Characterization of velvet family protein regulating fungal development and virulence of peach shoot blight fungus Diaporthe amygdali

Peach shoot blight, caused by Diaporthe amygdali, leads to annual yield losses of 20-50% in China’s peach industry; however, the molecular mechanisms remain poorly understood. Velvet family proteins, fungal-specific regulatory factors involved in development and virulence, are functionally diverse across plant pathogenic fungi, yet their roles in D. amygdali have not been characterized. Here, we identified and functionally characterized four velvet family proteins in D. amygdali: DaVeA, DaVelB, DaVelC and DaVosA. Transcriptomic analyses revealed that DaVEA, DaVELB and DaVELC exhibited significantly higher expression during the asexual (conidiation) stage; notably, DaVEA and DaVELB maintained markedly upregulated expression at invasive stages. Targeted single-gene deletion demonstrated that loss of DaVEA and DaVELB severely impaired vegetative growth, enhanced melanin accumulation, abolished asexual reproduction, reduced the pathogenicity on peach twigs or fruits, altered colony surface hydrophobicity, and compromised stress tolerance. In contrast, deletion of DaVELC and DaVOSA affected only asexual reproduction process, with no significant impact on other phenotypes. Further mechanistic investigation showed that DaVEA and DaVELB single-gene deletion mutants completely failed to penetrate the plant cell wall, and exhibited substantial downregulation of genes encoding cell wall-degrading enzymes and fusicoccin synthesis, a phytotoxic diterpene implicated in virulence. These results collectively expand the functional repertoire of velvet proteins in fungal phytopathogens and identify DaVeA and DaVelB as promising molecular targets for developing novel control strategies against peach shoot blight.