BIOCIDAL PROTEINS

Abstract: Biocidal proteins isolated from seeds have been characterized, in particular proteins isolated from members of the Brassicaceae, Compositae and Leguminosae families including Raphanus, Brassica, Sinapis, Arabidopsis, Dahlia, Cnicus, Lathyrus and Clitoria. The proteins show a wide range of antifungal activity and some are active against Gram-positive bacteria. All share a common amino acid sequence. DNA encoding the proteins has been isolated and incorporated into vectors. Plants transformed with this DNA may be produced. The proteins find commercial application as antifungal or antibacterial agents; transformed plants will show increased disease-resistance.

Description: BIOCIDAL PROTEINS This invention relates to biocidal proteins, processes for their manufacture and use, and DNA sequences coding for them. In particular, it relates to antimicrobial proteins isolated from seeds such as those of members of the Brassicaceae, Compositae or Leguminosae families. in this context, antimicrobial proteins are defined as proteins possessing at least one of the following activities: antifungal activity (which may include anti-yeast activity); antibacterial activity. Activity includes a range of antagonistic effects such as partial inhibition or death. The Brassicaceae is a large family of herbs and shrubs which grow widely in tropical, sub-tropical and temperate regions. The Family Brassicaceae is also known as the "Cruciferae". Raphanus sativus (radish) belongs to this family and is cultivated widely as a vegetable. Dahlia belongs to the Compositae and has been extensively cultivated as an ornamental garden plant. A number of hybrids are commercially available, belonging to the Dahlia merckii or Dahlia variablis species. Cnicus benedictus, another Compositae, is a native plant of the Mediterranean regions and was once used as a tonic and a cure for gout. Lathyrus and Clitoria belong to the Leguminosae family. Lathyrus has been extensively cultivated as an ornamental garden plant, the most widely known being the sweet pea plant, Lathyrus odoratus. The genus Clitoria is less well known to European gardeners; Clitoria ternatea was originally introduced from the East Indies in the 1800s. Although plants normally grow on substrates that are extremely rich in fungal organisms, infection remains a rare event. To keep out potential invaders, plants produce a wide array of antifungal compounds, either in a constitutive or an inducible manner. The best studied of these are phytoalexins which are secondary metabolites with a broad antimicrobial activity spectrum that are specifically synthesised upon perception of appropriate defence-related signal molecules. The production of phytoalexins depends on the tranεcriptional activation of a series of genes encoding enzymes of the phytoalexin biosynthetic pathway. During the last decade, however, it has become increasingly clear that some plant proteins can play a more direct role in the control of phytopathogenic fungi. Several classes of proteins with antifungal properties have now been identified, including chitinases, beta-l,3-glucanases, chitin-binding lectins, zeamatinε, thionins and ribosome-inactivating proteins. These proteins have gained considerable attention as they could potentially be used as biocontrol agents. The chitinases and beta-l,3-glucanases have weak activities by themselves, and are only inhibitory to plant pathogens when applied in combination (Mauch et al, 1988, Plant Physiol, 88, 936-942). The chitin-binding lectins can also be classified as rather weak antifungal factors (Broekaert et al, 1989, Science, 245, 1100-1102; Van Parijs et al, 1991, Planta, 183, 258-264). Zeamatin is a more potent antifungal protein but its activity is strongly reduced by the presence of ions at physiological concentrations (Roberts and Selitnermikoff, 1990, G Gen Microbiol, 136, 2150-2155). Finally, thionins and riboso e-inactivating proteins are potentially hazardous since they are known to be toxic for human cells (Carrasco et al, 1981, Eur J Biochem, 116, 185-189; Vernon et al, 1985,.Arch Biochem Biophys, 238, 18-29; Stirpe and Barbieri, 1986, FEBS Lett, 195, 1-8) . We have now purified a new class of potent antimicrobial proteins with broad spectrum activity against plant pathogenic fungi and with some antibacterial activity, moderate sensitivity to ions and apparent low toxicity for cultured human cells. According to the present invention, we provide antimicrobial proteins capable of being isolated from seeds and in particular from members of the Brassicaceae, the Compositae or the Leguminosae families including Raphanus, Brassica, Sinapis, Arabidopsis, Dahlia, Cnicus, Lathyrus or Clitoria. In further aspects, this invention comprises a vector containing a DNA sequence coding for a protein according to the invention. The DNA may be cloned or transformed into a biological system allowing expression of the encoded protein. The invention also comprises plants transformed with recombinant DNA encoding an antimicrobial protein according to the invention. The invention also comprises a process of combating fungi or bacteria whereby they are exposed to the proteins according to the invention. A new class of potent antimicrobial proteins has been isolated from seeds of the Brassicaceae, the Compositae, and the Leguminosae. Similar proteins may be found in other plant families, genera and species. The class includes proteins which share a common amino acid sequence and which show activity against a range of plant pathogenic fungi. The antimicrobial proteins isolated from seeds of Raphanus sativus (radish) include two protein factors, hereafter called Rs-AFPl (Raphanus sativus - Antifungal Protein 1) and Rs-AFP2 (Raphanus sativus - Antifungal Protein 2) respectively. Both are oligomeric proteins, composed of identical 5 kDa subunits. Both proteins are highly basic and have pi values above 10. Similar antifungal proteins have been isolated from other Brassicaceae, including Brassica napus (Bn-AFPs), Brassica rapa (Br-AFPs), Sinapis alba (Sa-AFPs) and Arabidopsis thaliana (At-AFPl). The antimicrobial proteins isolated from seeds of Dahlia and Cnicus include four protein factors, hereafter called Dm-AMPl (Dahlia erckii - Antimicrobial Protein 1), Dm-AMP2 (Dahlia merckii - Antimicrobial Protein 2), Cb-AMPl (Cnicus benedictus - Antimicrobial Protein 1) and Cb-AMP2 (Cnicus benedictus - Antimicrobial Protein 2) respectively. The Dm-AMP proteins may be isolated from seed of the Dahlia genus. The Cb-AMP proteins may be isolated from seed of the Cnicus genus. All four proteins are closely related and are composed of 5 kDa subunits arranged as oligomeric structures. All four proteins are highly basic. The antimicrobial proteins isolated from seeds of Lathyrus and Clitoria include three protein factors, hereafter called Lc-AFP (Lathyrus cicera - Antifungal Protein), Ct-AMPl (Clitoria ternatea - Antimicrobial Protein 1) and Ct-AMP2 (Clitoria ternatea - Antimicrobial Protein 2) respectively. Lc-AFP may be isolated from seed of the Lathyrus genus. The Ct-AMP proteins may be isolated from seed of the Clitoria genus. All three proteins are composed of 5 kDa subunits arranged as oligomeric structures and are highly basic. N-terminal amino acid sequence determination has shown that the above proteins isolated from the Brassicaceae, Compositae and Leguminosae are closely related and can be classified as a single protein family. Between the different plant families, the protein sequences are approximately 50% identical. These sequences enable manufacture of the proteins by chemical synthesis using a standard peptide synthesiser. The antimicrobial proteins are partially homologous to the predicted protein products of the Fusarium-induced genes pl39 and pl230 in pea (Pisum sativum - a member of the Leguminosae family) as described by Chiang and Hadwiger, 1991 (Mol Plant Microbe Interact, 4, 324-331). This homology is shared with the predicted protein product of the pSASlO gene from cowpea (Vigna unguiculata - another legume) as described by Iεhibashi e_t e (Plant Mol Biol, 1990, 15, 59-64). The antimicrobial proteins are also partially homologous with the predicted protein product of gene pl322 in potato (Solanum tuberosum - a member of the Solanaceae family) as described by Stiekema et al, 1988 (Plant Mol Biol, 11, 255-269). Nothing is known about the biological properties of the proteins encoded by genes pI39, pl230, pSASlO or pl322 as only the cDNA has been studied. However, the pl39, pl230 and pl322 genes are switched on after challenge to the plant by a disease or other stress. It has been proposed that the pSASlO gene encodes a protein involved in germination. Due to their sequence similarity with the antimicrobial proteins of the invention, the proteins encoded by the pl39, pl230, pSASlO or pl322 genes may be useful as fungicides or as antibiotics. The antimicrobial protein sequences show a lower degree of partial homology with the sequences of a group of small α-amylase inhibitors found in the following members of the Gramineae: sorghum (Bloch and Richardson, 1991, FEBS Lett, 279:101-104), wheat (Colitta et al, 1990, FEBS Lett, 270:191-194) and barley (Mendez et al, 1990 Eur J Biochem, 194:533-539). Such proteins, including SIα2 from sorghum and γ-1-purothionin from wheat, are known to inhibit insect α-amylase and are toxic to insect larvae. It is not known if these α-amylaεe inhibitors show any antifungal or other antimicrobial activity: no other data on their biological activity has been reported. Due to their sequence similarity with the antimicrobial proteins of the invention, the α-amylase inhibitor proteins may be useful as fungicides or as antibiotics. A third antifungal protein has been isolated from radish seeds, hereafter called Rs-nsLTP (Raphanus sativus non-specific lipid transfer protein). It is a dimeric protein, composed of two identical 9 kDa subunits. Amino