arabidopsis myc transcription factors are the target of ... · pathogenesis-related genes (little...

Arabidopsis MYC Transcription Factors Are the Target ofHormonal Salicylic Acid/Jasmonic Acid Cross Talk inResponse to Pieris brassicae Egg Extract1[OPEN]

André Schmiesing, Aurélia Emonet, Caroline Gouhier-Darimont, and Philippe Reymond*

Department of Plant Molecular Biology, University of Lausanne, CH-1015 Lausanne, Switzerland

ORCID IDs: 0000-0002-7682-655X (A.E.); 0000-0002-3341-6200 (P.R.).

Arabidopsis (Arabidopsis thaliana) plants recognize insect eggs and activate the salicylic acid (SA) pathway. As a consequence,expression of defense genes regulated by the jasmonic acid (JA) pathway is suppressed and larval performance is enhanced. Crosstalk between defense signaling pathways is common in plant-pathogen interactions, but the molecular mechanism mediating thisphenomenon is poorly understood. Here, we demonstrate that egg-induced SA/JA antagonism works independently of theAPETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factor ORA59, which controls the ERF branch of theJA pathway. In addition, treatment with egg extract did not enhance expression or stability of JASMONATE ZIM-domaintranscriptional repressors, and SA/JA cross talk did not involve JASMONATE ASSOCIATED MYC2-LIKEs, which are negativeregulators of the JA pathway. Investigating the stability of MYC2, MYC3, and MYC4, three basic helix-loop-helix transcriptionfactors that additively control jasmonate-related defense responses, we found that egg extract treatment strongly diminished MYCprotein levels in an SA-dependent manner. Furthermore, we identified WRKY75 as a novel and essential factor controlling SA/JAcross talk. These data indicate that insect eggs target the MYC branch of the JA pathway and uncover an unexpected modulation ofSA/JA antagonism depending on the biological context in which the SA pathway is activated.

Plants face a multitude of enemies with differentvirulence or feeding strategies. During evolution,plants have developed distinct defense mechanisms tospecifically respond to these attacks. In turn, microbialpathogens and insects have evolved countermeasuresto interfere with plant defenses, engaging in an armsrace that culminated in a diversification of plant rec-ognition systems on one side and a vast repertoire ofpathogen effectors on the other side (Dangl and Jones,2001). The plant hormones salicylic acid (SA), ethylene(ET), and jasmonic acid (JA) are key regulators of signaltransduction pathways that are activated after recog-nition of the invader and that lead to expression ofdefense genes. Biotroph pathogens generally trigger theSA pathway, necrotrophs a combination of ET and JApathways, whereas feeding insects mostly induce theJA pathway (Pieterse et al., 2012). However, these sig-naling pathways are tightly interconnected to allow afine control of resource utilization for growth and rapid

adaptation to a changing environment. Evidence hasaccumulated over recent years that plant enemies takeadvantage of this signal cross talk to evade or suppressdefenses (Pieterse et al., 2012).

SA plays a central role in disease resistance, bothlocally and systemically. It accumulates strongly inleaves upon perception of microbe-associated molecu-lar patterns and controls the genetic program respon-sible for the synthesis of pathogenesis-related proteinsor antimicrobial compounds. SA also is required forprotecting undamaged parts of the plant against fur-ther infections, a process called systemic acquired re-sistance (Vlot et al., 2009). One essential componentof the SA pathway is the transcriptional coactivatorNON EXPRESSOR OF PR GENES1 (NPR1). Upon SAaccumulation and change of cellular redox state, NPR1monomerizes and translocates to the nucleus, where itinteracts with TGA transcription factors that bind to thepromoter of SA-responsive genes (Fu and Dong, 2013).Among those, WRKY transcription factors influencepositively and negatively the expression of down-stream defense genes (Eulgem and Somssich, 2007).

The JA pathway is involved in different processes,including plant growth, development, and responses tobiotic and abiotic stresses (Howe and Jander, 2008;Browse, 2009). JA levels rise in response towounding orherbivory, triggering large changes in gene expression(Reymond et al., 2000, 2004; De Vos et al., 2005; Devotoet al., 2005; Ehlting et al., 2008). Signaling events re-sponsible for this transcriptional reprogramming arerelatively well understood. Upon stress, JA is syn-thesized from membrane-derived fatty acids through

1 This work was supported by the Swiss National Science Foun-dation (grant no. 31003A_149286 to P.R.).

* 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:Philippe Reymond ([email protected]).

A.S., A.E., and P.R. conceived the research plans; A.S., A.E., andC.G.-D. performed the experiments; P.R. wrote the article with con-tributions of all the authors.

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several steps, including oxygenation by lipoxygenases(LOXs), cyclization, andb-oxidation. JA is then conjugatedto Ile to form JA-Ile, the bioactive molecule that is per-ceived by a receptor complex containing CORONATINE-INSENSITIVE1 (COI1) and JASMONATE ZIM-domain(JAZ) repressors (Chini et al., 2007; Thines et al., 2007;Fonseca et al., 2009; Yan et al., 2007). COI1 is part ofan SCF-type E3 ubiquitin ligase complex that mediatesdegradation of JAZ proteins by the proteasome. In unsti-mulated plants, JAZ repressors inhibit the activity oftranscription factors by interacting with the adaptor pro-tein NINJA and the corepressor TOPLESS (Pauwels et al.,2010). JA-Ile accumulation leads to degradation of JAZs,allowing transcription of JA-responsive genes, includingthe marker VEGETATIVE STORAGE PROTEIN2 (VSP2).Known direct targets of JAZ repressors are closely relatedbHLH factors, MYC2, MYC3, and MYC4 (Fernández-Calvo et al., 2011). These three MYCs interact with MYBproteins to regulate defense against insect herbivory bybinding to a G-box motif found in the promoter of glu-cosinolate biosynthesis genes (Schweizer et al., 2013).Another subgroup of bHLH factors includes JASMO-NATE ASSOCIATED MYC2-LIKEs (JAMs), which arenegative regulators of the JA pathway. JAMs bind toMYCtarget sequences and act as transcription repressors,allowing fine regulation of defense gene expression (Songet al., 2013; Sasaki-Sekimoto et al., 2013).The JA pathway consists of two distinct branches that

