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Enhanced Protection Against Fungal Attack by Constitutive Co–expression of Chitinase and Glucanase Genes in Transgenic Tobacco


Plants respond to pathogen attack by the induction of a battery of defenses, suggesting that different protective mechanisms may have complementary roles in the overall expression of disease resistance. We have investigated possible functional interactions between two different hydrolytic enzymes, chitinase and glucanase, by constitutive co-expression in transgenic tobacco of genes encoding the rice RCH10 basic chitinase and the alfalfa AGLU1 acidic glucanase. Hybrid plants were generated by crossing transgenic parental lines exhibiting strong constitutive expression of cauliflower mosaic virus (CaMV) 35S enhancer / RCH10 and CaMV 35S double promoter / AGLU1 gene fusions, respectively. Evaluation of disease development in these hybrids, heterozygous for each transgene, and in homozygous selfed progeny, showed that combination of the two transgenes gave substantially greater protection against the fungal pathogen Cercospora nicotianae, causal agent of frogeye, than either transgene alone. Productive interactions between chitinase and glucanase transgenes in vivo point to combinatorial expression of antimicrobial genes as an effective approach to engineering enhanced crop protection against fungal disease.

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  1. Lamb, C.J., Lawton, M.A., Dron, M. and Dixon, R.A. 1989. Signals and transduction mechanisms for activation of plant defenses against microbial attack. Cell 56: 215–224.

    Article  CAS  Google Scholar 

  2. Bowles, D.J. 1990. Defense-related proteins in higher plants. Annu. Rev. Biochem. 59: 873–907.

    Article  CAS  Google Scholar 

  3. Kiedrowski, S., Kawalleck, P., Hahlbrock, K., Somssich, I.E. and Dangl, J. 1992. Rapid activation of a novel plant gene is strictly dependent on the Arabidopsis RPM1 disease resistance locus. EMBO J. 11: 1359–1369.

    Article  Google Scholar 

  4. Lamb, C.J., Ryals, J.A., Ward, E.R. and Dixon, R.A. 1992. Emerging strategies for enhancing resistance to microbial pathogens. Bio/Technology 10: 1436–1445.

    CAS  PubMed  Google Scholar 

  5. Mauch, F., Mauch-Mani, B. and Boller, T. 1988. Antifungal hydrolases in pea tissue. II. Inhibition of fungal growth by combinations of chitinase and β-1,3-glucanase. Plant Physiol. 88: 936–942.

    Article  CAS  Google Scholar 

  6. Broglie, K., Chet, I., Holliday, M., Cressman, R., Biddle, P., Knowlton, C., Mauvais, C.J. and Broglie, R. 1991. Transgenic plants with enhanced resistance to the fungal pathogen Rhizoctonia solani. Science 254: 1194–1197.

    Article  CAS  Google Scholar 

  7. Zhu, Q. and Lamb, C.J. 1991. Isolation and characterization of a rice gene encoding a basic chitinase. Mol. Gen. Genet. 226: 289–296.

    Article  CAS  Google Scholar 

  8. Zhu, Q., Doerner, P.W. and Lamb, C.J. 1993. Stress induction and developmental regulation of a rice chitinase promoter in transgenic tobacco. Plant J. 3: 203–212.

    Article  CAS  Google Scholar 

  9. Maher, E.A., Lamb, C.J. and Dixon, R.A. 1994. Stress responses in alfalfa (Medicago sativa L). XVII. Identification of multiple hydrolases and molecular characterization of an acidic glucanase. Physiol. Mol. Plant Pathol. 43: 329–342.

    Article  Google Scholar 

  10. Jones, J.D.G., Dunsmuir, P. and Bedbrook, J. 1985. High level expression of introduced chimaeric genes in regenerated transformed plants. EMBO J. 4: 2411–2418.

    Article  CAS  Google Scholar 

  11. Jefferson, R.A., Kavanagh, T.A. and Bevan, M.W. 1987. GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6: 3901–3907.

    Article  CAS  Google Scholar 

  12. Rogers, S.G., Horsch, R.B. and Fraley, R.T. 1986. Gene transfer in plants: Production of transformed plants using Ti-plasmid vectors. Methods Enzymol. 118: 627–640.

    Article  CAS  Google Scholar 

  13. Kay, R., Chan, A., Daly, M. and McPherson, J. 1987. Duplication of CaMV 35S promoter sequences creates a strong enhancer for plant genes. Science 236: 1299–1302.

    Article  CAS  Google Scholar 

  14. Shew, H.D. and Lucas, G.B. 1991. Compendium of tobacco diseases. APS Press, The American Phytopathological Society, St. Paul, MN.

  15. Yoshikawa, M., Tsuda, M. and Takeuchi, Y. 1993. Resistance to fungal diseases in transgenic tobacco plants expressing the phytoalexin elicitor-releasing factor, β-1,3-endoglucanase from soybean. Naturwissenschaften 80: 417–420.

