Figure 1.

14-3-3 interacts with NtCDPK1. A, 14-3-3 interacts with NtCDPK1 in an autophosphorylation- and Ca²⁺-dependent manner. GST and nonphosphorylated or autophosphorylated GST-NtCDPK1 were immobilized on glutathione beads and incubated with His-14-3-3 in the presence of Ca²⁺ or EGTA (EG). NtCDPK1-bound proteins were subjected to SDS-PAGE, followed by immunoblot analysis with anti-14-3-3 antibody for the detection of His-14-3-3. GST fusion proteins were visualized by Coomassie Brilliant Blue (CBB) staining. The values below the top gel indicate the relative strength levels of signals after standardization using the signals of Coomassie Brilliant Blue staining of GST or GST-NtCDPK1 as a loading control. The value of GST as a negative control was set to 1. This experiment was repeated three times with similar results. B, Statistical analysis of the data shown in A. The bar graph represents means and sd (n = 3). A two-way ANOVA was used to separate the effects of Ca²⁺, autophosphorylation, and their combination. Ca²⁺ main effect (P < 0.05), autophosphorylation main effect (P < 0.05), and Ca²⁺ × autophosphorylation interaction effect (P < 0.05) were detected. Different letters above the bars indicate significant differences among the treatments (P < 0.001, Tukey’s honestly significant difference test). C, BiFC analysis reveals in vivo interaction between NtCDPK1 and 14-3-3. BiFC constructs were delivered into leaf cells of tobacco by particle bombardment. After 24 h, the cells were visualized by epifluorescence microscopy. Coexpression of YFP^N-14-3-3 and NtCDPK1-YFP^C (top) and coexpression of YFP^N-14-3-3 and YFP^C (bottom) are shown. Reconstituted YFP fluorescence (left), RFP fluorescence as a control for transfection efficiency (center), and bright-field images (right) are shown. This experiment was repeated three times with similar results. Bars = 50 µm. D, Coimmunoprecipitation (Co-IP) assay also reveals the in vivo interaction between NtCDPK1 and 14-3-3. Leaf cell extracts from a transgenic plant overexpressing NtCDPK1-GFP were immunoprecipitated with anti-NtCDPK1 or anti-His antibody. Anti-His antibody was used as a negative control for immunoprecipitation (IP). Coprecipitated 14-3-3 with NtCDPK1-GFP was detected by immunoblot analysis with anti-14-3-3 antibody. NtCDPK1-GFP was detected by anti-NtCDPK1 antibody. [See online article for color version of this figure.]