www.sciencemag.org/cgi/content/full/310/5751/1180/DC1

Supporting Online Material for

Pre- and Postinvasion Defenses Both Contribute to Nonhost Resistance in Arabidopsis

Volker Lipka, Jan Dittgen, Pawel Bednarek, Riyaz Bhat, Marcel Wiermer, Monica Stein, Jörn Landtag, Wolfgang Brandt, Sabine Rosahl, Dierk Scheel, Francisco Llorente,

Antonio Molina, Jane Parker, Shauna Somerville, Paul Schulze-Lefert*

*To whom correspondence should be addressed. E-mail: schlef@mpiz-koeln.mpg.de

Published 18 November 2005, Science 310, 1180 (2005) DOI: 10.1126/science.1119409

This PDF file includes:

Materials and Methods Figs. S1 to S3

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Supporting Online Material

Materials and Methods

Plant Lines and Growth Conditions

The following mutant and transgenic lines were used for pathogenicity assays and/or to generate double and triple mutant combinations (all Col-0 background except where indicated): Ws-0 eds1-1 (1), La-er eds1-2 (2), ein2-1, ein4-1 (3), Etr1-1 (4), jar1-1 (5), NahG (6), npr1-1 (7), pad4-1 (8), Ws-0 pad4-5 (9), pen1-1 (10), sag101-1, sag101-2 (11). Plants to be infected with B. graminis or E. pisi were grown in growth chambers at 20-23°C with a 12 h photoperiod and a light intensity of about 150 µE m–2 s–1 on a turf substrate mix (Stender Substrate, Wesel-Schermbeck) containing 0,001% Confidor WG70 (Bayer, Leverkusen). For infection experiments with P. infestans, Arabidopsis plants were grown in a phytochamber with 8 h of light [200 µE] at 22 °C, 16 h dark at 20°C and 60% humidity for 4-5 weeks. For P. cucumerina infections, seeds were surface-sterilized, sown on square (12.5 cm x 12.5 cm) petri dishes containing MS basal salt mixture medium with 0,8% phytagel (Sigma, St. Louis, MO, USA), transferred to a phytochamber and grown as described previously (12). Arabidopsis pen2 mutant screen M2 populations were derived by ethylmethane sulphonate treatment of Arabidopsis Columbia (Col-0 or Col-3, gl1). In the screen yielding pen2-1, M2 plants were inoculated with B. g. hordei K1, and 72 h later examined with ultraviolet light (excitation filter 365/12 nm; dichroic mirror 400 LP) to monitor the autofluorescence resulting from the hypersensitive response-like cell death as a consequence of fungal entry. In the screen yielding pen2-3, M2 populations were inoculated with B. g. hordei isolate CR3, leaves were harvested after 48 h and stained with aniline blue. Callose deposition in response to penetration was monitored via epifluorescence microscopy. The Arabidopsis thaliana PEN2 T-DNA insertion line pen2-2 (GABI-KAT 134C04) was provided by Bernd Weisshaar (Max-Planck-Institute for Plant Breeding Research) and had been generated in the GABI-Kat program (http://www.mpiz-koeln.mpg.de/GABI-Kat/GABI-Kat_homepage.html). Oligonucleotide sequences used for T-DNA insertion detection were 5'-CTCTTTGGAACTGCTTCATCTTCT-3' and

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5'-CCCATTTGGACGTGAATGTAGACAC-3' (PEN2 specific and T-DNA left border specific, respectively). PEN2 cloning PEN2 was mapped using a Columbia pen2-1 X Landsberg erecta F2 population of 864 individuals using standard polymerase chain reaction (PCR)-based marker techniques. New markers within the fine mapping interval were generated using the CEREON database of Col and Ler polymorphisms (13). Derived oligonucleotide sequences used for fine mapping were as follows: F18O19: 5’-GCGACGGAGGCGATAATGCT-3’