acid sequence determination has identified the 43 N-terminal residueε of Rs-nsLTP, and has shown it to be homologous with non-εpecific lipid tranεfer proteins isolated from other plantε (Arondel and Kader, 1990, Experientia, 46:579-585) but not with the other antimicrobial proteinε discussed above. The Rs-nsLTP sequence enables manufacture of the protein by chemical syntheεis using a εtandard peptide synthesiεer. Knowledge of their primary εtructure, enables the production of DNA constructs encoding the antimicrobial proteinε. The DNA sequence may be predicted from the known amino acid sequence or the sequence may be isolated from plant-derived DNA libraries. Oligonucleotide probes may be derived from the known amino acid sequence and used to screen a cDNA library for cDNA clones encoding some or all of the protein. cDNA clones encoding the Rs-AFPs have been isolated in this way and sequenced. These same oligonucleotide probes or cDNA clones may be uεed to iεolate the actual AFP, AMP or Rs-nsLTP gene(s) by screening genomic DNA libraries. Such genomic clones may include control sequences operating in the plant genome. Thus it is alεo poεsible to isolate promoter sequences which may be uεed to drive expreεεion of the antimicrobial (or other) proteinε. Theεe promoterε may be particularly responsive to environmental conditions (such as the presence of a fungal pathogen). DNA encoding the antimicrobial proteins (which may be a cDNA clone, a genomic DNA clone or DNA manufactured using a εtandard nucleic acid synthesiεer) can then be cloned into a biological εyεtem which allowε expreεεion of the proteinε. Hence the proteinε can be produced in a suitable micro-organism or cultured cell, extracted and isolated for uεe. Suitable micro-organiεmε include Eεcherichia coli and Saccharomyces cerevisiae. The genetic material can also be cloned into a virus or bacteriophage. Suitable cells include cultured insect cells and cultured mammalian cells. The DNA can also be transformed by known.methods into any plant species, so that the antimicrobial proteinε are expreεsed within the plant. Plant cells according to the invention may be transformed with constructs of the invention according to a variety of known methods (Agrobacte-rium Ti plaεmidε, electroporation, microinjection, microprojectile gun, etc). The transformed cells may then in suitable cases be regenerated into whole plants in which the new nuclear material iε εtably incorporated into the genome. Both transformed onocot and dicot plants may be obtained in this way, although the latter are usually more eaεy to regenerate. Exampleε 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. The AFP, AMP and Rs-nsLTP proteins show a wide range of antifungal activity, including anti-yeast activity, and the AMPs are also active againεt Gram poεitive bacteria. The proteinε are useful aε fungicides or antibiotics. Exposure of a plant pathogen to an antimicrobial protein may be achieved by application of the protein to plant parts using standard agricultural techniques (eg spraying). The proteins may also be used to combat fungal or bacterial disease by expresεion within plant bodieε. All the antimicrobial proteinε show surprisingly high activity: they inhibit the growth of a variety of plant pathogenic fungi at submicromolar doses. Antifungal activity of the AMPs is only partially dependent on the ionic conditions. The antifungal effect of the AFPs is not affected by K ions at physiological concentrations (50 mM) . The antifungal effect of Rs-AFPl, but not Rε-AFP2, iε antagoniεed by Ca + at physiological concentrations (1 mM). Rs-nsLTP also inhibits growth of a variety of plant pathogenic fungi, but is less potent and more salt εenεitive that the AFPε. The antimicrobial proteins can be isolated and purified from appropriate seedε, εyntheεiεed artificially from their known amino acid sequence, or produced within a εuitable micro-organiεm by expression of recombinant DNA. The proteins may also be expressed within a tranεgenic plant. The invention may be further understood by reference to the drawings, in which: Figure 1 shows the cation exchange chromatogram for the Raphanus antifungal proteinε and the asεociated graph of fungal growth inhibition. Figure 2A εhowε the HPLC profile of purified Rε-AFPl. Figure 2B showε the HPLC profile of purified RS-AFP2 and Rs-nsLTP. Figure 3 εhowε native gel electrophoreεiε and bio-zymography of the purified Rε-AFPl and RS-AFP2. Figure 4 shows the cation exchange chromatogram for the jB napus antifungal proteinε and " the aεεociated graph of fungal growth inhibition. Figure 5 shows the HPLC profile of purified 13 napus antifungal proteins. Figure 6 shows the cation exchange chromatogram for 13 rapa antifungal proteins and the asεociated graph of fungal growth inhibition. Figure 7 εhowε the HPLC profile of purified B_ rapa antifungal proteinε. Figure 8 εhowε the cation exchange chromatogram for £> alba antifungal proteinε and the associated graph of fungal growth inhibition. Figure 9 showε the HPLC profile of purified S_ alba antifungal proteinε. Figure 10 εhows the cation exchange chromatogram for A thaliana antifungal protein and the associated graph of fungal growth inhibition. Figure 11 shows the HPLC profile of purified A thaliana antifungal proteins. Figure 12 showε the cation exchange chromatogram for the baεic extract of Dahlia merckii and the correεponding graph of antifungal activity. Figure 13 εhowε the reverεe-phaεe HPLC profile of purified Dm-AMPl and Dm-AMP2. Figure 14 showε the cation exchange chromatogram for the basic extract of Cnicus benedictus and the correεponding graph of antifungal activity. Figure 15 shows the reverse-phaεe HPLC profile of purified Cb-AMPl. Figure 16 shows the reverse-phase HPLC profile of purified Cb-AMP2. Figure 17 showε the cation exchange chromatogram for the basic extract of Lathyrus and the corresponding graph of antifungal activity. Figure 18 showε the reverεe-phaεe HPLC profile of purified Lc-AFP. Figure 19 shows the cation exchange chromatogram for the baεic extract of Clitoria and the correεponding graph of antifungal activity. Figure 20 shows the reverse-phase HPLC profile of purified Ct-AMPl. Figure 21 shows the reverse-phase HPLC profile of purified Ct-AMP2. Figure 22 shows the SDS-PAGE analysis of the purified Rs-AFPs. Figure 23 shows the SDS-PAGE analysiε of the purified Rε-nsLTP. Figure 24 εhowε the SDS-PAGE analyεiε of the purified Dm-AMPε. Figure 25 εhowε the SDS-PAGE analysis of the purified Cb-AMPs. Figure 26 εhowε the SDS-PAGE analyεis of the purified Lc-AFP and Ct-AMPs. Figure 27 showε the amino acid εequenceε of Rε-AFPl, RS-AFP2 and the related Brasεicaceae proteins. Figure 28 showε the amino acid εequenceε of the Dm-AMPs and the Cb-AMPs. Figure 29 shows the amino acid sequeπceε of Lc-AFP and Ct-AMPl. Figure 30 shows the alignment of the amino acid sequences of Rs-AFPl, Dm-AMPl, the Cb-AMPs, Lc-AFP, Ct-AMPl, εorghum SIα2, wheat γl purothionin, and the predicted productε of the pea genes p!230 and pl39, of the cowpea gene pSASlO, and of the potato gene p322. Figure 31A showε predicted DNA εequenceε for the Dm-AMP and Cb-AMP geneε. Figure 31B shows predicted DNA sequences for the Lc-AFP and Ct-AMPl geneε. Figure 32 εhowε the amino acid εequence of Rε-nsLTP. Figure 33 showε the alignment of the amino acid sequences of Rs-nεLTP and variouε plant non-εpecific lipid tranεfer proteinε. Figure 34 iε a graph of Rε-AFP2 ^n vivo activity. Figure 35 εhowε the full length cDNA εequence of Rε-AFPl. Figure 36 εhows the truncated cDNA sequence of Rs-AFP2. Figure 37 shows the full length DNA sequence of PCR assisted site directed mutagenesiε of Rε-AFP2. Figure 38 εhowε the expression vector pFRG7. Figure 39 showε the expreεεion vector pFRG8. The following Exampleε illuεtrate the invention. EXAMPLE 1 Antifungal and antibacterial activity assays. Antifungal activity waε meaεured by microεpectrophotometry aε previously described (Broekaert, 1990, FEMS Microbiol Lett, 69:55-60). Routinely, testε were performed with 20 μl of a (filter-sterilized) test εolution and 80 μl of a 4 εuεpension of fungal εporeε (2 x 10 εporeε/ml) in half strength potato dextroεe broth (1/2 PDB). Some teεtε were performed uεing a εuspension of mycelium fragmentε in a synthetic growth medium. The synthetic growth medium consiεted of K~HP0, (2.5 mM), MgS0 4 (50 μM ) , CaCl 2 (50 μM ) , FeS0 4 (5 μM ) , CoCl- (0.1 μM ) , CuSO^ (0.1 μM) , Na-MoO. (2 μM ) , H-B0 3 (0.5 μM ) , KI (0.1 μM ) , ZnS0 4 (0.5 μM ) , MnS0 4 (0.1 μM) , glucose (10 g/1), asparagine (1 g/1), methionine (20 mg/1) , myo-inositol (2 mg/1) , biotin (0.2 mg/1), thiamine-HCl (1 mg/1), and pyridoxine-HCl (0.2 mg/1). Control microcultureε contained 20 μl of sterile diεtilled water and 80 μl of the fungal εuεpenεion. Unless otherwise stated the test organiε waε Fusarium culmorum (εtrain IMI 180420) and incubation waε done at 25°C for 48 hourε. Percent growth inhibition iε defined aε 100 timeε the ratio of the corrected abεorbance of the control microculture minuε the corrected abεorbance of the teεt microculture over the corrected absorbance at 595 nm of the control microculture. The corrected absorbance values equal the absorbance at 595 nm of the culture measured after 48 hourε minuε the absorbance at 595 nm measured after 30 min. Antibacterial activity waε meaεured microεpectrophotometrically aε follows. A bacterial εuεpension was prepared by inoculating soft nutrient agarose (tryptone, 10 g/1; Seaplaque agarose (FMC), 5 g/1). Aliquots (80 μl ) of the ς bacterial suspenεion (10 colony forming units per ml) were added to filter-sterilized εamples (20 μl ) in flat-bottom 96-well microplates. The abεorbance at 595 nm of the culture was measured with the aid of a icroplate reader after 30 minutes and 24 hours of incubation at 28°C. Percent growth inhibition was calculated as described above for the antifungal activity aεεay. EXAMPLE 2 Extraction of the baεic protein fraction