Minisymposium 6: Epigenetics

Abs # 16001: An endogenous suppressor of RNA silencing in tobacco

Presenter:	Endres, Matthew W, endres@biol.sc.edu
Authors	Endres, Matthew W (A)   Ge, Xin  (A)   Appel, Bryan  (A)   Bowman, Lewis H (A)   Vance, Vicki B (A)
Affiliations:	(A): University of South Carolina

RNA silencing is a conserved RNA degradation mechanism that targets invading nucleic acids such as viruses, transgenes or transposons. In both plants and animals, RNA silencing has evolved, at least in part, as an antiviral defense pathway. Consistent with the antiviral role of RNA silencing, many viruses encode suppressors of silencing. Here we report the identification of a tobacco protein (termed ntRAV) that interacts with a plant viral suppressor of silencing called helper component proteinase (HC-Pro) in the yeast two-hybrid system. NtRAV is related to the Arabidopsis RAV family of transcription factors. The ntRAV/HC-Pro interaction raised the possibility that ntRAV plays a role in HC-Pro suppression of silencing. 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 (6b5) that is silenced in response to a sense-transgene encoding GUS. Initiation of GUS silencing in the 6b5 line occurs developmentally: the transgene is expressed in young seedlings, but declines with the onset of silencing at around three weeks after germination. Using histochemical staining and northern blot analyses, we find that ectopic over-expression of ntRAV delays the developmental onset of GUS silencing in leaves of the 6b5 line by about two weeks. However, once begun, the pattern of RNA silencing is similar in ntRAV over-expressing lines and wt controls, beginning in veins of older leaves and spreading into adjacent blade tissue. Interestingly, the pattern of accumulation of endogenous ntRAV mRNA parallels that of the silenced GUS mRNA, high in young seedlings and rapidly declining at two to three weeks after germination. Together, these data suggest that ntRAV encodes an endogenous suppressor of RNA silencing that acts early in development to block RNA silencing. Consistent with these data, our preliminary results suggest that the onset of GUS silencing is accelerated in an ntRAV silenced tobacco line. Earlier work identified a calmodulin-related protein (termed rgsCaM) that interacts with HC-Pro and impairs virus-induced RNA silencing when over-expressed in N. benthamiana. Interestingly, yeast two-hybrid studies indicate that rgsCaM and ntRAV both interact with the same E3 ubiquitin ligase. This information leads us to speculate that HC-Pro suppression of silencing may be mediated by rgsCaM and ntRAV via interactions with the ubiquitin pathway.