Abstract
BackgroundDe novo DNA methylation triggered by short interfering RNAs is called RNA-directed DNA methylation (RdDM). Transcriptional gene silencing (TGS) through RdDM can be induced using a viral vector. We have previously induced RdDM on the 35S promoter in the green fluorescent protein (GFP)-expressing Nicotiana benthamiana line 16c using the cucumber mosaic virus vector. The GFP fluorescence phenotype segregated into two types, “red” and “orange” in the first self-fertilized (S1) progeny plants by the difference in degree of recovery from TGS on GFP expression. In the second self-fertilized generation (S2 plants), the phenotypes again segregated. Explaining what generates the red and orange types could answer a very important question in epigenetics: How is the robustness of TGS maintained after RdDM induction?ResultsIn bisulfite sequencing analyses, we found a significant difference in the overall promoter hypermethylation pattern between the red and orange types in S1 plants but little difference in S2 plants. Therefore, we assumed that methylation at some specific cytosine residues might be important in determining the two phenotypes. To find the factor that discriminates stable, robust TGS from the unstable TGS with incomplete inheritance, we analyzed the direct effect of methylated cytosine residues on TGS. Because it has not yet been demonstrated that DNA methylation at a few specific cytosine residues on known sequence elements can indeed determine TGS robustness, we newly developed a method by which we can directly evaluate the effect of specific methylation on promoter activity. In this assay, we found that the effects of the specific cytosine methylation on TGS differed between the plus- and minus-strands.ConclusionsWe found two distinct phenotypes, the stable and unstable TGS in the progenies of virus-induced TGS plants. Our bisulfite sequencing analyses suggested that methylation at some specific cytosine residues in the 35S promoter played a role in determining whether stable or unstable TGSs are induced. Using the developed method, we inferred that DNA methylation heterogeneity in and between the plus- and minus-strands can differentially determine TGS.
Highlights
De novo DNA methylation triggered by short interfering RNAs is called RNA-directed DNA methylation (RdDM)
When we inoculated the green fluorescent protein (GFP)-expressing transgenic Nicotiana benthamiana (16c) with the cucumber mosaic virus (CMV)-A1 constructs containing various sizes of the 35S promoter sequences, we found that the GFP expression levels were downregulated due to RdDM and that the sizes of the virus-integrated sequences were important for DNA methylation [39]
The S1 progeny plants from A1–208-inoculated plants segregated into two phenotypes: red type, which lost GFP fluorescence and orange type, which was intermediate between S1:345 and 16c (Figs. 1b and 2)
Summary
De novo DNA methylation triggered by short interfering RNAs is called RNA-directed DNA methylation (RdDM). DRM2 is required for de novo DNA methylation, which is triggered by short-interfering RNAs (siRNAs) [6]. SiRNAs guide DRM2 to the target sequences on the genomic DNA and directs de novo DNA methylation through RNA-directed DNA methylation (RdDM) [7,8,9,10,11,12,13,14,15,16,17,18], in which two plant-specific RNA polymerases, Pol IV and Pol V play essential roles. A cascade of enzyme reactions (Pol IV-RDR2-DCL3) produces 24-nt siRNAs in concert, whereas several additional small RNAs seem to be involved in RdDM through the non-canonical pathway [19]
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