WO2006080032A2

WO2006080032A2 - Antifungal protein

Info

Publication number: WO2006080032A2
Application number: PCT/IN2006/000035
Authority: WO
Inventors: Venga Siva Reddy; Afif Hassari; Aparna Isalm
Original assignee: Internationl Centre For Genetic
Priority date: 2005-01-31
Filing date: 2006-01-31
Publication date: 2006-08-03
Also published as: WO2006080032A3

Abstract

For the first time, a small cystine rich protein (AFPl-Ca) secreted from the germinating seeds of a legume plant, chickpea (Cicer arietinum, L) with antifungal activity has been identified, purified and characterized the encoding gene cloned. The purified protein inhibited the growth of Pythium aphanidermatum mycelium at an IC50 value of 12 µg/ml. The SDS-PAGE analysis indicated the presence of disulfide bridges and a prior treatment of purified AFPl-Ca with a reducing agent, β- mercaptoethanol, abolished the antifungal activity completely. The complete gene coding for the AFPl-Ca was isolated, DNA sequenced and the deduced amino acid sequence revealed that the gene code for (74) amino acids; (27) amino acids of signal peptide and (47) amino acids of mature protein.

Description

ANTIFUNGAL PROTEIN Field of invention

The present invention relates to a cystene rich protein having antifungal activity. More particularly, the present invention relates to a cystene rich protein isolated from the germinating seeds of deer arietinum, L possessing antifungal activity. The invention relates to isolation and purification of said protein from the germinating seeds of Cicer arietinum, L and cloning of the encoding genes into a vector. The present invention also relates to transgenic plants incoporating such vectors which are resistant to fungal attack by Pythium aphanidematum and methods of making such transgenic plants. Background of the invention

In the absence of an adaptive immune system, plants have evolved constitutive or inducible antimicrobial defense mechanisms. The hypersensitive response is an inducible mechanism characterized by localized cell and tissue death at the site of infection and induction of intense metabolic alterations in the cells surrounding necrotic lesion (Hammond-Kosack and Jones 1996, Baker et al. 1997). These local responses often trigger non specific resistance throughout the plant, known as systemic acquired resistance (Ryals et al. 1996, Sticker et al. 1997). Once plant perceives the microorganisms, some dynamic defense mechanisms are triggered. Examples of these are: (1) cell wall reinforcement by the deposition and crosslinking of polysaccharides, proteins, glycoproteins and insoluble phenolics (Fritig and Legrand 1998); (2) stimulation of antimicrobial low molecular weight secondary metabolites known as phytoalexins (Van Etten et al. 1989; Maher et al. 1994); and (3) accumulation of broad range of defense related proteins (PR) and peptides (Fritig and Legrand 1998, Melchers and Stuiver 2000).

During the last decade, several classes of proteins and peptides with antifungal and antibacterial properties have been identified. These include chitinase (Schlumbaum et al. 1986; Broekaert et al. 1988), β-l,3-glucanages (Mauch et al. 1988), ribosome-inactivate proteins (Roberts et al. 1986; Leah et al. 1991), thionins (Bohlmann and Apel 1991), chitin-binding lectins (Broekaert et al. 1989; Van Parijs et al. 1991), permatins (Vigers et al. 1991) and non specific lipid transfer protein (Terras et al. 1992 a). Before germination, plant seeds have effective physical barriers (hard coat and low water content) against fungal and bacterial Invasion. During germination, the protection is relied mainly upon the secretion of antimicrobial compounds including proteins (Terras et al. 1992 b).

It is now well recognized that many organisms, including plants, utilize peptides as a component of their host defense strategies (Hancock and Lehrer. Trends In Biotechnology 16:82-88 (1998). Many of these peptides have a broad- spectrum activity and are known to be active against Gram-negative and Gram- positive bacteria, fungi and protozoa. Several claims have been made in respect pest resistance in crop plants due to over expression of antimicrobial peptides with antimicrobial activity hi transgenic plants. However, published reports describing transgenic tobacco plants expressing antimicrobial peptides reveal generally disappointing results (Florak et ah, Transgenic Res. 4:132-141 (1995)). In most cases, the antimicrobial peptides failed to accumulate to significant amounts within the plant cell as rapid degradation of the peptide was observed. For this reason, and also due to serious concerns about potentially phytotoxic effects exerted by the antimicrobial peptides when expressed in plants, plant scientists have not aggressively pursued this technology.

According to United States Patent 6,235,973 (Smith, et al. May 22, 2001) antimicrobial peptides can be classified into many categories based upon their structure (e.g., linear vs. cyclic), their size (20-45 amino acids) and their source (e.g., insect, amphibian, plant). However, despite their apparent diversity, numerous defense-related peptides are stated to have the common features of being highly basic and being capable of forming amphipathic structures. These unifying features suggest that most peptides appear to act by a direct lysis of the pathogenic cell membrane. Their basic structure facilitates their interaction with the cell membrane, and their amphipathic nature allow them to be incorporated into the membrane ultimately disrupting its structure.

Frog skin secretions of the African clawed frog, Xenopus laevis are reported to be a rich source of antibiotic peptides (Bevins and Zasloff Ann. Rev. Biochem. 59:395-414 (1990)). Known peptides include magainins, PGL.sup.a, xenopsin and caerulein. Magainins 1 and 2 are very closely related; each are 23 residues in length, contain no cysteine, and form an amphipathic .alpha, helix. PGL.sup.a is a small peptide processed from a larger precursor and is both cationic and amphipathic in nature (Andreu et al., Eur. J. Biochem. 149:531-535 (1985)). It has the somewhat unusual feature of containing a COOH-terminal amide group rather than the expected carboxyl group. According to US patent No. 5,254,537, magainin 2 and PGL.sup.a can interact synergistically with one another to exert enhanced levels of antibacterial activity Insects have also been demonstrated to possess a variety of defense-related peptides (Boman and Hultmark. Ann. Rev. Biochem. 41:103-126 (1987)). Cecropins from moths and flies are slightly larger than the frog-derived peptides (31-39 residues), are basic due to the presence of multiple arginine and lysine residues, and therefore interact strongly with the negatively charged lipid bilayers, Studies of these peptides have shown that they form an N-terminal .alpha.-helical region connected by a hinge region to a C-terminal .alpha.-helical domain.

Other antimicrobial peptides have been isolated from radish and feature a more complex three-dimensional structure which includes cysteine-stablized triple anti-parallel .beta, sheets with an .alpha. -helix. Terras et ah, (1995) reported very good levels of protection against infection by Alternaria in transgenic tobacco which overexpressed the radish AFP2 protein. However, a threshold level of AFP2 peptide (which was not easily obtained) in the transgenic plants was required to detect any significant level of disease resistance. Similarly, other plant species like mustard (βrassica juncea), cabbage (B. oleracea "capitata"), cauliflower (B. oleracea "botrytis") and knolkhol (B. oleracea "gongyloges") have shown inhibition halo zone surrounding the germinating seeds in a bioassay.

