Fig. 4: Roles of Osp24 and TaFROG in modulating TaSnRK1α stability.

a Western blots of the mixtures of equal amount of TaSnRK1α-HIS and total proteins isolated from wheat heads of cultivar Xiaoyan22 inoculated with the wild type PH-1 (XY22 + WT) or osp24 mutant (XY22 + osp24) incubated for the indicated times after the addition of 10 mM ATP were detected with the anti-HIS or anti-actin (loading control) antibody. b Western blots of the mixtures of equal amount of TaSnRK1α-HIS and Osp24-GST incubated for the indicated times were detected with an anti-HIS or anti-GST antibody. Degradation of TaSnRK1α-HIS by Osp24 was not observed. c Recombinant TaSnRK1α-HIS and Osp24-GST proteins were incubated with equal amounts of total proteins isolated from wheat head (XY22) in the presence of 10 mM ATP and used for western blot analyses with the indicated antibodies. d Degradation of TaSnRK1α by total proteins isolated from wheat head inoculated with PH-1 (XY22 + PH-1) was inhibited by proteasome inhibitor MG132. e Western blots of the indicated protein mixtures (Input) or proteins co-purified with TaSnRK1α-HIS from these protein mixtures (HIS IP) were detected with the anti-20S, anti-RBX1, anti-HIS, and anti-GST antibodies. f Yeast transformants expressing the indicated Osp24 or TaFROG bait construct and prey constructs of the N-terminal (1–266 aa) or C-terminal (267–499 aa) region of TaSnRK1α (TaSnRK1α-N and TaSnRK1α-C) were assayed for growth on SD-Trp-Leu-His plates and LacZ activity. g Western blots of the indicated protein mixtures (Input) or proteins co-purified with Osp24-GST from these mixtures (GST IP) were detected with the anti-GST, anti-HIS, or anti-S-tag antibody. The amount of TaSnRK1α-HIS proteins co-immunoprecipitated with Osp24-GST was reduced by the addition of increasing concentrations of TaFROG-S-tag proteins. h Representative wheat heads of cultivar KN199 and its TaFROG overexpressing transgenic line (TaFROG OE) infected with PH-1 were photographed at 8 dpi. i Mean and standard deviation (SD) of the disease index of PH-1 on the labeled wheat lines were estimated from three independent experiments (n = 3) with at least 10 infected wheat heads in each replicate. Different letters indicate significant differences based on ANOVA analysis followed by Duncan’s multiple range test (P = 0.05). j Cell-free degradation assays with recombinant TaSnRK1α-HIS incubated with equal amounts of total proteins isolated from wheat heads of KN199 and TaFROG OE transgenic lines infected by PH-1 for the indicated time after addition of 10 mM ATP. k Western blots of the indicated protein mixtures (Input) or proteins co-purified with TaSnRK1α-HIS from these protein mixtures (HIS IP) were detected with the anti-20S, anti-RBX1, and anti-HIS antibodies. For a, c, d, e, j, and k, detection with an anti-actin antibody was used as the control.