JP2003507010A - Synthetic peptide - Google Patents

Abstract

  (57) [Summary] A modified cysteine-containing antimicrobial peptide derived from plant defensin, wherein the modification (a) introduces one or more cysteine residues and / or (b) replaces or modifies one or more cysteine residues, Such a peptide, comprising blocking the ability to form a disulfide bridge. The modified peptides can have improved antimicrobial activity.

Description

Detailed Description of the Invention

The present invention relates to antibacterial peptides, methods for producing and using the same, and DNAs encoding them.

[0002] Plant defensins are small protein species that exhibit antibacterial activity (Broekaert et al., Plant Physiol,
108: 1353-1358 (1995), Crit. Rev. Pla
nt Sci (1997)). The first two antibacterial proteins (
Rs-AFP1 and Rs-AFP2) are radish (Raphanus sa)
(Terras et al. J. Biol. Che.
m. 267: 15301-15309 (1992)). Since then, many related proteins have been isolated from many plant species (Broekaert et al.,
Plant Physiol. 108: 1353-1358 (1995),
Osborn et al., FEBS Lett. 368: 257-262 (1995)
)). The common structural motifs of the plant defensin family are three antiparallel beta chains that are stabilized by alpha helices and highly conserved cysteine bridges. Other conserved amino acids include Gly-13, Gly-34, aromatic residues at position 11, and Glu-29.
(Broekaert et al., Plant Physiol. 108).
: 1353-1358 (1995)). Plant defensins are hyphal branching (hyp
with or without an increase in hal branching (eg Rs-AFPs) (eg Aesculus hippocasta).
num) seed-derived Ah-AMP1 (Osborn et al. FEBS Lett.
368: 257-262 (1995))) causes a decrease in hyphal elongation. The third subclass of plant defensins inhibits alpha-amylase. The mechanism of fungal growth inhibition is unknown, but the fungus Neurospora crassa
a) Plant defensins were found to cause an increased Ca ²⁺ influx when added to mycelium (Thevissen et al. J. Biol. Chem. 27).
1: 15018-15025 (1996)).

Rs-AFPs are isolated from Raphanus sativus seeds or leaves, consist of 50 or 51 amino acids, and belong to the plant defensin protein family. Four highly homologous Rs-AFPs have been isolated (Rs-AFP1 to 4). The structure of Rs-AFP1 consists of three beta chains and one alpha helix and is stabilized by four cystine bridges (Fant et al. J. Mol. Biol., 279: 257-.
270 (1998)). Isolation from biological sources yields small amounts of material, and chemical synthesis of bioactive proteins is complex. Thus, small peptides deduced from native sequences that are still biologically active are not only important tools for studying structure-function relationships, but are also commercially interesting targets.

Cysteine, which is conserved in natural proteins, is essential for the secondary structure of the protein. It has been previously described that activity is retained but generally diminished in certain cases where all cysteines in the peptide are replaced (WO 97 /
21815).

The cysteine in the 19-residue peptide from the beta-2-beta-3 loop can be replaced with alpha-aminobutyric acid (Abu) and retained activity. It is possible and sometimes even more effective. Similar 19-mer peptides, which were forced to adopt a hairpin structure by the introduction of one or two disulfide bridges, were also found to have high antifungal activity.

Accordingly, in a first aspect, the invention provides a modified cysteine-containing antimicrobial peptide derived from a plant defensin, wherein said modification introduces (a) one or more cysteine residues, and / or (b) ) Substituting or altering one or more cysteine residues to provide a disulfide bridge (disulphide br)
ids) are provided, which comprises blocking said ability to form said peptides.

As used herein, “modified cysteine-containing antimicrobial peptide (modified)
systemine-containing antimicrobial pe
The expression "peptide""has at least one cysteine residue, which residue is modified as described when compared to the corresponding naturally occurring sequence from which the peptide is derived. Refers to. The peptide preferably has antibacterial activity that is enhanced to some extent when compared to the corresponding naturally occurring sequence from which the peptide is derived.

A cysteine-containing antimicrobial peptide according to the invention comprises the addition of one or more cysteine residues to said peptide and / or the modification of one or more, and preferably not all naturally occurring cysteine residues within said peptide. A cysteine residue is blocked or modified such that it is unable to form a disulfide bridge when said peptide is compared to the peptide or protein from which the peptide is derived. , Is characterized by an altered ability to form disulfide bridges.

The addition of one or more cysteine residues to the peptide and / or modification of one or more but not all naturally occurring cysteine residues in the peptide is due to the formation of new unnatural disulfide bridges, and / or Alternatively, removal of some of the existing disulfide bridges in the native peptide or protein from which the peptide is derived leads to the disruption of the peptide's natural disulfide bridge formation.

As used herein, the term non-natural disulfide bridge refers to a disulfide bridge that does not naturally occur in the natural peptide or protein from which the peptide is derived. Peptides can be derived from larger peptides.

As used herein, the term “plant defensin” has antibacterial activity and also has the following characteristic structural features: 4, 15, 21, 25, 36, 45, 47 and 51.
Cysteine residue at positions 4 and 51, 15 and 36, 21 and 45,
And disulfide bridge formation between cysteines at positions 25 and 47; aromatic amino acid residue 4 amino acids upstream from cysteine at position 15; glycine residue 2 amino acids upstream from cysteine at position 15; glutamic acid residue 36 amino acids upstream from position 36 It is used to indicate a protein that also has a group and a glycine residue 2 amino acids upstream of cysteine at position 36. Here, the position of the cysteine residue is defined relative to the Rs-AFP1 sequence as well as its homologues, active variants and derivatives. In some plant defensins, positions 1 and 3, positions 5 and 14, 26 and 35,
And the portion between positions 37 and 44 may differ in length from 1 to 3 amino acids, which does not affect the overall characteristic cysteine motif described above. This characteristic structural feature of plant defensins is as follows:

[0012]

[Chemical 1]

(SEQ ID NO: 1) wherein a is an aromatic amino acid (F, W, Y), C is cysteine, E is glutamic acid and G is glycine, and an unspecified amino acid or amino acid Flock
Indicated by periods.

For example, the amino acid sequence of plant defensin Rs-AFP2 is shown as SEQ ID NO: 2, and the amino acid sequence of Rs-AFP1 is shown as SEQ ID NO: 3.

[0015]

[Chemical 2]

[0016] (SEQ ID NO: 2)

[0017]

[Chemical 3]

(SEQ ID NO: 3) The expression “homologues” as used herein refers to any peptide having some amino acids in common with a given sequence. Suitably at least 60% of the amino acids are 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%. Amino acids of will be similar to the corresponding amino acids in the given sequence.

