Spore Density Determines Infection Strategy by the …...Spore Density Determines Infection Strategy...

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Spore Density Determines Infection Strategy by the Plant Pathogenic Fungus Plectosphaerella cucumerina 1[OPEN] Pierre Pétriacq, Joost H.M. Stassen, and Jurriaan Ton* Plant Environmental Signaling Group (P.P., J.H.M.S., J.T.) and biOMICS Facility (P.P.), Department of Animal and Plant Sciences, University of Shefeld, S10 2TN Shefeld, United Kingdom ORCID IDs: 0000-0001-8151-7420 (P.P.); 0000-0001-5483-325X (J.H.M.S.); 0000-0002-8512-2802 (J.T.). Necrotrophic and biotrophic pathogens are resisted by different plant defenses. While necrotrophic pathogens are sensitive to jasmonic acid (JA)-dependent resistance, biotrophic pathogens are resisted by salicylic acid (SA)- and reactive oxygen species (ROS)-dependent resistance. Although many pathogens switch from biotrophy to necrotrophy during infection, little is known about the signals triggering this transition. This study is based on the observation that the early colonization pattern and symptom development by the ascomycete pathogen Plectosphaerella cucumerina (P. cucumerina) vary between inoculation methods. Using the Arabidopsis (Arabidopsis thaliana) defense response as a proxy for infection strategy, we examined whether P. cucumerina alternates between hemibiotrophic and necrotrophic lifestyles, depending on initial spore density and distribution on the leaf surface. Untargeted metabolome analysis revealed profound differences in metabolic defense signatures upon different inoculation methods. Quantication of JA and SA, marker gene expression, and cell death conrmed that infection from high spore densities activates JA-dependent defenses with excessive cell death, while infection from low spore densities induces SA-dependent defenses with lower levels of cell death. Phenotyping of Arabidopsis mutants in JA, SA, and ROS signaling conrmed that P. cucumerina is differentially resisted by JA- and SA/ROS-dependent defenses, depending on initial spore density and distribution on the leaf. Furthermore, in situ staining for early callose deposition at the infection sites revealed that necrotrophy by P. cucumerina is associated with elevated host defense. We conclude that P. cucumerina adapts to early-acting plant defenses by switching from a hemibiotrophic to a necrotrophic infection program, thereby gaining an advantage of immunity-related cell death in the host. Plant pathogens are often classied as necrotrophic or biotrophic, depending on their infection strategy (Glazebrook, 2005; Nishimura and Dangl, 2010). Necrotrophic pathogens kill living host cells and use the decayed plant tissue as a substrate to colonize the plant, whereas biotrophic pathogens parasitize living plant cells by employing effector molecules that sup- press the host immune system (Pel and Pieterse, 2013). Despite this binary classication, the majority of path- ogenic microbes employ a hemibiotrophic infection strategy, which is characterized by an initial biotrophic phase followed by a necrotrophic infection strategy at later stages of infection (Perfect and Green, 2001). The pathogenic fungi Magnaporthe grisea, Sclerotinia sclerotiorum, and Mycosphaerella graminicola, the oomycete Phytophthora infestans , and the bacterial pathogen Pseudomonas syringae are examples of hemibiotrophic plant pathogens (Perfect and Green, 2001; Koeck et al., 2011; van Kan et al., 2014; Kabbage et al., 2015). Despite considerable progress in our understanding of plant resistance to necrotrophic and biotrophic pathogens (Glazebrook, 2005; Mengiste, 2012; Lai and Mengiste, 2013), recent debate highlights the dynamic and complex interplay between plant-pathogenic mi- crobes and their hosts, which is raising concerns about the use of infection strategies as a static tool to classify plant pathogens. For instance, the fungal genus Botrytis is often labeled as an archetypal necrotroph, even though there is evidence that it can behave as an en- dophytic fungus with a biotrophic lifestyle (van Kan et al., 2014). The rice blast fungus Magnaporthe oryzae, which is often classied as a hemibiotrophic leaf path- ogen (Perfect and Green, 2001; Koeck et al., 2011), can adopt a purely biotrophic lifestyle when infecting root tissues (Marcel et al., 2010). It remains unclear which signals are responsible for the switch from biotrophy to necrotrophy and whether these signals rely solely on the physiological state of the pathogen, or whether host-derived signals play a role as well (Kabbage et al., 2015). The plant hormones salicylic acid (SA) and jasmonic acid (JA) play a central role in the activation of plant defenses (Glazebrook, 2005; Pieterse et al., 2009, 2012). 1 This work was supported by the European Research Council (grant no. 309944PrimeAPlant to J.T.) and the Leverhulme Trust (grant no. RL2012042 to J.T.). * Address correspondence to j.ton@shefeld.ac.uk. The author responsible for distribution of materials integral to the ndings presented in this article in accordance with the policy de- scribed in the Instructions for Authors (www.plantphysiol.org) is: Jurriaan Ton (j.ton@shefeld.ac.uk). J.T. and P.P. conceived the original research plans; J.T. supervised the experiments; P.P. performed most of the experiments; J.T. and J.H.M.S. provided technical assistance to P.P.; P.P., J.T., and J.H.M.S. designed the experiments and analyzed the data; P.P. and J.T. con- ceived the project and wrote the article with contributions of all the authors; J.T. supervised and complemented the writing. [OPEN] Articles can be viewed without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.15.00551 Plant Physiology Ò , April 2016, Vol. 170, pp. 23252339, www.plantphysiol.org Ó 2016 American Society of Plant Biologists. All Rights Reserved. 2325 www.plantphysiol.org on June 20, 2020 - Published by Downloaded from Copyright © 2016 American Society of Plant Biologists. All rights reserved.

Transcript of Spore Density Determines Infection Strategy by the …...Spore Density Determines Infection Strategy...

Spore Density Determines Infection Strategy by the PlantPathogenic Fungus Plectosphaerella cucumerina1[OPEN]

Pierre Pétriacq, Joost H.M. Stassen, and Jurriaan Ton*

Plant Environmental Signaling Group (P.P., J.H.M.S., J.T.) and biOMICS Facility (P.P.), Department of Animaland Plant Sciences, University of Sheffield, S10 2TN Sheffield, United Kingdom

ORCID IDs: 0000-0001-8151-7420 (P.P.); 0000-0001-5483-325X (J.H.M.S.); 0000-0002-8512-2802 (J.T.).

Necrotrophic and biotrophic pathogens are resisted by different plant defenses. While necrotrophic pathogens are sensitive to jasmonicacid (JA)-dependent resistance, biotrophic pathogens are resisted by salicylic acid (SA)- and reactive oxygen species (ROS)-dependentresistance. Although many pathogens switch from biotrophy to necrotrophy during infection, little is known about the signalstriggering this transition. This study is based on the observation that the early colonization pattern and symptom development bythe ascomycete pathogen Plectosphaerella cucumerina (P. cucumerina) vary between inoculation methods. Using the Arabidopsis(Arabidopsis thaliana) defense response as a proxy for infection strategy, we examined whether P. cucumerina alternates betweenhemibiotrophic and necrotrophic lifestyles, depending on initial spore density and distribution on the leaf surface. Untargetedmetabolome analysis revealed profound differences in metabolic defense signatures upon different inoculation methods.Quantification of JA and SA, marker gene expression, and cell death confirmed that infection from high spore densities activatesJA-dependent defenses with excessive cell death, while infection from low spore densities induces SA-dependent defenses with lowerlevels of cell death. Phenotyping of Arabidopsis mutants in JA, SA, and ROS signaling confirmed that P. cucumerina is differentiallyresisted by JA- and SA/ROS-dependent defenses, depending on initial spore density and distribution on the leaf. Furthermore, in situstaining for early callose deposition at the infection sites revealed that necrotrophy by P. cucumerina is associated with elevated hostdefense. We conclude that P. cucumerina adapts to early-acting plant defenses by switching from a hemibiotrophic to a necrotrophicinfection program, thereby gaining an advantage of immunity-related cell death in the host.

Plant pathogens are often classified as necrotrophicor biotrophic, depending on their infection strategy(Glazebrook, 2005; Nishimura and Dangl, 2010).Necrotrophic pathogens kill living host cells and usethe decayed plant tissue as a substrate to colonize theplant, whereas biotrophic pathogens parasitize livingplant cells by employing effector molecules that sup-press the host immune system (Pel and Pieterse, 2013).Despite this binary classification, the majority of path-ogenic microbes employ a hemibiotrophic infectionstrategy, which is characterized by an initial biotrophicphase followed by a necrotrophic infection strategyat later stages of infection (Perfect and Green, 2001).

