relative to the heterozygote, potentially equivalent to a loss of function mutation. The presence in maize of two other second largest subunits, including NRPD2/E2b that is 94% identical to NRPD2/E2a, provides a further complication and could also contribute to different phenotypes between Mop2-1 heterozygotes and homozy- gotes if the Mop2-1 encoded NRPD2/E2a protein competes with the other subunits for complex binding. This could lead to gain of function phenotypes, in which the Mop2-1 encoded NRPD2/E2a subunit is partially poisoning complexes that normally carry the NRPD2/E2b, c subunits. Competition would be postulated to be most effective in homozygous Mop2-1 plants where the dosage of the mutant subunit is highest. As stated previously, detailed biochemical analyses with each of the subunits differentially tagged will be necessary to begin to distinguish among possible models. New insights on roles of Pol-IV/Pol-V–like polymerases in b1 paramutation As b1 paramutation is the most extensively characterized system at the molecular level, we will limit our discussion to the b1 system. The Mop2-1 mutation prevents b1 paramutation when heterozy- gous and releases silencing of B’ when homozygous indicating Pol- IV/Pol-V-like RNA polymerases are required for b1 paramuta- tion. While the requirement for a Pol-IV-like polymerase for b1 paramutation was previously demonstrated in studies with a mutation in the large subunit most similar to Pol-IV in Arabidopsis [21,31], our results provide further clarification on potential roles for Pol-IV/Pol-V-like complexes at distinct steps in paramutation. Mop2-1 reduces siRNA production, characteristic of a Pol-IV- like mutation, but does not reduce transcription from the b1 tandem repeats, suggesting that NRPD2/E2a containing RNA polymerase complexes do not significantly contribute to b1 repeat transcription. Transcription from the b1 repeats is sensitive to actinomycinD, indicating that these transcripts are produced from DNA templates (Arteaga-Vazquez et al., in preparation). In Arabidopsis, Pol-V has been shown to use DNA as a template to produce non-coding transcripts [45]. Transcripts produced from Pol-V in Arabidopsis are rare [45], and if a similar situation exists in maize, differences between presumed Pol-V mediated tran- scription in wild type and homozygous Mop2-1 plants might be difficult to detect with nuclear run-ons, especially if most of the b1 repeat transcription is mediated by Pol-II. Transcription from the b1 repeats is highly sensitive to alpha-amanitin consistent with Pol-II being the best candidate for the polymerase performing the major transcription of the non-coding b1 repeats (Arteaga- Vazquez et al., in preparation). Further molecular and biochem- ical characterization of b1 repeat transcripts will determine whether these transcripts are polyadenylated, and chromatin immunoprecipitation assays with tagged Pol IV and Pol V complexes will determine if either physically interact with the b1 tandem repeats mediating paramutation. Our observation that silenced alleles are not up-regulated in Mop2-1 heterozygous plants, in spite of the dramatic reduction in b1 tandem repeat siRNAs, suggests that the major role for the tandem repeat siRNAs is not to maintain silencing. Instead, we hypothesize that the tandem repeat siRNAs may mediate the allele communication that establishes the heritable silent state; a process that is fully disrupted in Mop2-1 heterozygotes. Use of the Mop2-1 plants in future experiments may enable us to separate mechanisms operating at the initial establishment of paramutation from those operating subsequently to maintain silencing. Previ- ously this has not been possible as establishment can only be observed if it is heritably maintained, so mutations defective in maintaining silencing will also appear defective in establishment. mop2 Is Required for Multiple Silencing Processes However, mutations that do not relieve silencing, but do prevent the establishment of paramutation such as Mop2-1 (when heterozygous) provide a system to investigate mechanisms for establishment. Materials and Methods Generation of Mop2-1 and mop2-2 mutations The Mop2-1 mutation was generated by treating Gl2 B’ Wt; Pl-Rh; r-g (inbred W23 background) pollen with ethyl methane- sulfonate [74]. Treated pollen was used to pollinate gl2 B-I wt; Pl- Rh; r-g [W23/K55] ears, producing M1 seed (Figure 1A). In wild- type stocks, B’ will always paramutate B-I, and all progeny will be light. Thus, rare plants with B-I pigmentation levels may indicate the presence of a dominant mutation preventing paramutation. Presence of the recessive gl2 and wt markers on the B-I chromosome enabled rapid identification of self-pollination contaminant offspring. Pollen from the exceptional dark plant (KK1238-1) was crossed onto gl2 B’ wt ears (Figure S1). The mop2-2 mutation was isolated in an independent EMS screen in which B-I pollen was treated with EMS [74] and placed on silks of B’ plants. Resulting F1 plants were screened for dominant mutation phenotypes (none found) and self pollinated, and F2 progeny were screened for dark plants. Mapping of the Mop2-1 mutation Initial linkage of Mop2-1 relative to the b1, gl2 and wt loci was noticed in the test cross of the original Mop2-1 dark plant with + gl2 B’ wt tester (Figure S1). Of the 15 progeny plants, 12 plants inherited parental 2S chromosomes from the original mutant plant, three plants exhibited phenotypes consistent with recombi- nation, two between Mop2-1 and Gl2, and one between B-I* and wt (Figure S1). This result indicated that the Mop2-1 mutation is distinct from b1 and located distal of Gl2. The map position of the Mop2-1 mutation was subsequently confirmed and refined using simple sequence repeat (SSR) molecular markers (www.maizegdb. org) and a larger number of plants (data not shown). Two markers tightly linked to Mop2-1, bnlg1117 and bnlg1338, were used to routinely follow Mop2-1 segregation prior to cloning. PCR products were resolved using 4% Super Fine Resolution agarose gels. To further refine the location of Mop2, a large mapping population was generated by crossing homozygous Mop2-1 plants with B73, the inbred sequenced for the maize genome project that is also highly polymorphic relative to the Mop2-1 stock. The F1 was then backcrossed to homozygous Mop2-1, the resulting seed planted, and DNA was extracted from 1308 dark plants, the phenotype expected for homozygous Mop2-1. The resulting samples were screened with polymorphic markers on chromosome 2S (available upon request) to first determine the boundaries of the Mop2-1 interval (13 BACs) and then to further map it to within a two BAC interval on FPC contig 69 (Figure 2). Bioinformatics to identify candidate genes Examination of gene models within the two BAC interval (NCBI accessions AC1911113.2 and AC213986.2) revealed the presence of 21 putative protein encoding genes (Release 3b.50, February, 2009), including the AC191113.2_FGT037 gene model, which is predicted to encode the second largest subunit of a RNA polymerase most similar to rice gene SJNBa0063C18.1 (OsNrpd2a). The gene model AC191113.2_FGT037 was refined using FGENESH+ and Arabidopsis NRPD2/NRPE2 and OsNrpd2a genes as guides. The refined model corresponded well to the NRPDB101 gene model from www.chromdb.org, and was experimentally verified by PCR amplification of the full length PLoS Genetics | www.plosgenetics.org 14 November 2009 | Volume 5 | Issue 11 | e1000725