United States Patent 5,689,043, granted to Broekaert , et al., on November 18, 1997 teaches characterization of biocidal proteins isolated from seeds of members of the Brassicaceae, Compositae and Leguminosae families including Raphanus, Brassica, Sinapis, Arabidopsis, Dahlia, Cnicus, Lathyrus and Clitoria. The proteins are reported show a wide range of antifungal activity. All are reported to share a common amino acid sequence. This patent teaches isolation of DNA encoding the proteins and incorporation thereof into vectors.

According to the above-mentioned United States Patent in question, the new class of potent antimicrobial proteins isolated from seeds of the Brassicaceae, the Compositae, and the Leguminosae 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) is reported to include two protein factors, hereafter called Rs-AFPl (Raphanus sativus— Antifungal Protein 1) and R.S-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 are reported to 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 are reported to include four protein factors referred to as Dm-AMPl (Dahlia merckii— 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 are isolated from seed of the Dahlia genus. The Cb-AMP proteins may be isolated from seed of the Cnicus genus. All four proteins are reported to be closely related and are composed of 5 kDa sub units arranged as oligomeric structures. All four proteins are highly basic.

The antimicrobial proteins isolated from seeds of Lathyrus and Clitoria include three protein factors, referred to the above-mentioned US patent as 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.

According to said US Patent No. 5,689,043, N-terminal amino acid sequence determination revealed that the above proteins isolated from the Brassicaceae, Compositae and Leguminosae were closely related and could be classified as a single protein family. Between the different plant families, the protein sequences are approximately 50% identical.

The antimicrobial proteins are partially homologous to the predicted protein products of the Fusarium—induced genes pI39 and pI230 in pea (Pisum sativum~a member of the Leguminosae family) as described by Chiang and Hadwiger, 1991 (MoI Plant Microbe Interact, 4, 324-331). This homology is shared with the predicted protein product of the pSASIO gene from cowpea (Vigna unguiculata— another legume) as described by Ishibashi et al (Plant MoI Biol, 1990, 15, 59-64). The antimicrobial proteins are also partially homologous with the predicted protein product of gene pI322 in potato (Solanum tuberosum— a member of the Solanaceae family) as described by Stiekema et al, 1988 (Plant MoI Biol, 11,255-269). Nothing is known about the biological properties of the proteins encoded by genes pI39, pI230, pSASIO or pI322 as only the cDNA has been studied. However, the pI39, pI230 and pI322 genes are switched on after challenge to the plant by a disease or other stress. It has been proposed that the pSASIO gene encodes a protein involved in germination. However, the US Patent 5,689,043 does suggest that due to their sequence similarity with the antimicrobial' proteins of the invention, the proteins encoded by the pI39, pI230, pSASIO or pI322 genes may be useful as fungicides or as antibiotics.

However, to the applicant's knowledge, identification, purification and characterization of a small protein with an antifungal activity, particularly against Pythium aphanidermatum, from the germinating seeds of legumes have never been reported. Summary of the invention

The present invention is directed to the identification, purification and characterization of a small protein with an antifungal activity from the seeds of chickpea (Cicer arietinum, L). The N-terminal and internal amino acids sequences have been determined. Degenerate primers, designed on the basis of internal amino acids sequence, were used to isolate gene coding the full length AFPl-Ca. The alignment of deduced amino acids with known antifungal proteins indicated the presence of a distinct class of antifungal proteins in legumes that were not described earlier. In particular, the protein isolated from the germinating seeds of chickpea Cicer arietinum, L was particularly effective against growth of Pythium aphanidermatum.

Prior art is replete with evidence of germinating seeds of several plant varieties secreting antifungal substances. In the present invention, a preliminary screening was first carried out to identify the plant species that secrete antifungal compounds from the germinating seeds. While the plant species mustard (Brassica junced), cabbage (B. oleracea "capitata"), cauliflower (B. oleracea "botrytis"), knolkhol (B. oleracea "gongyloges"), radish (Raphanus sativus) have shown the inhibition halo zone surrounding the germinating seeds in a bioassay, fennel (Nigella damascenά), lady finger (Hibiscus esculentus), did not show any activity. Thereafter, several legumes were screen for antifungal activity in the germinating seeds. As expected, green gram (Vigna radiatq), Accacia catechu, methi (Trigonella foenum-graecum), pea (Pisum sativum), chickpea (Cicer arietinum), cowpea (Vigna unguuiculatά) did not reveal any antifungal activity at all. However, most surprisingly and unexpectedly, chickpea showed a remarkable antifungal activity. Tq eliminate possible release of antifungal compounds from endogenous microorganisms, seeds were surface sterilized thoroughly before their germination. In order to characterize the nature of the antifungal compound(s) released from chickpea germinating seeds, the seed imbibed solution was subjected to various biochemical and molecular analyses. The antifungal activity was not affected by heat treatment at 85⁰C for 15 min and by the presence of SDS up to 0.5%. On the other hand, the antifungal activity was abolished completely by a pretreatment of seed imbibed solution with proteases (pronase E, chymotrypsin, proteinase K), indicating that the antifungal activity could be due to the secretion of protein(s) from the germinating seeds.

The applicants have now for the first time, isolated, purified and characterized a small cystine rich protein (AFPl-Ca) secreted from the germinating seeds of a legume plant, chickpea (Cicer arietinum, L) with antifungal activity and cloned the encoding gene. The purified protein was found to successfully inhibit the growth of Pythium aphanidermatum mycelium at an IC₅₀ value of 12 μg/ml. The

SDS-PAGE analysis indicated the presence of disulfide bridges and a prior treatment of purified AFPl-Ca with a reducing agent, β-mercaptoethanol, abolished the antifungal activity completely. The complete gene coding for the AFPl-Ca was isolated, DNA sequenced and the deduced amino acid sequence revealed a gene code for 74 amino acids - 27 amino acid s of signal peptide and 47 amino acids of mature protein. The homology search suggested that the AFPl-Ca belong to a group of cysteine-rich antifungal proteins that play an important role in host defense mechanism. The AFPl-Ca showed high homology with the DR39 (70%) and D230

(71%) proteins from pea and lOKD-protein from cowpea (88%). However, the germinating seeds of neither cowpea or pea showed any antifungal activity nor the function of DR39, D230 and lOKD-protein's has been determined earlier. On the other hand, AFPl-Ca showed significantly low homology (13 to 29%) with several previously reported antifungal proteins. The phylogenetic tree generated on the basis of amino acid homology suggest that the AFPl-Ca along with DR39, D230 and lOKD-protein, all from the family Legumenacea, represent a distinct new class of defense proteins. The Sequence structures of the AFP-Ca gene of the present invention are as follows:

