Plant Cell-2014-Song-263-79

Interaction between MYC2and ETHYLENE INSENSITIVE3 Modulates Antagonism between Jasmonate and Ethylene Signaling in Arabidopsis C W

Susheng Song,a Huang Huang,a Hua Gao,a Jiaojiao Wang,a Dewei Wu,a Xili Liu,b Shuhua Yang,c Qingzhe Zhai,d Chuanyou Li,d Tiancong Qi,a,1and Daoxin Xie a,1,2

a Tsinghua-Peking Center for Life Sciences,MOE Key Laboratory of Bioinformatics,School of Life Sciences,Tsinghua University, Beijing100084,China

b Department of Plant Pathology,China Agricultural University,Beijing100193,China

c College of Biological Sciences,China Agricultural University,Beijing100193,China

d Stat

e Key Laboratory o

f Plant Genomics,National Centre for Plant Gene Research,Institute of Genetics and Developmental Biology, Chinese Academy of Sciences,Beijing100101,China

Plants have evolved sophisticated mechanisms for integration of endogenous and exogenous signals to adapt to the changing environment.Both the phytohormones jasmonate(JA)and ethylene(ET)regulate plant growth,development,and defense.In addition to synergistic regulation of root hair development and resistance to necrotrophic fungi,JA and ET act antagonistically to regulate gene expression,apical hook curvature,and plant defense against insect attack.However,the molecular mechanism for such antagonism between JA and ET signaling remains unclear.Here,we demonstrate that interaction between the JA-activated transcription factor MYC2and the ET-stabilized transcription factor ETHYLENE-INSENSITIVE3(EIN3) modulates JA and ET signaling antagonism in Arabidopsis thaliana.MYC2interacts with EIN3to attenuate the transcriptional activity of EIN3and repress ET-enhanced apical hook curvature.Conversely,EIN3interacts with and represses MYC2to inhibit JA-induced expression of wound-responsive genes and herbivory-inducible genes and to attenuate JA-regulated plant defense against generalist herbivores.Coordinated regulation of plant responses in both antagonistic and synergistic manners would help plants adapt to?uctuating environments.

INTRODUCTION

Sessile plants have evolved sophisticated mechanisms for in-tegration of endogenous and exogenous signals to regulate their growth,development,and defense responses,which bene?ts their survival in the changing environment.Both ethylene(ET) and jasmonate(JA)are essential plant hormones that regulate various plant developmental processes and perse defense re-sponses(Kieber,1997;Bleecker and Kende,2000;Guo and Ecker,2004;Broekaert et al.,2006;Howe and Jander,2008; Browse,2009;Shan et al.,2012;Wasternack and Hause,2013). ET signal is perceived by its receptors ETHYLENE RESPONSE1 (ETR1),ETR2,ETHYLENE RESPONSE SENSOR1(ERS1),ERS2, and ETHYLENE INSENSITIVE4(EIN4)(Hua and Meyerowitz, 1998)to repress CONSTITUTIVE TRIPLE RESPONSE1(CTR1) (Kieber et al.,1993),which activates EIN2(Alonso et al.,1999;Ju et al.,2012;Qiao et al.,2012;Wen et al.,2012)and subsequently stabilizes EIN3and EIN3-LIKE1(EIL1)(Chao et al.,1997;Guo and Ecker,2003;Potuschak et al.,2003;Gagne et al.,2004)to me-diate various ET responses,including hypocotyl growth(Zhong et al.,2012),apical hook formation(Knight et al.,1910;An et al., 2012),root growth(Ortega-Martínez et al.,2007;R?zicka et al., 2007),?owering(Ogawara et al.,2003;Achard et al.,2007),fruit ripening(Burg and Burg,1962;Theologis et al.,1992),leaf se-nescence(Gepstein and Thimann,1981;Li et al.,2013),freezing tolerance(Shi et al.,2012),and resistance against pathogen in-fection(Alonso et al.,2003;Chen et al.,2009).

JA plays essential roles in the regulation of plant development and defense.Upon perception of JA signal(Fonseca et al.,2009; Yan et al.,2009;Sheard et al.,2010),the F-box protein CORO-NATINE INSENSITIVE1(COI1)(Xie et al.,1998;Yan et al.,2009) recruits the JASMONATE ZIM-DOMAIN(JAZ)proteins(Chini et al.,2007;Thines et al.,2007;Yan et al.,2007)for degradation, which leads to the release of various downstream factors,in-cluding MYC2/JASMONATE INSENSITIVE1(JIN1),MYC3,and MYC4(Cheng et al.,2011;Fernández-Calvo et al.,2011;Niu et al.,2011),as well as WD-repeat/bHLH/MYB complex(Qi et al., 2011),MYB21,MYB24,and MYB57(Mandaokar et al.,2006; Song et al.,2011)and the IIId bHLH factors(Nakata et al.,2013; Song et al.,2013b),which regulate perse JA-mediated func-tions.These functions include root growth(Dathe et al.,1981; Chen et al.,2011),apical hook formation(Turner et al.,2002),?owering(Robson et al.,2010),stamen development(McConn and Browse,1996;Song et al.,2011,2013a),leaf senescence

1These authors contributed equally to this work.

2Address correspondence to daoxinlab@1a639f5dccbff121dc36833b.

The author responsible for distribution of materials integral to the?ndings

presented in this article in accordance with the policy described in the

Instructions for Authors(1a639f5dccbff121dc36833b)is:Daoxin Xie(daoxinlab@

1a639f5dccbff121dc36833b).

C Some?gures in this article are displayed in color online but in black and

white in the print edition.

W Online version contains Web-only data.

1a639f5dccbff121dc36833b/cgi/doi/10.1105/tpc.113.120394

The Plant Cell,Vol.26:263–279,January2014,1a639f5dccbff121dc36833b?2014American Society of Plant Biologists.All rights reserved.

(Ueda and Kato,1980;Shan et al.,2011),secondary metabolism (De Geyter et al.,2012;Schweizer et al.,2013),drought responses (Seo et al.,2011),wounding responses(Mason and Mullet,1990; Acosta et al.,2013;Mousavi et al.,2013),and defense against pathogen infection(Thomma et al.,1998;Vijayan et al.,1998; Melotto et al.,2006;Rowe et al.,2010;Yang et al.,2012;Zheng et al.,2012)and insect attack(McConn et al.,1997;Hu et al., 2013a).

Previous studies showed that both JA and ET concomitantly and synergistically regulate plant defense against necrotrophic fungi(Penninckx et al.,1998;Thomma et al.,1998;Thomma et al., 1999)and root hair development(Zhu et al.,2006).It is so far reported that such JA-ET signaling synergy is mediated by de-repression of ET-stabilized EIN3and EIL1:JAZ proteins directly interact with and repress EIN3/EIL1,while JA induces JAZ degradation to derepress EIN3and EIL1(Zhu et al.,2011).JA-induced EIN3and EIL1activation(Zhu et al.,2011)and ET-induced EIN3and EIL1stabilization(Guo and Ecker,2003; Potuschak et al.,2003;Gagne et al.,2004)mediate JA and ET signaling synergy in the regulation of root hair development and resistance against necrotrophic fungal infection.

