Tryptophan-derived secondary metabolites in Arabidopsisthaliana confer non-host resistance to necrotrophicPlectosphaerella cucumerina fungi

Andrea Sanchez-Vallet1,†, Brisa Ramos1,†, Paweł Bednarek2, Gemma Lopez1, Mariola Pislewska-Bednarek2,

Paul Schulze-Lefert2 and Antonio Molina1,*

1Centro de Biotecnologıa y Genomica de Plantas (UPM-INIA), Universidad Politecnica de Madrid, Campus Montegancedo,

E-28223-Pozuelo de Alarcon, Madrid, Spain, and2Department of Plant Microbe Interactions, Max Planck Institut fur Zuchtungsforschung, Carl-von-Linne-Weg 10, D-50829 Koln,

Germany

Received 1 March 2010; revised 23 March 2010; accepted 31 March 2010; published online 14 May 2010.*For correspondence (fax +34 917157721; e-mail antonio.molina@upm.es).†These authors contributed equally to this work.

SUMMARY

A defence pathway contributing to non-host resistance to biotrophic fungi in Arabidopsis involves the

synthesis and targeted delivery of the tryptophan (trp)-derived metabolites indol glucosinolates (IGs) and

camalexin at pathogen contact sites. We have examined whether these metabolites are also rate-limiting for

colonization by necrotrophic fungi. Inoculation of Arabidopsis with adapted or non-adapted isolates of the

ascomycete Plectosphaerella cucumerina triggers the accumulation of trp-derived metabolites. We found that

their depletion in cyp79B2 cyp79B3 mutants renders Arabidopsis fully susceptible to each of three tested non-

adapted P. cucumerina isolates, and super-susceptible to an adapted P. cucumerina isolate. This assigns a key

role to trp-derived secondary metabolites in limiting the growth of both non-adapted and adapted

necrotrophic fungi. However, 4-methoxy-indol-3-ylmethylglucosinolate, which is generated by the P450

monooxygenase CYP81F2, and hydrolyzed by PEN2 myrosinase, together with the antimicrobial camalexin

play a minor role in restricting the growth of the non-adapted necrotrophs. This contrasts with a major role of

these two trp-derived phytochemicals in limiting invasive growth of non-adapted biotrophic powdery mildew

fungi, thereby implying the existence of other unknown trp-derived metabolites in resistance responses to

non-adapted necrotrophic P. cucumerina. Impaired defence to non-adapted P. cucumerina, but not to the non-

adapted biotrophic fungus Erysiphe pisi, on cyp79B2 cyp79B3 plants is largely restored in the irx1 background,

which shows a constitutive accumulation of antimicrobial peptides. Our findings imply differential contribu-

tions of antimicrobials in non-host resistance to necrotrophic and biotrophic pathogens.

Keywords: indolglucosinolate, plant innate immunity, necrotrophic fungi, antifungal compounds,

non-adapted fungi.

INTRODUCTION

Plants are constantly exposed to a wide variety of potentially

pathogenic microorganisms, but only a small proportion, the

host-adapted pathogens, are able to successfully colonize a

plant species and cause disease. Host range changes are rare

in nature, and for this reason the mechanisms underlying

‘durable resistance’ in non-host plants are of growing inter-

est in plant–microbe interaction studies. Non-host plants

were shown to respond to inoculation with non-adapted

pathogens with inducible responses that appear to be shared

with host defence responses in interactions with adapted

pathogens (Heath, 2000; Thordal-Christensen, 2003).

Non-host resistance in Arabidopsis confers immunity to

non-adapted biotrophic fungi, such as powdery mildews

that colonize barley or pea in nature (Collins et al., 2003;

Lipka et al., 2005; Bednarek et al., 2009). Several Arabidopsis

pen mutants (pen1, pen2 and pen3) show an increased

penetration of these non-adapted biotrophic fungi in leaf

epidermal cells, a process that is critical for nutrient uptake

ª 2010 The Authors 115Journal compilation ª 2010 Blackwell Publishing Ltd

The Plant Journal (2010) 63, 115–127 doi: 10.1111/j.1365-313X.2010.04224.x

by these pathogens (Collins et al., 2003). The PEN1 syntaxin

is involved in a vesicle-mediated secretory defence pathway

that seems to be specific for biotrophic fungi (Collins et al.,

2003; Kwon et al., 2008). PEN2 is a myrosinase that accu-

mulates underneath powdery mildew contact sites, where

the enzyme initiates the metabolism of a group of trypto-

phan (trp)-derived compounds, known as indole glucosino-

lates (IGs), to release potential antimicrobial products (Lipka

et al., 2005; Bednarek et al., 2009). Specifically, 4-methoxy-

indol-3-ylmethyl glucosinolate (4MI3G), synthesized by

CYP81F2, is the biologically relevant substrate of PEN2

activity against non-adapted powdery mildews (Bednarek

et al., 2009; Pfalz et al., 2009). Formation of the PEN2-depen-

dent bioactive end product(s) relies on high glutathione

levels, as PAD2, encoding a c-glutamylcysteine synthetase

(Parisy et al., 2007), is indispensable for the formation of

indol-3-ylmethylamine (I3A) and raphanusamic acid (RA), as

well as extracellular growth termination of the non-adapted

powdery mildews (Bednarek et al., 2009). Interestingly,

under particular stress conditions the pad2-1 mutant, which

contains approximately 20% residual glutathione, accumu-

lates reduced levels of the trp-derived phytoalexin camalexin

(3-thiazol-2¢-yl-indole), IGs and aliphatic glucosinolates (Gs),

and is super-susceptible to infection by adapted fungi,

oomycete and bacteria (Glazebrook and Ausubel, 1994;

Roetschi et al., 2001; Schlaeppi et al., 2008; Bednarek et al.,

2009). PEN3 encodes a pleiotropic drug resistance (PDR)

ATP-binding cassette (ABC) transporter implicated in the

secretion of antimicrobial products, including those result-

ing from PEN2 activity (Stein et al., 2006). The PEN2/PEN3-

dependent extracellular defence is of particular interest

because this pathway restricts the growth of a broader

spectrum of microbial pathogens, including biotrophic and

necrotrophic fungi, and oomycetes (Lipka et al., 2005; Stein

et al., 2006; Bednarek et al., 2009; Maeda et al., 2009).

In addition to the PEN2/PEN3-dependent defence path-

way, the phytoalexin camalexin that is essential for Arabid-

opsis resistance to several adapted necrotrophic fungi

(Thomma et al., 1999a; van Wees et al., 2003; Bohman et al.,

2004; Denby et al., 2004; Kliebenstein et al., 2005; Glawisch-

nig, 2007; Van Baarlen et al., 2007), was found to be active in

the post-invasive defence barrier in non-host resistance to

powdery mildews (Bednarek et al., 2009). The pad3 mutant,

impaired in the last step of camalexin biosynthesis (Schu-

hegger et al., 2006), had the same infection phenotype as

wild-type plants, but the double mutant pen2 pad3 was not

only impaired in penetration resistance, but also supported

extensive hyphal growth of the non-adapted fungus Erysi-

phe pisi (Bednarek et al., 2009). The pen2 pad3 infection

phenotype was comparable with that of the double mutant

cyp79B2 cyp79B3, which is defective in the P450 monoox-

ygenase-catalyzed conversion of tryptophan to indole-3-

acetaldoxime (IAOX), a precursor of most known trp-derived

products, including IGs, such as 4MI3G and camalexin (Zhao

et al., 2002; Bednarek et al., 2009). Together, these data

indicate that an important component of Arabidopsis non-

host resistance to biotrophic powdery mildew fungi is the

accumulation and sequential action of PEN2-derived IGs

hydrolysis products and camalexin (Bednarek et al., 2009).

The majority of necrotrophic fungi, such as Botrytis

cinerea or Sclerotinia sclerotiorum, have a broad host range,

although for several well-studied necrothrophs, like Coch-

liobolus and Alternaria spp., a narrower host range has been

described (van Kan, 2006). Some ascomycete fungi, such as

the necrotrophs Plectosphaerella cucumerina or B. cinerea,

can proliferate and develop on dead and decaying tissues,

causing severe disease in a wide range of plants, including

Arabidopsis (Cramer and Lawrence, 2004; Bolton et al.,

2006; van Kan, 2006; Williamson et al., 2007).

Our current knowledge of the molecular basis of non-host

resistance to non-adapted necrotrophic pathogens is scarce.

By contrast, growth restriction of adapted necrotrophs in

Arabidopsis was found to be multigenic (Denby et al., 2004;

Llorente et al., 2005), and to depend on the precise regula-

tion of the ethylene (ET), jasmonate (JA), salicylic acid (SA)

or auxin signalling pathways, as well as the b subunit of

heterotrimeric G-protein (AGB1; Thomma et al., 1998,

1999b; Berrocal-Lobo et al., 2002; Ferrari et al., 2003;

Llorente et al., 2005; Hernandez-Blanco et al., 2007; Llorente

et al., 2008). Plant cell wall structure and composition are

also determinants for the success of necrotrophic fungal

colonization (Somerville et al., 2004). The irregular xylem

(irx) mutants, impaired in the cellulose synthase (CESA)

subunits specifically required for secondary cell wall

formation, showed enhanced resistance to both adapted

necrotrophic (e.g. P. cucumerina) and biotrophic (e.g.