5’-CAAAGTGATGACTTCAGATACA-3’ F6E13: 5’-GCAGGGGTTGGGGATTGATAACGAC-3’ 5’-GAAAATCCATGTAGTTAGACAACATG-3’ F4I1C: 5’-GACTGGCTTGAAGCCCATTGTAAG-3’ 5’-GCACCAAGCATGTGTTTCTCAGGA-3’ SNP1: 5’-CTGATCACCATTTTGAGAAGAAATG-3’ 5’-GATGGTCCTGATGGTAGCTTATCC-3’ polymorphism revealed with Hpy188I SNP4: 5’-GATTCATCGCCGTAGCTGTGTGACCG-3’ 5’-AGATGATAAAGAGTTTATGATCTG-3’ polymorphism revealed with MnlI F16B22: 5’-TCTCTATCCACAAGTTAGATGT-3’ 5’-CTCAACTTCTCAGAATCGTTCC-3’ F4L23: 5’-CTGAAACTGATGGAACCAGCGATG-3’ 5’-CAGTGCGTACATATACGAACGTG-3’ F4I18: 5’-GAAGATTTGATGGGCTTTTTGTGC-3’ 5’-ATATCTCTTTTCTGTTAGGTAGAAC-3’ Pathogenicity Assays and Microscopic Analysis 3-4 week-old plants were inoculated with B. graminis (Isolate K1) and E. pisi (Birmingham Isolate) using a settling tower. The E. pisi isolate was kindly provided by Tim Carver (Aberystwyth, UK). Leaves were fixed and cleared in an ethanolic solution containing 6.7% phenol, 6.7% lactic acid, 13.3% glycerol, 6.7% H20. Visualization of epidermal cell death by fluorescence macroscopy was performed as described (10). Pathogen invasion and cell death were scored microscopically under UV-light using callose encasements of haustorium initials and dead cells as well as cell death autofluorescence as markers. Callose was stained with Aniline Blue (0.01% in an aqueous solution containing 150 mM KH2PO4, pH 9.5). Two to three repetitions, scoring at least 400 sites per time point and genotype, were performed.

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Epiphytic fungal growth was visualized by staining of fungal structures with an ethanolic solution containing 0.6% Coomassie Blue. Scanning electron microscopy was performed as described previously (14). P. infestans isolate 208, transformed with the GFP gene under the control of the ham34 promoter from Bremia lactucae (208m2; kindly provided by Felix Mauch, Fribourg, Switzerland) (15) was grown on oat-bean medium at 18°C in the dark. 10-12 day old mycelium was incubated with 10 ml of sterile destilled water at 4°C for 3 hours. After filtration, zoospores were counted and the appropriate concentration of the zoospore suspension was adjusted. Arabidopsis plants were inoculated with 10 µl drops of the zoospore suspension. After inoculation, plants were incubated in a phytochamber with 16 h of light [200 µE] at 20°C, 8 h dark at 18°C and 100% humidity. Trypan blue staining was performed according to (16) using 0.1% trypan blue in lactophenol / ethanol solution. Leaves were cleared by incubation in chloral hydrate (2.5 g/ml). Inoculation experiments with P. cucumerina were carried out by spraying ten day-old plants with a spore suspension (2x105 spores/ml) of the fungus (1.5 ml/plate). Mock inoculations were carried out by spraying the plants with sterile water. After inoculation, plants were kept under the same growing conditions and 14 days later the relative reduction of plant Fresh Weight (± Standard Deviation) caused by the fungal infection was calculated as described (12). At least 30 plants per genotype were inoculated and the experiment was repeated four times. For statistical analysis of the data a two tailed Student´s t test was performed Hydrogen peroxide was visualized by staining with diaminobenzidine (2 mg/ml) according to (17). Leaves were cleared by incubation in chloral hydrate (2.5 g/ml) Molecular Modeling To find homologous proteins with known three-dimensional structures to the sequence of PEN2, a 3d-pssm search were applied (18). As the most promising template structure for homology modelling a CYANOGENIC BETA-GLUCOSIDASE (pdb-code 1cbg) (19) with a sequence identity of 48% and a PSSM-E value of 3.27e-16 was found. However, this was limited to the N-terminal 490 amino acid residues. No structure could be identified for the C-terminal tail of 64 amino acid residues of the target protein. Therefore, another search was performed using the last 180 residues of the protein. HUMAN INTERLEUKIN-6 (pdb-code:1alu) (20) was identified as structure with the longest alignment of 157 residues and a sequence identity of 20%. Only the very last 19 amino acid residues could not be modelled. The sequences were aligned using the blosum 45-matrix (21, 22). Since the alignment of all three structures (the target and the two templates) in once did not result in an alignment suggested by 3d-pssm, two separate models were constructed. Both structures (1cbg and 1alu) were then used separately as

