DNA sequences encoding antifungal proteins

Abstract

The present invention provides DNA sequences encoding antifungal peptides which comprise at least six amino acid residues identical to a run of amino acid residues found between position 21 and position 51 of the Rs-AFP2 antifungal protein sequence (SEQ ID NO: 35) or a substantially homologous protein. The peptides are useful for combating fungal diseases in agricultureal, pharmaceutical or perservative applications

Description

[0001] This application is a divisional of U.S. application Ser. No. 09/077,948 filed Aug. 7, 1998, which is a 371 of PCT/GB96/03068, filed Dec. 12, 1996, which claims the benefit of foreign application Serial Nos. GB9545255.3, filed Dec. 13, 1995, and GB9606552.9, filed Mar. 28, 1996, all of which are incorporated herein by reference in their entirety.

[0002] This invention relates to antifungal proteins, processes for their manufacture and use, and DNA sequences encoding them.

[0003] In this context, antifungal proteins are defined as proteins or peptides possessing antifungal activity. Activity includes a range of antagonistic effects such as partial inhibition or death.

[0004] A wide range of antifungal proteins with activity against plant pathogenic fungi have been isolated from certain plant species. We have previously described a class of antifungal proteins capable of isolation from radish and other plant species. These proteins are described in the following publications which are specifically incorporated herein by reference: International Patent Application Publication Number WO93/05153 published 18 Mar. 1993; Terras FRG et al, 1992, J Biol Chem, 267:15301-15309; Terras et al, 1993, FEBS Lett, 316:233-240; Terras et al, 1995, Plant Cell, 7:573-588. The class includes Rs-AFP1 (antifungal protein 1), Rs-AFP2, Rs-AFP3 and Rs-AFP4 from Raphanus sativus and homologous proteins such as Bn-AFP1 and Bn-AFP2 from Brassica napus, Br-AFP1 and Br-AFP2 from Brassica rapa, Sa-AFP1 and Sa-AFP2 from Sinapis alba, At-AFP1 from Arabidopsis thaliana, Dm-AMP1 and Dm-AMP2 from Dahlia merckii, Cb-AMP1 and Cb-AMP2 from Cnicus benedictus, Lc-AFP from Lathyrus cicera, Ct-AMP1 and Ct-AMP2 from Clitoria ternatea. The proteins specifically inhibit a range of fungi and may be used as fungicides for agricultural or pharmaceutical or preservative purposes.

[0005] It has been proposed that this class of antifungal proteins should be named plant defensins (Terras F. R. G. et al 1995, Plant Cell, 7 573-583) and these proteins have in common a similar motif of conserved cysteines and glycines (Broekaert W. F. et al 1995, Plant Physiol. 108 1353-1358).

[0006] FIG. 1 shows the amino acid sequences of the protein Rs-AFP1 (SEQ ID NO: 34) and the substantially homologous proteins Rs-AFP2 (SEQ ID NO: 35), Rs-AFP3 (SEQ ID NO: 36), Rs-AFP4 (SEQ ID NO: 37), Br-AFP1 (SEQ ID NO: 38), Br-AFP2 (SEQ ID NO: 39), Bn-AFP1 (SEQ ID NO: 40), Bn-AFP2 (SEQ ID NO: 41), Sa-AFP1 (SEQ ID NO: 42), Sa-AFP2 (SEQ ID NO: 43) and At-AFP1 (SEQ ID NO: 44) which are small 5 kDa polypeptides that are highly basic and rich in cysteine. FIG. 1 numbers the positions of the amino acid residues: the dash (-) at the start of the Rs-AFP3 (SEQ ID NO: 36) sequence indicates a gap introduced for maximum alignment. The sequences shown for Br-AFP1 (SEQ ID NO: 38), Br-AFP2 (SEQ ID NO: 39), Bn-AFP1 (SEQ ID NO: 40), Bn-AFP2 (SEQ ID NO: 41), Sa-AFP1 (SEQ ID NO: 42), Sa-AFP2 (SEQ ID NO: 43) and At-AFP1 (SEQ ID NO: 44) are not complete: only the N-terminal sequences are shown. The question mark (?) in the Br-AFP2 (SEQ ID NO: 39) sequence indicates a non-standard amino acid which the sequencing could not assign and which is thought to be a post-translational modification on one of the standard amino acid residues.

[0007] Further examples of antifungal plant defensins are described in International Patent Application Publication Number WO95/18229 published 6 Jul. 1995 which is specifically incorporated herein by reference. These examples include Hs-AFP1 (SEQ ID NO: 56), an antifungal protein capable of isolation from seeds of Heuchera species and AH-AMP1 (SEQ ID NO: 57), an antimicrobial protein capable of isolation from seeds of Aesculus hippocastanum. The proteins specifically inhibit a range of fungi and may be used as fungicides for agricultural or pharmaceutical or preservative purposes.

[0008] FIG. 9 shows the amino acid sequences of the proteins HS-AFP1 (SEQ ID NO: 56) and Ah-AMP1 (SEQ ID NO: 57). FIG. 9 numbers the positions of the amino acid residues. The Hs-AFP1 sequence shows 48% sequence identity with Rs-AFP1. The Ah-AMP1 sequence shows 54% sequence identity with Rs-AFP1. Hs-AFP1 shows 52% identity to Ah-AMP1 on the amino acid sequence level.

[0009] The primary structures of the two antifungal protein isoforms capable of isolation from radish seeds, Rs-AFP1 (SEQ ID NO: 34) and Rs-AFP2 (SEQ ID NO: 35), only differ at two positions: the glutamic acid residue (E) at position 5 in Rs-AFP1 is a glutamine residue (Q) in Rs-AFP2, and the asparagine residue (N) at position 27 in Rs-AFP 1 is substituted by an arginine residue (R) in Rs-AFP2. As a result, Rs-AFP2 has a higher net positive charge (+2) at physiological pH. Although both Rs-AFPs are 94% identical at the amino acid sequence level, Rs-AFP2 is two- to thirty-fold more active than Rs-AFP1 on various fungi and shows an increased salt-tolerence. The proteins Rs-AFP3 (SEQ ID NO: 36) and Rs-AFP4 (SEQ ID NO: 37) are found in radish leaves following localized fungal infection. The induced leaf proteins are homologous to Rs-AFP1 and Rs-AFP2 and exert similar antifungal activity in vitro.

[0010] The cDNA encoding Rs-AFP1 (SEQ ID NO: 45) encodes a preprotein with a signal peptide followed by the mature protein. The cDNA sequence is shown in FIG. 2. Saccharomyces cerevisiae can be used as a vector for the production and secretion of Rs-AFP2 (Vilas Alves et al, FEBS Lett, 1994, 348:228-232). Plant-derivable “wild-type” Rs-AFP2 can be correctly processed and secreted by yeast when expressed as a N-terminal fusion to the yeast mating factor &agr;1 (MF&agr;1) preprosequence. The Rs-AFP2 protein does not have adverse effects on yeast even at concentrations as high as 500 &mgr;g/ml.

[0011] We now provide new potent antifungal peptides based on the structure of the Rs-AFPs and related plant defensins.

[0012] According to the first aspect of the present invention there is provided an antifungal peptide which comprises at least six amino acid residues identical to a run of amino acid residues found between position 21 and position 51 of the Rs-AFP2 sequence shown in FIG. 1 (SEQ ID NO: 35, amino acids 21-51) or of substantially homologous protein sequences.

[0013] Proteins which are substantially homologous to the Rs-AFP2 protein include the proteins Rs-AFP1 (SEQ ID NO: 34), Rs-AFP3 (SEQ ID NO: 36), Rs-AFP4 (SEQ ID NO: 37), Br-AFP1 (SEQ ID NO: 38), Br-AFP2 (SEQ ID NO: 39), Bn-AFP1 (SEQ ID NO: 40), Bn-AFP2 (SEQ ID NO: 41), Sa-AFP1 (SEQ ID NO: 42), Sa-AFP2 (SEQ ID NO: 43) and At-AFP1 (SEQ ID NO: 44) shown in FIG. 1 and Hs-AFP2 (SEQ ID NO: 56), Ah-AMP1 (SEQ ID NO: 57) and Dm-AMP1 (SEQ ID NO: 58) shown in FIG. 9. Proteins which are substantially homologous have an amino acid sequence with at least 40% sequence identity to any of the sequences shown in FIGS. 1 and 9, and preferably at least 60% identity; and most preferably at least 80% identity.

[0014] Antifungal peptides according to the invention include especially peptides derived from the beta-2 strand/turn/beta-3 strand region of Rs-AFP2 and substantially homologous antifungal protein sequences. Preferred antifungal peptides according to the invention include the 6-mer, 9-mer and 12-mer, 13-mer, 14-mer, 15-mer, 16-mer, 17-mer, 18-mer, 19-mer and 20-mer, and most especially the 18-mer, 19-mer, and 20-mer peptides described in the accompanying examples, figures and tables especially Example 11 and FIGS. 10 to 13.

[0015] Antifungal peptides according to the invention include the following peptides: a peptide comprising fifteen amino acid residues identical to a run of fifteen amino acid residues found between position 21 and position 35 of the Rs-AFP2 sequence shown in FIG. 1 and having the sequence: CKNQCIRLEKARHGS (SEQ ID NO: 1); a peptide comprising fifteen amino acid residues identical to a run of fifteen amino acid residues found between position 25 and position 39 of the Rs-AFP2 sequence shown in FIG. 1 and having the sequence: CIRLEKARHGSCNYV (SEQ ID NO: 2); a peptide comprising fifteen amino acid residues identical to a run of fifteen amino acid residues found between position 29 and position 43 of the Rs-AFP2 sequence shown in FIG. 1 and having the sequence: EKARHGSCNYVPAH (SEQ ID NO: 3); a peptide comprising fifteen amino acid residues identical to a run of fifteen amino acid residues found between position 33 and position 47 of the Rs-AFP2 sequence shown in FIG. 1 and having the sequence: HGSCNYVFPAHKCIC (SEQ ID NO: 4); a peptide comprising ten amino acid residues identical to a run often amino acid residues found between position 36 and position 45 of the Rs-AFP2 sequence shown in FIG. 1 and having the sequence: CNYVFPAHKC (SEQ ID NO: 5); a peptide comprising six amino acid residues identical to a run of six amino acid residues found between position 40 and position 45 of the Rs-AFP2 sequence shown in FIGS. 1 and 15 having the sequence: FPAHKC (SEQ ID NO: 6); a peptide comprising six amino acid residues identical to a run of six amino acid residues found between position 42 and position 47 of the Rs-AFP2 sequence shown in FIG. 1 and having the sequence: AHKCIC (SEQ ID NO: 7); a peptide comprising six amino acid residues identical to a run of six amino acid residues found between position 43 and position 48 of the Rs-AFP2 sequence shown in FIG. 1 and having the sequence: HKCICY (SEQ ID NO: 8); a peptide comprising nine amino acid residues identical to a run of nine amino acid residues found between position 24 and position 32 of the Rs-AFP2 sequence shown in FIG. 1 and having the sequence: QCIRLEKAR (SEQ ID NO: 9); a peptide comprising nine amino acid residues identical to a run of nine amino acid residues found between position 25 and position 33 of the Rs-AFP2 sequence shown in FIG. 1 and having the sequence: CIRLEKARH (SEQ ID NO: 10); a peptide comprising nine amino acid residues identical to a run of nine amino acid residues found between position 32 and position 40 of the Rs-AFP2 sequence shown in FIG. 1 and 30 having the sequence: RHGSCNYVF (SEQ ID NO: 11); a peptide comprising nine amino acid residues identical to a run of nine amino acid residues found between position 36 and position 44 of the Rs-AFP2 sequence shown in FIG. 1 and having the sequence: CNYVFPAHK (SEQ ID NO: 12); a peptide comprising nine amino acid residues identical to a run of nine amino acid residues found between position 40 and position 48 of the Rs-AFP2 sequence shown in FIG. 1 and having the sequence: FPAHKCICY (SEQ ID NO: 13); a peptide comprising nine amino acid residues identical to a run of nine amino acid residues found between position 41 and position 49 of the Rs-AFP2 sequence shown in FIG. 1 and having the sequence: PAHKCICYF (SEQ ID NO: 14); a peptide comprising nine amino acid residues identical to a run of nine amino acid residues found between position 42 and position 50 of the Rs-AFP2 sequence shown in FIG. 1 and having the sequence: AHKCICYFP (SEQ ID NO: 15); a peptide comprising nine amino acid residues identical to a run of nine amino acid residues found between position 43 and position 51 of the Rs-AFP2 sequence shown in FIG. 1 and 15 having the sequence: HKCICYFPC (SEQ ID NO: 16); a peptide comprising twelve amino acid residues identical to a run of twelve amino acid residues found between position 25 and position 36 of the Rs-AFP2 sequence shown in FIG. 1 and having the sequence: CIRLEKARHGSC (SEQ ID NO: 17); a peptide comprising twelve amino acid residues identical to a run of twelve amino acid residues found between position 29 and position 40 of the Rs-AFP2 sequence shown in FIG. 1 and having the sequence: EKARHGSCNYVF (SEQ ID NO: 18); a peptide comprising twelve amino acid residues identical to a run of twelve amino acid residues found between position 30 and position 41 of the Rs-AFP2 sequence shown in FIG. 1 and having the sequence: KARHGSCNYVFP (SEQ ID NO: 19); a peptide comprising twelve amino acid residues identical to a run of twelve amino acid residues found between position 32 and position 43 of the Rs-AFP2 sequence shown in FIG. 1 and having the sequence: RHGSCNYVFPAH (SEQ ID NO: 20); a peptide composing twelve amino acid residues identical to a run of twelve amino acid residues found between position 33 and position 44 of the Rs-AFP2 sequence shown in FIG. 1 and having the sequence: HGSCNYVFPAHK (SEQ ID NO: 21); a peptide comprising nineteen amino acid residues identical to a run of nineteen amino acid residues found between position 31 and position 49 of the Rs-AFP2 sequence shown in FIG. 1 and having the sequence: ARHGSCNYVFPAHKCICYF (SEQ ID NO: 22).

[0016] We have found that the presence of an arginine residue at position 27 and a phenylalanine residue at position 40; a lysine residue at position 30 and a histidine residue at position 43 or an arginine residue at position 32 and a lysine residue at position 44 is particularly advantageous in Rs-AFP2 (SEQ ID NO: 35) based peptides. We have also found that antifungal peptides based on the Rs-AFP2 sequence (SEQ ID NO: 35) with an N-terminal amino acid selected from the group lysine at position 30, alanine at position 31, arginine at position 32 or histidine at position 33 and a C-terminal amino acid comprising either a tyrosine residue at position 48 or a phenylalanine residue at position 49 are particularly active. These antifungal peptides form a further embodiment of the invention.

[0017] The invention also provides an antifungal peptide which comprises at least six amino acid residues identical to a run of amino acid residues found between position 30 and position 48 of the Ah-AMP1 (SEQ ID NO: 57) sequence or the HS-AFP1 sequence (SEQ ID NO: 56) shown in FIG. 9. Such antifungal peptides include a peptide comprising nineteen amino acid residues identical to the run of nineteen amino acid residues found between position 30 and position 48 of the Ah-AMP 1 sequence (SEQ ID NO: 57) shown in FIG. 9 and having the sequence: ASHGACHKRENWKCFCYF (SEQ ID NO: 23). The invention also provides a peptide comprising nineteen amino acid residues found between position 30 and position 48 of the Dm-AMP1 sequence (SEQ ID NO: 58) shown in FIG. 9 and having the sequence AAHGACHVRNGKHMCFCYF (SEQ ID NO: 24).

[0018] Peptides derived from the regions defined herein of the Rs-AFP plant defensins exhibit antifungal activity. Such peptides may be easier to synthesise than the full length plant defensin while retaining antifungal activity. DNA sequences encoding the peptides may also be more suitable for transformation into biological hosts.

[0019] An antifungal peptide according to the invention may be manufactured from its known amino acid sequence by chemical synthesis using a standard peptide synthesiser, or produced within a suitable organism (for example, a micro-organism or plant) by expression of recombinant DNA. The antifungal peptide is useful as a fungicide and may be used for agricultural or pharmaceutical or other applications. The antifungal peptide may be used in combination with one or more of the antifungal proteins or with one or more other antifungal peptides of the present invention. For example, an antifungal composition comprising one of the above-mentioned fifteen-mer peptides plus the Rs-AFP2 (SEQ ID NO: 35) or Rs-AFP 1 (SEQ ID NO: 34) protein may show enhanced activity.

[0020] Knowledge of its primary structure enables manufacture of the antifungal peptide, or parts thereof, by chemical synthesis using a standard peptide synthesiser. It also enables production of DNA constructs encoding the antifungal peptide.

[0021] The invention further provides a DNA sequence encoding an antifungal peptide according to the invention. The DNA sequence may be predicted from the known amino acid sequence and DNA encoding the peptide may be manufactured using a standard nucleic acid synthesiser.

[0022] The DNA sequence encoding the antifungal peptide may be incorporated into a DNA construct or vector in combination with suitable regulatory sequences (promoter, terminator, transit peptide, etc). For some applications, the DNA sequence encoding the antifungal peptide may be inserted within a coding region expressing another protein to form an antifungal fusion protein or may be used to replace a domain of a protein to give that protein antifungal activity. The DNA sequence may be placed under the control of a homologous or heterologous promoter which may be a constitutive or an inducible promoter (stimulated by, for example, environmental conditions, presence of a pathogen, presence of a chemical). The transit peptide may be homologous or heterologous to the antifungal protein and will be chosen to ensure secretion to the desired organelle or to the extracellular space. The transit peptide is preferably that naturally associated with the antifungal protein of interest. Such a DNA construct may be cloned or transformed into a biological system which allows expression of the encoded peptide or an active part of the peptide. Suitable biological systems include micro-organisms (for example, bacteria such as Escherichia coli, Pseudomonas and endophytes such as Clavibacter xyli subsp. cynodontis (Cxc); yeast; viruses; bacteriophages; etc), cultured cells (such as insect cells, mammalian cells) and plants. In some cases, the expressed peptide may subsequently be extracted and isolated for use.

[0023] An antifungal peptide according to the invention is useful for combating fungal diseases in plants. The invention further provides a process of combating fungi whereby they are exposed to an antifungal peptide according to the invention. The antifungal peptide may be used in the form of a composition.

[0024] For pharmaceutical applications, the antifungal peptide (including any product derived from it) may be used as a fungicide to treat mammalian infections (for example, to combat yeasts such as Candida).

[0025] An antifungal peptide (including any product derived from it) according to the invention may also be used as a preservative (for example, as a food additive).

[0026] For agricultural applications, the antifungal peptide may be used to improve the disease-resistance or disease-tolerance of crops either during the life of the plant or for post-harvest crop protection. Pathogens exposed to the peptides are inhibited. The antifungal peptide may eradicate a pathogen already established on the plant or may protect the plant from future pathogen attack. The eradicant effect of the peptide is particularly advantageous.

[0027] Exposure of a plant pathogen to an antifungal peptide may be achieved in various ways, for example:

[0028] (a) The isolated peptide may be applied to plant parts or to the soil or other growth medium surrounding the roots of the plants or to the seed of the plant before it is sown using standard agricultural techniques (such as spraying).

