5S rDNA Silencing by siRNAs Figure 6. Model for 5S rDNA methylation and aberrant transcript silencing. A canonical RNA polymerase (e.g., Pol III) is assumed to initiate at the 5S rRNA gene promoter, transcribe through the 5S rRNA gene (120 bp, gray), and continue into the intergenic spacer (IGS, white). Concurrently, dsRNA corresponding to the IGS is generated in a Pol IV and RDR2-dependent manner. DCL3 processes dsRNA into 24-nt siRNAs, with DCL2 and DCL4 being alternate enzymes at this step. RNA-directed DNA methylation (RdDM) resulting from this process is thought to require DRM2. DDM1/MET1 maintenance of silent chromatin and CG methylation represses aberrant IGS transcription, limiting production of siRNA precursors and, by consequence, attenuating RdDM. IGS-derived siRNAs may also guide cleavage of nascent IGS transcripts. doi:10.1371/journal.pone.0005932.g006 heterochromatin maintenance, normal growth and development, and protecting the genome from transposon reactivation. Materials and Methods Mutant plant strains nrpd1a-3 (nrpd1), nrpd1b-11 (nrpe1), nrpd2a-1 (nrpd2) are SALK T- DNA insertion lines described in Onodera et al. (2005) and Pontes et al. (2006) [7,19]. rdr2-1 and rdr6-15 are described in Xie et al. (2004) and Allen et al. (2005), respectively [18,74]. dcl2, dcl3 and dcl4 double and triple mutant combinations are described in Blevins et al. (2006) [62], with the constituent alleles being dcl2-5 of Akbergenov et al. (2006) [75], dcl3-1 of Xie et al. (2004) [18] and dcl4-2 of Xie et al. (2005) [76]. dcl3-4 is a GABI T-DNA insertion described by Daxinger et al. (2008) [77]. ddm1-2 used for crosses is described in Vongs et al. (1993) [39], and met1-3 in Saze et al. (2003) [41]. Genetic crosses Quadruple mutant dcl1/2/3/4 material was obtained by crossing dcl1-9 (caf, [78]) to the triple homozygous mutant dcl2 (2/2) dcl3 (2/2) dcl4 (2/2). F2 progeny of the genotype dcl1- 9(+/2) dcl2 (2/2) dcl3 (2/2) dcl4 (2/2) were then selected and self-fertilized. About 25% of resulting F3 progeny were quadruple homozygous d1/2/3/4. dcl1-9 used in the above crosses had been introgressed into Col-0 from Ler/Ws backgrounds by 56 outcrossing. Double mutants with ddm1-2 were generated as follows: nrpd1, rdr2 and dcl3 were individually crossed to ddm1. F2 plants homozygous for both ddm1 and each RNA silencing mutation were then self-fertilized for two generations and then analyzed with ddm1 and wild-type lines propagated in parallel. Bioinformatic analysis Sequences of 283 individual 5S rDNA repeats (File S1)–from Yeast Artificial Chromosomes (YACs) 4E4, 6A1, 7E7, 7G3, 9A5, 9D3 and 11A3 of the CIC project [58], and representing arrays from chromosomes 3, 4 and 5 [32]–were retrieved from TIGR Plant Repeat Databases [79]. Small RNA datasets of Rajagopalan et al. (2006) and Kasschau et al. (2007) were retrieved from GEO (http://www.ncbi.nlm.nih.gov/geo) via accession numbers GSM118372 and GSM154370. Sequencing reads from each tissue were combined and filtered to obtain 20–25 nt small RNAs. BLAST (NCBI v2.2.16) was used to query reads to individual 5S rDNA sequences. A Perl script (incorporating Bioperl module Bio::SimpleAlign) converted the 59-end positions of small RNA hits to individual repeats into coordinates along a multiple alignment of all repeats generated using Clustal W [80] (File S2). Tallies of small RNA hits were weighted by number of sequencing reads and collected in 10 nt bins, using the Microsoft Excel 2003 Chart functionality for map generation. Small RNA distribution analyses and maps for leaves, inflorescences and seedlings are presented in Table S2, Figure 1A and Figure S2. RT-PCR RNA was isolated using RNeasy Mini Columns (Qiagen). In brief, 100 mg of mixed-stage inflorescences were ground in liquid nitrogen and processed following the manufacturer’s protocol. On-column DNase I digestion was performed (Qiagen), in addition to a second digestion after elution (DNase I Amplification Grade, Invitrogen). Following the protocol of Wierzbicki et al. (2008) [23], 400 ng RNA were subjected to gene-specific RT-PCR. To detect 5S LT1 and 5S LT2, primer 5SUNIV2 (R) was used for reverse transcription (SuperScript III, Invitrogen; 50uC for 30 min.). The RT enzyme was then inactivated (70uC for 15 min.), primer RTPCR5S1 (F) added and PCR amplification performed (Platinum Taq, Invitrogen; 52uC annealing, 30 s elongation, 35 cycles). See Vaillant et al. (2006) for 5S rDNA primer sequences [52]. ACT2 controls were amplified in separate reactions using primers described in Blevins et al. (2006) [62] and different cycling parameters (57uC annealing, 40 s elongation, 25 cycles). Reactions were subjected to agarose gel electrophoresis and stained with ethidium bromide. PLoS ONE | www.plosone.org 8 June 2009 | Volume 4 | Issue 6 | e5932