In addition to their synergistic regulation,JA and ET also act antagonistically in regulating expression of wound-responsive genes(Rojo et al.,1999;Lorenzo et al.,2004)and metabolite biosynthetic genes(Mikkelsen et al.,2003).JA represses apical hook formation(Turner et al.,2002)and positively regulates plant defense against insect attack(Fernández-Calvo et al.,2011; Schweizer et al.,2013),while ET functions oppositely(Guzmán and Ecker,1990;Mewis et al.,2005,2006;Bodenhausen and Reymond,2007).However,the molecular mechanism for such antagonism between JA and ET signaling remains unclear.In this study,we show that MYC2interacts with EIN3and EIL1to re-press the transcriptional activity of EIN3and EIL1in Arabidopsis thaliana and consequently to inhibit ET-regulated apical hook formation;similarly,we found that EIN3and EIL1interact with and repress MYC2,further attenuate JA-induced expression of wound-responsive genes and herbivory-inducible genes,and in-hibit plant defense against insect attack.This molecular,bio-chemical,and genetic evidence reveals that interactions of the JA-activated transcription factor MYC2with the ET-stabilized transcription factors(EIN3and EIL1)repress their respective transcriptional activities to modulate JA and ET signaling antag-onism,which provides insights into how plants integrate various phytohormone signals to coordinately regulate plant development, growth,and defense.

RESULTS

MYC2,MYC3,and MYC4Function Redundantly to Mediate JA-Inhibited Apical Hook Curvature

The formation of the apical hook helps cotyledons and meristem tissues protrude from the soil without being damaged.Previous studies showed that ET induces hook curvature(Guzmán and Ecker,1990),whereas JA antagonizes the ET pathway that functions in apical hook formation in etiolated Arabidopsis seed-lings(Turner et al.,2002).Consistent with previous observations (Turner et al.,2002),1-aminocyclopropane-1-carboxylic acid (ACC),the ET biosynthesis precursor,enhanced the apical hook curvature,while JA obviously suppressed the ET-enhanced hook curvature of the dark-grown Arabidopsis wild-type seedlings (Figure1A);the coi1-1mutant exhibited an exaggerated apical hook curvature(Figure1A).As expected,the mutants with ET overproduction(ethylene overproducer1[eto1-1])or constitutive ET responses(ctr1-1)exhibited constitutive exaggerated hook curvature,while the mutants de?cient in ET signaling(ein2-1and ein3-1eil1-3)displayed obvious reduction in hook curvature (Figure1A).We further found that the exaggerated hook cur-vature in eto1-1and ctr1-1was clearly inhibited by JA(Figure 1A),which indicates that JA functions downstream of ETO1and CTR1to repress ET-regulated hook curvature.

HOOKLESS1(HLS1)is a central positive regulator of apical hook development(Lehman et al.,1996;An et al.,2012)(Figures 1A and1B).The mutants eto1-1and ctr1-1,with high level of HLS1expression,exhibited exaggerated hook curvature,while the mutant ein3-1eil1-3,with low levels of HLS1expression, displayed reduced hook curvature(Figures1A and1B).ACC treatment induced HLS1expression and exaggerated apical hook curvature in the wild type,and ACC-induced HLS1expression and hook formation were obviously repressed by JA treatment (Figures1A and1C).These results suggest that JA represses HLS1expression to inhibit ET-enhanced hook formation and imply that JA acts upstream of HLS1to repress hook curvature. To genetically verify whether JA acts upstream of HLS1,we further generated the double mutant coi1-2hls1-1and the triple mutant coi1-2ein3eil1via genetic cross of coi1-2with hls1-1or ein3eil1.The results in Figure2showed that the coi1-2exhibited an exaggerated hook curvature,while no hook was formed in coi1-2hls1-1and hls1-1.Similar data were also observed for coi1-2ein3eil1(Figure2).Suppression of the exaggerated hook curvature in coi1-2by the hls1-1and ein3eil1mutations suggests that COI1acts upstream of the EIN3/EIL1-HLS1cascade to regulate apical hook formation.

To identify the key components responsible for repression of hook curvature in JA signaling pathway,we examined apical hook phenotypes in JA signaling mutants.As expected,the coi1-1mutant exhibited an exaggerated apical hook curvature (Figure3A)(Turner et al.,2002).JAZ1D3A transgenic plants, with high levels of JAZ proteins(Thines et al.,2007),also displayed an exaggerated apical hook curvature(Figure1A). Among the key transcription factors targeted by JAZ proteins, MYB21/MYB24/MYB57(Song et al.,2011)and WD-repeat/ bHLH/MYB complex(Qi et al.,2011)are not involved in the suppression of hook formation,as the myb21myb24myb57 and gl3egl3tt8mutants exhibited wild-type-like hook curva-ture(Figure3A).

Interestingly,the myc2single mutant exhibited a mildly exaggerated apical hook curvature compared with the wild type(Figure3A);the apical hook curvature was clearly en-hanced in the double mutants myc2myc3and myc2myc4 (Figure3A),while the triple mutant myc2myc3myc4dis-played the strongest apical hook curvature(Figure3A),which is similar to that observed in the coi1mutant(Figure3A).The hook curvature of the single or double mutants(myc2,myc2 myc3,and myc2myc4)could be further inhibited by JA

264The Plant Cell

treatment,whereas the triple mutant (myc2myc3myc4)was completely insensitive to JA-inhibited hook curvature (Figure 3A).Furthermore,JA was unable to repress ACC-enhanced hook curvature in myc2myc3myc4(Figure 3A).Consistent with the exaggerated hook curvature,the expression of HLS1was upregulated in the mutants myc2,myc2myc3,myc2myc4,and myc2myc3myc4(Figures 3B).Furthermore,ACC-enhanced HLS1expression in myc2myc3myc4was not repressed by JA treatment (Figures 3C).

Taken together,the results in Figure 3suggest that MYC2,MYC3,and MYC4function redundantly to mediate JA-inhibited hook curvature.

MYC2,MYC3,and MYC4Interact with EIN3and EIL1Having shown that MYC2,MYC3,and MYC4function re-dundantly to repress HLS1expression and mediate JA inhibition of ET-enhanced hook curvature (Figure 3),we further found that MYC2,MYC3,and MYC4were able to interact with EIN3and EIL1(Figure 4),activators of HLS1(An et al.,2012).

The yellow ?uorescent protein (YFP)–based bimolecular ?uo-rescence complementation (BiFC)assays showed that coex-pression of EIN3-nYFP (fusion of EIN3with N-terminal fragment of YFP)or EIL1-nYFP with cYFP-MYC2(fusion of MYC2with C-terminal fragment of YFP),cYFP-MYC3,or cYFP-MYC4

produced

Figure 1.JA Suppresses the ET-induced Apical Hook Formation.

