Fig. 3: CPK1 physically interacts with CNGC5/6/9.
a Y-2H results show the interactions of CPK1 with CNGC5/6/9. b Luciferase complementation analysis in N. benthamiana leaves show the protein interactions of CPK1 with CNGC5/6/9, but no interaction of WRKY72 with CNGC5/6/9. c Immuno-blot assay data show the expression of CPK1, WRKY72, and CNGC5/6/9 in N. benthamiana leaves, supporting the luciferase complementation results. d BiFC assays in Xenopus laevis oocytes show the interactions of CPK1 with CNGC5/6/9 and the subcellular localization of CNGC5/6/9 in the plasma membrane of oocytes. The Y-2H assay was performed using the mbSUS system, and the CDS of CPK1 and CNGC5/6/9 were cloned into the vectors pNXgate32-3HA (Nub) and pMetYCgate (Cub), respectively. For luciferase complementation assay in N. benthamiana leaves, the CDS of CPK1 was cloned into the modified vector pCambia1300-nLUC, the CDS of CNGC5/6/9 were cloned into the modified vector pCambia1300-cLUC. Agrobacterium (strain GV3101) carrying the nLUC and cLUC vectors were co-injected into N. benthamiana leaves. The leaves were sprayed with 1 mM/L luciferin in the dark 48 hours after the injection, and photos were taken 7 min later. For the BiFC assay in Xenopus laevis oocytes, the CDS of CPK1 and CNGC5/6/9 were cloned into the vectors pGEMKN-YFPC and pGEMKN-YFPN, respectively. cRNA was transcribed in vitro. A total of volume of 50 nL of the cRNA mixture containing 25 ng of each vector was micro-injected into the oocytes. The oocytes were incubated in the ND96 solution for 2-3 days at 16 ± 0.5 °C, and images of the oocytes were captured under a confocal microscope. Three biological replicates were performed for Y2-H, Luciferase complementation, and immune-blot assays. 5 oocytes were analyzed in BiFC assay for each experiment in (d). Scale bars, 0.1 mm (d). W72 denotes WRKY72 in (b) and (c). Source data are provided as a Source Data file.