from Raphanus sativus seeds. Ammonium εulphate fractionation of proteinε precipitating in the interval of 30 to 70% relative εaturation waε followed by heat treatment to remove heat-labile proteinε, and by iεolation of the baεic protein fraction (pl>9) by passage over a Q-Sepharose (Pharmacia) anion exchange column equilibrated at pH 9. The detailed methods are described below. One kg of R sativus seeds (obtained from Aveve, Belgium) was ground in a coffee mill and the reεulting meal waε extracted for 2 hours at 4°C with 2 litres of an ice-cold extraction buffer containing 10 mM NaH 2 P0 4 , 15 mM a 2 HP0 4 , 100 mM KCI, 2 mM EDTA, 2 mM thiourea, and 1 mM PMSF. The homogenate was squeezed through cheesecloth and clarified by centrifugation (30 min at 7,000 x g) . Solid ammonium sulphate was added to the εupernatant to obtain 30% relative εaturation and the precipitate formed after standing overnight at room temperature waε removed by centrifugation (30 min at 7,000 x g). The εupernatant was adjusted to 70% relative ammonium sulphate saturation and the precipitate formed overnight at room temperature collected by.centrifugation (30 min at 7,000 x g). After redisεolving the pellet in 400 ml distilled water the solution was heated at 80°C for 15 min. The coagulated inεoluble material waε removed by centrifugation (30 min at 7,000 x g) and the supernatant was dialyzed extensively against distilled water using tubing (SpectralPor, Spectrum, USA) with a molecular weight cut off of 1,000 Da. After dialysis the solution waε adjuεted to 50 mM Triε-HCl (pH 9) by addition of the ten-fold concentrated buffer, and εubεequently paεεed over a Q-Sepharose Faεt Flow (Pharmacia, Uppεala, Sweden) column (12 x 5 cm) in equilibrium with 50 mM Triε-HCl (pH 9). The protein fraction paεsed through the column was dialyzed extensively againεt distilled water and adjusted to 50 mM sodium N-morpholinoethaneεulphonic acid (Na-MES), pH6, by addition of the ten-fold concentrated buffer. Thiε material repreεentε the baεic heat-εtable protein fraction of R sativus seedε. Its further chromatographic purification is described in Example 3. EXAMPLE 3 Purification of antifungal proteins f om R sativus seeds. The starting material for the isolation of the R sativuε antifungal proteins was the basic heat-stable protein fraction extracted from the mature seedε as in Example 2. These proteins were further separated by cation exchange chromatography, as shown in Figure 1. About 150 mg of the baεic heat-εtable protein fraction diεεolved in 50 mM sodium MES (pH 6) was applied on a S-Sepharose High Performance (Pharmacia) column (10 x 1.6 cm) previouεly equilibrated with the εodiu MES buffer. The column waε eluted at 2.5 ml\min with a linear gradient of 1000 ml from 0 to 500 mM NaCl in 50 mM εodium MES buffer (pH 6). The eluate waε monitored for protein by online meaεurement of the abεorbance at 280 nm (resultε εhown in the lower panel of Figure 1) and collected in 10 ml fractionε. Of theεe fractionε, 20 μl was tested in the microspectrophotometric antifungal activity assay described in Example 1 uεing either the εynthetic growth medium (Medium A: results shown as full lines in the upper panel of Figure 1) or the same medium supplemented with 1 mM CaCl- and 50 mM KCI (Medium B: results shown as daεhed lineε in the upper panel of Figure 1). Upon fractionation, the mixture yielded a broad peak repreεenting the unbound fraction, two well reεolved peakε (peak 1 and peak 2) eluting around 100 and 200 mM NaCl respectively, and a group of five non-resolved peakε (peakε 3 to 7) eluting between 250 and 450 mM NaCl. No antifungal activity was asεociated with the unbound fraction, whereas all bound peak fractions diεplayed antifungal activity when aεεayed in medium A. However, teεtε performed in medium B only indicated growth inhibition for the fractionε correεponding to peaks 1 and 2, respectively. It appears therefore that the antifungal activity of these fractions is less salt-dependent than that of the fractions from peaks 3 to 7. The fractions εhowing antifungal activity in growth medium B (peakε 1 and 2) were further purified by reverεed-phaεe chromatography. About 1 mg amounts of peak 1 material (Figure 2A) and peak 2 material (Figure 2B) were loaded on a Pep-S (porous silica C 2 /C lβ , Pharmacia) column (25 x 0.93 cm) in equilibrium with 0.1% TFA. The column was eluted at 5 ml/min with a linear gradient of 200 ml from 0.1% trifluoroacetic acid (TFA) to 40% acetonitrile/0.1% TFA. The eluate was monitored for protein by online measurement of the absorption at 214 nm. Five ml fractionε of the eluate were collected, vacuum-dried, and finally dissolved in 0.5 ml distilled water of which 10 μl was used in a- microεpectrophotometric antifungal activity assay. Figure 2A and Figure 2B show the HPLC profiles of purified peak 1 and peak 2 material respectively. The lower panelε show monitoring of the eluate for protein by measurement of the abεorbance at 214 nm. Resultε of the microεpectrophotometric antifungal activity aεεay in medium A (full line) and medium B (daεhed line) are εhown in the upper panelε. The material from peak 1 yielded a single major peak eluting at 30% acetonitrile and co-eluting with the antifungal activity in both medium A and medium B. The active factor iεolated from thiε peak iε called Rε-AFPl (Raphanus sativus antifungal protein 1). The peak 2 material, on the other hand, resolved into two major peakε eluting at 30% and 33% acetonitrile reεpectively. The peak eluting at 3)% acetonitrile waε active in both medium A and medium B, whereaε the peak eluting at 33% was active only in medium A. • The active factor purified from the 30% acetonitrile peak is called Rε-AFP2 (Raphanuε εativus antifungal protein 2), and that from the 33% acetonitrile peak is designated Rs-nεLTP (Raphanuε εativuε non-specific lipid transfer protein) because of its homology with non-specific lipid transfer proteins isolated from other plant specieε (εee Example 13). EXAMPLE 4 Purity of the iεolated Rε-AFPε. The purity of the iεolated antifungal proteinε waε verified by native cathodic gel electrophoreεiε followed by protein εtaining (Figure 3, left panel) and iji εitu detection of antifungal activity uεing a bio-zymographic technique (Figure 3, right panel) . Native cathodic gel electrophoreεiε and bio-zymography were done aε previouεly described (De Bolle et al, 1991, Electrophoresiε, 12, 442-444) with εome modificationε. Electrophoresiε was performed on continuous 10% acrylamide gels containing 60 mM Tris/70 mM MES (pH 7). The electrophoresis buffer consiεted of 100 mM L-hiεtidine/41 mM MES (pH 6.5) Gelε were cooled at 10°C during electrophoresiε. The εampleε contained 20% glycerol, 0.0025% methylene blue, and 10 μg of purified Rε-AFPl or 20 μg of RS-AFP2. Proteins were detected by silver-εtaining of a diffusion blot prepared from the gel (Kovarik et al, 1987, Folia Biological, 33, 253-257). The gel was overlaid with a soft agar gel (De Bolle et al, 1991, Electrophoresis, 12, 442-444) containing viable Trichoderma hamatum spores and incubated at 25°C for 3 days. Figure 3 (left panel) showε that Rs-AFPl (lanes 1) and RS-AFP2 (laneε 2) migrate aε εingle protein bandε after cathodic gel electrophoreεiε. Moreover, the antifungal activity co-migrated exactly with the protein bandε in the gel (right panel). Theεe results indicate that the isolated factors are highly pure and that the antifungal activity is not attributable to minor conta inantε. EXAMPLE 5 Antifungal proteinε related to Rε-AFPε from other species of Brassicaceae. Using the purification procedure described in Example 3, we have isolated antifungal proteinε from other Brassicaceae, including Brassica napuε, Brasεica rapa, Sinapiε alba and Arabidopsis thaliana. Figure 4 shows the cation exchange chromatogram for antifungal protein isolated from B napus, and the asεociated graph of fungal growth inhibition (upper panel). Figure 5 εhowε the HPLC profile of the purified 13 napuε antifungal proteinε, isolated from peak 1 (Bn-AFPl, left panel) and peak 2 (Bn-AFP2, right panel). Figure 6 εhowε the cation exchange chromatogram for antifungal protein isolated from B_ rapa, and the associated graph of fungal growth inhibition (upper panel). Figure 7 shows the HPLC profile of the purified 13 rapa antifungal proteins, iεolated from peak 1 (Br-AFPl, left panel) and peak 2 (Br-AFP2, right panel). Figure 8 εhowε the cation exchange chromatogram for antifungal protein iεolated from alba, and the associated graph of fungal growth inhibition (upper panel). Figure 9 showε the HPLC profile of the purified £5 alba antifungal proteinε, iεolated from peak 1 (Sa-AFPl, left panel) and peak 2 (Sa-AFP2, right panel). Figure 10 εhowε the cation exchange chromatogram for antifungal protein iεolated from A thaliana, and the aεεociated graph of fungal growth inhibition (upper panel). Figure 11 εhowε the HPLC profile of the purified A thaliana antifungal proteinε, iεolated from peak 1 (At-AFPl). All theεe antifungal proteinε behave similarly to Rε-AFPl and RS-AFP2 with reεpect to their SDS-PAGE and iεoelectric focuεing pattern (aε deεcribed in Example 5). EXAMPLE 6 Extraction of the baεic protein fraction from Dahlia merckii, Cnicus benedictus, Lathyrus cicera and Clitoria ternatea seeds. Five hundred gramε of D merckii or C benedictuε or Clitoria ternatea seeds (purchased from Chiltern Seeds, Cumbria, UK) or Lathyruε cicera seeds (from Instituto Botanico Univerεitade Coimbra, Portugal) were ground in a coffee mill and the resulting meal waε extracted for 2 hours at 4°C with 2 litres of an ice-cold extraction buffer containing 10 mM NaH 2 P0 4 , 15 mM Na 2 HP0 4 , 100 mM KCl, 2 mM EDTA and 1 mM benzamidine. The resulting homogenate was squeezed through cheesecloth and clarified by centrifugation (30 min at 7,000 x g). Solid ammonium sulphate waε added to the εupernatant to obtain 75% relative εaturation and the precipitate allowed to form by