Interaction between MYC2 and ETHYLENE INSENSITIVE3 modulates antagonism between jasmonate and ethylene signaling in Arabidopsis

Abstract

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 MYC2 and the ET-stabilized transcription factor ETHYLENE-INSENSITIVE3 (EIN3) modulates JA and ET signaling antagonism in Arabidopsis thaliana. MYC2 interacts with EIN3 to attenuate the transcriptional activity of EIN3 and repress ET-enhanced apical hook curvature. Conversely, EIN3 interacts with and represses MYC2 to 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 fluctuating environments.

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, JAZ1Δ3A, eto1-1, ctr1-1, ein2-1, ein3-1 ein1-3 (ein3 eil1), and hls1-1 grown in the dark on MS medium supplied without (Mock) or with 5 μM MeJA (JA), 10 μM ACC, or 10 μM ACC plus 5 μM MeJA (ACC+JA). (B) Real-time PCR analysis for HLS1 in 4-d-old etiolated Col-0 (WT), eto1-1, ctr1-1, and ein3 eil1. Actin8 was used as the internal control. Data are means (±sd) of three biological replicates. Lowercase letters indicate significant differences by one-way ANOVA analysis with SAS software (P < 0.05). (C) Real-time PCR analysis for HLS1 in 4-d-old etiolated Col-0 (WT) treated with mock, 100 μM MeJA (JA), 100 μM ACC, or 100 μM ACC plus 100 μM MeJA (ACC+JA) for 6 h. Actin8 was used as the internal control. Data are means (±sd) of three biological replicates. Lowercase letters indicate significant differences by one-way ANOVA analysis with SAS software (P < 0.05).

Figure 2.

COI1 Acts Upstream of EIN3/EIL1 and HLS1 in Regulation of Apical Hook Formation. The hook phenotypes of 4-d-old etiolated Arabidopsis seedlings Col-0 (WT), coi1-2, ein3 eil1, coi1-2 ein3 eil1, hls1-1, and coi1-2 hls1-1 grown in the dark on MS medium supplied without (Mock) or with 5 μM MeJA (JA), 10 μM ACC, or 10 μM ACC plus 5 μM MeJA (ACC+JA).

Figure 3.

MYC2, MYC3, and MYC4 Function 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), myc2-2 myc3 (myc2/3), myc2-2 myc4 (myc2/4), myc2-2 myc3 myc4 (myc2/3/4), myb21 myb24 myb57 (myb21/24/57), and gl3 egl3 tt8 grown in the dark on MS medium supplied without (Mock) or with 5 μM MeJA (JA), 10 μM ACC, or 10 μM ACC plus 5 μM MeJA (ACC+JA). (B) Real-time PCR analysis for HLS1 in the indicated 4-d-old etiolated seedlings. Actin8 was used as the internal control. Data are means (±sd) of three biological replicates. Lowercase letters indicate significant differences by one-way ANOVA analysis with SAS software (P < 0.05). (C) Real-time PCR analysis for HLS1 in the 4-d-old etiolated Col-0 (WT) and myc2-2 myc3 myc4 (myc2/3/4) treated with mock, 100 μM MeJA (JA), 100 μM ACC, or 100 μM ACC plus 100 μM MeJA (ACC+JA) for 6 h. Actin8 was used as the internal control. Data are means (±sd) of three biological replicates. Lowercase letters indicate significant differences by one-way ANOVA analysis with SAS software (P < 0.05).

Figure 4.

MYC2, MYC3, and MYC4 Interact with EIN3 and EIL1. (A) BiFC assay to detect the interactions of MYC2, MYC3, and MYC4 with EIN3 and EIL1. EIN3 and EIL1 were fused with the N-terminal fragment of YFP (nYFP) to form EIN3-nYFP and EIL1-nYFP, respectively. MYC2, MYC3, and MYC4 were fused with the C-terminal fragment of YFP (cYFP) to generate cYFP-MYC2, cYFP-MYC3, and cYFP-MYC4. YFP fluorescence was detected in N. benthamiana leaves coinfiltrated with the combination of indicated constructs. The positions of nuclei were shown by 4′,6-diamidino-2-phenylindole (DAPI) staining. (B) In vitro pull-down assay to verify the interaction of MYC2 with EIN3. The purified MBP and MBP-MYC2 fusion protein were incubated with the total protein from N. benthamiana leaves with transient expression of flag-EIN3. Bound proteins were washed, separated on SDS-PAGE, and immunoblotted with the anti-flag antibody (α-flag; top panel). The input lane shows the protein level of flag-EIN3 expressed in leaves of N. benthamiana. The positions of purified MBP and MBP-MYC2 separated on SDS-PAGE are marked with asterisks (bottom panel; stained by Coomassie blue). (C) Co-IP assay to verify the interaction of MYC2 with EIN3 in planta. The flag-EIN3 was coexpressed without (Control) or with myc-MYC2 or myc-COI1 in the N. benthamiana leaves. The total protein extracts from the N. benthamiana leaves with transient expression of flag-EIN3, flag-EIN3 plus myc-MYC2, or flag-EIN3 plus myc-COI1 were immunoprecipitated with the anti-c-myc antibody-conjugated agarose and were further detected by immunoblot using anti-flag antibody and anti-c-myc antibody.

Figure 5.

