling and 363

possibly MAPK activationmdashby macerozyme is substantially altered in gae1 gae6 This 364

suggests that release of soluble pectin fragments possibly OGs by pectinolytic 365

enzymes might be reduced in gae1 gae6 plants while recognition of OGs is intact 366

367

Following B cinerea Infection Production of Soluble Pectin and Deposition of 368

Callose Are Reduced in gae1 gae6 Plants 369

Although pad3 plants were more susceptible to the B cinerea isolates Fresa 525 370

and Gallo 1 which also grew better on gae1 gae6 growth of the isolate Pepper was 371

similar on Col-0 and pad3 (Supplemental Figure 9A) However we did not detect any 372

significant changes in camalexin production after treatment with B cinerea isolate Gallo 373

1 and Fresa 525 in gae1 gae6 (Figure 8D) Neither did we detect changes in PAD3 374

expression after treatment with any B cinerea isolate in gae1 gae6 (Figure 8E) 375

Additionally SID2 and PR1 were similarly induced by all B cinerea isolates in both Col-376

0 and gae1 gae6 (Figure 9E and F) and sid2 plants which are deficient for pathogen-377

induced SA biosynthesis did not show any differences in relative B cinerea growth 378

(Supplemental Figure 9B) Expression of JAZ10 was induced more strongly by Gallo 1 379

treatment in gae1 gae6 than in wild-type Col-0 but not by Fresa 525 or Pepper (Fig 380

10C) while dde2 plants deficient for JA biosynthesis showed enhanced susceptibility to 381

all B cinerea isolates tested (Supplemental Figure 9B) Curiously exogenous 382

application of MeJA did not lead to altered relative fungal growth of B cinerea 383

(Supplemental Figure 9C) and cev1 isoxaben resistant 1 (ixr1) plants which have 384

constitutively high JA levels (Ellis and Turner 2001) did not show any changes in 385

susceptibility to the B cinerea isolates tested (Supplemental Figure 9D) Hence neither 386

Page 22 of 54

differences in B cinerea isolate susceptibility to camalexin nor altered SA or JA 387

signaling by themselves explained the enhanced susceptibility of gae1 gae6 to these B 388

cinerea isolates 389

Hydrogen peroxide accumulation in response to B cinerea isolates Fresa 525 390

Gallo 1 and Pepper was not obviously altered in gae1 gae6 plants (Supplemental Figure 391

14) However callose deposition upon treatment with B cinerea isolate Gallo 1 was 392

strongly reduced in gae1 gae6 (Figure 11) It was previously shown that OG treatment 393

induces callose deposition (Denoux et al 2008) suggesting that the reduction in 394

callose deposition might be due to reduced OG release in gae1 gae6 It is possible that 395

this reduction in callose deposition contributes to the reduced immunity of gae1 gae6 396

plants to B cinerea isolate Gallo 1 397

Because macerozyme could be used to release soluble likely low molecular 398

weight pectin from isolated cell walls we next investigated whether infection with B 399

cinerea was accompanied by accumulation of soluble pectin as well We prepared cell 400

walls (AIR fraction) from leaves infected with B cinerea incubated these cell walls with 401

water and measured uronic acid content of the extracts B cinerea treatment did in fact 402

lead to increased accumulation of soluble pectin in wild-type Col-0 plants when 403

compared to mock-treated samples (Figure 12) No such increased accumulation of 404

water-soluble pectin fractions was observed in gae1 gae6 (Figure 12) 405

406

Macerozyme-Induced Pattern-Triggered Immunity against B cinerea Is Abolished 407

in gae1 gae6 408

It was previously shown that OG treatment can induce patternndashtriggered 409

immunity (PTI) to B cinerea (Ferrari et al 2007) We tested macerozyme treatment to 410

determine whether it could also induce PTI to the B cinerea isolate Gallo 1 (Figure 13) 411

Indeed pretreatment with 001 macerozyme for 24 h led to reduced B cinerea growth 412

in wild-type Col-0 but not gae1 gae6 plants (Figure 13A) Interestingly mock and 413

macerozyme pretreated gae1 gae6 plants exhibited similar growth of B cinerea Gallo 1 414

as did macerozyme-treated Col-0 plants This result is likely due to activation of JA 415

signaling in gae1 gae6 during the pretreatment procedure (Figure 13C and D) OG-416

induced PTI was observed in both genotypes when using a high dose of OGs (Figure 417

Page 23 of 54

13B) whereas a lower dose of OGs only induced PTI in Col-0 (Supplemental Figure 418

15) This suggests that OG perception in gae1 gae6 at least above a certain threshold 419

is unaltered while release of immunity-activating pectin fragments possibly OGs is 420

421 reduced We conclude that immune responses induced by macerozyme treatment

422 which do not occur in gae1 gae6 plants are important for plant immunity to the B

423 cinerea isolate Gallo 1

Page 24 of 54

____________________________________________________________________________

Page 25 of 54

DISCUSSION 426

Here we showed that loss of GAE1 and GAE6 leads to a dramatic reduction in 427

pectin content in the cell wall (Figure 4) likely causing the brittleness of leaves that we 428

observed (Figure 3) Petioles and midribs of gae1 gae6 plants broke easily when bent 429

and inoculation of leaves with deionized water resulted in increased ion leakage 430

possibly due either to microscopic fractures of the cell wall induced by inoculation stress 431

or to a reduced capability of these walls to withstand increased turgor pressure The 432

increased brittleness was associated with altered cell wall composition In our analysis 433

the GalA content of gae1 gae6 leaf walls was reduced by more than 40 and the total 434

uronic acids by over 60 (Figure 4 and Supplemental Figure 5) This is a major change 435

in the overall cell wall composition considering that pectin makes up about 50 of the 436

plant cell walls found in Arabidopsis leaves (Zablackis et al 1995 Nobuta et al 2007 437

Harholt et al 2010) Interestingly quasimodo1 (qua1) plants which have a mutation in 438

a glycosyltransferase family 8 gene are reduced in GalA by 25 and show reduced 439

binding of HG specific antibodies (Bouton et al 2002) 440

Furthermore qua2 plants which have a mutation in a putative methyltransferase 441

have about 13 less total GalA and lack 50 of cell wall HG (Mouille et al 2007) 442

Likewise cgr2 cgr3 plants carrying mutations in another class of Golgi-localized 443

putative methyl-transferases have leaf cell walls that are reduced by about 40 in 444

uronic acid (Kim et al 2015) All three mutants are severely dwarfed (Bouton et al 445

2002 Mouille et al 2007 Kim et al 2015) Additionally qua1 and qua2 mutants show 446

a distorted overall shape likely due to reduced cell cohesion and are extremely fragile 447

(Bouton et al 2002 Mouille et al 2007) By contrast gae1 gae6 plants with a similar 448

strong reduction in GalA and total uronic acids have an overall normal shape and are 449

only slightly smaller than wild-type Col-0 (Supplemental Figure 4) In the wall analysis 450

performed here we did not detect changes in other wall polysaccharides or structures 451

that might compensate for the reduction of pectin in gae1 gae6 It is possible the severe 452

morphological alterations in qua2 and cgr2 cgr3 plants are due to reduction in pectin 453

methylesterification which is unaffected in gae1 gae6 It was speculated that quality 454

control mechanisms in the Golgi prevent secretion of inefficiently esterified pectin in 455

cgr2 cgr3 (Kim et al 2015) suggesting that the Golgi of these plants might over-456

Page 26 of 54

accumulate pectin and that this may contribute to the severe dwarf phenotype of these 457

plants The gae1 gae6 plants described here provide an opportunity to study the effects 458

of reduction of pectin in plants that do not show severe morphological phenotypes 459

We found that gae1 gae6 plants are also compromised in resistance to B 460

cinerea and Pma ES4326 indicating that loss of pectin reduces immunity to these 461

pathogens (Figure 2 and 6) We found that gae1 gae6 plants were slightly more 462

susceptible to Pma ES4326 but showed a strong increase in susceptibility to specific B 463

cinerea isolates This was not unexpected because B cinerea is known to interact with 464

pectin as part of its infection strategy It secretes various pectin-degrading enzymes 465

early during infection and requires some of these enzymes including PG1 PG2 and 466

PME1 for full virulence (ten Have et al 1998 Valette-Collet et al 2003 Kars et al 467

2005b Espino et al 2010) Additionally Arabidopsis plants expressing an antisense 468

construct of the polygalacturonase inhibitor PGIP1 are significantly more susceptible to 469

B cinerea (Ferrari et al 2006) On the other hand Pma ES4326 is not known to470

require cell wall degrading enzymes for full virulence even though it encodes two 471

pectate lyase genes in its genome (PMA4326_07094 and PMA4326_04621 472

wwwbacteriaensemblorg) B cinerea isolates are also known to be genetically quite 473

diverse in their polygalacturonase (PG) loci and genetic variability in PG2 is associated 474

with varying growth on pectin containing media (Rowe and Kliebenstein 2007) 475

Furthermore the contribution of PG1 and PG2 to B cinerea virulence seems to depend 476

on the specific B cinerea isolate and the host species (Kars et al 2005a Zhang and 477

Van Kan 2013) B cinerea isolates are also known to be differently sensitive to the anti-478

microbial secondary metabolite camalexin (Kliebenstein et al 2005) In line with these 479

observations the susceptibility of gae1 gae6 plants differed depending on the specific 480

B cinerea isolate Whereas Fresa 525 Gallo 1 UK Razz and KT grew better in gae1481

gae6 we detected no difference in Acacia Apple 517 DN Grape Pepper or Rasp 482

growth (Figure 6) 483

B cinerea was shown to metabolize pectin and to possibly utilize GalA as a484

carbon source during in planta growth (Zhang et al 2011 Zhang and Van Kan 2013) 485

Our data show that gae1 gae6 plants which are reduced in GalA content did allow 486

enhanced growth of specific B cinerea isolates but no reduction in growth of any B 487

Page 27 of 54

cinerea isolates tested was found This suggests that utilization of pectin as a carbon 488

source was not a contributing factor to alterations in B cinerea susceptibility It is 489

possible that changes in the cell wall composition affect the structural integrity of the 490

wall and affect hyphal penetration of specific B cinerea isolates However OGs (small 491

HG fragments) are likely released during the infection process (Ferrari et al 2013) 492

OGs function as DAMPs and initiate immune responses in Arabidopsis (Ferrari et al 493

2013) Treatment of Arabidopsis with OGs having a degree of polymerization (DP) of 494

10-15 sugar subunits was previously shown to enhance resistance to B cinerea (Ferrari495

et al 2007) We showed that pretreatment with OGs or macerozyme induced enhanced 496

immunity to B cinerea isolate Gallo 1 in wild-type Col-0 but only OGs and not 497

macerozyme induced immunity in gae1 gae6 (Figure 13) Our data suggest gae1 gae6 498

plants which have reduced pectin content may release less OGs and that this 499

reduction leads to the increased susceptibility to B cinerea 500

Consistently we found that macerozyme treatment released less soluble pectin 501

from gae1 gae6 cell walls than from wild-type walls Fractions containing soluble pectin 502

released from gae1 gae6 were also less active in inducing PAD3 gene expression than 503

the fractions released from wild-type walls (Figure 7) We also showed that less soluble 504

pectin accumulated in gae1 gae6 than in wild-type plants 48 h after treatment with B 505

cinerea (Figure 12) 506

We observed that loss of pectin leads to alterations in A thaliana immune 507

signaling Macerozyme treatment induced expression of PAD3 and the SA marker 508

genes PR1 and SID2 in wild-type Col-0 but not in gae1 gae6 whereas OG treatment 509

led to increased PAD3 and SID2 expression in both wild-type and gae1 gae6 (Figure 8 510

and 9) This suggests that although release of active soluble pectin in these plants 511

might be altered OG recognition is intact In fact cell walls isolated from qua1 another 512

mutant known to be reduced in HG released less OGs (DP1-6) than did wild-type cell 513

walls upon endopolygalacturonase treatment (Bouton et al 2002) This was also true 514

for gae1 gae6 cell walls as macerozyme treatment of isolated gae1 gae6 cell walls 515

released less soluble pectin than treatment of Col-0 cell walls Because the fraction 516

from gae1 gae6 plants was also less active in inducing PAD3 expression (Figure 7) it 517

seems possible that biologically active OGs were the active component inducing PAD3 518

Page 28 of 54

expression Interestingly expression of the JA marker genes JAZ5 and JAZ10 was 519

higher in gae1 gae6 than in wild-type mock-inoculated plants but was comparable to 520

wild-type in untreated plants suggesting that JA signaling in gae1 gae6 is 521

hyperresponsive (Figure 10 and Supplemental Figure 11) This induction of JA signaling 522

might be due to wounding stress incurred during the inoculation process JA and SA 523

signaling generally act antagonistically (Pieterse et al 2012) Increased JA signaling 524

activity might hence explain why mock- and macerozyme-treated gae1 gae6 plants 525

exhibited reduced expression of the SA marker PR1 and why OG treatment induced 526

expression of SID2 another SA marker to a lower extent in gae1 gae6 than in wild-type 527

plants (Figure 9) 528

No single immune signaling component was found to be the sole contributor to 529

the compromised immunity of gae1 gae6 to specific B cinerea isolates such as Gallo 1 530

and Fresa 525 These isolates grew better on pad3 while growth of Pepper another B 531

cinerea isolate that did not show altered virulence on gae1 gae6 was unaltered 532

(Supplemental Figure 9) This suggested that differences in camalexin sensitivity of 533

these B cinerea isolates might account for the differences in gae1 gae6 susceptibility 534

However camalexin accumulation and PAD3 expression was the same for wild-type 535

Col-0 and gae1 gae6 for the B cinerea isolates Fresa 525 and Gallo 1 (Figure 8) which 536

showed enhanced growth on gae1 gae6 plants It should be noted that B cinerea 537

virulence is often estimated by comparing lesion sizes Lesion size and measurements 538

of relative fungal growth by qPCR do not always show the same results For example 539

although Denby et al (2004) showed that Pepper causes larger lesions on pad3 than on 540

wild-type this study showed that relative fungal growth of Pepper on pad3 was 541

unaltered 542

We observed that callose deposition was strongly reduced in gae1 gae6 plants 543

72 h after treatment with B cinerea isolate Gallo 1 (Figure 11) possibly due to reduced 544

release of active soluble pectin Interestingly OG-induced PTI to an unspecified isolate 545

of B cinerea was intact in the pmr4 mutant which does not produce callose (Galletti et 546

al 2008) Due to the high SA levels in pmr4 plants (Nishimura et al 2003) and the fact 547

that different B cinerea isolates vary both in induction of and reaction to defense 548

responses (Kliebenstein et al 2005 Rowe et al 2010) we could not determine 549

Page 29 of 54

whether reduced callose deposition causes the increased susceptibility of gae1 gae6 to 550

B cinerea isolate Gallo 1551

We did not observe any changes in B cinerea growth either on cev1 ixr1 plants 552

which are known to have constitutively high JA and OPDA levels (Ellis et al 2002b) or 553

on plants exogenously treated with MeJA (Supplemental Figure 9) Because plants 554

were kept uncovered for 3 h after MeJA treatment it is unclear how much MeJA was 555

bioavailable in the plant However JAZ10 expression was induced 3 h after spraying 556

with MeJA (Supplemental Figure 10) Because pathogen growth generally depends on 557

the combination of a multitude of immune signaling events we conclude that multiple 558

changes to the immune signaling in gae1 gae6 together account for the altered B 559

cinerea growth phenotypes detected 560

We showed that expression of GAE1 and GAE6 is repressed by the bacterial 561

hemibiotroph Pma ES4326 This repression did not occur in an eds1 or pad4 562

background or in pbs3 at the 24-h time point (Figure 1 and Supplemental Figure 1) 563

During the Arabidopsis - Pma ES4326 interaction the expression of many defense 564

related genes is altered in a PAD4EDS1 dependent fashion (Wang et al 2008) Some 565

of these expression changes require SA CBP60g and SARD1 are thought to act 566

downstream of PAD4 and EDS1 but upstream of SA-dependent responses (Wang et 567

al 2011) PBS3 is also thought to act downstream of PAD4 and EDS1 and upstream of 568

SA but the set of genes whose expression it affects is only partially overlapping with 569

those affected by CBP60gSARD1 (Wang et al 2008 Wang et al 2011) Thus GAE1 570

and GAE6 repression is regulated by the part of the immune signaling network that 571

requires PAD4 EDS1 and PBS3 but is independent of CBP60gSARD1 and SA 572

Because EDS1 PAD4 and PBS3 are important components of the plant immune 573

signaling network (Wiermer et al 2005 Nobuta et al 2007) one might conclude that 574

repression of GAE1 and GAE6 may be part of the plant immune response to Pma 575

ES4326 B cinerea treatment also reduced GAE1 and GAE6 expression but this was 576

mostly independent of PAD4 (Figure 6) while both Pma ES4326 and B cinerea repress 577

GAE1 and GAE6 expression the host signaling mechanisms responsible likely differ 578

In summary our data show that Arabidopsis GAE1 and GAE6 are required for 579

pectin biosynthesis leaf flexibility and immunity to Pma ES4326 and to specific B 580

Page 30 of 54

cinerea isolates We used macerozyme a commercial pectinase to mimic the effect of 581

B cinerea pectinases Macerozyme treatment released less pectin from gae1 gae6 cell582

walls and induced less immune signaling in gae1 gae6 plants Less water soluble pectin 583

also accumulated in B cinerea infected gae1 gae6 plants and macerozyme-induced 584

immunity to B cinerea isolate Gallo 1 was reduced in these plants We conclude that 585

the reduced pectin content in these mutants may lead to reduced release of soluble 586

pectin including active OGs during pathogen attack and that this compromises 587

immunity 588

589

590

Page 31 of 54

METHODS 591

Plant Materials and Growth Conditions 592

Wild-type Columbia (Col-0) and all mutant plants (in Col-0 background) were 593

grown on sterilized BM2 germinating mix (Berger Inc Quebec Canada) in a controlled 594

environment chamber (Conviron Pembina ND USA) with a 12-h photoperiod under 595

100 microM m-2 s-1 fluorescent illumination at 22degC and 75 relative humidity Germplasm 596

used is described in the Accession Numbers section at the end of Methods 597

598

Data Analysis and Replicates 599

For biological replicates samples were obtained from separate plant tissues and 600

in many cases from plants grown in different flats In some experiments multiple 601

technical replicates were measured per biological replicate For statistical analysis all 602

technical replicates were averaged and the averages were considered one biological 603

replicate Independent experiments were performed at different times and often 604

consisted of multiple biological replicates per experiment 605

Data analysis was performed using mixed linear models in the R programming 606

environment (Bates et al 2015) unless otherwise indicated Generally genotypes 607

treatments and time points were used as fixed effects and replicate-specific effects like 608

different pots flats etc were considered random effects We reported p-values 609

obtained from the mixed linear model when less than 10 comparisons were performed 610

and q-values for more than 10 comparisons to correct for multiple comparison testing 611

Q-values were calculated using the Benjamini-Hochberg procedure (Benjamini and612

Hochberg 1995) unless specified otherwise 613

614

Pathogen Strains Growth Conditions and Pathogen Growth Assays 615

Growth of Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) and 616

bacterial growth assays were performed as previously described (Bethke et al 2014) 617

For qRT-PCR assays four week-old plants were inoculated with bacteria (OD600 = 618

0002) or mock (5 mM MgSO4) At least four leaves from two plants were collected at 619

the indicated time points per biological replicate 620

Page 32 of 54

Prior to each experiment Botrytis cinerea isolates were grown on 1x PDA 621

medium (Difco) for 10 d at room temperature All isolates used have been described 622

previously (Rowe and Kliebenstein 2007) The spores were washed from the surface of 623

the plate using an inoculation medium (1x Gamborgrsquos B-5 basal salt mixture (Sigma-624

Aldrich G5768) 2 (wv) glucose 10 mM phosphate buffer pH 64) and fungal hyphae 625

were removed from the suspension by filtering through four layers of cheesecloth The 626

concentration of spores was determined using a hemocytometer and adjusted to 25 x 627

105 spores mL-1 Four week-old Arabidopsis plants were inoculated by placing one 10-628

microL droplet of the B cinerea spore solution or inoculation medium (mock) on the adaxial 629

leaf surface of fully expanded leaves Inoculated plants were kept at 100 relative 630

humidity Infection sites were collected at various time points using a cork borer For 631

qRT-PCR assays at least four leaf discs from one plant were combined for each 632

biological sample For B cinerea growth assays at least 12 leaf discs from three to six 633

plants were pooled per biological sample Relative fungal growth was determined by 634

measuring the abundance of a fungal gene relative to a plant gene as described below 635

In brief DNA from infected tissue was extracted and equal amounts of total DNA were 636

used to perform qPCR reactions using the B cinerea cutinase A and the Arabidopsis 637

SK11 gene respectively Primers were described in (Gachon and Saindrenan 2004) 638

and are listed in Supplemental Table 1 Two technical replicates were combined for 639

each biological replicate B cinerea is a necrotrophic fungal pathogen and may destroy 640

plant DNA as disease progresses However decreases in the amount of plant DNA 641

over the course of our experiments were only observed at late time points with very 642

susceptible plants so the qPCR assay is generally a reasonable estimate of fungal 643

biomass (Supplemental Figure 16) 644

645

Treatment with Macerozyme or OGs 646

For PTI assays four week-old plants either were inoculated with 001 (wv) 647

macerozyme R-10 (Yakult) a Rhizopus polygalacturonase (EC 32115) that possesses 648

high pectinase and hemicellulase activity or were mock inoculated using heat-649

inactivated macerozyme Other similar plants were either inoculated with 250 or 500 microg 650

mL-1 OGs or mock inoculated with water One d later plants were infected with B 651

Page 33 of 54

cinerea and samples were collected immediately (0 dpi) and two or three d later To 652

investigate the role of MeJA on B cinerea growth plants were sprayed with 1mM MeJA 653

in water with 001 (vv) Tween 20 Covers were removed so that leaves could dry off 654

before plants were inoculated with B cinerea three h later Samples were harvested at 655

the indicated time points For expression analysis and MAPK activation assays plants 656

were either inoculated with 100 microg mL-1 OGs or mock inoculated with water OGs used 657

for experiments described in Figures 8 to 10 and Supplemental Figure 12 were a kind 658

gift from Simone Ferrari OGs used in experiments described in Figure 13 and 659

Supplemental Figures 11 13 and 15 were prepared as previously described (Kohorn et 660

al 2014) 661

662

Expression Analysis 663

Leaf tissue from inoculated plants was harvested at the indicated time points 664

flash frozen pulverized and RNA extracted using Trizol (Invitrogen) Quantitative RT-665

PCR was performed using the SuperScript III Platinum SYBR Green One-Step 666

quantitative RT-PCR kit (Invitrogen) and a Lightcycler 480 Real-Time PCR system 667

(Roche) as previously described (Truman and Glazebrook 2012) In brief equal 668

amounts of total RNA and a gene specific primer were used for each reaction The 669

crossing point (Cp) was calculated using the 2nd derivative max method provided with 670

the Lightcycler software for each amplification curve Each reaction was run with two 671

technical replicates and the Cp values for these replicates were averaged ACTIN2 was 672

used as a stably expressed reference gene At least three biological replicates each 673

were performed and analyzed using a mixed-linear model RT-PCR was performed to 674

verify T-DNA insertion lines using the One-Step RT-PCR kit (Qiagen) according to the 675

manufacturerrsquos instructions The primers used can be found in Supplemental Table 1 676

677

Electrolyte Leakage Assay 678

Fully expanded leaves of four week-old plants were inoculated with water (Milli-Q 679

grade) using a needleless syringe Leaf discs were collected using a cork borer and 680

three leaf discs from one leaf were placed with the adaxial surface down onto 2 mL 681

water in a 12-well cell culture plate Conductivity was measured using a Horiba B-173 682

Page 34 of 54

conductivity meter after 30 min and then the water was replaced Conductivity was 683

measured again 30 min later (1 h total) as indicated in Figure 3C Six biological 684

replicates each were performed in three independent experiments and means and SEs 685

were calculated Two-sided t-tests were performed for each time point 686

687

DAB and Aniline Blue Staining 688

Leaves from four week-old plants were either mock inoculated with B cinerea 689

inoculation medium or inoculated with 10 microL droplets of a B cinerea spore solution (25 690

x 105 spores mL-1) of the indicated isolates Samples were collected 72 h later For 33prime-691

diaminobenzidine (DAB) staining two to five inoculated leaves were vacuum infiltrated 692

with 33prime-Diaminobenzidine (1 mg mL-1 in water pH 38) solution and stained overnight 693

in the dark Leaves were destained using 100 ethanol at 65degC and then transferred to 694

50 (vv) glycerol and photographed 695

Callose was visualized by staining leaves with aniline blue as previously 696

described (Adam and Somerville 1996) Briefly leaves were vacuum infiltrated with 697

alcoholic lactophenol (one volume of phenolglycerollactic acidwater (1111) mixed 698

with two volumes of ethanol) and destained at 65degC Next leaves were moved through 699

an alcohol gradient (50 20 and 10 (vv) ethanol) into water The destained leaves 700

were stained for 30 min in 150 mM K2HPO4 (pH 95) with 001 (wv) aniline blue 701

Samples were mounted in water and examined using a Nikon Eclipse Ni-U microscope 702

at 10x magnification using a DAPI filter (excitation at 325-375 nm emission at 435-485 703

nm) For callose quantification six 200 microm x 200 microm squares were counted for each 704

sample type Callose deposits per mm2 were calculated and data analyzed using a t-705

test Note that there is an inherent uncertainty associated with such counts because 706

callose deposits may not all be in focus at the same time 707

708

MAPK Activity Assay 709

Fully expanded leaves of four week-old plants were inoculated with 001 710

macerozyme 100 microg mL-1 OGs 100 nM flg22 or water (mock) flg22 a peptide from 711

bacterial flagella that induces immune responses was used as a positive control for 712

Page 35 of 54

MAPK activation (Asai et al 2002) and was purchased from EZBiolab Inc (Westfield 713

IN USA) Samples were harvested 10 min later and immediately frozen in liquid 714

nitrogen Protein was extracted using an extraction buffer containing 25 mM Tris pH 78 715

75 mM NaCl 1 mM DTT 1 mM NaF 05mM Na3VO4 01 (vv) Tween 20 one 716

cOmplete Mini protease inhibitor cocktail tablet (Roche) and one PhosSTOP 717

phosphatase inhibitor cocktail tablet (Roche) per 10 mL buffer 15 microg of total protein 718

were electrophoresed on a 10 SDS-polyacrylamide gel and then blotted onto a PVDF 719

membrane (Bio-Rad 162-0177) Activated MAPKs were detected using p4442 MAPK 720

(Erk12) antibody (9102S Cell Signal 12500 in TBST (20 mM Tris pH 75 150 mM 721

NaCl 01 (vv) Tween 20)) Anti-Rabbit IgG-HRP (115000 in TBST A6154 Sigma) 722

and ECL Plus substrate (Pierce 32132) MPK3 and MPK6 were detected with an anti-723

MPK3 antibody (M8318 Sigma 12000 in TBST and 3 (wv) milk) or an anti-MPK6 724

antibody (A7104 Sigma 16000 in TBST and 3 (wv) milk) respectively The 725

secondary antibody and detection were the same as for the p4442 antibody Five 726

independent experiments were performed 727

728

Preparation of Alcohol Insoluble Residue (AIR) Pectin Extraction and Uronic Acid 729

Measurements 730

Fully expanded leaves from four week-old plants that were incubated for 48 h in 731

the dark to reduce starch were harvested flash frozen and pulverized AIR was 732

extracted by washing ground material twice in 70 (vv) ethanol three times in a 733

mixture of chloroform and methanol (11 vv) and once in acetone (Gille et al 2009) 734

and was then air dried Cell wall pectin was extracted from AIR with cell wall extraction 735

buffer (50 mM Trizma 50 mM CDTA pH 72) at 95degC for 15 min Samples were 736

homogenized using a paint shaker (Harbil 5G-HD) and glass beads (3 mm diameter) 737

For dot blot experiments 500 microL of cell wall extraction buffer were used per 10 mg of 738

AIR Debris was precipitated by centrifugation for 10 min at 10000 g 739

Total uronic acid content of pectin extracted as described above using 500 microL of 740

cell wall extraction buffer per 1 mg of AIR was measured as described previously 741

(Filisetti-Cozzi and Carpita 1991 van den Hoogen et al 1998) First 36 microL of pectin 742

extract were mixed with 4 microL of 4 M sulfamic acid Then 200 microL of sulfuric acid 743

Page 36 of 54

containing 120 mM sodium tetraborate was added and samples were incubated at 80degC 744

for 1 h Following cooling on ice the optical density of samples was measured at 490 745

nm Next 40 microL of m-hydroxydiphenyl reagent (100 microL of m-hydroxydiphenyl in dimethyl 746

sulfoxide at 100 mgmL mixed with 49 mL 80 (vv) sulfuric acid just before use) was 747

added and the samples were mixed The optical density at 490 nm was measured 748

again The optical density before m-hydroxydiphenyl reagent addition was subtracted 749

from the optical density measured after addition of the dye The concentration of uronic 750

acid was calculated using known amounts of GalA as a standard 751

752

Release of Pectin from Arabidopsis Cell Walls Using Macerozyme 753

AIR preparations from leaves of four week-old plants were treated with 025 754

(wv) macerozyme R-10 (Yakult) or water (mock) at a concentration of 100 microL per mg 755

AIR Samples were incubated for 3 h at room temperature on a vortex shaker and 756

centrifuged for 10 min at 12000 g Total uronic acid concentration of 5-fold diluted 757

supernatants was determined for two technical replicates each The concentration of 758

uronic acid was calculated using known amounts of GalA as a standard For 759

subsequent qRT-PCR experiments supernatants from multiple extractions were 760

combined boiled for 20 min to inactivate macerozyme diluted 10-fold with water and 761

inoculated into four week-old Col-0 plants Heat-inactivated 10-fold diluted macerozyme 762

solutions were used as mock treatment Samples were collected 3 h later and PAD3 763

expression was determined 764

765

Release of Pectin from Arabidopsis Leaves Infected with B cinerea 766

Fully expanded leaves of four week-old plants were either inoculated with up to 767

five 10 microL droplets of B cinerea spore solution (25 x 105 spores mL-1) or mock 768

inoculated with B cinerea inoculation medium For each biological replicate leaves with 769

a combined count of at least 20 infection sites were collected 48 h later AIR was 770

extracted as described above but plants were not dark-treated or freeze-dried and the 771

first ethanol step was performed using 85 (vv) ethanol To extract soluble pectin 772

fragments AIR samples were mixed with water at a concentration of 20 microL per infection 773

site incubated for 7 h at room temperature on a vortex shaker and centrifuged for 10 774

Page 37 of 54

min at 12000 g Total uronic acid concentration of supernatants was determined for two 775

technical replicates each The concentration of uronic acid was calculated using known 776

amounts of GalA as a standard 777

778

Measurement of Uronic Acids Neutral Monosaccharides and Cellulose Content in 779

Cell Walls 780

Neutral sugars and uronic acids from non-cellulosic polysaccharides in cell walls 781

(AIR) were released by trifluoroacetic acid (TFA) hydrolysis Approximately 1 mg of AIR 782

was hydrolyzed with 2 M TFA at 121degC for 90 min The TFA-soluble material was split 783

into two equal parts one for uronic acid and one for neutral sugar determination The 784

uronic acid content was determined by analyzing the hydrolyzate using a CarboPac 785

PA200 anion-exchange column with an ICS-3000 Dionex chromatography system 786

(Dionex Sunnyvale CA USA) The elution profile consisted of a linear gradient of 50 to 787

200 mM sodium acetate in 01 M NaOH in 10 min at 04 mL per min Alditol acetate 788

derivatives of neutral sugars were produced as described (York et al 1985) In brief 789

monosaccharides in the hydrolyzate were reduced with NaBH4 and peracetylated with 790

acetic anhydride and pyridine at 121degC for 20 min The generated alditol acetates were 791

analyzed by GC-MS Crystalline cellulose was measured according to Updegraff (1969) 792

After hydrolysis of noncellulosic polysaccharides from AIR with the Updegraff reagent 793

(acetic acid nitric acids water 812 (vv) ) the remaining pellet was hydrolyzed in 794

72 sulfuric acid The resulting glucose quantity was determined by the anthrone 795

method (Scott and Melvin 1953) 796

797

Dot Blot Experiments 798

Pectin solutions extracted using 50 microL of cell wall extraction buffer per mg AIR 799

were serially diluted and nitrocellulose membranes were spotted with 1 microL of the diluted 800

pectin solutions Membranes were dried overnight blocked with 5 milk (wv) in 1x 801

PBS (8 g L-1 NaCl 02 g L-1 KCl 144 g L-1 Na2HPO4 024 g L-1 KH2PO4 pH 74) and 802

probed with LM5 (Jones et al 1997) LM6 (Willats et al 1998) LM8 (Willats et al 803

2004) LM19 and LM20 (Verhertbruggen et al 2009a) or CCRC-M7 (Steffan et al 804

Page 38 of 54

1995) antibodies LM series antibodies were diluted 1250 and CCRC-M7 was diluted 805

1500 in 5 milk powder (Nestle Carnation) in 1 x PBS For LM series antibodies a 806

goat anti-Rat HRP conjugated antibody (Bethyl A110-105P 15000 diluted) and for 807

CCRC-M7 an anti-Mouse IgG HRP conjugate (Promega W402B 12500 diluted) was 808

used as secondary antibody in 5 milk powder in 1x PBS LM series antibodies were 809

obtained from plant probes (wwwplantprobesnet) and CCRC-M7 from CarboSource 810

(wwwcarbosourcenet) Membranes were washed after incubation with the primary and 811

secondary antibodies with 1x PBS Dot blots were developed using the ECL system 812

(GE Healthcare Amersham ECL prime) For quantification of signals dot blot results on 813

x-ray films were photographed using a CCD camera and intensities were measured814

using Image Jrsquos Integrated Density function Locally adjusted background was 815

subtracted As the dynamic range of the signal on an x-ray film was narrow non-816

linearity in the measured values was corrected for each genotype in each biological 817

replicate using the values from different dilutions in two technical replicates We noticed 818

that the residuals were not normally distributed and performed Box-Cox power 819

transformation (Box and Cox 1964) to obtain normally distributed values for the 820

subsequent statistical analysis of the data For LM19 Box-Cox selected power 821

transformation was done to the power of 038th and for LM20 to the power 075th After 822

correction of the transformed measured values with the effect of the biological 823

replicates the values were fit to a linear model with the Arabidopsis genotype as the 824

fixed effect This linear model was used both to generate the plots in Figures 5B and D 825

and to test significant differences 826

827

Camalexin Measurements 828

Four week-old plants were infected with B cinerea and samples (three leaf disks 829

from one plant each) were harvested at the indicated time points flash frozen and 830

pulverized Each sample was extracted with 300 microL of 90 methanol (vv) 50 microL of the 831

extract was run on an Agilent Lichrocart 250-4 RP18e 5-microm column using an Agilent 832

1100 series HPLC Camalexin was detected using a DiodeArray (DAD) at 330 nm and 833

with a fluorescence detector at emission 318 nmexcitation 385 nm (Agilent) Separation 834

was achieved using the following program with aqueous acetonitrile 5 min gradient 835

Page 39 of 54

from 63 to 69 acetonitrile 30 s gradient from 69 to 99 acetonitrile 2 min at 99 836

acetonitrile and a post-run equilibration of 35 min at 63 acetonitrile (Denby et al 837

2004 Kliebenstein et al 2005) Purified camalexin was used to produce a standard 838

curve to identify and quantitate camalexin 839

840

Accession Numbers 841

Microarray data used can be accessed at NCBI Gene Expression Omnnibus 842

(httpwwwncbinlmnihgovgeo) GSE18978 Sequence information for all Arabidopsis 843

genes described in this article can be found in TAIR (wwwarabidopsisorg) using the 844

following accession numbers PAD3 (CYP71B15 At3g26830) SID2 (ICS1 At1g74710) 845

GAE1 (At4g30440) GAE2 (At1g02000) GAE3 (At4g00110) GAE4 (At2g45310) GAE5 846

(At4g12250) GAE6 (At3g23820) PR1 (At2g14610) JAZ5 (At1g17380) JAZ10 847

(At5g13220) ACTIN2 (At3g18780) PDF12a (At5g44420) PDF12b (At2g26020) 848

AtSK11 (At5g26751) The sequence of the B cinerea cutinase Bc-CutA (Z69264) can 849

be found in the DNA Data Bank of Japan (httpgetentryddbjnigacjp) Germplasm 850

used included dde2-2 (At5g42650 (von Malek et al 2002)) pad3-1 (At3g26830 (Zhou 851

et al 1999)) pad4-1 (At3g52430 (Jirage et al 1999)) sid2-2 (At1g74710 (Wildermuth 852

et al 2001)) pbs3-2 (At5g13320 SALK_018225 (Nobuta et al 2007)) cbp60a-1 853

(At5g62570 SALK_124410 (Truman et al 2013)) cbp60g-1 sard1-2 (At5g26920 854

SALK_023199 At1g73805 SALK_052422 (Wang et al 2011) cbp60a-1 cbp60g-1 855

sard1-2 (Truman et al 2013) gae1-1 (At4g30440 SALK_085554) gae6-1 (At3g23280 856

SALK_104454) gae6-2 (At3g23280 SALK_017191) ixr1-1 cev1 (At5g05170 (Ellis 857

and Turner 2001 Scheible et al 2001)) eds1 plants were derived by introgression of 858

the Ler eds1-2 allele into Col-0 that contains two copies of EDS1 (At3g48090 859

At3g48080 (Bartsch et al 2006) mpk6-2 (At2g43790 SALK_073907 (Liu and Zhang 860

2004)) mpk3-DG (At3g45640 a fast neutron deletion mutant (Miles et al 2005)) and 861

npr1-1 (At1g64280 (Cao et al 1997)) T-DNA insertion lines were part of the SALK 862

collection (Alonso et al 2003) and were obtained from the Arabidopsis Biological 863

Resource Center 864

865

Supplemental Data 866

Page 40 of 54

Supplemental Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 867

Expression Does Not Require CBP60s or SID2 868

Supplemental Figure 2 Expression of GAE Family Members uponTreatment with Pma 869

ES4326 870

Supplemental Figure 3 Characterization of T-DNA Insertions in GAE1 and GAE6 871

Supplemental Figure 4 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves and 872

Are Slightly Smaller than Wild-Type Col-0 Plants 873

Supplemental Figure 5 Cell Walls of gae1 gae6 Plants Contain Less Uronic Acid than 874

Wild-Type Col-0 Cell Walls 875

Supplemental Figure 6 Antibodies Raised Against the RGI or XG Components of 876

Pectin Show No Difference in Binding to gae Mutant and Col-0 Cell Walls 877

Supplemental Figure 7 The Effect of B cinerea Treatment on the Expression of GAE 878

Family Genes 879

Supplemental Figure 8 Treatment with 001 Macerozyme Does Not Cause Any 880

Visible Damage to Col-0 Plants 881

Supplemental Figure 9 B cinerea Growth on Plants with Defects in Immune 882

Signaling 883

Supplemental Figure 10 MeJA-Induced Expression of the JA marker gene JAZ10 884

Supplemental Figure 11 The Expression of the JA Marker Gene JAZ5 Is Highly 885

Responsive in gae1 gae6 Plants 886

Supplemental Figure 12 The Expression of PDF12 Is Unaltered in gae1 gae6 Plants 887

Supplemental Figure 13 Macerozyme Treatment Activates MPK3 and MPK6 888

Supplemental Figure 14 B cinerea-Induced ROS Production in Wild-Type Col-0 and 889

gae1 gae6 Plants 890

Supplemental Figure 15 OG-Induced PTI Using 250 microg mL-1 OGs 891

Supplemental Figure 16 Abundance of Plant DNA in B cinerea Treated Plants 892

Supplemental Table 1 Primers used in this study 893

894

ACKNOWLEDGEMENTS 895

We thank William Truman for helpful discussions Simone Ferrari (Sapienza 896

Universita di Roma) for the generous gift of OGs Bruna Bucciarelli (USDA-ARS) for 897

Page 41 of 54

help with fluorescence microscopy the Arabidopsis Biological Resource Center for T-898

DNA insertion lines Paul Knoxrsquos lab (University of Leeds) for LM5 LM6 LM8 LM19 899

and LM20 antibodies and the Complex Carbohydrate Resource Center (University of 900

Georgia) for the CCRC-M7 antibody Most of this work was funded by the Division of 901

Chemical Sciences Geosciences and Biosciences Office of Basic Energy Sciences of 902

the US Department of Energy through Grant DE-FG02-05ER15670 to JG This work 903

was also supported by NSF awards IOS 1339125 and IOS 1021861 to DJK NSF 904

award IOS 1121425 to F K and the USDA National Institute of Food and Agriculture 905

Hatch project number CA-D-PLS-7033-H to DJK 906

907

AUTHOR CONTRIBUTIONS 908

GB and JG designed the research GB GX AT BL NES and NH 909

performed research DJK provided tools GB GX MP AD BL RAH and FK 910

analyzed data GB and JG wrote the paper with input from all authors 911

912

FIGURE LEGENDS 913

Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 Expression 914

Requires PAD4 EDS1 and PBS3 915

(A) Expression of GAE1 in leaves of wild-type Col-0 and mutant plants Expression was916

measured 24 or 32 h after either inoculation with Pma ES4326 (OD600 = 0002) or mock 917

inoculation with 5 mM MgSO4 and was then normalized to the level of ACTIN2 Data 918

from three biological replicates were combined using a mixed linear model Bars 919

represent mean log2-ratios to ACTIN2 plusmn SE Letters indicate significantly different 920

groups with q lt 005 where 005 represents the maximum false discovery rate at which 921

the test may be called significant 922

(B) Expression of GAE6 measured as described in (A)923

924

Figure 2 Susceptibility of gae6 and gae1 gae6 Plants to Pma ES4326 Is Increased 925

Relative to Wild-Type Col-0 926

Leaves of plants of the indicated genotypes were inoculated with Pma ES4326 (OD600 = 927

00002) as described in Methods Bacterial titers in leaves were determined either 928

Page 42 of 54

immediately (0 dpi) or after three days (3 dpi) Bars represent the mean plusmn SE of four 929

independent experiments each with 4 or 12 biological replicates at 0 and 3 dpi 930

respectively Data were combined using a mixed linear model Asterisks indicate 931

significant differences from Col-0 wild-type (q lt 005) Susceptible pad4 plants were 932

included as a positive control 933

934

Figure 3 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves 935

(A) Depiction of the assay for brittleness of gae mutant leaves Fully expanded leaves of936

four to five week-old plants were bent by 180ordm so that the adaxial surface of the leaf was 937

parallel with the soil Broken leaf petioles or midribs were considered brittle 938

(B) Quantification of broken midribs or petioles For each genotype 66 to 82 leaves 939

were analyzed for brittleness as described in (A) Bars represent the ratio of broken to 940

total number of leaves tested Results were analyzed using Fisherrsquos exact test Letters 941

indicate significantly different groups at p lt 005 942

(C) Conductivity measurements Leaves of four week-old plants were inoculated with943

deionized water and then leaf discs were cut out and placed on deionized water 944

Conductivity was measured at the indicated time points After 30 min the water was 945

replaced with fresh water (30 min f w) to account for wounding-related ion leakage from 946

the edges of the leaf discs Three independent experiments with six biological replicates 947

each were performed and data were analyzed using a t-test for each time point P-948

values were corrected using the Bonferroni method Data show means plusmn SE asterisks 949

indicate significant differences from wild-type Col-0 at q lt 001 950

951

Figure 4 Cell Walls of gae6 and gae1 gae6 Plants Are Reduced in GalA 952

(A) to (D) Levels of GalA (A) GlcA (B) cellulose (C) and neutral sugars (D) in alcohol953

insoluble residue (AIR) extracted from leaves of four week-old plants of the indicated 954

genotypes Bars represent means plusmn SE of four biological replicates combined using a 955

mixed linear model Letters indicate significantly different groups (p lt 005) 956

957

Figure 5 Cell Walls of gae1 gae6 Plants Are Reduced in Homogalacturonan 958

Page 43 of 54

Dot blot analyses with anti-LM19 and anti-LM20 antibodies Pectin was extracted from 959

AIR prepared from the indicated genotypes using 1 microL of extraction buffer per 10 microg of 960

AIR Pectin was serially diluted and one microL of undiluted 13 diluted and 19 diluted 961

samples were spotted on a nitrocellulose membrane Three biological replicates were 962

were spotted twice to obtain two technical replicates each All replicates produced 963

similar results 964

(A) Representative dot blot using anti-LM19 antibody965

(B) Relative signal intensities of the spots in the dot blot in (A)966

(C) Representative dot blot using anti-LM20 antibody967

(D) Relative signal intensities of the LM20 dot blot shown in (C)968

Relative signal intensities were measured using Image J software Measurements from 969

the three biological replicates were combined using a linear model after Box-Cox power 970

transformation as described in Methods Bars represent means plusmn SE Asterisks indicate 971

a difference from wild-type Col-0 at p lt 005 972

973

Figure 6 Growth of B cinerea on gae1 gae6 Plants and the Effect of B cinerea on 974

Expression of GAE1 and GAE6 975

(A) Growth of B cinerea isolates Leaves of Col-0 and gae1 gae6 plants were976

inoculated with 25 x 105 spores mL-1 of the B cinerea isolates indicated in the figure 977

Samples were collected two (2 dpi) and three (3 dpi) days later Bars represent means plusmn 978

SE of six biological replicates for each fungus combined using a mixed linear model 979

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 980

(B) and (C) Expression of GAE1 (B) and GAE6 (C) 48 h after inoculation with the B981

cinerea isolates indicated Expression levels were measured by qRT-PCR Bars 982

represent mean log2-ratios to ACTIN2 plusmn SE of three biological replicates combined 983

using a mixed linear model Letters indicate significantly different groups (q lt 005) 984

985

Figure 7 Levels of Soluble Pectin and Expression of PAD3 in Fractions Produced 986

Following Macerozyme Treatment of gae1 gae6 Cell Walls 987

(A) Schematic overview of the experimental procedure for (B) and (C)988

Page 44 of 54

(B) Uronic acid measurements Alcohol-insoluble residue (AIR) from leave tissue of four989

week-old wild-type Col-0 or gae1 gae6 plants was treated with water or 025 990

macerozyme and assayed for uronic acid levels Data from 11 biological replicates were 991

combined using a mixed linear model Bars represent means plusmn SE Letters indicate 992

significant differences (p lt 005) 993

(C) Expression of PAD3 in Col-0 plants treated with the fractions from (B) Extracts from994

(B) were heat inactivated pooled and diluted 5-fold with water Pooled extracts were995

inoculated into four week-old Col-0 plants PAD3 expression was measured 3 h after 996

inoculation and normalized to the level of ACTIN2 expression Data from four biological 997

replicates were combined using a mixed linear model Bars represent mean log2-ratios 998

to ACTIN2 plusmn SE Letters indicate significant differences (p lt 005) 999

1000

Figure 8 Macerozyme-Induced PAD3 Expression Is Absent in gae1 gae6 1001

(A) Expression of PAD3 after macerozyme treatment Four week-old plants were either1002

inoculated with 001 macerozyme or mock inoculated with water and PAD3 1003

expression was measured 3 h later by qRT-PCR Data from four biological replicates 1004

were combined using a mixed linear model Mean log2-ratios to ACTIN2 plusmn SE were 1005

plotted Letters indicate significantly different groups (p lt 005) 1006

(B) Expression of PAD3 after OG treatment PAD3 expression was measured as1007

described in (A) 3 h after inoculation with 100 microg mL-1 OGs or mock inoculation with 1008

water Data are from six biological replicates 1009

(C) PAD3 expression in untreated plants Three biological replicates were performed1010

and data analyzed as described in (A) 1011

(D) Camalexin accumulation after B cinerea treatment Camalexin accumulation was1012

measured in four week-old plants inoculated with the B cinerea strains shown in the 1013

figure Eight biological replicates each were combined using a mixed linear model 1014

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 1015

(E) PAD3 expression 28 h after treatment with the indicated B cinerea strains Data1016

from six biological replicates were collected and analyzed as described in (A) Data in 1017

all figure parts represent means plusmn SE Letters indicate significantly different groups (q lt 1018

005) 1019

Page 45 of 54

1020

Figure 9 Macerozyme-Induced Expression of the SA Marker Genes SID2 and PR1 1021

Is Abolished in gae1 gae6 Plants 1022

(A) to (H) Expression of PR1 (A) (C) (E) and (G) or SID2 (B) (D) (F) and (H) in four1023

week-old Col-0 and gae1 gae6 plants Data were combined using a mixed linear model 1024

Bars represent mean log2-ratios of expression vs ACTIN2 plusmn SE for all figure parts 1025

Letters indicate significantly different groups at p lt 005 for panels (A) to (D) (G) and 1026

(H) and qlt005 for panels (E) to (F) 1027

(A) and (B) Plants were treated with 001 macerozyme or mock treated with water1028

and samples were collected 3 h later Data from four biological replicates are shown 1029

(C) and (D) Plants were inoculated with 100 microg mL-1 OGs or mock treated with water1030

and samples collected 3 h later Data from six biological replicates are shown 1031

(E) and (F) Plants were inoculated with the indicated B cinerea isolates or mock1032

inoculated with B cinerea inoculation medium and samples collected 48 h later Data 1033

from eight biological replicates are shown 1034

(G) and (H) Expression in untreated plants Three biological replicates were performed1035

1036

Figure 10 The Expression of the JA Marker Gene JAZ10 is Highly Responsive in 1037

gae1 gae6 Plants 1038

(A) to (D) JAZ10 expression in four week-old plants Data were combined using a mixed1039

linear model Mean log2-ratios to ACTIN2 plusmn SE are shown Letters indicate significantly 1040

different groups at p lt 005 for panels (A) (B) and (D) and q lt 005 for panel (C) 1041

(A) Plants were inoculated with 001 macerozyme or mock inoculated using water and1042

samples were collected 3 h later Data from four biological replicates are shown 1043

(B) Plants were inoculated with 100 microg mL-1 OGs or mock inoculated using water and1044

samples collected 3 h later Data are from six biological replicates 1045

(C) JAZ10 expression 48 h after treatment with the indicated B cinerea strains Data1046

are from six biological replicates 1047

(D) JAZ10 expression in untreated plants Data are from three biological replicates1048

1049

Figure 11 Gallo 1-Induced Callose Deposition Is Reduced in gae1 gae6 Plants 1050

Page 46 of 54

(A) Callose deposition in four week-old Col-0 and gae1 gae6 plants Plants were1051

inoculated with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) or mock inoculated 1052

with B cinerea inoculation medium and samples were collected after 72 h Callose was 1053

stained with aniline blue and visualized using a Nikon Eclipse Ni-U microscope with a 1054

DAPI filter Three leaves were stained per treatment Scale bars are 100 microm Asterisks 1055

indicate fungal hyphae arrows indicate representative callose spots 1056

(B) Quantification of callose deposition Callose deposits were counted in 200 microm x 2001057

microm squares laid out across the images shown here Bars represent means plusmn SE from 1058

six squares each Letters indicate significantly different groups (p lt 005) according to a 1059

t-test analysis1060

1061

Figure 12 Less Soluble Pectin Accumulates in B cinerea-Infected gae1 gae6 1062

Leaves 1063

Four week-old Col-0 and gae1 gae6 plants were inoculated with 10 microL droplets of the 1064

indicated B cinerea isolate (25 x 105 spores mL-1) or mock inoculated with B cinerea 1065

inoculation medium Leaves were collected 48 h later AIR was extracted from leaf 1066

samples and water added (20 microL infection site) to extract soluble pectin fragments 1067

Samples were vortexed for 7 h centrifuged at 12000 g and the uronic acid 1068

concentration of the supernatant was measured Data from nine biological replicates 1069

were combined using a mixed linear model Means plusmn SE are shown Letters indicate 1070

significant differences (q lt 005) 1071

1072

Figure 13 Macerozyme-Induced PTI Is Lost in gae1 gae6 While OG-Induced PTI Is 1073

Unaltered 1074

(A) Macerozyme-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1075

inoculated with 001 macerozyme or mock inoculated with boiled macerozyme and 1076

were infected with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) 24 h later Samples 1077

were collected immediately (0 dpi) and after two (2 dpi) and three (3 dpi) days Data 1078

show mean plusmn SE of six to 14 biological replicates each combined using a linear model 1079

Letters indicate significantly different groups (q lt 005) 1080

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

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al 1994 Steffan et al 1995 Pattathil et al 2010) (Supplemental Figure 6) 280

Collectively these cell wall composition data show that gae1 gae6 plants are reduced in 281

HG Due to the strong proportional reduction of HG in the walls levels of all other 282

components should be higher However Rha indicative of RGI was not higher in gae1 283

gae6 suggesting that RGI may also be reduced in gae1 gae6 walls 284

285

GAE1 and GAE6 Are Required for Immunity to Specific B cinerea Isolates 286

We hypothesized that plant immunity to B cinerea might be affected by pectin 287

content in the host cell wall because this pathogen requires polygalacturonases and 288

pectin methylesterases for full virulence (ten Have et al 1998 Valette-Collet et al 289

2003 Kars et al 2005b Espino et al 2010) Because Bcinerea is genetically diverse 290

and has a high diversity of polygalacturonases (Rowe and Kliebenstein 2007) we 291

measured relative fungal growth at both 2 and 3 d post-inoculation (dpi) using ten 292

different B cinerea isolates (Figure 6) The B cinerea isolates Fresa 525 and Gallo 1 293

showed enhanced virulence on gae1 gae6 plants at both time points whereas KT and 294

UK Razz showed enhanced virulence in gae1 gae6 plants at 3 but not 2 d dpi (Figure 295

6A) We did not detect any significant changes in relative fungal growth between wild-296

type Col-0 and gae1 gae6 plants when using Acacia Apple 517 DN Grape Pepper or 297

Rasp isolates of B cinerea Therefore we continued our study using Gallo 1 and Fresa 298

525 We also included Pepper for comparison as this isolate has been used in a large-299

scale analysis of plant gene expression in response to B cinerea infection (Windram et 300

al 2012) 301

We next investigated whether expression of GAE1 and GAE6 was affected by 302

challenge with B cinerea Inoculation of wild-type Col-0 plants with any isolate 303

repressed GAE1 and GAE6 expression (Figure 6B and C) and this repression was 304

generally independent of PAD4 (Figure 6B and C) Expression of GAE2 and GAE3 was 305

unaltered by B cinerea treatment GAE4 was repressed only by Fresa 525 treatment 306

while GAE5 expression was enhanced with all three isolates tested (Supplemental 307

Figure 7) As seen for infection with Pma ES4326 the expression of GAE1 and GAE6 308

was repressed upon B cinerea treatment This indicates a role for GAE1 and GAE6 in 309

the immune response to B cinerea 310

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Page 16 of 54

Because the amount of pectin substrate for pathogen polygalacturonases is 313

reduced we hypothesized that immunity phenotypes of gae1 gae6 plants may result 314

from reduced generation of OGs by these enzymes In turn the reduced OGs could 315

lead to reduced immune responses To test this we used macerozyme a commercial 316

pectinase as a proxy for pathogen pectinases We isolated cell walls as a subcellular 317

fraction known as alcohol insoluble residue (AIR Gille et al 2009) This was treated 318

with macerozyme and tested for release of soluble likely low molecular weight pectin 319

fragments (Figure 7) Treatment with 025 macerozyme released significantly more 320

uronic acid from wild-type Col-0 AIR relative to gae1 gae6 AIR (Figure 7B) To test 321

activity of these pectin-containing solutions we infiltrated them into wild-type Col-0 322

plants and measured PAD3 expression PAD3 is required for synthesis of the 323

phytoalexin camalexin (Zhou et al 1999) and its expression is known to be induced by 324

OG treatment (Ferrari et al 2007) Fractions from macerozyme treated Col-0 walls 325

induced significantly more PAD3 expression than fractions derived from gae1 gae6 326

walls suggesting that macerozyme treatment did indeed release more active soluble 327

uronic acid possibly OGs from wild-type Col-0 walls than from gae1 gae6 walls (Figure 328

7C) 329

Next we studied the effect of macerozyme treatment on expression of PAD3 in 330

intact plants (Figure 8) This treatment did not lead to any visible leaf damage 331

(Supplemental Figure 8) In wild-type Col-0 PAD3 expression was induced three h after 332

treatment with 001 macerozyme (Figure 8A) By contrast no macerozyme-induced 333

induction of PAD3 expression was detected in gae1 gae6 (Figure 8A) However OG 334

treatment induced the expression of PAD3 in both genotypes indicating that gae1 gae6 335

plants do respond to OGs (Figure 8B) Macerozyme treatment also induced expression 336

of the SA marker genes PATHOGENESIS RELATED 1 (PR1) and SID2 in Col-0 but 337

not in gae1 gae6 (Figure 9 panel A and B) PR1 was not significantly induced by 338

purified OGs (Figure 9C) at the time and concentration tested but SID2 was induced by 339

OG treatment in both genotypes (Figure 9D) However the induction was stronger in 340

wild-type Col-0 In addition the JA markers JASMONATE-ZIM-DOMAIN PROTEIN 5 341

(JAZ5) and JAZ10 were induced by macerozyme and OG treatment in both Col-0 and 342

gae1 gae6 (Figure 10A and Supplemental Figure 11) Interestingly expression of JAZ5 343

312 Pectinase-Induced Immune Signaling Is Altered in gae1 gae6 Plants

Page 17 of 54

Page 18 of 54

Page 19 of 54

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344

345

346

and JAZ10 was already strongly induced in gae1 gae6 after mock inoculation (Figure 10

and Supplemental Figure 11) We observed no hyperresponsive induction by mock

treatment of PLANT DEFENSIN 12 (PDF12) a necrotrophy and late JA signaling

347 marker gene (Supplemental Figure 12A-D) PDF12 was also not induced by

348 macerozyme or OG treatments (Supplemental Figure 12A-D) This suggests that early

349 JA signaling is induced by mock-treatment in gae1 gae6 Because it is known that JA

350 and SA signaling act antagonistically (Pieterse et al 2012) the strong up-regulation of

351 JA-induced immune signaling in gae1 gae6 might explain the lower OG-induced SID2

352 expression in this genotype

353 To test whether other immune responses are altered in gae1 gae6 plants we

354 analyzed Mitogen-activated protein kinase (MAPK) activity following macerozyme and

355 OG treatments OGs are known to activate the MAPKs MPK3 and MPK6 in Arabidopsis

Page 21 of 54

(Galletti et al 2011) Macerozyme treatment activated MPK3 and MPK6 in wild-type 356

Col-0 whereas MAPK activation in gae1 gae6 was slightly reduced in four out of five 357

experiments performed (Supplemental Figure 13) By contrast OG-induced MAPK 358

activity was similar or increased in gae1 gae6 in four out of five experiments 359

(Supplemental Figure 13) Additionally mock treatment induced MAPK activity in gae1 360

gae6 more strongly than it did in wild-type Col-0 in four out of five experiments 361

performed (Fig S13) Collectively these results illustrate that activation of several 362

immune signaling componentsmdashcamalexin synthesis SA and JA signaling and 363

possibly MAPK activationmdashby macerozyme is substantially altered in gae1 gae6 This 364

suggests that release of soluble pectin fragments possibly OGs by pectinolytic 365

enzymes might be reduced in gae1 gae6 plants while recognition of OGs is intact 366

367

Following B cinerea Infection Production of Soluble Pectin and Deposition of 368

Callose Are Reduced in gae1 gae6 Plants 369

Although pad3 plants were more susceptible to the B cinerea isolates Fresa 525 370

and Gallo 1 which also grew better on gae1 gae6 growth of the isolate Pepper was 371

similar on Col-0 and pad3 (Supplemental Figure 9A) However we did not detect any 372

significant changes in camalexin production after treatment with B cinerea isolate Gallo 373

1 and Fresa 525 in gae1 gae6 (Figure 8D) Neither did we detect changes in PAD3 374

expression after treatment with any B cinerea isolate in gae1 gae6 (Figure 8E) 375

Additionally SID2 and PR1 were similarly induced by all B cinerea isolates in both Col-376

0 and gae1 gae6 (Figure 9E and F) and sid2 plants which are deficient for pathogen-377

induced SA biosynthesis did not show any differences in relative B cinerea growth 378

(Supplemental Figure 9B) Expression of JAZ10 was induced more strongly by Gallo 1 379

treatment in gae1 gae6 than in wild-type Col-0 but not by Fresa 525 or Pepper (Fig 380

10C) while dde2 plants deficient for JA biosynthesis showed enhanced susceptibility to 381

all B cinerea isolates tested (Supplemental Figure 9B) Curiously exogenous 382

application of MeJA did not lead to altered relative fungal growth of B cinerea 383

(Supplemental Figure 9C) and cev1 isoxaben resistant 1 (ixr1) plants which have 384

constitutively high JA levels (Ellis and Turner 2001) did not show any changes in 385

susceptibility to the B cinerea isolates tested (Supplemental Figure 9D) Hence neither 386

Page 22 of 54

differences in B cinerea isolate susceptibility to camalexin nor altered SA or JA 387

signaling by themselves explained the enhanced susceptibility of gae1 gae6 to these B 388

cinerea isolates 389

Hydrogen peroxide accumulation in response to B cinerea isolates Fresa 525 390

Gallo 1 and Pepper was not obviously altered in gae1 gae6 plants (Supplemental Figure 391

14) However callose deposition upon treatment with B cinerea isolate Gallo 1 was 392

strongly reduced in gae1 gae6 (Figure 11) It was previously shown that OG treatment 393

induces callose deposition (Denoux et al 2008) suggesting that the reduction in 394

callose deposition might be due to reduced OG release in gae1 gae6 It is possible that 395

this reduction in callose deposition contributes to the reduced immunity of gae1 gae6 396

plants to B cinerea isolate Gallo 1 397

Because macerozyme could be used to release soluble likely low molecular 398

weight pectin from isolated cell walls we next investigated whether infection with B 399

cinerea was accompanied by accumulation of soluble pectin as well We prepared cell 400

walls (AIR fraction) from leaves infected with B cinerea incubated these cell walls with 401

water and measured uronic acid content of the extracts B cinerea treatment did in fact 402

lead to increased accumulation of soluble pectin in wild-type Col-0 plants when 403

compared to mock-treated samples (Figure 12) No such increased accumulation of 404

water-soluble pectin fractions was observed in gae1 gae6 (Figure 12) 405

406

Macerozyme-Induced Pattern-Triggered Immunity against B cinerea Is Abolished 407

in gae1 gae6 408

It was previously shown that OG treatment can induce patternndashtriggered 409

immunity (PTI) to B cinerea (Ferrari et al 2007) We tested macerozyme treatment to 410

determine whether it could also induce PTI to the B cinerea isolate Gallo 1 (Figure 13) 411

Indeed pretreatment with 001 macerozyme for 24 h led to reduced B cinerea growth 412

in wild-type Col-0 but not gae1 gae6 plants (Figure 13A) Interestingly mock and 413

macerozyme pretreated gae1 gae6 plants exhibited similar growth of B cinerea Gallo 1 414

as did macerozyme-treated Col-0 plants This result is likely due to activation of JA 415

signaling in gae1 gae6 during the pretreatment procedure (Figure 13C and D) OG-416

induced PTI was observed in both genotypes when using a high dose of OGs (Figure 417

Page 23 of 54

13B) whereas a lower dose of OGs only induced PTI in Col-0 (Supplemental Figure 418

15) This suggests that OG perception in gae1 gae6 at least above a certain threshold 419

is unaltered while release of immunity-activating pectin fragments possibly OGs is 420

421 reduced We conclude that immune responses induced by macerozyme treatment

422 which do not occur in gae1 gae6 plants are important for plant immunity to the B

423 cinerea isolate Gallo 1

Page 24 of 54

____________________________________________________________________________

Page 25 of 54

DISCUSSION 426

Here we showed that loss of GAE1 and GAE6 leads to a dramatic reduction in 427

pectin content in the cell wall (Figure 4) likely causing the brittleness of leaves that we 428

observed (Figure 3) Petioles and midribs of gae1 gae6 plants broke easily when bent 429

and inoculation of leaves with deionized water resulted in increased ion leakage 430

possibly due either to microscopic fractures of the cell wall induced by inoculation stress 431

or to a reduced capability of these walls to withstand increased turgor pressure The 432

increased brittleness was associated with altered cell wall composition In our analysis 433

the GalA content of gae1 gae6 leaf walls was reduced by more than 40 and the total 434

uronic acids by over 60 (Figure 4 and Supplemental Figure 5) This is a major change 435

in the overall cell wall composition considering that pectin makes up about 50 of the 436

plant cell walls found in Arabidopsis leaves (Zablackis et al 1995 Nobuta et al 2007 437

Harholt et al 2010) Interestingly quasimodo1 (qua1) plants which have a mutation in 438

a glycosyltransferase family 8 gene are reduced in GalA by 25 and show reduced 439

binding of HG specific antibodies (Bouton et al 2002) 440

Furthermore qua2 plants which have a mutation in a putative methyltransferase 441

have about 13 less total GalA and lack 50 of cell wall HG (Mouille et al 2007) 442

Likewise cgr2 cgr3 plants carrying mutations in another class of Golgi-localized 443

putative methyl-transferases have leaf cell walls that are reduced by about 40 in 444

uronic acid (Kim et al 2015) All three mutants are severely dwarfed (Bouton et al 445

2002 Mouille et al 2007 Kim et al 2015) Additionally qua1 and qua2 mutants show 446

a distorted overall shape likely due to reduced cell cohesion and are extremely fragile 447

(Bouton et al 2002 Mouille et al 2007) By contrast gae1 gae6 plants with a similar 448

strong reduction in GalA and total uronic acids have an overall normal shape and are 449

only slightly smaller than wild-type Col-0 (Supplemental Figure 4) In the wall analysis 450

performed here we did not detect changes in other wall polysaccharides or structures 451

that might compensate for the reduction of pectin in gae1 gae6 It is possible the severe 452

morphological alterations in qua2 and cgr2 cgr3 plants are due to reduction in pectin 453

methylesterification which is unaffected in gae1 gae6 It was speculated that quality 454

control mechanisms in the Golgi prevent secretion of inefficiently esterified pectin in 455

cgr2 cgr3 (Kim et al 2015) suggesting that the Golgi of these plants might over-456

Page 26 of 54

accumulate pectin and that this may contribute to the severe dwarf phenotype of these 457

plants The gae1 gae6 plants described here provide an opportunity to study the effects 458

of reduction of pectin in plants that do not show severe morphological phenotypes 459

We found that gae1 gae6 plants are also compromised in resistance to B 460

cinerea and Pma ES4326 indicating that loss of pectin reduces immunity to these 461

pathogens (Figure 2 and 6) We found that gae1 gae6 plants were slightly more 462

susceptible to Pma ES4326 but showed a strong increase in susceptibility to specific B 463

cinerea isolates This was not unexpected because B cinerea is known to interact with 464

pectin as part of its infection strategy It secretes various pectin-degrading enzymes 465

early during infection and requires some of these enzymes including PG1 PG2 and 466

PME1 for full virulence (ten Have et al 1998 Valette-Collet et al 2003 Kars et al 467

2005b Espino et al 2010) Additionally Arabidopsis plants expressing an antisense 468

construct of the polygalacturonase inhibitor PGIP1 are significantly more susceptible to 469

B cinerea (Ferrari et al 2006) On the other hand Pma ES4326 is not known to470

require cell wall degrading enzymes for full virulence even though it encodes two 471

pectate lyase genes in its genome (PMA4326_07094 and PMA4326_04621 472

wwwbacteriaensemblorg) B cinerea isolates are also known to be genetically quite 473

diverse in their polygalacturonase (PG) loci and genetic variability in PG2 is associated 474

with varying growth on pectin containing media (Rowe and Kliebenstein 2007) 475

Furthermore the contribution of PG1 and PG2 to B cinerea virulence seems to depend 476

on the specific B cinerea isolate and the host species (Kars et al 2005a Zhang and 477

Van Kan 2013) B cinerea isolates are also known to be differently sensitive to the anti-478

microbial secondary metabolite camalexin (Kliebenstein et al 2005) In line with these 479

observations the susceptibility of gae1 gae6 plants differed depending on the specific 480

B cinerea isolate Whereas Fresa 525 Gallo 1 UK Razz and KT grew better in gae1481

gae6 we detected no difference in Acacia Apple 517 DN Grape Pepper or Rasp 482

growth (Figure 6) 483

B cinerea was shown to metabolize pectin and to possibly utilize GalA as a484

carbon source during in planta growth (Zhang et al 2011 Zhang and Van Kan 2013) 485

Our data show that gae1 gae6 plants which are reduced in GalA content did allow 486

enhanced growth of specific B cinerea isolates but no reduction in growth of any B 487

Page 27 of 54

cinerea isolates tested was found This suggests that utilization of pectin as a carbon 488

source was not a contributing factor to alterations in B cinerea susceptibility It is 489

possible that changes in the cell wall composition affect the structural integrity of the 490

wall and affect hyphal penetration of specific B cinerea isolates However OGs (small 491

HG fragments) are likely released during the infection process (Ferrari et al 2013) 492

OGs function as DAMPs and initiate immune responses in Arabidopsis (Ferrari et al 493

2013) Treatment of Arabidopsis with OGs having a degree of polymerization (DP) of 494

10-15 sugar subunits was previously shown to enhance resistance to B cinerea (Ferrari495

et al 2007) We showed that pretreatment with OGs or macerozyme induced enhanced 496

immunity to B cinerea isolate Gallo 1 in wild-type Col-0 but only OGs and not 497

macerozyme induced immunity in gae1 gae6 (Figure 13) Our data suggest gae1 gae6 498

plants which have reduced pectin content may release less OGs and that this 499

reduction leads to the increased susceptibility to B cinerea 500

Consistently we found that macerozyme treatment released less soluble pectin 501

from gae1 gae6 cell walls than from wild-type walls Fractions containing soluble pectin 502

released from gae1 gae6 were also less active in inducing PAD3 gene expression than 503

the fractions released from wild-type walls (Figure 7) We also showed that less soluble 504

pectin accumulated in gae1 gae6 than in wild-type plants 48 h after treatment with B 505

cinerea (Figure 12) 506

We observed that loss of pectin leads to alterations in A thaliana immune 507

signaling Macerozyme treatment induced expression of PAD3 and the SA marker 508

genes PR1 and SID2 in wild-type Col-0 but not in gae1 gae6 whereas OG treatment 509

led to increased PAD3 and SID2 expression in both wild-type and gae1 gae6 (Figure 8 510

and 9) This suggests that although release of active soluble pectin in these plants 511

might be altered OG recognition is intact In fact cell walls isolated from qua1 another 512

mutant known to be reduced in HG released less OGs (DP1-6) than did wild-type cell 513

walls upon endopolygalacturonase treatment (Bouton et al 2002) This was also true 514

for gae1 gae6 cell walls as macerozyme treatment of isolated gae1 gae6 cell walls 515

released less soluble pectin than treatment of Col-0 cell walls Because the fraction 516

from gae1 gae6 plants was also less active in inducing PAD3 expression (Figure 7) it 517

seems possible that biologically active OGs were the active component inducing PAD3 518

Page 28 of 54

expression Interestingly expression of the JA marker genes JAZ5 and JAZ10 was 519

higher in gae1 gae6 than in wild-type mock-inoculated plants but was comparable to 520

wild-type in untreated plants suggesting that JA signaling in gae1 gae6 is 521

hyperresponsive (Figure 10 and Supplemental Figure 11) This induction of JA signaling 522

might be due to wounding stress incurred during the inoculation process JA and SA 523

signaling generally act antagonistically (Pieterse et al 2012) Increased JA signaling 524

activity might hence explain why mock- and macerozyme-treated gae1 gae6 plants 525

exhibited reduced expression of the SA marker PR1 and why OG treatment induced 526

expression of SID2 another SA marker to a lower extent in gae1 gae6 than in wild-type 527

plants (Figure 9) 528

No single immune signaling component was found to be the sole contributor to 529

the compromised immunity of gae1 gae6 to specific B cinerea isolates such as Gallo 1 530

and Fresa 525 These isolates grew better on pad3 while growth of Pepper another B 531

cinerea isolate that did not show altered virulence on gae1 gae6 was unaltered 532

(Supplemental Figure 9) This suggested that differences in camalexin sensitivity of 533

these B cinerea isolates might account for the differences in gae1 gae6 susceptibility 534

However camalexin accumulation and PAD3 expression was the same for wild-type 535

Col-0 and gae1 gae6 for the B cinerea isolates Fresa 525 and Gallo 1 (Figure 8) which 536

showed enhanced growth on gae1 gae6 plants It should be noted that B cinerea 537

virulence is often estimated by comparing lesion sizes Lesion size and measurements 538

of relative fungal growth by qPCR do not always show the same results For example 539

although Denby et al (2004) showed that Pepper causes larger lesions on pad3 than on 540

wild-type this study showed that relative fungal growth of Pepper on pad3 was 541

unaltered 542

We observed that callose deposition was strongly reduced in gae1 gae6 plants 543

72 h after treatment with B cinerea isolate Gallo 1 (Figure 11) possibly due to reduced 544

release of active soluble pectin Interestingly OG-induced PTI to an unspecified isolate 545

of B cinerea was intact in the pmr4 mutant which does not produce callose (Galletti et 546

al 2008) Due to the high SA levels in pmr4 plants (Nishimura et al 2003) and the fact 547

that different B cinerea isolates vary both in induction of and reaction to defense 548

responses (Kliebenstein et al 2005 Rowe et al 2010) we could not determine 549

Page 29 of 54

whether reduced callose deposition causes the increased susceptibility of gae1 gae6 to 550

B cinerea isolate Gallo 1551

We did not observe any changes in B cinerea growth either on cev1 ixr1 plants 552

which are known to have constitutively high JA and OPDA levels (Ellis et al 2002b) or 553

on plants exogenously treated with MeJA (Supplemental Figure 9) Because plants 554

were kept uncovered for 3 h after MeJA treatment it is unclear how much MeJA was 555

bioavailable in the plant However JAZ10 expression was induced 3 h after spraying 556

with MeJA (Supplemental Figure 10) Because pathogen growth generally depends on 557

the combination of a multitude of immune signaling events we conclude that multiple 558

changes to the immune signaling in gae1 gae6 together account for the altered B 559

cinerea growth phenotypes detected 560

We showed that expression of GAE1 and GAE6 is repressed by the bacterial 561

hemibiotroph Pma ES4326 This repression did not occur in an eds1 or pad4 562

background or in pbs3 at the 24-h time point (Figure 1 and Supplemental Figure 1) 563

During the Arabidopsis - Pma ES4326 interaction the expression of many defense 564

related genes is altered in a PAD4EDS1 dependent fashion (Wang et al 2008) Some 565

of these expression changes require SA CBP60g and SARD1 are thought to act 566

downstream of PAD4 and EDS1 but upstream of SA-dependent responses (Wang et 567

al 2011) PBS3 is also thought to act downstream of PAD4 and EDS1 and upstream of 568

SA but the set of genes whose expression it affects is only partially overlapping with 569

those affected by CBP60gSARD1 (Wang et al 2008 Wang et al 2011) Thus GAE1 570

and GAE6 repression is regulated by the part of the immune signaling network that 571

requires PAD4 EDS1 and PBS3 but is independent of CBP60gSARD1 and SA 572

Because EDS1 PAD4 and PBS3 are important components of the plant immune 573

signaling network (Wiermer et al 2005 Nobuta et al 2007) one might conclude that 574

repression of GAE1 and GAE6 may be part of the plant immune response to Pma 575

ES4326 B cinerea treatment also reduced GAE1 and GAE6 expression but this was 576

mostly independent of PAD4 (Figure 6) while both Pma ES4326 and B cinerea repress 577

GAE1 and GAE6 expression the host signaling mechanisms responsible likely differ 578

In summary our data show that Arabidopsis GAE1 and GAE6 are required for 579

pectin biosynthesis leaf flexibility and immunity to Pma ES4326 and to specific B 580

Page 30 of 54

cinerea isolates We used macerozyme a commercial pectinase to mimic the effect of 581

B cinerea pectinases Macerozyme treatment released less pectin from gae1 gae6 cell582

walls and induced less immune signaling in gae1 gae6 plants Less water soluble pectin 583

also accumulated in B cinerea infected gae1 gae6 plants and macerozyme-induced 584

immunity to B cinerea isolate Gallo 1 was reduced in these plants We conclude that 585

the reduced pectin content in these mutants may lead to reduced release of soluble 586

pectin including active OGs during pathogen attack and that this compromises 587

immunity 588

589

590

Page 31 of 54

METHODS 591

Plant Materials and Growth Conditions 592

Wild-type Columbia (Col-0) and all mutant plants (in Col-0 background) were 593

grown on sterilized BM2 germinating mix (Berger Inc Quebec Canada) in a controlled 594

environment chamber (Conviron Pembina ND USA) with a 12-h photoperiod under 595

100 microM m-2 s-1 fluorescent illumination at 22degC and 75 relative humidity Germplasm 596

used is described in the Accession Numbers section at the end of Methods 597

598

Data Analysis and Replicates 599

For biological replicates samples were obtained from separate plant tissues and 600

in many cases from plants grown in different flats In some experiments multiple 601

technical replicates were measured per biological replicate For statistical analysis all 602

technical replicates were averaged and the averages were considered one biological 603

replicate Independent experiments were performed at different times and often 604

consisted of multiple biological replicates per experiment 605

Data analysis was performed using mixed linear models in the R programming 606

environment (Bates et al 2015) unless otherwise indicated Generally genotypes 607

treatments and time points were used as fixed effects and replicate-specific effects like 608

different pots flats etc were considered random effects We reported p-values 609

obtained from the mixed linear model when less than 10 comparisons were performed 610

and q-values for more than 10 comparisons to correct for multiple comparison testing 611

Q-values were calculated using the Benjamini-Hochberg procedure (Benjamini and612

Hochberg 1995) unless specified otherwise 613

614

Pathogen Strains Growth Conditions and Pathogen Growth Assays 615

Growth of Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) and 616

bacterial growth assays were performed as previously described (Bethke et al 2014) 617

For qRT-PCR assays four week-old plants were inoculated with bacteria (OD600 = 618

0002) or mock (5 mM MgSO4) At least four leaves from two plants were collected at 619

the indicated time points per biological replicate 620

Page 32 of 54

Prior to each experiment Botrytis cinerea isolates were grown on 1x PDA 621

medium (Difco) for 10 d at room temperature All isolates used have been described 622

previously (Rowe and Kliebenstein 2007) The spores were washed from the surface of 623

the plate using an inoculation medium (1x Gamborgrsquos B-5 basal salt mixture (Sigma-624

Aldrich G5768) 2 (wv) glucose 10 mM phosphate buffer pH 64) and fungal hyphae 625

were removed from the suspension by filtering through four layers of cheesecloth The 626

concentration of spores was determined using a hemocytometer and adjusted to 25 x 627

105 spores mL-1 Four week-old Arabidopsis plants were inoculated by placing one 10-628

microL droplet of the B cinerea spore solution or inoculation medium (mock) on the adaxial 629

leaf surface of fully expanded leaves Inoculated plants were kept at 100 relative 630

humidity Infection sites were collected at various time points using a cork borer For 631

qRT-PCR assays at least four leaf discs from one plant were combined for each 632

biological sample For B cinerea growth assays at least 12 leaf discs from three to six 633

plants were pooled per biological sample Relative fungal growth was determined by 634

measuring the abundance of a fungal gene relative to a plant gene as described below 635

In brief DNA from infected tissue was extracted and equal amounts of total DNA were 636

used to perform qPCR reactions using the B cinerea cutinase A and the Arabidopsis 637

SK11 gene respectively Primers were described in (Gachon and Saindrenan 2004) 638

and are listed in Supplemental Table 1 Two technical replicates were combined for 639

each biological replicate B cinerea is a necrotrophic fungal pathogen and may destroy 640

plant DNA as disease progresses However decreases in the amount of plant DNA 641

over the course of our experiments were only observed at late time points with very 642

susceptible plants so the qPCR assay is generally a reasonable estimate of fungal 643

biomass (Supplemental Figure 16) 644

645

Treatment with Macerozyme or OGs 646

For PTI assays four week-old plants either were inoculated with 001 (wv) 647

macerozyme R-10 (Yakult) a Rhizopus polygalacturonase (EC 32115) that possesses 648

high pectinase and hemicellulase activity or were mock inoculated using heat-649

inactivated macerozyme Other similar plants were either inoculated with 250 or 500 microg 650

mL-1 OGs or mock inoculated with water One d later plants were infected with B 651

Page 33 of 54

cinerea and samples were collected immediately (0 dpi) and two or three d later To 652

investigate the role of MeJA on B cinerea growth plants were sprayed with 1mM MeJA 653

in water with 001 (vv) Tween 20 Covers were removed so that leaves could dry off 654

before plants were inoculated with B cinerea three h later Samples were harvested at 655

the indicated time points For expression analysis and MAPK activation assays plants 656

were either inoculated with 100 microg mL-1 OGs or mock inoculated with water OGs used 657

for experiments described in Figures 8 to 10 and Supplemental Figure 12 were a kind 658

gift from Simone Ferrari OGs used in experiments described in Figure 13 and 659

Supplemental Figures 11 13 and 15 were prepared as previously described (Kohorn et 660

al 2014) 661

662

Expression Analysis 663

Leaf tissue from inoculated plants was harvested at the indicated time points 664

flash frozen pulverized and RNA extracted using Trizol (Invitrogen) Quantitative RT-665

PCR was performed using the SuperScript III Platinum SYBR Green One-Step 666

quantitative RT-PCR kit (Invitrogen) and a Lightcycler 480 Real-Time PCR system 667

(Roche) as previously described (Truman and Glazebrook 2012) In brief equal 668

amounts of total RNA and a gene specific primer were used for each reaction The 669

crossing point (Cp) was calculated using the 2nd derivative max method provided with 670

the Lightcycler software for each amplification curve Each reaction was run with two 671

technical replicates and the Cp values for these replicates were averaged ACTIN2 was 672

used as a stably expressed reference gene At least three biological replicates each 673

were performed and analyzed using a mixed-linear model RT-PCR was performed to 674

verify T-DNA insertion lines using the One-Step RT-PCR kit (Qiagen) according to the 675

manufacturerrsquos instructions The primers used can be found in Supplemental Table 1 676

677

Electrolyte Leakage Assay 678

Fully expanded leaves of four week-old plants were inoculated with water (Milli-Q 679

grade) using a needleless syringe Leaf discs were collected using a cork borer and 680

three leaf discs from one leaf were placed with the adaxial surface down onto 2 mL 681

water in a 12-well cell culture plate Conductivity was measured using a Horiba B-173 682

Page 34 of 54

conductivity meter after 30 min and then the water was replaced Conductivity was 683

measured again 30 min later (1 h total) as indicated in Figure 3C Six biological 684

replicates each were performed in three independent experiments and means and SEs 685

were calculated Two-sided t-tests were performed for each time point 686

687

DAB and Aniline Blue Staining 688

Leaves from four week-old plants were either mock inoculated with B cinerea 689

inoculation medium or inoculated with 10 microL droplets of a B cinerea spore solution (25 690

x 105 spores mL-1) of the indicated isolates Samples were collected 72 h later For 33prime-691

diaminobenzidine (DAB) staining two to five inoculated leaves were vacuum infiltrated 692

with 33prime-Diaminobenzidine (1 mg mL-1 in water pH 38) solution and stained overnight 693

in the dark Leaves were destained using 100 ethanol at 65degC and then transferred to 694

50 (vv) glycerol and photographed 695

Callose was visualized by staining leaves with aniline blue as previously 696

described (Adam and Somerville 1996) Briefly leaves were vacuum infiltrated with 697

alcoholic lactophenol (one volume of phenolglycerollactic acidwater (1111) mixed 698

with two volumes of ethanol) and destained at 65degC Next leaves were moved through 699

an alcohol gradient (50 20 and 10 (vv) ethanol) into water The destained leaves 700

were stained for 30 min in 150 mM K2HPO4 (pH 95) with 001 (wv) aniline blue 701

Samples were mounted in water and examined using a Nikon Eclipse Ni-U microscope 702

at 10x magnification using a DAPI filter (excitation at 325-375 nm emission at 435-485 703

nm) For callose quantification six 200 microm x 200 microm squares were counted for each 704

sample type Callose deposits per mm2 were calculated and data analyzed using a t-705

test Note that there is an inherent uncertainty associated with such counts because 706

callose deposits may not all be in focus at the same time 707

708

MAPK Activity Assay 709

Fully expanded leaves of four week-old plants were inoculated with 001 710

macerozyme 100 microg mL-1 OGs 100 nM flg22 or water (mock) flg22 a peptide from 711

bacterial flagella that induces immune responses was used as a positive control for 712

Page 35 of 54

MAPK activation (Asai et al 2002) and was purchased from EZBiolab Inc (Westfield 713

IN USA) Samples were harvested 10 min later and immediately frozen in liquid 714

nitrogen Protein was extracted using an extraction buffer containing 25 mM Tris pH 78 715

75 mM NaCl 1 mM DTT 1 mM NaF 05mM Na3VO4 01 (vv) Tween 20 one 716

cOmplete Mini protease inhibitor cocktail tablet (Roche) and one PhosSTOP 717

phosphatase inhibitor cocktail tablet (Roche) per 10 mL buffer 15 microg of total protein 718

were electrophoresed on a 10 SDS-polyacrylamide gel and then blotted onto a PVDF 719

membrane (Bio-Rad 162-0177) Activated MAPKs were detected using p4442 MAPK 720

(Erk12) antibody (9102S Cell Signal 12500 in TBST (20 mM Tris pH 75 150 mM 721

NaCl 01 (vv) Tween 20)) Anti-Rabbit IgG-HRP (115000 in TBST A6154 Sigma) 722

and ECL Plus substrate (Pierce 32132) MPK3 and MPK6 were detected with an anti-723

MPK3 antibody (M8318 Sigma 12000 in TBST and 3 (wv) milk) or an anti-MPK6 724

antibody (A7104 Sigma 16000 in TBST and 3 (wv) milk) respectively The 725

secondary antibody and detection were the same as for the p4442 antibody Five 726

independent experiments were performed 727

728

Preparation of Alcohol Insoluble Residue (AIR) Pectin Extraction and Uronic Acid 729

Measurements 730

Fully expanded leaves from four week-old plants that were incubated for 48 h in 731

the dark to reduce starch were harvested flash frozen and pulverized AIR was 732

extracted by washing ground material twice in 70 (vv) ethanol three times in a 733

mixture of chloroform and methanol (11 vv) and once in acetone (Gille et al 2009) 734

and was then air dried Cell wall pectin was extracted from AIR with cell wall extraction 735

buffer (50 mM Trizma 50 mM CDTA pH 72) at 95degC for 15 min Samples were 736

homogenized using a paint shaker (Harbil 5G-HD) and glass beads (3 mm diameter) 737

For dot blot experiments 500 microL of cell wall extraction buffer were used per 10 mg of 738

AIR Debris was precipitated by centrifugation for 10 min at 10000 g 739

Total uronic acid content of pectin extracted as described above using 500 microL of 740

cell wall extraction buffer per 1 mg of AIR was measured as described previously 741

(Filisetti-Cozzi and Carpita 1991 van den Hoogen et al 1998) First 36 microL of pectin 742

extract were mixed with 4 microL of 4 M sulfamic acid Then 200 microL of sulfuric acid 743

Page 36 of 54

containing 120 mM sodium tetraborate was added and samples were incubated at 80degC 744

for 1 h Following cooling on ice the optical density of samples was measured at 490 745

nm Next 40 microL of m-hydroxydiphenyl reagent (100 microL of m-hydroxydiphenyl in dimethyl 746

sulfoxide at 100 mgmL mixed with 49 mL 80 (vv) sulfuric acid just before use) was 747

added and the samples were mixed The optical density at 490 nm was measured 748

again The optical density before m-hydroxydiphenyl reagent addition was subtracted 749

from the optical density measured after addition of the dye The concentration of uronic 750

acid was calculated using known amounts of GalA as a standard 751

752

Release of Pectin from Arabidopsis Cell Walls Using Macerozyme 753

AIR preparations from leaves of four week-old plants were treated with 025 754

(wv) macerozyme R-10 (Yakult) or water (mock) at a concentration of 100 microL per mg 755

AIR Samples were incubated for 3 h at room temperature on a vortex shaker and 756

centrifuged for 10 min at 12000 g Total uronic acid concentration of 5-fold diluted 757

supernatants was determined for two technical replicates each The concentration of 758

uronic acid was calculated using known amounts of GalA as a standard For 759

subsequent qRT-PCR experiments supernatants from multiple extractions were 760

combined boiled for 20 min to inactivate macerozyme diluted 10-fold with water and 761

inoculated into four week-old Col-0 plants Heat-inactivated 10-fold diluted macerozyme 762

solutions were used as mock treatment Samples were collected 3 h later and PAD3 763

expression was determined 764

765

Release of Pectin from Arabidopsis Leaves Infected with B cinerea 766

Fully expanded leaves of four week-old plants were either inoculated with up to 767

five 10 microL droplets of B cinerea spore solution (25 x 105 spores mL-1) or mock 768

inoculated with B cinerea inoculation medium For each biological replicate leaves with 769

a combined count of at least 20 infection sites were collected 48 h later AIR was 770

extracted as described above but plants were not dark-treated or freeze-dried and the 771

first ethanol step was performed using 85 (vv) ethanol To extract soluble pectin 772

fragments AIR samples were mixed with water at a concentration of 20 microL per infection 773

site incubated for 7 h at room temperature on a vortex shaker and centrifuged for 10 774

Page 37 of 54

min at 12000 g Total uronic acid concentration of supernatants was determined for two 775

technical replicates each The concentration of uronic acid was calculated using known 776

amounts of GalA as a standard 777

778

Measurement of Uronic Acids Neutral Monosaccharides and Cellulose Content in 779

Cell Walls 780

Neutral sugars and uronic acids from non-cellulosic polysaccharides in cell walls 781

(AIR) were released by trifluoroacetic acid (TFA) hydrolysis Approximately 1 mg of AIR 782

was hydrolyzed with 2 M TFA at 121degC for 90 min The TFA-soluble material was split 783

into two equal parts one for uronic acid and one for neutral sugar determination The 784

uronic acid content was determined by analyzing the hydrolyzate using a CarboPac 785

PA200 anion-exchange column with an ICS-3000 Dionex chromatography system 786

(Dionex Sunnyvale CA USA) The elution profile consisted of a linear gradient of 50 to 787

200 mM sodium acetate in 01 M NaOH in 10 min at 04 mL per min Alditol acetate 788

derivatives of neutral sugars were produced as described (York et al 1985) In brief 789

monosaccharides in the hydrolyzate were reduced with NaBH4 and peracetylated with 790

acetic anhydride and pyridine at 121degC for 20 min The generated alditol acetates were 791

analyzed by GC-MS Crystalline cellulose was measured according to Updegraff (1969) 792

After hydrolysis of noncellulosic polysaccharides from AIR with the Updegraff reagent 793

(acetic acid nitric acids water 812 (vv) ) the remaining pellet was hydrolyzed in 794

72 sulfuric acid The resulting glucose quantity was determined by the anthrone 795

method (Scott and Melvin 1953) 796

797

Dot Blot Experiments 798

Pectin solutions extracted using 50 microL of cell wall extraction buffer per mg AIR 799

were serially diluted and nitrocellulose membranes were spotted with 1 microL of the diluted 800

pectin solutions Membranes were dried overnight blocked with 5 milk (wv) in 1x 801

PBS (8 g L-1 NaCl 02 g L-1 KCl 144 g L-1 Na2HPO4 024 g L-1 KH2PO4 pH 74) and 802

probed with LM5 (Jones et al 1997) LM6 (Willats et al 1998) LM8 (Willats et al 803

2004) LM19 and LM20 (Verhertbruggen et al 2009a) or CCRC-M7 (Steffan et al 804

Page 38 of 54

1995) antibodies LM series antibodies were diluted 1250 and CCRC-M7 was diluted 805

1500 in 5 milk powder (Nestle Carnation) in 1 x PBS For LM series antibodies a 806

goat anti-Rat HRP conjugated antibody (Bethyl A110-105P 15000 diluted) and for 807

CCRC-M7 an anti-Mouse IgG HRP conjugate (Promega W402B 12500 diluted) was 808

used as secondary antibody in 5 milk powder in 1x PBS LM series antibodies were 809

obtained from plant probes (wwwplantprobesnet) and CCRC-M7 from CarboSource 810

(wwwcarbosourcenet) Membranes were washed after incubation with the primary and 811

secondary antibodies with 1x PBS Dot blots were developed using the ECL system 812

(GE Healthcare Amersham ECL prime) For quantification of signals dot blot results on 813

x-ray films were photographed using a CCD camera and intensities were measured814

using Image Jrsquos Integrated Density function Locally adjusted background was 815

subtracted As the dynamic range of the signal on an x-ray film was narrow non-816

linearity in the measured values was corrected for each genotype in each biological 817

replicate using the values from different dilutions in two technical replicates We noticed 818

that the residuals were not normally distributed and performed Box-Cox power 819

transformation (Box and Cox 1964) to obtain normally distributed values for the 820

subsequent statistical analysis of the data For LM19 Box-Cox selected power 821

transformation was done to the power of 038th and for LM20 to the power 075th After 822

correction of the transformed measured values with the effect of the biological 823

replicates the values were fit to a linear model with the Arabidopsis genotype as the 824

fixed effect This linear model was used both to generate the plots in Figures 5B and D 825

and to test significant differences 826

827

Camalexin Measurements 828

Four week-old plants were infected with B cinerea and samples (three leaf disks 829

from one plant each) were harvested at the indicated time points flash frozen and 830

pulverized Each sample was extracted with 300 microL of 90 methanol (vv) 50 microL of the 831

extract was run on an Agilent Lichrocart 250-4 RP18e 5-microm column using an Agilent 832

1100 series HPLC Camalexin was detected using a DiodeArray (DAD) at 330 nm and 833

with a fluorescence detector at emission 318 nmexcitation 385 nm (Agilent) Separation 834

was achieved using the following program with aqueous acetonitrile 5 min gradient 835

Page 39 of 54

from 63 to 69 acetonitrile 30 s gradient from 69 to 99 acetonitrile 2 min at 99 836

acetonitrile and a post-run equilibration of 35 min at 63 acetonitrile (Denby et al 837

2004 Kliebenstein et al 2005) Purified camalexin was used to produce a standard 838

curve to identify and quantitate camalexin 839

840

Accession Numbers 841

Microarray data used can be accessed at NCBI Gene Expression Omnnibus 842

(httpwwwncbinlmnihgovgeo) GSE18978 Sequence information for all Arabidopsis 843

genes described in this article can be found in TAIR (wwwarabidopsisorg) using the 844

following accession numbers PAD3 (CYP71B15 At3g26830) SID2 (ICS1 At1g74710) 845

GAE1 (At4g30440) GAE2 (At1g02000) GAE3 (At4g00110) GAE4 (At2g45310) GAE5 846

(At4g12250) GAE6 (At3g23820) PR1 (At2g14610) JAZ5 (At1g17380) JAZ10 847

(At5g13220) ACTIN2 (At3g18780) PDF12a (At5g44420) PDF12b (At2g26020) 848

AtSK11 (At5g26751) The sequence of the B cinerea cutinase Bc-CutA (Z69264) can 849

be found in the DNA Data Bank of Japan (httpgetentryddbjnigacjp) Germplasm 850

used included dde2-2 (At5g42650 (von Malek et al 2002)) pad3-1 (At3g26830 (Zhou 851

et al 1999)) pad4-1 (At3g52430 (Jirage et al 1999)) sid2-2 (At1g74710 (Wildermuth 852

et al 2001)) pbs3-2 (At5g13320 SALK_018225 (Nobuta et al 2007)) cbp60a-1 853

(At5g62570 SALK_124410 (Truman et al 2013)) cbp60g-1 sard1-2 (At5g26920 854

SALK_023199 At1g73805 SALK_052422 (Wang et al 2011) cbp60a-1 cbp60g-1 855

sard1-2 (Truman et al 2013) gae1-1 (At4g30440 SALK_085554) gae6-1 (At3g23280 856

SALK_104454) gae6-2 (At3g23280 SALK_017191) ixr1-1 cev1 (At5g05170 (Ellis 857

and Turner 2001 Scheible et al 2001)) eds1 plants were derived by introgression of 858

the Ler eds1-2 allele into Col-0 that contains two copies of EDS1 (At3g48090 859

At3g48080 (Bartsch et al 2006) mpk6-2 (At2g43790 SALK_073907 (Liu and Zhang 860

2004)) mpk3-DG (At3g45640 a fast neutron deletion mutant (Miles et al 2005)) and 861

npr1-1 (At1g64280 (Cao et al 1997)) T-DNA insertion lines were part of the SALK 862

collection (Alonso et al 2003) and were obtained from the Arabidopsis Biological 863

Resource Center 864

865

Supplemental Data 866

Page 40 of 54

Supplemental Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 867

Expression Does Not Require CBP60s or SID2 868

Supplemental Figure 2 Expression of GAE Family Members uponTreatment with Pma 869

ES4326 870

Supplemental Figure 3 Characterization of T-DNA Insertions in GAE1 and GAE6 871

Supplemental Figure 4 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves and 872

Are Slightly Smaller than Wild-Type Col-0 Plants 873

Supplemental Figure 5 Cell Walls of gae1 gae6 Plants Contain Less Uronic Acid than 874

Wild-Type Col-0 Cell Walls 875

Supplemental Figure 6 Antibodies Raised Against the RGI or XG Components of 876

Pectin Show No Difference in Binding to gae Mutant and Col-0 Cell Walls 877

Supplemental Figure 7 The Effect of B cinerea Treatment on the Expression of GAE 878

Family Genes 879

Supplemental Figure 8 Treatment with 001 Macerozyme Does Not Cause Any 880

Visible Damage to Col-0 Plants 881

Supplemental Figure 9 B cinerea Growth on Plants with Defects in Immune 882

Signaling 883

Supplemental Figure 10 MeJA-Induced Expression of the JA marker gene JAZ10 884

Supplemental Figure 11 The Expression of the JA Marker Gene JAZ5 Is Highly 885

Responsive in gae1 gae6 Plants 886

Supplemental Figure 12 The Expression of PDF12 Is Unaltered in gae1 gae6 Plants 887

Supplemental Figure 13 Macerozyme Treatment Activates MPK3 and MPK6 888

Supplemental Figure 14 B cinerea-Induced ROS Production in Wild-Type Col-0 and 889

gae1 gae6 Plants 890

Supplemental Figure 15 OG-Induced PTI Using 250 microg mL-1 OGs 891

Supplemental Figure 16 Abundance of Plant DNA in B cinerea Treated Plants 892

Supplemental Table 1 Primers used in this study 893

894

ACKNOWLEDGEMENTS 895

We thank William Truman for helpful discussions Simone Ferrari (Sapienza 896

Universita di Roma) for the generous gift of OGs Bruna Bucciarelli (USDA-ARS) for 897

Page 41 of 54

help with fluorescence microscopy the Arabidopsis Biological Resource Center for T-898

DNA insertion lines Paul Knoxrsquos lab (University of Leeds) for LM5 LM6 LM8 LM19 899

and LM20 antibodies and the Complex Carbohydrate Resource Center (University of 900

Georgia) for the CCRC-M7 antibody Most of this work was funded by the Division of 901

Chemical Sciences Geosciences and Biosciences Office of Basic Energy Sciences of 902

the US Department of Energy through Grant DE-FG02-05ER15670 to JG This work 903

was also supported by NSF awards IOS 1339125 and IOS 1021861 to DJK NSF 904

award IOS 1121425 to F K and the USDA National Institute of Food and Agriculture 905

Hatch project number CA-D-PLS-7033-H to DJK 906

907

AUTHOR CONTRIBUTIONS 908

GB and JG designed the research GB GX AT BL NES and NH 909

performed research DJK provided tools GB GX MP AD BL RAH and FK 910

analyzed data GB and JG wrote the paper with input from all authors 911

912

FIGURE LEGENDS 913

Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 Expression 914

Requires PAD4 EDS1 and PBS3 915

(A) Expression of GAE1 in leaves of wild-type Col-0 and mutant plants Expression was916

measured 24 or 32 h after either inoculation with Pma ES4326 (OD600 = 0002) or mock 917

inoculation with 5 mM MgSO4 and was then normalized to the level of ACTIN2 Data 918

from three biological replicates were combined using a mixed linear model Bars 919

represent mean log2-ratios to ACTIN2 plusmn SE Letters indicate significantly different 920

groups with q lt 005 where 005 represents the maximum false discovery rate at which 921

the test may be called significant 922

(B) Expression of GAE6 measured as described in (A)923

924

Figure 2 Susceptibility of gae6 and gae1 gae6 Plants to Pma ES4326 Is Increased 925

Relative to Wild-Type Col-0 926

Leaves of plants of the indicated genotypes were inoculated with Pma ES4326 (OD600 = 927

00002) as described in Methods Bacterial titers in leaves were determined either 928

Page 42 of 54

immediately (0 dpi) or after three days (3 dpi) Bars represent the mean plusmn SE of four 929

independent experiments each with 4 or 12 biological replicates at 0 and 3 dpi 930

respectively Data were combined using a mixed linear model Asterisks indicate 931

significant differences from Col-0 wild-type (q lt 005) Susceptible pad4 plants were 932

included as a positive control 933

934

Figure 3 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves 935

(A) Depiction of the assay for brittleness of gae mutant leaves Fully expanded leaves of936

four to five week-old plants were bent by 180ordm so that the adaxial surface of the leaf was 937

parallel with the soil Broken leaf petioles or midribs were considered brittle 938

(B) Quantification of broken midribs or petioles For each genotype 66 to 82 leaves 939

were analyzed for brittleness as described in (A) Bars represent the ratio of broken to 940

total number of leaves tested Results were analyzed using Fisherrsquos exact test Letters 941

indicate significantly different groups at p lt 005 942

(C) Conductivity measurements Leaves of four week-old plants were inoculated with943

deionized water and then leaf discs were cut out and placed on deionized water 944

Conductivity was measured at the indicated time points After 30 min the water was 945

replaced with fresh water (30 min f w) to account for wounding-related ion leakage from 946

the edges of the leaf discs Three independent experiments with six biological replicates 947

each were performed and data were analyzed using a t-test for each time point P-948

values were corrected using the Bonferroni method Data show means plusmn SE asterisks 949

indicate significant differences from wild-type Col-0 at q lt 001 950

951

Figure 4 Cell Walls of gae6 and gae1 gae6 Plants Are Reduced in GalA 952

(A) to (D) Levels of GalA (A) GlcA (B) cellulose (C) and neutral sugars (D) in alcohol953

insoluble residue (AIR) extracted from leaves of four week-old plants of the indicated 954

genotypes Bars represent means plusmn SE of four biological replicates combined using a 955

mixed linear model Letters indicate significantly different groups (p lt 005) 956

957

Figure 5 Cell Walls of gae1 gae6 Plants Are Reduced in Homogalacturonan 958

Page 43 of 54

Dot blot analyses with anti-LM19 and anti-LM20 antibodies Pectin was extracted from 959

AIR prepared from the indicated genotypes using 1 microL of extraction buffer per 10 microg of 960

AIR Pectin was serially diluted and one microL of undiluted 13 diluted and 19 diluted 961

samples were spotted on a nitrocellulose membrane Three biological replicates were 962

were spotted twice to obtain two technical replicates each All replicates produced 963

similar results 964

(A) Representative dot blot using anti-LM19 antibody965

(B) Relative signal intensities of the spots in the dot blot in (A)966

(C) Representative dot blot using anti-LM20 antibody967

(D) Relative signal intensities of the LM20 dot blot shown in (C)968

Relative signal intensities were measured using Image J software Measurements from 969

the three biological replicates were combined using a linear model after Box-Cox power 970

transformation as described in Methods Bars represent means plusmn SE Asterisks indicate 971

a difference from wild-type Col-0 at p lt 005 972

973

Figure 6 Growth of B cinerea on gae1 gae6 Plants and the Effect of B cinerea on 974

Expression of GAE1 and GAE6 975

(A) Growth of B cinerea isolates Leaves of Col-0 and gae1 gae6 plants were976

inoculated with 25 x 105 spores mL-1 of the B cinerea isolates indicated in the figure 977

Samples were collected two (2 dpi) and three (3 dpi) days later Bars represent means plusmn 978

SE of six biological replicates for each fungus combined using a mixed linear model 979

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 980

(B) and (C) Expression of GAE1 (B) and GAE6 (C) 48 h after inoculation with the B981

cinerea isolates indicated Expression levels were measured by qRT-PCR Bars 982

represent mean log2-ratios to ACTIN2 plusmn SE of three biological replicates combined 983

using a mixed linear model Letters indicate significantly different groups (q lt 005) 984

985

Figure 7 Levels of Soluble Pectin and Expression of PAD3 in Fractions Produced 986

Following Macerozyme Treatment of gae1 gae6 Cell Walls 987

(A) Schematic overview of the experimental procedure for (B) and (C)988

Page 44 of 54

(B) Uronic acid measurements Alcohol-insoluble residue (AIR) from leave tissue of four989

week-old wild-type Col-0 or gae1 gae6 plants was treated with water or 025 990

macerozyme and assayed for uronic acid levels Data from 11 biological replicates were 991

combined using a mixed linear model Bars represent means plusmn SE Letters indicate 992

significant differences (p lt 005) 993

(C) Expression of PAD3 in Col-0 plants treated with the fractions from (B) Extracts from994

(B) were heat inactivated pooled and diluted 5-fold with water Pooled extracts were995

inoculated into four week-old Col-0 plants PAD3 expression was measured 3 h after 996

inoculation and normalized to the level of ACTIN2 expression Data from four biological 997

replicates were combined using a mixed linear model Bars represent mean log2-ratios 998

to ACTIN2 plusmn SE Letters indicate significant differences (p lt 005) 999

1000

Figure 8 Macerozyme-Induced PAD3 Expression Is Absent in gae1 gae6 1001

(A) Expression of PAD3 after macerozyme treatment Four week-old plants were either1002

inoculated with 001 macerozyme or mock inoculated with water and PAD3 1003

expression was measured 3 h later by qRT-PCR Data from four biological replicates 1004

were combined using a mixed linear model Mean log2-ratios to ACTIN2 plusmn SE were 1005

plotted Letters indicate significantly different groups (p lt 005) 1006

(B) Expression of PAD3 after OG treatment PAD3 expression was measured as1007

described in (A) 3 h after inoculation with 100 microg mL-1 OGs or mock inoculation with 1008

water Data are from six biological replicates 1009

(C) PAD3 expression in untreated plants Three biological replicates were performed1010

and data analyzed as described in (A) 1011

(D) Camalexin accumulation after B cinerea treatment Camalexin accumulation was1012

measured in four week-old plants inoculated with the B cinerea strains shown in the 1013

figure Eight biological replicates each were combined using a mixed linear model 1014

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 1015

(E) PAD3 expression 28 h after treatment with the indicated B cinerea strains Data1016

from six biological replicates were collected and analyzed as described in (A) Data in 1017

all figure parts represent means plusmn SE Letters indicate significantly different groups (q lt 1018

005) 1019

Page 45 of 54

1020

Figure 9 Macerozyme-Induced Expression of the SA Marker Genes SID2 and PR1 1021

Is Abolished in gae1 gae6 Plants 1022

(A) to (H) Expression of PR1 (A) (C) (E) and (G) or SID2 (B) (D) (F) and (H) in four1023

week-old Col-0 and gae1 gae6 plants Data were combined using a mixed linear model 1024

Bars represent mean log2-ratios of expression vs ACTIN2 plusmn SE for all figure parts 1025

Letters indicate significantly different groups at p lt 005 for panels (A) to (D) (G) and 1026

(H) and qlt005 for panels (E) to (F) 1027

(A) and (B) Plants were treated with 001 macerozyme or mock treated with water1028

and samples were collected 3 h later Data from four biological replicates are shown 1029

(C) and (D) Plants were inoculated with 100 microg mL-1 OGs or mock treated with water1030

and samples collected 3 h later Data from six biological replicates are shown 1031

(E) and (F) Plants were inoculated with the indicated B cinerea isolates or mock1032

inoculated with B cinerea inoculation medium and samples collected 48 h later Data 1033

from eight biological replicates are shown 1034

(G) and (H) Expression in untreated plants Three biological replicates were performed1035

1036

Figure 10 The Expression of the JA Marker Gene JAZ10 is Highly Responsive in 1037

gae1 gae6 Plants 1038

(A) to (D) JAZ10 expression in four week-old plants Data were combined using a mixed1039

linear model Mean log2-ratios to ACTIN2 plusmn SE are shown Letters indicate significantly 1040

different groups at p lt 005 for panels (A) (B) and (D) and q lt 005 for panel (C) 1041

(A) Plants were inoculated with 001 macerozyme or mock inoculated using water and1042

samples were collected 3 h later Data from four biological replicates are shown 1043

(B) Plants were inoculated with 100 microg mL-1 OGs or mock inoculated using water and1044

samples collected 3 h later Data are from six biological replicates 1045

(C) JAZ10 expression 48 h after treatment with the indicated B cinerea strains Data1046

are from six biological replicates 1047

(D) JAZ10 expression in untreated plants Data are from three biological replicates1048

1049

Figure 11 Gallo 1-Induced Callose Deposition Is Reduced in gae1 gae6 Plants 1050

Page 46 of 54

(A) Callose deposition in four week-old Col-0 and gae1 gae6 plants Plants were1051

inoculated with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) or mock inoculated 1052

with B cinerea inoculation medium and samples were collected after 72 h Callose was 1053

stained with aniline blue and visualized using a Nikon Eclipse Ni-U microscope with a 1054

DAPI filter Three leaves were stained per treatment Scale bars are 100 microm Asterisks 1055

indicate fungal hyphae arrows indicate representative callose spots 1056

(B) Quantification of callose deposition Callose deposits were counted in 200 microm x 2001057

microm squares laid out across the images shown here Bars represent means plusmn SE from 1058

six squares each Letters indicate significantly different groups (p lt 005) according to a 1059

t-test analysis1060

1061

Figure 12 Less Soluble Pectin Accumulates in B cinerea-Infected gae1 gae6 1062

Leaves 1063

Four week-old Col-0 and gae1 gae6 plants were inoculated with 10 microL droplets of the 1064

indicated B cinerea isolate (25 x 105 spores mL-1) or mock inoculated with B cinerea 1065

inoculation medium Leaves were collected 48 h later AIR was extracted from leaf 1066

samples and water added (20 microL infection site) to extract soluble pectin fragments 1067

Samples were vortexed for 7 h centrifuged at 12000 g and the uronic acid 1068

concentration of the supernatant was measured Data from nine biological replicates 1069

were combined using a mixed linear model Means plusmn SE are shown Letters indicate 1070

significant differences (q lt 005) 1071

1072

Figure 13 Macerozyme-Induced PTI Is Lost in gae1 gae6 While OG-Induced PTI Is 1073

Unaltered 1074

(A) Macerozyme-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1075

inoculated with 001 macerozyme or mock inoculated with boiled macerozyme and 1076

were infected with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) 24 h later Samples 1077

were collected immediately (0 dpi) and after two (2 dpi) and three (3 dpi) days Data 1078

show mean plusmn SE of six to 14 biological replicates each combined using a linear model 1079

Letters indicate significantly different groups (q lt 005) 1080

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

Parsed CitationsAdam L and Somerville SC (1996) Genetic characterization of five powdery mildew disease resistance loci in Arabidopsisthaliana Plant J 9 341-356

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Bouton S Leboeuf E Mouille G Leydecker MT Talbotec J Granier F Lahaye M Hofte H and Truong HN (2002)Quasimodo1 encodes a putative membrane-bound glycosyltransferase required for normal pectin synthesis and cell adhesion inArabidopsis Plant Cell 14 2577-2590

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Cao H Glazebrook J Clarke JD Volko S and Dong XN (1997) The Arabidopsis NPR1 gene that controls systemic acquiredresistance encodes a novel protein containing ankyrin repeats Cell 88 57-63

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Carpita N (2010) Update on Mechanisms of Plant Cell Wall Biosynthesis How plants make cellulose and other (1-gt4)-beta-D-glycans Plant Physiol

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Chisholm ST Coaker G Day B and Staskawicz BJ (2006) Host-microbe interactions Shaping the evolution of the plantimmune response Cell 124 803-814

Page 14 of 54

al 1994 Steffan et al 1995 Pattathil et al 2010) (Supplemental Figure 6) 280

Collectively these cell wall composition data show that gae1 gae6 plants are reduced in 281

HG Due to the strong proportional reduction of HG in the walls levels of all other 282

components should be higher However Rha indicative of RGI was not higher in gae1 283

gae6 suggesting that RGI may also be reduced in gae1 gae6 walls 284

285

GAE1 and GAE6 Are Required for Immunity to Specific B cinerea Isolates 286

We hypothesized that plant immunity to B cinerea might be affected by pectin 287

content in the host cell wall because this pathogen requires polygalacturonases and 288

pectin methylesterases for full virulence (ten Have et al 1998 Valette-Collet et al 289

2003 Kars et al 2005b Espino et al 2010) Because Bcinerea is genetically diverse 290

and has a high diversity of polygalacturonases (Rowe and Kliebenstein 2007) we 291

measured relative fungal growth at both 2 and 3 d post-inoculation (dpi) using ten 292

different B cinerea isolates (Figure 6) The B cinerea isolates Fresa 525 and Gallo 1 293

showed enhanced virulence on gae1 gae6 plants at both time points whereas KT and 294

UK Razz showed enhanced virulence in gae1 gae6 plants at 3 but not 2 d dpi (Figure 295

6A) We did not detect any significant changes in relative fungal growth between wild-296

type Col-0 and gae1 gae6 plants when using Acacia Apple 517 DN Grape Pepper or 297

Rasp isolates of B cinerea Therefore we continued our study using Gallo 1 and Fresa 298

525 We also included Pepper for comparison as this isolate has been used in a large-299

scale analysis of plant gene expression in response to B cinerea infection (Windram et 300

al 2012) 301

We next investigated whether expression of GAE1 and GAE6 was affected by 302

challenge with B cinerea Inoculation of wild-type Col-0 plants with any isolate 303

repressed GAE1 and GAE6 expression (Figure 6B and C) and this repression was 304

generally independent of PAD4 (Figure 6B and C) Expression of GAE2 and GAE3 was 305

unaltered by B cinerea treatment GAE4 was repressed only by Fresa 525 treatment 306

while GAE5 expression was enhanced with all three isolates tested (Supplemental 307

Figure 7) As seen for infection with Pma ES4326 the expression of GAE1 and GAE6 308

was repressed upon B cinerea treatment This indicates a role for GAE1 and GAE6 in 309

the immune response to B cinerea 310

Page 15 of 54

Page 16 of 54

Because the amount of pectin substrate for pathogen polygalacturonases is 313

reduced we hypothesized that immunity phenotypes of gae1 gae6 plants may result 314

from reduced generation of OGs by these enzymes In turn the reduced OGs could 315

lead to reduced immune responses To test this we used macerozyme a commercial 316

pectinase as a proxy for pathogen pectinases We isolated cell walls as a subcellular 317

fraction known as alcohol insoluble residue (AIR Gille et al 2009) This was treated 318

with macerozyme and tested for release of soluble likely low molecular weight pectin 319

fragments (Figure 7) Treatment with 025 macerozyme released significantly more 320

uronic acid from wild-type Col-0 AIR relative to gae1 gae6 AIR (Figure 7B) To test 321

activity of these pectin-containing solutions we infiltrated them into wild-type Col-0 322

plants and measured PAD3 expression PAD3 is required for synthesis of the 323

phytoalexin camalexin (Zhou et al 1999) and its expression is known to be induced by 324

OG treatment (Ferrari et al 2007) Fractions from macerozyme treated Col-0 walls 325

induced significantly more PAD3 expression than fractions derived from gae1 gae6 326

walls suggesting that macerozyme treatment did indeed release more active soluble 327

uronic acid possibly OGs from wild-type Col-0 walls than from gae1 gae6 walls (Figure 328

7C) 329

Next we studied the effect of macerozyme treatment on expression of PAD3 in 330

intact plants (Figure 8) This treatment did not lead to any visible leaf damage 331

(Supplemental Figure 8) In wild-type Col-0 PAD3 expression was induced three h after 332

treatment with 001 macerozyme (Figure 8A) By contrast no macerozyme-induced 333

induction of PAD3 expression was detected in gae1 gae6 (Figure 8A) However OG 334

treatment induced the expression of PAD3 in both genotypes indicating that gae1 gae6 335

plants do respond to OGs (Figure 8B) Macerozyme treatment also induced expression 336

of the SA marker genes PATHOGENESIS RELATED 1 (PR1) and SID2 in Col-0 but 337

not in gae1 gae6 (Figure 9 panel A and B) PR1 was not significantly induced by 338

purified OGs (Figure 9C) at the time and concentration tested but SID2 was induced by 339

OG treatment in both genotypes (Figure 9D) However the induction was stronger in 340

wild-type Col-0 In addition the JA markers JASMONATE-ZIM-DOMAIN PROTEIN 5 341

(JAZ5) and JAZ10 were induced by macerozyme and OG treatment in both Col-0 and 342

gae1 gae6 (Figure 10A and Supplemental Figure 11) Interestingly expression of JAZ5 343

312 Pectinase-Induced Immune Signaling Is Altered in gae1 gae6 Plants

Page 17 of 54

Page 18 of 54

Page 19 of 54

Page 20 of 54

344

345

346

and JAZ10 was already strongly induced in gae1 gae6 after mock inoculation (Figure 10

and Supplemental Figure 11) We observed no hyperresponsive induction by mock

treatment of PLANT DEFENSIN 12 (PDF12) a necrotrophy and late JA signaling

347 marker gene (Supplemental Figure 12A-D) PDF12 was also not induced by

348 macerozyme or OG treatments (Supplemental Figure 12A-D) This suggests that early

349 JA signaling is induced by mock-treatment in gae1 gae6 Because it is known that JA

350 and SA signaling act antagonistically (Pieterse et al 2012) the strong up-regulation of

351 JA-induced immune signaling in gae1 gae6 might explain the lower OG-induced SID2

352 expression in this genotype

353 To test whether other immune responses are altered in gae1 gae6 plants we

354 analyzed Mitogen-activated protein kinase (MAPK) activity following macerozyme and

355 OG treatments OGs are known to activate the MAPKs MPK3 and MPK6 in Arabidopsis

Page 21 of 54

(Galletti et al 2011) Macerozyme treatment activated MPK3 and MPK6 in wild-type 356

Col-0 whereas MAPK activation in gae1 gae6 was slightly reduced in four out of five 357

experiments performed (Supplemental Figure 13) By contrast OG-induced MAPK 358

activity was similar or increased in gae1 gae6 in four out of five experiments 359

(Supplemental Figure 13) Additionally mock treatment induced MAPK activity in gae1 360

gae6 more strongly than it did in wild-type Col-0 in four out of five experiments 361

performed (Fig S13) Collectively these results illustrate that activation of several 362

immune signaling componentsmdashcamalexin synthesis SA and JA signaling and 363

possibly MAPK activationmdashby macerozyme is substantially altered in gae1 gae6 This 364

suggests that release of soluble pectin fragments possibly OGs by pectinolytic 365

enzymes might be reduced in gae1 gae6 plants while recognition of OGs is intact 366

367

Following B cinerea Infection Production of Soluble Pectin and Deposition of 368

Callose Are Reduced in gae1 gae6 Plants 369

Although pad3 plants were more susceptible to the B cinerea isolates Fresa 525 370

and Gallo 1 which also grew better on gae1 gae6 growth of the isolate Pepper was 371

similar on Col-0 and pad3 (Supplemental Figure 9A) However we did not detect any 372

significant changes in camalexin production after treatment with B cinerea isolate Gallo 373

1 and Fresa 525 in gae1 gae6 (Figure 8D) Neither did we detect changes in PAD3 374

expression after treatment with any B cinerea isolate in gae1 gae6 (Figure 8E) 375

Additionally SID2 and PR1 were similarly induced by all B cinerea isolates in both Col-376

0 and gae1 gae6 (Figure 9E and F) and sid2 plants which are deficient for pathogen-377

induced SA biosynthesis did not show any differences in relative B cinerea growth 378

(Supplemental Figure 9B) Expression of JAZ10 was induced more strongly by Gallo 1 379

treatment in gae1 gae6 than in wild-type Col-0 but not by Fresa 525 or Pepper (Fig 380

10C) while dde2 plants deficient for JA biosynthesis showed enhanced susceptibility to 381

all B cinerea isolates tested (Supplemental Figure 9B) Curiously exogenous 382

application of MeJA did not lead to altered relative fungal growth of B cinerea 383

(Supplemental Figure 9C) and cev1 isoxaben resistant 1 (ixr1) plants which have 384

constitutively high JA levels (Ellis and Turner 2001) did not show any changes in 385

susceptibility to the B cinerea isolates tested (Supplemental Figure 9D) Hence neither 386

Page 22 of 54

differences in B cinerea isolate susceptibility to camalexin nor altered SA or JA 387

signaling by themselves explained the enhanced susceptibility of gae1 gae6 to these B 388

cinerea isolates 389

Hydrogen peroxide accumulation in response to B cinerea isolates Fresa 525 390

Gallo 1 and Pepper was not obviously altered in gae1 gae6 plants (Supplemental Figure 391

14) However callose deposition upon treatment with B cinerea isolate Gallo 1 was 392

strongly reduced in gae1 gae6 (Figure 11) It was previously shown that OG treatment 393

induces callose deposition (Denoux et al 2008) suggesting that the reduction in 394

callose deposition might be due to reduced OG release in gae1 gae6 It is possible that 395

this reduction in callose deposition contributes to the reduced immunity of gae1 gae6 396

plants to B cinerea isolate Gallo 1 397

Because macerozyme could be used to release soluble likely low molecular 398

weight pectin from isolated cell walls we next investigated whether infection with B 399

cinerea was accompanied by accumulation of soluble pectin as well We prepared cell 400

walls (AIR fraction) from leaves infected with B cinerea incubated these cell walls with 401

water and measured uronic acid content of the extracts B cinerea treatment did in fact 402

lead to increased accumulation of soluble pectin in wild-type Col-0 plants when 403

compared to mock-treated samples (Figure 12) No such increased accumulation of 404

water-soluble pectin fractions was observed in gae1 gae6 (Figure 12) 405

406

Macerozyme-Induced Pattern-Triggered Immunity against B cinerea Is Abolished 407

in gae1 gae6 408

It was previously shown that OG treatment can induce patternndashtriggered 409

immunity (PTI) to B cinerea (Ferrari et al 2007) We tested macerozyme treatment to 410

determine whether it could also induce PTI to the B cinerea isolate Gallo 1 (Figure 13) 411

Indeed pretreatment with 001 macerozyme for 24 h led to reduced B cinerea growth 412

in wild-type Col-0 but not gae1 gae6 plants (Figure 13A) Interestingly mock and 413

macerozyme pretreated gae1 gae6 plants exhibited similar growth of B cinerea Gallo 1 414

as did macerozyme-treated Col-0 plants This result is likely due to activation of JA 415

signaling in gae1 gae6 during the pretreatment procedure (Figure 13C and D) OG-416

induced PTI was observed in both genotypes when using a high dose of OGs (Figure 417

Page 23 of 54

13B) whereas a lower dose of OGs only induced PTI in Col-0 (Supplemental Figure 418

15) This suggests that OG perception in gae1 gae6 at least above a certain threshold 419

is unaltered while release of immunity-activating pectin fragments possibly OGs is 420

421 reduced We conclude that immune responses induced by macerozyme treatment

422 which do not occur in gae1 gae6 plants are important for plant immunity to the B

423 cinerea isolate Gallo 1

Page 24 of 54

____________________________________________________________________________

Page 25 of 54

DISCUSSION 426

Here we showed that loss of GAE1 and GAE6 leads to a dramatic reduction in 427

pectin content in the cell wall (Figure 4) likely causing the brittleness of leaves that we 428

observed (Figure 3) Petioles and midribs of gae1 gae6 plants broke easily when bent 429

and inoculation of leaves with deionized water resulted in increased ion leakage 430

possibly due either to microscopic fractures of the cell wall induced by inoculation stress 431

or to a reduced capability of these walls to withstand increased turgor pressure The 432

increased brittleness was associated with altered cell wall composition In our analysis 433

the GalA content of gae1 gae6 leaf walls was reduced by more than 40 and the total 434

uronic acids by over 60 (Figure 4 and Supplemental Figure 5) This is a major change 435

in the overall cell wall composition considering that pectin makes up about 50 of the 436

plant cell walls found in Arabidopsis leaves (Zablackis et al 1995 Nobuta et al 2007 437

Harholt et al 2010) Interestingly quasimodo1 (qua1) plants which have a mutation in 438

a glycosyltransferase family 8 gene are reduced in GalA by 25 and show reduced 439

binding of HG specific antibodies (Bouton et al 2002) 440

Furthermore qua2 plants which have a mutation in a putative methyltransferase 441

have about 13 less total GalA and lack 50 of cell wall HG (Mouille et al 2007) 442

Likewise cgr2 cgr3 plants carrying mutations in another class of Golgi-localized 443

putative methyl-transferases have leaf cell walls that are reduced by about 40 in 444

uronic acid (Kim et al 2015) All three mutants are severely dwarfed (Bouton et al 445

2002 Mouille et al 2007 Kim et al 2015) Additionally qua1 and qua2 mutants show 446

a distorted overall shape likely due to reduced cell cohesion and are extremely fragile 447

(Bouton et al 2002 Mouille et al 2007) By contrast gae1 gae6 plants with a similar 448

strong reduction in GalA and total uronic acids have an overall normal shape and are 449

only slightly smaller than wild-type Col-0 (Supplemental Figure 4) In the wall analysis 450

performed here we did not detect changes in other wall polysaccharides or structures 451

that might compensate for the reduction of pectin in gae1 gae6 It is possible the severe 452

morphological alterations in qua2 and cgr2 cgr3 plants are due to reduction in pectin 453

methylesterification which is unaffected in gae1 gae6 It was speculated that quality 454

control mechanisms in the Golgi prevent secretion of inefficiently esterified pectin in 455

cgr2 cgr3 (Kim et al 2015) suggesting that the Golgi of these plants might over-456

Page 26 of 54

accumulate pectin and that this may contribute to the severe dwarf phenotype of these 457

plants The gae1 gae6 plants described here provide an opportunity to study the effects 458

of reduction of pectin in plants that do not show severe morphological phenotypes 459

We found that gae1 gae6 plants are also compromised in resistance to B 460

cinerea and Pma ES4326 indicating that loss of pectin reduces immunity to these 461

pathogens (Figure 2 and 6) We found that gae1 gae6 plants were slightly more 462

susceptible to Pma ES4326 but showed a strong increase in susceptibility to specific B 463

cinerea isolates This was not unexpected because B cinerea is known to interact with 464

pectin as part of its infection strategy It secretes various pectin-degrading enzymes 465

early during infection and requires some of these enzymes including PG1 PG2 and 466

PME1 for full virulence (ten Have et al 1998 Valette-Collet et al 2003 Kars et al 467

2005b Espino et al 2010) Additionally Arabidopsis plants expressing an antisense 468

construct of the polygalacturonase inhibitor PGIP1 are significantly more susceptible to 469

B cinerea (Ferrari et al 2006) On the other hand Pma ES4326 is not known to470

require cell wall degrading enzymes for full virulence even though it encodes two 471

pectate lyase genes in its genome (PMA4326_07094 and PMA4326_04621 472

wwwbacteriaensemblorg) B cinerea isolates are also known to be genetically quite 473

diverse in their polygalacturonase (PG) loci and genetic variability in PG2 is associated 474

with varying growth on pectin containing media (Rowe and Kliebenstein 2007) 475

Furthermore the contribution of PG1 and PG2 to B cinerea virulence seems to depend 476

on the specific B cinerea isolate and the host species (Kars et al 2005a Zhang and 477

Van Kan 2013) B cinerea isolates are also known to be differently sensitive to the anti-478

microbial secondary metabolite camalexin (Kliebenstein et al 2005) In line with these 479

observations the susceptibility of gae1 gae6 plants differed depending on the specific 480

B cinerea isolate Whereas Fresa 525 Gallo 1 UK Razz and KT grew better in gae1481

gae6 we detected no difference in Acacia Apple 517 DN Grape Pepper or Rasp 482

growth (Figure 6) 483

B cinerea was shown to metabolize pectin and to possibly utilize GalA as a484

carbon source during in planta growth (Zhang et al 2011 Zhang and Van Kan 2013) 485

Our data show that gae1 gae6 plants which are reduced in GalA content did allow 486

enhanced growth of specific B cinerea isolates but no reduction in growth of any B 487

Page 27 of 54

cinerea isolates tested was found This suggests that utilization of pectin as a carbon 488

source was not a contributing factor to alterations in B cinerea susceptibility It is 489

possible that changes in the cell wall composition affect the structural integrity of the 490

wall and affect hyphal penetration of specific B cinerea isolates However OGs (small 491

HG fragments) are likely released during the infection process (Ferrari et al 2013) 492

OGs function as DAMPs and initiate immune responses in Arabidopsis (Ferrari et al 493

2013) Treatment of Arabidopsis with OGs having a degree of polymerization (DP) of 494

10-15 sugar subunits was previously shown to enhance resistance to B cinerea (Ferrari495

et al 2007) We showed that pretreatment with OGs or macerozyme induced enhanced 496

immunity to B cinerea isolate Gallo 1 in wild-type Col-0 but only OGs and not 497

macerozyme induced immunity in gae1 gae6 (Figure 13) Our data suggest gae1 gae6 498

plants which have reduced pectin content may release less OGs and that this 499

reduction leads to the increased susceptibility to B cinerea 500

Consistently we found that macerozyme treatment released less soluble pectin 501

from gae1 gae6 cell walls than from wild-type walls Fractions containing soluble pectin 502

released from gae1 gae6 were also less active in inducing PAD3 gene expression than 503

the fractions released from wild-type walls (Figure 7) We also showed that less soluble 504

pectin accumulated in gae1 gae6 than in wild-type plants 48 h after treatment with B 505

cinerea (Figure 12) 506

We observed that loss of pectin leads to alterations in A thaliana immune 507

signaling Macerozyme treatment induced expression of PAD3 and the SA marker 508

genes PR1 and SID2 in wild-type Col-0 but not in gae1 gae6 whereas OG treatment 509

led to increased PAD3 and SID2 expression in both wild-type and gae1 gae6 (Figure 8 510

and 9) This suggests that although release of active soluble pectin in these plants 511

might be altered OG recognition is intact In fact cell walls isolated from qua1 another 512

mutant known to be reduced in HG released less OGs (DP1-6) than did wild-type cell 513

walls upon endopolygalacturonase treatment (Bouton et al 2002) This was also true 514

for gae1 gae6 cell walls as macerozyme treatment of isolated gae1 gae6 cell walls 515

released less soluble pectin than treatment of Col-0 cell walls Because the fraction 516

from gae1 gae6 plants was also less active in inducing PAD3 expression (Figure 7) it 517

seems possible that biologically active OGs were the active component inducing PAD3 518

Page 28 of 54

expression Interestingly expression of the JA marker genes JAZ5 and JAZ10 was 519

higher in gae1 gae6 than in wild-type mock-inoculated plants but was comparable to 520

wild-type in untreated plants suggesting that JA signaling in gae1 gae6 is 521

hyperresponsive (Figure 10 and Supplemental Figure 11) This induction of JA signaling 522

might be due to wounding stress incurred during the inoculation process JA and SA 523

signaling generally act antagonistically (Pieterse et al 2012) Increased JA signaling 524

activity might hence explain why mock- and macerozyme-treated gae1 gae6 plants 525

exhibited reduced expression of the SA marker PR1 and why OG treatment induced 526

expression of SID2 another SA marker to a lower extent in gae1 gae6 than in wild-type 527

plants (Figure 9) 528

No single immune signaling component was found to be the sole contributor to 529

the compromised immunity of gae1 gae6 to specific B cinerea isolates such as Gallo 1 530

and Fresa 525 These isolates grew better on pad3 while growth of Pepper another B 531

cinerea isolate that did not show altered virulence on gae1 gae6 was unaltered 532

(Supplemental Figure 9) This suggested that differences in camalexin sensitivity of 533

these B cinerea isolates might account for the differences in gae1 gae6 susceptibility 534

However camalexin accumulation and PAD3 expression was the same for wild-type 535

Col-0 and gae1 gae6 for the B cinerea isolates Fresa 525 and Gallo 1 (Figure 8) which 536

showed enhanced growth on gae1 gae6 plants It should be noted that B cinerea 537

virulence is often estimated by comparing lesion sizes Lesion size and measurements 538

of relative fungal growth by qPCR do not always show the same results For example 539

although Denby et al (2004) showed that Pepper causes larger lesions on pad3 than on 540

wild-type this study showed that relative fungal growth of Pepper on pad3 was 541

unaltered 542

We observed that callose deposition was strongly reduced in gae1 gae6 plants 543

72 h after treatment with B cinerea isolate Gallo 1 (Figure 11) possibly due to reduced 544

release of active soluble pectin Interestingly OG-induced PTI to an unspecified isolate 545

of B cinerea was intact in the pmr4 mutant which does not produce callose (Galletti et 546

al 2008) Due to the high SA levels in pmr4 plants (Nishimura et al 2003) and the fact 547

that different B cinerea isolates vary both in induction of and reaction to defense 548

responses (Kliebenstein et al 2005 Rowe et al 2010) we could not determine 549

Page 29 of 54

whether reduced callose deposition causes the increased susceptibility of gae1 gae6 to 550

B cinerea isolate Gallo 1551

We did not observe any changes in B cinerea growth either on cev1 ixr1 plants 552

which are known to have constitutively high JA and OPDA levels (Ellis et al 2002b) or 553

on plants exogenously treated with MeJA (Supplemental Figure 9) Because plants 554

were kept uncovered for 3 h after MeJA treatment it is unclear how much MeJA was 555

bioavailable in the plant However JAZ10 expression was induced 3 h after spraying 556

with MeJA (Supplemental Figure 10) Because pathogen growth generally depends on 557

the combination of a multitude of immune signaling events we conclude that multiple 558

changes to the immune signaling in gae1 gae6 together account for the altered B 559

cinerea growth phenotypes detected 560

We showed that expression of GAE1 and GAE6 is repressed by the bacterial 561

hemibiotroph Pma ES4326 This repression did not occur in an eds1 or pad4 562

background or in pbs3 at the 24-h time point (Figure 1 and Supplemental Figure 1) 563

During the Arabidopsis - Pma ES4326 interaction the expression of many defense 564

related genes is altered in a PAD4EDS1 dependent fashion (Wang et al 2008) Some 565

of these expression changes require SA CBP60g and SARD1 are thought to act 566

downstream of PAD4 and EDS1 but upstream of SA-dependent responses (Wang et 567

al 2011) PBS3 is also thought to act downstream of PAD4 and EDS1 and upstream of 568

SA but the set of genes whose expression it affects is only partially overlapping with 569

those affected by CBP60gSARD1 (Wang et al 2008 Wang et al 2011) Thus GAE1 570

and GAE6 repression is regulated by the part of the immune signaling network that 571

requires PAD4 EDS1 and PBS3 but is independent of CBP60gSARD1 and SA 572

Because EDS1 PAD4 and PBS3 are important components of the plant immune 573

signaling network (Wiermer et al 2005 Nobuta et al 2007) one might conclude that 574

repression of GAE1 and GAE6 may be part of the plant immune response to Pma 575

ES4326 B cinerea treatment also reduced GAE1 and GAE6 expression but this was 576

mostly independent of PAD4 (Figure 6) while both Pma ES4326 and B cinerea repress 577

GAE1 and GAE6 expression the host signaling mechanisms responsible likely differ 578

In summary our data show that Arabidopsis GAE1 and GAE6 are required for 579

pectin biosynthesis leaf flexibility and immunity to Pma ES4326 and to specific B 580

Page 30 of 54

cinerea isolates We used macerozyme a commercial pectinase to mimic the effect of 581

B cinerea pectinases Macerozyme treatment released less pectin from gae1 gae6 cell582

walls and induced less immune signaling in gae1 gae6 plants Less water soluble pectin 583

also accumulated in B cinerea infected gae1 gae6 plants and macerozyme-induced 584

immunity to B cinerea isolate Gallo 1 was reduced in these plants We conclude that 585

the reduced pectin content in these mutants may lead to reduced release of soluble 586

pectin including active OGs during pathogen attack and that this compromises 587

immunity 588

589

590

Page 31 of 54

METHODS 591

Plant Materials and Growth Conditions 592

Wild-type Columbia (Col-0) and all mutant plants (in Col-0 background) were 593

grown on sterilized BM2 germinating mix (Berger Inc Quebec Canada) in a controlled 594

environment chamber (Conviron Pembina ND USA) with a 12-h photoperiod under 595

100 microM m-2 s-1 fluorescent illumination at 22degC and 75 relative humidity Germplasm 596

used is described in the Accession Numbers section at the end of Methods 597

598

Data Analysis and Replicates 599

For biological replicates samples were obtained from separate plant tissues and 600

in many cases from plants grown in different flats In some experiments multiple 601

technical replicates were measured per biological replicate For statistical analysis all 602

technical replicates were averaged and the averages were considered one biological 603

replicate Independent experiments were performed at different times and often 604

consisted of multiple biological replicates per experiment 605

Data analysis was performed using mixed linear models in the R programming 606

environment (Bates et al 2015) unless otherwise indicated Generally genotypes 607

treatments and time points were used as fixed effects and replicate-specific effects like 608

different pots flats etc were considered random effects We reported p-values 609

obtained from the mixed linear model when less than 10 comparisons were performed 610

and q-values for more than 10 comparisons to correct for multiple comparison testing 611

Q-values were calculated using the Benjamini-Hochberg procedure (Benjamini and612

Hochberg 1995) unless specified otherwise 613

614

Pathogen Strains Growth Conditions and Pathogen Growth Assays 615

Growth of Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) and 616

bacterial growth assays were performed as previously described (Bethke et al 2014) 617

For qRT-PCR assays four week-old plants were inoculated with bacteria (OD600 = 618

0002) or mock (5 mM MgSO4) At least four leaves from two plants were collected at 619

the indicated time points per biological replicate 620

Page 32 of 54

Prior to each experiment Botrytis cinerea isolates were grown on 1x PDA 621

medium (Difco) for 10 d at room temperature All isolates used have been described 622

previously (Rowe and Kliebenstein 2007) The spores were washed from the surface of 623

the plate using an inoculation medium (1x Gamborgrsquos B-5 basal salt mixture (Sigma-624

Aldrich G5768) 2 (wv) glucose 10 mM phosphate buffer pH 64) and fungal hyphae 625

were removed from the suspension by filtering through four layers of cheesecloth The 626

concentration of spores was determined using a hemocytometer and adjusted to 25 x 627

105 spores mL-1 Four week-old Arabidopsis plants were inoculated by placing one 10-628

microL droplet of the B cinerea spore solution or inoculation medium (mock) on the adaxial 629

leaf surface of fully expanded leaves Inoculated plants were kept at 100 relative 630

humidity Infection sites were collected at various time points using a cork borer For 631

qRT-PCR assays at least four leaf discs from one plant were combined for each 632

biological sample For B cinerea growth assays at least 12 leaf discs from three to six 633

plants were pooled per biological sample Relative fungal growth was determined by 634

measuring the abundance of a fungal gene relative to a plant gene as described below 635

In brief DNA from infected tissue was extracted and equal amounts of total DNA were 636

used to perform qPCR reactions using the B cinerea cutinase A and the Arabidopsis 637

SK11 gene respectively Primers were described in (Gachon and Saindrenan 2004) 638

and are listed in Supplemental Table 1 Two technical replicates were combined for 639

each biological replicate B cinerea is a necrotrophic fungal pathogen and may destroy 640

plant DNA as disease progresses However decreases in the amount of plant DNA 641

over the course of our experiments were only observed at late time points with very 642

susceptible plants so the qPCR assay is generally a reasonable estimate of fungal 643

biomass (Supplemental Figure 16) 644

645

Treatment with Macerozyme or OGs 646

For PTI assays four week-old plants either were inoculated with 001 (wv) 647

macerozyme R-10 (Yakult) a Rhizopus polygalacturonase (EC 32115) that possesses 648

high pectinase and hemicellulase activity or were mock inoculated using heat-649

inactivated macerozyme Other similar plants were either inoculated with 250 or 500 microg 650

mL-1 OGs or mock inoculated with water One d later plants were infected with B 651

Page 33 of 54

cinerea and samples were collected immediately (0 dpi) and two or three d later To 652

investigate the role of MeJA on B cinerea growth plants were sprayed with 1mM MeJA 653

in water with 001 (vv) Tween 20 Covers were removed so that leaves could dry off 654

before plants were inoculated with B cinerea three h later Samples were harvested at 655

the indicated time points For expression analysis and MAPK activation assays plants 656

were either inoculated with 100 microg mL-1 OGs or mock inoculated with water OGs used 657

for experiments described in Figures 8 to 10 and Supplemental Figure 12 were a kind 658

gift from Simone Ferrari OGs used in experiments described in Figure 13 and 659

Supplemental Figures 11 13 and 15 were prepared as previously described (Kohorn et 660

al 2014) 661

662

Expression Analysis 663

Leaf tissue from inoculated plants was harvested at the indicated time points 664

flash frozen pulverized and RNA extracted using Trizol (Invitrogen) Quantitative RT-665

PCR was performed using the SuperScript III Platinum SYBR Green One-Step 666

quantitative RT-PCR kit (Invitrogen) and a Lightcycler 480 Real-Time PCR system 667

(Roche) as previously described (Truman and Glazebrook 2012) In brief equal 668

amounts of total RNA and a gene specific primer were used for each reaction The 669

crossing point (Cp) was calculated using the 2nd derivative max method provided with 670

the Lightcycler software for each amplification curve Each reaction was run with two 671

technical replicates and the Cp values for these replicates were averaged ACTIN2 was 672

used as a stably expressed reference gene At least three biological replicates each 673

were performed and analyzed using a mixed-linear model RT-PCR was performed to 674

verify T-DNA insertion lines using the One-Step RT-PCR kit (Qiagen) according to the 675

manufacturerrsquos instructions The primers used can be found in Supplemental Table 1 676

677

Electrolyte Leakage Assay 678

Fully expanded leaves of four week-old plants were inoculated with water (Milli-Q 679

grade) using a needleless syringe Leaf discs were collected using a cork borer and 680

three leaf discs from one leaf were placed with the adaxial surface down onto 2 mL 681

water in a 12-well cell culture plate Conductivity was measured using a Horiba B-173 682

Page 34 of 54

conductivity meter after 30 min and then the water was replaced Conductivity was 683

measured again 30 min later (1 h total) as indicated in Figure 3C Six biological 684

replicates each were performed in three independent experiments and means and SEs 685

were calculated Two-sided t-tests were performed for each time point 686

687

DAB and Aniline Blue Staining 688

Leaves from four week-old plants were either mock inoculated with B cinerea 689

inoculation medium or inoculated with 10 microL droplets of a B cinerea spore solution (25 690

x 105 spores mL-1) of the indicated isolates Samples were collected 72 h later For 33prime-691

diaminobenzidine (DAB) staining two to five inoculated leaves were vacuum infiltrated 692

with 33prime-Diaminobenzidine (1 mg mL-1 in water pH 38) solution and stained overnight 693

in the dark Leaves were destained using 100 ethanol at 65degC and then transferred to 694

50 (vv) glycerol and photographed 695

Callose was visualized by staining leaves with aniline blue as previously 696

described (Adam and Somerville 1996) Briefly leaves were vacuum infiltrated with 697

alcoholic lactophenol (one volume of phenolglycerollactic acidwater (1111) mixed 698

with two volumes of ethanol) and destained at 65degC Next leaves were moved through 699

an alcohol gradient (50 20 and 10 (vv) ethanol) into water The destained leaves 700

were stained for 30 min in 150 mM K2HPO4 (pH 95) with 001 (wv) aniline blue 701

Samples were mounted in water and examined using a Nikon Eclipse Ni-U microscope 702

at 10x magnification using a DAPI filter (excitation at 325-375 nm emission at 435-485 703

nm) For callose quantification six 200 microm x 200 microm squares were counted for each 704

sample type Callose deposits per mm2 were calculated and data analyzed using a t-705

test Note that there is an inherent uncertainty associated with such counts because 706

callose deposits may not all be in focus at the same time 707

708

MAPK Activity Assay 709

Fully expanded leaves of four week-old plants were inoculated with 001 710

macerozyme 100 microg mL-1 OGs 100 nM flg22 or water (mock) flg22 a peptide from 711

bacterial flagella that induces immune responses was used as a positive control for 712

Page 35 of 54

MAPK activation (Asai et al 2002) and was purchased from EZBiolab Inc (Westfield 713

IN USA) Samples were harvested 10 min later and immediately frozen in liquid 714

nitrogen Protein was extracted using an extraction buffer containing 25 mM Tris pH 78 715

75 mM NaCl 1 mM DTT 1 mM NaF 05mM Na3VO4 01 (vv) Tween 20 one 716

cOmplete Mini protease inhibitor cocktail tablet (Roche) and one PhosSTOP 717

phosphatase inhibitor cocktail tablet (Roche) per 10 mL buffer 15 microg of total protein 718

were electrophoresed on a 10 SDS-polyacrylamide gel and then blotted onto a PVDF 719

membrane (Bio-Rad 162-0177) Activated MAPKs were detected using p4442 MAPK 720

(Erk12) antibody (9102S Cell Signal 12500 in TBST (20 mM Tris pH 75 150 mM 721

NaCl 01 (vv) Tween 20)) Anti-Rabbit IgG-HRP (115000 in TBST A6154 Sigma) 722

and ECL Plus substrate (Pierce 32132) MPK3 and MPK6 were detected with an anti-723

MPK3 antibody (M8318 Sigma 12000 in TBST and 3 (wv) milk) or an anti-MPK6 724

antibody (A7104 Sigma 16000 in TBST and 3 (wv) milk) respectively The 725

secondary antibody and detection were the same as for the p4442 antibody Five 726

independent experiments were performed 727

728

Preparation of Alcohol Insoluble Residue (AIR) Pectin Extraction and Uronic Acid 729

Measurements 730

Fully expanded leaves from four week-old plants that were incubated for 48 h in 731

the dark to reduce starch were harvested flash frozen and pulverized AIR was 732

extracted by washing ground material twice in 70 (vv) ethanol three times in a 733

mixture of chloroform and methanol (11 vv) and once in acetone (Gille et al 2009) 734

and was then air dried Cell wall pectin was extracted from AIR with cell wall extraction 735

buffer (50 mM Trizma 50 mM CDTA pH 72) at 95degC for 15 min Samples were 736

homogenized using a paint shaker (Harbil 5G-HD) and glass beads (3 mm diameter) 737

For dot blot experiments 500 microL of cell wall extraction buffer were used per 10 mg of 738

AIR Debris was precipitated by centrifugation for 10 min at 10000 g 739

Total uronic acid content of pectin extracted as described above using 500 microL of 740

cell wall extraction buffer per 1 mg of AIR was measured as described previously 741

(Filisetti-Cozzi and Carpita 1991 van den Hoogen et al 1998) First 36 microL of pectin 742

extract were mixed with 4 microL of 4 M sulfamic acid Then 200 microL of sulfuric acid 743

Page 36 of 54

containing 120 mM sodium tetraborate was added and samples were incubated at 80degC 744

for 1 h Following cooling on ice the optical density of samples was measured at 490 745

nm Next 40 microL of m-hydroxydiphenyl reagent (100 microL of m-hydroxydiphenyl in dimethyl 746

sulfoxide at 100 mgmL mixed with 49 mL 80 (vv) sulfuric acid just before use) was 747

added and the samples were mixed The optical density at 490 nm was measured 748

again The optical density before m-hydroxydiphenyl reagent addition was subtracted 749

from the optical density measured after addition of the dye The concentration of uronic 750

acid was calculated using known amounts of GalA as a standard 751

752

Release of Pectin from Arabidopsis Cell Walls Using Macerozyme 753

AIR preparations from leaves of four week-old plants were treated with 025 754

(wv) macerozyme R-10 (Yakult) or water (mock) at a concentration of 100 microL per mg 755

AIR Samples were incubated for 3 h at room temperature on a vortex shaker and 756

centrifuged for 10 min at 12000 g Total uronic acid concentration of 5-fold diluted 757

supernatants was determined for two technical replicates each The concentration of 758

uronic acid was calculated using known amounts of GalA as a standard For 759

subsequent qRT-PCR experiments supernatants from multiple extractions were 760

combined boiled for 20 min to inactivate macerozyme diluted 10-fold with water and 761

inoculated into four week-old Col-0 plants Heat-inactivated 10-fold diluted macerozyme 762

solutions were used as mock treatment Samples were collected 3 h later and PAD3 763

expression was determined 764

765

Release of Pectin from Arabidopsis Leaves Infected with B cinerea 766

Fully expanded leaves of four week-old plants were either inoculated with up to 767

five 10 microL droplets of B cinerea spore solution (25 x 105 spores mL-1) or mock 768

inoculated with B cinerea inoculation medium For each biological replicate leaves with 769

a combined count of at least 20 infection sites were collected 48 h later AIR was 770

extracted as described above but plants were not dark-treated or freeze-dried and the 771

first ethanol step was performed using 85 (vv) ethanol To extract soluble pectin 772

fragments AIR samples were mixed with water at a concentration of 20 microL per infection 773

site incubated for 7 h at room temperature on a vortex shaker and centrifuged for 10 774

Page 37 of 54

min at 12000 g Total uronic acid concentration of supernatants was determined for two 775

technical replicates each The concentration of uronic acid was calculated using known 776

amounts of GalA as a standard 777

778

Measurement of Uronic Acids Neutral Monosaccharides and Cellulose Content in 779

Cell Walls 780

Neutral sugars and uronic acids from non-cellulosic polysaccharides in cell walls 781

(AIR) were released by trifluoroacetic acid (TFA) hydrolysis Approximately 1 mg of AIR 782

was hydrolyzed with 2 M TFA at 121degC for 90 min The TFA-soluble material was split 783

into two equal parts one for uronic acid and one for neutral sugar determination The 784

uronic acid content was determined by analyzing the hydrolyzate using a CarboPac 785

PA200 anion-exchange column with an ICS-3000 Dionex chromatography system 786

(Dionex Sunnyvale CA USA) The elution profile consisted of a linear gradient of 50 to 787

200 mM sodium acetate in 01 M NaOH in 10 min at 04 mL per min Alditol acetate 788

derivatives of neutral sugars were produced as described (York et al 1985) In brief 789

monosaccharides in the hydrolyzate were reduced with NaBH4 and peracetylated with 790

acetic anhydride and pyridine at 121degC for 20 min The generated alditol acetates were 791

analyzed by GC-MS Crystalline cellulose was measured according to Updegraff (1969) 792

After hydrolysis of noncellulosic polysaccharides from AIR with the Updegraff reagent 793

(acetic acid nitric acids water 812 (vv) ) the remaining pellet was hydrolyzed in 794

72 sulfuric acid The resulting glucose quantity was determined by the anthrone 795

method (Scott and Melvin 1953) 796

797

Dot Blot Experiments 798

Pectin solutions extracted using 50 microL of cell wall extraction buffer per mg AIR 799

were serially diluted and nitrocellulose membranes were spotted with 1 microL of the diluted 800

pectin solutions Membranes were dried overnight blocked with 5 milk (wv) in 1x 801

PBS (8 g L-1 NaCl 02 g L-1 KCl 144 g L-1 Na2HPO4 024 g L-1 KH2PO4 pH 74) and 802

probed with LM5 (Jones et al 1997) LM6 (Willats et al 1998) LM8 (Willats et al 803

2004) LM19 and LM20 (Verhertbruggen et al 2009a) or CCRC-M7 (Steffan et al 804

Page 38 of 54

1995) antibodies LM series antibodies were diluted 1250 and CCRC-M7 was diluted 805

1500 in 5 milk powder (Nestle Carnation) in 1 x PBS For LM series antibodies a 806

goat anti-Rat HRP conjugated antibody (Bethyl A110-105P 15000 diluted) and for 807

CCRC-M7 an anti-Mouse IgG HRP conjugate (Promega W402B 12500 diluted) was 808

used as secondary antibody in 5 milk powder in 1x PBS LM series antibodies were 809

obtained from plant probes (wwwplantprobesnet) and CCRC-M7 from CarboSource 810

(wwwcarbosourcenet) Membranes were washed after incubation with the primary and 811

secondary antibodies with 1x PBS Dot blots were developed using the ECL system 812

(GE Healthcare Amersham ECL prime) For quantification of signals dot blot results on 813

x-ray films were photographed using a CCD camera and intensities were measured814

using Image Jrsquos Integrated Density function Locally adjusted background was 815

subtracted As the dynamic range of the signal on an x-ray film was narrow non-816

linearity in the measured values was corrected for each genotype in each biological 817

replicate using the values from different dilutions in two technical replicates We noticed 818

that the residuals were not normally distributed and performed Box-Cox power 819

transformation (Box and Cox 1964) to obtain normally distributed values for the 820

subsequent statistical analysis of the data For LM19 Box-Cox selected power 821

transformation was done to the power of 038th and for LM20 to the power 075th After 822

correction of the transformed measured values with the effect of the biological 823

replicates the values were fit to a linear model with the Arabidopsis genotype as the 824

fixed effect This linear model was used both to generate the plots in Figures 5B and D 825

and to test significant differences 826

827

Camalexin Measurements 828

Four week-old plants were infected with B cinerea and samples (three leaf disks 829

from one plant each) were harvested at the indicated time points flash frozen and 830

pulverized Each sample was extracted with 300 microL of 90 methanol (vv) 50 microL of the 831

extract was run on an Agilent Lichrocart 250-4 RP18e 5-microm column using an Agilent 832

1100 series HPLC Camalexin was detected using a DiodeArray (DAD) at 330 nm and 833

with a fluorescence detector at emission 318 nmexcitation 385 nm (Agilent) Separation 834

was achieved using the following program with aqueous acetonitrile 5 min gradient 835

Page 39 of 54

from 63 to 69 acetonitrile 30 s gradient from 69 to 99 acetonitrile 2 min at 99 836

acetonitrile and a post-run equilibration of 35 min at 63 acetonitrile (Denby et al 837

2004 Kliebenstein et al 2005) Purified camalexin was used to produce a standard 838

curve to identify and quantitate camalexin 839

840

Accession Numbers 841

Microarray data used can be accessed at NCBI Gene Expression Omnnibus 842

(httpwwwncbinlmnihgovgeo) GSE18978 Sequence information for all Arabidopsis 843

genes described in this article can be found in TAIR (wwwarabidopsisorg) using the 844

following accession numbers PAD3 (CYP71B15 At3g26830) SID2 (ICS1 At1g74710) 845

GAE1 (At4g30440) GAE2 (At1g02000) GAE3 (At4g00110) GAE4 (At2g45310) GAE5 846

(At4g12250) GAE6 (At3g23820) PR1 (At2g14610) JAZ5 (At1g17380) JAZ10 847

(At5g13220) ACTIN2 (At3g18780) PDF12a (At5g44420) PDF12b (At2g26020) 848

AtSK11 (At5g26751) The sequence of the B cinerea cutinase Bc-CutA (Z69264) can 849

be found in the DNA Data Bank of Japan (httpgetentryddbjnigacjp) Germplasm 850

used included dde2-2 (At5g42650 (von Malek et al 2002)) pad3-1 (At3g26830 (Zhou 851

et al 1999)) pad4-1 (At3g52430 (Jirage et al 1999)) sid2-2 (At1g74710 (Wildermuth 852

et al 2001)) pbs3-2 (At5g13320 SALK_018225 (Nobuta et al 2007)) cbp60a-1 853

(At5g62570 SALK_124410 (Truman et al 2013)) cbp60g-1 sard1-2 (At5g26920 854

SALK_023199 At1g73805 SALK_052422 (Wang et al 2011) cbp60a-1 cbp60g-1 855

sard1-2 (Truman et al 2013) gae1-1 (At4g30440 SALK_085554) gae6-1 (At3g23280 856

SALK_104454) gae6-2 (At3g23280 SALK_017191) ixr1-1 cev1 (At5g05170 (Ellis 857

and Turner 2001 Scheible et al 2001)) eds1 plants were derived by introgression of 858

the Ler eds1-2 allele into Col-0 that contains two copies of EDS1 (At3g48090 859

At3g48080 (Bartsch et al 2006) mpk6-2 (At2g43790 SALK_073907 (Liu and Zhang 860

2004)) mpk3-DG (At3g45640 a fast neutron deletion mutant (Miles et al 2005)) and 861

npr1-1 (At1g64280 (Cao et al 1997)) T-DNA insertion lines were part of the SALK 862

collection (Alonso et al 2003) and were obtained from the Arabidopsis Biological 863

Resource Center 864

865

Supplemental Data 866

Page 40 of 54

Supplemental Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 867

Expression Does Not Require CBP60s or SID2 868

Supplemental Figure 2 Expression of GAE Family Members uponTreatment with Pma 869

ES4326 870

Supplemental Figure 3 Characterization of T-DNA Insertions in GAE1 and GAE6 871

Supplemental Figure 4 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves and 872

Are Slightly Smaller than Wild-Type Col-0 Plants 873

Supplemental Figure 5 Cell Walls of gae1 gae6 Plants Contain Less Uronic Acid than 874

Wild-Type Col-0 Cell Walls 875

Supplemental Figure 6 Antibodies Raised Against the RGI or XG Components of 876

Pectin Show No Difference in Binding to gae Mutant and Col-0 Cell Walls 877

Supplemental Figure 7 The Effect of B cinerea Treatment on the Expression of GAE 878

Family Genes 879

Supplemental Figure 8 Treatment with 001 Macerozyme Does Not Cause Any 880

Visible Damage to Col-0 Plants 881

Supplemental Figure 9 B cinerea Growth on Plants with Defects in Immune 882

Signaling 883

Supplemental Figure 10 MeJA-Induced Expression of the JA marker gene JAZ10 884

Supplemental Figure 11 The Expression of the JA Marker Gene JAZ5 Is Highly 885

Responsive in gae1 gae6 Plants 886

Supplemental Figure 12 The Expression of PDF12 Is Unaltered in gae1 gae6 Plants 887

Supplemental Figure 13 Macerozyme Treatment Activates MPK3 and MPK6 888

Supplemental Figure 14 B cinerea-Induced ROS Production in Wild-Type Col-0 and 889

gae1 gae6 Plants 890

Supplemental Figure 15 OG-Induced PTI Using 250 microg mL-1 OGs 891

Supplemental Figure 16 Abundance of Plant DNA in B cinerea Treated Plants 892

Supplemental Table 1 Primers used in this study 893

894

ACKNOWLEDGEMENTS 895

We thank William Truman for helpful discussions Simone Ferrari (Sapienza 896

Universita di Roma) for the generous gift of OGs Bruna Bucciarelli (USDA-ARS) for 897

Page 41 of 54

help with fluorescence microscopy the Arabidopsis Biological Resource Center for T-898

DNA insertion lines Paul Knoxrsquos lab (University of Leeds) for LM5 LM6 LM8 LM19 899

and LM20 antibodies and the Complex Carbohydrate Resource Center (University of 900

Georgia) for the CCRC-M7 antibody Most of this work was funded by the Division of 901

Chemical Sciences Geosciences and Biosciences Office of Basic Energy Sciences of 902

the US Department of Energy through Grant DE-FG02-05ER15670 to JG This work 903

was also supported by NSF awards IOS 1339125 and IOS 1021861 to DJK NSF 904

award IOS 1121425 to F K and the USDA National Institute of Food and Agriculture 905

Hatch project number CA-D-PLS-7033-H to DJK 906

907

AUTHOR CONTRIBUTIONS 908

GB and JG designed the research GB GX AT BL NES and NH 909

performed research DJK provided tools GB GX MP AD BL RAH and FK 910

analyzed data GB and JG wrote the paper with input from all authors 911

912

FIGURE LEGENDS 913

Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 Expression 914

Requires PAD4 EDS1 and PBS3 915

(A) Expression of GAE1 in leaves of wild-type Col-0 and mutant plants Expression was916

measured 24 or 32 h after either inoculation with Pma ES4326 (OD600 = 0002) or mock 917

inoculation with 5 mM MgSO4 and was then normalized to the level of ACTIN2 Data 918

from three biological replicates were combined using a mixed linear model Bars 919

represent mean log2-ratios to ACTIN2 plusmn SE Letters indicate significantly different 920

groups with q lt 005 where 005 represents the maximum false discovery rate at which 921

the test may be called significant 922

(B) Expression of GAE6 measured as described in (A)923

924

Figure 2 Susceptibility of gae6 and gae1 gae6 Plants to Pma ES4326 Is Increased 925

Relative to Wild-Type Col-0 926

Leaves of plants of the indicated genotypes were inoculated with Pma ES4326 (OD600 = 927

00002) as described in Methods Bacterial titers in leaves were determined either 928

Page 42 of 54

immediately (0 dpi) or after three days (3 dpi) Bars represent the mean plusmn SE of four 929

independent experiments each with 4 or 12 biological replicates at 0 and 3 dpi 930

respectively Data were combined using a mixed linear model Asterisks indicate 931

significant differences from Col-0 wild-type (q lt 005) Susceptible pad4 plants were 932

included as a positive control 933

934

Figure 3 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves 935

(A) Depiction of the assay for brittleness of gae mutant leaves Fully expanded leaves of936

four to five week-old plants were bent by 180ordm so that the adaxial surface of the leaf was 937

parallel with the soil Broken leaf petioles or midribs were considered brittle 938

(B) Quantification of broken midribs or petioles For each genotype 66 to 82 leaves 939

were analyzed for brittleness as described in (A) Bars represent the ratio of broken to 940

total number of leaves tested Results were analyzed using Fisherrsquos exact test Letters 941

indicate significantly different groups at p lt 005 942

(C) Conductivity measurements Leaves of four week-old plants were inoculated with943

deionized water and then leaf discs were cut out and placed on deionized water 944

Conductivity was measured at the indicated time points After 30 min the water was 945

replaced with fresh water (30 min f w) to account for wounding-related ion leakage from 946

the edges of the leaf discs Three independent experiments with six biological replicates 947

each were performed and data were analyzed using a t-test for each time point P-948

values were corrected using the Bonferroni method Data show means plusmn SE asterisks 949

indicate significant differences from wild-type Col-0 at q lt 001 950

951

Figure 4 Cell Walls of gae6 and gae1 gae6 Plants Are Reduced in GalA 952

(A) to (D) Levels of GalA (A) GlcA (B) cellulose (C) and neutral sugars (D) in alcohol953

insoluble residue (AIR) extracted from leaves of four week-old plants of the indicated 954

genotypes Bars represent means plusmn SE of four biological replicates combined using a 955

mixed linear model Letters indicate significantly different groups (p lt 005) 956

957

Figure 5 Cell Walls of gae1 gae6 Plants Are Reduced in Homogalacturonan 958

Page 43 of 54

Dot blot analyses with anti-LM19 and anti-LM20 antibodies Pectin was extracted from 959

AIR prepared from the indicated genotypes using 1 microL of extraction buffer per 10 microg of 960

AIR Pectin was serially diluted and one microL of undiluted 13 diluted and 19 diluted 961

samples were spotted on a nitrocellulose membrane Three biological replicates were 962

were spotted twice to obtain two technical replicates each All replicates produced 963

similar results 964

(A) Representative dot blot using anti-LM19 antibody965

(B) Relative signal intensities of the spots in the dot blot in (A)966

(C) Representative dot blot using anti-LM20 antibody967

(D) Relative signal intensities of the LM20 dot blot shown in (C)968

Relative signal intensities were measured using Image J software Measurements from 969

the three biological replicates were combined using a linear model after Box-Cox power 970

transformation as described in Methods Bars represent means plusmn SE Asterisks indicate 971

a difference from wild-type Col-0 at p lt 005 972

973

Figure 6 Growth of B cinerea on gae1 gae6 Plants and the Effect of B cinerea on 974

