Expression of genes involved in germination, conidiogenesis and pathogenesis in Metarhizium anisopliae using quantitative real-time RT-PCR

Characterization of genes involved in germination, conidiogenesis and insect pathogenesis is an important step in identifying methods to increase the efficacy of Metarhizium anisopliae, a commercially important entomopathogenic fungus. Real-time RT-PCR is a sensitive, reproducible and quantitative method to study gene expression. However, it requires reliable reference gene transcripts for normalization. In this study, six putative housekeeping genes (act, gpd, 18sRNA, tef, try and ubi) were investigated as reliable reference genes. Transcripts from tef, gpd and try were found to be the most suitable reference genes for real-time RT-PCR analysis of genes expressed during germination, conidiogenesis and pathogenesis. Using these as reference genes, the relative expression levels of a virulence gene, a subtilisin-like protease (pr1), a regulator of G protein signaling gene involved in conidiogenesis (cag8), the nitrogen response regulator gene (nrr1), and a hydrophobin gene (ssga) were studied. None of these transcripts could be detected in the early stages of insect pathogenesis. The nitrogen response regulator, nrr1, was consistently expressed during all developmental stages. Expression levels of cag8 increased significantly in the later stages of conidiogenesis on insect cadavers. The expression level of ssga during conidiogenesis was significantly higher than that in mycelia during vegetative growth in nutrient rich media. The pr1 gene was expressed during fungal conidiation on the insect cadaver. This study acts as a foundation for investigating the transcriptional levels of genes expressed during germination, conidiogenesis and pathogenesis of M. anisopliae using real-time RT-PCR.