callus relative to the other two genes. Given this difference in expression and that the phylogenetic analyses suggested nrpd2/e2c represents a more ancient duplication, it is the most likely candidate for a distinct function relative to nrpd2/e2a,b. It is striking that the two most similar genes, nrpd2/e2a and nrpd2/e2b share similar expression patterns across all tissues tested, yet our results demonstrate that a recessive mutation in nrpd2/e2a, mop2-2, has paramutation defects. This demonstrates that at least with respect to paramutation, the nrpd2/e2a gene is unlikely to be functionally redundant with nrpd2/e2b. This hypothesis is support- ed by studies from the Hollick lab in which recessive mutations in the same gene were isolated in forward genetic screens for pl1 paramutation (Stonaker et al, this issue). These three genes may not be functionally redundant either because they function in distinct complexes or because there are differences in their expression patterns not detectable with quantitative RT-PCR in complex multi-cellular tissues. Expression differences among different cell types within organs, distinct cell layers within the same tissue, differences in subcellular location, or even locus specific distribution are all possible explanations for the lack of functional redundancy. Given the high degree of similarity among all three genes, the generation of transgenic plants with differentially tagged genes as well as mutations in the other two subunits will enable these hypotheses to be distinguished. Mop2-1 may be disrupting both Pol-IV–like and Pol-V– like complexes The loss of siRNAs from repetitive elements throughout the genome and from the tandem repeats that are required for b1 paramutation is consistent with Mop2-1 disrupting a Pol-IV like complex. When siRNAs are produced independently of Pol-IV as they are in many pIR transgene experiments [41], i.e. potentially in our case when the strong Pol-II promoter from the maize ubiquitin1 gene is used to produce inverted repeat transcripts, one can also examine the potential for Mop2-1 to reduce Pol-V like function, downstream of siRNA biogenesis. In unpublished data we have shown that the pIR transgenes we used do not require the endogenous siRNA biogenesis pathway for silencing, as the mop1-1 mutation (in the RDR orthologous to RDR2), which dramatically reduces 24 nt siRNAs does not prevent pIR silencing (M. Cigan and V. Chandler, unpublished data). In Mop2-1 homozygotes pIR-mediated silencing is relieved, yet there are similar levels of siRNAs from the pIR transgenes in fertile homozygotes relative to sterile heterozygotes. This result is consistent with Mop2-1 also acting downstream of siRNA biogenesis, potentially by disrupting a Pol-V like complex. However, it is possible that Mop2-1 may only be acting through a Pol-IV-like complex. For example, Mop2-1 may partially impair secondary siRNA production from the pIR transgenes, which might partially reduce transcriptional silencing resulting in fertility. This model can account for Mop2-1 effects entirely through Pol-IV deficiencies. If Mop2-1 does alter both Pol-IV and Pol-V functions, it could be because like Arabidopsis, the wild type nrpd2/e2a encoded subunit functions in both Pol-IV-like and Pol-V-like RNA polymerase complexes. Alternatively, the Mop2-1 muta- tion may confer a gain of function phenotype that enables the mutant subunit to interact with and disrupt complexes the wild type subunit normally does not form. The observation that loss of function mutations in nrpd2/e2a also cause a dramatic reduction in global siRNAs (Stonaker et al, this issue), suggests that at a minimum NRPD2/E2a functions in a Pol-IV like complex. mop2 Is Required for Multiple Silencing Processes Model for the dominance of Mop2-1 and potential explanations for dependence of certain phenotypes on Mop2-1 dosage The Mop2-1 mutation’s effects on paramutation at multiple loci, pIR-mediated silencing, and plant growth and development depend on the dosage of the Mop2-1 allele with some phenotypes more variable than others. Certain phenotypes were observed with high penetrance when Mop2-1 was heterozygous (dominant for prevention of b1 and pl1 paramutation, reduction in global and b1 tandem repeat siRNAs); some phenotypes required Mop2-1 to be homozygous (recessive for release of B’ silencing and prevention of p1 paramutation); and still other phenotypes were seen in Mop2-1 heterozygotes, but were much stronger in Mop2-1 homozygotes (semi-dominant for prevention of r1 paramutation, disrupting pIR- mediated silencing of Ms45 and 5126, release of Pl’ silencing, further reduction of b1 tandem repeat siRNAs, and many developmental phenotypes). There were also phenotypes that Mop2-1 did not alter (no release of P1-rr’ silencing and no disruption of pIR-mediated silencing of pg47). Below we discuss a model for Mop2-1 dominance, based on data from similar mutations in the second largest subunit of E.coli RNA Polymerase, and suggest explanations for the dependence of specific pheno- types on Mop2-1 dosage. The GEME motif mutated in Mop2-1 is nearly invariant among all second largest subunits in all polymerases from organisms ranging from bacteria, fungi, animals, and plants. The Mop2-1 mutation changes the second glutamic acid residue (E1076) of the GEME motif to a lysine. In RpoB (the second largest subunit of E.coli RNA polymerase) the GEME motif is located within the ‘‘anchor’’ region required for interaction with the clamp fold within the largest polymerase subunit [72]. The clamp swings open to produce a larger opening of the cleft that permits entry of promoter DNA and subsequent initiation of transcription. Extensive mutagenesis of the second largest subunit, rpoB in E.coli revealed that mutations within all four GEME motif amino acids result in dominant phenotypes when the mutant subunit is produced from a plasmid at similar levels to the chromosomal encoded non-mutant copy [56]. Substitutions in this region produced a RNA polymerase that competed with the wild type RNA polymerase complex potentially because the mutant polymerase was blocked after transcription initiation [56,73]. The strength of the dominant phenotypes varied depending on the specific substitution. The extreme conservation of the GEME motif [56] and the results in E.coli, lead us to hypothesize that a similar molecular mechanism contributes to the dominant phenotype of the Mop2-1 mutation. Our model is that NRPD2/E2a proteins with the Mop2-1 mutation associate normally with the same largest subunit(s) (NRPD1 and potentially NRPE1, or both) that the wild type subunit associates with, forming mutant polymerase com- plexes that are functionally defective, but that efficiently compete with wild type polymerase complexes. This model predicts that the relative dosage of mutant and wild type subunits would influence the number of functional complexes available. Assuming that the Mop2-1 encoded protein is expressed equivalently to the wild type, a Mop2-1 heterozygote should have equal amounts of wild type and mutant proteins, such that phenotypes that are particularly sensitive to Pol-IV or Pol-V dosage would be altered in the heterozygote. Moreover, processes that require both Pol-IV and Pol-V complexes might show more dramatic phenotypes than processes that require either Pol-IV or Pol-V alone, if there are additive consequences of partial loss of each complex. This model further predicts that a Mop2-1 homozygote, which would have no wild type NRPD2/E2, would produce a stronger phenotype PLoS Genetics | www.plosgenetics.org 13 November 2009 | Volume 5 | Issue 11 | e1000725