Figure 6. The Mop2-1 mutation alters paramutation at pl1 and r1. (A) Mop2-1 prevents pl1 paramutation. Paramutation occurs when paramutable Pl-Rh, specifying dark red anther pigment and paramuta- genic Pl’, conferring light speckled anther pigment are brought together in a wild type background (18 individuals). In contrast, when paramutable Pl-Rh is exposed to paramutagenic Pl’ in the Mop2-1/+ background Pl’ fails to paramutate Pl-Rh and dark anthered plants are observed (17 individuals). (B) Mop2-1 effect on Pl’ silencing. Silencing associated with Pl’ paramutation was assayed in progeny from crosses between Pl’; Mop2-1 B’/+ B’ and Pl’; Mop2-1 B’ plants. Histograms show distribution of anther color scores [3] in which lightest pigment is 1 and solid red pigment is 7. The number of plants in each class is shown above the corresponding bar. The majority of Mop2-1 homozygotes have dark anthers (anther scores 5–7) suggesting Pl’ silencing is relieved, while in most Mop2-1/+ heterozygotes Pl’ silencing is maintained (anther scores 1–4). The presence of a few plants with light anthers among Mop2-1 homozygotes suggests that Mop2-1 is not completely penetrant in relieving Pl’ silencing. As reversion of Pl’ to Pl- Rh is never observed in wild type plants [3], the observation of some Mop2-1/+ plants with dark anthers suggest Mop2-1 is partially dominant for relieving Pl’ silencing. (C) Effect of Mop2-1 on preventing r1 mop2 Is Required for Multiple Silencing Processes paramutation. Details of the crosses are in Figure S5. The paramutable R-r allele (solid or dark mottled seed) was exposed to the paramuta- genic R-st allele (stippled seed) in the presence of the Mop2-1 mutation (homozygous or heterozygous) or wild type. In wild type, R-st paramutates R-r (designated as R-r’) and lighter mottled seed color is observed. In Mop2-1 heterozygotes and homozygotes, R-st fails to fully paramutate R-r, resulting in medium mottled or darkly mottled to solid seed color, respectively. Colorless r alleles and solid colored R-sc alleles were pigmentation standards. doi:10.1371/journal.pgen.1000725.g006 by different pIR transgenes may use distinct mechanisms can not be eliminated. Taken together, these results suggest that a mutation in nrpd2/e2a disrupts pIR-mediated silencing, but not at all loci. Mop2-1 heterozygotes and homozygotes have similar levels of Ms45D1pIR and 5126pIR transgene transcripts and siRNAs In Arabidopsis, Pol-IV is required for siRNA production, whereas Pol-V primarily acts downstream of siRNA production generating non-coding transcripts and helping to direct chromatin modifying enzymes to target loci [41,42,45,70]. To gain insight into possible mechanisms by which the Mop2-1 mutation disrupts pIR-mediated silencing in maize, we assayed transcript levels of the Ms45D1pIR and 5126pIR transgenes and the targeted endogenous genes in Mop2-1 plants. Results demonstrated that endogenous Ms45 and 5126 transcripts were absent in sterile +/+ or Mop2-1/+ plants consistent with silencing and that they were present in fertile Mop2-1/Mop2-1 plants (upper panels in Figure 7D and 7E), consistent with Mop2-1 preventing silencing. However, the levels of the endogenous genes’ transcripts in Mop2-1/Mop2-1 plants were not as high as in non-transgenic plants (upper panels in Figure 7D and 7E), indicating that partial silencing was still occurring even in homozygous Mop2-1 plants, although clearly enough transcription of the endogenous genes was occurring to restore fertility. Hybridization with the Nos spacer fragment probe [67], which is unique to the Ms45pD1IR and 5126pIR transgenes, revealed that transgene transcript levels, while variable, were similar between sterile (+/+ or Mop2-1/+) and fertile (Mop2-1/ Mop2-1) plants (Figure 7D and 7E). Thus, Mop2-1 is not acting to reduce the pool of transgenic transcripts that serve as precursors for siRNA production and silencing. Because the Mop2-1 mutation reduced levels of total endoge- nous siRNAs and the b1 853 bp repeat specific siRNAs, we tested whether Mop2-1 might also dramatically reduce the siRNAs produced from the Ms45pD1IR and 5126pIR transgenes. Because we did not have wild type transgenic lines that were isogenic with the Mop2-1 heterozygotes and homozygotes, we compared the transgene siRNA levels in sterile and breaker heterozygous Mop2-1/+ plants to those in fertile homozygous Mop2-1/Mop2-1 plants. If the ability of Mop2-1 to relieve silencing was due solely to reduced siRNA biogenesis, we would expect few transgenic siRNAs to be seen in fertile homozygotes. Results in Figure 7F demonstrated that 24 nt siRNAs are produced at similar levels from the Ms45D1pIR transgene in both fertile Mop2-1 homozy- gous plants and sterile Mop2-1/+ heterozygous plants. In the 5126pIR transgenic plants transgene siRNAs were also detected in both breaker Mop2-1 heterozygotes and fertile homozygotes, although two out of three Mop2-1 heterozygotes had approxi- mately two fold higher siRNA levels relative to homozygotes (Figure 7G). These results demonstrated that the ability of the Mop2-1 mutation to restore male fertility in the Ms45pD1IR and 5126pIR transgenic plants was not simply because it eliminated PLoS Genetics | www.plosgenetics.org 10 November 2009 | Volume 5 | Issue 11 | e1000725