mop2 Is Required for Multiple Silencing Processes Figure 5. siRNA levels and transcription from the B’ tandem repeats that mediate b1 paramutation in Mop2-1. (A) Global siRNA levels in Mop2-1 plants. The small RNA fraction was isolated from young ears (3–5 cm long) of Mop2-1/Mop2-1, Mop2-1/+, and +/+ plants and ,100 ug samples were separated on a 15% denaturing polyacrylamide gel and stained with SyberGold. (B) Northern blot analysis of the siRNA fraction from young ears. The b1 tandem repeat probe used for hybridization is indicated in (C). Staining with SyberGold is shown, which served as a loading control. (C) Drawing of a portion of the B’ tandem repeats (black arrows) and the sequence immediately downstream (open rectangle). The position of the probes used are shown; the paired arrows indicate RNA probes used in the run-on analysis (D), while the gray arrowhead indicates the position of the DNA::LNA (locking nucleic acid) oligonucleotide used for the Northern blot analysis (B). (D) Results of nuclear run-on analysis of transcription within the seven B’ tandem repeats in young ears. Letters indicate forward (F) or reverse (R) transcription, respectively, in relation to this drawing. 2 Transcription levels were normalized to the transcription levels of the Ubiquitin2 gene, measured as mean counts per mm (Materials and Methods). Two other independent experiments gave similar results (data not shown). doi:10.1371/journal.pgen.1000725.g005 Mop2-1 disrupts paramutation at multiple loci Paramutation has been well characterized at three other maize loci: pl1, p1 and r1, all encoding transcription factors that activate pigment synthesis [4,7,9,66]. We were interested in determining whether Mop2-1 disrupted paramutation at these loci and, if so, whether disruption was similar to b1 paramutation, i.e., when heterozygous Mop2-1 prevented paramutation and when homo- zygous it increased the expression of silenced alleles. Table 1 summarizes the alleles for each gene used in each experiment and the results. For each of these paramutation systems, genetic backgrounds were available that enabled the monitoring of paramutation through changes in pigment levels (Materials and Methods). To investigate paramutation at each locus, Mop2-1 was introduced into the appropriate genetic background (Figure 6, Table 2, Figure S4, and Figure S5) and pigment was monitored in the appropriate tissues. The results at the pl1 locus resembled b1 paramutation; Mop2-1 was dominant for preventing pl1 paramutation (Figure 6A) and it relieved the silencing of the paramutated Pl’ allele (Figure 6B). However, the levels of increased expression of the paramutant Pl’ allele were variable suggesting Mop2-1 was partially dominant (Figure 6B). In contrast to what was observed at the b1 and pl1 loci, Mop2-1 prevented p1 paramutation only when homozygous and there was no change on the expression of the silenced allele even after multiple generations of exposure to Mop2-1 (Table 1 and Table 2). At the third locus tested, r1, Mop2-1 was semi-dominant for preventing paramutation (Figure 6C). These results demon- PLoS Genetics | www.plosgenetics.org 8 November 2009 | Volume 5 | Issue 11 | e1000725