Genomic Sequence of AFP-Ca gene (Seq ID 1)

i ΆTGGACAAGA AATCΆCTAGC TGGCTTGTGC TTCCTCTTCC TCGTTCTCTT

TGTTGCAcgt

61 aagattaatt aactacatat atactgcabt tgtatatatt atgaaatata tatttaaata

121 atattatata tatgcaGAAG AAATAGCGGT GAGTGAAGCA GCGAGGTGTG AGAATTTGGC

181 TGATACATAC AGGGGACCAT GTTTCACAAC TGGTAGCTGT GATGATCACT GTAAGAACAA

241 AGAGCATTTA GTTAGCGGCA GATGCAGGGA TGACTTTCGT TGTTGGTGCA CCAAAAA.TTG 301 TTAA

cDNA Sequence of AFP-Ca gene (Seq.ID 2)

1 ATGGACAAGA AATCACTAGC TGGCTTGTGC TTCCTCTTCC TCGTTCTCTT

TGTTGCAGAA

61 GAAATAGCGG TGAGTGAAGC AGCGAGGTGT GAGAATTTGG CTGATACATA CAGGGGACCA 121 TGTTTCACAA CTGGTAGCTG TGATGATCΆC TGTAAGAACA AAGAGCATTT

AGTTAGCGGC

181 AGATGCAGGG ATGACTTTCG TTGTTGGTGC ACCAΆΆAATT GTTAA

Peptide sequence of AFP-Ca gene (Seq ID.3^")

IMDKKSLAGLC FLFLVLFVAE EIAVSEA27 ARCENLADTY RGPCFTTGSC DDHCKNKEHL VSGRCRDDFR CWCTKNC74

Accordingly, the present invention provides a new class of antimicrobial proteins capable of being isolated from germinating seeds of a legume plant, Cicer αretinum, L. m another aspect, the present invention provides a full length gene encoding the above-mentioned new class of antifungal proteins. In yet another aspect, the present invention relates to 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 antifungal protein according to the invention. The invention also comprises a process of combating fungi whereby they are exposed to the proteins according to the invention.

The antifungal proteins can be isolated and purified from the germinating seeds of Cicer arietinum, synthesised artificially from their known amino acid sequence, or produced within a suitable micro-organism by expression of recombinant DNA. Knowledge of their primary 'structure, enables the production of DNA constructs encoding the antimicrobial proteins. The DNA sequence may be predicted from the known amino acid sequence or the sequence may be isolated from plant-derived DNA libraries.

DNA encoding the antimicrobial proteins (which may be a cDNA clone, a genomic DNA clone or DNA manufactured using a standard nucleic acid synthesiser) can be cloned into a biological system, which allows expression of the proteins. This makes it possible to produce the proteins in a suitable micro-organism or cultured cell, extracted and isolated for use. Suitable micro-organisms include Escherichia 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 proteins are expressed within the plant. The proteins may also be expressed within a transgenic plant.

Plant cells according to the invention may be transformed with constructs of the invention according to a variety of known methods (Agrobacterium Ti plasmids, electroporation, 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 monocot and dicot plants may be obtained in this way, although the latter are usually more easy to regenerate.

Detailed description

One of the host defense mechanism in plants involve the release of low molecular weight cysteine-rich proteins from the germinating seeds to fight against the attack of soil born micro-organisms by creating a micro-environment zone. In order to identify the antifungal compounds released from the germinating seeds, the applicants have used a bioassay described earlier by Terras et al (1995) in which seeds were germinated directly on a potato dextrose agar medium that also supported the growth of a fungus Pythium aphanidermatum. Among the plant species that were screened by the applicants, mustard, cabbage, cauliflower, knolkhol and radish, showed antifungal activity and the results were in agreement with the previously published reports. Out of six legume plant seeds (methi. green gram, accacia, pea, chickpea and cowpea) screened by the applicants, only chickpea was positive for antifugal activity in the assay conditions of the present invention.

. Unlike the previously reported methods where seeds were ground to release the antifungal proteins for purification (Terras et al. 1992 b), in the present invention, the seeds were imbibed in water for the secretion of antifungal protein(s). This method of the invention not only avoided the contamination of other proteins, especially the seed storage proteins but also prevented the simultaneous release of . proteases from the broken cells that can potentially degrade the AFPl-Ca. As a consequence, it was possible for the first time to purify the AFPl-Ca to homogeneity by a simple two step procedure when compared to previously published methods for similar proteins.

The complete sequence of the mature AFPl-Ca revealed the presence of eight cysteine residues that could potentially form four disulfide bridges. Indeed, the migration of purified AFPl-Ca on SDS-PAGE after the incubation with a reducing agent, β-mercaptoethanol, confirmed the presence of disulfide bridges in the secreted protein that might play an essential role in the biological function. Also, the bioassay using reduced and unreduced protein confirmed the functional significance of these disulfide bridges as the disruption of these disulfide bonds inactivated the protein completely. The AFPl-Ca was active even after a treating at 85⁰C for 15 minutes and again, it is believed that disulfide bonds might be playing an important role in its stability. Proteins that function in extracellular environment are known to often contain disulfide bridges.

The blast search using the full sequence of AFPl-Ca revealed the presence of high homology with the previously reported protein sequences from cowpea and pea, both the species belong to the family, Leguminaceae. The eight cysteine residues present in the lOKD-protein were conserved in the AFPl-Ca suggesting a structural similarity between these two proteins. The gene coding for 10KD-protein expressed during the seed development and the mRNA was shown to be "stored" in the quiescent seeds. In the absence of an experimental evidence for its function, it was suggested that the synthesis of 10KD-protein is required for the seed germination and no role was suggested in fungal resistance.

The AFPl-Ca sequence showed 71% and 70% homology with the predicted amino acids sequence of mature D230 and DR39 proteins of pea, respectively. The pI39 and pI230 genes coding for DR39 and D230 proteins, respectively, were shown to get induced upon the infection of Fusarium solani, implicating a functional role for these proteins in host defense mechanism against fungal disease. However, no direct evidence was provided for their antifungal activity. Also, no information was available with regard to the expression of these proteins in the germinating seeds. The presence of these highly homologous proteins in cowpea and pea prompted the applicants to reconfirm the absence of antifungal activity that were observed earlier in their preliminary screening. Surprisingly, neither the cowpea nor the pea showed any antifungal activity in the bioassay conditions of the present invention. It should be mentioned here that other legumes (accacia, green gram, and methi) also did not show any antifungal activity in the bioassay of the present invention. It is possible that the PI 39 and PI230 genes are induced in response to fungal infection only in leaves and not in the seeds. In radish, while Rs-AFPl and Rs-AFP2 genes express in seeds, the highly homologous Rs-AFP3 and Rs-AFP4 genes expressed only in leaves (Terras et al. 1995). Also, it is possible that the expression of DR39 and D230 proteins in the germinating seeds may be too low to be detected using the bioassay that was adopted in the invention. The alignment of AFPl-Ca sequence showed very low homology (13-29%) to several other antifungal proteins. Although, the presence of eight cysteine residues in a highly conserved position suggest high structural similarity among these molecules, the phylogenic tree suggest that the AFPl-Ca along with DR39, D230 and 10KD-protein, all from the family Legumenaceae, represent a distinct class of defense proteins.

