PVY 6K1 interacted with Nb14-3-3h. A) Co-IP analysis of the interaction between 6K1 and Nb14-3-3h. The 6K1-eGFP was coexpressed with Nb14-3-3h-Myc in N. benthamiana leaves, and the infiltrated leaves were harvested at 3 dpi. Total proteins were extracted and immunoprecipitated with GFP-Trap beads from individual samples followed by protein gel analysis using a GFP-specific or Myc-specific antibody. B) Analysis of the interaction between 6K1 and Nb14-3-3h in vitro. Purified eGFP-Nb14-3-3h and eGFP were coincubated with 6K1-GST or GST in different combinations. After being immunoprecipitated with GST-Trap beads, the precipitate was analyzed through Western blot using a GFP-specific or GST-specific antibody. C) BiFC analysis of the interaction between Nb14-3-3h and 6K1 in N. benthamiana leaves systematically infected with PVY-mCherry. Nb14-3-3h-nYFP and CUS-cYFP or GUS-nYFP and 6K1-cYFP were coexpressed as negative controls. The mCherry fluorescence was used to monitor the virus infection. The infiltrated leaves were examined under a confocal microscope at 2 dpi. The experiments were repeated 3 times independently with similar results. Scale bars = 20 μm. D) Confocal images showed that Nb14-3-3h-DsRed colocalized with 6K1-GFP. The Agrobacterium culture harboring Nb14-3-3h-DsRed was inoculated to the PVY-6K1-GFP systematically infected leaves, and the pictures were taken at 2 dpi. Scale bars = 20 μm. The second row showed a magnification of dotted rectangle regions in the first row. E) Co-IP analysis of the interaction between 6K1-eGFP and Nb14-3-3h^S63E-Myc, Nb14-3-3h^QQR-Myc, or Nb14-3-3h^Y89Q-Myc. The N. benthamiana leaf tissues coexpressing various combinations of proteins were harvested at 3 dpi. Total proteins were extracted and immunoprecipitated with GFP-Trap beads. The input and IP samples were then analyzed through Western blot assay using a Myc-specific or GFP-specific antibody. F) GST pull-down analysis of the interaction between 6K1 and 3 dimerization defective mutants. Purified 6K1-GST or GST was incubated with eGFP-Nb14-3-3h, eGFP-Nb14-3-3h^S63E, eGFP-Nb14-3-3h^QQR, eGFP-Nb14-3-3h^Y89Q, or eGFP. After immunoprecipitation with GST-Trap beads, the IP and input samples were analyzed through Western blot assay using a GST or GFP antibody.