Expression of GAE1 and GAE6 975

(A) Growth of B cinerea isolates Leaves of Col-0 and gae1 gae6 plants were976

inoculated with 25 x 105 spores mL-1 of the B cinerea isolates indicated in the figure 977

Samples were collected two (2 dpi) and three (3 dpi) days later Bars represent means plusmn 978

SE of six biological replicates for each fungus combined using a mixed linear model 979

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 980

(B) and (C) Expression of GAE1 (B) and GAE6 (C) 48 h after inoculation with the B981

cinerea isolates indicated Expression levels were measured by qRT-PCR Bars 982

represent mean log2-ratios to ACTIN2 plusmn SE of three biological replicates combined 983

using a mixed linear model Letters indicate significantly different groups (q lt 005) 984

985

Figure 7 Levels of Soluble Pectin and Expression of PAD3 in Fractions Produced 986

Following Macerozyme Treatment of gae1 gae6 Cell Walls 987

(A) Schematic overview of the experimental procedure for (B) and (C)988

Page 44 of 54

(B) Uronic acid measurements Alcohol-insoluble residue (AIR) from leave tissue of four989

week-old wild-type Col-0 or gae1 gae6 plants was treated with water or 025 990

macerozyme and assayed for uronic acid levels Data from 11 biological replicates were 991

combined using a mixed linear model Bars represent means plusmn SE Letters indicate 992

significant differences (p lt 005) 993

(C) Expression of PAD3 in Col-0 plants treated with the fractions from (B) Extracts from994

(B) were heat inactivated pooled and diluted 5-fold with water Pooled extracts were995

inoculated into four week-old Col-0 plants PAD3 expression was measured 3 h after 996

inoculation and normalized to the level of ACTIN2 expression Data from four biological 997

replicates were combined using a mixed linear model Bars represent mean log2-ratios 998

to ACTIN2 plusmn SE Letters indicate significant differences (p lt 005) 999

1000

Figure 8 Macerozyme-Induced PAD3 Expression Is Absent in gae1 gae6 1001

(A) Expression of PAD3 after macerozyme treatment Four week-old plants were either1002

inoculated with 001 macerozyme or mock inoculated with water and PAD3 1003

expression was measured 3 h later by qRT-PCR Data from four biological replicates 1004

were combined using a mixed linear model Mean log2-ratios to ACTIN2 plusmn SE were 1005

plotted Letters indicate significantly different groups (p lt 005) 1006

(B) Expression of PAD3 after OG treatment PAD3 expression was measured as1007

described in (A) 3 h after inoculation with 100 microg mL-1 OGs or mock inoculation with 1008

water Data are from six biological replicates 1009

(C) PAD3 expression in untreated plants Three biological replicates were performed1010

and data analyzed as described in (A) 1011

(D) Camalexin accumulation after B cinerea treatment Camalexin accumulation was1012

measured in four week-old plants inoculated with the B cinerea strains shown in the 1013

figure Eight biological replicates each were combined using a mixed linear model 1014

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 1015

(E) PAD3 expression 28 h after treatment with the indicated B cinerea strains Data1016

from six biological replicates were collected and analyzed as described in (A) Data in 1017

all figure parts represent means plusmn SE Letters indicate significantly different groups (q lt 1018

005) 1019

Page 45 of 54

1020

Figure 9 Macerozyme-Induced Expression of the SA Marker Genes SID2 and PR1 1021

Is Abolished in gae1 gae6 Plants 1022

(A) to (H) Expression of PR1 (A) (C) (E) and (G) or SID2 (B) (D) (F) and (H) in four1023

week-old Col-0 and gae1 gae6 plants Data were combined using a mixed linear model 1024

Bars represent mean log2-ratios of expression vs ACTIN2 plusmn SE for all figure parts 1025

Letters indicate significantly different groups at p lt 005 for panels (A) to (D) (G) and 1026

(H) and qlt005 for panels (E) to (F) 1027

(A) and (B) Plants were treated with 001 macerozyme or mock treated with water1028

and samples were collected 3 h later Data from four biological replicates are shown 1029

(C) and (D) Plants were inoculated with 100 microg mL-1 OGs or mock treated with water1030

and samples collected 3 h later Data from six biological replicates are shown 1031

(E) and (F) Plants were inoculated with the indicated B cinerea isolates or mock1032

inoculated with B cinerea inoculation medium and samples collected 48 h later Data 1033

from eight biological replicates are shown 1034

(G) and (H) Expression in untreated plants Three biological replicates were performed1035

1036

Figure 10 The Expression of the JA Marker Gene JAZ10 is Highly Responsive in 1037

gae1 gae6 Plants 1038

(A) to (D) JAZ10 expression in four week-old plants Data were combined using a mixed1039

linear model Mean log2-ratios to ACTIN2 plusmn SE are shown Letters indicate significantly 1040

different groups at p lt 005 for panels (A) (B) and (D) and q lt 005 for panel (C) 1041

(A) Plants were inoculated with 001 macerozyme or mock inoculated using water and1042

samples were collected 3 h later Data from four biological replicates are shown 1043

(B) Plants were inoculated with 100 microg mL-1 OGs or mock inoculated using water and1044

samples collected 3 h later Data are from six biological replicates 1045

(C) JAZ10 expression 48 h after treatment with the indicated B cinerea strains Data1046

are from six biological replicates 1047

(D) JAZ10 expression in untreated plants Data are from three biological replicates1048

1049

Figure 11 Gallo 1-Induced Callose Deposition Is Reduced in gae1 gae6 Plants 1050

Page 46 of 54

(A) Callose deposition in four week-old Col-0 and gae1 gae6 plants Plants were1051

inoculated with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) or mock inoculated 1052

with B cinerea inoculation medium and samples were collected after 72 h Callose was 1053

stained with aniline blue and visualized using a Nikon Eclipse Ni-U microscope with a 1054

DAPI filter Three leaves were stained per treatment Scale bars are 100 microm Asterisks 1055

indicate fungal hyphae arrows indicate representative callose spots 1056

(B) Quantification of callose deposition Callose deposits were counted in 200 microm x 2001057

microm squares laid out across the images shown here Bars represent means plusmn SE from 1058

six squares each Letters indicate significantly different groups (p lt 005) according to a 1059

t-test analysis1060

1061

Figure 12 Less Soluble Pectin Accumulates in B cinerea-Infected gae1 gae6 1062

Leaves 1063

Four week-old Col-0 and gae1 gae6 plants were inoculated with 10 microL droplets of the 1064

indicated B cinerea isolate (25 x 105 spores mL-1) or mock inoculated with B cinerea 1065

inoculation medium Leaves were collected 48 h later AIR was extracted from leaf 1066

samples and water added (20 microL infection site) to extract soluble pectin fragments 1067

Samples were vortexed for 7 h centrifuged at 12000 g and the uronic acid 1068

concentration of the supernatant was measured Data from nine biological replicates 1069

were combined using a mixed linear model Means plusmn SE are shown Letters indicate 1070

significant differences (q lt 005) 1071

1072

Figure 13 Macerozyme-Induced PTI Is Lost in gae1 gae6 While OG-Induced PTI Is 1073

Unaltered 1074

(A) Macerozyme-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1075

inoculated with 001 macerozyme or mock inoculated with boiled macerozyme and 1076

were infected with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) 24 h later Samples 1077

were collected immediately (0 dpi) and after two (2 dpi) and three (3 dpi) days Data 1078

show mean plusmn SE of six to 14 biological replicates each combined using a linear model 1079

Letters indicate significantly different groups (q lt 005) 1080

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

Parsed CitationsAdam L and Somerville SC (1996) Genetic characterization of five powdery mildew disease resistance loci in Arabidopsisthaliana Plant J 9 341-356

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Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk RGadrinab C Heller C Jeske A Koesema E Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt MHazari N Hom E Karnes M Mulholland C Ndubaku R Schmidt I Guzman P Aguilar-Henonin L Schmid M Weigel DCarter DE Marchand T Risseeuw E Brogden D Zeko A Crosby WL Berry CC and Ecker JR (2003) Genome-wideinsertional mutagenesis of Arabidopsis thaliana Science 301 653-657

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Asai T Tena G Plotnikova J Willmann MR Chiu WL Gomez-Gomez L Boller T Ausubel FM and Sheen J (2002) MAPkinase signalling cascade in Arabidopsis innate immunity Nature 415 977-983

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Bates D Machler M Bolker BM and Walker SC (2015) Fitting Linear Mixed-Effects Models Using lme4 J Stat Softw 67 1-48Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Benjamini Y and Hochberg Y (1995) Controlling the False Discovery Rate - a Practical and Powerful Approach to MultipleTesting J Roy Stat Soc B Met 57 289-300

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Bethke G Grundman RE Sreekanta S Truman W Katagiri F and Glazebrook J (2014) Arabidopsis PECTINMETHYLESTERASEs Contribute to Immunity against Pseudomonas syringae Plant Physiol 164 1093-1107

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Bouton S Leboeuf E Mouille G Leydecker MT Talbotec J Granier F Lahaye M Hofte H and Truong HN (2002)Quasimodo1 encodes a putative membrane-bound glycosyltransferase required for normal pectin synthesis and cell adhesion inArabidopsis Plant Cell 14 2577-2590

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Cao H Glazebrook J Clarke JD Volko S and Dong XN (1997) The Arabidopsis NPR1 gene that controls systemic acquiredresistance encodes a novel protein containing ankyrin repeats Cell 88 57-63

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Carpita N (2010) Update on Mechanisms of Plant Cell Wall Biosynthesis How plants make cellulose and other (1-gt4)-beta-D-glycans Plant Physiol

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Chisholm ST Coaker G Day B and Staskawicz BJ (2006) Host-microbe interactions Shaping the evolution of the plantimmune response Cell 124 803-814

Page 15 of 54

Page 16 of 54

Because the amount of pectin substrate for pathogen polygalacturonases is 313

reduced we hypothesized that immunity phenotypes of gae1 gae6 plants may result 314

from reduced generation of OGs by these enzymes In turn the reduced OGs could 315

lead to reduced immune responses To test this we used macerozyme a commercial 316

pectinase as a proxy for pathogen pectinases We isolated cell walls as a subcellular 317

fraction known as alcohol insoluble residue (AIR Gille et al 2009) This was treated 318

with macerozyme and tested for release of soluble likely low molecular weight pectin 319

fragments (Figure 7) Treatment with 025 macerozyme released significantly more 320

uronic acid from wild-type Col-0 AIR relative to gae1 gae6 AIR (Figure 7B) To test 321

activity of these pectin-containing solutions we infiltrated them into wild-type Col-0 322

plants and measured PAD3 expression PAD3 is required for synthesis of the 323

phytoalexin camalexin (Zhou et al 1999) and its expression is known to be induced by 324

OG treatment (Ferrari et al 2007) Fractions from macerozyme treated Col-0 walls 325

induced significantly more PAD3 expression than fractions derived from gae1 gae6 326

walls suggesting that macerozyme treatment did indeed release more active soluble 327

uronic acid possibly OGs from wild-type Col-0 walls than from gae1 gae6 walls (Figure 328

7C) 329

Next we studied the effect of macerozyme treatment on expression of PAD3 in 330

intact plants (Figure 8) This treatment did not lead to any visible leaf damage 331

(Supplemental Figure 8) In wild-type Col-0 PAD3 expression was induced three h after 332

treatment with 001 macerozyme (Figure 8A) By contrast no macerozyme-induced 333

induction of PAD3 expression was detected in gae1 gae6 (Figure 8A) However OG 334

treatment induced the expression of PAD3 in both genotypes indicating that gae1 gae6 335

plants do respond to OGs (Figure 8B) Macerozyme treatment also induced expression 336

of the SA marker genes PATHOGENESIS RELATED 1 (PR1) and SID2 in Col-0 but 337

not in gae1 gae6 (Figure 9 panel A and B) PR1 was not significantly induced by 338

purified OGs (Figure 9C) at the time and concentration tested but SID2 was induced by 339

OG treatment in both genotypes (Figure 9D) However the induction was stronger in 340

wild-type Col-0 In addition the JA markers JASMONATE-ZIM-DOMAIN PROTEIN 5 341

(JAZ5) and JAZ10 were induced by macerozyme and OG treatment in both Col-0 and 342

gae1 gae6 (Figure 10A and Supplemental Figure 11) Interestingly expression of JAZ5 343

312 Pectinase-Induced Immune Signaling Is Altered in gae1 gae6 Plants

Page 17 of 54

Page 18 of 54

Page 19 of 54

Page 20 of 54

344

345

346

and JAZ10 was already strongly induced in gae1 gae6 after mock inoculation (Figure 10

and Supplemental Figure 11) We observed no hyperresponsive induction by mock

treatment of PLANT DEFENSIN 12 (PDF12) a necrotrophy and late JA signaling

347 marker gene (Supplemental Figure 12A-D) PDF12 was also not induced by

348 macerozyme or OG treatments (Supplemental Figure 12A-D) This suggests that early

349 JA signaling is induced by mock-treatment in gae1 gae6 Because it is known that JA

350 and SA signaling act antagonistically (Pieterse et al 2012) the strong up-regulation of

351 JA-induced immune signaling in gae1 gae6 might explain the lower OG-induced SID2

352 expression in this genotype

353 To test whether other immune responses are altered in gae1 gae6 plants we

354 analyzed Mitogen-activated protein kinase (MAPK) activity following macerozyme and

355 OG treatments OGs are known to activate the MAPKs MPK3 and MPK6 in Arabidopsis

Page 21 of 54

(Galletti et al 2011) Macerozyme treatment activated MPK3 and MPK6 in wild-type 356

Col-0 whereas MAPK activation in gae1 gae6 was slightly reduced in four out of five 357

experiments performed (Supplemental Figure 13) By contrast OG-induced MAPK 358

activity was similar or increased in gae1 gae6 in four out of five experiments 359

(Supplemental Figure 13) Additionally mock treatment induced MAPK activity in gae1 360

gae6 more strongly than it did in wild-type Col-0 in four out of five experiments 361

performed (Fig S13) Collectively these results illustrate that activation of several 362

immune signaling componentsmdashcamalexin synthesis SA and JA signaling and 363

possibly MAPK activationmdashby macerozyme is substantially altered in gae1 gae6 This 364

suggests that release of soluble pectin fragments possibly OGs by pectinolytic 365

enzymes might be reduced in gae1 gae6 plants while recognition of OGs is intact 366

367

Following B cinerea Infection Production of Soluble Pectin and Deposition of 368

Callose Are Reduced in gae1 gae6 Plants 369

Although pad3 plants were more susceptible to the B cinerea isolates Fresa 525 370

and Gallo 1 which also grew better on gae1 gae6 growth of the isolate Pepper was 371

similar on Col-0 and pad3 (Supplemental Figure 9A) However we did not detect any 372

significant changes in camalexin production after treatment with B cinerea isolate Gallo 373

1 and Fresa 525 in gae1 gae6 (Figure 8D) Neither did we detect changes in PAD3 374

expression after treatment with any B cinerea isolate in gae1 gae6 (Figure 8E) 375

Additionally SID2 and PR1 were similarly induced by all B cinerea isolates in both Col-376

0 and gae1 gae6 (Figure 9E and F) and sid2 plants which are deficient for pathogen-377

induced SA biosynthesis did not show any differences in relative B cinerea growth 378

(Supplemental Figure 9B) Expression of JAZ10 was induced more strongly by Gallo 1 379

treatment in gae1 gae6 than in wild-type Col-0 but not by Fresa 525 or Pepper (Fig 380

10C) while dde2 plants deficient for JA biosynthesis showed enhanced susceptibility to 381

all B cinerea isolates tested (Supplemental Figure 9B) Curiously exogenous 382

application of MeJA did not lead to altered relative fungal growth of B cinerea 383

(Supplemental Figure 9C) and cev1 isoxaben resistant 1 (ixr1) plants which have 384

constitutively high JA levels (Ellis and Turner 2001) did not show any changes in 385

susceptibility to the B cinerea isolates tested (Supplemental Figure 9D) Hence neither 386

Page 22 of 54

differences in B cinerea isolate susceptibility to camalexin nor altered SA or JA 387

signaling by themselves explained the enhanced susceptibility of gae1 gae6 to these B 388

cinerea isolates 389

Hydrogen peroxide accumulation in response to B cinerea isolates Fresa 525 390

Gallo 1 and Pepper was not obviously altered in gae1 gae6 plants (Supplemental Figure 391

14) However callose deposition upon treatment with B cinerea isolate Gallo 1 was 392

strongly reduced in gae1 gae6 (Figure 11) It was previously shown that OG treatment 393

induces callose deposition (Denoux et al 2008) suggesting that the reduction in 394

callose deposition might be due to reduced OG release in gae1 gae6 It is possible that 395

this reduction in callose deposition contributes to the reduced immunity of gae1 gae6 396

plants to B cinerea isolate Gallo 1 397

Because macerozyme could be used to release soluble likely low molecular 398

weight pectin from isolated cell walls we next investigated whether infection with B 399

cinerea was accompanied by accumulation of soluble pectin as well We prepared cell 400

walls (AIR fraction) from leaves infected with B cinerea incubated these cell walls with 401

water and measured uronic acid content of the extracts B cinerea treatment did in fact 402

lead to increased accumulation of soluble pectin in wild-type Col-0 plants when 403

compared to mock-treated samples (Figure 12) No such increased accumulation of 404

water-soluble pectin fractions was observed in gae1 gae6 (Figure 12) 405

406

Macerozyme-Induced Pattern-Triggered Immunity against B cinerea Is Abolished 407

in gae1 gae6 408

It was previously shown that OG treatment can induce patternndashtriggered 409

immunity (PTI) to B cinerea (Ferrari et al 2007) We tested macerozyme treatment to 410

determine whether it could also induce PTI to the B cinerea isolate Gallo 1 (Figure 13) 411

Indeed pretreatment with 001 macerozyme for 24 h led to reduced B cinerea growth 412

in wild-type Col-0 but not gae1 gae6 plants (Figure 13A) Interestingly mock and 413

macerozyme pretreated gae1 gae6 plants exhibited similar growth of B cinerea Gallo 1 414

as did macerozyme-treated Col-0 plants This result is likely due to activation of JA 415

signaling in gae1 gae6 during the pretreatment procedure (Figure 13C and D) OG-416

induced PTI was observed in both genotypes when using a high dose of OGs (Figure 417

Page 23 of 54

13B) whereas a lower dose of OGs only induced PTI in Col-0 (Supplemental Figure 418

15) This suggests that OG perception in gae1 gae6 at least above a certain threshold 419

is unaltered while release of immunity-activating pectin fragments possibly OGs is 420

421 reduced We conclude that immune responses induced by macerozyme treatment

422 which do not occur in gae1 gae6 plants are important for plant immunity to the B

423 cinerea isolate Gallo 1

Page 24 of 54

____________________________________________________________________________

Page 25 of 54

DISCUSSION 426

Here we showed that loss of GAE1 and GAE6 leads to a dramatic reduction in 427

pectin content in the cell wall (Figure 4) likely causing the brittleness of leaves that we 428

observed (Figure 3) Petioles and midribs of gae1 gae6 plants broke easily when bent 429

and inoculation of leaves with deionized water resulted in increased ion leakage 430

possibly due either to microscopic fractures of the cell wall induced by inoculation stress 431

or to a reduced capability of these walls to withstand increased turgor pressure The 432

increased brittleness was associated with altered cell wall composition In our analysis 433

the GalA content of gae1 gae6 leaf walls was reduced by more than 40 and the total 434

uronic acids by over 60 (Figure 4 and Supplemental Figure 5) This is a major change 435

in the overall cell wall composition considering that pectin makes up about 50 of the 436

plant cell walls found in Arabidopsis leaves (Zablackis et al 1995 Nobuta et al 2007 437

Harholt et al 2010) Interestingly quasimodo1 (qua1) plants which have a mutation in 438

a glycosyltransferase family 8 gene are reduced in GalA by 25 and show reduced 439

binding of HG specific antibodies (Bouton et al 2002) 440

Furthermore qua2 plants which have a mutation in a putative methyltransferase 441

have about 13 less total GalA and lack 50 of cell wall HG (Mouille et al 2007) 442

Likewise cgr2 cgr3 plants carrying mutations in another class of Golgi-localized 443

putative methyl-transferases have leaf cell walls that are reduced by about 40 in 444

uronic acid (Kim et al 2015) All three mutants are severely dwarfed (Bouton et al 445

2002 Mouille et al 2007 Kim et al 2015) Additionally qua1 and qua2 mutants show 446

a distorted overall shape likely due to reduced cell cohesion and are extremely fragile 447

(Bouton et al 2002 Mouille et al 2007) By contrast gae1 gae6 plants with a similar 448

strong reduction in GalA and total uronic acids have an overall normal shape and are 449

only slightly smaller than wild-type Col-0 (Supplemental Figure 4) In the wall analysis 450

performed here we did not detect changes in other wall polysaccharides or structures 451

that might compensate for the reduction of pectin in gae1 gae6 It is possible the severe 452

morphological alterations in qua2 and cgr2 cgr3 plants are due to reduction in pectin 453

methylesterification which is unaffected in gae1 gae6 It was speculated that quality 454

control mechanisms in the Golgi prevent secretion of inefficiently esterified pectin in 455

cgr2 cgr3 (Kim et al 2015) suggesting that the Golgi of these plants might over-456

Page 26 of 54

accumulate pectin and that this may contribute to the severe dwarf phenotype of these 457

plants The gae1 gae6 plants described here provide an opportunity to study the effects 458

of reduction of pectin in plants that do not show severe morphological phenotypes 459

We found that gae1 gae6 plants are also compromised in resistance to B 460

cinerea and Pma ES4326 indicating that loss of pectin reduces immunity to these 461

pathogens (Figure 2 and 6) We found that gae1 gae6 plants were slightly more 462

susceptible to Pma ES4326 but showed a strong increase in susceptibility to specific B 463

cinerea isolates This was not unexpected because B cinerea is known to interact with 464

pectin as part of its infection strategy It secretes various pectin-degrading enzymes 465

early during infection and requires some of these enzymes including PG1 PG2 and 466

PME1 for full virulence (ten Have et al 1998 Valette-Collet et al 2003 Kars et al 467

2005b Espino et al 2010) Additionally Arabidopsis plants expressing an antisense 468

construct of the polygalacturonase inhibitor PGIP1 are significantly more susceptible to 469

B cinerea (Ferrari et al 2006) On the other hand Pma ES4326 is not known to470

require cell wall degrading enzymes for full virulence even though it encodes two 471

pectate lyase genes in its genome (PMA4326_07094 and PMA4326_04621 472

wwwbacteriaensemblorg) B cinerea isolates are also known to be genetically quite 473

diverse in their polygalacturonase (PG) loci and genetic variability in PG2 is associated 474

with varying growth on pectin containing media (Rowe and Kliebenstein 2007) 475

Furthermore the contribution of PG1 and PG2 to B cinerea virulence seems to depend 476

on the specific B cinerea isolate and the host species (Kars et al 2005a Zhang and 477

Van Kan 2013) B cinerea isolates are also known to be differently sensitive to the anti-478

microbial secondary metabolite camalexin (Kliebenstein et al 2005) In line with these 479

observations the susceptibility of gae1 gae6 plants differed depending on the specific 480

B cinerea isolate Whereas Fresa 525 Gallo 1 UK Razz and KT grew better in gae1481

gae6 we detected no difference in Acacia Apple 517 DN Grape Pepper or Rasp 482

growth (Figure 6) 483

B cinerea was shown to metabolize pectin and to possibly utilize GalA as a484

carbon source during in planta growth (Zhang et al 2011 Zhang and Van Kan 2013) 485

Our data show that gae1 gae6 plants which are reduced in GalA content did allow 486

enhanced growth of specific B cinerea isolates but no reduction in growth of any B 487

Page 27 of 54

cinerea isolates tested was found This suggests that utilization of pectin as a carbon 488

source was not a contributing factor to alterations in B cinerea susceptibility It is 489

possible that changes in the cell wall composition affect the structural integrity of the 490

wall and affect hyphal penetration of specific B cinerea isolates However OGs (small 491

HG fragments) are likely released during the infection process (Ferrari et al 2013) 492

OGs function as DAMPs and initiate immune responses in Arabidopsis (Ferrari et al 493

2013) Treatment of Arabidopsis with OGs having a degree of polymerization (DP) of 494

10-15 sugar subunits was previously shown to enhance resistance to B cinerea (Ferrari495

et al 2007) We showed that pretreatment with OGs or macerozyme induced enhanced 496

immunity to B cinerea isolate Gallo 1 in wild-type Col-0 but only OGs and not 497

macerozyme induced immunity in gae1 gae6 (Figure 13) Our data suggest gae1 gae6 498

plants which have reduced pectin content may release less OGs and that this 499

reduction leads to the increased susceptibility to B cinerea 500

Consistently we found that macerozyme treatment released less soluble pectin 501

from gae1 gae6 cell walls than from wild-type walls Fractions containing soluble pectin 502

released from gae1 gae6 were also less active in inducing PAD3 gene expression than 503

the fractions released from wild-type walls (Figure 7) We also showed that less soluble 504

pectin accumulated in gae1 gae6 than in wild-type plants 48 h after treatment with B 505

cinerea (Figure 12) 506

We observed that loss of pectin leads to alterations in A thaliana immune 507

signaling Macerozyme treatment induced expression of PAD3 and the SA marker 508

genes PR1 and SID2 in wild-type Col-0 but not in gae1 gae6 whereas OG treatment 509

led to increased PAD3 and SID2 expression in both wild-type and gae1 gae6 (Figure 8 510

and 9) This suggests that although release of active soluble pectin in these plants 511

might be altered OG recognition is intact In fact cell walls isolated from qua1 another 512

mutant known to be reduced in HG released less OGs (DP1-6) than did wild-type cell 513

walls upon endopolygalacturonase treatment (Bouton et al 2002) This was also true 514

for gae1 gae6 cell walls as macerozyme treatment of isolated gae1 gae6 cell walls 515

released less soluble pectin than treatment of Col-0 cell walls Because the fraction 516

from gae1 gae6 plants was also less active in inducing PAD3 expression (Figure 7) it 517

seems possible that biologically active OGs were the active component inducing PAD3 518

Page 28 of 54

expression Interestingly expression of the JA marker genes JAZ5 and JAZ10 was 519

higher in gae1 gae6 than in wild-type mock-inoculated plants but was comparable to 520

wild-type in untreated plants suggesting that JA signaling in gae1 gae6 is 521

hyperresponsive (Figure 10 and Supplemental Figure 11) This induction of JA signaling 522

might be due to wounding stress incurred during the inoculation process JA and SA 523

signaling generally act antagonistically (Pieterse et al 2012) Increased JA signaling 524

activity might hence explain why mock- and macerozyme-treated gae1 gae6 plants 525

exhibited reduced expression of the SA marker PR1 and why OG treatment induced 526

expression of SID2 another SA marker to a lower extent in gae1 gae6 than in wild-type 527

plants (Figure 9) 528

No single immune signaling component was found to be the sole contributor to 529

the compromised immunity of gae1 gae6 to specific B cinerea isolates such as Gallo 1 530

and Fresa 525 These isolates grew better on pad3 while growth of Pepper another B 531

cinerea isolate that did not show altered virulence on gae1 gae6 was unaltered 532

(Supplemental Figure 9) This suggested that differences in camalexin sensitivity of 533

these B cinerea isolates might account for the differences in gae1 gae6 susceptibility 534

However camalexin accumulation and PAD3 expression was the same for wild-type 535

Col-0 and gae1 gae6 for the B cinerea isolates Fresa 525 and Gallo 1 (Figure 8) which 536

showed enhanced growth on gae1 gae6 plants It should be noted that B cinerea 537

virulence is often estimated by comparing lesion sizes Lesion size and measurements 538

of relative fungal growth by qPCR do not always show the same results For example 539

although Denby et al (2004) showed that Pepper causes larger lesions on pad3 than on 540

wild-type this study showed that relative fungal growth of Pepper on pad3 was 541

unaltered 542

We observed that callose deposition was strongly reduced in gae1 gae6 plants 543

72 h after treatment with B cinerea isolate Gallo 1 (Figure 11) possibly due to reduced 544

release of active soluble pectin Interestingly OG-induced PTI to an unspecified isolate 545

of B cinerea was intact in the pmr4 mutant which does not produce callose (Galletti et 546

al 2008) Due to the high SA levels in pmr4 plants (Nishimura et al 2003) and the fact 547

that different B cinerea isolates vary both in induction of and reaction to defense 548

responses (Kliebenstein et al 2005 Rowe et al 2010) we could not determine 549

Page 29 of 54

whether reduced callose deposition causes the increased susceptibility of gae1 gae6 to 550

B cinerea isolate Gallo 1551

We did not observe any changes in B cinerea growth either on cev1 ixr1 plants 552

which are known to have constitutively high JA and OPDA levels (Ellis et al 2002b) or 553

on plants exogenously treated with MeJA (Supplemental Figure 9) Because plants 554

were kept uncovered for 3 h after MeJA treatment it is unclear how much MeJA was 555

bioavailable in the plant However JAZ10 expression was induced 3 h after spraying 556

with MeJA (Supplemental Figure 10) Because pathogen growth generally depends on 557

the combination of a multitude of immune signaling events we conclude that multiple 558

changes to the immune signaling in gae1 gae6 together account for the altered B 559

cinerea growth phenotypes detected 560

We showed that expression of GAE1 and GAE6 is repressed by the bacterial 561

hemibiotroph Pma ES4326 This repression did not occur in an eds1 or pad4 562

background or in pbs3 at the 24-h time point (Figure 1 and Supplemental Figure 1) 563

During the Arabidopsis - Pma ES4326 interaction the expression of many defense 564

related genes is altered in a PAD4EDS1 dependent fashion (Wang et al 2008) Some 565

of these expression changes require SA CBP60g and SARD1 are thought to act 566

downstream of PAD4 and EDS1 but upstream of SA-dependent responses (Wang et 567

al 2011) PBS3 is also thought to act downstream of PAD4 and EDS1 and upstream of 568

SA but the set of genes whose expression it affects is only partially overlapping with 569

those affected by CBP60gSARD1 (Wang et al 2008 Wang et al 2011) Thus GAE1 570

and GAE6 repression is regulated by the part of the immune signaling network that 571

requires PAD4 EDS1 and PBS3 but is independent of CBP60gSARD1 and SA 572

Because EDS1 PAD4 and PBS3 are important components of the plant immune 573

signaling network (Wiermer et al 2005 Nobuta et al 2007) one might conclude that 574

repression of GAE1 and GAE6 may be part of the plant immune response to Pma 575

ES4326 B cinerea treatment also reduced GAE1 and GAE6 expression but this was 576

mostly independent of PAD4 (Figure 6) while both Pma ES4326 and B cinerea repress 577

GAE1 and GAE6 expression the host signaling mechanisms responsible likely differ 578

In summary our data show that Arabidopsis GAE1 and GAE6 are required for 579

pectin biosynthesis leaf flexibility and immunity to Pma ES4326 and to specific B 580

Page 30 of 54

cinerea isolates We used macerozyme a commercial pectinase to mimic the effect of 581

B cinerea pectinases Macerozyme treatment released less pectin from gae1 gae6 cell582

walls and induced less immune signaling in gae1 gae6 plants Less water soluble pectin 583

also accumulated in B cinerea infected gae1 gae6 plants and macerozyme-induced 584

immunity to B cinerea isolate Gallo 1 was reduced in these plants We conclude that 585

the reduced pectin content in these mutants may lead to reduced release of soluble 586

pectin including active OGs during pathogen attack and that this compromises 587

immunity 588

589

590

Page 31 of 54

METHODS 591

Plant Materials and Growth Conditions 592

Wild-type Columbia (Col-0) and all mutant plants (in Col-0 background) were 593

grown on sterilized BM2 germinating mix (Berger Inc Quebec Canada) in a controlled 594

environment chamber (Conviron Pembina ND USA) with a 12-h photoperiod under 595

100 microM m-2 s-1 fluorescent illumination at 22degC and 75 relative humidity Germplasm 596

used is described in the Accession Numbers section at the end of Methods 597

598

Data Analysis and Replicates 599

For biological replicates samples were obtained from separate plant tissues and 600

in many cases from plants grown in different flats In some experiments multiple 601

technical replicates were measured per biological replicate For statistical analysis all 602

technical replicates were averaged and the averages were considered one biological 603

replicate Independent experiments were performed at different times and often 604

consisted of multiple biological replicates per experiment 605

Data analysis was performed using mixed linear models in the R programming 606

environment (Bates et al 2015) unless otherwise indicated Generally genotypes 607

treatments and time points were used as fixed effects and replicate-specific effects like 608

different pots flats etc were considered random effects We reported p-values 609

obtained from the mixed linear model when less than 10 comparisons were performed 610

and q-values for more than 10 comparisons to correct for multiple comparison testing 611

Q-values were calculated using the Benjamini-Hochberg procedure (Benjamini and612

Hochberg 1995) unless specified otherwise 613

614

Pathogen Strains Growth Conditions and Pathogen Growth Assays 615

Growth of Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) and 616

bacterial growth assays were performed as previously described (Bethke et al 2014) 617

For qRT-PCR assays four week-old plants were inoculated with bacteria (OD600 = 618

0002) or mock (5 mM MgSO4) At least four leaves from two plants were collected at 619

the indicated time points per biological replicate 620

Page 32 of 54

Prior to each experiment Botrytis cinerea isolates were grown on 1x PDA 621

medium (Difco) for 10 d at room temperature All isolates used have been described 622

previously (Rowe and Kliebenstein 2007) The spores were washed from the surface of 623

the plate using an inoculation medium (1x Gamborgrsquos B-5 basal salt mixture (Sigma-624

Aldrich G5768) 2 (wv) glucose 10 mM phosphate buffer pH 64) and fungal hyphae 625

were removed from the suspension by filtering through four layers of cheesecloth The 626

concentration of spores was determined using a hemocytometer and adjusted to 25 x 627

105 spores mL-1 Four week-old Arabidopsis plants were inoculated by placing one 10-628

microL droplet of the B cinerea spore solution or inoculation medium (mock) on the adaxial 629

leaf surface of fully expanded leaves Inoculated plants were kept at 100 relative 630

humidity Infection sites were collected at various time points using a cork borer For 631

qRT-PCR assays at least four leaf discs from one plant were combined for each 632

biological sample For B cinerea growth assays at least 12 leaf discs from three to six 633

plants were pooled per biological sample Relative fungal growth was determined by 634

measuring the abundance of a fungal gene relative to a plant gene as described below 635

In brief DNA from infected tissue was extracted and equal amounts of total DNA were 636

used to perform qPCR reactions using the B cinerea cutinase A and the Arabidopsis 637

SK11 gene respectively Primers were described in (Gachon and Saindrenan 2004) 638

and are listed in Supplemental Table 1 Two technical replicates were combined for 639

each biological replicate B cinerea is a necrotrophic fungal pathogen and may destroy 640

plant DNA as disease progresses However decreases in the amount of plant DNA 641

over the course of our experiments were only observed at late time points with very 642

susceptible plants so the qPCR assay is generally a reasonable estimate of fungal 643

biomass (Supplemental Figure 16) 644

645

Treatment with Macerozyme or OGs 646

For PTI assays four week-old plants either were inoculated with 001 (wv) 647

macerozyme R-10 (Yakult) a Rhizopus polygalacturonase (EC 32115) that possesses 648

high pectinase and hemicellulase activity or were mock inoculated using heat-649

inactivated macerozyme Other similar plants were either inoculated with 250 or 500 microg 650

mL-1 OGs or mock inoculated with water One d later plants were infected with B 651

Page 33 of 54

cinerea and samples were collected immediately (0 dpi) and two or three d later To 652

investigate the role of MeJA on B cinerea growth plants were sprayed with 1mM MeJA 653

in water with 001 (vv) Tween 20 Covers were removed so that leaves could dry off 654

before plants were inoculated with B cinerea three h later Samples were harvested at 655

the indicated time points For expression analysis and MAPK activation assays plants 656

were either inoculated with 100 microg mL-1 OGs or mock inoculated with water OGs used 657

for experiments described in Figures 8 to 10 and Supplemental Figure 12 were a kind 658

gift from Simone Ferrari OGs used in experiments described in Figure 13 and 659

Supplemental Figures 11 13 and 15 were prepared as previously described (Kohorn et 660

al 2014) 661

662

Expression Analysis 663

Leaf tissue from inoculated plants was harvested at the indicated time points 664

flash frozen pulverized and RNA extracted using Trizol (Invitrogen) Quantitative RT-665

PCR was performed using the SuperScript III Platinum SYBR Green One-Step 666

quantitative RT-PCR kit (Invitrogen) and a Lightcycler 480 Real-Time PCR system 667

(Roche) as previously described (Truman and Glazebrook 2012) In brief equal 668

amounts of total RNA and a gene specific primer were used for each reaction The 669

crossing point (Cp) was calculated using the 2nd derivative max method provided with 670

the Lightcycler software for each amplification curve Each reaction was run with two 671

technical replicates and the Cp values for these replicates were averaged ACTIN2 was 672

used as a stably expressed reference gene At least three biological replicates each 673

were performed and analyzed using a mixed-linear model RT-PCR was performed to 674

verify T-DNA insertion lines using the One-Step RT-PCR kit (Qiagen) according to the 675

manufacturerrsquos instructions The primers used can be found in Supplemental Table 1 676

677

Electrolyte Leakage Assay 678

Fully expanded leaves of four week-old plants were inoculated with water (Milli-Q 679

grade) using a needleless syringe Leaf discs were collected using a cork borer and 680

three leaf discs from one leaf were placed with the adaxial surface down onto 2 mL 681

water in a 12-well cell culture plate Conductivity was measured using a Horiba B-173 682

Page 34 of 54

conductivity meter after 30 min and then the water was replaced Conductivity was 683

measured again 30 min later (1 h total) as indicated in Figure 3C Six biological 684

replicates each were performed in three independent experiments and means and SEs 685

were calculated Two-sided t-tests were performed for each time point 686

687

DAB and Aniline Blue Staining 688

Leaves from four week-old plants were either mock inoculated with B cinerea 689

inoculation medium or inoculated with 10 microL droplets of a B cinerea spore solution (25 690

x 105 spores mL-1) of the indicated isolates Samples were collected 72 h later For 33prime-691

diaminobenzidine (DAB) staining two to five inoculated leaves were vacuum infiltrated 692

with 33prime-Diaminobenzidine (1 mg mL-1 in water pH 38) solution and stained overnight 693

in the dark Leaves were destained using 100 ethanol at 65degC and then transferred to 694

50 (vv) glycerol and photographed 695

Callose was visualized by staining leaves with aniline blue as previously 696

described (Adam and Somerville 1996) Briefly leaves were vacuum infiltrated with 697

alcoholic lactophenol (one volume of phenolglycerollactic acidwater (1111) mixed 698

with two volumes of ethanol) and destained at 65degC Next leaves were moved through 699

an alcohol gradient (50 20 and 10 (vv) ethanol) into water The destained leaves 700

were stained for 30 min in 150 mM K2HPO4 (pH 95) with 001 (wv) aniline blue 701

Samples were mounted in water and examined using a Nikon Eclipse Ni-U microscope 702

at 10x magnification using a DAPI filter (excitation at 325-375 nm emission at 435-485 703

nm) For callose quantification six 200 microm x 200 microm squares were counted for each 704

sample type Callose deposits per mm2 were calculated and data analyzed using a t-705

test Note that there is an inherent uncertainty associated with such counts because 706

callose deposits may not all be in focus at the same time 707

708

MAPK Activity Assay 709

Fully expanded leaves of four week-old plants were inoculated with 001 710

macerozyme 100 microg mL-1 OGs 100 nM flg22 or water (mock) flg22 a peptide from 711

bacterial flagella that induces immune responses was used as a positive control for 712

Page 35 of 54

MAPK activation (Asai et al 2002) and was purchased from EZBiolab Inc (Westfield 713

IN USA) Samples were harvested 10 min later and immediately frozen in liquid 714

nitrogen Protein was extracted using an extraction buffer containing 25 mM Tris pH 78 715

75 mM NaCl 1 mM DTT 1 mM NaF 05mM Na3VO4 01 (vv) Tween 20 one 716

cOmplete Mini protease inhibitor cocktail tablet (Roche) and one PhosSTOP 717

phosphatase inhibitor cocktail tablet (Roche) per 10 mL buffer 15 microg of total protein 718

were electrophoresed on a 10 SDS-polyacrylamide gel and then blotted onto a PVDF 719

membrane (Bio-Rad 162-0177) Activated MAPKs were detected using p4442 MAPK 720

(Erk12) antibody (9102S Cell Signal 12500 in TBST (20 mM Tris pH 75 150 mM 721

NaCl 01 (vv) Tween 20)) Anti-Rabbit IgG-HRP (115000 in TBST A6154 Sigma) 722

and ECL Plus substrate (Pierce 32132) MPK3 and MPK6 were detected with an anti-723

MPK3 antibody (M8318 Sigma 12000 in TBST and 3 (wv) milk) or an anti-MPK6 724

antibody (A7104 Sigma 16000 in TBST and 3 (wv) milk) respectively The 725

secondary antibody and detection were the same as for the p4442 antibody Five 726

independent experiments were performed 727

728

Preparation of Alcohol Insoluble Residue (AIR) Pectin Extraction and Uronic Acid 729

Measurements 730

Fully expanded leaves from four week-old plants that were incubated for 48 h in 731

the dark to reduce starch were harvested flash frozen and pulverized AIR was 732

extracted by washing ground material twice in 70 (vv) ethanol three times in a 733

mixture of chloroform and methanol (11 vv) and once in acetone (Gille et al 2009) 734

and was then air dried Cell wall pectin was extracted from AIR with cell wall extraction 735

buffer (50 mM Trizma 50 mM CDTA pH 72) at 95degC for 15 min Samples were 736

homogenized using a paint shaker (Harbil 5G-HD) and glass beads (3 mm diameter) 737

For dot blot experiments 500 microL of cell wall extraction buffer were used per 10 mg of 738

AIR Debris was precipitated by centrifugation for 10 min at 10000 g 739

Total uronic acid content of pectin extracted as described above using 500 microL of 740

cell wall extraction buffer per 1 mg of AIR was measured as described previously 741

(Filisetti-Cozzi and Carpita 1991 van den Hoogen et al 1998) First 36 microL of pectin 742

extract were mixed with 4 microL of 4 M sulfamic acid Then 200 microL of sulfuric acid 743

Page 36 of 54

containing 120 mM sodium tetraborate was added and samples were incubated at 80degC 744

for 1 h Following cooling on ice the optical density of samples was measured at 490 745

nm Next 40 microL of m-hydroxydiphenyl reagent (100 microL of m-hydroxydiphenyl in dimethyl 746

sulfoxide at 100 mgmL mixed with 49 mL 80 (vv) sulfuric acid just before use) was 747

added and the samples were mixed The optical density at 490 nm was measured 748

again The optical density before m-hydroxydiphenyl reagent addition was subtracted 749

from the optical density measured after addition of the dye The concentration of uronic 750

acid was calculated using known amounts of GalA as a standard 751

752

Release of Pectin from Arabidopsis Cell Walls Using Macerozyme 753

AIR preparations from leaves of four week-old plants were treated with 025 754

(wv) macerozyme R-10 (Yakult) or water (mock) at a concentration of 100 microL per mg 755

AIR Samples were incubated for 3 h at room temperature on a vortex shaker and 756

centrifuged for 10 min at 12000 g Total uronic acid concentration of 5-fold diluted 757

supernatants was determined for two technical replicates each The concentration of 758

uronic acid was calculated using known amounts of GalA as a standard For 759

subsequent qRT-PCR experiments supernatants from multiple extractions were 760

combined boiled for 20 min to inactivate macerozyme diluted 10-fold with water and 761

inoculated into four week-old Col-0 plants Heat-inactivated 10-fold diluted macerozyme 762

solutions were used as mock treatment Samples were collected 3 h later and PAD3 763

expression was determined 764

765

Release of Pectin from Arabidopsis Leaves Infected with B cinerea 766

Fully expanded leaves of four week-old plants were either inoculated with up to 767

five 10 microL droplets of B cinerea spore solution (25 x 105 spores mL-1) or mock 768

inoculated with B cinerea inoculation medium For each biological replicate leaves with 769

a combined count of at least 20 infection sites were collected 48 h later AIR was 770

extracted as described above but plants were not dark-treated or freeze-dried and the 771

first ethanol step was performed using 85 (vv) ethanol To extract soluble pectin 772

fragments AIR samples were mixed with water at a concentration of 20 microL per infection 773

site incubated for 7 h at room temperature on a vortex shaker and centrifuged for 10 774

Page 37 of 54

min at 12000 g Total uronic acid concentration of supernatants was determined for two 775

technical replicates each The concentration of uronic acid was calculated using known 776

amounts of GalA as a standard 777

778

Measurement of Uronic Acids Neutral Monosaccharides and Cellulose Content in 779

Cell Walls 780

Neutral sugars and uronic acids from non-cellulosic polysaccharides in cell walls 781

(AIR) were released by trifluoroacetic acid (TFA) hydrolysis Approximately 1 mg of AIR 782

was hydrolyzed with 2 M TFA at 121degC for 90 min The TFA-soluble material was split 783

into two equal parts one for uronic acid and one for neutral sugar determination The 784

uronic acid content was determined by analyzing the hydrolyzate using a CarboPac 785

PA200 anion-exchange column with an ICS-3000 Dionex chromatography system 786

(Dionex Sunnyvale CA USA) The elution profile consisted of a linear gradient of 50 to 787

200 mM sodium acetate in 01 M NaOH in 10 min at 04 mL per min Alditol acetate 788

derivatives of neutral sugars were produced as described (York et al 1985) In brief 789

monosaccharides in the hydrolyzate were reduced with NaBH4 and peracetylated with 790

acetic anhydride and pyridine at 121degC for 20 min The generated alditol acetates were 791

analyzed by GC-MS Crystalline cellulose was measured according to Updegraff (1969) 792

After hydrolysis of noncellulosic polysaccharides from AIR with the Updegraff reagent 793

(acetic acid nitric acids water 812 (vv) ) the remaining pellet was hydrolyzed in 794

72 sulfuric acid The resulting glucose quantity was determined by the anthrone 795

method (Scott and Melvin 1953) 796

797

Dot Blot Experiments 798

Pectin solutions extracted using 50 microL of cell wall extraction buffer per mg AIR 799

were serially diluted and nitrocellulose membranes were spotted with 1 microL of the diluted 800

pectin solutions Membranes were dried overnight blocked with 5 milk (wv) in 1x 801

PBS (8 g L-1 NaCl 02 g L-1 KCl 144 g L-1 Na2HPO4 024 g L-1 KH2PO4 pH 74) and 802

probed with LM5 (Jones et al 1997) LM6 (Willats et al 1998) LM8 (Willats et al 803

2004) LM19 and LM20 (Verhertbruggen et al 2009a) or CCRC-M7 (Steffan et al 804

Page 38 of 54

1995) antibodies LM series antibodies were diluted 1250 and CCRC-M7 was diluted 805

1500 in 5 milk powder (Nestle Carnation) in 1 x PBS For LM series antibodies a 806

goat anti-Rat HRP conjugated antibody (Bethyl A110-105P 15000 diluted) and for 807

CCRC-M7 an anti-Mouse IgG HRP conjugate (Promega W402B 12500 diluted) was 808

used as secondary antibody in 5 milk powder in 1x PBS LM series antibodies were 809

obtained from plant probes (wwwplantprobesnet) and CCRC-M7 from CarboSource 810

(wwwcarbosourcenet) Membranes were washed after incubation with the primary and 811

secondary antibodies with 1x PBS Dot blots were developed using the ECL system 812

(GE Healthcare Amersham ECL prime) For quantification of signals dot blot results on 813

x-ray films were photographed using a CCD camera and intensities were measured814

using Image Jrsquos Integrated Density function Locally adjusted background was 815

subtracted As the dynamic range of the signal on an x-ray film was narrow non-816

linearity in the measured values was corrected for each genotype in each biological 817

replicate using the values from different dilutions in two technical replicates We noticed 818

that the residuals were not normally distributed and performed Box-Cox power 819

transformation (Box and Cox 1964) to obtain normally distributed values for the 820

subsequent statistical analysis of the data For LM19 Box-Cox selected power 821

transformation was done to the power of 038th and for LM20 to the power 075th After 822

correction of the transformed measured values with the effect of the biological 823

replicates the values were fit to a linear model with the Arabidopsis genotype as the 824

fixed effect This linear model was used both to generate the plots in Figures 5B and D 825

and to test significant differences 826

827

Camalexin Measurements 828

Four week-old plants were infected with B cinerea and samples (three leaf disks 829

from one plant each) were harvested at the indicated time points flash frozen and 830

pulverized Each sample was extracted with 300 microL of 90 methanol (vv) 50 microL of the 831

extract was run on an Agilent Lichrocart 250-4 RP18e 5-microm column using an Agilent 832

1100 series HPLC Camalexin was detected using a DiodeArray (DAD) at 330 nm and 833

with a fluorescence detector at emission 318 nmexcitation 385 nm (Agilent) Separation 834

was achieved using the following program with aqueous acetonitrile 5 min gradient 835

Page 39 of 54

from 63 to 69 acetonitrile 30 s gradient from 69 to 99 acetonitrile 2 min at 99 836

acetonitrile and a post-run equilibration of 35 min at 63 acetonitrile (Denby et al 837

2004 Kliebenstein et al 2005) Purified camalexin was used to produce a standard 838

curve to identify and quantitate camalexin 839

840

Accession Numbers 841

Microarray data used can be accessed at NCBI Gene Expression Omnnibus 842

(httpwwwncbinlmnihgovgeo) GSE18978 Sequence information for all Arabidopsis 843

genes described in this article can be found in TAIR (wwwarabidopsisorg) using the 844

following accession numbers PAD3 (CYP71B15 At3g26830) SID2 (ICS1 At1g74710) 845

GAE1 (At4g30440) GAE2 (At1g02000) GAE3 (At4g00110) GAE4 (At2g45310) GAE5 846

(At4g12250) GAE6 (At3g23820) PR1 (At2g14610) JAZ5 (At1g17380) JAZ10 847

(At5g13220) ACTIN2 (At3g18780) PDF12a (At5g44420) PDF12b (At2g26020) 848

AtSK11 (At5g26751) The sequence of the B cinerea cutinase Bc-CutA (Z69264) can 849

be found in the DNA Data Bank of Japan (httpgetentryddbjnigacjp) Germplasm 850

used included dde2-2 (At5g42650 (von Malek et al 2002)) pad3-1 (At3g26830 (Zhou 851

et al 1999)) pad4-1 (At3g52430 (Jirage et al 1999)) sid2-2 (At1g74710 (Wildermuth 852

et al 2001)) pbs3-2 (At5g13320 SALK_018225 (Nobuta et al 2007)) cbp60a-1 853

(At5g62570 SALK_124410 (Truman et al 2013)) cbp60g-1 sard1-2 (At5g26920 854

SALK_023199 At1g73805 SALK_052422 (Wang et al 2011) cbp60a-1 cbp60g-1 855

sard1-2 (Truman et al 2013) gae1-1 (At4g30440 SALK_085554) gae6-1 (At3g23280 856

SALK_104454) gae6-2 (At3g23280 SALK_017191) ixr1-1 cev1 (At5g05170 (Ellis 857

and Turner 2001 Scheible et al 2001)) eds1 plants were derived by introgression of 858

the Ler eds1-2 allele into Col-0 that contains two copies of EDS1 (At3g48090 859

At3g48080 (Bartsch et al 2006) mpk6-2 (At2g43790 SALK_073907 (Liu and Zhang 860

2004)) mpk3-DG (At3g45640 a fast neutron deletion mutant (Miles et al 2005)) and 861

npr1-1 (At1g64280 (Cao et al 1997)) T-DNA insertion lines were part of the SALK 862

collection (Alonso et al 2003) and were obtained from the Arabidopsis Biological 863

Resource Center 864

865

Supplemental Data 866

Page 40 of 54

Supplemental Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 867

Expression Does Not Require CBP60s or SID2 868

Supplemental Figure 2 Expression of GAE Family Members uponTreatment with Pma 869

ES4326 870

Supplemental Figure 3 Characterization of T-DNA Insertions in GAE1 and GAE6 871

Supplemental Figure 4 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves and 872

Are Slightly Smaller than Wild-Type Col-0 Plants 873

Supplemental Figure 5 Cell Walls of gae1 gae6 Plants Contain Less Uronic Acid than 874

Wild-Type Col-0 Cell Walls 875

Supplemental Figure 6 Antibodies Raised Against the RGI or XG Components of 876

Pectin Show No Difference in Binding to gae Mutant and Col-0 Cell Walls 877

Supplemental Figure 7 The Effect of B cinerea Treatment on the Expression of GAE 878

Family Genes 879

Supplemental Figure 8 Treatment with 001 Macerozyme Does Not Cause Any 880

Visible Damage to Col-0 Plants 881

Supplemental Figure 9 B cinerea Growth on Plants with Defects in Immune 882

Signaling 883

Supplemental Figure 10 MeJA-Induced Expression of the JA marker gene JAZ10 884

Supplemental Figure 11 The Expression of the JA Marker Gene JAZ5 Is Highly 885

Responsive in gae1 gae6 Plants 886

Supplemental Figure 12 The Expression of PDF12 Is Unaltered in gae1 gae6 Plants 887

Supplemental Figure 13 Macerozyme Treatment Activates MPK3 and MPK6 888

Supplemental Figure 14 B cinerea-Induced ROS Production in Wild-Type Col-0 and 889

gae1 gae6 Plants 890

Supplemental Figure 15 OG-Induced PTI Using 250 microg mL-1 OGs 891

Supplemental Figure 16 Abundance of Plant DNA in B cinerea Treated Plants 892

Supplemental Table 1 Primers used in this study 893

894

ACKNOWLEDGEMENTS 895

We thank William Truman for helpful discussions Simone Ferrari (Sapienza 896

Universita di Roma) for the generous gift of OGs Bruna Bucciarelli (USDA-ARS) for 897

Page 41 of 54

help with fluorescence microscopy the Arabidopsis Biological Resource Center for T-898

DNA insertion lines Paul Knoxrsquos lab (University of Leeds) for LM5 LM6 LM8 LM19 899

and LM20 antibodies and the Complex Carbohydrate Resource Center (University of 900

Georgia) for the CCRC-M7 antibody Most of this work was funded by the Division of 901

Chemical Sciences Geosciences and Biosciences Office of Basic Energy Sciences of 902

the US Department of Energy through Grant DE-FG02-05ER15670 to JG This work 903

was also supported by NSF awards IOS 1339125 and IOS 1021861 to DJK NSF 904

award IOS 1121425 to F K and the USDA National Institute of Food and Agriculture 905

Hatch project number CA-D-PLS-7033-H to DJK 906

907

AUTHOR CONTRIBUTIONS 908

GB and JG designed the research GB GX AT BL NES and NH 909

performed research DJK provided tools GB GX MP AD BL RAH and FK 910

analyzed data GB and JG wrote the paper with input from all authors 911

912

FIGURE LEGENDS 913

Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 Expression 914

Requires PAD4 EDS1 and PBS3 915

(A) Expression of GAE1 in leaves of wild-type Col-0 and mutant plants Expression was916

measured 24 or 32 h after either inoculation with Pma ES4326 (OD600 = 0002) or mock 917

inoculation with 5 mM MgSO4 and was then normalized to the level of ACTIN2 Data 918

from three biological replicates were combined using a mixed linear model Bars 919

represent mean log2-ratios to ACTIN2 plusmn SE Letters indicate significantly different 920

groups with q lt 005 where 005 represents the maximum false discovery rate at which 921

the test may be called significant 922

(B) Expression of GAE6 measured as described in (A)923

924

Figure 2 Susceptibility of gae6 and gae1 gae6 Plants to Pma ES4326 Is Increased 925

Relative to Wild-Type Col-0 926

Leaves of plants of the indicated genotypes were inoculated with Pma ES4326 (OD600 = 927

00002) as described in Methods Bacterial titers in leaves were determined either 928

Page 42 of 54

immediately (0 dpi) or after three days (3 dpi) Bars represent the mean plusmn SE of four 929

independent experiments each with 4 or 12 biological replicates at 0 and 3 dpi 930

respectively Data were combined using a mixed linear model Asterisks indicate 931

significant differences from Col-0 wild-type (q lt 005) Susceptible pad4 plants were 932

included as a positive control 933

934

Figure 3 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves 935

(A) Depiction of the assay for brittleness of gae mutant leaves Fully expanded leaves of936

four to five week-old plants were bent by 180ordm so that the adaxial surface of the leaf was 937

parallel with the soil Broken leaf petioles or midribs were considered brittle 938

(B) Quantification of broken midribs or petioles For each genotype 66 to 82 leaves 939

were analyzed for brittleness as described in (A) Bars represent the ratio of broken to 940

total number of leaves tested Results were analyzed using Fisherrsquos exact test Letters 941

indicate significantly different groups at p lt 005 942

(C) Conductivity measurements Leaves of four week-old plants were inoculated with943

deionized water and then leaf discs were cut out and placed on deionized water 944

Conductivity was measured at the indicated time points After 30 min the water was 945

replaced with fresh water (30 min f w) to account for wounding-related ion leakage from 946

the edges of the leaf discs Three independent experiments with six biological replicates 947

each were performed and data were analyzed using a t-test for each time point P-948

values were corrected using the Bonferroni method Data show means plusmn SE asterisks 949

indicate significant differences from wild-type Col-0 at q lt 001 950

951

Figure 4 Cell Walls of gae6 and gae1 gae6 Plants Are Reduced in GalA 952

(A) to (D) Levels of GalA (A) GlcA (B) cellulose (C) and neutral sugars (D) in alcohol953

insoluble residue (AIR) extracted from leaves of four week-old plants of the indicated 954

genotypes Bars represent means plusmn SE of four biological replicates combined using a 955

mixed linear model Letters indicate significantly different groups (p lt 005) 956

957

Figure 5 Cell Walls of gae1 gae6 Plants Are Reduced in Homogalacturonan 958

Page 43 of 54

Dot blot analyses with anti-LM19 and anti-LM20 antibodies Pectin was extracted from 959

AIR prepared from the indicated genotypes using 1 microL of extraction buffer per 10 microg of 960

AIR Pectin was serially diluted and one microL of undiluted 13 diluted and 19 diluted 961

samples were spotted on a nitrocellulose membrane Three biological replicates were 962

were spotted twice to obtain two technical replicates each All replicates produced 963

similar results 964

(A) Representative dot blot using anti-LM19 antibody965

(B) Relative signal intensities of the spots in the dot blot in (A)966

(C) Representative dot blot using anti-LM20 antibody967

(D) Relative signal intensities of the LM20 dot blot shown in (C)968

Relative signal intensities were measured using Image J software Measurements from 969

the three biological replicates were combined using a linear model after Box-Cox power 970

transformation as described in Methods Bars represent means plusmn SE Asterisks indicate 971

a difference from wild-type Col-0 at p lt 005 972

973

Figure 6 Growth of B cinerea on gae1 gae6 Plants and the Effect of B cinerea on 974

Expression of GAE1 and GAE6 975

(A) Growth of B cinerea isolates Leaves of Col-0 and gae1 gae6 plants were976

inoculated with 25 x 105 spores mL-1 of the B cinerea isolates indicated in the figure 977

Samples were collected two (2 dpi) and three (3 dpi) days later Bars represent means plusmn 978

SE of six biological replicates for each fungus combined using a mixed linear model 979

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 980

(B) and (C) Expression of GAE1 (B) and GAE6 (C) 48 h after inoculation with the B981

cinerea isolates indicated Expression levels were measured by qRT-PCR Bars 982

represent mean log2-ratios to ACTIN2 plusmn SE of three biological replicates combined 983

using a mixed linear model Letters indicate significantly different groups (q lt 005) 984

985

Figure 7 Levels of Soluble Pectin and Expression of PAD3 in Fractions Produced 986

Following Macerozyme Treatment of gae1 gae6 Cell Walls 987

(A) Schematic overview of the experimental procedure for (B) and (C)988

Page 44 of 54

(B) Uronic acid measurements Alcohol-insoluble residue (AIR) from leave tissue of four989

week-old wild-type Col-0 or gae1 gae6 plants was treated with water or 025 990

macerozyme and assayed for uronic acid levels Data from 11 biological replicates were 991

combined using a mixed linear model Bars represent means plusmn SE Letters indicate 992

significant differences (p lt 005) 993

(C) Expression of PAD3 in Col-0 plants treated with the fractions from (B) Extracts from994

(B) were heat inactivated pooled and diluted 5-fold with water Pooled extracts were995

inoculated into four week-old Col-0 plants PAD3 expression was measured 3 h after 996

inoculation and normalized to the level of ACTIN2 expression Data from four biological 997

replicates were combined using a mixed linear model Bars represent mean log2-ratios 998

to ACTIN2 plusmn SE Letters indicate significant differences (p lt 005) 999

1000

Figure 8 Macerozyme-Induced PAD3 Expression Is Absent in gae1 gae6 1001

(A) Expression of PAD3 after macerozyme treatment Four week-old plants were either1002

inoculated with 001 macerozyme or mock inoculated with water and PAD3 1003

expression was measured 3 h later by qRT-PCR Data from four biological replicates 1004

were combined using a mixed linear model Mean log2-ratios to ACTIN2 plusmn SE were 1005

plotted Letters indicate significantly different groups (p lt 005) 1006

(B) Expression of PAD3 after OG treatment PAD3 expression was measured as1007

described in (A) 3 h after inoculation with 100 microg mL-1 OGs or mock inoculation with 1008

water Data are from six biological replicates 1009

(C) PAD3 expression in untreated plants Three biological replicates were performed1010

and data analyzed as described in (A) 1011

(D) Camalexin accumulation after B cinerea treatment Camalexin accumulation was1012

measured in four week-old plants inoculated with the B cinerea strains shown in the 1013

figure Eight biological replicates each were combined using a mixed linear model 1014

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 1015

(E) PAD3 expression 28 h after treatment with the indicated B cinerea strains Data1016

from six biological replicates were collected and analyzed as described in (A) Data in 1017

all figure parts represent means plusmn SE Letters indicate significantly different groups (q lt 1018

005) 1019

Page 45 of 54

1020

Figure 9 Macerozyme-Induced Expression of the SA Marker Genes SID2 and PR1 1021

Is Abolished in gae1 gae6 Plants 1022

(A) to (H) Expression of PR1 (A) (C) (E) and (G) or SID2 (B) (D) (F) and (H) in four1023

week-old Col-0 and gae1 gae6 plants Data were combined using a mixed linear model 1024

Bars represent mean log2-ratios of expression vs ACTIN2 plusmn SE for all figure parts 1025

Letters indicate significantly different groups at p lt 005 for panels (A) to (D) (G) and 1026

(H) and qlt005 for panels (E) to (F) 1027

(A) and (B) Plants were treated with 001 macerozyme or mock treated with water1028

and samples were collected 3 h later Data from four biological replicates are shown 1029

(C) and (D) Plants were inoculated with 100 microg mL-1 OGs or mock treated with water1030

and samples collected 3 h later Data from six biological replicates are shown 1031

(E) and (F) Plants were inoculated with the indicated B cinerea isolates or mock1032

inoculated with B cinerea inoculation medium and samples collected 48 h later Data 1033

from eight biological replicates are shown 1034

(G) and (H) Expression in untreated plants Three biological replicates were performed1035

1036

Figure 10 The Expression of the JA Marker Gene JAZ10 is Highly Responsive in 1037

gae1 gae6 Plants 1038

(A) to (D) JAZ10 expression in four week-old plants Data were combined using a mixed1039

linear model Mean log2-ratios to ACTIN2 plusmn SE are shown Letters indicate significantly 1040

different groups at p lt 005 for panels (A) (B) and (D) and q lt 005 for panel (C) 1041

(A) Plants were inoculated with 001 macerozyme or mock inoculated using water and1042

samples were collected 3 h later Data from four biological replicates are shown 1043

(B) Plants were inoculated with 100 microg mL-1 OGs or mock inoculated using water and1044

samples collected 3 h later Data are from six biological replicates 1045

(C) JAZ10 expression 48 h after treatment with the indicated B cinerea strains Data1046

are from six biological replicates 1047

(D) JAZ10 expression in untreated plants Data are from three biological replicates1048

1049

Figure 11 Gallo 1-Induced Callose Deposition Is Reduced in gae1 gae6 Plants 1050

Page 46 of 54

(A) Callose deposition in four week-old Col-0 and gae1 gae6 plants Plants were1051

inoculated with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) or mock inoculated 1052

with B cinerea inoculation medium and samples were collected after 72 h Callose was 1053

stained with aniline blue and visualized using a Nikon Eclipse Ni-U microscope with a 1054

DAPI filter Three leaves were stained per treatment Scale bars are 100 microm Asterisks 1055

indicate fungal hyphae arrows indicate representative callose spots 1056

(B) Quantification of callose deposition Callose deposits were counted in 200 microm x 2001057

microm squares laid out across the images shown here Bars represent means plusmn SE from 1058

six squares each Letters indicate significantly different groups (p lt 005) according to a 1059

t-test analysis1060

1061

Figure 12 Less Soluble Pectin Accumulates in B cinerea-Infected gae1 gae6 1062

Leaves 1063

Four week-old Col-0 and gae1 gae6 plants were inoculated with 10 microL droplets of the 1064

indicated B cinerea isolate (25 x 105 spores mL-1) or mock inoculated with B cinerea 1065

inoculation medium Leaves were collected 48 h later AIR was extracted from leaf 1066

samples and water added (20 microL infection site) to extract soluble pectin fragments 1067

Samples were vortexed for 7 h centrifuged at 12000 g and the uronic acid 1068

concentration of the supernatant was measured Data from nine biological replicates 1069

were combined using a mixed linear model Means plusmn SE are shown Letters indicate 1070

significant differences (q lt 005) 1071

1072

Figure 13 Macerozyme-Induced PTI Is Lost in gae1 gae6 While OG-Induced PTI Is 1073

Unaltered 1074

(A) Macerozyme-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1075

inoculated with 001 macerozyme or mock inoculated with boiled macerozyme and 1076

were infected with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) 24 h later Samples 1077

were collected immediately (0 dpi) and after two (2 dpi) and three (3 dpi) days Data 1078

show mean plusmn SE of six to 14 biological replicates each combined using a linear model 1079

Letters indicate significantly different groups (q lt 005) 1080

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

Parsed CitationsAdam L and Somerville SC (1996) Genetic characterization of five powdery mildew disease resistance loci in Arabidopsisthaliana Plant J 9 341-356

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Albersheim P Jones TM and English PD (1969) Biochemistry of the cell wall in relation to infective processes Annu RevPhytopathol 7 171-194

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Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk RGadrinab C Heller C Jeske A Koesema E Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt MHazari N Hom E Karnes M Mulholland C Ndubaku R Schmidt I Guzman P Aguilar-Henonin L Schmid M Weigel DCarter DE Marchand T Risseeuw E Brogden D Zeko A Crosby WL Berry CC and Ecker JR (2003) Genome-wideinsertional mutagenesis of Arabidopsis thaliana Science 301 653-657

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Asai T Tena G Plotnikova J Willmann MR Chiu WL Gomez-Gomez L Boller T Ausubel FM and Sheen J (2002) MAPkinase signalling cascade in Arabidopsis innate immunity Nature 415 977-983

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Bartsch M Gobbato E Bednarek P Debey S Schultze JL Bautor J and Parker JE (2006) Salicylic acid-independentENHANCED DISEASE SUSCEPTIBILITY1 signaling in Arabidopsis immunity and cell death is regulated by the monooxygenaseFMO1 and the nudix hydrolase NUDT7 Plant Cell 18 1038-1051

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Bates D Machler M Bolker BM and Walker SC (2015) Fitting Linear Mixed-Effects Models Using lme4 J Stat Softw 67 1-48Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Benjamini Y and Hochberg Y (1995) Controlling the False Discovery Rate - a Practical and Powerful Approach to MultipleTesting J Roy Stat Soc B Met 57 289-300

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Bethke G Grundman RE Sreekanta S Truman W Katagiri F and Glazebrook J (2014) Arabidopsis PECTINMETHYLESTERASEs Contribute to Immunity against Pseudomonas syringae Plant Physiol 164 1093-1107

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Bouton S Leboeuf E Mouille G Leydecker MT Talbotec J Granier F Lahaye M Hofte H and Truong HN (2002)Quasimodo1 encodes a putative membrane-bound glycosyltransferase required for normal pectin synthesis and cell adhesion inArabidopsis Plant Cell 14 2577-2590

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Box GEP and Cox DR (1964) An analysis of transformations Journal of the Royal Statistical Society Series B 26 211-252Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Cao H Glazebrook J Clarke JD Volko S and Dong XN (1997) The Arabidopsis NPR1 gene that controls systemic acquiredresistance encodes a novel protein containing ankyrin repeats Cell 88 57-63

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Carpita N (2010) Update on Mechanisms of Plant Cell Wall Biosynthesis How plants make cellulose and other (1-gt4)-beta-D-glycans Plant Physiol

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Chisholm ST Coaker G Day B and Staskawicz BJ (2006) Host-microbe interactions Shaping the evolution of the plantimmune response Cell 124 803-814

Page 16 of 54

Because the amount of pectin substrate for pathogen polygalacturonases is 313

reduced we hypothesized that immunity phenotypes of gae1 gae6 plants may result 314

from reduced generation of OGs by these enzymes In turn the reduced OGs could 315

lead to reduced immune responses To test this we used macerozyme a commercial 316

pectinase as a proxy for pathogen pectinases We isolated cell walls as a subcellular 317

fraction known as alcohol insoluble residue (AIR Gille et al 2009) This was treated 318

with macerozyme and tested for release of soluble likely low molecular weight pectin 319

fragments (Figure 7) Treatment with 025 macerozyme released significantly more 320

uronic acid from wild-type Col-0 AIR relative to gae1 gae6 AIR (Figure 7B) To test 321

activity of these pectin-containing solutions we infiltrated them into wild-type Col-0 322

plants and measured PAD3 expression PAD3 is required for synthesis of the 323

phytoalexin camalexin (Zhou et al 1999) and its expression is known to be induced by 324

OG treatment (Ferrari et al 2007) Fractions from macerozyme treated Col-0 walls 325

induced significantly more PAD3 expression than fractions derived from gae1 gae6 326

walls suggesting that macerozyme treatment did indeed release more active soluble 327

uronic acid possibly OGs from wild-type Col-0 walls than from gae1 gae6 walls (Figure 328

7C) 329

Next we studied the effect of macerozyme treatment on expression of PAD3 in 330

intact plants (Figure 8) This treatment did not lead to any visible leaf damage 331

(Supplemental Figure 8) In wild-type Col-0 PAD3 expression was induced three h after 332

treatment with 001 macerozyme (Figure 8A) By contrast no macerozyme-induced 333

induction of PAD3 expression was detected in gae1 gae6 (Figure 8A) However OG 334

treatment induced the expression of PAD3 in both genotypes indicating that gae1 gae6 335

plants do respond to OGs (Figure 8B) Macerozyme treatment also induced expression 336

of the SA marker genes PATHOGENESIS RELATED 1 (PR1) and SID2 in Col-0 but 337

not in gae1 gae6 (Figure 9 panel A and B) PR1 was not significantly induced by 338

purified OGs (Figure 9C) at the time and concentration tested but SID2 was induced by 339

OG treatment in both genotypes (Figure 9D) However the induction was stronger in 340

wild-type Col-0 In addition the JA markers JASMONATE-ZIM-DOMAIN PROTEIN 5 341

(JAZ5) and JAZ10 were induced by macerozyme and OG treatment in both Col-0 and 342

gae1 gae6 (Figure 10A and Supplemental Figure 11) Interestingly expression of JAZ5 343

312 Pectinase-Induced Immune Signaling Is Altered in gae1 gae6 Plants

Page 17 of 54

Page 18 of 54

Page 19 of 54

Page 20 of 54

344

345

346

and JAZ10 was already strongly induced in gae1 gae6 after mock inoculation (Figure 10

and Supplemental Figure 11) We observed no hyperresponsive induction by mock

treatment of PLANT DEFENSIN 12 (PDF12) a necrotrophy and late JA signaling

347 marker gene (Supplemental Figure 12A-D) PDF12 was also not induced by

348 macerozyme or OG treatments (Supplemental Figure 12A-D) This suggests that early

349 JA signaling is induced by mock-treatment in gae1 gae6 Because it is known that JA

350 and SA signaling act antagonistically (Pieterse et al 2012) the strong up-regulation of

351 JA-induced immune signaling in gae1 gae6 might explain the lower OG-induced SID2

352 expression in this genotype

353 To test whether other immune responses are altered in gae1 gae6 plants we

354 analyzed Mitogen-activated protein kinase (MAPK) activity following macerozyme and

355 OG treatments OGs are known to activate the MAPKs MPK3 and MPK6 in Arabidopsis

Page 21 of 54

(Galletti et al 2011) Macerozyme treatment activated MPK3 and MPK6 in wild-type 356

Col-0 whereas MAPK activation in gae1 gae6 was slightly reduced in four out of five 357

experiments performed (Supplemental Figure 13) By contrast OG-induced MAPK 358

activity was similar or increased in gae1 gae6 in four out of five experiments 359

(Supplemental Figure 13) Additionally mock treatment induced MAPK activity in gae1 360

gae6 more strongly than it did in wild-type Col-0 in four out of five experiments 361

performed (Fig S13) Collectively these results illustrate that activation of several 362

immune signaling componentsmdashcamalexin synthesis SA and JA signaling and 363

possibly MAPK activationmdashby macerozyme is substantially altered in gae1 gae6 This 364

suggests that release of soluble pectin fragments possibly OGs by pectinolytic 365

enzymes might be reduced in gae1 gae6 plants while recognition of OGs is intact 366

367

Following B cinerea Infection Production of Soluble Pectin and Deposition of 368

Callose Are Reduced in gae1 gae6 Plants 369

Although pad3 plants were more susceptible to the B cinerea isolates Fresa 525 370

and Gallo 1 which also grew better on gae1 gae6 growth of the isolate Pepper was 371

similar on Col-0 and pad3 (Supplemental Figure 9A) However we did not detect any 372

significant changes in camalexin production after treatment with B cinerea isolate Gallo 373

1 and Fresa 525 in gae1 gae6 (Figure 8D) Neither did we detect changes in PAD3 374

expression after treatment with any B cinerea isolate in gae1 gae6 (Figure 8E) 375

Additionally SID2 and PR1 were similarly induced by all B cinerea isolates in both Col-376

0 and gae1 gae6 (Figure 9E and F) and sid2 plants which are deficient for pathogen-377

induced SA biosynthesis did not show any differences in relative B cinerea growth 378

(Supplemental Figure 9B) Expression of JAZ10 was induced more strongly by Gallo 1 379

treatment in gae1 gae6 than in wild-type Col-0 but not by Fresa 525 or Pepper (Fig 380

10C) while dde2 plants deficient for JA biosynthesis showed enhanced susceptibility to 381

all B cinerea isolates tested (Supplemental Figure 9B) Curiously exogenous 382

application of MeJA did not lead to altered relative fungal growth of B cinerea 383

(Supplemental Figure 9C) and cev1 isoxaben resistant 1 (ixr1) plants which have 384

constitutively high JA levels (Ellis and Turner 2001) did not show any changes in 385

susceptibility to the B cinerea isolates tested (Supplemental Figure 9D) Hence neither 386

Page 22 of 54

differences in B cinerea isolate susceptibility to camalexin nor altered SA or JA 387

signaling by themselves explained the enhanced susceptibility of gae1 gae6 to these B 388

cinerea isolates 389

Hydrogen peroxide accumulation in response to B cinerea isolates Fresa 525 390

Gallo 1 and Pepper was not obviously altered in gae1 gae6 plants (Supplemental Figure 391

14) However callose deposition upon treatment with B cinerea isolate Gallo 1 was 392

strongly reduced in gae1 gae6 (Figure 11) It was previously shown that OG treatment 393

induces callose deposition (Denoux et al 2008) suggesting that the reduction in 394

callose deposition might be due to reduced OG release in gae1 gae6 It is possible that 395

this reduction in callose deposition contributes to the reduced immunity of gae1 gae6 396

plants to B cinerea isolate Gallo 1 397

Because macerozyme could be used to release soluble likely low molecular 398

weight pectin from isolated cell walls we next investigated whether infection with B 399

cinerea was accompanied by accumulation of soluble pectin as well We prepared cell 400

walls (AIR fraction) from leaves infected with B cinerea incubated these cell walls with 401

water and measured uronic acid content of the extracts B cinerea treatment did in fact 402

lead to increased accumulation of soluble pectin in wild-type Col-0 plants when 403

compared to mock-treated samples (Figure 12) No such increased accumulation of 404

water-soluble pectin fractions was observed in gae1 gae6 (Figure 12) 405

406

Macerozyme-Induced Pattern-Triggered Immunity against B cinerea Is Abolished 407

in gae1 gae6 408

It was previously shown that OG treatment can induce patternndashtriggered 409

immunity (PTI) to B cinerea (Ferrari et al 2007) We tested macerozyme treatment to 410

determine whether it could also induce PTI to the B cinerea isolate Gallo 1 (Figure 13) 411

Indeed pretreatment with 001 macerozyme for 24 h led to reduced B cinerea growth 412

in wild-type Col-0 but not gae1 gae6 plants (Figure 13A) Interestingly mock and 413

macerozyme pretreated gae1 gae6 plants exhibited similar growth of B cinerea Gallo 1 414

as did macerozyme-treated Col-0 plants This result is likely due to activation of JA 415

signaling in gae1 gae6 during the pretreatment procedure (Figure 13C and D) OG-416

induced PTI was observed in both genotypes when using a high dose of OGs (Figure 417

Page 23 of 54

13B) whereas a lower dose of OGs only induced PTI in Col-0 (Supplemental Figure 418

15) This suggests that OG perception in gae1 gae6 at least above a certain threshold 419

is unaltered while release of immunity-activating pectin fragments possibly OGs is 420

421 reduced We conclude that immune responses induced by macerozyme treatment

422 which do not occur in gae1 gae6 plants are important for plant immunity to the B

423 cinerea isolate Gallo 1

Page 24 of 54

____________________________________________________________________________

Page 25 of 54

DISCUSSION 426

Here we showed that loss of GAE1 and GAE6 leads to a dramatic reduction in 427

pectin content in the cell wall (Figure 4) likely causing the brittleness of leaves that we 428

observed (Figure 3) Petioles and midribs of gae1 gae6 plants broke easily when bent 429

and inoculation of leaves with deionized water resulted in increased ion leakage 430

possibly due either to microscopic fractures of the cell wall induced by inoculation stress 431

or to a reduced capability of these walls to withstand increased turgor pressure The 432

increased brittleness was associated with altered cell wall composition In our analysis 433

the GalA content of gae1 gae6 leaf walls was reduced by more than 40 and the total 434

uronic acids by over 60 (Figure 4 and Supplemental Figure 5) This is a major change 435

in the overall cell wall composition considering that pectin makes up about 50 of the 436

plant cell walls found in Arabidopsis leaves (Zablackis et al 1995 Nobuta et al 2007 437

Harholt et al 2010) Interestingly quasimodo1 (qua1) plants which have a mutation in 438

a glycosyltransferase family 8 gene are reduced in GalA by 25 and show reduced 439

binding of HG specific antibodies (Bouton et al 2002) 440

Furthermore qua2 plants which have a mutation in a putative methyltransferase 441

have about 13 less total GalA and lack 50 of cell wall HG (Mouille et al 2007) 442

Likewise cgr2 cgr3 plants carrying mutations in another class of Golgi-localized 443

putative methyl-transferases have leaf cell walls that are reduced by about 40 in 444

uronic acid (Kim et al 2015) All three mutants are severely dwarfed (Bouton et al 445

2002 Mouille et al 2007 Kim et al 2015) Additionally qua1 and qua2 mutants show 446

a distorted overall shape likely due to reduced cell cohesion and are extremely fragile 447

(Bouton et al 2002 Mouille et al 2007) By contrast gae1 gae6 plants with a similar 448

strong reduction in GalA and total uronic acids have an overall normal shape and are 449

only slightly smaller than wild-type Col-0 (Supplemental Figure 4) In the wall analysis 450

performed here we did not detect changes in other wall polysaccharides or structures 451

that might compensate for the reduction of pectin in gae1 gae6 It is possible the severe 452

morphological alterations in qua2 and cgr2 cgr3 plants are due to reduction in pectin 453

methylesterification which is unaffected in gae1 gae6 It was speculated that quality 454

control mechanisms in the Golgi prevent secretion of inefficiently esterified pectin in 455

cgr2 cgr3 (Kim et al 2015) suggesting that the Golgi of these plants might over-456

Page 26 of 54

accumulate pectin and that this may contribute to the severe dwarf phenotype of these 457

plants The gae1 gae6 plants described here provide an opportunity to study the effects 458

of reduction of pectin in plants that do not show severe morphological phenotypes 459

We found that gae1 gae6 plants are also compromised in resistance to B 460

cinerea and Pma ES4326 indicating that loss of pectin reduces immunity to these 461

pathogens (Figure 2 and 6) We found that gae1 gae6 plants were slightly more 462

susceptible to Pma ES4326 but showed a strong increase in susceptibility to specific B 463

cinerea isolates This was not unexpected because B cinerea is known to interact with 464

pectin as part of its infection strategy It secretes various pectin-degrading enzymes 465

early during infection and requires some of these enzymes including PG1 PG2 and 466

PME1 for full virulence (ten Have et al 1998 Valette-Collet et al 2003 Kars et al 467

2005b Espino et al 2010) Additionally Arabidopsis plants expressing an antisense 468

construct of the polygalacturonase inhibitor PGIP1 are significantly more susceptible to 469

B cinerea (Ferrari et al 2006) On the other hand Pma ES4326 is not known to470

require cell wall degrading enzymes for full virulence even though it encodes two 471

pectate lyase genes in its genome (PMA4326_07094 and PMA4326_04621 472

wwwbacteriaensemblorg) B cinerea isolates are also known to be genetically quite 473

diverse in their polygalacturonase (PG) loci and genetic variability in PG2 is associated 474

with varying growth on pectin containing media (Rowe and Kliebenstein 2007) 475

Furthermore the contribution of PG1 and PG2 to B cinerea virulence seems to depend 476

on the specific B cinerea isolate and the host species (Kars et al 2005a Zhang and 477

Van Kan 2013) B cinerea isolates are also known to be differently sensitive to the anti-478

microbial secondary metabolite camalexin (Kliebenstein et al 2005) In line with these 479

observations the susceptibility of gae1 gae6 plants differed depending on the specific 480

B cinerea isolate Whereas Fresa 525 Gallo 1 UK Razz and KT grew better in gae1481

gae6 we detected no difference in Acacia Apple 517 DN Grape Pepper or Rasp 482

growth (Figure 6) 483

B cinerea was shown to metabolize pectin and to possibly utilize GalA as a484

carbon source during in planta growth (Zhang et al 2011 Zhang and Van Kan 2013) 485

Our data show that gae1 gae6 plants which are reduced in GalA content did allow 486

enhanced growth of specific B cinerea isolates but no reduction in growth of any B 487

Page 27 of 54

cinerea isolates tested was found This suggests that utilization of pectin as a carbon 488

source was not a contributing factor to alterations in B cinerea susceptibility It is 489

possible that changes in the cell wall composition affect the structural integrity of the 490

wall and affect hyphal penetration of specific B cinerea isolates However OGs (small 491

HG fragments) are likely released during the infection process (Ferrari et al 2013) 492

OGs function as DAMPs and initiate immune responses in Arabidopsis (Ferrari et al 493

2013) Treatment of Arabidopsis with OGs having a degree of polymerization (DP) of 494

10-15 sugar subunits was previously shown to enhance resistance to B cinerea (Ferrari495

et al 2007) We showed that pretreatment with OGs or macerozyme induced enhanced 496

immunity to B cinerea isolate Gallo 1 in wild-type Col-0 but only OGs and not 497

macerozyme induced immunity in gae1 gae6 (Figure 13) Our data suggest gae1 gae6 498

plants which have reduced pectin content may release less OGs and that this 499

reduction leads to the increased susceptibility to B cinerea 500

Consistently we found that macerozyme treatment released less soluble pectin 501

from gae1 gae6 cell walls than from wild-type walls Fractions containing soluble pectin 502

released from gae1 gae6 were also less active in inducing PAD3 gene expression than 503

the fractions released from wild-type walls (Figure 7) We also showed that less soluble 504

pectin accumulated in gae1 gae6 than in wild-type plants 48 h after treatment with B 505

cinerea (Figure 12) 506

We observed that loss of pectin leads to alterations in A thaliana immune 507

signaling Macerozyme treatment induced expression of PAD3 and the SA marker 508

genes PR1 and SID2 in wild-type Col-0 but not in gae1 gae6 whereas OG treatment 509

led to increased PAD3 and SID2 expression in both wild-type and gae1 gae6 (Figure 8 510

and 9) This suggests that although release of active soluble pectin in these plants 511

might be altered OG recognition is intact In fact cell walls isolated from qua1 another 512

mutant known to be reduced in HG released less OGs (DP1-6) than did wild-type cell 513

walls upon endopolygalacturonase treatment (Bouton et al 2002) This was also true 514

for gae1 gae6 cell walls as macerozyme treatment of isolated gae1 gae6 cell walls 515

released less soluble pectin than treatment of Col-0 cell walls Because the fraction 516

from gae1 gae6 plants was also less active in inducing PAD3 expression (Figure 7) it 517

seems possible that biologically active OGs were the active component inducing PAD3 518

Page 28 of 54

expression Interestingly expression of the JA marker genes JAZ5 and JAZ10 was 519

higher in gae1 gae6 than in wild-type mock-inoculated plants but was comparable to 520

wild-type in untreated plants suggesting that JA signaling in gae1 gae6 is 521

hyperresponsive (Figure 10 and Supplemental Figure 11) This induction of JA signaling 522

might be due to wounding stress incurred during the inoculation process JA and SA 523

signaling generally act antagonistically (Pieterse et al 2012) Increased JA signaling 524

activity might hence explain why mock- and macerozyme-treated gae1 gae6 plants 525

exhibited reduced expression of the SA marker PR1 and why OG treatment induced 526

expression of SID2 another SA marker to a lower extent in gae1 gae6 than in wild-type 527

plants (Figure 9) 528

No single immune signaling component was found to be the sole contributor to 529

the compromised immunity of gae1 gae6 to specific B cinerea isolates such as Gallo 1 530

and Fresa 525 These isolates grew better on pad3 while growth of Pepper another B 531

cinerea isolate that did not show altered virulence on gae1 gae6 was unaltered 532

(Supplemental Figure 9) This suggested that differences in camalexin sensitivity of 533

these B cinerea isolates might account for the differences in gae1 gae6 susceptibility 534

However camalexin accumulation and PAD3 expression was the same for wild-type 535

Col-0 and gae1 gae6 for the B cinerea isolates Fresa 525 and Gallo 1 (Figure 8) which 536

showed enhanced growth on gae1 gae6 plants It should be noted that B cinerea 537

virulence is often estimated by comparing lesion sizes Lesion size and measurements 538

of relative fungal growth by qPCR do not always show the same results For example 539

although Denby et al (2004) showed that Pepper causes larger lesions on pad3 than on 540

wild-type this study showed that relative fungal growth of Pepper on pad3 was 541

unaltered 542

We observed that callose deposition was strongly reduced in gae1 gae6 plants 543

72 h after treatment with B cinerea isolate Gallo 1 (Figure 11) possibly due to reduced 544

release of active soluble pectin Interestingly OG-induced PTI to an unspecified isolate 545

of B cinerea was intact in the pmr4 mutant which does not produce callose (Galletti et 546

al 2008) Due to the high SA levels in pmr4 plants (Nishimura et al 2003) and the fact 547

that different B cinerea isolates vary both in induction of and reaction to defense 548

responses (Kliebenstein et al 2005 Rowe et al 2010) we could not determine 549

Page 29 of 54

whether reduced callose deposition causes the increased susceptibility of gae1 gae6 to 550

B cinerea isolate Gallo 1551

We did not observe any changes in B cinerea growth either on cev1 ixr1 plants 552

which are known to have constitutively high JA and OPDA levels (Ellis et al 2002b) or 553

on plants exogenously treated with MeJA (Supplemental Figure 9) Because plants 554

were kept uncovered for 3 h after MeJA treatment it is unclear how much MeJA was 555

bioavailable in the plant However JAZ10 expression was induced 3 h after spraying 556

with MeJA (Supplemental Figure 10) Because pathogen growth generally depends on 557

the combination of a multitude of immune signaling events we conclude that multiple 558

changes to the immune signaling in gae1 gae6 together account for the altered B 559

cinerea growth phenotypes detected 560

We showed that expression of GAE1 and GAE6 is repressed by the bacterial 561

hemibiotroph Pma ES4326 This repression did not occur in an eds1 or pad4 562

background or in pbs3 at the 24-h time point (Figure 1 and Supplemental Figure 1) 563

During the Arabidopsis - Pma ES4326 interaction the expression of many defense 564

related genes is altered in a PAD4EDS1 dependent fashion (Wang et al 2008) Some 565

of these expression changes require SA CBP60g and SARD1 are thought to act 566

downstream of PAD4 and EDS1 but upstream of SA-dependent responses (Wang et 567

al 2011) PBS3 is also thought to act downstream of PAD4 and EDS1 and upstream of 568

SA but the set of genes whose expression it affects is only partially overlapping with 569

those affected by CBP60gSARD1 (Wang et al 2008 Wang et al 2011) Thus GAE1 570

and GAE6 repression is regulated by the part of the immune signaling network that 571

requires PAD4 EDS1 and PBS3 but is independent of CBP60gSARD1 and SA 572

Because EDS1 PAD4 and PBS3 are important components of the plant immune 573

signaling network (Wiermer et al 2005 Nobuta et al 2007) one might conclude that 574

repression of GAE1 and GAE6 may be part of the plant immune response to Pma 575

ES4326 B cinerea treatment also reduced GAE1 and GAE6 expression but this was 576

mostly independent of PAD4 (Figure 6) while both Pma ES4326 and B cinerea repress 577

GAE1 and GAE6 expression the host signaling mechanisms responsible likely differ 578

In summary our data show that Arabidopsis GAE1 and GAE6 are required for 579

pectin biosynthesis leaf flexibility and immunity to Pma ES4326 and to specific B 580

Page 30 of 54

cinerea isolates We used macerozyme a commercial pectinase to mimic the effect of 581

B cinerea pectinases Macerozyme treatment released less pectin from gae1 gae6 cell582

walls and induced less immune signaling in gae1 gae6 plants Less water soluble pectin 583

also accumulated in B cinerea infected gae1 gae6 plants and macerozyme-induced 584

immunity to B cinerea isolate Gallo 1 was reduced in these plants We conclude that 585

the reduced pectin content in these mutants may lead to reduced release of soluble 586

pectin including active OGs during pathogen attack and that this compromises 587

immunity 588

589

590

Page 31 of 54

METHODS 591

Plant Materials and Growth Conditions 592

Wild-type Columbia (Col-0) and all mutant plants (in Col-0 background) were 593

grown on sterilized BM2 germinating mix (Berger Inc Quebec Canada) in a controlled 594

environment chamber (Conviron Pembina ND USA) with a 12-h photoperiod under 595

100 microM m-2 s-1 fluorescent illumination at 22degC and 75 relative humidity Germplasm 596

used is described in the Accession Numbers section at the end of Methods 597

598

Data Analysis and Replicates 599

For biological replicates samples were obtained from separate plant tissues and 600

in many cases from plants grown in different flats In some experiments multiple 601

technical replicates were measured per biological replicate For statistical analysis all 602

technical replicates were averaged and the averages were considered one biological 603

replicate Independent experiments were performed at different times and often 604

consisted of multiple biological replicates per experiment 605

Data analysis was performed using mixed linear models in the R programming 606

environment (Bates et al 2015) unless otherwise indicated Generally genotypes 607

treatments and time points were used as fixed effects and replicate-specific effects like 608

different pots flats etc were considered random effects We reported p-values 609

obtained from the mixed linear model when less than 10 comparisons were performed 610

and q-values for more than 10 comparisons to correct for multiple comparison testing 611

Q-values were calculated using the Benjamini-Hochberg procedure (Benjamini and612

Hochberg 1995) unless specified otherwise 613

614

Pathogen Strains Growth Conditions and Pathogen Growth Assays 615

Growth of Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) and 616

bacterial growth assays were performed as previously described (Bethke et al 2014) 617

For qRT-PCR assays four week-old plants were inoculated with bacteria (OD600 = 618

0002) or mock (5 mM MgSO4) At least four leaves from two plants were collected at 619

the indicated time points per biological replicate 620

Page 32 of 54

Prior to each experiment Botrytis cinerea isolates were grown on 1x PDA 621

medium (Difco) for 10 d at room temperature All isolates used have been described 622

previously (Rowe and Kliebenstein 2007) The spores were washed from the surface of 623

the plate using an inoculation medium (1x Gamborgrsquos B-5 basal salt mixture (Sigma-624

Aldrich G5768) 2 (wv) glucose 10 mM phosphate buffer pH 64) and fungal hyphae 625

were removed from the suspension by filtering through four layers of cheesecloth The 626

concentration of spores was determined using a hemocytometer and adjusted to 25 x 627

105 spores mL-1 Four week-old Arabidopsis plants were inoculated by placing one 10-628

microL droplet of the B cinerea spore solution or inoculation medium (mock) on the adaxial 629

leaf surface of fully expanded leaves Inoculated plants were kept at 100 relative 630

humidity Infection sites were collected at various time points using a cork borer For 631

qRT-PCR assays at least four leaf discs from one plant were combined for each 632

biological sample For B cinerea growth assays at least 12 leaf discs from three to six 633

plants were pooled per biological sample Relative fungal growth was determined by 634

measuring the abundance of a fungal gene relative to a plant gene as described below 635

In brief DNA from infected tissue was extracted and equal amounts of total DNA were 636

used to perform qPCR reactions using the B cinerea cutinase A and the Arabidopsis 637

SK11 gene respectively Primers were described in (Gachon and Saindrenan 2004) 638

and are listed in Supplemental Table 1 Two technical replicates were combined for 639

each biological replicate B cinerea is a necrotrophic fungal pathogen and may destroy 640

plant DNA as disease progresses However decreases in the amount of plant DNA 641

over the course of our experiments were only observed at late time points with very 642

susceptible plants so the qPCR assay is generally a reasonable estimate of fungal 643

biomass (Supplemental Figure 16) 644

645

Treatment with Macerozyme or OGs 646

For PTI assays four week-old plants either were inoculated with 001 (wv) 647

macerozyme R-10 (Yakult) a Rhizopus polygalacturonase (EC 32115) that possesses 648

high pectinase and hemicellulase activity or were mock inoculated using heat-649

inactivated macerozyme Other similar plants were either inoculated with 250 or 500 microg 650

mL-1 OGs or mock inoculated with water One d later plants were infected with B 651

Page 33 of 54

cinerea and samples were collected immediately (0 dpi) and two or three d later To 652

investigate the role of MeJA on B cinerea growth plants were sprayed with 1mM MeJA 653

in water with 001 (vv) Tween 20 Covers were removed so that leaves could dry off 654

before plants were inoculated with B cinerea three h later Samples were harvested at 655

the indicated time points For expression analysis and MAPK activation assays plants 656

were either inoculated with 100 microg mL-1 OGs or mock inoculated with water OGs used 657

for experiments described in Figures 8 to 10 and Supplemental Figure 12 were a kind 658

gift from Simone Ferrari OGs used in experiments described in Figure 13 and 659

Supplemental Figures 11 13 and 15 were prepared as previously described (Kohorn et 660

al 2014) 661

662

Expression Analysis 663

Leaf tissue from inoculated plants was harvested at the indicated time points 664

flash frozen pulverized and RNA extracted using Trizol (Invitrogen) Quantitative RT-665

PCR was performed using the SuperScript III Platinum SYBR Green One-Step 666

quantitative RT-PCR kit (Invitrogen) and a Lightcycler 480 Real-Time PCR system 667

(Roche) as previously described (Truman and Glazebrook 2012) In brief equal 668

amounts of total RNA and a gene specific primer were used for each reaction The 669

crossing point (Cp) was calculated using the 2nd derivative max method provided with 670

the Lightcycler software for each amplification curve Each reaction was run with two 671

technical replicates and the Cp values for these replicates were averaged ACTIN2 was 672

used as a stably expressed reference gene At least three biological replicates each 673

were performed and analyzed using a mixed-linear model RT-PCR was performed to 674

verify T-DNA insertion lines using the One-Step RT-PCR kit (Qiagen) according to the 675

manufacturerrsquos instructions The primers used can be found in Supplemental Table 1 676

677

Electrolyte Leakage Assay 678

Fully expanded leaves of four week-old plants were inoculated with water (Milli-Q 679

grade) using a needleless syringe Leaf discs were collected using a cork borer and 680

three leaf discs from one leaf were placed with the adaxial surface down onto 2 mL 681

water in a 12-well cell culture plate Conductivity was measured using a Horiba B-173 682

Page 34 of 54

conductivity meter after 30 min and then the water was replaced Conductivity was 683

measured again 30 min later (1 h total) as indicated in Figure 3C Six biological 684

replicates each were performed in three independent experiments and means and SEs 685

were calculated Two-sided t-tests were performed for each time point 686

687

DAB and Aniline Blue Staining 688

Leaves from four week-old plants were either mock inoculated with B cinerea 689

inoculation medium or inoculated with 10 microL droplets of a B cinerea spore solution (25 690

x 105 spores mL-1) of the indicated isolates Samples were collected 72 h later For 33prime-691

diaminobenzidine (DAB) staining two to five inoculated leaves were vacuum infiltrated 692

with 33prime-Diaminobenzidine (1 mg mL-1 in water pH 38) solution and stained overnight 693

in the dark Leaves were destained using 100 ethanol at 65degC and then transferred to 694

50 (vv) glycerol and photographed 695

Callose was visualized by staining leaves with aniline blue as previously 696

described (Adam and Somerville 1996) Briefly leaves were vacuum infiltrated with 697

alcoholic lactophenol (one volume of phenolglycerollactic acidwater (1111) mixed 698

with two volumes of ethanol) and destained at 65degC Next leaves were moved through 699

an alcohol gradient (50 20 and 10 (vv) ethanol) into water The destained leaves 700

were stained for 30 min in 150 mM K2HPO4 (pH 95) with 001 (wv) aniline blue 701

Samples were mounted in water and examined using a Nikon Eclipse Ni-U microscope 702

at 10x magnification using a DAPI filter (excitation at 325-375 nm emission at 435-485 703

nm) For callose quantification six 200 microm x 200 microm squares were counted for each 704

sample type Callose deposits per mm2 were calculated and data analyzed using a t-705

test Note that there is an inherent uncertainty associated with such counts because 706

callose deposits may not all be in focus at the same time 707

708

MAPK Activity Assay 709

Fully expanded leaves of four week-old plants were inoculated with 001 710

macerozyme 100 microg mL-1 OGs 100 nM flg22 or water (mock) flg22 a peptide from 711

bacterial flagella that induces immune responses was used as a positive control for 712

Page 35 of 54

MAPK activation (Asai et al 2002) and was purchased from EZBiolab Inc (Westfield 713

IN USA) Samples were harvested 10 min later and immediately frozen in liquid 714

nitrogen Protein was extracted using an extraction buffer containing 25 mM Tris pH 78 715

75 mM NaCl 1 mM DTT 1 mM NaF 05mM Na3VO4 01 (vv) Tween 20 one 716

cOmplete Mini protease inhibitor cocktail tablet (Roche) and one PhosSTOP 717

phosphatase inhibitor cocktail tablet (Roche) per 10 mL buffer 15 microg of total protein 718

were electrophoresed on a 10 SDS-polyacrylamide gel and then blotted onto a PVDF 719

membrane (Bio-Rad 162-0177) Activated MAPKs were detected using p4442 MAPK 720

(Erk12) antibody (9102S Cell Signal 12500 in TBST (20 mM Tris pH 75 150 mM 721

NaCl 01 (vv) Tween 20)) Anti-Rabbit IgG-HRP (115000 in TBST A6154 Sigma) 722

and ECL Plus substrate (Pierce 32132) MPK3 and MPK6 were detected with an anti-723

MPK3 antibody (M8318 Sigma 12000 in TBST and 3 (wv) milk) or an anti-MPK6 724

antibody (A7104 Sigma 16000 in TBST and 3 (wv) milk) respectively The 725

secondary antibody and detection were the same as for the p4442 antibody Five 726

independent experiments were performed 727

728

Preparation of Alcohol Insoluble Residue (AIR) Pectin Extraction and Uronic Acid 729

Measurements 730

Fully expanded leaves from four week-old plants that were incubated for 48 h in 731

the dark to reduce starch were harvested flash frozen and pulverized AIR was 732

extracted by washing ground material twice in 70 (vv) ethanol three times in a 733

mixture of chloroform and methanol (11 vv) and once in acetone (Gille et al 2009) 734

and was then air dried Cell wall pectin was extracted from AIR with cell wall extraction 735

buffer (50 mM Trizma 50 mM CDTA pH 72) at 95degC for 15 min Samples were 736

homogenized using a paint shaker (Harbil 5G-HD) and glass beads (3 mm diameter) 737

For dot blot experiments 500 microL of cell wall extraction buffer were used per 10 mg of 738

AIR Debris was precipitated by centrifugation for 10 min at 10000 g 739

Total uronic acid content of pectin extracted as described above using 500 microL of 740

cell wall extraction buffer per 1 mg of AIR was measured as described previously 741

(Filisetti-Cozzi and Carpita 1991 van den Hoogen et al 1998) First 36 microL of pectin 742

extract were mixed with 4 microL of 4 M sulfamic acid Then 200 microL of sulfuric acid 743

Page 36 of 54

containing 120 mM sodium tetraborate was added and samples were incubated at 80degC 744

for 1 h Following cooling on ice the optical density of samples was measured at 490 745

nm Next 40 microL of m-hydroxydiphenyl reagent (100 microL of m-hydroxydiphenyl in dimethyl 746

sulfoxide at 100 mgmL mixed with 49 mL 80 (vv) sulfuric acid just before use) was 747

added and the samples were mixed The optical density at 490 nm was measured 748

again The optical density before m-hydroxydiphenyl reagent addition was subtracted 749

from the optical density measured after addition of the dye The concentration of uronic 750

acid was calculated using known amounts of GalA as a standard 751

752

Release of Pectin from Arabidopsis Cell Walls Using Macerozyme 753

AIR preparations from leaves of four week-old plants were treated with 025 754

(wv) macerozyme R-10 (Yakult) or water (mock) at a concentration of 100 microL per mg 755

AIR Samples were incubated for 3 h at room temperature on a vortex shaker and 756

centrifuged for 10 min at 12000 g Total uronic acid concentration of 5-fold diluted 757

supernatants was determined for two technical replicates each The concentration of 758

uronic acid was calculated using known amounts of GalA as a standard For 759

subsequent qRT-PCR experiments supernatants from multiple extractions were 760

combined boiled for 20 min to inactivate macerozyme diluted 10-fold with water and 761

inoculated into four week-old Col-0 plants Heat-inactivated 10-fold diluted macerozyme 762

solutions were used as mock treatment Samples were collected 3 h later and PAD3 763

expression was determined 764

765

Release of Pectin from Arabidopsis Leaves Infected with B cinerea 766

Fully expanded leaves of four week-old plants were either inoculated with up to 767

five 10 microL droplets of B cinerea spore solution (25 x 105 spores mL-1) or mock 768

inoculated with B cinerea inoculation medium For each biological replicate leaves with 769

a combined count of at least 20 infection sites were collected 48 h later AIR was 770

extracted as described above but plants were not dark-treated or freeze-dried and the 771

first ethanol step was performed using 85 (vv) ethanol To extract soluble pectin 772

fragments AIR samples were mixed with water at a concentration of 20 microL per infection 773

site incubated for 7 h at room temperature on a vortex shaker and centrifuged for 10 774

Page 37 of 54

min at 12000 g Total uronic acid concentration of supernatants was determined for two 775

technical replicates each The concentration of uronic acid was calculated using known 776

amounts of GalA as a standard 777

778

Measurement of Uronic Acids Neutral Monosaccharides and Cellulose Content in 779

Cell Walls 780

Neutral sugars and uronic acids from non-cellulosic polysaccharides in cell walls 781

(AIR) were released by trifluoroacetic acid (TFA) hydrolysis Approximately 1 mg of AIR 782

was hydrolyzed with 2 M TFA at 121degC for 90 min The TFA-soluble material was split 783

into two equal parts one for uronic acid and one for neutral sugar determination The 784

uronic acid content was determined by analyzing the hydrolyzate using a CarboPac 785

PA200 anion-exchange column with an ICS-3000 Dionex chromatography system 786

(Dionex Sunnyvale CA USA) The elution profile consisted of a linear gradient of 50 to 787

200 mM sodium acetate in 01 M NaOH in 10 min at 04 mL per min Alditol acetate 788

derivatives of neutral sugars were produced as described (York et al 1985) In brief 789

monosaccharides in the hydrolyzate were reduced with NaBH4 and peracetylated with 790

acetic anhydride and pyridine at 121degC for 20 min The generated alditol acetates were 791

analyzed by GC-MS Crystalline cellulose was measured according to Updegraff (1969) 792

After hydrolysis of noncellulosic polysaccharides from AIR with the Updegraff reagent 793

(acetic acid nitric acids water 812 (vv) ) the remaining pellet was hydrolyzed in 794

72 sulfuric acid The resulting glucose quantity was determined by the anthrone 795

method (Scott and Melvin 1953) 796

797

Dot Blot Experiments 798

Pectin solutions extracted using 50 microL of cell wall extraction buffer per mg AIR 799

were serially diluted and nitrocellulose membranes were spotted with 1 microL of the diluted 800

pectin solutions Membranes were dried overnight blocked with 5 milk (wv) in 1x 801

PBS (8 g L-1 NaCl 02 g L-1 KCl 144 g L-1 Na2HPO4 024 g L-1 KH2PO4 pH 74) and 802

probed with LM5 (Jones et al 1997) LM6 (Willats et al 1998) LM8 (Willats et al 803

2004) LM19 and LM20 (Verhertbruggen et al 2009a) or CCRC-M7 (Steffan et al 804

Page 38 of 54

1995) antibodies LM series antibodies were diluted 1250 and CCRC-M7 was diluted 805

1500 in 5 milk powder (Nestle Carnation) in 1 x PBS For LM series antibodies a 806

goat anti-Rat HRP conjugated antibody (Bethyl A110-105P 15000 diluted) and for 807

CCRC-M7 an anti-Mouse IgG HRP conjugate (Promega W402B 12500 diluted) was 808

used as secondary antibody in 5 milk powder in 1x PBS LM series antibodies were 809

obtained from plant probes (wwwplantprobesnet) and CCRC-M7 from CarboSource 810

(wwwcarbosourcenet) Membranes were washed after incubation with the primary and 811

secondary antibodies with 1x PBS Dot blots were developed using the ECL system 812

(GE Healthcare Amersham ECL prime) For quantification of signals dot blot results on 813

x-ray films were photographed using a CCD camera and intensities were measured814

using Image Jrsquos Integrated Density function Locally adjusted background was 815

subtracted As the dynamic range of the signal on an x-ray film was narrow non-816

linearity in the measured values was corrected for each genotype in each biological 817

replicate using the values from different dilutions in two technical replicates We noticed 818

that the residuals were not normally distributed and performed Box-Cox power 819

transformation (Box and Cox 1964) to obtain normally distributed values for the 820

subsequent statistical analysis of the data For LM19 Box-Cox selected power 821

transformation was done to the power of 038th and for LM20 to the power 075th After 822

correction of the transformed measured values with the effect of the biological 823

replicates the values were fit to a linear model with the Arabidopsis genotype as the 824

fixed effect This linear model was used both to generate the plots in Figures 5B and D 825

and to test significant differences 826

827

Camalexin Measurements 828

Four week-old plants were infected with B cinerea and samples (three leaf disks 829

from one plant each) were harvested at the indicated time points flash frozen and 830

pulverized Each sample was extracted with 300 microL of 90 methanol (vv) 50 microL of the 831

extract was run on an Agilent Lichrocart 250-4 RP18e 5-microm column using an Agilent 832

1100 series HPLC Camalexin was detected using a DiodeArray (DAD) at 330 nm and 833

with a fluorescence detector at emission 318 nmexcitation 385 nm (Agilent) Separation 834

was achieved using the following program with aqueous acetonitrile 5 min gradient 835

Page 39 of 54

from 63 to 69 acetonitrile 30 s gradient from 69 to 99 acetonitrile 2 min at 99 836

acetonitrile and a post-run equilibration of 35 min at 63 acetonitrile (Denby et al 837

2004 Kliebenstein et al 2005) Purified camalexin was used to produce a standard 838

curve to identify and quantitate camalexin 839

840

Accession Numbers 841

Microarray data used can be accessed at NCBI Gene Expression Omnnibus 842

(httpwwwncbinlmnihgovgeo) GSE18978 Sequence information for all Arabidopsis 843

genes described in this article can be found in TAIR (wwwarabidopsisorg) using the 844

following accession numbers PAD3 (CYP71B15 At3g26830) SID2 (ICS1 At1g74710) 845

GAE1 (At4g30440) GAE2 (At1g02000) GAE3 (At4g00110) GAE4 (At2g45310) GAE5 846

(At4g12250) GAE6 (At3g23820) PR1 (At2g14610) JAZ5 (At1g17380) JAZ10 847

(At5g13220) ACTIN2 (At3g18780) PDF12a (At5g44420) PDF12b (At2g26020) 848

AtSK11 (At5g26751) The sequence of the B cinerea cutinase Bc-CutA (Z69264) can 849

be found in the DNA Data Bank of Japan (httpgetentryddbjnigacjp) Germplasm 850

used included dde2-2 (At5g42650 (von Malek et al 2002)) pad3-1 (At3g26830 (Zhou 851

et al 1999)) pad4-1 (At3g52430 (Jirage et al 1999)) sid2-2 (At1g74710 (Wildermuth 852

et al 2001)) pbs3-2 (At5g13320 SALK_018225 (Nobuta et al 2007)) cbp60a-1 853

(At5g62570 SALK_124410 (Truman et al 2013)) cbp60g-1 sard1-2 (At5g26920 854

SALK_023199 At1g73805 SALK_052422 (Wang et al 2011) cbp60a-1 cbp60g-1 855

sard1-2 (Truman et al 2013) gae1-1 (At4g30440 SALK_085554) gae6-1 (At3g23280 856

SALK_104454) gae6-2 (At3g23280 SALK_017191) ixr1-1 cev1 (At5g05170 (Ellis 857

and Turner 2001 Scheible et al 2001)) eds1 plants were derived by introgression of 858

the Ler eds1-2 allele into Col-0 that contains two copies of EDS1 (At3g48090 859

At3g48080 (Bartsch et al 2006) mpk6-2 (At2g43790 SALK_073907 (Liu and Zhang 860

2004)) mpk3-DG (At3g45640 a fast neutron deletion mutant (Miles et al 2005)) and 861

npr1-1 (At1g64280 (Cao et al 1997)) T-DNA insertion lines were part of the SALK 862

collection (Alonso et al 2003) and were obtained from the Arabidopsis Biological 863

Resource Center 864

865

Supplemental Data 866

Page 40 of 54

Supplemental Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 867

Expression Does Not Require CBP60s or SID2 868

Supplemental Figure 2 Expression of GAE Family Members uponTreatment with Pma 869

ES4326 870

Supplemental Figure 3 Characterization of T-DNA Insertions in GAE1 and GAE6 871

Supplemental Figure 4 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves and 872

Are Slightly Smaller than Wild-Type Col-0 Plants 873

Supplemental Figure 5 Cell Walls of gae1 gae6 Plants Contain Less Uronic Acid than 874

Wild-Type Col-0 Cell Walls 875

Supplemental Figure 6 Antibodies Raised Against the RGI or XG Components of 876

Pectin Show No Difference in Binding to gae Mutant and Col-0 Cell Walls 877

Supplemental Figure 7 The Effect of B cinerea Treatment on the Expression of GAE 878

Family Genes 879

Supplemental Figure 8 Treatment with 001 Macerozyme Does Not Cause Any 880

Visible Damage to Col-0 Plants 881

Supplemental Figure 9 B cinerea Growth on Plants with Defects in Immune 882

Signaling 883

Supplemental Figure 10 MeJA-Induced Expression of the JA marker gene JAZ10 884

Supplemental Figure 11 The Expression of the JA Marker Gene JAZ5 Is Highly 885

Responsive in gae1 gae6 Plants 886

Supplemental Figure 12 The Expression of PDF12 Is Unaltered in gae1 gae6 Plants 887

Supplemental Figure 13 Macerozyme Treatment Activates MPK3 and MPK6 888

Supplemental Figure 14 B cinerea-Induced ROS Production in Wild-Type Col-0 and 889

gae1 gae6 Plants 890

Supplemental Figure 15 OG-Induced PTI Using 250 microg mL-1 OGs 891

Supplemental Figure 16 Abundance of Plant DNA in B cinerea Treated Plants 892

Supplemental Table 1 Primers used in this study 893

894

ACKNOWLEDGEMENTS 895

We thank William Truman for helpful discussions Simone Ferrari (Sapienza 896

Universita di Roma) for the generous gift of OGs Bruna Bucciarelli (USDA-ARS) for 897

Page 41 of 54

help with fluorescence microscopy the Arabidopsis Biological Resource Center for T-898

DNA insertion lines Paul Knoxrsquos lab (University of Leeds) for LM5 LM6 LM8 LM19 899

and LM20 antibodies and the Complex Carbohydrate Resource Center (University of 900

Georgia) for the CCRC-M7 antibody Most of this work was funded by the Division of 901

Chemical Sciences Geosciences and Biosciences Office of Basic Energy Sciences of 902

the US Department of Energy through Grant DE-FG02-05ER15670 to JG This work 903

was also supported by NSF awards IOS 1339125 and IOS 1021861 to DJK NSF 904

award IOS 1121425 to F K and the USDA National Institute of Food and Agriculture 905

Hatch project number CA-D-PLS-7033-H to DJK 906

907

AUTHOR CONTRIBUTIONS 908

GB and JG designed the research GB GX AT BL NES and NH 909

performed research DJK provided tools GB GX MP AD BL RAH and FK 910

analyzed data GB and JG wrote the paper with input from all authors 911

912

FIGURE LEGENDS 913

Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 Expression 914

Requires PAD4 EDS1 and PBS3 915

(A) Expression of GAE1 in leaves of wild-type Col-0 and mutant plants Expression was916

measured 24 or 32 h after either inoculation with Pma ES4326 (OD600 = 0002) or mock 917

inoculation with 5 mM MgSO4 and was then normalized to the level of ACTIN2 Data 918

from three biological replicates were combined using a mixed linear model Bars 919

represent mean log2-ratios to ACTIN2 plusmn SE Letters indicate significantly different 920

groups with q lt 005 where 005 represents the maximum false discovery rate at which 921

the test may be called significant 922

(B) Expression of GAE6 measured as described in (A)923

924

Figure 2 Susceptibility of gae6 and gae1 gae6 Plants to Pma ES4326 Is Increased 925

Relative to Wild-Type Col-0 926

Leaves of plants of the indicated genotypes were inoculated with Pma ES4326 (OD600 = 927

00002) as described in Methods Bacterial titers in leaves were determined either 928

Page 42 of 54

immediately (0 dpi) or after three days (3 dpi) Bars represent the mean plusmn SE of four 929

independent experiments each with 4 or 12 biological replicates at 0 and 3 dpi 930

respectively Data were combined using a mixed linear model Asterisks indicate 931

significant differences from Col-0 wild-type (q lt 005) Susceptible pad4 plants were 932

included as a positive control 933

934

Figure 3 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves 935

(A) Depiction of the assay for brittleness of gae mutant leaves Fully expanded leaves of936

four to five week-old plants were bent by 180ordm so that the adaxial surface of the leaf was 937

parallel with the soil Broken leaf petioles or midribs were considered brittle 938

(B) Quantification of broken midribs or petioles For each genotype 66 to 82 leaves 939

were analyzed for brittleness as described in (A) Bars represent the ratio of broken to 940

total number of leaves tested Results were analyzed using Fisherrsquos exact test Letters 941

indicate significantly different groups at p lt 005 942

(C) Conductivity measurements Leaves of four week-old plants were inoculated with943

deionized water and then leaf discs were cut out and placed on deionized water 944

Conductivity was measured at the indicated time points After 30 min the water was 945

replaced with fresh water (30 min f w) to account for wounding-related ion leakage from 946

the edges of the leaf discs Three independent experiments with six biological replicates 947

each were performed and data were analyzed using a t-test for each time point P-948

values were corrected using the Bonferroni method Data show means plusmn SE asterisks 949

indicate significant differences from wild-type Col-0 at q lt 001 950

951

Figure 4 Cell Walls of gae6 and gae1 gae6 Plants Are Reduced in GalA 952

(A) to (D) Levels of GalA (A) GlcA (B) cellulose (C) and neutral sugars (D) in alcohol953

insoluble residue (AIR) extracted from leaves of four week-old plants of the indicated 954

genotypes Bars represent means plusmn SE of four biological replicates combined using a 955

mixed linear model Letters indicate significantly different groups (p lt 005) 956

957

Figure 5 Cell Walls of gae1 gae6 Plants Are Reduced in Homogalacturonan 958

Page 43 of 54

Dot blot analyses with anti-LM19 and anti-LM20 antibodies Pectin was extracted from 959

AIR prepared from the indicated genotypes using 1 microL of extraction buffer per 10 microg of 960

AIR Pectin was serially diluted and one microL of undiluted 13 diluted and 19 diluted 961

samples were spotted on a nitrocellulose membrane Three biological replicates were 962

were spotted twice to obtain two technical replicates each All replicates produced 963

similar results 964

(A) Representative dot blot using anti-LM19 antibody965

(B) Relative signal intensities of the spots in the dot blot in (A)966

(C) Representative dot blot using anti-LM20 antibody967

(D) Relative signal intensities of the LM20 dot blot shown in (C)968

Relative signal intensities were measured using Image J software Measurements from 969

the three biological replicates were combined using a linear model after Box-Cox power 970

transformation as described in Methods Bars represent means plusmn SE Asterisks indicate 971

a difference from wild-type Col-0 at p lt 005 972

973

Figure 6 Growth of B cinerea on gae1 gae6 Plants and the Effect of B cinerea on 974

Expression of GAE1 and GAE6 975

(A) Growth of B cinerea isolates Leaves of Col-0 and gae1 gae6 plants were976

inoculated with 25 x 105 spores mL-1 of the B cinerea isolates indicated in the figure 977

Samples were collected two (2 dpi) and three (3 dpi) days later Bars represent means plusmn 978

SE of six biological replicates for each fungus combined using a mixed linear model 979

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 980

(B) and (C) Expression of GAE1 (B) and GAE6 (C) 48 h after inoculation with the B981

cinerea isolates indicated Expression levels were measured by qRT-PCR Bars 982

represent mean log2-ratios to ACTIN2 plusmn SE of three biological replicates combined 983

using a mixed linear model Letters indicate significantly different groups (q lt 005) 984

985

Figure 7 Levels of Soluble Pectin and Expression of PAD3 in Fractions Produced 986

Following Macerozyme Treatment of gae1 gae6 Cell Walls 987

(A) Schematic overview of the experimental procedure for (B) and (C)988

Page 44 of 54

(B) Uronic acid measurements Alcohol-insoluble residue (AIR) from leave tissue of four989

week-old wild-type Col-0 or gae1 gae6 plants was treated with water or 025 990

macerozyme and assayed for uronic acid levels Data from 11 biological replicates were 991

combined using a mixed linear model Bars represent means plusmn SE Letters indicate 992

significant differences (p lt 005) 993

(C) Expression of PAD3 in Col-0 plants treated with the fractions from (B) Extracts from994

(B) were heat inactivated pooled and diluted 5-fold with water Pooled extracts were995

inoculated into four week-old Col-0 plants PAD3 expression was measured 3 h after 996

inoculation and normalized to the level of ACTIN2 expression Data from four biological 997

replicates were combined using a mixed linear model Bars represent mean log2-ratios 998

to ACTIN2 plusmn SE Letters indicate significant differences (p lt 005) 999

1000

Figure 8 Macerozyme-Induced PAD3 Expression Is Absent in gae1 gae6 1001

(A) Expression of PAD3 after macerozyme treatment Four week-old plants were either1002

inoculated with 001 macerozyme or mock inoculated with water and PAD3 1003

expression was measured 3 h later by qRT-PCR Data from four biological replicates 1004

were combined using a mixed linear model Mean log2-ratios to ACTIN2 plusmn SE were 1005

plotted Letters indicate significantly different groups (p lt 005) 1006

(B) Expression of PAD3 after OG treatment PAD3 expression was measured as1007

described in (A) 3 h after inoculation with 100 microg mL-1 OGs or mock inoculation with 1008

water Data are from six biological replicates 1009

(C) PAD3 expression in untreated plants Three biological replicates were performed1010

and data analyzed as described in (A) 1011

(D) Camalexin accumulation after B cinerea treatment Camalexin accumulation was1012

measured in four week-old plants inoculated with the B cinerea strains shown in the 1013

figure Eight biological replicates each were combined using a mixed linear model 1014

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 1015

(E) PAD3 expression 28 h after treatment with the indicated B cinerea strains Data1016

from six biological replicates were collected and analyzed as described in (A) Data in 1017

all figure parts represent means plusmn SE Letters indicate significantly different groups (q lt 1018

005) 1019

Page 45 of 54

1020

Figure 9 Macerozyme-Induced Expression of the SA Marker Genes SID2 and PR1 1021

Is Abolished in gae1 gae6 Plants 1022

(A) to (H) Expression of PR1 (A) (C) (E) and (G) or SID2 (B) (D) (F) and (H) in four1023

week-old Col-0 and gae1 gae6 plants Data were combined using a mixed linear model 1024

Bars represent mean log2-ratios of expression vs ACTIN2 plusmn SE for all figure parts 1025

Letters indicate significantly different groups at p lt 005 for panels (A) to (D) (G) and 1026

(H) and qlt005 for panels (E) to (F) 1027

(A) and (B) Plants were treated with 001 macerozyme or mock treated with water1028

and samples were collected 3 h later Data from four biological replicates are shown 1029

(C) and (D) Plants were inoculated with 100 microg mL-1 OGs or mock treated with water1030

and samples collected 3 h later Data from six biological replicates are shown 1031

(E) and (F) Plants were inoculated with the indicated B cinerea isolates or mock1032

inoculated with B cinerea inoculation medium and samples collected 48 h later Data 1033

from eight biological replicates are shown 1034

(G) and (H) Expression in untreated plants Three biological replicates were performed1035

1036

Figure 10 The Expression of the JA Marker Gene JAZ10 is Highly Responsive in 1037

gae1 gae6 Plants 1038

(A) to (D) JAZ10 expression in four week-old plants Data were combined using a mixed1039

linear model Mean log2-ratios to ACTIN2 plusmn SE are shown Letters indicate significantly 1040

different groups at p lt 005 for panels (A) (B) and (D) and q lt 005 for panel (C) 1041

(A) Plants were inoculated with 001 macerozyme or mock inoculated using water and1042

samples were collected 3 h later Data from four biological replicates are shown 1043

(B) Plants were inoculated with 100 microg mL-1 OGs or mock inoculated using water and1044

samples collected 3 h later Data are from six biological replicates 1045

(C) JAZ10 expression 48 h after treatment with the indicated B cinerea strains Data1046

are from six biological replicates 1047

(D) JAZ10 expression in untreated plants Data are from three biological replicates1048

1049

Figure 11 Gallo 1-Induced Callose Deposition Is Reduced in gae1 gae6 Plants 1050

Page 46 of 54

(A) Callose deposition in four week-old Col-0 and gae1 gae6 plants Plants were1051

inoculated with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) or mock inoculated 1052

with B cinerea inoculation medium and samples were collected after 72 h Callose was 1053

stained with aniline blue and visualized using a Nikon Eclipse Ni-U microscope with a 1054

DAPI filter Three leaves were stained per treatment Scale bars are 100 microm Asterisks 1055

indicate fungal hyphae arrows indicate representative callose spots 1056

(B) Quantification of callose deposition Callose deposits were counted in 200 microm x 2001057

microm squares laid out across the images shown here Bars represent means plusmn SE from 1058

six squares each Letters indicate significantly different groups (p lt 005) according to a 1059

t-test analysis1060

1061

Figure 12 Less Soluble Pectin Accumulates in B cinerea-Infected gae1 gae6 1062

Leaves 1063

Four week-old Col-0 and gae1 gae6 plants were inoculated with 10 microL droplets of the 1064

indicated B cinerea isolate (25 x 105 spores mL-1) or mock inoculated with B cinerea 1065

inoculation medium Leaves were collected 48 h later AIR was extracted from leaf 1066

samples and water added (20 microL infection site) to extract soluble pectin fragments 1067

Samples were vortexed for 7 h centrifuged at 12000 g and the uronic acid 1068

concentration of the supernatant was measured Data from nine biological replicates 1069

were combined using a mixed linear model Means plusmn SE are shown Letters indicate 1070

significant differences (q lt 005) 1071

1072

Figure 13 Macerozyme-Induced PTI Is Lost in gae1 gae6 While OG-Induced PTI Is 1073

Unaltered 1074

(A) Macerozyme-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1075

inoculated with 001 macerozyme or mock inoculated with boiled macerozyme and 1076

were infected with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) 24 h later Samples 1077

were collected immediately (0 dpi) and after two (2 dpi) and three (3 dpi) days Data 1078

show mean plusmn SE of six to 14 biological replicates each combined using a linear model 1079

Letters indicate significantly different groups (q lt 005) 1080

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

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Cao H Glazebrook J Clarke JD Volko S and Dong XN (1997) The Arabidopsis NPR1 gene that controls systemic acquiredresistance encodes a novel protein containing ankyrin repeats Cell 88 57-63

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Chisholm ST Coaker G Day B and Staskawicz BJ (2006) Host-microbe interactions Shaping the evolution of the plantimmune response Cell 124 803-814

Page 17 of 54

Page 18 of 54

Page 19 of 54

Page 20 of 54

344

345

346

and JAZ10 was already strongly induced in gae1 gae6 after mock inoculation (Figure 10

and Supplemental Figure 11) We observed no hyperresponsive induction by mock

treatment of PLANT DEFENSIN 12 (PDF12) a necrotrophy and late JA signaling

347 marker gene (Supplemental Figure 12A-D) PDF12 was also not induced by

348 macerozyme or OG treatments (Supplemental Figure 12A-D) This suggests that early

349 JA signaling is induced by mock-treatment in gae1 gae6 Because it is known that JA

350 and SA signaling act antagonistically (Pieterse et al 2012) the strong up-regulation of

351 JA-induced immune signaling in gae1 gae6 might explain the lower OG-induced SID2

352 expression in this genotype

353 To test whether other immune responses are altered in gae1 gae6 plants we

354 analyzed Mitogen-activated protein kinase (MAPK) activity following macerozyme and

355 OG treatments OGs are known to activate the MAPKs MPK3 and MPK6 in Arabidopsis

Page 21 of 54

(Galletti et al 2011) Macerozyme treatment activated MPK3 and MPK6 in wild-type 356

Col-0 whereas MAPK activation in gae1 gae6 was slightly reduced in four out of five 357

experiments performed (Supplemental Figure 13) By contrast OG-induced MAPK 358

activity was similar or increased in gae1 gae6 in four out of five experiments 359

(Supplemental Figure 13) Additionally mock treatment induced MAPK activity in gae1 360

gae6 more strongly than it did in wild-type Col-0 in four out of five experiments 361

performed (Fig S13) Collectively these results illustrate that activation of several 362

immune signaling componentsmdashcamalexin synthesis SA and JA signaling and 363

possibly MAPK activationmdashby macerozyme is substantially altered in gae1 gae6 This 364

suggests that release of soluble pectin fragments possibly OGs by pectinolytic 365

enzymes might be reduced in gae1 gae6 plants while recognition of OGs is intact 366

367

Following B cinerea Infection Production of Soluble Pectin and Deposition of 368

Callose Are Reduced in gae1 gae6 Plants 369

Although pad3 plants were more susceptible to the B cinerea isolates Fresa 525 370

and Gallo 1 which also grew better on gae1 gae6 growth of the isolate Pepper was 371

similar on Col-0 and pad3 (Supplemental Figure 9A) However we did not detect any 372

significant changes in camalexin production after treatment with B cinerea isolate Gallo 373

1 and Fresa 525 in gae1 gae6 (Figure 8D) Neither did we detect changes in PAD3 374

expression after treatment with any B cinerea isolate in gae1 gae6 (Figure 8E) 375

Additionally SID2 and PR1 were similarly induced by all B cinerea isolates in both Col-376

0 and gae1 gae6 (Figure 9E and F) and sid2 plants which are deficient for pathogen-377

induced SA biosynthesis did not show any differences in relative B cinerea growth 378

(Supplemental Figure 9B) Expression of JAZ10 was induced more strongly by Gallo 1 379

treatment in gae1 gae6 than in wild-type Col-0 but not by Fresa 525 or Pepper (Fig 380

10C) while dde2 plants deficient for JA biosynthesis showed enhanced susceptibility to 381

all B cinerea isolates tested (Supplemental Figure 9B) Curiously exogenous 382

application of MeJA did not lead to altered relative fungal growth of B cinerea 383

(Supplemental Figure 9C) and cev1 isoxaben resistant 1 (ixr1) plants which have 384

constitutively high JA levels (Ellis and Turner 2001) did not show any changes in 385

susceptibility to the B cinerea isolates tested (Supplemental Figure 9D) Hence neither 386

Page 22 of 54

differences in B cinerea isolate susceptibility to camalexin nor altered SA or JA 387

signaling by themselves explained the enhanced susceptibility of gae1 gae6 to these B 388

cinerea isolates 389

Hydrogen peroxide accumulation in response to B cinerea isolates Fresa 525 390

Gallo 1 and Pepper was not obviously altered in gae1 gae6 plants (Supplemental Figure 391

14) However callose deposition upon treatment with B cinerea isolate Gallo 1 was 392

strongly reduced in gae1 gae6 (Figure 11) It was previously shown that OG treatment 393

induces callose deposition (Denoux et al 2008) suggesting that the reduction in 394

callose deposition might be due to reduced OG release in gae1 gae6 It is possible that 395

this reduction in callose deposition contributes to the reduced immunity of gae1 gae6 396

plants to B cinerea isolate Gallo 1 397

Because macerozyme could be used to release soluble likely low molecular 398

weight pectin from isolated cell walls we next investigated whether infection with B 399

cinerea was accompanied by accumulation of soluble pectin as well We prepared cell 400

walls (AIR fraction) from leaves infected with B cinerea incubated these cell walls with 401

water and measured uronic acid content of the extracts B cinerea treatment did in fact 402

lead to increased accumulation of soluble pectin in wild-type Col-0 plants when 403

compared to mock-treated samples (Figure 12) No such increased accumulation of 404

water-soluble pectin fractions was observed in gae1 gae6 (Figure 12) 405

406

Macerozyme-Induced Pattern-Triggered Immunity against B cinerea Is Abolished 407

in gae1 gae6 408

It was previously shown that OG treatment can induce patternndashtriggered 409

immunity (PTI) to B cinerea (Ferrari et al 2007) We tested macerozyme treatment to 410

determine whether it could also induce PTI to the B cinerea isolate Gallo 1 (Figure 13) 411

Indeed pretreatment with 001 macerozyme for 24 h led to reduced B cinerea growth 412

in wild-type Col-0 but not gae1 gae6 plants (Figure 13A) Interestingly mock and 413

macerozyme pretreated gae1 gae6 plants exhibited similar growth of B cinerea Gallo 1 414

as did macerozyme-treated Col-0 plants This result is likely due to activation of JA 415

signaling in gae1 gae6 during the pretreatment procedure (Figure 13C and D) OG-416

induced PTI was observed in both genotypes when using a high dose of OGs (Figure 417

Page 23 of 54

13B) whereas a lower dose of OGs only induced PTI in Col-0 (Supplemental Figure 418

15) This suggests that OG perception in gae1 gae6 at least above a certain threshold 419

is unaltered while release of immunity-activating pectin fragments possibly OGs is 420

421 reduced We conclude that immune responses induced by macerozyme treatment

422 which do not occur in gae1 gae6 plants are important for plant immunity to the B

423 cinerea isolate Gallo 1

Page 24 of 54

____________________________________________________________________________

Page 25 of 54

DISCUSSION 426

Here we showed that loss of GAE1 and GAE6 leads to a dramatic reduction in 427

pectin content in the cell wall (Figure 4) likely causing the brittleness of leaves that we 428

observed (Figure 3) Petioles and midribs of gae1 gae6 plants broke easily when bent 429

and inoculation of leaves with deionized water resulted in increased ion leakage 430

possibly due either to microscopic fractures of the cell wall induced by inoculation stress 431

or to a reduced capability of these walls to withstand increased turgor pressure The 432

increased brittleness was associated with altered cell wall composition In our analysis 433

the GalA content of gae1 gae6 leaf walls was reduced by more than 40 and the total 434

uronic acids by over 60 (Figure 4 and Supplemental Figure 5) This is a major change 435

in the overall cell wall composition considering that pectin makes up about 50 of the 436

plant cell walls found in Arabidopsis leaves (Zablackis et al 1995 Nobuta et al 2007 437

Harholt et al 2010) Interestingly quasimodo1 (qua1) plants which have a mutation in 438

a glycosyltransferase family 8 gene are reduced in GalA by 25 and show reduced 439

binding of HG specific antibodies (Bouton et al 2002) 440

Furthermore qua2 plants which have a mutation in a putative methyltransferase 441

have about 13 less total GalA and lack 50 of cell wall HG (Mouille et al 2007) 442

Likewise cgr2 cgr3 plants carrying mutations in another class of Golgi-localized 443

putative methyl-transferases have leaf cell walls that are reduced by about 40 in 444

uronic acid (Kim et al 2015) All three mutants are severely dwarfed (Bouton et al 445

2002 Mouille et al 2007 Kim et al 2015) Additionally qua1 and qua2 mutants show 446

a distorted overall shape likely due to reduced cell cohesion and are extremely fragile 447

(Bouton et al 2002 Mouille et al 2007) By contrast gae1 gae6 plants with a similar 448

strong reduction in GalA and total uronic acids have an overall normal shape and are 449

only slightly smaller than wild-type Col-0 (Supplemental Figure 4) In the wall analysis 450

performed here we did not detect changes in other wall polysaccharides or structures 451

that might compensate for the reduction of pectin in gae1 gae6 It is possible the severe 452

morphological alterations in qua2 and cgr2 cgr3 plants are due to reduction in pectin 453

methylesterification which is unaffected in gae1 gae6 It was speculated that quality 454

control mechanisms in the Golgi prevent secretion of inefficiently esterified pectin in 455

cgr2 cgr3 (Kim et al 2015) suggesting that the Golgi of these plants might over-456

Page 26 of 54

accumulate pectin and that this may contribute to the severe dwarf phenotype of these 457

plants The gae1 gae6 plants described here provide an opportunity to study the effects 458

of reduction of pectin in plants that do not show severe morphological phenotypes 459

We found that gae1 gae6 plants are also compromised in resistance to B 460

cinerea and Pma ES4326 indicating that loss of pectin reduces immunity to these 461

pathogens (Figure 2 and 6) We found that gae1 gae6 plants were slightly more 462

susceptible to Pma ES4326 but showed a strong increase in susceptibility to specific B 463

cinerea isolates This was not unexpected because B cinerea is known to interact with 464

pectin as part of its infection strategy It secretes various pectin-degrading enzymes 465

early during infection and requires some of these enzymes including PG1 PG2 and 466

PME1 for full virulence (ten Have et al 1998 Valette-Collet et al 2003 Kars et al 467

2005b Espino et al 2010) Additionally Arabidopsis plants expressing an antisense 468

construct of the polygalacturonase inhibitor PGIP1 are significantly more susceptible to 469

B cinerea (Ferrari et al 2006) On the other hand Pma ES4326 is not known to470

require cell wall degrading enzymes for full virulence even though it encodes two 471

pectate lyase genes in its genome (PMA4326_07094 and PMA4326_04621 472

wwwbacteriaensemblorg) B cinerea isolates are also known to be genetically quite 473

diverse in their polygalacturonase (PG) loci and genetic variability in PG2 is associated 474

with varying growth on pectin containing media (Rowe and Kliebenstein 2007) 475

Furthermore the contribution of PG1 and PG2 to B cinerea virulence seems to depend 476

on the specific B cinerea isolate and the host species (Kars et al 2005a Zhang and 477

Van Kan 2013) B cinerea isolates are also known to be differently sensitive to the anti-478

microbial secondary metabolite camalexin (Kliebenstein et al 2005) In line with these 479

observations the susceptibility of gae1 gae6 plants differed depending on the specific 480

B cinerea isolate Whereas Fresa 525 Gallo 1 UK Razz and KT grew better in gae1481

gae6 we detected no difference in Acacia Apple 517 DN Grape Pepper or Rasp 482

growth (Figure 6) 483

B cinerea was shown to metabolize pectin and to possibly utilize GalA as a484

carbon source during in planta growth (Zhang et al 2011 Zhang and Van Kan 2013) 485

Our data show that gae1 gae6 plants which are reduced in GalA content did allow 486

enhanced growth of specific B cinerea isolates but no reduction in growth of any B 487

Page 27 of 54

cinerea isolates tested was found This suggests that utilization of pectin as a carbon 488

source was not a contributing factor to alterations in B cinerea susceptibility It is 489

possible that changes in the cell wall composition affect the structural integrity of the 490

wall and affect hyphal penetration of specific B cinerea isolates However OGs (small 491

HG fragments) are likely released during the infection process (Ferrari et al 2013) 492

OGs function as DAMPs and initiate immune responses in Arabidopsis (Ferrari et al 493

2013) Treatment of Arabidopsis with OGs having a degree of polymerization (DP) of 494

10-15 sugar subunits was previously shown to enhance resistance to B cinerea (Ferrari495

et al 2007) We showed that pretreatment with OGs or macerozyme induced enhanced 496

immunity to B cinerea isolate Gallo 1 in wild-type Col-0 but only OGs and not 497

macerozyme induced immunity in gae1 gae6 (Figure 13) Our data suggest gae1 gae6 498

plants which have reduced pectin content may release less OGs and that this 499

reduction leads to the increased susceptibility to B cinerea 500

Consistently we found that macerozyme treatment released less soluble pectin 501

from gae1 gae6 cell walls than from wild-type walls Fractions containing soluble pectin 502

released from gae1 gae6 were also less active in inducing PAD3 gene expression than 503

the fractions released from wild-type walls (Figure 7) We also showed that less soluble 504

pectin accumulated in gae1 gae6 than in wild-type plants 48 h after treatment with B 505

cinerea (Figure 12) 506

We observed that loss of pectin leads to alterations in A thaliana immune 507

signaling Macerozyme treatment induced expression of PAD3 and the SA marker 508

genes PR1 and SID2 in wild-type Col-0 but not in gae1 gae6 whereas OG treatment 509

led to increased PAD3 and SID2 expression in both wild-type and gae1 gae6 (Figure 8 510

and 9) This suggests that although release of active soluble pectin in these plants 511

might be altered OG recognition is intact In fact cell walls isolated from qua1 another 512

mutant known to be reduced in HG released less OGs (DP1-6) than did wild-type cell 513

walls upon endopolygalacturonase treatment (Bouton et al 2002) This was also true 514

for gae1 gae6 cell walls as macerozyme treatment of isolated gae1 gae6 cell walls 515

released less soluble pectin than treatment of Col-0 cell walls Because the fraction 516

from gae1 gae6 plants was also less active in inducing PAD3 expression (Figure 7) it 517

seems possible that biologically active OGs were the active component inducing PAD3 518

Page 28 of 54

expression Interestingly expression of the JA marker genes JAZ5 and JAZ10 was 519

higher in gae1 gae6 than in wild-type mock-inoculated plants but was comparable to 520

wild-type in untreated plants suggesting that JA signaling in gae1 gae6 is 521

hyperresponsive (Figure 10 and Supplemental Figure 11) This induction of JA signaling 522

might be due to wounding stress incurred during the inoculation process JA and SA 523

signaling generally act antagonistically (Pieterse et al 2012) Increased JA signaling 524

activity might hence explain why mock- and macerozyme-treated gae1 gae6 plants 525

exhibited reduced expression of the SA marker PR1 and why OG treatment induced 526

expression of SID2 another SA marker to a lower extent in gae1 gae6 than in wild-type 527

plants (Figure 9) 528

No single immune signaling component was found to be the sole contributor to 529

the compromised immunity of gae1 gae6 to specific B cinerea isolates such as Gallo 1 530

and Fresa 525 These isolates grew better on pad3 while growth of Pepper another B 531

cinerea isolate that did not show altered virulence on gae1 gae6 was unaltered 532

(Supplemental Figure 9) This suggested that differences in camalexin sensitivity of 533

these B cinerea isolates might account for the differences in gae1 gae6 susceptibility 534

However camalexin accumulation and PAD3 expression was the same for wild-type 535

Col-0 and gae1 gae6 for the B cinerea isolates Fresa 525 and Gallo 1 (Figure 8) which 536

showed enhanced growth on gae1 gae6 plants It should be noted that B cinerea 537

virulence is often estimated by comparing lesion sizes Lesion size and measurements 538

of relative fungal growth by qPCR do not always show the same results For example 539

although Denby et al (2004) showed that Pepper causes larger lesions on pad3 than on 540

wild-type this study showed that relative fungal growth of Pepper on pad3 was 541

unaltered 542

We observed that callose deposition was strongly reduced in gae1 gae6 plants 543

72 h after treatment with B cinerea isolate Gallo 1 (Figure 11) possibly due to reduced 544

release of active soluble pectin Interestingly OG-induced PTI to an unspecified isolate 545

of B cinerea was intact in the pmr4 mutant which does not produce callose (Galletti et 546

al 2008) Due to the high SA levels in pmr4 plants (Nishimura et al 2003) and the fact 547

that different B cinerea isolates vary both in induction of and reaction to defense 548

responses (Kliebenstein et al 2005 Rowe et al 2010) we could not determine 549

Page 29 of 54

whether reduced callose deposition causes the increased susceptibility of gae1 gae6 to 550

B cinerea isolate Gallo 1551

We did not observe any changes in B cinerea growth either on cev1 ixr1 plants 552

which are known to have constitutively high JA and OPDA levels (Ellis et al 2002b) or 553

on plants exogenously treated with MeJA (Supplemental Figure 9) Because plants 554

were kept uncovered for 3 h after MeJA treatment it is unclear how much MeJA was 555

bioavailable in the plant However JAZ10 expression was induced 3 h after spraying 556

with MeJA (Supplemental Figure 10) Because pathogen growth generally depends on 557

the combination of a multitude of immune signaling events we conclude that multiple 558

changes to the immune signaling in gae1 gae6 together account for the altered B 559

cinerea growth phenotypes detected 560

We showed that expression of GAE1 and GAE6 is repressed by the bacterial 561

hemibiotroph Pma ES4326 This repression did not occur in an eds1 or pad4 562

background or in pbs3 at the 24-h time point (Figure 1 and Supplemental Figure 1) 563

During the Arabidopsis - Pma ES4326 interaction the expression of many defense 564

related genes is altered in a PAD4EDS1 dependent fashion (Wang et al 2008) Some 565

of these expression changes require SA CBP60g and SARD1 are thought to act 566

downstream of PAD4 and EDS1 but upstream of SA-dependent responses (Wang et 567

al 2011) PBS3 is also thought to act downstream of PAD4 and EDS1 and upstream of 568

SA but the set of genes whose expression it affects is only partially overlapping with 569

those affected by CBP60gSARD1 (Wang et al 2008 Wang et al 2011) Thus GAE1 570

and GAE6 repression is regulated by the part of the immune signaling network that 571

requires PAD4 EDS1 and PBS3 but is independent of CBP60gSARD1 and SA 572

Because EDS1 PAD4 and PBS3 are important components of the plant immune 573

signaling network (Wiermer et al 2005 Nobuta et al 2007) one might conclude that 574

repression of GAE1 and GAE6 may be part of the plant immune response to Pma 575

ES4326 B cinerea treatment also reduced GAE1 and GAE6 expression but this was 576

mostly independent of PAD4 (Figure 6) while both Pma ES4326 and B cinerea repress 577

GAE1 and GAE6 expression the host signaling mechanisms responsible likely differ 578

In summary our data show that Arabidopsis GAE1 and GAE6 are required for 579

pectin biosynthesis leaf flexibility and immunity to Pma ES4326 and to specific B 580

Page 30 of 54

cinerea isolates We used macerozyme a commercial pectinase to mimic the effect of 581

B cinerea pectinases Macerozyme treatment released less pectin from gae1 gae6 cell582

walls and induced less immune signaling in gae1 gae6 plants Less water soluble pectin 583

also accumulated in B cinerea infected gae1 gae6 plants and macerozyme-induced 584

immunity to B cinerea isolate Gallo 1 was reduced in these plants We conclude that 585

the reduced pectin content in these mutants may lead to reduced release of soluble 586

pectin including active OGs during pathogen attack and that this compromises 587

immunity 588

589

590

Page 31 of 54

METHODS 591

Plant Materials and Growth Conditions 592

Wild-type Columbia (Col-0) and all mutant plants (in Col-0 background) were 593

grown on sterilized BM2 germinating mix (Berger Inc Quebec Canada) in a controlled 594

environment chamber (Conviron Pembina ND USA) with a 12-h photoperiod under 595

100 microM m-2 s-1 fluorescent illumination at 22degC and 75 relative humidity Germplasm 596

used is described in the Accession Numbers section at the end of Methods 597

598

Data Analysis and Replicates 599

For biological replicates samples were obtained from separate plant tissues and 600

in many cases from plants grown in different flats In some experiments multiple 601

technical replicates were measured per biological replicate For statistical analysis all 602

technical replicates were averaged and the averages were considered one biological 603

replicate Independent experiments were performed at different times and often 604

consisted of multiple biological replicates per experiment 605

Data analysis was performed using mixed linear models in the R programming 606

environment (Bates et al 2015) unless otherwise indicated Generally genotypes 607

treatments and time points were used as fixed effects and replicate-specific effects like 608

different pots flats etc were considered random effects We reported p-values 609

obtained from the mixed linear model when less than 10 comparisons were performed 610

and q-values for more than 10 comparisons to correct for multiple comparison testing 611

Q-values were calculated using the Benjamini-Hochberg procedure (Benjamini and612

Hochberg 1995) unless specified otherwise 613

614

Pathogen Strains Growth Conditions and Pathogen Growth Assays 615

Growth of Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) and 616

bacterial growth assays were performed as previously described (Bethke et al 2014) 617

For qRT-PCR assays four week-old plants were inoculated with bacteria (OD600 = 618

0002) or mock (5 mM MgSO4) At least four leaves from two plants were collected at 619

the indicated time points per biological replicate 620

Page 32 of 54

Prior to each experiment Botrytis cinerea isolates were grown on 1x PDA 621

medium (Difco) for 10 d at room temperature All isolates used have been described 622

previously (Rowe and Kliebenstein 2007) The spores were washed from the surface of 623

the plate using an inoculation medium (1x Gamborgrsquos B-5 basal salt mixture (Sigma-624

Aldrich G5768) 2 (wv) glucose 10 mM phosphate buffer pH 64) and fungal hyphae 625

were removed from the suspension by filtering through four layers of cheesecloth The 626

concentration of spores was determined using a hemocytometer and adjusted to 25 x 627

105 spores mL-1 Four week-old Arabidopsis plants were inoculated by placing one 10-628

microL droplet of the B cinerea spore solution or inoculation medium (mock) on the adaxial 629

leaf surface of fully expanded leaves Inoculated plants were kept at 100 relative 630

humidity Infection sites were collected at various time points using a cork borer For 631

qRT-PCR assays at least four leaf discs from one plant were combined for each 632

biological sample For B cinerea growth assays at least 12 leaf discs from three to six 633

plants were pooled per biological sample Relative fungal growth was determined by 634

measuring the abundance of a fungal gene relative to a plant gene as described below 635

In brief DNA from infected tissue was extracted and equal amounts of total DNA were 636

used to perform qPCR reactions using the B cinerea cutinase A and the Arabidopsis 637

SK11 gene respectively Primers were described in (Gachon and Saindrenan 2004) 638

and are listed in Supplemental Table 1 Two technical replicates were combined for 639

each biological replicate B cinerea is a necrotrophic fungal pathogen and may destroy 640

plant DNA as disease progresses However decreases in the amount of plant DNA 641

over the course of our experiments were only observed at late time points with very 642

susceptible plants so the qPCR assay is generally a reasonable estimate of fungal 643

biomass (Supplemental Figure 16) 644

645

Treatment with Macerozyme or OGs 646

For PTI assays four week-old plants either were inoculated with 001 (wv) 647

macerozyme R-10 (Yakult) a Rhizopus polygalacturonase (EC 32115) that possesses 648

high pectinase and hemicellulase activity or were mock inoculated using heat-649

inactivated macerozyme Other similar plants were either inoculated with 250 or 500 microg 650

mL-1 OGs or mock inoculated with water One d later plants were infected with B 651

Page 33 of 54

cinerea and samples were collected immediately (0 dpi) and two or three d later To 652

investigate the role of MeJA on B cinerea growth plants were sprayed with 1mM MeJA 653

in water with 001 (vv) Tween 20 Covers were removed so that leaves could dry off 654

before plants were inoculated with B cinerea three h later Samples were harvested at 655

the indicated time points For expression analysis and MAPK activation assays plants 656

were either inoculated with 100 microg mL-1 OGs or mock inoculated with water OGs used 657

for experiments described in Figures 8 to 10 and Supplemental Figure 12 were a kind 658

gift from Simone Ferrari OGs used in experiments described in Figure 13 and 659

Supplemental Figures 11 13 and 15 were prepared as previously described (Kohorn et 660

al 2014) 661

662

Expression Analysis 663

Leaf tissue from inoculated plants was harvested at the indicated time points 664

flash frozen pulverized and RNA extracted using Trizol (Invitrogen) Quantitative RT-665

PCR was performed using the SuperScript III Platinum SYBR Green One-Step 666

quantitative RT-PCR kit (Invitrogen) and a Lightcycler 480 Real-Time PCR system 667

(Roche) as previously described (Truman and Glazebrook 2012) In brief equal 668

amounts of total RNA and a gene specific primer were used for each reaction The 669

crossing point (Cp) was calculated using the 2nd derivative max method provided with 670

the Lightcycler software for each amplification curve Each reaction was run with two 671

technical replicates and the Cp values for these replicates were averaged ACTIN2 was 672

used as a stably expressed reference gene At least three biological replicates each 673

were performed and analyzed using a mixed-linear model RT-PCR was performed to 674

verify T-DNA insertion lines using the One-Step RT-PCR kit (Qiagen) according to the 675

manufacturerrsquos instructions The primers used can be found in Supplemental Table 1 676

677

Electrolyte Leakage Assay 678

Fully expanded leaves of four week-old plants were inoculated with water (Milli-Q 679

grade) using a needleless syringe Leaf discs were collected using a cork borer and 680

three leaf discs from one leaf were placed with the adaxial surface down onto 2 mL 681

water in a 12-well cell culture plate Conductivity was measured using a Horiba B-173 682

Page 34 of 54

conductivity meter after 30 min and then the water was replaced Conductivity was 683

measured again 30 min later (1 h total) as indicated in Figure 3C Six biological 684

replicates each were performed in three independent experiments and means and SEs 685

were calculated Two-sided t-tests were performed for each time point 686

687

DAB and Aniline Blue Staining 688

Leaves from four week-old plants were either mock inoculated with B cinerea 689

inoculation medium or inoculated with 10 microL droplets of a B cinerea spore solution (25 690

x 105 spores mL-1) of the indicated isolates Samples were collected 72 h later For 33prime-691

diaminobenzidine (DAB) staining two to five inoculated leaves were vacuum infiltrated 692

with 33prime-Diaminobenzidine (1 mg mL-1 in water pH 38) solution and stained overnight 693

in the dark Leaves were destained using 100 ethanol at 65degC and then transferred to 694

50 (vv) glycerol and photographed 695

Callose was visualized by staining leaves with aniline blue as previously 696

described (Adam and Somerville 1996) Briefly leaves were vacuum infiltrated with 697

alcoholic lactophenol (one volume of phenolglycerollactic acidwater (1111) mixed 698

with two volumes of ethanol) and destained at 65degC Next leaves were moved through 699

an alcohol gradient (50 20 and 10 (vv) ethanol) into water The destained leaves 700

were stained for 30 min in 150 mM K2HPO4 (pH 95) with 001 (wv) aniline blue 701

Samples were mounted in water and examined using a Nikon Eclipse Ni-U microscope 702

at 10x magnification using a DAPI filter (excitation at 325-375 nm emission at 435-485 703

nm) For callose quantification six 200 microm x 200 microm squares were counted for each 704

sample type Callose deposits per mm2 were calculated and data analyzed using a t-705

test Note that there is an inherent uncertainty associated with such counts because 706

callose deposits may not all be in focus at the same time 707

708

MAPK Activity Assay 709

Fully expanded leaves of four week-old plants were inoculated with 001 710

macerozyme 100 microg mL-1 OGs 100 nM flg22 or water (mock) flg22 a peptide from 711

bacterial flagella that induces immune responses was used as a positive control for 712

Page 35 of 54

MAPK activation (Asai et al 2002) and was purchased from EZBiolab Inc (Westfield 713

IN USA) Samples were harvested 10 min later and immediately frozen in liquid 714

nitrogen Protein was extracted using an extraction buffer containing 25 mM Tris pH 78 715

75 mM NaCl 1 mM DTT 1 mM NaF 05mM Na3VO4 01 (vv) Tween 20 one 716

cOmplete Mini protease inhibitor cocktail tablet (Roche) and one PhosSTOP 717

phosphatase inhibitor cocktail tablet (Roche) per 10 mL buffer 15 microg of total protein 718

were electrophoresed on a 10 SDS-polyacrylamide gel and then blotted onto a PVDF 719

membrane (Bio-Rad 162-0177) Activated MAPKs were detected using p4442 MAPK 720

(Erk12) antibody (9102S Cell Signal 12500 in TBST (20 mM Tris pH 75 150 mM 721

NaCl 01 (vv) Tween 20)) Anti-Rabbit IgG-HRP (115000 in TBST A6154 Sigma) 722

and ECL Plus substrate (Pierce 32132) MPK3 and MPK6 were detected with an anti-723

MPK3 antibody (M8318 Sigma 12000 in TBST and 3 (wv) milk) or an anti-MPK6 724

antibody (A7104 Sigma 16000 in TBST and 3 (wv) milk) respectively The 725

secondary antibody and detection were the same as for the p4442 antibody Five 726

independent experiments were performed 727

728

Preparation of Alcohol Insoluble Residue (AIR) Pectin Extraction and Uronic Acid 729

Measurements 730

Fully expanded leaves from four week-old plants that were incubated for 48 h in 731

the dark to reduce starch were harvested flash frozen and pulverized AIR was 732

extracted by washing ground material twice in 70 (vv) ethanol three times in a 733

mixture of chloroform and methanol (11 vv) and once in acetone (Gille et al 2009) 734

and was then air dried Cell wall pectin was extracted from AIR with cell wall extraction 735

buffer (50 mM Trizma 50 mM CDTA pH 72) at 95degC for 15 min Samples were 736

homogenized using a paint shaker (Harbil 5G-HD) and glass beads (3 mm diameter) 737

For dot blot experiments 500 microL of cell wall extraction buffer were used per 10 mg of 738

AIR Debris was precipitated by centrifugation for 10 min at 10000 g 739

Total uronic acid content of pectin extracted as described above using 500 microL of 740

cell wall extraction buffer per 1 mg of AIR was measured as described previously 741

(Filisetti-Cozzi and Carpita 1991 van den Hoogen et al 1998) First 36 microL of pectin 742

extract were mixed with 4 microL of 4 M sulfamic acid Then 200 microL of sulfuric acid 743

Page 36 of 54

containing 120 mM sodium tetraborate was added and samples were incubated at 80degC 744

for 1 h Following cooling on ice the optical density of samples was measured at 490 745

nm Next 40 microL of m-hydroxydiphenyl reagent (100 microL of m-hydroxydiphenyl in dimethyl 746

sulfoxide at 100 mgmL mixed with 49 mL 80 (vv) sulfuric acid just before use) was 747

added and the samples were mixed The optical density at 490 nm was measured 748

again The optical density before m-hydroxydiphenyl reagent addition was subtracted 749

from the optical density measured after addition of the dye The concentration of uronic 750

acid was calculated using known amounts of GalA as a standard 751

752

Release of Pectin from Arabidopsis Cell Walls Using Macerozyme 753

AIR preparations from leaves of four week-old plants were treated with 025 754

(wv) macerozyme R-10 (Yakult) or water (mock) at a concentration of 100 microL per mg 755

AIR Samples were incubated for 3 h at room temperature on a vortex shaker and 756

centrifuged for 10 min at 12000 g Total uronic acid concentration of 5-fold diluted 757

supernatants was determined for two technical replicates each The concentration of 758

uronic acid was calculated using known amounts of GalA as a standard For 759

subsequent qRT-PCR experiments supernatants from multiple extractions were 760

combined boiled for 20 min to inactivate macerozyme diluted 10-fold with water and 761

inoculated into four week-old Col-0 plants Heat-inactivated 10-fold diluted macerozyme 762

solutions were used as mock treatment Samples were collected 3 h later and PAD3 763

expression was determined 764

765

Release of Pectin from Arabidopsis Leaves Infected with B cinerea 766

Fully expanded leaves of four week-old plants were either inoculated with up to 767

five 10 microL droplets of B cinerea spore solution (25 x 105 spores mL-1) or mock 768

inoculated with B cinerea inoculation medium For each biological replicate leaves with 769

a combined count of at least 20 infection sites were collected 48 h later AIR was 770

extracted as described above but plants were not dark-treated or freeze-dried and the 771

first ethanol step was performed using 85 (vv) ethanol To extract soluble pectin 772

fragments AIR samples were mixed with water at a concentration of 20 microL per infection 773

site incubated for 7 h at room temperature on a vortex shaker and centrifuged for 10 774

Page 37 of 54

min at 12000 g Total uronic acid concentration of supernatants was determined for two 775

technical replicates each The concentration of uronic acid was calculated using known 776

amounts of GalA as a standard 777

778

Measurement of Uronic Acids Neutral Monosaccharides and Cellulose Content in 779

Cell Walls 780

Neutral sugars and uronic acids from non-cellulosic polysaccharides in cell walls 781

(AIR) were released by trifluoroacetic acid (TFA) hydrolysis Approximately 1 mg of AIR 782

was hydrolyzed with 2 M TFA at 121degC for 90 min The TFA-soluble material was split 783

into two equal parts one for uronic acid and one for neutral sugar determination The 784

uronic acid content was determined by analyzing the hydrolyzate using a CarboPac 785

PA200 anion-exchange column with an ICS-3000 Dionex chromatography system 786

(Dionex Sunnyvale CA USA) The elution profile consisted of a linear gradient of 50 to 787

200 mM sodium acetate in 01 M NaOH in 10 min at 04 mL per min Alditol acetate 788

derivatives of neutral sugars were produced as described (York et al 1985) In brief 789

monosaccharides in the hydrolyzate were reduced with NaBH4 and peracetylated with 790

acetic anhydride and pyridine at 121degC for 20 min The generated alditol acetates were 791

analyzed by GC-MS Crystalline cellulose was measured according to Updegraff (1969) 792

After hydrolysis of noncellulosic polysaccharides from AIR with the Updegraff reagent 793

(acetic acid nitric acids water 812 (vv) ) the remaining pellet was hydrolyzed in 794

72 sulfuric acid The resulting glucose quantity was determined by the anthrone 795

method (Scott and Melvin 1953) 796

797

Dot Blot Experiments 798

Pectin solutions extracted using 50 microL of cell wall extraction buffer per mg AIR 799

were serially diluted and nitrocellulose membranes were spotted with 1 microL of the diluted 800

pectin solutions Membranes were dried overnight blocked with 5 milk (wv) in 1x 801

PBS (8 g L-1 NaCl 02 g L-1 KCl 144 g L-1 Na2HPO4 024 g L-1 KH2PO4 pH 74) and 802

probed with LM5 (Jones et al 1997) LM6 (Willats et al 1998) LM8 (Willats et al 803

2004) LM19 and LM20 (Verhertbruggen et al 2009a) or CCRC-M7 (Steffan et al 804

Page 38 of 54

1995) antibodies LM series antibodies were diluted 1250 and CCRC-M7 was diluted 805

1500 in 5 milk powder (Nestle Carnation) in 1 x PBS For LM series antibodies a 806

goat anti-Rat HRP conjugated antibody (Bethyl A110-105P 15000 diluted) and for 807

CCRC-M7 an anti-Mouse IgG HRP conjugate (Promega W402B 12500 diluted) was 808

used as secondary antibody in 5 milk powder in 1x PBS LM series antibodies were 809

obtained from plant probes (wwwplantprobesnet) and CCRC-M7 from CarboSource 810

(wwwcarbosourcenet) Membranes were washed after incubation with the primary and 811

secondary antibodies with 1x PBS Dot blots were developed using the ECL system 812

(GE Healthcare Amersham ECL prime) For quantification of signals dot blot results on 813

x-ray films were photographed using a CCD camera and intensities were measured814

using Image Jrsquos Integrated Density function Locally adjusted background was 815

subtracted As the dynamic range of the signal on an x-ray film was narrow non-816

linearity in the measured values was corrected for each genotype in each biological 817

replicate using the values from different dilutions in two technical replicates We noticed 818

that the residuals were not normally distributed and performed Box-Cox power 819

transformation (Box and Cox 1964) to obtain normally distributed values for the 820

subsequent statistical analysis of the data For LM19 Box-Cox selected power 821

transformation was done to the power of 038th and for LM20 to the power 075th After 822

correction of the transformed measured values with the effect of the biological 823

replicates the values were fit to a linear model with the Arabidopsis genotype as the 824

fixed effect This linear model was used both to generate the plots in Figures 5B and D 825

and to test significant differences 826

827

Camalexin Measurements 828

Four week-old plants were infected with B cinerea and samples (three leaf disks 829

from one plant each) were harvested at the indicated time points flash frozen and 830

pulverized Each sample was extracted with 300 microL of 90 methanol (vv) 50 microL of the 831

extract was run on an Agilent Lichrocart 250-4 RP18e 5-microm column using an Agilent 832

1100 series HPLC Camalexin was detected using a DiodeArray (DAD) at 330 nm and 833

with a fluorescence detector at emission 318 nmexcitation 385 nm (Agilent) Separation 834

was achieved using the following program with aqueous acetonitrile 5 min gradient 835

Page 39 of 54

from 63 to 69 acetonitrile 30 s gradient from 69 to 99 acetonitrile 2 min at 99 836

acetonitrile and a post-run equilibration of 35 min at 63 acetonitrile (Denby et al 837

