Viral and endogenous suppressors of RNA silencing in Arabidopsis small RNA pathways

Post-transcriptional gene silencing (PTGS) is a mechanism in which double-stranded RNA (dsRNA) or otherwise aberrant RNA is processed to short interfering RNAs (siRNAs) which guide the sequence-specific degradation of homologous mRNA. Subsequent attempts to clone siRNAs have led to the identification of a number of related small RNA-mediated pathways. The most notable of these, microRNAs (miRNAs) have been shown to regulate a variety of endogenous processes in nearly all eukaryotic organisms. Moreover, small RNA cloning in plants has also detected other pathways that utilize components of both the siRNA and miRNA pathways.; Shortly after the discovery of PTGS, certain plant viruses were shown to encode suppressors of gene silencing. One such viral suppressor, the potyviral helper-component protease (P1/HC-Pro) has been shown to alter small RNA pathways in plants. Specifically, P1/HC-Pro suppresses PTGS by elimination or reduction of siRNAs. P1/HC-pro was also demonstrated to interfere with miRNA-directed mRNA target cleavage in plants. In at least one case, though, plant miRNAs regulate gene expression through translational repression. Therefore, we were interested to determine whether P1/HC-Pro was able to suppress this type of miRNA-directed mRNA regulation. We employed the Agrobacterium transient expression system to investigate the effects of P1/HC-Pro on translational repression. We confirmed the earlier report that P1/HC-Pro is a suppressor of miRNA-directed cleavage. However, P1/HC-Pro was unable to interfere with mRNA degradation resulting from miRNA-mediated translational repression. Our results indicate that plant miRNAs induce degradation of target messages through two different pathways which can be differentiated by sensitivity to P1/HC-Pro. Moreover, examination of target mRNA poly-A tails points to a translational repression-induced degradation mechanism that involves mRNA deadenylation and/or decapping.; Yeast two-hybrid identification of HC-Pro interacting proteins has also led to the discovery of an endogenous suppressor of RNA silencing, rgs-CaM (regulator of gene silencing, calmodulin-like protein) in tobacco. Like HC-Pro, rgs-CaM has been shown to suppress gene silencing triggered by plant viruses. We were interested to determine whether rgs-CaM, like HC-Pro, could also suppress silencing triggered by a transgene. Using an Arabidopsis plant line silenced for the GUS reporter gene, we were able to demonstrate that rgs-CaM is capable of suppression of this type of gene silencing. Furthermore, our results indicate that rgs-CaM suppression of transgene silencing is dependent on the levels of rgs-CaM ectopic expression in Arabidopsis.