A dominant mutation in mediator of paramutation2, one of three second-largest subunits of a plant-specific RNA polymerase, disrupts multiple siRNA silencing processes

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.

Figure 1. The Mop2-1 mutation prevents b1 paramutation and relieves B' silencing.

(A) The EMS mutagenesis screen used to isolate Mop2-1. The stocks carry distinct alleles for two genes that are linked and flank b1 on chromosome 2S; glossy2 (gl2) and white tip (wt). The glossy and white tip phenotypes are only visible in young seedlings and thus are not apparent in the mature plants shown. B'* is used to indicate a B-I allele that was paramutated to B' in wild type plants. B-I* is used to signify a B-I allele exposed to B' in the presence of Mediator of paramutation2 (Mop2-1), which prevents paramutation resulting in the B-I phenotype. Mop2-1 is shown linked to GL2 based on subsequent analysis (Figure S1). (B) Frequencies of plants with different pigmentation levels [Lt (light), Md (medium), and Dk (dark)] in progeny segregating Mop2-1/Mop2-1 and Mop2-1/+. The photo shows that Mop2-1 B'/+ B' pigmentation remains light (left plant), indistinguishable from B' in wild type. In contrast, homozygous Mop2-1 B' plants have increased pigmentation (right plant). For (C) and (D), to test the heritability of the increased B' pigmentation that is observed in homozygous Mop2-1 B' plants (D) and to generate larger numbers of progeny to examine the penetrance of Mop2-1/+ on preventing B-I* paramutation (C) dark Mop2-1 B' plants were crossed with +/+ tester carrying B-I/B-P, (Figure S2). The resulting Mop2-1 B'/+ B-P (C) and Mop2-1 B'/+ B-I (D) progeny were scored for light (Lt), medium (Md) and dark (Dk) pigment. (E) Testcrosses to assay whether B-I* segregates unchanged from + B-I*/Mop2-1 B' are diagramed in Figure S2. Plant pigmentation is shown from the +B-I*/+ B-P (parental class) and +B'/+B-P (recombinant class).

Figure 2. Mop2-1 encodes a second-largest subunit of a plant-specific RNA polymerase.

(A) Location of the Mop2-1 interval on chromosome 2S is shown (FPC contigs 69–70). Polymorphic markers (indicated above the contigs) were used for mapping. The number of recombinants over the total number of plants screened are shown in parenthesis next to each marker. (B) An expanded map of the Mop2-1 interval localized to two BACs. The dashed line is used for the AC213986 BAC because at the time of the publication it consisted of more then 30 unordered fragments. Predicted gene models within the two BACs were obtained from www.maizesequence.org. The predicted position and orientation of the nrpd2/e2 gene (based on synteny with rice) is indicated. (C) Exons and introns of the nrpd2/e2 gene based on alignment of genomic and cDNA sequences. The exons 1–7, translation start and stop, and polyadenylation sites are indicated. The positions of the G to A transitions in the Mop2-1 and mop2-2 alleles are shown. Location of the domains conserved with Pol-II RPB2 are shown below the exons. Domains were identified using the BLASTP program at http://pfam.sanger.ac.uk/search.

Figure 3. Mop2-1 and mop2-2 mutations are located in highly conserved motifs of the NRPD2/E2 proteins.

(A) and (B) show excerpts from edited alignments of the second largest subunits of RNA polymerases with the positions of the Mop2-1 and mop2-2 mutations shown. Mutations in the single functional NRPD2/E2 gene in Arabidopsis are also shown, drd2-12 and -9 . Species designations are: Ec-Escherichia coli, Sc-Saccharomyces cerevisiae, At-Arabidopsis thaliana, Os-Oryza sativa, and Zm-Zea mays. The full alignment is in Figure S3. (C) Un-rooted radial bootstrap neighbor-joining phylogenetic tree of the second largest subunits of RNA polymerases. Species designations are shown in Table S1. (D) A portion of the phylogenetic tree shown in (C) with an expanded traditional view of the Pol-IV/Pol-V branch is shown. Bootstrap values are at the base of each branch.

Figure 4. Maize nrpd2/e2 genes are differentially expressed.

Gene-specific primers and quantitative RT–PCR was used to analyze the RNA expression patterns of each of the three nrpd2/e2 genes in multiple maize tissues from the B73 inbred line and HiII and BMS callus. Details on the developmental stages of the tissues are in Materials and Methods. Expression was normalized to actin1 expression levels.

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. Transcription levels were normalized to the transcription levels of the Ubiquitin2 gene, measured as mean counts per mm² (Materials and Methods). Two other independent experiments gave similar results (data not shown).

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 paramutagenic 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 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 , 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 paramutation. Details of the crosses are in Figure S5. The paramutable R-r allele (solid or dark mottled seed) was exposed to the paramutagenic 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.

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 Ms45Δ1pIR 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 Ms45Δ1pIR and 5126pIR transgenic siRNAs in heterozygous and homozygous Mop2-1 plants and non transgenic controls (−/−). 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.