2004 Kliebenstein et al 2005) Purified camalexin was used to produce a standard 838

curve to identify and quantitate camalexin 839

840

Accession Numbers 841

Microarray data used can be accessed at NCBI Gene Expression Omnnibus 842

(httpwwwncbinlmnihgovgeo) GSE18978 Sequence information for all Arabidopsis 843

genes described in this article can be found in TAIR (wwwarabidopsisorg) using the 844

following accession numbers PAD3 (CYP71B15 At3g26830) SID2 (ICS1 At1g74710) 845

GAE1 (At4g30440) GAE2 (At1g02000) GAE3 (At4g00110) GAE4 (At2g45310) GAE5 846

(At4g12250) GAE6 (At3g23820) PR1 (At2g14610) JAZ5 (At1g17380) JAZ10 847

(At5g13220) ACTIN2 (At3g18780) PDF12a (At5g44420) PDF12b (At2g26020) 848

AtSK11 (At5g26751) The sequence of the B cinerea cutinase Bc-CutA (Z69264) can 849

be found in the DNA Data Bank of Japan (httpgetentryddbjnigacjp) Germplasm 850

used included dde2-2 (At5g42650 (von Malek et al 2002)) pad3-1 (At3g26830 (Zhou 851

et al 1999)) pad4-1 (At3g52430 (Jirage et al 1999)) sid2-2 (At1g74710 (Wildermuth 852

et al 2001)) pbs3-2 (At5g13320 SALK_018225 (Nobuta et al 2007)) cbp60a-1 853

(At5g62570 SALK_124410 (Truman et al 2013)) cbp60g-1 sard1-2 (At5g26920 854

SALK_023199 At1g73805 SALK_052422 (Wang et al 2011) cbp60a-1 cbp60g-1 855

sard1-2 (Truman et al 2013) gae1-1 (At4g30440 SALK_085554) gae6-1 (At3g23280 856

SALK_104454) gae6-2 (At3g23280 SALK_017191) ixr1-1 cev1 (At5g05170 (Ellis 857

and Turner 2001 Scheible et al 2001)) eds1 plants were derived by introgression of 858

the Ler eds1-2 allele into Col-0 that contains two copies of EDS1 (At3g48090 859

At3g48080 (Bartsch et al 2006) mpk6-2 (At2g43790 SALK_073907 (Liu and Zhang 860

2004)) mpk3-DG (At3g45640 a fast neutron deletion mutant (Miles et al 2005)) and 861

npr1-1 (At1g64280 (Cao et al 1997)) T-DNA insertion lines were part of the SALK 862

collection (Alonso et al 2003) and were obtained from the Arabidopsis Biological 863

Resource Center 864

865

Supplemental Data 866

Page 40 of 54

Supplemental Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 867

Expression Does Not Require CBP60s or SID2 868

Supplemental Figure 2 Expression of GAE Family Members uponTreatment with Pma 869

ES4326 870

Supplemental Figure 3 Characterization of T-DNA Insertions in GAE1 and GAE6 871

Supplemental Figure 4 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves and 872

Are Slightly Smaller than Wild-Type Col-0 Plants 873

Supplemental Figure 5 Cell Walls of gae1 gae6 Plants Contain Less Uronic Acid than 874

Wild-Type Col-0 Cell Walls 875

Supplemental Figure 6 Antibodies Raised Against the RGI or XG Components of 876

Pectin Show No Difference in Binding to gae Mutant and Col-0 Cell Walls 877

Supplemental Figure 7 The Effect of B cinerea Treatment on the Expression of GAE 878

Family Genes 879

Supplemental Figure 8 Treatment with 001 Macerozyme Does Not Cause Any 880

Visible Damage to Col-0 Plants 881

Supplemental Figure 9 B cinerea Growth on Plants with Defects in Immune 882

Signaling 883

Supplemental Figure 10 MeJA-Induced Expression of the JA marker gene JAZ10 884

Supplemental Figure 11 The Expression of the JA Marker Gene JAZ5 Is Highly 885

Responsive in gae1 gae6 Plants 886

Supplemental Figure 12 The Expression of PDF12 Is Unaltered in gae1 gae6 Plants 887

Supplemental Figure 13 Macerozyme Treatment Activates MPK3 and MPK6 888

Supplemental Figure 14 B cinerea-Induced ROS Production in Wild-Type Col-0 and 889

gae1 gae6 Plants 890

Supplemental Figure 15 OG-Induced PTI Using 250 microg mL-1 OGs 891

Supplemental Figure 16 Abundance of Plant DNA in B cinerea Treated Plants 892

Supplemental Table 1 Primers used in this study 893

894

ACKNOWLEDGEMENTS 895

We thank William Truman for helpful discussions Simone Ferrari (Sapienza 896

Universita di Roma) for the generous gift of OGs Bruna Bucciarelli (USDA-ARS) for 897

Page 41 of 54

help with fluorescence microscopy the Arabidopsis Biological Resource Center for T-898

DNA insertion lines Paul Knoxrsquos lab (University of Leeds) for LM5 LM6 LM8 LM19 899

and LM20 antibodies and the Complex Carbohydrate Resource Center (University of 900

Georgia) for the CCRC-M7 antibody Most of this work was funded by the Division of 901

Chemical Sciences Geosciences and Biosciences Office of Basic Energy Sciences of 902

the US Department of Energy through Grant DE-FG02-05ER15670 to JG This work 903

was also supported by NSF awards IOS 1339125 and IOS 1021861 to DJK NSF 904

award IOS 1121425 to F K and the USDA National Institute of Food and Agriculture 905

Hatch project number CA-D-PLS-7033-H to DJK 906

907

AUTHOR CONTRIBUTIONS 908

GB and JG designed the research GB GX AT BL NES and NH 909

performed research DJK provided tools GB GX MP AD BL RAH and FK 910

analyzed data GB and JG wrote the paper with input from all authors 911

912

FIGURE LEGENDS 913

Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 Expression 914

Requires PAD4 EDS1 and PBS3 915

(A) Expression of GAE1 in leaves of wild-type Col-0 and mutant plants Expression was916

measured 24 or 32 h after either inoculation with Pma ES4326 (OD600 = 0002) or mock 917

inoculation with 5 mM MgSO4 and was then normalized to the level of ACTIN2 Data 918

from three biological replicates were combined using a mixed linear model Bars 919

represent mean log2-ratios to ACTIN2 plusmn SE Letters indicate significantly different 920

groups with q lt 005 where 005 represents the maximum false discovery rate at which 921

the test may be called significant 922

(B) Expression of GAE6 measured as described in (A)923

924

Figure 2 Susceptibility of gae6 and gae1 gae6 Plants to Pma ES4326 Is Increased 925

Relative to Wild-Type Col-0 926

Leaves of plants of the indicated genotypes were inoculated with Pma ES4326 (OD600 = 927

00002) as described in Methods Bacterial titers in leaves were determined either 928

Page 42 of 54

immediately (0 dpi) or after three days (3 dpi) Bars represent the mean plusmn SE of four 929

independent experiments each with 4 or 12 biological replicates at 0 and 3 dpi 930

respectively Data were combined using a mixed linear model Asterisks indicate 931

significant differences from Col-0 wild-type (q lt 005) Susceptible pad4 plants were 932

included as a positive control 933

934

Figure 3 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves 935

(A) Depiction of the assay for brittleness of gae mutant leaves Fully expanded leaves of936

four to five week-old plants were bent by 180ordm so that the adaxial surface of the leaf was 937

parallel with the soil Broken leaf petioles or midribs were considered brittle 938

(B) Quantification of broken midribs or petioles For each genotype 66 to 82 leaves 939

were analyzed for brittleness as described in (A) Bars represent the ratio of broken to 940

total number of leaves tested Results were analyzed using Fisherrsquos exact test Letters 941

indicate significantly different groups at p lt 005 942

(C) Conductivity measurements Leaves of four week-old plants were inoculated with943

deionized water and then leaf discs were cut out and placed on deionized water 944

Conductivity was measured at the indicated time points After 30 min the water was 945

replaced with fresh water (30 min f w) to account for wounding-related ion leakage from 946

the edges of the leaf discs Three independent experiments with six biological replicates 947

each were performed and data were analyzed using a t-test for each time point P-948

values were corrected using the Bonferroni method Data show means plusmn SE asterisks 949

indicate significant differences from wild-type Col-0 at q lt 001 950

951

Figure 4 Cell Walls of gae6 and gae1 gae6 Plants Are Reduced in GalA 952

(A) to (D) Levels of GalA (A) GlcA (B) cellulose (C) and neutral sugars (D) in alcohol953

insoluble residue (AIR) extracted from leaves of four week-old plants of the indicated 954

genotypes Bars represent means plusmn SE of four biological replicates combined using a 955

mixed linear model Letters indicate significantly different groups (p lt 005) 956

957

Figure 5 Cell Walls of gae1 gae6 Plants Are Reduced in Homogalacturonan 958

Page 43 of 54

Dot blot analyses with anti-LM19 and anti-LM20 antibodies Pectin was extracted from 959

AIR prepared from the indicated genotypes using 1 microL of extraction buffer per 10 microg of 960

AIR Pectin was serially diluted and one microL of undiluted 13 diluted and 19 diluted 961

samples were spotted on a nitrocellulose membrane Three biological replicates were 962

were spotted twice to obtain two technical replicates each All replicates produced 963

similar results 964

(A) Representative dot blot using anti-LM19 antibody965

(B) Relative signal intensities of the spots in the dot blot in (A)966

(C) Representative dot blot using anti-LM20 antibody967

(D) Relative signal intensities of the LM20 dot blot shown in (C)968

Relative signal intensities were measured using Image J software Measurements from 969

the three biological replicates were combined using a linear model after Box-Cox power 970

transformation as described in Methods Bars represent means plusmn SE Asterisks indicate 971

a difference from wild-type Col-0 at p lt 005 972

973

Figure 6 Growth of B cinerea on gae1 gae6 Plants and the Effect of B cinerea on 974

Expression of GAE1 and GAE6 975

(A) Growth of B cinerea isolates Leaves of Col-0 and gae1 gae6 plants were976

inoculated with 25 x 105 spores mL-1 of the B cinerea isolates indicated in the figure 977

Samples were collected two (2 dpi) and three (3 dpi) days later Bars represent means plusmn 978

SE of six biological replicates for each fungus combined using a mixed linear model 979

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 980

(B) and (C) Expression of GAE1 (B) and GAE6 (C) 48 h after inoculation with the B981

cinerea isolates indicated Expression levels were measured by qRT-PCR Bars 982

represent mean log2-ratios to ACTIN2 plusmn SE of three biological replicates combined 983

using a mixed linear model Letters indicate significantly different groups (q lt 005) 984

985

Figure 7 Levels of Soluble Pectin and Expression of PAD3 in Fractions Produced 986

Following Macerozyme Treatment of gae1 gae6 Cell Walls 987

(A) Schematic overview of the experimental procedure for (B) and (C)988

Page 44 of 54

(B) Uronic acid measurements Alcohol-insoluble residue (AIR) from leave tissue of four989

week-old wild-type Col-0 or gae1 gae6 plants was treated with water or 025 990

macerozyme and assayed for uronic acid levels Data from 11 biological replicates were 991

combined using a mixed linear model Bars represent means plusmn SE Letters indicate 992

significant differences (p lt 005) 993

(C) Expression of PAD3 in Col-0 plants treated with the fractions from (B) Extracts from994

(B) were heat inactivated pooled and diluted 5-fold with water Pooled extracts were995

inoculated into four week-old Col-0 plants PAD3 expression was measured 3 h after 996

inoculation and normalized to the level of ACTIN2 expression Data from four biological 997

replicates were combined using a mixed linear model Bars represent mean log2-ratios 998

to ACTIN2 plusmn SE Letters indicate significant differences (p lt 005) 999

1000

Figure 8 Macerozyme-Induced PAD3 Expression Is Absent in gae1 gae6 1001

(A) Expression of PAD3 after macerozyme treatment Four week-old plants were either1002

inoculated with 001 macerozyme or mock inoculated with water and PAD3 1003

expression was measured 3 h later by qRT-PCR Data from four biological replicates 1004

were combined using a mixed linear model Mean log2-ratios to ACTIN2 plusmn SE were 1005

plotted Letters indicate significantly different groups (p lt 005) 1006

(B) Expression of PAD3 after OG treatment PAD3 expression was measured as1007

described in (A) 3 h after inoculation with 100 microg mL-1 OGs or mock inoculation with 1008

water Data are from six biological replicates 1009

(C) PAD3 expression in untreated plants Three biological replicates were performed1010

and data analyzed as described in (A) 1011

(D) Camalexin accumulation after B cinerea treatment Camalexin accumulation was1012

measured in four week-old plants inoculated with the B cinerea strains shown in the 1013

figure Eight biological replicates each were combined using a mixed linear model 1014

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 1015

(E) PAD3 expression 28 h after treatment with the indicated B cinerea strains Data1016

from six biological replicates were collected and analyzed as described in (A) Data in 1017

all figure parts represent means plusmn SE Letters indicate significantly different groups (q lt 1018

005) 1019

Page 45 of 54

1020

Figure 9 Macerozyme-Induced Expression of the SA Marker Genes SID2 and PR1 1021

Is Abolished in gae1 gae6 Plants 1022

(A) to (H) Expression of PR1 (A) (C) (E) and (G) or SID2 (B) (D) (F) and (H) in four1023

week-old Col-0 and gae1 gae6 plants Data were combined using a mixed linear model 1024

Bars represent mean log2-ratios of expression vs ACTIN2 plusmn SE for all figure parts 1025

Letters indicate significantly different groups at p lt 005 for panels (A) to (D) (G) and 1026

(H) and qlt005 for panels (E) to (F) 1027

(A) and (B) Plants were treated with 001 macerozyme or mock treated with water1028

and samples were collected 3 h later Data from four biological replicates are shown 1029

(C) and (D) Plants were inoculated with 100 microg mL-1 OGs or mock treated with water1030

and samples collected 3 h later Data from six biological replicates are shown 1031

(E) and (F) Plants were inoculated with the indicated B cinerea isolates or mock1032

inoculated with B cinerea inoculation medium and samples collected 48 h later Data 1033

from eight biological replicates are shown 1034

(G) and (H) Expression in untreated plants Three biological replicates were performed1035

1036

Figure 10 The Expression of the JA Marker Gene JAZ10 is Highly Responsive in 1037

gae1 gae6 Plants 1038

(A) to (D) JAZ10 expression in four week-old plants Data were combined using a mixed1039

linear model Mean log2-ratios to ACTIN2 plusmn SE are shown Letters indicate significantly 1040

different groups at p lt 005 for panels (A) (B) and (D) and q lt 005 for panel (C) 1041

(A) Plants were inoculated with 001 macerozyme or mock inoculated using water and1042

samples were collected 3 h later Data from four biological replicates are shown 1043

(B) Plants were inoculated with 100 microg mL-1 OGs or mock inoculated using water and1044

samples collected 3 h later Data are from six biological replicates 1045

(C) JAZ10 expression 48 h after treatment with the indicated B cinerea strains Data1046

are from six biological replicates 1047

(D) JAZ10 expression in untreated plants Data are from three biological replicates1048

1049

Figure 11 Gallo 1-Induced Callose Deposition Is Reduced in gae1 gae6 Plants 1050

Page 46 of 54

(A) Callose deposition in four week-old Col-0 and gae1 gae6 plants Plants were1051

inoculated with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) or mock inoculated 1052

with B cinerea inoculation medium and samples were collected after 72 h Callose was 1053

stained with aniline blue and visualized using a Nikon Eclipse Ni-U microscope with a 1054

DAPI filter Three leaves were stained per treatment Scale bars are 100 microm Asterisks 1055

indicate fungal hyphae arrows indicate representative callose spots 1056

(B) Quantification of callose deposition Callose deposits were counted in 200 microm x 2001057

microm squares laid out across the images shown here Bars represent means plusmn SE from 1058

six squares each Letters indicate significantly different groups (p lt 005) according to a 1059

t-test analysis1060

1061

Figure 12 Less Soluble Pectin Accumulates in B cinerea-Infected gae1 gae6 1062

Leaves 1063

Four week-old Col-0 and gae1 gae6 plants were inoculated with 10 microL droplets of the 1064

indicated B cinerea isolate (25 x 105 spores mL-1) or mock inoculated with B cinerea 1065

inoculation medium Leaves were collected 48 h later AIR was extracted from leaf 1066

samples and water added (20 microL infection site) to extract soluble pectin fragments 1067

Samples were vortexed for 7 h centrifuged at 12000 g and the uronic acid 1068

concentration of the supernatant was measured Data from nine biological replicates 1069

were combined using a mixed linear model Means plusmn SE are shown Letters indicate 1070

significant differences (q lt 005) 1071

1072

Figure 13 Macerozyme-Induced PTI Is Lost in gae1 gae6 While OG-Induced PTI Is 1073

Unaltered 1074

(A) Macerozyme-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1075

inoculated with 001 macerozyme or mock inoculated with boiled macerozyme and 1076

were infected with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) 24 h later Samples 1077

were collected immediately (0 dpi) and after two (2 dpi) and three (3 dpi) days Data 1078

show mean plusmn SE of six to 14 biological replicates each combined using a linear model 1079

Letters indicate significantly different groups (q lt 005) 1080

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

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Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk RGadrinab C Heller C Jeske A Koesema E Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt MHazari N Hom E Karnes M Mulholland C Ndubaku R Schmidt I Guzman P Aguilar-Henonin L Schmid M Weigel DCarter DE Marchand T Risseeuw E Brogden D Zeko A Crosby WL Berry CC and Ecker JR (2003) Genome-wideinsertional mutagenesis of Arabidopsis thaliana Science 301 653-657

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Asai T Tena G Plotnikova J Willmann MR Chiu WL Gomez-Gomez L Boller T Ausubel FM and Sheen J (2002) MAPkinase signalling cascade in Arabidopsis innate immunity Nature 415 977-983

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Cao H Glazebrook J Clarke JD Volko S and Dong XN (1997) The Arabidopsis NPR1 gene that controls systemic acquiredresistance encodes a novel protein containing ankyrin repeats Cell 88 57-63

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Carpita N (2010) Update on Mechanisms of Plant Cell Wall Biosynthesis How plants make cellulose and other (1-gt4)-beta-D-glycans Plant Physiol

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Chisholm ST Coaker G Day B and Staskawicz BJ (2006) Host-microbe interactions Shaping the evolution of the plantimmune response Cell 124 803-814

Page 18 of 54

Page 19 of 54

Page 20 of 54

344

345

346

and JAZ10 was already strongly induced in gae1 gae6 after mock inoculation (Figure 10

and Supplemental Figure 11) We observed no hyperresponsive induction by mock

treatment of PLANT DEFENSIN 12 (PDF12) a necrotrophy and late JA signaling

347 marker gene (Supplemental Figure 12A-D) PDF12 was also not induced by

348 macerozyme or OG treatments (Supplemental Figure 12A-D) This suggests that early

349 JA signaling is induced by mock-treatment in gae1 gae6 Because it is known that JA

350 and SA signaling act antagonistically (Pieterse et al 2012) the strong up-regulation of

351 JA-induced immune signaling in gae1 gae6 might explain the lower OG-induced SID2

352 expression in this genotype

353 To test whether other immune responses are altered in gae1 gae6 plants we

354 analyzed Mitogen-activated protein kinase (MAPK) activity following macerozyme and

355 OG treatments OGs are known to activate the MAPKs MPK3 and MPK6 in Arabidopsis

Page 21 of 54

(Galletti et al 2011) Macerozyme treatment activated MPK3 and MPK6 in wild-type 356

Col-0 whereas MAPK activation in gae1 gae6 was slightly reduced in four out of five 357

experiments performed (Supplemental Figure 13) By contrast OG-induced MAPK 358

activity was similar or increased in gae1 gae6 in four out of five experiments 359

(Supplemental Figure 13) Additionally mock treatment induced MAPK activity in gae1 360

gae6 more strongly than it did in wild-type Col-0 in four out of five experiments 361

performed (Fig S13) Collectively these results illustrate that activation of several 362

immune signaling componentsmdashcamalexin synthesis SA and JA signaling and 363

possibly MAPK activationmdashby macerozyme is substantially altered in gae1 gae6 This 364

suggests that release of soluble pectin fragments possibly OGs by pectinolytic 365

enzymes might be reduced in gae1 gae6 plants while recognition of OGs is intact 366

367

Following B cinerea Infection Production of Soluble Pectin and Deposition of 368

Callose Are Reduced in gae1 gae6 Plants 369

Although pad3 plants were more susceptible to the B cinerea isolates Fresa 525 370

and Gallo 1 which also grew better on gae1 gae6 growth of the isolate Pepper was 371

similar on Col-0 and pad3 (Supplemental Figure 9A) However we did not detect any 372

significant changes in camalexin production after treatment with B cinerea isolate Gallo 373

1 and Fresa 525 in gae1 gae6 (Figure 8D) Neither did we detect changes in PAD3 374

expression after treatment with any B cinerea isolate in gae1 gae6 (Figure 8E) 375

Additionally SID2 and PR1 were similarly induced by all B cinerea isolates in both Col-376

0 and gae1 gae6 (Figure 9E and F) and sid2 plants which are deficient for pathogen-377

induced SA biosynthesis did not show any differences in relative B cinerea growth 378

(Supplemental Figure 9B) Expression of JAZ10 was induced more strongly by Gallo 1 379

treatment in gae1 gae6 than in wild-type Col-0 but not by Fresa 525 or Pepper (Fig 380

10C) while dde2 plants deficient for JA biosynthesis showed enhanced susceptibility to 381

all B cinerea isolates tested (Supplemental Figure 9B) Curiously exogenous 382

application of MeJA did not lead to altered relative fungal growth of B cinerea 383

(Supplemental Figure 9C) and cev1 isoxaben resistant 1 (ixr1) plants which have 384

constitutively high JA levels (Ellis and Turner 2001) did not show any changes in 385

susceptibility to the B cinerea isolates tested (Supplemental Figure 9D) Hence neither 386

Page 22 of 54

differences in B cinerea isolate susceptibility to camalexin nor altered SA or JA 387

signaling by themselves explained the enhanced susceptibility of gae1 gae6 to these B 388

cinerea isolates 389

Hydrogen peroxide accumulation in response to B cinerea isolates Fresa 525 390

Gallo 1 and Pepper was not obviously altered in gae1 gae6 plants (Supplemental Figure 391

14) However callose deposition upon treatment with B cinerea isolate Gallo 1 was 392

strongly reduced in gae1 gae6 (Figure 11) It was previously shown that OG treatment 393

induces callose deposition (Denoux et al 2008) suggesting that the reduction in 394

callose deposition might be due to reduced OG release in gae1 gae6 It is possible that 395

this reduction in callose deposition contributes to the reduced immunity of gae1 gae6 396

plants to B cinerea isolate Gallo 1 397

Because macerozyme could be used to release soluble likely low molecular 398

weight pectin from isolated cell walls we next investigated whether infection with B 399

cinerea was accompanied by accumulation of soluble pectin as well We prepared cell 400

walls (AIR fraction) from leaves infected with B cinerea incubated these cell walls with 401

water and measured uronic acid content of the extracts B cinerea treatment did in fact 402

lead to increased accumulation of soluble pectin in wild-type Col-0 plants when 403

compared to mock-treated samples (Figure 12) No such increased accumulation of 404

water-soluble pectin fractions was observed in gae1 gae6 (Figure 12) 405

406

Macerozyme-Induced Pattern-Triggered Immunity against B cinerea Is Abolished 407

in gae1 gae6 408

It was previously shown that OG treatment can induce patternndashtriggered 409

immunity (PTI) to B cinerea (Ferrari et al 2007) We tested macerozyme treatment to 410

determine whether it could also induce PTI to the B cinerea isolate Gallo 1 (Figure 13) 411

Indeed pretreatment with 001 macerozyme for 24 h led to reduced B cinerea growth 412

in wild-type Col-0 but not gae1 gae6 plants (Figure 13A) Interestingly mock and 413

macerozyme pretreated gae1 gae6 plants exhibited similar growth of B cinerea Gallo 1 414

as did macerozyme-treated Col-0 plants This result is likely due to activation of JA 415

signaling in gae1 gae6 during the pretreatment procedure (Figure 13C and D) OG-416

induced PTI was observed in both genotypes when using a high dose of OGs (Figure 417

Page 23 of 54

13B) whereas a lower dose of OGs only induced PTI in Col-0 (Supplemental Figure 418

15) This suggests that OG perception in gae1 gae6 at least above a certain threshold 419

is unaltered while release of immunity-activating pectin fragments possibly OGs is 420

421 reduced We conclude that immune responses induced by macerozyme treatment

422 which do not occur in gae1 gae6 plants are important for plant immunity to the B

423 cinerea isolate Gallo 1

Page 24 of 54

____________________________________________________________________________

Page 25 of 54

DISCUSSION 426

Here we showed that loss of GAE1 and GAE6 leads to a dramatic reduction in 427

pectin content in the cell wall (Figure 4) likely causing the brittleness of leaves that we 428

observed (Figure 3) Petioles and midribs of gae1 gae6 plants broke easily when bent 429

and inoculation of leaves with deionized water resulted in increased ion leakage 430

possibly due either to microscopic fractures of the cell wall induced by inoculation stress 431

or to a reduced capability of these walls to withstand increased turgor pressure The 432

increased brittleness was associated with altered cell wall composition In our analysis 433

the GalA content of gae1 gae6 leaf walls was reduced by more than 40 and the total 434

uronic acids by over 60 (Figure 4 and Supplemental Figure 5) This is a major change 435

in the overall cell wall composition considering that pectin makes up about 50 of the 436

plant cell walls found in Arabidopsis leaves (Zablackis et al 1995 Nobuta et al 2007 437

Harholt et al 2010) Interestingly quasimodo1 (qua1) plants which have a mutation in 438

a glycosyltransferase family 8 gene are reduced in GalA by 25 and show reduced 439

binding of HG specific antibodies (Bouton et al 2002) 440

Furthermore qua2 plants which have a mutation in a putative methyltransferase 441

have about 13 less total GalA and lack 50 of cell wall HG (Mouille et al 2007) 442

Likewise cgr2 cgr3 plants carrying mutations in another class of Golgi-localized 443

putative methyl-transferases have leaf cell walls that are reduced by about 40 in 444

uronic acid (Kim et al 2015) All three mutants are severely dwarfed (Bouton et al 445

2002 Mouille et al 2007 Kim et al 2015) Additionally qua1 and qua2 mutants show 446

a distorted overall shape likely due to reduced cell cohesion and are extremely fragile 447

(Bouton et al 2002 Mouille et al 2007) By contrast gae1 gae6 plants with a similar 448

strong reduction in GalA and total uronic acids have an overall normal shape and are 449

only slightly smaller than wild-type Col-0 (Supplemental Figure 4) In the wall analysis 450

performed here we did not detect changes in other wall polysaccharides or structures 451

that might compensate for the reduction of pectin in gae1 gae6 It is possible the severe 452

morphological alterations in qua2 and cgr2 cgr3 plants are due to reduction in pectin 453

methylesterification which is unaffected in gae1 gae6 It was speculated that quality 454

control mechanisms in the Golgi prevent secretion of inefficiently esterified pectin in 455

cgr2 cgr3 (Kim et al 2015) suggesting that the Golgi of these plants might over-456

Page 26 of 54

accumulate pectin and that this may contribute to the severe dwarf phenotype of these 457

plants The gae1 gae6 plants described here provide an opportunity to study the effects 458

of reduction of pectin in plants that do not show severe morphological phenotypes 459

We found that gae1 gae6 plants are also compromised in resistance to B 460

cinerea and Pma ES4326 indicating that loss of pectin reduces immunity to these 461

pathogens (Figure 2 and 6) We found that gae1 gae6 plants were slightly more 462

susceptible to Pma ES4326 but showed a strong increase in susceptibility to specific B 463

cinerea isolates This was not unexpected because B cinerea is known to interact with 464

pectin as part of its infection strategy It secretes various pectin-degrading enzymes 465

early during infection and requires some of these enzymes including PG1 PG2 and 466

PME1 for full virulence (ten Have et al 1998 Valette-Collet et al 2003 Kars et al 467

2005b Espino et al 2010) Additionally Arabidopsis plants expressing an antisense 468

construct of the polygalacturonase inhibitor PGIP1 are significantly more susceptible to 469

B cinerea (Ferrari et al 2006) On the other hand Pma ES4326 is not known to470

require cell wall degrading enzymes for full virulence even though it encodes two 471

pectate lyase genes in its genome (PMA4326_07094 and PMA4326_04621 472

wwwbacteriaensemblorg) B cinerea isolates are also known to be genetically quite 473

diverse in their polygalacturonase (PG) loci and genetic variability in PG2 is associated 474

with varying growth on pectin containing media (Rowe and Kliebenstein 2007) 475

Furthermore the contribution of PG1 and PG2 to B cinerea virulence seems to depend 476

on the specific B cinerea isolate and the host species (Kars et al 2005a Zhang and 477

Van Kan 2013) B cinerea isolates are also known to be differently sensitive to the anti-478

microbial secondary metabolite camalexin (Kliebenstein et al 2005) In line with these 479

observations the susceptibility of gae1 gae6 plants differed depending on the specific 480

B cinerea isolate Whereas Fresa 525 Gallo 1 UK Razz and KT grew better in gae1481

gae6 we detected no difference in Acacia Apple 517 DN Grape Pepper or Rasp 482

growth (Figure 6) 483

B cinerea was shown to metabolize pectin and to possibly utilize GalA as a484

carbon source during in planta growth (Zhang et al 2011 Zhang and Van Kan 2013) 485

Our data show that gae1 gae6 plants which are reduced in GalA content did allow 486

enhanced growth of specific B cinerea isolates but no reduction in growth of any B 487

Page 27 of 54

cinerea isolates tested was found This suggests that utilization of pectin as a carbon 488

source was not a contributing factor to alterations in B cinerea susceptibility It is 489

possible that changes in the cell wall composition affect the structural integrity of the 490

wall and affect hyphal penetration of specific B cinerea isolates However OGs (small 491

HG fragments) are likely released during the infection process (Ferrari et al 2013) 492

OGs function as DAMPs and initiate immune responses in Arabidopsis (Ferrari et al 493

2013) Treatment of Arabidopsis with OGs having a degree of polymerization (DP) of 494

10-15 sugar subunits was previously shown to enhance resistance to B cinerea (Ferrari495

et al 2007) We showed that pretreatment with OGs or macerozyme induced enhanced 496

immunity to B cinerea isolate Gallo 1 in wild-type Col-0 but only OGs and not 497

macerozyme induced immunity in gae1 gae6 (Figure 13) Our data suggest gae1 gae6 498

plants which have reduced pectin content may release less OGs and that this 499

reduction leads to the increased susceptibility to B cinerea 500

Consistently we found that macerozyme treatment released less soluble pectin 501

from gae1 gae6 cell walls than from wild-type walls Fractions containing soluble pectin 502

released from gae1 gae6 were also less active in inducing PAD3 gene expression than 503

the fractions released from wild-type walls (Figure 7) We also showed that less soluble 504

pectin accumulated in gae1 gae6 than in wild-type plants 48 h after treatment with B 505

cinerea (Figure 12) 506

We observed that loss of pectin leads to alterations in A thaliana immune 507

signaling Macerozyme treatment induced expression of PAD3 and the SA marker 508

genes PR1 and SID2 in wild-type Col-0 but not in gae1 gae6 whereas OG treatment 509

led to increased PAD3 and SID2 expression in both wild-type and gae1 gae6 (Figure 8 510

and 9) This suggests that although release of active soluble pectin in these plants 511

might be altered OG recognition is intact In fact cell walls isolated from qua1 another 512

mutant known to be reduced in HG released less OGs (DP1-6) than did wild-type cell 513

walls upon endopolygalacturonase treatment (Bouton et al 2002) This was also true 514

for gae1 gae6 cell walls as macerozyme treatment of isolated gae1 gae6 cell walls 515

released less soluble pectin than treatment of Col-0 cell walls Because the fraction 516

from gae1 gae6 plants was also less active in inducing PAD3 expression (Figure 7) it 517

seems possible that biologically active OGs were the active component inducing PAD3 518

Page 28 of 54

expression Interestingly expression of the JA marker genes JAZ5 and JAZ10 was 519

higher in gae1 gae6 than in wild-type mock-inoculated plants but was comparable to 520

wild-type in untreated plants suggesting that JA signaling in gae1 gae6 is 521

hyperresponsive (Figure 10 and Supplemental Figure 11) This induction of JA signaling 522

might be due to wounding stress incurred during the inoculation process JA and SA 523

signaling generally act antagonistically (Pieterse et al 2012) Increased JA signaling 524

activity might hence explain why mock- and macerozyme-treated gae1 gae6 plants 525

exhibited reduced expression of the SA marker PR1 and why OG treatment induced 526

expression of SID2 another SA marker to a lower extent in gae1 gae6 than in wild-type 527

plants (Figure 9) 528

No single immune signaling component was found to be the sole contributor to 529

the compromised immunity of gae1 gae6 to specific B cinerea isolates such as Gallo 1 530

and Fresa 525 These isolates grew better on pad3 while growth of Pepper another B 531

cinerea isolate that did not show altered virulence on gae1 gae6 was unaltered 532

(Supplemental Figure 9) This suggested that differences in camalexin sensitivity of 533

these B cinerea isolates might account for the differences in gae1 gae6 susceptibility 534

However camalexin accumulation and PAD3 expression was the same for wild-type 535

Col-0 and gae1 gae6 for the B cinerea isolates Fresa 525 and Gallo 1 (Figure 8) which 536

showed enhanced growth on gae1 gae6 plants It should be noted that B cinerea 537

virulence is often estimated by comparing lesion sizes Lesion size and measurements 538

of relative fungal growth by qPCR do not always show the same results For example 539

although Denby et al (2004) showed that Pepper causes larger lesions on pad3 than on 540

wild-type this study showed that relative fungal growth of Pepper on pad3 was 541

unaltered 542

We observed that callose deposition was strongly reduced in gae1 gae6 plants 543

72 h after treatment with B cinerea isolate Gallo 1 (Figure 11) possibly due to reduced 544

release of active soluble pectin Interestingly OG-induced PTI to an unspecified isolate 545

of B cinerea was intact in the pmr4 mutant which does not produce callose (Galletti et 546

al 2008) Due to the high SA levels in pmr4 plants (Nishimura et al 2003) and the fact 547

that different B cinerea isolates vary both in induction of and reaction to defense 548

responses (Kliebenstein et al 2005 Rowe et al 2010) we could not determine 549

Page 29 of 54

whether reduced callose deposition causes the increased susceptibility of gae1 gae6 to 550

B cinerea isolate Gallo 1551

We did not observe any changes in B cinerea growth either on cev1 ixr1 plants 552

which are known to have constitutively high JA and OPDA levels (Ellis et al 2002b) or 553

on plants exogenously treated with MeJA (Supplemental Figure 9) Because plants 554

were kept uncovered for 3 h after MeJA treatment it is unclear how much MeJA was 555

bioavailable in the plant However JAZ10 expression was induced 3 h after spraying 556

with MeJA (Supplemental Figure 10) Because pathogen growth generally depends on 557

the combination of a multitude of immune signaling events we conclude that multiple 558

changes to the immune signaling in gae1 gae6 together account for the altered B 559

cinerea growth phenotypes detected 560

We showed that expression of GAE1 and GAE6 is repressed by the bacterial 561

hemibiotroph Pma ES4326 This repression did not occur in an eds1 or pad4 562

background or in pbs3 at the 24-h time point (Figure 1 and Supplemental Figure 1) 563

During the Arabidopsis - Pma ES4326 interaction the expression of many defense 564

related genes is altered in a PAD4EDS1 dependent fashion (Wang et al 2008) Some 565

of these expression changes require SA CBP60g and SARD1 are thought to act 566

downstream of PAD4 and EDS1 but upstream of SA-dependent responses (Wang et 567

al 2011) PBS3 is also thought to act downstream of PAD4 and EDS1 and upstream of 568

SA but the set of genes whose expression it affects is only partially overlapping with 569

those affected by CBP60gSARD1 (Wang et al 2008 Wang et al 2011) Thus GAE1 570

and GAE6 repression is regulated by the part of the immune signaling network that 571

requires PAD4 EDS1 and PBS3 but is independent of CBP60gSARD1 and SA 572

Because EDS1 PAD4 and PBS3 are important components of the plant immune 573

signaling network (Wiermer et al 2005 Nobuta et al 2007) one might conclude that 574

repression of GAE1 and GAE6 may be part of the plant immune response to Pma 575

ES4326 B cinerea treatment also reduced GAE1 and GAE6 expression but this was 576

mostly independent of PAD4 (Figure 6) while both Pma ES4326 and B cinerea repress 577

GAE1 and GAE6 expression the host signaling mechanisms responsible likely differ 578

In summary our data show that Arabidopsis GAE1 and GAE6 are required for 579

pectin biosynthesis leaf flexibility and immunity to Pma ES4326 and to specific B 580

Page 30 of 54

cinerea isolates We used macerozyme a commercial pectinase to mimic the effect of 581

B cinerea pectinases Macerozyme treatment released less pectin from gae1 gae6 cell582

walls and induced less immune signaling in gae1 gae6 plants Less water soluble pectin 583

also accumulated in B cinerea infected gae1 gae6 plants and macerozyme-induced 584

immunity to B cinerea isolate Gallo 1 was reduced in these plants We conclude that 585

the reduced pectin content in these mutants may lead to reduced release of soluble 586

pectin including active OGs during pathogen attack and that this compromises 587

immunity 588

589

590

Page 31 of 54

METHODS 591

Plant Materials and Growth Conditions 592

Wild-type Columbia (Col-0) and all mutant plants (in Col-0 background) were 593

grown on sterilized BM2 germinating mix (Berger Inc Quebec Canada) in a controlled 594

environment chamber (Conviron Pembina ND USA) with a 12-h photoperiod under 595

100 microM m-2 s-1 fluorescent illumination at 22degC and 75 relative humidity Germplasm 596

used is described in the Accession Numbers section at the end of Methods 597

598

Data Analysis and Replicates 599

For biological replicates samples were obtained from separate plant tissues and 600

in many cases from plants grown in different flats In some experiments multiple 601

technical replicates were measured per biological replicate For statistical analysis all 602

technical replicates were averaged and the averages were considered one biological 603

replicate Independent experiments were performed at different times and often 604

consisted of multiple biological replicates per experiment 605

Data analysis was performed using mixed linear models in the R programming 606

environment (Bates et al 2015) unless otherwise indicated Generally genotypes 607

treatments and time points were used as fixed effects and replicate-specific effects like 608

different pots flats etc were considered random effects We reported p-values 609

obtained from the mixed linear model when less than 10 comparisons were performed 610

and q-values for more than 10 comparisons to correct for multiple comparison testing 611

Q-values were calculated using the Benjamini-Hochberg procedure (Benjamini and612

Hochberg 1995) unless specified otherwise 613

614

Pathogen Strains Growth Conditions and Pathogen Growth Assays 615

Growth of Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) and 616

bacterial growth assays were performed as previously described (Bethke et al 2014) 617

For qRT-PCR assays four week-old plants were inoculated with bacteria (OD600 = 618

0002) or mock (5 mM MgSO4) At least four leaves from two plants were collected at 619

the indicated time points per biological replicate 620

Page 32 of 54

Prior to each experiment Botrytis cinerea isolates were grown on 1x PDA 621

medium (Difco) for 10 d at room temperature All isolates used have been described 622

previously (Rowe and Kliebenstein 2007) The spores were washed from the surface of 623

the plate using an inoculation medium (1x Gamborgrsquos B-5 basal salt mixture (Sigma-624

Aldrich G5768) 2 (wv) glucose 10 mM phosphate buffer pH 64) and fungal hyphae 625

were removed from the suspension by filtering through four layers of cheesecloth The 626

concentration of spores was determined using a hemocytometer and adjusted to 25 x 627

105 spores mL-1 Four week-old Arabidopsis plants were inoculated by placing one 10-628

microL droplet of the B cinerea spore solution or inoculation medium (mock) on the adaxial 629

leaf surface of fully expanded leaves Inoculated plants were kept at 100 relative 630

humidity Infection sites were collected at various time points using a cork borer For 631

qRT-PCR assays at least four leaf discs from one plant were combined for each 632

biological sample For B cinerea growth assays at least 12 leaf discs from three to six 633

plants were pooled per biological sample Relative fungal growth was determined by 634

measuring the abundance of a fungal gene relative to a plant gene as described below 635

In brief DNA from infected tissue was extracted and equal amounts of total DNA were 636

used to perform qPCR reactions using the B cinerea cutinase A and the Arabidopsis 637

SK11 gene respectively Primers were described in (Gachon and Saindrenan 2004) 638

and are listed in Supplemental Table 1 Two technical replicates were combined for 639

each biological replicate B cinerea is a necrotrophic fungal pathogen and may destroy 640

plant DNA as disease progresses However decreases in the amount of plant DNA 641

over the course of our experiments were only observed at late time points with very 642

susceptible plants so the qPCR assay is generally a reasonable estimate of fungal 643

biomass (Supplemental Figure 16) 644

645

Treatment with Macerozyme or OGs 646

For PTI assays four week-old plants either were inoculated with 001 (wv) 647

macerozyme R-10 (Yakult) a Rhizopus polygalacturonase (EC 32115) that possesses 648

high pectinase and hemicellulase activity or were mock inoculated using heat-649

inactivated macerozyme Other similar plants were either inoculated with 250 or 500 microg 650

mL-1 OGs or mock inoculated with water One d later plants were infected with B 651

Page 33 of 54

cinerea and samples were collected immediately (0 dpi) and two or three d later To 652

investigate the role of MeJA on B cinerea growth plants were sprayed with 1mM MeJA 653

in water with 001 (vv) Tween 20 Covers were removed so that leaves could dry off 654

before plants were inoculated with B cinerea three h later Samples were harvested at 655

the indicated time points For expression analysis and MAPK activation assays plants 656

were either inoculated with 100 microg mL-1 OGs or mock inoculated with water OGs used 657

for experiments described in Figures 8 to 10 and Supplemental Figure 12 were a kind 658

gift from Simone Ferrari OGs used in experiments described in Figure 13 and 659

Supplemental Figures 11 13 and 15 were prepared as previously described (Kohorn et 660

al 2014) 661

662

Expression Analysis 663

Leaf tissue from inoculated plants was harvested at the indicated time points 664

flash frozen pulverized and RNA extracted using Trizol (Invitrogen) Quantitative RT-665

PCR was performed using the SuperScript III Platinum SYBR Green One-Step 666

quantitative RT-PCR kit (Invitrogen) and a Lightcycler 480 Real-Time PCR system 667

(Roche) as previously described (Truman and Glazebrook 2012) In brief equal 668

amounts of total RNA and a gene specific primer were used for each reaction The 669

crossing point (Cp) was calculated using the 2nd derivative max method provided with 670

the Lightcycler software for each amplification curve Each reaction was run with two 671

technical replicates and the Cp values for these replicates were averaged ACTIN2 was 672

used as a stably expressed reference gene At least three biological replicates each 673

were performed and analyzed using a mixed-linear model RT-PCR was performed to 674

verify T-DNA insertion lines using the One-Step RT-PCR kit (Qiagen) according to the 675

manufacturerrsquos instructions The primers used can be found in Supplemental Table 1 676

677

Electrolyte Leakage Assay 678

Fully expanded leaves of four week-old plants were inoculated with water (Milli-Q 679

grade) using a needleless syringe Leaf discs were collected using a cork borer and 680

three leaf discs from one leaf were placed with the adaxial surface down onto 2 mL 681

water in a 12-well cell culture plate Conductivity was measured using a Horiba B-173 682

Page 34 of 54

conductivity meter after 30 min and then the water was replaced Conductivity was 683

measured again 30 min later (1 h total) as indicated in Figure 3C Six biological 684

replicates each were performed in three independent experiments and means and SEs 685

were calculated Two-sided t-tests were performed for each time point 686

687

DAB and Aniline Blue Staining 688

Leaves from four week-old plants were either mock inoculated with B cinerea 689

inoculation medium or inoculated with 10 microL droplets of a B cinerea spore solution (25 690

x 105 spores mL-1) of the indicated isolates Samples were collected 72 h later For 33prime-691

diaminobenzidine (DAB) staining two to five inoculated leaves were vacuum infiltrated 692

with 33prime-Diaminobenzidine (1 mg mL-1 in water pH 38) solution and stained overnight 693

in the dark Leaves were destained using 100 ethanol at 65degC and then transferred to 694

50 (vv) glycerol and photographed 695

Callose was visualized by staining leaves with aniline blue as previously 696

described (Adam and Somerville 1996) Briefly leaves were vacuum infiltrated with 697

alcoholic lactophenol (one volume of phenolglycerollactic acidwater (1111) mixed 698

with two volumes of ethanol) and destained at 65degC Next leaves were moved through 699

an alcohol gradient (50 20 and 10 (vv) ethanol) into water The destained leaves 700

were stained for 30 min in 150 mM K2HPO4 (pH 95) with 001 (wv) aniline blue 701

Samples were mounted in water and examined using a Nikon Eclipse Ni-U microscope 702

at 10x magnification using a DAPI filter (excitation at 325-375 nm emission at 435-485 703

nm) For callose quantification six 200 microm x 200 microm squares were counted for each 704

sample type Callose deposits per mm2 were calculated and data analyzed using a t-705

test Note that there is an inherent uncertainty associated with such counts because 706

callose deposits may not all be in focus at the same time 707

708

MAPK Activity Assay 709

Fully expanded leaves of four week-old plants were inoculated with 001 710

macerozyme 100 microg mL-1 OGs 100 nM flg22 or water (mock) flg22 a peptide from 711

bacterial flagella that induces immune responses was used as a positive control for 712

Page 35 of 54

MAPK activation (Asai et al 2002) and was purchased from EZBiolab Inc (Westfield 713

IN USA) Samples were harvested 10 min later and immediately frozen in liquid 714

nitrogen Protein was extracted using an extraction buffer containing 25 mM Tris pH 78 715

75 mM NaCl 1 mM DTT 1 mM NaF 05mM Na3VO4 01 (vv) Tween 20 one 716

cOmplete Mini protease inhibitor cocktail tablet (Roche) and one PhosSTOP 717

phosphatase inhibitor cocktail tablet (Roche) per 10 mL buffer 15 microg of total protein 718

were electrophoresed on a 10 SDS-polyacrylamide gel and then blotted onto a PVDF 719

membrane (Bio-Rad 162-0177) Activated MAPKs were detected using p4442 MAPK 720

(Erk12) antibody (9102S Cell Signal 12500 in TBST (20 mM Tris pH 75 150 mM 721

NaCl 01 (vv) Tween 20)) Anti-Rabbit IgG-HRP (115000 in TBST A6154 Sigma) 722

and ECL Plus substrate (Pierce 32132) MPK3 and MPK6 were detected with an anti-723

MPK3 antibody (M8318 Sigma 12000 in TBST and 3 (wv) milk) or an anti-MPK6 724

antibody (A7104 Sigma 16000 in TBST and 3 (wv) milk) respectively The 725

secondary antibody and detection were the same as for the p4442 antibody Five 726

independent experiments were performed 727

728

Preparation of Alcohol Insoluble Residue (AIR) Pectin Extraction and Uronic Acid 729

Measurements 730

Fully expanded leaves from four week-old plants that were incubated for 48 h in 731

the dark to reduce starch were harvested flash frozen and pulverized AIR was 732

extracted by washing ground material twice in 70 (vv) ethanol three times in a 733

mixture of chloroform and methanol (11 vv) and once in acetone (Gille et al 2009) 734

and was then air dried Cell wall pectin was extracted from AIR with cell wall extraction 735

buffer (50 mM Trizma 50 mM CDTA pH 72) at 95degC for 15 min Samples were 736

homogenized using a paint shaker (Harbil 5G-HD) and glass beads (3 mm diameter) 737

For dot blot experiments 500 microL of cell wall extraction buffer were used per 10 mg of 738

AIR Debris was precipitated by centrifugation for 10 min at 10000 g 739

Total uronic acid content of pectin extracted as described above using 500 microL of 740

cell wall extraction buffer per 1 mg of AIR was measured as described previously 741

(Filisetti-Cozzi and Carpita 1991 van den Hoogen et al 1998) First 36 microL of pectin 742

extract were mixed with 4 microL of 4 M sulfamic acid Then 200 microL of sulfuric acid 743

Page 36 of 54

containing 120 mM sodium tetraborate was added and samples were incubated at 80degC 744

for 1 h Following cooling on ice the optical density of samples was measured at 490 745

nm Next 40 microL of m-hydroxydiphenyl reagent (100 microL of m-hydroxydiphenyl in dimethyl 746

sulfoxide at 100 mgmL mixed with 49 mL 80 (vv) sulfuric acid just before use) was 747

added and the samples were mixed The optical density at 490 nm was measured 748

again The optical density before m-hydroxydiphenyl reagent addition was subtracted 749

from the optical density measured after addition of the dye The concentration of uronic 750

acid was calculated using known amounts of GalA as a standard 751

752

Release of Pectin from Arabidopsis Cell Walls Using Macerozyme 753

AIR preparations from leaves of four week-old plants were treated with 025 754

(wv) macerozyme R-10 (Yakult) or water (mock) at a concentration of 100 microL per mg 755

AIR Samples were incubated for 3 h at room temperature on a vortex shaker and 756

centrifuged for 10 min at 12000 g Total uronic acid concentration of 5-fold diluted 757

supernatants was determined for two technical replicates each The concentration of 758

uronic acid was calculated using known amounts of GalA as a standard For 759

subsequent qRT-PCR experiments supernatants from multiple extractions were 760

combined boiled for 20 min to inactivate macerozyme diluted 10-fold with water and 761

inoculated into four week-old Col-0 plants Heat-inactivated 10-fold diluted macerozyme 762

solutions were used as mock treatment Samples were collected 3 h later and PAD3 763

expression was determined 764

765

Release of Pectin from Arabidopsis Leaves Infected with B cinerea 766

Fully expanded leaves of four week-old plants were either inoculated with up to 767

five 10 microL droplets of B cinerea spore solution (25 x 105 spores mL-1) or mock 768

inoculated with B cinerea inoculation medium For each biological replicate leaves with 769

a combined count of at least 20 infection sites were collected 48 h later AIR was 770

extracted as described above but plants were not dark-treated or freeze-dried and the 771

first ethanol step was performed using 85 (vv) ethanol To extract soluble pectin 772

fragments AIR samples were mixed with water at a concentration of 20 microL per infection 773

site incubated for 7 h at room temperature on a vortex shaker and centrifuged for 10 774

Page 37 of 54

min at 12000 g Total uronic acid concentration of supernatants was determined for two 775

technical replicates each The concentration of uronic acid was calculated using known 776

amounts of GalA as a standard 777

778

Measurement of Uronic Acids Neutral Monosaccharides and Cellulose Content in 779

Cell Walls 780

Neutral sugars and uronic acids from non-cellulosic polysaccharides in cell walls 781

(AIR) were released by trifluoroacetic acid (TFA) hydrolysis Approximately 1 mg of AIR 782

was hydrolyzed with 2 M TFA at 121degC for 90 min The TFA-soluble material was split 783

into two equal parts one for uronic acid and one for neutral sugar determination The 784

uronic acid content was determined by analyzing the hydrolyzate using a CarboPac 785

PA200 anion-exchange column with an ICS-3000 Dionex chromatography system 786

(Dionex Sunnyvale CA USA) The elution profile consisted of a linear gradient of 50 to 787

200 mM sodium acetate in 01 M NaOH in 10 min at 04 mL per min Alditol acetate 788

derivatives of neutral sugars were produced as described (York et al 1985) In brief 789

monosaccharides in the hydrolyzate were reduced with NaBH4 and peracetylated with 790

acetic anhydride and pyridine at 121degC for 20 min The generated alditol acetates were 791

analyzed by GC-MS Crystalline cellulose was measured according to Updegraff (1969) 792

After hydrolysis of noncellulosic polysaccharides from AIR with the Updegraff reagent 793

(acetic acid nitric acids water 812 (vv) ) the remaining pellet was hydrolyzed in 794

72 sulfuric acid The resulting glucose quantity was determined by the anthrone 795

method (Scott and Melvin 1953) 796

797

Dot Blot Experiments 798

Pectin solutions extracted using 50 microL of cell wall extraction buffer per mg AIR 799

were serially diluted and nitrocellulose membranes were spotted with 1 microL of the diluted 800

pectin solutions Membranes were dried overnight blocked with 5 milk (wv) in 1x 801

PBS (8 g L-1 NaCl 02 g L-1 KCl 144 g L-1 Na2HPO4 024 g L-1 KH2PO4 pH 74) and 802

probed with LM5 (Jones et al 1997) LM6 (Willats et al 1998) LM8 (Willats et al 803

2004) LM19 and LM20 (Verhertbruggen et al 2009a) or CCRC-M7 (Steffan et al 804

Page 38 of 54

1995) antibodies LM series antibodies were diluted 1250 and CCRC-M7 was diluted 805

1500 in 5 milk powder (Nestle Carnation) in 1 x PBS For LM series antibodies a 806

goat anti-Rat HRP conjugated antibody (Bethyl A110-105P 15000 diluted) and for 807

CCRC-M7 an anti-Mouse IgG HRP conjugate (Promega W402B 12500 diluted) was 808

used as secondary antibody in 5 milk powder in 1x PBS LM series antibodies were 809

obtained from plant probes (wwwplantprobesnet) and CCRC-M7 from CarboSource 810

(wwwcarbosourcenet) Membranes were washed after incubation with the primary and 811

secondary antibodies with 1x PBS Dot blots were developed using the ECL system 812

(GE Healthcare Amersham ECL prime) For quantification of signals dot blot results on 813

x-ray films were photographed using a CCD camera and intensities were measured814

using Image Jrsquos Integrated Density function Locally adjusted background was 815

subtracted As the dynamic range of the signal on an x-ray film was narrow non-816

linearity in the measured values was corrected for each genotype in each biological 817

replicate using the values from different dilutions in two technical replicates We noticed 818

that the residuals were not normally distributed and performed Box-Cox power 819

transformation (Box and Cox 1964) to obtain normally distributed values for the 820

subsequent statistical analysis of the data For LM19 Box-Cox selected power 821

transformation was done to the power of 038th and for LM20 to the power 075th After 822

correction of the transformed measured values with the effect of the biological 823

replicates the values were fit to a linear model with the Arabidopsis genotype as the 824

fixed effect This linear model was used both to generate the plots in Figures 5B and D 825

and to test significant differences 826

827

Camalexin Measurements 828

Four week-old plants were infected with B cinerea and samples (three leaf disks 829

from one plant each) were harvested at the indicated time points flash frozen and 830

pulverized Each sample was extracted with 300 microL of 90 methanol (vv) 50 microL of the 831

extract was run on an Agilent Lichrocart 250-4 RP18e 5-microm column using an Agilent 832

1100 series HPLC Camalexin was detected using a DiodeArray (DAD) at 330 nm and 833

with a fluorescence detector at emission 318 nmexcitation 385 nm (Agilent) Separation 834

was achieved using the following program with aqueous acetonitrile 5 min gradient 835

Page 39 of 54

from 63 to 69 acetonitrile 30 s gradient from 69 to 99 acetonitrile 2 min at 99 836

acetonitrile and a post-run equilibration of 35 min at 63 acetonitrile (Denby et al 837

2004 Kliebenstein et al 2005) Purified camalexin was used to produce a standard 838

curve to identify and quantitate camalexin 839

840

Accession Numbers 841

Microarray data used can be accessed at NCBI Gene Expression Omnnibus 842

(httpwwwncbinlmnihgovgeo) GSE18978 Sequence information for all Arabidopsis 843

genes described in this article can be found in TAIR (wwwarabidopsisorg) using the 844

following accession numbers PAD3 (CYP71B15 At3g26830) SID2 (ICS1 At1g74710) 845

GAE1 (At4g30440) GAE2 (At1g02000) GAE3 (At4g00110) GAE4 (At2g45310) GAE5 846

(At4g12250) GAE6 (At3g23820) PR1 (At2g14610) JAZ5 (At1g17380) JAZ10 847

(At5g13220) ACTIN2 (At3g18780) PDF12a (At5g44420) PDF12b (At2g26020) 848

AtSK11 (At5g26751) The sequence of the B cinerea cutinase Bc-CutA (Z69264) can 849

be found in the DNA Data Bank of Japan (httpgetentryddbjnigacjp) Germplasm 850

used included dde2-2 (At5g42650 (von Malek et al 2002)) pad3-1 (At3g26830 (Zhou 851

et al 1999)) pad4-1 (At3g52430 (Jirage et al 1999)) sid2-2 (At1g74710 (Wildermuth 852

et al 2001)) pbs3-2 (At5g13320 SALK_018225 (Nobuta et al 2007)) cbp60a-1 853

(At5g62570 SALK_124410 (Truman et al 2013)) cbp60g-1 sard1-2 (At5g26920 854

SALK_023199 At1g73805 SALK_052422 (Wang et al 2011) cbp60a-1 cbp60g-1 855

sard1-2 (Truman et al 2013) gae1-1 (At4g30440 SALK_085554) gae6-1 (At3g23280 856

SALK_104454) gae6-2 (At3g23280 SALK_017191) ixr1-1 cev1 (At5g05170 (Ellis 857

and Turner 2001 Scheible et al 2001)) eds1 plants were derived by introgression of 858

the Ler eds1-2 allele into Col-0 that contains two copies of EDS1 (At3g48090 859

At3g48080 (Bartsch et al 2006) mpk6-2 (At2g43790 SALK_073907 (Liu and Zhang 860

2004)) mpk3-DG (At3g45640 a fast neutron deletion mutant (Miles et al 2005)) and 861

npr1-1 (At1g64280 (Cao et al 1997)) T-DNA insertion lines were part of the SALK 862

collection (Alonso et al 2003) and were obtained from the Arabidopsis Biological 863

Resource Center 864

865

Supplemental Data 866

Page 40 of 54

Supplemental Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 867

Expression Does Not Require CBP60s or SID2 868

Supplemental Figure 2 Expression of GAE Family Members uponTreatment with Pma 869

ES4326 870

Supplemental Figure 3 Characterization of T-DNA Insertions in GAE1 and GAE6 871

Supplemental Figure 4 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves and 872

Are Slightly Smaller than Wild-Type Col-0 Plants 873

Supplemental Figure 5 Cell Walls of gae1 gae6 Plants Contain Less Uronic Acid than 874

Wild-Type Col-0 Cell Walls 875

Supplemental Figure 6 Antibodies Raised Against the RGI or XG Components of 876

Pectin Show No Difference in Binding to gae Mutant and Col-0 Cell Walls 877

Supplemental Figure 7 The Effect of B cinerea Treatment on the Expression of GAE 878

Family Genes 879

Supplemental Figure 8 Treatment with 001 Macerozyme Does Not Cause Any 880

Visible Damage to Col-0 Plants 881

Supplemental Figure 9 B cinerea Growth on Plants with Defects in Immune 882

Signaling 883

Supplemental Figure 10 MeJA-Induced Expression of the JA marker gene JAZ10 884

Supplemental Figure 11 The Expression of the JA Marker Gene JAZ5 Is Highly 885

Responsive in gae1 gae6 Plants 886

Supplemental Figure 12 The Expression of PDF12 Is Unaltered in gae1 gae6 Plants 887

Supplemental Figure 13 Macerozyme Treatment Activates MPK3 and MPK6 888

Supplemental Figure 14 B cinerea-Induced ROS Production in Wild-Type Col-0 and 889

gae1 gae6 Plants 890

Supplemental Figure 15 OG-Induced PTI Using 250 microg mL-1 OGs 891

Supplemental Figure 16 Abundance of Plant DNA in B cinerea Treated Plants 892

Supplemental Table 1 Primers used in this study 893

894

ACKNOWLEDGEMENTS 895

We thank William Truman for helpful discussions Simone Ferrari (Sapienza 896

Universita di Roma) for the generous gift of OGs Bruna Bucciarelli (USDA-ARS) for 897

Page 41 of 54

help with fluorescence microscopy the Arabidopsis Biological Resource Center for T-898

DNA insertion lines Paul Knoxrsquos lab (University of Leeds) for LM5 LM6 LM8 LM19 899

and LM20 antibodies and the Complex Carbohydrate Resource Center (University of 900

Georgia) for the CCRC-M7 antibody Most of this work was funded by the Division of 901

Chemical Sciences Geosciences and Biosciences Office of Basic Energy Sciences of 902

the US Department of Energy through Grant DE-FG02-05ER15670 to JG This work 903

was also supported by NSF awards IOS 1339125 and IOS 1021861 to DJK NSF 904

award IOS 1121425 to F K and the USDA National Institute of Food and Agriculture 905

Hatch project number CA-D-PLS-7033-H to DJK 906

907

AUTHOR CONTRIBUTIONS 908

GB and JG designed the research GB GX AT BL NES and NH 909

performed research DJK provided tools GB GX MP AD BL RAH and FK 910

analyzed data GB and JG wrote the paper with input from all authors 911

912

FIGURE LEGENDS 913

Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 Expression 914

Requires PAD4 EDS1 and PBS3 915

(A) Expression of GAE1 in leaves of wild-type Col-0 and mutant plants Expression was916

measured 24 or 32 h after either inoculation with Pma ES4326 (OD600 = 0002) or mock 917

inoculation with 5 mM MgSO4 and was then normalized to the level of ACTIN2 Data 918

from three biological replicates were combined using a mixed linear model Bars 919

represent mean log2-ratios to ACTIN2 plusmn SE Letters indicate significantly different 920

groups with q lt 005 where 005 represents the maximum false discovery rate at which 921

the test may be called significant 922

(B) Expression of GAE6 measured as described in (A)923

924

Figure 2 Susceptibility of gae6 and gae1 gae6 Plants to Pma ES4326 Is Increased 925

Relative to Wild-Type Col-0 926

Leaves of plants of the indicated genotypes were inoculated with Pma ES4326 (OD600 = 927

00002) as described in Methods Bacterial titers in leaves were determined either 928

Page 42 of 54

immediately (0 dpi) or after three days (3 dpi) Bars represent the mean plusmn SE of four 929

independent experiments each with 4 or 12 biological replicates at 0 and 3 dpi 930

respectively Data were combined using a mixed linear model Asterisks indicate 931

significant differences from Col-0 wild-type (q lt 005) Susceptible pad4 plants were 932

included as a positive control 933

934

Figure 3 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves 935

(A) Depiction of the assay for brittleness of gae mutant leaves Fully expanded leaves of936

four to five week-old plants were bent by 180ordm so that the adaxial surface of the leaf was 937

parallel with the soil Broken leaf petioles or midribs were considered brittle 938

(B) Quantification of broken midribs or petioles For each genotype 66 to 82 leaves 939

were analyzed for brittleness as described in (A) Bars represent the ratio of broken to 940

total number of leaves tested Results were analyzed using Fisherrsquos exact test Letters 941

indicate significantly different groups at p lt 005 942

(C) Conductivity measurements Leaves of four week-old plants were inoculated with943

deionized water and then leaf discs were cut out and placed on deionized water 944

Conductivity was measured at the indicated time points After 30 min the water was 945

replaced with fresh water (30 min f w) to account for wounding-related ion leakage from 946

the edges of the leaf discs Three independent experiments with six biological replicates 947

each were performed and data were analyzed using a t-test for each time point P-948

values were corrected using the Bonferroni method Data show means plusmn SE asterisks 949

indicate significant differences from wild-type Col-0 at q lt 001 950

951

Figure 4 Cell Walls of gae6 and gae1 gae6 Plants Are Reduced in GalA 952

(A) to (D) Levels of GalA (A) GlcA (B) cellulose (C) and neutral sugars (D) in alcohol953

insoluble residue (AIR) extracted from leaves of four week-old plants of the indicated 954

genotypes Bars represent means plusmn SE of four biological replicates combined using a 955

mixed linear model Letters indicate significantly different groups (p lt 005) 956

957

Figure 5 Cell Walls of gae1 gae6 Plants Are Reduced in Homogalacturonan 958

Page 43 of 54

Dot blot analyses with anti-LM19 and anti-LM20 antibodies Pectin was extracted from 959

AIR prepared from the indicated genotypes using 1 microL of extraction buffer per 10 microg of 960

AIR Pectin was serially diluted and one microL of undiluted 13 diluted and 19 diluted 961

samples were spotted on a nitrocellulose membrane Three biological replicates were 962

were spotted twice to obtain two technical replicates each All replicates produced 963

similar results 964

(A) Representative dot blot using anti-LM19 antibody965

(B) Relative signal intensities of the spots in the dot blot in (A)966

(C) Representative dot blot using anti-LM20 antibody967

(D) Relative signal intensities of the LM20 dot blot shown in (C)968

Relative signal intensities were measured using Image J software Measurements from 969

the three biological replicates were combined using a linear model after Box-Cox power 970

transformation as described in Methods Bars represent means plusmn SE Asterisks indicate 971

a difference from wild-type Col-0 at p lt 005 972

973

Figure 6 Growth of B cinerea on gae1 gae6 Plants and the Effect of B cinerea on 974

Expression of GAE1 and GAE6 975

(A) Growth of B cinerea isolates Leaves of Col-0 and gae1 gae6 plants were976

inoculated with 25 x 105 spores mL-1 of the B cinerea isolates indicated in the figure 977

Samples were collected two (2 dpi) and three (3 dpi) days later Bars represent means plusmn 978

SE of six biological replicates for each fungus combined using a mixed linear model 979

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 980

(B) and (C) Expression of GAE1 (B) and GAE6 (C) 48 h after inoculation with the B981

cinerea isolates indicated Expression levels were measured by qRT-PCR Bars 982

represent mean log2-ratios to ACTIN2 plusmn SE of three biological replicates combined 983

using a mixed linear model Letters indicate significantly different groups (q lt 005) 984

985

Figure 7 Levels of Soluble Pectin and Expression of PAD3 in Fractions Produced 986

Following Macerozyme Treatment of gae1 gae6 Cell Walls 987

(A) Schematic overview of the experimental procedure for (B) and (C)988

Page 44 of 54

(B) Uronic acid measurements Alcohol-insoluble residue (AIR) from leave tissue of four989

week-old wild-type Col-0 or gae1 gae6 plants was treated with water or 025 990

macerozyme and assayed for uronic acid levels Data from 11 biological replicates were 991

combined using a mixed linear model Bars represent means plusmn SE Letters indicate 992

significant differences (p lt 005) 993

(C) Expression of PAD3 in Col-0 plants treated with the fractions from (B) Extracts from994

(B) were heat inactivated pooled and diluted 5-fold with water Pooled extracts were995

inoculated into four week-old Col-0 plants PAD3 expression was measured 3 h after 996

inoculation and normalized to the level of ACTIN2 expression Data from four biological 997

replicates were combined using a mixed linear model Bars represent mean log2-ratios 998

to ACTIN2 plusmn SE Letters indicate significant differences (p lt 005) 999

1000

Figure 8 Macerozyme-Induced PAD3 Expression Is Absent in gae1 gae6 1001

(A) Expression of PAD3 after macerozyme treatment Four week-old plants were either1002

inoculated with 001 macerozyme or mock inoculated with water and PAD3 1003

expression was measured 3 h later by qRT-PCR Data from four biological replicates 1004

were combined using a mixed linear model Mean log2-ratios to ACTIN2 plusmn SE were 1005

plotted Letters indicate significantly different groups (p lt 005) 1006

(B) Expression of PAD3 after OG treatment PAD3 expression was measured as1007

described in (A) 3 h after inoculation with 100 microg mL-1 OGs or mock inoculation with 1008

water Data are from six biological replicates 1009

(C) PAD3 expression in untreated plants Three biological replicates were performed1010

and data analyzed as described in (A) 1011

(D) Camalexin accumulation after B cinerea treatment Camalexin accumulation was1012

measured in four week-old plants inoculated with the B cinerea strains shown in the 1013

figure Eight biological replicates each were combined using a mixed linear model 1014

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 1015

(E) PAD3 expression 28 h after treatment with the indicated B cinerea strains Data1016

from six biological replicates were collected and analyzed as described in (A) Data in 1017

all figure parts represent means plusmn SE Letters indicate significantly different groups (q lt 1018

005) 1019

Page 45 of 54

1020

Figure 9 Macerozyme-Induced Expression of the SA Marker Genes SID2 and PR1 1021

Is Abolished in gae1 gae6 Plants 1022

(A) to (H) Expression of PR1 (A) (C) (E) and (G) or SID2 (B) (D) (F) and (H) in four1023

week-old Col-0 and gae1 gae6 plants Data were combined using a mixed linear model 1024

Bars represent mean log2-ratios of expression vs ACTIN2 plusmn SE for all figure parts 1025

Letters indicate significantly different groups at p lt 005 for panels (A) to (D) (G) and 1026

(H) and qlt005 for panels (E) to (F) 1027

(A) and (B) Plants were treated with 001 macerozyme or mock treated with water1028

and samples were collected 3 h later Data from four biological replicates are shown 1029

(C) and (D) Plants were inoculated with 100 microg mL-1 OGs or mock treated with water1030

and samples collected 3 h later Data from six biological replicates are shown 1031

(E) and (F) Plants were inoculated with the indicated B cinerea isolates or mock1032

inoculated with B cinerea inoculation medium and samples collected 48 h later Data 1033

from eight biological replicates are shown 1034

(G) and (H) Expression in untreated plants Three biological replicates were performed1035

1036

Figure 10 The Expression of the JA Marker Gene JAZ10 is Highly Responsive in 1037

gae1 gae6 Plants 1038

(A) to (D) JAZ10 expression in four week-old plants Data were combined using a mixed1039

linear model Mean log2-ratios to ACTIN2 plusmn SE are shown Letters indicate significantly 1040

different groups at p lt 005 for panels (A) (B) and (D) and q lt 005 for panel (C) 1041

(A) Plants were inoculated with 001 macerozyme or mock inoculated using water and1042

samples were collected 3 h later Data from four biological replicates are shown 1043

(B) Plants were inoculated with 100 microg mL-1 OGs or mock inoculated using water and1044

samples collected 3 h later Data are from six biological replicates 1045

(C) JAZ10 expression 48 h after treatment with the indicated B cinerea strains Data1046

are from six biological replicates 1047

(D) JAZ10 expression in untreated plants Data are from three biological replicates1048

1049

Figure 11 Gallo 1-Induced Callose Deposition Is Reduced in gae1 gae6 Plants 1050

Page 46 of 54

(A) Callose deposition in four week-old Col-0 and gae1 gae6 plants Plants were1051

inoculated with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) or mock inoculated 1052

with B cinerea inoculation medium and samples were collected after 72 h Callose was 1053

stained with aniline blue and visualized using a Nikon Eclipse Ni-U microscope with a 1054

DAPI filter Three leaves were stained per treatment Scale bars are 100 microm Asterisks 1055

indicate fungal hyphae arrows indicate representative callose spots 1056

(B) Quantification of callose deposition Callose deposits were counted in 200 microm x 2001057

microm squares laid out across the images shown here Bars represent means plusmn SE from 1058

six squares each Letters indicate significantly different groups (p lt 005) according to a 1059

t-test analysis1060

1061

Figure 12 Less Soluble Pectin Accumulates in B cinerea-Infected gae1 gae6 1062

Leaves 1063

Four week-old Col-0 and gae1 gae6 plants were inoculated with 10 microL droplets of the 1064

indicated B cinerea isolate (25 x 105 spores mL-1) or mock inoculated with B cinerea 1065

inoculation medium Leaves were collected 48 h later AIR was extracted from leaf 1066

samples and water added (20 microL infection site) to extract soluble pectin fragments 1067

Samples were vortexed for 7 h centrifuged at 12000 g and the uronic acid 1068

concentration of the supernatant was measured Data from nine biological replicates 1069

were combined using a mixed linear model Means plusmn SE are shown Letters indicate 1070

significant differences (q lt 005) 1071

1072

Figure 13 Macerozyme-Induced PTI Is Lost in gae1 gae6 While OG-Induced PTI Is 1073

Unaltered 1074

(A) Macerozyme-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1075

inoculated with 001 macerozyme or mock inoculated with boiled macerozyme and 1076

were infected with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) 24 h later Samples 1077

were collected immediately (0 dpi) and after two (2 dpi) and three (3 dpi) days Data 1078

show mean plusmn SE of six to 14 biological replicates each combined using a linear model 1079

Letters indicate significantly different groups (q lt 005) 1080

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

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Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk RGadrinab C Heller C Jeske A Koesema E Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt MHazari N Hom E Karnes M Mulholland C Ndubaku R Schmidt I Guzman P Aguilar-Henonin L Schmid M Weigel DCarter DE Marchand T Risseeuw E Brogden D Zeko A Crosby WL Berry CC and Ecker JR (2003) Genome-wideinsertional mutagenesis of Arabidopsis thaliana Science 301 653-657

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Bethke G Grundman RE Sreekanta S Truman W Katagiri F and Glazebrook J (2014) Arabidopsis PECTINMETHYLESTERASEs Contribute to Immunity against Pseudomonas syringae Plant Physiol 164 1093-1107

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Bouton S Leboeuf E Mouille G Leydecker MT Talbotec J Granier F Lahaye M Hofte H and Truong HN (2002)Quasimodo1 encodes a putative membrane-bound glycosyltransferase required for normal pectin synthesis and cell adhesion inArabidopsis Plant Cell 14 2577-2590

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Cao H Glazebrook J Clarke JD Volko S and Dong XN (1997) The Arabidopsis NPR1 gene that controls systemic acquiredresistance encodes a novel protein containing ankyrin repeats Cell 88 57-63

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Carpita N (2010) Update on Mechanisms of Plant Cell Wall Biosynthesis How plants make cellulose and other (1-gt4)-beta-D-glycans Plant Physiol

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Chisholm ST Coaker G Day B and Staskawicz BJ (2006) Host-microbe interactions Shaping the evolution of the plantimmune response Cell 124 803-814

Page 19 of 54

Page 20 of 54

344

345

346

and JAZ10 was already strongly induced in gae1 gae6 after mock inoculation (Figure 10

and Supplemental Figure 11) We observed no hyperresponsive induction by mock

treatment of PLANT DEFENSIN 12 (PDF12) a necrotrophy and late JA signaling

347 marker gene (Supplemental Figure 12A-D) PDF12 was also not induced by

348 macerozyme or OG treatments (Supplemental Figure 12A-D) This suggests that early

349 JA signaling is induced by mock-treatment in gae1 gae6 Because it is known that JA

350 and SA signaling act antagonistically (Pieterse et al 2012) the strong up-regulation of

351 JA-induced immune signaling in gae1 gae6 might explain the lower OG-induced SID2

352 expression in this genotype

353 To test whether other immune responses are altered in gae1 gae6 plants we

354 analyzed Mitogen-activated protein kinase (MAPK) activity following macerozyme and

355 OG treatments OGs are known to activate the MAPKs MPK3 and MPK6 in Arabidopsis

Page 21 of 54

(Galletti et al 2011) Macerozyme treatment activated MPK3 and MPK6 in wild-type 356

Col-0 whereas MAPK activation in gae1 gae6 was slightly reduced in four out of five 357

experiments performed (Supplemental Figure 13) By contrast OG-induced MAPK 358

activity was similar or increased in gae1 gae6 in four out of five experiments 359

(Supplemental Figure 13) Additionally mock treatment induced MAPK activity in gae1 360

gae6 more strongly than it did in wild-type Col-0 in four out of five experiments 361

performed (Fig S13) Collectively these results illustrate that activation of several 362

immune signaling componentsmdashcamalexin synthesis SA and JA signaling and 363

possibly MAPK activationmdashby macerozyme is substantially altered in gae1 gae6 This 364

suggests that release of soluble pectin fragments possibly OGs by pectinolytic 365

enzymes might be reduced in gae1 gae6 plants while recognition of OGs is intact 366

367

Following B cinerea Infection Production of Soluble Pectin and Deposition of 368

Callose Are Reduced in gae1 gae6 Plants 369

Although pad3 plants were more susceptible to the B cinerea isolates Fresa 525 370

and Gallo 1 which also grew better on gae1 gae6 growth of the isolate Pepper was 371

similar on Col-0 and pad3 (Supplemental Figure 9A) However we did not detect any 372

significant changes in camalexin production after treatment with B cinerea isolate Gallo 373

1 and Fresa 525 in gae1 gae6 (Figure 8D) Neither did we detect changes in PAD3 374

expression after treatment with any B cinerea isolate in gae1 gae6 (Figure 8E) 375

Additionally SID2 and PR1 were similarly induced by all B cinerea isolates in both Col-376

0 and gae1 gae6 (Figure 9E and F) and sid2 plants which are deficient for pathogen-377

induced SA biosynthesis did not show any differences in relative B cinerea growth 378

(Supplemental Figure 9B) Expression of JAZ10 was induced more strongly by Gallo 1 379

treatment in gae1 gae6 than in wild-type Col-0 but not by Fresa 525 or Pepper (Fig 380

10C) while dde2 plants deficient for JA biosynthesis showed enhanced susceptibility to 381

all B cinerea isolates tested (Supplemental Figure 9B) Curiously exogenous 382

application of MeJA did not lead to altered relative fungal growth of B cinerea 383

(Supplemental Figure 9C) and cev1 isoxaben resistant 1 (ixr1) plants which have 384

constitutively high JA levels (Ellis and Turner 2001) did not show any changes in 385

susceptibility to the B cinerea isolates tested (Supplemental Figure 9D) Hence neither 386

Page 22 of 54

differences in B cinerea isolate susceptibility to camalexin nor altered SA or JA 387

signaling by themselves explained the enhanced susceptibility of gae1 gae6 to these B 388

cinerea isolates 389

Hydrogen peroxide accumulation in response to B cinerea isolates Fresa 525 390

Gallo 1 and Pepper was not obviously altered in gae1 gae6 plants (Supplemental Figure 391

14) However callose deposition upon treatment with B cinerea isolate Gallo 1 was 392

strongly reduced in gae1 gae6 (Figure 11) It was previously shown that OG treatment 393

induces callose deposition (Denoux et al 2008) suggesting that the reduction in 394

callose deposition might be due to reduced OG release in gae1 gae6 It is possible that 395

this reduction in callose deposition contributes to the reduced immunity of gae1 gae6 396

plants to B cinerea isolate Gallo 1 397

Because macerozyme could be used to release soluble likely low molecular 398

weight pectin from isolated cell walls we next investigated whether infection with B 399

cinerea was accompanied by accumulation of soluble pectin as well We prepared cell 400

walls (AIR fraction) from leaves infected with B cinerea incubated these cell walls with 401

water and measured uronic acid content of the extracts B cinerea treatment did in fact 402

lead to increased accumulation of soluble pectin in wild-type Col-0 plants when 403

compared to mock-treated samples (Figure 12) No such increased accumulation of 404

water-soluble pectin fractions was observed in gae1 gae6 (Figure 12) 405

406

Macerozyme-Induced Pattern-Triggered Immunity against B cinerea Is Abolished 407

in gae1 gae6 408

It was previously shown that OG treatment can induce patternndashtriggered 409

immunity (PTI) to B cinerea (Ferrari et al 2007) We tested macerozyme treatment to 410

determine whether it could also induce PTI to the B cinerea isolate Gallo 1 (Figure 13) 411

Indeed pretreatment with 001 macerozyme for 24 h led to reduced B cinerea growth 412

in wild-type Col-0 but not gae1 gae6 plants (Figure 13A) Interestingly mock and 413

macerozyme pretreated gae1 gae6 plants exhibited similar growth of B cinerea Gallo 1 414

as did macerozyme-treated Col-0 plants This result is likely due to activation of JA 415

signaling in gae1 gae6 during the pretreatment procedure (Figure 13C and D) OG-416

induced PTI was observed in both genotypes when using a high dose of OGs (Figure 417

Page 23 of 54

13B) whereas a lower dose of OGs only induced PTI in Col-0 (Supplemental Figure 418

15) This suggests that OG perception in gae1 gae6 at least above a certain threshold 419

is unaltered while release of immunity-activating pectin fragments possibly OGs is 420

421 reduced We conclude that immune responses induced by macerozyme treatment

422 which do not occur in gae1 gae6 plants are important for plant immunity to the B

423 cinerea isolate Gallo 1

Page 24 of 54

____________________________________________________________________________

Page 25 of 54

DISCUSSION 426

Here we showed that loss of GAE1 and GAE6 leads to a dramatic reduction in 427

pectin content in the cell wall (Figure 4) likely causing the brittleness of leaves that we 428

observed (Figure 3) Petioles and midribs of gae1 gae6 plants broke easily when bent 429

and inoculation of leaves with deionized water resulted in increased ion leakage 430

possibly due either to microscopic fractures of the cell wall induced by inoculation stress 431

or to a reduced capability of these walls to withstand increased turgor pressure The 432

increased brittleness was associated with altered cell wall composition In our analysis 433

the GalA content of gae1 gae6 leaf walls was reduced by more than 40 and the total 434

uronic acids by over 60 (Figure 4 and Supplemental Figure 5) This is a major change 435

in the overall cell wall composition considering that pectin makes up about 50 of the 436

plant cell walls found in Arabidopsis leaves (Zablackis et al 1995 Nobuta et al 2007 437

Harholt et al 2010) Interestingly quasimodo1 (qua1) plants which have a mutation in 438

a glycosyltransferase family 8 gene are reduced in GalA by 25 and show reduced 439

binding of HG specific antibodies (Bouton et al 2002) 440

Furthermore qua2 plants which have a mutation in a putative methyltransferase 441

have about 13 less total GalA and lack 50 of cell wall HG (Mouille et al 2007) 442

Likewise cgr2 cgr3 plants carrying mutations in another class of Golgi-localized 443

putative methyl-transferases have leaf cell walls that are reduced by about 40 in 444

uronic acid (Kim et al 2015) All three mutants are severely dwarfed (Bouton et al 445

2002 Mouille et al 2007 Kim et al 2015) Additionally qua1 and qua2 mutants show 446

a distorted overall shape likely due to reduced cell cohesion and are extremely fragile 447

(Bouton et al 2002 Mouille et al 2007) By contrast gae1 gae6 plants with a similar 448

strong reduction in GalA and total uronic acids have an overall normal shape and are 449

only slightly smaller than wild-type Col-0 (Supplemental Figure 4) In the wall analysis 450

performed here we did not detect changes in other wall polysaccharides or structures 451

that might compensate for the reduction of pectin in gae1 gae6 It is possible the severe 452

morphological alterations in qua2 and cgr2 cgr3 plants are due to reduction in pectin 453

methylesterification which is unaffected in gae1 gae6 It was speculated that quality 454

control mechanisms in the Golgi prevent secretion of inefficiently esterified pectin in 455

cgr2 cgr3 (Kim et al 2015) suggesting that the Golgi of these plants might over-456

Page 26 of 54

accumulate pectin and that this may contribute to the severe dwarf phenotype of these 457

plants The gae1 gae6 plants described here provide an opportunity to study the effects 458

of reduction of pectin in plants that do not show severe morphological phenotypes 459

We found that gae1 gae6 plants are also compromised in resistance to B 460

cinerea and Pma ES4326 indicating that loss of pectin reduces immunity to these 461

pathogens (Figure 2 and 6) We found that gae1 gae6 plants were slightly more 462

susceptible to Pma ES4326 but showed a strong increase in susceptibility to specific B 463

cinerea isolates This was not unexpected because B cinerea is known to interact with 464

pectin as part of its infection strategy It secretes various pectin-degrading enzymes 465

early during infection and requires some of these enzymes including PG1 PG2 and 466

PME1 for full virulence (ten Have et al 1998 Valette-Collet et al 2003 Kars et al 467

2005b Espino et al 2010) Additionally Arabidopsis plants expressing an antisense 468

construct of the polygalacturonase inhibitor PGIP1 are significantly more susceptible to 469

B cinerea (Ferrari et al 2006) On the other hand Pma ES4326 is not known to470

require cell wall degrading enzymes for full virulence even though it encodes two 471

pectate lyase genes in its genome (PMA4326_07094 and PMA4326_04621 472

wwwbacteriaensemblorg) B cinerea isolates are also known to be genetically quite 473

diverse in their polygalacturonase (PG) loci and genetic variability in PG2 is associated 474

with varying growth on pectin containing media (Rowe and Kliebenstein 2007) 475

Furthermore the contribution of PG1 and PG2 to B cinerea virulence seems to depend 476

on the specific B cinerea isolate and the host species (Kars et al 2005a Zhang and 477

Van Kan 2013) B cinerea isolates are also known to be differently sensitive to the anti-478

microbial secondary metabolite camalexin (Kliebenstein et al 2005) In line with these 479

observations the susceptibility of gae1 gae6 plants differed depending on the specific 480

B cinerea isolate Whereas Fresa 525 Gallo 1 UK Razz and KT grew better in gae1481

gae6 we detected no difference in Acacia Apple 517 DN Grape Pepper or Rasp 482

growth (Figure 6) 483

B cinerea was shown to metabolize pectin and to possibly utilize GalA as a484

carbon source during in planta growth (Zhang et al 2011 Zhang and Van Kan 2013) 485

Our data show that gae1 gae6 plants which are reduced in GalA content did allow 486

enhanced growth of specific B cinerea isolates but no reduction in growth of any B 487

Page 27 of 54

cinerea isolates tested was found This suggests that utilization of pectin as a carbon 488

source was not a contributing factor to alterations in B cinerea susceptibility It is 489

possible that changes in the cell wall composition affect the structural integrity of the 490

wall and affect hyphal penetration of specific B cinerea isolates However OGs (small 491

HG fragments) are likely released during the infection process (Ferrari et al 2013) 492

OGs function as DAMPs and initiate immune responses in Arabidopsis (Ferrari et al 493

2013) Treatment of Arabidopsis with OGs having a degree of polymerization (DP) of 494

10-15 sugar subunits was previously shown to enhance resistance to B cinerea (Ferrari495

et al 2007) We showed that pretreatment with OGs or macerozyme induced enhanced 496

immunity to B cinerea isolate Gallo 1 in wild-type Col-0 but only OGs and not 497

macerozyme induced immunity in gae1 gae6 (Figure 13) Our data suggest gae1 gae6 498

plants which have reduced pectin content may release less OGs and that this 499

reduction leads to the increased susceptibility to B cinerea 500

Consistently we found that macerozyme treatment released less soluble pectin 501

from gae1 gae6 cell walls than from wild-type walls Fractions containing soluble pectin 502

released from gae1 gae6 were also less active in inducing PAD3 gene expression than 503

the fractions released from wild-type walls (Figure 7) We also showed that less soluble 504

pectin accumulated in gae1 gae6 than in wild-type plants 48 h after treatment with B 505

cinerea (Figure 12) 506

We observed that loss of pectin leads to alterations in A thaliana immune 507

signaling Macerozyme treatment induced expression of PAD3 and the SA marker 508

genes PR1 and SID2 in wild-type Col-0 but not in gae1 gae6 whereas OG treatment 509

led to increased PAD3 and SID2 expression in both wild-type and gae1 gae6 (Figure 8 510

and 9) This suggests that although release of active soluble pectin in these plants 511

might be altered OG recognition is intact In fact cell walls isolated from qua1 another 512

mutant known to be reduced in HG released less OGs (DP1-6) than did wild-type cell 513

walls upon endopolygalacturonase treatment (Bouton et al 2002) This was also true 514

for gae1 gae6 cell walls as macerozyme treatment of isolated gae1 gae6 cell walls 515

released less soluble pectin than treatment of Col-0 cell walls Because the fraction 516

from gae1 gae6 plants was also less active in inducing PAD3 expression (Figure 7) it 517

seems possible that biologically active OGs were the active component inducing PAD3 518

Page 28 of 54

expression Interestingly expression of the JA marker genes JAZ5 and JAZ10 was 519

higher in gae1 gae6 than in wild-type mock-inoculated plants but was comparable to 520

wild-type in untreated plants suggesting that JA signaling in gae1 gae6 is 521

hyperresponsive (Figure 10 and Supplemental Figure 11) This induction of JA signaling 522

might be due to wounding stress incurred during the inoculation process JA and SA 523

signaling generally act antagonistically (Pieterse et al 2012) Increased JA signaling 524

activity might hence explain why mock- and macerozyme-treated gae1 gae6 plants 525

exhibited reduced expression of the SA marker PR1 and why OG treatment induced 526

expression of SID2 another SA marker to a lower extent in gae1 gae6 than in wild-type 527

plants (Figure 9) 528

No single immune signaling component was found to be the sole contributor to 529

the compromised immunity of gae1 gae6 to specific B cinerea isolates such as Gallo 1 530

and Fresa 525 These isolates grew better on pad3 while growth of Pepper another B 531

cinerea isolate that did not show altered virulence on gae1 gae6 was unaltered 532

(Supplemental Figure 9) This suggested that differences in camalexin sensitivity of 533

these B cinerea isolates might account for the differences in gae1 gae6 susceptibility 534

However camalexin accumulation and PAD3 expression was the same for wild-type 535

Col-0 and gae1 gae6 for the B cinerea isolates Fresa 525 and Gallo 1 (Figure 8) which 536

showed enhanced growth on gae1 gae6 plants It should be noted that B cinerea 537

virulence is often estimated by comparing lesion sizes Lesion size and measurements 538

of relative fungal growth by qPCR do not always show the same results For example 539

although Denby et al (2004) showed that Pepper causes larger lesions on pad3 than on 540

wild-type this study showed that relative fungal growth of Pepper on pad3 was 541

unaltered 542

We observed that callose deposition was strongly reduced in gae1 gae6 plants 543

72 h after treatment with B cinerea isolate Gallo 1 (Figure 11) possibly due to reduced 544

release of active soluble pectin Interestingly OG-induced PTI to an unspecified isolate 545

of B cinerea was intact in the pmr4 mutant which does not produce callose (Galletti et 546

al 2008) Due to the high SA levels in pmr4 plants (Nishimura et al 2003) and the fact 547

that different B cinerea isolates vary both in induction of and reaction to defense 548

responses (Kliebenstein et al 2005 Rowe et al 2010) we could not determine 549

Page 29 of 54

whether reduced callose deposition causes the increased susceptibility of gae1 gae6 to 550

B cinerea isolate Gallo 1551

We did not observe any changes in B cinerea growth either on cev1 ixr1 plants 552

which are known to have constitutively high JA and OPDA levels (Ellis et al 2002b) or 553

on plants exogenously treated with MeJA (Supplemental Figure 9) Because plants 554

were kept uncovered for 3 h after MeJA treatment it is unclear how much MeJA was 555

bioavailable in the plant However JAZ10 expression was induced 3 h after spraying 556

with MeJA (Supplemental Figure 10) Because pathogen growth generally depends on 557

the combination of a multitude of immune signaling events we conclude that multiple 558

changes to the immune signaling in gae1 gae6 together account for the altered B 559

cinerea growth phenotypes detected 560

We showed that expression of GAE1 and GAE6 is repressed by the bacterial 561

hemibiotroph Pma ES4326 This repression did not occur in an eds1 or pad4 562

background or in pbs3 at the 24-h time point (Figure 1 and Supplemental Figure 1) 563

During the Arabidopsis - Pma ES4326 interaction the expression of many defense 564

related genes is altered in a PAD4EDS1 dependent fashion (Wang et al 2008) Some 565

of these expression changes require SA CBP60g and SARD1 are thought to act 566

downstream of PAD4 and EDS1 but upstream of SA-dependent responses (Wang et 567

al 2011) PBS3 is also thought to act downstream of PAD4 and EDS1 and upstream of 568

SA but the set of genes whose expression it affects is only partially overlapping with 569

those affected by CBP60gSARD1 (Wang et al 2008 Wang et al 2011) Thus GAE1 570

and GAE6 repression is regulated by the part of the immune signaling network that 571

requires PAD4 EDS1 and PBS3 but is independent of CBP60gSARD1 and SA 572

Because EDS1 PAD4 and PBS3 are important components of the plant immune 573

signaling network (Wiermer et al 2005 Nobuta et al 2007) one might conclude that 574

repression of GAE1 and GAE6 may be part of the plant immune response to Pma 575

ES4326 B cinerea treatment also reduced GAE1 and GAE6 expression but this was 576

mostly independent of PAD4 (Figure 6) while both Pma ES4326 and B cinerea repress 577

GAE1 and GAE6 expression the host signaling mechanisms responsible likely differ 578

In summary our data show that Arabidopsis GAE1 and GAE6 are required for 579

pectin biosynthesis leaf flexibility and immunity to Pma ES4326 and to specific B 580

Page 30 of 54

cinerea isolates We used macerozyme a commercial pectinase to mimic the effect of 581

B cinerea pectinases Macerozyme treatment released less pectin from gae1 gae6 cell582

walls and induced less immune signaling in gae1 gae6 plants Less water soluble pectin 583

also accumulated in B cinerea infected gae1 gae6 plants and macerozyme-induced 584

immunity to B cinerea isolate Gallo 1 was reduced in these plants We conclude that 585

the reduced pectin content in these mutants may lead to reduced release of soluble 586

pectin including active OGs during pathogen attack and that this compromises 587

immunity 588

589

590

Page 31 of 54

METHODS 591

Plant Materials and Growth Conditions 592

Wild-type Columbia (Col-0) and all mutant plants (in Col-0 background) were 593

grown on sterilized BM2 germinating mix (Berger Inc Quebec Canada) in a controlled 594

environment chamber (Conviron Pembina ND USA) with a 12-h photoperiod under 595

100 microM m-2 s-1 fluorescent illumination at 22degC and 75 relative humidity Germplasm 596

used is described in the Accession Numbers section at the end of Methods 597

598

Data Analysis and Replicates 599

For biological replicates samples were obtained from separate plant tissues and 600

in many cases from plants grown in different flats In some experiments multiple 601

technical replicates were measured per biological replicate For statistical analysis all 602

technical replicates were averaged and the averages were considered one biological 603

replicate Independent experiments were performed at different times and often 604

consisted of multiple biological replicates per experiment 605

Data analysis was performed using mixed linear models in the R programming 606

environment (Bates et al 2015) unless otherwise indicated Generally genotypes 607

treatments and time points were used as fixed effects and replicate-specific effects like 608

different pots flats etc were considered random effects We reported p-values 609

obtained from the mixed linear model when less than 10 comparisons were performed 610

and q-values for more than 10 comparisons to correct for multiple comparison testing 611

Q-values were calculated using the Benjamini-Hochberg procedure (Benjamini and612

Hochberg 1995) unless specified otherwise 613

614

Pathogen Strains Growth Conditions and Pathogen Growth Assays 615

Growth of Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) and 616

bacterial growth assays were performed as previously described (Bethke et al 2014) 617

For qRT-PCR assays four week-old plants were inoculated with bacteria (OD600 = 618

0002) or mock (5 mM MgSO4) At least four leaves from two plants were collected at 619

the indicated time points per biological replicate 620

Page 32 of 54

Prior to each experiment Botrytis cinerea isolates were grown on 1x PDA 621

medium (Difco) for 10 d at room temperature All isolates used have been described 622

previously (Rowe and Kliebenstein 2007) The spores were washed from the surface of 623

the plate using an inoculation medium (1x Gamborgrsquos B-5 basal salt mixture (Sigma-624

Aldrich G5768) 2 (wv) glucose 10 mM phosphate buffer pH 64) and fungal hyphae 625

were removed from the suspension by filtering through four layers of cheesecloth The 626

concentration of spores was determined using a hemocytometer and adjusted to 25 x 627

105 spores mL-1 Four week-old Arabidopsis plants were inoculated by placing one 10-628

microL droplet of the B cinerea spore solution or inoculation medium (mock) on the adaxial 629

leaf surface of fully expanded leaves Inoculated plants were kept at 100 relative 630

humidity Infection sites were collected at various time points using a cork borer For 631

qRT-PCR assays at least four leaf discs from one plant were combined for each 632

biological sample For B cinerea growth assays at least 12 leaf discs from three to six 633

plants were pooled per biological sample Relative fungal growth was determined by 634

measuring the abundance of a fungal gene relative to a plant gene as described below 635

In brief DNA from infected tissue was extracted and equal amounts of total DNA were 636

used to perform qPCR reactions using the B cinerea cutinase A and the Arabidopsis 637

SK11 gene respectively Primers were described in (Gachon and Saindrenan 2004) 638

and are listed in Supplemental Table 1 Two technical replicates were combined for 639

each biological replicate B cinerea is a necrotrophic fungal pathogen and may destroy 640

plant DNA as disease progresses However decreases in the amount of plant DNA 641

over the course of our experiments were only observed at late time points with very 642

susceptible plants so the qPCR assay is generally a reasonable estimate of fungal 643

biomass (Supplemental Figure 16) 644

645

Treatment with Macerozyme or OGs 646

For PTI assays four week-old plants either were inoculated with 001 (wv) 647

macerozyme R-10 (Yakult) a Rhizopus polygalacturonase (EC 32115) that possesses 648

high pectinase and hemicellulase activity or were mock inoculated using heat-649

inactivated macerozyme Other similar plants were either inoculated with 250 or 500 microg 650

mL-1 OGs or mock inoculated with water One d later plants were infected with B 651

Page 33 of 54

cinerea and samples were collected immediately (0 dpi) and two or three d later To 652

investigate the role of MeJA on B cinerea growth plants were sprayed with 1mM MeJA 653

in water with 001 (vv) Tween 20 Covers were removed so that leaves could dry off 654

before plants were inoculated with B cinerea three h later Samples were harvested at 655

the indicated time points For expression analysis and MAPK activation assays plants 656

were either inoculated with 100 microg mL-1 OGs or mock inoculated with water OGs used 657

for experiments described in Figures 8 to 10 and Supplemental Figure 12 were a kind 658

gift from Simone Ferrari OGs used in experiments described in Figure 13 and 659

Supplemental Figures 11 13 and 15 were prepared as previously described (Kohorn et 660

al 2014) 661

662

Expression Analysis 663

Leaf tissue from inoculated plants was harvested at the indicated time points 664

flash frozen pulverized and RNA extracted using Trizol (Invitrogen) Quantitative RT-665

PCR was performed using the SuperScript III Platinum SYBR Green One-Step 666

quantitative RT-PCR kit (Invitrogen) and a Lightcycler 480 Real-Time PCR system 667

(Roche) as previously described (Truman and Glazebrook 2012) In brief equal 668

amounts of total RNA and a gene specific primer were used for each reaction The 669

crossing point (Cp) was calculated using the 2nd derivative max method provided with 670

the Lightcycler software for each amplification curve Each reaction was run with two 671

technical replicates and the Cp values for these replicates were averaged ACTIN2 was 672

used as a stably expressed reference gene At least three biological replicates each 673

were performed and analyzed using a mixed-linear model RT-PCR was performed to 674

verify T-DNA insertion lines using the One-Step RT-PCR kit (Qiagen) according to the 675

manufacturerrsquos instructions The primers used can be found in Supplemental Table 1 676

677

Electrolyte Leakage Assay 678

Fully expanded leaves of four week-old plants were inoculated with water (Milli-Q 679

grade) using a needleless syringe Leaf discs were collected using a cork borer and 680

three leaf discs from one leaf were placed with the adaxial surface down onto 2 mL 681

water in a 12-well cell culture plate Conductivity was measured using a Horiba B-173 682

Page 34 of 54

conductivity meter after 30 min and then the water was replaced Conductivity was 683

measured again 30 min later (1 h total) as indicated in Figure 3C Six biological 684

replicates each were performed in three independent experiments and means and SEs 685

were calculated Two-sided t-tests were performed for each time point 686

687

DAB and Aniline Blue Staining 688

Leaves from four week-old plants were either mock inoculated with B cinerea 689

inoculation medium or inoculated with 10 microL droplets of a B cinerea spore solution (25 690

x 105 spores mL-1) of the indicated isolates Samples were collected 72 h later For 33prime-691

diaminobenzidine (DAB) staining two to five inoculated leaves were vacuum infiltrated 692

with 33prime-Diaminobenzidine (1 mg mL-1 in water pH 38) solution and stained overnight 693

in the dark Leaves were destained using 100 ethanol at 65degC and then transferred to 694

50 (vv) glycerol and photographed 695

Callose was visualized by staining leaves with aniline blue as previously 696

described (Adam and Somerville 1996) Briefly leaves were vacuum infiltrated with 697

alcoholic lactophenol (one volume of phenolglycerollactic acidwater (1111) mixed 698

with two volumes of ethanol) and destained at 65degC Next leaves were moved through 699

an alcohol gradient (50 20 and 10 (vv) ethanol) into water The destained leaves 700

were stained for 30 min in 150 mM K2HPO4 (pH 95) with 001 (wv) aniline blue 701

Samples were mounted in water and examined using a Nikon Eclipse Ni-U microscope 702

at 10x magnification using a DAPI filter (excitation at 325-375 nm emission at 435-485 703

nm) For callose quantification six 200 microm x 200 microm squares were counted for each 704

sample type Callose deposits per mm2 were calculated and data analyzed using a t-705

test Note that there is an inherent uncertainty associated with such counts because 706

callose deposits may not all be in focus at the same time 707

708

MAPK Activity Assay 709

Fully expanded leaves of four week-old plants were inoculated with 001 710

macerozyme 100 microg mL-1 OGs 100 nM flg22 or water (mock) flg22 a peptide from 711

bacterial flagella that induces immune responses was used as a positive control for 712

Page 35 of 54

MAPK activation (Asai et al 2002) and was purchased from EZBiolab Inc (Westfield 713

IN USA) Samples were harvested 10 min later and immediately frozen in liquid 714

nitrogen Protein was extracted using an extraction buffer containing 25 mM Tris pH 78 715

75 mM NaCl 1 mM DTT 1 mM NaF 05mM Na3VO4 01 (vv) Tween 20 one 716

cOmplete Mini protease inhibitor cocktail tablet (Roche) and one PhosSTOP 717

phosphatase inhibitor cocktail tablet (Roche) per 10 mL buffer 15 microg of total protein 718

were electrophoresed on a 10 SDS-polyacrylamide gel and then blotted onto a PVDF 719

membrane (Bio-Rad 162-0177) Activated MAPKs were detected using p4442 MAPK 720

(Erk12) antibody (9102S Cell Signal 12500 in TBST (20 mM Tris pH 75 150 mM 721

NaCl 01 (vv) Tween 20)) Anti-Rabbit IgG-HRP (115000 in TBST A6154 Sigma) 722

and ECL Plus substrate (Pierce 32132) MPK3 and MPK6 were detected with an anti-723

MPK3 antibody (M8318 Sigma 12000 in TBST and 3 (wv) milk) or an anti-MPK6 724

antibody (A7104 Sigma 16000 in TBST and 3 (wv) milk) respectively The 725

secondary antibody and detection were the same as for the p4442 antibody Five 726

independent experiments were performed 727

728

Preparation of Alcohol Insoluble Residue (AIR) Pectin Extraction and Uronic Acid 729

Measurements 730

Fully expanded leaves from four week-old plants that were incubated for 48 h in 731

the dark to reduce starch were harvested flash frozen and pulverized AIR was 732

extracted by washing ground material twice in 70 (vv) ethanol three times in a 733

mixture of chloroform and methanol (11 vv) and once in acetone (Gille et al 2009) 734

and was then air dried Cell wall pectin was extracted from AIR with cell wall extraction 735

buffer (50 mM Trizma 50 mM CDTA pH 72) at 95degC for 15 min Samples were 736

homogenized using a paint shaker (Harbil 5G-HD) and glass beads (3 mm diameter) 737

For dot blot experiments 500 microL of cell wall extraction buffer were used per 10 mg of 738

AIR Debris was precipitated by centrifugation for 10 min at 10000 g 739

Total uronic acid content of pectin extracted as described above using 500 microL of 740

cell wall extraction buffer per 1 mg of AIR was measured as described previously 741

(Filisetti-Cozzi and Carpita 1991 van den Hoogen et al 1998) First 36 microL of pectin 742

extract were mixed with 4 microL of 4 M sulfamic acid Then 200 microL of sulfuric acid 743

Page 36 of 54

containing 120 mM sodium tetraborate was added and samples were incubated at 80degC 744

for 1 h Following cooling on ice the optical density of samples was measured at 490 745

nm Next 40 microL of m-hydroxydiphenyl reagent (100 microL of m-hydroxydiphenyl in dimethyl 746

sulfoxide at 100 mgmL mixed with 49 mL 80 (vv) sulfuric acid just before use) was 747

added and the samples were mixed The optical density at 490 nm was measured 748

again The optical density before m-hydroxydiphenyl reagent addition was subtracted 749

from the optical density measured after addition of the dye The concentration of uronic 750

acid was calculated using known amounts of GalA as a standard 751

752

Release of Pectin from Arabidopsis Cell Walls Using Macerozyme 753

AIR preparations from leaves of four week-old plants were treated with 025 754

(wv) macerozyme R-10 (Yakult) or water (mock) at a concentration of 100 microL per mg 755

AIR Samples were incubated for 3 h at room temperature on a vortex shaker and 756

centrifuged for 10 min at 12000 g Total uronic acid concentration of 5-fold diluted 757

supernatants was determined for two technical replicates each The concentration of 758

uronic acid was calculated using known amounts of GalA as a standard For 759

subsequent qRT-PCR experiments supernatants from multiple extractions were 760

combined boiled for 20 min to inactivate macerozyme diluted 10-fold with water and 761

inoculated into four week-old Col-0 plants Heat-inactivated 10-fold diluted macerozyme 762

solutions were used as mock treatment Samples were collected 3 h later and PAD3 763

expression was determined 764

765

Release of Pectin from Arabidopsis Leaves Infected with B cinerea 766

Fully expanded leaves of four week-old plants were either inoculated with up to 767

five 10 microL droplets of B cinerea spore solution (25 x 105 spores mL-1) or mock 768

inoculated with B cinerea inoculation medium For each biological replicate leaves with 769

a combined count of at least 20 infection sites were collected 48 h later AIR was 770

extracted as described above but plants were not dark-treated or freeze-dried and the 771

first ethanol step was performed using 85 (vv) ethanol To extract soluble pectin 772

fragments AIR samples were mixed with water at a concentration of 20 microL per infection 773

site incubated for 7 h at room temperature on a vortex shaker and centrifuged for 10 774

Page 37 of 54

min at 12000 g Total uronic acid concentration of supernatants was determined for two 775

technical replicates each The concentration of uronic acid was calculated using known 776

amounts of GalA as a standard 777

778

Measurement of Uronic Acids Neutral Monosaccharides and Cellulose Content in 779

Cell Walls 780

Neutral sugars and uronic acids from non-cellulosic polysaccharides in cell walls 781

(AIR) were released by trifluoroacetic acid (TFA) hydrolysis Approximately 1 mg of AIR 782

was hydrolyzed with 2 M TFA at 121degC for 90 min The TFA-soluble material was split 783

into two equal parts one for uronic acid and one for neutral sugar determination The 784

uronic acid content was determined by analyzing the hydrolyzate using a CarboPac 785

PA200 anion-exchange column with an ICS-3000 Dionex chromatography system 786

(Dionex Sunnyvale CA USA) The elution profile consisted of a linear gradient of 50 to 787

200 mM sodium acetate in 01 M NaOH in 10 min at 04 mL per min Alditol acetate 788

derivatives of neutral sugars were produced as described (York et al 1985) In brief 789

monosaccharides in the hydrolyzate were reduced with NaBH4 and peracetylated with 790

acetic anhydride and pyridine at 121degC for 20 min The generated alditol acetates were 791

analyzed by GC-MS Crystalline cellulose was measured according to Updegraff (1969) 792

After hydrolysis of noncellulosic polysaccharides from AIR with the Updegraff reagent 793

(acetic acid nitric acids water 812 (vv) ) the remaining pellet was hydrolyzed in 794

72 sulfuric acid The resulting glucose quantity was determined by the anthrone 795

method (Scott and Melvin 1953) 796

797

Dot Blot Experiments 798

Pectin solutions extracted using 50 microL of cell wall extraction buffer per mg AIR 799

were serially diluted and nitrocellulose membranes were spotted with 1 microL of the diluted 800

pectin solutions Membranes were dried overnight blocked with 5 milk (wv) in 1x 801

PBS (8 g L-1 NaCl 02 g L-1 KCl 144 g L-1 Na2HPO4 024 g L-1 KH2PO4 pH 74) and 802

probed with LM5 (Jones et al 1997) LM6 (Willats et al 1998) LM8 (Willats et al 803

2004) LM19 and LM20 (Verhertbruggen et al 2009a) or CCRC-M7 (Steffan et al 804

Page 38 of 54

1995) antibodies LM series antibodies were diluted 1250 and CCRC-M7 was diluted 805

1500 in 5 milk powder (Nestle Carnation) in 1 x PBS For LM series antibodies a 806

goat anti-Rat HRP conjugated antibody (Bethyl A110-105P 15000 diluted) and for 807

CCRC-M7 an anti-Mouse IgG HRP conjugate (Promega W402B 12500 diluted) was 808

used as secondary antibody in 5 milk powder in 1x PBS LM series antibodies were 809

obtained from plant probes (wwwplantprobesnet) and CCRC-M7 from CarboSource 810

(wwwcarbosourcenet) Membranes were washed after incubation with the primary and 811

secondary antibodies with 1x PBS Dot blots were developed using the ECL system 812

(GE Healthcare Amersham ECL prime) For quantification of signals dot blot results on 813

x-ray films were photographed using a CCD camera and intensities were measured814

using Image Jrsquos Integrated Density function Locally adjusted background was 815

subtracted As the dynamic range of the signal on an x-ray film was narrow non-816

linearity in the measured values was corrected for each genotype in each biological 817

replicate using the values from different dilutions in two technical replicates We noticed 818

that the residuals were not normally distributed and performed Box-Cox power 819

transformation (Box and Cox 1964) to obtain normally distributed values for the 820

subsequent statistical analysis of the data For LM19 Box-Cox selected power 821

transformation was done to the power of 038th and for LM20 to the power 075th After 822

correction of the transformed measured values with the effect of the biological 823

replicates the values were fit to a linear model with the Arabidopsis genotype as the 824

fixed effect This linear model was used both to generate the plots in Figures 5B and D 825

and to test significant differences 826

827

Camalexin Measurements 828

Four week-old plants were infected with B cinerea and samples (three leaf disks 829

from one plant each) were harvested at the indicated time points flash frozen and 830

pulverized Each sample was extracted with 300 microL of 90 methanol (vv) 50 microL of the 831

extract was run on an Agilent Lichrocart 250-4 RP18e 5-microm column using an Agilent 832

1100 series HPLC Camalexin was detected using a DiodeArray (DAD) at 330 nm and 833

with a fluorescence detector at emission 318 nmexcitation 385 nm (Agilent) Separation 834

was achieved using the following program with aqueous acetonitrile 5 min gradient 835

Page 39 of 54

from 63 to 69 acetonitrile 30 s gradient from 69 to 99 acetonitrile 2 min at 99 836

acetonitrile and a post-run equilibration of 35 min at 63 acetonitrile (Denby et al 837

2004 Kliebenstein et al 2005) Purified camalexin was used to produce a standard 838

curve to identify and quantitate camalexin 839

840

Accession Numbers 841

Microarray data used can be accessed at NCBI Gene Expression Omnnibus 842

(httpwwwncbinlmnihgovgeo) GSE18978 Sequence information for all Arabidopsis 843

genes described in this article can be found in TAIR (wwwarabidopsisorg) using the 844

following accession numbers PAD3 (CYP71B15 At3g26830) SID2 (ICS1 At1g74710) 845

GAE1 (At4g30440) GAE2 (At1g02000) GAE3 (At4g00110) GAE4 (At2g45310) GAE5 846

(At4g12250) GAE6 (At3g23820) PR1 (At2g14610) JAZ5 (At1g17380) JAZ10 847

(At5g13220) ACTIN2 (At3g18780) PDF12a (At5g44420) PDF12b (At2g26020) 848

AtSK11 (At5g26751) The sequence of the B cinerea cutinase Bc-CutA (Z69264) can 849

be found in the DNA Data Bank of Japan (httpgetentryddbjnigacjp) Germplasm 850

used included dde2-2 (At5g42650 (von Malek et al 2002)) pad3-1 (At3g26830 (Zhou 851

et al 1999)) pad4-1 (At3g52430 (Jirage et al 1999)) sid2-2 (At1g74710 (Wildermuth 852

et al 2001)) pbs3-2 (At5g13320 SALK_018225 (Nobuta et al 2007)) cbp60a-1 853

(At5g62570 SALK_124410 (Truman et al 2013)) cbp60g-1 sard1-2 (At5g26920 854

SALK_023199 At1g73805 SALK_052422 (Wang et al 2011) cbp60a-1 cbp60g-1 855

sard1-2 (Truman et al 2013) gae1-1 (At4g30440 SALK_085554) gae6-1 (At3g23280 856

SALK_104454) gae6-2 (At3g23280 SALK_017191) ixr1-1 cev1 (At5g05170 (Ellis 857

and Turner 2001 Scheible et al 2001)) eds1 plants were derived by introgression of 858

the Ler eds1-2 allele into Col-0 that contains two copies of EDS1 (At3g48090 859

At3g48080 (Bartsch et al 2006) mpk6-2 (At2g43790 SALK_073907 (Liu and Zhang 860

2004)) mpk3-DG (At3g45640 a fast neutron deletion mutant (Miles et al 2005)) and 861

npr1-1 (At1g64280 (Cao et al 1997)) T-DNA insertion lines were part of the SALK 862

collection (Alonso et al 2003) and were obtained from the Arabidopsis Biological 863

Resource Center 864

865

Supplemental Data 866

Page 40 of 54

Supplemental Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 867

Expression Does Not Require CBP60s or SID2 868

Supplemental Figure 2 Expression of GAE Family Members uponTreatment with Pma 869

ES4326 870

Supplemental Figure 3 Characterization of T-DNA Insertions in GAE1 and GAE6 871

Supplemental Figure 4 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves and 872

Are Slightly Smaller than Wild-Type Col-0 Plants 873

Supplemental Figure 5 Cell Walls of gae1 gae6 Plants Contain Less Uronic Acid than 874

Wild-Type Col-0 Cell Walls 875

Supplemental Figure 6 Antibodies Raised Against the RGI or XG Components of 876

Pectin Show No Difference in Binding to gae Mutant and Col-0 Cell Walls 877

Supplemental Figure 7 The Effect of B cinerea Treatment on the Expression of GAE 878

Family Genes 879

Supplemental Figure 8 Treatment with 001 Macerozyme Does Not Cause Any 880

Visible Damage to Col-0 Plants 881

Supplemental Figure 9 B cinerea Growth on Plants with Defects in Immune 882

Signaling 883

Supplemental Figure 10 MeJA-Induced Expression of the JA marker gene JAZ10 884

Supplemental Figure 11 The Expression of the JA Marker Gene JAZ5 Is Highly 885

Responsive in gae1 gae6 Plants 886

Supplemental Figure 12 The Expression of PDF12 Is Unaltered in gae1 gae6 Plants 887

Supplemental Figure 13 Macerozyme Treatment Activates MPK3 and MPK6 888

Supplemental Figure 14 B cinerea-Induced ROS Production in Wild-Type Col-0 and 889

gae1 gae6 Plants 890

Supplemental Figure 15 OG-Induced PTI Using 250 microg mL-1 OGs 891

Supplemental Figure 16 Abundance of Plant DNA in B cinerea Treated Plants 892

Supplemental Table 1 Primers used in this study 893

894

ACKNOWLEDGEMENTS 895

We thank William Truman for helpful discussions Simone Ferrari (Sapienza 896

Universita di Roma) for the generous gift of OGs Bruna Bucciarelli (USDA-ARS) for 897

Page 41 of 54

help with fluorescence microscopy the Arabidopsis Biological Resource Center for T-898

DNA insertion lines Paul Knoxrsquos lab (University of Leeds) for LM5 LM6 LM8 LM19 899

and LM20 antibodies and the Complex Carbohydrate Resource Center (University of 900

Georgia) for the CCRC-M7 antibody Most of this work was funded by the Division of 901

Chemical Sciences Geosciences and Biosciences Office of Basic Energy Sciences of 902

the US Department of Energy through Grant DE-FG02-05ER15670 to JG This work 903

was also supported by NSF awards IOS 1339125 and IOS 1021861 to DJK NSF 904

award IOS 1121425 to F K and the USDA National Institute of Food and Agriculture 905

Hatch project number CA-D-PLS-7033-H to DJK 906

907

AUTHOR CONTRIBUTIONS 908

GB and JG designed the research GB GX AT BL NES and NH 909

performed research DJK provided tools GB GX MP AD BL RAH and FK 910

analyzed data GB and JG wrote the paper with input from all authors 911

912

FIGURE LEGENDS 913

Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 Expression 914

Requires PAD4 EDS1 and PBS3 915

(A) Expression of GAE1 in leaves of wild-type Col-0 and mutant plants Expression was916

measured 24 or 32 h after either inoculation with Pma ES4326 (OD600 = 0002) or mock 917

inoculation with 5 mM MgSO4 and was then normalized to the level of ACTIN2 Data 918

from three biological replicates were combined using a mixed linear model Bars 919

represent mean log2-ratios to ACTIN2 plusmn SE Letters indicate significantly different 920

groups with q lt 005 where 005 represents the maximum false discovery rate at which 921

the test may be called significant 922

(B) Expression of GAE6 measured as described in (A)923

924

Figure 2 Susceptibility of gae6 and gae1 gae6 Plants to Pma ES4326 Is Increased 925

Relative to Wild-Type Col-0 926

Leaves of plants of the indicated genotypes were inoculated with Pma ES4326 (OD600 = 927

00002) as described in Methods Bacterial titers in leaves were determined either 928

Page 42 of 54

immediately (0 dpi) or after three days (3 dpi) Bars represent the mean plusmn SE of four 929

independent experiments each with 4 or 12 biological replicates at 0 and 3 dpi 930

respectively Data were combined using a mixed linear model Asterisks indicate 931

significant differences from Col-0 wild-type (q lt 005) Susceptible pad4 plants were 932

included as a positive control 933

934

Figure 3 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves 935

(A) Depiction of the assay for brittleness of gae mutant leaves Fully expanded leaves of936

four to five week-old plants were bent by 180ordm so that the adaxial surface of the leaf was 937

parallel with the soil Broken leaf petioles or midribs were considered brittle 938

(B) Quantification of broken midribs or petioles For each genotype 66 to 82 leaves 939

were analyzed for brittleness as described in (A) Bars represent the ratio of broken to 940

total number of leaves tested Results were analyzed using Fisherrsquos exact test Letters 941

indicate significantly different groups at p lt 005 942

(C) Conductivity measurements Leaves of four week-old plants were inoculated with943

deionized water and then leaf discs were cut out and placed on deionized water 944

Conductivity was measured at the indicated time points After 30 min the water was 945

replaced with fresh water (30 min f w) to account for wounding-related ion leakage from 946

the edges of the leaf discs Three independent experiments with six biological replicates 947

each were performed and data were analyzed using a t-test for each time point P-948

values were corrected using the Bonferroni method Data show means plusmn SE asterisks 949

indicate significant differences from wild-type Col-0 at q lt 001 950

951

Figure 4 Cell Walls of gae6 and gae1 gae6 Plants Are Reduced in GalA 952

(A) to (D) Levels of GalA (A) GlcA (B) cellulose (C) and neutral sugars (D) in alcohol953

insoluble residue (AIR) extracted from leaves of four week-old plants of the indicated 954

genotypes Bars represent means plusmn SE of four biological replicates combined using a 955

mixed linear model Letters indicate significantly different groups (p lt 005) 956

957

Figure 5 Cell Walls of gae1 gae6 Plants Are Reduced in Homogalacturonan 958

Page 43 of 54

Dot blot analyses with anti-LM19 and anti-LM20 antibodies Pectin was extracted from 959

AIR prepared from the indicated genotypes using 1 microL of extraction buffer per 10 microg of 960

AIR Pectin was serially diluted and one microL of undiluted 13 diluted and 19 diluted 961

samples were spotted on a nitrocellulose membrane Three biological replicates were 962

were spotted twice to obtain two technical replicates each All replicates produced 963

similar results 964

(A) Representative dot blot using anti-LM19 antibody965

(B) Relative signal intensities of the spots in the dot blot in (A)966

(C) Representative dot blot using anti-LM20 antibody967

(D) Relative signal intensities of the LM20 dot blot shown in (C)968

Relative signal intensities were measured using Image J software Measurements from 969

the three biological replicates were combined using a linear model after Box-Cox power 970

transformation as described in Methods Bars represent means plusmn SE Asterisks indicate 971

a difference from wild-type Col-0 at p lt 005 972

973

Figure 6 Growth of B cinerea on gae1 gae6 Plants and the Effect of B cinerea on 974

Expression of GAE1 and GAE6 975

(A) Growth of B cinerea isolates Leaves of Col-0 and gae1 gae6 plants were976

inoculated with 25 x 105 spores mL-1 of the B cinerea isolates indicated in the figure 977

Samples were collected two (2 dpi) and three (3 dpi) days later Bars represent means plusmn 978

SE of six biological replicates for each fungus combined using a mixed linear model 979

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 980

(B) and (C) Expression of GAE1 (B) and GAE6 (C) 48 h after inoculation with the B981

cinerea isolates indicated Expression levels were measured by qRT-PCR Bars 982

represent mean log2-ratios to ACTIN2 plusmn SE of three biological replicates combined 983

using a mixed linear model Letters indicate significantly different groups (q lt 005) 984

985

Figure 7 Levels of Soluble Pectin and Expression of PAD3 in Fractions Produced 986

Following Macerozyme Treatment of gae1 gae6 Cell Walls 987

(A) Schematic overview of the experimental procedure for (B) and (C)988

Page 44 of 54

(B) Uronic acid measurements Alcohol-insoluble residue (AIR) from leave tissue of four989

week-old wild-type Col-0 or gae1 gae6 plants was treated with water or 025 990

macerozyme and assayed for uronic acid levels Data from 11 biological replicates were 991

combined using a mixed linear model Bars represent means plusmn SE Letters indicate 992

significant differences (p lt 005) 993

(C) Expression of PAD3 in Col-0 plants treated with the fractions from (B) Extracts from994

(B) were heat inactivated pooled and diluted 5-fold with water Pooled extracts were995

inoculated into four week-old Col-0 plants PAD3 expression was measured 3 h after 996

inoculation and normalized to the level of ACTIN2 expression Data from four biological 997

replicates were combined using a mixed linear model Bars represent mean log2-ratios 998

to ACTIN2 plusmn SE Letters indicate significant differences (p lt 005) 999

1000

Figure 8 Macerozyme-Induced PAD3 Expression Is Absent in gae1 gae6 1001

(A) Expression of PAD3 after macerozyme treatment Four week-old plants were either1002

inoculated with 001 macerozyme or mock inoculated with water and PAD3 1003

expression was measured 3 h later by qRT-PCR Data from four biological replicates 1004

were combined using a mixed linear model Mean log2-ratios to ACTIN2 plusmn SE were 1005

plotted Letters indicate significantly different groups (p lt 005) 1006

(B) Expression of PAD3 after OG treatment PAD3 expression was measured as1007

described in (A) 3 h after inoculation with 100 microg mL-1 OGs or mock inoculation with 1008

water Data are from six biological replicates 1009

(C) PAD3 expression in untreated plants Three biological replicates were performed1010

and data analyzed as described in (A) 1011

(D) Camalexin accumulation after B cinerea treatment Camalexin accumulation was1012

measured in four week-old plants inoculated with the B cinerea strains shown in the 1013

figure Eight biological replicates each were combined using a mixed linear model 1014

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 1015

(E) PAD3 expression 28 h after treatment with the indicated B cinerea strains Data1016

from six biological replicates were collected and analyzed as described in (A) Data in 1017

all figure parts represent means plusmn SE Letters indicate significantly different groups (q lt 1018

005) 1019

Page 45 of 54

1020

Figure 9 Macerozyme-Induced Expression of the SA Marker Genes SID2 and PR1 1021

Is Abolished in gae1 gae6 Plants 1022

(A) to (H) Expression of PR1 (A) (C) (E) and (G) or SID2 (B) (D) (F) and (H) in four1023

week-old Col-0 and gae1 gae6 plants Data were combined using a mixed linear model 1024

Bars represent mean log2-ratios of expression vs ACTIN2 plusmn SE for all figure parts 1025

Letters indicate significantly different groups at p lt 005 for panels (A) to (D) (G) and 1026

(H) and qlt005 for panels (E) to (F) 1027

(A) and (B) Plants were treated with 001 macerozyme or mock treated with water1028

and samples were collected 3 h later Data from four biological replicates are shown 1029

(C) and (D) Plants were inoculated with 100 microg mL-1 OGs or mock treated with water1030

and samples collected 3 h later Data from six biological replicates are shown 1031

(E) and (F) Plants were inoculated with the indicated B cinerea isolates or mock1032

inoculated with B cinerea inoculation medium and samples collected 48 h later Data 1033

from eight biological replicates are shown 1034

(G) and (H) Expression in untreated plants Three biological replicates were performed1035

1036

Figure 10 The Expression of the JA Marker Gene JAZ10 is Highly Responsive in 1037

gae1 gae6 Plants 1038

(A) to (D) JAZ10 expression in four week-old plants Data were combined using a mixed1039

linear model Mean log2-ratios to ACTIN2 plusmn SE are shown Letters indicate significantly 1040

different groups at p lt 005 for panels (A) (B) and (D) and q lt 005 for panel (C) 1041

(A) Plants were inoculated with 001 macerozyme or mock inoculated using water and1042

samples were collected 3 h later Data from four biological replicates are shown 1043

(B) Plants were inoculated with 100 microg mL-1 OGs or mock inoculated using water and1044

samples collected 3 h later Data are from six biological replicates 1045

(C) JAZ10 expression 48 h after treatment with the indicated B cinerea strains Data1046

are from six biological replicates 1047

(D) JAZ10 expression in untreated plants Data are from three biological replicates1048

1049

Figure 11 Gallo 1-Induced Callose Deposition Is Reduced in gae1 gae6 Plants 1050

Page 46 of 54

(A) Callose deposition in four week-old Col-0 and gae1 gae6 plants Plants were1051

inoculated with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) or mock inoculated 1052

with B cinerea inoculation medium and samples were collected after 72 h Callose was 1053

stained with aniline blue and visualized using a Nikon Eclipse Ni-U microscope with a 1054

DAPI filter Three leaves were stained per treatment Scale bars are 100 microm Asterisks 1055

indicate fungal hyphae arrows indicate representative callose spots 1056

(B) Quantification of callose deposition Callose deposits were counted in 200 microm x 2001057

microm squares laid out across the images shown here Bars represent means plusmn SE from 1058

six squares each Letters indicate significantly different groups (p lt 005) according to a 1059

t-test analysis1060

1061

Figure 12 Less Soluble Pectin Accumulates in B cinerea-Infected gae1 gae6 1062

Leaves 1063

Four week-old Col-0 and gae1 gae6 plants were inoculated with 10 microL droplets of the 1064

indicated B cinerea isolate (25 x 105 spores mL-1) or mock inoculated with B cinerea 1065

inoculation medium Leaves were collected 48 h later AIR was extracted from leaf 1066

samples and water added (20 microL infection site) to extract soluble pectin fragments 1067

Samples were vortexed for 7 h centrifuged at 12000 g and the uronic acid 1068

concentration of the supernatant was measured Data from nine biological replicates 1069

were combined using a mixed linear model Means plusmn SE are shown Letters indicate 1070

significant differences (q lt 005) 1071

1072

Figure 13 Macerozyme-Induced PTI Is Lost in gae1 gae6 While OG-Induced PTI Is 1073

Unaltered 1074

(A) Macerozyme-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1075

inoculated with 001 macerozyme or mock inoculated with boiled macerozyme and 1076

were infected with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) 24 h later Samples 1077

were collected immediately (0 dpi) and after two (2 dpi) and three (3 dpi) days Data 1078

show mean plusmn SE of six to 14 biological replicates each combined using a linear model 1079

Letters indicate significantly different groups (q lt 005) 1080

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

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Cao H Glazebrook J Clarke JD Volko S and Dong XN (1997) The Arabidopsis NPR1 gene that controls systemic acquiredresistance encodes a novel protein containing ankyrin repeats Cell 88 57-63

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Chisholm ST Coaker G Day B and Staskawicz BJ (2006) Host-microbe interactions Shaping the evolution of the plantimmune response Cell 124 803-814

Page 20 of 54

344

345

346

and JAZ10 was already strongly induced in gae1 gae6 after mock inoculation (Figure 10

and Supplemental Figure 11) We observed no hyperresponsive induction by mock

treatment of PLANT DEFENSIN 12 (PDF12) a necrotrophy and late JA signaling

347 marker gene (Supplemental Figure 12A-D) PDF12 was also not induced by

348 macerozyme or OG treatments (Supplemental Figure 12A-D) This suggests that early

349 JA signaling is induced by mock-treatment in gae1 gae6 Because it is known that JA

350 and SA signaling act antagonistically (Pieterse et al 2012) the strong up-regulation of

351 JA-induced immune signaling in gae1 gae6 might explain the lower OG-induced SID2

352 expression in this genotype

353 To test whether other immune responses are altered in gae1 gae6 plants we

354 analyzed Mitogen-activated protein kinase (MAPK) activity following macerozyme and

355 OG treatments OGs are known to activate the MAPKs MPK3 and MPK6 in Arabidopsis

Page 21 of 54

(Galletti et al 2011) Macerozyme treatment activated MPK3 and MPK6 in wild-type 356

Col-0 whereas MAPK activation in gae1 gae6 was slightly reduced in four out of five 357

experiments performed (Supplemental Figure 13) By contrast OG-induced MAPK 358

activity was similar or increased in gae1 gae6 in four out of five experiments 359

(Supplemental Figure 13) Additionally mock treatment induced MAPK activity in gae1 360

gae6 more strongly than it did in wild-type Col-0 in four out of five experiments 361

performed (Fig S13) Collectively these results illustrate that activation of several 362

immune signaling componentsmdashcamalexin synthesis SA and JA signaling and 363

possibly MAPK activationmdashby macerozyme is substantially altered in gae1 gae6 This 364

suggests that release of soluble pectin fragments possibly OGs by pectinolytic 365

enzymes might be reduced in gae1 gae6 plants while recognition of OGs is intact 366

367

Following B cinerea Infection Production of Soluble Pectin and Deposition of 368

Callose Are Reduced in gae1 gae6 Plants 369

Although pad3 plants were more susceptible to the B cinerea isolates Fresa 525 370

and Gallo 1 which also grew better on gae1 gae6 growth of the isolate Pepper was 371

similar on Col-0 and pad3 (Supplemental Figure 9A) However we did not detect any 372

significant changes in camalexin production after treatment with B cinerea isolate Gallo 373

1 and Fresa 525 in gae1 gae6 (Figure 8D) Neither did we detect changes in PAD3 374

expression after treatment with any B cinerea isolate in gae1 gae6 (Figure 8E) 375

Additionally SID2 and PR1 were similarly induced by all B cinerea isolates in both Col-376

0 and gae1 gae6 (Figure 9E and F) and sid2 plants which are deficient for pathogen-377

induced SA biosynthesis did not show any differences in relative B cinerea growth 378

(Supplemental Figure 9B) Expression of JAZ10 was induced more strongly by Gallo 1 379

treatment in gae1 gae6 than in wild-type Col-0 but not by Fresa 525 or Pepper (Fig 380

10C) while dde2 plants deficient for JA biosynthesis showed enhanced susceptibility to 381

all B cinerea isolates tested (Supplemental Figure 9B) Curiously exogenous 382

application of MeJA did not lead to altered relative fungal growth of B cinerea 383

(Supplemental Figure 9C) and cev1 isoxaben resistant 1 (ixr1) plants which have 384

constitutively high JA levels (Ellis and Turner 2001) did not show any changes in 385

susceptibility to the B cinerea isolates tested (Supplemental Figure 9D) Hence neither 386

Page 22 of 54

differences in B cinerea isolate susceptibility to camalexin nor altered SA or JA 387

signaling by themselves explained the enhanced susceptibility of gae1 gae6 to these B 388

cinerea isolates 389

Hydrogen peroxide accumulation in response to B cinerea isolates Fresa 525 390

Gallo 1 and Pepper was not obviously altered in gae1 gae6 plants (Supplemental Figure 391

14) However callose deposition upon treatment with B cinerea isolate Gallo 1 was 392

strongly reduced in gae1 gae6 (Figure 11) It was previously shown that OG treatment 393

induces callose deposition (Denoux et al 2008) suggesting that the reduction in 394

callose deposition might be due to reduced OG release in gae1 gae6 It is possible that 395

this reduction in callose deposition contributes to the reduced immunity of gae1 gae6 396

plants to B cinerea isolate Gallo 1 397

Because macerozyme could be used to release soluble likely low molecular 398

weight pectin from isolated cell walls we next investigated whether infection with B 399

cinerea was accompanied by accumulation of soluble pectin as well We prepared cell 400

walls (AIR fraction) from leaves infected with B cinerea incubated these cell walls with 401

water and measured uronic acid content of the extracts B cinerea treatment did in fact 402

lead to increased accumulation of soluble pectin in wild-type Col-0 plants when 403

compared to mock-treated samples (Figure 12) No such increased accumulation of 404

water-soluble pectin fractions was observed in gae1 gae6 (Figure 12) 405

406

Macerozyme-Induced Pattern-Triggered Immunity against B cinerea Is Abolished 407

in gae1 gae6 408

It was previously shown that OG treatment can induce patternndashtriggered 409

immunity (PTI) to B cinerea (Ferrari et al 2007) We tested macerozyme treatment to 410

determine whether it could also induce PTI to the B cinerea isolate Gallo 1 (Figure 13) 411

Indeed pretreatment with 001 macerozyme for 24 h led to reduced B cinerea growth 412

in wild-type Col-0 but not gae1 gae6 plants (Figure 13A) Interestingly mock and 413

macerozyme pretreated gae1 gae6 plants exhibited similar growth of B cinerea Gallo 1 414

as did macerozyme-treated Col-0 plants This result is likely due to activation of JA 415

signaling in gae1 gae6 during the pretreatment procedure (Figure 13C and D) OG-416

induced PTI was observed in both genotypes when using a high dose of OGs (Figure 417

Page 23 of 54

13B) whereas a lower dose of OGs only induced PTI in Col-0 (Supplemental Figure 418

15) This suggests that OG perception in gae1 gae6 at least above a certain threshold 419

is unaltered while release of immunity-activating pectin fragments possibly OGs is 420

421 reduced We conclude that immune responses induced by macerozyme treatment

422 which do not occur in gae1 gae6 plants are important for plant immunity to the B

423 cinerea isolate Gallo 1

Page 24 of 54

____________________________________________________________________________

Page 25 of 54

DISCUSSION 426

Here we showed that loss of GAE1 and GAE6 leads to a dramatic reduction in 427

pectin content in the cell wall (Figure 4) likely causing the brittleness of leaves that we 428

observed (Figure 3) Petioles and midribs of gae1 gae6 plants broke easily when bent 429

and inoculation of leaves with deionized water resulted in increased ion leakage 430

possibly due either to microscopic fractures of the cell wall induced by inoculation stress 431

or to a reduced capability of these walls to withstand increased turgor pressure The 432

increased brittleness was associated with altered cell wall composition In our analysis 433

the GalA content of gae1 gae6 leaf walls was reduced by more than 40 and the total 434

uronic acids by over 60 (Figure 4 and Supplemental Figure 5) This is a major change 435

in the overall cell wall composition considering that pectin makes up about 50 of the 436

plant cell walls found in Arabidopsis leaves (Zablackis et al 1995 Nobuta et al 2007 437

Harholt et al 2010) Interestingly quasimodo1 (qua1) plants which have a mutation in 438

a glycosyltransferase family 8 gene are reduced in GalA by 25 and show reduced 439

binding of HG specific antibodies (Bouton et al 2002) 440

Furthermore qua2 plants which have a mutation in a putative methyltransferase 441

have about 13 less total GalA and lack 50 of cell wall HG (Mouille et al 2007) 442

Likewise cgr2 cgr3 plants carrying mutations in another class of Golgi-localized 443

putative methyl-transferases have leaf cell walls that are reduced by about 40 in 444

uronic acid (Kim et al 2015) All three mutants are severely dwarfed (Bouton et al 445

2002 Mouille et al 2007 Kim et al 2015) Additionally qua1 and qua2 mutants show 446

a distorted overall shape likely due to reduced cell cohesion and are extremely fragile 447

(Bouton et al 2002 Mouille et al 2007) By contrast gae1 gae6 plants with a similar 448

strong reduction in GalA and total uronic acids have an overall normal shape and are 449

only slightly smaller than wild-type Col-0 (Supplemental Figure 4) In the wall analysis 450

performed here we did not detect changes in other wall polysaccharides or structures 451

that might compensate for the reduction of pectin in gae1 gae6 It is possible the severe 452

morphological alterations in qua2 and cgr2 cgr3 plants are due to reduction in pectin 453

methylesterification which is unaffected in gae1 gae6 It was speculated that quality 454

control mechanisms in the Golgi prevent secretion of inefficiently esterified pectin in 455

cgr2 cgr3 (Kim et al 2015) suggesting that the Golgi of these plants might over-456

Page 26 of 54

accumulate pectin and that this may contribute to the severe dwarf phenotype of these 457

plants The gae1 gae6 plants described here provide an opportunity to study the effects 458

of reduction of pectin in plants that do not show severe morphological phenotypes 459

We found that gae1 gae6 plants are also compromised in resistance to B 460

cinerea and Pma ES4326 indicating that loss of pectin reduces immunity to these 461

pathogens (Figure 2 and 6) We found that gae1 gae6 plants were slightly more 462

susceptible to Pma ES4326 but showed a strong increase in susceptibility to specific B 463

cinerea isolates This was not unexpected because B cinerea is known to interact with 464

pectin as part of its infection strategy It secretes various pectin-degrading enzymes 465

early during infection and requires some of these enzymes including PG1 PG2 and 466

PME1 for full virulence (ten Have et al 1998 Valette-Collet et al 2003 Kars et al 467

2005b Espino et al 2010) Additionally Arabidopsis plants expressing an antisense 468

construct of the polygalacturonase inhibitor PGIP1 are significantly more susceptible to 469

B cinerea (Ferrari et al 2006) On the other hand Pma ES4326 is not known to470

require cell wall degrading enzymes for full virulence even though it encodes two 471

pectate lyase genes in its genome (PMA4326_07094 and PMA4326_04621 472

wwwbacteriaensemblorg) B cinerea isolates are also known to be genetically quite 473

diverse in their polygalacturonase (PG) loci and genetic variability in PG2 is associated 474

with varying growth on pectin containing media (Rowe and Kliebenstein 2007) 475

Furthermore the contribution of PG1 and PG2 to B cinerea virulence seems to depend 476

on the specific B cinerea isolate and the host species (Kars et al 2005a Zhang and 477

Van Kan 2013) B cinerea isolates are also known to be differently sensitive to the anti-478

microbial secondary metabolite camalexin (Kliebenstein et al 2005) In line with these 479

observations the susceptibility of gae1 gae6 plants differed depending on the specific 480

B cinerea isolate Whereas Fresa 525 Gallo 1 UK Razz and KT grew better in gae1481

gae6 we detected no difference in Acacia Apple 517 DN Grape Pepper or Rasp 482

growth (Figure 6) 483

B cinerea was shown to metabolize pectin and to possibly utilize GalA as a484

carbon source during in planta growth (Zhang et al 2011 Zhang and Van Kan 2013) 485

Our data show that gae1 gae6 plants which are reduced in GalA content did allow 486

enhanced growth of specific B cinerea isolates but no reduction in growth of any B 487

Page 27 of 54

cinerea isolates tested was found This suggests that utilization of pectin as a carbon 488

source was not a contributing factor to alterations in B cinerea susceptibility It is 489

possible that changes in the cell wall composition affect the structural integrity of the 490

wall and affect hyphal penetration of specific B cinerea isolates However OGs (small 491

HG fragments) are likely released during the infection process (Ferrari et al 2013) 492

OGs function as DAMPs and initiate immune responses in Arabidopsis (Ferrari et al 493

2013) Treatment of Arabidopsis with OGs having a degree of polymerization (DP) of 494

10-15 sugar subunits was previously shown to enhance resistance to B cinerea (Ferrari495

et al 2007) We showed that pretreatment with OGs or macerozyme induced enhanced 496

immunity to B cinerea isolate Gallo 1 in wild-type Col-0 but only OGs and not 497

macerozyme induced immunity in gae1 gae6 (Figure 13) Our data suggest gae1 gae6 498

plants which have reduced pectin content may release less OGs and that this 499

reduction leads to the increased susceptibility to B cinerea 500

Consistently we found that macerozyme treatment released less soluble pectin 501

from gae1 gae6 cell walls than from wild-type walls Fractions containing soluble pectin 502

released from gae1 gae6 were also less active in inducing PAD3 gene expression than 503

the fractions released from wild-type walls (Figure 7) We also showed that less soluble 504

pectin accumulated in gae1 gae6 than in wild-type plants 48 h after treatment with B 505

cinerea (Figure 12) 506

We observed that loss of pectin leads to alterations in A thaliana immune 507

signaling Macerozyme treatment induced expression of PAD3 and the SA marker 508

genes PR1 and SID2 in wild-type Col-0 but not in gae1 gae6 whereas OG treatment 509

led to increased PAD3 and SID2 expression in both wild-type and gae1 gae6 (Figure 8 510

and 9) This suggests that although release of active soluble pectin in these plants 511

might be altered OG recognition is intact In fact cell walls isolated from qua1 another 512

mutant known to be reduced in HG released less OGs (DP1-6) than did wild-type cell 513

walls upon endopolygalacturonase treatment (Bouton et al 2002) This was also true 514

for gae1 gae6 cell walls as macerozyme treatment of isolated gae1 gae6 cell walls 515

released less soluble pectin than treatment of Col-0 cell walls Because the fraction 516

from gae1 gae6 plants was also less active in inducing PAD3 expression (Figure 7) it 517

seems possible that biologically active OGs were the active component inducing PAD3 518

Page 28 of 54

expression Interestingly expression of the JA marker genes JAZ5 and JAZ10 was 519

higher in gae1 gae6 than in wild-type mock-inoculated plants but was comparable to 520

wild-type in untreated plants suggesting that JA signaling in gae1 gae6 is 521

hyperresponsive (Figure 10 and Supplemental Figure 11) This induction of JA signaling 522

might be due to wounding stress incurred during the inoculation process JA and SA 523

signaling generally act antagonistically (Pieterse et al 2012) Increased JA signaling 524

activity might hence explain why mock- and macerozyme-treated gae1 gae6 plants 525

exhibited reduced expression of the SA marker PR1 and why OG treatment induced 526

expression of SID2 another SA marker to a lower extent in gae1 gae6 than in wild-type 527

plants (Figure 9) 528

No single immune signaling component was found to be the sole contributor to 529

the compromised immunity of gae1 gae6 to specific B cinerea isolates such as Gallo 1 530

and Fresa 525 These isolates grew better on pad3 while growth of Pepper another B 531

cinerea isolate that did not show altered virulence on gae1 gae6 was unaltered 532

(Supplemental Figure 9) This suggested that differences in camalexin sensitivity of 533

these B cinerea isolates might account for the differences in gae1 gae6 susceptibility 534

However camalexin accumulation and PAD3 expression was the same for wild-type 535

Col-0 and gae1 gae6 for the B cinerea isolates Fresa 525 and Gallo 1 (Figure 8) which 536

showed enhanced growth on gae1 gae6 plants It should be noted that B cinerea 537

virulence is often estimated by comparing lesion sizes Lesion size and measurements 538

of relative fungal growth by qPCR do not always show the same results For example 539

although Denby et al (2004) showed that Pepper causes larger lesions on pad3 than on 540

wild-type this study showed that relative fungal growth of Pepper on pad3 was 541

unaltered 542

We observed that callose deposition was strongly reduced in gae1 gae6 plants 543

72 h after treatment with B cinerea isolate Gallo 1 (Figure 11) possibly due to reduced 544

release of active soluble pectin Interestingly OG-induced PTI to an unspecified isolate 545

of B cinerea was intact in the pmr4 mutant which does not produce callose (Galletti et 546

al 2008) Due to the high SA levels in pmr4 plants (Nishimura et al 2003) and the fact 547

that different B cinerea isolates vary both in induction of and reaction to defense 548

responses (Kliebenstein et al 2005 Rowe et al 2010) we could not determine 549

Page 29 of 54

whether reduced callose deposition causes the increased susceptibility of gae1 gae6 to 550

B cinerea isolate Gallo 1551

We did not observe any changes in B cinerea growth either on cev1 ixr1 plants 552

which are known to have constitutively high JA and OPDA levels (Ellis et al 2002b) or 553

on plants exogenously treated with MeJA (Supplemental Figure 9) Because plants 554

were kept uncovered for 3 h after MeJA treatment it is unclear how much MeJA was 555

bioavailable in the plant However JAZ10 expression was induced 3 h after spraying 556

with MeJA (Supplemental Figure 10) Because pathogen growth generally depends on 557

the combination of a multitude of immune signaling events we conclude that multiple 558

changes to the immune signaling in gae1 gae6 together account for the altered B 559

cinerea growth phenotypes detected 560

We showed that expression of GAE1 and GAE6 is repressed by the bacterial 561

hemibiotroph Pma ES4326 This repression did not occur in an eds1 or pad4 562

background or in pbs3 at the 24-h time point (Figure 1 and Supplemental Figure 1) 563

During the Arabidopsis - Pma ES4326 interaction the expression of many defense 564

related genes is altered in a PAD4EDS1 dependent fashion (Wang et al 2008) Some 565

of these expression changes require SA CBP60g and SARD1 are thought to act 566

downstream of PAD4 and EDS1 but upstream of SA-dependent responses (Wang et 567

al 2011) PBS3 is also thought to act downstream of PAD4 and EDS1 and upstream of 568

SA but the set of genes whose expression it affects is only partially overlapping with 569

those affected by CBP60gSARD1 (Wang et al 2008 Wang et al 2011) Thus GAE1 570

and GAE6 repression is regulated by the part of the immune signaling network that 571

requires PAD4 EDS1 and PBS3 but is independent of CBP60gSARD1 and SA 572

Because EDS1 PAD4 and PBS3 are important components of the plant immune 573

signaling network (Wiermer et al 2005 Nobuta et al 2007) one might conclude that 574

repression of GAE1 and GAE6 may be part of the plant immune response to Pma 575

ES4326 B cinerea treatment also reduced GAE1 and GAE6 expression but this was 576

mostly independent of PAD4 (Figure 6) while both Pma ES4326 and B cinerea repress 577

GAE1 and GAE6 expression the host signaling mechanisms responsible likely differ 578

In summary our data show that Arabidopsis GAE1 and GAE6 are required for 579

pectin biosynthesis leaf flexibility and immunity to Pma ES4326 and to specific B 580

Page 30 of 54

cinerea isolates We used macerozyme a commercial pectinase to mimic the effect of 581

B cinerea pectinases Macerozyme treatment released less pectin from gae1 gae6 cell582

walls and induced less immune signaling in gae1 gae6 plants Less water soluble pectin 583

also accumulated in B cinerea infected gae1 gae6 plants and macerozyme-induced 584

immunity to B cinerea isolate Gallo 1 was reduced in these plants We conclude that 585

the reduced pectin content in these mutants may lead to reduced release of soluble 586

pectin including active OGs during pathogen attack and that this compromises 587

immunity 588

589

590

Page 31 of 54

METHODS 591

Plant Materials and Growth Conditions 592

Wild-type Columbia (Col-0) and all mutant plants (in Col-0 background) were 593

grown on sterilized BM2 germinating mix (Berger Inc Quebec Canada) in a controlled 594

environment chamber (Conviron Pembina ND USA) with a 12-h photoperiod under 595

100 microM m-2 s-1 fluorescent illumination at 22degC and 75 relative humidity Germplasm 596

used is described in the Accession Numbers section at the end of Methods 597

598

Data Analysis and Replicates 599

For biological replicates samples were obtained from separate plant tissues and 600

in many cases from plants grown in different flats In some experiments multiple 601

technical replicates were measured per biological replicate For statistical analysis all 602

technical replicates were averaged and the averages were considered one biological 603

replicate Independent experiments were performed at different times and often 604

consisted of multiple biological replicates per experiment 605

Data analysis was performed using mixed linear models in the R programming 606

environment (Bates et al 2015) unless otherwise indicated Generally genotypes 607

treatments and time points were used as fixed effects and replicate-specific effects like 608

different pots flats etc were considered random effects We reported p-values 609

obtained from the mixed linear model when less than 10 comparisons were performed 610

and q-values for more than 10 comparisons to correct for multiple comparison testing 611

Q-values were calculated using the Benjamini-Hochberg procedure (Benjamini and612

Hochberg 1995) unless specified otherwise 613

614

Pathogen Strains Growth Conditions and Pathogen Growth Assays 615

Growth of Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) and 616

bacterial growth assays were performed as previously described (Bethke et al 2014) 617

For qRT-PCR assays four week-old plants were inoculated with bacteria (OD600 = 618

0002) or mock (5 mM MgSO4) At least four leaves from two plants were collected at 619

the indicated time points per biological replicate 620

Page 32 of 54

Prior to each experiment Botrytis cinerea isolates were grown on 1x PDA 621

medium (Difco) for 10 d at room temperature All isolates used have been described 622

previously (Rowe and Kliebenstein 2007) The spores were washed from the surface of 623

the plate using an inoculation medium (1x Gamborgrsquos B-5 basal salt mixture (Sigma-624

Aldrich G5768) 2 (wv) glucose 10 mM phosphate buffer pH 64) and fungal hyphae 625

were removed from the suspension by filtering through four layers of cheesecloth The 626

concentration of spores was determined using a hemocytometer and adjusted to 25 x 627

105 spores mL-1 Four week-old Arabidopsis plants were inoculated by placing one 10-628

microL droplet of the B cinerea spore solution or inoculation medium (mock) on the adaxial 629

leaf surface of fully expanded leaves Inoculated plants were kept at 100 relative 630

humidity Infection sites were collected at various time points using a cork borer For 631

qRT-PCR assays at least four leaf discs from one plant were combined for each 632

biological sample For B cinerea growth assays at least 12 leaf discs from three to six 633

plants were pooled per biological sample Relative fungal growth was determined by 634

measuring the abundance of a fungal gene relative to a plant gene as described below 635

In brief DNA from infected tissue was extracted and equal amounts of total DNA were 636

used to perform qPCR reactions using the B cinerea cutinase A and the Arabidopsis 637

SK11 gene respectively Primers were described in (Gachon and Saindrenan 2004) 638

and are listed in Supplemental Table 1 Two technical replicates were combined for 639

each biological replicate B cinerea is a necrotrophic fungal pathogen and may destroy 640

plant DNA as disease progresses However decreases in the amount of plant DNA 641

over the course of our experiments were only observed at late time points with very 642

susceptible plants so the qPCR assay is generally a reasonable estimate of fungal 643

biomass (Supplemental Figure 16) 644

645

Treatment with Macerozyme or OGs 646

For PTI assays four week-old plants either were inoculated with 001 (wv) 647

macerozyme R-10 (Yakult) a Rhizopus polygalacturonase (EC 32115) that possesses 648

high pectinase and hemicellulase activity or were mock inoculated using heat-649

inactivated macerozyme Other similar plants were either inoculated with 250 or 500 microg 650

mL-1 OGs or mock inoculated with water One d later plants were infected with B 651

Page 33 of 54

cinerea and samples were collected immediately (0 dpi) and two or three d later To 652

investigate the role of MeJA on B cinerea growth plants were sprayed with 1mM MeJA 653

in water with 001 (vv) Tween 20 Covers were removed so that leaves could dry off 654

before plants were inoculated with B cinerea three h later Samples were harvested at 655

the indicated time points For expression analysis and MAPK activation assays plants 656

were either inoculated with 100 microg mL-1 OGs or mock inoculated with water OGs used 657

for experiments described in Figures 8 to 10 and Supplemental Figure 12 were a kind 658

gift from Simone Ferrari OGs used in experiments described in Figure 13 and 659

Supplemental Figures 11 13 and 15 were prepared as previously described (Kohorn et 660

al 2014) 661

662

Expression Analysis 663

Leaf tissue from inoculated plants was harvested at the indicated time points 664

flash frozen pulverized and RNA extracted using Trizol (Invitrogen) Quantitative RT-665

PCR was performed using the SuperScript III Platinum SYBR Green One-Step 666

quantitative RT-PCR kit (Invitrogen) and a Lightcycler 480 Real-Time PCR system 667

(Roche) as previously described (Truman and Glazebrook 2012) In brief equal 668

amounts of total RNA and a gene specific primer were used for each reaction The 669

crossing point (Cp) was calculated using the 2nd derivative max method provided with 670

the Lightcycler software for each amplification curve Each reaction was run with two 671

technical replicates and the Cp values for these replicates were averaged ACTIN2 was 672

used as a stably expressed reference gene At least three biological replicates each 673

were performed and analyzed using a mixed-linear model RT-PCR was performed to 674

verify T-DNA insertion lines using the One-Step RT-PCR kit (Qiagen) according to the 675

manufacturerrsquos instructions The primers used can be found in Supplemental Table 1 676

677

Electrolyte Leakage Assay 678

Fully expanded leaves of four week-old plants were inoculated with water (Milli-Q 679

grade) using a needleless syringe Leaf discs were collected using a cork borer and 680

three leaf discs from one leaf were placed with the adaxial surface down onto 2 mL 681

water in a 12-well cell culture plate Conductivity was measured using a Horiba B-173 682

Page 34 of 54

conductivity meter after 30 min and then the water was replaced Conductivity was 683

measured again 30 min later (1 h total) as indicated in Figure 3C Six biological 684

replicates each were performed in three independent experiments and means and SEs 685

were calculated Two-sided t-tests were performed for each time point 686

687

DAB and Aniline Blue Staining 688

Leaves from four week-old plants were either mock inoculated with B cinerea 689

inoculation medium or inoculated with 10 microL droplets of a B cinerea spore solution (25 690

x 105 spores mL-1) of the indicated isolates Samples were collected 72 h later For 33prime-691

diaminobenzidine (DAB) staining two to five inoculated leaves were vacuum infiltrated 692

with 33prime-Diaminobenzidine (1 mg mL-1 in water pH 38) solution and stained overnight 693

in the dark Leaves were destained using 100 ethanol at 65degC and then transferred to 694

50 (vv) glycerol and photographed 695

Callose was visualized by staining leaves with aniline blue as previously 696

described (Adam and Somerville 1996) Briefly leaves were vacuum infiltrated with 697

alcoholic lactophenol (one volume of phenolglycerollactic acidwater (1111) mixed 698

with two volumes of ethanol) and destained at 65degC Next leaves were moved through 699

an alcohol gradient (50 20 and 10 (vv) ethanol) into water The destained leaves 700

were stained for 30 min in 150 mM K2HPO4 (pH 95) with 001 (wv) aniline blue 701

Samples were mounted in water and examined using a Nikon Eclipse Ni-U microscope 702

at 10x magnification using a DAPI filter (excitation at 325-375 nm emission at 435-485 703

nm) For callose quantification six 200 microm x 200 microm squares were counted for each 704

sample type Callose deposits per mm2 were calculated and data analyzed using a t-705

test Note that there is an inherent uncertainty associated with such counts because 706

callose deposits may not all be in focus at the same time 707

708

MAPK Activity Assay 709

Fully expanded leaves of four week-old plants were inoculated with 001 710

macerozyme 100 microg mL-1 OGs 100 nM flg22 or water (mock) flg22 a peptide from 711

bacterial flagella that induces immune responses was used as a positive control for 712

Page 35 of 54

MAPK activation (Asai et al 2002) and was purchased from EZBiolab Inc (Westfield 713

IN USA) Samples were harvested 10 min later and immediately frozen in liquid 714

nitrogen Protein was extracted using an extraction buffer containing 25 mM Tris pH 78 715

75 mM NaCl 1 mM DTT 1 mM NaF 05mM Na3VO4 01 (vv) Tween 20 one 716

cOmplete Mini protease inhibitor cocktail tablet (Roche) and one PhosSTOP 717

phosphatase inhibitor cocktail tablet (Roche) per 10 mL buffer 15 microg of total protein 718

were electrophoresed on a 10 SDS-polyacrylamide gel and then blotted onto a PVDF 719

membrane (Bio-Rad 162-0177) Activated MAPKs were detected using p4442 MAPK 720

(Erk12) antibody (9102S Cell Signal 12500 in TBST (20 mM Tris pH 75 150 mM 721

NaCl 01 (vv) Tween 20)) Anti-Rabbit IgG-HRP (115000 in TBST A6154 Sigma) 722

and ECL Plus substrate (Pierce 32132) MPK3 and MPK6 were detected with an anti-723

MPK3 antibody (M8318 Sigma 12000 in TBST and 3 (wv) milk) or an anti-MPK6 724

antibody (A7104 Sigma 16000 in TBST and 3 (wv) milk) respectively The 725

secondary antibody and detection were the same as for the p4442 antibody Five 726

independent experiments were performed 727

728

Preparation of Alcohol Insoluble Residue (AIR) Pectin Extraction and Uronic Acid 729

Measurements 730

Fully expanded leaves from four week-old plants that were incubated for 48 h in 731

the dark to reduce starch were harvested flash frozen and pulverized AIR was 732

extracted by washing ground material twice in 70 (vv) ethanol three times in a 733

mixture of chloroform and methanol (11 vv) and once in acetone (Gille et al 2009) 734

and was then air dried Cell wall pectin was extracted from AIR with cell wall extraction 735

buffer (50 mM Trizma 50 mM CDTA pH 72) at 95degC for 15 min Samples were 736

homogenized using a paint shaker (Harbil 5G-HD) and glass beads (3 mm diameter) 737

For dot blot experiments 500 microL of cell wall extraction buffer were used per 10 mg of 738

AIR Debris was precipitated by centrifugation for 10 min at 10000 g 739

Total uronic acid content of pectin extracted as described above using 500 microL of 740

cell wall extraction buffer per 1 mg of AIR was measured as described previously 741

(Filisetti-Cozzi and Carpita 1991 van den Hoogen et al 1998) First 36 microL of pectin 742

extract were mixed with 4 microL of 4 M sulfamic acid Then 200 microL of sulfuric acid 743

Page 36 of 54

containing 120 mM sodium tetraborate was added and samples were incubated at 80degC 744

for 1 h Following cooling on ice the optical density of samples was measured at 490 745

nm Next 40 microL of m-hydroxydiphenyl reagent (100 microL of m-hydroxydiphenyl in dimethyl 746

sulfoxide at 100 mgmL mixed with 49 mL 80 (vv) sulfuric acid just before use) was 747

added and the samples were mixed The optical density at 490 nm was measured 748

again The optical density before m-hydroxydiphenyl reagent addition was subtracted 749

from the optical density measured after addition of the dye The concentration of uronic 750

acid was calculated using known amounts of GalA as a standard 751

752

Release of Pectin from Arabidopsis Cell Walls Using Macerozyme 753

AIR preparations from leaves of four week-old plants were treated with 025 754

(wv) macerozyme R-10 (Yakult) or water (mock) at a concentration of 100 microL per mg 755

AIR Samples were incubated for 3 h at room temperature on a vortex shaker and 756

centrifuged for 10 min at 12000 g Total uronic acid concentration of 5-fold diluted 757

supernatants was determined for two technical replicates each The concentration of 758

uronic acid was calculated using known amounts of GalA as a standard For 759

subsequent qRT-PCR experiments supernatants from multiple extractions were 760

combined boiled for 20 min to inactivate macerozyme diluted 10-fold with water and 761

inoculated into four week-old Col-0 plants Heat-inactivated 10-fold diluted macerozyme 762

solutions were used as mock treatment Samples were collected 3 h later and PAD3 763

expression was determined 764

765

Release of Pectin from Arabidopsis Leaves Infected with B cinerea 766

Fully expanded leaves of four week-old plants were either inoculated with up to 767

five 10 microL droplets of B cinerea spore solution (25 x 105 spores mL-1) or mock 768

inoculated with B cinerea inoculation medium For each biological replicate leaves with 769

a combined count of at least 20 infection sites were collected 48 h later AIR was 770

extracted as described above but plants were not dark-treated or freeze-dried and the 771

first ethanol step was performed using 85 (vv) ethanol To extract soluble pectin 772

fragments AIR samples were mixed with water at a concentration of 20 microL per infection 773

site incubated for 7 h at room temperature on a vortex shaker and centrifuged for 10 774

Page 37 of 54

min at 12000 g Total uronic acid concentration of supernatants was determined for two 775

technical replicates each The concentration of uronic acid was calculated using known 776

amounts of GalA as a standard 777

778

Measurement of Uronic Acids Neutral Monosaccharides and Cellulose Content in 779

Cell Walls 780

Neutral sugars and uronic acids from non-cellulosic polysaccharides in cell walls 781

(AIR) were released by trifluoroacetic acid (TFA) hydrolysis Approximately 1 mg of AIR 782

was hydrolyzed with 2 M TFA at 121degC for 90 min The TFA-soluble material was split 783

into two equal parts one for uronic acid and one for neutral sugar determination The 784

uronic acid content was determined by analyzing the hydrolyzate using a CarboPac 785

PA200 anion-exchange column with an ICS-3000 Dionex chromatography system 786

(Dionex Sunnyvale CA USA) The elution profile consisted of a linear gradient of 50 to 787

200 mM sodium acetate in 01 M NaOH in 10 min at 04 mL per min Alditol acetate 788

derivatives of neutral sugars were produced as described (York et al 1985) In brief 789

monosaccharides in the hydrolyzate were reduced with NaBH4 and peracetylated with 790

acetic anhydride and pyridine at 121degC for 20 min The generated alditol acetates were 791

analyzed by GC-MS Crystalline cellulose was measured according to Updegraff (1969) 792

After hydrolysis of noncellulosic polysaccharides from AIR with the Updegraff reagent 793

(acetic acid nitric acids water 812 (vv) ) the remaining pellet was hydrolyzed in 794

72 sulfuric acid The resulting glucose quantity was determined by the anthrone 795

method (Scott and Melvin 1953) 796

797

Dot Blot Experiments 798

Pectin solutions extracted using 50 microL of cell wall extraction buffer per mg AIR 799

were serially diluted and nitrocellulose membranes were spotted with 1 microL of the diluted 800

pectin solutions Membranes were dried overnight blocked with 5 milk (wv) in 1x 801

PBS (8 g L-1 NaCl 02 g L-1 KCl 144 g L-1 Na2HPO4 024 g L-1 KH2PO4 pH 74) and 802

probed with LM5 (Jones et al 1997) LM6 (Willats et al 1998) LM8 (Willats et al 803

2004) LM19 and LM20 (Verhertbruggen et al 2009a) or CCRC-M7 (Steffan et al 804

Page 38 of 54

1995) antibodies LM series antibodies were diluted 1250 and CCRC-M7 was diluted 805

1500 in 5 milk powder (Nestle Carnation) in 1 x PBS For LM series antibodies a 806

goat anti-Rat HRP conjugated antibody (Bethyl A110-105P 15000 diluted) and for 807

CCRC-M7 an anti-Mouse IgG HRP conjugate (Promega W402B 12500 diluted) was 808

used as secondary antibody in 5 milk powder in 1x PBS LM series antibodies were 809

obtained from plant probes (wwwplantprobesnet) and CCRC-M7 from CarboSource 810

(wwwcarbosourcenet) Membranes were washed after incubation with the primary and 811

secondary antibodies with 1x PBS Dot blots were developed using the ECL system 812

(GE Healthcare Amersham ECL prime) For quantification of signals dot blot results on 813

x-ray films were photographed using a CCD camera and intensities were measured814

using Image Jrsquos Integrated Density function Locally adjusted background was 815

subtracted As the dynamic range of the signal on an x-ray film was narrow non-816

linearity in the measured values was corrected for each genotype in each biological 817

replicate using the values from different dilutions in two technical replicates We noticed 818

that the residuals were not normally distributed and performed Box-Cox power 819

transformation (Box and Cox 1964) to obtain normally distributed values for the 820

subsequent statistical analysis of the data For LM19 Box-Cox selected power 821

transformation was done to the power of 038th and for LM20 to the power 075th After 822

correction of the transformed measured values with the effect of the biological 823

replicates the values were fit to a linear model with the Arabidopsis genotype as the 824

fixed effect This linear model was used both to generate the plots in Figures 5B and D 825

and to test significant differences 826

827

Camalexin Measurements 828

Four week-old plants were infected with B cinerea and samples (three leaf disks 829

from one plant each) were harvested at the indicated time points flash frozen and 830

pulverized Each sample was extracted with 300 microL of 90 methanol (vv) 50 microL of the 831

extract was run on an Agilent Lichrocart 250-4 RP18e 5-microm column using an Agilent 832