standing overnight at 4°C. Following centrifugation at 7,000 x g for 30 minutes, the precipitate was rediεsolved in a minimal volume of distilled water and dialyzed extensively against distilled water uεing benzoylated cellulose tubing (Sigma, St Louis, MO). After dialyεiε the εolution waε adjuεted to 50 mM NH 4 Ac (pH 9) by addition of the ten-fold concentrated buffer and paεsed over a Q-Sepharose Faεt Flow (Pharmacia, Uppεala, Sweden) column (12 x 5 cm) equilibrated in 50 mM NH 4 Ac (pH 9). The protein fraction which passed through the column waε adjuεted to pH6 with acetic acid. This material repreεents the baεic (pl>9) protein fraction of the εeedε. The fractionε were further purified as described in Examples 7, 8, 9 and 10. EXAMPLE 7 Purification of antimicrobial proteinε from Dahlia merckii seedε. The starting material for the isolation of the D merckii antimicrobial proteins waε the baεic protein fraction extracted from the mature εeedε aε in Example 6. Proteinε were further purified by cation exchange chromatography of thiε extract. Approximately 500 ml of the baεic protein fraction waε applied to a S-Sepharoεe High Performance (Pharmacia) column (10 x 1.6 cm) equilibrated in 50 mM NH 4 Ac, pH 6.0. The column was eluted at 3.0 ml\min with a linear gradient of 50-750 ml H 4 Ac, pH 6.0 over 325 minutes. The eluate was monitored for protein by online measurement of the absorbance at 280 nm (results shown in the lower panel of Figure 12) and collected in 10 ml fractions. Samples from each fraction were asεayed for antifungal activity aε deεcribed in Example 1 (reεultε εhown in the upper panel of Figure 12). Following chromatography, the extract yielded a broad peak of activity eluting at around 250 mM NH.Ac. The fractionε εhowing antifungal activity were pooled and further purified by reverse-phase HPLC. About 3 mg amounts of the peak were loaded on a PEP-S (porous εilica C 2^ C 1 ' - >narmac i a ) column (25 x 0.4 cm) equilibrated with 0.1% TFA (trifluoracetic acid). The column waε developed at 1 ml/min with a linear gradient of 0.1% TFA to 100% acetonitrile/0.1% TFA over 100 minutes. The eluate was monitored for protein by online measurement of the absorption at 280 nm (resultε εhown in the lower panel of Figure 13). One ml fractionε were collected, vacuum-dried, and disεolved in 0.5 ml diεtilled water. 10 μl from each fraction waε assayed for antifungal activity ( resultε εhown in the upper panel of Figure 13). The material yielded two well-reεolved peakε of activity, eluting at 18% and 22% acetonitrile. Theεe represent the purified proteins Dm-AMPl and Dm-AMP2 respectively. EXAMPLE 8 Purification of antimicrobial proteins from Cnicus benedictus seedε. The procedure deεcribed in Example 7 waε followed uεing the baεic extract from Cnicuε benedictus seedε. Following chromatography on the S-Sepharoεe High Performance column, the Cnicuε extract yielded two peakε of antifungal activity eluting at approximately 250 mM (peak 1) and 500 mM (peak 2) NH 4 Ac (reεultε εhown in Figure 14). Active fractionε were pooled for each peak and further purified on reverεe-phaεe HPLC aε deεcribed in Example 7. Reεultε for peak 1 are εhown in Figure 15: it yielded an active factor eluting at 18% acetonitrile which iε designated Cb- MPl. Similarly peak 2 eluted to a single peak of activity which is designated Cb-AMP2 (results shown in Figure 16) . EXAMPLE 9 Purification of antifungal protein from Lathyrus cicera seeds. The procedure described in Example 7 was followed using the basic extract from Lathyrus cicera seedε. Following chromatography on the S-Sepharose High Performance column, the Lathyrus extract yielded a single peak of antifungal activity eluting at approximately 160 mM NH 4 Ac (results shown in Figure 17). Active fractions were pooled and further purified on reverse-phase HPLC aε described in Example 7. Results for peak 1 are shown in Figure 18: it yielded an active factor eluting at 22% acetonitrile which is designated Lc-AFP. EXAMPLE 10 Purification of antimicrobial proteins from Clitoria ternatea seedε. The procedure deεcribed in Example 7 waε followed uεing the baεic extract from Clitoria ternatea εeedε. Following chromatography on the S—Sepharoεe High Performance column, the Clitoria extract yielded two partially reεolved peakε of antifungal activity eluting between 260 mM and 400 mM NH 4 Ac (reεultε εhown in Figure 19). Active fractionε were pooled for each peak and further purified on reverεe-phaεe HPLC as described in Example T. Resultε for peak 1 are εhown in Figure 20: it yielded an active factor eluting at approximately 18% acetonitrile which iε deεignated Ct-AMPl. Similarly peak 2 yielded an active factor eluting at approximately 18% acetonitrile which iε deεignated Ct-AMP2 (reεultε εhown in Figure 21). EXAMPLE 11 Molecular structure of the purified antimicrobial, proteinε. The molecular structure of the purified antimicrobial proteins waε further analysed. Sodium dodecyl εulphate polyacrylamide gel electrophoreεiε (SDS-PAGE) waε performed on precaεt commercial gelε (PhaεtGel High Density from Pharmacia) using a PhastSystem (Pharmacia) electrophoreεis apparatus. The sample buffer contained 200 M Tris-HCl (pH 8.3), 1% (w/v) SDS, 1 mM EDTA, 0.005% bromophenol blue and, unless otherwise stated, 1% (w/v) dithioerythritol (DTE). Proteins were fixed after electrophoresiε in 12.5% glutaraldehyde and εilver-εtained according to Heukeεhoven and Dernick (1985, Electrophoreεiε, 6, 103-112). The Rs-AFPs were analysed by SDS-PAGE. After reduction with β-mercaptoethanol and modification of the cysteine reεidueε by S-pyridylethylation, both Rs-AFPl and RS-AFP2 show single bands with an apparent molecular maεε of about 5 kDa. After εimple reduction without further cyεteine derivatiεation, the 5 kDa band is always accompanied by a 16 kDa band at variable yields, which may represent an oligomeric form of the 5 kDa protein resiεted during electrophoreεiε. Unreduced Rs-AFPl and RS-AFP2 migrate aε εingle bands of 20 kDa and 17 kDa, respectively. These results show that the native Rs-AFPε are oligomeric proteinε, consisting of dimers, trimers or tetramers of the 5 kDa polypeptide. The oligomeric structure appears to be stabilised by disulphide linkageε. Figure 22 shows the SDS-PAGE analysiε of the purified Rs-AFPs: lane 1 is unreduced Rs-AFPl, lane 2 is reduced and S-pyridylethylated Rs-AFPl, lane 3 iε unreduced Rε-AFP2, lane % is reduced and S-pyridylethylated Rs-AFP2. Two hundred nanogramε of the proteinε were separated on the gels. Lane M showε myoglobin fragments used as molecular weight markers (Pharmacia) with the following sizeε: 17 kDa, 14.5 kDa, 8 kDa, 6 kDa, and 2.5 kDa. SDS-PAGE analyεis of Rs-nεLTP after reduction with DTE yielded a single 9 kDa band. The unreduced Rs-nsLTP migrated as a single 18 kDa band. It appears therefore that Rε-nεLTP iε a dimeric protein (2 9 kDa) stabilised by disulphide bridges. Figure 23 showε the SDS-PAGE analyεis of the purified Rs-nsLTP: Lane 1 is unreduced Rε-nεLTP, lane 2 is reduced Rs-nsLTP. Molecular weight markers (lane M) are as in Figure 22. Free cysteine thiol groupε of the Rs-AFPs were asseεsed qualitatively aε followε. Hundred μq amounts of reduced or unreduced proteins were dissolved in 6 M guanidinium-Cl containing 100 mM sodium phosphate buffer (pH 7) and 1 mM EDTA. The mixtures were allowed to react with 5,5'-dithionitrobenzoic acid and monitored for release of nitrothiobenzoate as described by Creighton (1989, Protein structure, a practical approach, 155-167). Reduction of the proteins waε done by addition of Tris-HCl (pH 8.6) to 100 mM and dithioerythritol to 30 mM, followed by incubation at 45°C for 1 hour. The proteinε were εeparated from the exceεε reagentε by reverεed-phaεe chromatography on a C 2 /C»g silica column. The unreduced Rs-AFPs did not contain free cysteine thiol groups, whereas the reduced proteins did, indicating that all cysteine reεidueε participate in diεulphide bonds. The pi values of Rs-AFPl and Rs-AFP2 were determined by isoelectric focusing and found to be higher than 10 for both proteins. Iεoelectric focuεing was performed on precast Immobiline Dry Strips (Pharmacia) rehydrated in 8 M urea, using marker proteinε in the pi range from 4.7 to 10.6 (Pharmacia) . Figure 24 showε the SDS-PAGE analyεiε of the two purified proteinε from Dahlia; Figure 25 shows the SDS-PAGE analysiε of the two purified Cnicus proteinε. Molecular weight markers were run aε reference proteinε (lane M: 29 kDa, 20.1 kDa, 13.2 kDa and 5.2 kDa). When reduced with DTT, all four proteinε run aε 5/6 kDa bandε (Figure 24, laneε 2 and 4; Figure 25, laneε 2 and 4). In their unreduced form, the purified proteinε run aε oligomerε. Unreduced Dm-AMPl runε as a 24 kDa protein (Figure 24, lane 1) and Dm-AMP2 as a 17 kDa protein (Figure 24, lane 3). Similarly, unreduced Cb-AMPl runs as a single band of 30 kDa (Figure 25, lane 1) and Cb-AMP2 aε a band of 18 kDa (Figure 25, lane 3) . Figure 26 εhowε the SDS-PAGE analyεiε of the two purified proteinε from Clitoria and the purified proteinε form Lathyruε. Molecular weight markers were run as reference proteinε (lane M: 29 kDa, 20.1 kDa, 13.2 kDa and 5.2 kDa). When reduced with DTT, all three proteins run as 5/6 kDa bands (Figure 26: lane 2, Ct-AMPl; lane 4, Ct-AMP2; lane 6, Lc-AFP). In their unreduced form, the purified proteins run as oligomers. Unreduced Ct-AMPl and Ct-AMP2 run as proteins of approximately 15 kDa (Figure 26, lanes 1 and 3 respectively) whereas unreduced Lc-AFP runs as an approximately 12 kDa protein (Figure 26, lane 5). EXAMPLE 12 Amino acid sequencing of the Rs-AFPs and related proteins. Cysteine residues of the antifungal proteins were modified by S-pyridylethylation using the method of Fullmer (1984, Anal Biochem, 142, 336-341). Reagents were removed by HPLC on a Pep-S (porous silica C 2 /C.g) (Pharmacia) column (25 x 0.4 cm) . The S-pyridylethylated proteins were recovered by eluting the column with a linear gradient from 