Identification of Pns6, a putative movement protein of RRSV, as a silencing suppressor

Abstract

RNA silencing is a potent antiviral response in plants. As a counterdefense, most plant and some animal viruses encode RNA silencing suppressors. In this study, we showed that Pns6, a putative movement protein of Rice ragged stunt virus (RRSV), exhibited silencing suppressor activity in coinfiltration assays with the reporter green fluorescent protein (GFP) in transgenic Nicotiana benthamiana line 16c. Pns6 of RRSV suppressed local silencing induced by sense RNA but had no effect on that induced by dsRNA. Deletion of a region involved in RNA binding abolished the silencing suppressor activity of Pns6. Further, expression of Pns6 enhanced Potato virus × pathogenicity in N. benthamiana. Collectively, these results suggested that RRSV Pns6 functions as a virus suppressor of RNA silencing that targets an upstream step of the dsRNA formation in the RNA silencing pathway. This is the first silencing suppressor to be identified from the genus Oryzavirus.

Figure 1

Schematic representation of plasmids used in this study.

Figure 2

Suppression of local GFP silencing by RRSV Pns6. (A-F) N. benthamiana line 16c plants were coinfiltrated with Agrobacterium spp. (Agro.) mixtures carrying 35S-GFP and the individual constructs indicated in each image. GFP fluorescence was viewed under longwavelength UV light at 7 days postinfiltration (dpi). (G) Northern blot analysis of the steady-state levels of GFP mRNA and siRNA extracted from different infiltrated patches shown in panel A to F. 28 S rRNA and t RNA were used as loading controls for detection of GFP mRNA and GFP siRNA respectively.

Figure 3

RRSV Pns6 can not inhibit local silencing induced by dsRNA (A-C) N. benthamiana line 16c plants were coinfiltrated with Agrobacterium spp. (Agro.) mixtures carrying 35S-dsGFP and the individual constructs indicated in each image. GFP fluorescence was viewed under longwavelength UV light at 7 days postinfiltration (dpi). (D) Northern blot analysis of the steady-state levels of GFP mRNA extracted from different infiltrated patches shown in panel A to C. The bottom gel shows rRNA with ethidium bromide staining as a loading control.

Figure 4

RRSV Pns6 enhances pathogenicity of chimeric PVX. (A) plants infected by PVXΔS6 or PVXâ–³S6 show mild disease symptoms as a few scattered chlorotic speckles, whereas leaves infected with PVX-S6 show more severe symptoms. (B) RNA gel blot analysis of accumulation of PVX genomic (gRNA) and subgenomic mRNAs (sgRNA1 to sgRNA3) at 9 and 18 dpi. The bottom gel shows rRNA with ethidium bromide staining as a loading control.