Host-dependent suppression of RNA silencing mediated by the viral suppressor p19 in potato

Abstract

p19 protein encoded by tomato bushy stunt virus (TBSV) is known as a suppressor of RNA silencing via inhibition of small RNA-guided cleavage in plants. In this study, we generated TBSVp19-expressing patatin-RNAi transgenic potatoes to identify the inhibitory mechanisms of RNA silencing mediated by TBSVp19. In TBSVp19-expressing patatin-RNAi lines, reduction of patatin-derived siRNA accumulation and complementation of patatin transcripts were detected in comparison with the non-TBSVp19-expressing patatin-RNAi line, suggesting that TBSVp19 suppresses the siRNA-mediated silencing pathway. Interestingly, no apparent effect on the accumulation of miRNA168 and other miRNAs was detected in TBSVp19-expressing lines; previous studies reported that p19 induced the accumulation of both miRNA168 and its target Argonaute 1 (AGO1) mRNA, but suppressed AGO1 translation via up-regulation of miRNA168 in Arabidopsis. In addition, the expression of Argonaute 1 (AGO1-1 and AGO1-2) and Dicer-like 1 (DCL1) was not significantly altered in p19-expressing lines. Interestingly, no translational inhibition of AGO1 mediated by p19 was detected. These results suggest that p19 suppresses siRNA-mediated silencing in potato, but may not affect miRNA-mediated silencing, possibly due to the host-dependent manner of p19 activity.

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

“…However, it was shown that miR168 was unaffected in tomato plants infected with turnip crinkle virus, cucumber mosaic virus and tobacco rattle virus (40). In addition, transgenic expression of Tombusvirus p19 VSR in potato had no effect on miR168 level, and no AGO1 control was observed (41). These data indicate that virus-mediated miR168 induction requires host-dependent specific factors.…”

Utility of the P19 suppressor of gene‐silencing protein for production of therapeutic antibodies in Nicotiana expression hosts

“…However, whether the use of P19 would perturb the silencing of XT and FT in DXTFT mutants has not been investigated yet. Based on previous reports on the cellular targets of P19, including a documented case of interference with the siRNA-silencing pathway in a transgenic plant line (Ahn et al, 2011), we expected P19 to interfere with the RNAi-induced gene silencing of XT and FT in glycomodified N. benthamiana. Surprisingly, mAbs expressed in DXTFT with and without P19 were in both cases completely devoid of xylose and fucose residues within the harvesting period of 5-7 days dpi (see Figure S1).…”

“…Studies on TBSV mutants with lowered levels of P19 have shown that a high titre of the protein is critical for exerting its biological activity (Qiu et al, 2002;Scholthof et al, 1999). Despite P19's nonspecific siRNA binding, the effects brought about by this protein show host-specificity (Ahn et al, 2011;Angel et al, 2011;Siddiqui et al, 2008).…”

“…For the most part, glycomodified plants have been created through RNAi gene-silencing technology, mainly owing to the existence of multiple endogenous fucosyltransferase and xylosyltransferase genes in most plants (Cox et al, 2006;Sourrouille et al, 2008;Strasser et al, 2008). Although it is generally believed that P19 cannot reverse established gene-silencing pathways (Scholthof, 2006), interference in the siRNA pathway by P19 becomes a concern when RNAi-generated genetic backgrounds are to be used as expression hosts for producing therapeutic proteins, primarily because there are not reports in the literature on using P19 with an RNAi-based glycomodified host, but also since in an unrelated case, P19 was shown to repress the knockdown of a previously established RNAi transgenic line (Ahn et al, 2011). With the above-discussed results in mind, we present a series of experiments that demonstrate how P19 can be used effectively to enhance the expression of a recombinant therapeutic glycoprotein, such as a biosimilar therapeutic antibody, in plant-based expression systems.…”

Involvement of Eukaryotic Small RNA Pathways in Host Defense and Viral Pathogenesis

“…The helper component protease of tobacco etch virus (TEV) is able to interact with ds-siRNA and prevent strand separation [73]. The p19 protein of tomato bushy stunt virus (TBSV) blocks RISC loading by binding small dsRNA duplexes [74]. It was recently been shown, however, that some Nicotiana plants have developed counter mechanisms to p19 RISC interference [75].…”