Fig. 4: CPK1 interacts with the N termini of CNGC5/6/9.

a, b The interactions of CPK1 with the N termini of CNGC5/6/9 were observed using the Y2-H assay (a) and firefly luciferase complementation assay in N. benthamiana leaves (b). The expression of CPK1 and the N termini of CNGC5/6/9 in N. benthamiana leaves was verified by immuno-blot assay (c). The interactions of CPK1 to the N termini of CNGC5/6/9 were further verified by in vitro Co-IP: CNGC5-N (d), CNGC6-N (e), and CNGC9-N (f). The Y2-H assay was performed using the classical GAL4 Y2-H system. For the in vitro Co-IP assay, the CDS of CPK1 and CNGC5-N, CNGC6-N, CNGC9-N were cloned into the vectors 1305-UBQ10: Myc and 1305-UBQ10-Venus-Flag, respectively, downstream the UBQ10 promoter. The vectors were co-transformed into N. benthamiana leaves, and the combined expression of mCherry-Myc and CNGC9-N-Venus-Flag was used as control. The fused proteins were extracted, and the antibodies against the Myc and Flag tags were used for immuno-blot analysis. Three biological replicates were conducted for all experiments. Abbreviations: C5 (CNGC5), C6 (CNGC6), C9 (CNGC9), C14 (CNGC14), and TM (transmembrane) in (a), W72 (WRKY72), 5 N (CNGC5-N), 6 N (CNGC6-N), and 9 N (CNGC9-N) in (b), C5-N-Ve (CNGC5-N-Venus) in (d), C6-N-Ve (CNGC6-N-Venus) in (e), and C9-N-Ve (CNGC9-N-Venus) in (f). Source data are provided as a Source Data file.