Proteomic Analysis of a Hypervirulent Mutant of the Insect-Pathogenic Fungus Metarhizium anisopliae Reveals Changes in Pathogenicity and Terpenoid Pathways

Metarhizium anisopliae is a commercialized entomopathogenic fungus widely used for the control of insect pests. Significant efforts have been expended to screen and/or select for isolates that display increased virulence toward target insect hosts. UV-induced mutagenesis has resulted in the isolation of a number of hypervirulent M. anisopliae mutants; however, the underlying mechanisms that have led to the desired phenotype have yet to be characterized. Here, we performed a comparative proteomic analysis of an M. anisopliae UV-induced hypervirulent mutant (MaUV-HV) and its wild-type parent using tandem mass tag (TMT)-based quantitative proteomics. A total of 842 differentially abundant proteins were identified, with 360 being more abundant in the hypervirulent mutant and 482 in the wild-type parent. In terms of differential abundance, the critical pathways affected included those involved in secondary metabolite production, virulence, and stress response. In addition, a number of genes involved in terpenoid biosynthesis pathways were identified as significantly mutated in the MaUV-HV strain. In particular, mutations in the farnesyl pyrophosphate synthase (FPPS1) and geranylgeranyl diphosphate synthase (GGPPS5) genes were seen. The effects of the FPPS1 mutation were confirmed via the construction and characterization of a targeted gene knockout strain (ΔMaFPPS1). The overall effects of the mutations were increased resistance to UV stress, faster growth, and increased virulence. These results provide mechanistic insights and new avenues for modulating fungal virulence in efforts to increase the biological control potential of insect-pathogenic fungi. IMPORTANCE The mechanisms that underlie and contribute to microbial (fungal) virulence are known to be varied; however, the identification of contributing pathways beyond known virulence factors remains difficult. Using TMT-based proteomic analyses, changes in the proteomes of an M. anisopliae hypervirulent mutant and its wild-type parent were determined. These data revealed alterations in pathogenicity, stress, and growth/developmental pathways, as well as pathways not previously known to affect virulence. These include terpenoid pathways that can be manipulated to increase the efficacy of fungal insect biological control agents for increased sustainable pest control.