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Tobacco-expressed Brassica juncea chitinase BjCHI1 shows antifungal activity in vitro

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Abstract

We have previously isolated a Brassica juncea cDNA encoding BjCHI1, a novel chitinase with two chitin-binding domains, and have shown that its mRNA is induced by wounding and methyl jasmonate treatment (K.-J. Zhao and M.-L. Chye, Plant Mol. Biol. 40 (1999) 1009–1018). By the presence of two chitin-binding domains, BjCHI1 resembles the precursor of UDA (Urtica dioica agglutinin) but, unlike UDA, BjCHI1 retains its chitinase catalytic domain after post-translational processing. Here, we indicate the role of BjCHI1 in plant defense by demonstrating its mRNA induction upon Aspergillus niger infection or caterpillar Pieris rapae (L.) feeding. To further investigate the biological properties of BjCHI1, we transformed tobacco with a construct expressing the BjCHI1 cDNA from the CaMV 35S promoter. Subsequently, we purified BjCHI1 from the resultant transgenic R0 plants using a regenerated chitin column followed by fast protein liquid chromatography (FPLC). Also, the significance of the second chitin-binding domain in BjCHI1 was investigated by raising transgenic tobacco plants expressing BjCHI2, a deletion derivative of BjCHI1 lacking one chitin-binding domain. Colorimetric chitinase assays at 25 °C, pH 5, showed no significant differences between the activities of BjCHI1 and BjCHI2, suggesting that chitinase activity, due to the catalytic domain, is not enhanced by the presence of a second chitin-binding domain. Both BjCHI1 and BjCHI2 show in vitro anti-fungal activity toward Trichoderma viride, causing reductions in hyphal diameter, hyphal branching and conidia size.

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References

  • Beintema, J.J. and Peumans, W.J. 1992. The primary structure of stinging nettle (Urtica dioica) agglutinin, A two-domain member of the hevein family. FEBS Lett. 299: 131–134.

    Google Scholar 

  • Boller, T. 1985. Induction of hydrolases as a defense reaction against pathogens. In: J.L. Key and T. Kosuge (Eds) Cellular and Molecular Biology of Plant Stress, Alan R. Liss, New York, pp. 247–262.

    Google Scholar 

  • Boller, T. 1992. Biochemical analysis of chitinase and ?-1,3-glucanases. In: S.J. Gurr, M.J. Mcpherson and D.J. Bowles (Eds) Molecular Plant Pathology: A Practical Approach, vol. II, IRL Press, Oxford, pp. 23–30.

    Google Scholar 

  • 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.

    Google Scholar 

  • Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248–254.

    Google Scholar 

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

    Google Scholar 

  • Broekaert, W.F., Van Parijs, J., Leyns, F., Joos, H. and Peumans, W.J. 1989. A chitin-binding lectin from stinging nettle rhizomes with antifungal properties. Science 245: 1100–1102.

    Google Scholar 

  • Büchter, R., Strömberg, A., Schmelzer, E. and Kombrink, E. 1997. Primary structure and expression of acidic (class II) chitinase in potato. Plant Mol. Biol. 35: 749–761.

    Google Scholar 

  • Chrispeels, M.J. and Raikhel, N.V. 1991. Lectin, lectin genes and their role in plant defense. Plant Cell 3: 1–9.

    Google Scholar 

  • Collinge, D.B., Kragh, K.M., Mikkelsen, J.D., Nielsen, K.K., Rasmussen, U. and Vad, K. 1993. Plant chitinases. Plant J. 3: 31–40.

    Google Scholar 

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

    Google Scholar 

  • Does, M.P., Houterman, P.M., Dekker, H.L. and Cornelissen, B.J.C. 1999. Processing, targeting and antifungal activity of stinging nettle agglutinin in transgenic tobacco. Plant Physiol. 120: 421–431.

    Google Scholar 

  • Fitchette-Laine, A.-C., Denmat, L.-A., Lerouge, P. and Faye, L. 1998. Analysis of N-and O-glycosylation of plant proteins. In: C. Cunningham and A.J.R. Porter (Eds) Methods in Biotechnology, vol. 3. Recombinant Proteins from Plants. Humana Press, Totowa, NJ, pp. 271–290.

    Google Scholar 

  • Gooday, G.W. 1999. Aggressive and defensive roles for chitinases. In: P. Jollès and R.A.A. Muzzarelli (Eds) Chitin and Chitinases, Birkhäuser, Basel, Switzerland, pp. 157–170.

