Changes in Translatable MRNA Species Associated With Nutrient Deprivation and Protease Synthesis in Metarhizium Anisopliae

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Journal of General Microbiology



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We have studied the regulation of the extracellular chymoelastase protease (Prl) of the fungal entomopathogen Metarhiziurn anisopliae. This enzyme is involved in the penetration of insect cuticle by Metarhiziurn and other entomopathogenic fungi. Changes in mRNA sequences during starvation-induced synthesis of chymoelastase were investigatedbycomparingpoly(A+)RNAsandtheircomplementaryDNAinrapidly growingornutrient-deprived cultures of Metarhiziurn. Hybrid-selected translation revealed that three novel polypeptides (41, 40.2 and 29.8kDa) were produced rapidly (<1h) during nutrient deprivation; the most intensely translated species (41 kDa) was identified as the primary translation product of P r l by examination with anti-Prl-IgG. Measurement of starvation-specific RNA levels by dot-blot hybridization with [32PlcDNAshowed 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 a t the level of transcription. Immunoblot analysis with anti-Prl-IgC showed that P r l protein increased in concert with the appearance of P r l transcripts and active enzyme. Two very short lived precursors of Prl, the primary translation product (41 kDa) and an intermediate (40.8 kDa) in addition to mature P r l (30 kDa) were detected by immunoprecipitation of 13sS]methionine-labelledcell extracts with anti-Prl-IgG. Pulse-labelling experiments demonstrated that a time of about 7 min was required for (3sS]methionineto be processed into extracellular P r l . These results suggest that regulation of P r l gene expression occurs during starvation which, coupled with fast processing, allows very rapid secretion of Prl.Isolates of four other entomopathogenic fungi (Verticilfiurnlecanii, Beauveria bassiana, Tolypocladiurnniveurn and Paecilornycesfarinosus) produced Prl-type enzymes during nutrient deprivation, suggesting our results may have widespread application in understanding entomopathogenicity.

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