Fig. 1. Characterization of orphan genes encoding secreted proteins in Fusarium graminearum.

a Comparative analysis of the length of proteins encoded by orphan and non-orphan genes with the Wilcoxon rank-sum test (p < 2.2e−16). b Comparative analysis of the expression levels of orphan and non-orphan genes with the Wilcoxon rank-sum test (p < 2.2e−16). For each gene, the maximum gene expression level was estimated from its transcripts per kilobase million (TPM) in RNA-seq data of conidia, vegetative hyphae, perithecia, and infected wheat heads. c Length and Cys (cysteine) content (%) of mature orphan secretory proteins (OSPs). The three OSPs important for virulence are in red. d Representative images of 3-day-old PDA cultures and 8-dpf (8 days post-fertilization) mating plates of the wild-type strain PH-1 (WT) and the osp24, osp25, and osp44 deletion mutants. e Representative images of wheat heads infected with marked strains were photographed at 14 dpi. f The disease index of WT and three osp mutants. Error bar represents standard deviation (SD) from mean (marked with black dots on the bars) of three independent experiments (n = 3) with at least 10 wheat heads examined in each experiment. Different letters indicate significant differences based on ANOVA analysis followed by Duncan’s multiple range test (P = 0.05). g Mean and standard deviations of DON levels in diseased wheat spikelets inoculated with WT or three osp mutants based on data from three biological replicates (n = 3). No significant differences was observed based on ANOVA analysis followed by Duncan’s multiple range test (P = 0.05). h Distribution of the OSP genes (blue vertical bars) on the four chromosomes of F. graminearum. Red bars and arrowheads indicate the positions of OSP24, OSP25, and OSP44. i Expression levels of the indicated OSP genes based on their TPMs in RNA-seq data of conidia and infected wheat heads²⁰ sampled at 1-dpi, 2-dpi, or 3-dpi. Error bar represents SD from three biological replicates (n = 3).