Abstract
Agroinfiltration assay using green fluorescent protein (GFP)-expressing Nicotiana benthamiana line 16c is a powerful method for screening of putative plant virus-encoded gene silencing suppressors. This method allows the investigator to know whether the putative viral suppressor inhibits silencing in a cell (local silencing) and/or spreading of silencing throughout a plant (systemic silencing). Additionally, grafting experiments using transgenic plants expressing the suppressor and the GFP will indicate whether the suppressor blocks systemic silencing steps, which include the production of a silencing signal in a silenced cell, and the cell-to-cell and long-distance movement of a silencing signal throughout a plant. Here, we describe methods and techniques of an agroinfiltration assay and grafting experiments, which were used for the characterization of Apple chlorotic leaf spot virus 50 kDa movement protein as a gene silencing suppressor. This protocol should allow the investigator to characterize putative plant virus-encoded gene silencing suppressors.
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Acknowledgments
We thank Dr. David C. Baulcombe, Sainsbury Laboratory, Norwich, UK for providing the GFP-expressing transgenic N. benthamiana line 16c, and Prof. Masato Ikegami, Tohoku University, Miyagi, Japan for providing pBI-GFP and pBI-dsGFP. This work was supported in part by a grant-in-aid for the 21st Century Center of Excellence Program from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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Yaegashi, H., Isogai, M., Yoshikawa, N. (2012). Characterization of Plant Virus-Encoded Gene Silencing Suppressors. In: Watson, J., Wang, MB. (eds) Antiviral Resistance in Plants. Methods in Molecular Biology, vol 894. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-882-5_8
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DOI: https://doi.org/10.1007/978-1-61779-882-5_8
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