Systematic Identification of the GH28 Gene Family in the Pathogenic Fungus Cytospora pyri and Functional Verification of the Candidate Virulence Gene VP1G_08835

Abstract

Fragrant pear canker, caused by Cytospora pyri, is a major branch disease that reduces the productivity and longevity of Korla fragrant pear orchards in Xinjiang, China. During infection and lesion expansion in woody tissues, C. pyri must breach the host cell wall barrier, in which pectin degradation plays a central role by weakening intercellular adhesion and disrupting tissue integrity. Members of glycoside hydrolase family 28 (GH28), represented mainly by polygalacturonases and other pectin-degrading enzymes, are closely involved in fungal invasion and colonization. However, the composition and virulence-related functions of this gene family encoding for these enzymes in C. pyri remain unclear. In this study, GH28 genes were systematically identified and comparatively analyzed in C. pyri and closely related Cytospora species. Infection-stage expression profiling and functional validation were then performed to assess their roles in pathogenicity. A total of 73 GH28 genes were identified across five Cytospora species, including 15 in C. pyri. Most C. pyri GH28 proteins were predicted to be acidic, hydrophilic, extracellularly secreted proteins carrying conserved motifs. Phylogenetic analysis showed that C. pyri GH28 members were closely related to homologs from C. mali. Genomic distribution analysis revealed that these genes were dispersed across multiple scaffolds, with no obvious tandem duplication events. RT-qPCR analysis showed that all seven candidate GH28 genes were induced during infection of fragrant pear branches, with VP1G_08835 and VP1G_03209 exhibiting strong expression responses at 6 dpi. Functional validation further showed that deletion of VP1G_08835 impaired vegetative growth and reduced lesion length on detached pear branches by 26.62% compared with the wild type, whereas complementation restored these phenotypes. These findings demonstrate that GH28 genes participate in C. pyri infection and identify VP1G_08835 as an important GH28 member required for normal growth and contributing to virulence.