Fig. 6: Interaction, subcellular localization and functional model of RACB and RIPb in planta.

a Bimolecular fluorescence complementation (BiFC) assays with single epidermal cells transiently transformed with split-YFP constructs. Images represent typical cell recordings of a minimum of 20 cells per experiment and from two independent transformation experiments with similar results. Images represent z-stacks of 25 confocal sections, each with a 1.5–2 µm increment. Scale bar = 50 µm. b Quantification of BiFC signals from images was performed with constant settings. Signal intensity (mean fluorescence intensity [MFI]) was measured over the whole cell. The ratio between split-YFP and free mCherry signal was calculated. Signals were measured in 10 cells for each construct. Letters indicate significance by one-way ANOVA (Tukey’s multiple comparison test; P = 0.05). c Yeast-two-hybrid assays with wild-type RACB and CA RACB in bait plasmids and RIPb constructs in prey plasmids. As negative control, empty vectors (EV) were used. For transformation control, yeast was dropped on SD medium without leucine (-Leu) and tryptophan (-Trp). For the selection of positive interactions, SD medium was used additionally lacking adenine (-Ade) and histidine (-His). d Model of prenylated dimeric RACB-RIPb at the membrane. RACB is anchored through its C-terminal prenylated cysteine 194. RIPb is depicted in yellow, RACB in gray. e View from top down the membrane. The C-terminus of RIPb (yellow) binds to the membrane surface. The gray spheres in the membrane represent the phosphate moieties. f Electrostatic surface coloring of the RACB-RIPb complex indicates a positive charge on RACB and RIPb, facilitating membrane attachment.