running head: expression of fungal pg decreases ......2007/12/07 · lionetti, daniela bellincampi,...

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Running head: Expression of fungal PG decreases susceptibility to pathogens.

Corresponding author: Giulia De Lorenzo. Dipartimento di Biologia Vegetale, Università

degli Studi di Roma La Sapienza, 00185 Rome, Italy; Phone: +39-06-49912454 ; fax:

+39-06-49912446 ; e-mail: [email protected].

Journal research area: Plants Interacting with Other Organisms

Plant Physiology Preview. Published on December 7, 2007, as DOI:10.1104/pp.107.109686

Copyright 2007 by the American Society of Plant Biologists

https://plantphysiol.orgDownloaded on May 5, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

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Transgenic Expression of a Fungal Endo-Polygalacturonase Increases Plant

Resistance to Pathogens and Reduces Auxin Sensitivity.1

Simone Ferrari2, Roberta Galletti2, Daniela Pontiggia2, Cinzia Manfredini, Vincenzo

Lionetti, Daniela Bellincampi, Felice Cervone, Giulia De Lorenzo*

Dipartimento di Biologia Vegetale, Università degli Studi di Roma La Sapienza, Piazzale

Aldo Moro 5, 00185 Roma, Italy.



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FOOTNOTES 1 This work was supported by the Ministero dell’Università e della Ricerca (Fondo per gli

Investimenti della Ricerca di Base RBNE01KZE7 and PRIN 2005052297), by the

Giovanni Armenise-Harvard Foundation and the Institute Pasteur-Fondazione Cenci

Bolognetti. 2 These authors contributed equally to this work.

*Corresponding author: Giulia De Lorenzo. Fax: +39-06-49912446 ; e-mail:

[email protected].



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ABSTRACT

Polygalacturonases (PGs), enzymes that hydrolyze the homogalacturonan (HGA) of the

plant cell wall, are virulence factors of several phytopathogenic fungi and bacteria. On

the other hand, PGs may activate defence responses by releasing oligogalacturonides

(OGs), perceived by the plant cell as host-associated molecular patterns. Tobacco and

Arabidopsis plants expressing a fungal PG (PG plants) have a reduced content of HGA.

Here we show that PG plants are more resistant to microbial pathogens and have

constitutively activated defence responses. Interestingly, either in tobacco PG or wild

type plants treated with OGs, resistance to fungal infection is suppressed by exogenous

auxin, whereas sensitivity to auxin of PG plants is reduced in different bioassays. The

altered plant defence responses and auxin sensitivity in PG plants may reflect an

increased accumulation of OGs and subsequent antagonism of auxin action.

Alternatively, it may be a consequence of perturbations of cellular physiology and

elevated defence status as a result of altered cell wall architecture.



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INTRODUCTION

The plant cell wall possesses mechanical features determining strength and

plasticity of a tissue and signalling properties affecting expansion, growth and

development (Carpita and McCann, 2000). The cell wall also represents a barrier against

invading microorganisms. During early stages of infection, pathogens produce enzymes

which degrade the various components of the wall, thus releasing compounds that are

used as carbon sources. The major components of primary cell walls of higher plants are

complex polysaccharides; increasing evidence indicates that these molecules contribute

to disease resistance not just as mechanical barriers but also as sensors for incoming

infections (Vorwerk et al., 2004). For example, partial degradation of homogalacturonan

(HGA) by fungal endo-polygalacturonases (PGs) releases oligogalacturonides with a

degree of polymerization between 10 and 15 (OGs) that show elicitor activity (Cervone et

al., 1987a; Cervone et al., 1987b; Cervone et al., 1989). Treatment with OGs causes the

accumulation of reactive oxygen species (ROS), the biosynthesis of phytoalexins (Hahn

et al., 1981) and the expression of pathogenesis-related (PR) proteins (Davis and

Hahlbrock, 1987; Broekaert and Pneumas, 1988) in several plant species. In Arabidopsis,

OGs induce the expression of several defence genes and proteins Ferrari et al., 2003b;

Casasoli et al., 2007; Ferrari et al., 2007), including AtPGIP1 and PAD3, which encode,

respectively, an inhibitor of fungal PGs and the cytochrome P450 CYP71B15 that

catalyzes the last step of camalexin biosynthesis. Exogenous OGs protect Arabidopsis

and grapevine leaves against the necrotrophic pathogen Botrytis cinerea (Aziz et al.,

2004; Ferrari et al., 2007). In analogy with the role of hyaluronan fragments in animal

cells, OGs may be regarded as host-associated molecular patterns involved in the innate

immunity (Stern et al., 2006; Taylor and Gallo, 2006).

Besides inducing defence responses, OGs can also affect several aspects of plant

growth and development. In particular, a number of reports from our laboratory indicate

that exogenously added OGs are able to antagonize the action of auxin, as in the case of

pea stem elongation (Branca et al., 1988), tobacco adventitious root formation

(Bellincampi et al., 1993) and pericycle cell differentiation (Altamura et al., 1998). OGs

prevent rhizogenesis in tobacco leaf explants expressing the Agrobacterium tumefaciens

rolB gene by inhibiting the auxin-induced expression of the transgene (Bellincampi et al.,



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1996). Furthermore, in tobacco OGs inhibit the induction of the late auxin-responsive

genes Nt114, rolB and rolD (Mauro et al., 2002). In cucumber seedlings, OGs allow for a

more rapid recovery of root growth in auxin-treated roots (Spiro et al., 2002). To date, the

mechanism underlying the antagonistic effect of OGs on auxin-induced responses is not

known.

We have previously generated tobacco and Arabidopsis transgenic lines (hereafter

referred to as PG plants) expressing an attenuated version of PGII of Aspergillus niger

(Capodicasa et al., 2004). PG plants of both species accumulate the enzyme in their

tissues and display morphological alterations, such as dwarfism and slightly curled

leaves, but no other severe aberrations (Capodicasa et al., 2004). These phenotypes are

dependent on the enzyme activity, since expression of an inactive form of A. niger PGII

has no obvious impact on Arabidopsis development. Moreover, crossing tobacco PG

plants with a line expressing high levels of the bean polygalacturonase-inhibiting protein

PvPGIP2, an inhibitor of A. niger PGII, completely blocked PG activity and reverted the

dwarf phenotype. The observed morphological alterations are associated to a reduced

content of HGA in both tobacco and Arabidopsis, suggesting that the phenotype of PG

plants is due to an enhanced degradation of HGA (Capodicasa et al., 2004). Interestingly,

tobacco PG plants show no alterations of ion-mediated increase in xylem hydraulic

conductivity (Nardini et al., 2007) and have photosyntesis rate and stomatal conductance

similar to those of wild-type (WT) plants (Galletti R. and Pontiggia D., unpublished).

