Modified antimicrobial peptides

Abstract

Disclosed are antimicrobial defensin peptide variants comprising modified C-terminal fragments of defensins and nucleic acids encoding same. Also disclosed are compositions comprising the defensin variant peptides and methods of their use to control microbial infections in plants and vertebrate subjects, as well as contamination of livestock feed and food.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Patent Application No. 63 / 365,928, filed June 6, 2022, and U.S. Patent Application No. 63 / 202,559, filed June 16, 2021, each of which is incorporated by reference in its entirety herein.

[0002] Statement of Government Support This invention was made with Government support under National Science Foundation EAGER Award Number 1955461 awarded by the National Science Foundation. The Government has certain rights in this invention.

[0003] Array List The sequence listing contained in the file name "P13511WO00_ST25", which is 294,870 bytes as measured on a Windows operating system and was created on June 15, 2022 and submitted electronically herewith, is incorporated by reference in its entirety herein. [Background technology]

[0004] Protecting agriculturally important crops from pathogenic microorganisms (e.g., fungi or oomycetes) is important to improving crop yields. Microbial infections are particularly problematic in humid climates and can be of great concern during crop storage, and such infections can result in spoilage and contamination of food or feed with microbial toxins. Unfortunately, current growing methods, harvesting and storage systems can promote plant pathogen infections.

[0005] Control of plant pathogens is further complicated by the need to simultaneously control multiple microorganisms of separate genera, such as Alternaria, Ascochyta, Aphenomyces, Botrytis, Cercospora, Colletotrichum, Diplodia, Erysiphe, Fusarium, Gaeumanomyces, Helminthosporium, Leptosphaeria, Macrophomina, Magnaporthe, Nectria, Peronospora, Phoma, Phakopsora, and others. Microorganisms such as S. sora, Phymatotrichum, Phytophthora, Plasmopara, Podosphaera, Puccinia, Pythium, Pyrenophora, Pyricularia, Rhizoctonia, Sclerotium, Sclerotinia, Septoria, Thielaviopsis, Uncinula, Venturia, and Verticillium species are all recognized plant pathogens.

[0006] Certain microorganisms (eg, fungi or oomycetes, including molds, yeasts and dimorphic fungi) can also be pathogens of a variety of vertebrate animals, including humans, livestock, companion animals, fish, and the like. Microorganisms including dermatophytes, Aspergillus, Candida, Cryptococcus, Coccidiomyces, Penicillium, Rhizopus, Apophysomyces, Cunninghamella, Saksenaea, Rhizomucor, Syncephalostrum, Cokeromyces, Actinomucor, Pythium, Fusarium, Histoplasmosis or Blastomyces species are also important vertebrate pathogens.

[0007] A group of proteins known as defensins have been shown to inhibit plant pathogens. Defensins were previously identified as small cysteine-rich peptides of approximately 45-54 amino acids that constitute a key component of plant innate immunity (Thomma et al., 2002; Lay and Anderson, 2005; Vriens et al., 2014). Widely distributed in plants, defensins vary widely in amino acid composition. However, they all have a compact shape stabilized by either four or five intramolecular disulfide bonds. Plant defensins have previously been characterized as containing a conserved gamma core peptide that contains a conserved GXCX3-9C (X is any amino acid) sequence (Sagaram et al., 2011; Lacerda et al., 2014). The three-dimensional structure of the previously characterized gamma core peptide consists of two antiparallel β-sheets with an interpolated turn region (ibid.). The antimicrobial activity of certain defensins has been correlated with the presence of positively charged amino acid residues in the gamma core peptide (Spelbrink et al., Plant Physiol., 2004; Sagaram et al., 2013).

[0008] Plant defensins have been widely studied for their role in plant defense. Some plant defensins inhibit the growth of a wide range of microorganisms at micromolar concentrations (Broekaert et al. 1995; Broekaert et al. 1997; da Silva Conceicao and Broekaert, 1999) and confer strong resistance to microbial pathogens when expressed in transgenic plants (da Silva Conceicao and Broekaert, 1999; Thomma et al., 2002; Lay and Anderson, 2005). Two small cysteine-rich proteins, Rs-AFP1 and Rs-AFP2, isolated from radish seeds inhibited the growth of many pathogenic microorganisms when the pure proteins were added to in vitro antimicrobial assay media (U.S. Patent No. 5,538,525). Transgenic tobacco plants containing the gene encoding the Rs-AFP2 protein were found to be more resistant to microbial attack than non-transformed plants.

[0009] Defensin genes have also been identified in the legume Medicago truncatula (Hanks et al., 2005). The cloned MtDef2 protein was shown to have little or no antimicrobial activity through in vitro experiments (Spelbrink et al., 2004). The Medicago truncatula defensin proteins MtDef4 (U.S. Pat. No. 7,825,297; incorporated herein by reference in its entirety) and MtDef5 (WO2014179260 and U.S. Pat. App. Pub. No. 20160208278; both incorporated herein by reference in their entirety) have antimicrobial activity. The C-terminal 16 amino acid GMA4-C peptide of the MtDef4 defensin protein inhibited Fusarium graminearum at concentrations as low as 3 μM ( Sagaram et al., 2011 ).

[0010] Plant defensins with potent antifungal activity in vitro often fail to confer effective disease resistance in planta. This limits their commercial development as antifungals in transgenic crops. Antifungal plant defensins are generally cationic, and cationic residues in their sequences are thought to initiate passage through the fungal cell envelope by electrostatic interactions with the anionic fungal cell membrane (Kerenga et al., 2019). Potassium (K+) is an essential macronutrient and the most abundant cation in plants. The concentration of K+ in the cytoplasm of plant cells is consistently 100-200 mM (Shabala and Pottosin, 2010) and 10-200 mM in the apoplast (White and Karley, 2010). Calcium is an essential secondary micronutrient, and its concentration can range from 0.1% to 6% of the plant dry weight (Broadley et al., 2003). The concentration of sodium (Na+) in plants ranges from 0.001% to 8% (Marschner, 1995). Na+ is an essential micronutrient for plants in saline soils.