[0029] The peptide may have been extracted from plant tissue or chemically synthesised or extracted from micro-organisms genetically modified to express the peptide. The peptide may be applied to plants or to the plant growth medium in the form of a composition comprising the peptide in admixture with a solid or liquid diluent and optionally various adjuvants such as surface-active agents. Solid compositions may be in the form of dispersible powders, granules, or grains.

[0030] (b) A composition comprising a micro-organism genetically modified to express the antifungal peptide may be applied to a plant or the soil in which a plant grows.

[0031] (c) An endophyte genetically modified to express the antifungal peptide may be introduced into the plant tissue (for example, via a seed treatment process).

[0032] An endophyte is defined as a micro-organism having the ability to enter into non-pathogenic endosymbiotic relationships with a plant host. A method of endophyte-enhanced protection of plants has been described in a series of patent applications by Crop Genetics International Corporation (for example, International Application Publication Number WO90/13224, European Patent Publication Number EP-125468-B1, International Application Publication Number WO91/10363, International Application Publication Number WO87/03303). The endophyte may be genetically modified to produce agricultural chemicals. International Patent Application Publication Number WO94/16076 (ZENECA Limited) describes the use of endophytes which have been genetically modified to express a plant-derived antifungal peptide.

[0033] (d) DNA encoding an antifungal peptide may be introduced into the plant genome so that the peptide is expressed within the plant body (the DNA may be cDNA, genomic DNA or DNA manufactured using a standard nucleic acid synthesiser).

[0034] Exposure of a plant pathogen to an antifungal composition comprising an antifungal peptide plus an antifungal protein may be achieved by delivering the protein as well as the peptide as described above. For example, both one of the above-mentioned fifteen-mer peptides plus Rs-AFP2 (SEQ ID NO: 35) or Rs-AFP1 (SEQ ID NO: 34) could be simultaneously applied to plant parts or simultaneously expressed within the plant body.

[0035] Plant cells may be transformed with recombinant DNA constructs according to a variety of known methods (Agrobacterium Ti plasmids, electrop oration, microinjection, microprojectile gun, etc). The transformed cells may then in suitable cases be regenerated into whole plants in which the new nuclear material is stably incorporated into the genome. Both transformed monocotyledonous and dicotyledonous plants may be obtained in this way, although the latter are usually more easy to regenerate. Some of the progeny of these primary transformants will inherit the recombinant DNA encoding the antifungal peptide(s).

[0036] The invention further provides a plant having improved resistance to a fungal pathogen and containing recombinant DNA which expresses an antifungal peptide according to the invention. Such a plant may be used as a parent in standard plant breeding crosses to develop hybrids and lines having improved fungal resistance.

[0037] Recombinant DNA is DNA, preferably heterologous, which has been introduced into the plant or its ancestors by transformation. The recombinant DNA encodes an antifungal peptide expressed for delivery to a site of pathogen attack (such as the leaves). The DNA may encode an active subunit of an antifungal peptide.

[0038] A pathogen may be any fungus growing on, in or near the plant. In this context, improved resistance is defined as enhanced tolerance to a fungal pathogen when compared to a wild-type plant. Resistance may vary from a slight increase in tolerance to the effects of the pathogen (where the pathogen in partially inhibited) to total resistance so that the plant is unaffected by the presence of pathogen (where the pathogen is severely inhibited or killed). An increased level of resistance against a particular pathogen or resistance against a wider spectrum of pathogens may both constitute an improvement in resistance. Transgenic plants (or plants derived therefrom) showing improved resistance are selected following plant transformation or subsequent crossing.

[0039] Where the antifungal peptide is expressed within a transgenic plant or its progeny, the fungus is exposed to the peptide at the site of pathogen attack on the plant. In particular, by use of appropriate gene regulatory sequences, the peptide may be produced in vivo when and where it will be most effective. For example, the peptide may be produced within parts of the plant where it is not normally expressed in quantity but where disease resistance is important (such as in the leaves).

[0040] Examples of genetically modified plants which may be produced include field crops, cereals, fruit and vegetables such as: canola, sunflower, tobacco, sugarbeet, cotton, soya, maize, wheat, barley, rice, sorghum, tomatoes, mangoes, peaches, apples, pears, strawberries, bananas, melons, potatoes, carrot, lettuce, cabbage, onion.

[0041] We have surprisingly found that when the peptides according to the invention are mixed with full length Rs-AFP2 (SEQ ID NO: 35) a synergistic effect is observed where the antifungal activity of the mixture is better than that observed with the protein or the peptide on its own.

[0042] In a further aspect the invention provides an antifungal composition comprising a peptide according to the invention and Rs-AFP1 (SEQ ID NO: 34) or Rs-AFP2 (SEQ ID NO: 35).

[0043] The invention also extends to DNA constructs encoding both the antifungal peptide and Rs-AFP1 or Rs-AFP2, and to the use of said peptide mixtures in antifungal compositions for pharmaceutical, agricultural, and preservative applications.

[0044] The invention will now be described by way of example only, with reference to the following drawings wherein:

[0045] FIG. 1 shows the amino acid sequences of some plant defensins. In FIG. 1, the proteins shown have the following sequence identifications: Rs-AFP1—SEQ ID NO: 34; Rs-AFP2—SEQ ID NO: 35; Rs-AFP3—SEQ ID NO: 36; Rs-AFP4—SEQ ID NO: 37; Br-AFP1—SEQ ID NO: 38; Br-AFP2—SEQ ID NO: 39; Bn-AFP1—SEQ ID NO: 40; Bn-AFP2—SEQ ID NO: 41; Sa-AFP1—SEQ ID NO: 42; Sa-AFP2—SEQ ID NO: 43; AtAFP1—SEQ ID NO: 44.

[0046] FIG. 2 shows the nucleotide sequence of the cDNA encoding Rs-AFP1 (SEQ ID NO: 45).

[0047] FIG. 3 shows the amino acid sequences of the 15-mer Rs-AFP2 peptides. In FIG. 3, Rs-AFP2 is SEQ ID NO: 35 and the peptides have the following sequence identifications: Peptide 1—SEQ D NO: 35, amino acids 1-15; Peptide 2—SEQ ID NO: 35, amino acids 5-19; Peptide 3—SEQ ID NO: 35, amino acids 9-23; Peptide 4—SEQ ID NO: 35, amino acids 13-27; Peptide 5—SEQ ID NO: 35, amino acids 17-31; Peptide 6—SEQ ID NO: 1; Peptide 7—SEQ ID NO: 2; Peptide 8—SEQ ID NO: 3; Peptide 9—SEQ ID NO: 4; Peptide 10—SEQ ID NO: 35, amino acids 37-51.

[0048] FIG. 4 is a diagram illustrating the sets of 6-, 9- and 12-mer Rs-AFP2 peptides. In FIG. 4, the peptides have the following sequence identifications: 6-mer peptides—Peptide 1—SEQ ID NO: 133; Peptide 2—SEQ ID NO: 134; Peptide 3—SEQ ID NO: 135; Peptide 45—SEQ ID NO: 129; Peptide 46—SEQ ID NO: 130. 9-mer peptides—Peptide 1—SEQ ID NO: 136; Peptide 2—SEQ ID NO: 137; Peptide 3—SEQ ID NO: 138; Peptide 42—SEQ ID NO: 15; Peptide 43—SEQ ID NO: 16. 12-mer peptides—Peptide 1—SEQ ID NO: 139; Peptide 2—SEQ ID NO: 140; Peptide 3—SEQ ID NO: 141; Peptide 39—SEQ ID NO: 131; Peptide 40—SEQ ID NO: 132. Rs AFP2 is SEQ ID NO: 35.

[0049] FIG. 5 is a graphical representation showing the antifungal activity of the Rs-AFP2-based 6-, 9- and 12-mer peptides. In FIG. 5, the peptides have the following sequence identifications: (The corresponding peptide numbers refer to the number of the overlapping Rs-AFP2 peptide shown in Example 1 (b).) 6-mer peptides—Peptide 1—SEQ ID NO: 133; Peptide 31—SEQ ID NO: 35, amino acids 31-36; Peptide 32—SEQ ID NO: 35, amino acids 32-37; Peptide 40—SEQ ID NO: 35, amino acids 40-45; Peptide 42—SEQ ID NO: 35, amino acids 42-47; Peptide 43—SEQ ID NO: 35, amino acids 43-48; 9-mer peptides—Peptide 19—SEQ ID NO: 35, amino acids 19-27 (corresponds to Peptide 65); Peptide 20—SEQ ID NO: 35, amino acids 20-28 (corresponds to Peptide 66); Peptide 24—SEQ ID NO: 9 (corresponds to Peptide 70); Peptide 25—SEQ ID NO: 35, amino acids 25-33 (corresponds to Peptide 71); Peptide 28—SEQ ID NO: 35, amino acids 28-36 (corresponds to Peptide 74); Peptide 30—SEQ ID NO: 35, amino acids 30-38 (corresponds to Peptide 76); Peptide 31: SEQ ID NO: 35, amino acids 31-39 (corresponds to Peptide 77); Peptide 32—SEQ ID NO: 11 (corresponds to Peptide 78); Peptide 36—SEQ ID NO: 35, amino acids 3644 (corresponds to Peptide 82); Peptide 37—SEQ ID NO: 35, amino acids 3745 (corresponds to Peptide 83); Peptide 38—SEQ ID NO: 35, amino acids 38-46 (corresponds to Peptide 84); Peptide 39—SEQ ID NO: 35, amino acids 3947 (corresponds to Peptide 85); Peptide 40—SEQ ID NO: 13 (corresponds to Peptide 86); Peptide 41—SEQ ID NO: 14 (corresponds to Peptide 87); Peptide 42—SEQ ID NO: 15 (corresponds to Peptide 88); Peptide 43—SEQ ID NO: 16 (corresponds to Peptide 89); 12-mer peptides: Peptide 17—SEQ ID NO: 35, amino acids 17-28 (corresponds to Peptide 106); Peptide 20—SEQ ID NO: 35, amino acids 20-31 (corresponds to Peptide 109); Peptide 21—SEQ ID NO: 35, amino acids 21-32 (corresponds to Peptide 110); Peptide 22—SEQ ID NO: 35, amino acids 22-33 (corresponds to Peptide 111); Peptide 23—SEQ ID NO: 35, amino acids 23-34 (corresponds to Peptide 112); Peptide 24—SEQ ID NO: 35, amino acids 24-35 (corresponds to Peptide 113); Peptide 26—SEQ ID NO: 35, amino acids 26-37 (corresponds to Peptide 115); Peptide 27—SEQ ID NO: 35, amino acids 27-38 (corresponds to Peptide 116); Peptide 28—SEQ ID NO: 35, amino acids 28-39 (corresponds to Peptide 117); Peptide 29—SEQ ID NO: 18 (corresponds to Peptide 118); Peptide 30—SEQ ID NO: 19 (corresponds to Peptide 119); Peptide 31—SEQ ID NO: 20 (corresponds to Peptide 120); Peptide 34—SEQ ID NO: 35, amino acids 3445 (corresponds to Peptide 123); Peptides 35—SEQ ID NO: 35, amino acids 35-46 (corresponds to Peptide 124); Peptide 36—SEQ ID NO: 35, amino acids 3647 (corresponds to Peptide 125); Peptide 37—SEQ ID NO: 35, amino acids 3748 (corresponds to Peptide 126); Peptide 25—SEQ ID NO: 17 (corresponds to Peptide 114); Peptide 32—SEQ ID NO: 21 (corresponds to Peptide 121); Peptide 33—SEQ ID NO: 35, amino acids 33-44 (corresponds with Peptide 122).

[0050] FIG. 6 is a diagram summarising all the active Rs-AFP2-based 6-mer, 9-mer, 12-mer and 15-mer peptides. In FIG. 6, the peptides have the following sequence identifications: (The corresponding peptide numbers refer to the number of the overlapping Rs-AFP2 peptide shown in Example 1(b).) 6-mer peptides: Peptide 1—SEQ ID NO: 133; Peptide 31—SEQ ID NO: 35, amino acids 31-36; Peptide 32—SEQ ID NO: 35, amino acids 32-37; Peptide 40—SEQ ID NO: 35, amino acids 40-45; Peptide 42—SEQ ID NO: 35, amino acids 42-47; Peptide 43—SEQ ID NO: 35, amino acids 43-48. 9-mer peptides: Peptide 19—SEQ ID NO: 35, amino acids 19-27 (corresponds to Peptide 65); Peptide 20—SEQ ID NO: 35, amino acids 20-28 (corresponds to Peptide 66); Peptide 24- SEQ ID NO: 9 (corresponds to Peptide 70); Peptide 25—SEQ ID NO: 35, amino acids 25-33 (corresponds to Peptide 71); Peptide 28—SEQ ID NO: 35, amino acids 28-36, (corresponds to Peptide 74); Peptide 30—SEQ ID NO: 35, amino acids 30-38 (corresponds to Peptide 76); Peptide 31—SEQ ID NO: 35, amino acids 31-39 (corresponds to Peptide 77); Peptide 32—SEQ ID NO: 11 (corresponds to Peptide 78); Peptide 36—SEQ ID NO: 35, amino acids 3644 (corresponds to Peptide 82); Peptide 37—SEQ ID NO: 35, amino acids 37-45 (corresponds to Peptide 83); Peptide 38—SEQ ID NO: 35, amino acids 38-46 (corresponds to Peptide 84); Peptide 39—SEQ ID NO: 35, amino acids 39-47 (corresponds to Peptide 85); Peptide 40—SEQ ID NO: 13 (corresponds to Peptide 86); Peptide 41—SEQ ID NO: 14 (corresponds to Peptide 87); Peptide 42—SEQ ID NO: 15 (corresponds to Peptide 88); Peptide 43—SEQ ID NO: 16 (corresponds to Peptide 89). 12-mer peptides: Peptide 17—SEQ ID NO: 35, amino acids 17-28 (corresponds to Peptide 106); Peptide 20—SEQ ID NO: 35, amino acids 20-31 (corresponds to Peptide 109); Peptide 21—SEQ ID NO: 35, amino acids 21-32 (corresponds to Peptide 110); Peptide 22—SEQ ID NO: 35, amino acids 22-33 (corresponds to Peptide 111); Peptide 23—SEQ ID NO: 35, amino acids 23-34 (corresponds to Peptide 112); Peptide 24—SEQ ID NO: 35, amino acids 24-35 (corresponds 113); Peptide 26—SEQ ID NO: 35, amino acids 26-37 (corresponds to Peptide 115); Peptide 27—SEQ ID NO: 35, amino acids 27-38 (corresponds to Peptide 116); Peptide 28—SEQ ID NO: 35, amino acids 28-39 (corresponds to Peptide 117); Peptide 29—SEQ ID NO: 18 (corresponds to Peptide 118); Peptide 30—SEQ ID NO: 19 (corresponds to Peptide 119); Peptide 31—SEQ ID NO: 20 (corresponds to Peptide 120); Peptide 34—SEQ ID NO: 35, amino acids 3445 (corresponds to Peptide 123); Peptide 35—SEQ ID NO: 35, amino acids 3546 (corresponds to Peptide 124); Peptide 36—SEQ ID NO: 35, amino acids 36-47 (corresponds to Peptide 125); Peptide 37—SEQ ID NO: 35, amino acids 37-48 (corresponds to Peptide 126); Peptide 25—SEQ ID NO: 17 (corresponds to Peptide 114); Peptide 32—SEQ ID NO: 21 (corresponds to Peptide 121); Peptide 33—SEQ ID NO: 35, amino acids 33-44 (corresponds with Peptide 122). 15-mer peptides—Peptide 1—SEQ ID NO: 35, amino acids 1-15; Peptide 5 (Peptide 17 in FIG. 6)—SEQ ID NO: 35, amino acids 17-3 1; Peptide 6 (Peptide 21 in FIG. 6)—SEQ ID NO: 1; Peptide 7 (Peptide 25 in FIG. 6)—SEQ ID NO: 2; Peptide 8 (Peptide 33 in FIG. 6)—SEQ ID NO: 3; Peptide 9 (Peptide 37 in FIG. 6)—SEQ ID NO: 4.

[0051] FIG. 7 is a graphical representation showing the antifungal activity of the Rs-AFP1 -based 6-, 9- and 12-mer peptides. In FIG. 7, the peptides have the following sequence identifications: (The corresponding peptide numbers refer to the number of the overlapping Rs-AFP1 peptides shown in Example 1 (c).) 6-mer peptides: Peptide 31—SEQ ID NO: 34, amino acids 31-36; Peptide 42—SEQ ID NO: 34, amino acids 42-47; Peptide 43—SEQ ID NO: 34, amino acids 43-48; Peptide 44—SEQ ID NO: 34, amino acids 44-49. The 9-mer peptides have the following sequence identifications: Peptide 28—SEQ ID NO: 34, amino acids 28-36 (corresponds to Peptide 74); Peptide 30—SEQ ID NO: 34, amino acids 30-38 (corresponds to Peptide 76); Peptide 31—SEQ ID NO: 34, amino acids 34-42 (corresponds to Peptide 77); Peptide 32—SEQ ID NO: 34, amino acids 3240 (corresponds to Peptide 78); Peptide 36—SEQ ID NO: 34, amino acids 3644 (corresponds to Peptide 82; Peptide 37—SEQ ID NO: 34, amino acids 37-45 (corresponds to Peptide 83); Peptide 38—SEQ ID NO: 34, amino acids 3846 (corresponds to Peptide 84); Peptide 39—SEQ ID NO: 34, amino acids 39-47 (corresponds to Peptide 85); Peptide 40—SEQ ID NO: 34, amino acids 40-48 (corresponds to Peptide 86); Peptide 41—SEQ ID NO: 34, amino acids 41-49 (corresponds to Peptide 87); Peptide 42—SEQ ID NO: 34, amino acids 42-50 (corresponds to Peptide 88); Peptide 43 SEQ ID NO: 34, amino acids 43-51 (corresponds to Peptide 89). The 12-mer peptides have the following sequence identifications: Peptide 21—SEQ ID NO: 34, amino acids 21-32 (corresponds to Peptide 110); Peptide 24—SEQ ID NO: 34, amino acids 24-35 (corresponds to Peptide 113); Peptide 25—SEQ ID NO: 34, amino acids 25-36 (corresponds to Peptide 114); Peptide 27—SEQ ID NO: 34, amino acids 27-38 (corresponds to Peptide 116); Peptide 28—SEQ ID NO: 34, amino acids 28-39 (corresponds to Peptide 117); Peptide 29—SEQ ID NO: 34, amino acids 2940 (corresponds to Peptide 118); Peptide 30—SEQ ID NO: 34, amino acids 30-41 (corresponds to Peptide 119); Peptide 31—SEQ ID NO: 34, amino acids 31-42 (corresponds to Peptide 120); Peptide 32—SEQ ID NO: 34, amino acids 32-43 (corresponds to Peptide 121); Peptide 33—SEQ ID NO: 34, amino acids 33-44 (corresponds to Peptide 122); Peptide 34—SEQ ID NO: 34, amino acids 34-45 (corresponds to Peptide 123); Peptide 35—SEQ ID NO: 34, amino acids 35-46 (corresponds to Peptide 124); Peptide 36—SEQ ID NO: 34, amino acids 36-47 (corresponds to Peptide 125); Peptide 37—SEQ ID NO: 34, amino acids 37-48 (corresponds to Peptide 126); Peptide 38—SEQ ID NO: 34, amino acids 38-19 (corresponds to Peptide 127); Peptide 39—SEQ ID NO: 34, amino acids 39-50 (corresponds to Peptide 128).

