Running head: Oligogalacturonide-mediated resistance · 2007. 3. 23. · (reviewed in Nurnberger and Brunner, 2002). Pathogen-secreted hydrolytic enzymes that degrade host cell wall

Running head:

Oligogalacturonide-mediated resistance

Corresponding author:

Simone Ferrari

Università degli Studi di Padova

Dipartimento Territorio e Sistemi Agro-forestali

Viale dell'Università 16 - 35020 Legnaro (PD) - Italy

Phone +39-049-8272894

Fax +39-049-8272890

E-mail: [email protected]

Journal research area: Plants Interacting with Other Organisms

Plant Physiology Preview. Published on March 23, 2007, as DOI:10.1104/pp.107.095596

Copyright 2007 by the American Society of Plant Biologists

https://plantphysiol.orgDownloaded on December 19, 2020. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

https://plantphysiol.org

Title:

Resistance to Botrytis cinerea Induced in Arabidopsis thaliana by Elicitors is Independent of

Salicylic Acid, Ethylene or Jasmonate Signaling but Requires PAD3.

Authors:

Simone Ferrari, Roberta Galletti, Carine Denoux, Giulia De Lorenzo, Frederick M. Ausubel, Julia

Dewdney

Dipartimento Territorio e Sistemi Agro-Forestali, Università degli Studi di Padova, c/o Agripolis,

Viale dell'Università, 23 - 35020 Legnaro (PD), Italy (S.F.); Department of Genetics, Harvard

Medical School, and Department of Molecular Biology, Massachusetts General Hospital, Boston,

MA 02114 USA (C.D., F.M.A., J.D.); Dipartimento di Biologia Vegetale, Università di Roma “La

Sapienza”, Piazzale Aldo Moro 5, 00185 Rome, Italy (R.G., G.D.L.).



FOOTNOTES

This work was supported by the Giovanni Armenise – Harvard Foundation, the Institute Pasteur –

Fondazione Cenci Bolognetti, by MIUR FIRB 2001 and MIUR COFIN 2002 grants awarded to

G.D.L., and by NSF grant DBI-0114783 and NIH grant GM48707 awarded to F.M.A.

Corresponding author: Simone Ferrari (e-mail: [email protected]; fax: +39-049-8272890).



ABSTRACT

Oligogalacturonides (OGs) released from plant cell walls by pathogen polygalacturonases induce a

variety of host defense responses. Here we show that in Arabidopsis thaliana, OGs increase

resistance to the necrotrophic fungal pathogen Botrytis cinerea independently of jasmonate (JA),

salicylic acid (SA) and ethylene (ET) mediated signaling. Microarray analysis showed that about

50% of the genes regulated by OGs, including genes encoding enzymes involved in secondary

metabolism, show a similar change of expression during B. cinerea infection. In particular,

expression of Phytoalexin Deficient 3 (PAD3) is strongly up-regulated by both OGs and infection

independently of SA, JA and ET. OG treatments do not enhance resistance to B. cinerea in the pad3

mutant, nor in ups1, a mutant with severely impaired PAD3 expression in response to OGs.

Similarly to OGs, the bacterial flagellin peptide elicitor flg22 also enhanced resistance to B. cinerea

in a PAD3-dependent manner, independently of SA, JA and ET. This work suggests, therefore, that

elicitors released from the cell wall during pathogen infection contribute to basal resistance against

fungal pathogens through a signaling pathway also activated by pathogen-associated molecular

pattern molecules.



1

INTRODUCTION

Plants need to recognize invading pathogens in a timely manner in order to mount

appropriate defense responses. In the so-called “gene-for-gene” resistance, early

recognition of specific pathogen strains depends on complementary pairs of dominant

genes, one in the host and one in the pathogen. The outcome of this recognition is the

induction of a multitude of biochemical and physiological changes, including localized

programmed cell death (hypersensitive response), that restrict pathogen growth in the

host tissues. A loss of or a mutation in either the plant resistance (R) gene or in the

pathogen avirulence (avr) gene leads to a “compatible” interaction, resulting in disease

(Flor, 1971). Gene-for-gene resistance has been observed in interactions with many

biotrophic pathogens, including fungi, viruses, bacteria and nematodes (Hammond-

Kosack and Jones, 1997). In contrast, many necrotrophic fungal and bacterial pathogens

cause disease in a variety of plant species and resistance mediated by a single host

resistance gene is uncommon. Nevertheless, plants also recognize non-specific elicitors

that activate a battery of defense responses effective against a wide range of pathogens.

Some of these elicitors, referred to as pathogen-associated molecular patterns (PAMPs),

are derived from essential components of the pathogen cell wall (e.g. chitin, glucan) or

other macromolecular structures (e.g. the 22 amino acid flagellin peptide flg22)

(reviewed in Nurnberger and Brunner, 2002).

Pathogen-secreted hydrolytic enzymes that degrade host cell wall polymers are

also able to induce defense responses in plants. Among these, the most extensively

studied are endopolygalacturonases (PGs; EC 3.2.1.15). PGs cleave the α-(1→4) linkages

between D-galacturonic acid residues in non-methylated homogalacturonan, a major

component of pectin (De Lorenzo et al., 1997). PGs are important virulence factors for

necrotrophic soft rot-causing pathogens, including the fungus Botrytis cinerea (ten Have

et al., 1998). The elicitor activity of PGs in activating plant defense responses has been

demonstrated in many pathosystems (De Lorenzo et al., 1997). Hahn and colleagues first

showed that PGs are not directly responsible for the induction of plant defense responses,

but rather cause the release from the plant cell wall of the true elicitors, namely

oligogalacturonides with a degree of polymerization (DP) between 10 and 15 (OGs)



2

(Hahn, 1981). A functional catalytic site is required for elicitor activity of a

Colletotrichum lindemuthianum PG in tobacco (Boudart et al., 2003), supporting the

hypothesis that OGs mediate responses activated by PGs. In contrast, Poinssot and

colleagues reported that enzymatic activity is not required for elicitor activity of the B.

cinerea PG BcPG1 in grape cells (Poinssot et al., 2003), suggesting that, in some

biological systems, PGs themselves can be perceived and activate defense responses.

OGs elicit a variety of defense responses, including accumulation of phytoalexins

(Davis et al., 1986), glucanase and chitinase (Broekaert and Pneumas, 1988, Davis and

Hahlbrock, 1987), and phenylalanine ammonia lyase (PAL) (De Lorenzo et al., 1987).

Exogenous treatments with OGs protect grapevine leaves against B. cinerea infection in a

dose-dependent fashion (Aziz et al., 2004). Despite having been extensively studied, the

role of OGs in plant defense and their mode of action are still largely unknown.

