5S rDNA Silencing by siRNAs Figure 5. Simultaneous deficiency for DDM1 and siRNAs impairs plant growth. A) Rosette stage plants (21 days-post-germination) of lines deficient for heterochromatic siRNA biogenesis (nrpd1, rdr2 and dcl3) compared to corresponding double mutant combinations with ddm1. Four representative plants in each population (n = 18) were photographed. B) Fresh weight of aerial portion of plants from each population: plants were individually weighed and mean values plotted with standard errors (bars). doi:10.1371/journal.pone.0005932.g005 Discussion Silent chromatin structure is an attribute of tandem repeats in many eukaryotic genomes, including repeats in plants [12,68–70]. Arabidopsis 5S rRNA gene loci are interesting in that their cytosine methylation is both DDM1/MET1 and RNA dependent [7,18,39,54]. Earlier findings that DDM1 deficiency perturbs siRNA accumulation suggested that DDM1 might act directly in the RNA silencing pathway [7,14]. Indeed, our results show that silencing of aberrant 5S rDNA transcripts depends on siRNA biogenesis, in addition to DDM1. However, the contributions of siRNA biogenesis and DDM1 are genetically separable, suggesting that they involve independent mechanisms. DDM1 limits accumulation of siRNAs derived from the 5S rDNA IGS. Several findings indicate that DDM1 does not directly participate in siRNA production, but helps control transcription of RNAs that serve as precursors for siRNA production. For instance, siRNA overaccumulation persists upon out-cross of ddm1 mutants to wild-type plants. Moreover, mutants deficient for DDM1, MET1 or VIM methylcytosine-binding proteins gain ectopic (apparently RNA-directed) 5S rDNA methylation but display reduced CG methylation [54,71]. We therefore posit that a silent chromatin state, maintained by DDM1 and associated with CG methylation, represses production of IGS precursor RNAs that give rise to siRNAs directing DNA methylation. Detection of dsRNA interme- diates that are precursors of siRNA duplexes would help test this hypothesis. In addition, analysis of small RNA sequences from ddm1 (e.g., [49,72]) could determine whether particular 5S rDNA arrays contribute to siRNA overaccumulation. In our model (Figure 6) a canonical RNA polymerase (e.g., Pol III) is assumed to initiate at the 5S rRNA gene promoter and transcribe into the downstream IGS upon the loss of a silent chromatin state. Pol IV or Pol V transcription can be excluded as the source of these initial transcripts, because 5S LT1 accumu- lation was not abolished in nrpd1, nrpd2 or nrpe1 mutants. Concurrently, dsRNA corresponding to the IGS is generated in a Pol IV and RDR2-dependent manner. Multiple DCLs can digest these dsRNA substrates into IGS siRNAs, although DCL3 is overwhelmingly favored. Inhibition of IGS transcription by DDM1 and MET1 would explain why IGS siRNAs over- accumulate in mutants for these proteins. The increased siRNA titers in ddm1 correlate with hypermethylation of asymmetric CHH sites, and this increased methylation depends on Pol IV and RDR2. DRM2 is thought to mediate such RNA-directed DNA methylation (RdDM) [7,67,73], consistent with a report that ectopic 5S rDNA hypermethylation occurring in a met1 mutant requires DRM2 [54]. Derepression of aberrant 5S rDNA transcripts in ddm1 is clearly not a total reactivation, because mutants deficient for both DDM1 and siRNA biogenesis showed an additive effect on 5S LT1 derepression. This suggests that siRNA overaccumulation and resulting asymmetric DNA methylation may partially compensate for DDM1 deficiency. However, the enhanced RdDM observed in ddm1 and met1 is not sufficient to completely repress 5S LT1 transcripts [52]. Future analysis of 5S LT1 accumulation in mutants deficient for specialized Argonaute effector proteins, such as AGO1 and AGO4, could help address whether RNA silencing of 5S rDNA transcripts occurs by mechanisms other than RdDM. Beyond 5S rDNA transcript repression, RNA silencing proteins and DDM1 are both required for full condensation of 5S rDNA repeats in the nucleus. Previous studies implicated Pol IV and DDM1 individually in 5S locus condensation [7,51,56]. Our study shows that double deficiency for DDM1 and heterochromatic siRNAs causes more decondensation of 5S rDNA than the ddm1 mutation alone. Furthermore, the reduced biomass of double mutants nrpd1 ddm1 and rdr2 ddm1 implies that plant growth responds to both DDM1 and siRNA-dependent signals. This conclusion is consistent with the pronounced phenotype of the double mutant drm2 met1, in which both maintenance methylation and RdDM are impaired [54]. The siRNA-dependent pathway has recently been implicated in correcting hypomethylation of transposable element sequences induced by ddm1 [49]. Taken together, these findings suggest that the balancing of RNA silencing and DDM1/MET1 functions has significance for PLoS ONE | www.plosone.org 7 June 2009 | Volume 4 | Issue 6 | e5932