Subtilisin-like Pr1 proteases marking the evolution of pathogenicity in a wide-spectrum insect-pathogenic fungus

Subtilisin-like Pr1 proteases of insect-pathogenic fungi are a large family of extracellular cuticle-degrading enzymes that presumably determine a capability of hyphal invasion into insect hemocoel through normal cuticle infection, but remain poorly understood although often considered as virulence factors for genetic improvement of fungal potential against pests. Here, we report that not all of 11 Pr1 family members necessarily function in Beauveria bassiana, an ancient wide-spectrum pathogen evolved insect pathogenicity ~200 million years ago. These Pr1 proteases are phylogenetically similar to or distinct from 11 homologues (Pr1A-K) early named in Metarhizium anisopliae complex, a young entomopathogen lineage undergoing molecular evolution toward Pr1 diversification, and hence renamed Pr1A1/A2, Pr1B1-B3, Pr1 C, Pr1F1-F4,4 and Pr1 G, respectively. Multiple analyses of all single gene-deleted and rescued mutants led to the recognition of five conserved members (Pr1C, Pr1G, Pr1A2, Pr1B1, and Pr1B2) contributing significantly to the fungal pathogenicity to insect. The conserved Pr1 proteases were proven to function only in cuticle degradation, individually contribute 19-29% to virulence, but play no role in post-infection cellular events critical for fungal killing action. Six other Pr1 proteases were not functional at all in either cuticle degradation during host infection or virulence-related cellular events post-infection. Therefore, only the five conserved proteases are collectively required for, and hence mark evolution of, insect pathogenicity in B. bassiana. These findings provide the first referable base for insight into the evolution of Pr1 family members in different lineages of fungal insect pathogens.