Date of Award

1-1-2012

Document Type

Campus Access Dissertation

Department

Biological Sciences

First Advisor

Vicki Vance

Second Advisor

Lewis Bowman

Abstract

RNA silencing is a sequence-specific RNA degradation pathway that acts as an antiviral defense pathway. Viruses have evolved suppressors of RNA silencing, including the potyviral protein Helper Component-Proteinase (HC-Pro). Our focus on evaluating the mechanism of HC-Pro function has led us to the discovery of several endogenous plant proteins that facilitate suppression of RNA silencing. One of these proteins, REGULATOR OF GENE SILENCING CALMODULIN-LIKE PROTEIN (rgs-CaM), was the first endogenous silencing suppressor discovered over a decade ago. Here we show that the Arabidopsis homolog of rgs-CaM, CALMODULIN-LIKE38 (CML38) must be present as two functional gene copies in order for HC-Pro to suppress silencing of either a virus or a hairpin transgene. In addition, haploinsufficiency of CML38 results in a decrease in primary and secondary siRNAs in wildtype as well as HC-Pro-expressing plants. We determined that CML38 interacts with S-RIBONUCLEASE BINDING PROTEIN (SBP1), a RING-HC domain E3 ubiquitin ligase, which in turn also interacts with another endogenous suppressor of silencing, a transcription factor called RAV2. SBP1 is required for complete suppression of RNA silencing by HC-Pro and over-expression of SBP1 partially suppresses RNA silencing of a hairpin transgene, raising the possibility that regulation of RNA silencing may occur via the ubiquitin pathway. Since ubiquitination is primarily associated with protein degradation, we investigated whether suppression of silencing was correlated with protein accumulation. We found that HC-Pro-expressing Arabidopsis plants accumulate more RAV2 protein than wildtype plants. This work identifies additional components in the RNA silencing pathway and furthers our understanding of HC-Pro-mediated suppression of silencing.

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