Transient expression of coding and non-coding regions of PVY confer resistance to virus infection

Document Type : Original Research Papers

Authors

1 Department of Horticultural Sciences, Science and Research Branch, Islamic Azad University, Tehran, Iran

2 Department of Tissue Culture and Gene Transformation, Agricultural Biotechnology Research Institute of Iran, Karaj, Iran

3 Department of Agricultural Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran

4 Department of Plant Viruses, Iranian Research Institute of Plant Protection, Tehran, Iran

Abstract

One of the most efficient mechanisms by which plants protect themselves from invading viruses
is the specific RNA-dependent silencing pathway termed post-transcriptional gene silencing
(PTGS). In this mechanism, resistance to a virus is engineered through the expression of a
segment of the virus genomein transgenic plants. Potato VirusY (PVY) is one of the most
damaging viruses of potato, infecting most cultivars and causing significant yield losses
throughout the world. The present study was performed to compare the efficiency of three
construct containing different regions of 3′UTR (UR) and coat protein (CP) against PVY
infection. Expression of homologous hairpin RNA to PVY in potato plant was carried out by
transient gene expression of constructs with agro-infiltration followed by mechanical viral
infection. Results showed that successful production of siRNAs confer resistance to two PVY
strain. Comparison between transiently expressed constructs indicated that applying CP+UR
PVY hairpin RNA was the most efficient RNAi construct to confer resistance. Resistance was
found to have taken the form of immunity, since no viral particle could be detected in the upper
leaves as shown by ELISA assay and Northern hybridizations. To the best of our knowledge,
this is the first report on the application of 3'non-coding region of PVY in conferring complete
resistance against virus in potato.

