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Molecular Breeding

, Volume 14, Issue 2, pp 185–197 | Cite as

Generation of transgenic potato plants highly resistant to potato virus Y (PVY) through RNA silencing

  • Anastasia Missiou
  • Kriton Kalantidis
  • Alexandra Boutla
  • Sergia Tzortzakaki
  • Martin Tabler
  • Mina Tsagris
Article

Abstract

In this study we applied RNA silencing to engineer potato plants that are resistant to potato virus Y (PVY). We expressed double-stranded (ds) RNA derived from the 3′ terminal part of the coat protein gene of PVY, which is highly conserved in sequence amongst different PVY isolates, in transgenic potatoes of the commercial variety ‘Spunta’. Transgenic plants were analyzed for generation of transgene-derived short interfering RNAs (siRNAs) prior to virus inoculation. Twelve of fifteen transgenic lines produced siRNAs and were highly resistant to three strains of PVY, each belonging to three different subtypes of the virus (PVYN, PVYO and PVYNTN). Infection of transgenic plants with Potato virus X (PVX) simultaneously or prior to the challenge with PVY did not interfere with PVY-resistance.

Coat Protein gene PTGS PVY RNA silencing siRNA Solanum tuberosum Transgenic plants 

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References

  1. Altschul S.F., Madden T.L., Schaffer A.A., Zhang J., Zhang Z., Miller W. and Lipman D.J. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25: 3389–402.Google Scholar
  2. Anandalakshmi R., Pruss G.J., Ge X., Marathe R., Mallory A.C., Smith T.H. and Vance V.B. 1998. A viral suppressor of gene silencing in plants. Proc. Natl. Acad. Sci. USA 95(22): 13079–84.Google Scholar
  3. Baulcombe D. 2001. RNA silencing. Diced defence. Nature 409: 295–6.Google Scholar
  4. Beaujean A., Sangwan R., Lecardonnel A. and Sangwan-Norreel B. 1998. Agrobacterium-mediated transformation of three economically important potato cultivars using sliced internodal explants: an efficient protocol of transformation. J. of Exp. Botany 49: 1589–1595.Google Scholar
  5. Bernstein E., Caudy A.A., Hammond S.M. and Hannon G.J. 2001. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409: 363–6.Google Scholar
  6. Brigneti G., Voinnet O., Li W.X., Ji L.H., Ding S.W. and Baulcombe D.C. 1998. Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana. EMBO J. 17: 6739–46.Google Scholar
  7. Davies C., Hills G. and Baulcombe D.C. 1993. Sub-cellular localization of the 25-kDa protein encoded in the triple gene block of potato virus X. Virology 197: 166–75.Google Scholar
  8. Ditta G.S., Stanfield D., Corbin D., Helinski D.R. 1980. Broad host range cloning system for gram negative bacteria: Construction of a gene bank of Rhizobium meliloti. Proc. Natl. Acad. Sci. 77: 7347–7351.Google Scholar
  9. Dougherty W.G., Lindbo J.A., Smith H.A., Parks T.D., Swaney S. and Proebsting W.M. 1994. RNA-mediated virus resistance in transgenic plants: exploitation of a cellular pathway possibly involved in RNA degradation. Mol. Plant Microbe Interact. 7: 544–52.Google Scholar
  10. Dougherty W.G. and Parks T.D. 1995. Transgenes and gene suppression: telling us something new? Curr. Opin. Cell Biol. 7: 399–405.Google Scholar
  11. Elbashir S.M., LendeckelW. and Tuschl T. 2001. RNA interference is mediated by 21-and 22-nucleotide RNAs. Genes Dev. 15: 188–200.Google Scholar
  12. Esna-Ashari M. and Villiers T. 1998. Plant regeneration from tuber discs of potato Solanum tuberosumL._ using 6-benzylaminopurine (BAP)}. Potato Research 41: 371–382.Google Scholar
  13. Farinelli L., Malnoe P. and Collet G. Heterologous encapsidation of potato virus Y strain O (PVY-O) with the transgene coat protein of PVY strain N (PVY-N) in Solanum tuberosum. cv. Bintje. Biotechnology 10: 1020-1025.Google Scholar
  14. Fire A., Xu S., Montgomery M.K., Kostas S.A., Driver S.E. and Mello C.C. 1998. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391: 806–11.Google Scholar