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templates for homology modelling using the program MOE© (23). For each sequence ten models have been calculated by MOE (Molecular operating environment, Chemical Computing Group Inc., Montreal, Canada; www.chemcomp.com) and pre-optimized using the Charmm force field (24). All 10 models were checked with regard to stereochemical quality (Ramachandran plot) using PROCHECK (25). The best ones of both structures were optimized to a gradient smaller than 0.05 kcal/(mol*A). Within some loops, corresponding to gaps in the alignment, some residues appeared in disallowed regions of the Ramachandran plot. These conformations were manually modified accompanied by a subsequent short molecular dynamics simulation for the corresponding loops (10 ps), followed by re-optimization of the whole proteins. Finally, both structures, the N-terminal one of 490 residues and the C-terminal one consisting of 138 residues were spatially aligned with the overlapping identical residues occurring in both sequences and appropriate merged to one protein structure and optimized. The backbone dihedral angle distribution of all amino acid residues (Ramachandran Plot) showed 85.7 % in most favoured, 12.7 % in additional allowed and 1.7 % in generously allowed regions. All other stereochemical parameters were inside or even better in the quality expected for a structure with a resolution of 2.0 Å. The quality of the fold was inspected with PROSA (26) that showed nearly all residues in negative energy regions very similar to the template proteins. The only deviation occurred in a loop area around residues 190 till 210 indicating not a perfect fold for this area. Altogether the plot strongly indicates the principle correctness of the modelled structures. Figure 2 was generated with RasMol Version 2.6. PEN2 antibody and PEN2 localization The PEN2 antiserum was raised against the peptide 506EEKKESYGKQLLHSVQ521 in rabbit, affinity-purified (Eurogentec, Belgium), and used for protein blots at a dilution of 1:1000. General protein techniques and other antisera were as described (10). Generation of transgenic lines The binary PEN2 transformation constructs (vector backbone pAMPAT-MCS [AY436765]) contained RT-PCR amplified PEN2 cDNA coding sequence (oligonucleotide primers: 5’-CGAAAGCTTGCAACCAAAGATGGCACATCTTCAAAGA-3’ / 5’-CATGAATTCTCAATTATTAGCTCCTTTG-3’), flanked by 1213 bp of PEN2 5’ untranslated sequences and the 35S terminator. For site-directed mutagenesis of the codon leading to the replacement of Glu183 with Asp, the original wt sequence was exchanged with a PCR fragment (oligonucleotide primers: 5’-ACACTGTAGACCCACGGATCATTCATTGTGCACCA-3’ / 5’-CGAAAGCTTGCAACCAAAGATGGCACATCTTCAAAGA-3’) by AccI-HindIII digestion and ligation. For the deletion construct lacking the C-terminal 28 amino acids, the original wt sequence was exchanged with a PCR fragment (oligonucleotide primers: 5’-CAGAGTGGAGTTTCACATAT-3’ /

