that the nrpd2/e2a and nrpd2/e2b genes are more similar to the presumed rice ortholog OsNrpd2a, while maize nrpd2/e2c is more similar to the other rice gene OsNrpd2b (Figure 3C and 3D). The more similar genes, nrpd2/e2a and nrpd2/e2b are located in recently duplicated blocks within the maize genome, while the more diverged nrpd2/e2c gene is located in a more anciently duplicated block [57]. High conservation within all of the critical polymerase domains (Figure S3) suggested that all three nrpd2/e2 genes are likely to encode functional proteins. BLAST analysis indicated that nrpd2/e2a and nrpd2/e2b have multiple EST hits, whereas nrpd2/e2c had no significant EST hits in current databases. Lack of nrpd2/e2c ESTs could be either because of low expression, or because it is expressed in tissues under represented in the public EST datasets. To further explore the expression of all three genes, we carried out quantitative RT-PCR experiments using gene-specific primers. We detected expression of all nrpd2/e2 genes in a wide range of tissues, but there was quantitative variation among the genes (Figure 4). For all three genes, the highest expression was in immature tassel and the lowest expression was in endosperm. The expression of nrpd2/e2c was more elevated in pollen and two callus samples (HiII and BMS) relative to nrpd2/e2a and nrpd2/e2b genes. Taken together these results demonstrate that all three maize nrpd2/e2 genes are likely to be functional, in contrast with Arabidopsis where only one functional nrpd2/e2 gene exists. Mop2-1 reduces siRNAs levels, but does not alter transcription from the 853 bp tandem repeats that are required for b1 paramutation Mutations in the largest and second largest subunit of the Arabidopsis Pol-IV RNA polymerase cause dramatically reduced siRNA production [32,58,59]. Similarly, mutations in rmr6, which encodes the maize large subunit most similar to NRPD1 within the Pol-IV complex in Arabidopsis, show a dramatic reduction in siRNAs [31]. To determine if Mop2-1 might reduce the function of a Pol-IV-like complex in maize, siRNA levels in Mop2-1 were tested both globally and from the tandem repeats that mediate b1 paramutation. The small RNA fraction was isolated from Figure 4. Maize nrpd2/e2 genes are differentially expressed. Gene-specific primers and quantitative RT–PCR was used to analyze the RNA expression patterns of each of the three nrpd2/e2 genes in multiple maize tissues from the B73 inbred line and HiII and BMS callus. Details on the developmental stages of the tissues are in Materials and Methods. Expression was normalized to actin1 expression levels. doi:10.1371/journal.pgen.1000725.g004 mop2 Is Required for Multiple Silencing Processes immature ears, which are a rich source of RNA, separated on gels, and stained with SyberGold. Staining revealed that global siRNA levels were dramatically reduced in both heterozy- gous (Mop2-1/+) and homozygous (Mop2-1/Mop2-1) samples (Figure 5A), consistent with the dominant phenotype of Mop2-1. We next asked whether levels of siRNAs from the 853 bp tandem repeats (Arteaga-Vazquez et al., in preparation) mediating b1 paramutation [11] were altered in Mop2-1. In the wild type (+/+) background, two siRNA bands (prominent ,25 nt and faint ,35 nt) were detected in the B’ allele (Figure 5B), which carries seven tandem 853 bp repeats and causes paramutation. In contrast, in the B’ Mop2-1 samples there was a dramatic reduction of the 24 nt band, while the ,35 nt siRNAs appeared to increase (Figure 5B). Consistent with the dominant phenotypes that occur in Mop2-1, reduced levels of siRNAs were seen in both heterozygous and homozygous Mop2-1 individuals, although there was more reduction in 24 nt siRNAs in homozygotes. Future experiments to examine whether the reduction of 24 nt siRNAs in Mop2-1 is associated with reduced asymmetric (CHH) DNA methylation, as observed in Arabidopsis will be important to carry out. Small RNAs larger than 24 nt have been reported in multiple species. In the ciliate protozoan, Tetrahymena thermophila, 27–30 nt RNAs direct developmentally directed DNA elimination [60], in mammals and zebrafish 26–31 nt PIWI-interacting RNAs are present in the germline [61,62], and in Drosophila repeat associated RNAs direct retrotransposon and repetitive sequence silencing [63]. In Arabidopsis, the role of a specific 30 nt siRNA reported for Flowering Locus C is unknown [64] and 30–40 nt siRNAs are induced in response to pathogen infection or under specific growth conditions [65]. In our studies, the ,35 nt b1 tandem repeat RNAs were only observed when using the VC1658 LNA probe, one of four LNA probes for the b1 tandem repeats that we have used (data not shown). One possibility is that transcripts from the LNA-VC1658 region are stable enough to detect alternative processing that increases when the predominant 24 nt pathway is disrupted. Further studies will be required to determine if the presence of the ,35 nt siRNA class is significant. The reduction in tandem repeat 24 nt siRNA levels in Mop2-1 plants could theoretically be because Mop2-1 is causing a reduction in transcription of the tandem repeats or a defect in processing. Previously, we showed that the 853 bp tandem repeats that mediate b1 paramutation are transcribed [22]. To test whether transcription from the 853 bp repeats was altered in Mop2-1, we conducted nuclear run-on analyses from nuclei isolated from young ears, the same tissue used for siRNA analyses. The results presented in Figure 5D revealed no significant differences in transcription from the 853 bp repeats between wild type (+ B’) and homozygous Mop2-1 B’ samples. This result indicated that Mop2-1 did not disrupt transcription from the b1 tandem repeats and suggested that the lack of 24 nt siRNAs is caused by a defect downstream of transcription. The dramatic reduction in 24 nt siRNAs is consistent with mutations in the large subunit of the Pol- IV complex in Arabidopsis and maize supporting the hypothesis that the Mop2-1 mutation is disrupting the function of a Pol-IV- like complex in maize. Our observations that there is no change in the transcription from the b1 tandem repeats in Mop2-1 mutants, further suggests that the major polymerase(s) responsible for this transcription is unlikely to be Pol-IV. Consistent with that hypothesis, other experiments suggest that the polymerase responsible for the bulk of the b1 tandem repeat transcription is likely to be Pol-II as transcription is dramatically reduced with levels of alpha-amanitin that inhibit Pol-II (see Discussion). PLoS Genetics | www.plosgenetics.org 7 November 2009 | Volume 5 | Issue 11 | e1000725