[0011] Many plant defensins characterized to date lose their antifungal activity at high concentrations of monovalent and divalent cations, such as 100 mM KCl or 2 mM CaCl. However, the maize plant defensin ZmD32, which has a predicted charge of +10.1 at pH 7, exhibits inhibitory activity against Candida sp. and E. coli in the presence of 100 mM NaCl, while the Nicotiana benthamiana plant defensin NbD6, which has a predicted charge of +7.6 at pH 7, exhibits inhibitory activity against Candida albicans in the presence of 100 mM NaCl (Kerenga et al., 2019). Summary of the Invention [Means for solving the problem]

[0012] Peptides are provided that comprise the sequence XGXCXGFXXXX(F / W / Y)XXXXC (SEQ ID NO:1), wherein the peptide does not comprise the corresponding full-length sequence of a defensin peptide of SEQ ID NO:8, 16, 20, 23, 26, 29, 37, 38, 39, 40 or 41; optionally, the peptide comprises a modified gamma-core consensus sequence GXCX3-8(F / W / Y)(F / W / Y)(F / W / Y) as set forth in SEQ ID NO:33, GXCX3-9(F / W / Y)(F / W / Y)(F / W / Y) as set forth in SEQ ID NO:34, GXCX3-8(F / W / Y) (SEQ ID NO:43) or GXCX3-9(F / W / Y) (SEQ ID NO:44); and / or optionally, the C-terminal cysteine ​​residue or the C-terminal amino acid residue is amidated. A peptide having at least 75%, 82% or 94% sequence identity over the entire length of any one of SEQ ID NOs: 3, 4, 5 or 6 is not identical to SEQ ID NO: 8, and optionally any amino acid substitutions in said sequences improve or maintain a net positive charge at neutral pH and / or improve or maintain the hydrophobicity of the peptide, and optionally the C-terminal cysteine ​​residue is amidated. Peptides having at least 75%, 82% or 94% sequence identity over the entire length of SEQ ID NOs:7, 12, 13, 14, 15, 17-20, 22, 23, 25, 26, 28, 29, 31 and 32 (wherein the peptide is not identical to SEQ ID NOs:8, 16, 20, 23, 26, 29, 37, 38, 39, 40 or 41, optionally SEQ ID NO:5, SEQ ID NOs:13, 14, 15, 18, 19, 20, 22, 23, 26, 28, 29, 31 or 32 contain a disulfide bond between two cysteine ​​residues, optionally any substitutions which improve or maintain a net positive charge at neutral pH and / or improve or maintain the hydrophobicity of the peptide, and optionally the C-terminal cysteine ​​residue or the C-terminal amino acid residue is amidated) are provided.Defensin C-terminal peptide variants are provided that include conserved C1 and C4 cysteine ​​residues corresponding to the N-terminal and C-terminal cysteines of a reference defensin C-terminal peptide, where the conserved C2 and C3 cysteine ​​residues of the reference defensin C-terminal peptide are independently replaced with tryptophan, tyrosine, phenylalanine, leucine, valine, isoleucine, or methionine; optionally the defensin peptide variant has a net positive charge of at least 3, 3.5, 4, 5, or 6 and a hydrophobic amino acid content of at least 18%. Modified gamma-core consensus sequences GXCX3-8(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO: 33), GXCX3-9(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO: 34), GXCX3-8(F / W / Y) (SEQ ID NO: 43), GXCX3-9(F / W / Y) (SEQ ID NO: 44), GXCX3-8(F / W / Y / L / V / I / M) (SEQ ID NO: 45), GXCX3-10(F / GXCX3-8(L / V / I / M)(F / W / Y)(L / V / I / M)(SEQ ID NO:47), GXCX3-10(L / V / I / M)(F / W / Y)(L / V / I / M)(SEQ ID NO:48), GXCX3-8(F / W / Y / L / V / I / M)(F / W / Y)(F / W / Y / L / V / I / M)(SEQ ID NO:49), GXCX3-10(F / W / Y / L / V / I / M)(F / W / Y)(F / W / Y / L / V / I / M) (SEQ ID NO: 50), GXCX3-8(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M) (SEQ ID NO: 51), GXCX3-10(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO: 587), GXCX3-12(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO: 588), GXCX3-15(F / W / GXCX3-10(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(SEQ ID NO:52). In certain embodiments, the peptides exhibit antimicrobial activity, which optionally is one or more of antifungal or antibacterial activity.Also provided are compositions comprising any of the above-described peptides and an agriculturally, pharma- ceutical or veterinarily acceptable carrier, diluent or excipient.Also provided are compositions comprising any of the above-described peptides and a carrier, diluent or excipient for use in treating, preventing or inhibiting a microbial infection in a subject in need thereof.

[0013] Also provided is a method for (i) preventing or reducing crop damage due to plant pathogenic microorganisms or (ii) preventing contamination of a plant, plant part, seed, livestock feed derived therefrom or foodstuff derived therefrom with undesirable microorganisms, comprising the step of contacting a plant, plant seed or other part of said plant with an effective amount of any of the compositions described above, which composition optionally comprises an agriculturally acceptable carrier, diluent or excipient.

[0014] Also provided are plant parts, including seeds, at least partially coated with any of the above-mentioned compositions, where the composition optionally includes an agriculturally acceptable carrier, diluent or excipient.

[0015] Also provided is a medical device comprising a device and the composition described above, the medical device comprising at least one surface that is topically coated and / or immersed in the composition, the composition optionally comprising a pharma- ceutically or veterinarily acceptable carrier, diluent or excipient.

[0016] Also provided is a method for treating, preventing or inhibiting a microbial infection in a subject in need thereof comprising administering to said subject an effective amount of any of the above compositions, which composition optionally comprises a pharma- ceutically or veterinarily acceptable carrier, diluent or excipient. Also provided is the use of any of the above compositions in a method for treating, preventing or inhibiting a microbial or yeast infection in a subject in need thereof. Also provided is the use of any of the above first antimicrobial peptides or proteins in the manufacture of a medicament or composition for inhibiting a microbial or yeast infection in a subject in need thereof.

[0017] Recombinant polynucleotides comprising a polynucleotide, a peptide comprising any of the above-mentioned peptides, wherein a polynucleotide encoding an antimicrobial peptide is operably linked to a polynucleotide comprising a promoter that is heterologous to the polynucleotide encoding said first antimicrobial peptide, and optionally any amino acid substitutions in said sequence that improve or maintain the net positive charge and / or hydrophobicity of the peptide, are provided.

