Figure 4A BiFC co-localization analyses of αa and TGB1 in N. benthamiana leaf cells at 3 dpi. YFPn and YFPc was fused to the TGB1 and the αa proteins, respectively. Combinations of γb, γb3CS and the TBSV P19 control are shown above the micrographs. YFP signals were visualized by confocal microscopy at 3 dpi and depicted as a false-green color. Scale bars, 30 μm. B Quantification of the numbers of YFP foci per visual field (n = 10). Data information: In (A, , representative data are shown and three biological replicates had similar results. In (B, the box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles, whiskers indicate minimum and maximum values, central band indicates the median. Letters in the chart denote statistically significant differences among groups according to the Duncan's multiple range test (p < 0.05).C Co-IP assays to determine in vivo interactions between TGB1 and γb or γb3CS. N. benthamiana leaf tissues co-agroinfiltrated with RNAα, RNAγγb-GFP or RNAγ3CS-GFP, and TGB1-3xFlag plasmids were harvested at 3 dpi. Total proteins were immunoprecipitated with anti-Flag beads. Input and immunoprecipitated proteins (IP) were analyzed by Western blotting with anti-GFP or anti-Flag antibodies.D Confocal microscopy analyses showing RFP-TGB1 localization at the chloroplast periphery in N. benthamiana leaves agroinfiltrated with αa-GFP and CFP-γb plasmids. CFP-γb or CFP-γb3CS were co-expressed with RFP-TGB1 and αa-GFP in N. benthamiana leaves. Chloroplast autofluorescence was displayed in false pink color. The third and fourth rows show leaves treated with 400 μM 2-BP inhibitor or the DMSO control at 1 dpi. Two days later, confocal microscopy observations of RFP-TGB1, αa-GFP, and CFP-γb were performed. Figures on the right indicate the normalized fluorescence intensity of GFP, RFP, and CFP channels along the white dashed line in the merged confocal images. Scale bars, 10 μm. Data information: In , representative data are shown and three biological replicates had similar results.E Statistical analyses of the GFP and RFP co-localization in panel D. Pearson correlation coefficient (Pearson's r) of GFP and RFP at the periphery of the chloroplasts was measured with the ImageJ PSC colocalization tool (n = 8). F Relative percentages of RFP-TGB1 foci that co-localized with αa-GFP and CFP-γb at the periphery of the chloroplast images shown in panel D (n = 8). Data information: In E and F), the box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles, whiskers indicate minimum and maximum values, central band indicates the median. Letters in the chart denote statistically significant differences among groups according to the Duncan's multiple range test (p < 0.05).G Chloroplast extraction experiment for detection of TGB1 chloroplast localization. Plasmids harboring αa-GFP and RFP-TGB1 were co-infiltrated into N. benthamiana along with CFP-γb or CFP-γb3CS. Intact chloroplast preparations isolated from infiltrated N. benthamiana leaves at 4 dpi were subjected to Western blot analyses. RFP-TGB1 was analyzed with an anti-TGB1 antibody. The αa-GFP, CFP-γb, and CFP-γb3CS proteins were detected with an anti-GFP antibody. NTRC was the loading control for the chloroplast preparations Phosphoenolpyruvate carboxylase (PEPC) served as a control to assess cytosolic contamination of isolated chloroplasts. Data information: In G), representative data are shown and three biological replicates had similar results.