mop2 Is Required for Multiple Silencing Processes Table 1. Paramutation phenotypes at multiple loci in Mop2-1 plants. Locusa b1 pl1 p1 r1 Paramutagenic allele B’ (light plant) Pl’ (speckled anthers) P1-rr’ (patterned pericarp) R-st (stippled) Paramutable allele B-I (dark purple plant) Pl-Rh (purple anthers) P1-rr (red pericarp) R-r (purple seeds) Prevents paramutationb Releases silencingb dominant dominant recessive semi-dominant recessive semi-dominant noc no dominant phenotyped Data Figure1 Figure 6A, 6B Table 2 Figure 6C a At b1, pl1 and p1 the paramutagenic and paramutable alleles are epialleles, i.e. they have exactly the same DNA sequence, but distinct expression and chromatin states. In contrast, the paramutagenic R-st and paramutable R-r alleles are structurally distinct [for a review, see [7]]. When R-r is paramutated by R-st, it is referred to as R- r’; R-r and R-r’ are epialleles. b The prevention of paramutation is assayed when a paramutagenic and paramutable allele are combined in plants homozygous or heterozygous for Mop2-1. The test for release of silencing assays pigment levels when a previously paramutated (silenced) allele is exposed to heterozygous or homozygous Mop2-1. c Three sequential generations carrying P1-rr’ in Mop2-1 homozygotes (40 total ears) showed no increased expression of P1-rr’. d Mop2-1/+ did not release R-r’ silencing previously established by R-st. Tests with homozygous Mop2-1 were inconclusive because paramutated R-r’ reverted at high frequency to a highly expressed state similar to R-r regardless of the Mop2-1 genotype. doi:10.1371/journal.pgen.1000725.t001 strated that the Mop2-1 mutation differentially alters para- mutation at these loci. Potential reasons for this are explored in the Discussion. Mop2-1 is required for transcriptional silencing mediated by two transgenes expressing inverted repeat promoters Roles for the Arabidopsis Pol-IV and Pol-V polymerase complexes in transcriptional silencing mediated by siRNAs generated from the expression of promoters in inverted repeats (pIR) are well documented [67,68]. To test whether nrpd2/e2a might be involved in a similar transgene-mediated silencing system in maize, we tested whether the Mop2-1 mutation could prevent the silencing of two pIR-targeted loci (Figure 7A), each required for male fertility [67]. One of these genes, Ms45 is expressed in the anther tapetum during early vacuolate stage of pollen develop- ment and is required for microspore development [67]. In wild type backgrounds, targeting the Ms45 gene by the Ms45D1pIR inverted repeat transgene results in complete sterility; 100% of tassels do not extrude anthers and they fail to produce any pollen [67]. To test whether Mop2-1 disrupts silencing, transgenic Ms45D1pIR/- plants were used as females and pollinated with the homozygous Mop2-1 B’ stock (Figure S6). In the first generation, all plants were heterozygous (Mop2-1/+); if Mop2-1 disrupted this process as a dominant or semi-dominant mutation, then full or partial restoration of male fertility would be expected. Examination of the Mop2-1/+ plants revealed that although all plants remained male sterile, small shriveled anthers developed in many of the plants (data not shown). Because Ms45D1pIR leads to complete absence of anthers in wild type plants, improved anther development in Mop2-1/+ plants was a significant finding, and suggested Mop2-1 was semi-dominant. To test whether homozy- gous Mop2-1 plants would show a more dramatic relief of Ms45 silencing, transgenic plants were pollinated with the Mop2-1 B’ stock (Figure S6). The resulting herbicide resistant plants segregating heterozygous and homozygous Mop2-1 individuals were examined. Because the B’ allele was introduced together with the Mop2-1 mutation, dark plant pigmentation was used to initially identify Mop2-1 homozygous plants in the segregating families. If Mop2-1/Mop2-1 prevents pIR transgene-induced silencing of Ms45, dark plants would be expected to exhibit partial or complete restoration of male fertility. This expectation was met as 72% of the dark plants were fertile, 19% had small anthers that did not shed pollen (referred to as breakers), and only 9% were sterile (Figure 7B). These results indicated that Ms45D1pIR- induced silencing of the Ms45 gene was disrupted in the majority of the darkly pigmented plants, likely representing Mop2-1/Mop2-1 homozygotes. The majority of light B’ plants (60%), mostly representing Mop2-1/+, were completely sterile, but a few were fertile (9%) or had the breaker phenotype of extruded sterile anthers (31%) (Figure 7B). The detection of fertile plants among light B’ plants could result from cumulative effects of carrying Mop2-1/+ for two generations, or incomplete correspondence between the Mop2-1 genotype and release of B’ silencing. Both explanations are likely to be occurring because molecular genotyping revealed that 28/30 dark plants were homozygous and 28/33 of light plants were heterozygous for Mop2-1. Altogether, these results demonstrated that the Mop2-1 mutation can prevent the Ms45D1pIR transgene from silencing the endogenous Ms45 gene in a semi-dominant manner, with the strongest phenotypes observed in homozygotes. A second pIR-targeted locus was tested for whether Mop2-1 could disrupt silencing. The 5126 gene has also been demonstrat- ed to be expressed during microsporogenesis [67]. The 5126pIR- induced silencing of the 5126 gene results in complete male sterility, but a portion of tassels do show the breaker phenotype, i.e., 30% of the flowers on a tassel extrude small shrunken anthers that do not contain pollen [67]. Similar to the results with the Ms45D1pIR/2 transgenes, some improvement of anther devel- opment of the 5126pIR/2 transgenic plants was noted in the first generation of plants heterozygous for Mop2-1/+; unlike wild type backgrounds some plants extruded anthers that produced small amounts of pollen (unpublished data). After a backcross with the homozygous Mop2-1 stock (Figure S6), families segregating Mop2- 1/Mop2-1 and Mop2-1/+ progeny were examined. Almost half of the darkly pigmented Mop2-1 homozygous plants had fertile tassels (48%), while the other half had the breaker phenotype (Figure 7C). There were fertile plants (19%) among light B’ Mop2-1/+ heterozygous plants, although most showed the breaker phenotype (81%). These results indicate that the Mop2-1 mutation can disrupt pIR silencing at the 5126 locus in a semi-dominant manner, with increased activity as a homozygote. We also tested whether Mop2-1 disrupted pIR-mediated silencing of a third transgene, pg47pIR, which targets the pg47 locus, a gene that is highly expressed during pollen development [68,69]. In contrast to the results with Ms45D1pIR and 5126pIR, the Mop2-1 mutation did not reverse pg47pIR-mediated silencing of the pg47 gene family (Cigan M, unpublished data). The pg47 gene is expressed in pollen, a tissue where nrpd2/e2a is expressed at a very low level and nrpd2/e2c is expressed highly (Figure 4). Thus, we favor the hypothesis that nrpd2/e2a is not used extensively in the pollen, and therefore a mutation in it has no phenotype in this tissue. However, an alternative explanation that silencing induced PLoS Genetics | www.plosgenetics.org 9 November 2009 | Volume 5 | Issue 11 | e1000725