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. 2023 Jul 3;120(28):e2302226120. doi: 10.1073/pnas.2302226120

Fig. 5.

Fig. 5.

MED10b/MED7 mediate jasmonate-dependent transcription repression. (A) A Y2H assay result showing the interaction between NbJAZs and NbMED10b or between NbJAZs and NbMED7. The transformed yeast cells were grown on the SD/-TL and the SD/-TLHA plates, respectively. (B) Quantitative RT-PCR analysis results showing the expressions of representative marker genes involved in the JA signaling pathways in wild-type (WT) N. benthamiana Nbmed7 knockout, and the Nbmed10b knockout plants. The expression levels of NbActin in these samples were used as the internal controls. Data are presented as the means ± SE (three independent biological sample per treatment). (C) Quantitative RT-PCR analysis results showing the expressions of representative marker genes involved in the JA signaling pathways in N. benthamiana coexpressing Sw-5b with EV, NSm with EV, or Sw-5b with NSm. The expression levels of NbActin in these samples were used as the internal controls. Data are presented as the means ± SE (three independent biological samples per treatment). (D) Transient overexpression of NbMYC2 transcription factor activated the expression of luciferase (LUC) driven by the NbLOX2 promoter, and addition of NbJAZ1 into NbMYC2 reduced the expression of LUC. The luciferase activity (integrated optical density, IOD) in the treated leaves was quantified and shown in the right. Data are presented as the means ± SE (three independent biological samples per treatment). (E) Coexpression of NbJAZ1, or NbMED7 and NbJAZ1, or NbMED7, NbMED10b, and NbJAZ1, or NbMED7, NbMED10b, NbMED26, NbMED31, and NbJAZ1 with NbMYC2 reduced the expression of LUC. The luciferase activity (integrated optical density, IOD) in the treated leaves was quantified and shown in the right. Data are presented as the means ± SE (three independent biological samples per treatment). (F) Coexpression of Sw-5b CC with NbMYC2, NbJAZ1, NbMED7 and NbMED10b, or NbMYC2, NbJAZ1, NbMED7, NbMED10b, NbMED26, and NbMED31 activated the expression of LUC. The luciferase activity (integrated optical density, IOD) in the treated leaves was quantified and shown in the right. Data are presented as the means ± SE (three independent biological samples per treatment). (G) N. benthamiana plants were sprayed with DMSO or 100 µM MeJA. At 2 d post treatment, phytohormone-treated leaves were inoculated again with TSWV infectious clone [L(+)opt+M(–)opt+SR(+)eGFP] via agro-infiltration. The infiltrated N. benthamiana plant leaves were harvested at 60 hpai and imaged for eGFP fluorescence loci under an inverted fluorescence microscope (Scale bars, 800 μm.). (H) Western blot assay results showing the accumulation level of eGFP at 60 hpai in the infiltrated leaves shown in (G), using GFP antibody.