Therefore, PG plants, despite their growth defects, do not show alterations which are

typical of plants suffering abiotic stresses, such as water stress (Flexas and Medrano,

2002).

Since the signalling potential of pectin and pectin-derived fragments may be

critical for the outcome of a plant-pathogen interaction (Vorwerk et al., 2004), we have

investigated whether the modifications caused by the expression of PG affect responses

of plants to microbial pathogens. Here we show that PG plants exhibit enhanced

resistance to the necrotrophic fungal pathogen Botrytis cinerea and to the virulent

bacterial pathogen Pseudomonas syringae, and constitutive expression of defence

responses. Notably, tobacco PG plants show a susceptibility to B. cinerea similar to WT

plants when treated with auxin, as well as a reduced sensitivity to auxin in different



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bioassays. Furthermore, we show that auxin inhibits OG-induced resistance in WT

tobacco plants. The relationship between pectin degradation, auxin responses and defence

against pathogens is discussed.

RESULTS

PG plants are less susceptible to pathogen infection.

Tobacco PG plants were inoculated with B. cinerea and their susceptibility to the

fungus was compared to that of WT plants and plants expressing the bean PvPGIP2,

which inhibits several fungal PGs, including B. cinerea PG (PGIP2 plants; Leckie et al.,

1999; Capodicasa et al., 2004). Upon infection, WT plants exhibited typical soft rot

symptoms with rapidly expanding water-soaked lesions, whereas PG plants displayed

lesions with a reduced size (Fig. 1A). Furthermore, inoculation of PG plants resulted in

dry lesions unable to develop beyond the inoculation site in a significant number of cases

(Fig. 1B). PGIP2 plants also displayed reduced lesions that, unlike the lesions observed in

PG plants, were typically water-soaked (Fig. 1A and B). Leaves from plants obtained by

crossing line PG16 with the PGIP2 line (PG16×PGIP2 plants), expressing PGIP2 in large

excess with respect to PG and showing no detectable PG activity in their tissues

(Capodicasa et al., 2004), exhibited symptoms comparable to those developed on PGIP2

plants (Fig.1A and B).

Arabidopsis Wassiliewskija (Ws) PG plants also displayed a significant reduction

of lesion size after fungal inoculation (Fig 1C). Line PG1, expressing the highest levels of

PG (Capodicasa et al., 2004), developed smaller lesions (Fig. 1C) and showed a

significant reduction of the number of spreading lesions, compared to the other lines (Fig.

1D). Transcript levels of the B. cinerea actin gene Bcact were dramatically reduced in

inoculated PG1 plants (Supplementary Fig. S1), confirming that the reduced lesion

development mirrored a reduced fungal growth. On the other hand, leaves of a transgenic

line (PG201), expressing an inactive form of A. niger PGII (van Santen et al., 1999;

Federici et al., 2001), showed symptoms similar to those of WT plants (Fig. 1C and D),

indicating that enzymatic activity is required to confer enhanced resistance.



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To determine whether the enhanced resistance observed in PG plants is specific

for B. cinerea or is effective against other pathogens, leaves of tobacco WT and PG

plants were inoculated with a virulent strain of the bacterial pathogen Pseudomonas

syringae pv. tabaci. Infiltration with a bacterial suspension produced the collapse of the

inoculated area in WT plants but no visible symptoms in line PG16, and only very mild

symptoms in lines PG5 and 7 (Fig. 2). PGIP2 and PG16×PGIP2 plants showed symptoms

similar to those observed in WT plants (Fig. 2). This indicates that PvPGIP2 has no effect

on resistance to P. syringae and, more importantly, that inhibition of enzymatic activity

of PG by PvPGIP2 completely blocks PG-mediated resistance. An active and free

(uncomplexed) PG is therefore required to confer resistance to plants against organisms

as different as the fungal pathogen B. cinerea and the bacterial pathogen P. syringae.

PG plants have enhanced defence responses.

PGs may act not only as virulence factors of phytopathogenic microorganisms,

but may also induce defence responses, by either releasing endogenous OG elicitors or

affecting cell wall integrity (Hahn et al., 1981; Davis et al., 1986; Cervone et al., 1987a).

Staining of leaves with 3-3’-diaminobenzidine (DAB) revealed accumulation of H2O2 in

tobacco PG16 and PG7 and, to a lesser extent, in PG5 leaves (Fig. 3A). No significant

staining was observed in WT, PGIP2 and PG16×PGIP2 plants, indicating that

accumulation of H2O2 correlates with the level of active PG in the tissues. Similarly,

constitutive accumulation of H2O2 was observed in leaves of Arabidopsis PG1 and, to a

lesser extent, PG5 plants, whereas accumulation of H2O2 in Arabidopsis WT and PG201

plants was restricted to the severed petioles (Fig. 3B). Leaves of tobacco and Arabidopsis

PG plants exhibited a bright fluorescence when illuminated with UV light, possibly due

to the accumulation of secondary metabolites (Supplementary Fig. S2).

We also determined total peroxidase and 1,3-β-glucanase enzymatic activities,

which are known to be regulated by biotic stress (Van Loon and van Strien, 1999). Total

peroxidase activity was significantly higher in leaves of tobacco plants PG16 and PG7 as

compared to control plants, whereas in PG5 as well as in PG16×PGIP2 plants it was only

slightly, but still significantly, higher than in WT plants or plants expressing PvPGIP2

alone (Fig. 4A). Analysis of peroxidase activity in the intercellular washing fluids (IWFs)



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prepared from control and transgenic tobacco plants indicated that the increase of

peroxidase activity of PG plants was mainly due to extracellular isoforms (Fig. 4B).

Peroxidase activity was also significantly increased in leaves of Arabidopsis plants

belonging to line PG1, compared to the other genotypes (Fig. 4C). Basal 1,3-β-glucanase

activity was significantly higher only in tobacco and Arabidopsis lines expressing high

levels of PG (Fig. 5A and B).

