Characterization of velvet family proteins regulating fungal development and virulence in the peach shoot blight fungus <i>Diaporthe amygdali</i>

Peach shoot blight, caused by Diaporthe amygdali, is a serious challenge for China’s peach industry, contributing to annual yield losses of 20–50%. Despite its economic significance, the molecular mechanisms underlying D. amygdali pathogenicity remain poorly characterized. Velvet proteins are fungal-specific regulatory factors that exhibit functional diversity across plant pathogenic fungi; however, their roles in D. amygdali remain uncharacterized. Here, we identified and systematically characterized four velvet proteins in D. amygdali, including DaVeA, DaVelB, DaVelC, and DaVosA. Expression analysis revealed that DaVEA, DaVELB, and DaVELC exhibited significantly higher expression during the asexual stage; notably, DaVEA and DaVELB maintained markedly upregulated expression at the invasive stages. We further obtained single-gene velvet protein deletion mutants and their complemented strains, finding that deletion of DaVEA or DaVELB significantly reduced vegetative growth, abolished asexual reproduction, weakened the pathogenicity, increased melanin accumulation, altered colony surface hydrophobicity, and compromised stress tolerance. In contrast, deletion of DaVELC or DaVOSA affected only asexual reproduction. We further observed that deletion of DaVEA or DaVELB led to significant downregulation of the genes encoding cell wall-degrading enzymes and the fusicoccin biosynthetic gene cluster–a phytotoxic diterpene metabolite implicated in fungal virulence. Moreover, the four velvet proteins form complex interactions in D. amygdali. Our findings expand the functional repertoire of velvet proteins in fungal phytopathogens and clarify DaVeA and DaVelB as potential molecular targets for future studies aimed at developing control strategies.