1100 series HPLC Camalexin was detected using a DiodeArray (DAD) at 330 nm and 833

with a fluorescence detector at emission 318 nmexcitation 385 nm (Agilent) Separation 834

was achieved using the following program with aqueous acetonitrile 5 min gradient 835

Page 39 of 54

from 63 to 69 acetonitrile 30 s gradient from 69 to 99 acetonitrile 2 min at 99 836

acetonitrile and a post-run equilibration of 35 min at 63 acetonitrile (Denby et al 837

2004 Kliebenstein et al 2005) Purified camalexin was used to produce a standard 838

curve to identify and quantitate camalexin 839

840

Accession Numbers 841

Microarray data used can be accessed at NCBI Gene Expression Omnnibus 842

(httpwwwncbinlmnihgovgeo) GSE18978 Sequence information for all Arabidopsis 843

genes described in this article can be found in TAIR (wwwarabidopsisorg) using the 844

following accession numbers PAD3 (CYP71B15 At3g26830) SID2 (ICS1 At1g74710) 845

GAE1 (At4g30440) GAE2 (At1g02000) GAE3 (At4g00110) GAE4 (At2g45310) GAE5 846

(At4g12250) GAE6 (At3g23820) PR1 (At2g14610) JAZ5 (At1g17380) JAZ10 847

(At5g13220) ACTIN2 (At3g18780) PDF12a (At5g44420) PDF12b (At2g26020) 848

AtSK11 (At5g26751) The sequence of the B cinerea cutinase Bc-CutA (Z69264) can 849

be found in the DNA Data Bank of Japan (httpgetentryddbjnigacjp) Germplasm 850

used included dde2-2 (At5g42650 (von Malek et al 2002)) pad3-1 (At3g26830 (Zhou 851

et al 1999)) pad4-1 (At3g52430 (Jirage et al 1999)) sid2-2 (At1g74710 (Wildermuth 852

et al 2001)) pbs3-2 (At5g13320 SALK_018225 (Nobuta et al 2007)) cbp60a-1 853

(At5g62570 SALK_124410 (Truman et al 2013)) cbp60g-1 sard1-2 (At5g26920 854

SALK_023199 At1g73805 SALK_052422 (Wang et al 2011) cbp60a-1 cbp60g-1 855

sard1-2 (Truman et al 2013) gae1-1 (At4g30440 SALK_085554) gae6-1 (At3g23280 856

SALK_104454) gae6-2 (At3g23280 SALK_017191) ixr1-1 cev1 (At5g05170 (Ellis 857

and Turner 2001 Scheible et al 2001)) eds1 plants were derived by introgression of 858

the Ler eds1-2 allele into Col-0 that contains two copies of EDS1 (At3g48090 859

At3g48080 (Bartsch et al 2006) mpk6-2 (At2g43790 SALK_073907 (Liu and Zhang 860

2004)) mpk3-DG (At3g45640 a fast neutron deletion mutant (Miles et al 2005)) and 861

npr1-1 (At1g64280 (Cao et al 1997)) T-DNA insertion lines were part of the SALK 862

collection (Alonso et al 2003) and were obtained from the Arabidopsis Biological 863

Resource Center 864

865

Supplemental Data 866

Page 40 of 54

Supplemental Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 867

Expression Does Not Require CBP60s or SID2 868

Supplemental Figure 2 Expression of GAE Family Members uponTreatment with Pma 869

ES4326 870

Supplemental Figure 3 Characterization of T-DNA Insertions in GAE1 and GAE6 871

Supplemental Figure 4 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves and 872

Are Slightly Smaller than Wild-Type Col-0 Plants 873

Supplemental Figure 5 Cell Walls of gae1 gae6 Plants Contain Less Uronic Acid than 874

Wild-Type Col-0 Cell Walls 875

Supplemental Figure 6 Antibodies Raised Against the RGI or XG Components of 876

Pectin Show No Difference in Binding to gae Mutant and Col-0 Cell Walls 877

Supplemental Figure 7 The Effect of B cinerea Treatment on the Expression of GAE 878

Family Genes 879

Supplemental Figure 8 Treatment with 001 Macerozyme Does Not Cause Any 880

Visible Damage to Col-0 Plants 881

Supplemental Figure 9 B cinerea Growth on Plants with Defects in Immune 882

Signaling 883

Supplemental Figure 10 MeJA-Induced Expression of the JA marker gene JAZ10 884

Supplemental Figure 11 The Expression of the JA Marker Gene JAZ5 Is Highly 885

Responsive in gae1 gae6 Plants 886

Supplemental Figure 12 The Expression of PDF12 Is Unaltered in gae1 gae6 Plants 887

Supplemental Figure 13 Macerozyme Treatment Activates MPK3 and MPK6 888

Supplemental Figure 14 B cinerea-Induced ROS Production in Wild-Type Col-0 and 889

gae1 gae6 Plants 890

Supplemental Figure 15 OG-Induced PTI Using 250 microg mL-1 OGs 891

Supplemental Figure 16 Abundance of Plant DNA in B cinerea Treated Plants 892

Supplemental Table 1 Primers used in this study 893

894

ACKNOWLEDGEMENTS 895

We thank William Truman for helpful discussions Simone Ferrari (Sapienza 896

Universita di Roma) for the generous gift of OGs Bruna Bucciarelli (USDA-ARS) for 897

Page 41 of 54

help with fluorescence microscopy the Arabidopsis Biological Resource Center for T-898

DNA insertion lines Paul Knoxrsquos lab (University of Leeds) for LM5 LM6 LM8 LM19 899

and LM20 antibodies and the Complex Carbohydrate Resource Center (University of 900

Georgia) for the CCRC-M7 antibody Most of this work was funded by the Division of 901

Chemical Sciences Geosciences and Biosciences Office of Basic Energy Sciences of 902

the US Department of Energy through Grant DE-FG02-05ER15670 to JG This work 903

was also supported by NSF awards IOS 1339125 and IOS 1021861 to DJK NSF 904

award IOS 1121425 to F K and the USDA National Institute of Food and Agriculture 905

Hatch project number CA-D-PLS-7033-H to DJK 906

907

AUTHOR CONTRIBUTIONS 908

GB and JG designed the research GB GX AT BL NES and NH 909

performed research DJK provided tools GB GX MP AD BL RAH and FK 910

analyzed data GB and JG wrote the paper with input from all authors 911

912

FIGURE LEGENDS 913

Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 Expression 914

Requires PAD4 EDS1 and PBS3 915

(A) Expression of GAE1 in leaves of wild-type Col-0 and mutant plants Expression was916

measured 24 or 32 h after either inoculation with Pma ES4326 (OD600 = 0002) or mock 917

inoculation with 5 mM MgSO4 and was then normalized to the level of ACTIN2 Data 918

from three biological replicates were combined using a mixed linear model Bars 919

represent mean log2-ratios to ACTIN2 plusmn SE Letters indicate significantly different 920

groups with q lt 005 where 005 represents the maximum false discovery rate at which 921

the test may be called significant 922

(B) Expression of GAE6 measured as described in (A)923

924

Figure 2 Susceptibility of gae6 and gae1 gae6 Plants to Pma ES4326 Is Increased 925

Relative to Wild-Type Col-0 926

Leaves of plants of the indicated genotypes were inoculated with Pma ES4326 (OD600 = 927

00002) as described in Methods Bacterial titers in leaves were determined either 928

Page 42 of 54

immediately (0 dpi) or after three days (3 dpi) Bars represent the mean plusmn SE of four 929

independent experiments each with 4 or 12 biological replicates at 0 and 3 dpi 930

respectively Data were combined using a mixed linear model Asterisks indicate 931

significant differences from Col-0 wild-type (q lt 005) Susceptible pad4 plants were 932

included as a positive control 933

934

Figure 3 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves 935

(A) Depiction of the assay for brittleness of gae mutant leaves Fully expanded leaves of936

four to five week-old plants were bent by 180ordm so that the adaxial surface of the leaf was 937

parallel with the soil Broken leaf petioles or midribs were considered brittle 938

(B) Quantification of broken midribs or petioles For each genotype 66 to 82 leaves 939

were analyzed for brittleness as described in (A) Bars represent the ratio of broken to 940

total number of leaves tested Results were analyzed using Fisherrsquos exact test Letters 941

indicate significantly different groups at p lt 005 942

(C) Conductivity measurements Leaves of four week-old plants were inoculated with943

deionized water and then leaf discs were cut out and placed on deionized water 944

Conductivity was measured at the indicated time points After 30 min the water was 945

replaced with fresh water (30 min f w) to account for wounding-related ion leakage from 946

the edges of the leaf discs Three independent experiments with six biological replicates 947

each were performed and data were analyzed using a t-test for each time point P-948

values were corrected using the Bonferroni method Data show means plusmn SE asterisks 949

indicate significant differences from wild-type Col-0 at q lt 001 950

951

Figure 4 Cell Walls of gae6 and gae1 gae6 Plants Are Reduced in GalA 952

(A) to (D) Levels of GalA (A) GlcA (B) cellulose (C) and neutral sugars (D) in alcohol953

insoluble residue (AIR) extracted from leaves of four week-old plants of the indicated 954

genotypes Bars represent means plusmn SE of four biological replicates combined using a 955

mixed linear model Letters indicate significantly different groups (p lt 005) 956

957

Figure 5 Cell Walls of gae1 gae6 Plants Are Reduced in Homogalacturonan 958

Page 43 of 54

Dot blot analyses with anti-LM19 and anti-LM20 antibodies Pectin was extracted from 959

AIR prepared from the indicated genotypes using 1 microL of extraction buffer per 10 microg of 960

AIR Pectin was serially diluted and one microL of undiluted 13 diluted and 19 diluted 961

samples were spotted on a nitrocellulose membrane Three biological replicates were 962

were spotted twice to obtain two technical replicates each All replicates produced 963

similar results 964

(A) Representative dot blot using anti-LM19 antibody965

(B) Relative signal intensities of the spots in the dot blot in (A)966

(C) Representative dot blot using anti-LM20 antibody967

(D) Relative signal intensities of the LM20 dot blot shown in (C)968

Relative signal intensities were measured using Image J software Measurements from 969

the three biological replicates were combined using a linear model after Box-Cox power 970

transformation as described in Methods Bars represent means plusmn SE Asterisks indicate 971

a difference from wild-type Col-0 at p lt 005 972

973

Figure 6 Growth of B cinerea on gae1 gae6 Plants and the Effect of B cinerea on 974

Expression of GAE1 and GAE6 975

(A) Growth of B cinerea isolates Leaves of Col-0 and gae1 gae6 plants were976

inoculated with 25 x 105 spores mL-1 of the B cinerea isolates indicated in the figure 977

Samples were collected two (2 dpi) and three (3 dpi) days later Bars represent means plusmn 978

SE of six biological replicates for each fungus combined using a mixed linear model 979

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 980

(B) and (C) Expression of GAE1 (B) and GAE6 (C) 48 h after inoculation with the B981

cinerea isolates indicated Expression levels were measured by qRT-PCR Bars 982

represent mean log2-ratios to ACTIN2 plusmn SE of three biological replicates combined 983

using a mixed linear model Letters indicate significantly different groups (q lt 005) 984

985

Figure 7 Levels of Soluble Pectin and Expression of PAD3 in Fractions Produced 986

Following Macerozyme Treatment of gae1 gae6 Cell Walls 987

(A) Schematic overview of the experimental procedure for (B) and (C)988

Page 44 of 54

(B) Uronic acid measurements Alcohol-insoluble residue (AIR) from leave tissue of four989

week-old wild-type Col-0 or gae1 gae6 plants was treated with water or 025 990

macerozyme and assayed for uronic acid levels Data from 11 biological replicates were 991

combined using a mixed linear model Bars represent means plusmn SE Letters indicate 992

significant differences (p lt 005) 993

(C) Expression of PAD3 in Col-0 plants treated with the fractions from (B) Extracts from994

(B) were heat inactivated pooled and diluted 5-fold with water Pooled extracts were995

inoculated into four week-old Col-0 plants PAD3 expression was measured 3 h after 996

inoculation and normalized to the level of ACTIN2 expression Data from four biological 997

replicates were combined using a mixed linear model Bars represent mean log2-ratios 998

to ACTIN2 plusmn SE Letters indicate significant differences (p lt 005) 999

1000

Figure 8 Macerozyme-Induced PAD3 Expression Is Absent in gae1 gae6 1001

(A) Expression of PAD3 after macerozyme treatment Four week-old plants were either1002

inoculated with 001 macerozyme or mock inoculated with water and PAD3 1003

expression was measured 3 h later by qRT-PCR Data from four biological replicates 1004

were combined using a mixed linear model Mean log2-ratios to ACTIN2 plusmn SE were 1005

plotted Letters indicate significantly different groups (p lt 005) 1006

(B) Expression of PAD3 after OG treatment PAD3 expression was measured as1007

described in (A) 3 h after inoculation with 100 microg mL-1 OGs or mock inoculation with 1008

water Data are from six biological replicates 1009

(C) PAD3 expression in untreated plants Three biological replicates were performed1010

and data analyzed as described in (A) 1011

(D) Camalexin accumulation after B cinerea treatment Camalexin accumulation was1012

measured in four week-old plants inoculated with the B cinerea strains shown in the 1013

figure Eight biological replicates each were combined using a mixed linear model 1014

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 1015

(E) PAD3 expression 28 h after treatment with the indicated B cinerea strains Data1016

from six biological replicates were collected and analyzed as described in (A) Data in 1017

all figure parts represent means plusmn SE Letters indicate significantly different groups (q lt 1018

005) 1019

Page 45 of 54

1020

Figure 9 Macerozyme-Induced Expression of the SA Marker Genes SID2 and PR1 1021

Is Abolished in gae1 gae6 Plants 1022

(A) to (H) Expression of PR1 (A) (C) (E) and (G) or SID2 (B) (D) (F) and (H) in four1023

week-old Col-0 and gae1 gae6 plants Data were combined using a mixed linear model 1024

Bars represent mean log2-ratios of expression vs ACTIN2 plusmn SE for all figure parts 1025

Letters indicate significantly different groups at p lt 005 for panels (A) to (D) (G) and 1026

(H) and qlt005 for panels (E) to (F) 1027

(A) and (B) Plants were treated with 001 macerozyme or mock treated with water1028

and samples were collected 3 h later Data from four biological replicates are shown 1029

(C) and (D) Plants were inoculated with 100 microg mL-1 OGs or mock treated with water1030

and samples collected 3 h later Data from six biological replicates are shown 1031

(E) and (F) Plants were inoculated with the indicated B cinerea isolates or mock1032

inoculated with B cinerea inoculation medium and samples collected 48 h later Data 1033

from eight biological replicates are shown 1034

(G) and (H) Expression in untreated plants Three biological replicates were performed1035

1036

Figure 10 The Expression of the JA Marker Gene JAZ10 is Highly Responsive in 1037

gae1 gae6 Plants 1038

(A) to (D) JAZ10 expression in four week-old plants Data were combined using a mixed1039

linear model Mean log2-ratios to ACTIN2 plusmn SE are shown Letters indicate significantly 1040

different groups at p lt 005 for panels (A) (B) and (D) and q lt 005 for panel (C) 1041

(A) Plants were inoculated with 001 macerozyme or mock inoculated using water and1042

samples were collected 3 h later Data from four biological replicates are shown 1043

(B) Plants were inoculated with 100 microg mL-1 OGs or mock inoculated using water and1044

samples collected 3 h later Data are from six biological replicates 1045

(C) JAZ10 expression 48 h after treatment with the indicated B cinerea strains Data1046

are from six biological replicates 1047

(D) JAZ10 expression in untreated plants Data are from three biological replicates1048

1049

Figure 11 Gallo 1-Induced Callose Deposition Is Reduced in gae1 gae6 Plants 1050

Page 46 of 54

(A) Callose deposition in four week-old Col-0 and gae1 gae6 plants Plants were1051

inoculated with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) or mock inoculated 1052

with B cinerea inoculation medium and samples were collected after 72 h Callose was 1053

stained with aniline blue and visualized using a Nikon Eclipse Ni-U microscope with a 1054

DAPI filter Three leaves were stained per treatment Scale bars are 100 microm Asterisks 1055

indicate fungal hyphae arrows indicate representative callose spots 1056

(B) Quantification of callose deposition Callose deposits were counted in 200 microm x 2001057

microm squares laid out across the images shown here Bars represent means plusmn SE from 1058

six squares each Letters indicate significantly different groups (p lt 005) according to a 1059

t-test analysis1060

1061

Figure 12 Less Soluble Pectin Accumulates in B cinerea-Infected gae1 gae6 1062

Leaves 1063

Four week-old Col-0 and gae1 gae6 plants were inoculated with 10 microL droplets of the 1064

indicated B cinerea isolate (25 x 105 spores mL-1) or mock inoculated with B cinerea 1065

inoculation medium Leaves were collected 48 h later AIR was extracted from leaf 1066

samples and water added (20 microL infection site) to extract soluble pectin fragments 1067

Samples were vortexed for 7 h centrifuged at 12000 g and the uronic acid 1068

concentration of the supernatant was measured Data from nine biological replicates 1069

were combined using a mixed linear model Means plusmn SE are shown Letters indicate 1070

significant differences (q lt 005) 1071

1072

Figure 13 Macerozyme-Induced PTI Is Lost in gae1 gae6 While OG-Induced PTI Is 1073

Unaltered 1074

(A) Macerozyme-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1075

inoculated with 001 macerozyme or mock inoculated with boiled macerozyme and 1076

were infected with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) 24 h later Samples 1077

were collected immediately (0 dpi) and after two (2 dpi) and three (3 dpi) days Data 1078

show mean plusmn SE of six to 14 biological replicates each combined using a linear model 1079

Letters indicate significantly different groups (q lt 005) 1080

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

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Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk RGadrinab C Heller C Jeske A Koesema E Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt MHazari N Hom E Karnes M Mulholland C Ndubaku R Schmidt I Guzman P Aguilar-Henonin L Schmid M Weigel DCarter DE Marchand T Risseeuw E Brogden D Zeko A Crosby WL Berry CC and Ecker JR (2003) Genome-wideinsertional mutagenesis of Arabidopsis thaliana Science 301 653-657

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Asai T Tena G Plotnikova J Willmann MR Chiu WL Gomez-Gomez L Boller T Ausubel FM and Sheen J (2002) MAPkinase signalling cascade in Arabidopsis innate immunity Nature 415 977-983

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Bartsch M Gobbato E Bednarek P Debey S Schultze JL Bautor J and Parker JE (2006) Salicylic acid-independentENHANCED DISEASE SUSCEPTIBILITY1 signaling in Arabidopsis immunity and cell death is regulated by the monooxygenaseFMO1 and the nudix hydrolase NUDT7 Plant Cell 18 1038-1051

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Bates D Machler M Bolker BM and Walker SC (2015) Fitting Linear Mixed-Effects Models Using lme4 J Stat Softw 67 1-48Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Benjamini Y and Hochberg Y (1995) Controlling the False Discovery Rate - a Practical and Powerful Approach to MultipleTesting J Roy Stat Soc B Met 57 289-300

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Bethke G Grundman RE Sreekanta S Truman W Katagiri F and Glazebrook J (2014) Arabidopsis PECTINMETHYLESTERASEs Contribute to Immunity against Pseudomonas syringae Plant Physiol 164 1093-1107

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Bouton S Leboeuf E Mouille G Leydecker MT Talbotec J Granier F Lahaye M Hofte H and Truong HN (2002)Quasimodo1 encodes a putative membrane-bound glycosyltransferase required for normal pectin synthesis and cell adhesion inArabidopsis Plant Cell 14 2577-2590

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Box GEP and Cox DR (1964) An analysis of transformations Journal of the Royal Statistical Society Series B 26 211-252Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Cao H Glazebrook J Clarke JD Volko S and Dong XN (1997) The Arabidopsis NPR1 gene that controls systemic acquiredresistance encodes a novel protein containing ankyrin repeats Cell 88 57-63

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Carpita N (2010) Update on Mechanisms of Plant Cell Wall Biosynthesis How plants make cellulose and other (1-gt4)-beta-D-glycans Plant Physiol

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Chisholm ST Coaker G Day B and Staskawicz BJ (2006) Host-microbe interactions Shaping the evolution of the plantimmune response Cell 124 803-814

Page 21 of 54

(Galletti et al 2011) Macerozyme treatment activated MPK3 and MPK6 in wild-type 356

Col-0 whereas MAPK activation in gae1 gae6 was slightly reduced in four out of five 357

experiments performed (Supplemental Figure 13) By contrast OG-induced MAPK 358

activity was similar or increased in gae1 gae6 in four out of five experiments 359

(Supplemental Figure 13) Additionally mock treatment induced MAPK activity in gae1 360

gae6 more strongly than it did in wild-type Col-0 in four out of five experiments 361

performed (Fig S13) Collectively these results illustrate that activation of several 362

immune signaling componentsmdashcamalexin synthesis SA and JA signaling and 363

possibly MAPK activationmdashby macerozyme is substantially altered in gae1 gae6 This 364

suggests that release of soluble pectin fragments possibly OGs by pectinolytic 365

enzymes might be reduced in gae1 gae6 plants while recognition of OGs is intact 366

367

Following B cinerea Infection Production of Soluble Pectin and Deposition of 368

Callose Are Reduced in gae1 gae6 Plants 369

Although pad3 plants were more susceptible to the B cinerea isolates Fresa 525 370

and Gallo 1 which also grew better on gae1 gae6 growth of the isolate Pepper was 371

similar on Col-0 and pad3 (Supplemental Figure 9A) However we did not detect any 372

significant changes in camalexin production after treatment with B cinerea isolate Gallo 373

1 and Fresa 525 in gae1 gae6 (Figure 8D) Neither did we detect changes in PAD3 374

expression after treatment with any B cinerea isolate in gae1 gae6 (Figure 8E) 375

Additionally SID2 and PR1 were similarly induced by all B cinerea isolates in both Col-376

0 and gae1 gae6 (Figure 9E and F) and sid2 plants which are deficient for pathogen-377

induced SA biosynthesis did not show any differences in relative B cinerea growth 378

(Supplemental Figure 9B) Expression of JAZ10 was induced more strongly by Gallo 1 379

treatment in gae1 gae6 than in wild-type Col-0 but not by Fresa 525 or Pepper (Fig 380

10C) while dde2 plants deficient for JA biosynthesis showed enhanced susceptibility to 381

all B cinerea isolates tested (Supplemental Figure 9B) Curiously exogenous 382

application of MeJA did not lead to altered relative fungal growth of B cinerea 383

(Supplemental Figure 9C) and cev1 isoxaben resistant 1 (ixr1) plants which have 384

constitutively high JA levels (Ellis and Turner 2001) did not show any changes in 385

susceptibility to the B cinerea isolates tested (Supplemental Figure 9D) Hence neither 386

Page 22 of 54

differences in B cinerea isolate susceptibility to camalexin nor altered SA or JA 387

signaling by themselves explained the enhanced susceptibility of gae1 gae6 to these B 388

cinerea isolates 389

Hydrogen peroxide accumulation in response to B cinerea isolates Fresa 525 390

Gallo 1 and Pepper was not obviously altered in gae1 gae6 plants (Supplemental Figure 391

14) However callose deposition upon treatment with B cinerea isolate Gallo 1 was 392

strongly reduced in gae1 gae6 (Figure 11) It was previously shown that OG treatment 393

induces callose deposition (Denoux et al 2008) suggesting that the reduction in 394

callose deposition might be due to reduced OG release in gae1 gae6 It is possible that 395

this reduction in callose deposition contributes to the reduced immunity of gae1 gae6 396

plants to B cinerea isolate Gallo 1 397

Because macerozyme could be used to release soluble likely low molecular 398

weight pectin from isolated cell walls we next investigated whether infection with B 399

cinerea was accompanied by accumulation of soluble pectin as well We prepared cell 400

walls (AIR fraction) from leaves infected with B cinerea incubated these cell walls with 401

water and measured uronic acid content of the extracts B cinerea treatment did in fact 402

lead to increased accumulation of soluble pectin in wild-type Col-0 plants when 403

compared to mock-treated samples (Figure 12) No such increased accumulation of 404

water-soluble pectin fractions was observed in gae1 gae6 (Figure 12) 405

406

Macerozyme-Induced Pattern-Triggered Immunity against B cinerea Is Abolished 407

in gae1 gae6 408

It was previously shown that OG treatment can induce patternndashtriggered 409

immunity (PTI) to B cinerea (Ferrari et al 2007) We tested macerozyme treatment to 410

determine whether it could also induce PTI to the B cinerea isolate Gallo 1 (Figure 13) 411

Indeed pretreatment with 001 macerozyme for 24 h led to reduced B cinerea growth 412

in wild-type Col-0 but not gae1 gae6 plants (Figure 13A) Interestingly mock and 413

macerozyme pretreated gae1 gae6 plants exhibited similar growth of B cinerea Gallo 1 414

as did macerozyme-treated Col-0 plants This result is likely due to activation of JA 415

signaling in gae1 gae6 during the pretreatment procedure (Figure 13C and D) OG-416

induced PTI was observed in both genotypes when using a high dose of OGs (Figure 417

Page 23 of 54

13B) whereas a lower dose of OGs only induced PTI in Col-0 (Supplemental Figure 418

15) This suggests that OG perception in gae1 gae6 at least above a certain threshold 419

is unaltered while release of immunity-activating pectin fragments possibly OGs is 420

421 reduced We conclude that immune responses induced by macerozyme treatment

422 which do not occur in gae1 gae6 plants are important for plant immunity to the B

423 cinerea isolate Gallo 1

Page 24 of 54

____________________________________________________________________________

Page 25 of 54

DISCUSSION 426

Here we showed that loss of GAE1 and GAE6 leads to a dramatic reduction in 427

pectin content in the cell wall (Figure 4) likely causing the brittleness of leaves that we 428

observed (Figure 3) Petioles and midribs of gae1 gae6 plants broke easily when bent 429

and inoculation of leaves with deionized water resulted in increased ion leakage 430

possibly due either to microscopic fractures of the cell wall induced by inoculation stress 431

or to a reduced capability of these walls to withstand increased turgor pressure The 432

increased brittleness was associated with altered cell wall composition In our analysis 433

the GalA content of gae1 gae6 leaf walls was reduced by more than 40 and the total 434

uronic acids by over 60 (Figure 4 and Supplemental Figure 5) This is a major change 435

in the overall cell wall composition considering that pectin makes up about 50 of the 436

plant cell walls found in Arabidopsis leaves (Zablackis et al 1995 Nobuta et al 2007 437

Harholt et al 2010) Interestingly quasimodo1 (qua1) plants which have a mutation in 438

a glycosyltransferase family 8 gene are reduced in GalA by 25 and show reduced 439

binding of HG specific antibodies (Bouton et al 2002) 440

Furthermore qua2 plants which have a mutation in a putative methyltransferase 441

have about 13 less total GalA and lack 50 of cell wall HG (Mouille et al 2007) 442

Likewise cgr2 cgr3 plants carrying mutations in another class of Golgi-localized 443

putative methyl-transferases have leaf cell walls that are reduced by about 40 in 444

uronic acid (Kim et al 2015) All three mutants are severely dwarfed (Bouton et al 445

2002 Mouille et al 2007 Kim et al 2015) Additionally qua1 and qua2 mutants show 446

a distorted overall shape likely due to reduced cell cohesion and are extremely fragile 447

(Bouton et al 2002 Mouille et al 2007) By contrast gae1 gae6 plants with a similar 448

strong reduction in GalA and total uronic acids have an overall normal shape and are 449

only slightly smaller than wild-type Col-0 (Supplemental Figure 4) In the wall analysis 450

performed here we did not detect changes in other wall polysaccharides or structures 451

that might compensate for the reduction of pectin in gae1 gae6 It is possible the severe 452

morphological alterations in qua2 and cgr2 cgr3 plants are due to reduction in pectin 453

methylesterification which is unaffected in gae1 gae6 It was speculated that quality 454

control mechanisms in the Golgi prevent secretion of inefficiently esterified pectin in 455

cgr2 cgr3 (Kim et al 2015) suggesting that the Golgi of these plants might over-456

Page 26 of 54

accumulate pectin and that this may contribute to the severe dwarf phenotype of these 457

plants The gae1 gae6 plants described here provide an opportunity to study the effects 458

of reduction of pectin in plants that do not show severe morphological phenotypes 459

We found that gae1 gae6 plants are also compromised in resistance to B 460

cinerea and Pma ES4326 indicating that loss of pectin reduces immunity to these 461

pathogens (Figure 2 and 6) We found that gae1 gae6 plants were slightly more 462

susceptible to Pma ES4326 but showed a strong increase in susceptibility to specific B 463

cinerea isolates This was not unexpected because B cinerea is known to interact with 464

pectin as part of its infection strategy It secretes various pectin-degrading enzymes 465

early during infection and requires some of these enzymes including PG1 PG2 and 466

PME1 for full virulence (ten Have et al 1998 Valette-Collet et al 2003 Kars et al 467

2005b Espino et al 2010) Additionally Arabidopsis plants expressing an antisense 468

construct of the polygalacturonase inhibitor PGIP1 are significantly more susceptible to 469

B cinerea (Ferrari et al 2006) On the other hand Pma ES4326 is not known to470

require cell wall degrading enzymes for full virulence even though it encodes two 471

pectate lyase genes in its genome (PMA4326_07094 and PMA4326_04621 472

wwwbacteriaensemblorg) B cinerea isolates are also known to be genetically quite 473

diverse in their polygalacturonase (PG) loci and genetic variability in PG2 is associated 474

with varying growth on pectin containing media (Rowe and Kliebenstein 2007) 475

Furthermore the contribution of PG1 and PG2 to B cinerea virulence seems to depend 476

on the specific B cinerea isolate and the host species (Kars et al 2005a Zhang and 477

Van Kan 2013) B cinerea isolates are also known to be differently sensitive to the anti-478

microbial secondary metabolite camalexin (Kliebenstein et al 2005) In line with these 479

observations the susceptibility of gae1 gae6 plants differed depending on the specific 480

B cinerea isolate Whereas Fresa 525 Gallo 1 UK Razz and KT grew better in gae1481

gae6 we detected no difference in Acacia Apple 517 DN Grape Pepper or Rasp 482

growth (Figure 6) 483

B cinerea was shown to metabolize pectin and to possibly utilize GalA as a484

carbon source during in planta growth (Zhang et al 2011 Zhang and Van Kan 2013) 485

Our data show that gae1 gae6 plants which are reduced in GalA content did allow 486

enhanced growth of specific B cinerea isolates but no reduction in growth of any B 487

Page 27 of 54

cinerea isolates tested was found This suggests that utilization of pectin as a carbon 488

source was not a contributing factor to alterations in B cinerea susceptibility It is 489

possible that changes in the cell wall composition affect the structural integrity of the 490

wall and affect hyphal penetration of specific B cinerea isolates However OGs (small 491

HG fragments) are likely released during the infection process (Ferrari et al 2013) 492

OGs function as DAMPs and initiate immune responses in Arabidopsis (Ferrari et al 493

2013) Treatment of Arabidopsis with OGs having a degree of polymerization (DP) of 494

10-15 sugar subunits was previously shown to enhance resistance to B cinerea (Ferrari495

et al 2007) We showed that pretreatment with OGs or macerozyme induced enhanced 496

immunity to B cinerea isolate Gallo 1 in wild-type Col-0 but only OGs and not 497

macerozyme induced immunity in gae1 gae6 (Figure 13) Our data suggest gae1 gae6 498

plants which have reduced pectin content may release less OGs and that this 499

reduction leads to the increased susceptibility to B cinerea 500

Consistently we found that macerozyme treatment released less soluble pectin 501

from gae1 gae6 cell walls than from wild-type walls Fractions containing soluble pectin 502

released from gae1 gae6 were also less active in inducing PAD3 gene expression than 503

the fractions released from wild-type walls (Figure 7) We also showed that less soluble 504

pectin accumulated in gae1 gae6 than in wild-type plants 48 h after treatment with B 505

cinerea (Figure 12) 506

We observed that loss of pectin leads to alterations in A thaliana immune 507

signaling Macerozyme treatment induced expression of PAD3 and the SA marker 508

genes PR1 and SID2 in wild-type Col-0 but not in gae1 gae6 whereas OG treatment 509

led to increased PAD3 and SID2 expression in both wild-type and gae1 gae6 (Figure 8 510

and 9) This suggests that although release of active soluble pectin in these plants 511

might be altered OG recognition is intact In fact cell walls isolated from qua1 another 512

mutant known to be reduced in HG released less OGs (DP1-6) than did wild-type cell 513

walls upon endopolygalacturonase treatment (Bouton et al 2002) This was also true 514

for gae1 gae6 cell walls as macerozyme treatment of isolated gae1 gae6 cell walls 515

released less soluble pectin than treatment of Col-0 cell walls Because the fraction 516

from gae1 gae6 plants was also less active in inducing PAD3 expression (Figure 7) it 517

seems possible that biologically active OGs were the active component inducing PAD3 518

Page 28 of 54

expression Interestingly expression of the JA marker genes JAZ5 and JAZ10 was 519

higher in gae1 gae6 than in wild-type mock-inoculated plants but was comparable to 520

wild-type in untreated plants suggesting that JA signaling in gae1 gae6 is 521

hyperresponsive (Figure 10 and Supplemental Figure 11) This induction of JA signaling 522

might be due to wounding stress incurred during the inoculation process JA and SA 523

signaling generally act antagonistically (Pieterse et al 2012) Increased JA signaling 524

activity might hence explain why mock- and macerozyme-treated gae1 gae6 plants 525

exhibited reduced expression of the SA marker PR1 and why OG treatment induced 526

expression of SID2 another SA marker to a lower extent in gae1 gae6 than in wild-type 527

plants (Figure 9) 528

No single immune signaling component was found to be the sole contributor to 529

the compromised immunity of gae1 gae6 to specific B cinerea isolates such as Gallo 1 530

and Fresa 525 These isolates grew better on pad3 while growth of Pepper another B 531

cinerea isolate that did not show altered virulence on gae1 gae6 was unaltered 532

(Supplemental Figure 9) This suggested that differences in camalexin sensitivity of 533

these B cinerea isolates might account for the differences in gae1 gae6 susceptibility 534

However camalexin accumulation and PAD3 expression was the same for wild-type 535

Col-0 and gae1 gae6 for the B cinerea isolates Fresa 525 and Gallo 1 (Figure 8) which 536

showed enhanced growth on gae1 gae6 plants It should be noted that B cinerea 537

virulence is often estimated by comparing lesion sizes Lesion size and measurements 538

of relative fungal growth by qPCR do not always show the same results For example 539

although Denby et al (2004) showed that Pepper causes larger lesions on pad3 than on 540

wild-type this study showed that relative fungal growth of Pepper on pad3 was 541

unaltered 542

We observed that callose deposition was strongly reduced in gae1 gae6 plants 543

72 h after treatment with B cinerea isolate Gallo 1 (Figure 11) possibly due to reduced 544

release of active soluble pectin Interestingly OG-induced PTI to an unspecified isolate 545

of B cinerea was intact in the pmr4 mutant which does not produce callose (Galletti et 546

al 2008) Due to the high SA levels in pmr4 plants (Nishimura et al 2003) and the fact 547

that different B cinerea isolates vary both in induction of and reaction to defense 548

responses (Kliebenstein et al 2005 Rowe et al 2010) we could not determine 549

Page 29 of 54

whether reduced callose deposition causes the increased susceptibility of gae1 gae6 to 550

B cinerea isolate Gallo 1551

We did not observe any changes in B cinerea growth either on cev1 ixr1 plants 552

which are known to have constitutively high JA and OPDA levels (Ellis et al 2002b) or 553

on plants exogenously treated with MeJA (Supplemental Figure 9) Because plants 554

were kept uncovered for 3 h after MeJA treatment it is unclear how much MeJA was 555

bioavailable in the plant However JAZ10 expression was induced 3 h after spraying 556

with MeJA (Supplemental Figure 10) Because pathogen growth generally depends on 557

the combination of a multitude of immune signaling events we conclude that multiple 558

changes to the immune signaling in gae1 gae6 together account for the altered B 559

cinerea growth phenotypes detected 560

We showed that expression of GAE1 and GAE6 is repressed by the bacterial 561

hemibiotroph Pma ES4326 This repression did not occur in an eds1 or pad4 562

background or in pbs3 at the 24-h time point (Figure 1 and Supplemental Figure 1) 563

During the Arabidopsis - Pma ES4326 interaction the expression of many defense 564

related genes is altered in a PAD4EDS1 dependent fashion (Wang et al 2008) Some 565

of these expression changes require SA CBP60g and SARD1 are thought to act 566

downstream of PAD4 and EDS1 but upstream of SA-dependent responses (Wang et 567

al 2011) PBS3 is also thought to act downstream of PAD4 and EDS1 and upstream of 568

SA but the set of genes whose expression it affects is only partially overlapping with 569

those affected by CBP60gSARD1 (Wang et al 2008 Wang et al 2011) Thus GAE1 570

and GAE6 repression is regulated by the part of the immune signaling network that 571

requires PAD4 EDS1 and PBS3 but is independent of CBP60gSARD1 and SA 572

Because EDS1 PAD4 and PBS3 are important components of the plant immune 573

signaling network (Wiermer et al 2005 Nobuta et al 2007) one might conclude that 574

repression of GAE1 and GAE6 may be part of the plant immune response to Pma 575

ES4326 B cinerea treatment also reduced GAE1 and GAE6 expression but this was 576

mostly independent of PAD4 (Figure 6) while both Pma ES4326 and B cinerea repress 577

GAE1 and GAE6 expression the host signaling mechanisms responsible likely differ 578

In summary our data show that Arabidopsis GAE1 and GAE6 are required for 579

pectin biosynthesis leaf flexibility and immunity to Pma ES4326 and to specific B 580

Page 30 of 54

cinerea isolates We used macerozyme a commercial pectinase to mimic the effect of 581

B cinerea pectinases Macerozyme treatment released less pectin from gae1 gae6 cell582

walls and induced less immune signaling in gae1 gae6 plants Less water soluble pectin 583

also accumulated in B cinerea infected gae1 gae6 plants and macerozyme-induced 584

immunity to B cinerea isolate Gallo 1 was reduced in these plants We conclude that 585

the reduced pectin content in these mutants may lead to reduced release of soluble 586

pectin including active OGs during pathogen attack and that this compromises 587

immunity 588

589

590

Page 31 of 54

METHODS 591

Plant Materials and Growth Conditions 592

Wild-type Columbia (Col-0) and all mutant plants (in Col-0 background) were 593

grown on sterilized BM2 germinating mix (Berger Inc Quebec Canada) in a controlled 594

environment chamber (Conviron Pembina ND USA) with a 12-h photoperiod under 595

100 microM m-2 s-1 fluorescent illumination at 22degC and 75 relative humidity Germplasm 596

used is described in the Accession Numbers section at the end of Methods 597

598

Data Analysis and Replicates 599

For biological replicates samples were obtained from separate plant tissues and 600

in many cases from plants grown in different flats In some experiments multiple 601

technical replicates were measured per biological replicate For statistical analysis all 602

technical replicates were averaged and the averages were considered one biological 603

replicate Independent experiments were performed at different times and often 604

consisted of multiple biological replicates per experiment 605

Data analysis was performed using mixed linear models in the R programming 606

environment (Bates et al 2015) unless otherwise indicated Generally genotypes 607

treatments and time points were used as fixed effects and replicate-specific effects like 608

different pots flats etc were considered random effects We reported p-values 609

obtained from the mixed linear model when less than 10 comparisons were performed 610

and q-values for more than 10 comparisons to correct for multiple comparison testing 611

Q-values were calculated using the Benjamini-Hochberg procedure (Benjamini and612

Hochberg 1995) unless specified otherwise 613

614

Pathogen Strains Growth Conditions and Pathogen Growth Assays 615

Growth of Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) and 616

bacterial growth assays were performed as previously described (Bethke et al 2014) 617

For qRT-PCR assays four week-old plants were inoculated with bacteria (OD600 = 618

0002) or mock (5 mM MgSO4) At least four leaves from two plants were collected at 619

the indicated time points per biological replicate 620

Page 32 of 54

Prior to each experiment Botrytis cinerea isolates were grown on 1x PDA 621

medium (Difco) for 10 d at room temperature All isolates used have been described 622

previously (Rowe and Kliebenstein 2007) The spores were washed from the surface of 623

the plate using an inoculation medium (1x Gamborgrsquos B-5 basal salt mixture (Sigma-624

Aldrich G5768) 2 (wv) glucose 10 mM phosphate buffer pH 64) and fungal hyphae 625

were removed from the suspension by filtering through four layers of cheesecloth The 626

concentration of spores was determined using a hemocytometer and adjusted to 25 x 627

105 spores mL-1 Four week-old Arabidopsis plants were inoculated by placing one 10-628

microL droplet of the B cinerea spore solution or inoculation medium (mock) on the adaxial 629

leaf surface of fully expanded leaves Inoculated plants were kept at 100 relative 630

humidity Infection sites were collected at various time points using a cork borer For 631

qRT-PCR assays at least four leaf discs from one plant were combined for each 632

biological sample For B cinerea growth assays at least 12 leaf discs from three to six 633

plants were pooled per biological sample Relative fungal growth was determined by 634

measuring the abundance of a fungal gene relative to a plant gene as described below 635

In brief DNA from infected tissue was extracted and equal amounts of total DNA were 636

used to perform qPCR reactions using the B cinerea cutinase A and the Arabidopsis 637

SK11 gene respectively Primers were described in (Gachon and Saindrenan 2004) 638

and are listed in Supplemental Table 1 Two technical replicates were combined for 639

each biological replicate B cinerea is a necrotrophic fungal pathogen and may destroy 640

plant DNA as disease progresses However decreases in the amount of plant DNA 641

over the course of our experiments were only observed at late time points with very 642

susceptible plants so the qPCR assay is generally a reasonable estimate of fungal 643

biomass (Supplemental Figure 16) 644

645

Treatment with Macerozyme or OGs 646

For PTI assays four week-old plants either were inoculated with 001 (wv) 647

macerozyme R-10 (Yakult) a Rhizopus polygalacturonase (EC 32115) that possesses 648

high pectinase and hemicellulase activity or were mock inoculated using heat-649

inactivated macerozyme Other similar plants were either inoculated with 250 or 500 microg 650

mL-1 OGs or mock inoculated with water One d later plants were infected with B 651

Page 33 of 54

cinerea and samples were collected immediately (0 dpi) and two or three d later To 652

investigate the role of MeJA on B cinerea growth plants were sprayed with 1mM MeJA 653

in water with 001 (vv) Tween 20 Covers were removed so that leaves could dry off 654

before plants were inoculated with B cinerea three h later Samples were harvested at 655

the indicated time points For expression analysis and MAPK activation assays plants 656

were either inoculated with 100 microg mL-1 OGs or mock inoculated with water OGs used 657

for experiments described in Figures 8 to 10 and Supplemental Figure 12 were a kind 658

gift from Simone Ferrari OGs used in experiments described in Figure 13 and 659

Supplemental Figures 11 13 and 15 were prepared as previously described (Kohorn et 660

al 2014) 661

662

Expression Analysis 663

Leaf tissue from inoculated plants was harvested at the indicated time points 664

flash frozen pulverized and RNA extracted using Trizol (Invitrogen) Quantitative RT-665

PCR was performed using the SuperScript III Platinum SYBR Green One-Step 666

quantitative RT-PCR kit (Invitrogen) and a Lightcycler 480 Real-Time PCR system 667

(Roche) as previously described (Truman and Glazebrook 2012) In brief equal 668

amounts of total RNA and a gene specific primer were used for each reaction The 669

crossing point (Cp) was calculated using the 2nd derivative max method provided with 670

the Lightcycler software for each amplification curve Each reaction was run with two 671

technical replicates and the Cp values for these replicates were averaged ACTIN2 was 672

used as a stably expressed reference gene At least three biological replicates each 673

were performed and analyzed using a mixed-linear model RT-PCR was performed to 674

verify T-DNA insertion lines using the One-Step RT-PCR kit (Qiagen) according to the 675

manufacturerrsquos instructions The primers used can be found in Supplemental Table 1 676

677

Electrolyte Leakage Assay 678

Fully expanded leaves of four week-old plants were inoculated with water (Milli-Q 679

grade) using a needleless syringe Leaf discs were collected using a cork borer and 680

three leaf discs from one leaf were placed with the adaxial surface down onto 2 mL 681

water in a 12-well cell culture plate Conductivity was measured using a Horiba B-173 682

Page 34 of 54

conductivity meter after 30 min and then the water was replaced Conductivity was 683

measured again 30 min later (1 h total) as indicated in Figure 3C Six biological 684

replicates each were performed in three independent experiments and means and SEs 685

were calculated Two-sided t-tests were performed for each time point 686

687

DAB and Aniline Blue Staining 688

Leaves from four week-old plants were either mock inoculated with B cinerea 689

inoculation medium or inoculated with 10 microL droplets of a B cinerea spore solution (25 690

x 105 spores mL-1) of the indicated isolates Samples were collected 72 h later For 33prime-691

diaminobenzidine (DAB) staining two to five inoculated leaves were vacuum infiltrated 692

with 33prime-Diaminobenzidine (1 mg mL-1 in water pH 38) solution and stained overnight 693

in the dark Leaves were destained using 100 ethanol at 65degC and then transferred to 694

50 (vv) glycerol and photographed 695

Callose was visualized by staining leaves with aniline blue as previously 696

described (Adam and Somerville 1996) Briefly leaves were vacuum infiltrated with 697

alcoholic lactophenol (one volume of phenolglycerollactic acidwater (1111) mixed 698

with two volumes of ethanol) and destained at 65degC Next leaves were moved through 699

an alcohol gradient (50 20 and 10 (vv) ethanol) into water The destained leaves 700

were stained for 30 min in 150 mM K2HPO4 (pH 95) with 001 (wv) aniline blue 701

Samples were mounted in water and examined using a Nikon Eclipse Ni-U microscope 702

at 10x magnification using a DAPI filter (excitation at 325-375 nm emission at 435-485 703

nm) For callose quantification six 200 microm x 200 microm squares were counted for each 704

sample type Callose deposits per mm2 were calculated and data analyzed using a t-705

test Note that there is an inherent uncertainty associated with such counts because 706

callose deposits may not all be in focus at the same time 707

708

MAPK Activity Assay 709

Fully expanded leaves of four week-old plants were inoculated with 001 710

macerozyme 100 microg mL-1 OGs 100 nM flg22 or water (mock) flg22 a peptide from 711

bacterial flagella that induces immune responses was used as a positive control for 712

Page 35 of 54

MAPK activation (Asai et al 2002) and was purchased from EZBiolab Inc (Westfield 713

IN USA) Samples were harvested 10 min later and immediately frozen in liquid 714

nitrogen Protein was extracted using an extraction buffer containing 25 mM Tris pH 78 715

75 mM NaCl 1 mM DTT 1 mM NaF 05mM Na3VO4 01 (vv) Tween 20 one 716

cOmplete Mini protease inhibitor cocktail tablet (Roche) and one PhosSTOP 717

phosphatase inhibitor cocktail tablet (Roche) per 10 mL buffer 15 microg of total protein 718

were electrophoresed on a 10 SDS-polyacrylamide gel and then blotted onto a PVDF 719

membrane (Bio-Rad 162-0177) Activated MAPKs were detected using p4442 MAPK 720

(Erk12) antibody (9102S Cell Signal 12500 in TBST (20 mM Tris pH 75 150 mM 721

NaCl 01 (vv) Tween 20)) Anti-Rabbit IgG-HRP (115000 in TBST A6154 Sigma) 722

and ECL Plus substrate (Pierce 32132) MPK3 and MPK6 were detected with an anti-723

MPK3 antibody (M8318 Sigma 12000 in TBST and 3 (wv) milk) or an anti-MPK6 724

antibody (A7104 Sigma 16000 in TBST and 3 (wv) milk) respectively The 725

secondary antibody and detection were the same as for the p4442 antibody Five 726

independent experiments were performed 727

728

Preparation of Alcohol Insoluble Residue (AIR) Pectin Extraction and Uronic Acid 729

Measurements 730

Fully expanded leaves from four week-old plants that were incubated for 48 h in 731

the dark to reduce starch were harvested flash frozen and pulverized AIR was 732

extracted by washing ground material twice in 70 (vv) ethanol three times in a 733

mixture of chloroform and methanol (11 vv) and once in acetone (Gille et al 2009) 734

and was then air dried Cell wall pectin was extracted from AIR with cell wall extraction 735

buffer (50 mM Trizma 50 mM CDTA pH 72) at 95degC for 15 min Samples were 736

homogenized using a paint shaker (Harbil 5G-HD) and glass beads (3 mm diameter) 737

For dot blot experiments 500 microL of cell wall extraction buffer were used per 10 mg of 738

AIR Debris was precipitated by centrifugation for 10 min at 10000 g 739

Total uronic acid content of pectin extracted as described above using 500 microL of 740

cell wall extraction buffer per 1 mg of AIR was measured as described previously 741

(Filisetti-Cozzi and Carpita 1991 van den Hoogen et al 1998) First 36 microL of pectin 742

extract were mixed with 4 microL of 4 M sulfamic acid Then 200 microL of sulfuric acid 743

Page 36 of 54

containing 120 mM sodium tetraborate was added and samples were incubated at 80degC 744

for 1 h Following cooling on ice the optical density of samples was measured at 490 745

nm Next 40 microL of m-hydroxydiphenyl reagent (100 microL of m-hydroxydiphenyl in dimethyl 746

sulfoxide at 100 mgmL mixed with 49 mL 80 (vv) sulfuric acid just before use) was 747

added and the samples were mixed The optical density at 490 nm was measured 748

again The optical density before m-hydroxydiphenyl reagent addition was subtracted 749

from the optical density measured after addition of the dye The concentration of uronic 750

acid was calculated using known amounts of GalA as a standard 751

752

Release of Pectin from Arabidopsis Cell Walls Using Macerozyme 753

AIR preparations from leaves of four week-old plants were treated with 025 754

(wv) macerozyme R-10 (Yakult) or water (mock) at a concentration of 100 microL per mg 755

AIR Samples were incubated for 3 h at room temperature on a vortex shaker and 756

centrifuged for 10 min at 12000 g Total uronic acid concentration of 5-fold diluted 757

supernatants was determined for two technical replicates each The concentration of 758

uronic acid was calculated using known amounts of GalA as a standard For 759

subsequent qRT-PCR experiments supernatants from multiple extractions were 760

combined boiled for 20 min to inactivate macerozyme diluted 10-fold with water and 761

inoculated into four week-old Col-0 plants Heat-inactivated 10-fold diluted macerozyme 762

solutions were used as mock treatment Samples were collected 3 h later and PAD3 763

expression was determined 764

765

Release of Pectin from Arabidopsis Leaves Infected with B cinerea 766

Fully expanded leaves of four week-old plants were either inoculated with up to 767

five 10 microL droplets of B cinerea spore solution (25 x 105 spores mL-1) or mock 768

inoculated with B cinerea inoculation medium For each biological replicate leaves with 769

a combined count of at least 20 infection sites were collected 48 h later AIR was 770

extracted as described above but plants were not dark-treated or freeze-dried and the 771

first ethanol step was performed using 85 (vv) ethanol To extract soluble pectin 772

fragments AIR samples were mixed with water at a concentration of 20 microL per infection 773

site incubated for 7 h at room temperature on a vortex shaker and centrifuged for 10 774

Page 37 of 54

min at 12000 g Total uronic acid concentration of supernatants was determined for two 775

technical replicates each The concentration of uronic acid was calculated using known 776

amounts of GalA as a standard 777

778

Measurement of Uronic Acids Neutral Monosaccharides and Cellulose Content in 779

Cell Walls 780

Neutral sugars and uronic acids from non-cellulosic polysaccharides in cell walls 781

(AIR) were released by trifluoroacetic acid (TFA) hydrolysis Approximately 1 mg of AIR 782

was hydrolyzed with 2 M TFA at 121degC for 90 min The TFA-soluble material was split 783

into two equal parts one for uronic acid and one for neutral sugar determination The 784

uronic acid content was determined by analyzing the hydrolyzate using a CarboPac 785

PA200 anion-exchange column with an ICS-3000 Dionex chromatography system 786

(Dionex Sunnyvale CA USA) The elution profile consisted of a linear gradient of 50 to 787

200 mM sodium acetate in 01 M NaOH in 10 min at 04 mL per min Alditol acetate 788

derivatives of neutral sugars were produced as described (York et al 1985) In brief 789

monosaccharides in the hydrolyzate were reduced with NaBH4 and peracetylated with 790

acetic anhydride and pyridine at 121degC for 20 min The generated alditol acetates were 791

analyzed by GC-MS Crystalline cellulose was measured according to Updegraff (1969) 792

After hydrolysis of noncellulosic polysaccharides from AIR with the Updegraff reagent 793

(acetic acid nitric acids water 812 (vv) ) the remaining pellet was hydrolyzed in 794

72 sulfuric acid The resulting glucose quantity was determined by the anthrone 795

method (Scott and Melvin 1953) 796

797

Dot Blot Experiments 798

Pectin solutions extracted using 50 microL of cell wall extraction buffer per mg AIR 799

were serially diluted and nitrocellulose membranes were spotted with 1 microL of the diluted 800

pectin solutions Membranes were dried overnight blocked with 5 milk (wv) in 1x 801

PBS (8 g L-1 NaCl 02 g L-1 KCl 144 g L-1 Na2HPO4 024 g L-1 KH2PO4 pH 74) and 802

probed with LM5 (Jones et al 1997) LM6 (Willats et al 1998) LM8 (Willats et al 803

2004) LM19 and LM20 (Verhertbruggen et al 2009a) or CCRC-M7 (Steffan et al 804

Page 38 of 54

1995) antibodies LM series antibodies were diluted 1250 and CCRC-M7 was diluted 805

1500 in 5 milk powder (Nestle Carnation) in 1 x PBS For LM series antibodies a 806

goat anti-Rat HRP conjugated antibody (Bethyl A110-105P 15000 diluted) and for 807

CCRC-M7 an anti-Mouse IgG HRP conjugate (Promega W402B 12500 diluted) was 808

used as secondary antibody in 5 milk powder in 1x PBS LM series antibodies were 809

obtained from plant probes (wwwplantprobesnet) and CCRC-M7 from CarboSource 810

(wwwcarbosourcenet) Membranes were washed after incubation with the primary and 811

secondary antibodies with 1x PBS Dot blots were developed using the ECL system 812

(GE Healthcare Amersham ECL prime) For quantification of signals dot blot results on 813

x-ray films were photographed using a CCD camera and intensities were measured814

using Image Jrsquos Integrated Density function Locally adjusted background was 815

subtracted As the dynamic range of the signal on an x-ray film was narrow non-816

linearity in the measured values was corrected for each genotype in each biological 817

replicate using the values from different dilutions in two technical replicates We noticed 818

that the residuals were not normally distributed and performed Box-Cox power 819

transformation (Box and Cox 1964) to obtain normally distributed values for the 820

subsequent statistical analysis of the data For LM19 Box-Cox selected power 821

transformation was done to the power of 038th and for LM20 to the power 075th After 822

correction of the transformed measured values with the effect of the biological 823

replicates the values were fit to a linear model with the Arabidopsis genotype as the 824

fixed effect This linear model was used both to generate the plots in Figures 5B and D 825

and to test significant differences 826

827

Camalexin Measurements 828

Four week-old plants were infected with B cinerea and samples (three leaf disks 829

from one plant each) were harvested at the indicated time points flash frozen and 830

pulverized Each sample was extracted with 300 microL of 90 methanol (vv) 50 microL of the 831

extract was run on an Agilent Lichrocart 250-4 RP18e 5-microm column using an Agilent 832

1100 series HPLC Camalexin was detected using a DiodeArray (DAD) at 330 nm and 833

with a fluorescence detector at emission 318 nmexcitation 385 nm (Agilent) Separation 834

was achieved using the following program with aqueous acetonitrile 5 min gradient 835

Page 39 of 54

from 63 to 69 acetonitrile 30 s gradient from 69 to 99 acetonitrile 2 min at 99 836

acetonitrile and a post-run equilibration of 35 min at 63 acetonitrile (Denby et al 837

2004 Kliebenstein et al 2005) Purified camalexin was used to produce a standard 838

curve to identify and quantitate camalexin 839

840

Accession Numbers 841

Microarray data used can be accessed at NCBI Gene Expression Omnnibus 842

(httpwwwncbinlmnihgovgeo) GSE18978 Sequence information for all Arabidopsis 843

genes described in this article can be found in TAIR (wwwarabidopsisorg) using the 844

following accession numbers PAD3 (CYP71B15 At3g26830) SID2 (ICS1 At1g74710) 845

GAE1 (At4g30440) GAE2 (At1g02000) GAE3 (At4g00110) GAE4 (At2g45310) GAE5 846

(At4g12250) GAE6 (At3g23820) PR1 (At2g14610) JAZ5 (At1g17380) JAZ10 847

(At5g13220) ACTIN2 (At3g18780) PDF12a (At5g44420) PDF12b (At2g26020) 848

AtSK11 (At5g26751) The sequence of the B cinerea cutinase Bc-CutA (Z69264) can 849

be found in the DNA Data Bank of Japan (httpgetentryddbjnigacjp) Germplasm 850

used included dde2-2 (At5g42650 (von Malek et al 2002)) pad3-1 (At3g26830 (Zhou 851

et al 1999)) pad4-1 (At3g52430 (Jirage et al 1999)) sid2-2 (At1g74710 (Wildermuth 852

et al 2001)) pbs3-2 (At5g13320 SALK_018225 (Nobuta et al 2007)) cbp60a-1 853

(At5g62570 SALK_124410 (Truman et al 2013)) cbp60g-1 sard1-2 (At5g26920 854

SALK_023199 At1g73805 SALK_052422 (Wang et al 2011) cbp60a-1 cbp60g-1 855

sard1-2 (Truman et al 2013) gae1-1 (At4g30440 SALK_085554) gae6-1 (At3g23280 856

SALK_104454) gae6-2 (At3g23280 SALK_017191) ixr1-1 cev1 (At5g05170 (Ellis 857

and Turner 2001 Scheible et al 2001)) eds1 plants were derived by introgression of 858

the Ler eds1-2 allele into Col-0 that contains two copies of EDS1 (At3g48090 859

At3g48080 (Bartsch et al 2006) mpk6-2 (At2g43790 SALK_073907 (Liu and Zhang 860

2004)) mpk3-DG (At3g45640 a fast neutron deletion mutant (Miles et al 2005)) and 861

npr1-1 (At1g64280 (Cao et al 1997)) T-DNA insertion lines were part of the SALK 862

collection (Alonso et al 2003) and were obtained from the Arabidopsis Biological 863

Resource Center 864

865

Supplemental Data 866

Page 40 of 54

Supplemental Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 867

Expression Does Not Require CBP60s or SID2 868

Supplemental Figure 2 Expression of GAE Family Members uponTreatment with Pma 869

ES4326 870

Supplemental Figure 3 Characterization of T-DNA Insertions in GAE1 and GAE6 871

Supplemental Figure 4 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves and 872

Are Slightly Smaller than Wild-Type Col-0 Plants 873

Supplemental Figure 5 Cell Walls of gae1 gae6 Plants Contain Less Uronic Acid than 874

Wild-Type Col-0 Cell Walls 875

Supplemental Figure 6 Antibodies Raised Against the RGI or XG Components of 876

Pectin Show No Difference in Binding to gae Mutant and Col-0 Cell Walls 877

Supplemental Figure 7 The Effect of B cinerea Treatment on the Expression of GAE 878

Family Genes 879

Supplemental Figure 8 Treatment with 001 Macerozyme Does Not Cause Any 880

Visible Damage to Col-0 Plants 881

Supplemental Figure 9 B cinerea Growth on Plants with Defects in Immune 882

Signaling 883

Supplemental Figure 10 MeJA-Induced Expression of the JA marker gene JAZ10 884

Supplemental Figure 11 The Expression of the JA Marker Gene JAZ5 Is Highly 885

Responsive in gae1 gae6 Plants 886

Supplemental Figure 12 The Expression of PDF12 Is Unaltered in gae1 gae6 Plants 887

Supplemental Figure 13 Macerozyme Treatment Activates MPK3 and MPK6 888

Supplemental Figure 14 B cinerea-Induced ROS Production in Wild-Type Col-0 and 889

gae1 gae6 Plants 890

Supplemental Figure 15 OG-Induced PTI Using 250 microg mL-1 OGs 891

Supplemental Figure 16 Abundance of Plant DNA in B cinerea Treated Plants 892

Supplemental Table 1 Primers used in this study 893

894

ACKNOWLEDGEMENTS 895

We thank William Truman for helpful discussions Simone Ferrari (Sapienza 896

Universita di Roma) for the generous gift of OGs Bruna Bucciarelli (USDA-ARS) for 897

Page 41 of 54

help with fluorescence microscopy the Arabidopsis Biological Resource Center for T-898

DNA insertion lines Paul Knoxrsquos lab (University of Leeds) for LM5 LM6 LM8 LM19 899

and LM20 antibodies and the Complex Carbohydrate Resource Center (University of 900

Georgia) for the CCRC-M7 antibody Most of this work was funded by the Division of 901

Chemical Sciences Geosciences and Biosciences Office of Basic Energy Sciences of 902

the US Department of Energy through Grant DE-FG02-05ER15670 to JG This work 903

was also supported by NSF awards IOS 1339125 and IOS 1021861 to DJK NSF 904

award IOS 1121425 to F K and the USDA National Institute of Food and Agriculture 905

Hatch project number CA-D-PLS-7033-H to DJK 906

907

AUTHOR CONTRIBUTIONS 908

GB and JG designed the research GB GX AT BL NES and NH 909

performed research DJK provided tools GB GX MP AD BL RAH and FK 910

analyzed data GB and JG wrote the paper with input from all authors 911

912

FIGURE LEGENDS 913

Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 Expression 914

Requires PAD4 EDS1 and PBS3 915

(A) Expression of GAE1 in leaves of wild-type Col-0 and mutant plants Expression was916

measured 24 or 32 h after either inoculation with Pma ES4326 (OD600 = 0002) or mock 917

inoculation with 5 mM MgSO4 and was then normalized to the level of ACTIN2 Data 918

from three biological replicates were combined using a mixed linear model Bars 919

represent mean log2-ratios to ACTIN2 plusmn SE Letters indicate significantly different 920

groups with q lt 005 where 005 represents the maximum false discovery rate at which 921

the test may be called significant 922

(B) Expression of GAE6 measured as described in (A)923

924

Figure 2 Susceptibility of gae6 and gae1 gae6 Plants to Pma ES4326 Is Increased 925

Relative to Wild-Type Col-0 926

Leaves of plants of the indicated genotypes were inoculated with Pma ES4326 (OD600 = 927

00002) as described in Methods Bacterial titers in leaves were determined either 928

Page 42 of 54

immediately (0 dpi) or after three days (3 dpi) Bars represent the mean plusmn SE of four 929

independent experiments each with 4 or 12 biological replicates at 0 and 3 dpi 930

respectively Data were combined using a mixed linear model Asterisks indicate 931

significant differences from Col-0 wild-type (q lt 005) Susceptible pad4 plants were 932

included as a positive control 933

934

Figure 3 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves 935

(A) Depiction of the assay for brittleness of gae mutant leaves Fully expanded leaves of936

four to five week-old plants were bent by 180ordm so that the adaxial surface of the leaf was 937

parallel with the soil Broken leaf petioles or midribs were considered brittle 938

(B) Quantification of broken midribs or petioles For each genotype 66 to 82 leaves 939

were analyzed for brittleness as described in (A) Bars represent the ratio of broken to 940

total number of leaves tested Results were analyzed using Fisherrsquos exact test Letters 941

indicate significantly different groups at p lt 005 942

(C) Conductivity measurements Leaves of four week-old plants were inoculated with943

deionized water and then leaf discs were cut out and placed on deionized water 944

Conductivity was measured at the indicated time points After 30 min the water was 945

replaced with fresh water (30 min f w) to account for wounding-related ion leakage from 946

the edges of the leaf discs Three independent experiments with six biological replicates 947

each were performed and data were analyzed using a t-test for each time point P-948

values were corrected using the Bonferroni method Data show means plusmn SE asterisks 949

indicate significant differences from wild-type Col-0 at q lt 001 950

951

Figure 4 Cell Walls of gae6 and gae1 gae6 Plants Are Reduced in GalA 952

(A) to (D) Levels of GalA (A) GlcA (B) cellulose (C) and neutral sugars (D) in alcohol953

insoluble residue (AIR) extracted from leaves of four week-old plants of the indicated 954

genotypes Bars represent means plusmn SE of four biological replicates combined using a 955

mixed linear model Letters indicate significantly different groups (p lt 005) 956

957

Figure 5 Cell Walls of gae1 gae6 Plants Are Reduced in Homogalacturonan 958

Page 43 of 54

Dot blot analyses with anti-LM19 and anti-LM20 antibodies Pectin was extracted from 959

AIR prepared from the indicated genotypes using 1 microL of extraction buffer per 10 microg of 960

AIR Pectin was serially diluted and one microL of undiluted 13 diluted and 19 diluted 961

samples were spotted on a nitrocellulose membrane Three biological replicates were 962

were spotted twice to obtain two technical replicates each All replicates produced 963

similar results 964

(A) Representative dot blot using anti-LM19 antibody965

(B) Relative signal intensities of the spots in the dot blot in (A)966

(C) Representative dot blot using anti-LM20 antibody967

(D) Relative signal intensities of the LM20 dot blot shown in (C)968

Relative signal intensities were measured using Image J software Measurements from 969

the three biological replicates were combined using a linear model after Box-Cox power 970

transformation as described in Methods Bars represent means plusmn SE Asterisks indicate 971

a difference from wild-type Col-0 at p lt 005 972

973

Figure 6 Growth of B cinerea on gae1 gae6 Plants and the Effect of B cinerea on 974

Expression of GAE1 and GAE6 975

(A) Growth of B cinerea isolates Leaves of Col-0 and gae1 gae6 plants were976

inoculated with 25 x 105 spores mL-1 of the B cinerea isolates indicated in the figure 977

Samples were collected two (2 dpi) and three (3 dpi) days later Bars represent means plusmn 978

SE of six biological replicates for each fungus combined using a mixed linear model 979

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 980

(B) and (C) Expression of GAE1 (B) and GAE6 (C) 48 h after inoculation with the B981

cinerea isolates indicated Expression levels were measured by qRT-PCR Bars 982

represent mean log2-ratios to ACTIN2 plusmn SE of three biological replicates combined 983

using a mixed linear model Letters indicate significantly different groups (q lt 005) 984

985

Figure 7 Levels of Soluble Pectin and Expression of PAD3 in Fractions Produced 986

Following Macerozyme Treatment of gae1 gae6 Cell Walls 987

(A) Schematic overview of the experimental procedure for (B) and (C)988

Page 44 of 54

(B) Uronic acid measurements Alcohol-insoluble residue (AIR) from leave tissue of four989

week-old wild-type Col-0 or gae1 gae6 plants was treated with water or 025 990

macerozyme and assayed for uronic acid levels Data from 11 biological replicates were 991

combined using a mixed linear model Bars represent means plusmn SE Letters indicate 992

significant differences (p lt 005) 993

(C) Expression of PAD3 in Col-0 plants treated with the fractions from (B) Extracts from994

(B) were heat inactivated pooled and diluted 5-fold with water Pooled extracts were995

inoculated into four week-old Col-0 plants PAD3 expression was measured 3 h after 996

inoculation and normalized to the level of ACTIN2 expression Data from four biological 997

replicates were combined using a mixed linear model Bars represent mean log2-ratios 998

to ACTIN2 plusmn SE Letters indicate significant differences (p lt 005) 999

1000

Figure 8 Macerozyme-Induced PAD3 Expression Is Absent in gae1 gae6 1001

(A) Expression of PAD3 after macerozyme treatment Four week-old plants were either1002

inoculated with 001 macerozyme or mock inoculated with water and PAD3 1003

expression was measured 3 h later by qRT-PCR Data from four biological replicates 1004

were combined using a mixed linear model Mean log2-ratios to ACTIN2 plusmn SE were 1005

plotted Letters indicate significantly different groups (p lt 005) 1006

(B) Expression of PAD3 after OG treatment PAD3 expression was measured as1007

described in (A) 3 h after inoculation with 100 microg mL-1 OGs or mock inoculation with 1008

water Data are from six biological replicates 1009

(C) PAD3 expression in untreated plants Three biological replicates were performed1010

and data analyzed as described in (A) 1011

(D) Camalexin accumulation after B cinerea treatment Camalexin accumulation was1012

measured in four week-old plants inoculated with the B cinerea strains shown in the 1013

figure Eight biological replicates each were combined using a mixed linear model 1014

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 1015

(E) PAD3 expression 28 h after treatment with the indicated B cinerea strains Data1016

from six biological replicates were collected and analyzed as described in (A) Data in 1017

all figure parts represent means plusmn SE Letters indicate significantly different groups (q lt 1018

005) 1019

Page 45 of 54

1020

Figure 9 Macerozyme-Induced Expression of the SA Marker Genes SID2 and PR1 1021

Is Abolished in gae1 gae6 Plants 1022

(A) to (H) Expression of PR1 (A) (C) (E) and (G) or SID2 (B) (D) (F) and (H) in four1023

week-old Col-0 and gae1 gae6 plants Data were combined using a mixed linear model 1024

Bars represent mean log2-ratios of expression vs ACTIN2 plusmn SE for all figure parts 1025

Letters indicate significantly different groups at p lt 005 for panels (A) to (D) (G) and 1026

(H) and qlt005 for panels (E) to (F) 1027

(A) and (B) Plants were treated with 001 macerozyme or mock treated with water1028

and samples were collected 3 h later Data from four biological replicates are shown 1029

(C) and (D) Plants were inoculated with 100 microg mL-1 OGs or mock treated with water1030

and samples collected 3 h later Data from six biological replicates are shown 1031

(E) and (F) Plants were inoculated with the indicated B cinerea isolates or mock1032

inoculated with B cinerea inoculation medium and samples collected 48 h later Data 1033

from eight biological replicates are shown 1034

(G) and (H) Expression in untreated plants Three biological replicates were performed1035

1036

Figure 10 The Expression of the JA Marker Gene JAZ10 is Highly Responsive in 1037

gae1 gae6 Plants 1038

(A) to (D) JAZ10 expression in four week-old plants Data were combined using a mixed1039

linear model Mean log2-ratios to ACTIN2 plusmn SE are shown Letters indicate significantly 1040

different groups at p lt 005 for panels (A) (B) and (D) and q lt 005 for panel (C) 1041

(A) Plants were inoculated with 001 macerozyme or mock inoculated using water and1042

samples were collected 3 h later Data from four biological replicates are shown 1043

(B) Plants were inoculated with 100 microg mL-1 OGs or mock inoculated using water and1044

samples collected 3 h later Data are from six biological replicates 1045

(C) JAZ10 expression 48 h after treatment with the indicated B cinerea strains Data1046

are from six biological replicates 1047

(D) JAZ10 expression in untreated plants Data are from three biological replicates1048

1049

Figure 11 Gallo 1-Induced Callose Deposition Is Reduced in gae1 gae6 Plants 1050

Page 46 of 54

(A) Callose deposition in four week-old Col-0 and gae1 gae6 plants Plants were1051

inoculated with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) or mock inoculated 1052

with B cinerea inoculation medium and samples were collected after 72 h Callose was 1053

stained with aniline blue and visualized using a Nikon Eclipse Ni-U microscope with a 1054

DAPI filter Three leaves were stained per treatment Scale bars are 100 microm Asterisks 1055

indicate fungal hyphae arrows indicate representative callose spots 1056

(B) Quantification of callose deposition Callose deposits were counted in 200 microm x 2001057

microm squares laid out across the images shown here Bars represent means plusmn SE from 1058

six squares each Letters indicate significantly different groups (p lt 005) according to a 1059

t-test analysis1060

1061

Figure 12 Less Soluble Pectin Accumulates in B cinerea-Infected gae1 gae6 1062

Leaves 1063

Four week-old Col-0 and gae1 gae6 plants were inoculated with 10 microL droplets of the 1064

indicated B cinerea isolate (25 x 105 spores mL-1) or mock inoculated with B cinerea 1065

inoculation medium Leaves were collected 48 h later AIR was extracted from leaf 1066

samples and water added (20 microL infection site) to extract soluble pectin fragments 1067

Samples were vortexed for 7 h centrifuged at 12000 g and the uronic acid 1068

concentration of the supernatant was measured Data from nine biological replicates 1069

were combined using a mixed linear model Means plusmn SE are shown Letters indicate 1070

significant differences (q lt 005) 1071

1072

Figure 13 Macerozyme-Induced PTI Is Lost in gae1 gae6 While OG-Induced PTI Is 1073

Unaltered 1074

(A) Macerozyme-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1075

inoculated with 001 macerozyme or mock inoculated with boiled macerozyme and 1076

were infected with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) 24 h later Samples 1077

were collected immediately (0 dpi) and after two (2 dpi) and three (3 dpi) days Data 1078

show mean plusmn SE of six to 14 biological replicates each combined using a linear model 1079

Letters indicate significantly different groups (q lt 005) 1080

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

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Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk RGadrinab C Heller C Jeske A Koesema E Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt MHazari N Hom E Karnes M Mulholland C Ndubaku R Schmidt I Guzman P Aguilar-Henonin L Schmid M Weigel DCarter DE Marchand T Risseeuw E Brogden D Zeko A Crosby WL Berry CC and Ecker JR (2003) Genome-wideinsertional mutagenesis of Arabidopsis thaliana Science 301 653-657

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Asai T Tena G Plotnikova J Willmann MR Chiu WL Gomez-Gomez L Boller T Ausubel FM and Sheen J (2002) MAPkinase signalling cascade in Arabidopsis innate immunity Nature 415 977-983

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Bartsch M Gobbato E Bednarek P Debey S Schultze JL Bautor J and Parker JE (2006) Salicylic acid-independentENHANCED DISEASE SUSCEPTIBILITY1 signaling in Arabidopsis immunity and cell death is regulated by the monooxygenaseFMO1 and the nudix hydrolase NUDT7 Plant Cell 18 1038-1051

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Bates D Machler M Bolker BM and Walker SC (2015) Fitting Linear Mixed-Effects Models Using lme4 J Stat Softw 67 1-48Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Benjamini Y and Hochberg Y (1995) Controlling the False Discovery Rate - a Practical and Powerful Approach to MultipleTesting J Roy Stat Soc B Met 57 289-300

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Bethke G Grundman RE Sreekanta S Truman W Katagiri F and Glazebrook J (2014) Arabidopsis PECTINMETHYLESTERASEs Contribute to Immunity against Pseudomonas syringae Plant Physiol 164 1093-1107

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Bouton S Leboeuf E Mouille G Leydecker MT Talbotec J Granier F Lahaye M Hofte H and Truong HN (2002)Quasimodo1 encodes a putative membrane-bound glycosyltransferase required for normal pectin synthesis and cell adhesion inArabidopsis Plant Cell 14 2577-2590

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Box GEP and Cox DR (1964) An analysis of transformations Journal of the Royal Statistical Society Series B 26 211-252Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Cao H Glazebrook J Clarke JD Volko S and Dong XN (1997) The Arabidopsis NPR1 gene that controls systemic acquiredresistance encodes a novel protein containing ankyrin repeats Cell 88 57-63

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Carpita N (2010) Update on Mechanisms of Plant Cell Wall Biosynthesis How plants make cellulose and other (1-gt4)-beta-D-glycans Plant Physiol

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Chisholm ST Coaker G Day B and Staskawicz BJ (2006) Host-microbe interactions Shaping the evolution of the plantimmune response Cell 124 803-814

Page 22 of 54

differences in B cinerea isolate susceptibility to camalexin nor altered SA or JA 387

signaling by themselves explained the enhanced susceptibility of gae1 gae6 to these B 388

cinerea isolates 389

Hydrogen peroxide accumulation in response to B cinerea isolates Fresa 525 390

Gallo 1 and Pepper was not obviously altered in gae1 gae6 plants (Supplemental Figure 391

14) However callose deposition upon treatment with B cinerea isolate Gallo 1 was 392

strongly reduced in gae1 gae6 (Figure 11) It was previously shown that OG treatment 393

induces callose deposition (Denoux et al 2008) suggesting that the reduction in 394

callose deposition might be due to reduced OG release in gae1 gae6 It is possible that 395

this reduction in callose deposition contributes to the reduced immunity of gae1 gae6 396

plants to B cinerea isolate Gallo 1 397

Because macerozyme could be used to release soluble likely low molecular 398

weight pectin from isolated cell walls we next investigated whether infection with B 399

cinerea was accompanied by accumulation of soluble pectin as well We prepared cell 400

walls (AIR fraction) from leaves infected with B cinerea incubated these cell walls with 401

water and measured uronic acid content of the extracts B cinerea treatment did in fact 402

lead to increased accumulation of soluble pectin in wild-type Col-0 plants when 403

compared to mock-treated samples (Figure 12) No such increased accumulation of 404

water-soluble pectin fractions was observed in gae1 gae6 (Figure 12) 405

406

Macerozyme-Induced Pattern-Triggered Immunity against B cinerea Is Abolished 407

in gae1 gae6 408

It was previously shown that OG treatment can induce patternndashtriggered 409

immunity (PTI) to B cinerea (Ferrari et al 2007) We tested macerozyme treatment to 410

determine whether it could also induce PTI to the B cinerea isolate Gallo 1 (Figure 13) 411

Indeed pretreatment with 001 macerozyme for 24 h led to reduced B cinerea growth 412

in wild-type Col-0 but not gae1 gae6 plants (Figure 13A) Interestingly mock and 413

macerozyme pretreated gae1 gae6 plants exhibited similar growth of B cinerea Gallo 1 414

as did macerozyme-treated Col-0 plants This result is likely due to activation of JA 415

signaling in gae1 gae6 during the pretreatment procedure (Figure 13C and D) OG-416

induced PTI was observed in both genotypes when using a high dose of OGs (Figure 417

Page 23 of 54

13B) whereas a lower dose of OGs only induced PTI in Col-0 (Supplemental Figure 418

15) This suggests that OG perception in gae1 gae6 at least above a certain threshold 419

is unaltered while release of immunity-activating pectin fragments possibly OGs is 420

421 reduced We conclude that immune responses induced by macerozyme treatment

422 which do not occur in gae1 gae6 plants are important for plant immunity to the B

423 cinerea isolate Gallo 1

Page 24 of 54

____________________________________________________________________________

Page 25 of 54

DISCUSSION 426

Here we showed that loss of GAE1 and GAE6 leads to a dramatic reduction in 427

pectin content in the cell wall (Figure 4) likely causing the brittleness of leaves that we 428

observed (Figure 3) Petioles and midribs of gae1 gae6 plants broke easily when bent 429

and inoculation of leaves with deionized water resulted in increased ion leakage 430

possibly due either to microscopic fractures of the cell wall induced by inoculation stress 431

or to a reduced capability of these walls to withstand increased turgor pressure The 432

increased brittleness was associated with altered cell wall composition In our analysis 433

the GalA content of gae1 gae6 leaf walls was reduced by more than 40 and the total 434

uronic acids by over 60 (Figure 4 and Supplemental Figure 5) This is a major change 435

in the overall cell wall composition considering that pectin makes up about 50 of the 436

plant cell walls found in Arabidopsis leaves (Zablackis et al 1995 Nobuta et al 2007 437

Harholt et al 2010) Interestingly quasimodo1 (qua1) plants which have a mutation in 438

a glycosyltransferase family 8 gene are reduced in GalA by 25 and show reduced 439

binding of HG specific antibodies (Bouton et al 2002) 440

Furthermore qua2 plants which have a mutation in a putative methyltransferase 441

have about 13 less total GalA and lack 50 of cell wall HG (Mouille et al 2007) 442

Likewise cgr2 cgr3 plants carrying mutations in another class of Golgi-localized 443

putative methyl-transferases have leaf cell walls that are reduced by about 40 in 444

uronic acid (Kim et al 2015) All three mutants are severely dwarfed (Bouton et al 445

2002 Mouille et al 2007 Kim et al 2015) Additionally qua1 and qua2 mutants show 446

a distorted overall shape likely due to reduced cell cohesion and are extremely fragile 447

(Bouton et al 2002 Mouille et al 2007) By contrast gae1 gae6 plants with a similar 448

strong reduction in GalA and total uronic acids have an overall normal shape and are 449

only slightly smaller than wild-type Col-0 (Supplemental Figure 4) In the wall analysis 450

performed here we did not detect changes in other wall polysaccharides or structures 451

that might compensate for the reduction of pectin in gae1 gae6 It is possible the severe 452

morphological alterations in qua2 and cgr2 cgr3 plants are due to reduction in pectin 453

methylesterification which is unaffected in gae1 gae6 It was speculated that quality 454

control mechanisms in the Golgi prevent secretion of inefficiently esterified pectin in 455

cgr2 cgr3 (Kim et al 2015) suggesting that the Golgi of these plants might over-456

Page 26 of 54

accumulate pectin and that this may contribute to the severe dwarf phenotype of these 457

plants The gae1 gae6 plants described here provide an opportunity to study the effects 458

of reduction of pectin in plants that do not show severe morphological phenotypes 459

We found that gae1 gae6 plants are also compromised in resistance to B 460

cinerea and Pma ES4326 indicating that loss of pectin reduces immunity to these 461

pathogens (Figure 2 and 6) We found that gae1 gae6 plants were slightly more 462

susceptible to Pma ES4326 but showed a strong increase in susceptibility to specific B 463

cinerea isolates This was not unexpected because B cinerea is known to interact with 464

pectin as part of its infection strategy It secretes various pectin-degrading enzymes 465

early during infection and requires some of these enzymes including PG1 PG2 and 466

PME1 for full virulence (ten Have et al 1998 Valette-Collet et al 2003 Kars et al 467

2005b Espino et al 2010) Additionally Arabidopsis plants expressing an antisense 468

construct of the polygalacturonase inhibitor PGIP1 are significantly more susceptible to 469

B cinerea (Ferrari et al 2006) On the other hand Pma ES4326 is not known to470

require cell wall degrading enzymes for full virulence even though it encodes two 471

pectate lyase genes in its genome (PMA4326_07094 and PMA4326_04621 472

wwwbacteriaensemblorg) B cinerea isolates are also known to be genetically quite 473

diverse in their polygalacturonase (PG) loci and genetic variability in PG2 is associated 474

with varying growth on pectin containing media (Rowe and Kliebenstein 2007) 475

Furthermore the contribution of PG1 and PG2 to B cinerea virulence seems to depend 476

on the specific B cinerea isolate and the host species (Kars et al 2005a Zhang and 477

Van Kan 2013) B cinerea isolates are also known to be differently sensitive to the anti-478

microbial secondary metabolite camalexin (Kliebenstein et al 2005) In line with these 479

observations the susceptibility of gae1 gae6 plants differed depending on the specific 480

B cinerea isolate Whereas Fresa 525 Gallo 1 UK Razz and KT grew better in gae1481

gae6 we detected no difference in Acacia Apple 517 DN Grape Pepper or Rasp 482

growth (Figure 6) 483

B cinerea was shown to metabolize pectin and to possibly utilize GalA as a484

carbon source during in planta growth (Zhang et al 2011 Zhang and Van Kan 2013) 485

Our data show that gae1 gae6 plants which are reduced in GalA content did allow 486

enhanced growth of specific B cinerea isolates but no reduction in growth of any B 487

Page 27 of 54

cinerea isolates tested was found This suggests that utilization of pectin as a carbon 488

source was not a contributing factor to alterations in B cinerea susceptibility It is 489

possible that changes in the cell wall composition affect the structural integrity of the 490

wall and affect hyphal penetration of specific B cinerea isolates However OGs (small 491

HG fragments) are likely released during the infection process (Ferrari et al 2013) 492

OGs function as DAMPs and initiate immune responses in Arabidopsis (Ferrari et al 493

2013) Treatment of Arabidopsis with OGs having a degree of polymerization (DP) of 494

10-15 sugar subunits was previously shown to enhance resistance to B cinerea (Ferrari495

et al 2007) We showed that pretreatment with OGs or macerozyme induced enhanced 496

immunity to B cinerea isolate Gallo 1 in wild-type Col-0 but only OGs and not 497

macerozyme induced immunity in gae1 gae6 (Figure 13) Our data suggest gae1 gae6 498

plants which have reduced pectin content may release less OGs and that this 499

reduction leads to the increased susceptibility to B cinerea 500

Consistently we found that macerozyme treatment released less soluble pectin 501

from gae1 gae6 cell walls than from wild-type walls Fractions containing soluble pectin 502

released from gae1 gae6 were also less active in inducing PAD3 gene expression than 503

the fractions released from wild-type walls (Figure 7) We also showed that less soluble 504

pectin accumulated in gae1 gae6 than in wild-type plants 48 h after treatment with B 505

cinerea (Figure 12) 506

We observed that loss of pectin leads to alterations in A thaliana immune 507

signaling Macerozyme treatment induced expression of PAD3 and the SA marker 508

genes PR1 and SID2 in wild-type Col-0 but not in gae1 gae6 whereas OG treatment 509

led to increased PAD3 and SID2 expression in both wild-type and gae1 gae6 (Figure 8 510

and 9) This suggests that although release of active soluble pectin in these plants 511

might be altered OG recognition is intact In fact cell walls isolated from qua1 another 512

mutant known to be reduced in HG released less OGs (DP1-6) than did wild-type cell 513

walls upon endopolygalacturonase treatment (Bouton et al 2002) This was also true 514

for gae1 gae6 cell walls as macerozyme treatment of isolated gae1 gae6 cell walls 515

released less soluble pectin than treatment of Col-0 cell walls Because the fraction 516

from gae1 gae6 plants was also less active in inducing PAD3 expression (Figure 7) it 517

seems possible that biologically active OGs were the active component inducing PAD3 518

Page 28 of 54

expression Interestingly expression of the JA marker genes JAZ5 and JAZ10 was 519

higher in gae1 gae6 than in wild-type mock-inoculated plants but was comparable to 520

wild-type in untreated plants suggesting that JA signaling in gae1 gae6 is 521

hyperresponsive (Figure 10 and Supplemental Figure 11) This induction of JA signaling 522

might be due to wounding stress incurred during the inoculation process JA and SA 523

signaling generally act antagonistically (Pieterse et al 2012) Increased JA signaling 524

activity might hence explain why mock- and macerozyme-treated gae1 gae6 plants 525

exhibited reduced expression of the SA marker PR1 and why OG treatment induced 526

expression of SID2 another SA marker to a lower extent in gae1 gae6 than in wild-type 527

plants (Figure 9) 528

No single immune signaling component was found to be the sole contributor to 529

the compromised immunity of gae1 gae6 to specific B cinerea isolates such as Gallo 1 530

and Fresa 525 These isolates grew better on pad3 while growth of Pepper another B 531

cinerea isolate that did not show altered virulence on gae1 gae6 was unaltered 532

(Supplemental Figure 9) This suggested that differences in camalexin sensitivity of 533

these B cinerea isolates might account for the differences in gae1 gae6 susceptibility 534

However camalexin accumulation and PAD3 expression was the same for wild-type 535

Col-0 and gae1 gae6 for the B cinerea isolates Fresa 525 and Gallo 1 (Figure 8) which 536

showed enhanced growth on gae1 gae6 plants It should be noted that B cinerea 537

virulence is often estimated by comparing lesion sizes Lesion size and measurements 538

of relative fungal growth by qPCR do not always show the same results For example 539

although Denby et al (2004) showed that Pepper causes larger lesions on pad3 than on 540

wild-type this study showed that relative fungal growth of Pepper on pad3 was 541

unaltered 542

We observed that callose deposition was strongly reduced in gae1 gae6 plants 543

72 h after treatment with B cinerea isolate Gallo 1 (Figure 11) possibly due to reduced 544

release of active soluble pectin Interestingly OG-induced PTI to an unspecified isolate 545

of B cinerea was intact in the pmr4 mutant which does not produce callose (Galletti et 546

al 2008) Due to the high SA levels in pmr4 plants (Nishimura et al 2003) and the fact 547

that different B cinerea isolates vary both in induction of and reaction to defense 548

responses (Kliebenstein et al 2005 Rowe et al 2010) we could not determine 549

Page 29 of 54

whether reduced callose deposition causes the increased susceptibility of gae1 gae6 to 550

B cinerea isolate Gallo 1551

We did not observe any changes in B cinerea growth either on cev1 ixr1 plants 552

which are known to have constitutively high JA and OPDA levels (Ellis et al 2002b) or 553

on plants exogenously treated with MeJA (Supplemental Figure 9) Because plants 554

were kept uncovered for 3 h after MeJA treatment it is unclear how much MeJA was 555

bioavailable in the plant However JAZ10 expression was induced 3 h after spraying 556

with MeJA (Supplemental Figure 10) Because pathogen growth generally depends on 557

the combination of a multitude of immune signaling events we conclude that multiple 558

changes to the immune signaling in gae1 gae6 together account for the altered B 559

cinerea growth phenotypes detected 560

We showed that expression of GAE1 and GAE6 is repressed by the bacterial 561

hemibiotroph Pma ES4326 This repression did not occur in an eds1 or pad4 562

background or in pbs3 at the 24-h time point (Figure 1 and Supplemental Figure 1) 563

During the Arabidopsis - Pma ES4326 interaction the expression of many defense 564

related genes is altered in a PAD4EDS1 dependent fashion (Wang et al 2008) Some 565

of these expression changes require SA CBP60g and SARD1 are thought to act 566

downstream of PAD4 and EDS1 but upstream of SA-dependent responses (Wang et 567

al 2011) PBS3 is also thought to act downstream of PAD4 and EDS1 and upstream of 568

SA but the set of genes whose expression it affects is only partially overlapping with 569

those affected by CBP60gSARD1 (Wang et al 2008 Wang et al 2011) Thus GAE1 570

and GAE6 repression is regulated by the part of the immune signaling network that 571

requires PAD4 EDS1 and PBS3 but is independent of CBP60gSARD1 and SA 572

Because EDS1 PAD4 and PBS3 are important components of the plant immune 573

signaling network (Wiermer et al 2005 Nobuta et al 2007) one might conclude that 574

repression of GAE1 and GAE6 may be part of the plant immune response to Pma 575

ES4326 B cinerea treatment also reduced GAE1 and GAE6 expression but this was 576

mostly independent of PAD4 (Figure 6) while both Pma ES4326 and B cinerea repress 577

GAE1 and GAE6 expression the host signaling mechanisms responsible likely differ 578

In summary our data show that Arabidopsis GAE1 and GAE6 are required for 579

pectin biosynthesis leaf flexibility and immunity to Pma ES4326 and to specific B 580

Page 30 of 54

cinerea isolates We used macerozyme a commercial pectinase to mimic the effect of 581

B cinerea pectinases Macerozyme treatment released less pectin from gae1 gae6 cell582

walls and induced less immune signaling in gae1 gae6 plants Less water soluble pectin 583

also accumulated in B cinerea infected gae1 gae6 plants and macerozyme-induced 584

immunity to B cinerea isolate Gallo 1 was reduced in these plants We conclude that 585

the reduced pectin content in these mutants may lead to reduced release of soluble 586

pectin including active OGs during pathogen attack and that this compromises 587

immunity 588

589

590

Page 31 of 54

METHODS 591

Plant Materials and Growth Conditions 592

Wild-type Columbia (Col-0) and all mutant plants (in Col-0 background) were 593

grown on sterilized BM2 germinating mix (Berger Inc Quebec Canada) in a controlled 594

environment chamber (Conviron Pembina ND USA) with a 12-h photoperiod under 595

100 microM m-2 s-1 fluorescent illumination at 22degC and 75 relative humidity Germplasm 596

used is described in the Accession Numbers section at the end of Methods 597

598

Data Analysis and Replicates 599

For biological replicates samples were obtained from separate plant tissues and 600

in many cases from plants grown in different flats In some experiments multiple 601

technical replicates were measured per biological replicate For statistical analysis all 602

technical replicates were averaged and the averages were considered one biological 603

replicate Independent experiments were performed at different times and often 604

consisted of multiple biological replicates per experiment 605

Data analysis was performed using mixed linear models in the R programming 606

environment (Bates et al 2015) unless otherwise indicated Generally genotypes 607

treatments and time points were used as fixed effects and replicate-specific effects like 608

different pots flats etc were considered random effects We reported p-values 609

obtained from the mixed linear model when less than 10 comparisons were performed 610

and q-values for more than 10 comparisons to correct for multiple comparison testing 611

Q-values were calculated using the Benjamini-Hochberg procedure (Benjamini and612

Hochberg 1995) unless specified otherwise 613

614

Pathogen Strains Growth Conditions and Pathogen Growth Assays 615

Growth of Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) and 616

bacterial growth assays were performed as previously described (Bethke et al 2014) 617

For qRT-PCR assays four week-old plants were inoculated with bacteria (OD600 = 618

0002) or mock (5 mM MgSO4) At least four leaves from two plants were collected at 619

the indicated time points per biological replicate 620

Page 32 of 54

Prior to each experiment Botrytis cinerea isolates were grown on 1x PDA 621

medium (Difco) for 10 d at room temperature All isolates used have been described 622

previously (Rowe and Kliebenstein 2007) The spores were washed from the surface of 623

the plate using an inoculation medium (1x Gamborgrsquos B-5 basal salt mixture (Sigma-624

Aldrich G5768) 2 (wv) glucose 10 mM phosphate buffer pH 64) and fungal hyphae 625

were removed from the suspension by filtering through four layers of cheesecloth The 626

concentration of spores was determined using a hemocytometer and adjusted to 25 x 627

105 spores mL-1 Four week-old Arabidopsis plants were inoculated by placing one 10-628

microL droplet of the B cinerea spore solution or inoculation medium (mock) on the adaxial 629

leaf surface of fully expanded leaves Inoculated plants were kept at 100 relative 630

humidity Infection sites were collected at various time points using a cork borer For 631

qRT-PCR assays at least four leaf discs from one plant were combined for each 632

biological sample For B cinerea growth assays at least 12 leaf discs from three to six 633

plants were pooled per biological sample Relative fungal growth was determined by 634

measuring the abundance of a fungal gene relative to a plant gene as described below 635

In brief DNA from infected tissue was extracted and equal amounts of total DNA were 636

used to perform qPCR reactions using the B cinerea cutinase A and the Arabidopsis 637

SK11 gene respectively Primers were described in (Gachon and Saindrenan 2004) 638

and are listed in Supplemental Table 1 Two technical replicates were combined for 639

each biological replicate B cinerea is a necrotrophic fungal pathogen and may destroy 640

plant DNA as disease progresses However decreases in the amount of plant DNA 641

over the course of our experiments were only observed at late time points with very 642

susceptible plants so the qPCR assay is generally a reasonable estimate of fungal 643

biomass (Supplemental Figure 16) 644

645

Treatment with Macerozyme or OGs 646

For PTI assays four week-old plants either were inoculated with 001 (wv) 647

macerozyme R-10 (Yakult) a Rhizopus polygalacturonase (EC 32115) that possesses 648

high pectinase and hemicellulase activity or were mock inoculated using heat-649

inactivated macerozyme Other similar plants were either inoculated with 250 or 500 microg 650

mL-1 OGs or mock inoculated with water One d later plants were infected with B 651

Page 33 of 54

cinerea and samples were collected immediately (0 dpi) and two or three d later To 652

investigate the role of MeJA on B cinerea growth plants were sprayed with 1mM MeJA 653

in water with 001 (vv) Tween 20 Covers were removed so that leaves could dry off 654

before plants were inoculated with B cinerea three h later Samples were harvested at 655

the indicated time points For expression analysis and MAPK activation assays plants 656

were either inoculated with 100 microg mL-1 OGs or mock inoculated with water OGs used 657

for experiments described in Figures 8 to 10 and Supplemental Figure 12 were a kind 658

gift from Simone Ferrari OGs used in experiments described in Figure 13 and 659

Supplemental Figures 11 13 and 15 were prepared as previously described (Kohorn et 660

al 2014) 661

662

Expression Analysis 663

Leaf tissue from inoculated plants was harvested at the indicated time points 664

flash frozen pulverized and RNA extracted using Trizol (Invitrogen) Quantitative RT-665

PCR was performed using the SuperScript III Platinum SYBR Green One-Step 666

quantitative RT-PCR kit (Invitrogen) and a Lightcycler 480 Real-Time PCR system 667

(Roche) as previously described (Truman and Glazebrook 2012) In brief equal 668

amounts of total RNA and a gene specific primer were used for each reaction The 669

crossing point (Cp) was calculated using the 2nd derivative max method provided with 670

the Lightcycler software for each amplification curve Each reaction was run with two 671

technical replicates and the Cp values for these replicates were averaged ACTIN2 was 672

used as a stably expressed reference gene At least three biological replicates each 673

were performed and analyzed using a mixed-linear model RT-PCR was performed to 674

verify T-DNA insertion lines using the One-Step RT-PCR kit (Qiagen) according to the 675

manufacturerrsquos instructions The primers used can be found in Supplemental Table 1 676

677

Electrolyte Leakage Assay 678

Fully expanded leaves of four week-old plants were inoculated with water (Milli-Q 679

grade) using a needleless syringe Leaf discs were collected using a cork borer and 680

three leaf discs from one leaf were placed with the adaxial surface down onto 2 mL 681

water in a 12-well cell culture plate Conductivity was measured using a Horiba B-173 682

Page 34 of 54

conductivity meter after 30 min and then the water was replaced Conductivity was 683

measured again 30 min later (1 h total) as indicated in Figure 3C Six biological 684

replicates each were performed in three independent experiments and means and SEs 685

were calculated Two-sided t-tests were performed for each time point 686

687

DAB and Aniline Blue Staining 688

Leaves from four week-old plants were either mock inoculated with B cinerea 689

inoculation medium or inoculated with 10 microL droplets of a B cinerea spore solution (25 690

x 105 spores mL-1) of the indicated isolates Samples were collected 72 h later For 33prime-691

diaminobenzidine (DAB) staining two to five inoculated leaves were vacuum infiltrated 692

with 33prime-Diaminobenzidine (1 mg mL-1 in water pH 38) solution and stained overnight 693

in the dark Leaves were destained using 100 ethanol at 65degC and then transferred to 694

50 (vv) glycerol and photographed 695

Callose was visualized by staining leaves with aniline blue as previously 696

described (Adam and Somerville 1996) Briefly leaves were vacuum infiltrated with 697

alcoholic lactophenol (one volume of phenolglycerollactic acidwater (1111) mixed 698

with two volumes of ethanol) and destained at 65degC Next leaves were moved through 699

an alcohol gradient (50 20 and 10 (vv) ethanol) into water The destained leaves 700

were stained for 30 min in 150 mM K2HPO4 (pH 95) with 001 (wv) aniline blue 701

Samples were mounted in water and examined using a Nikon Eclipse Ni-U microscope 702

at 10x magnification using a DAPI filter (excitation at 325-375 nm emission at 435-485 703

nm) For callose quantification six 200 microm x 200 microm squares were counted for each 704

sample type Callose deposits per mm2 were calculated and data analyzed using a t-705

test Note that there is an inherent uncertainty associated with such counts because 706

callose deposits may not all be in focus at the same time 707

708

MAPK Activity Assay 709

Fully expanded leaves of four week-old plants were inoculated with 001 710

macerozyme 100 microg mL-1 OGs 100 nM flg22 or water (mock) flg22 a peptide from 711

bacterial flagella that induces immune responses was used as a positive control for 712

Page 35 of 54

MAPK activation (Asai et al 2002) and was purchased from EZBiolab Inc (Westfield 713

IN USA) Samples were harvested 10 min later and immediately frozen in liquid 714

nitrogen Protein was extracted using an extraction buffer containing 25 mM Tris pH 78 715

75 mM NaCl 1 mM DTT 1 mM NaF 05mM Na3VO4 01 (vv) Tween 20 one 716

cOmplete Mini protease inhibitor cocktail tablet (Roche) and one PhosSTOP 717

phosphatase inhibitor cocktail tablet (Roche) per 10 mL buffer 15 microg of total protein 718

were electrophoresed on a 10 SDS-polyacrylamide gel and then blotted onto a PVDF 719

membrane (Bio-Rad 162-0177) Activated MAPKs were detected using p4442 MAPK 720

(Erk12) antibody (9102S Cell Signal 12500 in TBST (20 mM Tris pH 75 150 mM 721

NaCl 01 (vv) Tween 20)) Anti-Rabbit IgG-HRP (115000 in TBST A6154 Sigma) 722

and ECL Plus substrate (Pierce 32132) MPK3 and MPK6 were detected with an anti-723

MPK3 antibody (M8318 Sigma 12000 in TBST and 3 (wv) milk) or an anti-MPK6 724

antibody (A7104 Sigma 16000 in TBST and 3 (wv) milk) respectively The 725

secondary antibody and detection were the same as for the p4442 antibody Five 726

independent experiments were performed 727

728

Preparation of Alcohol Insoluble Residue (AIR) Pectin Extraction and Uronic Acid 729

Measurements 730

Fully expanded leaves from four week-old plants that were incubated for 48 h in 731

the dark to reduce starch were harvested flash frozen and pulverized AIR was 732

extracted by washing ground material twice in 70 (vv) ethanol three times in a 733

mixture of chloroform and methanol (11 vv) and once in acetone (Gille et al 2009) 734

and was then air dried Cell wall pectin was extracted from AIR with cell wall extraction 735

buffer (50 mM Trizma 50 mM CDTA pH 72) at 95degC for 15 min Samples were 736

homogenized using a paint shaker (Harbil 5G-HD) and glass beads (3 mm diameter) 737

For dot blot experiments 500 microL of cell wall extraction buffer were used per 10 mg of 738

AIR Debris was precipitated by centrifugation for 10 min at 10000 g 739

Total uronic acid content of pectin extracted as described above using 500 microL of 740

cell wall extraction buffer per 1 mg of AIR was measured as described previously 741

(Filisetti-Cozzi and Carpita 1991 van den Hoogen et al 1998) First 36 microL of pectin 742

extract were mixed with 4 microL of 4 M sulfamic acid Then 200 microL of sulfuric acid 743

Page 36 of 54

containing 120 mM sodium tetraborate was added and samples were incubated at 80degC 744

for 1 h Following cooling on ice the optical density of samples was measured at 490 745

nm Next 40 microL of m-hydroxydiphenyl reagent (100 microL of m-hydroxydiphenyl in dimethyl 746

sulfoxide at 100 mgmL mixed with 49 mL 80 (vv) sulfuric acid just before use) was 747

added and the samples were mixed The optical density at 490 nm was measured 748

again The optical density before m-hydroxydiphenyl reagent addition was subtracted 749

from the optical density measured after addition of the dye The concentration of uronic 750

acid was calculated using known amounts of GalA as a standard 751

752

Release of Pectin from Arabidopsis Cell Walls Using Macerozyme 753

AIR preparations from leaves of four week-old plants were treated with 025 754

(wv) macerozyme R-10 (Yakult) or water (mock) at a concentration of 100 microL per mg 755

AIR Samples were incubated for 3 h at room temperature on a vortex shaker and 756

centrifuged for 10 min at 12000 g Total uronic acid concentration of 5-fold diluted 757

supernatants was determined for two technical replicates each The concentration of 758

uronic acid was calculated using known amounts of GalA as a standard For 759

subsequent qRT-PCR experiments supernatants from multiple extractions were 760

combined boiled for 20 min to inactivate macerozyme diluted 10-fold with water and 761

inoculated into four week-old Col-0 plants Heat-inactivated 10-fold diluted macerozyme 762

solutions were used as mock treatment Samples were collected 3 h later and PAD3 763

expression was determined 764

765

Release of Pectin from Arabidopsis Leaves Infected with B cinerea 766

Fully expanded leaves of four week-old plants were either inoculated with up to 767

five 10 microL droplets of B cinerea spore solution (25 x 105 spores mL-1) or mock 768

inoculated with B cinerea inoculation medium For each biological replicate leaves with 769

a combined count of at least 20 infection sites were collected 48 h later AIR was 770

extracted as described above but plants were not dark-treated or freeze-dried and the 771

first ethanol step was performed using 85 (vv) ethanol To extract soluble pectin 772

fragments AIR samples were mixed with water at a concentration of 20 microL per infection 773

site incubated for 7 h at room temperature on a vortex shaker and centrifuged for 10 774

Page 37 of 54

min at 12000 g Total uronic acid concentration of supernatants was determined for two 775

technical replicates each The concentration of uronic acid was calculated using known 776

amounts of GalA as a standard 777

778

Measurement of Uronic Acids Neutral Monosaccharides and Cellulose Content in 779

Cell Walls 780

Neutral sugars and uronic acids from non-cellulosic polysaccharides in cell walls 781

(AIR) were released by trifluoroacetic acid (TFA) hydrolysis Approximately 1 mg of AIR 782

was hydrolyzed with 2 M TFA at 121degC for 90 min The TFA-soluble material was split 783

into two equal parts one for uronic acid and one for neutral sugar determination The 784

uronic acid content was determined by analyzing the hydrolyzate using a CarboPac 785

PA200 anion-exchange column with an ICS-3000 Dionex chromatography system 786

(Dionex Sunnyvale CA USA) The elution profile consisted of a linear gradient of 50 to 787

200 mM sodium acetate in 01 M NaOH in 10 min at 04 mL per min Alditol acetate 788

derivatives of neutral sugars were produced as described (York et al 1985) In brief 789

monosaccharides in the hydrolyzate were reduced with NaBH4 and peracetylated with 790

acetic anhydride and pyridine at 121degC for 20 min The generated alditol acetates were 791

analyzed by GC-MS Crystalline cellulose was measured according to Updegraff (1969) 792

After hydrolysis of noncellulosic polysaccharides from AIR with the Updegraff reagent 793

(acetic acid nitric acids water 812 (vv) ) the remaining pellet was hydrolyzed in 794

72 sulfuric acid The resulting glucose quantity was determined by the anthrone 795

method (Scott and Melvin 1953) 796

797

Dot Blot Experiments 798

Pectin solutions extracted using 50 microL of cell wall extraction buffer per mg AIR 799

were serially diluted and nitrocellulose membranes were spotted with 1 microL of the diluted 800

pectin solutions Membranes were dried overnight blocked with 5 milk (wv) in 1x 801

PBS (8 g L-1 NaCl 02 g L-1 KCl 144 g L-1 Na2HPO4 024 g L-1 KH2PO4 pH 74) and 802

probed with LM5 (Jones et al 1997) LM6 (Willats et al 1998) LM8 (Willats et al 803

2004) LM19 and LM20 (Verhertbruggen et al 2009a) or CCRC-M7 (Steffan et al 804

Page 38 of 54

1995) antibodies LM series antibodies were diluted 1250 and CCRC-M7 was diluted 805

1500 in 5 milk powder (Nestle Carnation) in 1 x PBS For LM series antibodies a 806

goat anti-Rat HRP conjugated antibody (Bethyl A110-105P 15000 diluted) and for 807

CCRC-M7 an anti-Mouse IgG HRP conjugate (Promega W402B 12500 diluted) was 808

used as secondary antibody in 5 milk powder in 1x PBS LM series antibodies were 809

obtained from plant probes (wwwplantprobesnet) and CCRC-M7 from CarboSource 810

(wwwcarbosourcenet) Membranes were washed after incubation with the primary and 811

secondary antibodies with 1x PBS Dot blots were developed using the ECL system 812

(GE Healthcare Amersham ECL prime) For quantification of signals dot blot results on 813

x-ray films were photographed using a CCD camera and intensities were measured814

using Image Jrsquos Integrated Density function Locally adjusted background was 815

subtracted As the dynamic range of the signal on an x-ray film was narrow non-816

linearity in the measured values was corrected for each genotype in each biological 817

replicate using the values from different dilutions in two technical replicates We noticed 818

that the residuals were not normally distributed and performed Box-Cox power 819

transformation (Box and Cox 1964) to obtain normally distributed values for the 820

subsequent statistical analysis of the data For LM19 Box-Cox selected power 821

transformation was done to the power of 038th and for LM20 to the power 075th After 822

correction of the transformed measured values with the effect of the biological 823

replicates the values were fit to a linear model with the Arabidopsis genotype as the 824

fixed effect This linear model was used both to generate the plots in Figures 5B and D 825

and to test significant differences 826

827

Camalexin Measurements 828

Four week-old plants were infected with B cinerea and samples (three leaf disks 829

from one plant each) were harvested at the indicated time points flash frozen and 830

pulverized Each sample was extracted with 300 microL of 90 methanol (vv) 50 microL of the 831

extract was run on an Agilent Lichrocart 250-4 RP18e 5-microm column using an Agilent 832

1100 series HPLC Camalexin was detected using a DiodeArray (DAD) at 330 nm and 833

with a fluorescence detector at emission 318 nmexcitation 385 nm (Agilent) Separation 834

was achieved using the following program with aqueous acetonitrile 5 min gradient 835

Page 39 of 54

from 63 to 69 acetonitrile 30 s gradient from 69 to 99 acetonitrile 2 min at 99 836

acetonitrile and a post-run equilibration of 35 min at 63 acetonitrile (Denby et al 837

2004 Kliebenstein et al 2005) Purified camalexin was used to produce a standard 838

curve to identify and quantitate camalexin 839

840

Accession Numbers 841

Microarray data used can be accessed at NCBI Gene Expression Omnnibus 842

(httpwwwncbinlmnihgovgeo) GSE18978 Sequence information for all Arabidopsis 843

genes described in this article can be found in TAIR (wwwarabidopsisorg) using the 844

following accession numbers PAD3 (CYP71B15 At3g26830) SID2 (ICS1 At1g74710) 845

GAE1 (At4g30440) GAE2 (At1g02000) GAE3 (At4g00110) GAE4 (At2g45310) GAE5 846

(At4g12250) GAE6 (At3g23820) PR1 (At2g14610) JAZ5 (At1g17380) JAZ10 847

(At5g13220) ACTIN2 (At3g18780) PDF12a (At5g44420) PDF12b (At2g26020) 848

AtSK11 (At5g26751) The sequence of the B cinerea cutinase Bc-CutA (Z69264) can 849

be found in the DNA Data Bank of Japan (httpgetentryddbjnigacjp) Germplasm 850

used included dde2-2 (At5g42650 (von Malek et al 2002)) pad3-1 (At3g26830 (Zhou 851

et al 1999)) pad4-1 (At3g52430 (Jirage et al 1999)) sid2-2 (At1g74710 (Wildermuth 852

et al 2001)) pbs3-2 (At5g13320 SALK_018225 (Nobuta et al 2007)) cbp60a-1 853

(At5g62570 SALK_124410 (Truman et al 2013)) cbp60g-1 sard1-2 (At5g26920 854

SALK_023199 At1g73805 SALK_052422 (Wang et al 2011) cbp60a-1 cbp60g-1 855

sard1-2 (Truman et al 2013) gae1-1 (At4g30440 SALK_085554) gae6-1 (At3g23280 856

SALK_104454) gae6-2 (At3g23280 SALK_017191) ixr1-1 cev1 (At5g05170 (Ellis 857

and Turner 2001 Scheible et al 2001)) eds1 plants were derived by introgression of 858

the Ler eds1-2 allele into Col-0 that contains two copies of EDS1 (At3g48090 859

At3g48080 (Bartsch et al 2006) mpk6-2 (At2g43790 SALK_073907 (Liu and Zhang 860

2004)) mpk3-DG (At3g45640 a fast neutron deletion mutant (Miles et al 2005)) and 861

npr1-1 (At1g64280 (Cao et al 1997)) T-DNA insertion lines were part of the SALK 862

collection (Alonso et al 2003) and were obtained from the Arabidopsis Biological 863

Resource Center 864

865

Supplemental Data 866

Page 40 of 54

Supplemental Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 867

Expression Does Not Require CBP60s or SID2 868

Supplemental Figure 2 Expression of GAE Family Members uponTreatment with Pma 869

ES4326 870

Supplemental Figure 3 Characterization of T-DNA Insertions in GAE1 and GAE6 871

Supplemental Figure 4 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves and 872

Are Slightly Smaller than Wild-Type Col-0 Plants 873

Supplemental Figure 5 Cell Walls of gae1 gae6 Plants Contain Less Uronic Acid than 874

Wild-Type Col-0 Cell Walls 875

Supplemental Figure 6 Antibodies Raised Against the RGI or XG Components of 876

Pectin Show No Difference in Binding to gae Mutant and Col-0 Cell Walls 877

Supplemental Figure 7 The Effect of B cinerea Treatment on the Expression of GAE 878

Family Genes 879

Supplemental Figure 8 Treatment with 001 Macerozyme Does Not Cause Any 880

Visible Damage to Col-0 Plants 881

Supplemental Figure 9 B cinerea Growth on Plants with Defects in Immune 882

Signaling 883

Supplemental Figure 10 MeJA-Induced Expression of the JA marker gene JAZ10 884

Supplemental Figure 11 The Expression of the JA Marker Gene JAZ5 Is Highly 885

Responsive in gae1 gae6 Plants 886

Supplemental Figure 12 The Expression of PDF12 Is Unaltered in gae1 gae6 Plants 887

Supplemental Figure 13 Macerozyme Treatment Activates MPK3 and MPK6 888

Supplemental Figure 14 B cinerea-Induced ROS Production in Wild-Type Col-0 and 889

gae1 gae6 Plants 890

Supplemental Figure 15 OG-Induced PTI Using 250 microg mL-1 OGs 891

Supplemental Figure 16 Abundance of Plant DNA in B cinerea Treated Plants 892

Supplemental Table 1 Primers used in this study 893

894

ACKNOWLEDGEMENTS 895

We thank William Truman for helpful discussions Simone Ferrari (Sapienza 896

Universita di Roma) for the generous gift of OGs Bruna Bucciarelli (USDA-ARS) for 897

Page 41 of 54

help with fluorescence microscopy the Arabidopsis Biological Resource Center for T-898

DNA insertion lines Paul Knoxrsquos lab (University of Leeds) for LM5 LM6 LM8 LM19 899

and LM20 antibodies and the Complex Carbohydrate Resource Center (University of 900

Georgia) for the CCRC-M7 antibody Most of this work was funded by the Division of 901

Chemical Sciences Geosciences and Biosciences Office of Basic Energy Sciences of 902

the US Department of Energy through Grant DE-FG02-05ER15670 to JG This work 903

was also supported by NSF awards IOS 1339125 and IOS 1021861 to DJK NSF 904

award IOS 1121425 to F K and the USDA National Institute of Food and Agriculture 905

Hatch project number CA-D-PLS-7033-H to DJK 906

907

AUTHOR CONTRIBUTIONS 908

GB and JG designed the research GB GX AT BL NES and NH 909

performed research DJK provided tools GB GX MP AD BL RAH and FK 910

analyzed data GB and JG wrote the paper with input from all authors 911

912

FIGURE LEGENDS 913

Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 Expression 914

Requires PAD4 EDS1 and PBS3 915

(A) Expression of GAE1 in leaves of wild-type Col-0 and mutant plants Expression was916

measured 24 or 32 h after either inoculation with Pma ES4326 (OD600 = 0002) or mock 917

inoculation with 5 mM MgSO4 and was then normalized to the level of ACTIN2 Data 918

from three biological replicates were combined using a mixed linear model Bars 919

represent mean log2-ratios to ACTIN2 plusmn SE Letters indicate significantly different 920

groups with q lt 005 where 005 represents the maximum false discovery rate at which 921

the test may be called significant 922

(B) Expression of GAE6 measured as described in (A)923

924

Figure 2 Susceptibility of gae6 and gae1 gae6 Plants to Pma ES4326 Is Increased 925

Relative to Wild-Type Col-0 926

Leaves of plants of the indicated genotypes were inoculated with Pma ES4326 (OD600 = 927

00002) as described in Methods Bacterial titers in leaves were determined either 928

Page 42 of 54

immediately (0 dpi) or after three days (3 dpi) Bars represent the mean plusmn SE of four 929

independent experiments each with 4 or 12 biological replicates at 0 and 3 dpi 930

respectively Data were combined using a mixed linear model Asterisks indicate 931

significant differences from Col-0 wild-type (q lt 005) Susceptible pad4 plants were 932

included as a positive control 933

934

Figure 3 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves 935

(A) Depiction of the assay for brittleness of gae mutant leaves Fully expanded leaves of936

four to five week-old plants were bent by 180ordm so that the adaxial surface of the leaf was 937

parallel with the soil Broken leaf petioles or midribs were considered brittle 938

(B) Quantification of broken midribs or petioles For each genotype 66 to 82 leaves 939

were analyzed for brittleness as described in (A) Bars represent the ratio of broken to 940

total number of leaves tested Results were analyzed using Fisherrsquos exact test Letters 941

indicate significantly different groups at p lt 005 942

(C) Conductivity measurements Leaves of four week-old plants were inoculated with943

deionized water and then leaf discs were cut out and placed on deionized water 944

Conductivity was measured at the indicated time points After 30 min the water was 945

replaced with fresh water (30 min f w) to account for wounding-related ion leakage from 946

the edges of the leaf discs Three independent experiments with six biological replicates 947

each were performed and data were analyzed using a t-test for each time point P-948

values were corrected using the Bonferroni method Data show means plusmn SE asterisks 949

indicate significant differences from wild-type Col-0 at q lt 001 950

951

Figure 4 Cell Walls of gae6 and gae1 gae6 Plants Are Reduced in GalA 952

(A) to (D) Levels of GalA (A) GlcA (B) cellulose (C) and neutral sugars (D) in alcohol953

insoluble residue (AIR) extracted from leaves of four week-old plants of the indicated 954

genotypes Bars represent means plusmn SE of four biological replicates combined using a 955

mixed linear model Letters indicate significantly different groups (p lt 005) 956

957

Figure 5 Cell Walls of gae1 gae6 Plants Are Reduced in Homogalacturonan 958

Page 43 of 54

Dot blot analyses with anti-LM19 and anti-LM20 antibodies Pectin was extracted from 959

AIR prepared from the indicated genotypes using 1 microL of extraction buffer per 10 microg of 960

AIR Pectin was serially diluted and one microL of undiluted 13 diluted and 19 diluted 961

samples were spotted on a nitrocellulose membrane Three biological replicates were 962

were spotted twice to obtain two technical replicates each All replicates produced 963

similar results 964

(A) Representative dot blot using anti-LM19 antibody965

(B) Relative signal intensities of the spots in the dot blot in (A)966

(C) Representative dot blot using anti-LM20 antibody967

(D) Relative signal intensities of the LM20 dot blot shown in (C)968

Relative signal intensities were measured using Image J software Measurements from 969

the three biological replicates were combined using a linear model after Box-Cox power 970

transformation as described in Methods Bars represent means plusmn SE Asterisks indicate 971

a difference from wild-type Col-0 at p lt 005 972

973

Figure 6 Growth of B cinerea on gae1 gae6 Plants and the Effect of B cinerea on 974

Expression of GAE1 and GAE6 975

(A) Growth of B cinerea isolates Leaves of Col-0 and gae1 gae6 plants were976

inoculated with 25 x 105 spores mL-1 of the B cinerea isolates indicated in the figure 977

Samples were collected two (2 dpi) and three (3 dpi) days later Bars represent means plusmn 978

SE of six biological replicates for each fungus combined using a mixed linear model 979

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 980

(B) and (C) Expression of GAE1 (B) and GAE6 (C) 48 h after inoculation with the B981

cinerea isolates indicated Expression levels were measured by qRT-PCR Bars 982

represent mean log2-ratios to ACTIN2 plusmn SE of three biological replicates combined 983

using a mixed linear model Letters indicate significantly different groups (q lt 005) 984

985

Figure 7 Levels of Soluble Pectin and Expression of PAD3 in Fractions Produced 986

Following Macerozyme Treatment of gae1 gae6 Cell Walls 987

(A) Schematic overview of the experimental procedure for (B) and (C)988

Page 44 of 54

(B) Uronic acid measurements Alcohol-insoluble residue (AIR) from leave tissue of four989

week-old wild-type Col-0 or gae1 gae6 plants was treated with water or 025 990

macerozyme and assayed for uronic acid levels Data from 11 biological replicates were 991

combined using a mixed linear model Bars represent means plusmn SE Letters indicate 992

significant differences (p lt 005) 993

(C) Expression of PAD3 in Col-0 plants treated with the fractions from (B) Extracts from994

(B) were heat inactivated pooled and diluted 5-fold with water Pooled extracts were995

inoculated into four week-old Col-0 plants PAD3 expression was measured 3 h after 996

inoculation and normalized to the level of ACTIN2 expression Data from four biological 997

replicates were combined using a mixed linear model Bars represent mean log2-ratios 998

to ACTIN2 plusmn SE Letters indicate significant differences (p lt 005) 999

1000

Figure 8 Macerozyme-Induced PAD3 Expression Is Absent in gae1 gae6 1001

(A) Expression of PAD3 after macerozyme treatment Four week-old plants were either1002

inoculated with 001 macerozyme or mock inoculated with water and PAD3 1003

expression was measured 3 h later by qRT-PCR Data from four biological replicates 1004

were combined using a mixed linear model Mean log2-ratios to ACTIN2 plusmn SE were 1005

plotted Letters indicate significantly different groups (p lt 005) 1006

(B) Expression of PAD3 after OG treatment PAD3 expression was measured as1007

described in (A) 3 h after inoculation with 100 microg mL-1 OGs or mock inoculation with 1008

water Data are from six biological replicates 1009

(C) PAD3 expression in untreated plants Three biological replicates were performed1010

and data analyzed as described in (A) 1011

(D) Camalexin accumulation after B cinerea treatment Camalexin accumulation was1012

measured in four week-old plants inoculated with the B cinerea strains shown in the 1013

figure Eight biological replicates each were combined using a mixed linear model 1014

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 1015

(E) PAD3 expression 28 h after treatment with the indicated B cinerea strains Data1016

from six biological replicates were collected and analyzed as described in (A) Data in 1017

all figure parts represent means plusmn SE Letters indicate significantly different groups (q lt 1018

005) 1019

Page 45 of 54

1020

Figure 9 Macerozyme-Induced Expression of the SA Marker Genes SID2 and PR1 1021

Is Abolished in gae1 gae6 Plants 1022

(A) to (H) Expression of PR1 (A) (C) (E) and (G) or SID2 (B) (D) (F) and (H) in four1023

week-old Col-0 and gae1 gae6 plants Data were combined using a mixed linear model 1024

Bars represent mean log2-ratios of expression vs ACTIN2 plusmn SE for all figure parts 1025

Letters indicate significantly different groups at p lt 005 for panels (A) to (D) (G) and 1026

(H) and qlt005 for panels (E) to (F) 1027

(A) and (B) Plants were treated with 001 macerozyme or mock treated with water1028

and samples were collected 3 h later Data from four biological replicates are shown 1029

(C) and (D) Plants were inoculated with 100 microg mL-1 OGs or mock treated with water1030

and samples collected 3 h later Data from six biological replicates are shown 1031

(E) and (F) Plants were inoculated with the indicated B cinerea isolates or mock1032

inoculated with B cinerea inoculation medium and samples collected 48 h later Data 1033

from eight biological replicates are shown 1034

(G) and (H) Expression in untreated plants Three biological replicates were performed1035

1036

Figure 10 The Expression of the JA Marker Gene JAZ10 is Highly Responsive in 1037

gae1 gae6 Plants 1038

(A) to (D) JAZ10 expression in four week-old plants Data were combined using a mixed1039

linear model Mean log2-ratios to ACTIN2 plusmn SE are shown Letters indicate significantly 1040

different groups at p lt 005 for panels (A) (B) and (D) and q lt 005 for panel (C) 1041

(A) Plants were inoculated with 001 macerozyme or mock inoculated using water and1042

samples were collected 3 h later Data from four biological replicates are shown 1043

(B) Plants were inoculated with 100 microg mL-1 OGs or mock inoculated using water and1044

samples collected 3 h later Data are from six biological replicates 1045

(C) JAZ10 expression 48 h after treatment with the indicated B cinerea strains Data1046

are from six biological replicates 1047

(D) JAZ10 expression in untreated plants Data are from three biological replicates1048

1049

Figure 11 Gallo 1-Induced Callose Deposition Is Reduced in gae1 gae6 Plants 1050

Page 46 of 54

(A) Callose deposition in four week-old Col-0 and gae1 gae6 plants Plants were1051

inoculated with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) or mock inoculated 1052

with B cinerea inoculation medium and samples were collected after 72 h Callose was 1053

stained with aniline blue and visualized using a Nikon Eclipse Ni-U microscope with a 1054

DAPI filter Three leaves were stained per treatment Scale bars are 100 microm Asterisks 1055

indicate fungal hyphae arrows indicate representative callose spots 1056

(B) Quantification of callose deposition Callose deposits were counted in 200 microm x 2001057

microm squares laid out across the images shown here Bars represent means plusmn SE from 1058

six squares each Letters indicate significantly different groups (p lt 005) according to a 1059

t-test analysis1060

1061

Figure 12 Less Soluble Pectin Accumulates in B cinerea-Infected gae1 gae6 1062

Leaves 1063

Four week-old Col-0 and gae1 gae6 plants were inoculated with 10 microL droplets of the 1064

indicated B cinerea isolate (25 x 105 spores mL-1) or mock inoculated with B cinerea 1065

inoculation medium Leaves were collected 48 h later AIR was extracted from leaf 1066

samples and water added (20 microL infection site) to extract soluble pectin fragments 1067

Samples were vortexed for 7 h centrifuged at 12000 g and the uronic acid 1068

concentration of the supernatant was measured Data from nine biological replicates 1069

were combined using a mixed linear model Means plusmn SE are shown Letters indicate 1070

significant differences (q lt 005) 1071

1072

Figure 13 Macerozyme-Induced PTI Is Lost in gae1 gae6 While OG-Induced PTI Is 1073

Unaltered 1074

(A) Macerozyme-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1075

inoculated with 001 macerozyme or mock inoculated with boiled macerozyme and 1076

were infected with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) 24 h later Samples 1077

were collected immediately (0 dpi) and after two (2 dpi) and three (3 dpi) days Data 1078

show mean plusmn SE of six to 14 biological replicates each combined using a linear model 1079

Letters indicate significantly different groups (q lt 005) 1080

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

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Page 23 of 54

13B) whereas a lower dose of OGs only induced PTI in Col-0 (Supplemental Figure 418

15) This suggests that OG perception in gae1 gae6 at least above a certain threshold 419

is unaltered while release of immunity-activating pectin fragments possibly OGs is 420

421 reduced We conclude that immune responses induced by macerozyme treatment

422 which do not occur in gae1 gae6 plants are important for plant immunity to the B

423 cinerea isolate Gallo 1

Page 24 of 54

____________________________________________________________________________

Page 25 of 54

DISCUSSION 426

Here we showed that loss of GAE1 and GAE6 leads to a dramatic reduction in 427

pectin content in the cell wall (Figure 4) likely causing the brittleness of leaves that we 428

observed (Figure 3) Petioles and midribs of gae1 gae6 plants broke easily when bent 429

and inoculation of leaves with deionized water resulted in increased ion leakage 430

possibly due either to microscopic fractures of the cell wall induced by inoculation stress 431

or to a reduced capability of these walls to withstand increased turgor pressure The 432

increased brittleness was associated with altered cell wall composition In our analysis 433

the GalA content of gae1 gae6 leaf walls was reduced by more than 40 and the total 434

uronic acids by over 60 (Figure 4 and Supplemental Figure 5) This is a major change 435

in the overall cell wall composition considering that pectin makes up about 50 of the 436

plant cell walls found in Arabidopsis leaves (Zablackis et al 1995 Nobuta et al 2007 437

Harholt et al 2010) Interestingly quasimodo1 (qua1) plants which have a mutation in 438

a glycosyltransferase family 8 gene are reduced in GalA by 25 and show reduced 439

binding of HG specific antibodies (Bouton et al 2002) 440

Furthermore qua2 plants which have a mutation in a putative methyltransferase 441

have about 13 less total GalA and lack 50 of cell wall HG (Mouille et al 2007) 442

Likewise cgr2 cgr3 plants carrying mutations in another class of Golgi-localized 443

putative methyl-transferases have leaf cell walls that are reduced by about 40 in 444

uronic acid (Kim et al 2015) All three mutants are severely dwarfed (Bouton et al 445

2002 Mouille et al 2007 Kim et al 2015) Additionally qua1 and qua2 mutants show 446

a distorted overall shape likely due to reduced cell cohesion and are extremely fragile 447

(Bouton et al 2002 Mouille et al 2007) By contrast gae1 gae6 plants with a similar 448

strong reduction in GalA and total uronic acids have an overall normal shape and are 449

only slightly smaller than wild-type Col-0 (Supplemental Figure 4) In the wall analysis 450

performed here we did not detect changes in other wall polysaccharides or structures 451

that might compensate for the reduction of pectin in gae1 gae6 It is possible the severe 452

morphological alterations in qua2 and cgr2 cgr3 plants are due to reduction in pectin 453

methylesterification which is unaffected in gae1 gae6 It was speculated that quality 454

control mechanisms in the Golgi prevent secretion of inefficiently esterified pectin in 455

cgr2 cgr3 (Kim et al 2015) suggesting that the Golgi of these plants might over-456

Page 26 of 54

accumulate pectin and that this may contribute to the severe dwarf phenotype of these 457

plants The gae1 gae6 plants described here provide an opportunity to study the effects 458

of reduction of pectin in plants that do not show severe morphological phenotypes 459

We found that gae1 gae6 plants are also compromised in resistance to B 460

cinerea and Pma ES4326 indicating that loss of pectin reduces immunity to these 461

pathogens (Figure 2 and 6) We found that gae1 gae6 plants were slightly more 462

susceptible to Pma ES4326 but showed a strong increase in susceptibility to specific B 463

cinerea isolates This was not unexpected because B cinerea is known to interact with 464

pectin as part of its infection strategy It secretes various pectin-degrading enzymes 465

early during infection and requires some of these enzymes including PG1 PG2 and 466

PME1 for full virulence (ten Have et al 1998 Valette-Collet et al 2003 Kars et al 467

2005b Espino et al 2010) Additionally Arabidopsis plants expressing an antisense 468

construct of the polygalacturonase inhibitor PGIP1 are significantly more susceptible to 469

B cinerea (Ferrari et al 2006) On the other hand Pma ES4326 is not known to470

require cell wall degrading enzymes for full virulence even though it encodes two 471

pectate lyase genes in its genome (PMA4326_07094 and PMA4326_04621 472

wwwbacteriaensemblorg) B cinerea isolates are also known to be genetically quite 473

diverse in their polygalacturonase (PG) loci and genetic variability in PG2 is associated 474

with varying growth on pectin containing media (Rowe and Kliebenstein 2007) 475

Furthermore the contribution of PG1 and PG2 to B cinerea virulence seems to depend 476

on the specific B cinerea isolate and the host species (Kars et al 2005a Zhang and 477

Van Kan 2013) B cinerea isolates are also known to be differently sensitive to the anti-478

microbial secondary metabolite camalexin (Kliebenstein et al 2005) In line with these 479

observations the susceptibility of gae1 gae6 plants differed depending on the specific 480

B cinerea isolate Whereas Fresa 525 Gallo 1 UK Razz and KT grew better in gae1481

gae6 we detected no difference in Acacia Apple 517 DN Grape Pepper or Rasp 482

growth (Figure 6) 483

B cinerea was shown to metabolize pectin and to possibly utilize GalA as a484

carbon source during in planta growth (Zhang et al 2011 Zhang and Van Kan 2013) 485

Our data show that gae1 gae6 plants which are reduced in GalA content did allow 486

enhanced growth of specific B cinerea isolates but no reduction in growth of any B 487

Page 27 of 54

cinerea isolates tested was found This suggests that utilization of pectin as a carbon 488

source was not a contributing factor to alterations in B cinerea susceptibility It is 489

possible that changes in the cell wall composition affect the structural integrity of the 490

wall and affect hyphal penetration of specific B cinerea isolates However OGs (small 491

HG fragments) are likely released during the infection process (Ferrari et al 2013) 492

OGs function as DAMPs and initiate immune responses in Arabidopsis (Ferrari et al 493

2013) Treatment of Arabidopsis with OGs having a degree of polymerization (DP) of 494

10-15 sugar subunits was previously shown to enhance resistance to B cinerea (Ferrari495

et al 2007) We showed that pretreatment with OGs or macerozyme induced enhanced 496

immunity to B cinerea isolate Gallo 1 in wild-type Col-0 but only OGs and not 497

macerozyme induced immunity in gae1 gae6 (Figure 13) Our data suggest gae1 gae6 498

plants which have reduced pectin content may release less OGs and that this 499

reduction leads to the increased susceptibility to B cinerea 500

Consistently we found that macerozyme treatment released less soluble pectin 501

from gae1 gae6 cell walls than from wild-type walls Fractions containing soluble pectin 502

released from gae1 gae6 were also less active in inducing PAD3 gene expression than 503

the fractions released from wild-type walls (Figure 7) We also showed that less soluble 504

pectin accumulated in gae1 gae6 than in wild-type plants 48 h after treatment with B 505

cinerea (Figure 12) 506

We observed that loss of pectin leads to alterations in A thaliana immune 507

signaling Macerozyme treatment induced expression of PAD3 and the SA marker 508

genes PR1 and SID2 in wild-type Col-0 but not in gae1 gae6 whereas OG treatment 509

led to increased PAD3 and SID2 expression in both wild-type and gae1 gae6 (Figure 8 510

and 9) This suggests that although release of active soluble pectin in these plants 511

might be altered OG recognition is intact In fact cell walls isolated from qua1 another 512

mutant known to be reduced in HG released less OGs (DP1-6) than did wild-type cell 513

walls upon endopolygalacturonase treatment (Bouton et al 2002) This was also true 514

for gae1 gae6 cell walls as macerozyme treatment of isolated gae1 gae6 cell walls 515

released less soluble pectin than treatment of Col-0 cell walls Because the fraction 516

from gae1 gae6 plants was also less active in inducing PAD3 expression (Figure 7) it 517

seems possible that biologically active OGs were the active component inducing PAD3 518

Page 28 of 54

expression Interestingly expression of the JA marker genes JAZ5 and JAZ10 was 519

higher in gae1 gae6 than in wild-type mock-inoculated plants but was comparable to 520

wild-type in untreated plants suggesting that JA signaling in gae1 gae6 is 521

hyperresponsive (Figure 10 and Supplemental Figure 11) This induction of JA signaling 522

might be due to wounding stress incurred during the inoculation process JA and SA 523

signaling generally act antagonistically (Pieterse et al 2012) Increased JA signaling 524

activity might hence explain why mock- and macerozyme-treated gae1 gae6 plants 525

exhibited reduced expression of the SA marker PR1 and why OG treatment induced 526

expression of SID2 another SA marker to a lower extent in gae1 gae6 than in wild-type 527

plants (Figure 9) 528

No single immune signaling component was found to be the sole contributor to 529

the compromised immunity of gae1 gae6 to specific B cinerea isolates such as Gallo 1 530

and Fresa 525 These isolates grew better on pad3 while growth of Pepper another B 531

cinerea isolate that did not show altered virulence on gae1 gae6 was unaltered 532

(Supplemental Figure 9) This suggested that differences in camalexin sensitivity of 533

these B cinerea isolates might account for the differences in gae1 gae6 susceptibility 534

However camalexin accumulation and PAD3 expression was the same for wild-type 535

Col-0 and gae1 gae6 for the B cinerea isolates Fresa 525 and Gallo 1 (Figure 8) which 536

showed enhanced growth on gae1 gae6 plants It should be noted that B cinerea 537

virulence is often estimated by comparing lesion sizes Lesion size and measurements 538

of relative fungal growth by qPCR do not always show the same results For example 539

although Denby et al (2004) showed that Pepper causes larger lesions on pad3 than on 540

wild-type this study showed that relative fungal growth of Pepper on pad3 was 541

unaltered 542

We observed that callose deposition was strongly reduced in gae1 gae6 plants 543

72 h after treatment with B cinerea isolate Gallo 1 (Figure 11) possibly due to reduced 544

release of active soluble pectin Interestingly OG-induced PTI to an unspecified isolate 545

of B cinerea was intact in the pmr4 mutant which does not produce callose (Galletti et 546

al 2008) Due to the high SA levels in pmr4 plants (Nishimura et al 2003) and the fact 547

that different B cinerea isolates vary both in induction of and reaction to defense 548

responses (Kliebenstein et al 2005 Rowe et al 2010) we could not determine 549

Page 29 of 54

whether reduced callose deposition causes the increased susceptibility of gae1 gae6 to 550

B cinerea isolate Gallo 1551

We did not observe any changes in B cinerea growth either on cev1 ixr1 plants 552

which are known to have constitutively high JA and OPDA levels (Ellis et al 2002b) or 553

on plants exogenously treated with MeJA (Supplemental Figure 9) Because plants 554

were kept uncovered for 3 h after MeJA treatment it is unclear how much MeJA was 555

bioavailable in the plant However JAZ10 expression was induced 3 h after spraying 556

with MeJA (Supplemental Figure 10) Because pathogen growth generally depends on 557

the combination of a multitude of immune signaling events we conclude that multiple 558

changes to the immune signaling in gae1 gae6 together account for the altered B 559

cinerea growth phenotypes detected 560

We showed that expression of GAE1 and GAE6 is repressed by the bacterial 561

hemibiotroph Pma ES4326 This repression did not occur in an eds1 or pad4 562

background or in pbs3 at the 24-h time point (Figure 1 and Supplemental Figure 1) 563

During the Arabidopsis - Pma ES4326 interaction the expression of many defense 564

related genes is altered in a PAD4EDS1 dependent fashion (Wang et al 2008) Some 565

of these expression changes require SA CBP60g and SARD1 are thought to act 566

downstream of PAD4 and EDS1 but upstream of SA-dependent responses (Wang et 567

al 2011) PBS3 is also thought to act downstream of PAD4 and EDS1 and upstream of 568

SA but the set of genes whose expression it affects is only partially overlapping with 569

those affected by CBP60gSARD1 (Wang et al 2008 Wang et al 2011) Thus GAE1 570

and GAE6 repression is regulated by the part of the immune signaling network that 571

requires PAD4 EDS1 and PBS3 but is independent of CBP60gSARD1 and SA 572

Because EDS1 PAD4 and PBS3 are important components of the plant immune 573

signaling network (Wiermer et al 2005 Nobuta et al 2007) one might conclude that 574

repression of GAE1 and GAE6 may be part of the plant immune response to Pma 575

ES4326 B cinerea treatment also reduced GAE1 and GAE6 expression but this was 576

mostly independent of PAD4 (Figure 6) while both Pma ES4326 and B cinerea repress 577

GAE1 and GAE6 expression the host signaling mechanisms responsible likely differ 578

In summary our data show that Arabidopsis GAE1 and GAE6 are required for 579

pectin biosynthesis leaf flexibility and immunity to Pma ES4326 and to specific B 580

Page 30 of 54

cinerea isolates We used macerozyme a commercial pectinase to mimic the effect of 581

B cinerea pectinases Macerozyme treatment released less pectin from gae1 gae6 cell582

walls and induced less immune signaling in gae1 gae6 plants Less water soluble pectin 583

also accumulated in B cinerea infected gae1 gae6 plants and macerozyme-induced 584

immunity to B cinerea isolate Gallo 1 was reduced in these plants We conclude that 585

the reduced pectin content in these mutants may lead to reduced release of soluble 586

pectin including active OGs during pathogen attack and that this compromises 587

immunity 588

589

590

Page 31 of 54

METHODS 591

Plant Materials and Growth Conditions 592

Wild-type Columbia (Col-0) and all mutant plants (in Col-0 background) were 593

grown on sterilized BM2 germinating mix (Berger Inc Quebec Canada) in a controlled 594

environment chamber (Conviron Pembina ND USA) with a 12-h photoperiod under 595

100 microM m-2 s-1 fluorescent illumination at 22degC and 75 relative humidity Germplasm 596

used is described in the Accession Numbers section at the end of Methods 597

598

Data Analysis and Replicates 599

For biological replicates samples were obtained from separate plant tissues and 600

in many cases from plants grown in different flats In some experiments multiple 601

technical replicates were measured per biological replicate For statistical analysis all 602

technical replicates were averaged and the averages were considered one biological 603

replicate Independent experiments were performed at different times and often 604

consisted of multiple biological replicates per experiment 605

Data analysis was performed using mixed linear models in the R programming 606

environment (Bates et al 2015) unless otherwise indicated Generally genotypes 607

treatments and time points were used as fixed effects and replicate-specific effects like 608

different pots flats etc were considered random effects We reported p-values 609

obtained from the mixed linear model when less than 10 comparisons were performed 610

and q-values for more than 10 comparisons to correct for multiple comparison testing 611

Q-values were calculated using the Benjamini-Hochberg procedure (Benjamini and612

Hochberg 1995) unless specified otherwise 613

614

Pathogen Strains Growth Conditions and Pathogen Growth Assays 615

Growth of Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) and 616

bacterial growth assays were performed as previously described (Bethke et al 2014) 617

For qRT-PCR assays four week-old plants were inoculated with bacteria (OD600 = 618

0002) or mock (5 mM MgSO4) At least four leaves from two plants were collected at 619

the indicated time points per biological replicate 620

Page 32 of 54

Prior to each experiment Botrytis cinerea isolates were grown on 1x PDA 621

medium (Difco) for 10 d at room temperature All isolates used have been described 622

previously (Rowe and Kliebenstein 2007) The spores were washed from the surface of 623

the plate using an inoculation medium (1x Gamborgrsquos B-5 basal salt mixture (Sigma-624

Aldrich G5768) 2 (wv) glucose 10 mM phosphate buffer pH 64) and fungal hyphae 625

were removed from the suspension by filtering through four layers of cheesecloth The 626

concentration of spores was determined using a hemocytometer and adjusted to 25 x 627

105 spores mL-1 Four week-old Arabidopsis plants were inoculated by placing one 10-628

microL droplet of the B cinerea spore solution or inoculation medium (mock) on the adaxial 629

leaf surface of fully expanded leaves Inoculated plants were kept at 100 relative 630

humidity Infection sites were collected at various time points using a cork borer For 631

qRT-PCR assays at least four leaf discs from one plant were combined for each 632

biological sample For B cinerea growth assays at least 12 leaf discs from three to six 633

plants were pooled per biological sample Relative fungal growth was determined by 634

measuring the abundance of a fungal gene relative to a plant gene as described below 635

In brief DNA from infected tissue was extracted and equal amounts of total DNA were 636

used to perform qPCR reactions using the B cinerea cutinase A and the Arabidopsis 637

SK11 gene respectively Primers were described in (Gachon and Saindrenan 2004) 638

and are listed in Supplemental Table 1 Two technical replicates were combined for 639

each biological replicate B cinerea is a necrotrophic fungal pathogen and may destroy 640

plant DNA as disease progresses However decreases in the amount of plant DNA 641

over the course of our experiments were only observed at late time points with very 642

susceptible plants so the qPCR assay is generally a reasonable estimate of fungal 643

biomass (Supplemental Figure 16) 644

645

Treatment with Macerozyme or OGs 646

For PTI assays four week-old plants either were inoculated with 001 (wv) 647

macerozyme R-10 (Yakult) a Rhizopus polygalacturonase (EC 32115) that possesses 648

high pectinase and hemicellulase activity or were mock inoculated using heat-649

inactivated macerozyme Other similar plants were either inoculated with 250 or 500 microg 650

mL-1 OGs or mock inoculated with water One d later plants were infected with B 651

Page 33 of 54

cinerea and samples were collected immediately (0 dpi) and two or three d later To 652

investigate the role of MeJA on B cinerea growth plants were sprayed with 1mM MeJA 653

in water with 001 (vv) Tween 20 Covers were removed so that leaves could dry off 654

before plants were inoculated with B cinerea three h later Samples were harvested at 655

the indicated time points For expression analysis and MAPK activation assays plants 656

were either inoculated with 100 microg mL-1 OGs or mock inoculated with water OGs used 657

for experiments described in Figures 8 to 10 and Supplemental Figure 12 were a kind 658

gift from Simone Ferrari OGs used in experiments described in Figure 13 and 659

Supplemental Figures 11 13 and 15 were prepared as previously described (Kohorn et 660

al 2014) 661

662

Expression Analysis 663

Leaf tissue from inoculated plants was harvested at the indicated time points 664

flash frozen pulverized and RNA extracted using Trizol (Invitrogen) Quantitative RT-665

PCR was performed using the SuperScript III Platinum SYBR Green One-Step 666

quantitative RT-PCR kit (Invitrogen) and a Lightcycler 480 Real-Time PCR system 667

(Roche) as previously described (Truman and Glazebrook 2012) In brief equal 668

amounts of total RNA and a gene specific primer were used for each reaction The 669

crossing point (Cp) was calculated using the 2nd derivative max method provided with 670

the Lightcycler software for each amplification curve Each reaction was run with two 671

technical replicates and the Cp values for these replicates were averaged ACTIN2 was 672

used as a stably expressed reference gene At least three biological replicates each 673

were performed and analyzed using a mixed-linear model RT-PCR was performed to 674

verify T-DNA insertion lines using the One-Step RT-PCR kit (Qiagen) according to the 675

manufacturerrsquos instructions The primers used can be found in Supplemental Table 1 676

677

Electrolyte Leakage Assay 678

Fully expanded leaves of four week-old plants were inoculated with water (Milli-Q 679

grade) using a needleless syringe Leaf discs were collected using a cork borer and 680

three leaf discs from one leaf were placed with the adaxial surface down onto 2 mL 681

water in a 12-well cell culture plate Conductivity was measured using a Horiba B-173 682

Page 34 of 54

conductivity meter after 30 min and then the water was replaced Conductivity was 683

measured again 30 min later (1 h total) as indicated in Figure 3C Six biological 684

replicates each were performed in three independent experiments and means and SEs 685

were calculated Two-sided t-tests were performed for each time point 686

687

DAB and Aniline Blue Staining 688

Leaves from four week-old plants were either mock inoculated with B cinerea 689

inoculation medium or inoculated with 10 microL droplets of a B cinerea spore solution (25 690

x 105 spores mL-1) of the indicated isolates Samples were collected 72 h later For 33prime-691

diaminobenzidine (DAB) staining two to five inoculated leaves were vacuum infiltrated 692

with 33prime-Diaminobenzidine (1 mg mL-1 in water pH 38) solution and stained overnight 693

in the dark Leaves were destained using 100 ethanol at 65degC and then transferred to 694

50 (vv) glycerol and photographed 695

Callose was visualized by staining leaves with aniline blue as previously 696

described (Adam and Somerville 1996) Briefly leaves were vacuum infiltrated with 697

alcoholic lactophenol (one volume of phenolglycerollactic acidwater (1111) mixed 698

with two volumes of ethanol) and destained at 65degC Next leaves were moved through 699

an alcohol gradient (50 20 and 10 (vv) ethanol) into water The destained leaves 700

were stained for 30 min in 150 mM K2HPO4 (pH 95) with 001 (wv) aniline blue 701

Samples were mounted in water and examined using a Nikon Eclipse Ni-U microscope 702

at 10x magnification using a DAPI filter (excitation at 325-375 nm emission at 435-485 703

nm) For callose quantification six 200 microm x 200 microm squares were counted for each 704

sample type Callose deposits per mm2 were calculated and data analyzed using a t-705

test Note that there is an inherent uncertainty associated with such counts because 706

callose deposits may not all be in focus at the same time 707

708

MAPK Activity Assay 709

Fully expanded leaves of four week-old plants were inoculated with 001 710

macerozyme 100 microg mL-1 OGs 100 nM flg22 or water (mock) flg22 a peptide from 711

bacterial flagella that induces immune responses was used as a positive control for 712

Page 35 of 54

MAPK activation (Asai et al 2002) and was purchased from EZBiolab Inc (Westfield 713

IN USA) Samples were harvested 10 min later and immediately frozen in liquid 714

nitrogen Protein was extracted using an extraction buffer containing 25 mM Tris pH 78 715

75 mM NaCl 1 mM DTT 1 mM NaF 05mM Na3VO4 01 (vv) Tween 20 one 716

cOmplete Mini protease inhibitor cocktail tablet (Roche) and one PhosSTOP 717

phosphatase inhibitor cocktail tablet (Roche) per 10 mL buffer 15 microg of total protein 718

were electrophoresed on a 10 SDS-polyacrylamide gel and then blotted onto a PVDF 719

membrane (Bio-Rad 162-0177) Activated MAPKs were detected using p4442 MAPK 720

(Erk12) antibody (9102S Cell Signal 12500 in TBST (20 mM Tris pH 75 150 mM 721

NaCl 01 (vv) Tween 20)) Anti-Rabbit IgG-HRP (115000 in TBST A6154 Sigma) 722

and ECL Plus substrate (Pierce 32132) MPK3 and MPK6 were detected with an anti-723

MPK3 antibody (M8318 Sigma 12000 in TBST and 3 (wv) milk) or an anti-MPK6 724

antibody (A7104 Sigma 16000 in TBST and 3 (wv) milk) respectively The 725

secondary antibody and detection were the same as for the p4442 antibody Five 726

independent experiments were performed 727

728

Preparation of Alcohol Insoluble Residue (AIR) Pectin Extraction and Uronic Acid 729

Measurements 730

Fully expanded leaves from four week-old plants that were incubated for 48 h in 731

the dark to reduce starch were harvested flash frozen and pulverized AIR was 732

extracted by washing ground material twice in 70 (vv) ethanol three times in a 733

mixture of chloroform and methanol (11 vv) and once in acetone (Gille et al 2009) 734

and was then air dried Cell wall pectin was extracted from AIR with cell wall extraction 735

buffer (50 mM Trizma 50 mM CDTA pH 72) at 95degC for 15 min Samples were 736

homogenized using a paint shaker (Harbil 5G-HD) and glass beads (3 mm diameter) 737

For dot blot experiments 500 microL of cell wall extraction buffer were used per 10 mg of 738

AIR Debris was precipitated by centrifugation for 10 min at 10000 g 739

Total uronic acid content of pectin extracted as described above using 500 microL of 740

cell wall extraction buffer per 1 mg of AIR was measured as described previously 741

(Filisetti-Cozzi and Carpita 1991 van den Hoogen et al 1998) First 36 microL of pectin 742

extract were mixed with 4 microL of 4 M sulfamic acid Then 200 microL of sulfuric acid 743

Page 36 of 54

containing 120 mM sodium tetraborate was added and samples were incubated at 80degC 744

for 1 h Following cooling on ice the optical density of samples was measured at 490 745

nm Next 40 microL of m-hydroxydiphenyl reagent (100 microL of m-hydroxydiphenyl in dimethyl 746

sulfoxide at 100 mgmL mixed with 49 mL 80 (vv) sulfuric acid just before use) was 747

added and the samples were mixed The optical density at 490 nm was measured 748

again The optical density before m-hydroxydiphenyl reagent addition was subtracted 749

from the optical density measured after addition of the dye The concentration of uronic 750

acid was calculated using known amounts of GalA as a standard 751

752

Release of Pectin from Arabidopsis Cell Walls Using Macerozyme 753

AIR preparations from leaves of four week-old plants were treated with 025 754

(wv) macerozyme R-10 (Yakult) or water (mock) at a concentration of 100 microL per mg 755

AIR Samples were incubated for 3 h at room temperature on a vortex shaker and 756

centrifuged for 10 min at 12000 g Total uronic acid concentration of 5-fold diluted 757

supernatants was determined for two technical replicates each The concentration of 758

uronic acid was calculated using known amounts of GalA as a standard For 759

subsequent qRT-PCR experiments supernatants from multiple extractions were 760

combined boiled for 20 min to inactivate macerozyme diluted 10-fold with water and 761

inoculated into four week-old Col-0 plants Heat-inactivated 10-fold diluted macerozyme 762

solutions were used as mock treatment Samples were collected 3 h later and PAD3 763

expression was determined 764

765

Release of Pectin from Arabidopsis Leaves Infected with B cinerea 766

Fully expanded leaves of four week-old plants were either inoculated with up to 767

five 10 microL droplets of B cinerea spore solution (25 x 105 spores mL-1) or mock 768

inoculated with B cinerea inoculation medium For each biological replicate leaves with 769

a combined count of at least 20 infection sites were collected 48 h later AIR was 770

extracted as described above but plants were not dark-treated or freeze-dried and the 771

first ethanol step was performed using 85 (vv) ethanol To extract soluble pectin 772

fragments AIR samples were mixed with water at a concentration of 20 microL per infection 773

site incubated for 7 h at room temperature on a vortex shaker and centrifuged for 10 774

Page 37 of 54

min at 12000 g Total uronic acid concentration of supernatants was determined for two 775

technical replicates each The concentration of uronic acid was calculated using known 776

amounts of GalA as a standard 777

778

Measurement of Uronic Acids Neutral Monosaccharides and Cellulose Content in 779

Cell Walls 780

Neutral sugars and uronic acids from non-cellulosic polysaccharides in cell walls 781

(AIR) were released by trifluoroacetic acid (TFA) hydrolysis Approximately 1 mg of AIR 782

was hydrolyzed with 2 M TFA at 121degC for 90 min The TFA-soluble material was split 783

into two equal parts one for uronic acid and one for neutral sugar determination The 784

uronic acid content was determined by analyzing the hydrolyzate using a CarboPac 785

PA200 anion-exchange column with an ICS-3000 Dionex chromatography system 786

(Dionex Sunnyvale CA USA) The elution profile consisted of a linear gradient of 50 to 787

200 mM sodium acetate in 01 M NaOH in 10 min at 04 mL per min Alditol acetate 788

derivatives of neutral sugars were produced as described (York et al 1985) In brief 789

monosaccharides in the hydrolyzate were reduced with NaBH4 and peracetylated with 790

acetic anhydride and pyridine at 121degC for 20 min The generated alditol acetates were 791

analyzed by GC-MS Crystalline cellulose was measured according to Updegraff (1969) 792

After hydrolysis of noncellulosic polysaccharides from AIR with the Updegraff reagent 793

(acetic acid nitric acids water 812 (vv) ) the remaining pellet was hydrolyzed in 794

72 sulfuric acid The resulting glucose quantity was determined by the anthrone 795

method (Scott and Melvin 1953) 796

797

Dot Blot Experiments 798

Pectin solutions extracted using 50 microL of cell wall extraction buffer per mg AIR 799

were serially diluted and nitrocellulose membranes were spotted with 1 microL of the diluted 800

pectin solutions Membranes were dried overnight blocked with 5 milk (wv) in 1x 801

PBS (8 g L-1 NaCl 02 g L-1 KCl 144 g L-1 Na2HPO4 024 g L-1 KH2PO4 pH 74) and 802

probed with LM5 (Jones et al 1997) LM6 (Willats et al 1998) LM8 (Willats et al 803

2004) LM19 and LM20 (Verhertbruggen et al 2009a) or CCRC-M7 (Steffan et al 804

Page 38 of 54

1995) antibodies LM series antibodies were diluted 1250 and CCRC-M7 was diluted 805

1500 in 5 milk powder (Nestle Carnation) in 1 x PBS For LM series antibodies a 806

goat anti-Rat HRP conjugated antibody (Bethyl A110-105P 15000 diluted) and for 807

CCRC-M7 an anti-Mouse IgG HRP conjugate (Promega W402B 12500 diluted) was 808

used as secondary antibody in 5 milk powder in 1x PBS LM series antibodies were 809

obtained from plant probes (wwwplantprobesnet) and CCRC-M7 from CarboSource 810

(wwwcarbosourcenet) Membranes were washed after incubation with the primary and 811

secondary antibodies with 1x PBS Dot blots were developed using the ECL system 812

(GE Healthcare Amersham ECL prime) For quantification of signals dot blot results on 813

x-ray films were photographed using a CCD camera and intensities were measured814

using Image Jrsquos Integrated Density function Locally adjusted background was 815

subtracted As the dynamic range of the signal on an x-ray film was narrow non-816

linearity in the measured values was corrected for each genotype in each biological 817

replicate using the values from different dilutions in two technical replicates We noticed 818

that the residuals were not normally distributed and performed Box-Cox power 819

transformation (Box and Cox 1964) to obtain normally distributed values for the 820

subsequent statistical analysis of the data For LM19 Box-Cox selected power 821

transformation was done to the power of 038th and for LM20 to the power 075th After 822

correction of the transformed measured values with the effect of the biological 823

replicates the values were fit to a linear model with the Arabidopsis genotype as the 824

fixed effect This linear model was used both to generate the plots in Figures 5B and D 825

and to test significant differences 826

827

Camalexin Measurements 828

Four week-old plants were infected with B cinerea and samples (three leaf disks 829

from one plant each) were harvested at the indicated time points flash frozen and 830

pulverized Each sample was extracted with 300 microL of 90 methanol (vv) 50 microL of the 831

extract was run on an Agilent Lichrocart 250-4 RP18e 5-microm column using an Agilent 832

1100 series HPLC Camalexin was detected using a DiodeArray (DAD) at 330 nm and 833

with a fluorescence detector at emission 318 nmexcitation 385 nm (Agilent) Separation 834

was achieved using the following program with aqueous acetonitrile 5 min gradient 835

Page 39 of 54

from 63 to 69 acetonitrile 30 s gradient from 69 to 99 acetonitrile 2 min at 99 836

acetonitrile and a post-run equilibration of 35 min at 63 acetonitrile (Denby et al 837

2004 Kliebenstein et al 2005) Purified camalexin was used to produce a standard 838

curve to identify and quantitate camalexin 839

840

Accession Numbers 841

Microarray data used can be accessed at NCBI Gene Expression Omnnibus 842

(httpwwwncbinlmnihgovgeo) GSE18978 Sequence information for all Arabidopsis 843

genes described in this article can be found in TAIR (wwwarabidopsisorg) using the 844

following accession numbers PAD3 (CYP71B15 At3g26830) SID2 (ICS1 At1g74710) 845

GAE1 (At4g30440) GAE2 (At1g02000) GAE3 (At4g00110) GAE4 (At2g45310) GAE5 846

(At4g12250) GAE6 (At3g23820) PR1 (At2g14610) JAZ5 (At1g17380) JAZ10 847

(At5g13220) ACTIN2 (At3g18780) PDF12a (At5g44420) PDF12b (At2g26020) 848

AtSK11 (At5g26751) The sequence of the B cinerea cutinase Bc-CutA (Z69264) can 849

be found in the DNA Data Bank of Japan (httpgetentryddbjnigacjp) Germplasm 850

used included dde2-2 (At5g42650 (von Malek et al 2002)) pad3-1 (At3g26830 (Zhou 851

et al 1999)) pad4-1 (At3g52430 (Jirage et al 1999)) sid2-2 (At1g74710 (Wildermuth 852

et al 2001)) pbs3-2 (At5g13320 SALK_018225 (Nobuta et al 2007)) cbp60a-1 853

(At5g62570 SALK_124410 (Truman et al 2013)) cbp60g-1 sard1-2 (At5g26920 854

SALK_023199 At1g73805 SALK_052422 (Wang et al 2011) cbp60a-1 cbp60g-1 855

sard1-2 (Truman et al 2013) gae1-1 (At4g30440 SALK_085554) gae6-1 (At3g23280 856

SALK_104454) gae6-2 (At3g23280 SALK_017191) ixr1-1 cev1 (At5g05170 (Ellis 857

and Turner 2001 Scheible et al 2001)) eds1 plants were derived by introgression of 858

the Ler eds1-2 allele into Col-0 that contains two copies of EDS1 (At3g48090 859

At3g48080 (Bartsch et al 2006) mpk6-2 (At2g43790 SALK_073907 (Liu and Zhang 860

2004)) mpk3-DG (At3g45640 a fast neutron deletion mutant (Miles et al 2005)) and 861

npr1-1 (At1g64280 (Cao et al 1997)) T-DNA insertion lines were part of the SALK 862

collection (Alonso et al 2003) and were obtained from the Arabidopsis Biological 863

Resource Center 864

865

Supplemental Data 866

Page 40 of 54

Supplemental Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 867

Expression Does Not Require CBP60s or SID2 868

Supplemental Figure 2 Expression of GAE Family Members uponTreatment with Pma 869

ES4326 870

Supplemental Figure 3 Characterization of T-DNA Insertions in GAE1 and GAE6 871

Supplemental Figure 4 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves and 872

Are Slightly Smaller than Wild-Type Col-0 Plants 873

Supplemental Figure 5 Cell Walls of gae1 gae6 Plants Contain Less Uronic Acid than 874

Wild-Type Col-0 Cell Walls 875

Supplemental Figure 6 Antibodies Raised Against the RGI or XG Components of 876

Pectin Show No Difference in Binding to gae Mutant and Col-0 Cell Walls 877

Supplemental Figure 7 The Effect of B cinerea Treatment on the Expression of GAE 878

Family Genes 879

Supplemental Figure 8 Treatment with 001 Macerozyme Does Not Cause Any 880

Visible Damage to Col-0 Plants 881

Supplemental Figure 9 B cinerea Growth on Plants with Defects in Immune 882

Signaling 883

Supplemental Figure 10 MeJA-Induced Expression of the JA marker gene JAZ10 884

Supplemental Figure 11 The Expression of the JA Marker Gene JAZ5 Is Highly 885

Responsive in gae1 gae6 Plants 886

Supplemental Figure 12 The Expression of PDF12 Is Unaltered in gae1 gae6 Plants 887

Supplemental Figure 13 Macerozyme Treatment Activates MPK3 and MPK6 888

Supplemental Figure 14 B cinerea-Induced ROS Production in Wild-Type Col-0 and 889

gae1 gae6 Plants 890

Supplemental Figure 15 OG-Induced PTI Using 250 microg mL-1 OGs 891

Supplemental Figure 16 Abundance of Plant DNA in B cinerea Treated Plants 892

Supplemental Table 1 Primers used in this study 893

894

ACKNOWLEDGEMENTS 895

We thank William Truman for helpful discussions Simone Ferrari (Sapienza 896

Universita di Roma) for the generous gift of OGs Bruna Bucciarelli (USDA-ARS) for 897

Page 41 of 54

help with fluorescence microscopy the Arabidopsis Biological Resource Center for T-898

DNA insertion lines Paul Knoxrsquos lab (University of Leeds) for LM5 LM6 LM8 LM19 899

and LM20 antibodies and the Complex Carbohydrate Resource Center (University of 900

Georgia) for the CCRC-M7 antibody Most of this work was funded by the Division of 901

Chemical Sciences Geosciences and Biosciences Office of Basic Energy Sciences of 902

the US Department of Energy through Grant DE-FG02-05ER15670 to JG This work 903

was also supported by NSF awards IOS 1339125 and IOS 1021861 to DJK NSF 904

award IOS 1121425 to F K and the USDA National Institute of Food and Agriculture 905

Hatch project number CA-D-PLS-7033-H to DJK 906

907

AUTHOR CONTRIBUTIONS 908

GB and JG designed the research GB GX AT BL NES and NH 909

performed research DJK provided tools GB GX MP AD BL RAH and FK 910

analyzed data GB and JG wrote the paper with input from all authors 911

912

FIGURE LEGENDS 913

Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 Expression 914

Requires PAD4 EDS1 and PBS3 915

(A) Expression of GAE1 in leaves of wild-type Col-0 and mutant plants Expression was916

measured 24 or 32 h after either inoculation with Pma ES4326 (OD600 = 0002) or mock 917

inoculation with 5 mM MgSO4 and was then normalized to the level of ACTIN2 Data 918

from three biological replicates were combined using a mixed linear model Bars 919

represent mean log2-ratios to ACTIN2 plusmn SE Letters indicate significantly different 920

groups with q lt 005 where 005 represents the maximum false discovery rate at which 921

the test may be called significant 922

(B) Expression of GAE6 measured as described in (A)923

924

Figure 2 Susceptibility of gae6 and gae1 gae6 Plants to Pma ES4326 Is Increased 925

