pink cob pigment [4]. The highly paramutagenic P1-rr’/P1-rr’; P1.2b::GUS/- stock was used to assess Mop2-1 effects on p1 paramutation, by crossing with a Mop2-1 B’ P1-rr stock. The P1.2b::GUS transgene (transgenic event P2P147-37) carried the P1.2 enhancer fragment that is sufficient for paramutation, fused to the basal P1-rr promoter, the Adh1 (maize Alcohol dehydrogenase1 gene intron 1), the E.coli GUS gene and the PinII (potato Proteinase InhibitorII) 3’, along with a resistance gene for the BASTA herbicide [13]. Details of the crosses are presented in Figure S4. Genetic and molecular tests with Ms45D1pIR and 5126pIR transgenes The Ms45D1pIR and 5126pIR transgenic constructs and phenotypes upon silencing were previously described [67]. To assay whether the Mop2-1 mutation could disrupt pIR transgene induced silencing of the endogenous Ms45 and 5126 genes, Mop2-1 plants were crossed with four independent transgenic events each for Ms45D1pIR and 5126pIR. Herbicide resistant F1 plants were backcrossed to the Mop2-1 stock to generate a family segregating heterozygous or homozygous Mop2-1 plants. These were grown in the field and sprayed with herbicide to remove non transgenic plants, while the remaining transgenic plants were visually scored for plant color and male fertility at anthesis. Plants homozygous for the Mop2-1 mutations were initially identified using the dark plant color associated with increased expression of the B’ allele. For a subset of the Ms45D1pIR plants, the presence of the Mop2-1 mutation was confirmed by genotyping with molecular markers. Northern blot analysis of Ms45 and 5126 transcript levels were as previously described [67,84]. Conditions for small RNA Northern blots were the same as described below, using previously described probes for MS45 and 5126 transgene siRNA detection [67]. Northern blot analysis of siRNAs RNA was extracted from 3 g of immature ears (3–5 cm long) using TRIZOL reagent (Invitrogen), and the large RNA fraction was precipitated using 5% polyethyleneglycol MW 8000 [85]. The aqueous phase, enriched for the small RNA fraction, was subjected to phenol:chlorophorm:isoamyl-alcohol (24:1:1) extraction fol- lowed by ethanol precipitation. The pellet was resuspended in DEPC treated water. Approximately 100 ug of the small RNA fraction was loaded in each lane. RNA was electrophoresed on 15% denaturing UREA polyacrylamide gels, electroblotted onto GeneScreen Nylon membrane and immobilized using UV cross- linking. Blots were hybridized with a 32P end labeled DNA:LNA (DNA::Locking Nucleic Acid) oligonucleotide [86]. The DNA::LNA oligo (vc1658F, TGAA+CATCTT+GTCCA+GT- TAAAT+CACTGG+ACACC+GTGAC+AGCC+ACA; ‘‘+’’ pre- cedes an LNA base) was synthesized by Sigma-Proligo. For the U6 probe, DNA oligo (vc1969F, AGACATCCGATAAAATTG- GAACGATACAGA) was end labeled with 32P. Hybridization and image was processed using QuantityOne software (BioRad). Nuclear run-on analysis Approximately ,5 g of immature ears were used to extract nuclei as described [20]. The nuclei isolations and run on reactions were as described [22]. To prepare the b1 tandem repeat probes, PCR fragments carrying T3 promoter tails were used as templates for in vitro transcription with T3 RNA polymerase (Invitrogen), as recommended by the manufacturer. Sequences of the primers used to produce the b1 RNA probes are available upon request. The positive control, the Ubiqutin2 RNA probe, was as described mop2 Is Required for Multiple Silencing Processes [27]. Lambda phage genomic DNA, 100 ng per slot, was used as a negative control. Supporting Information Figure S1 Genetic mapping of the Mop2-1 mutation using phenotypic markers linked to the b1 locus on chromosome 2S. Asterisk denotes the B-I that was protected from paramutation in Mop2-1/+ plants. Red bars indicate the interval in which recombination occured in the previous generation. In testcross 1, 12 out of 15 progeny plants inherited parental combinations of phenotypic markers on chromosome 2. Analysis of phenotypes of the three recombinant plants indicates that Mop2-1 is located distal to the gl2 locus. Testcross 2 was carried out to score the presence of the Mop2-1 mutation. Found at: doi:10.1371/journal.pgen.1000725.s001 (0.10 MB PDF) Figure S2 Crossing schema for the genetic test used to assay Mop2-1/+ effects on preventing b1 paramutation and relief of B’ silencing. The B-I allele exposed to homozygous Mop2-1 is denoted by an asterisk. The red bar indicates the potential for recombination as the b1 and Mop2-1 loci are linked (27 cM). The B-Peru (B-P) allele of the b1 gene does not undergo paramutation. Weak plant pigment specified by B-P is convenient for observing B’ and B-I* phenotypes. Because B’ and B-I do not pigment seeds, the purple seed color specified by B-P is used for pre-planting segregation of B’/B-I* and B’/B-P seeds. If B-I* escaped paramutation in the previous generation, then accounting for the linkage between b1 and mop2, assuming absence of spontaneous paramutation of B-I* to B’, and 100% penetrance of the Mop2-1 mutation, 73% of dark + B-I*/+ B-P and 27% of light + B’/+ B-P progeny are expected. Found at: doi:10.1371/journal.pgen.1000725.s002 (0.10 MB PDF) Figure S3 Alignment of second largest subunits of RNA polymerases. Alignment was performed using MUSCLE, edited using GENEDOC, and shaded using BOXSHADE. Identical amino acids are shaded in black, while similar amino acids are shaded in gray. Conserved domains are underlined and indicated A though I [72]. The active site (metal B) is indicated by asterisks [72]. Positions of Mop2-1 and mop2-2 mutations are indicated above the alignment in blue. Positions of removed amino acids are indicated above the alignment in gray. Found at: doi:10.1371/journal.pgen.1000725.s003 (0.33 MB PDF) Figure S4 Crossing schema for experiment to test effect of Mop2-1 on p1 paramutation. The paramutagenic P1-rr’ allele has light patterned pericarp, while paramutable P1-rr has red pericarp pigment. The P1.2b::GUS transgene carried the highly para- mutagenic P2P147-37 integration event [13]. To assay whether the Mop2-1 mutation would prevent p1 paramutation, plants carrying the paramutagenic endogenous P1-rr’ allele and the P1.2b::GUS transgene were pollinated with the Mop2-1 P1-rr stock. Because the B’ allele was introduced together with the Mop2-1 mutation we used dark plant pigment for initial identification of Mop2-1 homozygous plants in segregating families, and subsequent molecular markers were used to verify the Mop2-1/Mop2-1 and Mop2-1/+ genotypes. Spraying with the BASTA herbicide eliminated non transgenic plants. In the F1 all transgenic plants had light pericarp color indicating that when Mop2-1 is heterozygous it does not p1 prevent paramutation. A backcross with the Mop2-1 stock was used to generate families in which the effect of homozygous Mop2-1 on preventing p1 paramutation was assayed. Results are presented in Table 2. Found at: doi:10.1371/journal.pgen.1000725.s004 (0.17 MB PDF) PLoS Genetics | www.plosgenetics.org 16 November 2009 | Volume 5 | Issue 11 | e1000725