0.1 % trifluoroacetic acid (TFA) to acetonitrile containing 0.1 % TFA. The reεulting protein fractionε were εubjected to amino acid εequence analyεiε in a 477A Protein Sequencer (Applied Bioεyεtems) with on-line detection of phenylthiohydantoin amino acid derivatives in a 120A Analyser (Applied Bioεyεtemε). Where necesεary due to the proteinε being blocked, treatment of the S-pyridylethylated proteinε with pyroglutamate amino peptidaεe was done according to the supplier's instructionε (Boehringer Mannheim, Mannheim, FRG) . The N-terminal amino acid εequence of Rs-AFPl and Rs-AFP2 was determined by automated Edman degradation, after treatment with pyroglutamate amino peptidaεe which cleaves off cyclic N-terminal gluta ate residues. Figure 27 shows the sequence of the first 44 N-terminal amino acids of Rε-AFPl and of the firεt 35 residues of RS-AFP2. The sequences of Rs-AFPl and RS-AFP2 differ at only two positions within the first 36 residue ' s. The replacement of a glutamic acid by a glutamine (position 4) and an asparagine by an arginine (poεition 27) in Rs-AFP2 are conεiεtent with the higher net poεitive charge of thiε protein relative to Rs-AFPl, which waε previously evidenced by cathodic gel electrophoresiε (Figure 3) and cation exchange chromatography (Figure 1). Rs-AFPl appears to be rich in cysteine and basic amino acids (5 and 9 respectively within the firεt 45 residues). The molecular mass of Rs-AFPl calculated on the basiε of the partial amino acid sequence (4964 Da) is very close to the value estimated by SDS-PAGE (about 5000 Da) which indicates that the determined sequence encompasses the major part of the protein. However, it is anticipated that Rs-AFPl contains at leaεt one more cyεteine, εince the absence of free thiol groups assumes an even number of cysteineε. Figure 27 alεo εhows the first 23 to 30 N-terminal amino acids of the Rs-AFP-like proteins isolated from other Braεsicaceae as described in Example 5 (Bn-AFPl, Bn-AFP2, Br-AFPl, Br-AFP2, Sa-AFPl, Sa-AFP2, At-AFPl) . All proteins were treated with pyroglutamate amino peptidase prior to εequencing but the cyεteine residues were not modified. Consequently, cysteine residues appear as blanks upon Edman degradation. Amino acids identical to the correεponding amino acidε in Rs-AFPl are shown by dots. It appears therefore that the Rs-AFP-like proteinε from other memberε of the family Braεεicaceae are identical or nearly identical to Rs-AFPl and RS-AFP2. Br-AFP2 containε an unidentified uncommon amino acid at poεition 11. Figure 28 εhowε the complete amino acid εequence for the peptideε Dm-AMPl, Cb-AMPl and Cb-AMP2. Shown alεo iε the εequence for the firεt 20 N-terminal amino acidε of Dm-AMP2. The sequences for Dm-AMPl and Dm-AMP2 differ at only one poεition (position 2) in these firεt 20 amino acids. Comparing the sequenceε for Cb-AMPl and Cb-AMP2, there are three changeε. The εubεtitution of an acidic reεidue (aεpartic acid at poεition 22) in Cb-AMPl for a neutral aεparagine in Cb-AMP2 and the εubεtitution of glutamine at poεition 23 for a baεic lyεine are conεiεtent with the higher net poεitive charge. Similarly, Cb-AMP2 alεo differε from Dm-AMPl at two poεitionε although the reεult iε the net gain of two poεitive chargeε. All four proteinε εhow εtriking similarity to the proteins iεolated from εeedε of the Braεεicaceae family. Alignment of the amino acid εequence for Rε-AFPl ( aphanuε Sativuε - Antifungal Protein 1) with the sequence for Dm-AMPl revealε that they have approximately 50% identical reεidueε. Figure 29 εhowε the complete amino acid εequence for the peptideε Lc-AFP and Ct-AMPl. Ct-AMP2 iε expected to be highly homologouε to Ct-AMPl. Both Lc-AFP and Ct-AMPl are also homologouε to the Compoεitae and Braεεicaceae proteinε. In particular Ct-AMPl is very homologous to the Dahlia peptide Dm-AMPl, having 35 identical reεidueε in its εequence. Homologieε can be found between thiε group of cloεely related proteinε and the productε encoded by two pea (Piεum εativum) genes, pl39 and pl230, which are specifically induced by the fungus Fusarium solani (Chiang and Hadwiger, 1991, Mol Plant Microbe Interact, 4, 324-331), and with the protein product of potato (Solanum tuberoεum) gene p322 (Stiekema et al, 1988, Plant Mol Biol, 11, 255-269). Nothing iε known about the biological propertieε of the proteins encoded by genes pl39, pl230 or p322. In addition, the Rs-AFP-like/Dahlia/Cnicuε/Lathyruε/Clitoria claεε of antimicrobial proteinε εhow homology to inhibitorε of inεect gut α-amylaεeε from Sorghum bicolor (Bloch and Richardεon, 1991, FEBS Lett, 279, 101-104), and alεo to γ-purothioninε from Triticum aestivum (Colilla et al, 1990, FEBS Lett, 270, 191-194) which inhibit iri vitro protein εyntheεiε in cell-free εyεtemε (Mendez et al, 1990, Eur J Biochem, 194, 533-539). Figure 30 εhows the alignment of the amino acid sequenceε of Rε-AFPl, Dm-AMPl, the Cb-AMPε, Lc-AFP, Ct-AMPl, the εorghum α-amylaεe inhibitor SIα2, wheat γl purothionin, and the predicted εequenceε of the mature protein productε of the Fuεarium-induced pea geneε pI230 and pI39, of the cowpea gene pSASlO, and of the potato gene p322. Sequence identitieε and conεerved changeε compared with Rε-AFPl are boxed. Conεerved changeε are conεidered aε εubεtitutionε within the amino acid homology groupε FWY, MILV, RHK, EDNQ, and PAGST. Gapε introduced for optimal alignment are represented by dasheε. Upon alignment of the sequences, all of the cysteines and most of the glycineε appear at conεerved positions, εuggeεting their importance with reεpect to structure and function of these pr.oteins. Also noteworthy are the conserved aromatic residueε at poεitionε 11 and 40. Figure 31A εhowε one of the poεεible DNA εequences of the genes encoding Dm-AMPl, Dm-AMP2, Cb-AMPl and Cb-AMP2. Similarly Figure 31B εhowε one of the poεεible DNA εequenceε of the genes encoding Lc-AFP and Ct-AMPl. Theεe gene εequenceε have been predicted from the known amino acid εequenceε using codons which commonly occur in dicotyledonouε plantε. The actual gene εequenceε within the εeed may differ due to the degeneracy of the genetic code. EXAMPLE 13 Amino acid sequencing of Rs-nsLTP. Amino acid εequencing of the Rε-nεLTP protein was carried out according to the description in Example 12. Figure 32 showε the firεt 43 N-terminal amino acidε of Rε-nεLTP of which the cyεteine reεidues were modified by S-pyridylethylation. In Figure 33 the sequence of Rs-nsLTP is aligned with the N-terminal sequenceε of non-εpecific lipid tranεfer proteinε isolated from Spinacia oleracea (So-nsLTP; Bernhard et al, 1990, Plant Physiol, 95, 164-170), Ricinus communis (Rc-nsLTP; Takiεhima et al, 1986, Biochim Biophyε Acta, 870, 248-255), Daucuε carota (Dc-nsLTP; Stenk et al, 1991, Plant Cell, 9, 907-921), Hordeum vulgare (Hv-nsLTP; Bernhard and Somerville, 1989, Arch Biochem Biophyε, 269, 695-697), and Zea mays (Zm-nsLTP; Tchang et al, 1988, J Biol Chem, 263, 16849-16855). Gaps introduced for optimal alignment of the sequenceε are indicated .by daεheε. Identical amino acidε and conserved subεtitutionε occurring in at leaεt 4 of the 6 εequenceε are boxed. Conεerved changes are considered as εubεtitutionε within the amino acid homology groupε FWY, MILV, RHK, EDNQ and PAGST. Rε-nsLTP εhowε 38 to 53% εequence identity with the non-specific lipid transport proteins from other plant sourceε. Non-εpecific lipid tranεport proteins are proteinε that can tranεlocate phospholipids or other apolar compounds between two membrane syεtemε. Theεe proteinε were previously thought to play a role in the transport of phoεpholipidε from endoplaεmic reticulum to cell and organelle me braneε (Arondel and Kaden, 1990, Experientia, 46, 579-585). However, recent evidence εhows that nsLTPs are located extra-cellularly, making their proposed function in membrane biogenesiε unlikely (Sterk et al, 1991, Plant Cell, 3, 907-921). EXAMPLE 14 Stability of the proteins' antifungal activity. Teεtε for antifungal activity were performed with 20 μl εampleε diluted five-fold with growth medium containing Fuεarium culmorum εporeε, according to the assay method given in Example 1. Untreated control samples consiεted of the teεt proteinε at 500 jug/ml in 10 mM εodium phoεphate buffer (pH 7). Heat εtability teεtε were performed by heating aliquotε of the test proteins for 10 minutes at different temperatures up to 100°C. Reduction of diεulphide bridgeε waε done by addition of dithiothreitol at 30 mM and Triε-HCl (pH 8.6) at 300 mM. The reagentε were removed by reverεed-phaεe chromatography. For digeεtionε, different proteaεeε were added at 100 //g/ml and incubated at 37°C for 16 hourε. The control treatment containing only the reagentε proved negative for antifungal activity after the reversed-phase chromatography εtep. The antifungal activity of all the purified proteins tested was resiεtant to heat treatmentε at up to 100°C for 10 minuteε. Reduction of their diεulphide bonds by dithiothreitol, however, completely abolished the antifungal activity. These diεulphide linkageε are eεεential for biological activity. Treatment of the Rε-AFP proteins with trypεin, chymotrypsin, proteinaεe K or pronase E reduced the antifungal activity by at leaεt 10-fold. EXAMPLE 15 Antifungal potency of the proteins. The antifungal potency of the purified proteinε waε assessed on different plant pathogenic fungi, using the aεεay deεcribed in Example 1. Growth of fungi, collection and harvest of fungal sporeε, and preparation of mycelial fragmentε were done aε previouεly described (Broekaert et al, 1990, FEMS Microbiol Lett, 69:55-60). The following fungal εtrains were used: Alternaria brassicola MUCL 20297, Ascochyta pisi MUCL 30164, Botrytis cinerea MUCL 30158, Cercospora beticola εtrain K897, Cladoεporium εphaeroεperum (K0791), Colletotrichum lindemuthianum MUCL 9577, Fuεarium culmorum IMI 180420, Fuεarium oxyεporum f.εp. piεi IMI 236441, Fuεarium oxyεporum f.