Systematic Identification of the Serine Protease Family (StSPs) and Functional Characterization of the Secretory...

Serine proteases represent a significant family of proteolytic enzymes, characterized by their serine-dependent catalytic mechanism. These enzymes are integral to various biological processes, including fungal growth, development, and pathogenicity. Despite their importance, the sequence characterization and expression patterns of this protein family in Setosphaeria turcica are not yet fully characterized and remain underexplored. A total of 74 putative serine protease family proteins (StSPs) were identified in S. turcica and classified into 12 subfamilies based on phylogenetic analysis. Structural domain analysis revealed that 24 StSPs contain signal peptides, of which five were experimentally validated as secretory proteins through yeast secretion assays. Expression profiling using RNA-seq data demonstrated that StSPs exhibit distinct expression patterns across different developmental and infection stages, with 61 genes showing high expression during critical infection phases. The expression levels of nine genes were validated via qRT-PCR, and the results were consistent with the RNA-seq data. Among these proteins, StSP8-4 demonstrated elevated expression during the course of fungal infection. Functional characterization of StSP8-4 OE and RNAi strains revealed that this gene plays a crucial role in maintaining fungal pathogenicity, although silencing did not impair conidium or hyphal development. These findings provide valuable insights for further research on serine protease genes in S. turcica.