M.F.Mette and W.Aufsatz contributed equally to this workDouble-stranded RNA induces a post-transcriptional gene silencing process, termed RNAi, in diverse organisms. It is shown here that transcriptional gene silencing accompanied by de novo methylation of a target promoter in plants can be triggered by a double-stranded RNA containing promoter sequences. Similar to the double-stranded RNA involved in RNAi, this promoter double-stranded RNA, which is synthesized in the nucleus, is partially cleaved into small RNAs~23 nucleotides in length. Both transcriptional and post-transcriptional gene silencing can thus be initiated by double-stranded RNAs that enter the same degradation pathway. The results also implicate double-stranded RNA in directing DNA methylation. Different constructs designed to produce doublestranded promoter RNA in various ways were evaluated for their ability to induce gene silencing in tobacco and Arabidopsis. RNA hairpins transcribed from inverted DNA repeats were the most effective trans-acting silencing signals. This strategy could be useful for transcriptionally downregulating genes in a variety of plants.

To analyze relationships between RNA signals, DNA methylation and chromatin modi®cations, we performed a genetic screen to recover Arabidopsis mutants defective in RNA-directed transcriptional silencing and methylation of a nopaline synthase promoter±neomycinphosphotransferase II (NOSpro± NPTII) target gene. Mutants were identi®ed by screening for recovery of kanamycin resistance in the presence of an unlinked silencing complex encoding NOSpro double-stranded RNA. One mutant, rts1 (RNA-mediated transcriptional silencing), displayed moderate recovery of NPTII gene expression and partial loss of methylation in the target NOSpro, predominantly at symmetrical C(N)Gs. The RTS1 gene was isolated by positional cloning and found to encode a putative histone deacetylase, HDA6. The more substantial decrease in methylation of symmetrical compared with asymmetrical cytosines in rts1 mutants suggests that HDA6 is dispensable for RNA-directed de novo methylation, which results in intermediate methylation of cytosines in all sequence contexts, but is necessary for reinforcing primarily C(N)G methylation induced by RNA. Because CG methylation in centromeric and rDNA repeats was not reduced in rts1 mutants, HDA6 might be specialized for the RNAdirected pathway of genome modi®cation.

In plants, double-stranded RNA that is processed to short RNAs Ϸ21-24 nt in length can trigger two types of epigenetic gene silencing. Posttranscriptional gene silencing, which is related to RNA interference in animals and quelling in fungi, involves targeted elimination of homologous mRNA in the cytoplasm. RNAdirected DNA methylation involves de novo methylation of almost all cytosine residues within a region of RNA-DNA sequence identity. RNA-directed DNA methylation is presumed to be responsible for the methylation observed in protein coding regions of posttranscriptionally silenced genes. Moreover, a type of transcriptional gene silencing and de novo methylation of homologous promoters in trans can occur if a double-stranded RNA contains promoter sequences. Although RNA-directed DNA methylation has been described so far only in plants, there is increasing evidence that RNA can also target genome modifications in other organisms. To understand how RNA directs methylation to identical DNA sequences and how changes in chromatin configuration contribute to initiating or maintaining DNA methylation induced by RNA, a promoter double-stranded RNA-mediated transcriptional gene silencing system has been established in Arabidopsis. A genetic analysis of this system is helping to unravel the relationships among RNA signals, DNA methylation, and chromatin structure.T he term ''RNA silencing'' refers to epigenetic gene silencing effects that are initiated by double-stranded RNA (dsRNA) (1). Discovered independently in plants, fungi, and animals, RNA silencing phenomena are revealing new ways to repress gene expression and to subdue transposable elements and viruses that produce dsRNA during their replication cycle (2-8). A fundamental step in RNA silencing pathways is cleavage of dsRNA into short RNAs (9), which are believed to act as guides for enzyme complexes that either degrade or modify homologous nucleic acids.The most familiar type of RNA silencing occurs primarily in the cytoplasm and is termed posttranscriptional gene silencing (PTGS) in plants, quelling in Neurospora, and RNA interference (RNAi) in animals. PTGS͞RNAi involves a dsRNA that is processed by an RNase III-like enzyme called Dicer into short interfering (si) RNAs 21-22 nt in length. The antisense siRNAs associate with a ribonuclease complex and guide sequencespecific degradation of complementary mRNAs (5-8).A second form of RNA silencing involves sequence-specific changes at the genome level. RNA-directed DNA methylation (RdDM) (10), which has been described so far only in plants, leads to de novo methylation of almost all cytosine residues within the region of sequence identity between the triggering RNA and the target DNA. Similarly to PTGS͞RNAi, RdDM requires a dsRNA that is cleaved to short RNAs Ϸ21-24 nt in length (11). It is not yet certain whether the short RNAs or dsRNA guide methylation of homologous DNA sequences, although the length of short RNAs is consistent with the minimum DNA target size of RdDM (Ϸ30 bp) (12).RdDM is assumed to be the ...