Finally, we examined the expression of genes potentially involved in pathogen

responses. Expression of two tobacco genes, EAS1/2, encoding a 5-epi-aristolochene

synthase required for the biosynthesis of the phytoalexin capsidiol (Facchini and

Chappell, 1992), and POX, a gene encoding an apoplastic anionic peroxidase (Diaz-De-

Leon et al., 1993), was clearly induced by B. cinerea infection in both WT and PG16

plants (Fig. 6A). However, expression of POX was detectable also in uninfected PG16

plants and increased earlier and to a greater extent in response to fungal infection (Fig.

6A). EAS1/2 mRNA levels were not constitutively expressed in PG16 plants, but

increased earlier and to a larger extent in response to fungal infection (Fig. 6A). In

Arabidopsis WT plants, expression of AtPGIP1, of Pathogenesis-Related protein 1 (PR-

1) and of the defensin gene PDF1.2 steadily increased up to two days after infection with

B. cinerea (Fig. 6B-D). The basal mRNA levels of all three genes were higher in PG1

plants than in WT plants (Fig. 6B-D). However, their expression differed after

inoculation with B. cinerea. AtPGIP1 expression increased at 1 dpi, but returned to basal

levels at 2 dpi (Fig. 6B). In contrast, transcripts of PR-1 and PDF1.2 showed only a mild

increase after fungal infection of PG plants (Fig. 6C-D). These findings are consistent

with previous data correlating the expression levels of both genes to fungal lesion

development rather than to basal resistance (Ferrari et al., 2003a).

A possible explanation for the enhanced expression of defence genes observed in

healthy PG plants is the accumulation of OG elicitors released from the cell wall by the

action of the fungal PG. We therefore determined whether the expression of these genes

is up-regulated by exogenous OGs. Both POX and EAS1/2 mRNA levels increased in

tobacco leaf explants treated for 4 hours with OGs (Fig. 7A and B). Similarly, AtPGIP1

and PR-1 transcripts accumulated in response to OGs in Arabidopsis WT seedlings (Fig.

8A and B), while no significant increase of PDF1.2 mRNA levels was observed (Fig.



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8C). Taken together, these data support the hypothesis that the constitutive expression of

at least some of the defence responses observed in PG plants is mediated by OGs released

by the fungal enzyme expressed in the transgenic plants.

Increased resistance of tobacco PG plants is abolished by auxin.

Because OGs have auxin-antagonistic activity and treatment with OGs leads to a

decreased sensitivity to auxin in tobacco plants (Branca et al., 1988; Bellincampi et al.,

1993; Bellincampi et al., 1996; Altamura et al., 1998; Mauro et al., 2002), a support for

the possible involvement of OGs in the phenotype of PG plants might be provided by the

ability of auxin to revert some of the phenotypic features, as well as by a decreased

sensitivity to auxin of the PG plants.

We therefore investigated whether the increased resistance against B. cinerea of

the tobacco PG plants could be reverted by exogenous auxin. Leaf discs from WT and

PG16 tobacco plants were treated with 3-indoleacetic acid (IAA) and then inoculated

with B. cinerea. Notably, auxin pre-treatment of PG16 leaf discs restored their

susceptibility to a level comparable to that of WT plants, whereas it did not significantly

increase the susceptibility of WT leaf discs (Fig. 9A). Interestingly, whereas pre-

treatment of WT leaf discs with exogenous OGs reduced lesion development after B.

cinerea inoculation, co-treatment with OGs and IAA did not produce any effect and the

susceptibility was comparable to that of untreated tissues (Fig. 9B).

We also evaluated the sensitivity to auxin of tobacco PG plants by analysing the

auxin-induced root formation in leaf explants. While a concentration of 0.57 µM of IAA

was sufficient to induce rhizogenesis in WT explants, no roots were observed in PG

explants treated with up to 1.7 µM IAA; however, at higher concentrations, IAA induced

root formation in PG explants in a dose-dependent fashion (Fig. 10A). The ability of PG

plants to respond to auxin was also tested using a root growth inhibition assay. WT

plantlets showed a significant reduction of primary root growth in the presence of 10-7 M

IAA, whereas concentrations of IAA up to 10-6 M were not effective in PG16 plants (Fig.

10B). However, when auxin concentration was increased to 10-5 M, a reduction of root

length was observed in PG16 plants, consistently with the hypothesis that they are not

completely resistant but only less sensitive to auxin.. Taken together, these results



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indicate that auxin-induced developmental and growth responses are impaired in PG

plants. This is not due to increased catabolism of IAA, since no significant IAA oxidase

activity could be detected in extracts from PG leaves (data not shown). We therefore

conclude that auxin signaling is partly compromised in PG plants.

Because high concentrations of IAA induce ethylene production in several plant

species (Abeles, 1966), and ethylene-controlled responses play a role in both resistance

against B. cinerea (Thomma et al., 1999; Diaz et al., 2002; Chague et al., 2006) and

inhibition of root development (Ortega-Martinez et al., 2007; Stepanova et al., 2007), we

investigated whether PG plants show increased ability to produce ethylene. Ethylene

produced by WT and PG16 leaf explants was measured after water or IAA treatment.

Water-treated WT or PG16 leaf explants did not release any detectable accumulation of

ethylene; in contrast, 1-aminocyclopropane-1-carboxylic acid (ACC) induced high levels

of ethylene in both genotypes, indicating that they have similar ability of producing this

hormone (Fig. 11). Furthermore, treatment with 100 µM IAA resulted in the

accumulation of lower, but significant and comparable levels of ethylene in WT and

PG16 explants (Fig. 11). OGs had no significant impact on ethylene production in water-

or IAA-treated WT tobacco explants (data not shown). A role of ethylene in the basal

resistance, in the IAA-mediated reversion of the resistant phenotype and in the reduced

ability to form adventitious roots in response to IAA of tobacco PG plants is therefore

unlikely.

DISCUSSION

In this paper we have shown that the expression of a fungal PG in Arabidopsis

and tobacco increases plant resistance to microbial pathogens. The resistant phenotype is

not exhibited by transgenic tobacco plants expressing both PG and its inhibitor PvPGIP2,

and by Arabidopsis plants expressing a mutagenized and inactive AnPGII (PG201),

indicating that resistance is dependent on the enzymatic activity of the expressed PG and

is likely a consequence of the degradation of the host pectin accomplished by the

enzyme. Interestingly, PG plants appear to have reduced sensitivity to exogenous auxin,

and auxin treatments revert their resistant phenotype. The data presented here allow to

consider the reasons that might explain why heterologous expression of a fungal PG



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increases plant resistance, providing a link between HGA degradation, auxin perception

and activation of defence responses.

