Independent parallel functions of p19 plant viral suppressor of RNA silencing required for effective suppressor activity

Abstract

Plant viruses ubiquitously mediate the induction of miR168 trough the activities of viral suppressors of RNA silencing (VSRs) controlling the accumulation of ARGONAUTE1 (AGO1), one of the main components of RNA silencing based host defence system. Here we used a mutant Tombusvirus p19 VSR (p19-3M) disabled in its main suppressor function, small interfering RNA (siRNA) binding, to investigate the biological role of VSR-mediated miR168 induction. Infection with the mutant virus carrying p19-3M VSR resulted in suppressed recovery phenotype despite the presence of free virus specific siRNAs. Analysis of the infected plants revealed that the mutant p19-3M VSR is able to induce miR168 level controlling the accumulation of the antiviral AGO1, and this activity is associated with the enhanced accumulation of viral RNAs. Moreover, saturation of the siRNA-binding capacity of p19 VSR mediated by defective interfering RNAs did not influence the miR168-inducing activity. Our data indicate that p19 VSR possesses two independent silencing suppressor functions, viral siRNA binding and the miR168-mediated AGO1 control, both of which are required to efficiently cope with the RNA-silencing based host defence. This finding suggests that p19 VSR protein evolved independent parallel capacities to block the host defence at multiple levels.

Homeologs of the Nicotiana benthamiana Antiviral ARGONAUTE1 Show Different Susceptibilities to microRNA168-Mediated Control

“…Plant virus infections correlate with a significant accumulation of miR168 in infected tissues (Zhang et al, 2006;Csorba et al, 2007;Havelda et al, 2008;Várallyay et al, 2010Várallyay et al, , 2014Lang et al, 2011). The reasons leading to this phenomenon are yet uncertain.…”

“…benthamiana is widely considered as a model host for virus-plant interaction studies, and it has also been successfully used in molecular studies of RNA silencingbased plant-virus interactions with tombusvirus systems. Here, we applied CymRSV because the functionality of miR168 in N. benthamiana during a CymRSV infection was convincingly demonstrated by Várallyay et al (2014). Indeed, we considered this virus system most adequate to dissect the miR168-directed regulation of the NbAGO1-1 mRNAs for the following reasons: (1) N. benthamiana plants show a strong accumulation of miR168 associated with a CymRSV infection ( Fig.…”

“…Indeed, we considered this virus system most adequate to dissect the miR168-directed regulation of the NbAGO1-1 mRNAs for the following reasons: (1) N. benthamiana plants show a strong accumulation of miR168 associated with a CymRSV infection ( Fig. 4B; Várallyay et al, 2010); (2) while not binding miR168 (Várallyay et al, 2014), the viral suppressor P19 is supposed to effectively sequester vsiRNAs and to impede their interference with endogenous and physiological AGO1 functionalities (at least during the initial stages of infection and spread); and (3) the level of NbAGO1-1 mRNA seems to increase in CymRSV-infected plants, but this is not associated with an accumulation of mRNA cleavage products (Várallyay et al, 2010). Earlier works detected miRNA-mediated NbAGO1 mRNA cleavage in virus-infected cells at a lower extent than in naive cells (Várallyay et al, 2010), and our findings are consistent with these data, if we consider solely the situation with NbAGO1-1H (Fig.…”

Modulation of host plant immunity by Tobamovirus proteins

“…The action of VSRs is based on different strategies: inhibiting DCL activity, binding to the viral dsRNAs and protecting them from the subsequent DCL processing, sequestering and/or degrading vsiRNAs, inactivating RISCs and also inhibiting the amplification and/or spread of the silencing signal (Mallory et al, 2002;Qiu et al, 2002;Vargason et al, 2003;Soitamo et al, 2011;Varallyay et al, 2014). In particular, the TMV replicase suppressor of silencing activity is mediated by the protection of viral transcripts from enzymes of the RNA silencing pathway rather than defeating them (Ding et al, 2004;Kurihara et al, 2007).…”

Tospovirus : induction and suppression of RNA silencing

“… Bind ds(sRNA) size selectively (Vargason et al, 2003;Lakatos et al, 2006;Rawlings et al, 2011)  inhibits the 3' methylation of sRNAs (Lozsa et al, 2008)  AGO1 repression (via miR168 upregulation) (Varallyay et al, 2014) (+)ssRNA Tombusviridae, Tombusvirus CymRSV P19…”