respond to different aggressors. In response to woundingor herbivorous insects, the MYC branch activates MYCtranscription factors and JA-responsive genes, includingVSP2. Necrotrophs utilize the ERF branch, which is con-trolled by APETALA2/ETHYLENE RESPONSE FAC-TOR (AP2/ERF) transcription factors, including ERF1and OCTADECANOID-RESPONSIVEARABIDOPSIS59(ORA59). The ERF branch requires the combined activa-tion of JA and ET pathways and induces the marker genePLANT DEFENSIN1.2 (PDF1.2; Pieterse et al., 2012).Upon perception by its receptors, ET stabilizes two pri-mary transcription factors, ETHYLENE-INSENSITIVE3(EIN3) and EIN3-LIKE1 (EIL1), through upstream activ-ity of the essential regulator EIN2 (Yoo et al., 2009). EIN3and EIL1 regulate the expression of most ET-responsivegenes, including ERF1 and possibly ORA59. It has re-cently been shown that JAZs interact with EIN3 and EIL1and repress their transcriptional activity, explaining thesynergism in JA/ET gene regulation (Zhu et al., 2011).Antagonism between SA, ET, and JA pathways has

been demonstrated in many plant species (Pieterseet al., 2012). It is suggested to help the plant prioritizedefense allocation toward the appropriate attacker byfine-tuning the induction of specific defense programswhen these hormones accumulate (Reymond andFarmer, 1998; Spoel and Dong, 2008). However, at-tackers can also exploit this antagonism for their ownbenefit. For example, activation of the JA pathway bythe biotroph pathogen Pseudomonas syringae pv tomatoresults in the inhibition of SA-dependent defensesthrough production of the virulence factor coronatine(COR), which is a JA-Ile analog (Brooks et al., 2005).

Another bacterial effector, HopX1 from P. syringae pvtabaci, degrades JAZs and thus promotes virulence byinhibiting SA defenses through activation of the JApathway (Gimenez-Ibanez et al., 2014). Relatively littleinformation is available on the molecular mechanismsof JA/SA cross talk, but it was recently found that CORinduces the expression of NAC transcriptions factorsthat inhibit SA biosynthesis (Zheng et al., 2012). Incontrast, antagonism of the SA signaling pathway onthe JA pathway has been extensively studied (Caarlset al., 2015). SA suppressed JA-dependent defense re-sponses in tomato (Solanum lycopersicum; Doares et al.,1995), tobacco (Nicotiana tabacum; Niki et al., 1998), andArabidopsis (Arabidopsis thaliana; Spoel et al., 2003).Consequently, stimulation of the SA pathway dimin-ished plant response to a necrotrophic fungus (Spoelet al., 2007) and a generalist herbivore (Cipollini et al.,2004). In Arabidopsis, treatments with SA or patho-gens that enhance SA levels inhibited expression ofherbivore-induced VSP2 and necrotroph-inducedPDF1.2 (Koornneef et al., 2008; Leon-Reyes et al.,2009). Pharmacological experiments with SA andmethyl jasmonate (MeJA) treatments have shown thatlow doses of SA exert a long-lasting (up to 96 h) in-hibitory effect on MeJA-induced PDF1.2 expression(Koornneef et al., 2008). This negative cross talk wasconserved in several accessions (Koornneef et al.,2008), occurred downstream of JA biosynthesis (Leon-Reyes et al., 2010a), and required cytosolic activity ofNPR1 (Spoel et al., 2003). Additional experimentsdemonstrated that ET can override NPR1 dependencyof SA-JA antagonism, indicating that biotic inductionof ET modulates the interaction between SA and JApathways (Leon-Reyes et al., 2010b). Two recent studiesdiscovered that SA treatment promotes ORA59 proteindegradation (Van der Does et al., 2013) and inhibitORA59 expression (Zander et al., 2014), providing thefirst clues on how gene expression from the ERF branchof the JA pathway is suppressed.

Eggs from herbivorous insects are frequently de-posited on plant leaves and represent a threat as theygive rise to feeding larvae. Evidence has accumulatedthat plants can recognize and respond to ovipositionby inducing direct and indirect defenses (Hilker andFatouros, 2015; Reymond, 2013). In Arabidopsis, in-sect eggs trigger cellular and molecular changes thatare very similar to those that are caused by infectionwith biotroph pathogens. Indeed, eggs from the spe-cialist lepidopteran herbivore Pieris brassicae inducea hypersensitive-like response, callose and reactiveoxygen species accumulation, and the induction ofpathogenesis-related genes (Little et al., 2007; Gouhier-Darimont et al., 2013). Expression of PATHOGENESIS-RELATED1 (PR1) is strongly up-regulated at the site ofoviposition in response to P. brassicae eggs but also inresponse to egg extracts from other insect species, in-cluding the generalist Spodoptera littoralis (Bruessowet al., 2010). Findings that SA accumulates to high levelsfollowing P. brassicae oviposition and that mutants inENHANCEDDISEASE SUSCEPTIBILITY1, SALICYLIC

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Insect-Induced SA/JA Cross Talk in Arabidopsis

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ACID-DEFICIENT2 (SID2), and NPR1 are deficient inegg-induced PR1 expression indicate that eggs acti-vate the SA pathway (Gouhier-Darimont et al., 2013;Bruessow et al., 2010). Interestingly, egg-induced SAwas shown to antagonize the JA pathway by inhibitingthe expression of insect-induced defense genes. WhenArabidopsis plants were treated with P. brassicae orS. littoralis egg extract, induction of JA-responsive genesby feeding larvae was strongly diminished. This effectwas abolished in sid2-1 mutant, indicating that egg-induced SA accumulation was responsible for thenegative cross talk (Bruessow et al., 2010). Furthermore,performance of S. littoralis larvae was enhanced onegg extract-treated plants, illustrating the potentialbenefit of activating SA/JA antagonism for the at-tacker (Bruessow et al., 2010).

Here, we further investigate how insect eggs sup-press the JA pathway. We provide evidence that theERF branch is not involved but that egg-induced SApromotes degradation of MYC transcription factors.This study reveals a molecular mechanism by whichinsect eggsmay favor the performance of their progeny,illustrating the intricate connection between defensesignaling pathways in plants.

RESULTS

Egg-Induced Suppression of the JA Pathway IsIndependent of ORA59 But Requires NPR1 and EIN2

We previously found that treatment of Arabidopsisplants with P. brassicae and S. littoralis egg extractsinhibited the expression of JA-responsive genes that areinduced by feeding larvae, including VSP2, LOX3, andMYROSINASE-ASSOCIATEDPROTEIN (MAP; Bruessowet al., 2010). This was accompanied by a better larvalperformance of the generalist herbivore S. littoralis butnot of the specialist P. brassicae, which is adapted toArabidopsis defensemetabolites. Since both effects werelost in sid2-1, we concluded that egg-induced SA accu-mulation suppressed JA-dependent defenses (Bruessowet al., 2010). To test if this negative SA/JA cross talkinvolves the ERF branch of the JA pathway, we treated4-week-old Arabidopsis ecotype Columbia (Col-0) andora59 plants with egg extract for 5 d and subsequentlychallenged themwith S. littoralis larvae for 48 h (analysisof gene expression) or 10 d (larval performance assay).Egg extract suppressed insect-induced expression ofVSP2 in both wild-type and ora59 plants (Fig. 1A). Inaddition, larvae gained significantly more weight onplants treated with egg extract compared with controlplants, irrespective of the genotype (Fig. 1B). Thus, theseresults demonstrate that ORA59 is not involved in egg-induced SA/JA cross talk.