PG plants have an altered pectin composition with a reduced galacturonic acid

and HGA content (Capodicasa et al., 2004). A direct negative effect of the lack of

consumable galacturonic acid on pathogen growth appears unlikely, because we did not

observe any significant reduction of growth when B. cinerea was cultivated in the

presence of cell walls from PG plants as the sole carbon source (data not shown). On the

other hand, PG plants constitutively express a number of defence responses, such as the

accumulation of UV-fluorescent metabolites, H2O2, β-1,3-glucanase and peroxidase, and

expression of defence-related genes that are normally induced only in the presence of

pathogens. High levels of H2O2 in PG plants are concomitant with an increased

peroxidase activity, which at least in tobacco corresponds to an anionic and apoplastic

enzyme possibly encoded by the constitutively expressed POX gene (see Fig. 6A). In the

presence of H2O2, peroxidases may generate free radicals that have direct antimicrobial

activity (Bolwell et al., 2002) or may modify the plant cell wall (Passardi et al., 2004).

On the other hand, H2O2 itself may be responsible for the induction of PR proteins

(Apostol et al., 1989; Chen et al., 1993). Whether the high peroxidase activity and

elevated H2O2 levels in PG plants directly contribute to their increased resistance to

pathogens still needs to be determined. Besides causing the constitutive activation of

defence responses, PG expression makes plants more prone to respond to infection.

Following B. cinerea inoculation tobacco PG plants accumulate more POX and EAS1/2

transcripts than WT plants, and Arabidopsis plants show enhanced expression of

AtPGIP1.

The enhanced expression of diverse defence responses is likely the ultimate

reason for their increased resistance to pathogens. We could not observe a perfect

correlation between the levels of a specific defence response and the degree of resistance

observed in independent PG lines, suggesting that multiple defence responses, activated

to different extents in different PG lines, contribute to the final resistant phenotype. The

observation that tobacco PG16×PGIP2 plants or Arabidopsis PG201 plant, which have no

PG activity, display neither altered expression of defence responses nor increased

resistance, indicate that these phenotypes are mediated by HGA degradation in PG plants,



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rather than by the recognition per se of the heterologous protein by plant cells. Enhanced

expression of basal and inducible defence responses and increased resistance to

pathogens were previously observed in plants expressing pectin-degrading enzymes.

Basal levels of polyphenoloxidase activity and induction of phenylalanine ammonia lyase

upon wounding are enhanced in transgenic potato tubers expressing a bacterial pectate

lyase (Wegener, 2002), and accumulation of β-1,3-glucanase and H2O2 is induced in

tobacco leaves by the transient expression of a PG from Colletotrichum lindemuthianum

(Boudart et al., 2003). Moreover, H2O2 accumulates in tomato plants that have increased

PG activity upon antisense expression of the regulatory subunit of a wound-inducible

endogenous PG (PGβS); these plants exhibit constitutive expression of wound-inducible

genes and enhanced resistance to insect attack (Orozco-Cardenas and Ryan, 2003).

One explanation for the observed phenotypes in the PG plants is that altered cell

wall integrity may be perceived as a signal of pathogen attack or mechanical damage,

leading to the induction of defence mechanisms (Humphrey et al., 2007). Links between

pectin and cytoplasm, mediated by specific cell wall-associated transmembrane proteins,

may allow the host cell to sense alterations caused by microbial pathogens and regulate

the activation of defence responses. The observation that the Arabidopsis wall-associated

kinase WAK1 (Decreux and Messiaen, 2005), which is able to bind polygalacturonic

acid, is induced by bacterial infection, and that this induction is required for survival

upon treatments with chemical inducers of resistance, supports this hypothesis (He et al.,

1998). Apoplastic proteins that have domains interacting with pectin also include a

peroxidase (Carpin et al., 2001) and PGIP (Spadoni et al., 2006); these proteins may

participate to perceive alterations of pectin structure and transmit this information across

the plasma membrane with mechanisms not yet investigated.

Indeed, increased disease resistance is observed in plants with alterations of cell

wall structural components other than pectin. For example, mutations in an Arabidopsis

cellulose synthase (CESA3) cause not only decreased cellulose content but also

constitutive expression of defence responses (Ellis et al., 2002; Cano-Delgado et al.,

2003). More recently, it has been reported that mutations in Arabidopsis cellulose

synthase genes confer resistance to different pathogens through a mechanism that is

independent of SA, ET and JA signaling (Hernandez-Blanco et al., 2007). Defects in the



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cuticle cause complete resistance to B. cinerea, and this resistance is also independent of

SA, ET and JA (Chassot et al., 2007). It is therefore likely that plants have evolved

mechanisms to perceive the presence of a potential pathogen by monitoring the integrity

of different components of their own cell wall. However, a direct mechanistic link

between modifications of these cell wall components and activation of defence responses

is still missing.

Another possible explanation for the enhanced resistance of plants with altered

cell wall components is that plant cells perceive molecules released in the apoplast as a

consequence of the hydrolysis of specific structural polymers, and this molecules act as

elicitors of defence responses. In particular, OGs generated upon degradation of HGA by

fungal PGs act as elicitors of defence responses in several plant systems (reviewed in

Ridley et al., 2001). It is possible that in planta expression of PG confers resistance to

pathogens because of the release of OGs or other wall-derived elicitors. Circumstantial

evidence supporting this hypothesis is presented here. Firstly, PG plants show enhanced

expression of defence responses similar to those observed in WT plants after addition of

exogenous OGs. ROS accumulation is a hallmark of OG-mediated responses (Ridley et

al., 2001; Aziz et al., 2004), and we could detected a dramatic increase of H2O2 levels in

both tobacco and Arabidopsis PG plants. Furthermore, we could demonstrate that

expression of EAS1/2 and POX is induced by both OGs and B. cinerea in tobacco, and

that their expression is increased in PG plants. In Arabidopsis PG plants, high basal levels

of three defence-related genes, AtPGIP1, PR-1 and PDF1.2, are also enhanced, compared

to the WT. Expression of AtPGIP1 was previously demonstrated to be responsive to OGs

(Ferrari et al., 2003b), and PR-1, a marker of SA-mediated defence responses (Delaney et

al., 1994), is also up-regulated by OGs in the Ws ecotype, though at later time points than

AtPGIP1. PDF1.2, which is not significantly regulated by OGs, but is responsive to

oxidative stress (Penninckx et al., 1996), may be high in PG plants because of the high

levels of H2O2 in their tissues. The reduced mRNA levels of both PR-1 and PDF1.2 in

inoculated PG plants reflect the fact that their expression correlates to fungal growth

(reduced in PG plants) rather than to plant resistance (Ferrari et al., 2003a; Chassot et al.,

2007). The observation that PG plants accumulate higher levels of mRNA corresponding

to genes that are responsive to OGs suggests that HGA alterations cause the activation of



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a defence-related pathway that is also activated by elicitors; this activation may be either

directly mediated by pectin fragments or by other mechanisms. It should be noted that the

levels of defence-related transcripts accumulating upon infection are dramatically higher

than those observed in either OG-treated WT plants or in untreated PG plants. This may

be due to the concomitant activation of multiple defence-related pathways during

pathogen infection and/or the massive release of different elicitors in infected tissues.