Thus, completely contrary to the prior art teachings, the present invention for the first time has identified, purified and characterized a small protein (AFPl-Ca) released from the germinating seeds of a legume plant, chickpea (Cicer arietinum, L) with antifungal activity. In addition, the gene coding for AFPl-Ca was cloned and sequenced. The alignment of deduced amino acids with previously reported antifungal proteins showed that AFPl-Ca along with lOKD-protein, D230 and DR39, all from the family Legumenacea, represent a distinct class of defense proteins.

However, the antifungal activity was proved so far only for AFPl-Ca. The gene coding for AFPl-Ca was cloned into a vector to transform tobacco and potato plants.

Stable plants were selected and the vector was incorporated therein by conventional techniques. Such transgenic plants overexpressing AFPl-Ca were subjected to In planta analysis. The transgenic plants containing the vector of the present invention showed a surprising and remarkable resistance to infection by Pythium aphanidermatum.

The present invention will now be described in with reference to the accompanying drawings wherein: Figure 1 shows bioassay for antifungal activity.

Figure 2 shows the electrophoretic analysis of proteins released from chickpea seeds.

Figure 3 discloses growth inhibition curve for Ca-AFP

Figure 4 shows the DNA sequence coding for CA-AFP along with the deduced amino acids. Figure 5 teaches the multi-sequence alignment of the N-terminal and the internal amino acids sequence from Ca-AFP with previously reported sequences.

Figure 6 depicts a dendrogram showing the phylogenetic relationship of various antifungal proteins reported from plants.

Figure 7 shows the sequence structures of the AFP-Ca gene; Figure 8 shows Northern Blot analysis showing the expression pattern of AFP-Ca in chickpea plant;

Figure 9 shows photographs showing appearance of Alternaria alternate mycelium growth four days after the inoculation with fungus spores on detached leaves from control and AFP-Ca expressing transgenic tobacco lines; Figure 10 shows effect on the Alternaria solani mycelium growth in the presence of leaf extracts from tobacco control and transgenic lines.

Figure 11 shows the sequence of the promoter controlling the expression of AFP-Ca gene. Important cis-acting elements as predicted by PlantCare (1-6) and Place (7-19) databases are shown.

Referring to Fig. 1, bioassay was conducted using various surface sterilized germinating seeds. Surface sterilised seeds were germinated on PDA medium inoculated with Pythium aphanidermatum to identify the release of antifungal compounds. Germinating chickpea seeds, are shown in Fig. IA while Fig. 1 B shows a petri dish inoculated only with fungus. In Fig 1C, the petri dish was inoculated with the fungus and grown in the presence of water (1), 100 μg of crude protein secreted from the chickpea after 24 hours inhibition in water at 4⁰C (2) and 20 μg of purified

Ca-AFP after SP sepharose and Phenyl sepharose chromatography (3). Fig. ID, IE and IF respectively show germinating cowpea seeds germinating pea seeds and germinating acacia seeds. It is clear from Fig. 1 that only germinating chick pea seeds exhibit any antifungal activity.

Figure 2 discloses the results of electrophoretic analysis of proteins released from chickpea seeds. In Fig. 2, 'A' discloses elution profile of protein fractions that showed antifungal activity after purification on SP sepharose column. Protein fractions were separated on 17% SDS-PAGE and stained with coomassie blue. 'M' represents molecular marker, 'C represents Flow through, lane 1 - 6 represent fraction numbers 13 - 18 that showed antifungal activity, 'F' shows secreted proteins loaded on the column. Fig 2B discloses silver stained gel showing elution profile of protein purified on Phenyl sepharose column. 'M' represents molecular marker, Lane 1 -3 represent fraction numbers 16 -18.

Fig 2C discloses Coomassie blue stained gel showing Ca-AFP treated with

(R) and without (UR) β-mercaptoethanol and separated on SDS-PAGE gel to identify the presence of disulfide bridges. The difference in migration between the treated and untreated protein can be clearly seen.

Figure 3 discloses the growth inhibition curve for Ca-AFP. The P. aphanidermatum was grown in the presence of various concentration of purified Ca-

AFP to deteSrmine the IC₅₀ value. The IC₅₀ is defined as the amount of Ca-AFP required to inhibit 50% growth of the fungus. The calculated ICsovalue for CA-AFP was found to be 12μg/ml.

Figure 4 discloses the DNA sequence coding for CA-AFP of the present invention along with the deduced amino acids. The arrow indicates the cleavage site. The sequenced N-terminal and internal amino acids of the mature peptide are under lined. These nucleotide and amino acid sequences are indicated below:

Nucleotide Sequence of the AFPl-Ca gene of the present invention (Sea. ID 4)

ATGGAGAAAAAATCGATTGCTGGCTTGTGC TTC CTCTTC

CTC GTTCTCTTTGTTGCA CAA GAA ATAGCG GTGAGTGAA GCA GCGAGGTGTGAGAAl¹TTGGCTGATACA¹IACAGGGGA CCATGTTTCACAACTGGTAGC TGTGATGAT CAC TGTAAG

AACAAAGAG CATTTAGTTAGCGGCAGATGCAGGGATGAC

TTTCGTTGTTGGTGCACCAAAAATTGTTAA

Amino acid Sequence of the AFPl-Ca gene of the present invention (Seq. ID 5)

M E K K S I A G L C F L F

L V L F V A Q E I A V S E

A A R C E N L A D T Y R G

P C F T T G S C . D D H C K

N K E H L V S G R C R D D

F R C W C T K N C

Figure 5 discloses the multi-sequence alignment of the N-terminal and the internal amino acids sequence from Ca-AFP with previously reported sequences. Sequences were aligned with the MacVector Clusta IW program. Amino acid residues conserved in all sequences are boxed together. AFPl-Ca (present invention); 10 KD-VGF from Vigna unguiculata (Ace. No. P18646, Ishibashiet al. 1990); D230- PEA (Q01783) and DR39-PEA (Q01784) from Pisum sativum (Chiang and Hadwiger 1991); P322-STF from Solarium tuberosum (P 20346, Stiekema et al. 1988)); AMPl-Ct from Clitoria ternatea (S66219, Osborn et α/.1995); AFPl-RSF (CAA65983, Terras et al. 1996, direct submission) and AFP2-RSF (P30230) from Raphanus sativa (Terras et al. 1995); AMPl-Ah (S66218) from Aesculus hippocastanum (Osborn et al. 1995), AMPl-Fb (B58445) and AMP2-Fb (A58445) from Vicia faba (Zhang and Lewis, 1997), THGl-Te (P20158) and THG2-Te (P20159) from Triticum aestivum (Mendez et al. 1990); SIAlphal-SOR (S69145), SIAlpha2-S0R (P21924) and SIAlpha3-S0R (P21925) from Sorghum bicolor (Bloch and Richardson 1991); AMPl-MJF (P25403), AMP2-MJF (P25404) from Mirabilis jalapa (Cammue et al. 1992); AMP-Mc (AAC19399) from Mesembryanthemum crystallinum (Direct submission, Michalowski and Bohnert 1998).