Relative to Wild-Type Col-0 926

Leaves of plants of the indicated genotypes were inoculated with Pma ES4326 (OD600 = 927

00002) as described in Methods Bacterial titers in leaves were determined either 928

Page 42 of 54

immediately (0 dpi) or after three days (3 dpi) Bars represent the mean plusmn SE of four 929

independent experiments each with 4 or 12 biological replicates at 0 and 3 dpi 930

respectively Data were combined using a mixed linear model Asterisks indicate 931

significant differences from Col-0 wild-type (q lt 005) Susceptible pad4 plants were 932

included as a positive control 933

934

Figure 3 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves 935

(A) Depiction of the assay for brittleness of gae mutant leaves Fully expanded leaves of936

four to five week-old plants were bent by 180ordm so that the adaxial surface of the leaf was 937

parallel with the soil Broken leaf petioles or midribs were considered brittle 938

(B) Quantification of broken midribs or petioles For each genotype 66 to 82 leaves 939

were analyzed for brittleness as described in (A) Bars represent the ratio of broken to 940

total number of leaves tested Results were analyzed using Fisherrsquos exact test Letters 941

indicate significantly different groups at p lt 005 942

(C) Conductivity measurements Leaves of four week-old plants were inoculated with943

deionized water and then leaf discs were cut out and placed on deionized water 944

Conductivity was measured at the indicated time points After 30 min the water was 945

replaced with fresh water (30 min f w) to account for wounding-related ion leakage from 946

the edges of the leaf discs Three independent experiments with six biological replicates 947

each were performed and data were analyzed using a t-test for each time point P-948

values were corrected using the Bonferroni method Data show means plusmn SE asterisks 949

indicate significant differences from wild-type Col-0 at q lt 001 950

951

Figure 4 Cell Walls of gae6 and gae1 gae6 Plants Are Reduced in GalA 952

(A) to (D) Levels of GalA (A) GlcA (B) cellulose (C) and neutral sugars (D) in alcohol953

insoluble residue (AIR) extracted from leaves of four week-old plants of the indicated 954

genotypes Bars represent means plusmn SE of four biological replicates combined using a 955

mixed linear model Letters indicate significantly different groups (p lt 005) 956

957

Figure 5 Cell Walls of gae1 gae6 Plants Are Reduced in Homogalacturonan 958

Page 43 of 54

Dot blot analyses with anti-LM19 and anti-LM20 antibodies Pectin was extracted from 959

AIR prepared from the indicated genotypes using 1 microL of extraction buffer per 10 microg of 960

AIR Pectin was serially diluted and one microL of undiluted 13 diluted and 19 diluted 961

samples were spotted on a nitrocellulose membrane Three biological replicates were 962

were spotted twice to obtain two technical replicates each All replicates produced 963

similar results 964

(A) Representative dot blot using anti-LM19 antibody965

(B) Relative signal intensities of the spots in the dot blot in (A)966

(C) Representative dot blot using anti-LM20 antibody967

(D) Relative signal intensities of the LM20 dot blot shown in (C)968

Relative signal intensities were measured using Image J software Measurements from 969

the three biological replicates were combined using a linear model after Box-Cox power 970

transformation as described in Methods Bars represent means plusmn SE Asterisks indicate 971

a difference from wild-type Col-0 at p lt 005 972

973

Figure 6 Growth of B cinerea on gae1 gae6 Plants and the Effect of B cinerea on 974

Expression of GAE1 and GAE6 975

(A) Growth of B cinerea isolates Leaves of Col-0 and gae1 gae6 plants were976

inoculated with 25 x 105 spores mL-1 of the B cinerea isolates indicated in the figure 977

Samples were collected two (2 dpi) and three (3 dpi) days later Bars represent means plusmn 978

SE of six biological replicates for each fungus combined using a mixed linear model 979

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 980

(B) and (C) Expression of GAE1 (B) and GAE6 (C) 48 h after inoculation with the B981

cinerea isolates indicated Expression levels were measured by qRT-PCR Bars 982

represent mean log2-ratios to ACTIN2 plusmn SE of three biological replicates combined 983

using a mixed linear model Letters indicate significantly different groups (q lt 005) 984

985

Figure 7 Levels of Soluble Pectin and Expression of PAD3 in Fractions Produced 986

Following Macerozyme Treatment of gae1 gae6 Cell Walls 987

(A) Schematic overview of the experimental procedure for (B) and (C)988

Page 44 of 54

(B) Uronic acid measurements Alcohol-insoluble residue (AIR) from leave tissue of four989

week-old wild-type Col-0 or gae1 gae6 plants was treated with water or 025 990

macerozyme and assayed for uronic acid levels Data from 11 biological replicates were 991

combined using a mixed linear model Bars represent means plusmn SE Letters indicate 992

significant differences (p lt 005) 993

(C) Expression of PAD3 in Col-0 plants treated with the fractions from (B) Extracts from994

(B) were heat inactivated pooled and diluted 5-fold with water Pooled extracts were995

inoculated into four week-old Col-0 plants PAD3 expression was measured 3 h after 996

inoculation and normalized to the level of ACTIN2 expression Data from four biological 997

replicates were combined using a mixed linear model Bars represent mean log2-ratios 998

to ACTIN2 plusmn SE Letters indicate significant differences (p lt 005) 999

1000

Figure 8 Macerozyme-Induced PAD3 Expression Is Absent in gae1 gae6 1001

(A) Expression of PAD3 after macerozyme treatment Four week-old plants were either1002

inoculated with 001 macerozyme or mock inoculated with water and PAD3 1003

expression was measured 3 h later by qRT-PCR Data from four biological replicates 1004

were combined using a mixed linear model Mean log2-ratios to ACTIN2 plusmn SE were 1005

plotted Letters indicate significantly different groups (p lt 005) 1006

(B) Expression of PAD3 after OG treatment PAD3 expression was measured as1007

described in (A) 3 h after inoculation with 100 microg mL-1 OGs or mock inoculation with 1008

water Data are from six biological replicates 1009

(C) PAD3 expression in untreated plants Three biological replicates were performed1010

and data analyzed as described in (A) 1011

(D) Camalexin accumulation after B cinerea treatment Camalexin accumulation was1012

measured in four week-old plants inoculated with the B cinerea strains shown in the 1013

figure Eight biological replicates each were combined using a mixed linear model 1014

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 1015

(E) PAD3 expression 28 h after treatment with the indicated B cinerea strains Data1016

from six biological replicates were collected and analyzed as described in (A) Data in 1017

all figure parts represent means plusmn SE Letters indicate significantly different groups (q lt 1018

005) 1019

Page 45 of 54

1020

Figure 9 Macerozyme-Induced Expression of the SA Marker Genes SID2 and PR1 1021

Is Abolished in gae1 gae6 Plants 1022

(A) to (H) Expression of PR1 (A) (C) (E) and (G) or SID2 (B) (D) (F) and (H) in four1023

week-old Col-0 and gae1 gae6 plants Data were combined using a mixed linear model 1024

Bars represent mean log2-ratios of expression vs ACTIN2 plusmn SE for all figure parts 1025

Letters indicate significantly different groups at p lt 005 for panels (A) to (D) (G) and 1026

(H) and qlt005 for panels (E) to (F) 1027

(A) and (B) Plants were treated with 001 macerozyme or mock treated with water1028

and samples were collected 3 h later Data from four biological replicates are shown 1029

(C) and (D) Plants were inoculated with 100 microg mL-1 OGs or mock treated with water1030

and samples collected 3 h later Data from six biological replicates are shown 1031

(E) and (F) Plants were inoculated with the indicated B cinerea isolates or mock1032

inoculated with B cinerea inoculation medium and samples collected 48 h later Data 1033

from eight biological replicates are shown 1034

(G) and (H) Expression in untreated plants Three biological replicates were performed1035

1036

Figure 10 The Expression of the JA Marker Gene JAZ10 is Highly Responsive in 1037

gae1 gae6 Plants 1038

(A) to (D) JAZ10 expression in four week-old plants Data were combined using a mixed1039

linear model Mean log2-ratios to ACTIN2 plusmn SE are shown Letters indicate significantly 1040

different groups at p lt 005 for panels (A) (B) and (D) and q lt 005 for panel (C) 1041

(A) Plants were inoculated with 001 macerozyme or mock inoculated using water and1042

samples were collected 3 h later Data from four biological replicates are shown 1043

(B) Plants were inoculated with 100 microg mL-1 OGs or mock inoculated using water and1044

samples collected 3 h later Data are from six biological replicates 1045

(C) JAZ10 expression 48 h after treatment with the indicated B cinerea strains Data1046

are from six biological replicates 1047

(D) JAZ10 expression in untreated plants Data are from three biological replicates1048

1049

Figure 11 Gallo 1-Induced Callose Deposition Is Reduced in gae1 gae6 Plants 1050

Page 46 of 54

(A) Callose deposition in four week-old Col-0 and gae1 gae6 plants Plants were1051

inoculated with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) or mock inoculated 1052

with B cinerea inoculation medium and samples were collected after 72 h Callose was 1053

stained with aniline blue and visualized using a Nikon Eclipse Ni-U microscope with a 1054

DAPI filter Three leaves were stained per treatment Scale bars are 100 microm Asterisks 1055

indicate fungal hyphae arrows indicate representative callose spots 1056

(B) Quantification of callose deposition Callose deposits were counted in 200 microm x 2001057

microm squares laid out across the images shown here Bars represent means plusmn SE from 1058

six squares each Letters indicate significantly different groups (p lt 005) according to a 1059

t-test analysis1060

1061

Figure 12 Less Soluble Pectin Accumulates in B cinerea-Infected gae1 gae6 1062

Leaves 1063

Four week-old Col-0 and gae1 gae6 plants were inoculated with 10 microL droplets of the 1064

indicated B cinerea isolate (25 x 105 spores mL-1) or mock inoculated with B cinerea 1065

inoculation medium Leaves were collected 48 h later AIR was extracted from leaf 1066

samples and water added (20 microL infection site) to extract soluble pectin fragments 1067

Samples were vortexed for 7 h centrifuged at 12000 g and the uronic acid 1068

concentration of the supernatant was measured Data from nine biological replicates 1069

were combined using a mixed linear model Means plusmn SE are shown Letters indicate 1070

significant differences (q lt 005) 1071

1072

Figure 13 Macerozyme-Induced PTI Is Lost in gae1 gae6 While OG-Induced PTI Is 1073

Unaltered 1074

(A) Macerozyme-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1075

inoculated with 001 macerozyme or mock inoculated with boiled macerozyme and 1076

were infected with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) 24 h later Samples 1077

were collected immediately (0 dpi) and after two (2 dpi) and three (3 dpi) days Data 1078

show mean plusmn SE of six to 14 biological replicates each combined using a linear model 1079

Letters indicate significantly different groups (q lt 005) 1080

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

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Page 24 of 54

____________________________________________________________________________

Page 25 of 54

DISCUSSION 426

Here we showed that loss of GAE1 and GAE6 leads to a dramatic reduction in 427

pectin content in the cell wall (Figure 4) likely causing the brittleness of leaves that we 428

observed (Figure 3) Petioles and midribs of gae1 gae6 plants broke easily when bent 429

and inoculation of leaves with deionized water resulted in increased ion leakage 430

possibly due either to microscopic fractures of the cell wall induced by inoculation stress 431

or to a reduced capability of these walls to withstand increased turgor pressure The 432

increased brittleness was associated with altered cell wall composition In our analysis 433

the GalA content of gae1 gae6 leaf walls was reduced by more than 40 and the total 434

uronic acids by over 60 (Figure 4 and Supplemental Figure 5) This is a major change 435

in the overall cell wall composition considering that pectin makes up about 50 of the 436

plant cell walls found in Arabidopsis leaves (Zablackis et al 1995 Nobuta et al 2007 437

Harholt et al 2010) Interestingly quasimodo1 (qua1) plants which have a mutation in 438

a glycosyltransferase family 8 gene are reduced in GalA by 25 and show reduced 439

binding of HG specific antibodies (Bouton et al 2002) 440

Furthermore qua2 plants which have a mutation in a putative methyltransferase 441

have about 13 less total GalA and lack 50 of cell wall HG (Mouille et al 2007) 442

Likewise cgr2 cgr3 plants carrying mutations in another class of Golgi-localized 443

putative methyl-transferases have leaf cell walls that are reduced by about 40 in 444

uronic acid (Kim et al 2015) All three mutants are severely dwarfed (Bouton et al 445

2002 Mouille et al 2007 Kim et al 2015) Additionally qua1 and qua2 mutants show 446

a distorted overall shape likely due to reduced cell cohesion and are extremely fragile 447

(Bouton et al 2002 Mouille et al 2007) By contrast gae1 gae6 plants with a similar 448

strong reduction in GalA and total uronic acids have an overall normal shape and are 449

only slightly smaller than wild-type Col-0 (Supplemental Figure 4) In the wall analysis 450

performed here we did not detect changes in other wall polysaccharides or structures 451

that might compensate for the reduction of pectin in gae1 gae6 It is possible the severe 452

morphological alterations in qua2 and cgr2 cgr3 plants are due to reduction in pectin 453

methylesterification which is unaffected in gae1 gae6 It was speculated that quality 454

control mechanisms in the Golgi prevent secretion of inefficiently esterified pectin in 455

cgr2 cgr3 (Kim et al 2015) suggesting that the Golgi of these plants might over-456

Page 26 of 54

accumulate pectin and that this may contribute to the severe dwarf phenotype of these 457

plants The gae1 gae6 plants described here provide an opportunity to study the effects 458

of reduction of pectin in plants that do not show severe morphological phenotypes 459

We found that gae1 gae6 plants are also compromised in resistance to B 460

cinerea and Pma ES4326 indicating that loss of pectin reduces immunity to these 461

pathogens (Figure 2 and 6) We found that gae1 gae6 plants were slightly more 462

susceptible to Pma ES4326 but showed a strong increase in susceptibility to specific B 463

cinerea isolates This was not unexpected because B cinerea is known to interact with 464

pectin as part of its infection strategy It secretes various pectin-degrading enzymes 465

early during infection and requires some of these enzymes including PG1 PG2 and 466

PME1 for full virulence (ten Have et al 1998 Valette-Collet et al 2003 Kars et al 467

2005b Espino et al 2010) Additionally Arabidopsis plants expressing an antisense 468

construct of the polygalacturonase inhibitor PGIP1 are significantly more susceptible to 469

B cinerea (Ferrari et al 2006) On the other hand Pma ES4326 is not known to470

require cell wall degrading enzymes for full virulence even though it encodes two 471

pectate lyase genes in its genome (PMA4326_07094 and PMA4326_04621 472

wwwbacteriaensemblorg) B cinerea isolates are also known to be genetically quite 473

diverse in their polygalacturonase (PG) loci and genetic variability in PG2 is associated 474

with varying growth on pectin containing media (Rowe and Kliebenstein 2007) 475

Furthermore the contribution of PG1 and PG2 to B cinerea virulence seems to depend 476

on the specific B cinerea isolate and the host species (Kars et al 2005a Zhang and 477

Van Kan 2013) B cinerea isolates are also known to be differently sensitive to the anti-478

microbial secondary metabolite camalexin (Kliebenstein et al 2005) In line with these 479

observations the susceptibility of gae1 gae6 plants differed depending on the specific 480

B cinerea isolate Whereas Fresa 525 Gallo 1 UK Razz and KT grew better in gae1481

gae6 we detected no difference in Acacia Apple 517 DN Grape Pepper or Rasp 482

growth (Figure 6) 483

B cinerea was shown to metabolize pectin and to possibly utilize GalA as a484

carbon source during in planta growth (Zhang et al 2011 Zhang and Van Kan 2013) 485

Our data show that gae1 gae6 plants which are reduced in GalA content did allow 486

enhanced growth of specific B cinerea isolates but no reduction in growth of any B 487

Page 27 of 54

cinerea isolates tested was found This suggests that utilization of pectin as a carbon 488

source was not a contributing factor to alterations in B cinerea susceptibility It is 489

possible that changes in the cell wall composition affect the structural integrity of the 490

wall and affect hyphal penetration of specific B cinerea isolates However OGs (small 491

HG fragments) are likely released during the infection process (Ferrari et al 2013) 492

OGs function as DAMPs and initiate immune responses in Arabidopsis (Ferrari et al 493

2013) Treatment of Arabidopsis with OGs having a degree of polymerization (DP) of 494

10-15 sugar subunits was previously shown to enhance resistance to B cinerea (Ferrari495

et al 2007) We showed that pretreatment with OGs or macerozyme induced enhanced 496

immunity to B cinerea isolate Gallo 1 in wild-type Col-0 but only OGs and not 497

macerozyme induced immunity in gae1 gae6 (Figure 13) Our data suggest gae1 gae6 498

plants which have reduced pectin content may release less OGs and that this 499

reduction leads to the increased susceptibility to B cinerea 500

Consistently we found that macerozyme treatment released less soluble pectin 501

from gae1 gae6 cell walls than from wild-type walls Fractions containing soluble pectin 502

released from gae1 gae6 were also less active in inducing PAD3 gene expression than 503

the fractions released from wild-type walls (Figure 7) We also showed that less soluble 504

pectin accumulated in gae1 gae6 than in wild-type plants 48 h after treatment with B 505

cinerea (Figure 12) 506

We observed that loss of pectin leads to alterations in A thaliana immune 507

signaling Macerozyme treatment induced expression of PAD3 and the SA marker 508

genes PR1 and SID2 in wild-type Col-0 but not in gae1 gae6 whereas OG treatment 509

led to increased PAD3 and SID2 expression in both wild-type and gae1 gae6 (Figure 8 510

and 9) This suggests that although release of active soluble pectin in these plants 511

might be altered OG recognition is intact In fact cell walls isolated from qua1 another 512

mutant known to be reduced in HG released less OGs (DP1-6) than did wild-type cell 513

walls upon endopolygalacturonase treatment (Bouton et al 2002) This was also true 514

for gae1 gae6 cell walls as macerozyme treatment of isolated gae1 gae6 cell walls 515

released less soluble pectin than treatment of Col-0 cell walls Because the fraction 516

from gae1 gae6 plants was also less active in inducing PAD3 expression (Figure 7) it 517

seems possible that biologically active OGs were the active component inducing PAD3 518

Page 28 of 54

expression Interestingly expression of the JA marker genes JAZ5 and JAZ10 was 519

higher in gae1 gae6 than in wild-type mock-inoculated plants but was comparable to 520

wild-type in untreated plants suggesting that JA signaling in gae1 gae6 is 521

hyperresponsive (Figure 10 and Supplemental Figure 11) This induction of JA signaling 522

might be due to wounding stress incurred during the inoculation process JA and SA 523

signaling generally act antagonistically (Pieterse et al 2012) Increased JA signaling 524

activity might hence explain why mock- and macerozyme-treated gae1 gae6 plants 525

exhibited reduced expression of the SA marker PR1 and why OG treatment induced 526

expression of SID2 another SA marker to a lower extent in gae1 gae6 than in wild-type 527

plants (Figure 9) 528

No single immune signaling component was found to be the sole contributor to 529

the compromised immunity of gae1 gae6 to specific B cinerea isolates such as Gallo 1 530

and Fresa 525 These isolates grew better on pad3 while growth of Pepper another B 531

cinerea isolate that did not show altered virulence on gae1 gae6 was unaltered 532

(Supplemental Figure 9) This suggested that differences in camalexin sensitivity of 533

these B cinerea isolates might account for the differences in gae1 gae6 susceptibility 534

However camalexin accumulation and PAD3 expression was the same for wild-type 535

Col-0 and gae1 gae6 for the B cinerea isolates Fresa 525 and Gallo 1 (Figure 8) which 536

showed enhanced growth on gae1 gae6 plants It should be noted that B cinerea 537

virulence is often estimated by comparing lesion sizes Lesion size and measurements 538

of relative fungal growth by qPCR do not always show the same results For example 539

although Denby et al (2004) showed that Pepper causes larger lesions on pad3 than on 540

wild-type this study showed that relative fungal growth of Pepper on pad3 was 541

unaltered 542

We observed that callose deposition was strongly reduced in gae1 gae6 plants 543

72 h after treatment with B cinerea isolate Gallo 1 (Figure 11) possibly due to reduced 544

release of active soluble pectin Interestingly OG-induced PTI to an unspecified isolate 545

of B cinerea was intact in the pmr4 mutant which does not produce callose (Galletti et 546

al 2008) Due to the high SA levels in pmr4 plants (Nishimura et al 2003) and the fact 547

that different B cinerea isolates vary both in induction of and reaction to defense 548

responses (Kliebenstein et al 2005 Rowe et al 2010) we could not determine 549

Page 29 of 54

whether reduced callose deposition causes the increased susceptibility of gae1 gae6 to 550

B cinerea isolate Gallo 1551

We did not observe any changes in B cinerea growth either on cev1 ixr1 plants 552

which are known to have constitutively high JA and OPDA levels (Ellis et al 2002b) or 553

on plants exogenously treated with MeJA (Supplemental Figure 9) Because plants 554

were kept uncovered for 3 h after MeJA treatment it is unclear how much MeJA was 555

bioavailable in the plant However JAZ10 expression was induced 3 h after spraying 556

with MeJA (Supplemental Figure 10) Because pathogen growth generally depends on 557

the combination of a multitude of immune signaling events we conclude that multiple 558

changes to the immune signaling in gae1 gae6 together account for the altered B 559

cinerea growth phenotypes detected 560

We showed that expression of GAE1 and GAE6 is repressed by the bacterial 561

hemibiotroph Pma ES4326 This repression did not occur in an eds1 or pad4 562

background or in pbs3 at the 24-h time point (Figure 1 and Supplemental Figure 1) 563

During the Arabidopsis - Pma ES4326 interaction the expression of many defense 564

related genes is altered in a PAD4EDS1 dependent fashion (Wang et al 2008) Some 565

of these expression changes require SA CBP60g and SARD1 are thought to act 566

downstream of PAD4 and EDS1 but upstream of SA-dependent responses (Wang et 567

al 2011) PBS3 is also thought to act downstream of PAD4 and EDS1 and upstream of 568

SA but the set of genes whose expression it affects is only partially overlapping with 569

those affected by CBP60gSARD1 (Wang et al 2008 Wang et al 2011) Thus GAE1 570

and GAE6 repression is regulated by the part of the immune signaling network that 571

requires PAD4 EDS1 and PBS3 but is independent of CBP60gSARD1 and SA 572

Because EDS1 PAD4 and PBS3 are important components of the plant immune 573

signaling network (Wiermer et al 2005 Nobuta et al 2007) one might conclude that 574

repression of GAE1 and GAE6 may be part of the plant immune response to Pma 575

ES4326 B cinerea treatment also reduced GAE1 and GAE6 expression but this was 576

mostly independent of PAD4 (Figure 6) while both Pma ES4326 and B cinerea repress 577

GAE1 and GAE6 expression the host signaling mechanisms responsible likely differ 578

In summary our data show that Arabidopsis GAE1 and GAE6 are required for 579

pectin biosynthesis leaf flexibility and immunity to Pma ES4326 and to specific B 580

Page 30 of 54

cinerea isolates We used macerozyme a commercial pectinase to mimic the effect of 581

B cinerea pectinases Macerozyme treatment released less pectin from gae1 gae6 cell582

walls and induced less immune signaling in gae1 gae6 plants Less water soluble pectin 583

also accumulated in B cinerea infected gae1 gae6 plants and macerozyme-induced 584

immunity to B cinerea isolate Gallo 1 was reduced in these plants We conclude that 585

the reduced pectin content in these mutants may lead to reduced release of soluble 586

pectin including active OGs during pathogen attack and that this compromises 587

immunity 588

589

590

Page 31 of 54

METHODS 591

Plant Materials and Growth Conditions 592

Wild-type Columbia (Col-0) and all mutant plants (in Col-0 background) were 593

grown on sterilized BM2 germinating mix (Berger Inc Quebec Canada) in a controlled 594

environment chamber (Conviron Pembina ND USA) with a 12-h photoperiod under 595

100 microM m-2 s-1 fluorescent illumination at 22degC and 75 relative humidity Germplasm 596

used is described in the Accession Numbers section at the end of Methods 597

598

Data Analysis and Replicates 599

For biological replicates samples were obtained from separate plant tissues and 600

in many cases from plants grown in different flats In some experiments multiple 601

technical replicates were measured per biological replicate For statistical analysis all 602

technical replicates were averaged and the averages were considered one biological 603

replicate Independent experiments were performed at different times and often 604

consisted of multiple biological replicates per experiment 605

Data analysis was performed using mixed linear models in the R programming 606

environment (Bates et al 2015) unless otherwise indicated Generally genotypes 607

treatments and time points were used as fixed effects and replicate-specific effects like 608

different pots flats etc were considered random effects We reported p-values 609

obtained from the mixed linear model when less than 10 comparisons were performed 610

and q-values for more than 10 comparisons to correct for multiple comparison testing 611

Q-values were calculated using the Benjamini-Hochberg procedure (Benjamini and612

Hochberg 1995) unless specified otherwise 613

614

Pathogen Strains Growth Conditions and Pathogen Growth Assays 615

Growth of Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) and 616

bacterial growth assays were performed as previously described (Bethke et al 2014) 617

For qRT-PCR assays four week-old plants were inoculated with bacteria (OD600 = 618

0002) or mock (5 mM MgSO4) At least four leaves from two plants were collected at 619

the indicated time points per biological replicate 620

Page 32 of 54

Prior to each experiment Botrytis cinerea isolates were grown on 1x PDA 621

medium (Difco) for 10 d at room temperature All isolates used have been described 622

previously (Rowe and Kliebenstein 2007) The spores were washed from the surface of 623

the plate using an inoculation medium (1x Gamborgrsquos B-5 basal salt mixture (Sigma-624

Aldrich G5768) 2 (wv) glucose 10 mM phosphate buffer pH 64) and fungal hyphae 625

were removed from the suspension by filtering through four layers of cheesecloth The 626

concentration of spores was determined using a hemocytometer and adjusted to 25 x 627

105 spores mL-1 Four week-old Arabidopsis plants were inoculated by placing one 10-628

microL droplet of the B cinerea spore solution or inoculation medium (mock) on the adaxial 629

leaf surface of fully expanded leaves Inoculated plants were kept at 100 relative 630

humidity Infection sites were collected at various time points using a cork borer For 631

qRT-PCR assays at least four leaf discs from one plant were combined for each 632

biological sample For B cinerea growth assays at least 12 leaf discs from three to six 633

plants were pooled per biological sample Relative fungal growth was determined by 634

measuring the abundance of a fungal gene relative to a plant gene as described below 635

In brief DNA from infected tissue was extracted and equal amounts of total DNA were 636

used to perform qPCR reactions using the B cinerea cutinase A and the Arabidopsis 637

SK11 gene respectively Primers were described in (Gachon and Saindrenan 2004) 638

and are listed in Supplemental Table 1 Two technical replicates were combined for 639

each biological replicate B cinerea is a necrotrophic fungal pathogen and may destroy 640

plant DNA as disease progresses However decreases in the amount of plant DNA 641

over the course of our experiments were only observed at late time points with very 642

susceptible plants so the qPCR assay is generally a reasonable estimate of fungal 643

biomass (Supplemental Figure 16) 644

645

Treatment with Macerozyme or OGs 646

For PTI assays four week-old plants either were inoculated with 001 (wv) 647

macerozyme R-10 (Yakult) a Rhizopus polygalacturonase (EC 32115) that possesses 648

high pectinase and hemicellulase activity or were mock inoculated using heat-649

inactivated macerozyme Other similar plants were either inoculated with 250 or 500 microg 650

mL-1 OGs or mock inoculated with water One d later plants were infected with B 651

Page 33 of 54

cinerea and samples were collected immediately (0 dpi) and two or three d later To 652

investigate the role of MeJA on B cinerea growth plants were sprayed with 1mM MeJA 653

in water with 001 (vv) Tween 20 Covers were removed so that leaves could dry off 654

before plants were inoculated with B cinerea three h later Samples were harvested at 655

the indicated time points For expression analysis and MAPK activation assays plants 656

were either inoculated with 100 microg mL-1 OGs or mock inoculated with water OGs used 657

for experiments described in Figures 8 to 10 and Supplemental Figure 12 were a kind 658

gift from Simone Ferrari OGs used in experiments described in Figure 13 and 659

Supplemental Figures 11 13 and 15 were prepared as previously described (Kohorn et 660

al 2014) 661

662

Expression Analysis 663

Leaf tissue from inoculated plants was harvested at the indicated time points 664

flash frozen pulverized and RNA extracted using Trizol (Invitrogen) Quantitative RT-665

PCR was performed using the SuperScript III Platinum SYBR Green One-Step 666

quantitative RT-PCR kit (Invitrogen) and a Lightcycler 480 Real-Time PCR system 667

(Roche) as previously described (Truman and Glazebrook 2012) In brief equal 668

amounts of total RNA and a gene specific primer were used for each reaction The 669

crossing point (Cp) was calculated using the 2nd derivative max method provided with 670

the Lightcycler software for each amplification curve Each reaction was run with two 671

technical replicates and the Cp values for these replicates were averaged ACTIN2 was 672

used as a stably expressed reference gene At least three biological replicates each 673

were performed and analyzed using a mixed-linear model RT-PCR was performed to 674

verify T-DNA insertion lines using the One-Step RT-PCR kit (Qiagen) according to the 675

manufacturerrsquos instructions The primers used can be found in Supplemental Table 1 676

677

Electrolyte Leakage Assay 678

Fully expanded leaves of four week-old plants were inoculated with water (Milli-Q 679

grade) using a needleless syringe Leaf discs were collected using a cork borer and 680

three leaf discs from one leaf were placed with the adaxial surface down onto 2 mL 681

water in a 12-well cell culture plate Conductivity was measured using a Horiba B-173 682

Page 34 of 54

conductivity meter after 30 min and then the water was replaced Conductivity was 683

measured again 30 min later (1 h total) as indicated in Figure 3C Six biological 684

replicates each were performed in three independent experiments and means and SEs 685

were calculated Two-sided t-tests were performed for each time point 686

687

DAB and Aniline Blue Staining 688

Leaves from four week-old plants were either mock inoculated with B cinerea 689

inoculation medium or inoculated with 10 microL droplets of a B cinerea spore solution (25 690

x 105 spores mL-1) of the indicated isolates Samples were collected 72 h later For 33prime-691

diaminobenzidine (DAB) staining two to five inoculated leaves were vacuum infiltrated 692

with 33prime-Diaminobenzidine (1 mg mL-1 in water pH 38) solution and stained overnight 693

in the dark Leaves were destained using 100 ethanol at 65degC and then transferred to 694

50 (vv) glycerol and photographed 695

Callose was visualized by staining leaves with aniline blue as previously 696

described (Adam and Somerville 1996) Briefly leaves were vacuum infiltrated with 697

alcoholic lactophenol (one volume of phenolglycerollactic acidwater (1111) mixed 698

with two volumes of ethanol) and destained at 65degC Next leaves were moved through 699

an alcohol gradient (50 20 and 10 (vv) ethanol) into water The destained leaves 700

were stained for 30 min in 150 mM K2HPO4 (pH 95) with 001 (wv) aniline blue 701

Samples were mounted in water and examined using a Nikon Eclipse Ni-U microscope 702

at 10x magnification using a DAPI filter (excitation at 325-375 nm emission at 435-485 703

nm) For callose quantification six 200 microm x 200 microm squares were counted for each 704

sample type Callose deposits per mm2 were calculated and data analyzed using a t-705

test Note that there is an inherent uncertainty associated with such counts because 706

callose deposits may not all be in focus at the same time 707

708

MAPK Activity Assay 709

Fully expanded leaves of four week-old plants were inoculated with 001 710

macerozyme 100 microg mL-1 OGs 100 nM flg22 or water (mock) flg22 a peptide from 711

bacterial flagella that induces immune responses was used as a positive control for 712

Page 35 of 54

MAPK activation (Asai et al 2002) and was purchased from EZBiolab Inc (Westfield 713

IN USA) Samples were harvested 10 min later and immediately frozen in liquid 714

nitrogen Protein was extracted using an extraction buffer containing 25 mM Tris pH 78 715

75 mM NaCl 1 mM DTT 1 mM NaF 05mM Na3VO4 01 (vv) Tween 20 one 716

cOmplete Mini protease inhibitor cocktail tablet (Roche) and one PhosSTOP 717

phosphatase inhibitor cocktail tablet (Roche) per 10 mL buffer 15 microg of total protein 718

were electrophoresed on a 10 SDS-polyacrylamide gel and then blotted onto a PVDF 719

membrane (Bio-Rad 162-0177) Activated MAPKs were detected using p4442 MAPK 720

(Erk12) antibody (9102S Cell Signal 12500 in TBST (20 mM Tris pH 75 150 mM 721

NaCl 01 (vv) Tween 20)) Anti-Rabbit IgG-HRP (115000 in TBST A6154 Sigma) 722

and ECL Plus substrate (Pierce 32132) MPK3 and MPK6 were detected with an anti-723

MPK3 antibody (M8318 Sigma 12000 in TBST and 3 (wv) milk) or an anti-MPK6 724

antibody (A7104 Sigma 16000 in TBST and 3 (wv) milk) respectively The 725

secondary antibody and detection were the same as for the p4442 antibody Five 726

independent experiments were performed 727

728

Preparation of Alcohol Insoluble Residue (AIR) Pectin Extraction and Uronic Acid 729

Measurements 730

Fully expanded leaves from four week-old plants that were incubated for 48 h in 731

the dark to reduce starch were harvested flash frozen and pulverized AIR was 732

extracted by washing ground material twice in 70 (vv) ethanol three times in a 733

mixture of chloroform and methanol (11 vv) and once in acetone (Gille et al 2009) 734

and was then air dried Cell wall pectin was extracted from AIR with cell wall extraction 735

buffer (50 mM Trizma 50 mM CDTA pH 72) at 95degC for 15 min Samples were 736

homogenized using a paint shaker (Harbil 5G-HD) and glass beads (3 mm diameter) 737

For dot blot experiments 500 microL of cell wall extraction buffer were used per 10 mg of 738

AIR Debris was precipitated by centrifugation for 10 min at 10000 g 739

Total uronic acid content of pectin extracted as described above using 500 microL of 740

cell wall extraction buffer per 1 mg of AIR was measured as described previously 741

(Filisetti-Cozzi and Carpita 1991 van den Hoogen et al 1998) First 36 microL of pectin 742

extract were mixed with 4 microL of 4 M sulfamic acid Then 200 microL of sulfuric acid 743

Page 36 of 54

containing 120 mM sodium tetraborate was added and samples were incubated at 80degC 744

for 1 h Following cooling on ice the optical density of samples was measured at 490 745

nm Next 40 microL of m-hydroxydiphenyl reagent (100 microL of m-hydroxydiphenyl in dimethyl 746

sulfoxide at 100 mgmL mixed with 49 mL 80 (vv) sulfuric acid just before use) was 747

added and the samples were mixed The optical density at 490 nm was measured 748

again The optical density before m-hydroxydiphenyl reagent addition was subtracted 749

from the optical density measured after addition of the dye The concentration of uronic 750

acid was calculated using known amounts of GalA as a standard 751

752

Release of Pectin from Arabidopsis Cell Walls Using Macerozyme 753

AIR preparations from leaves of four week-old plants were treated with 025 754

(wv) macerozyme R-10 (Yakult) or water (mock) at a concentration of 100 microL per mg 755

AIR Samples were incubated for 3 h at room temperature on a vortex shaker and 756

centrifuged for 10 min at 12000 g Total uronic acid concentration of 5-fold diluted 757

supernatants was determined for two technical replicates each The concentration of 758

uronic acid was calculated using known amounts of GalA as a standard For 759

subsequent qRT-PCR experiments supernatants from multiple extractions were 760

combined boiled for 20 min to inactivate macerozyme diluted 10-fold with water and 761

inoculated into four week-old Col-0 plants Heat-inactivated 10-fold diluted macerozyme 762

solutions were used as mock treatment Samples were collected 3 h later and PAD3 763

expression was determined 764

765

Release of Pectin from Arabidopsis Leaves Infected with B cinerea 766

Fully expanded leaves of four week-old plants were either inoculated with up to 767

five 10 microL droplets of B cinerea spore solution (25 x 105 spores mL-1) or mock 768

inoculated with B cinerea inoculation medium For each biological replicate leaves with 769

a combined count of at least 20 infection sites were collected 48 h later AIR was 770

extracted as described above but plants were not dark-treated or freeze-dried and the 771

first ethanol step was performed using 85 (vv) ethanol To extract soluble pectin 772

fragments AIR samples were mixed with water at a concentration of 20 microL per infection 773

site incubated for 7 h at room temperature on a vortex shaker and centrifuged for 10 774

Page 37 of 54

min at 12000 g Total uronic acid concentration of supernatants was determined for two 775

technical replicates each The concentration of uronic acid was calculated using known 776

amounts of GalA as a standard 777

778

Measurement of Uronic Acids Neutral Monosaccharides and Cellulose Content in 779

Cell Walls 780

Neutral sugars and uronic acids from non-cellulosic polysaccharides in cell walls 781

(AIR) were released by trifluoroacetic acid (TFA) hydrolysis Approximately 1 mg of AIR 782

was hydrolyzed with 2 M TFA at 121degC for 90 min The TFA-soluble material was split 783

into two equal parts one for uronic acid and one for neutral sugar determination The 784

uronic acid content was determined by analyzing the hydrolyzate using a CarboPac 785

PA200 anion-exchange column with an ICS-3000 Dionex chromatography system 786

(Dionex Sunnyvale CA USA) The elution profile consisted of a linear gradient of 50 to 787

200 mM sodium acetate in 01 M NaOH in 10 min at 04 mL per min Alditol acetate 788

derivatives of neutral sugars were produced as described (York et al 1985) In brief 789

monosaccharides in the hydrolyzate were reduced with NaBH4 and peracetylated with 790

acetic anhydride and pyridine at 121degC for 20 min The generated alditol acetates were 791

analyzed by GC-MS Crystalline cellulose was measured according to Updegraff (1969) 792

After hydrolysis of noncellulosic polysaccharides from AIR with the Updegraff reagent 793

(acetic acid nitric acids water 812 (vv) ) the remaining pellet was hydrolyzed in 794

72 sulfuric acid The resulting glucose quantity was determined by the anthrone 795

method (Scott and Melvin 1953) 796

797

Dot Blot Experiments 798

Pectin solutions extracted using 50 microL of cell wall extraction buffer per mg AIR 799

were serially diluted and nitrocellulose membranes were spotted with 1 microL of the diluted 800

pectin solutions Membranes were dried overnight blocked with 5 milk (wv) in 1x 801

PBS (8 g L-1 NaCl 02 g L-1 KCl 144 g L-1 Na2HPO4 024 g L-1 KH2PO4 pH 74) and 802

probed with LM5 (Jones et al 1997) LM6 (Willats et al 1998) LM8 (Willats et al 803

2004) LM19 and LM20 (Verhertbruggen et al 2009a) or CCRC-M7 (Steffan et al 804

Page 38 of 54

1995) antibodies LM series antibodies were diluted 1250 and CCRC-M7 was diluted 805

1500 in 5 milk powder (Nestle Carnation) in 1 x PBS For LM series antibodies a 806

goat anti-Rat HRP conjugated antibody (Bethyl A110-105P 15000 diluted) and for 807

CCRC-M7 an anti-Mouse IgG HRP conjugate (Promega W402B 12500 diluted) was 808

used as secondary antibody in 5 milk powder in 1x PBS LM series antibodies were 809

obtained from plant probes (wwwplantprobesnet) and CCRC-M7 from CarboSource 810

(wwwcarbosourcenet) Membranes were washed after incubation with the primary and 811

secondary antibodies with 1x PBS Dot blots were developed using the ECL system 812

(GE Healthcare Amersham ECL prime) For quantification of signals dot blot results on 813

x-ray films were photographed using a CCD camera and intensities were measured814

using Image Jrsquos Integrated Density function Locally adjusted background was 815

subtracted As the dynamic range of the signal on an x-ray film was narrow non-816

linearity in the measured values was corrected for each genotype in each biological 817

replicate using the values from different dilutions in two technical replicates We noticed 818

that the residuals were not normally distributed and performed Box-Cox power 819

transformation (Box and Cox 1964) to obtain normally distributed values for the 820

subsequent statistical analysis of the data For LM19 Box-Cox selected power 821

transformation was done to the power of 038th and for LM20 to the power 075th After 822

correction of the transformed measured values with the effect of the biological 823

replicates the values were fit to a linear model with the Arabidopsis genotype as the 824

fixed effect This linear model was used both to generate the plots in Figures 5B and D 825

and to test significant differences 826

827

Camalexin Measurements 828

Four week-old plants were infected with B cinerea and samples (three leaf disks 829

from one plant each) were harvested at the indicated time points flash frozen and 830

pulverized Each sample was extracted with 300 microL of 90 methanol (vv) 50 microL of the 831

extract was run on an Agilent Lichrocart 250-4 RP18e 5-microm column using an Agilent 832

1100 series HPLC Camalexin was detected using a DiodeArray (DAD) at 330 nm and 833

with a fluorescence detector at emission 318 nmexcitation 385 nm (Agilent) Separation 834

was achieved using the following program with aqueous acetonitrile 5 min gradient 835

Page 39 of 54

from 63 to 69 acetonitrile 30 s gradient from 69 to 99 acetonitrile 2 min at 99 836

acetonitrile and a post-run equilibration of 35 min at 63 acetonitrile (Denby et al 837

2004 Kliebenstein et al 2005) Purified camalexin was used to produce a standard 838

curve to identify and quantitate camalexin 839

840

Accession Numbers 841

Microarray data used can be accessed at NCBI Gene Expression Omnnibus 842

(httpwwwncbinlmnihgovgeo) GSE18978 Sequence information for all Arabidopsis 843

genes described in this article can be found in TAIR (wwwarabidopsisorg) using the 844

following accession numbers PAD3 (CYP71B15 At3g26830) SID2 (ICS1 At1g74710) 845

GAE1 (At4g30440) GAE2 (At1g02000) GAE3 (At4g00110) GAE4 (At2g45310) GAE5 846

(At4g12250) GAE6 (At3g23820) PR1 (At2g14610) JAZ5 (At1g17380) JAZ10 847

(At5g13220) ACTIN2 (At3g18780) PDF12a (At5g44420) PDF12b (At2g26020) 848

AtSK11 (At5g26751) The sequence of the B cinerea cutinase Bc-CutA (Z69264) can 849

be found in the DNA Data Bank of Japan (httpgetentryddbjnigacjp) Germplasm 850

used included dde2-2 (At5g42650 (von Malek et al 2002)) pad3-1 (At3g26830 (Zhou 851

et al 1999)) pad4-1 (At3g52430 (Jirage et al 1999)) sid2-2 (At1g74710 (Wildermuth 852

et al 2001)) pbs3-2 (At5g13320 SALK_018225 (Nobuta et al 2007)) cbp60a-1 853

(At5g62570 SALK_124410 (Truman et al 2013)) cbp60g-1 sard1-2 (At5g26920 854

SALK_023199 At1g73805 SALK_052422 (Wang et al 2011) cbp60a-1 cbp60g-1 855

sard1-2 (Truman et al 2013) gae1-1 (At4g30440 SALK_085554) gae6-1 (At3g23280 856

SALK_104454) gae6-2 (At3g23280 SALK_017191) ixr1-1 cev1 (At5g05170 (Ellis 857

and Turner 2001 Scheible et al 2001)) eds1 plants were derived by introgression of 858

the Ler eds1-2 allele into Col-0 that contains two copies of EDS1 (At3g48090 859

At3g48080 (Bartsch et al 2006) mpk6-2 (At2g43790 SALK_073907 (Liu and Zhang 860

2004)) mpk3-DG (At3g45640 a fast neutron deletion mutant (Miles et al 2005)) and 861

npr1-1 (At1g64280 (Cao et al 1997)) T-DNA insertion lines were part of the SALK 862

collection (Alonso et al 2003) and were obtained from the Arabidopsis Biological 863

Resource Center 864

865

Supplemental Data 866

Page 40 of 54

Supplemental Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 867

Expression Does Not Require CBP60s or SID2 868

Supplemental Figure 2 Expression of GAE Family Members uponTreatment with Pma 869

ES4326 870

Supplemental Figure 3 Characterization of T-DNA Insertions in GAE1 and GAE6 871

Supplemental Figure 4 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves and 872

Are Slightly Smaller than Wild-Type Col-0 Plants 873

Supplemental Figure 5 Cell Walls of gae1 gae6 Plants Contain Less Uronic Acid than 874

Wild-Type Col-0 Cell Walls 875

Supplemental Figure 6 Antibodies Raised Against the RGI or XG Components of 876

Pectin Show No Difference in Binding to gae Mutant and Col-0 Cell Walls 877

Supplemental Figure 7 The Effect of B cinerea Treatment on the Expression of GAE 878

Family Genes 879

Supplemental Figure 8 Treatment with 001 Macerozyme Does Not Cause Any 880

Visible Damage to Col-0 Plants 881

Supplemental Figure 9 B cinerea Growth on Plants with Defects in Immune 882

Signaling 883

Supplemental Figure 10 MeJA-Induced Expression of the JA marker gene JAZ10 884

Supplemental Figure 11 The Expression of the JA Marker Gene JAZ5 Is Highly 885

Responsive in gae1 gae6 Plants 886

Supplemental Figure 12 The Expression of PDF12 Is Unaltered in gae1 gae6 Plants 887

Supplemental Figure 13 Macerozyme Treatment Activates MPK3 and MPK6 888

Supplemental Figure 14 B cinerea-Induced ROS Production in Wild-Type Col-0 and 889

gae1 gae6 Plants 890

Supplemental Figure 15 OG-Induced PTI Using 250 microg mL-1 OGs 891

Supplemental Figure 16 Abundance of Plant DNA in B cinerea Treated Plants 892

Supplemental Table 1 Primers used in this study 893

894

ACKNOWLEDGEMENTS 895

We thank William Truman for helpful discussions Simone Ferrari (Sapienza 896

Universita di Roma) for the generous gift of OGs Bruna Bucciarelli (USDA-ARS) for 897

Page 41 of 54

help with fluorescence microscopy the Arabidopsis Biological Resource Center for T-898

DNA insertion lines Paul Knoxrsquos lab (University of Leeds) for LM5 LM6 LM8 LM19 899

and LM20 antibodies and the Complex Carbohydrate Resource Center (University of 900

Georgia) for the CCRC-M7 antibody Most of this work was funded by the Division of 901

Chemical Sciences Geosciences and Biosciences Office of Basic Energy Sciences of 902

the US Department of Energy through Grant DE-FG02-05ER15670 to JG This work 903

was also supported by NSF awards IOS 1339125 and IOS 1021861 to DJK NSF 904

award IOS 1121425 to F K and the USDA National Institute of Food and Agriculture 905

Hatch project number CA-D-PLS-7033-H to DJK 906

907

AUTHOR CONTRIBUTIONS 908

GB and JG designed the research GB GX AT BL NES and NH 909

performed research DJK provided tools GB GX MP AD BL RAH and FK 910

analyzed data GB and JG wrote the paper with input from all authors 911

912

FIGURE LEGENDS 913

Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 Expression 914

Requires PAD4 EDS1 and PBS3 915

(A) Expression of GAE1 in leaves of wild-type Col-0 and mutant plants Expression was916

measured 24 or 32 h after either inoculation with Pma ES4326 (OD600 = 0002) or mock 917

inoculation with 5 mM MgSO4 and was then normalized to the level of ACTIN2 Data 918

from three biological replicates were combined using a mixed linear model Bars 919

represent mean log2-ratios to ACTIN2 plusmn SE Letters indicate significantly different 920

groups with q lt 005 where 005 represents the maximum false discovery rate at which 921

the test may be called significant 922

(B) Expression of GAE6 measured as described in (A)923

924

Figure 2 Susceptibility of gae6 and gae1 gae6 Plants to Pma ES4326 Is Increased 925

Relative to Wild-Type Col-0 926

Leaves of plants of the indicated genotypes were inoculated with Pma ES4326 (OD600 = 927

00002) as described in Methods Bacterial titers in leaves were determined either 928

Page 42 of 54

immediately (0 dpi) or after three days (3 dpi) Bars represent the mean plusmn SE of four 929

independent experiments each with 4 or 12 biological replicates at 0 and 3 dpi 930

respectively Data were combined using a mixed linear model Asterisks indicate 931

significant differences from Col-0 wild-type (q lt 005) Susceptible pad4 plants were 932

included as a positive control 933

934

Figure 3 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves 935

(A) Depiction of the assay for brittleness of gae mutant leaves Fully expanded leaves of936

four to five week-old plants were bent by 180ordm so that the adaxial surface of the leaf was 937

parallel with the soil Broken leaf petioles or midribs were considered brittle 938

(B) Quantification of broken midribs or petioles For each genotype 66 to 82 leaves 939

were analyzed for brittleness as described in (A) Bars represent the ratio of broken to 940

total number of leaves tested Results were analyzed using Fisherrsquos exact test Letters 941

indicate significantly different groups at p lt 005 942

(C) Conductivity measurements Leaves of four week-old plants were inoculated with943

deionized water and then leaf discs were cut out and placed on deionized water 944

Conductivity was measured at the indicated time points After 30 min the water was 945

replaced with fresh water (30 min f w) to account for wounding-related ion leakage from 946

the edges of the leaf discs Three independent experiments with six biological replicates 947

each were performed and data were analyzed using a t-test for each time point P-948

values were corrected using the Bonferroni method Data show means plusmn SE asterisks 949

indicate significant differences from wild-type Col-0 at q lt 001 950

951

Figure 4 Cell Walls of gae6 and gae1 gae6 Plants Are Reduced in GalA 952

(A) to (D) Levels of GalA (A) GlcA (B) cellulose (C) and neutral sugars (D) in alcohol953

insoluble residue (AIR) extracted from leaves of four week-old plants of the indicated 954

genotypes Bars represent means plusmn SE of four biological replicates combined using a 955

mixed linear model Letters indicate significantly different groups (p lt 005) 956

957

Figure 5 Cell Walls of gae1 gae6 Plants Are Reduced in Homogalacturonan 958

Page 43 of 54

Dot blot analyses with anti-LM19 and anti-LM20 antibodies Pectin was extracted from 959

AIR prepared from the indicated genotypes using 1 microL of extraction buffer per 10 microg of 960

AIR Pectin was serially diluted and one microL of undiluted 13 diluted and 19 diluted 961

samples were spotted on a nitrocellulose membrane Three biological replicates were 962

were spotted twice to obtain two technical replicates each All replicates produced 963

similar results 964

(A) Representative dot blot using anti-LM19 antibody965

(B) Relative signal intensities of the spots in the dot blot in (A)966

(C) Representative dot blot using anti-LM20 antibody967

(D) Relative signal intensities of the LM20 dot blot shown in (C)968

Relative signal intensities were measured using Image J software Measurements from 969

the three biological replicates were combined using a linear model after Box-Cox power 970

transformation as described in Methods Bars represent means plusmn SE Asterisks indicate 971

a difference from wild-type Col-0 at p lt 005 972

973

Figure 6 Growth of B cinerea on gae1 gae6 Plants and the Effect of B cinerea on 974

Expression of GAE1 and GAE6 975

(A) Growth of B cinerea isolates Leaves of Col-0 and gae1 gae6 plants were976

inoculated with 25 x 105 spores mL-1 of the B cinerea isolates indicated in the figure 977

Samples were collected two (2 dpi) and three (3 dpi) days later Bars represent means plusmn 978

SE of six biological replicates for each fungus combined using a mixed linear model 979

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 980

(B) and (C) Expression of GAE1 (B) and GAE6 (C) 48 h after inoculation with the B981

cinerea isolates indicated Expression levels were measured by qRT-PCR Bars 982

represent mean log2-ratios to ACTIN2 plusmn SE of three biological replicates combined 983

using a mixed linear model Letters indicate significantly different groups (q lt 005) 984

985

Figure 7 Levels of Soluble Pectin and Expression of PAD3 in Fractions Produced 986

Following Macerozyme Treatment of gae1 gae6 Cell Walls 987

(A) Schematic overview of the experimental procedure for (B) and (C)988

Page 44 of 54

(B) Uronic acid measurements Alcohol-insoluble residue (AIR) from leave tissue of four989

week-old wild-type Col-0 or gae1 gae6 plants was treated with water or 025 990

macerozyme and assayed for uronic acid levels Data from 11 biological replicates were 991

combined using a mixed linear model Bars represent means plusmn SE Letters indicate 992

significant differences (p lt 005) 993

(C) Expression of PAD3 in Col-0 plants treated with the fractions from (B) Extracts from994

(B) were heat inactivated pooled and diluted 5-fold with water Pooled extracts were995

inoculated into four week-old Col-0 plants PAD3 expression was measured 3 h after 996

inoculation and normalized to the level of ACTIN2 expression Data from four biological 997

replicates were combined using a mixed linear model Bars represent mean log2-ratios 998

to ACTIN2 plusmn SE Letters indicate significant differences (p lt 005) 999

1000

Figure 8 Macerozyme-Induced PAD3 Expression Is Absent in gae1 gae6 1001

(A) Expression of PAD3 after macerozyme treatment Four week-old plants were either1002

inoculated with 001 macerozyme or mock inoculated with water and PAD3 1003

expression was measured 3 h later by qRT-PCR Data from four biological replicates 1004

were combined using a mixed linear model Mean log2-ratios to ACTIN2 plusmn SE were 1005

plotted Letters indicate significantly different groups (p lt 005) 1006

(B) Expression of PAD3 after OG treatment PAD3 expression was measured as1007

described in (A) 3 h after inoculation with 100 microg mL-1 OGs or mock inoculation with 1008

water Data are from six biological replicates 1009

(C) PAD3 expression in untreated plants Three biological replicates were performed1010

and data analyzed as described in (A) 1011

(D) Camalexin accumulation after B cinerea treatment Camalexin accumulation was1012

measured in four week-old plants inoculated with the B cinerea strains shown in the 1013

figure Eight biological replicates each were combined using a mixed linear model 1014

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 1015

(E) PAD3 expression 28 h after treatment with the indicated B cinerea strains Data1016

from six biological replicates were collected and analyzed as described in (A) Data in 1017

all figure parts represent means plusmn SE Letters indicate significantly different groups (q lt 1018

005) 1019

Page 45 of 54

1020

Figure 9 Macerozyme-Induced Expression of the SA Marker Genes SID2 and PR1 1021

Is Abolished in gae1 gae6 Plants 1022

(A) to (H) Expression of PR1 (A) (C) (E) and (G) or SID2 (B) (D) (F) and (H) in four1023

week-old Col-0 and gae1 gae6 plants Data were combined using a mixed linear model 1024

Bars represent mean log2-ratios of expression vs ACTIN2 plusmn SE for all figure parts 1025

Letters indicate significantly different groups at p lt 005 for panels (A) to (D) (G) and 1026

(H) and qlt005 for panels (E) to (F) 1027

(A) and (B) Plants were treated with 001 macerozyme or mock treated with water1028

and samples were collected 3 h later Data from four biological replicates are shown 1029

(C) and (D) Plants were inoculated with 100 microg mL-1 OGs or mock treated with water1030

and samples collected 3 h later Data from six biological replicates are shown 1031

(E) and (F) Plants were inoculated with the indicated B cinerea isolates or mock1032

inoculated with B cinerea inoculation medium and samples collected 48 h later Data 1033

from eight biological replicates are shown 1034

(G) and (H) Expression in untreated plants Three biological replicates were performed1035

1036

Figure 10 The Expression of the JA Marker Gene JAZ10 is Highly Responsive in 1037

gae1 gae6 Plants 1038

(A) to (D) JAZ10 expression in four week-old plants Data were combined using a mixed1039

linear model Mean log2-ratios to ACTIN2 plusmn SE are shown Letters indicate significantly 1040

different groups at p lt 005 for panels (A) (B) and (D) and q lt 005 for panel (C) 1041

(A) Plants were inoculated with 001 macerozyme or mock inoculated using water and1042

samples were collected 3 h later Data from four biological replicates are shown 1043

(B) Plants were inoculated with 100 microg mL-1 OGs or mock inoculated using water and1044

samples collected 3 h later Data are from six biological replicates 1045

(C) JAZ10 expression 48 h after treatment with the indicated B cinerea strains Data1046

are from six biological replicates 1047

(D) JAZ10 expression in untreated plants Data are from three biological replicates1048

1049

Figure 11 Gallo 1-Induced Callose Deposition Is Reduced in gae1 gae6 Plants 1050

Page 46 of 54

(A) Callose deposition in four week-old Col-0 and gae1 gae6 plants Plants were1051

inoculated with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) or mock inoculated 1052

with B cinerea inoculation medium and samples were collected after 72 h Callose was 1053

stained with aniline blue and visualized using a Nikon Eclipse Ni-U microscope with a 1054

DAPI filter Three leaves were stained per treatment Scale bars are 100 microm Asterisks 1055

indicate fungal hyphae arrows indicate representative callose spots 1056

(B) Quantification of callose deposition Callose deposits were counted in 200 microm x 2001057

microm squares laid out across the images shown here Bars represent means plusmn SE from 1058

six squares each Letters indicate significantly different groups (p lt 005) according to a 1059

t-test analysis1060

1061

Figure 12 Less Soluble Pectin Accumulates in B cinerea-Infected gae1 gae6 1062

Leaves 1063

Four week-old Col-0 and gae1 gae6 plants were inoculated with 10 microL droplets of the 1064

indicated B cinerea isolate (25 x 105 spores mL-1) or mock inoculated with B cinerea 1065

inoculation medium Leaves were collected 48 h later AIR was extracted from leaf 1066

samples and water added (20 microL infection site) to extract soluble pectin fragments 1067

Samples were vortexed for 7 h centrifuged at 12000 g and the uronic acid 1068

concentration of the supernatant was measured Data from nine biological replicates 1069

were combined using a mixed linear model Means plusmn SE are shown Letters indicate 1070

significant differences (q lt 005) 1071

1072

Figure 13 Macerozyme-Induced PTI Is Lost in gae1 gae6 While OG-Induced PTI Is 1073

Unaltered 1074

(A) Macerozyme-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1075

inoculated with 001 macerozyme or mock inoculated with boiled macerozyme and 1076

were infected with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) 24 h later Samples 1077

were collected immediately (0 dpi) and after two (2 dpi) and three (3 dpi) days Data 1078

show mean plusmn SE of six to 14 biological replicates each combined using a linear model 1079

Letters indicate significantly different groups (q lt 005) 1080

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

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Page 25 of 54

DISCUSSION 426

Here we showed that loss of GAE1 and GAE6 leads to a dramatic reduction in 427

pectin content in the cell wall (Figure 4) likely causing the brittleness of leaves that we 428

observed (Figure 3) Petioles and midribs of gae1 gae6 plants broke easily when bent 429

and inoculation of leaves with deionized water resulted in increased ion leakage 430

possibly due either to microscopic fractures of the cell wall induced by inoculation stress 431

or to a reduced capability of these walls to withstand increased turgor pressure The 432

increased brittleness was associated with altered cell wall composition In our analysis 433

the GalA content of gae1 gae6 leaf walls was reduced by more than 40 and the total 434

uronic acids by over 60 (Figure 4 and Supplemental Figure 5) This is a major change 435

in the overall cell wall composition considering that pectin makes up about 50 of the 436

plant cell walls found in Arabidopsis leaves (Zablackis et al 1995 Nobuta et al 2007 437

Harholt et al 2010) Interestingly quasimodo1 (qua1) plants which have a mutation in 438

a glycosyltransferase family 8 gene are reduced in GalA by 25 and show reduced 439

binding of HG specific antibodies (Bouton et al 2002) 440

Furthermore qua2 plants which have a mutation in a putative methyltransferase 441

have about 13 less total GalA and lack 50 of cell wall HG (Mouille et al 2007) 442

Likewise cgr2 cgr3 plants carrying mutations in another class of Golgi-localized 443

putative methyl-transferases have leaf cell walls that are reduced by about 40 in 444

uronic acid (Kim et al 2015) All three mutants are severely dwarfed (Bouton et al 445

2002 Mouille et al 2007 Kim et al 2015) Additionally qua1 and qua2 mutants show 446

a distorted overall shape likely due to reduced cell cohesion and are extremely fragile 447

(Bouton et al 2002 Mouille et al 2007) By contrast gae1 gae6 plants with a similar 448

strong reduction in GalA and total uronic acids have an overall normal shape and are 449

only slightly smaller than wild-type Col-0 (Supplemental Figure 4) In the wall analysis 450

performed here we did not detect changes in other wall polysaccharides or structures 451

that might compensate for the reduction of pectin in gae1 gae6 It is possible the severe 452

morphological alterations in qua2 and cgr2 cgr3 plants are due to reduction in pectin 453

methylesterification which is unaffected in gae1 gae6 It was speculated that quality 454

control mechanisms in the Golgi prevent secretion of inefficiently esterified pectin in 455

cgr2 cgr3 (Kim et al 2015) suggesting that the Golgi of these plants might over-456

Page 26 of 54

accumulate pectin and that this may contribute to the severe dwarf phenotype of these 457

plants The gae1 gae6 plants described here provide an opportunity to study the effects 458

of reduction of pectin in plants that do not show severe morphological phenotypes 459

We found that gae1 gae6 plants are also compromised in resistance to B 460

cinerea and Pma ES4326 indicating that loss of pectin reduces immunity to these 461

pathogens (Figure 2 and 6) We found that gae1 gae6 plants were slightly more 462

susceptible to Pma ES4326 but showed a strong increase in susceptibility to specific B 463

cinerea isolates This was not unexpected because B cinerea is known to interact with 464

pectin as part of its infection strategy It secretes various pectin-degrading enzymes 465

early during infection and requires some of these enzymes including PG1 PG2 and 466

PME1 for full virulence (ten Have et al 1998 Valette-Collet et al 2003 Kars et al 467

2005b Espino et al 2010) Additionally Arabidopsis plants expressing an antisense 468

construct of the polygalacturonase inhibitor PGIP1 are significantly more susceptible to 469

B cinerea (Ferrari et al 2006) On the other hand Pma ES4326 is not known to470

require cell wall degrading enzymes for full virulence even though it encodes two 471

pectate lyase genes in its genome (PMA4326_07094 and PMA4326_04621 472

wwwbacteriaensemblorg) B cinerea isolates are also known to be genetically quite 473

diverse in their polygalacturonase (PG) loci and genetic variability in PG2 is associated 474

with varying growth on pectin containing media (Rowe and Kliebenstein 2007) 475

Furthermore the contribution of PG1 and PG2 to B cinerea virulence seems to depend 476

on the specific B cinerea isolate and the host species (Kars et al 2005a Zhang and 477

Van Kan 2013) B cinerea isolates are also known to be differently sensitive to the anti-478

microbial secondary metabolite camalexin (Kliebenstein et al 2005) In line with these 479

observations the susceptibility of gae1 gae6 plants differed depending on the specific 480

B cinerea isolate Whereas Fresa 525 Gallo 1 UK Razz and KT grew better in gae1481

gae6 we detected no difference in Acacia Apple 517 DN Grape Pepper or Rasp 482

growth (Figure 6) 483

B cinerea was shown to metabolize pectin and to possibly utilize GalA as a484

carbon source during in planta growth (Zhang et al 2011 Zhang and Van Kan 2013) 485

Our data show that gae1 gae6 plants which are reduced in GalA content did allow 486

enhanced growth of specific B cinerea isolates but no reduction in growth of any B 487

Page 27 of 54

cinerea isolates tested was found This suggests that utilization of pectin as a carbon 488

source was not a contributing factor to alterations in B cinerea susceptibility It is 489

possible that changes in the cell wall composition affect the structural integrity of the 490

wall and affect hyphal penetration of specific B cinerea isolates However OGs (small 491

HG fragments) are likely released during the infection process (Ferrari et al 2013) 492

OGs function as DAMPs and initiate immune responses in Arabidopsis (Ferrari et al 493

2013) Treatment of Arabidopsis with OGs having a degree of polymerization (DP) of 494

10-15 sugar subunits was previously shown to enhance resistance to B cinerea (Ferrari495

et al 2007) We showed that pretreatment with OGs or macerozyme induced enhanced 496

immunity to B cinerea isolate Gallo 1 in wild-type Col-0 but only OGs and not 497

macerozyme induced immunity in gae1 gae6 (Figure 13) Our data suggest gae1 gae6 498

plants which have reduced pectin content may release less OGs and that this 499

reduction leads to the increased susceptibility to B cinerea 500

Consistently we found that macerozyme treatment released less soluble pectin 501

from gae1 gae6 cell walls than from wild-type walls Fractions containing soluble pectin 502

released from gae1 gae6 were also less active in inducing PAD3 gene expression than 503

the fractions released from wild-type walls (Figure 7) We also showed that less soluble 504

pectin accumulated in gae1 gae6 than in wild-type plants 48 h after treatment with B 505

cinerea (Figure 12) 506

We observed that loss of pectin leads to alterations in A thaliana immune 507

signaling Macerozyme treatment induced expression of PAD3 and the SA marker 508

genes PR1 and SID2 in wild-type Col-0 but not in gae1 gae6 whereas OG treatment 509

led to increased PAD3 and SID2 expression in both wild-type and gae1 gae6 (Figure 8 510

and 9) This suggests that although release of active soluble pectin in these plants 511

might be altered OG recognition is intact In fact cell walls isolated from qua1 another 512

mutant known to be reduced in HG released less OGs (DP1-6) than did wild-type cell 513

walls upon endopolygalacturonase treatment (Bouton et al 2002) This was also true 514

for gae1 gae6 cell walls as macerozyme treatment of isolated gae1 gae6 cell walls 515

released less soluble pectin than treatment of Col-0 cell walls Because the fraction 516

from gae1 gae6 plants was also less active in inducing PAD3 expression (Figure 7) it 517

seems possible that biologically active OGs were the active component inducing PAD3 518

Page 28 of 54

expression Interestingly expression of the JA marker genes JAZ5 and JAZ10 was 519

higher in gae1 gae6 than in wild-type mock-inoculated plants but was comparable to 520

wild-type in untreated plants suggesting that JA signaling in gae1 gae6 is 521

hyperresponsive (Figure 10 and Supplemental Figure 11) This induction of JA signaling 522

might be due to wounding stress incurred during the inoculation process JA and SA 523

signaling generally act antagonistically (Pieterse et al 2012) Increased JA signaling 524

activity might hence explain why mock- and macerozyme-treated gae1 gae6 plants 525

exhibited reduced expression of the SA marker PR1 and why OG treatment induced 526

expression of SID2 another SA marker to a lower extent in gae1 gae6 than in wild-type 527

plants (Figure 9) 528

No single immune signaling component was found to be the sole contributor to 529

the compromised immunity of gae1 gae6 to specific B cinerea isolates such as Gallo 1 530

and Fresa 525 These isolates grew better on pad3 while growth of Pepper another B 531

cinerea isolate that did not show altered virulence on gae1 gae6 was unaltered 532

(Supplemental Figure 9) This suggested that differences in camalexin sensitivity of 533

these B cinerea isolates might account for the differences in gae1 gae6 susceptibility 534

However camalexin accumulation and PAD3 expression was the same for wild-type 535

Col-0 and gae1 gae6 for the B cinerea isolates Fresa 525 and Gallo 1 (Figure 8) which 536

showed enhanced growth on gae1 gae6 plants It should be noted that B cinerea 537

virulence is often estimated by comparing lesion sizes Lesion size and measurements 538

of relative fungal growth by qPCR do not always show the same results For example 539

although Denby et al (2004) showed that Pepper causes larger lesions on pad3 than on 540

wild-type this study showed that relative fungal growth of Pepper on pad3 was 541

unaltered 542

We observed that callose deposition was strongly reduced in gae1 gae6 plants 543

72 h after treatment with B cinerea isolate Gallo 1 (Figure 11) possibly due to reduced 544

release of active soluble pectin Interestingly OG-induced PTI to an unspecified isolate 545

of B cinerea was intact in the pmr4 mutant which does not produce callose (Galletti et 546

al 2008) Due to the high SA levels in pmr4 plants (Nishimura et al 2003) and the fact 547

that different B cinerea isolates vary both in induction of and reaction to defense 548

responses (Kliebenstein et al 2005 Rowe et al 2010) we could not determine 549

Page 29 of 54

whether reduced callose deposition causes the increased susceptibility of gae1 gae6 to 550

B cinerea isolate Gallo 1551

We did not observe any changes in B cinerea growth either on cev1 ixr1 plants 552

which are known to have constitutively high JA and OPDA levels (Ellis et al 2002b) or 553

on plants exogenously treated with MeJA (Supplemental Figure 9) Because plants 554

were kept uncovered for 3 h after MeJA treatment it is unclear how much MeJA was 555

bioavailable in the plant However JAZ10 expression was induced 3 h after spraying 556

with MeJA (Supplemental Figure 10) Because pathogen growth generally depends on 557

the combination of a multitude of immune signaling events we conclude that multiple 558

changes to the immune signaling in gae1 gae6 together account for the altered B 559

cinerea growth phenotypes detected 560

We showed that expression of GAE1 and GAE6 is repressed by the bacterial 561

hemibiotroph Pma ES4326 This repression did not occur in an eds1 or pad4 562

background or in pbs3 at the 24-h time point (Figure 1 and Supplemental Figure 1) 563

During the Arabidopsis - Pma ES4326 interaction the expression of many defense 564

related genes is altered in a PAD4EDS1 dependent fashion (Wang et al 2008) Some 565

of these expression changes require SA CBP60g and SARD1 are thought to act 566

downstream of PAD4 and EDS1 but upstream of SA-dependent responses (Wang et 567

al 2011) PBS3 is also thought to act downstream of PAD4 and EDS1 and upstream of 568

SA but the set of genes whose expression it affects is only partially overlapping with 569

those affected by CBP60gSARD1 (Wang et al 2008 Wang et al 2011) Thus GAE1 570

and GAE6 repression is regulated by the part of the immune signaling network that 571

requires PAD4 EDS1 and PBS3 but is independent of CBP60gSARD1 and SA 572

Because EDS1 PAD4 and PBS3 are important components of the plant immune 573

signaling network (Wiermer et al 2005 Nobuta et al 2007) one might conclude that 574

repression of GAE1 and GAE6 may be part of the plant immune response to Pma 575

ES4326 B cinerea treatment also reduced GAE1 and GAE6 expression but this was 576

mostly independent of PAD4 (Figure 6) while both Pma ES4326 and B cinerea repress 577

GAE1 and GAE6 expression the host signaling mechanisms responsible likely differ 578

In summary our data show that Arabidopsis GAE1 and GAE6 are required for 579

pectin biosynthesis leaf flexibility and immunity to Pma ES4326 and to specific B 580

Page 30 of 54

cinerea isolates We used macerozyme a commercial pectinase to mimic the effect of 581

B cinerea pectinases Macerozyme treatment released less pectin from gae1 gae6 cell582

walls and induced less immune signaling in gae1 gae6 plants Less water soluble pectin 583

also accumulated in B cinerea infected gae1 gae6 plants and macerozyme-induced 584

immunity to B cinerea isolate Gallo 1 was reduced in these plants We conclude that 585

the reduced pectin content in these mutants may lead to reduced release of soluble 586

pectin including active OGs during pathogen attack and that this compromises 587

immunity 588

589

590

Page 31 of 54

METHODS 591

Plant Materials and Growth Conditions 592

Wild-type Columbia (Col-0) and all mutant plants (in Col-0 background) were 593

grown on sterilized BM2 germinating mix (Berger Inc Quebec Canada) in a controlled 594

environment chamber (Conviron Pembina ND USA) with a 12-h photoperiod under 595

100 microM m-2 s-1 fluorescent illumination at 22degC and 75 relative humidity Germplasm 596

used is described in the Accession Numbers section at the end of Methods 597

598

Data Analysis and Replicates 599

For biological replicates samples were obtained from separate plant tissues and 600

in many cases from plants grown in different flats In some experiments multiple 601

technical replicates were measured per biological replicate For statistical analysis all 602

technical replicates were averaged and the averages were considered one biological 603

replicate Independent experiments were performed at different times and often 604

consisted of multiple biological replicates per experiment 605

Data analysis was performed using mixed linear models in the R programming 606

environment (Bates et al 2015) unless otherwise indicated Generally genotypes 607

treatments and time points were used as fixed effects and replicate-specific effects like 608

different pots flats etc were considered random effects We reported p-values 609

obtained from the mixed linear model when less than 10 comparisons were performed 610

and q-values for more than 10 comparisons to correct for multiple comparison testing 611

Q-values were calculated using the Benjamini-Hochberg procedure (Benjamini and612

Hochberg 1995) unless specified otherwise 613

614

Pathogen Strains Growth Conditions and Pathogen Growth Assays 615

Growth of Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) and 616

bacterial growth assays were performed as previously described (Bethke et al 2014) 617

For qRT-PCR assays four week-old plants were inoculated with bacteria (OD600 = 618

0002) or mock (5 mM MgSO4) At least four leaves from two plants were collected at 619

the indicated time points per biological replicate 620

Page 32 of 54

Prior to each experiment Botrytis cinerea isolates were grown on 1x PDA 621

medium (Difco) for 10 d at room temperature All isolates used have been described 622

previously (Rowe and Kliebenstein 2007) The spores were washed from the surface of 623

the plate using an inoculation medium (1x Gamborgrsquos B-5 basal salt mixture (Sigma-624

Aldrich G5768) 2 (wv) glucose 10 mM phosphate buffer pH 64) and fungal hyphae 625

were removed from the suspension by filtering through four layers of cheesecloth The 626

concentration of spores was determined using a hemocytometer and adjusted to 25 x 627

105 spores mL-1 Four week-old Arabidopsis plants were inoculated by placing one 10-628

microL droplet of the B cinerea spore solution or inoculation medium (mock) on the adaxial 629

leaf surface of fully expanded leaves Inoculated plants were kept at 100 relative 630

humidity Infection sites were collected at various time points using a cork borer For 631

qRT-PCR assays at least four leaf discs from one plant were combined for each 632

biological sample For B cinerea growth assays at least 12 leaf discs from three to six 633

plants were pooled per biological sample Relative fungal growth was determined by 634

measuring the abundance of a fungal gene relative to a plant gene as described below 635

In brief DNA from infected tissue was extracted and equal amounts of total DNA were 636

used to perform qPCR reactions using the B cinerea cutinase A and the Arabidopsis 637

SK11 gene respectively Primers were described in (Gachon and Saindrenan 2004) 638

and are listed in Supplemental Table 1 Two technical replicates were combined for 639

each biological replicate B cinerea is a necrotrophic fungal pathogen and may destroy 640

plant DNA as disease progresses However decreases in the amount of plant DNA 641

over the course of our experiments were only observed at late time points with very 642

susceptible plants so the qPCR assay is generally a reasonable estimate of fungal 643

biomass (Supplemental Figure 16) 644

645

Treatment with Macerozyme or OGs 646

For PTI assays four week-old plants either were inoculated with 001 (wv) 647

macerozyme R-10 (Yakult) a Rhizopus polygalacturonase (EC 32115) that possesses 648

high pectinase and hemicellulase activity or were mock inoculated using heat-649

inactivated macerozyme Other similar plants were either inoculated with 250 or 500 microg 650

mL-1 OGs or mock inoculated with water One d later plants were infected with B 651

Page 33 of 54

cinerea and samples were collected immediately (0 dpi) and two or three d later To 652

investigate the role of MeJA on B cinerea growth plants were sprayed with 1mM MeJA 653

in water with 001 (vv) Tween 20 Covers were removed so that leaves could dry off 654

before plants were inoculated with B cinerea three h later Samples were harvested at 655

the indicated time points For expression analysis and MAPK activation assays plants 656

were either inoculated with 100 microg mL-1 OGs or mock inoculated with water OGs used 657

for experiments described in Figures 8 to 10 and Supplemental Figure 12 were a kind 658

gift from Simone Ferrari OGs used in experiments described in Figure 13 and 659

Supplemental Figures 11 13 and 15 were prepared as previously described (Kohorn et 660

al 2014) 661

662

Expression Analysis 663

Leaf tissue from inoculated plants was harvested at the indicated time points 664

flash frozen pulverized and RNA extracted using Trizol (Invitrogen) Quantitative RT-665

PCR was performed using the SuperScript III Platinum SYBR Green One-Step 666

quantitative RT-PCR kit (Invitrogen) and a Lightcycler 480 Real-Time PCR system 667

(Roche) as previously described (Truman and Glazebrook 2012) In brief equal 668

amounts of total RNA and a gene specific primer were used for each reaction The 669

crossing point (Cp) was calculated using the 2nd derivative max method provided with 670

the Lightcycler software for each amplification curve Each reaction was run with two 671

technical replicates and the Cp values for these replicates were averaged ACTIN2 was 672

used as a stably expressed reference gene At least three biological replicates each 673

were performed and analyzed using a mixed-linear model RT-PCR was performed to 674

verify T-DNA insertion lines using the One-Step RT-PCR kit (Qiagen) according to the 675

manufacturerrsquos instructions The primers used can be found in Supplemental Table 1 676

677

Electrolyte Leakage Assay 678

Fully expanded leaves of four week-old plants were inoculated with water (Milli-Q 679

grade) using a needleless syringe Leaf discs were collected using a cork borer and 680

three leaf discs from one leaf were placed with the adaxial surface down onto 2 mL 681

water in a 12-well cell culture plate Conductivity was measured using a Horiba B-173 682

Page 34 of 54

conductivity meter after 30 min and then the water was replaced Conductivity was 683

measured again 30 min later (1 h total) as indicated in Figure 3C Six biological 684

replicates each were performed in three independent experiments and means and SEs 685

were calculated Two-sided t-tests were performed for each time point 686

687

DAB and Aniline Blue Staining 688

Leaves from four week-old plants were either mock inoculated with B cinerea 689

inoculation medium or inoculated with 10 microL droplets of a B cinerea spore solution (25 690

x 105 spores mL-1) of the indicated isolates Samples were collected 72 h later For 33prime-691

diaminobenzidine (DAB) staining two to five inoculated leaves were vacuum infiltrated 692

with 33prime-Diaminobenzidine (1 mg mL-1 in water pH 38) solution and stained overnight 693

in the dark Leaves were destained using 100 ethanol at 65degC and then transferred to 694

50 (vv) glycerol and photographed 695

Callose was visualized by staining leaves with aniline blue as previously 696

described (Adam and Somerville 1996) Briefly leaves were vacuum infiltrated with 697

alcoholic lactophenol (one volume of phenolglycerollactic acidwater (1111) mixed 698

with two volumes of ethanol) and destained at 65degC Next leaves were moved through 699

an alcohol gradient (50 20 and 10 (vv) ethanol) into water The destained leaves 700

were stained for 30 min in 150 mM K2HPO4 (pH 95) with 001 (wv) aniline blue 701

Samples were mounted in water and examined using a Nikon Eclipse Ni-U microscope 702

at 10x magnification using a DAPI filter (excitation at 325-375 nm emission at 435-485 703

nm) For callose quantification six 200 microm x 200 microm squares were counted for each 704

sample type Callose deposits per mm2 were calculated and data analyzed using a t-705

test Note that there is an inherent uncertainty associated with such counts because 706

callose deposits may not all be in focus at the same time 707

708

MAPK Activity Assay 709

Fully expanded leaves of four week-old plants were inoculated with 001 710

macerozyme 100 microg mL-1 OGs 100 nM flg22 or water (mock) flg22 a peptide from 711

bacterial flagella that induces immune responses was used as a positive control for 712

Page 35 of 54

MAPK activation (Asai et al 2002) and was purchased from EZBiolab Inc (Westfield 713

IN USA) Samples were harvested 10 min later and immediately frozen in liquid 714

nitrogen Protein was extracted using an extraction buffer containing 25 mM Tris pH 78 715

75 mM NaCl 1 mM DTT 1 mM NaF 05mM Na3VO4 01 (vv) Tween 20 one 716

cOmplete Mini protease inhibitor cocktail tablet (Roche) and one PhosSTOP 717

phosphatase inhibitor cocktail tablet (Roche) per 10 mL buffer 15 microg of total protein 718

were electrophoresed on a 10 SDS-polyacrylamide gel and then blotted onto a PVDF 719

membrane (Bio-Rad 162-0177) Activated MAPKs were detected using p4442 MAPK 720

(Erk12) antibody (9102S Cell Signal 12500 in TBST (20 mM Tris pH 75 150 mM 721

NaCl 01 (vv) Tween 20)) Anti-Rabbit IgG-HRP (115000 in TBST A6154 Sigma) 722

and ECL Plus substrate (Pierce 32132) MPK3 and MPK6 were detected with an anti-723

MPK3 antibody (M8318 Sigma 12000 in TBST and 3 (wv) milk) or an anti-MPK6 724

antibody (A7104 Sigma 16000 in TBST and 3 (wv) milk) respectively The 725

secondary antibody and detection were the same as for the p4442 antibody Five 726

independent experiments were performed 727

728

Preparation of Alcohol Insoluble Residue (AIR) Pectin Extraction and Uronic Acid 729

Measurements 730

Fully expanded leaves from four week-old plants that were incubated for 48 h in 731

the dark to reduce starch were harvested flash frozen and pulverized AIR was 732

extracted by washing ground material twice in 70 (vv) ethanol three times in a 733

mixture of chloroform and methanol (11 vv) and once in acetone (Gille et al 2009) 734

and was then air dried Cell wall pectin was extracted from AIR with cell wall extraction 735

buffer (50 mM Trizma 50 mM CDTA pH 72) at 95degC for 15 min Samples were 736

homogenized using a paint shaker (Harbil 5G-HD) and glass beads (3 mm diameter) 737

For dot blot experiments 500 microL of cell wall extraction buffer were used per 10 mg of 738

AIR Debris was precipitated by centrifugation for 10 min at 10000 g 739

Total uronic acid content of pectin extracted as described above using 500 microL of 740

cell wall extraction buffer per 1 mg of AIR was measured as described previously 741

(Filisetti-Cozzi and Carpita 1991 van den Hoogen et al 1998) First 36 microL of pectin 742

extract were mixed with 4 microL of 4 M sulfamic acid Then 200 microL of sulfuric acid 743

Page 36 of 54

containing 120 mM sodium tetraborate was added and samples were incubated at 80degC 744

for 1 h Following cooling on ice the optical density of samples was measured at 490 745

nm Next 40 microL of m-hydroxydiphenyl reagent (100 microL of m-hydroxydiphenyl in dimethyl 746

sulfoxide at 100 mgmL mixed with 49 mL 80 (vv) sulfuric acid just before use) was 747

added and the samples were mixed The optical density at 490 nm was measured 748

again The optical density before m-hydroxydiphenyl reagent addition was subtracted 749

from the optical density measured after addition of the dye The concentration of uronic 750

acid was calculated using known amounts of GalA as a standard 751

752

Release of Pectin from Arabidopsis Cell Walls Using Macerozyme 753

AIR preparations from leaves of four week-old plants were treated with 025 754

(wv) macerozyme R-10 (Yakult) or water (mock) at a concentration of 100 microL per mg 755

AIR Samples were incubated for 3 h at room temperature on a vortex shaker and 756

centrifuged for 10 min at 12000 g Total uronic acid concentration of 5-fold diluted 757

supernatants was determined for two technical replicates each The concentration of 758

uronic acid was calculated using known amounts of GalA as a standard For 759

subsequent qRT-PCR experiments supernatants from multiple extractions were 760

combined boiled for 20 min to inactivate macerozyme diluted 10-fold with water and 761

inoculated into four week-old Col-0 plants Heat-inactivated 10-fold diluted macerozyme 762

solutions were used as mock treatment Samples were collected 3 h later and PAD3 763

expression was determined 764

765

Release of Pectin from Arabidopsis Leaves Infected with B cinerea 766

Fully expanded leaves of four week-old plants were either inoculated with up to 767

five 10 microL droplets of B cinerea spore solution (25 x 105 spores mL-1) or mock 768

inoculated with B cinerea inoculation medium For each biological replicate leaves with 769

a combined count of at least 20 infection sites were collected 48 h later AIR was 770

extracted as described above but plants were not dark-treated or freeze-dried and the 771

first ethanol step was performed using 85 (vv) ethanol To extract soluble pectin 772

fragments AIR samples were mixed with water at a concentration of 20 microL per infection 773

site incubated for 7 h at room temperature on a vortex shaker and centrifuged for 10 774

Page 37 of 54

min at 12000 g Total uronic acid concentration of supernatants was determined for two 775

technical replicates each The concentration of uronic acid was calculated using known 776

amounts of GalA as a standard 777

778

Measurement of Uronic Acids Neutral Monosaccharides and Cellulose Content in 779

Cell Walls 780

Neutral sugars and uronic acids from non-cellulosic polysaccharides in cell walls 781

(AIR) were released by trifluoroacetic acid (TFA) hydrolysis Approximately 1 mg of AIR 782

was hydrolyzed with 2 M TFA at 121degC for 90 min The TFA-soluble material was split 783

into two equal parts one for uronic acid and one for neutral sugar determination The 784

uronic acid content was determined by analyzing the hydrolyzate using a CarboPac 785

PA200 anion-exchange column with an ICS-3000 Dionex chromatography system 786

(Dionex Sunnyvale CA USA) The elution profile consisted of a linear gradient of 50 to 787

200 mM sodium acetate in 01 M NaOH in 10 min at 04 mL per min Alditol acetate 788

derivatives of neutral sugars were produced as described (York et al 1985) In brief 789

monosaccharides in the hydrolyzate were reduced with NaBH4 and peracetylated with 790

acetic anhydride and pyridine at 121degC for 20 min The generated alditol acetates were 791

analyzed by GC-MS Crystalline cellulose was measured according to Updegraff (1969) 792

After hydrolysis of noncellulosic polysaccharides from AIR with the Updegraff reagent 793

(acetic acid nitric acids water 812 (vv) ) the remaining pellet was hydrolyzed in 794

72 sulfuric acid The resulting glucose quantity was determined by the anthrone 795

method (Scott and Melvin 1953) 796

797

Dot Blot Experiments 798

Pectin solutions extracted using 50 microL of cell wall extraction buffer per mg AIR 799

were serially diluted and nitrocellulose membranes were spotted with 1 microL of the diluted 800

pectin solutions Membranes were dried overnight blocked with 5 milk (wv) in 1x 801

PBS (8 g L-1 NaCl 02 g L-1 KCl 144 g L-1 Na2HPO4 024 g L-1 KH2PO4 pH 74) and 802

probed with LM5 (Jones et al 1997) LM6 (Willats et al 1998) LM8 (Willats et al 803

2004) LM19 and LM20 (Verhertbruggen et al 2009a) or CCRC-M7 (Steffan et al 804

Page 38 of 54

1995) antibodies LM series antibodies were diluted 1250 and CCRC-M7 was diluted 805

1500 in 5 milk powder (Nestle Carnation) in 1 x PBS For LM series antibodies a 806

goat anti-Rat HRP conjugated antibody (Bethyl A110-105P 15000 diluted) and for 807

CCRC-M7 an anti-Mouse IgG HRP conjugate (Promega W402B 12500 diluted) was 808

used as secondary antibody in 5 milk powder in 1x PBS LM series antibodies were 809

obtained from plant probes (wwwplantprobesnet) and CCRC-M7 from CarboSource 810

(wwwcarbosourcenet) Membranes were washed after incubation with the primary and 811

secondary antibodies with 1x PBS Dot blots were developed using the ECL system 812

(GE Healthcare Amersham ECL prime) For quantification of signals dot blot results on 813

x-ray films were photographed using a CCD camera and intensities were measured814

using Image Jrsquos Integrated Density function Locally adjusted background was 815

subtracted As the dynamic range of the signal on an x-ray film was narrow non-816

linearity in the measured values was corrected for each genotype in each biological 817

replicate using the values from different dilutions in two technical replicates We noticed 818

that the residuals were not normally distributed and performed Box-Cox power 819

transformation (Box and Cox 1964) to obtain normally distributed values for the 820

subsequent statistical analysis of the data For LM19 Box-Cox selected power 821

transformation was done to the power of 038th and for LM20 to the power 075th After 822

correction of the transformed measured values with the effect of the biological 823

replicates the values were fit to a linear model with the Arabidopsis genotype as the 824

fixed effect This linear model was used both to generate the plots in Figures 5B and D 825

and to test significant differences 826

827

Camalexin Measurements 828

Four week-old plants were infected with B cinerea and samples (three leaf disks 829

from one plant each) were harvested at the indicated time points flash frozen and 830

pulverized Each sample was extracted with 300 microL of 90 methanol (vv) 50 microL of the 831

extract was run on an Agilent Lichrocart 250-4 RP18e 5-microm column using an Agilent 832

1100 series HPLC Camalexin was detected using a DiodeArray (DAD) at 330 nm and 833

with a fluorescence detector at emission 318 nmexcitation 385 nm (Agilent) Separation 834

was achieved using the following program with aqueous acetonitrile 5 min gradient 835

Page 39 of 54

from 63 to 69 acetonitrile 30 s gradient from 69 to 99 acetonitrile 2 min at 99 836

acetonitrile and a post-run equilibration of 35 min at 63 acetonitrile (Denby et al 837

2004 Kliebenstein et al 2005) Purified camalexin was used to produce a standard 838

curve to identify and quantitate camalexin 839

840

Accession Numbers 841

Microarray data used can be accessed at NCBI Gene Expression Omnnibus 842

(httpwwwncbinlmnihgovgeo) GSE18978 Sequence information for all Arabidopsis 843

genes described in this article can be found in TAIR (wwwarabidopsisorg) using the 844

following accession numbers PAD3 (CYP71B15 At3g26830) SID2 (ICS1 At1g74710) 845

GAE1 (At4g30440) GAE2 (At1g02000) GAE3 (At4g00110) GAE4 (At2g45310) GAE5 846

(At4g12250) GAE6 (At3g23820) PR1 (At2g14610) JAZ5 (At1g17380) JAZ10 847

(At5g13220) ACTIN2 (At3g18780) PDF12a (At5g44420) PDF12b (At2g26020) 848

AtSK11 (At5g26751) The sequence of the B cinerea cutinase Bc-CutA (Z69264) can 849

be found in the DNA Data Bank of Japan (httpgetentryddbjnigacjp) Germplasm 850

used included dde2-2 (At5g42650 (von Malek et al 2002)) pad3-1 (At3g26830 (Zhou 851

et al 1999)) pad4-1 (At3g52430 (Jirage et al 1999)) sid2-2 (At1g74710 (Wildermuth 852

et al 2001)) pbs3-2 (At5g13320 SALK_018225 (Nobuta et al 2007)) cbp60a-1 853

(At5g62570 SALK_124410 (Truman et al 2013)) cbp60g-1 sard1-2 (At5g26920 854

SALK_023199 At1g73805 SALK_052422 (Wang et al 2011) cbp60a-1 cbp60g-1 855

sard1-2 (Truman et al 2013) gae1-1 (At4g30440 SALK_085554) gae6-1 (At3g23280 856

SALK_104454) gae6-2 (At3g23280 SALK_017191) ixr1-1 cev1 (At5g05170 (Ellis 857

and Turner 2001 Scheible et al 2001)) eds1 plants were derived by introgression of 858

the Ler eds1-2 allele into Col-0 that contains two copies of EDS1 (At3g48090 859

At3g48080 (Bartsch et al 2006) mpk6-2 (At2g43790 SALK_073907 (Liu and Zhang 860

2004)) mpk3-DG (At3g45640 a fast neutron deletion mutant (Miles et al 2005)) and 861

npr1-1 (At1g64280 (Cao et al 1997)) T-DNA insertion lines were part of the SALK 862

collection (Alonso et al 2003) and were obtained from the Arabidopsis Biological 863

Resource Center 864

865

Supplemental Data 866

Page 40 of 54

Supplemental Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 867

Expression Does Not Require CBP60s or SID2 868

Supplemental Figure 2 Expression of GAE Family Members uponTreatment with Pma 869

ES4326 870

Supplemental Figure 3 Characterization of T-DNA Insertions in GAE1 and GAE6 871

Supplemental Figure 4 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves and 872

Are Slightly Smaller than Wild-Type Col-0 Plants 873

Supplemental Figure 5 Cell Walls of gae1 gae6 Plants Contain Less Uronic Acid than 874

Wild-Type Col-0 Cell Walls 875

Supplemental Figure 6 Antibodies Raised Against the RGI or XG Components of 876

Pectin Show No Difference in Binding to gae Mutant and Col-0 Cell Walls 877

Supplemental Figure 7 The Effect of B cinerea Treatment on the Expression of GAE 878

Family Genes 879

Supplemental Figure 8 Treatment with 001 Macerozyme Does Not Cause Any 880

Visible Damage to Col-0 Plants 881

Supplemental Figure 9 B cinerea Growth on Plants with Defects in Immune 882

Signaling 883

Supplemental Figure 10 MeJA-Induced Expression of the JA marker gene JAZ10 884

Supplemental Figure 11 The Expression of the JA Marker Gene JAZ5 Is Highly 885

Responsive in gae1 gae6 Plants 886

Supplemental Figure 12 The Expression of PDF12 Is Unaltered in gae1 gae6 Plants 887

Supplemental Figure 13 Macerozyme Treatment Activates MPK3 and MPK6 888

Supplemental Figure 14 B cinerea-Induced ROS Production in Wild-Type Col-0 and 889

gae1 gae6 Plants 890

Supplemental Figure 15 OG-Induced PTI Using 250 microg mL-1 OGs 891

Supplemental Figure 16 Abundance of Plant DNA in B cinerea Treated Plants 892

Supplemental Table 1 Primers used in this study 893

894

ACKNOWLEDGEMENTS 895

We thank William Truman for helpful discussions Simone Ferrari (Sapienza 896

Universita di Roma) for the generous gift of OGs Bruna Bucciarelli (USDA-ARS) for 897

Page 41 of 54

help with fluorescence microscopy the Arabidopsis Biological Resource Center for T-898

DNA insertion lines Paul Knoxrsquos lab (University of Leeds) for LM5 LM6 LM8 LM19 899

and LM20 antibodies and the Complex Carbohydrate Resource Center (University of 900

Georgia) for the CCRC-M7 antibody Most of this work was funded by the Division of 901

Chemical Sciences Geosciences and Biosciences Office of Basic Energy Sciences of 902

the US Department of Energy through Grant DE-FG02-05ER15670 to JG This work 903

was also supported by NSF awards IOS 1339125 and IOS 1021861 to DJK NSF 904

award IOS 1121425 to F K and the USDA National Institute of Food and Agriculture 905

Hatch project number CA-D-PLS-7033-H to DJK 906

907

AUTHOR CONTRIBUTIONS 908

GB and JG designed the research GB GX AT BL NES and NH 909

performed research DJK provided tools GB GX MP AD BL RAH and FK 910

analyzed data GB and JG wrote the paper with input from all authors 911

912

FIGURE LEGENDS 913

Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 Expression 914

Requires PAD4 EDS1 and PBS3 915

(A) Expression of GAE1 in leaves of wild-type Col-0 and mutant plants Expression was916

measured 24 or 32 h after either inoculation with Pma ES4326 (OD600 = 0002) or mock 917

inoculation with 5 mM MgSO4 and was then normalized to the level of ACTIN2 Data 918

from three biological replicates were combined using a mixed linear model Bars 919

represent mean log2-ratios to ACTIN2 plusmn SE Letters indicate significantly different 920

groups with q lt 005 where 005 represents the maximum false discovery rate at which 921

the test may be called significant 922

(B) Expression of GAE6 measured as described in (A)923

924

Figure 2 Susceptibility of gae6 and gae1 gae6 Plants to Pma ES4326 Is Increased 925

Relative to Wild-Type Col-0 926

Leaves of plants of the indicated genotypes were inoculated with Pma ES4326 (OD600 = 927

00002) as described in Methods Bacterial titers in leaves were determined either 928

Page 42 of 54

immediately (0 dpi) or after three days (3 dpi) Bars represent the mean plusmn SE of four 929

independent experiments each with 4 or 12 biological replicates at 0 and 3 dpi 930

respectively Data were combined using a mixed linear model Asterisks indicate 931

significant differences from Col-0 wild-type (q lt 005) Susceptible pad4 plants were 932

included as a positive control 933

934

Figure 3 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves 935

(A) Depiction of the assay for brittleness of gae mutant leaves Fully expanded leaves of936

four to five week-old plants were bent by 180ordm so that the adaxial surface of the leaf was 937

parallel with the soil Broken leaf petioles or midribs were considered brittle 938

(B) Quantification of broken midribs or petioles For each genotype 66 to 82 leaves 939

were analyzed for brittleness as described in (A) Bars represent the ratio of broken to 940

total number of leaves tested Results were analyzed using Fisherrsquos exact test Letters 941

indicate significantly different groups at p lt 005 942

(C) Conductivity measurements Leaves of four week-old plants were inoculated with943

deionized water and then leaf discs were cut out and placed on deionized water 944

Conductivity was measured at the indicated time points After 30 min the water was 945

replaced with fresh water (30 min f w) to account for wounding-related ion leakage from 946

the edges of the leaf discs Three independent experiments with six biological replicates 947

each were performed and data were analyzed using a t-test for each time point P-948

values were corrected using the Bonferroni method Data show means plusmn SE asterisks 949

indicate significant differences from wild-type Col-0 at q lt 001 950

951

Figure 4 Cell Walls of gae6 and gae1 gae6 Plants Are Reduced in GalA 952

(A) to (D) Levels of GalA (A) GlcA (B) cellulose (C) and neutral sugars (D) in alcohol953

insoluble residue (AIR) extracted from leaves of four week-old plants of the indicated 954

genotypes Bars represent means plusmn SE of four biological replicates combined using a 955

mixed linear model Letters indicate significantly different groups (p lt 005) 956

957

Figure 5 Cell Walls of gae1 gae6 Plants Are Reduced in Homogalacturonan 958

Page 43 of 54

Dot blot analyses with anti-LM19 and anti-LM20 antibodies Pectin was extracted from 959

AIR prepared from the indicated genotypes using 1 microL of extraction buffer per 10 microg of 960

AIR Pectin was serially diluted and one microL of undiluted 13 diluted and 19 diluted 961

samples were spotted on a nitrocellulose membrane Three biological replicates were 962

were spotted twice to obtain two technical replicates each All replicates produced 963

similar results 964

(A) Representative dot blot using anti-LM19 antibody965

(B) Relative signal intensities of the spots in the dot blot in (A)966

(C) Representative dot blot using anti-LM20 antibody967

(D) Relative signal intensities of the LM20 dot blot shown in (C)968

Relative signal intensities were measured using Image J software Measurements from 969

the three biological replicates were combined using a linear model after Box-Cox power 970

transformation as described in Methods Bars represent means plusmn SE Asterisks indicate 971

a difference from wild-type Col-0 at p lt 005 972

973

Figure 6 Growth of B cinerea on gae1 gae6 Plants and the Effect of B cinerea on 974

Expression of GAE1 and GAE6 975

(A) Growth of B cinerea isolates Leaves of Col-0 and gae1 gae6 plants were976

inoculated with 25 x 105 spores mL-1 of the B cinerea isolates indicated in the figure 977

Samples were collected two (2 dpi) and three (3 dpi) days later Bars represent means plusmn 978

SE of six biological replicates for each fungus combined using a mixed linear model 979

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 980

(B) and (C) Expression of GAE1 (B) and GAE6 (C) 48 h after inoculation with the B981

cinerea isolates indicated Expression levels were measured by qRT-PCR Bars 982

represent mean log2-ratios to ACTIN2 plusmn SE of three biological replicates combined 983

using a mixed linear model Letters indicate significantly different groups (q lt 005) 984

985

Figure 7 Levels of Soluble Pectin and Expression of PAD3 in Fractions Produced 986

Following Macerozyme Treatment of gae1 gae6 Cell Walls 987

(A) Schematic overview of the experimental procedure for (B) and (C)988

Page 44 of 54

(B) Uronic acid measurements Alcohol-insoluble residue (AIR) from leave tissue of four989

week-old wild-type Col-0 or gae1 gae6 plants was treated with water or 025 990

macerozyme and assayed for uronic acid levels Data from 11 biological replicates were 991

combined using a mixed linear model Bars represent means plusmn SE Letters indicate 992

significant differences (p lt 005) 993

(C) Expression of PAD3 in Col-0 plants treated with the fractions from (B) Extracts from994

(B) were heat inactivated pooled and diluted 5-fold with water Pooled extracts were995

inoculated into four week-old Col-0 plants PAD3 expression was measured 3 h after 996

inoculation and normalized to the level of ACTIN2 expression Data from four biological 997

replicates were combined using a mixed linear model Bars represent mean log2-ratios 998

to ACTIN2 plusmn SE Letters indicate significant differences (p lt 005) 999

1000

Figure 8 Macerozyme-Induced PAD3 Expression Is Absent in gae1 gae6 1001

(A) Expression of PAD3 after macerozyme treatment Four week-old plants were either1002

inoculated with 001 macerozyme or mock inoculated with water and PAD3 1003

expression was measured 3 h later by qRT-PCR Data from four biological replicates 1004

were combined using a mixed linear model Mean log2-ratios to ACTIN2 plusmn SE were 1005

plotted Letters indicate significantly different groups (p lt 005) 1006

(B) Expression of PAD3 after OG treatment PAD3 expression was measured as1007

described in (A) 3 h after inoculation with 100 microg mL-1 OGs or mock inoculation with 1008

water Data are from six biological replicates 1009

(C) PAD3 expression in untreated plants Three biological replicates were performed1010

and data analyzed as described in (A) 1011

(D) Camalexin accumulation after B cinerea treatment Camalexin accumulation was1012

measured in four week-old plants inoculated with the B cinerea strains shown in the 1013

figure Eight biological replicates each were combined using a mixed linear model 1014

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 1015

(E) PAD3 expression 28 h after treatment with the indicated B cinerea strains Data1016

from six biological replicates were collected and analyzed as described in (A) Data in 1017

all figure parts represent means plusmn SE Letters indicate significantly different groups (q lt 1018

005) 1019

Page 45 of 54

1020

Figure 9 Macerozyme-Induced Expression of the SA Marker Genes SID2 and PR1 1021

Is Abolished in gae1 gae6 Plants 1022

(A) to (H) Expression of PR1 (A) (C) (E) and (G) or SID2 (B) (D) (F) and (H) in four1023

week-old Col-0 and gae1 gae6 plants Data were combined using a mixed linear model 1024

Bars represent mean log2-ratios of expression vs ACTIN2 plusmn SE for all figure parts 1025

Letters indicate significantly different groups at p lt 005 for panels (A) to (D) (G) and 1026

(H) and qlt005 for panels (E) to (F) 1027

(A) and (B) Plants were treated with 001 macerozyme or mock treated with water1028

and samples were collected 3 h later Data from four biological replicates are shown 1029

(C) and (D) Plants were inoculated with 100 microg mL-1 OGs or mock treated with water1030

and samples collected 3 h later Data from six biological replicates are shown 1031

(E) and (F) Plants were inoculated with the indicated B cinerea isolates or mock1032

inoculated with B cinerea inoculation medium and samples collected 48 h later Data 1033

from eight biological replicates are shown 1034

(G) and (H) Expression in untreated plants Three biological replicates were performed1035

1036

Figure 10 The Expression of the JA Marker Gene JAZ10 is Highly Responsive in 1037

gae1 gae6 Plants 1038

(A) to (D) JAZ10 expression in four week-old plants Data were combined using a mixed1039

linear model Mean log2-ratios to ACTIN2 plusmn SE are shown Letters indicate significantly 1040

different groups at p lt 005 for panels (A) (B) and (D) and q lt 005 for panel (C) 1041

(A) Plants were inoculated with 001 macerozyme or mock inoculated using water and1042

samples were collected 3 h later Data from four biological replicates are shown 1043

(B) Plants were inoculated with 100 microg mL-1 OGs or mock inoculated using water and1044

samples collected 3 h later Data are from six biological replicates 1045

(C) JAZ10 expression 48 h after treatment with the indicated B cinerea strains Data1046

are from six biological replicates 1047

(D) JAZ10 expression in untreated plants Data are from three biological replicates1048

1049

Figure 11 Gallo 1-Induced Callose Deposition Is Reduced in gae1 gae6 Plants 1050

Page 46 of 54

(A) Callose deposition in four week-old Col-0 and gae1 gae6 plants Plants were1051

inoculated with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) or mock inoculated 1052

with B cinerea inoculation medium and samples were collected after 72 h Callose was 1053

stained with aniline blue and visualized using a Nikon Eclipse Ni-U microscope with a 1054

DAPI filter Three leaves were stained per treatment Scale bars are 100 microm Asterisks 1055

indicate fungal hyphae arrows indicate representative callose spots 1056

(B) Quantification of callose deposition Callose deposits were counted in 200 microm x 2001057

microm squares laid out across the images shown here Bars represent means plusmn SE from 1058

six squares each Letters indicate significantly different groups (p lt 005) according to a 1059

t-test analysis1060

1061

Figure 12 Less Soluble Pectin Accumulates in B cinerea-Infected gae1 gae6 1062

Leaves 1063

Four week-old Col-0 and gae1 gae6 plants were inoculated with 10 microL droplets of the 1064

indicated B cinerea isolate (25 x 105 spores mL-1) or mock inoculated with B cinerea 1065

inoculation medium Leaves were collected 48 h later AIR was extracted from leaf 1066

samples and water added (20 microL infection site) to extract soluble pectin fragments 1067

Samples were vortexed for 7 h centrifuged at 12000 g and the uronic acid 1068

concentration of the supernatant was measured Data from nine biological replicates 1069

were combined using a mixed linear model Means plusmn SE are shown Letters indicate 1070

significant differences (q lt 005) 1071

1072

Figure 13 Macerozyme-Induced PTI Is Lost in gae1 gae6 While OG-Induced PTI Is 1073

Unaltered 1074

(A) Macerozyme-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1075

inoculated with 001 macerozyme or mock inoculated with boiled macerozyme and 1076

were infected with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) 24 h later Samples 1077

were collected immediately (0 dpi) and after two (2 dpi) and three (3 dpi) days Data 1078

show mean plusmn SE of six to 14 biological replicates each combined using a linear model 1079

Letters indicate significantly different groups (q lt 005) 1080

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

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Page 26 of 54

accumulate pectin and that this may contribute to the severe dwarf phenotype of these 457

plants The gae1 gae6 plants described here provide an opportunity to study the effects 458

of reduction of pectin in plants that do not show severe morphological phenotypes 459

We found that gae1 gae6 plants are also compromised in resistance to B 460

cinerea and Pma ES4326 indicating that loss of pectin reduces immunity to these 461

pathogens (Figure 2 and 6) We found that gae1 gae6 plants were slightly more 462

susceptible to Pma ES4326 but showed a strong increase in susceptibility to specific B 463

cinerea isolates This was not unexpected because B cinerea is known to interact with 464

pectin as part of its infection strategy It secretes various pectin-degrading enzymes 465

early during infection and requires some of these enzymes including PG1 PG2 and 466

PME1 for full virulence (ten Have et al 1998 Valette-Collet et al 2003 Kars et al 467

2005b Espino et al 2010) Additionally Arabidopsis plants expressing an antisense 468

construct of the polygalacturonase inhibitor PGIP1 are significantly more susceptible to 469

B cinerea (Ferrari et al 2006) On the other hand Pma ES4326 is not known to470

require cell wall degrading enzymes for full virulence even though it encodes two 471

pectate lyase genes in its genome (PMA4326_07094 and PMA4326_04621 472

wwwbacteriaensemblorg) B cinerea isolates are also known to be genetically quite 473

diverse in their polygalacturonase (PG) loci and genetic variability in PG2 is associated 474

with varying growth on pectin containing media (Rowe and Kliebenstein 2007) 475

Furthermore the contribution of PG1 and PG2 to B cinerea virulence seems to depend 476

on the specific B cinerea isolate and the host species (Kars et al 2005a Zhang and 477

Van Kan 2013) B cinerea isolates are also known to be differently sensitive to the anti-478

microbial secondary metabolite camalexin (Kliebenstein et al 2005) In line with these 479

observations the susceptibility of gae1 gae6 plants differed depending on the specific 480

B cinerea isolate Whereas Fresa 525 Gallo 1 UK Razz and KT grew better in gae1481

gae6 we detected no difference in Acacia Apple 517 DN Grape Pepper or Rasp 482

growth (Figure 6) 483

B cinerea was shown to metabolize pectin and to possibly utilize GalA as a484

carbon source during in planta growth (Zhang et al 2011 Zhang and Van Kan 2013) 485

Our data show that gae1 gae6 plants which are reduced in GalA content did allow 486

enhanced growth of specific B cinerea isolates but no reduction in growth of any B 487

Page 27 of 54

cinerea isolates tested was found This suggests that utilization of pectin as a carbon 488

source was not a contributing factor to alterations in B cinerea susceptibility It is 489

possible that changes in the cell wall composition affect the structural integrity of the 490

wall and affect hyphal penetration of specific B cinerea isolates However OGs (small 491

HG fragments) are likely released during the infection process (Ferrari et al 2013) 492

OGs function as DAMPs and initiate immune responses in Arabidopsis (Ferrari et al 493

2013) Treatment of Arabidopsis with OGs having a degree of polymerization (DP) of 494

10-15 sugar subunits was previously shown to enhance resistance to B cinerea (Ferrari495

et al 2007) We showed that pretreatment with OGs or macerozyme induced enhanced 496

immunity to B cinerea isolate Gallo 1 in wild-type Col-0 but only OGs and not 497

macerozyme induced immunity in gae1 gae6 (Figure 13) Our data suggest gae1 gae6 498

plants which have reduced pectin content may release less OGs and that this 499

reduction leads to the increased susceptibility to B cinerea 500

Consistently we found that macerozyme treatment released less soluble pectin 501

from gae1 gae6 cell walls than from wild-type walls Fractions containing soluble pectin 502

released from gae1 gae6 were also less active in inducing PAD3 gene expression than 503

the fractions released from wild-type walls (Figure 7) We also showed that less soluble 504

pectin accumulated in gae1 gae6 than in wild-type plants 48 h after treatment with B 505

cinerea (Figure 12) 506

We observed that loss of pectin leads to alterations in A thaliana immune 507

signaling Macerozyme treatment induced expression of PAD3 and the SA marker 508

genes PR1 and SID2 in wild-type Col-0 but not in gae1 gae6 whereas OG treatment 509

led to increased PAD3 and SID2 expression in both wild-type and gae1 gae6 (Figure 8 510

and 9) This suggests that although release of active soluble pectin in these plants 511

might be altered OG recognition is intact In fact cell walls isolated from qua1 another 512

mutant known to be reduced in HG released less OGs (DP1-6) than did wild-type cell 513

walls upon endopolygalacturonase treatment (Bouton et al 2002) This was also true 514

for gae1 gae6 cell walls as macerozyme treatment of isolated gae1 gae6 cell walls 515

released less soluble pectin than treatment of Col-0 cell walls Because the fraction 516

from gae1 gae6 plants was also less active in inducing PAD3 expression (Figure 7) it 517

seems possible that biologically active OGs were the active component inducing PAD3 518

Page 28 of 54

expression Interestingly expression of the JA marker genes JAZ5 and JAZ10 was 519

higher in gae1 gae6 than in wild-type mock-inoculated plants but was comparable to 520

wild-type in untreated plants suggesting that JA signaling in gae1 gae6 is 521

hyperresponsive (Figure 10 and Supplemental Figure 11) This induction of JA signaling 522

might be due to wounding stress incurred during the inoculation process JA and SA 523

signaling generally act antagonistically (Pieterse et al 2012) Increased JA signaling 524

activity might hence explain why mock- and macerozyme-treated gae1 gae6 plants 525

exhibited reduced expression of the SA marker PR1 and why OG treatment induced 526

expression of SID2 another SA marker to a lower extent in gae1 gae6 than in wild-type 527

plants (Figure 9) 528

No single immune signaling component was found to be the sole contributor to 529

the compromised immunity of gae1 gae6 to specific B cinerea isolates such as Gallo 1 530

and Fresa 525 These isolates grew better on pad3 while growth of Pepper another B 531

cinerea isolate that did not show altered virulence on gae1 gae6 was unaltered 532

(Supplemental Figure 9) This suggested that differences in camalexin sensitivity of 533

these B cinerea isolates might account for the differences in gae1 gae6 susceptibility 534

However camalexin accumulation and PAD3 expression was the same for wild-type 535

Col-0 and gae1 gae6 for the B cinerea isolates Fresa 525 and Gallo 1 (Figure 8) which 536

showed enhanced growth on gae1 gae6 plants It should be noted that B cinerea 537

virulence is often estimated by comparing lesion sizes Lesion size and measurements 538

of relative fungal growth by qPCR do not always show the same results For example 539

although Denby et al (2004) showed that Pepper causes larger lesions on pad3 than on 540

wild-type this study showed that relative fungal growth of Pepper on pad3 was 541

unaltered 542

We observed that callose deposition was strongly reduced in gae1 gae6 plants 543

72 h after treatment with B cinerea isolate Gallo 1 (Figure 11) possibly due to reduced 544

release of active soluble pectin Interestingly OG-induced PTI to an unspecified isolate 545

of B cinerea was intact in the pmr4 mutant which does not produce callose (Galletti et 546

al 2008) Due to the high SA levels in pmr4 plants (Nishimura et al 2003) and the fact 547

that different B cinerea isolates vary both in induction of and reaction to defense 548

responses (Kliebenstein et al 2005 Rowe et al 2010) we could not determine 549

Page 29 of 54

whether reduced callose deposition causes the increased susceptibility of gae1 gae6 to 550

B cinerea isolate Gallo 1551

We did not observe any changes in B cinerea growth either on cev1 ixr1 plants 552

which are known to have constitutively high JA and OPDA levels (Ellis et al 2002b) or 553

on plants exogenously treated with MeJA (Supplemental Figure 9) Because plants 554

were kept uncovered for 3 h after MeJA treatment it is unclear how much MeJA was 555

bioavailable in the plant However JAZ10 expression was induced 3 h after spraying 556

with MeJA (Supplemental Figure 10) Because pathogen growth generally depends on 557

the combination of a multitude of immune signaling events we conclude that multiple 558

changes to the immune signaling in gae1 gae6 together account for the altered B 559

cinerea growth phenotypes detected 560

We showed that expression of GAE1 and GAE6 is repressed by the bacterial 561

hemibiotroph Pma ES4326 This repression did not occur in an eds1 or pad4 562

background or in pbs3 at the 24-h time point (Figure 1 and Supplemental Figure 1) 563

During the Arabidopsis - Pma ES4326 interaction the expression of many defense 564

related genes is altered in a PAD4EDS1 dependent fashion (Wang et al 2008) Some 565

of these expression changes require SA CBP60g and SARD1 are thought to act 566

downstream of PAD4 and EDS1 but upstream of SA-dependent responses (Wang et 567

al 2011) PBS3 is also thought to act downstream of PAD4 and EDS1 and upstream of 568

SA but the set of genes whose expression it affects is only partially overlapping with 569

those affected by CBP60gSARD1 (Wang et al 2008 Wang et al 2011) Thus GAE1 570

and GAE6 repression is regulated by the part of the immune signaling network that 571

requires PAD4 EDS1 and PBS3 but is independent of CBP60gSARD1 and SA 572

Because EDS1 PAD4 and PBS3 are important components of the plant immune 573

signaling network (Wiermer et al 2005 Nobuta et al 2007) one might conclude that 574

repression of GAE1 and GAE6 may be part of the plant immune response to Pma 575

ES4326 B cinerea treatment also reduced GAE1 and GAE6 expression but this was 576

mostly independent of PAD4 (Figure 6) while both Pma ES4326 and B cinerea repress 577

GAE1 and GAE6 expression the host signaling mechanisms responsible likely differ 578

In summary our data show that Arabidopsis GAE1 and GAE6 are required for 579

pectin biosynthesis leaf flexibility and immunity to Pma ES4326 and to specific B 580

Page 30 of 54

cinerea isolates We used macerozyme a commercial pectinase to mimic the effect of 581

B cinerea pectinases Macerozyme treatment released less pectin from gae1 gae6 cell582

walls and induced less immune signaling in gae1 gae6 plants Less water soluble pectin 583

also accumulated in B cinerea infected gae1 gae6 plants and macerozyme-induced 584

immunity to B cinerea isolate Gallo 1 was reduced in these plants We conclude that 585

the reduced pectin content in these mutants may lead to reduced release of soluble 586

pectin including active OGs during pathogen attack and that this compromises 587

immunity 588

589

590

Page 31 of 54

METHODS 591

Plant Materials and Growth Conditions 592

Wild-type Columbia (Col-0) and all mutant plants (in Col-0 background) were 593

grown on sterilized BM2 germinating mix (Berger Inc Quebec Canada) in a controlled 594

environment chamber (Conviron Pembina ND USA) with a 12-h photoperiod under 595

100 microM m-2 s-1 fluorescent illumination at 22degC and 75 relative humidity Germplasm 596

used is described in the Accession Numbers section at the end of Methods 597

598

Data Analysis and Replicates 599

For biological replicates samples were obtained from separate plant tissues and 600

in many cases from plants grown in different flats In some experiments multiple 601

technical replicates were measured per biological replicate For statistical analysis all 602

technical replicates were averaged and the averages were considered one biological 603

replicate Independent experiments were performed at different times and often 604

consisted of multiple biological replicates per experiment 605

Data analysis was performed using mixed linear models in the R programming 606

environment (Bates et al 2015) unless otherwise indicated Generally genotypes 607

treatments and time points were used as fixed effects and replicate-specific effects like 608

different pots flats etc were considered random effects We reported p-values 609

obtained from the mixed linear model when less than 10 comparisons were performed 610

and q-values for more than 10 comparisons to correct for multiple comparison testing 611

Q-values were calculated using the Benjamini-Hochberg procedure (Benjamini and612

Hochberg 1995) unless specified otherwise 613

614

Pathogen Strains Growth Conditions and Pathogen Growth Assays 615

Growth of Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) and 616

bacterial growth assays were performed as previously described (Bethke et al 2014) 617

For qRT-PCR assays four week-old plants were inoculated with bacteria (OD600 = 618

0002) or mock (5 mM MgSO4) At least four leaves from two plants were collected at 619

the indicated time points per biological replicate 620

Page 32 of 54

Prior to each experiment Botrytis cinerea isolates were grown on 1x PDA 621

medium (Difco) for 10 d at room temperature All isolates used have been described 622

previously (Rowe and Kliebenstein 2007) The spores were washed from the surface of 623

the plate using an inoculation medium (1x Gamborgrsquos B-5 basal salt mixture (Sigma-624

Aldrich G5768) 2 (wv) glucose 10 mM phosphate buffer pH 64) and fungal hyphae 625

were removed from the suspension by filtering through four layers of cheesecloth The 626

concentration of spores was determined using a hemocytometer and adjusted to 25 x 627

105 spores mL-1 Four week-old Arabidopsis plants were inoculated by placing one 10-628

microL droplet of the B cinerea spore solution or inoculation medium (mock) on the adaxial 629

leaf surface of fully expanded leaves Inoculated plants were kept at 100 relative 630

humidity Infection sites were collected at various time points using a cork borer For 631

qRT-PCR assays at least four leaf discs from one plant were combined for each 632

biological sample For B cinerea growth assays at least 12 leaf discs from three to six 633

plants were pooled per biological sample Relative fungal growth was determined by 634

measuring the abundance of a fungal gene relative to a plant gene as described below 635

In brief DNA from infected tissue was extracted and equal amounts of total DNA were 636

used to perform qPCR reactions using the B cinerea cutinase A and the Arabidopsis 637

SK11 gene respectively Primers were described in (Gachon and Saindrenan 2004) 638

and are listed in Supplemental Table 1 Two technical replicates were combined for 639

each biological replicate B cinerea is a necrotrophic fungal pathogen and may destroy 640

plant DNA as disease progresses However decreases in the amount of plant DNA 641

over the course of our experiments were only observed at late time points with very 642

susceptible plants so the qPCR assay is generally a reasonable estimate of fungal 643

biomass (Supplemental Figure 16) 644

645

Treatment with Macerozyme or OGs 646

For PTI assays four week-old plants either were inoculated with 001 (wv) 647

macerozyme R-10 (Yakult) a Rhizopus polygalacturonase (EC 32115) that possesses 648

high pectinase and hemicellulase activity or were mock inoculated using heat-649

inactivated macerozyme Other similar plants were either inoculated with 250 or 500 microg 650

mL-1 OGs or mock inoculated with water One d later plants were infected with B 651

Page 33 of 54

cinerea and samples were collected immediately (0 dpi) and two or three d later To 652

investigate the role of MeJA on B cinerea growth plants were sprayed with 1mM MeJA 653

in water with 001 (vv) Tween 20 Covers were removed so that leaves could dry off 654

before plants were inoculated with B cinerea three h later Samples were harvested at 655

the indicated time points For expression analysis and MAPK activation assays plants 656

were either inoculated with 100 microg mL-1 OGs or mock inoculated with water OGs used 657

for experiments described in Figures 8 to 10 and Supplemental Figure 12 were a kind 658

gift from Simone Ferrari OGs used in experiments described in Figure 13 and 659

Supplemental Figures 11 13 and 15 were prepared as previously described (Kohorn et 660

al 2014) 661

662

Expression Analysis 663

Leaf tissue from inoculated plants was harvested at the indicated time points 664

flash frozen pulverized and RNA extracted using Trizol (Invitrogen) Quantitative RT-665

PCR was performed using the SuperScript III Platinum SYBR Green One-Step 666

quantitative RT-PCR kit (Invitrogen) and a Lightcycler 480 Real-Time PCR system 667

(Roche) as previously described (Truman and Glazebrook 2012) In brief equal 668

amounts of total RNA and a gene specific primer were used for each reaction The 669

crossing point (Cp) was calculated using the 2nd derivative max method provided with 670

the Lightcycler software for each amplification curve Each reaction was run with two 671

technical replicates and the Cp values for these replicates were averaged ACTIN2 was 672

used as a stably expressed reference gene At least three biological replicates each 673

were performed and analyzed using a mixed-linear model RT-PCR was performed to 674

verify T-DNA insertion lines using the One-Step RT-PCR kit (Qiagen) according to the 675

manufacturerrsquos instructions The primers used can be found in Supplemental Table 1 676

677

Electrolyte Leakage Assay 678

Fully expanded leaves of four week-old plants were inoculated with water (Milli-Q 679

grade) using a needleless syringe Leaf discs were collected using a cork borer and 680

three leaf discs from one leaf were placed with the adaxial surface down onto 2 mL 681

water in a 12-well cell culture plate Conductivity was measured using a Horiba B-173 682

Page 34 of 54

conductivity meter after 30 min and then the water was replaced Conductivity was 683

measured again 30 min later (1 h total) as indicated in Figure 3C Six biological 684

replicates each were performed in three independent experiments and means and SEs 685

were calculated Two-sided t-tests were performed for each time point 686

687

DAB and Aniline Blue Staining 688

Leaves from four week-old plants were either mock inoculated with B cinerea 689

inoculation medium or inoculated with 10 microL droplets of a B cinerea spore solution (25 690

x 105 spores mL-1) of the indicated isolates Samples were collected 72 h later For 33prime-691

diaminobenzidine (DAB) staining two to five inoculated leaves were vacuum infiltrated 692

with 33prime-Diaminobenzidine (1 mg mL-1 in water pH 38) solution and stained overnight 693

in the dark Leaves were destained using 100 ethanol at 65degC and then transferred to 694

50 (vv) glycerol and photographed 695

Callose was visualized by staining leaves with aniline blue as previously 696

described (Adam and Somerville 1996) Briefly leaves were vacuum infiltrated with 697

alcoholic lactophenol (one volume of phenolglycerollactic acidwater (1111) mixed 698

with two volumes of ethanol) and destained at 65degC Next leaves were moved through 699

an alcohol gradient (50 20 and 10 (vv) ethanol) into water The destained leaves 700

were stained for 30 min in 150 mM K2HPO4 (pH 95) with 001 (wv) aniline blue 701

Samples were mounted in water and examined using a Nikon Eclipse Ni-U microscope 702

at 10x magnification using a DAPI filter (excitation at 325-375 nm emission at 435-485 703

nm) For callose quantification six 200 microm x 200 microm squares were counted for each 704

sample type Callose deposits per mm2 were calculated and data analyzed using a t-705

test Note that there is an inherent uncertainty associated with such counts because 706

callose deposits may not all be in focus at the same time 707

708

MAPK Activity Assay 709

Fully expanded leaves of four week-old plants were inoculated with 001 710

macerozyme 100 microg mL-1 OGs 100 nM flg22 or water (mock) flg22 a peptide from 711

bacterial flagella that induces immune responses was used as a positive control for 712

Page 35 of 54

MAPK activation (Asai et al 2002) and was purchased from EZBiolab Inc (Westfield 713

IN USA) Samples were harvested 10 min later and immediately frozen in liquid 714

nitrogen Protein was extracted using an extraction buffer containing 25 mM Tris pH 78 715

75 mM NaCl 1 mM DTT 1 mM NaF 05mM Na3VO4 01 (vv) Tween 20 one 716

cOmplete Mini protease inhibitor cocktail tablet (Roche) and one PhosSTOP 717

phosphatase inhibitor cocktail tablet (Roche) per 10 mL buffer 15 microg of total protein 718

were electrophoresed on a 10 SDS-polyacrylamide gel and then blotted onto a PVDF 719

membrane (Bio-Rad 162-0177) Activated MAPKs were detected using p4442 MAPK 720

(Erk12) antibody (9102S Cell Signal 12500 in TBST (20 mM Tris pH 75 150 mM 721

NaCl 01 (vv) Tween 20)) Anti-Rabbit IgG-HRP (115000 in TBST A6154 Sigma) 722

and ECL Plus substrate (Pierce 32132) MPK3 and MPK6 were detected with an anti-723

MPK3 antibody (M8318 Sigma 12000 in TBST and 3 (wv) milk) or an anti-MPK6 724

antibody (A7104 Sigma 16000 in TBST and 3 (wv) milk) respectively The 725

secondary antibody and detection were the same as for the p4442 antibody Five 726

independent experiments were performed 727

728

Preparation of Alcohol Insoluble Residue (AIR) Pectin Extraction and Uronic Acid 729

Measurements 730

Fully expanded leaves from four week-old plants that were incubated for 48 h in 731

the dark to reduce starch were harvested flash frozen and pulverized AIR was 732

extracted by washing ground material twice in 70 (vv) ethanol three times in a 733

mixture of chloroform and methanol (11 vv) and once in acetone (Gille et al 2009) 734

and was then air dried Cell wall pectin was extracted from AIR with cell wall extraction 735

buffer (50 mM Trizma 50 mM CDTA pH 72) at 95degC for 15 min Samples were 736

homogenized using a paint shaker (Harbil 5G-HD) and glass beads (3 mm diameter) 737

For dot blot experiments 500 microL of cell wall extraction buffer were used per 10 mg of 738

AIR Debris was precipitated by centrifugation for 10 min at 10000 g 739

Total uronic acid content of pectin extracted as described above using 500 microL of 740

cell wall extraction buffer per 1 mg of AIR was measured as described previously 741

(Filisetti-Cozzi and Carpita 1991 van den Hoogen et al 1998) First 36 microL of pectin 742

extract were mixed with 4 microL of 4 M sulfamic acid Then 200 microL of sulfuric acid 743

Page 36 of 54

containing 120 mM sodium tetraborate was added and samples were incubated at 80degC 744

for 1 h Following cooling on ice the optical density of samples was measured at 490 745

nm Next 40 microL of m-hydroxydiphenyl reagent (100 microL of m-hydroxydiphenyl in dimethyl 746

sulfoxide at 100 mgmL mixed with 49 mL 80 (vv) sulfuric acid just before use) was 747

added and the samples were mixed The optical density at 490 nm was measured 748

again The optical density before m-hydroxydiphenyl reagent addition was subtracted 749

from the optical density measured after addition of the dye The concentration of uronic 750

acid was calculated using known amounts of GalA as a standard 751

752

Release of Pectin from Arabidopsis Cell Walls Using Macerozyme 753

AIR preparations from leaves of four week-old plants were treated with 025 754

(wv) macerozyme R-10 (Yakult) or water (mock) at a concentration of 100 microL per mg 755

AIR Samples were incubated for 3 h at room temperature on a vortex shaker and 756

centrifuged for 10 min at 12000 g Total uronic acid concentration of 5-fold diluted 757

supernatants was determined for two technical replicates each The concentration of 758

uronic acid was calculated using known amounts of GalA as a standard For 759

subsequent qRT-PCR experiments supernatants from multiple extractions were 760

combined boiled for 20 min to inactivate macerozyme diluted 10-fold with water and 761

inoculated into four week-old Col-0 plants Heat-inactivated 10-fold diluted macerozyme 762

solutions were used as mock treatment Samples were collected 3 h later and PAD3 763

expression was determined 764

765

Release of Pectin from Arabidopsis Leaves Infected with B cinerea 766

Fully expanded leaves of four week-old plants were either inoculated with up to 767

five 10 microL droplets of B cinerea spore solution (25 x 105 spores mL-1) or mock 768

inoculated with B cinerea inoculation medium For each biological replicate leaves with 769

a combined count of at least 20 infection sites were collected 48 h later AIR was 770

extracted as described above but plants were not dark-treated or freeze-dried and the 771

first ethanol step was performed using 85 (vv) ethanol To extract soluble pectin 772

fragments AIR samples were mixed with water at a concentration of 20 microL per infection 773

site incubated for 7 h at room temperature on a vortex shaker and centrifuged for 10 774

Page 37 of 54

min at 12000 g Total uronic acid concentration of supernatants was determined for two 775

technical replicates each The concentration of uronic acid was calculated using known 776

amounts of GalA as a standard 777

778

Measurement of Uronic Acids Neutral Monosaccharides and Cellulose Content in 779

Cell Walls 780

Neutral sugars and uronic acids from non-cellulosic polysaccharides in cell walls 781

(AIR) were released by trifluoroacetic acid (TFA) hydrolysis Approximately 1 mg of AIR 782

was hydrolyzed with 2 M TFA at 121degC for 90 min The TFA-soluble material was split 783

into two equal parts one for uronic acid and one for neutral sugar determination The 784

uronic acid content was determined by analyzing the hydrolyzate using a CarboPac 785

PA200 anion-exchange column with an ICS-3000 Dionex chromatography system 786

(Dionex Sunnyvale CA USA) The elution profile consisted of a linear gradient of 50 to 787

200 mM sodium acetate in 01 M NaOH in 10 min at 04 mL per min Alditol acetate 788

derivatives of neutral sugars were produced as described (York et al 1985) In brief 789

monosaccharides in the hydrolyzate were reduced with NaBH4 and peracetylated with 790

acetic anhydride and pyridine at 121degC for 20 min The generated alditol acetates were 791

analyzed by GC-MS Crystalline cellulose was measured according to Updegraff (1969) 792

After hydrolysis of noncellulosic polysaccharides from AIR with the Updegraff reagent 793

(acetic acid nitric acids water 812 (vv) ) the remaining pellet was hydrolyzed in 794

72 sulfuric acid The resulting glucose quantity was determined by the anthrone 795

method (Scott and Melvin 1953) 796

797

Dot Blot Experiments 798

Pectin solutions extracted using 50 microL of cell wall extraction buffer per mg AIR 799

were serially diluted and nitrocellulose membranes were spotted with 1 microL of the diluted 800

pectin solutions Membranes were dried overnight blocked with 5 milk (wv) in 1x 801

PBS (8 g L-1 NaCl 02 g L-1 KCl 144 g L-1 Na2HPO4 024 g L-1 KH2PO4 pH 74) and 802

probed with LM5 (Jones et al 1997) LM6 (Willats et al 1998) LM8 (Willats et al 803

2004) LM19 and LM20 (Verhertbruggen et al 2009a) or CCRC-M7 (Steffan et al 804

Page 38 of 54

1995) antibodies LM series antibodies were diluted 1250 and CCRC-M7 was diluted 805

1500 in 5 milk powder (Nestle Carnation) in 1 x PBS For LM series antibodies a 806

goat anti-Rat HRP conjugated antibody (Bethyl A110-105P 15000 diluted) and for 807

CCRC-M7 an anti-Mouse IgG HRP conjugate (Promega W402B 12500 diluted) was 808

used as secondary antibody in 5 milk powder in 1x PBS LM series antibodies were 809

obtained from plant probes (wwwplantprobesnet) and CCRC-M7 from CarboSource 810

(wwwcarbosourcenet) Membranes were washed after incubation with the primary and 811

secondary antibodies with 1x PBS Dot blots were developed using the ECL system 812

(GE Healthcare Amersham ECL prime) For quantification of signals dot blot results on 813

x-ray films were photographed using a CCD camera and intensities were measured814

using Image Jrsquos Integrated Density function Locally adjusted background was 815

subtracted As the dynamic range of the signal on an x-ray film was narrow non-816

linearity in the measured values was corrected for each genotype in each biological 817

replicate using the values from different dilutions in two technical replicates We noticed 818

that the residuals were not normally distributed and performed Box-Cox power 819

transformation (Box and Cox 1964) to obtain normally distributed values for the 820

subsequent statistical analysis of the data For LM19 Box-Cox selected power 821

transformation was done to the power of 038th and for LM20 to the power 075th After 822

correction of the transformed measured values with the effect of the biological 823

replicates the values were fit to a linear model with the Arabidopsis genotype as the 824

fixed effect This linear model was used both to generate the plots in Figures 5B and D 825

and to test significant differences 826

827

Camalexin Measurements 828

Four week-old plants were infected with B cinerea and samples (three leaf disks 829

from one plant each) were harvested at the indicated time points flash frozen and 830

pulverized Each sample was extracted with 300 microL of 90 methanol (vv) 50 microL of the 831

extract was run on an Agilent Lichrocart 250-4 RP18e 5-microm column using an Agilent 832

1100 series HPLC Camalexin was detected using a DiodeArray (DAD) at 330 nm and 833

with a fluorescence detector at emission 318 nmexcitation 385 nm (Agilent) Separation 834

was achieved using the following program with aqueous acetonitrile 5 min gradient 835

Page 39 of 54

from 63 to 69 acetonitrile 30 s gradient from 69 to 99 acetonitrile 2 min at 99 836

acetonitrile and a post-run equilibration of 35 min at 63 acetonitrile (Denby et al 837

2004 Kliebenstein et al 2005) Purified camalexin was used to produce a standard 838

curve to identify and quantitate camalexin 839

840

Accession Numbers 841

Microarray data used can be accessed at NCBI Gene Expression Omnnibus 842

(httpwwwncbinlmnihgovgeo) GSE18978 Sequence information for all Arabidopsis 843

genes described in this article can be found in TAIR (wwwarabidopsisorg) using the 844

following accession numbers PAD3 (CYP71B15 At3g26830) SID2 (ICS1 At1g74710) 845

GAE1 (At4g30440) GAE2 (At1g02000) GAE3 (At4g00110) GAE4 (At2g45310) GAE5 846

(At4g12250) GAE6 (At3g23820) PR1 (At2g14610) JAZ5 (At1g17380) JAZ10 847

(At5g13220) ACTIN2 (At3g18780) PDF12a (At5g44420) PDF12b (At2g26020) 848

AtSK11 (At5g26751) The sequence of the B cinerea cutinase Bc-CutA (Z69264) can 849

be found in the DNA Data Bank of Japan (httpgetentryddbjnigacjp) Germplasm 850

used included dde2-2 (At5g42650 (von Malek et al 2002)) pad3-1 (At3g26830 (Zhou 851

et al 1999)) pad4-1 (At3g52430 (Jirage et al 1999)) sid2-2 (At1g74710 (Wildermuth 852

et al 2001)) pbs3-2 (At5g13320 SALK_018225 (Nobuta et al 2007)) cbp60a-1 853

(At5g62570 SALK_124410 (Truman et al 2013)) cbp60g-1 sard1-2 (At5g26920 854

SALK_023199 At1g73805 SALK_052422 (Wang et al 2011) cbp60a-1 cbp60g-1 855

sard1-2 (Truman et al 2013) gae1-1 (At4g30440 SALK_085554) gae6-1 (At3g23280 856

SALK_104454) gae6-2 (At3g23280 SALK_017191) ixr1-1 cev1 (At5g05170 (Ellis 857

and Turner 2001 Scheible et al 2001)) eds1 plants were derived by introgression of 858

the Ler eds1-2 allele into Col-0 that contains two copies of EDS1 (At3g48090 859

At3g48080 (Bartsch et al 2006) mpk6-2 (At2g43790 SALK_073907 (Liu and Zhang 860

2004)) mpk3-DG (At3g45640 a fast neutron deletion mutant (Miles et al 2005)) and 861

npr1-1 (At1g64280 (Cao et al 1997)) T-DNA insertion lines were part of the SALK 862

collection (Alonso et al 2003) and were obtained from the Arabidopsis Biological 863

Resource Center 864

865

Supplemental Data 866

Page 40 of 54

Supplemental Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 867

Expression Does Not Require CBP60s or SID2 868

Supplemental Figure 2 Expression of GAE Family Members uponTreatment with Pma 869

ES4326 870

Supplemental Figure 3 Characterization of T-DNA Insertions in GAE1 and GAE6 871

Supplemental Figure 4 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves and 872

Are Slightly Smaller than Wild-Type Col-0 Plants 873

Supplemental Figure 5 Cell Walls of gae1 gae6 Plants Contain Less Uronic Acid than 874

Wild-Type Col-0 Cell Walls 875

Supplemental Figure 6 Antibodies Raised Against the RGI or XG Components of 876

Pectin Show No Difference in Binding to gae Mutant and Col-0 Cell Walls 877

Supplemental Figure 7 The Effect of B cinerea Treatment on the Expression of GAE 878

Family Genes 879

Supplemental Figure 8 Treatment with 001 Macerozyme Does Not Cause Any 880

Visible Damage to Col-0 Plants 881

Supplemental Figure 9 B cinerea Growth on Plants with Defects in Immune 882

Signaling 883

Supplemental Figure 10 MeJA-Induced Expression of the JA marker gene JAZ10 884

Supplemental Figure 11 The Expression of the JA Marker Gene JAZ5 Is Highly 885

Responsive in gae1 gae6 Plants 886

Supplemental Figure 12 The Expression of PDF12 Is Unaltered in gae1 gae6 Plants 887

Supplemental Figure 13 Macerozyme Treatment Activates MPK3 and MPK6 888

Supplemental Figure 14 B cinerea-Induced ROS Production in Wild-Type Col-0 and 889

gae1 gae6 Plants 890

Supplemental Figure 15 OG-Induced PTI Using 250 microg mL-1 OGs 891

Supplemental Figure 16 Abundance of Plant DNA in B cinerea Treated Plants 892

Supplemental Table 1 Primers used in this study 893

894

ACKNOWLEDGEMENTS 895

We thank William Truman for helpful discussions Simone Ferrari (Sapienza 896

Universita di Roma) for the generous gift of OGs Bruna Bucciarelli (USDA-ARS) for 897

Page 41 of 54

help with fluorescence microscopy the Arabidopsis Biological Resource Center for T-898

DNA insertion lines Paul Knoxrsquos lab (University of Leeds) for LM5 LM6 LM8 LM19 899

and LM20 antibodies and the Complex Carbohydrate Resource Center (University of 900

Georgia) for the CCRC-M7 antibody Most of this work was funded by the Division of 901

Chemical Sciences Geosciences and Biosciences Office of Basic Energy Sciences of 902

the US Department of Energy through Grant DE-FG02-05ER15670 to JG This work 903

was also supported by NSF awards IOS 1339125 and IOS 1021861 to DJK NSF 904

award IOS 1121425 to F K and the USDA National Institute of Food and Agriculture 905

Hatch project number CA-D-PLS-7033-H to DJK 906

907

AUTHOR CONTRIBUTIONS 908

GB and JG designed the research GB GX AT BL NES and NH 909

performed research DJK provided tools GB GX MP AD BL RAH and FK 910

analyzed data GB and JG wrote the paper with input from all authors 911

912

FIGURE LEGENDS 913

Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 Expression 914

Requires PAD4 EDS1 and PBS3 915

(A) Expression of GAE1 in leaves of wild-type Col-0 and mutant plants Expression was916

measured 24 or 32 h after either inoculation with Pma ES4326 (OD600 = 0002) or mock 917

inoculation with 5 mM MgSO4 and was then normalized to the level of ACTIN2 Data 918

from three biological replicates were combined using a mixed linear model Bars 919

represent mean log2-ratios to ACTIN2 plusmn SE Letters indicate significantly different 920

groups with q lt 005 where 005 represents the maximum false discovery rate at which 921

the test may be called significant 922

(B) Expression of GAE6 measured as described in (A)923

924

Figure 2 Susceptibility of gae6 and gae1 gae6 Plants to Pma ES4326 Is Increased 925

Relative to Wild-Type Col-0 926

Leaves of plants of the indicated genotypes were inoculated with Pma ES4326 (OD600 = 927

00002) as described in Methods Bacterial titers in leaves were determined either 928

Page 42 of 54

immediately (0 dpi) or after three days (3 dpi) Bars represent the mean plusmn SE of four 929

independent experiments each with 4 or 12 biological replicates at 0 and 3 dpi 930

respectively Data were combined using a mixed linear model Asterisks indicate 931

significant differences from Col-0 wild-type (q lt 005) Susceptible pad4 plants were 932

included as a positive control 933

934

Figure 3 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves 935

(A) Depiction of the assay for brittleness of gae mutant leaves Fully expanded leaves of936

four to five week-old plants were bent by 180ordm so that the adaxial surface of the leaf was 937

parallel with the soil Broken leaf petioles or midribs were considered brittle 938

(B) Quantification of broken midribs or petioles For each genotype 66 to 82 leaves 939

were analyzed for brittleness as described in (A) Bars represent the ratio of broken to 940

total number of leaves tested Results were analyzed using Fisherrsquos exact test Letters 941

indicate significantly different groups at p lt 005 942

(C) Conductivity measurements Leaves of four week-old plants were inoculated with943

deionized water and then leaf discs were cut out and placed on deionized water 944

Conductivity was measured at the indicated time points After 30 min the water was 945

replaced with fresh water (30 min f w) to account for wounding-related ion leakage from 946

the edges of the leaf discs Three independent experiments with six biological replicates 947

each were performed and data were analyzed using a t-test for each time point P-948

values were corrected using the Bonferroni method Data show means plusmn SE asterisks 949

indicate significant differences from wild-type Col-0 at q lt 001 950

951

Figure 4 Cell Walls of gae6 and gae1 gae6 Plants Are Reduced in GalA 952

(A) to (D) Levels of GalA (A) GlcA (B) cellulose (C) and neutral sugars (D) in alcohol953

insoluble residue (AIR) extracted from leaves of four week-old plants of the indicated 954

genotypes Bars represent means plusmn SE of four biological replicates combined using a 955

mixed linear model Letters indicate significantly different groups (p lt 005) 956

957

Figure 5 Cell Walls of gae1 gae6 Plants Are Reduced in Homogalacturonan 958

Page 43 of 54

Dot blot analyses with anti-LM19 and anti-LM20 antibodies Pectin was extracted from 959

AIR prepared from the indicated genotypes using 1 microL of extraction buffer per 10 microg of 960

AIR Pectin was serially diluted and one microL of undiluted 13 diluted and 19 diluted 961

samples were spotted on a nitrocellulose membrane Three biological replicates were 962

were spotted twice to obtain two technical replicates each All replicates produced 963

similar results 964

(A) Representative dot blot using anti-LM19 antibody965

(B) Relative signal intensities of the spots in the dot blot in (A)966

(C) Representative dot blot using anti-LM20 antibody967

(D) Relative signal intensities of the LM20 dot blot shown in (C)968

Relative signal intensities were measured using Image J software Measurements from 969

the three biological replicates were combined using a linear model after Box-Cox power 970

transformation as described in Methods Bars represent means plusmn SE Asterisks indicate 971

a difference from wild-type Col-0 at p lt 005 972

973

Figure 6 Growth of B cinerea on gae1 gae6 Plants and the Effect of B cinerea on 974

Expression of GAE1 and GAE6 975

(A) Growth of B cinerea isolates Leaves of Col-0 and gae1 gae6 plants were976

inoculated with 25 x 105 spores mL-1 of the B cinerea isolates indicated in the figure 977

Samples were collected two (2 dpi) and three (3 dpi) days later Bars represent means plusmn 978

SE of six biological replicates for each fungus combined using a mixed linear model 979

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 980

(B) and (C) Expression of GAE1 (B) and GAE6 (C) 48 h after inoculation with the B981

cinerea isolates indicated Expression levels were measured by qRT-PCR Bars 982

represent mean log2-ratios to ACTIN2 plusmn SE of three biological replicates combined 983

using a mixed linear model Letters indicate significantly different groups (q lt 005) 984

985

Figure 7 Levels of Soluble Pectin and Expression of PAD3 in Fractions Produced 986

Following Macerozyme Treatment of gae1 gae6 Cell Walls 987

(A) Schematic overview of the experimental procedure for (B) and (C)988

Page 44 of 54

(B) Uronic acid measurements Alcohol-insoluble residue (AIR) from leave tissue of four989

week-old wild-type Col-0 or gae1 gae6 plants was treated with water or 025 990

macerozyme and assayed for uronic acid levels Data from 11 biological replicates were 991

combined using a mixed linear model Bars represent means plusmn SE Letters indicate 992

significant differences (p lt 005) 993

(C) Expression of PAD3 in Col-0 plants treated with the fractions from (B) Extracts from994

(B) were heat inactivated pooled and diluted 5-fold with water Pooled extracts were995

inoculated into four week-old Col-0 plants PAD3 expression was measured 3 h after 996

inoculation and normalized to the level of ACTIN2 expression Data from four biological 997

replicates were combined using a mixed linear model Bars represent mean log2-ratios 998

to ACTIN2 plusmn SE Letters indicate significant differences (p lt 005) 999

1000

Figure 8 Macerozyme-Induced PAD3 Expression Is Absent in gae1 gae6 1001

(A) Expression of PAD3 after macerozyme treatment Four week-old plants were either1002

inoculated with 001 macerozyme or mock inoculated with water and PAD3 1003

expression was measured 3 h later by qRT-PCR Data from four biological replicates 1004

were combined using a mixed linear model Mean log2-ratios to ACTIN2 plusmn SE were 1005

plotted Letters indicate significantly different groups (p lt 005) 1006

(B) Expression of PAD3 after OG treatment PAD3 expression was measured as1007

described in (A) 3 h after inoculation with 100 microg mL-1 OGs or mock inoculation with 1008

water Data are from six biological replicates 1009

(C) PAD3 expression in untreated plants Three biological replicates were performed1010

and data analyzed as described in (A) 1011

(D) Camalexin accumulation after B cinerea treatment Camalexin accumulation was1012

measured in four week-old plants inoculated with the B cinerea strains shown in the 1013

figure Eight biological replicates each were combined using a mixed linear model 1014

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 1015

(E) PAD3 expression 28 h after treatment with the indicated B cinerea strains Data1016

from six biological replicates were collected and analyzed as described in (A) Data in 1017

all figure parts represent means plusmn SE Letters indicate significantly different groups (q lt 1018

005) 1019

Page 45 of 54

1020

Figure 9 Macerozyme-Induced Expression of the SA Marker Genes SID2 and PR1 1021

Is Abolished in gae1 gae6 Plants 1022

(A) to (H) Expression of PR1 (A) (C) (E) and (G) or SID2 (B) (D) (F) and (H) in four1023

week-old Col-0 and gae1 gae6 plants Data were combined using a mixed linear model 1024

Bars represent mean log2-ratios of expression vs ACTIN2 plusmn SE for all figure parts 1025

Letters indicate significantly different groups at p lt 005 for panels (A) to (D) (G) and 1026

(H) and qlt005 for panels (E) to (F) 1027

(A) and (B) Plants were treated with 001 macerozyme or mock treated with water1028

and samples were collected 3 h later Data from four biological replicates are shown 1029

(C) and (D) Plants were inoculated with 100 microg mL-1 OGs or mock treated with water1030

and samples collected 3 h later Data from six biological replicates are shown 1031

(E) and (F) Plants were inoculated with the indicated B cinerea isolates or mock1032

inoculated with B cinerea inoculation medium and samples collected 48 h later Data 1033

from eight biological replicates are shown 1034

(G) and (H) Expression in untreated plants Three biological replicates were performed1035

1036

Figure 10 The Expression of the JA Marker Gene JAZ10 is Highly Responsive in 1037

gae1 gae6 Plants 1038

(A) to (D) JAZ10 expression in four week-old plants Data were combined using a mixed1039

linear model Mean log2-ratios to ACTIN2 plusmn SE are shown Letters indicate significantly 1040

different groups at p lt 005 for panels (A) (B) and (D) and q lt 005 for panel (C) 1041

(A) Plants were inoculated with 001 macerozyme or mock inoculated using water and1042

samples were collected 3 h later Data from four biological replicates are shown 1043

(B) Plants were inoculated with 100 microg mL-1 OGs or mock inoculated using water and1044

samples collected 3 h later Data are from six biological replicates 1045

(C) JAZ10 expression 48 h after treatment with the indicated B cinerea strains Data1046

are from six biological replicates 1047

(D) JAZ10 expression in untreated plants Data are from three biological replicates1048

1049

Figure 11 Gallo 1-Induced Callose Deposition Is Reduced in gae1 gae6 Plants 1050

Page 46 of 54

(A) Callose deposition in four week-old Col-0 and gae1 gae6 plants Plants were1051

inoculated with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) or mock inoculated 1052

with B cinerea inoculation medium and samples were collected after 72 h Callose was 1053

stained with aniline blue and visualized using a Nikon Eclipse Ni-U microscope with a 1054

DAPI filter Three leaves were stained per treatment Scale bars are 100 microm Asterisks 1055

indicate fungal hyphae arrows indicate representative callose spots 1056

(B) Quantification of callose deposition Callose deposits were counted in 200 microm x 2001057

microm squares laid out across the images shown here Bars represent means plusmn SE from 1058

six squares each Letters indicate significantly different groups (p lt 005) according to a 1059

t-test analysis1060

1061

Figure 12 Less Soluble Pectin Accumulates in B cinerea-Infected gae1 gae6 1062

Leaves 1063

Four week-old Col-0 and gae1 gae6 plants were inoculated with 10 microL droplets of the 1064

indicated B cinerea isolate (25 x 105 spores mL-1) or mock inoculated with B cinerea 1065

inoculation medium Leaves were collected 48 h later AIR was extracted from leaf 1066

samples and water added (20 microL infection site) to extract soluble pectin fragments 1067

Samples were vortexed for 7 h centrifuged at 12000 g and the uronic acid 1068

concentration of the supernatant was measured Data from nine biological replicates 1069

were combined using a mixed linear model Means plusmn SE are shown Letters indicate 1070

significant differences (q lt 005) 1071

1072

Figure 13 Macerozyme-Induced PTI Is Lost in gae1 gae6 While OG-Induced PTI Is 1073

Unaltered 1074

(A) Macerozyme-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1075

inoculated with 001 macerozyme or mock inoculated with boiled macerozyme and 1076

were infected with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) 24 h later Samples 1077

were collected immediately (0 dpi) and after two (2 dpi) and three (3 dpi) days Data 1078

show mean plusmn SE of six to 14 biological replicates each combined using a linear model 1079

Letters indicate significantly different groups (q lt 005) 1080

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

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Page 27 of 54

cinerea isolates tested was found This suggests that utilization of pectin as a carbon 488

source was not a contributing factor to alterations in B cinerea susceptibility It is 489

possible that changes in the cell wall composition affect the structural integrity of the 490

wall and affect hyphal penetration of specific B cinerea isolates However OGs (small 491

HG fragments) are likely released during the infection process (Ferrari et al 2013) 492

OGs function as DAMPs and initiate immune responses in Arabidopsis (Ferrari et al 493

2013) Treatment of Arabidopsis with OGs having a degree of polymerization (DP) of 494

10-15 sugar subunits was previously shown to enhance resistance to B cinerea (Ferrari495

et al 2007) We showed that pretreatment with OGs or macerozyme induced enhanced 496

immunity to B cinerea isolate Gallo 1 in wild-type Col-0 but only OGs and not 497

macerozyme induced immunity in gae1 gae6 (Figure 13) Our data suggest gae1 gae6 498

plants which have reduced pectin content may release less OGs and that this 499

reduction leads to the increased susceptibility to B cinerea 500

Consistently we found that macerozyme treatment released less soluble pectin 501

from gae1 gae6 cell walls than from wild-type walls Fractions containing soluble pectin 502

released from gae1 gae6 were also less active in inducing PAD3 gene expression than 503

the fractions released from wild-type walls (Figure 7) We also showed that less soluble 504

pectin accumulated in gae1 gae6 than in wild-type plants 48 h after treatment with B 505

cinerea (Figure 12) 506

We observed that loss of pectin leads to alterations in A thaliana immune 507

signaling Macerozyme treatment induced expression of PAD3 and the SA marker 508

genes PR1 and SID2 in wild-type Col-0 but not in gae1 gae6 whereas OG treatment 509

led to increased PAD3 and SID2 expression in both wild-type and gae1 gae6 (Figure 8 510

and 9) This suggests that although release of active soluble pectin in these plants 511

might be altered OG recognition is intact In fact cell walls isolated from qua1 another 512

mutant known to be reduced in HG released less OGs (DP1-6) than did wild-type cell 513

walls upon endopolygalacturonase treatment (Bouton et al 2002) This was also true 514

for gae1 gae6 cell walls as macerozyme treatment of isolated gae1 gae6 cell walls 515

released less soluble pectin than treatment of Col-0 cell walls Because the fraction 516

from gae1 gae6 plants was also less active in inducing PAD3 expression (Figure 7) it 517

seems possible that biologically active OGs were the active component inducing PAD3 518

Page 28 of 54

expression Interestingly expression of the JA marker genes JAZ5 and JAZ10 was 519

higher in gae1 gae6 than in wild-type mock-inoculated plants but was comparable to 520

wild-type in untreated plants suggesting that JA signaling in gae1 gae6 is 521

hyperresponsive (Figure 10 and Supplemental Figure 11) This induction of JA signaling 522

might be due to wounding stress incurred during the inoculation process JA and SA 523

signaling generally act antagonistically (Pieterse et al 2012) Increased JA signaling 524

activity might hence explain why mock- and macerozyme-treated gae1 gae6 plants 525

exhibited reduced expression of the SA marker PR1 and why OG treatment induced 526

expression of SID2 another SA marker to a lower extent in gae1 gae6 than in wild-type 527

plants (Figure 9) 528

No single immune signaling component was found to be the sole contributor to 529

the compromised immunity of gae1 gae6 to specific B cinerea isolates such as Gallo 1 530

and Fresa 525 These isolates grew better on pad3 while growth of Pepper another B 531

cinerea isolate that did not show altered virulence on gae1 gae6 was unaltered 532

(Supplemental Figure 9) This suggested that differences in camalexin sensitivity of 533

these B cinerea isolates might account for the differences in gae1 gae6 susceptibility 534

However camalexin accumulation and PAD3 expression was the same for wild-type 535

Col-0 and gae1 gae6 for the B cinerea isolates Fresa 525 and Gallo 1 (Figure 8) which 536

showed enhanced growth on gae1 gae6 plants It should be noted that B cinerea 537

virulence is often estimated by comparing lesion sizes Lesion size and measurements 538

of relative fungal growth by qPCR do not always show the same results For example 539

although Denby et al (2004) showed that Pepper causes larger lesions on pad3 than on 540

wild-type this study showed that relative fungal growth of Pepper on pad3 was 541

unaltered 542

We observed that callose deposition was strongly reduced in gae1 gae6 plants 543

72 h after treatment with B cinerea isolate Gallo 1 (Figure 11) possibly due to reduced 544

release of active soluble pectin Interestingly OG-induced PTI to an unspecified isolate 545

of B cinerea was intact in the pmr4 mutant which does not produce callose (Galletti et 546

al 2008) Due to the high SA levels in pmr4 plants (Nishimura et al 2003) and the fact 547

that different B cinerea isolates vary both in induction of and reaction to defense 548

responses (Kliebenstein et al 2005 Rowe et al 2010) we could not determine 549

Page 29 of 54

whether reduced callose deposition causes the increased susceptibility of gae1 gae6 to 550

B cinerea isolate Gallo 1551

We did not observe any changes in B cinerea growth either on cev1 ixr1 plants 552

which are known to have constitutively high JA and OPDA levels (Ellis et al 2002b) or 553

on plants exogenously treated with MeJA (Supplemental Figure 9) Because plants 554

were kept uncovered for 3 h after MeJA treatment it is unclear how much MeJA was 555

bioavailable in the plant However JAZ10 expression was induced 3 h after spraying 556

with MeJA (Supplemental Figure 10) Because pathogen growth generally depends on 557

the combination of a multitude of immune signaling events we conclude that multiple 558

changes to the immune signaling in gae1 gae6 together account for the altered B 559

cinerea growth phenotypes detected 560

We showed that expression of GAE1 and GAE6 is repressed by the bacterial 561

hemibiotroph Pma ES4326 This repression did not occur in an eds1 or pad4 562

background or in pbs3 at the 24-h time point (Figure 1 and Supplemental Figure 1) 563

During the Arabidopsis - Pma ES4326 interaction the expression of many defense 564

related genes is altered in a PAD4EDS1 dependent fashion (Wang et al 2008) Some 565

of these expression changes require SA CBP60g and SARD1 are thought to act 566

downstream of PAD4 and EDS1 but upstream of SA-dependent responses (Wang et 567

al 2011) PBS3 is also thought to act downstream of PAD4 and EDS1 and upstream of 568

SA but the set of genes whose expression it affects is only partially overlapping with 569

those affected by CBP60gSARD1 (Wang et al 2008 Wang et al 2011) Thus GAE1 570

and GAE6 repression is regulated by the part of the immune signaling network that 571

requires PAD4 EDS1 and PBS3 but is independent of CBP60gSARD1 and SA 572

Because EDS1 PAD4 and PBS3 are important components of the plant immune 573

signaling network (Wiermer et al 2005 Nobuta et al 2007) one might conclude that 574

repression of GAE1 and GAE6 may be part of the plant immune response to Pma 575

ES4326 B cinerea treatment also reduced GAE1 and GAE6 expression but this was 576

mostly independent of PAD4 (Figure 6) while both Pma ES4326 and B cinerea repress 577

GAE1 and GAE6 expression the host signaling mechanisms responsible likely differ 578

In summary our data show that Arabidopsis GAE1 and GAE6 are required for 579

pectin biosynthesis leaf flexibility and immunity to Pma ES4326 and to specific B 580

Page 30 of 54

cinerea isolates We used macerozyme a commercial pectinase to mimic the effect of 581

B cinerea pectinases Macerozyme treatment released less pectin from gae1 gae6 cell582

walls and induced less immune signaling in gae1 gae6 plants Less water soluble pectin 583

also accumulated in B cinerea infected gae1 gae6 plants and macerozyme-induced 584

immunity to B cinerea isolate Gallo 1 was reduced in these plants We conclude that 585

the reduced pectin content in these mutants may lead to reduced release of soluble 586

pectin including active OGs during pathogen attack and that this compromises 587

immunity 588

589

590

Page 31 of 54

METHODS 591

Plant Materials and Growth Conditions 592

Wild-type Columbia (Col-0) and all mutant plants (in Col-0 background) were 593

grown on sterilized BM2 germinating mix (Berger Inc Quebec Canada) in a controlled 594

environment chamber (Conviron Pembina ND USA) with a 12-h photoperiod under 595

100 microM m-2 s-1 fluorescent illumination at 22degC and 75 relative humidity Germplasm 596

used is described in the Accession Numbers section at the end of Methods 597

598

Data Analysis and Replicates 599

For biological replicates samples were obtained from separate plant tissues and 600

in many cases from plants grown in different flats In some experiments multiple 601

technical replicates were measured per biological replicate For statistical analysis all 602

technical replicates were averaged and the averages were considered one biological 603

replicate Independent experiments were performed at different times and often 604

consisted of multiple biological replicates per experiment 605

Data analysis was performed using mixed linear models in the R programming 606

environment (Bates et al 2015) unless otherwise indicated Generally genotypes 607

treatments and time points were used as fixed effects and replicate-specific effects like 608

different pots flats etc were considered random effects We reported p-values 609

obtained from the mixed linear model when less than 10 comparisons were performed 610

and q-values for more than 10 comparisons to correct for multiple comparison testing 611

Q-values were calculated using the Benjamini-Hochberg procedure (Benjamini and612

Hochberg 1995) unless specified otherwise 613

614

Pathogen Strains Growth Conditions and Pathogen Growth Assays 615

Growth of Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) and 616

bacterial growth assays were performed as previously described (Bethke et al 2014) 617

For qRT-PCR assays four week-old plants were inoculated with bacteria (OD600 = 618

0002) or mock (5 mM MgSO4) At least four leaves from two plants were collected at 619

the indicated time points per biological replicate 620

Page 32 of 54

Prior to each experiment Botrytis cinerea isolates were grown on 1x PDA 621

medium (Difco) for 10 d at room temperature All isolates used have been described 622

previously (Rowe and Kliebenstein 2007) The spores were washed from the surface of 623

the plate using an inoculation medium (1x Gamborgrsquos B-5 basal salt mixture (Sigma-624

Aldrich G5768) 2 (wv) glucose 10 mM phosphate buffer pH 64) and fungal hyphae 625

were removed from the suspension by filtering through four layers of cheesecloth The 626

concentration of spores was determined using a hemocytometer and adjusted to 25 x 627

105 spores mL-1 Four week-old Arabidopsis plants were inoculated by placing one 10-628

microL droplet of the B cinerea spore solution or inoculation medium (mock) on the adaxial 629

leaf surface of fully expanded leaves Inoculated plants were kept at 100 relative 630

humidity Infection sites were collected at various time points using a cork borer For 631

qRT-PCR assays at least four leaf discs from one plant were combined for each 632

biological sample For B cinerea growth assays at least 12 leaf discs from three to six 633

plants were pooled per biological sample Relative fungal growth was determined by 634

measuring the abundance of a fungal gene relative to a plant gene as described below 635

In brief DNA from infected tissue was extracted and equal amounts of total DNA were 636

used to perform qPCR reactions using the B cinerea cutinase A and the Arabidopsis 637

SK11 gene respectively Primers were described in (Gachon and Saindrenan 2004) 638

and are listed in Supplemental Table 1 Two technical replicates were combined for 639

each biological replicate B cinerea is a necrotrophic fungal pathogen and may destroy 640

plant DNA as disease progresses However decreases in the amount of plant DNA 641

over the course of our experiments were only observed at late time points with very 642

susceptible plants so the qPCR assay is generally a reasonable estimate of fungal 643

biomass (Supplemental Figure 16) 644

645

Treatment with Macerozyme or OGs 646

For PTI assays four week-old plants either were inoculated with 001 (wv) 647

macerozyme R-10 (Yakult) a Rhizopus polygalacturonase (EC 32115) that possesses 648

high pectinase and hemicellulase activity or were mock inoculated using heat-649

inactivated macerozyme Other similar plants were either inoculated with 250 or 500 microg 650

mL-1 OGs or mock inoculated with water One d later plants were infected with B 651

Page 33 of 54

cinerea and samples were collected immediately (0 dpi) and two or three d later To 652

investigate the role of MeJA on B cinerea growth plants were sprayed with 1mM MeJA 653

in water with 001 (vv) Tween 20 Covers were removed so that leaves could dry off 654

before plants were inoculated with B cinerea three h later Samples were harvested at 655

the indicated time points For expression analysis and MAPK activation assays plants 656

were either inoculated with 100 microg mL-1 OGs or mock inoculated with water OGs used 657

for experiments described in Figures 8 to 10 and Supplemental Figure 12 were a kind 658

gift from Simone Ferrari OGs used in experiments described in Figure 13 and 659

Supplemental Figures 11 13 and 15 were prepared as previously described (Kohorn et 660

al 2014) 661

662

Expression Analysis 663

Leaf tissue from inoculated plants was harvested at the indicated time points 664

flash frozen pulverized and RNA extracted using Trizol (Invitrogen) Quantitative RT-665