As used herein, the term “similar” is used to indicate sequences that, when aligned, have similar (identical or conservatively substituted) amino acids at similar positions or regions. Where the same or conservatively substituted amino acids are those that do not alter the activity or function of the protein when compared to the starting protein. For example, two amino acid sequences that are at least 85% similar to each other allow gaps of up to 3 with the exception that when the gaps are optimally aligned so that no more than a total of 15 amino acid residues are affected. It has a sequence with at least 85% similar (identical or conservatively substituted) amino acid residues at similar positions. The degree of similarity can be determined using methods known in the art (eg, Wilbur, WJ and Lipman, DJ “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. et al. And Miller W. et al. "Optimal Alignments in Line
ar Space ". Comput. Appl. Biosci. 4: 1.
1-17 (1988)). One program that can be used to determine the degree of similarity is MegAlign Lipman-Pear.
son pair method (using default parameters) and the program is La
As part of the sergene system, DNAstar Inc, 1228, Se
lfpark Street, Madison, Wisconsin, 5
3715, USA.

Amino acids that differ from the basic sequence may have conservative or non-conservative substitutions. Conservative substitutions should be understood to mean that an amino acid has been replaced with an amino acid having broadly similar chemical properties. In particular, conservative substitutions can be made between the following groups of amino acids: (i) alanine, serine, glycine and threonine; (ii) glutamic acid and aspartic acid; (iii) arginine and lysine; (iv) Asparagine and glutamine; (v) Isoleucine, leucine, valine and methionine; (vi) Phenylalanine, tyrosine and tryptophan.

[0021] In general, conservative substitutions will likely not destroy the antibacterial properties of the compound, rather than non-conservative substitutions. Suitable homologues can be determined, for example, by testing the antimicrobial properties of the peptide using routine methods, as exemplified below.

The term “variant” is used herein (other than in the sequence listing), experimentally generated variants, or related, as can be identified by molecular genetic techniques. Includes family members of naturally occurring peptides. Such techniques are described, for example, in US Pat. No. 5,605, the contents of which are incorporated herein.
, 793, US Pat. No. 5,811,238 and US Pat. No. 5,830,7.
No. 21. In essence, this technique is based on Escherichia coli (Escherich
ia coli) and the expression of the parental gene in a microbial expression system. The particular system chosen has to be validated and calibrated to ensure that the biologically active peptide is expressed, which can be easily achieved using in vivo bioassays. It is possible. The gene, or preferably a collection of related genes from different species, may be subjected to mutagenesis polymerase chain reaction (PCR) as is known in the art. Fragmentation of the product using PCR and subsequent repair leads to a series of chimeric genes that are reconstructed from the parental mutants. These chimeras may then be expressed in microbial systems and screened in the usual fashion to determine active mutants, which may then be isolated and sequenced. Repetition of this molecularly evolved DNA shuffling cycle can lead to the progressive enhancement of desirable genetic properties. The advantage of a technique of this nature is that it allows a wide range of different mutations, including multiple mutation block exchanges, to be produced and screened.

Other variants can be identified or identified using a bioinformatics system. Examples of such systems are described in W. R. Pearson and D.M.
J. Lipman PNAS (1988) 85: 2444-2488 FAS.
The TA method. This method provides a quick and easy method for comparing protein sequences and detecting levels of similarity, and is a standard tool used by molecular biologists. Such similar sequences are compared from natural sources, through molecular evolution, or by synthetic methods, and using this method to arrive at an “opt score” that indicates the level of similarity between proteins. Can be obtained.

[0024] Certain variants of the invention have the FASTA selection score (F) for any one of the sequences of the proteins in the antimicrobial compositions of the invention described herein as follows:
ASTA version 3.0t82, as defined according to Nov. 1, 1997). A variant of the invention comprises an antibacterial amino acid sequence for Rs-AFP1 or 2 which has a FASTA selection score of 300 or more (as defined according to FASTA version 3.0t82, Nov. 1, 1997). Will contain protein.

The term “derivative” relates to an antibacterial protein that has been modified, for example by using known chemical or biological methods. Preferably, the modified peptides of the present invention are naturally occurring plant defensins modified according to the present invention.

Modification of the defensins according to the invention by the introduction of one or more cysteine residues is particularly preferred. Furthermore, the modification of defensins according to the invention by the introduction of one or more cysteine residues in combination with the modification of one or more cysteine residues that block the ability to form disulfide bridges is particularly preferred.

In this and all further aspects of the invention, the cysteine-containing antimicrobial peptide is preferably a published International Patent Application Nos. WO 93/05153 and WO 95/95, the comments of which are incorporated herein. 18229, group Rs-AFP1, Rs-AFP2, Rs-AFP3, Rs-AFP, fully described in 18229.
4, Br-AFP1, Br-AFP2, Bn-AFP1, Bn-AFP2, Sa
-AFP1, Sa-AFP2 and At-AFP1 and Hs-AFP1, Ah
-AMP1 and Dm-AMP1, from Aly-AFP and Alf-AFP, fully described in published international patent applications WO 97/37024 and WO 98/26083, the descriptions of which are incorporated herein by reference. It is derived from the selected plant defensin. More preferably, the cysteine-containing antimicrobial peptide is of group Rs-
It is from a plant defensin selected from AFP1 or Rs-AFP2, and most preferably from Rs-AFP2. The peptide is a beta-2 chain / turn / beta-3 chain region of plant defensins (beta-2 strand).
More preferably, it is derived from / turn / beta-3 strand region). The secondary structural element of RsAFP1 is described by Fant et al. (J. Mol.
Biol. 279: 257-270 (1998)).

[0028] In a particularly preferred embodiment, the present invention provides a modified cysteine-containing antimicrobial peptide derived from the beta-2 chain / turn / beta-3 chain region of plant defensin, wherein the modification is (a) 1 or more. Introducing a cysteine residue, and / or (b)
There is provided said peptide comprising substituting or modifying one or more cysteine residues to block the ability to form disulfide bridges.

The beta-2 chain / turn / beta-3 chain region of plant defensins can be determined by analysis of primary amino acid sequence information, and is generally the fourth and eighth cysteine residues. Is expected to be located between. For example, Rs-AFP
In 1 and Rs-AFP2, this region occurs between positions 21 and 51 of the sequence, and more precisely between positions 30 and 51 of the sequence.

In a preferred embodiment, the present invention provides two Rs-AFP1 or Rs-AFP2.
A modified cysteine-containing antimicrobial peptide derived from positions 1 to 51, wherein said modification introduces (a) one or more cysteine residues and / or (b) replaces one or more cysteine residues. There is provided said peptide comprising or modified to block its ability to form disulfide bridges.

In a further preferred embodiment, the invention provides Rs-AFP1 or Rs-AF.
A modified cysteine-containing antimicrobial peptide derived from positions 30 to 51 of P2, wherein said modification introduces (a) one or more cysteine residues, and / or (b)
There is provided said peptide comprising substituting or modifying one or more cysteine residues to block the ability to form disulfide bridges.