Figure 6 shows a dendrogram showing the phylogenetic relationship of various antifungal proteins reported from plants. Based on the homology, the antifungal proteins can be divided into four (A-D) major groups. The Ca-AFP, 10KX⁾-VGF, D230-PEA and DR39-PEA, all from legume plants, with considerable sequence homology among themselves (70 - 88%) separated from the rest of the antifungal proteins that shared very low homology (19-30%).

Among the four proteins from legumes, the antifungal activity was demonstrated so far only for AFPl-Ca in the present invention. Figure 7 shows the sequence structures of the AFP-Ca gene. The genomic clone of the AFP-Ca was obtained following polymerase chain reaction approach and using the AFP-Ca genomic forward primer ATGGACAAGA AATCACTAGC (Seq ED 6) and AFP-Ca genomic reverse primer TTAACAATTTTTGGTGC (Seq ID T). The PCR product was cloned into pGEMT-E vector (Promega (USA) and sequenced. The sequence of the genomic clone of AFP-Ca and its organization is shown in the figure. Sequence analysis revealed the presence of a small intron (italics). The AFP-Ca gene representing the cDNA clone and the translated amino acids sequences are presented in fig. B and C, respectively. From the translated sequence, it can be found that the AFP- Ca has 27 amino acids long signal peptide at its N-terminal end. It should be noted here that the peptide sequences obtained from the mature purified protein matched with the predicted amino acids from the cDNA clone, indicating that the genomic clone indeed represent the antifungal peptide that we have isolated and characterized in this study.

Figure 8 shows Northern blot analysis showing the expression pattern of AFP- Ca in chickpea plant. C: control plant (without any treatment), D: drought inducing (Kept without watering for 72 hours). S: salt NaCl inducing (treated with 100 mM NaCl for 72 hours), SA: salicylic acid inducing (treated with 2 mM salicylic acid for 72 hours), MJ: Methyl Jasmonate (treated with 2 mM methyl jasmonate for 72 hours).

Figure 9 shows Appearance of Alternaria solani mycelium growth four days after the inoculation with fungus spores on detached leaves from control and AFP-Ca expressing transgenic tobacco lines #111, #114, #107 (T2 generation). Expression of AFP-Ca vary in different lines depending on the site of integration. Tobacco is not considered as a normal host for Alternaria solani. However, artificial inoculation can 5 show disease symptoms. WI: leaf is wounded by sterile blade before inoculation. I: inoculation on the leaf without wounding. NI: un-inoculated leaf.

Figure 10 shows effect on the Alternaria solani mycelium growth in the presence of leaf extracts from tobacco control and transgenic lines. In this figure LEC: leaf extract from Control plant; LE-107, LE-111 and LE-114: leaf extract from lό transgenic lines (T2 generation) 107, 11 and 114 respectively.

Figure 11 shows the sequence of the promoter (Seq ID 7) controlling the expression of AFP-Ca gene. Important cis-acting elements as predicted by PlantCare (1- 6) and Place (7-19) databases are shown.

Transcription start with a score of 0.98 is shown in larger font: prediction was

15 done by the software 1999 NNPP (Promoter Prediction by Neural Network) version 2.2 at the web site: "http://www.fruitfly.org/seq_tools/promoter.html" Cis-acting element from PlantCare database:

1-CAAT-box : common cis-acting element in promoter and enhancer regions; 2-ERE: ethylene-responsive element (ATTTCAAA) ; 3 -HSE: cis-acting element involved in

20 heat stress responsiveness ; 4-TATA-box: core promoter element around -30 of transcription start ; 5-GCN4-motif: cis-regulatory element involved in endosperm expression ; 6-Prolamin-box: cis-acting regulatory element associated with GCN4 Cis-acting element from PLACE database: 7-GTGA motif: cis-regulatory elements within the promoter of the tobacco late pollen

25 gene glO ; 8-DOFCOREZM: endosperm specific Dof protein that binds to prolamin box ; 9-NTBBF IARROLB: Required for tissue-specific expression and auxin induction ; 10-OSE2ROOTNODULE: motifs of organ-specific elements (OSE) characteristic of the promoters activated in infected cells of root nodules ; 11 -MYCATERDl: necessary for expression of erdl (early responsive to dehydration) in dehydrated Arabidopsis; 12- 0 MYCCONSENSUSAT: MYC recognition site(CANNTG) found in the promoters of the dehydration-responsive gene rd22 and many other genes in Arabidopsis; Binding site of ATMYC2 (previously known as rd22BPl) ; 13-SEBFCONSSTPRlOA: Binding site (YTGTCWC) of the potato silencing element binding factor (SEBF) gene found in promoter of pathogenesis-related gene (PR-IOa) ; 14.Ebox/ABRE motif: (TGHAAARK) Conserved in many napA storage-protein gene promoters; may be important for high activity of the napA promoter ; 15-CANBNNAPA: Core of "(CA)n element" in storage protein genes in Brasica napus; embryo- and endosperm-specific transcription of napin(storage protein) gene, napA; seed specificity; activator andrepressor ; 16-GT1GMSCAM4:(GAAAAA) "GT-I motif found in the promoter of soybean (Glycine max) CaM isoform, SCaM-4; Plays a role in pathogen- and salt- induced SCaM-4 gene expression ; 17.I.BOX:(GATAAG) "I-box"; Conserved sequence upstream of light-regulated genes; Sequence found in Lhe promoter region of rbcS of tomato and Arabidopsis ; 18-WBOXATNPR1: (TTGAC) found in promoter of Arabidopsis thaliana (A.t.) NPRl gene. They were recognized specifically by salicylic acid (SA)-induced WRKY DNA binding proteins; 19-Alfinl: (GTGTGT) Alfinl may play a role in the regulated expression of PRP2 in alfalfa roots and contribute to salt tolerance in these plants. Its target is PRP2 gene (proline-rich cell wall protein), calli overexpressing Alfinl were more resistant to growth inhibition by 171 mM NaCl.

In order to identify the AFP-Ca gene expression, plants were treated with various agents for 24 hours and RNA was isolated from roots and leaf tissue. The RNA was separated in denaturing agarose gel and blotted on to nylon membrane. Blot was probed with the AFP-Ca coding region. It can be seen that expression is more in root tissue in comparison to leaf tissue. Also it can be noted that the expression is more when treated with salt and under drought conditions. This data suggests that the expression of the AFP-Ca gene is induced in response to various signal molecules that are known to be involved in plant defense mechanisms.