Golovinomyces cichoracearum) fungi (Hernandez-Blanco

et al., 2007). The irx1-mediated broad spectrum resistance

may rely on the constitutive accumulation of antibiotic

compounds, as revealed by comparative transcriptome

analyses (Hernandez-Blanco et al., 2007).

In this study, we first identified three non-adapted P. cu-

cumerina isolates that were unable to colonize Arabidopsis

Col-0 wild-type plants. Using this pathosystem, we found

that in addition to the biosynthesis of camalexin and

CYP81F2/PEN2-derived metabolites, the engagement of

additional uncharacterized trp-derived compounds is

required for effective non-host resistance to necrotrophs.

The results presented here also suggest a differential

contribution of distinct sets of antimicrobials in non-host

resistance to necrotrophic and biotrophic pathogens.

RESULTS

Plectosphaerella cucumerina non-adapted isolates fail

to colonize Arabidopsis plants

We selected three P. cucumerina isolates (Pc1187, Pc2127

and Pc2125) that colonise Nicotiana tabacinum, Viola spp.

116 Andrea Sanchez-Vallet et al.

ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 63, 115–127

and the wheat bulb fly (Delia coarctata), respectively, in

nature. Their virulence on Arabidopsis plants (Col-0) was

compared with that of P. cucumerina BMM (PcBMM), a vir-

ulent isolate on Arabidopsis (Ton and Mauch-Mani, 2004).

From these isolates we obtained the DNA sequences of the

complete internal transcribed spacer 1 and 2 (ITS1 and

ITS2), and the ribosomal 5.8S RNA located between these

ITSs. A phylogenetic tree of these sequences and those from

other plant pathogenic fungi showed that the four isolates

formed a single group, with 100% bootstrap support, and

that they have a close relationship with other fungal patho-

gens, such as B. cinerea and Verticillium dahliae (Figure S1;

Pitt et al., 2004).

Three-week-old Arabidopsis Col-0 leaves were sprayed

either with water or a spore suspension (4 · 106 spores

ml)1) of the virulent PcBMM or isolates Pc1187, Pc2127 and

Pc2125. The infection progression was examined at differ-

ent hours/days post-inoculation (hpi/dpi) by trypan blue

staining (TB) of the inoculated leaves, determination of

fungal biomass by quantitative real-time PCR (qRT-PCR) of

the P. cucumerina b-tubulin gene and by macroscopic

evaluation of the disease rating (DR) of the inoculated

plants. TB staining at 12 hpi revealed similar spore germi-

nation rates on the leaf surface, but differences at 20 hpi,

with PcBMM having longer hyphae than Pc1187, Pc2127

and Pc2125 (Figure 1a, and data not shown). The sparse

hyphal growth of the putative non-adapted isolates was not

associated with a host cell death response at fungal contact

sites with plant cells (not shown). Fungal biomass at 3 and

5 dpi was lower in plants inoculated with Pc1187, Pc2127

and Pc2125, and increased over time only in the plants

inoculated with PcBMM (Figure 1b, and data not shown).

There was a positive correlation between fungal biomass

and macroscopic disease symptoms (Figure 1c,d). As

Pc1187, Pc2127 and Pc2125 also failed to colonize an

additional Arabidopsis accession (La-0) and the er-1 mutant,

that is impaired in the ERECTA (ER) receptor-like kinase

required for resistance to PcBMM (Llorente et al., 2005),

we considered these fungi as non-adapted pathogens of

Arabidopsis.

Tryptophan-derived metabolites contribute to Arabidopsis

resistance to non-adapted P. cucumerina isolates

To determine whether trp-derived metabolites exert a rate-

limiting role during pathogenesis of necrotrophic P. cu-

cumerina, we compared the ability of Pc1187, Pc2127 and

PcBMM to colonize Arabidopsis Col-0 wild-type plants and

mutant lines blocked either in the accumulation (e.g.

cyp79B2 cyp79B3, pad3, pen2, cyp81F2 and pen2 pad3) or

extracellular release (e.g. pen3) of these phytochemicals.

Fungal growth of PcBMM, Pc1187 and Pc2127 was dramat-

ically enhanced on the cyp79B2 cyp79B3 mutant line (Fig-

ure 2a,b), which fails to accumulate most known trp-derived

metabolites, including IGs and camalexin (Zhao et al., 2002;

Bednarek et al., 2009; Pfalz et al., 2009). In this mutant, the

disease symptoms caused by the three isolates increased

over time, leading to leaf tissue collapse (Figure 2c, and data

not shown). This provides genetic evidence that Arabidopsis

determines the outcome of attempted colonization by the

non-adapted P. cucumerina isolates.

(a)

(d)

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Figure 1. The Plectosphaerella cucumerina isolates Pc1187 and Pc2127 are

non-adapted necrotrophic fungi on Arabidopsis wild-type plants.

Wild-type plants (Col-0) were spray inoculated with a fungal suspension

(4 · 106 spores ml)1) of the virulent PcBMM isolate or the non-adapted

isolates Pc1187 and Pc2127. (a) Lactophenol Trypan Blue staining of inocu-

lated leaves at 12 and 20 h post-inoculation (hpi). Scale bar: 20 lm.

(b) Relative quantification of fungal DNA (P. cucumerina b-tubulin) on Col-0

plants at 3 and 5 days post inoculation (pdi). Values are represented as n-fold

fungal DNA levels relative to wild-type plants at 3 dpi with PcBMM. Data

represent the averages (�SDs) of two replicates.

(c) Average disease rating (DR � SD) at 5 and 7 dpi of plants inoculated with

different P. cucumerina isolates. DR values vary between 0 (no symptoms)

and 5 (dead plants). The letters indicate significantly different statistical

groups (ANOVA, P £ 0.05, Bonferroni’s test). Experiments in (b) and (c) are one

of three performed that gave similar results.

(d) Mock and inoculated Col-0 plants with different P. cucumerina isolates at

10 dpi.

Arabidopsis non-host resistance to necrotrophic fungi 117

ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 63, 115–127

To evaluate which of these metabolites was responsible

for limiting P. cucumerina growth, we analyzed infection

phenotypes on the camalexin-deficient pad3 mutant (Glaze-

brook and Ausubel, 1994). Wild-type and pad3 plants

displayed similar macroscopic disease symptoms, and

indistinguishable fungal growth, upon inoculation with

either adapted or non-adapted P. cucumerina (Figure 2a–c).

Plants lacking CYP81F2 and PEN2, which are blocked,

respectively, in the biosynthesis or hydrolysis of 4MI3G

(Bednarek et al., 2009; Pfalz et al., 2009), were found to be

super-susceptible to the adapted PcBMM isolate (Figure

2a–c; Lipka et al., 2005). Unexpectedly, in interactions with

the non-adapted isolates the infection phenotype of these

mutants was indistinguishable from that of wild-type plants

(Figure 2a–c), suggesting that IG-derived metabolites and

camalexin are dispensable for fungal growth termination. To

test the possibility that these compounds act in a function-

ally redundant manner, we examined pen2 pad3 double

mutant plants. These plants supported significantly

enhanced hyphal growth of the non-adapted and adapted

P. cucumerina isolates, and showed more severe disease

symptoms than Col-0 plants. However, fungal growth was

still clearly lower compared with cyp79B2 cyp79B3 plants

(Figure 2a–c).

Next, we tested the P. cucumerina isolates on pad2 plants,

in which c-glutamylcysteine synthetase activity is impaired

(c)(a)

(b)

0

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Figure 2. Tryptophan-derived metabolites are essential for Arabidopsis resistance to non-adapted Plectosphaerella cucumerina isolates.

Plants of the shown genotypes were spray inoculated with spores of the indicated P. cucumerina isolates. (a) Quantitative real-time PCR (qRT-PCR) quantification of

fungal DNA (Pc b-tubulin) at 5 days post-inoculation (dpi). Values (�SDs) are represented as the average of the n-fold fungal DNA levels, relative to wild-type (Col-0)

plants.

(b) Average disease rating (DR � SD) of the indicated genotypes at 5 dpi. DR varies between 0 (no symptoms) and 5 (dead plant). The asterisks indicate values

statistically different from those of Col-0 plants for each isolate (ANOVA P £ 0.05, Bonferroni’s test). The experiments were performed three times with similar results.

(c) Lactophenol Trypan Blue staining of inoculated leaves at 24 hpi. Scale bar: 20 lm.

118 Andrea Sanchez-Vallet et al.

ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 63, 115–127

and lower levels of trp derivatives accumulate upon infec-

tion with a diverse range of pathogens (Glazebrook and

Ausubel, 1994; Parisy et al., 2007; Schlaeppi et al., 2008;

Bednarek et al., 2009). Notably, pad2 plants supported

enhanced fungal growth of the adapted and non-adapted

isolates (Figure 2a–c). However, fungal growth limitation of

the latter isolates was essentially unaffected in pen3 plants

(Figure 2; Stein et al., 2006). This contrasts with a major

contribution of PEN3 in limiting hyphal growth of the

adapted PcBMM isolate (Figure 2a,b). In summary, these

results suggest that the accumulation and delivery of

IG-derived compounds and camalexin are required for full

resistance to non-adapted P. cucumerina. However, this

must involve other yet unknown trp-derived metabolites

than those acting against biotrophic non-adapted powdery

mildews (Bednarek et al., 2009).