(A)The hook phenotypes of 4-d-old etiolated Arabidopsis seedlings Columbia-0(Col-0;WT),coi1-1,JAZ1D 3A ,eto1-1,ctr1-1,ein2-1,ein3-1ein1-3(ein3eil1),and hls1-1grown in the dark on MS medium supplied without (Mock)or with 5m M MeJA (JA),10m M ACC,or 10m M ACC plus 5m M MeJA (ACC+JA).

(B)Real-time PCR analysis for HLS1in 4-d-old etiolated Col-0(WT),eto1-1,ctr1-1,and ein3eil1.Actin8was used as the internal control.Data are means (6SD )of three biological replicates.Lowercase letters indicate signi ?cant differences by one-way ANOVA analysis with SAS software (P <0.05).(C)Real-time PCR analysis for HLS1in 4-d-old etiolated Col-0(WT)treated with mock,100m M MeJA (JA),100m M ACC,or 100m M ACC plus 100m M MeJA (ACC+JA)for 6h.Actin8was used as the internal control.Data are means (6SD )of three biological replicates.Lowercase letters indicate signi ?cant differences by one-way ANOVA analysis with SAS software (P <0.05).

MYC2-EIN3Mediates JA-ET Antagonism 265

strong YFP signals in the nuclei (Figure 4A),while the negative controls did not (Supplemental Figure 1),demonstrating that MYC2,MYC3,and MYC4interact with EIN3and EIL1.

We also used pull-down assays to representatively examine the interaction of MYC2with EIN3(Figure 4B).Puri ?ed maltose binding protein (MBP)–fused MYC2(MBP-MYC2)resin was in-cubated with total protein from Nicotiana benthamiana leaves with transient expression of ?ag-tagged EIN3(?ag-EIN3)and separated by SDS-PAGE for immunoblotting with anti-?ag an-tibody.As shown in Figure 4B,the MBP-MYC2resin could pull down ?ag-EIN3,suggesting that MYC2interacts with EIN3.Furthermore,we performed coimmunoprecipitation (Co-IP)as-says to examine the interaction between MYC2and EIN3in planta.The ?ag-EIN3was coexpressed with myc-tagged MYC2(myc-MYC2)or myc-COI1,respectively,in leaves of N.benthamiana ,and

the total proteins were then used for coimmunoprecipitation.The results showed that ?ag-EIN3was indeed coimmunoprecipitated with myc-MYC2(Figure 4C),but not with the control protein myc-COI1(Figure 4C).Taken together,the BiFC assay,pull-down assay,and Co-IP assay consistently demonstrate that MYC2,MYC3,and MYC4interact with EIN3and EIL1(Figure 4).

MYC2Inhibits Transcriptional Activity of EIN3and EIL1Having shown that MYC2,MYC3,and MYC4interact with EIN3and EIL1,we then investigated whether such interactions affect the transcriptional activity of EIN3and EIL1using an Arabidopsis mesophyll protoplast transfection-based transcriptional activity assay (Hellens et al.,2005).

A previous study showed that EIN3could bind to the pro-moter of HLS1to activate its expression,leading to hook cur-vature (An et al.,2012).We ?rst examined whether MYC2affects the in ?uence of EIN3on HLS1transcription.As expected (An et al.,2012),expression of EIN3dramatically activated the ex-pression of LUC driven by the HLS1promoter (Figures 5A and 5B).However,coexpression of MYC2with EIN3signi ?cantly re-pressed EIN3-activated P HLS1-LUC activity (Figure 5B).Similarly,expression of EIL1activated P HLS1-LUC activity,whereas coex-pression of MYC2repressed EIL1-activated P HLS1-LUC activity (Figures 5A and 5C).The results in Figures 4and 5A to 5C demonstrate that MYC2interacts with EIN3and EIL1to interfere with their effect on the transcription of HLS1.

Having shown that MYC2suppresses the effect of EIN3and EIL1on HLS1transcription,we further examined whether MYC2could repress the effects of EIN3and EIL1on the transcription of another target gene,ETHYLENE RESPONSE FACTOR1(ERF1)(Solano et al.,1998),a key transcription factor that activates the expression of PDF1.2to induce resistance against necrotrophic pathogens (Préet al.,2008;Zarei et al.,2011).As shown in Figures 5D and 5E,overexpression of EIN3activated the ERF1promoter that controlled expression of the LUC gene (P ERF1-LUC ),whereas such EIN3-activated P ERF1-LUC expression was obviously repressed by coexpression of MYC2(Figures 5D and 5E).Furthermore,we found that expression of EIL1also acti-vated P ERF1-LUC activity,while coexpression of MYC2repressed the EIL1-activated P ERF1-LUC activity (Figures 5D and 5F).Taken together (Figures 4and 5),these results demonstrate that MYC2interacts with EIN3and EIL1to attenuate their effect on the transcription of their target genes HLS1and ERF1.

Disruption of EIN3and EIL1Suppresses Exaggerated Apical Hook Formation and Resistance against a Necrotrophic Pathogen in myc2

In agreement with the observation that MYC2represses the transcriptional activity of EIN3and EIL1,abolishment of MYC2in planta is expected to derepress EIN3and EIL1,which would further activate the expression of HLS1(essential for hook cur-vature)and ERF1(vital for resistance against Botrytis cinerea ).Indeed,the myc2mutants (e.g.,myc2myc3,myc2myc4,and myc2myc3myc4)showed increased expression of HLS1(Figures 3B,3C,and 6B),and the myc2mutant exhibited elevated expression of defensive genes,such as ERF1,

OCTADECANOID-RESPONSIVE

Figure 2.COI1Acts Upstream of EIN3/EIL1and HLS1in Regulation of Apical Hook Formation.

The hook phenotypes of 4-d-old etiolated Arabidopsis seedlings Col-0(WT),coi1-2,ein3eil1,coi1-2ein3eil1,hls1-1,and coi1-2hls1-1grown in the dark on MS medium supplied without (Mock)or with 5m M MeJA (JA),10m M ACC,or 10m M ACC plus 5m M MeJA (ACC+JA).

266The Plant Cell

ARABIDOPSIS AP2/ERF59(ORA59)(ERF1homolog),and their

target gene PLANT DEFENSIN1.2(PDF1.2)(Figure 7C).Consistent

with their gene expression patterns,the myc2-related mutants

showed exaggerated hook formation (Figures 3A and 6A)and the

myc2mutant displayed increased resistance against B.cinerea

(Figures 7A and 7B)(Lorenzo et al.,2004).These results suggest

that mutation in MYC2releases EIN3and EIL1to further activate

the expression of HLS1and ERF1,which are vital for hook cur-

vature and disease resistance.To examine whether ein3eil1is able to suppress the exag-gerated hooks in the myc2-related mutants,we generated the myc2ein3eil1and myc2myc3myc4ein3eil1mutants via crossing myc2-related mutants with the ein3eil1mutant.The results in Figure 6A show that the exaggerated hook curvature in myc2-related mutants was repressed by ein3eil1(Figure 6A).Consistently,the elevated expression of HLS1in myc2and myc2myc3myc4was abolished in myc2ein3eil1and myc2myc3myc4ein3eil1(Figure 6B).Furthermore,the expression

of Figure 3.MYC2,MYC3,and MYC4Function Redundantly to Mediate the JA-Inhibited Apical Hook Formation.