Specific embodiments of the present invention will now be illustrated with the following non-limiting Examples, which where applicable will also refer to the accompanying drawings mentioned above. Example 1 Purification and characterization of antifungal protein (AFPl-Ca)

Seeds of Cicer arietinum L, were imbibed in water for the secretion of antifungal proteins. The solution obtained from the incubation of seeds for 24 hours at 4°C was fractionated by 75% ammonium sulfate. This was followed by a cation exchange (SP sepharose) and hydrophobic interaction chromatography column (phenyl sepharose). The fractions that showed antifungal activity were analyzed on SDS-PAGE and stained with silver nitrate. The results are shown in Fig. 2. It can be seen from figure 2A that the SP-sepharose fractions having the peak activity contained a prominent low molecular weight (~5 kDa) protein. This low molecular weight protein got purified to homogeneity after phenyl sepharose purification step as shown in Fig. 2B. The highly purified AFPl-Ca migrated slowly on SDS-PAGE when compared to the reduced form of protein as can be seen from Fig. 2C indicating the presence of one or more disulfide bridges. The IC50 value for the purified peptide was found to be 12 μg/ml in the bioassay conditions of the present invention as will be explained hereinafter. The reduced form of AFPl-Ca was completely inactive even at 200 μg/ml concentration. The requirement of monovelant (NaCl and KCl) and divelent (CaC12 and MgC12) ion concentrations was found to be 5 mM and 5OmM, respectively. The AFPl-Ca retained its activity even after heat treatment of 80°C for 15 min. Example 2 Protein sequence of the purified AFPl-Ca and isolation of the encoding gene

In order to isolate the encoding gene, the N-terminal and an internal amino acid of the highly purified AFPl-Ca was determined. The N-terminal and the internal sequence consisted of ARCENLAATYRGPCF (Seq ID 8) and EHLVSGR (Seq ID 9) amino acids, respectively. Two degenerative primers designed on the basis of these amino acids sequences were used to PCR amplify the DNA representing the internal fragment of the gene and sequenced. This sequence information was used to obtain the full length gene coding for the AFPl-Ca protein along with a putative signal peptide. The complete DNA sequence of the amplified fragment along with the deduced amino acids is shown in figure 4. The sequence analysis suggested that the open reading frame (ORF) encode 74 amino acids that include a 27 amino acid of signal peptide and a 47 amino acids of mature peptide with antifungal activity. The predicted molecular mass of the mature peptide is 5416.65 kDa. The deduced amino acids sequence confirmed the earlier micro-sequencing data of the purified peptide of the present invention. The mature AFPl-Ca contained eight cysteines that could form four disulfide bridges, again confirming the earlier observation on the migration pattern of reduced and unreduced peptide in SDS-PAGE gels as shown in Fig. 2. Figure 5 shows the multiple alignment of AFPl-Ca to different previously reported antifungal proteins. The AFPl-Ca showed high homology with the DR39 (70%) and D230 (71%) proteins from pea and lOKD-protein from cowpea (88%). On the other

, hand, AFPl-Ca showed very low homology (13 to 29%) with several previously reported antifungal proteins as can be seen from Fig. 5. The PCR amplified product using the genomic DNA as a template confirmed lack of any introns in the gene coding for AFPl-Ca.

Despite the high homology of AFPl-Ca to lOKD-protein from cowpea and DR39 and D230 proteins from pea, the antifungal activity of these three proteins could not be observed neither in bioassay adopted in the present invention from the germinating seeds υf oυwpea or pea (Fig. 1), nor in the earlier studies (Ishibashi et ah 1990; Chiang and Hadwiger 1991). A notable feature of the amino acids sequence homology was the presence of eight cysteine residues at highly conserved position. Also two conserved glycine residue at positions 39 and 60 were present. In addition, one glutamic acid was conserved at position 60 in all antifungal peptides except in DR39, AMPl-MJF, AMP2-MJF and AMP-Mc. The phylogenic tree generated on the basis of amino acids homology divided all antifungal proteins into four separate groups as shown in Fig, 6. The AFPl-Ca along with DR39, D230 and lOKD-protein, all from the family Legumenaceae, represented a distinct class of defense proteins. EXAMPLE 3 Bioassay to determine the antifungal activity of the proteins of the present invention

Mature and dry seeds of mustard (Brassica campestris), cabbage (B. oleracea "capitata"), cauliflower (B. oleracea "botrytis"), knolkhol (B. oleracea "gongyloges"), radish (Raphanus sativus), fennel (Nigella damascena), lady's finger (Hibiscus esculentus), methi (Trigonella foenum-graecum), green gram (Vigna radiata), Accacia catechu, pea (Pisum sativum), chickpea (Cicer arietinum), cowpea (Vigna unguuiculata) were tested for their antifungal activity during the seed germination. After the initial screening using a bioassay, chickpea seeds were selected for the purification of the antifungal protein. The fungus Pythium aphanidermatum was used to test the antifungal activity. The antifungal assay conducted was essentially as described previously

(Terras et al. 1995). Seeds were surface sterilized by 4% sodium hypochlorite solution for 20 min followed by five washes of 5 min each in sterile water. The fungus P. aphanidermatum was grown on a half strength potato dextrose agar (PDA) medium. With a sterile cark borer, a mycellial plug of 4 mm in diameter was removed from a plate colony and placed mycelium-side down in the center of a 9 cm Petri dish containing half strength PDA. Sterilized seeds or protein extracts were incubated for a total period of 3-5 days at 26°C±2°C and examined for zones of growth inhibition.

Purification of antifungal peptide from C. arietinum

Two kilograms of chickpea seeds were washed extensively with running tap water, sterilized with commercial bleach and finally washed with sterile distilled water 5 times. These seeds were imbibed in 2 liters υf distilled water for 24 hours at

» 4°C. The solution was clarified by centrifugation at 600Og for 20 min. Powdered solid ammonium sulfate was added to the supernatant until 75% relative saturation was reached. The precipitate formed overnight at 4⁰C under gentle stirring was collected by centrifugation at 800Og and dissolved in 100 ml distilled water. The solution was heated at 80°C for 15min, clarified and extensively dialyzed against water using dialyses tubing with a molecular mass cut-off of 3000 Da. The dialyzed solution was filter sterilized (0.45 μm), adjusted to 50 mM NaCl and 25 mM MES (pH 4.7), and loaded on a cation-exchange column (SP-seahorse) equilibrated with 25 mM MES (pH 4.7) and 50 mM NaCl. Bound proteins were eluted with a linear gradient of 50 mM to 1.0 M NaCl (pH 4.7). Fractions that showed antifungal activity in the bioassay were pooled and further purified by hydrophobic interaction chromatography column (phenyl-sepharose). (The dialysed proteins were loaded on a phenyl-sepharose (10 ml bed volume) previously equilibrated with 2.0 M ammonium sulfate in 0.1 M potassium phosphate buffer (pH 7.4). The bound proteins were eluted with a linear gradient of 2.0 M to 0 M NH₄Ac (pH 7.4) and the protein fractions were tested for their antifungal activity.

