Chitinase, DNA coding therefor and plants containing same

Details Chitinase, DNA coding therefor and plants containing same Chitinase, DNA coding therefor and plants containing same

1996-02-15

1999-11-30

Prouty, Rebecca E.

Drug, bio-affecting and body treating compositions

Enzyme or coenzyme containing

Hydrolases

435200, 435209, A61K 3847, C12N 924, C12N 942

Patent

active

059938080

DESCRIPTION:

BRIEF SUMMARY
FIELD OF THE INVENTION

The present invention relates to chitinases from plant origin, DNA coding therefor, and plants which have been transformed to contain and express said recombinant polynucleotides to produce or overproduce said chitinase. The invention further provides methods to protect plants from pathogen attack, as well as plants obtainable thereby. The invention further comprises antipathogenic compositions containing as active ingredient a chitinase according to the invention.


STATE OF THE ART

Plants react to stresses such as attack by pathogens, chemical treatment and wounding, by accumulating a large number of proteins. One of the best studied plant responses is the induced expression of so-called pathogenesis-related proteins, PR-proteins, (Bowles, D. 1990, Annu. Rev. Biochem. 59, 873-907); Linthorst, H. J. M., 1991, Crit. Rev. Plant Sci. 10, 123-150).
PR-proteins can be classified into at least five families (PR-1 to PR-5) based on structure and/or activity. In tobacco, within each family of PR-proteins both intracellular (class-I) and extracellular (Class-II) isoforms are distinguished.
Among the PR-proteins are chitinases, which constitute the PR-3 family (Legrand M., et al, 1987, Proc. Natl. Acad. Sci. USA, 84, 6750-6754). The class-I chitinases possess antifungal activity (Broekaert W. F. et al, 1988, Physiol. Mol. Plant Pathol. 33, 319-331), especially in combination with .beta.-1,3-glucanase (Schlumbaum A. et al, 1986, Nature 324, 365-367), classified as the PR-2 family (Kauffmann S. et al, 1987, EMBO J. 6, 3209-3212).
The occurrence of chitinases and the cloning of their corresponding cDNAs has been described for a large number of plant species, both dicotyledonous and monocotyledonous species (for a recent review, vide Collinge D. B. et al, 1993, The Plant Journal 3(1), 31-40). Chitinases are classified according to their primary structure into three classes (Shinshi H. et al, 1990, Plant Mol. Biol. 14, 357-368). Recently chitinases have been found that do not fit into this classification: these chitinases have been proposed to form class-IV. The class I chitinases are characterised by the presence of an approximately 40 amino acid cysteine rich domain.
The class-II chitinases resemble the class-I chitinases, inter alia by their amino acid sequence homology, but the former lack a cystein-rich domain.
The class-III chitinases do not share sequence similarity with chitinases of either of the classes I or II. The class-IV chitinases, as proposed by Collinge et al. supra, comprise sugar beet chitinase IV (Mikkelsen J. D. et al, 1992, In Advances in Chitin and Chitosan (Brine, C. J. Sandford, P. A. and Zikakis, J. P. eds. Amsterdam Elsevier, in press) and the basic rape chitinase ChB4 (Rasmussen U. et al, 1992, Planta, 187, 328-334), as well as the acidic bean PR4 chitinase (Margis-Pinheiro M. et al, 1991, Plant. Mol. Biol. 17, 247-253), show sequence similarity with the class I chitinases, contain a N-terminal cystein-rich domain, but are smaller than the class-I chitinases due to a number of deletions. The molecular weights of plant chitinases range on average from about 26 to about 36 kDa.
By cloning the genes coding for PR-proteins, altering their expression patterns by placing the genes under the control of non-natural, e.g. consitutive, regulatory elements, and reintroducing those constructs in plants, it is possible to reduce the susceptibility of plants to pathogen-attack, in particular fungal attack (European patent application 0 440 304 A1; EP 0 460 753). Simultaneous expression in transgenic plants of class-I chitinases and .beta.-1,3-glucanases produces a marked enhancement of fungal resistance in transgenic plants, showing the value of combining antipathogenic proteins.
There is a continuous need for the identification of new chitinases for use in antifungal compositions and/or for the cloning of cDNAs or genes encoding such chitinases, to study the effect of expressing these cDNAs or genes in transgenic plants and evaluate the susceptibility of said plants to pathogenic

REFERENCES:
C.G. Bae et al. "Purification and Characterization of Two Acidic Chitinases Having and Lacking N-Terminal Cysteine-Rich Domain From Root of Rice (Oryza sativa L.)", Mol. Cells 3(3), 1993.
A. Elghaouth et al., "Glucanohydrolases and Inhibitory Activity to Botrytis-cinerea in Extracts From Strawberry Fruits", Can. J. Plant Pathology 13(4): 315-320, Dec. 1991.
E. Delcampillo et al. "Identification and Kinetics of Accumulation of Proteins Induced by Ethylene in Bean Abscission Zones", Plant Pathology 98(3): 955-961, Mar. 1992.
Fritig, B. et al. "Virus Induced Glycanhydrolases . . . " NATO ASI Series, vol. H.36, Signal Molecules in Plants & Plant-Microbe Interactions, Springer-Verlag Berlin, Heidelberg 1989.
Melchers, L.S. "A new class of tobacco . . . ".
van Huijsduijenen, R.A.M. et al. "cDNA cloning of six . . . " EMBO Journal, vol. 5, No. 9, pp. 2057-2061, 1986.
Schuler, et al Proteins: Structure, Function and Genetics 9 180-190 (1991) A Workbench for Multiple Alignment Construction and Analysis.
van Loon, et al, Plant Molecular Biology Reporter 12 (3) 245-264 (1994) Genetic Resources: Recommendations for Naming Plant Pathogenesis-Related Proteins.
Bol, J.F. "Structure and Expression of Plant . . . " Chapter 11, pp. 201-221.
Heitz, T. et al. "Molecular Characterization . . . " Mol. Gen. Genet (1994) Springer Verlag 1994.

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