AU723474B2 - Antimicrobial proteins - Google Patents

Description

WO 98/27805 PCT/AU97/00874 ANTIMICROBIAL PROTEINS TECHNICAL FIELD This invention relates to isolated proteins which exert inhibitory activity on the growth of fungi and bacteria, which fungi and bacteria include some microbial pathogens of plants and animals.

The invention also relates to recombinant genes which include sequences encoding the proteins, the expression products of which recombinant genes can contribute to plant cells or cells of other organism's defence against invasion by microbial pathogens. The invention further relates to the use of the proteins and/or genes encoding the proteins for the control of microbes in human and veterinary clinical conditions.

BACKGROUND ART 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. etal. [1986] Biochim. Biophys. Acta 880:161-170); 3-1,3glucanases (Manners, J.D. et al. [1973] Phytochemistry 12:547-553); thionins (Bolmann, H. et al.

[1988] EMBO J. 7:1559-1565 and Feradez 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] FEBSLetts. 291:127-131); PRI-type WO 98/27805 PCT/AU97/00874 2 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] FEBSLetts. 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, Jolm 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 P- 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 PRI -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, NL, 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 WO 98/27805 PCT/AU97/00874 3 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 a-helical and P-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 ao-helix and the P-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 Figure 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.

SUMMARY OF THE INVENTION According to a first embodiment of the invention, there is provided a protein fragment having antimicrobial activity, wherein said protein fragment is selected from: a polypeptide having an amino acid sequence selected from: residues 29 to 73 of SEQ ID NO: 1 residues 74 to 116 of SEQ ID NO: 1 residues 117 to 185 of SEQ ID NO: 1 residues 186 to 248 of SEQ ID NO: 1 residues 29 to 73 of SEQ ID NO: 3 WO 98/27805 PCT/AU97/00874 4 residues 74 to 116 of SEQ ID NO: 3 residues 117 to 185 of SEQ ID NO: 3 residues 186 to 248 of SEQ ID NO: 3 residues 1 to 32 of SEQ ID NO: residues 33 to 75 of SEQ ID NO: residues 76 to 144 of SEQ ID NO: residues 145 to 210 of SEQ ID NO: residues 34 to 80 of SEQ ID NO: 7 residues 81 to 140 of SEQ ID NO: 7 residues 33 to 79 of SEQ ID NO: 8 residues 80 to 119 of SEQ ID NO: 8 residues 120 to 161 of SEQ ID NO: 8 residues 32 to 91 of SEQ ID NO: 21 residues 25 to 84 of SEQ ID NO: 22 residues 29 to 94 of SEQ ID NO: 24 residues 31 to 85 of SEQ ID NO: residues 1 to 23 of SEQ ID NO: 26 residues 1 to 17 of SEQ ID NO: 27 residues I to 28 of SEQ ID NO: 28; (ii) a homologue of (iii) a polypeptide containing a relative cysteine spacing of C-2X-C-3X-C-( 10-12)X-C-3X- C-3X-C wherein X is any amino acid residue, and C is cysteine; (iv) a polypeptide containing a relative cysteine and tyrosine/phenylalanine spacing of Z- 2X-C-3X-C-(10-12)X-C-3X-C-3X-Z wherein X is any amino acid residue, and C is cysteine, and Z is tyrosine or phenylalanine; a polypeptide containing a relative cysteine spacing of C-3X-C-( 10-12)X-C-3X-C wherein X is any amino acid residue, and C is cysteine; (vi) a polypeptide with substantially the same spacing of positively charged residues relative to the spacing of cysteine residues as and (vii) a fragment of the polypeptide of any one of(i) to (vi) which has substantially the same antimicrobial activity as According to a second embodiment of the invention, there is provided a protein containing at least one polypeptide fragment according to the first embodiment, wherein said polypeptide fragment WO 98/27805 PCT/AU97/00874 has a sequence selected from within a sequence comprising SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: According to a third embodiment of the invention, there is provided a protein having a sequence selected from SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: According to a fourth embodiment of the invention, there is provided an isolated or synthetic DNA encoding a protein according to the first embodiment According to a fifth embodiment of the invention, there is provided a DNA construct which includes a DNA according to the fourth embodiment operatively linked to elements for the expression of said encoded protein.

According to a sixth embodiment of the invention, there is provided a transgenic plant harbouring a DNA construct according to the fifth embodiment.

According to a seventh embodiment of the invention, there is provided reproductive material of a transgenic plant according to the sixth embodiment.

According to an eighth embodiment of the invention, there is provided a composition comprising an antimicrobial protein according to the first embodiment together with an agriculturally-acceptable carrier diluent or excipient.

According to a ninth embodiment of the invention, there is provided a composition comprising an antimicrobial protein according to the first embodiment together with an pharmaceuticallyacceptable carrier diluent or excipient.

According to a tenth embodiment of the invention, there is provided a method of controlling microbial infestation of a plant, the method comprising: i) treating said plant with an antimicrobial protein according to the first embodiment or a composition according to the eighth embodiment; or ii) introducing a DNA construct according to the fifth embodiment into said plant.

According to an eleventh embodiment of the invention, there is provided a method of controlling microbial infestation of a mammalian animal, the method comprising treating the animal with an antimicrobial protein according to the first embodiment or a composition according to the ninth embodiment.

According to a twelfth embodiment of the invention, there is provided a method of preparing an antimicrobial protein, which method comprises the steps of: a) obtaining or designing an amino acid sequence which forms a helix-turn-helix structure; b) replacing individual residues to achieve substantially the same distribution of positively charged residues and cysteine residues as in one or more of the amino acid sequences shown in Figure 4; WO 98/27805 PCT/AU97/00874 6 c) synthesising a protein comprising said amino acid sequence chemically or by recombinant DNA techniques in liquid culture; and d) if necessary, forming disulphide linkages between said cysteine residues.

Other embodiments of the invention include methods for producing antimicrobial protein.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the results of cation-exchange chromatography of the basic protein fraction of a Macadamia integrifolia extract with the results of a bioassay for antimicrobial activity shown for fractions in the region of MiAMP2c elution.

Figure 2 shows the results of including 1 mM Ca 2 in a parallel bioassay of fractions from the cation-exchange separation.

Figure 3 shows a reverse-phase HPLC profile of highly inhibitory fractions containing MiAMP2c from the cation-exchange separation in Figure 1 and 2 together with growth inhibition exhibited by the HPLC fractions.

Figure 4 shows the amino acid sequences of MiAMP2a, b, c and d and protein fragments derived from other seed storage proteins which contain regions of homology to the MiAMP2 series of antimicrobial proteins.

Figure 5 shows an example of a synthetic nucleotide sequence which can be used for the expression and secretion of MiAMP2c in transgenic plants.

Figure 6 shows the alignment of clones 1-3 from macadamia containing MiAMP2a, b, c and d subunits together with sequences from cocoa and cotton vicilin seed storage proteins which exhibit significant homology to the macadamia clones.

Figure 7 displays a series of secondary structure predictions for MiAMP2c.

Figure 8 shows a three-dimensional model of the MiAMP2c protein.

Figure 9 shows stained SDS-PAGE gels of protein fractions at various stages in the expression and purification of TcAMPI(Theobroma cacao subunit MiAMP2a, MiAMP2b, MiAMP2c and MiAMP2d expressed in E.coli liquid culture.

Figure 10 shows the reverse-phase HPLC purification of cocoa subunit 2 (TcAMP2) after the initial purification step using Ni-NTA media.

Figure 11 shows a western blot of crude protein extracts from various plant species using rabbit antiserum raised to MiAMP2c.

Figure 12 shows a cation-exchange fractionation of the Stenocarpus sinuatus basic protein fraction along with the accompanying western blot which shows the presence of immunologicallyrelated proteins in a range of fractions.

WO 98/27805 PCT/AU97/00874 7 Figure 13 shows a reverse-phase HPLC separation of Stenocarpus sinuatus cation-exchange fractions which had previously reacted with MiAMP2c antibodies (see Figure 14). A western blot is also presented which reveals the presence of putative MiAMP2c homologues in individual HPLC fractions.

Figure 14 is a map of the binary vector pPCV91 -MiAMP2c as an example of a vector that can be used to express these antimicrobial proteins in transgenic plants.

Figure 15 shows a western blot to detect MiAMP2c expressed in transgenic tobacco plants.

BEST MODE AND OTHER MODES FOR CARRYING OUT THE INVENTION The following abbreviations are used hereafter: EDTA ethylenediaminetetraacetic acid IPTG Isopropyl-p-D-thiogalactopyranoside MeCN methyl cyanide (acetonitrile) Mi Macadamia integrifolia MiAMP2 Macadamia integrifolia antimicrobial protein series number 2 Ni-NTA Nickel-nitrilotriacetic acid chromatography media ND not determined PCR polymerase chain reaction PMSF phenylmethylsulphonyl fluoride SDS-PAGE sodium-dodecylsulphate polyacrylamide gel electrophoresis TFA trifluoroacetate The term homologue is used herein to denote any polypeptide having substantial similarity in composition and sequence to the polypeptide used as the reference. The homologue of a reference polypeptide will contain key elements such as cysteine or other residues spaced at identical intervals such that a substantially similar three-dimensional global structure is adopted by the homologue as compared to the reference. The homologue will also exhibit substantially the same antimicrobial activity as the reference protein.

The present inventors have identified a new class of proteins with antimicrobial activity.

Prototype proteins can be isolated from seeds of Macadamia integrifolia. The invention thus provides antimicrobial proteins per se and also DNA sequences encoding these antimicrobial proteins.

The invention also provides amino acid sequences of proteins which are homologous to the prototype antimicrobial proteins from Macadamia integrifolia. Thus, in addition to the antimicrobial proteins from Macadamia, this invention also provides amino acid sequences of homologues from other species which have hitherto been unrecognized as having antimicrobial activity.

WO 98/27805 PCT/AU97/00874 8 While the first antimicrobial protein in the present series was isolated directly from Macadamia integrifolia, additional antimicrobial proteins were identified through cloning efforts, homology searches and subsequent antimicrobial testing of the encoded proteins after expression in and purification from liquid culture. After the first protein from this series was purified from macadamia and termed MiAMP2, clones were obtained which encoded a preproprotein containing MiAMP2. This large protein (666 amino acids), represented by several almost identical clones, contained four adjacent regions with significant similarity to the purified antimicrobial protein fragment (MiAMP2) which itself was found to lie within region three in the cloned nucleotide sequence; hence the purified antimicrobial protein is termed MiAMP2c. Other fragments contained in the 666-amino-acid clone are termed MiAMP2a, b and d as per their locations in the cloned nucleotide sequence. Several other sequences with significant homology to the MiAMP2a, b, c, and d protein fragments were then identifed in the Entrez data base. These homologous sequences were contained within larger seed storage proteins from cotton and cocoa which sequences had not been previously described as containing antimicrobial protein sequences or as exhibiting antimicrobial activity. Fragments of larger seed storage proteins containing sequences homologous to MiAMP2c were tested and are here demonstrated to exhibit antimicrobial activity. Thus, the inventors have established a process for obtaining antimicrobial protein fragments from larger seed storage proteins.

In the light of these findings, it is evident that fragments of other seed storage proteins containing sequences similar to the proteins described will also exhibit antimicrobial activity.

In particular, the 47-amino-acid TcAMP1 (for Theobroma cacao antimicrobial protein 1) and the 60-amino-acid TcAMP2 sequences were derived from a cocoa vicilin seed storage protein gene sequence (which contains 525 amino acids) (Spencer, M.E. and Hodge R. [1992] Planta 186:567- 576). These derived fragments were then expressed in liquid culture. Cocoa vicilin fragments thus expressed and subsequently purified (Examples 10 and 11), were shown to be antimicrobial (Example 15). This is the first report that fragments of the cocoa vicilin protein possess antimicrobial activity. Pools of sequences containing fragments homologous to the MiAMP2c apparently released from cotton vicilin seed storage protein have been shown to possess antimicrobial activity (Chung, R. P.T. et al. [1997] Plant Science 127:1-16). This finding is clearly embodied in sequences disclosed in this application.

In addition to showing that cocoa-vicilin-derived fragments exhibit antimicrobial activity, there is herein described additional proteins which exhibit antimicrobial activity. For example, there is described below proteins from Stenocarpus sinuatus which are of similar size to MiAMP2 subunits, react with MiAMP2c antiserum, and contain sequences homologous to MiAMP2 proteins (see Figure Based on the evidence provided herein, sequences homologous to the MiAMP2c WO 98/27805 PCT/AU97/00874 9 subunit MiAMP2a, b, d; TcAMP1; TcAMP2; and cotton fragments 1, 2 and 3-see Figure 4) constitute proteins which contain the fragment with antimicrobial activity. The antimicrobial activity of MiAMP2 fragments from macadamia, and the TcAMP and 2 fragments from cocoa, is exemplified below. R. P. T. Chung et al. (Plant Science 127:1-16 [1997]) have demonstrated that the cotton fragments exhibit antimicrobial activity. Other antimicrobial proteins can also be derived from seed storage proteins such as peanut allergen Ara h (Burks, A.W. et al. [1995] J Clin. Invest.

96 1715-1721), maize globulin (Belanger, F. C. and Kriz, A. L.[1991] Genetics 129 863- 872), barley globulin (Heck, G. R. et al. [1993] Mol. Gen. Genet. 239 209-218), and soybean conglycinin (Sebastiani, F. L. et al. [1990] Plant Mol. Biol. 15 197-201), all of which contain the same key elements which are present in the sequences which are here shown to exhibit antimicrobial activity.

The proteins which contain regions of sequence homologous to MiAMP2 (as in Figure 4) can be used to construct nucleotide sequences encoding 1) the active fragments of larger proteins, or 2) fusions of multiple antimicrobial fragments. This can be done using standard codon tables and cloning methods as described in laboratory manuals such as Current Protocols in Molecular Biology (copyright 1987-1995 edited by Ausubel F. M. et aL and published by John Wiley Sons, Inc., printed in the USA). Subsequently, these can be expressed in liquid culture for purification and testing, or the sequences can be expressed in transgenic plants after placing them in appropriate expression vectors.

The antimicrobial proteins per se will manifest a particular three-dimensional structure which may be determined using X-ray crystallography or nuclear magnetic resonance techniques. This structure will be responsible in large part for the antimicrobial activity of the protein. The sequence of the protein can also be subjected to structure prediction algorithms to assess whether any secondary structure elements are likely to be exhibited by the protein (see Example 8 and Figure 7).

Secondary structures, thus predicted, can then be used to model three-dimensional global structures.

Although three-dimensional structure prediction is not feasible for most proteins, the secondary structure predictions for MiAMP2c were sufficiently simple and clear that a three-dimensional model structure has been obtained for the MiAMP2c protein. Homologues exhibiting the same cysteine spacing and other key elements will also adopt the same three-dimensional structure.

Example 8 shows that the structure most likely to be adopted by MiAMP2c (and homologues) is a helix-turn-helix structure stabilised by at least two disulfide bridges connecting the two antiparallel a-helical segments (see Figure Additional stabilisation can be provided by an extra disulfide bridge as in MiAMP2b) or by a hydrophobic ring-stacking interaction between tyrosine and/or phenylalanine residues MiAMP2a and MiAMP2c), each located on the same face of the a- WO 98/27805 PCT/AU97/00874 helical segments as the normally present cysteine residues which participate in the 2 disulfide linkages mentioned above. NMR signals exhibited by MiAMP2c are consistent with the threedimensional global model produced from the secondary-structure predictions mentioned above.

It will be appreciated that one skilled in the art could take a protein with known structure, alter the sequence significantly, and yet retain the overall three-dimensional shape and antimicrobial activity of the protein. One aspect of the structure that most likely could not be altered without seriously affecting the structure (and, therefore, the activity of the protein) is the content and spacing of the cysteine residues since this would disrupt the formation of disulfide bonds which are critical to a) maintaining the overall structure of the protein and/or b) making the protein more resistant to denaturation and proteolysis (stabilizing the protein structure). In particular, it is essential that cysteine residues reside on one face of the helix in which they are contained. This can best be accomplished by maintaining a three-residue spacing between the cysteine residues within each helix, but, can also be accomplished with a two-residue interval between the cysteine residues provided the cysteines on the other helical segment are separated by three residues C-X-X-C-X- X-X-C-nX-C-X-X-X-C-X-X-X-C where C is cysteine, X is any amino acid, and n is the number of residues forming a turn between the two a-helical segments). Aromatic tyrosine (or phenylalanine) residues can also function to add stability to the protein structure if they are located on the same face of the helix as the cysteine side chains. This can be accomplished by providing appropriate spacing of two or three residues between the aromatic residue and the proximate cysteine residue Z-X- X-C-X-X-X-C-nX-C-X-X-X-C-X-X-X-Z where Z is tyrosine or phenylalanine).

The distribution of positive (and negative) charges on the various surfaces of the protein will also serve a critical role in determining the structure and activity of the protein. In particular, the distribution of positively-charged residues in an a-helical region of a protein can result in positive charges lying on one face of the helix or may result in the charged residues being concentrated in some particular portion of the molecule. An alternative distribution of positively charged residues is for them to project into the solvent in a radial orientation to the core of the protein. This orientation is predicted for several of the MiAMP2 homologues (data not shown). The spacing which is required for positioning of the residues on one face of the helix or the spacing required to accomplish a radial orientation from the core can easily be determined by one skilled in the art using a helical wheel plot with the sequence of interest. A helical wheel plot uses the fact that, in a-helices, each turn of the helix is composed of 3.6 residues on average. This number translates to 1000 of rotational translation per residue making it possible to construct a plot showing the distribution of side chains in a helical region. Figure 8 shows how the spacing of charged residues can lead to most of the WO 98/27805 PCT/AU97/00874 11 positively charged side chains being localised on one face of the helix. It will be appreciated by one of skill in the art that positive charges are conferred by arginine and lysine residues.

In order for the protein to develop into a helix-turn-helix structure, it is also necessary to have particular residues that favor ac-helix formation and that also favor a turn structure in the middle portion of the amino acid sequence (and disfavor a helical structure in the turn region). This can be accomplished by a proline residue or residues in the middle of the turn segment as seen with many of the MiAMP2 homologues. When proline is not present, glycine can also contribute to breaking a continuous helix structure, and inducing the formation of a turn at this position. In one case TcAMP it appears that serine may be taking on this role. It will be appreciated that the residues in this region of the protein will usually favor the fomation of a turn structure; residues which fulfill this requirement include proline, glycine, serine, and aspartic acid; but, other residues are also allowed.