The pathogenic fungi Magnaporthe grisea, Sclerotiniasclerotiorum, andMycosphaerella graminicola, the oomycetePhytophthora infestans, and the bacterial pathogenPseudomonas syringae are examples of hemibiotrophicplant pathogens (Perfect and Green, 2001; Koeck et al.,2011; van Kan et al., 2014; Kabbage et al., 2015).

Despite considerable progress in our understandingof plant resistance to necrotrophic and biotrophicpathogens (Glazebrook, 2005; Mengiste, 2012; Lai andMengiste, 2013), recent debate highlights the dynamicand complex interplay between plant-pathogenic mi-crobes and their hosts, which is raising concerns aboutthe use of infection strategies as a static tool to classifyplant pathogens. For instance, the fungal genus Botrytisis often labeled as an archetypal necrotroph, eventhough there is evidence that it can behave as an en-dophytic fungus with a biotrophic lifestyle (van Kanet al., 2014). The rice blast fungus Magnaporthe oryzae,which is often classified as a hemibiotrophic leaf path-ogen (Perfect and Green, 2001; Koeck et al., 2011), canadopt a purely biotrophic lifestyle when infecting roottissues (Marcel et al., 2010). It remains unclear whichsignals are responsible for the switch from biotrophy tonecrotrophy and whether these signals rely solely onthe physiological state of the pathogen, or whetherhost-derived signals play a role as well (Kabbage et al.,2015).

The plant hormones salicylic acid (SA) and jasmonicacid (JA) play a central role in the activation of plantdefenses (Glazebrook, 2005; Pieterse et al., 2009, 2012).

1 This work was supported by the European Research Council(grant no. 309944–Prime–A–Plant to J.T.) and the Leverhulme Trust(grant no. RL–2012–042 to J.T.).

* Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Jurriaan Ton ([email protected]).

J.T. and P.P. conceived the original research plans; J.T. supervisedthe experiments; P.P. performed most of the experiments; J.T. andJ.H.M.S. provided technical assistance to P.P.; P.P., J.T., and J.H.M.S.designed the experiments and analyzed the data; P.P. and J.T. con-ceived the project and wrote the article with contributions of all theauthors; J.T. supervised and complemented the writing.

[OPEN] Articles can be viewed without a subscription.www.plantphysiol.org/cgi/doi/10.1104/pp.15.00551

Plant Physiology�, April 2016, Vol. 170, pp. 2325–2339, www.plantphysiol.org � 2016 American Society of Plant Biologists. All Rights Reserved. 2325 www.plantphysiol.orgon June 20, 2020 - Published by Downloaded from

Copyright © 2016 American Society of Plant Biologists. All rights reserved.

The first evidence that biotrophic and necrotrophicpathogens are resisted by different immune responsescame fromThomma et al. (1998), who demonstrated thatArabidopsis (Arabidopsis thaliana) genotypes impaired inSA signaling show enhanced susceptibility to the bio-trophic pathogen Hyaloperonospora arabidopsidis (formerlyknown as Peronospora parastitica), while JA-insensitivegenotypes were more susceptible to the necrotrophicfungus Alternaria brassicicola. In subsequent years, thedifferential effectiveness of SA- and JA-dependentdefense mechanisms has been confirmed in differentplant-pathogen interactions, while additional planthormones, such as ethylene, abscisic acid (ABA),auxins, and cytokinins, have emerged as regulatorsof SA- and JA-dependent defenses (Bari and Jones,2009; Cao et al., 2011; Pieterse et al., 2012). Moreover,SA- and JA-dependent defense pathways havebeen shown to act antagonistically on each other,which allows plants to prioritize an appropriatedefense response to attack by biotrophic pathogens,necrotrophic pathogens, or herbivores (Koornneefand Pieterse, 2008; Pieterse et al., 2009; Verhageet al., 2010).

In addition to plant hormones, reactive oxygen species(ROS) play an important regulatory role in plant de-fenses (Torres et al., 2006; Lehmann et al., 2015). Withinminutes after the perception of pathogen-associatedmolecular patterns, NADPH oxidases and apoplasticperoxidases generate early ROS bursts (Torres et al.,2002; Daudi et al., 2012; O’Brien et al., 2012), which ac-tivate downstreamdefense signaling cascades (Apel andHirt, 2004; Torres et al., 2006; Miller et al., 2009; Mittleret al., 2011; Lehmann et al., 2015). ROS play an importantregulatory role in the deposition of callose (Luna et al.,2011; Pastor et al., 2013) and can also stimulate SA-dependent defenses (Chaouch et al., 2010; Yun andChen, 2011; Wang et al., 2014; Mammarella et al., 2015).However, the spread of SA-induced apoptosis duringhyperstimulation of the plant immune system is con-tained by the ROS-generating NADPH oxidase RBOHD(Torres et al., 2005), presumably to allow for the suffi-cient generation of SA-dependent defense signals fromliving cells that are adjacent to apoptotic cells. Nitricoxide (NO) plays an additional role in the regulationof SA/ROS-dependent defense (Trapet et al., 2015).This gaseous molecule can stimulate ROS produc-tion and cell death in the absence of SA while pre-venting excessive ROS production at high cellularSA levels via S-nitrosylation of RBOHD (Yun et al.,2011). Recently, it was shown that pathogen-inducedaccumulation of NO and ROS promotes the produc-tion of azelaic acid, a lipid derivative that primesdistal plants for SA-dependent defenses (Wang et al.,2014). Hence, NO, ROS, and SA are intertwined ina complex regulatory network to mount local andsystemic resistance against biotrophic pathogens.Interestingly, pathogens with a necrotrophic life-style can benefit from ROS/SA-dependent defensesand associated cell death (Govrin and Levine, 2000).For instance, Kabbage et al. (2013) demonstrated that

S. sclerotiorum utilizes oxalic acid to repress oxidativedefense signaling during initial biotrophic colonization,but it stimulates apoptosis at later stages to advancenecrotrophic colonization. Moreover, SA-induced re-pression of JA-dependent resistance not only benefitsnecrotrophic pathogens but also hemibiotrophic patho-gens after having switched from biotrophy to necrotro-phy (Glazebrook, 2005; Pieterse et al., 2009, 2012).

Plectosphaerella cucumerina ((P. cucumerina, anamorphPlectosporum tabacinum) anamorph Plectosporum tabacinum)is a filamentous ascomycete fungus that can survive sap-rophytically in soil by decomposing plant material (Palmet al., 1995). The fungus can cause suddendeath and blightdisease in a variety of crops (Chen et al., 1999; Harringtonet al., 2000). Because P. cucumerina can infect Arabidopsisleaves, the P. cucumerina-Arabidopsis interaction hasemerged as a popular model system in which to studyplant defense reactions to necrotrophic fungi (Berrocal-Lobo et al., 2002; Ton and Mauch-Mani, 2004; Carlucciet al., 2012; Ramos et al., 2013). Various studies haveshown thatArabidopsis deploys awide range of inducibledefense strategies against P. cucumerina, including JA-,SA-, ABA-, and auxin-dependent defenses, glucosinolates(Tierens et al., 2001; Sánchez-Vallet et al., 2010; Gamiret al., 2014; Pastor et al., 2014), callose deposition (García-Andrade et al., 2011; Gamir et al., 2012, 2014; Sánchez-Vallet et al., 2012), and ROS (Tierens et al., 2002;Sánchez-Vallet et al., 2010; Barna et al., 2012; Gamir et al.,2012, 2014; Pastor et al., 2014). Recent metabolomicsstudies have revealed large-scale metabolic changes inP. cucumerina-infected Arabidopsis, presumably tomobilize chemical defenses (Sánchez-Vallet et al.,2010; Gamir et al., 2014; Pastor et al., 2014). Furthermore,various chemical agents have been reported to induceresistance against P. cucumerina. These chemicals includeb-amino-butyric acid, which primes callose depositionand SA-dependent defenses, benzothiadiazole (BTH orBion; Görlach et al., 1996; Ton and Mauch-Mani, 2004),which activates SA-related defenses (Lawton et al., 1996;Ton and Mauch-Mani, 2004; Gamir et al., 2014; Lunaet al., 2014), JA (Ton and Mauch-Mani, 2004), and ABA,which primes ROS and callose deposition (Ton andMauch-Mani, 2004; Pastor et al., 2013). However, amongall these studies, there is increasing controversy aboutthe exact signaling pathways and defense responsescontributing to plant resistance against P. cucumerina.While it is clear that JA and ethylene contribute to basalresistance against the fungus, the exact roles of SA, ABA,and ROS in P. cucumerina resistance vary betweenstudies (Thomma et al., 1998; Ton and Mauch-Mani,2004; Sánchez-Vallet et al., 2012; Gamir et al., 2014).

