Molecular cloning of a novel subtilisin-like protease (Pr1A) gene from the biocontrol fungus Isaria farinosa

Fungal proteases, such as subtilisin-like serine protease (Pr1) and trypsin-like serine protease (Pr2), play an important role in penetration through the host cuticle in entomopathogenic fungi, including Isaria farinosa. In the present study, one gene of I. farinosa subtilisin-like protease (Ifa-pr1A), showing high homology to a Beauveria bassiana cuticle-degrading protease, was amplified by rapid amplification of cDNA ends PCR. The resulting full-length cDNA displayed an open reading frame (ORF) of 960 bp, encoding a protein of 319 amino acids. The Ifa-Pr1A protein was expressed in Escherichia coli to verify its protease activity. The recombinant Ifa-Pr1A protein exhibited high enzymatic activity according to the enzyme assay using a synthetic substrate. The expression profiles of Ifa-pr1A and Ifa-pr1H (another subtilisin-like protease gene from I. Farinosa) were analyzed at different induction times by adding cuticle material to the media. Quantitative reverse transcriptase PCR (RT-PCR) analysis revealed that Ifa-pr1A transcripts increased 58,800-fold at 12 h post-induction, whereas Ifa-pr1H transcripts peaked at 12 h with only an 11-fold increase, indicating that Ifa-pr1A may be a major gene of cuticle-degrading proteases in I. farinosa. Furthermore, Ifa-pr1A gene expression under in vivo conditions showed a clear upward trend during the early stages of the infection process. These results suggest that Pr1A cloned from I. farinosa is a potential virulence factor for the development of engineered biopesticides.