WO2001009174A2

WO2001009174A2 - Plant defensin variants

Info

Publication number: WO2001009174A2
Application number: PCT/GB2000/002941
Authority: WO
Inventors: Geertruida Afina Posthuma; Wilhelmus Martinus Maria Schaaper; Lolke Sijtsma; Aart Van Amerongen; Franky Fant; Frans Alois Mekania Borremans
Original assignee: Syngenta Limited
Priority date: 1999-08-02
Filing date: 2000-07-31
Publication date: 2001-02-08
Also published as: EP1206557A2; AU6579100A; GB9918155D0; WO2001009174A3; JP2003507009A; CA2378432A1

Abstract

An antimicrobial protein or peptide derived from a plant defensin, or a derivative thereof, characterised in that said protein or peptide comprises one or more of the following replacement amino acid residues selected from the group consisting of: (i) a tryptophan residue at position 32; (ii) a valine, leucine, isoleucine, tryptophan, phenylalanine, lysine, arginine, tyrosine, methionine, cysteine or histidine residue at position 34; (iii) an isoleucine, tryptophan, lysine, arginine, valine, leucine, phenylalanine or histidine residue at position 35; (iv) a tryptophan residue at position 36; (v) a tryptophan, glycine, threonine, tyrosine, glutamine, lysine, arginine, phenylalanine or histidine residue at position 37; (vi) a leucine, isoleucine, tryptophan, phenylalanine, valine or cysteine residue at position 38; (vii) a leucine, isoleucine, tryptophan, phenylalanine, methionine, lysine, arginine, tyrosine or histidine residue at position 39; (viii) a tryptophan residue at position 40; (ix) an isoleucine, a tryptophan, phenylalanine, serine, threonine, tyrosine, glutamine, asparagine, lysine, arginine, histidine at position 41; and/or (x) a valine, leucine, isoleucine, tryptophan, phenylalanine, tyrosine, asparagine, lysine, arginine, serine or threonine residue at position 42; where said amino acid residues are not found naturally at said positions in the antimicrobial protein or peptide, with the proviso that the antimicrobial proteins do not comprise only a replacement arginine residue at position 37, 39 or 42. These proteins or peptides are useful in agriculture and pharmaceuticals.

Description

PROTEINS AND PEPTIDES This invention relates to antimicrobial proteins and peptides, processes for their manufacture and use, and DNA sequences encoding them.

In this context, antimicrobial proteins and peptides are defined as proteins and peptides possessing antifungal and/or antibacterial activity and/or antiviral activity. Activity includes a range of antagonistic effects such as partial inhibition or death.

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

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

As used herein the term "plant defensin" is used to denote those proteins having antimicrobial activity, especially antifungal activity or antifungal and antibacterial activity and also having the following characteristic structural features: a cysteine residue at positions 4, 15, 21, 25, 36, 45, 47 and 51; disulphide bridge formation between the cysteines at positions 4 and 51, 15 and 36, 21 and 45 and 25 and 47; an aromatic amino acid residue 4 amino acids upstream from the cysteine at position 15, a glycine residue 2 amino acids upstream from the cysteine at position 15, a glutamic acid residue 7 amino acids upstream from the cysteine at position 36, and a glycine residue 2 amino acids upstream of the cysteine at position 36 wherein the positions of the cysteine residues are defined relative to the amino acid sequence of Rs-AFPl as shown in SEQ ID NO 1, and homologues, active variants and derivatives thereof.

QKLCERPSGTWSGVCGNNNACKNQCINLEKARHGSCNYVFPAHKCICYFPC (SEQ ID NO 1)

In some plant defensins the segments between positions 1 and 3, 5 and 14, 26 and 35 and 37 and 44 may vary in length by one to three amino acids but this does not affect the overall characteristic cysteine motif described above. This characteristic structural feature of the plant defensins can be summarised as follows:

...C a.G.C C...C...E....G.C C.C...C (SEQ ID NO 2) where a is an aromatic amino acid (F,W,Y), C represents cysteine, E represents glutamic acid and G is glycine and unspecified amino acids or groups of amino acids are represented by stops. The expression "homologues" as used herein refers to any peptide which has some amino acids in common with the given sequence. Suitably at least 60% of the amino acids will be similar, more suitably at least 70%, preferably at least 80%, more preferably at least 90% and most preferably at least 95%. 96%, 97% or 98% of amino acids will be similar to the corresponding amino acid in the given sequence. As used herein the term "similar" is used to denote sequences which when aligned have similar (identical or conservatively replaced) amino acids in like positions or regions, where identical or conservatively replaced amino acids are those which do not alter the activity or function of the protein as compared to the starting protein. For example, two amino acid sequences with at least 85% similarity to each other have at least 85%o similar (identical or conservatively replaced) amino acid residues in a like position when aligned optimally allowing for up to 3 gaps, with the proviso that in respect of the gaps a total of not more than 15 amino acid residues is affected. The degree of similarity may be determined using methods well known in the art (see, for example, Wilbur, W.J. and Lipman, D.J. "Rapid Similarity Searches of Nucleic Acid and Protein Data Banks." Proceedings of the National Academy of Sciences USA 80, 726-730 (1983) and Myers E.and Miller W. "Optimal Alignments in Linear Space". Comput. Appl. Biosci. 4:11-17(1988)). One programme which may be used in determining the degree of similarity is the MegAlign Lipman-Pearson one pair method (using default parameters) which can be obtained from DNAstar Inc. 1228, Selfpark Street, Madison, Wisconsin, 53715, USA as part of the Lasergene system.

