Fig. 3: TaCAT2 interacted with TaSnRK1α through phosphorylation.

a TaCAT2 interacted with TaSnRK1α in the yeast cell by Y2H. Different concentrations of the yeast transformants were grown on medium lacking SD/Trp-Leu and SD/Trp-Leu-His-Ade plates, respectively; AD-T + BD-53 and AD-T + BD-lam were used as positive control and negative control, respectively. b TaCAT2 interacted with TaSnRK1α in Nicotiana. benthamiana leaves by firefly luciferase complementation assay. SnRK1α-cLUC/PAP6-nLUC was positive control, and empty vectors were negative controls. c TaCAT2 interacted with TaSnRK1α by in vitro GST Pull-down. Marked protein mixtures (Input) or proteins co-purified with TaCAT2-His from the mixtures (pull-down assay) were detected with anti-His and anti-GST antibodies by western blot, respectively. The experiment was independently repeated three times. d The TaSnRK1α interacted with the catalase domain at the N-terminal covering the Ser214Thr site but did not interact with the immune-responsive domain at the C-terminal by Y2H. Different concentrations of the yeast transformants were grown on a medium lacking SD/ Trp-Leu and SD/Trp-Leu-His-Ade plates; AD-T + BD-53 was the positive control, and AD-T + BD-lam was the negative control. e, f TaCAT2^Ser214 was phosphorylated by TaSnRK1α in vitro and in vivo, respectively. Immunoblots detected that the phosphorylation level of TaCAT2^Ser214 with the addition of both TaSnRK1α and GRIK1 showed a stronger signal than that with the individual addition of TaSnRK1α or GRIK1 in vitro (e). Immunoblots detected that the phosphorylation level of TaCAT2^Ser214 was increased after adding TaSnRK1α using an anti-Flag antibody and Pan Phospho-S/T antibody in vivo (f). g, h Differential phosphorylation levels were detected among TaCAT2^Ser214, TaCAT2^Thr214 and TaCAT2^Ala214 after phosphorylation by TaSnRK1α in vitro and in vivo, respectively. Immunoblots detected higher phosphorylation levels of TaCAT2^Ser214 than TaCAT2^Thr214 and TaCAT2^Ala214 by TaSnRK1α and GRIK1 with Pan Phospho-S/T antibody in vitro; Different variants of TaCAT2 were detected with anti-His antibody; TaSnRK1α and GRIK1 were detected with anti-GST antibody (g). Immunoblots detected higher phosphorylation level of TaCAT2^Ser214 than TaCAT2^Thr214 and TaCAT2^Ala214 by TaSnRK1α with Pan Phospho-S/T antibody in vivo; Different variants of TaCAT2 were detected with anti-Flag antibody; TaSnRK1α was detected with anti-GFP antibody (h). All experiments were independently repeated three times.