[0052] FIG. 8 is a diagram summarising all the active Rs-AFP1-based 6-mer, 9-mer and 12-mer peptides. In FIG. 8, the peptides have the following sequence identifications: (The corresponding peptide numbers refer to the number of the overlapping Rs-AFP1 peptides shown in Example 1 (c).) 6-mer peptides: Peptide 31—SEQ ID NO: 34, amino acids 31-36; Peptide 42—SEQ ID NO: 34, amino acids 4247; Peptide 43—SEQ ID NO: 34, amino acids 43-48; Peptide 44—SEQ ID NO: 34, amino acids 44-49. The 9-mer peptides have the following sequence identifications: Peptide 28—SEQ ID NO: 34, amino acids 28-36 (corresponds to Peptide 74); Peptide 30—SEQ ID NO: 34, amino acids 30-38 (corresponds to Peptide 76); Peptide 31—SEQ ID NO: 34, amino acids 34-42 (corresponds to Peptide 77); Peptide 32—SEQ ID NO: 34, amino acids 3240 (corresponds to Peptide 78); Peptide 36—SEQ ID NO: 34, amino acids 36-44 (corresponds to Peptide 82); Peptide 37—SEQ ID NO: 34, amino acids 37-45 (corresponds to Peptide 83); Peptide 38—SEQ ID NO: 34, amino acids 3846 (corresponds to Peptide 84); Peptide 39—SEQ ID NO: 34, amino acids 39-47 (corresponds to Peptide 85); Peptide 40—SEQ ID NO: 34, amino acids 40-48 (corresponds to Peptide 86); Peptide 41—SEQ ID NO: 34, amino acids 41-49 (corresponds to Peptide 87); Peptide 42—SEQ ID NO: 34, amino acids 42-50 (corresponds to Peptide 88); Peptide 43—SEQ ID NO: 34, amino acids 43-51 (corresponds to Peptide 89). The 12-mer peptides have the following sequence identifications: Peptide 21—SEQ ID NO: 34, amino acids 21-32 (corresponds to Peptide 110); Peptide 24—SEQ ID NO: 34, amino acids 24-35 (corresponds to Peptide 113); Peptide 25—SEQ ID NO: 34, amino acids 25-36 (corresponds to Peptide 114); Peptide 27—SEQ ID NO: 34, amino acids 27-38 (corresponds to Peptide 116); Peptide 28—SEQ ID NO: 34, amino acids 28-39 (corresponds to Peptide 117); Peptide 29—SEQ ID NO: 34, amino acids 29-40 (corresponds to Peptide 118); Peptide 30—SEQ ID NO: 34, amino acids 3041 (corresponds to Peptide 119); Peptide 31 SEQ ID NO: 34, amino acids 31-42 (corresponds to Peptide 120); Peptide 32—SEQ ID NO: 34, amino acids 32-43 (corresponds to Peptide 121); Peptide 33—SEQ D NO: 34, amino acids 33-44 (corresponds to Peptide 122); Peptide 34—SEQ ID NO: 34, amino acids 34-45 (corresponds to Peptide 123); Peptide 35—SEQ ID NO: 34, amino acids 35-46 (corresponds to Peptide 124); Peptide 36—SEQ ID NO: 34, amino acids 36-47 (corresponds to Peptide 125); Peptide 37—SEQ ID NO: 34, amino acids 37-48 (corresponds to Peptide 126); Peptide 38—SEQ ID NO: 34, amino acids 38-49 (corresponds to Peptide 127); Peptide 39—SEQ ID NO: 34, amino acids 39-50 (corresponds to Peptide 128). RsAFP2 15-mer peptides—Peptide 1-SEQ ID NO: 35, amino acids 1-15; Peptide 17—SEQ ID NO: 35, amino acids 17-31; Peptide 21—SEQ ID NO: 1; Peptide 25—SEQ ID NO: 2; Peptide 29—SEQ ID NO: 3; Peptide 33—SEQ ID NO: 4; Peptide 37—SEQ ID NO: 35, amino acids 37-51.

[0053] FIG. 9 shows the amino acid sequences of the proteins Hs-AFP1 (SEQ ID NO: 56), Ah-AMP1 (SEQ ID NO: 57) and Dm-AMP1 (SEQ ID NO: 58).

[0054] FIG. 10a is a diagram summarising active Rs-AFP2-based 13-mer, 14-mer and 15-mer peptides. In FIG. 10a the peptides have the following sequence identifications: 1 SEQ ID NO: PEPTIDE SEQ ID NO: 59 IRLEKARHGSBNY SEQ ID NO: 60 RLEKARHGSBNYV SEQ ID NO: 61 LEKARHGSBNYVF SEQ ID NO: 62 EKARKGSBNYVFP SEQ ID NO: 63 KARHGSBNYVFPA SEQ ID NO: 64 ARHGSBNYVFPAH SEQ ID NO: 65 RHGSBNYVFPAHK SEQ ID NO: 66 HGSBNYVFPAHKB SEQ ID NO: 67 GSBNYVFPAHKBI SEQ ID NO: 68 SBNYVFPAHKBIB SEQ ID NO: 69 BNYVFPAHKBIBY SEQ ID NO: 70 NYVFPAHKBIBYF SEQ ID NO: 71 IRLEKARHGSBNYV SEQ ID NO: 72 RLEKARHGSBNYVF SEQ ID NO: 73 LEKARHGSBNYVFP SEQ ID NO: 74 EKARHGSBNYVFPA SEQ ID NO: 75 KARHGSBNYVFPAH SEQ ID NO: 76 ARHGSBNYVFPAHK SEQ ID NO: 77 RHGSBNYVFPAHKB SEQ ID NO: 78 HGSBNYVFPAHKBI SEQ ID NO: 79 HGSBNYVFPAHKBIB SEQ ID NO: 80 SBNYVFPAHKBIBY SEQ ID NO: 81 BNYVFPAHKBIBYF SEQ ID NO: 82 IRLEKARHGSBNYVF SEQ ID NO: 83 RLEKARHGSBNYVFP SEQ ID NO: 84 LEKARHGSBNYVFPA SEQ ID NO: 85 EKARHGSBNYVFPAH SEQ ID NO: 86 KARHGSBNYVFPAHK SEQ ID NO: 87 ARHGSBNYVFPAHKB SEQ ID NO: 88 RHGSBNYVFPAHKBI SEQ ID NO: 89 HGSBNYVFPAHKBIB SEQ ID NO: 90 GSBNYVFPAHKBIBY SEQ ID NO: 91 SBNYVFPAHKBIBYF

[0055] FIG. 10b is a diagram summarising active Rs-AFP2-based 16-mer, 17-mer and 18-mer peptides. In FIG. 10b, the peptides have the following sequence identifications: 2 SEQ ID NO: PEPTIDE SEQ ID NO: 92 IRLEKARHGSBNYVFP SEQ ID NO: 93 RLEKARHGSBNYVFPA SEQ ID NO: 94 LEKARHGSBNYVFPAH SEQ ID NO: 95 EKARHGSBNYVFPAHK SEQ ID NO: 96 KARHGSBNYVFPAHKB SEQ ID NO: 97 ARHGSBNYVFPAHKBI SEQ ID NO: 98 RHGSBNYVFPAHKBIB SEQ ID NO: 99 HGSBNYVFPAHKBIBY SEQ ID NO: 100 GSBNYVFPAHKBIBYF SEQ ID NO: 101 IRLEKARHGSBNYVFPA SEQ ID NO: 102 RLEKARHGSBNYVFPAH SEQ ID NO: 103 LEKARHGSBNYVFPAHK SEQ ID NO: 104 EKARHGSBNYVFPAHKB SEQ ID NO: 105 KARHGSBNYVFPAHKBI SEQ ID NO: 106 ARHGSBNYVFPAHKBIB SEQ ID NO: 107 RHGSBNYVFPAHKBIBY SEQ ID NO: 108 HGSBNYVFPAHKBIBYF SEQ ID NO: 109 IRLEKARHGSBNYVFPAH SEQ ID NO: 110 RLEKARHGSBNYVFPAHK SEQ ID NO: 111 LEKARHGSBNYVFPAHKB SEQ ID NO: 112 EKARHGSBNYVFPAHKBI SEQ ID NO: 113 KARHGSBNYVFPAHKBIB SEQ ID NO: 114 ARHGSBNYVFPAHKBIBY SEQ ID NO: 115 RHGSBNYVFPAHKBIBYF

[0056] FIG. 10c is a diagram summarising active Rs-AFP2-based 19-mer and 20 mer peptides. In FIG. 10c, the peptides have the following sequence identifications: 3 SEQ ID NO: PEPTIDE SEQ ID NO: 116 IRLEKARHGSBNYVFPAHK SEQ ID NO: 117 RLEKARHGSBNYVFPAHKB SEQ ID NO: 118 LEKARHGSBNYVFPAHKBI SEQ ID NO: 119 EKARHGSBNYVFPAHKBIB SEQ ID NO: 120 KARHGSBNYVFPAHKBIBY SEQ ID NO: 121 ARHGSBNYVFPAHKBIBYF SEQ ID NO: 122 IRLEKARHGSBNYVFPAHKB SEQ ID NO: 123 RLEKARHGSBNYVFPAHKBI SEQ ID NO: 124 LEKARHGSBNYVFPAHKBIB SEQ ID NO: 125 EKARHGSBNYVFPAHKIBIBY SEQ ID NO: 126 KARHGSBNYVFPAHKBIBYF

[0057] FIG. 11a is a graphical representation showing the antifungal activity of Rs-AFP2-based 13-20-mers with same N-terminal residue Ile26-Ala31. In FIG. 11a, the 20 peptides have the following sequence identifications: 4 upper left graph (peptides having an N-terminal Ile26) Y-SEQ ID NO: 59 A-SEQ ID NO: 101 V-SEQ ID NO: 71 H-SEQ ID NO: 109 F-SEQ ID NO: 82 K-SEQ ID NO: 116 P-SEQ ID NO: 92 B-SEQ ID NO: 122 upper right graph (peptides having an N-terminal Arg27) V-SEQ ID NO: 60 H-SEQ ID NO: 102 F-SEQ ID NO: 72 K-SEQ ID NO: 110 P-SEQ ID NO: 83 B-SEQ ID NO: 117 A-SEQ ID NO: 93 I-SEQ ID NO: 123 middle left graph (peptides having an N-terminal Leu28) F-SEQ ID NO: 61 K-SEQ ID NO: 103 P-SEQ ID NO: 73 B-SEQ ID NO: 111 A-SEQ ID NO: 84 I-SEQ ID NO: 118 H-SEQ ID NO: 94 B-SEQ ID NO: 123 middle right graph (peptides having an N-terminal Glu29) P-SEQ ID NO: 62 B-SEQ ID NO: 104 A-SEQ ID NO: 74 I-SEQ ID NO: 112 H-SEQ ID NO: 85 B-SEQ ID NO: 119 K-SEQ ID NO: 95 Y-SEQ ID NO: 125 lower left graph (peptides having an N-terminal Lys30) A-SEQ ID NO: 63 I-SEQ ID NO: 105 H-SEQ ID NO: 75 B-SEQ ID NO: 113 K-SEQ ID NO: 86 Y-SEQ ID NO: 120 B-SEQ ID NO: 96 F-SEQ ID NO: 126 lower right graph (peptides having an N-terminal Ala31) H-SEQ ID NO: 64 B-SEQ ID NO: 106 K-SEQ ID NO: 76 Y-SEQ ID NO: 114 B-SEQ ID NO: 87 F-SEQ ID NO: 121 I-SEQ ID NO: 97

[0058] FIG. 11b is a graphical representation showing the antifungal activity of Rs-AFP2-based 13-20-mers with same N-terminal residue Arg32-Asn37. In FIG. 11b, the peptides have the following sequence identifications: 5 upper left graph (peptides having an N-terminal Arg32) K-SEQ ID NO: 65 B-SEQ ID NO: 98 B-SEQ ID NO: 77 Y-SEQ ID NO: 107 I-SEQ ID NO: 88 F-SEQ ID NO: 115 upper right graph (peptides having an N-terminal His33) B-SEQ ID NO: 66 Y-SEQ ID NO: 99 I-SEQ ID NO: 78 F-SEQ ID NO: 108 B-SEQ ID NO: 89 middle left graph (peptides having an N-terminal Gly34) I-SEQ ID NO: 67 Y-SEQ ID NO: 90 B-SEQ ID NO: 79 F-SEQ ID NO: 100 middle right graph (peptides having an N-terminal Ser35) B-SEQ ID NO: 68 Y-SEQ ID NO: 80 F-SEQ ID NO: 91 lower left graph (peptides having an N-terminal B36) Y-SEQ ID NO: 69 F-SEQ ID NO: 81 lower right graph (peptides having an N-terminal Asn37) F-SEQ ID NO: 70.

[0059] FIG. 12a is a graphical representation showing the antifungal activity of Rs-AFP2-based 13-20-mers with same C-terminal residue Tyr38-His43. In FIG. 12a, the peptides have the following sequence identifications: 6 upper left graph (peptides having a C-terminal Tyr38) I-SEQ ID NO: 59 upper right graph (peptides having a C-terminal Val39) I-SEQ ID NO: 71 R-SEQ ID NO: 60 middle left graph (peptides having a C-terminal Phe40) I-SEQ ID NO: 82 R-SEQ ID NO: 72 L-SEQ ID NO: 61 middle right graph (peptides having a C-terminal Pro41) I-SEQ ID NO: 92 L-SEQ ID NO: 73 R-SEQ ID NO: 83 E-SEQ ID NO: 62 lower left graph (peptides having a C-terminal Ala42) I-SEQ ID NO: 101 E-SEQ ID NO: 74 R-SEQ ID NO: 93 K-SEQ ID NO: 63 L-SEQ ID NO: 84 lower right graph (peptides having a C-terminal His43) I-SEQ ID NO: 109 E-SEQ ID NO: 85 R-SEQ ID NO: 102 K-SEQ ID NO: 75 L-SEQ ID NO: 94 A-SEQ ID NO: 64

[0060] FIG. 12b is a graphical representation showing the antifungal activity of Rs-AFP2-based 13-20-mers with same C-terminal residue Lys44-Phe49. In FIG. 12b, the peptides have the following sequence identifications: 7 upper left graph (peptides having a C-terminal Lys44) I-SEQ ID NO: 116 K-SEQ ID NO: 86 R-SEQ ID NO: 110 A-SEQ ID NO: 76 L-SEQ ID NO: 103 R-SEQ ID NO: 63 E-SEQ ID NO: 95 upper right graph (peptides having a C-terminal B45) I-SEQ ID NO: 122 K-SEQ ID NO: 96 R-SEQ ID NO: 117 A-SEQ ID NO: 87 L-SEQ ID NO: 111 R-SEQ ID NO: 77 E-SEQ ID NO: 104 H-SEQ ID NO: 64 middle left graph (peptides having a C-terminal Ile46) R-SEQ ID NO: 123 A-SEQ ID NO: 97 L-SEQ ID NO: 118 R-SEQ ID NO: 88 E-SEQ ID NO: 112 H-SEQ ID NO: 78 K-SEQ ID NO: 105 G-SEQ ID NO: 67 middle right graph (peptides having a C-terminal B47) L-SEQ ID NO: 124 R-SEQ ID NO: 98 E-SEQ ID NO: 119 H-SEQ ID NO: 89 K-SEQ ID NO: 113 G-SEQ ID NO: 79 A-SEQ ID NO: 106 S-SEQ ID NO: 68 lower left graph (peptides having a C-terminal Tyr48) E-SEQ ID NO: 125 H-SEQ ID NO: 99 K-SEQ ID NO: 120 G-SEQ ID NO: 90 A-SEQ ID NO: 114 S-SEQ ID NO: 80 R-SEQ ID NO: 107 B-SEQ ID NO: 69 lower right graph (peptides having a C-terminal Phe49) K-SEQ ID NO: 126 G-SEQ ID NO: 100 A-SEQ ID NO: 121 S-SEQ ID NO: 91 R-SEQ ID NO: 115 B-SEQ ID NO: 81 H-SEQ ID NO: 108 N-SEQ ID NO: 70

[0061] FIG. 13 is an analysis of the results with overlapping 13-20-mer peptides within the region of Ile26-Phe49 from Rs-AFP2 (SEQ ID NO: 35, amino acids 26-49). In FIG. 13, the peptides have the following sequence identifications: His33=>Phe49—SEQ ID NO: 108; Ile 26<=Val 39—SEQ ID NO: 71; Arg 32 +Lys44—SEQ ID NO: 65; Arg27+Phe40—SEQ ID NO: 72; Lys30+His43—SEQ ID NO: 75; Arg32+Tyr48—SEQ ID NO: 107; His33+Phe49—SEQ ID NO: 108; Arg32+Phe49—SEQ ID NO: 115; Ala31+Phe49—SEQ ID NO: 121; Lys30+Phe49—SEQ ID NO: 126.

EXAMPLE 1

Production of Synthetic Peptides

[0062] Split peptides were synthesised by the PEPSCAN method. MPS peptides were synthesised by a Multiple Peptide Synthesis. All peptides were blocked at the amino terminal residue by an acetyl group and at the carboxy terminal residue by a carboxamide group.

[0063] PEPSCAN-split (C-terminal beta-alanine-amide). Radiation grafted polyethylene pins were functionalised with amino groups. Glycolic acid was coupled using dicyclohexylcarbodiimide (DCC) and after washing Boc-beta-alanine was coupled using DCC and dimethylaminopyridine (DMAP) as catalyst. In a block with pins, ten overlapping 15-mer peptides of AFP2 were synthesised simultaneously using Fmoc-amino acids and overnight couplings with DCC/Hydroxybenzotriazole (HOBt) as coupling method. The peptides were deprotected with a mixture of trifluoroacetic acid/phenol/thioanisole/water/ethanedithiol 10/0.75/0.5/0.5/0.25 (cleavage mixture B), then washed, dried, and finally cleaved from the pins using ammonia. This procedure yields peptides up to about 1 mg with C-terminal beta-alanine-amide.

[0064] PEPSCAN-split (C-terminal amide). Radiation grafted polyethylene pins were functionalised with hydroxyl groups. Boc-beta-alanine was coupled using DCC and DMAP as catalyst, the Boc group was removed with TFA and Fmoc-2,4-dimethoxy4′(carboxymethyloxy) benzhydrylamine (Rink linker) was coupled using the DCC/HOBt method. Next, 46 6-mer, 43 9-mer and 40 12-mer peptides from AFP2 were synthesised as described above in blocks containing 96 pins. After washing and drying the peptides were deprotected and cleaved with mixture B. The cleavage mixture was evaporated, extracted twice with diethylether, and lyophilised twice from water. This procedure yields peptides up to about 1 mg with a C-terminal amide.