Amino acids which differ from the basic sequence may be conservatively or non- conservatively substituted. A conservative substitution is to be understood to mean that the amino acid is replaced with an amino acid with broadly similar chemical properties. In particular conservative substitutions may be made between amino acids with the following groups:

(a) Alanine, Serine, Glycine and Threonine;

(b) Glutamic acid and Aspartic acid;

(c) Arginine and Lysine; (d) Asparagine and Glutamine;

(e) Isoleucine, Leucine, Valine and Methionine;

(f) Phenylalanine, Tyrosine and Tryptophan.

In general, more conservative than non-conservative substitutions will be possible without destroying the antimicrobial properties of the compounds. Suitable homologues may be determined by testing antimicrobial properties of the peptide using routine methods, for example as illustrated hereinafter.

The term "variant" as used herein includes experimentally generated variants or members of a family of related naturally-occurring peptides as may be identified by molecular genetic techniques. Such techniques are described for example in US Patent No. 5,605,793, US Patent No. 5,811,238 and US Patent No 5,830,721, the content of which is incorporated herein by reference. In essence this technique involves expression of the parental gene in a microbial expression system such as Escherichia coli. The particular system selected must be validated and calibrated to ensure that biologically active peptides are expressed, which may be readily achieved using an in vivo bioassay. The gene, or preferably a collection of related genes from different species, may be subject to mutagenic polymerase chain reaction (PCR) as is known in the art. Fragmentation of the products and subsequent repair using PCR leads to a series of chimeric genes reconstructed from parental variants. These chimeras are then expressed in the microbial system which can be screened in the usual way to determine active mutants, which may then be isolated and sequenced. Reiteration of this molecular evolution DNA shuffling cycle may lead to progressive enhancement of the desired gene properties. The advantage of a technique of this nature is that it allows a wide range of different mutations, including multi-mutation block exchanges, to be produced and screened.

Other variants may be identified or defined using bioinformatics systems. An example of such a system is the FASTA method of W.R. Pearson and D.J. Lipman PNAS (1988) 85:2444-2488. This method provides a rapid and easy method for comparing protein sequences and detecting levels of similarity and is a standard tool, used by molecular biologists. Such similar sequences may be obtained from natural sources, through molecular evolution or by synthetic methods and comparisons made using this method to arrive at "opt scores" which are indicative of the level of similarity between the proteins.

Particular variants of the invention will comprise antimicrobial proteins with an amino acid sequence with a FASTA opt score (as defined in accordance with FASTA version 3.0t82 November 1, 1997) against any one of the sequences of the antimicrobial proteins of the invention described herein as follows. Variants of the invention will comprise antimicrobial proteins with an amino acid sequence with a FASTA opt score (as defined in accordance with FASTA version 3.0t82 November 1, 1997) of greater than or equal to 300 against Rs-AFPl or 2.

The term "derivative" relates to antimicrobial proteins which have been modified for example by using known chemical or biological methods. The expression "protein or peptide derived from a plant defensin" used herein includes derivatives. A particular derivative is one in which cysteine residues are replaced by α-aminobutyric acid.

Further examples of antifungal plant defensins are described in International Patent Application Publication Number WO95/18229 published 6 July 1995 which is specifically incorporated herein by reference. These examples include Hs-AFPl, an antifungal protein capable of isolation from seeds of Heuchera species and Ah-AMPl, an antimicrobial protein capable of isolation from seeds of Aesculus hippocastanum. The proteins specifically inhibit a range of fungi and may be used as fungicides for agricultural or pharmaceutical or preservative purposes.

The primary structures of the two antifungal protein isoforms capable of isolation from radish seeds, Rs-AFPl (SEQ ID NO 1) and Rs-AFP2 (SEQ ID NO 3), only differ at two positions: the glutamic acid residue (E) at position 5 in Rs-AFPl is a glutamine residue (Q) in Rs-AFP2, and the asparagine residue (N) at position 27 in Rs-AFPl is substituted by an arginine residue (R) in Rs-AFP2. As a result, Rs-AFP2 has a higher net positive charge (+2) at physiological pH. Although both Rs-AFPs are 94% identical at the amino acid sequence level, Rs-AFP2 is two- to thirty-fold more active than Rs-AFPl on various fungi and shows an increased salt-tolerence.

The proteins Rs-AFP3 and Rs-AFP4 are found in radish leaves following localized fungal infection. The induced leaf proteins are homologous to Rs-AFPl and Rs-AFP2 and exert similar antifungal activity in vitro.

