Fig. 2. Functions of Osp24 and its signal peptide and cysteine residues.

a Infection cushions formed by the wild-type strain PH-1 (WT) and osp24 deletion mutant on wheat lemma at 2 dpi were examined by SEM under ×800 amplification. Scale bar, 10 µm. b. Thick sections of infected wheat heads were examined for invasive hyphae (red arrowheads) in the rachis tissues at 5 dpi. Scale bar, 20 µm. c Relative biomass of F. graminearum in infected wheat heads at 5 dpi was determined by qPCR. Mean and standard deviation were estimated with data from three (n = 3) independent biological replicates (marked as black dots on the bar). The asterisk * indicates significant differences (P = 0.05) based on Bootstrap analysis. d The positions of eight cysteine residues in Osp24 and the predicted intra-molecular disulfide bond between C94 and C105. SP, signal peptide. e The yeast suc2 mutant YTK12 and its transformants expressing the empty vector pSUC2 or vectors with the signal peptide from Osp24 and Avr1 (positive control) were assayed for growth on CMD-W or YPRAA plates and invertase activity in TTC medium. f Representative images of wheat heads infected with PH-1, osp24 mutant, and the osp24/OSP24 and osp24/OSP24^ΔSP transformants were photographed at 14 dpi. g Wheat coleoptiles were infected with PH-1 expressing the Osp24:mCherry:NLS construct and examined for mCherry signals in plant cells. Scale bar, 20 µm. h Representative images of wheat heads infected with PH-1, osp24 mutant, and transformants of osp24 expressing OSP24 mutant alleles carrying the indicated C-to-A mutations were photographed at 14 dpi. i Western blots of mixtures of equal amounts of total proteins isolated from wheat heads of cultivar Xiaoyan 22 (XY22) and recombinant Osp24-GST, Osp24^C94A-GST, or Osp24^C105A–GST proteins incubated for the indicated times after the addition of 10 mM ATP were detected with an anti-GST antibody. Detection with an anti-actin antibody was used as a loading control. j Different concentrations (cells/ml) of yeast transformants expressing the indicated bait and prey constructs were assayed for growth on SD-Trp-Leu-His plates and LacZ activity. k Transient expression of Osp24 suppressed programmed cell death triggered by BAX or INF1. At the indicated spots, N. benthamiana leaves were infiltrated with Agrobacterium cells expressing GFP/BAX/INF1 alone or infiltrated with BAX/INF1-expressing cells at 18 h after infiltration with GFP or Osp24 first. Representative leaves were photographed 5 days after infiltration.