A variety of plant defense responses against microbial pathogens are regulated by

the signaling molecules salicylic acid (SA), jasmonates (JA) and ethylene (ET) (for

review, see Feys and Parker, 2000). These signaling pathways have been exensively

studied, but a major unanswered question is how the SA, JA and ET signaling pathways

are related to the signaling pathways activated by OGs and other PAMPs. Although

mutants impaired in the responses mediated by SA, JA and ET show enhanced disease

symptoms upon infection with B. cinerea (Alonso et al., 2003, Ferrari et al., 2003a,

Thomma et al., 1998, Thomma et al., 1999), treatment with OGs or infection with B.

cinerea induce the expression of AtPGIP1, which encodes an Arabidopsis inhibitor of

fungal PGs, independently of these secondary signaling molecules (Ferrari et al., 2003b).

Since expression of AtPGIP1 is required for full resistance to B. cinerea (Ferrari et al.,

2006), we speculated that a SA-, JA- and ET-independent defense pathway induced by

OGs during fungal infection may actively contribute to plant defense. To investigate this

hypothesis, we performed global transcription profiling and susceptibility assays in

Arabidopsis plants treated with exogenous elicitors or inoculated with B. cinerea. Our

results suggest that the expression of many defense-related genes are induced by OGs

released during pathogen infection and that responses induced independently of SA, JA

and ET, and in particular the expression of genes involved in the production of

antimicrobial compounds, participate to restrict the growth of B. cinerea.



3

RESULTS

OGs induce local and systemic resistance to B. cinerea.

To assess the ability of OGs to enhance Arabidopsis resistance to fungal infection,

adult plants were sprayed with OGs and inoculated with B. cinerea 24 h after the

treatment. Leaves from plants treated with OGs showed significantly delayed progression

of the infection, compared to control-treated plants (Supplemental Figure S1 and Fig.

1A). Similar results were obtained when plants were inoculated 48 or 72 hours after OG

treatment (not shown). To determine whether OGs also induce systemic resistance, lower

rosette leaves were injected with water or OGs and, after 72 h, upper leaves were

inoculated with B. cinerea. The average area of necrotic lesions in systemic leaves of

plants pre-treated with OGs was significantly smaller than in control plants (Fig. 1B).

These results indicate that OGs induce both local and systemic resistance against Botrytis

infection in Arabidopsis plants.

Role of SA, JA and ET in OG-mediated resistance

To determine the role of SA, JA or ET in OG-induced resistance, we sprayed OGs

on mutants or transgenic plants affected in the perception or transduction of these

hormone signals, and inoculated them with B. cinerea 24 h later. The Arabidopsis lines

used were transgenic plants expressing the nahG gene, and therefore unable to

accumulate SA (Gaffney et al., 1993), plants carrying single mutations in the NPR1,

EIN2, or JAR1 genes impaired in signaling mediated by SA, ET or JA, respectively (Cao

et al., 1994, Guzman and Ecker, 1990, Staswick et al., 1992), or a triple mutant carrying a

mutation in each of these genes (npr1ein2jar1) and therefore unable to respond to any of

the hormones (Clarke et al., 2000). As described previously (Ferrari et al., 2003a,

Thomma et al., 1999), ein2 mutant and nahG transgenic plants are more susceptible to B.

cinerea. Nevertheless, OG pre-treatments of these mutants, as well as the other mutants

tested, significantly reduced lesion size (Fig. 2A and B). To further confirm that OG-

mediated resistance is JA-independent, we also treated B. cinerea-susceptible coi1

homozygous mutant plants (Thomma et al., 1998) with OGs . Two days after inoculation,



4

lesions in control coi1 plants were more than five-fold larger than in wt control plants;

however, OG-pretreated coi1 plants were significantly more resistant to B. cinerea,

similar to the highly susceptible ein2 mutant (Fig. 2C). Taken together, these results

indicate that OGs increase Arabidopsis resistance to B. cinerea through the activation of

defense responses that are independent of SA, JA and ET, but that SA, JA, and ET are

also involved in defense pathways that confer resistance to B. cinerea.

Changes in gene expression in Arabidopsis plants treated with oligogalacturonides

or inoculated with B. cinerea.

To identify Arabidopsis genes that may be involved in OG-mediated resistance

to B. cinerea, we analyzed the transcriptome of plants inoculated with the fungus and

compared it to the transcription profile of 10-day-old seedlings treated with OGs for 1h or

3h. For the analysis of infected plants, rosette leaves were inoculated with a B. cinerea

spore suspension or with sterile medium and harvested after 18 or 48 hours. At the early

time point of infection, no macroscopic lesions were observed at the site of inoculation,

though staining of fungal hyphae with trypan blue revealed that the spores had

germinated and started growing on the leaf surface (data not shown). After 48 hours, the

infected leaves showed water-soaked lesions about 3-4 mm in width that are typical of

soft rot disease. Total RNA from control or treated samples from two or three

independent infection or elicitor-treatment experiments, respectively, was analyzed using

the Affymetrix ATH1 GeneChip™ DNA microarray, which contains probe sets

corresponding to more than 22,000 putative open reading frames. Original raw data for

each experiment are available at NASC

(http://affymetrix.arabidopsis.info/narrays/experimentbrowse.pl) and

http://ausubellab.mgh.harvard.edu/imds (under experiment names “Botrytis cinerea

infection, 18 and 48 hpi” and “Comparison of response to Flg22 and OGs elicitors”). For

each probe set, mean expression fold-change, signal intensities, and P-values were

calculated after normalization (see Supplemental Tables S1 and S2). Only probe sets

showing a statistically significant difference (P ≤ 0.01) between control and experimental

treatments anda mean fold-change ≥ 2.0 were considered for further analysis.



5

As summarized in Table 1, at 48 hours post inoculation (hpi), when typical water-

soaked lesions were visible, more than ten times as many genes showed significant

changes in transcription than at 18 hpi. Interestingly, 588 out of 1299 genes up-regulated

by OGs after either 1h or 3h were also up-regulated by B. cinerea at either of the two

time points analyzed (Fig. 3). Similarly, 316 out of 577 genes significantly repressed by

OGs at either 1h or 3h were also down-regulated during fungal infection, indicating that

about half of the genes responsive to OGs show a significant change of expression in the

same direction upon B. cinerea attack (Fig. 3) (a complete list of the genes co-regulated

by OGs and B. cinerea can be found in Supplemental Table S3).

We have also compared the transcript profile of OG-treated seedlings to the

available data on the profile induced by crab shell chitin (CSC) and chitin octamers

(Ramonell et al., 2005), using the same threshold (expression change ≥ 1.5-fold). Out of

1002 genes whose expression is induced by both CSC and octamers after 30 minutes of

treatment, 697 were also up-regulated by OGs after either 1h or 3h, whereas 84 genes out

of 312 chitin-repressed genes were significantly down-regulated by OGs (Supplemental

Table S4). In contrast, only 6 out of 68 genes induced specifically by octamers, and 39

out of 238 genes repressed only by octamers, were also induced or repressed by OGs,

respectively.

