Resistance to Botrytis cinerea Induced in - Plant Physiology

Resistance to Botrytis cinerea Induced in Arabidopsis byElicitors Is Independent of Salicylic Acid, Ethylene,or Jasmonate Signaling But RequiresPHYTOALEXIN DEFICIENT31[W]

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

Dipartimento Territorio e Sistemi Agro-Forestali, Universita degli Studi di Padova, 23–35020 Legnaro, Italy(S.F.); Department of Genetics, Harvard Medical School, and Department of Molecular Biology, MassachusettsGeneral Hospital, Boston, Massachusetts 02114 (C.D., F.M.A., J.D.); and Dipartimento di Biologia Vegetale,Universita di Roma La Sapienza, 00185 Rome, Italy (R.G., G.D.L.)

Oligogalacturonides (OGs) released from plant cell walls by pathogen polygalacturonases induce a variety of host defenseresponses. Here we show that in Arabidopsis (Arabidopsis thaliana), OGs increase resistance to the necrotrophic fungalpathogen 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 insecondary metabolism, show a similar change of expression during B. cinerea infection. In particular, expression ofPHYTOALEXIN DEFICIENT3 (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 or in underinducer after pathogen and stress1, a mutantwith severely impaired PAD3 expression in response to OGs. Similarly to OGs, the bacterial flagellin peptide elicitor flg22 alsoenhanced 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 pathogensthrough a signaling pathway also activated by pathogen-associated molecular pattern molecules.

Plants need to recognize invading pathogens in atimely manner to mount appropriate defense responses.In the so-called gene-for-gene resistance, early recog-nition of specific pathogen strains depends on comple-mentary pairs of dominant genes, one in the host andone in the pathogen. The outcome of this recognition isthe induction of a multitude of biochemical and phys-iological changes, including localized programmed celldeath (hypersensitive response), that restrict pathogengrowth in the host tissues. A loss of or a mutation ineither the plant resistance gene or in the pathogen

avirulence gene leads to a compatible interaction,resulting in disease (Flor, 1971). Gene-for-gene resis-tance has been observed in interactions with manybiotrophic pathogens, including fungi, viruses, bac-teria, and nematodes (Hammond-Kosack and Jones,1997). In contrast, many necrotrophic fungal andbacterial pathogens cause disease in a variety of plantspecies and resistance mediated by a single hostresistance gene is uncommon. Nevertheless, plantsalso recognize nonspecific elicitors that activate abattery of defense responses effective against a widerange of pathogens. Some of these elicitors, referred toas pathogen-associated molecular patterns (PAMPs),are derived from essential components of the patho-gen cell wall (e.g. chitin, glucan) or other macromo-lecular structures (e.g. the 22-amino acid flagellinpeptide flg22; for review, see Nurnberger and Brunner,2002).

Pathogen-secreted hydrolytic enzymes that degradehost cell wall polymers are also able to induce defenseresponses in plants. Among these, the most exten-sively studied are endopolygalacturonases (PGs; EC3.2.1.15). PGs cleave the a-(1 / 4) linkages betweenD-GalA residues in nonmethylated homogalacturonan,a major component of pectin (De Lorenzo et al., 1997).PGs are important virulence factors for necrotrophicsoft rot-causing pathogens, including the fungusBotrytis cinerea (ten Have et al., 1998). The elicitor

1 This work was supported by the Giovanni Armenise-HarvardFoundation, the Institute Pasteur-Fondazione Cenci Bolognetti, byMinistero dell’Universita e della Ricerca Fondo per gli Investimentidella Ricerca di Base 2001 and Cofinanziamento 2002 grants awardedto G.D.L., by the European Union (grant no. 23044 ‘‘Nutra-Snacks’’ toS.F.), and by the National Science Foundation (grant no. DBI–0114783to F.M.A.) and the National Institutes of Health (grant no. GM48707to F.M.A.).

* Corresponding author; e-mail [email protected]; fax 39–049–8272890.

The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Simone Ferrari ([email protected]).

[W] The online version of this article contains Web-only data.www.plantphysiol.org/cgi/doi/10.1104/pp.107.095596

Plant Physiology, May 2007, Vol. 144, pp. 367–379, www.plantphysiol.org � 2007 American Society of Plant Biologists 367

Dow

nloaded from https://academ

ic.oup.com/plphys/article/144/1/367/6106622 by guest on 14 January 2022

activity of PGs in activating plant defense responseshas been demonstrated in many pathosystems (DeLorenzo et al., 1997). Hahn and colleagues first showedthat PGs are not directly responsible for the inductionof plant defense responses, but rather cause the releasefrom the plant cell wall of the true elicitors, namelyoligogalacturonides (OGs) with a degree of polymeri-zation between 10 and 15 (OGs; Hahn, 1981). A func-tional catalytic site is required for elicitor activity of aColletotrichum lindemuthianum PG in tobacco (Nicotianatabacum; Boudart et al., 2003), supporting the hypoth-esis that OGs mediate responses activated by PGs. Incontrast, Poinssot and colleagues reported that enzy-matic activity is not required for elicitor activity of theB. cinerea PG BcPG1 in grape (Vitis vinifera) cells(Poinssot et al., 2003), suggesting that in some biolog-ical systems, PGs themselves can be perceived andactivate defense responses.

OGs elicit a variety of defense responses, includingaccumulation of phytoalexins (Davis et al., 1986),glucanase and chitinase (Davis and Hahlbrock, 1987;Broekaert and Pneumas, 1988), and Phe ammonialyase (PAL; De Lorenzo et al., 1987). Exogenous treat-ments with OGs protect grapevine leaves against B.cinerea infection in a dose-dependent fashion (Azizet al., 2004). Despite having been extensively studied,the role of OGs in plant defense and their mode ofaction are still largely unknown.

A variety of plant defense responses against micro-bial pathogens are regulated by the signaling mole-cules salicylic acid (SA), jasmonates (JAs), andethylene (ET; for review, see Feys and Parker, 2000).These signaling pathways have been exensively stud-ied, but a major unanswered question is how the SA,JA, and ET signaling pathways are related to thesignaling pathways activated by OGs and otherPAMPs. Although mutants impaired in the responsesmediated by SA, JA, and ET show enhanced diseasesymptoms upon infection with B. cinerea (Thommaet al., 1998, 1999; Alonso et al., 2003; Ferrari et al.,2003a), treatment with OGs or infection with B. cinereainduce the expression of AtPGIP1, which encodes anArabidopsis (Arabidopsis thaliana) inhibitor of fungalPGs, independently of these secondary signaling mol-ecules (Ferrari et al., 2003b). Since expression ofAtPGIP1 is required for full resistance to B. cinerea(Ferrari et al., 2006), we speculated that a SA-, JA-, andET-independent defense pathway induced by OGsduring fungal infection may actively contribute toplant defense. To investigate this hypothesis, weperformed global transcription profiling and suscep-tibility assays in Arabidopsis plants treated with ex-ogenous elicitors or inoculated with B. cinerea. Ourresults suggest that the expression of many defense-related genes is induced by OGs released duringpathogen infection and that responses induced inde-pendently of SA, JA, and ET, and in particular the expres-sion of genes involved in the production of antimicrobialcompounds, participate to restrict the growth of B.cinerea.