It has previously been shown (WO97/21814 and WO97/21815) that peptides derived from the regions defined herein of the Rs-AFP plant defensins exhibit antimicrobial activity. WO97/21814 discloses that some specific mutations to the wild- type sequence may be made in the peptides whilst retaining activity which may in fact be enhanced. Such peptides may be easier to synthesise than the full length plant defensin while retaining antifungal or antifungal and antibacterial activity. DNA sequences encoding the peptides may also be more suitable for transformation into biological hosts. In a first aspect the invention provides an antimicrobial protein or peptide derived from a plant defensin characterised in that said protein or peptide comprises one or more of the following replacement amino acid residues selected from the group consisting of: (i) a tryptophan residue at position 32; (ii) a valine, leucine, isoleucine, tryptophan, phenylalanine, lysine, arginine, tyrosine, methionine, cysteine or histidine residue at position 34; (iii) an isoleucine, tryptophan, lysine, arginine, valine, leucine, phenylalanine or histidine residue at position 35; (iv) a tryptophan residue at position 36; (v) a tryptophan, glycine, threonine, tyrosine, glutamine, lysine, arginine, phenylalanine or histidine residue at position 37; (vi) a leucine, isoleucine, tryptophan, phenylalanine, valine or cysteine residue at position 38; (vii) a leucine, isoleucine, tryptophan, phenylalanine, methionine, lysine, arginine, tyrosine or histidine residue at position 39; (viii) a tryptophan residue at position 40;

(ix) an isoleucine, a tryptophan, phenylalanine, serine, threonine, tyrosine, glutamine, asparagine, lysine, arginine, histidine at position 41; and/or (x) a valine, leucine, isoleucine, tryptophan, phenylalanine, tyrosine, asparagine, lysine, arginine, serine or threonine residue at position 42: where said amino acid residues are not found naturally at said positions in the antimicrobial protein or peptide, with the proviso that the antimicrobial proteins do not comprise only a replacement arginine residue at position 37, 39 or 42.

Suitably, where the antimicrobial proteins comprise any basic amino acid residue at position 39, they include at least one further replacement residue. Suitably, the same criteria applies to the peptides derived from the proteins.

In other embodiments, where the antimicrobial proteins or peptides comprise any of the above mentioned replacements at positions 37, 39 or 42, they suitably include at least one further replacement.

Suitably also, the protein or peptide of the invention has enhanced antimicrobial activity as compared to the plant defensin from which they are derived.

The amino acid positions mentioned above correspond to the amino acid positions found in the full length amino acid sequence of Rs-AFP2 as shown in SEQ ID NO 3 or to the equivalent positions in defensins from different sources when optimally aligned with the Rs-AFP2 sequence.

QKLCQRPSGTWSGVCGNNNACKNQCIRLEKARHGSCNYVFPAHKCICYFPC (SEQ ID NO 3)

The equivalent position will be readily apparent to the man skilled in the art when the sequences are optimally aligned to maximise sequence similarity and colinearity relative to the characteristic pattern of the cysteine residues in the defensin sequence. The equivalent position will be readily apparent to the man skilled in the art based on the positioning of amino acid residues relative to the characteristic pattern of cysteine residues in the defensin sequence.

The replacement amino acid residue at position 34 is preferably selected from the group consisting of a valine, leucine, isoleucine, tryptophan, phenylalanine, methionine, cysteine, lysine, histidine or tyrosine residue, more preferably from the group consisting of a valine, leucine, isoleucine, tryptophan, phenylalanine, methionine, lysine or histidine residue and is most preferably selected from the group consisting of a valine, leucine or isoleucine residue.

The replacement residue at position 35 is preferably selected from the group consisting of a valine, leucine, isoleucine, trytophan, phenylalanine, lysine, arginine, or a histidine residue, and most preferably selected from the group consisting of a leucine, isoleucine, arginine, histidine or a phenylalanine residue.

The replacement residue at position 37 is preferably selected from the group consisting of a tryptophan, glycine, threonine, tyrosine, glutamine, lysine, arginine, histidine, or phenylalanine residue and more preferably from the group consisting of a tryptophan, tyrosine, lysine, arginine, or histidine residue and is most preferably selected from the group consisting of a tryptophan. arginine or histidine residue.

The replacement residues at position 38 are preferably selected from the group consisting of a leucine, isoleucine, tryptophan, phenylalanine, cysteine or a valine residue; more preferably from the group consisting of a leucine, tryptophan or phenylalanine residue.

The replacement residue at position 39 is preferably selected from the group consisting of a leucine, isoleucine, tryptophan, phenylalanine, methionine, lysine, arginine, histidine, or tyrosine residue, more preferably from the group consisting of a tryptophan, lysine, arginine or a histidine residue and is most preferably selected from the group consisting of an arginine or a histidine residue.

The replacement residue at position 41 is preferably selected from the group consisting of an isoleucine, tryptophan, phenylalanine, serine, threonine, tyrosine, glutamine, asparagine, lysine, arginine or histidine residue, and is more preferably selected from the group consisting of an isoleucine, tryptophan, phenylalanine, tyrosine, asparagine, lysine, arginine or histidine residue and is most preferably selected from the group consisting of a tryptophan, phenylalanine, lysine, arginine or histidine residue. The replacement residue at position 42 is preferably selected from the group consisting of a valine, leucine, isoleucine, tryptophan, phenylalanine, serine, threonine, tyrosine, asparagine, lysine or arginine residue and is more preferably selected from the group consisting of a tryptophan, phenylalanine, tyrosine, lysine or an arginine residue and is most preferably selected from the group consisting of a lysine residue or an arginine residue.