[0065] Multiple Peptide Synthesis. We used a Hamilton Microlab 2200 to synthesise up to 40 peptides simultaneously at 15-30 &mgr;mol scale. The Hamilton Microlab 2200 was programmed to deliver washing solvents and reagents to a rack with 20 or 40 individual 4 ml columns with filter containing resin for peptide synthesis. The columns were drained automatically after each step by vacuum. The coupling cycle was based on Fmoc/2-(1H benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) chemistry (Fields et al, Peptide Research 4, 199195-101) using double coupling steps. Peptides were deprotected and cleaved in two hours using 1.5 ml of mixture B and then precipitated twice by adding hexane/diethylether 1/1. The precipitate was dried and lyophilised from water/acetonitrile.

[0066] (a) Overlapping 15-mer Peptides Based on the Rs-AFP2 Protein

[0067] A set often split and MPS peptides were synthesised based on the primary sequence of the Rs-AFP2 protein. The sequences of the MPS peptides are shown in FIG. 3; the split peptides correspond to the MPS peptides with an additional C-terminal beta alanine (an extra linker that was only used in the first PEPSCAN split synthesis). Each peptide consisted of fifteen amino acid residues identical to a run of amino acids in the Rs-AFP2 sequence:

[0068] PEPTIDE 1 had the sequence of Rs-AFP2 from amino acid position 1 to position 15 (SEQ ID NO: 35, amino acids 1-15);

[0069] PEPTIDE 2 had the sequence of Rs-AFP2 from amino acid position 5 to position 19 (SEQ ID NO: 35, amino acids 5-19);

[0070] PEPTIDE 3 had the sequence of Rs-AFP2 from amino acid position 9 to position 23 (SEQ ID NO: 35, amino acids 9-23);

[0071] PEPTIDE 4 had the sequence of Rs-AFP2 from amino acid position 13 to position 27 (SEQ ID NO: 35, amino acids 13-27);

[0072] PEPTIDE 5 had the sequence of Rs-AFP2 from amino acid position 17 to position 31 (SEQ ID NO: 35, amino acids 17-31;

[0073] PEPTIDE 6 had the sequence of Rs-AFP2 from amino acid position 21 to position 35 (SEQ ID NO: 1);

[0074] PEPTIDE 7 had the sequence of Rs-AFP2 from amino acid position 25 to position 39 (SEQ ID NO: 2);

[0075] PEPTIDE 8 had the sequence of Rs-AFP2 from amino acid position 29 to position 43 (SEQ ID NO: 3);

[0076] PEPTIDE 9 had the sequence of Rs-AFP2 from amino acid position 33 to position 47 (SEQ ID NO: 4);

[0077] PEPTIDE 10 had the sequence of Rs-AFP2 from amino acid position 37 to position 51 (SEQ ID NO: 35, amino acids 37-51).

[0078] Overlapping 6-,9- and 12-mer Peptides Based on the Rs-AFP2 Protein

[0079] Three sets of overlapping split peptides were synthesised by the PEPSCAN method based on the primary sequence of the Rs-AFP2 protein.

[0080] In the first set, each peptide consisted of six amino acid residues identical to a run of amino acids in the Rs-AFP2 sequence (SEQ ID NO: 35). The set of forty-six 6-mer peptides (numbered 1 to 46) covered the entire Rs-AFP2 sequence. For example, PEPTIDE 1 had the sequence of Rs-AFP2 (SEQ ID NO: 35) from amino acid position 1 to position 6; PEPTIDE 2 had the sequence of Rs-AFP2 (SEQ ID NO: 35) from amino acid position 2 to position 7; PEPTIDE 3 had the sequence of Rs-AFP2 (SEQ ID NO: 35) from amino acid position 3 to position 8; PEPTIDE 45 (SEQ ID NO: 35) had the sequence of Rs-AFP2 (SEQ ID NO: 35) from amino acid position 45 to position 50; PEPTIDE 46 had the sequence of Rs-AFP2 (SEQ ID NO: 35) from amino acid position 46 to position 51. The sequence identifications of each of the peptides is set out below. 8 Rs-AFP2 6-mer peptides Peptide SEQ ID NO: 1 SEQ ID NO: 133 2 SEQ ID NO: 134 3 SEQ ID NO: 135 4 SEQ ID NO: 35, amino acids 4-9 5 SEQ ID NO: 35, amino acids 5-10 6 SEQ ID NO: 35, amino acids 6-11 7 SEQ ID NO: 35, amino acids 7-12 8 SEQ ID NO: 35, amino acids 8-13 9 SEQ ID NO: 35, amino acids 9-14 10 SEQ ID NO: 35, amino acids 10-15 11 SEQ ID NO: 35, amino acids 11-16 12 SEQ ID NO: 35, amino acids 12-17 13 SEQ ID NO: 35, amino acids 13-18 14 SEQ ID NO: 35, amino acids 14-19 15 SEQ ID NO: 35, amino acids 15-20 16 SEQ ID NO: 35, amino acids 16-21 17 SEQ ID NO: 35, amino acids 17-22 18 SEQ ID NO: 35, amino acids 18-23 19 SEQ ID NO: 35, amino acids 19-24 20 SEQ ID NO: 35, amino acids 20-25 21 SEQ ID NO: 35, amino acids 21-26 22 SEQ ID NO: 35, amino acids 22-27 23 SEQ ID NO: 35, amino acids 23-28 24 SEQ ID NO: 35, amino acids 24-29 25 SEQ ID NO: 35, amino acids 25-30 26 SEQ ID NO: 35, amino acids 26-31 27 SEQ ID NO: 35, amino acids 27-32 28 SEQ ID NO: 35, amino acids 28-33 29 SEQ ID NO: 35, amino acids 29-34 30 SEQ ID NO: 35, amino acids 30-35 31 SEQ ID NO: 35, amino acids 31-36 32 SEQ ID NO: 35, amino acids 32-37 33 SEQ ID NO: 35, amino acids 33-38 34 SEQ ID NO: 35, amino acids 34-39 35 SEQ ID NO: 35, amino acids 35-40 36 SEQ ID NO: 35, amino acids 36-41 37 SEQ ID NO: 35, amino acids 37-42 38 SEQ ID NO: 35, amino acids 38-43 39 SEQ ID NO: 35, amino acids 39-44 40 SEQ ID NO: 6 41 SEQ ID NO: 35, amino acids 41-46 42 SEQ ID NO: 7 43 SEQ ID NO: 8 44 SEQ ID NO: 35, amino acids 44-49 45 SEQ ID NO: 129 46 SEQ ID NO: 130

[0081] In the second set, each peptide consisted of nine amino acid residues identical to a run of amino acids in the Rs-AFP2 sequence (SEQ ID NO: 35). The set of forty-three 9-mer peptides (numbered 47 to 89) covered the entire Rs-AFP2 (SEQ ID NO: 35) sequence. For example, PEPTIDE 47 had the sequence of Rs-AFP2 (SEQ ID NO: 35) from amino acid position 1 to position 9; PEPTIDE 48 had the sequence of Rs-AFP2 (SEQ ID NO: 35) from amino acid position 2 to position 10; PEPTIDE 49 had the sequence of Rs-AFP2 (SEQ ID NO: 35) from amino acid position 3 to position 11; PEPTIDE 88 had the sequence of Rs-AFP2 (SEQ ID NO: 35) from amino acid position 42 to position 50; PEPTIDE 89 had the sequence of Rs-AFP-2 (SEQ ID NO: 35) from amino acid position 43 to position 51. The sequence identifications of each of the peptides is set out below. 9 Rs-AFP2 9-mer peptides Peptide SEQ ID NO: 47 SEQ ID NO: 136 48 SEQ ID NO: 137 49 SEQ ID NO: 138 50 SEQ ID NO: 35, amino acids 4-12 51 SEQ ID NO: 35, amino acids 5-13 52 SEQ ID NO: 35, amino acids 6-14 53 SEQ ID NO: 35, amino acids 7-15 54 SEQ ID NO: 35, amino acids 8-16 55 SEQ ID NO: 35, amino acids 9-17 56 SEQ ID NO: 35, amino acids 10-18 57 SEQ ID NO: 35, amino acids 11-19 58 SEQ ID NO: 35, amino acids 12-20 59 SEQ ID NO: 35, amino acids 13-21 60 SEQ ID NO: 35, amino acids 14-22 61 SEQ ID NO: 35, amino acids 15-23 62 SEQ ID NO: 35, amino acids 16-24 63 SEQ ID NO: 35, amino acids 17-25 64 SEQ ID NO: 35, amino acids 18-26 65 SEQ ID NO: 35, amino acids 19-27 66 SEQ ID NO: 35, amino acids 20-28 67 SEQ ID NO: 35, amino acids 21-29 68 SEQ ID NO: 35, amino acids 22-30 69 SEQ ID NO: 35, amino acids 23-31 70 SEQ ID NO: 9 71 SEQ ID NO: 10 72 SEQ ID NO: 35, amino acids 26-34 73 SEQ ID NO: 35, amino acids 27-35 74 SEQ ID NO: 35, amino acids 28-36 75 SEQ ID NO: 35, amino acids 29-37 76 SEQ ID NO: 35, amino acids 30-38 77 SEQ ID NO: 35, amino acids 31-39 78 SEQ ID NO: 11 79 SEQ ID NO: 35, amino acids 33-41 80 SEQ ID NO: 35, amino acids 34-42 81 SEQ ID NO: 35, amino acids 35-43 82 SEQ ID NO: 12 83 SEQ ID NO: 35, amino acids 37-45 84 SEQ ID NO: 35, amino acids 38-46 85 SEQ ID NO: 35, amino acids 39-47 86 SEQ ID NO: 13 87 SEQ ID NO: 14 88 SEQ ID NO: 15 89 SEQ ID NO: 16

[0082] In the third set, each peptide consisted of twelve amino acid residues identical to a run of amino acids in the Rs-AFP2 sequence (SEQ ID NO: 35). The set of forty 12-mer peptides (numbered 90 to 129) covered the entire Rs-AFP2 sequence. For example, PEPTIDE 90 had the sequence of Rs-AFP2 (SEQ ID NO: 35) from amino acid position 1 to position 12; PEPTIDE 91 hand the sequence of Rs-AFP2 (SEQ ID NO: 35) from amino acid position 2 to position 13; PEPTIDE 92 had the sequence of Rs-AFP2 (SEQ ID NO: 35) from amino acid position 3 to position 14; PEPTIDE 128 had the sequence of Rs-AFP2 (SEQ ID NO: 35) from amino acid position 39 to position 50; PEPTIDE 129 had the sequence of Rs-AFP2 (SEQ ID NO: 35) from amino acid position 40 to position 51. The sequence identifications of each of the peptides is set out below. 10 Rs-AFP2 12-mer peptides Peptide SEQ ID NO: 90 SEQ ID NO: 139 91 SEQ ID NO: 140 92 SEQ ID NO: 141 93 SEQ ID NO: 35, amino acids 4-15 94 SEQ ID NO: 35, amino acids 5-16 95 SEQ ID NO: 35, amino acids 6-17 96 SEQ ID NO: 35, amino acids 7-18 97 SEQ ID NO: 35, amino acids 8-19 98 SEQ ID NO: 35, amino acids 9-20 99 SEQ ID NO: 35, amino acids 10-21 100 SEQ ID NO: 35, amino acids 11-22 101 SEQ ID NO: 35, amino acids 12-23 102 SEQ ID NO: 35, amino acids 13-24 103 SEQ ID NO: 35, amino acids 14-25 104 SEQ ID NO: 35, amino acids 15-26 105 SEQ ID NO: 35, amino acids 16-27 106 SEQ ID NO: 35, amino acids 17-28 107 SEQ ID NO: 35, amino acids 18-29 108 SEQ ID NO: 35, amino acids 19-30 109 SEQ ID NO: 35, amino acids 20-31 110 SEQ ID NO: 35, amino acids 21-32 111 SEQ ID NO: 35, amino acids 22-33 112 SEQ ID NO: 35, amino acids 23-34 113 SEQ ID NO: 35, amino acids 24-35 114 SEQ ID NO: 17 115 SEQ ID NO: 35, amino acids 26-37 116 SEQ ID NO: 35, amino acids 27-38 117 SEQ ID NO: 35, amino acids 28-39 118 SEQ ID NO: 18 119 SEQ ID NO: 19 120 SEQ ID NO: 20 121 SEQ ID NO: 21 122 SEQ ID NO: 35, amino acids 33-44 123 SEQ ID NO: 35, amino acids 34-45 124 SEQ ID NO: 35, amino acids 35-46 125 SEQ ID NO: 35, amino acids 36-47 126 SEQ ID NO: 35, amino acids 37-48 127 SEQ ID NO: 35, amino acids 38-49 128 SEQ ID NO: 131 129 SEQ ID NO: 132

[0083] FIG. 4 is a visual representation of the sets of overlapping 6-, 9- and 12-mer peptides based on the sequence of Rs-AFP2.

[0084] Overlapping 6-,9- and 12-mer Peptides Based on the Rs-AFP1 Protein

[0085] Three sets of overlapping split peptides were synthesised by the PEPSCAN method based on the primary sequence of the Rs-AFP1 protein (SEQ ID NO: 34).

[0086] In the first set, each peptide consisted of six amino acid residues identical to a run of amino acids in the Rs-AFP1 sequence (SEQ ID NO: 34). The set of forty-six 6-mer peptides (numbered 1 to 46) covered the entire Rs-AFP1 sequence (SEQ ID NO: 34). For example, PEPTIDE 1 had the sequence of Rs-AFP1 (SEQ ID NO: 34) from amino acid position 1 to position 6; PEPTIDE 2 had the sequence of Rs-AFP1 (SEQ ID NO: 34) from 40 amino acid position 2 to position 7; PEPTIDE 3 had the sequence of Rs-AFP1 (SEQ ID NO: 34) from amino acid position 3 to position 8; PEPTIDE 45 had the sequence of Rs-AFP1 (SEQ ID NO: 34) from amino acid position 45 to position 50; PEPTIDE 46 had the sequence of Rs-AFP1 (SEQ ID NO: 34) from amino acid position 46 to position 51. The sequence identifications of each of the set out below. 11 Rs-AFP1 6-mer peptides Peptide SEQ ID NO: 1 SEQ ID NO: 34, amino acids 1-6 2 SEQ ID NO: 34, amino acids 2-7 3 SEQ ID NO: 34, amino acids 3-8 4 SEQ ID NO: 34, amino acids 4-9 5 SEQ ID NO: 34, amino acids 5-10 6 SEQ ID NO: 34, amino acids 6-11 7 SEQ ID NO: 34, amino acids 7-12 8 SEQ ID NO: 34, amino acids 8-13 9 SEQ ID NO: 34, amino acids 9-14 10 SEQ ID NO: 34, amino acids 10-15 11 SEQ ID NO: 34, amino acids 11-16 12 SEQ ID NO: 34, amino acids 12-17 13 SEQ ID NO: 34, amino acids 13-18 14 SEQ ID NO: 34, amino acids 14-19 15 SEQ ID NO: 34, amino acids 15-20 16 SEQ ID NO: 34, amino acids 16-21 17 SEQ ID NO: 34, amino acids 17-22 18 SEQ ID NO: 34, amino acids 18-23 19 SEQ ID NO: 34, amino acids 19-24 20 SEQ ID NO: 34, amino acids 20-25 21 SEQ ID NO: 34, amino acids 21-26 22 SEQ ID NO: 34, amino acids 22-27 23 SEQ ID NO: 34, amino acids 23-28 24 SEQ ID NO: 34, amino acids 24-29 25 SEQ ID NO: 34, amino acids 25-30 26 SEQ ID NO: 34, amino acids 26-31 27 SEQ ID NO: 34, amino acids 27-32 28 SEQ ID NO: 34, amino acids 28-33 29 SEQ ID NO: 34, amino acids 29-34 30 SEQ ID NO: 34, amino acids 30-35 31 SEQ ID NO: 34, amino acids 31-36 32 SEQ ID NO: 34, amino acids 32-37 33 SEQ ID NO: 34, amino acids 33-38 34 SEQ ID NO: 34, amino acids 34-39 35 SEQ ID NO: 34, amino acids 35-40 36 SEQ ID NO: 34, amino acids 36-41 37 SEQ ID NO: 34, amino acids 37-42 38 SEQ ID NO: 34, amino acids 38-43 39 SEQ ID NO: 34, amino acids 39-44 40 SEQ ID NO: 34, amino acids 40-45 41 SEQ ID NO: 34, amino acids 41-46 42 SEQ ID NO: 34, amino acids 42-47 43 SEQ ID NO: 34, amino acids 43-48 44 SEQ ID NO: 34, amino acids 44-49 45 SEQ ID NO: 34, amino acids 45-50 46 SEQ ID NO: 34, amino acids 46-51

[0087] In the second set, each peptide consisted of nine amino acid residues identical to a run of amino acids in the Rs-AFP1 sequence (SEQ ID NO: 34). The set of forty-three 9-mer peptides (numbered 47 to 89) covered the entire Rs-AFP1 sequence (SEQ ID NO: 34). For example, PEPTIDE 47 had the sequence of Rs-AFP1 (SEQ ID NO: 34) from amino acid position 1 to position 9; PEPTIDE 48 had the sequence of Rs-AFP1 (SEQ ID NO: 34) from amino acid position 2 to position 10; PEPTIDE 49 had the sequence of Rs-AFP1 (SEQ ID NO: 34) from amino acid position 3 to position 11; PEPTIDE 88 had the sequence of Rs-AFP1 (SEQ ID NO:34) from amino acid position 42 to position 50; PEPTIDE 89 had the sequence of Rs-AFP1 SEQ ID NO: 34) from amino acid position 43 to position 51. The sequence identifications of each of the 9-mer peptides is set out below. 12 Rs-AFP1 9-mer peptides Peptide SEQ ID NO: Peptide SEQ ID NO: 47 SEQ ID NO: 34, 69 SEQ ID NO: 34, amino acids 1-9 amino acids 23-31 48 SEQ ID NO: 34, 70 SEQ ID NO: 34, amino acids 2-10 amino acids 24-32 49 SEQ ID NO: 71 SEQ ID NO: 34, 34,amino acids 3-11 amino acids 25-33 50 SEQ ID NO: 34, 72 SEQ ID NO: 34, amino acids 4-12 amino acids 26-34 51 SEQ ID NO: 34, 73 SEQ ID NO: 34, amino acids 5-13 amino acids 27-35 52 SEQ ID NO: 34, 74 SEQ ID NO: 34, amino acids 6-14 amino acids 28-36 53 SEQ ID NO: 34, 75 SEQ ID NO: 34, amino acids 7-15 amino acids 29-37 54 SEQ ID NO: 34, 76 SEQ ID NO: 34, amino acids 8-16 amino acids 30-38 55 SEQ ID NO: 34, 77 SEQ ID NO: 34, amino acids 9-17 amino acids 31-39 56 SEQ ID NO: 34, 78 SEQ ID NO: 34, amino acids 10-18 amino acids 32-40 57 SEQ ID NO: 34, 79 SEQ ID NO: 34, amino acids 11-19 amino acids 33-41 58 SEQ ID NO: 34, 80 SEQ ID NO: 34, amino acids 12-20 amino acids 34-42 59 SEQ ID NO: 34, 81 SEQ ID NO: 34, amino acids 13-21 amino acids 35-43 60 SEQ ID NO: 34, 82 SEQ ID NO: 34, amino acids 14-22 amino acids 36-44 61 SEQ ID NO: 34, 83 SEQ ID NO: 34, amino acids 15-23 amino acids 37-45 62 SEQ ID NO: 34, 84 SEQ ID NO: 34, amino acids 16-24 amino acids 38-46 63 SEQ ID NO: 34, 85 SEQ ID NO: 34, amino acids 17-25 amino acids 39-47 64 SEQ ID NO: 34, 86 SEQ ID NO: 34, amino acids 18-26 amino acids 40-48 65 SEQ ID NO: 34, 87 SEQ ID NO: 34, amino acids 19-27 amino acids 41-49 66 SEQ ED NO: 34, 88 SEQ ID NO: 34, amino acids 20-28 amino acids 42-50 67 SEQ ID NO: 34, 89 SEQ ID NO: 34, amino acids 21-29 amino acids 43-51 68 SEQ ID NO: 34, amino acids 22-30

