mop2 Is Required for Multiple Silencing Processes Figure 3. Mop2-1 and mop2-2 mutations are located in highly conserved motifs of the NRPD2/E2 proteins. (A) and (B) show excerpts from edited alignments of the second largest subunits of RNA polymerases with the positions of the Mop2-1 and mop2-2 mutations shown. Mutations in the single functional NRPD2/E2 gene in Arabidopsis are also shown, drd2-12 and -9 [41]. Species designations are: Ec-Escherichia coli, Sc-Saccharomyces cerevisiae, At-Arabidopsis thaliana, Os-Oryza sativa, and Zm-Zea mays. The full alignment is in Figure S3. (C) Un-rooted radial bootstrap neighbor-joining phylogenetic tree of the second largest subunits of RNA polymerases. Species designations are shown in Table S1. (D) A portion of the phylogenetic tree shown in (C) with an expanded traditional view of the Pol-IV/Pol-V branch is shown. Bootstrap values are at the base of each branch. doi:10.1371/journal.pgen.1000725.g003 hypothesis that mop2-2 disrupts B’ silencing as a homozygote, similar to Mop2-1. Further experiments demonstrate that unlike Mop2-1, mop2-2 is a recessive mutation as the establishment of paramutation is not prevented in heterozygotes (data not shown). Two mutations isolated in Arabidopsis NRPD2/E2 are in the same domains as Mop2-1 and mop2-2 (Figure 3A), but as only homozygous phenotypes are reported [41], it is not clear if the Arabidopsis mutations also have dominant or semi- dominant phenotypes. Maize has three expressed genes that encode a second- largest subunit of Pol-IV/Pol-V–like polymerase in maize BLAST searches of the maize genome revealed that maize encodes three nrpd2/e2 genes. In addition to the mop2 nrpd2/e2 gene, designated nrpd2/e2a, located on chromosome 2S, there are two genes on chromosome 10: nrpd2/e2b on 10L, FPC Contig 418 (94% identity and 97% similarity to nrpd2/e2a); and nrpd2/e2c on 10S, FPC Contig 401 (67% identity and 79% similarity to nrpd2/e2a). Phylogenetic analysis demonstrated PLoS Genetics | www.plosgenetics.org 6 November 2009 | Volume 5 | Issue 11 | e1000725