Fig. 4
Analysis of the interaction between N and NbUBR7 in vivo and in vitro. a Subcellular localization of NbUBR7. NbUBR7 was fused to citrine (NbUBR7-citrine) followed by agroinfiltration. Confocal analysis was performed at 2 dpi. NbUBR7 is present in the cytoplasm and in the nucleus (left panel). The rectangular region on top right of the image indicated a lower gain value image to confirm the nuclear localization of NbUBR7. Scale bar represents 10 µm. NbUBR7 expression was confirmed by western blot analysis using antibodies against MYC tag (right panel). Empty vector served as the control. The molecular weight size markers in kDa are indicated at the left. b BiFC analysis of the interaction between NbUBR7 and N as well as its different domains. NbUBR7 fused to C-terminus of citrine (NbUBR7YC) was coexpressed with native promoter-driven full-length N (gN), TIR domain deleted N (gNΔTIR) or TIR domain alone (N-TIR) fused to N-terminus of citrine in the N. benthamiana leaves. Confocal analysis was performed at 2 dpi. gNYN and p50U1YC served as the positive control. Scale bars = 10 µm. c In vitro GST-pull down assay to examine the interaction between NbUBR7 and the TIR domain of N. The purified GFP-tagged TIR domain of N or C-terminal region of the respiratory burst oxidase homolog D (RBOHD) were incubated with GST-tagged NbUBR7. After being pulled-down with glutathione-sepharose beads, the proteins were detected by Western blot (WB) with anti-GFP or anti-GST antibodies. Source data are provided as a Source Data file