This study is based on the observation that the dis-ease phenotype during P. cucumerina infection differsaccording to the inoculation method used. We provideevidence that the fungus follows a hemibiotrophic in-fection strategy when infecting from relatively lowspore densities on the leaf surface. By contrast, whenchallenged by localized host defense to relatively highspore densities, the fungus switches to a necrotrophicinfection program. Our study has uncovered a novel

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strategy by which plant-pathogenic fungi can take ad-vantage of the early immune response in the host plant.

RESULTS

Different Disease Phenotypes by P. cucumerina afterDroplet and Spray Inoculation

P. cucumerina spore suspensions of similar density (106

spores mL21) were applied as droplets or spray ontoleaves of 5-week-old Arabidopsis, after which diseasesymptoms were monitored over an 11-d period. Al-though similar amounts of inoculum were applied todroplet- and spray-inoculated leaves (Supplemental Fig.S1), disease phenotypes differedmarkedly between bothinoculation methods (Supplemental Fig. S2). Droplet-inoculated leaves started developing localized necrosisunder the sites of inoculum application between 5 and7 d after inoculation, followed by the occurrence of achlorotic halo around the slowly expanding central ne-crosis (Fig. 1A; Supplemental Fig. S2). Before the onset ofmacroscopic symptoms at 4 d after droplet inoculation,

microscopy analysis of Trypan Blue-stained leavesrevealed that fungal colonization coincided with singlecell death, which progressed into widespread cell deathand tissue necrosis by 7 dai (Fig. 1B). This cell death-associated colonization pattern is consistent with anecrotrophic lifestyle of the fungus. When spores wereapplied by spray inoculation, leaves developed smallwater-soaked lesions by 5 dai, which progressed intoextensive chlorosis and necrosis between 7 and 11 dai(Fig. 1B). Interestingly, fungal colonization at 4 d afterspray inoculation extended beyond the water-soakedlesions and was not associated with cell death (Fig.1B). At 7 d after spray inoculation, fungal colonization inspray-inoculated leaves increasingly coincided with celldeath, although still to a lower extent than observed atlocal sites of droplet-inoculated leaves (Fig. 1B). Hence,colonization patterns by droplet- and spray-inoculatedP. cucumerina differ during the earlier stages (4 dai),whereas colonization at later stages (7 dai) coincideswith cell death and necrosis, irrespective of inoculationmethod. Consequently, subsequent experiments to as-sess Arabidopsis resistance against P. cucumerina were

Figure 1. Disease phenotypes after dropletand spray inoculation of 5-week-old Arabi-dopsis with P. cucumerina spores (106 sporesmL21). A, Disease progression in droplet- andspray-inoculated leaves at different days afterinoculation (dai). B, Hyphal colonization andcell death in Trypan Blue-stained leaves at 4and 7 d after droplet and spray inoculation.Red arrows indicate cell death.

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performed at 7 dai and were based on quantifications ofnecrotic lesion diameters and numbers of necrotic leavesafter droplet and spray inoculation, respectively.

Droplet and Spray Inoculation of P. cucumerina ElicitDistinct Plant Metabolic Profiles

To determine global metabolome patterns precedingthe differential disease phenotypes of droplet- andspray-inoculated leaves, we performed broad-spectrummetabolic profiling by matrix-assisted laser-desorptionionization-quadrupole-time of flight (MALDI-Q-TOF)mass spectrometry. This method enables accurate mass-to-charge ratio (m/z) detection from a wide range ofplant metabolites (Ye et al., 2013; Ernst et al., 2014). Inorder to account for early metabolic signaling events,leaves were collected at the early time point of 2 dai.Methanol extracts from four biologically replicated leafsamples were analyzed in negative (MALDI2) andpositive (MALDI+) ionization mode in order to accountfor both anion- and cation-forming metabolites. Ions(m/z) showing statistically significant responses to in-fection treatment (ANOVA; P , 0.01) ranged from 5%in MALDI2 to 8.7% in MALDI+, out of a total of 7,046and 34,171 detected ions, respectively (Fig. 2). PCA ofstatistically significant ions between treatments showeda clear separation between inoculation methods in bothionization modes (Fig. 2). Furthermore, bidirectional

orthogonal partial least square discriminant analysis(O2PLS-DA) prior to statistical filtering yielded similarseparation patterns between treatments (SupplementalFig. S3). Because O2PLS-DA constitutes a supervisedmultivariate analysis that projects predictive variationbetween treatments, we also carried out unsupervisedPCA of unfiltered data to obtain a measure of globalmetabolic impacts of treatments (Supplemental Fig. S4).Although PCA of MALDI+ data only showed separa-tion of samples from spray-inoculated plants, PCA ofMALDI2 data revealed separation between all treatments(Supplemental Fig. S4). In the latter case, pathogen-inoculated samples showed the strongest separationbetween droplet and spray application in compari-son with the corresponding mock-inoculated samples(Supplemental Fig. S4). This indicates that the majormetabolic impacts are caused by differential pathogeneffects between the inoculation methods, rather thanthe different inoculation methods themselves. Subse-quently, we performed a two-way ANOVA (P, 0.01)of the entire data set in order to identify ions dis-playing a statistically significant interaction betweeninoculation method and P. cucumerina, yielding 115and 704 ions in MALDI2 and MALDI+, respectively.This selection of ions represents the fraction of me-tabolites that responds differently to droplet and sprayinoculation with P. cucumerina, which was retained forfurther analysis.

Figure 2. Metabolic profiling by MALDI-Q-TOFanalysis of P. cucumerina-infected Arabidopsis leavesfollowing spray and droplet inoculation. Principalcomponent analysis (PCA) of ions (m/z values) thatshow a statistically significant difference betweentreatments (n = 4; ANOVA, P , 0.01). Five-week-oldleaves were collected at 2 d after mock or P. cucu-merina inoculation (106 spores mL21). Leaf extractswere analyzed byMALDI-Q-TOF in negative (MALDI2)and positive (MALDI+) ionization mode. Pie chartsshow statistically significant (red) and nonsignificant(black) fractions of all detected ions. Each data point inthe PCA plots represents the profile of one biologicallyreplicated sample, which was derived from the averageof six technical replicates. Percentages of variationexplained by each principal component (PC) are shownin parentheses.

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Metabolic Pathway Reconstruction of Droplet- andSpray-Inoculated Leaves

The relatively soft ionization bymatrix-assisted laser-desorption ionization (MALDI) in combination withaccurate mass detection by quadrupole-time of flightallows for the putative identification of metabolites. Tocharacterize the selection of 819 differentially producedions between droplet- and spray-inoculated leaves, m/zvalues were corrected for adducts and carbon isotopesusing MarVis software (Kaever et al., 2012; Gamir et al.,2014; Pastor et al., 2014). Hierarchical cluster analysis ofcorresponding mass predictions confirmed the contrast-ing intensity profiles between droplet and spray inocu-lation (Supplemental Fig. S5). Although the identity ofsingle ions remains uncertain, when two or more puta-tive compounds map to the same metabolic pathway,this represents a plausible case for differential activity ofthis pathway. Therefore, we only selected compoundsfor which there was at least one other compoundmapping to the same plant-specific metabolic path-way. Putative identities were obtained by cross ref-erencing predicted masses against publicly availabledatabases (ChemSpider,METLIN,MassBank, PubChem,KEGG, AraCyc, and MetaCyc; Smith et al., 2005;Williams, 2008;Horai et al., 2010), afterwhich ionswith arelatively high mass accuracy fit (0.05 D) were re-tained for fold change analysis (Table I; SupplementalFig. S6; Supplemental Table S1). The analysis anno-tated three ions to JAmetabolism (JA,m2H: 209.9645;12-oxo-phytodienoic acid [OPDA], m+H: 293.1700;and jasmonyl-isoleucine [JA-Ile], m+H: 324.2425),which all showed statistically significant levels of in-duction after droplet inoculation but not after spray

inoculation (Table I; Supplemental Fig. S6).Hence, dropletinoculation with P. cucumerina boosts JA biosynthesis,which is consistentwith the notion that the fungus followsa necrotrophic infection strategy. Conversely, spray in-oculation induced two ions matching different SA deriv-atives (salicyl alcohol/catechol monomethyl ether,m+H: 125.0156; and benzoic acid/salicyl aldehyde,m2H: 121.0451) and 10 ions corresponding to differentglucosinolate-related metabolites (Table I; SupplementalFig. S6; Supplemental Table S1). Nine of these ions weredetected in MALDI2 mode and identified as (interme-diates of) aliphatic glucosinolates (Botting et al., 2002).The analysis in MALDI+ mode identified one additionalbreakdown product of indole-derived glucosinolates(indole-3-acetonitrile, m+H: 157.1203). The fact that mul-tiple ions could be assigned to either SA or glucosinolatemetabolism indicates that spray-inoculated P. cucumerinaenhances the activity of SA- and glucosinolate-relatedmetabolism. This host defense profile is consistentwith a (hemi)biotrophic infection strategy by the fungus(Glazebrook, 2005; Bednarek et al., 2009).

