A Dominant Mutation in mediator of paramutation2 , One of Three Second-Largest Subunits of a Plant-Specific RNA Polymerase, Disrupts Multiple siRNA Silencing Processes Lyudmila Sidorenko1, Jane E. Dorweiler2, A. Mark Cigan 3 , Mario Arteaga-Vazquez1, Meenal Vyas1, Jerry Kermicle4, Diane Jurcin1, Jan Brzeski1, Yu Cai1, Vicki L. Chandler1* 1 Department of Plant Sciences, University of Arizona, Tucson, Arizona, United States of America, 2 Department of Biological Sciences, Marquette University, Milwaukee, Wisconsin, United States of America, 3 Pioneer Hi-Bred International, Johnston, Iowa, United States of America, 4 Genetics Department, University of Wisconsin, Madison, Wisconsin, United States of America Abstract Paramutation involves homologous sequence communication that leads to meiotically heritable transcriptional silencing. We demonstrate that mop2 (mediator of paramutation2), which alters paramutation at multiple loci, encodes a gene similar to Arabidopsis NRPD2/E2, the second-largest subunit of plant-specific RNA polymerases IV and V. In Arabidopsis, Pol-IV and Pol-V play major roles in RNA–mediated silencing and a single second-largest subunit is shared between Pol-IV and Pol-V. Maize encodes three second-largest subunit genes: all three genes potentially encode full length proteins with highly conserved polymerase domains, and each are expressed in multiple overlapping tissues. The isolation of a recessive paramutation mutation in mop2 from a forward genetic screen suggests limited or no functional redundancy of these three genes. Potential alternative Pol-IV/Pol-V–like complexes could provide maize with a greater diversification of RNA–mediated transcriptional silencing machinery relative to Arabidopsis. Mop2-1 disrupts paramutation at multiple loci when heterozygous, whereas previously silenced alleles are only up-regulated when Mop2-1 is homozygous. The dramatic reduction in b1 tandem repeat siRNAs, but no disruption of silencing in Mop2-1 heterozygotes, suggests the major role for tandem repeat siRNAs is not to maintain silencing. Instead, we hypothesize the tandem repeat siRNAs mediate the establishment of the heritable silent state—a process fully disrupted in Mop2-1 heterozygotes. The dominant Mop2-1 mutation, which has a single nucleotide change in a domain highly conserved among all polymerases (E. coli to eukaryotes), disrupts both siRNA biogenesis (Pol-IV–like) and potentially processes downstream (Pol-V–like). These results suggest either the wild-type protein is a subunit in both complexes or the dominant mutant protein disrupts both complexes. Dominant mutations in the same domain in E. coli RNA polymerase suggest a model for Mop2-1 dominance: complexes containing Mop2-1 subunits are non-functional and compete with wild-type complexes. Citation: Sidorenko L, Dorweiler JE, Cigan AM, Arteaga-Vazquez M, Vyas M, et al. (2009) A Dominant Mutation in mediator of paramutation2, One of Three Second- Largest Subunits of a Plant-Specific RNA Polymerase, Disrupts Multiple siRNA Silencing Processes. PLoS Genet 5(11): e1000725. doi:10.1371/journal. pgen.1000725 Editor: Gregory P. Copenhaver, The University of North Carolina at Chapel Hill, United States of America Received August 9, 2009; Accepted October 15, 2009; Published November 20, 2009 Copyright: 2009 Sidorenko et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding was provided by a NIH Award DP1OD575 to VLC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: chandler@ag.arizona.edu Introduction Paramutation, an interaction between specific alleles that leads to a heritable change of expression of one allele, was first described for the maize red1 (r1) gene [1]. Subsequently three more regulatory genes of the flavonoid biosynthetic pathway, b1 (Booster1), pl1 (plant color1), and p1 (pericarp color1) [2–4], and a gene involved in phytic acid biosynthesis [5] were shown to undergo paramutation in maize. Paramutation-like phenomena have also been reported in other plants, fungi, and animals [for a review, see [6–8]]. Paramutation terminology defines alleles that induce silencing as paramutagenic and alleles that become silenced as paramutable. Once silenced (paramutated), alleles are designated with an apostrophe to signify their paramutant state. In addition to becoming heritably silenced, paramutant alleles also acquire the ability to silence na¨ıve paramutable alleles. Paramutant and paramutable states often have different stabilities, which can potentially be reversible depending on the locus [for a review, see [7,9,10]]. Most alleles of a locus do not participate in paramutation. Key sequences mediating paramutation have been identified for two systems, b1 [11,12] and p1 [4,13]. Recombination mapping between alleles that do and do not participate in b1 paramutation defined a specific sequence that when tandemly repeated is absolutely required for paramutation [11,12]. Characterization of these repeats revealed that the paramutable and paramutagenic alleles have identical DNA sequences and numbers of repeats, but differ in their chromatin structure demonstrating that paramuta- tion is epigenetic and associated with changes in chromatin [11]. Transgenic approaches were used to identify sequences within p1 sufficient to mediate paramutation. These sequences lie within a direct repeat flanking the p1 alleles that participate in paramuta- tion [4,13]. At the r1 locus, paramutagenic alleles contain direct PLoS Genetics | www.plosgenetics.org 1 November 2009 | Volume 5 | Issue 11 | e1000725