The DNA sequences reported here are an extremely powerful tool which can be used to obtain homologous genes from other species. Using the DNA sequences, one skilled in the art can design and synthesise oligonucleotide probes which can be used to screen cDNA libraries from other species of plants for the presence of genes encoding antimicrobial proteins homologous to the ones described here. This would simply involve construction of a cDNA library and subsequent screening of the library using as the oligonucleotide probe one or part of one of the sequences reported here (such as sequence ID. No. 2 or the PCR fragment described in Example Other oligonucleotide sequences coding for proteins homologous to MiAMP2 can also be used for this purpose DNA sequences corresponding to cotton and cocoa vicilins). Making and screening of a cDNA library can be carried out by purchasing a kit for said purpose from Stratagene) or by following well established protocols described in available DNA cloning manuals (see Current Protocols in Molecular Biology, supra). It is relatively straight forward to construct libraries of various species and to specifically isolate vicilin homologues which are similar to the Macadamia, cotton, or cocoa vicilins by using a simple DNA hybridization technique to screen such libraries. Once cloned, these vicilin-related sequences can then be examined for the presence of MiAMP2-like subunits. Such subunits can easily be expressed in E. coli using the system described in Examples 10 and 11.

Subsequently, these proteins can also be expressed in transgenic.

Genes, or fragments thereof, under the control of a constitutive or inducible promoter, can then be cloned into a biological system which allows expression of the protein encoded thereby.

Transformation methods allowing for the protein to be expressed in a variety of systems are known.

The protein can thus be expressed in any suitable system for the purpose of producing the protein for further use. Suitable hosts for the expression of the protein include E. coli, fungal cells, insect cells, WO 98/27805 PCT/AU97/00874 12 mammalian cells, and plants. Standard methods for expressing proteins in such hosts are described in a variety of texts including section 16 (Protein Expression) of Current Protocols in Molecular Biology (supra).

Plant cells can be transformed with DNA constructs of the invention according to a variety of known methods (Agrobacterium, Ti plasmids, electroporation, micro-injections, micro-projectile gun, and the like). DNA sequences encoding the Macadamia integrifolia antimicrobial protein subunits fragments a, b, c, or d from the MiAMP2 clones) as well as DNA coding for other homologues can be used in conjunction with a DNA sequence encoding a preprotein from which the mature protein is produced. This preprotein can contain a native or synthetic signal peptide sequence which will target the protein to a particular cell compartment the apoplast or the vacuole).

These coding sequences can be ligated to a plant promoter sequence that will ensure strong expression in plant cells. This promoter sequence might ensure strong constitutive expression of the protein in most or all plant cells, it may be a promoter which ensures expression in specific tissues or cells that are susceptible to microbial infection and it may also be a promoter which ensures strong induction of expression during the infection process. These types of gene cassettes will also include a transcription termination and polyadenylation sequence 3' of the antimicrobial protein coding region to ensure efficient production and stabilisation of the mRNA encoding the antimicrobial proteins. It is possible that efficient expression of the antimicrobial proteins disclosed herein might be facilitated by inclusion of their individual DNA sequences into a sequence encoding a much larger protein which is processed in planta to produce one or more active MiAMP2-like fragments.

Gene cassettes encoding the MiAMP2 series antimicrobial proteins MiAMP2a, b, c, or d; or all of the subunits together; or the entire MiAMP2 clone) or homologues of the MiAMP2 proteins as described above can then be expressed in plant cells using two common methods. Firstly, the gene cassettes can be ligated into binary vectors carrying: i) left and right border sequences that flank the T-DNA of the Agrobacterium tumefaciens Ti plasmid; ii) a suitable selectable marker gene for the selection of antibiotic resistant plant cells; iii) origins of replication that function in either A.

tumefaciens or Escherichia coli; and iv) antibiotic resistance genes that allow selection of plasmidcarrying cells of A. tumefaciens and E. coli. This binary vector carrying the chimaeric MiAMP2 encoding gene can be introduced by either electroporation or triparental mating into A. tumefaciens strains carrying disarmed Ti plasmids such as strains LBA4404, GV3101, and AGL1 or into A.

rhizogenes strains such as A4 or NCCP1885. These Agrobacterium strains can then be co-cultivated with suitable plant explants or intact plant tissue and the transformed plant cells and/or regenerants selected using antibiotic resistance.

WO 98/27805 PCT/AU97/00874 13 A second method of gene transfer to plants can be achieved by direct insertion of the gene in target plant cells. For example, an MiAMP2-encoding gene cassette can be co-precipitated onto gold or tungsten particles along with a plasmid encoding a chimaeric gene for antibiotic resistance in plants. The tungsten particles can be accelerated using a fast flow of helium gas and the particles allowed to bombard a suitable plant tissue. This can be an embryogenic cell culture, a plant explant, a callus tissue or cell suspension or an intact meristem. Plants can be recovered using the antibiotic resistance gene for selection and antibodies used to detect plant cells expressing the MiAMP2 proteins or related fragments.

The expression of MiAMP2 proteins in the transgenic plants can be detected using either antibodies raised to the protein(s) or using antimicrobial bioassays. These and other related methods for the expression of MiAMP2 proteins or fragments thereof in plants are described in Plant Molecular Biology (2nd ed., edited by Gelvin, S.B. and Schilperoort, 1994, published by Kluwer Academic Publishers, Dordrecht, The Netherlands) Both monocotyledonous and dicotyledonous plants can be transformed and regenerated.

Examples of genetically modified plants include maize, banana, peanut, field peas, sunflower, tomato, canola, tobacco, wheat, barley, oats, potato, soybeans, cotton, carnations, roses, sorghum.

These, as well as other agricultural plants can be transformed with the antimicrobial genes such that they would exhibit a greater degree of resistance to pathogen attack. Alternatively, the proteins can be used for the control of diseases by topological application.

The invention also relates to application of antimicrobial protein in the control of pathogens of mammals, including humans. The protein can be used either in topological or intravenous applications for the control of microbial infections.

As indicated above in the description of the tenth embodiment, the invention includes within its scope the preparation of antimicrobial proteins based on the prototype MiAMP2 series of proteins. New sequences can be designed from the MiAMP2 amino acid sequences which substantially retain the distribution of positively charged residues relative to cysteine residues as found in the MiAMP2 proteins. The new sequence can be synthesised or expressed from a gene encoding the sequence in an appropriate host cell. Suitable methods for such procedures have been described above. Expression of the new protein in a genetically engineered cell will typically result in a product having a correct three-dimensional structure, including correctly formed disulphide linkages between cysteine residues. However, even if the protein is chemically synthesised, methods are known in the art for further processing of the protein to break undesireable disulfide bridges and form the bridges between the desired cysteine residues to give the desired three-dimensional structure should this be necessary.

WO 98/27805 PCT/AU97/00874 14 Macadamia integrifolia antimicrobial proteins series number 2 As indicated above, a new series of potent antimicrobial proteins has been identified in the seeds of Macadamia integrifolia. The proteins collectivelly are called the MiAMP2 series of antimicrobial proteins (or MiAMP2 proteins) because they are all found on one large preproprotein which is processed into smaller subunits, each exhibiting antimicrobial activity; they represent the second class of antimicrobial proteins isolated from Macadamia integrifolia. Each protein fragment of the series has a characteristic pI value. MiAMP2a, b, c, and d subunits as shown in Figure 4 have predicted pi values of 4.4, 4.6, 11.5, and 11.6 respectively (predicted using raw sequence data without the His tag or cleavage sequences associated with expression of fragments in the vector pET 6b), and contain two sets of CXXXC motifs which are important in stabilising the threedimensional structure of the protein through the formation of disulfide bonds. Additionally, the proteins contain either an added set of aromatic (tyrosine/phenylalanine) residues or an added set of cysteine residues located at positions which would give more stability to the helix-turn-helix structure as described above and in Example 8.

The amino acid sequences of the MiAMP2 series of proteins share significant homology with fragments of previously described proteins in sequence databases (Swiss Prot and Non-redundant databases) searched using the BLASTP algorithm (Altschul, S.F. et al. [1990] J. Mol. Biol. 215:403).

In particular, MiAMP2a, b, c and d sequences exhibit significant similarity with regions of cocoa vicilin and cotton vicilin (as seen in Figure Some similarity is also seen with fragments from other seed storage proteins of peanut (Burks, A. W, et al. [1995] J. Clin. Invest. 96 1715-1721), maize (Belanger, F. C. and Kriz, A. L.[1991] Genetics 129 863-872), barley (Heck, G. R. et al.

[1993] Mol. Gen. Genet. 239 209-218), and soybean (Sebastiani, F. L. et al. [1990] Plant Mol.

Biol. 15 197-201). Although, in some cases the homology is not extremely high (for example, 18% identity between MiAMP2a and cotton subunit 1; see Figure the spacing of the main four cysteine residues is conserved in all subunits and homologues. In addition, both cotton and cocoa vicilin-derived subunits retain the conserved tyrosine or phenylalanine residues as additional stabilisers of the tertiary structure. The cotton and cocoa vicilins with 525 and 590 amino acids, respectively, are much larger proteins than MiAMP2c (47 amino acids) (see Figures 4 and 6).

Although MiAMP2 subunits also share some homology with MBP-1 antimicrobial protein from maize (Duvick, J.P. et al. (1992) J Biol Chem 267:18814-20) the number of residues between the CXXXC motifs is 13 which puts MBP-1 outside the specifications for the spacing given here in this application. MBP-1 is also a smaller protein (33 amino acids), overall, than the sequences claimed here and there is no evidence available the MBP-1 is derived from a larger seed storage protein other than some similarity with a portion of miaze globulin protein. However, MBP-1 cannot be derived WO 98/27805 PCT/AU97/00874 from from the maize globulin since maize globulin contains 10 residues between the two CXXXC motifs while MBP-1 contains 13. The alignments in Figures 4 and 6 show the similarity in cysteine spacing between MiAMP2 subunits and the cocoa and cotton vicilin-derived molecules. The cysteine and the aromatic tyrosine/phenylalanine residues in Figures 4 and 6 are highlighted with bold underlined text. Figure 4 also shows the alignment of additional proteins which can be expressed in liquid culture and shown to exhibit antimicrobial activity.

All of the MiAMP2 homologues that have been tested exhibit antifungal activity. MiAMP2 homologues show very significant inhibition of fungal growth at concentrations as low as 2 [ig/ml for some of the pathogens/microbes against which the proteins were tested. Thus they can be used to provide protection against several plant diseases. MiAMP2 homologues can be used as fungicides or antibiotics by application to plant parts. The proteins can also be used to inhibit growth of pathogens by expressing them in transgenic plants. The proteins can also be used for the control of human pathogens by topological application or intravenous injection. One characteristic of the proteins is that inhibition of some microbes is suppressed by the presence of Ca 2 (1 mM). An example of this effect is provided for MiAMP2c subunit in Table 1.

Some of the MiAMP2 proteins and homologues could also function as insect control agents.

Since some of the proteins are extremely basic pi 11.5 for MiAMP2c and d subunits), they would maintain a strong net-positive charge even in the highly alkaline environment of an insect gut.

This strong net-positive charge would enable it to interact with negatively charged structures within the gut. This interaction may lead to inefficient feeding, slowing of growth, and possibly death of the insect pest.

Non-limiting examples of the invention follow.

Example 1 Extraction of Basic Protein from Macadamia integrifolia Seeds Twenty five kilograms of Mi nuts (purchased from the Macadamia Nut Factory, Queensland, Australia) were ground in a food processor (The Big Oscar, Sunbeam) and the resulting meal was extracted for 2-4 hours at 4 0 C with 50 L of an ice-cold extraction buffer containing 10 mM NaH2PO4, 15 mM Na2HPO4, 100 mM KCI, 2 mM EDTA, 0.75% polyvinylpolypyrolidone, and mM phenylmethylsulfonyl fluoride (PMSF). The resulting homogenate was run through a kitchen strainer to remove larger particulate material and then further clarified by centrifugation (4000 rpm for 15 min) in a large capacity centrifuge. Solid ammonium sulphate was added to the supernatant to obtain 30% relative saturation and the precipitate allowed to form overnight with stirring at 4 0

C.

Following centrifugation at 4000 rpm for 30 min, the supematant was taken and ammonium sulphate added to achieve 70% relative saturation. The solution was allowed to precipitate overnight and then WO 98/27805 PCT/AU97/00874 16 centrifuged at 4000 rpm for 30 min in order to collect the precipitated protein fraction. The precipitated protein was resuspended in a minimal volume of extraction buffer and centrifuged once again (13,000 rpm x 30 min) to remove the any insoluble material yet remaining. After dialysis mM ethanolamine pH 9.0, 2 mM EDTA and 1 mM PMSF) to remove residual ammonium sulphate, the protein solution was passed through a Q-Sepharose Fast Flow column (5 x 12 cm) previously equilibrated with 10 mM ethanolamine (pH 2 mM in EDTA). The collected flowthrough from this column represents the basic (pl protein fraction of the seeds. This fraction was further purified as described in Example 3.

Example 2 Antifungal and Antibacterial Activity Assays In general, bioassays to assess antifungal and antibacterial activity were carried out in 96-well microtitre plates. Typically, the test organism was suspended in a synthetic growth medium consisting of K2HPO4 (2.5 mM), MgSO4 (50 CaCl2 (50 tM), FeSO4 (5 RM), CoCl2 (0.1 uM), CuSO4 (0.1 gM), Na2MoO4 (2 tM), H-3B03 (0.5 uM), KI (0.1 ZnSO4 (0.5 gM), MnSO4 (0.1 uM), glucose (10 asparagine (1 methionine (20 mg/L), myo-inositol (2 mg/L), biotin (0.2 mg/L), thiamine-HCI (1 mg/L) and pyridoxine-HCL (0.2 mg/L). The test organism consisted of bacterial cells, fungal spores (50,000 spores/ml) or fungal mycelial fragments (produced by blending a hyphal mass from a culture of the fungus to be tested and then filtering through a fine mesh to remove larger hyphal masses). Fifty microlitres of the test organism suspended in medium was placed into each well of the microtitre plate. A further 50 pl of the test antimicrobial solution was added to appropriate wells. To deal with well-to-well variability in the bioassay, 4 replicates of each test solution were done. Sixteen wells from each 96-well plate were used as controls for comparison with the test solutions.

Unless otherwise stated, incubation was at 25 0 C for 48 hours. All fungi including yeast were grown at 25C. E. coli were grown at 37 0 C and other bacteria were bioassayed at 28 0 C. Percent growth inhibition was measured by following the absorbance at 600 nm of growing cultures over various time intervals and is defined as 100 times the ratio of the average change in absorbance in the control wells minus the change in absorbance in the test well divided by the average change in absorbance at 600 nm for the control wells [(avg change in control wells change in test well) (avg change in control wells)] x 100). Typically, measurements were taken at 24 hour intervals and the period from 24-48 hours was used for %Inhibition measurements.

WO 98/27805 PCT/AU97/00874 17 Example 3 Purification of antimicrobial protein from Macadamia integrifolia basic protein fraction The starting material for the isolation of the Mi antimicrobial protein was the basic fraction extracted from the mature seeds as described above in Example 1. This protein was further purified by cation exchange chromatography as shown in Figure 1.

About 4 g of the basic protein fraction dissolved in 20 mM sodium succinate (pH 4) was applied to an S-Sepharose High Performance column (5 X 60 cm) (Pharmacia) previously equilibrated with the succinate buffer. The column was eluted at 17 ml/min with a linear gradient of L from 0 to 2 M NaCI in 20 mM sodium succinate (pH The eluate was monitored for protein by on-line measurement of the absorbance at 280 nm and collected in 200 ml fractions. Portions of each fraction were subsequently tested in the antifungal activity assay against Phytopthora cryptogea at a concentration of 100 gg/ml in the presence and absence of 1 mM Ca 2 Results of bioassays are included in Figures la and Ib where the elution gradient is shown as a solid line and the shaded bars represent %Inhibition. The Figure la assays were conducted without added Ca 2 while 1 mM Ca 2 was included in the Figure Ib assays. Fractionation yielded a number of unresolved peaks eluting between 0.05 and 2 M NaC. A peak eluting at about 16 hours into the separation (fractions 90-92) showed significant antimicrobial activity.

Fractions showing significant antimicrobial activity were further purified by reversed-phase chromatography. Aliquots of fractions 90-92 were loaded onto a Pep-S (C 2

/C

18 column (25 x 0.93 cm) (Pharmacia) equilibrated with 95% H20/5% MeCN/0.1% TFA The column was eluted at 3 ml/min with a 240 ml linear gradient (80 min) from 100%A to 100%B H20/95% MeCN/0.1% TFA). Individual peaks were collected, vacuum dried three times in order to remove traces of TFA, and subsequently resuspended in 500 microlitres of milli-Q water (Millipore Corporation water purification system) for use in bioassays as described in Example 2. Figure 2 shows the HPLC profile of purified fraction 92 from the cation-exchange separation shown in Figures 1 and 2. Protein elution was monitored at 214 nm. The acetonitrile gradient is shown by the straight line. Individual peaks were bioassayed for antimicrobial activity: the bars in Figure 3 show the inhibition corresponding to 15 .tg/ml of material from each of the fractions. The active protein elutes at approximately 27 min MeCN/0.1%TFA) and is called MiAMP2c.

Example 4 Purity of Isolated MiAMP2c The purity of the isolated antimicrobial protein was verified by native SDS-PAGE followed by staining with coomassie blue protein staining solution. Electrophoresis was performed on a 10-20% tricine gradient gel (Novex) as per the manufacturers recommendations (100 V, 1-2 hour separation WO 98/27805 PCT/AU97/00874 18 time). Under these conditions the purified MiAMP2c migrates as a single discrete band (<10 kDa in size). The detection of a single major band in the SDS-PAGE analysis together with single peaks eluting in the cation-exchange and reversed-phase separations (not shown), gives strong indication that the MiAMP2c preparation is greater than 95% pure and therefore the activity of the preparation was almost certainly due to the MiAMP2c alone and not to a minor contaminating component. A clean signal in mass spectrometric analysis (Example 5 below) also supports this conclusion.

Example Mass Spectroscopic Analysis of MiAMP2c Purified MiAMP2c was submitted for mass spectroscopic analysis. Approximately 1 gg of protein in solution was used for testing. Analysis showed the protein to have a molecular weight of 6216.8 Da 2 Da. Additionally, the protein was subjected to reduction of disulfide bonds with dithiothreitol and alkylation with 4-vinylpyridine. The product of this reduction/alkylation was then submitted for mass spectroscopic analysis and was shown to have gained 427 mass units (i.e.

molecular weight was increased by approximately 4 X 106 Da). The gain in mass indicated that four 4-vinylpyridine groups had reacted with the reduced protein, demonstrating that the protein contains a total of 4 cysteine residues. The cysteine content has also been subsequently confirmed through amino acid sequencing.

Example 6 Amino Acid Sequence of MiAMP2c Protein Approximately 1 jg of the pure protein which had been reduced and alkylated was subjected to Automated Edman degradation N-terminal sequencing. In the first sequencing run, the sequence of the first 39 residues was determined. Subsequently, approximately 1 mg of MiAMP2c was reacted with Cyanogen Bromide which cleaved the protein on the C-terminal side of Methionine-26.

