Russian Journal of Genetics

, Volume 40, Issue 4, pp 425–430 | Cite as

Analysis of the Proteins in the Leaves of Transgenic Tobacco Plants Expressing Double-Stranded RNA Upon Viral Infection

  • T. V. Korostyleva
  • T. I. Odintsova
  • G. V. Kozlovskaya
  • V. A. Pukhalskiy


Generation of transgenic tobacco plants, producing double-stranded RNA with no homology to tobacco genome sequences is reported. The RNA synthesis is mediated by a construct containing an inverted repeat of the pBR322 tetracycline-resistance gene fragment under control of the 35S CaMV promoter. Analysis of the resistance of transgenic plants to the tobacco mosaic virus revealed the changes in the protein spectra of the infected plants. The 25- and 30-kDa proteins found were not detected in the extracts of normal plants. Amino acid sequencing of the 30-kDa peptide with subsequent computer database search revealed the homology of this protein to the hydrolases belonging to the group of plant β-glucanases. The role of the novel polypeptides in an increase of the resistance of transgenic plants to TMV, and also the possibility of the regulation of their expression by nonhomologous dsRNA are discussed.


Transgenic Plant Polypeptide Mosaic Virus Database Search Gene Fragment 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Beachy, R.N., Mechanisms and Applications of Pathogen-Derived Resistance in Transgenic Plants, Curr. Opin. Biotechnol., 1997, vol. 8, pp. 215-220.Google Scholar
  2. 2.
    Wintermantel, W.M. and Zaitlin, M., Transgene Translatability Increases Effectiveness of Replicase-Mediated Resistance to Cucumber Mosaic Virus, J. Gen. Virol., 2000, vol. 81, pp. 587-595.Google Scholar
  3. 3.
    Seppanen, P., Puska, R., Honkanen, J., et al., Movement Protein-Derived Resistance to Triple Gene Block-Containing Plant Viruses, J. Gen. Virol., 1997, vol. 78, pp. 1241-1246.Google Scholar
  4. 4.
    Bejarno, E.R., Day, A.G., Paranjape, V., and Lichtenstein, C.P., Antisense Genes as Tools to Engineer Virus Resistance in Plants, Biochem. Soc. Trans., 1992, vol. 20, pp. 757-761.Google Scholar
  5. 5.
    Magbanua, Z.V., Wilde, H.D., Roberts, J.K., et al., Field Resistance to Tomato Spotted Wilt Virus in Transgenic Peanut (Arachis hypogaea L.) Expressing an Antisense Nucleocapsid Gene Sequence, Mol. Breed., 2000, vol. 6, pp. 227-236.Google Scholar
  6. 6.
    Jan, F.-J., Fagoaga, C., Pang, S.-Z., and Gonsales, D., A Minimum Length of N Gene Sequence in Transgenic Plants Is Required for RNA-Mediated Tospovirus Resistance, J. Gen. Virol., 2000, vol. 81, pp. 235-242.Google Scholar
  7. 7.
    Sijen, T., Wellink, J., Hiriart, J.B., and Van Kammen, A., RNA-Mediated Virus Resistance: Role of Repeated Transgenes and Delineation of Targeted Regions, Plant Cell, 1996, vol. 8, pp. 2277-2294.Google Scholar
  8. 8.
    Waterhouse, P.M., Graham, M.W., and Wang, M.-B., Virus Resistance and Gene Silencing in Plants Can Be Induced by Simultaneous Expression of Sense and Antisense RNA, Proc. Natl. Acad. Sci. USA, 1998, vol. 95,no. 23, pp. 13 959-13 964.Google Scholar
  9. 9.
    Bistritskaite, G.B. and Stasyavichute, Z.B., Induction of Plant Resistance to Tobacco Mosaic Virus and Potato Virus X with the Use of Double-Stranded RNAs Isolated from Yeast Saccharomyces, Tr. Akad. Nauk Lit. SSR, Ser. V, 1986, vol. 4,no. 86, pp. 3-8.Google Scholar
  10. 10.
    Kumar, M. and Carmichael, G.G., Antisense RNA: Function and Fate of Duplex RNA in Cells of Higher Eukaryotes, Microbiol. Mol. Biol. Rev., 1998, vol. 62,no. 4, pp. 1415-1434.Google Scholar
  11. 11.
    Crum, C.J., Hu, J., Hiddinga, H.J., and Roth, D.A., Tobacco Mosaic Virus Infection Stimulates the Phosphorylation of a Plant Protein Associated with Double-Stranded RNA-Dependent Protein Kinase Activity, J. Biol. Chem., 1988, vol. 263,no. 26, pp. 13 440-13 443.Google Scholar
  12. 12.
    Langland, J.O., Langland, L.A., Browning, K.S., and Roth, D.A., Phosphorylation of Plant Eukaryotic Initiation Factor 2 by the Plant-Encoded Double-Stranded RNA-Dependent Protein Kinase, PPKR, and Inhibition of Protein Synthesis in Vitro, J. Biol. Chem., 1996, vol. 271, pp. 4539-4544.Google Scholar
  13. 13.
