Figure 6.

SEC11 Binding to SYP121 in Vivo Is Suppressed by the L128R Site Mutation.

rBiFC analysis of nYFP fusions of SEC11 and SEC11^L128R and their interaction with SYP121^ΔC and SYP121^{ΔC,L185A,D186A}.

(A) rBiFC images collected from tobacco transformed using the pBiFCt-2in1-NC tricistronic vector (schematic above). Images are (left to right) YFP (BiFC) fluorescence, RFP fluorescence as a cell marker, and bright-field. Constructs (top to bottom) included coding sequences for nYFP-SEC11 and nYFP-SEC11^L128R with either the empty cassette (control) or X-cYFP fusions with SYP121^ΔC and SYP121^{ΔC,L185A,D186A}. Images for SEC11^L128R alone were similar to those for SEC11 and have been omitted for simplicity. Bar = 40 μm. Immunoblot analysis verifying expression of the fusion proteins and RFP marker as indicated (left). Expression of fusion constructs SEC11 and SEC11^L128R was detected using αSEC11 antibody (Assaad et al., 2001), of SYP121^ΔC and SYP121^{ΔC,L185A,D186A} using αSYP121 antibody (Tyrrell et al., 2007), and of RFP using αRFP antibody.

(B) Means ± se of rBiFC data from five independent experiments, each with 30 images taken at positions selected at random over the leaf surface including the images in (A). rBiFC fluorescence ratios were calculated from the mean fluorescence intensities determined from each YFP/RFP image pair after background subtraction. Significance of difference is indicated by letters (P < 0.02).