PCR was performed using the SuperScript III Platinum SYBR Green One-Step 666

quantitative RT-PCR kit (Invitrogen) and a Lightcycler 480 Real-Time PCR system 667

(Roche) as previously described (Truman and Glazebrook 2012) In brief equal 668

amounts of total RNA and a gene specific primer were used for each reaction The 669

crossing point (Cp) was calculated using the 2nd derivative max method provided with 670

the Lightcycler software for each amplification curve Each reaction was run with two 671

technical replicates and the Cp values for these replicates were averaged ACTIN2 was 672

used as a stably expressed reference gene At least three biological replicates each 673

were performed and analyzed using a mixed-linear model RT-PCR was performed to 674

verify T-DNA insertion lines using the One-Step RT-PCR kit (Qiagen) according to the 675

manufacturerrsquos instructions The primers used can be found in Supplemental Table 1 676

677

Electrolyte Leakage Assay 678

Fully expanded leaves of four week-old plants were inoculated with water (Milli-Q 679

grade) using a needleless syringe Leaf discs were collected using a cork borer and 680

three leaf discs from one leaf were placed with the adaxial surface down onto 2 mL 681

water in a 12-well cell culture plate Conductivity was measured using a Horiba B-173 682

Page 34 of 54

conductivity meter after 30 min and then the water was replaced Conductivity was 683

measured again 30 min later (1 h total) as indicated in Figure 3C Six biological 684

replicates each were performed in three independent experiments and means and SEs 685

were calculated Two-sided t-tests were performed for each time point 686

687

DAB and Aniline Blue Staining 688

Leaves from four week-old plants were either mock inoculated with B cinerea 689

inoculation medium or inoculated with 10 microL droplets of a B cinerea spore solution (25 690

x 105 spores mL-1) of the indicated isolates Samples were collected 72 h later For 33prime-691

diaminobenzidine (DAB) staining two to five inoculated leaves were vacuum infiltrated 692

with 33prime-Diaminobenzidine (1 mg mL-1 in water pH 38) solution and stained overnight 693

in the dark Leaves were destained using 100 ethanol at 65degC and then transferred to 694

50 (vv) glycerol and photographed 695

Callose was visualized by staining leaves with aniline blue as previously 696

described (Adam and Somerville 1996) Briefly leaves were vacuum infiltrated with 697

alcoholic lactophenol (one volume of phenolglycerollactic acidwater (1111) mixed 698

with two volumes of ethanol) and destained at 65degC Next leaves were moved through 699

an alcohol gradient (50 20 and 10 (vv) ethanol) into water The destained leaves 700

were stained for 30 min in 150 mM K2HPO4 (pH 95) with 001 (wv) aniline blue 701

Samples were mounted in water and examined using a Nikon Eclipse Ni-U microscope 702

at 10x magnification using a DAPI filter (excitation at 325-375 nm emission at 435-485 703

nm) For callose quantification six 200 microm x 200 microm squares were counted for each 704

sample type Callose deposits per mm2 were calculated and data analyzed using a t-705

test Note that there is an inherent uncertainty associated with such counts because 706

callose deposits may not all be in focus at the same time 707

708

MAPK Activity Assay 709

Fully expanded leaves of four week-old plants were inoculated with 001 710

macerozyme 100 microg mL-1 OGs 100 nM flg22 or water (mock) flg22 a peptide from 711

bacterial flagella that induces immune responses was used as a positive control for 712

Page 35 of 54

MAPK activation (Asai et al 2002) and was purchased from EZBiolab Inc (Westfield 713

IN USA) Samples were harvested 10 min later and immediately frozen in liquid 714

nitrogen Protein was extracted using an extraction buffer containing 25 mM Tris pH 78 715

75 mM NaCl 1 mM DTT 1 mM NaF 05mM Na3VO4 01 (vv) Tween 20 one 716

cOmplete Mini protease inhibitor cocktail tablet (Roche) and one PhosSTOP 717

phosphatase inhibitor cocktail tablet (Roche) per 10 mL buffer 15 microg of total protein 718

were electrophoresed on a 10 SDS-polyacrylamide gel and then blotted onto a PVDF 719

membrane (Bio-Rad 162-0177) Activated MAPKs were detected using p4442 MAPK 720

(Erk12) antibody (9102S Cell Signal 12500 in TBST (20 mM Tris pH 75 150 mM 721

NaCl 01 (vv) Tween 20)) Anti-Rabbit IgG-HRP (115000 in TBST A6154 Sigma) 722

and ECL Plus substrate (Pierce 32132) MPK3 and MPK6 were detected with an anti-723

MPK3 antibody (M8318 Sigma 12000 in TBST and 3 (wv) milk) or an anti-MPK6 724

antibody (A7104 Sigma 16000 in TBST and 3 (wv) milk) respectively The 725

secondary antibody and detection were the same as for the p4442 antibody Five 726

independent experiments were performed 727

728

Preparation of Alcohol Insoluble Residue (AIR) Pectin Extraction and Uronic Acid 729

Measurements 730

Fully expanded leaves from four week-old plants that were incubated for 48 h in 731

the dark to reduce starch were harvested flash frozen and pulverized AIR was 732

extracted by washing ground material twice in 70 (vv) ethanol three times in a 733

mixture of chloroform and methanol (11 vv) and once in acetone (Gille et al 2009) 734

and was then air dried Cell wall pectin was extracted from AIR with cell wall extraction 735

buffer (50 mM Trizma 50 mM CDTA pH 72) at 95degC for 15 min Samples were 736

homogenized using a paint shaker (Harbil 5G-HD) and glass beads (3 mm diameter) 737

For dot blot experiments 500 microL of cell wall extraction buffer were used per 10 mg of 738

AIR Debris was precipitated by centrifugation for 10 min at 10000 g 739

Total uronic acid content of pectin extracted as described above using 500 microL of 740

cell wall extraction buffer per 1 mg of AIR was measured as described previously 741

(Filisetti-Cozzi and Carpita 1991 van den Hoogen et al 1998) First 36 microL of pectin 742

extract were mixed with 4 microL of 4 M sulfamic acid Then 200 microL of sulfuric acid 743

Page 36 of 54

containing 120 mM sodium tetraborate was added and samples were incubated at 80degC 744

for 1 h Following cooling on ice the optical density of samples was measured at 490 745

nm Next 40 microL of m-hydroxydiphenyl reagent (100 microL of m-hydroxydiphenyl in dimethyl 746

sulfoxide at 100 mgmL mixed with 49 mL 80 (vv) sulfuric acid just before use) was 747

added and the samples were mixed The optical density at 490 nm was measured 748

again The optical density before m-hydroxydiphenyl reagent addition was subtracted 749

from the optical density measured after addition of the dye The concentration of uronic 750

acid was calculated using known amounts of GalA as a standard 751

752

Release of Pectin from Arabidopsis Cell Walls Using Macerozyme 753

AIR preparations from leaves of four week-old plants were treated with 025 754

(wv) macerozyme R-10 (Yakult) or water (mock) at a concentration of 100 microL per mg 755

AIR Samples were incubated for 3 h at room temperature on a vortex shaker and 756

centrifuged for 10 min at 12000 g Total uronic acid concentration of 5-fold diluted 757

supernatants was determined for two technical replicates each The concentration of 758

uronic acid was calculated using known amounts of GalA as a standard For 759

subsequent qRT-PCR experiments supernatants from multiple extractions were 760

combined boiled for 20 min to inactivate macerozyme diluted 10-fold with water and 761

inoculated into four week-old Col-0 plants Heat-inactivated 10-fold diluted macerozyme 762

solutions were used as mock treatment Samples were collected 3 h later and PAD3 763

expression was determined 764

765

Release of Pectin from Arabidopsis Leaves Infected with B cinerea 766

Fully expanded leaves of four week-old plants were either inoculated with up to 767

five 10 microL droplets of B cinerea spore solution (25 x 105 spores mL-1) or mock 768

inoculated with B cinerea inoculation medium For each biological replicate leaves with 769

a combined count of at least 20 infection sites were collected 48 h later AIR was 770

extracted as described above but plants were not dark-treated or freeze-dried and the 771

first ethanol step was performed using 85 (vv) ethanol To extract soluble pectin 772

fragments AIR samples were mixed with water at a concentration of 20 microL per infection 773

site incubated for 7 h at room temperature on a vortex shaker and centrifuged for 10 774

Page 37 of 54

min at 12000 g Total uronic acid concentration of supernatants was determined for two 775

technical replicates each The concentration of uronic acid was calculated using known 776

amounts of GalA as a standard 777

778

Measurement of Uronic Acids Neutral Monosaccharides and Cellulose Content in 779

Cell Walls 780

Neutral sugars and uronic acids from non-cellulosic polysaccharides in cell walls 781

(AIR) were released by trifluoroacetic acid (TFA) hydrolysis Approximately 1 mg of AIR 782

was hydrolyzed with 2 M TFA at 121degC for 90 min The TFA-soluble material was split 783

into two equal parts one for uronic acid and one for neutral sugar determination The 784

uronic acid content was determined by analyzing the hydrolyzate using a CarboPac 785

PA200 anion-exchange column with an ICS-3000 Dionex chromatography system 786

(Dionex Sunnyvale CA USA) The elution profile consisted of a linear gradient of 50 to 787

200 mM sodium acetate in 01 M NaOH in 10 min at 04 mL per min Alditol acetate 788

derivatives of neutral sugars were produced as described (York et al 1985) In brief 789

monosaccharides in the hydrolyzate were reduced with NaBH4 and peracetylated with 790

acetic anhydride and pyridine at 121degC for 20 min The generated alditol acetates were 791

analyzed by GC-MS Crystalline cellulose was measured according to Updegraff (1969) 792

After hydrolysis of noncellulosic polysaccharides from AIR with the Updegraff reagent 793

(acetic acid nitric acids water 812 (vv) ) the remaining pellet was hydrolyzed in 794

72 sulfuric acid The resulting glucose quantity was determined by the anthrone 795

method (Scott and Melvin 1953) 796

797

Dot Blot Experiments 798

Pectin solutions extracted using 50 microL of cell wall extraction buffer per mg AIR 799

were serially diluted and nitrocellulose membranes were spotted with 1 microL of the diluted 800

pectin solutions Membranes were dried overnight blocked with 5 milk (wv) in 1x 801

PBS (8 g L-1 NaCl 02 g L-1 KCl 144 g L-1 Na2HPO4 024 g L-1 KH2PO4 pH 74) and 802

probed with LM5 (Jones et al 1997) LM6 (Willats et al 1998) LM8 (Willats et al 803

2004) LM19 and LM20 (Verhertbruggen et al 2009a) or CCRC-M7 (Steffan et al 804

Page 38 of 54

1995) antibodies LM series antibodies were diluted 1250 and CCRC-M7 was diluted 805

1500 in 5 milk powder (Nestle Carnation) in 1 x PBS For LM series antibodies a 806

goat anti-Rat HRP conjugated antibody (Bethyl A110-105P 15000 diluted) and for 807

CCRC-M7 an anti-Mouse IgG HRP conjugate (Promega W402B 12500 diluted) was 808

used as secondary antibody in 5 milk powder in 1x PBS LM series antibodies were 809

obtained from plant probes (wwwplantprobesnet) and CCRC-M7 from CarboSource 810

(wwwcarbosourcenet) Membranes were washed after incubation with the primary and 811

secondary antibodies with 1x PBS Dot blots were developed using the ECL system 812

(GE Healthcare Amersham ECL prime) For quantification of signals dot blot results on 813

x-ray films were photographed using a CCD camera and intensities were measured814

using Image Jrsquos Integrated Density function Locally adjusted background was 815

subtracted As the dynamic range of the signal on an x-ray film was narrow non-816

linearity in the measured values was corrected for each genotype in each biological 817

replicate using the values from different dilutions in two technical replicates We noticed 818

that the residuals were not normally distributed and performed Box-Cox power 819

transformation (Box and Cox 1964) to obtain normally distributed values for the 820

subsequent statistical analysis of the data For LM19 Box-Cox selected power 821

transformation was done to the power of 038th and for LM20 to the power 075th After 822

correction of the transformed measured values with the effect of the biological 823

replicates the values were fit to a linear model with the Arabidopsis genotype as the 824

fixed effect This linear model was used both to generate the plots in Figures 5B and D 825

and to test significant differences 826

827

Camalexin Measurements 828

Four week-old plants were infected with B cinerea and samples (three leaf disks 829

from one plant each) were harvested at the indicated time points flash frozen and 830

pulverized Each sample was extracted with 300 microL of 90 methanol (vv) 50 microL of the 831

extract was run on an Agilent Lichrocart 250-4 RP18e 5-microm column using an Agilent 832

1100 series HPLC Camalexin was detected using a DiodeArray (DAD) at 330 nm and 833

with a fluorescence detector at emission 318 nmexcitation 385 nm (Agilent) Separation 834

was achieved using the following program with aqueous acetonitrile 5 min gradient 835

Page 39 of 54

from 63 to 69 acetonitrile 30 s gradient from 69 to 99 acetonitrile 2 min at 99 836

acetonitrile and a post-run equilibration of 35 min at 63 acetonitrile (Denby et al 837

2004 Kliebenstein et al 2005) Purified camalexin was used to produce a standard 838

curve to identify and quantitate camalexin 839

840

Accession Numbers 841

Microarray data used can be accessed at NCBI Gene Expression Omnnibus 842

(httpwwwncbinlmnihgovgeo) GSE18978 Sequence information for all Arabidopsis 843

genes described in this article can be found in TAIR (wwwarabidopsisorg) using the 844

following accession numbers PAD3 (CYP71B15 At3g26830) SID2 (ICS1 At1g74710) 845

GAE1 (At4g30440) GAE2 (At1g02000) GAE3 (At4g00110) GAE4 (At2g45310) GAE5 846

(At4g12250) GAE6 (At3g23820) PR1 (At2g14610) JAZ5 (At1g17380) JAZ10 847

(At5g13220) ACTIN2 (At3g18780) PDF12a (At5g44420) PDF12b (At2g26020) 848

AtSK11 (At5g26751) The sequence of the B cinerea cutinase Bc-CutA (Z69264) can 849

be found in the DNA Data Bank of Japan (httpgetentryddbjnigacjp) Germplasm 850

used included dde2-2 (At5g42650 (von Malek et al 2002)) pad3-1 (At3g26830 (Zhou 851

et al 1999)) pad4-1 (At3g52430 (Jirage et al 1999)) sid2-2 (At1g74710 (Wildermuth 852

et al 2001)) pbs3-2 (At5g13320 SALK_018225 (Nobuta et al 2007)) cbp60a-1 853

(At5g62570 SALK_124410 (Truman et al 2013)) cbp60g-1 sard1-2 (At5g26920 854

SALK_023199 At1g73805 SALK_052422 (Wang et al 2011) cbp60a-1 cbp60g-1 855

sard1-2 (Truman et al 2013) gae1-1 (At4g30440 SALK_085554) gae6-1 (At3g23280 856

SALK_104454) gae6-2 (At3g23280 SALK_017191) ixr1-1 cev1 (At5g05170 (Ellis 857

and Turner 2001 Scheible et al 2001)) eds1 plants were derived by introgression of 858

the Ler eds1-2 allele into Col-0 that contains two copies of EDS1 (At3g48090 859

At3g48080 (Bartsch et al 2006) mpk6-2 (At2g43790 SALK_073907 (Liu and Zhang 860

2004)) mpk3-DG (At3g45640 a fast neutron deletion mutant (Miles et al 2005)) and 861

npr1-1 (At1g64280 (Cao et al 1997)) T-DNA insertion lines were part of the SALK 862

collection (Alonso et al 2003) and were obtained from the Arabidopsis Biological 863

Resource Center 864

865

Supplemental Data 866

Page 40 of 54

Supplemental Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 867

Expression Does Not Require CBP60s or SID2 868

Supplemental Figure 2 Expression of GAE Family Members uponTreatment with Pma 869

ES4326 870

Supplemental Figure 3 Characterization of T-DNA Insertions in GAE1 and GAE6 871

Supplemental Figure 4 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves and 872

Are Slightly Smaller than Wild-Type Col-0 Plants 873

Supplemental Figure 5 Cell Walls of gae1 gae6 Plants Contain Less Uronic Acid than 874

Wild-Type Col-0 Cell Walls 875

Supplemental Figure 6 Antibodies Raised Against the RGI or XG Components of 876

Pectin Show No Difference in Binding to gae Mutant and Col-0 Cell Walls 877

Supplemental Figure 7 The Effect of B cinerea Treatment on the Expression of GAE 878

Family Genes 879

Supplemental Figure 8 Treatment with 001 Macerozyme Does Not Cause Any 880

Visible Damage to Col-0 Plants 881

Supplemental Figure 9 B cinerea Growth on Plants with Defects in Immune 882

Signaling 883

Supplemental Figure 10 MeJA-Induced Expression of the JA marker gene JAZ10 884

Supplemental Figure 11 The Expression of the JA Marker Gene JAZ5 Is Highly 885

Responsive in gae1 gae6 Plants 886

Supplemental Figure 12 The Expression of PDF12 Is Unaltered in gae1 gae6 Plants 887

Supplemental Figure 13 Macerozyme Treatment Activates MPK3 and MPK6 888

Supplemental Figure 14 B cinerea-Induced ROS Production in Wild-Type Col-0 and 889

gae1 gae6 Plants 890

Supplemental Figure 15 OG-Induced PTI Using 250 microg mL-1 OGs 891

Supplemental Figure 16 Abundance of Plant DNA in B cinerea Treated Plants 892

Supplemental Table 1 Primers used in this study 893

894

ACKNOWLEDGEMENTS 895

We thank William Truman for helpful discussions Simone Ferrari (Sapienza 896

Universita di Roma) for the generous gift of OGs Bruna Bucciarelli (USDA-ARS) for 897

Page 41 of 54

help with fluorescence microscopy the Arabidopsis Biological Resource Center for T-898

DNA insertion lines Paul Knoxrsquos lab (University of Leeds) for LM5 LM6 LM8 LM19 899

and LM20 antibodies and the Complex Carbohydrate Resource Center (University of 900

Georgia) for the CCRC-M7 antibody Most of this work was funded by the Division of 901

Chemical Sciences Geosciences and Biosciences Office of Basic Energy Sciences of 902

the US Department of Energy through Grant DE-FG02-05ER15670 to JG This work 903

was also supported by NSF awards IOS 1339125 and IOS 1021861 to DJK NSF 904

award IOS 1121425 to F K and the USDA National Institute of Food and Agriculture 905

Hatch project number CA-D-PLS-7033-H to DJK 906

907

AUTHOR CONTRIBUTIONS 908

GB and JG designed the research GB GX AT BL NES and NH 909

performed research DJK provided tools GB GX MP AD BL RAH and FK 910

analyzed data GB and JG wrote the paper with input from all authors 911

912

FIGURE LEGENDS 913

Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 Expression 914

Requires PAD4 EDS1 and PBS3 915

(A) Expression of GAE1 in leaves of wild-type Col-0 and mutant plants Expression was916

measured 24 or 32 h after either inoculation with Pma ES4326 (OD600 = 0002) or mock 917

inoculation with 5 mM MgSO4 and was then normalized to the level of ACTIN2 Data 918

from three biological replicates were combined using a mixed linear model Bars 919

represent mean log2-ratios to ACTIN2 plusmn SE Letters indicate significantly different 920

groups with q lt 005 where 005 represents the maximum false discovery rate at which 921

the test may be called significant 922

(B) Expression of GAE6 measured as described in (A)923

924

Figure 2 Susceptibility of gae6 and gae1 gae6 Plants to Pma ES4326 Is Increased 925

Relative to Wild-Type Col-0 926

Leaves of plants of the indicated genotypes were inoculated with Pma ES4326 (OD600 = 927

00002) as described in Methods Bacterial titers in leaves were determined either 928

Page 42 of 54

immediately (0 dpi) or after three days (3 dpi) Bars represent the mean plusmn SE of four 929

independent experiments each with 4 or 12 biological replicates at 0 and 3 dpi 930

respectively Data were combined using a mixed linear model Asterisks indicate 931

significant differences from Col-0 wild-type (q lt 005) Susceptible pad4 plants were 932

included as a positive control 933

934

Figure 3 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves 935

(A) Depiction of the assay for brittleness of gae mutant leaves Fully expanded leaves of936

four to five week-old plants were bent by 180ordm so that the adaxial surface of the leaf was 937

parallel with the soil Broken leaf petioles or midribs were considered brittle 938

(B) Quantification of broken midribs or petioles For each genotype 66 to 82 leaves 939

were analyzed for brittleness as described in (A) Bars represent the ratio of broken to 940

total number of leaves tested Results were analyzed using Fisherrsquos exact test Letters 941

indicate significantly different groups at p lt 005 942

(C) Conductivity measurements Leaves of four week-old plants were inoculated with943

deionized water and then leaf discs were cut out and placed on deionized water 944

Conductivity was measured at the indicated time points After 30 min the water was 945

replaced with fresh water (30 min f w) to account for wounding-related ion leakage from 946

the edges of the leaf discs Three independent experiments with six biological replicates 947

each were performed and data were analyzed using a t-test for each time point P-948

values were corrected using the Bonferroni method Data show means plusmn SE asterisks 949

indicate significant differences from wild-type Col-0 at q lt 001 950

951

Figure 4 Cell Walls of gae6 and gae1 gae6 Plants Are Reduced in GalA 952

(A) to (D) Levels of GalA (A) GlcA (B) cellulose (C) and neutral sugars (D) in alcohol953

insoluble residue (AIR) extracted from leaves of four week-old plants of the indicated 954

genotypes Bars represent means plusmn SE of four biological replicates combined using a 955

mixed linear model Letters indicate significantly different groups (p lt 005) 956

957

Figure 5 Cell Walls of gae1 gae6 Plants Are Reduced in Homogalacturonan 958

Page 43 of 54

Dot blot analyses with anti-LM19 and anti-LM20 antibodies Pectin was extracted from 959

AIR prepared from the indicated genotypes using 1 microL of extraction buffer per 10 microg of 960

AIR Pectin was serially diluted and one microL of undiluted 13 diluted and 19 diluted 961

samples were spotted on a nitrocellulose membrane Three biological replicates were 962

were spotted twice to obtain two technical replicates each All replicates produced 963

similar results 964

(A) Representative dot blot using anti-LM19 antibody965

(B) Relative signal intensities of the spots in the dot blot in (A)966

(C) Representative dot blot using anti-LM20 antibody967

(D) Relative signal intensities of the LM20 dot blot shown in (C)968

Relative signal intensities were measured using Image J software Measurements from 969

the three biological replicates were combined using a linear model after Box-Cox power 970

transformation as described in Methods Bars represent means plusmn SE Asterisks indicate 971

a difference from wild-type Col-0 at p lt 005 972

973

Figure 6 Growth of B cinerea on gae1 gae6 Plants and the Effect of B cinerea on 974

Expression of GAE1 and GAE6 975

(A) Growth of B cinerea isolates Leaves of Col-0 and gae1 gae6 plants were976

inoculated with 25 x 105 spores mL-1 of the B cinerea isolates indicated in the figure 977

Samples were collected two (2 dpi) and three (3 dpi) days later Bars represent means plusmn 978

SE of six biological replicates for each fungus combined using a mixed linear model 979

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 980

(B) and (C) Expression of GAE1 (B) and GAE6 (C) 48 h after inoculation with the B981

cinerea isolates indicated Expression levels were measured by qRT-PCR Bars 982

represent mean log2-ratios to ACTIN2 plusmn SE of three biological replicates combined 983

using a mixed linear model Letters indicate significantly different groups (q lt 005) 984

985

Figure 7 Levels of Soluble Pectin and Expression of PAD3 in Fractions Produced 986

Following Macerozyme Treatment of gae1 gae6 Cell Walls 987

(A) Schematic overview of the experimental procedure for (B) and (C)988

Page 44 of 54

(B) Uronic acid measurements Alcohol-insoluble residue (AIR) from leave tissue of four989

week-old wild-type Col-0 or gae1 gae6 plants was treated with water or 025 990

macerozyme and assayed for uronic acid levels Data from 11 biological replicates were 991

combined using a mixed linear model Bars represent means plusmn SE Letters indicate 992

significant differences (p lt 005) 993

(C) Expression of PAD3 in Col-0 plants treated with the fractions from (B) Extracts from994

(B) were heat inactivated pooled and diluted 5-fold with water Pooled extracts were995

inoculated into four week-old Col-0 plants PAD3 expression was measured 3 h after 996

inoculation and normalized to the level of ACTIN2 expression Data from four biological 997

replicates were combined using a mixed linear model Bars represent mean log2-ratios 998

to ACTIN2 plusmn SE Letters indicate significant differences (p lt 005) 999

1000

Figure 8 Macerozyme-Induced PAD3 Expression Is Absent in gae1 gae6 1001

(A) Expression of PAD3 after macerozyme treatment Four week-old plants were either1002

inoculated with 001 macerozyme or mock inoculated with water and PAD3 1003

expression was measured 3 h later by qRT-PCR Data from four biological replicates 1004

were combined using a mixed linear model Mean log2-ratios to ACTIN2 plusmn SE were 1005

plotted Letters indicate significantly different groups (p lt 005) 1006

(B) Expression of PAD3 after OG treatment PAD3 expression was measured as1007

described in (A) 3 h after inoculation with 100 microg mL-1 OGs or mock inoculation with 1008

water Data are from six biological replicates 1009

(C) PAD3 expression in untreated plants Three biological replicates were performed1010

and data analyzed as described in (A) 1011

(D) Camalexin accumulation after B cinerea treatment Camalexin accumulation was1012

measured in four week-old plants inoculated with the B cinerea strains shown in the 1013

figure Eight biological replicates each were combined using a mixed linear model 1014

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 1015

(E) PAD3 expression 28 h after treatment with the indicated B cinerea strains Data1016

from six biological replicates were collected and analyzed as described in (A) Data in 1017

all figure parts represent means plusmn SE Letters indicate significantly different groups (q lt 1018

005) 1019

Page 45 of 54

1020

Figure 9 Macerozyme-Induced Expression of the SA Marker Genes SID2 and PR1 1021

Is Abolished in gae1 gae6 Plants 1022

(A) to (H) Expression of PR1 (A) (C) (E) and (G) or SID2 (B) (D) (F) and (H) in four1023

week-old Col-0 and gae1 gae6 plants Data were combined using a mixed linear model 1024

Bars represent mean log2-ratios of expression vs ACTIN2 plusmn SE for all figure parts 1025

Letters indicate significantly different groups at p lt 005 for panels (A) to (D) (G) and 1026

(H) and qlt005 for panels (E) to (F) 1027

(A) and (B) Plants were treated with 001 macerozyme or mock treated with water1028

and samples were collected 3 h later Data from four biological replicates are shown 1029

(C) and (D) Plants were inoculated with 100 microg mL-1 OGs or mock treated with water1030

and samples collected 3 h later Data from six biological replicates are shown 1031

(E) and (F) Plants were inoculated with the indicated B cinerea isolates or mock1032

inoculated with B cinerea inoculation medium and samples collected 48 h later Data 1033

from eight biological replicates are shown 1034

(G) and (H) Expression in untreated plants Three biological replicates were performed1035

1036

Figure 10 The Expression of the JA Marker Gene JAZ10 is Highly Responsive in 1037

gae1 gae6 Plants 1038

(A) to (D) JAZ10 expression in four week-old plants Data were combined using a mixed1039

linear model Mean log2-ratios to ACTIN2 plusmn SE are shown Letters indicate significantly 1040

different groups at p lt 005 for panels (A) (B) and (D) and q lt 005 for panel (C) 1041

(A) Plants were inoculated with 001 macerozyme or mock inoculated using water and1042

samples were collected 3 h later Data from four biological replicates are shown 1043

(B) Plants were inoculated with 100 microg mL-1 OGs or mock inoculated using water and1044

samples collected 3 h later Data are from six biological replicates 1045

(C) JAZ10 expression 48 h after treatment with the indicated B cinerea strains Data1046

are from six biological replicates 1047

(D) JAZ10 expression in untreated plants Data are from three biological replicates1048

1049

Figure 11 Gallo 1-Induced Callose Deposition Is Reduced in gae1 gae6 Plants 1050

Page 46 of 54

(A) Callose deposition in four week-old Col-0 and gae1 gae6 plants Plants were1051

inoculated with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) or mock inoculated 1052

with B cinerea inoculation medium and samples were collected after 72 h Callose was 1053

stained with aniline blue and visualized using a Nikon Eclipse Ni-U microscope with a 1054

DAPI filter Three leaves were stained per treatment Scale bars are 100 microm Asterisks 1055

indicate fungal hyphae arrows indicate representative callose spots 1056

(B) Quantification of callose deposition Callose deposits were counted in 200 microm x 2001057

microm squares laid out across the images shown here Bars represent means plusmn SE from 1058

six squares each Letters indicate significantly different groups (p lt 005) according to a 1059

t-test analysis1060

1061

Figure 12 Less Soluble Pectin Accumulates in B cinerea-Infected gae1 gae6 1062

Leaves 1063

Four week-old Col-0 and gae1 gae6 plants were inoculated with 10 microL droplets of the 1064

indicated B cinerea isolate (25 x 105 spores mL-1) or mock inoculated with B cinerea 1065

inoculation medium Leaves were collected 48 h later AIR was extracted from leaf 1066

samples and water added (20 microL infection site) to extract soluble pectin fragments 1067

Samples were vortexed for 7 h centrifuged at 12000 g and the uronic acid 1068

concentration of the supernatant was measured Data from nine biological replicates 1069

were combined using a mixed linear model Means plusmn SE are shown Letters indicate 1070

significant differences (q lt 005) 1071

1072

Figure 13 Macerozyme-Induced PTI Is Lost in gae1 gae6 While OG-Induced PTI Is 1073

Unaltered 1074

(A) Macerozyme-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1075

inoculated with 001 macerozyme or mock inoculated with boiled macerozyme and 1076

were infected with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) 24 h later Samples 1077

were collected immediately (0 dpi) and after two (2 dpi) and three (3 dpi) days Data 1078

show mean plusmn SE of six to 14 biological replicates each combined using a linear model 1079

Letters indicate significantly different groups (q lt 005) 1080

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

Parsed CitationsAdam L and Somerville SC (1996) Genetic characterization of five powdery mildew disease resistance loci in Arabidopsisthaliana Plant J 9 341-356

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Cao H Glazebrook J Clarke JD Volko S and Dong XN (1997) The Arabidopsis NPR1 gene that controls systemic acquiredresistance encodes a novel protein containing ankyrin repeats Cell 88 57-63

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Page 28 of 54

expression Interestingly expression of the JA marker genes JAZ5 and JAZ10 was 519

higher in gae1 gae6 than in wild-type mock-inoculated plants but was comparable to 520

wild-type in untreated plants suggesting that JA signaling in gae1 gae6 is 521

hyperresponsive (Figure 10 and Supplemental Figure 11) This induction of JA signaling 522

might be due to wounding stress incurred during the inoculation process JA and SA 523

signaling generally act antagonistically (Pieterse et al 2012) Increased JA signaling 524

activity might hence explain why mock- and macerozyme-treated gae1 gae6 plants 525

exhibited reduced expression of the SA marker PR1 and why OG treatment induced 526

expression of SID2 another SA marker to a lower extent in gae1 gae6 than in wild-type 527

plants (Figure 9) 528

No single immune signaling component was found to be the sole contributor to 529

the compromised immunity of gae1 gae6 to specific B cinerea isolates such as Gallo 1 530

and Fresa 525 These isolates grew better on pad3 while growth of Pepper another B 531

cinerea isolate that did not show altered virulence on gae1 gae6 was unaltered 532

(Supplemental Figure 9) This suggested that differences in camalexin sensitivity of 533

these B cinerea isolates might account for the differences in gae1 gae6 susceptibility 534

However camalexin accumulation and PAD3 expression was the same for wild-type 535

Col-0 and gae1 gae6 for the B cinerea isolates Fresa 525 and Gallo 1 (Figure 8) which 536

showed enhanced growth on gae1 gae6 plants It should be noted that B cinerea 537

virulence is often estimated by comparing lesion sizes Lesion size and measurements 538

of relative fungal growth by qPCR do not always show the same results For example 539

although Denby et al (2004) showed that Pepper causes larger lesions on pad3 than on 540

wild-type this study showed that relative fungal growth of Pepper on pad3 was 541

unaltered 542

We observed that callose deposition was strongly reduced in gae1 gae6 plants 543

72 h after treatment with B cinerea isolate Gallo 1 (Figure 11) possibly due to reduced 544

release of active soluble pectin Interestingly OG-induced PTI to an unspecified isolate 545

of B cinerea was intact in the pmr4 mutant which does not produce callose (Galletti et 546

al 2008) Due to the high SA levels in pmr4 plants (Nishimura et al 2003) and the fact 547

that different B cinerea isolates vary both in induction of and reaction to defense 548

responses (Kliebenstein et al 2005 Rowe et al 2010) we could not determine 549

Page 29 of 54

whether reduced callose deposition causes the increased susceptibility of gae1 gae6 to 550

B cinerea isolate Gallo 1551

We did not observe any changes in B cinerea growth either on cev1 ixr1 plants 552

which are known to have constitutively high JA and OPDA levels (Ellis et al 2002b) or 553

on plants exogenously treated with MeJA (Supplemental Figure 9) Because plants 554

were kept uncovered for 3 h after MeJA treatment it is unclear how much MeJA was 555

bioavailable in the plant However JAZ10 expression was induced 3 h after spraying 556

with MeJA (Supplemental Figure 10) Because pathogen growth generally depends on 557

the combination of a multitude of immune signaling events we conclude that multiple 558

changes to the immune signaling in gae1 gae6 together account for the altered B 559

cinerea growth phenotypes detected 560

We showed that expression of GAE1 and GAE6 is repressed by the bacterial 561

hemibiotroph Pma ES4326 This repression did not occur in an eds1 or pad4 562

background or in pbs3 at the 24-h time point (Figure 1 and Supplemental Figure 1) 563

During the Arabidopsis - Pma ES4326 interaction the expression of many defense 564

related genes is altered in a PAD4EDS1 dependent fashion (Wang et al 2008) Some 565

of these expression changes require SA CBP60g and SARD1 are thought to act 566

downstream of PAD4 and EDS1 but upstream of SA-dependent responses (Wang et 567

al 2011) PBS3 is also thought to act downstream of PAD4 and EDS1 and upstream of 568

SA but the set of genes whose expression it affects is only partially overlapping with 569

those affected by CBP60gSARD1 (Wang et al 2008 Wang et al 2011) Thus GAE1 570

and GAE6 repression is regulated by the part of the immune signaling network that 571

requires PAD4 EDS1 and PBS3 but is independent of CBP60gSARD1 and SA 572

Because EDS1 PAD4 and PBS3 are important components of the plant immune 573

signaling network (Wiermer et al 2005 Nobuta et al 2007) one might conclude that 574

repression of GAE1 and GAE6 may be part of the plant immune response to Pma 575

ES4326 B cinerea treatment also reduced GAE1 and GAE6 expression but this was 576

mostly independent of PAD4 (Figure 6) while both Pma ES4326 and B cinerea repress 577

GAE1 and GAE6 expression the host signaling mechanisms responsible likely differ 578

In summary our data show that Arabidopsis GAE1 and GAE6 are required for 579

pectin biosynthesis leaf flexibility and immunity to Pma ES4326 and to specific B 580

Page 30 of 54

cinerea isolates We used macerozyme a commercial pectinase to mimic the effect of 581

B cinerea pectinases Macerozyme treatment released less pectin from gae1 gae6 cell582

walls and induced less immune signaling in gae1 gae6 plants Less water soluble pectin 583

also accumulated in B cinerea infected gae1 gae6 plants and macerozyme-induced 584

immunity to B cinerea isolate Gallo 1 was reduced in these plants We conclude that 585

the reduced pectin content in these mutants may lead to reduced release of soluble 586

pectin including active OGs during pathogen attack and that this compromises 587

immunity 588

589

590

Page 31 of 54

METHODS 591

Plant Materials and Growth Conditions 592

Wild-type Columbia (Col-0) and all mutant plants (in Col-0 background) were 593

grown on sterilized BM2 germinating mix (Berger Inc Quebec Canada) in a controlled 594

environment chamber (Conviron Pembina ND USA) with a 12-h photoperiod under 595

100 microM m-2 s-1 fluorescent illumination at 22degC and 75 relative humidity Germplasm 596

used is described in the Accession Numbers section at the end of Methods 597

598

Data Analysis and Replicates 599

For biological replicates samples were obtained from separate plant tissues and 600

in many cases from plants grown in different flats In some experiments multiple 601

technical replicates were measured per biological replicate For statistical analysis all 602

technical replicates were averaged and the averages were considered one biological 603

replicate Independent experiments were performed at different times and often 604

consisted of multiple biological replicates per experiment 605

Data analysis was performed using mixed linear models in the R programming 606

environment (Bates et al 2015) unless otherwise indicated Generally genotypes 607

treatments and time points were used as fixed effects and replicate-specific effects like 608

different pots flats etc were considered random effects We reported p-values 609

obtained from the mixed linear model when less than 10 comparisons were performed 610

and q-values for more than 10 comparisons to correct for multiple comparison testing 611

Q-values were calculated using the Benjamini-Hochberg procedure (Benjamini and612

Hochberg 1995) unless specified otherwise 613

614

Pathogen Strains Growth Conditions and Pathogen Growth Assays 615

Growth of Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) and 616

bacterial growth assays were performed as previously described (Bethke et al 2014) 617

For qRT-PCR assays four week-old plants were inoculated with bacteria (OD600 = 618

0002) or mock (5 mM MgSO4) At least four leaves from two plants were collected at 619

the indicated time points per biological replicate 620

Page 32 of 54

Prior to each experiment Botrytis cinerea isolates were grown on 1x PDA 621

medium (Difco) for 10 d at room temperature All isolates used have been described 622

previously (Rowe and Kliebenstein 2007) The spores were washed from the surface of 623

the plate using an inoculation medium (1x Gamborgrsquos B-5 basal salt mixture (Sigma-624

Aldrich G5768) 2 (wv) glucose 10 mM phosphate buffer pH 64) and fungal hyphae 625

were removed from the suspension by filtering through four layers of cheesecloth The 626

concentration of spores was determined using a hemocytometer and adjusted to 25 x 627

105 spores mL-1 Four week-old Arabidopsis plants were inoculated by placing one 10-628

microL droplet of the B cinerea spore solution or inoculation medium (mock) on the adaxial 629

leaf surface of fully expanded leaves Inoculated plants were kept at 100 relative 630

humidity Infection sites were collected at various time points using a cork borer For 631

qRT-PCR assays at least four leaf discs from one plant were combined for each 632

biological sample For B cinerea growth assays at least 12 leaf discs from three to six 633

plants were pooled per biological sample Relative fungal growth was determined by 634

measuring the abundance of a fungal gene relative to a plant gene as described below 635

In brief DNA from infected tissue was extracted and equal amounts of total DNA were 636

used to perform qPCR reactions using the B cinerea cutinase A and the Arabidopsis 637

SK11 gene respectively Primers were described in (Gachon and Saindrenan 2004) 638

and are listed in Supplemental Table 1 Two technical replicates were combined for 639

each biological replicate B cinerea is a necrotrophic fungal pathogen and may destroy 640

plant DNA as disease progresses However decreases in the amount of plant DNA 641

over the course of our experiments were only observed at late time points with very 642

susceptible plants so the qPCR assay is generally a reasonable estimate of fungal 643

biomass (Supplemental Figure 16) 644

645

Treatment with Macerozyme or OGs 646

For PTI assays four week-old plants either were inoculated with 001 (wv) 647

macerozyme R-10 (Yakult) a Rhizopus polygalacturonase (EC 32115) that possesses 648

high pectinase and hemicellulase activity or were mock inoculated using heat-649

inactivated macerozyme Other similar plants were either inoculated with 250 or 500 microg 650

mL-1 OGs or mock inoculated with water One d later plants were infected with B 651

Page 33 of 54

cinerea and samples were collected immediately (0 dpi) and two or three d later To 652

investigate the role of MeJA on B cinerea growth plants were sprayed with 1mM MeJA 653

in water with 001 (vv) Tween 20 Covers were removed so that leaves could dry off 654

before plants were inoculated with B cinerea three h later Samples were harvested at 655

the indicated time points For expression analysis and MAPK activation assays plants 656

were either inoculated with 100 microg mL-1 OGs or mock inoculated with water OGs used 657

for experiments described in Figures 8 to 10 and Supplemental Figure 12 were a kind 658

gift from Simone Ferrari OGs used in experiments described in Figure 13 and 659

Supplemental Figures 11 13 and 15 were prepared as previously described (Kohorn et 660

al 2014) 661

662

Expression Analysis 663

Leaf tissue from inoculated plants was harvested at the indicated time points 664

flash frozen pulverized and RNA extracted using Trizol (Invitrogen) Quantitative RT-665

PCR was performed using the SuperScript III Platinum SYBR Green One-Step 666

quantitative RT-PCR kit (Invitrogen) and a Lightcycler 480 Real-Time PCR system 667

(Roche) as previously described (Truman and Glazebrook 2012) In brief equal 668

amounts of total RNA and a gene specific primer were used for each reaction The 669

crossing point (Cp) was calculated using the 2nd derivative max method provided with 670

the Lightcycler software for each amplification curve Each reaction was run with two 671

technical replicates and the Cp values for these replicates were averaged ACTIN2 was 672

used as a stably expressed reference gene At least three biological replicates each 673

were performed and analyzed using a mixed-linear model RT-PCR was performed to 674

verify T-DNA insertion lines using the One-Step RT-PCR kit (Qiagen) according to the 675

manufacturerrsquos instructions The primers used can be found in Supplemental Table 1 676

677

Electrolyte Leakage Assay 678

Fully expanded leaves of four week-old plants were inoculated with water (Milli-Q 679

grade) using a needleless syringe Leaf discs were collected using a cork borer and 680

three leaf discs from one leaf were placed with the adaxial surface down onto 2 mL 681

water in a 12-well cell culture plate Conductivity was measured using a Horiba B-173 682

Page 34 of 54

conductivity meter after 30 min and then the water was replaced Conductivity was 683

measured again 30 min later (1 h total) as indicated in Figure 3C Six biological 684

replicates each were performed in three independent experiments and means and SEs 685

were calculated Two-sided t-tests were performed for each time point 686

687

DAB and Aniline Blue Staining 688

Leaves from four week-old plants were either mock inoculated with B cinerea 689

inoculation medium or inoculated with 10 microL droplets of a B cinerea spore solution (25 690

x 105 spores mL-1) of the indicated isolates Samples were collected 72 h later For 33prime-691

diaminobenzidine (DAB) staining two to five inoculated leaves were vacuum infiltrated 692

with 33prime-Diaminobenzidine (1 mg mL-1 in water pH 38) solution and stained overnight 693

in the dark Leaves were destained using 100 ethanol at 65degC and then transferred to 694

50 (vv) glycerol and photographed 695

Callose was visualized by staining leaves with aniline blue as previously 696

described (Adam and Somerville 1996) Briefly leaves were vacuum infiltrated with 697

alcoholic lactophenol (one volume of phenolglycerollactic acidwater (1111) mixed 698

with two volumes of ethanol) and destained at 65degC Next leaves were moved through 699

an alcohol gradient (50 20 and 10 (vv) ethanol) into water The destained leaves 700

were stained for 30 min in 150 mM K2HPO4 (pH 95) with 001 (wv) aniline blue 701

Samples were mounted in water and examined using a Nikon Eclipse Ni-U microscope 702

at 10x magnification using a DAPI filter (excitation at 325-375 nm emission at 435-485 703

nm) For callose quantification six 200 microm x 200 microm squares were counted for each 704

sample type Callose deposits per mm2 were calculated and data analyzed using a t-705

test Note that there is an inherent uncertainty associated with such counts because 706

callose deposits may not all be in focus at the same time 707

708

MAPK Activity Assay 709

Fully expanded leaves of four week-old plants were inoculated with 001 710

macerozyme 100 microg mL-1 OGs 100 nM flg22 or water (mock) flg22 a peptide from 711

bacterial flagella that induces immune responses was used as a positive control for 712

Page 35 of 54

MAPK activation (Asai et al 2002) and was purchased from EZBiolab Inc (Westfield 713

IN USA) Samples were harvested 10 min later and immediately frozen in liquid 714

nitrogen Protein was extracted using an extraction buffer containing 25 mM Tris pH 78 715

75 mM NaCl 1 mM DTT 1 mM NaF 05mM Na3VO4 01 (vv) Tween 20 one 716

cOmplete Mini protease inhibitor cocktail tablet (Roche) and one PhosSTOP 717

phosphatase inhibitor cocktail tablet (Roche) per 10 mL buffer 15 microg of total protein 718

were electrophoresed on a 10 SDS-polyacrylamide gel and then blotted onto a PVDF 719

membrane (Bio-Rad 162-0177) Activated MAPKs were detected using p4442 MAPK 720

(Erk12) antibody (9102S Cell Signal 12500 in TBST (20 mM Tris pH 75 150 mM 721

NaCl 01 (vv) Tween 20)) Anti-Rabbit IgG-HRP (115000 in TBST A6154 Sigma) 722

and ECL Plus substrate (Pierce 32132) MPK3 and MPK6 were detected with an anti-723

MPK3 antibody (M8318 Sigma 12000 in TBST and 3 (wv) milk) or an anti-MPK6 724

antibody (A7104 Sigma 16000 in TBST and 3 (wv) milk) respectively The 725

secondary antibody and detection were the same as for the p4442 antibody Five 726

independent experiments were performed 727

728

Preparation of Alcohol Insoluble Residue (AIR) Pectin Extraction and Uronic Acid 729

Measurements 730

Fully expanded leaves from four week-old plants that were incubated for 48 h in 731

the dark to reduce starch were harvested flash frozen and pulverized AIR was 732

extracted by washing ground material twice in 70 (vv) ethanol three times in a 733

mixture of chloroform and methanol (11 vv) and once in acetone (Gille et al 2009) 734

and was then air dried Cell wall pectin was extracted from AIR with cell wall extraction 735

buffer (50 mM Trizma 50 mM CDTA pH 72) at 95degC for 15 min Samples were 736

homogenized using a paint shaker (Harbil 5G-HD) and glass beads (3 mm diameter) 737

For dot blot experiments 500 microL of cell wall extraction buffer were used per 10 mg of 738

AIR Debris was precipitated by centrifugation for 10 min at 10000 g 739

Total uronic acid content of pectin extracted as described above using 500 microL of 740

cell wall extraction buffer per 1 mg of AIR was measured as described previously 741

(Filisetti-Cozzi and Carpita 1991 van den Hoogen et al 1998) First 36 microL of pectin 742

extract were mixed with 4 microL of 4 M sulfamic acid Then 200 microL of sulfuric acid 743

Page 36 of 54

containing 120 mM sodium tetraborate was added and samples were incubated at 80degC 744

for 1 h Following cooling on ice the optical density of samples was measured at 490 745

nm Next 40 microL of m-hydroxydiphenyl reagent (100 microL of m-hydroxydiphenyl in dimethyl 746

sulfoxide at 100 mgmL mixed with 49 mL 80 (vv) sulfuric acid just before use) was 747

added and the samples were mixed The optical density at 490 nm was measured 748

again The optical density before m-hydroxydiphenyl reagent addition was subtracted 749

from the optical density measured after addition of the dye The concentration of uronic 750

acid was calculated using known amounts of GalA as a standard 751

752

Release of Pectin from Arabidopsis Cell Walls Using Macerozyme 753

AIR preparations from leaves of four week-old plants were treated with 025 754

(wv) macerozyme R-10 (Yakult) or water (mock) at a concentration of 100 microL per mg 755

AIR Samples were incubated for 3 h at room temperature on a vortex shaker and 756

centrifuged for 10 min at 12000 g Total uronic acid concentration of 5-fold diluted 757

supernatants was determined for two technical replicates each The concentration of 758

uronic acid was calculated using known amounts of GalA as a standard For 759

subsequent qRT-PCR experiments supernatants from multiple extractions were 760

combined boiled for 20 min to inactivate macerozyme diluted 10-fold with water and 761

inoculated into four week-old Col-0 plants Heat-inactivated 10-fold diluted macerozyme 762

solutions were used as mock treatment Samples were collected 3 h later and PAD3 763

expression was determined 764

765

Release of Pectin from Arabidopsis Leaves Infected with B cinerea 766

Fully expanded leaves of four week-old plants were either inoculated with up to 767

five 10 microL droplets of B cinerea spore solution (25 x 105 spores mL-1) or mock 768

inoculated with B cinerea inoculation medium For each biological replicate leaves with 769

a combined count of at least 20 infection sites were collected 48 h later AIR was 770

extracted as described above but plants were not dark-treated or freeze-dried and the 771

first ethanol step was performed using 85 (vv) ethanol To extract soluble pectin 772

fragments AIR samples were mixed with water at a concentration of 20 microL per infection 773

site incubated for 7 h at room temperature on a vortex shaker and centrifuged for 10 774

Page 37 of 54

min at 12000 g Total uronic acid concentration of supernatants was determined for two 775

technical replicates each The concentration of uronic acid was calculated using known 776

amounts of GalA as a standard 777

778

Measurement of Uronic Acids Neutral Monosaccharides and Cellulose Content in 779

Cell Walls 780

Neutral sugars and uronic acids from non-cellulosic polysaccharides in cell walls 781

(AIR) were released by trifluoroacetic acid (TFA) hydrolysis Approximately 1 mg of AIR 782

was hydrolyzed with 2 M TFA at 121degC for 90 min The TFA-soluble material was split 783

into two equal parts one for uronic acid and one for neutral sugar determination The 784

uronic acid content was determined by analyzing the hydrolyzate using a CarboPac 785

PA200 anion-exchange column with an ICS-3000 Dionex chromatography system 786

(Dionex Sunnyvale CA USA) The elution profile consisted of a linear gradient of 50 to 787

200 mM sodium acetate in 01 M NaOH in 10 min at 04 mL per min Alditol acetate 788

derivatives of neutral sugars were produced as described (York et al 1985) In brief 789

monosaccharides in the hydrolyzate were reduced with NaBH4 and peracetylated with 790

acetic anhydride and pyridine at 121degC for 20 min The generated alditol acetates were 791

analyzed by GC-MS Crystalline cellulose was measured according to Updegraff (1969) 792

After hydrolysis of noncellulosic polysaccharides from AIR with the Updegraff reagent 793

(acetic acid nitric acids water 812 (vv) ) the remaining pellet was hydrolyzed in 794

72 sulfuric acid The resulting glucose quantity was determined by the anthrone 795

method (Scott and Melvin 1953) 796

797

Dot Blot Experiments 798

Pectin solutions extracted using 50 microL of cell wall extraction buffer per mg AIR 799

were serially diluted and nitrocellulose membranes were spotted with 1 microL of the diluted 800

pectin solutions Membranes were dried overnight blocked with 5 milk (wv) in 1x 801

PBS (8 g L-1 NaCl 02 g L-1 KCl 144 g L-1 Na2HPO4 024 g L-1 KH2PO4 pH 74) and 802

probed with LM5 (Jones et al 1997) LM6 (Willats et al 1998) LM8 (Willats et al 803

2004) LM19 and LM20 (Verhertbruggen et al 2009a) or CCRC-M7 (Steffan et al 804

Page 38 of 54

1995) antibodies LM series antibodies were diluted 1250 and CCRC-M7 was diluted 805

1500 in 5 milk powder (Nestle Carnation) in 1 x PBS For LM series antibodies a 806

goat anti-Rat HRP conjugated antibody (Bethyl A110-105P 15000 diluted) and for 807

CCRC-M7 an anti-Mouse IgG HRP conjugate (Promega W402B 12500 diluted) was 808

used as secondary antibody in 5 milk powder in 1x PBS LM series antibodies were 809

obtained from plant probes (wwwplantprobesnet) and CCRC-M7 from CarboSource 810

(wwwcarbosourcenet) Membranes were washed after incubation with the primary and 811

secondary antibodies with 1x PBS Dot blots were developed using the ECL system 812

(GE Healthcare Amersham ECL prime) For quantification of signals dot blot results on 813

x-ray films were photographed using a CCD camera and intensities were measured814

using Image Jrsquos Integrated Density function Locally adjusted background was 815

subtracted As the dynamic range of the signal on an x-ray film was narrow non-816

linearity in the measured values was corrected for each genotype in each biological 817

replicate using the values from different dilutions in two technical replicates We noticed 818

that the residuals were not normally distributed and performed Box-Cox power 819

transformation (Box and Cox 1964) to obtain normally distributed values for the 820

subsequent statistical analysis of the data For LM19 Box-Cox selected power 821

transformation was done to the power of 038th and for LM20 to the power 075th After 822

correction of the transformed measured values with the effect of the biological 823

replicates the values were fit to a linear model with the Arabidopsis genotype as the 824

fixed effect This linear model was used both to generate the plots in Figures 5B and D 825

and to test significant differences 826

827

Camalexin Measurements 828

Four week-old plants were infected with B cinerea and samples (three leaf disks 829

from one plant each) were harvested at the indicated time points flash frozen and 830

pulverized Each sample was extracted with 300 microL of 90 methanol (vv) 50 microL of the 831

extract was run on an Agilent Lichrocart 250-4 RP18e 5-microm column using an Agilent 832

1100 series HPLC Camalexin was detected using a DiodeArray (DAD) at 330 nm and 833

with a fluorescence detector at emission 318 nmexcitation 385 nm (Agilent) Separation 834

was achieved using the following program with aqueous acetonitrile 5 min gradient 835

Page 39 of 54

from 63 to 69 acetonitrile 30 s gradient from 69 to 99 acetonitrile 2 min at 99 836

acetonitrile and a post-run equilibration of 35 min at 63 acetonitrile (Denby et al 837

2004 Kliebenstein et al 2005) Purified camalexin was used to produce a standard 838

curve to identify and quantitate camalexin 839

840

Accession Numbers 841

Microarray data used can be accessed at NCBI Gene Expression Omnnibus 842

(httpwwwncbinlmnihgovgeo) GSE18978 Sequence information for all Arabidopsis 843

genes described in this article can be found in TAIR (wwwarabidopsisorg) using the 844

following accession numbers PAD3 (CYP71B15 At3g26830) SID2 (ICS1 At1g74710) 845

GAE1 (At4g30440) GAE2 (At1g02000) GAE3 (At4g00110) GAE4 (At2g45310) GAE5 846

(At4g12250) GAE6 (At3g23820) PR1 (At2g14610) JAZ5 (At1g17380) JAZ10 847

(At5g13220) ACTIN2 (At3g18780) PDF12a (At5g44420) PDF12b (At2g26020) 848

AtSK11 (At5g26751) The sequence of the B cinerea cutinase Bc-CutA (Z69264) can 849

be found in the DNA Data Bank of Japan (httpgetentryddbjnigacjp) Germplasm 850

used included dde2-2 (At5g42650 (von Malek et al 2002)) pad3-1 (At3g26830 (Zhou 851

et al 1999)) pad4-1 (At3g52430 (Jirage et al 1999)) sid2-2 (At1g74710 (Wildermuth 852

et al 2001)) pbs3-2 (At5g13320 SALK_018225 (Nobuta et al 2007)) cbp60a-1 853

(At5g62570 SALK_124410 (Truman et al 2013)) cbp60g-1 sard1-2 (At5g26920 854

SALK_023199 At1g73805 SALK_052422 (Wang et al 2011) cbp60a-1 cbp60g-1 855

sard1-2 (Truman et al 2013) gae1-1 (At4g30440 SALK_085554) gae6-1 (At3g23280 856

SALK_104454) gae6-2 (At3g23280 SALK_017191) ixr1-1 cev1 (At5g05170 (Ellis 857

and Turner 2001 Scheible et al 2001)) eds1 plants were derived by introgression of 858

the Ler eds1-2 allele into Col-0 that contains two copies of EDS1 (At3g48090 859

At3g48080 (Bartsch et al 2006) mpk6-2 (At2g43790 SALK_073907 (Liu and Zhang 860

2004)) mpk3-DG (At3g45640 a fast neutron deletion mutant (Miles et al 2005)) and 861

npr1-1 (At1g64280 (Cao et al 1997)) T-DNA insertion lines were part of the SALK 862

collection (Alonso et al 2003) and were obtained from the Arabidopsis Biological 863

Resource Center 864

865

Supplemental Data 866

Page 40 of 54

Supplemental Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 867

Expression Does Not Require CBP60s or SID2 868

Supplemental Figure 2 Expression of GAE Family Members uponTreatment with Pma 869

ES4326 870

Supplemental Figure 3 Characterization of T-DNA Insertions in GAE1 and GAE6 871

Supplemental Figure 4 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves and 872

Are Slightly Smaller than Wild-Type Col-0 Plants 873

Supplemental Figure 5 Cell Walls of gae1 gae6 Plants Contain Less Uronic Acid than 874

Wild-Type Col-0 Cell Walls 875

Supplemental Figure 6 Antibodies Raised Against the RGI or XG Components of 876

Pectin Show No Difference in Binding to gae Mutant and Col-0 Cell Walls 877

Supplemental Figure 7 The Effect of B cinerea Treatment on the Expression of GAE 878

Family Genes 879

Supplemental Figure 8 Treatment with 001 Macerozyme Does Not Cause Any 880

Visible Damage to Col-0 Plants 881

Supplemental Figure 9 B cinerea Growth on Plants with Defects in Immune 882

Signaling 883

Supplemental Figure 10 MeJA-Induced Expression of the JA marker gene JAZ10 884

Supplemental Figure 11 The Expression of the JA Marker Gene JAZ5 Is Highly 885

Responsive in gae1 gae6 Plants 886

Supplemental Figure 12 The Expression of PDF12 Is Unaltered in gae1 gae6 Plants 887

Supplemental Figure 13 Macerozyme Treatment Activates MPK3 and MPK6 888

Supplemental Figure 14 B cinerea-Induced ROS Production in Wild-Type Col-0 and 889

gae1 gae6 Plants 890

Supplemental Figure 15 OG-Induced PTI Using 250 microg mL-1 OGs 891

Supplemental Figure 16 Abundance of Plant DNA in B cinerea Treated Plants 892

Supplemental Table 1 Primers used in this study 893

894

ACKNOWLEDGEMENTS 895

We thank William Truman for helpful discussions Simone Ferrari (Sapienza 896

Universita di Roma) for the generous gift of OGs Bruna Bucciarelli (USDA-ARS) for 897

Page 41 of 54

help with fluorescence microscopy the Arabidopsis Biological Resource Center for T-898

DNA insertion lines Paul Knoxrsquos lab (University of Leeds) for LM5 LM6 LM8 LM19 899

and LM20 antibodies and the Complex Carbohydrate Resource Center (University of 900

Georgia) for the CCRC-M7 antibody Most of this work was funded by the Division of 901

Chemical Sciences Geosciences and Biosciences Office of Basic Energy Sciences of 902

the US Department of Energy through Grant DE-FG02-05ER15670 to JG This work 903

was also supported by NSF awards IOS 1339125 and IOS 1021861 to DJK NSF 904

award IOS 1121425 to F K and the USDA National Institute of Food and Agriculture 905

Hatch project number CA-D-PLS-7033-H to DJK 906

907

AUTHOR CONTRIBUTIONS 908

GB and JG designed the research GB GX AT BL NES and NH 909

performed research DJK provided tools GB GX MP AD BL RAH and FK 910

analyzed data GB and JG wrote the paper with input from all authors 911

912

FIGURE LEGENDS 913

Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 Expression 914

Requires PAD4 EDS1 and PBS3 915

(A) Expression of GAE1 in leaves of wild-type Col-0 and mutant plants Expression was916

measured 24 or 32 h after either inoculation with Pma ES4326 (OD600 = 0002) or mock 917

inoculation with 5 mM MgSO4 and was then normalized to the level of ACTIN2 Data 918

from three biological replicates were combined using a mixed linear model Bars 919

represent mean log2-ratios to ACTIN2 plusmn SE Letters indicate significantly different 920

groups with q lt 005 where 005 represents the maximum false discovery rate at which 921

the test may be called significant 922

(B) Expression of GAE6 measured as described in (A)923

924

Figure 2 Susceptibility of gae6 and gae1 gae6 Plants to Pma ES4326 Is Increased 925

Relative to Wild-Type Col-0 926

Leaves of plants of the indicated genotypes were inoculated with Pma ES4326 (OD600 = 927

00002) as described in Methods Bacterial titers in leaves were determined either 928

Page 42 of 54

immediately (0 dpi) or after three days (3 dpi) Bars represent the mean plusmn SE of four 929

independent experiments each with 4 or 12 biological replicates at 0 and 3 dpi 930

respectively Data were combined using a mixed linear model Asterisks indicate 931

significant differences from Col-0 wild-type (q lt 005) Susceptible pad4 plants were 932

included as a positive control 933

934

Figure 3 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves 935

(A) Depiction of the assay for brittleness of gae mutant leaves Fully expanded leaves of936

four to five week-old plants were bent by 180ordm so that the adaxial surface of the leaf was 937

parallel with the soil Broken leaf petioles or midribs were considered brittle 938

(B) Quantification of broken midribs or petioles For each genotype 66 to 82 leaves 939

were analyzed for brittleness as described in (A) Bars represent the ratio of broken to 940

total number of leaves tested Results were analyzed using Fisherrsquos exact test Letters 941

indicate significantly different groups at p lt 005 942

(C) Conductivity measurements Leaves of four week-old plants were inoculated with943

deionized water and then leaf discs were cut out and placed on deionized water 944

Conductivity was measured at the indicated time points After 30 min the water was 945

replaced with fresh water (30 min f w) to account for wounding-related ion leakage from 946

the edges of the leaf discs Three independent experiments with six biological replicates 947

each were performed and data were analyzed using a t-test for each time point P-948

values were corrected using the Bonferroni method Data show means plusmn SE asterisks 949

indicate significant differences from wild-type Col-0 at q lt 001 950

951

Figure 4 Cell Walls of gae6 and gae1 gae6 Plants Are Reduced in GalA 952

(A) to (D) Levels of GalA (A) GlcA (B) cellulose (C) and neutral sugars (D) in alcohol953

insoluble residue (AIR) extracted from leaves of four week-old plants of the indicated 954

genotypes Bars represent means plusmn SE of four biological replicates combined using a 955

mixed linear model Letters indicate significantly different groups (p lt 005) 956

957

Figure 5 Cell Walls of gae1 gae6 Plants Are Reduced in Homogalacturonan 958

Page 43 of 54

Dot blot analyses with anti-LM19 and anti-LM20 antibodies Pectin was extracted from 959

AIR prepared from the indicated genotypes using 1 microL of extraction buffer per 10 microg of 960

AIR Pectin was serially diluted and one microL of undiluted 13 diluted and 19 diluted 961

samples were spotted on a nitrocellulose membrane Three biological replicates were 962

were spotted twice to obtain two technical replicates each All replicates produced 963

similar results 964

(A) Representative dot blot using anti-LM19 antibody965

(B) Relative signal intensities of the spots in the dot blot in (A)966

(C) Representative dot blot using anti-LM20 antibody967

(D) Relative signal intensities of the LM20 dot blot shown in (C)968

Relative signal intensities were measured using Image J software Measurements from 969

the three biological replicates were combined using a linear model after Box-Cox power 970

transformation as described in Methods Bars represent means plusmn SE Asterisks indicate 971

a difference from wild-type Col-0 at p lt 005 972

973

Figure 6 Growth of B cinerea on gae1 gae6 Plants and the Effect of B cinerea on 974

Expression of GAE1 and GAE6 975

(A) Growth of B cinerea isolates Leaves of Col-0 and gae1 gae6 plants were976

inoculated with 25 x 105 spores mL-1 of the B cinerea isolates indicated in the figure 977

Samples were collected two (2 dpi) and three (3 dpi) days later Bars represent means plusmn 978

SE of six biological replicates for each fungus combined using a mixed linear model 979

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 980

(B) and (C) Expression of GAE1 (B) and GAE6 (C) 48 h after inoculation with the B981

cinerea isolates indicated Expression levels were measured by qRT-PCR Bars 982

represent mean log2-ratios to ACTIN2 plusmn SE of three biological replicates combined 983

using a mixed linear model Letters indicate significantly different groups (q lt 005) 984

985

Figure 7 Levels of Soluble Pectin and Expression of PAD3 in Fractions Produced 986

Following Macerozyme Treatment of gae1 gae6 Cell Walls 987

(A) Schematic overview of the experimental procedure for (B) and (C)988

Page 44 of 54

(B) Uronic acid measurements Alcohol-insoluble residue (AIR) from leave tissue of four989

week-old wild-type Col-0 or gae1 gae6 plants was treated with water or 025 990

macerozyme and assayed for uronic acid levels Data from 11 biological replicates were 991

combined using a mixed linear model Bars represent means plusmn SE Letters indicate 992

significant differences (p lt 005) 993

(C) Expression of PAD3 in Col-0 plants treated with the fractions from (B) Extracts from994

(B) were heat inactivated pooled and diluted 5-fold with water Pooled extracts were995

inoculated into four week-old Col-0 plants PAD3 expression was measured 3 h after 996

inoculation and normalized to the level of ACTIN2 expression Data from four biological 997

replicates were combined using a mixed linear model Bars represent mean log2-ratios 998

to ACTIN2 plusmn SE Letters indicate significant differences (p lt 005) 999

1000

Figure 8 Macerozyme-Induced PAD3 Expression Is Absent in gae1 gae6 1001

(A) Expression of PAD3 after macerozyme treatment Four week-old plants were either1002

inoculated with 001 macerozyme or mock inoculated with water and PAD3 1003

expression was measured 3 h later by qRT-PCR Data from four biological replicates 1004

were combined using a mixed linear model Mean log2-ratios to ACTIN2 plusmn SE were 1005

plotted Letters indicate significantly different groups (p lt 005) 1006

(B) Expression of PAD3 after OG treatment PAD3 expression was measured as1007

described in (A) 3 h after inoculation with 100 microg mL-1 OGs or mock inoculation with 1008

water Data are from six biological replicates 1009

(C) PAD3 expression in untreated plants Three biological replicates were performed1010

and data analyzed as described in (A) 1011

(D) Camalexin accumulation after B cinerea treatment Camalexin accumulation was1012

measured in four week-old plants inoculated with the B cinerea strains shown in the 1013

figure Eight biological replicates each were combined using a mixed linear model 1014

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 1015

(E) PAD3 expression 28 h after treatment with the indicated B cinerea strains Data1016

from six biological replicates were collected and analyzed as described in (A) Data in 1017

all figure parts represent means plusmn SE Letters indicate significantly different groups (q lt 1018

005) 1019

Page 45 of 54

1020

Figure 9 Macerozyme-Induced Expression of the SA Marker Genes SID2 and PR1 1021

Is Abolished in gae1 gae6 Plants 1022

(A) to (H) Expression of PR1 (A) (C) (E) and (G) or SID2 (B) (D) (F) and (H) in four1023

week-old Col-0 and gae1 gae6 plants Data were combined using a mixed linear model 1024

Bars represent mean log2-ratios of expression vs ACTIN2 plusmn SE for all figure parts 1025

Letters indicate significantly different groups at p lt 005 for panels (A) to (D) (G) and 1026

(H) and qlt005 for panels (E) to (F) 1027

(A) and (B) Plants were treated with 001 macerozyme or mock treated with water1028

and samples were collected 3 h later Data from four biological replicates are shown 1029

(C) and (D) Plants were inoculated with 100 microg mL-1 OGs or mock treated with water1030

and samples collected 3 h later Data from six biological replicates are shown 1031

(E) and (F) Plants were inoculated with the indicated B cinerea isolates or mock1032

inoculated with B cinerea inoculation medium and samples collected 48 h later Data 1033

from eight biological replicates are shown 1034

(G) and (H) Expression in untreated plants Three biological replicates were performed1035

1036

Figure 10 The Expression of the JA Marker Gene JAZ10 is Highly Responsive in 1037

gae1 gae6 Plants 1038

(A) to (D) JAZ10 expression in four week-old plants Data were combined using a mixed1039

linear model Mean log2-ratios to ACTIN2 plusmn SE are shown Letters indicate significantly 1040

different groups at p lt 005 for panels (A) (B) and (D) and q lt 005 for panel (C) 1041

(A) Plants were inoculated with 001 macerozyme or mock inoculated using water and1042

samples were collected 3 h later Data from four biological replicates are shown 1043

(B) Plants were inoculated with 100 microg mL-1 OGs or mock inoculated using water and1044

samples collected 3 h later Data are from six biological replicates 1045

(C) JAZ10 expression 48 h after treatment with the indicated B cinerea strains Data1046

are from six biological replicates 1047

(D) JAZ10 expression in untreated plants Data are from three biological replicates1048

1049

Figure 11 Gallo 1-Induced Callose Deposition Is Reduced in gae1 gae6 Plants 1050

Page 46 of 54

(A) Callose deposition in four week-old Col-0 and gae1 gae6 plants Plants were1051

inoculated with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) or mock inoculated 1052

with B cinerea inoculation medium and samples were collected after 72 h Callose was 1053

stained with aniline blue and visualized using a Nikon Eclipse Ni-U microscope with a 1054

DAPI filter Three leaves were stained per treatment Scale bars are 100 microm Asterisks 1055

indicate fungal hyphae arrows indicate representative callose spots 1056

(B) Quantification of callose deposition Callose deposits were counted in 200 microm x 2001057

microm squares laid out across the images shown here Bars represent means plusmn SE from 1058

six squares each Letters indicate significantly different groups (p lt 005) according to a 1059

t-test analysis1060

1061

Figure 12 Less Soluble Pectin Accumulates in B cinerea-Infected gae1 gae6 1062

Leaves 1063

Four week-old Col-0 and gae1 gae6 plants were inoculated with 10 microL droplets of the 1064

indicated B cinerea isolate (25 x 105 spores mL-1) or mock inoculated with B cinerea 1065

inoculation medium Leaves were collected 48 h later AIR was extracted from leaf 1066

samples and water added (20 microL infection site) to extract soluble pectin fragments 1067

Samples were vortexed for 7 h centrifuged at 12000 g and the uronic acid 1068

concentration of the supernatant was measured Data from nine biological replicates 1069

were combined using a mixed linear model Means plusmn SE are shown Letters indicate 1070

significant differences (q lt 005) 1071

1072

Figure 13 Macerozyme-Induced PTI Is Lost in gae1 gae6 While OG-Induced PTI Is 1073

Unaltered 1074

(A) Macerozyme-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1075

inoculated with 001 macerozyme or mock inoculated with boiled macerozyme and 1076

were infected with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) 24 h later Samples 1077

were collected immediately (0 dpi) and after two (2 dpi) and three (3 dpi) days Data 1078

show mean plusmn SE of six to 14 biological replicates each combined using a linear model 1079

Letters indicate significantly different groups (q lt 005) 1080

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

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Page 29 of 54

whether reduced callose deposition causes the increased susceptibility of gae1 gae6 to 550

B cinerea isolate Gallo 1551

We did not observe any changes in B cinerea growth either on cev1 ixr1 plants 552

which are known to have constitutively high JA and OPDA levels (Ellis et al 2002b) or 553

on plants exogenously treated with MeJA (Supplemental Figure 9) Because plants 554

were kept uncovered for 3 h after MeJA treatment it is unclear how much MeJA was 555

bioavailable in the plant However JAZ10 expression was induced 3 h after spraying 556

with MeJA (Supplemental Figure 10) Because pathogen growth generally depends on 557

the combination of a multitude of immune signaling events we conclude that multiple 558

changes to the immune signaling in gae1 gae6 together account for the altered B 559

cinerea growth phenotypes detected 560

We showed that expression of GAE1 and GAE6 is repressed by the bacterial 561

hemibiotroph Pma ES4326 This repression did not occur in an eds1 or pad4 562

background or in pbs3 at the 24-h time point (Figure 1 and Supplemental Figure 1) 563

During the Arabidopsis - Pma ES4326 interaction the expression of many defense 564

related genes is altered in a PAD4EDS1 dependent fashion (Wang et al 2008) Some 565

of these expression changes require SA CBP60g and SARD1 are thought to act 566

downstream of PAD4 and EDS1 but upstream of SA-dependent responses (Wang et 567

al 2011) PBS3 is also thought to act downstream of PAD4 and EDS1 and upstream of 568

SA but the set of genes whose expression it affects is only partially overlapping with 569

those affected by CBP60gSARD1 (Wang et al 2008 Wang et al 2011) Thus GAE1 570

and GAE6 repression is regulated by the part of the immune signaling network that 571

requires PAD4 EDS1 and PBS3 but is independent of CBP60gSARD1 and SA 572

Because EDS1 PAD4 and PBS3 are important components of the plant immune 573

signaling network (Wiermer et al 2005 Nobuta et al 2007) one might conclude that 574

repression of GAE1 and GAE6 may be part of the plant immune response to Pma 575

ES4326 B cinerea treatment also reduced GAE1 and GAE6 expression but this was 576

mostly independent of PAD4 (Figure 6) while both Pma ES4326 and B cinerea repress 577

GAE1 and GAE6 expression the host signaling mechanisms responsible likely differ 578

In summary our data show that Arabidopsis GAE1 and GAE6 are required for 579

pectin biosynthesis leaf flexibility and immunity to Pma ES4326 and to specific B 580

Page 30 of 54

cinerea isolates We used macerozyme a commercial pectinase to mimic the effect of 581

B cinerea pectinases Macerozyme treatment released less pectin from gae1 gae6 cell582

walls and induced less immune signaling in gae1 gae6 plants Less water soluble pectin 583

also accumulated in B cinerea infected gae1 gae6 plants and macerozyme-induced 584

immunity to B cinerea isolate Gallo 1 was reduced in these plants We conclude that 585

the reduced pectin content in these mutants may lead to reduced release of soluble 586

pectin including active OGs during pathogen attack and that this compromises 587

immunity 588

589

590

Page 31 of 54

METHODS 591

Plant Materials and Growth Conditions 592

Wild-type Columbia (Col-0) and all mutant plants (in Col-0 background) were 593

grown on sterilized BM2 germinating mix (Berger Inc Quebec Canada) in a controlled 594

environment chamber (Conviron Pembina ND USA) with a 12-h photoperiod under 595

100 microM m-2 s-1 fluorescent illumination at 22degC and 75 relative humidity Germplasm 596

used is described in the Accession Numbers section at the end of Methods 597

598

Data Analysis and Replicates 599

For biological replicates samples were obtained from separate plant tissues and 600

in many cases from plants grown in different flats In some experiments multiple 601

technical replicates were measured per biological replicate For statistical analysis all 602

technical replicates were averaged and the averages were considered one biological 603

replicate Independent experiments were performed at different times and often 604

consisted of multiple biological replicates per experiment 605

Data analysis was performed using mixed linear models in the R programming 606

environment (Bates et al 2015) unless otherwise indicated Generally genotypes 607

treatments and time points were used as fixed effects and replicate-specific effects like 608

different pots flats etc were considered random effects We reported p-values 609

obtained from the mixed linear model when less than 10 comparisons were performed 610

and q-values for more than 10 comparisons to correct for multiple comparison testing 611

Q-values were calculated using the Benjamini-Hochberg procedure (Benjamini and612

Hochberg 1995) unless specified otherwise 613

614

Pathogen Strains Growth Conditions and Pathogen Growth Assays 615

Growth of Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) and 616

bacterial growth assays were performed as previously described (Bethke et al 2014) 617

For qRT-PCR assays four week-old plants were inoculated with bacteria (OD600 = 618

0002) or mock (5 mM MgSO4) At least four leaves from two plants were collected at 619

the indicated time points per biological replicate 620

Page 32 of 54

Prior to each experiment Botrytis cinerea isolates were grown on 1x PDA 621

medium (Difco) for 10 d at room temperature All isolates used have been described 622

previously (Rowe and Kliebenstein 2007) The spores were washed from the surface of 623

the plate using an inoculation medium (1x Gamborgrsquos B-5 basal salt mixture (Sigma-624

Aldrich G5768) 2 (wv) glucose 10 mM phosphate buffer pH 64) and fungal hyphae 625

were removed from the suspension by filtering through four layers of cheesecloth The 626

concentration of spores was determined using a hemocytometer and adjusted to 25 x 627

105 spores mL-1 Four week-old Arabidopsis plants were inoculated by placing one 10-628

microL droplet of the B cinerea spore solution or inoculation medium (mock) on the adaxial 629

leaf surface of fully expanded leaves Inoculated plants were kept at 100 relative 630

humidity Infection sites were collected at various time points using a cork borer For 631

qRT-PCR assays at least four leaf discs from one plant were combined for each 632

biological sample For B cinerea growth assays at least 12 leaf discs from three to six 633

plants were pooled per biological sample Relative fungal growth was determined by 634

measuring the abundance of a fungal gene relative to a plant gene as described below 635

In brief DNA from infected tissue was extracted and equal amounts of total DNA were 636

used to perform qPCR reactions using the B cinerea cutinase A and the Arabidopsis 637

SK11 gene respectively Primers were described in (Gachon and Saindrenan 2004) 638

and are listed in Supplemental Table 1 Two technical replicates were combined for 639

each biological replicate B cinerea is a necrotrophic fungal pathogen and may destroy 640

plant DNA as disease progresses However decreases in the amount of plant DNA 641

over the course of our experiments were only observed at late time points with very 642

susceptible plants so the qPCR assay is generally a reasonable estimate of fungal 643

biomass (Supplemental Figure 16) 644

645

Treatment with Macerozyme or OGs 646

For PTI assays four week-old plants either were inoculated with 001 (wv) 647

macerozyme R-10 (Yakult) a Rhizopus polygalacturonase (EC 32115) that possesses 648

high pectinase and hemicellulase activity or were mock inoculated using heat-649

inactivated macerozyme Other similar plants were either inoculated with 250 or 500 microg 650

mL-1 OGs or mock inoculated with water One d later plants were infected with B 651

Page 33 of 54

cinerea and samples were collected immediately (0 dpi) and two or three d later To 652

investigate the role of MeJA on B cinerea growth plants were sprayed with 1mM MeJA 653

in water with 001 (vv) Tween 20 Covers were removed so that leaves could dry off 654

before plants were inoculated with B cinerea three h later Samples were harvested at 655

the indicated time points For expression analysis and MAPK activation assays plants 656

were either inoculated with 100 microg mL-1 OGs or mock inoculated with water OGs used 657

for experiments described in Figures 8 to 10 and Supplemental Figure 12 were a kind 658

gift from Simone Ferrari OGs used in experiments described in Figure 13 and 659

Supplemental Figures 11 13 and 15 were prepared as previously described (Kohorn et 660

al 2014) 661

662

Expression Analysis 663

Leaf tissue from inoculated plants was harvested at the indicated time points 664

flash frozen pulverized and RNA extracted using Trizol (Invitrogen) Quantitative RT-665

PCR was performed using the SuperScript III Platinum SYBR Green One-Step 666

quantitative RT-PCR kit (Invitrogen) and a Lightcycler 480 Real-Time PCR system 667

(Roche) as previously described (Truman and Glazebrook 2012) In brief equal 668

amounts of total RNA and a gene specific primer were used for each reaction The 669

crossing point (Cp) was calculated using the 2nd derivative max method provided with 670

the Lightcycler software for each amplification curve Each reaction was run with two 671

technical replicates and the Cp values for these replicates were averaged ACTIN2 was 672

used as a stably expressed reference gene At least three biological replicates each 673

were performed and analyzed using a mixed-linear model RT-PCR was performed to 674

verify T-DNA insertion lines using the One-Step RT-PCR kit (Qiagen) according to the 675

manufacturerrsquos instructions The primers used can be found in Supplemental Table 1 676

677

Electrolyte Leakage Assay 678

Fully expanded leaves of four week-old plants were inoculated with water (Milli-Q 679

grade) using a needleless syringe Leaf discs were collected using a cork borer and 680

three leaf discs from one leaf were placed with the adaxial surface down onto 2 mL 681

water in a 12-well cell culture plate Conductivity was measured using a Horiba B-173 682

Page 34 of 54

conductivity meter after 30 min and then the water was replaced Conductivity was 683

measured again 30 min later (1 h total) as indicated in Figure 3C Six biological 684

replicates each were performed in three independent experiments and means and SEs 685

were calculated Two-sided t-tests were performed for each time point 686

687

DAB and Aniline Blue Staining 688

Leaves from four week-old plants were either mock inoculated with B cinerea 689

inoculation medium or inoculated with 10 microL droplets of a B cinerea spore solution (25 690

x 105 spores mL-1) of the indicated isolates Samples were collected 72 h later For 33prime-691

diaminobenzidine (DAB) staining two to five inoculated leaves were vacuum infiltrated 692

with 33prime-Diaminobenzidine (1 mg mL-1 in water pH 38) solution and stained overnight 693

in the dark Leaves were destained using 100 ethanol at 65degC and then transferred to 694

50 (vv) glycerol and photographed 695

Callose was visualized by staining leaves with aniline blue as previously 696

described (Adam and Somerville 1996) Briefly leaves were vacuum infiltrated with 697

alcoholic lactophenol (one volume of phenolglycerollactic acidwater (1111) mixed 698

with two volumes of ethanol) and destained at 65degC Next leaves were moved through 699

an alcohol gradient (50 20 and 10 (vv) ethanol) into water The destained leaves 700

were stained for 30 min in 150 mM K2HPO4 (pH 95) with 001 (wv) aniline blue 701

Samples were mounted in water and examined using a Nikon Eclipse Ni-U microscope 702

at 10x magnification using a DAPI filter (excitation at 325-375 nm emission at 435-485 703

nm) For callose quantification six 200 microm x 200 microm squares were counted for each 704

sample type Callose deposits per mm2 were calculated and data analyzed using a t-705

test Note that there is an inherent uncertainty associated with such counts because 706

callose deposits may not all be in focus at the same time 707

708

MAPK Activity Assay 709

Fully expanded leaves of four week-old plants were inoculated with 001 710

macerozyme 100 microg mL-1 OGs 100 nM flg22 or water (mock) flg22 a peptide from 711

bacterial flagella that induces immune responses was used as a positive control for 712

Page 35 of 54

MAPK activation (Asai et al 2002) and was purchased from EZBiolab Inc (Westfield 713

IN USA) Samples were harvested 10 min later and immediately frozen in liquid 714

nitrogen Protein was extracted using an extraction buffer containing 25 mM Tris pH 78 715

75 mM NaCl 1 mM DTT 1 mM NaF 05mM Na3VO4 01 (vv) Tween 20 one 716

cOmplete Mini protease inhibitor cocktail tablet (Roche) and one PhosSTOP 717

phosphatase inhibitor cocktail tablet (Roche) per 10 mL buffer 15 microg of total protein 718

were electrophoresed on a 10 SDS-polyacrylamide gel and then blotted onto a PVDF 719

membrane (Bio-Rad 162-0177) Activated MAPKs were detected using p4442 MAPK 720

(Erk12) antibody (9102S Cell Signal 12500 in TBST (20 mM Tris pH 75 150 mM 721

NaCl 01 (vv) Tween 20)) Anti-Rabbit IgG-HRP (115000 in TBST A6154 Sigma) 722

and ECL Plus substrate (Pierce 32132) MPK3 and MPK6 were detected with an anti-723

MPK3 antibody (M8318 Sigma 12000 in TBST and 3 (wv) milk) or an anti-MPK6 724

antibody (A7104 Sigma 16000 in TBST and 3 (wv) milk) respectively The 725

secondary antibody and detection were the same as for the p4442 antibody Five 726

independent experiments were performed 727

728

Preparation of Alcohol Insoluble Residue (AIR) Pectin Extraction and Uronic Acid 729

Measurements 730

Fully expanded leaves from four week-old plants that were incubated for 48 h in 731

the dark to reduce starch were harvested flash frozen and pulverized AIR was 732

extracted by washing ground material twice in 70 (vv) ethanol three times in a 733

mixture of chloroform and methanol (11 vv) and once in acetone (Gille et al 2009) 734

and was then air dried Cell wall pectin was extracted from AIR with cell wall extraction 735

buffer (50 mM Trizma 50 mM CDTA pH 72) at 95degC for 15 min Samples were 736

homogenized using a paint shaker (Harbil 5G-HD) and glass beads (3 mm diameter) 737

For dot blot experiments 500 microL of cell wall extraction buffer were used per 10 mg of 738

AIR Debris was precipitated by centrifugation for 10 min at 10000 g 739

Total uronic acid content of pectin extracted as described above using 500 microL of 740

cell wall extraction buffer per 1 mg of AIR was measured as described previously 741

(Filisetti-Cozzi and Carpita 1991 van den Hoogen et al 1998) First 36 microL of pectin 742

extract were mixed with 4 microL of 4 M sulfamic acid Then 200 microL of sulfuric acid 743

Page 36 of 54

containing 120 mM sodium tetraborate was added and samples were incubated at 80degC 744

for 1 h Following cooling on ice the optical density of samples was measured at 490 745

nm Next 40 microL of m-hydroxydiphenyl reagent (100 microL of m-hydroxydiphenyl in dimethyl 746

sulfoxide at 100 mgmL mixed with 49 mL 80 (vv) sulfuric acid just before use) was 747

added and the samples were mixed The optical density at 490 nm was measured 748

again The optical density before m-hydroxydiphenyl reagent addition was subtracted 749

from the optical density measured after addition of the dye The concentration of uronic 750

acid was calculated using known amounts of GalA as a standard 751

752

Release of Pectin from Arabidopsis Cell Walls Using Macerozyme 753

AIR preparations from leaves of four week-old plants were treated with 025 754

(wv) macerozyme R-10 (Yakult) or water (mock) at a concentration of 100 microL per mg 755

AIR Samples were incubated for 3 h at room temperature on a vortex shaker and 756

centrifuged for 10 min at 12000 g Total uronic acid concentration of 5-fold diluted 757

supernatants was determined for two technical replicates each The concentration of 758

uronic acid was calculated using known amounts of GalA as a standard For 759

subsequent qRT-PCR experiments supernatants from multiple extractions were 760

combined boiled for 20 min to inactivate macerozyme diluted 10-fold with water and 761

inoculated into four week-old Col-0 plants Heat-inactivated 10-fold diluted macerozyme 762

solutions were used as mock treatment Samples were collected 3 h later and PAD3 763

expression was determined 764

765

Release of Pectin from Arabidopsis Leaves Infected with B cinerea 766

Fully expanded leaves of four week-old plants were either inoculated with up to 767

five 10 microL droplets of B cinerea spore solution (25 x 105 spores mL-1) or mock 768

inoculated with B cinerea inoculation medium For each biological replicate leaves with 769

a combined count of at least 20 infection sites were collected 48 h later AIR was 770

extracted as described above but plants were not dark-treated or freeze-dried and the 771

first ethanol step was performed using 85 (vv) ethanol To extract soluble pectin 772

fragments AIR samples were mixed with water at a concentration of 20 microL per infection 773

site incubated for 7 h at room temperature on a vortex shaker and centrifuged for 10 774

Page 37 of 54

min at 12000 g Total uronic acid concentration of supernatants was determined for two 775

technical replicates each The concentration of uronic acid was calculated using known 776

amounts of GalA as a standard 777

778

Measurement of Uronic Acids Neutral Monosaccharides and Cellulose Content in 779

Cell Walls 780

Neutral sugars and uronic acids from non-cellulosic polysaccharides in cell walls 781

(AIR) were released by trifluoroacetic acid (TFA) hydrolysis Approximately 1 mg of AIR 782

was hydrolyzed with 2 M TFA at 121degC for 90 min The TFA-soluble material was split 783

into two equal parts one for uronic acid and one for neutral sugar determination The 784

uronic acid content was determined by analyzing the hydrolyzate using a CarboPac 785

PA200 anion-exchange column with an ICS-3000 Dionex chromatography system 786

(Dionex Sunnyvale CA USA) The elution profile consisted of a linear gradient of 50 to 787

200 mM sodium acetate in 01 M NaOH in 10 min at 04 mL per min Alditol acetate 788

derivatives of neutral sugars were produced as described (York et al 1985) In brief 789

monosaccharides in the hydrolyzate were reduced with NaBH4 and peracetylated with 790

acetic anhydride and pyridine at 121degC for 20 min The generated alditol acetates were 791

analyzed by GC-MS Crystalline cellulose was measured according to Updegraff (1969) 792

After hydrolysis of noncellulosic polysaccharides from AIR with the Updegraff reagent 793

(acetic acid nitric acids water 812 (vv) ) the remaining pellet was hydrolyzed in 794

72 sulfuric acid The resulting glucose quantity was determined by the anthrone 795

method (Scott and Melvin 1953) 796

797

Dot Blot Experiments 798

Pectin solutions extracted using 50 microL of cell wall extraction buffer per mg AIR 799

were serially diluted and nitrocellulose membranes were spotted with 1 microL of the diluted 800

pectin solutions Membranes were dried overnight blocked with 5 milk (wv) in 1x 801

PBS (8 g L-1 NaCl 02 g L-1 KCl 144 g L-1 Na2HPO4 024 g L-1 KH2PO4 pH 74) and 802

probed with LM5 (Jones et al 1997) LM6 (Willats et al 1998) LM8 (Willats et al 803

2004) LM19 and LM20 (Verhertbruggen et al 2009a) or CCRC-M7 (Steffan et al 804

Page 38 of 54

1995) antibodies LM series antibodies were diluted 1250 and CCRC-M7 was diluted 805

1500 in 5 milk powder (Nestle Carnation) in 1 x PBS For LM series antibodies a 806

goat anti-Rat HRP conjugated antibody (Bethyl A110-105P 15000 diluted) and for 807

CCRC-M7 an anti-Mouse IgG HRP conjugate (Promega W402B 12500 diluted) was 808

used as secondary antibody in 5 milk powder in 1x PBS LM series antibodies were 809

obtained from plant probes (wwwplantprobesnet) and CCRC-M7 from CarboSource 810

(wwwcarbosourcenet) Membranes were washed after incubation with the primary and 811

secondary antibodies with 1x PBS Dot blots were developed using the ECL system 812

(GE Healthcare Amersham ECL prime) For quantification of signals dot blot results on 813

x-ray films were photographed using a CCD camera and intensities were measured814

using Image Jrsquos Integrated Density function Locally adjusted background was 815

subtracted As the dynamic range of the signal on an x-ray film was narrow non-816

linearity in the measured values was corrected for each genotype in each biological 817

replicate using the values from different dilutions in two technical replicates We noticed 818

that the residuals were not normally distributed and performed Box-Cox power 819

transformation (Box and Cox 1964) to obtain normally distributed values for the 820

subsequent statistical analysis of the data For LM19 Box-Cox selected power 821

transformation was done to the power of 038th and for LM20 to the power 075th After 822

correction of the transformed measured values with the effect of the biological 823

replicates the values were fit to a linear model with the Arabidopsis genotype as the 824

fixed effect This linear model was used both to generate the plots in Figures 5B and D 825

and to test significant differences 826

827

Camalexin Measurements 828

Four week-old plants were infected with B cinerea and samples (three leaf disks 829

from one plant each) were harvested at the indicated time points flash frozen and 830

pulverized Each sample was extracted with 300 microL of 90 methanol (vv) 50 microL of the 831

extract was run on an Agilent Lichrocart 250-4 RP18e 5-microm column using an Agilent 832

1100 series HPLC Camalexin was detected using a DiodeArray (DAD) at 330 nm and 833

with a fluorescence detector at emission 318 nmexcitation 385 nm (Agilent) Separation 834

was achieved using the following program with aqueous acetonitrile 5 min gradient 835

Page 39 of 54

from 63 to 69 acetonitrile 30 s gradient from 69 to 99 acetonitrile 2 min at 99 836

acetonitrile and a post-run equilibration of 35 min at 63 acetonitrile (Denby et al 837

2004 Kliebenstein et al 2005) Purified camalexin was used to produce a standard 838

curve to identify and quantitate camalexin 839

840

Accession Numbers 841

Microarray data used can be accessed at NCBI Gene Expression Omnnibus 842

(httpwwwncbinlmnihgovgeo) GSE18978 Sequence information for all Arabidopsis 843

genes described in this article can be found in TAIR (wwwarabidopsisorg) using the 844

following accession numbers PAD3 (CYP71B15 At3g26830) SID2 (ICS1 At1g74710) 845

GAE1 (At4g30440) GAE2 (At1g02000) GAE3 (At4g00110) GAE4 (At2g45310) GAE5 846

(At4g12250) GAE6 (At3g23820) PR1 (At2g14610) JAZ5 (At1g17380) JAZ10 847

(At5g13220) ACTIN2 (At3g18780) PDF12a (At5g44420) PDF12b (At2g26020) 848

AtSK11 (At5g26751) The sequence of the B cinerea cutinase Bc-CutA (Z69264) can 849

be found in the DNA Data Bank of Japan (httpgetentryddbjnigacjp) Germplasm 850

used included dde2-2 (At5g42650 (von Malek et al 2002)) pad3-1 (At3g26830 (Zhou 851

et al 1999)) pad4-1 (At3g52430 (Jirage et al 1999)) sid2-2 (At1g74710 (Wildermuth 852

et al 2001)) pbs3-2 (At5g13320 SALK_018225 (Nobuta et al 2007)) cbp60a-1 853

(At5g62570 SALK_124410 (Truman et al 2013)) cbp60g-1 sard1-2 (At5g26920 854

SALK_023199 At1g73805 SALK_052422 (Wang et al 2011) cbp60a-1 cbp60g-1 855

sard1-2 (Truman et al 2013) gae1-1 (At4g30440 SALK_085554) gae6-1 (At3g23280 856

SALK_104454) gae6-2 (At3g23280 SALK_017191) ixr1-1 cev1 (At5g05170 (Ellis 857

and Turner 2001 Scheible et al 2001)) eds1 plants were derived by introgression of 858

the Ler eds1-2 allele into Col-0 that contains two copies of EDS1 (At3g48090 859

At3g48080 (Bartsch et al 2006) mpk6-2 (At2g43790 SALK_073907 (Liu and Zhang 860

2004)) mpk3-DG (At3g45640 a fast neutron deletion mutant (Miles et al 2005)) and 861

npr1-1 (At1g64280 (Cao et al 1997)) T-DNA insertion lines were part of the SALK 862

collection (Alonso et al 2003) and were obtained from the Arabidopsis Biological 863

Resource Center 864

865

Supplemental Data 866

Page 40 of 54

Supplemental Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 867

Expression Does Not Require CBP60s or SID2 868

Supplemental Figure 2 Expression of GAE Family Members uponTreatment with Pma 869

ES4326 870

Supplemental Figure 3 Characterization of T-DNA Insertions in GAE1 and GAE6 871

Supplemental Figure 4 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves and 872

Are Slightly Smaller than Wild-Type Col-0 Plants 873

Supplemental Figure 5 Cell Walls of gae1 gae6 Plants Contain Less Uronic Acid than 874

Wild-Type Col-0 Cell Walls 875

Supplemental Figure 6 Antibodies Raised Against the RGI or XG Components of 876

Pectin Show No Difference in Binding to gae Mutant and Col-0 Cell Walls 877

Supplemental Figure 7 The Effect of B cinerea Treatment on the Expression of GAE 878

Family Genes 879

Supplemental Figure 8 Treatment with 001 Macerozyme Does Not Cause Any 880

Visible Damage to Col-0 Plants 881

Supplemental Figure 9 B cinerea Growth on Plants with Defects in Immune 882

Signaling 883

Supplemental Figure 10 MeJA-Induced Expression of the JA marker gene JAZ10 884

Supplemental Figure 11 The Expression of the JA Marker Gene JAZ5 Is Highly 885

Responsive in gae1 gae6 Plants 886

Supplemental Figure 12 The Expression of PDF12 Is Unaltered in gae1 gae6 Plants 887

Supplemental Figure 13 Macerozyme Treatment Activates MPK3 and MPK6 888

Supplemental Figure 14 B cinerea-Induced ROS Production in Wild-Type Col-0 and 889

gae1 gae6 Plants 890

Supplemental Figure 15 OG-Induced PTI Using 250 microg mL-1 OGs 891

Supplemental Figure 16 Abundance of Plant DNA in B cinerea Treated Plants 892

Supplemental Table 1 Primers used in this study 893

894

ACKNOWLEDGEMENTS 895

We thank William Truman for helpful discussions Simone Ferrari (Sapienza 896

Universita di Roma) for the generous gift of OGs Bruna Bucciarelli (USDA-ARS) for 897

Page 41 of 54

help with fluorescence microscopy the Arabidopsis Biological Resource Center for T-898

DNA insertion lines Paul Knoxrsquos lab (University of Leeds) for LM5 LM6 LM8 LM19 899

and LM20 antibodies and the Complex Carbohydrate Resource Center (University of 900

Georgia) for the CCRC-M7 antibody Most of this work was funded by the Division of 901

Chemical Sciences Geosciences and Biosciences Office of Basic Energy Sciences of 902

the US Department of Energy through Grant DE-FG02-05ER15670 to JG This work 903

was also supported by NSF awards IOS 1339125 and IOS 1021861 to DJK NSF 904

award IOS 1121425 to F K and the USDA National Institute of Food and Agriculture 905

Hatch project number CA-D-PLS-7033-H to DJK 906

907

AUTHOR CONTRIBUTIONS 908

GB and JG designed the research GB GX AT BL NES and NH 909

performed research DJK provided tools GB GX MP AD BL RAH and FK 910

analyzed data GB and JG wrote the paper with input from all authors 911

912

FIGURE LEGENDS 913

Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 Expression 914

Requires PAD4 EDS1 and PBS3 915

(A) Expression of GAE1 in leaves of wild-type Col-0 and mutant plants Expression was916

measured 24 or 32 h after either inoculation with Pma ES4326 (OD600 = 0002) or mock 917

inoculation with 5 mM MgSO4 and was then normalized to the level of ACTIN2 Data 918

from three biological replicates were combined using a mixed linear model Bars 919

represent mean log2-ratios to ACTIN2 plusmn SE Letters indicate significantly different 920

groups with q lt 005 where 005 represents the maximum false discovery rate at which 921

the test may be called significant 922

(B) Expression of GAE6 measured as described in (A)923

924

Figure 2 Susceptibility of gae6 and gae1 gae6 Plants to Pma ES4326 Is Increased 925

Relative to Wild-Type Col-0 926

Leaves of plants of the indicated genotypes were inoculated with Pma ES4326 (OD600 = 927

00002) as described in Methods Bacterial titers in leaves were determined either 928

Page 42 of 54

immediately (0 dpi) or after three days (3 dpi) Bars represent the mean plusmn SE of four 929

independent experiments each with 4 or 12 biological replicates at 0 and 3 dpi 930

respectively Data were combined using a mixed linear model Asterisks indicate 931

significant differences from Col-0 wild-type (q lt 005) Susceptible pad4 plants were 932

included as a positive control 933

934

Figure 3 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves 935

(A) Depiction of the assay for brittleness of gae mutant leaves Fully expanded leaves of936

four to five week-old plants were bent by 180ordm so that the adaxial surface of the leaf was 937

parallel with the soil Broken leaf petioles or midribs were considered brittle 938

(B) Quantification of broken midribs or petioles For each genotype 66 to 82 leaves 939

were analyzed for brittleness as described in (A) Bars represent the ratio of broken to 940

total number of leaves tested Results were analyzed using Fisherrsquos exact test Letters 941

indicate significantly different groups at p lt 005 942

(C) Conductivity measurements Leaves of four week-old plants were inoculated with943

deionized water and then leaf discs were cut out and placed on deionized water 944

Conductivity was measured at the indicated time points After 30 min the water was 945

replaced with fresh water (30 min f w) to account for wounding-related ion leakage from 946

the edges of the leaf discs Three independent experiments with six biological replicates 947

each were performed and data were analyzed using a t-test for each time point P-948

values were corrected using the Bonferroni method Data show means plusmn SE asterisks 949

indicate significant differences from wild-type Col-0 at q lt 001 950

951

Figure 4 Cell Walls of gae6 and gae1 gae6 Plants Are Reduced in GalA 952

(A) to (D) Levels of GalA (A) GlcA (B) cellulose (C) and neutral sugars (D) in alcohol953

insoluble residue (AIR) extracted from leaves of four week-old plants of the indicated 954

genotypes Bars represent means plusmn SE of four biological replicates combined using a 955

mixed linear model Letters indicate significantly different groups (p lt 005) 956

957

Figure 5 Cell Walls of gae1 gae6 Plants Are Reduced in Homogalacturonan 958

Page 43 of 54

Dot blot analyses with anti-LM19 and anti-LM20 antibodies Pectin was extracted from 959

AIR prepared from the indicated genotypes using 1 microL of extraction buffer per 10 microg of 960

AIR Pectin was serially diluted and one microL of undiluted 13 diluted and 19 diluted 961

samples were spotted on a nitrocellulose membrane Three biological replicates were 962

were spotted twice to obtain two technical replicates each All replicates produced 963

similar results 964

(A) Representative dot blot using anti-LM19 antibody965

(B) Relative signal intensities of the spots in the dot blot in (A)966

(C) Representative dot blot using anti-LM20 antibody967

(D) Relative signal intensities of the LM20 dot blot shown in (C)968

Relative signal intensities were measured using Image J software Measurements from 969

the three biological replicates were combined using a linear model after Box-Cox power 970

transformation as described in Methods Bars represent means plusmn SE Asterisks indicate 971

a difference from wild-type Col-0 at p lt 005 972

973

Figure 6 Growth of B cinerea on gae1 gae6 Plants and the Effect of B cinerea on 974

Expression of GAE1 and GAE6 975

(A) Growth of B cinerea isolates Leaves of Col-0 and gae1 gae6 plants were976

inoculated with 25 x 105 spores mL-1 of the B cinerea isolates indicated in the figure 977

Samples were collected two (2 dpi) and three (3 dpi) days later Bars represent means plusmn 978

SE of six biological replicates for each fungus combined using a mixed linear model 979

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 980

(B) and (C) Expression of GAE1 (B) and GAE6 (C) 48 h after inoculation with the B981

cinerea isolates indicated Expression levels were measured by qRT-PCR Bars 982

represent mean log2-ratios to ACTIN2 plusmn SE of three biological replicates combined 983

using a mixed linear model Letters indicate significantly different groups (q lt 005) 984

985

Figure 7 Levels of Soluble Pectin and Expression of PAD3 in Fractions Produced 986

Following Macerozyme Treatment of gae1 gae6 Cell Walls 987

(A) Schematic overview of the experimental procedure for (B) and (C)988

Page 44 of 54

(B) Uronic acid measurements Alcohol-insoluble residue (AIR) from leave tissue of four989

week-old wild-type Col-0 or gae1 gae6 plants was treated with water or 025 990

macerozyme and assayed for uronic acid levels Data from 11 biological replicates were 991

combined using a mixed linear model Bars represent means plusmn SE Letters indicate 992

significant differences (p lt 005) 993

(C) Expression of PAD3 in Col-0 plants treated with the fractions from (B) Extracts from994

(B) were heat inactivated pooled and diluted 5-fold with water Pooled extracts were995

inoculated into four week-old Col-0 plants PAD3 expression was measured 3 h after 996

inoculation and normalized to the level of ACTIN2 expression Data from four biological 997

replicates were combined using a mixed linear model Bars represent mean log2-ratios 998

to ACTIN2 plusmn SE Letters indicate significant differences (p lt 005) 999

1000

Figure 8 Macerozyme-Induced PAD3 Expression Is Absent in gae1 gae6 1001

(A) Expression of PAD3 after macerozyme treatment Four week-old plants were either1002

inoculated with 001 macerozyme or mock inoculated with water and PAD3 1003

expression was measured 3 h later by qRT-PCR Data from four biological replicates 1004

were combined using a mixed linear model Mean log2-ratios to ACTIN2 plusmn SE were 1005

plotted Letters indicate significantly different groups (p lt 005) 1006

(B) Expression of PAD3 after OG treatment PAD3 expression was measured as1007

described in (A) 3 h after inoculation with 100 microg mL-1 OGs or mock inoculation with 1008

water Data are from six biological replicates 1009

(C) PAD3 expression in untreated plants Three biological replicates were performed1010

and data analyzed as described in (A) 1011

(D) Camalexin accumulation after B cinerea treatment Camalexin accumulation was1012

measured in four week-old plants inoculated with the B cinerea strains shown in the 1013

figure Eight biological replicates each were combined using a mixed linear model 1014

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 1015

(E) PAD3 expression 28 h after treatment with the indicated B cinerea strains Data1016

from six biological replicates were collected and analyzed as described in (A) Data in 1017

all figure parts represent means plusmn SE Letters indicate significantly different groups (q lt 1018

005) 1019

Page 45 of 54

1020

Figure 9 Macerozyme-Induced Expression of the SA Marker Genes SID2 and PR1 1021

Is Abolished in gae1 gae6 Plants 1022

(A) to (H) Expression of PR1 (A) (C) (E) and (G) or SID2 (B) (D) (F) and (H) in four1023

week-old Col-0 and gae1 gae6 plants Data were combined using a mixed linear model 1024

Bars represent mean log2-ratios of expression vs ACTIN2 plusmn SE for all figure parts 1025

Letters indicate significantly different groups at p lt 005 for panels (A) to (D) (G) and 1026

(H) and qlt005 for panels (E) to (F) 1027

(A) and (B) Plants were treated with 001 macerozyme or mock treated with water1028

and samples were collected 3 h later Data from four biological replicates are shown 1029

(C) and (D) Plants were inoculated with 100 microg mL-1 OGs or mock treated with water1030

and samples collected 3 h later Data from six biological replicates are shown 1031

(E) and (F) Plants were inoculated with the indicated B cinerea isolates or mock1032

inoculated with B cinerea inoculation medium and samples collected 48 h later Data 1033

from eight biological replicates are shown 1034

(G) and (H) Expression in untreated plants Three biological replicates were performed1035

1036

Figure 10 The Expression of the JA Marker Gene JAZ10 is Highly Responsive in 1037

gae1 gae6 Plants 1038

(A) to (D) JAZ10 expression in four week-old plants Data were combined using a mixed1039

linear model Mean log2-ratios to ACTIN2 plusmn SE are shown Letters indicate significantly 1040

different groups at p lt 005 for panels (A) (B) and (D) and q lt 005 for panel (C) 1041

(A) Plants were inoculated with 001 macerozyme or mock inoculated using water and1042

samples were collected 3 h later Data from four biological replicates are shown 1043

(B) Plants were inoculated with 100 microg mL-1 OGs or mock inoculated using water and1044

samples collected 3 h later Data are from six biological replicates 1045

(C) JAZ10 expression 48 h after treatment with the indicated B cinerea strains Data1046

are from six biological replicates 1047

(D) JAZ10 expression in untreated plants Data are from three biological replicates1048

1049

Figure 11 Gallo 1-Induced Callose Deposition Is Reduced in gae1 gae6 Plants 1050

Page 46 of 54

(A) Callose deposition in four week-old Col-0 and gae1 gae6 plants Plants were1051

inoculated with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) or mock inoculated 1052

with B cinerea inoculation medium and samples were collected after 72 h Callose was 1053

stained with aniline blue and visualized using a Nikon Eclipse Ni-U microscope with a 1054

DAPI filter Three leaves were stained per treatment Scale bars are 100 microm Asterisks 1055

indicate fungal hyphae arrows indicate representative callose spots 1056

(B) Quantification of callose deposition Callose deposits were counted in 200 microm x 2001057

microm squares laid out across the images shown here Bars represent means plusmn SE from 1058

six squares each Letters indicate significantly different groups (p lt 005) according to a 1059

t-test analysis1060

1061

Figure 12 Less Soluble Pectin Accumulates in B cinerea-Infected gae1 gae6 1062

Leaves 1063

Four week-old Col-0 and gae1 gae6 plants were inoculated with 10 microL droplets of the 1064

indicated B cinerea isolate (25 x 105 spores mL-1) or mock inoculated with B cinerea 1065

inoculation medium Leaves were collected 48 h later AIR was extracted from leaf 1066

samples and water added (20 microL infection site) to extract soluble pectin fragments 1067

Samples were vortexed for 7 h centrifuged at 12000 g and the uronic acid 1068

concentration of the supernatant was measured Data from nine biological replicates 1069

were combined using a mixed linear model Means plusmn SE are shown Letters indicate 1070

significant differences (q lt 005) 1071

1072

Figure 13 Macerozyme-Induced PTI Is Lost in gae1 gae6 While OG-Induced PTI Is 1073

Unaltered 1074

(A) Macerozyme-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1075

inoculated with 001 macerozyme or mock inoculated with boiled macerozyme and 1076

were infected with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) 24 h later Samples 1077

were collected immediately (0 dpi) and after two (2 dpi) and three (3 dpi) days Data 1078

show mean plusmn SE of six to 14 biological replicates each combined using a linear model 1079

Letters indicate significantly different groups (q lt 005) 1080

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

Parsed CitationsAdam L and Somerville SC (1996) Genetic characterization of five powdery mildew disease resistance loci in Arabidopsisthaliana Plant J 9 341-356

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Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk RGadrinab C Heller C Jeske A Koesema E Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt MHazari N Hom E Karnes M Mulholland C Ndubaku R Schmidt I Guzman P Aguilar-Henonin L Schmid M Weigel DCarter DE Marchand T Risseeuw E Brogden D Zeko A Crosby WL Berry CC and Ecker JR (2003) Genome-wideinsertional mutagenesis of Arabidopsis thaliana Science 301 653-657

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Asai T Tena G Plotnikova J Willmann MR Chiu WL Gomez-Gomez L Boller T Ausubel FM and Sheen J (2002) MAPkinase signalling cascade in Arabidopsis innate immunity Nature 415 977-983

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Bartsch M Gobbato E Bednarek P Debey S Schultze JL Bautor J and Parker JE (2006) Salicylic acid-independentENHANCED DISEASE SUSCEPTIBILITY1 signaling in Arabidopsis immunity and cell death is regulated by the monooxygenaseFMO1 and the nudix hydrolase NUDT7 Plant Cell 18 1038-1051

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Bethke G Grundman RE Sreekanta S Truman W Katagiri F and Glazebrook J (2014) Arabidopsis PECTINMETHYLESTERASEs Contribute to Immunity against Pseudomonas syringae Plant Physiol 164 1093-1107

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Bouton S Leboeuf E Mouille G Leydecker MT Talbotec J Granier F Lahaye M Hofte H and Truong HN (2002)Quasimodo1 encodes a putative membrane-bound glycosyltransferase required for normal pectin synthesis and cell adhesion inArabidopsis Plant Cell 14 2577-2590

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Cao H Glazebrook J Clarke JD Volko S and Dong XN (1997) The Arabidopsis NPR1 gene that controls systemic acquiredresistance encodes a novel protein containing ankyrin repeats Cell 88 57-63

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Page 30 of 54

cinerea isolates We used macerozyme a commercial pectinase to mimic the effect of 581

B cinerea pectinases Macerozyme treatment released less pectin from gae1 gae6 cell582

walls and induced less immune signaling in gae1 gae6 plants Less water soluble pectin 583

also accumulated in B cinerea infected gae1 gae6 plants and macerozyme-induced 584

immunity to B cinerea isolate Gallo 1 was reduced in these plants We conclude that 585

the reduced pectin content in these mutants may lead to reduced release of soluble 586

pectin including active OGs during pathogen attack and that this compromises 587

immunity 588

589

590

Page 31 of 54

METHODS 591

Plant Materials and Growth Conditions 592

Wild-type Columbia (Col-0) and all mutant plants (in Col-0 background) were 593

grown on sterilized BM2 germinating mix (Berger Inc Quebec Canada) in a controlled 594

environment chamber (Conviron Pembina ND USA) with a 12-h photoperiod under 595

100 microM m-2 s-1 fluorescent illumination at 22degC and 75 relative humidity Germplasm 596

used is described in the Accession Numbers section at the end of Methods 597

598

Data Analysis and Replicates 599

For biological replicates samples were obtained from separate plant tissues and 600

in many cases from plants grown in different flats In some experiments multiple 601

technical replicates were measured per biological replicate For statistical analysis all 602

technical replicates were averaged and the averages were considered one biological 603

replicate Independent experiments were performed at different times and often 604

consisted of multiple biological replicates per experiment 605

Data analysis was performed using mixed linear models in the R programming 606

environment (Bates et al 2015) unless otherwise indicated Generally genotypes 607

treatments and time points were used as fixed effects and replicate-specific effects like 608

different pots flats etc were considered random effects We reported p-values 609

obtained from the mixed linear model when less than 10 comparisons were performed 610

and q-values for more than 10 comparisons to correct for multiple comparison testing 611

Q-values were calculated using the Benjamini-Hochberg procedure (Benjamini and612

Hochberg 1995) unless specified otherwise 613

614

Pathogen Strains Growth Conditions and Pathogen Growth Assays 615

Growth of Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) and 616

bacterial growth assays were performed as previously described (Bethke et al 2014) 617

For qRT-PCR assays four week-old plants were inoculated with bacteria (OD600 = 618

0002) or mock (5 mM MgSO4) At least four leaves from two plants were collected at 619

the indicated time points per biological replicate 620

Page 32 of 54

Prior to each experiment Botrytis cinerea isolates were grown on 1x PDA 621

medium (Difco) for 10 d at room temperature All isolates used have been described 622

previously (Rowe and Kliebenstein 2007) The spores were washed from the surface of 623

the plate using an inoculation medium (1x Gamborgrsquos B-5 basal salt mixture (Sigma-624

Aldrich G5768) 2 (wv) glucose 10 mM phosphate buffer pH 64) and fungal hyphae 625

were removed from the suspension by filtering through four layers of cheesecloth The 626

concentration of spores was determined using a hemocytometer and adjusted to 25 x 627

105 spores mL-1 Four week-old Arabidopsis plants were inoculated by placing one 10-628

microL droplet of the B cinerea spore solution or inoculation medium (mock) on the adaxial 629

leaf surface of fully expanded leaves Inoculated plants were kept at 100 relative 630

humidity Infection sites were collected at various time points using a cork borer For 631

qRT-PCR assays at least four leaf discs from one plant were combined for each 632

biological sample For B cinerea growth assays at least 12 leaf discs from three to six 633

plants were pooled per biological sample Relative fungal growth was determined by 634

measuring the abundance of a fungal gene relative to a plant gene as described below 635

In brief DNA from infected tissue was extracted and equal amounts of total DNA were 636

used to perform qPCR reactions using the B cinerea cutinase A and the Arabidopsis 637

SK11 gene respectively Primers were described in (Gachon and Saindrenan 2004) 638

and are listed in Supplemental Table 1 Two technical replicates were combined for 639

each biological replicate B cinerea is a necrotrophic fungal pathogen and may destroy 640

plant DNA as disease progresses However decreases in the amount of plant DNA 641

over the course of our experiments were only observed at late time points with very 642

susceptible plants so the qPCR assay is generally a reasonable estimate of fungal 643

biomass (Supplemental Figure 16) 644

645

Treatment with Macerozyme or OGs 646

For PTI assays four week-old plants either were inoculated with 001 (wv) 647

macerozyme R-10 (Yakult) a Rhizopus polygalacturonase (EC 32115) that possesses 648

high pectinase and hemicellulase activity or were mock inoculated using heat-649

inactivated macerozyme Other similar plants were either inoculated with 250 or 500 microg 650

mL-1 OGs or mock inoculated with water One d later plants were infected with B 651

Page 33 of 54

cinerea and samples were collected immediately (0 dpi) and two or three d later To 652

investigate the role of MeJA on B cinerea growth plants were sprayed with 1mM MeJA 653

in water with 001 (vv) Tween 20 Covers were removed so that leaves could dry off 654

before plants were inoculated with B cinerea three h later Samples were harvested at 655

the indicated time points For expression analysis and MAPK activation assays plants 656

were either inoculated with 100 microg mL-1 OGs or mock inoculated with water OGs used 657

for experiments described in Figures 8 to 10 and Supplemental Figure 12 were a kind 658

gift from Simone Ferrari OGs used in experiments described in Figure 13 and 659

Supplemental Figures 11 13 and 15 were prepared as previously described (Kohorn et 660

al 2014) 661

662

Expression Analysis 663

Leaf tissue from inoculated plants was harvested at the indicated time points 664

flash frozen pulverized and RNA extracted using Trizol (Invitrogen) Quantitative RT-665

PCR was performed using the SuperScript III Platinum SYBR Green One-Step 666

quantitative RT-PCR kit (Invitrogen) and a Lightcycler 480 Real-Time PCR system 667

(Roche) as previously described (Truman and Glazebrook 2012) In brief equal 668

amounts of total RNA and a gene specific primer were used for each reaction The 669

crossing point (Cp) was calculated using the 2nd derivative max method provided with 670

the Lightcycler software for each amplification curve Each reaction was run with two 671

technical replicates and the Cp values for these replicates were averaged ACTIN2 was 672

used as a stably expressed reference gene At least three biological replicates each 673

were performed and analyzed using a mixed-linear model RT-PCR was performed to 674

verify T-DNA insertion lines using the One-Step RT-PCR kit (Qiagen) according to the 675

manufacturerrsquos instructions The primers used can be found in Supplemental Table 1 676

677

Electrolyte Leakage Assay 678

Fully expanded leaves of four week-old plants were inoculated with water (Milli-Q 679

grade) using a needleless syringe Leaf discs were collected using a cork borer and 680

three leaf discs from one leaf were placed with the adaxial surface down onto 2 mL 681

water in a 12-well cell culture plate Conductivity was measured using a Horiba B-173 682

Page 34 of 54

conductivity meter after 30 min and then the water was replaced Conductivity was 683

measured again 30 min later (1 h total) as indicated in Figure 3C Six biological 684

replicates each were performed in three independent experiments and means and SEs 685

were calculated Two-sided t-tests were performed for each time point 686

687

DAB and Aniline Blue Staining 688

Leaves from four week-old plants were either mock inoculated with B cinerea 689

inoculation medium or inoculated with 10 microL droplets of a B cinerea spore solution (25 690

x 105 spores mL-1) of the indicated isolates Samples were collected 72 h later For 33prime-691

diaminobenzidine (DAB) staining two to five inoculated leaves were vacuum infiltrated 692

with 33prime-Diaminobenzidine (1 mg mL-1 in water pH 38) solution and stained overnight 693

in the dark Leaves were destained using 100 ethanol at 65degC and then transferred to 694

50 (vv) glycerol and photographed 695

Callose was visualized by staining leaves with aniline blue as previously 696

described (Adam and Somerville 1996) Briefly leaves were vacuum infiltrated with 697

alcoholic lactophenol (one volume of phenolglycerollactic acidwater (1111) mixed 698

with two volumes of ethanol) and destained at 65degC Next leaves were moved through 699

an alcohol gradient (50 20 and 10 (vv) ethanol) into water The destained leaves 700

were stained for 30 min in 150 mM K2HPO4 (pH 95) with 001 (wv) aniline blue 701

Samples were mounted in water and examined using a Nikon Eclipse Ni-U microscope 702

at 10x magnification using a DAPI filter (excitation at 325-375 nm emission at 435-485 703

nm) For callose quantification six 200 microm x 200 microm squares were counted for each 704

sample type Callose deposits per mm2 were calculated and data analyzed using a t-705

test Note that there is an inherent uncertainty associated with such counts because 706

callose deposits may not all be in focus at the same time 707

708

MAPK Activity Assay 709

Fully expanded leaves of four week-old plants were inoculated with 001 710

macerozyme 100 microg mL-1 OGs 100 nM flg22 or water (mock) flg22 a peptide from 711

bacterial flagella that induces immune responses was used as a positive control for 712

Page 35 of 54

MAPK activation (Asai et al 2002) and was purchased from EZBiolab Inc (Westfield 713

IN USA) Samples were harvested 10 min later and immediately frozen in liquid 714

nitrogen Protein was extracted using an extraction buffer containing 25 mM Tris pH 78 715

75 mM NaCl 1 mM DTT 1 mM NaF 05mM Na3VO4 01 (vv) Tween 20 one 716

cOmplete Mini protease inhibitor cocktail tablet (Roche) and one PhosSTOP 717

phosphatase inhibitor cocktail tablet (Roche) per 10 mL buffer 15 microg of total protein 718

were electrophoresed on a 10 SDS-polyacrylamide gel and then blotted onto a PVDF 719

membrane (Bio-Rad 162-0177) Activated MAPKs were detected using p4442 MAPK 720

(Erk12) antibody (9102S Cell Signal 12500 in TBST (20 mM Tris pH 75 150 mM 721

NaCl 01 (vv) Tween 20)) Anti-Rabbit IgG-HRP (115000 in TBST A6154 Sigma) 722

and ECL Plus substrate (Pierce 32132) MPK3 and MPK6 were detected with an anti-723

MPK3 antibody (M8318 Sigma 12000 in TBST and 3 (wv) milk) or an anti-MPK6 724

antibody (A7104 Sigma 16000 in TBST and 3 (wv) milk) respectively The 725

secondary antibody and detection were the same as for the p4442 antibody Five 726

independent experiments were performed 727

728

Preparation of Alcohol Insoluble Residue (AIR) Pectin Extraction and Uronic Acid 729

Measurements 730

Fully expanded leaves from four week-old plants that were incubated for 48 h in 731

the dark to reduce starch were harvested flash frozen and pulverized AIR was 732

extracted by washing ground material twice in 70 (vv) ethanol three times in a 733

mixture of chloroform and methanol (11 vv) and once in acetone (Gille et al 2009) 734

and was then air dried Cell wall pectin was extracted from AIR with cell wall extraction 735

buffer (50 mM Trizma 50 mM CDTA pH 72) at 95degC for 15 min Samples were 736

homogenized using a paint shaker (Harbil 5G-HD) and glass beads (3 mm diameter) 737

For dot blot experiments 500 microL of cell wall extraction buffer were used per 10 mg of 738

AIR Debris was precipitated by centrifugation for 10 min at 10000 g 739

Total uronic acid content of pectin extracted as described above using 500 microL of 740

cell wall extraction buffer per 1 mg of AIR was measured as described previously 741

(Filisetti-Cozzi and Carpita 1991 van den Hoogen et al 1998) First 36 microL of pectin 742

extract were mixed with 4 microL of 4 M sulfamic acid Then 200 microL of sulfuric acid 743

Page 36 of 54

containing 120 mM sodium tetraborate was added and samples were incubated at 80degC 744

for 1 h Following cooling on ice the optical density of samples was measured at 490 745

nm Next 40 microL of m-hydroxydiphenyl reagent (100 microL of m-hydroxydiphenyl in dimethyl 746

sulfoxide at 100 mgmL mixed with 49 mL 80 (vv) sulfuric acid just before use) was 747

added and the samples were mixed The optical density at 490 nm was measured 748

again The optical density before m-hydroxydiphenyl reagent addition was subtracted 749

from the optical density measured after addition of the dye The concentration of uronic 750

acid was calculated using known amounts of GalA as a standard 751

752

Release of Pectin from Arabidopsis Cell Walls Using Macerozyme 753

AIR preparations from leaves of four week-old plants were treated with 025 754

(wv) macerozyme R-10 (Yakult) or water (mock) at a concentration of 100 microL per mg 755

AIR Samples were incubated for 3 h at room temperature on a vortex shaker and 756

centrifuged for 10 min at 12000 g Total uronic acid concentration of 5-fold diluted 757

supernatants was determined for two technical replicates each The concentration of 758

uronic acid was calculated using known amounts of GalA as a standard For 759

subsequent qRT-PCR experiments supernatants from multiple extractions were 760

combined boiled for 20 min to inactivate macerozyme diluted 10-fold with water and 761

inoculated into four week-old Col-0 plants Heat-inactivated 10-fold diluted macerozyme 762

solutions were used as mock treatment Samples were collected 3 h later and PAD3 763

expression was determined 764

765

Release of Pectin from Arabidopsis Leaves Infected with B cinerea 766

Fully expanded leaves of four week-old plants were either inoculated with up to 767

five 10 microL droplets of B cinerea spore solution (25 x 105 spores mL-1) or mock 768

inoculated with B cinerea inoculation medium For each biological replicate leaves with 769

a combined count of at least 20 infection sites were collected 48 h later AIR was 770

extracted as described above but plants were not dark-treated or freeze-dried and the 771

first ethanol step was performed using 85 (vv) ethanol To extract soluble pectin 772

fragments AIR samples were mixed with water at a concentration of 20 microL per infection 773

site incubated for 7 h at room temperature on a vortex shaker and centrifuged for 10 774

Page 37 of 54

min at 12000 g Total uronic acid concentration of supernatants was determined for two 775

technical replicates each The concentration of uronic acid was calculated using known 776

amounts of GalA as a standard 777

778

Measurement of Uronic Acids Neutral Monosaccharides and Cellulose Content in 779

Cell Walls 780

Neutral sugars and uronic acids from non-cellulosic polysaccharides in cell walls 781

(AIR) were released by trifluoroacetic acid (TFA) hydrolysis Approximately 1 mg of AIR 782

was hydrolyzed with 2 M TFA at 121degC for 90 min The TFA-soluble material was split 783

into two equal parts one for uronic acid and one for neutral sugar determination The 784

uronic acid content was determined by analyzing the hydrolyzate using a CarboPac 785

PA200 anion-exchange column with an ICS-3000 Dionex chromatography system 786

(Dionex Sunnyvale CA USA) The elution profile consisted of a linear gradient of 50 to 787

200 mM sodium acetate in 01 M NaOH in 10 min at 04 mL per min Alditol acetate 788

derivatives of neutral sugars were produced as described (York et al 1985) In brief 789

monosaccharides in the hydrolyzate were reduced with NaBH4 and peracetylated with 790

acetic anhydride and pyridine at 121degC for 20 min The generated alditol acetates were 791

analyzed by GC-MS Crystalline cellulose was measured according to Updegraff (1969) 792

After hydrolysis of noncellulosic polysaccharides from AIR with the Updegraff reagent 793

(acetic acid nitric acids water 812 (vv) ) the remaining pellet was hydrolyzed in 794

72 sulfuric acid The resulting glucose quantity was determined by the anthrone 795

method (Scott and Melvin 1953) 796

797

Dot Blot Experiments 798

Pectin solutions extracted using 50 microL of cell wall extraction buffer per mg AIR 799

were serially diluted and nitrocellulose membranes were spotted with 1 microL of the diluted 800

pectin solutions Membranes were dried overnight blocked with 5 milk (wv) in 1x 801

PBS (8 g L-1 NaCl 02 g L-1 KCl 144 g L-1 Na2HPO4 024 g L-1 KH2PO4 pH 74) and 802

probed with LM5 (Jones et al 1997) LM6 (Willats et al 1998) LM8 (Willats et al 803

2004) LM19 and LM20 (Verhertbruggen et al 2009a) or CCRC-M7 (Steffan et al 804

Page 38 of 54

1995) antibodies LM series antibodies were diluted 1250 and CCRC-M7 was diluted 805

1500 in 5 milk powder (Nestle Carnation) in 1 x PBS For LM series antibodies a 806

goat anti-Rat HRP conjugated antibody (Bethyl A110-105P 15000 diluted) and for 807

CCRC-M7 an anti-Mouse IgG HRP conjugate (Promega W402B 12500 diluted) was 808

used as secondary antibody in 5 milk powder in 1x PBS LM series antibodies were 809

obtained from plant probes (wwwplantprobesnet) and CCRC-M7 from CarboSource 810

(wwwcarbosourcenet) Membranes were washed after incubation with the primary and 811

secondary antibodies with 1x PBS Dot blots were developed using the ECL system 812

(GE Healthcare Amersham ECL prime) For quantification of signals dot blot results on 813

x-ray films were photographed using a CCD camera and intensities were measured814

using Image Jrsquos Integrated Density function Locally adjusted background was 815

subtracted As the dynamic range of the signal on an x-ray film was narrow non-816

linearity in the measured values was corrected for each genotype in each biological 817

replicate using the values from different dilutions in two technical replicates We noticed 818

that the residuals were not normally distributed and performed Box-Cox power 819

transformation (Box and Cox 1964) to obtain normally distributed values for the 820

subsequent statistical analysis of the data For LM19 Box-Cox selected power 821

transformation was done to the power of 038th and for LM20 to the power 075th After 822

correction of the transformed measured values with the effect of the biological 823

replicates the values were fit to a linear model with the Arabidopsis genotype as the 824

fixed effect This linear model was used both to generate the plots in Figures 5B and D 825

and to test significant differences 826

827

Camalexin Measurements 828

Four week-old plants were infected with B cinerea and samples (three leaf disks 829

from one plant each) were harvested at the indicated time points flash frozen and 830

pulverized Each sample was extracted with 300 microL of 90 methanol (vv) 50 microL of the 831

extract was run on an Agilent Lichrocart 250-4 RP18e 5-microm column using an Agilent 832

1100 series HPLC Camalexin was detected using a DiodeArray (DAD) at 330 nm and 833

with a fluorescence detector at emission 318 nmexcitation 385 nm (Agilent) Separation 834

was achieved using the following program with aqueous acetonitrile 5 min gradient 835

Page 39 of 54

from 63 to 69 acetonitrile 30 s gradient from 69 to 99 acetonitrile 2 min at 99 836

acetonitrile and a post-run equilibration of 35 min at 63 acetonitrile (Denby et al 837

2004 Kliebenstein et al 2005) Purified camalexin was used to produce a standard 838

curve to identify and quantitate camalexin 839

840

Accession Numbers 841

Microarray data used can be accessed at NCBI Gene Expression Omnnibus 842

(httpwwwncbinlmnihgovgeo) GSE18978 Sequence information for all Arabidopsis 843

genes described in this article can be found in TAIR (wwwarabidopsisorg) using the 844

following accession numbers PAD3 (CYP71B15 At3g26830) SID2 (ICS1 At1g74710) 845

GAE1 (At4g30440) GAE2 (At1g02000) GAE3 (At4g00110) GAE4 (At2g45310) GAE5 846

(At4g12250) GAE6 (At3g23820) PR1 (At2g14610) JAZ5 (At1g17380) JAZ10 847

(At5g13220) ACTIN2 (At3g18780) PDF12a (At5g44420) PDF12b (At2g26020) 848

AtSK11 (At5g26751) The sequence of the B cinerea cutinase Bc-CutA (Z69264) can 849

be found in the DNA Data Bank of Japan (httpgetentryddbjnigacjp) Germplasm 850

used included dde2-2 (At5g42650 (von Malek et al 2002)) pad3-1 (At3g26830 (Zhou 851

et al 1999)) pad4-1 (At3g52430 (Jirage et al 1999)) sid2-2 (At1g74710 (Wildermuth 852

et al 2001)) pbs3-2 (At5g13320 SALK_018225 (Nobuta et al 2007)) cbp60a-1 853

(At5g62570 SALK_124410 (Truman et al 2013)) cbp60g-1 sard1-2 (At5g26920 854

SALK_023199 At1g73805 SALK_052422 (Wang et al 2011) cbp60a-1 cbp60g-1 855

sard1-2 (Truman et al 2013) gae1-1 (At4g30440 SALK_085554) gae6-1 (At3g23280 856

SALK_104454) gae6-2 (At3g23280 SALK_017191) ixr1-1 cev1 (At5g05170 (Ellis 857

and Turner 2001 Scheible et al 2001)) eds1 plants were derived by introgression of 858

the Ler eds1-2 allele into Col-0 that contains two copies of EDS1 (At3g48090 859

At3g48080 (Bartsch et al 2006) mpk6-2 (At2g43790 SALK_073907 (Liu and Zhang 860

2004)) mpk3-DG (At3g45640 a fast neutron deletion mutant (Miles et al 2005)) and 861

npr1-1 (At1g64280 (Cao et al 1997)) T-DNA insertion lines were part of the SALK 862

collection (Alonso et al 2003) and were obtained from the Arabidopsis Biological 863

Resource Center 864

865

Supplemental Data 866

Page 40 of 54

Supplemental Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 867

Expression Does Not Require CBP60s or SID2 868

Supplemental Figure 2 Expression of GAE Family Members uponTreatment with Pma 869

ES4326 870

Supplemental Figure 3 Characterization of T-DNA Insertions in GAE1 and GAE6 871

Supplemental Figure 4 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves and 872

Are Slightly Smaller than Wild-Type Col-0 Plants 873

Supplemental Figure 5 Cell Walls of gae1 gae6 Plants Contain Less Uronic Acid than 874

Wild-Type Col-0 Cell Walls 875

Supplemental Figure 6 Antibodies Raised Against the RGI or XG Components of 876

Pectin Show No Difference in Binding to gae Mutant and Col-0 Cell Walls 877

Supplemental Figure 7 The Effect of B cinerea Treatment on the Expression of GAE 878

Family Genes 879

Supplemental Figure 8 Treatment with 001 Macerozyme Does Not Cause Any 880

Visible Damage to Col-0 Plants 881

Supplemental Figure 9 B cinerea Growth on Plants with Defects in Immune 882

Signaling 883

Supplemental Figure 10 MeJA-Induced Expression of the JA marker gene JAZ10 884

Supplemental Figure 11 The Expression of the JA Marker Gene JAZ5 Is Highly 885

Responsive in gae1 gae6 Plants 886

Supplemental Figure 12 The Expression of PDF12 Is Unaltered in gae1 gae6 Plants 887

Supplemental Figure 13 Macerozyme Treatment Activates MPK3 and MPK6 888

Supplemental Figure 14 B cinerea-Induced ROS Production in Wild-Type Col-0 and 889

gae1 gae6 Plants 890

Supplemental Figure 15 OG-Induced PTI Using 250 microg mL-1 OGs 891

Supplemental Figure 16 Abundance of Plant DNA in B cinerea Treated Plants 892

Supplemental Table 1 Primers used in this study 893

894

ACKNOWLEDGEMENTS 895

We thank William Truman for helpful discussions Simone Ferrari (Sapienza 896

Universita di Roma) for the generous gift of OGs Bruna Bucciarelli (USDA-ARS) for 897

Page 41 of 54

help with fluorescence microscopy the Arabidopsis Biological Resource Center for T-898

DNA insertion lines Paul Knoxrsquos lab (University of Leeds) for LM5 LM6 LM8 LM19 899

and LM20 antibodies and the Complex Carbohydrate Resource Center (University of 900

Georgia) for the CCRC-M7 antibody Most of this work was funded by the Division of 901

Chemical Sciences Geosciences and Biosciences Office of Basic Energy Sciences of 902

the US Department of Energy through Grant DE-FG02-05ER15670 to JG This work 903

was also supported by NSF awards IOS 1339125 and IOS 1021861 to DJK NSF 904

award IOS 1121425 to F K and the USDA National Institute of Food and Agriculture 905

Hatch project number CA-D-PLS-7033-H to DJK 906

907

AUTHOR CONTRIBUTIONS 908

GB and JG designed the research GB GX AT BL NES and NH 909

performed research DJK provided tools GB GX MP AD BL RAH and FK 910

analyzed data GB and JG wrote the paper with input from all authors 911

912

FIGURE LEGENDS 913

Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 Expression 914

Requires PAD4 EDS1 and PBS3 915

(A) Expression of GAE1 in leaves of wild-type Col-0 and mutant plants Expression was916

measured 24 or 32 h after either inoculation with Pma ES4326 (OD600 = 0002) or mock 917

inoculation with 5 mM MgSO4 and was then normalized to the level of ACTIN2 Data 918

from three biological replicates were combined using a mixed linear model Bars 919

represent mean log2-ratios to ACTIN2 plusmn SE Letters indicate significantly different 920

groups with q lt 005 where 005 represents the maximum false discovery rate at which 921

the test may be called significant 922

(B) Expression of GAE6 measured as described in (A)923

924

Figure 2 Susceptibility of gae6 and gae1 gae6 Plants to Pma ES4326 Is Increased 925

Relative to Wild-Type Col-0 926

Leaves of plants of the indicated genotypes were inoculated with Pma ES4326 (OD600 = 927

00002) as described in Methods Bacterial titers in leaves were determined either 928

Page 42 of 54

immediately (0 dpi) or after three days (3 dpi) Bars represent the mean plusmn SE of four 929

independent experiments each with 4 or 12 biological replicates at 0 and 3 dpi 930

respectively Data were combined using a mixed linear model Asterisks indicate 931

significant differences from Col-0 wild-type (q lt 005) Susceptible pad4 plants were 932

included as a positive control 933

934

Figure 3 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves 935

(A) Depiction of the assay for brittleness of gae mutant leaves Fully expanded leaves of936

four to five week-old plants were bent by 180ordm so that the adaxial surface of the leaf was 937

parallel with the soil Broken leaf petioles or midribs were considered brittle 938

(B) Quantification of broken midribs or petioles For each genotype 66 to 82 leaves 939

were analyzed for brittleness as described in (A) Bars represent the ratio of broken to 940

total number of leaves tested Results were analyzed using Fisherrsquos exact test Letters 941

indicate significantly different groups at p lt 005 942

(C) Conductivity measurements Leaves of four week-old plants were inoculated with943

deionized water and then leaf discs were cut out and placed on deionized water 944

Conductivity was measured at the indicated time points After 30 min the water was 945

replaced with fresh water (30 min f w) to account for wounding-related ion leakage from 946

the edges of the leaf discs Three independent experiments with six biological replicates 947

each were performed and data were analyzed using a t-test for each time point P-948

values were corrected using the Bonferroni method Data show means plusmn SE asterisks 949

indicate significant differences from wild-type Col-0 at q lt 001 950

951

Figure 4 Cell Walls of gae6 and gae1 gae6 Plants Are Reduced in GalA 952

(A) to (D) Levels of GalA (A) GlcA (B) cellulose (C) and neutral sugars (D) in alcohol953

insoluble residue (AIR) extracted from leaves of four week-old plants of the indicated 954

genotypes Bars represent means plusmn SE of four biological replicates combined using a 955

mixed linear model Letters indicate significantly different groups (p lt 005) 956

957

Figure 5 Cell Walls of gae1 gae6 Plants Are Reduced in Homogalacturonan 958

Page 43 of 54

Dot blot analyses with anti-LM19 and anti-LM20 antibodies Pectin was extracted from 959

AIR prepared from the indicated genotypes using 1 microL of extraction buffer per 10 microg of 960

AIR Pectin was serially diluted and one microL of undiluted 13 diluted and 19 diluted 961

samples were spotted on a nitrocellulose membrane Three biological replicates were 962

were spotted twice to obtain two technical replicates each All replicates produced 963

similar results 964

(A) Representative dot blot using anti-LM19 antibody965

(B) Relative signal intensities of the spots in the dot blot in (A)966

(C) Representative dot blot using anti-LM20 antibody967

(D) Relative signal intensities of the LM20 dot blot shown in (C)968

Relative signal intensities were measured using Image J software Measurements from 969

the three biological replicates were combined using a linear model after Box-Cox power 970

transformation as described in Methods Bars represent means plusmn SE Asterisks indicate 971

a difference from wild-type Col-0 at p lt 005 972

973

Figure 6 Growth of B cinerea on gae1 gae6 Plants and the Effect of B cinerea on 974

Expression of GAE1 and GAE6 975

(A) Growth of B cinerea isolates Leaves of Col-0 and gae1 gae6 plants were976

inoculated with 25 x 105 spores mL-1 of the B cinerea isolates indicated in the figure 977

Samples were collected two (2 dpi) and three (3 dpi) days later Bars represent means plusmn 978

SE of six biological replicates for each fungus combined using a mixed linear model 979

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 980

(B) and (C) Expression of GAE1 (B) and GAE6 (C) 48 h after inoculation with the B981

cinerea isolates indicated Expression levels were measured by qRT-PCR Bars 982

represent mean log2-ratios to ACTIN2 plusmn SE of three biological replicates combined 983

using a mixed linear model Letters indicate significantly different groups (q lt 005) 984

985

Figure 7 Levels of Soluble Pectin and Expression of PAD3 in Fractions Produced 986

Following Macerozyme Treatment of gae1 gae6 Cell Walls 987

(A) Schematic overview of the experimental procedure for (B) and (C)988

Page 44 of 54

(B) Uronic acid measurements Alcohol-insoluble residue (AIR) from leave tissue of four989

week-old wild-type Col-0 or gae1 gae6 plants was treated with water or 025 990

macerozyme and assayed for uronic acid levels Data from 11 biological replicates were 991

combined using a mixed linear model Bars represent means plusmn SE Letters indicate 992

significant differences (p lt 005) 993

(C) Expression of PAD3 in Col-0 plants treated with the fractions from (B) Extracts from994

(B) were heat inactivated pooled and diluted 5-fold with water Pooled extracts were995

inoculated into four week-old Col-0 plants PAD3 expression was measured 3 h after 996

inoculation and normalized to the level of ACTIN2 expression Data from four biological 997

replicates were combined using a mixed linear model Bars represent mean log2-ratios 998

to ACTIN2 plusmn SE Letters indicate significant differences (p lt 005) 999

1000

Figure 8 Macerozyme-Induced PAD3 Expression Is Absent in gae1 gae6 1001

(A) Expression of PAD3 after macerozyme treatment Four week-old plants were either1002

inoculated with 001 macerozyme or mock inoculated with water and PAD3 1003

expression was measured 3 h later by qRT-PCR Data from four biological replicates 1004

were combined using a mixed linear model Mean log2-ratios to ACTIN2 plusmn SE were 1005

plotted Letters indicate significantly different groups (p lt 005) 1006

(B) Expression of PAD3 after OG treatment PAD3 expression was measured as1007

described in (A) 3 h after inoculation with 100 microg mL-1 OGs or mock inoculation with 1008

water Data are from six biological replicates 1009

(C) PAD3 expression in untreated plants Three biological replicates were performed1010

and data analyzed as described in (A) 1011

(D) Camalexin accumulation after B cinerea treatment Camalexin accumulation was1012

measured in four week-old plants inoculated with the B cinerea strains shown in the 1013

figure Eight biological replicates each were combined using a mixed linear model 1014

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 1015

(E) PAD3 expression 28 h after treatment with the indicated B cinerea strains Data1016

from six biological replicates were collected and analyzed as described in (A) Data in 1017

all figure parts represent means plusmn SE Letters indicate significantly different groups (q lt 1018

005) 1019

Page 45 of 54

1020

Figure 9 Macerozyme-Induced Expression of the SA Marker Genes SID2 and PR1 1021

Is Abolished in gae1 gae6 Plants 1022

(A) to (H) Expression of PR1 (A) (C) (E) and (G) or SID2 (B) (D) (F) and (H) in four1023

week-old Col-0 and gae1 gae6 plants Data were combined using a mixed linear model 1024

Bars represent mean log2-ratios of expression vs ACTIN2 plusmn SE for all figure parts 1025

Letters indicate significantly different groups at p lt 005 for panels (A) to (D) (G) and 1026

(H) and qlt005 for panels (E) to (F) 1027

(A) and (B) Plants were treated with 001 macerozyme or mock treated with water1028

and samples were collected 3 h later Data from four biological replicates are shown 1029

(C) and (D) Plants were inoculated with 100 microg mL-1 OGs or mock treated with water1030

and samples collected 3 h later Data from six biological replicates are shown 1031

(E) and (F) Plants were inoculated with the indicated B cinerea isolates or mock1032

inoculated with B cinerea inoculation medium and samples collected 48 h later Data 1033

from eight biological replicates are shown 1034

(G) and (H) Expression in untreated plants Three biological replicates were performed1035

1036

Figure 10 The Expression of the JA Marker Gene JAZ10 is Highly Responsive in 1037

gae1 gae6 Plants 1038

(A) to (D) JAZ10 expression in four week-old plants Data were combined using a mixed1039

linear model Mean log2-ratios to ACTIN2 plusmn SE are shown Letters indicate significantly 1040

different groups at p lt 005 for panels (A) (B) and (D) and q lt 005 for panel (C) 1041

(A) Plants were inoculated with 001 macerozyme or mock inoculated using water and1042

samples were collected 3 h later Data from four biological replicates are shown 1043

(B) Plants were inoculated with 100 microg mL-1 OGs or mock inoculated using water and1044

samples collected 3 h later Data are from six biological replicates 1045

(C) JAZ10 expression 48 h after treatment with the indicated B cinerea strains Data1046

are from six biological replicates 1047

(D) JAZ10 expression in untreated plants Data are from three biological replicates1048

1049

Figure 11 Gallo 1-Induced Callose Deposition Is Reduced in gae1 gae6 Plants 1050

Page 46 of 54

(A) Callose deposition in four week-old Col-0 and gae1 gae6 plants Plants were1051

inoculated with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) or mock inoculated 1052

with B cinerea inoculation medium and samples were collected after 72 h Callose was 1053

stained with aniline blue and visualized using a Nikon Eclipse Ni-U microscope with a 1054

DAPI filter Three leaves were stained per treatment Scale bars are 100 microm Asterisks 1055

indicate fungal hyphae arrows indicate representative callose spots 1056

(B) Quantification of callose deposition Callose deposits were counted in 200 microm x 2001057

microm squares laid out across the images shown here Bars represent means plusmn SE from 1058

six squares each Letters indicate significantly different groups (p lt 005) according to a 1059

t-test analysis1060

1061

Figure 12 Less Soluble Pectin Accumulates in B cinerea-Infected gae1 gae6 1062

Leaves 1063

Four week-old Col-0 and gae1 gae6 plants were inoculated with 10 microL droplets of the 1064

indicated B cinerea isolate (25 x 105 spores mL-1) or mock inoculated with B cinerea 1065

inoculation medium Leaves were collected 48 h later AIR was extracted from leaf 1066

samples and water added (20 microL infection site) to extract soluble pectin fragments 1067

Samples were vortexed for 7 h centrifuged at 12000 g and the uronic acid 1068

concentration of the supernatant was measured Data from nine biological replicates 1069

were combined using a mixed linear model Means plusmn SE are shown Letters indicate 1070

significant differences (q lt 005) 1071

1072

Figure 13 Macerozyme-Induced PTI Is Lost in gae1 gae6 While OG-Induced PTI Is 1073

Unaltered 1074

(A) Macerozyme-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1075

inoculated with 001 macerozyme or mock inoculated with boiled macerozyme and 1076

were infected with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) 24 h later Samples 1077

were collected immediately (0 dpi) and after two (2 dpi) and three (3 dpi) days Data 1078

show mean plusmn SE of six to 14 biological replicates each combined using a linear model 1079

Letters indicate significantly different groups (q lt 005) 1080

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

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Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk RGadrinab C Heller C Jeske A Koesema E Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt MHazari N Hom E Karnes M Mulholland C Ndubaku R Schmidt I Guzman P Aguilar-Henonin L Schmid M Weigel DCarter DE Marchand T Risseeuw E Brogden D Zeko A Crosby WL Berry CC and Ecker JR (2003) Genome-wideinsertional mutagenesis of Arabidopsis thaliana Science 301 653-657

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Cao H Glazebrook J Clarke JD Volko S and Dong XN (1997) The Arabidopsis NPR1 gene that controls systemic acquiredresistance encodes a novel protein containing ankyrin repeats Cell 88 57-63

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Carpita N (2010) Update on Mechanisms of Plant Cell Wall Biosynthesis How plants make cellulose and other (1-gt4)-beta-D-glycans Plant Physiol

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Chisholm ST Coaker G Day B and Staskawicz BJ (2006) Host-microbe interactions Shaping the evolution of the plantimmune response Cell 124 803-814

Page 31 of 54

METHODS 591

Plant Materials and Growth Conditions 592

Wild-type Columbia (Col-0) and all mutant plants (in Col-0 background) were 593

grown on sterilized BM2 germinating mix (Berger Inc Quebec Canada) in a controlled 594

environment chamber (Conviron Pembina ND USA) with a 12-h photoperiod under 595

100 microM m-2 s-1 fluorescent illumination at 22degC and 75 relative humidity Germplasm 596

used is described in the Accession Numbers section at the end of Methods 597

598

Data Analysis and Replicates 599

For biological replicates samples were obtained from separate plant tissues and 600

in many cases from plants grown in different flats In some experiments multiple 601

technical replicates were measured per biological replicate For statistical analysis all 602

technical replicates were averaged and the averages were considered one biological 603

replicate Independent experiments were performed at different times and often 604

consisted of multiple biological replicates per experiment 605

Data analysis was performed using mixed linear models in the R programming 606

environment (Bates et al 2015) unless otherwise indicated Generally genotypes 607

treatments and time points were used as fixed effects and replicate-specific effects like 608

different pots flats etc were considered random effects We reported p-values 609

obtained from the mixed linear model when less than 10 comparisons were performed 610

and q-values for more than 10 comparisons to correct for multiple comparison testing 611

Q-values were calculated using the Benjamini-Hochberg procedure (Benjamini and612

Hochberg 1995) unless specified otherwise 613

614

Pathogen Strains Growth Conditions and Pathogen Growth Assays 615

Growth of Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) and 616

bacterial growth assays were performed as previously described (Bethke et al 2014) 617

For qRT-PCR assays four week-old plants were inoculated with bacteria (OD600 = 618

0002) or mock (5 mM MgSO4) At least four leaves from two plants were collected at 619

the indicated time points per biological replicate 620

Page 32 of 54

Prior to each experiment Botrytis cinerea isolates were grown on 1x PDA 621

medium (Difco) for 10 d at room temperature All isolates used have been described 622

previously (Rowe and Kliebenstein 2007) The spores were washed from the surface of 623

the plate using an inoculation medium (1x Gamborgrsquos B-5 basal salt mixture (Sigma-624

Aldrich G5768) 2 (wv) glucose 10 mM phosphate buffer pH 64) and fungal hyphae 625

were removed from the suspension by filtering through four layers of cheesecloth The 626

concentration of spores was determined using a hemocytometer and adjusted to 25 x 627

105 spores mL-1 Four week-old Arabidopsis plants were inoculated by placing one 10-628

microL droplet of the B cinerea spore solution or inoculation medium (mock) on the adaxial 629

leaf surface of fully expanded leaves Inoculated plants were kept at 100 relative 630

humidity Infection sites were collected at various time points using a cork borer For 631

qRT-PCR assays at least four leaf discs from one plant were combined for each 632

biological sample For B cinerea growth assays at least 12 leaf discs from three to six 633

plants were pooled per biological sample Relative fungal growth was determined by 634

measuring the abundance of a fungal gene relative to a plant gene as described below 635

In brief DNA from infected tissue was extracted and equal amounts of total DNA were 636

used to perform qPCR reactions using the B cinerea cutinase A and the Arabidopsis 637

SK11 gene respectively Primers were described in (Gachon and Saindrenan 2004) 638

and are listed in Supplemental Table 1 Two technical replicates were combined for 639

each biological replicate B cinerea is a necrotrophic fungal pathogen and may destroy 640

plant DNA as disease progresses However decreases in the amount of plant DNA 641

over the course of our experiments were only observed at late time points with very 642

susceptible plants so the qPCR assay is generally a reasonable estimate of fungal 643

biomass (Supplemental Figure 16) 644

645

Treatment with Macerozyme or OGs 646

For PTI assays four week-old plants either were inoculated with 001 (wv) 647

macerozyme R-10 (Yakult) a Rhizopus polygalacturonase (EC 32115) that possesses 648

high pectinase and hemicellulase activity or were mock inoculated using heat-649

inactivated macerozyme Other similar plants were either inoculated with 250 or 500 microg 650

mL-1 OGs or mock inoculated with water One d later plants were infected with B 651

Page 33 of 54

cinerea and samples were collected immediately (0 dpi) and two or three d later To 652

investigate the role of MeJA on B cinerea growth plants were sprayed with 1mM MeJA 653

in water with 001 (vv) Tween 20 Covers were removed so that leaves could dry off 654

before plants were inoculated with B cinerea three h later Samples were harvested at 655

the indicated time points For expression analysis and MAPK activation assays plants 656

were either inoculated with 100 microg mL-1 OGs or mock inoculated with water OGs used 657

for experiments described in Figures 8 to 10 and Supplemental Figure 12 were a kind 658

gift from Simone Ferrari OGs used in experiments described in Figure 13 and 659

Supplemental Figures 11 13 and 15 were prepared as previously described (Kohorn et 660

al 2014) 661

662

Expression Analysis 663

Leaf tissue from inoculated plants was harvested at the indicated time points 664

flash frozen pulverized and RNA extracted using Trizol (Invitrogen) Quantitative RT-665

PCR was performed using the SuperScript III Platinum SYBR Green One-Step 666

quantitative RT-PCR kit (Invitrogen) and a Lightcycler 480 Real-Time PCR system 667

(Roche) as previously described (Truman and Glazebrook 2012) In brief equal 668

amounts of total RNA and a gene specific primer were used for each reaction The 669

crossing point (Cp) was calculated using the 2nd derivative max method provided with 670

the Lightcycler software for each amplification curve Each reaction was run with two 671

technical replicates and the Cp values for these replicates were averaged ACTIN2 was 672

used as a stably expressed reference gene At least three biological replicates each 673

were performed and analyzed using a mixed-linear model RT-PCR was performed to 674

verify T-DNA insertion lines using the One-Step RT-PCR kit (Qiagen) according to the 675

manufacturerrsquos instructions The primers used can be found in Supplemental Table 1 676

677

Electrolyte Leakage Assay 678

Fully expanded leaves of four week-old plants were inoculated with water (Milli-Q 679

grade) using a needleless syringe Leaf discs were collected using a cork borer and 680

three leaf discs from one leaf were placed with the adaxial surface down onto 2 mL 681

water in a 12-well cell culture plate Conductivity was measured using a Horiba B-173 682

Page 34 of 54

conductivity meter after 30 min and then the water was replaced Conductivity was 683

measured again 30 min later (1 h total) as indicated in Figure 3C Six biological 684

replicates each were performed in three independent experiments and means and SEs 685

were calculated Two-sided t-tests were performed for each time point 686

687

DAB and Aniline Blue Staining 688

Leaves from four week-old plants were either mock inoculated with B cinerea 689

inoculation medium or inoculated with 10 microL droplets of a B cinerea spore solution (25 690

x 105 spores mL-1) of the indicated isolates Samples were collected 72 h later For 33prime-691

diaminobenzidine (DAB) staining two to five inoculated leaves were vacuum infiltrated 692

with 33prime-Diaminobenzidine (1 mg mL-1 in water pH 38) solution and stained overnight 693

in the dark Leaves were destained using 100 ethanol at 65degC and then transferred to 694

50 (vv) glycerol and photographed 695

Callose was visualized by staining leaves with aniline blue as previously 696

described (Adam and Somerville 1996) Briefly leaves were vacuum infiltrated with 697

alcoholic lactophenol (one volume of phenolglycerollactic acidwater (1111) mixed 698

with two volumes of ethanol) and destained at 65degC Next leaves were moved through 699

an alcohol gradient (50 20 and 10 (vv) ethanol) into water The destained leaves 700

were stained for 30 min in 150 mM K2HPO4 (pH 95) with 001 (wv) aniline blue 701

Samples were mounted in water and examined using a Nikon Eclipse Ni-U microscope 702

at 10x magnification using a DAPI filter (excitation at 325-375 nm emission at 435-485 703

nm) For callose quantification six 200 microm x 200 microm squares were counted for each 704

sample type Callose deposits per mm2 were calculated and data analyzed using a t-705

test Note that there is an inherent uncertainty associated with such counts because 706

callose deposits may not all be in focus at the same time 707

708

MAPK Activity Assay 709

Fully expanded leaves of four week-old plants were inoculated with 001 710

macerozyme 100 microg mL-1 OGs 100 nM flg22 or water (mock) flg22 a peptide from 711

bacterial flagella that induces immune responses was used as a positive control for 712

Page 35 of 54

MAPK activation (Asai et al 2002) and was purchased from EZBiolab Inc (Westfield 713

IN USA) Samples were harvested 10 min later and immediately frozen in liquid 714

nitrogen Protein was extracted using an extraction buffer containing 25 mM Tris pH 78 715

75 mM NaCl 1 mM DTT 1 mM NaF 05mM Na3VO4 01 (vv) Tween 20 one 716

cOmplete Mini protease inhibitor cocktail tablet (Roche) and one PhosSTOP 717

phosphatase inhibitor cocktail tablet (Roche) per 10 mL buffer 15 microg of total protein 718

were electrophoresed on a 10 SDS-polyacrylamide gel and then blotted onto a PVDF 719

membrane (Bio-Rad 162-0177) Activated MAPKs were detected using p4442 MAPK 720

(Erk12) antibody (9102S Cell Signal 12500 in TBST (20 mM Tris pH 75 150 mM 721

NaCl 01 (vv) Tween 20)) Anti-Rabbit IgG-HRP (115000 in TBST A6154 Sigma) 722

and ECL Plus substrate (Pierce 32132) MPK3 and MPK6 were detected with an anti-723

MPK3 antibody (M8318 Sigma 12000 in TBST and 3 (wv) milk) or an anti-MPK6 724

antibody (A7104 Sigma 16000 in TBST and 3 (wv) milk) respectively The 725

secondary antibody and detection were the same as for the p4442 antibody Five 726

independent experiments were performed 727

728

Preparation of Alcohol Insoluble Residue (AIR) Pectin Extraction and Uronic Acid 729

Measurements 730

Fully expanded leaves from four week-old plants that were incubated for 48 h in 731

the dark to reduce starch were harvested flash frozen and pulverized AIR was 732

extracted by washing ground material twice in 70 (vv) ethanol three times in a 733

mixture of chloroform and methanol (11 vv) and once in acetone (Gille et al 2009) 734

and was then air dried Cell wall pectin was extracted from AIR with cell wall extraction 735

buffer (50 mM Trizma 50 mM CDTA pH 72) at 95degC for 15 min Samples were 736

homogenized using a paint shaker (Harbil 5G-HD) and glass beads (3 mm diameter) 737

For dot blot experiments 500 microL of cell wall extraction buffer were used per 10 mg of 738

AIR Debris was precipitated by centrifugation for 10 min at 10000 g 739

Total uronic acid content of pectin extracted as described above using 500 microL of 740

cell wall extraction buffer per 1 mg of AIR was measured as described previously 741

(Filisetti-Cozzi and Carpita 1991 van den Hoogen et al 1998) First 36 microL of pectin 742

extract were mixed with 4 microL of 4 M sulfamic acid Then 200 microL of sulfuric acid 743

Page 36 of 54

containing 120 mM sodium tetraborate was added and samples were incubated at 80degC 744

for 1 h Following cooling on ice the optical density of samples was measured at 490 745

nm Next 40 microL of m-hydroxydiphenyl reagent (100 microL of m-hydroxydiphenyl in dimethyl 746

sulfoxide at 100 mgmL mixed with 49 mL 80 (vv) sulfuric acid just before use) was 747

added and the samples were mixed The optical density at 490 nm was measured 748

again The optical density before m-hydroxydiphenyl reagent addition was subtracted 749

from the optical density measured after addition of the dye The concentration of uronic 750

acid was calculated using known amounts of GalA as a standard 751

752

Release of Pectin from Arabidopsis Cell Walls Using Macerozyme 753

AIR preparations from leaves of four week-old plants were treated with 025 754

(wv) macerozyme R-10 (Yakult) or water (mock) at a concentration of 100 microL per mg 755

AIR Samples were incubated for 3 h at room temperature on a vortex shaker and 756

centrifuged for 10 min at 12000 g Total uronic acid concentration of 5-fold diluted 757

supernatants was determined for two technical replicates each The concentration of 758

uronic acid was calculated using known amounts of GalA as a standard For 759

subsequent qRT-PCR experiments supernatants from multiple extractions were 760

combined boiled for 20 min to inactivate macerozyme diluted 10-fold with water and 761

inoculated into four week-old Col-0 plants Heat-inactivated 10-fold diluted macerozyme 762

solutions were used as mock treatment Samples were collected 3 h later and PAD3 763

expression was determined 764

765

Release of Pectin from Arabidopsis Leaves Infected with B cinerea 766

Fully expanded leaves of four week-old plants were either inoculated with up to 767

five 10 microL droplets of B cinerea spore solution (25 x 105 spores mL-1) or mock 768

inoculated with B cinerea inoculation medium For each biological replicate leaves with 769

a combined count of at least 20 infection sites were collected 48 h later AIR was 770

extracted as described above but plants were not dark-treated or freeze-dried and the 771

first ethanol step was performed using 85 (vv) ethanol To extract soluble pectin 772

fragments AIR samples were mixed with water at a concentration of 20 microL per infection 773

site incubated for 7 h at room temperature on a vortex shaker and centrifuged for 10 774

Page 37 of 54

min at 12000 g Total uronic acid concentration of supernatants was determined for two 775

technical replicates each The concentration of uronic acid was calculated using known 776

amounts of GalA as a standard 777

778

Measurement of Uronic Acids Neutral Monosaccharides and Cellulose Content in 779

Cell Walls 780

Neutral sugars and uronic acids from non-cellulosic polysaccharides in cell walls 781

(AIR) were released by trifluoroacetic acid (TFA) hydrolysis Approximately 1 mg of AIR 782

was hydrolyzed with 2 M TFA at 121degC for 90 min The TFA-soluble material was split 783

into two equal parts one for uronic acid and one for neutral sugar determination The 784

uronic acid content was determined by analyzing the hydrolyzate using a CarboPac 785

PA200 anion-exchange column with an ICS-3000 Dionex chromatography system 786

(Dionex Sunnyvale CA USA) The elution profile consisted of a linear gradient of 50 to 787

200 mM sodium acetate in 01 M NaOH in 10 min at 04 mL per min Alditol acetate 788

derivatives of neutral sugars were produced as described (York et al 1985) In brief 789

monosaccharides in the hydrolyzate were reduced with NaBH4 and peracetylated with 790

acetic anhydride and pyridine at 121degC for 20 min The generated alditol acetates were 791

analyzed by GC-MS Crystalline cellulose was measured according to Updegraff (1969) 792

After hydrolysis of noncellulosic polysaccharides from AIR with the Updegraff reagent 793

(acetic acid nitric acids water 812 (vv) ) the remaining pellet was hydrolyzed in 794

72 sulfuric acid The resulting glucose quantity was determined by the anthrone 795

method (Scott and Melvin 1953) 796

797

Dot Blot Experiments 798

Pectin solutions extracted using 50 microL of cell wall extraction buffer per mg AIR 799

were serially diluted and nitrocellulose membranes were spotted with 1 microL of the diluted 800

pectin solutions Membranes were dried overnight blocked with 5 milk (wv) in 1x 801

PBS (8 g L-1 NaCl 02 g L-1 KCl 144 g L-1 Na2HPO4 024 g L-1 KH2PO4 pH 74) and 802

probed with LM5 (Jones et al 1997) LM6 (Willats et al 1998) LM8 (Willats et al 803

2004) LM19 and LM20 (Verhertbruggen et al 2009a) or CCRC-M7 (Steffan et al 804

Page 38 of 54

1995) antibodies LM series antibodies were diluted 1250 and CCRC-M7 was diluted 805

1500 in 5 milk powder (Nestle Carnation) in 1 x PBS For LM series antibodies a 806

goat anti-Rat HRP conjugated antibody (Bethyl A110-105P 15000 diluted) and for 807

CCRC-M7 an anti-Mouse IgG HRP conjugate (Promega W402B 12500 diluted) was 808

used as secondary antibody in 5 milk powder in 1x PBS LM series antibodies were 809

obtained from plant probes (wwwplantprobesnet) and CCRC-M7 from CarboSource 810

(wwwcarbosourcenet) Membranes were washed after incubation with the primary and 811

secondary antibodies with 1x PBS Dot blots were developed using the ECL system 812