[0088] In the third set, each peptide consisted of twelve amino acid residues identical to a run of amino acids in the Rs-AFP1 sequence (SEQ ID NO: 34). The set of forty 12-mer peptides (numbered 90 to 129) covered the entire Rs-AFP1 (SEQ ID NO: 34) sequence. For example, PEPTIDE 90 had the sequence of Rs-AFP1 (SEQ ID NO: 34) from amino acid position 1 to position 12; PEPTIDE 91. had the sequence of Rs-AFP1 (SEQ ID NO: 34) from amino acid position 2 to position 13; PEPTIDE 92 had the sequence of Rs-AFP 1 (SEQ ID NO: 34) from amino acid position 3 to position 14; PEPTIDE 128 had the sequence of Rs-AFP1 (SEQ ID NO: 34) from amino acid position 39 to position 50; PEPTIDE 129 had the sequence of Rs-AFP I (SEQ ID NO: 34) from amino acid position 40 to position 51. The sequence identifications of each of the 12-mer peptides is set out below. 13 Rs-AFP1 12-mer peptide Peptide SEQ ID NO: Peptide SEQ ID NO: 90 SEQ ID NO: 34, 110 SEQ ID NO: 34, amino acids 1-12 amino acids 21-32 91 SEQ ID NO: 34, 111 SEQ ID NO: 34, amino acids 2-13 amino acids 22-33 92 SEQ ID NO: 34, 112 SEQ ID NO: 34, amino acids 3-14 amino acids 23-34 93 SEQ ID NO: 34, 113 SEQ ID NO: 34, amino acids 4-15 amino acids 24-35 94 SEQ ID NO: 34, 114 SEQ ID NO: 34, amino acids 5-16 amino acids 25-36 95 SEQ ID NO: 34, 115 SEQ ID NO: 34, amino acids 6-17 amino acids 26-37 96 SEQ ID NO: 34, 116 SEQ ID NO: 34, amino acids 7-18 amino acids 27-38 97 SEQ ID NO: 34, 117 SEQ ID NO: 34, amino acids 8-19 amino acids 28-39 98 SEQ ID NO: 34, 118 SEQ ID NO: 34, amino acids 9-20 amino acids 29-40 99 SEQ ID NO: 34, 119 SEQ ID NO: 34, amino acids 10-21 amino acids 30-41 100 SEQ ID NO: 34, 120 SEQ ID NO: 34, amino acids 11-22 amino acids 31-42 101 SEQ ID NO: 34, 121 SEQ ID NO: 34, amino acids 12-23 amino acids 32-43 102 SEQ ID NO: 34, 122 SEQ ID NO: 34, amino acids 13-24 amino acids 33-44 103 SEQ ID NO: 34, 123 SEQ ID NO: 34, amino acids 14-25 amino acids 34-45 104 SEQ ID NO: 34, 124 SEQ ID NO: 34, amino acids 15-26 amino acids 35-46 105 SEQ ID NO: 34, 125 SEQ ID NO: 34, amino acids 16-27 amino acids 36-47 106 SEQ ID NO: 34, 126 SEQ ID NO: 34, amino acids 17-28 amino acids 37-48 107 SEQ ID NO: 34, 127 SEQ ID NO: 34, amino acids 18-29 amino acids 38-49 108 SEQ ID NO: 34, 128 SEQ ID NO: 34, amino acids 19-30 amino acids 39-50 109 SEQ ID NO: 34, 129 SEQ ID NO: 34, amino acids 20-31 amino acids 40-51

[0089] (d) Loop 1 Peptide Based on the Rs-AFP2 Protein

[0090] A further cyclic Rs-AFP2-based MPS peptide was synthesised. The loop 1 20 peptide consisted often amino acid residues identical to the Rs-AFP2 sequence between the cysteine residue at position 36 and the cysteine residue at position 45. The loop 1 peptide has the following sequence: CNYVFPAHKC (SEQ ID NO: 28). The peptide was cyclised via the two cysteines.

[0091] (e) 19-mer Peptides Based on the Rs-AFP2 Protein

[0092] Two further MPS peptides were synthesised.

[0093] Peptide G1 consisted of nineteen amino acid residues and had a sequence identical to the primary sequence of the Rs-AFP2 protein between positions 31 and 49. Peptide G1 has the sequence: ARHGSCNYVFPAHKCICYF (SEQ ID NO: 25).

[0094] Peptide G2 consisted of nineteen amino acid residues and had a sequence based on the primary sequence of the Rs-AFP2 protein between positions 31 and 49. To prevent dimerization or cyclization of the peptide, cysteine residues were replaced by alpha-aminobutyric acid (identified by the symbol B). Alpha-aminobutyric acid is a derivative with a side chain consisting of —CH2—CH3 which cannot form disulphide bonds. Peptide G2 has the sequence: ARHGSBNYVFPAHYBIBYF (SEQ ID NO: 26).

[0095] (f) Peptide J1, Based on the Ah-AMP1 Protein

[0096] A further MPS peptide was synthesised. Peptide J1 consisted of nineteen amino acid residues and had a sequence based on the primary sequence of Ah-AMP1, shown in FIG. 9 between positions 30 and 48. To prevent dimerization or cyclization of the peptide, cysteine residues were replaced by alpha-aminobutyric acid (identified by the symbol B). Peptide J1 had the following sequence: ASHGABHKRENHWKBFBYF (SEQ ID NO: 27).

[0097] (g) Peptide Handling and Storage

[0098] Peptides insoluble in water were dissolved in 50% acetonitrile: 50% acetonitrile was added to the peptide to give a stock solution which could be further diluted with water for fungal growth assays. Fungal growth was not affected by the presence of acetonitrile at the maximum concentration tested (20% v/v in the test well).

[0099] Split 15-mer peptides were dissolved in sterile milli-Q water to a final concentration of 5 mg/ml. MPS 15-mer peptides were dissolved to a final concentration between 4 and 10 mg/ml using acetonitrile as solvent for those peptides insoluble in water. Split 6-, 9- and 12-mer Rs-AFP 1 peptides were dissolved to a final concentration of 2 mg/ml in 20% acetonitrile. Split 6-, 9- and 12-mer Rs-AFP2 peptides were dissolved to a final concentration of 2 mg/ml in sterile water except for peptides numbers 1 (SEQ ID NO: 133) and 83 (SEQ ID NO: 35, amino acids 37-45) which were dissolved in 50% acetonitrile and numbers 3, 4, 5, 25, 47, 52, 64, 69, 70, 73, 74, 77, 85 and 93 which were dissolved in 20% acetonitrile. Both Rs-AFP1 and Rs-AFP2 peptides were freeze-dried just before weighing. The loop 1 MPS peptide was completely soluble in water and dissolved at a concentration of 2 mg/ml. The foregoing peptides have the following sequence identifications:

[0100] Peptide 3—SEQ ID NO: 135; Peptide 69—SEQ ID NO: 35, amino acids 23-3 1; Peptide 4—SEQ ID NO: 35, amino acids 4-9; Peptide 70—SEQ ID NO: 9; Peptide 5—SEQ ID NO: 35, amino acids 5-10; Peptide 73—SEQ ID NO: 35, amino acids 27-35; Peptide 25—SEQ ID NO: 35, amino acids 25-30; Peptide 74—SEQ ID NO: 35, amino acids 28-36; Peptide 47 SEQ ID NO: 136; Peptide 77 SEQ ID NO: 35, amino acids 31-39; Peptide 52—SEQ ID NO: 35, amino acids 6-14; Peptide 85—SEQ ID NO: 35, amino acids 39-47; Peptide 64—SEQ ID NO: 35, amino acids 18-26; Peptide 93—SEQ ID NO: 35, amino acids 4-15; loop 1—SEQ ID NO: 28

[0101] De-aerated water and solvents were used to avoid peptide oxidation. Acetonitrile was deoxygenated with nitrogen; water was de-aerated by boiling for 20 minutes. Peptide solutions were stored at −20° C. under an atmosphere of nitrogen gas to avoid oxidation. Peptides that had been refrigerated were allowed to warm to room temperature before opening of the vials so as to avoid absorption of water.

EXAMPLE 2

[0102] Bioassays for Antifungal Activity: Methodology

[0103] The following fungal strains were used: Alternaria brassicicola (MUCL 20297), Ascophyta pisi (MUCL 30164), Botrytis cinerea (MUCL 30158), Fusarium culmorum (IMI 180420) and Verticillium dahliae (MUCL 19210).

[0104] Fungi were grown on six cereal agar plates at room temperature and under white fluorescent light except for F culmorum and V dahliae which were grown in the dark. The duration of the spore harvest varied between 10 and 25 days depending on the strain. Spores were collected as follows. Five to ten ml of sterile milli-Q water was poured into each dish and the surface of the agar was rubbed with a sterile spatula to obtain a suspension containing mycelium and spores. This suspension was filtered through a sterile glasswool-plugged funnel and the filtrate containing the spores was collected in a sterile polypropylene centrifuge tube. The A brassicicola spores were suspended in sterile 0.1 Tween 20 (Merk, 822184) due to their hydrophobic nature. The spore suspensions were then washed twice by centrifugation at 2,400×g for 15 minutes and resuspended in a small volume of steril milli-Q water. The spore density was determined in a counting chamber and then adjusted to 4×107 spores per ml. Aliquots of the spore suspension were transferred to sterile microtubes and an equal volume of sterile 50% glycerol (Merck, 4091) was added to each tube so that a final spore suspension of 2×107 spores per ml in 25% glycerol was obtained. After careful mixing of the stock, the spore suspension was transferred in 100 &mgr;l aliquots to sterile microtubes and stored at −80° C.

[0105] Antifungal activity was measured by photospectrometry as described by Broekaert et al, (1990, FEMS Microbiol Lett, 69:55-60). Tests were performed by adding 20 &mgr;l of test solution and 80 &mgr;l of a fungal spore suspension (2×104 spores/ml) per well in a sterile flat-bottom 96-well microtiterplate. The spore suspension was prepared by diluting the stock spore suspension (107 spores/ml in 25% glycerol) 1:1000 in half-strength potato dextrose broth. A positive growth control consisting of eight wells containing 20 &mgr;l of sterile milli-Q water and 80 &mgr;l of the fungal spore suspension was included in each test. The microtiterplates were incubated (with the lid on) in an aerated place at room temperature and under conditions of darkness for all test organisms.

[0106] After 30 minutes of incubation (when the spores are settled on the well bottom) the optical density was measured in a microplate reader (Bio Rad 3550-UV) at 595 nm. Incubation of the microtiterplates continued until the optical density of the control microculture was between 0.250 and 0.500, which took approximately 72 hours (96 hours for V dahliae). Percentage of growth inhibition (% GI) was estimated as 100 times the ratio of the corrected absorbance of the control microculture minus the corrected absorbance of the test microculture over the corrected absorbance of the control microculture at 595 nm. The corrected absorbance values equal the absorbance at 595 nm of the culture measured after 72 or 96 hours minus the absorbance at 595 nm measured after 30 minutes.

% GI=[(Acontrol−Atest/Acontrol]×100

where Acontrol=(A72/96h−A30min)control

Atest=(A72/96h−A30min)test.

[0107] The IC50 value is defined as the concentration that gives a 50% growth inhibition after 72 or 96 hours of incubation. The minimum inhibitory concentration (MIC) corresponds to the minimum concentration that gives 100% growth inhibition after 72 or 96 hours incubation.

[0108] The composition of the different culture media used in the bioassays is given below:

[0109] Six Cereal Agar (6CA)

[0110] 20 g Bambix (Nutricia), 15 g Agar Technical (Oxoid L13), 1 l milli-Q water, sterilized for 15 minutes at 121° C.

[0111] SMF

[0112] 285 mg (2.5 mM K+) K2HPO4.3H2O, 12.5 mg (50 &mgr;M Mg2+) MgSO4.7H2O, 7.3 mg (50 &mgr;M Ca2+) CaCl2.2H2O, 1.14 mg (5 &mgr;MFe2+) FeSO4.7H2O, 0.023 mg (0.1 &mgr;M Co2+) CoCl2.6H2O, 0.024 mg (0.1 &mgr;M Cu2+) CuSO4.5H2O, 0.24 mg (2 &mgr;M Na+) Na2MoO4.2H2O, 0.03 mg (0.5 &mgr;M BO3+) H3BO3, 0.01 mg (0.1 &mgr;M K+)Kl, 0.14 mg (0.5 &mgr;M Zn+) ZnSO4.7H2O, 0.01 mg (0.1 &mgr;M Mn2+) MnSO4.1H2O, 10 g glucose, 1 g asparagine, 20 mg methionine, 20 mg myo-inositol, 2 mg biotine, 10 mg thiamine-HCl, 2 mg piridoxine, 1 l milli-Q water; sterilized by filtration on 0.22 &mgr;m filters and stored at 4° C.

[0113] SMF+

[0114] SMF medium supplemented with 1 mM Ca2+ and 50 mM K+.

[0115] Half Strength Potato Dextrose Broth (½ PDB)

[0116] 12 g PDB (Difco 0549-01-7), 1 l milli-Q water; sterilized for 15 minutes at 121° C.

[0117] {fraction (1/16)} Potato Dextrose Broth ({fraction (1/16)} PDB)

[0118] 1.5 g PDB (Difco 0549-01-7), 1 l milli-Q water; sterilized for 15 minutes at 121° C.

EXAMPLE 3

[0119] Antifungal Activity of the 15-mer Peptides

[0120] The split peptides were tested for their antifungal activity on F. culmorum. Each peptide was tested in a twofold dilution series starting from 500 &mgr;g/ml down to 3.9 &mgr;g/ml and each test was carried out in duplicate. The results given in Table 1 are a combination of microscopic analysis and optical density determination. Only peptides 6 (SEQ ID NO: 1), 7 (SEQ ID NO: 2), 8 (SEQ ID NO: 3) and 9 (SEQ ID NO: 4) showed a clear antifungal activity with minimum inhibitory concentration (MIC) values of around 30 &mgr;g/ml (peptide 6 (SEQ ID NO: 1)), 60 &mgr;g/ml (peptide 7 (SEQ ID NO: 2)) and 15 &mgr;g/ml (peptides 8 (SEQ ID NO: 3) and 9 (SEQ ID NO: 4)). Due to the partial solubility of the peptides, the initial test concentrations are only approximations and hence the MIC values are approximations.

[0121] The MPS peptides were tested on five different fungal strains: A. brassiciciola, A. pisi, B. cinerea, F. culmorum, and V. dahliae. Each peptide was tested in a twofold dilution series starting from 500 &mgr;g/ml down to 3.9 &mgr;g/ml. The test was carried out five times with F. culmorum and once with the other fungi. The same stock dry peptide was used for all tests but the peptide solutions were different, one solution being used for a maximum of three tests. The results shown in Table 1 correspond to the medium value for the various tests: the variability of results between tests was of an order of two. The sensitivity of the different fungi to the presence of peptides varied, V. dahliae being the most sensitive and B. cinerea being the least sensitive. However, peptides 6 (SEQ ID NO: 1), 7 (SEQ ID NO: 2), 8 (SEQ ID NO: 3) and 9 (SEQ ID NO: 4) were the most potent antifungal peptides on all the test organisms with MIC values varying from 31.25 to 250 &mgr;g/ml depending on the fungus. 14 MIC VALUES (&mgr;g/ml) F CULMORUM A V PEPTIDE Split MPS BRASSICICOLA A PISI B CINEREA DAHLIAE 1 250 250-500 250 >500 >500 250 2 >500 >500 >500 >500 >500 >500 3 >500 250 >500 >500 500 500 4 >500 125 500 >500 250 125 5 250-500 125-250 125 500 500 250 6 31.25 62.5 62.5 125 250 31.25 7 31.25-62.5  62.5 62.5 250 250 31.25 8 15.625 31.25-62.5  31.25 125 250 31.25 9 15.625 31.25-62.5  62.5 250 250 31.25 10 125-250 500 >500 >500 >500 250 Rs-AFP2 10 5-10 10 20-40 >40 10

[0122] In Table 1, peptides 1-10 have the following sequence identifications: Peptide 1—SEQ ID NO: 35, amino acids 1-15; Peptide 2—SEQ ID NO: 35, amino acids 5-19; Peptide 3—SEQ ID NO: 35, amino acids 9-23; Peptide 4—SEQ ID NO: 35, amino acids 13-27; Peptide 5—SEQ ID NO: 35, amino acids 17-31; Peptide 6—SEQ ID 5 NO: 1; Peptide 7—SEQ ID NO: 2; Peptide 8—SEQ ID NO: 3; Peptide 9—SEQ ID NO: 4; Peptide 10—SEQ ID NO: 35, amino acids 37-51; Rs-AFP—SEQ ID NO: 35.

[0123] Peptides 6 (SEQ ID NO: 1), 7 (SEQ ID NO: 2),8 (SEQ ID NO: 3) and 9 (SEQ ID NO: 4) each comprise fifteen amino acid residues identical to a run of fifteen amino acid residues found between position 21 and position 47 of the Rs-AFP2 (SEQ ID NO: 35) sequence shown in FIG. 1. These tests show that peptides 6 (SEQ ID NO: 1), 7 (SEQ ID NO: 2), 8 (SEQ ID NO: 3) and 9 (SEQ ID NO: 4) have antifungal activity.

