RNA–DNA Interactions and DNA Methylation in Post-Transcriptional Gene Silencing

Abstract

Post-transcriptional gene silencing (PTGS) is a homology-dependent process that reduces cytoplasmic RNA levels. In several experimental systems, there is also an association of PTGS with methylation of DNA. To investigate this association, we used plants carrying a transgene encoding the green fluorescent protein (GFP). Gene silencing was induced using potato virus X RNA vectors carrying parts of the coding sequence or the promoter of the GFP transgene. In each instance, homology-based, RNA-directed methylation was associated with silencing. When the GFP -transcribed region was targeted, PTGS affected both transgene and viral RNA levels. When methylation was targeted to a promoter region, transgene RNA levels were reduced; however, viral RNA levels were unaffected. For comparison, we induced PTGS of the gene encoding the endogenous ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) small subunit ( rbcS ) by inoculation with potato virus X-rbcS. In this example, no methylation of the rbcS DNA was associated with the reduction in rbcS transcript levels, and viral RNA levels were unaffected. Finally, we investigated DNA methylation by using GFP -transformed plants in which PTGS was induced by localized introduction of a T-DNA carrying GFP sequences. In these plants, there was methylation of a GFP transgene associated with systemic spread of a gene-silencing signal from the infiltrated part of the plant. This transgene methylation was not affected when systemic PTGS was blocked by suppressors of silencing encoded by potato virus Y and cucumber mosaic virus. Combined, these data support an epigenetic model of PTGS in which transgene methylation is associated with an RNA-DNA interaction that ensures that PTGS is maintained. INTRODUCTIONPost-transcriptional gene silencing (PTGS) is a genetic control mechanism that operates at the level of sequence-specific RNA degradation and acts against transgenes, endogenous genes, and viruses (reviewed in Depicker and Van Montagu, 1997;Stam et al., 1997a; van den Boogaart et al., 1998). Although PTGS originally was identified in plants, recent evidence suggests that a similar mechanism exists in animals (Fire et al., 1998;Kennerdell and Carthew, 1998).In transgenic plants, particularly in lines that carry multiple transgene copies, PTGS may be initiated spontaneously (Hobbs et al., 1993;Goodwin et al., 1996;Stam et al., 1997b). PTGS also can be triggered by viruses carrying homology to nuclear sequences (Lindbo et al., 1993;Kumagai et al., 1995;Jones et al., 1998a;Kjemtrup et al., 1998;Ruiz et al., 1998), by using biolistic Palauqui and Balzergue, 1999) or Agrobacteriummediated delivery of DNA (Voinnet and Baulcombe, 1997). These observations have led to proposals that ectopic interactions between homologous nucleic acids lead to the onset of PTGS Selker, 1999).Several studies have indicated an association between PTGS and coding region methylation in plants (Ingelbrecht et al., 1994;English et al., 1996;Sijen et al., 1996; van Houdt et al., 1997). Indeed, methylation may p...

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