In order to group genes up-regulated by both OGs and B. cinerea according to

their predicted functions, we identified Functional Catalogue (FunCat) terms (Ruepp et

al., 2004) associated with each gene using the MIPS Arabidopsis thaliana Data Base

(http://mips.gsf.de/proj/thal/db/index.html). For this analysis, only probes with an

annotated locus identifier were used. For each expression category, frequencies of genes

in a given FunCat group were compared with the frequency found for all genes

represented on the array (Table 2). As expected, the most represented categories included

genes involved in cell rescue and defense, genes whose classification is ambiguous, genes

involved in cell communication and signaling, and genes encoding proteins implicated in

primary and secondary metabolism.

A majority of the genes involved in secondary metabolism that are induced by

OGs and Botrytis and identified through the MAtDB database encode enzymes

implicated in amino acid metabolism and phenylpropanoid biosynthesis (Table 2).



6

However, we noticed that a number of genes involved in the metabolism of tryptophan-

derived secondary compounds, like PAD3 (Zhou et al., 1999) and CYP79B2 (Hull et al.,

2000), were included in the “not yet clear-cut” category. We therefore manually compiled

a list of 66 Arabidopsis genes previously implicated in secondary metabolism (full list is

available in the Supplemental Table S5). Expression of 36 of these genes appeared

significantly induced or repressed by at least one treatment (Table 3). In particular,

several genes encoding enzymes of the shikimate and the phenylpropanoid pathways

(with the exception of flavonoid biosynthesis) and enzymes involved in the biosynthesis

of tryptophan and of indole compounds were up-regulated by both OGs and B. cinerea,

whereas most genes proposed to encode enzymes involved in the biosynthesis of aliphatic

glucosinolates, like CYP79F1, REF2 and UGT74C1 (Gachon et al., 2005, Hansen et al.,

2001, Hemm et al., 2003), were repressed or not significantly affected by OGs or fungal

infection (Table 3, Fig. 4A and B). Interestingly, mRNA levels of ASA1, ASB2, IGPS,

CYP79B2 and PAD3, which are all involved in the biosynthesis of indolic compounds

(Glawischnig et al., 2004, Hull et al., 2000, Zhao and Last, 1996, Zhou et al., 1999, Zook,

1998), increase at both 18h and 48 h after infection and by OGs after 3 hours of treatment

(Table 3). With the exception of PAD3, which encodes the cytochrome P450 enzyme

CYP71B15 that catalyzes the last step of camalexin biosynthesis (Schuhegger et al.,

2006), the other genes encoding enzymes in the indole pathway may also be involved in

the biosynthesis of glucosinolates or auxin. However, genes specifically required for IAA

metabolism, including a nitrilase gene and ILR1, encoding an IAA-conjugate hydrolase

(Bartel and Fink, 1995), were not up-regulated by OGs, although they were induced by B.

cinerea (Table 3 and Fig. 4A and B). Because pad3 plants are extremely susceptible to B.

cinerea infection (Ferrari et al., 2003a), it appears likely that OG-mediated activation of

PAD3 and other genes involved in the biosynthesis of camalexin may contribute to OG-

induced resistance against B. cinerea.

PAD3 is expressed independently of SA, JA and ET and is required for OG-induced

resistance against B. cinerea.

Since PAD3 is required for basal resistance to B. cinerea strains sensitive to

camalexin (Ferrari et al., 2003a, Kliebenstein et al., 2005), and microarray experiments



7

showed that PAD3 expression is induced by both OGs and B. cinerea (this work), we

further investigated OG-mediated induction of PAD3 expression in adult plants. A

transient accumulation of PAD3 mRNA, with a peak at 3 to 6 hours after treatment, was

observed by quantitative RT-PCR in rosette leaves sprayed with OGs (Fig. 5A).

Interestingly, the increase in PAD3 transcript levels after B. cinerea inoculation of leaves

was not attenuated in nahG, ein2, npr1, jar1 or coi1 plants (Fig. 5B), indicating that

PAD3 expression during fungal infection is also independent of SA, JA and ET.

Consistently, PAD3 mRNA levels in wt and npr1ein2jar1 seedlings treated with OGs for

1h and 3 h were not significantly different (Fig. 5C). OG treatments were also similarly

effective in inducing PAD3 expression in single ein2, npr1 and jar1 seedlings, as

determined by semi-quantitative RT-PCR (data not shown). Furthermore, mRNA levels

of PAD3 in coi1 seedlings treated with OGs were even higher than in wt seedlings (Fig.

5D). These results indicate that OGs are able to increase PAD3 expression independently

of SA, JA and ET.

Since protection against B. cinerea infection mediated by OG pre-treatment is also

independent of SA-, JA- and ET-mediated signaling (Fig. 2A-C), PAD3 may be

important for OG-induced resistance to this pathogen. To test this hypothesis, we

inoculated OG-treated wild type or pad3 plants with B. cinerea. Strikingly, control and

OG-treated pad3 plants showed similar lesion development, whereas, in accordance with

our previous results, OG-treated wt plants were more resistant to infection (Fig. 6). To

further confirm the role of PAD3 in OG-induced resistance, we analyzed the ups1

(underinducer after pathogen and stress 1) mutant, which is defective in tryptophan

biosynthetic pathway regulation and accumulates low levels of camalexin and of PAD3

mRNA in response to fungal infection and oxidative stress (Denby et al., 2005). As

shown in Fig. 6, ups1 plants were not protected by OGs against B. cinerea. Consistent

with our hypothesis that PAD3 expression is required for OG-induced resistance, PAD3

expression in OG-treated ups1 seedlings was strongly reduced (Fig. 5C), in contrast to

the triple npr1ein2jar1 mutant. Interestingly, the bacterial flagellar peptide elicitor flg22,

which is structurally unrelated to OGs, was also effective in protecting Arabidopsis wild

type plants, as well as npr1ein2jar1 triple mutant plants, against B. cinerea, and this

protection was also dependent on PAD3 and UPS1 (Fig. 7A-B). In one experiment we



8

observed a slight reduction of symptoms in flg22-treated ups1 plants (data not shown),

but this result could not be replicated, and in two independent experiments flg22 pre-

treatments failed to protect ups1 against B. cinerea infection (Fig. 7B).

Since PAD3 is required for elicitor-induced resistance against B. cinerea, we

expected that OGs could increase camalexin levels in Arabidopsis adult plants.

Suprisingly, however, HPLC analysis failed to reveal significant camalexin accumulation

up to 24 hours after treatment with the elicitor, compared to control-treated plants (data

not shown). Therefore, OG-induced resistance does not seem to be directly due to an

increase in the levels of camalexin in leaf tissues before pathogen inoculation.