Plectosphaerella cucumerina non-adapted isolates induce

the accumulation of trp-derived metabolites

The transcriptional modulation of Arabidopsis genes

encoding key enzymes of secondary metabolite biosynthetic

pathways was determined by qRT-PCR upon plant inocula-

tion with the adapted (PcBMM), or non-adapted (Pc1187 or

Pc2127), necrotrophic fungi. The genes analysed encoded

CYP81F2, CYP79B2, CYP79B3, PAD3 and PAD2 proteins, and

also ASA1, involved in the biosynthesis of tryptophan

(Niyogi and Fink, 1992). Expression levels of these genes,

except PAD2, in Col-0 plants significantly increased at 1 and

3 days after inoculation with both adapted and non-adapted

isolates, compared with their expression in mock-treated

plants (Figure 3). With a few exceptions, the transcriptional

activation was similar upon inoculation with either adapted

or non-adapted isolates (Figure 3). Finally, the expression

of PAD2 varied slightly only 3 days after challenge with

PcBMM (Figure 3).

We examined whether the observed transcriptional

upregulation correlates with enhanced trp-derived metab-

olite levels during fungal colonization. We performed a

comparative metabolite profiling experiment of leaf

extracts of wild-type plants (Col-0), and the pen2-3, pen3-

1, pad2-1 and cyp79B2 cyp79B3 mutant lines. At 1 dpi a

slight but significant increase in the accumulation of

4MI3G and camalexin occurs in Col-0 plants inoculated

with the adapted and non-adapted isolates (Figure 4). In

contrast, a significant depletion of indol-3-ylmethyl-

glucosinolate (I3G), the precursor of 4MI3G, was found in

the inoculated plants, whereas a significant reduction of

1-methoxy-indol-3-ylmethylglucosinolate (1MI3G) levels

was observed only upon challenge with Pc2127 (Figure 4).

In line with the enhanced accumulation of 4MI3G, higher

levels of I3A and RA, the products of PEN2 myrosinase

activity on IGs, were also detected in Col-0 plants inocu-

lated with the P. cucumerina isolates (Figure 4). The levels

of another indolic compound, 6-OH-indole-3-carboxylic

acid glucopyranoside (6-OGlc-I3CA; Hagemeier et al.,

2001; Bednarek et al., 2009; Bottcher et al., 2009) increased

in the inoculated Col-0 plants (Figure 4). As expected, I3G,

4MI3G, I3A, 6-OGlc-I3CA and camalexin were undetectable

in the inoculated cyp79B2 cyp79B3 plants, whereas the

accumulation of RA was lower compared with Col-0 plants

(Figure 4). These data further demonstrate the trp origin of

P. cucumerina-triggered accumulation of IGs, camalexin

and I3A (Bednarek et al., 2009). In addition, this points

to CYP79B2/CYP79B3-generated IAOx as the precursor of

6-OGlc-I3CA.

PEN2 has been shown to function in planta as pathogen-

inducible myrosinase that initiates IG metabolism, leading

to I3A and RA (Bednarek et al., 2009). Consistent with this,

4MI3G levels were higher and the concentration of I3A was

0

50

100

150

200

250

300

350

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Figure 3. Plectosphaerella cucumerina-stimulated expression of genes

encoding proteins involved in the biosynthesis of trp-derived metabolites.

Wild-type plants (Col-0) were spray inoculated with the indicated P. cucume-

rina isolates, as described in Figure 1. Expression of the indicated genes was

quantified by quantitative real-time PCR (qRT-PCR) at 1 and 3 days post-

inoculation (dpi). Values were normalized to Arabidopsis UBIQUITIN21

expression levels, and are represented as n-fold compared with the mock-

treated plants. Bars represent the average (�SD) of two technical replicates.

Asterisks indicate statistically significant differences with mock-treated

plants, and triangles indicate statistically significant differences with

PcBMM-inoculated plants (ANOVA P £ 0.05, Bonferroni’s test). Data are from

one out of three independent experiments, which gave similar results.

Arabidopsis non-host resistance to necrotrophic fungi 119

ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 63, 115–127

lower in pen2-3 plants than in wild-type Col-0 upon

pathogen challenge (Figure 4). RA levels were also lower

in mock-treated and in PcBMM- and Pc2127-inoculated

plants (Figure 4). Therefore, PEN2 is suggested to hydro-

lyse 4MI3G preferentially in P. cucumerina-challenged tis-

sue, similar to data reported upon inoculation with the

biotrophic E. pisi fungus (Bednarek et al., 2009). Unexpect-

edly, the concentrations of camalexin and 6-OGlc-I3CA

were higher in pen2-3 than in Col-0 plants challenged with

either adapted or non-adapted P. cucumerina isolates (Fig-

ure 4). In line with the proposed function of PEN3 as

transporter of trp-derived metabolites, the concentration of

RA, 4MI3G, 6-OGlc-I3CA and camalexin was higher in

pen3-1 than in Col-0 plants (Figure 4). In contrast to

previous studies with other microbial pathogens and

insects (Glazebrook and Ausubel, 1994; Schlaeppi et al.,

2008), pad2 plants displayed higher concentrations of

4MI3G and similar levels of camalexin than wild-type plants

(Figure 4). However, the concentration of I3A and RA in the

inoculated mutant was lower compared with wild-type

plants, further supporting the role of glutathione in the

biosynthesis of these PEN2-dependent and IG-derived

products (Bednarek et al., 2009).

The IG-derived metabolites and camalexin have

antimicrobial activity in vitro against P. cucumerina

The IG-derived metabolites have been suggested to act as

signalling molecules downstream from the perception of a

microbe-associated-molecular pattern (MAMP), as well as

broad-spectrum antimicrobials against fungal pathogens in

Arabidopsis (Bednarek et al., 2009; Clay et al., 2009). Cam-

alexin and diverse products of Gs degradation were previ-

ously shown to be toxic to a wide range of pathogens

(Tierens et al., 2001; Sellam et al., 2007). We analysed the

in vitro antifungal activity of camalexin and the IG-derived

products from PEN2 activity (e.g. I3A and RA) against the

P. cucumerina isolates used in this study, and determined

their effective concentrations causing 50% inhibition (EC50).

All the compounds tested inhibited spore germination and

hyphal elongation of the three P. cucumerina isolates (Fig-

ure S2), with EC50 values that were similar for the adapted

and non-adapted isolates (e.g. EC50 values for PcBMM were

30.27 � 11.79 lM for camalexin, 183.08 � 90.14 lM for I3A

and 366.74 � 116.74 lM for RA). The antifungal activity

(EC50) of these metabolites was weaker than those of known

plant antibiotic peptides (Molina et al., 1993a,b), such as

thionins (THs) and lipid transfer proteins (LTPs) (e.g. EC50

values for PcBMM of 0.47 � 0.19 and 3.6 � 0.5 lM, respec-

tively). No significant difference was found between the

antifungal activities of these compounds against the adap-

ted or non-adapted isolates tested (Figure S2). Thus, there is

no evidence that the adapted P. cucumerina isolate has

evolved specific mechanisms to tolerate higher concentra-

tions of trp derivatives and antibiotic peptides.

100

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1.4

1.6 I3A

Col-0 pen2-3 pen3-1 pad2-1 cyp79B2cyp79B3

0

2

4

6

8

10

12

14

nmol

g–1

FW

nmol

g–1

FW

nmol

g–1

FW

nmol

g–1

FW

nmol

g–1

FW

nmol

g–1

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nmol

g–1

FW

RA

0

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10

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25

306-OGlc-I3CA

0

5

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251MI3G

0

5

10

15

20

25

30

35 Camalexin

0102030405060708090 I3G

PcBMMPc1187Pc2127

Mock

Figure 4. Determination of tryptophan-derivative content in Arabidopsis

plants inoculated with adapted and non-adapted Plectosphaerella cucume-

rina.

Average relative content (nmol per g fresh weight, �SD) of the indicated

metabolites in mock or PcBMM-, Pc1187- or Pc2127-treated plants at 1 day

post-inoculation (dpi). Two-tailed Student’s t-test for pairwise comparison of

infected and non-infected plants (*P £ 0.05; +P £ 0.1), and mutant and wild-

type plants (.P £ 0.05; ,P £ 0.1), was performed. The analyses were repeated

three times, and similar results were obtained each time.

120 Andrea Sanchez-Vallet et al.

ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 63, 115–127

Impaired defence to non-adapted P. cucumerina in

cyp79B cyp79B3 plants is largely restored in the irx1-6

mutant background

The irx1-6 cell wall mutant exhibits an enhanced and broad-

spectrum resistance to pathogens compared with Col-0

wild-type plants. Based on comparative microarray analyses

of irx1-6 and Col-0 plants, this phenotype was proposed to

be in part caused by the constitutive accumulation of

antimicrobials, such as trp-derived metabolites and anti-

microbial peptides (Hernandez-Blanco et al., 2007). The

determination of trp derivatives in irx1-6 and Col-0 plants

demonstrated that irx1-6 plants accumulated constitutively

higher levels of I3G, 1MI3G and 4MI3G than Col-0 plants.