εp. lycopersici MUCL 909, Mycosphaerella fijienεiε var fijiensis IMI 105378, Nectria haematococca Collection Van Etten 160-2-2, Penicillium digitatum (K0879), Phoma betae MUCL 9916, Pyrenophora tritici-repentis MUCL 30217, Pyricularia oryzae MUCL 30166, Rhizoctonia solani CBS 207-84, Sclerotinia εclerotianum MUCL 30163, Septoria nodorum MUCL 30111, Septoria tritici (K1097D), Trichoderma hamatum MUCL 29736, Trichoderma viride (K1127), Verticillium albo-atrum (K0937), Verticillium dahliae MUCL 19210, Venturia inaequalis MUCL 15927. For C beticola, R solani, S_ εclerotianum, S_ nodorum and M fijiensis, mycelial fragments were used as inoculum, whereas all other fungi were inoculated as εporeε. Serial dilutionε of the antifungal proteinε were applied to the fungi, either uεing growth medium A or medium B. The percent growth inhibition waε meaεured by microεpectrophotometry. The concentration required for 50% growth inhibition after 48 h of incubation (IC CQ value) waε calculated from the doεe-reponεe curveε. The I 5 n valueε for the εlow growing fungi nodorum and V inaequalis was measured after 5 and 15 days of incubation respectively. The reεultε for Rε-AFPl and Rε-AFP2 are summarised in Table 1. TABLE 1 ANTIFUNGAL ACTIVITY of Rs-AFPl and RS-AFP2 ND not determined The concentration of Rε-AFPs required for 50% growth inhibition in medium A varied from 0.3 g/ml to over 100 /g/ml, depending on the test organiεm. The antifungal potency of Rs-AFPl is generally slightly lower than that of Rs-AFP2 in medium A. The difference in antifungal potency between Rε-AFPl and Rε-AFP2 iε more pronounced for the teεtε performed in medium B. Rε-AFPl only inhibitε 4 out of 17 fungi by more than 50% at concentrationε below 100 g/ml, whereaε R8-AFP2 iε inhibitory on 11 out of 18 fungi at this concentration. For εome fungi, εuch aε culmorum and C beticola, the IC 5Q value of R8-AFP2 measured in medium A is comparable to that obtained in medium B. On other fungi, such as £ oxyεporum f.sp. pisi, the ICς 0 value of RS-AFP2 iε increaεed from 2 /g/ml in medium A to over 100 / /g/ml in medium B. The antifungal potency of the Rε-AFP-like proteinε from B napuε, B rapa, £ alba and A thaliana waε compared to that of Rε-AFPl and Rε-AFP2 uεing five different teεt fungi. The reεultε of theεe experiments are shown in Table 2. With the exception of Br-AFP2, all proteins had specific activities comparable to that of the Rs-AFPε. The fact that Br-AFP2 iε on average 20-fold leεε active than the related species may be related to the observation that Br-AFP2 has an uncommon amino acid at position 11 (see Figure 27) whereas the Rs-AFPs and related proteins all have an aromatic residue at this poεition (εee Figure 30). When tested in medium B, RS-AFP2 appears to be the most potent protein, eεpecially on the fungus F_ culmorum. TABLE 2 Antifungal Activity of Rs-AFP-like proteins from Brassica rapa, Brassica napus, Sinapis alba and Arabidopsis thaliana IC C Q ( g/ml) in medium A Fungus Rs-AFPl RS-AFP2 Br-AFPl Br-AFP2 Bn-AFPl Bn-AFP2 Sa-AFPl S&-AFP2 At-AFPl A brassicola, 15 2 3 75 0, 60 1.20 1.2 4. 10 2 1.50 >100 2 2 1.8 3. 3, 90 2 2 38 2 80 2.10 4 2, 3 2 1.80 42 1 ,30 1.50 6 2, 3 0 0.25 3 0, ,35 0.25 0.5 0, 0.25 1 0.80 15 1,,20 1 1.5 1, 1.50 ICC (//g/ml) in medium B Fungus Rs-AFPl RS-AFP2 Br-AFPl Br-AFP2 Bn-AFPl Bn-AFP2 Sa-AFPl Sa-AFP2 At-AFPl The antifungal potency of Rε-nεLTP iε εhown in Table 3. On moεt fungi Rε-nεLTP iε 10 to 20 fold leεs potent relative to RS-AFP2. Rs-nsLTP also appears to be highly salt-εenεitive. None of the 13 fungi teεted are inhibited by Rε-nεLTP in Medium B at concentrations below 100/g/ml. The reεultε for the Compoεitae proteinε are εummariεed in Table 4. The concentration of antimicrobial proteinε required for 50% growth inhibition in medium A varied from 0.3 g/ml to over 100 /g/ml, depending on the test organism. In general, the antifungal potency of the proteins was in the order: Cb-AMP2 > Cb-AMPl > Dm-AMPl > Dm-AMP2. The differences in activity between the proteins is more pronounced in medium B, with Cb-AMP2 showing the beεt εalt tolerance. Dm-AMPl and Dm-AMP2 only inhibit the growth of 6 out of 11 fungi by more than 50% at concentrations below 100 g/ml, whereas the two Cnicus proteins inhibit the growth of 7 out of 8 fungi when asεayed in medium B. Table 5 εum ariεeε the reεultε for the antimicrobial proteins isolated from Leguminoεae εeedε. Theεe proteinε are active, although their activity iε εomewhat lower than that of the Rε-AFPε and Compoεitae proteins, especially when aεεayed in high εalt buffer, Medium B. in particular, the activity of Lc-AFP iε markedly lower and comparable to the activity of Br-AFP2. The amino acid sequence of Lc-AFP also εhowε a εubεtitution at position 11 which iε normally tryptophan (Figureε 27 and 29) . The high levelε of antifungal activitieε demonεtrated ^n vitro by each of the purified proteins suggeεt that they may play a role in the defence of εeedε or εeedlingε againεt fungal attack. TABLE 4 ANTIFUNGAL ACTIVITY of the Dm-AMPε and the Cb-AMPε FUNGUS IC 50 ( / /g/ml) ND = not determined TABLE 5 ANTIFUNGAL ACTIVITY of the Ct-AMPε and Lc-AFP FUNGUS ιc 50 ( g/ml) Medium A Medium B Ct-AMPl Ct-AMP2 Lc-AFP Ct-AMPl Ct-AMP2 Lc-AFP >150 >150 >200 >150 50 >200 75 50 >200 >150 >150 >200 >150 >150 >200 >150 60 >200 >150 >150 >200 >150 100 >200 40 12 >200 EXAMPLE 16 Effect of ionε on antifungal activity. The effect of ionε on the antifungal activity of the Rε-AFPε and Rε-nεLTP was examined in more detail. The IC*- 0 values of Rs-AFPl, Rε-AFP2 and Rs-nsLTP on F_ culmorum and T hamatum were meaεured in five different media. The reference medium was the synthetic growth medium described in Example 1 which contains a total of 2.5 mM monovalent cations and 0.1 mM divalent cations. The four other media contained 10 M KCI, 50 mM KCI, 1 mM CaCl 2 or 5 mM CaCl 2 in supplement, respectively. For the purpose of comparison, theεe teεtε were performed in parallel with β-purothionin, an antifungal protein from wheat εeedε (iεolated as described in Redman and Fisher, 1969, J Sci Food Agric, 20, 427-432) and MJ-AMP2, an antifungal protein from Mirabilis jalapa seedε (Cammue et al, 1992, J Biol Chem, 267, 2228-2233). Table 6 εhowε the reεultε of the antifungal activity aεεayε in the preεence of K and Ca . Addition of KCI at up to 50 mM did not affect the antifungal activity of either Rε-AFPl or Rε-AFP2. CaCl 2 at ImM had no effect on RS-AFP2 but increaεed the IC*- 0 value of Rε-AFPl by about four-fold (ie, Ca + reduced the antifungal activity of Rs-AFPl). CaCl 2 at 5mM al oεt completely inactivated Rε-AFPl while itε effect on RS-AFP2 varied from a εlight increaεe in IC- Q for F_ culmorum to complete inactivation for T hamatum. Addition of KCl at 50 mM decreaεes the activity of Rs-nεLTP by more than 30-fold with both teεt fungi. In compariεon, the IC*- Q value of β-purothionin TABLE 6 , ? . VARIATIONS IN ANTIFUNGAL ACTIVITY IN THE PRESENCE OF K + AND CA + Funguε Antifungal IC50 ( g/ml) protein Reference medium supplement: None lOmM K 50mM K ImM Ca 2+ 5mM Ca 2+ increaεed by about 7-fold in the preεence of 5 mM CaCl 2 - MJ-AMP2 appeared to be highly εenεitive to the presence of salts, since itε IC ςo valueε increaεed by about 10-fold upon addition of either 1 mM CaCl 2 or 50 mM KCl. Theεe reεultε show that the Rs-AFP.ε are antagoniεed by divalent cationε. Rε-AFPl iε much more sensitive to the presence of divalent cations than RS-AFP2. Rs-nsLTP is clearly more εalt-senεitive than either Rε-AFPl or Rε-AFP2. The antagoniεtic effect of cationε appearε to be εtrongly dependent on the teεt organiεm. EXAMPLE 17 Effect of the purified antimicrobial proteinε on the growth of the yeast, Saccharomyces cerevisiae. The purified proteins were teεted for their effect on Saccharomyceε cereviεiae. The method uεed waε εimilar to the antifungal aεεay deεcribed in Example 1 except that the growth medium waε YPD (lOg/l yeast extract, 20g/l bactopeptone, 20g/l glucose) with 0.5% seaplaque agarose. When asεayed at levelε of 250 / /g/ml, none of the purified Braεεicaceae proteinε had an effect on the growth of Saccharomyceε cereviεiae (strain Spl) . Similarly, Lc-AFP did not inhibit the growth of S_ cerevisiae (strain JRY188) at a concentration The Compoεitae and Clitoria peptides were active against the growth of j3 cerevisiae (εtrain JRY188). Theεe reεults are shown in Table 7. Of the six peptides, the two Clitoria peptides, Ct-AMPl and Ct-AMP2 εhowed the higheεt level of activity. TABLE 7 ACTIVITY OF Dm-AMPs, Cb-AMPs and Ct-AMPε on YEAST Protein —50 (•"-' nil) Dm-AMPl 50 Dm-AMP2 50 Cb-AMPl 30 Cb-AMP2 20 Ct-AMPl 18 Ct-AMP2 9 EXAMPLE 18 Effect of the purified antimicrobial proteinε on bacteria. The antibacterial effect of the purified proteinε was aεεeεεed on Agrobacterium tumefaciens C58, Alcaligenes eutrophus, Azoεpirillum brasilense Sp7, Bacillus megaterium ATCC 13632, Erwinia carotovora strain 3912, Escherichia coli εtrain HB101, Pεeudomonas solanacearum εtrain K60 and Sarcina lutea ATCC 9342, uεing the aεsay deεcribed in Example 1. Rε-AFP2 cauεed 50% inhibition in B megaterium at 200 / g/ml, but had no effect on the other bacteria at concentrationε up to 500 /g/ml. The Compoεitae peptideε Dm-AMPl, Dm-AMP2, Cb-AMPl and Cb-AMP2 εhowed activity only on B megaterium where they inhibited growth to 50% at concentrations of 180, 40, 80 and 32 / /g/ml reεpectively. Rs-AFPl, Bn-AFPε, Br-AFP2, Sa-AFPε, Ct-AMP8 and Lc-AFP had no effect on any of the bacteria at concentrationε up to 500 / /g/ml. Reεultε εhpw that in general theεe proteinε poεεeεs only weak antibacterial activity. EXAMPLE 19 Effect of the purified antifungal proteins on cultured human cells. Human cell toxicity assays were performed either on umbilical vein endothelial cells (Aleεεi et al, 1988, Eur J Biochem, 175, 531-540) or εkin-muεcle fibroblasts (Van Damme et al, 1987, Eur J Immunol, 17, l 7) cultured in 96-well microplateε. The growth medium was replaced by 80//1 of serum-free medium (Optimem 1 for endothelial cells or Eagle's minimal