Preferably, the protein or peptide of the invention includes at least one replacement in group (iii), (iv), (v), (vi), (vii), (viii), (ix) or (x) above, and most preferably at least one of the replacements listed in group (iii), (iv),(v), (vii), (ix) or (x) above. In this and all further aspects of the invention the antimicrobial protein or peptide derived therefrom is preferably a modified plant defensin selected from the group Rs-AFPl, Rs-AFP2, Rs-AFP3, Rs-AFP4. Br-AFPl, Br-AFP2, Bn-AFPl, Bn-AFP2, Sa-AFPl, Sa-AFP2 and At- AFP 1 and Hs-AFPl , Ah-AMPl and Dm-AMPl which are fully described in Published International Patent Applications Nos. WO 93/05153 and WO 95/18229 the teachings of which are incorporated herein by reference, Aly-AFP and Alf-AFP which are fully described in Published International Patent Applications Nos. WO 97/37024 and WO 98/26083 the teachings of which are incorporated herein by reference. The antimicrobial protein or peptide derived therefrom is more preferably a plant defensin selected from the group Rs-AFPl or Rs-AFP2, and is most preferably derived from Rs-AFP2.

In a particularly preferred embodiment of the invention the plant defensin is Rs- AFPl or especially Rs-AFP2.

In a further preferred embodiment of the invention, where the proteins and peptides contain one or more cysteines, these may be replaced by an alpha-aminobutyric acid group.

It is particularly preferred that the peptides and proteins of the invention are derived from plant defensins having substantially similar activity to Rs-AFP2, and which show at least 40%, 50%, 60%, 70%, 80%, or 85% sequence similarity, more preferably at least 90%) sequence similarity and most preferably at least 95% sequence similarity to Rs- AFP2.

Antimicrobial proteins which show sequence similarity to the Rs-AFP2 protein include the proteins Rs-AFPl, Rs-AFP3, Rs-AFP4, Br-AFPl, Br-AFP2, Bn-AFPl, Bn-AFP2, Sa-AFPl, Sa-AFP2 and At-AFPl and Hs-AFP2, Ah-AMPl and Dm-AMPl. Further sequence information on the above-mentioned proteins is provided in Published International Patent Applications Nos. WO 93/05153 and WO 95/18229 the teachings of which are incorporated herein by reference. For the purpose of the present invention a conservative replacement is defined as one which does not alter the activity/function of the protein when compared with the unmodified protein.

The antimicrobial peptides of the invention are preferably at least six amino acid residues long, more preferably greater than 10 amino acid residues long preferably 12 amino acids long, most preferably 19 amino acids or longer, especially 20 amino acids in length. Short peptides will contain at least one modified residue as described above. Where this includes modified residues 37, 39 or 42, the peptide suitably contains more than one such modification.

In a particularly preferred embodiment of the first aspect the peptide is derived from the beta-2 strand/turn/beta-3 strand region of a plant defensin, as defined by the three-dimentional structure characterization of these proteins (Bruix et al. 1995 Biochemistry 32, 715-724; Fant et al. 1998, J. Mol. Biol. 279, 257-270. Fant et al., (1999), Proteins: Structure, Function, and Genetics 37 (3), 388-403).

The beta-2 strand/turn/beta-3 strand region of a plant defensin may be determined by analysis of the primary amino acid sequence information and generally is predicted to be located between the fourth and the eighth cysteine residue. For example in Rs-AFPl and Rs-AFP2 this occurs between positions 21 to 51 of the sequence, and more precisely at positions 30 to 51 of the sequence. The antimicrobial peptides of the invention are preferably derived from position 21 to 51 of the Rs-AFP2 sequence, preferably from position 30 to 51 of the sequence, more preferably from position 30 to 49 or position 32 to 43 of the Rs-AFP2 sequence.

The number of replacement residues within a protein or peptide according to the invention is preferably no greater than 10 i.e 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 replacement residues and is more preferably from 1 to 6 residues i.e. 1, 2, 3, 4, 5, or 6 replacement residues.

We have found that the antimicrobial proteins and peptides according to the invention show activity and are particularly useful against a broad spectrum of fungi and have particularly advantageous antifungal activity in the presence of salts as evidenced by their activity in SMF+ medium. The proteins and peptides of the invention may also be useful in combating bacterial infections. This is described in more detail in the accompanying examples and figures. An antimicrobial protein or peptide according to the invention may be manufactured from its known amino acid sequence by chemical synthesis using standard peptide chemistry, or produced within a suitable organism (for example, a micro-organism or plant) by expression of recombinant DNA. The antimicrobial peptide is useful as a fungicide and may be used for agricultural or pharmaceutical or other applications. The antimicrobial peptides and proteins may be used in combination with one or more of the antimicrobial proteins or with one or more other antimicrobial peptides of the present invention.

Knowledge of its primary structure enables manufacture of the antimicrobial protein, peptide, or parts thereof, by chemical synthesis using standard peptide chemistry. It also enables production of DNA constructs encoding the antimicrobial peptide or protein.

The invention further provides a DNA sequence encoding an antimicrobial peptide or protein according to the invention. The DNA sequence may be predicted from the known amino acid sequence and DNA encoding the peptide or protein may be manufactured using a standard nucleic acid synthesiser.

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

An antimicrobial peptide or protein according to the invention is useful for combating fungal and bacterial diseases in plants. The invention further provides a process of combating microbial infection whereby microbes are exposed to an antimicrobial peptide or protein according to the invention. The antimicrobial peptide or protein may be used in the form of a composition, for example in combination with a suitable carrier or diluent. For example, for agricultural use, compositions of the invention may be in the form of either a dilute composition which is ready for immediate use, or a concentrated compositions which require dilution before use, usually with water. Liquid compositions may contain other conventional components such as surface-active agents, dispersants etc.