DISCUSSION

Activation of the plant innate immune system can be mediated, as in animals, by

molecular patterns conserved among different pathogen species but not present in host

cells. However, in plants, some host-derived low molecular weight elicitors may also be

released during an infection as a consequence of pathogen activity. These endogenous

elicitors, like true PAMPs, may trigger defense responses that contribute to basal

resistance. OGs represent the best characterized endogenous elicitors and have been

extensively studied since their identification about 25 years ago (Hahn et al., 1981). In

this paper we have investigated what responses activated by OGs may be involved in

Arabidopsis defense against the fungal pathogen B. cinerea. Treatment with exogenous

OGs enhances resistance against B. cinerea in grape (Aziz et al., 2004) and Arabidopsis

leaves (this work). Since B. cinerea secretes large amounts of polygalacturonases during

tissue invasion (reviewed in Kars and van Kan, 2004), it is likely that OGs transiently

accumulate at the interface between fungal hyphae and plant tissues. However, detection

of OGs in the apoplast of infected plants is technically challenging. Recently, An and

colleagues have employed MALDI-Fourier transform and MALDI-TOF mass

spectrometry to identify cell wall pectin-derived oligosaccharides generated through the

breakdown of homogalacturonan pectins in B. cinerea-infected tomato fruits (An et al.,

2005). This study suggests that pectic fragments with a degree of polymerization of about

16 accumulate around the lesions caused by this fungus. However, a conclusive

demonstration that OGs with elicitor activity accumulate to significant levels during



9

infection is still lacking, and their role in basal resistance to pathogens must be inferred

from indirect evidence.

We have carried out a full-genome expression analysis of Arabidopsis plants

treated with OGs or infected with B. cinerea, in order to determine the extent of overlap

between transcriptional responses induced by these two treatments. Previously, Navarro

and colleagues, comparing microarray data obtained from flg22-treated cell cultures and

seedlings, and data obtained from bacterial-inoculated rosette leaves, found a limited

overlap (7%) in the compatible interaction and a more consistent overlap (34%) with a

incompatible interaction (Navarro et al., 2004). Our results indicate that about half of the

Arabidopsis genes affected by OG treatment display a similar behavior after fungal

infection, suggesting that at least part of the responses activated by B. cinerea are

mediated, directly or indirectly, by the accumulation of OGs or other elicitors able to

activate the same signaling pathway, and that these responses are not suppressed by the

fungus. The genes induced by both OGs and B. cinerea infection not only encode

defense-related proteins, but also enzymes implicated in primary and secondary

metabolism. The transcriptional activation of some of these genes during fungal infection

may result in the accumulation of antimicrobial proteins or low molecular weight

compounds, which are able to restrict fungal growth. In the case of B. cinerea, it has been

previously shown that strains sensitive to camalexin are partially restricted in planta by

this phytoalexin, since pad3 and other mutants impaired in camalexin accumulation are

more susceptible to infection than the parental lines (Ferrari et al., 2003a, Kliebenstein et

al., 2005). Camalexin may therefore play a major role in the reduction in B. cinerea

growth observed in OG-treated plants. Furthermore, OG-induced resistance and PAD3

expression are both largely independent of SA, ET and JA. OGs (and B. cinerea)

therefore appear to activate an SA-, ET- and JA-independent signaling pathway that

regulates PAD3 expression and other defense responses effective against B. cinerea. This

conclusion is consistent with the previous observation that the expression of AtPGIP1,

another OG-responsive gene, in Botrytis-inoculated plants, is also independent of SA, ET

and JA (Ferrari et al., 2003b). Similarly, expression of two Arabidopsis chitin-inducible

genes was shown to be independent of SA, ET and JA (Zhang et al., 2002). More

recently, Raacke and colleagues found that yeast cells also induce resistance against B.



10

cinerea in Arabidopsis, and that this resistance is independent of SA, JA and ET (Raacke

et al., 2006b). Interestingly, the authors observed that this resistance is also independent

of PAD3; however, it is not known whether the fungal strain they used is sensitive to

camalexin, and it is therefore difficult to draw any conclusion on the role of camalexin in

yeast-induced resistance. Furthermore, yeast treatment, in contrast to OGs, and also to

flg22, induces resistance against P. syringae through an SA-dependent mechanism

(Raacke et al., 2006a), indicating that additional defense responses are activated by yeast

cells, compared to chemically defined elicitors. A recent paper describes the expression

profile of nahG, ein2 and coi1 plants inoculated with B. cinerea, and demonstrates that

the ZFAR1 gene, encoding a zinc finger proteins containing ankyrin repeats, is expressed

independently of SA, ET and JA and is required for basal local resistance against this

fungus (AbuQamar et al., 2006). Interestingly, ZFAR1 is also induced by OGs in our

microarray experiments, and determinining whether this gene is also required for OG-

induced resistance could provide further insights in its role in elicitor-mediated signaling.

Furthermore, the isolation of mutants impaired in OG-dependent responses will confirm

the role of these elicitors in defense against pathogens.

The data presented in this manuscript and in previous publications (Ferrari et al.,

2003a) indicate that PAD3-mediated resistance elicited by OGs (and other PAMPs) is one

of several independent signaling pathways that contribute to basal resistance. Thus in SA,

JA and ET signaling mutants, PAMP-mediated resistance is still operating such that OG

treatment will result in partial resistance as shown in Figure 2. Additional evidence that

basal resistance is likely dependent on additional mechanisms, besides the PAMP-

dependent activation of defense responses, is that fungal infection is able to activate

multiple defense-related pathways, as indicated by the increased PR-1 and PDF1.2

expression in response to B. cinerea (Ferrari et al., 2003a). The cumulative effect of these

responses results in the overall level of resistance observed in wild type plants, and the

relative contribution of each pathway appears evident only when specific mutants are

assayed. Similarly, basal defense against Pseudomonas syringae requires SA (Cao et al.,

1997, Delaney et al., 1994), even though flg22-mediate resistance also occurs in lines

impaired in this pathway (Zipfel et al., 2004). We have also shown here that flg22, like

OGs, induces resistance against B. cinerea independently of SA, ET and JA, and that



11

flg22-mediated resistance requires PAD3 and UPS1. This would suggest that both OGs,

flg22 and possibly other PAMPs act through similar or convergent signaling pathways. A

comparison of our microarray data to those previously reported on chitin-treated

Arabidopsis seedlings (Ramonell et al., 2005) revealed a remarkable overlap (about 70%)

between genes induced by OGs and genes induced by chitin. Considering that the

experimental conditions used in each analysis were variable with regards to age of

seedlings and time of elicitation, this observation is quite remarkable and supports the

hypothesis that a large portion of responses mediated by PAMPs and by OGs are

activated through a common signaling pathway, which is largely independent of SA, ET

and JA. In contrast, transcriptional changes specifically induced by chitin octamers are

quite distinct from those induced by OGs, supporting the hypothesis that a distinct

signaling pathway is mediated by short fragments (Ramonell et al., 2005).