RESULTS

OGs Induce Local and Systemic Resistance to B. cinerea

To assess the ability of OGs to enhance Arabidopsisresistance to fungal infection, adult plants weresprayed with OGs and inoculated with B. cinerea 24 hafter the treatment. Leaves from plants treated withOGs showed significantly delayed progression of theinfection, compared to control-treated plants (Supple-mental Fig. S1; Fig. 1A). Similar results were obtainedwhen plants were inoculated 48 or 72 h after OGtreatment (data not shown). To determine whetherOGs also induce systemic resistance, lower rosetteleaves were injected with water or OGs and, after 72 h,upper leaves were inoculated with B. cinerea. The

Figure 1. Induction of resistance to B. cinerea by OGs in Arabidopsisplants. A, Lesion development in Arabidopsis Col-0 plants inoculatedwith B. cinerea 24 h after treatment with a control solution (whitecircles) or with OGs (black circles). Lesion areas were measured at theindicated times. B, Lesion development in systemic leaves of wild-typeplants pretreated with OGs. Lower leaves were infiltrated with OGs(black bars) or water (white bars) and upper, untreated leaves werecollected after 72 h and inoculated with B. cinerea. Lesion areas weremeasured 48 h after inoculation. Values are means 6 SE of at least 12lesions. Asterisks indicate statistically significant differences betweencontrol and OG-treated plants, according to Student’s t test (*, P , 0.05;***, P , 0.01).

Ferrari et al.

368 Plant Physiol. Vol. 144, 2007

Dow



average area of necrotic lesions in systemic leaves ofplants pretreated with OGs was significantly smallerthan in control plants (Fig. 1B). These results indicatethat OGs induce both local and systemic resistanceagainst 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-inducedresistance, we sprayed OGs on mutants or transgenicplants affected in the perception or transduction ofthese hormone signals and inoculated them with B.cinerea 24 h later. The Arabidopsis lines used weretransgenic plants expressing the salicylate hydroxylasenahG gene and therefore unable to accumulate SA(Gaffney et al., 1993), plants carrying single mutationsin the NONEXPRESSOR OF PATHOGENESIS-RELATEDGENES1 (NPR1), ETHYLENE-INSENSITIVE2 (EIN2), orJASMONATE RESISTANT1 (JAR1) genes impairedin signaling mediated by SA, ET, or JA, respectively(Guzman and Ecker, 1990; Staswick et al., 1992; Caoet al., 1994), or a triple mutant carrying a mutation ineach of these genes (npr1ein2jar1) and therefore un-able to respond to any of the hormones (Clarke et al.,2000). As described previously (Thomma et al., 1999;Ferrari et al., 2003a), ein2 mutant and nahG transgenicplants are more susceptible to B. cinerea. Neverthe-less, OG pretreatments of these mutants, as well as ofthe other mutants tested, significantly reduced lesionsize (Fig. 2, A and B). To further confirm that OG-mediated resistance is JA independent, we also treatedB. cinerea-susceptible coronatine-insensitive1 (coi1) ho-mozygous mutant plants (Thomma et al., 1998) withOGs. Two days after inoculation, lesions in control coi1plants were more than 5-fold larger than in wild-typecontrol plants; however, OG-pretreated coi1 plantswere significantly more resistant to B. cinerea, similarto the highly susceptible ein2 mutant (Fig. 2C). Takentogether, these results indicate that OGs increaseArabidopsis resistance to B. cinerea through the acti-vation of defense responses that are independent ofSA, JA, and ET, but that SA, JA, and ET are alsoinvolved in defense pathways that confer resistanceto B. cinerea.

Changes in Gene Expression in Arabidopsis PlantsTreated with OGs or Inoculated with B. cinerea

To identify Arabidopsis genes that may be involvedin OG-mediated resistance to B. cinerea, we analyzedthe transcriptome of plants inoculated with the fungusand compared it to the transcription profile of 10-d-oldseedlings treated with OGs for 1 or 3 h. For the analysisof infected plants, rosette leaves were inoculated witha B. cinerea spore suspension or with sterile mediumand harvested after 18 or 48 h. At the early time pointof infection, no macroscopic lesions were observed atthe site of inoculation, though staining of fungalhyphae with trypan blue revealed that the sporeshad germinated and started growing on the leaf sur-face (data not shown). After 48 h, the infected leavesshowed water-soaked lesions about 3 to 4 mm inwidth that are typical of soft rot disease. Total RNAfrom control or treated samples from two or three inde-pendent infection or elicitor-treatment experiments,respectively, was analyzed using the Affymetrix ATH1

Figure 2. Inductionof resistance toB.cinereabyOGsinmutants impairedin SA, JA, or ET signaling. A, Lesion area in Arabidopsis Col-0 (WT), ein2,and nahG plants treated with a control solution (white bars) or OGs (blackbars) and inoculatedwithB. cinerea24 hafter treatment. Lesionareasweremeasured 48 h after inoculation. B, Lesion area in Arabidopsis Col-0 (WT),jar1, npr1, and npr1ein2jar1 (nej ) plants treated with a control solution(white bars) or OGs (black bars) and inoculated with B. cinerea 24 h aftertreatment. Lesion areas were measured 48 h after inoculation. C, Lesionarea in Arabidopsis Col-0 (WT) or homozygous coi1 plants treated with acontrol solution (white bars) or OGs (black bars) and inoculated with B.cinerea 24 h after treatment. Lesion areas were measured 48 h afterinoculation. Values are means 6 SE of at least 12 lesions. Asterisks indicatestatistically significant differences between control and OG-treated plants,according to Student’s t test (*, P , 0.05; ***, P , 0.01).

Oligogalacturonide-Mediated Resistance

Plant Physiol. Vol. 144, 2007 369

Dow



GeneChip DNA microarray, which contains probe setscorresponding to more than 22,000 putative openreading frames. Original raw data for each experimentare available at the Nottingham Arabidopsis StockCentre (http://affymetrix.arabidopsis.info/narrays/experimentbrowse.pl) and http://ausubellab.mgh.harvard.edu/imds (under experiment names ‘‘Botrytiscinerea infection, 18 and 48 hpi’’ and ‘‘Comparison ofresponse to Flg22 and OGs elicitors’’). For each probeset, mean expression fold-change, signal intensities,and P values were calculated after normalization (seeSupplemental Tables S1 and S2). Only probe sets show-ing a statistically significant difference (P # 0.01)between control and experimental treatments and amean fold-change $2.0 were considered for furtheranalysis.

As summarized in Table I, at 48 h post inoculation(hpi), when typical water-soaked lesions were visible,more than 10 times as many genes showed significantchanges in transcription than at 18 hpi. Interestingly,588 out of 1,299 genes up-regulated by OGs after either1 h or 3 h were also up-regulated by B. cinerea at eitherof the two time points analyzed (Fig. 3). Similarly, 316out of 577 genes significantly repressed by OGs ateither 1 h or 3 h were also down-regulated duringfungal infection, indicating that about half of the genesresponsive to OGs show a significant change of ex-pression in the same direction upon B. cinerea attack(Fig. 3; a complete list of the genes coregulated by OGsand 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 profileinduced by crab shell chitin and chitin octamers(Ramonell et al., 2005), using the same threshold (ex-pression change $1.5-fold). Out of 1,002 genes whoseexpression is induced by both crab shell chitin andoctamers after 30 min of treatment, 697 were also up-regulated by OGs after either 1 or 3 h, whereas 84 genesout of 312 chitin-repressed genes were significantlydown-regulated by OGs (Supplemental Table S4). Incontrast, only six out of 68 genes induced specifically byoctamers and 39 out of 238 genes repressed only byoctamers were also induced or repressed by OGs,respectively.

To group genes up-regulated by both OGs and B.cinerea according to their predicted functions, we iden-tified Functional Catalogue (FunCat) terms (Rueppet al., 2004) associated with each gene using theMunich Information Center for Protein Sequences

(MIPS) Arabidopsis Data Base (MAtDB; http://mips.gsf.de/proj/thal/db/index.html). For this anal-ysis, only probes with an annotated locus identifierwere used. For each expression category, frequenciesof genes in a given FunCat group were compared withthe frequency found for all genes represented on thearray (Table II). As expected, the most representedcategories included genes involved in cell rescue anddefense, genes whose classification is ambiguous,genes involved in cell communication and signaling,and genes encoding proteins implicated in primaryand secondary metabolism.