A second line of evidence suggesting that pectic fragments with elicitor activity

accumulate in PG plants is that auxin reverts the susceptibility to B. cinerea of tobacco

PG plants to WT levels, and also suppresses OG-induced protection against this fungus in

WT plants. Furthermore, auxin-dependent responses are impaired in tobacco PG plants,

and our previous work has shown that OGs suppress different responses regulated by

auxin in tobacco (Bellincampi et al., 1993; Bellincampi et al., 1996; Mauro et al., 2002).

In particular, tobacco PG plants show reduced sensitivity to IAA in experiments of

rhizogenesis in leaf explants and of inhibition of primary root growth. This reduced

sensitivity does not appear to depend on IAA degradation, as suggested by the absence of

IAA oxidase activity in PG plants.

We also show that tobacco PG plants do not differ from WT plants in their ability

to produce ethylene, and, on the other hand, OGs do not induce significant production of

ethylene in WT plants. This suggests that a role of this hormone in the basal resistance, as

well as in the IAA-mediated reversion of the resistant phenotype and in the reduced

ability to form adventitious roots in response to IAA is unlikely.

Other mechanisms, such as an alteration of the biomechanical properties of the

cell wall may be responsible for the observed responses of the PG plants. However, no

obvious correlation between auxin-induced wall loosening and auxin-mediated reversion

of the resistant phenotype of PG explants could be observed. Indeed, IAA did not

increase susceptibility in WT explants, which showed curling in response to auxin,

whereas it did so in PG explants that did not show any curling (data not shown).

Therefore, it may be argued that the loosening of the wall that leads to curling in WT

explants has no apparent effect on the susceptibility to B. cinerea, while the increased

susceptibility of PG explants in the presence of auxin occurs in the absence of significant

wall loosening/curling. Furthermore, since expansion of discs from PG plants in water is



16

similar or even higher than that of discs from WT plants (Galletti, R and Pontiggia, D,

unpublished), an alteration of biomechanical properties (for example, more rigid cell

walls) that can be compensated by treatment with auxin is probably not the reason for the

resistant phenotype and the reduced ability to form adventitious roots. On the other hand,

the ability of auxin to revert the altered responses of PG plants also suggest that

irreversible wall modifications such as lignification or cross-links are unlikely to be

involved.

Auxin and elicitors can have opposite effects on defence gene expression. For

instance, IAA inhibits induction of PR proteins in tobacco protoplasts treated with

elicitors from the cell wall of the oomycete Phytophthora megasperma glycinea

(Jouanneau et al., 1991) and inhibits the expression of the wound-inducible pinII gene

(Kernan and Thornburg, 1989). IAA secreted by Pseudomonas savastanoi has the ability

to suppress the hypersensitive response (Robinette and Matthysse, 1990), whereas the

expression of the pathogen-induced gene CEV-1 correlates with a defect of perception of

auxin (Mayda et al., 2000). Importantly, it has been reported that the flagellin-derived

peptide flg22 represses auxin signaling through the induction of a miRNA directed

against the auxin receptors TIR1, AFB2 and AFB3, and this leads to an increased

resistance to bacterial infection (Navarro et al., 2006). It is possible that release of the

auxin-mediated inhibition of defence gene expression is a crucial step in the transduction

pathway activated by general elicitors, including OGs. According to this model, OGs, by

negatively regulating auxin signalling, allow for increased expression of defence

responses. However, our observations imply that, if the phenotype of the PG plants is not

due to accumulation of OGs but to the perception of a defective wall structure, this

second mechanism also involves a cross-talk with auxin. Perception of cell wall-derived

fragments and of alterations of cell wall biomechanical properties may all converge in a

common signalling pathway (Humphrey et al., 2007), that features a negative cross-talk

with the auxin response pathway.

In conclusion, we have shown that the expression of a fungal PG and the

subsequent degradation of HGA increases resistance of plants to microbial pathogens,

likely through a pre-activation of plant defence responses, and that auxin reverts the

enhanced resistance to fungal infection. Our data also indicate that in muro pectin



17

degradation by PG leads to decreased auxin sensitivity. This is reminiscent of the effects

observed in untransformed plants treated with OGs, suggesting a possible role of these

pectic fragments in the resistant phenotypes of PG plants. Alternatively, an altered cell

wall architecture may be directly perceived by the plant cell as a signal of the presence of

a pathogen, leading to an increased activation of defence responses. The molecular

mechanisms linking pectin degradation, auxin signalling and activation of defence

responses still remain to be investigated. We believe that this line of research will provide

new insights in the regulation of plant defence responses during plant-pathogen

interactions.

METHODS

Transgenic lines, plant growth and treatments.

Generation of transgenic Arabidopsis thaliana Wassiliewskija and Nicotiana

tabacum Petit Havana-SR1 plants expressing PG or PvPGIP2 and their cross were

previously described (Capodicasa et al., 2004). The PG expressed in these plants had a

point mutation (N178D) which has an estimated activity about twenty-fold lower than the

native A. niger PGII (data not shown). The expression of a PG with a reduced enzymatic

activity allowed to generate viable plants. Arabidopsis line PG201 expresses an inactive

version of A. niger PGII with a point mutation in the catalytic site (D201N) that causes

complete loss of enzymatic activity (van Santen et al., 1999; Federici et al., 2001). These

plants express a level of the inactive enzyme comparable to that observed in line PG5, as

estimated by immunoblot analysis (data not shown), and show no obvious developmental

defects (Capodicasa et al., 2004). Arabidopsis plants were grown in growth chambers at

22°C and 70% of relative humidity, with a 12 h photoperiod (100 µmol m-2 s-1 of

fluorescent light). Tobacco plants were grown in a greenhouse at 23°C and 60% of

relative humidity, with a 16 h photoperiod (130 µmol m2 s-1).