Consistent with a role of PAD3 in OG-mediated resistance, induction of PAD3

expression in response to OGs is dramatically reduced in ups1 plants, which are also not

protected by elicitor treatments. In contrast to PAD3, UPS1 appears to encode a

regulatory protein required for the expression of different defense genes activated by

reactive oxygen species (Denby et al., 2005). OGs released during fungal infection may

activate the expression of PAD3 in a UPS1-dependent manner through the activation of a

localized oxidative burst. It is however important to note that expression of PDF1.2 and

PR-1 is also partially compromised in the ups1 mutant (Denby et al., 2005). Therefore,

the lack of OG-induced resistance in this genotype may due to loss of multiple defense

responses beside PAD3 expression.

All genetic evidence and expression data presented in this paper point to the

PAD3-dependent accumulation of camalexin as a major determinant of elicitor-induced

resistance against B. cinerea in Arabidopsis plants. However, no significant increase of

camalexin levels could be detected in plants sprayed with OGs compared to control-

treated plants. AgNO3 treatment resulted in a significant accumulation of camalexin in wt

adult leaves (data not shown), indicating that camalexin could be detected under our

experimental conditions. Therefore, although OG-induced resistance is PAD3-dependent,

it does not seem to be directly due to an increase in the levels of camalexin in leaf tissues

before pathogen inoculation. This result is surprising because PAD3 catalyzes the last



12

step in camalexin biosynthesis (Glawischnig et al., 2004). One possible explanation for

this discrepancy is that elicitor treatments do not induce camalexin accumulation

directly, but rather prime plants to synthesize more phytoalexin, or to do it more quickly,

after pathogen infection. It was previously shown that some chemicals can increase

resistance against pathogen infection by priming plant tissues to activate defense

responses more efficiently after inoculation. For instance, pre-treatments of parsley cells

with benzothiadiazole (BTH) results in enhanced production of coumarin and augmented

expression of genes encoding enzymes involved in phytoalexin biosynthesis after

inoculation (Katz et al., 1998). Arabidopsis plants pre-treated with β-amino-butyric acid

(BABA) show increased accumulation of callose at the site of infection (Ton and Mauch-

Mani, 2004). In the latter case, camalexin is not involved in the observed enhanced

resistance, since BABA-treated wt and pad3 plants are similarly protected against

necrotrophic pathogens, and, after inoculation, camalexin accumulates in primed plants to

lower levels than in control plants (Ton and Mauch-Mani, 2004). Experiments are in

progress to determine whether elicitors like OGs and flg22 are able to prime Arabidopsis

plants to produce more camalexin in response to B. cinerea. An alternative hypothesis is

is that PAD3 is involved in the biosynthesis of an unknown antimicrobial compound.

These compounds could either be degradation products of camalexin, or other indolic

compounds whose accumulation is affected by the pad3 and ups1 mutations. To verify

these hypotheses, a comprehensive analysis of secondary metabolites accumulating in

wild type and mutant plant treated with elicitors and/or infected with B. cinerea is

needed. Interestingly, 16 different indolic compounds were previously identified in

Arabidopsis, several of them being induced by pathogen infection (Bednarek et al.,

2005). Their role in defense against different pathogens, their levels in response to

elicitors and the impact of the pad3 mutation on their accumulation is still largely

unknown.

In conclusion, we have shown that OGs activate the expression of Arabidopsis

responses effective against B. cinerea through a pathway that is independent of the well-

characterized defense-related signaling molecules SA, JA and ET. Among these

responses, the expression of PAD3 and possibly other genes involved in the biosynthesis

of secondary metabolites play a major role in determining the enhanced resistance against



13

B. cinerea observed in OG-treated plants. The outcome of the interaction between

Arabidopsis and this fungus is mediated mainly by the levels of secondary metabolites in

the host and the sensitivity of the pathogen to such compounds (Kliebenstein et al.,

2005). Therefore, the activation of genes involved in secondary metabolism by cell wall

fragments released at the site of infection likely represents an effective mechanism to

restrict fungal growth. The dissection of the OG-activated transduction pathway and the

identification of the effectors induced by this and other elicitors will provide further

insights in the molecular mechanisms regulating the plant innate immune response.

MATERIALS AND METHODS

Upon request, all novel materials described in this publication will be made available in a

timely manner for non-commercial research purposes, subject to the requisite permission

from any third-party owners of all or parts of the material. Obtaining such permission, if

necessary, will be the responsibility of the requestor.

Plant treatments.

Arabidopsis accession Col-0 was obtained from G. Redei and A.R. Kranz

(Arabidopsis Information Service, Frankfurt, Germany); ups1 seeds were a kind gift of K.

Denby (University of Cape Town, South Africa). The triple npr1ein2jar1 mutant was

obtained from X. Dong (Duke University, Durham, NC, USA). Heterozygous coi1-

1/COI1-1 seeds were a kind gift from J. Turner (University of East Anglia, Norwich,

UK). Plants were grown on Metromix 200 medium (Scott, Marysville, OH) in a Percival

AR66 growth chamber at 22oC, relative humidity of 70%, and a 12 hour photoperiod with

light provided by Philips Hi-Vision white fluorescent lamps at an intensity of 120 µE m-2

s-1. Plants were fertilized weekly with 0.5× Hoagland solution.

OGs with a degree of polymerization of 10 to 15 were kindly provided by G.

Salvi (University of Rome “La Sapienza”, Italy). For elicitor treatments in adult plants, a

solution containing 200 µg ml-1 OGs or 5 µM flg22 and 0.01% Silwet L-77 (OSi

Specialties, Inc., Sisterville, WV, USA) was uniformly sprayed on 4-week-plants until

run-off (approximately 1 ml for each plant). Plants were then covered with transparent



14

plastic sheet, placed back in the growth chamber, and the plastic cover removed after 3 to

4 hours.

For expression profiling following OG treatments in seedlings, seeds were

sterilized and germinated in 12-well plates (approximately 15 seeds per well) containing

1 ml per well Murashige-Skoog (MS) medium (Life Technologies, Rockville, MD, USA)

(Murashige and Skoog, 1962) supplemented with 0.5% sucrose and Gamborg B5

vitamins. Plates were incubated at 22°C with a 16 hour photoperiod and a light intensity

of 100 µE m-2 s-1. After 8 days, the medium was replaced with 1ml of fresh medium. At

10 days, 50 µg ml-1 OGs or an equivalent volume of water was added to the medium. For

each biological replicate, about 15 seedlings were harvested, briefly blotted dry, and

immediately frozen in liquid nitrogen. For assays of PAD3 expression in seedlings, seeds

were sterilized and germinated in 24-well plates (approximately 15 seeds per well)

containing 1 ml per well Murashige-Skoog (MS) medium (Life Technologies, Rockville,

MD, USA) (Murashige and Skoog, 1962) supplemented with 0.5% sucrose and Gamborg

B5 vitamins. Plates were incubated at 22°C with a 16 hour photoperiod and a light

intensity of 100 µE m-2 s-1. After 10 days, the medium was replaced with 1ml of fresh

medium with or without 200 µg ml-1 OGs. For each biological replicate, about 10

seedlings from each of three separate wells (fresh weight: 100 to 200 mg) were harvested,

briefly blotted dry, pooled and immediately frozen in liquid nitrogen. The effectiveness

of the treatments was assessed by measuring H2O2 released in the medium (data not

shown). For treatment of coi1 seedlings, heterozygous COI1/coi1 seeds were first

germinated on agar plates containing 30 µM MeJA, and, after 8 days of growth, resistant

seedlings were transferred to liquid MS medium and treated with OGs two days later. As

a control, wt seedlings were grown for 8 days on agar plates and then transferred to liquid

MS medium.

