Mechanism of StLAC2 and StLAC6 Differentially Affecting Setosphaeria turcica Based on Non-Targeted Metabonomics Analysis

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.