Role of Two G-Protein alpha Subunits in Vegetative Growth, Cell Wall Integrity, and Virulence of the Entomopathogenic Fungus Metarhizium robertsii

Heterotrimeric G-proteins are crucial for fungal growth and differentiation. The alpha subunits of heterotrimeric G-proteins play an essential role in controlling signal transduction. However, the function of G-protein alpha subunits in entomopathogenic fungi remains poorly understood. Two group II G alpha subunits (MrGPA2 and MrGPA4) were characterized in the entomopathogenic fungus, Metarhizium robertsii. Bioinformatics analysis showed that the relationship between MrGPA2 and MrGPA4 was closer than that of other MrGPAs. Both green fluorescent protein (GFP)-tagged MrGPA2 and MrGPA4 were localized at the cytoplasm. Furthermore, increment MrGpa2 increment MrGpa4 double mutants showed remarkably reduced vegetative growth compared to the wild-type and single-mutant strains, which was accompanied by the downregulation of several growth-related genes, such as ssk2, pbs2, stuA, hog1, and ac. Only the increment MrGpa2 increment MrGpa4 double mutant was sensitive to Congo red stress. The insect bioassay demonstrated significantly attenuated virulence for the increment MrGpa2 increment MrGpa4 double mutant compared to the wild-type and single-mutant strains. Further analysis indicated that double deletion of MrGpa2 and MrGpa4 had no effect on appressorium formation but suppressed the expression levels of several virulence-related genes in the insect hemocoel. These findings demonstrate that MrGpa2 and MrGpa4 exhibit functional redundancy and contribute to the vegetative growth, stress tolerance, and pest control potential in M. robertsii.