Enhancement of RNA-directed DNA methylation of a transgene by simultaneously downregulating a ROS1 ortholog using a virus vector in Nicotiana benthamiana

Shungo Otagaki,Chikara Masuta,Megumi Kasai,Akira Kanazawa

doi:10.3389/fgene.2013.00044

Abstract

Cytosine methylation can be induced by double-stranded RNAs through the RNA-directed DNA methylation (RdDM) pathway. A DNA glycosylase REPRESSOR OF SILENCING 1 (ROS1) participates in DNA demethylation in Arabidopsis and may possibly counteract RdDM. Here, we isolated an ortholog of ROS1 (NbROS1) from Nicotiana benthamiana and examined the antagonistic activity of NbROS1 against virus-induced RdDM by simultaneously inducing RdDM and NbROS1 knockdown using a vector based on Cucumber mosaic virus. Plants were inoculated with a virus that contained a portion of the Cauliflower mosaic virus 35S promoter, which induced RdDM of the promoter integrated in the plant genome and transcriptional silencing of the green fluorescent protein gene driven by the promoter. Plants were also inoculated with a virus that contained a portion of NbROS1, which induced downregulation of NbROS1. Simultaneous induction of RdDM and NbROS1 knockdown resulted in an increase in the level of cytosine methylation of the target promoter. These results provide evidence for the presence of antagonistic activity of NbROS1 against virus-induced RdDM and suggest that the simultaneous induction of promoter-targeting RdDM and NbROS1 knockdown by a virus vector is useful as a tool to enhance targeted DNA methylation.

Highlights

Nucleotide-sequence-specific interactions mediated by RNA have a role in the control of gene expression via diverse pathways of RNA silencing, which involve either RNA-guided RNA degradation or epigenetic modification of the genome (Baulcombe, 2004; Brodersen and Voinnet, 2006)
We found that NbROS1 is expressed in vegetative tissues in N. benthamiana as in Arabidopsis
A recent model for the mechanism(s) of localization of REPRESSOR OF SILENCING 1 (ROS1) protein on a target site involves the sliding of ROS1 protein along DNA (Ponferrada-Marín et al, 2012), which may fit the notion that ROS1 randomly finds target sites on genomic DNA

Summary

Introduction

Nucleotide-sequence-specific interactions mediated by RNA have a role in the control of gene expression via diverse pathways of RNA silencing, which involve either RNA-guided RNA degradation or epigenetic modification of the genome (Baulcombe, 2004; Brodersen and Voinnet, 2006). When dsRNAs corresponding to a gene promoter are synthesized, RdDM of the promoter and transcriptional gene silencing (TGS) can be induced. Such promoter-targeted gene silencing has been used to modify gene expression in plants (Jones et al, 2001; Sijen et al, 2001; Cigan et al, 2005; Heilersig et al, 2006; Okano et al, 2008; Kanazawa et al, 2011a,b; Otagaki et al, 2011; Kasai and Kanazawa, 2013) and other organisms (Hawkins and Morris, 2008; Suzuki and Kelleher, 2009)

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Results

Conclusion