Differential Induction of SA- and JA-Dependent Defensesin Spray- and Droplet-Inoculated Leaves

To confirm the differential involvement of SA and JAin plant defense to spray- and droplet-inoculated P.cucumerina, we quantified levels of JA and SA at 2 and 5dai by ultra-performance liquid chromatography-quadrupole time of flight-tandem mass spectrometry(UPLC-Q-TOF-MS/MS; Glauser et al., 2014). In agree-ment with ourMALDI-Q-TOF results (Table I), droplet-inoculated P. cucumerina enhanced endogenous JA

Table I. Pathway reconstruction of m/z values that respond differently to droplet and spray inoculation with P. cucumerina

Accurate m/z

Valuea Adduct Predicted Massb Putative Compound PathwaycFold Change,

DropletdFold Change,

Sprayd VIP Rankinge

450.0536 m2H 451.0641 5-Methylsulfinylpentyl glucosinolate GS 0.10 7.89 36462.0879 m2H 463.0952 7-Methylthioheptyl glucosinolate GS NI 25.61 61428.1081 m2H 427.1087 7-Octenyl glucosinolate GS 0.10 6.27 135476.1009 m2H 477.1161 8-Methylthiooctyl glucosinolate GS NI 489.39 16434.0212 m2H 435.0285 Glucoberteroin GS 0.08 400.96 136420.0445 m2H 421.0518 Glucoerucin GS 0.07 491.81 9492.0947 m2H 493.1020 Glucohirsutin GS NI 14.87 10436.0375 m2H 437.0448 Glucoraphanin GS 0.11 7.19 2388.0424 m2H 389.0497 Progoitrin GS 0.02 447.61 1,028157.1203 m+H 156.1130 3-Indoleacetonitrile GS NI 11.66 905293.1700 m+H 292.1627 12-OPDA JA metabolism 19,640.00 NI 788209.9645 m2H 210.9718 JA JA metabolism 4.23 NI 15324.2425 m+H 323.2352 JA-Ile JA signaling 55.09 NI 303125.0156 m+H 124.0083 Salicyl alcohol/catechol monomethyl ether SA degradation 0.32 2,933.40 4,962121.0451 m2H 122.0524 Benzoic acid/salicyl aldehyde SA metabolism 0.04 435.41 244

aThe m/z values were detected by MALDI-Q-TOF analysis. Values were selected (1) when showing a statistically significant interaction betweeninoculation method and P. cucumerina and (2) if at least one additional mass prediction could be annotated to the same metabolic pathway. bThem/z values were corrected for adducts and/or for carbon isotopes (m/z tolerance = 0.05 D) using MarVis software. cMass predictions were crossreferenced against publicly available databases (ChemSpider, METLIN, MassBank, PubChem, KEGG, AraCyc, and MetaCyc) and selected at a massaccuracy fit of 0.05 D. GS, Glucosinolates. dFold change values of m/z values were determined using MetaboAnalyst software. NI, No statis-tically significant induction or repression by P. cucumerina. eVIP, Variable importance for projection ranking, derived from the loading plot of theO2PLS-DA analysis (Supplemental Fig. S3).

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levels, whereas spray-inoculated P. cucumerina en-hanced SA levels (Fig. 3A). Although not statisticallysignificant (P = 0.063, Student’s t test), spray inoculationweakly stimulated JA production at 5 dai, suggestingthat the pathogen is switching to a necrotrophic infec-tion program at this stage of infection. To test whetherthe differential production of JA and SA translates todownstream defense gene expression, we quantifiedinduction levels of JA- and SA-inducible marker genesat 2 and 5 dai, using reverse transcription-quantitativePCR analysis. At 2 dai, spray-inoculated P. cucumerinasignificantly induced the transcription of the SA-dependent genes PR1, PR5,WRKY70 (Li et al., 2004), andICS1 (Wildermuth et al., 2001; Fig. 3B; SupplementalFig. S7). Conversely, droplet-inoculated P. cucumerinainduced PR1 expression to significantly lower levelsand failed to induce PR5, WRKY70, and ICS1 (Fig. 3B;Supplemental Fig. S7). At 5 dai, droplet-inoculated P.cucumerina induced the expression of the JA-dependentmarker gene VSP2, whereas spray inoculation failed toinduce this gene at either time point (Fig. 3B). Re-markably, the relatively strong induction of PR1 andWRKY70 at 2 d after spray inoculation was severelyreduced by 5 dai (Fig. 3B), despite the fact that SA levelshad increased further (Fig. 3A). This incongruity be-tween SA accumulation and SA response suggestssuppression of the SA response by spray-inoculatedP. cucumerina.

The Effectiveness of SA-Induced Defense againstP. cucumerina Depends on Inoculation Method

Treatment of Arabidopsis with JA enhances basalresistance against necrotrophic pathogens, whereasapplication of SA, or its functional analog BTH (Görlachet al., 1996), induces resistance against biotrophicpathogens (Ton et al., 2002). To test whether the ob-served differences in SA and JA elicitation after sprayand droplet inoculation with P. cucumerina (Fig. 3) re-late to the differential effectiveness of the correspond-ing defense responses, 5-week-old plants were soildrenched with water, 100 mM JA, or 300 mM BTH andinoculated 2 d later (Fig. 4). Pretreatment with JAinduced a statistically significant reduction of lesiondiameter at 7 d after droplet inoculation, which isconsistent with the notion that P. cucumerina follows anecrotrophic lifestyle. Conversely, pretreatment withBTH failed to reduce lesion diameter (Fig. 4A). Both JAand BTH were effective in reducing the number ofdiseased leaves following spray inoculation withP. cucumerina (Fig. 4B). This indicates that JA- and

Figure 3. Production and activity of SA and JA in droplet- and spray-inoculated leaves. A, UPLC-Q-TOF-MS/MS quantification of SA and JAin leaves from 5-week-old plants at 2 and 5 dai. Shown are averagevalues 6 SD in ng g21 dry weight (DW; n = 4). Asterisks indicate statis-tically significant differences relative to corresponding mock treatments

(Student’s t test, a = 0.05). B, Reverse transcription-quantitative PCRanalysis of JA-dependent transcription of VSP2 and SA-dependent tran-scription of PR1 and WRKY70 in P. cucumerina-inoculated leaves at2 and 5 dai. Data represent average fold induction values 6 SD (n = 4)relative to transcription levels in mock-treated leaves. Asterisks indicatestatistically significant differences in gene induction between droplet-and spray-inoculated leaves (Student’s t test, a = 0.05).

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SA-inducible defenses are effective against spray-inoculated P. cucumerina, which is consistent with ahemibiotrophic lifestyle by the fungus.

The Intensity of Callose Deposition and Cell DeathDepends on P. cucumerina Spore Density

To further characterize the differential immune re-sponse to droplet- and spray-inoculated P. cucumerina,we quantified callose deposition at 3 dai using AnilineBlue staining. Droplet-inoculated leaves showed dra-matically higher levels of callose deposition than spray-inoculated leaves, in which levels of callose were justmarginally higher than in mock-inoculated leaves (Fig.5A). Furthermore, the relatively high callose depositionat the sites of droplet inoculation were spreading to thesurrounding leaf tissues, suggesting spatially activedefense signaling (Fig. 5A). To link the differential cal-lose response to numbers of germinating fungal sporeson the leaf surface, leaves were double stained withAniline Blue and Calcofluor at 3 dai. Despite using

similar inoculum densities of 106 spores mL21, dropletinoculation resulted in a 15-fold higher spore density onthe leaf surface compared with spray inoculation (Fig.5B; Supplemental Fig. S8). Hence, localized applicationof relatively high spore densities after droplet inocula-tion elicits high levels of callose deposition, whereasapplication of evenly distributed spores at lower den-sity after spray inoculation elicits relatively low levelsof callose elicitation. Consistent with the outcome ofPR1 and WRKY70 gene expression profiling (Fig. 3B),these results suggest that spray-inoculated P. cucumerinasuppresses plant immune responses, which is charac-teristic of biotrophic infections (Glazebrook, 2005; Douand Zhou, 2012).