Upon PcBMM infection (24 hpi), 4MI3G levels increased

further, and I3G and 1MI3G were consumed (Figure S3). In

contrast, the level of camalexin was similar in irx1-6 and

Col-0 plants (Figure S3). These data demonstrate directly

that irx1-6 plants accumulate enhanced levels of IGs. The

contribution of biosynthesis or extracellular release of the

trp derivatives in irx1-mediated resistance was analysed

by generating irx1-6 cyp79B2 cyp79B3 and irx1-6 pen3-1

mutants, respectively. Remarkably, the growth of the tested

non-adapted Pc2127 and Pc1187, and adapted PcBMM, iso-

lates was significantly reduced in irx1-6 cyp79B2 cyp79B3

plants compared with the cyp79B2 cyp792B3 double mutant

(Figure 5a–c). Similarly, the fungal growth of the non-

adapted isolates and the severity of disease symptoms in

irx1-6 pen3-1 plants were lower than in the pen3-1 single

mutant (Figure 5a–c). These data demonstrate that trp

derivatives are largely dispensable for full resistance in the

irx1 background. This points to the existence of functionally

redundant defence mechanisms that are active in this

mutant background.

To examine whether irx1-mediated defence mechanisms

specifically act in non-host resistance to necrotrophic

cyp79B2cyp79B30

2

4

6

8

10

12

40

60

80

100

Pcb

tub

(n-f

old

WT

)

b aab

c,d

aaa,ba,b

d

bb b,c

c c

e

c

d

(c)(a)

irx1-6cyp79B2cyp79B3

Col-0 irx1-6 irx1-6pen3-1

pen3-1 cyp79B2cyp79B3

dd

d

0

1

2

3

4

5

Dis

ease

rat

ing

a

b

a

a,b

a

b

c

ab

b

b,c

bb

c,d

c c

7 dpiPcBMM

Pc1187

Pc2127

(b)

Col-0

irx1-6

irx1-6cyp79B2cyp79B3

irx1-6pen3-1

PcBMM Pc1187 Pc2127

pen3-1

5 dpiPcBMM

Pc1187

Pc2127

Figure 5. The irx1-6 mutation partially restores resistance to adapted and non-adapted Plectosphaerella cucumerina isolates of cyp79B2 cyp79B3 and pen3-1

mutants.

Plants from the indicated genotypes were spray inoculated with a spore suspension (4 · 106 spores ml)1) of P. cucumerina BMM (PcBMM), or the non-adapted

fungi Pc1187 or Pc2127. (a) Quantification of fungal DNA (P. cucumerina b-tubulin) by quantitative real-time PCR (qRT-PCR) at 5 days post-inoculation (dpi). Values

(�SDs) are represented as the averages of fungal DNA levels relative to Col-0 plants. The letters indicate significantly different statistical groups for each fungal

isolate (ANOVA P £ 0.05, Bonferroni’s test).

(b) Average disease rating (DR � SD) of the indicated genotypes at 7 dpi. DR varies between 0 (no symptoms) and 5 (dead plant). The letters indicate significantly

different statistical groups for each fungal isolate (ANOVA P £ 0.05, Bonferroni’s test). Data in (a) and (b) are from one of three independent experiments performed,

which gave similar results.

(c) Lactophenol Trypan Blue staining of inoculated leaves at 24 hpi. Scale bar: 20 lm.

Arabidopsis non-host resistance to necrotrophic fungi 121

ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 63, 115–127

P. cucumerina, we tested the infection phenotype of wild-

type Col-0 plants and various irx1-containing genotypes

with the non-adapted biotrophic fungus E. pisi (Bednarek

et al., 2009). As shown in Figure 6, E. pisi entry rates in Col-0

and irx1-6 mutant plants were statistically indistinguishable.

Of note, the E. pisi entry rates on irx1-6 pen3-1 and irx1-6

cyp79B2 cyp79B3 plants were significantly higher than

those observed in the corresponding pen3-1 and cyp79B2

cyp79B3 parental lines (Figure 6), which, as reported, sup-

ported higher entry rates than Col-0 plants (Stein et al., 2006;

Bednarek et al., 2009). These data suggest that irx1-medi-

ated defence against necrotrophic P. cucumerina negatively

interferes with pre-invasive non-host resistance responses

to the non-adapted biotrophic fungal pathogen.

Function of SA, JA and ET signalling pathways in

Arabidopsis non-host resistance to non-adapted

P. cucumerina fungi

In Arabidopsis, the SA, JA and ET signalling pathways are

essential to restrict the growth of the adapted PcBMM

isolate, as well as other necrotrophic fungal pathogens

(Berrocal-Lobo et al., 2002; Llorente et al., 2005). To test

whether any of these defence pathways contribute to the

early termination of fungal pathogenesis in interactions with

non-adapted P. cucumerina, we determined the expression

of defence marker genes of the SA (PR1), ET/JA (PDF1.2), ET

(PR4) and JA (THI2.1) signalling pathways upon inoculation

of wild-type plants. Their expression was upregulated at 1

and 3 dpi in Col-0 plants inoculated with either non-adapted

or adapted P. cucumerina (Figure 7a). We next determined

the expression of these genes in cyp79B2 cyp79B3 plants

following pathogen challenge. The activation of PR1,

PDF1.2, PR4 and THI2.1 at 1 dpi was retained in cy-

p79B2 cyp79B3 plants (Figure 7b). Of note, PR1 and PDF1.2

upregulation was even enhanced in the double mutant,

compared with Col-0 plants, which is consistent with the

idea that the expression level of these genes correlates with

the level of fungal colonization. Thus, the activation of the

Col-0 irx1-6 pen3-10

20

40

60

80

100

Fun

gal e

ntry

rat

e (%

)

a

a

b

irx1-6pen3-1

c,d

irx1-6cyp79B2cyp79B3

d

cyp79B2cyp79B3

c

Figure 6. Impaired defence of cyp79B2 cyp79B3 and pen3 single mutants to

the non-adapted biotrophic fungus Erysiphe pisi is enhanced by the irx1-6

mutation.

Frequency of E. pisi entry at interaction sites on Arabidopsis genotypes

scored 72 h after inoculation with conidiospores. Values are averages of two

experiments (�SDs). Letters indicate statistical differences with wild-type

plants (ANOVA P £ 0.05, Bonferroni’s test).

(a)

(b)

n-fo

ld m

ock

expr

essi

on

0

10

20

30

40 PR-4

*

*

*

*

*

*

0

THI2.1

10

20

30

40

50

60

70

* **

*

* *

PcBMM Pc1187 Pc2127

n-fo

ld m

ock

expr

essi

on

0

200

400

600

800

1000

1200 PDF1.2

*

*

*

*

*

*PcBMM Pc1187 Pc2127

0

20 000

40 000

60 000

80 000PR-1

*

**

*

*

*

1 dpi3 dpi

1 dpi3 dpi

1 dpi3 dpi

1 dpi3 dpi

n-fo

ld U

BC

21 e

xpre

ssio

n

PR1

0

2

4

6

8

10

12Col-0

cyp79B2cyp79B3

*

*

*

*

PDF1.2

0

10

20

30

40

50

60

70

80

*

* * THI2.1

0.00

0.05

0.10

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0.25

**

Mock BMM 1187 2127 Mock BMM 1187 2127

90PR4

0

10

20

30

40

50

60

70

80

*

**

n-fo

ld U

BC

21 e

xpre

ssio

n

Pc Pc

Figure 7. Expression of ethylene (ET), jasmonate (JA) and salicylic acid

(SA)-related defence genes is induced in Arabidopsis upon infection with

non-adapted Plectosphaerella cucumerina fungi.

(a) Wild-type Col-0 plants were treated with the indicated P. cucumerina

isolates, as described in Figure 1. Expression of the indicated defence-related

genes was determined by quantitative real-time PCR (qRT-PCR) at 1 and

3 days post-inoculation (dpi). Values are represented as n-fold increased

expression, compared with mock-treated plants. Asterisks indicate expres-

sion values that are statistically different from those of mock-treated plants,

and triangles represent values that are statistically different from PcBMM-

challenged plants (ANOVA P £ 0.05, Bonferroni’s test).

(b) Expression of the indicated defence-related genes in Col-0 (grey bars)

and cyp79B2 cyp79B3 (black bars) plants 1 dpi with P. cucumerina isolates.

Expression values are normalized to UBIQUITIN21 (UBC21). Asterisks indicate

expression values statistically different from those of wild-type plants (ANOVA

P £ 0.05, Bonferroni’s test). These experiments were repeated three times

with similar results.

122 Andrea Sanchez-Vallet et al.

ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 63, 115–127

SA, JA and ET signalling pathways occurs independently of

the pathogen-inducible accumulation of trp-derived sec-

ondary metabolites, and is not impaired in cyp79B2 cyp79B3

mutants.

We also examined whether non-adapted P. cucumerina

were able to colonize Arabidopsis mutant lines blocked in

the synthesis of either SA (NahG) or JA (aos), or in ET

signalling (ein2-1). The Pc1187 and Pc2127 isolates failed to

colonize the NahG and ein2-1 plants, and caused slight

disease symptoms in aos mutant plants (Figure S4). As

described previously (Berrocal-Lobo et al., 2002; Llorente

et al., 2005), the severity of disease symptoms caused by the

adapted PcBMM in ein2-1, NahG and aos plants was higher

than that observed in Col-0 plants (Figure S4). These data

indicate that the SA and ET signalling pathways are either

dispensable for resistance to the non-adapted P. cucumerina

isolates or act in a functionally redundant manner, whereas

the JA signalling pathway may have a minor role in

restricting the pathogenesis of these non-adapted fungi.