EXAMPLE 4

[0124] Antifungal Activity of the 6-, 9- and 12-mer Rs-AFP2 Peptides

[0125] The 6-, 9- and 12-mer Rs-AFP2 split peptides were tested for their antifungal activity on F culmorum. Each peptide was tested in a twofold dilution series starting from 400 &mgr;g/ml dow to 3.1 &mgr;g/ml using the medium ½PDB. Rs-AFP2 (SEQ ID NO: 35) was used in all the plates as a positive control, in a twofold dilution series starting from 40 &mgr;g/ml down to 0.31 &mgr;g/ml. The tests were carried out in duplicate for the 6-mer peptides (numbered 1 to 46) and for the 9-mer peptides (numbered 47 to 89) and in triplicate for the 12-mer peptides (numbered 90 to 129). Tables 2, 3 and 4 show the results for the active peptides only. 15 TABLE 2 6-MER PEPTIDES MIC IC50 PEPTIDE (&mgr;g/ml) (&mgr;g/ml) 1 400 268 31 400 297 32 400 279 40 100 72 42 100 83 43 50 28

[0126] The peptides in Table 2 have the following sequence identifications:

[0127] Peptide 1—SEQ ID NO: 133; Peptide 31—SEQ ID NO: 35, amino acids 31-36; Peptide 32—SEQ ID NO: 35, amino acids 32-37; Peptide 40 SEQ ID NO: 6; Peptide 42—SEQ ID NO: 7; Peptide 43—SEQ ID NO: 8. 16 TABLE 3 9-MER PEPTIDES MIC IC50 PEPTIDE (mg/ml) (&mgr;g/ml) 65 200 144 66 150 105 70 100 78 71 100 66 74 400 270 76 300 162 77 300 189 78 400 284 82 100 77 83 250 175 84 400 175 85 200 121 86 400 144 87 50 39 88 50 35 89 400 242

[0128] The peptides in Table 2 have the following sequence identifications: Peptide 65—SEQ ID NO: 35, amino acids 19-27; Peptide 66—SEQ ID NO: 35, amino acids 20-28; Peptide 70—SEQ ID NO: 9; Peptide 71—SEQ ID NO: 10; Peptide 74 SEQ ID NO: 35, amino acids 28-36; Peptide 76—SEQ ID NO: 35, amino acids 30-38; Peptide 77—SEQ ID NO: 35, amino acids 31-39; Peptide 78—SEQ ID NO: 11; Peptide 82—SEQ ID NO: 12; Peptide 83—SEQ ID NO: 35, amino acids 37-45; Peptide 84—SEQ ID NO: 35, amino acids 38-46; Peptide 85—SEQ ID NO: 35, amino acids 39-47; Peptide 86—SEQ ID NO: 13; Peptide 87—SEQ ID NO: 14; Peptide 88—SEQ ID NO: 15; Peptide 89—SEQ ID NO: 16. 17 TABLE 4 12-MER PEPTIDES MIC IC50 PEPTIDE (&mgr;g/ml) (&mgr;g/ml) 106 150 116 108 200 150 109 150 106 110 200 148 111 200 126 112 200 149 113 200 127 114 100 74 115 300 203 116 400 202 117 >400 327 118 200 154 119 50 37 120 200 139 121 50 33 122 50 30 123 300 280 124 200 135 125 400 265 126 300 201 128 >400 344

[0129] In Table 4, the peptides have the following sequence identifications: Peptide 106—SEQ ID NO: 35, amino acids 17-28; Peptide 108—SEQ ID NO: 35, amino acids 19-30; Peptide 109—SEQ ID NO: 35, amino acids 20-31; Peptide 110—SEQ ID NO: 5, amino acids 21-32; Peptide 111—SEQ ID NO: 35, amino acids 22-33; Peptide 112—SEQ ID NO: 35, amino acids 23-34; Peptide 113—SEQ ID NO: 35, amino acids 24-35; Peptide 114—SEQ ID NO: 17; Peptide 115—SEQ ID NO: 35, amino acids 26-37; Peptide 116—SEQ ID NO: 35, amino acids 27-38; Peptide 117—SEQ ID NO: 35, amino acids 28-39; Peptide 118—SEQ ID NO: 18; Peptide 119—SEQ ID NO: 19; Peptide 120—SEQ ID NO: 20; Peptide 21—SEQ ID NO: 21; Peptide 122—SEQ ID NO: 35, amino acids 33-44; Peptide 123—SEQ ID NO: 35, amino acids 34-45; Peptide 124—SEQ ID NO: 35, amino acids 35-46; Peptide 125—SEQ ID NO: 35, amino acids 36-47; Peptide 126—SEQ ID NO: 35, amino acids 37-48; Peptide 127—SEQ ID NO: 35, amino acids 38-49; Peptide 128—SEQ ID NO: 131.

[0130] FIG. 5 is a graphical representation of the results showing the antifungal activity of the Rs-AFP2-based 6-, 9- and 12-mer peptides. Active peptides are indicated by bars in the graphs of IC50 value (&mgr;g/ml) against peptide number which are given for each set of peptides. In the graph for the 9-mer peptide set, peptides are numbered according to their N-terminal amino acid so that, for example, peptide number 1 in the FIG. 5 9-mer graph corresponds to peptide 47 (SEQ ID NO: 136) and peptide number 43 in the FIG. 5 9-mer graph corresponds to peptide number 89 (SEQ ID NO: 16) in Table 3. Similarly, in the graph for the 12-mer peptide set, peptides are numbered according to their N-terminal amino acid so that, for example, peptide number 1 in the FIG. 5 12-mer graph corresponds to peptide 90 (SEQ ID NO: 139) and peptide number 39 in the FIG. 5 12-mer graph corresponds to peptide number 128 (SEQ ID NO: 131) in Table 4.

[0131] FIG. 6 is a diagram summarising all the active 6-mer, 9-mer and 12-mer peptides. Peptides are once again numbered according to their N-terminal amino acid. Each of the active peptides has been categorised according to its IC50 value, as follows.

[0132] In the 6-mer peptide set, peptide numbers 1, 31 and 32 have an IC50 value between 100 and 300 &mgr;g/ml while peptide numbers 40, 42 and 43 have an IC50 value less than 100 &mgr;g/ml. The peptides in the 6-mer set have the following sequence identifications: Peptide 1—SEQ ID NO: 133; Peptide 31—SEQ ID NO: 35, amino acids 31-36; Peptide 32—SEQ ID NO: 35, amino acids 32-37; Peptide 40—SEQ ID NO: 6; Peptide 42—SEQ ID NO: 7; Peptide 43—SEQ ID NO: 8.

[0133] In the 9-mer peptide set, peptide numbers 19, 20, 28, 30 to 32, 37 to 40 and 43 have an IC50 value between 100 and 300 &mgr;g/ml while peptide numbers 24, 25, 36, 41 and 42 have an IC50 value less than 100 &mgr;g/ml (equivalent to peptides 70, 71, 82, 87 and 88 in Table 3). The peptides in the 9-mer set in FIG. 6 have the following sequence identifications: (Corresponding peptide numbers refer to the number of the overlapping RsAFP2 peptide shown in Example 1(b).) Peptide 19—SEQ ID NO: 35, amino acids 19-27 (corresponds to Peptide 65); Peptide 20—SEQ ID NO: 35, amino acids 20-28 (corresponds to Peptide 66); Peptide 24—SEQ ID NO: 9 (corresponds to Peptide 70); Peptide 25—SEQ ID NO: 35, amino acids 25-33 (corresponds to Peptide 71); Peptide 28—SEQ ID NO: 35, amino acids 28-36 (corresponds to Peptide 74); Peptide 30—SEQ ID NO: 35, amino acids 30-38 (corresponds to Peptide 76); Peptide 31—SEQ ID NO: 35, amino acids 31-39 (corresponds to Peptide 77); Peptide 32—SEQ ID NO: 11 (corresponds to Peptide 78); Peptide 36—SEQ ID NO: 35, amino acids 36-44 (corresponds to Peptide 82); Peptide 37—SEQ ID NO: 35, amino acids 37-45 (corresponds to Peptide 83); Peptide 38—SEQ ID NO: 35, amino acids 38-48 (corresponds to Peptide 84); Peptide 39—SEQ ID NO: 35, amino acids 39-47 (corresponds to Peptide 85); Peptide 40—SEQ ID NO: 13 (corresponds to Peptide 86); Peptide 41—SEQ ID NO: 14 (corresponds to Peptide 87); Peptide 42—SEQ ID NO: 15 (corresponds to Peptide 88); Peptide 43—SEQ ID NO: 16 (corresponds to Peptide 89).

[0134] In the 12-mer peptide set, peptide numbers 28 and 39 have an IC50 value between 300 and 400 &mgr;g/ml, peptide numbers 17, 20 to 24, 26, 27, 29, 31 and 34 to 37 have an IC50 value between 100 and 300 &mgr;g/ml while peptides 25, 30, 32 and 33 have an IC50 value less than 100 &mgr;g/ml (equivalent to peptides 114, 119, 121 and 122 in Table 4). In the 12-mer peptide set, the peptides have the following sequence identifications: (Corresponding peptide numbers refer to the number of the overlapping Rs-AFP2 peptide shown in Example 1(b).) Peptide 17—SEQ ID NO: 35, amino acids 17-28 (corresponds to Peptide 106); Peptide 20—SEQ ID NO: 35, amino acids 20-31 (corresponds to Peptide 109); Peptide 21—SEQ ID NO: 35, amino acids 21-32 (corresponds to Peptide 110); Peptide 22—SEQ ID NO: 35, amino acids 22-33 (corresponds to Peptide 111); Peptide 23—SEQ ID NO: 35, amino acids 23-34 (corresponds to Peptide 112); Peptide 24—SEQ ID NO: 35, amino acids 24-35 (corresponds to Peptide 113); Peptide 26—SEQ ID NO: 35, amino acids 26-37 (corresponds to Peptide 115); Peptide 27—SEQ ID NO: 35, amino acids 27-38 (corresponds to Peptide 116); Peptide 28—SEQ ID NO: 35, amino acids 28-39 (corresponds to Peptide 117); Peptide 29—SEQ ID NO: 18 (corresponds to Peptide 118); Peptide 30—SEQ ID NO: 19 (corresponds to Peptide 119); Peptide 31—SEQ ID NO: 20 (corresponds to Peptide 120); Peptide 34—SEQ ID NO: 35, amino acids 34-45 (corresponds to Peptide 123); Peptide 35—SEQ ID NO: 35, amino acids 35-46 (corresponds to Peptide 124); Peptide 36—SEQ ID NO: 35, amino acids 36-47 (corresponds to Peptide 125); Peptide 37—SEQ ID NO: 35 (corresponds to Peptide 126); Peptide 25—SEQ ID NO: 17 (corresponds to Peptide 114); Peptide 30—SEQ ID NO: 19 (corresponds to Peptide 119); Peptide 32—SEQ ID NO: 21 (corresponds to Peptide 121); Peptide 33—SEQ ID NO: 35, amino acids 33-44 (corresponds with Peptide 122).

[0135] FIG. 6 also shows the active 15-mer peptides. Peptide 1 (N-terminal amino acid corresponding to position 1 in the Rs-AFP2 sequence (SEQ ID NO: 35, amino acids 1-15)), peptide 5 (SEQ ID NO: 35, amino acids 17-31) (N-terminal amino acid corresponding to position 17 in the Rs-AFP2 sequence) and peptide 10 (SEQ ID NO: 35, amino acids 37-51) (N-terminal amino acid corresponding to position 37 in the Rs-AFP2 sequence) have an IC50 value between 100 and 300 &mgr;g/ml. Peptide 6 (SEQ ID NO: 1) (N-terminal amino acid corresponding to position 21 in the Rs-AFP2 sequence), peptide 7 (SEQ ID NO: 2) (N-terminal amino acid corresponding to position 25 in the Rs-AFP2 sequence), peptide 8 (SEQ ID NO: 3) (N-terminal amino acid corresponding to position 29 in the Rs-AFP2 sequence) and peptide 9 (SEQ ID NO: 4) (N-terminal amino acid corresponding to position 33 in the Rs-AFP2 sequence) have an IC50 value less than 100 &mgr;g/ml.

[0136] The antifungal activity of the peptides is reduced by the presence of inorganic salts (1 mM CaCl2 or 50 mM KCl) in the growth medium. The antagonistic effect of cations has previously been reported for Rs-AFPs (Terras et al, 1992, J Biol Chem. 267:1-9) although the cation sensitivity seems to vary largely with the test fungus used.

EXAMPLE 5

[0137] Antifungal Activity of the 6-,9- and 12-mer Rs-AFP1 Peptides

[0138] The 6-, 9- and 12-mer Rs-AFP1 (SEQ ID NO: 34) split peptides were tested for their antifungal activity on F culmorurn and on Ascophyta pisi. Each peptide was tested in a twofold dilution series starting form 400 &mgr;g/ml down to 3.1 &mgr;g/ml using the medium ½ PDB. Rs-AFP2 was used in all the plates as a positive control, in a twofold dilution series starting from 40 &mgr;g/ml down to 0.31 &mgr;g/ml. The tests were carried out in duplicate for F culmorum and once for A p si. The MIC and IC50 values are the average values of two or three experiments, the results being a combination of microscopic analysis and optical density determination. Tables 5, 6 and 7 show the results for the active peptides only. The 6-mer peptides are numbered 1 to 46, the 9-mer peptides are numbered 47 to 89 and the 12-mer peptides are numbered 90 to 129. 18 TABLE 5 6-MER PEPTIDES F CULMORUM A PISI MIC IC50 MIC IC50 PEPTIDE (tg/ml) (&mgr;g/ml) (&mgr;g/ml) (&mgr;g/ml) 31 400 266 — — 42 100 61   400 267 43 75 38 >400 340 44 >400 376 >400 400

[0139] In Table 5, the peptides have the following sequence identifications: Peptide 31—SEQ ID NO: 34, amino acids 31-36; Peptide 42—SEQ ID NO: 34, amino acids 42-47; Peptide 43—SEQ ID NO: 34 amino acids 43-48; Peptide 44—SEQ ID NO: 34, amino acids 44-49. 19 TABLE 6 9-MER PEPTIDES F CULMORUM A PISI MIC IC MIC IC PEPTIDE (&mgr;g/ml) (&mgr;g/ml) (&mgr;g/ml) (&mgr;g/ml) 74 >400 370 — — 76 250 169 >400 342 77 300 193 200 143 78 100 69 100 70 82 150 113 400 265 83 400 330 — — 84 250 145 — — 85 200 121 400 184 86 250 196 200 80 87 37.5 28 50 31 88 37.5 21 25 18 89 400 233 100 70

[0140] In Table 6, the 9-mer peptides have the following sequence identifications: Peptide 74—SEQ ID NO: 34, amino acids 28-36; Peptide 76—SEQ ID NO: 34, amino acids 30-38; Peptide 77—SEQ ID NO: 34, amino acids 31-39; Peptide 78—SEQ ID NO: 34, amino acids 32-40; Peptide 82—SEQ ID NO: 34, amino acids 36-44; Peptide 83—SEQ ID NO: 34, amino acids 37-45; Peptide 84; —SEQ ID NO: 34, amino acids 38-46; Peptide 85—SEQ ID NO: 34, amino acids 39-47; Peptide 86—SEQ ID NO: 34, amino acids 4048; Peptide 87—SEQ ID NO: 34, amino acids 41-49; Peptide 88—SEQ ID NO: 34, amino acids 42-50; Peptide 89—SEQ ID NO: 34, amino acids 43-51. 20 TABLE 7 12-MER PEPTIDES F CULMORUM A PISI MIC IC50 MIC IC50 PEPTIDE (&mgr;g/ml) (&mgr;g/ml) (&mgr;g/ml) (&mgr;g/ml) 110 400 223 — — 113 400 362 — — 114 400 328 >400 318 116 400 359 — — 117 400 347 400 293 118 100 63 200 91 119 50 33 100 66 120 150 105 200 126 121 50 38 100 55 122 50 35 100 74 123 300 266 >400 349 124 300 213 >400 318 125 >400 350 400 281 126 300 189 400 298 127 >400 354 400 302 128 >400 320 400 181 129 — — 400 311

[0141] In Table 7, the 12-mer peptides have the following sequence identifications: Peptide 110—SEQ ID NO: 34, amino acids 21-32; Peptide 113—SEQ ID NO: 34, amino acids 24-35; Peptide 114—SEQ ID NO: 34, amino acids 25-36; Peptide 116—SEQ ID NO: 34, amino acids 27-38; Peptide 117—SEQ ID NO: 34, amino acids 28-39; Peptide 118—SEQ ID NO: 34, amino acids 29-40; Peptide 119—SEQ ID NO: 34, amino acids 30-41; Peptide 120—SEQ ID NO: 34, amino acids 31-42; Peptide 121—SEQ ID NO: 34, amino acids 32-43; Peptide 122—SEQ ID NO: 34, amino acids 33-44; Peptide 123—SEQ ID NO: 34, amino acids 34-45l Peptide 124—SEQ ID NO: 34, amino acids 35-46; Peptide 125—SEQ ID NO: 34, amino acids 36-47; Peptide 126 SEQ ID NO: 34, amino acids 37-48; Peptide 127—SEQ ID NO: 34, amino acids 38-49; Peptide 128—SEQ ID NO: 34, amino acids 39-50; Peptide 129 SEQ ID NO: 34, amino acids 40-51.

[0142] FIG. 7 is a graphical representation of the results showing the antifungal activity of the Rs-AFP1-based 6-, 9- and 12-mer peptides on F culmorum. Active peptides are indicated by bars in the graphs of IC50 value (&mgr;g/ml) against peptide number which are given the graph for the 9-mer peptide set, peptides are numbered according to their N-terminal amino acid so that, for example, peptide number 1 in the FIG. 7 9-mer graph corresponds to peptide 47 and peptide number 43 in the FIG. 7 9-mer graph corresponds to peptide number 89 in Table 6. Similarly, in the graph for the 12-mer peptide set, peptides are numbered according to their N-terminal amino acid so that, for example, peptide number 1 in the FIG. 7 12-mer graph corresponds to peptide 90 and peptide number 39 in the FIG. 7 12-mer graph corresponds to peptide number 128 in Table 7.