While cell death can contribute to resistance againstbiotrophic pathogens (Glazebrook, 2005), it can facili-tate infection by necrotrophic pathogens (Govrin andLevine, 2000; Kabbage et al., 2013). Supported by ourobservation that early colonization of droplet-inoculatedP. cucumerina coincides with microscopic cell death (Fig.1B),wehypothesized that the exaggerated early immuneresponse to high spore densities (Fig. 5A) leads to in-creased cell death that facilitates necrotrophic infection.Therefore, we monitored levels of cell death inductionfrom 3 to 7 dai by quantifying electrolyte leakage.In comparison with spray-inoculated leaves, droplet-inoculated leaves showed consistently higher levels ofcell death induction (Fig. 5C), which is consistent witha necrotrophic lifestyle. To confirm that these differ-ences were determined by the density of germinatingspores on the leaf surface, rather than the inoculationmethod itself, we increased the density of the sprayinoculum from 106 to 108 spores mL21. This 100-foldincrease resulted in a 48-fold increase in the density ofgerminating spores at 3 dai (Fig. 5B), suggesting thata larger proportion of spores from the high-densityspray inoculum had failed to anneal and had beenwashed off during the staining procedure. Never-theless, this 48-fold increase in spore density on theleaf surface was sufficient to trigger a similarly rapidcell death response to leaves that had been dropletinoculated with 106 spores mL21 (Fig. 5C). Together,these results indicate that the exaggerated immuneresponse to high densities of droplet-inoculatedP. cucumerina spores results in enhanced cell death,which in turn facilitates a necrotrophic lifestyle bythe fungus (Govrin and Levine, 2000; Kabbage et al.,2013).

Spore Density Determines the Contrasting Effectiveness ofJA- and SA-Dependent Defenses after Droplet andSpray Inoculation

To further examine the relationship between sporedensity and the effectiveness of JA, SA, and ROS-dependent defenses, we profiled basal resistance levelsin the JA biosynthesis mutant 12-oxophytodienoate reduc-tase3 (opr3; Schaller et al., 2000; Stintzi and Browse, 2000;Chehab et al., 2011), the JA-insensitive mutant jar1-1(Staswick et al., 1992, 2002), the SA biosynthesis mutant

Figure 4. Quantification of BTH- and JA-induced resistance in wild-type Arabidopsis (Columbia-0 [Col-0]) against P. cucumerina followingspray (A) or droplet (B) inoculation. Five-week-old plants were soildrenched with water (Control), BTH (300 mM), or JA (100 mM). Two daysafter chemical treatments, plants were inoculated with P. cucumerina(106 spores mL21). Symptoms were quantified at 7 dai by lesion diam-eter (A) or percentage of diseased leaves (B). Values shown representmeans6 SD (n = 6–12). Different letters represent statistically significantdifferences between conditions (Student’s t test, a = 0.05).

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sid2-1 (Wildermuth et al., 2001), and the rbohD mutant,which is affected in pathogen-associated molecularpattern-induced ROS production (Torres et al., 2002). At7 d after droplet inoculation with P. cucumerina, the opr3and jar1-1 mutants had developed significantly largerlesion diameters than wild-type Col-0 plants, whereassid2-1 and rbohD displayed smaller lesion diameterscompared with Col-0 plants (Fig. 6A). This profile of mu-tant phenotypes indicates effectiveness of JA-dependentdefenses, which is consistent with a necrotrophic infec-tion strategy. Conversely, when P. cucumerina was ap-plied by spray inoculation, opr3 and jar1-1 developedwild-type levels of disease, whereas sid2-1 and rbohDshowed statistically higher levels of disease in compari-son with Col-0 plants (Fig. 6B). This mutant profile in-dicates effectiveness of SA- and ROS-dependent basaldefenses, which is consistent with a biotrophic infectionstrategy (Torres et al., 2002; Glazebrook, 2005). To de-termine whether the biotrophic lifestyle of P. cucumerinais suppressed beyond a certain threshold of spores perleaf area, we increased the density of the spray inocula-tion from 106 to 108 spores mL21. Strikingly, this changein spore density resulted in a necrotrophic profile ofmutant disease phenotypes: the opr3 and jar1-1 mutantsshowed enhanced disease susceptibility in comparisonwith Col-0, while sid2-1 and rbohD showed similar orreduced levels of susceptibility in comparisonwith Col-0,respectively (Fig. 6C). To verify the role of SA signalingand to examine the role of glucosinolate-dependentdefense in spray-inoculated P. cucumerina, we testedtwo additional Arabidopsis mutants: the pad4-1 mutant,which is impaired in SA-dependent signaling (Glazebrooket al., 1997; Jirage et al., 1999), and the pen2-1 mutant,which is affected in the hydrolysis of indole glucosi-nolates (Bednarek et al., 2009; Clay et al., 2009). Com-pared with the Col-0 wild type, both mutants showedincreased resistance to P. cucumerina after droplet inoc-ulation (Supplemental Fig. S9A), but increased sus-ceptibility after spray inoculation (SupplementalFig. S9B). Increasing the spray inoculum densityfrom 106 to 108 spores mL21 reverted the enhanced dis-ease susceptibility towild-type levels (Supplemental Fig.S9C). In the context of the experimental results presentedabove, these results not only confirm the spore density-dependent role of SA-dependent defense against thefungus, but they also indicate that PEN2-dependentglucosinolate defense only contributes to basal resis-tance against biotrophic P. cucumerina.

DISCUSSION

This study is based on the observation thatP. cucumerinacauses markedly different disease phenotypes in Arabi-dopsis after droplet and spray inoculation of the leaves

Figure 5. Relationship between callose deposition, cell death, andP. cucumerina spore density on the leaf surface. A, Relative callosequantities in droplet- and spray-inoculated leaves of 5-week-old plants.Leaveswere stained by Calcofluor/Aniline Blue at 3 dai (106 spores mL21)and analyzed by UV epifluorescence microscopy. Callose was quanti-fied from digital microscopy photographs. Shown are mean areas ofcallose per leaf relative to total leaf area 6 SD (n = 24). Callose afterdroplet inoculation with P. cucumerina spores was quantified within(droplet-local) and outside (droplet-distal) the spore-containing area onthe leaf surface. B, Spore density of germinating P. cucumerina at 3 dai.Shown are average numbers of P. cucumerina spores mm22 leaf area6 SD

(n = 24). Different letters indicate statistically significant differences(multiple Student’s t test, a = 0.05). C, Time course of cell death oc-currence in mock- and P. cucumerina-inoculated leaves. Cell deathwas quantified by relative levels of electrolyte leakage from water-suspended leaves. Shown are average levels of conductivity6 SD (n = 4)

relative to the maximum level of conductivity after subsequent tissuelysis (set at 100%). Different letters indicate statistically significant dif-ferences at each time point between P. cucumerina-inoculated samples(multiple Student’s t test, a = 0.05).

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(Fig. 1). These differences in virulence and disease phe-notype prompted us to investigatewhether P. cucumerinais capable of employing different infection strategies.Since Arabidopsis responds with different defensemechanisms to necrotrophic and biotrophic pathogens(Glazebrook, 2005; Pieterse et al., 2012),we characterizedthe nature and effectiveness of the Arabidopsis defenseresponse to droplet- and spray-inoculated P. cucumerina.

Our results have shown that droplet- and spray-inoculated P. cucumerina provoke profoundly differentdefense responses between 2 and 7 dai (Table I; Figs. 2, 3,and 5; Supplemental Table S1; Supplemental Fig. S7),which were not associated with noticeable differences infungal colonization (Fig. 1B; Supplemental Fig. S8). Todate, previous studies of the Arabidopsis-P. cucumerinainteraction employed different inoculation methods,which might explain the controversy about the contri-bution of SA- and ABA-dependent defense againstP. cucumerina (Ton and Mauch-Mani, 2004; Sánchez-Vallet et al., 2012; Gamir et al., 2014). Therefore, we urgeextra caution when using the Arabidopsis-P. cucumerinapathosystem as a model for studying interactions be-tween plants and necrotrophic fungi.