(A)The hook phenotypes of 4-d-old etiolated Arabidopsis seedlings Col-0(WT),myc2-2(myc2),myc2-2myc3(myc2/3),myc2-2myc4(myc2/4),myc2-2myc3myc4(myc2/3/4),myb21myb24myb57(myb21/24/57),and gl3egl3tt8grown in the dark on MS medium supplied without (Mock)or with 5m M MeJA (JA),10m M ACC,or 10m M ACC plus 5m M MeJA (ACC+JA).

(B)Real-time PCR analysis for HLS1in the indicated 4-d-old etiolated seedlings.Actin8was used as the internal control.Data are means (6SD )of three biological replicates.Lowercase letters indicate signi ?cant differences by one-way ANOVA analysis with SAS software (P <0.05).

(C)Real-time PCR analysis for HLS1in the 4-d-old etiolated Col-0(WT)and myc2-2myc3myc4(myc2/3/4)treated with mock,100m M MeJA (JA),100m M ACC,or 100m M ACC plus 100m M MeJA (ACC+JA)for 6h.Actin8was used as the internal control.Data are means (6SD )of three biological replicates.Lowercase letters indicate signi ?cant differences by one-way ANOVA analysis with SAS software (P <0.05).

MYC2-EIN3Mediates JA-ET Antagonism 267

HLS1in the myc2myc3myc4ein3eil1mutant was not affected

by JA and/or ACC treatment (Figure 6C).Taken together (Figures

3to 6),these results show that MYC2,MYC3,and MYC4interact

with and attenuate EIN3and EIL1to repress hook curvature.

We further investigated whether ein3eil1could suppress the

increased disease resistance against necrotrophic pathogen B.cinerea in myc2.As shown in Figures 7A and 7B,after in-oculation with spores of B.cinerea ,the myc2mutant exhibited disease resistance,as indicated by the smaller lesion size com-pared with the wild type,which is similar with previous studies demonstrating that MYC2negatively regulates resistance against B.cinerea (Lorenzo et al.,2004;Zhai et al.,2013).The ein3

eil1Figure 4.MYC2,MYC3,and MYC4Interact with EIN3and EIL1.

(A)BiFC assay to detect the interactions of MYC2,MYC3,and MYC4with EIN3and EIL1.EIN3and EIL1were fused with the N-terminal fragment of YFP (nYFP)to form EIN3-nYFP and EIL1-nYFP,respectively.MYC2,MYC3,and MYC4were fused with the C-terminal fragment of YFP (cYFP)to generate cYFP-MYC2,cYFP-MYC3,and cYFP-MYC4.YFP ?uorescence was detected in N.benthamiana leaves coin ?ltrated with the combination of indicated constructs.The positions of nuclei were shown by 49,6-diamidino-2-phenylindole (DAPI)staining.

(B)In vitro pull-down assay to verify the interaction of MYC2with EIN3.The puri ?ed MBP and MBP-MYC2fusion protein were incubated with the total protein from N.benthamiana leaves with transient expression of ?ag-EIN3.Bound proteins were washed,separated on SDS-PAGE,and immunoblotted with the anti-?ag antibody (a -?ag;top panel).The input lane shows the protein level of ?ag-EIN3expressed in leaves of N.benthamiana .The positions of puri ?ed MBP and MBP-MYC2separated on SDS-PAGE are marked with asterisks (bottom panel;stained by Coomassie blue).

(C)Co-IP assay to verify the interaction of MYC2with EIN3in planta.The ?ag-EIN3was coexpressed without (Control)or with myc-MYC2or myc-COI1in the N.benthamiana leaves.The total protein extracts from the N.benthamiana leaves with transient expression of ?ag-EIN3,?ag-EIN3plus myc-MYC2,or ?ag-EIN3plus myc-COI1were immunoprecipitated with the anti-c-myc antibody-conjugated agarose and were further detected by im-munoblot using anti-?ag antibody and anti-c-myc antibody.

268The Plant Cell

double mutant displayed susceptibility,as indicated by the larger

lesion size compared with the wild type (Figures 7A and 7B),

con ?rming that EIN3and EIL1are required for resistance against B.

cinerea (Alonso et al.,2003;Zhu et al.,2011).Similar to ein3eil1,the

myc2ein3eil1triple mutant also exhibited larger lesion size (Figures

7A and 7B),demonstrating that ein3eil1blocked the elevated

resistance against B.cinerea in myc2.Consistently,the upregu-

lated expression of defense genes ERF1,ORA59,and PDF1.2

(Zarei et al.,2011)in myc2was blocked by the ein3eil1mutations

(Figure 7C).Taken together (Figures 4,5,and 7),these results

showed that MYC2interacts with and attenuates EIN3and EIL1to

repress resistance against the necrotrophic pathogen B.cinerea .In summary,the results in Figures 3to 7collectively demon-strate that MYC2interacts with and represses EIN3and EIL1to regulate apical hook formation and resistance against the ne-crotrophic pathogen B.cinerea .EIN3and EIL1Attenuate the Transcriptional Activation Function of MYC2to Repress Plant Defense against Insect Attack Having shown MYC2interacts with and represses EIN3and EIL1to attenuate hook formation and disease resistance (Fig-ures 3to 7),we next explored whether EIN3and EIL1

conversely Figure 5.MYC2Represses Transcriptional Activity of EIN3and EIL1.

(A)The schematic diagram shows the constructs used in the transient transcriptional activity assays of (B)and (C).

(B)and (C)Transient transcriptional activity assays show that activation of HLS1promoter by EIN3(B)and EIL1(C)is repressed by MYC2.The P HLS1-LUC reporter was cotransformed with the indicated constructs.The LUC/REN ratio represents the P HLS1-LUC activity relative to the internal control (REN driven by 35S promoter).Data are means (6SD )of three biological replicates.Asterisks represent Student ’s t test signi ?cance between EIN3and EIN3+MYC2or EIL1and EIL1+MYC2samples (**P <0.01).

(D)The schematic diagram shows the constructs used in the transient transcriptional activity assays of (E)and (F).

(E)and (F)Transient transcriptional activity assays show that activation of ERF1promoter by EIN3(E)and EIL1(F)is repressed by MYC2.The P ERF1-LUC reporter was cotransformed with the indicated constructs.Data are means (6SD )of three biological replicates.Asterisks represent Student ’s t test signi ?cance between EIN3and EIN3+MYC2or EIL1and EIL1+MYC2samples (**P <0.01).

MYC2-EIN3Mediates JA-ET Antagonism 269

Figure 6.Mutations in EIN3and EIL1Block the Exaggerated Hook Curvature of myc2and myc2myc3myc4.