5

5’-TCGGAATTCTCAACTGTCTTTAATCGAATG-3’) by EcoRI-Eco105I digestion and ligation. For insertion of GFP between the predicted globular PEN2 enzyme and the C-terminal extension two PEN2 C-terminal fragments were PCR amplified (oligonucleotide primer pairs: 5’-GTTATTACGTATGGTCATTGC-3’ / 5’-CTCTGGTACCATCTGGAATATCTCCGGAGGCTTGATCAAACCTCAAGAACTC-3’ [nucleotide 1337 to 1488 of the cDNA] and 5’-CAAGGGTACCGAGGCCCAAGAAGACGATTCTTCGACG-3’ / 5’-GCAGGAATTCTCAATTATTAGCTCCTTTGAAG-3’ [nucleotide1486 to 1683 of the cDNA]). After digestion with SnaBI-KpnI and KpnI-EcoRI, respectively, the original WT sequence (nucleotide 1337 to 1683 of the cDNA) was exchanged with these fragments via SnaBI-EcoRI restriction and ligation. The resulting construct harbours BseAI and KpnI restriction sites which were used for in-frame insertion of PCR amplified smRS-GFP (27); oligonucleotide primers: 5’-GGATTCCGGAATGAGTAAAGGAGAAGAACTTTTC-3’ / 5’-CTCTGGTACCTTTGTATAGTTCATCCATGCC-3’) by BseAI-KpnI digestion and ligation. All constructs were confirmed by sequencing. Agrobacterium tumefaciens strain GV3101 (pMP90RK) was transformed with plasmids by electroporation, and used for stable transformation of Arabidopsis pen2-1 mutants. Confocal laser-scanning microscopy Analysis of intracellular fluorescence was performed by confocal laser-scanning microscopy using a LSM 510 META microscopy system (Zeiss, Germany) or Leica TCS SP2 AOBS system (Leica, Germany) equipped with Argon ion and He-Ne lasers. The 488-nm Argon ion laser line was used to excite GFP while 543 nm (Zeiss) or 561 nm (Leica) He-Ne line was used to excite dsRed and/or propidium iodide. Images were collected in the multi-channel mode and the overlay images were generated using the software supplied with the respective microscope. The peroxisomal marker protein is described in (28), the Golgi marker protein in (29), and the mitochondrial marker in (30). Detection of mutant alleles and transgenes Genomic DNA was extracted from young leaves of Arabidopsis as described (31). Oligonucleotide sequences for PCR-based detection of mutant alleles or transgenes were as follows: eds1-2: 5’-ACACAAGGGTGATGCGAGACA-3’

5’-CGGAATTCTGCAGTCAGGTATCTGTTATTTCATCCAT-3’ NahG: 5’-ATGAAAAACAATAAACTTGGCTTGC-3’

5’-GCGTCGATGAAATCCGCCCG-3’

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npr1-1: 5’-AATCCGAGGGGATATACGGTGCC-3’ 5’-AAGAGGGAGGAACATCT CTAGGAAT-3’ polymorphism revealed with MnlI

pad4-1: 5’-GCGATGCATCAGAAGAG-3’ 5’-TTAGCCCAAAAGCAAGTATC-3’ polymorphism revealed with BsmFI

pen1-1: 5’-CAACGAAACACTCTCTTCATGTCACGC-3’ 5’-CATCAATTTCTTCCTGAGAC-3’

polymorphism revealed with MluI pen2-1: 5’-TTTGGAACTGCTTCATCTTCTTATCAGG-3’ 5’-CCTGTACAAGAAAT CAATCACAGATCTTCA-3’

polymorphism revealed with BspPI sag101 T-DNA insert: 5’-GGTGCAGCAAAACCCACACTTTTACTTC-3’

5’-CACGC GTCCGAAGATCTTGGAGATACATA-3’ sag101 wild-type allele: 5’-CACGCGTCCGAAGATCTTGGAGATACATA-3’

5’-AC TTCCGGGTGTTCATAAACTCGGTCAAG-3’

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Supplementary Figure Legends

Fig. S1. (A) Light microscopic view of a P. infestans sporeling on an Arabidopsis wild-

type leaf two days post inoculation (upper panel). Failure of oomycete pathogen entry

into an attacked epidermal cell correlates with deposition of callose underneath the

appressorium (stained with aniline blue; right half). Scale bar, 25 µm. ap, appressorium;

ec, empty cyst, gt, germ tube; pa, papilla; nh, non-invasive hypha. Invasive growth of P.

infestans on pen2-1 (lower panel). Invasive infection structures become encased in

autofluorescent material (right half). Scale bar, 25 µm. ap, appressorium; ec, empty cyst;

gt, germ tube; ih, invasive hypha; enc, autofluorescent encasement. (B) Epidermal cell

death-associated autofluorescence of B. g. hordei inoculated leaves under UV light

excitiation at five days post spore inoculation. (C) Phytophthora infestans droplet

inoculations (5x105 zoospores/ml) reveal enhanced cell death-associated

autofluorescence (upper panel) and accumulation of hydrogen peroxide (middle panel;

