Attenuated mutants of Potato virus Y necrotic strain produced by nitrous acid treatment and mutagenesis-in-tissue culture methods
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We produced attenuated mutants of Potato virus Y necrotic strain not only by nitrous acid treatment but also by a novel method, probably unique to plant viruses, which we call the “mutagenesis-in-tissue culture method”. This relies on the natural or experimental generation of virus sequence variants within an infected plant, and then isolating the mutants by serially cloning them in plants. A total of fifteen attenuated mutants were obtained and studied. Nucleotide and amino acid sequences of the genomes of the attenuated mutant populations were compared with those parental severe isolates, and the amino acid changes in relevant genomic regions for viral attenuation were inferred. Many of the mutations were located in the 5’ half of the genome; 65 % were located in the protein 1 (P1) and helper component proteinase protein (HC-Pro) encoding regions. Amino acid changes mostly involved simultaneous changes in two or more protein encoding regions, one of which was often in the HC-Pro encoding region. The attenuated mutants M-MY10 and N-NA10 were effective in cross-protection against the original severe isolate NTND6.
KeywordsAttenuated mutant Mutagenesis-in-tissue culture Nitrous acid treatment PVY
Potato virus Y
We thank Yasuhiro Tomitaka, Hiromi Kajiyama, Sadayuki Akaishi, Hiroko Soda, Ryoko Koga, Mutsumi Ikematsu, Yuki Honda and Huy Duc Nguyen (Saga University, Japan) for their careful technical assistance and Adrian Gibbs (Canberra, Australia) for very kindly reading the manuscript. We thank Tetsuo Maoka (NARO Hokkaido Agricultural Research Center, Japan), Masataka Chaya (Nagasaki Prefectural Government, Japan), Noboru Ohnishi and Kanji Mamiya (Japan Agribio Co. Ltd., Japan) for helpful discussions. This work was supported by Grant-in-Aid for Research project for utilizing advanced technologies in Agriculture, Forestry and Fisheries no. 18061 from The Ministry of Agriculture, Forestry and Fisheries of Japan.
- Domingo, E., Escarmis, C., Menéndez-Arias, L., Perales, C., Herrera, M., Novella, S., & Holland, J. J. (2008). Viral quasispecies: dynamics, interactions, and pathogenesis: Origin and Evolution of Viruses 2nd edn. (In E. Domingo, C. R. Parrish and J. J. Holland (Eds.), (pp. 87–118). Elsevier Academic Press.)Google Scholar
- Hall, T. A. (1999). BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium, Ser., 41, 95–98.Google Scholar
- King, A. M. Q., Adams, M. J., Carstens, E. B., & Lefkowitz, E. J. (2012). Virus taxonomy, ninth report of the International Committee on Taxonomy of Viruses. San Diego, CA, USA, Elsevier Academic Press, p 1327.Google Scholar
- Ogawa, T., Nakagawa, A., Hataya, T., & Ohshima, K. (2012). The genetic structure of populations of Potato virus Y in Japan; based on the analysis of 20 full genomic sequences. Journal of Phytopathology. doi: 10.1111/j.1439-0434.2012.01959.x.
- Ohshima, K., Yamaguchi, Y., Hirota, R., Hamamoto, T., Tomimura, K., Tan, Z., Sano, T., Azuhata, F., Walsh, J. A., Fletcher, J., Chen, J., Gera, A., & Gibbs, A. (2002). Molecular evolution of Turnip mosaic virus: evidence of host adaptation, genetic recombination and geographical spread. Journal of General Virology, 83, 1511–1521.PubMedGoogle Scholar
- Shiboleth, Y. M., Haronsky, E., Leibman, D., Arazi, T., Wassenegger, M., Whitham, S. A., Gaba, V., & Gal-On, A. (2007). The conserved FRNK box in HC-Pro, a plant viral suppressor of gene silencing, is required for small RNA binding and mediates symptom development. Journal of Virology, 81, 13135–13148.PubMedCrossRefGoogle Scholar
- Yambao, M. L. M., Yagihashi, H., Sekiguchi, H., Sekiguchi, T., Sasaki, T., Sato, M., Atsumi, G., Tacahashi, Y., Nakahara, K. S., & Uyeda, I. (2008). Point mutations in helper component protease of clover yellow vein virus are associated with the attenuation of RNA-silencing suppression activity and symptom expression in broad bean. Archives of Virology, 153, 105–115.PubMedCrossRefGoogle Scholar