(GE Healthcare Amersham ECL prime) For quantification of signals dot blot results on 813

x-ray films were photographed using a CCD camera and intensities were measured814

using Image Jrsquos Integrated Density function Locally adjusted background was 815

subtracted As the dynamic range of the signal on an x-ray film was narrow non-816

linearity in the measured values was corrected for each genotype in each biological 817

replicate using the values from different dilutions in two technical replicates We noticed 818

that the residuals were not normally distributed and performed Box-Cox power 819

transformation (Box and Cox 1964) to obtain normally distributed values for the 820

subsequent statistical analysis of the data For LM19 Box-Cox selected power 821

transformation was done to the power of 038th and for LM20 to the power 075th After 822

correction of the transformed measured values with the effect of the biological 823

replicates the values were fit to a linear model with the Arabidopsis genotype as the 824

fixed effect This linear model was used both to generate the plots in Figures 5B and D 825

and to test significant differences 826

827

Camalexin Measurements 828

Four week-old plants were infected with B cinerea and samples (three leaf disks 829

from one plant each) were harvested at the indicated time points flash frozen and 830

pulverized Each sample was extracted with 300 microL of 90 methanol (vv) 50 microL of the 831

extract was run on an Agilent Lichrocart 250-4 RP18e 5-microm column using an Agilent 832

1100 series HPLC Camalexin was detected using a DiodeArray (DAD) at 330 nm and 833

with a fluorescence detector at emission 318 nmexcitation 385 nm (Agilent) Separation 834

was achieved using the following program with aqueous acetonitrile 5 min gradient 835

Page 39 of 54

from 63 to 69 acetonitrile 30 s gradient from 69 to 99 acetonitrile 2 min at 99 836

acetonitrile and a post-run equilibration of 35 min at 63 acetonitrile (Denby et al 837

2004 Kliebenstein et al 2005) Purified camalexin was used to produce a standard 838

curve to identify and quantitate camalexin 839

840

Accession Numbers 841

Microarray data used can be accessed at NCBI Gene Expression Omnnibus 842

(httpwwwncbinlmnihgovgeo) GSE18978 Sequence information for all Arabidopsis 843

genes described in this article can be found in TAIR (wwwarabidopsisorg) using the 844

following accession numbers PAD3 (CYP71B15 At3g26830) SID2 (ICS1 At1g74710) 845

GAE1 (At4g30440) GAE2 (At1g02000) GAE3 (At4g00110) GAE4 (At2g45310) GAE5 846

(At4g12250) GAE6 (At3g23820) PR1 (At2g14610) JAZ5 (At1g17380) JAZ10 847

(At5g13220) ACTIN2 (At3g18780) PDF12a (At5g44420) PDF12b (At2g26020) 848

AtSK11 (At5g26751) The sequence of the B cinerea cutinase Bc-CutA (Z69264) can 849

be found in the DNA Data Bank of Japan (httpgetentryddbjnigacjp) Germplasm 850

used included dde2-2 (At5g42650 (von Malek et al 2002)) pad3-1 (At3g26830 (Zhou 851

et al 1999)) pad4-1 (At3g52430 (Jirage et al 1999)) sid2-2 (At1g74710 (Wildermuth 852

et al 2001)) pbs3-2 (At5g13320 SALK_018225 (Nobuta et al 2007)) cbp60a-1 853

(At5g62570 SALK_124410 (Truman et al 2013)) cbp60g-1 sard1-2 (At5g26920 854

SALK_023199 At1g73805 SALK_052422 (Wang et al 2011) cbp60a-1 cbp60g-1 855

sard1-2 (Truman et al 2013) gae1-1 (At4g30440 SALK_085554) gae6-1 (At3g23280 856

SALK_104454) gae6-2 (At3g23280 SALK_017191) ixr1-1 cev1 (At5g05170 (Ellis 857

and Turner 2001 Scheible et al 2001)) eds1 plants were derived by introgression of 858

the Ler eds1-2 allele into Col-0 that contains two copies of EDS1 (At3g48090 859

At3g48080 (Bartsch et al 2006) mpk6-2 (At2g43790 SALK_073907 (Liu and Zhang 860

2004)) mpk3-DG (At3g45640 a fast neutron deletion mutant (Miles et al 2005)) and 861

npr1-1 (At1g64280 (Cao et al 1997)) T-DNA insertion lines were part of the SALK 862

collection (Alonso et al 2003) and were obtained from the Arabidopsis Biological 863

Resource Center 864

865

Supplemental Data 866

Page 40 of 54

Supplemental Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 867

Expression Does Not Require CBP60s or SID2 868

Supplemental Figure 2 Expression of GAE Family Members uponTreatment with Pma 869

ES4326 870

Supplemental Figure 3 Characterization of T-DNA Insertions in GAE1 and GAE6 871

Supplemental Figure 4 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves and 872

Are Slightly Smaller than Wild-Type Col-0 Plants 873

Supplemental Figure 5 Cell Walls of gae1 gae6 Plants Contain Less Uronic Acid than 874

Wild-Type Col-0 Cell Walls 875

Supplemental Figure 6 Antibodies Raised Against the RGI or XG Components of 876

Pectin Show No Difference in Binding to gae Mutant and Col-0 Cell Walls 877

Supplemental Figure 7 The Effect of B cinerea Treatment on the Expression of GAE 878

Family Genes 879

Supplemental Figure 8 Treatment with 001 Macerozyme Does Not Cause Any 880

Visible Damage to Col-0 Plants 881

Supplemental Figure 9 B cinerea Growth on Plants with Defects in Immune 882

Signaling 883

Supplemental Figure 10 MeJA-Induced Expression of the JA marker gene JAZ10 884

Supplemental Figure 11 The Expression of the JA Marker Gene JAZ5 Is Highly 885

Responsive in gae1 gae6 Plants 886

Supplemental Figure 12 The Expression of PDF12 Is Unaltered in gae1 gae6 Plants 887

Supplemental Figure 13 Macerozyme Treatment Activates MPK3 and MPK6 888

Supplemental Figure 14 B cinerea-Induced ROS Production in Wild-Type Col-0 and 889

gae1 gae6 Plants 890

Supplemental Figure 15 OG-Induced PTI Using 250 microg mL-1 OGs 891

Supplemental Figure 16 Abundance of Plant DNA in B cinerea Treated Plants 892

Supplemental Table 1 Primers used in this study 893

894

ACKNOWLEDGEMENTS 895

We thank William Truman for helpful discussions Simone Ferrari (Sapienza 896

Universita di Roma) for the generous gift of OGs Bruna Bucciarelli (USDA-ARS) for 897

Page 41 of 54

help with fluorescence microscopy the Arabidopsis Biological Resource Center for T-898

DNA insertion lines Paul Knoxrsquos lab (University of Leeds) for LM5 LM6 LM8 LM19 899

and LM20 antibodies and the Complex Carbohydrate Resource Center (University of 900

Georgia) for the CCRC-M7 antibody Most of this work was funded by the Division of 901

Chemical Sciences Geosciences and Biosciences Office of Basic Energy Sciences of 902

the US Department of Energy through Grant DE-FG02-05ER15670 to JG This work 903

was also supported by NSF awards IOS 1339125 and IOS 1021861 to DJK NSF 904

award IOS 1121425 to F K and the USDA National Institute of Food and Agriculture 905

Hatch project number CA-D-PLS-7033-H to DJK 906

907

AUTHOR CONTRIBUTIONS 908

GB and JG designed the research GB GX AT BL NES and NH 909

performed research DJK provided tools GB GX MP AD BL RAH and FK 910

analyzed data GB and JG wrote the paper with input from all authors 911

912

FIGURE LEGENDS 913

Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 Expression 914

Requires PAD4 EDS1 and PBS3 915

(A) Expression of GAE1 in leaves of wild-type Col-0 and mutant plants Expression was916

measured 24 or 32 h after either inoculation with Pma ES4326 (OD600 = 0002) or mock 917

inoculation with 5 mM MgSO4 and was then normalized to the level of ACTIN2 Data 918

from three biological replicates were combined using a mixed linear model Bars 919

represent mean log2-ratios to ACTIN2 plusmn SE Letters indicate significantly different 920

groups with q lt 005 where 005 represents the maximum false discovery rate at which 921

the test may be called significant 922

(B) Expression of GAE6 measured as described in (A)923

924

Figure 2 Susceptibility of gae6 and gae1 gae6 Plants to Pma ES4326 Is Increased 925

Relative to Wild-Type Col-0 926

Leaves of plants of the indicated genotypes were inoculated with Pma ES4326 (OD600 = 927

00002) as described in Methods Bacterial titers in leaves were determined either 928

Page 42 of 54

immediately (0 dpi) or after three days (3 dpi) Bars represent the mean plusmn SE of four 929

independent experiments each with 4 or 12 biological replicates at 0 and 3 dpi 930

respectively Data were combined using a mixed linear model Asterisks indicate 931

significant differences from Col-0 wild-type (q lt 005) Susceptible pad4 plants were 932

included as a positive control 933

934

Figure 3 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves 935

(A) Depiction of the assay for brittleness of gae mutant leaves Fully expanded leaves of936

four to five week-old plants were bent by 180ordm so that the adaxial surface of the leaf was 937

parallel with the soil Broken leaf petioles or midribs were considered brittle 938

(B) Quantification of broken midribs or petioles For each genotype 66 to 82 leaves 939

were analyzed for brittleness as described in (A) Bars represent the ratio of broken to 940

total number of leaves tested Results were analyzed using Fisherrsquos exact test Letters 941

indicate significantly different groups at p lt 005 942

(C) Conductivity measurements Leaves of four week-old plants were inoculated with943

deionized water and then leaf discs were cut out and placed on deionized water 944

Conductivity was measured at the indicated time points After 30 min the water was 945

replaced with fresh water (30 min f w) to account for wounding-related ion leakage from 946

the edges of the leaf discs Three independent experiments with six biological replicates 947

each were performed and data were analyzed using a t-test for each time point P-948

values were corrected using the Bonferroni method Data show means plusmn SE asterisks 949

indicate significant differences from wild-type Col-0 at q lt 001 950

951

Figure 4 Cell Walls of gae6 and gae1 gae6 Plants Are Reduced in GalA 952

(A) to (D) Levels of GalA (A) GlcA (B) cellulose (C) and neutral sugars (D) in alcohol953

insoluble residue (AIR) extracted from leaves of four week-old plants of the indicated 954

genotypes Bars represent means plusmn SE of four biological replicates combined using a 955

mixed linear model Letters indicate significantly different groups (p lt 005) 956

957

Figure 5 Cell Walls of gae1 gae6 Plants Are Reduced in Homogalacturonan 958

Page 43 of 54

Dot blot analyses with anti-LM19 and anti-LM20 antibodies Pectin was extracted from 959

AIR prepared from the indicated genotypes using 1 microL of extraction buffer per 10 microg of 960

AIR Pectin was serially diluted and one microL of undiluted 13 diluted and 19 diluted 961

samples were spotted on a nitrocellulose membrane Three biological replicates were 962

were spotted twice to obtain two technical replicates each All replicates produced 963

similar results 964

(A) Representative dot blot using anti-LM19 antibody965

(B) Relative signal intensities of the spots in the dot blot in (A)966

(C) Representative dot blot using anti-LM20 antibody967

(D) Relative signal intensities of the LM20 dot blot shown in (C)968

Relative signal intensities were measured using Image J software Measurements from 969

the three biological replicates were combined using a linear model after Box-Cox power 970

transformation as described in Methods Bars represent means plusmn SE Asterisks indicate 971

a difference from wild-type Col-0 at p lt 005 972

973

Figure 6 Growth of B cinerea on gae1 gae6 Plants and the Effect of B cinerea on 974

Expression of GAE1 and GAE6 975

(A) Growth of B cinerea isolates Leaves of Col-0 and gae1 gae6 plants were976

inoculated with 25 x 105 spores mL-1 of the B cinerea isolates indicated in the figure 977

Samples were collected two (2 dpi) and three (3 dpi) days later Bars represent means plusmn 978

SE of six biological replicates for each fungus combined using a mixed linear model 979

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 980

(B) and (C) Expression of GAE1 (B) and GAE6 (C) 48 h after inoculation with the B981

cinerea isolates indicated Expression levels were measured by qRT-PCR Bars 982

represent mean log2-ratios to ACTIN2 plusmn SE of three biological replicates combined 983

using a mixed linear model Letters indicate significantly different groups (q lt 005) 984

985

Figure 7 Levels of Soluble Pectin and Expression of PAD3 in Fractions Produced 986

Following Macerozyme Treatment of gae1 gae6 Cell Walls 987

(A) Schematic overview of the experimental procedure for (B) and (C)988

Page 44 of 54

(B) Uronic acid measurements Alcohol-insoluble residue (AIR) from leave tissue of four989

week-old wild-type Col-0 or gae1 gae6 plants was treated with water or 025 990

macerozyme and assayed for uronic acid levels Data from 11 biological replicates were 991

combined using a mixed linear model Bars represent means plusmn SE Letters indicate 992

significant differences (p lt 005) 993

(C) Expression of PAD3 in Col-0 plants treated with the fractions from (B) Extracts from994

(B) were heat inactivated pooled and diluted 5-fold with water Pooled extracts were995

inoculated into four week-old Col-0 plants PAD3 expression was measured 3 h after 996

inoculation and normalized to the level of ACTIN2 expression Data from four biological 997

replicates were combined using a mixed linear model Bars represent mean log2-ratios 998

to ACTIN2 plusmn SE Letters indicate significant differences (p lt 005) 999

1000

Figure 8 Macerozyme-Induced PAD3 Expression Is Absent in gae1 gae6 1001

(A) Expression of PAD3 after macerozyme treatment Four week-old plants were either1002

inoculated with 001 macerozyme or mock inoculated with water and PAD3 1003

expression was measured 3 h later by qRT-PCR Data from four biological replicates 1004

were combined using a mixed linear model Mean log2-ratios to ACTIN2 plusmn SE were 1005

plotted Letters indicate significantly different groups (p lt 005) 1006

(B) Expression of PAD3 after OG treatment PAD3 expression was measured as1007

described in (A) 3 h after inoculation with 100 microg mL-1 OGs or mock inoculation with 1008

water Data are from six biological replicates 1009

(C) PAD3 expression in untreated plants Three biological replicates were performed1010

and data analyzed as described in (A) 1011

(D) Camalexin accumulation after B cinerea treatment Camalexin accumulation was1012

measured in four week-old plants inoculated with the B cinerea strains shown in the 1013

figure Eight biological replicates each were combined using a mixed linear model 1014

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 1015

(E) PAD3 expression 28 h after treatment with the indicated B cinerea strains Data1016

from six biological replicates were collected and analyzed as described in (A) Data in 1017

all figure parts represent means plusmn SE Letters indicate significantly different groups (q lt 1018

005) 1019

Page 45 of 54

1020

Figure 9 Macerozyme-Induced Expression of the SA Marker Genes SID2 and PR1 1021

Is Abolished in gae1 gae6 Plants 1022

(A) to (H) Expression of PR1 (A) (C) (E) and (G) or SID2 (B) (D) (F) and (H) in four1023

week-old Col-0 and gae1 gae6 plants Data were combined using a mixed linear model 1024

Bars represent mean log2-ratios of expression vs ACTIN2 plusmn SE for all figure parts 1025

Letters indicate significantly different groups at p lt 005 for panels (A) to (D) (G) and 1026

(H) and qlt005 for panels (E) to (F) 1027

(A) and (B) Plants were treated with 001 macerozyme or mock treated with water1028

and samples were collected 3 h later Data from four biological replicates are shown 1029

(C) and (D) Plants were inoculated with 100 microg mL-1 OGs or mock treated with water1030

and samples collected 3 h later Data from six biological replicates are shown 1031

(E) and (F) Plants were inoculated with the indicated B cinerea isolates or mock1032

inoculated with B cinerea inoculation medium and samples collected 48 h later Data 1033

from eight biological replicates are shown 1034

(G) and (H) Expression in untreated plants Three biological replicates were performed1035

1036

Figure 10 The Expression of the JA Marker Gene JAZ10 is Highly Responsive in 1037

gae1 gae6 Plants 1038

(A) to (D) JAZ10 expression in four week-old plants Data were combined using a mixed1039

linear model Mean log2-ratios to ACTIN2 plusmn SE are shown Letters indicate significantly 1040

different groups at p lt 005 for panels (A) (B) and (D) and q lt 005 for panel (C) 1041

(A) Plants were inoculated with 001 macerozyme or mock inoculated using water and1042

samples were collected 3 h later Data from four biological replicates are shown 1043

(B) Plants were inoculated with 100 microg mL-1 OGs or mock inoculated using water and1044

samples collected 3 h later Data are from six biological replicates 1045

(C) JAZ10 expression 48 h after treatment with the indicated B cinerea strains Data1046

are from six biological replicates 1047

(D) JAZ10 expression in untreated plants Data are from three biological replicates1048

1049

Figure 11 Gallo 1-Induced Callose Deposition Is Reduced in gae1 gae6 Plants 1050

Page 46 of 54

(A) Callose deposition in four week-old Col-0 and gae1 gae6 plants Plants were1051

inoculated with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) or mock inoculated 1052

with B cinerea inoculation medium and samples were collected after 72 h Callose was 1053

stained with aniline blue and visualized using a Nikon Eclipse Ni-U microscope with a 1054

DAPI filter Three leaves were stained per treatment Scale bars are 100 microm Asterisks 1055

indicate fungal hyphae arrows indicate representative callose spots 1056

(B) Quantification of callose deposition Callose deposits were counted in 200 microm x 2001057

microm squares laid out across the images shown here Bars represent means plusmn SE from 1058

six squares each Letters indicate significantly different groups (p lt 005) according to a 1059

t-test analysis1060

1061

Figure 12 Less Soluble Pectin Accumulates in B cinerea-Infected gae1 gae6 1062

Leaves 1063

Four week-old Col-0 and gae1 gae6 plants were inoculated with 10 microL droplets of the 1064

indicated B cinerea isolate (25 x 105 spores mL-1) or mock inoculated with B cinerea 1065

inoculation medium Leaves were collected 48 h later AIR was extracted from leaf 1066

samples and water added (20 microL infection site) to extract soluble pectin fragments 1067

Samples were vortexed for 7 h centrifuged at 12000 g and the uronic acid 1068

concentration of the supernatant was measured Data from nine biological replicates 1069

were combined using a mixed linear model Means plusmn SE are shown Letters indicate 1070

significant differences (q lt 005) 1071

1072

Figure 13 Macerozyme-Induced PTI Is Lost in gae1 gae6 While OG-Induced PTI Is 1073

Unaltered 1074

(A) Macerozyme-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1075

inoculated with 001 macerozyme or mock inoculated with boiled macerozyme and 1076

were infected with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) 24 h later Samples 1077

were collected immediately (0 dpi) and after two (2 dpi) and three (3 dpi) days Data 1078

show mean plusmn SE of six to 14 biological replicates each combined using a linear model 1079

Letters indicate significantly different groups (q lt 005) 1080

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

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Page 32 of 54

Prior to each experiment Botrytis cinerea isolates were grown on 1x PDA 621

medium (Difco) for 10 d at room temperature All isolates used have been described 622

previously (Rowe and Kliebenstein 2007) The spores were washed from the surface of 623

the plate using an inoculation medium (1x Gamborgrsquos B-5 basal salt mixture (Sigma-624

Aldrich G5768) 2 (wv) glucose 10 mM phosphate buffer pH 64) and fungal hyphae 625

were removed from the suspension by filtering through four layers of cheesecloth The 626

concentration of spores was determined using a hemocytometer and adjusted to 25 x 627

105 spores mL-1 Four week-old Arabidopsis plants were inoculated by placing one 10-628

microL droplet of the B cinerea spore solution or inoculation medium (mock) on the adaxial 629

leaf surface of fully expanded leaves Inoculated plants were kept at 100 relative 630

humidity Infection sites were collected at various time points using a cork borer For 631

qRT-PCR assays at least four leaf discs from one plant were combined for each 632

biological sample For B cinerea growth assays at least 12 leaf discs from three to six 633

plants were pooled per biological sample Relative fungal growth was determined by 634

measuring the abundance of a fungal gene relative to a plant gene as described below 635

In brief DNA from infected tissue was extracted and equal amounts of total DNA were 636

used to perform qPCR reactions using the B cinerea cutinase A and the Arabidopsis 637

SK11 gene respectively Primers were described in (Gachon and Saindrenan 2004) 638

and are listed in Supplemental Table 1 Two technical replicates were combined for 639

each biological replicate B cinerea is a necrotrophic fungal pathogen and may destroy 640

plant DNA as disease progresses However decreases in the amount of plant DNA 641

over the course of our experiments were only observed at late time points with very 642

susceptible plants so the qPCR assay is generally a reasonable estimate of fungal 643

biomass (Supplemental Figure 16) 644

645

Treatment with Macerozyme or OGs 646

For PTI assays four week-old plants either were inoculated with 001 (wv) 647

macerozyme R-10 (Yakult) a Rhizopus polygalacturonase (EC 32115) that possesses 648

high pectinase and hemicellulase activity or were mock inoculated using heat-649

inactivated macerozyme Other similar plants were either inoculated with 250 or 500 microg 650

mL-1 OGs or mock inoculated with water One d later plants were infected with B 651

Page 33 of 54

cinerea and samples were collected immediately (0 dpi) and two or three d later To 652

investigate the role of MeJA on B cinerea growth plants were sprayed with 1mM MeJA 653

in water with 001 (vv) Tween 20 Covers were removed so that leaves could dry off 654

before plants were inoculated with B cinerea three h later Samples were harvested at 655

the indicated time points For expression analysis and MAPK activation assays plants 656

were either inoculated with 100 microg mL-1 OGs or mock inoculated with water OGs used 657

for experiments described in Figures 8 to 10 and Supplemental Figure 12 were a kind 658

gift from Simone Ferrari OGs used in experiments described in Figure 13 and 659

Supplemental Figures 11 13 and 15 were prepared as previously described (Kohorn et 660

al 2014) 661

662

Expression Analysis 663

Leaf tissue from inoculated plants was harvested at the indicated time points 664

flash frozen pulverized and RNA extracted using Trizol (Invitrogen) Quantitative RT-665

PCR was performed using the SuperScript III Platinum SYBR Green One-Step 666

quantitative RT-PCR kit (Invitrogen) and a Lightcycler 480 Real-Time PCR system 667

(Roche) as previously described (Truman and Glazebrook 2012) In brief equal 668

amounts of total RNA and a gene specific primer were used for each reaction The 669

crossing point (Cp) was calculated using the 2nd derivative max method provided with 670

the Lightcycler software for each amplification curve Each reaction was run with two 671

technical replicates and the Cp values for these replicates were averaged ACTIN2 was 672

used as a stably expressed reference gene At least three biological replicates each 673

were performed and analyzed using a mixed-linear model RT-PCR was performed to 674

verify T-DNA insertion lines using the One-Step RT-PCR kit (Qiagen) according to the 675

manufacturerrsquos instructions The primers used can be found in Supplemental Table 1 676

677

Electrolyte Leakage Assay 678

Fully expanded leaves of four week-old plants were inoculated with water (Milli-Q 679

grade) using a needleless syringe Leaf discs were collected using a cork borer and 680

three leaf discs from one leaf were placed with the adaxial surface down onto 2 mL 681

water in a 12-well cell culture plate Conductivity was measured using a Horiba B-173 682

Page 34 of 54

conductivity meter after 30 min and then the water was replaced Conductivity was 683

measured again 30 min later (1 h total) as indicated in Figure 3C Six biological 684

replicates each were performed in three independent experiments and means and SEs 685

were calculated Two-sided t-tests were performed for each time point 686

687

DAB and Aniline Blue Staining 688

Leaves from four week-old plants were either mock inoculated with B cinerea 689

inoculation medium or inoculated with 10 microL droplets of a B cinerea spore solution (25 690

x 105 spores mL-1) of the indicated isolates Samples were collected 72 h later For 33prime-691

diaminobenzidine (DAB) staining two to five inoculated leaves were vacuum infiltrated 692

with 33prime-Diaminobenzidine (1 mg mL-1 in water pH 38) solution and stained overnight 693

in the dark Leaves were destained using 100 ethanol at 65degC and then transferred to 694

50 (vv) glycerol and photographed 695

Callose was visualized by staining leaves with aniline blue as previously 696

described (Adam and Somerville 1996) Briefly leaves were vacuum infiltrated with 697

alcoholic lactophenol (one volume of phenolglycerollactic acidwater (1111) mixed 698

with two volumes of ethanol) and destained at 65degC Next leaves were moved through 699

an alcohol gradient (50 20 and 10 (vv) ethanol) into water The destained leaves 700

were stained for 30 min in 150 mM K2HPO4 (pH 95) with 001 (wv) aniline blue 701

Samples were mounted in water and examined using a Nikon Eclipse Ni-U microscope 702

at 10x magnification using a DAPI filter (excitation at 325-375 nm emission at 435-485 703

nm) For callose quantification six 200 microm x 200 microm squares were counted for each 704

sample type Callose deposits per mm2 were calculated and data analyzed using a t-705

test Note that there is an inherent uncertainty associated with such counts because 706

callose deposits may not all be in focus at the same time 707

708

MAPK Activity Assay 709

Fully expanded leaves of four week-old plants were inoculated with 001 710

macerozyme 100 microg mL-1 OGs 100 nM flg22 or water (mock) flg22 a peptide from 711

bacterial flagella that induces immune responses was used as a positive control for 712

Page 35 of 54

MAPK activation (Asai et al 2002) and was purchased from EZBiolab Inc (Westfield 713

IN USA) Samples were harvested 10 min later and immediately frozen in liquid 714

nitrogen Protein was extracted using an extraction buffer containing 25 mM Tris pH 78 715

75 mM NaCl 1 mM DTT 1 mM NaF 05mM Na3VO4 01 (vv) Tween 20 one 716

cOmplete Mini protease inhibitor cocktail tablet (Roche) and one PhosSTOP 717

phosphatase inhibitor cocktail tablet (Roche) per 10 mL buffer 15 microg of total protein 718

were electrophoresed on a 10 SDS-polyacrylamide gel and then blotted onto a PVDF 719

membrane (Bio-Rad 162-0177) Activated MAPKs were detected using p4442 MAPK 720

(Erk12) antibody (9102S Cell Signal 12500 in TBST (20 mM Tris pH 75 150 mM 721

NaCl 01 (vv) Tween 20)) Anti-Rabbit IgG-HRP (115000 in TBST A6154 Sigma) 722

and ECL Plus substrate (Pierce 32132) MPK3 and MPK6 were detected with an anti-723

MPK3 antibody (M8318 Sigma 12000 in TBST and 3 (wv) milk) or an anti-MPK6 724

antibody (A7104 Sigma 16000 in TBST and 3 (wv) milk) respectively The 725

secondary antibody and detection were the same as for the p4442 antibody Five 726

independent experiments were performed 727

728

Preparation of Alcohol Insoluble Residue (AIR) Pectin Extraction and Uronic Acid 729

Measurements 730

Fully expanded leaves from four week-old plants that were incubated for 48 h in 731

the dark to reduce starch were harvested flash frozen and pulverized AIR was 732

extracted by washing ground material twice in 70 (vv) ethanol three times in a 733

mixture of chloroform and methanol (11 vv) and once in acetone (Gille et al 2009) 734

and was then air dried Cell wall pectin was extracted from AIR with cell wall extraction 735

buffer (50 mM Trizma 50 mM CDTA pH 72) at 95degC for 15 min Samples were 736

homogenized using a paint shaker (Harbil 5G-HD) and glass beads (3 mm diameter) 737

For dot blot experiments 500 microL of cell wall extraction buffer were used per 10 mg of 738

AIR Debris was precipitated by centrifugation for 10 min at 10000 g 739

Total uronic acid content of pectin extracted as described above using 500 microL of 740

cell wall extraction buffer per 1 mg of AIR was measured as described previously 741

(Filisetti-Cozzi and Carpita 1991 van den Hoogen et al 1998) First 36 microL of pectin 742

extract were mixed with 4 microL of 4 M sulfamic acid Then 200 microL of sulfuric acid 743

Page 36 of 54

containing 120 mM sodium tetraborate was added and samples were incubated at 80degC 744

for 1 h Following cooling on ice the optical density of samples was measured at 490 745

nm Next 40 microL of m-hydroxydiphenyl reagent (100 microL of m-hydroxydiphenyl in dimethyl 746

sulfoxide at 100 mgmL mixed with 49 mL 80 (vv) sulfuric acid just before use) was 747

added and the samples were mixed The optical density at 490 nm was measured 748

again The optical density before m-hydroxydiphenyl reagent addition was subtracted 749

from the optical density measured after addition of the dye The concentration of uronic 750

acid was calculated using known amounts of GalA as a standard 751

752

Release of Pectin from Arabidopsis Cell Walls Using Macerozyme 753

AIR preparations from leaves of four week-old plants were treated with 025 754

(wv) macerozyme R-10 (Yakult) or water (mock) at a concentration of 100 microL per mg 755

AIR Samples were incubated for 3 h at room temperature on a vortex shaker and 756

centrifuged for 10 min at 12000 g Total uronic acid concentration of 5-fold diluted 757

supernatants was determined for two technical replicates each The concentration of 758

uronic acid was calculated using known amounts of GalA as a standard For 759

subsequent qRT-PCR experiments supernatants from multiple extractions were 760

combined boiled for 20 min to inactivate macerozyme diluted 10-fold with water and 761

inoculated into four week-old Col-0 plants Heat-inactivated 10-fold diluted macerozyme 762

solutions were used as mock treatment Samples were collected 3 h later and PAD3 763

expression was determined 764

765

Release of Pectin from Arabidopsis Leaves Infected with B cinerea 766

Fully expanded leaves of four week-old plants were either inoculated with up to 767

five 10 microL droplets of B cinerea spore solution (25 x 105 spores mL-1) or mock 768

inoculated with B cinerea inoculation medium For each biological replicate leaves with 769

a combined count of at least 20 infection sites were collected 48 h later AIR was 770

extracted as described above but plants were not dark-treated or freeze-dried and the 771

first ethanol step was performed using 85 (vv) ethanol To extract soluble pectin 772

fragments AIR samples were mixed with water at a concentration of 20 microL per infection 773

site incubated for 7 h at room temperature on a vortex shaker and centrifuged for 10 774

Page 37 of 54

min at 12000 g Total uronic acid concentration of supernatants was determined for two 775

technical replicates each The concentration of uronic acid was calculated using known 776

amounts of GalA as a standard 777

778

Measurement of Uronic Acids Neutral Monosaccharides and Cellulose Content in 779

Cell Walls 780

Neutral sugars and uronic acids from non-cellulosic polysaccharides in cell walls 781

(AIR) were released by trifluoroacetic acid (TFA) hydrolysis Approximately 1 mg of AIR 782

was hydrolyzed with 2 M TFA at 121degC for 90 min The TFA-soluble material was split 783

into two equal parts one for uronic acid and one for neutral sugar determination The 784

uronic acid content was determined by analyzing the hydrolyzate using a CarboPac 785

PA200 anion-exchange column with an ICS-3000 Dionex chromatography system 786

(Dionex Sunnyvale CA USA) The elution profile consisted of a linear gradient of 50 to 787

200 mM sodium acetate in 01 M NaOH in 10 min at 04 mL per min Alditol acetate 788

derivatives of neutral sugars were produced as described (York et al 1985) In brief 789

monosaccharides in the hydrolyzate were reduced with NaBH4 and peracetylated with 790

acetic anhydride and pyridine at 121degC for 20 min The generated alditol acetates were 791

analyzed by GC-MS Crystalline cellulose was measured according to Updegraff (1969) 792

After hydrolysis of noncellulosic polysaccharides from AIR with the Updegraff reagent 793

(acetic acid nitric acids water 812 (vv) ) the remaining pellet was hydrolyzed in 794

72 sulfuric acid The resulting glucose quantity was determined by the anthrone 795

method (Scott and Melvin 1953) 796

797

Dot Blot Experiments 798

Pectin solutions extracted using 50 microL of cell wall extraction buffer per mg AIR 799

were serially diluted and nitrocellulose membranes were spotted with 1 microL of the diluted 800

pectin solutions Membranes were dried overnight blocked with 5 milk (wv) in 1x 801

PBS (8 g L-1 NaCl 02 g L-1 KCl 144 g L-1 Na2HPO4 024 g L-1 KH2PO4 pH 74) and 802

probed with LM5 (Jones et al 1997) LM6 (Willats et al 1998) LM8 (Willats et al 803

2004) LM19 and LM20 (Verhertbruggen et al 2009a) or CCRC-M7 (Steffan et al 804

Page 38 of 54

1995) antibodies LM series antibodies were diluted 1250 and CCRC-M7 was diluted 805

1500 in 5 milk powder (Nestle Carnation) in 1 x PBS For LM series antibodies a 806

goat anti-Rat HRP conjugated antibody (Bethyl A110-105P 15000 diluted) and for 807

CCRC-M7 an anti-Mouse IgG HRP conjugate (Promega W402B 12500 diluted) was 808

used as secondary antibody in 5 milk powder in 1x PBS LM series antibodies were 809

obtained from plant probes (wwwplantprobesnet) and CCRC-M7 from CarboSource 810

(wwwcarbosourcenet) Membranes were washed after incubation with the primary and 811

secondary antibodies with 1x PBS Dot blots were developed using the ECL system 812

(GE Healthcare Amersham ECL prime) For quantification of signals dot blot results on 813

x-ray films were photographed using a CCD camera and intensities were measured814

using Image Jrsquos Integrated Density function Locally adjusted background was 815

subtracted As the dynamic range of the signal on an x-ray film was narrow non-816

linearity in the measured values was corrected for each genotype in each biological 817

replicate using the values from different dilutions in two technical replicates We noticed 818

that the residuals were not normally distributed and performed Box-Cox power 819

transformation (Box and Cox 1964) to obtain normally distributed values for the 820

subsequent statistical analysis of the data For LM19 Box-Cox selected power 821

transformation was done to the power of 038th and for LM20 to the power 075th After 822

correction of the transformed measured values with the effect of the biological 823

replicates the values were fit to a linear model with the Arabidopsis genotype as the 824

fixed effect This linear model was used both to generate the plots in Figures 5B and D 825

and to test significant differences 826

827

Camalexin Measurements 828

Four week-old plants were infected with B cinerea and samples (three leaf disks 829

from one plant each) were harvested at the indicated time points flash frozen and 830

pulverized Each sample was extracted with 300 microL of 90 methanol (vv) 50 microL of the 831

extract was run on an Agilent Lichrocart 250-4 RP18e 5-microm column using an Agilent 832

1100 series HPLC Camalexin was detected using a DiodeArray (DAD) at 330 nm and 833

with a fluorescence detector at emission 318 nmexcitation 385 nm (Agilent) Separation 834

was achieved using the following program with aqueous acetonitrile 5 min gradient 835

Page 39 of 54

from 63 to 69 acetonitrile 30 s gradient from 69 to 99 acetonitrile 2 min at 99 836

acetonitrile and a post-run equilibration of 35 min at 63 acetonitrile (Denby et al 837

2004 Kliebenstein et al 2005) Purified camalexin was used to produce a standard 838

curve to identify and quantitate camalexin 839

840

Accession Numbers 841

Microarray data used can be accessed at NCBI Gene Expression Omnnibus 842

(httpwwwncbinlmnihgovgeo) GSE18978 Sequence information for all Arabidopsis 843

genes described in this article can be found in TAIR (wwwarabidopsisorg) using the 844

following accession numbers PAD3 (CYP71B15 At3g26830) SID2 (ICS1 At1g74710) 845

GAE1 (At4g30440) GAE2 (At1g02000) GAE3 (At4g00110) GAE4 (At2g45310) GAE5 846

(At4g12250) GAE6 (At3g23820) PR1 (At2g14610) JAZ5 (At1g17380) JAZ10 847

(At5g13220) ACTIN2 (At3g18780) PDF12a (At5g44420) PDF12b (At2g26020) 848

AtSK11 (At5g26751) The sequence of the B cinerea cutinase Bc-CutA (Z69264) can 849

be found in the DNA Data Bank of Japan (httpgetentryddbjnigacjp) Germplasm 850

used included dde2-2 (At5g42650 (von Malek et al 2002)) pad3-1 (At3g26830 (Zhou 851

et al 1999)) pad4-1 (At3g52430 (Jirage et al 1999)) sid2-2 (At1g74710 (Wildermuth 852

et al 2001)) pbs3-2 (At5g13320 SALK_018225 (Nobuta et al 2007)) cbp60a-1 853

(At5g62570 SALK_124410 (Truman et al 2013)) cbp60g-1 sard1-2 (At5g26920 854

SALK_023199 At1g73805 SALK_052422 (Wang et al 2011) cbp60a-1 cbp60g-1 855

sard1-2 (Truman et al 2013) gae1-1 (At4g30440 SALK_085554) gae6-1 (At3g23280 856

SALK_104454) gae6-2 (At3g23280 SALK_017191) ixr1-1 cev1 (At5g05170 (Ellis 857

and Turner 2001 Scheible et al 2001)) eds1 plants were derived by introgression of 858

the Ler eds1-2 allele into Col-0 that contains two copies of EDS1 (At3g48090 859

At3g48080 (Bartsch et al 2006) mpk6-2 (At2g43790 SALK_073907 (Liu and Zhang 860

2004)) mpk3-DG (At3g45640 a fast neutron deletion mutant (Miles et al 2005)) and 861

npr1-1 (At1g64280 (Cao et al 1997)) T-DNA insertion lines were part of the SALK 862

collection (Alonso et al 2003) and were obtained from the Arabidopsis Biological 863

Resource Center 864

865

Supplemental Data 866

Page 40 of 54

Supplemental Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 867

Expression Does Not Require CBP60s or SID2 868

Supplemental Figure 2 Expression of GAE Family Members uponTreatment with Pma 869

ES4326 870

Supplemental Figure 3 Characterization of T-DNA Insertions in GAE1 and GAE6 871

Supplemental Figure 4 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves and 872

Are Slightly Smaller than Wild-Type Col-0 Plants 873

Supplemental Figure 5 Cell Walls of gae1 gae6 Plants Contain Less Uronic Acid than 874

Wild-Type Col-0 Cell Walls 875

Supplemental Figure 6 Antibodies Raised Against the RGI or XG Components of 876

Pectin Show No Difference in Binding to gae Mutant and Col-0 Cell Walls 877

Supplemental Figure 7 The Effect of B cinerea Treatment on the Expression of GAE 878

Family Genes 879

Supplemental Figure 8 Treatment with 001 Macerozyme Does Not Cause Any 880

Visible Damage to Col-0 Plants 881

Supplemental Figure 9 B cinerea Growth on Plants with Defects in Immune 882

Signaling 883

Supplemental Figure 10 MeJA-Induced Expression of the JA marker gene JAZ10 884

Supplemental Figure 11 The Expression of the JA Marker Gene JAZ5 Is Highly 885

Responsive in gae1 gae6 Plants 886

Supplemental Figure 12 The Expression of PDF12 Is Unaltered in gae1 gae6 Plants 887

Supplemental Figure 13 Macerozyme Treatment Activates MPK3 and MPK6 888

Supplemental Figure 14 B cinerea-Induced ROS Production in Wild-Type Col-0 and 889

gae1 gae6 Plants 890

Supplemental Figure 15 OG-Induced PTI Using 250 microg mL-1 OGs 891

Supplemental Figure 16 Abundance of Plant DNA in B cinerea Treated Plants 892

Supplemental Table 1 Primers used in this study 893

894

ACKNOWLEDGEMENTS 895

We thank William Truman for helpful discussions Simone Ferrari (Sapienza 896

Universita di Roma) for the generous gift of OGs Bruna Bucciarelli (USDA-ARS) for 897

Page 41 of 54

help with fluorescence microscopy the Arabidopsis Biological Resource Center for T-898

DNA insertion lines Paul Knoxrsquos lab (University of Leeds) for LM5 LM6 LM8 LM19 899

and LM20 antibodies and the Complex Carbohydrate Resource Center (University of 900

Georgia) for the CCRC-M7 antibody Most of this work was funded by the Division of 901

Chemical Sciences Geosciences and Biosciences Office of Basic Energy Sciences of 902

the US Department of Energy through Grant DE-FG02-05ER15670 to JG This work 903

was also supported by NSF awards IOS 1339125 and IOS 1021861 to DJK NSF 904

award IOS 1121425 to F K and the USDA National Institute of Food and Agriculture 905

Hatch project number CA-D-PLS-7033-H to DJK 906

907

AUTHOR CONTRIBUTIONS 908

GB and JG designed the research GB GX AT BL NES and NH 909

performed research DJK provided tools GB GX MP AD BL RAH and FK 910

analyzed data GB and JG wrote the paper with input from all authors 911

912

FIGURE LEGENDS 913

Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 Expression 914

Requires PAD4 EDS1 and PBS3 915

(A) Expression of GAE1 in leaves of wild-type Col-0 and mutant plants Expression was916

measured 24 or 32 h after either inoculation with Pma ES4326 (OD600 = 0002) or mock 917

inoculation with 5 mM MgSO4 and was then normalized to the level of ACTIN2 Data 918

from three biological replicates were combined using a mixed linear model Bars 919

represent mean log2-ratios to ACTIN2 plusmn SE Letters indicate significantly different 920

groups with q lt 005 where 005 represents the maximum false discovery rate at which 921

the test may be called significant 922

(B) Expression of GAE6 measured as described in (A)923

924

Figure 2 Susceptibility of gae6 and gae1 gae6 Plants to Pma ES4326 Is Increased 925

Relative to Wild-Type Col-0 926

Leaves of plants of the indicated genotypes were inoculated with Pma ES4326 (OD600 = 927

00002) as described in Methods Bacterial titers in leaves were determined either 928

Page 42 of 54

immediately (0 dpi) or after three days (3 dpi) Bars represent the mean plusmn SE of four 929

independent experiments each with 4 or 12 biological replicates at 0 and 3 dpi 930

respectively Data were combined using a mixed linear model Asterisks indicate 931

significant differences from Col-0 wild-type (q lt 005) Susceptible pad4 plants were 932

included as a positive control 933

934

Figure 3 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves 935

(A) Depiction of the assay for brittleness of gae mutant leaves Fully expanded leaves of936

four to five week-old plants were bent by 180ordm so that the adaxial surface of the leaf was 937

parallel with the soil Broken leaf petioles or midribs were considered brittle 938

(B) Quantification of broken midribs or petioles For each genotype 66 to 82 leaves 939

were analyzed for brittleness as described in (A) Bars represent the ratio of broken to 940

total number of leaves tested Results were analyzed using Fisherrsquos exact test Letters 941

indicate significantly different groups at p lt 005 942

(C) Conductivity measurements Leaves of four week-old plants were inoculated with943

deionized water and then leaf discs were cut out and placed on deionized water 944

Conductivity was measured at the indicated time points After 30 min the water was 945

replaced with fresh water (30 min f w) to account for wounding-related ion leakage from 946

the edges of the leaf discs Three independent experiments with six biological replicates 947

each were performed and data were analyzed using a t-test for each time point P-948

values were corrected using the Bonferroni method Data show means plusmn SE asterisks 949

indicate significant differences from wild-type Col-0 at q lt 001 950

951

Figure 4 Cell Walls of gae6 and gae1 gae6 Plants Are Reduced in GalA 952

(A) to (D) Levels of GalA (A) GlcA (B) cellulose (C) and neutral sugars (D) in alcohol953

insoluble residue (AIR) extracted from leaves of four week-old plants of the indicated 954

genotypes Bars represent means plusmn SE of four biological replicates combined using a 955

mixed linear model Letters indicate significantly different groups (p lt 005) 956

957

Figure 5 Cell Walls of gae1 gae6 Plants Are Reduced in Homogalacturonan 958

Page 43 of 54

Dot blot analyses with anti-LM19 and anti-LM20 antibodies Pectin was extracted from 959

AIR prepared from the indicated genotypes using 1 microL of extraction buffer per 10 microg of 960

AIR Pectin was serially diluted and one microL of undiluted 13 diluted and 19 diluted 961

samples were spotted on a nitrocellulose membrane Three biological replicates were 962

were spotted twice to obtain two technical replicates each All replicates produced 963

similar results 964

(A) Representative dot blot using anti-LM19 antibody965

(B) Relative signal intensities of the spots in the dot blot in (A)966

(C) Representative dot blot using anti-LM20 antibody967

(D) Relative signal intensities of the LM20 dot blot shown in (C)968

Relative signal intensities were measured using Image J software Measurements from 969

the three biological replicates were combined using a linear model after Box-Cox power 970

transformation as described in Methods Bars represent means plusmn SE Asterisks indicate 971

a difference from wild-type Col-0 at p lt 005 972

973

Figure 6 Growth of B cinerea on gae1 gae6 Plants and the Effect of B cinerea on 974

Expression of GAE1 and GAE6 975

(A) Growth of B cinerea isolates Leaves of Col-0 and gae1 gae6 plants were976

inoculated with 25 x 105 spores mL-1 of the B cinerea isolates indicated in the figure 977

Samples were collected two (2 dpi) and three (3 dpi) days later Bars represent means plusmn 978

SE of six biological replicates for each fungus combined using a mixed linear model 979

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 980

(B) and (C) Expression of GAE1 (B) and GAE6 (C) 48 h after inoculation with the B981

cinerea isolates indicated Expression levels were measured by qRT-PCR Bars 982

represent mean log2-ratios to ACTIN2 plusmn SE of three biological replicates combined 983

using a mixed linear model Letters indicate significantly different groups (q lt 005) 984

985

Figure 7 Levels of Soluble Pectin and Expression of PAD3 in Fractions Produced 986

Following Macerozyme Treatment of gae1 gae6 Cell Walls 987

(A) Schematic overview of the experimental procedure for (B) and (C)988

Page 44 of 54

(B) Uronic acid measurements Alcohol-insoluble residue (AIR) from leave tissue of four989

week-old wild-type Col-0 or gae1 gae6 plants was treated with water or 025 990

macerozyme and assayed for uronic acid levels Data from 11 biological replicates were 991

combined using a mixed linear model Bars represent means plusmn SE Letters indicate 992

significant differences (p lt 005) 993

(C) Expression of PAD3 in Col-0 plants treated with the fractions from (B) Extracts from994

(B) were heat inactivated pooled and diluted 5-fold with water Pooled extracts were995

inoculated into four week-old Col-0 plants PAD3 expression was measured 3 h after 996

inoculation and normalized to the level of ACTIN2 expression Data from four biological 997

replicates were combined using a mixed linear model Bars represent mean log2-ratios 998

to ACTIN2 plusmn SE Letters indicate significant differences (p lt 005) 999

1000

Figure 8 Macerozyme-Induced PAD3 Expression Is Absent in gae1 gae6 1001

(A) Expression of PAD3 after macerozyme treatment Four week-old plants were either1002

inoculated with 001 macerozyme or mock inoculated with water and PAD3 1003

expression was measured 3 h later by qRT-PCR Data from four biological replicates 1004

were combined using a mixed linear model Mean log2-ratios to ACTIN2 plusmn SE were 1005

plotted Letters indicate significantly different groups (p lt 005) 1006

(B) Expression of PAD3 after OG treatment PAD3 expression was measured as1007

described in (A) 3 h after inoculation with 100 microg mL-1 OGs or mock inoculation with 1008

water Data are from six biological replicates 1009

(C) PAD3 expression in untreated plants Three biological replicates were performed1010

and data analyzed as described in (A) 1011

(D) Camalexin accumulation after B cinerea treatment Camalexin accumulation was1012

measured in four week-old plants inoculated with the B cinerea strains shown in the 1013

figure Eight biological replicates each were combined using a mixed linear model 1014

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 1015

(E) PAD3 expression 28 h after treatment with the indicated B cinerea strains Data1016

from six biological replicates were collected and analyzed as described in (A) Data in 1017

all figure parts represent means plusmn SE Letters indicate significantly different groups (q lt 1018

005) 1019

Page 45 of 54

1020

Figure 9 Macerozyme-Induced Expression of the SA Marker Genes SID2 and PR1 1021

Is Abolished in gae1 gae6 Plants 1022

(A) to (H) Expression of PR1 (A) (C) (E) and (G) or SID2 (B) (D) (F) and (H) in four1023

week-old Col-0 and gae1 gae6 plants Data were combined using a mixed linear model 1024

Bars represent mean log2-ratios of expression vs ACTIN2 plusmn SE for all figure parts 1025

Letters indicate significantly different groups at p lt 005 for panels (A) to (D) (G) and 1026

(H) and qlt005 for panels (E) to (F) 1027

(A) and (B) Plants were treated with 001 macerozyme or mock treated with water1028

and samples were collected 3 h later Data from four biological replicates are shown 1029

(C) and (D) Plants were inoculated with 100 microg mL-1 OGs or mock treated with water1030

and samples collected 3 h later Data from six biological replicates are shown 1031

(E) and (F) Plants were inoculated with the indicated B cinerea isolates or mock1032

inoculated with B cinerea inoculation medium and samples collected 48 h later Data 1033

from eight biological replicates are shown 1034

(G) and (H) Expression in untreated plants Three biological replicates were performed1035

1036

Figure 10 The Expression of the JA Marker Gene JAZ10 is Highly Responsive in 1037

gae1 gae6 Plants 1038

(A) to (D) JAZ10 expression in four week-old plants Data were combined using a mixed1039

linear model Mean log2-ratios to ACTIN2 plusmn SE are shown Letters indicate significantly 1040

different groups at p lt 005 for panels (A) (B) and (D) and q lt 005 for panel (C) 1041

(A) Plants were inoculated with 001 macerozyme or mock inoculated using water and1042

samples were collected 3 h later Data from four biological replicates are shown 1043

(B) Plants were inoculated with 100 microg mL-1 OGs or mock inoculated using water and1044

samples collected 3 h later Data are from six biological replicates 1045

(C) JAZ10 expression 48 h after treatment with the indicated B cinerea strains Data1046

are from six biological replicates 1047

(D) JAZ10 expression in untreated plants Data are from three biological replicates1048

1049

Figure 11 Gallo 1-Induced Callose Deposition Is Reduced in gae1 gae6 Plants 1050

Page 46 of 54

(A) Callose deposition in four week-old Col-0 and gae1 gae6 plants Plants were1051

inoculated with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) or mock inoculated 1052

with B cinerea inoculation medium and samples were collected after 72 h Callose was 1053

stained with aniline blue and visualized using a Nikon Eclipse Ni-U microscope with a 1054

DAPI filter Three leaves were stained per treatment Scale bars are 100 microm Asterisks 1055

indicate fungal hyphae arrows indicate representative callose spots 1056

(B) Quantification of callose deposition Callose deposits were counted in 200 microm x 2001057

microm squares laid out across the images shown here Bars represent means plusmn SE from 1058

six squares each Letters indicate significantly different groups (p lt 005) according to a 1059

t-test analysis1060

1061

Figure 12 Less Soluble Pectin Accumulates in B cinerea-Infected gae1 gae6 1062

Leaves 1063

Four week-old Col-0 and gae1 gae6 plants were inoculated with 10 microL droplets of the 1064

indicated B cinerea isolate (25 x 105 spores mL-1) or mock inoculated with B cinerea 1065

inoculation medium Leaves were collected 48 h later AIR was extracted from leaf 1066

samples and water added (20 microL infection site) to extract soluble pectin fragments 1067

Samples were vortexed for 7 h centrifuged at 12000 g and the uronic acid 1068

concentration of the supernatant was measured Data from nine biological replicates 1069

were combined using a mixed linear model Means plusmn SE are shown Letters indicate 1070

significant differences (q lt 005) 1071

1072

Figure 13 Macerozyme-Induced PTI Is Lost in gae1 gae6 While OG-Induced PTI Is 1073

Unaltered 1074

(A) Macerozyme-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1075

inoculated with 001 macerozyme or mock inoculated with boiled macerozyme and 1076

were infected with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) 24 h later Samples 1077

were collected immediately (0 dpi) and after two (2 dpi) and three (3 dpi) days Data 1078

show mean plusmn SE of six to 14 biological replicates each combined using a linear model 1079

Letters indicate significantly different groups (q lt 005) 1080

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

Parsed CitationsAdam L and Somerville SC (1996) Genetic characterization of five powdery mildew disease resistance loci in Arabidopsisthaliana Plant J 9 341-356

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Albersheim P Jones TM and English PD (1969) Biochemistry of the cell wall in relation to infective processes Annu RevPhytopathol 7 171-194

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk RGadrinab C Heller C Jeske A Koesema E Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt MHazari N Hom E Karnes M Mulholland C Ndubaku R Schmidt I Guzman P Aguilar-Henonin L Schmid M Weigel DCarter DE Marchand T Risseeuw E Brogden D Zeko A Crosby WL Berry CC and Ecker JR (2003) Genome-wideinsertional mutagenesis of Arabidopsis thaliana Science 301 653-657

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Asai T Tena G Plotnikova J Willmann MR Chiu WL Gomez-Gomez L Boller T Ausubel FM and Sheen J (2002) MAPkinase signalling cascade in Arabidopsis innate immunity Nature 415 977-983

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bartsch M Gobbato E Bednarek P Debey S Schultze JL Bautor J and Parker JE (2006) Salicylic acid-independentENHANCED DISEASE SUSCEPTIBILITY1 signaling in Arabidopsis immunity and cell death is regulated by the monooxygenaseFMO1 and the nudix hydrolase NUDT7 Plant Cell 18 1038-1051

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bates D Machler M Bolker BM and Walker SC (2015) Fitting Linear Mixed-Effects Models Using lme4 J Stat Softw 67 1-48Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Benjamini Y and Hochberg Y (1995) Controlling the False Discovery Rate - a Practical and Powerful Approach to MultipleTesting J Roy Stat Soc B Met 57 289-300

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bethke G Grundman RE Sreekanta S Truman W Katagiri F and Glazebrook J (2014) Arabidopsis PECTINMETHYLESTERASEs Contribute to Immunity against Pseudomonas syringae Plant Physiol 164 1093-1107

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bouton S Leboeuf E Mouille G Leydecker MT Talbotec J Granier F Lahaye M Hofte H and Truong HN (2002)Quasimodo1 encodes a putative membrane-bound glycosyltransferase required for normal pectin synthesis and cell adhesion inArabidopsis Plant Cell 14 2577-2590

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Box GEP and Cox DR (1964) An analysis of transformations Journal of the Royal Statistical Society Series B 26 211-252Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Cao H Glazebrook J Clarke JD Volko S and Dong XN (1997) The Arabidopsis NPR1 gene that controls systemic acquiredresistance encodes a novel protein containing ankyrin repeats Cell 88 57-63

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Carpita N (2010) Update on Mechanisms of Plant Cell Wall Biosynthesis How plants make cellulose and other (1-gt4)-beta-D-glycans Plant Physiol

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Chisholm ST Coaker G Day B and Staskawicz BJ (2006) Host-microbe interactions Shaping the evolution of the plantimmune response Cell 124 803-814

Page 33 of 54

cinerea and samples were collected immediately (0 dpi) and two or three d later To 652

investigate the role of MeJA on B cinerea growth plants were sprayed with 1mM MeJA 653

in water with 001 (vv) Tween 20 Covers were removed so that leaves could dry off 654

before plants were inoculated with B cinerea three h later Samples were harvested at 655

the indicated time points For expression analysis and MAPK activation assays plants 656

were either inoculated with 100 microg mL-1 OGs or mock inoculated with water OGs used 657

for experiments described in Figures 8 to 10 and Supplemental Figure 12 were a kind 658

gift from Simone Ferrari OGs used in experiments described in Figure 13 and 659

Supplemental Figures 11 13 and 15 were prepared as previously described (Kohorn et 660

al 2014) 661

662

Expression Analysis 663

Leaf tissue from inoculated plants was harvested at the indicated time points 664

flash frozen pulverized and RNA extracted using Trizol (Invitrogen) Quantitative RT-665

PCR was performed using the SuperScript III Platinum SYBR Green One-Step 666

quantitative RT-PCR kit (Invitrogen) and a Lightcycler 480 Real-Time PCR system 667

(Roche) as previously described (Truman and Glazebrook 2012) In brief equal 668

amounts of total RNA and a gene specific primer were used for each reaction The 669

crossing point (Cp) was calculated using the 2nd derivative max method provided with 670

the Lightcycler software for each amplification curve Each reaction was run with two 671

technical replicates and the Cp values for these replicates were averaged ACTIN2 was 672

used as a stably expressed reference gene At least three biological replicates each 673

were performed and analyzed using a mixed-linear model RT-PCR was performed to 674

verify T-DNA insertion lines using the One-Step RT-PCR kit (Qiagen) according to the 675

manufacturerrsquos instructions The primers used can be found in Supplemental Table 1 676

677

Electrolyte Leakage Assay 678

Fully expanded leaves of four week-old plants were inoculated with water (Milli-Q 679

grade) using a needleless syringe Leaf discs were collected using a cork borer and 680

three leaf discs from one leaf were placed with the adaxial surface down onto 2 mL 681

water in a 12-well cell culture plate Conductivity was measured using a Horiba B-173 682

Page 34 of 54

conductivity meter after 30 min and then the water was replaced Conductivity was 683

measured again 30 min later (1 h total) as indicated in Figure 3C Six biological 684

replicates each were performed in three independent experiments and means and SEs 685

were calculated Two-sided t-tests were performed for each time point 686

687

DAB and Aniline Blue Staining 688

Leaves from four week-old plants were either mock inoculated with B cinerea 689

inoculation medium or inoculated with 10 microL droplets of a B cinerea spore solution (25 690

x 105 spores mL-1) of the indicated isolates Samples were collected 72 h later For 33prime-691

diaminobenzidine (DAB) staining two to five inoculated leaves were vacuum infiltrated 692

with 33prime-Diaminobenzidine (1 mg mL-1 in water pH 38) solution and stained overnight 693

in the dark Leaves were destained using 100 ethanol at 65degC and then transferred to 694

50 (vv) glycerol and photographed 695

Callose was visualized by staining leaves with aniline blue as previously 696

described (Adam and Somerville 1996) Briefly leaves were vacuum infiltrated with 697

alcoholic lactophenol (one volume of phenolglycerollactic acidwater (1111) mixed 698

with two volumes of ethanol) and destained at 65degC Next leaves were moved through 699

an alcohol gradient (50 20 and 10 (vv) ethanol) into water The destained leaves 700

were stained for 30 min in 150 mM K2HPO4 (pH 95) with 001 (wv) aniline blue 701

Samples were mounted in water and examined using a Nikon Eclipse Ni-U microscope 702

at 10x magnification using a DAPI filter (excitation at 325-375 nm emission at 435-485 703

nm) For callose quantification six 200 microm x 200 microm squares were counted for each 704

sample type Callose deposits per mm2 were calculated and data analyzed using a t-705

test Note that there is an inherent uncertainty associated with such counts because 706

callose deposits may not all be in focus at the same time 707

708

MAPK Activity Assay 709

Fully expanded leaves of four week-old plants were inoculated with 001 710

macerozyme 100 microg mL-1 OGs 100 nM flg22 or water (mock) flg22 a peptide from 711

bacterial flagella that induces immune responses was used as a positive control for 712

Page 35 of 54

MAPK activation (Asai et al 2002) and was purchased from EZBiolab Inc (Westfield 713

IN USA) Samples were harvested 10 min later and immediately frozen in liquid 714

nitrogen Protein was extracted using an extraction buffer containing 25 mM Tris pH 78 715

75 mM NaCl 1 mM DTT 1 mM NaF 05mM Na3VO4 01 (vv) Tween 20 one 716

cOmplete Mini protease inhibitor cocktail tablet (Roche) and one PhosSTOP 717

phosphatase inhibitor cocktail tablet (Roche) per 10 mL buffer 15 microg of total protein 718

were electrophoresed on a 10 SDS-polyacrylamide gel and then blotted onto a PVDF 719

membrane (Bio-Rad 162-0177) Activated MAPKs were detected using p4442 MAPK 720

(Erk12) antibody (9102S Cell Signal 12500 in TBST (20 mM Tris pH 75 150 mM 721

NaCl 01 (vv) Tween 20)) Anti-Rabbit IgG-HRP (115000 in TBST A6154 Sigma) 722

and ECL Plus substrate (Pierce 32132) MPK3 and MPK6 were detected with an anti-723

MPK3 antibody (M8318 Sigma 12000 in TBST and 3 (wv) milk) or an anti-MPK6 724

antibody (A7104 Sigma 16000 in TBST and 3 (wv) milk) respectively The 725

secondary antibody and detection were the same as for the p4442 antibody Five 726

independent experiments were performed 727

728

Preparation of Alcohol Insoluble Residue (AIR) Pectin Extraction and Uronic Acid 729

Measurements 730

Fully expanded leaves from four week-old plants that were incubated for 48 h in 731

the dark to reduce starch were harvested flash frozen and pulverized AIR was 732

extracted by washing ground material twice in 70 (vv) ethanol three times in a 733

mixture of chloroform and methanol (11 vv) and once in acetone (Gille et al 2009) 734

and was then air dried Cell wall pectin was extracted from AIR with cell wall extraction 735

buffer (50 mM Trizma 50 mM CDTA pH 72) at 95degC for 15 min Samples were 736

homogenized using a paint shaker (Harbil 5G-HD) and glass beads (3 mm diameter) 737

For dot blot experiments 500 microL of cell wall extraction buffer were used per 10 mg of 738

AIR Debris was precipitated by centrifugation for 10 min at 10000 g 739

Total uronic acid content of pectin extracted as described above using 500 microL of 740

cell wall extraction buffer per 1 mg of AIR was measured as described previously 741

(Filisetti-Cozzi and Carpita 1991 van den Hoogen et al 1998) First 36 microL of pectin 742

extract were mixed with 4 microL of 4 M sulfamic acid Then 200 microL of sulfuric acid 743

Page 36 of 54

containing 120 mM sodium tetraborate was added and samples were incubated at 80degC 744

for 1 h Following cooling on ice the optical density of samples was measured at 490 745

nm Next 40 microL of m-hydroxydiphenyl reagent (100 microL of m-hydroxydiphenyl in dimethyl 746

sulfoxide at 100 mgmL mixed with 49 mL 80 (vv) sulfuric acid just before use) was 747

added and the samples were mixed The optical density at 490 nm was measured 748

again The optical density before m-hydroxydiphenyl reagent addition was subtracted 749

from the optical density measured after addition of the dye The concentration of uronic 750

acid was calculated using known amounts of GalA as a standard 751

752

Release of Pectin from Arabidopsis Cell Walls Using Macerozyme 753

AIR preparations from leaves of four week-old plants were treated with 025 754

(wv) macerozyme R-10 (Yakult) or water (mock) at a concentration of 100 microL per mg 755

AIR Samples were incubated for 3 h at room temperature on a vortex shaker and 756

centrifuged for 10 min at 12000 g Total uronic acid concentration of 5-fold diluted 757

supernatants was determined for two technical replicates each The concentration of 758

uronic acid was calculated using known amounts of GalA as a standard For 759

subsequent qRT-PCR experiments supernatants from multiple extractions were 760

combined boiled for 20 min to inactivate macerozyme diluted 10-fold with water and 761

inoculated into four week-old Col-0 plants Heat-inactivated 10-fold diluted macerozyme 762

solutions were used as mock treatment Samples were collected 3 h later and PAD3 763

expression was determined 764

765

Release of Pectin from Arabidopsis Leaves Infected with B cinerea 766

Fully expanded leaves of four week-old plants were either inoculated with up to 767

five 10 microL droplets of B cinerea spore solution (25 x 105 spores mL-1) or mock 768

inoculated with B cinerea inoculation medium For each biological replicate leaves with 769

a combined count of at least 20 infection sites were collected 48 h later AIR was 770

extracted as described above but plants were not dark-treated or freeze-dried and the 771

first ethanol step was performed using 85 (vv) ethanol To extract soluble pectin 772

fragments AIR samples were mixed with water at a concentration of 20 microL per infection 773

site incubated for 7 h at room temperature on a vortex shaker and centrifuged for 10 774

Page 37 of 54

min at 12000 g Total uronic acid concentration of supernatants was determined for two 775

technical replicates each The concentration of uronic acid was calculated using known 776

amounts of GalA as a standard 777

778

Measurement of Uronic Acids Neutral Monosaccharides and Cellulose Content in 779

Cell Walls 780

Neutral sugars and uronic acids from non-cellulosic polysaccharides in cell walls 781

(AIR) were released by trifluoroacetic acid (TFA) hydrolysis Approximately 1 mg of AIR 782

was hydrolyzed with 2 M TFA at 121degC for 90 min The TFA-soluble material was split 783

into two equal parts one for uronic acid and one for neutral sugar determination The 784

uronic acid content was determined by analyzing the hydrolyzate using a CarboPac 785

PA200 anion-exchange column with an ICS-3000 Dionex chromatography system 786

(Dionex Sunnyvale CA USA) The elution profile consisted of a linear gradient of 50 to 787

200 mM sodium acetate in 01 M NaOH in 10 min at 04 mL per min Alditol acetate 788

derivatives of neutral sugars were produced as described (York et al 1985) In brief 789

monosaccharides in the hydrolyzate were reduced with NaBH4 and peracetylated with 790

acetic anhydride and pyridine at 121degC for 20 min The generated alditol acetates were 791

analyzed by GC-MS Crystalline cellulose was measured according to Updegraff (1969) 792

After hydrolysis of noncellulosic polysaccharides from AIR with the Updegraff reagent 793

(acetic acid nitric acids water 812 (vv) ) the remaining pellet was hydrolyzed in 794

72 sulfuric acid The resulting glucose quantity was determined by the anthrone 795

method (Scott and Melvin 1953) 796

797

Dot Blot Experiments 798

Pectin solutions extracted using 50 microL of cell wall extraction buffer per mg AIR 799

were serially diluted and nitrocellulose membranes were spotted with 1 microL of the diluted 800

pectin solutions Membranes were dried overnight blocked with 5 milk (wv) in 1x 801

PBS (8 g L-1 NaCl 02 g L-1 KCl 144 g L-1 Na2HPO4 024 g L-1 KH2PO4 pH 74) and 802

probed with LM5 (Jones et al 1997) LM6 (Willats et al 1998) LM8 (Willats et al 803

2004) LM19 and LM20 (Verhertbruggen et al 2009a) or CCRC-M7 (Steffan et al 804

Page 38 of 54

1995) antibodies LM series antibodies were diluted 1250 and CCRC-M7 was diluted 805

1500 in 5 milk powder (Nestle Carnation) in 1 x PBS For LM series antibodies a 806

goat anti-Rat HRP conjugated antibody (Bethyl A110-105P 15000 diluted) and for 807

CCRC-M7 an anti-Mouse IgG HRP conjugate (Promega W402B 12500 diluted) was 808

used as secondary antibody in 5 milk powder in 1x PBS LM series antibodies were 809

obtained from plant probes (wwwplantprobesnet) and CCRC-M7 from CarboSource 810

(wwwcarbosourcenet) Membranes were washed after incubation with the primary and 811

secondary antibodies with 1x PBS Dot blots were developed using the ECL system 812

(GE Healthcare Amersham ECL prime) For quantification of signals dot blot results on 813

x-ray films were photographed using a CCD camera and intensities were measured814

using Image Jrsquos Integrated Density function Locally adjusted background was 815

subtracted As the dynamic range of the signal on an x-ray film was narrow non-816

linearity in the measured values was corrected for each genotype in each biological 817

replicate using the values from different dilutions in two technical replicates We noticed 818

that the residuals were not normally distributed and performed Box-Cox power 819

transformation (Box and Cox 1964) to obtain normally distributed values for the 820

subsequent statistical analysis of the data For LM19 Box-Cox selected power 821

transformation was done to the power of 038th and for LM20 to the power 075th After 822

correction of the transformed measured values with the effect of the biological 823

replicates the values were fit to a linear model with the Arabidopsis genotype as the 824

fixed effect This linear model was used both to generate the plots in Figures 5B and D 825

and to test significant differences 826

827

Camalexin Measurements 828

Four week-old plants were infected with B cinerea and samples (three leaf disks 829

from one plant each) were harvested at the indicated time points flash frozen and 830

pulverized Each sample was extracted with 300 microL of 90 methanol (vv) 50 microL of the 831

extract was run on an Agilent Lichrocart 250-4 RP18e 5-microm column using an Agilent 832

1100 series HPLC Camalexin was detected using a DiodeArray (DAD) at 330 nm and 833

with a fluorescence detector at emission 318 nmexcitation 385 nm (Agilent) Separation 834

was achieved using the following program with aqueous acetonitrile 5 min gradient 835

Page 39 of 54

from 63 to 69 acetonitrile 30 s gradient from 69 to 99 acetonitrile 2 min at 99 836

acetonitrile and a post-run equilibration of 35 min at 63 acetonitrile (Denby et al 837

2004 Kliebenstein et al 2005) Purified camalexin was used to produce a standard 838

curve to identify and quantitate camalexin 839

840

Accession Numbers 841

Microarray data used can be accessed at NCBI Gene Expression Omnnibus 842

(httpwwwncbinlmnihgovgeo) GSE18978 Sequence information for all Arabidopsis 843

genes described in this article can be found in TAIR (wwwarabidopsisorg) using the 844

following accession numbers PAD3 (CYP71B15 At3g26830) SID2 (ICS1 At1g74710) 845

GAE1 (At4g30440) GAE2 (At1g02000) GAE3 (At4g00110) GAE4 (At2g45310) GAE5 846

(At4g12250) GAE6 (At3g23820) PR1 (At2g14610) JAZ5 (At1g17380) JAZ10 847

(At5g13220) ACTIN2 (At3g18780) PDF12a (At5g44420) PDF12b (At2g26020) 848

AtSK11 (At5g26751) The sequence of the B cinerea cutinase Bc-CutA (Z69264) can 849

be found in the DNA Data Bank of Japan (httpgetentryddbjnigacjp) Germplasm 850

used included dde2-2 (At5g42650 (von Malek et al 2002)) pad3-1 (At3g26830 (Zhou 851

et al 1999)) pad4-1 (At3g52430 (Jirage et al 1999)) sid2-2 (At1g74710 (Wildermuth 852

et al 2001)) pbs3-2 (At5g13320 SALK_018225 (Nobuta et al 2007)) cbp60a-1 853

(At5g62570 SALK_124410 (Truman et al 2013)) cbp60g-1 sard1-2 (At5g26920 854

SALK_023199 At1g73805 SALK_052422 (Wang et al 2011) cbp60a-1 cbp60g-1 855

sard1-2 (Truman et al 2013) gae1-1 (At4g30440 SALK_085554) gae6-1 (At3g23280 856

SALK_104454) gae6-2 (At3g23280 SALK_017191) ixr1-1 cev1 (At5g05170 (Ellis 857

and Turner 2001 Scheible et al 2001)) eds1 plants were derived by introgression of 858

the Ler eds1-2 allele into Col-0 that contains two copies of EDS1 (At3g48090 859

At3g48080 (Bartsch et al 2006) mpk6-2 (At2g43790 SALK_073907 (Liu and Zhang 860

2004)) mpk3-DG (At3g45640 a fast neutron deletion mutant (Miles et al 2005)) and 861

npr1-1 (At1g64280 (Cao et al 1997)) T-DNA insertion lines were part of the SALK 862

collection (Alonso et al 2003) and were obtained from the Arabidopsis Biological 863

Resource Center 864

865

Supplemental Data 866

Page 40 of 54

Supplemental Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 867

Expression Does Not Require CBP60s or SID2 868

Supplemental Figure 2 Expression of GAE Family Members uponTreatment with Pma 869

ES4326 870

Supplemental Figure 3 Characterization of T-DNA Insertions in GAE1 and GAE6 871

Supplemental Figure 4 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves and 872

Are Slightly Smaller than Wild-Type Col-0 Plants 873

Supplemental Figure 5 Cell Walls of gae1 gae6 Plants Contain Less Uronic Acid than 874

Wild-Type Col-0 Cell Walls 875

Supplemental Figure 6 Antibodies Raised Against the RGI or XG Components of 876

Pectin Show No Difference in Binding to gae Mutant and Col-0 Cell Walls 877

Supplemental Figure 7 The Effect of B cinerea Treatment on the Expression of GAE 878

Family Genes 879

Supplemental Figure 8 Treatment with 001 Macerozyme Does Not Cause Any 880

Visible Damage to Col-0 Plants 881

Supplemental Figure 9 B cinerea Growth on Plants with Defects in Immune 882

Signaling 883

Supplemental Figure 10 MeJA-Induced Expression of the JA marker gene JAZ10 884

Supplemental Figure 11 The Expression of the JA Marker Gene JAZ5 Is Highly 885

Responsive in gae1 gae6 Plants 886

Supplemental Figure 12 The Expression of PDF12 Is Unaltered in gae1 gae6 Plants 887

Supplemental Figure 13 Macerozyme Treatment Activates MPK3 and MPK6 888

Supplemental Figure 14 B cinerea-Induced ROS Production in Wild-Type Col-0 and 889

gae1 gae6 Plants 890

Supplemental Figure 15 OG-Induced PTI Using 250 microg mL-1 OGs 891

Supplemental Figure 16 Abundance of Plant DNA in B cinerea Treated Plants 892

Supplemental Table 1 Primers used in this study 893

894

ACKNOWLEDGEMENTS 895

We thank William Truman for helpful discussions Simone Ferrari (Sapienza 896

Universita di Roma) for the generous gift of OGs Bruna Bucciarelli (USDA-ARS) for 897

Page 41 of 54

help with fluorescence microscopy the Arabidopsis Biological Resource Center for T-898

DNA insertion lines Paul Knoxrsquos lab (University of Leeds) for LM5 LM6 LM8 LM19 899

and LM20 antibodies and the Complex Carbohydrate Resource Center (University of 900

Georgia) for the CCRC-M7 antibody Most of this work was funded by the Division of 901

Chemical Sciences Geosciences and Biosciences Office of Basic Energy Sciences of 902

the US Department of Energy through Grant DE-FG02-05ER15670 to JG This work 903

was also supported by NSF awards IOS 1339125 and IOS 1021861 to DJK NSF 904

award IOS 1121425 to F K and the USDA National Institute of Food and Agriculture 905

Hatch project number CA-D-PLS-7033-H to DJK 906

907

AUTHOR CONTRIBUTIONS 908

GB and JG designed the research GB GX AT BL NES and NH 909

performed research DJK provided tools GB GX MP AD BL RAH and FK 910

analyzed data GB and JG wrote the paper with input from all authors 911

912

FIGURE LEGENDS 913

Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 Expression 914

Requires PAD4 EDS1 and PBS3 915

(A) Expression of GAE1 in leaves of wild-type Col-0 and mutant plants Expression was916

measured 24 or 32 h after either inoculation with Pma ES4326 (OD600 = 0002) or mock 917

inoculation with 5 mM MgSO4 and was then normalized to the level of ACTIN2 Data 918

from three biological replicates were combined using a mixed linear model Bars 919

represent mean log2-ratios to ACTIN2 plusmn SE Letters indicate significantly different 920

groups with q lt 005 where 005 represents the maximum false discovery rate at which 921

the test may be called significant 922

(B) Expression of GAE6 measured as described in (A)923

924

Figure 2 Susceptibility of gae6 and gae1 gae6 Plants to Pma ES4326 Is Increased 925

Relative to Wild-Type Col-0 926

Leaves of plants of the indicated genotypes were inoculated with Pma ES4326 (OD600 = 927

00002) as described in Methods Bacterial titers in leaves were determined either 928

Page 42 of 54

immediately (0 dpi) or after three days (3 dpi) Bars represent the mean plusmn SE of four 929

independent experiments each with 4 or 12 biological replicates at 0 and 3 dpi 930

respectively Data were combined using a mixed linear model Asterisks indicate 931

significant differences from Col-0 wild-type (q lt 005) Susceptible pad4 plants were 932

included as a positive control 933

934

Figure 3 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves 935

(A) Depiction of the assay for brittleness of gae mutant leaves Fully expanded leaves of936

four to five week-old plants were bent by 180ordm so that the adaxial surface of the leaf was 937

parallel with the soil Broken leaf petioles or midribs were considered brittle 938

(B) Quantification of broken midribs or petioles For each genotype 66 to 82 leaves 939

were analyzed for brittleness as described in (A) Bars represent the ratio of broken to 940

total number of leaves tested Results were analyzed using Fisherrsquos exact test Letters 941

indicate significantly different groups at p lt 005 942

(C) Conductivity measurements Leaves of four week-old plants were inoculated with943

deionized water and then leaf discs were cut out and placed on deionized water 944

Conductivity was measured at the indicated time points After 30 min the water was 945

replaced with fresh water (30 min f w) to account for wounding-related ion leakage from 946

the edges of the leaf discs Three independent experiments with six biological replicates 947

each were performed and data were analyzed using a t-test for each time point P-948

values were corrected using the Bonferroni method Data show means plusmn SE asterisks 949

indicate significant differences from wild-type Col-0 at q lt 001 950

951

Figure 4 Cell Walls of gae6 and gae1 gae6 Plants Are Reduced in GalA 952

(A) to (D) Levels of GalA (A) GlcA (B) cellulose (C) and neutral sugars (D) in alcohol953

insoluble residue (AIR) extracted from leaves of four week-old plants of the indicated 954

genotypes Bars represent means plusmn SE of four biological replicates combined using a 955

mixed linear model Letters indicate significantly different groups (p lt 005) 956

957

Figure 5 Cell Walls of gae1 gae6 Plants Are Reduced in Homogalacturonan 958

Page 43 of 54

Dot blot analyses with anti-LM19 and anti-LM20 antibodies Pectin was extracted from 959

AIR prepared from the indicated genotypes using 1 microL of extraction buffer per 10 microg of 960

AIR Pectin was serially diluted and one microL of undiluted 13 diluted and 19 diluted 961

samples were spotted on a nitrocellulose membrane Three biological replicates were 962

were spotted twice to obtain two technical replicates each All replicates produced 963

similar results 964

(A) Representative dot blot using anti-LM19 antibody965

(B) Relative signal intensities of the spots in the dot blot in (A)966

(C) Representative dot blot using anti-LM20 antibody967

(D) Relative signal intensities of the LM20 dot blot shown in (C)968

Relative signal intensities were measured using Image J software Measurements from 969

the three biological replicates were combined using a linear model after Box-Cox power 970

transformation as described in Methods Bars represent means plusmn SE Asterisks indicate 971

a difference from wild-type Col-0 at p lt 005 972

973

Figure 6 Growth of B cinerea on gae1 gae6 Plants and the Effect of B cinerea on 974

Expression of GAE1 and GAE6 975

(A) Growth of B cinerea isolates Leaves of Col-0 and gae1 gae6 plants were976

inoculated with 25 x 105 spores mL-1 of the B cinerea isolates indicated in the figure 977

Samples were collected two (2 dpi) and three (3 dpi) days later Bars represent means plusmn 978

SE of six biological replicates for each fungus combined using a mixed linear model 979

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 980

(B) and (C) Expression of GAE1 (B) and GAE6 (C) 48 h after inoculation with the B981

cinerea isolates indicated Expression levels were measured by qRT-PCR Bars 982

represent mean log2-ratios to ACTIN2 plusmn SE of three biological replicates combined 983

using a mixed linear model Letters indicate significantly different groups (q lt 005) 984

985

Figure 7 Levels of Soluble Pectin and Expression of PAD3 in Fractions Produced 986

Following Macerozyme Treatment of gae1 gae6 Cell Walls 987

(A) Schematic overview of the experimental procedure for (B) and (C)988

Page 44 of 54

(B) Uronic acid measurements Alcohol-insoluble residue (AIR) from leave tissue of four989

week-old wild-type Col-0 or gae1 gae6 plants was treated with water or 025 990

macerozyme and assayed for uronic acid levels Data from 11 biological replicates were 991

combined using a mixed linear model Bars represent means plusmn SE Letters indicate 992

significant differences (p lt 005) 993

(C) Expression of PAD3 in Col-0 plants treated with the fractions from (B) Extracts from994

(B) were heat inactivated pooled and diluted 5-fold with water Pooled extracts were995

inoculated into four week-old Col-0 plants PAD3 expression was measured 3 h after 996

inoculation and normalized to the level of ACTIN2 expression Data from four biological 997

replicates were combined using a mixed linear model Bars represent mean log2-ratios 998

to ACTIN2 plusmn SE Letters indicate significant differences (p lt 005) 999

1000

Figure 8 Macerozyme-Induced PAD3 Expression Is Absent in gae1 gae6 1001

(A) Expression of PAD3 after macerozyme treatment Four week-old plants were either1002

inoculated with 001 macerozyme or mock inoculated with water and PAD3 1003

expression was measured 3 h later by qRT-PCR Data from four biological replicates 1004

were combined using a mixed linear model Mean log2-ratios to ACTIN2 plusmn SE were 1005

plotted Letters indicate significantly different groups (p lt 005) 1006

(B) Expression of PAD3 after OG treatment PAD3 expression was measured as1007

described in (A) 3 h after inoculation with 100 microg mL-1 OGs or mock inoculation with 1008

water Data are from six biological replicates 1009

(C) PAD3 expression in untreated plants Three biological replicates were performed1010

and data analyzed as described in (A) 1011

(D) Camalexin accumulation after B cinerea treatment Camalexin accumulation was1012

measured in four week-old plants inoculated with the B cinerea strains shown in the 1013

figure Eight biological replicates each were combined using a mixed linear model 1014

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 1015

(E) PAD3 expression 28 h after treatment with the indicated B cinerea strains Data1016

from six biological replicates were collected and analyzed as described in (A) Data in 1017

all figure parts represent means plusmn SE Letters indicate significantly different groups (q lt 1018

005) 1019

Page 45 of 54

1020

Figure 9 Macerozyme-Induced Expression of the SA Marker Genes SID2 and PR1 1021

Is Abolished in gae1 gae6 Plants 1022

(A) to (H) Expression of PR1 (A) (C) (E) and (G) or SID2 (B) (D) (F) and (H) in four1023

week-old Col-0 and gae1 gae6 plants Data were combined using a mixed linear model 1024

Bars represent mean log2-ratios of expression vs ACTIN2 plusmn SE for all figure parts 1025

Letters indicate significantly different groups at p lt 005 for panels (A) to (D) (G) and 1026

(H) and qlt005 for panels (E) to (F) 1027

(A) and (B) Plants were treated with 001 macerozyme or mock treated with water1028

and samples were collected 3 h later Data from four biological replicates are shown 1029

(C) and (D) Plants were inoculated with 100 microg mL-1 OGs or mock treated with water1030

and samples collected 3 h later Data from six biological replicates are shown 1031

(E) and (F) Plants were inoculated with the indicated B cinerea isolates or mock1032

inoculated with B cinerea inoculation medium and samples collected 48 h later Data 1033

from eight biological replicates are shown 1034

(G) and (H) Expression in untreated plants Three biological replicates were performed1035

1036

Figure 10 The Expression of the JA Marker Gene JAZ10 is Highly Responsive in 1037

gae1 gae6 Plants 1038

(A) to (D) JAZ10 expression in four week-old plants Data were combined using a mixed1039

linear model Mean log2-ratios to ACTIN2 plusmn SE are shown Letters indicate significantly 1040

different groups at p lt 005 for panels (A) (B) and (D) and q lt 005 for panel (C) 1041

(A) Plants were inoculated with 001 macerozyme or mock inoculated using water and1042

samples were collected 3 h later Data from four biological replicates are shown 1043

(B) Plants were inoculated with 100 microg mL-1 OGs or mock inoculated using water and1044

samples collected 3 h later Data are from six biological replicates 1045

(C) JAZ10 expression 48 h after treatment with the indicated B cinerea strains Data1046

are from six biological replicates 1047

(D) JAZ10 expression in untreated plants Data are from three biological replicates1048

1049

Figure 11 Gallo 1-Induced Callose Deposition Is Reduced in gae1 gae6 Plants 1050

Page 46 of 54

(A) Callose deposition in four week-old Col-0 and gae1 gae6 plants Plants were1051

inoculated with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) or mock inoculated 1052

with B cinerea inoculation medium and samples were collected after 72 h Callose was 1053

stained with aniline blue and visualized using a Nikon Eclipse Ni-U microscope with a 1054

DAPI filter Three leaves were stained per treatment Scale bars are 100 microm Asterisks 1055

indicate fungal hyphae arrows indicate representative callose spots 1056

(B) Quantification of callose deposition Callose deposits were counted in 200 microm x 2001057

microm squares laid out across the images shown here Bars represent means plusmn SE from 1058

six squares each Letters indicate significantly different groups (p lt 005) according to a 1059

t-test analysis1060

1061

Figure 12 Less Soluble Pectin Accumulates in B cinerea-Infected gae1 gae6 1062

Leaves 1063

Four week-old Col-0 and gae1 gae6 plants were inoculated with 10 microL droplets of the 1064

indicated B cinerea isolate (25 x 105 spores mL-1) or mock inoculated with B cinerea 1065

inoculation medium Leaves were collected 48 h later AIR was extracted from leaf 1066

samples and water added (20 microL infection site) to extract soluble pectin fragments 1067

Samples were vortexed for 7 h centrifuged at 12000 g and the uronic acid 1068

concentration of the supernatant was measured Data from nine biological replicates 1069

were combined using a mixed linear model Means plusmn SE are shown Letters indicate 1070

significant differences (q lt 005) 1071

1072

Figure 13 Macerozyme-Induced PTI Is Lost in gae1 gae6 While OG-Induced PTI Is 1073

Unaltered 1074

(A) Macerozyme-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1075

inoculated with 001 macerozyme or mock inoculated with boiled macerozyme and 1076

were infected with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) 24 h later Samples 1077

were collected immediately (0 dpi) and after two (2 dpi) and three (3 dpi) days Data 1078

show mean plusmn SE of six to 14 biological replicates each combined using a linear model 1079

Letters indicate significantly different groups (q lt 005) 1080

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

Parsed CitationsAdam L and Somerville SC (1996) Genetic characterization of five powdery mildew disease resistance loci in Arabidopsisthaliana Plant J 9 341-356

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Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk RGadrinab C Heller C Jeske A Koesema E Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt MHazari N Hom E Karnes M Mulholland C Ndubaku R Schmidt I Guzman P Aguilar-Henonin L Schmid M Weigel DCarter DE Marchand T Risseeuw E Brogden D Zeko A Crosby WL Berry CC and Ecker JR (2003) Genome-wideinsertional mutagenesis of Arabidopsis thaliana Science 301 653-657

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Asai T Tena G Plotnikova J Willmann MR Chiu WL Gomez-Gomez L Boller T Ausubel FM and Sheen J (2002) MAPkinase signalling cascade in Arabidopsis innate immunity Nature 415 977-983

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Bartsch M Gobbato E Bednarek P Debey S Schultze JL Bautor J and Parker JE (2006) Salicylic acid-independentENHANCED DISEASE SUSCEPTIBILITY1 signaling in Arabidopsis immunity and cell death is regulated by the monooxygenaseFMO1 and the nudix hydrolase NUDT7 Plant Cell 18 1038-1051

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Bates D Machler M Bolker BM and Walker SC (2015) Fitting Linear Mixed-Effects Models Using lme4 J Stat Softw 67 1-48Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Benjamini Y and Hochberg Y (1995) Controlling the False Discovery Rate - a Practical and Powerful Approach to MultipleTesting J Roy Stat Soc B Met 57 289-300

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Bethke G Grundman RE Sreekanta S Truman W Katagiri F and Glazebrook J (2014) Arabidopsis PECTINMETHYLESTERASEs Contribute to Immunity against Pseudomonas syringae Plant Physiol 164 1093-1107

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Bouton S Leboeuf E Mouille G Leydecker MT Talbotec J Granier F Lahaye M Hofte H and Truong HN (2002)Quasimodo1 encodes a putative membrane-bound glycosyltransferase required for normal pectin synthesis and cell adhesion inArabidopsis Plant Cell 14 2577-2590

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Box GEP and Cox DR (1964) An analysis of transformations Journal of the Royal Statistical Society Series B 26 211-252Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Cao H Glazebrook J Clarke JD Volko S and Dong XN (1997) The Arabidopsis NPR1 gene that controls systemic acquiredresistance encodes a novel protein containing ankyrin repeats Cell 88 57-63

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Carpita N (2010) Update on Mechanisms of Plant Cell Wall Biosynthesis How plants make cellulose and other (1-gt4)-beta-D-glycans Plant Physiol

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Chisholm ST Coaker G Day B and Staskawicz BJ (2006) Host-microbe interactions Shaping the evolution of the plantimmune response Cell 124 803-814

Page 34 of 54

conductivity meter after 30 min and then the water was replaced Conductivity was 683

measured again 30 min later (1 h total) as indicated in Figure 3C Six biological 684

replicates each were performed in three independent experiments and means and SEs 685

were calculated Two-sided t-tests were performed for each time point 686

687

DAB and Aniline Blue Staining 688

Leaves from four week-old plants were either mock inoculated with B cinerea 689

inoculation medium or inoculated with 10 microL droplets of a B cinerea spore solution (25 690

x 105 spores mL-1) of the indicated isolates Samples were collected 72 h later For 33prime-691

diaminobenzidine (DAB) staining two to five inoculated leaves were vacuum infiltrated 692

with 33prime-Diaminobenzidine (1 mg mL-1 in water pH 38) solution and stained overnight 693

in the dark Leaves were destained using 100 ethanol at 65degC and then transferred to 694

50 (vv) glycerol and photographed 695

Callose was visualized by staining leaves with aniline blue as previously 696

described (Adam and Somerville 1996) Briefly leaves were vacuum infiltrated with 697

alcoholic lactophenol (one volume of phenolglycerollactic acidwater (1111) mixed 698

with two volumes of ethanol) and destained at 65degC Next leaves were moved through 699

an alcohol gradient (50 20 and 10 (vv) ethanol) into water The destained leaves 700

were stained for 30 min in 150 mM K2HPO4 (pH 95) with 001 (wv) aniline blue 701

Samples were mounted in water and examined using a Nikon Eclipse Ni-U microscope 702

at 10x magnification using a DAPI filter (excitation at 325-375 nm emission at 435-485 703

nm) For callose quantification six 200 microm x 200 microm squares were counted for each 704

sample type Callose deposits per mm2 were calculated and data analyzed using a t-705

test Note that there is an inherent uncertainty associated with such counts because 706

callose deposits may not all be in focus at the same time 707

708

MAPK Activity Assay 709

Fully expanded leaves of four week-old plants were inoculated with 001 710

macerozyme 100 microg mL-1 OGs 100 nM flg22 or water (mock) flg22 a peptide from 711

bacterial flagella that induces immune responses was used as a positive control for 712

Page 35 of 54

MAPK activation (Asai et al 2002) and was purchased from EZBiolab Inc (Westfield 713

IN USA) Samples were harvested 10 min later and immediately frozen in liquid 714

nitrogen Protein was extracted using an extraction buffer containing 25 mM Tris pH 78 715

75 mM NaCl 1 mM DTT 1 mM NaF 05mM Na3VO4 01 (vv) Tween 20 one 716

cOmplete Mini protease inhibitor cocktail tablet (Roche) and one PhosSTOP 717

phosphatase inhibitor cocktail tablet (Roche) per 10 mL buffer 15 microg of total protein 718

were electrophoresed on a 10 SDS-polyacrylamide gel and then blotted onto a PVDF 719

membrane (Bio-Rad 162-0177) Activated MAPKs were detected using p4442 MAPK 720

(Erk12) antibody (9102S Cell Signal 12500 in TBST (20 mM Tris pH 75 150 mM 721

NaCl 01 (vv) Tween 20)) Anti-Rabbit IgG-HRP (115000 in TBST A6154 Sigma) 722

and ECL Plus substrate (Pierce 32132) MPK3 and MPK6 were detected with an anti-723

MPK3 antibody (M8318 Sigma 12000 in TBST and 3 (wv) milk) or an anti-MPK6 724

antibody (A7104 Sigma 16000 in TBST and 3 (wv) milk) respectively The 725

secondary antibody and detection were the same as for the p4442 antibody Five 726

independent experiments were performed 727

728

Preparation of Alcohol Insoluble Residue (AIR) Pectin Extraction and Uronic Acid 729

Measurements 730

Fully expanded leaves from four week-old plants that were incubated for 48 h in 731

the dark to reduce starch were harvested flash frozen and pulverized AIR was 732

extracted by washing ground material twice in 70 (vv) ethanol three times in a 733

mixture of chloroform and methanol (11 vv) and once in acetone (Gille et al 2009) 734

and was then air dried Cell wall pectin was extracted from AIR with cell wall extraction 735

buffer (50 mM Trizma 50 mM CDTA pH 72) at 95degC for 15 min Samples were 736

homogenized using a paint shaker (Harbil 5G-HD) and glass beads (3 mm diameter) 737

For dot blot experiments 500 microL of cell wall extraction buffer were used per 10 mg of 738

AIR Debris was precipitated by centrifugation for 10 min at 10000 g 739

Total uronic acid content of pectin extracted as described above using 500 microL of 740

cell wall extraction buffer per 1 mg of AIR was measured as described previously 741

(Filisetti-Cozzi and Carpita 1991 van den Hoogen et al 1998) First 36 microL of pectin 742

extract were mixed with 4 microL of 4 M sulfamic acid Then 200 microL of sulfuric acid 743

Page 36 of 54

containing 120 mM sodium tetraborate was added and samples were incubated at 80degC 744

for 1 h Following cooling on ice the optical density of samples was measured at 490 745

nm Next 40 microL of m-hydroxydiphenyl reagent (100 microL of m-hydroxydiphenyl in dimethyl 746

sulfoxide at 100 mgmL mixed with 49 mL 80 (vv) sulfuric acid just before use) was 747

added and the samples were mixed The optical density at 490 nm was measured 748

again The optical density before m-hydroxydiphenyl reagent addition was subtracted 749

from the optical density measured after addition of the dye The concentration of uronic 750

acid was calculated using known amounts of GalA as a standard 751

752

Release of Pectin from Arabidopsis Cell Walls Using Macerozyme 753

AIR preparations from leaves of four week-old plants were treated with 025 754

(wv) macerozyme R-10 (Yakult) or water (mock) at a concentration of 100 microL per mg 755

AIR Samples were incubated for 3 h at room temperature on a vortex shaker and 756

centrifuged for 10 min at 12000 g Total uronic acid concentration of 5-fold diluted 757

supernatants was determined for two technical replicates each The concentration of 758

uronic acid was calculated using known amounts of GalA as a standard For 759

subsequent qRT-PCR experiments supernatants from multiple extractions were 760

combined boiled for 20 min to inactivate macerozyme diluted 10-fold with water and 761

inoculated into four week-old Col-0 plants Heat-inactivated 10-fold diluted macerozyme 762

solutions were used as mock treatment Samples were collected 3 h later and PAD3 763

expression was determined 764

765

Release of Pectin from Arabidopsis Leaves Infected with B cinerea 766

Fully expanded leaves of four week-old plants were either inoculated with up to 767

five 10 microL droplets of B cinerea spore solution (25 x 105 spores mL-1) or mock 768

inoculated with B cinerea inoculation medium For each biological replicate leaves with 769

a combined count of at least 20 infection sites were collected 48 h later AIR was 770

extracted as described above but plants were not dark-treated or freeze-dried and the 771

first ethanol step was performed using 85 (vv) ethanol To extract soluble pectin 772

fragments AIR samples were mixed with water at a concentration of 20 microL per infection 773

site incubated for 7 h at room temperature on a vortex shaker and centrifuged for 10 774

Page 37 of 54

min at 12000 g Total uronic acid concentration of supernatants was determined for two 775

technical replicates each The concentration of uronic acid was calculated using known 776

amounts of GalA as a standard 777

778

Measurement of Uronic Acids Neutral Monosaccharides and Cellulose Content in 779

Cell Walls 780

Neutral sugars and uronic acids from non-cellulosic polysaccharides in cell walls 781

(AIR) were released by trifluoroacetic acid (TFA) hydrolysis Approximately 1 mg of AIR 782

was hydrolyzed with 2 M TFA at 121degC for 90 min The TFA-soluble material was split 783

into two equal parts one for uronic acid and one for neutral sugar determination The 784

uronic acid content was determined by analyzing the hydrolyzate using a CarboPac 785

PA200 anion-exchange column with an ICS-3000 Dionex chromatography system 786

(Dionex Sunnyvale CA USA) The elution profile consisted of a linear gradient of 50 to 787

200 mM sodium acetate in 01 M NaOH in 10 min at 04 mL per min Alditol acetate 788

derivatives of neutral sugars were produced as described (York et al 1985) In brief 789

monosaccharides in the hydrolyzate were reduced with NaBH4 and peracetylated with 790

acetic anhydride and pyridine at 121degC for 20 min The generated alditol acetates were 791

analyzed by GC-MS Crystalline cellulose was measured according to Updegraff (1969) 792

After hydrolysis of noncellulosic polysaccharides from AIR with the Updegraff reagent 793

(acetic acid nitric acids water 812 (vv) ) the remaining pellet was hydrolyzed in 794

72 sulfuric acid The resulting glucose quantity was determined by the anthrone 795

method (Scott and Melvin 1953) 796

797

Dot Blot Experiments 798

Pectin solutions extracted using 50 microL of cell wall extraction buffer per mg AIR 799

were serially diluted and nitrocellulose membranes were spotted with 1 microL of the diluted 800

pectin solutions Membranes were dried overnight blocked with 5 milk (wv) in 1x 801

PBS (8 g L-1 NaCl 02 g L-1 KCl 144 g L-1 Na2HPO4 024 g L-1 KH2PO4 pH 74) and 802

probed with LM5 (Jones et al 1997) LM6 (Willats et al 1998) LM8 (Willats et al 803

2004) LM19 and LM20 (Verhertbruggen et al 2009a) or CCRC-M7 (Steffan et al 804

Page 38 of 54

1995) antibodies LM series antibodies were diluted 1250 and CCRC-M7 was diluted 805

1500 in 5 milk powder (Nestle Carnation) in 1 x PBS For LM series antibodies a 806

goat anti-Rat HRP conjugated antibody (Bethyl A110-105P 15000 diluted) and for 807

CCRC-M7 an anti-Mouse IgG HRP conjugate (Promega W402B 12500 diluted) was 808

used as secondary antibody in 5 milk powder in 1x PBS LM series antibodies were 809

obtained from plant probes (wwwplantprobesnet) and CCRC-M7 from CarboSource 810

(wwwcarbosourcenet) Membranes were washed after incubation with the primary and 811

secondary antibodies with 1x PBS Dot blots were developed using the ECL system 812

(GE Healthcare Amersham ECL prime) For quantification of signals dot blot results on 813

x-ray films were photographed using a CCD camera and intensities were measured814

using Image Jrsquos Integrated Density function Locally adjusted background was 815

subtracted As the dynamic range of the signal on an x-ray film was narrow non-816

linearity in the measured values was corrected for each genotype in each biological 817

replicate using the values from different dilutions in two technical replicates We noticed 818

that the residuals were not normally distributed and performed Box-Cox power 819

transformation (Box and Cox 1964) to obtain normally distributed values for the 820

subsequent statistical analysis of the data For LM19 Box-Cox selected power 821

transformation was done to the power of 038th and for LM20 to the power 075th After 822

correction of the transformed measured values with the effect of the biological 823

replicates the values were fit to a linear model with the Arabidopsis genotype as the 824

fixed effect This linear model was used both to generate the plots in Figures 5B and D 825

and to test significant differences 826

827

Camalexin Measurements 828

Four week-old plants were infected with B cinerea and samples (three leaf disks 829

from one plant each) were harvested at the indicated time points flash frozen and 830

pulverized Each sample was extracted with 300 microL of 90 methanol (vv) 50 microL of the 831

extract was run on an Agilent Lichrocart 250-4 RP18e 5-microm column using an Agilent 832

1100 series HPLC Camalexin was detected using a DiodeArray (DAD) at 330 nm and 833

with a fluorescence detector at emission 318 nmexcitation 385 nm (Agilent) Separation 834

was achieved using the following program with aqueous acetonitrile 5 min gradient 835

Page 39 of 54

from 63 to 69 acetonitrile 30 s gradient from 69 to 99 acetonitrile 2 min at 99 836

acetonitrile and a post-run equilibration of 35 min at 63 acetonitrile (Denby et al 837

2004 Kliebenstein et al 2005) Purified camalexin was used to produce a standard 838

curve to identify and quantitate camalexin 839

840

Accession Numbers 841

Microarray data used can be accessed at NCBI Gene Expression Omnnibus 842

(httpwwwncbinlmnihgovgeo) GSE18978 Sequence information for all Arabidopsis 843

genes described in this article can be found in TAIR (wwwarabidopsisorg) using the 844

following accession numbers PAD3 (CYP71B15 At3g26830) SID2 (ICS1 At1g74710) 845

GAE1 (At4g30440) GAE2 (At1g02000) GAE3 (At4g00110) GAE4 (At2g45310) GAE5 846

(At4g12250) GAE6 (At3g23820) PR1 (At2g14610) JAZ5 (At1g17380) JAZ10 847

(At5g13220) ACTIN2 (At3g18780) PDF12a (At5g44420) PDF12b (At2g26020) 848

AtSK11 (At5g26751) The sequence of the B cinerea cutinase Bc-CutA (Z69264) can 849

be found in the DNA Data Bank of Japan (httpgetentryddbjnigacjp) Germplasm 850

used included dde2-2 (At5g42650 (von Malek et al 2002)) pad3-1 (At3g26830 (Zhou 851

et al 1999)) pad4-1 (At3g52430 (Jirage et al 1999)) sid2-2 (At1g74710 (Wildermuth 852

et al 2001)) pbs3-2 (At5g13320 SALK_018225 (Nobuta et al 2007)) cbp60a-1 853

(At5g62570 SALK_124410 (Truman et al 2013)) cbp60g-1 sard1-2 (At5g26920 854

SALK_023199 At1g73805 SALK_052422 (Wang et al 2011) cbp60a-1 cbp60g-1 855

sard1-2 (Truman et al 2013) gae1-1 (At4g30440 SALK_085554) gae6-1 (At3g23280 856

SALK_104454) gae6-2 (At3g23280 SALK_017191) ixr1-1 cev1 (At5g05170 (Ellis 857

and Turner 2001 Scheible et al 2001)) eds1 plants were derived by introgression of 858

the Ler eds1-2 allele into Col-0 that contains two copies of EDS1 (At3g48090 859

At3g48080 (Bartsch et al 2006) mpk6-2 (At2g43790 SALK_073907 (Liu and Zhang 860

2004)) mpk3-DG (At3g45640 a fast neutron deletion mutant (Miles et al 2005)) and 861

npr1-1 (At1g64280 (Cao et al 1997)) T-DNA insertion lines were part of the SALK 862

collection (Alonso et al 2003) and were obtained from the Arabidopsis Biological 863

Resource Center 864

865

Supplemental Data 866

Page 40 of 54

Supplemental Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 867

Expression Does Not Require CBP60s or SID2 868

Supplemental Figure 2 Expression of GAE Family Members uponTreatment with Pma 869

ES4326 870

Supplemental Figure 3 Characterization of T-DNA Insertions in GAE1 and GAE6 871

Supplemental Figure 4 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves and 872

Are Slightly Smaller than Wild-Type Col-0 Plants 873

Supplemental Figure 5 Cell Walls of gae1 gae6 Plants Contain Less Uronic Acid than 874

Wild-Type Col-0 Cell Walls 875

Supplemental Figure 6 Antibodies Raised Against the RGI or XG Components of 876

Pectin Show No Difference in Binding to gae Mutant and Col-0 Cell Walls 877

Supplemental Figure 7 The Effect of B cinerea Treatment on the Expression of GAE 878

Family Genes 879

Supplemental Figure 8 Treatment with 001 Macerozyme Does Not Cause Any 880

Visible Damage to Col-0 Plants 881

Supplemental Figure 9 B cinerea Growth on Plants with Defects in Immune 882

Signaling 883

Supplemental Figure 10 MeJA-Induced Expression of the JA marker gene JAZ10 884

Supplemental Figure 11 The Expression of the JA Marker Gene JAZ5 Is Highly 885

Responsive in gae1 gae6 Plants 886

Supplemental Figure 12 The Expression of PDF12 Is Unaltered in gae1 gae6 Plants 887

Supplemental Figure 13 Macerozyme Treatment Activates MPK3 and MPK6 888

Supplemental Figure 14 B cinerea-Induced ROS Production in Wild-Type Col-0 and 889

gae1 gae6 Plants 890

Supplemental Figure 15 OG-Induced PTI Using 250 microg mL-1 OGs 891

Supplemental Figure 16 Abundance of Plant DNA in B cinerea Treated Plants 892

Supplemental Table 1 Primers used in this study 893

894

ACKNOWLEDGEMENTS 895

We thank William Truman for helpful discussions Simone Ferrari (Sapienza 896

Universita di Roma) for the generous gift of OGs Bruna Bucciarelli (USDA-ARS) for 897

Page 41 of 54

help with fluorescence microscopy the Arabidopsis Biological Resource Center for T-898

DNA insertion lines Paul Knoxrsquos lab (University of Leeds) for LM5 LM6 LM8 LM19 899

and LM20 antibodies and the Complex Carbohydrate Resource Center (University of 900

Georgia) for the CCRC-M7 antibody Most of this work was funded by the Division of 901

Chemical Sciences Geosciences and Biosciences Office of Basic Energy Sciences of 902

the US Department of Energy through Grant DE-FG02-05ER15670 to JG This work 903

was also supported by NSF awards IOS 1339125 and IOS 1021861 to DJK NSF 904

award IOS 1121425 to F K and the USDA National Institute of Food and Agriculture 905

Hatch project number CA-D-PLS-7033-H to DJK 906

907

AUTHOR CONTRIBUTIONS 908

GB and JG designed the research GB GX AT BL NES and NH 909

performed research DJK provided tools GB GX MP AD BL RAH and FK 910

analyzed data GB and JG wrote the paper with input from all authors 911

912

FIGURE LEGENDS 913

Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 Expression 914

Requires PAD4 EDS1 and PBS3 915

(A) Expression of GAE1 in leaves of wild-type Col-0 and mutant plants Expression was916

measured 24 or 32 h after either inoculation with Pma ES4326 (OD600 = 0002) or mock 917

inoculation with 5 mM MgSO4 and was then normalized to the level of ACTIN2 Data 918

from three biological replicates were combined using a mixed linear model Bars 919

represent mean log2-ratios to ACTIN2 plusmn SE Letters indicate significantly different 920

groups with q lt 005 where 005 represents the maximum false discovery rate at which 921

the test may be called significant 922

(B) Expression of GAE6 measured as described in (A)923

924

Figure 2 Susceptibility of gae6 and gae1 gae6 Plants to Pma ES4326 Is Increased 925

Relative to Wild-Type Col-0 926

Leaves of plants of the indicated genotypes were inoculated with Pma ES4326 (OD600 = 927

00002) as described in Methods Bacterial titers in leaves were determined either 928

Page 42 of 54

immediately (0 dpi) or after three days (3 dpi) Bars represent the mean plusmn SE of four 929

independent experiments each with 4 or 12 biological replicates at 0 and 3 dpi 930

respectively Data were combined using a mixed linear model Asterisks indicate 931

significant differences from Col-0 wild-type (q lt 005) Susceptible pad4 plants were 932

included as a positive control 933

934

Figure 3 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves 935

(A) Depiction of the assay for brittleness of gae mutant leaves Fully expanded leaves of936

four to five week-old plants were bent by 180ordm so that the adaxial surface of the leaf was 937

parallel with the soil Broken leaf petioles or midribs were considered brittle 938

(B) Quantification of broken midribs or petioles For each genotype 66 to 82 leaves 939

were analyzed for brittleness as described in (A) Bars represent the ratio of broken to 940

total number of leaves tested Results were analyzed using Fisherrsquos exact test Letters 941

indicate significantly different groups at p lt 005 942

(C) Conductivity measurements Leaves of four week-old plants were inoculated with943

deionized water and then leaf discs were cut out and placed on deionized water 944

Conductivity was measured at the indicated time points After 30 min the water was 945

replaced with fresh water (30 min f w) to account for wounding-related ion leakage from 946

the edges of the leaf discs Three independent experiments with six biological replicates 947

each were performed and data were analyzed using a t-test for each time point P-948

values were corrected using the Bonferroni method Data show means plusmn SE asterisks 949

indicate significant differences from wild-type Col-0 at q lt 001 950

951

Figure 4 Cell Walls of gae6 and gae1 gae6 Plants Are Reduced in GalA 952

(A) to (D) Levels of GalA (A) GlcA (B) cellulose (C) and neutral sugars (D) in alcohol953

insoluble residue (AIR) extracted from leaves of four week-old plants of the indicated 954

genotypes Bars represent means plusmn SE of four biological replicates combined using a 955

mixed linear model Letters indicate significantly different groups (p lt 005) 956

957

Figure 5 Cell Walls of gae1 gae6 Plants Are Reduced in Homogalacturonan 958

Page 43 of 54

Dot blot analyses with anti-LM19 and anti-LM20 antibodies Pectin was extracted from 959

AIR prepared from the indicated genotypes using 1 microL of extraction buffer per 10 microg of 960

AIR Pectin was serially diluted and one microL of undiluted 13 diluted and 19 diluted 961

samples were spotted on a nitrocellulose membrane Three biological replicates were 962

were spotted twice to obtain two technical replicates each All replicates produced 963

similar results 964

(A) Representative dot blot using anti-LM19 antibody965

(B) Relative signal intensities of the spots in the dot blot in (A)966

(C) Representative dot blot using anti-LM20 antibody967

(D) Relative signal intensities of the LM20 dot blot shown in (C)968

Relative signal intensities were measured using Image J software Measurements from 969

the three biological replicates were combined using a linear model after Box-Cox power 970

transformation as described in Methods Bars represent means plusmn SE Asterisks indicate 971

a difference from wild-type Col-0 at p lt 005 972

973

Figure 6 Growth of B cinerea on gae1 gae6 Plants and the Effect of B cinerea on 974

Expression of GAE1 and GAE6 975

(A) Growth of B cinerea isolates Leaves of Col-0 and gae1 gae6 plants were976

inoculated with 25 x 105 spores mL-1 of the B cinerea isolates indicated in the figure 977

Samples were collected two (2 dpi) and three (3 dpi) days later Bars represent means plusmn 978

SE of six biological replicates for each fungus combined using a mixed linear model 979

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 980

(B) and (C) Expression of GAE1 (B) and GAE6 (C) 48 h after inoculation with the B981

cinerea isolates indicated Expression levels were measured by qRT-PCR Bars 982

represent mean log2-ratios to ACTIN2 plusmn SE of three biological replicates combined 983

using a mixed linear model Letters indicate significantly different groups (q lt 005) 984

985

Figure 7 Levels of Soluble Pectin and Expression of PAD3 in Fractions Produced 986

Following Macerozyme Treatment of gae1 gae6 Cell Walls 987

(A) Schematic overview of the experimental procedure for (B) and (C)988

Page 44 of 54

(B) Uronic acid measurements Alcohol-insoluble residue (AIR) from leave tissue of four989

week-old wild-type Col-0 or gae1 gae6 plants was treated with water or 025 990

macerozyme and assayed for uronic acid levels Data from 11 biological replicates were 991

combined using a mixed linear model Bars represent means plusmn SE Letters indicate 992

significant differences (p lt 005) 993

(C) Expression of PAD3 in Col-0 plants treated with the fractions from (B) Extracts from994

(B) were heat inactivated pooled and diluted 5-fold with water Pooled extracts were995

inoculated into four week-old Col-0 plants PAD3 expression was measured 3 h after 996

inoculation and normalized to the level of ACTIN2 expression Data from four biological 997

replicates were combined using a mixed linear model Bars represent mean log2-ratios 998

to ACTIN2 plusmn SE Letters indicate significant differences (p lt 005) 999

1000

Figure 8 Macerozyme-Induced PAD3 Expression Is Absent in gae1 gae6 1001

(A) Expression of PAD3 after macerozyme treatment Four week-old plants were either1002

inoculated with 001 macerozyme or mock inoculated with water and PAD3 1003

expression was measured 3 h later by qRT-PCR Data from four biological replicates 1004

were combined using a mixed linear model Mean log2-ratios to ACTIN2 plusmn SE were 1005

plotted Letters indicate significantly different groups (p lt 005) 1006

(B) Expression of PAD3 after OG treatment PAD3 expression was measured as1007

described in (A) 3 h after inoculation with 100 microg mL-1 OGs or mock inoculation with 1008

water Data are from six biological replicates 1009

(C) PAD3 expression in untreated plants Three biological replicates were performed1010

and data analyzed as described in (A) 1011

(D) Camalexin accumulation after B cinerea treatment Camalexin accumulation was1012

measured in four week-old plants inoculated with the B cinerea strains shown in the 1013

figure Eight biological replicates each were combined using a mixed linear model 1014

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 1015

(E) PAD3 expression 28 h after treatment with the indicated B cinerea strains Data1016

from six biological replicates were collected and analyzed as described in (A) Data in 1017

all figure parts represent means plusmn SE Letters indicate significantly different groups (q lt 1018

005) 1019

Page 45 of 54

1020

Figure 9 Macerozyme-Induced Expression of the SA Marker Genes SID2 and PR1 1021

Is Abolished in gae1 gae6 Plants 1022

(A) to (H) Expression of PR1 (A) (C) (E) and (G) or SID2 (B) (D) (F) and (H) in four1023

week-old Col-0 and gae1 gae6 plants Data were combined using a mixed linear model 1024

Bars represent mean log2-ratios of expression vs ACTIN2 plusmn SE for all figure parts 1025

Letters indicate significantly different groups at p lt 005 for panels (A) to (D) (G) and 1026

(H) and qlt005 for panels (E) to (F) 1027

(A) and (B) Plants were treated with 001 macerozyme or mock treated with water1028

and samples were collected 3 h later Data from four biological replicates are shown 1029

(C) and (D) Plants were inoculated with 100 microg mL-1 OGs or mock treated with water1030

and samples collected 3 h later Data from six biological replicates are shown 1031

(E) and (F) Plants were inoculated with the indicated B cinerea isolates or mock1032

inoculated with B cinerea inoculation medium and samples collected 48 h later Data 1033

from eight biological replicates are shown 1034

(G) and (H) Expression in untreated plants Three biological replicates were performed1035

1036

Figure 10 The Expression of the JA Marker Gene JAZ10 is Highly Responsive in 1037

gae1 gae6 Plants 1038

(A) to (D) JAZ10 expression in four week-old plants Data were combined using a mixed1039

linear model Mean log2-ratios to ACTIN2 plusmn SE are shown Letters indicate significantly 1040

different groups at p lt 005 for panels (A) (B) and (D) and q lt 005 for panel (C) 1041

(A) Plants were inoculated with 001 macerozyme or mock inoculated using water and1042

samples were collected 3 h later Data from four biological replicates are shown 1043

(B) Plants were inoculated with 100 microg mL-1 OGs or mock inoculated using water and1044

samples collected 3 h later Data are from six biological replicates 1045

(C) JAZ10 expression 48 h after treatment with the indicated B cinerea strains Data1046

are from six biological replicates 1047

(D) JAZ10 expression in untreated plants Data are from three biological replicates1048

1049

Figure 11 Gallo 1-Induced Callose Deposition Is Reduced in gae1 gae6 Plants 1050

Page 46 of 54

(A) Callose deposition in four week-old Col-0 and gae1 gae6 plants Plants were1051

inoculated with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) or mock inoculated 1052

with B cinerea inoculation medium and samples were collected after 72 h Callose was 1053

stained with aniline blue and visualized using a Nikon Eclipse Ni-U microscope with a 1054

DAPI filter Three leaves were stained per treatment Scale bars are 100 microm Asterisks 1055

indicate fungal hyphae arrows indicate representative callose spots 1056

(B) Quantification of callose deposition Callose deposits were counted in 200 microm x 2001057

microm squares laid out across the images shown here Bars represent means plusmn SE from 1058

six squares each Letters indicate significantly different groups (p lt 005) according to a 1059

t-test analysis1060

1061

Figure 12 Less Soluble Pectin Accumulates in B cinerea-Infected gae1 gae6 1062

Leaves 1063

Four week-old Col-0 and gae1 gae6 plants were inoculated with 10 microL droplets of the 1064

indicated B cinerea isolate (25 x 105 spores mL-1) or mock inoculated with B cinerea 1065

inoculation medium Leaves were collected 48 h later AIR was extracted from leaf 1066

samples and water added (20 microL infection site) to extract soluble pectin fragments 1067

Samples were vortexed for 7 h centrifuged at 12000 g and the uronic acid 1068

concentration of the supernatant was measured Data from nine biological replicates 1069

were combined using a mixed linear model Means plusmn SE are shown Letters indicate 1070

significant differences (q lt 005) 1071

1072

Figure 13 Macerozyme-Induced PTI Is Lost in gae1 gae6 While OG-Induced PTI Is 1073

Unaltered 1074

(A) Macerozyme-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1075

inoculated with 001 macerozyme or mock inoculated with boiled macerozyme and 1076

were infected with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) 24 h later Samples 1077

were collected immediately (0 dpi) and after two (2 dpi) and three (3 dpi) days Data 1078

show mean plusmn SE of six to 14 biological replicates each combined using a linear model 1079

Letters indicate significantly different groups (q lt 005) 1080

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

Parsed CitationsAdam L and Somerville SC (1996) Genetic characterization of five powdery mildew disease resistance loci in Arabidopsisthaliana Plant J 9 341-356

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Albersheim P Jones TM and English PD (1969) Biochemistry of the cell wall in relation to infective processes Annu RevPhytopathol 7 171-194

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk RGadrinab C Heller C Jeske A Koesema E Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt MHazari N Hom E Karnes M Mulholland C Ndubaku R Schmidt I Guzman P Aguilar-Henonin L Schmid M Weigel DCarter DE Marchand T Risseeuw E Brogden D Zeko A Crosby WL Berry CC and Ecker JR (2003) Genome-wideinsertional mutagenesis of Arabidopsis thaliana Science 301 653-657

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Asai T Tena G Plotnikova J Willmann MR Chiu WL Gomez-Gomez L Boller T Ausubel FM and Sheen J (2002) MAPkinase signalling cascade in Arabidopsis innate immunity Nature 415 977-983

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bartsch M Gobbato E Bednarek P Debey S Schultze JL Bautor J and Parker JE (2006) Salicylic acid-independentENHANCED DISEASE SUSCEPTIBILITY1 signaling in Arabidopsis immunity and cell death is regulated by the monooxygenaseFMO1 and the nudix hydrolase NUDT7 Plant Cell 18 1038-1051

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bates D Machler M Bolker BM and Walker SC (2015) Fitting Linear Mixed-Effects Models Using lme4 J Stat Softw 67 1-48Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Benjamini Y and Hochberg Y (1995) Controlling the False Discovery Rate - a Practical and Powerful Approach to MultipleTesting J Roy Stat Soc B Met 57 289-300

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bethke G Grundman RE Sreekanta S Truman W Katagiri F and Glazebrook J (2014) Arabidopsis PECTINMETHYLESTERASEs Contribute to Immunity against Pseudomonas syringae Plant Physiol 164 1093-1107

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bouton S Leboeuf E Mouille G Leydecker MT Talbotec J Granier F Lahaye M Hofte H and Truong HN (2002)Quasimodo1 encodes a putative membrane-bound glycosyltransferase required for normal pectin synthesis and cell adhesion inArabidopsis Plant Cell 14 2577-2590

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Box GEP and Cox DR (1964) An analysis of transformations Journal of the Royal Statistical Society Series B 26 211-252Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Cao H Glazebrook J Clarke JD Volko S and Dong XN (1997) The Arabidopsis NPR1 gene that controls systemic acquiredresistance encodes a novel protein containing ankyrin repeats Cell 88 57-63

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Carpita N (2010) Update on Mechanisms of Plant Cell Wall Biosynthesis How plants make cellulose and other (1-gt4)-beta-D-glycans Plant Physiol

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Chisholm ST Coaker G Day B and Staskawicz BJ (2006) Host-microbe interactions Shaping the evolution of the plantimmune response Cell 124 803-814

Page 35 of 54

MAPK activation (Asai et al 2002) and was purchased from EZBiolab Inc (Westfield 713

IN USA) Samples were harvested 10 min later and immediately frozen in liquid 714

nitrogen Protein was extracted using an extraction buffer containing 25 mM Tris pH 78 715

75 mM NaCl 1 mM DTT 1 mM NaF 05mM Na3VO4 01 (vv) Tween 20 one 716

cOmplete Mini protease inhibitor cocktail tablet (Roche) and one PhosSTOP 717

phosphatase inhibitor cocktail tablet (Roche) per 10 mL buffer 15 microg of total protein 718

were electrophoresed on a 10 SDS-polyacrylamide gel and then blotted onto a PVDF 719

membrane (Bio-Rad 162-0177) Activated MAPKs were detected using p4442 MAPK 720

(Erk12) antibody (9102S Cell Signal 12500 in TBST (20 mM Tris pH 75 150 mM 721

NaCl 01 (vv) Tween 20)) Anti-Rabbit IgG-HRP (115000 in TBST A6154 Sigma) 722

and ECL Plus substrate (Pierce 32132) MPK3 and MPK6 were detected with an anti-723

MPK3 antibody (M8318 Sigma 12000 in TBST and 3 (wv) milk) or an anti-MPK6 724

antibody (A7104 Sigma 16000 in TBST and 3 (wv) milk) respectively The 725

secondary antibody and detection were the same as for the p4442 antibody Five 726

independent experiments were performed 727

728

Preparation of Alcohol Insoluble Residue (AIR) Pectin Extraction and Uronic Acid 729

Measurements 730

Fully expanded leaves from four week-old plants that were incubated for 48 h in 731

the dark to reduce starch were harvested flash frozen and pulverized AIR was 732

extracted by washing ground material twice in 70 (vv) ethanol three times in a 733

mixture of chloroform and methanol (11 vv) and once in acetone (Gille et al 2009) 734

and was then air dried Cell wall pectin was extracted from AIR with cell wall extraction 735

buffer (50 mM Trizma 50 mM CDTA pH 72) at 95degC for 15 min Samples were 736

homogenized using a paint shaker (Harbil 5G-HD) and glass beads (3 mm diameter) 737

For dot blot experiments 500 microL of cell wall extraction buffer were used per 10 mg of 738

AIR Debris was precipitated by centrifugation for 10 min at 10000 g 739

Total uronic acid content of pectin extracted as described above using 500 microL of 740

cell wall extraction buffer per 1 mg of AIR was measured as described previously 741

(Filisetti-Cozzi and Carpita 1991 van den Hoogen et al 1998) First 36 microL of pectin 742

extract were mixed with 4 microL of 4 M sulfamic acid Then 200 microL of sulfuric acid 743

Page 36 of 54

containing 120 mM sodium tetraborate was added and samples were incubated at 80degC 744

for 1 h Following cooling on ice the optical density of samples was measured at 490 745

nm Next 40 microL of m-hydroxydiphenyl reagent (100 microL of m-hydroxydiphenyl in dimethyl 746

sulfoxide at 100 mgmL mixed with 49 mL 80 (vv) sulfuric acid just before use) was 747

added and the samples were mixed The optical density at 490 nm was measured 748

again The optical density before m-hydroxydiphenyl reagent addition was subtracted 749

from the optical density measured after addition of the dye The concentration of uronic 750

acid was calculated using known amounts of GalA as a standard 751

752

Release of Pectin from Arabidopsis Cell Walls Using Macerozyme 753

AIR preparations from leaves of four week-old plants were treated with 025 754

(wv) macerozyme R-10 (Yakult) or water (mock) at a concentration of 100 microL per mg 755

AIR Samples were incubated for 3 h at room temperature on a vortex shaker and 756

centrifuged for 10 min at 12000 g Total uronic acid concentration of 5-fold diluted 757

supernatants was determined for two technical replicates each The concentration of 758

uronic acid was calculated using known amounts of GalA as a standard For 759

subsequent qRT-PCR experiments supernatants from multiple extractions were 760

combined boiled for 20 min to inactivate macerozyme diluted 10-fold with water and 761

inoculated into four week-old Col-0 plants Heat-inactivated 10-fold diluted macerozyme 762

solutions were used as mock treatment Samples were collected 3 h later and PAD3 763

expression was determined 764

765

Release of Pectin from Arabidopsis Leaves Infected with B cinerea 766

Fully expanded leaves of four week-old plants were either inoculated with up to 767

five 10 microL droplets of B cinerea spore solution (25 x 105 spores mL-1) or mock 768

inoculated with B cinerea inoculation medium For each biological replicate leaves with 769

a combined count of at least 20 infection sites were collected 48 h later AIR was 770

extracted as described above but plants were not dark-treated or freeze-dried and the 771

first ethanol step was performed using 85 (vv) ethanol To extract soluble pectin 772

fragments AIR samples were mixed with water at a concentration of 20 microL per infection 773

site incubated for 7 h at room temperature on a vortex shaker and centrifuged for 10 774

Page 37 of 54

min at 12000 g Total uronic acid concentration of supernatants was determined for two 775

technical replicates each The concentration of uronic acid was calculated using known 776

amounts of GalA as a standard 777

778

Measurement of Uronic Acids Neutral Monosaccharides and Cellulose Content in 779

Cell Walls 780

Neutral sugars and uronic acids from non-cellulosic polysaccharides in cell walls 781

(AIR) were released by trifluoroacetic acid (TFA) hydrolysis Approximately 1 mg of AIR 782

was hydrolyzed with 2 M TFA at 121degC for 90 min The TFA-soluble material was split 783

into two equal parts one for uronic acid and one for neutral sugar determination The 784

uronic acid content was determined by analyzing the hydrolyzate using a CarboPac 785

PA200 anion-exchange column with an ICS-3000 Dionex chromatography system 786

(Dionex Sunnyvale CA USA) The elution profile consisted of a linear gradient of 50 to 787

200 mM sodium acetate in 01 M NaOH in 10 min at 04 mL per min Alditol acetate 788

derivatives of neutral sugars were produced as described (York et al 1985) In brief 789

monosaccharides in the hydrolyzate were reduced with NaBH4 and peracetylated with 790

acetic anhydride and pyridine at 121degC for 20 min The generated alditol acetates were 791

analyzed by GC-MS Crystalline cellulose was measured according to Updegraff (1969) 792

After hydrolysis of noncellulosic polysaccharides from AIR with the Updegraff reagent 793

(acetic acid nitric acids water 812 (vv) ) the remaining pellet was hydrolyzed in 794

72 sulfuric acid The resulting glucose quantity was determined by the anthrone 795

method (Scott and Melvin 1953) 796

797

Dot Blot Experiments 798

Pectin solutions extracted using 50 microL of cell wall extraction buffer per mg AIR 799

were serially diluted and nitrocellulose membranes were spotted with 1 microL of the diluted 800

pectin solutions Membranes were dried overnight blocked with 5 milk (wv) in 1x 801

PBS (8 g L-1 NaCl 02 g L-1 KCl 144 g L-1 Na2HPO4 024 g L-1 KH2PO4 pH 74) and 802

probed with LM5 (Jones et al 1997) LM6 (Willats et al 1998) LM8 (Willats et al 803

2004) LM19 and LM20 (Verhertbruggen et al 2009a) or CCRC-M7 (Steffan et al 804

Page 38 of 54

1995) antibodies LM series antibodies were diluted 1250 and CCRC-M7 was diluted 805

1500 in 5 milk powder (Nestle Carnation) in 1 x PBS For LM series antibodies a 806

goat anti-Rat HRP conjugated antibody (Bethyl A110-105P 15000 diluted) and for 807

CCRC-M7 an anti-Mouse IgG HRP conjugate (Promega W402B 12500 diluted) was 808

used as secondary antibody in 5 milk powder in 1x PBS LM series antibodies were 809

obtained from plant probes (wwwplantprobesnet) and CCRC-M7 from CarboSource 810

(wwwcarbosourcenet) Membranes were washed after incubation with the primary and 811

secondary antibodies with 1x PBS Dot blots were developed using the ECL system 812

(GE Healthcare Amersham ECL prime) For quantification of signals dot blot results on 813

x-ray films were photographed using a CCD camera and intensities were measured814

using Image Jrsquos Integrated Density function Locally adjusted background was 815

subtracted As the dynamic range of the signal on an x-ray film was narrow non-816

linearity in the measured values was corrected for each genotype in each biological 817

replicate using the values from different dilutions in two technical replicates We noticed 818

that the residuals were not normally distributed and performed Box-Cox power 819

transformation (Box and Cox 1964) to obtain normally distributed values for the 820

subsequent statistical analysis of the data For LM19 Box-Cox selected power 821

transformation was done to the power of 038th and for LM20 to the power 075th After 822

correction of the transformed measured values with the effect of the biological 823

replicates the values were fit to a linear model with the Arabidopsis genotype as the 824

fixed effect This linear model was used both to generate the plots in Figures 5B and D 825

and to test significant differences 826

827

Camalexin Measurements 828

Four week-old plants were infected with B cinerea and samples (three leaf disks 829

from one plant each) were harvested at the indicated time points flash frozen and 830

pulverized Each sample was extracted with 300 microL of 90 methanol (vv) 50 microL of the 831

extract was run on an Agilent Lichrocart 250-4 RP18e 5-microm column using an Agilent 832

1100 series HPLC Camalexin was detected using a DiodeArray (DAD) at 330 nm and 833

with a fluorescence detector at emission 318 nmexcitation 385 nm (Agilent) Separation 834

was achieved using the following program with aqueous acetonitrile 5 min gradient 835

Page 39 of 54

from 63 to 69 acetonitrile 30 s gradient from 69 to 99 acetonitrile 2 min at 99 836

acetonitrile and a post-run equilibration of 35 min at 63 acetonitrile (Denby et al 837

2004 Kliebenstein et al 2005) Purified camalexin was used to produce a standard 838

curve to identify and quantitate camalexin 839

840

Accession Numbers 841

Microarray data used can be accessed at NCBI Gene Expression Omnnibus 842

(httpwwwncbinlmnihgovgeo) GSE18978 Sequence information for all Arabidopsis 843

genes described in this article can be found in TAIR (wwwarabidopsisorg) using the 844

following accession numbers PAD3 (CYP71B15 At3g26830) SID2 (ICS1 At1g74710) 845

GAE1 (At4g30440) GAE2 (At1g02000) GAE3 (At4g00110) GAE4 (At2g45310) GAE5 846

(At4g12250) GAE6 (At3g23820) PR1 (At2g14610) JAZ5 (At1g17380) JAZ10 847

(At5g13220) ACTIN2 (At3g18780) PDF12a (At5g44420) PDF12b (At2g26020) 848

AtSK11 (At5g26751) The sequence of the B cinerea cutinase Bc-CutA (Z69264) can 849

be found in the DNA Data Bank of Japan (httpgetentryddbjnigacjp) Germplasm 850

used included dde2-2 (At5g42650 (von Malek et al 2002)) pad3-1 (At3g26830 (Zhou 851

et al 1999)) pad4-1 (At3g52430 (Jirage et al 1999)) sid2-2 (At1g74710 (Wildermuth 852

et al 2001)) pbs3-2 (At5g13320 SALK_018225 (Nobuta et al 2007)) cbp60a-1 853

(At5g62570 SALK_124410 (Truman et al 2013)) cbp60g-1 sard1-2 (At5g26920 854

SALK_023199 At1g73805 SALK_052422 (Wang et al 2011) cbp60a-1 cbp60g-1 855

sard1-2 (Truman et al 2013) gae1-1 (At4g30440 SALK_085554) gae6-1 (At3g23280 856

SALK_104454) gae6-2 (At3g23280 SALK_017191) ixr1-1 cev1 (At5g05170 (Ellis 857

and Turner 2001 Scheible et al 2001)) eds1 plants were derived by introgression of 858

the Ler eds1-2 allele into Col-0 that contains two copies of EDS1 (At3g48090 859

At3g48080 (Bartsch et al 2006) mpk6-2 (At2g43790 SALK_073907 (Liu and Zhang 860

2004)) mpk3-DG (At3g45640 a fast neutron deletion mutant (Miles et al 2005)) and 861

npr1-1 (At1g64280 (Cao et al 1997)) T-DNA insertion lines were part of the SALK 862

collection (Alonso et al 2003) and were obtained from the Arabidopsis Biological 863

Resource Center 864

865

Supplemental Data 866

Page 40 of 54

Supplemental Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 867

Expression Does Not Require CBP60s or SID2 868

Supplemental Figure 2 Expression of GAE Family Members uponTreatment with Pma 869

ES4326 870

Supplemental Figure 3 Characterization of T-DNA Insertions in GAE1 and GAE6 871

Supplemental Figure 4 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves and 872

Are Slightly Smaller than Wild-Type Col-0 Plants 873

Supplemental Figure 5 Cell Walls of gae1 gae6 Plants Contain Less Uronic Acid than 874

Wild-Type Col-0 Cell Walls 875

Supplemental Figure 6 Antibodies Raised Against the RGI or XG Components of 876

Pectin Show No Difference in Binding to gae Mutant and Col-0 Cell Walls 877

Supplemental Figure 7 The Effect of B cinerea Treatment on the Expression of GAE 878

Family Genes 879

Supplemental Figure 8 Treatment with 001 Macerozyme Does Not Cause Any 880

Visible Damage to Col-0 Plants 881

Supplemental Figure 9 B cinerea Growth on Plants with Defects in Immune 882

Signaling 883

Supplemental Figure 10 MeJA-Induced Expression of the JA marker gene JAZ10 884

Supplemental Figure 11 The Expression of the JA Marker Gene JAZ5 Is Highly 885

Responsive in gae1 gae6 Plants 886

Supplemental Figure 12 The Expression of PDF12 Is Unaltered in gae1 gae6 Plants 887

Supplemental Figure 13 Macerozyme Treatment Activates MPK3 and MPK6 888

Supplemental Figure 14 B cinerea-Induced ROS Production in Wild-Type Col-0 and 889

gae1 gae6 Plants 890

Supplemental Figure 15 OG-Induced PTI Using 250 microg mL-1 OGs 891

Supplemental Figure 16 Abundance of Plant DNA in B cinerea Treated Plants 892

Supplemental Table 1 Primers used in this study 893

894

ACKNOWLEDGEMENTS 895

We thank William Truman for helpful discussions Simone Ferrari (Sapienza 896

Universita di Roma) for the generous gift of OGs Bruna Bucciarelli (USDA-ARS) for 897

Page 41 of 54

help with fluorescence microscopy the Arabidopsis Biological Resource Center for T-898

DNA insertion lines Paul Knoxrsquos lab (University of Leeds) for LM5 LM6 LM8 LM19 899

and LM20 antibodies and the Complex Carbohydrate Resource Center (University of 900

Georgia) for the CCRC-M7 antibody Most of this work was funded by the Division of 901

Chemical Sciences Geosciences and Biosciences Office of Basic Energy Sciences of 902

the US Department of Energy through Grant DE-FG02-05ER15670 to JG This work 903

was also supported by NSF awards IOS 1339125 and IOS 1021861 to DJK NSF 904

award IOS 1121425 to F K and the USDA National Institute of Food and Agriculture 905

Hatch project number CA-D-PLS-7033-H to DJK 906

907

AUTHOR CONTRIBUTIONS 908

GB and JG designed the research GB GX AT BL NES and NH 909

performed research DJK provided tools GB GX MP AD BL RAH and FK 910

analyzed data GB and JG wrote the paper with input from all authors 911

912

FIGURE LEGENDS 913

Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 Expression 914

Requires PAD4 EDS1 and PBS3 915

(A) Expression of GAE1 in leaves of wild-type Col-0 and mutant plants Expression was916

measured 24 or 32 h after either inoculation with Pma ES4326 (OD600 = 0002) or mock 917

inoculation with 5 mM MgSO4 and was then normalized to the level of ACTIN2 Data 918

from three biological replicates were combined using a mixed linear model Bars 919

represent mean log2-ratios to ACTIN2 plusmn SE Letters indicate significantly different 920

groups with q lt 005 where 005 represents the maximum false discovery rate at which 921

the test may be called significant 922

(B) Expression of GAE6 measured as described in (A)923

924

Figure 2 Susceptibility of gae6 and gae1 gae6 Plants to Pma ES4326 Is Increased 925

Relative to Wild-Type Col-0 926

Leaves of plants of the indicated genotypes were inoculated with Pma ES4326 (OD600 = 927

00002) as described in Methods Bacterial titers in leaves were determined either 928

Page 42 of 54

immediately (0 dpi) or after three days (3 dpi) Bars represent the mean plusmn SE of four 929

independent experiments each with 4 or 12 biological replicates at 0 and 3 dpi 930

respectively Data were combined using a mixed linear model Asterisks indicate 931

significant differences from Col-0 wild-type (q lt 005) Susceptible pad4 plants were 932

included as a positive control 933

934

Figure 3 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves 935

(A) Depiction of the assay for brittleness of gae mutant leaves Fully expanded leaves of936

four to five week-old plants were bent by 180ordm so that the adaxial surface of the leaf was 937

parallel with the soil Broken leaf petioles or midribs were considered brittle 938

(B) Quantification of broken midribs or petioles For each genotype 66 to 82 leaves 939

were analyzed for brittleness as described in (A) Bars represent the ratio of broken to 940

total number of leaves tested Results were analyzed using Fisherrsquos exact test Letters 941

indicate significantly different groups at p lt 005 942

(C) Conductivity measurements Leaves of four week-old plants were inoculated with943

deionized water and then leaf discs were cut out and placed on deionized water 944

Conductivity was measured at the indicated time points After 30 min the water was 945

replaced with fresh water (30 min f w) to account for wounding-related ion leakage from 946

the edges of the leaf discs Three independent experiments with six biological replicates 947

each were performed and data were analyzed using a t-test for each time point P-948

values were corrected using the Bonferroni method Data show means plusmn SE asterisks 949

indicate significant differences from wild-type Col-0 at q lt 001 950

951

Figure 4 Cell Walls of gae6 and gae1 gae6 Plants Are Reduced in GalA 952

(A) to (D) Levels of GalA (A) GlcA (B) cellulose (C) and neutral sugars (D) in alcohol953

insoluble residue (AIR) extracted from leaves of four week-old plants of the indicated 954

genotypes Bars represent means plusmn SE of four biological replicates combined using a 955

mixed linear model Letters indicate significantly different groups (p lt 005) 956

957

Figure 5 Cell Walls of gae1 gae6 Plants Are Reduced in Homogalacturonan 958

Page 43 of 54

Dot blot analyses with anti-LM19 and anti-LM20 antibodies Pectin was extracted from 959

AIR prepared from the indicated genotypes using 1 microL of extraction buffer per 10 microg of 960

AIR Pectin was serially diluted and one microL of undiluted 13 diluted and 19 diluted 961

samples were spotted on a nitrocellulose membrane Three biological replicates were 962

were spotted twice to obtain two technical replicates each All replicates produced 963

similar results 964

(A) Representative dot blot using anti-LM19 antibody965

(B) Relative signal intensities of the spots in the dot blot in (A)966

(C) Representative dot blot using anti-LM20 antibody967

(D) Relative signal intensities of the LM20 dot blot shown in (C)968

Relative signal intensities were measured using Image J software Measurements from 969

the three biological replicates were combined using a linear model after Box-Cox power 970

transformation as described in Methods Bars represent means plusmn SE Asterisks indicate 971

a difference from wild-type Col-0 at p lt 005 972

973

Figure 6 Growth of B cinerea on gae1 gae6 Plants and the Effect of B cinerea on 974

Expression of GAE1 and GAE6 975

(A) Growth of B cinerea isolates Leaves of Col-0 and gae1 gae6 plants were976

inoculated with 25 x 105 spores mL-1 of the B cinerea isolates indicated in the figure 977

Samples were collected two (2 dpi) and three (3 dpi) days later Bars represent means plusmn 978

SE of six biological replicates for each fungus combined using a mixed linear model 979

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 980

(B) and (C) Expression of GAE1 (B) and GAE6 (C) 48 h after inoculation with the B981

cinerea isolates indicated Expression levels were measured by qRT-PCR Bars 982

represent mean log2-ratios to ACTIN2 plusmn SE of three biological replicates combined 983

using a mixed linear model Letters indicate significantly different groups (q lt 005) 984

985

Figure 7 Levels of Soluble Pectin and Expression of PAD3 in Fractions Produced 986

Following Macerozyme Treatment of gae1 gae6 Cell Walls 987

(A) Schematic overview of the experimental procedure for (B) and (C)988

Page 44 of 54

(B) Uronic acid measurements Alcohol-insoluble residue (AIR) from leave tissue of four989

week-old wild-type Col-0 or gae1 gae6 plants was treated with water or 025 990

macerozyme and assayed for uronic acid levels Data from 11 biological replicates were 991

combined using a mixed linear model Bars represent means plusmn SE Letters indicate 992

significant differences (p lt 005) 993

(C) Expression of PAD3 in Col-0 plants treated with the fractions from (B) Extracts from994

(B) were heat inactivated pooled and diluted 5-fold with water Pooled extracts were995

inoculated into four week-old Col-0 plants PAD3 expression was measured 3 h after 996

inoculation and normalized to the level of ACTIN2 expression Data from four biological 997

replicates were combined using a mixed linear model Bars represent mean log2-ratios 998

to ACTIN2 plusmn SE Letters indicate significant differences (p lt 005) 999

1000

Figure 8 Macerozyme-Induced PAD3 Expression Is Absent in gae1 gae6 1001

(A) Expression of PAD3 after macerozyme treatment Four week-old plants were either1002

inoculated with 001 macerozyme or mock inoculated with water and PAD3 1003

expression was measured 3 h later by qRT-PCR Data from four biological replicates 1004

were combined using a mixed linear model Mean log2-ratios to ACTIN2 plusmn SE were 1005

plotted Letters indicate significantly different groups (p lt 005) 1006

(B) Expression of PAD3 after OG treatment PAD3 expression was measured as1007

described in (A) 3 h after inoculation with 100 microg mL-1 OGs or mock inoculation with 1008

water Data are from six biological replicates 1009

(C) PAD3 expression in untreated plants Three biological replicates were performed1010

and data analyzed as described in (A) 1011

(D) Camalexin accumulation after B cinerea treatment Camalexin accumulation was1012

measured in four week-old plants inoculated with the B cinerea strains shown in the 1013

figure Eight biological replicates each were combined using a mixed linear model 1014

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 1015

(E) PAD3 expression 28 h after treatment with the indicated B cinerea strains Data1016

from six biological replicates were collected and analyzed as described in (A) Data in 1017

all figure parts represent means plusmn SE Letters indicate significantly different groups (q lt 1018

005) 1019

Page 45 of 54

1020

Figure 9 Macerozyme-Induced Expression of the SA Marker Genes SID2 and PR1 1021

Is Abolished in gae1 gae6 Plants 1022

(A) to (H) Expression of PR1 (A) (C) (E) and (G) or SID2 (B) (D) (F) and (H) in four1023

week-old Col-0 and gae1 gae6 plants Data were combined using a mixed linear model 1024

Bars represent mean log2-ratios of expression vs ACTIN2 plusmn SE for all figure parts 1025

Letters indicate significantly different groups at p lt 005 for panels (A) to (D) (G) and 1026

(H) and qlt005 for panels (E) to (F) 1027

(A) and (B) Plants were treated with 001 macerozyme or mock treated with water1028

and samples were collected 3 h later Data from four biological replicates are shown 1029

(C) and (D) Plants were inoculated with 100 microg mL-1 OGs or mock treated with water1030

and samples collected 3 h later Data from six biological replicates are shown 1031

(E) and (F) Plants were inoculated with the indicated B cinerea isolates or mock1032

inoculated with B cinerea inoculation medium and samples collected 48 h later Data 1033

from eight biological replicates are shown 1034

(G) and (H) Expression in untreated plants Three biological replicates were performed1035

1036

Figure 10 The Expression of the JA Marker Gene JAZ10 is Highly Responsive in 1037

gae1 gae6 Plants 1038

(A) to (D) JAZ10 expression in four week-old plants Data were combined using a mixed1039

linear model Mean log2-ratios to ACTIN2 plusmn SE are shown Letters indicate significantly 1040

different groups at p lt 005 for panels (A) (B) and (D) and q lt 005 for panel (C) 1041

(A) Plants were inoculated with 001 macerozyme or mock inoculated using water and1042

samples were collected 3 h later Data from four biological replicates are shown 1043

(B) Plants were inoculated with 100 microg mL-1 OGs or mock inoculated using water and1044

samples collected 3 h later Data are from six biological replicates 1045

(C) JAZ10 expression 48 h after treatment with the indicated B cinerea strains Data1046

are from six biological replicates 1047

(D) JAZ10 expression in untreated plants Data are from three biological replicates1048

1049

Figure 11 Gallo 1-Induced Callose Deposition Is Reduced in gae1 gae6 Plants 1050

Page 46 of 54

(A) Callose deposition in four week-old Col-0 and gae1 gae6 plants Plants were1051

inoculated with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) or mock inoculated 1052

with B cinerea inoculation medium and samples were collected after 72 h Callose was 1053

stained with aniline blue and visualized using a Nikon Eclipse Ni-U microscope with a 1054

DAPI filter Three leaves were stained per treatment Scale bars are 100 microm Asterisks 1055

indicate fungal hyphae arrows indicate representative callose spots 1056

(B) Quantification of callose deposition Callose deposits were counted in 200 microm x 2001057

microm squares laid out across the images shown here Bars represent means plusmn SE from 1058

six squares each Letters indicate significantly different groups (p lt 005) according to a 1059

t-test analysis1060

1061

Figure 12 Less Soluble Pectin Accumulates in B cinerea-Infected gae1 gae6 1062

Leaves 1063

Four week-old Col-0 and gae1 gae6 plants were inoculated with 10 microL droplets of the 1064

indicated B cinerea isolate (25 x 105 spores mL-1) or mock inoculated with B cinerea 1065

inoculation medium Leaves were collected 48 h later AIR was extracted from leaf 1066

samples and water added (20 microL infection site) to extract soluble pectin fragments 1067

Samples were vortexed for 7 h centrifuged at 12000 g and the uronic acid 1068

concentration of the supernatant was measured Data from nine biological replicates 1069

were combined using a mixed linear model Means plusmn SE are shown Letters indicate 1070

significant differences (q lt 005) 1071

1072

Figure 13 Macerozyme-Induced PTI Is Lost in gae1 gae6 While OG-Induced PTI Is 1073

Unaltered 1074

(A) Macerozyme-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1075

inoculated with 001 macerozyme or mock inoculated with boiled macerozyme and 1076

were infected with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) 24 h later Samples 1077

were collected immediately (0 dpi) and after two (2 dpi) and three (3 dpi) days Data 1078

show mean plusmn SE of six to 14 biological replicates each combined using a linear model 1079

Letters indicate significantly different groups (q lt 005) 1080

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

Parsed CitationsAdam L and Somerville SC (1996) Genetic characterization of five powdery mildew disease resistance loci in Arabidopsisthaliana Plant J 9 341-356

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Albersheim P Jones TM and English PD (1969) Biochemistry of the cell wall in relation to infective processes Annu RevPhytopathol 7 171-194

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Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk RGadrinab C Heller C Jeske A Koesema E Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt MHazari N Hom E Karnes M Mulholland C Ndubaku R Schmidt I Guzman P Aguilar-Henonin L Schmid M Weigel DCarter DE Marchand T Risseeuw E Brogden D Zeko A Crosby WL Berry CC and Ecker JR (2003) Genome-wideinsertional mutagenesis of Arabidopsis thaliana Science 301 653-657

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Asai T Tena G Plotnikova J Willmann MR Chiu WL Gomez-Gomez L Boller T Ausubel FM and Sheen J (2002) MAPkinase signalling cascade in Arabidopsis innate immunity Nature 415 977-983

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bartsch M Gobbato E Bednarek P Debey S Schultze JL Bautor J and Parker JE (2006) Salicylic acid-independentENHANCED DISEASE SUSCEPTIBILITY1 signaling in Arabidopsis immunity and cell death is regulated by the monooxygenaseFMO1 and the nudix hydrolase NUDT7 Plant Cell 18 1038-1051

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bates D Machler M Bolker BM and Walker SC (2015) Fitting Linear Mixed-Effects Models Using lme4 J Stat Softw 67 1-48Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Benjamini Y and Hochberg Y (1995) Controlling the False Discovery Rate - a Practical and Powerful Approach to MultipleTesting J Roy Stat Soc B Met 57 289-300

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bethke G Grundman RE Sreekanta S Truman W Katagiri F and Glazebrook J (2014) Arabidopsis PECTINMETHYLESTERASEs Contribute to Immunity against Pseudomonas syringae Plant Physiol 164 1093-1107

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bouton S Leboeuf E Mouille G Leydecker MT Talbotec J Granier F Lahaye M Hofte H and Truong HN (2002)Quasimodo1 encodes a putative membrane-bound glycosyltransferase required for normal pectin synthesis and cell adhesion inArabidopsis Plant Cell 14 2577-2590

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Box GEP and Cox DR (1964) An analysis of transformations Journal of the Royal Statistical Society Series B 26 211-252Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Cao H Glazebrook J Clarke JD Volko S and Dong XN (1997) The Arabidopsis NPR1 gene that controls systemic acquiredresistance encodes a novel protein containing ankyrin repeats Cell 88 57-63

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Carpita N (2010) Update on Mechanisms of Plant Cell Wall Biosynthesis How plants make cellulose and other (1-gt4)-beta-D-glycans Plant Physiol

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Chisholm ST Coaker G Day B and Staskawicz BJ (2006) Host-microbe interactions Shaping the evolution of the plantimmune response Cell 124 803-814

Page 36 of 54

containing 120 mM sodium tetraborate was added and samples were incubated at 80degC 744

for 1 h Following cooling on ice the optical density of samples was measured at 490 745

nm Next 40 microL of m-hydroxydiphenyl reagent (100 microL of m-hydroxydiphenyl in dimethyl 746

sulfoxide at 100 mgmL mixed with 49 mL 80 (vv) sulfuric acid just before use) was 747

added and the samples were mixed The optical density at 490 nm was measured 748

again The optical density before m-hydroxydiphenyl reagent addition was subtracted 749

from the optical density measured after addition of the dye The concentration of uronic 750

acid was calculated using known amounts of GalA as a standard 751

752

Release of Pectin from Arabidopsis Cell Walls Using Macerozyme 753

AIR preparations from leaves of four week-old plants were treated with 025 754

(wv) macerozyme R-10 (Yakult) or water (mock) at a concentration of 100 microL per mg 755

AIR Samples were incubated for 3 h at room temperature on a vortex shaker and 756

centrifuged for 10 min at 12000 g Total uronic acid concentration of 5-fold diluted 757

supernatants was determined for two technical replicates each The concentration of 758

uronic acid was calculated using known amounts of GalA as a standard For 759

subsequent qRT-PCR experiments supernatants from multiple extractions were 760

combined boiled for 20 min to inactivate macerozyme diluted 10-fold with water and 761

inoculated into four week-old Col-0 plants Heat-inactivated 10-fold diluted macerozyme 762

solutions were used as mock treatment Samples were collected 3 h later and PAD3 763

expression was determined 764

765

Release of Pectin from Arabidopsis Leaves Infected with B cinerea 766

Fully expanded leaves of four week-old plants were either inoculated with up to 767

five 10 microL droplets of B cinerea spore solution (25 x 105 spores mL-1) or mock 768

inoculated with B cinerea inoculation medium For each biological replicate leaves with 769

a combined count of at least 20 infection sites were collected 48 h later AIR was 770

extracted as described above but plants were not dark-treated or freeze-dried and the 771

first ethanol step was performed using 85 (vv) ethanol To extract soluble pectin 772

fragments AIR samples were mixed with water at a concentration of 20 microL per infection 773

site incubated for 7 h at room temperature on a vortex shaker and centrifuged for 10 774

Page 37 of 54

min at 12000 g Total uronic acid concentration of supernatants was determined for two 775

technical replicates each The concentration of uronic acid was calculated using known 776

amounts of GalA as a standard 777

778

Measurement of Uronic Acids Neutral Monosaccharides and Cellulose Content in 779

Cell Walls 780

Neutral sugars and uronic acids from non-cellulosic polysaccharides in cell walls 781

(AIR) were released by trifluoroacetic acid (TFA) hydrolysis Approximately 1 mg of AIR 782

was hydrolyzed with 2 M TFA at 121degC for 90 min The TFA-soluble material was split 783

into two equal parts one for uronic acid and one for neutral sugar determination The 784

uronic acid content was determined by analyzing the hydrolyzate using a CarboPac 785

PA200 anion-exchange column with an ICS-3000 Dionex chromatography system 786

(Dionex Sunnyvale CA USA) The elution profile consisted of a linear gradient of 50 to 787

200 mM sodium acetate in 01 M NaOH in 10 min at 04 mL per min Alditol acetate 788

derivatives of neutral sugars were produced as described (York et al 1985) In brief 789

monosaccharides in the hydrolyzate were reduced with NaBH4 and peracetylated with 790

acetic anhydride and pyridine at 121degC for 20 min The generated alditol acetates were 791

analyzed by GC-MS Crystalline cellulose was measured according to Updegraff (1969) 792

After hydrolysis of noncellulosic polysaccharides from AIR with the Updegraff reagent 793

(acetic acid nitric acids water 812 (vv) ) the remaining pellet was hydrolyzed in 794

72 sulfuric acid The resulting glucose quantity was determined by the anthrone 795

method (Scott and Melvin 1953) 796

797

Dot Blot Experiments 798

Pectin solutions extracted using 50 microL of cell wall extraction buffer per mg AIR 799

were serially diluted and nitrocellulose membranes were spotted with 1 microL of the diluted 800

pectin solutions Membranes were dried overnight blocked with 5 milk (wv) in 1x 801

PBS (8 g L-1 NaCl 02 g L-1 KCl 144 g L-1 Na2HPO4 024 g L-1 KH2PO4 pH 74) and 802

probed with LM5 (Jones et al 1997) LM6 (Willats et al 1998) LM8 (Willats et al 803

2004) LM19 and LM20 (Verhertbruggen et al 2009a) or CCRC-M7 (Steffan et al 804

Page 38 of 54

1995) antibodies LM series antibodies were diluted 1250 and CCRC-M7 was diluted 805

1500 in 5 milk powder (Nestle Carnation) in 1 x PBS For LM series antibodies a 806

goat anti-Rat HRP conjugated antibody (Bethyl A110-105P 15000 diluted) and for 807

CCRC-M7 an anti-Mouse IgG HRP conjugate (Promega W402B 12500 diluted) was 808

used as secondary antibody in 5 milk powder in 1x PBS LM series antibodies were 809

obtained from plant probes (wwwplantprobesnet) and CCRC-M7 from CarboSource 810

(wwwcarbosourcenet) Membranes were washed after incubation with the primary and 811

secondary antibodies with 1x PBS Dot blots were developed using the ECL system 812

(GE Healthcare Amersham ECL prime) For quantification of signals dot blot results on 813

x-ray films were photographed using a CCD camera and intensities were measured814

using Image Jrsquos Integrated Density function Locally adjusted background was 815

subtracted As the dynamic range of the signal on an x-ray film was narrow non-816

linearity in the measured values was corrected for each genotype in each biological 817

replicate using the values from different dilutions in two technical replicates We noticed 818

that the residuals were not normally distributed and performed Box-Cox power 819

transformation (Box and Cox 1964) to obtain normally distributed values for the 820

subsequent statistical analysis of the data For LM19 Box-Cox selected power 821

transformation was done to the power of 038th and for LM20 to the power 075th After 822

correction of the transformed measured values with the effect of the biological 823

replicates the values were fit to a linear model with the Arabidopsis genotype as the 824

fixed effect This linear model was used both to generate the plots in Figures 5B and D 825

and to test significant differences 826

827

Camalexin Measurements 828

Four week-old plants were infected with B cinerea and samples (three leaf disks 829

from one plant each) were harvested at the indicated time points flash frozen and 830

pulverized Each sample was extracted with 300 microL of 90 methanol (vv) 50 microL of the 831

extract was run on an Agilent Lichrocart 250-4 RP18e 5-microm column using an Agilent 832

1100 series HPLC Camalexin was detected using a DiodeArray (DAD) at 330 nm and 833

with a fluorescence detector at emission 318 nmexcitation 385 nm (Agilent) Separation 834

was achieved using the following program with aqueous acetonitrile 5 min gradient 835

Page 39 of 54

from 63 to 69 acetonitrile 30 s gradient from 69 to 99 acetonitrile 2 min at 99 836

acetonitrile and a post-run equilibration of 35 min at 63 acetonitrile (Denby et al 837

2004 Kliebenstein et al 2005) Purified camalexin was used to produce a standard 838

curve to identify and quantitate camalexin 839

840

Accession Numbers 841

Microarray data used can be accessed at NCBI Gene Expression Omnnibus 842

(httpwwwncbinlmnihgovgeo) GSE18978 Sequence information for all Arabidopsis 843

genes described in this article can be found in TAIR (wwwarabidopsisorg) using the 844

following accession numbers PAD3 (CYP71B15 At3g26830) SID2 (ICS1 At1g74710) 845

GAE1 (At4g30440) GAE2 (At1g02000) GAE3 (At4g00110) GAE4 (At2g45310) GAE5 846

(At4g12250) GAE6 (At3g23820) PR1 (At2g14610) JAZ5 (At1g17380) JAZ10 847

(At5g13220) ACTIN2 (At3g18780) PDF12a (At5g44420) PDF12b (At2g26020) 848

AtSK11 (At5g26751) The sequence of the B cinerea cutinase Bc-CutA (Z69264) can 849

be found in the DNA Data Bank of Japan (httpgetentryddbjnigacjp) Germplasm 850

used included dde2-2 (At5g42650 (von Malek et al 2002)) pad3-1 (At3g26830 (Zhou 851

et al 1999)) pad4-1 (At3g52430 (Jirage et al 1999)) sid2-2 (At1g74710 (Wildermuth 852

et al 2001)) pbs3-2 (At5g13320 SALK_018225 (Nobuta et al 2007)) cbp60a-1 853

(At5g62570 SALK_124410 (Truman et al 2013)) cbp60g-1 sard1-2 (At5g26920 854

SALK_023199 At1g73805 SALK_052422 (Wang et al 2011) cbp60a-1 cbp60g-1 855

sard1-2 (Truman et al 2013) gae1-1 (At4g30440 SALK_085554) gae6-1 (At3g23280 856

SALK_104454) gae6-2 (At3g23280 SALK_017191) ixr1-1 cev1 (At5g05170 (Ellis 857

and Turner 2001 Scheible et al 2001)) eds1 plants were derived by introgression of 858

the Ler eds1-2 allele into Col-0 that contains two copies of EDS1 (At3g48090 859

At3g48080 (Bartsch et al 2006) mpk6-2 (At2g43790 SALK_073907 (Liu and Zhang 860

2004)) mpk3-DG (At3g45640 a fast neutron deletion mutant (Miles et al 2005)) and 861

npr1-1 (At1g64280 (Cao et al 1997)) T-DNA insertion lines were part of the SALK 862

collection (Alonso et al 2003) and were obtained from the Arabidopsis Biological 863

Resource Center 864

865

Supplemental Data 866

Page 40 of 54

Supplemental Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 867

Expression Does Not Require CBP60s or SID2 868

Supplemental Figure 2 Expression of GAE Family Members uponTreatment with Pma 869

ES4326 870

Supplemental Figure 3 Characterization of T-DNA Insertions in GAE1 and GAE6 871

Supplemental Figure 4 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves and 872

Are Slightly Smaller than Wild-Type Col-0 Plants 873

Supplemental Figure 5 Cell Walls of gae1 gae6 Plants Contain Less Uronic Acid than 874

Wild-Type Col-0 Cell Walls 875

Supplemental Figure 6 Antibodies Raised Against the RGI or XG Components of 876

Pectin Show No Difference in Binding to gae Mutant and Col-0 Cell Walls 877

Supplemental Figure 7 The Effect of B cinerea Treatment on the Expression of GAE 878

Family Genes 879

Supplemental Figure 8 Treatment with 001 Macerozyme Does Not Cause Any 880

Visible Damage to Col-0 Plants 881

Supplemental Figure 9 B cinerea Growth on Plants with Defects in Immune 882

Signaling 883

Supplemental Figure 10 MeJA-Induced Expression of the JA marker gene JAZ10 884

Supplemental Figure 11 The Expression of the JA Marker Gene JAZ5 Is Highly 885

Responsive in gae1 gae6 Plants 886

Supplemental Figure 12 The Expression of PDF12 Is Unaltered in gae1 gae6 Plants 887

Supplemental Figure 13 Macerozyme Treatment Activates MPK3 and MPK6 888

Supplemental Figure 14 B cinerea-Induced ROS Production in Wild-Type Col-0 and 889

gae1 gae6 Plants 890

Supplemental Figure 15 OG-Induced PTI Using 250 microg mL-1 OGs 891

Supplemental Figure 16 Abundance of Plant DNA in B cinerea Treated Plants 892

Supplemental Table 1 Primers used in this study 893

894

ACKNOWLEDGEMENTS 895

We thank William Truman for helpful discussions Simone Ferrari (Sapienza 896

Universita di Roma) for the generous gift of OGs Bruna Bucciarelli (USDA-ARS) for 897

Page 41 of 54

help with fluorescence microscopy the Arabidopsis Biological Resource Center for T-898

DNA insertion lines Paul Knoxrsquos lab (University of Leeds) for LM5 LM6 LM8 LM19 899

and LM20 antibodies and the Complex Carbohydrate Resource Center (University of 900

Georgia) for the CCRC-M7 antibody Most of this work was funded by the Division of 901

Chemical Sciences Geosciences and Biosciences Office of Basic Energy Sciences of 902

the US Department of Energy through Grant DE-FG02-05ER15670 to JG This work 903

was also supported by NSF awards IOS 1339125 and IOS 1021861 to DJK NSF 904

award IOS 1121425 to F K and the USDA National Institute of Food and Agriculture 905

Hatch project number CA-D-PLS-7033-H to DJK 906

907

AUTHOR CONTRIBUTIONS 908

GB and JG designed the research GB GX AT BL NES and NH 909

performed research DJK provided tools GB GX MP AD BL RAH and FK 910

analyzed data GB and JG wrote the paper with input from all authors 911

912

FIGURE LEGENDS 913

Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 Expression 914

Requires PAD4 EDS1 and PBS3 915

(A) Expression of GAE1 in leaves of wild-type Col-0 and mutant plants Expression was916

measured 24 or 32 h after either inoculation with Pma ES4326 (OD600 = 0002) or mock 917

inoculation with 5 mM MgSO4 and was then normalized to the level of ACTIN2 Data 918

from three biological replicates were combined using a mixed linear model Bars 919

represent mean log2-ratios to ACTIN2 plusmn SE Letters indicate significantly different 920

groups with q lt 005 where 005 represents the maximum false discovery rate at which 921

the test may be called significant 922

(B) Expression of GAE6 measured as described in (A)923

924

Figure 2 Susceptibility of gae6 and gae1 gae6 Plants to Pma ES4326 Is Increased 925

Relative to Wild-Type Col-0 926

Leaves of plants of the indicated genotypes were inoculated with Pma ES4326 (OD600 = 927

00002) as described in Methods Bacterial titers in leaves were determined either 928

Page 42 of 54

immediately (0 dpi) or after three days (3 dpi) Bars represent the mean plusmn SE of four 929

independent experiments each with 4 or 12 biological replicates at 0 and 3 dpi 930

respectively Data were combined using a mixed linear model Asterisks indicate 931

significant differences from Col-0 wild-type (q lt 005) Susceptible pad4 plants were 932

included as a positive control 933

934

Figure 3 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves 935

(A) Depiction of the assay for brittleness of gae mutant leaves Fully expanded leaves of936

four to five week-old plants were bent by 180ordm so that the adaxial surface of the leaf was 937

parallel with the soil Broken leaf petioles or midribs were considered brittle 938

(B) Quantification of broken midribs or petioles For each genotype 66 to 82 leaves 939

were analyzed for brittleness as described in (A) Bars represent the ratio of broken to 940

total number of leaves tested Results were analyzed using Fisherrsquos exact test Letters 941

indicate significantly different groups at p lt 005 942

(C) Conductivity measurements Leaves of four week-old plants were inoculated with943

deionized water and then leaf discs were cut out and placed on deionized water 944