DISCUSSION

Most of the characterized plant necrotrophic fungi have a

broad host range and can colonize phylogenetically diverse

plant species. However, for some necrotrophs specific,

compatible interactions with particular plant hosts have

been described (van Kan, 2006). The plant genetic basis

determining host restriction of necrotrophic fungi are

poorly understood. Here, we selected three isolates of the

ascomycete necrotrophic fungus P. cucumerina (Pc1187,

Pc2127 and Pc2125), which colonize different plant species

in nature, but fail to infect Arabidopsis Col-0 wild-type

plants after spore germination. This contrasts with the

sustained hyphal growth of the adapted, virulent

P. cucumerina (PcBMM) isolate (Berrocal-Lobo et al., 2002;

Ton and Mauch-Mani, 2004). Using this pathosystem, we

have shown that trp-derived metabolites are essential for

limiting Arabidopsis colonization by non-adapted and

adapted necrotrophic fungi. Notably, a comprehensive

depletion of trp-derived metabolites, as it occurs in cy-

p79B2 cyp79B3 plants, dramatically enhances the growth of

the non-adapted P. cucumerina isolates that sporulate on

leaf tissues, rendering these plants fully susceptible (Fig-

ure 2, and data not shown). This demonstrates that the

depletion of a single class of phytochemicals is sufficient to

render Arabidopsis plants as hosts for these pathogens.

This differs from a previous study in which the concurrent

impairment of pre-invasive (pen2) and post-invasive

(pad4 sag101) non-host resistance layers was necessary to

make Arabidopsis a host for the biotrophic E. pisi and

Blumeria graminis f.sp. hordei fungi (Lipka et al., 2005).

The function of trp-derived metabolites in plant defence

responses is supported by the observed upregulation of

genes encoding enzymes involved in their biosynthesis (e.g.

ASA1, CYP79B2, CYP79B3, CYP81F2 and PAD3) following

challenge with the non-adapted or adapted P. cucumerina

isolates (Figure 3). Accordingly, the accumulation of RA and

trp-derived metabolites, such as 4MI3G, I3A, 6-OGlc-I3CA

and camalexin, was enhanced in the inoculated plants

(Figure 4). The levels of these compounds were similar in

plants challenged with the adapted or non-adapted isolates,

although hyphal growth of the host-adapted PcBMM on

leaves was unrestricted, and the number of plant cells in

contact with this fungus was higher (Figure 4). In addition to

genes related to trp-derived metabolism, the expression of

marker genes of defence signalling pathways (Glazebrook,

2005; Robert-Seilaniantz et al., 2007; Grant and Jones, 2009),

such as PR-1, PR-4, PDF1.2 and THI2.1, was also upregulat-

ed upon inoculation with the non-adapted P. cucumerina

isolates, indicating that the SA, ET and JA pathways were

positively triggered by these pathogens. With a few excep-

tions, the immune responses activated by the non-adapted

isolates overlapped with those induced by the adapted

PcBMM, as the same subset of genes was similarly

upregulated upon infection. These results are in line with

those observed in Arabidopsis plants challenged with other

non-adapted fungi, such as powdery mildews, rusts and

Alternaria alternata (Zimmerli et al., 2004; Narusaka et al.,

2005; Shafiei et al., 2007). Consistent with the activation

of a defence response, a reduction of plant growth was

observed in the inoculated Col-0 wild-type plants (Fig-

ure 1e, and data not shown), indicating that activation of

non-host resistance entails an associated fitness cost, as

described for other immune responses (Zimmerli et al.,

2004).

The biosynthesis and targeted delivery of trp derivatives

are essential for Arabidopsis immune responses to non-

adapated and adapted powdery mildews (Bednarek et al.,

2009; Consonni et al., 2010). Specifically, PEN2 and CYP81F2

activities were demonstrated to be critical for the effective

resistance against non-adapted powdery mildews, other

non-adapted fungal pathogens, like Phytophthora infestans

and Magnaporthe grisea, as well as the adapted P. cucume-

rina BMM isolate (Lipka et al., 2005; Bednarek et al., 2009;

Maeda et al., 2009). Unexpectedly, we found that resistance

responses to the non-adapted P. cucumerina isolates were

unaffected in pen2 and cyp81F2 mutants (Figure 2). Simi-

larly, camalexin, which constitutes a post-invasive barrier

against non-adapted powdery mildews (Bednarek et al.,

2009), and mediates plant resistance to necrotrophic patho-

gens, such as Alternaria brassicicola and B. cinerea (Thom-

ma et al., 1999a; Kliebenstein et al., 2005; Nafisi et al., 2007),

was not essential for non-host resistance to the non-adapted

isolates (Figure 2). Although the disruption of the biosyn-

thetic pathway of camalexin in the pen2 background

(pen2 pad3 mutant) significantly enhanced the susceptibility

of the parental lines to non-adapted and host-adapted

necrotrophs (Figure 2), P. cucumerina growth on pen2 pad3

plants was still much lower than on cyp79B2 cyp79B3 plants.

Arabidopsis non-host resistance to necrotrophic fungi 123

ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 63, 115–127

This is in marked contrast with the comparable growth of

non-adapted biotrophic powdery mildews on these two

double mutants (Bednarek et al., 2009). This strongly sug-

gests that, in addition to camalexin and the PEN2-derived

products, other trp derivatives must be required for Arabid-

opsis non-host resistance to P. cucumerina (Figure 2). One

candidate group of compounds comprises indole-3-carbox-

ylic acid derivatives, of which 6-OGlc-I3CA has been shown

here to accumulate in wild-type plants upon inoculation with

the adapted and non-adapted P. cucumerina isolates (Fig-

ure 4). Indole-3-carboxylate derivatives could exert a role in

host cell wall fortification, as some of these have been

detected as pathogen-induced conjugates of the plant cell

wall (Hagemeier et al., 2001; Tan et al., 2004; Bednarek et al.,

2005). Unfortunately, mutants specifically impaired in the

accumulation of 6-OH-I3CA have not yet been identified.

Compelling evidence has been recently obtained for indole-

derived compounds in the establishment and maintenance

of systemic acquired immunity in Arabidopsis, thereby

underlining a key role of this compound class in diverse

forms of immunity (Truman et al., 2010).

As in non-host resistance to biotrophic powdery mildew

fungi, pad2 plants supported enhanced growth of the non-

adapted P. cucumerina isolates (Figure 2). Remarkably,

the concentrations of camalexin and IGs in the mutant

increased, as in the wild type, upon inoculation with the

non-adapted necrotrophs (Figure 4). This contrasts with the

reduction in the levels of camalexin, aliphatic Gs and IGs in

pad2 plants under particular stress conditions or pathogen

infection (Glazebrook and Ausubel, 1994; Roetschi et al.,

2001; Schlaeppi et al., 2008; Bednarek et al., 2009). The

reduced levels of these compounds are believed to explain

the enhanced susceptibility phenotype of this mutant to

non-adapted biotrophs and several adapted fungi, oomycete

and bacteria. The levels of PEN2-dependent metabolites, I3A

and RA, were reduced in P. cucumerina-challenged pad2-1

plants, further supporting the potential function of glutathi-

one as a cysteine donor in the synthesis of RA (Bednarek

et al., 2009). We hypothesize that the enhanced susceptibil-

ity of pad2 plants to non-adapted P. cucumerina isolates, in

conjunction with increases in the levels of camalexin and

iGSs, but full susceptibility of cyp79B2 cyp79B3 plants,

reflects the demand for glutathione in the biosynthesis

and/or metabolism of unknown resistance-conferring trp-

derived metabolites. Alternatively, glutathione may exert a

function in immunity that is completely independent from

trp metabolism.

PEN3 activity, which is essential for resistance to the non-

adapted powdery mildews and to P. cucumerina BMM

(Stein et al., 2006; Bednarek et al., 2009), is dispensable for

resistance to the tested non-adapted necrotrophs. However,

macroscopic lesions were more severe in pen3 plants

(Figure 2b). The pen3 plants also develop chlorotic lesions

upon E. cichoracearum inoculation that leads to the activa-

tion of the SA-signalling pathway (Stein et al., 2006;

Figure 6), and are presumably the result of an enhanced

intracellular accumulation of toxic compounds. Consistent

with this, pen3 plants inoculated with P. cucumerina

showed an increased level of trp-derived metabolites

(Figure 4).

The IG-derived metabolites have been suggested to act as

signalling molecules in MAMP-triggered immunity (Clay

et al., 2009). In addition, an insect-deterrent function for

IG-derived compounds and antimicrobial activity for cama-

lexin and diverse products of G metabolism have been

described (Tierens et al., 2001; Sellam et al., 2007). Here, we

have shown that RA, I3A and camalexin display antimicrobial

activity in vitro against the P. cucumerina isolates tested

(Figure S2). The EC50 values of these metabolites for adapted

and non-adapted isolates were similar, indicating that non-

host resistance to P. cucumerina cannot be explained by a

differential sensitivity of these fungal isolates to these

pathogen-induced compounds. Camalexin was the most

active of the compounds tested, with EC50 values compara-

ble with those described for A. brassicicola and Pseudomo-

nas syringae pv. maculicola (Figure S2; Rogers et al., 1996;

Thomma et al., 1999a). The combinatorial activity of these

metabolites and antimicrobial peptides (e.g. THs and LTPs)

against P. cucumerina isolates was tested, and found to be

additive rather than synergistic (not shown). This could

indicate that trp-derived metabolites inhibit fungal growth

by a mechanism similar to that described for plant antimi-

crobial peptides (Caaveiro et al., 1997). It remains a possi-

bility that the host-adapted P. cucumerina evolved

detoxification mechanisms that are specific to the unknown,

but functionally most important, trp-derived metabolites.

