Strict De Novo Methylation of the 35S Enhancer Sequence in Gentian

A novel transgene silencing phenomenon was found in the ornamental plant, gentian (Gentiana triflora × G. scabra), in which the introduced Cauliflower mosaic virus (CaMV) 35S promoter region was strictly methylated, irrespective of the transgene copy number and integrated loci. Transgenic tobacco having the same vector did not show the silencing behavior. Not only unmodified, but also modified 35S promoters containing a 35S enhancer sequence were found to be highly methylated in the single copy transgenic gentian lines. The 35S core promoter (−90)-introduced transgenic lines showed a small degree of methylation, implying that the 35S enhancer sequence was involved in the methylation machinery. The rigorous silencing phenomenon enabled us to analyze methylation in a number of the transgenic lines in parallel, which led to the discovery of a consensus target region for de novo methylation, which comprised an asymmetric cytosine (CpHpH; H is A, C or T) sequence. Consequently, distinct footprints of de novo methylation were detected in each (modified) 35S promoter sequence, and the enhancer region (−148 to −85) was identified as a crucial target for de novo methylation. Electrophoretic mobility shift assay (EMSA) showed that complexes formed in gentian nuclear extract with the −149 to −124 and −107 to −83 region probes were distinct from those of tobacco nuclear extracts, suggesting that the complexes might contribute to de novo methylation. Our results provide insights into the phenomenon of sequence- and species- specific gene silencing in higher plants

An attempt to detect siRNA-mediated genomic DNA modification by artificially induced mismatch siRNA in Arabidopsis.

Although tremendous progress has been made in recent years in identifying molecular mechanisms of small interfering RNA (siRNA) functions in higher plants, the possibility of direct interaction between genomic DNA and siRNA remains an enigma. Such an interaction was proposed in the 'RNA cache' hypothesis, in which a mutant allele is restored based on template-directed gene conversion. To test this hypothesis, we generated transgenic Arabidopsis thaliana plants conditionally expressing a hairpin dsRNA construct of a mutated acetolactate synthase (mALS) gene coding sequence, which confers chlorsulfuron resistance, in the presence of dexamethasone (DEX). In the transgenic plants, suppression of the endogenous ALS mRNA expression as well as 21-nt mALS siRNA expression was detected after DEX treatment. After screening >100,000 progeny of the mALS siRNA-induced plants, no chlorsulfuron-resistant progeny were obtained. Further experiments using transgenic calli also showed that DEX-induced expression of mALS siRNA did not affect the number of chlorsulfuron-resistant calli. No trace of cytosine methylation of the genomic ALS region corresponding to the dsRNA region was observed in the DEX-treated calli. These results do not necessarily disprove the 'RNA cache' hypothesis, but indicate that an RNAi machinery for ALS mRNA suppression does not alter the ALS locus, either genetically or epigenetically

Phenotypes of transgenic <i>Arabidopsis</i> plants expressing <i>mALS</i> siRNA after DEX treatment.

<p>(A) Beta-glucuronidase (GUS) expression in root tissues of the transgenic plant lines #3, #6 and #12 after treatment with (+) or without (-) DEX. (B) Relative mRNA expression of the <i>ALS</i> gene in the wild-type (WT) and transgenic (#6) plants with or without (-) DEX treatment. <i>ALS</i> mRNA expression was normalized to the expression level of <i>Act8</i>. Experiments were replicated three times. (C) Expression of siRNA derived from <i>mALS</i> dsRNA in the transgenic plants (lines #3, #4, #6 and #12) after DEX treatment. DEX was applied for 2 days. Ethidium bromide staining of tRNA is shown as a loading control. (D) Effect on <i>mALS</i> siRNA expression of subculturing onto medium containing DEX in the #6 transgenic plants. Plants cultured after one (S1) to three (S3) rounds of subculturing or without subculturing (no S) for different periods (indicated as days after treatment) were analyzed.</p

Selection of chlorsulfuron-resistant selfed progeny of the transgenic plants treated with or without DEX.

<p>Seeds were sown on MS medium containing 100 nM chlorsulfuron. CS^S, number of chlorsulfuron-sensitive seedlings. CS^R, number of chlorsulfuron-resistant seedlings.</p

Effect of DEX treatment on the number of chlorsulfuron-resistant colonies in the transgenic calli.

<p>CS^R, number of chlorsulfuron-resistant colonies.</p

Schematic diagram of mutated acetolactate synthase (<i>mALS</i>) dsRNA induction vector and possible consequence of endogenous <i>ALS</i> mRNA degradation and hypothetical modification of the genomic <i>ALS</i> sequence.

<p>The <i>mALS</i> dsRNA is transcribed from the pOpOff2mALSir binary vector (upper) after dexamethasone (DEX) treatment, which activates LhGR transcription factor targeting the pOp6 bidirectional promoter. The <i>mALS</i> dsRNA is processed into siRNAs, which in turn compose an RNA-induced silencing complex (RISC) and the endogenous <i>ALS</i> mRNA is expected to be degraded by the RNAi machinery. At the same time, accumulation of <i>mALS</i> siRNA may be guided to the complementary genomic <i>ALS</i> locus (lower), resulting in <i>ALS</i> mutation, which would confer chlorsulfuron resistance. The black circles on the <i>ALS</i> locus represent hypothetical DNA methylation caused by RNA-dependent DNA methylation (RdDM).</p