A Branched Pathway for Transgene-Induced RNA Silencing in Plants

Abstract

In plants, RNA silencing can be induced by highly transcribed sense transgenes (S-PTGS) or by transgene loci producing double-stranded RNA (dsRNA) due to the presence of inverted repeats (IR-PTGS). Both phenomena correlate with accumulation of 21-25 nt sense and anti-sense RNA homologous to the silent gene and with methylation of the coding sequence. We have challenged IR-PTGS with four viruses known to inhibit S-PTGS: CMV, TuMV, TVCV, and TCV ( this work) and in sgs2, sgs3, and ago1 mutants impaired in S-PTGS. Surprisingly, whereas the four viruses inhibit IR-PTGS, IR-PTGS and methylation of a GUS trangene and IR-PTGS of three endogeneous genes occur in the sgs2, sgs3, and ago1 mutations. Based on these results, we propose a branched pathway for RNA silencing in plants. RNA silencing would occur via the action of dsRNA produced either via the action of SGS2 (also known as SDE1), SGS3, and AGO1 on the S-PTGS branch or by transgenes arranged as inverted repeats on the IR-PTGS branch. Moreover, transgene methylation would result from production or action of dsRNA, since it does not require SGS2/SDE1, SGS3, and AGO1.

Viral Virulence Protein Suppresses RNA Silencing–Mediated Defense but Upregulates the Role of MicroRNA in Host Gene Expression[W]

“…p69 Does Not Inhibit PTGS Induced by IR-RNA Transgenes RNA silencing induced by IR-RNA transgenes is distinct to sense and virus RNA silencing because neither the cellular nor viral RdRP is involved in the formation of the dsRNA inducer from a nuclear self-complementary IR-RNA transcript (Beclin et al, 2002). Thus, we next determined if P69 interfered with silencing of the phytoene desaturase (PDS) gene mediated by an IR-RNA transgene (IRPDS), which caused a dominant photobleaching phenotype.…”

“…These include a cellular RNA-dependent RNA polymerase (RdRP) homolog (SGS2/SDE1), AGO1, an RNA helicase gene (SDE3), and genes that encode a coiled-coil protein (SGS3) and a novel protein (HEN1) (Fagard et al, 2000;Mourrain et al, 2000;Boutet et al, 2003). Current models place these genes upstream of dsRNA because none of these genes are required for IR-RNA-induced PTGS (Dalmay et al, 2001;Beclin et al, 2002). In A. thaliana, both Dicer-like 1 (DCL1) and HEN1 are involved in the production of miRNAs (Park et al, 2002;Reinhart et al, 2002), and there is evidence that miRNAs function as siRNAs to specify cleavage of their mRNA targets (Llave et al, 2002;Rhoades et al, 2002;Kasschau et al, 2003).…”

Developmental Defects Mediated by the P1/HC-Pro Potyviral Silencing Suppressor Are Not Due to Misregulation of AUXIN RESPONSE FACTOR 8

“…For the purpose of the molecular analysis, the P1/HC-Pro line we used for the cross contained two additional transgenes that provide an assay for silencing suppression (Mlotshwa et al, 2008). These additional loci are a sense uidA transgene (GUS) encoding b-glucuronidase and a hairpin transgene (hpGUS) that posttranscriptionally silences the GUS locus (Béclin et al, 2002). Expression of each of the three transgenes in this P1/HC-Pro line is under the control of the cauliflower mosaic virus 35S promoter.…”

The Function of RNAi in Plant Development

“…RDR6 and SGS3 were originally identified in screens for mutants that block the posttranscriptional silencing of transgenes (Dalmay et al 2000;Mourrain et al 2000). RDR6 was proposed to be responsible for the transformation of mRNA into double-stranded RNA based on its similarity to a tomato RNA-dependent RNA polymerase and the observation that it is required for the silencing of transgenes that produce "sense" transcripts, but not transgenes that have been engineered to produce a hairpin transcript (Beclin et al 2002). Additional evi- -1, sgs3-11, rdr6-11, and dcl4-2.…”

“…SGS3 is a plant-specific protein of unknown function. Although mutations in these genes have identical effects on transgene silencing (Beclin et al 2002) and have identical morphological phenotypes (Peragine et al 2004), they have opposite effects on the accumulation of the miR173-generated cleavage fragments of TAS2 and TAS1a (Yoshikawa et al 2005). sgs3 decreases the accumulation of these fragments, and rdr6 produces a dramatic increase in their accumulation.…”