A majority of the genes involved in secondarymetabolism that are induced by OGs and Botrytisand identified through the MAtDB database encodeenzymes implicated in amino acid metabolism andphenylpropanoid biosynthesis (Table II). However, wenoticed that a number of genes involved in the me-tabolism of Trp-derived secondary compounds, likePHYTOALEXIN DEFICIENT3 (PAD3; Zhou et al.,1999) and CYP79B2 (Hull et al., 2000), were includedin the not yet clear-cut category. We therefore manu-ally compiled a list of 66 Arabidopsis genes previouslyimplicated in secondary metabolism (full list is avail-able in the Supplemental Table S5). Expression of 36 ofthese genes appeared significantly induced or re-pressed by at least one treatment (Table III). In partic-ular, several genes encoding enzymes of the shikimateand the phenylpropanoid pathways (with the excep-tion of flavonoid biosynthesis) and enzymes involvedin the biosynthesis of Trp and of indole compoundswere up-regulated by both OGs and B. cinerea, whereasmost genes proposed to encode enzymes involved inthe biosynthesis of aliphatic glucosinolates, likeCYP79F1, REF2, and UGT74C1 (Hansen et al., 2001;Hemm et al., 2003; Gachon et al., 2005), were repressedor not significantly affected by OGs or fungal infection(Table III; Fig. 4, A and B). Interestingly, mRNA levelsof ASA1, ASB2, IGPS, CYP79B2, and PAD3, which areall involved in the biosynthesis of indolic compounds(Zhao and Last, 1996; Zook, 1998; Zhou et al., 1999;Hull et al., 2000; Glawischnig et al., 2004), increase atboth 18 and 48 h after infection and by OGs after 3 h oftreatment (Table III). With the exception of PAD3, whichencodes the cytochrome P450 enzyme CYP71B15 thatcatalyzes the last step of camalexin biosynthesis(Schuhegger et al., 2006), the other genes encodingenzymes in the indole pathway may also be involvedin the biosynthesis of glucosinolates or auxin. How-ever, genes specifically required for indole-3-aceticacid (IAA) metabolism, including a nitrilase geneand ILR1, encoding an IAA-conjugate hydrolase (Barteland Fink, 1995), were not up-regulated by OGs, al-though they were induced by B. cinerea (Table III; Fig.4, A and B). Because pad3 plants are extremely sus-ceptible to B. cinerea infection (Ferrari et al., 2003a),it appears likely that OG-mediated activation ofPAD3 and other genes involved in the biosynthesisof camalexin may contribute to OG-induced resistanceagainst B. cinerea.

Table I. Number of genes showing altered expression in response toB. cinerea infection

Time Induceda Represseda

h

18 153 148 1,942 2,871

aNumber of genes reproducibly showing $2.0-fold induction orrepression in all the analyzed replicate samples (P # 0.01).

Ferrari et al.


Dow



PAD3 Is Expressed Independently of SA, JA, and ET andIs 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 exper-iments showed that PAD3 expression is induced byboth OGs and B. cinerea (this work), we further inves-tigated OG-mediated induction of PAD3 expression inadult plants. A transient accumulation of PAD3 mRNA,with a peak at 3 to 6 h after treatment, was observed byquantitative reverse transcription (RT)-PCR in rosetteleaves sprayed with OGs (Fig. 5A). Interestingly, theincrease in PAD3 transcript levels after B. cinereainoculation of leaves was not attenuated in nahG,ein2, npr1, jar1, or coi1 plants (Fig. 5B), indicating thatPAD3 expression during fungal infection is also inde-pendent of SA, JA, and ET. Consistently, PAD3 mRNAlevels in wild-type and npr1ein2jar1 seedlings treated

with OGs for 1 and 3 h were not significantly different(Fig. 5C). OG treatments were also similarly effectivein inducing PAD3 expression in single ein2, npr1, andjar1 seedlings, as determined by semiquantitative RT-PCR (data not shown). Furthermore, mRNA levels ofPAD3 in coi1 seedlings treated with OGs were evenhigher than in wild-type seedlings (Fig. 5D). Theseresults indicate that OGs are able to increase PAD3expression independently of SA, JA, and ET.

Since protection against B. cinerea infection medi-ated by OG pretreatment is also independent of SA-,JA-, and ET-mediated signaling (Fig. 2, A–C), PAD3may be important for OG-induced resistance to thispathogen. To test this hypothesis, we inoculated OG-treated wild-type or pad3 plants with B. cinerea. Strik-ingly, control and OG-treated pad3 plants showedsimilar lesion development, whereas, in accordancewith our previous results, OG-treated wild-type plantswere more resistant to infection (Fig. 6). To furtherconfirm the role of PAD3 in OG-induced resistance, weanalyzed the underinducer after pathogen and stress1(ups1) mutant, which is defective in Trp biosyntheticpathway regulation and accumulates low levels ofcamalexin and of PAD3 mRNA in response to fungalinfection and oxidative stress (Denby et al., 2005). Asshown in Figure 6, ups1 plants were not protected byOGs against B. cinerea. Consistent with our hypothesisthat PAD3 expression is required for OG-inducedresistance, PAD3 expression in OG-treated ups1 seed-lings was strongly reduced (Fig. 5C), in contrast to thetriple npr1ein2jar1 mutant. Interestingly, the bacterialflagellar peptide elicitor flg22, which is structurallyunrelated to OGs, was also effective in protectingArabidopsis wild-type plants, as well as npr1ein2jar1triple mutant plants, against B. cinerea, and this pro-tection was also dependent on PAD3 and UPS1 (Fig. 7,A and B). In one experiment we observed a slightreduction of symptoms in flg22-treated ups1 plants(data not shown), but this result could not be repli-cated, and in two independent experiments flg22pretreatments failed to protect ups1 against B. cinereainfection (Fig. 7B).

Figure 3. Overlap between OG- and fungal infection-induced tran-scriptional changes. Venn diagram of the number of overlapping andnonoverlapping genes in response to OGs or B. cinerea infection. OG-up, Genes induced 2.0-fold or more (P # 0.01) after 1 or 3 h oftreatment with OGs; OG-down, genes repressed 2.0-fold or more (P #

0.01) after 1 or 3 h of treatment with OGs; Bc-up, genes induced 2.0-fold or more (P # 0.01) after 18 or 48 h of inoculation with B. cinerea;Bc-down, genes repressed 2.0-fold or more (P # 0.01) after 18 or 48 hof inoculation with B. cinerea. In parentheses is indicated the totalnumber of genes belonging to each category.

Table II. Functional categories significantly overrepresented among OG- 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 3 1026

Classification not yet clear cut 50 (8.6) 1,238 (4.6) 2.5 3 1025

Cellular communication/signal transduction 35 (6.0) 825 (3.1) 1.7 3 1024

Metabolism 57 (9.8) 1,722 (6.5) 1.2 3 1023

Amino acid metabolism 13 (2.2) 237 (0.9) 2.0 3 1023

Metabolism of the Cys-aromatic group 4 (0.7) 29 (0.1) 3.5 3 1023

Secondary metabolism 18 (3.1) 336 (1.3) 4.9 3 1024

Biosynthesis of phenylpropanoids 7 (1.2) 69 (0.3) 7.7 3 1024

aFunctional categories according to MIPS FunCat database. bNumber of genes induced $2.0-fold by both OGs and B. cinerea and present inthe indicated category. In parentheses is indicated the percentage of total OG- and B. cinerea-induced genes that match the category. cTotalnumber of Arabidopsis genes in the indicated functional category. In parentheses is indicated the percentage of genes in the Arabidopsis genome thatmatch the category. The comparison was done to the Arabidopsis thaliana MAtDB containing 26,642 annotated genes (http://mips.gsf.de/projects/funcat). dP value of significance of distribution of OG- and Botrytis-induced genes in the indicated category, compared to distribution in thecomplete Arabidopsis dataset. Only categories significantly overrepresented (P , 0.001) are indicated in the table.



