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Summary: We have studied the regulation of the extracellular chymoelastase protease (PrI) of the fungal entomopathogen Metarhizium anisopliae. This enzyme is involved in the penetration of insect cuticle by Metarhizium and other entomopathogenic fungi. Changes in mRNA sequences during starvation-induced synthesis of chymoelastase were investigated by comparing poly(A+)RNAs and their complementary DNA in rapidly growing or nutrient-deprived cultures of Metarhizium. Hybrid-selected translation revealed that three novel polypeptides (41, 40·2 and 29·8 kDa) were produced rapidly (<1 h) during nutrient deprivation; the most intensely translated species (41 kDa) was identified as the primary translation product of Pr1 by examination with anti-Pr1-IgG. Measurement of starvation-specific RNA levels by dot-blot hybridization with [32P]cDNA showed that transcripts were not present in spores but were produced when the formation against a hard surface of infection structures was induced by depletion of endogenous nutrient reserves. Starvation-specific mRNA species failed to appear when new RNA synthesis was blocked with actinomycin D, indicating that control is at the level of transcription. Immunoblot analysis with anti-Pr1-IgG showed that Pr1 protein increased in concert with the appearance of Pr1 transcripts and active enzyme. Two very short lived precursors of Pr1, the primary translation product (41 kDa) and an intermediate (40·8 kDa) in addition to mature Pr1 (30 kDa) were detected by immunoprecipitation of [35S]methionine-labelled cell extracts with anti-Pr1-IgG. Pulse-labelling experiments demonstrated that a time of about 7 min was required for [35S]methionine to be processed into extracellular Pr1. These results suggest that regulation of Pr1 gene expression occurs during starvation which, coupled with fast processing, allows very rapid secretion of Pr1. Isolates of four other entomopathogenic fungi (Verticillium lecanii, Beauveria bassiana, Tolypocladium niveum and Paecilomyces farinosus) produced Pr1-type enzymes during nutrient deprivation, suggesting our results may have widespread application in understanding entomopathogenicity.

Changes in translatable mRNA species associated with nutrient deprivation and protease synthesis in Metarhizium anisopliae