Fig. 3: Interaction of Osp24 with TaSnRK1α and function of TaSnRK1α in wheat resistance against F. graminearum.

a Yeast two-hybrid assays to detect the interaction of Osp24 (Bait) with TaSnRK1α (Prey). Different concentrations of the labeled yeast transformants were assayed for growth on SD-Trp-Leu-His plates and LacZ activity. b BiFC assays for the interaction of Osp24 with TaSnRK1α. Leaves of N. benthamiana were agroinfiltrated with a mixture of A. tumefaciens strains expressing the Osp24-nYFP and TaSnRK1α-cYFP constructs. YFP signals were observed at 2 days post‐agroinfiltration. Infiltration with Agrobacterium expressing the TaSnRK1α or Osp24 construct alone was used as the negative control. No YFP signals was observed in these negative controls. Scale bar, 20 µm. c Verification of the Osp24–TaSnRK1α interaction by GST pull down assays. Western blots of the marked protein mixtures (Input) or proteins co-purified with TaSnRK1α-HIS from these mixtures (GST IP) were detected with the anti-HIS and anti-GST antibodies. d Yeast transformants expressing the TaSnRK1α (prey) and marked full-length or truncated Osp24 (bait) were assayed for growth on SD-Trp-Leu-His plates and LacZ activities. SP signal peptide. e Representative wheat heads of cultivar KN199 and its transgenic lines expressing the TaSnRK1α overexpression (TaSnRK1α OE 5 and 6) or silencing (TaSnRK1α RNAi 2 and 12) construct were drop-inoculated with PH-1 and photographed at 7 dpi. f 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 experiment. Different letters indicate significant differences based on ANOVA analysis followed by Duncan’s multiple range test (P = 0.05). g Thick sections of the rachises of wheat heads of KN199 and its TaSnRK1α OE or TaSnRK1α RNAi transgenic line inoculated with PH-1 were examined for invasive hyphae (red arrowheads) at 5 dpi. Scale bar, 50 µm. h Relative biomass of F. graminearum was determined by qPCR in infected wheat heads of KN199 and labeled transgenic lines sampled at 5 dpi. Mean and standard deviation were estimated with data from three (n = 3) independent biological replicates (indicated with black dots on the bar). *Indicates significant differences (P = 0.05) based on Bootstrap analysis.