Fig. 7. VPY interacts with the PAM-localized kinase, KINASE2.
a Colonized root expressing KIN2pro:GUS. Scale bar, 100 μm. Representative image, n = 5 transgenic root systems and 15 infection units. b–e Cells containing arbuscules from roots expressing KIN2pro:KIN2-GFP. KIN2-GFP locates at the periarbuscular membrane and plasma membranes in cells with arbuscules at all stages of development and degeneration. Scale bars are 10 microns. Representative image of n = 60 arbuscule-containing cells. f Co-immunoprecipitation assay with VPY and KIN2. VPY-GFP, or controls, Medtr8g056020-GFP (Mt8g056020-GFP) or GFP were co-expressed in N. benthamiana with KIN2-HA. Proteins were immunoprecipitated using GFP trap magnetic agarose beads and the presence of KIN2-HA was assayed via western blot. This experiment was repeated once with similar results. g in vitro phosphorylation assay with KIN2. KIN2, or its catalytically inactive variant, KIN2K70N were expressed in E.coli. Partially purified proteins were mixed with [32 P]γ -ATP, and auto-phosphorylation and trans-phosphorylation of myelin basic protein (MBP) or VPY was assessed. KIN2 shows kinase activity but did not phosphorylate VPY. This experiment was repeated five times with similar results.