Inoculation with B. cinerea for the microarray experiments was conducted on 4-

week-old plants by placing four 5 µl droplets of a spore suspension (5x105 conidia ml-1)

in 24 g l-1 potato dextrose broth on each rosette leaf (two fully expanded leaves per

plant). Inoculated plants were covered with a transparent plastic dome, to maintain high

humidity, and returned to the growth chamber. For each biological replicate, inoculated



15

leaves from three different plants (corresponding to about 200 mg fresh weight) were

harvested, pooled and immediately frozen in liquid nitrogen.

Inoculation of adult plants for the pathogenicity assays was conducted on

detached leaves, as previously described (Ferrari et al., 2003a). Homozygous coi1-1/coi1-

1 plants were identified after fungal infection by their sterile phenotype (Feys et al.,

1994). For induction of systemic resistance, OGs (200 µg ml-1 in sterile distilled water) or

water were infiltrated in two lower rosette leaves using a needle-less syringe. After 72

hours, upper, untreated fully expanded leaves were detached and inoculated with B.

cinerea.

Microarray hybridization.

For the OG experiment, three biological replicates for each treatment were

analyzed. For fungal infection, two replicates were analyzed. Total RNA was extracted

from each sample using the Qiagen RNeasy Plant RNA Miniprep kit (Qiagen, Valencia,

CA); samples were split in two before homogenization and re-pooled before loading on

the RNA-binding column. RNA quality was assessed by determining the A260/280 ratio of

RNA in Tris buffer and by checking the integrity of RNA on an Agilent 2100

Bioanalyzer (Agilent Technologies, www.agilent.com). Target labeling and microarray

hybridizations were performed according to the protocol given in the Affymetrix

GeneChip Expression Analysis Technical Manual 701025 rev 1 (for details, see

Supplemental Methods S1). Arrays were scanned using an Affymetrix GeneArray®

2500 Scanner and Affymetrix MicroArray Suite v5.0 software. Original raw data for each

experiment are available at NASC

(http://affymetrix.arabidopsis.info/narrays/experimentbrowse.pl) and

http://ausubellab.mgh.harvard.edu/imds (under experiment names “Botrytis cinerea

infection, 18 and 48 hpi” and “Comparison of response to Flg22 and OGs elicitors”).

Data analysis

To assess the quality of each hybridization, we used Affymetrix MicroArray Suite

v5.0 analysis software for reports of background intensity, signal to noise ratio, scaling

factor for global normalization, and ratios of intensity between 3’ and 5’ probe sets for



16

selected genes. Further data analysis was performed with Rosetta Resolver v3.2 Gene

Expression Data Analysis System (Rosetta Inpharmatics, Kirkland, WA, USA), using

Affymetrix .CEL files of array feature intensities and standard deviations as input.

Determination of absolute intensity values, propagation of error and P-values, and

normalization for comparing arrays in the Resolver system have been described in

Waring et al., 2001, and are summarized below. For each probe set, comprised of

multiple perfect match (PM) and mismatch (MM) probe pairs, an intensity difference

between each PM and corresponding MM was calculated. Probe pairs which differed by

more than 3 standard deviations from the mean PM-MM difference for the probe set were

considered outliers and were not included in the final calculation of the mean PM-MM

intensity difference. Calculation of the probability that a gene is present in the set of

transcripts being analyzed was based on the intensities of negative control genes. To

increase detection sensitivity, data from three biological replicates per OG treatment, and

two biological replicates per B. cinerea treatment were combined. For each array,

average intensities, associated intensity errors, and P-values were calculated for each

probe set. For calculating average intensity from replicate samples, arrays were scaled to

mean intensity, intensity values were transformed for homogenous variance, non-linear

error correction was performed, and probe set average intensities computed taking into

account measurement error calculations. P-values were calculated and intensity

transformed back to the original scale. Ratios of treated versus control intensities were

computed by calculating baseline mean background and signal, calculating ratio P-values,

and building simple ratios. One-way error-weighted ANOVA was used to identify

differentially expressed genes for each time point, using a threshold of P ≤ 0.01. Error-

weighted ANOVA has two inputs, expression level and measurement error associated

with the expression level, which provides additional information that yields more reliable

variance estimates when the number of replicates is small. Multiple testing correction

was performed using q-value. Only genes for which the absolute fold-change between

treated and control samples was greater than or equal to 2 were considered to be up- or

down-regulated. Gene annotation and assignment to functional categories were based on

The Institute for Genomic Research (TIGR) Arabidopsis thaliana Genome Annotation

Database (http://www.tigr.org/tdb/e2k1/ath1/ath1.shtml), the MIPS Functional Categories



17

Database (http://mips.gsf.de/projects/funcat) (Ruepp et al., 2004) and, for secondary

metabolism genes, the available literature (Gachon et al., 2005, Kliebenstein et al., 2005)

(see also Supplemental Table S5). Graphic representation of the expression of secondary

metabolism genes was performed using MapMan software (Thimm et al., 2004).

RNA analysis

Total RNA was prepared using the Trizol reagent (Life Technologies, Inc.,

Gaithersburg, MD, USA). RNA gel blots were prepared and hybridized with single-

stranded radioactive probes as previously described Rogers and Ausubel, 1997. Blots

were washed twice with 1% SDS, 2× SSC at 65°C for 45 minutes and images were taken

with a Phosphorimager (Molecular Dynamics, Sunnyvale, CA, USA) after overnight

exposure. The template used to generate the PAD3 probes was amplified by polymerase

chain reaction (PCR) from Arabidopsis Col-0 genomic DNA as previously described

(Zhou et al., 1999). For quantitative RT-PCR analysis, RNA was treated with RQ1

DNase (Promega) and first-strand cDNA was synthesized using ImProm-II Reverse

Transcriptase (Promega) according to the manufacturer’s guide. Real-Time PCR analysis

was performed using an I-Cycler (Biorad) according to the manufacturer’s guide. 2 µl

cDNA (corresponding to 120 ng of total RNA) were amplified in 30 µl reaction mix

containing IQ SYBR Green Supermix (Biorad) and 0.4 mM of each primer. Primer

sequences were the following: 5’-CCGGTGAATCTTGAGAGAGCC-3’ and 5’-

GATCAGCTCGGTCATTCCCC-3’ (PAD3); 5’-GGAAGAAGAAGACTTACACC-3’

and 5’-AGTCCACACTTACCACAGTA -3’ (UBQ5). Relative expression of the RT-

PCR products was determined using the ∆∆Ct method (Livak and Schmittgen, 2001).