One or more cysteine residues may be added at any position within the peptide sequence.
The presence of these cysteine residues, under appropriate conditions, allows the formation of unnatural disulfide bridges within the peptide so as to modify the conformation of the peptide, leading to a stronger and less flexible structure. To do. It may also result in improved biological activity, as can be seen in the examples herein.

The disulfide bridge may be an intermolecular disulfide bridge, ie two different protein intermolecular bridges, or an intramolecular disulfide bridge, ie one.
It may be a crosslink that occurs within one peptide molecule. The formation of intermolecular disulfide bridges is especially preferred. The formation of intermolecular disulfide bridges is
merization) and dimer formation, and is particularly preferred, especially when the peptide is a hexapeptide or a 15-mer peptide. The preferred position of the cysteine residue (s) to be added is determined by considering the NMR model of the peptide and selecting the positions where the two ends of the natural loop are brought together. This is particularly appropriate if the peptide is sufficiently long to form a complete loop structure, and this will typically be 17,18,19,20-mers and longer peptides.

Those skilled in the art will appreciate that if the peptide is modified to have only one cysteine residue available for disulfide bridge formation, the disulfide bridge that will be formed is an intermolecular disulfide bridge. It will be clear, and it will be clear that the peptide structure will be a dimer.

We have also shown that when one or more naturally occurring cysteine residues are replaced by side-chain blocking cysteines or by closely related amino acids, such as alpha aminobutyric acid residues, non-naturally occurring cysteine residues It was also found that the addition can enhance the activity of the peptide, which is a preferred aspect of the invention. However, in an alternative embodiment, the cysteine residue may be replaced by another naturally occurring amino acid by site-directed mutagenesis using known methods or, preferably, by simply synthesizing a peptide with the desired modified structure. May be modified to This may be preferred if it is desired to express the peptide in plants, for example as discussed below.

In a preferred embodiment of the first aspect of the present invention one or more cysteines are added to the N-terminus and / or C-terminus of the peptide. Peptides to which cysteine is added or cysteine modified are preferably at least 4, 5 or 6 amino acids in length, preferably at least 6 amino acids in length, more preferably at least 10 amino acids in length, and most preferably at least length. It is 15, 17, 19, 20 amino acids or more.

The cysteine added to the peptide may be in the D and / or L configuration,
And, according to the present invention, preferably 1, 2, 3 or 4 cysteines are added to the peptide. According to the present invention, one or more naturally occurring cysteine residues in the peptide may be replaced with D-cysteine.

In a further aspect, the invention comprises one or more cysteine residues in the D configuration,
Provided is a plant defensin or a peptide derivative thereof. The naturally occurring cysteine residue may be blocked with a blocking group, such as iodoacetamide, or with an alpha aminobutyric acid group, such that the formation of naturally occurring disulfide bridges with the amino acid residue is inhibited. ,
Alternatively, it may be replaced by an alternative amino acid acid.

In a particularly preferred embodiment of all aspects of the invention the antimicrobial peptide comprises the residue H
Includes GS and / or HKY. In a further aspect, the invention provides a method of making an antimicrobial peptide more robust, comprising (a) introducing one or more cysteine residues and / or (b) replacing one or more cysteine residues, or Provided is a method of modifying a peptide structure by modifying and blocking the ability to form disulfide bridges.

In a preferred further aspect, the present invention is a method of making an antimicrobial peptide more robust, which comprises (a) introducing one or more cysteine residues and / or (b) 1.
Provided is a method, which comprises modifying peptide structure by substituting or modifying cysteine residues, but not all, to block the ability to form disulfide bridges.

As used herein, the term “rigid” means that the peptide is less flexible in solution and that the addition or removal of the disulfide bridge (s) precludes certain natural conformations. It is used to indicate that Thus, in general, the structure will include elevated levels of intramolecular or intermolecular crosslinks and the elimination of linear structures that are incapable of forming intermolecular crosslinks. Properly,
This has the effect of enhancing the desired activity of the peptide, eg antibacterial activity. The antimicrobial peptide is preferably derived from the plant defensins, as described herein.

In a further aspect, the invention provides a method of improving the antibacterial activity of an antimicrobial peptide derived from a plant defensin, comprising (a) introducing one or more cysteine residues and / or (b) Provided is a method comprising altering the natural disulfide bridge pattern of a peptide by substituting or modifying some, but not all, cysteine residues and blocking the ability to form disulfide bridges.

[0043] Antifungal proteins exhibiting the characteristic structural attributes of plant defensins are fully described in published international patent applications WO 93/05153 and WO 95/18229, the comments of which are incorporated herein. The protein Rs-AFP1, described.
Rs-AFP3, Rs-AFP4, Br-AFP1, Br-AFP2, Bn-A
FP1, Bn-AFP2, Sa-AFP1, Sa-AFP2 and At-AFP
1 and Hs-AFP1, Ah-AMP1 and Dm-AMP1. It is particularly preferred that the peptides of the invention are derived from proteins that have activities substantially similar to Rs-AFP2 and Rs-AFP1. Peptides derived from plant defensins are described in Published International Patent Application No. WO 9
7/21815, and the peptides described therein, introduce one or more additional cysteine residues and / or modify one or more but not all cysteine residues to form disulfide bonds. Particularly useful starting peptides for modifications in accordance with the invention, including blocking potency.

Antimicrobial peptides according to the invention include, in particular, those from the beta-2 chain / turn / beta-3 chain region of Rs-AFP2 and proteins with substantially similar activity, such as homologues and variants. Peptides are included.

Antimicrobial peptides derived from the regions defined herein of Rs-AFP plant defensins exhibit antifungal activity. While retaining antifungal activity, such peptides may be easier to synthesize than full-length plant defensins. The DNA sequence encoding the peptide may also be more suitable for transformation into a biological host.

The antimicrobial peptides according to the present invention may be prepared from known amino acid sequences using suitable techniques, such as by chemical synthesis, or by expression of recombinant DNA in suitable organisms (eg microorganisms or (Plant). The antimicrobial peptides are useful as fungicides and may be used in agricultural or pharmaceutical or other applications. The antimicrobial peptide may be used in combination with one or more other antimicrobial peptides of the invention.

Knowing the primary structure allows the production of antimicrobial peptides, or parts thereof, by chemical synthesis. One of ordinary skill in the art will recognize that if the peptides contain chemical blocking groups as described herein, these will require synthesis or modification by chemical routes. When appropriate, the production of DNA constructs encoding antimicrobial peptides is also possible.

The invention further provides a DNA sequence encoding an antimicrobial peptide according to the invention. DNA sequences can be predicted from known amino acid sequences,
The DNA encoding the peptide can then be produced using a standard nucleic acid synthesizer.