Untargeted metabolic profiling by MALDI-Q-TOF(Table I; Supplemental Fig. S6; Supplemental Table S1)followed by targeted hormone quantification usingUPLC-Q-TOF-MS/MS (Fig. 3A) revealed that dropletinoculation of P. cucumerina spores enhances JA bio-synthesis. This defense hormone is synthesized fromthe 13-lipoxygenase pathway, which involves the pro-duction of OPDA that is converted into JA by a series ofoxidation steps (Howe and Schilmiller, 2002). JA itselfrequires conjugation to Ile before it can activate thedownstream response pathway via COI1-dependentinactivation of transcriptional JAZ activators that re-press JA response genes (Chini et al., 2007; Thines et al.,2007). The fact that our MALDI-Q-TOF profiling ofdroplet-inoculated leaves identified three different ionsannotating to OPDA, JA and JA-Ile and that targetedUPLC-Q-TOF-MS/MS analysis confirmed increased JAlevels at different time points after droplet inoculation(Fig. 3A), provides strong evidence that JA biosynthesisis induced by droplet-inoculated P. cucumerina. Thisconclusion is further supported by our finding thatdroplet inoculation induces the transcription of theJA-dependent VSP2 (Fig. 3B). The JA response isactivated by damage-associated molecular patterns(Ballaré, 2011), which are typically formed during celllysis by mycotoxins from necrotrophic fungi. To de-termine whether JA-dependent defenses are effectiveagainst droplet-inoculated P. cucumerina, we quantifiedinduced resistance in JA-treated wild-type plants andbasal resistance in JA signaling mutants. While exoge-nous application of JA enhanced resistance in wild-typeplants (Fig. 4A), the opr3 and jar1-1 mutants showedreduced levels of basal resistance to droplet-inoculatedP. cucumerina (Fig. 6A). Thus, droplet inoculation ofP. cucumerina not only elicits JA production and JA-dependent gene induction; we also demonstrated thatthe expression of JA-dependent plant defense is effec-tive against droplet-inoculated P. cucumerina. Since JA-dependent defenses are elicited by and effective againstnecrotrophic pathogens (Ton et al., 2002; Pieterse et al.,2012; Lai andMengiste, 2013), we conclude that droplet-inoculated P. cucumerina behaves like a necrotrophicfungus on Arabidopsis (Fig. 7).

Spray inoculation with P. cucumerina spores re-sulted in a different disease phenotype than droplet

Figure 6. The contributions of SA, JA, and ROS to basal resistanceagainst P. cucumerina depend on inoculation method and spore den-sity. Basal resistance was determined at 7 dai in the wild-type Col-0, theSA induction mutant sid2-1, the JA-deficient mutant opr3, the JA-insensitive mutant jar1-1, and the ROS production mutant rbohD. A,Average lesion diameters6 SD (n = 6–12) in leaves of droplet-inoculatedplants (106 spores mL21). B, Average percentages of diseased leaves 6 SD

(n = 6–12) in spray-inoculated plants (106 spores mL21). C, Averagepercentages of diseased leaves 6 SD (n = 6–12) in spray-inoculatedplants (108 spores mL21). Different letters indicate statistically significantdifferences (multiple Student’s t test, a = 0.05) between conditions.

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inoculation (Fig. 1; Supplemental Fig. S2), even thoughlevels of fungal colonization appeared similar betweenboth inoculation methods (Fig. 1B; Supplemental Fig.S8). The metabolic profile of spray-inoculated leaveswas markedly different from that of droplet-inoculatedleaves (Table I; Fig. 2; Supplemental Figs. S3–S6;Supplemental Table S1). Selection of candidate metab-olites that were specifically induced by spray-inoculatedP. cucumerina identified two SA derivatives and 10 inter-mediates or breakdown products of glucosinolates (TableI; Supplemental Table S1; Supplemental Fig. S6). Fur-thermore, targeted UPLC-Q-TOF-MS/MS analysis con-firmed that spray-inoculated leaves increase endogenousSA levels at 2 and 5 dai (Fig. 3A). Hence, spray-inoculatedP. cucumerina elicits an SA- and glucosinolate-related de-fense response in Arabidopsis. Glucosinolates can con-tribute to defense against (hemi)biotrophic pathogens(Bednarek and Osbourn, 2009; Schlaeppi et al., 2010). In-deed, pen2-1 plants, which are affected in glucosinolate-dependent defense (Bednarek et al., 2009; Clay et al.,2009), only showed enhanced disease susceptibility whenspray inoculated with 106 spores mL21, which was

reverted to wild-type levels of susceptibility whenspray inoculated with a 100-fold higher spore den-sity (Supplemental Fig. S9). The combination ofSA- and glucosinolate-dependent defenses was re-cently reported to act against the (hemi)biotrophicoomycete Phytophthora capsici (Wang et al., 2013). Inaddition, enhanced production of aliphatic glucosino-lates upon spray inoculation of Arabidopsis withP. cucumerina has been reported previously (Kułak andBednarek, 2014), illustrating the reproducibility ofthis response. Moreover, a recent study reported thatendogenous JA levels remain unaltered at 2 d afterspraying 5-week-old Arabidopsis (accession Col-0) withP. cucumerina spores, while levels of SA, glucosinolates,flavonoids, benzoic acid, and 3-indoleacetonitrile wereinduced (Pastor et al., 2014). Clearly, these results fullysupport our analysis of spray-inoculated plants (Table I;Fig. 3A; Supplemental Fig. S7) and challenge the generalassumption that P. cucumerina acts like a necrotrophicpathogen.

Recent technical advances in the analysis ofglucosinolates by MALDI-mass spectrometry imaging

Figure 7. Model of the spore density-dependent infection strategy by P. cucumerina. High spore densities at localized leaf areasafter droplet inoculation (left) trigger a strong pathogen-associated molecular pattern-triggered immunity (PTI) response in thehost plant. In response, P. cucumerina activates a necrotrophic infection program that might be facilitated by the production of amycotoxin(s). Furthermore, by employing a necrotrophic infection strategy, the fungus gains an advantage from immunity-relatedcell death. Under these conditions, the host plant activates JA-dependent defenses to mount resistance against the necrotrophicinfection strategy of the fungus. Conversely, the homogenous distribution of spores at low density after spray inoculation (right)triggers a relatively weak PTI response in the host plant. As a consequence, P. cucumerina is capable of employing a biotrophicinfection program that might depend on immune-suppressing effectors. Under these conditions, the host plant activates SA-dependent basal defenses to mount resistance against the biotrophic infection strategy by the fungus. To gain an advantage fromthe SA-induced suppression of JA-dependent defenses in the host plant under these conditions, the fungus switches to necrotrophyduring the later stages of the infection.

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(Shroff et al., 2015) may provide further perspective onthe spatial-temporal distribution of glucosinolates inP. cucumerina-infected Arabidopsis. We also showed thatspray-inoculated P. cucumerina induces the transcriptionof SA-dependent PR1, PR5, WRKY70, and ICS1 at 2 dai(Fig. 3B; Supplemental Fig. S7), demonstrating that theobserved induction of SA biosynthesis translates into en-hanced gene response activity. Interestingly, however, theinduction of PR1 andWRKY70was no longer apparent at5 d after spray inoculation (Fig. 3B), suggesting that spray-inoculated P. cucumerina represses the initial surge in SAsignaling to advance infection. The suppression of SA-dependent defense signaling is characteristic of infectionby (hemi)biotrophic pathogens such as P. syringae (Choiet al., 2012). Finally, we showed that spray-inoculatedP. cucumerina is resisted by SA-dependent defenses: pre-treatmentwithBTH induced resistance inwild-typeplants(Fig. 5B), while the SA biosynthesis mutant sid2-1 and thesignaling mutant pad4-1 exhibited enhanced susceptibilityto the spray-inoculated fungus (Fig. 6B; Supplemental Fig.S9). Considering that (hemi)biotrophic pathogens aresensitive to SA-dependent plant defenses (Thomma et al.,1998; Ton et al., 2002), these results reinforce the notionthat P. cucumerina employs a biotrophic infection strategyduring the early stages of infection following spray inoc-ulation (Fig. 7).Apart fromdifferences in the elicitation of JA-, SA-, and

glucosinolate-dependent defenses, the characterizationof callose deposition and cell death revealed further dis-parities in the plant response to droplet- and spray-inoculated P. cucumerina. Droplet-inoculated leavesdepositeddramatically higher levels of callose at 3dai thanspray-inoculated leaves (Fig. 5A), which resulted in en-hanced levels of cell death (Figs. 1B and 5C). Since calloseand cell death are genuine markers for PTI (Glazebrook,2005; Luna et al., 2011; Lai and Mengiste, 2013), we con-clude that droplet-inoculated P. cucumerina triggers a rel-atively strong PTI response. Moreover, the transition froma relatively strong callose response to an extensive celldeath response would benefit the necrotrophic infectionstrategy of the fungus. Previous studies about the controlof cell death during infection by S. sclerotiorum havehighlighted the significance of apoptosis for necrotrophiccolonization by this fungus (Kabbage et al., 2013, 2015). Bycontrast, spray-inoculated P. cucumerina triggered rela-tively low levels of callose deposition and cell death (Figs.1B and 5). These results indicate that spray-inoculatedP. cucumerina is able to suppress PTI, which is character-istic of biotrophic pathogens (Glazebrook, 2005; Dou andZhou, 2012).A plausible explanation for the differences in P.

