Evidence for a Common Toolbox Based on Necrotrophy in a Fungal Lineage Spanning Necrotrophs, Biotrophs, Endophytes, Host Generalists and Specialists

The Sclerotiniaceae (Ascomycotina, Leotiomycetes) is a relatively recently evolved lineage of necrotrophic host generalists, and necrotrophic or biotrophic host specialists, some latent or symptomless. We hypothesized that they inherited a basic toolbox of genes for plant symbiosis from their common ancestor. Maintenance and evolutionary diversification of symbiosis could require selection on toolbox genes or on timing and magnitude of gene expression. The genes studied were chosen because their products have been previously investigated as pathogenicity factors in the Sclerotiniaceae. They encode proteins associated with cell wall degradation: acid protease 1 (acp1), aspartyl protease (asps), and polygalacturonases (pg1, pg3, pg5, pg6), and the oxalic acid (OA) pathway: a zinc finger transcription factor (pac1), and oxaloacetate acetylhydrolase (oah), catalyst in OA production, essential for full symptom production in Sclerotinia sclerotiorum. Site-specific likelihood analyses provided evidence for purifying selection in all 8 pathogenicity-related genes. Consistent with an evolutionary arms race model, positive selection was detected in 5 of 8 genes. Only generalists produced large, proliferating disease lesions on excised Arabidopsis thaliana leaves and oxalic acid by 72 hours in vitro. In planta expression of oah was 10-300 times greater among the necrotrophic host generalists than necrotrophic and biotrophic host specialists; pac1 was not differentially expressed. Ability to amplify 6/8 pathogenicity related genes and produce oxalic acid in all genera are consistent with the common toolbox hypothesis for this gene sample. That our data did not distinguish biotrophs from necrotrophs is consistent with 1) a common toolbox based on necrotrophy and 2) the most conservative interpretation of the 3-locus housekeeping gene phylogeny - a baseline of necrotrophy from which forms of biotrophy emerged at least twice. Early oah overexpression likely expands the host range of necrotrophic generalists in the Sclerotiniaceae, while specialists and biotrophs deploy oah, or other as-yet-unknown toolbox genes, differently.