    Lu, C. and Fedoroff, N., A Mutation in the Arabidopsis HYL1 Gene Encoding a dsRNA-Binding Protein Affects Responses to Abscisic Acid, Auxin, and Cytokinin, Plant Cell, 2000, vol. 12, pp. 2351-2365.Google Scholar
  14. 14.
    Smirnov, S.P., Krasheninnikova, L.V., and Pukhal'skii, V.A., The Effect of Double-Stranded RNA Synthesis on the Resistance of Transgenic Tobacco Plants to the Tobacco Mosaic Virus, Dokl. Akad. Nauk, 1993, vol. 331,no. 2, pp. 241-245.Google Scholar
  15. 15.
    Plant Genetic Transformation and Gene Expression: A Laboratory Manual, Draper, J., Scott, R., Armitage, P., and Walden, R., Eds., Oxford: Blackwell, 1988.Google Scholar
  16. 16.
    Herrington, S. and MacGie, J., Molekulyarnaya klinicheskaya diagnostika. Metody (Molecular Clinical Diagnostics: Methods), Moscow: Mir, 1999.Google Scholar
  17. 17.
    Fraser, C.M., Kerlavage, A.R., Mariani, A.P., and Venter, J.C., Structural Analysis of Purified β-Adrenergic Receptors, Proteins, 1987, vol. 2, pp. 34-41.Google Scholar
  18. 18.
    Okazawa, K., Sato, Y., Nakagava, T., et al., Molecular Cloning and cDNA Sequencing of Endo Xyloglucan Transferase, a Novel Class of Glicosyltransferase That Mediates Molecular Grafting between Matrix Polysaccharides in Plant Cell Walls, J. Biol. Chem., 1993, vol. 268, pp. 25 364-25 368.Google Scholar
  19. 19.
    Takeuchi, Y., Yoshikawa, M., Takeba, G., et al., Molecular Cloning and Ethylene Induction of mRNA Encoding a Phytoalexin Elicitor-Releasing Factor, β-1,3-Endoglucanase, in Soybean, Plant Physiol., 1990, vol. 93, pp. 673-682.Google Scholar
  20. 20.
    Beffa, R. and Meins, F.J., Pathogenesis-Related Function of Plant β-1,3-Glucanases Investigated by Antisense Transformation—A Review, Gene, 1996, vol. 179, pp. 97-103.Google Scholar
  21. 21.
    Linthorst, H.J., Melchers, L.S., Mayer, A., et al., Analysis of Gene Families Encoding Acidic and Basic β-1,3-Glucanases of Tobacco, Proc. Natl. Acad. Sci. USA, 1990, vol. 87, pp. 8756-8760.Google Scholar
  22. 22.
    Bucher, G.L., Tarina, C., Heinlein, M., et al., Local Expression of Enzymatically Active Class I β-1,3-Glucanase Enhances Symptoms of TMV Infection in Tobacco, Plant J., 2001, vol. 28, pp. 361-369.Google Scholar
  23. 23.
    Hennig, J., Dewey, R.E., Cutt, J.R., and Klessig, D.F., Pathogen, Salicylic Acid and Developmental Dependent Expression of a β-1,3-Glucanase Gus Gene Fusion in Transgenic Tobacco Plants, Plant J., 1993, vol. 4, pp. 481-493.Google Scholar
  24. 24.
    Cote, F., Cutt, J.R., Asselin, A., and Klessig, D.F., Pathogenesis-Related Acidic β-1,3-Glucanase Genes of Tobacco Are Regulated by Both Stress and Developmental Signals, Mol. Plant-Microbe Interact., 1991, vol. 4, pp. 173-181.Google Scholar
  25. 25.
    Delp, G. and Palva, E.T., A Novel Flower-Specific Arabidopsis Gene Related to Both Pathogen-Induced and Developmentally Regulated Plant β-1,3-Glucanase Genes, Plant Mol. Biol., 1999, vol. 39, pp. 565-575.Google Scholar
  26. 26.
    Obregon, P., Martin, R., Sanz, A., and Castresana, C., Activation of Defense-Related Genes during Senescence: A Correlation between Gene Expression and Cellular Damage, Plant Mol. Biol., 2001, vol. 46, pp. 67-77.Google Scholar
  27. 27.
    Del Campillo, E., Multiple Endo-1,4-β-D-Glucanase (Cellulase) Genes in Arabidopsis, Curr. Top. Dev. Biol., 1999, vol. 46, pp. 39-61.Google Scholar
  28. 28.
    Kalaitzis, P., Hong, S.B., Solomos, T., and Tucker, M.L., Molecular Characterization of a Tomato Endo-β-1,4-Glucanase Gene Expressed in Mature Pistils, Abscission Zones and Fruit, Plant Cell Physiol., 1999, vol. 40, pp. 905-908.Google Scholar
  29. 29.
    Beffa, R.S., Neunaus, J.-M., and Meins, Jr., Physiological Compensation in Antisense Transformants: Specific Induction of an “Ersatz” Glucan Endo-1,3-β-Glucosidase in Plants Infected with Necrotizing Viruses, Proc. Natl. Acad. Sci. USA, 1993, vol. 90, pp. 8792-8796.Google Scholar

Copyright information

© MAIK “Nauka/Interperiodica” 2004

Authors and Affiliations

  • T. V. Korostyleva
    • 1
  • T. I. Odintsova
    • 1
  • G. V. Kozlovskaya
    • 1
  • V. A. Pukhalskiy
    • 1
  1. 1.Vavilov Institute of General GeneticsRussian Academy of SciencesMoscowRussia

Personalised recommendations