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Inhibition of Binding of Tomato Yellow Leaf Curl Virus Rep to its Replication Origin by Artificial Zinc-Finger Protein

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Abstract

Previously we demonstrated that inhibition of replication-associated protein (Rep) binding to its replication origin by artificial zinc-finger proteins (AZPs) is a powerful method to prevent plant virus infection in vivo. In the present study, we applied the AZP technology to Tomato yellow leaf curl virus (TYLCV), which is a limiting factor in tomato cultivation worldwide. First, we determined 5′-ATCGGTGT ATCGGTGT-3′ in the 195-bp intergenic region of the TYLCV-Israel strain, a strain reported first among TYLCV strains, as the Rep-binding site by gel shift assays. We then constructed a 6-finger AZP that bound to a 19-bp DNA including the Rep-binding site. We demonstrated that the binding affinity of the AZP was >1,000-fold greater than that of Rep and that the AZP inhibited Rep binding completely in vitro. Because the binding capability of the AZP was same as that of the AZP previously designed for geminivirus-resistant Arabidopsis thaliana, we predict that the present AZP will prevent TYLCV infection in vivo.

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Acknowledgments

We thank Tadayuki Imanaka and Haruyuki Atomi for the use of their DNA sequencer. This work was supported by a grant from the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) to T.S.

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Authors and Affiliations

  1. Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyotodaigaku-Katsura, Nishikyo-ku, Kyoto, 615-8510, Japan

    Tomoaki Mori, Kosuke Takenaka, Fumiya Domoto, Yasuhiro Aoyama & Takashi Sera

  2. Department of Applied Chemistry and Biotechnology, Graduate School of Natural Science and Technology, Okayama University, Tsushima-Naka, Kita-ku, Okayama, 700-8530, Japan

    Tomoaki Mori & Takashi Sera

  3. Department of Molecular Chemistry and Biochemistry, Faculty of Science and Engineering, Doshisha University, 1-3 Tatara-Miyakodani, Kyotanabe, Kyoto, 610-0321, Japan

    Yasuhiro Aoyama

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  1. Tomoaki Mori
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  2. Kosuke Takenaka
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  3. Fumiya Domoto
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  4. Yasuhiro Aoyama
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  5. Takashi Sera
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Correspondence to Takashi Sera.

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Mori, T., Takenaka, K., Domoto, F. et al. Inhibition of Binding of Tomato Yellow Leaf Curl Virus Rep to its Replication Origin by Artificial Zinc-Finger Protein. Mol Biotechnol 54, 198–203 (2013). https://doi.org/10.1007/s12033-012-9552-5

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  • DOI: https://doi.org/10.1007/s12033-012-9552-5

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