To further investigate which molecular players medi-ate suppression of the JApathway byP. brassicae eggs,weestablished a simplified protocol where 12-d-old Arabi-dopsis were treated with egg extract for 24 h and sub-sequentlywoundedwith forceps tomimic herbivory andactivate the JA pathway (Fig. 2A). Similar to older plants

challenged with larvae, treatment with egg extractstrongly suppressed wound-induced VSP2 expression(Fig. 2B). This suppression was also observed for LOX3and MAP (Supplemental Fig. S1). In contrast, consecu-tive egg extract and wound treatments did not suppressPDF1.2 expression, confirming that egg-induced SA/JAcross talk does not target the ERF branch but more likelythe MYC branch of JA pathway (Fig. 2C). To verify thattreatment with egg extract can substitute for the lesscontrollable natural oviposition, plants were placed in acage with P. brassicae females until one egg batch per leafwas laid, and 24 h later leaves were wounded for 3 hand collected for gene expression analysis. In a parallelexperiment, oviposited plants were challenged withS. littoralis larvae for 10d and larval performance assessed.Natural oviposition suppressed wound-induced VSP2expression and stimulated S. littoralis larval growth to a

Figure 1. Egg extract-induced SA/JA cross talk does not depend onORA59. A, Expression of insect-responsive VSP2 was measured by real-time PCR. Four-week-old plants were treated for 5 d with egg extract (EE),challenged for 48 h with S. littoralis larvae (S.l.), or subjected to eggtreatment followed by herbivory (EE + S.l.). Untreated plants were used ascontrols (C). Values 6 SE are the mean of two biological replicates andnormalized to the reference gene SAND. Different letters within eachgenotype indicate significant differences at P , 0.05 (Tukey’s highlysignificant difference test). B, Freshly hatched S. littoralis larvae wereplaced on Col-0 or ora59 plants, and larval weight was measured after10 d of feeding. White bars indicate untreated control plants challengedwith insects. Black bars indicate plants treated with egg extract for 5 dbefore insect challenge. The number of larvae used in each experimentis shown within each bar. Error bars indicate SE. Significant differencesbetween control and treated plants are indicated (Student’s t test; **P ,0.01; ***P , 0.001). This experiment was repeated once with similarresults.

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similar extent as egg extract (Supplemental Fig. S2).These results are in line with our previous observationsthat SA accumulation, defense gene expression, andphysiological changes occur in response to natural ovi-position or application of egg extract (Little et al., 2007;Bruessow et al., 2010; Reymond, 2013).Early experiments with SA and MeJA treatments

showed that NPR1 was determinant for SA/JA crosstalk, but it was later found that ET can override NPR1function and abolishes this effect (Spoel et al., 2003;Leon-Reyes et al., 2009). We previously observed thategg-induced SA/JA cross talk was independent ofNPR1. Expression of JA-responsive genes was stillsuppressed by egg extract in npr1-1, and enhancedperformance of S. littoralis larvaewas retained (Bruessowet al., 2010). To explore whether ET and NPR1 collec-tively modulate egg-induced cross talk, we used thenpr1-1 ein2-1 double mutant. In contrast to Col-0, sup-pression of wound-induced JA-regulated gene expres-sion and enhanced larval performance were absent innpr1-1 ein2-1 (Fig. 2; Supplemental Fig. S1). Thus, thecombined action of NPR1 and ET signaling is necessaryto regulate SA/JA antagonism for both ERF and MYCbranches of the JA pathway (Leon-Reyes et al., 2009;this study).

Egg-Induced SA/JA Antagonism Does Not Act throughJAZs and JAMs

To further assess how egg-induced SA antagonizes theMYC branch and lowers VSP2 expression, we hypothe-sized that enhanced expression of JAZ repressors and/orstabilization of these proteins could play a role. JAZ10.4,a splice variant of JAZ10, lacks the Jas domain that allowsbinding to COI1 and therefore is resistant to JA-induceddegradation (Chung andHowe, 2009).Wemonitored theexpression of JAZ10.4 in cross-talk conditions and com-pared it to the expression of JAZ10.1, which contains a Jasdomain. Contrary to our expectations, expression ofneither variant was enhanced by a consecutive treatmentof egg extract andwounding. Both genes rather showed asimilar profile as VSP2, with a strong induction bywounding alone and a reduced induction, although notsignificant, when plants were pretreated with egg extract(Fig. 3, A and B). We next performed assays with Ara-bidopsis lines stably expressing 35S:JAZ1-GUS and 35S:JAZ10-GUS constructs. Plants were subjected to treat-ments with egg extract, wounding, or both, and levels ofJAZ1 and JAZ10were assessed bywestern blot with anti-GUS antibody or by measuring GUS activity. JAZ1 andJAZ10 levels were similar in control and egg extract-treated plants, but declined in singly wounded or egg

Figure 2. Egg extract-induced SA/JA cross talk is abolished in npr1-1 ein2-1mutant. A, Experimental scheme used with 12-d-oldseedlings. Onemicroliter of egg extract (EE) was applied underneath the first true leaf. After 24 h, the leaf waswounded twicewithforceps and harvested after 3 h (EE + W). Controls consisted of untreated plants (C), plants only treated with egg extract (EE), orplants only wounded (W). B and C, Expression of VSP2 and PDF1.2 was measured by real-time PCR. Twelve-day-old seedlingswere subjected to the treatments described in A. Values 6 SE are the mean of three biological replicates and normalized to thereference gene SAND. Different letters within each genotype indicate significant differences at P , 0.05 (Tukey’s highly sig-nificant difference test). D, Freshly hatched S. littoralis larvaewere placed on 4-week-old Col-0 or npr1-1 ein2-1 plants, and larvalweight was measured after 10 d of feeding. White bars indicate untreated control plants challenged with insects (C). Black barsindicate plants treated with egg extract (EE) for 5 d before insect challenge. The number of larvae used in each experiment isshown within the bars. Error bars indicate SE. Significant differences between control and treated plants are indicated (Student’st test; ***P , 0.001; n.s., not significant). This experiment was repeated twice with similar results.