ACKNOWLEDGMENTS

We are grateful to Jennifer Couget and Paul Grosu (Bauer Center for Genomics

Research, Harvard University, Cambridge, MA, USA) for assistance with microarray

hybridization and data analysis.



18

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24

FIGURE LEGENDS

Figure 1. Induction of resistance to B. cinerea by oligogalacturonides in Arabidopsis

plants. A, Lesion development in Arabidopsis Col-0 plants inoculated with B. cinerea 24

hours after treatment with a control solution (empty circles) or with OGs (black circles).

Lesion areas were measured at the indicated times. B, Lesion development in systemic

leaves of wild-type plants pre-treated with OGs. Lower leaves were infiltrated with OGs

or water and upper, untreated leaves were collected after 72 hours and inoculated with B.

cinerea. Lesion areas were measured 48 hours after inoculation. Values are means ± SE

of at least 12 lesions. Asterisks indicate statistically significant differences between

control and OG-treated plants, according to Student’s t-test (*, P < 0.05; ***, P < 0.01).

Figure 2. Induction of resistance to B. cinerea by oligogalacturonides in mutants

impaired in salicylic acid, jasmonate or ethylene signalling. A, Lesion area in

Arabidopsis Col-0 (WT), ein2 and nahG, plants treated with a control solution (empty

bars) or OGs (black bars) and inoculated with B. cinerea 24 hours after treatment. Lesion

areas were measured 48 hours after inoculation. B, Lesion area in Arabidopsis Col-0

(WT), jar1, npr1 and npr1ein2jar1 (nej) plants treated with a control solution (empty

bars) or OGs (black bars) and inoculated with B. cinerea 24 hours after treatment. Lesion

areas were measured 48 hours after inoculation. C, Lesion area in Arabidopsis Col-0

(WT) or homozygous coi1 plants treated with a control solution (empty bars) or OGs

(black bars) and inoculated with B. cinerea 24 hours after treatment. Lesion areas were

measured 48 hours after inoculation. Values are means ± SE of at least 12 lesions.

Asterisks indicate statistically significant differences between control and OG-treated

plants, according to Student’s t-test (*, P < 0.05; ***, P < 0.01).

Figure 3. Overlap between oligogalacturonide- and fungal infection-induced

transcriptional changes. Venn diagram of the number of overlapping and non-overlapping

genes in response to oligogalacturonides or B. cinerea infection. OG-up, genes induced

2.0-fold or more (P ≤ 0.01) after 1h or 3h of treatment with OGs; OG-down, genes

repressed 2.0-fold or more (P ≤ 0.01) after 1h or 3h of treatment with OGs; Bc-up, genes



25

induced 2.0-fold or more (P ≤ 0.01) after 18h or 48h of inoculation with B. cinerea; Bc-

down, genes repressed 2.0-fold or more (P ≤ 0.01) after 18h or 48h of inoculation with B.

cinerea. In parentheses is indicated the total number of genes belonging to each category.

Figure 4. Expression of genes involved in secondary metabolism in response to

oligogalacturonides and fungal infection. The scheme summarizes the relationships

between the shikimate, tryptophan, phenylalanine, phenylpropanoids, flavonoids,

camalexin, indole glucosinolates and aliphatic glucosinolates biosynthetic pathways and

the levels of expression of selected genes in each pathway, portrayed with MapMan

software. The number of small squares next to each pathway or portion of pathway

indicates how many genes present in the manually compiled list (for details, see Table 3)

and assigned to that pathway showed a 2.0-fold or greater change of expression (P ≤

0.01) in response to OGs (A) at 1h (left squares) or 3h (right squares) or Botrytis cinerea

infection (B) at 18 h (left squares) or 48 h (right squares). Blue squares represent genes

showing increased expression, red squares represent genes showing decreased

expression. Colour intensity indicates the extent of change, expressed as log2 of the mean

ratio between treated and control samples (see scale). Gray dots indicate that none of the

genes in the pathway are significantly induced or repressed by the indicated treatment.

Figure 5. Expression of PAD3 in response to oligogalacturonides and infection. A, Adult

Col-0 plants were sprayed with OGs and total RNA was extracted from rosette leaves

harvested at the indicated times (hours). PAD3 expression in each sample was determined

by Real Time RT-PCR and normalized to the expression of UBQ5. Bars indicate average

expression ± SD of two replicates, relative to the expression in untreated Col-0 plants. B,

Col-0 (WT), nahG, coi1, npr1, ein2, and jar1 adult plants were inoculated with B.

cinerea and total RNA was extracted from inoculated leaves at the indicated times (days

post infection). PAD3 expression was determined by RNA blot analysis. UBQ5

expression confirmed equal loading of the samples (not shown). C, Col-0 (WT),

ein2npr1jar1 (nej) and ups1 seedlings were treated with OGs and total RNA was

extracted at the indicated times (hours). PAD3 expression was analyzed by Real Time

RT-PCR and normalized using the expression of the UBQ5 gene. Bars indicate average



26

expression ± SD of two replicates, relative to the expression in untreated Col-0 plants. D,

Col-0 (WT) and coi1 seedlings were treated with OGs and total RNA was extracted at the

indicated times (hours). Expression of PAD3 and UBQ5 was analyzed by semi-

quantitative RT-PCR.

Figure 6. Induction of resistance to B. cinerea by oligogalacturonides in wild type plants

and in mutants impaired in camalexin production. Lesion area in Arabidopsis Col-0

(WT), pad3 and ups1 plants treated with a control solution (empty bars) or OGs (black

bars) and inoculated with B. cinerea 24 hours after treatment. Lesion areas were

measured 48 hours after inoculation. Values are means ± SE of at least 12 lesions.

Asterisks indicate statistically significant differences between control and OG-treated

plants, according to Student’s t-test (***, P < 0.01). The experiment was repeated three

times with similar results.

Figure 7. Induction of resistance to B. cinerea by flg22. A, Lesion area in Arabidopsis

Col-0 (WT), ein2npr1jar1 (nej) and pad3 plants treated with a control solution (empty

bars), or flg22 (black bars) and inoculated with B. cinerea 24 hours after treatment. B,

Lesion area in Arabidopsis Col-0 (WT), pad3 and ups1 plants treated with a control

solution (empty bars), or flg22 (black bars) and inoculated with B. cinerea 24 hours after

treatment. Lesion areas were measured 48 hours after inoculation. Values are means ± SE

of at least 12 lesions. Asterisks indicate statistically significant differences between

control and flg22-treated plants, according to Student’s t-test (***, P < 0.01). This

experiment was repeated twice with similar results.



