Exploring the biochemical and biological functions of copper radical oxidases in the vascular wilt phytopathogen Verticillium dahliae

Plant pathogenic fungi pose a significant threat to global food security. Hence, discovering targeted (bio)chemical methods to combat plant diseases is a critical research avenue. Recent work has implicated copper radical oxidases (CROs) from Auxiliary Activity Family 5 (AA5) in fungal morphogenesis and pathogenesis, including in appressorium penetration by foliar phytopathogens in the genera Colletotrichum and Magnaporthe . We identified orthologous AA5-encoding genes in Verticillium species; however, it was not clear that the functions of the corresponding CROs would be conserved in these vascular wilt pathogens. Using Verticillium dahliae JR2 as an exemplar, recombinant protein production and detailed enzyme kinetic analyses revealed predominant aryl-alcohol oxidase activity of the AA5 subfamily 2 (AA5_2) homolog, and predominant aldehyde oxidase activity of the AA5 subfamily 1 (AA5_1) homolog. Transcriptomics and reverse genetics experiments on both CROs and three putative peroxidases/catalases (known activators of CROs) in V. dahliae JR2 failed to indicate direct roles in phytopathogenesis. Taken together, the data indicate that the biological roles of CROs and AA5_2 members, in particular, are not broadly conserved between foliar and vascular wilt phytopathogens. However, the biochemical characterization of an AA5_1 aldehyde oxidase and an AA5_2 aryl-alcohol oxidase from V. dahliae , as well as an AA5_2 ortholog from V. longisporum VL43, provides a foundation for further functional elucidation, as well as new biocatalysts for biotechnological applications. IMPORTANCE Plant pathogens constitute a considerable burden to human society by attacking crops and reducing agricultural yields. Oxidative enzymes are often key weapons in the arsenal deployed by phytopathogens in the effort to breach cell walls and extract nutrients. Here, the characterization of the biochemical specificities of copper radical oxidases (CROs) from Verticillium wilt/stripe fungi defines a range of possible alcohol and aldehyde substrates in vivo and outlines the potential of these enzymes for their biotechnological application for chemical valorization. Although specific gene knockouts did not reveal a biological function for these CROs, we now know that the molecular mechanism of CRO-mediated pathogenesis previously observed in foliar phytopathogens from the genera Colletotrichum and Magnaporthe is not conserved in the vascular wilt pathogen Verticillium .