The soil-borne fungal pathogen Verticillium dahliae causes devastating vascular wilt disease in numerous crops, including cotton. In this study, we reveal that VdSOX1, a highly conserved sarcosine oxidase gene, is significantly upregulated during host infection and plays a multifaceted role in fungal physiology and pathogenicity. Functional deletion of VdSOX1 leads to increased fungal virulence, accompanied by enhanced microsclerotia formation, elevated carbon source utilization, and pronounced upregulation of effector genes, including over 50 predicted secreted proteins genes. Moreover, the VdSOX1 knockout strains suppress the expression of key defense-related transcription factors in cotton, such as WRKY, MYB, AP2/ERF, and GRAS families, thereby impairing host immune responses. Transcriptomic analyses confirm that VdSOX1 orchestrates a broad metabolic reprogramming that links nutrient acquisition to immune evasion. Our findings identify VdSOX1 as a central regulator that promotes V. dahliae virulence by upregulating effector gene expression and suppressing host immune responses, offering novel insights into the molecular basis of host–pathogen interactions and highlighting potential targets for disease management.

The soil-borne fungus Verticillium dahliae is a devastating pathogen responsible for substantial losses in cotton production. This study elucidated the key functions of VdARO2 and VdCPC1 in fungal pathogenicity. VdARO2 encodes a Chalmoic acid synthase involved in the biosynthesis of aromatic amino acids, while VdCPC1 is a central regulator of amino acid starvation response and reveals a key regulatory relationship between VdARO2 and VdCPC1 to jointly control fungal virulence. We demonstrate that both genes are essential for growth, conidiation, and microsclerotia formation in V. dahliae. The VdΔaro2 mutant exhibited severe developmental defects and a complete loss of microsclerotia production, accompanied by widespread transcriptional dysregulation. Disruption of VdARO2 significantly upregulated VdCPC1, triggering a compensatory starvation response that nonetheless failed to restore pathogenicity. Silencing VdCPC1 similarly impaired fungal development and attenuated virulence. Our findings reveal a crucial regulatory axis in which VdARO2 and VdCPC1 coordinate metabolic homeostasis and stress adaptation to facilitate host colonization, thereby identifying promising targets for the control of Verticillium wilt.