Solid compositions may be in the form of granules, or dusting powders wherein the active ingredient is mixed with a finely divided solid diluent, e.g. kaolin, bentonite. kieselguhr, dolomite, calcium carbonate, talc, powdered magnesia. Fuller's earth and gypsum. They may also be in the form of dispersible powders or grains, comprising a wetting agent to facilitate the dispersion of the powder or grains in liquid. Solid compositions in the form of a powder may be applied as foliar dusts.

In a preferred embodiment the invention provides a method of combating fungal infection by exposing said fungi to an antimicrobial peptide according to the invention. In a further preferred embodiment the invention provides a method of combating bacterial infection by exposing said bacteria to an antimicrobial peptide according to the invention.

For pharmaceutical applications, the antimicrobial peptide or protein (including any product derived from it) may be used as a fungicide to treat mammalian infections (for example, to combat yeasts such as Candida). Suitably, the peptide or protein is in the form of a composition comprising a carrier or diluent, which will be a pharmaceutically acceptable carrier or diluent as is conventional in the art.

Pharmaceutical compositions of the invention may be in a form suitable for oral use (for example as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), for topical use (for example as creams, ointments, gels, or aqueous or oily solutions or suspensions), for administration by inhalation (for example as a finely divided powder or a liquid aerosol), for administration by insufflation (for example as a finely divided powder) or for parenteral administration (for example as a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular or intramuscular dosing or as a suppository for rectal dosing). For further information on Formulation the reader is referred to Chapter 25.2 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990.

An antimicrobial peptide or protein (including any product derived from it ) according to the invention may also be used as a preservative (for example, as a food or cosmetic additive). Again, suitable compositions may comprise acceptable carriers or diluents.

For agricultural applications, the antimicrobial peptide or protein may be used to improve the disease-resistance or disease-tolerance of crops either during the life of the plant or for post-harvest crop protection. Pathogens exposed to the peptide or proteins are inhibited. The antimicrobial peptide or protein may eradicate a pathogen already established on the plant or may protect the plant from future pathogen attack. The eradicant effect of the peptide or protein is particularly advantageous. Exposure of a plant pathogen to an antimicrobial peptide or protein may be achieved in various ways, for example:

(a) The isolated peptide or protein may be applied to plant parts or to the soil or other growth medium surrounding the roots of the plants or to the seed of the plant before it is sown using standard agricultural techniques (such as spraying). The peptide or protein may have been extracted from plant tissue or chemically synthesised or extracted from micro-organisms genetically modified to express the peptide or protein. The peptide or protein may be applied to plants or to the plant growth medium in the form of a composition comprising the peptide or protein in admixture with a solid or liquid diluent and optionally various adjuvants such as surface-active agents. Solid compositions may be in the form of dispersible powders, granules, or grains.

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

(c) An endophyte genetically modified to express the antimicrobial peptide or protein may be introduced into the plant tissue (for example, via a seed treatment process). An endophyte is defined as a micro-organism having the ability to enter into non-pathogenic endosymbiotic relationships with a plant host. A method of endophyte-enhanced protection of plants has been described in a series of patent applications by Crop Genetics International Corporation (for example, International Application Publication Number WO90/13224, European Patent Publication Number EP-125468-B1, International Application Publication Number WO91/10363,

International Application Publication Number WO87/03303). The endophyte may be genetically modified to produce agricultural chemicals. International Patent Application Publication Number WO94/ 16076 (ZENECA Limited) describes the use of endophytes which have been genetically modified to express a plant-derived antimicrobial peptide or protein.

(d) DNA encoding an antimicrobial peptide or protein may be introduced into the plant genome so that the peptide or protein is expressed within the plant body (the DNA may be cDNA, genomic DNA or DNA manufactured using a standard nucleic acid synthesiser). Exposure of a plant pathogen to an antimicrobial composition comprising an antimicrobial peptide or protein according to the invention plus an antimicrobial protein may be achieved by delivering the protein as well as the peptide or protein as described above. For example, both one of the above-mentioned peptides or proteins according to the invention plus Rs-AFP2 or Rs-AFPl could be simultaneously applied to plant parts or simultaneously expressed within the plant body. We have discovered a synergistic effect when peptides derived from Rs-AFP2, such as derivatives having cysteine residues replaced with α-aminobutyric acid, and/or in particular those having replacement residues according to the invention are mixed with the full length natural protein and this forms a further aspect of the invention. This is described more fully in the examples herein.

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

The invention further provides a plant having improved resistance to a microbial pathogen and containing recombinant DNA which expresses an antimicrobial peptide or protein according to the invention. Such a plant may be used as a parent in standard plant breeding crosses to develop hybrids and lines having improved microbial resistance.

In a preferred embodiment the invention further provides a plant having improved resistance to a fungal pathogen and containing recombinant DNA which expresses an antimicrobial peptide or protein according to the invention.

In a further preferred embodiment the invention further provides a plant having improved resistance to a bacterial pathogen and containing recombinant DNA which expresses an antimicrobial peptide or protein according to the invention.