The DNA sequence encoding the antimicrobial peptide may be combined with appropriate regulatory sequences (promoter, terminator, transit peptide, etc.) and incorporated into a DNA construct or vector. For some applications, a DNA sequence encoding an antimicrobial peptide may be inserted within the coding region that expresses another protein to form an antimicrobial fusion protein, or the domain of the protein may be replaced to replace that protein. It may be used to provide antibacterial activity. The DNA sequence may be placed under the control of a homologous or heterologous promoter, which may be a constitutive or inducible promoter (eg stimulated by environmental conditions, the presence of pathogens, the presence of chemicals). The transit peptide may be homologous or heterologous to the antimicrobial protein and will be selected to ensure secretion to the desired organelle or into the extracellular space. The transit peptide is preferably naturally associated with the antimicrobial protein of interest. Such DNA constructs may be cloned or transformed into a biological system that allows expression of the encoded peptide, or active portion of the peptide. Suitable biological systems include microorganisms such as bacteria such as E. coli, Pseudomonas.
nas) and endophytes such as Claviobacter xyli susp. cynodontis.
) (Cxc); yeast; virus; bacteriophage etc.), cultured cells (eg insect cells, mammalian cells) and plants. In some cases, the expressed peptides may be subsequently extracted and isolated for use.

The invention further provides the use of a peptide according to any of the preceding claims in the treatment or prevention of microbial infections, preferably fungal infections. The antimicrobial peptides according to the present invention are useful in plants to combat microbial diseases. The invention further provides a method of combating fungi by exposing the fungus to an antimicrobial peptide according to the invention. The antimicrobial peptide may be used in the form of a composition.

The antimicrobial peptide or protein may be used in the form of a composition, eg in combination with a suitable carrier or diluent. For example, for agricultural use, the compositions of the invention may be in the form of either a ready-to-use dilute composition or a concentrated composition, usually requiring dilution prior to use with water. Good. The liquid composition may contain other conventional components such as surfactants, dispersants and the like.

The solid composition may be a solid diluent in which the granules or active ingredient is finely divided, such as kaolin, bentonite, kieselguhr, dolomite, calcium carbonate, talc, powdered magnesia, Fuller's earth. e
dust), mixed with arch and gypsum
It may be in the form of r). They may also be in the form of dispersible powders or particles which contain wetting agents which facilitate the dispersion of the powders or particles in the liquid. The solid composition in powder form may be applied as a foliar dust.

For pharmaceutical applications, antimicrobial peptides (including any products derived therefrom) may be used as fungicides to treat mammalian infections (eg Candida (Ca).
ndida) to combat yeasts).

The pharmaceutical composition of the present invention may be used for oral use (eg tablets, lozenges).
), Hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), topical use (eg creams, ointments, gels,
Or aqueous or oily solutions or suspensions), administration by inhalation (eg as finely divided powder or liquid aerosol), administration by dusting (eg as finely divided powder) or parenteral administration (eg intravenous, subcutaneous, It may be in a form suitable as a sterile aqueous or oily solution for intramuscular or intramuscular administration, or as a suppository for rectal administration. For more information on prescribing, see Co
mpregensive Medicinal Chemistry (Corw
in Hansch; Supervisor), Pergamon Press 199
0, Volume 5, Chapter 25.2.

The antimicrobial peptides according to the invention (including any products derived therefrom) may also be used as preservatives (eg as food or cosmetic additives). For agricultural applications, antimicrobial peptides may be used to improve disease resistance or resistance of crops for crop protection during plant life or after harvest. Pathogens exposed to the peptide are inhibited. The antimicrobial peptide eradicates already established pathogens in the plant or protects the plant from future pathogen attack. The eradication effect of the peptides is particularly advantageous.

Exposure of the plant pathogen to the antimicrobial peptide may be accomplished in a variety of ways, including: (a) the isolated peptide on plant parts or on soil or other growth medium surrounding the roots of the plant. Alternatively, it may be applied to the seeds of the plant before being sown using standard agricultural techniques (eg spraying).

The peptide may be extracted from plant tissue or chemically synthesized or extracted from a microorganism that has been genetically modified to express the peptide. The peptide
It may be applied to the plant or to the plant growth medium in the form of a composition comprising the peptide mixed with a solid or liquid diluent, and optionally various adjuvants such as surfactants. The solid composition may be in the form of a dispersible powder, granules or particles. (B) a composition comprising a microorganism genetically modified to express an antimicrobial peptide,
It may be applied to the plant or the soil in which it grows. (C) Endophytic plants (endo) genetically modified to express antimicrobial peptides
Phyte) may be introduced into plant tissues (eg, via seed treatment methods).

An endoparasitic plant is defined as a microorganism that has the ability to enter into a non-pathogenic endosymbiotic relationship with a plant host. A method of protecting endophytes from plants is described in Crop Genetics.
s International Corporation for a series of patent applications (eg International Application Publication No. WO 90/13224, European Patent Publication EP-1
25468-B1, International Application Publication No. WO 91/10363, International Application Publication No. WO 87/03303). Endophytic plants can be genetically modified to produce agrochemicals. International Patent Application Publication No. WO 94/16076 (ZENECA Limited) describes the use of endophytic plants that have been genetically modified to express antimicrobial peptides of plant origin.

(D) A DNA encoding an antimicrobial peptide may be introduced into a plant genome so that the peptide is expressed in the plant body (DNA may be cDNA, genomic DNA or a standard nucleic acid synthesizer. It may be DNA produced by the method).

Plant cells may be transformed with recombinant DNA constructs according to various known methods (Agrobacterium Ti plasmid, electroporation, microinjection, microprojectile). gun))).
The invention extends to plant cells which have been transformed with a DNA construct according to the invention. The transformed cells may then be regenerated into whole plants, where appropriate, with new nuclear components stably integrated into the genome. Both transformed monocots and dicots can be obtained in this way, although the latter are usually easier to regenerate. Some of the progeny of these primary transformants will inherit the recombinant DNA encoding the antimicrobial peptide (s).

We have found that the antimicrobial peptides according to the invention show activity against a wide range of phytopathogenic fungi and are particularly useful. The peptides of the invention may also be useful in combating bacterial infections.

The present invention further provides plants having improved resistance to microbial pathogens, in particular fungal pathogens, and containing recombinant DNA expressing an antimicrobial peptide according to the invention. Such plants may be used as parents in standard plant breeding crosses to develop hybrids and strains with improved fungal resistance.

Recombinant DNA is preferably heterologous DNA introduced into a plant or its ancestors by transformation. The recombinant DNA encodes an antimicrobial peptide that is expressed for delivery to the pathogen attack site (eg, leaf). The DNA may encode the active subunit of an antimicrobial peptide.

