Spreading of RNA Targeting and DNA Methylation in RNA Silencing Requires Transcription of the Target Gene and a Putative RNA-Dependent RNA Polymerase

TLDR

RNA silencing is a sequence‑specific RNA degradation process that follows the recognition of double‑stranded RNA. Virus vectors carrying GFP fragments trigger RNA silencing that spreads from the initiator region to adjacent 5′ and 3′ regions of the GFP gene, accompanied by DNA methylation, and this spreading requires transcription of the transgene and the RNA‑dependent RNA polymerase SDE1/SGS2, which generates double‑stranded RNA from the target RNA; endogenous genes such as Rubisco and phytoene desaturase do not show such spreading or methylation.

Abstract

RNA silencing is a sequence-specific RNA degradation process that follows the recognition of double-stranded RNA. Here, we show that virus vectors carrying parts of a green fluorescent protein (GFP) transgene targeted RNA silencing in Nicotiana benthamiana and Arabidopsis against the entire GFP RNA. These results indicate that there was spreading of RNA targeting from the initiator region into the adjacent 5' and 3' regions of the target gene. Spreading was accompanied by methylation of the corresponding GFP DNA. It also was dependent on transcription of the transgene and on the putative RNA-dependent RNA polymerase, SDE1/SGS2. These findings indicate that SDE1/SGS2 produces double-stranded RNA using the target RNA as a template. RNA silencing of ribulose-1,5-bisphosphate carboxylase/oxygenase and phytoene desaturase was not associated with the spreading of RNA targeting or DNA methylation, indicating that these endogenous RNAs are not templates for SDE1/SGS2.