Two secretory T2 RNases from a fungal pathogen target distinct insect cell transmembrane proteins to cause cytotoxicity

Fungal pathogens produce secretory ribonuclease (RNase) T2 proteins during infection, which contribute to fungal virulence via their enzyme functions in degradation of host cell RNA. However, the details of those proteins entering the host cells are unclear. Our previous study demonstrated that the two secretory RNase T2 members, BbRNT2 and BbTrv, produced by the insect fungal pathogen Beauveria bassiana, caused cytotoxic damage to insect cells and contributed to fungal virulence. Here, the Spodoptera frugiperda ovarian epithelial cells (sf9 cells) were used as models to investigate the interactions of the two fungus-produced RNase T2 proteins with the insect cells. Two transmembrane proteins, an ABC transporter (SfABCG) and an Innexin 7-like protein (Sfinx), were identified from the sf9 cells as interacting with BbRNT2 and BbTrv, respectively, through protein immunoprecipitation, yeast-two hybrid tests and protein pull-down assays. Although a slight decrease in the sf9 cell viability was examined by transfection of RNA interference of SfABCG or Sfinx, the transfected cells displayed a dramatically decreased sensitivity to BbRNT2 or BbTrv, suggesting the requirement of the two transmembrane proteins for BbRNT2 and BbTrv to enter the insect cells. These results reveal a mechanism of the cytotoxic molecules, T2 RNases, produced by the fungal pathogen, entering the insect cells via interaction with specific insect cell transmembrane proteins and causing cytotoxic damage.