(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
