S-palmitoylation is a dynamically reversible post-translational protein modification that exerts a key regulatory role in biological processes such as signal transduction, metabolic homeostasis, and protein localization and transport in organisms. However, its function in Setosphaeria turcica and its impact on the pathogenicity of this fungus remain unclear. This study aimed to investigate the role of the palmitoyl transferase StPFA5 in regulating the growth, development and pathogenicity of S. turcica, and to lay a foundation for elucidating the molecular mechanisms underlying S. turcica growth and pathogenic processes mediated by S-palmitoylation

StPFA5 was identified via homologous alignment and a phylogenetic tree was constructed. RT-qPCR was performed to determine the expression level of StPFA5 during the fungal infection process. The knockout mutant and complementation strain of StPFA5 were generated using homologous recombination technology. For the WT, ∆StPFA5 and C.∆StPFA5 strains, the growth rate and pathogenicity were determined, and hyphal morphology and hyphal germination were observed. Mycelial melanin was extracted by acid precipitation and alkaline dissolution, and the expression levels of melanin synthesis-related genes were detected by RT-qPCR. In addition, all strains were inoculated on PDA plates containing Congo red and CFW to assess the effect of StPFA5 on the cell wall integrity of S. turcica. Furthermore, proteomic and S-palmitoylomic analyses combined with database retrieval were conducted to identify differentially modified S-palmitoylation sites and their corresponding proteins in the WT and ∆StPFA5 strains, followed by functional annotation of these proteins.

StPFA5 shared the highest homology (80.35%) with PFA5 from Cochliobolus heterostrophus. Compared with the WT strain, ∆StPFA5 exhibited reduced colony growth rate, abnormal hyphal morphology and significantly decreased pathogenicity, and these phenotypic defects were restored to the WT level in the C.∆StPFA5 strain. Meanwhile, the melanin content in the mycelia of ∆StPFA5 was decreased, the expression of melanin synthesis-related genes was significantly down-regulated, and the sensitivity of ∆StPFA5 to cell wall stress was reduced. S-palmitoylomic analysis revealed that knockout of StPFA5 led to the downregulation of modification levels at 448 S-palmitoylation sites, and the proteins containing these downregulated sites were significantly enriched in biological pathways including amino acid anabolism, carbon metabolism, glycolysis, cell cycle and chromosome condensation. Combined with the phenotypic characteristics of ∆StPFA5, StPFA5 may affect the growth, development and pathogenicity of S. turcica by regulating the S-palmitoylation levels of proteins associated with these pathways.

The palmitoyl transferase StPFA5 promotes the growth, development and melanin synthesis of S. turcica by regulating the S-palmitoylation modification levels of target proteins, and exerts a positive regulatory effect on the pathogenicity of this fungus.

As a polyphenol oxidase, laccase plays an important role in fungal growth, development and secondary metabolism. A plurality of laccase genes are encoded in the genome of Setosphaeria turcica, among which StLAC2 and StLAC6 have differential effects on the growth, development, and pathogenicity of S. turcica.

To clarify the differential mechanisms of StLAC2 and StLAC6 on S. turcica and explore new targets for developing new fungicides and disease control strategies by mining differential metabolites.

StLAC6 was connected with pHZ100-GFP plasmid by seamless cloning, and the complementary expression vector of StLAC6 was constructed. Using PEG-mediated protoplast transformation method, the constructed vector was transferred into the protoplast of StLAC6 gene knockout mutant, and the positive transformants were identified by PCR, RT-qPCR and GFP fluorescence verification, and the StLAC6 revertant strain was successfully constructed. The effects of knocking out and reverting StLAC2 and StLAC6 on melanin synthesis and oxidation resistance in and out of S. turcica were analyzed. Taking wild-type (WT), StLAC2 and StLAC6 gene knockout mutants as experimental materials, the differential metabolites were analyzed by non-targeted metabonomics, and the mechanism of the differential action of StLAC2 and StLAC6 was analyzed by KEGG.

StLAC2 and StLAC6 have differential effects on melanin synthesis in mycelium and secreted into culture medium, and StLAC2 also affects antioxidant activity of S. turcica. Metabolomic analysis found that compared with the WT strain of S. turcica, there were more differential metabolites in the mycelium or secreted into the culture medium after knocking out StLAC2, and KEGG analysis showed that the differential metabolites were mainly lipids, especially phospholipids. Meanwhile, the absence of the StLAC2 caused down-regulation of various flavonoids and polyphenols. The contents of intermediates of the 1, 8-dihydroxynaphthalene melanin biosynthesis pathway, scytalone and vermelone, significantly increased in ΔStLAC2 and decreased in ΔStLAC6.

The StLAC2 participates in melanin polymerization, the StLAC6 negatively regulates melanin biosynthesis in S. turcica, and the differential effects of StLAC2 and StLAC6 affect lipid metabolism and intermediates of the melanin biosynthesis pathway in S. turcica. The absence of StLAC2 caused down-regulation of various flavonoids and polyphenols, leading to decreased antioxidant activity.