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

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

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

Examples of genetically modified plants which may be produced include field crops, cereals, fruit and vegetables such as: canola, sunflower, tobacco, sugarbeet, cotton, soya, maize, wheat, barley, rice, sorghum, tomatoes, mangoes, peaches, apples, pears, strawberries, bananas, melons, potatoes, carrot, lettuce, cabbage, onion. The invention will now be described by way of example only, with reference to the following drawings wherein:

Figure 1 : shows a histogram analysis of the antifungal activity measured against the fungus Fusarium culmorum of peptides from Rs-AFP with replacement residues at positions 32 to 37 in buffer Vi PDB. The antifungal activity is expressed as % relative to the activity of the reference peptide *RHGSCNYVFPAH#.

Figure 2 : shows a histogram analysis of peptides from Rs-AFP with replacement residues at positions 38 to 43 in buffer Vi PDB. The antifungal activity is expressed as % relative to the activity of the reference peptide *RHGSCNYVFPAH#. Figure 3 : shows a histogram analysis of peptides from Rs-AFP with replacement residues at positions 32 to 37 in buffer SMF+ pH5. The antifungal activity is expressed as % relative to the activity of the reference peptide *RHGSCNYVFPAH#. Figure 4 : shows a histogram analysis of peptides from Rs-AFP with replacement residues at positions 38 to 43 in buffer SMF+ pH5. The antifungal activity is expressed as % relative to the activity of the reference peptide *RHGSCNYVFPAH#. Figure 5 : shows a histogram analysis of peptides from Rs-AFP with replacement residues at positions 38 to 43 in buffer SMF+ pH7. The antifungal activity is expressed as % relative to the activity of the reference peptide *RHGSCNYVFPAH#. Figure 6 : shows a histogram analysis of peptides from Rs-AFP with replacement residues at positions 32 to 47 in buffer SMF+ pH7. The antifungal activity is expressed as % relative to the activity of the reference peptide *RHGSCNYNFPAH#. EXAMPLES Materials. Ν-methylpyrrolidone (ΝMP) and piperidine were peptide synthesis grade and obtained from Perkin Elmer/ ABI (Warrington, UK). Dimethylformamide (DMF), dicyclohexylcarbodiimide (DCC), Ν-hydroxybenzotriazole (HOBt), diisopropylethylamine (DIEA), trifluoroacetic acid (TFA), thioanisole (TA), ethanedithiol (EDT), dimethylsulfoxide (DMSO), and dimethylamino pyridine (DMAP) were pro- analysis grade and were obtained from Merck (Darmstad, Germany). Diethylether was purified over a column of activated basic aluminumoxide and DIEA was distilled twice over ninhydrin and potassium hydroxide before use. Amino acid derivatives and resins were obtained from Saxon Biochemicals (Hannover, Germany).

For analytical HPLC we used two Waters pumps model 510, a Waters gradient controller model 680, a Waters WISP 712 autoinjector, and a Waters 991 photodiode array detector. Products were analysed in a linear gradient from water with 0.1% TFA to 60%) acetonitrile/water with 0.1% TFA in 60 min on a Waters Delta Pak C18-100A (3.9x150 mm, 5 μm) column at lml/min. Preparative HPLC was carried out using a Waters Prep 4000 liquid chromatograph, equipped with a Waters RCM module with two PrepPak cartridges plus guard cartridge (40x210mm or 25x210mm) filled with delta-Pak C18-100A (15 μm) material. Peptides were detected at 230 nm using a Waters 486 spectrophotometer with a preparative cell. Amino acid analysis was performed using a Waters Pico-Tag system, after hydrolysis in a Pico-Tag workstation using 6Ν HC 1 with 1%) phenol at 150 °C for 1 hour, and derivatization with phenylisothiocyanate. PEPSCAN-split. Radiation grafted polyethylene pins were functionalized with hydroxyl groups. Boc-β-alanine was coupled using DCC and DMAP as catalyst, the Boc group was removed with TFA and after careful washing Fmoc-2,4-dimethoxy-4' (carboxymethyloxy)-benzhydrylamine (Rink Linker, Bachem, Laufelfingen, Switzerland) was coupled using the DOC/HOBt method. Next, 240 dodecapeptides from AFP2 were synthesized simultaneously using standard Fmoc-chemistry and overnight couplings with DCC/HOBt as coupling method. After coupling of the last amino acid the Fmoc group was removed with 30% piperidine/DMF and the peptides were acetylated with aceticanhydride. After washing and drying, the peptides were deprotected and cleaved from the pins with TFA/phenol/TA/water/EDT 10/0.75/0. 5/0.5/0.25. The cleavage mixtures were evaporated, extracted twice with diethylether, and lyophilized twice from water. This procedure yields peptides up to about lmg with a C-terminal amide. Multi-synthesis (MPS). A Hamilton Microlab 2200 was programmed to deliver washing solvents and reagents to a rack with 40 individual 4ml columns with filter, containing 30 μmol of resin for peptide synthesis. The columns were drained after each step by vacuum. The next coupling cycle was based on Fmoc chemistry using double coupling steps: 1. NMP wash (lml)

2. 30% (v/v) piperidine/NMP (3 min, 0.5ml)

3. 30% (v/v) piperidine/NMP (17 min, 0.5ml)

4. NMP wash (5x1 ml)

5. double coupling (2x30 min) 6. NMP wash (2x1 ml)

Coupling step: Fmoc-amino acid in NMP (0.4M, 0.25ml), 0.22ml of 0,45M HBTU/HOBt in DMF, and 0.2ml of 2M DIEA in NMP were transferred to the reaction vessel and allowed to react for 30 min. Then the reaction mixture was drained and the coupling procedure was repeated once.

