Functional Characterization of PCWDEs VdCut1 and VdPL16 Reveals Their Critical Roles in Verticillium dahliae Pathogenicity

Abstract Verticillium wilt, a soil-borne fungal disease caused by Verticillium dahliae, is a major threat to agricultural production. The plant cell wall serves as the primary barrier against the invasion of fungal pathogens. To facilitate successful infection, many pathogens encode plant cell wall-degrading enzymes (PCWDEs) to degrade cell wall components. In this study, we conducted a genome-wide analysis of PCWDE gene families in two V.dahliae strains, VdSHZ-4 and VdSHZ-9, with different pathogenicity. VdSHZ-4 and VdSHZ-9 genomes were found to contain 415 and 396 PCWDE genes, respectively. We successfully isolated two key enzymes from V. dahliae: the cutinase VdCut1 and the pectin lyase VdPL16. To investigate the functions of these two genes, we constructed VdCut1 and VdPL16 deletion and complementation mutants and examined their roles in hyphal growth, conidiation, and pathogenicity toward cotton Verticillium wilt. Results showed that deletion of VdCut1 or VdPL16 suppressed colony growth and reduced conidial production, and it also impaired the utilization of sucrose and galactose and markedly increased sensitivity to abiotic stresses such as sodium dodecyl sulfate (SDS) and sorbitol. The VdCut1 deletion mutant completely lost the ability to penetrate cellophane membranes, whereas the VdPL16 deletion mutant exhibited significantly reduced penetration capacity. Both deletions markedly attenuated virulence, with the loss of VdCut1 having the more pronounced effect. In planta colonization assays revealed that the VdCut1 mutant accumulated much less biomass in host tissues than the wild-type strain and the VdPL16 mutant. These findings demonstrate that VdCut1 and VdPL16 play critical roles in V.dahliae pathogenicity by regulating hyphal growth and conidiation. The results deepen our understanding of the molecular mechanisms by which this pathogen degrades plant cell walls and provide a theoretical basis for antifungal strategies that target plant cell wall-degrading enzymes (PCWDEs). Author summary The plant cell wall is the first line of defense against fungal invasion, and Verticillium dahliae can secrete plant cell wall-degrading enzymes (PCWDEs) to break this barrier—which is a key step for its successful infection. However, the specific PCWDEs that drive the pathogenicity of V. dahliae and their mechanisms of action remain incompletely understood. Here, we conducted a comparative analysis of carbohydrate-active enzymes (CAZymes) in the genomes of two V. dahliae strains— (same cotton field origin, high pathogenicity VdSHZ-9 and weak pathogenicity VdSHZ-4). We then focused on key PCWDE genes: VdCut1 (encoding a cutinase) and VdPL16 (encoding a pectin lyase), which showed differential pathogenicity associations between the two strains. Using gene deletion mutants and complementation strains, We fund that VdCut1 and VdPL16 support V. dahliae’s basic biological functions and hyphal penetration, and that the deletion of these genes significantly reduces the pathogenicity of V. dahliae. These findings provide a basis for future research on the mechanism underlying the pathogenic variation of V. dahliae and identify the genes encoding PCWDEs as potential targets for developing control strategies against cotton Verticillium wilt. Competing Interest Statement The authors have declared that no competing interests exist.