Up-regulation of Pr1, a subtilisin-like protease, during conidiation in the insect pathogen Metarhizium anisopliae

Metarhizium anisopliae exhibits two different developmental patterns under nutrient-deprived conditions: appressorium formation in early stages and conidiation in late stages of pathogenesis in its insect hosts. In this study we isolated genes enriched during mature conidial production under nutrient-deprived conditions in M. anisopliae by using the method of suppression subtractive hybridization. We sequence-identified seven conidiation-associated genes (cag) in M. anisopliae. One of the genes, cag7, encoded an extracellular subtilisin-like protease, Pr1, that plays a fundamental role in cuticular protein degradation. Reverse-transcription polymerase chain reaction (RT-PCR) analysis confirmed that cag cDNAs are expressed during the development of mature conidia under nutrient-deprived conditions. RT-PCR analysis was also performed for Pr1 during infection of greater wax moth larvae (Galleria mellonella). Results showed up-regulation of Pr1 in the infected insect as the mycelia emerge and produce conidia on the surface of the cadaver. It is well documented that Pr1 is produced during the initial stages of transcuticular penetration by M. anisopliae. Here we show that Pr1 is also up-regulated during the final stages of pathogenesis as the fungus emerges from the dead host and subsequently conidiates on the cadaver.