(A)The hook phenotypes of 4-d-old etiolated Arabidopsis Col-0(WT),myc2-2(myc2),jin1-2myc3myc4(myc2/3/4),myc2-2ein3eil1(myc2ein3eil1),jin1-2myc3myc4ein3eil1(myc2/3/4ein3eil1),and ein3eil1grown in the dark on MS medium supplied without (Mock)or with 5m M MeJA (JA),10m M ACC,or 10m M ACC plus 5m M MeJA (ACC+JA).

(B)Real-time PCR analysis for HLS1in the indicated 4-d-old etiolated seedlings.Actin8was used as the internal control.Data are means (6SD )of three biological replicates.Lowercase letters indicate signi ?cant differences by one-way ANOVA analysis with SAS software (P <0.05).

(C)Real-time PCR analysis for HLS1in the indicated 4-d-old etiolated seedlings treated with mock,100m M MeJA (JA),100m M ACC,or 100m M ACC plus 100m M MeJA (JA+ACC)for 6h.Actin8was used as the internal control.Data are means (6SD )of three biological replicates.Lowercase letters indicate signi ?cant differences by one-way ANOVA analysis with SAS software (P <0.05).270The Plant Cell

affect the transcriptional function of MYC2using the GAL4DNA binding domain (GAL4DB)and its binding site [GAL4(4X)-D1-3(4X)-GUS]–based Arabidopsis protoplast transient expression system (Tiwari et al.,2001).

The MYC2gene was fused with GAL4DB under the control of 35S promoter to generate the effector GAL4DB-MYC2.The b -glucuronidase (GUS )gene driven by four copies of GAL4DNA binding sites [GAL4(4x)-D1-3(4x)]was used as a reporter,whereas the LUC gene under the control of 35S promoter was used as the internal control (Figure 8A).Similar with previous observations (Pauwels et al.,2010;Song et al.,2013b),expression of GAL4DB-MYC2clearly increased the GUS/LUC ratio (Figure 8B).However,coexpression of EIN3or EIL1with GAL4BD-MYC2obviously re-duced the GUS/LUC ratio (Figure 8B),suggesting that EIN3and EIL1attenuate the transcriptional activation function of MYC2.To further verify that the EIN3and EIL1repress the tran-scriptional activation function of MYC2,we investigated whether abolishment of EIN3and EIL1in planta would derepress MYC2to enhance the expression of MYC2-regulated genes.Consis-tent with previous studies (Lorenzo et al.,2004;Fernández-Calvo et al.,2011;Schweizer et al.,2013),our results showed that MYC2upregulated JA-induced expression of the wound-responsive genes VEGETATIVE STORAGE PROTEIN1(VSP1),VSP2,and TYROSINE AMINOTRANSFERASE3(TAT3)(Figure 9A)and the herbivore-inducible genes CYP79B3,BRANCHED-CHAIN AMINOTRANSFERASE4(BCAT4),and BILE ACID TRANS-PORTER5(BAT5)(Figure 9B),which are required for the bio-synthesis of the secondary metabolites glucosinolates (Zhao et al.,2002;Kliebenstein et al.,2005;Schweizer et al.,2013).Interestingly,the double mutant ein3eil1exhibited upregulated expression of these wound-responsive genes (VSP1,VSP2,and TAT3)as well as herbivory-inducible genes (CYP79B3,BCAT4,and BAT5)when treated with (or even without)JA compared with the wild type (Figures 9A and 9B).Consistent with the expression levels

of

Figure 7.Mutations in EIN3and EIL1Repress the Enhanced Resistance against Necrotrophic Pathogen Botrytis cinerea in myc2.

(A)Symptoms on detached rosette leaves from 3-week-old plants of Col-0(WT),myc2-2,ein3eil1,and myc2-2ein3eil1at day 2after inoculation with mock or B.cinerea (B.c )spores.

(B)The lesion sizes on rosette leaves at day 2after inoculation with B.cinerea spores.Data are means (6SD )of three biological replicates.Asterisks represent Student ’s t test signi ?cance compared with the wild type (**P <0.01).

(C)Quantitative real-time PCR analysis of ERF1,PDF1.2,and ORA59in 12-d-old wild type,myc2-2,ein3eil1,and myc2-2ein3eil1treated with mock or 100m M MeJA (JA)for 6h.Actin8was used as the internal control.Data are means (6SD )of three biological replicates.Different letters indicate signi ?cant differences by one-way ANOVA analysis with SAS software (P <0.05).Capital letters correspond with each other,and lowercase letters correspond with each other.

MYC2-EIN3Mediates JA-ET Antagonism 271

wound-responsive and herbivory-inducible genes (Figures 9A and 9B),the myc2myc3myc4triple mutant,which was almost com-pletely devoid of glucosinolates (Schweizer et al.,2013),exhibited susceptibility to the generalist herbivores Spodoptera littoralis (Schweizer et al.,2013)and Spodoptera exigua (Figures 9C and 9D),while plant defense against these generalist herbivores was enhanced in the ET-signaling mutants ein3eil1(Figures 9C and 9D),etr1,and ein2(Stotz et al.,2000;Mewis et al.,2005,2006;Bod-enhausen and Reymond,2007).These results demonstrate that the abolishment of EIN3and EIL1derepresses MYC2,which enhances the expression of wound-responsive and herbivore-inducible genes and elevates plant defenses against generalist herbivores.

Further comparison of the gene expression pattern among the double mutant ein3eil1,the pentuple mutant myc2myc3myc4ein3eil1,and the triple mutant myc2myc3myc4showed that JA-induced expression of VSP1,VSP2,TAT3,CYP79B3,BCAT4,and BAT5was signi ?cantly elevated in ein3eil1,whereas such elevated gene expression was obviously repressed by the myc2myc3myc4mutations (Figures 9A and 9B).Consistently,plant defense against insect attack was enhanced in ein3eil1,but disrupted by the myc2myc3myc4mutations (Figures 9C and 9D).These results suggest that mutations in MYC2,MYC3,and MYC4abolish the enhanced expression of wound/herbivore-inducible genes and plant defense against insect attack in ein3eil1.

Taken together (Figures 3to 9),we demonstrated that the interaction between the JA-activated transcription factors (MYC2,MYC3,and MYC4)and the ET-stabilized transcription factors (EIN3and EIL1)represses their respective transcriptional activities to modulate the JA and ET signaling antagonism.EIN3and EIL1interact with and repress MYC2,MYC3,and MYC4to attenuate JA-induced expression of wound-responsive and herbivore-inducible genes and to repress plant defense against the generalist herbivores S.littoralis and S.exigua (Figures 4,8,and 9).Con-versely,MYC2interacts with and represses EIN3and EIL1to inhibit hook formation and disease resistance against a ne-crotrophic pathogen (Figures 3to 7).