[0143] In FIG. 7, the 6-mer peptides have the following sequence identifications: (The corresponding peptide numbers refer to the number of the overlapping Rs-AFP1 peptide shown in Example 1(c).) Peptide 31—SEQ ID NO: 34, amino acids 31-36; Peptide 42—SEQ ID NO: 34, amino acids 42-47; Peptide 43—SEQ ID NO: 34, amino acids 43-48; Peptide 44—SEQ ID NO: 34, amino acids 44-49. The 9-mer peptides have the following sequence identifications: Peptide 28—SEQ ID NO: 34, amino acids 28-36 (corresponds to Peptide 74); Peptide 30—SEQ ID NO: 34, amino acids 30-38 (corresponds to Peptide 76); Peptide 31—SEQ ID NO: 34, amino acids 34-42 (corresponds to Peptide 77); Peptide 32—SEQ ID NO: 34, amino acids 32-40 (corresponds to Peptide 78); Peptide 36—SEQ ID NO: 34, amino acids 36-44 (corresponds to Peptide 82); Peptide 37—SEQ ID NO: 34, amino acids 37-45 (corresponds to Peptide 83); Peptide 38—SEQ ID NO: 34, amino acids 38-46 (corresponds to Peptide 84); Peptide 39—SEQ ID NO: 34, amino acids 39-47 (corresponds to Peptide 85); Peptide 40—SEQ ID NO: 34, amino acids 40-48 (corresponds to Peptide 86); Peptide 41—SEQ ID NO: 34, amino acids 41-49 (corresponds to Peptide 87); Peptide 42—SEQ ID NO: 34, amino acids 42-50 (corresponds to Peptide 88); Peptide 43, amino acids 43-51 (corresponds to Peptide 89). The 12-mer peptides have the following sequence identifications: Peptide 21—SEQ ID NO: 34, amino acids 21-32 (corresponds to Peptide 110); Peptide 24—SEQ ID NO: 34, amino acids 24-35 (corresponds to Peptide 113); Peptide 25—SEQ ID NO: 34, amino acids 25-36 (corresponds to Peptide 114); Peptide 27—SEQ ID NO: 34, amino acids 27-38 (corresponds to Peptide 116); Peptide 28—SEQ ID NO: 34, amino acids 28-39 (corresponds to Peptide 117); Peptide 29—SEQ ID NO: 34, amino acids 29-40 (corresponds to Peptide 118); Peptide 30—SEQ ID NO: 34, amino acids 30-41 (corresponds to Peptide 119); Peptide 31—SEQ ID NO: 34, amino acids 31-42 (corresponds to Peptide 120); Peptide 32—SEQ ID NO: 34, amino acids 32-43 (corresponds to Peptide 121); Peptide 33—SEQ ID NO: 34, amino acids 33-44 (corresponds to Peptide 122); Peptide 34—SEQ ID NO: 34, amino acids 34-45 (corresponds to Peptide 124); Peptide 36—SEQ ID NO: 34, amino acids 36-47 (corresponds to Peptide 125); Peptide 37—SEQ ID NO: 34, amino acids 37-48 (corresponds to Peptide 126); Peptide 38—SEQ ID NO: 34, amino acids 38-49 (corresponds to Peptide 127); Peptide 39—SEQ ID NO: 34, amino acids 39-50 (corresponds to Peptide 128).

[0144] FIG. 8 is a diagram summarising the 6-mer, 9-mer and 12-mer peptides which are active on F culmorum. Peptides are once again numbered according to their 15 N-terminal amino acid. Each of the active peptides has been categorised according to its IC50 value, as follows.

[0145] In the 6-mer peptide set, peptide number 44 (SEQ ID NO: 34, amino acids 44-49) has an IC50 value between 300 and 400 &mgr;g/ml, peptide number 31 (SEQ ID NO: 34, amino acids 31-36) has an IC50 value between 100 and 300 &mgr;g/ml while peptide numbers 42 (SEQ ID NO: 34, amino acids 42-46) and 43 (SEQ ID NO: 34, amino acids 43-47) have an IC50 value less than 100 &mgr;g/ml.

[0146] In the 9-mer peptide set, peptide numbers 28 (SEQ ID NO: 34, amino acids 28-36) and 37 (SEQ ID NO: 34, amino acids 37-45)have an IC50 value between 300 and 400 &mgr;g/ml, peptide numbers 30 (SEQ ID NO: 34, amino acids 30-38),31 (SEQ ID NO: 34, amino acids 31-39),36 (SEQ ID NO: 34, amino acids 36-44),38 (SEQ ID NO: 34, amino acids 38-46) to 40 (SEQ ID NO: 34, amino acids 40-48) and 43 (SEQ ID NO: 34, amino acids 43-51) have an IC50 value between 100 and 300 &mgr;g/ml while peptide numbers 32 (SEQ ID NO: 34, amino acids 32-40),41 (SEQ ID NO: 34, amino acids 41-49) and 42 (SEQ ID NO: 34, amino acids 42-50) have an IC50 value less than 100 &mgr;g/ml (equivalent to peptides 78, 87 and 88 in Table 6). On A pisi two further 9-mer peptides have an IC50 value less than 100 &mgr;g/ml: peptide number 40 (SEQ ID NO: 34, amino acids 40-48) (equivalent to peptide 86 in Table 6) and peptide number 43 (SEQ ID NO: 34, amino acids 43-51) (equivalent to peptide 89 in Table 6).

[0147] In the 12-mer peptide set, peptide numbers 24 to 28, 36, 38 and 39 have an IC50 value between 300 and 400 &mgr;g/ml, peptide numbers 21, 31, 34, 35 and 37 have an IC50 value between 100 and 300 &mgr;g/ml while peptides 29, 30, 32 and 33 have an IC50 value less than 100 &mgr;g/ml (equivalent to peptides 118, 119, 121 and 122 in Table 7). The 12-mer peptides referred to above have the following sequence identifications: Peptide 24—SEQ ID NO: 34, amino acids 24-35; Peptide 25—SEQ ID NO: 34, amino acids 25-36; Peptide 26—SEQ ID NO: 34, amino acids 26-37; Peptide 27—SEQ ID NO: 34, amino acids 27-38; Peptide 28—SEQ ID NO: 34, amino acids 28-39; Peptide 36—SEQ ID NO: 34, amino acids 36-47; Peptide 38—SEQ ID NO: 34, amino acids 38-49; Peptide 39—SEQ ID NO: 34, amino acids 39-50; Peptide 21—SEQ ID NO: 34, amino acids 21-32; Peptide 31—SEQ ID NO: 34, amino acids 31-42; Peptide 34—SEQ ID NO: 34, amino acids 34-45; Peptide 35—SEQ ID NO: 34, amino acids 35-46; Peptide 37—SEQ ID NO: 34, amino acids 37-48; Peptide 29—SEQ ID NO: 34, amino acids 29-40; Peptide 30—SEQ ID NO: 34, amino acids 30-41; Peptide 32—SEQ ID NO: 34, amino acids 32-43; Peptide 33—SEQ ID NO: 34, amino acids 33-44.

[0148] FIG. 8 also shows the active Rs-AFP2-based 15-mer peptides as a comparison. Peptide 1 (SEQ ID NO: 35, amino acids 1 -15) (N-terminal amino acid corresponding to position 1 in the Rs-AFP2 sequence), peptide 5 (SEQ ID NO: 35, amino acids 17-3 1) (N-terminal amino acid corresponding to position 17 in the Rs-AFP2 sequence) and peptide 10 (SEQ ID NO: 35, amino acids 37-51 (N-terminal amino acid corresponding to position 37 in the Rs-AFP2 sequence) have an IC50 value between 100 and 300 &mgr;g/ml. Peptide 6 (SEQ ID NO: 1) (N-terminal amino acid corresponding to position 21 in the Rs-AFP2 sequence), peptide 7 (SEQ ID NO: 2) (N-terminal amino acid corresponding to position 25 in the Rs-AFP2 sequence), peptide 8 (SEQ ID NO: 3) (N-terminal amino acid corresponding to position 29 in the Rs-AFP2 sequence) and peptide 9 (SEQ ID NO: 4) (N-terminal amino acid corresponding to position 33 in the Rs-AFP2 sequence) have an, IC50 value less than 100 &mgr;g/ml.

EXAMPLE 6

[0149] Antifungal Activity of the Loop 1 Peptide Based on the Rs-AFP2 Protein

[0150] The Loop 1 peptide consists often amino acid residues and has the following sequence: CNYVFPAHKC (SEQ ID NO: 28). The two cysteines are cyclised. Table 8 shows the antifungal activity (as MIC values) of the Loop 1 peptide compared to the activity of the 15 -mer Rs-AFP2-based peptides numbered 1 to 10. Table 9 shows the antifungal activity (as IC50 values) of the Loop 1 (SEQ ID NO: 28) peptide compared to the activity of the most active 15-mer peptides. Activity was measured in {fraction (1/16)}th PDB against F culmorum. In Tables 8 and 9, the peptides have the following sequence identifications: Peptide 1—SEQ ID NO: 35, amino acids 1-15; Peptide 2—SEQ ID NO: 35, amino acids 5-19; Peptide 3—SEQ ID NO: 35 amino acids 9-23; Peptide 4—SEQ ID NO: 35, amino acids 13-27; Peptide 5—SEQ ID NO: 35, amino acids 17-31; Peptide 6—SEQ ID NO: 1; Peptide 7—SEQ ID NO: 2; Peptide 8—SEQ ID NO: 3; Peptide 9—SEQ ID NO: 4; Peptide 10—SEQ ID NO: 35, amino acids 37-51; Loop 1—SEQ ID NO: 28. 21 TABLE 8 PEPTIDE ANTIFUNGAL ACTIVITY, MIC &mgr;g/ml 1 180 2 >330 3 180 4 150 5 180 6 45 7 45 8 22.5 9 17.5 10 100 Loop 1 2.5

[0151] 22 TABLE 9 PEPTIDE ANTIFUNGAL ACTIVITY, IC50 (&mgr;g/ml) 6 12 7 5 8 6 9 10 Loop 1 4

EXAMPLE 7

[0152] Antifungal Activity of the 19-mer Peptides G1 and G2 Based on the Rs-AFP2 Protein and the 19-mer Peptide JI Based on the Ah-AMP1 Protein

[0153] Peptide G1 consisted of nineteen amino acid residues and had the sequence: ARHGSCNYVFPAHKCICYF (SEQ ID NO: 25). This peptide was conceived based on the observations that the most active 12-mer and 15-mer peptides fall within this stretch of amino acids and on the observation that this stretch of amino acids corresponds to a beta strand/beta-turn/beta-strand region in the three dimensional model of Rs-AFP1 (Fant F. et al (1994) Abstract of the 12th European Experimental NMR Conference, p247). Peptide G2 consisted of nineteen amino acid residues and had the sequence: ARHGSBNYVFPAHKBIBYF (SEQ ID NO: 26), where the symbol “B” represents an alpha-aminobutyric acid residue. Peptide J1 consisted of nineteen amino acid residues and had the sequence: ASHGABHKRENHWKBFBYF (SEQ ID NO: 27) where the symbol “B” represents an alpha-aminobutyric acid residue.

[0154] Antifungal activity tests were carried out once in ½ PDB and once in {fraction (1/16)} PDB. Table 10 gives the MIC and IC50 values. 23 TABLE 10 MEDIUM: ½ PDB MEDIUM: {fraction (1/16)} PDB PEPTIDE MIC (&mgr;g/ml) IC50 (&mgr;g/ml) MIC (&mgr;g/ml) IC50 (&mgr;g/ml) G1 25 19 12.5 10 G2 25 17 12.5 10 J1 12.5 9 6.25 5 Peptide G1 - SEQ ID NO: 25; Peptide G2 - SEQ ID NO: 26; Peptide J1 - SEQ ID NO: 27.

[0155] The replacement of cysteine residues in G1 (SEQ ID NO: 25) by alpha-amino butyric acid to give peptide G2 (SEQ ID NO: 26) does not affect antifungal activity. Peptide J1 (SEQ ID NO: 27)was even more active than peptides G1 (SEQ ID NO: 25) and G2 (SEQ ID NO: 26).

EXAMPLE 8

[0156] Antifungal Activity of Combinations of Rs-AFP2 and the 15-mer Peptides

[0157] The 15-mer peptides were tested for their ability to affect the antifungal activity of the Rs-AFP2 protein (SEQ ID NO: 35). Each peptide was added at a subinhibitory concentration (20 &mgr;g/ml ) to a twofold dilution series of Rs-AFP2 (SEQ ID NO: 35) ranging from 20 &mgr;g/ml down to 0.15 &mgr;g/ml. In the control series only water was added.

[0158] The target fungus was F culmorum and the growth medium was SMF. In order to exclude the effect of differential binding of the peptides to the microplate wells, the plates were coated with bovine serum albumin. Table 11 shows the relative specific antifungal activity of the Rs-AFO2/peptide combinations in comparison to the antifungal activity of Rs-AFP2 (SEQ ID NO: 35). The relative specific activity is defined as one hundred times the MIC of Rs-AFP2 divided by the MIC of the Rs-AFP2/peptide combination. Data are based on duplicate tests. 24 TABLE 11 Peptide Added Relative Specific Antifungal Activity — 100 1 310 2 120 3 180 4 180 5 180 6 300 7 350 8 470 9 450 10  250

[0159] In Table 11, the peptides have the following sequence identifications: Peptide 1—SEQ ID NO: 35, amino acids 1-15; Peptide 2—SEQ ID NO: 35, amino acids 5-19; Peptide 3—SEQ ID NO: 35, amino acids 9-23; Peptide 4—SEQ ID NO: 35, amino acids 13-27; Peptide 5—SEQ ID NO: 35, amino acids 17-31; Peptide 6—SEQ ID NO: 1; Peptide 7—SEQ ID NO: 2; Peptide 8—SEQ ID NO: 3; Peptide 9—SEQ ID NO: 4; Peptide 10—SEQ ID NO: 35, amino acids 37-51.

[0160] Except for peptide 2 (SEQ ID NO: 35, amino acids 5-19), presence of the peptides resulted in increased antifungal activity of Rs-AFP2 (SEQ ID NO: 35). Peptides causing the strongest increase of the antifungal activity were 1, 6, 7, 8, 9 and (to a lesser extent) 10 (Peptide 1—SEQ ID - NO: 35, amino acids 1-15; Peptide 6—SEQ ID NO: 1; Peptide 7—SEQ ID NO: 2; Peptide 8—SEQ D NO: 3; Peptide 9—SEQ ID NO: 4; Peptide 10—SEQ ID NO: 35, amino acids 37-5 1). These peptides potentiated the activity of Rs-AFP2 from 3 to 5-fold. When added to Rs-AFP1 in a similar assay, the peptides caused a comparable enhancement of the antifungal activity.

[0161] The antifungal activity tests show that in most cases the Rs-AFP/peptide combination has an increase in activity compared to the activity of Rs-AFP or the individual peptide when used alone. This increase in activity may be an enhancement due to synergistic interactions between the protein and peptide. For example, protein/peptide hetero-oligomers may be forming resulting in a complex with higher activity. Synergistic interactions between related but non-identical vertebrate antimicrobial peptides have been previously reported (Mor et al, 1994, J Biol Chem, 269,31635-31641). Alternatively, the Rs-AFP protein and the synthetic peptides may have differing modes of action so that their simultaneous action at two distinct sites results in a synergistic antifungal effect.

EXAMPLE 9

[0162] Antifungal Activity of the 15-mer Peptides in the Presence of Inorganic Cations

[0163] In order to evaluate the sensitivity of the Rs-AFP2-derived 15-mer peptides to the presence of salts, different concentrations of a divalent (Ca2+) and a monovalent (K+) cation were added to the growth medium in the antifungal activity assay on F culmorum. Table 12 shows the results, expressed in MIC values for peptides 1 to 10 and for Rs-AFP2.

[0164] For peptides 1 to 5 (Peptide 1—SEQ ID NO: 35, amino acids 1-15; Peptide 2-—SEQ ID NO: 35, amino acids 5-19; Peptide 3—SEQ ID NO: 35, amino acids 9-23; Peptide 4—SEQ ID NO: 35, amino acids 13-27; Peptide 5—SEQ ID NO: 35, amino acids 17-31) and peptide 10 (SEQ ID NO: 35, amino acids 37-51), which have weaker antifungal activity in ½ PDB, the addition of salts at all concentrations tested caused a nearly complete loss of the antifungal activity. The addition of 10 mM KCl did not significantly affect the antifungal activity of the more active peptides 6 to 8 (Peptide 6—SEQ ID NO: 1; Peptide 7—SEQ ID NO: 2; Peptide 8—SEQ ID NO: 3). The active peptide 9 (SEQ ID NO: 4) did, however, show a marked increase in its MIC value (from 30 to 250 &mgr;g/ml) although it was still more active than peptides 1 to 5 and 10. In the presence of 50 mM KCI, the MIC values of peptides 6 and 7 increased by twofold, whereas those of peptides 8 and 9 increased by about 16-fold and 8-fold, respectively. The addition of CaCl2 had a greater effect. When CaCl2 was present in the growth medium at a concentration of 1 mM, most peptides lost their activity although peptides 6 and 7 still inhibited growth of the test fungus at 250 &mgr;g/ml. At 5 mM CaCl2, none of the peptides was active at concentrations below 500 &mgr;g/ml. 25 TABLE 12 MIC (&mgr;g/ml) ½ PDB supplemented with: 50 mM KCl; PEPTIDE ½ PDB 10 mM KCl 50 mM KCl 1 mM CaCl2 5 mM CaCl2 1 mM CaCl2 1 250 500 >500 >500 >500 >500 2 >500 >500 >500 >500 >500 >500 3 250 >500 >500 500 500 500 4 125 500 500 500 500 500 5 250 500 500 500 500 500 6 60 60 125 250 500 250 7 60 60 125 250 500 500 8 30 60 500 500 >500 >500 9 30 250 250 500 500 500 10 250 500 500 500 500 500 AFP 3 3 3 6 6 6

[0165] In Table 12, the peptides have the following sequence identifications: Peptide 1—SEQ ID NO: 35, amino acids 1-15; Peptide 2—SEQ ID NO: 35, amino acids 5-9; Peptide 3—SEQ ID NO 35, amino acids 9-23; Peptide 4—SEQ ID NO: 35, amino acids 13-27; Peptide 5—SEQ ID NO: 35, amino acids 17-31; Peptide 6—SEQ ID NO: 1; Peptide 7—SEQ ID NO: 2; Peptide 8—SEQ ID NO: 3; Peptide 9—SEQ ID NO: 4; Peptide 10—SEQ ID NO: 35, amino acids 37-51, AFP2—SEQ ID NO: 35.

EXAMPLE 10

[0166] Comparison of Linear Loop Peptides of Rs-AFP. Ah-AMP1 and Dm-AMP1 in Media ½PDB

[0167] SMF+ pH5 and SMF+ pH7 (MPS peptidess

[0168] Several &bgr;2-&bgr;3 loop peptides of Rs-AFP, Ah-AMP1 and Dm-AMP I were tested and compared in one experiment. In ½ PDB the various Rs-AFP peptides showed similar activities (Table 13).

[0169] Substituting the cysteine residues by alpha-aminobutyric acid resulted in reduced activities. The addition of an extra lysine residue at the N-terminus, corresponding to Lys30 in Rs-AFP decreased the influence of the higher ionic strength on the antifungal activity even further. 26 TABLE 13 Antifungal activity of linear &bgr;2-&bgr;3 loop peptides from Rs-AFP2, Ah-AMP1 and DmAMP1 in media ½ PDB, SMF + pH5 and SMF + pH7. CODE IC50 (&mgr;g/ml) PEPTIDE SEQUENCE 1/2 PDB SMF + pH5 SMF + pH7 G1 Rs-AFP  ARHGSCNYVFPAHKCICYF 17.6 ± 1.1 17.5 ± 2.3  12.5 ± 0.4  (SEQ ID NO: 25) G2 Rs-AFP  ARHGSBNYVFPAHKBIBYF 16.0 ± 1.2 50.0 ± 6.0 >400 (SEQ ID NO: 26) N1 Rs-AFP  KARHGSBNYVFPAHKBIBYF 14.0 ± 6.2 27.0 ± 7.7 142.6 ± 13.6 (SEQ ID NO: 29) J1 Ah-AMP1 ASHGABHKRENHWKBIBYF  9.1 ± 0.6   47 ± 33  173.4 ± 0.2  (SEQ ID NO: 27) N5 Dm-AMP1 AAHGABHVRNGKHMBFBYF  8.0 ± 0.4 20.9 ± 1.8  24.2 ± 0.2  (SEQ ID NO: 30) Rs-AFP2    (SEQ ID NO: 35)  3.3 ± 0.0  5.2 ± 1.2   6.1 ± 1.3  Fusarium culmorum (2 × 104 spores/ml); duplo experiments; B = alpha-aminobutyric acid

[0170] In ½ PDB the Ah-AMP1 and Dm-AMP1 loop peptides (J1- SEQ ID NO: 27; N5—SEQ ID NO: 30) showed an almost two-fold higher activity as compared to their Rs-AFP counterpart, i.e., G2 (SEQ ID NO: 26). Furthermore, the influence of salts was less pronounced for AhAMP 1 (SEQ ID NO: 57) than for G2 (SEQ ID NO: 26), whereas the activity of Dm-AMP 1 (SEQ ID NO: 58) was only slightly decreased, even in SMF+ pH7.