    Article  CAS  Google Scholar 

  16. Neuhaus, J.-M., Ahl-Goy, P., Hinz, U., Flores, U. and Meins, F. 1991. High level expression of a tobacco chitinase gene in Nicotiana sylvestris. Susceptibility of transgenic tobacco plants to Cercospora nicotianae. Plant Mol. Biol. 16: 141–151.

    Article  CAS  Google Scholar 

  17. Alexander, D., Goodman, R.M., Gut-Rella, M., Glascock, C., Weymann, K., Friedrich, L., Maddox, D., Ahl-Goy, P., Luntz, T., Ward, E. and Ryals, J. 1993. Increased tolerance to two oomycete pathogens in transgenic tobacco expressing pathogenesis-related protein 1a. Proc. Natl. Acad. Sci. USA 90: 7327–7331.

    Article  CAS  Google Scholar 

  18. Van den Elzen, P.J.M., Jongedijk, E., Melchers, L.S. and Cornelissen, B.J.C. 1993. Virus and fungal resistance: from laboratory to field. Phil. Trans. R. Soc. London. B 342: 271–278.

    Article  Google Scholar 

  19. Benhamou, N., Broglie, K., Chet, I. and Broglie, R. 1993. Cytology of infection of 35S-bean chitinase transgenic canola plants by Rhizoctonia solani: cytochemical aspects of chitin breakdown in vivo. Plant J. 4: 295–305.

    Article  CAS  Google Scholar 

  20. Dixon, R.A. and Lamb, C.J. 1990. Molecular communication in interactions between plants and microbial pathogens. Annu. Rev. Plant Physiol. Plant Mol. Biol. 41: 339–367.

    Article  CAS  Google Scholar 

  21. Meins, F., Neuhaus, J.-M., Sperisen, C. and Ryals, J. 1992. The primary structure of plant pathogenesis-related glucanohydrolases and their genes, p. 246–282. In: Plant Gene Research. Genes Involved in Plant Defense. T.P. Boiler and F. Meins, Jr. (Eds.). Springer-Verlag, Vienna.

    Google Scholar 

  22. Leah, R., Tommerup, H., Svendsen, I. and Mundy, J. 1991. Biochemical and molecular characterization of three anti-fungal proteins from barley seed. J. Biol. Chem. 266: 1564–1573.

    CAS  Google Scholar 

  23. Terras, F.R.G., Schoofs, H.M.E., Thevissen, K., Osborn, R.W., Vanderlyden, J., Cammue, B.P.A. and Broekaert, W.F. 1993. Synergistic enhancement of the antifungal activity of wheat and barley thionins by radish and oilseed rape 2S albumins and by barley trypsin inhibitors. Plant Physiol. 103: 1311–1319.

    Article  CAS  Google Scholar 

  24. Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473.

    Article  CAS  Google Scholar 

  25. Edwards, K., Johnstone, C. and Thompson, C. 1991. A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucl. Acids Res. 19: 1349.

    Article  CAS  Google Scholar 

  26. Dellaporta, S.L., Wood, J. and Hicks, J.B. 1983. A plant DNA minipreparation. Version II. Plant Mol. Biol. Rep. 1: 19–21.

    Article  CAS  Google Scholar 

  27. Sambrook, J., Fritsch, E.F. and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual, 2nd Edn., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

  28. Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B. and Erlich, H.A. 1988. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 489–491.

    Article  Google Scholar 

  29. Chomczynski, P. and Sacchi, N. 1989. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156–159.

    Article  Google Scholar 

  30. Boller, T., Gehri, A., Mauch, F. and Vögeli, U. 1983. Chitinase in bean leaves: Induction by ethylene, purification, properties and possible function. Planta 157: 22–31.

    Article  CAS  Google Scholar 

  31. Pan, S.Q., Ye, X.S. and Kuć, J. 1991. A technique for detection of chitinase, β-1,3-glucanase and protein pattern after a single separation using polyacryl-amide gel electrophoresis or isoelectricfocusing. Phytopathology 81: 970–974.

    Article  CAS  Google Scholar 

  32. Mauch, F., Hadwiger, L.A. and Boller, T. 1984. Ethylene: symptom, not signal for the induction of chitinase and β-1,3-glucanase in pea pods by pathogens and elicitors. Plant Physiol. 76: 607–611.

    Article  CAS  Google Scholar 

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Correspondence to Chris J. Lamb.

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Zhu, Q., Maher, E., Masoud, S. et al. Enhanced Protection Against Fungal Attack by Constitutive Co–expression of Chitinase and Glucanase Genes in Transgenic Tobacco. Nat Biotechnol 12, 807–812 (1994).

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