DAB staining) on pen2-1 mutant plants compared to WT 3 days post zoospore

inoculation. Enhanced cell death at inoculation sites of pen2-1 leaves is visualized by

trypan blue staining of cells that are committed to die (bottom panel). (D) Enhanced

disease susceptibility of pen2-2 plants to the necrotrophic ascomycete P. cucumerina

scored by fresh weight reduction at 14 days post spore inoculation. Since defense

responses to P. cucumerina are SA-dependent, fresh weight reduction of a NahG

expressing line was used as positive control.

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Fig. S2. (A) Map-based cloning and structure of the PEN2 locus. CAPS and SSLP

marker analysis located pen2 in a 7.4 cM target interval bordered by markers F18O19 (on

BAC F18O19) and F4I18 (on BAC F4I18) on the bottom of chromosome 3. To fine-map

PEN2, 864 F2 plants from a cross between pen2-1 (in Col-3, gl1 background) and Ler

were screened for individuals with recombination break points between these markers.

Twenty-four recombinants were recovered and their genotypes at the PEN2 locus were

deduced by scoring F3 progeny for entry resistance to B. g. hordei. Figures in brackets are

the numbers of plants with recombinations between PEN2 and the indicated marker in

telomeric (t) or centromeric (c) direction. Use of additional CAPS and SSLP-like markers

allowed delimitation to an interval of approximately 18 kb (bordered by markers SNP1

and SNP4 on BAC F4I1) harbouring two predicted ORFs (At2g44480 and At2g44490).

Sequencing of pen2-1 and pen2-3 derived cDNAs and genomic sequences revealed single

stop codon mutations at the indicated positions of the coding regions of At2g44490,

whereas no sequence polymorphism was found for At2g44480. The T-DNA insertion in

mutant pen2-2 is located in the sixth intron of At2g44490. (B) Comparison of PEN2 with

F1GHs from Trifolium repens (TrCBG) and Zea mays (ZmGlu1). Amino acid residues

thought to confer catalytic activity, glycone binding, and aglycone specificity in ZmGlu1

are indicated by #, ∆, and *, respectively. The conserved F1GH N-terminal signature is

boxed. The peptide sequence used to generate a PEN2-specific antibody is underlined.

The low complexity region in the unique C-terminus is indicated by †. The C-terminal

deletion in PEN2∆28 expressing transgenic lines is indicated by an open triangle (∆28).

Positions of stop codon mutations and the T-DNA insertion in pen2 mutant alleles are

labeled by vertical lines and a black triangle, respectively. (C) Immunoblot analysis using

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a PEN2-specific antibody. A signal corresponding to the predicted size of PEN2 is

detected in crude leaf protein extracts derived from WT and pen1-1 plants but is absent in

all pen2 mutants. Ponceau S detection of RuBisCo (ribulose-1,5-bisphosphate-

carboxylase-oxygenase) on the membrane used as protein loading control. (D) Schematic

representation of transgene expression cassettes introduced in pen2-1 mutant plants.

Acronyms for each construct are shown on the left. Gene expression is driven by native 5'

regulatory sequences (orange box; PPEN2). The wild-type cDNA is indicated by a white

box. Wild-type glutamic acids participating in catalysis are indicated by E183 and E398,

respectively. The red-coloured D183 indicates the exchange of the wild-type glutamate in

amino acid position 183 to aspartic acid, rendering the expressed protein catalytically

inactive. The full-length wild-type C-terminal extension (C-t.) is indicated by a yellow

box.