cucumerina behavior after spray and droplet inoculationcomes from our analysis of spore densities on the leafsurface. Although both inocula had equal spore densi-ties, droplet inoculation resulted in 15-fold higher sporedensities on the leaf area than spray inoculation (Fig.5B; Supplemental Fig. S8). We attribute this differ-ence to evaporation of the inoculation droplet, caus-ing the congregation of spores to confined leaf areas(Supplemental Fig. S10). In support of this hypothesis,

lowering the initial spore density in the droplet inocu-lum resulted in smaller areas of similar spore density onthe leaf surface (data not shown). By contrast, sprayinoculation with P. cucumerina spores resulted in anequal distribution of spores over the entire leaf area,resulting in overall lower spore densities per surfaceleaf area (Fig. 5B; Supplemental Fig. S8). Enhancing thespore density in the spray inoculum changed the basalresistance profile of Arabidopsis signaling mutantsfrom a biotrophic signature to a necrotrophic signature(Fig. 6, B and C; Supplemental Fig. S9), indicating ashift from SA/ROS/glucosinolate-dependent resis-tance at 106 spores mL21 to JA-dependent resistanceat 108 spores mL21. Hence, increased spore densitieson the leaf surface favor a necrotrophic infection strat-egy by P. cucumerina. Considering the relatively strongPTI response to droplet inoculation (Fig. 5), we concludethat P. cucumerina changes from a default (hemi)bio-trophic infection strategy to a necrotrophic infectionstrategy when challenged with a strong host immuneresponse to localized areas of high spore density (Fig. 7).This situation can occur in nature when spores are scat-tered in rain drops and concentrated onto the leaf surfaceafter water evaporation. The ability of P. cucumerina toswitch from a hemibiotrophic to a necrotrophic lifestyleenables the fungus to take advantage of cell death fromhyperstimulation of the plant immune system.

Apart from the hyperstimulation of PTI, necrotrophicfungi typically rely on the production of mycotoxins toinduce host necrosis (Christensen and Kolomiets, 2011;Woloshuk and Shim, 2013; Ameye et al., 2015). Futureresearch is required to verify whether the necrotrophicbehavior of droplet-inoculated P. cucumerina depends onmycotoxin production and whether such mycotoxinproduction is stimulated by early immune responses ofthe host plant. Recently, Dobón et al. (2015) identifiedseveral P. cucumerina- and Botrytis cinerea-inducibletranscription factors that contribute to disease suscepti-bility, indicating that P. cucumerina is indeed capable ofthe suppression of host immune responses.Whether suchprovirulence factors are induced by P. cucumerina effec-tors during biotrophic colonization to suppress SA-,ROS-, and glucosinolate-dependent defenses awaits fur-ther research. The outcome of our study has shown thatthe Arabidopsis-P. cucumerina pathosystem is moremultifaceted and versatile than a mere necrotrophicplant-pathogen interaction. At the same time, our studyhas opened new opportunities to deepen our under-standing of the signals and molecular mechanisms me-diating the fungal switch from biotrophy to necrotrophy.Understanding these processes will allow for the devel-opment of new strategies to control fungal diseases,which continue to threaten food security and biodiversityin the 21st century (Fisher et al., 2012).

CONCLUSION

Our study has demonstrated that the plant-pathogenic fungus P. cucumerina can alter its infectionstrategy depending on the initial spore density on the

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leaf surface and the corresponding plant immune re-sponse. The fungus can switch from a hemibiotrophicto a necrotrophic lifestyle in response to a strong hostimmune response during the onset of infection. Thisadaptation enables the fungus to take full advantage ofdefense-related cell death (Fig. 7). Considering that theArabidopsis-P. cucumerina interaction has emerged as apopular model system for studying plant interactionswith necrotrophic fungi (Sánchez-Vallet et al., 2010,2012; Gamir et al., 2012, 2014; Ramos et al., 2013; Pastoret al., 2014), our study urges extra caution when inter-preting experimental results from this pathosystem.

MATERIALS AND METHODS

Chemicals and Reagents

All chemicals used in this studywere purchased from Sigma-Aldrich, exceptJA and JA-Ile, which were obtained from OlChemim (http://www.olchemim.cz/), and BTH containing 50% benzo(1,2,3)thiadiazole-7-carbothioic acid-S-methyl ester, from Syngenta (http://www3.syngenta.com).

Statistical Analyses

Univariate analyses for the statistical significance of differences in diseaseresistance, callose deposition, spore density, hormone quantifications, geneexpression, and conductivity were performed in Excel using multiple com-parisons of Student’s t tests (a = 0.05). MeV software (version 4.9; Raychaudhuriet al., 2000; http://www.tm4.org/mev.html) was used for one- and two-wayANOVA (P, 0.01) of MALDI-Q-TOF data. A Student’s t test was also appliedto selected putative markers from MALDI-Q-TOF analysis to compare withtwo-way ANOVA P values (Supplemental Table S1). Details for multivariateanalyses (PCA and O2PLS-DA) of MALDI-Q-TOF data are described below.

Arabidopsis Genotypes and Growth Conditions

Experiments were performed with Arabidopsis (Arabidopsis thaliana) wild-type accession Col-0 and Col-0 mutant lines sid2-1, opr3, jar1-1, and rbohD(Stintzi and Browse, 2000; Wildermuth et al., 2001; Staswick et al., 2002; Torreset al., 2002). After 2 d of stratification (dark, 4°C, and 100% relative humidity[RH]), seeds were planted in a 1:1 soil:sandmixture and kept at 100% RH undera light/dark photoperiod of 8.5/15.5 h at 130 mmol m22 s21 and a day/nighttemperature of 20°C/18°C. Two weeks after germination, seedlings weretransferred to individual 60-mL pots containing jiffy7 peat pellets (http://www.gardensupplydirect.co.uk; Ramírez et al., 2009) and cultivated at 70%RHin the same climate chamber. All experiments were performed with 5- to6-week-old plants that were watered twice per week.

Plectosphaerella cucumerina Resistance Assays

P. cucumerina was grown on potato dextrose agar (PDA) as described (Tonand Mauch-Mani, 2004). Spores were collected and suspended in water.Droplet inoculation was performed by applying 6-mL droplets of spore sus-pension at the indicated concentrations (106 or 108 spores mL21) to six to eightfully expanded leaves. The resistance of droplet-inoculated plants was quan-tified by average lesion diameters from 12 to 20 plants per treatment as de-scribed (Ton and Mauch-Mani, 2004). If no lesion could be detected on adroplet-inoculated leaf, it was given a score of zero. Spray inoculation of en-tire plants (12–20 plants per treatment) was performed at the indicated sporedensities (106 or 108 spores mL21) using a hand-held 30-mL bottle sprayingdevice (Mestall). To ensure homogenous spore dispersion on the leaves, sprayinoculum was supplemented with 0.01% (v/v) Silwet. The resistance of spray-inoculated plants was determined by the percentage of leaves with visibledisease symptoms as described (Llorente et al., 2005; Sánchez-Vallet et al.,2010). Following inoculation, plants were kept in closed trays at 100% RH.Unless stated otherwise, disease was scored at 7 dai. Each experiment to de-termine basal resistance was performed at least three times with similar results.To assess induced resistance, 5-week-old plants (Col-0) were soil drenchedwith

demineralized water, Bion (300 mM BTH), or JA (100 mM) as described (Ton andMauch-Mani, 2004) and inoculated with P. cucumerina 2 d later. Induced re-sistance assays were repeated twice with similar results.

Sampling of Plant Material

All samples were collected from plants that had been grown in the sameclimate chamber and that had been inoculated when 5 weeks old. Samples formetabolic and transcriptomic analyses consisted of fully expanded leaves ofsimilar developmental stage that were taken at 2 and 5 dai. Each biologicallyreplicated sample consisted of four leaves that had been collected from differentplants (n = 4). To quantify cell death by leaf conductivity measurements, leafdiscs were taken at corresponding time points of infection from the inoculationsites showing macroscopic disease symptoms (middle top area on the half leaf).Each biologically replicated measurement was based on six leaf discs fromleaves of different plants (n = 4). For all experiments, leaf sampleswere collectedat 3 h after the start of the photoperiod to exclude bias from circadian clock- orlight-dependent responses.