After the coupling of the last amino acid, the Fmoc group was removed with 30%) piperidine/NMP, the peptides were washed, acetylated in 30 min using NMP/acetic anhydride/DIEA 10/1/0.1, washed again, dried, and deprotected and cleaved in 2 hr with 1.5ml of TFA/phenol/TA/water/EDTA 10/0.75/0.5/0.5/0.25. The cleavage mixture was filtered, the resin was washed with 0.5ml TFA, and the peptide was precipitated by adding 13ml hexane/diethylether 1/1. After centrifugation the precipitate was extracted again with hexane/diethylether. The precipitate was dried and lyophilized from water/acetonitrile 1/1.

Bioassavs. The data (IC50) shown in the tables were all concentrations in μg/ml that give 50% growth inhibition of mycelium grown from spore suspensions of Fusarium culmorum after 72 hours at room temperature in a medium of half-strength potato dextrose broth (1/2 PDB, from Difco) at pH 5.8 or medium SMF + pH5 or SMF + pH7. Bioassays were performed as described in Terras et al. 1992. J. Biol. Chem. 267: 15301- 15309). Medium SMF+pH5 consists of medium SMF (Cammue et al. 1992, J. Biol. Chem 267: 2228-2233) with addition of ImM CaCl₂, 50mM KC1 and lOmM MES (final pH adjusted to 5.0). Medium SMF+pH7 is identical to SMF+pH5 except that lOmM MES is replaced by lOmM. Tris and that the pH is adjusted to 7.0. Example 1

It has previously been demonstrated that synthetic peptides corresponding to parts of the sequence of plant defensins show antifungal activity (De Samblanx et al. Peptide Research, 1996, 9: 262-268 and Published International Patent Applications WO97/21814 and WO97/21815).

We have chosen to synthesise a 12-mer peptide corresponding to the RsAFP2 sequence from position 32 to 43. This peptide with sequence RHGSCNYVFPAH (SEQ ID NO 4) was found to inhibit 50% of the growth of F. culmorum in three different media, '/_ PDB, SMF+pH5 and SMF+pH at concentration 57μg/ml, 400μg/ml and 400μg/ml, respectively. In order to optimise the antifungal potency of this peptide, amino acid replacement net tests were performed whereby every residue at each position was replaced by any of 19 other amino acids. The antifungal activity of these substitution variants was determined against F. culmorum in the media Vi PDB, SMF+pH5 and SMF+pH7. The activity data are presented in figures 1 to 6 where thay are expressed relative to the antifungal activity of the reference peptide RHGSCNYVFPAH. (SEQ ID NO 4). As can be seen in figures 1 to 6, the activity of some of the substitution variants was increased up to 20-fold compared to that of the reference peptide.

Example 2 We have found that improved antifungal activity can be observed in longer (>19 residues) peptide derivatives of RsAFP2 by replacing the cysteines by alpha- isoaminobutyric acid. We have now synthesised a 20-mer peptide corresponding to the RsAFP2 sequence from position 30 to 49 whereby each of the three cysteines was substituted by alpha-aminobutyric acid. This peptide is called MBN01. MBN01 inhibits growth of F. culmorum by 50% in media Vi PDB, SMF+pH5and

SMF+pH7 at concentration of 5.8, 21.8 and 70 μg/ml, respectively (table 1). Variants of MBN01 were synthesised in which either one, two, three, four or six residues were substituted. The substitutions were chosen based on the data obtained for the 12-mer series described above. The antifungal activity of the 20-mer variants against F. culmorum determined in three different media (1/2 PDB, SMF+pH5 and SMF+pH7) is presented in table 1. Some of the peptides in particular peptides MBY32, MBY33, MBZOl, MBZ02 and MBY10 showed strongly improved activity in all three media relative to MBNOl.

Table 1. Antifungal activity of single- and multi-substitution peptides of Rs-AFP2 (30-49).

Multiple Peptide Synthesis at 30 umol scale:

* = acetyl

# = amide

B = alpha-aminobutyric acid; substitution amino acids are underlined.

Synergistic effects of combinations of Rs-AFP2 and peptide MBO06 or peptide MBY10 We have done a checkerboard titration of Rs-AFP2 and two peptides: MBQ06 of SEQ ID NO 5 (identical to MBNOl see above) or peptide MBY10 (identical to MBNOl except for the following substitutions; Ser35=>Arg, Asn37=>Arg, Tyr38=>Phe, Val39=>Arg, Phe40=>Trp, Ala42=>Tyr). Thus MBY10 is of SEQ ID NO 31 shown above. Native protein and peptide were diluted in a microtitre plate and tested for antifungal activity against Fusarium culmorum in various media. In Table II synergy scores are indicated as a function of the molar amounts of native protein and peptide in each well. Synergy has been tested in three media: V PDB, SMF+pH5 and SMF+pH7.

Table II Synergistic effects of combinations of Rs-AFP2 and peptide

MBQ06 or peptide MBYIO, in media i PDB, SMF+pH5 and SMF+pH7

Amounts of protein peptide are indicated in pmol/lOOul. Test fungus was Fusarium culmorum (2 x 10⁴ spores.ml)

0: No synergy effect was observed.

