suppresses siRNA precursors, we crossed homozygous nrpd1 (2/ 2) and ddm1 (2/2) lines to wild type Col-0, generating F1 plants that were heterozygous for nrpd1 (+/2) or ddm1 (+/2). Re- introduction of a functional NRPD1 allele in this manner restored siR1003 production to wild-type levels, as expected based on previous complementation and out-cross experiments [5,19]. In contrast, reintroduction of a functional DDM1 allele failed to reduce siR1003 overaccumulation (Figure 2D). Persistent siRNA overaccumulation and CG hypomethylation in ddm1 heterozygotes (Figure S3) suggests that an epigenetic state correlated with CG methylation, rather than expression of DDM1 per se, represses IGS siRNA precursor production. Next, we tested whether elevated IGS siRNA levels are associated with enhanced RNA-directed DNA methylation (RdDM). Using Southern blot analysis we assayed cytosine methylation in asymmetric sequence contexts (CHH), which largely results from RdDM [35,67]. After digestion with Alu I, which occurs once per 5S gene repeat, 5S rDNA arrays appear on Southern blots as a ladder of bands, each separated by 500-bp intervals. Longer than unit-length (500 bp) fragments reflect Alu I site methylation, which is more prominent in ddm1 and met1 than in wild type or siRNA biogenesis mutants (nrpd1, rdr2 and dcl3) (Figure 2E). Methylation is reduced in nrpd1 and rdr2 mutants, indicating that siRNA biogenesis is important for Alu I site methylation. However, dcl3 mutants resemble wild-type plants suggesting that other dicers compensate for the loss of 24-nt siRNAs in dcl3 mutants (see Figure 2A). Collectively, the observed increase in DNA methylation in met1 and ddm1 mutants is consistent with overproduction of siRNAs leading to increased de novo methylation of asymmetric sites. An analogous finding has been reported for siRNA overaccumulation and 5S rDNA hypermethylation in met1 [54]. 5S rDNA Silencing by siRNAs Overlapping Pol IV and DDM1 silencing effects are genetically independent To further test whether hypermethylation in ddm1 is siRNA- dependent, we generated the double mutants nrpd1 ddm1 and rdr2 ddm1, in addition to dcl3 ddm1 mentioned above. The three double mutants were subjected to Southern blot analysis, alongside wild type and ddm1 controls. Long Alu I fragments typical of ddm1 mutants were absent in nrpd1 ddm1 and rdr2 ddm1 (Figure 3A), indicating that Alu I site hypermethylation in ddm1 is indeed siRNA-dependent (i.e., RdDM). dcl3 ddm1 showed 5S rDNA methylation similar to ddm1, again consistent with production of abundant siRNAs of variable lengths in this double mutant (Figure 2C). Analysis of Hae III methylation, used to test asymmetric 5S rDNA methylation in previous studies [19,54], yielded identical results (Figure 3A). An additional assay using 5S LT1-specific primers, in which PCR amplification indicated DNA methylation status (Figure 3A), also supports the hypothesis that the hypermethylation is siRNA-dependent. Analysis of 5S aberrant transcripts revealed that 5S LT1 accumulates to higher levels in nrpd1 ddm1 and rdr2 ddm1 double mutants than in ddm1 alone; this indicates that both DDM1 maintenance of silent chromatin and siRNA-dependent silencing contribute to suppression of aberrant transcripts (Figure 3B). Because siRNA and DDM1 deficiencies are additive in effect, they apparently act in separate pathways. Deficiency for DDM1 and siRNAs enhances 5S rDNA decondensation and slows plant growth The previously observed localization of 5S rDNA outside chromocenters in ddm1 nuclei implies that 5S rDNA arrays are partially decondensed in ddm1 [51]. Using DNA-FISH we Figure 3. Effect of combined DDM1 and siRNA deficiency on 5S rDNA methylation and aberrant transcripts. A) 5S rDNA hypermethylation in ddm1 is siRNA-dependent: Southern blot comparison of AluI and HaeIII-digested genomic DNA isolated from inflorescences of wild type (WT), ddm1, and double mutant lines nrpd1 ddm1, rdr2 ddm1, and dcl3 ddm1 (top panel). The probe is the same as in Figure 2E. Dilutions of the above digests were also assayed by PCR using 5S LT1 primers (bottom panel). Samples to which no restriction enzyme was added are controls (no digest). B) 5S LT1 is silenced by two overlapping processes: RNA samples from inflorescences of WT, ddm1 and the double mutant panel were analyzed by one-step RT-PCR, performed as described in Figure 1C. Control reactions were performed with ACT2 primers; reverse transcriptase was omitted from duplicate 5S LT1 and ACT2 reactions (no RT). doi:10.1371/journal.pone.0005932.g003 PLoS ONE | www.plosone.org 5 June 2009 | Volume 4 | Issue 6 | e5932