Functional comparison of RNA silencing suppressor between the p5 protein of rice grassy stunt virus and the p3 protein of rice stripe virus

•RGSVp5 encoded by the viral-sense strand of RNA5 functioned as a VSR.•RGSVp5 suppressed S-PTGS, IR-PTGS and systemic silencing.•In contrast to RSVp3, RGSVp5 did not have any binding activity with siRNA duplexes.•RGSVp5 was localized in the cytoplasm; RSVp3 was localized in the nucleus plus the cytoplasm.•The two tenuiviral RNA silencing suppressors differed in function.

Rice grassy stunt virus (RGSV) is a member of the genus Tenuivirus, which includes rice stripe virus (RSV), as the type species. A viral suppressor of RNA silencing (VSR) of RGSV has not been identified, whereas the p3 protein of RSV (RSVp3) encoded by the viral-sense (v) strand of RNA3 has been reported to act as a VSR. In this study, we examined the VSR function of the p5 protein of RGSV (RGSVp5), encoded by vRNA5. Expecting it to correspond to the vRNA3 of RSV, we compared the VSR function of RGSVp5 with that of RSVp3. In an Agrobacterium-mediated transient expression assay using a transgenic line of Nicotiana benthamiana that expressed green fluorescent protein and the wild type, RGSVp5 suppressed sense transgene-mediated post-transcriptional gene silencing (S-PTGS), inverted-repeat (IR) transgene-induced PTGS (IR-PTGS), and the systemic spread of GFP silencing, as in the case with RSVp3. By contrast, a gel mobility shift assay revealed that RGSVp5 did not have any distinct binding activity with 21-, 22-, or 24-nucleotide small interfering RNA (siRNA) duplexes, whereas RSVp3 binds to all three sizes of siRNA duplexes. Furthermore, the transiently expressed p5 protein fused with GFP was dispersed mainly in the cytoplasm, whereas the GFP-fused p3 protein of RSV was localized both in the nucleus and in the cytoplasm. Our results suggest that RGSVp5 functions as a VSR but that the suppression mechanism of RNA silencing and the subcellular localization of RGSVp5 differ from those of the analogous VSR, RSVp3, even in the same genus.