5S rDNA Silencing by siRNAs analyzed nuclei of the ddm1 mutant in genetic combination with nrpd1, rdr2 or dcl3. In the majority of wild type nuclei, 5S rDNA colocalized predominantly with heterochromatic domains known as chromocenters, which stain intensely with DAPI (Figure 4A). In contrast, decondensation of 5S rDNA was evident in ddm1 nuclei, resulting in smaller and more numerous signals. Analysis of 5S rDNA signals colocalized with DAPI-stained chromocenters confirmed that ddm1 nuclei contained more 5S rDNA outside chromocenters (52%, n = 158) than did wild-type nuclei (23%, n = 134). Importantly, nrpd1 ddm1 and rdr2 ddm1 double mutants displayed FISH signal outside chromocenters more frequently (68%, n = 186 and 73%, n = 215, respectively) than did ddm1, indicating an additive effect of these mutations on 5S rDNA decondensation (Figure 4B). These observed differences were all statistically significant (P,0.05, see Table S1). By contrast, 5S rDNA decondensation status in dcl3 ddm1 (58% outside, n = 190) was not distinguishable from ddm1. Interestingly, mutants showing pronounced 5S rDNA decon- densation also exhibited growth deficiencies; this was particularly evident in the double mutants nrpd1 ddm1 and rdr2 ddm1 (Figure 5A). The single mutants nrpd1, rdr2, dcl3 and ddm1 showed no appreciable effect on fresh weight (Figure 5B). In contrast, ddm1 nrpd1 and ddm1 rdr2 double mutant individuals were smaller and weighed, on average, 50% that of ddm1 or wild-type plants (Figure 5). The overall stature and fresh weight of ddm1 dcl3 plants were similar to those of ddm1 plants. Figure 4. Both DDM1 and siRNAs are required for proper 5S rDNA condensation. A) 5S rDNA localization in interphase nuclei: Fluorescent in situ hybridization (red) was performed with a probe for 5S rDNA. DNA was stained with DAPI (white). The size bar corresponds to 5 mm. The white arrow in the wild type (WT) images indicates 5S rDNA colocalized with a DAPI-stained chromocenter; this colocalization was observed in a majority of WT nuclei (see panel B). In contrast, a majority of nuclei from ddm1 and the double mutants nrpd1 ddm1, rdr2 ddm1 and dcl3 ddm1 showed 5S rDNA localization outside chromocenters (arrows in mutant panels). B) Nuclei from each genotype were scored for 5S rDNA colocalization with chromocenters (white bars), as compared to 5S rDNA not colocalized with chromocenters (black bars). Differences observed between 5S rDNA localization outside chromocenters in ddm1 nuclei, compared to in nrpd1 ddm1 or rdr2 ddm1 nuclei are statistically significant (*). Numbers of nuclei scored: WT (n = 134), ddm1 (n = 158), nrpd1 ddm1 (n = 186), rdr2 ddm1 (n = 215), dcl3 ddm1 (n = 190). doi:10.1371/journal.pone.0005932.g004 PLoS ONE | www.plosone.org 6 June 2009 | Volume 4 | Issue 6 | e5932