The C-terminal fragment generated by the cleavage reaction was purified by reversed-phase HPLC and sequenced, yielding the remaining sequence of MiAMP2c residues 27-47). The full amino acid sequence is RQRDP QQQYE QCQER CQRHE TEPRH MQTCQ QRCER RYEKE KRKQQ KR and represents amino acids 118 to 164 of clone 3 from Example 9 (see Figure 6 and SEQUENCE ID NO: In the figure, cysteine residues are in bold type and underlined to facilitate recognition of the spacing patterns. Depending on the number of disulfide bonds that are formed, the protein mass will range from 6215.6 to 6219.6 Da. This is in close agreement with the mass of 6216.8 2 Da obtained by mass spectrometric analysis (Example The measured mass closely approximates the predicted mass of MiAMP2c in a two-disulfide form as is expected to be the case.

WO 98/27805 PCT/AU97/00874 19 Example 7 Synthetic DNA Sequence Coding for MiAMP2c with a leader peptide Using standard codon tables it is possible to reverse-translate the protein sequences to obtain DNA sequences that will code for the antimicrobial proteins. The software program MacVector S 5 4.5.3 was used to enter the protein sequence and obtain a degenerate nucleotide sequence. A codon usage table for tobacco was referenced in order to pick codons that would be adequately represented in tobacco for purposes of obtaining high expression in this test plant. A 30 amino-acid leader peptide was also designed to ensure efficient processing of the signal peptide and secretion of the peptide extracellularly. For this purpose, the method of Von Hiejne was used to evaluate a series of possible leader sequences for probability of cleavage at the correct position [Von Hiejne, G.(1 986) Nucleic Acids Research 14(11): 4683-4690]. In particular, the amino acid sequence MAWFH VSVCN AVFVV IIIIM LLMFV PVVRG (Sequence ID. No. 11) was found to give an optimal probability of correct processing of the signal peptide immediately following the G (Gly) of this leader sequence. A 5' untranslated region from tobacco mosaic virus was also added to this synthetic gene to promote higher translational efficiency [Dowson, et al. (1994) Plant Mol. Biol. Rep.

12(4):347-357]. The synthetic gene also contains restriction sites at the 5' and 3' ends and immediately 5' of the start ATG for efficient cloning and subcloning procedures. Figure 5 shows a synthetic DNA sequence suitable for use in plant expression experiments. In this Figure, the arrow shows where translation is initiated and the triangular symbol indicates the point of cleavage of the signal peptide.

Example 8 Structure prediction of MiAMP2c Protein Using sequence analysis algorithms, putative secondary structure motifs can be assigned to the protein. Five different algorithms were used to predict whether a-helices, p-sheets, or turns can occur in the MiAMP2c protein (Figure Methods were obtained from the following sources: DPM method, Deleage, and Roux, B. (1987) Prot. Eng. 1:289-294; SOPMA method, Geourjon, and Deleage, G. (1994) Prot. Eng. 7:157-164; Gibrat method, Gibrat, Gamier, and Robson, B.(1987) J.Mol.Biol. 198:425-443; Levin method, Levin, Robson, and Gamier, J. (1986) FEBS Lett. 205:303-308; and PhD method, Rost, And Sander, C. (1994) Proteins 19:55-72.

Figure 7 shows the predicted locations of a-helices, P-sheets and turns. The following symbols have been used in Figure 7: C, coil (unstructured); H, alpha helix; E, 3- sheet; and S, turn. Underlined residues are those which were predicted to exhibit an a-helical structure by at least 2 separate structure prediction methods; these are represented as helices in Figure 8.

WO 98/27805 PCT/AU97/00874 It is clear from the secondary structure predictions that the protein is highly a-helical. While secondary structure prediction is often difficult and inaccurate, this particular prediction gives a clear indication of the structure of the protein. Examination of the secondary-structure predictions show a clear preponderance of two a-helical regions broken by a stretch of about 5-8 residues. This is highly suggestive of a helix-turn-helix motif.

Helical wheel analysis of the MiAMP2c amino acid sequence shows that cysteine residues with a CXXXC spacing will be aligned on one face of the helix in which they are located. Since the cysteines are involved in disulfide bond formation, the cysteine side chains in one helix must form covalent bonds with the cysteine side chains located on the other helical segment. When the helical segments are arranged in such a way as to bring the cysteine side chains from each respective helix into proximity with the other cysteine side chains, the resulting three-dimensional structure is shown in Figure 8. This structure exhibits a remarkable distribution of positively charge residues on one face of the protein comprised of two helices held together by two disulfide bonds. Figure 8 shows how the spacing of positively charged residues in helical regions of this molecule will cause these side chains to lie on one face of the helix. The positively charged residues are the dark side chains outlined in black. Other dark side chains represent acidic residues. A proline residue (grey colour marked with a is located at the extreme left end of the molecule in the turn region. Solid black lines show where disulfide bonds connect the two helices. The dotted line shows where the two aromatic hydrophobic residues interact to add stability to the helix-turn-helix structure.

This helix-turn-helix structure will be adopted by all MiAMP2 homologues containing the same cysteine spacing and residues with helix and turn-forming propensities. Other MiAMP2 fragment sequences can be superimposed onto the global structure shown in figure 8. The overall structure will remain essentially the same but the charge distribution will vary according to the sequences involved. In the case of MiAMP2b, the dotted line would represent an added disulfide bridge instead of a hydrophobic interaction.

Example 9 cDNA cloning of genes corresponding to MiAMP2c PCR Amplification of a genomic fragment of the MiAMP2c gene Using the reverse-translated nucleotide sequences, degenerate primers were made for use in PCR reactions with genomic DNA from Macadamia. Primer JPM17 sequence was 5' CAG CAG CAG TAT GAG CAG TG 3' and primer JPM20 degenerate sequence was 5' TTT TTC GTA TTC GCA 3' (SEQ ID NOS: 12 and 13). Primers JPM17 and were used in PCR amplifications carried out for 30 cycles with 30 sec at 950C, 1 min at 50°C, and 1 min at 72°C. PCR products with sizes close to those which were expected were directly sequenced WO 98/27805 PCT/AU97/00874 21 (ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit from Perkin Elmer Corporation) after excising DNA bands from agarose gels and purifying them using a Qiagen DNA clean-up kit. Using this approach, it was possible to amplify a fragment of DNA of approximately 100 bp. Direct sequencing of this nucleotide fragment yielded the nucleotide sequence corresponding to a portion of the amino acid sequence of the antimicrobial protein MiAMP2c (amino acids 7-39 of Figure The partial nucleotide sequence obtained from the above-mentioned fragment excluding the primer sequences was 5' TCA GAA GCG CTG CCA ACG GCG CGA GAC AGA GCC ACG ACA CAT GCA AAT TTG TCA ACA ACG C 3' (corresponding to base pairs 264 to 324 in SEQ ID NO: This sequence can be used for a variety of purposes including screening of cDNA and genomic libraries for clones of MiAMP2 homologues or design of specific primers for PCR amplification reactions.

Messenger RNA isolation from Macadamia nut kernels Fifty-eight grams of Macadamia nut kernels were ground to powder under liquid nitrogen using a mortar and pestel. RNA from ground material was then purified using a Guanidine thiocyanate/Cesium chloride technique (Current Protocols in Molecular Biology, supra). Using this method approximately 5 mg of total RNA was isolated. Messenger RNA was then purified from total RNA using a spun column mRNA purification kit (Pharmacia).

cDNA library construction A cDNA library was constructed in a lambda ZAP vector using a library kit from Stratagene.

A total of 6 reactions were performed using 25 micrograms of messenger RNA. First and second strand cDNA synthesis was performed using MMLV Reverse transcriptase and DNA Polymerase I, respectively. After blunting the cDNA with Pfu DNA Polymerase, Eco RI linker adapters were ligated to the DNA. DNA was then kinased using T4 polynucleotide kinase and the DNA subsequently digested with Xho I restriction endonuclease. At this point cDNA material was fractionated according to size using a sephacryl-S500 column supplied with the kit. DNA was then ligated into the lambda ZAP vector. The vector containing ligated insert was then packaged into lambda phage (Gigapack III packaging extract from Stratagene).

Screening of library The library constructed above was then plated and screened in XLl-blue E.coli bacterial lawns growing in top agarose. Plaques containing individual clones were isolated by lifting onto Hybond N+ membranes (Amersham LIFE SCIENCE), hybridizing to a radiolabeled version of the genomic DNA fragment amplified above, imaging of the blot, and picking of possitive clones for the next round of screeing. After secondary and tertiary screening, plaques were sufficiently isolated to allow WO 98/27805 PCT/AU97/00874 22 picking of single clones. Several clones were obtained, and subsequently the pBK-CMV vector portion from the larger lambda vector was excised.

Sequence of MiAMP2c cDNA clones Vectors (pBK-CMV) containing putative MiAMP2c clones were sequenced to obtain the DNA sequence of the cloned inserts. Seven clones were partially sequenced and an additional three clones were fully sequenced (see SEQ ID NOS: 2, 4 and 6 for DNA sequences of the macadamia clones).

Translation of the DNA sequences showed that the full length clones encoded highly similar proteins of 666 amino acids. Figure 6 shows that these proteins have substantial similarity to vicilin seedstrorage proteins from cocoa and cotton. Stars show positions of conserved identities and dots show positions of conserved similarities. Examination of the protein sequences revealed that the exact MiAMP2c sequence is found within the translated protein sequence of clone 3 at amino acid positions 118 to 164 (see Figure clones 1 and 2 contained sequences differing from MiAMP2c by 2 residues and 3 residues, respectively, out of 47 amino acids total in the MiAMP2c sequence.

The translation products of the full-length clones clones 1 and 2) consist of a short signal peptide from residues 1 to 28, a hydrophilic region from residues 29 to -246, and then two segments stretching from residues -246 to 666 with a stretch of acidic residues separating them at positions 542-546.

Significantly, the hydrophilic region containing the sequence for MiAMP2c, also contains 3 additional segments which are very similar to MiAMP2 (termed MiAMP2a, b and These 4 segments (found between residues 28 and -246) are separated by stretches in which approximately four out of five residues are acidic (usually glutamic acid). These acidic stretches occur at positions 64-68, 111-115, 171-174, and 241-246 and appear to delineate processing sites for cleavage of the 666-residue preproprotein into smaller functional fragments (acidic stretches delineating cleavage sites are shown as bold characters in Figure All four MiAMP2-like segments of the protein contain 2 doublets of cysteine residues separated by 10-12 residues to give the following pattern C- X-X-X-C-(10-12X)-C-X-X-X-C where X is any amino acid, and C is cysteine. All four segments are expected to form helix-turn-helix motifs as decribed in Example 8 above. It is clear that the cysteines in these locations will form disulfide bridges that stabilize the structure of the proteins by holding the two helical portions together.

The predicted helix-turn-helix motifs can be further stabilized in several ways. The first method of stabilization is exemplified in segments 1 and 3 residues 29-63 and 118-170, respectively, of the 666-residue Macadamia vicilin-like protein). These segments is the are stabilized by a hydrophobic ring-stacking interaction between two aromatic residues (one on each ahelical segment); this is normally accomplished with tyrosine residues but phenylalanine is also WO 98/27805 PCT/AU97/00874 23 used. As with the cysteine residues, the location of these aromatic residues in the predicted cc-helical segments is critical if they are to offer stabilization to the helix-tur-helix structure. In segments 1 and 3, the aromatic residues are 2 and 3 residues removed from the cysteine doublets as shown here: 10-12X)-C-X-X-X-C-X-X-X-Z where C is cysteine and Z is usually tyrosine c 5 but can be substituted with phenylalanine as is done in segment 1.

The second way to stabilize the helix-turn-helix fragment is by using an added disulfide bridge as seen in fragment 2 (residues 71-110). This is accomplished by placing additional cysteine residues 2 and 3 residues removed from the cysteine doublets as shown here: nX-C-X-X-C-X-X-X- C-(10-1 2 X)-C-X-X-X-C-X-X-X-C-nX. This is the only report that the inventors know of where a helix-turn-helix domain in an antimicrobial protein is stabilized by three disulfide bridges. While segment 4 (residues 175-241) does not contain the extra disulfide bridge or the hydrophobic ringstacking stabilization, it is probably stabilized by means of weaker ionic and or hydrogen bonding interactions.

Example Vectors for liquid culture expression of MiAMP2 and homologues PCR primers flanking the nucleotide region coding for MiAMP2c were engineered to contain restriction sites for Nde I and Bam HI (corresponding to the 5' and 3' ends of the coding region, respectively; Primer JPM31 sequence: 5' A CAC CAT ATG CGA CAA CGT GAT CC Primer JPM32 sequence: 3' C GTT GTT TTC TCT ATT CCT AGG GTT G SEQ ID NOS: 14 and These primers were then used to amplify the coding region of MiAMP2c DNA. The PCR product from this amplification was then digested with Nde I and Bar HI and ligated into a pETI 7b vector (Novagen Studier, F. W. et al. [1986] J. Mol. Biol. 189:113) with the coding region in-frame to produce the vector pET17-MiAMP2c.

A similar approach to the one above was used to construct vectors carrying the coding sequences of MiAMP2c homologues MiAMP2a, b, and d as well as Tc AMP1, and TcAMP2).

To construct the expression vectors for fragments a, b and d in MiAMP2 clone 1, specific PCR primers incorporating the Nde I and Barn HI sites were designed to amplify the fragments of interest.

The products were then digested with the appropriate restriction enzymes and ligated into the Nde I/Ban HI sites of a pETI 6b vector [Novagen] containing a His tag and a Factor Xa cleavage site (amino acid sequence MGHHH HHHHH HHSSG HIEGR HM, SEQ ID NO: 16). The protein products expressed from the pET16b vector is a fusion to the antimicrobial protein. The coding sequences for MiAMP2-like subunits from cocoa (Figure 4, TcAMPI and TcAMP2) were obtained from the published DNA sequence of the cocoa vicilin gene (Spencer, M. E. and Hodge R. 1992] Planta 186:567-576). Two MiAMP2-like fragments within the cocoa vicilin gene were located at WO 98/27805 PCT/AU97/00874 24 the 5' end (corresponding to the residues shown in Figure and two sets of complimentary oligonucleotides corresponding to the desired coding sequences were designed. The complimentary oligonucleotides (90 to -100 bases) corresponding to each cocoa subunit contained a 20bp overlap and also contained the Nde I and Bam HI restriction endonuclease cut sites.

For TcAMP, the following nucleotides were synthesised: TcAMPI forward oligo 5' GGGAATTCCA TATGTATGAG

CGTGATCCTC

GACAGCAATA CGAGCAATGC

CAGAGGCGAT

GCGAGTCGGA AGCGACTGAA GAAAGGGAGC 3'; TcAMP1 reverse oligo 5' GAAGCGACTG AAGAAAGGGA

GCAAGAGCAG

TGTGAACAAC GCTGTGAAAG

GGAGTACAAG

GAGCAGCAGA GACAGCAATA GGGATCCACA C 3'.

For TcAMP2, the following oligonucleotides were used: TcAMP2 forward oligo 5' GGGAATTCCA TATGCTTCAA

AGGCAATACC

AGCAATGTCA AGGGCGTTGT CAAGAGCAAC AACAGGGGCA GAGAGAGCAG CAGCAGTGCC AGAGAAAATG C 3'; TcAMP2 reverse oligo 5' GTGTGGATCC CTAGCTCCTA TTTTTTTTGT GATTATGGTA ATTCTCGTGC TCGCCTCTCT CTTGTTCCTT ATATTGCTCC CAGCATTTTC TCTGGCACTG CT 3'.

The oligonucleotide sets were added to individual PCR amplification reactions in order make individual PCR fragments containing the desired coding region. Since initial PCR amplifications gave fuzzy bands, reamplification of the original products was carried out using new 20mer primers (complimentary to the 5'ends of the forward and reverse oligonucleotides shown above) designed to amplify the entire coding region of the cocoa subunits. Once amplified, the PCR products were restriction digested with the appropriate enzymes and ligated into the vector pET16b as above. This procedure was carried out for both cocoa fragments with similarities to MiAMP2c (shown in Figure 4).

Example 11 Expression in E.coli and purification of MiAMP2c and homologues Starter cultures (50 ml) of E.coli strain BL21 (Grodberg, J. [1988] J. Bacteriol. 170:1245) transformed with the appropriate pET construct (Example 10) were added to 500ml of NZCYM media (Current Protocols in Molecular Biology, supra) and cultured to an optical density of 0.6 (600 nm) and induced with the addition of 0.4 or 1.0 mM IPTG depending on whether pETI7b WO 98/27805 PCT/AU97/00874 (containing a T7 promoter) or pETI6b (containing a His tag fusion and a T7 promoter/lac operator) vector was being used. After cells were induced, cultures were allowed to grow for 4 hours before harvesting. Aliquots of the growing cultures were removed at timed intervals and protein extracts run on an SDS-PAGE gel to follow the expression levels of MiAMP2 and homologues in the 5 cultures. Fragments being expressed with a Histidine tag in the pET 6b vector), were harvested by centrifuging induced cell cultures at 5000g for 10 minutes. Cell pellets were resuspended and broken by stirring for one hour in 6 M Guanidine-HC1, buffered with 100 mM sodium phosphate and mM Tris at pH 8.0. Broken cell suspensions were centrifuged at 10,000g for 20-30 minutes to settle the cellular debris. Supernatants were removed to fresh tubes and 500 mg of Ni-NTA fast flow resin (Qiagen) was added to each supternatant. After gentle mixing at 4 0 C for 30-60 minutes, the suspension was loaded into a small column, rinsed two times with 8 M Urea (pH 8.0 and then pH 6.3) and subsequently, the protein was eluted using 8 M Urea pH 4.5. Protein fractions thus obtained were substantially pure but were further purified using an 9.3 x 250 mm C2/C18 reverse phase column (Pharmacia) and 75 minute gradient from 5% to 50% acetonitrile (0.1 TFA) flowing at 3 ml/min (data not shown).

All of the MiAMP2c homologues (except MiAMP2c which was expressed in pETI 7b) were expressed in the pET 6b vector containing the Histidine tag. While induction of the MiAMP2c culture proceded as above, the rest of the purification was somewhat different. In this case, MiAMP2c-expressing cells were harvested by centrifugation but were then resuspended in phosphate buffer (100 mM, pH 7.0 containing 10 mM EDTA and 1 mM PMSF) and broken open using a French press instrument. Cellular debris containing MiAMP2c inclusion bodies was solubilized using a 6 M Guanidine-HC1, 10 mM MES pH 6.0 buffer. Soluble material was then recovered after centrifugation to remove insoluble debris remaining from the solubilization step. Guanidine-HCI soluble material was then dialyzed against 10 mM MES pH 6.0 containing PMSF (1 mM) and EDTA (10 mM). Cation-exchange fractionation was carried out as described in Example 3 except on a smaller scale after the dialysis step. Subsequently, the major eluting protein from the cationexchange columnm, which was MiAMP2c, was then further purified using reverse phase HPLC as described in Example 3.