DISCUSSION

Land plants live in ?xed location,often encounter environmental stresses,and maintain plasticity in growth and development to adapt to the ?uctuating environment by integrating multiple signals including the endogenous phytohormone signals JA and ET.JA and ET act synergistically to defend against necrotrophic pathogen infection and to promote root hair development (Penninckx et al.,1996;Zhu et al.,2006)via a synergistic reg-ulatory model in which JA induces degradation of JAZ proteins and derepresses ET-stabilized EIN3and EIL1,which interact with JAZs (Zhu et al.,2011).However,such a synergistic regu-latory model is inconsistent with the antagonistic roles of JA and ET signaling in many important processes.For example,JA antagonizes ET to repress apical hook formation (e.g.,exag-gerated hook formation in the coi1mutant)(Figures 1and 2)(Turner et al.,2002),whereas ET antagonizes JA to repress the expression of wound-responsive genes (VSP1,VSP2,and TAT3)and herbivore-inducible genes (CYP79B3,BCAT4,and BAT5)(Figures 9A and 9B)(Rojo et al.,1999;Mikkelsen et al.,2003)and to attenuate plant defense against generalist herbivores (Figures 9C and 9D)(Stotz et al.,2000;Mewis et al.,2005,2006;Bodenhausen and Reymond,2007).

This study reveals a mechanism underlying antagonism be-tween JA and ET signaling.Molecular,biochemical,and genetic evidence suggest an antagonistic regulatory model in which interaction between MYC2,key transcription factor in the JA pathway,and EIN3and EIL1,master transcription factors in the ET pathway,modulates the JA-ET signaling antagonism (Figure 10).MYC2interacts with and represses EIN3and EIL1to inhibit their effects on the transcription of HLS1and ERF1,which re-presses ET-regulated apical hook formation (Figures 10A)and resistance to necrotrophic pathogen (Figures 10B).Conversely,EIN3and EIL1interact with and attenuate MYC2,MYC3,and MYC4to inhibit the expression of wound-responsive and her-bivore-inducible genes and to repress JA-regulated plant

defense

Figure 8.EIN3and EIL1Antagonize the Transcriptional Activation Function of MYC2.

(A)The schematic diagram shows the constructs used in the transient expression assays.

(B)Transient expression assays show that MYC2acts as a transcriptional activator,while EIN3and EIL1attenuate the transcriptional activation function of MYC2.Data are means (6SD )of three biological replicates.Asterisks represent Student ’s t test signi ?cance between MYC2and MYC2+EIN3or MYC2+EIL1samples (**P <0.01).

272The Plant Cell

Figure 9.Mutations in MYC2,MYC3,and MYC4Block the Enhanced Defense against Insect Attack in ein3eil1.

(A)and (B)Real-time PCR analysis for VSP1,VSP2,TAT3,CYP79B3,BCAT4,and BAT5in the 12-d-old seedlings Col-0(WT),jin1-2myc3myc4(myc2/3/4),jin1-2myc3myc4ein3eil1(myc2/3/4ein3eil1),and ein3eil1treated with mock or 100m M MeJA (JA)for 6h.Actin8was used as the internal control.Data are means (6SD )of three biological replicates.Different letters indicate signi ?cant differences by one-way ANOVA analysis with SAS software (P <0.05).Capital letters compare with each other,and lowercase letters compare with each other.

(C)Photograph of S.exigua larvae before feeding (0d)and 7d after feeding (7d)with wild-type,ein3eil1,jin1-2myc3myc4(myc2/3/4),or jin1-2myc3myc4ein3eil1(myc2/3/4ein3eil1)plants.Bars =1mm.

(D)Larval weight of S .exigua reared on wild-type,ein3eil1,jin1-2myc3myc4(myc2/3/4),or jin1-2myc3myc4ein3eil1(myc2/3/4ein3eil1)plants for 7d.Ten larvae as one sample were weighed together to obtain one datum for average weight.Fifty larvae (?ve independent samples)for each genotype in each biological experiment were used.Values are means (6SD )from three biological replicates.Lowercase letters indicate signi ?cant differences by one-way ANOVA analysis with SAS software (P <0.05).

[See online article for color version of this ?gure.]MYC2-EIN3Mediates JA-ET Antagonism 273

against the generalist herbivores S.littoralis and S.exigua (Figure 10A).

Consistent with this antagonistic regulatory model,in the myc2mutant,the absence of MYC2fails to repress the tran-scriptional activity of EIN3and EIL1,leads to the activation of EIN3/EIL1-regulated gene expression (HLS1,ERF1,ORA59,and PDF1.2)and results in enhanced apical hook formation and plant resistance against B.cinerea infection (Figures 3to 7and 10).On the other hand,in the ET signaling mutants (e.g.,ein3eil1and ein2),the absence of EIN3and EIL1enables MYC2,MYC3,and MYC4to induce the expression of wound-responsive genes (VSP1,VSP2,and TAT3)(Figure 9A)(Rojo et al.,1999;Lorenzo et al.,2004)and herbivore-inducible genes (CYP79B3,BCAT4,and BAT5),and enhances plant defense against the herbivores S.littoralis and S.exigua (Figures 4,8,9,and 10A)(Stotz et al.,2000;Mewis et al.,2005,2006;Bodenhausen and Reymond,2007).In wild-type plants,antagonistic regulation between the ET-stabilized transcription factors (EIN3and EIL1)and the JA-activated transcription factors (MYC2,MYC3,and MYC4)would lead to suitable expression of MYC2-dependent genes (VSP1,VSP2,TAT3,CYP79B3,BCAT4,and BAT5)and EIN3-regulated genes (HLS1,ERF1,ORA59,and PDF1.2),resulting in proper plant responses,such as hook formation and defense against the herbivores S.littoralis and S.exigua .

MYC2functions as a key transcription factor to positively regulate perse JA responses (Kazan and Manners,2013),in-cluding root growth (Boter et al.,2004;Lorenzo et al.,2004),secondary metabolism (Dombrecht et al.,2007;Hong et al.,2012;Schweizer et al.,2013),wound response,and plant defense against insect attack (Zhang and Turner,2008;Fernández-Calvo et al.,2011;Schweizer et al.,2013).Surprisingly,MYC2also acts as a negative regulator to repress JA-mediated plant resistance to necrotrophic fungi and pathogenesis-related gene expression (e.g.,PDF1.2)(Anderson et al.,2004;Lorenzo et al.,2004;Zhai et al.,2013).Such MYC2-regulated susceptibility to necrotrophic fungi seems incompatible with the previously reported synergistic model (Zhu et al.,2011).Our results provide a mechanistic un-derstanding of the long-standing question of how MYC2represses JA-regulated plant resistance against necrotrophic fungi:MYC2interacts with and represses EIN3and EIL1,which inhibits ex-pression of the EIN3/EIL1-dependent defense genes (ERF1,ORA59,and PDF1.2)and consequently depresses plant resistance against necrotrophic pathogen

infection.