For elicitor treatments on Arabidopsis seedlings, seeds were surface-sterilized and

germinated in multiwell plates (approximately 10 seeds per well) containing 1 ml per

well of 1X Murashige-Skoog (MS) medium (Sigma, Milano, Italy) (Murashige and

Skoog, 1962) supplemented with 0.5% sucrose. Plates were incubated at 22°C with a 16



18

hour photoperiod and a light intensity of 100 µmol m-2 s-1. After 8 days, the medium was

replaced and after two additional days treatments with 100 mg l-1 OGs or water were

performed (Ferrari et al., 2003b). Tobacco leaf explants (5×10 mm) were obtained from

apical leaves of 4-week-old SR1 plants, washed six times for 30 min with 0.25× MS

medium containing 2% sucrose and incubated in the same medium supplemented with 10

µg ml-1 OGs. The OGs used in this work were a pool with average DP of 10-15, prepared

as previously described (Bellincampi et al., 2000).

Ethylene measurements.

About 20-30 tobacco leaf explants (about 500 mg fresh weight) were prepared

from 7-week-old tobacco leaves avoiding the midrib and extensively washed with sterile

distilled water. The explants were placed in sealed 500 ml flasks containing 100 ml of

sterile water alone or supplemented with 250 µM ACC, 100 µM IAA sodium salt or 200

mg l-1 OGs. Gas samples were withdrawn from the flasks after 24 h and analyzed with a

gas chromatographer equipped with a flame ionization detection system (Carlo Erba,

Milano, Italy). Chromatographic separations were carried out on a Porapak Q 80-100

mesh column (4 mm i.d., 1.5 ml). The flow rate of the carrier gas (N2; 0.80 kg cm-2) was

40 ml min−1. Column, injector and detector temperatures were 30°C, 50°C and 120°C,

respectively. Heating rate was 18°C min−1 to 170°C. Air flow was 1.60 kg cm-2,

hydrogen flow was 0.70 kg cm-2. Ethylene concentration in each sample was normalized

using dry weight of the leaf explants.

Pathogen growth and infection.

Botrytis cinerea (a kind gift of J. Plotnikova, Massachusetts General Hospital,

USA) was grown for 7-10 days at 22°C under constant light on 20 g l-1 malt extract, 10 g

l-1 mycological peptone and 15 g l-1 agar until sporulation. Conidia were collected with

10 ml of sterile water containing 0.05% Tween 20, filtered with sterile glass wool and

centrifuged for 5 min at 5000×g. Before plant inoculation, fresh spores were resuspended

in 24 g l-1 potato dextrose broth and incubated for 3 hours at room temperature to allow

uniform germination. Inoculation of Arabidopsis detached leaves was performed as

previously described (Ferrari et al., 2003b). Fully developed leaves of 8-week-old



19

tobacco plants were detached and placed in large Petri dishes, with the petioles embedded

in 0.5% agar, and inoculated with a spore suspension in 24 g l-1 potato dextrose broth

(106 spores ml-1). Six droplets of spore suspension (10 µl each) were placed on the

adaxial surface of each leaf. Plates containing the inoculated leaves were wrapped with a

transparent plastic film and incubated in a growth chamber at 22°C under fluorescent

light with a 16 h photoperiod. Disease progress was scored as described by ten Have et

al., 1998. Results were analyzed by one way ANOVA followed by Tukey’s Student range

test. For leaf disc infections, discs (10 mm of diameter) were cut from surface-sterilized

fully expanded tobacco leaves and placed floating in 12-well-plates containing MS

medium supplemented with 0.5% sucrose and water, 100 µM IAA or 200 mg l-1 OGs (1

ml per well; final ethanol concentration in all samples was 0.01%). After three hours, the

discs were drop-inoculated with a B. cinerea spore suspension (105 spores ml-1) and

incubated in a growth chamber as above indicated. Lesion area was scored 24 hours after

inoculation. EtOH alone had no significant effect on lesion size (data not shown).

P. syringae pv tabaci DC3000 was cultured in King’s B broth at 28°C for 2 days,

and a bacterial suspension was prepared in 10 mM MgCl2 (5×104 colony-forming units

cm-2). Challenge inoculation was performed by infiltration of the bacterial suspension

using a 1-ml syringe without a needle.

Determination of hydrogen peroxide content, glucanase, peroxidase and IAA

oxidase activity and visualization of UV-fluorescent compounds.

For H2O2 visualization, leaves were cut from adult plants using a razor blade and

dipped for 12 h in a solution containing 1 mg ml-1 of 3-3’-diaminobenzidine-HCl (DAB),

pH 5.0. Chlorophyll was extracted for 10 min with boiling ethanol and for 2 h with

ethanol at room temperature prior to photography (Orozco-Cardenas and Ryan, 1999).

For total protein extraction, frozen leaves or stems were homogenized in 1M

NaCl, 20 mM sodium acetate, pH 4.7, incubated under gentle shaking for 1 h and

centrifuged for 20 min at 10,000×g. IWFs were prepared from tobacco stems using 0.3 M

NaCl, 20 mM sodium acetate pH 4.7 buffer, as previously described (Terry and Bonner,

1980). After IWF extraction, stem sections were homogenized with 1M NaCl, 20 mM

sodium acetate, pH 4.7, as described above for total protein extraction, in order to obtain



20

intracellular proteins. Total protein concentration was determined by the Bradford

method (Bradford, 1976).

Activity of beta-1,3-glucanase was determined incubating 50 µg and 150 µg of

tobacco or Arabidopsis total proteins, respectively, at 37°C with 2 mg ml-1 laminarin

(Sigma) in 50 mM sodium acetate pH 5.2. The produced reducing sugars were

determined colorimetrically with dinitrosalicylic acid (Sigma) (Miller, 1959). Peroxidase

activity was measured using a modified version of the method described in Smith and

Barker, 1988. Enzymatic activity was determined by the change in absorbance at 515 nm

due to the oxidation of aminoantipyrine and 3,5-dicloro-2-idroxibenzensulphonic acid.

IAA oxidase activity was determined as described in Pressey, 1990.

The presence of UV-fluorescent compounds was visualized by photographing

detached leaves under UV light (λ=320 nm) using the "ImageMaster" VDS (Pharmacia

Biotech).

RNA gel blot analysis.

Tobacco leaf discs of 5 mm of radius around the site of inoculation were frozen in

liquid nitrogen, homogenized, and total RNA was extracted with Tri-reagent (Sigma).