  15. Glais L., Tribodet M. and Kerlan C. 2002. Genomic variability in Potato potyvirus Y (PVY): evidence that PVY(N)W and PVY(NTN) variants are single to multiple recombinants between PVY(O) and PVY(N) isolates. Arch. Virol. 147: 363–78.Google Scholar
  16. Gleave A.P. 1992. A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome. Plant Mol. Biol. 20: 1203–7.Google Scholar
  17. Hamilton A.J. and Baulcombe D.C. 1999. A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286: 950–2.Google Scholar
  18. Hamilton A., Voinnet O., Chappell L. and Baulcombe D. 2002. Two classes of short interfering RNA in RNA silencing. EMBO J. 21: 4671–9.Google Scholar
  19. Hammond S.M., Bernstein E., Beach D. and Hannon G.J. 2000. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophilacells. Nature 404: 293–6.Google Scholar
  20. Hassairi A., Masmoudi K., Albouy J., Robaglia C., Julien M. and Ellouz R. 1998. Transformation of two potato cultivars 'Spunta' and 'Claustar' (Solanum tuberosum) with lettuce mosaic virus coat protein gene and heterologous immunity to potato virus Y. Plant Science 136: 31-42.Google Scholar
  21. Holen T., Amarzguioui M., Wiiger M.T., Babaie E. and Prydz H. 2002. Positional effects of short interfering RNAs targeting the human coagulation trigger Tissue Factor. Nucleic Acids Res. 30: 1757–66.Google Scholar
  22. Hutvagner G. and Zamore P.D. 2002. RNAi: nature abhors a double-strand. Curr. Opin. Genet. Dev. 12: 225–32.Google Scholar
  23. Kalantidis K., Psaradakis S., Tabler M. and Tsagris M. 2002. The occurrence of CMV-specific short Rnas in transgenic tobacco expressing virus-derived double-stranded RNA is indicative of resistance to the virus. Mol. Plant Microbe Interact. 15: 826–33.Google Scholar
  24. Kaniewski W., Lawson G., Sammons B., Haley L., Hart J., Delannay X. and Tumer N.E. 1990. Field resistance of transgenic Russet Burbank potato to effects of infection by potato virus X and potato virus Y. Biotechnology 8: 750–754.Google Scholar
  25. Lawson G., Kaniewski W., Haley L., Rozman R., Newell C., Sanders P. and Tumer N.E. 1990. Engineering resistance to mixed virus infection in a commercial potato cultivar: resistance to potato virus X and potato virus Y in transgenic Russet Burbank. Biotechnology 8: 1277–134.Google Scholar
  26. Maki-Valkama T., Pehu T., Santala A., Valkonen J.P., Koivu K., Lehto K. and Pehu E. 2000a. High level of resistance to potato virus Y expressing P1 sequence in antisense orientation in transgenic potato. Molecular Breeding 6: 95–104.Google Scholar
  27. Maki-Valkama T., Valkonen J.P., Kreuze J.F. and Pehu E. 2000b. HC-Pro Suppression of Transgene Silencing Eliminates the Small RNAs but Not Transgene Methylation or the Mobile Signal. Plant Cell 13: 571–83.Google Scholar
  28. Malnoe P., Farinelli L., Collet G. and Reust W. 1994. Small-scale field tests with transgenic potato, cv. Bintje, to test the resistance to primary and secondary infections with potato virus Y. Plant Mol. Biol. 25: 963–975.Google Scholar
  29. Mitsuhara I., Shirasawa-Seo N., Iwai T., Nakamura S., Honkura R. and Ohashi Y. 2002. Release From Post-transcriptional Gene Silencing by Cell Proliferation in Transgenic Tobacco Plants: Possible Mechanism for Noninheritance of the Silencing. Genetics 160: 343–352.Google Scholar
  30. Okamoto D., Nielsen S.V.S., Albrechtsen M. and Borkhardt B. 1996. General resistance against potato virus Y introduced into a commercial potato cultivar by genetic transformation with PVYN coat protein. Potato Res. 39: 271–282.Google Scholar
  31. Papaefthimiou I., Hamilton A., Denti M., Baulcombe D., Tsagris M. and Tabler M. 2001. Replicating potato spindle tuber viroid RNA is accompanied by short RNA fragments that are characteristic of post-transcriptional gene silencing. Nucleic Acids Res. 29: 2395–2400.Google Scholar