[0018] Provided is a plant nuclear or plastid genome comprising a polynucleotide encoding an antimicrobial peptide, including any of the above-described peptides, wherein the polynucleotide is heterologous to the nuclear or plastid genome, and the polynucleotide is operably linked to an endogenous promoter of the nuclear or plastid genome.

[0019] Plant parts, including cells, plants and seeds, which contain the recombinant polynucleotides or genomes described above are provided. [Brief description of the drawings]

[0020] [Figure 1A-1B]1A and 1B show a drop inoculation assay in Nicotiana benthamiana leaves. Four-week-old leaves of N. benthamiana were used for the drop inoculation assay against B. cinerea with different concentrations of GMA4C peptide. 1A. Pictorial illustration and photographs of the drop inoculation assay were taken after 48 hours of incubation using CropReporter, both in white light. Damaged areas are shown in red in the CropReporter images. Data for the GMA4C_AC wild-type control (SEQ ID NO:2) are in the left-most panel (labeled "GMA4C"), data for GMA4c_V1A (SEQ ID NO:3) are in the second panel from the left, data for GMA4c_V2A (SEQ ID NO:4) are in the third panel from the left, and data for GMA4c_V3A (SEQ ID NO:5) are in the fourth panel from the left. 1B, Lesions were measured 48 hours later using ImageJ software and their sizes are shown graphically. [Diagram 2] Figure 2 shows an alignment of a non-limiting subset of reference defensin C-terminal peptides containing the conserved C1, C2, C3 and C4 cysteines (bold). The conserved gamma core peptide is underlined. [Diagram 3] Figure 3 shows an alignment of a non-limiting subset of reference defensin C-terminal peptides provided herein that contain the conserved C1, C2, C3 and C4 cysteines (bold), the gamma-core consensus and various modified gamma-core consensus sequences. The conserved gamma-core peptides are underlined and are absent from the modified gamma-core consensus peptides. [Figure 4] FIG. 4 shows a table of data involving the antimicrobial activity of various GMA4C defensin peptide variants. [Figure 5A-5B]5A,B show the results of a drop inoculation assay showing the antifungal activity of GMA4C_V9 (SEQ ID NO:581) and GMA4C_V10 peptides (SEQ ID NO:582). GMA4C_V10 completely suppresses Botrytis symptoms at 6 μM. In FIG. 5B, the leftmost bar is for the untreated control ("B. cinerea"), the middle bar is GMA4C_V9 (SEQ ID NO:581) treatment, and the rightmost bar is GMA4C_V10 (SEQ ID NO:582) treatment. At 3 μM and 1.5 μM, GMA4C_V10 is more effective than GMA4C_V9 in reducing Botrytis symptoms. [Figure 6A-6B] 6A,B show the results of a drop inoculation assay showing the antifungal activity of GMAOe1C_WT (SEQ ID NO: 577), GMAOe1C_V3 (SEQ ID NO: 578) and GMAOe1C_V4 (SEQ ID NO: 579). In FIG. 6B, the leftmost bar is for the untreated control ("B. cinerea"), the second bar from the left is for the GMAOe1C_WT treatment, the third bar from the left is for the GMAOe1C_V3 treatment, and the fourth bar from the left is for the GMAOe1C_V4 treatment. GMAOe1C_V3 and GMAOe1C_V4 at 3 μM and 6 μM completely suppress the symptoms of gray mold, while GMAOe1C_WT is only effective at 6 μM. At a concentration of 1.5 μM, GMAOe1C_V3 is more effective than GMAOe1C_V4 or GMAOe1C_WT. [Figure 7A-7B] Figures 7A,B show the results of a drop inoculation assay showing the antifungal activity of GMA1C_V1 (SEQ ID NO: 583) and GMA1C_V2 (SEQ ID NO: 584) against B. cinerea. In Figure 7B, the leftmost bar is for the treatment control ("B. cinerea"), the middle bar is for the GMA1C_V1 (SEQ ID NO: 583) treatment, and the rightmost bar is for the GMA1C_V2 (SEQ ID NO: 584) treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] definition The term "and / or", as used herein, should be construed as a specific disclosure of each of the two specified features or components with or without the other. Thus, when the term "and / or" is used herein in a phrase such as "A and / or B", it is intended to include "A and B", "A or B", "A" (single) and "B" (single). Similarly, when the term "and / or" is used in a phrase such as "A, B and / or C", it is intended to include each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0022] As used herein, the terms "correspond," "corresponding," and the like, when used in reference to amino acid positions, mutations, and / or substitutions in any peptide (e.g., a defensin variant peptide) relative to a reference peptide sequence (e.g., a reference defensin C-terminal peptide sequence including SEQ ID NOs: 8, 16, 20, 23, 26, 29, 37, 38, 39, 40, 41, or 89-122), all refer to amino acid residues in a peptide sequence that have the same position in the peptide as the residue in the reference amino acid sequence when the peptide is aligned with the reference sequence. In certain embodiments, this alignment is of the four conserved cysteine ​​residues of the defensin C-terminal peptides of the defensin variant peptide and the reference defensin C-terminal peptide sequence (e.g., as shown in Figures 2 and 3).

[0023] As used herein, the words "include", "includes" and "including" are to be interpreted as having at least the properties they refer to without excluding any further unspecified properties.

[0024] Where a term is provided in the singular, other embodiments are also provided that are described by the plural of that term.

[0025] The phrase "antimicrobial peptide", as used herein, refers to peptides that exhibit any one or more of the following characteristics: inhibiting microbial cell growth, killing microbial cells, disrupting or delaying a stage of the microbial life cycle such as spore germination, sporulation or conjugation, and / or disrupting microbial cell infection, invasion or spread in plants or other susceptible subjects, including humans, livestock, poultry, fish or companion animals (e.g., dogs or cats).

[0026] As used herein, the terms "acidic" or "anionic" are used interchangeably to refer to amino acids such as aspartic acid and glutamic acid.

