Molecular Characterizations of Transgenic Nicotiana Benthamiana Plants Resistant to Red Clover Necrotic Mosaic Virus and Effects of Mixed Infections with Potato Virus Y on RNAi-Mediated Resistance

Engineered resistance mediated by RNA interference to control viral diseases in plants has shown great promise. However, the discovery that most known plant viruses encode RNAi suppressors which interfere with RNAi raised the issue to whether this type of engineered resistance can be durable in the presence of heterologous viruses in mixed infection. The overall goal of this study was to investigate the mechanism of suppression of RNAi-mediated resistance in transgenic plants in the presence of a virus carrying a strong suppressor of RNAi. Nicotiana benthamiana plants were transformed with a 1.2 kb from the 5' end of RCNMV RNA-1. Transgenic resistant lines were obtained. Resistance in two different transgenic lines was shown to be mediated by two different types of RNAi: constitutive RNAi in D2 line induced by doubles-stranded (ds) transgene transcripts and virus-induced RNAi in B1 line. We demonstrated that PVY differentially affected RNAi-mediated resistance in the two lines. D2 line is completely immune to RCNMV infection. D2 line contained multiple copies of the 1.2 kb transgene which are rearranged and produced dsRNAs. PVY did not break the resistance in this transgenic line however data showed that PVY interfered with RNAi which correlated to an increase of the 1.2 kb transgene mRNA. In addition, PVY infection induced accumulation of 21 nt siRNAs and did not alter the transcription of the transgene. In contrast, PVY infection suppressed resistance mediated by virus-induced RNAi in B1 line. B1 contains a single copy of the1.2 kb transgene and is initially susceptible to RCNMV infection however became resistant to RCNMV in newly merging leaves after 14 days post inoculation. PVY infection did not affect the accumulation of the 1.2 kb transgene mRNA nor the accumulation of 21 nt siRNA corresponding to the transgene. The differential effect of PVY infection on the two RNAi-mediated resistances in the two transgenic lines suggests that properly designed resistant plants might withstand mixed virus infections and the presence of a strong suppressor of RNAi. In addition, the differential effect of PVY on RNAi suggests that parallel but distinct pathways are involved in dsRNA-induced, virus-induced, and sense RNAi