A single transgene locus triggers both transcriptional and post-transcriptional silencing through double-stranded RNA production

Abstract

Silencing of a target locus by an unlinked silencing locus can result from transcription inhibition (transcriptional gene silencing; TGS) or mRNA degradation (post-transcriptional gene silencing; PTGS), owing to the production of double-stranded RNA (dsRNA) corresponding to promoter or transcribed sequences, respectively. The involvement of distinct cellular components in each process suggests that dsRNA-induced TGS and PTGS likely result from the diversiWcation of an ancient common mechanism. However, a strict comparison of TGS and PTGS has been diYcult to achieve because it generally relies on the analysis of distinct silencing loci. We describe a single transgene locus that triggers both TGS and PTGS, owing to the production of dsRNA corresponding to promoter and transcribed sequences of diVerent target genes. We describe mutants and epigenetic variants derived from this locus and propose a model for the production of dsRNA. Also, we show that PTGS, but not TGS, is graft-transmissible, which together with the sensitivity of PTGS, but not TGS, to RNA viruses that replicate in the cytoplasm, suggest that the nuclear compartmentalization of TGS is responsible for cell-autonomy. In contrast, we contribute local and systemic traYcking of silencing signals and sensitivity to viruses to the cytoplasmic steps of PTGS and to ampliWcation steps that require high levels of target mRNAs.

Genome-Wide Analysis of the RNA-DEPENDENT RNA POLYMERASE6/DICER-LIKE4 Pathway inArabidopsisReveals Dependency on miRNA- and tasiRNA-Directed Targeting

“…Other siRNA classes function at the posttranscriptional level. Silencing of viral RNA and transgene transcripts involves RDR6/DCL4-dependent siRNAs (21 nucleotides) and, for at least some viruses, DCL2-dependent siRNAs (22 nucleotides) (Waterhouse et al, 2001;Xie et al, 2004;Bouche et al, 2006;Deleris et al, 2006;Fusaro et al, 2006;Waterhouse and Fusaro, 2006;Mourrain et al, 2007). Dual siRNA biogenesis pathways may be an important adaptation during antiviral defense due to the ability of viruses to suppress individual pathways (Deleris et al, 2006).…”

Epimutant Induction as a New Plant Breeding Technology

“…PTGS signals such as siRNA can be delivered over extended distances through the sieve tube (Mourrain et al 2007), usually from source to sink, following the direction of phloem flow (Tournier et al 2006). Short-distance spreading of PTGS signals via plasmodesmata is limited to within 10-15 cells (Himber et al 2003) unless the target transcript works as a template for RNA-dependent RNA polymerase 6 (RDR6), in which case the PTGS can spread through the entire plant by transitivity (Brosnan et al 2007).…”

“…Although it has been considered that the TGS triggered by transgene-derived siRNA is not graft-transmissible (Mlotshwa et al 2002, Mourrain et al 2007), Molnar et al (2010) demonstrated that transgene-derived siRNA moved across the graft union using an Arabidopsis mutant in which siRNA biogenesis was blocked. They provided evidence that a 24-nucleotide mobile siRNA from an endogenous gene could direct epigenetic DNA methylation in the genome of the recipient cells.…”

Engineering metabolic pathways in plants by multigene transformation

“…Other studies have indicated that repetitive use of the same promoter can encourage (although probably not directly trigger) transgene silencing (Mourrain et al, 2007). This observation may reflect several underlying factors, such as the presence of potential secondary structures that could interact in trans to promote de novo methylation, or the intrinsic activity of the promoters generating enough mRNA to saturate the polyadenylation machinery of the cell, allowing the formation of hairpin RNAs.…”