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extract-treated and wounded plants, underlying theknown JA-Ile-dependent degradation of JAZs by SCFCOI1

complex. Similarly, GUS activity was lower after wound-ing or after consecutive egg extract and woundtreatment (Supplemental Fig. S3). Thus, suppressionof JA-responsive genes in response to egg-inducedSA/JA cross talk is not explained by enhanced JAZexpression or stabilized JAZs.

With the recent discovery that the MYC branch of theJA pathway is negatively regulated by JAMs, we nexthypothesized that egg-induced SA could enhance theactivity of these transcriptional repressors. To addressthis hypothesis, we made use of the quadruple JAMmutant bhlh3 bhlh13 bhlh14 bhlh17, which was shownto exhibit a significant increase in JA-regulated gene

expression and resistance to herbivory (Song et al.,2013). VSP2 was strongly induced by wounding in themutant, but this inductionwas significantly diminishedwith egg extract pretreatment, ruling out a role for thesefactors in SA/JA cross talk (Fig. 3C).

Egg-Induced SA Modulates MYC2, MYC3, andMYC4 Accumulation

Since SA targets ORA59 protein stability in the ERFbranch of the JA pathway (Van der Does et al., 2013), wethen investigated whether a similar process occurs inthe MYC branch. Twelve-day-old plants were treatedwith egg extract, wounding, or a combination of both,and accumulation of MYC2 was monitored by westernblot with anti-MYC2 antibody. Treatment with eggextract had a marked effect on MYC2 protein accumu-lation, which was severely diminished compared withcontrol levels (Fig. 4A). MYC2 levels were increased afterwounding compared with control plants, but, strikingly,were again almost undetectable in the consecutive eggextract and wound treatment. To see if this egg-triggeredinhibition of MYC2 accumulation was dependent on SA,we subjected sid2-1 plants to the same cross-talk condi-tions. In clear contrast with wild-type plants, egg extracttreatment alone or in combination with wounding wereunable to lower MYC2 levels in this SA-deficient mutant(Fig. 4A). This is consistent with our previous reportshowing that egg-induced inhibition of JA-responsivedefense gene expression and enhanced larval perfor-mance are abolished in sid2-1 (Bruessow et al., 2010).Thus, egg-induced SA/JA cross talk operates throughMYC2 protein stability, and this process is analogous tothe negative effect of SA on ORA59 accumulation. Inaddition, use of the proteasome inhibitor MG132 pre-vented egg-induced MYC2 degradation, implying a rolefor the 26S proteasome in the control of MYC2 stability(Fig. 4B).

Since inhibition of wound-induced VSP2 expressionby egg extract treatment was abolished in npr1-1 ein2-1(Fig. 2), we postulated that this absence of SA/JA an-tagonism may be explained by a failure to degradeMYC2. On the contrary, MYC2 protein levels were stillhighly reduced in response to egg extract treatment innpr1-1 ein2-1plants, with orwithoutwounding (Fig. 4C).

To further substantiate our findings on MYC2, wemeasured the effect of egg extract on MYC3 and MYC4stability. Because no antibodies against these proteinsare available, we used 35S:MYC3-GFP and 35S:MYC4-GFP Arabidopsis lines. Similar to MYC2, levels ofMYC3-GFP andMYC4-GFP were significantly reducedin plants treated with egg extract, and this effect wasconserved in consecutive egg extract and wound treat-ments (Supplemental Fig. S4).

Finally, to test if egg-induced SA modulates MYCprotein levels by inhibition of transcriptional activity,we measured MYC2, MYC3, and MYC4 expression incross-talk conditions. MYC2 expression was similar incontrol and egg extract-treated plants, whereas it wasinduced by wounding and reduced by a consecutive

Figure 3. Role of JAZ10 splice variants and JAM negative regulators. Aand B, Expression of JAZ10.1 (A) and JAZ10.4 (B) was measured by real-time PCR in 12-d-old Col-0 seedlings subjected to single or consecutiveegg extract (EE) and wound (W) treatments. Untreated plants were usedas controls (C). C, Expression of VSP2 was measured in the quadrupleJAMmutant (bhlh3 bhlh13 bhlh14 bhlh17). Values6 SE are the mean ofthree biological replicates and normalized to the reference gene SAND.Different letters indicate significant differences at P , 0.05 (Tukey’shighly significant difference test).


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egg extract and wound treatment, like VSP2. In con-trast, MYC3 and MYC4 showed similar expressionlevels in all conditions (Supplemental Fig. S5). It thusappears that there is no strict correlation betweenMYCexpression and protein levels, suggesting that egg-induced SA regulates MYC protein levels through an-other process.

WRKY75 Regulates Egg-Induced SA/JA Cross Talk

We previously noticed that WRKY75 was highly in-duced by P. brassicae oviposition (Little et al., 2007).Since several WRKYs have been reported to modulateSA/JA antagonism (Caarls et al., 2015), this promptedus to test the involvement of this factor in response toegg extract treatment. The Arabidopsis T-DNA inser-tion mutant wrky75-25 (Encinas-Villarejo et al., 2009)and a 35S:WRKY75 overexpressor line (Van Aken et al.,2013) were treated with P. brassicae egg extract andsubsequently wounded. Controls consisted of no treat-ment or wounding alone. Expression of VSP2 wasstrongly up-regulated by wounding in 35S:WRKY75,but, contrary to Col-0, this induction was not sup-pressed by egg extract pretreatment. Indeed, woundingor egg extract treatment followed by wounding in-duced VSP2 to similar levels (Fig. 5A). In wrky75-25,expression of both genes was not induced bywoundingand was not different between all treatments (Fig. 5A).In line with these results, enhanced S. littoralis larvalperformance after egg extract pretreatment was not

observed in 35S:WRKY75. In addition, wrky75-25 mu-tant allowed much more growth of S. littoralis com-pared with Col-0, and this effect was even morepronounced in egg extract-treated plants (Fig. 5B).

Since wound-induced VSP2 expression was notinhibited by egg extract treatment in 35S:WRKY75plants, we then tested whether overexpression ofWRKY75 was interfering with egg-triggered MYC2degradation. However, MYC2 protein levels were stillstrongly reduced in response to egg extract treatment orin response to consecutive egg extract and woundtreatments in 35S:WRKY75 plants (Fig. 5C).

