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| Neutral trehalase (Ntl) is a trehalose hydrolyzing enzyme found in many organisms, including the entomopathogenic fungus Metarhizium anisopliae. To determine ntl’s role in thermotolerance and virulence of fungal conidiospores, we constructed an ntl RNA interference (RNAi) vector with dual promoter system and an over-expression vector, which could randomly integrate into the fungal genomic DNA. Compared to the wild-type, ntl mRNA reduced to 35-66% in RNAi mutants, while increased to 2-3 folds in the over-expression mutants. In RNAi conidiospores accumulated more trehalose, had less trehalase activity, and were much more tolerable to heat stress than the wild-type. Opposite effects were found in conidiospores of over-expression mutants compared to RNAi mutants. Pathogenicity was not altered with the changes in ntl mRNA and activity compared to the wild-type. These results indicated that ntl controlled trehalose concentration in M. anisopliae by degrading trehalose, which indirectly increased conidiospore thermotolerance. These results offered a new strategy to increased conidiospore thermotolerance of entomopathogenic fungus without affecting its virulence. Additionally, the dual promoter system developed for RNAi was proved as an efficient tool for functional analysis of entomopathogenic fungal genes. |
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| Keywords:neutral trehalase, thermotolerance, dual promoter RNAi system, trehalose, virulence |
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