Dow



Since PAD3 is required for elicitor-induced resis-tance against B. cinerea, we expected that OGs couldincrease camalexin levels in Arabidopsis adult plants.Surprisingly, however, HPLC analysis failed to revealsignificant camalexin accumulation up to 24 h after

treatment with the elicitor, compared to control-treatedplants (data not shown). Therefore, OG-induced resis-tance does not seem to be directly due to an increase inthe levels of camalexin in leaf tissues before pathogeninoculation.

Table III. Secondary metabolism genes that change expression in response to OGs or B. cinerea infection

Descriptiona Transcript ID Probe Set ID Fold-Changeb

OG 1 h OG 3 h Bc 18 h Bc 48 h

Shikimate pathwayDHS1; 2-dehydro-3-deoxyphosphoheptonate aldolase At4g39980 252831_at 2.7 3.1 4.25-Enolpyruvylshikimate-3-P synthase At1g48860 246627_s_at 2.3 2.1CS; chorismate synthase At1g48850 245832_at 2.8 3.0

Anthranilate biosynthesisASB2; anthranilate synthase b-chain At5g57890 247864_s_at 4.7 5.0 2.8 6.0ASA1; anthranilate synthase component I-1 precursor At5g05730 250738_at 4.5 5.2 3.4 6.8

Anthranilate glucosylationUGT74F2; anthranilate glucosyltransferase At2g43820 260567_at 6.6

Tryptophan biosynthesisIGPS; putative indole-3-glycerol phosphate synthase At2g04400 263807_at 3.0 6.6 2.8 5.4PAT1; anthranilate phosphoribosyltransferase At5g17990 250014_at 3.5 2.2 3.4IGPS1; indole-3-glycerol phosphate synthase At5g48220 248688_at 23.3

IAOx biosynthesisCYP79B2 At4g39950 252827_at 4.6 5.5 4.8 8.7CYP79B3 At2g22330 264052_at 2.6

Glucosinolate biosynthesis (general)UGT74B1 At1g24100 264873_at 2.5SUR1/C-S lyase At2g20610 263714_at 2.9

IAA metabolismILR1; IAA conjugate hydrolase At3g02875 258610_at 8.7NIT3; nitrilase At3g44320 252677_at 4.0

Camalexin biosynthesisPAD3/CYP71B15; camalexin biosynthesis At3g26830 258277_at 11.5 14.6 10.1

Phenylpropanoids/1-early steps4CL1; 4-coumarate:CoA ligase At1g51680 256186_at 4.0PAL1; phenylanine ammonia lyase At2g37040 263845_at 3.6 3.5 2.84CL4; 4-coumarate:CoA ligase At5g45000 248971_at 3.6CH4/REF3; cinnamate-4-hydroxylase At2g30490 267470_at 3.0 3.24CL2; 4-coumarate:CoA ligase At3g21240 258047_at 2.7 2.7 2.8PAL2; phenylanine ammonia lyase At3g53260 251984_at 2.5 2.0 5.2PAL3; phenylanine ammonia lyase At5g04230 245690_at 23.8

Flavonoid biosynthesisUGT78D1; flavonol-3-rhamnosyltransferase At1g30530 261804_at 24.1 22.6UGT73C6; flavonol-7-O-glucosyltransferase At2g36790 265200_s_at 6.3

Phenylpropanoids/2-late stepsCCR2; putative cinnamoyl-CoA reductase At1g80820 261899_at 19.0 26.9CCR; putative cinnamoyl-CoA reductase At5g14700 250149_at 2.2 7.3ELI3-2/CAD-B2/AtCAD8; cinnamyl-alcohol dehydrogenase At4g37990 252984_at 31.5CCOMT1; putative caffeoyl-CoA O-methyltransferase At4g34050 253276_at 2.0FAH1/F5H1; ferulate-5-hydroxylase 1 At4g36220 253088_at 4.8 8.0CAD1/AtCAD9; cinnamyl-alcohol dehydrogenase At4g39330 252943_at 28.1OMT1/COMT1; O-methyltransferase 1 At5g54160 248200_at 2.7 2.5

Aliphatic glucosinolate biosynthesisUGT74C1 At2g31790 263477_at 22.2 26.4CYP79F1 At1g16410 262717_s_at 22.8REF2/CYP83A1 At4g13770 254687_at 25.3

Indole glucosinolate biosynthesisSUR2/CYP83B1 At4g31500 253534_at 4.6 5.5

aAnnotation based on The Institute for Genomic Research Arabidopsis 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). bMean expressionfold-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.

Ferrari et al.


Dow



DISCUSSION

Activation of the plant innate immune system can bemediated, as in animals, by molecular patterns con-served among different pathogen species but notpresent in host cells. However, in plants, some host-derived low Mr elicitors may also be released duringan infection as a consequence of pathogen activity.These endogenous elicitors, like true PAMPs, maytrigger defense responses that contribute to basalresistance. OGs represent the best characterized en-dogenous elicitors and have been extensively studiedsince their identification about 25 years ago (Hahnet al., 1981). In this article we have investigated whatresponses activated by OGs may be involved inArabidopsis defense against the fungal pathogen B.cinerea. Treatment with exogenous OGs enhances re-sistance against B. cinerea in grape (Aziz et al., 2004)and Arabidopsis leaves (this work). Since B. cinereasecretes large amounts of polygalacturonases duringtissue invasion (for review, see Kars and van Kan,2004), it is likely that OGs transiently accumulate atthe interface between fungal hyphae and plant tis-sues. However, detection of OGs in the apoplast ofinfected plants is technically challenging. Recently, Anand colleagues have employed matrix-assisted laser-desorption ionization-Fourier transform and matrix-assisted laser-desorption ionization-time of flight massspectrometry to identify cell wall pectin-derived oli-gosaccharides generated through the breakdown ofhomogalacturonan pectins in B. cinerea-infected tomato(Solanum lycopersicum) fruits (An et al., 2005). This studysuggests that pectic fragments with a degree of po-lymerization of about 16 accumulate around the le-sions caused by this fungus. However, a conclusivedemonstration that OGs with elicitor activity accumu-late to significant levels during infection is still lacking,and their role in basal resistance to pathogens must beinferred from indirect evidence.

We have carried out a full-genome expression anal-ysis of Arabidopsis plants treated with OGs or infectedwith B. cinerea to determine the extent of overlapbetween transcriptional responses induced by thesetwo treatments. Previously, Navarro and colleagues,comparing microarray data obtained from flg22-treated cell cultures and seedlings, and data obtainedfrom bacterial-inoculated rosette leaves, found a limitedoverlap (7%) in the compatible interaction and a moreconsistent overlap (34%) with an incompatible inter-action (Navarro et al., 2004). Our results indicate thatabout half of the Arabidopsis genes affected by OGtreatment display a similar behavior after fungal in-fection, suggesting that at least part of the responsesactivated by B. cinerea are mediated, directly or indi-rectly, by the accumulation of OGs or other elicitorsable to activate the same signaling pathway, and thatthese responses are not suppressed by the fungus. Thegenes induced by both OGs and B. cinerea infectionnot only encode defense-related proteins, but also en-zymes implicated in primary and secondary metabo-