MYC2 Represses Transcriptional Activity of EIN3 and 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 HLS1 promoter 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 (±sd) of three biological replicates. Asterisks represent Student’s t test significance between EIN3 and EIN3+MYC2 or EIL1 and EIL1+MYC2 samples (**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 ERF1 promoter by EIN3 (E) and EIL1 (F) is repressed by MYC2. The P_ERF1-LUC reporter was cotransformed with the indicated constructs. Data are means (±sd) of three biological replicates. Asterisks represent Student’s t test significance between EIN3 and EIN3+MYC2 or EIL1 and EIL1+MYC2 samples (**P < 0.01).

Figure 6.

Mutations in EIN3 and EIL1 Block the Exaggerated Hook Curvature of myc2 and myc2 myc3 myc4. (A) The hook phenotypes of 4-d-old etiolated Arabidopsis Col-0 (WT), myc2-2 (myc2), jin1-2 myc3 myc4 (myc2/3/4), myc2-2 ein3 eil1 (myc2 ein3 eil1), jin1-2 myc3 myc4 ein3 eil1 (myc2/3/4 ein3 eil1), and ein3 eil1 grown in the dark on MS medium supplied without (Mock) or with 5 μM MeJA (JA), 10 μM ACC, or 10 μM ACC plus 5 μM MeJA (ACC+JA). (B) Real-time PCR analysis for HLS1 in the indicated 4-d-old etiolated seedlings. Actin8 was used as the internal control. Data are means (±sd) of three biological replicates. Lowercase letters indicate significant differences by one-way ANOVA analysis with SAS software (P < 0.05). (C) Real-time PCR analysis for HLS1 in the indicated 4-d-old etiolated seedlings treated with mock, 100 μM MeJA (JA), 100 μM ACC, or 100 μM ACC plus 100 μM MeJA (JA+ACC) for 6 h. Actin8 was used as the internal control. Data are means (±sd) of three biological replicates. Lowercase letters indicate significant differences by one-way ANOVA analysis with SAS software (P < 0.05).

Figure 7.

Mutations in EIN3 and EIL1 Repress 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, ein3 eil1, and myc2-2 ein3 eil1 at day 2 after inoculation with mock or B. cinerea (B.c) spores. (B) The lesion sizes on rosette leaves at day 2 after inoculation with B. cinerea spores. Data are means (±sd) of three biological replicates. Asterisks represent Student’s t test significance compared with the wild type (**P < 0.01). (C) Quantitative real-time PCR analysis of ERF1, PDF1.2, and ORA59 in 12-d-old wild type, myc2-2, ein3 eil1, and myc2-2 ein3 eil1 treated with mock or 100 μM MeJA (JA) for 6 h. Actin8 was used as the internal control. Data are means (±sd) of three biological replicates. Different letters indicate significant 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.

Figure 8.

EIN3 and EIL1 Antagonize 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 MYC2 acts as a transcriptional activator, while EIN3 and EIL1 attenuate the transcriptional activation function of MYC2. Data are means (±sd) of three biological replicates. Asterisks represent Student’s t test significance between MYC2 and MYC2+EIN3 or MYC2+EIL1 samples (**P < 0.01).

Figure 9.

Mutations in MYC2, MYC3, and MYC4 Block the Enhanced Defense against Insect Attack in ein3 eil1. (A) and (B) Real-time PCR analysis for VSP1, VSP2, TAT3, CYP79B3, BCAT4, and BAT5 in the 12-d-old seedlings Col-0 (WT), jin1-2 myc3 myc4 (myc2/3/4), jin1-2 myc3 myc4 ein3 eil1 (myc2/3/4 ein3 eil1), and ein3 eil1 treated with mock or 100 μM MeJA (JA) for 6 h. Actin8 was used as the internal control. Data are means (±sd) of three biological replicates. Different letters indicate significant 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 (0 d) and 7 d after feeding (7 d) with wild-type, ein3 eil1, jin1-2 myc3 myc4 (myc2/3/4), or jin1-2 myc3 myc4 ein3 eil1 (myc2/3/4ein3 eil1) plants. Bars = 1 mm. (D) Larval weight of S. exigua reared on wild-type, ein3 eil1, jin1-2 myc3 myc4 (myc2/3/4), or jin1-2 myc3 myc4 ein3 eil1 (myc2/3/4ein3 eil1) plants for 7 d. Ten larvae as one sample were weighed together to obtain one datum for average weight. Fifty larvae (five independent samples) for each genotype in each biological experiment were used. Values are means (±sd) from three biological replicates. Lowercase letters indicate significant differences by one-way ANOVA analysis with SAS software (P < 0.05). [See online article for color version of this figure.]

Figure 10.

A Simplified Model for JA and ET Signaling Antagonism. (A) Model for JA and ET antagonistic action in regulating hook curvature, wounding, and defense against insect attack. In response to JA signaling, SCF^COI1 recruits JAZs for ubiquitination and degradation. MYC2, MYC3, and MYC4 (indicated as MYC2) are then released to interact with and repress EIN3 and EIL1 (indicated as EIN3), which leads to attenuation of ET-enhanced hook curvature. ET signal inactivates the ET receptors (indicated as ETR1) and the negative regulator CTR1 to mediate EIN2 translocation into nucleus and to stabilize EIN3 and EIL1. EIN3 and EIL1 then interact with and repress MYC2, MYC3, and MYC4 to 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 defense 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 MYC2 interact with and repress ET-stabilized EIN3 and EIL1 (indicated as EIN3). In response to JA signaling, JAZ proteins are degraded to derepress EIN3/EIL1, leading to the increased disease resistance against necrotrophic pathogen B. cinerea (indicated as disease resistance) (Zhu et al., 2011). Meanwhile, JA-induced JAZ degradation releases MYC2, which counteracts EIN3 and EIL1 to 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 MYC2 to 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 figure.]