SDS-PAGE analysis and amino acids sequencing of AFPl-Ca

After each purification step, the protein fractions that showed antifungal activity was analyzed on 17% SDS-PAGE (Laemmli 1970) and silver stained. The sample buffer contained 62.5 mM Tris-HCl (pH 6.8), 10% (v/v) glycerol, 1% (w/v) SDS, 5% (v/v) β-mercaptoethanol and 1% dithiothreitol (DTT). The DTT and β- mercaptoethanol were omitted for the analyses of unreduced form of protein. For micro-sequencing, the purified protein was separated on SDS-PAGE under reducing conditions and blotted onto a PVDF membrane (Millipore) for 1 hour at 50V using the transfer buffer containing 10 mM 3-(cyclohexylamins)-l-propanesulfonic acid (CAPS) with 10% (v/v) methanol. Protein was detected on the blot by Amidoblack staining. The unique peptide band exihibiting the antifungal activity was excised from the blot and the N-terminal and an internal sequences were determined by a sequencer (ABI Model 492A) at the University of Massachusetts Medical school, U.S.A. Cloning of the cDNA coding for AFPl-Ca

On the basis of the N-terminal and internal amino acids sequence of AFPl- Ca, two degenerative primers AFPNlI (5¹ TGYGARAAYYTIGCIGAIACITA 3') (Seq K) 10) and AFPCl (5¹ CKNCCNSWNACNARRTGYCT 3') (Seq ID 11) were designed to amplify an internal fragment of the gene using seed specific cDNA as a template in a polymerase chain reaction (PCR). Total RNA was extracted from chickpea seed as described by Cheng and Seemann (1998). The first strand was synthesized from total RNA using the SupertScript™ preamplifϊcation system of Gibco BRL. The PCR amplified DNA fragment was cloned into pGEM-T vector (Promega) and sequenced. The alignment of the partial gene sequence to previous known antifungal proteins showed high homology to 10KD-protein of cowpea (Ishibashi et al. 1990), D230 and DR39 proteins of pea (Chiang and Hadwiger 1991). Based on the homology, a third degenerative primer AFPPEAC (5'ATTCGAGCT CTTARCARTTKTYNGTRCACCARCAS¹) (Seq ID 12) was designed and used in combination with AFPNlI to amplify the gene coding for the mature AFPl-Ca. The amplified fragment was cloned into pGEM-T vector and sequenced. Finally, the full length gene that codes for the mature AFPl-Ca along with its signal peptide was isolated using a fourth degenerative primer AFPN3 (5¹CTAGGARAARAARTCIATHGCS') (Seq ID 13) and AFPC2 primer (5⁽ TTAACAATTTTTGTGCACCAAC 3') (Seq ID 14). The full length gene was cloned into pGEM-T vector and both the strands of the DNA sequenced completely.

References Baker, B., Zambryski, P., Staskawicz, B., and Dinesh-Kumar, S. P. 1997. Signaling in plant-microbe interactions. Science 276:726-733.

Bloch, C. Jr., and Richardson, M. 1991. A new family of small (5 kDa) protein inhibitors of insect alpha-amylases from seeds or sorghum (Sorghum bicolar (L) Moench) have sequence homologies with wheat gamma-purothionins FEBS Lett.

279:101-104.

Bohlmann, H., and Apel, K. 1991. Thionins. Annu. Rev. Plant Physiol. Plant MoI.

Biol. 42:227-240. Broekaert, W. F., Van Parijis, J., Allen, A. K., and Pneumans, W. J. 1988.

Comparison of some molecular, enzymatic and antifungal properties of chitinases from thorn-apple, tobacco and wheat. Physiol. MoI. Plant Pathol. 33:319-331.

Broekaert, W. F., Van Parijs, J., Ceyns, F., Soos, H., and Peuman, W. J. 1989. A chitin-binding lectin from stinging nettle rhizomes with antifungal properties. Science 245:1100-1102. '

Cammue, B. P. A., De Boelle M. F. C, Terras, F. R. G., Jo Van Damme, P. P., Rees,

S. B., Vanderleyden, J., and Broekaert, W. F. 1992. Isolation and characterization of a novel class of plant antimicrobial peptides from Mirabilis jalapa L. seeds. J. Biol.

Chem. 267:2228-2233 Cheng SH., Seemann JR. 1998. Extraction and purification of RNA from plant tissue enriched in polysaccharides. In Methods in Molecular Biology . VoI 86: 27-32

Chiang, C. C, and Hadwiger L. A. 1991. The Fusarium solani-induced expression of pea gene family encoding high cyteine content proteins of a pea gene family encoding high cysteine content proteins. MoI. Plant. Microb. Interact.4:324-331. Fritig, B., Heitz, T. H., and Legrand, M. 1998. Antimicrobial proteins in induced plant defense. Curr. Opinion in immunology 10:16-22.

Hammond-kosack, K. E., and Jones, J. D. G. 1996. Resistance gene-dependent response. Plant Cell 8:1773-1791.

Ishibashi, N., Yamauchi, D., and Minamikawa, T. 1990. Stored mRNA in cotyledons of Vigna unguiculata seeds: nucleotide sequuencing of cloned cDNA for a stored mRNA and induction of its synthesis by precocious germination. Plant MoI. Biol.

15:59-64.

Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685. Leah, R., Tommerup, H., Svendsen, T., and Mundy, J. 1991. Biochemical and molecular characterization of three barley seed proteins with antifungal properties. J. Biol. Chem. 266:1564-1573.

Maher, E. A., Bate, N. J., Ni, W., Elkin,Y., Dixon, R. A., and Lamb, C. J. 1994. Increased disease susceptibility of transgenic tobacco plants with suppressed levels of preformed phenylpropanoid products. Pro. Natl. Acad. Sci. USA 250:1002-1004.

Mauch, F., Mauch-Mani, B., and Boiler, T. 1988. Antifungal hydrolases in pea tissue. π Inhibition of fungal growth by combination of chitinase and _-l,3-ghicanase. Plant Physiol. 88:447-457.

Melchers, L. S., and Stuiver, M. 2000. Novel genes for disease-resistance breeding.

Curr. Opin. Plant Biol. 3:147-152.

Mendez, E., Moreno, A., Collila, F., Pelaez, F., Limas, G. G., Mendez, R., Soriano,

F., Salinas, M., and DeHaro, C. 1990. Primary structure and inhibition of protein synthesis in eukaryotic cell-free system of a novel thionin, gamma-hordothioninj from barley endosperm. Eur. J. Biochem. 194 :533-539.