Figure 9 shows the SDS-PAGE gel analysis of the various purification stages obtained following induction with IPTG and subsequent purification of expressed proteins. Samples analysed during the TcAMPI purification were are as follows: lane 1, molecular weight markers; lane 2, Ni- NTA non-binding fraction; lane 3, rinse of Ni-NTA resin with pH 8 urea; lane 4, rinse of Ni-NTA resin with pH 6.3 urea; lane 5, elution of TcAMP1 with pH 4.5 urea; and lane 6, second elution of TcAMPI with pH 4.5 urea. TcAMP2 was purified in a similar manner and was also subjected to WO 98/27805 PCT/AU97/00874 26 reverse-phase HPLC to further purify the fraction eluting from the Ni-NTA resin. Figure 10 shows the reverse phase purification of cocoa subunit number 2 (TcAMP2).

SDS-PAGE gel analysis of the MiAMP2a, b, and d fragment purification is shown in the second panel of Figure 9. Lane contents are as follows: lane 1, molecular weight markers; lane 2, MiAMP2a pre-induced cellular extractp; lane 3, MiAMP2a IPTG induced cellular extract; lane 4, MiAMP2a Ni-NTA non-binding fraction; lane 5, MiAMP2a elution from Ni-NTA; lane 6, MiAMP2b pre-induced cellular extract; lane 7, MiAMP2b IPTG induced cellular extract; lane 8, MiAMP2b Ni-NTA non-binding fraction; lane 9, MiAMP2b elution from Ni-NTA; lane MiAMP2d pre-induced cellular extract; lane 11, MiAMP2d IPTG induced cellular extract; lane 12, MiAMP2d Ni-NTA non-binding fraction; and lane 13, MiAMP2d elution from Ni-NTA.

Using the vectors described in Example 10, MiAMP2c, and 5 homologues MiAMP2a, MiAMP2b, MiAMP2d, TcAMPI and TcAMP2) were all expressed, purified and tested for antimicrobial activity. The approach taken above can be applied to all of the antimicrobial fragments described in Figure 4. Purified fragments can then be tested for specific inhibition agains microbial pathogens of interest.

Example 12 Detection of MiAMP2 homologues in other species using antibodies raised to MiAMP2c Rabbits were immunised intramuscularly according to standard protocols with MiAMP2 conjugated to diphtheria toxoid suspended in Fruends incomplete adjuvent. Serum was harvested from the animals at regular intervals after giving the animal added doses of MiAMP2 adjuvent to boost the immune response. Approximately 100 ml of serum were collected and used for screening of crude extracts obtained from several plant seeds. One hundred gram quantities of seeds were ground and extracted to obtain a crude extract as in Example 1. Aliquots of protein were separated on SDS-PAGE gels and the gels were then blotted onto nitrocellulose membrane for subsequent detection of antibody reacting proteins. The membranes were incubated with MiAMP2c rabbit primary antibodies, washed and then incubated with alkaline phosphatase-conjugated goat anti-rabbit IgG for colorimetric detection of antigenic bands using the chemical 5-bromo-4-chloro-3-indolyl phosphate nitroblue tetrazolium substrate system (Schleicher and Schuell). Figure 11 shows that various other species contain immunologically-related proteins of similar size to MiAMP2c. Lanes 1-15 contain the extracts from the following species: 1) Stenocarpus sinuatus, 2) Stenocarpus loading) 3) Restio tremulus, 4) Mesomalaena tetragona, 5) Nitraria billardieri, 6) Petrophile canescens, 7) Synaphae acutiloba, 8) Dryandraformosa, 9) Lambertia inermis, Stirlingia latifolia, 11) Xylomelum angustifolium, 12) Conospermum bracteosum, 13) Conospermum triplinernium, 14) Molecular weight marker, 15) Macacamia integrifolia pure MiAMP2c. Lanes 1- WO 98/27805 PCT/AU97/00874 27 13 contain a variety of species, some of which show the presence of antigenically related proteins of a similar size to MiAMP2c. Other bands exhibiting higher molecular weights probably represent the larger precursor seed storage proteins from which the antimcrobial proteins are derived.

Antigenically-related proteins can be seen in lanes 1, 2, 4, 6, 7, 8, 9, and 11-13.

Bioassays were also performed using crude extracts from various Proteaceae species.

Specifically, extracts from Banksia robur, Banksia canei, Hakea gibbosa, Stenocarpus sinuatus, and Stirlingia latifolia have all been shown to exhibit antimicrobial activity. This activity may derive from MiAMP2 homologues since these species are related to Macadamia.

Example 13 Purification of MiAMP2c homologues in another species using antibodies raised to MiAMP2c Based on the detection of immunologically related proteins in other species of the family Proteaceae and the presence of antimicrobial activity in crude extracts, Stenocarpus sinuatis was chosen for a large scale fractionation experiment in an attempt to isolate MiAMP2c homologues.

Five kg of S.sinuatus seed was frozen in liquid nitrogen and ground in a food processor (Big Oscaar Sunbeam). The ground seed was immediately placed into 12 L of 50 mM H2S0 4 extraction buffer and extracted at 4 0 C for 1 hour with stirring. The slurry was then centrifuged for 20 min at 10,000 g to remove particulate matter. The supernatant was then adjusted to pH 9 using a 50mM ammonia solution. PMSF and EDTA were added to final concentrations of 1 and 10 mM respectively.

The crude protein extract was applied to an anion exchange column (Amberlite IRA-938, Rohm and Haas) (3cmx90cm) equilibrated with 50 mM NH4Ac pH 9.0 at a flow rate of 40 ml/min.

The unbound protein comprising the basic protein fraction was collected and used in the subsequent purification steps.

The basic protein fraction was adjusted to pH 5.5 with acetic acid and then applied at ml/minute over 12 h to a SP-Sepharose Fast Flow (Pharmacia) Column (5cm x 60cm) preequilibrated with 25mM ammonium acetate. The column was then washed for 3.5 h with 25 mM Acetate pH 5.5. Elution of bound proteins was achieved by applying a linear gradient of NH4Ac from 25 mM to 2.0 M (pH 5.5) at 10 ml/min over 10 h. Absorbance of the eluate was observed at 280 nm and 100 ml fractions collected (see Figure 12).

Cation-exchange fractions that cross-reacted with the antiserum (fractions 14-28, Figure 12) were then further purified by reverse phase chromatography. Cross-reacting fractions were loaded onto a 7 [pm Cl 8 reverse phase column (Brownlee) equilibrated with 90% H20, 10% acetonitrile and 0.1% Trifluoroacetic acid (TFA)(=100%A). Bound proteins were eluted with a linear gradient from 100%A to 100%B H20, 95% acetonitrile, 0.08% TFA). The absorbance of the eluted proteins was monitored at 214nm and 2 80nm. The eluted proteins were dried under vacuum and resuspended WO 98/27805 PCT/AU97/00874 28 in water three times to remove traces of TFA from the samples. Reverse phase protein elution fractions 20 to 61 were analysed by pooling 2 adjacent fractions and performing a western blot analysis (see Figure 13). Fractions 22-41 gave a weak positive reaction and fractions 42-57 gave a strong positive reaction to the anti-MiAMP2c antiserum. Fractions that showed antifungal activity against S.sclerotiorum at 50 Rg/ml and 10 gg/ml are indicated by arrows on the chromatogram.

Using the approach above, several active fractions (termed SsAMPI and SsAMP2) were obtained which were assessed for their antifungal activity against Sclerotinia sclerotiorum, Alternaria brassicola, Leptosphaeria maculans, Verticilium dahliae and Fusarium oxysporum.

Bioassays were carried out as described in Example 2 and results shown in Example 15. Another fragment which reacted with MiAMP2 antiserum was purified and sequenced (SsAMP3) but insufficient protein was available for characterisation of antimicrobial activity. Partial sequences obtained from these proteins are shown in Figure 4 (SEQ ID NOS: 26, 27 and 28). Full sequencing of the peptides or cloning of cDNAs encoding the seed storage proteins from this species will reveal the extent of homology between these peptides and MiAMP2-series homologues.

Example 14 Synthesis of small fragments of MiAMP2c In an effort to determine if the full MiAMP2c molecule was absolutely necessary for the protein to exhibit antimicrobial activity, two separate peptides were chemically synthesized by Auspep Pty. Ltd. (Australia). For each peptide, the cysteine residues were changed to alanine residues so that disulfide bonds were no longer capable of being formed between two separate protein chains. Tyrosine residues were also changed to alanine since it was expected that tyrosine also participated in the helix-turn-helix stabilization and this would not be needed in the synthetic peptides lacking one of the helices. Alanine is also favorable to the formation of alpha-helices so it should not interfere with the native helical structure to a large degree. Peptide one is comprised of 22 amino acids from 118 to 139 in the amino acid sequence of clone 3 (sequence: RQRDP QQQAE QAQKR AQRRE TE, SEQUENCE ID NO: Peptide 2 is 25 amino acids in length and runs from 140 to 164 in clone 3 (sequence: PRHMQ IAQQR AERRA EKEKR KQQKR, SEQ ID NO: Peptides 1 and 2 are labeled MiAMP2c pepl and MiAMP2c pep2 respectively. These peptides were resuspended in Milli-Q water and bioassayed against a number of fungi. As seen in Table 2, peptide 2 has inhibitory activity against a variety of fungi whereas peptide 1 exhibited little or no activity.

Mixtures of peptide 1 and peptide 2 exhibit similar levels of activity as seen with peptide 2 alone indicating that only peptide 2 is exhibiting activity. The fact that peptide 2 exhibits antimicrobial activity in the absence of the helix-turn-helix structure exhibited by MiAMP2c reveals that the helixturn-helix structure is not absolutely necessary for the peptides to retain activity. Nevertheless, WO 98/27805 PCT/AU97/00874 29 peptide 2 did not exhibit the same degree of activity on a molar basis as MiAMP2c (whole fragment) indicating that the helix-turn-helix structure is important for maximal expression of antimicrobial activity by the fragments involved. It is also expected that the helix-turn-helix structure will confer greater stability to the MiAMP2 homologues, thus rendering these proteins less susceptible to proteolytic cleavage and other forms of degredation. Greater stability would lead to maintaining antimicrobial activity over a longer period of time.

Example Antifungal activity of MiAMP2c homologues and fragment(s) MiAMP2c and each of the various MiAMP2 homologues were tested against a variety of fungi as concentrations ranging from 2 to 50 ptg/ml. Table 1 shows the IC50 value of pure MiAMP2c against various fungi and bacteria. In the table, the indicates that 50% inhibition of the fungus was not achieved at 50 fig/ml which was the highest concentration tested. The abbreviation "ND" indicates that the test was not performed or that results could not be interpreted. The antimicrobial activity of MiAMP2c was also tested in the presence of 1 mM Ca 2 in the test medium and the values for these tests are given in the right-hand column. As can be seen in the table, the inhibitory activity of MiAMP2c is greatly reduced (although not eliminated) in the presence of Ca 2 Table 1 Concentrations of MiAMP2c at which 50% inhibition of growth was observed Organism IC50 (Ug/ml) IC50 Ca 2 (ig/ml) Alternaria helianthi 5-10

ND

Candida alhicans >50 Ceratocystis paradoxa 20-50 Cercospora nicotianae 5-10 5-10 Clavibacter michiganensis 50 Chalara elegans 2-5 10-20 Fusarium oxysporum 10 20-50 Sclerotinia sclerotiorum 20-50 Phytophthora cryptogea 5-10 10-25 Phytophthora parasitica nicotiana 10-20 WO 98/27805 PCT/AU97/00874 Verticillium dahliae 5-10 Ralstonia solanacearum >50 Pseudomonas syringae tabaci >50 Saccharomyces cerevisiae 20-50 Escherichia coli >50 Table 2 shows the the antimicrobial activity of various homologues and fragments of MiAMP2c. In the table, the following abbreviations are used: Ab, Alternaria brassicola; Cp: Ceratocystis paradoxa; Foc: Fusarium oxysporum; Lm: Leptosphaeria maculans; Ss: Sclerotinia sclerotiorum; Vd: Verticillium dahliae. The indicates that concentrations higher than ug/ml were not tested so that an IC50 value could not be established. A blank space indicates that the test was not performed or that results could not be interpreted.

The TcAMPI and 2 used for the results presented in Table 2 were derived from cocoa vicilin (Examples 10 and 11). SsAMPI and 2 show reactivity with MiAMP2c antibodies and also exhibit antimicrobial activity as seen in the table below. The versions of MiAMP2a, b and d as well as TcAMPI and TcAMP2 tested in the bioassays all contain a His tag fusion resulting from expression in the vector pET 6b. MiAMP2c pepl and 2 are the N and C terminal regions, respectively, of MiAMP2c antimicrobial peptide as specified in Example 14 above. The concentration value listed for 'MiAMP2c pep 1+2' is the concentration of each individual peptide in the mixture. It should be remembered that MiAMP2c pepl and pep2 are both about V 2 the size of MiAMP2c; comparisons of the activity of these peptides with the MiAMP2c protein should, therefore, be made on a molar basis rather than on a strict ptg/ml concentration basis. Peptides were only tested in media A which did not contain added Ca 2 Table 2

IC

50 values ((tg/ml) of MiAMP2 related proteins against various fungi Peptide tested Fungus used in bioassy Ab Cp Foc Lm Ss Vd MiAMP2a 5-10 2.5-5 5-10 MiAMP2b 2.5 2.5 5-10 MiAMP2c 20-50 10 20-50 5-10 MiAMP2d 5 2.5 5-10 MiAMP2c pepl 100 WO 98/27805 PCT/AU97/00874 31 MiAMP2c pep2 10-20 10-20 50 10-20 MiAMP2c pepl+2 10-25 TcAMPI 10 5-10 2-5 10 5-20 TcAMP2 5-10 5-10 2-5 5 5-20 SsAMP1 20-50 20-50 20-50 10-20 SsAMP2 20-50 >50 >50 >50 It is worthy of note that while the TcAMPI and 2 sequences are readily available in the public data bases, no antimicrobial activity had ever been assigned to them. These sequences were derived from much larger proteins involved in seed storage functions. The inventors have thus described a completely new activity for a small portion of the overall cocoa vicilin molecules. The activity of cotton fragments 1, 2, and 3 has been exemplified by other authors (Chung, R. P.T. et al. [1997] Plant Science 127:1-16).

Example 16 Construction of the plant transfomation vector PCV91-MiAMP2c The expression vector pPCV91-MiAMP2c (Figure 14) contains the full coding region of the MiAMP2c (Example 7) DNA flanked at it 5' end by the strong constitutive promoter of 35S RNA from the cauliflower mosaic virus (pCaMV35S) (Odel et al., [1985] Nature 313: 810-812) with a quadruple-repeat enhancer element (e-35S) to allow for high transcriptional activity (Kay et al.

[1987] Science 236:1299-1302). The coding region of MiAMP2c DNA is flanked at its 3' end by the polyadenylation sequence of 35S RNA of the cauliflower mosaic virus (pA35S). The plasmid backbone of this vector is the plasmid pPCV91 (Walden, R. et al. [1990] Methods Mol. Cell. Biol.

1:175-194). The plasmid also contains other elements useful for plant transformation such as an ampicillin resistance gene (bla) and a hygromycin resistance gene (hph) driven by the nos promoter (pnos). These and other features allow for selection in various cloning and transformation procedures. The plasmid pPCV91-MiAMP2c was constructed as follows: A cloned fragment encoding MiAMP2c (Example 7) was digested using restriction enzymes to release the MiAMP2c gene fragment containing a synthetic leader sequence.. The binary vector pPCV91 was digested with the restriction enzyme Bar HI. Both the MiAMP2c DNA fragment containing and the binary vector were ligated using T4 DNA ligase to produce pPCV91-MiAMP2c binary vector for plant transformation (Figure 12).

Using this approach, other homologues of MiAMP2c can be expressed in plants. Not only can individual homologues be expressed, but they may be expressed in combination with other proteins as fusion proteins or as portions of larger precursor proteins. For example, it is possible to express WO 98/27805 PCT/AU97/00874 32 the N-terminal region of MiAMP2 clone 1 (amino acids 1 to -246) which contains a signal peptide and the hydrophilic region containing four antimicrobial segments. Transgenic plants can then be assessed to examine whether the individual fragments are being processed into the expected fragments by the processing machinery already present in the plant cells. It is also possible to express the entire MiAMP2 clone 1 (amino acids 1 to 666) and to examine the processing of the entire protein when expressed in transgenic plants. Homologous regions from other sequences can also be used in multiple combinations with, for example, ten (10) or more MiAMP2-like fragments expressed as one large fusion protein with acidic cleavage sites located as proper locations between each of the fragments. As well as linking MiAMP2 fragments together, it would also be possible to link MiAMP2 fragments to other useful proteins for expression in plants.

Example 17 Transgenic plants expressing MiAMP2c (or related fragments) The disarmed Agrobacterium tumefaciens strain GV3101 (pMP90RK) (Koncz, Cs.[1986] Mol.

Gen. Genet. 204:383-396) was transformed with the vector pPCV91 -MiAMP2c (Example 16) using the method of Walkerpeach et al. (Plant Mol. Biol. Manual B1: 1-19 [1994]) adapted from Van Haute et al (EMBO J. 2:411-417 1983]).

Tobacco transformation was carried out using leaf discs of Nicotiana tabacum based on the method of Horsch et al. (Science 227:1229-1231 [1985]) and co-culturing strains containing pPCV91 -MiAMP2c. After co-cultivation ofAgrobacterium and tobacco leaf disks, transgenic plants (transfomed with pPCV91-MiAMP2c) were regenerated on media containing 50 aig/ml hygromycin and 500 pg/ml Cefotaxime. These transgenic plants were analysed for expression of the newlyintroduced genes using standard western blotting techniques (Figure 15). Figure 15 shows a western blot of extracts from trangenic tobacco carrying the construct for MiAMP2c from example 16. Lane 1 contains pure MiAMP2c as a standard, lanes 2 and 3 contain extracts from transgenic plants canying the pPCV91-MiAMP2c construct. As can be see in the figure, faint bands are present at approximately the correct molecular weight, indicating that the transgenic plants appear to be expressing the MiAMP2c protein. Plants capable of constitutive expression of the introduced genes may be selected and self-pollinated to give seed. Fl seedlings of the transgenic plants may be further analysed.

Example 18 MiAMP2c Homologues Every homologue of MiAMP2c that has been tested has exhibited some antimicrobial activity.

This evidence indicates that other homologues will also exhibit antimicrobial activity. These homologues include fragments from 1) peanut (Burks, A.W. et al. [1995] J. Clin. Invest. 96 WO 98/27805 PCT/AU97/00874 33 1715-1721), 2) maize (Belanger, F.C. and Kriz, A.L.[1991] Genetics 129 863-872), 3) barley (Heck, G.R. et al. [1993] Mol. Gen. Genet. 239 209-218), and 4) soybean (Sebastiani, F.L. et al. [1990] Plant Mol. Biol. 15 197-201). (see SEQ IDNOS: 21, 22, 24, and 25). Other sequences derived from seed storage proteins of the 7S class are also expected to yield homologues of MiAMP2 proteins.

