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 µm. 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).