# Systematic Identification of the Serine Protease Family (StSPs) and Functional Characterization of the Secretory Protein StSP8-4 for Pathogenicity in Setosphaeria turcica

## TL;DR

This study identifies and characterizes serine protease genes in Setosphaeria turcica, finding that one gene, StSP8-4, is crucial for causing corn leaf blight disease.

## Contribution

The study systematically identifies and functionally characterizes the serine protease family in Setosphaeria turcica, revealing a key pathogenicity gene.

## Key findings

- 74 serine protease genes were identified and classified into 12 subfamilies in Setosphaeria turcica.
- StSP8-4 is essential for pathogenicity but not for normal fungal growth.
- RNA-seq and qRT-PCR confirmed high expression of StSP genes during infection stages.

## Abstract

Northern corn leaf blight, a global foliar disease caused by Setosphaeria turcica, poses a serious threat to corn yield and quality. Current control strategies rely mainly on breeding resistant varieties and applying chemical agents. However, frequent variation in the pathogen often leads to rapid loss of cultivar resistance, while the emergence of pesticide resistance results in increased chemical usage, causing environmental pollution. This study investigates a key enzyme family in S. turcica—serine proteases—to elucidate their genetic composition and functions. We identified 74 genes in this family and classified them into 12 subfamilies, with five confirmed as secretory proteins. Most genes showed high expression during infection, particularly StSP8-4, which exhibited significantly elevated expression during pathogenesis. Functional experiments demonstrated that this gene is essential for fungal pathogenicity without affecting normal growth. These findings provide new insights into the pathogenic mechanisms of northern corn leaf blight and offer potential value for developing environmentally friendly disease control strategies.

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.