Alternatively, these unknown trp-derived metabolites may

have a novel defence signalling function, rather than an

in vivo antimicrobial activity. In this scenario, the host-

adapted PcBMM fungus may have evolved the means to

block the corresponding Arabidopsis immune response

pathway(s).

The relevance of antimicrobial compounds in plant

immunity may be supported by the broad-spectrum resis-

tance of the Arabidopsis irx1-6/cesa8 secondary cell wall

mutant. Defence activation in irx1-6 plants could result in an

enhanced accumulation of trp-derived metabolites and

antimicrobial peptides. Indeed, the irx1-6 plants accumu-

lated constitutively higher levels of I3G, 1MI3G and 4MI3G

than Col-0 plants, and upon P. cucumerina BMM infection,

4MI3G levels increased further (Figure S3). The biosynthesis

and delivery of IGs in irx1-6 plants was disrupted by

generating the irx1-6 cyp79B2 cyp79B3 and irx1-6 pen3-1

mutants, respectively. Remarkably, the growth of non-

adapted and adapted isolates in these mutants was almost

comparable with wild-type plants, pointing to the existence

of additional trp-independent resistance components in this

mutant background (Figure 5). In contrast, disrupting IRX1

124 Andrea Sanchez-Vallet et al.

ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 63, 115–127

in cyp79B2 cyp79B3 and pen3 backgrounds enhanced their

susceptibility to the non-adapted powdery mildew E. pisi

(Figure 6), indicating a negative crosstalk between the

trp-independent and -dependent defences.

Immune responses of wild-type Arabidopsis upon inocu-

lation with the non-adapted P. cucumerina isolates suggest

an enhanced activation of the SA, ET and JA signalling

pathways, that, like the b-subunit of heterotrimeric G-protein

(AGB1) and the ER receptor-like kinase are essential for

growth restriction of the host-adapted PcBMM isolate

(Berrocal-Lobo et al., 2002; Llorente et al., 2005). However,

the SA and ET pathways, and AGB1 and ER, were shown to

be dispensable or to act in a functionally redundant manner

in interactions with the non-adapted P. cucumerina isolates

(Figure S4, and data not shown). Similar results for

hormone signalling have been obtained for interactions

between Arabidopsis and Leptosphaeria maculans, whereas

non-host resistance to Magnaporthe oryzae was found to be

slightly impaired in the agb1 mutant (Bohman et al., 2004;

Park et al., 2009). Besides, the JA signalling pathway seems

to contribute to non-host resistance against P. cucumerina,

as shown by the slightly enhanced susceptibility of the aos

mutant, which is impaired in JA biosynthesis (Figure S4). JA

signalling controls the accumulation of iGSs upon treatment

of Arabidopsis with JAs or a culture filtrate from Erwinia

carotovora (Brader et al., 2001; Mikkelsen et al., 2003; Jost

et al., 2005). In line with these data, we found that the

constitutive expression of CYP79B2 in aos plants was lower

than in the Col-0 wild-type (not shown), suggesting that the

enhanced susceptibility of the aos mutant to non-adapted

P. cucumerina isolates could be in part explained by its

lower content of trp derivatives. These findings suggest that,

despite trp-derived metabolites being the main executioners

and/or signalling molecules of Arabidopsis resistance to

non-adapted P. cucumerina, a complex interplay of other

defence signalling pathways becomes engaged in the

control of these necrotrophic fungi.

EXPERIMENTAL PROCEDURES

Biological material and growth conditions

Arabidopsis thaliana plants were grown in phytochambers on asterilized mixture of soil and vermiculite (3:1; Hernandez-Blancoet al., 2007), with a 10-h day/14-h night photoperiod, a temperatureof 22�C day/20�C night and 50% relative humidity. Light intensitywas fixed to 120–150 lmol m)2 s)1, according to Weigel andGlazebrook (2002). The following lines, in the Col-0 background,were used: pen2-2, pen2-3 (Lipka et al., 2005), pen3-1 (Stein et al.,2006), cyp81F2-1 (Bednarek et al., 2009), cyp79B2 cyp79B3 (Zhaoet al., 2002), pad2-1 (Parisy et al., 2007), pad3-1 (Zhou et al., 1999),pen2-1 pad3-1 (Bednarek et al., 2009), irx1-6 (Hernandez-Blancoet al., 2007), NahG (Delaney et al., 1994) and ein2-1 (Guzman andEcker, 1990). The aos mutant is in Col-6 (gl1; Park et al., 2002). TheP. cucumerina BMM isolate was provided by Dr B. Mauch-Mani(University of Fribourg, Switzerland; Tierens et al., 2001), and theP. cucumerina isolates Pc2127, Pc1187 and Pc2125 were from theDSMZ collection (http://www.dsmz.de). In vitro fungal growth and

the collection of spores were carried out as reported by Hernandez-Blanco et al. (2007).

Fungal inoculation assays

Three-week-old Arabidopsis plants were inoculated with a sporesuspension (4 · 106 spores ml)1) of the different P. cucumerinaisolates. Disease progression was estimated by determining theaverage DR (0–5), trypan blue staining and relative quantification offungal DNA by qRT-PCR, as described by Sanchez-Rodriguez et al.(2009). Inoculation with E. pisi (Birmingham isolate) and determi-nation of entry rates was performed as described by Bednarek et al.(2009). For all of the fungal inoculations, at least three independentexperiments were performed. Statistical differences amongArabidopsis genotypes were determined by one-way analysis ofvariance and Bonferroni post hoc test, as previously reported(Sanchez-Rodriguez et al., 2009).

Gene expression analyses and fungal biomass

determination

For gene expression analysis, RNA extractions from P. cucumerina-infected or mock-treated plants were performed; for fungal DNAquantification, DNA from infected plants was extracted (Llorenteet al., 2005). Oligonucleotides were designed using PRIMER

EXPRESS v2.0 (Applied Biosystems, http://www.appliedbiosys-tems.com; Table S1). qRT-PCR analyses were performed as previ-ously reported, with 0.3 lM of each primer (Hernandez-Blanco et al.,2007), using the FS Universal SYBR GreenMasterRox (Roche, http://www.roche.com) and the described amplification conditions (San-chez-Rodriguez et al., 2009). The plant UBIQUITIN21 (At5G25760)gene was used to normalize and calculate the change in cyclethreshold (DCt) value. The relative expression ratio was determinedwith the equation 2)DDC

t (Rieu and Powers, 2009), using the relativequantification application of the sequence detector software (v1.4;Applied Biosystems; Rieu and Powers, 2009). The qRT-PCR resultsare mean values (�SDs) from two technical replicates. Differencesin expression ratios (DCt) among the samples were analysed byANOVA or Student’s t-test using STATGRAPHICS.

Quantification of trp derivatives

Plant samples were collected at 24 hpi and frozen in liquid nitrogen.Extraction and HPLC analysis of tryptophan derivatives was per-formed as previously described (Bednarek et al., 2009). Each linewas tested in at least three independent experiments.

In vitro antimicrobial tests

Inhibition tests of P. cucumerina spore were carried out in sterilemicrotiter plates, as described by Molina et al. (1993b). Differentconcentrations of purified secondary metabolites (Bednarek et al.,2009) and peptides (Molina et al., 1993a,b) were analysed. Plateswere incubated at 28�C, and pictures of hyphal growth were taken atdifferent time points.

Generation and selection if irx1-6 cyp79B2 cyp79B3 and

irx1-6 pen3-1 mutants

These mutants were generated by standard genetic crossesfollowed by identification of the mutant alleles. Genotyping of theirx1-6 and pen3-1 mutations were confirmed by sequencing themutations in the PCR products (irx1-6, 5¢-CATGTGCTGTTGGGT-AGGAATC-3¢ and 5¢-CATAGAGAATGTGTTTGATGATG-3¢; pen3-1,5¢-CTCGTCACTGATTATACTCTC-3¢ and 5¢-TGAGGTGAACGATTTG-TTGC-3¢). The T-DNA insertion mutations in cyp79B2 cyp79B3 wereidentified as described (Zhao et al., 2002).

Arabidopsis non-host resistance to necrotrophic fungi 125

ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 63, 115–127

Plectosphaerella cucumerina ITS1 and ITS2 GenBank

numbers

PcBMM, GU724979; Pc1187, GU724980; Pc2125, GU724981; Pc2127,GU724982.

ACKNOWLEDGEMENTS

Work in the laboratory of AM was supported by the Spanish Min-isterio de Ciencia e Innovacion (MICINN) [grants EUI2008-03728(BALANCE) and BIO2006-00488]. AS-V was a PhD fellow from theMinisterio de Educacion y Ciencia (MEC). BR was an Ayudante fromUniversidad Salamanca. Work in the laboratory of PS-L was sup-ported by the German Bundesministerium fur Bildung und For-schung (BMBF) (Plant-KBBE grant: BALANCE) and the Max PlanckSociety.