[0027] As used herein, the term "amino acid" refers to an organic compound containing amino (-NH3) and carboxylate (-CO2) functional groups, along with a side chain (R group) specific to each amino acid. Amino acid residues in a polypeptide are hereby designated, in certain instances, by the single-letter amino acid code: G-glycine (Gly); P-proline (Pro); A-alanine (Ala); V-valine (Val); L-leucine (Leu); I-isoleucine (Ile); M-methionine (Met); C-cysteine ​​(Cys); F-phenylalanine (Phe); Y-tyrosine (Tyr); W-tryptophan (Trp); H-histidine (His); K-lysine (Lys); R-arginine (Arg); Q-glutamine (Gln); N-asparagine (Asn); E-glutamic acid (Glu); D-aspartic acid (Asp); S-serine (Ser); or T-threonine (Thr).

[0028] As used herein, the terms "basic" and "cationic" are used interchangeably to refer to amino acids such as arginine, histidine and lysine.

[0029] As used herein, the phrase "cation tolerant" refers to a defensin peptide or defensin peptide variant that exhibits equivalent in vitro antifungal or antimicrobial activity or up to about a 1.5-fold, 2-fold, 3-fold or 4-fold reduction in in vitro antifungal or antimicrobial activity in the presence of 100 mM KCl or 100 mM NaCl when compared to the antifungal activity of the defensin peptide or defensin peptide variant in the absence of KCl or NaCl.

[0030] As used herein, the phrase "consensus sequence" refers to an amino acid, DNA, or RNA sequence generated by aligning two or more homologous sequences and deriving a new sequence that has, at each position in the resulting sequence, either a conserved or a series of alternative amino acid, deoxyribonucleic acid, or ribonucleic acid residues of the homologous sequences.

[0031] The phrases "effective against microbial damage", "effective or controlling microbial damage" or "controlling microbial damage" as used herein refer to the reduction of damage to a crop plant or crop plant product due to infection by a microbial pathogen. More generally, these phrases refer to the reduction of adverse effects caused by the presence of a pathogenic microorganism in a crop plant. Adverse effects of microbial growth are understood to include any type of plant tissue damage or necrosis, any type of plant yield reduction, any reduction in the value of the crop plant product, and / or the production of undesirable microbial metabolic products or microbial growth by-products, including mycotoxins.

[0032] The phrase "defensin peptide" is used herein to refer to peptides that contain a conserved gamma core peptide. Plant defensins have previously been characterized as containing the conserved GXCX3-9C gamma core peptide sequence (SEQ ID NO: 9), where X is any amino acid residue (Lacerda et al.), or the conserved GXCX3-10C variant gamma core peptide sequence (SEQ ID NO: 10), where X is any amino acid residue. In certain embodiments, the defensin peptides disclosed herein may also include non-canonical defensin gamma core peptides, including GXCX3-12C (SEQ ID NO: 590) or GXCX3-15C (SEQ ID NO: 591). Thus, as used in this disclosure, a plant defensin or C-terminal peptide comprising a defensin or fragment thereof may comprise the conserved GXCX3-9C (SEQ ID NO: 9), GXCX3-10C (SEQ ID NO: 10), GXCX3-12C (SEQ ID NO: 590) or GXCX3-15C (SEQ ID NO: 591) gamma core peptide sequence, where X is any amino acid residue. Defensin peptides include proteins that are antimicrobial, can permeabilize plasma membranes, can bind phospholipids, can bind sphingolipids, or exhibit any combination of these properties. Defensin peptides can be natural or non-natural (e.g., synthetic and / or chimeric).

[0033] The phrase "defensin peptide variant" refers to (i) at least one amino acid substitution in the conserved gamma core peptide and the parent defensin peptide of SEQ ID NO: 9 or 10; or (ii) the modified gamma core variant sequences GXCX3-8(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO: 33), GXCX3-9(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO: 34), GXCX3-8(F / W / Y) (SEQ ID NO: 43), GXCX3-9(F / W / Y)(SEQ ID NO: 44), GXCX3-8(F / W / Y)(SEQ ID NO: 45), GXCX3-9(F / W / Y)(SEQ ID NO: 46), GXCX3-8(F / W / Y)(SEQ ID NO: 47), GXCX3-9(F / W / Y)(SEQ ID NO: 48), GXCX3-8(F / W / Y)(SEQ ID NO: 49), GXCX3-9(F / W / Y)(SEQ ID NO: 50), GXCX3-8(F / W / Y)(SEQ ID NO: 51), GXCX3-9(F / W / Y)(SEQ ID NO: 52), GXCX3-8(F / W / Y)(SEQ ID NO: 53), GXCX3-9(F / W / Y)(SEQ ID NO: 54), GXCX3-8(F / W / Y)(SEQ ID NO: 55), GXCX3-8(F / W / Y)(SEQ ID NO: 56), GXCX3-8(F / W / Y)(SEQ ID NO: 57), GXCX3-8(F / W / Y)(SEQ ID NO: 59), GXCX3-9(F No. 44), GXCX3-8(F / W / Y / L / V / I / M) (SEQ ID NO: 45), GXCX3-10(F / W / Y / L / V / I / M) (SEQ ID NO: 46), GXCX3-8(L / V / I / M)(F / W / Y)(L / V / I / M) (SEQ ID NO: 47), GXCX3-10(L / V / I / M)(F / W / Y)(L / V / I / M) (SEQ ID NO: 48), GXCX3-8(F / W / Y / L / V / I / M)(F / W / Y)(F / W / Y / L / V / I / M) (SEQ ID NO: 49), GXCX3-10(F / W / Y / L / V / I / GXCX3-8(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(SEQ ID NO:51), GXCX3-10(F / W / Y)(F / W / Y)(F / W / Y)(SEQ ID NO:587), GXCX3-12(F / W / Y)(F / W / Y)(F / W / Y)(SEQ ID NO:588), GXCX3-15(F / W / Y)(F / W / Y)(F / W / Y)((SEQ ID NO:589) or GXCX3-10(F / W (F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M) (SEQ ID NO: 52). In certain embodiments, the defensin peptide variants provided herein are less than a full-length defensin peptide (e.g., a peptide comprising, consisting essentially of, or consisting of (i) 30 amino acid residues or less; or (ii) between 15, 16, or 17 and 30 amino acid residues).