eεεential medium (EMEM) for fibroblaεts, both from GIBCO), to which 20 μl of a filter-steriliεed teεt solution was added. The cells were further incubated for 24 hours at 37°C under a 5% C0 2 atmoεphere with 100 % relative humidity. The viability of the cellε waε assessed microscopically after staining with trypane blue (400 mg/1 in phosphate buffered saline, PBS) for 10 minutes. Alternatively, cellε were stained with neutral red (56 mg/1 in PBS) for 2 hourε at 37°C. Cellε were lysed in acidic ethanol (100 mM sodium citrate, pH 4, containing 50% ethanol) and εcored for releaεe of the dye by microεpectrophotometry at 540 nm. The Rε-AFPε and Rs-nsLTP were evaluated for their potential toxic effects using this assay. When added at up to 500 //g/ml to either cultured human umbilical vein endothelial " cells or human skin-muscle fibroblastε, neither Rs-AFPl, Rs-AFP2, nor Rε-nεLTP affected cell viability after 24 h of incubation. In. contraεt, β-purothionin adminiεtered at 50 /g/ml decreased the viability of both cell types by more than 90%. EXAMPLE 20 Anti-fungal activity of the Rs-AFPs against foliar disease: in vivo test Rs-AFP2 was tested against the εugarbeet foliar diεeaεe Cercoεpora beticola (εtrain E897) uεing the following method. Sugarbeet plantε were grown in John Inneε potting compoεt ( No. 1 or 2 ) in 4cm diameter mini-potε. The protein preparation waε formulated immediately prior to uεe by diεεolving in εterile diεtilled water and diluting to the appropriate concentration. The formulation waε applied to the plantε aε a foliar εpray. The εpray waε applied to maximum diεcrete droplet retention. Plantε were treated with the protein one day prior to inoculation with the diεeaεe which waε applied aε a foliar spray at a concentration of 50000 sporeε/ml. Plantε were kept in an humidity chamber for 48 hours and then transferred to the glasεhouse. Diseaεe waε aεεeεεed following a further incubation of 8 dayε. Reεultε are εhown in Figure 34. The commercially available fungicide hexaconazole waε used aε a εtandard. R8-AFP2 gave good control of the disease and the concentration giving 50% control waε approximately 15 μM . In comparison hexaconazole gave 50% disease control when applied at approximately 7 μM . This confirms that the protein can act as an effective fungicide i_n vivo and that its activity is on a molar basiε comparable to the chemical εtandard. EXAMPLE 21 Molecular cloning of Rε-AFPl and Rε-AFP2 cDNAs From outdoor grown Raphanuε εativuε plants, seeds at 6 different developmental stages were collected, frozen in liquid nitrogen and stored at -80°C. After pulverisation, total RNA was extracted from 15 g of a mixture of the 6 different developmental stageε, uεing the method of De Vrieε et al (1988, Plant Molecular Biology Manual, B6, 1- 13) with the exception that 6 ml of a 1:2 phenol:RNA extraction buffer mixture and 2 ml of chloroform were uεed per g of tiεsue. Poly (A) + mRNA was purified by affinity chromatography on oligo(dT)-celluloεe aε deεcribed by Siflow e_ aj. (1979, Biochemiεtry 18, 2725-2731) yielding about 10 μg of poly(A) + RNA per g of tiεεue. Double εtranded cDNAε were prepared from 1,5 μg of poly(A) + RNA according to Gubler and Hoffman (1983, Gene 25, 263-269) and ligated to EcoRI/Not adaptors using the cDNA Synthesiε Kit of Pharmacia. The cDNAs were cloned into the lambda ZAPII phage vector (Stratagene) according to the manufacturers instructionε. A DNA probe for εcreening the cDNA library was produced by polymerase chain reaction (PCR) as follows. Two degenerate oligonucleotides were synthesised: OWB15 (5'AAAGAATTCAARYTNTGYSARMGNCC 3') and OWB17 (5'AAAGAATTCRTGNGCNGGRAANACRTARTTRC 3'). OWB15 correspondε to amino acidε 2 to 7 of Rε-AFPl and has a sense orientation. OWB17 corresponds to amino acids 36 to 43 of Rs-AFPl and has an antisense orientation. Both primers have the AAAGAATTC (i.e. AAA followed by the EcoRI recognition sequence) sequence at their 5' ends. PCR was performed with the Tag polymeraεe under εtandard conditions (Sambrook e_t al, 1989, Molecular Cloning, Cold Spring Harbor Laboratory Press) using OWB15 and OWB17 as amplimerε and 25 ng of cDNA as target DNA. The temperature programme included an initial step at 94°C for 5 min, 30 cycles (94°C for 1 min; 45°C for 2 min, 72°C for 3 min) and a final step at 72°C for 10 min. The 144 bp PCR amplification product waε purified on a 3% agaroεe (NuSieve, FMC) gel. This PCR product was partially reamplified using the senεe degenerate oligonucleotide OWB16 (5'AAAGAATTCGGNACNTGGWSNGGNGTNTG 3') and OWB17. OWB16 alεo haε the AAAGAATTC extension at its 5' end. This 123 bp PCR amplification product was again purified on a 3% agaroεe (NuSieve, FMC) gel and reamplified by PCR under the same conditions except that the reaction mixture contained 130 μM dTTP and 70 μM digoxigenin-11-dUTP inεtead of 200 μM dTTP. The digoxigenin-labeled PCR product was purified on a 3% NuSieve agarose gel. About 10,000 plaque forming unitε of the lambda ZAPII cDNA library were screened with the digoxigenin-labeled PCR product by _irι situ plaque hybridiεation uεing nylon membranes (Hybond-N, Amersham) . Membranes were air-dried and DNA was crosslinked to the membranes under UV light (0.15 2 J/cm ) . Hybridisation waε performed for 16 h at 64°C in 5 x SSC, 1 % blocking reagent (Boehringer Mannheim), 0.1 % N-lauroylsarcosine, 0.02 % sodium dodecylsulphate containing 10 ng/ml of heat denatured digoxigenin-labeled probe. Non- specifically bound probe was removed by rinsing two times 5 min in 2 x SSC / 0.1 % SDS at 25°C and two times 15 min in 0.1 x SSC / 0.1 % SDS at 60°C. Detection of the probe was done using anti- digoxigenin antibodies linked to alkaline phosphatase (Boehringer Mannheim) and its substrate 5-bromo-4-chloro-3-indolyl phosphate (Boehringer Mannheim) according to the manufacturers instructionε. Positive plaques were purified by two additional εcreening roundε with the εame probe under the same conditions. Insertε from purified plaques were excised iri vivo into the pBluescript phagemid form with the aid of the helper phage R408. The inεertε from 22 different poεitive clones were excised by EcoRI digeεtion and their εizes compared by agarose gel electrophoresis. Four clones had an insert of approximately 400 bp, the other 18 positive clones contained insertε ranging between approximately 250 and 300 bp. The four cloneε with the 400 bp inserts and six cloneε with the smaller insertε were εubjected to nucleotide sequence analysis. The clones with the largest insert all had an open reading frame of 80 amino acidε correεponding to Rε-AFPl, aε could be determined by compariεon to the experimental N- terminal amino acid sequenceε (see Example 12). The 243 bp open reading frames code for the mature Rε-AFPl (50 amino acidε) preceded by a putative 29 amino acid εignal εequence obeying the (-1,-3) rule (von Heijne 1985, Mol. Biol. 184, 99-105). These full-length cDNA clones only differed from each other in the length of their 5' and 3' end untranslated regions. Five of the cloneε with the smallest insert were partially identical to the full-length Rs-AFPl cDNA clones except that they were truncated at their 5' endε. The remaining clone waε identified aε a 5' truncated R8-AFP2 cDNA clone by comparing the deduced and the experimentally determined amino acid sequenceε. When comparing the full-length Rs-AFPl cDNA clone pFRGl (Fig. 35) and the truncated RS-AFP2 cDNA clone pFRG2 (Fig. 36), it can be εeen that the codon usage is εlightly different and that the 3' end untranslated region of the RS-AFP2 cDNA iε longer than the one of the Rε-AFPl cDNA. Finally, both the Rε-AFPl and the Rε-AFP2 cDNA clones have at least two polyadenylation signals. Figure 35 showε the nucleotide εequence and the deduced amino acid sequence of the full-length Rs- AFPl cDNA clone pFRGl. The putative εignal εequence iε underlined and the εequence of the mature Rs-AFPl is boxed. Figure 36 showε the nucleotide εequence and the deduced amino acid εequence of the 5' truncated Rε-AFP2 cDNA clone pFRG2. In order to obtain a full-length Rε-AFP2 cDNA, another approach waε followed : PCR waε performed under standard conditions using the antisenεe oligonucleotide OWB23 (5ΆTAGAATTCGACGTGAGCTTATCATCTTATTATCCG 3') in combination with the M13 universal primer at one hand and the M13 reverse primer at the other hand. The laεt 30 nucleotideε of OWB23 form the inverted complementary sequence of the part of the 3' untranslated region immediately flanking the poly-A tail of pFRG2 (see Fig. 36). This εequence is extended to the 5' end with the GAATTC EcoRI recognition site preceded by the nucleotides 'ATA' . ς Aε a template, either 2 μg of total cDNA or 10 recombinant phages were uεed. In both caεeε, 3 εeparate reactionε were εet up. Prior to amplification, phages were lysed by an initial step in the PCR temperature programme of 5 min at 99°C to liberate the phage DNA. The size of the amplification products was determined by electrophoresiε on a 3% agaroεe (NuSieve, FMC) gel. Products were obtained with sizeε correεponding to inεerts of 280 to 300 bp. Thus, it can be concluded that no full-length Rs-AFP2 cDNA cloneε seem to be preεent in the cDNA library. EXAMPLE 22 Mutagenesis of Rs-AFPl cDNA to Rε-AFP2 DNA As can be deduced from the experimentally determined N-terminal εequenceε (εee Example 12) and the nucleotide εequenceε (εee Example 21), Rs- AFPl and RS-AFP2 only differ in two amino acids as stated in Example 12. As the antifungal potency of Rε-AFP2 is εignificantly higher than that of Rε AFP1 (see Table 1) and a full-length cDNA clone of the RS-AFP2 is not available, the Rs-AFPl cDNA was transformed into the RS-AFP2 nucleotide εequence by PCR-aεsisted εite-directed mutagenesis according to the method of E. Merino e_t a (1992, BioTechniques 12, 508-510). The following oligonucleotideε were used : OWB28 (5'CTTGGCCTTTGGCACAACTTC 3'), OWB29 (5'GCTTTCTCAAGTCTAATGCAC 3' ) , OWB30 (5 r AACTCGAGCTGCAGTGTCGACCTATTAACAAGGAAAGTAGC 