The pathogen can be any fungus that grows on, in or near the plant. In this context, improved resistance is defined as increased resistance to fungal pathogens when compared to wild type plants. Resistance is from a slight increase in resistance to the influence of the pathogen (partial inhibition of the pathogen) to a complete resistance in which the presence of the pathogen does not affect the plant (pathogens are severely inhibited or killed) ) Can be diverse. Both resistance to specific pathogens or increased levels of resistance to a wide range of pathogens can constitute improved resistance. Transgenic plants (or plants derived therefrom) that exhibit improved resistance are selected after plant transformation or subsequent crosses.

When the antimicrobial peptide is expressed in the transgenic plant or its progeny, the fungus is exposed to the peptide at the site of pathogen attack on the plant. In particular,
Through the use of appropriate genetic control sequences, peptides can be produced in vivo, where they will be most effective, and where they will be most effective. For example, peptides can be produced in plant parts (eg leaves) where disease resistance is important, although not usually expressed in some amount.

Examples of genetically modified plants that may be produced include farm crops, cereals, fruits and vegetables, such as: canola, sunflower,
Tobacco, sugar beet, cotton
on), soybean, maize, wheat (where)
t), barley (barley), rice (rice), sorghum (sorghu)
m), tomatoes (tomatoes), mangoes (mangoes), peaches (pe)
aches), apples (apples), pears (pears), strawberries (strawberries), bananas (bananas), melons (melo).
ns), potatoes, carrots, lettuce, cabbage, onions.

The antimicrobial peptide MBG03 CRHGSBN described more fully herein.
YVFPAHKBIBYC (SEQ ID NO: 21), MBG04 CRHGScNYV
FPAHKcIBYC (SEQ ID NO: 22), MBN06 CRHGSCNYVFP
AHKCIBYC (SEQ ID NO: 25), MCH32 and MCH32x HKBI
BYC (SEQ ID NO: 4), MCH33 and MCH33x CHKBIBY (SEQ ID NO: 5), MCH37 and MCH37x HKCIBY (SEQ ID NO: 7), MC
H38 and MCH38x HKBICY (SEQ ID NO: 8), MCI28 and M
CI28x CHKBIBYC (SEQ ID NO: 10), MCI10 and MCI10
x CHGSBNYVFPAHKBIBC (SEQ ID NO: 11), MCI11 and MCI11 x CHGSBNYVFPAHKBIB (SEQ ID NO: 12), MCI1
2 and MCI12x HGSBNYVFPAHKBIBC (SEQ ID NO: 13),
MCI15 and MCI15x HGSCNYVFPAHKBIB (SEQ ID NO: 1
5), MCI16 and MCI16x HGSBNYVFPAHKCIB (SEQ ID NO: 16) and MCI17 and MCI17x HGSBNYVFPAHKB
IC (SEQ ID NO: 17) is particularly preferred. In the above, the lower case "c" is D-
Represents cysteine.

According to the present invention, the most particularly preferred peptide is MBG03 CRHGSB
NYVFPAHKBIBYC (SEQ ID NO: 21), MBG04 CRHGScNY
VFPAHKcIBYC (SEQ ID NO: 22), MBN06 CRHGSCNYVF
PAHKCIBYC (SEQ ID NO: 25), MCH32x HKBIBYC (SEQ ID NO: 4), MCH38 and MCH38x HKBICY (SEQ ID NO: 8), MCI
12x HGSBNYVFPAHKBIBC (SEQ ID NO: 13), MCI16 and MCI16x HGSBNYVFPAHKCIB (SEQ ID NO: 16) and MC
I17 and MCI17x HGSBNYVFPAHKBIC (SEQ ID NO: 17)
Is.

The invention will now be described by way of example only, with reference to the following examples.

[0070]

【Example】

Experimental material. N-methylpyrrolidone (NMP), dimethylformamide (DMF),
N-hydroxybenzotriazole (HOBt), 2- (1H-benzotriazol-1-yl) -1,1,3,3, -tetramethyluronium hexafluoro-phosphate (HBTU) and piperidine are peptide synthesis grades. And obtained from Perkin Elmer / ABI (Warrington, UK). Acetonitrile (ACN) is a gradient grade, diisopropylethylamine (DIEA), trifluoroacetic acid (TFA), thioanisole (TA), phenol, and ethanedithiol (EDT) are synthetic grades.
synthesis grade) and obtained from Merck (Darmstadt, Germany). Prior to use, diethyl ether was purified on a column of activated basic aluminum oxide and DIEA was distilled twice over ninhydrin and potassium hydroxide. Fmoc-amino acid derivative and resin (4- (2 ', 4'
-Dimethoxyphenyl-Fmoc-aminomethyl) phenoxy resin (Rink resin)) was obtained from Saxon Biochemicals (Hannover, Germany).

Analytical HPLC. Because of the analytical HPLC, we have 2
Waters Pump Model 510, Waters Grader Model 68
0, Waters WISP 712 Automatic Injector, and Waters
A 991 photodiode array detector was used. The product is 0.1% (v /
v) Water containing TFA, 60% (v / v) acetonitrile / water containing 0.1% (v / v) TFA in a linear gradient for 60 minutes, Waters Delta Pak.
1 ml / on a C18-100A (3.9 × 150 mm, 5 mm) column
Analyzed in minutes.

Amino acid analysis. Amino acid analysis was performed using a Waters Pico-Tag system after hydrolysis with 6N HCl in a Pico-Tag workstation for 1 hour at 150 ° C. and derivatization with phenylisothiocyanate.

Preparative HPLC. Preparative HPLC was performed using Delta-P
AK C18-100A (15 mm) component-filled guard cartridge (4
Waters Pre with Waters RCM module containing two PrepPak cartridges (0x210mm or 25x210mm)
p4000 liquid chromatograph. Peptides are collected in preparative cells
Detection was carried out at 230 nm using a Waters 486 spectrophotometer with an eparative cell).

Molecular modeling. Molecular modeling was performed using SYBYL molecular modeling software, version 6.1 (Tripos Associates, St. Louis, Mo., USA), using Silicon Graphics Iris Indi.
Performed on a go Elan workstation (Silicon Graphics, Mountain View, CA, USA). Energy minimization is Syb
The yl 6.1 Tripos force field was used (Clark et al. Science.
e 273: 458-463 (1989)).

Multi-peptide synthesis. We have a Hamilton Microlab 2200 (
Up to 40 peptides were simultaneously synthesized on a 30 mmol scale using Reno, Nevada, USA. 20 individual 4 ml with filters programmed Hamilton Microlab 2200 and containing resin for peptide synthesis
The wash solvent and reagents were delivered to two racks containing columns. The column was automatically dried by vacuum after each step. Coupling period
cycle), using a 40-minute double coupling step, Fmoc / HBT
U chemistry (Fields et al., Peptide Res. 4: 9).
5-101 (1991)). After coupling the last amino acid, 30% (v / v
) Piperidine / NMP was used for 3 and 15 minutes to remove the Fmoc group. The peptide was washed with NMP (5 times) and NMP / acetic anhydride / DIEA (10/1/0
． 1; v / v / v) for 30 minutes, successively washed with NMP and ethanol and then dried. Deprotect the peptide, and 1.
5 ml TFA / phenol / TA / water / EDTA (10 / 0.75 / 0.5
/0.5/0.25; v / w / v / v / v /) for 2 hours and then
Precipitated twice by adding hexane / diethyl ether (1/1; v / v). The precipitate was dried and lyophilized from water / acetonitrile (1/1; v / v).