SUPPORTING INFORMATION

Additional Supporting Information may be found in the onlineversion of this article:Figure S1. Neighbour-joining tree of ribosomal DNA sequences(ITS1-5.8rRNA-ITS2) from Plectosphaerella cucumerina (Pc) iso-lates.Figure S2. Tryptophan-derived metabolites inhibit in vitro Plectosp-haerella cucumerina growth.Figure S3. Determination of tryptophan-derived metabolites con-tent in irx1-6 and wild-type plants upon infection by Plectosphae-rella cucumerina.Figure S4. Contribution of the ethylene (ET), jasmonate (JA) andsalicylic acid (SA) signalling pathways to Arabidopsis non-hostresistance against non-adapted Plectosphaerella cucumerina fungi.Table S1. Oligonucleotides used for quantitative real-time PCR(qRT-PCR) analyses.Please note: As a service to our authors and readers, this journalprovides supporting information supplied by the authors. Suchmaterials are peer-reviewed and may be re-organized for onlinedelivery, but are not copy-edited or typeset. Technical supportissues arising from supporting information (other than missingfiles) should be addressed to the authors.

REFERENCES

Bednarek, P., Schneider, B., Svatos, A., Oldham, N.J. and Hahlbrock, K. (2005)

Structural complexity, differential response to infection, and tissue speci-

ficity of indolic and phenylpropanoid secondary metabolism in Arabidop-

sis roots. Plant Physiol. 138, 1058–1070.

Bednarek, P., Pislewska-Bednarek, M., Svatos, A. et al. (2009) A glucosinolate

metabolism pathway in living plant cells mediates broad-spectrum anti-

fungal defense. Science, 323, 101–106.

Berrocal-Lobo, M., Molina, A. and Solano, R. (2002) Constitutive expression of

ETHYLENE-RESPONSE-FACTOR1 in Arabidopsis confers resistance to

several necrotrophic fungi. Plant J. 29, 23–32.

Bohman, S., Staal, J., Thomma, B.P.H.J., Wang, M. and Dixelius, C. (2004)

Characterisation of an Arabidopsis-Leptosphaeria maculans pathosys-

tem: resistance partially requires camalexin biosynthesis and is inde-

pendent of salicylic acid, ethylene and jasmonic acid signalling. Plant J.

37, 9–20.

Bolton, M.D., Thomma, B.P.H.J. and Nelson, B.D. (2006) Sclerotinia sclero-

tiorum (Lib.) de Bary: biology and molecular traits of a cosmopolitan

pathogen. Mol. Plant Pathol. 7, 1–16.

Bottcher, C., Westphal, L., Schmotz, C., Prade, E., Scheel, D. and Glawischnig,

E. (2009) The multifunctional enzyme CYP71B15 (PHYTOALEXIN DEFI-

CIENT3) converts cysteine-indole-3-acetonitrile to camalexin in the indole-

3-acetonitrile metabolic network of Arabidopsis thaliana. Plant Cell, 21,

1830–1841.

Brader, G., Tas, E. and Palva, E.T. (2001) Jasmonate-dependent induction of

indole glucosinolates in Arabidopsis by culture filtrates of the nonspecific

pathogen Erwinia carotovora. Plant Physiol. 126, 849–860.

Caaveiro, J.M., Molina, A., Gonzalez-Manas, J.M., Rodriguez-Palenzuela, P.,

Garcia-Olmedo, F. and Goni, F.M. (1997) Differential effects of five types of

antipathogenic plant peptides on model membranes. FEBS Lett. 410, 338–

342.

Clay, N.K., Adio, A.M., Denoux, C., Jander, G. and Ausubel, F.M. (2009)

Glucosinolate metabolites required for an Arabidopsis innate immune

response. Science, 323, 95–101.

Collins, N.C., Thordal-Christensen, H., Lipka, V. et al. (2003) SNARE-protein-

mediated disease resistance at the plant cell wall. Nature, 425, 973–977.

Consonni, C., Bednarek, P., Humphry, M., Francocci, F., Ferrari, S., Harzen, A.,

Ver Loren van Themaat, E. and Panstruga, R. (2010) Tryptophan-derived

metabolites are required for antifungal defence in the Arabidopsis thaliana

mlo2 mutant. Plant Physiol., 152, 1544–1561.

Cramer, R.A. and Lawrence, C.B. (2004) Identification of Alternaria brassici-

cola genes expressed in planta during pathogenesis of Arabidopsis thali-

ana. Fungal Genet. Biol. 41, 115–128.

Delaney, T.P., Uknes, S., Vernooij, B. et al. (1994) A central role of salicylic

acid in plant disease resistance. Science, 266, 1247–1250.

Denby, K.J., Kumar, P. and Kliebenstein, D.J. (2004) Identification of Botrytis

cinerea susceptibility loci in Arabidopsis thaliana. Plant J. 38, 473–486.

Domsch, K.H., Gams, W. and Anderson, T.H. (2007) Compendium of Soil

Fungi, 2nd edn. Eching: IHW Verlag.

Ferrari, S., Plotnikova, J.M., Lorenzo, G.D. and Ausubel, F.M. (2003) Arabid-

opsis local resistance to Botrytis cinerea involves salicylic acid and cama-

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

193–205.

Glawischnig, E. (2007) Camalexin. Phytochemistry, 68, 401–406.

Glazebrook, J. (2005) Contrasting mechanisms of defense against biotrophic

and necrotrophic pathogens. Annu. Rev. Phytopathol. 43, 205–227.

Glazebrook, J. and Ausubel, F.M. (1994) Isolation of phytoalexin-deficient

mutants of Arabidopsis thaliana and characterization of their interactions

with bacterial pathogens. Proc. Natl. Acad. Sci. USA, 91, 8955–8959.

Grant, M.R. and Jones, J.D. (2009) Hormone (dis)harmony moulds plant

health and disease. Science, 324, 750–752.

Guzman, P. and Ecker, J.R. (1990) Exploiting the triple response of Arabid-

opsis to identify ethylene-related mutants. Plant Cell, 2, 513–523.

Hagemeier, J., Schneider, B., Oldham, N.J. and Hahlbrock, K. (2001) Accu-

mulation of soluble and wall-bound indolic metabolites in Arabidopsis

thaliana leaves infected with virulent or avirulent Pseudomonas syringae

pathovar tomato strains. Proc. Natl. Acad. Sci. USA, 98, 753–758.

Heath, M.C. (2000) Nonhost resistance and nonspecific plant defenses. Curr.

Opin. Plant Biol. 3, 315–319.

Hernandez-Blanco, C., Feng, D.X., Hu, J. et al. (2007) Impairment of cellulose

synthases required for Arabidopsis secondary cell wall formation enhances

disease resistance. Plant Cell, 19, 890–903.

Jost, R., Altschmied, L., Bloem, E. et al. (2005) Expression profiling of meta-

bolic genes in response to methyl jasmonate reveals regulation of genes of

primary and secondary sulfur-related pathways in Arabidopsis thaliana.

Photosynth. Res. 86, 491–508.

van Kan, J.A.L. (2006) Licensed to kill: the lifestyle of a necrotrophic plant

pathogen. Trends Plant Sci., 11, 247–253.

Kliebenstein, D.J., Rowe, H.C. and Denby, K.J. (2005) Secondary metabolites

influence Arabidopsis/Botrytis interactions: variation in host production

and pathogen sensitivity. Plant J. 44, 25–36.

Kwon, C., Neu, C., Pajonk, S. et al. (2008) Co-option of a default secretory

pathway for plant immune responses. Nature, 451, 835–840.

Lipka, V., Dittgen, J., Bednarek, P. et al. (2005) Pre- and postinvasion defenses

both contribute to nonhost resistance in Arabidopsis. Science, 310, 1180–

1183.

Llorente, F., Alonso-Blanco, C., Sanchez-Rodriguez, C., Jorda, L. and Molina,

A. (2005) ERECTA receptor-like kinase and heterotrimeric G protein from

Arabidopsis are required for resistance to the necrotrophic fungus Plec-

tosphaerella cucumerina. Plant J. 43, 165–180.

Llorente, F., Muskett, P., Sanchez-Vallet, A., Lopez, G., Ramos, B., Sanchez-

Rodriguez, C., Jorda, L., Parker, J. and Molina, A. (2008) Repression of the

auxin response pathway increases Arabidopsis susceptibility to necro-

trophic fungi. Mol. Plant, 1, 496.

Maeda, K., Houjyou, Y., Komatsu, T., Hori, H., Kodaira, T. and Ishikawa, A.

(2009) AGB1 and PMR5 contribute to PEN2-mediated preinvasion resis-

tance to Magnaporthe oryzae in Arabidopsis thaliana. Mol. Plant-Microbe

Interact. 22, 1331–1340.

126 Andrea Sanchez-Vallet et al.

ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 63, 115–127

Mikkelsen, M.D., Petersen, B.L., Glawischnig, E., Jensen, A.B., Andreasson, E.

and Halkier, B.A. (2003) Modulation of CYP79 genes and glucosinolate

profiles in Arabidopsis by defense signaling pathways. Plant Physiol. 131,

298–308.

Molina, A., Ahl Goy, P., Fraile, A., Sanchez-Monge, R. and Garcia-Olmedo, F.

(1993a) Inhibition of bacterial and fungal plant pathogens by thionins of

types I and II. Plant Sci. 92, 166–177.

Molina, A., Segura, A. and Garcia-Olmedo, F. (1993b) Lipid transfer proteins

(nsLTPs) from barley and maize leaves are potent inhibitors of bacterial and

fungal plant pathogens. FEBS Lett. 316, 119–122.

