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.