The Arabidopsis genome encodes four Dicer-like (DCL) proteins, two of which contain putative nuclear localization signals. This suggests one or more nuclear pathways for processing double-stranded (ds) RNA in plants. To study the subcellular location of processing of nuclear-encoded dsRNA involved in transcriptional silencing, we examined short interfering (si) RNA and micro (mi) RNA accumulation in transgenic Arabidopsis expressing nuclear and cytoplasmic variants of P19, a viral protein that suppresses posttranscriptional gene silencing. P19 binds specifically to DCL-generated 21-to 25-nucleotide (nt) dsRNAs with 2-nt 3Ј overhangs and reportedly suppresses the accumulation of all size classes of siRNA. Nuclear P19 resulted in a significant reduction of 21-to 22-nt siRNAs and a 21-nt miRNA, but had a lesser effect on 24-nt siRNAs. Cytoplasmic P19 did not decrease the quantity but resulted in a 2-nt truncation of siRNAs and miRNA. This suggests that the direct products of DCL cleavage of dsRNA precursors of 21-to 22-nt siRNAs and miRNA are present in the nucleus, where their accumulation is partially repressed, and in the cytoplasm, where both normal sized and truncated forms accumulate. DCL1, which contains two putative nuclear localization signals, is required for miRNA production but not siRNA production. DCL1-green fluorescent protein fusion proteins localize to nuclei in transient expression assays, indicating that DCL1 is a nuclear protein. The results are consistent with a model in which dsRNA precursors of miRNAs and at least some 21-to 22-nt siRNAs are processed in the nucleus, the former by nuclear DCL1 and the latter by an unknown nuclear DCL."RNA silencing" is the suppression of gene expression through nucleotide (nt) sequence-specific interactions that are mediated by RNA (Voinnet, 2002). RNA silencing is triggered by double-stranded (ds) RNA that is processed by an RNase III activity termed Dicer into short RNAs 21 to 25 nt in length (Hannon, 2002;Zamore, 2002). In plants, RNA silencing can act at the posttranscriptional and transcriptional levels (Cerutti, 2003). The 21-to 22-nt short interfering (si) RNAs and micro (mi) RNAs silence genes posttranscriptionally by targeting cognate mRNAs for degradation by an endonuclease complex (Llave et al., 2002a;Tang et al., 2003). A longer class of siRNAs in plants (24-26 nt) has been implicated in directing homologous DNA methylation and in systemic silencing (Hamilton et al., 2002). RNA-directed DNA methylation (RdDM) can lead to transcriptional gene silencing (TGS) if promoter sequences are targeted by homologous RNA (Mette et al., 1999(Mette et al., , 2000Jones et al., 1999Jones et al., , 2001Sijen et al., 2001;Aufsatz et al., 2002aAufsatz et al., , 2002b.The length and functional diversity of short RNAs in plants are reflected in the multiplicity of Dicer-like (DCL) activities. In contrast to genomes of other organisms, which encode one (human, mouse, fission yeast [Schizosaccharomyces pombe], and Caenorhabditis elegans) or two (fruitfly [Drosophila mel...

We used a transgene system to study spreading of RNAdirected DNA methylation (RdDM) during transcriptional gene silencing in Arabidopsis thaliana. Forward and reverse genetics approaches using this system delineated a stepwise pathway for the biogenesis of secondary siRNAs and unidirectional spreading of methylation from an upstream enhancer element into downstream sequences. Trans-acting, hairpin-derived primary siRNAs induce primary RdDM, independently of an enhancer-associated 'nascent' RNA, at the target enhancer region. Primary RdDM is a key step in the pathway because it attracts the secondary siRNA-generating machinery, including RNA polymerase IV, RNA-dependent RNA polymerase2 and Dicer-like3 (DCL3). These factors act in a turnover pathway involving a nascent RNA, which normally accumulates stably in non-silenced plants, to produce cis-acting secondary siRNAs that induce methylation in the downstream region. The identification of DCL3 in a forward genetic screen for silencing-defective mutants demonstrated a strict requirement for 24-nt siRNAs to direct methylation. A similar stepwise process for spreading of DNA methylation may occur in mammalian genomes, which are extensively transcribed in upstream regulatory regions.

