Virus‐mediated efficient induction of epigenetic modifications of endogenous genes with phenotypic changes in plants

Abstract

SUMMARYGene silencing through transcriptional repression can be induced by targeting double-stranded RNA (dsRNA) to a gene promoter. It has been reported that a transgene was silenced by targeting dsRNA to the promoter, and the silenced state was inherited to the progeny plant even after removal of the silencing inducer from cells. In contrast, no plant has been produced that harbors silenced endogenous gene after removal of promotertargeting dsRNA. Here, we show that heritable gene silencing can be induced by targeting dsRNA to the endogenous gene promoters in petunia and tomato plants, using the Cucumber mosaic virus (CMV)-based vector. We found that efficient silencing of endogenous genes depends on the function of the 2b protein encoded in the vector virus, which has the ability to facilitate epigenetic modifications through the transport of short interfering RNA to nucleus. Bisulfite sequencing analyses on the targeted promoter in the virus-infected and its progeny plants revealed that cytosine methylation was found not only at CG or CNG but also at CNN sites. The observed inheritance of asymmetric DNA methylation is quite unique, suggesting that plants have a mechanism to maintain even asymmetric methylation. This CMV-based gene silencing system provides a useful tool to artificially modify DNA methylation in plant genomes and elucidate the mechanism for epigenetic controls.

Virus-Induced Necrosis Is a Consequence of Direct Protein-Protein Interaction between a Viral RNA-Silencing Suppressor and a Host Catalase

“…Recently, 2b was found to bind to Argonaute1 (AGO1), necessary for the plant antiviral machinery, and to AGO4, necessary for the DNA methylation process that controls heterochromatin formation and transcription (Zhang et al, 2006;González et al, 2010). In addition, 2b was also reported to directly bind to small interfering RNAs (Goto et al, 2007;Kanazawa et al, 2011).…”

<i>Peanut stunt virus</i>-induced gene silencing in white lupin (<i>Lupinus albus</i>)

“…host plant gene fragments were introduced just downstream of a stop codon of the 2b ORF (Figure 1), consistent with the method used to develop CMV vectors to prevent the deleterious effects of extra fragments on systemic viral infection (Hong et al 2012;Kanazawa et al 2011;Otagaki et al 2006). Plants can be inoculated with PSV and CMV by rubbing the leaves with virus solution.…”

Induction of RNA-directed DNA methylation and heritable transcriptional gene silencing as a tool to engineer novel traits in plants

“…Subsequently, we reported for the first time that TGS of endogenous genes can be induced by the vector in petunia and tomato plants and that the induced TGS is heritable (Kanazawa et al 2011a). The TGS resulted in phenotypic changes and was accompanied by epigenetic changes including cytosine methylation and histone modification.…”

“…When RdDM and TGS of plant endogenous genes were induced by the CMV vector, an altered state of histone modification, the presence of dimethylation at Lys9 of histone H3 and the absence of acetylation of histone H3, was detected and the altered state was inherited by the progeny (Kanazawa et al 2011a). The altered state of histone modification was maintained in the progeny even when the frequency of cytosine methylation decreased.…”

“…We also found that promoter-targeted TGS was induced more efficiently in both virus-infected plants and dsRNAtransfected protoplasts when the 2b gene is expressed. Thus, efficient TGS depends on the function of the 2b protein, which has the ability to facilitate epigenetic modifications through the transport of siRNA to the nucleus (Kanazawa et al 2011a). …”