lism. The transcriptional activation of some of thesegenes during fungal infection may result in the accu-mulation of antimicrobial proteins or low Mr com-pounds, which are able to restrict fungal growth. In thecase of B. cinerea, it has been previously shown thatstrains sensitive to camalexin are partially restricted inplanta by this phytoalexin, since pad3 and other mu-tants impaired in camalexin accumulation are moresusceptible to infection than the parental lines (Ferrariet al., 2003a; Kliebenstein et al., 2005).Camalexin maytherefore play a major role in the reduction in B. cinereagrowth observed in OG-treated plants. Furthermore,OG-induced resistance and PAD3 expression are bothlargely independent of SA, ET, and JA. OGs (and B.cinerea) therefore appear to activate an SA-, ET-, andJA-independent signaling pathway that regulatesPAD3 expression and other defense responses effec-tive against B. cinerea. This conclusion is consistentwith the previous observation that the expression ofAtPGIP1, another OG-responsive gene in Botrytis-inoculated plants, is also independent of SA, ET, andJA (Ferrari et al., 2003b). Similarly, expression of twoArabidopsis chitin-inducible genes was shown to beindependent of SA, ET, and JA (Zhang et al., 2002).More recently, Raacke and colleagues found that yeast(Saccharomyces cerevisiae) cells also induce resistanceagainst B. cinerea in Arabidopsis, and that this resis-tance is independent of SA, JA, and ET (Raacke et al.,2006b). Interestingly, the authors observed that thisresistance is also independent of PAD3; however, it isnot known whether the fungal strain they used issensitive to camalexin, and it is therefore difficult todraw any conclusion on the role of camalexin in yeast-induced resistance. Furthermore, yeast treatment, incontrast to OGs, and also to flg22, induces resistanceagainst P. syringae through an SA-dependent mecha-nism (Raacke et al., 2006a), indicating that additionaldefense responses are activated by yeast cells, com-pared to chemically defined elicitors. A recent articledescribes the expression profile of nahG, ein2, and coi1plants inoculated with B. cinerea, and demonstratesthat the ZFAR1 gene, encoding a zinc finger proteincontaining ankyrin repeats, is expressed indepen-dently of SA, ET, and JA and is required for basallocal resistance against this fungus (AbuQamar et al.,2006). Interestingly, ZFAR1 is also induced by OGs inour microarray experiments, and determining whetherthis gene is also required for OG-induced resistancecould provide further insights in its role in elicitor-mediated signaling. Furthermore, the isolation of mu-tants impaired in OG-dependent responses willconfirm the role of these elicitors in defense againstpathogens.

The data presented in this manuscript and in pre-vious publications (Ferrari et al., 2003a) indicate thatPAD3-mediated resistance elicited by OGs (and otherPAMPs) is one of several independent signaling path-ways that contribute to basal resistance. Thus in SA,JA, and ET signaling mutants, PAMP-mediated resis-tance is still operating such that OG treatment will



Dow



result in partial resistance as shown in Figure 2.Additional evidence that basal resistance is likelydependent on additional mechanisms, besides thePAMP-dependent activation of defense responses, isthat fungal infection is able to activate multiple defense-

related pathways, as indicated by the increased PR-1and PDF1.2 expression in response to B. cinerea (Ferrariet al., 2003a). The cumulative effect of these responsesresults in the overall level of resistance observed inwild-type plants, and the relative contribution of each

Figure 4. Expression of genes in-volved in secondary metabolism inresponse to OGs and fungal in-fection. The scheme summarizesthe relationships between theshikimate, Trp, Phe, phenylpropa-noids, flavonoids, camalexin, in-dole glucosinolates, and aliphaticglucosinolates biosynthetic path-ways and the levels of expressionof selected genes in each pathway,portrayed with MapMan software.The number of small squares nextto each pathway or portion of path-way indicates how many genespresent in the manually compiledlist (for details, see Table III) andassigned to that pathway showed a2.0-fold or greater change of ex-pression (P # 0.01) in response toOGs (A) at 1 h (left squares) or 3 h(right squares) or B. cinerea infec-tion (B) at 18 h (left squares) or 48 h(right squares). Red squares repre-sent genes showing increased ex-pression, blue squares representgenes showing decreased expres-sion. Color intensity indicates theextent of change, expressed as log2

of the mean ratio between treatedand control samples (see scale).Gray dots indicate that none of thegenes in the pathway are signifi-cantly induced or repressed by theindicated treatment.

Ferrari et al.


Dow



pathway appears evident only when specific mutantsare assayed. Similarly, basal defense against Pseudomo-nas syringae requires SA (Delaney et al., 1994; Cao et al.,1997), even though flg22-mediated resistance also oc-curs 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 ofSA, ET, and JA, and that flg22-mediated resistancerequires PAD3 and UPS1. This would suggest that OGs,flg22, and possibly other PAMPs act through similar orconvergent signaling pathways. A comparison of ourmicroarray data to those previously reported on chitin-treated Arabidopsis seedlings (Ramonell et al., 2005)revealed a remarkable overlap (about 70%) betweengenes induced by OGs and genes induced by chitin.Considering that the experimental conditions used ineach analysis were variable with regards to age ofseedlings and time of elicitation, this observation isquite remarkable and supports the hypothesis that alarge portion of responses mediated by PAMPs and byOGs are activated through a common signaling path-way, which is largely independent of SA, ET, and JA. Incontrast, transcriptional changes specifically inducedby chitin octamers are quite distinct from those in-duced by OGs, supporting the hypothesis that a dis-tinct signaling pathway is mediated by short fragments(Ramonell et al., 2005).

Consistent with a role of PAD3 in OG-mediatedresistance, induction of PAD3 expression in responseto OGs is dramatically reduced in ups1 plants, whichare also not protected by elicitor treatments. In contrastto PAD3, UPS1 appears to encode a regulatory proteinrequired for the expression of different defense genesactivated by reactive oxygen species (Denby et al.,2005). OGs released during fungal infection may acti-vate the expression of PAD3 in a UPS1-dependentmanner through the activation of a localized oxidativeburst. It is, however, important to note that expressionof PDF1.2 and PR-1 is also partially compromised inthe ups1 mutant (Denby et al., 2005). Therefore, thelack of OG-induced resistance in this genotype maydue to loss of multiple defense responses beside PAD3expression.

All genetic evidence and expression data presentedin this article point to the PAD3-dependent accumula-tion of camalexin as a major determinant of elicitor-induced resistance against B. cinerea in Arabidopsisplants. However, no significant increase of camalexinlevels could be detected in plants sprayed with OGscompared to control-treated plants. AgNO3 treatmentresulted in a significant accumulation of camalexin inwild-type adult leaves (data not shown), indicatingthat camalexin could be detected under our experi-mental conditions. Therefore, although OG-inducedresistance is PAD3 dependent, it does not seem to bedirectly due to an increase in the levels of camalexinin leaf tissues before pathogen inoculation. This resultis surprising because PAD3 catalyzes the last stepin camalexin biosynthesis (Glawischnig et al., 2004).One possible explanation for this discrepancy is that

Figure 5. Expression of PAD3 in response to OGs and infection. A,Adult Col-0 plants were sprayed with OGs and total RNA was extractedfrom rosette leaves harvested at the indicated times (hours). PAD3expression in each sample was determined by real-time RT-PCR andnormalized to the expression of UBQ5. Bars indicate average expres-sion 6 SD of two replicates, relative to the expression in untreated Col-0plants. B, Col-0 (WT), nahG, coi1, npr1, ein2, and jar1 adult plantswere inoculated with B. cinerea and total RNA was extracted frominoculated leaves at the indicated times (days post infection). PAD3expression was determined by RNA-blot analysis. UBQ5 expressionconfirmed equal loading of the samples (data not shown). C, Col-0(WT), ein2npr1jar1 (nej), and ups1 seedlings were treated with OGsand total RNA was extracted at the indicated times (hours). PAD3expression was analyzed by real-time RT-PCR and normalized usingthe expression of the UBQ5 gene. Bars indicate average expression 6

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 RNAwas extracted at the indicated times (hours). Expression of PAD3 andUBQ5 was analyzed by semiquantitative RT-PCR.