    Google Scholar 

  • Grison, R., Grezes-Besset, B., Schneider, M., Lucante, N., Olsen, L., Leguay, J.-J. and Toppan, A. 1996. Field tolerance to fungal pathogens of Brassica napus constitutively expressing a chimeric chitinase gene. Nature Biotechnol. 14: 643–646.

    Google Scholar 

  • Horsch, R.B., Fry, J.E., Hoffmann, N.L., Eichholz, D., Rogers, S.G. and Fraley, R.T. 1985. A simple and genetic method for transferring genes into plants. Science 227: 1227–1231.

    Google Scholar 

  • Iseli, B., Boller, T. and Neuhaus, J.-M. 1993. The N-terminal cysteine-rich domain of tobacco class I chitinase is essential for chitin binding but not for catalytic or antifungal activity. Plant Physiol. 103: 221–226.

    Google Scholar 

  • Kellmann, J.W., Kleinow, T., Engelhardt, K., Philipp, C., Wegener, D., Schell, J. and Schreier, P.H. 1996. Characterization of two class II chitinase genes from peanut and expression studies in transgenic tobacco plants. Plant Mol. Biol. 30: 351–358.

    Google Scholar 

  • Kush, A., Goyvaerts, E., Chye, M.-L. and Chua, N.-H. 1990. Laticifer specific gene expression of Hevea brasiliensis (rubber tree). Proc. Natl. Acad. Sci. USA 87: 1787–1790.

    Google Scholar 

  • Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.

    Google Scholar 

  • Lerner, D.R. and Raikhel, N.V. 1992. The gene for stinging nettle lectin (Urtica dioica agglutinin) encodes both a lectin and a chitinase. J. Biol. Chem. 267: 11085–11091.

    Google Scholar 

  • Lin, W., Anuratha, C.S., Datta, K., Potrykus, I., Muthukrishnan, S. and Datta, S.K. 1995. Genetic engineering of rice for resistance to sheath blight. Bio/technology 13: 686–691.

    Google Scholar 

  • Mauch, F., Mauch-Mani, B. and Boller, T. 1988. Antifungal hydrolases in pea tissue. Plant Physiol. 88: 936–942.

    Google Scholar 

  • Mayer, R.T., Shapiro, J.P., Berdis, E., Hearn, C.J., McCollum, T.G., McDonald, R.E. and Doostdar, H. 1995. Citrus rootstock responses to herbivory by larvae of the sugarcane rootstock borer weevil (Diaprepes abbreviatus). Physiol. Plant. 94: 164–173.

    Google Scholar 

  • Melchers, L.S., Apotheker-de Groot, M., van der Knaap, J.A., Ponstein, A.S., Sela-Buurlage, M.B, Bol, J.F., Cornelissen, B.J.C., van den Elzen, P.J.M. and Linthorst, H.J.M. 1994. A new class of tobacco chitinases homologous to bacterial exo-chitinases displays antifungal activity. Plant J. 5: 469–480.

    Google Scholar 

  • Nagy, F., Kay, S.A. and Chua, N.H. 1988. Analysis of gene expression in transgenic plants. In: S.B. Gelvin and R.A. Schilperoort (Eds) Plant Molecular Biology Manual, Kluwer Academic Publishers, Dordrecht, Netherlands, pp. B4: 1-29.

    Google Scholar 

  • Neuhaus, J.M. Fritig, B., Linthorst, H.J.M., Meins, F., Mikkelsen, J.D. and Ryals, J. 1996. A revised nomenclature for chitinase genes. Plant Mol. Biol. Rep. 14: 102–104.

    Google Scholar 

  • Neuhaus, J.M., Sticher, L., Meins, F. and Boller, T. 1991. A short C-terminal sequence is necessary and sufficient for the targeting of chitinase to the plant vacuole. Proc. Natl. Acad. Sci. USA 88: 10362–10366.

    Google Scholar 

  • Peumans, W.J. and Van Damme, E.J.M. 1995. Lectins as plant defense proteins. Plant Physiol. 109: 347–352.

    Google Scholar 

  • Rasmussen, U., Bojsen, K. and Collinge, D.B. 1992. Cloning and characterization of a pathogen-induced chitinase in Brassica napus. Plant Mol. Biol. 20: 277–287.

    Google Scholar 

  • Roberts, W.K. and Selitrennikoff, C.P. 1988. Plant and bacterial chitinases differ in antifungal activity. J. Gen. Microbiol. 134: 169–176.