Nafisi, M., Goregaoker, S., Botanga, C.J., Glawischnig, E., Olsen, C.E., Halkier,

B.A. and Glazebrook, J. (2007) Arabidopsis cytochrome P450 monooxy-

genase 71A13 catalyzes the conversion of indole-3-acetaldoxime in cama-

lexin synthesis. Plant Cell, 19, 2039–2052.

Narusaka, Y., Narusaka, M., Seki, M., Ishida, J., Shinozaki, K., Nan, Y., Park, P.,

Shiraishi, T. and Kobayashi, M. (2005) Cytological and molecular analyses

of non-host resistance of Arabidopsis thaliana to Alternaria alternata. Mol.

Plant Pathol. 6, 615–627.

Niyogi, K.K. and Fink, G.R. (1992) Two anthranilate synthase genes in Ara-

bidopsis: defense-related regulation of the tryptophan pathway. Plant Cell,

4, 721–733.

Parisy, V., Poinssot, B., Owsianowski, L., Buchala, A., Glazebrook, J. and

Mauch, F. (2007) Identification of PAD2 as a gamma-glutamylcysteine

synthetase highlights the importance of glutathione in disease resistance

of Arabidopsis. Plant J. 49, 159–172.

Park, J.-H., Halitschke, R., Kim, H.B., Baldwin, I.T., Feldmann, K.A. and

Feyereisen, R. (2002) A knock-out mutation in allene oxide synthase results

in male sterility and defective wound signal transduction in Arabidopsis

due to a block in jasmonic acid biosynthesis. Plant J. 31, 1–12.

Park, J.-Y., Jin, J., Lee, Y.-W., Kang, S. and Lee, Y.-H. (2009) Rice blast fungus

(Magnaporthe oryzae) infects Arabidopsis via a mechanism distinct from

that required for the infection of rice. Plant Physiol. 149, 474–486.

Pfalz, M., Vogel, H. and Kroymann, J. (2009) The gene controlling the indole

glucosinolate modifier1 quantitative trait locus alters indole glucosinolate

structures and aphid resistance in Arabidopsis. Plant Cell, 21, 985–999.

Pitt, W.M., Goodwin, S.B., Ash, G.J., Cother, N.J. and Cother, E.J. (2004)

Plectosporium alismatis comb. nov. a new placement for the Alismataceae

pathogen Rhynchosporium alismatis. Mycol. Res., 108, 775–780.

Rieu, I. and Powers, S.J. (2009) Real-time quantitative RT-PCR: design, cal-

culations, and statistics. Plant Cell, 21, 1031–1033.

Robert-Seilaniantz, A., Navarro, L., Bari, R. and Jones, J.D.G. (2007) Patho-

logical hormone imbalances. Curr. Opin. Plant Biol. 10, 372–379.

Roetschi, A., Si-Ammour, A., Belbahri, L., Mauch, F. and Mauch-Mani, B.

(2001) Characterization of an Arabidopsis-Phytophthora pathosystem:

resistance requires a functional PAD2 gene and is independent of salicylic

acid, ethylene and jasmonic acid signalling. Plant J. 28, 293–305.

Rogers, E.E., Glazebrook, J. and Ausubel, F.M. (1996) Mode of action of the

Arabidopsis thaliana phytoalexin camalexin and its role in Arabidopsis-

pathogen interactions. Mol. Plant Microbe Interact. 9, 748–757.

Sanchez-Rodriguez, C., Estevez, J.M., Llorente, F., Hernandez-Blanco, C.,

Jorda, L., Pagan, I., Berrocal, M., Marco, Y., Somerville, S. and Molina, A.

(2009) The ERECTA receptor-like kinase regulates cell wall mediated

resistance to pathogens in Arabidopsis thaliana. Mol. Plant-Microbe

Interact. 22, 953–963.

Schlaeppi, K., Bodenhausen, N., Buchala, A., Mauch, F. and Reymond, P.

(2008) The glutathione-deficient mutant pad2-1 accumulates lower

amounts of glucosinolates and is more susceptible to the insect herbivore

Spodoptera littoralis. Plant J. 55, 774–786.

Schuhegger, R., Nafisi, M., Mansourova, M., Petersen, B.L., Olsen, C.E.,

Svatos, A., Halkier, B.A. and Glawischnig, E. (2006) CYP71B15 (PAD3) cat-

alyzes the final step in camalexin biosynthesis. Plant Physiol. 141, 1248–

1254.

Sellam, A., Iacomi-Vasilescu, B., Hudhomme, P. and Simoneau, P. (2007)

In vitro antifungal activity of brassinin, camalexin and two isothiocyanates

against the crucifer pathogens Alternaria brassicicola and Alternaria

brassicae. Plant Pathol. 56, 296–301.

Shafiei, R., Hang, C., Kang, J.-G. and Loake, G.J. (2007) Identification of loci

controlling non-host disease resistance in Arabidopsis against the leaf rust

pathogen Puccinia triticina. Mol. Plant Pathol. 8, 773–784.

Somerville, C., Bauer, S., Brininstool, G. et al. (2004) Toward a systems ap-

proach to understanding plant cell walls. Science, 306, 2206–2211.

Stein, M., Dittgen, J., Sanchez-Rodriguez, C., Hou, B.H., Molina, A.,

Schulze-Lefert, P., Lipka, V. and Somerville, S. (2006) Arabidopsis PEN3/

PDR8, an ATP binding cassette transporter, contributes to nonhost resis-

tance to inappropriate pathogens that enter by direct penetration. Plant

Cell, 18, 731–746.

Tan, J., Bednarek, P., Liu, J., Schneider, B., Svatos, A. and Hahlbrock, K. (2004)

Universally occurring phenylpropanoid and species-specific indolic

metabolites in infected and uninfected Arabidopsis thaliana roots and

leaves. Phytochemistry, 65, 691–699.

Thomma, B.P., Eggermont, K., Penninckx, I.A., Mauch-Mani, B., Vogelsang,

R., Cammue, B.P. and Broekaert, W.F. (1998) Separate jasmonate-depen-

dent 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, B.P., Nelissen, I., Eggermont, K. and Broekaert, W.F. (1999a)

Deficiency in phytoalexin production causes enhanced susceptibility of

Arabidopsis thaliana to the fungus Alternaria brassicicola. Plant J. 19,

163–171.

Thomma, B.P.H.J., Eggermont, K., Tierens, K.F.M.J. and Broekaert, W.F.

(1999b) Requirement of functional ethylene-insensitive 2 gene for efficient

resistance of Arabidopsis to infection by Botrytis cinerea. Plant Physiol.

121, 1093–1101.

Thordal-Christensen, H. (2003) Fresh insights into processes of nonhost

resistance. Curr. Opin. Plant Biol. 6, 351–357.

Tierens, K.F., Thomma, B.P., Brouwer, M., Schmidt, J., Kistner, K., Porzel, A.,

Mauch-Mani, B., Cammue, B.P. and Broekaert, W.F. (2001) Study of the role

of antimicrobial glucosinolate-derived isothiocyanates in resistance of

Arabidopsis to microbial pathogens. Plant Physiol. 125, 1688–1699.

Ton, J. and Mauch-Mani, B. (2004) b-amino-butyric acid-induced resistance

against necrotrophic pathogens is based on ABA-dependent priming for

callose. Plant J. 38, 119–130.

Truman, W.M., Bennett, M.H., Turnbull, C.G.N. and Grant, M.R. (2010) Ara-

bidopsis auxin mutants are compromised in sytemic acquired resistance

and exhibit aberrant accumulation of various indolic compounds. Plant

Physiol. 152, 1562–1573.

Van Baarlen, P., Woltering, E.J., Staats, M. and Van Kan, J.A. (2007) Histo-

chemical and genetic analysis of host and non-host interactions of Ara-

bidopsis with three Botrytis species: an important role for cell death

control. Mol. Plant Pathol. 8, 41.

van Wees, S.C.M., Chang, H.-S., Zhu, T. and Glazebrook, J. (2003) Charac-

terization of the early response of Arabidopsis to Alternaria brassicicola

infection using expression profiling. Plant Physiol. 132, 606–617.

Weigel, D. and Glazebrook, J. (2002) Arabidopsis, a Laboratory Manual. New

York: Cold Spring Harbor.

Williamson, B., Tudzynski, B., Tudzynski, P. and Kan, J.A.L.V. (2007)

Botrytis cinerea: the cause of grey mould disease. Mol. Plant Pathol. 8,

561–580.

Zhao, Y., Hull, A.K., Gupta, N.R., Goss, K.A., Alonso, J., Ecker, J.R., Normanly,

J., Chory, J. and Celenza, J.L. (2002) Trp-dependent auxin biosynthesis in

Arabidopsis: involvement of cytochrome P450s CYP79B2 and CYP79B3.

Genes Dev. 16, 3100–3112.

Zhou, N., Tootle, T.L. and Glazebrook, J. (1999) Arabidopsis PAD3, a gene

required for camalexin biosynthesis, encodes a putative cytochrome P450

monooxygenase. Plant Cell, 11, 2419–2428.

Zimmerli, L., Stein, M., Lipka, V., Schulze-Lefert, P. and Somerville, S. (2004)

Host and non-host pathogens elicit different jasmonate/ethylene responses

in Arabidopsis. Plant J. 40, 633–646.

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