[0034] The phrase "reference defensin C-terminal peptide" is used herein to refer to a less than full-length defensin peptide that includes the conserved GXCX3-9C gamma core peptide sequence (SEQ ID NO: 9), the conserved GXCX3-10C variant gamma core peptide sequence (SEQ ID NO: 10), the non-canonical GXCX3-12C (SEQ ID NO: 590) gamma core sequence or the non-canonical GXCX3-15C (SEQ ID NO: 591) gamma core sequence and two additional conserved cysteine ​​residues located C-terminal to the gamma core peptide sequence (wherein the cysteines located closest to the N-terminus of the reference defensin C-terminal peptide correspond to the cysteines located closest to the N-terminus of the gamma core sequence of SEQ ID NO: 9 or SEQ ID NO: 10). Examples of reference defensin C-terminal peptides include MtDef4 C-terminal peptide (SEQ ID NO: 8), MtDef5A C-terminal peptide (SEQ ID NO: 16), MtDef5B C-terminal peptide (SEQ ID NO: 41), MtDef6 peptide (SEQ ID NO: 21), OeDef1 C-terminal peptide (SEQ ID NO: 24), OeDef7 C-terminal peptide (SEQ ID NO: 27), SbDef1 C-terminal peptide (SEQ ID NO: 30), NaD1 C-terminal peptide (SEQ ID NO: 37), RsAFP2 C-terminal peptide (SEQ ID NO: 38), DmAMP1 C-terminal peptide (SEQ ID NO: 39), and HsAFP1 (SEQ ID NO: 40). Other examples of reference defensin C-terminal peptides include SEQ ID NOs: 89-122. Thus, the reference defensin C-terminal peptide contains four conserved cysteine ​​residues, designated herein as C1 (most N-terminal in both peptides and in the gamma core peptide sequence), C2 (located C-terminal to C1 in the gamma core peptide sequence), C3 (C-terminal to C2) and C4 (C-terminal to C3). An alignment of a non-limiting subset of reference defensin C-terminal peptides containing the conserved gamma core peptide sequence and the C1, C2, C3 and C4 cysteines is shown in Figure 2.An alignment of a non-limiting subset of reference defensin C-terminal peptides including the conserved gamma-core peptide sequence and the gamma-core consensus including the C1, C2, C3 and C4 cysteines, the C1 and C2 cysteines, and certain modified gamma-core consensus sequences provided herein is shown in Figure 3. Other reference defensin C-terminal peptides including those contained in the full-length defensin peptides of SEQ ID NOs: 123-434 and 436-576.

[0035] As used herein, the terms "edit," "editing," "edited," and the like refer to processes or products in which insertions, deletions, and / or nucleotide substitutions are introduced into a genome. Such processes include methods of inducing homology directed repair and / or non-homologous end joining at one or more sites in the genome.

[0036] The phrases "gene-edited plant" or "edited plant" are used herein to refer to a plant that contains one or more nucleotide insertions, deletions, substitutions, or any combination thereof in the genomic DNA of the plant. Such gene-edited plants can be constructed by techniques including CRISPR / Cas endonuclease-mediated editing, meganuclease-mediated editing, engineered zinc finger endonuclease-mediated editing, and the like.

[0037] The term "heterologous", as used herein, in reference to a second polynucleotide operably linked to a first polynucleotide, refers to: (i) a second polynucleotide that is derived from a source different from that of the first polynucleotide; (ii) a second polynucleotide that is derived from the same source as the first polynucleotide, where the first, second or both polynucleotide sequences have been modified from their original form; (iii) a second polynucleotide that is positioned in an order and / or orientation or in a genomic location or environment relative to the first polynucleotide that is different from the order and / or orientation in the genomic location or environment of the first and second polynucleotides in the native cell; or (iv) the second polynucleotide does not occur in the native cell that contains the first polynucleotide. Heterologous polynucleotides include polynucleotides that facilitate transcription (e.g., promoter and enhancer elements), transcript abundance (e.g., introns, 5'UTR and 3'UTR), translation, or combinations thereof, as well as polynucleotides that encode defensin peptide variants or defensin peptides, spacer peptides, or localization peptides. In certain embodiments, the nuclear or plastid genome may comprise a first polynucleotide and a second polynucleotide is heterologous to the nuclear or plastid genome. A "heterologous" polynucleotide that enhances transcription, transcript amount, translation, or a combination thereof, and a polynucleotide that encodes a defensin peptide variant or a defensin peptide, a spacer peptide, or a localization peptide may be autologous to the cell, but may be located in a different order and / or orientation in a genomic location or environment than in the native cell. A polynucleotide that enhances transcription, transcript amount, translation, or a combination thereof, and a polynucleotide that encodes a defensin peptide variant or a defensin peptide, a spacer peptide, or a localization may be heterologous to another polynucleotide when the polynucleotides are not operably linked to each other in the native cell. A heterologous peptide or protein includes a peptide or protein that is not found in a cell or organism because it does not occur in the cell or organism in nature.Thus, a heterologous peptide or protein includes a peptide or protein that is localized to or expressed in a subcellular or extracellular location or tissue that is different from the subcellular or extracellular location or tissue in which the peptide is found in the cell or organism when it occurs in nature. A heterologous polynucleotide includes a polynucleotide that is not found in a cell or organism when it occurs in nature.

[0038] The phrases "inhibit the growth of plant pathogenic microorganisms", "inhibit microbial growth" and the like, as used herein, refer to a method that results in any measurable reduction in microbial growth, including any measurable reduction in the number and / or extent of microbial cells, spores, conidia, or mycelium. As used herein, "inhibit the growth of plant pathogenic microorganisms" is also understood to include any measurable reduction in the adverse effects caused by microbial growth in plants. Adverse effects of microbial growth in plants include any type of plant tissue damage or necrosis, any type of reduction in plant yield, any reduction in the value of crop plant products, and / or the production of undesirable microbial metabolic products or microbial growth by-products, including mycotoxins. As used herein, the phrases "inhibit microbial growth" and the like, unless otherwise indicated, may include inhibition in plants, humans, or animals.

