RNA silencing is a mechanism used by eukaryotes to defend them self against viruses. When encountering an external or foreign double-stranded RNA (dsRNA), plants activate their machinery to assemble an RNA induced gene silencing complex (RISC) in which Argonautes (AGOs) play an important role. Once activated plant use RISC to survey for those sequences identical to the original dsRNA and cleave these into small fragments. This study provides evidence that RNA silencing against Tomato bushy stunt virus (TBSV) can depend on plant age in Nicotiana benthamiana (Nb) by using TBSV mutants that do not express P19 (TGdp19 or 157). I show that NbAGO2 plays role in older plants during viral silencing by measuring mRNA and comparing treatments. These results indicated that NbAGO2 is up-regulated after TBSV inoculation and that 6 week old plants have a better ability to silence TBSV compared to younger plants. By using transgenic plants expressing a dsRNA NbAGO2 hairpin for downregulation of NbAGO2, my studies provide evidence that NbAGO2 is required for viral silencing by conferring several layers of protection. This was demonstrated by infecting NbAGO2-hairpin transgenic plants with different TBSV variants, different Tombusvirus members and non-Tombusvirus. The results showed that reduced levels of NbAGO2 enhanced viral infection in general, these infections compromised plant integrity, TBSV not expressing the coat protein yielded severe systemic infections not otherwise observed, and TBSV not expressing P19 (TGdp19 and TBSV-157) caused more severe infections compared to controls. Additionally, my studies validate the use of two well-known viruses that express suppressors to avoid the effects of silencing by NbAGO2 or other components. This was shown for Tobacco mosaic virus (TMV) and TBSV that were used as viral vectors to express and co-express green fluorescent protein (GFP) and red fluorescent protein (RFP) in N. benthamiana and tomato. The results demonstrated that the vectors accumulate in the same plant and leaves, and even in the same cells, providing a tool for fast expression of potentially biomedically or otherwise valuable oligomeric proteins.
