Fig. 6: Determination of key amino acids that are responsible for CP-14-3-3a interaction.

a Schematic representation of CP truncated mutants used for BiFC assays. b BiFC analysis of regions within CP that interacted with 14-3-3a. Wild-type or truncated CP mutants fused to N-terminus of YFP and 14-3-3a fused to C-terminus were transiently co-expressed in N. benthamiana leaves. Confocal analysis was performed at 3 dpi. Representative results of at least three independent experiments are shown. Scale bars = 50 µm. c Multiple sequence alignment of the C-terminus of CPs from Betanecroviruses. The 14-3-3-binding-like motif is shaded in gray, and the putative 14-3-3-binding site is highlighted in red. d BiFC analysis of the interaction between wild-type or mutant CP and 14-3-3a. Wild-type or mutant CP and 14-3-3a fused to N or C-terminus of YFP were transiently co-expressed in N. benthamiana leaves. Confocal analysis was performed at 3 dpi. Representative results of at least three independent experiments are shown. Scale bars = 50 µm. e Co-IP analysis of the interaction between wild-type or mutant CP and 14-3-3a. N. benthamiana leaves transiently expressing combinations of different proteins were harvested at 3 dpi. Total proteins were immunoprecipitated with anti-Flag beads and detected by western blot with an anti-CP or anti-Flag antibody. f GST pull-down assay to detect the interaction between wild-type or mutant CP and 14-3-3a. Purified His-tagged CP or its derivatives was incubated with GST-14-3-3a or GST protein. His-GFP protein served as the negative control. After incubation with glutathione-Sepharose beads, the pull-down products were analyzed by western blot with an anti-His or anti-GST antibody. For panels e and f, the experiments were repeated three times with similar results.