Absence of SA/JA cross talk in 35S:WRKY75 couldbe due to impaired egg-induced SA signaling. We an-alyzed the expression of genes that are strongly up-regulated by oviposition (Little et al., 2007). The SAmarker gene PR1, a chitinase gene (CHIT), and a trypsininhibitor gene (TI) were all markedly up-regulated byegg extract treatment or by a consecutive egg extractand wound treatment. This up-regulation was similarin Col-0, 35S:WRKY75, and wrky75-25, indicating thatWRKY75 does not block the SA pathway but rather actsat another level (Supplemental Fig. S6).

DISCUSSION

Cross talk between defense signaling pathways hasbeen the subject of intense research in recent years. It isthought to represent a way to optimize resource allo-cation to properly fight pathogens or insect attackers. In

Figure 4. MYC2 protein is degraded in response toegg extract treatment. A and C, Immunoblotanalysis of MYC2 in Col-0, sid2-1, and npr1-1ein2-1 12-d-old seedlings subjected to single orconsecutive egg extract (EE) and wound (W)treatments. Untreated plants were used as controls(C). B, Immunoblot analysis of MYC2 in Col-0plants in the presence of the proteasome inhibitorMG132. Protein molecular mass is shown on theleft. Immunoblot of ACTIN and Ponceau S-stainedRubisco are shown as loading controls. All experi-ments were repeated twice with similar results.




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contrast, plant enemies also can hijack cross talk to theirown benefit.We recently discovered that insect eggs arerecognized by Arabidopsis. Egg-induced SA accumu-lation antagonized expression of JA-related genes,which resulted in enhanced larval performance of ageneralist herbivore (Bruessow et al., 2010). In thiswork, we decided to investigate the molecular mecha-nisms by which insect eggs target the JA pathway.

ORA59 is a key transcription factor that controls ex-pression of defense genes belonging to the ERF branchof the JA pathway, including the marker gene PDF1.2.We find here that egg-induced SA/JA cross talk stilloccurs in ora59mutant. In addition, induction of PDF1.2after wounding was not subjected to SA/JA antag-onism by insect eggs. The ERF branch is involved inresistance to necrotroph pathogens and requires si-multaneous activation of ET and JA signaling pathways(Lorenzo et al., 2003). The distinctMYC branch of the JApathway is triggered by wounding in herbivory andregulates VSP2 expression (Pieterse et al., 2012). In-deed, JA is not able to induce VSP2 in the myc2 myc3myc4 triplemutant (Fernández-Calvo et al., 2011). Thus,our results strongly indicate that insect eggs target the

MYCbranch and not the ERF branch of the JA pathway.It is intriguing that egg-induced SA accumulation leadsto a specific SA/JA cross talk that only affects onebranch of the JA pathway. In experiments where in-ducers of the SA pathway were either the biotrophicpathogen Hyaloperonospora parasitica or exogenous ap-plication of SA, it was shown that MeJA up-regulationof PDF1.2 was suppressed (Koornneef et al., 2008).Thus, the biological context in which SA is induced islikely to influence the outcome of SA/JA cross talk andfurther investigation on additional factors responsiblefor this specificity will be necessary.

Several hypotheses were then tested to explain howegg-induced SA accumulation antagonizes defensegene expression controlled by theMYCbranch of the JApathway. We found that SA did not increase the ex-pression of the nondegradable splice variant JAZ10.4and did not stabilize JAZ1 and JAZ10 protein levels.JAZ10.4 variant was previously shown to bind toMYC2 but not to COI1, and Arabidopsis plants over-expressing JAZ10.4 exhibited JA-insensitive pheno-types, indicative of a reduced JA signaling pathway(Chung and Howe, 2009). JAZ10.4 and JAZ8, another

Figure 5. WRKY75 regulates SA/JA cross talk. A,Expression of VSP2 was measured by real-timePCR in Col-0, WRKY75 overexpressing line, andwrky75-25 T-DNA mutant. Twelve-day-old seed-lings were subjected to single or consecutive eggextract (EE) and wound (W) treatments. Untreatedplants were used as controls (C). Values 6 SE arethe mean of three biological replicates and nor-malized to the reference gene SAND. Differentletters indicate significant differences at P , 0.05(Tukey’s highly significant difference test). B,Freshly hatched S. littoralis larvae were placed on4-week-old plants, and larval weight was mea-sured after 10 d of feeding. White bars indicateuntreated control plants challenged with insects(C). Black bars indicate plants treated with eggextract (EE) for 5 d before insect challenge. Thenumber of larvae used in each experiment isshown within each bar. Error bars indicate SE.Significant differences between control and trea-ted plants are indicated (Student’s t test; ***P ,0.001; *P , 0.05; n.s., not significant). This ex-periment was repeated twice with similar results.C, Immunoblot analysis of MYC2 in 12-d-oldCol-0 and WRKY75 overexpressing line subjectedto the same treatments as in A. Immunoblot ofACTIN and Ponceau S-stained Rubisco are shownas loading controls. This experiment was repeatedonce with similar results.


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transcriptional repressor that is not degraded by theJA-Ile-SCFCOI1 complex (Shyu et al., 2012), are thoughtto constitute a negative feedback loop that preventsexacerbated activation of JA responses. In line withexperiments using exogenous application of SA (Vander Does et al., 2013), we conclude that egg-inducedSA/JA cross talk is not regulated by increased expres-sion of JAZ10.4 or stabilization of JAZ repressors.Recently, a subclass of bHLH factors closely related

to MYC2, MYC3, and MYC4 was shown to antagonizeMYC activity. bHLH3, bHLH13, bLHL14, and bHLH17are repressors that bind to cis-regulatory elements ofMYC target genes and inhibit transcription (Fonsecaet al., 2014; Song et al., 2013; Sasaki-Sekimoto et al.,2013). We testedwhether these factors could participatein the suppression of JA responses mediated by egg-induced SA. VSP2 expression was still inhibited by acombined egg extract and wound treatment in thequadruple bhlh3 bhlh13 bhlh14 bhlh17mutant, ruling outthe involvement of this family of repressors in egg-induced SA/JA cross talk. It would be interesting totest their role in the ERF branch of the JA pathway, butaltogether our data and those of Van der Does et al.(2013) point to separate mechanisms to modulate JA-dependent transcriptional responses. On one hand,stabilized JAZs and bHLH repressors counteract theactivation of JA responses to fine-tune defense anddevelopment. On the other hand, accumulation of SA inresponse to biotrophs and insect eggs interferes with JAdefense gene expression via other routes.Assessing MYC protein stability, we discovered that