RNA was separated on agarose-formaldehyde gel and transferred to a nylon membrane as

previously described (Ferrari et al., 2003b). Equal RNA loading and transfer were

verified by staining the membrane with a solution containing 0.03% methylene blue

(w/v) and 0.3 M sodium acetate, pH 4.7. Pre-hybridization was performed for 6 h at 42°C

in 50% formamide, 6× SSPE, 1× Denhardt’s solution, 0.1% SDS and 250 µg ml-1

denatured harring sperm DNA. Filters were hybridized overnight at 42°C using the

following solution: 50% formamide, 6× SSPE, 1× Denhardt’s solution, 0.1% SDS, 5%

dextran sulphate, 100 µg ml-1 of denatured harring sperm DNA and 100 ng of denatured

probe. 32P-labelled probes were prepared by PCR; as templates for the tobacco probes,

two DNA fragments, corresponding respectively to a region of a tobacco peroxidase gene

(POX) (GeneBank Accession L02124; Diaz-De-Leon et al., 1993) conserved also in

different anionic peroxidase genes, and to the EAS1 and EAS2 genes (EAS1/2) (Facchini

and Chappell, 1992), were amplified from cDNA of infected tobacco leaves, using the

following primers: 5’-TAATGTAGGTGGCAGGAGG-3’ (EAS1/2 forward), 5’-



21

CTAGGAATATCACTATTAGC-3’ (EAS1/2 reverse), 5’-

ACATCGTATTTGGGCATG-3’ (POX forward), 5’-GTTGCAATTTGTCCCCT-3’

(POX reverse). Probes were purified using ProbeQuantTM G-50 micro-columns

(Amersham Pharmacia Biotech). Hybridized blots were washed once with 2× SSC, 0.1%

SDS at room temperature and twice with 0.1× SSC, 0.1% SDS at 65°C. Images were

taken after overnight exposure using a phosphorimager (Typhoon 9200, Amersham).

Real-Time RT-PCR analysis

Total RNA was was extracted with Tri-reagent (Sigma) and treated with Turbo-

DNase I (Ambion). First-strand cDNA was synthesized using ImProm-II Reverse

Transcriptase (Promega). Real-Time PCR analysis was performed using an Iq-Cycler

(Biorad) according to the manufacturer’s guide. 2 µl cDNA (corresponding to 120 ng of

total RNA) were amplified in 30 µl of reaction mix containing IQ SYBR Green Supermix

(Biorad) and 0.4 mM of each primer. Primer sequences for POX and EAS1/2 are

described above. The tobacco actin gene (Tob66, accession number U60491) was

amplified using the following primers: 5’-CTGCCATGTATGTTGCTATT-3’ and 5’-

AGTCTCCAACTCTTGCTCAT-3’. The primers for PDF1.2, PR-1, AtPGIP1 and UBQ5

were previously described (Penninckx et al., 1996; Rogers and Ausubel, 1997; Ferrari et

al., 2006). The primers utilized for the amplification of the B. cinerea Bcact actin gene

(accession number AJ000335) were the following: 5’-AAGTGTGATGTTGATGTCC-3’

and 5’-CTGTTGGAAAGTAGACAAAG-3’. Relative expression of the RT-PCR

products was determined using a modified version of the Pfaffl method (Pfaffl, 2001;

Ferrari et al., 2006).

Auxin responses in tobacco leaf explants and seedlings.

For rhizogenesis of leaf explants, the second apical leaves from tobacco plants

grown for 4 weeks on soil were harvested and surface-sterilized. Ten explants of about

0.4×0.8 cm were excised in correspondence of the midrib vein and placed, abaxial side

down, in Petri dishes containing 10 ml of sterile MS medium (pH 5.7) supplemented with

2% sucrose, 0.8% plant agar and IAA at the indicated concentrations. Leaf explants were

incubated at 25°C under low intensity light (60 µE m-2 s-2) for 15 days. For seedling



22

assays, seeds were sterilized and germinated in 0.5X MS liquid medium supplemented

with 2% sucrose, pH 5.8. After one week, seedlings were transferred to full-strength MS

medium, pH 5.7, supplemented with 2% sucrose, 1.5% plant agar and IAA. Primary root

length was measured after 12 days of growth at 22°C with a photoperiod of 16 hours.

Supplemental Data

The following materials are available in the online version of this article:

Supplemental Figure S1. Expression of the Botrytis cinerea Bcact gene in PG plants

after infection.

Supplemental Figure S2. Accumulation of UV-fluorescent compounds in PG plants.

Supplemental Figures Legends.

ACKNOWLEDGEMENTS

We wish to thank Gianni Salvi for the preparation of OGs and Lucia Tufano and

Lorenzo Mariotti for assistance. We are grateful to Francesco Loreto and Francesco Brilli

(Istituto di Biologia Agro-ambientale e Forestale, Consiglio Nazionale delle Ricerche) for

their help in the analysis of physiological parameters and in the gas chromatography

experiments.

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FIGURE LEGENDS

Figure 1. Resistance of PG plants to fungal infection. Development of symptoms in

tobacco (A, B) and Arabidopsis (C, D) untransformed (WT) and PG plants inoculated

with B. cinerea is shown. Fully expanded leaves from untransformed tobacco plants

(WT), from three independent transgenic lines expressing PG (PG5, PG7 and PG16),

from plants overexpressing P. vulgaris PvPGIP2 (PGIP2) or generated by crossing line

PG16 with the line expressing PvPGIP2 (PG16×PGIP2) were inoculated with B. cinerea.

Lesion area (A) and percentage of expanding lesions (B) were measured after 6 days.

Bars in (A) indicate average lesion area ± standard error (SE; n >36); this experiment was

repeated four times with similar results. Bars in (B) represent the average percentage of

spreading lesions ± SE of five experiments (n >36 in each experiments). Adult rosette

leaves from untransformed Arabidopsis plants (WT) and from transgenic plants

expressing PG (PG1 and PG5) or an inactive version of the A. niger PGII (PG201) were

inoculated with B. cinerea and lesion size was determined after 2 days (C) whereas the

number of expanding lesions was determined after 3 days (D). Bars in (C) indicate the

average lesion area ± SE (n >12); this experiment was repeated three times with similar

results. Bars in (D) represent the average percentage of spreading lesions ± standard error

of three experiments (n >12 in each experiment). Different letters represent data sets

significantly different, according to ANOVA analysis followed by Tukey’s test (P <

0.01).