Dow



elicitor treatments do not induce camalexin accumula-tion directly, but rather prime plants to synthesize morephytoalexin, or to do it more quickly, after pathogeninfection. It was previously shown that some chemi-cals can increase resistance against pathogen infectionby priming plant tissues to activate defense responsesmore efficiently after inoculation. For instance, pre-treatments of parsley (Petroselinum crispum) cells withbenzothiadiazole results in enhanced production ofcoumarin and augmented expression of genes encod-ing enzymes involved in phytoalexin biosynthesisafter inoculation (Katz et al., 1998). Arabidopsis plantspretreated with b-amino-butyric acid show increasedaccumulation of callose at the site of infection (Ton andMauch-Mani, 2004). In the latter case, camalexin isnot involved in the observed enhanced resistance,since b-amino-butyric acid-treated wild-type and pad3plants are similarly protected against necrotrophicpathogens, and, after inoculation, camalexin accumu-lates in primed plants to lower levels than in controlplants (Ton and Mauch-Mani, 2004). Experiments arein progress to determine whether elicitors like OGsand flg22 are able to prime Arabidopsis plants to pro-duce more camalexin in response to B. cinerea. An al-ternative hypothesis is that PAD3 is involved in thebiosynthesis of an unknown antimicrobial compound.These compounds could either be degradation prod-ucts of camalexin, or other indolic compounds whoseaccumulation is affected by the pad3 and ups1 muta-tions. To verify these hypotheses, a comprehensiveanalysis of secondary metabolites accumulating inwild-type and mutant plant treated with elicitorsand/or infected with B. cinerea is needed. Interestingly,16 different indolic compounds were previously iden-tified in Arabidopsis, several of them being induced by

pathogen infection (Bednarek et al., 2005). Their role indefense against different pathogens, their levels inresponse to elicitors, and the impact of the pad3 mu-tation on their accumulation are still largely unknown.

In conclusion, we have shown that OGs activate theexpression of Arabidopsis responses effective againstB. cinerea through a pathway that is independent of thewell-characterized defense-related signaling mole-cules SA, JA, and ET. Among these responses, the ex-pression of PAD3 and possibly other genes involved inthe biosynthesis of secondary metabolites plays amajor role in determining the enhanced resistanceagainst B. cinerea observed in OG-treated plants. The

Figure 7. Induction of resistance to B. cinerea by flg22. A, Lesion areain Arabidopsis Col-0 (WT), ein2npr1jar1 (nej ), and pad3 plants treatedwith a control solution (white bars) or flg22 (black bars) and inoculatedwith B. cinerea 24 h after treatment. B, Lesion area in Arabidopsis Col-0(WT), pad3, and ups1 plants treated with a control solution (white bars)or flg22 (black bars) and inoculated with B. cinerea 24 h aftertreatment. Lesion areas were measured 48 h after inoculation. Valuesare means 6 SE of at least 12 lesions. Asterisks indicate statisticallysignificant differences between control and flg22-treated plants, ac-cording to Student’s t test (***, P , 0.01). This experiment was repeatedtwice with similar results.

Figure 6. Induction of resistance to B. cinerea by OGs in wild-typeplants and in mutants impaired in camalexin production. Lesion area inArabidopsis Col-0 (WT), pad3, and ups1 plants treated with a controlsolution (white bars) or OGs (black bars) and inoculated with B. cinerea24 h after treatment. Lesion areas were measured 48 h after inoculation.Values are means 6 SE of at least 12 lesions. Asterisks indicatestatistically significant differences between control and OG-treatedplants, according to Student’s t test (***, P , 0.01). The experiment wasrepeated three times with similar results.

Ferrari et al.


Dow



outcome of the interaction between Arabidopsis andthis fungus is mediated mainly by the levels of sec-ondary metabolites in the host and the sensitivity ofthe pathogen to such compounds (Kliebenstein et al.,2005). Therefore, the activation of genes involved insecondary metabolism by cell wall fragments releasedat the site of infection likely represents an effectivemechanism to restrict fungal growth. The dissection ofthe OG-activated transduction pathway and the iden-tification of the effectors induced by this and otherelicitors will provide further insights in the molecularmechanisms regulating the plant innate immune re-sponse.

MATERIALS AND METHODS

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

made available in a timely manner for noncommercial 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 respon-

sibility of the requestor.

Plant Treatments

Arabidopsis (Arabidopsis thaliana) accession Columbia-0 (Col-0) was ob-

tained from G. Redei and A.R. Kranz (Arabidopsis Information Service,

Frankfurt); 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). 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) in a Percival AR66 growth chamber at

22�C, relative humidity of 70%, and a 12 h photoperiod with light provided by

Philips Hi-Vision white fluorescent lamps at an intensity of 120 mE m22 s21.

Plants were fertilized weekly with 0.53 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 mg mL21 OGs or 5 mM flg22 and 0.01%

Silwet L-77 (OSi Specialties) was uniformly sprayed on 4-week-old plants

until run off (approximately 1 mL for each plant). Plants were then covered

with transparent plastic sheet, placed back in the growth chamber, and the

plastic cover removed after 3 to 4 h.

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 and Skoog medium (Life Technologies;

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

vitamins. Plates were incubated at 22�C with a 16 h photoperiod and a light

intensity of 100 mE m22 s21. After 8 d, the medium was replaced with 1 mL of

fresh medium. At 10 d, 50 mg mL21 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 and Skoog medium (Life Technologies; Murashige and Skoog,

1962) supplemented with 0.5% Suc and Gamborg B5 vitamins. Plates were

incubated at 22�C with a 16 h photoperiod and a light intensity of 100 mE m22

s21. After 10 d, the medium was replaced with 1 mL of fresh medium with or

without 100 mg mL21 OGs. For each biological replicate, about 10 seedlings

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

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

effectiveness of the treatments was assessed by measuring hydrogen peroxide

released in the medium (data not shown). For treatment of coi1 seedlings,

heterozygous COI1/coi1 seeds were first germinated on agar plates containing

30 mM methyl jasmonate, and, after 8 d of growth, resistant seedlings were

transferred to liquid Murashige and Skoog medium and treated with OGs 2 d

later. As a control, wild-type seedlings were grown for 8 d on agar plates and

then transferred to liquid Murashige and Skoog medium.

Inoculation with Botrytis cinerea for the microarray experiments was

conducted on 4-week-old plants by placing four 5 mL droplets of a spore

suspension (5 3 105 conidia mL21) in 24 g L21 potato (Solanum tuberosum)

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 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

mg mL21 in sterile distilled water) or water were infiltrated in two lower

rosette leaves using a needleless syringe. After 72 h, 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); samples were split in two before homogenization and repooled

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 per-

formed according to the protocol given in the Affymetrix GeneChip Expres-

sion 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 the Nottingham Arabidopsis Stock Centre

(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 Micro-

Array 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 selected genes. Further data analysis was

performed with Rosetta Resolver v3.2 Gene Expression Data Analysis system

(Rosetta Inpharmatics), using Affymetrix.CEL files of array feature intensities

and SDs 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 correspond-

ing MM was calculated. Probe pairs that differed by more than 3 SDs 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 com-

bined. 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, nonlinear 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 expres-

sion 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



Dow



Research Arabidopsis Genome Annotation Database (http://www.tigr.org/

tdb/e2k1/ath1/ath1.shtml), the MIPS Functional Categories 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). 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, 23 SSC at 65�C for 45 min, and images were taken with a

Phosphorimager (Molecular Dynamics) after overnight exposure. The tem-

plate used to generate the PAD3 probes was amplified by 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 (Bio-Rad) according to the

manufacturer’s guide. Two microliters of cDNA (corresponding to 120 ng of

total RNA) were amplified in 30 mL reaction mix containing IQ SYBR Green

Supermix (Bio-Rad) and 0.4 mM of each primer. Primer sequences were the

following: 5#-CCGGTGAATCTTGAGAGAGCC-3# and 5#-GATCAGCTCG-

GTCATTCCCC-3# (PAD3); 5#-GGAAGAAGAAGACTTACACC-3# and 5#-AGT-

CCACACTTACCACAGTA-3# (UBQ5). Relative expression of the RT-PCR products

was determined using the DDCt method (Livak and Schmittgen, 2001).