Keywords

Main Subjects


  1. Baulcombe, D. (2004) RNA silencing in plants. Nature., 431, 356–63.
  2. Brodersen, P. and Voinnet, O. (2006) The diversity of RNA silencing pathways in plants. Trends Genet., 22, 268–280.
  3. Chen, Y.K., Lohuis, D., Goldbach, R. and Prins, M. (2004) High frequency induction of RNA-mediated resistance against Cucumber mosaic virus using inverted repeat constructs. Mol. Breeding, 14, 215-226.
  4. Di Nicola-Negri, E., Brunetti, A., Tavazza, M. and Ilardi, V. (2005). Hairpin RNA-mediated silencing of Plum pox virus P1 and HC-Pro genes for efficient and predictable resistance to the virus. Transgenic Res., 14, 989–94.
  5. Fuchs, M. and Gonsalves D. (2007). Safety of virus-resistant transgenic plants two decades after their introduction: lessons from realistic field risk assessment studies. Annu. Rev. Phytopathol.,45, 173–202.
  6. Gaba, V.,  Rosner, A., Maslenin, L., Leibman, D., Singer, S., Kukurt, E., Shiboleth, Y.M. and Gal-On, A. (2010) Hairpin-based virus resistance depends on the sequence similarity between challenge virus and discrete, highly accumulating siRNA species. Eur. J. Plant Pathol.,128, 153–164.
  7. Hamilton, A., Voinnet, O., Chappell, L. and Baulcombe, D. (2002) Two classes of short interfering RNA in RNA silencing. EMBO J., 21, 4671–9.
  8. Hammond, SM., Bernstein, E., Beach, D., Hannon, G.J. (2000) An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature, 404, 293–296.
  9. Hirai, S., Kodama, H. (2008) RNAi vectors for manipulation of gene expression in higher plants. Open Plant Sci. J., 2, 21–30.
  10. 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, 826–833.
  11. 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 Xand potato virus Y. Biotech., 8, 750–754.
  12. Krubphachaya, P., Juricek, M. and Kertbundit, S. (2007) Induction of RNA-mediated resistance to papaya ringspot virus type W. J. Biochem. Mol. Biol., 40, 404–411.
  13. 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: resistanceto potato virus X and potato virus Y in transgenic Russet Burbank. Biotech.,8, 1277–134.
  14. Lindbo, J.A. and Dougherty, W.G. (2005) Plant pathology and RNAi: a brief history. Annu. Rev. Phytopathol.,43, 191–204.
  15. Liu, Y., Schiff, M., Dinesh-Kumar, S.P. (2002) Virus induced gene silencing in tomato. Plant J., 31, 777–786.
  16. MacDiarmid, R. (2005) RNA silencing in productive virus infections. Annu. Rev. Phytopathol., 43, 523–44.
  17. Maki-Valkama, T., Pehu, T., Santala, A., Valkonen, J.P.T., Koivu, K., Lehto, K. and Pehu, E. (2000) High level of resistance to potato virus Y by expressing P1 sequence in antisense orientation in transgenic potato. Mol. Breeding, 6, 95-104.
  18. McCue, K.F.,  Ponciano, G.,  Rockhold, D.R.,   Whitworth, J.L.,  Gray, S.M., Fofanov, Y. and Belknap, W.R. (2012) Generation of PVY coat protein siRNAs in transgenic potatoes resistant to PVY. Am. J. Pot. Res., 89, 374–383.
  19. Missiou, A., Kalantidis, K., Boutla, A., Tzortzakaki, S., Tabler, M. and Tsagris, M. (2004) Generation of transgenic potato plants highly resistant to potato virus Y (PVY) through RNA silencing. Mol. breeding, 14, 185-197.
  20. Nelson, R., McCormick, S.M., Delannay, X., Dube, P. and Layton, J. (1988) Virus tolerance, plant growth and field performance of transgenic tomato plants expressing the coat protein from tobacco mosaic virus. Bio.Technology, 6, 403–9.
  21. Patil, B.L., Ogwok, E., Wagaba, H., Mohammed, I., Ydav, S.J., Bagewadi, B., Taylor, N.J., Kreuze, J.F., Maruthi, M.N., Alicai, T. and Fauquet, C.M. (2011) RNAi-mediated resistance to diverse isolates belonging to two virus species involved in cassava brown streak disease. Mol. Plant Pathol., 12, 31–41.
  22. Powell-Abel, P., Nelson, R.S., De, B., Hoffmann, N. and Rogers, S.G. 1986. Delay of disease development in transgenic plants that express the tobacco mosaic virus coat protein gene. Science, 232, 738–43.
  23. Qu, F., Ren, T. and Morris, T.J. (2003) The coat protein of turnip crincle virus suppresses posttranscriptional gene silencing at an early initiation step. J. Virol., 77, 511-522.
  24. Sambrook, J. and Russell, D. W. (2001) Molecular cloning. Cold Spring Harbor, New York.
  25. Sanford, J.C. and Johnston, S.A. (1985) The concept of parasite-derived resistance deriving resistance genes from the parasite’s own genome. J. Theor. Biol., 113, 395–405.
  26. Smith, N.A. and Eamens A. (2012) Isolation and detection of small RNAs from plant tissues. In Watson, J.M., Wang, M.B. (eds), Antiviral resistance in plants: methods and protocols. Methods Mol. Biol., 894, 155–172.
  27. Smith, N.A., Singh, S.P., Wang, M.B., Stoutjesdijk, P., Green, A. and Waterhouse, P.M. (2000) Total silencing by intron-spliced hairpin RNA. Nature, 407, 319-320.
  28. Tenllado, F. and  Díaz-Ruíz, J.R. (2001) Double-Stranded RNA-Mediated Interference with Plant Virus Infection. Journal of virology,75.
  29. Tepfer, M. (2002) Risk assessment of virus-resistant transgenic plants. Annu. Rev. Phytopathol.,40, 467–91.
  30. Thomas, C.L., Leh, V., Lederer, C. and Maule, A.J. (2003) Turnip crincle virus coat protein mediates suppression of RNA silencing in Nicotiana benthamiana. Virology, 306, 33-41.
  31. Vargas, M., Martínez-García, B., Díaz-Ruíz, J.R. and Tenllado, F. (2008) Transient expression of homologous hairpin RNA interferes with PVY transmission by aphids. Virology J., 5, 42.
  32. Voinnet, O. (2001). RNA silencing as a plant immune system against viruses. Trends Genet.,17, 449–59.
  33. Waterhouse, P.M., Wang, M.B. and Lough, T. (2001) Gene silencing as an adaptive defence against viruses. Nature, 411, 834–842.
  34. Yadav, S.J., Ogwok, E., Wagaba, H., Patil, B.L., Bagewadi, B., Alicia, T., Gaitan-Solis, E., Taylor, N.J. and Fauquet, C.M. (2011) RNAi mediated resistance to cassava brown streak Uganda virus in transgenic cassava. Mol. Plant Pathol.,12, 677–687.
  35. Zhang, P., Vanderschuren, H., Futterer, J. and Gruissem, W. (2005) Resistance to cassava mosaic disease in transgenic cassava expressing antisense RNAs targeting virus replication genes. Plant Biotechnol. J., 3, 385–397.