WO 98/27805 PCT/AU97/00874 34 SEQUENCE LISTINGS GENERAL INFORMATION: (i)APPLICANT: NAME: COOPERATIVE RESEARCH CENTRE FOR TROPICAL PLANT

PATHOLOGY

STREET: The University of Queensland CITY: St Lucia STATE: Queensland COUNTRY: Australia POSTAL CODE (ZIP): 4067 (ii) TITLE OF INVENTION: Antimicrobial Protein (iii) NUMBER OF SEQUENCES: 28 (iv) COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (EPO) INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 666 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi ORIGINAL SOURCE: ORGANISM: Macadamia integrifolia TISSUE TYPE: Seeds (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: Met Ala Ile Asn Thr Ser Asn Leu Cys Ser Leu Leu Phe Leu Leu Ser 1 5 10 Leu Phe Leu Leu Ser Thr Thr Val Ser Leu Ala Glu Ser Glu Phe Asp 25 Arg Gin Glu Tyr Glu Glu Cys Lys Arg Gln Cys Met Gin Leu Glu Thr 35 40 Ser Gly Gin Met Arg Arg Cys Val Ser Gin Cys Asp Lys Arg Phe Glu 55 Glu Asp Ile Asp Trp Ser Lys Tyr Asp Asn Gin Glu Asp Pro Gin Thr WO 98/27805 PTA9/07 PCT/AU97/00874 Gin Cys Gin Gin Cys Gin Arg Arg Cys Arg Gin Cys Thr 145 Gin Giu Gin Gin Arg 225 Gin Arg Leu Tyr Thr 305 Ala Cys Asn Gin Giu Gin 130 Cys Gin Giu Tyr His 210 Gly Giu Ser Giu Arg 290 His Len Gly Arg Gin Gin 115 Lys Gin Lys Arg Gin 195 Gin Giy Giy Len Asn 275 Len Len Lys Asp Asp 355 Gin 100 Tyr His Gin Arg Met 180 Asp Cys Asp Giu Ser 260 Phe Val Asp Met Val1 340 Asn Tyr Asn Cys Arg Tyr 165 Lys Cys Gin Met Giu 245 Thr Tyr Len Ala Ile 325 Ile Asn Cys Arg Gin Cys 150 Giu Gin Arg Len Met 230 Gin Arg Giy Len Asp 310 His Arg Gin Gin Gin Arg 135 Gin Giu Giu Arg Arg 215 Asn Gin Phe Arg Giu 295 Ala His Ile Arg Arg Arg 120 Arg Arg Gin Asp Arg 200 Cys Pro Ser Arg Ser 280 Ala Ile Asp Pro Len 360 Arg 105 Asp Gin Arg Gin Asn 185 Cys Arg Gin Asp Thr 265 Lys Asn Len Asn Ala 345 His Arg 90 Cys Pro Thr Tyr Arg 170 Lys Gin Giu Arg Asn 250 Giu Len Pro Leu Arg 330 Giy Ile Gin Gin Gin Lys Gin Ile Gin Gin Gin 125 Gin Pro Arg 140 Gin Lys Gin 155 Gin Asp Gin Arg Asp Pro Gin Gin Gin 205 Gin Gin Arg 220 Gly Giy Ser 235 Pro Tyr Tyr Gin Gly His Len Arg Ala 285 Asn Ala Phe 300 Vai Ile Gly 315 Gin Ser Tyr Thr Thr Phe Ala Lys Phe 365 Ser Gly Cys Giu 110 Tyr Gin His Met Lys Arg Gin Lys 175 Gin Gin 190 Pro Arg Gin His Gly Arg Phe Asp 255 Ile Ser 270 Len Lys Vai Len Gly Arg Asn Len 335 Tyr Len 350 Len Gin Pro Gin Gin Gin Lys 160 Tyr Arg Gin Gly Tyr 240 Gin Val1 Asn Pro Giy 320 Gin Ile Thr WO 98/27805 PCT/AU97/00874 36 Ile Ser Thr Pro Gly Gin Tyr Lys Giu Phe Phe Pro Ala Gly Gly Gin 370 375 380 Asn Pro Giu Pro Tyr Leu Ser Thr Phe Ser Lys Glu Ile Leu Giu Ala 385 390 395 400 Ala Leu Asn Thr Gin Thr Giu Lys Leu Arg Gly Val Phe Gly Gin Gin 405 410 415 Arg Glu Gly Val Ile Ile Arg Ala Ser Gin Giu Gin Ile Arg Giu Leu 420 425 430 Thr Arg Asp Asp Ser Giu Ser Arg His Trp His Ile Arg Arg Gly Gly 435 440 445 Giu Ser Ser Arg Gly Pro Tyr Asn Leu Phe Asn Lys Arg Pro Leu Tyr 450 455 460 Ser Asn Lys Tyr Gly Gin Ala Tyr Giu Val Lys Pro Glu Asp Tyr Arg 465 470 475 480 Gin Leu Gin Asp Met Asp Leu Ser Val Phe Ile Ala Asn Val Thr Gin 485 490 495 Gly Ser Met Met Gly Pro Phe Phe Asn Thr Arg Ser Thr Lys Val Val 500 505 510 Val Val Ala Ser Gly Glu Ala Asp Val Glu Met Ala Cys Pro His Leu 515 520 525 Ser Gly Ary His Gly Gly Arg Giy Gly Gly Lys Arg His Giu Glu Giu 530 535 540 Giu Asp Val His Tyr Giu Gin Val Arg Ala Arg Leu Ser Lys Arg Giu 545 550 555 560 Ala Ile Val Val Leu Ala Gly His Pro Val Val Phe Val Ser Ser Gly 565 570 575 Asn Glu Asn Leu Leu Leu Phe Ala Phe Gly Ile Asn Ala Gin Asn Asn 580 585 590 His Glu Asn Phe Leu Ala Gly Arg Giu Arg Asn Val Leu Gin Gin Ile 595 600 605 Glu Pro Gin Ala Met Giu Leu Ala Phe Ala Ala Pro Arg Lys Glu Val 610 615 620 Giu Glu Ser Phe Asn Ser Gin Asp Gin Ser Ile Phe Phe Pro Gly Pro 625 630 635 640 Arg Gin His Gin Gin Gin Ser Pro Arg Ser Thr Lys Gin Gin Gin Pro 645 650 655 Leu Val Ser Ile Leu Asp Phe Val Gly Phe 660 665 WO 98/27805 PCT/AU97/00874 37 INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 2171 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: Macadamia integrifolia TISSUE TYPE: Seeds (ix) FEATURE: NAME/KEY: sigpeptide LOCATION:l.

FEATURE:

NAME/KEY: mat peptide LOCATION:86. .1999 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: ATGGCGATCA ATACATCAAA TTTATGTTCT CTTCTCTTTC TCCTTTCACT CTTCCTTCTG

TCTACGACAG

CGGCAATGCA

AAGAGATTTG

GAATGCCAAC

TACTGCCAAC

GATCCACAGC

CGTCACATGC

CAACAAAAGA

AAGGAAGAAG

TGCGAACAAC

AGGCAACACG

GAGGAGGGAG

TGTCTCTTGC

TGCAGTTGGA

AAGAGGATAT

AATGCCAGAG

GACGCTGCAA

AGCAATACGA

AAACATGTCA

GATATGAAGA

ATAACAAACG

AGGAGCCACG

GCCGAGGTGG

AAGAGGAGCA

TGAAAGTGAA

GACATCAGGC

AGATTGGTCT

GCGATGCAGG

GGAAATATGT

GCAATGTCAG

ACAACGCTGC

GCAACAACGT

CGATCCACAA

TCAGCAGCAC

CGATATGATG

AAGCGACAAC

TTTGACAGGC

CAGATGCGTC

AAGTATGATA

CAGCAGGAGA

GAAGAAGAAG

AAGCACTGCC

GAGAGGAGAT

GAAGACGAAG

CAAAGAGAGT

CAGTGCCAGC

AACCCTCAGA

CCCTACTACT

TCAGTTCTGG

AGGAATATGA

GGTGTGTGAG

ACCAAGAGGA

GTGGCCCACG

AAGAATATAA

AACGGCGCGA

ATGAAAAGGA

AGAAATATGA

ACGAAGACTG

TAAGATGCCG

GGGGAGGCAG

TCGACGAACG

AGAACTTCTA

GGAGTGCAAA

TCAGTGCGAT

TCCTCAGACG

TCAGCAACAA

CCGACAACGT

GACAGAGCCA

GAAACGTAAG

AGAGCGAATG

CCGGAGGCGC

AGAGCAGCAG

CGGCAGATAC

AAGCTTAAGT

TGGTAGATCC

120 180 240 300 360 420 480 540 600 660 720 780 840 ACAAGGTTCA GGACCGAGGA AGGCCACATC AAGCTTCTAC GCGCACTAAA AAACTATCGC TTGGTGCTCC TCGAGGCTAA CCCCAACGCC WO 98/27805 WO 9827805PCT/AU.97/00874

TTCGTGCTCC

GCCCTCAAAA

ATCAGAATCC

CACATAGCCA

GCTGGAGGCC

GCGCTCAACA

ATAATTAGGG

CACTGGCATA

AGGCCACTGT

CAACTCCAAG

GGTCCCTTCT

GTGGAAATGG

CATGAGGAGG

GCCATTGTTG

CTGCTTTTTG

GAGAGGALACG

AGGAAAGAGG

AGGCAGCACC

CTGGACTTCG

TAGCTCCTAG

CTACCCACTT

TGATCCACCA

CAGCTGGAAC

AGTTCTTACA

AAAACCCAGA

CACAAACAGA

CGTCACAGGA

TAAGGAGAGG

ACTCCAACAA

ACATGGACTT

TCAACACTAG

CATGCCCTCA

AAGAGGATGT

TTCTGGCAGG

CATTTGGAAT

TGCTGCAGCA

TAGAAGAGTC

AGCAACAGTC

TTGGCTTCT.A

CTCGGTGTAT

GGATGCAGAT

CGACAACAGA

CACATTCTAC

GACCATATCC

GCCGTACCTC

GAAGCTGCGT

GCAGATCAGG

TGGTGAATCA

ATACGGTCAA

ATCGGTTTTC

GTCTACAAAG

CTTGTCGGGA

GCACTATGAG

TCATCCCGTC

CAATGCCCAA

GATAGAGCCA

ATTTAACAGC

GCCCCGCTCC

AAGTTCCACA

GAGAGTGGTA

GCCATTCTCT

GAATCCTACA

TTAATCAACC

ACTCCTGGCC

AGTACCTTCA

GGGGTGTTTG

GAGTTGACTC

AGCAGGGGAC

GCCTACGAAG

ATAGCCAACG

GTGGTAGTGG

AGACACGGCG

CAGGTTAGAG

GTCTTCGTTT

AACAACCACG

CAGGCAATGG

CAGGACCAGT

ACCAAGCAAC

AAAAAGAGTG

AGAGACTAAG

TGGTCATAGG

ACCTCGAGTG

GAGACAACAA

AATACAAGGA

GCAAAGAGAT

GACAGCAAAG

GAGATGACTC

CTTACAATCT

TCAAACCTGA

TCACCCAGGG

TGGCTAGTGG

GCCGCGGTGG

CACGTTTGTC

CATCCGGAAA

AGAACTTCCT

AGCTAGCGTT

CTATCTTCTT

AACAGCCTCT

TGTTATGTAG

ACGCTAAATC

AGGGAGAGGA

TGGAGACGTA

CGAGAGGCTC

ATTCTTCCC.A

TCTCGAGGCT

GGAGGGAGTG

AGAGTCACGA

GTTCAACAAA~

GGACTACAGG

ATCCATGATG

AGAGGCAGAT

AGGAAAAAGG

GAAGAGAGAG

CGAGAACCTG

CGCGGGGAGA

TGCCGCTCCA

TCCTGGGCCC

CGTCTCCATT

TATAGGTTAG

CCTAAGTAAC

960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2171 TAACCTGGCG AGCTTGCGTG TATGCAAATA AAGAGGAACA GCTTTCCAAC TTTAAAAAAA AAAAAA A INFORMATION FOR SEQ ID NO: 3: SEQUENCE CHARACTERISTICS: LENGTH: 666 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein WO 98/27805 WO 9827805PCT/AU97/00874 39 (vi) ORIGINAL SOURCE: ORGANISM: Macadamia integrifolia TISSUE TYPE: Seeds (ix) FEATURE: NAME/KEY: sigpeptide LOCATION:i. .28 (ix) FEATURE: NAME/KEY: mat peptide LOCATION:29. .666 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: Met Ala Ile Asn Thr Ser Asn Leu Cys Ser Leu Leu Arg Ser Giu Asp Arg Giu Cys Thr 145 Gin Giu Giu Leu Glu Gin Ile Gin Gin Giu 115 Glu Gin Lys Arg Glu 195 Leu Tyr Met Asp Gin Gin 100 Tyr Arg Gin Arg Met 180 Asp Ser Giu Arg Trp Cys Tyr Asn Cys Arg Tyr 165 Lys Cys Thr Giu Arg Ser 70 Gin Cys Arg Gin Cys 150 Giu Giu Arg Thr Cys Cys Lys Arg Gin Gin Arg 135 Giu Giu Giu Arg Arg 215 Vali Lys Val Tyr Arg Arg Arg 120 His Arg Gin Asp Arg 200 Ser 25 Arg Ser Asp Cys Arg 105 Asp Giu Arg Gin Asn 185 Cys 10 Leu Gin Gin Asn Arg 90 Cys Pro Thr Tyr Arg 170 Lys Giu Ala Cys Cys Gin 75 Gin Lys Gin Giu Giu 155 Glu Arg Gin Leu Giu Met Asp Asp Gin Giu Gin Pro 140 Lys Asp Asp Gin Gin 220 Leu Giu Leu Arg Pro Ser Cys 110 Tyr His Lys Giu Gin 190 Pro Leu is Phe Giu Phe Gin Gly Glu Glu Met Arg Lys 175 Gin Arg Ser Asp Thr Giu Thr Pro Giu Gin Gin Lys 160 Tyr Arg Gin Gin Tyr 210 Gin Cys Gin Arg Cys Arg Glu Gin Arg Gin His Gly WO 98/27805 WO 9827805PCT/AU97/00874 Arg 225 Giu Arg Leu Tyr Thr 305 Ala Cys Asn Ile Asn 385 Ala Arg Thr Glu Ser 465 Gin Gly Val Gly Giu Ser Giu Arg 290 His Leu Gly Arg Ser 370 Pro Leu Giu Arg Ser 450 Asn Leu Ser Val Gly Gly Leu Asn 275 Leu Leu Lys Asp Asp 355 Thr Glu Asn Gly Asp 435 Ser Lys Gin Met Ala Asp Giu Ser 260 Phe Val1 Asp Met Val1 340 Asn Pro Pro Thr Val1 420 Asp Arg Tyr Asp Met 500 Ser Leu Ile 230 Giu Lys 245 Thr Arg Tyr Gly Leu Leu Ala Asp 310 Ile His 325 Ile Arg Asn Giu Gly Gin Tyr Leu 390 Gin Aia 405 Ile Ile Ser Giu Gly Pro Giy Gin 470 Met Asp 485 Gly Pro Gly Giu Asn Pro Gin Ser Phe Arg Arg Ser 280 Giu Ala 295 Ala Ile Arg Asp Ile Pro Arg Leu 360 Tyr Lys 375 Ser Thr Giu Arg Ser Ala Ser Arg 440 Tyr Asn 455 Ala Tyr Val Ser Phe Phe Ala Asp Gin Arg Asp Asn 250 Thr Giu 265 Lys Leu Asn Pro Leu Leu Asn Arg 330 Ala Gly 345 His Ile Glu Phe Phe Ser Leu Arg 410 Ser Gin 425 Arg Trp Leu Phe Glu Val Val Phe 490 Asn Thr 505 Val Glu Gly 235 Pro Giu Leu Asn Val1 315 Giu Thr Ala Phe Lys 395 Gly Giu His Asn Lys 475 Ile Arg Met Gly Tyr Gly Arg Ala 300 Thr Ser Thr Lys Pro 380 Glu Val Gin Ile Lys 460 Pro Ala Ser Ala Ser Tyr His Ala 285 Phe Gly Tyr Phe Phe 365 Al a Ile Leu Ile Arg 445 Arg Giu Asn Thr Cys Gly Phe Ile 270 Leu Val Gly Asn Tyr 350 Leu Gly Leu Giy Arg 430 Arg Pro Asp Ile Lys £510 Pro Arg Asp 255 Ser Lys Leu Arg Leu 335 Leu Gin Gly Giu Gin 415 Giu Gly Leu Tyr Thr 495 Val1 His Tyr 240 Giu Val Asn Pro Gly 320 Giu le Thr Gin Ala 400 Gin Leu Gly Tyr Arg 480 Gin Val Leu WO 98/27805 PCT/AU97/00874 Ser Gly 530 Glu Asp 545 Ala Ile Asn Glu His Glu Glu Pro 610 Glu Glu 625 Arg Gin Leu Val 515 Arg His Val His Val Val Asn Leu 580 Asn Phe 595 Gin Ala Leu Phe His Gin Ser Ile 660 Gly Tyr Pro 565 Leu Leu Met Asn Gin 645 Leu Gly Glu 550 Val Leu Ala Glu Ser 630 Gin Asp Arg 535 Gin Gly Phe Gly Leu 615 Gin Ser Phe 520 Arg Val His Ala Arg 600 Ala Asp Ser Val Gly Lys Pro Phe 585 Glu Phe Glu Arg Gly Gly Lys Ala Arg 555 Val Val 570 Gly Ile Arg Asn Ala Ala Ser lie 635 Ser Thr 650 Phe Arg 540 Leu Phe Asn Val Pro 620 Phe Lys 525 His Ser Val Ala Leu 605 Arg Phe Gin Glu Lys Ser Gin 590 Gin Lys Pro Gin Glu Arg Ser 575 Asn Gin Glu Gly Gin 655 Glu Glu 560 Gly Asn Ile Val Pro 640 Pro INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: LENGTH: 2171 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: Macadamia integrifolia TISSUE TYPE: Seeds (ix) FEATURE: NAME/KEY: sig_peptide LOCATION:1..86 (ix) FEATURE: NAME/KEY: mat peptide LOCATION:87..1999 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: ATGGCGATCA ATACATCAAA TTTATGTTCT CTTCTCTTTC TCCTTTCCCT CTTCCTTCTG WO 98/27805 WO 9827805PCT/AU97/00874