[0039] The phrase "percent identity" or "sequence identity," as used herein, refers to the number of elements in sequences (i.e., amino acids or nucleotides) that are identical within two DNA, RNA segments of a defined length in an alignment that maximizes the number of identical elements, and is calculated by dividing the number of identical elements by the total number of elements in the defined length of the aligned segments and multiplying by 100.

[0040] The phrase "transgenic" refers to an organism or its progeny, in which the nuclear or organellar genome of the organism or the progeny organism's DNA contains an insertion of an exogenous DNA molecule that is 10 or more nucleotides in length. The phrase "transgenic plant" refers to a plant or its progeny, in which the nuclear or plastid genome of the plant or the progeny plant's DNA contains the introduction of an exogenous DNA molecule that is 10 or more nucleotides in length. Such introduced exogenous DNA molecules can be natural, non-natural (e.g., synthetic and / or chimeric), from heterologous or autologous sources.

[0041] To the extent that any of the foregoing definitions does not match a definition provided in any patent or non-patent reference incorporated herein by reference, any patent or non-patent reference cited herein, or found elsewhere, it is understood that the foregoing definitions are used herein.

[0042] Further explanation Antimicrobial peptides and proteins, referred to as defensin peptide variants, are provided herein. The antimicrobial peptides and proteins can be applied directly to plants, livestock feed, or food; they can be applied to plants in the form of microorganisms that produce the defensin peptide variants or proteins, or the plants can be genetically edited to produce the defensin peptide variants or proteins. The present disclosure also relates to recombinant or edited polynucleotides, microorganisms and plants transformed with recombinant or edited polynucleotides, plants that contain genetically edited nuclear or plastid genomes that encode the defensin peptide variants and proteins, and compositions that include the defensin peptide variants and proteins that are useful in controlling pathogenic microorganisms, including plant pathogenic microorganisms. In certain embodiments, defensin variant proteins that include two defensin peptide variants or a defensin peptide variant and another peptide (including a defensin peptide variant or a defensin peptide) can provide improved inhibition of microbial growth when compared to proteins that contain only one of the antimicrobial peptides found in the defensin variant protein. In certain embodiments, the defensin peptide variants and proteins provided herein are cation-tolerant. Such cation-tolerant defensins may be more effective than cation-sensitive defensins in providing effective control of plant pathogenic microorganisms in transgenic crops. The cation-tolerant defensins provided herein may function in the normal cation-rich physiological environment of plant tissues (e.g., inhibiting plant pathogenic microorganisms, including fungal pathogens). The cation-tolerant defensins provided herein may also function in the normal cation-rich physiological environment of a subject (e.g., a human or animal) infected with a pathogenic microorganism (e.g., inhibiting pathogenic microorganisms, including fungal pathogens).

[0043] Provided herein is a recombinant polynucleotide comprising a polynucleotide encoding a first antifungal peptide operably linked to a polynucleotide comprising a promoter heterologous to the polynucleotide encoding the first antifungal protein. In certain embodiments, the first antifungal peptide is a defensin peptide variant.

[0044] The defensin peptide variant has the sequence: Xaa1-G-Xaa3-C-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Xaa11-Xaa12-Xaa13-Xaa14-Xaa15-Xaa16-Xaa17-Xaa18-Xaa19-Xaa20-Xaa21-Xaa22-Xaa23, where Xaa1 is F, G or absent. Xaa3 is A, G, Y, H, S, R or Dab; ​​Xaa5 is H, L, S, N, R, K or Dab; ​​Xaa6, Xaa7, Xaa8, Xaa9, Xaa10, Xaa11, Xaa12, Xaa13, Xaa14 and Xaa15 are each independently R, K, H, N, D, F, G, Q, P, A, I, L, V, Y, S or Dab; , one or more of Xaa9, Xaa10, Xaa11, Xaa12, Xaa13, Xaa14, and / or Xaa15 may be absent; Xaa16 is C, V, W, Y, or F; Xaa17 is L, I, W, Y, or F; Xaa18 is C, V, W, Y, or F; Xaa19 is K, T, F, W, or Y; Xaa20 is R, K, T, Xaa21 is N, H, K, R, P, Q, I, F, W or Y; Xaa22 is N, H, K, P, Q, I, F, W, Y or absent; Xaa23 is C, optionally amidated, and Dab is diaminobutyric acid) (SEQ ID NO:1). In certain embodiments, a peptide comprising SEQ ID NO:1 may further comprise one or more additional amino acids present C-terminally to the C-terminus of SEQ ID NO:1, wherein said C-terminal amino acid is optionally amidated. In certain embodiments, a peptide comprising SEQ ID NO:1 may comprise a modified gamma core variant sequence GXCX3-8(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO:33), GXCX3-9(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO:34).GXCX3-8(F / W / Y) (SEQ ID NO:43) or GXCX3-9(F / W / Y) (SEQ ID NO:44).Defensin peptide variants also include peptides encompassed by the sequence: Xaa1-GRC-Xaa5-GFRRR(F / W / Y)(F / W / Y)(F / W / Y)XXXC-(NH2), where Xaa1 is F, G or absent; Xaa5 is R or K; Xaa14 is T, F, W or Y; Xaa15 is R, K or Dab; ​​Xaa16 is I, F, W or Y; Xaa17 is C, optionally amidated, where Dab is diaminobutyric acid (SEQ ID NO: 14). The defensin peptide variant has the sequence Xaa1-G-Xaa2-C-Xaa5-Xaa6-Xaa7-GF-Xaa10-Xaa11-Xaa12-(F / W / Y)(F / W / Y)(F / W / Y)-Xaa16-Xaa17-Xaa18-C-(NH2), where Xaa1 is F, G or absent, Xaa3 is A, F, W, Y or absent; Xaa5 is H, R or K; and Xaa6 is Xaa7 is Q, R or K; Xaa10 is G, R or K; Xaa11 is F, W, Y, R or K; Xaa12 is A, F, W, Y, R or K; Xaa16 is F, W, Y, R or K; Xaa17 is R or K; Xaa18 is R or K; the C-terminal cysteine ​​is optionally amidated) (SEQ ID NO: 17). The defensin variant peptides provided herein do not include the corresponding full-length sequence of the wild-type defensin peptide of SEQ ID NO: 8, 16, 20, 23, 26, 29, 37, 38, 39, 40 or 41. Table 1 shows the defensin peptide variants of SEQ ID NO: 1, 3-7 11, 13 and 14 and the GMA4C peptide of SEQ ID NO: 2 and SEQ ID NO: 8. In certain embodiments, the defensin peptide variants described above (i) have 30 amino acid residues or less; or (ii) comprise, consist essentially of, or consist of 15, 16, or 17-30 amino acid residues.