Peptide cyclization. In the case of air oxidation; peptide (MBG03 or MBG04)
Was dissolved in 1% (w / v) ammonium bicarbonate / water at 0.5 mg / ml and stirred slowly. Sample for HPLC analysis at several intervals,
The amount of free thiol groups was then determined by Ellman (Ellman Biochem.
Biophys. 82: 70-77 (1959)), 5,5'-
After the reaction with dithiobis (2-nitrobenzoic acid), UV was measured at 412 nm. In the case of DMSO oxidation, the peptide (MBG03 or MBG04) was added to 20% (
v / v) 0.5 mg / in DMSO / 0.1 M phosphate buffer (pH 5)
Dissolved in ml. Samples were taken for HPLC analysis and Ellman test at several intervals.

Antifungal activity. Antifungal activity is determined by twofold serial dilution of the peptide (twofold se).
Rial dilutions and the fungus Fusarium krumorum (Fusa)
rum culrum (IMI 180420) or other fungal spore suspensions in microtiter plates (Broekaert et al.).
FEMS Microbiol. Lett. 69: 55059 (1990)
). Half-strength potato dextrose broth (1/2 PDB, pH 5.8)
, Difco) medium at room temperature for 72 hours, after which the fungal growth was monitored microscopically and spectrophotometrically. In some experiments, Medium 1/2 PDB was buffered by the addition of 10 mM MES at pH 5.0 as indicated.

[0078]   Alternatively, SMF (Cammue et al. J. Biol. Chem. 267).
2228-2233 (1992)), 1 mM CaCl ₂ , 50 mM KCl and 10 mM Tris, and pH 7
． A synthetic medium adjusted to 0 was used. This medium is called SMF + pH7. Cultivation
The local SMF + pH5 added 10 mM MES instead of 10 mM Tris.
Identical to SMF + pH7 except added and adjusted pH to 5.0
. Results are from Cammue et al. (J. Biol. Chem. 267, 222.
8-2233 (1992)), 50% growth inhibition (IC50).
Is expressed as the concentration of μg / ml.

Antibacterial activity . Antibacterial activity is demonstrated by Cammue et al. (J. Biol. Chem.
267, 2228-2233 (1992)).
The medium consisted of either 1% tryptone and 0.5% low melting point agarose (TA medium) or TA medium supplemented with 1 mM CaCl ₂ and 50 mM KCl (TA + medium).

The results are expressed as the concentration of μg / ml giving 50% growth inhibition (IC ₅₀ ). Ca ²⁺ influx assay. Red bread mold (Neurospora crassa)
And Fusarium culmorum is 0
． 1/2 supplemented with 5 mCi [ ³ H] N-acetyl-D-glucosamine / ml
In PDB, in a 100 ml Erlenmeyer flask placed on a rotary shaker, 3 each
were grown in inoculation density of x 10 ⁵ and 5 x 10 ⁴ spores / ml (The
vissen et al. Biol. Chem, 271: 15018-150.
25 (1996)). After incubation at 22 ° C for 20 hours, 2 mCi
/ Ml ⁴⁵ CaCl ₂ was added along with the peptide. After an appropriate incubation time, a 250 ml sample is taken and a MultiScreen vacuum filtration manifold (Millipore, Bedford, MA, USA).
Transferred to the wells of the MultiScreen Durapore 96-well filtration plate (Millipore) placed above. After filtration, collect the mycelium 250
It was washed 4 times with ml 10 mM CaCl ₂ . The membrane containing mycelium was
Punch manually using a screen punch tip (Millipore) and liquid scintillation counter (Wallac 1410, Pharma).
(Cia, Uppsala, Sweden) for ³ H and ⁴⁵ Ca. The background count was negligible. ^To correlate ³ H counts with biomass, 10 ml culture samples were pre-weighed with Mi.
Filtered on an Ilopore glass fiber filter, dried in a vacuum oven, re-weighed and counted for ³ H.

Results It was previously demonstrated that a synthetic peptide corresponding to a part of the sequence of the plant defensin RsAFP2 exhibits antifungal activity (De Samblanx et al. Pept.
ide Research 1996, 9: 262-268 + published international patent application WO 97/21815).

Since these peptides without exception contain at least one cysteine residue,
They are capable of forming intramolecular or intermolecular disulfide bridges by air oxidation under the test conditions. This can result in the formation of linear monomers, cyclic monomers and multimeric compounds, making it difficult to understand which of these compounds contribute the most to antifungal activity. There is. Two series of synthetic peptides were synthesized. The first series contains the two cysteines, R at positions 43-48
A hexameric peptide called MCH 39 (6-me corresponding to the sAFP2 sequence)
r peptide). The derivative had one or two of the cysteines replaced with alpha-aminobutyric acid or had additional cysteines at the N-terminus, C-terminus, or both ends. After synthesis, fractions containing either linear monomeric peptides, cyclic monomeric peptides or dimeric peptides were separated and in 3 different media, 1/2 PDB, SMF + pH5 or SMF + pH7,
Tested for antifungal activity against Fusarium krumorum (Table 1). The only peptide that completely lacks antifungal activity is the cysteine-free peptide MCH36
Met. This indicates that these small peptides require cysteine to exert antifungal activity. In all cases, the fractions containing dimers (due to the formation of intermolecular disulfide bridges) had stronger antifungal efficacy than the linear monomeric form. Of particular note is the strong antifungal potency of the compounds found by autodimerization of the hexameric peptide MCH38 containing a single cysteine.

A second set of derivatives contains RsAF at positions 33-47 containing three cysteines.
Produced on the basis of the 15-mer peptide MCI14, which corresponds to the P2 sequence. Again, in this case too, one or more cysteines were replaced with alpha-aminobutyric acid, or extra cysteines were added at the N-terminus, C-terminus or both ends. Data on the antifungal activity of this peptide series are shown in Table 2. Again, the only peptide in this series that has no antifungal activity is one that lacks cysteine at all (M
CI 18).

A 19-mer peptide (MBG01) was synthesized whose sequence corresponds to the Rs-AFP2 sequence between Ala-31 and Phe-49. This sequence was chosen to include the beta-2 chain / betaturn / beta3-chain region of RsAFP2. The concentration of MBG01 (IC ₅₀ ) required for 50% growth inhibition of the fungus Fusarium krumorum was 3
3 μg / ml, which is about 7 times higher than the IC ₅₀ value of Rs-AFP2 itself.

