Fig. 5: MED16 antagonizes MBRs-mediated MED25 degradation.
a Yeast three-hybrid (Y3H) assays showing that addition of MBR1, MBR2 dramatically reduced the MED16–MED25 interaction (Top three panels) and addition of MED16 reduced the MED25–MBR1/2 interactions (Bottom four panels). (Top three panels) Yeast cells cotransformed with pGADT7-MED16 and pBridge-MED25-MBR1,2 were dropped onto SD/-Trp/-Leu (SD/-2) and SD/-Trp/-Leu/-Ade/-His (SD/-4) media to assess the MED25–MED16 interaction. The cotransformed yeast cells were dropped onto SD/-Trp/-Leu/-Ade/-His/-Met (SD/-5) medium to induce expression of MBR1 or MBR2. (Bottom four panels) Yeast cells cotransformed with pGADT7-MBR1, 2 and pBridge-MED25-MED16 were dropped onto SD/-2 and SD/-4 media to assess the MED25/MBR1,2 interactions. The cotransformed yeast cells were dropped onto SD/-5 medium to induce expression of MED16. AD, activation domain fusion; BD, binding domain fusion. b In vitro quantitative pull-down assays showing that MED16 competes with MBR2 for binding to MED25. For each sample, the amounts of MED25-Flag and GST-MBR2C were equal, and His-MED16 was added according to the indicated gradient. GST-MBR2C was pulled down by MED25-Flag immobilized on an anti-Flag resin. Proteins were eluted and analyzed using an anti-Flag antibody. The white asterisk indicates the position of His-MED16 (upper) and His-TF (lower). c MED16 prevents MED25 from degradation in planta. MED25-myc was infiltrated with MBR2-Flag and/or MED16-GFP co-expression. d Co-IP assays of MED25 and MYC2 in the MYC2-myc/WT and MYC2-myc/med16-3 plants. Ten-day-old MYC2-myc/WT and MYC2-myc/med16-3 seedlings were treated with 100 mM MeJA for the indicated times. Proteins extracted from MYC2-myc/WT and MYC2-myc/med16-3 plants were immunoprecipitated using an anti-myc antibody and immunoblotted using an anti-MED25. In (a–d), all experiments were repeated three times, and similar results were obtained. e Proposed working model for the mechanistic roles of MED16 coordinates with MED25 in regulating JA-induced activation of MYC2. MED16 competes with MBRs for binding to vWF-A domain of MED25, thereby recruiting MED25 into Mediator complex, which impairs the interaction ability of MBRs with MED25 and stabilizes MED25. In addition, MED16 also promotes hormone-dependent enhancement of protein interactions between MYC2 and MED25, thereby activating JA-responsive gene expression. Source data are provided as a Source Data file.
