transcript and sequencing (NCBI GQ453405). The corrected AC191113.2_FGT037 gene model was renamed nrpd2/e2a. Please note that the order of fragments within the AC191113 in Figure 2 is different from that shown www.maizesequence.org at the time of publication. We reordered fragments based on additional sequence information that indicated positions of overlapping fragments within the AC191113 and neighboring BACs (not shown). Sequencing of the candidate gene in Mop2-1 and mop2-2 stocks A custom BAC library was constructed from Mop2-1 genomic DNA with the assistance of the Arizona Genomic Institute (Tucson, AZ). Nylon filters with printed DNA from this library were hybridized with a probe unique to the 3’ UTR of nrpd2/e2a (available upon request). One of the positive clones that contained the full length nrpd2/e2a gene was used as a template to PCR amplify all predicted exons (primer sequences available upon request). The resulting PCR fragments were sequenced in both directions using the core sequencing facility at University of Arizona (Tucson, AZ). Consistent with an EMS-induced mutation, a G to A transition was identified in the nrpd2/e2a gene, within an absolutely conserved motif in exon 7. To identify additional mutations, we sequenced the exons of the nrpd2/e2a gene in three newly isolated EMS-induced b1 paramutation mutants. Exons of the nrpd2/e2a gene were PCR amplified and amplicons were sequenced in both directions for each candidate mutant. One of the new mutants was found to carry a G to A transition in a conserved domain in exon 6, consistent with an EMS induced mutation. This mutation was named mop2-2. The nrpd2/e2a gene was sequenced in a total of 12 dark plants (mop2-2 homozygotes) and all carried the same lesion, indicating that the lesion segregated with the mutant paramutation phenotype. Sequence alignment and phylogenetic analysis Sequence alignment was carried out using MUSCLE [75], manually edited in GENEDOC 2.6.04. Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 4 software [76]. Bootstrap neighbor-joining method with 1000 replicates was used to generate the phylogenic trees. Protein sequences of the maize second largest subunits of Pol-I (ZmNRPA2) and Pol-II (ZmNRPB2a, ZmNRPB2b) were predicted using FGENESH+ software and corresponding Arabidopsis proteins as guides (Figure S7). To obtain genomic sequence suitable for protein prediction of maize nrpd2/e2b, the gap in the AC212557 sequence was PCR amplified and sequenced. FGENESH+ (http://linux1.softberry.com/berry.phtml) was used to generate the gene model, which is equivalent to GRMZM2G146935_T02 at www.maizesequence.org. The maize nrpd2/e2c gene model was predicted from the AC203335.4 BAC sequence using FGE- NESH+ and rice OsNrpd2b as a guide, which was equivalent to GRMZM2G133512_T01 at www.maizesequence.org. The qual- ity of the resulting gene models was inspected using the ClustalX multiple sequence alignment program [77]. For the alignment shown in Figure S3 and the phylogenetic tree shown in Figure 3C and 3D, sequences of the second largest subunits were provided by the Pikaard lab or retrieved from NCBI. The complete list of the genes used for the phylogenetic analysis is in Table S1. mop2 Is Required for Multiple Silencing Processes RNA was treated with DNAseI (Invitrogen) and acid phenol (Ambion) to remove DNA contamination. First stand synthesis was carried out using oligo(dT) primers and 10 ug of total RNA. Superscript III First Strand Synthesis System (Invitrogen) was used according to manufacturer’s recommendations at 55 C for 1 hour. About 200 ng of cDNA were used for each quantitative PCR assay on the Bio-Rad MyIQ Real-Time PCR machine and quantified using My-IQ software (Bio-Rad). Expression of nrpd2/e2 genes was normalized to actin1 expression. Sequences of gene-specific primers are available upon request. All plant materials were from the B73 inbred with the exception of the Black Mexican Sweet (BMS) tissue culture cells and the HiII callus cells. All tissues were collected in the morning, 2–4 hours after sunrise. At the time of collection all plant tissues were immediately frozen in liquid nitrogen. Seedlings were germinated for 6 days after imbibing in paper towels at 16 day/8 night photo period at 20C and tissue before the first leaf emerged from the coleoptile. Root tissue was from seedlings grown in a vermiculite/ soil mixture, 10 days after emergence (2–3 leaf stage). Seedling roots were washed before freezing. Husk, silks, immature ears, immature tassels, and pollen were collected from field grown plants. Husk and silks were collected simultaneously on the first day of silk emergence. Immature ears and tassels were collected when they were 1.5 cm to 2.5 cm in length. Pollen was collected in the morning from tassels on the first day of shedding. Pollen was filtered through fine metal mesh filters to remove debris before freezing. Endosperm and embryos were collected from greenhouse grown plants at 12 and 14 days after pollination (DAP). HiIIAxB type II embryogenic callus was maintained on N6 media with 1.0 mg/L of 2, 4-dichlorophenoxyacetic acid with sub-culturing every two weeks as described [78]. The BMS callus suspension culture used in these experiments was acquired from C. Armstrong (Monsanto Company, St. Louis, MO) in 2001. BMS cultures were maintained in N6 media supplemented with 1.5 mg/L of 2, 4- dichlorophenoxyacetic acid with monthly subcultures to a fresh media. Both the HiII and BMS cultures were cultured in the dark at 26–28uC. b1 and pl1 genetic stocks The gl2 b wt, Pl, r-g (inbred K55 background), the B-I Pl r-g (inbred W23 background) and the B’ Pl r-g (inbred K55 background) stocks were originally obtained from E.H. Coe, Jr. (University of Missouri, Columbia). Paramutagenic Pl’ allele and paramutable Pl-Rhoades (Pl-Rh) were previously described [3]. r1 genetic stocks and tests with Mop2-1 The phenotypes and paramutation properties of the R-st, R-r and r alleles were previously described [79,80]. Paramutation at r1 is typically assayed in the aleurone, the outer cell layer of the endosperm, where purple anthocyanin pigments accumulate in the highly expressed R-r allele; reduced pigmentation is observed when R-r is paramutated to R-r’ by R-st. The fully colored R-sc allele [79,81] was used as a positive seed color control (Figure 6C). Both heterozygous and homozygous Mop2-1 plants were assayed for effects on r1 paramutation (Figure S5), although only a small number of Mop2-1 homozygotes could be assayed because these plants have reduced fertility. To quantify changes in seed color occurring during paramutation, the relative color was determined as described [82]. RT–PCR analysis of the maize nrpd2/e2 genes For expression analysis of the maize nrpd2/e2 genes total RNA was extracted from tissues flash frozen in liquid nitrogen using the Trizol protocol as described by manufacturer (Invitrogen). Total p1 genetic stocks and tests with Mop2-1 The paramutable P1-rr stock, the standard P1-rr4B2 allele of the p1 gene [83], has red pericarp and red cob pigmentation, while the silenced P1-rr’ allele has lightly patterned or colorless pericarp and PLoS Genetics | www.plosgenetics.org 15 November 2009 | Volume 5 | Issue 11 | e1000725