Evidence implying only unprimed RdRP activity during transitive gene silencing in plants.

RNA silencing is a sequence-specific RNA degradation mechanism found in most eukaryotes, where small cleavage products (siRNAs) of double stranded RNA (dsRNA) mediate silencing of genes with sequence identity to the dsRNA inducer. In several systems, silencing has been found to spread from the dsRNA inducer sequence into upstream or downstream regions of the target RNA, a phenomenon termed transitive silencing. In nematodes, silencing spreads only in the 3'-5' direction along the target mRNA by siRNAs serving as primers for cRNA synthesis by RNA-dependent RNA polymerase. In plants, transitive silencing is seen in both directions suggesting that at least some cRNA synthesis occurs by un-primed initiation at the 3' end of mRNAs. Replicating plant viruses trigger an RNA silencing defence response that degrades the viral RNA, thus tempering the virus infection. Likewise, fragments of plant genes inserted into a virus will become targets for degradation, leading to virus-induced gene silencing (VIGS) of the homologous plant mRNAs. We have analyzed the spreading of gene silencing in VIGS experiments using a transgene and two endogenous genes as targets. In Nicotiana benthamiana plants expressing a beta-glucuronidase (GUS) transgene, a Potato virus X vector carrying a 5' fragment of the GUS gene induced silencing which spread to downstream regions of the transgene mRNA including the 3'-untranslated region. Conversely, silencing induced by a 3' fragment spread only for a limited distance in the 3'-5' direction. Silencing induced by a central GUS gene fragment spread only into downstream regions. Similar analyses using the endogenous plant genes, magnesium chelatase subunit I (ChlI) and an RNase L inhibitor homologue (RLIh), revealed no spreading along target sequences. This implies that transitive silencing in plants occurs by un-primed cRNA synthesis from the 3' end of targeted (transgene) transcripts, and not by siRNA-primed cRNA synthesis.