Conductivity was measured at the indicated time points After 30 min the water was 945

replaced with fresh water (30 min f w) to account for wounding-related ion leakage from 946

the edges of the leaf discs Three independent experiments with six biological replicates 947

each were performed and data were analyzed using a t-test for each time point P-948

values were corrected using the Bonferroni method Data show means plusmn SE asterisks 949

indicate significant differences from wild-type Col-0 at q lt 001 950

951

Figure 4 Cell Walls of gae6 and gae1 gae6 Plants Are Reduced in GalA 952

(A) to (D) Levels of GalA (A) GlcA (B) cellulose (C) and neutral sugars (D) in alcohol953

insoluble residue (AIR) extracted from leaves of four week-old plants of the indicated 954

genotypes Bars represent means plusmn SE of four biological replicates combined using a 955

mixed linear model Letters indicate significantly different groups (p lt 005) 956

957

Figure 5 Cell Walls of gae1 gae6 Plants Are Reduced in Homogalacturonan 958

Page 43 of 54

Dot blot analyses with anti-LM19 and anti-LM20 antibodies Pectin was extracted from 959

AIR prepared from the indicated genotypes using 1 microL of extraction buffer per 10 microg of 960

AIR Pectin was serially diluted and one microL of undiluted 13 diluted and 19 diluted 961

samples were spotted on a nitrocellulose membrane Three biological replicates were 962

were spotted twice to obtain two technical replicates each All replicates produced 963

similar results 964

(A) Representative dot blot using anti-LM19 antibody965

(B) Relative signal intensities of the spots in the dot blot in (A)966

(C) Representative dot blot using anti-LM20 antibody967

(D) Relative signal intensities of the LM20 dot blot shown in (C)968

Relative signal intensities were measured using Image J software Measurements from 969

the three biological replicates were combined using a linear model after Box-Cox power 970

transformation as described in Methods Bars represent means plusmn SE Asterisks indicate 971

a difference from wild-type Col-0 at p lt 005 972

973

Figure 6 Growth of B cinerea on gae1 gae6 Plants and the Effect of B cinerea on 974

Expression of GAE1 and GAE6 975

(A) Growth of B cinerea isolates Leaves of Col-0 and gae1 gae6 plants were976

inoculated with 25 x 105 spores mL-1 of the B cinerea isolates indicated in the figure 977

Samples were collected two (2 dpi) and three (3 dpi) days later Bars represent means plusmn 978

SE of six biological replicates for each fungus combined using a mixed linear model 979

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 980

(B) and (C) Expression of GAE1 (B) and GAE6 (C) 48 h after inoculation with the B981

cinerea isolates indicated Expression levels were measured by qRT-PCR Bars 982

represent mean log2-ratios to ACTIN2 plusmn SE of three biological replicates combined 983

using a mixed linear model Letters indicate significantly different groups (q lt 005) 984

985

Figure 7 Levels of Soluble Pectin and Expression of PAD3 in Fractions Produced 986

Following Macerozyme Treatment of gae1 gae6 Cell Walls 987

(A) Schematic overview of the experimental procedure for (B) and (C)988

Page 44 of 54

(B) Uronic acid measurements Alcohol-insoluble residue (AIR) from leave tissue of four989

week-old wild-type Col-0 or gae1 gae6 plants was treated with water or 025 990

macerozyme and assayed for uronic acid levels Data from 11 biological replicates were 991

combined using a mixed linear model Bars represent means plusmn SE Letters indicate 992

significant differences (p lt 005) 993

(C) Expression of PAD3 in Col-0 plants treated with the fractions from (B) Extracts from994

(B) were heat inactivated pooled and diluted 5-fold with water Pooled extracts were995

inoculated into four week-old Col-0 plants PAD3 expression was measured 3 h after 996

inoculation and normalized to the level of ACTIN2 expression Data from four biological 997

replicates were combined using a mixed linear model Bars represent mean log2-ratios 998

to ACTIN2 plusmn SE Letters indicate significant differences (p lt 005) 999

1000

Figure 8 Macerozyme-Induced PAD3 Expression Is Absent in gae1 gae6 1001

(A) Expression of PAD3 after macerozyme treatment Four week-old plants were either1002

inoculated with 001 macerozyme or mock inoculated with water and PAD3 1003

expression was measured 3 h later by qRT-PCR Data from four biological replicates 1004

were combined using a mixed linear model Mean log2-ratios to ACTIN2 plusmn SE were 1005

plotted Letters indicate significantly different groups (p lt 005) 1006

(B) Expression of PAD3 after OG treatment PAD3 expression was measured as1007

described in (A) 3 h after inoculation with 100 microg mL-1 OGs or mock inoculation with 1008

water Data are from six biological replicates 1009

(C) PAD3 expression in untreated plants Three biological replicates were performed1010

and data analyzed as described in (A) 1011

(D) Camalexin accumulation after B cinerea treatment Camalexin accumulation was1012

measured in four week-old plants inoculated with the B cinerea strains shown in the 1013

figure Eight biological replicates each were combined using a mixed linear model 1014

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 1015

(E) PAD3 expression 28 h after treatment with the indicated B cinerea strains Data1016

from six biological replicates were collected and analyzed as described in (A) Data in 1017

all figure parts represent means plusmn SE Letters indicate significantly different groups (q lt 1018

005) 1019

Page 45 of 54

1020

Figure 9 Macerozyme-Induced Expression of the SA Marker Genes SID2 and PR1 1021

Is Abolished in gae1 gae6 Plants 1022

(A) to (H) Expression of PR1 (A) (C) (E) and (G) or SID2 (B) (D) (F) and (H) in four1023

week-old Col-0 and gae1 gae6 plants Data were combined using a mixed linear model 1024

Bars represent mean log2-ratios of expression vs ACTIN2 plusmn SE for all figure parts 1025

Letters indicate significantly different groups at p lt 005 for panels (A) to (D) (G) and 1026

(H) and qlt005 for panels (E) to (F) 1027

(A) and (B) Plants were treated with 001 macerozyme or mock treated with water1028

and samples were collected 3 h later Data from four biological replicates are shown 1029

(C) and (D) Plants were inoculated with 100 microg mL-1 OGs or mock treated with water1030

and samples collected 3 h later Data from six biological replicates are shown 1031

(E) and (F) Plants were inoculated with the indicated B cinerea isolates or mock1032

inoculated with B cinerea inoculation medium and samples collected 48 h later Data 1033

from eight biological replicates are shown 1034

(G) and (H) Expression in untreated plants Three biological replicates were performed1035

1036

Figure 10 The Expression of the JA Marker Gene JAZ10 is Highly Responsive in 1037

gae1 gae6 Plants 1038

(A) to (D) JAZ10 expression in four week-old plants Data were combined using a mixed1039

linear model Mean log2-ratios to ACTIN2 plusmn SE are shown Letters indicate significantly 1040

different groups at p lt 005 for panels (A) (B) and (D) and q lt 005 for panel (C) 1041

(A) Plants were inoculated with 001 macerozyme or mock inoculated using water and1042

samples were collected 3 h later Data from four biological replicates are shown 1043

(B) Plants were inoculated with 100 microg mL-1 OGs or mock inoculated using water and1044

samples collected 3 h later Data are from six biological replicates 1045

(C) JAZ10 expression 48 h after treatment with the indicated B cinerea strains Data1046

are from six biological replicates 1047

(D) JAZ10 expression in untreated plants Data are from three biological replicates1048

1049

Figure 11 Gallo 1-Induced Callose Deposition Is Reduced in gae1 gae6 Plants 1050

Page 46 of 54

(A) Callose deposition in four week-old Col-0 and gae1 gae6 plants Plants were1051

inoculated with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) or mock inoculated 1052

with B cinerea inoculation medium and samples were collected after 72 h Callose was 1053

stained with aniline blue and visualized using a Nikon Eclipse Ni-U microscope with a 1054

DAPI filter Three leaves were stained per treatment Scale bars are 100 microm Asterisks 1055

indicate fungal hyphae arrows indicate representative callose spots 1056

(B) Quantification of callose deposition Callose deposits were counted in 200 microm x 2001057

microm squares laid out across the images shown here Bars represent means plusmn SE from 1058

six squares each Letters indicate significantly different groups (p lt 005) according to a 1059

t-test analysis1060

1061

Figure 12 Less Soluble Pectin Accumulates in B cinerea-Infected gae1 gae6 1062

Leaves 1063

Four week-old Col-0 and gae1 gae6 plants were inoculated with 10 microL droplets of the 1064

indicated B cinerea isolate (25 x 105 spores mL-1) or mock inoculated with B cinerea 1065

inoculation medium Leaves were collected 48 h later AIR was extracted from leaf 1066

samples and water added (20 microL infection site) to extract soluble pectin fragments 1067

Samples were vortexed for 7 h centrifuged at 12000 g and the uronic acid 1068

concentration of the supernatant was measured Data from nine biological replicates 1069

were combined using a mixed linear model Means plusmn SE are shown Letters indicate 1070

significant differences (q lt 005) 1071

1072

Figure 13 Macerozyme-Induced PTI Is Lost in gae1 gae6 While OG-Induced PTI Is 1073

Unaltered 1074

(A) Macerozyme-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1075

inoculated with 001 macerozyme or mock inoculated with boiled macerozyme and 1076

were infected with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) 24 h later Samples 1077

were collected immediately (0 dpi) and after two (2 dpi) and three (3 dpi) days Data 1078

show mean plusmn SE of six to 14 biological replicates each combined using a linear model 1079

Letters indicate significantly different groups (q lt 005) 1080

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

Parsed CitationsAdam L and Somerville SC (1996) Genetic characterization of five powdery mildew disease resistance loci in Arabidopsisthaliana Plant J 9 341-356

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Albersheim P Jones TM and English PD (1969) Biochemistry of the cell wall in relation to infective processes Annu RevPhytopathol 7 171-194

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Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk RGadrinab C Heller C Jeske A Koesema E Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt MHazari N Hom E Karnes M Mulholland C Ndubaku R Schmidt I Guzman P Aguilar-Henonin L Schmid M Weigel DCarter DE Marchand T Risseeuw E Brogden D Zeko A Crosby WL Berry CC and Ecker JR (2003) Genome-wideinsertional mutagenesis of Arabidopsis thaliana Science 301 653-657

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Asai T Tena G Plotnikova J Willmann MR Chiu WL Gomez-Gomez L Boller T Ausubel FM and Sheen J (2002) MAPkinase signalling cascade in Arabidopsis innate immunity Nature 415 977-983

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bartsch M Gobbato E Bednarek P Debey S Schultze JL Bautor J and Parker JE (2006) Salicylic acid-independentENHANCED DISEASE SUSCEPTIBILITY1 signaling in Arabidopsis immunity and cell death is regulated by the monooxygenaseFMO1 and the nudix hydrolase NUDT7 Plant Cell 18 1038-1051

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bates D Machler M Bolker BM and Walker SC (2015) Fitting Linear Mixed-Effects Models Using lme4 J Stat Softw 67 1-48Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Benjamini Y and Hochberg Y (1995) Controlling the False Discovery Rate - a Practical and Powerful Approach to MultipleTesting J Roy Stat Soc B Met 57 289-300

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bethke G Grundman RE Sreekanta S Truman W Katagiri F and Glazebrook J (2014) Arabidopsis PECTINMETHYLESTERASEs Contribute to Immunity against Pseudomonas syringae Plant Physiol 164 1093-1107

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bouton S Leboeuf E Mouille G Leydecker MT Talbotec J Granier F Lahaye M Hofte H and Truong HN (2002)Quasimodo1 encodes a putative membrane-bound glycosyltransferase required for normal pectin synthesis and cell adhesion inArabidopsis Plant Cell 14 2577-2590

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Box GEP and Cox DR (1964) An analysis of transformations Journal of the Royal Statistical Society Series B 26 211-252Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Cao H Glazebrook J Clarke JD Volko S and Dong XN (1997) The Arabidopsis NPR1 gene that controls systemic acquiredresistance encodes a novel protein containing ankyrin repeats Cell 88 57-63

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Carpita N (2010) Update on Mechanisms of Plant Cell Wall Biosynthesis How plants make cellulose and other (1-gt4)-beta-D-glycans Plant Physiol

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Chisholm ST Coaker G Day B and Staskawicz BJ (2006) Host-microbe interactions Shaping the evolution of the plantimmune response Cell 124 803-814

Page 37 of 54

min at 12000 g Total uronic acid concentration of supernatants was determined for two 775

technical replicates each The concentration of uronic acid was calculated using known 776

amounts of GalA as a standard 777

778

Measurement of Uronic Acids Neutral Monosaccharides and Cellulose Content in 779

Cell Walls 780

Neutral sugars and uronic acids from non-cellulosic polysaccharides in cell walls 781

(AIR) were released by trifluoroacetic acid (TFA) hydrolysis Approximately 1 mg of AIR 782

was hydrolyzed with 2 M TFA at 121degC for 90 min The TFA-soluble material was split 783

into two equal parts one for uronic acid and one for neutral sugar determination The 784

uronic acid content was determined by analyzing the hydrolyzate using a CarboPac 785

PA200 anion-exchange column with an ICS-3000 Dionex chromatography system 786

(Dionex Sunnyvale CA USA) The elution profile consisted of a linear gradient of 50 to 787

200 mM sodium acetate in 01 M NaOH in 10 min at 04 mL per min Alditol acetate 788

derivatives of neutral sugars were produced as described (York et al 1985) In brief 789

monosaccharides in the hydrolyzate were reduced with NaBH4 and peracetylated with 790

acetic anhydride and pyridine at 121degC for 20 min The generated alditol acetates were 791

analyzed by GC-MS Crystalline cellulose was measured according to Updegraff (1969) 792

After hydrolysis of noncellulosic polysaccharides from AIR with the Updegraff reagent 793

(acetic acid nitric acids water 812 (vv) ) the remaining pellet was hydrolyzed in 794

72 sulfuric acid The resulting glucose quantity was determined by the anthrone 795

method (Scott and Melvin 1953) 796

797

Dot Blot Experiments 798

Pectin solutions extracted using 50 microL of cell wall extraction buffer per mg AIR 799

were serially diluted and nitrocellulose membranes were spotted with 1 microL of the diluted 800

pectin solutions Membranes were dried overnight blocked with 5 milk (wv) in 1x 801

PBS (8 g L-1 NaCl 02 g L-1 KCl 144 g L-1 Na2HPO4 024 g L-1 KH2PO4 pH 74) and 802

probed with LM5 (Jones et al 1997) LM6 (Willats et al 1998) LM8 (Willats et al 803

2004) LM19 and LM20 (Verhertbruggen et al 2009a) or CCRC-M7 (Steffan et al 804

Page 38 of 54

1995) antibodies LM series antibodies were diluted 1250 and CCRC-M7 was diluted 805

1500 in 5 milk powder (Nestle Carnation) in 1 x PBS For LM series antibodies a 806

goat anti-Rat HRP conjugated antibody (Bethyl A110-105P 15000 diluted) and for 807

CCRC-M7 an anti-Mouse IgG HRP conjugate (Promega W402B 12500 diluted) was 808

used as secondary antibody in 5 milk powder in 1x PBS LM series antibodies were 809

obtained from plant probes (wwwplantprobesnet) and CCRC-M7 from CarboSource 810

(wwwcarbosourcenet) Membranes were washed after incubation with the primary and 811

secondary antibodies with 1x PBS Dot blots were developed using the ECL system 812

(GE Healthcare Amersham ECL prime) For quantification of signals dot blot results on 813

x-ray films were photographed using a CCD camera and intensities were measured814

using Image Jrsquos Integrated Density function Locally adjusted background was 815

subtracted As the dynamic range of the signal on an x-ray film was narrow non-816

linearity in the measured values was corrected for each genotype in each biological 817

replicate using the values from different dilutions in two technical replicates We noticed 818

that the residuals were not normally distributed and performed Box-Cox power 819

transformation (Box and Cox 1964) to obtain normally distributed values for the 820

subsequent statistical analysis of the data For LM19 Box-Cox selected power 821

transformation was done to the power of 038th and for LM20 to the power 075th After 822

correction of the transformed measured values with the effect of the biological 823

replicates the values were fit to a linear model with the Arabidopsis genotype as the 824

fixed effect This linear model was used both to generate the plots in Figures 5B and D 825

and to test significant differences 826

827

Camalexin Measurements 828

Four week-old plants were infected with B cinerea and samples (three leaf disks 829

from one plant each) were harvested at the indicated time points flash frozen and 830

pulverized Each sample was extracted with 300 microL of 90 methanol (vv) 50 microL of the 831

extract was run on an Agilent Lichrocart 250-4 RP18e 5-microm column using an Agilent 832

1100 series HPLC Camalexin was detected using a DiodeArray (DAD) at 330 nm and 833

with a fluorescence detector at emission 318 nmexcitation 385 nm (Agilent) Separation 834

was achieved using the following program with aqueous acetonitrile 5 min gradient 835

Page 39 of 54

from 63 to 69 acetonitrile 30 s gradient from 69 to 99 acetonitrile 2 min at 99 836

acetonitrile and a post-run equilibration of 35 min at 63 acetonitrile (Denby et al 837

2004 Kliebenstein et al 2005) Purified camalexin was used to produce a standard 838

curve to identify and quantitate camalexin 839

840

Accession Numbers 841

Microarray data used can be accessed at NCBI Gene Expression Omnnibus 842

(httpwwwncbinlmnihgovgeo) GSE18978 Sequence information for all Arabidopsis 843

genes described in this article can be found in TAIR (wwwarabidopsisorg) using the 844

following accession numbers PAD3 (CYP71B15 At3g26830) SID2 (ICS1 At1g74710) 845

GAE1 (At4g30440) GAE2 (At1g02000) GAE3 (At4g00110) GAE4 (At2g45310) GAE5 846

(At4g12250) GAE6 (At3g23820) PR1 (At2g14610) JAZ5 (At1g17380) JAZ10 847

(At5g13220) ACTIN2 (At3g18780) PDF12a (At5g44420) PDF12b (At2g26020) 848

AtSK11 (At5g26751) The sequence of the B cinerea cutinase Bc-CutA (Z69264) can 849

be found in the DNA Data Bank of Japan (httpgetentryddbjnigacjp) Germplasm 850

used included dde2-2 (At5g42650 (von Malek et al 2002)) pad3-1 (At3g26830 (Zhou 851

et al 1999)) pad4-1 (At3g52430 (Jirage et al 1999)) sid2-2 (At1g74710 (Wildermuth 852

et al 2001)) pbs3-2 (At5g13320 SALK_018225 (Nobuta et al 2007)) cbp60a-1 853

(At5g62570 SALK_124410 (Truman et al 2013)) cbp60g-1 sard1-2 (At5g26920 854

SALK_023199 At1g73805 SALK_052422 (Wang et al 2011) cbp60a-1 cbp60g-1 855

sard1-2 (Truman et al 2013) gae1-1 (At4g30440 SALK_085554) gae6-1 (At3g23280 856

SALK_104454) gae6-2 (At3g23280 SALK_017191) ixr1-1 cev1 (At5g05170 (Ellis 857

and Turner 2001 Scheible et al 2001)) eds1 plants were derived by introgression of 858

the Ler eds1-2 allele into Col-0 that contains two copies of EDS1 (At3g48090 859

At3g48080 (Bartsch et al 2006) mpk6-2 (At2g43790 SALK_073907 (Liu and Zhang 860

2004)) mpk3-DG (At3g45640 a fast neutron deletion mutant (Miles et al 2005)) and 861

npr1-1 (At1g64280 (Cao et al 1997)) T-DNA insertion lines were part of the SALK 862

collection (Alonso et al 2003) and were obtained from the Arabidopsis Biological 863

Resource Center 864

865

Supplemental Data 866

Page 40 of 54

Supplemental Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 867

Expression Does Not Require CBP60s or SID2 868

Supplemental Figure 2 Expression of GAE Family Members uponTreatment with Pma 869

ES4326 870

Supplemental Figure 3 Characterization of T-DNA Insertions in GAE1 and GAE6 871

Supplemental Figure 4 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves and 872

Are Slightly Smaller than Wild-Type Col-0 Plants 873

Supplemental Figure 5 Cell Walls of gae1 gae6 Plants Contain Less Uronic Acid than 874

Wild-Type Col-0 Cell Walls 875

Supplemental Figure 6 Antibodies Raised Against the RGI or XG Components of 876

Pectin Show No Difference in Binding to gae Mutant and Col-0 Cell Walls 877

Supplemental Figure 7 The Effect of B cinerea Treatment on the Expression of GAE 878

Family Genes 879

Supplemental Figure 8 Treatment with 001 Macerozyme Does Not Cause Any 880

Visible Damage to Col-0 Plants 881

Supplemental Figure 9 B cinerea Growth on Plants with Defects in Immune 882

Signaling 883

Supplemental Figure 10 MeJA-Induced Expression of the JA marker gene JAZ10 884

Supplemental Figure 11 The Expression of the JA Marker Gene JAZ5 Is Highly 885

Responsive in gae1 gae6 Plants 886

Supplemental Figure 12 The Expression of PDF12 Is Unaltered in gae1 gae6 Plants 887

Supplemental Figure 13 Macerozyme Treatment Activates MPK3 and MPK6 888

Supplemental Figure 14 B cinerea-Induced ROS Production in Wild-Type Col-0 and 889

gae1 gae6 Plants 890

Supplemental Figure 15 OG-Induced PTI Using 250 microg mL-1 OGs 891

Supplemental Figure 16 Abundance of Plant DNA in B cinerea Treated Plants 892

Supplemental Table 1 Primers used in this study 893

894

ACKNOWLEDGEMENTS 895

We thank William Truman for helpful discussions Simone Ferrari (Sapienza 896

Universita di Roma) for the generous gift of OGs Bruna Bucciarelli (USDA-ARS) for 897

Page 41 of 54

help with fluorescence microscopy the Arabidopsis Biological Resource Center for T-898

DNA insertion lines Paul Knoxrsquos lab (University of Leeds) for LM5 LM6 LM8 LM19 899

and LM20 antibodies and the Complex Carbohydrate Resource Center (University of 900

Georgia) for the CCRC-M7 antibody Most of this work was funded by the Division of 901

Chemical Sciences Geosciences and Biosciences Office of Basic Energy Sciences of 902

the US Department of Energy through Grant DE-FG02-05ER15670 to JG This work 903

was also supported by NSF awards IOS 1339125 and IOS 1021861 to DJK NSF 904

award IOS 1121425 to F K and the USDA National Institute of Food and Agriculture 905

Hatch project number CA-D-PLS-7033-H to DJK 906

907

AUTHOR CONTRIBUTIONS 908

GB and JG designed the research GB GX AT BL NES and NH 909

performed research DJK provided tools GB GX MP AD BL RAH and FK 910

analyzed data GB and JG wrote the paper with input from all authors 911

912

FIGURE LEGENDS 913

Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 Expression 914

Requires PAD4 EDS1 and PBS3 915

(A) Expression of GAE1 in leaves of wild-type Col-0 and mutant plants Expression was916

measured 24 or 32 h after either inoculation with Pma ES4326 (OD600 = 0002) or mock 917

inoculation with 5 mM MgSO4 and was then normalized to the level of ACTIN2 Data 918

from three biological replicates were combined using a mixed linear model Bars 919

represent mean log2-ratios to ACTIN2 plusmn SE Letters indicate significantly different 920

groups with q lt 005 where 005 represents the maximum false discovery rate at which 921

the test may be called significant 922

(B) Expression of GAE6 measured as described in (A)923

924

Figure 2 Susceptibility of gae6 and gae1 gae6 Plants to Pma ES4326 Is Increased 925

Relative to Wild-Type Col-0 926

Leaves of plants of the indicated genotypes were inoculated with Pma ES4326 (OD600 = 927

00002) as described in Methods Bacterial titers in leaves were determined either 928

Page 42 of 54

immediately (0 dpi) or after three days (3 dpi) Bars represent the mean plusmn SE of four 929

independent experiments each with 4 or 12 biological replicates at 0 and 3 dpi 930

respectively Data were combined using a mixed linear model Asterisks indicate 931

significant differences from Col-0 wild-type (q lt 005) Susceptible pad4 plants were 932

included as a positive control 933

934

Figure 3 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves 935

(A) Depiction of the assay for brittleness of gae mutant leaves Fully expanded leaves of936

four to five week-old plants were bent by 180ordm so that the adaxial surface of the leaf was 937

parallel with the soil Broken leaf petioles or midribs were considered brittle 938

(B) Quantification of broken midribs or petioles For each genotype 66 to 82 leaves 939

were analyzed for brittleness as described in (A) Bars represent the ratio of broken to 940

total number of leaves tested Results were analyzed using Fisherrsquos exact test Letters 941

indicate significantly different groups at p lt 005 942

(C) Conductivity measurements Leaves of four week-old plants were inoculated with943

deionized water and then leaf discs were cut out and placed on deionized water 944

Conductivity was measured at the indicated time points After 30 min the water was 945

replaced with fresh water (30 min f w) to account for wounding-related ion leakage from 946

the edges of the leaf discs Three independent experiments with six biological replicates 947

each were performed and data were analyzed using a t-test for each time point P-948

values were corrected using the Bonferroni method Data show means plusmn SE asterisks 949

indicate significant differences from wild-type Col-0 at q lt 001 950

951

Figure 4 Cell Walls of gae6 and gae1 gae6 Plants Are Reduced in GalA 952

(A) to (D) Levels of GalA (A) GlcA (B) cellulose (C) and neutral sugars (D) in alcohol953

insoluble residue (AIR) extracted from leaves of four week-old plants of the indicated 954

genotypes Bars represent means plusmn SE of four biological replicates combined using a 955

mixed linear model Letters indicate significantly different groups (p lt 005) 956

957

Figure 5 Cell Walls of gae1 gae6 Plants Are Reduced in Homogalacturonan 958

Page 43 of 54

Dot blot analyses with anti-LM19 and anti-LM20 antibodies Pectin was extracted from 959

AIR prepared from the indicated genotypes using 1 microL of extraction buffer per 10 microg of 960

AIR Pectin was serially diluted and one microL of undiluted 13 diluted and 19 diluted 961

samples were spotted on a nitrocellulose membrane Three biological replicates were 962

were spotted twice to obtain two technical replicates each All replicates produced 963

similar results 964

(A) Representative dot blot using anti-LM19 antibody965

(B) Relative signal intensities of the spots in the dot blot in (A)966

(C) Representative dot blot using anti-LM20 antibody967

(D) Relative signal intensities of the LM20 dot blot shown in (C)968

Relative signal intensities were measured using Image J software Measurements from 969

the three biological replicates were combined using a linear model after Box-Cox power 970

transformation as described in Methods Bars represent means plusmn SE Asterisks indicate 971

a difference from wild-type Col-0 at p lt 005 972

973

Figure 6 Growth of B cinerea on gae1 gae6 Plants and the Effect of B cinerea on 974

Expression of GAE1 and GAE6 975

(A) Growth of B cinerea isolates Leaves of Col-0 and gae1 gae6 plants were976

inoculated with 25 x 105 spores mL-1 of the B cinerea isolates indicated in the figure 977

Samples were collected two (2 dpi) and three (3 dpi) days later Bars represent means plusmn 978

SE of six biological replicates for each fungus combined using a mixed linear model 979

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 980

(B) and (C) Expression of GAE1 (B) and GAE6 (C) 48 h after inoculation with the B981

cinerea isolates indicated Expression levels were measured by qRT-PCR Bars 982

represent mean log2-ratios to ACTIN2 plusmn SE of three biological replicates combined 983

using a mixed linear model Letters indicate significantly different groups (q lt 005) 984

985

Figure 7 Levels of Soluble Pectin and Expression of PAD3 in Fractions Produced 986

Following Macerozyme Treatment of gae1 gae6 Cell Walls 987

(A) Schematic overview of the experimental procedure for (B) and (C)988

Page 44 of 54

(B) Uronic acid measurements Alcohol-insoluble residue (AIR) from leave tissue of four989

week-old wild-type Col-0 or gae1 gae6 plants was treated with water or 025 990

macerozyme and assayed for uronic acid levels Data from 11 biological replicates were 991

combined using a mixed linear model Bars represent means plusmn SE Letters indicate 992

significant differences (p lt 005) 993

(C) Expression of PAD3 in Col-0 plants treated with the fractions from (B) Extracts from994

(B) were heat inactivated pooled and diluted 5-fold with water Pooled extracts were995

inoculated into four week-old Col-0 plants PAD3 expression was measured 3 h after 996

inoculation and normalized to the level of ACTIN2 expression Data from four biological 997

replicates were combined using a mixed linear model Bars represent mean log2-ratios 998

to ACTIN2 plusmn SE Letters indicate significant differences (p lt 005) 999

1000

Figure 8 Macerozyme-Induced PAD3 Expression Is Absent in gae1 gae6 1001

(A) Expression of PAD3 after macerozyme treatment Four week-old plants were either1002

inoculated with 001 macerozyme or mock inoculated with water and PAD3 1003

expression was measured 3 h later by qRT-PCR Data from four biological replicates 1004

were combined using a mixed linear model Mean log2-ratios to ACTIN2 plusmn SE were 1005

plotted Letters indicate significantly different groups (p lt 005) 1006

(B) Expression of PAD3 after OG treatment PAD3 expression was measured as1007

described in (A) 3 h after inoculation with 100 microg mL-1 OGs or mock inoculation with 1008

water Data are from six biological replicates 1009

(C) PAD3 expression in untreated plants Three biological replicates were performed1010

and data analyzed as described in (A) 1011

(D) Camalexin accumulation after B cinerea treatment Camalexin accumulation was1012

measured in four week-old plants inoculated with the B cinerea strains shown in the 1013

figure Eight biological replicates each were combined using a mixed linear model 1014

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 1015

(E) PAD3 expression 28 h after treatment with the indicated B cinerea strains Data1016

from six biological replicates were collected and analyzed as described in (A) Data in 1017

all figure parts represent means plusmn SE Letters indicate significantly different groups (q lt 1018

005) 1019

Page 45 of 54

1020

Figure 9 Macerozyme-Induced Expression of the SA Marker Genes SID2 and PR1 1021

Is Abolished in gae1 gae6 Plants 1022

(A) to (H) Expression of PR1 (A) (C) (E) and (G) or SID2 (B) (D) (F) and (H) in four1023

week-old Col-0 and gae1 gae6 plants Data were combined using a mixed linear model 1024

Bars represent mean log2-ratios of expression vs ACTIN2 plusmn SE for all figure parts 1025

Letters indicate significantly different groups at p lt 005 for panels (A) to (D) (G) and 1026

(H) and qlt005 for panels (E) to (F) 1027

(A) and (B) Plants were treated with 001 macerozyme or mock treated with water1028

and samples were collected 3 h later Data from four biological replicates are shown 1029

(C) and (D) Plants were inoculated with 100 microg mL-1 OGs or mock treated with water1030

and samples collected 3 h later Data from six biological replicates are shown 1031

(E) and (F) Plants were inoculated with the indicated B cinerea isolates or mock1032

inoculated with B cinerea inoculation medium and samples collected 48 h later Data 1033

from eight biological replicates are shown 1034

(G) and (H) Expression in untreated plants Three biological replicates were performed1035

1036

Figure 10 The Expression of the JA Marker Gene JAZ10 is Highly Responsive in 1037

gae1 gae6 Plants 1038

(A) to (D) JAZ10 expression in four week-old plants Data were combined using a mixed1039

linear model Mean log2-ratios to ACTIN2 plusmn SE are shown Letters indicate significantly 1040

different groups at p lt 005 for panels (A) (B) and (D) and q lt 005 for panel (C) 1041

(A) Plants were inoculated with 001 macerozyme or mock inoculated using water and1042

samples were collected 3 h later Data from four biological replicates are shown 1043

(B) Plants were inoculated with 100 microg mL-1 OGs or mock inoculated using water and1044

samples collected 3 h later Data are from six biological replicates 1045

(C) JAZ10 expression 48 h after treatment with the indicated B cinerea strains Data1046

are from six biological replicates 1047

(D) JAZ10 expression in untreated plants Data are from three biological replicates1048

1049

Figure 11 Gallo 1-Induced Callose Deposition Is Reduced in gae1 gae6 Plants 1050

Page 46 of 54

(A) Callose deposition in four week-old Col-0 and gae1 gae6 plants Plants were1051

inoculated with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) or mock inoculated 1052

with B cinerea inoculation medium and samples were collected after 72 h Callose was 1053

stained with aniline blue and visualized using a Nikon Eclipse Ni-U microscope with a 1054

DAPI filter Three leaves were stained per treatment Scale bars are 100 microm Asterisks 1055

indicate fungal hyphae arrows indicate representative callose spots 1056

(B) Quantification of callose deposition Callose deposits were counted in 200 microm x 2001057

microm squares laid out across the images shown here Bars represent means plusmn SE from 1058

six squares each Letters indicate significantly different groups (p lt 005) according to a 1059

t-test analysis1060

1061

Figure 12 Less Soluble Pectin Accumulates in B cinerea-Infected gae1 gae6 1062

Leaves 1063

Four week-old Col-0 and gae1 gae6 plants were inoculated with 10 microL droplets of the 1064

indicated B cinerea isolate (25 x 105 spores mL-1) or mock inoculated with B cinerea 1065

inoculation medium Leaves were collected 48 h later AIR was extracted from leaf 1066

samples and water added (20 microL infection site) to extract soluble pectin fragments 1067

Samples were vortexed for 7 h centrifuged at 12000 g and the uronic acid 1068

concentration of the supernatant was measured Data from nine biological replicates 1069

were combined using a mixed linear model Means plusmn SE are shown Letters indicate 1070

significant differences (q lt 005) 1071

1072

Figure 13 Macerozyme-Induced PTI Is Lost in gae1 gae6 While OG-Induced PTI Is 1073

Unaltered 1074

(A) Macerozyme-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1075

inoculated with 001 macerozyme or mock inoculated with boiled macerozyme and 1076

were infected with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) 24 h later Samples 1077

were collected immediately (0 dpi) and after two (2 dpi) and three (3 dpi) days Data 1078

show mean plusmn SE of six to 14 biological replicates each combined using a linear model 1079

Letters indicate significantly different groups (q lt 005) 1080

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

Parsed CitationsAdam L and Somerville SC (1996) Genetic characterization of five powdery mildew disease resistance loci in Arabidopsisthaliana Plant J 9 341-356

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Albersheim P Jones TM and English PD (1969) Biochemistry of the cell wall in relation to infective processes Annu RevPhytopathol 7 171-194

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk RGadrinab C Heller C Jeske A Koesema E Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt MHazari N Hom E Karnes M Mulholland C Ndubaku R Schmidt I Guzman P Aguilar-Henonin L Schmid M Weigel DCarter DE Marchand T Risseeuw E Brogden D Zeko A Crosby WL Berry CC and Ecker JR (2003) Genome-wideinsertional mutagenesis of Arabidopsis thaliana Science 301 653-657

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Asai T Tena G Plotnikova J Willmann MR Chiu WL Gomez-Gomez L Boller T Ausubel FM and Sheen J (2002) MAPkinase signalling cascade in Arabidopsis innate immunity Nature 415 977-983

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bartsch M Gobbato E Bednarek P Debey S Schultze JL Bautor J and Parker JE (2006) Salicylic acid-independentENHANCED DISEASE SUSCEPTIBILITY1 signaling in Arabidopsis immunity and cell death is regulated by the monooxygenaseFMO1 and the nudix hydrolase NUDT7 Plant Cell 18 1038-1051

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bates D Machler M Bolker BM and Walker SC (2015) Fitting Linear Mixed-Effects Models Using lme4 J Stat Softw 67 1-48Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Benjamini Y and Hochberg Y (1995) Controlling the False Discovery Rate - a Practical and Powerful Approach to MultipleTesting J Roy Stat Soc B Met 57 289-300

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bethke G Grundman RE Sreekanta S Truman W Katagiri F and Glazebrook J (2014) Arabidopsis PECTINMETHYLESTERASEs Contribute to Immunity against Pseudomonas syringae Plant Physiol 164 1093-1107

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bouton S Leboeuf E Mouille G Leydecker MT Talbotec J Granier F Lahaye M Hofte H and Truong HN (2002)Quasimodo1 encodes a putative membrane-bound glycosyltransferase required for normal pectin synthesis and cell adhesion inArabidopsis Plant Cell 14 2577-2590

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Box GEP and Cox DR (1964) An analysis of transformations Journal of the Royal Statistical Society Series B 26 211-252Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Cao H Glazebrook J Clarke JD Volko S and Dong XN (1997) The Arabidopsis NPR1 gene that controls systemic acquiredresistance encodes a novel protein containing ankyrin repeats Cell 88 57-63

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Carpita N (2010) Update on Mechanisms of Plant Cell Wall Biosynthesis How plants make cellulose and other (1-gt4)-beta-D-glycans Plant Physiol

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Chisholm ST Coaker G Day B and Staskawicz BJ (2006) Host-microbe interactions Shaping the evolution of the plantimmune response Cell 124 803-814

Page 38 of 54

1995) antibodies LM series antibodies were diluted 1250 and CCRC-M7 was diluted 805

1500 in 5 milk powder (Nestle Carnation) in 1 x PBS For LM series antibodies a 806

goat anti-Rat HRP conjugated antibody (Bethyl A110-105P 15000 diluted) and for 807

CCRC-M7 an anti-Mouse IgG HRP conjugate (Promega W402B 12500 diluted) was 808

used as secondary antibody in 5 milk powder in 1x PBS LM series antibodies were 809

obtained from plant probes (wwwplantprobesnet) and CCRC-M7 from CarboSource 810

(wwwcarbosourcenet) Membranes were washed after incubation with the primary and 811

secondary antibodies with 1x PBS Dot blots were developed using the ECL system 812

(GE Healthcare Amersham ECL prime) For quantification of signals dot blot results on 813

x-ray films were photographed using a CCD camera and intensities were measured814

using Image Jrsquos Integrated Density function Locally adjusted background was 815

subtracted As the dynamic range of the signal on an x-ray film was narrow non-816

linearity in the measured values was corrected for each genotype in each biological 817

replicate using the values from different dilutions in two technical replicates We noticed 818

that the residuals were not normally distributed and performed Box-Cox power 819

transformation (Box and Cox 1964) to obtain normally distributed values for the 820

subsequent statistical analysis of the data For LM19 Box-Cox selected power 821

transformation was done to the power of 038th and for LM20 to the power 075th After 822

correction of the transformed measured values with the effect of the biological 823

replicates the values were fit to a linear model with the Arabidopsis genotype as the 824

fixed effect This linear model was used both to generate the plots in Figures 5B and D 825

and to test significant differences 826

827

Camalexin Measurements 828

Four week-old plants were infected with B cinerea and samples (three leaf disks 829

from one plant each) were harvested at the indicated time points flash frozen and 830

pulverized Each sample was extracted with 300 microL of 90 methanol (vv) 50 microL of the 831

extract was run on an Agilent Lichrocart 250-4 RP18e 5-microm column using an Agilent 832

1100 series HPLC Camalexin was detected using a DiodeArray (DAD) at 330 nm and 833

with a fluorescence detector at emission 318 nmexcitation 385 nm (Agilent) Separation 834

was achieved using the following program with aqueous acetonitrile 5 min gradient 835

Page 39 of 54

from 63 to 69 acetonitrile 30 s gradient from 69 to 99 acetonitrile 2 min at 99 836

acetonitrile and a post-run equilibration of 35 min at 63 acetonitrile (Denby et al 837

2004 Kliebenstein et al 2005) Purified camalexin was used to produce a standard 838

curve to identify and quantitate camalexin 839

840

Accession Numbers 841

Microarray data used can be accessed at NCBI Gene Expression Omnnibus 842

(httpwwwncbinlmnihgovgeo) GSE18978 Sequence information for all Arabidopsis 843

genes described in this article can be found in TAIR (wwwarabidopsisorg) using the 844

following accession numbers PAD3 (CYP71B15 At3g26830) SID2 (ICS1 At1g74710) 845

GAE1 (At4g30440) GAE2 (At1g02000) GAE3 (At4g00110) GAE4 (At2g45310) GAE5 846

(At4g12250) GAE6 (At3g23820) PR1 (At2g14610) JAZ5 (At1g17380) JAZ10 847

(At5g13220) ACTIN2 (At3g18780) PDF12a (At5g44420) PDF12b (At2g26020) 848

AtSK11 (At5g26751) The sequence of the B cinerea cutinase Bc-CutA (Z69264) can 849

be found in the DNA Data Bank of Japan (httpgetentryddbjnigacjp) Germplasm 850

used included dde2-2 (At5g42650 (von Malek et al 2002)) pad3-1 (At3g26830 (Zhou 851

et al 1999)) pad4-1 (At3g52430 (Jirage et al 1999)) sid2-2 (At1g74710 (Wildermuth 852

et al 2001)) pbs3-2 (At5g13320 SALK_018225 (Nobuta et al 2007)) cbp60a-1 853

(At5g62570 SALK_124410 (Truman et al 2013)) cbp60g-1 sard1-2 (At5g26920 854

SALK_023199 At1g73805 SALK_052422 (Wang et al 2011) cbp60a-1 cbp60g-1 855

sard1-2 (Truman et al 2013) gae1-1 (At4g30440 SALK_085554) gae6-1 (At3g23280 856

SALK_104454) gae6-2 (At3g23280 SALK_017191) ixr1-1 cev1 (At5g05170 (Ellis 857

and Turner 2001 Scheible et al 2001)) eds1 plants were derived by introgression of 858

the Ler eds1-2 allele into Col-0 that contains two copies of EDS1 (At3g48090 859

At3g48080 (Bartsch et al 2006) mpk6-2 (At2g43790 SALK_073907 (Liu and Zhang 860

2004)) mpk3-DG (At3g45640 a fast neutron deletion mutant (Miles et al 2005)) and 861

npr1-1 (At1g64280 (Cao et al 1997)) T-DNA insertion lines were part of the SALK 862

collection (Alonso et al 2003) and were obtained from the Arabidopsis Biological 863

Resource Center 864

865

Supplemental Data 866

Page 40 of 54

Supplemental Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 867

Expression Does Not Require CBP60s or SID2 868

Supplemental Figure 2 Expression of GAE Family Members uponTreatment with Pma 869

ES4326 870

Supplemental Figure 3 Characterization of T-DNA Insertions in GAE1 and GAE6 871

Supplemental Figure 4 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves and 872

Are Slightly Smaller than Wild-Type Col-0 Plants 873

Supplemental Figure 5 Cell Walls of gae1 gae6 Plants Contain Less Uronic Acid than 874

Wild-Type Col-0 Cell Walls 875

Supplemental Figure 6 Antibodies Raised Against the RGI or XG Components of 876

Pectin Show No Difference in Binding to gae Mutant and Col-0 Cell Walls 877

Supplemental Figure 7 The Effect of B cinerea Treatment on the Expression of GAE 878

Family Genes 879

Supplemental Figure 8 Treatment with 001 Macerozyme Does Not Cause Any 880

Visible Damage to Col-0 Plants 881

Supplemental Figure 9 B cinerea Growth on Plants with Defects in Immune 882

Signaling 883

Supplemental Figure 10 MeJA-Induced Expression of the JA marker gene JAZ10 884

Supplemental Figure 11 The Expression of the JA Marker Gene JAZ5 Is Highly 885

Responsive in gae1 gae6 Plants 886

Supplemental Figure 12 The Expression of PDF12 Is Unaltered in gae1 gae6 Plants 887

Supplemental Figure 13 Macerozyme Treatment Activates MPK3 and MPK6 888

Supplemental Figure 14 B cinerea-Induced ROS Production in Wild-Type Col-0 and 889

gae1 gae6 Plants 890

Supplemental Figure 15 OG-Induced PTI Using 250 microg mL-1 OGs 891

Supplemental Figure 16 Abundance of Plant DNA in B cinerea Treated Plants 892

Supplemental Table 1 Primers used in this study 893

894

ACKNOWLEDGEMENTS 895

We thank William Truman for helpful discussions Simone Ferrari (Sapienza 896

Universita di Roma) for the generous gift of OGs Bruna Bucciarelli (USDA-ARS) for 897

Page 41 of 54

help with fluorescence microscopy the Arabidopsis Biological Resource Center for T-898

DNA insertion lines Paul Knoxrsquos lab (University of Leeds) for LM5 LM6 LM8 LM19 899

and LM20 antibodies and the Complex Carbohydrate Resource Center (University of 900

Georgia) for the CCRC-M7 antibody Most of this work was funded by the Division of 901

Chemical Sciences Geosciences and Biosciences Office of Basic Energy Sciences of 902

the US Department of Energy through Grant DE-FG02-05ER15670 to JG This work 903

was also supported by NSF awards IOS 1339125 and IOS 1021861 to DJK NSF 904

award IOS 1121425 to F K and the USDA National Institute of Food and Agriculture 905

Hatch project number CA-D-PLS-7033-H to DJK 906

907

AUTHOR CONTRIBUTIONS 908

GB and JG designed the research GB GX AT BL NES and NH 909

performed research DJK provided tools GB GX MP AD BL RAH and FK 910

analyzed data GB and JG wrote the paper with input from all authors 911

912

FIGURE LEGENDS 913

Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 Expression 914

Requires PAD4 EDS1 and PBS3 915

(A) Expression of GAE1 in leaves of wild-type Col-0 and mutant plants Expression was916

measured 24 or 32 h after either inoculation with Pma ES4326 (OD600 = 0002) or mock 917

inoculation with 5 mM MgSO4 and was then normalized to the level of ACTIN2 Data 918

from three biological replicates were combined using a mixed linear model Bars 919

represent mean log2-ratios to ACTIN2 plusmn SE Letters indicate significantly different 920

groups with q lt 005 where 005 represents the maximum false discovery rate at which 921

the test may be called significant 922

(B) Expression of GAE6 measured as described in (A)923

924

Figure 2 Susceptibility of gae6 and gae1 gae6 Plants to Pma ES4326 Is Increased 925

Relative to Wild-Type Col-0 926

Leaves of plants of the indicated genotypes were inoculated with Pma ES4326 (OD600 = 927

00002) as described in Methods Bacterial titers in leaves were determined either 928

Page 42 of 54

immediately (0 dpi) or after three days (3 dpi) Bars represent the mean plusmn SE of four 929

independent experiments each with 4 or 12 biological replicates at 0 and 3 dpi 930

respectively Data were combined using a mixed linear model Asterisks indicate 931

significant differences from Col-0 wild-type (q lt 005) Susceptible pad4 plants were 932

included as a positive control 933

934

Figure 3 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves 935

(A) Depiction of the assay for brittleness of gae mutant leaves Fully expanded leaves of936

four to five week-old plants were bent by 180ordm so that the adaxial surface of the leaf was 937

parallel with the soil Broken leaf petioles or midribs were considered brittle 938

(B) Quantification of broken midribs or petioles For each genotype 66 to 82 leaves 939

were analyzed for brittleness as described in (A) Bars represent the ratio of broken to 940

total number of leaves tested Results were analyzed using Fisherrsquos exact test Letters 941

indicate significantly different groups at p lt 005 942

(C) Conductivity measurements Leaves of four week-old plants were inoculated with943

deionized water and then leaf discs were cut out and placed on deionized water 944

Conductivity was measured at the indicated time points After 30 min the water was 945

replaced with fresh water (30 min f w) to account for wounding-related ion leakage from 946

the edges of the leaf discs Three independent experiments with six biological replicates 947

each were performed and data were analyzed using a t-test for each time point P-948

values were corrected using the Bonferroni method Data show means plusmn SE asterisks 949

indicate significant differences from wild-type Col-0 at q lt 001 950

951

Figure 4 Cell Walls of gae6 and gae1 gae6 Plants Are Reduced in GalA 952

(A) to (D) Levels of GalA (A) GlcA (B) cellulose (C) and neutral sugars (D) in alcohol953

insoluble residue (AIR) extracted from leaves of four week-old plants of the indicated 954

genotypes Bars represent means plusmn SE of four biological replicates combined using a 955

mixed linear model Letters indicate significantly different groups (p lt 005) 956

957

Figure 5 Cell Walls of gae1 gae6 Plants Are Reduced in Homogalacturonan 958

Page 43 of 54

Dot blot analyses with anti-LM19 and anti-LM20 antibodies Pectin was extracted from 959

AIR prepared from the indicated genotypes using 1 microL of extraction buffer per 10 microg of 960

AIR Pectin was serially diluted and one microL of undiluted 13 diluted and 19 diluted 961

samples were spotted on a nitrocellulose membrane Three biological replicates were 962

were spotted twice to obtain two technical replicates each All replicates produced 963

similar results 964

(A) Representative dot blot using anti-LM19 antibody965

(B) Relative signal intensities of the spots in the dot blot in (A)966

(C) Representative dot blot using anti-LM20 antibody967

(D) Relative signal intensities of the LM20 dot blot shown in (C)968

Relative signal intensities were measured using Image J software Measurements from 969

the three biological replicates were combined using a linear model after Box-Cox power 970

transformation as described in Methods Bars represent means plusmn SE Asterisks indicate 971

a difference from wild-type Col-0 at p lt 005 972

973

Figure 6 Growth of B cinerea on gae1 gae6 Plants and the Effect of B cinerea on 974

Expression of GAE1 and GAE6 975

(A) Growth of B cinerea isolates Leaves of Col-0 and gae1 gae6 plants were976

inoculated with 25 x 105 spores mL-1 of the B cinerea isolates indicated in the figure 977

Samples were collected two (2 dpi) and three (3 dpi) days later Bars represent means plusmn 978

SE of six biological replicates for each fungus combined using a mixed linear model 979

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 980

(B) and (C) Expression of GAE1 (B) and GAE6 (C) 48 h after inoculation with the B981

cinerea isolates indicated Expression levels were measured by qRT-PCR Bars 982

represent mean log2-ratios to ACTIN2 plusmn SE of three biological replicates combined 983

using a mixed linear model Letters indicate significantly different groups (q lt 005) 984

985

Figure 7 Levels of Soluble Pectin and Expression of PAD3 in Fractions Produced 986

Following Macerozyme Treatment of gae1 gae6 Cell Walls 987

(A) Schematic overview of the experimental procedure for (B) and (C)988

Page 44 of 54

(B) Uronic acid measurements Alcohol-insoluble residue (AIR) from leave tissue of four989

week-old wild-type Col-0 or gae1 gae6 plants was treated with water or 025 990

macerozyme and assayed for uronic acid levels Data from 11 biological replicates were 991

combined using a mixed linear model Bars represent means plusmn SE Letters indicate 992

significant differences (p lt 005) 993

(C) Expression of PAD3 in Col-0 plants treated with the fractions from (B) Extracts from994

(B) were heat inactivated pooled and diluted 5-fold with water Pooled extracts were995

inoculated into four week-old Col-0 plants PAD3 expression was measured 3 h after 996

inoculation and normalized to the level of ACTIN2 expression Data from four biological 997

replicates were combined using a mixed linear model Bars represent mean log2-ratios 998

to ACTIN2 plusmn SE Letters indicate significant differences (p lt 005) 999

1000

Figure 8 Macerozyme-Induced PAD3 Expression Is Absent in gae1 gae6 1001

(A) Expression of PAD3 after macerozyme treatment Four week-old plants were either1002

inoculated with 001 macerozyme or mock inoculated with water and PAD3 1003

expression was measured 3 h later by qRT-PCR Data from four biological replicates 1004

were combined using a mixed linear model Mean log2-ratios to ACTIN2 plusmn SE were 1005

plotted Letters indicate significantly different groups (p lt 005) 1006

(B) Expression of PAD3 after OG treatment PAD3 expression was measured as1007

described in (A) 3 h after inoculation with 100 microg mL-1 OGs or mock inoculation with 1008

water Data are from six biological replicates 1009

(C) PAD3 expression in untreated plants Three biological replicates were performed1010

and data analyzed as described in (A) 1011

(D) Camalexin accumulation after B cinerea treatment Camalexin accumulation was1012

measured in four week-old plants inoculated with the B cinerea strains shown in the 1013

figure Eight biological replicates each were combined using a mixed linear model 1014

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 1015

(E) PAD3 expression 28 h after treatment with the indicated B cinerea strains Data1016

from six biological replicates were collected and analyzed as described in (A) Data in 1017

all figure parts represent means plusmn SE Letters indicate significantly different groups (q lt 1018

005) 1019

Page 45 of 54

1020

Figure 9 Macerozyme-Induced Expression of the SA Marker Genes SID2 and PR1 1021

Is Abolished in gae1 gae6 Plants 1022

(A) to (H) Expression of PR1 (A) (C) (E) and (G) or SID2 (B) (D) (F) and (H) in four1023

week-old Col-0 and gae1 gae6 plants Data were combined using a mixed linear model 1024

Bars represent mean log2-ratios of expression vs ACTIN2 plusmn SE for all figure parts 1025

Letters indicate significantly different groups at p lt 005 for panels (A) to (D) (G) and 1026

(H) and qlt005 for panels (E) to (F) 1027

(A) and (B) Plants were treated with 001 macerozyme or mock treated with water1028

and samples were collected 3 h later Data from four biological replicates are shown 1029

(C) and (D) Plants were inoculated with 100 microg mL-1 OGs or mock treated with water1030

and samples collected 3 h later Data from six biological replicates are shown 1031

(E) and (F) Plants were inoculated with the indicated B cinerea isolates or mock1032

inoculated with B cinerea inoculation medium and samples collected 48 h later Data 1033

from eight biological replicates are shown 1034

(G) and (H) Expression in untreated plants Three biological replicates were performed1035

1036

Figure 10 The Expression of the JA Marker Gene JAZ10 is Highly Responsive in 1037

gae1 gae6 Plants 1038

(A) to (D) JAZ10 expression in four week-old plants Data were combined using a mixed1039

linear model Mean log2-ratios to ACTIN2 plusmn SE are shown Letters indicate significantly 1040

different groups at p lt 005 for panels (A) (B) and (D) and q lt 005 for panel (C) 1041

(A) Plants were inoculated with 001 macerozyme or mock inoculated using water and1042

samples were collected 3 h later Data from four biological replicates are shown 1043

(B) Plants were inoculated with 100 microg mL-1 OGs or mock inoculated using water and1044

samples collected 3 h later Data are from six biological replicates 1045

(C) JAZ10 expression 48 h after treatment with the indicated B cinerea strains Data1046

are from six biological replicates 1047

(D) JAZ10 expression in untreated plants Data are from three biological replicates1048

1049

Figure 11 Gallo 1-Induced Callose Deposition Is Reduced in gae1 gae6 Plants 1050

Page 46 of 54

(A) Callose deposition in four week-old Col-0 and gae1 gae6 plants Plants were1051

inoculated with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) or mock inoculated 1052

with B cinerea inoculation medium and samples were collected after 72 h Callose was 1053

stained with aniline blue and visualized using a Nikon Eclipse Ni-U microscope with a 1054

DAPI filter Three leaves were stained per treatment Scale bars are 100 microm Asterisks 1055

indicate fungal hyphae arrows indicate representative callose spots 1056

(B) Quantification of callose deposition Callose deposits were counted in 200 microm x 2001057

microm squares laid out across the images shown here Bars represent means plusmn SE from 1058

six squares each Letters indicate significantly different groups (p lt 005) according to a 1059

t-test analysis1060

1061

Figure 12 Less Soluble Pectin Accumulates in B cinerea-Infected gae1 gae6 1062

Leaves 1063

Four week-old Col-0 and gae1 gae6 plants were inoculated with 10 microL droplets of the 1064

indicated B cinerea isolate (25 x 105 spores mL-1) or mock inoculated with B cinerea 1065

inoculation medium Leaves were collected 48 h later AIR was extracted from leaf 1066

samples and water added (20 microL infection site) to extract soluble pectin fragments 1067

Samples were vortexed for 7 h centrifuged at 12000 g and the uronic acid 1068

concentration of the supernatant was measured Data from nine biological replicates 1069

were combined using a mixed linear model Means plusmn SE are shown Letters indicate 1070

significant differences (q lt 005) 1071

1072

Figure 13 Macerozyme-Induced PTI Is Lost in gae1 gae6 While OG-Induced PTI Is 1073

Unaltered 1074

(A) Macerozyme-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1075

inoculated with 001 macerozyme or mock inoculated with boiled macerozyme and 1076

were infected with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) 24 h later Samples 1077

were collected immediately (0 dpi) and after two (2 dpi) and three (3 dpi) days Data 1078

show mean plusmn SE of six to 14 biological replicates each combined using a linear model 1079

Letters indicate significantly different groups (q lt 005) 1080

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

Parsed CitationsAdam L and Somerville SC (1996) Genetic characterization of five powdery mildew disease resistance loci in Arabidopsisthaliana Plant J 9 341-356

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Albersheim P Jones TM and English PD (1969) Biochemistry of the cell wall in relation to infective processes Annu RevPhytopathol 7 171-194

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk RGadrinab C Heller C Jeske A Koesema E Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt MHazari N Hom E Karnes M Mulholland C Ndubaku R Schmidt I Guzman P Aguilar-Henonin L Schmid M Weigel DCarter DE Marchand T Risseeuw E Brogden D Zeko A Crosby WL Berry CC and Ecker JR (2003) Genome-wideinsertional mutagenesis of Arabidopsis thaliana Science 301 653-657

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Asai T Tena G Plotnikova J Willmann MR Chiu WL Gomez-Gomez L Boller T Ausubel FM and Sheen J (2002) MAPkinase signalling cascade in Arabidopsis innate immunity Nature 415 977-983

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bartsch M Gobbato E Bednarek P Debey S Schultze JL Bautor J and Parker JE (2006) Salicylic acid-independentENHANCED DISEASE SUSCEPTIBILITY1 signaling in Arabidopsis immunity and cell death is regulated by the monooxygenaseFMO1 and the nudix hydrolase NUDT7 Plant Cell 18 1038-1051

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bates D Machler M Bolker BM and Walker SC (2015) Fitting Linear Mixed-Effects Models Using lme4 J Stat Softw 67 1-48Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Benjamini Y and Hochberg Y (1995) Controlling the False Discovery Rate - a Practical and Powerful Approach to MultipleTesting J Roy Stat Soc B Met 57 289-300

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bethke G Grundman RE Sreekanta S Truman W Katagiri F and Glazebrook J (2014) Arabidopsis PECTINMETHYLESTERASEs Contribute to Immunity against Pseudomonas syringae Plant Physiol 164 1093-1107

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bouton S Leboeuf E Mouille G Leydecker MT Talbotec J Granier F Lahaye M Hofte H and Truong HN (2002)Quasimodo1 encodes a putative membrane-bound glycosyltransferase required for normal pectin synthesis and cell adhesion inArabidopsis Plant Cell 14 2577-2590

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Box GEP and Cox DR (1964) An analysis of transformations Journal of the Royal Statistical Society Series B 26 211-252Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Cao H Glazebrook J Clarke JD Volko S and Dong XN (1997) The Arabidopsis NPR1 gene that controls systemic acquiredresistance encodes a novel protein containing ankyrin repeats Cell 88 57-63

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Carpita N (2010) Update on Mechanisms of Plant Cell Wall Biosynthesis How plants make cellulose and other (1-gt4)-beta-D-glycans Plant Physiol

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Chisholm ST Coaker G Day B and Staskawicz BJ (2006) Host-microbe interactions Shaping the evolution of the plantimmune response Cell 124 803-814

Page 39 of 54

from 63 to 69 acetonitrile 30 s gradient from 69 to 99 acetonitrile 2 min at 99 836

acetonitrile and a post-run equilibration of 35 min at 63 acetonitrile (Denby et al 837

2004 Kliebenstein et al 2005) Purified camalexin was used to produce a standard 838

curve to identify and quantitate camalexin 839

840

Accession Numbers 841

Microarray data used can be accessed at NCBI Gene Expression Omnnibus 842

(httpwwwncbinlmnihgovgeo) GSE18978 Sequence information for all Arabidopsis 843

genes described in this article can be found in TAIR (wwwarabidopsisorg) using the 844

following accession numbers PAD3 (CYP71B15 At3g26830) SID2 (ICS1 At1g74710) 845

GAE1 (At4g30440) GAE2 (At1g02000) GAE3 (At4g00110) GAE4 (At2g45310) GAE5 846

(At4g12250) GAE6 (At3g23820) PR1 (At2g14610) JAZ5 (At1g17380) JAZ10 847

(At5g13220) ACTIN2 (At3g18780) PDF12a (At5g44420) PDF12b (At2g26020) 848

AtSK11 (At5g26751) The sequence of the B cinerea cutinase Bc-CutA (Z69264) can 849

be found in the DNA Data Bank of Japan (httpgetentryddbjnigacjp) Germplasm 850

used included dde2-2 (At5g42650 (von Malek et al 2002)) pad3-1 (At3g26830 (Zhou 851

et al 1999)) pad4-1 (At3g52430 (Jirage et al 1999)) sid2-2 (At1g74710 (Wildermuth 852

et al 2001)) pbs3-2 (At5g13320 SALK_018225 (Nobuta et al 2007)) cbp60a-1 853

(At5g62570 SALK_124410 (Truman et al 2013)) cbp60g-1 sard1-2 (At5g26920 854

SALK_023199 At1g73805 SALK_052422 (Wang et al 2011) cbp60a-1 cbp60g-1 855

sard1-2 (Truman et al 2013) gae1-1 (At4g30440 SALK_085554) gae6-1 (At3g23280 856

SALK_104454) gae6-2 (At3g23280 SALK_017191) ixr1-1 cev1 (At5g05170 (Ellis 857

and Turner 2001 Scheible et al 2001)) eds1 plants were derived by introgression of 858

the Ler eds1-2 allele into Col-0 that contains two copies of EDS1 (At3g48090 859

At3g48080 (Bartsch et al 2006) mpk6-2 (At2g43790 SALK_073907 (Liu and Zhang 860

2004)) mpk3-DG (At3g45640 a fast neutron deletion mutant (Miles et al 2005)) and 861

npr1-1 (At1g64280 (Cao et al 1997)) T-DNA insertion lines were part of the SALK 862

collection (Alonso et al 2003) and were obtained from the Arabidopsis Biological 863

Resource Center 864

865

Supplemental Data 866

Page 40 of 54

Supplemental Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 867

Expression Does Not Require CBP60s or SID2 868

Supplemental Figure 2 Expression of GAE Family Members uponTreatment with Pma 869

ES4326 870

Supplemental Figure 3 Characterization of T-DNA Insertions in GAE1 and GAE6 871

Supplemental Figure 4 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves and 872

Are Slightly Smaller than Wild-Type Col-0 Plants 873

Supplemental Figure 5 Cell Walls of gae1 gae6 Plants Contain Less Uronic Acid than 874

Wild-Type Col-0 Cell Walls 875

Supplemental Figure 6 Antibodies Raised Against the RGI or XG Components of 876

Pectin Show No Difference in Binding to gae Mutant and Col-0 Cell Walls 877

Supplemental Figure 7 The Effect of B cinerea Treatment on the Expression of GAE 878

Family Genes 879

Supplemental Figure 8 Treatment with 001 Macerozyme Does Not Cause Any 880

Visible Damage to Col-0 Plants 881

Supplemental Figure 9 B cinerea Growth on Plants with Defects in Immune 882

Signaling 883

Supplemental Figure 10 MeJA-Induced Expression of the JA marker gene JAZ10 884

Supplemental Figure 11 The Expression of the JA Marker Gene JAZ5 Is Highly 885

Responsive in gae1 gae6 Plants 886

Supplemental Figure 12 The Expression of PDF12 Is Unaltered in gae1 gae6 Plants 887

Supplemental Figure 13 Macerozyme Treatment Activates MPK3 and MPK6 888

Supplemental Figure 14 B cinerea-Induced ROS Production in Wild-Type Col-0 and 889

gae1 gae6 Plants 890

Supplemental Figure 15 OG-Induced PTI Using 250 microg mL-1 OGs 891

Supplemental Figure 16 Abundance of Plant DNA in B cinerea Treated Plants 892

Supplemental Table 1 Primers used in this study 893

894

ACKNOWLEDGEMENTS 895

We thank William Truman for helpful discussions Simone Ferrari (Sapienza 896

Universita di Roma) for the generous gift of OGs Bruna Bucciarelli (USDA-ARS) for 897

Page 41 of 54

help with fluorescence microscopy the Arabidopsis Biological Resource Center for T-898

DNA insertion lines Paul Knoxrsquos lab (University of Leeds) for LM5 LM6 LM8 LM19 899

and LM20 antibodies and the Complex Carbohydrate Resource Center (University of 900

Georgia) for the CCRC-M7 antibody Most of this work was funded by the Division of 901

Chemical Sciences Geosciences and Biosciences Office of Basic Energy Sciences of 902

the US Department of Energy through Grant DE-FG02-05ER15670 to JG This work 903

was also supported by NSF awards IOS 1339125 and IOS 1021861 to DJK NSF 904

award IOS 1121425 to F K and the USDA National Institute of Food and Agriculture 905

Hatch project number CA-D-PLS-7033-H to DJK 906

907

AUTHOR CONTRIBUTIONS 908

GB and JG designed the research GB GX AT BL NES and NH 909

performed research DJK provided tools GB GX MP AD BL RAH and FK 910

analyzed data GB and JG wrote the paper with input from all authors 911

912

FIGURE LEGENDS 913

Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 Expression 914

Requires PAD4 EDS1 and PBS3 915

(A) Expression of GAE1 in leaves of wild-type Col-0 and mutant plants Expression was916

measured 24 or 32 h after either inoculation with Pma ES4326 (OD600 = 0002) or mock 917

inoculation with 5 mM MgSO4 and was then normalized to the level of ACTIN2 Data 918

from three biological replicates were combined using a mixed linear model Bars 919

represent mean log2-ratios to ACTIN2 plusmn SE Letters indicate significantly different 920

groups with q lt 005 where 005 represents the maximum false discovery rate at which 921

the test may be called significant 922

(B) Expression of GAE6 measured as described in (A)923

924

Figure 2 Susceptibility of gae6 and gae1 gae6 Plants to Pma ES4326 Is Increased 925

Relative to Wild-Type Col-0 926

Leaves of plants of the indicated genotypes were inoculated with Pma ES4326 (OD600 = 927

00002) as described in Methods Bacterial titers in leaves were determined either 928

Page 42 of 54

immediately (0 dpi) or after three days (3 dpi) Bars represent the mean plusmn SE of four 929

independent experiments each with 4 or 12 biological replicates at 0 and 3 dpi 930

respectively Data were combined using a mixed linear model Asterisks indicate 931

significant differences from Col-0 wild-type (q lt 005) Susceptible pad4 plants were 932

included as a positive control 933

934

Figure 3 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves 935

(A) Depiction of the assay for brittleness of gae mutant leaves Fully expanded leaves of936

four to five week-old plants were bent by 180ordm so that the adaxial surface of the leaf was 937

parallel with the soil Broken leaf petioles or midribs were considered brittle 938

(B) Quantification of broken midribs or petioles For each genotype 66 to 82 leaves 939

were analyzed for brittleness as described in (A) Bars represent the ratio of broken to 940

total number of leaves tested Results were analyzed using Fisherrsquos exact test Letters 941

indicate significantly different groups at p lt 005 942

(C) Conductivity measurements Leaves of four week-old plants were inoculated with943

deionized water and then leaf discs were cut out and placed on deionized water 944

Conductivity was measured at the indicated time points After 30 min the water was 945

replaced with fresh water (30 min f w) to account for wounding-related ion leakage from 946

the edges of the leaf discs Three independent experiments with six biological replicates 947

each were performed and data were analyzed using a t-test for each time point P-948

values were corrected using the Bonferroni method Data show means plusmn SE asterisks 949

indicate significant differences from wild-type Col-0 at q lt 001 950

951

Figure 4 Cell Walls of gae6 and gae1 gae6 Plants Are Reduced in GalA 952

(A) to (D) Levels of GalA (A) GlcA (B) cellulose (C) and neutral sugars (D) in alcohol953

insoluble residue (AIR) extracted from leaves of four week-old plants of the indicated 954

genotypes Bars represent means plusmn SE of four biological replicates combined using a 955

mixed linear model Letters indicate significantly different groups (p lt 005) 956

957

Figure 5 Cell Walls of gae1 gae6 Plants Are Reduced in Homogalacturonan 958

Page 43 of 54

Dot blot analyses with anti-LM19 and anti-LM20 antibodies Pectin was extracted from 959

AIR prepared from the indicated genotypes using 1 microL of extraction buffer per 10 microg of 960

AIR Pectin was serially diluted and one microL of undiluted 13 diluted and 19 diluted 961

samples were spotted on a nitrocellulose membrane Three biological replicates were 962

were spotted twice to obtain two technical replicates each All replicates produced 963

similar results 964

(A) Representative dot blot using anti-LM19 antibody965

(B) Relative signal intensities of the spots in the dot blot in (A)966

(C) Representative dot blot using anti-LM20 antibody967

(D) Relative signal intensities of the LM20 dot blot shown in (C)968

Relative signal intensities were measured using Image J software Measurements from 969

the three biological replicates were combined using a linear model after Box-Cox power 970

transformation as described in Methods Bars represent means plusmn SE Asterisks indicate 971

a difference from wild-type Col-0 at p lt 005 972

973

Figure 6 Growth of B cinerea on gae1 gae6 Plants and the Effect of B cinerea on 974

Expression of GAE1 and GAE6 975

(A) Growth of B cinerea isolates Leaves of Col-0 and gae1 gae6 plants were976

inoculated with 25 x 105 spores mL-1 of the B cinerea isolates indicated in the figure 977

Samples were collected two (2 dpi) and three (3 dpi) days later Bars represent means plusmn 978

SE of six biological replicates for each fungus combined using a mixed linear model 979

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 980

(B) and (C) Expression of GAE1 (B) and GAE6 (C) 48 h after inoculation with the B981

cinerea isolates indicated Expression levels were measured by qRT-PCR Bars 982

represent mean log2-ratios to ACTIN2 plusmn SE of three biological replicates combined 983

using a mixed linear model Letters indicate significantly different groups (q lt 005) 984

985

Figure 7 Levels of Soluble Pectin and Expression of PAD3 in Fractions Produced 986

Following Macerozyme Treatment of gae1 gae6 Cell Walls 987

(A) Schematic overview of the experimental procedure for (B) and (C)988

Page 44 of 54

(B) Uronic acid measurements Alcohol-insoluble residue (AIR) from leave tissue of four989

week-old wild-type Col-0 or gae1 gae6 plants was treated with water or 025 990

macerozyme and assayed for uronic acid levels Data from 11 biological replicates were 991

combined using a mixed linear model Bars represent means plusmn SE Letters indicate 992

significant differences (p lt 005) 993

(C) Expression of PAD3 in Col-0 plants treated with the fractions from (B) Extracts from994

(B) were heat inactivated pooled and diluted 5-fold with water Pooled extracts were995

inoculated into four week-old Col-0 plants PAD3 expression was measured 3 h after 996

inoculation and normalized to the level of ACTIN2 expression Data from four biological 997

replicates were combined using a mixed linear model Bars represent mean log2-ratios 998

to ACTIN2 plusmn SE Letters indicate significant differences (p lt 005) 999

1000

Figure 8 Macerozyme-Induced PAD3 Expression Is Absent in gae1 gae6 1001

(A) Expression of PAD3 after macerozyme treatment Four week-old plants were either1002

inoculated with 001 macerozyme or mock inoculated with water and PAD3 1003

expression was measured 3 h later by qRT-PCR Data from four biological replicates 1004

were combined using a mixed linear model Mean log2-ratios to ACTIN2 plusmn SE were 1005

plotted Letters indicate significantly different groups (p lt 005) 1006

(B) Expression of PAD3 after OG treatment PAD3 expression was measured as1007

described in (A) 3 h after inoculation with 100 microg mL-1 OGs or mock inoculation with 1008

water Data are from six biological replicates 1009

(C) PAD3 expression in untreated plants Three biological replicates were performed1010

and data analyzed as described in (A) 1011

(D) Camalexin accumulation after B cinerea treatment Camalexin accumulation was1012

measured in four week-old plants inoculated with the B cinerea strains shown in the 1013

figure Eight biological replicates each were combined using a mixed linear model 1014

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 1015

(E) PAD3 expression 28 h after treatment with the indicated B cinerea strains Data1016

from six biological replicates were collected and analyzed as described in (A) Data in 1017

all figure parts represent means plusmn SE Letters indicate significantly different groups (q lt 1018

005) 1019

Page 45 of 54

1020

Figure 9 Macerozyme-Induced Expression of the SA Marker Genes SID2 and PR1 1021

Is Abolished in gae1 gae6 Plants 1022

(A) to (H) Expression of PR1 (A) (C) (E) and (G) or SID2 (B) (D) (F) and (H) in four1023

week-old Col-0 and gae1 gae6 plants Data were combined using a mixed linear model 1024

Bars represent mean log2-ratios of expression vs ACTIN2 plusmn SE for all figure parts 1025

Letters indicate significantly different groups at p lt 005 for panels (A) to (D) (G) and 1026

(H) and qlt005 for panels (E) to (F) 1027

(A) and (B) Plants were treated with 001 macerozyme or mock treated with water1028

and samples were collected 3 h later Data from four biological replicates are shown 1029

(C) and (D) Plants were inoculated with 100 microg mL-1 OGs or mock treated with water1030

and samples collected 3 h later Data from six biological replicates are shown 1031

(E) and (F) Plants were inoculated with the indicated B cinerea isolates or mock1032

inoculated with B cinerea inoculation medium and samples collected 48 h later Data 1033

from eight biological replicates are shown 1034

(G) and (H) Expression in untreated plants Three biological replicates were performed1035

1036

Figure 10 The Expression of the JA Marker Gene JAZ10 is Highly Responsive in 1037

gae1 gae6 Plants 1038

(A) to (D) JAZ10 expression in four week-old plants Data were combined using a mixed1039

linear model Mean log2-ratios to ACTIN2 plusmn SE are shown Letters indicate significantly 1040

different groups at p lt 005 for panels (A) (B) and (D) and q lt 005 for panel (C) 1041

(A) Plants were inoculated with 001 macerozyme or mock inoculated using water and1042

samples were collected 3 h later Data from four biological replicates are shown 1043

(B) Plants were inoculated with 100 microg mL-1 OGs or mock inoculated using water and1044

samples collected 3 h later Data are from six biological replicates 1045

(C) JAZ10 expression 48 h after treatment with the indicated B cinerea strains Data1046

are from six biological replicates 1047

(D) JAZ10 expression in untreated plants Data are from three biological replicates1048

1049

Figure 11 Gallo 1-Induced Callose Deposition Is Reduced in gae1 gae6 Plants 1050

Page 46 of 54

(A) Callose deposition in four week-old Col-0 and gae1 gae6 plants Plants were1051

inoculated with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) or mock inoculated 1052

with B cinerea inoculation medium and samples were collected after 72 h Callose was 1053

stained with aniline blue and visualized using a Nikon Eclipse Ni-U microscope with a 1054

DAPI filter Three leaves were stained per treatment Scale bars are 100 microm Asterisks 1055

indicate fungal hyphae arrows indicate representative callose spots 1056

(B) Quantification of callose deposition Callose deposits were counted in 200 microm x 2001057

microm squares laid out across the images shown here Bars represent means plusmn SE from 1058

six squares each Letters indicate significantly different groups (p lt 005) according to a 1059

t-test analysis1060

1061

Figure 12 Less Soluble Pectin Accumulates in B cinerea-Infected gae1 gae6 1062

Leaves 1063

Four week-old Col-0 and gae1 gae6 plants were inoculated with 10 microL droplets of the 1064

indicated B cinerea isolate (25 x 105 spores mL-1) or mock inoculated with B cinerea 1065

inoculation medium Leaves were collected 48 h later AIR was extracted from leaf 1066

samples and water added (20 microL infection site) to extract soluble pectin fragments 1067

Samples were vortexed for 7 h centrifuged at 12000 g and the uronic acid 1068

concentration of the supernatant was measured Data from nine biological replicates 1069

were combined using a mixed linear model Means plusmn SE are shown Letters indicate 1070

significant differences (q lt 005) 1071

1072

Figure 13 Macerozyme-Induced PTI Is Lost in gae1 gae6 While OG-Induced PTI Is 1073

Unaltered 1074

(A) Macerozyme-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1075

inoculated with 001 macerozyme or mock inoculated with boiled macerozyme and 1076

were infected with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) 24 h later Samples 1077

were collected immediately (0 dpi) and after two (2 dpi) and three (3 dpi) days Data 1078

show mean plusmn SE of six to 14 biological replicates each combined using a linear model 1079

Letters indicate significantly different groups (q lt 005) 1080

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

Parsed CitationsAdam L and Somerville SC (1996) Genetic characterization of five powdery mildew disease resistance loci in Arabidopsisthaliana Plant J 9 341-356

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Albersheim P Jones TM and English PD (1969) Biochemistry of the cell wall in relation to infective processes Annu RevPhytopathol 7 171-194

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk RGadrinab C Heller C Jeske A Koesema E Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt MHazari N Hom E Karnes M Mulholland C Ndubaku R Schmidt I Guzman P Aguilar-Henonin L Schmid M Weigel DCarter DE Marchand T Risseeuw E Brogden D Zeko A Crosby WL Berry CC and Ecker JR (2003) Genome-wideinsertional mutagenesis of Arabidopsis thaliana Science 301 653-657

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Asai T Tena G Plotnikova J Willmann MR Chiu WL Gomez-Gomez L Boller T Ausubel FM and Sheen J (2002) MAPkinase signalling cascade in Arabidopsis innate immunity Nature 415 977-983

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bartsch M Gobbato E Bednarek P Debey S Schultze JL Bautor J and Parker JE (2006) Salicylic acid-independentENHANCED DISEASE SUSCEPTIBILITY1 signaling in Arabidopsis immunity and cell death is regulated by the monooxygenaseFMO1 and the nudix hydrolase NUDT7 Plant Cell 18 1038-1051

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bates D Machler M Bolker BM and Walker SC (2015) Fitting Linear Mixed-Effects Models Using lme4 J Stat Softw 67 1-48Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Benjamini Y and Hochberg Y (1995) Controlling the False Discovery Rate - a Practical and Powerful Approach to MultipleTesting J Roy Stat Soc B Met 57 289-300

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bethke G Grundman RE Sreekanta S Truman W Katagiri F and Glazebrook J (2014) Arabidopsis PECTINMETHYLESTERASEs Contribute to Immunity against Pseudomonas syringae Plant Physiol 164 1093-1107

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bouton S Leboeuf E Mouille G Leydecker MT Talbotec J Granier F Lahaye M Hofte H and Truong HN (2002)Quasimodo1 encodes a putative membrane-bound glycosyltransferase required for normal pectin synthesis and cell adhesion inArabidopsis Plant Cell 14 2577-2590

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Box GEP and Cox DR (1964) An analysis of transformations Journal of the Royal Statistical Society Series B 26 211-252Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Cao H Glazebrook J Clarke JD Volko S and Dong XN (1997) The Arabidopsis NPR1 gene that controls systemic acquiredresistance encodes a novel protein containing ankyrin repeats Cell 88 57-63

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Carpita N (2010) Update on Mechanisms of Plant Cell Wall Biosynthesis How plants make cellulose and other (1-gt4)-beta-D-glycans Plant Physiol

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Chisholm ST Coaker G Day B and Staskawicz BJ (2006) Host-microbe interactions Shaping the evolution of the plantimmune response Cell 124 803-814

Page 40 of 54

Supplemental Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 867

Expression Does Not Require CBP60s or SID2 868

Supplemental Figure 2 Expression of GAE Family Members uponTreatment with Pma 869

ES4326 870

Supplemental Figure 3 Characterization of T-DNA Insertions in GAE1 and GAE6 871

Supplemental Figure 4 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves and 872

Are Slightly Smaller than Wild-Type Col-0 Plants 873

Supplemental Figure 5 Cell Walls of gae1 gae6 Plants Contain Less Uronic Acid than 874

Wild-Type Col-0 Cell Walls 875

Supplemental Figure 6 Antibodies Raised Against the RGI or XG Components of 876

Pectin Show No Difference in Binding to gae Mutant and Col-0 Cell Walls 877

Supplemental Figure 7 The Effect of B cinerea Treatment on the Expression of GAE 878

Family Genes 879

Supplemental Figure 8 Treatment with 001 Macerozyme Does Not Cause Any 880

Visible Damage to Col-0 Plants 881

Supplemental Figure 9 B cinerea Growth on Plants with Defects in Immune 882

Signaling 883

Supplemental Figure 10 MeJA-Induced Expression of the JA marker gene JAZ10 884

Supplemental Figure 11 The Expression of the JA Marker Gene JAZ5 Is Highly 885

Responsive in gae1 gae6 Plants 886

Supplemental Figure 12 The Expression of PDF12 Is Unaltered in gae1 gae6 Plants 887

Supplemental Figure 13 Macerozyme Treatment Activates MPK3 and MPK6 888

Supplemental Figure 14 B cinerea-Induced ROS Production in Wild-Type Col-0 and 889

gae1 gae6 Plants 890

Supplemental Figure 15 OG-Induced PTI Using 250 microg mL-1 OGs 891

Supplemental Figure 16 Abundance of Plant DNA in B cinerea Treated Plants 892

Supplemental Table 1 Primers used in this study 893

894

ACKNOWLEDGEMENTS 895

We thank William Truman for helpful discussions Simone Ferrari (Sapienza 896

Universita di Roma) for the generous gift of OGs Bruna Bucciarelli (USDA-ARS) for 897

Page 41 of 54

help with fluorescence microscopy the Arabidopsis Biological Resource Center for T-898

DNA insertion lines Paul Knoxrsquos lab (University of Leeds) for LM5 LM6 LM8 LM19 899

and LM20 antibodies and the Complex Carbohydrate Resource Center (University of 900

Georgia) for the CCRC-M7 antibody Most of this work was funded by the Division of 901

Chemical Sciences Geosciences and Biosciences Office of Basic Energy Sciences of 902

the US Department of Energy through Grant DE-FG02-05ER15670 to JG This work 903

was also supported by NSF awards IOS 1339125 and IOS 1021861 to DJK NSF 904

award IOS 1121425 to F K and the USDA National Institute of Food and Agriculture 905

Hatch project number CA-D-PLS-7033-H to DJK 906

907

AUTHOR CONTRIBUTIONS 908

GB and JG designed the research GB GX AT BL NES and NH 909

performed research DJK provided tools GB GX MP AD BL RAH and FK 910

analyzed data GB and JG wrote the paper with input from all authors 911

912

FIGURE LEGENDS 913

Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 Expression 914

Requires PAD4 EDS1 and PBS3 915

(A) Expression of GAE1 in leaves of wild-type Col-0 and mutant plants Expression was916

measured 24 or 32 h after either inoculation with Pma ES4326 (OD600 = 0002) or mock 917

inoculation with 5 mM MgSO4 and was then normalized to the level of ACTIN2 Data 918

from three biological replicates were combined using a mixed linear model Bars 919

represent mean log2-ratios to ACTIN2 plusmn SE Letters indicate significantly different 920

groups with q lt 005 where 005 represents the maximum false discovery rate at which 921

the test may be called significant 922

(B) Expression of GAE6 measured as described in (A)923

924

Figure 2 Susceptibility of gae6 and gae1 gae6 Plants to Pma ES4326 Is Increased 925

Relative to Wild-Type Col-0 926

Leaves of plants of the indicated genotypes were inoculated with Pma ES4326 (OD600 = 927

00002) as described in Methods Bacterial titers in leaves were determined either 928

Page 42 of 54

immediately (0 dpi) or after three days (3 dpi) Bars represent the mean plusmn SE of four 929

independent experiments each with 4 or 12 biological replicates at 0 and 3 dpi 930

respectively Data were combined using a mixed linear model Asterisks indicate 931

significant differences from Col-0 wild-type (q lt 005) Susceptible pad4 plants were 932

included as a positive control 933

934

Figure 3 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves 935

(A) Depiction of the assay for brittleness of gae mutant leaves Fully expanded leaves of936

four to five week-old plants were bent by 180ordm so that the adaxial surface of the leaf was 937

parallel with the soil Broken leaf petioles or midribs were considered brittle 938

(B) Quantification of broken midribs or petioles For each genotype 66 to 82 leaves 939

were analyzed for brittleness as described in (A) Bars represent the ratio of broken to 940

total number of leaves tested Results were analyzed using Fisherrsquos exact test Letters 941

indicate significantly different groups at p lt 005 942

(C) Conductivity measurements Leaves of four week-old plants were inoculated with943

deionized water and then leaf discs were cut out and placed on deionized water 944

Conductivity was measured at the indicated time points After 30 min the water was 945

replaced with fresh water (30 min f w) to account for wounding-related ion leakage from 946

the edges of the leaf discs Three independent experiments with six biological replicates 947

each were performed and data were analyzed using a t-test for each time point P-948

values were corrected using the Bonferroni method Data show means plusmn SE asterisks 949

indicate significant differences from wild-type Col-0 at q lt 001 950

951

Figure 4 Cell Walls of gae6 and gae1 gae6 Plants Are Reduced in GalA 952

(A) to (D) Levels of GalA (A) GlcA (B) cellulose (C) and neutral sugars (D) in alcohol953

insoluble residue (AIR) extracted from leaves of four week-old plants of the indicated 954

genotypes Bars represent means plusmn SE of four biological replicates combined using a 955

mixed linear model Letters indicate significantly different groups (p lt 005) 956

957

Figure 5 Cell Walls of gae1 gae6 Plants Are Reduced in Homogalacturonan 958

Page 43 of 54

Dot blot analyses with anti-LM19 and anti-LM20 antibodies Pectin was extracted from 959

AIR prepared from the indicated genotypes using 1 microL of extraction buffer per 10 microg of 960

AIR Pectin was serially diluted and one microL of undiluted 13 diluted and 19 diluted 961

samples were spotted on a nitrocellulose membrane Three biological replicates were 962

were spotted twice to obtain two technical replicates each All replicates produced 963

similar results 964

(A) Representative dot blot using anti-LM19 antibody965

(B) Relative signal intensities of the spots in the dot blot in (A)966

(C) Representative dot blot using anti-LM20 antibody967

(D) Relative signal intensities of the LM20 dot blot shown in (C)968

Relative signal intensities were measured using Image J software Measurements from 969

the three biological replicates were combined using a linear model after Box-Cox power 970

transformation as described in Methods Bars represent means plusmn SE Asterisks indicate 971

a difference from wild-type Col-0 at p lt 005 972

973

Figure 6 Growth of B cinerea on gae1 gae6 Plants and the Effect of B cinerea on 974

Expression of GAE1 and GAE6 975

(A) Growth of B cinerea isolates Leaves of Col-0 and gae1 gae6 plants were976

inoculated with 25 x 105 spores mL-1 of the B cinerea isolates indicated in the figure 977

Samples were collected two (2 dpi) and three (3 dpi) days later Bars represent means plusmn 978

SE of six biological replicates for each fungus combined using a mixed linear model 979

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 980

(B) and (C) Expression of GAE1 (B) and GAE6 (C) 48 h after inoculation with the B981

cinerea isolates indicated Expression levels were measured by qRT-PCR Bars 982

represent mean log2-ratios to ACTIN2 plusmn SE of three biological replicates combined 983

using a mixed linear model Letters indicate significantly different groups (q lt 005) 984

985

Figure 7 Levels of Soluble Pectin and Expression of PAD3 in Fractions Produced 986

Following Macerozyme Treatment of gae1 gae6 Cell Walls 987

(A) Schematic overview of the experimental procedure for (B) and (C)988

Page 44 of 54

(B) Uronic acid measurements Alcohol-insoluble residue (AIR) from leave tissue of four989

week-old wild-type Col-0 or gae1 gae6 plants was treated with water or 025 990

macerozyme and assayed for uronic acid levels Data from 11 biological replicates were 991

combined using a mixed linear model Bars represent means plusmn SE Letters indicate 992

significant differences (p lt 005) 993

(C) Expression of PAD3 in Col-0 plants treated with the fractions from (B) Extracts from994

(B) were heat inactivated pooled and diluted 5-fold with water Pooled extracts were995

inoculated into four week-old Col-0 plants PAD3 expression was measured 3 h after 996

inoculation and normalized to the level of ACTIN2 expression Data from four biological 997

replicates were combined using a mixed linear model Bars represent mean log2-ratios 998

to ACTIN2 plusmn SE Letters indicate significant differences (p lt 005) 999

1000

Figure 8 Macerozyme-Induced PAD3 Expression Is Absent in gae1 gae6 1001

(A) Expression of PAD3 after macerozyme treatment Four week-old plants were either1002

inoculated with 001 macerozyme or mock inoculated with water and PAD3 1003

expression was measured 3 h later by qRT-PCR Data from four biological replicates 1004

were combined using a mixed linear model Mean log2-ratios to ACTIN2 plusmn SE were 1005

plotted Letters indicate significantly different groups (p lt 005) 1006

(B) Expression of PAD3 after OG treatment PAD3 expression was measured as1007

described in (A) 3 h after inoculation with 100 microg mL-1 OGs or mock inoculation with 1008

water Data are from six biological replicates 1009

(C) PAD3 expression in untreated plants Three biological replicates were performed1010

and data analyzed as described in (A) 1011

(D) Camalexin accumulation after B cinerea treatment Camalexin accumulation was1012

measured in four week-old plants inoculated with the B cinerea strains shown in the 1013

figure Eight biological replicates each were combined using a mixed linear model 1014

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 1015

(E) PAD3 expression 28 h after treatment with the indicated B cinerea strains Data1016

from six biological replicates were collected and analyzed as described in (A) Data in 1017

all figure parts represent means plusmn SE Letters indicate significantly different groups (q lt 1018

005) 1019

Page 45 of 54

1020

Figure 9 Macerozyme-Induced Expression of the SA Marker Genes SID2 and PR1 1021

Is Abolished in gae1 gae6 Plants 1022

(A) to (H) Expression of PR1 (A) (C) (E) and (G) or SID2 (B) (D) (F) and (H) in four1023

week-old Col-0 and gae1 gae6 plants Data were combined using a mixed linear model 1024

Bars represent mean log2-ratios of expression vs ACTIN2 plusmn SE for all figure parts 1025

Letters indicate significantly different groups at p lt 005 for panels (A) to (D) (G) and 1026

(H) and qlt005 for panels (E) to (F) 1027

(A) and (B) Plants were treated with 001 macerozyme or mock treated with water1028

and samples were collected 3 h later Data from four biological replicates are shown 1029

(C) and (D) Plants were inoculated with 100 microg mL-1 OGs or mock treated with water1030

and samples collected 3 h later Data from six biological replicates are shown 1031

(E) and (F) Plants were inoculated with the indicated B cinerea isolates or mock1032

inoculated with B cinerea inoculation medium and samples collected 48 h later Data 1033

from eight biological replicates are shown 1034

(G) and (H) Expression in untreated plants Three biological replicates were performed1035

1036

Figure 10 The Expression of the JA Marker Gene JAZ10 is Highly Responsive in 1037

gae1 gae6 Plants 1038

(A) to (D) JAZ10 expression in four week-old plants Data were combined using a mixed1039

linear model Mean log2-ratios to ACTIN2 plusmn SE are shown Letters indicate significantly 1040

different groups at p lt 005 for panels (A) (B) and (D) and q lt 005 for panel (C) 1041

(A) Plants were inoculated with 001 macerozyme or mock inoculated using water and1042

samples were collected 3 h later Data from four biological replicates are shown 1043

(B) Plants were inoculated with 100 microg mL-1 OGs or mock inoculated using water and1044

samples collected 3 h later Data are from six biological replicates 1045

(C) JAZ10 expression 48 h after treatment with the indicated B cinerea strains Data1046

are from six biological replicates 1047

(D) JAZ10 expression in untreated plants Data are from three biological replicates1048

1049

Figure 11 Gallo 1-Induced Callose Deposition Is Reduced in gae1 gae6 Plants 1050

Page 46 of 54

(A) Callose deposition in four week-old Col-0 and gae1 gae6 plants Plants were1051

inoculated with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) or mock inoculated 1052

with B cinerea inoculation medium and samples were collected after 72 h Callose was 1053

stained with aniline blue and visualized using a Nikon Eclipse Ni-U microscope with a 1054

DAPI filter Three leaves were stained per treatment Scale bars are 100 microm Asterisks 1055

indicate fungal hyphae arrows indicate representative callose spots 1056

(B) Quantification of callose deposition Callose deposits were counted in 200 microm x 2001057

microm squares laid out across the images shown here Bars represent means plusmn SE from 1058

six squares each Letters indicate significantly different groups (p lt 005) according to a 1059

t-test analysis1060

1061

Figure 12 Less Soluble Pectin Accumulates in B cinerea-Infected gae1 gae6 1062

Leaves 1063

Four week-old Col-0 and gae1 gae6 plants were inoculated with 10 microL droplets of the 1064

indicated B cinerea isolate (25 x 105 spores mL-1) or mock inoculated with B cinerea 1065

inoculation medium Leaves were collected 48 h later AIR was extracted from leaf 1066

samples and water added (20 microL infection site) to extract soluble pectin fragments 1067

Samples were vortexed for 7 h centrifuged at 12000 g and the uronic acid 1068

concentration of the supernatant was measured Data from nine biological replicates 1069

were combined using a mixed linear model Means plusmn SE are shown Letters indicate 1070

significant differences (q lt 005) 1071

1072

Figure 13 Macerozyme-Induced PTI Is Lost in gae1 gae6 While OG-Induced PTI Is 1073

Unaltered 1074

(A) Macerozyme-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1075

inoculated with 001 macerozyme or mock inoculated with boiled macerozyme and 1076

were infected with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) 24 h later Samples 1077

were collected immediately (0 dpi) and after two (2 dpi) and three (3 dpi) days Data 1078

show mean plusmn SE of six to 14 biological replicates each combined using a linear model 1079

Letters indicate significantly different groups (q lt 005) 1080

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

Parsed CitationsAdam L and Somerville SC (1996) Genetic characterization of five powdery mildew disease resistance loci in Arabidopsisthaliana Plant J 9 341-356

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Albersheim P Jones TM and English PD (1969) Biochemistry of the cell wall in relation to infective processes Annu RevPhytopathol 7 171-194

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk RGadrinab C Heller C Jeske A Koesema E Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt MHazari N Hom E Karnes M Mulholland C Ndubaku R Schmidt I Guzman P Aguilar-Henonin L Schmid M Weigel DCarter DE Marchand T Risseeuw E Brogden D Zeko A Crosby WL Berry CC and Ecker JR (2003) Genome-wideinsertional mutagenesis of Arabidopsis thaliana Science 301 653-657

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Asai T Tena G Plotnikova J Willmann MR Chiu WL Gomez-Gomez L Boller T Ausubel FM and Sheen J (2002) MAPkinase signalling cascade in Arabidopsis innate immunity Nature 415 977-983

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bartsch M Gobbato E Bednarek P Debey S Schultze JL Bautor J and Parker JE (2006) Salicylic acid-independentENHANCED DISEASE SUSCEPTIBILITY1 signaling in Arabidopsis immunity and cell death is regulated by the monooxygenaseFMO1 and the nudix hydrolase NUDT7 Plant Cell 18 1038-1051

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bates D Machler M Bolker BM and Walker SC (2015) Fitting Linear Mixed-Effects Models Using lme4 J Stat Softw 67 1-48Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Benjamini Y and Hochberg Y (1995) Controlling the False Discovery Rate - a Practical and Powerful Approach to MultipleTesting J Roy Stat Soc B Met 57 289-300

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bethke G Grundman RE Sreekanta S Truman W Katagiri F and Glazebrook J (2014) Arabidopsis PECTINMETHYLESTERASEs Contribute to Immunity against Pseudomonas syringae Plant Physiol 164 1093-1107

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bouton S Leboeuf E Mouille G Leydecker MT Talbotec J Granier F Lahaye M Hofte H and Truong HN (2002)Quasimodo1 encodes a putative membrane-bound glycosyltransferase required for normal pectin synthesis and cell adhesion inArabidopsis Plant Cell 14 2577-2590

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Box GEP and Cox DR (1964) An analysis of transformations Journal of the Royal Statistical Society Series B 26 211-252Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Cao H Glazebrook J Clarke JD Volko S and Dong XN (1997) The Arabidopsis NPR1 gene that controls systemic acquiredresistance encodes a novel protein containing ankyrin repeats Cell 88 57-63

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Carpita N (2010) Update on Mechanisms of Plant Cell Wall Biosynthesis How plants make cellulose and other (1-gt4)-beta-D-glycans Plant Physiol

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Chisholm ST Coaker G Day B and Staskawicz BJ (2006) Host-microbe interactions Shaping the evolution of the plantimmune response Cell 124 803-814

Page 41 of 54

help with fluorescence microscopy the Arabidopsis Biological Resource Center for T-898

DNA insertion lines Paul Knoxrsquos lab (University of Leeds) for LM5 LM6 LM8 LM19 899

and LM20 antibodies and the Complex Carbohydrate Resource Center (University of 900

Georgia) for the CCRC-M7 antibody Most of this work was funded by the Division of 901

Chemical Sciences Geosciences and Biosciences Office of Basic Energy Sciences of 902

the US Department of Energy through Grant DE-FG02-05ER15670 to JG This work 903

was also supported by NSF awards IOS 1339125 and IOS 1021861 to DJK NSF 904

award IOS 1121425 to F K and the USDA National Institute of Food and Agriculture 905

Hatch project number CA-D-PLS-7033-H to DJK 906

907

AUTHOR CONTRIBUTIONS 908

GB and JG designed the research GB GX AT BL NES and NH 909

performed research DJK provided tools GB GX MP AD BL RAH and FK 910

analyzed data GB and JG wrote the paper with input from all authors 911

912

FIGURE LEGENDS 913

Figure 1 Pma ES4326-Induced Repression of GAE1 and GAE6 Expression 914

Requires PAD4 EDS1 and PBS3 915

(A) Expression of GAE1 in leaves of wild-type Col-0 and mutant plants Expression was916

measured 24 or 32 h after either inoculation with Pma ES4326 (OD600 = 0002) or mock 917

inoculation with 5 mM MgSO4 and was then normalized to the level of ACTIN2 Data 918

from three biological replicates were combined using a mixed linear model Bars 919

represent mean log2-ratios to ACTIN2 plusmn SE Letters indicate significantly different 920

groups with q lt 005 where 005 represents the maximum false discovery rate at which 921

the test may be called significant 922

(B) Expression of GAE6 measured as described in (A)923

924

Figure 2 Susceptibility of gae6 and gae1 gae6 Plants to Pma ES4326 Is Increased 925

Relative to Wild-Type Col-0 926

Leaves of plants of the indicated genotypes were inoculated with Pma ES4326 (OD600 = 927

00002) as described in Methods Bacterial titers in leaves were determined either 928

Page 42 of 54

immediately (0 dpi) or after three days (3 dpi) Bars represent the mean plusmn SE of four 929

independent experiments each with 4 or 12 biological replicates at 0 and 3 dpi 930

respectively Data were combined using a mixed linear model Asterisks indicate 931

significant differences from Col-0 wild-type (q lt 005) Susceptible pad4 plants were 932

included as a positive control 933

934

Figure 3 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves 935

(A) Depiction of the assay for brittleness of gae mutant leaves Fully expanded leaves of936

four to five week-old plants were bent by 180ordm so that the adaxial surface of the leaf was 937

parallel with the soil Broken leaf petioles or midribs were considered brittle 938

(B) Quantification of broken midribs or petioles For each genotype 66 to 82 leaves 939

were analyzed for brittleness as described in (A) Bars represent the ratio of broken to 940

total number of leaves tested Results were analyzed using Fisherrsquos exact test Letters 941

indicate significantly different groups at p lt 005 942

(C) Conductivity measurements Leaves of four week-old plants were inoculated with943

deionized water and then leaf discs were cut out and placed on deionized water 944

Conductivity was measured at the indicated time points After 30 min the water was 945

replaced with fresh water (30 min f w) to account for wounding-related ion leakage from 946

the edges of the leaf discs Three independent experiments with six biological replicates 947

each were performed and data were analyzed using a t-test for each time point P-948

values were corrected using the Bonferroni method Data show means plusmn SE asterisks 949

indicate significant differences from wild-type Col-0 at q lt 001 950

951

Figure 4 Cell Walls of gae6 and gae1 gae6 Plants Are Reduced in GalA 952

(A) to (D) Levels of GalA (A) GlcA (B) cellulose (C) and neutral sugars (D) in alcohol953

insoluble residue (AIR) extracted from leaves of four week-old plants of the indicated 954

genotypes Bars represent means plusmn SE of four biological replicates combined using a 955

mixed linear model Letters indicate significantly different groups (p lt 005) 956

957

Figure 5 Cell Walls of gae1 gae6 Plants Are Reduced in Homogalacturonan 958

Page 43 of 54

Dot blot analyses with anti-LM19 and anti-LM20 antibodies Pectin was extracted from 959

AIR prepared from the indicated genotypes using 1 microL of extraction buffer per 10 microg of 960

AIR Pectin was serially diluted and one microL of undiluted 13 diluted and 19 diluted 961

samples were spotted on a nitrocellulose membrane Three biological replicates were 962

were spotted twice to obtain two technical replicates each All replicates produced 963

similar results 964

(A) Representative dot blot using anti-LM19 antibody965

(B) Relative signal intensities of the spots in the dot blot in (A)966

(C) Representative dot blot using anti-LM20 antibody967

(D) Relative signal intensities of the LM20 dot blot shown in (C)968

Relative signal intensities were measured using Image J software Measurements from 969

the three biological replicates were combined using a linear model after Box-Cox power 970

transformation as described in Methods Bars represent means plusmn SE Asterisks indicate 971

a difference from wild-type Col-0 at p lt 005 972

973

Figure 6 Growth of B cinerea on gae1 gae6 Plants and the Effect of B cinerea on 974

Expression of GAE1 and GAE6 975

(A) Growth of B cinerea isolates Leaves of Col-0 and gae1 gae6 plants were976

inoculated with 25 x 105 spores mL-1 of the B cinerea isolates indicated in the figure 977

Samples were collected two (2 dpi) and three (3 dpi) days later Bars represent means plusmn 978

SE of six biological replicates for each fungus combined using a mixed linear model 979

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 980

(B) and (C) Expression of GAE1 (B) and GAE6 (C) 48 h after inoculation with the B981

cinerea isolates indicated Expression levels were measured by qRT-PCR Bars 982

represent mean log2-ratios to ACTIN2 plusmn SE of three biological replicates combined 983

using a mixed linear model Letters indicate significantly different groups (q lt 005) 984

985

Figure 7 Levels of Soluble Pectin and Expression of PAD3 in Fractions Produced 986

Following Macerozyme Treatment of gae1 gae6 Cell Walls 987

(A) Schematic overview of the experimental procedure for (B) and (C)988

Page 44 of 54

(B) Uronic acid measurements Alcohol-insoluble residue (AIR) from leave tissue of four989

week-old wild-type Col-0 or gae1 gae6 plants was treated with water or 025 990

macerozyme and assayed for uronic acid levels Data from 11 biological replicates were 991

combined using a mixed linear model Bars represent means plusmn SE Letters indicate 992

significant differences (p lt 005) 993

(C) Expression of PAD3 in Col-0 plants treated with the fractions from (B) Extracts from994

(B) were heat inactivated pooled and diluted 5-fold with water Pooled extracts were995

inoculated into four week-old Col-0 plants PAD3 expression was measured 3 h after 996

inoculation and normalized to the level of ACTIN2 expression Data from four biological 997

replicates were combined using a mixed linear model Bars represent mean log2-ratios 998

to ACTIN2 plusmn SE Letters indicate significant differences (p lt 005) 999

1000

Figure 8 Macerozyme-Induced PAD3 Expression Is Absent in gae1 gae6 1001

(A) Expression of PAD3 after macerozyme treatment Four week-old plants were either1002

inoculated with 001 macerozyme or mock inoculated with water and PAD3 1003

expression was measured 3 h later by qRT-PCR Data from four biological replicates 1004

were combined using a mixed linear model Mean log2-ratios to ACTIN2 plusmn SE were 1005

plotted Letters indicate significantly different groups (p lt 005) 1006

(B) Expression of PAD3 after OG treatment PAD3 expression was measured as1007

described in (A) 3 h after inoculation with 100 microg mL-1 OGs or mock inoculation with 1008

water Data are from six biological replicates 1009

(C) PAD3 expression in untreated plants Three biological replicates were performed1010

and data analyzed as described in (A) 1011

(D) Camalexin accumulation after B cinerea treatment Camalexin accumulation was1012

measured in four week-old plants inoculated with the B cinerea strains shown in the 1013

figure Eight biological replicates each were combined using a mixed linear model 1014

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 1015

(E) PAD3 expression 28 h after treatment with the indicated B cinerea strains Data1016

from six biological replicates were collected and analyzed as described in (A) Data in 1017

all figure parts represent means plusmn SE Letters indicate significantly different groups (q lt 1018

005) 1019

Page 45 of 54

1020

Figure 9 Macerozyme-Induced Expression of the SA Marker Genes SID2 and PR1 1021

Is Abolished in gae1 gae6 Plants 1022

(A) to (H) Expression of PR1 (A) (C) (E) and (G) or SID2 (B) (D) (F) and (H) in four1023

week-old Col-0 and gae1 gae6 plants Data were combined using a mixed linear model 1024

Bars represent mean log2-ratios of expression vs ACTIN2 plusmn SE for all figure parts 1025

Letters indicate significantly different groups at p lt 005 for panels (A) to (D) (G) and 1026

(H) and qlt005 for panels (E) to (F) 1027

(A) and (B) Plants were treated with 001 macerozyme or mock treated with water1028

and samples were collected 3 h later Data from four biological replicates are shown 1029

(C) and (D) Plants were inoculated with 100 microg mL-1 OGs or mock treated with water1030

and samples collected 3 h later Data from six biological replicates are shown 1031

(E) and (F) Plants were inoculated with the indicated B cinerea isolates or mock1032

inoculated with B cinerea inoculation medium and samples collected 48 h later Data 1033

from eight biological replicates are shown 1034

(G) and (H) Expression in untreated plants Three biological replicates were performed1035

1036

Figure 10 The Expression of the JA Marker Gene JAZ10 is Highly Responsive in 1037

gae1 gae6 Plants 1038

(A) to (D) JAZ10 expression in four week-old plants Data were combined using a mixed1039

linear model Mean log2-ratios to ACTIN2 plusmn SE are shown Letters indicate significantly 1040

different groups at p lt 005 for panels (A) (B) and (D) and q lt 005 for panel (C) 1041

(A) Plants were inoculated with 001 macerozyme or mock inoculated using water and1042

samples were collected 3 h later Data from four biological replicates are shown 1043

(B) Plants were inoculated with 100 microg mL-1 OGs or mock inoculated using water and1044

samples collected 3 h later Data are from six biological replicates 1045

(C) JAZ10 expression 48 h after treatment with the indicated B cinerea strains Data1046

are from six biological replicates 1047

(D) JAZ10 expression in untreated plants Data are from three biological replicates1048

1049

Figure 11 Gallo 1-Induced Callose Deposition Is Reduced in gae1 gae6 Plants 1050

Page 46 of 54

(A) Callose deposition in four week-old Col-0 and gae1 gae6 plants Plants were1051

inoculated with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) or mock inoculated 1052

with B cinerea inoculation medium and samples were collected after 72 h Callose was 1053

stained with aniline blue and visualized using a Nikon Eclipse Ni-U microscope with a 1054

DAPI filter Three leaves were stained per treatment Scale bars are 100 microm Asterisks 1055

indicate fungal hyphae arrows indicate representative callose spots 1056

(B) Quantification of callose deposition Callose deposits were counted in 200 microm x 2001057

microm squares laid out across the images shown here Bars represent means plusmn SE from 1058

six squares each Letters indicate significantly different groups (p lt 005) according to a 1059

t-test analysis1060

1061

Figure 12 Less Soluble Pectin Accumulates in B cinerea-Infected gae1 gae6 1062

Leaves 1063

Four week-old Col-0 and gae1 gae6 plants were inoculated with 10 microL droplets of the 1064

indicated B cinerea isolate (25 x 105 spores mL-1) or mock inoculated with B cinerea 1065

inoculation medium Leaves were collected 48 h later AIR was extracted from leaf 1066

samples and water added (20 microL infection site) to extract soluble pectin fragments 1067

Samples were vortexed for 7 h centrifuged at 12000 g and the uronic acid 1068

concentration of the supernatant was measured Data from nine biological replicates 1069

were combined using a mixed linear model Means plusmn SE are shown Letters indicate 1070

significant differences (q lt 005) 1071

1072

Figure 13 Macerozyme-Induced PTI Is Lost in gae1 gae6 While OG-Induced PTI Is 1073

Unaltered 1074

(A) Macerozyme-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1075

inoculated with 001 macerozyme or mock inoculated with boiled macerozyme and 1076

were infected with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) 24 h later Samples 1077

were collected immediately (0 dpi) and after two (2 dpi) and three (3 dpi) days Data 1078

show mean plusmn SE of six to 14 biological replicates each combined using a linear model 1079

Letters indicate significantly different groups (q lt 005) 1080

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

Parsed CitationsAdam L and Somerville SC (1996) Genetic characterization of five powdery mildew disease resistance loci in Arabidopsisthaliana Plant J 9 341-356

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Albersheim P Jones TM and English PD (1969) Biochemistry of the cell wall in relation to infective processes Annu RevPhytopathol 7 171-194

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk RGadrinab C Heller C Jeske A Koesema E Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt MHazari N Hom E Karnes M Mulholland C Ndubaku R Schmidt I Guzman P Aguilar-Henonin L Schmid M Weigel DCarter DE Marchand T Risseeuw E Brogden D Zeko A Crosby WL Berry CC and Ecker JR (2003) Genome-wideinsertional mutagenesis of Arabidopsis thaliana Science 301 653-657

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Asai T Tena G Plotnikova J Willmann MR Chiu WL Gomez-Gomez L Boller T Ausubel FM and Sheen J (2002) MAPkinase signalling cascade in Arabidopsis innate immunity Nature 415 977-983

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bartsch M Gobbato E Bednarek P Debey S Schultze JL Bautor J and Parker JE (2006) Salicylic acid-independentENHANCED DISEASE SUSCEPTIBILITY1 signaling in Arabidopsis immunity and cell death is regulated by the monooxygenaseFMO1 and the nudix hydrolase NUDT7 Plant Cell 18 1038-1051

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bates D Machler M Bolker BM and Walker SC (2015) Fitting Linear Mixed-Effects Models Using lme4 J Stat Softw 67 1-48Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Benjamini Y and Hochberg Y (1995) Controlling the False Discovery Rate - a Practical and Powerful Approach to MultipleTesting J Roy Stat Soc B Met 57 289-300

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bethke G Grundman RE Sreekanta S Truman W Katagiri F and Glazebrook J (2014) Arabidopsis PECTINMETHYLESTERASEs Contribute to Immunity against Pseudomonas syringae Plant Physiol 164 1093-1107

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bouton S Leboeuf E Mouille G Leydecker MT Talbotec J Granier F Lahaye M Hofte H and Truong HN (2002)Quasimodo1 encodes a putative membrane-bound glycosyltransferase required for normal pectin synthesis and cell adhesion inArabidopsis Plant Cell 14 2577-2590

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Box GEP and Cox DR (1964) An analysis of transformations Journal of the Royal Statistical Society Series B 26 211-252Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Cao H Glazebrook J Clarke JD Volko S and Dong XN (1997) The Arabidopsis NPR1 gene that controls systemic acquiredresistance encodes a novel protein containing ankyrin repeats Cell 88 57-63

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Carpita N (2010) Update on Mechanisms of Plant Cell Wall Biosynthesis How plants make cellulose and other (1-gt4)-beta-D-glycans Plant Physiol

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Chisholm ST Coaker G Day B and Staskawicz BJ (2006) Host-microbe interactions Shaping the evolution of the plantimmune response Cell 124 803-814

Page 42 of 54

immediately (0 dpi) or after three days (3 dpi) Bars represent the mean plusmn SE of four 929

independent experiments each with 4 or 12 biological replicates at 0 and 3 dpi 930

respectively Data were combined using a mixed linear model Asterisks indicate 931

significant differences from Col-0 wild-type (q lt 005) Susceptible pad4 plants were 932

included as a positive control 933

934

Figure 3 Mutant gae6 and gae1 gae6 Plants Have Brittle Leaves 935

(A) Depiction of the assay for brittleness of gae mutant leaves Fully expanded leaves of936

four to five week-old plants were bent by 180ordm so that the adaxial surface of the leaf was 937

parallel with the soil Broken leaf petioles or midribs were considered brittle 938

(B) Quantification of broken midribs or petioles For each genotype 66 to 82 leaves 939

were analyzed for brittleness as described in (A) Bars represent the ratio of broken to 940

total number of leaves tested Results were analyzed using Fisherrsquos exact test Letters 941

indicate significantly different groups at p lt 005 942

(C) Conductivity measurements Leaves of four week-old plants were inoculated with943

deionized water and then leaf discs were cut out and placed on deionized water 944

Conductivity was measured at the indicated time points After 30 min the water was 945

replaced with fresh water (30 min f w) to account for wounding-related ion leakage from 946

the edges of the leaf discs Three independent experiments with six biological replicates 947

each were performed and data were analyzed using a t-test for each time point P-948

values were corrected using the Bonferroni method Data show means plusmn SE asterisks 949

indicate significant differences from wild-type Col-0 at q lt 001 950

951

Figure 4 Cell Walls of gae6 and gae1 gae6 Plants Are Reduced in GalA 952

(A) to (D) Levels of GalA (A) GlcA (B) cellulose (C) and neutral sugars (D) in alcohol953

insoluble residue (AIR) extracted from leaves of four week-old plants of the indicated 954

genotypes Bars represent means plusmn SE of four biological replicates combined using a 955

mixed linear model Letters indicate significantly different groups (p lt 005) 956

957

Figure 5 Cell Walls of gae1 gae6 Plants Are Reduced in Homogalacturonan 958

Page 43 of 54

Dot blot analyses with anti-LM19 and anti-LM20 antibodies Pectin was extracted from 959

AIR prepared from the indicated genotypes using 1 microL of extraction buffer per 10 microg of 960

AIR Pectin was serially diluted and one microL of undiluted 13 diluted and 19 diluted 961

samples were spotted on a nitrocellulose membrane Three biological replicates were 962

were spotted twice to obtain two technical replicates each All replicates produced 963

similar results 964

(A) Representative dot blot using anti-LM19 antibody965

(B) Relative signal intensities of the spots in the dot blot in (A)966

(C) Representative dot blot using anti-LM20 antibody967

(D) Relative signal intensities of the LM20 dot blot shown in (C)968

Relative signal intensities were measured using Image J software Measurements from 969

the three biological replicates were combined using a linear model after Box-Cox power 970

transformation as described in Methods Bars represent means plusmn SE Asterisks indicate 971

a difference from wild-type Col-0 at p lt 005 972

973

Figure 6 Growth of B cinerea on gae1 gae6 Plants and the Effect of B cinerea on 974

Expression of GAE1 and GAE6 975

(A) Growth of B cinerea isolates Leaves of Col-0 and gae1 gae6 plants were976

inoculated with 25 x 105 spores mL-1 of the B cinerea isolates indicated in the figure 977

Samples were collected two (2 dpi) and three (3 dpi) days later Bars represent means plusmn 978

SE of six biological replicates for each fungus combined using a mixed linear model 979

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 980

(B) and (C) Expression of GAE1 (B) and GAE6 (C) 48 h after inoculation with the B981

cinerea isolates indicated Expression levels were measured by qRT-PCR Bars 982

represent mean log2-ratios to ACTIN2 plusmn SE of three biological replicates combined 983

using a mixed linear model Letters indicate significantly different groups (q lt 005) 984

985

Figure 7 Levels of Soluble Pectin and Expression of PAD3 in Fractions Produced 986

Following Macerozyme Treatment of gae1 gae6 Cell Walls 987

(A) Schematic overview of the experimental procedure for (B) and (C)988

Page 44 of 54

(B) Uronic acid measurements Alcohol-insoluble residue (AIR) from leave tissue of four989

week-old wild-type Col-0 or gae1 gae6 plants was treated with water or 025 990

macerozyme and assayed for uronic acid levels Data from 11 biological replicates were 991

combined using a mixed linear model Bars represent means plusmn SE Letters indicate 992

significant differences (p lt 005) 993

(C) Expression of PAD3 in Col-0 plants treated with the fractions from (B) Extracts from994

(B) were heat inactivated pooled and diluted 5-fold with water Pooled extracts were995

inoculated into four week-old Col-0 plants PAD3 expression was measured 3 h after 996

inoculation and normalized to the level of ACTIN2 expression Data from four biological 997

replicates were combined using a mixed linear model Bars represent mean log2-ratios 998

to ACTIN2 plusmn SE Letters indicate significant differences (p lt 005) 999

1000

Figure 8 Macerozyme-Induced PAD3 Expression Is Absent in gae1 gae6 1001

(A) Expression of PAD3 after macerozyme treatment Four week-old plants were either1002

inoculated with 001 macerozyme or mock inoculated with water and PAD3 1003

expression was measured 3 h later by qRT-PCR Data from four biological replicates 1004

were combined using a mixed linear model Mean log2-ratios to ACTIN2 plusmn SE were 1005

plotted Letters indicate significantly different groups (p lt 005) 1006

(B) Expression of PAD3 after OG treatment PAD3 expression was measured as1007

described in (A) 3 h after inoculation with 100 microg mL-1 OGs or mock inoculation with 1008

water Data are from six biological replicates 1009

(C) PAD3 expression in untreated plants Three biological replicates were performed1010

and data analyzed as described in (A) 1011

(D) Camalexin accumulation after B cinerea treatment Camalexin accumulation was1012

measured in four week-old plants inoculated with the B cinerea strains shown in the 1013

figure Eight biological replicates each were combined using a mixed linear model 1014

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 1015

(E) PAD3 expression 28 h after treatment with the indicated B cinerea strains Data1016

from six biological replicates were collected and analyzed as described in (A) Data in 1017

all figure parts represent means plusmn SE Letters indicate significantly different groups (q lt 1018

005) 1019

Page 45 of 54

1020

Figure 9 Macerozyme-Induced Expression of the SA Marker Genes SID2 and PR1 1021

Is Abolished in gae1 gae6 Plants 1022

(A) to (H) Expression of PR1 (A) (C) (E) and (G) or SID2 (B) (D) (F) and (H) in four1023

week-old Col-0 and gae1 gae6 plants Data were combined using a mixed linear model 1024

Bars represent mean log2-ratios of expression vs ACTIN2 plusmn SE for all figure parts 1025

Letters indicate significantly different groups at p lt 005 for panels (A) to (D) (G) and 1026

(H) and qlt005 for panels (E) to (F) 1027

(A) and (B) Plants were treated with 001 macerozyme or mock treated with water1028

and samples were collected 3 h later Data from four biological replicates are shown 1029

(C) and (D) Plants were inoculated with 100 microg mL-1 OGs or mock treated with water1030

and samples collected 3 h later Data from six biological replicates are shown 1031

(E) and (F) Plants were inoculated with the indicated B cinerea isolates or mock1032

inoculated with B cinerea inoculation medium and samples collected 48 h later Data 1033

from eight biological replicates are shown 1034

(G) and (H) Expression in untreated plants Three biological replicates were performed1035

1036

Figure 10 The Expression of the JA Marker Gene JAZ10 is Highly Responsive in 1037

gae1 gae6 Plants 1038

(A) to (D) JAZ10 expression in four week-old plants Data were combined using a mixed1039

linear model Mean log2-ratios to ACTIN2 plusmn SE are shown Letters indicate significantly 1040

different groups at p lt 005 for panels (A) (B) and (D) and q lt 005 for panel (C) 1041

(A) Plants were inoculated with 001 macerozyme or mock inoculated using water and1042

samples were collected 3 h later Data from four biological replicates are shown 1043

(B) Plants were inoculated with 100 microg mL-1 OGs or mock inoculated using water and1044

samples collected 3 h later Data are from six biological replicates 1045

(C) JAZ10 expression 48 h after treatment with the indicated B cinerea strains Data1046

are from six biological replicates 1047

(D) JAZ10 expression in untreated plants Data are from three biological replicates1048

1049

Figure 11 Gallo 1-Induced Callose Deposition Is Reduced in gae1 gae6 Plants 1050

Page 46 of 54

(A) Callose deposition in four week-old Col-0 and gae1 gae6 plants Plants were1051

inoculated with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) or mock inoculated 1052

with B cinerea inoculation medium and samples were collected after 72 h Callose was 1053

stained with aniline blue and visualized using a Nikon Eclipse Ni-U microscope with a 1054

DAPI filter Three leaves were stained per treatment Scale bars are 100 microm Asterisks 1055

indicate fungal hyphae arrows indicate representative callose spots 1056

(B) Quantification of callose deposition Callose deposits were counted in 200 microm x 2001057

microm squares laid out across the images shown here Bars represent means plusmn SE from 1058

six squares each Letters indicate significantly different groups (p lt 005) according to a 1059

t-test analysis1060

1061

Figure 12 Less Soluble Pectin Accumulates in B cinerea-Infected gae1 gae6 1062

Leaves 1063

Four week-old Col-0 and gae1 gae6 plants were inoculated with 10 microL droplets of the 1064

indicated B cinerea isolate (25 x 105 spores mL-1) or mock inoculated with B cinerea 1065

inoculation medium Leaves were collected 48 h later AIR was extracted from leaf 1066

samples and water added (20 microL infection site) to extract soluble pectin fragments 1067

Samples were vortexed for 7 h centrifuged at 12000 g and the uronic acid 1068

concentration of the supernatant was measured Data from nine biological replicates 1069

were combined using a mixed linear model Means plusmn SE are shown Letters indicate 1070

significant differences (q lt 005) 1071

1072

Figure 13 Macerozyme-Induced PTI Is Lost in gae1 gae6 While OG-Induced PTI Is 1073

Unaltered 1074

(A) Macerozyme-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1075

inoculated with 001 macerozyme or mock inoculated with boiled macerozyme and 1076

were infected with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) 24 h later Samples 1077

were collected immediately (0 dpi) and after two (2 dpi) and three (3 dpi) days Data 1078

show mean plusmn SE of six to 14 biological replicates each combined using a linear model 1079

Letters indicate significantly different groups (q lt 005) 1080

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

Parsed CitationsAdam L and Somerville SC (1996) Genetic characterization of five powdery mildew disease resistance loci in Arabidopsisthaliana Plant J 9 341-356

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Albersheim P Jones TM and English PD (1969) Biochemistry of the cell wall in relation to infective processes Annu RevPhytopathol 7 171-194

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk RGadrinab C Heller C Jeske A Koesema E Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt MHazari N Hom E Karnes M Mulholland C Ndubaku R Schmidt I Guzman P Aguilar-Henonin L Schmid M Weigel DCarter DE Marchand T Risseeuw E Brogden D Zeko A Crosby WL Berry CC and Ecker JR (2003) Genome-wideinsertional mutagenesis of Arabidopsis thaliana Science 301 653-657

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Asai T Tena G Plotnikova J Willmann MR Chiu WL Gomez-Gomez L Boller T Ausubel FM and Sheen J (2002) MAPkinase signalling cascade in Arabidopsis innate immunity Nature 415 977-983

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bartsch M Gobbato E Bednarek P Debey S Schultze JL Bautor J and Parker JE (2006) Salicylic acid-independentENHANCED DISEASE SUSCEPTIBILITY1 signaling in Arabidopsis immunity and cell death is regulated by the monooxygenaseFMO1 and the nudix hydrolase NUDT7 Plant Cell 18 1038-1051

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bates D Machler M Bolker BM and Walker SC (2015) Fitting Linear Mixed-Effects Models Using lme4 J Stat Softw 67 1-48Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Benjamini Y and Hochberg Y (1995) Controlling the False Discovery Rate - a Practical and Powerful Approach to MultipleTesting J Roy Stat Soc B Met 57 289-300

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bethke G Grundman RE Sreekanta S Truman W Katagiri F and Glazebrook J (2014) Arabidopsis PECTINMETHYLESTERASEs Contribute to Immunity against Pseudomonas syringae Plant Physiol 164 1093-1107

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bouton S Leboeuf E Mouille G Leydecker MT Talbotec J Granier F Lahaye M Hofte H and Truong HN (2002)Quasimodo1 encodes a putative membrane-bound glycosyltransferase required for normal pectin synthesis and cell adhesion inArabidopsis Plant Cell 14 2577-2590

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Box GEP and Cox DR (1964) An analysis of transformations Journal of the Royal Statistical Society Series B 26 211-252Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Cao H Glazebrook J Clarke JD Volko S and Dong XN (1997) The Arabidopsis NPR1 gene that controls systemic acquiredresistance encodes a novel protein containing ankyrin repeats Cell 88 57-63

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Carpita N (2010) Update on Mechanisms of Plant Cell Wall Biosynthesis How plants make cellulose and other (1-gt4)-beta-D-glycans Plant Physiol

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Chisholm ST Coaker G Day B and Staskawicz BJ (2006) Host-microbe interactions Shaping the evolution of the plantimmune response Cell 124 803-814

Page 43 of 54

Dot blot analyses with anti-LM19 and anti-LM20 antibodies Pectin was extracted from 959

AIR prepared from the indicated genotypes using 1 microL of extraction buffer per 10 microg of 960

AIR Pectin was serially diluted and one microL of undiluted 13 diluted and 19 diluted 961

samples were spotted on a nitrocellulose membrane Three biological replicates were 962

were spotted twice to obtain two technical replicates each All replicates produced 963

similar results 964

(A) Representative dot blot using anti-LM19 antibody965

(B) Relative signal intensities of the spots in the dot blot in (A)966

(C) Representative dot blot using anti-LM20 antibody967

(D) Relative signal intensities of the LM20 dot blot shown in (C)968

Relative signal intensities were measured using Image J software Measurements from 969

the three biological replicates were combined using a linear model after Box-Cox power 970

transformation as described in Methods Bars represent means plusmn SE Asterisks indicate 971

a difference from wild-type Col-0 at p lt 005 972

973

Figure 6 Growth of B cinerea on gae1 gae6 Plants and the Effect of B cinerea on 974

Expression of GAE1 and GAE6 975

(A) Growth of B cinerea isolates Leaves of Col-0 and gae1 gae6 plants were976

inoculated with 25 x 105 spores mL-1 of the B cinerea isolates indicated in the figure 977

Samples were collected two (2 dpi) and three (3 dpi) days later Bars represent means plusmn 978

SE of six biological replicates for each fungus combined using a mixed linear model 979

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 980

(B) and (C) Expression of GAE1 (B) and GAE6 (C) 48 h after inoculation with the B981

cinerea isolates indicated Expression levels were measured by qRT-PCR Bars 982

represent mean log2-ratios to ACTIN2 plusmn SE of three biological replicates combined 983

using a mixed linear model Letters indicate significantly different groups (q lt 005) 984

985

Figure 7 Levels of Soluble Pectin and Expression of PAD3 in Fractions Produced 986

Following Macerozyme Treatment of gae1 gae6 Cell Walls 987

(A) Schematic overview of the experimental procedure for (B) and (C)988

Page 44 of 54

(B) Uronic acid measurements Alcohol-insoluble residue (AIR) from leave tissue of four989

week-old wild-type Col-0 or gae1 gae6 plants was treated with water or 025 990

macerozyme and assayed for uronic acid levels Data from 11 biological replicates were 991

combined using a mixed linear model Bars represent means plusmn SE Letters indicate 992

significant differences (p lt 005) 993

(C) Expression of PAD3 in Col-0 plants treated with the fractions from (B) Extracts from994

(B) were heat inactivated pooled and diluted 5-fold with water Pooled extracts were995

inoculated into four week-old Col-0 plants PAD3 expression was measured 3 h after 996

inoculation and normalized to the level of ACTIN2 expression Data from four biological 997

replicates were combined using a mixed linear model Bars represent mean log2-ratios 998

to ACTIN2 plusmn SE Letters indicate significant differences (p lt 005) 999

1000

Figure 8 Macerozyme-Induced PAD3 Expression Is Absent in gae1 gae6 1001

(A) Expression of PAD3 after macerozyme treatment Four week-old plants were either1002

inoculated with 001 macerozyme or mock inoculated with water and PAD3 1003

expression was measured 3 h later by qRT-PCR Data from four biological replicates 1004

were combined using a mixed linear model Mean log2-ratios to ACTIN2 plusmn SE were 1005

plotted Letters indicate significantly different groups (p lt 005) 1006

(B) Expression of PAD3 after OG treatment PAD3 expression was measured as1007

described in (A) 3 h after inoculation with 100 microg mL-1 OGs or mock inoculation with 1008

water Data are from six biological replicates 1009

(C) PAD3 expression in untreated plants Three biological replicates were performed1010

and data analyzed as described in (A) 1011

(D) Camalexin accumulation after B cinerea treatment Camalexin accumulation was1012

measured in four week-old plants inoculated with the B cinerea strains shown in the 1013

figure Eight biological replicates each were combined using a mixed linear model 1014

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 1015

(E) PAD3 expression 28 h after treatment with the indicated B cinerea strains Data1016

from six biological replicates were collected and analyzed as described in (A) Data in 1017

all figure parts represent means plusmn SE Letters indicate significantly different groups (q lt 1018

005) 1019

Page 45 of 54

1020

Figure 9 Macerozyme-Induced Expression of the SA Marker Genes SID2 and PR1 1021

Is Abolished in gae1 gae6 Plants 1022

(A) to (H) Expression of PR1 (A) (C) (E) and (G) or SID2 (B) (D) (F) and (H) in four1023

week-old Col-0 and gae1 gae6 plants Data were combined using a mixed linear model 1024

Bars represent mean log2-ratios of expression vs ACTIN2 plusmn SE for all figure parts 1025

Letters indicate significantly different groups at p lt 005 for panels (A) to (D) (G) and 1026

(H) and qlt005 for panels (E) to (F) 1027

(A) and (B) Plants were treated with 001 macerozyme or mock treated with water1028

and samples were collected 3 h later Data from four biological replicates are shown 1029

(C) and (D) Plants were inoculated with 100 microg mL-1 OGs or mock treated with water1030

and samples collected 3 h later Data from six biological replicates are shown 1031

(E) and (F) Plants were inoculated with the indicated B cinerea isolates or mock1032

inoculated with B cinerea inoculation medium and samples collected 48 h later Data 1033

from eight biological replicates are shown 1034

(G) and (H) Expression in untreated plants Three biological replicates were performed1035

1036

Figure 10 The Expression of the JA Marker Gene JAZ10 is Highly Responsive in 1037

gae1 gae6 Plants 1038

(A) to (D) JAZ10 expression in four week-old plants Data were combined using a mixed1039

linear model Mean log2-ratios to ACTIN2 plusmn SE are shown Letters indicate significantly 1040

different groups at p lt 005 for panels (A) (B) and (D) and q lt 005 for panel (C) 1041

(A) Plants were inoculated with 001 macerozyme or mock inoculated using water and1042

samples were collected 3 h later Data from four biological replicates are shown 1043

(B) Plants were inoculated with 100 microg mL-1 OGs or mock inoculated using water and1044

samples collected 3 h later Data are from six biological replicates 1045

(C) JAZ10 expression 48 h after treatment with the indicated B cinerea strains Data1046

are from six biological replicates 1047

(D) JAZ10 expression in untreated plants Data are from three biological replicates1048

1049

Figure 11 Gallo 1-Induced Callose Deposition Is Reduced in gae1 gae6 Plants 1050

Page 46 of 54

(A) Callose deposition in four week-old Col-0 and gae1 gae6 plants Plants were1051

inoculated with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) or mock inoculated 1052

with B cinerea inoculation medium and samples were collected after 72 h Callose was 1053

stained with aniline blue and visualized using a Nikon Eclipse Ni-U microscope with a 1054

DAPI filter Three leaves were stained per treatment Scale bars are 100 microm Asterisks 1055

indicate fungal hyphae arrows indicate representative callose spots 1056

(B) Quantification of callose deposition Callose deposits were counted in 200 microm x 2001057

microm squares laid out across the images shown here Bars represent means plusmn SE from 1058

six squares each Letters indicate significantly different groups (p lt 005) according to a 1059

t-test analysis1060

1061

Figure 12 Less Soluble Pectin Accumulates in B cinerea-Infected gae1 gae6 1062

Leaves 1063

Four week-old Col-0 and gae1 gae6 plants were inoculated with 10 microL droplets of the 1064

indicated B cinerea isolate (25 x 105 spores mL-1) or mock inoculated with B cinerea 1065

inoculation medium Leaves were collected 48 h later AIR was extracted from leaf 1066

samples and water added (20 microL infection site) to extract soluble pectin fragments 1067

Samples were vortexed for 7 h centrifuged at 12000 g and the uronic acid 1068

concentration of the supernatant was measured Data from nine biological replicates 1069

were combined using a mixed linear model Means plusmn SE are shown Letters indicate 1070

significant differences (q lt 005) 1071

1072

Figure 13 Macerozyme-Induced PTI Is Lost in gae1 gae6 While OG-Induced PTI Is 1073

Unaltered 1074

(A) Macerozyme-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1075

inoculated with 001 macerozyme or mock inoculated with boiled macerozyme and 1076

were infected with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) 24 h later Samples 1077

were collected immediately (0 dpi) and after two (2 dpi) and three (3 dpi) days Data 1078

show mean plusmn SE of six to 14 biological replicates each combined using a linear model 1079

Letters indicate significantly different groups (q lt 005) 1080

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

Parsed CitationsAdam L and Somerville SC (1996) Genetic characterization of five powdery mildew disease resistance loci in Arabidopsisthaliana Plant J 9 341-356

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Albersheim P Jones TM and English PD (1969) Biochemistry of the cell wall in relation to infective processes Annu RevPhytopathol 7 171-194

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk RGadrinab C Heller C Jeske A Koesema E Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt MHazari N Hom E Karnes M Mulholland C Ndubaku R Schmidt I Guzman P Aguilar-Henonin L Schmid M Weigel DCarter DE Marchand T Risseeuw E Brogden D Zeko A Crosby WL Berry CC and Ecker JR (2003) Genome-wideinsertional mutagenesis of Arabidopsis thaliana Science 301 653-657

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Asai T Tena G Plotnikova J Willmann MR Chiu WL Gomez-Gomez L Boller T Ausubel FM and Sheen J (2002) MAPkinase signalling cascade in Arabidopsis innate immunity Nature 415 977-983

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bartsch M Gobbato E Bednarek P Debey S Schultze JL Bautor J and Parker JE (2006) Salicylic acid-independentENHANCED DISEASE SUSCEPTIBILITY1 signaling in Arabidopsis immunity and cell death is regulated by the monooxygenaseFMO1 and the nudix hydrolase NUDT7 Plant Cell 18 1038-1051

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bates D Machler M Bolker BM and Walker SC (2015) Fitting Linear Mixed-Effects Models Using lme4 J Stat Softw 67 1-48Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Benjamini Y and Hochberg Y (1995) Controlling the False Discovery Rate - a Practical and Powerful Approach to MultipleTesting J Roy Stat Soc B Met 57 289-300

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bethke G Grundman RE Sreekanta S Truman W Katagiri F and Glazebrook J (2014) Arabidopsis PECTINMETHYLESTERASEs Contribute to Immunity against Pseudomonas syringae Plant Physiol 164 1093-1107

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bouton S Leboeuf E Mouille G Leydecker MT Talbotec J Granier F Lahaye M Hofte H and Truong HN (2002)Quasimodo1 encodes a putative membrane-bound glycosyltransferase required for normal pectin synthesis and cell adhesion inArabidopsis Plant Cell 14 2577-2590

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Box GEP and Cox DR (1964) An analysis of transformations Journal of the Royal Statistical Society Series B 26 211-252Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Cao H Glazebrook J Clarke JD Volko S and Dong XN (1997) The Arabidopsis NPR1 gene that controls systemic acquiredresistance encodes a novel protein containing ankyrin repeats Cell 88 57-63

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Carpita N (2010) Update on Mechanisms of Plant Cell Wall Biosynthesis How plants make cellulose and other (1-gt4)-beta-D-glycans Plant Physiol

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Chisholm ST Coaker G Day B and Staskawicz BJ (2006) Host-microbe interactions Shaping the evolution of the plantimmune response Cell 124 803-814

Page 44 of 54

(B) Uronic acid measurements Alcohol-insoluble residue (AIR) from leave tissue of four989

week-old wild-type Col-0 or gae1 gae6 plants was treated with water or 025 990

macerozyme and assayed for uronic acid levels Data from 11 biological replicates were 991

combined using a mixed linear model Bars represent means plusmn SE Letters indicate 992

significant differences (p lt 005) 993

(C) Expression of PAD3 in Col-0 plants treated with the fractions from (B) Extracts from994

(B) were heat inactivated pooled and diluted 5-fold with water Pooled extracts were995

inoculated into four week-old Col-0 plants PAD3 expression was measured 3 h after 996

inoculation and normalized to the level of ACTIN2 expression Data from four biological 997

replicates were combined using a mixed linear model Bars represent mean log2-ratios 998

to ACTIN2 plusmn SE Letters indicate significant differences (p lt 005) 999

1000

Figure 8 Macerozyme-Induced PAD3 Expression Is Absent in gae1 gae6 1001

(A) Expression of PAD3 after macerozyme treatment Four week-old plants were either1002

inoculated with 001 macerozyme or mock inoculated with water and PAD3 1003

expression was measured 3 h later by qRT-PCR Data from four biological replicates 1004

were combined using a mixed linear model Mean log2-ratios to ACTIN2 plusmn SE were 1005

plotted Letters indicate significantly different groups (p lt 005) 1006

(B) Expression of PAD3 after OG treatment PAD3 expression was measured as1007

described in (A) 3 h after inoculation with 100 microg mL-1 OGs or mock inoculation with 1008

water Data are from six biological replicates 1009

(C) PAD3 expression in untreated plants Three biological replicates were performed1010

and data analyzed as described in (A) 1011

(D) Camalexin accumulation after B cinerea treatment Camalexin accumulation was1012

measured in four week-old plants inoculated with the B cinerea strains shown in the 1013

figure Eight biological replicates each were combined using a mixed linear model 1014

Asterisks indicate significant differences from wild-type Col-0 for each isolate (p lt 005) 1015

(E) PAD3 expression 28 h after treatment with the indicated B cinerea strains Data1016

from six biological replicates were collected and analyzed as described in (A) Data in 1017

all figure parts represent means plusmn SE Letters indicate significantly different groups (q lt 1018

005) 1019

Page 45 of 54

1020

Figure 9 Macerozyme-Induced Expression of the SA Marker Genes SID2 and PR1 1021

Is Abolished in gae1 gae6 Plants 1022

(A) to (H) Expression of PR1 (A) (C) (E) and (G) or SID2 (B) (D) (F) and (H) in four1023

week-old Col-0 and gae1 gae6 plants Data were combined using a mixed linear model 1024

Bars represent mean log2-ratios of expression vs ACTIN2 plusmn SE for all figure parts 1025

Letters indicate significantly different groups at p lt 005 for panels (A) to (D) (G) and 1026

(H) and qlt005 for panels (E) to (F) 1027

(A) and (B) Plants were treated with 001 macerozyme or mock treated with water1028

and samples were collected 3 h later Data from four biological replicates are shown 1029

(C) and (D) Plants were inoculated with 100 microg mL-1 OGs or mock treated with water1030

and samples collected 3 h later Data from six biological replicates are shown 1031

(E) and (F) Plants were inoculated with the indicated B cinerea isolates or mock1032

inoculated with B cinerea inoculation medium and samples collected 48 h later Data 1033

from eight biological replicates are shown 1034

(G) and (H) Expression in untreated plants Three biological replicates were performed1035

1036

Figure 10 The Expression of the JA Marker Gene JAZ10 is Highly Responsive in 1037

gae1 gae6 Plants 1038

(A) to (D) JAZ10 expression in four week-old plants Data were combined using a mixed1039

linear model Mean log2-ratios to ACTIN2 plusmn SE are shown Letters indicate significantly 1040

different groups at p lt 005 for panels (A) (B) and (D) and q lt 005 for panel (C) 1041

(A) Plants were inoculated with 001 macerozyme or mock inoculated using water and1042

samples were collected 3 h later Data from four biological replicates are shown 1043

(B) Plants were inoculated with 100 microg mL-1 OGs or mock inoculated using water and1044

samples collected 3 h later Data are from six biological replicates 1045

(C) JAZ10 expression 48 h after treatment with the indicated B cinerea strains Data1046

are from six biological replicates 1047

(D) JAZ10 expression in untreated plants Data are from three biological replicates1048

1049

Figure 11 Gallo 1-Induced Callose Deposition Is Reduced in gae1 gae6 Plants 1050

Page 46 of 54

(A) Callose deposition in four week-old Col-0 and gae1 gae6 plants Plants were1051

inoculated with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) or mock inoculated 1052

with B cinerea inoculation medium and samples were collected after 72 h Callose was 1053

stained with aniline blue and visualized using a Nikon Eclipse Ni-U microscope with a 1054

DAPI filter Three leaves were stained per treatment Scale bars are 100 microm Asterisks 1055

indicate fungal hyphae arrows indicate representative callose spots 1056

(B) Quantification of callose deposition Callose deposits were counted in 200 microm x 2001057

microm squares laid out across the images shown here Bars represent means plusmn SE from 1058

six squares each Letters indicate significantly different groups (p lt 005) according to a 1059

t-test analysis1060

1061

Figure 12 Less Soluble Pectin Accumulates in B cinerea-Infected gae1 gae6 1062

Leaves 1063

Four week-old Col-0 and gae1 gae6 plants were inoculated with 10 microL droplets of the 1064

indicated B cinerea isolate (25 x 105 spores mL-1) or mock inoculated with B cinerea 1065

inoculation medium Leaves were collected 48 h later AIR was extracted from leaf 1066

samples and water added (20 microL infection site) to extract soluble pectin fragments 1067

Samples were vortexed for 7 h centrifuged at 12000 g and the uronic acid 1068

concentration of the supernatant was measured Data from nine biological replicates 1069

were combined using a mixed linear model Means plusmn SE are shown Letters indicate 1070

significant differences (q lt 005) 1071

1072

Figure 13 Macerozyme-Induced PTI Is Lost in gae1 gae6 While OG-Induced PTI Is 1073

Unaltered 1074

(A) Macerozyme-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1075

inoculated with 001 macerozyme or mock inoculated with boiled macerozyme and 1076

were infected with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) 24 h later Samples 1077

were collected immediately (0 dpi) and after two (2 dpi) and three (3 dpi) days Data 1078

show mean plusmn SE of six to 14 biological replicates each combined using a linear model 1079

Letters indicate significantly different groups (q lt 005) 1080

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

Parsed CitationsAdam L and Somerville SC (1996) Genetic characterization of five powdery mildew disease resistance loci in Arabidopsisthaliana Plant J 9 341-356

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Albersheim P Jones TM and English PD (1969) Biochemistry of the cell wall in relation to infective processes Annu RevPhytopathol 7 171-194

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk RGadrinab C Heller C Jeske A Koesema E Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt MHazari N Hom E Karnes M Mulholland C Ndubaku R Schmidt I Guzman P Aguilar-Henonin L Schmid M Weigel DCarter DE Marchand T Risseeuw E Brogden D Zeko A Crosby WL Berry CC and Ecker JR (2003) Genome-wideinsertional mutagenesis of Arabidopsis thaliana Science 301 653-657

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Asai T Tena G Plotnikova J Willmann MR Chiu WL Gomez-Gomez L Boller T Ausubel FM and Sheen J (2002) MAPkinase signalling cascade in Arabidopsis innate immunity Nature 415 977-983

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bartsch M Gobbato E Bednarek P Debey S Schultze JL Bautor J and Parker JE (2006) Salicylic acid-independentENHANCED DISEASE SUSCEPTIBILITY1 signaling in Arabidopsis immunity and cell death is regulated by the monooxygenaseFMO1 and the nudix hydrolase NUDT7 Plant Cell 18 1038-1051

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bates D Machler M Bolker BM and Walker SC (2015) Fitting Linear Mixed-Effects Models Using lme4 J Stat Softw 67 1-48Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Benjamini Y and Hochberg Y (1995) Controlling the False Discovery Rate - a Practical and Powerful Approach to MultipleTesting J Roy Stat Soc B Met 57 289-300

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bethke G Grundman RE Sreekanta S Truman W Katagiri F and Glazebrook J (2014) Arabidopsis PECTINMETHYLESTERASEs Contribute to Immunity against Pseudomonas syringae Plant Physiol 164 1093-1107

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bouton S Leboeuf E Mouille G Leydecker MT Talbotec J Granier F Lahaye M Hofte H and Truong HN (2002)Quasimodo1 encodes a putative membrane-bound glycosyltransferase required for normal pectin synthesis and cell adhesion inArabidopsis Plant Cell 14 2577-2590

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Box GEP and Cox DR (1964) An analysis of transformations Journal of the Royal Statistical Society Series B 26 211-252Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Cao H Glazebrook J Clarke JD Volko S and Dong XN (1997) The Arabidopsis NPR1 gene that controls systemic acquiredresistance encodes a novel protein containing ankyrin repeats Cell 88 57-63

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Carpita N (2010) Update on Mechanisms of Plant Cell Wall Biosynthesis How plants make cellulose and other (1-gt4)-beta-D-glycans Plant Physiol

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Chisholm ST Coaker G Day B and Staskawicz BJ (2006) Host-microbe interactions Shaping the evolution of the plantimmune response Cell 124 803-814

Page 45 of 54

1020

Figure 9 Macerozyme-Induced Expression of the SA Marker Genes SID2 and PR1 1021

Is Abolished in gae1 gae6 Plants 1022

(A) to (H) Expression of PR1 (A) (C) (E) and (G) or SID2 (B) (D) (F) and (H) in four1023

week-old Col-0 and gae1 gae6 plants Data were combined using a mixed linear model 1024

Bars represent mean log2-ratios of expression vs ACTIN2 plusmn SE for all figure parts 1025

Letters indicate significantly different groups at p lt 005 for panels (A) to (D) (G) and 1026

(H) and qlt005 for panels (E) to (F) 1027

(A) and (B) Plants were treated with 001 macerozyme or mock treated with water1028

and samples were collected 3 h later Data from four biological replicates are shown 1029

(C) and (D) Plants were inoculated with 100 microg mL-1 OGs or mock treated with water1030

and samples collected 3 h later Data from six biological replicates are shown 1031

(E) and (F) Plants were inoculated with the indicated B cinerea isolates or mock1032

inoculated with B cinerea inoculation medium and samples collected 48 h later Data 1033

from eight biological replicates are shown 1034

(G) and (H) Expression in untreated plants Three biological replicates were performed1035

1036

Figure 10 The Expression of the JA Marker Gene JAZ10 is Highly Responsive in 1037

gae1 gae6 Plants 1038

(A) to (D) JAZ10 expression in four week-old plants Data were combined using a mixed1039

linear model Mean log2-ratios to ACTIN2 plusmn SE are shown Letters indicate significantly 1040

different groups at p lt 005 for panels (A) (B) and (D) and q lt 005 for panel (C) 1041

(A) Plants were inoculated with 001 macerozyme or mock inoculated using water and1042

samples were collected 3 h later Data from four biological replicates are shown 1043

(B) Plants were inoculated with 100 microg mL-1 OGs or mock inoculated using water and1044

samples collected 3 h later Data are from six biological replicates 1045

(C) JAZ10 expression 48 h after treatment with the indicated B cinerea strains Data1046

are from six biological replicates 1047

(D) JAZ10 expression in untreated plants Data are from three biological replicates1048

1049

Figure 11 Gallo 1-Induced Callose Deposition Is Reduced in gae1 gae6 Plants 1050

Page 46 of 54

(A) Callose deposition in four week-old Col-0 and gae1 gae6 plants Plants were1051

inoculated with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) or mock inoculated 1052

with B cinerea inoculation medium and samples were collected after 72 h Callose was 1053

stained with aniline blue and visualized using a Nikon Eclipse Ni-U microscope with a 1054

DAPI filter Three leaves were stained per treatment Scale bars are 100 microm Asterisks 1055

indicate fungal hyphae arrows indicate representative callose spots 1056

(B) Quantification of callose deposition Callose deposits were counted in 200 microm x 2001057

microm squares laid out across the images shown here Bars represent means plusmn SE from 1058

six squares each Letters indicate significantly different groups (p lt 005) according to a 1059

t-test analysis1060

1061

Figure 12 Less Soluble Pectin Accumulates in B cinerea-Infected gae1 gae6 1062

Leaves 1063

Four week-old Col-0 and gae1 gae6 plants were inoculated with 10 microL droplets of the 1064

indicated B cinerea isolate (25 x 105 spores mL-1) or mock inoculated with B cinerea 1065

inoculation medium Leaves were collected 48 h later AIR was extracted from leaf 1066

samples and water added (20 microL infection site) to extract soluble pectin fragments 1067

Samples were vortexed for 7 h centrifuged at 12000 g and the uronic acid 1068

concentration of the supernatant was measured Data from nine biological replicates 1069

were combined using a mixed linear model Means plusmn SE are shown Letters indicate 1070

significant differences (q lt 005) 1071

1072

Figure 13 Macerozyme-Induced PTI Is Lost in gae1 gae6 While OG-Induced PTI Is 1073

Unaltered 1074

(A) Macerozyme-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1075

inoculated with 001 macerozyme or mock inoculated with boiled macerozyme and 1076

were infected with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) 24 h later Samples 1077

were collected immediately (0 dpi) and after two (2 dpi) and three (3 dpi) days Data 1078

show mean plusmn SE of six to 14 biological replicates each combined using a linear model 1079

Letters indicate significantly different groups (q lt 005) 1080

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

Parsed CitationsAdam L and Somerville SC (1996) Genetic characterization of five powdery mildew disease resistance loci in Arabidopsisthaliana Plant J 9 341-356

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Albersheim P Jones TM and English PD (1969) Biochemistry of the cell wall in relation to infective processes Annu RevPhytopathol 7 171-194

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk RGadrinab C Heller C Jeske A Koesema E Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt MHazari N Hom E Karnes M Mulholland C Ndubaku R Schmidt I Guzman P Aguilar-Henonin L Schmid M Weigel DCarter DE Marchand T Risseeuw E Brogden D Zeko A Crosby WL Berry CC and Ecker JR (2003) Genome-wideinsertional mutagenesis of Arabidopsis thaliana Science 301 653-657

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Asai T Tena G Plotnikova J Willmann MR Chiu WL Gomez-Gomez L Boller T Ausubel FM and Sheen J (2002) MAPkinase signalling cascade in Arabidopsis innate immunity Nature 415 977-983

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bartsch M Gobbato E Bednarek P Debey S Schultze JL Bautor J and Parker JE (2006) Salicylic acid-independentENHANCED DISEASE SUSCEPTIBILITY1 signaling in Arabidopsis immunity and cell death is regulated by the monooxygenaseFMO1 and the nudix hydrolase NUDT7 Plant Cell 18 1038-1051

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bates D Machler M Bolker BM and Walker SC (2015) Fitting Linear Mixed-Effects Models Using lme4 J Stat Softw 67 1-48Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Benjamini Y and Hochberg Y (1995) Controlling the False Discovery Rate - a Practical and Powerful Approach to MultipleTesting J Roy Stat Soc B Met 57 289-300

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bethke G Grundman RE Sreekanta S Truman W Katagiri F and Glazebrook J (2014) Arabidopsis PECTINMETHYLESTERASEs Contribute to Immunity against Pseudomonas syringae Plant Physiol 164 1093-1107

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bouton S Leboeuf E Mouille G Leydecker MT Talbotec J Granier F Lahaye M Hofte H and Truong HN (2002)Quasimodo1 encodes a putative membrane-bound glycosyltransferase required for normal pectin synthesis and cell adhesion inArabidopsis Plant Cell 14 2577-2590

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Box GEP and Cox DR (1964) An analysis of transformations Journal of the Royal Statistical Society Series B 26 211-252Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Cao H Glazebrook J Clarke JD Volko S and Dong XN (1997) The Arabidopsis NPR1 gene that controls systemic acquiredresistance encodes a novel protein containing ankyrin repeats Cell 88 57-63

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Carpita N (2010) Update on Mechanisms of Plant Cell Wall Biosynthesis How plants make cellulose and other (1-gt4)-beta-D-glycans Plant Physiol

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Chisholm ST Coaker G Day B and Staskawicz BJ (2006) Host-microbe interactions Shaping the evolution of the plantimmune response Cell 124 803-814

Page 46 of 54

(A) Callose deposition in four week-old Col-0 and gae1 gae6 plants Plants were1051

inoculated with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) or mock inoculated 1052

with B cinerea inoculation medium and samples were collected after 72 h Callose was 1053

stained with aniline blue and visualized using a Nikon Eclipse Ni-U microscope with a 1054

DAPI filter Three leaves were stained per treatment Scale bars are 100 microm Asterisks 1055

indicate fungal hyphae arrows indicate representative callose spots 1056

(B) Quantification of callose deposition Callose deposits were counted in 200 microm x 2001057

microm squares laid out across the images shown here Bars represent means plusmn SE from 1058

six squares each Letters indicate significantly different groups (p lt 005) according to a 1059

t-test analysis1060

1061

Figure 12 Less Soluble Pectin Accumulates in B cinerea-Infected gae1 gae6 1062

Leaves 1063

Four week-old Col-0 and gae1 gae6 plants were inoculated with 10 microL droplets of the 1064

indicated B cinerea isolate (25 x 105 spores mL-1) or mock inoculated with B cinerea 1065

inoculation medium Leaves were collected 48 h later AIR was extracted from leaf 1066

samples and water added (20 microL infection site) to extract soluble pectin fragments 1067

Samples were vortexed for 7 h centrifuged at 12000 g and the uronic acid 1068

concentration of the supernatant was measured Data from nine biological replicates 1069

were combined using a mixed linear model Means plusmn SE are shown Letters indicate 1070

significant differences (q lt 005) 1071

1072

Figure 13 Macerozyme-Induced PTI Is Lost in gae1 gae6 While OG-Induced PTI Is 1073

Unaltered 1074

(A) Macerozyme-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1075

inoculated with 001 macerozyme or mock inoculated with boiled macerozyme and 1076

were infected with B cinerea isolate Gallo 1 (25 x 105 spores mL-1) 24 h later Samples 1077

were collected immediately (0 dpi) and after two (2 dpi) and three (3 dpi) days Data 1078

show mean plusmn SE of six to 14 biological replicates each combined using a linear model 1079

Letters indicate significantly different groups (q lt 005) 1080

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

Parsed CitationsAdam L and Somerville SC (1996) Genetic characterization of five powdery mildew disease resistance loci in Arabidopsisthaliana Plant J 9 341-356

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Albersheim P Jones TM and English PD (1969) Biochemistry of the cell wall in relation to infective processes Annu RevPhytopathol 7 171-194

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk RGadrinab C Heller C Jeske A Koesema E Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt MHazari N Hom E Karnes M Mulholland C Ndubaku R Schmidt I Guzman P Aguilar-Henonin L Schmid M Weigel DCarter DE Marchand T Risseeuw E Brogden D Zeko A Crosby WL Berry CC and Ecker JR (2003) Genome-wideinsertional mutagenesis of Arabidopsis thaliana Science 301 653-657

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Asai T Tena G Plotnikova J Willmann MR Chiu WL Gomez-Gomez L Boller T Ausubel FM and Sheen J (2002) MAPkinase signalling cascade in Arabidopsis innate immunity Nature 415 977-983

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bartsch M Gobbato E Bednarek P Debey S Schultze JL Bautor J and Parker JE (2006) Salicylic acid-independentENHANCED DISEASE SUSCEPTIBILITY1 signaling in Arabidopsis immunity and cell death is regulated by the monooxygenaseFMO1 and the nudix hydrolase NUDT7 Plant Cell 18 1038-1051

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bates D Machler M Bolker BM and Walker SC (2015) Fitting Linear Mixed-Effects Models Using lme4 J Stat Softw 67 1-48Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Benjamini Y and Hochberg Y (1995) Controlling the False Discovery Rate - a Practical and Powerful Approach to MultipleTesting J Roy Stat Soc B Met 57 289-300

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bethke G Grundman RE Sreekanta S Truman W Katagiri F and Glazebrook J (2014) Arabidopsis PECTINMETHYLESTERASEs Contribute to Immunity against Pseudomonas syringae Plant Physiol 164 1093-1107

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bouton S Leboeuf E Mouille G Leydecker MT Talbotec J Granier F Lahaye M Hofte H and Truong HN (2002)Quasimodo1 encodes a putative membrane-bound glycosyltransferase required for normal pectin synthesis and cell adhesion inArabidopsis Plant Cell 14 2577-2590

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Box GEP and Cox DR (1964) An analysis of transformations Journal of the Royal Statistical Society Series B 26 211-252Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Cao H Glazebrook J Clarke JD Volko S and Dong XN (1997) The Arabidopsis NPR1 gene that controls systemic acquiredresistance encodes a novel protein containing ankyrin repeats Cell 88 57-63

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Carpita N (2010) Update on Mechanisms of Plant Cell Wall Biosynthesis How plants make cellulose and other (1-gt4)-beta-D-glycans Plant Physiol

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Chisholm ST Coaker G Day B and Staskawicz BJ (2006) Host-microbe interactions Shaping the evolution of the plantimmune response Cell 124 803-814

Page 47 of 54

(B) OG-induced PTI in Col-0 and gae1 gae6 plants Four week-old plants were1081

inoculated with 500 microg mL-1 OGs and infected with B cinerea as described in (A) Six to 1082

eight biological experiments were performed 1083

(C) and (D) Expression of JAZ5 (C) and JAZ10 (D) was measured in untreated plants or1084

24 h after inoculation with water (mock) 001 macerozyme or 250 microg mL-1 OGs Data 1085

show mean log2-ratios to ACTIN2 plusmn SE from three biological replicates combined using 1086

a mixed linear model Letters indicate significantly different groups (q lt 005) The q-1087

value for comparison of JAZ10 expression in wild-type and mutant after macerozyme 1088

treatment is 0056 1089

1090

1091

Parsed CitationsAdam L and Somerville SC (1996) Genetic characterization of five powdery mildew disease resistance loci in Arabidopsisthaliana Plant J 9 341-356

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Albersheim P Jones TM and English PD (1969) Biochemistry of the cell wall in relation to infective processes Annu RevPhytopathol 7 171-194

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk RGadrinab C Heller C Jeske A Koesema E Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt MHazari N Hom E Karnes M Mulholland C Ndubaku R Schmidt I Guzman P Aguilar-Henonin L Schmid M Weigel DCarter DE Marchand T Risseeuw E Brogden D Zeko A Crosby WL Berry CC and Ecker JR (2003) Genome-wideinsertional mutagenesis of Arabidopsis thaliana Science 301 653-657

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Asai T Tena G Plotnikova J Willmann MR Chiu WL Gomez-Gomez L Boller T Ausubel FM and Sheen J (2002) MAPkinase signalling cascade in Arabidopsis innate immunity Nature 415 977-983

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bartsch M Gobbato E Bednarek P Debey S Schultze JL Bautor J and Parker JE (2006) Salicylic acid-independentENHANCED DISEASE SUSCEPTIBILITY1 signaling in Arabidopsis immunity and cell death is regulated by the monooxygenaseFMO1 and the nudix hydrolase NUDT7 Plant Cell 18 1038-1051

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bates D Machler M Bolker BM and Walker SC (2015) Fitting Linear Mixed-Effects Models Using lme4 J Stat Softw 67 1-48Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Benjamini Y and Hochberg Y (1995) Controlling the False Discovery Rate - a Practical and Powerful Approach to MultipleTesting J Roy Stat Soc B Met 57 289-300

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bethke G Grundman RE Sreekanta S Truman W Katagiri F and Glazebrook J (2014) Arabidopsis PECTINMETHYLESTERASEs Contribute to Immunity against Pseudomonas syringae Plant Physiol 164 1093-1107

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bouton S Leboeuf E Mouille G Leydecker MT Talbotec J Granier F Lahaye M Hofte H and Truong HN (2002)Quasimodo1 encodes a putative membrane-bound glycosyltransferase required for normal pectin synthesis and cell adhesion inArabidopsis Plant Cell 14 2577-2590

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Box GEP and Cox DR (1964) An analysis of transformations Journal of the Royal Statistical Society Series B 26 211-252Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Cao H Glazebrook J Clarke JD Volko S and Dong XN (1997) The Arabidopsis NPR1 gene that controls systemic acquiredresistance encodes a novel protein containing ankyrin repeats Cell 88 57-63

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Carpita N (2010) Update on Mechanisms of Plant Cell Wall Biosynthesis How plants make cellulose and other (1-gt4)-beta-D-glycans Plant Physiol

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Chisholm ST Coaker G Day B and Staskawicz BJ (2006) Host-microbe interactions Shaping the evolution of the plantimmune response Cell 124 803-814

Parsed CitationsAdam L and Somerville SC (1996) Genetic characterization of five powdery mildew disease resistance loci in Arabidopsisthaliana Plant J 9 341-356

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Albersheim P Jones TM and English PD (1969) Biochemistry of the cell wall in relation to infective processes Annu RevPhytopathol 7 171-194

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Alonso JM Stepanova AN Leisse TJ Kim CJ Chen H Shinn P Stevenson DK Zimmerman J Barajas P Cheuk RGadrinab C Heller C Jeske A Koesema E Meyers CC Parker H Prednis L Ansari Y Choy N Deen H Geralt MHazari N Hom E Karnes M Mulholland C Ndubaku R Schmidt I Guzman P Aguilar-Henonin L Schmid M Weigel DCarter DE Marchand T Risseeuw E Brogden D Zeko A Crosby WL Berry CC and Ecker JR (2003) Genome-wideinsertional mutagenesis of Arabidopsis thaliana Science 301 653-657

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Asai T Tena G Plotnikova J Willmann MR Chiu WL Gomez-Gomez L Boller T Ausubel FM and Sheen J (2002) MAPkinase signalling cascade in Arabidopsis innate immunity Nature 415 977-983

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bartsch M Gobbato E Bednarek P Debey S Schultze JL Bautor J and Parker JE (2006) Salicylic acid-independentENHANCED DISEASE SUSCEPTIBILITY1 signaling in Arabidopsis immunity and cell death is regulated by the monooxygenaseFMO1 and the nudix hydrolase NUDT7 Plant Cell 18 1038-1051

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bates D Machler M Bolker BM and Walker SC (2015) Fitting Linear Mixed-Effects Models Using lme4 J Stat Softw 67 1-48Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Benjamini Y and Hochberg Y (1995) Controlling the False Discovery Rate - a Practical and Powerful Approach to MultipleTesting J Roy Stat Soc B Met 57 289-300

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bethke G Grundman RE Sreekanta S Truman W Katagiri F and Glazebrook J (2014) Arabidopsis PECTINMETHYLESTERASEs Contribute to Immunity against Pseudomonas syringae Plant Physiol 164 1093-1107

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bouton S Leboeuf E Mouille G Leydecker MT Talbotec J Granier F Lahaye M Hofte H and Truong HN (2002)Quasimodo1 encodes a putative membrane-bound glycosyltransferase required for normal pectin synthesis and cell adhesion inArabidopsis Plant Cell 14 2577-2590

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Box GEP and Cox DR (1964) An analysis of transformations Journal of the Royal Statistical Society Series B 26 211-252Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Cao H Glazebrook J Clarke JD Volko S and Dong XN (1997) The Arabidopsis NPR1 gene that controls systemic acquiredresistance encodes a novel protein containing ankyrin repeats Cell 88 57-63

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Carpita N (2010) Update on Mechanisms of Plant Cell Wall Biosynthesis How plants make cellulose and other (1-gt4)-beta-D-glycans Plant Physiol

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Chisholm ST Coaker G Day B and Staskawicz BJ (2006) Host-microbe interactions Shaping the evolution of the plantimmune response Cell 124 803-814

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DOI 101105tpc1500404 originally published online January 26 2016Plant Cell

Hillmer Fumiaki Katagiri Daniel J Kliebenstein Markus Pauly and Jane GlazebrookGerit Bethke Amanda Thao Guangyan Xiong Baohua Li Nicole E Soltis Noriyuki Hatsugai Rachel A

Pectin Biosynthesis is Critical for Cell Wall Integrity and Immunity in Arabidopsis thaliana

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