Fig. S3. (A) Vesicle-like bodies (VLBs) identified in Fig.2C co-localize with an RFP

marker protein targeted to peroxisomes. The left panel shows PEN2-GFP-tagged VLBs,

the middle panel RFP-tagged peroxisomes. The right panel shows an overlay of left and

middle panels. Note that PEN2-GFP forms a halo around the peroxisome matrix marked

by RFP. Scale bar, 1 µm. (B) Subcellular fractionation of PEN2. Immunoblot analysis of

crude leaf protein extracts and microsomal fractions. Total leaf microsomes of wild-type

Arabidopsis were separated by differential centrifugation in 20,000 and 100,000 g

fractions (20 K and 100 K, respectively; S, supernatant; P, microsomal pellet). Note the

presence of PEN2 in both supernatant and microbody pellets of 20 K (and lower amounts

in 100 K microsomal fractions). Plasma membrane vesicles were enriched from total leaf

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microsomes by using the two-phase partitioning method. A duplicate blot was probed

with an antibody for the plasma membrane (PM)-resident H+-ATPase to demonstrate

enrichment of PM vesicles. The seemingly different electrophoretic forms of PEN2 in

microsomal membrane and soluble fractions do not represent PEN2 variants since mixing

experiments of the respective fractions detected consistently only one molecular species.

The apparent PEN2 mobility differences in soluble (S) and membrane (P) fractions are

likely due to the highly diverse protein composition (see Ponceau S staining) as well as

the presence of lipids in the membrane fractions. (C) Epiphytic E. pisi growth on leaves

of WT and the indicated mutant genotypes seven days post conidiospore inoculation.

Light microscopic pictures were taken after visualization of fungal structures using

Coomassie brilliant blue. Inserts show close up views of E. pisi conidiophores on the

respective genotypes. Scale bar, 200 µm. (D) Scanning electron microscopy of cryo-fixed

B. g. hordei (left) and E. pisi (right) conidiophores seven days post spore inoculation on

pen2-1 pad4-1 sag101-2 Arabidopsis leaves. Scale bar, 100 µm. bc=conidiophore basal

cell, a characteristic morphological feature of B. g. hordei; ic=incipient conidiophore

differentiation; mc=mature conidiophore.

WT pen1-1 pen2-2 NahG0

20

40

60

80

100

fresh

wei

ghtr

educ

tion

(%)

D

A

WT pen1-1

pen2-1pen1-1pen2-1

B

WT

ap

ec

gt

nh pa

pen2-1

ap

ec

ih

gt

enc

C WT pen2-1

fluor

esce

nce

mic

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DA

B s

tain

ing

tryp

anbl

uest

aini

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Figure S1

F6E13 (1c)

SNP1 (1c) SNP4 (1t)

F18O19F18O19

F6E13 F16B22

F4L23 F4I18

F4I1 T13E15

T14P1 F17K2

nga168 GBF3

F18O19 (2c)

F4I1C (7c) F16B

22 (1

t)

F4L2

3 (14

t)

F4I18

(2t)

Centromere Telomere

At2g44480 At2g44490

pen2-1* = stop

*

pen2-2T-DNA

*

pen2-3* = stop

A

B

Cpen2-1 pen2-2 pen2-3 pen1-1WT

αPEN2

RuBisCo

D PPEN2::PEN2 C-t.E183 E398PPEN2

PPEN2::pen2E183D C-t.D183 E398PPEN2

PPEN2::pen2∆28 E183 E398PPEN2

AtPEN2 -------------------------------MAHLQRTFPTEMSK----------------------------GRASFPKGFLFGTASSSYQYEGAVNEGARGQSVWDHFSNRF 55TrCBG -------------------------------FKPLPISFDDFSDL----------------------------NRSCFAPGFVFGTASSAFQYEGAAFEDGKGPSIWDTFTHKY 55ZmGLU1 MAPLLAAAMNHAAAHPGLRSHLVGPNNESFSRHHLPSSSPQSSKRRCNLSFTTRSARVGSQNGVQMLSPSEIPQRDWFPSDFTFGAATSAYQIEGAWNEDGKGESNWDHFCHNH 114

AtPEN2 PHRISDSSDGNVAVDFYHRYKEDIKRMKDINMDSFRLSIAWPRVLPYGKRDRGVSEEGIKFYNDVIDELLANEITPLVTIFHWDIPQDLEDEYGGFLS---EQIIDDFRDYASL 166TrCBG PEKIKDRTNGDVAIDEYHRYKEDIGIMKDMNLDAYRFSISWPRVLPKGKLSGGVNREGINYYNNLINEVLANGMQPYVTLFHWDVPQALEDEYRGFLG---RNIVDDFRDYAEL 166ZmGLU1 PERILDGSNSDIGANSYHMYKTDVRLLKEMGMDAYRFSISWPRILPKGTKEGGINPDGIKYYRNLINLLLENGIEPYVTIFHWDVPQALEEKYGGFLDKSHKSIVEDYTYFAKV 230

