Fig. 6
TMV p50 effector interferes with the interaction between NbUBR7 and the TIR domain of N. a BiFC analysis of the interaction between p50U1 and NbUBR7. The constructs used for co-infiltration were indicated on the upper left of each panel. GUSYC served as a control for the specificity of associations involving p50U1YN. Scale bars = 10 µm. The expression of p50U1YN and NbUBR7YC were confirmed by western blot analysis (Supplementary Fig. 13b). b Co-IP analysis of the interaction between p50U1 and NbUBR7. HA-tagged UBR7 or citrine was coexpressed with TAP-tagged p50 in the N. benthamiana leaves. Protein extracts from the infiltrated leaf tissues were incubated with anti-HA antibody-conjugated agarose beads. Protein extracts (input) or immunoprecipitated (IP) complexes were separated by SDS-PAGE and probed with anti-MYC or anti-HA antibodies. p50-TAP was co-precipitated with NbUBR7-HA, but not with Citrine-HA. Molecular size markers in kDa are indicated on the left. c BiFC competition analysis showed the inhibitory effect of p50 on the interactions between NbUBR7 and N or NbUBR7 and TIR domain. BiFC constructs together with the empty vector control or the p50-TAP were coinfiltrated into the N. benthamiana leaves. Confocal analysis was performed at 2 dpi. Scale bars = 10 µm. Reconstituted citrine fluorescence intensity was quantified using the Image J software and p50-TAP-infitrated groups were used as the normalizer (=1). Error bars represent standard deviation from the mean (n = 3). Asterisk indicates statistically significant difference between vector and p50-TAP groups (Student’s t-test, **P = 0.004 for upper right panel and **P = 0.007 for bottom right panel). The expression of NbUBR7YC and p50-TAP were confirmed by western blot analysis (Supplementary Fig. 13c). d In vitro competitive GST pull-down assay showed the binding of NbUBR7 to TIR domain was inhibited with the addition of an increasing amount of p50. GST-tagged NbUBR7 or GFP-tagged TIR domain were expressed and purified from E. coli. These two proteins were pulled down with glutathione-agarose in the presence of an increasing concentration of p50 (0.1, 1, 2, 4, 8 µg). Pull-down samples were analyzed by western blot (WB) with anti-GST or anti-GFP antibodies. CBB staining of the increasing amounts of p50 protein is shown in the bottom panel. Source data are provided as a Source Data file
