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
