Copper Radical Oxidases Contribute to Virulence in Sclerotinia sclerotiorum

Sclerotinia sclerotiorum is a broad-spectrum soilborne fungal pathogen, causing substantial yield losses in crops worldwide. Auxiliary Activity Family 5 (AA5) copper radical oxidases (CROs) are members of the carbohydrate active enzymes (CAZymes), valued for their potential as biocatalysts. Although they were reported to contribute to fungal development and disease progression in some phytopathogenic fungi such as Colletotrichum graminicola, the roles of AA5 enzymes in S. sclerotiorum remain largely unexplored. Here, we systematically investigated S. sclerotiorum AA5 genes through reverse genetics and biochemical approaches. Phylogenetic analysis grouped the S. sclerotiorum AA5 proteins into two subfamilies, including three subfamily 1 (AA5_1) members and one subfamily 2 (AA5_2) member. Enzymatic characterization revealed that one AA5_1 enzyme, SsAA5c, exhibited the highest catalytic specificity towards D-glyceraldehyde compared with methylglyoxal and glycerol. In addition, a predicted peroxidase gene (SsPX1) was identified in tandem with the putative AA5_2 gene, SsAA5d. Phenotypic analysis of gene deletion mutants demonstrated that these genes differentially affect fungal development and virulence. While their exact physiological substrates await identification, our findings highlight important roles of AA5 oxidases and the associated peroxidase in the biology of S. sclerotiorum, providing insights that may contribute to future improved disease management strategies.