spontaneous paramutation of B-I* to B’) or increased expression of +B’/+B-P recombinants. Independent of these hypotheses, the significant number of dark B-I* plants segregating demonstrates that the presence of one Mop2-1 mutant allele in the previous generation can prevent paramutation. This finding is in sharp contrast to wild type backgrounds in which exceptional B-I-like plants have never been observed in thousands heterozygous B’/B-I plants grown over decades of experiments [12,54,55]. Mop2 encodes the second largest subunit of a plant- specific RNA polymerase The B’ and B-I stocks were differentially marked with two genes linked to b1 (Figure 1A), which enabled following the original chromosomes carrying B’ and B-I in subsequent generations. Presence of these markers enabled the initial observation that the Mop2-1 mutation was loosely linked to B’, distal of glossy2 (Figure S1). Screening of a large mapping population (Materials and Methods) further located the Mop2-1 mutation to the 3.6 cM (13 BACs) interval spanning FPC Contigs 69 and 70 (Release 3b.50, February, 2009) (Figure 2A). Using additional molecular markers, the interval was further reduced to 1.5 cM, which consisted of two BAC clones (,400 kb) on FPC Contig 69 (Figure 2B). Analysis of putative genes in this interval revealed a strong candidate, a nrpd2/ e2 gene, closely related to the Arabidopsis NRPD2/NRPE2 gene mop2 Is Required for Multiple Silencing Processes encoding the second largest subunit of the Pol-IV and Pol-V plant specific RNA polymerases. Arabidopsis NRPD2/NRPE2 is in- volved in regulating several epigenetic gene silencing phenomena [32,33,41,42]. Sequencing of the nrpd2/e2 gene from this interval in the Mop2-1 mutant revealed a transition mutation of guanine to adenine (G to A) relative to the progenitor allele, consistent with an EMS-induced mutation (Figure 2C). This change in DNA sequence led to a missense mutation of glutamic acid to lysine (E1079K), within the GEME motif, which is absolutely conserved [56] in Pol-I, Pol-II, Pol-III, and Pol-IV/Pol-V related polymerases from E.coli to higher eukaryotes (Figure 3A). The high conservation of the mutated residue strongly suggested that this change in Mop2-1 would produce a mutant phenotype. The hypothesis that mop2 is a nrpd2/e2 gene was supported by the isolation of a second allele of mop2 from an independent screen (Materials and Methods). The second allele, designated mop2-2, carries a G to A transition mutation relative to its progenitor, which is consistent with an EMS induced mutation. This mutation changes a glycine to arginine, (G1026R, Figure 2C) within another highly conserved domain (Figure 3B), distinct from that mutated in Mop2-1. Co- segregation analysis revealed that all plants homozygous for the mop2-2 lesion had a dark plant phenotype consistent with the Figure 2. Mop2-1 encodes a second-largest subunit of a plant-specific RNA polymerase. (A) Location of the Mop2-1 interval on chromosome 2S is shown (FPC contigs 69–70). Polymorphic markers (indicated above the contigs) were used for mapping. The number of recombinants over the total number of plants screened are shown in parenthesis next to each marker. (B) An expanded map of the Mop2-1 interval localized to two BACs. The dashed line is used for the AC213986 BAC because at the time of the publication it consisted of more then 30 unordered fragments. Predicted gene models within the two BACs were obtained from www.maizesequence.org. The predicted position and orientation of the nrpd2/e2 gene (based on synteny with rice) is indicated. (C) Exons and introns of the nrpd2/e2 gene based on alignment of genomic and cDNA sequences. The exons 1–7, translation start and stop, and polyadenylation sites are indicated. The positions of the G to A transitions in the Mop2-1 and mop2-2 alleles are shown. Location of the domains conserved with Pol-II RPB2 are shown below the exons. Domains were identified using the BLASTP program at http://pfam.sanger.ac.uk/search. doi:10.1371/journal.pgen.1000725.g002 PLoS Genetics | www.plosgenetics.org 5 November 2009 | Volume 5 | Issue 11 | e1000725