A hairpin RNA construct residing in an intron efficiently triggered RNA‐directed DNA methylation in tobacco

Abstract

SUMMARYSo far, conventional hairpin RNA (hpRNA) constructs consisting of an inverted repeat (IR) of target promoters directly introduced into an expression cassette have been used to mediate de novo DNA methylation. Transcripts of such constructs resemble mRNA molecules, and are likely to be exported to the cytoplasm. The presence of hpRNAs in the cytoplasm and the nucleus may account for the simultaneous activation of posttranscriptional gene silencing (PTGS) and RNA-directed DNA methylation (RdDM). We hypothesized that by retaining hpRNAs in the nucleus, efficient induction of only RdDM may be achieved. Thus, we introduced into tobacco a transgene containing an intron into which an IR of a target promoter was inserted. The intronic hpRNA initiated highly specific cis-and trans-methylation, but did not induce PTGS. No spreading of methylation into sequences flanking the region of homology between the hpRNA and the target DNA was detectable. The efficient methylation-directing activity of the intronic hpRNA may indicate a previously unrecognized role of introns, potentially regulating gene expression at the transcriptional level.

Plant siRNAs from introns mediate DNA methylation of host genes

“…However, based on the observation that these 24-nt siRNAs displayed a heterogeneous distribution along sirtron loci and showed no exact correlation with the methylation density ( Fig. 2B,D; Supplemental Data Set S2), the sirtron-derived 24-nt siRNAs may be involved in the amplification and maintenance of DNA methylation rather than triggering this process (Dalakouras et al 2009). This notion could also be applied to the 24-nt siRNAs derived from the dual-coding introns (Fig.…”

“…In addition, an intronic hairpin-structured RNA (int-hpRNA) construct was reported to be capable of initiating cis-and trans-methylation by producing 24-nt siRNAs in tobacco (Dalakouras et al 2009). …”

4. Epigenetik in der Pflanzenzüchtung

“…Fast alle der Umwelt-/Stress-bedingten epigenetischen Veränderungen werden nicht vererbt, sondern wieder zurückgesetzt, sobald die äußeren Bedingungen sich normalisiert haben (Paszkowski/Grossniklaus, 2011;Pecinka/Mittelsten Scheid, 2012 (Dalakouras et al, 2009), wodurch die Promotoraktivität und damit die Expression der entsprechenden Gene inhibiert werden kann. 16 Nach Selbstungen solcher transgenen Linien entstehen unter anderem auch Nachkommen, die die dsRNA-transkribierenden Transgene verloren haben (Auskreuzen).…”

An endogene-resembling transgene is resistant to DNA methylation and systemic silencing

“…Agrobacterium tumefaciens (strain GV3101) containing the binary vector pPCV702SM-GpG (hpF) 67 was used for N. benthamiana leaf agroinfiltration at an optical density (OD) of 1.0, as previously described.…”