treatment with egg extract strongly diminished MYC2,MYC3, and MYC4 levels. This effect was due to SAaccumulation since MYC2 was not degraded in sid2-1.The fact that all three MYCs were degraded by eggextract treatment is likely explained by their partiallyredundant activity. They are known to form homo- andheterodimers and additively control the expression ofdefense genes and resistance to herbivory (Schweizeret al., 2013; Fernández-Calvo et al., 2011). Interestingly,a study reported recently that ORA59 is degradedby exogenous SA application, providing a molecularmechanism for how SA/JA cross talk targets the ERFbranch of the JA pathway (Van der Does et al., 2013). Itis striking that two seemingly different SA/JA crosstalks target different transcription factors. How suchspecificity is achieved will require further work, but itis tempting to speculate that some components areshared.We found that MYC2, MYC3, and MYC4 expression

was similar in control and egg extract-treated plants.However, contrary to MYC3 and MYC4, MYC2 wasinduced by wounding, and this induction was reducedwhen plants were pretreated with egg extract. Inter-estingly, the promoter of MYC2 contains three G-boxelements (Promomer; http://bbc.botany.utoronto.ca).G-box is a known binding site of MYC2, MYC3, andMYC4 (Fernández-Calvo et al., 2011; Godoy et al.,2011). MYC2 thus seems to be part of a positive feed-back loop and is subjected to SA/JA cross talk. We

cannot rule out that suppression of MYC2 expressioncontributes overall to the antagonistic effect of egg-induced SA accumulation, but our data rather suggestthat MYC protein degradation is an essential compo-nent of this phenomenon.

Regulated proteolysis is an important component ofplant signaling pathways (Sadanandom et al., 2012).Besides JAZs that are targeted for degradation uponactivation of the JA pathway by the SCFCOI1 complex,MYCs were also shown to be unstable proteins. Forexample, the circadian clock regulates MYC2 proteinlevel. MYC2 accumulates during the day and is de-graded during the night (Shin et al., 2012). MYC2,MYC3, and MYC4 are stabilized by light, via the actionof red and blue light photoreceptors, and also byJA treatment (Chico et al., 2014). Phosphorylation-dependent proteolysis of MYC2 was reported to beessential for its transcriptional activity (Zhai et al.,2013). In all these cases, MYC protein degradation wasmediated by the proteasome. Recently, it was shownthat MYC2 is polyubiquitinated by PLANT U-BOXPROTEIN10 (PUB10), which belongs to the U-boxfamily of E3 ligases (Jung et al., 2015). There thus isgrowing evidence that the JA pathway is tightly coor-dinated with other environmental inputs and that partof this regulation operates at the translational level.Here, we provide evidence that egg-induced SA/JAcross talk also targets MYC protein stability throughthe proteasome. Further research will be necessary toidentify factors that control MYC and ORA59 degra-dation in the context of SA-mediated JA antagonism.

NPR1 is required for SA-mediated suppression of theERF branch of the JA pathway, although this effect canbe modulated by ET (Spoel et al., 2003; Leon-Reyeset al., 2009). For egg-induced SA/JA cross talk, wepreviously found that suppression of JA-responsivegenes and enhanced larval performance was conservedin npr1-1 (Bruessow et al., 2010) and ein2-1 (Bruessow,2011) single mutants. Interestingly, we show here thategg-induced SA/JA cross talk was abolished in thenpr1-1 ein2-1 double mutant, implying that NPR1 andET are collectively necessary to suppress the MYCbranch. Antagonism between ET and JA pathways hasbeen reported previously. For example, ET perceptionnegatively affected expression of wound-regulatedgenes (Rojo et al., 1999), and ein2-1 plants were moreresistant to herbivory (Bodenhausen and Reymond,2007). At the molecular level, EIN3 and EIL, which arekey transcription factors of the ET pathway, interactedwith and repressedMYC2 transcriptional activity (Songet al., 2014). Here, we show that PDF1.2 expression wasinduced by egg extract treatment and that this induc-tion was abolished in npr1-1 ein2-1, suggesting that theET pathway is activated by insect eggs. However, thefinding that MYC2 protein degradation in response toegg extract treatment still occurred in npr1-1 ein2-1strongly suggests that NPR1 and the ET pathwaymodulate JA defenses independently of MYC factorsand not through EIN3/EIL1-mediated inhibition ofMYC activity.




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In this study, we identified WRKY75 as a novelregulator of egg-induced SA/JA cross talk. Over-expression of WRKY75 abolished egg extract-mediatedsuppression of VSP2 expression and did not lead toenhanced larval performance. In contrast, MYC2 wasstill degraded by egg extract treatment in 35S:WRKY75,similar to experiments with npr1-1 ein2-1. Since wound-activation of VSP2 was strongly diminished in wrky75-25 and maintained after egg extract treatment in 35S:WRKY75, our results are consistent with a positive roleof WRKY75 in the control of the JA-regulated genesindependently of MYC2. WRKY75 was previouslyshown to play a role in plant defense against biotrophsand necrotrophs (Encinas-Villarejo et al., 2009;Windramet al., 2012; Chen et al., 2013). Noteworthy, WRKY75overexpression was accompanied by elevated expres-sion of JA-responsive genes, including PDF1.2,VSP1, andLOX2, and enhanced resistance against the necrotrophSclerotinia sclerotiorum (Chen et al., 2013). In support ofthese findings, aWRKYDNAbindingmotif (W-box)wasenriched in genes that were responsive to SA/JA crosstalk (Van der Does et al., 2013).

Interestingly, we found that induction ofWRKY75 byegg extract treatment was more pronounced in npr1-1ein2-1 than in Col-0 (Supplemental Fig. S7), suggestinga link between NPR1/ET and WRKY75. We propose amodel (Fig. 6) where insect eggs inhibit JA-dependentdefense gene expression in two independent ways.First, SA accumulation leads to proteasome-dependentMYC degradation. Second, a combined action of NPR1and the ET pathway negatively regulates WRKY75 ac-tivity. This model explains why a similar loss of crosstalk was observed in npr1-1 ein2-1 and 35S:WRKY75,independent of MYC2 protein degradation. Indeed, wepostulate that a release of WRKY75 inhibition byNPR1/ET or overexpression of WRKY75 compensatesfor the reduced expression of JA-regulated genes,which results from MYC degradation. Noteworthy,egg-induced SA accumulation leads to enhanced lar-val performance and is believed to be a strategyevolved by insects to attenuate JA-dependent defenses(Bruessow et al., 2010). Oviposition-induced WRKY75expression may have evolved by the plant to coun-teract egg-induced cross talk. In turn, that NPR1/ETnegatively control WRKY75 expression could repre-sent a strategy to reinforce SA/JA cross talk, in anongoing arms race between insects and plants. HowNPR1, ET signaling, andWRKY75 are connected at themolecular level will represent an interesting challengefor future work.

