Functional Characterization of the Subtilase Gene <i>Cg043</i> Downregulated by 4-Ethyl-1,2-dimethoxybenzene in the Growth and Pathogenicity of <i>Colletotrichum gloeosporioides</i>

<i>Colletotrichum gloeosporioides</i>, the causative agent of anthracnose, poses a significant threat to agricultural production. Previous studies identified 4-ethyl-1,2-dimethoxybenzene as a potent antifungal compound that downregulates the expression of the subtilase gene <i>Cg043</i>, although the underlying molecular mechanism remains unclear. Here, we generated <i>Cg043</i> knockout mutants (Δ<i>Cg043</i>) and found that their sensitivity to 4-ethyl-1,2-dimethoxybenzene was significantly reduced, identifying <i>Cg043</i> as a key molecular target. Phenotypic assays and transcriptomic analyses revealed that <i>Cg043</i> downregulation inhibits hyphal growth, spore production, and germination while impairing cell wall and membrane integrity and reducing pathogenicity. Furthermore, functional verification of the signal peptide and subcellular localization analysis confirmed that Cg043 is a secreted protein specifically localized to the plant cell nucleus, suggesting its role in virulence. These findings elucidate a novel antifungal mechanism by which 4-ethyl-1,2-dimethoxybenzene suppresses the growth, development, and pathogenicity of <i>C. gloeosporioides</i> via <i>Cg043</i> downregulation, highlighting a promising molecular target for sustainable anthracnose management.