# * *AtPEN2 CFERFGDRVSLWCTMNEPWVYSVAGYDTGRKAPGRCSKYVNGASVAGMSGYEAYIVSHNMLLAHAEAVEVFR-KCDHIKNGQIGIAHNPLWYEPYDPSDPDDVEGCNRAMDFML 279TrCBG CFKEFGDRVKHWITLNEPWGVSMNAYAYGTFAPGRCSDWLKLNCTGGDSGREPYLAAHYQLLAHAAAARLYKTKYQASQNGIIGITLVSHWFEPAS-KEKADVDAAKRGLDFML 279ZmGLU1 CFDNFGDKVKNWLTFNEPQTFTSFSYGTGVFAPGRCSPGLDCAYPTGNSLVEPYTAGHNILLAHAEAVDLYN-KHYKRDDTRIGLAFDVMGRVPYG-TSFLDKQAEERSWDINL 340

*AtPEN2 GWHQHPTACGDYPETMKKSVGDRLPSFTPEQSKKLIGSCDYVGINYYSSLFVKSIKHVDPTQPTWRTDQGVDWMKTNI-DGKQIAKQGGSEWSFTYPTGLRNILKYVKKTYGNP 392TrCBG GWFMHPLTKGRYPESMRYLVRKRLPKFSTEESKELTGSFDFLGLNYYSSYYAAKAPRIPNARPAIQTDSLIN-ATFEH-NGKPLGPMAASSWLCIYPQGIRKLLLYVKNHYNNP 389ZmGLU1 GWFLEPVVRGDYPFSMRSLARERLPFFKDEQKEKLAGSYNMLGLNYYTSRFSKNIDISPNYSPVLNTDDAYASQEVNGPDGKPIGPPMGNPWIYMYPEGLKDLLMIMKNKYGNP 454

# ∆ ∆∆* _AtPEN2 PILITENGYGEVAEQSQSLYMYNPSIDTERLEYIEGHIHAIHQAIHEDGVRVEGYYVWSLLDNFEWNSGYGVRYGLYYIDYKDGLRRYPKMSALWLKEFLRFDQEDDSSTSKKE 506TrCBG VIYITENGRNEFNDP--TLSLQESLLDTPRIDYYYRHLYYVLTAIG-DGVNVKGYFAWSLFDNMEWDSGYTVRFGLVFVDFKNNLKRHPKLSAHWFKSFLKK------------ 490ZmGLU1 PIYITENGIGDVDTKETPLPMEAALNDYKRLDYIQRHIATLKESID-LGSNVQGYFAWSLLDNFEWFAGFTERYGIVYVDRNNNCTRYMKESAKWLKEFNTAKKPSKKILTPA- 566

_______________AtPEN2 EKKESYGKQLLHSVQDSQFVHSIKDSGALPAVLGSLFVVSATVGTSLFFKGANN 560TrCBG ------------------------------------------------------ZmGLU1 ------------------------------------------------------

loop A

loop B

loop C

loop D

AtPEN2 -------------------------------MAHLQRTFPTEMSK----------------------------GRASFPKGFLFGTASSSYQYEGAVNEGARGQSVWDHFSNRF 55TrCBG -------------------------------FKPLPISFDDFSDL----------------------------NRSCFAPGFVFGTASSAFQYEGAAFEDGKGPSIWDTFTHKY 55ZmGLU1 MAPLLAAAMNHAAAHPGLRSHLVGPNNESFSRHHLPSSSPQSSKRRCNLSFTTRSARVGSQNGVQMLSPSEIPQRDWFPSDFTFGAATSAYQIEGAWNEDGKGESNWDHFCHNH 114