  32. Pehu T., Maki-Valkama T., Valkonen J.P., Koivu K., Lehto K. and Pehu E. 1995. Potato plants transformed with a potato virus Y P1 gene sequence are resistant to PVY-O. Am. Potato J. 72: 523–532.Google Scholar
  33. Rook and Lindsey K. 1998. Methods in molecular virology. In: Foster G. and Taylor S. (eds), Plant Virology Protocols, Vol. 81, pp. 353–359. Humana Press, New Jersey, USA.Google Scholar
  34. Sambrook J, Fritsch E.F., Maniatis T, 1989. Molecular cloning: A Laboratory Manual, 1 ed. Cold Spring Harbour: CSH, USA.Google Scholar
  35. Schauer S.E., Jacobsen S.E., Meinke D.W. and Ray A. 2002. DICER-LIKE1: blind men and elephants in Arabidopsis development. Trends Plant Sci. 7: 487–491.Google Scholar
  36. Smith H.A., Powers H., Swaney S., Brown C. and Dougherty W.G. 1995. Transgenic potato virus Y resistance in potato: evidence for an RNA-mediated cellular response. Phytopathology 85: 864–870.Google Scholar
  37. Smith N.A., Singh S.P., Wang M.B., Stoutjesdijk P.A., Green A.G. and Waterhouse P.M. 2000. Total silencing by intron-spliced hairpin RNAs. Nature 407: 319–20.Google Scholar
  38. Solomon-Blackburn R. and Barker H. 2001. Breeding virus resistant potatoes (solanum tuberosum): a review of traditional and molecular approaches. Heredity 86: 17-35.Google Scholar
  39. Tacke E., Salamini F. and Rohde W. 1996. Genetic engineering of potato for broad-spectrum protection against virus infection. Nature Biotechnology 14: 1597–1601.Google Scholar
  40. Vance V. and Vaucheret H. 2001. RNA silencing in plants-defense and counterdefense. Science 292: 2277–80.Google Scholar
  41. Vance V.B. 1991. Replication of potato virus X RNA is altered in coinfections with potato virus Y. Virology 182: 486–94.Google Scholar
  42. Vance V.B., Berger P.H., Carrington J.C., Hunt A.G., and Shi X.M. 1995. 5' proximinal potyviral sequences mediate potato virus X/potyviral synergistic disease in transgenic tobacco. Virology 206: 583-90.Google Scholar
  43. Vaucheret H., Beclin C. and Fagard M. 2001. Post-transcriptional gene silencing in plants. J. Cell Sci. 114: 3083–91.Google Scholar
  44. Vaucheret H. and Fagard M. 2001. Transcriptional gene silencing in plants: targets, inducers and regulators. Trends Genet. 17: 29–35.Google Scholar
  45. Voinnet O. 2001. RNA silencing as a plant immune system against viruses. Trends Genet. 17: 449–59.Google Scholar
  46. Voinnet O., Lederer C. and Baulcombe D.C. 2000. A viral movement protein prevents spread of the gene silencing signal in Nicotiana benthamiana. Cell 103: 157–67.Google Scholar
  47. Voinnet O., Pinto Y.M., and Baulcombe D.C. 1999. Suppression of gene silencing: a general strategy used by diverse DNA and RNA viruses of plants. Proc. Natl. Acad. Sci. USA 96: 14147–52.Google Scholar
  48. Wang M.B., Abbot D.C. and Waterhouse P.M. 2000. A single copy of a virus-derived transgene encoding hairpin RNA gives immunity to barley yellow dwarf virus. Molecular Plant Pathology 1: 347–356.Google Scholar
  49. Waterhouse P.M., Graham M.W. and Wang M.B. 1998. Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proc. Natl. Acad. Sci. USA 95: 13959–64.Google Scholar
  50. Waterhouse P.M., Wang M. and Finnegan E.J. 2001a. Role of short RNAs in gene silencing. Trends Plant Sci. 6: 297–301.Google Scholar
  51. Waterhouse P.M., Wang M.B. and Lough T. 2001b. Gene silencing as an adaptive defense against viruses. Nature 411: 834–842.Google Scholar

Copyright information

© Kluwer Academic Publishers 2004

Authors and Affiliations

  • Anastasia Missiou
    • 1
  • Kriton Kalantidis
    • 2
  • Alexandra Boutla
    • 3
    • 2
  • Sergia Tzortzakaki
    • 2
  • Martin Tabler
    • 2
  • Mina Tsagris
    • 3
    • 2
  1. 1.Department of BiologyUniversity of CreteCreteGreece
  2. 2.Institute of Molecular Biology and BiotechnologyFoundation for Research and Technology – HellasCreteGreece
  3. 3.Department of BiologyUniversity of CreteCreteGreece

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