Michalowski, CB., Bohnert HJ., Antimicrobial peptide 1 from common ice plant

Mesembryanthemum crystallium. Unpublished. Accession AAC19399

Osborn RW., De Samlanx GW., Thevissen K., Goderis L, Torrekens S., Van Leuven F., Attenborough S., Rees SB., Broekaert WF., 1995. Isolation and characterisation of plant defensins from seeds of Asteraceae, Fabaceae, Hippocastanaceae and saxifragaceae. FEBS. Lett. ,368, 2: 257-262

Roberts, W. K., and Selitrennikof, C. P. 1986. Isolation and partial characterization of two antifungal proteins from barley. Biochim. Biophys. Acta 880:161-170. Ryals, J. A., Neuenschwander, U. H., Willits, M. G., Molina, A., Steiner, H. Y., and

Hant, M. D. 1996. Systemic acquired resistance. Plant [Cell 8:1809-1819.

Schlumbaum, A., Mauch, F., Vogeli, V., and Boiler, T. 1986. Plant chitinases are potent inhibitors of fungal growth. Nature 324:365-367.

Schulz, G. E., and Schirmer, R. H. 1979. Pages 51-55 in: Principles of protein ^' struucture. C. R. Cator, ed. Springer- Verlag, New York.

Sticher, L., Manch-Mani, B., and Metraux, J. P. 1997. Systemic acquired resistance.

Annu. Rev. Phytopathol. 35:235-270.

Stiekema, W.J., Heidekamp, F., Dirkse, W.G., Van Beckum, J., de Haan, P., ten

Bosch, C, and Louwerse, J. D. 1988. Molecular cloning and analysis of four potato tubers mRNAs. Plant MoI. Biol. 11 :255-269

Terras, F. R. G., Eggermont, K., Raikhel, N. V., Osborn, R. W., Kester, A., Rees, S.

B., Torrekens, S., Van Leuven, F., Vanderleyden, J., Cammue, B. P. A., and

Broekaert, W. F. 1995. Small cysteine-rich antifungal proteins from radish: their role in host defense. Plant Cell 7:573-588.

Terras, F. R. G., Goderis, I. J., Van Leuven, F., Vanderleyden, J., Cammue, B. P. A., and Broekaert, W. F. 1992a. In vitro antifungal activity of a radish (Raphanus sativus L.) seed protein homologous to nonspecific lipid transfer proteins. Plant Physiol. 100:1055-1058.

Terras, F. R. G., Schoofs, H. M. E., De Bolle, M. F. G., Van Leuven, F., Rees, S. B., Vanderleyden, J., Cammue, B. P. A., and Broekaert, W.F. 1992b. Analysis of two novel classes of plant antifungal proteins from radish (Raphanus sativus L.) seeds. J. Biol. Chem. 267:15301-15309. Van Etten, H. D., Matthews, D. E., Matthews, P. S. 1989. Phytoalexin detoxification importance for pathogenicity and practical implications. Annu. Rev. phytopathol. 27:143-164.

Van Parijs, J., Broekaert, W. F., Goldstein, I. J., and Peuman, W. J. 1991. Antifungal protein from rubber tree (Hevea brasiliensis) latex. Planta 183:258-264. Vigers, AJ ., Robbert, W. K., and Selitrennikoff, C. P. 1991. A new family of plant antifungal proteins. MoI. Plant. Microb. Interact. 4:315-323.

Zhong Y., Lewis K., 1997. Fabatins: new antimicrobial plant peptides. FEMS Microbiol. Lett., 149, 1: 59-64

Claims

Claims:

1. An antifungal protein having the amino acid sequence as shown in Seq ID 4.

2. An antifungal protein as claimed in claim 1 wherein the N-terminal thereof has the following amino acids ARCENLAATYRGPCF as shown in Seq ID 8.

3. An antifungal protein as claimed in claim 2 wherein the internal sequence thereof has the following amino acids EHLVSGR as shown in Seq ID 9.

4. An antifungal protein as claimed in any preceding claim wherein said protein is isolated from the germinating seeds of Cicer arietinum, L.

5. An antifungal protein as claimed in any preceding claim wherein said protein is effective against the fungus Pythium aphanidermatum.

6. A recombinant DNA sequence encoding an antimicrobial protein having an amino acid sequence as shown in SEQ ID NO: 5 and a nucleotide sequence shown in Seq ID 4.

7. A DNA sequence as claimed in claim 6 which is a cDNA.

8. A DNA sequence as claimed in claim 6 which is genomic DNA.

9. A DNA sequence as claimed in claim 3 which is isolated from a plant genome.

10. A DNA sequence as claimed in claim 6 wherein said amino acid sequence has gene code for 74 amino acids.

11. A DNA sequence as claimed in claim 10, wherein said amino acids comprise 27 amino acids of signal peptide and 47 amino acids of mature protein.

12. A vector containing a DNA sequence as claimed in claim 6.

13. A cultured cell comprising recombinant DNA as claimed in claim 6 such that the encoded protein is expressed.

14. A cultured cell as claimed in claim 11 which is a micro-organism.

15. A cultured cell system as claimed in claim 6 which is a plant cell.

16. A plant transformed with recombinant DNA as claimed in claim 1.

17. A method of isolating an antifungal protein from the seeds of Cicer arietinum, L. which comprises incubating said seeds in water for a period sufficient to initiate germination, removing the solution and treating it with ammonium sulphate to form a precipitate, separating the precipitate and dissolving it in water, subjecting the solution so formed to clarification followed by cation-exchange, and separating the bound proteins

18. A method as claimed in claim 17 wherein said seeds are incubated in water for 24 hrs.

19. A method as claimed in claim 18, wherein said incubation is carried out in distilled water at about 4⁰C.

20. A method as claimed in any one of claims 17 to 19 wherein, prior to treatment with centrifugation, the solution is clarified.

21. A method as claimed in claim 20 wherein said clarification is carried out at by centrifugation at 600Og for 20 minutes.

22. A method as claimed in claim any preceding claim 17 to 21 wherein the precipitate is collected by centrifugation at 8000g.and dissolved in distilled water.

23. A method as claimed in claim any preceding claim 17 to 22 wherein said solution is heated to 80⁰C prior to clarification.

24. A method as claimed in claim 23 wherein said clarified solution is dialysed and filter sterilized.

25. A method as claimed in any preceding claim 17 to 24 wherein said cation exchange is carried out in a SP-sepharose cation-exchange column equilibrated with 25 mM MES and 5O mM NaCl.

26. A method as claimed in claim 25, wherein after cation-exchange, the bound proteins are eluted with a linear gradient of 50 mM to 1.0 M Nacl at a pH of 4.7 to obtain purified proteins.

27. A method as claimed in claim 26 wherein said purified proteins are further purified by hydrophobic interaction in a phenyl-sepharose chromatography column.

28. AFPl-Ca proteins as claimed in any one of claims 1 to 5 having antifungal activity.

29. AFPl-Ca proteins as claimed in claim 28 having activity against Pythium aphanidermatum.

30. AFPl-Ca proteins as claimed in claim 28 or 29 capable of being isolated from germinating seeds of Cicer arietinum, L.

31. A method of combating the fungus Pythium apahnidermatum which comprises exposing said fungus or plants infected by said fungus to the proteins as claimed in any one of claims 1 to 5.