Antifungal preparations, and process for making such preparation

Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai

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514 2, 530370, A61K 3578

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053896092

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BRIEF SUMMARY
TECHNICAL FIELD

The invention is related to inhibition of fungal growth using proteins that are isolated from plants. The invention provides processes for the preparation of such proteins in active form, as well as functional formulations thereof.
The invention also comprises plants with reduced susceptibility to fungi, characterized in that a gene or genes encoding such a protein are expressed constitutively or in a specifically regulated fashion in one or more parts of the plant, as well as processes for obtaining such plants.


BACKGROUND OF THE ART

Plant proteins with antifungal activity are known. A chitinase purified from bean causes inhibition of the growth of the fungus Trichoderma viride (Schlumbaum et el., (1986), Nature 324, 365-367).
A pea chitinase with a growth inhibitory effect on Trichoderma viride in agar plate tests is described by Mauch et al., (1988, Plant Physiol. 88, 936-942). This enzyme, however, only has a limited effect on for instance the ascomycete Cladosporium cucumerinum, and no effect on the growth of inter alia the Oomycetes Phytophthora cactorum, Pythium apahanidermatum, and Pythium ultimum. Hence, an important disadvantage of this enzyme is its limited working range. In a similar test it was established that .beta.-1,3-glucanase has a growth inhibitory effect on Fusarium solani f.sp. pisi.
A preparation with a hydrolytic effect on isolated cell walls of Verticillium alboatrum, comprising a combination of a purified endo-.beta.-1,3-glucanase from tomato and an exo-.beta.-1,3-glucanase from fungal origin is described by Young & Pegg (1982, Physiol. Plant Pathol. 21, 411-423). Both enzymes had no effect on their own.
Several thionines, inter alia from leaves of barley, maize, wheat, rye, and several dicotyledonous plants, showing a significant antifungal effect in in vitro tests, are described by Bohlmann, H. et al., (1988, EMBO J. 7, 1559-1565).
Furthermore, plant proteins with an enhancing effect on the fungus-inhibitory activity of antibiotics are described in International Patent Application PCT/US88/03420. These plant proteins are generally designated as Synergistic Antifungal Proteins or SAFPs. SAFPs are used in combination with polyoxines and nikkomycines, that are active on their own against phytopathogenic fungi; in combination with SAFPs improvements of the effectivity can be achieved in the order of 10 to 100. SAFPs have no antifungal effect on their own.
In plants, the synthesis of chitinases and glucanases, as well as a large number of different so-called pathogenesis-related (PR-) proteins, is known to be accompanied by a phenomenon known as the hypersensitive response, which is inter alia triggered by an incompatible plant pathogen. This hypersensitive response eventually results in resistance of the plant against a broad range of pathogens. Similarly, the synthesis of PR-proteins can be induced by a number of biotic and abiotic factors, such as fragments of fungal cell walls, chemical inducers, such as salicylate and the like, which also results in a broad pathogen-resistance of the plant. This resistance obtained through induction either by an incompatible pathogen or a biotic or abiotic factor, or chemical substance, is called `induced resistance`. Although still very much has to be learned about induced resistance and the role of these PR-proteins, some classification has been done. In tobacco, it seems that at least 5 classes of PR-proteins are induced upon treatment with tobacco mosaic virus (TMV). This classification is based on features such as molecular weight, serological relationship, amino-acid sequence homology, and if known, enzymatic activity. Within these classes a division can be made into intracellular and extracellular proteins, which except for their cellular localisation in the plant, correspond to each other with respect to the features just mentioned (vide for overview, Bol J. F. et al., 1990, Annu. Rev. Phytopathol. 28, 113-138.). Since these proteins are believed to be somehow involved in pathogen resistance, a great deal of effort is

REFERENCES:
Fischer, Willi et al., Physiological and Molecular Plant Pathology, vol. 35, pp. 67-83, 1989.
Grosset, Jean et al., Plant Physiol., vol. 92, pp. 520-527, 1990.
Takeda, Satomi et al., Plant Cell Physiol., vol. 31 (2), pp. 215-221, 1990.
Neale, Alan et al., The Plant Cell, vol. 2, pp. 673-684 (1990 Jul.).
Richardson, Michael et al., Nature, vol. 327, pp. 432-434, 1987.
Mirelman, David et al, Nature, vol. 256, pp. 414-416, 1975.
Singh, Narendra et al., Plant Physiology, vol. 85, pp. 529-536, 1987.
King, Gretchen et al, Plant Molecular Biology, vol. 10, pp. 401-412 (1988).
Journal of Cellular . . . Biology, Suppl. 14E, 16th-22nd Apr. 1990, p. 276, Abstract No. R 108: R. Broglie et al.: "Chitinase . . . Pathogen".
Proc. Natl. Acad. Sci. USA, vol. 86, Dec. 1989, pp. 9871-9875: Johnson et al.: "Expression of Proteinase Inhibitors I and III . . . Larvae".
The Embo Journal, vol. 7, No. 6, Jun. 1988, pp. 1559-1565, IRL Press LTT Oxford, GM: Bohlmann et al.: "Leaf-Specific Thionins . . . Plants".
Plant Molecular Biology, vol. 9, 1987, pp. 389-410, Martinus Nijhoff Publishers, Dordrecht, NL: D. B. Collinge et al.: "Plant . . . Pathogens".
Plant Cell Physiol., vol. 31, No. 2, 1990, pp. 215-221: S Takeda et al.: "Characterization of Polypeptides that . . . Cells".
Physiological Plant Pathology, vol. 15, No. 3, Nov. 1979, pp. 321-330, Academic Press Inc., London, GB: McIntyre et al.: "Induction of . . . Virus".
Plant Physiol., vol. 90, Jul. 1989, pp. 1096-1011: N. K. Singh et al.: "Molecular Cloning . . . Potential" *The Whole Article*.
Biochimica et Biophysica Acta, vol. 880, 1986, pp. 161-170, Elsevier Science Publishers B.V. (Biomedical Div): Roberts et al.: "Isolation . . . Barley".
Physiological and Molecular Plant Pathology, vol. 35, 1989, pp. 53-65, Acad. Press Ltd: Christ et al.: "Pathogenesis-Related . . . Resistance".
Nature, vol. 324, No. 6095, 27th Nov. 1986, pp. 365-367, Neptune, N.J., US A. Schlumbaum et al.: "Plant Chitinases are Potent Inhibitors . . . Growth".
Chemical Abstracts, vol. 110, 1989, p. 451, Abstract No. 4848V, F. Mauch et al.: "Antifungal Hydrolases in Pea Tissue. II . . . Glucanase".

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