AUTHOR=Otagaki Shungo , Kasai Megumi , Masuta Chikara , Kanazawa Akira 

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

JOURNAL=Frontiers in Genetics

VOLUME=4

YEAR=2013

URL=https://www.frontiersin.org/journals/genetics/articles/10.3389/fgene.2013.00044

DOI=10.3389/fgene.2013.00044

ISSN=1664-8021

ABSTRACT=<p>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 <italic>Arabidopsis</italic> and may possibly counteract RdDM. Here, we isolated an ortholog of <italic>ROS1</italic> (<italic>NbROS1</italic>) from <italic>Nicotiana benthamiana</italic> and examined the antagonistic activity of NbROS1 against virus-induced RdDM by simultaneously inducing RdDM and <italic>NbROS1</italic> knockdown using a vector based on <italic>Cucumber mosaic virus</italic>. Plants were inoculated with a virus that contained a portion of the <italic>Cauliflower mosaic virus</italic> 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 <italic>NbROS1</italic>, which induced downregulation of <italic>NbROS1</italic>. Simultaneous induction of RdDM and <italic>NbROS1</italic> 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 <italic>NbROS1 </italic>knockdown by a virus vector is useful as a tool to enhance targeted DNA methylation.</p>