AtPEN2 PHRISDSSDGNVAVDFYHRYKEDIKRMKDINMDSFRLSIAWPRVLPYGKRDRGVSEEGIKFYNDVIDELLANEITPLVTIFHWDIPQDLEDEYGGFLS---EQIIDDFRDYASL 166TrCBG PEKIKDRTNGDVAIDEYHRYKEDIGIMKDMNLDAYRFSISWPRVLPKGKLSGGVNREGINYYNNLINEVLANGMQPYVTLFHWDVPQALEDEYRGFLG---RNIVDDFRDYAEL 166ZmGLU1 PERILDGSNSDIGANSYHMYKTDVRLLKEMGMDAYRFSISWPRILPKGTKEGGINPDGIKYYRNLINLLLENGIEPYVTIFHWDVPQALEEKYGGFLDKSHKSIVEDYTYFAKV 230

# * *AtPEN2 CFERFGDRVSLWCTMNEPWVYSVAGYDTGRKAPGRCSKYVNGASVAGMSGYEAYIVSHNMLLAHAEAVEVFR-KCDHIKNGQIGIAHNPLWYEPYDPSDPDDVEGCNRAMDFML 279TrCBG CFKEFGDRVKHWITLNEPWGVSMNAYAYGTFAPGRCSDWLKLNCTGGDSGREPYLAAHYQLLAHAAAARLYKTKYQASQNGIIGITLVSHWFEPAS-KEKADVDAAKRGLDFML 279ZmGLU1 CFDNFGDKVKNWLTFNEPQTFTSFSYGTGVFAPGRCSPGLDCAYPTGNSLVEPYTAGHNILLAHAEAVDLYN-KHYKRDDTRIGLAFDVMGRVPYG-TSFLDKQAEERSWDINL 340

*AtPEN2 GWHQHPTACGDYPETMKKSVGDRLPSFTPEQSKKLIGSCDYVGINYYSSLFVKSIKHVDPTQPTWRTDQGVDWMKTNI-DGKQIAKQGGSEWSFTYPTGLRNILKYVKKTYGNP 392TrCBG GWFMHPLTKGRYPESMRYLVRKRLPKFSTEESKELTGSFDFLGLNYYSSYYAAKAPRIPNARPAIQTDSLIN-ATFEH-NGKPLGPMAASSWLCIYPQGIRKLLLYVKNHYNNP 389ZmGLU1 GWFLEPVVRGDYPFSMRSLARERLPFFKDEQKEKLAGSYNMLGLNYYTSRFSKNIDISPNYSPVLNTDDAYASQEVNGPDGKPIGPPMGNPWIYMYPEGLKDLLMIMKNKYGNP 454

# ∆ ∆∆* _AtPEN2 PILITENGYGEVAEQSQSLYMYNPSIDTERLEYIEGHIHAIHQAIHEDGVRVEGYYVWSLLDNFEWNSGYGVRYGLYYIDYKDGLRRYPKMSALWLKEFLRFDQEDDSSTSKKE 506TrCBG VIYITENGRNEFNDP--TLSLQESLLDTPRIDYYYRHLYYVLTAIG-DGVNVKGYFAWSLFDNMEWDSGYTVRFGLVFVDFKNNLKRHPKLSAHWFKSFLKK------------ 490ZmGLU1 PIYITENGIGDVDTKETPLPMEAALNDYKRLDYIQRHIATLKESID-LGSNVQGYFAWSLLDNFEWFAGFTERYGIVYVDRNNNCTRYMKESAKWLKEFNTAKKPSKKILTPA- 566

_______________AtPEN2 EKKESYGKQLLHSVQDSQFVHSIKDSGALPAVLGSLFVVSATVGTSLFFKGANN 560TrCBG ------------------------------------------------------ZmGLU1 ------------------------------------------------------

loop Aloop A

loop B

loop Cloop C

loop Dloop D

∆28†††††

††††††††††

pen2-3 stop

pen2-1 stop

pen2-2 T-DNA insertion

Figure S2

B

crud

eex

tract S

20 K P

20 K S

100 K P

100 K PM

αPEN2

RuBisCo

αPM-ATPase

A

sag101

pen2 pad4 sag101pen2 pad4pen2 pen2 sag101

pad4WT pad4 sag101eds1

pen2 eds1

C

D

mc

ic

bc

mc

Figure S3

11

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