A broadly conserved fungal alcohol oxidase (AOX) facilitates fungal invasion of plants

Alcohol oxidases are ecologically important enzymes which facilitate a number of plant-fungal interactions. Within Ascomycota they are primarily associated with methylotrophy, as a peroxisomal alcohol oxidase (AOX) catalyzing the conversion of methanol to formaldehyde in methylotrophic yeast. In this study we demonstrate that AOX orthologs are phylogenetically conserved proteins which are common in the genomes of non-methylotrophic, plant-associating fungi. Additionally, AOX orthologs are highly expressed during infection in a range of diverse pathosystems. To study the role of AOX in plant colonization, AOX knockout mutants were generated in the broad host range pathogen Sclerotinia sclerotiorum. Disease assays in soybean showed that these mutants had a significant virulence defect as evidenced by markedly reduced stem lesions and mortality rates. Chemical genomics suggest that SsAOX may function as an aromatic alcohol oxidase, and growth assays demonstrate that ?SsAOX is incapable of properly utilizing plant extract as a nutrient source. Profiling of known aromatic alcohols point towards the monolignol coniferyl alcohol (CoA) as a possible substrate for SsAOX. As CoA and other monolignols are ubiquitous among land plants, the presence of highly conserved AOX orthologs throughout Ascomycota imply that this is a broadly conserved protein used by ascomycete fungi during plant colonization. Overall design: WT (1980) and ?SsAOX S. sclerotiorum was grown in 1% glucose minimal media (GMM) for three days, before being transferred to fresh 1% GMM containing 1% soybean stem extract. Cultures were grown for an additional 4 hours before being flash frozen in liquid nitrogen and subjected to RNA extraction using a Maxwell RSC Plant RNA kit.