FWA locus, the silenced allele is not paramutagenic; it can not silence an active allele as reviewed in [40]. In addition, maize contains significant levels of a new class of 22 nt heterochromatic RNAs [34], suggesting greater complexity with these processes in maize. Clearly, further studies of paramutation in maize are needed to understand how the paramutagenic and paramutable alleles communicate to set up and heritably maintain RNA- mediated transcriptional silencing. In this report we describe the identification of mop2 and show that it is required for paramutation at multiple loci. Map-based cloning demonstrated that mop2 encodes a second largest subunit of plant specific RNA polymerases similar to the NRPD2/NRPE2 subunit shared by Pol-IV and Pol-V in Arabidopsis. Unlike Arabidopsis, which encodes a single functional gene, maize encodes three closely related genes, all of which appear to encode full length proteins and show significant overlapping expression in a variety of tissues. We report on a number of additional gene silencing phenotypes of an EMS-induced dominant mutant allele of mop2, Mop2-1, which has a single nucleotide change in a domain highly conserved among all polymerases ranging from E.coli to higher eukaryotes. Our results suggest that Mop2-1 is disrupting siRNA biogenesis and may be disrupting chromatin targeting properties of small RNAs in maize, and that its ability to disrupt epigenetic processes varies with dosage. Models for Mop2-1 dominance and implications of our findings for mechanisms of b1 paramutation are discussed. Results Dominant mutation, mediator of paramutation2-1, prevents b1 paramutation and activates previously silenced B’ alleles Paramutation at b1 involves two alleles, paramutable B-I (B-Intense) and paramutagenic B’ [54]. Phenotypes of B-I and B’ are easily distinguished; the highly expressed paramutable B-I allele specifies high levels of purple anthocyanin pigment in most of the above ground organs (sheath, husk, and tassel), while the low expressed paramutagenic B’ allele confers light speckled plant pigmentation (Figure 1A). The B-I allele is unstable and spontaneous paramutation to the low expressed B’ state occurs at variable frequencies ranging from 0.1 to .50%, depending on the stock. In contrast to B-I, the silenced B’ state is very stable and no change to higher expression has been observed in wild types backgrounds in many thousands of plants examined [12,54,55]. In addition to being very stable, the B’ state is highly paramutagenic, when B-I and B’ are combined in an heterozygote, B’ always paramutates B-I resulting in all F1 progeny having light plant pigment [54]. In addition, the newly paramutated B’ allele (B-I in the previous generation) is as efficient as the parental B’ allele at causing paramutation of na¨ıve B-I alleles [54]. The absolute penetrance of b1 paramutation in wild type backgrounds, such that B’ always changes the B-I allele to B’, was exploited to identify mutations required for b1 paramutation. For this genetic screen B’ pollen was treated with the chemical mutagen, ethyl methanesulfonate (EMS), and used to fertilize B-I ears (Figure 1A and Materials and Methods). The resulting M1 progeny were screened for rare dark purple plants, which would appear if B’ failed to paramutate B-I, presumably due to the presence of a dominant mutation that prevented paramutation. One such exceptional dark plant was found among ,7300 M1 plants (Figure 1A) and the putative mutation this plant carried was named Mediator of paramutation2-1 (Mop2-1). The B-I allele that escaped paramutation was designated B-I* to indicate its exposure to B’, but that it remained B-I (Figure 1A). In subsequent mop2 Is Required for Multiple Silencing Processes generations the presence of a single copy of the Mop2-1 mutation continued to protect B-I from paramutation by a newly introduced B’ allele and this protection occurred independent of whether Mop2-1 was transmitted through the male or female (Figure S1 and Figure S2; data not shown). From the initial experiments it was apparent that when Mop2-1 is heterozygous B’ silencing is not relieved, as these plants are lightly pigmented (Figure 1B and Figure S1). It was also apparent that Mop2-1 was loosely linked to b1 (Figure S1). To examine whether the Mop2-1 mutation when homozygous might relieve B’ silencing, a family segregating Mop2-1 B’/Mop2-1 B’ and Mop2-1 B’/+ B’ plants was developed (Figure S2). If B’ silencing was relieved in Mop2-1 homozygous plants, then such plants would be expected to be darker relative to Mop2-1 B’/+ B’ siblings. This expectation was met as the majority of homozygous Mop2-1 plants (69%) showed increased pigmentation (Chart in Figure 1B), but none were as dark as B-I (Photo in Figure 1B). The remaining homozygous Mop2-1 plants had medium dark (28%) or light (2%) pigment (Figure 1B) suggesting that the ability of Mop2-1/Mop2-1 to relieve B’ silencing was not fully penetrant. To determine whether the increased expression of the B’ allele observed in homozygous Mop2-1 plants was a heritable change in the absence of Mop2-1, darkly pigmented Mop2-1 B’ homozygous plants were crossed with the B-I/B-P tester (Figure S2). The B-Peru (B-P) allele does not participate in paramutation and confers essentially no plant color, which provides an excellent background for scoring B’ and B-I pigmentation. Examination of Mop2-1 B’/+ B-P progeny revealed no dark plants (Figure 1C). The majority of plants had light B’ pigment (87%), indicating that in these individuals B’ was efficiently re-silenced in the presence of the wild type allele. The presence of some medium dark plants (13%) suggested that increased expression could be weakly heritable. Taken together, these results demonstrate that only when the Mop2-1 mutation is homozygous is B’ silencing relieved, unlike preventing paramutation where a single copy of the Mop2-1 mutation was sufficient to prevent B’ from silencing B-I. The crosses described in Figure S2 were also used to assess the penetrance of the Mop2-1/+ effects on b1 paramutation in a larger population of plants. Analysis of the Mop2-1 B’/+ B-I * progeny demonstrated that 96% of these plants were darkly pigmented indicative of no paramutation and demonstrating that Mop2-1 acts in a dominant and highly penetrant manner to prevent paramutation (Figure 1D). There were a few (5/126) Mop2-1 B’/+ B-I * plants that had a medium dark phenotype. These could result from either spontaneous paramutation of B-I* to B’, incomplete penetrance of Mop2-1/+ in preventing paramutation, or both. To confirm that B-I* segregates phenotypically unchanged from B’ in Mop2-1/+ plants, a backcross to the B-I/B-P stock was performed (Figure S2). Phenotypic and molecular markers were used to identify the + B-I*/+ B-P plants that are informative for B-I* heritability. If B-I* escaped paramutation in the previous generation, then, after accounting for recombination between the linked b1 and mop2 loci, assuming full Mop2-1 penetrance, and absence of spontaneous paramutation (Figure S2), 73% should be parental (+ B-I*/+ B-P; dark plants) and 27% recombinant (+B’/ +B-P; light plants). Results presented in Figure 1E demonstrate that light plants (22%), likely representing the B’/B-P progeny, were observed at a frequency close to the expected 27% (x2 = 0.53, P = 0.46). The frequency of dark plants (53%) was lower than expected (x2 = 7.7, P = 0.005), with medium dark plants observed at 24%. As there are no molecular markers to distinguish B’ from the B-I, the medium dark plants could theoretically represent reduced expression of B-I* (because of PLoS Genetics | www.plosgenetics.org 3 November 2009 | Volume 5 | Issue 11 | e1000725