A Vicious Cycle RNA Silencing and DNA Methylation in Plants

came, several years ago, from a study with a plant RNA Several new studies have stimulated intense interest in viroid in tobacco (Wassenegger et al., 1994). In this work, understanding the mechanism and evolutionary signifia transgenic tobacco strain was engineered with either cance of RNA silencing, the targeted degradation of replication-proficient or replication-deficient viroid geRNA (reviewed in Sharp, 2001). RNA silencing is typically nomes present on the transgene. Uniquely in the replicatriggered by double-stranded RNA (dsRNA). The dsRNA tion-proficient lines, the viroid transgene DNA became trigger is diced into very small species of 21–25 nt methylated. Sequences in the tobacco genome can also (siRNAs), which then guide sequence-specific cleavage be methylated by infection with RNA viruses containing of other homologous RNAs, such as messenger RNAs. homologous segments, although only transgene targets Thus, both the trigger and target RNAs are ultimately have been successfully methylated by this means to destroyed. This mechanism occurs in eukaryotic organdate (Jones et al., 1999; Thomas et al., 2001). These isms as diverse as the laboratory plant Arabidopsis thaliresults tie in with the observation from several plant ana, nematode worms, and mice. It is speculated that transgene systems that transgenes experiencing RNA RNA silencing exists as a defense against invasive silencing often display methylation within the coding dsRNA species such as RNA viruses. In fact, plants can sequences of the transgene. The interpretation of these recover from infection by RNA viruses via RNA silencing results is that one or more aberrant RNA species pro(Ratcliff et al., 1999). RNA silencing might also have duced by RNA viruses or transgenes—dsRNA, siRNAs, a role in developmental regulation. Furthermore, RNA or some other unusual form—actually contact genomic silencing induced by injected dsRNA or dsRNA exDNA and provide a signal for methylation of the conpressed from a transgene provides a powerful tool for tacted region (Figure 1; Jones et al., 1999). In this way, reverse genetics in animals and plants. RNA-directed DNA methylation can be thought of as a Plant RNA silencing displays certain unique features violation of the “central dogma” that biological informabeyond this common framework. For example, some tion flow moves from DNA to RNA. plant transgene constructs that would not obviously The idea that siRNAs might direct DNA methylation produce dsRNA nonetheless produce siRNAs and trigas well as RNA degradation was proposed to account ger RNA silencing. For this reason, it is thought that in for observations from a two-component transgene RNA plants, other “aberrant” RNAs exist that can be prosilencing system developed in Arabidopsis (Dalmay et cessed into siRNAs. In addition, plant RNA silencing is al., 2000a). The first component in this system, the trigfrequently accompanied by DNA cytosine methylation ger RNA for silencing, is generated from a replicationof the silenced gene (reviewed in Wassenegger, 2000). proficient plant RNA virus genome expressed from a The general observation from plant transgene RNA sisingle-copy “amplicon” transgene. This transgene can lencing systems is that affected DNA sequences can produce various viral RNA replication intermediates inbecome methylated over the regions that are homolocluding dsRNA species. The amplicon viral genome has gous to the aberrant RNA silencing trigger. been engineered to include a gene encoding green fluoMethylation is usually diagnostic of heterochromatin rescent protein (GFP) as a reporter. Arabidopsis plants formation—that is, the modification and remodeling of carrying the amplicon display a weak RNA silencing DNA-associated proteins into a condensed chromatin response against the amplicon sequences, with a low complex. When methylation occurs in the promoters of level of amplicon siRNAs but also a low level of steadygenes, transcription initiation is blocked. However, when state viral RNAs. However, there is no detectable methmethylation occurs in the coding sequences of genes, ylation over the amplicon sequences. The second comsuch as plant transgenes undergoing RNA silencing, ponent, the target for RNA silencing, is a single-copy