Initiation, establishment, and maintenance of heritable MuDR transposon silencing in maize are mediated by distinct factors

Abstract

Paramutation and transposon silencing are two epigenetic phenomena that have intrigued and puzzled geneticists for decades. Each involves heritable changes in gene activity without changes in DNA sequence. Here we report the cloning of a gene whose activity is required for the maintenance of both silenced transposons and paramutated color genes in maize. We show that this gene, Mop1 (Mediator of paramutation1) codes for a putative RNA-dependent RNA polymerase, whose activity is required for the production of small RNAs that correspond to the MuDR transposon sequence. We also demonstrate that although Mop1 is required to maintain MuDR methylation and silencing, it is not required for the initiation of heritable silencing. In contrast, we present evidence that a reduction in the transcript level of a maize homolog of the nucleosome assembly protein 1 histone chaperone can reduce the heritability of MuDR silencing. Together, these data suggest that the establishment and maintenance of MuDR silencing have distinct requirements.

Figure 1. Cloning of the Mop1 Gene

(A) Southern blot depicting a family segregating for either wild-type (WT) or mop1–1 heterozygous individuals (m/+). Individuals were genotyped using the SSR marker umc1541, which has been previously mapped to within one centimorgan from the mop1 locus. Arrows denote both Mu1.7 transposon elements that are only present in individuals containing the mop1–1 allele. The two other bands in the middle of the blot found in all lanes denote the Mu1 element in the a1-mum2 allele that is present in all individuals in this family. DNA was digested with NcoI restriction enzyme. (B) Depicted is a map of ZmRDR2 (not to scale). The Mu insertion is depicted here as a gray inverted triangle. Primers (indicated as arrows) correspond to the canonical sequence of ZmRDR2/RDR101 exon 4 (primer 1) and the Mu1.7 terminal inverted repeat sequence (primer 2). Primers flanking the insertion used for RT-PCR portrayed in figure 2 are designated primer 3 and primer 4. The indicated sizes are those obtained from amplification and sequencing of these products. Primers used to amplify sequences 3′ of the insertion were primer 4 and primer 5. The region sequenced in mop1–1, mop1–2, and minimal line wild-type Mop1 is indicated by the black bar above exon 2. (C) PCR of a family segregating for mop1–1 homozygote (m/m), heterozygote (m/+), and WT individuals using primers corresponding to ZmRDR2 exon 4 and Mu1.7 as depicted in Figure 1B. Individuals carrying the mop1–1 allele (m/m and m/+) give rise to the expected 470-bp amplicon, indicating the presence of a Mu1.7 insertion at the mutant allele. Conversely, the WT individuals do not give rise to this amplicon, suggesting that the mop1–1 allele is the result of a Mu1.7 insertion in exon 4 of ZmRDR2.

Figure 2. The ZmRDR2 mRNA Transcript Is Altered in mop1–1 Mutants

(A) RT-PCR of a family segregating for the mop1–1 allele. Primers used were those corresponding to the ZmRDR2 sequences and that spanned intron 3 as well as the Mu1.7 insertion in exon 4 (primers 3 and 4 in Figure 1). Individuals carrying the WT allele (m/+) give rise to an 854-bp cDNA amplicon (as shown in Figure 1B); those that are mop1–1 homozygous (m/m) do not, suggesting that the ZmRDR2 RNA transcript is impaired in mop1–1 homozygous mutant individuals. The DNA control sample gives rise to a 1335-bp amplicon. (B) RT-PCR of WT and mop1–1 homozygous individuals using a primer from exon 4 and a primer corresponding to Mu1.7 (primers 1 and 2 as shown in Figure 1B). The mop1–1 homozygotes give rise to a 470-bp amplicon whereas the WT individuals do not, suggesting that in mop1–1 homozygotes, the ZmRDR2 mRNA is transcribed but the transcript includes the Mu1.7 insertion and is thus nonfunctional. aat was used a control.