*: The sum of the percentage growth inhibition of the native protein and the peptide of the individual inhibitory activity of one of them was above 80%. +: The growth inhibition was 20% - 40%) (+), 40% - 60% (++), 60% - 80% (+++), 80% - 100% (++++) more than the sum of the individual growth inhibition percentages of Rs-AFP2 and MBY10 or MBQ06.

Synergy was scored as growth inhibition more than 20% higher than the sum of Rs-AFP2 and peptide or of one of the individual components. Peptide MBY10 showed activity in all media, whereas peptide MBQ06 only showed inhibitory activity in V PDB. In all cases some combinations of peptide (MBY10 or MBQ06) and Rs-AFP2 resulted in additional inhibitory activity. MBQ06 that did not have inhibitory activity in SMF+ media was still able to increase growth inhibition in combination with sub-inhibitory amounts of Rs-AFP2.

Claims

1. An antimicrobial protein or peptide derived from a plant defensin, or a derivative thereof, characterised in that said protein or peptide comprises one or more of the following replacement amino acid residues selected from the group consisting of:

(i) a tryptophan residue at position 32;

(ii) a valine, leucine, isoleucine, tryptophan, phenylalanine, lysine, arginine, tyrosine, methionine, cysteine or histidine residue at position 34; (iii) an isoleucine, tryptophan, lysine, arginine, valine, leucine, phenylalanine or histidine residue at position 35;

(iv) a tryptophan residue at position 36; (v) a tryptophan, glycine. threonine, tyrosine, glutamine, lysine, arginine, phenylalanine or histidine residue at position 37; (vi) a leucine, isoleucine, tryptophan, phenylalanine, valine or cysteine residue at position 38 ;

(vii) a leucine, isoleucine, tryptophan, phenylalanine, methionine, lysine, arginine, tyrosine or histidine residue at position 39; (viii) a tryptophan residue at position 40;

(ix) an isoleucine, a tryptophan, phenylalanine, serine, threonine, tyrosine, glutamine, asparagine, lysine, arginine. histidine at position 41 ; and/or

(x) a valine, leucine. isoleucine, tryptophan. phenylalanine, tyrosine, asparagine, lysine, arginine, serine or threonine residue at position 42: where said amino acid residues are not found naturally at said positions in the antimicrobial protein or peptide, with the proviso that the antimicrobial proteins do not comprise only a replacement arginine residue at position 37, 39 or 42.

2. An antimicrobial protein or peptide according to claim 1 which contains at least one replacement in group (iii), (iv), (v), (vi), (vii), (viii), (ix) or (x) as defined in claim 1.

3. An antimicrobial protein or peptide according to claim 2 which contains at least one replacements listed in group (iii), (iv),(v), (vii), (ix) or (x) as defined in claim 1.

4. An antimicrobial protein or peptide according to any one of the preceding claims which contains one or more alpha-aminobutyric acid group as a replacement for a cysteine residue.

5. An antimicrobial protein or peptide according to any one of the preceding claims which comprises at least six amino acid residues.

6. An antimicrobial protein or peptide according to claim 5 which comprises at least 19 amino acids.

7. An antimicrobial protein or peptide according to any one of the preceding claims which are derived from plant defensins having substantially similar activity to Rs- AFP2, and which show at least 40%, sequence similarity to Rs-AFP2.

8. An antimicrobial protein or peptide according to claim 7 which are derived from Rs-AFPl, Rs-AFP3, Rs-AFP4, Br-AFPl, Br-AFP2, Bn-AFPl, Bn-AFP2,

Sa-AFPl, Sa-AFP2 and At- AFP 1 and Hs-AFP2, Ah-AMPl or Dm- AMP 1

9. An antimicrobial protein or peptide according claim 8 which is derived from Rs- AFP2.

10. An antimicrobial protein or peptide according to claim 9 wherein said peptide is derived from position 21 to 51 of the Rs-AFP2 sequence.

11. A combination of an antimicrobial protein or peptides according to any one of the preceding claims and one or more different the antimicrobial proteins or with one or more other antimicrobial proteins or peptides according to any one of the preceding claims.

12. A synergistic antimicrobial combination comprising a plant defensin and a peptide derived from a plant defensin.

13. A synergistic antimicrobial combination according to claim 12 wherein said peptide is a peptide according to any one of claims 1 to 10.

14. A synergistic combination according to claim 12 or claim 13 wherein said plant defensin is Rs-AFP2.

15. A process of combating fungi whereby they are exposed to an antifungal peptide or protein according to any one of claims 1 to 10, or a combination according to any one of claims 11 to 14.

16. A composition comprising an antimicrobial protein or peptide according to any one of claims 1 to 10, or a combination according to any one of claims 11 to 14, in combination with a carrier or diluent.

17. A plant having improved resistance to a fungal or microbial pathogen and containing recombinant DNA which expresses an antimicrobial peptide or protein according to any one of claims 1 to 10.

18. A nucleic acid which encodes an antimicrobial protein or peptide according to any one of claims 1 to 10.

19. A vector comprising a nucleic acid according to claim 18.

20. A vector according to claim 19 which is a plant transformation vector.

21. Use of a protein or peptide according to any of claims 1 to 10 in the treatment or prevention of microbial infections.

2. Use according to claim 21 wherein the microbial infection is a fungal infection.