Skip to main content

Advertisement

Log in

Attenuated mutants of Potato virus Y necrotic strain produced by nitrous acid treatment and mutagenesis-in-tissue culture methods

  • Published:
European Journal of Plant Pathology Aims and scope Submit manuscript

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

Abbreviations

PVY:

Potato virus Y

References

  • Beczner, L., Horvath, J., Romhanyi, I., & Forster, H. (1984). Studies on the etiology of tuber necrotic ringspot disease in potato. Potato Research, 27, 339–352.

    Article  Google Scholar 

  • Bukovinszki, A., Gotz, R., Johansen, E., Maiss, E., & Balazs, E. (2007). The role of the coat protein region in symptom formation on Physalis floridana varies between PVY strains. Virus Research, 127, 122–125.

    Article  PubMed  CAS  Google Scholar 

  • Bull, J. J., Badgett, M. R., Wichman, H. A., Huelsenbeck, J. P., Hillis, D. M., Gulati, A., Ho, C., & Molineux, I. J. (1997). Exceptional convergent evolution in a virus. Genetics, 147, 1497–1507.

    PubMed  CAS  Google Scholar 

  • Chiang, C. H., Lee, C. Y., Wang, C. H., Jan, F. J., Lin, S. S., Chen, T. C., Raja, J. A. J., & Yeh, S. D. (2007). Genetic analysis of an attenuated Papaya ringspot virus strain applied for cross-protection. European Journal of Plant Pathology, 118, 333–348.

    Article  CAS  Google Scholar 

  • Chrzanowska, M. (1991). New isolates of the necrotic strain of potato virus Y (PVYN) found recently in Poland. Potato Research, 34, 179–182.

    Article  Google Scholar 

  • Chung, B. Y. W., Miller, W. A., Atkins, J. F., & Firth, A. E. (2008). An overlapping essential gene in the Potyviridae. Proceedings of the National Academy of Sciences of the United States of America, 105, 5897–5902.

    Article  PubMed  CAS  Google Scholar 

  • Clark, M. F., & Adams, A. N. (1977). Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. Journal of General Virology, 34, 475–485.

    Article  PubMed  CAS  Google Scholar 

  • Desbiez, C., Girard, M., & Lecoq, H. (2010). A novel natural mutation in HC-Pro responsible for mild symptomatology of Zucchini yellow mosaic virus (ZYMV, Potyvirus) in cucurbits. Archives of Virology, 155, 397–401.

    Article  PubMed  CAS  Google Scholar 

  • 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.)

  • Drake, J. W., & Holland, J. J. (1999). Mutation rates among RNA viruses. Proceedings of the National Academy of Sciences of the United States of America, 96, 13910–13913.

    Article  PubMed  CAS  Google Scholar 

  • French, R., & Stenger, D. C. (2005). Population structure within lineages of Wheat streak mosaic virus derived from a common founding event exhibits stochastic variation inconsistent with the deterministic quasi-species model. Virology, 343, 179–189.

    Article  PubMed  CAS  Google Scholar 

  • Gibbs, A. J., & Ohshima, K. (2010). Potyviruses and the digital revolution. Annual Review of Phytopathology, 48, 205–223.

    Article  PubMed  CAS  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.

    CAS  Google Scholar 

  • Hu, X., Meacham, T., Ewing, L., Gray, S. M., & Karasev, A. V. (2009). A novel recombinant strain of Potato virus Y suggests a new viral genetic determinant of vein necrosis in tobacco. Virus Research, 143, 68–76.

    Article  PubMed  CAS  Google Scholar 

  • Hu, X., Nie, X., He, C., & Xiong, X. (2011). Differential pathogenicity of two different recombinant PVYNTN isolates in Physalis floridana is likely determined by the coat protein gene. Virology Journal, 8, 207.

    Article  PubMed  Google Scholar 

  • Ji, J. P., & Loeb, L. A. (1992). Fidelity of HIV-1 reverse transcriptase copying RNA in vitro. Biochemistry, 31, 954–958.

    Article  PubMed  CAS  Google Scholar 

  • Kosaka, Y., & Fukunishi, T. (1993). Attenuated isolates of soybean mosaic virus derived at a low temperature. Plant Disease, 77, 882–886.

    Article  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.

  • Le Romancer, M., Kerlan, C., & Nedellec, M. (1994). Biological characterisation of various geographical isolates of potato virus Y inducing superficial necrosis on potato tubers. Plant Pathology, 43, 138–144.

    Article  Google Scholar 

  • Lecoq, H., Lemaire, J. M., & Wipf-Scheibel, C. (1991). Control of zucchini yellow mosaic virus in squash by cross protection. Plant Disease, 75, 208–211.