27

Table 1. Number of genes showing altered expression in response to Botrytis cinerea

infection.

Time (hours) Induceda Represseda

18 153 1

48 1,942 2,871 a Number of genes reproducibly showing ≥ 2.0-fold induction or repression in all the

analyzed replicate samples (P ≤ 0.01).



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Table 2. Functional categories significantly over-represented among oligogalacturonide-

and B. cinerea-induced genes.

FUNCTIONAL CATEGORYa

Gene

matchesb Totalc P-valued

CELL RESCUE, DEFENSE AND

VIRULENCE 35 (6.0) 567 (2.1) 1.2×10-6

CLASSIFICATION NOT YET CLEAR-CUT 50 (8.6) 1,238 (4.6) 2.5 ×10-5

CELLULAR COMMUNICATION/SIGNAL

TRANSDUCTION 35 (6.0) 825 (3.1) 1.7 ×10-4

METABOLISM 57 (9.8) 1,722 (6.5) 1.2 ×10-3

amino acid metabolism 13 (2.2) 237 (0.9) 2.0 ×10-3

metabolism of the cysteine - aromatic group 4 (0.7) 29 (0.1) 3.5 ×10-3

secondary metabolism 18 (3.1) 336 (1.3) 4.9 ×10-4

biosynthesis of phenylpropanoids 7 (1.2) 69 (0.3) 7.7 ×10-4 a Functional categories according to MIPS FunCat database. b Number of genes induced ≥ 2.0-fold by both OGs and B. cinerea and present in the

indicated category. In parentheses is indicated the percentage of total OG- and B.cinerea-

induced genes that match the category.

c Total number of Arabidopsis genes in the indicated functional category. In parentheses

is indicated the percentage of genes in the Arabidopsis genome that match the category.

The comparison was done to the “Arabidopsis thaliana MAtDB” containing 26,642

annotated genes (http://mips.gsf.de/projects/funcat).

d P-value of significance of distribution of OG- and Botrytis-induced genes in the

indicated category, compared to distribution in the complete Arabidopsis dataset. Only

categories significantly over-represented (P<0.001) are indicated in the table.



29

Table 3. Secondary metabolism genes that change expression in response to

oligogalacturonides or B. cinerea infection.

Descriptiona Transcript ID Probe Set ID Fold changeb

OG

1h

OG

3h

Bc

18h

Bc

48h

Shikimate Pathway

DHS1; 2-dehydro-3-

deoxyphosphoheptonate aldolase At4g39980 252831_at 2.7 3.1 4.2

5-enolpyruvylshikimate-3-

phosphate synthase At1g48860 246627_s_at 2.3 2.1

CS; chorismate synthase At1g48850 245832_at 2.8 3.0

Anthranilate biosynthesis

ASB2; anthranilate synthase beta

chain At5g57890 247864_s_at 4.7 5.0 2.8 6.0

ASA1; anthranilate synthase

component I-1 precursor At5g05730 250738_at 4.5 5.2 3.4 6.8

Anthranilate glucosylation

UGT74F2; anthranilate

glucosyltransferase At2g43820 260567_at 6.6

Tryptophan biosynthesis

IGPS; putative indole-3-glycerol

phosphate synthase At2g04400 263807_at 3.0 6.6 2.8 5.4

PAT1; anthranilate

phosphoribosyltransferase At5g17990 250014_at 3.5 2.2 3.4

IGPS1; indole-3-glycerol

phosphate synthase At5g48220 248688_at -3.3

IAOx biosynthesis

CYP79B2 At4g39950 252827_at 4.6 5.5 4.8 8.7



30

CYP79B3 At2g22330 264052_at 2.6

Glucosinolate biosynthesis-general

UGT74B1 At1g24100 264873_at 2.5

SUR1/C-S lyase At2g20610 263714_at 2.9

IAA metabolism

ILR1; IAA conjugate hydrolase At3g02875 258610_at 8.7

NIT3; nitrilase At3g44320 252677_at 4.0

Camalexin biosynthesis

PAD3/CYP71B15; camalexin

biosynthesis At3g26830 258277_at 11.5 14.6 10.1

Phenylpropanoids/1-early steps

4CL1; 4-coumarate:CoA ligase At1g51680 256186_at 4.0

PAL1; phenylanine ammonia

lyase At2g37040 263845_at 3.6 3.5 2.8

4CL4; 4-coumarate:CoA ligase At5g45000 248971_at 3.6

CH4/REF3; cinnamate-4-

hydroxylase At2g30490 267470_at 3.0 3.2

4CL2; 4-coumarate:CoA ligase At3g21240 258047_at 2.7 2.7 2.8


lyase At3g53260 251984_at 2.5 2.0 5.2


lyase At5g04230 245690_at -3.8

Flavonoid biosynthesis

UGT78D1; flavonol-3-

rhamnosyltransferase At1g30530 261804_at -4.1 -2.6

UGT73C6; flavonol-7-O-

glucosyltransferase At2g36790 265200_s_at 6.3

Phenylpropanoids/2-late steps

CCR2; putative cinnamoyl-CoA At1g80820 261899_at 19.0 26.9



31

reductase

CCR; putative cinnamoyl-CoA

reductase At5g14700 250149_at 2.2 7.3

ELI3-2/CAD-B2/AtCAD8;

cinnamyl-alcohol dehydrogenase At4g37990 252984_at 31.5

CCOMT1; putative caffeoyl-

CoA O-methyltransferase At4g34050 253276_at 2.0

FAH1/F5H1; ferulate-5-

hydroxylase 1 At4g36220 253088_at 4.8 8.0

CAD1/AtCAD9; cinnamyl-

alcohol dehydrogenase At4g39330 252943_at -8.1

OMT1/COMT1; O-

methyltransferase 1 At5g54160 248200_at 2.7 2.5

Aliphatic glucosinolate biosynthesis

UGT74C1 At2g31790 263477_at -2.2 -6.4

CYP79F1 At1g16410 262717_s_at -2.8

REF2/CYP83A1 At4g13770 254687_at -5.3

Indole glucosinolate biosynthesis

SUR2/CYP83B1 At4g31500 253534_at 4.6 5.5 a Annotation based on The Institute for Genomic Research (TIGR) Arabidopsis thaliana

Genome Annotation Database, the MIPS Functional Categories Database

(http://mips.gsf.de/projects/funcat) or the available literature (for complete list and references,

see Supplemental Table S5). b Mean expression fold-change of probe sets is indicated only when change is significant (P ≤

0.01) and ≥ 2.0, and signal intensity is ≥ 0.1 for at least one treatment.

















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Running head: Oligogalacturonide-mediated resistance · 2007. 3. 23. · (reviewed in Nurnberger and Brunner, 2002). Pathogen-secreted hydrolytic enzymes that degrade host cell wall