Besides NPR1 and ET signaling, several proteins havebeen reported to modulate SA/JA cross talk in Arabi-dopsis. MAP KINASE4 is a negative regulator of the SApathway and a positive regulator of the JA pathway(Petersen et al., 2000). Multiple TGA transcription factormutants tga235 and tga2356 are blocked in SA-mediatedinhibition of JA (Zander et al., 2010; Leon-Reyes et al.,2010b). For WRKY transcription factors, wrky50 andwrky51 mutants do not show SA suppression ofJA-mediated PDF1.2 expression (Gao et al., 2011).

Overexpression of WRKY70 enhanced the expression ofSA-related genes and suppressed JA-responsive geneexpression (Li et al., 2004); however, a wrky70 mutantwas not impaired in SA/JA cross talk (Leon-Reyes et al.,2010b). Finally, JA and SA induced WRKY62 down-stream of NPR1. WRKY62 overexpression attenuatedJA-responsive gene expression, whereas awrky62mutanthad increased JA-responsive gene expression (Mao et al.,2007). WRKY75 thus adds to the list of regulators ofSA/JA cross talk. Whether these factors have a direct orindirect role in controlling the SA/JA antagonism,whether they specificallymediate suppression of the ERFor MYC branch of the JA pathway, and by whichmechanism they exert their function are, however, stilllargely unknown.

Manipulation of plant defense signaling pathways byattackers is the subject of intense research. In recentyears, evidence has accumulated that different insects

Figure 6. Model for the regulation of egg-induced SA/JA cross talk. SAaccumulates upon oviposition and the expression of SA- and NPR1-dependent genes (e.g. PR1) is activated (Little et al., 2007; Bruessowet al., 2010, Gouhier-Darimont et al., 2013). In addition, egg-inducedSA accumulation is known to inhibit larvae-induced expression of JA-dependent genes (e.g.VSP2; Bruessow et al., 2010). Here, we show thatthis SA/JA antagonism is mediated by proteasome-dependent degra-dation of MYC3, MYC3, andMYC4 (MYCs) transcription factors, whichregulate the expression of JA-responsive genes in response to herbivory.We identified another level of regulation by the transcription factorWRKY75, whose expression is induced by eggs. Overexpression ofWRKY75 compensates for MYC degradation by inducing VSP2 ex-pression. On the contrary, NPR1 and EIN2 collectively inhibit WRKY75activity, leading to an attenuated VSP2 expression in response to her-bivory. The balanced positive and negative effects of oviposition onWRKY75 may underlie an ongoing arms race between eggs that inhibitJA-dependent defenses for the benefit of larvae and the plant that tries toovercome this inhibition. Finally, we show that ET controls egg-inducedPDF1.2 expression synergistically with JA-induced PDF1.2 expressionand that the ERF branch of the JA pathway is not subjected to egg-induced SA/JA antagonism. This model postulates that insect eggs trig-ger ET accumulation, but this has to be experimentally validated.


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use SA/JA cross talk to specifically inhibit plant defensesthat are detrimental for their own development. InArabidopsis, the silverleaf whitefly Bemisia tabaci wasshown to activate the SA pathway and repress the ex-pression of JA-dependent defenses, to which it is highlysensitive (Zarate et al., 2007). In Nicotiana attenuata, oralsecretions from the beat armyworm Spodoptera exiguatriggered SA accumulation, which resulted in lower JAlevels, although larval performance was not evaluated(Diezel et al., 2009). In tomato, themealybug Phenacoccussolenopsis activated the SA pathway, which in turndown-regulated the expression of JA-responsive genes.This effect was lost in SA-deficient plants and ledto enhanced nymphal performance (Zhang et al., 2015).As a stunning indirect cross-talk manipulation, theColorado potato beetle (Leptinotarsa decemlineata) wasreported to exploit orally secreted bacteria. Symbiontsbelonging to three genera activated SA accumulationand expression of SA-regulated geneswhile suppressingJA responses. Antibiotic-treated larvae did not causesuch response and gained less weight on tomato plants(Chung et al., 2013). Our finding that insect egg-inducedactivation of the SA pathway leads to diminished MYCprotein levels and therefore suppresses JA-dependentdefense gene expression provides a molecular mecha-nism that may be conserved between all these examples.It will be interesting in the future to explore whethermanipulation of SA/JA cross talk by insects is widelyspread in nature and whether some plants have evolvedstrategies to counteract this phenomenon.

MATERIALS AND METHODS

Plant and Insect Growth Conditions

Arabidopsis (Arabidopsis thaliana) wild type (Col-0), mutants, and transgeniclines were grown in soil in a growth chamber (21°C, 10/14 h light/dark cycle,100 mmol m22 s21, 60% relative humidity) as described previously (Reymondet al., 2000). Genotypes of npr1-1 ein2-1, wrky75-25, and 35S:WRKY75 wereconfirmed (Supplemental Fig. S8).

Spodoptera littoralis (Egyptian cotton worm) eggs were obtained from Syn-genta (Stein AG). Pieris brassicae (large white butterfly) was reared in a green-house as described previously (Schlaeppi et al., 2008). Hundreds of P. brassicaeeggs were regularly collected on cabbage leaves 0 to 72 h after oviposition. Theywere gently crushed with a pestle in an Eppendorf tube without addition of anyliquid. After centrifugation (15,000g, 3min), the supernatant (“egg extract”) wasstored at 220°C. To standardize egg extract application, all egg extracts werecombined to constitute a bulk sample that was used for the treatments.

Cross-Talk Experiments

For experimentswith 4-week-oldplants, two spots of 2mLof egg extractwereapplied onto each leaf. This amount corresponds to two egg batches of 20 eggseach. In total, two leaves each from 20 plants were treated. After 5 d, egg extractwas removed with a paintbrush, and two freshly hatched S. littoralis larvaewere placed onto each plant. Plants were placed in transparent plastic boxes in agrowth room as described previously (Bodenhausen and Reymond, 2007).After 2 d of feeding, treated leaves from four plants were harvested for RNAanalysis, and the remaining plants were left with larvae for another 8 d. At theend of the experiment, larvae were collected and weighed on a precision bal-ance (XP205DR; Mettler-Toledo). Controls consisted of untreated and/oruninfested plants.

