Microbial diseases of plants are a significant problem to the agricultural and horticultural industries. Plant diseases in general cause millions of tonnes of crop losses annually with fungal and bacterial diseases responsible for significant portions of these losses. One possible way of combating fungal and bacterial diseases is to provide transgenic plants capable of expressing a protein or proteins which in some way increase the resistance of the plant to pathogen attack. A simple strategy is to first identify a protein with antimicrobial activity in vitro, to clone or synthesise the DNA sequence encoding the protein, to make a chimaeric gene construct for efficient expression of the protein in plants, to transfer this gene to transgenic plants and to assess the effect of the introduced gene on resistance to microbial pathogens by comparison with control plants.
The first and most important step in the strategy for disease control described above is to identify, characterise and describe a protein with strong antimicrobial activity. In recent years, many different plant proteins with antimicrobial and/or antifungal activity have been identified and described. These proteins have been categorised into several classes according to either their presumed mode of action and/or their amino acid sequence homologies. These classes include the following: chitinases (Roberts, W. K. et al. [1986] Biochim. Biophys. Acta 880:161–170); β-1,3-glucanases (Manners, J. D. et al. [1973] Phytochemistry 12:547–553); thionins (Bolmann, H. et al. [1988] EMBO J. 7:1559–1565 and Fernadez de Caleya, R. et al. [1972] Appl. Microbiol. 23:998–1000); permatins (Roberts, W. K. et al. [1990] J. Gen. Microbiol. 136:1771–1778 and Vigers, A. J. et al. [1991] Mol. Plant-Microbe Interact. 4:315–323); ribosome-inactivating proteins (Roberts, W. K. et al. [1986] Biochim. Biophys. Acta 880:161–170 and Leah, R. et al. [1991] J. Biol. Chem. 266:1564–1573); plant defensins (Terras, F. R. G. et al. [1995] The Plant Cell 7:573–588); chitin binding proteins (De Bolle, M. F. C. et al. [1992] Plant Mol. Biol. 22:1187–1190 and Van Parijs, J. et al. [1991] Planta 183:258–264); thaumatin-like, or osmotin-like proteins (Woloshuk, C. P. et al. [1991] The Plant Cell 3:619–628 and Hejgaard, J. [1991] FEBS Letts. 291:127–131); PR1-typ proteins (Niderman, T. et al. [1995] Plant Physiol. 108:17–27.) and the non-specific lipid transfer proteins (Terras, F. R. G. et al. [1992] Plant Physiol. 100:1055–1058 and Molina, A. et al. [1993] FEBS Letts. 3166:119–122). Another class of antimicrobial proteins from plants is the knottin or knottin-like antimicrobial proteins (Cammue, B. P. A. et al. [1992] J. Biol. Chem. 67:2228–2233; Broekaert W. F. et al. (1997) Crit. Rev. in Plant Sci. 16(3):297–323). A class of antimicrobial proteins termed 4-cysteine proteins has also been reported in the literature which class includes Maize Basic Protein (MBP-1) (Duvick, J. P. et al. [1992] J. Biol. Chem. 267:18114–18120). A novel antimicrobial protein which does not fit into any previously described class of antimicrobial proteins has also been isolated from the seeds of Macadamia integrifolia termed MiAMP1 (Marcus, J. P. et al. [1997] Eur. J. Biochem. 244:743–749). In addition, plants are not the sole source of antimicrobial proteins and there are many reports of the isolation of antimicrobial proteins from animal and microbial cells (reviewed in Gabay, J. E. [1994] Science 264:373–374 and in “Antimicrobial peptides” [1994] CIBA Foundation Symposium 186, John Wiley and Sons Publ., Chichester, UK).
There is evidence that the ectopic expression of genes encoding proteins that have in vitro antimicrobial activity in transgenic plants can result in increased resistance to microbial pathogens. Examples of this engineered resistance include transgenic plants expressing genes encoding: a plant chitinase, either alone (Broglie, K. et al. [1991] Science 254:1194–1197) or in combination with a β-1,3-glucanase (Van den Elzen, P. J. M. et al. [1993] Phil. Trans. Roy. Soc. 342:271–278); a plant defensin (Terras, F. R. G. et al. [1995] The Plant Cell 7:573–588); an osmotin-like protein (Liu, D. et al. [1994] Proc. Natl. Acad. Sci. USA 91:1888–1892); a PR1-class protein (Alexander, D. et al. [1993] Proc. Natl. Acad. Sci. USA 90:7327–7331) and a ribosome-inactivating protein (Logemann, J. et al. [1992] Bio/Technology 10:305–308).
Although the potential use of antimicrobial proteins for engineering disease resistance in transgenic plants has been described extensively, there are other applications which are worthy of mention. Firstly, highly potent antimicrobial proteins can be used for the control of plant disease by direct application (De Bolle, M. F. C. et al. [1993] in Mechanisms of Plant Defence Responses, B. Fritig and M. Legrand eds., Kluwer Acad. Publ., Dordrecht, N L, pp. 433–436). In addition, antimicrobial peptides have potential therapeutic applications in human and veterinary medicine. Although this has not been described for peptides of plant origin it is being actively explored with peptides from animals and has reached clinical trials (Jacob, L. and Zasloff, M. [1994] in “Antimicrobial Peptides”, CIBA Foundation Symposium 186, John Wiley and Sons Publ., Chichester, UK, pp. 197–223).
Antimicrobial proteins exhibit a variety of three-dimensional structures which will determine in large part the activity which they manifest. Many of the global structures exhibited by these proteins have been determined (Broekaert W. F. et al. (1997) Crit. Rev. in Plant Sci. 16(3):297–323). A large factor in determining the stability of these proteins is the presence of disulfide bridges between various cysteines located in α-helical and β-sheet regions. Many peptides with toxic activity such as conotoxin are well known to be stabilized by disulfide bridges (see for example Hill, J. M. et al. (1996) Biochemistry 35(27): 8824–8835). In the case of the conotoxin referenced above, a compact structure is formed consisting of a helix, a small-hairpin, a cis-hydroxyproline, and several turns. The molecule is stabilized by three disulfide bonds, two of which connect the α-helix and the β-sheet, forming a solid structural core. Interestingly, eight arginine and lysine side chains in this molecule project into the solvent in a radial orientation relative to the core of the molecule. These cationic side chains form potential sites of interaction with anionic sites on pathogen membranes (Hill, J. M. et al. supra).
The invention described herein constitutes previously undiscovered and thus novel proteins with antimicrobial activity. These proteins can be isolated from Macadamia integrifolia (Mi) seeds or from cotton or cocoa seeds. In addition, protein fragments which are antifungal can be derived from larger seed storage proteins containing regions of substantial similarity to the antimicrobial proteins from macadamia described here. Examples of seed storage proteins which contain regions similar to the proteins which have been purified can be seen in FIG. 4. Macadamia integrifolia belongs to the family Proteaceae. M. integrifolia, also known as Bauple Nut or Queensland Nut, is considered by some to be the world's best edible nut. Cotton (Gossypium hirsutum) belongs to the family Malvaceae and is cultivated extensively for its fiber. Cocoa (Threobroma cacao) belongs to the family Sterculiaceae and is used around the world for a wide variety of cocoa products.
The fact that both the macadamia and cocoa antimicrobial proteins are found in edible portions of these plants makes these peptides attractive for use in genetic engineering for disease resistance since trangenic plants expressing these proteins are unlikely to show added toxicity. Proteins may also be safe for human and veterinary use.