Two secretory T2 RNases contribute to virulence of an insect fungal pathogen due to cytotoxicity and activation of host immune response

Abstract

Abstract The RNase T2 family are ubiquitously distributed in all kingdoms and involved in a variety of physical functions. RNase T2 members are secreted by several pathogens or parasites during infection, playing various roles in pathogen-host interaction. However, functions of those members in biocontrol microbes targeting their hosts are still unknown. Here, two secretory RNase T2 member, BbRNT2 and BbTrv, were found to be important virulence factors in an insect fungal pathogen, Beauveria bassiana. Although no significant effect on fungal virulence was cause by disruption of either BbRNT2 or BbTrv, significantly decreased virulence was examined in double gene disruption mutants. Overexpression of a single gene or both genes dramatically enhanced virulence, while few fungal cells proliferated in hemocoel, accompanying with dramatical reduction in hemocytes as compared to the wild type strain treatments. These results appeared to be due to cytotoxicity of the secreted proteins or excessive activation of the insect immune response. In vitro assays using yeast-expressed proteins verified the cytotoxicity of BbRNT2 and BbTrv against insect cells, including hemocytes from Galleria mellonella larvae and sf9 cells from Spodoptera frugiperda. Those effects of the two proteins depended on their RNases enzyme activities and glycosylation modification. Moreover, the two ribonucleases could also trigger humoral immune responses, with stimulation of reactive oxygen species levels, as well as phenol oxidase, lysozyme and nitric oxide synthase activities. These data demonstrate important roles of two secretory RNase T2 members in virulence of insect fungal pathogens, displaying prospects for improvement of biocontrol agents.