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
