mop2 Is Required for Multiple Silencing Processes Figure 7. Mop2-1 disrupts pIR transgene-mediated silencing at the Ms45 and 5126 loci. (A) Outline of the experiment with the pIR transgenes. The strong constitutive Ubiquitin1 promoter was used to transcribe inverted repeats containing Ms45 and 5126 promoter fragments, which results in siRNA that target promoters of the endogenous Ms45 and 5126 genes for silencing. Silencing of the Ms45 and 5126 genes leads to male sterility in wild type plants. (B) Disruption of pIR silencing of the Ms45 locus in Mop2-1 plants. The chart shows frequencies of fertile, breaker (extruded anthers), and sterile plants among dark (homozygous) and light (heterozygous) Mop2-1 plants. The number of plants in each group is shown above each bar. (C) Disruption of pIR silencing of the 5126 locus in Mop2-1 plants. Frequencies of fertile and breaker phenotypes are shown, with the number of plants in each group above each bar. (D) and (E) show Northern blot analyses on samples from non transgenic siblings (first two lanes) and from Ms45D1pIR and 5126pIR transgenic lines, respectively (remaining lanes). PolyA enriched RNA samples from anthers containing quartet/early uninucleate microspores were used for Northern blot analysis. Tassel phenotypes are indicated with F for fertile and S for sterile. Probes used for hybridization are indicated on the left of each panel. Actin1 was used as a loading control. (F) and (G) show results from Northern blot analyses for Ms45D1pIR and 5126pIR transgenic siRNAs in heterozygous and homozygous Mop2-1 plants and non transgenic controls (2/2). Hybridization with the U6 probe and SyberGold staining of rRNA served as loading controls. The numbers below each lane indicate the quantification of the transgene siRNA levels normalized to U6. Tassel phenotypes are indicated: S for sterile, B for breaker and F for fertile. doi:10.1371/journal.pgen.1000725.g007 PLoS Genetics | www.plosgenetics.org 12 November 2009 | Volume 5 | Issue 11 | e1000725