Figure 3. Mop1 Is Implicated in Small RNA Processing in the Immature Ear

(A) Small RNA Northern blot of a family segregating for the mop1–1 allele. All individuals carrying the WT allele of Mop1–1 (m/+: mop1/+ and +/+: WT), including active MuDR, contain small 26- and 24-nt RNAs for corresponding to mudrA and small 24-nt RNAs corresponding to mudrB. None of the individuals that are homozygous for the mop1–1 mutant (m/m) carry small RNAs corresponding to either mudrA or mudrB, suggesting that the Mop1/ZmRDR2 gene is involved in the processing of MuDR small RNAs. Shown below is a loading control. The same quantity from each RNA sample used for the blot was run in an ethidium-stained gel and photographed. (B) RT-PCR of embryo, leaf, and immature ear cDNA of individuals wild type for Mop1–1 using primers corresponding to ZmRDR2. The right-hand figure depicts much higher levels of ZmRDR2 transcript in embryo and immature ear tissue than in the leaves (L2, leaf 2; L8, leaf 8). The left-hand picture is the cDNA control using primers specific to aat.

Figure 4. Prevention of Mutator Silencing by Muk

(A) Shown at the top are the progeny of a cross Muk; mop1/+ × MuDR/−; mop1/mop1, which are subsequently crossed to the wild-type a1-mum2 tester that lacks both MuDR and Muk. Those individuals that contain MuDR but not Muk give rise to a significant percentage of spotted kernels when outcrossed; conversely, individuals that carry Muk and MuDR, regardless of whether they are mop1 homozygous, give rise to few spotted kernels when outcrossed, suggesting that the mop1 homozygous mutant does not prevent Muk silencing initiation. The images at the bottom show plant color suppression on the left due to active MuDR causing suppression of a1-mum2 expression; on the right is the expected dark plant color indicative of MuDR inactivation and lack of Mutator suppression at the a1-mum2 color allele. (B) A small RNA Northern blot of RNA collected from leaf 2 (L2) of individuals heterozygous for active MuDR. The individual on the left does not contain Muk and is wild-type for Mop1. The other two individuals carry Muk and are mop1–1 homozygous mutants (m/m).

Figure 5. The NAP1 RNAi Mutants Prevent Mutator Silencing by Muk

(A) Diagram of the crosses performed to generate the prophylactic experiment to examine the effects of various RNAi knockdown mutants on the process of Mutator silencing by Muk. The images are examples of ears derived from plants either lacking (left) or carrying the NFA104 transgene (right). (B) Percent spotted progeny kernels from individuals either carrying the NFA104 transgene (left) or not (right). All individuals carry both MuDR and Muk. Individuals that carry the NFA104 transgene on average have a higher percentage of spotted kernels compared to individuals that do not, suggesting that the NFA104 transgene can prevent MuDR silencing by Muk. (C) Percent spotted progeny kernels from individuals either carrying (left) or lacking (right) the NFA101 transgene. All individuals carry both MuDR and Muk. Individuals that carry the transgene on average have a higher percentage of kernel spotting versus individuals that do not carry the transgene.

Figure 6. Knockdown of the Endogenous NFA104 Transcript by the NFA104 Transgene Is Correlated with Kernel Spotting in MuDR;Muk F1 Progeny

RT-PCR of the endogenous NFA104 transcript in individual progeny from the cross NFA104; MuDR × Muk. NFA104 transgenic individuals (T) (lanes 1–5) in which the endogenous NFA104 transcript has been lost give rise to a significant percentage of spotted kernels (% spotting) when outcrossed to a1-mum2. Transgenic individuals where the endogenous NFA104 transcript is present (lanes 6 and 7) give rise to few spotted kernels when crossed to a1-mum2. Individuals not carrying the transgene (lanes 8–10) express the endogenous NFA104 transcript and give rise to few spotted kernels when crossed to a1-mum2. All individuals carry both MuDR (except for lane 11) and Muk.

Figure 7. NFA104 Does Not Prevent Methylation of Mu1 TIRs in a Muk Background

Shown is a Southern blot of DNA from individuals segregating for the NFA104 transgene (T) and MuDR (*), digested with methyl-sensitive HinfI and probed with a Mu1 fragment. All individuals carry Muk. AT, active tester control. Bands at the top of the blot represent Mu1 insertions that occurred in a previous generation.