Since MBG01 contains three cysteine residues, it is possible to form a variety of intramolecular and intermolecular disulfide bridges by aerial oxidation under test conditions.
Therefore, MBG01 may exist as a mixture of diverse multimers and cyclic monomers, and it is difficult to assess which form is actually responsible for the antifungal effect. To avoid these problems caused by unregulated cystine formation, an analog of MBG01 was synthesized in which all three cysteine residues were replaced with alpha aminobutyric acid (MBG02). Surprisingly, MBG
02 was more potent than MBG01 and showed an IC ₅₀ value of 8 μg / ml (Table 3).

Two of MBG01, both forced to adopt a stronger loop-like structure
Two further analogs were synthesized. Peptide MBG03 is identical to peptide MBG02 (including three alpha-aminobutyric acid residues) except that the terminal amino acid residue was alanine and phenylalanine was replaced with cysteine. Peptide MBG04 contains alpha-aminobutyric acid residues at positions 36 and 45 with two D-
Identical to MBG03 except replaced with cysteine. Energy minimization calculations indicate that a cystine bridge may be formed between the two cysteines of MBG03, while the peptide may adopt a conformation close to that of the corresponding loop of Rs-AFP2. showed that. Similarly, by molecular modeling, MB
Two cystine bridges may be formed between each of the two L-cysteines and two D-cysteines of G04, and the resulting conformation is:
It was shown to be possible in terms of energy. Cystine crosslink formation in MBG03 was performed under two different conditions: aerial oxidation at pH 8 and pH 5-6.
DMSO oxidation at. Both oxidations are based on the Ellman test (Ellman
Biochem. Biophys. 82: 70-77 (1959)), completed within 40 hours. HPLC analysis showed that air oxidation at pH 8 allowed reorganization into the undesired multimeric product, whereas DMSO oxidation yielded one major product. Oxidation of MBG04 introducing two cystine bridges was performed under the same conditions as MBG03. Again, air oxidation at pH 8 produced a complex mixture of undesired products, whereas DMSO oxidation at pH 5-6 resulted in 1
This yielded two main products. MBG03 and MBG04 were purified using preparative HPLC and tested for antifungal efficacy (Table 3). MBG03 and MBG
04 is F. It was found to be active against Kurumolum, 1 each
IC ₅₀ values of 7 and 15 μg / ml.

Rs-AFP2 has been previously shown to mediate Ca ²⁺ entry into fungal hyphae treated with this protein (Thevissen et al. J. Biol.
Chem, 271: 15018-15025 (1996)). Therefore, R
s-AFP2 and the synthetic 19-mer peptides MBG01 and MBG02 were used for pregerminated F. They were tested side by side in a Ca ²⁺ influx assay against Krumrum mycelia. The peptide MAT02 (Table 3), which was previously found to correspond to the Rs-AFP2 sequences of Gln-5 to Glu-29 and lacks antifungal activity, was included as a negative control. MAT02 was found to be inactive in the Ca ²⁺ influx assay. In contrast, MBG01, MBG02, and Rs-AFP2 showed 0.
At 3 mM and above, it caused a dramatically increased Ca ²⁺ influx. The dose for the half maximal effect is MBG01
, MBG02 and Rs-AFP2 were about 2-4 mM.

In a previous study using PEPSCAN analysis, we found that the activity of Rs-AFP2 was
It was found to be located mainly in a loop sequence consisting of a beta 2 chain, a beta turn near Pro-41 and an antiparallel beta 3 chain (De Samblanx et al. Pe.
ptide Res. 9 (1996) 262-268). In this study, we used molecular modeling based on NMR-derived 3D structural data of Rs-AFP1 (
Fant et al. Mol. Biol. 279, 257-270 (199
8)), the optimum length of the loop sequence was defined. Based on these results, we have increased the rigidity by introducing one or two cysteine bridges.
) Was synthesized.

Since three cysteines in the loop sequence are not required for biological activity, we used two of these and the terminal amino acid residues to create a tighter loop structure. We have replaced Ala-31 and Phe-49 with cysteine to build a single intramolecular disulfide bridge (MBG03), or additionally, replaced Cys-36 and Cys-45 with D-cysteine to give two molecules. An internal disulfide bridge was formed (MBG04). D-cysteine is a side chain sulfhydryl (in the model
The sulphuryl) groups were chosen because they are located closer to each other as compared to L-cysteine and after energy minimization of the bicyclic compound, there is very little effect on the structure. The antifungal activity of both cyclic peptides was similar to that of the linear peptides. This indicates that the conformations of all four loop peptides were either very similar to the native structure or had two disulfide bridges but were still sufficiently flexible to exert activity against fungal hyphae. means.

[0090] TABLE 1 Rs-AFP2 ^a result medium 1/2 of synthesis and antifungal activity of loop constructs PDB, SMF = pH5 (both 10 mM MES buffer) and SMF + pH7 (10 mM Tris / HCl buffer) Rs in -Peptides based on AFP active hexamers (regions 42-48)

[0091]

[Table 1]

[0092]   Peptides were tested against Fusarium krumorum spores (2 x 10 ^Four Spores / ml); mean of duplicate experiments after 72 hours of incubation (n = 2)
). Optical density of 100% -control wells (no peptide added) growth is shown.^*
= Acetyl, # = amide, B = alpha-aminobutyric acid; lin = linear; dim
= Dimer; cyc = cyclic; ox = ambiguous oxidation products; f = fraction number.   Table 2   Medium 1/2 PDB, SMF + pH5 (both 10 mM MES buffer)
And Rs-AFP in SMF + pH7 (10 mM Tris / HCl buffer)
β2-β3 15 to 17-mer loop peptide

[0093]

[Table 2]

[0094]   Peptides were tested against Fusarium krumorum spores (2 x 10 ^Four Spores / ml); mean of duplicate experiments after 72 hours of incubation (n = 2)
). Optical density of 100% -control wells (no peptide added) growth is shown.^*
= Acetyl, # = amide, B = alpha-aminobutyric acid; lin = linear; dim
= Dimer; cyc = cyclic; ox = ambiguous oxidation products; f = fraction number.   Table 3 Results of Synthesis and antifungal activity of loop constructs Rs-AFP2 ^a

[0095]

[Table 3]

Multipeptide synthesis on the ^a 15 (MAT02) or 30 mmol scale. ^{b *} = acetyl, # = amide, B = alpha-aminobutyric acid, c = D-Cys. ^c % of peak area as estimated from HPLC at 215 nm.

^D Concentration giving 50% inhibition of Fusarium krumorum in unbuffered medium (unit: μg / ml). ^e Cyclized and purified product.