TCAACGACAG

CGGCAATGCA

AAGAGATTTG

GATTGCCAAC

TACTGCCAAC

GATCCACAGC

CGTCACATGC

CAACAAAAGA

AAGGAAGAAG

TGCGAACAAC

AGGCAACACG

GAGGAGGGAG

ACAAGGTTCA

AAGCTTCTAC

TTCGTGCTCC

GCCCTCAAAA

ATCAGAATCC

CACATAGCCA

GCTGGAGGCC

GCGCTCAACA

ATAATTAGTG

CGCTGGCATA

AGGCCACTGT

CAACTCCAAG

GGTCCCTTCT

GTGGAAATGG

CATGAGGAGG

TGTCTCTTGC

TGCAGTTGGA

AAGAGGATAT

AATGCCAGAG

GACGCTGCAA

AGCAATACGA

AAACATGTCA

GATATGAAGA

ATAACAAACG

AGGAGCCACG

GCCGAGGTGG

AAGAGAAGCA

GGACCGAGGA

GCGCACTAAA

CTACCCACTT

TGATCCACCG

CAGCTGGAAC

AGTTCTTACA

AAAACCCAGA

CACAAGCAGA

CGTCACAGGA

TAAGGAGAGG

ACTCCAACAA

ACATGGACGT

TCAACACTAG

CATGCCCTCA

AAGAGGATGT

TGAAAGTGAA

GACATCAGGC

AGATTGGTCT

GCGATGCAGG

GGAAATATGT

GCAATGTCAG

ACAACGCTGC

GCAACAACGT

CGATCCACAA

TCAGCAGTAC

TGATTTGATT

AAGCGACAAC

AGGCCACATC

AAACTATCGC

GGACGCAGAT

TGACAACAGA

CACATTCTAC

GACCATATCC

GCCGTACCTC

GAGGCTGCGT

GCAGATCAGG

TGGTGAATCA

ATACGGTCAA

ATCGGTTTTC

GTCTACAAAG

CTTGTCGGGA

GCACTATGAG

TTTGACAGGC

CAGATGCGTC

AAGTATGATA

CAGCAGGAGA

GAAGAAGAAG

GAGCGCTGCC

GAGAGGAGAT

GAAGACGAAG

CAAAGAGAGT

CAGTGCCAGC

AACCCTCAGA

CCCTACTACT

TCAGTTCTGG

TTGGTGCTCC

GCCATTCTCT

GAATCCTACA

TTAATCAACC

ACTCCTGGCC

AGTACCTTCA

GGGGTGCTTG

GAGTTGACTC

AGCAGGGGAC

GCCTACGAALG

ATAGCCAACA

GTGGTAGTGG

AGACACGGCG

CAGGTTAAAG

AGGAATATGA

GGTGTGTGAG

ACCAAGACGA

GTGGCCCACG

AAGAATATAA

AACGGCACGA

ATGAAAAGGA

AGAAATATGA

ACGAAGACTG

GAAGATGCCG

GGGGAGGCAG

TCGACGAACG

AGALACTTCTA

TCGAGGCTAA

TGGTCACCGG

ACCTCGAGTG

GAGACAACAA

AATACAAGGA

GCAAAGAGAT

GACAGCAAAG

GAGATGACTC

CTTACAATCT

TCAAACCTGA

TCACCCAGGG

TGGCTAGTGG

GCCGCCGTGG

CACGTTTGTC

GGAGTGCAAA

TCAGTGCGAT

TCCTCAGACG

TCAGCAACAA

CCGACAACGT

GACAGAGCCA

GAAACGTAAG

AGAGCGAATG

CCGGAGGCGC

AGAGCAGCAG

CGGCAGATAC

AAGCTTAAGT

TGGTAGATCC

CCCCAACGCC

AGGGAGAGGA

TGGAGACGTA

CGAGAGGCTC

ATTCTTCCCA

TCTCGAGGCT

GGAGGGAGTG

AGAGTCACGA

GTTCAACAAA

GGACTACAGG

ATCCATGATG

AGAGGCAGAT

AGGGAAAAGG

GAAGAGAGAG

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 GCCATTGTTG TTCCGGTAGG TCATCCCGTC GTCTTCGTTT CATCCGGAAA CGAGAACCTG WO 98/27805 WO 9827805PCT/AU97/00874 CTGCTTTTTG CATTTGGAAT GAGAGGAACG TGCTGCAGCA.

AGGAAAGAGG TAGAAGAGTT AGGCAGCACC AGCAACAGTC CTGGACTTCG TTGGCTTCTA TAGCTCCTAG CTCGGTGTAT TAACCTGGCG AGCTTGCGTG AAAA~A~AA A

CAATGCCCAA

GATAGAGCCA

ATTTAACAGC

TTCCCGCTCC

AAGTTCTACA

GCGAGTGGTA

TATGCAAATA

AACAACCACG

CAGGCAATGG

CAGGACGAGT

ACCAAGCAAC

AAAAAGAGTG

AGAGACCAAG

AAGAGGAACA

AGAACTTCCT

AGCTAGCGTT

CTATCTTCTT

AACAGCCTCT

TGTTATGTAG

ACGCTAAATC

GCTTTCCAAC

CGCGGGGAGA

TGCCGCTCCA

TCCTGGGCCC

CGTCTCCATT

TATAGGTTAG

CCTAAGTAAC

TTTAAAAAAA

1800 1860 1920 1980 2040 2100 2160 2171 INFORMATION FOR SEQ ID NO: Wi SEQUENCE CHARACTERISTICS: LENGTH: 625 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: Macadamia integrifolia TISSUE TYPE: Seeds (ix) FEATURE: NAME/KEY: partial mat peptide LOCATION:1. .625 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Gin Cys Met Gin Leu Glu Thr Ser Gly Gin Met 1 5 10 Gin Cys Asp Lys Arg Phe Glu Giu Asp Ile Asp 25 Asn Gin Glu Asp Pro Gin Thr Giu Cys Gin Gin 40 Arg Gin Gin Giu Ser Asp Pro Arg Gin Gin Gin 55 Cys Lys Giu Ile Cys Glu Giu Giu Giu Giu Tyr 70 75 Pro Gin Gin Gin Tyr Giu Gin Cys Gin Lys Arg Arg Arg Cys Val Ser Trp Ser Lys Tyr Asp Cys Gin Arg Arg Cys Tyr Cys Asn Arg Cys Gin Gin Arg Arg Gin Arg Asp Arg Arg Giu WO 98/27805 WO 9827805PCT/AU97/00874 Thr Giu Pro Arg His Met Gin Ile Cys Gin Gin Arg Cys Glu Tyr Arg Lys 145 Giu 165 Glu Arg Asn Giu 230 Leu Pro Leu Arg Gly 310 Ile Phe Ser Arg Giu Giu 130 Arg Gin Gin Gly Pro 215 Giu Leu Asn Val Giu 295 Thr Ala Phe Lys Gly 375 Lys 115 Asp Asp Gin Gin Gly 200 Tyr Gly Arg Ala Ile 280 Ser Thr Lys Pro Giu 360 Val 100 Giu Glu Pro Giu Arg 185 Ser Tyr His Ala Phe 265 Gly Tyr Phe Phe Al a 345 Ile Leu Lys Giu Gin Pro 170 Gin Gly Phe Ile Leu 250 Vai Gly Asn Tyr Leu 330 Gly Leu Gly Arg Lys Gin 150 Arg His Arg Asp Ser 235 Lys Leu Arg Leu Leu 315 Gin Gly Giu Gin Lys Tyr 135 Arg Leu Gly Tyr Giu 220 Val Asn Pro Gly Giu 300 Ile Thr Gin Ala Gln 380 Gin 120 Giu Giu Gin Arg Giu 205 Arg Leu Tyr Thr Ala 285 Cys Asn Ile Asn Ala 365 Arg 105 Gin Glu Tyr Tyr Gly 190 Giu Ser Glu Arg His 270 Leu Gly Arg Ser Pro 350 Leu Glu Lys Arg Glu Gin 175 Gly Gly Leu Asn Leu 255 Leu Lys Asp Asp Thr 335 Glu Asn Gly Arg Met Asp 155 Cys Asp Giu Ser Phe 240 Val1 Asp Met Val1 Asn 320 Pro Pro Thr Val Tyr Lys 140 Cys Gin Leu Glu Thr 225 Tyr Leu Ala Ile Ile 305 Asn Gly Tyr Gin Ile 385 110 Giu Gly Arg Arg Asn 195 Gin Phe Arg Giu Ala 275 Arg Ile Arg Tyr, Ser 355 G lu Arg Arg Gin Asp His Cys 180 Pro Ser Arg Ser Ala 260 Ile Asp Pro Leu Lys 340 Thr Arg Ala Arg Gin Asn Cys 160 Gin Gin Asp Thr Lys 245 Asn Leu Asn Ala His 325 Glu Phe Leu Ser Gin Giu Gin Ile Arg Giu Leu Thr Arg Asp Asp Ser Giu Ser Arg Arg 390 395 400 405 WO 98/27805 WO 9827805PCT/A1J97/00874 Trp His Ile Arg Arg Gly Gly Glu 410 Phe Val1 Phe Thr 480 Glu Gly Ala Val1 Gly 565 Arg Ala Ser Ser Asn Lys Ile 455 Arg Met Lys Arg Val1 550 Ile Asn Ala Ile Thr Lys Pro 440 Al a Ser Ala Arg Leu 535 Phe Asn Val Ser Phe 615 Lys Arg 425 Giu Asn Thr Cys His 520 Ser Val Ala Leu Arg 600 Phe Gin Pro Asp Ile Lys Pro 505 Giu Lys Ser Gin Gin 585 Lys Pro Gin Tyr Arg Gin 460 Val Leu Glu Giu Giy 555 Asn Ile Vai Pro Pro Ser Gin 445 Gly Val1 Ser Glu Ala 540 Asn His Giu Giu Arg 620 Leu Ser Asn 430 Leu Ser Val1 Gly Glu 525 Ile Giu Glu Pro Glu 605 Gin Val Ser 415 Lys Gin Met Ala Arg 510 Val Val1 Asn Asn Gin 590 Leu His Ser Arg Tyr Asp Met Ser 490 His His Val1 Leu Phe 575 Ala Phe Gin Ile Gly Gly Met Gly 470 Gly Gly Tyr Leu Leu 560 Leu Met Asn Gin Leu 640 Pro Gin Asp 450 Pro Giu Giy Glu Ala 545 Len Ala Giu Ser Gin 625 Asp Tyr Ala 435 Val Phe Ala Arg Gin 530 Gly Phe Gly Leu Gin 610 Ser Phe Asn 420 Tyr Ser Phe Asp Gly 515 Val1 His Al a Arg Ala 595 Asp Pro Val Leu Glu Val Asn Val1 500 Gly Arg Pro Phe Gin 580 Phe Gin Arg Gly 630 INFORMATION FOR SEQ ID NO: 6: (i)SEQUENCE CHARACTERISTICS: LENGTH: 2140 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE- ORGANISM: Macadamia integrifolia WO 98/27805 WO 9827805PCT/AU97/00874 TISSUE TYPE: Seeds

FEATURE:

NAME/KEY: partial mat peptide LOCATION:l. .1875 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: CAATGCATGC AGTTAGAGAC ATCAGGCCAG ATGCGTCGGT GTGTGAGTCA GTGCGATAAG

AGATTTGAAG

TGCCAACAAT

TGCCAACGAC

CCACAGCAGC

CACATGCAAA

CAAAAGAGAT

GAAGGAGATA

GAACAACAGG

CAACACGGCC

GAGGGAGAAG

AGGTTCAGGA

CTTCTACGCG

GTGCTCCCTA

CTCAAAATGA

AGAATCCCAG

ATAGCCAAGT

GGAGGCCAAA

CTCAACACAC

ATTAGGGCGT

TGGCATATAA

CCACTGTACT

CTCCA-AGACA

AGGATATAGA

GCCAGAGGCG

GCTGCAAGGA

AATACGAGCA

TATGTCAACA

ATGAAGAGCA

ACAAACGCGA

AGCCACGTCT

GAGGTGGCGA

AGAAGCAAAG

CCGAGGAAGG

CACTAAAAAA

CCCACTTGGA

TCCACCGTGA

CTGGAACCAC

TCTTACAGAC

ACCCAGAGCC

AAACAGAGAG

CACAGGAGCA

GGAGAGGTGG

CCAACAAATA

TGGACGTATC

TTGGTCTAAG

ATGCAGGCAG

AATATGTGAA

ATGTCAGAAG

ACGCTGCGAG

ACAACGTGAA

TCCACAACAA

GCAGTACCAG

TTTGATGAAC

CGACAACCCC

CCACATCTCA

CTATCGCTTG

TGCAGATGCC

CAACAGAGAA

ATTCTACTTA

CATATCCACT

GTACCTCAGT

GCTGCGTGGG

GATCAGGGAG

TGAATCAAGC

CGGTCAAGCC

AGTTTTCATA

TATGATAACC

CAGGAGAGTG

GAAGAAGAAG

CGCTGCCAAC

AGGAGATATG

GACGAAGAGA

AGAGAGTACG

TGCCAGCGAA

CCTCAGAGGG

TACTACTTCG

GTTCTGGAGA.

GTGCTCCTCG

ATTCTCTTGG

TCCTACAACC

ATCAACCGAG

CCTGGCCAAT

ACCTTCAGCA

GTGCTTGGAC!

TTGACTCGAG

AGGGGACCTT

TACGAAGTCA

GCCAACATCA

AAGAGGATCC

ACCCACGTCA

AATATAACCG

GGCGCGAGAC

AAAAGGAGAA

AATATGAAGA

AAGACTGCCG

GATGCCAAGA

GAGGCAGCGG

ACGAACGAAG

ACTTCTATGG

AGGCTAACCC

TCATCGGAGG

TCGAGTGTGG

ACAACAACGA

ACAAGGAATT

AAGAGATTCT

AGCAAAGGGA

ATGACTCAGA

ACAATCTGTT

AACCTGAGGA

CCCAGGGATC

TCAGACGGAA

GCAACAATAC

ACAACGTGAT

AGAGCCACGT

ACGTAAGCAA

GCGAATGAAG

GCGGCACTGC

GCAGCAGAGG

CAGATACGAG

CTTAAGTACA

TAGATCCAAG

CAACGCCTTC

GAGAGGAGCC

AGACGTAATC

GAGGCTCCAC

CTTCCCAGCT

CGAGGCTGCG

GGGAGTGATA

GTCACGACGC

CAACAAAAGG

CTACAGGCAA

CATGATGGGT

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 CCCTTCTTCA. ACACTAGGTC TACAAAGGTG GTAGTGGTGG CTAGTGGAGA GGCAGATGTG WO 98/27805 WO 9827805PCT/AU97/00874

