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A technique to reduce DNA methylation in a sequence-specific manner by using a ribozyme-expressing cucumber mosaic virus vector

Reika Isoda: Research Faculty of Agriculture, Hokkaido University

<div>Host epigenetic status has been found to be modified by some plant virus infections. For example, cucumber mosaic virus (CMV) has been shown to affect genome-wide DNA methylation in infected plants. We have been studying the interactions between CMV and host in epigenetic modifications. Taking advantage of our findings on CMV, we here tried to use the CMV vector to develop a technique to induce demethylation in a sequence-specific manner. We first induced transcriptional gene silencing (TGS) to the 35S promoter upstream of the GFP gene in the transgenic 16C plants; TGS has been stably maintained through at least several generations (GFP-TGS). Short RNAs and scaffold RNAs (scRNAs) are known to play a role in guiding the DNA methylation complex to the target sequence. We thus tried to destroy short RNAs and scRNAs by sequence-specific hammer-head ribozyme (Rz) cleavage. An Rz expressed through the CMV vector was designed to cut scRNAs before the transcription start site (TSS). When we inoculated the CMV vector with an Rz onto the GFP-TGS plants, we found that DNA methylation levels were decreased over the 35S promoter and especially around the TSS, resulting in recovery of GFP fluorescence. To confirm whether the designed Rzs were really functional, we conducted <em>in vitro</em> cleavage and <em>in vivo</em> sensor experiments. These results together indicate that we could successfully develop a technique for DNA demethylation by using an Rz-expressing CMV vector.</div>