    Article  Google Scholar 

  • Nishiguchi, M., & Kobayashi, K. (2011). Attenuated plant viruses: preventing virus diseases and understanding the molecular mechanism. Journal of General Plant Pathology, 77, 221–229.

    Article  Google Scholar 

  • Ogawa, T., Tomitaka, Y., Nakagawa, A., & Ohshima, K. (2008). Genetic structure of a population of Potato virus Y inducing potato tuber necrotic ringspot disease in Japan; comparison with North American and European populations. Virus Research, 131, 199–212.

    Article  PubMed  CAS  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., Sako, K., Hiraishi, C., Nakagawa, A., Matsuo, K., Ogawa, T., Shikata, E., & Sako, N. (2000). Potato tuber necrotic ringspot disease occurring in Japan: its association with Potato virus Y necrotic strain. Plant Disease, 84, 1109–1115.

    Article  CAS  Google Scholar 

  • 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.

    PubMed  CAS  Google Scholar 

  • Ohshima, K., Akaishi, S., Kajiyama, H., Koga, R., & Gibbs, A. J. (2010). Evolutionary trajectory of turnip mosaic virus populations adapting to a new host. Journal of General Virology, 91, 788–801.

    Article  PubMed  CAS  Google Scholar 

  • Schubert, J., Fomitcheva, V., & Sztangret-Wiśniewska, J. (2007). Differentiation of Potato virus Y strains using improved sets of diagnostic PCR-primers. Journal of Virological Methods, 140, 66–74.

    Article  PubMed  CAS  Google 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.

    Article  PubMed  CAS  Google Scholar 

  • Singh, R. P., Valkonen, J. P. T., Gray, S. M., Boonham, N., Jones, R. A. C., Kerlan, C., & Schubert, J. (2008). Discussion paper: the naming of Potato virus Y strains infecting potato. Archives of Virology, 153, 1–13.

    Article  PubMed  CAS  Google Scholar 

  • Steinhauer, D. A., Domingo, E., & Holland, J. J. (1992). Lack of evidence for proofreading mechanisms associated with an RNA virus polymerase. Gene, 122, 281–288.

    Article  PubMed  CAS  Google Scholar 

  • Takaki, F., Sano, T., & Yamashita, K. (2006). The complete nucleotide sequence of attenuated onion yellow dwarf virus: a natural potyvirus deletion mutant lacking the N-terminal 92 amino acids of HC-Pro. Archives of Virology, 151, 1439–1445.

    Article  PubMed  CAS  Google Scholar 

  • Tan, Z., Gibbs, A. J., Tomitaka, Y., Sánchez, F., Ponz, F., & Ohshima, K. (2005). Mutations in Turnip mosaic virus genomes that have adapted to Raphanus sativus. Journal of General Virology, 86, 501–510.

    Article  PubMed  CAS  Google Scholar 

  • Tribodet, M., Glais, L., Kerlan, C., & Jacquot, E. (2005). Characterization of Potato virus Y (PVY) molecular determinants involved in the vein necrosis symptom induced by PVYN isolates in infected Nicotiana tabacum cv. Xanthi. Journal of General Virology, 86, 2101–2105.

    Article  CAS  Google Scholar 

  • Urcuqui-Inchima, S., Haenni, A. L., & Bernardi, F. (2001). Potyvirus proteins: a wealth of functions. Virus Reseach, 74, 157–175.

    Article  CAS  Google Scholar 

  • Yamasaki, S., Sakai, J., Fuji, S., Kamisoyama, S., Emoto, K., Ohshima, K., & Hanada, K. (2010). Comparisons among isolates of Sweet potato feathery mottle virus using complete genomic RNA sequences. Archives of Virology, 155, 795–800.

    Article  PubMed  CAS  Google 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.

    Article  PubMed  CAS  Google Scholar 

  • Yeh, S. D., & Gonsalves, D. (1984). Evaluation of induced mutants of papaya ringspot virus for control by cross protection. Phytopathology, 74, 1086–1091.

    Article  Google Scholar 

Download references

Acknowledgements

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.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Kazusato Ohshima.

Electronic supplementary materials

Below is the link to the electronic supplementary material.

ESM 1

(PDF 315 kb)

ESM 2

(PDF 255 kb)

ESM 3

(PDF 223 kb)

ESM 4

(PDF 260 kb)

ESM 5

(PDF 294 kb)

ESM 6

(PDF 198 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ogawa, T., Nakamura, S., Sayama, M. et al. Attenuated mutants of Potato virus Y necrotic strain produced by nitrous acid treatment and mutagenesis-in-tissue culture methods. Eur J Plant Pathol 135, 745–760 (2013). https://doi.org/10.1007/s10658-012-0115-1

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10658-012-0115-1

Keywords

Navigation