Anti-viral RNAi and its suppression in plants.

As a defense against viral infection, plants are thought to use RNA-induced silencing complexes (RISCs) to target and cleave viral RNA. To counteract this, some viruses have evolved proteins to inhibit RISC-mediated activity, thus ensuring continued virulence. This research focused on the study and analysis of the anti-viral RNAi response to various viruses in plants to gain an understanding of how the plant defense operates on the molecular and biochemical levels. Nicotiana benthamiana plants were infected with Tomato bushy stunt virus (TBSV) and Tobacco rattle virus (TRV). These plants were subjected to column chromatography methods, and fractions contained a virus-specific ribonuclease activity, co-eluting with small interfering RNAs (siRNA), that was shown to be sensitive to inhibition with EDTA and enhanced by the addition of divalent metal cations. This ribonuclease activity co-purified with proteins that contained a domain from the hallmark RISC protein Argonaute family. To further study host responses to viral infection, monocots were infected with Panicum mosaic virus (PMV) and satellite panicum mosaic virus (SPMV) and also were subjected to column chromatography following infection. Preliminary studies show that fractions contained ribonuclease activity as well as siRNAs and proteins containing an Argonaute domain. Additionally, silencing suppressors have been directly implicated in interfering with RNAi pathways in plants. Studies involving Agrobacterium- and virus-vectored cDNA to express green fluorescent protein (GFP) were used to establish that cointroduced suppressors of RNAi can extend the production of a foreign protein for enhancement of biotechnological applications. It was found that the hordeivirus protein ?b contributes to enhancement of expression for the foreign protein GFP early in the infection, while the potyvirus protein HcPro and tombusvirus protein P19 enhance and extend protein production later in the infection.