One of the functions of RNA silencing in plants is antiviral defense. A hallmark of RNA silencing is spreading of the silenced state through the plant. Little is known about the nature of the systemic silencing signal and the proteins required for its production, transport, and reception in plant tissues. Here, we show that the RNA-dependent RNA polymerase RDR6 in Nicotiana benthamiana is involved in defense against potato virus X at the level of systemic spreading and in exclusion of the virus from the apical growing point. It has no effect on primary replication and cell-to-cell movement of the virus and does not contribute significantly to the formation of virus-derived small interfering (si) RNA in a fully established potato virus X infection. In grafting experiments, the RDR6 homolog was required for the ability of a cell to respond to, but not to produce or translocate, the systemic silencing signal. Taking these findings together, we suggest a model of virus defense in which RDR6 uses incoming silencing signal to generate double-stranded RNA precursors of secondary siRNA. According to this idea, the secondary siRNAs mediate RNA silencing as an immediate response that slows down the systemic spreading of the virus into the growing point and newly emerging leaves.Endogenous RNA-dependent RNA polymerase (RDR) activity was demonstrated in plants more than 30 years ago (Astier-Manifacier and Cornuet, 1974), but its function remained unclear for many years. More recently, RDRs were proposed as part of an RNA silencing system (Lindbo et al., 1993) related to RNA interference in animals and quelling in fungi. Consistent with this idea, the SILENCING DEFECTIVE 1 (SDE1)/SUPPRESSOR OF GENE SILENCING 2 (SGS2) locus was identified as a requirement for certain examples of transgene RNA silencing in Arabidopsis (Arabidopsis thaliana; Dalmay et al., 2000;Mourrain et al., 2000). Following a more recent nomenclature, we will henceforth refer to SDE1/SGS2 as RDR6 .A likely biochemical role of RDRs in RNA silencing is to produce double-stranded (ds) RNA that is cleaved by RNase III-like enzymes called Dicer (DCR) in animals and Dicer-like (DCL) in plants (Bernstein et al., 2001;Golden et al., 2002;Carmell and Hannon, 2004). The resulting 21 to 24 nucleotide small interfering (si) RNAs are then recruited as the specificity determinants of silencing effector complexes that also include argonaute (AGO) proteins. The in vitro activity of RDR from Lycopersicum esculentum (Schiebel et al., 1993a(Schiebel et al., , 1993b(Schiebel et al., , 1998, Neurospora crassa (Makeyev and Bamford, 2002), and Schizosaccharomyces pombe (Motamedi et al., 2004) is consistent with a role in dsRNA synthesis.There are several RNA silencing pathways (Baulcombe, 2004), and, correspondingly, there are multiple genes for RDR, DCR, DCL, and AGO proteins in many organisms. In some instances, when the initiator of silencing is a viral genome (Dalmay et al., 2000) or a gene that is transcribed into an RNA with ds regions (Beclin et al., 2002), there is no kno...

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 doublestranded 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. INTRODUCTIONRNA silencing is a nucleotide sequence-specific process of RNA degradation in higher plants (post-transcriptional gene silencing), animals (RNA interference [RNAi]), and fungi (quelling) as well as in unicellular eukaryotic algae. In higher plants, a natural role of RNA silencing is to protect against viruses (Covey et al., 1997;Ratcliff et al., 1997;Al-Kaff et al., 1998;Hamilton and Baulcombe, 1999). A role in genome protection also is likely because there is enhanced transposon mobility in RNA silencing-defective mutants of Chlamydomonas reinhardtii and Caenorhabditis elegans and because DNA transposition is suppressed by RNA silencing in Drosophila melanogaster (Jensen et al., 1999;Ketting and Plasterk, 2000;Wu-Scharf et al., 2000). Double-stranded RNA (dsRNA) is a potent activator of RNA silencing in C. elegans , D. melanogaster , and mammals (Fire et al., 1998; Zamore et al., 2000;Elbashir et al., 2001). Biochemical analyses of RNA silencing in D. melanogaster have shown that an RNase III (DICER) cleaves the dsRNAs into 21-to 25-nucleotide RNAs (siRNAs) that then associate with a second RNase in an RNA-induced silencing complex (RISC) (Hammond et al., 2000;Bernstein et al., 2001). RISC cleaves target single-stranded RNAs (ssRNAs) at a site that is complementary to the (antisense) siRNA. Thus, siRNAs provide sequence specificity to the RNA degradation process (Elbashir et al., 2001). Plant DICER and RISC have not yet been identified. However, siRNAs are present in plants ( Hamilton and Baulcombe, 1999), suggesting that mechanisms are conserved across kingdoms.In plants, RNA silencing is activated by viral RNAs that replicate via double-stranded intermediates and by transgenes with inverted repeat (IR) structures that produce dsRNA (Chuang and Meyerowitz, 2000;Smith et al., 2000;Waterhouse et al., 2001). Single-copy transgenes without IR structures also can activate RNA silencing (Elmayan and Vaucheret, 1996;Jorgensen et al., 1996). In these cases, it is possible that promoters in the transgene and the flanking plant DNA result in the transcrip...

SummaryA severe lignin mutant, irx4, has been identi®ed in Arabidopsis thaliana as a result of its collapsed xylem phenotype. In contrast to previously described irx mutants, irx4 plants have 50% less lignin than wildtype plants, whilst the cellulose and hemicellulose content remained unchanged. These alterations in the composition of irx4 secondary cell walls had a dramatic effect on the morphology and architecture of the walls, which expand to ®ll most of the cell, and also on the physical properties of irx4 stems. Further analysis indicated that the irx4 mutation occurred in a cinnamoyl-CoA reductase (CCR) gene within a highly conserved intron splice site sequence of intron 2. As a result, CCR mRNA transcripts were incorrectly spliced. Transgenic plants expressing an IRX3 promoter±CCR cDNA construct were used to generate a series of plants with varying degrees of lignin content in order to assess the role of lignin content in determining the physical properties of Arabidopsis stems.

RNA-triggered events can lead to heritable changes in gene expression, and it is possible that initiation of other epigenetic phenomena such as trans-silencing and paramutation may have an RNA component.