GAAATGGCAT

GAGGAGGAAG

ATTGTTGTTC

CTTTTTGCAT

AGGAACGTGC

AAAGAGGTAG

CAGCACCAGC

GACTTCGTTG

CTCCTAGCTC

CCTGGCGAGC

TTTTTTTTTT

AAAAAAA

GCCCTCACTT

AGGAGGTGCA

TGGCAGGTCA

TTGGAATCAA

TGCAGCAGAT

AAGAGTTATT

AACAGTCGCC

GCTTCTAAAG

GGTGTATGAG

TTGCGTGTAT

TTTTTTCTTT

GTCGGGAAGA

CTATGAGCAG

TCCCGTCGTC

TGCCCAAAAC

AGAGCCACAG

TAACAGCCAG

CCGCTCCACC

TTCTACAAAA

AGTGGTAAGA

GCAAATAAAG

CTTTTTCTTA

CACGGCGGCC

GTTAGAGCAC

TTCGTTTCAT

AACCACGAGA

GCAATGGAGC

GACGAGTCTA

AAGCAACAAC

AAGAGTGTGT

GACTAAGACG

AGGAACAGCT

AGAAATAAAC

GCGGTGGAGG

GTTTGTC!GAA

CCGGAAACGA

ACTTCCTCGC

TAGCGTTTGC

TCTTCTTTCC

AGCCTCTCGT

TATGTAGTAT

CTAAATCCCT

TTCCAACTTT

GAACGTAGAT

GAAAAGGCAT

GAGAGAGGCC

AAACCTGCTG

GGGGAGAGAG

CGCTTCAAGG

TGGGCCCAGG

CTCCATTCTG,

AGGTTAGTAG

AAGTAACTAA

AGAAAGCTCT

TGCGGCTCAA

1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2140 INFORMATION FOR SEQ ID NO: 7: SEQUENCE CHARACTERISTICS: LENGTH: 525 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: Theobroma cacao TISSUE TYPE: Seeds (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: Met Val Ile Ser Lys Ser Pro Phe Ile Val Leu Ile Phe 1 5 10 Leu Ser Phe Ala Leu Leu Cys Ser Gly Val Ser Ala Tyr 25 Gin Tyr Glu Arg Asp Pro Arg Gln Gin Tyr Glu Gin Cys 40 Cys Glu Ser Glu Ala Thr Glu Glu Arg Glu Gln Glu Gin 55 Arg Cys Glu Arg Glu Tyr Lys Glu Gin Gin Arg Gin Gin 70 Ser Leu Leu Gly Arg Lys Gin Arg Arg Cys Glu Gin Glu Glu WO 98/27805 WO 9827805PCT/AU97/00874 48 Leu Gin Arg Gin Tyr Gin Gin Cys Gin Gly Arq Cys Gin Giu Gin Gin 90 Gin Gly Gin Arg Giu Gin Gin Gin Cys Gin Arg Lys Cys Trp Giu Gin 100 105 110 Tyr Lys Giu Gin Giu Arg Gly Giu His Giu Asn Tyr His Asn His Lys 115 120 125 Lys Asn Arg Ser Giu Giu Giu Giu Giy Gin Gin Arg Asn Asn Pro Tyr 130 135 140 Tyr Phe Pro Lys Arg Arg Ser Phe Gin Thr Arg Phe Arg Asp Giu Giu 145 150 155 160 Giy Asn Phe Lys Ile Leu Gin Arg Phe Ala Glu Asn Ser Pro Pro Leu 165 170 175 Lys Giy Ile Asn Asp Tyr Arg Leu Aia Met Phe Giu Ala Asa Pro Asn 180 185 190 Thr Phe Ile Leu Pro His His Cys Asp Ala Giu Ala Ile Tyr Phe Vai 195 200 205 Thr Asn Gly Lys Gly Thr Ile Thr Phe Val Thr His Giu Asn Lys Giu 210 215 220 Ser Tyr Asn Val Gin Arg Gly Thr Val Val Ser Vai Pro Aia Gly Ser 225 230 235 240 Thr Val Tyr Val Val Ser Gin Asp Asn Gin Giu Lys Leu Thr Ile Ala 245 250 255 Val Leu Ala Leu Pro Vai Asn Ser Pro Gly Lys Tyr Giu Leu Phe Phe 260 265 270 Pro Ala Gly Asn Asn Lys Pro Giu Ser Tyr Tyr Gly Ala Phe Ser Tyr 275 280 285 Giu Val Leu Giu Thr Val Phe Asn Thr Gin Arg Giu Lys Leu Giu Giu 290 295 300 Ile Leu Giu Glu Gin Arg Giy Gin Lys Arg Gin Gin Gly Gin Gin Gly 305 310 315 320 Met Phe Arg Lys Ala Lys Pro Giu Gin Ile Arg Aia Ile Ser Gin Gin 325 330 335 Ala Thr Ser Pro Arg His Arg Gly Giy Giu Arg Leu Ala Ile Asn Leu 340 345 350 Leu Ser Gin Ser Pro Val Tyr Ser Asn Gin Asn Gly Arg Phe Phe Giu 355 360 365 Ala Cys Pro Glu Asp Phe Ser Gin Phe Gin Asn Met Asp Val Ala Val 370 375 380 WO 98/27805 PCT/AU97/00874 49 Ser Ala Phe Lys Leu Asn Gin Gly Ala Ile Phe Val Pro His Tyr Asn 385 390 395 400 Ser Lys Ala Thr Phe Val Val Phe Val Thr Asp Gly Tyr Gly Tyr Ala 405 410 415 Gin Met Ala Cys Pro His Leu Ser Arg Gin Ser Gin Gly Ser Gin Ser 420 425 430 Gly Arg Gin Asp Arg Arg Glu Gin Glu Glu Glu Ser Glu Glu Glu Thr 435 440 445 Phe Gly Glu Phe Gin Gin Val Lys Ala Pro Leu Ser Pro Gly Asp Val 450 455 460 Phe Val Ala Pro Ala Gly His Ala Val Thr Phe Phe Ala Ser Lys Asp 465 470 475 480 Gin Pro Leu Asn Ala Val Ala Phe Gly Leu Asn Ala Gin Asn Asn Gin 485 490 495 Arg Ile Phe Leu Ala Gly Arg Pro Phe Phe Leu Asn His Lys Gin Asn 500 505 510 Thr Asn Val Ile Lys Phe Thr Val Lys Ala Ser Ala Tyr 515 520 525 INFORMATION FOR SEQ ID NO: 8: SEQUENCE CHARACTERISTICS: LENGTH: 590 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: Gossypium hirsutum TISSUE TYPE: Seeds (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Met Val Arg Asn Lys Ser Ala Cys Val Val Leu Leu Phe Ser Leu Phe 1 5 10 Leu Ser Phe Gly Leu Leu Cys Ser Ala Lys Asp Phe Pro Gly Arg Arg 20 25 Gly Asp Asp Asp Pro Pro Lys Arg Tyr Glu Asp Cys Arg Arg Arg Cys 40 Glu Trp Asp Thr Arg Gly Gin Lys Glu Gin Gin Gin Cys Glu Glu Ser 55 WO 98/27805 WO 9827805PCT/AU97/00874 Cys Giu Gin Phe Gin Vai 145 Arg Gin Phe Arg Giu 225 Lys His Val1 Lys Phe 305 Arg Giu Gin Lys Asp Giu Giu His 130 Arg Giu Ser Arg His 210 Aia Ile Giu Pro Leu 290 Giu Aia Gin Giy Ser Pro Giu Gin 115 Cys Giu Giu His Giu 195 Pro Asn Tyr Asn Ala 275 Ile Giu Phe Leu Gin Gin Gin Arg 100 Giu His Cys Giu Asn 180 Giu Ile Pro Leu Lys 260 Gly Ile Phe Ser Asp 340 Giy Tyr Arg Gin Gin Gin Arg Aia 165 Pro His Leu Asn Val1 245 Giu Ser Ala Phe Arg 325 Giu Met Giy Giu 70 Arg Tyr Gin Pro Gin Gin Gin Glu 135 Giu Lys 150 Giu Giu Phe His Gly Asn Arg Giy 215 Thr Phe 230 Thr Asn Ser Tyr Thr Vai Vai Leu 295 Pro Ala 310 Giu Ile Leu Phe Phe Arg Lys Giu Gin Ser 120 Gin Tyr Giu Phe Phe 200 Ile Vali Giy Asn Tyr 280 His Gly Leu Giy Lys Asp Giu Cys 105 Gin Arg Gin Giu His 185 Arg Asn Leu Arg Ile 265 Leu Arg Ser Giu Gly 345 Ala Gin Cys 90 Gin Arg Pro Giu Thr 170 Arg Vali Giu Pro Giy 250 Vai Ala Pro Gin Pro 330 Arg Ser Gin 75 Gin Gin Gin Giu Asn 155 Giu Arg Leu Phe His 235 Thr Pro Asn Val1 Arg 315 Al a Gin Gin Gin Gin Arg Phe Lys 140 Pro Giu Ser Gin Arg 220 His Leu Giy Gin Asn 300 Pro Phe Ser Glu Arg Glu Cys Gin 125 Lys Trp Gly Phe Arg 205 Leu Cys Thr Vali Asp 285 Asn Gin Asn Arg Gin His Cys Leu 110 Giu Gin Arg Glu Gin 190 Phe Ser Asp Phe Vai 270 Asn Pro Ser Thr Arg 350 Ile Arg Arg Lys Cys Gin Giy Gin 175 Ser Ala Ile Ala Leu 255 Vai Lys Gly Tyr Arg 335 Arg Arg Pro Gin Arg Gin Cys Giu 160 Giu Arg Ser Leu Glu 240 Thr Lys Giu Gin Leu 320 Ser Gin Ala WO 98/27805 WO 9827805PCT/AU97/00874 Leu Ser 370 Ala Phe 385 Arg Phe Asn Val Pro His Asn Gly 450 Tyr Giu 465 Giu Giu Arg Gly Ala Ser Gin Asn 530 Ile Asn 545 Gly Val Giu Ser 355 Gin Asn Phe Thr Tyr 435 Tyr Giu Arg Asp Gin 515 Ile His Ser Tyr Glu Aia Leu Leu Giu Aia 405 Val Ser 420 Asn Ser Ala Giu Glu Giu Ar Ser 485 Ile Phe 500 Asn Gin Asn Pro Val Arg Ser Arg 565 Phe Val Thr Ser 390 Cys Ala Lys Met Glu 470 Gly Val Asn Asp Gin 550 Leu S er Ser 375 Gin Pro Leu Aia Val 455 Giu Gin Val1 Leu His 535 Trp Val1 Arg 360 Pro Thr Pro Gin Thr 440 Ser Asp Tyr Pro Arg 520 Asn Asp Asp Gin Arg Pro Giu Leu 425 Phe Pro Giu Arg Aia 505 Met Gin Ser Giu Arg 585 Glu Arg Phe 410 Asn Vai His Giu Lys 490 Asn Thr Arg Gin Ile 570 Gin Lys Tyr 395 Arg Gin Ile Leu Giu 475 Ile Phe Gly Ile Ala 555 Phe Arg Ser 380 Ser Gin Giy Leu Pro 460 Giu Arg Pro Phe Phe 540 Lys Asn Ala 365 Gly Asn Leu Ser Val1 445 Arg Gin Ser Val1 Gly 525 Vali Giu Ser Ser Giu Gin Arg Ile 430 Thr Gin Giu Arg Thr Leu Aia Leu Asn Giu 590 Arg Asn Asp 415 Phe Giu Ser Gin Leu 495 Phe Tyr Gly Ala Pro 575 Phe Giy 400 Ile Val Gly Ser Giu 480 Ser Val Asn.

Lys Phe 560 Gin INFORM4ATION FOR SEQ ID NO: 9: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 22 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: WO 98/27805 PCT/AU97/00874 52 Arg Gin Arg Asp Pro Gin Gin Gin Ala Glu Gin Ala Gin Lys Arg Ala 1 5 10 Gin Arg Arg Glu Thr Glu INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 25 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Pro Arg His Met Gin Ile Ala Gin Gin Arg Ala Glu Arg Arg Ala Glu 1 5 10 Lys Glu Lys Arg Lys Gin Gin Lys Arg INFORMATION FOR SEQ ID NO: 11: SEQUENCE CHARACTERISTICS: LENGTH: 30 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: Met Ala Trp Phe His Val Ser Val Cys Asn Ala Val Phe Val Val Ile 1 5 10 Ile Ile Ile Met Leu Leu Met Phe Val Pro Val Val Arg Gly 25 INFORMATION FOR SEQ ID NO: 12: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: nucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: WO 98/27805 PCT/AU97/00874 53 CAGCAGCAGT ATGAGCAGTG INFORMATION FOR SEQ ID NO: 13: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: TTTTTCGTAK CKKCKTTCGC A 21 INFORMATION FOR SEQ ID NO: 14: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: ACACCATATG CGACAACGTG ATCC 24 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CGTTGTTTTC TCTATTCCTA GGGTTG 26 INFORMATION FOR SEQ ID NO: 16: SEQUENCE CHARACTERISTICS: LENGTH: 22 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear WO 98/27805 PCT/AU97/00874 54 (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: Met Gly His His His His His His His His His His Ser Ser Gly His 1 5 10 Ile Glu Gly Arg His Met -4 INFORMATION FOR SEQ ID NO: 17: SEQUENCE CHARACTERISTICS: LENGTH: 90 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17 GGGAATTCCA TATGTATGAG CGTGATCCTC GACAGCAATA CGAGCAATGC CAGAGGCGAT GCGAGTCGGA AGCGACTGAA GAAAGGGAGC INFORMATION FOR SEQ ID NO: 18 SEQUENCE CHARACTERISTICS: LENGTH: 91 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: GAAGCGACTG AAGAAAGGGA GCAAGAGCAG TGTGAACAAC GCTGTGAAAG GGAGTACAAG GAGCAGCAGA GACAGCAATA GGGATCCACA C 91 INFORMATION FOR SEQ ID NO: 19 SEQUENCE CHARACTERISTICS: LENGTH: 101 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA WO 98/27805 PCT/AU97/00874 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: GGGAATTCCA TATGCTTCAA AGGCAATACC AGCAATGTCA AGGGCGTTGT CAAGAGCAAC AACAGGGGCA GAGAGAGCAG CAGCAGTGCC AGAGAAAATG C INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 102 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GTGTGGATCC CTAGCTCCTA TTTTTTTTGT GATTATGGTA ATTCTCGTGC TCGCCTCTCT CTTGTTCCTT ATATTGCTCC CAGCATTTTC TCTGGCACTG CT 101 102 INFORMATION FOR SEQ ID NO: 21: SEQUENCE CHARACTERISTICS: LENGTH: 42 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: Peanut TISSUE TYPE: Seeds (xi) SEQUENCE DESCRIPTION: SEQ Met Arg Gly Arg Val Ser Pro Leu Met 1 5 Leu Ala Ser Val Ser Ala Thr Gin Ala ID NO: 21: Leu Leu Leu 10 Lys Ser Pro Gin Ser Cys Cys 25 Leu Gly Ile Leu Val Tyr Arg Lys Thr Gin Gin Glu Pro Thr Lys Leu Glu Glu Asn Pro Asp Asp Leu Ala Gin Arg Cys 40 Lys Gin Lys Tyr Asp 65 Ala 55 Tyr Cys Glu Ser Arg Cys Asp Thr Gly Ala Thr Pro Arg Cys Asn Gin Arg WO 98/27805 PTA9/07 PCT/AU97/00874 56 Pro Pro Gly Giu Arg Thr Arg Gly Arg Gin Pro Gly Asp Tyr Asp Asp Asp Glu Asp Gly 145 Glu 165 Ser Phe Val Asp 230 Val1 Ala Asp Pro Ser 310 Ala Gly Asp Arg Arg Pro Arg 115 Trp Arg 130 Arg Giu Giu Thr Thr Arg Asp Gin 200 Gin Ile 215 Ala Asp Ala Asn Leu Arg Asn Gin 280 Gly Gin 295 Tyr Leu Giu Phe Giu Gin Asn Glu 360 Gin 100 Giu Arg Gly Ser Tyr 185 Arg Giu Asn Gly Ile 265 Asn Phe Gin Asn Giu 345 Gly Pro Arg Pro Giu Arg 170 Gly Ser Ala Ile Asn 250 Pro Leu Glu Gly Giu 330 Glu Val1 Arg Arg Giu Arg Ser His 135 Gin Glu 150 Asn Asn Asn Gin Lys Gin Arg Pro 220 Leu Val 235 Asn Arg Ser Gly Arg Val Asp Phe 300 Phe Ser 315 Ile Arg Arg Gly Ile Val Glu Giu 120 Gin Trp Pro Asn Phe 205 Asn Ile Lys Phe Ala 285 Phe Arg Arg Gin Lys 365 Giu 105 Glu Gin Gly Phe Gly 190 Gin Thr Gin Ser Ile 270 Lys Pro Asn Val Arg 350 Val 90 Gly Gly Asp Trp Pro Arg Thr Pro 155 Tyr Phe 175 Arg Ile Asn Leu Leu Val Gin Gly 240 Phe Asn 255 Ser Tyr Ile Ser Ala Ser Thr Leu 320 Leu Leu 335 Arg Arg Ser Lys Arg Trp Arg Gin 125 Lys Ile 140 Gly Ser Pro Ser Arg Val Gin Asn 210 Leu Pro 225 Gin Ala Leu Asp Ile Leu Met Pro 290 Ser Arg 305 Giu Ala Giu Giu Ser Thr Giu His 370 Gly Pro 110 Pro Arg Arg Pro Giu Val Arg Arg 180 Leu Gin 195 His Arg Lys His Thr Val Giu Gly 260 Asn Arg 275 Val Asn Asp Gin Aia Phe..