Figure 10.A Simpli ?ed Model for JA and ET Signaling Antagonism.(A)Model for JA and ET antagonistic action in regulating hook curva-ture,wounding,and defense against insect attack.In response to JA signaling,SCF COI1recruits JAZs for ubiquitination and degradation.MYC2,MYC3,and MYC4(indicated as MYC2)are then released to interact with and repress EIN3and EIL1(indicated as EIN3),which leads to attenuation of ET-enhanced hook curvature.ET signal in-activates the ET receptors (indicated as ETR1)and the negative reg-ulator CTR1to mediate EIN2translocation into nucleus and to stabilize EIN3and EIL1.EIN3and EIL1then interact with and repress MYC2,MYC3,and MYC4to inhibit expression of wound responsive genes (e.g.,VSP1,VSP2,and TAT3)and herbivory-inducible genes (e.g.,CYP79B3,BCAT4,and BAT5)and suppress JA-regulated plant de-fense against generalist herbivores S.littoralis and S.exigua (indicated as wound and defense).

(B)Model for JA and ET crosstalk in regulating plant resistance against necrotrophic pathogen.JAZs and MYC2interact with and repress ET-stabilized EIN3and EIL1(indicated as EIN3).In response to JA signaling,JAZ proteins are degraded to derepress EIN3/EIL1,leading to the in-creased disease resistance against necrotrophic pathogen B .cinerea (indicated as disease resistance)(Zhu et al.,2011).Meanwhile,JA-induced JAZ degradation releases MYC2,which counteracts EIN3and EIL1to prevent excessive disease resistance responses.In addition,other factors,including CYP79B3,which is required for biosynthesis of camalexin (Glawischnig et al.,2004;Kliebenstein et al.,2005),may be also regulated by MYC2to modulate disease resistance.Regulation of plant resistance against B .cinerea might be complicated and modulated by the coordinated action of synergistic and antagonistic mechanisms.[See online article for color version of this ?gure.]

274The Plant Cell

Coordinated regulation of plant responses in both antago-nistic and synergistic manners would help plants adapt to ?uctuating environments.Plant resistance against necrotro-phic fungi might be modulated by a balance between the pre-viously described synergistic mechanism(interaction of JAZs with EIN3)(Zhu et al.,2011)(Figure10B)and our antagonistic regulation model(interaction between MYC2and EIN3)(Figure 10B).Future research should focus on identifying protein do-mains essential for the interaction between MYC2and EIN3and clarifying whether reciprocal repression between MYC2and EIN3occurs on promoters of its target genes or disrupts the binding of MYC2or EIN3to its respective target promoters, which would help advance our understanding of the reciprocal regulation of the transcriptional functions of MYC2and EIN3. JA and ET exhibit opposite effects on many other plant re-sponses.JA enhances anthocyanin accumulation(Qi et al.,2011) and freezing tolerance(Hu et al.,2013b),inhibits seed germination (Miersch et al.,2008),hypocotyl elongation in the light(Chen et al.,2013)and the ozone-induced spreading of cell death (Rao et al.,2000;Tuominen et al.,2004),and delays?owering (Robson et al.,2010).Conversely,ET suppresses anthocyanin accumulation(Jeong et al.,2010)and freezing tolerance(Shi et al., 2012)and enhances seed germination(Linkies et al.,2009;Linkies and Leubner-Metzger,2012),hypocotyl elongation in the light (Zhong et al.,2012),the ozone-induced spreading of cell death (Overmyer et al.,2000),and?owering(Ogawara et al.,2003).It would be interesting to investigate whether these ET-JA antago-nistic actions are mediated by similar interactions between their respective master transcription factors in the JA and ET pathways.

METHODS

Plant Materials and Growth Conditions

The Arabidopsis thaliana mutants coi1-1(Xie et al.,1998),coi1-2(Xu et al., 2002),JAZ1D3A(Thines et al.,2007),myc2-2(Boter et al.,2004),jin1-2 (Lorenzo et al.,2004),myc3(GK445B11)(Fernández-Calvo et al.,2011), myc4(GK491E10)(Fernández-Calvo et al.,2011),jin1-2myc3myc4 (Fernández-Calvo et al.,2011),eto1-1(Guzmán and Ecker,1990),ctr1-1 (Kieber et al.,1993),ein2-1(Alonso et al.,1999),hls1-1(Lehman et al., 1996),ein3-1(Chao et al.,1997),eil1-3/Salk_049679(Binder et al.,2007), myb21myb24myb57(Cheng et al.,2009),and gl3egl3tt8(Qi et al.,2011) were previously described.The higher order mutants myc2-2myc3, myc2-2myc4,myc2-2myc3myc4,ein3-1eil1-3,coi1-2ein3-1eil1-3, coi1-2hls1-1,myc2-2ein3eil1-3,and jin1-2myc3myc4ein3-1eil1-3were generated by genetic crosses using standard techniques.

The Arabidopsis seeds were sterilized with20%bleach,plated on Murashige and Skoog(MS)medium,chilled at4°C for3d,and then transferred to a growth room under a16-h(20to24°C)/8-h(16to19°C) light/dark photoperiod.For hook phenotype analysis,seeds were ster-ilized,chilled,and transferred to a growth chamber at22°C in the dark for 4d.Nicotiana benthamiana was grown in a growth room under a16-h (25to28°C)/8-h(22to25°C)light/dark cycle.

BiFC Assay

For the BiFC assays,the full-length coding sequence(CDS)of Arabi-dopsis EIN3,EIL1,MYC2,MYC3,and MYC4were cloned into the binary nYFP or cYFP vector through the Gateway system(Invitrogen)(Qi et al., 2011).Primer pairs used for the generation of constructs are listed in Supplemental Table1.BiFC assays were performed as previously de-scribed(Qi et al.,2011).Equal concentrations and volumes of re-suspended Agrobacterium tumefaciens strain GV3101harboring the indicated nYFP or cYFP vectors in in?ltration buffer(0.2mM acetosyr-ingone,10mM MgCl

2

,and10mM MES)were mixed and coin?ltrated into leaves of N.benthamiana using a needleless syringe.Two days after in?ltration,the YFP signal was observed using a Zeiss confocal micro-scope(LSM710).Four hours before observation,100m M MG132was in?ltrated into the leaves of N.benthamiana.

Pull-Down Assay

MBP-MYC2(Chen et al.,2011)and MBP proteins were puri?ed from Es-cherichia coli using MBP af?nity chromatography according to Qi et al. (2011).The full-length CDS of EIN3was cloned into the modi?ed pCam-bia1300vector under the control of35S promoter for fusion with three?ag tags to generate?ag-EIN3.Agrobacterium strain harboring?ag-EIN3was in?ltrated into N.benthamiana leaves.After50h,5g of N.benthamiana leaves transiently expressing?ag-EIN3were harvested for total protein extraction in RB buffer(100mM NaCl,50mM Tris-Cl,pH7.8,25mM imidazole,0.1%[v/v]Tween20,10%[v/v]glycerol,EDTA-free complete miniprotease inhibitor cocktail,and20mM2-mercaptoethanol).The ex-tracted total protein was concentrated in centrifugal?lter tubes(Millipore)to 400μL.Coomassie Brilliant Blue was used to con?rm the protein amount. About50μg of puri?ed MBP and MBP-MYC2was incubated with120m L of amylose resin beads for2h at4°C.These amylose resin beads were then washed?ve times with1mL of RB buffer and incubated with200m L of concentrated total proteins containing?ag-EIN3for2h at4°C.After washing?ve times with1mL RB buffer,the mixture was resuspended in SDS loading buffer,boiled for5min,separated on15%SDS-PAGE,and immunoblotted using1:1000dilution for anti-?ag antibody(Abmart).