    Google Scholar 

  • Saharan, G.S. 1993. Disease resistance. In: K.S. Labana, S.S. Banga and S.K. Banga (Eds) Breeding Oilseed Brassicas, Springer-Verlag, Berlin, pp. 181–205.

    Google Scholar 

  • Sambrook, J., Fritsch, E. F. and Maniatis, T. 1989. Molecular Cloning: a Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Plainview, NY.

    Google Scholar 

  • Schlumbaum, A., Mauch, F., Vögeli, U. and Boller, T. 1986. Plant chitinases are potent inhibitors of fungal growth. Nature 324: 365–367.

    Google Scholar 

  • Soedjanaatmadja, U.M.S., Subroto, T. and Beintema, J.J. 1995. Processed products of the hevein precursor in the latex of the rubber tree (Hevea brasiliensis). FEBS Lett. 363: 211–213.

    Google Scholar 

  • Sticher, L., Hofsteenge, J., Neuhaus, J.-M., Boller, T. and Meins, F. Jr. 1993. Posttranslational processing of a new class of hydroxyproline-containing proteins. Plant Physiol. 101: 1239–1247.

    Google Scholar 

  • Thomas K.R., Davis, B. and Mills, J. 1979. The effect of betaglucuronidase and chitinase on the cell wall of Aspergillus niger and Aspergillus fumigatus. Microbios 25: 111–123.

    Google Scholar 

  • Thomma, B.P.H.J., Eggermont, K., Penninckx, I.A.M.A., Mauch-Mani, B., Vogelsang, R., Cammue, B.P.A. and Broekaert, W.F. 1998. Separate jasmonate-dependent and salicylate-dependent defense-response pathways in Arabidopsis are essential for resistance to distinct microbial pathogens. Proc. Natl. Acad. Sci. USA 95: 15107–15111.

    Google Scholar 

  • Van Parijs, J., Broekaert, W. F., Goldstein, I. J. and Peumans W. J. 1991. Hevein: an antifungal protein from rubber-tree (Hevea brasiliensis) latex. Planta 183: 258–264.

    Google Scholar 

  • Vijayan, P., Shockey, J., Levesque, C.A., Cook, R.J. and Browse, J. 1998. A role for jasmonate in pathogen defense of Arabidopsis. Proc. Natl. Acad. Sci. 95: 7209–7214.

    Google Scholar 

  • Wirth, S. and Wolf, G.A. 1990. Dye-labeled substrates for the assay and detection of chitinase and lysozyme activity. J. Microbiol. Meth. 12: 197–205.

    Google Scholar 

  • Wright, H.T., Sandrasegaram, G. and Wright, C.S. 1991. Evolution of a family of N-acetylglucosamine-binding proteins containing the disulfide-rich domain of wheat germ agglutinin. J.Mol. Evol. 33: 283–294.

    Google Scholar 

  • Wu, S., Kriz, A.L. and Widholm, J.M. 1994. Molecular analysis of two cDNA clones encoding acidic class I chitinase in maize. Plant Physiol. 105: 1097–1105.

    Google Scholar 

  • Zhao, K.J. and Chye, M.L. 1999. Methyl jasmonate induces expression of a novel Brassica juncea chitinase with two chitin-binding domains. Plant Mol. Biol. 40: 1009–1018.

    Google Scholar 

  • Zhu, Q., Maher, E.A., Masoud, S., Dixon, R.A. and Lamb, C.J. 1994. Enhanced protection against fungal attack by constitutive co-expression of chitinase and glucanase genes in trangenic tobacco. Bio/technology 12: 807–812.

    Google Scholar 

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Author notes

  1. Kai-Jun Zhao

    Present address: Chinese Academy of Agricultural Sciences, Institute of Crop Breeding and Cultivation, Zhongguancun South Street, Beijing , 100081, China

Authors and Affiliations

  1. Department of Botany, University of Hong Kong, Pokfulam Road, Hong Kong, China

    King-Leung Fung & Mee-Len Chye

  2. Biotechnology Research Center, Zhongshan University, Guangzhou , 510275, China

    Zhu-Mei He

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  1. King-Leung Fung
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Fung, KL., Zhao, KJ., He, ZM. et al. Tobacco-expressed Brassica juncea chitinase BjCHI1 shows antifungal activity in vitro . Plant Mol Biol 50, 283–294 (2002). https://doi.org/10.1023/A:1016067200148

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