NASCArrays Experiment Reference Numbers for microarray data are

NASCARRAYS-409 and NASCARRAYS-167.

Supplemental Data

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

Supplemental Figure S1. Lesion development in control and OG-treated

leaves.

Supplemental Table S1. B. cinerea infection microarray full dataset.

Supplemental Table S2. OG treatment microarray full dataset.

Supplemental Table S3. Genes coregulated by OGs and infection.

Supplemental Table S4. Comparison between OG- and chitin-regulated

genes.

Supplemental Table S5. Secondary metabolism gene expression and

references.

Supplemental Materials and Methods S1. Microarray hybridization

details.

ACKNOWLEDGMENTS

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

Genomics Research, Harvard University, Cambridge, MA) for assistance with

microarray hybridization and data analysis.

Received January 8, 2007; accepted March 20, 2007; published March 23, 2007.

LITERATURE CITED

AbuQamar S, Chen X, Dhawan R, Bluhm B, Salmeron J, Lam S, Dietrich

RA, Mengiste T (2006) Expression profiling and mutant analysis reveals

complex regulatory networks involved in Arabidopsis response to

Botrytis infection. Plant J 48: 28–44

Alonso JM, Stepanova AN, Solano R, Wisman E, Ferrari S, Ausubel FM,

Ecker JR (2003) Five components of the ethylene-response pathway

identified in a screen for weak ethylene-insensitive mutants in Arabi-

dopsis. Proc Natl Acad Sci USA 100: 2992–2997

An HJ, Lurie S, Greve LC, Rosenquist D, Kirmiz C, Labavitch JM,

Lebrilla CB (2005) Determination of pathogen-related enzyme action by

mass spectrometry analysis of pectin breakdown products of plant cell

walls. Anal Biochem 338: 71–82

Aziz A, Heyraud A, Lambert B (2004) Oligogalacturonide signal trans-

duction, induction of defense-related responses and protection of

grapevine against Botrytis cinerea. Planta 218: 767–774

Bartel B, Fink GR (1995) ILR1, an amidohydrolase that releases active

indole-3-acetic acid from conjugates. Science 268: 1745–1748

Bednarek P, Schneider B, Svatos A, Oldham NJ, Hahlbrock K (2005)

Structural complexity, differential response to infection, and tissue

specificity of indolic and phenylpropanoid secondary metabolism in

Arabidopsis roots. Plant Physiol 138: 1058–1070

Boudart G, Charpentier M, Lafitte C, Martinez Y, Jauneau A, Gaulin E,

Esquerre-Tugaye MT, Dumas B (2003) Elicitor activity of a fungal

endopolygalacturonase in tobacco requires a functional catalytic site

and cell wall localization. Plant Physiol 131: 93–101

Broekaert WF, Pneumas WJ (1988) Pectic polysaccharides elicit chitinase

accumulation in tobacco. Physiol Plant 74: 740–744

Cao H, Bowling SA, Gordon AS, Dong X (1994) Characterization of an

Arabidopsis mutant that is nonresponsive to inducers of systemic

acquired resistance. Plant Cell 6: 1583–1592

Cao H, Glazebrook J, Clarke JD, Volko S, Dong X (1997) The Arabidopsis

NPR1 gene that controls systemic acquired resistance encodes a novel

protein containing ankyrin repeats. Cell 88: 57–63

Clarke JD, Volko SM, Ledford H, Ausubel FM, Dong X (2000) Roles of

salicylic acid, jasmonic acid, and ethylene in cpr-induced resistance in

Arabidopsis. Plant Cell 12: 2175–2190

Davis KR, Darvill AG, Albersheim P, Dell A (1986) Host-pathogen

interactions. XXIX. Oligogalacturonides released from sodium poly-

pectate by endopolygalacturonic acid lyase are elicitors of phytoalexins

in soybean. Plant Physiol 80: 568–577

Davis KR, Hahlbrock K (1987) Induction of defense responses in cultured

parsley cells by plant cell wall fragments. Plant Physiol 85: 1286–1290

De Lorenzo G, Castoria R, Bellincampi D, Cervone F (1997) Fungal

invasion enzymes and their inhibition. In G Carroll and P Tudzynski,

eds, The Mycota. Springer-Verlag, Berlin, pp 61–83

De Lorenzo G, Ranucci A, Bellincampi D, Salvi G, Cervone F (1987)

Elicitation of phenylalanine ammonia-lyase in Daucus carota by oligo-

galacturonides released from sodium polypectate by homogenous poly-

galacturonase. Plant Sci 51: 147–150

Delaney TP, Uknes S, Vernooij B, Friederich L, Weymann K, Negrotto

D, Gaffney T, Gut-Rella M, Kessmann H, Ward E, et al (1994) A

central role of salicylic acid in plant disease resistance. Science 266:

1247–1250

Denby KJ, Jason LJ, Murray SL, Last RL (2005) ups1, an Arabidopsis

thaliana camalexin accumulation mutant defective in multiple defence

signalling pathways. Plant J 41: 673–684

Ferrari S, Galletti R, Vairo D, Cervone F, De Lorenzo G (2006) Anti-

sense expression of the Arabidopsis thaliana AtPGIP1 gene reduces

polygalacturonase-inhibiting protein accumulation and enhances sus-

ceptibility to Botrytis cinerea. Mol Plant Microbe Interact 19: 931–936

Ferrari S, Plotnikova JM, De Lorenzo G, Ausubel FM (2003a) Arabidopsis

local resistance to Botrytis cinerea involves salicylic acid and camalexin

and requires EDS4 and PAD2, but not SID2, EDS5 or PAD4. Plant J 35:

193–205

Ferrari S, Vairo D, Ausubel FM, Cervone F, De Lorenzo G (2003b)

Tandemly duplicated Arabidopsis genes that encode polygalacturonase-

inhibiting proteins are regulated coordinately by different signal transduc-

tion pathways in response to fungal infection. Plant Cell 15: 93–106

Feys BF, Benedetti CE, Penfold CN, Turner JG (1994) Arabidopsis mutants

selected for resistance to the phytotoxin coronatine are male sterile,

insensitive to methyl jasmonate, and resistant to a bacterial pathogen.

Plant Cell 6: 751–759

Feys BJ, Parker JE (2000) Interplay of signaling pathways in plant disease

resistance. Trends Genet 16: 449–455

Flor HH (1971) Current status of the gene-for-gene concept. Annu Rev

Phytopathol 9: 275–296

Gachon CM, Langlois-Meurinne M, Henry Y, Saindrenan P (2005) Tran-

scriptional co-regulation of secondary metabolism enzymes in Arabi-

dopsis: functional and evolutionary implications. Plant Mol Biol 58:

229–245

Gaffney T, Friederich L, Vernooij B, Negrotto D, Nye G, Uknes S, Ward E,

Kessmann H, Ryals J (1993) Requirement of salycilic acid for the

induction of systemic acquired resistance. Science 261: 754–756

Ferrari et al.


Dow



Glawischnig E, Hansen BG, Olsen CE, Halkier BA (2004) Camalexin is

synthesized from indole-3-acetaldoxime, a key branching point between

primary and secondary metabolism in Arabidopsis. Proc Natl Acad Sci

USA 101: 8245–8250

Guzman P, Ecker JR (1990) Exploiting the triple response of Arabidopsis to

identify ethylene-related mutants. Plant Cell 2: 513–523

Hahn MG (1981) Fragments of plant and fungal cell wall polysaccharides

elicit the accumulation of phytoalexins in plants. PhD thesis. University

of Colorado, Boulder, CO

Hahn MG, Darvill AG, Albersheim P (1981) Host-pathogen interactions.