Co-IP

N.benthamiana leaves were in?ltrated with Agrobacterium strains har-boring?ag-EIN3,?ag-EIN3with myc-MYC2(Zhai et al.,2013),or?ag-EIN3with myc-COI1(Yan et al.,2013).Two days after in?ltration,3g of agroin?ltrated leaves for each combination was collected and homoge-nized in Co-IP buffer containing50mM Tris-HCl,pH7.5,100mM NaCl, 2mM DTT,0.1%Tween20,1mM phenylmethylsulfonyl?uoride,50mM MG132,and complete protease inhibitor cocktail(Roche)and centrifuged twice at16,000g at4°C.The supernatant was concentrated to400m L and incubated with the agarose-conjugated anti-myc matrix(Abmart)for2h (4°C,with rotation),then washed three times with1mL of immunopre-cipitation buffer.After denaturation in100m L of SDS loading buffer,the samples were loaded into15%SDS-PAGE gels,subjected to gel elec-trophoresis,transferred to polyvinylidene?uoride membranes(Millipore), and immunoblotted with anti-?ag antibody(Abmart)and anti-myc anti-body,respectively.

Protoplast Transfection Assay

For the transient transcriptional activity assay,constructs harboring the LUC gene under the control of the;1500-bp or;1523-bp promoter sequences of HLS1or ERF1,respectively,in the pGreenII0800-LUC vector were generated as reporters(Hellens et al.,2005).The renilla luciferase(REN)gene under the control of35S promoter in the pGreenII 0800-LUC vector was used as the internal control.The CDS sequences of EIN3,EIL1,and MYC2were cloned into the pGreenII62-SK vector under the control of the35S promoter and were used as effectors.All primers used for making these constructs are listed in Supplemental Table1. Arabidopsis mesophyll protoplast was prepared and transfected as previously described(Yoo et al.,2007).The?re?y LUC and REN activities

MYC2-EIN3Mediates JA-ET Antagonism275

were measured using the Dual-Luciferase Reporter Assay System (Promega).LUC/REN ratios were presented.

For transient expression assay,the CDS of MYC2fused with GAL4DB (GAL4DB-MYC2)under the control of35S promoter was used as effector. The GUS gene under the control of four copies of upstream GAL4DNA binding sites[GAL4(4x)-D1-3(4x)]was used as the reporter(Tiwari et al.,2001; Zhu et al.,2008).The?re?y LUC gene under the control of35S promoter was the internal control.Primers used for plasmid construction are shown in Supplemental Table1.The preparation and subsequent transfection of Arabidopsis mesophyll protoplasts were performed as described previously(Yoo et al.,2007).The GUS/LUC ratios were presented. Infection with Pathogen

Detached leave from3-week-old plant were inoculated with5m L spores of Botrytis cinerea(SCL2-4,isolated from tomato in2011,Shanghai)(Song et al.,2013b)(105spores/mL)suspended in potato dextrose broth(with potato dextrose broth alone as the control),placed in Petri dishes with0.8% agar,and covered with lids.The lesion diameter from eight leaves for each genotype exhibiting disease symptoms was measured2d after inoculation. Insect Defense Assay with Spodoptera exigua

Newly hatched S.exigua larvae were placed on3-week-old plants(10-h-light/14-h-dark photoperiod)of each genotype for7d of feeding.Ten surviving larvae were weighted as one sample to obtain one datum for average weight.Fifty surviving larvae(?ve independent samples in total) for each genotype in each biological experiment were used.The ex-periment was repeated for three biological replicates.

Quantitative Real-Time PCR

For Figures1B,3B,and6B,Arabidopsis seedlings were grown on MS medium at22°C in the dark for4d.For Figures1C,3C,and6C,seedlings were grown on MS medium at22°C in the dark for4d and then were treated with mock,100m M methyl jasmonate(MeJA),100m M ACC,or 100m M MeJA plus100m M ACC for6h.For Figures7C,9A,and9B, seedlings were grown on MS medium for12d under a16-h(20to24°C)/ 8-h(16to19°C)light/dark photoperiod and then were treated with mock or100m M MeJA for6h.These materials were harvested for RNA ex-traction and subsequent reverse transcription.Real-time PCR analyses were performed using the ABI7500real-time PCR system with the RealMasterMix(SYBR Green I)(Takara)as described previously(Qi et al., 2011).The primers for real-time PCR analysis are presented in Supplemental Table2online.ACTIN8was used as the internal control.The experiment was repeated for three biological replicates.

Accession Numbers

The Arabidopsis Genome Initiative numbers for genes mentioned in this article are as follows:COI1(AT2G39940),MYC2(AT1G32640),MYC3(AT5G46760), MYC4(AT4G17880),JAZ1(AT1G19180),ETO1(At3g51770),CTR1 (AT5G03730),EIN2(AT5G03280),EIN3(AT3G20770),EIL1(AT2G27050), ERF1(AT3G23240),HLS1(AT4G37580),PDF1.2(AT5G44420),MYB21 (At3g27810),MYB24(At5g40350),MYB57(At3g01530),TT8(AT4G09820), GL3(AT5G41315),EGL3(AT1G63650),VSP1(AT5G24780),VSP2 (AT5G24770),TAT3(AT2G24850),ORA59(AT1G06160),CYP79B3 (At2g22330),BCAT4(At3g19710),BAT5(At4g12030),and ACTIN8 (AT1G49240).

Supplemental Data

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

Supplemental Figure1.The Negative Controls for the BiFC Experiments.

Supplemental Table1.Primers Used for Vector Construction.

Supplemental Table2.Primers Used for Quantitative Real-Time PCR Analysis.

ACKNOWLEDGMENTS

We thank Roberto Solano,Joseph Ecker,and John Browse for the mutants and Qilian Qin for S.exigua eggs.The work was supported by the Ministry of Science and Technology(973Program2011CB915404) and the National Science Foundation of China(31230008and91017012). AUTHOR CONTRIBUTIONS

S.S.,T.Q.,and D.X.designed the research.S.S.,H.H.,H.G.,J.W.,D.W., Q.Z.,and T.Q.performed research.S.S.,X.L.,S.Y.,C.L.,T.Q.,and D.X. analyzed data.S.S.,T.Q.,and D.X.wrote the article.

Received November7,2013;revised December9,2013;accepted December13,2013;published January7,2014.

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DOI 10.1105/tpc.113.120394

; originally published online January 7, 2014; 2014;26;263-279Plant Cell Zhai, Chuanyou Li, Tiancong Qi and Daoxin Xie Susheng Song, Huang Huang, Hua Gao, Jiaojiao Wang, Dewei Wu, Xili Liu, Shuhua Yang, Qingzhe Arabidopsis Jasmonate and Ethylene Signaling in Interaction between MYC2 and ETHYLENE INSENSITIVE3 Modulates Antagonism between

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