The deletion of a major facilitator superfamily gene <i>VdMFS2</i> results in enhanced pathogenicity of <i>Verticillium dahliae</i> to cotton

ABSTRACT Among soil-borne phytopathogenic fungi, Verticillium dahliae is recognized as a devastating causal agent of cotton Verticillium wilt. Elucidation of molecular interactions between V. dahliae and cotton plants remains critical for disease control strategy development. A highly expressed major facilitator superfamily (MFS) transporter gene (VDAG_09088, designated VdMFS2 ) was identified through previous RNA sequencing (RNA-seq) analysis of V. dahliae exposed to cotton root exudates. Functional characterization was performed using host-induced gene silencing (HIGS), targeted gene knockout, and heterologous expression in Saccharomyces cerevisiae . Additionally, the secreted proteins of wild-type and VdMFS2 deletion mutant (Δ VdMFS2-4 ) were compared. It was found that VdMFS2 silencing enhances the pathogenicity of V. dahliae to cotton. The Δ VdMFS2-4 and Δ VdMFS2-5 mutants displayed accelerated colony growth rates, enhanced carbon source utilization ability, reduced leucine (Leu) and valine (Val) utilization ability, increased stress tolerance, increased host penetration ability, and accompanied by elevated virulence. Proteomic profiling identified 94 upregulated secreted proteins in the mutant, including 54 canonically secreted proteins (CSPs) and 40 non-canonically secreted proteins (N-SCPs). Notably, 23 carbohydrate-active enzymes (CAZymes) and 18 small cysteine-rich proteins (SCRPs) were detected among the CSPs, demonstrating collective contributions to pathogenicity enhancement. Taken together, VdMFS2 was demonstrated to function as a negative growth, development, and stress resistance regulator of V. dahliae , capable of transporting certain amino acids. Gene disruption triggered compensatory secretion of virulence effectors, particularly CAZymes and SCRPs, ultimately leading to enhanced virulence of V. dahliae . IMPORTANCE The knockout of VdMFS2 can inhibit the proliferation of mycelia and the germination of spores of V. dahliae while enhancing its pathogenicity to cotton. This study provides new insights into the role of major facilitator superfamily (MFS) transporters in V. dahliae and offers more precise clues for elucidating the impact of MFS transporters on this fungus.