MALDI-Q-TOF Mass Spectrometry

Metabolites were obtained by methanol extraction (95% with 0.1% formicacid, v/v) as described (Pétriacq et al., 2012; Luna et al., 2014). High-resolutionfull-scan mass spectrometry was performed with a Synapt G2 HDMS Q-TOFmass spectrometer (Waters) interfaced with a MALDI ionization head (Lunaet al., 2014). To increase the numbers of ionized analytes, two different matriceswere used, depending on the polarity of the ionization. Samples were mixedwith matrix solution at 50:50 (v/v). For positive ionization mode, the matrixconsisted of 5 mg mL21 a-cyano-4-hydroxycinnamic acid (Sigma-Aldrich) in a99.9:0.1 (v/v) mixture of methanol and trifluoroacetic acid, and ionswere collectedat the following mass spectrometry settings: sample plate 2 V, extraction grid 3 V,hexapole 1 V, and aperture 5 V. For negative ionizationmode, thematrix consistedof 10 mg mL21 9-aminoacridine (Sigma-Aldrich) in methanol, and ions were col-lected at the following mass spectrometry parameters: sample plate 0 V, extraction12 V, hexapole 9 V, and aperture 3 V. For each sample, six technical replicates werespotted on a 96-well MALDI plate. Red phosphorus in acetone was used to cali-brate the time of flight detector. Sulfadimethoxine (Sigma-Aldrich) was used as thelockmass during each run (one in four spotted samples).MALDIwas poweredby asolid-state laser, emitting at 355 nm with a repetition rate of 2.5 kHz.

Analysis of MALDI-Q-TOF Data

MALDI-Q-TOF continuum data were combined, background subtracted,and centered usingMassLynx version 4.1 software (Waters). Output data for allsamples were binned using the MALDIquant R package (strict mode with atolerance of 0.02 D; Gibb and Strimmer, 2012). The resulting peak lists werecorrected for total ion current and dry weight of the sample. Six technicalreplicates of each biological sample were averaged for further analysis. Dif-ferent multivariate analyses were conducted. Prior to PCA, corrected data foreach ion were normalized to the average intensity from all samples using MeVversion 4.9 (Raychaudhuri et al., 2000) and filtered using anANOVA (P, 0.01).In order to get a wider exploratory approach, features were not corrected forfalse discovery rate. PCA on filtered data (Fig. 2) was performed using Multi-base plugin software in Excel (http://www.numericaldynamics.com). In par-allel, unfiltered rawMALDI-Q-TOF data were analyzed using PCA inMeV anda supervised O2PLS-DA. O2PLS-DA was performed using SIMCA version13.0.3 (http://www.umetrics.com/products/simca) with Pareto scaling andlog transformation. Goodness to fit, goodness of prediction, and cross-validation ANOVA were satisfactory for MALDI2 (R2X = 0.65, Q2 = 0.94,cross-validation ANOVA P = 7.02 3 1026) and MALDI+ (R2X = 0.68, Q2 = 0.92,cross-validation ANOVA P = 1.81 3 1023). Variable importance for projectionrankings were determined on O2PLS-DA with Pareto scaling and without logtransformation in order to keep bigger differences between variables. MarVisFilter, Cluster, and Pathway allowed for adduct and isotope corrections, clus-tering of metabolites (m/z tolerance = 0.05 D), and screening metabolite iden-tities using the KEGG, AraCyc, and MetaCyc databases (http://marvis.gobics.de/; Kaever et al., 2012). Mass spectra of putatively identified metabolites(benzoic acid, JA, JA-Ile, and SA) were verified by comparing against corre-sponding standards, using UPLC-Q-TOF-MS/MS with electrospray ionizationinterface, as described previously (Gamir et al., 2014). Fold changes of se-lected metabolites were determined using MetaboAnalyst (http://www.metaboanalyst.ca).

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SA and JA Quantification by UPLC-Q-TOF-MS/MS Analysis

Phytohormones were double extracted from frozen leaf material (10 mg dryweight) in a total volume of 1.5 mL of ethyl acetate that was spiked with iso-topically labeled standards, as describedpreviously (Glauser et al., 2014).UPLC-Q-TOF-MS/MS was used to quantify absolute amounts of hormones based onthe following fragmentation: SA, 137→93; and JA, 209→59 (Glauser et al., 2014).Each biologically replicated sample consisted of four pooled leaves of similarsize and age from different plants.

Gene Expression Analyses

Col-0 plants were either droplet or spray inoculated with P. cucumerina(106 spores mL21) or treated with the corresponding control (water for droplet andwater + 0.01% [v/v] Silwet for spray). Four leaves fromdifferent plantswerepooled(n= 4),flash frozen in liquid nitrogen at 2 and 5dai, then stored at280°Cuntil RNAextraction, reverse transcriptase conversion, and quantitative PCR analyses, as de-scribed previously (Luna et al., 2014). PCR amplification of PR1 (At2g14610), PR5(At1g75040), WKY70 (At3g56400), ICS1 (At1g74710), and VSP2 (At5g24770) wasperformed using previously described gene-specific primers (Gouhier-Darimontet al., 2013; Gruner et al., 2013; Li et al., 2013). Relative transcript quantities werecalculated according to (1 + E)DCt, where Ct represents cycle threshold and DCt =Ct(sample) 2 Ct(calibrator sample), and normalized to (1 + E)DCt values of threereference genes, At1g13440, At2g28390, and At5g25760 (Czechowski et al., 2005).

Cell Death Measurements

Cell death was determined by conductivity measurements from electrolyteleakage as reported (Pike et al., 1998). Five-week-old Col-0 plants were dropletor spray inoculated with water (60.01% [v/v] Silwet) or P. cucumerina (106 or108 spores mL21). At various time points after inoculation (0 to 7 dai), six discs(1.2 cm2) from similarly aged leaves were collected from different plants, usinga cork borer (n = 4). Discs were agitated in 5 mL of double-deionized sterilewater at room temperature for 2 h on an orbital shaker (200 rpm). Conductivitywas measured in the balanced bathing solution using a CMD 500 WPA con-ductivity meter. To express levels of electrolyte leakage relative to maximumelectrolyte leakage from lysed tissue (set at 100%), samples were boiled for15 min and remeasured for total conductivity. Cell death was expressed as thepercentage of electrolyte leakage between initial and total electrolyte leakage.Three independent experiments were conducted with comparable results.

Trypan Blue Staining

Leaves (n = 8–12) were collected at 4 and 7 dai and cleared in 70% (v/v)ethanol prior to lactophenol-Trypan Blue staining, as described previously (Tonand Mauch-Mani, 2004). Fungal colonization and associated cell death wereexamined using anOlympus BX51microscope at different magnifications (403,1003, and 4003). Photographs of representative colonization patterns weretaken at 4003 magnification. The presented images were compiled from pho-tographs at five different focal planes that were merged at 70% transparencyusing Photoshop CS4 (Adobe).

Aniline Blue and Calcofluor Staining

Callose deposition relative to germinating fungal spores was performed inleaves from 5-week-old Col-0 plants at 3 d of droplet or spray inoculation withwater or P. cucumerina (106 spores mL21). Aniline Blue-Calcofluor doublestaining of spores were performed as described previously (Ton and Mauch-Mani, 2004; Luna et al., 2011). Leaves were analyzed by epifluorescence mi-croscopy (Olympus BX51). For each treatment, four similarly aged leaves of sixplants per condition were considered (n = 24). Levels of callose deposition werequantified using Photoshop CS4 (Adobe) and expressed relative to total leafarea as described (Luna et al., 2011).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Droplet and spray inoculation result in similaramounts of inoculum on the leaf.

Supplemental Figure S2. Differential disease phenotypes after droplet andspray inoculation.

Supplemental Figure S3. Supervised multivariate analysis of metabolicprofiles.

Supplemental Figure S4. Unsupervised principal component analysis ofmetabolic profiles.

Supplemental Figure S5. Hierarchical cluster analysis of metabolic profiles.

Supplemental Figure S6. Differential regulation of JA, SA, and glucosinolatemetabolites.

Supplemental Figure S7. Gene expression analysis of PR5 and ICS1.

Supplemental Figure S8. Epi-fluorescence microscopy of callose deposi-tion and spore distribution.

Supplemental Figure S9. Basal resistance of pad4-1 and pen2 after dropletand spray inoculation.

Supplemental Figure S10. Model explaining the link between inoculationmethod and spore density.

Supplemental Table S1. Statistical analysis of differentially respondingm/z values.

ACKNOWLEDGMENTS

We thank Dr. Heather Walker (biOMICS Facility, University of Sheffield)and Dr. Gaétan Glauser (University of Neuchâtel) for useful advice regardingmass spectrometry experiments.

Received May 19, 2015; accepted January 31, 2016; published February 3, 2016.

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A Novel Infection Strategy by Plectosphaerella cucumerina

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