^{F in} 10 mM MES buffer medium nd = undecided

[Sequence list]

─────────────────────────────────────────────────── ─── Continued front page    (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K E, LS, MW, MZ, SD, SL, SZ, TZ, UG , ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, C A, CH, CN, CR, CU, CZ, DE, DK, DM , DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, K E, KG, KP, KR, KZ, LC, LK, LR, LS , LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, R U, SD, SE, SG, SI, SK, SL, TJ, TM , TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor SKY Perfect, Wilhelms Martina             Su, Maria             Netherlands Nuel-8200 Arbele             -Ristat, Postbus 65, Dale             Oh Institute Vol de             Eelhouderidge en Dealgazo             Height (72) Inventor Saizuma, Lorke             Netherlands Nuel-6700 Aahwa             ー Henningen, P / O Box             17, Bonesse Stoke 59, Dee Luo             ー Institute Vol Ag             Rotenokgish Ondalzouk (72) Inventor Van Amelongen, Art             Netherlands Nuel-6700 Aahwa             ー Henningen, P / O Box             17, Bonesse Stoke 59, Dee Luo             ー Institute Vol Ag             Rotenokgish Ondalzouk (72) Inventor Phanto and Frankie             Belgium BE-9000 Ghent, Sint             Pietersney Westrath, 25, Yu             Niversity of Gent (72) Inventor Borremans, France Alois Mechani             A             Belgium BE-9000 Ghent, Sint             Pietersney Westrath, 25, Yu             Niversity of Gent (72) Inventor Leeds, Sarah Bronwen             UK RG 42 6 Ewaiba             Kusher, Bracknell, Gerott             Hill Research Station (72) Inventor Osborne, Rupert William             UK N4 47 UH             Norwich, Cornie Lane, Know             Switch Research Park, Plant Bar             Iotech Limited F-term (reference) 2B030 AA02 AB03 AD08 CD07 CD09                 4B024 AA01 BA80 CA01 GA11 HA06                 4H011 AA01 AA03 BA01 BB06 BB21                       DA13 DH11                 4H045 AA10 BA10 BA50 CA30 DA83                       EA29 EA50 FA74

Claims (21)

[Claims] 1. A modified cysteine-containing antimicrobial peptide derived from a plant defensin, wherein the modification introduces (a) one or more cysteine residues and / or (b) replaces one or more cysteine residues. The peptide, comprising or modified to block its ability to form disulfide bridges. 2. The modified cysteine-containing antimicrobial peptide according to claim 1, which is derived from a naturally occurring plant defensin. 3. The peptide is a beta-2 chain / turn / of plant defensin.
The modified cysteine-containing antimicrobial peptide according to claim 1 or 2, which is derived from the beta-3 chain region. 4. A method according to any one of claims 1 to 3, which is derived from a plant defensin having an activity substantially similar to Rs-AFP2 and which exhibits at least 40% sequence similarity to Rs-AFP2. Modified cysteine-containing antimicrobial peptide of. 5. Rs-AFP1, Rs-AFP3, Rs-AFP4, Br
-AFP1, Br-AFP2, Bn-AFP1, Bn-AFP2, Sa-AFP
1, Sa-AFP2 and At-AFP1 and Hs-AFP2, Ah-AMP
The modified cysteine-containing antimicrobial peptide according to claim 4, which is derived from 1 or Dm-AMP1. 6. The modified cysteine-containing antimicrobial peptide according to claim 5, which is derived from Rs-AFP2 or Rs-AFP1. 7. The modified cysteine-containing antimicrobial peptide according to claim 6, which is derived from positions 21 to 51 of Rs-AFP1 or Rs-AFP2. 8. The modified cysteine-containing antimicrobial peptide according to claim 7, wherein the peptide is derived from positions 21 to 51 of the Rs-AFP2 sequence. 9. The modified cysteine-containing antimicrobial peptide according to any one of claims 1 to 8, wherein one or more cysteines are added to the N-terminal and / or C-terminal of the peptide. 10. The modified cysteine-containing antimicrobial peptide of claim 1, and MBG03 CRHGSBNYVFPAHKBIBYC (SEQ ID NO: 21).
), MBG04 CRHGScNYVFPAHKcIBYC (SEQ ID NO: 22),
MBN06 CRHGSCNYVFPAHKCIBYC (SEQ ID NO: 25), MC
H32 and MCH32x HKBIBYC (SEQ ID NO: 4), MCH33 and MCH33x CHKBIBY (SEQ ID NO: 5), MCH37 and MCH37x
HKCIBY (SEQ ID NO: 7), MCH38 and MCH38x HKBICY
(SEQ ID NO: 8), MCI28 and MCI28x CHKBIBYC (SEQ ID NO: 10), MCI10 and MCI10x CHGSBNYVFPAHKBIBC
(SEQ ID NO: 11), MCI11 and MCI11x CHGSBNYVFPAH
KBIB (SEQ ID NO: 12), MCI12 and MCI12x HGSBNYVF
PAHKBIBC (SEQ ID NO: 13), MCI15 and MCI15x HGSC
NYVFPAHKBIB (SEQ ID NO: 15), MCI16 and MCI16xH
GSBNYVFPAHKCIB (SEQ ID NO: 16) and MCI17 and MCI
A modified cysteine-containing antimicrobial peptide selected from 17x HGSBNYVFPAHKBIC (SEQ ID NO: 17). 11. An antimicrobial peptide comprising a plant defensin or a peptide derivative thereof, which comprises one or more cysteine residues in the D configuration. 12. A method for strengthening an antimicrobial peptide, which comprises (a) 1.
Introducing the above cysteine residues, and / or (b) substituting or modifying one or more cysteine residues to modify the peptide structure by blocking the ability to form disulfide bridges, The method. 13. The method of claim 12, wherein the antimicrobial peptide is made more robust by modifying one or more but not all cysteine residues to block its ability to form disulfide bridges. 14. A method for improving the antibacterial activity of an antibacterial peptide derived from plant defensins, which comprises (a) introducing one or more cysteine residues, and / or (
b) A method comprising altering the natural disulfide bridge pattern of a peptide by substituting or modifying one or more but not all cysteine residues to block the ability to form disulfide bridges. 15. A method of combating a fungus by exposing the fungus to the antimicrobial peptide of any one of claims 1-10. 16. A composition comprising the antimicrobial peptide of any one of claims 1-10. 17. Having improved resistance to fungal or microbial pathogens,
A plant comprising a recombinant DNA expressing the antimicrobial peptide according to any one of claims 1 to 10, wherein the peptide comprises an unmodified amino acid. 18. A method according to claims 1 to 1 in the treatment or prevention of microbial infections.
Use of the peptide according to any of 0. 19. The use according to claim 18, wherein the microbial infection is a fungal infection. 20. A DNA sequence encoding the antimicrobial peptide according to any one of claims 1-10. 21. A vector comprising the DNA sequence of claim 17.