[0045] [Table 1]

[0046]

Table 2

[0047]

Table 3

[0048] In certain embodiments, the defensin peptide variants of the disclosure are characterized in that they contain a defensin gamma-core peptide, which is involved in the antifungal activity of plant defensins. Gamma-core peptides generally contain a net positive charge and have at least one hydrophobic amino acid. In certain embodiments, the defensin peptide variants may contain a gamma-core consensus sequence of GXCX3-9C, where X is any amino acid (SEQ ID NO: 9). In certain embodiments, the defensin peptide variants contain a gamma-core peptide with a variant gamma-core consensus sequence of GXCX3-10C, where X is any amino acid (SEQ ID NO: 10). In certain embodiments, the defensin peptide variants contain a gamma-core consensus sequence of GXCX3-9C (SEQ ID NO: 9) or GXCX3-10C (SEQ ID NO: 10); where X is preferentially selected from cationic and / or hydrophobic amino acids. In certain embodiments, the defensin peptide variant comprises SEQ ID NO:1 and includes the gamma-core consensus sequence of GXCX3-9C (SEQ ID NO:9) or GXCX3-10C (SEQ ID NO:10); where X is preferentially selected from cationic and / or hydrophobic amino acids, and where any of the variable (Xaa) amino acid residues of SEQ ID NO:1 include amino acid residues that maintain or increase the number of cationic and / or hydrophobic amino acids found in SEQ ID NOs:3-7. It is believed that the gamma-core peptide is involved in phospholipid- and / or sphingolipid-binding, while specific amino acids outside of the gamma-core motif are also involved in phospholipid- and sphingolipid-binding. With respect to SEQ ID NO:1, the sequence between the first cysteine ​​(C1) and the second cysteine ​​(C2) from the amino terminus (or the corresponding region in certain embodiments in the defensin peptide variant) also contributes to antimicrobial activity.

[0049] Defensin peptide variants, including the variant defensin C-terminal peptides provided herein, can also include one or more substitutions of a conserved cysteine ​​residue of a wild-type defensin C-terminal peptide sequence. The conserved cysteine ​​residues that can be substituted can correspond to C1, C2, C3, or C4 in the defensin C-terminal peptide, where C1 is the most N-terminal cysteine, C2 is the second most N-terminal cysteine, C3 is the third most N-terminal cysteine, and C4 is the most C-terminal cysteine; see FIG. 2). A non-limiting subset of reference defensin C-terminal peptides containing the conserved cysteines at C1, C2, C3 and C4 include the GMA4C peptide of SEQ ID NO:8, the MtDef5A peptide of SEQ ID NO:16, the MtDef5A peptide of SEQ ID NO:41, the MtDef6 peptide of SEQ ID NO:20, the OeDef1 peptide of SEQ ID NO:23, the OeDef7 peptide of SEQ ID NO:26, the SbDef1 peptide of SEQ ID NO:29, the NaD1 C-terminal peptide (SEQ ID NO:37), the RsAFP2 C-terminal peptide (SEQ ID NO:38), the DmAMP1 C-terminal peptide (SEQ ID NO:39) and the HsAFP1 (SEQ ID NO:40), which may be substituted at least at the C2 and / or C3 positions to obtain defensin peptide variants. Further non-limiting reference defensin C-terminal peptides containing the conserved cysteines at C1, C2, C3 and C4 include the defensin C-terminal peptides set forth in SEQ ID NOs:89-122. Additional non-limiting reference defensin C-terminal peptides containing the conserved cysteines at C1, C2, C3 and C4 include the defensin C-terminal peptides contained in the full-length defensin peptides of SEQ ID NOs: 123-434 and 436-576.

