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

Fig. 3.

Fig. 3.

MED10b interacts with MED7, which also negatively regulates Sw-5b-mediated defense. (A) A Y2H assay result showing the interaction between NbMED10b and NbMED7, NbMED26, or NbMED31. The transformed yeast cells were grown on the SD/-TL and the SD/-TLHA dropout plates, respectively. (B) GST pull-down assay results showing the interaction between the GST-NbMED10b and 6×His-NbMED7. 6×His-NbMED7, 6×His-MBP, and GST-NbMED10b were expressed individually in E. coli and then purified. The purified 6×His-NbMED7 or 6×His-MBP was incubated with GST-NbMED10b followed by the pull-down assay using glutathione-sepharose beads. The blots were probed with an anti-GST- or anti-His-specific antibody. (C) Co-IP assay results showing the interaction between the FLAG-NbMED10b and YFP-NbMED7. The FLAG-NbMED10b was used to coimmunoprecipitate YFP-NbMED7 from N. benthamiana leaf extracts. The blots were probed with an anti-FLAG or an anti-YFP antibody. (D) The NbMED7-silenced or nonsilenced Sw-5b transgenic N. benthamiana plants were inoculated with TSWV-infected crude leaf extracts. The inoculated leaves were photographed at 3 dpi. The number and size of HR loci in these inoculated leaves were determined. (E) The accumulation level of TSWV RNA in the inoculated leaves shown in (D) was determined through qRT-PCR using the TSWV N gene–specific primers at 3 d post TSWV inoculation. The expression levels of NbActin in the assayed samples were used as the internal controls. Data are presented as the means ± SE (three biological samples per treatment). (F) The TSWV-inoculated NbMED7-silenced or nonsilenced Sw-5b transgenic N. benthamiana plants were photographed at 14 d post TSWV inoculation. (G) Detection of TSWV RNA in the systemic leaves of the TSWV-inoculated NbMED7-silenced or nonsilenced Sw-5b transgenic N. benthamiana plants through RT-PCR using TSWV N gene–specific primers. The systemic leaves of the TSWV-infected N. benthamiana plants were used as the positive control. The systemic leaves of the mock-inoculated plants were used as the negative controls. The expression levels of NbActin in the assayed samples were used as the internal controls. (H) A photograph showing 12-wk-old wild-type N. benthamiana plant and an Nbmed7 knockout mutant plant. (I) Western blot analysis of TSWV N protein accumulation in the TSWV-inoculated leaves of the wild-type N. benthamiana plant or Nbmed7 mutant plant at 5 dpi using an N protein–specific antibody.