Previous work has suggested that de novo methylation of plant nuclear genes can be triggered by an RNA-DNA interaction. To test whether transcription of a promoter would induce de novo methylation and silencing of unlinked genes driven by the same promoter, a chimeric 'gene' consisting of a nopaline synthase promoter (NOSpro) positioned downstream of the cauliflower mosaic virus 35S promoter (35Spro) and flanked at the 3Ј end by a NOS terminator (NOSter) was constructed and introduced into the genome of a plant that normally expresses an unmethylated NOSpro-neomycinphosphotransferase (nptII) gene. Transformants were tested for kanamycin resistance and NOSpro RNA synthesis. Most produced a fulllength polyadenylated NOSpro RNA, which did not induce silencing or methylation at the NOSpro-nptII target gene. One, however, contained truncated nonpolyadenylated NOSpro RNA; in this plant, the NOSpro-nptII gene became silenced and methylated in the NOSpro region. Molecular analysis of the NOSpro silencing locus revealed two incomplete copies of the 35Spro-NOSpro gene arranged as an inverted repeat with NOSpro sequences at the center. Reducing NOSpro transcription by crossing a 35Spro-silencing locus partially reactivated nptII gene expression and decreased NOSpro methylation at the target locus, thus implicating aberrant NOSpro RNA in this transsilencing phenomenon.

Although integration of viral DNA into host chromosomes occurs regularly in bacteria and animals, there are few reported cases in plants, and these involve insertion at only one or a few sites. Here, we report that pararetrovirus-like sequences have integrated repeatedly into tobacco chromosomes, attaining a copy number of Ϸ10 3 . Insertion apparently occurred by illegitimate recombination. From the sequences of 22 independent insertions recovered from a healthy plant, an 8-kilobase genome encoding a previously uncharacterized pararetrovirus that does not contain an integrase function could be assembled. Preferred boundaries of the viral inserts may correspond to recombinogenic gaps in open circular viral DNA. An unusual feature of the integrated viral sequences is a variable tandem repeat cluster, which might reflect defective genomes that preferentially recombine into plant DNA. The recurrent invasion of pararetroviral DNA into tobacco chromosomes demonstrates that viral sequences can contribute significantly to plant genome evolution. M ost plant viruses have single-stranded RNA genomes. Only two classes of plant viruses, caulimoviruses and badnaviruses, contain genomes of double-stranded DNA (1). Because these double-stranded DNA viruses use a virally encoded reverse transcriptase to replicate their genomes, they, together with vertebrate hepadnaviruses, are classified as pararetroviruses to distinguish them from true retroviruses, which have RNA genomes. Retroviruses have not yet been conclusively identified in plants, although recent findings of retrotransposons that encode envelope-like proteins suggest that they might exist (2-4). Pararetrovirus replication in plants proceeds by nuclear transcription of a slightly greater than genome-length RNA with terminal repeats that is generated by the host RNA polymerase II. This is followed by reverse transcription in the cytoplasm of the terminally redundant RNA, which also serves as an mRNA for viral proteins (5). Although retrovirus DNA integrates into host chromosomes by means of a virally encoded integrase (6), pararetroviruses generally lack the gene for this enzyme, and integration is not required for virus replication. However, pararetroviral DNA can in principle integrate into host DNA, as exemplified by mammalian hepatitis B (hepadna)virus, which has been found integrated into host chromosomes in hepatic tissue, where it is associated with liver carcinomas (7). Until recently, there were no data suggesting comparable integration of pararetroviral sequences into plant DNA.In contrast to bacterial and animal viruses, plant viral sequences are generally thought to integrate rarely, if at all, into host genomes. One well characterized example concerns a single insertion of sequences related to a geminivirus, which has a single-stranded circular DNA genome, into tobacco nuclear DNA (8-10). Although the geminivirus case has been considered exceptional, several recent reports prompt a reconsideration of the possibility that plant pararetrovirus DNA might integr...

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