For experimentswith natural oviposition, 4-week-old plantswere placed in acage containing P. brassicae females for a maximum of 8 h. As soon as one eggbatchwas deposited on one leaf per plant, plants were removed and transferred

in a growth chamber for 5 d. Then, oviposited leaves were wounded twice witha forceps. Three hours later, treated leaves were collected for RNA analysis. Forinsect bioassay, each oviposited plant was challenged with two freshly hatchedS. littoralis larvae for 10 d, after which larval weight was measured.

For experiments with 12-d-old seedlings, 1 mL of egg extract was depositedunder the first true leaf from 12 seedlings. After 24 h, treated leaves werewounded twice with a forceps. Three hours later, whole shoots were cut fromthe root system with scissors and collected for RNA or immunoblot analyses.Other samples consisted of seedlings only treated with egg extract, onlywounded, or untreated.

Treatment with MG132

Arabidopsis seedlings were germinated and grown on sterile mesh inMurashige and Skoog plates at 22°C under a 16-h-light/8-h-dark cycle in aphytotron. Twelve-day-old seedlings were transferred on the mesh to newplates containing 4 mL of liquid Murashige and Skoog. Seedlings were treatedwith 1mL of egg extract, and themediumwas supplementedwith 50mMMG132(Sigma-Aldrich) for 1 d. Treatmentwith dimethyl sulfoxidewas used as control.Afterward, seedlings were wounded with forceps for 3 h, and leaves werecollected for immunoblot analyses.

Quantitative Real-Time PCR

Geneexpressionanalysiswasperformedaccording toapreviouslypublishedprotocol (Hilfiker et al., 2014). Samples were grinded in liquid nitrogen, andtotal RNAwas extracted using RNeasy Plant Mini Kit and treated with DNaseI(Qiagen). Afterward, cDNA was synthesized from RNA using M-MLV reversetranscriptase (Invitrogen). Quantitative real-time PCR reactions were per-formed using Brilliant III Ultra Fast SYBR-Green QPCR Master Mix on anMx3000P real-time PCR instrument (Agilent) with the following program:95°C for 3 min, then 40 cycles of 10 s at 95°C and 20 s at 60°C. Values werenormalized to the housekeeping gene SAND. Gene-specific primers are listedin Supplemental Table S1 online.

Protein Gel Blotting Analysis

Frozen seedling samples were homogenized by vortexing 1 min in 100 mL ofextraction buffer (50 mM Tris-HCl, pH 7.4, 80 mM NaCl, 0.1% [v/v] Tween 20,10% [v/v] glycerol, 1 mM DTT, 1% [w/v] Complete Mini Protease inhibitorcocktail [Roche]) in a TissueLyser II (Qiagen)with small plastic beads, centrifugedat 4°C for 10 min (15,000g). Supernatant was collected, and samples containing60 mg of proteins in 53 loading buffer (125 mM Tris-HCl, pH 6.8, 5% [w/v] SDS,25% [v/v] glycerol, 25% [v/v] b-mercaptoethanol, 3.13 mg bromophenolblue/5 mL buffer) were boiled at 100°C for 2 min, cooled on ice, and centrifuged.Proteins were run into SDS-PAGE gel and transferred onto a nitrocellulosemembrane (Bio-Rad). The membrane was subsequently stained with Ponceau Ssolution (Sigma). Then, membranes were destained with water and blocked with13TBS (1M Tris, 3 MNaCl) 2% (w/v)milk powder solution for 50min.After threewashes with TTBS (0.05% [v/v] Tween 20, 13 TBS), membranes were incubatedovernight with anti-GFP-horseradish peroxidase (HRP; 1:3000; Miltenyi Biotech),anti-MYC2 (1:7000, produced in rabbit; Abiocode), anti-GUS (1:2000, produced inrabbit; Sigma-Aldrich), or antiactin (1:3000, produced in mouse; Sigma-Aldrich).Membranes were washed in TTBS at room temperature and, when required, in-cubatedwith secondary antibody antirabbit IgG-HRPor antimouse IgG-HRP for atleast 2 h (1:3000; Cell Signaling Technology).Membraneswere incubated for 2minwith the westernBright Sirius HRP Kit (Witec). Signal detection was performedwith ImageQuant LAS4000 mini Luminescent Image Analyzer (GE Healthcare).

Histochemical Staining

GUS staining was performed as described previously (Little et al., 2007).

Accession Numbers

Sequence data from this article can be found in The Arabidopsis InformationResource (www.arabidopsis.org) under the following accession numbers:MYC2 (At1g32640), MYC3 (At5g46760), MYC4 (At4g17880), JAZ1 (At1g19180),JAZ10 (At5g13220), PDF1.2 (At5g44420), ORA59 (At1g06160), NPR1(At1g64280), EIN2 (At5g03280), VSP2 (At5g24770), WRKY75 (At5g13080),LOX3 (At1g17420), MAP (At1g54010), BHLH3 (At4g16430), BHLH13




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(At1g01260), BHLH14 (At4g00870), BHLH17 (At2g46510), PR1 (At2g14610),CHIT (At2g43570), and TI (At1g73260).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Expression of JA marker genes during eggextract-induced SA/JA cross talk.

Supplemental Figure S2. Oviposition-induced SA/JA cross talk is abol-ished in npr1-1 ein2-1 mutant.

Supplemental Figure S3. Egg extract treatment does not stabilize JAZproteins.

Supplemental Figure S4. MYC3 and MYC4 proteins are degraded in re-sponse to egg extract treatment.

Supplemental Figure S5. Expression of MYC2, MYC3, and MYC4 in SA/JA cross talk conditions.

Supplemental Figure S6. Expression of oviposition-inducible genes.

Supplemental Figure S7. Expression of WRKY75 in SA/JA cross-talk con-ditions.

Supplemental Figure S8. Genotyping of npr1-1 ein2-1, 35S:WRKY75, andwrky75-25 lines.

Supplemental Table S1. List of primers for quantitative PCR analyses.

ACKNOWLEDGMENTS

We thank Blaise Tissot for maintenance of the plants, and Roland Reist andOliver Kindler (Syngenta, Stein, Switzerland) for providing S. littoralis eggs. Wethank Xinnian Dong for providing npr1-1 ein2-1, Martin Hülskamp forwrky75-25, Olivier Van Aken for 35S:WRKY75, Christiane Gatz for ora59, GreggHowe for 35S:JAZ1-GUS, Ted Farmer for 35S:JAZ10-GUS, Roberto Solano for 35S:MYC3-GFP and 35S:MYC4-GFP, and Susheng Song for bhlh3 bhlh13 bhlh14 bhlh17.

Received January 9, 2016; accepted February 12, 2016; published February 16,2016.

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