Figure 2. Resistance of tobacco PG plants to bacterial infection. Disease symptoms of

(from top to bottom and from left to right) tobacco untransformed plants (WT) and

transgenic PG5, PG7, PG16, PG16×PGIP2 and PGIP2 plants injected with a virulent

strain of P. syringae pv. tabaci. Inoculated leaves of the indicated genotypes were

photographed 5 days after inoculation. This experiment was repeated twice with similar

results.

Figure 3. Accumulation of hydrogen peroxide in PG plants. A, DAB staining of detached

leaves from (from left to right) tobacco untransformed (WT) and transgenic PG5, PG7,



30

PG16, PG×PGIP2 and PGIP2. B, DAB staining of detached leaves from (from left to

right) Arabidopsis untransformed (WT) and transgenic PG201, PG5 and PG1 plants.

Figure 4. Peroxidase activity in PG plants. A, Peroxidase activity in total protein extracts

from leaves of tobacco untransformed (WT) and transgenic PG5, PG7, PG16, PGIP2 and

PG×PGIP2 plants. B, Peroxidase activity in intercellular washing fluids (IWFs, black

bars) and intracellular proteins extracted after recovery of IWFs (empty bars) from the

same tobacco lines shown in (A). C, Peroxidase activity in total protein extracts from

leaves of Arabidopsis untransformed (WT) and transgenic PG201, PG5 and PG1 plants.

Bars indicate the average activity, expressed as enzymatic units per mg of total proteins,

of three samples ± SE. Different letters represent data sets significantly different,

according to ANOVA analysis followed by Tukey’s test (P < 0.01).

Figure 5. Glucanase activity in PG plants. A, Levels of β-1,3-glucanase activity,

expressed as enzymatic units per mg of total proteins, in leaves of tobacco untransformed

(WT) and transgenic PG5, PG7, PG16, PG×PGIP2 and PGIP2 plants. Bars indicate

average activity of three independent samples ± SE. B, Levels of β-1,3-glucanase

activity, expressed as enzymatic units per mg of total proteins, in leaves of Arabidopsis

untransformed (WT) and transgenic PG201, PG5 and PG1 plants. Bars indicate the

average activity of three independent samples ± SE. Different letters represent data sets

significantly different, according to ANOVA analysis followed by Tukey’s test (P <

0.01).

Figure 6. Expression of defence genes in PG plants after infection with Botrytis cinerea.

A, Fully expanded leaves from tobacco untransformed (WT) and transgenic PG16 (PG)

plants were inoculated with B. cinerea and total RNA was extracted at the indicated time

points (days post infection). The RNA gel blot was hybridized with the indicated probes.

Equal loading was verified by methylene blue staining of ribosomal RNA (rRNA). B-D,

Fully expanded leaves from Arabidopsis untransformed (WT, empty bars) or transgenic

PG1 (PG, black bars) plants were inoculated with B. cinerea and total RNA was extracted

at the indicated time points (DPI, days post infection). The expression of AtPGIP1 (B),



31

PR-1 (C) and PDF1.2 (D) was analysed by Real Time RT-PCR and normalized using the

expression of UBQ5 in each sample. The insets in C and D show gene expression in

untreated leaves (n.d., not detectable). Bars represent the average expression ± SD of two

replicates.

Figure 7. Regulation of defence gene expression by OGs in tobacco plants. Real Time

RT-PCR analysis of the expression of EAS1/2 (A) and POX (B) in untransformed

tobacco leaf explants treated for the indicated times with water (control, empty bars) or

OGs (OG, black bars). The expression of each gene was normalized using the expression

of the actin gene Tob66 in each sample. Bars represent the average gene expression ± SD

of two replicates.

Figure 8. Regulation of defence gene expression by OGs in Arabidopsis plants. Real

Time RT-PCR analysis of the expression of AtPGIP1 (A), PR-1 (B) and PDF1.2 (C) in

untransformed Arabidopsis seedlings treated for the indicated times with OGs. The

expression of each gene was normalized using the expression of UBQ5 in each sample.

Bars represent the average gene expression ± SD of two replicates.

Figure 9. Susceptibility to Botrytis cinerea in tobacco PG plants treated with auxin. A,

Leaf discs from tobacco untransformed (WT, empty bars) or transgenic PG16 (PG, black

bars) plants were incubated for three hours in liquid medium containing water (control)

or 100 µM IAA (IAA) and inoculated with B. cinerea. Lesion area was measured after 24

hours. Bars indicate average area ± SE of at least ten lesions. Asterisks indicate

statistically significant difference between lesions in WT and transgenic plants (***, P <

0.01). This experiment was repeated three times with similar results. B, Leaf discs from

untransformed tobacco plants were incubated for three hours in liquid medium containing

water (control), 100 µM IAA (IAA), 200 µg ml-1 OGs (OG) or both (OG + IAA) and

inoculated with B. cinerea. Lesion area was measured after 24 hours. Bars indicate the

average area ± SE of at least ten lesions. Different letters indicate data sets significantly

different, according to ANOVA followed by Tukey’s test (P< 0.05). This experiment was

repeated twice with similar results.



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Figure 10. Auxin sensitivity of tobacco PG plants. A, Leaf explants from tobacco

untransformed (WT, empty squares) and transgenic PG16 (PG, black squares) plants

were treated with the indicated concentrations of IAA for 15 days and the number of

explants forming roots was measured. Each data point represents the average percentage

of explants forming roots ± SD calculated in three independent experiments (n> 8 in each

experiment). B, Length of primary roots of tobacco untransformed (WT) and transgenic

PG16 (PG) plants grown for 12 days on solid medium containing the indicated IAA

concentrations. Bars represent the average length of at least eight plants ± SE. Asterisks

indicate statistically significant difference between treated and control samples, according

to the Student’s t-test (P < 0.01). This experiment was repeated twice with similar results.

Figure 11. Ethylene production in tobacco PG leaf explants. Leaf explants from tobacco

untransformed (WT, empty bars) and transgenic PG16 (PG, black bars) plants were

incubated with water, 250 µM 1-aminocyclopropane-1-carboxylic acid (ACC) or 100 µM

IAA (IAA) in sealed flasks. Ethylene accumulating in the flask was determined by gas

chromatography. Bars indicate ethylene concentration after 24 hours of treatment. No

detectable accumulation of ethylene could be observed in control samples. This

experiment was repeated twice with similar results.



running head: expression of fungal pg decreases ......2007/12/07 · lionetti, daniela bellincampi,...

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