Asn Ala 340 Arg Ser 355 Val Gin Ala Giu Giu Arg 160 Phe Arg Ile Ala Thr 245 His His Thr Ser Asn 325 Gly Ser Glu Leu Thr Lys His Ala Lys Ser Val Ser Lys Lys Giy Ser Glu Giu Giu 375 380 385 WO 98/27805 PCT/AU97/00874 57 Asp Ile Thr Asn Pro Ile Asn Leu Arg Asp Gly Glu Pro Asp Leu Ser 390 395 400 405 Asn Asn Phe Gly Arg Leu Phe Glu Val Lys Pro Asp Lys Lys Asn Pro 410 415 420 Gin Leu Gin Asp Leu Asp Met Met Leu Thr Cys Val Glu Ile Lys Glu 425 430 435 Gly Ala Leu Met Leu Pro His Phe Asn Ser Lys Ala Met Val Ile Val 440 445 450 Val Val Asn Lys Gly Thr Gly Asn Leu Glu Leu Val Ala Val Arg Lys 455 460 470 Glu Gin Gin Gin Arg Gly Arg Arg Glu Gin Glu Trp Glu Glu Glu Glu 480 485 490 500 Glu Asp Glu Glu Glu Glu Gly Ser Asn Arg Glu Val Arg Arg Tyr Thr 505 510 515 Ala Arg Leu Lys Glu Gly Asp Val Phe Ile Met Pro Ala Ala His Pro 520 525 530 Val Ala Ile Asn Ala Ser Ser Glu Leu His Leu Leu Gly Phe Gly Ile 535 540 545 Asn Ala Glu Asn Asn His Arg Ile Phe Leu Ala Gly Asp Lys Asp Asn 550 555 560 Val Ile Asp Gin Ile Glu Lys Gin Ala Lys Asp Leu Ala Phe Pro Gly 565 570 575 580 Ser Gly Glu Gin Val Glu Lys Leu Ile Lys Asn Gin Arg Glu Ser His 585 590 595 Phe Val Ser Ala Arg Pro Gin Ser Gin Ser Pro Ser Ser Pro Glu Lys 600 605 610 Glu Asp Gin Glu Glu Glu Asn Gin Gly Gly Lys Gly Pro Leu Leu Ser 615 620 625 Ile Leu Lys Ala Phe Asn 630 INFORMATION FOR SEQ ID NO: 22: SEQUENCE CHARACTERISTICS: LENGTH: 46 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein WO 98/27805 58 (vi) ORIGINAL SOURCE: ORGANISM: Maize TISSUE TYPE: Seeds (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: PCT/AU97/00874 Met Val Ser Ala Arg Ile Val Val Ala Gly Trp Giu Glu Asp Arg Ser Ala 145 Ile 165 Gly Pro Leu Gin 230 Ser Ala Giy His Lys Gly Arg Ser Arg 130 Asn Cys Giu Ala Val 215 Phe Phe Ala His 35 Gin Arg Ser Arg Giu 115 Leu Pro Tyr Arg Gly 200 Ile Phe Ser Val Lys Arg Gin Ser Pro 100 Gin Leu Arg Vali Arg 185 Ala Thr Phe Lys Ala Ser Pro Giu Giu Tyr Gly Arg Ser Al a 170 Ser Val Lys Gly Ser Ser Gly Arg Arg 70 Asp Val Ser Gly Phe 150 Giu Tyr Thr Ile Pro 235 Ile Ser Gin Cys 55 Ser Giu Phe Leu Ile 135 Val1 Gly Thr Tyr Leu 220 Gly Gin Trp Cys 40 Leu Arg Arg Asp Arg 120 Arg Val1 Giu Ile Leu 205 His Gly Arg Leu Giu 25 Val Giu His Giu Arg 105 Val Asp Pro Gly Lys 190 Ala Thr Arg Ala Leu Asp Arg Gin Giu Gin 90 Arg Leu Tyr Ser Val 175 Gin Asn Ile Asn Ala Ala Asp Arg Cys Ala 75 Giu Ser Arg Arg His 155 Val1 Gly Thr Ser Pro 240 Tyr Thr Asn Cys Arg Asp Lys Phe Pro Val 140 Thr Thr His Asp Val 225 Glu Lys Leu His Giu Glu Asp Giu Arg Phe 125 Ala Asp Thr Val1 Gly 210 Pro Ser Thr Leu His Asp Giu Arg Lys Arg 110 Asp Val Ala Ile Phe 195 Arg Gly Phe S er Cys His Arg Giu Ser Gin Val1 Giu Leu His Giu 180 Val1 Lys Giu Leu Ser Ala His Pro Arg Gly Lys Val1 Val Giu Cys 160 Asn Ala Lys Phe Ser 245 Asp 250 260 Arg Leu Giu Arg Leu Phe Gly Arg His Gly Gin Asp Lys Gly Ile Ile WO 98/27805 WO 9827805PCT/AU97/00874 Val1 Glu Arg 310 His Glu Ser Asn Gly 390 Glu His Ala Ile Ala 480 Leu Arg Giu Arg Glu 565 Arg Gly 295 Gly Gly His Ala Gly 375 Gly Glu Thr Gly Val1 455 Gly Ser Arg Giu Gly 550 Arg 265 Ala Thr 280 Gly His Pro Tyr Gin Leu Asp Val 345 Pro Leu 360 Lys Gly Glu Ser Giu Glu Ile Arg 425 His Pro 440 Cys Phe Ala Asp Phe Ala Glu Lys 520 Arg Giu 535 Giu Arg Giu Gly Giu Gly Ser Tyr 330 Ser Phe Tyr Glu Ser 410 Ala Phe Giu Asn Ser 505 Gly Gin Glu Arg Glu Pro Leu 315 Giu Val1 Asn Ala Arg 395 Ser Arg Val Val Val1 485 Lys Phe Giu Arg His 570 Gin His 300 Leu Ala Ser Thr Glu 380 Glu Giu Leu Ala His 460 Leu Ala Leu Glu His 555 Gly Thr 285 Trp Asp Asp Phe Arg 365 Ile Arg Glu Ser Val 445 Ala Gin Giu Pro Glu 540 Gly Gly 270 Arg Pro Gin Ala Ala 350 Ser Val Asp Gin Pro 430 Ala Asp Lys Glu Gly 525 Glu Arg Arg Glu Leu Arg Arg 335 Asn Phe Cys Lys Glu 415 Gly Ser Arg Leu Val1 510 Pro Arg Glu Glu Leu Pro Pro 320 Ser Ile Lys Pro Gly 400 Glu Thr Arg Asn Asp 490 Asp Glu Glu Glu Glu 575 Arg Pro 305 Ser Phe Thr Ile His 385 Arg Ala Ala Asp Giu 470 Arg Giu Giu Giu Arg 560 Arg Arg 290 Phe Ile His Ala Ala 370 Arg Arg Giy Phe Ser 450 Lys Val Val Ser Arg 545 Giu Glu 275 His Gly Ala Asp Gly 355 Tyr Gin Ser Gin Val 435 Asn Val Ala Leu Gly 530 His Lys Glu Ala Glu Asn Leu 340 Ser Val1 Ser Glu Gly 420 Val1 Leu Phe Lys Gly 515s Gly Gly Glu Glu WO 98/27805 PCT/AU97/00874 Arg His Gly Arg Gly Arg Arg Glu Glu Val Ala Glu Thr Leu Met Arg 585 590 595 Met Val Thr Ala Arg Met 600 INFORMATION FOR SEQ ID NO: 23: SEQUENCE CHARACTERISTICS: LENGTH: 33 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: Maize TISSUE TYPE: Seeds (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: Arg Ser Gly Arg Gly Glu Cys Arg Arg Gin Cys Leu Arg Arg His Glu 1 5 10 Gly Gln Pro Trp Glu Thr Gln Glu Cys Met Arg Arg Cys Arg Arg Arg 25 Gly INFORMATION FOR SEQ ID NO: 24: SEQUENCE CHARACTERISTICS: LENGTH: 42 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: Barley TISSUE TYPE: Seeds (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: Met Ala Thr Arg Ala Lys Ala Thr Ile Pro Leu Leu Phe Leu Leu Gly 1 5 10 Thr Ser Leu Leu Phe Ala Ala Ala Val Ser Ala Ser His Asp Asp Glu 25 Asp Asp Arg Arg Gly Gly His Ser Leu Gin Gin Cys Val Gin Arg Cys 40 WO 98/27805 WO 9827805PCT/AU97/00874 Arg Arg Gly Gly Gly Gly Gly 145 Gly 165 Gly Ile Gin Met 230 Asp GinU Val Arg Lys 310 Arg Leu Gin Asp Arg Arg Arg Arg 130 Arg Arg Asp Ile Val1 215 Giu Gly Asn Ala Lys 295 Phe Val Phe *Giu Asp Giy Giy Gly 115 Gly Gly Ser Gin 200 Ser Vali Val1 Gly Pro 280 Leu Gin Leu Asn Arg Gin Arg Arg 100 Arg His Arg Arg Arg 185 Ser Arg Asn Gly Gin 265 Ala Val Phe Arg Gin Pro Gin Gly Gly Giy Gly Giy Gly 170 Arg Asp Leu Pro Tyr 250 Lys Giy Ile Leu Ala 330 Arg Arg Gin 70 Trp Arg Arg Arg Arg 150 Arg Pro His Leu Arg 235 Val Arg Ser Ala Ser 315 Ala Gin Tyr 55 His His His His His 135 His Arg Tyr Gly Arg 220 Ala Ala Ser Ile Lys 300 Val Phe Gly Ser Gly Gly Gly Gly 120 Gly Gly Giy Val1 Phe 205 Gly Phe Gin Tyr Met 285 Ile Lys Lys Gin His Arg Gin Gin 105 Giu Gin Gin Giu Phe 190 Val Ile Val1 Gly Thr 270 His Leu Pro Thr Giu Ala Arg Cys Val Gin Giu Cys His Giy 90 Giy Gly Gly Giy Gly 175 Gly Arg Arg Val1 Gin 255 Vai Leu His Leu Ser 335 Lys Gin 75 Giu Gin Giu Giu Glu 155 Gin Pro Al a Asp Pro 240 Gly Lys Al a Thr Leu 320 Asp Thr Gin Arg Arg Arg Arg 140 Arg Arg Arg Leu Tyr 225 Giy Val Gin Asn Ile 305 Ala Gin Arg Giu Gin Glu Gin 125 Giu Giu Asp Ser Arg 210 Arg Phe Len Gdy Thr 290 Ser Ser Arg Ser Giu Gin Giu 110 Giu Gin Giu Giu Phe 195 Pro Val Thr Thr Asp 275 Asp Vai Leu Len Vl Gin Gin Gin Gin Gin Gin Gin 180 Arg Phe Aia Asp Val 260 Val1 Gly Pro Ser Gin 340 Ser Gin His His Arg Arg Giu 160 Gin Arg Asp Ile Ala 245 Ile Ile Arg Gly Lys 325 Arg Ile WO 98/27805 WO 9827805PCT/AU97/00874 62 345 350 355 Val Arg Ala Ser Glu Glu Gin Leu Arg Glu Leu Arg Arg Glu Ala Ala 360 365 370 Glu Gly Gly Gin Gly His Arg Trp, Pro Leu Pro Pro Phe Arg Gly Asp 375 380 385 Ser Arg Asp Thr Phe Asn Leu Leu Glu Gin Arg Pro Lys le Ala Asn 390 395 400 405 Arg His Gly Arg Leu Tyr Glu Ala Asp Ala Arg Ser Phe His Ala Leu 410 415 420 Ala Asn Gin Asp Val Arg Val Ala Val Ala Asn Ile Thr Pro Gly Ser 425 430 435 Met Thr Ala Pro Tyr Leu Asn Thr Gin Ser Phe Lys Leu Ala Val Val 440 445 450 Leu Giu Gly Giu Gly Giu Val Gin Ile Val Cys Pro His Leu Gly Arg 455 460 470 Giu Ser Giu Ser Giu Arg Giu His Gly Lys Gly Arg Arg Arg Glu Giu 480 485 490 500 Giu Giu Asp Asp Gin Arg Gin Gin Arg Arg Arg Gly Ser Glu Ser Giu 505 510 515 Ser Giu Giu Glu Glu Glu Gin Gin Arg Tyr Glu Thr Val Arg Ala Arg 520 525 530 Val Ser Arg Gly Ser Ala Phe Val Val Pro Pro Gly His Pro Val Val 535 540 545 Giu Ile Ser Ser Ser Gin Gly Ser Ser Asn Leu Gin Val Val Cys Phe 550 555 560 Giu Ile Asn Ala Giu Arg Asn Glu Arg Val Trp Leu Ala Gly Arg Asn 565 570 575 580 Asn Val Ile Gly Lys Leu Gly Ser Pro Ala Gin Glu Leu Thr Phe Gly 585 590 595 Arg Pro Ala Arg Giu Val Gin Giu Val Phe Arg Ala Gin Asp Gin Asp 600 605 610 Glu Gly Phe Val Ala Gly Pro Giu Gin Gin Ser Arg Giu Gin Glu Gin 615 620 625 Giu Gin Glu Arg His Arg Arg Arg Gly Asp Arg Gly Arg Gly Asp Giu 630 635 640 Ala Val Giu Thr Phe Leu Arg Met Ala Thr Gly Ala le 645 650 655 WO 98/27805 PCT/AU97/00874 63 INFORMATION FOR SEQ ID NO: Wi SEQUENCE CHARACTERISTICS: LENGTH: 55 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: Soybean (Glycine max) TISSUE TYPE: Seeds (Xi) SEQUENCE DESCRIPTION: SEQ Met Met Arg Ala Arg Phe Pro Leu Ala Pro Tyr Lys Gin Glu Gln Glu Asp 145 Gin 165 Arg Ser Lys Arg Giu His Asp Glu Giu 130 Giu Lys Glu Val1 His 35 Asn Giu Pro Giu Glu 115 Lys Glu Glu Ser Ser Asn Gin Cys Giu Gin 100 Giu Arg Gin Giu Glu 185 Val Lys Ala Giu Arg Pro His Gly Asp Arg 170 Glu Ser Cys Cys Glu 70 Giu Arg Glu Giu Glu 150 Asn Ser Phe Leu His Gly Pro Pro Gin Lys 135 Arg Glu Glu Gly Gin 40 Ala Glu Gin Ile Arg 120 Gly Gin Giu Asp Leu Ile 25 Ser Arg Ile Gin Pro 105 Glu Ser Phe Giu Ser 190 ID NO: Leu Leu Gly Ala Tyr Trp Cys Asn Ser Cys Asn Leu Pro Arg Pro 75 Pro Gly Glu 90 Phe Pro Arg Giu Gin Giu Glu Glu Giu 140 Pro Phe Pro 155 Asp Giu Asp 175 Giu Leu Arg Leu Glu Glu Leu Arg Lys Pro Trp 125 Asp Arg Glu Arg Val Lys Arg Lys Pro Glu Gin 110 Pro Glu Pro Giu His 195 Phe Leu Giu Asn Asp Ser Val Giu Arg Pro Giu Asp Pro Arg Arg Lys Asp Giu Pro His 160 Gin Gin 180 Lys Asn Lys Asn Pro Phe Leu Phe Gly Ser Asn Arg Phe Giu Thr Leu Phe Lys u u 205 WO 98/27805 PCT/AU97/00874 64 Asn Gin Tyr Gly Arg Ile Arg Val Leu Gin Arg Phe Asn Gin Arg Ser 215 220 225 Pro Gin Leu Gin Asn Leu Arg Asp Tyr Arg Ile Leu Giu Phe Asn Ser 230 235 240 245 Lys Pro Asn Thr Leu Leu Leu Pro Asn His Aia Asp Aia Asp Tyr Leu 250 255 260 Ile Val Ile Leu Asn Gly Thr Ala Ile Leu Ser Leu Val Asn Asn Asp *265 270 275 Asp Arg Asp Ser Tyr Arg Leu Gin Ser Gly Asp Ala Leu Arg Val Pro 280 285 290 Ser Gly Thr Thr Tyr Tyr Val Val Asn Pro Asp Asn Asn Giu Asn Leu 295 300 305 Arg Leu Ile Thr Leu Ala Ile Pro Val Asn Lys Pro Gly Arg Phe Giu 310 315 320 325 Ser Phe Phe Leu Ser Ser Thr Giu Ala Gin Gin Ser Tyr Leu Gin Gly 330 335 340 Phe Ser Arg Asn Ile Leu Giu Ala Ser Tyr Asp Thr Lys Phe Giu Giu 345 350 355 Ile Asn Lys Val Leu Phe Ser Arg Giu Giu Gly Gin Gin Gin Gly Giu 360 365 370 Gin Arg Leu Gin Giu Ser Vai Ile Vai Gu Ile Ser Lys Giu Gin Ile 375 380 385 Arg Ala Leu Ser Lys Arg Ala Lys Ser Ser Ser Arg Lys Thr Ile Ser 390 395 400 405 Ser Giu Asp Lys Pro Phe Asn Leu Arg Ser Arg Asp Pro Ile Tyr Ser 410 415 420 Asn Lys Leu Giy Lys Phe Phe Giu Ile Thr Pro Giu Lys Asn Pro Gin 425 430 435 Leu Arg Asp Leu Asp Ile Phe Leu Ser Ile Val Asp Met Asn GiuGiy 440 445 450 Ala Leu Leu Leu Pro His Phe Asn Ser Lys Ala Ile Vai Ile Leu Val 455 460 470 Ile Asn Giu Gly Asp Ala Asn Ile Giu Leu Val Gly Leu Lys Giu Gin 480 485 490 500 Gin Gin Giu Gin Gin Gin Giu Giu Gin Pro Leu Giu Val Arg Lys Tyr 505 510 515 Arg Ala Glu Leu Ser Glu Gin Asp Ile Phe Val Ile Pro Ala Giy Tyr 520 525 530 WO 98/27805 PCT/AU97/00874 Pro Val Val 535 Val Asn Ala Thr Ser 540 Asn Leu Asn Phe Phe 545 Ala Ile Gly S Ile Asn 550 Ala Glu Asn Asn Gin 555 Arg Asn Phe Leu Gly Ser Gin Asp Val Ile Ser Gin Ile 570 Pro Ser Gin Val Gin 575 Glu Leu Ala Phe Gly Ser Ala Gin Ala 585 Val Glu Lys Leu Leu 590 Lys Asn Gin Arg Glu Ser 595 Tyr Phe Val Arg Lys Gly 615 Asp 600 Ala Gin Pro Lys Lys 605 Lys Glu Glu Gly Asn Lys Gly 610 Pro Leu Ser Ser Ile 620 Leu Arg Ala Phe Tyr 625 INFORMATION FOR SEQ ID NO: 26: SEQUENCE CHARACTERISTICS: LENGTH: 23 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: Stenocarpus sinuatus TISSUE TYPE: Seeds (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: Val Lys Glu Asp His Gin Phe Glu Thr Arg Gly Glu Ile Leu Glu Cys 1 5 10 Tyr Arg Leu Cys Gin Gln Gin (28) INFORMATION FOR SEQ ID NO: 27: SEQUENCE CHARACTERISTICS: LENGTH: 17 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: Stenocarpus sinuatus TISSUE TYPE: Seeds WO 98/27805 PCT/AU97/00874 66 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: Gin Lys His Arg Ser Gin Ile Leu Gly Cys Tyr Leu Xxx cys Gin Gin 1 5 10 Leu INFORMATION FOR SEQ ID NO: 28: SEQUENCE CHARACTERISTICS: LENGTH: 28 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: Stenocarpus sinuatus TISSUE TYPE: Seeds (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: Leu Asp Pro Ile Arg Gin Gin Gin Leu Cys Gin Met Arg Cys Gin Gin 1 5 10 Gin Glu Lys Asp Pro Arg Gin Gin Gin Gin Cys Lys 20

Claims (12)

1. selected from: CLAIMS A protein fragment having antimicrobial activity, wherein said protein fragment is a polypeptide having an amino acid sequence selected from: residues 29 to 73 of SEQ ID NO: 1 residues 74 to 116 of SEQ ID NO: 1 residues 117 to 185 of SEQ ID NO: 1 residues 186 to 248 of SEQ ID NO: 1 residues 29 to 73 of SEQ ID NO: 3 residues 74 to 116 of SEQ ID NO: 3 residues 117 to 185 of SEQ ID NO: 3 residues 186 to 248 of SEQ ID NO: 3 residues 1 to 32 of SEQ ID NO: residues 33 to 75 of SEQ ID NO: residues 76 to 144 of SEQ ID NO: residues 145 to 210 of SEQ ID NO: residues 34 to 80 of SEQ ID NO: 7 residues 81 to 140 of SEQ ID NO: 7 residues 33 to 79 of SEQ ID NO: 8 residues 80 to 119 of SEQ ID NO: 8 residues 120 to 161 of SEQ ID NO: 8 residues 32 to 91 of SEQ ID NO: 21 residues 25 to 84 of SEQ ID NO: 22 residues 29 to 94 of SEQ ID NO: 24 residues 31 to 85 of SEQ ID NO: residues 1 to 23 of SEQ ID NO: 26 residues 1 to 17 of SEQ ID NO: 27 residues I to 28 of SEQ ID NO: 28; (ii) a homologue of (iii) a polypeptide containing a relative cysteine spacing of C-2X-C-3X-C-(10-12)X-C-3X- C-3X-C wherein X is any amino acid residue, and C is cysteine; (iv) a polypeptide containing a relative cysteine and tyrosine/phenylalanine spacing of Z- 2X-C-3X-C-(10-12)X-C-3X-C-3X-Z wherein X is any amino acid residue, and C is cysteine, and Z is tyrosine or phenylalanine; AMENDED SHEET IPEA/AU 68 a polypeptide containing a relative cysteine spacing of C-3X-C-(10-12)X-C-3X-C wherein X is any amino acid residue, and C is cysteine; (vi) a polypeptide with substantially the same spacing of positively charged residues relative to the spacing of cysteine residues of any one of the polypeptides of(i); and (vii) a fragment of the polypeptide of any one of to (vi) which has substantially the same antimicrobial activity as

2. A protein containing at least one polypeptide fragment according to claim 1, wherein said polypeptide fragment has a sequence selected from within a sequence comprising SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO:

3. A protein having a sequence selected from SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO:

4. An isolated or synthetic DNA encoding a polypeptide fragment according to claim 1. The DNA according to claim 4, wherein said DNA has a sequence selected from SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6.

6. A DNA construct which includes a DNA according to claim 4 operatively linked to elements for the expression of said encoded protein.

7. A transgenic plant harbouring a DNA construct according to claim 6.

8. The transgenic plant according to claim 7, wherein said plant is a monocotyledonous plant or a dicotyledonous plant. 20 9. The transgenic plant according to claim 7, wherein said plant is selected from maize, banana, peanut, field peas, sunflower, tomato, canola, tobacco, wheat, barley, oats, potato, soybeans, cotton, carnations, roses, or sorghum. Reproductive material of a transgenic plant according to claim 7.

11. A composition comprising an antimicrobial protein according to claim 1 together with 25 an agriculturally-acceptable carrier diluent or excipient.

12. A composition comprising an antimicrobial protein according to claim 1 together with an pharmaceutically-acceptable carrier diluent or excipient.

13. A method of controlling microbial infestation of a plant, the method comprising: i) treating said plant with an antimicrobial protein according to claim 1 or a composition according to claim 11; or ii) introducing a DNA construct according to claim 6 into said plant.

14. A method of controlling microbial infestation of a mammalian animal, the method comprising treating the animal with an antimicrobial protein according to claim 1 or a composition according to claim 12. WO 98/27805 PCT/AU97/00874 69 The method of claim 14, wherein said mammalian animal is a human.

16. A method of preparing an antimicrobial protein, which method comprises the steps of: a) obtaining or designing an amino acid sequence which forms a helix-turn-helix structure; b) replacing individual residues to achieve substantially the same distribution of positively charged residues and cysteine residues as in one or more of the amino acid sequences shown in Figure 4; c) synthesising a protein comprising said amino acid sequence chemically or by recombinant DNA techniques in liquid culture; and d) if necessary, forming disulphide linkages between said cysteine residues.