EXAMPLE 11

[0171] Antifungal Activity of Overlapping 13- to 20-mer Pe tides from the Rs-AFP2 Primary Amino Acid Sequence Ile26 to Phe49 (SEQ ID NO: 35, Amino Acids 26-49) in Media ½ PDB, SMF+ pH5 and SMF+ pH7 (MPS Peptides)

[0172] The most active region of Rs-AFP is located at the &bgr;2-strand/turn/&bgr;3-strand region. To inventory in more detail the contribution of the amino acids in this region to the antifungal activity a set of overlapping 13- to 20-mer peptides was synthesised and tested on Fusarium culmorum. In this set cysteine residues were replaced by alpha-aminobutyric acid.

[0173] A graphical representation of the results with the overlapping 13- to 20-mer peptides is shown in FIGS. 10a (13- to 15-mers), 10b (16- to 18-mers) and 10c (19- and 20-mers). In the set 13and 14-mer peptides two activity areas can be seen: one around the His33-Gly34-Ser35 sequence (SEQ ID NO: 35, amino acids 33, 34 and 35) and the other around Tyr38-Val 39-Phe4O (SEQ ID NO: 35, amino acids 38,39 and 40). From-the set of 15-mers onwards these activity areas turn to one activity region, although the IC50 values differ in relation to the size and composition of the particular peptides. In FIG. 10a, the peptides have the following sequence identifications: 27 SEQ ID NO: PEPTIDE SEQ ID NO:59 IRLEKARHGSBNY SEQ ID NO:60 RLEKARHGSBNYV SEQ ID NO:61 LEKARHGSBNYVF SEQ ID NO:62 EKARKGSBNYVFP SEQ ID NO:63 KARHGSBNYVFPA SEQ ID NO:64 ARHGSBNYVFPAH SEQ ID NO:65 RHGSBNYVFPAHK SEQ ID NO:66 HGSBNYVFPAHKB SEQ ID NO:67 GSBNYVFPAHKBI SEQ ID NO:68 SBNYVFPAHKBIB SEQ ID NO:69 BNYVFPAHKBIBY SEQ ID NO:70 NYVFPAHKBIBYF SEQ ID NO:71 IRLEKARHGSBNYV SEQ ID NO:72 RLEKARHGSBNYVF SEQ ID NO:73 LEKARHGSBNYVFP SEQ ID NO:74 EKARHGSBNYVFPA SEQ ID NO:75 KARHGSBNYVFPAH SEQ ID NO:76 ARHGSBNYVFPAHK SEQ ID NO:77 RHGSBNYVFPAHKB SEQ ID NO:78 HGSBNYVFPAHKBI SEQ ID NO:79 GSBNYVFPAHKBIB SEQ ID NO:80 SBNYVFPAHKBIBY SEQ ID NO:81 BNYVFPAHKBIBYF SEQ ID NO:82 IRLEKARHGSBNYVF SEQ ID NO:83 RLEKARHGSBNYVFP SEQ ID NO:84 LEKARHGSBNYVFPA SEQ ID NO:85 EKARHGSBNYVFPAH SEQ ID NO:86 KARHGSBNYVFPAHK SEQ ID NO:87 ARHGSBNYVFPAHKB SEQ ID NO:88 RHGSBNYVFPAHKBI SEQ ID NO:89 HGSBNYVFPAHKBIB SEQ ID NO:90 GSBNYVFPAHKBIBY SEQ ID NO:91 SBNYVFPAHKBIBYF

[0174] In FIG. 10b, the peptides have the following sequence identifications: 28 SEQ ID NO: PEPTIDE SEQ ID NO:92 IRLEKARHGSBNYVFP SEQ ID NO:93 RLEKARHGSBNYVFPA SEQ ID NO:94 LEKARHGSBNYVFPAH SEQ ID NO:95 EKARHGSBNYVFPAHK SEQ ID NO:96 KARHGSBNYVFPAHKB SEQ ID NO:97 ARHGSBNYVFPAHKBI SEQ ID NO:98 RHGSBNYVFPAHKBIB SEQ ID NO:99 HGSBNYVFPAHKBIBY SEQ ID NO:100 GSBNYVFPAHKBIBYF SEQ ID NO:101 IRLEKARHGSBNYVFPA SEQ ID NO:102 RLEKARHGSBNYVFPAH SEQ ID NO:103 LEKARHGSBNYVFPAHK SEQ ID NO:104 EKARHGSBNYVFPAHKB SEQ ID NO:105 KARHGSBNYVFPABYBI SEQ ID NO:106 ARHGSBNYVFPAHKBIB SEQ ID NO:107 RHGSBNYVFPAHKBIBY SEQ ID NO:108 HGSBNYVFPAHKBIBYF SEQ ID NO:109 IRLEKARHGSBNYVFPAH SEQ ID NO:110 RLEKARHGSBNYVFPAHK SEQ ID NO:111 LEKARHGSBNYVFPAHKB SEQ ID NO:112 EKARHGSBNYVFPAHKBI SEQ ID NO:113 KARHGSBNYVFPAHKBTB SEQ ID NO:114 ARHGSBNYVFPAHKBIBY SEQ ID NO:115 RHGSBNYVFPAHKBIBYF

[0175] In FIG. 10c, the peptides have the following sequence identifications: 29 SEQ ID NO: PEPTIDE SEQ ID NO:116 IRLEKARHGSBNYVFPAHK SEQ ID NO:117 RLEKARHGSBNYVFPAHKB SEQ ID NO:118 LEKARHGSBNYVFPAHKBI SEQ ID NO:119 EKARHGSBNYVFPAHKBIB SEQ ID NO:120 KARHGSBNYVFPAHKBIBY SEQ ID NO:121 ARHGSBNYVFPAHKBIBYF SEQ ID NO:122 IRLEKARHGSBNYVFPAHKB SEQ ID NO:123 RLEKARHGSBNYVFPAHKBI SEQ ID NO:124 EKARHGSBNYVFPAHKBIBY SEQ ID NO:125 EKARHGSBNYVFPAHKBIBY SEQ ID NO:126 KARHGSBNYVFPAHKBIBYF

[0176] In FIGS. 11a and 11b all 13-to 20-mer peptides with the same N-terminal amino acid have been clustered. In FIG. 11a, the peptides have the following sequence identifications: 30 upper left graph (peptides having an N-terminal Ile26) Y-SEQ ID NO: 59 A-SEQ ID NO: 101 V-SEQ ID NO: 71 H-SEQ ID NO: 109 F-SEQ ID NO: 82 K-SEQ ID NO: 116 P-SEQ ID NO: 92 B-SEQ ID NO: 122 upper right graph (peptides having an N-terminal Arg27) V-SEQ ID NO: 60 H-SEQ ID NO: 102 F-SEQ ID NO: 72 K-SEQ ID NO: 110 P-SEQ ID NO: 83 B-SEQ ID NO: 117 A-SEQ ID NO: 93 I-SEQ ID NO: 123 middle left graph (peptides having an N-terminal Leu28) F-SEQ ID NO: 61 K-SEQ ID NO: 103 P-SEQ ID NO: 73 B-SEQ ID NO: 111 A-SEQ ID NO: 84 I-SEQ ID NO: 118 H-SEQ ID NO: 94 B-SEQ ID NO: 124 middle right graph (peptides having an N-terminal Glu29) P-SEQ ID NO: 62 B-SEQ ID NO: 104 A-SEQ ID NO: 74 I-SEQ ID NO: 112 H-SEQ ID NO: 85 B-SEQ ID NO: 119 K-SEQ ID NO: 95 Y-SEQ ID NO: 125 lower left graph (peptides having an N-terminal Lys30) A-SEQ ID NO: 63 I-SEQ ID NO: 105 H-SEQ ID NO: 75 B-SEQ ID NO: 113 K-SEQ ID NO: 86 Y-SEQ ID NO: 120 B-SEQ ID NO: 96 F-SEQ ID NO: 126 lower right graph (peptides having an N-terminal Ala31) H-SEQ ID NO: 64 B-SEQ ID NO: 106 K-SEQ ID NO: 76 Y-SEQ ID NO: 114 B-SEQ ID NO: 87 F-SEQ ID NO: 121 1-SEQ ID NO: 97

[0177] In FIG. 11b, the peptides have the following sequence identifications: 31 upper left graph (peptides having an N-terminal Arg32) K - SEQ ID NO: 65 B - SEQ ID NO: 98 B - SEQ ID NO: 77 Y - SEQ ID NO: 107 I - SEQ ID NO: 88 F - SEQ ID NO: 115 upper right graph (peptides having an N-terminal His33) B - SEQ ID NO: 66 Y - SEQ ID NO: 99 I - SEQ ID NO: 78 F - SEQ ID NO: 108 B - SEQ ID NO: 89 middle left graph (peptides having an N-terminal Gly34) I - SEQ ID NO: 67 Y - SEQ ID NO: 90 B - SEQ ID NO: 79 F - SEQ ID NO: 100 middle right graph (peptides having an N-terminal Ser35) B - SEQ ID NO: 68 Y - SEQ ID NO: 80 F - SEQ ID NO: 91 lower left graph (peptides having an N-terminal B36) Y - SEQ ID NO: 69 F - SEQ ID NO: 81 lower left graph (peptides having an N-terminal Asn37) F - SEQ ID NO: 70

[0178] Clustering of all peptides with identical C-terminal residues can be seen in FIGS. 12a and 12b. In FIG. 12a, the peptides have the following sequence identifications: 32 upper left graph (peptides having a C-terminal Tyr38) I-SEQ ID NO: 59 upper right graph (peptides having a C-terminal Val39) I-SEQ ID NO: 71 R-SEQ ID NO: 60 middle left graph (peptides having a C-terminal Phe40) I-SEQ ID NO: 82 R-SEQ ID NO: 72 L-SEQ ID NO: 61 middle right graph (peptides having a C-terminal Pro41) I-SEQ ID NO: 92 L-SEQ ID NO: 73 R-SEQ ID NO: 83 E-SEQ ID NO: 62 lower left graph (peptides having a C-terminal Ala42) I-SEQ ID NO: 101 E-SEQ ID NO: 74 R-SEQ ID NO: 93 K-SEQ ID NO: 63 L-SEQ ID NO: 84 lower right graph (peptides having a C-terminal His43) I-SEQ ID NO: 109 E-SEQ ID NO: 85 R-SEQ ID NO: 102 K-SEQ ID NO: 75 L-SEQ ID NO: 94 A-SEQ ID NO: 64

[0179] In FIG. 12b, the peptides have the following sequence identifications:

[0180] upper left graph (peptides having a C-terminal Lys44) 33 upper left graph (peptides having a C-terminal Lys44) I-SEQ ID NO: 116 K-SEQ ID NO: 86 R SEQ ID NO: 110 A-SEQ ID NO: 76 L-SEQ ID NO: 103 R-SEQ ID NO: 63 E-SEQ ID NO: 95 upper right graph (peptides having a C-terminal B45) I-SEQ ID NO: 122 K-SEQ ID NO: 96 R-SEQ ID NO: 117 A-SEQ ID NO: 87 L-SEQ ID NO: 111 R-SEQ ID NO: 77 E-SEQ ID NO: 104 H-SEQ ID NO: 64 middle left graph (peptides having a C-terminal Ile46) R-SEQ ID NO: 123 A-SEQ ID NO: 97 L-SEQ ID NO: 118 R-SEQ ID NO: 88 E-SEQ ID NO: 112 H-SEQ ID NO: 78 K-SEQ ID NO: 105 G-SEQ ID NO: 67 middle right graph (peptides having a C-terminal B47) L-SEQ ID NO: 124 R-SEQ ID NO: 98 E-SEQ ID NO: 119 H-SEQ ID NO: 89 K-SEQ ID NO: 113 G-SEQ ID NO: 79 A-SEQ ID NO: 106 S-SEQ ID NO: 68 lower left graph (peptides having a C-terminal Tyr48) E-SEQ ID NO: 125 H-SEQ ID NO: 99 K-SEQ ID NO: 120 G-SEQ ID NO: 90 A-SEQ ID NO: 114 S-SEQ ID NO: 80 R-SEQ ID NO: 107 B-SEQ ID NO: 69 lower right graph (peptides having a C-terminal Phe49) K-SEQ ID NO: 126 G-SEQ ID NO: 100 A-SEQ ID NO: 121 S-SEQ ID NO: 91 R-SEQ ID NO: 115 B-SEQ ID NO: 81 H-SEQ ID NO: 108 N-SEQ ID NO: 70

[0181] The addition of a particular amino acid to the C- or N-terminal side, respectively, can turn a nonactive peptide into a very active one, e.g., addition of Arg32 to the 16-mer His33-Tyr48 (SEQ ID NO: 99) which forms the peptide of SEQ ID NO: 107. A summary of this evaluation is shown in FIG. 13. In FIG. 13, the peptides have the following identifications: His33=>Phe49—SEQ ID NO: 108; Ile 26<=Val39—SEQ ID NO: 71; Arg 32+Lys 44—SEQ ID NO: 65; Arg27+Phe4O SEQ ID NO: 72; Lys30+His43—SEQ ID NO: 75; Arg32+Tyr48—SEQ ID NO: 107; His33+Phe49 —. SEQ ID NO: 108; Arg32+Phe49—SEQ ID NO: 115; Ala31+Phe49—SEQ ID NO: 121; Lys30+Phe49—SEQ ID NO: 126. In the region Ile26 to Phe49 (SEQ ID NO: 35, amino acids 26-49) the 13- to 20-mer peptides from His33 onwards are less active. Similarly, peptides from Ile26 up to Val39 (SEQ ID NO: 35, amino acids 26-39) are less active. Minimal requirements for active peptides are the presence of Arg27 and Phe40, Lys30 and His43, or Arg32 and Lys44. Very active peptides start with Lys30, Ala3 1, Arg32 or His33 and end with Tyr48 or Phe49.

[0182] In SMF+ pH5 and pH7 media the activity of the peptides was substantially reduced. However, as is shown in Table 14, some longer peptides do show activity even at higher ionic strength and increased pH value. Again, the presence of Phe49 and Ala31, Arg32, His33 is necessary. In medium ½ PDB these 18- to 20-mer peptides are only a factor 5 to 8 less potent than Rs-AFP2 when molar quantities are compared. 34 TABLE 14 Antifungal activity of Rs-AFP2 based 18-, 19- and 20-mer peptides with Phe49 as the C-terminal residue in media ½ PDB,SNF ± pH5 and SMF + pH7. IC50 (&mgr;g/ml) CODE/PEPTIDE SEQUENCE ½ PDB SMF + pH5 SMF + pH7 P02   18-mer RHGSBNYVFPAHKBIBYF 8.9  69 ± 34  133 ± 6   001   19-mer ARHGSBNYVFPAHKBIBYF 5.6 102 ± 2   159 ± 5   Q06   20-mer KARHGSBNYVFPAHKBIBYF 4.8  31 ± 17   62 ± 8   Rs-AFP2 2.5 ± 0.8 7.2 ± 0.6 3.7 ± 0.6 Fusarium culmorum (2 × 104 spores/ml); duplo experiments, except for ½ PDB: in the first experiment the peptides were diluted up to 12.5 gg/ml, a dilution that appeared to be insufficient to score the IC50 value. B = alpha-aminobutyric acid. Peptide PO2 - SEQ ID NO: 31; Peptide 001 - SEQ ID NO: 32; Peptide Q06 - SEQ ID NO: 33

[0183]

Claims

1. An isolated DNA sequence encoding an antifungal peptide, wherein said DNA sequence is selected from the group consisting of:

(a) a DNA sequence wherein said antifungal peptide consists of at least six amino acid residues identical to a run of amino acid residues found between position 21 and 51 of SEQ ID NO: 35; and

(b) a DNA sequence that comprises (a) and further comprises a DNA sequence that encodes Rs-AFP1 (SEQ ID NO: 34) or Rs-AFP2 (SEQ ID NO: 35).

2. The isolated DNA sequence of claim 1, wherein said antifungal peptide consists of at least six amino acid residues identical to a run of amino acid residues found between position 21 and 51 of SEQ ID NO: 35.

3. The isolated DNA sequence of claim 1, wherein said DNA sequence encodes a peptide which consists of at least six amino acid residues identical to a run of amino acid residues found between position 21 and 51 of SEQ ID NO: 35 and encodes Rs-AFP1 (SEQ ID NO: 34) or Rs-AFP2 (SEQ ID NO: 35).

4. A recombinant vector comprising the DNA sequence of claim 1.

5. A non-human biological system comprising the recombinant vector of claim 4.

6. The biological system of claim 5, which is a plant cell.

7. A transgenic plant comprising the transgenic plant cell of claim 6.

8. The transgenic plant of claim 7, wherein said plant has improved resistance to a fungal pathogen.

9. Transgenic seed from the transgenic plant of claim 8.

10. A method of producing an antifungal peptide comprising:

(a) obtaining the biological system of claim 5;

(b) growing the biological system under conditions which allow the expression of the antifungal peptide; and

(c) isolating the antifungal peptide.

11. A method for making a transgenic plant capable of producing an antifungal peptide, comprising:

(a) stably integrating the DNA sequence of claim 1 into the genome of plant cells; and

(b) regenerating stably transformed plants from said transformed plant cells, wherein said stably transformed plants produce at least one antifungal peptide.

12. A transgenic plant made by the method of claim 11.

13. Transgenic seed from the transgenic plant of claim 12.

14. A method of producing hybrid plant seed comprising crossing the transgenic plant of claim 7 with a plant having a different genotype.

15. Hybrid seed produced by the method of claim 14.

16. A hybrid plant produced by growing the hybrid seed of claim 15.

17. A method of producing a hybrid plant with improved fungal resistance comprising:

(a) crossing a first transgenic parent plant comprising a DNA sequence that encodes an antifungal peptide consisting of at least six amino acid residues identical to a run of amino acid residues found between position 21 and 51 of SEQ ID NO: 35 with a second transgenic parent plant comprising a DNA sequence that encodes SEQ ID NO: 34 or SEQ ID NO: 35;

(b) harvesting the resultant first generation hybrid seed;

(c) planting said hybrid seed under conditions which allow said seed to germinate and grow into hybrid plants; and

(d) testing said hybrid plants for improved fungal resistance.

18. A hybrid plant produced by the method of claim 17.