Fig 5. Analyses of regions responsible for γb and TGB1 protein interactions.
(A) BiFC analyses of TGB1 and truncated γb protein mutants. Co-expression of the N-terminal halves of YFP-fused γb or truncated derivatives and TGB1-YFPc under control of the 35S promoter in N. benthamiana leaves. The subscript numbers show γb amino acids used for BiFC assays. YFP signals were visualized by confocal microscopy at 3 dpi. Scale bars, 10 μm. (B) GST pull-down assays of interactions between TGB1 and different truncated γb mutant proteins. The His-tagged TGB1 protein was incubated with different GST-tagged γb variants. After incubation with glutathione agarose beads, the pull-down products were analyzed by Western blotting with anti-His or anti-GST antibody. Sizes (in kDa) of molecular weight markers and the antibodies used for detection are shown on the left. The white arrows indicate the target bands. (C) Y2H assays of yeast transformants expressing γb or truncated TGB1 mutants as BD or AD fusions. Various combinations of yeast two-hybrid vectors are indicated on the left. A dilution series (10−1, 10−2, 10−3, and 10−4) of yeast cells were spotted on yeast synthetic drop-out media (SD/-Trp-Leu or SD/-Trp-Leu-His-Ade) supplemented with X-α-Gal. Interactions of γb and TGB1 confirmed by Y2H assays serve as a positive control. (D) Confocal Analyses of cell-to-cell movement of BSMV γb mutant derivatives. A. tumefaciens harboring pCB301-α, pCB301-β or various pCB301-γ-derivative constructs were co-infiltrated into the N. benthamiana leaves, and analyses were performed at 3 dpi. The dfBSMVBM26 mutant is a control. Scale bars, 100 μm. The white arrow indicates the direction of BSMV movement. (E) Quantification of BSMV movement efficiency shown in panel D. The green (infiltrated and peripheral BSMV invaded regions) and red fluorescent (Agrobacterium infiltrated regions) areas were measured by ImageJ software (n = 7). The Y-axis indicates the relative sizes of the green areas in comparison with the red-colored areas. Different letters in the chart denote statistically significant differences among different groups according to the Duncan’s multiple range test (P < 0.05). (F) Western anti-GFP antibody blotting to detect GFP accumulation in leaf regions adjacent to the red-colored areas shown in Figure 5D. Actin immunoblots shown below are loading controls. Leaf samples for Western blots were excised under a Leica stereo fluorescent microscope as in Fig 4D. Sizes (in kDa) of molecular weight markers are shown on the left and antibodies used for detection are shown on the right of each panel.