3' ) , OWBSδ^ (5'GGAATAGCCGATCGAGATCTAGGAAACAGCTATGACCATG 3' ) , OWB36 (5'GGAATAGCCGATCGAGATCTAGGA3' ) . The first mutation (glutamate into glutamine at position 5 of the mature protein) was introduced by performing PCR with the Pfu polymerase (Stratagene) uεing OWB35 (this is the M13 universal _•primer with a 5' tag sequence) and OWB28 (the firεt antiεenεe mutageneεiε primer) aε amplimerε and 100 ng of the Kpnl-digeεted pFRGl cDNA aε target DNA. MgCl, waε added to the amplification mixture to a final concentration of " 50 mM. The temperature programme included an initial εtep at 94°C for 5 min, 30 cycleε (94°C for 1 min, 45°C for 2 min, 72°C for 3 min) and a final εtep at 72°C for 10 min. In a εecond εtep, thiε PCR product waε uεed as a megaprimer and extended by the Pfu polymerase using 50 ng of the Kpnl-digested pFRGl cDNA aε the target DNA. The temperature programme included an initial εtep at 94°C for 5 min followed by a 5 cycles extension (94°C for 1 min, 50°C for 1 min, 72°C for 1 min). Then OWB29 (the antiεenεe primer introducing the second mutation, from asparagine to arginine at poεition 27 of the mature protein) and OWB36 (which iε identical to the 5' tag εequence of OWB35) were added, followed by PCR amplification by the Pfu-polymeraεe aε deεcribed for the introduction of the firεt mutation. To get a full- length Rε-AFP2 nucleotide εequence, the procedure outlined in the second step was repeated though using the oligonucleotide primers OWB36 and OWB30 (which introduces a second εtop codon followed by the Sai , Pεtl and Xhol reεtriction εiteε, thus also eliminating the 3' end untranslated region of the Rs-AFPl cDNA clone pFRGl). The final PCR product was cut with BamHI (occurring in the polylinker of the pBluescript phagemid pFRGl) and Sail, subcloned in pEMBLl8+ (pre-digested with the εame restriction enzymes) and subjected to nucleotide sequence analyεiε. Figure 37 εhowε the nucleotide sequence and the derived amino acid sequence of the full-length Rs-AFP2 DNA clone pFRG4 obtained by PCR-aεεisted site-directed mutagenesiε of the Rε-AFPl cDNA clone pFRGl. The putative εignal εequence iε underlined and the sequence of the mature Rs-AFP2 is boxed. EXAMPLE 23 Construction of the expression vector pFRG7 The expression vector pFRG7 (Figure 38; SP - signal peptide, MP - mature protein) contains the full coding region of the RS-AFP2 DNA flanked at itε 5' end by the εtrong constitutive promoter of the 35S RNA of the cauliflower mosaic virus (Odell et al, 1985, Nature 313, 810-812) with a duplicated enhancer element to allow for high transcriptional activity (Kay et al, 1987, Science 236, 1299-1302). The coding region of the RS-AFP2 DNA iε flanked at itε 3' end εide by the polyadenylation εequence of 35S RNA of the cauliflower moεaic viruε (CaMV35S). The plaεmid backbone of thiε vector iε the phagemid pUC120 (Vieira and Meεεing 1987, Methodε Enzymol. 153, 3-11). pFRG7 waε conεtructed aε followε : clone pFRG4 which consisted of the Rs—AFP2 DNA (Figure 37) cloned into the BamHI / Sai εiteε of pEMBLl8+, Boehringer). The 298 bp BamHI / Sail fragment waε εubcloned into the expreεεion vector pFAJ3002 which waε pre-digeεted with BamHI and Sail. pFAJ3002 iε a derivative of the expreεεion vector pFFl9 (Timmermanε et. ^1_, 1990, J. Biotechnol. 14, 333-344) of which the unique EcoRI εite is replaced by a Hindlll εite. EXAMPLE 24 Conεtruction of the plant tranεformation vector pFRGβ The expreεεion vector pFRG7 waε digested with Hindlll and the fragment containing the Rs-AFP2 DNA expreεεion caεεette waε εubcloned into the unique Hindlll εite of pBinl9Ri. pBinl9Ri is a modified version of the plant tranεformation vector pBinl9 (Bevan 1984, Nucleic Acidε Reεearch 12, 8711-8721) wherein the unique EcoRI and Hindlll εiteε are εwitched and the defective nptll expreεsion casεette (Yenofεky et al. 1990, Proc. Natl. Acad. Sci. USA 87: 3435-3439) iε introduced. The new plant tranεformation vector iε deεignated pFRG8 (Figure 39) . EXAMPLE 25 Plant Tranεformation The diεarmed Agrobacterium tumefaciens strain LBA4404 (pAL4404) (Hoekema e_t a_l, 1983, Nature 303, 179-180) waε tranεformed with the vector pFRGδ uεing the method of de Framond e_t a_l (BioTechnology 1, 262-269). Tobacco tranεformation waε carried out using leaf discε of Nicotiana tabacum Samsun based on the method of Horεch e_t a_l (1985, Science 227, 1229- 1231) and co-culturing with Agrobacterium εtrainε containing pFRGβ. Co-cultivation waε carried out under selection preεεure of 100 /g/ml kanamycin. Transgenic plants (transformed with pFRGδ) were regenerated on media containing 100 / /g/ml kanamycin. Theεe transgenic plants may be analysed for expresεion of the newly introduced geneε uεing εtandard western blotting techniques. Plantε capable of conεtitutive expreεεion of the introduced geneε may be εelected and εelf- pollinated to give εeed. Fl εeedlingε of the tranεgenic plantε may be further analyεed.

Claims: CLAIMS An antimicrobial protein having εubεtantially the amino acid sequence shown in Figures 27 to 29 or in. Figure 32. A protein aε claimed in claim 1 which iε an oligomer and which compriseε at leaεt one polypeptide having εubεtantially the amino acid εequence εhown in Figureε 27 to 29 or in Figure 32. A protein aε claimed in claim 1 or claim 2 which iε capable of being iεolated from a plant seed. A protein as claimed in claim 3 which iε capable of being iεolated from a εeed of the family Braεεicaceae or of the family Compoεitae or of the family Leguminoεae. A protein aε claimed in claim 4 which iε iεolated from Raphanuε, Brassica, Sinapis, Arabidopεiε, Dahlia, Cnicuε, Lathyruε or Clitoria. A pure protein Rε-AFPl, capable of being iεolated from Raphanuε εeed. A pure protein Rε-AFP2, capable of being isolated from Raphanus seed. A pure protein Rs-nsLTP, capable of being isolated from Raphanus seed. Pure proteins Bn-AFPl, Bn-AFP2, Br-AFPl and Br-AFP2, capable of being isolated from Brassica .εeed. Pure proteinε Sa-AFPl and Sa-AFP2, capable of being iεolated from Sinapis seed. A pure protein At-AFPl, capable of being isolated from Arabidopsiε εeed. A pure protein Dm-AMPl, capable of being isolated from Dahlia εeed. A pure protein Dm-AMP2, capable of being isolated from Dahlia seed. A pure protein Cb-AMPl, capable of being isolated from Cnicus εeed. A pure protein Cb-AMP2, capable of being iεolated from Cnicuε εeed. A pure protein Lc-AFP, capable of being iεolated from Lathyruε εeed. A pure protein Ct-AMPl, capable of being iεolated from Clitoria εeed. A pure protein Ct-AMP2, capable of being iεolated from Clitoria εeed. 19 A protein as claimed in any of claimε 1 to 18 which iε εynthetic. 20 A recombinant DNA εequence encoding a protein as claimed in any of claims 1-19. 21 A DNA εequence aε claimed in claim 20 which iε a cDNA. 22 A DNA εequence aε claimed in claim 20 which iε genomic DNA. 23 A DNA εequence aε claimed in claim 22 which iε isolated from a plant genome. 24 A DNA sequence as claimed in claim 23 which includes a promoter sequence. 25 A promoter sequence obtainable from a gene encoding a protein as claimed in any of claims 1-19. 26 A vector containing a DNA sequence as claimed in claim 20. 27 A biological syεtem including recombinant DNA aε claimed in claim 20 εuch that the encoded protein iε expreεεed. 28 A biological εyεtem aε claimed in claim 27 which iε a micro-organism. 29 A biological syεtem as claimed in claim 27 which iε a plant. 30 An antimicrobial protein produced by expression of recombinant DNA as claimed in claim 20. 31 A plant transformed with recombinant DNA as claimed .in claim 20. 32 A plant aε claimed in claim 26 in which the recombinant DNA encodeε at leaεt one of the following proteins: Rs-AFPl, Rs-AFP2, Rs-nsLTP, Bn-AFPl, Bn-AFP2, Br-AFPl, Br-AFP2, Sa-AFPl, Sa-AFP2, At-AFPl, Dm-AMPl, Dm-AMP2, Cb-AMPl, Cb-AMP2, Lc-AFP, Ct-AMPl, Ct-AMP2. 33 Seeds and progeny of a plant as claimed in claim 31 or claim 32. 34 A compoεition containing at least one of the proteinε as claimed in any of claims 1 to 19 or claim 30. 35 A process of combating fungi or bacteria which comprises exposure to a protein or composition as claimed in any of claims 1 to 19, claim 30 or claim 34. 36 A procesε of combating fungi or bacteria which comprises exposure to a protein encoded by pea gene pl39, by pea gene pl230, by cowpea gene pSASlO, or by potato gene pl322. 37 A procesε of combating fungi or bacteria which compriεeε expoεure to SIα2, γ-1-purothionin, or another α-amylaεe inhibitor protein. 38 An extraction proceεε for producing a protein aε claimed in any of claimε 1 to 19 or claim 30 from organic material containing them which compriεes submitting the organic material to 5 maceration and solvent extraction. _. 39 An extraction procesε aε claimed in claim 38 where the protein iε εubεequently purified by centrifugation, chromatography and dialyεiε. 10 40 An extraction proceεε aε claimed in either claim 38 or claim 39 where the organic matter compriεes seedε of Raphanuε, Brassica, Sinapis, Arabidopsis, Dahlia, Cnicuε, Lathyruε 15 or Clitoria. 41 An extraction proceεε aε claimed in either claim 38 or claim 39 where the organic matter compriεeε a biological εyεtem aε claimed in 20 claim 27. 42 A proceεs for producing a protein aε claimed in any of claimε 1 to 19 which compriseε chemical εyntheεiε of the protein. 25 43 A proceεs for producing a protein aε claimed in any of claimε 1 to 19 which comprises expresεion of a recombinant DNA εequence encoding the protein.

Inventors: BROEKAERT WILLEM FRANS, CAMMUE BRUNO PHILIPPE ANGELO, OSBORN RUPERT WILLIAM, REES SARAH BRONWEN, TERRAS FRANKY RAYMOND GERARD, VANDERLEYDEN JOZEF, BROEKAERT, WILLEM, FRANS, CAMMUE, BRUNO, PHILIPPE, ANGELO, OSBORN, RUPERT, WILLIAM, REES, SARAH, BRONWEN, TERRAS, FRANKY, RAYMOND, GERARD, VANDERLEYDEN, JOZEF
Applicants: ICI PLC, IMPERIAL CHEMICAL INDUSTRIES PLC
Application year: 1992
Main classification: C12N5
IPC codes: A01H1, A01H5, A01N65, C07K14, C12N1, C12N5, C12N15, C12P21