XIX. The endogenous elicitor, a fragment of plant cell wall polysaccha-

ride that elicits phytoalexin accumulation in soy beans. Plant Physiol 68:

1161–1169

Hammond-Kosack KE, Jones JDG (1997) Plant disease resistance genes.

Annu Rev Plant Physiol Plant Mol Biol 48: 575–607

Hansen CH, Wittstock U, Olsen CE, Hick AJ, Pickett JA, Halkier BA

(2001) Cytochrome p450 CYP79F1 from arabidopsis catalyzes the con-

version of dihomomethionine and trihomomethionine to the corre-

sponding aldoximes in the biosynthesis of aliphatic glucosinolates. J

Biol Chem 276: 11078–11085

Hemm MR, Ruegger MO, Chapple C (2003) The Arabidopsis ref2 mutant

is defective in the gene encoding CYP83A1 and shows both phenyl-

propanoid and glucosinolate phenotypes. Plant Cell 15: 179–194

Hull AK, Vij R, Celenza JL (2000) Arabidopsis cytochrome P450s that

catalyze the first step of tryptophan-dependent indole-3-acetic acid

biosynthesis. Proc Natl Acad Sci USA 97: 2379–2384

Kars I, van Kan JA (2004) Extracellular enzymes and metabolites involved

in pathogenesis of Botrytis. In Y Elad, B Williamson, P Tudzynski, N

Delen, eds, Botrytis: Biology, Pathology and Control. Kluwer Academic

Publishers, Dordrecht, The Netherlands, pp 99–118

Katz VA, Thulke OU, Conrath U (1998) A benzothiadiazole primes parsley

cells for augmented elicitation of defense responses. Plant Physiol 117:

1333–1339

Kliebenstein DJ, Rowe HC, Denby KJ (2005) Secondary metabolites

influence Arabidopsis/Botrytis interactions: variation in host produc-

tion and pathogen sensitivity. Plant J 44: 25–36

Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data

using real-time quantitative PCR and the 2(-DDC(T)). Methods 25:

402–408

Murashige T, Skoog F (1962) Revised medium for rapid growth and

bioassays with tobacco cultures. Physiol Plant 15: 437–479

Navarro L, Zipfel C, Rowland O, Keller I, Robatzek S, Boller T, Jones JD

(2004) The transcriptional innate immune response to flg22: interplay

and overlap with Avr gene-dependent defense responses and bacterial

pathogenesis. Plant Physiol 135: 1113–1128

Nurnberger T, Brunner F (2002) Innate immunity in plants and animals:

emerging parallels between the recognition of general elicitors and

pathogen-associated molecular patterns. Curr Opin Plant Biol 5:

318–324

Poinssot B, Vandelle E, Bentejac M, Adrian M, Levis C, Brygoo Y, Garin J,

Sicilia F, Coutos-Thevenot P, Pugin A (2003) The endopolygalact-

uronase 1 from Botrytis cinerea activates grapevine defense reactions

unrelated to its enzymatic activity. Mol Plant Microbe Interact 16:

553–564

Raacke IC, von Rad U, Mueller MJ, Berger S (2006a) Yeast increases

resistance in Arabidopsis against Pseudomonas syringae and Botrytis

cinerea by salicylic acid-dependent as well as -independent mecha-

nisms. Mol Plant Microbe Interact 19: 1138–1146

Raacke IC, von Rad U, Mueller MJ, Berger S (2006b) Yeast increases

resistance in Arabidopsis against Pseudomonas syringae and Botrytis

cinerea by salicylic acid-dependent as well as -independent mecha-

nisms. Mol Plant Microbe Interact 19: 1138–1146

Ramonell K, Berrocal-Lobo M, Koh S, Wan J, Edwards H, Stacey G,

Somerville S (2005) Loss-of-function mutations in chitin responsive

genes show increased susceptibility to the powdery mildew pathogen

Erysiphe cichoracearum. Plant Physiol 138: 1027–1036

Rogers EE, Ausubel FM (1997) Arabidopsis enhanced disease susceptibil-

ity mutants exhibit enhanced susceptibility to several bacterial patho-

gens and alterations in PR-1 gene expression. Genetics 146: 381–392

Ruepp A, Zollner A, Maier D, Albermann K, Hani J, Mokrejs M, Tetko I,

Guldener U, Mannhaupt G, Munsterkotter M, et al (2004) The FunCat,

a functional annotation scheme for systematic classification of proteins

from whole genomes. Nucleic Acids Res 32: 5539–5545

Schuhegger R, Nafisi M, Mansourova M, Petersen BL, Olsen CE, Svatos

A, Halkier BA, Glawischnig E (2006) CYP71B15 (PAD3) catalyzes the

final step in camalexin biosynthesis. Plant Physiol 141: 1248–1254

Staswick PE, Su W, Howell SH (1992) Methyl jasmonate inhibition of root

growth and induction of a leaf protein are decreased in an Arabidopsis

thaliana mutant. Proc Natl Acad Sci USA 89: 6837–6840

ten Have A, Mulder W, Visser J, van Kan JA (1998) The endopolygalact-

uronase gene Bcpg1 is required for full virulence of Botrytis cinerea. Mol

Plant Microbe Interact 11: 1009–1016

Thimm O, Blasing O, Gibon Y, Nagel A, Meyer S, Kruger P, Selbig J,

Muller LA, Rhee SY, Stitt M (2004) MAPMAN: a user-driven tool to

display genomics data sets onto diagrams of metabolic pathways and

other biological processes. Plant J 37: 914–939

Thomma BP, Eggermont K, Penninckx I, Mauch-Mani B, Vogelsang R,

Cammue BPA, Broekaert WF (1998) Separate jasmonate-dependent and

salicylate-dependent defense-response pathways in arabidopsis are

essential for resistance to distinct microbial pathogens. Proc Natl

Acad Sci USA 95: 15107–15111

Thomma BP, Eggermont K, Tierens KF, Broekaert WF (1999) Requirement

of functional ethylene-insensitive 2 gene for efficient resistance of

Arabidopsis to infection by Botrytis cinerea. Plant Physiol 121: 1093–1102

Ton J, Mauch-Mani B (2004) Beta-amino-butyric acid-induced resistance

against necrotrophic pathogens is based on ABA-dependent priming for

callose. Plant J 38: 119–130

Waring JF, Jolly RA, Ciurlionis R, Lum PY, Praestgaard JT, Morfitt DC,

Buratto B, Roberts C, Schadt E, Ulrich RG (2001) Clustering of hepa-

totoxins based on mechanism of toxicity using gene expression profiles.

Toxicol Appl Pharmacol 175: 28–42

Zhang B, Ramonell K, Somerville S, Stacey G (2002) Characterization of

early, chitin-induced gene expression in Arabidopsis. Mol Plant Microbe

Interact 15: 963–970

Zhao J, Last RL (1996) Coordinate regulation of the tryptophan biosyn-

thetic pathway and indolic phytoalexin accumulation in Arabidopsis.

Plant Cell 8: 2235–2244

Zhou N, Tootle TL, Glazebrook J (1999) Arabidopsis PAD3, a gene

required for camalexin biosynthesis, encodes a putative cytochrome

P450 monooxygenase. Plant Cell 11: 2419–2428

Zipfel C, Robatzek S, Navarro L, Oakeley EJ, Jones JD, Felix G, Boller T

(2004) Bacterial disease resistance in Arabidopsis through flagellin

perception. Nature 428: 764–767

Zook M (1998) Biosynthesis of camalexin from tryptophan pathway inter-

mediates in cell-suspension cultures of Arabidopsis. Plant Physiol 118:

1389–1393



Dow



Documents

Resistance to Botrytis cinerea Induced in - Plant Physiology