Thesis (Ph.D.)--University of South Carolina, 2009
RNA silencing is a conserved RNA degradation mechanism that targets invading nucleic acids such as viruses, transgenes or transposons. HC-Pro is a plant viral protein that impairs RNA silencing, acting in a counter-defensive role. Here we report the identification of a tobacco protein (termed ntRAV) that interacts with HC-Pro in the yeast two-hybrid system. ntRAV shows relatedness to the Arabidopsis thaliana RAV family of transcription factors. Endogenous ntRAV mRNA accumulates to very high levels in young tobacco plants, then rapidly declines at around three weeks post-germination to a nearly undetectable level. To determine if ntRAV plays a role in RNA silencing, we produced transgenic tobacco lines that either over-express or are silenced for ntRAV and crossed these lines to a transgenic line (line 6b5) that is silenced in response to a sense-transgene encoding GUS. Using histochemical staining and northern blot analyses, we find that ectopic over-expression of ntRAV delays the developmental onset of GUS silencing in both the blade and the vein tissues of 6b5 leaves. Although delayed by about two weeks, once begun, the pattern of RNA silencing is similar in ntRAV over-expressing lines and wt controls. In both cases, silencing begins in the veinal tissues of older leaves and spreads into adjacent blade tissue over the next two weeks. In contrast to the ntRAV over-expressing lines, GUS RNA silencing is enhanced in the older leaves of adult 6b5 plants that have reduced ntRAV expression. We hypothesize that ntRAV encodes an endogenous suppressor of RNA silencing that may act early in development to regulate the developmental onset of RNA silencing. We next investigated the possible role of ntRAV in viral protein mediated suppression of RNA silencing. Established viral suppressors of RNA silencing, HC-Pro (TEV) and P38 (TCV) have been shown to alter siRNA biogenesis originating from the stem of a hairpin construct, as well as restore the accumulation of the transcript targeted for degradation by the hairpin siRNAs. We obtained insertional knockout lines of a homologous gene, EDF2/RAV2 (AT1g68840) in Arabidopsis. By crossing the rav2 knockout line to a plant expressing both P1/HC-Pro and the hairpin silencing system we show that P1/HC-Pro and P38 no longer mediate siRNA biosynthesis or target degradation. Additionally, we show that HC-Pro can no longer suppress virus-induced gene silencing (VIGS) in the absence of RAV2. Taken together these data suggest that RAV2/EDF2 is an endogenous suppressor of silencing which is plays a key role in viral suppression of silencing. To answer the question of how RAV2 works in HC-Pro suppression of silencing we employed whole genome tiling arrays. Analysis of these arrays indicated that HC-Pro is altering a complex network of stress and defense pathways which might play a role in suppression of silencing and we also find that HC-Pro requires RAV2 for a subset of these alterations
