Fig. 6: PRAF and GNOM proteins physically interact.

a Diagrams depict the domain structure of PRAF4 and GNOM, respectively. “N”, the N-terminus; “C”, the C-terminus. Dashed red box, identified PRAF-GNOM interacting domains. b Pairwise yeast two-hybrid assays show PRAF_FYVE-CC interacts with GNOM_C. Bait, GNOM_N (left) or GNOM_C (right) fused with BD. Prey, subdomains of PRAF4 fused with AD and “-” indicates AD only. “Test” means interaction assays on synthetic dropout media (-LTH). “Control” means yeast growth in rich media (-LT). Auto-activity of the bait protein fusions were suppressed by the addition of 3-AT. The result represents three biological repeats. c BiFC assays in N. benthamiana leaf epidermal cells show interactions of PRAF4 (left) or PRAF8 (right) with GNOM. nYFP N-terminal YFP, cYFP C-terminal YFP. Complemented YFP signals were converted to the ImageJ’s Fire LUT mode. Data represent results of three independent experiments. d In vivo co-IP assays test the interaction between PRAF9 and GNOM. 5- to 7-day-old seedlings co-expressing 35S::GNOM-Myc with PRAF9p::PRAF9-YFP or BASLp::GFP were used for the co-IP experiment. The numbers indicate protein sizes (kDa). The result represents three biological repeats. e Individual protein expression and co-localization of mCherry-PRAF8 (magenta) and GNOM-GFP (green) in stomatal lineage cells. Note the changes of GNOM alone vs. when co-expressed with PRAF8. Protein co-localization is calculated as PCC values (cyan). n = 29 stomatal lineage cells counted. f Co-localization of the BiFC PRAF8-GNOM interaction signals (green) with the WAVE endomembrane markers (magenta) in N. benthamiana leaf epidermal cells. Data represent the results of three independent experiments. Overlapping signals (white circles) were identified between PRAF8-GNOM BiFC with RabC1 (uncharacterized membrane compartments) and RabE1d (Golgi/endosomes) but not with the LE/PVC marker RabF2b. Insets show enlarged views of overlapping signals between BiFC and the WAVE marker. (z), z-stacked confocal images. Scale bars are as indicated (μm).