[0050] In certain embodiments, the defensin peptide variants provided herein lack the canonical gamma-core consensus sequence of GXCX3-9C (SEQ ID NO: 9) or GXCX3-10C (SEQ ID NO: 10). In certain embodiments, the defensin peptide variants provided herein comprise a substitution of tryptophan, tyrosine, or phenylalanine for the C-terminal cysteine ​​residue of the gamma-core consensus sequence of SEQ ID NO: 9 or 10 (e.g., C2 in reference SEQ ID NOs: 8, 16, 20, 23, 26, 29, 37, 38, 39, 40, 41, or 89-122). In certain embodiments, the defensin peptide variants provided herein comprise substitutions of both the C2 and C3 residues corresponding to the C2 and C3 residues in the reference C-terminal defensin peptide (e.g., SEQ ID NOs: 8, 16, 20, 23, 26, 29, 37, 38, 39, 40, 41, or 89-122). A GMA4C variant with valine substitutions at C2 and C3 is set forth in SEQ ID NO: 5. A GMA4C variant with tryptophan substitutions at C2 and C3 is set forth in SEQ ID NO: 13. In certain embodiments, the defensin peptide variants provided herein comprise tryptophan, tyrosine, or phenylalanine substitutions at the C2 and C3 residues corresponding to the C2 and C3 residues in a reference C-terminal defensin peptide (e.g., SEQ ID NOs: 8, 16, 20, 23, 26, 29, 37, 38, 39, 40, 41, or 89-122). In certain embodiments, the defensin peptide variants provided herein may further comprise tryptophan, tyrosine, or phenylalanine residues at amino acid positions located between the C2 and C3 residues in a reference C-terminal defensin peptide (e.g., SEQ ID NOs: 8, 16, 20, 23, 26, 29, 37, 38, 39, 40, 41, 89-122).Defensin peptide variants provided herein that contain tryptophan, tyrosine, or phenylalanine substitutions of the C2 and C3 residues corresponding to the C2 and C3 residues in a reference C-terminal defensin peptide include modified gamma core variant sequences GXCX3-8(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO: 33), GXCX3-9(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO: 34), GXCX3-10(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO: 587), GXCX3-11(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO: 590), GXCX3-12(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO: 592), GXCX3-13(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO: 593), GXCX3-14(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO: 594), GXCX3-15(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO: 595), GXCX3-16(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO: 596), GXCX3-17(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO: 597), GXCX3-18(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO: 598), GXCX3-19(F / W / Y)(F / W / Y) GXCX3-12(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO: 588), GXCX3-15(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO: 589), GXCX3-8(F / W / Y) (SEQ ID NO: 43) or GXCX3-9(F / W / Y) (SEQ ID NO: 44). In certain embodiments, the above-mentioned defensin peptide variants comprise, consist essentially of, or consist of (i) 30 amino acid residues or less; or (ii) between 15, 16, or 17 and 30 amino acid residues.In certain embodiments, the defensin peptide variants described above comprise, consist essentially of, or consist of a peptide corresponding to the C-terminus of a defensin peptide containing the conserved cysteines at C1, C2, C3, and C4 (e.g., as contained in the full-length defensin peptides of SEQ ID NOs: 8, 16, 20, 23, 26, 29, 37, 38, 39, 40, 41, 89-122, or SEQ ID NOs: 123-434 and 436-576), wherein the gamma core variant sequence GXCX3-8(F / W / Y)(F / W / Y) is modified as a result of tryptophan, tyrosine, or phenylalanine substitutions of the C2 and C3 residues corresponding to the C2 and C3 residues in the reference C-terminal defensin peptide. Defensin variant peptides can be obtained that include GXCX3-9(F / W / Y)(F / W / Y)(F / W / Y)(SEQ ID NO:34), GXCX3-10(F / W / Y)(F / W / Y)(F / W / Y)(SEQ ID NO:587), GXCX3-12(F / W / Y)(F / W / Y)(F / W / Y)(SEQ ID NO:588), GXCX3-15(F / W / Y)(F / W / Y)(F / W / Y)((SEQ ID NO:589), GXCX3-8(F / W / Y)(SEQ ID NO:43) or GXCX3-9(F / W / Y)(SEQ ID NO:44), optionally including peptides having exactly two cysteine ​​residues corresponding to the conserved C1 and C4 cysteines of the reference defensin C-terminal peptide.

[0051] In certain embodiments, additional defensin peptide variants are provided that lack the canonical gamma-core consensus sequence of GXCX3-9C (SEQ ID NO: 9) or GXCX3-10C (SEQ ID NO: 10). In certain embodiments, the defensin peptide variants provided herein comprise a substitution of tryptophan, tyrosine, phenylalanine, leucine, isoleucine, valine, or methionine for the C-terminal cysteine ​​residue of the gamma-core consensus sequence of SEQ ID NO: 9 or 10 (e.g., C2 contained in reference SEQ ID NOs: 8, 16, 20, 23, 26, 29, 37, 38, 39, 40, 41, or 89-122 or in the full-length defensin peptides of SEQ ID NOs: 123-434 and 436-576). In certain embodiments, the defensin peptide variants provided herein include independent substitutions of both the C2 and C3 residues corresponding to the C2 and C3 residues in a reference C-terminal defensin peptide (e.g., contained within the full-length defensin peptides of SEQ ID NOs: 8, 16, 20, 23, 26, 29, 37, 38, 39, 40, 41, or 89-122, or SEQ ID NOs: 123-434 and 436-576) with tryptophan, tyrosine, phenylalanine, leucine, isoleucine, valine, or methionine residues. In certain embodiments, the defensin peptide variants provided herein include substitutions of C2 and C3 residues corresponding to the C2 and C3 residues in a reference C-terminal defensin peptide (e.g., contained within the full-length defensin peptides of SEQ ID NOs: 8, 16, 20, 23, 26, 29, 37, 38, 39, 40, 41, or 89-122, or SEQ ID NOs: 123-434 and 436-576) with tryptophan, tyrosine, phenylalanine, leucine, isoleucine, valine, or methionine residues.In certain embodiments, the defensin peptide variants provided herein may further comprise a tryptophan, tyrosine, phenylalanine, leucine, isoleucine, valine or methionine residue at an amino acid position located between the C2 and C3 residues in a reference C-terminal defensin peptide (e.g., contained within the full-length defensin peptides of SEQ ID NOs: 8, 16, 20, 23, 26, 29, 37, 38, 39, 40, 41, 89-122, or SEQ ID NOs: 123-434 and 436-576). Defensin peptide variants as provided herein that contain independent tryptophan, tyrosine, phenylalanine, leucine, isoleucine, valine or methionione substitutions of the C2 and C3 residues corresponding to the C2 and C3 residues in a reference C-terminal defensin peptide result in modified gamma core variant sequences GXCX3-10(L / V / I / M)(F / W / Y)(L / V / I / M) (SEQ ID NO: 48), GXCX3-8(F / W / Y / L / V / I / M)(F / W / Y)(L / V / I / M) (SEQ ID NO: 49), GXCX3-9(F / W / Y / L / V / I / M)(F / W / Y)(L / V / I / M) (SEQ ID NO: 50), GXCX3-10(L / V / I / M)(F / W / Y)(L / V / I / M) (SEQ ID NO: 51), GXCX3-11(L / V / I / M)(F / W / Y)(L / V / I / M) (SEQ ID NO: 52), GXCX3-12(F / W / Y / L / V / I / M)(F / W / Y)(L / V / I / M) (SEQ ID NO: 53), GXCX3-13(F / W / Y / L / V / I / M)(F / W / Y)(L / V / I / M) (SEQ ID NO: 54), GXCX3-14(F / W / Y / L / V / I / M)(F / W / Y)(L / V / I / M) (SEQ ID NO: 55), GXCX3-15(F / W / Y / L / V / I / M)(F / W / Y)(L / V / I / M) (SEQ ID NO: 56), GXCX3-16(F / W / )(F / W / Y / L / V / I / M) (SEQ ID NO: 49), GXCX3-10(F / W / Y / L / V / I / M)(F / W / Y)(F / W / Y / L / V / I / M) (SEQ ID NO: 50), GXCX3-8(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M) (SEQ ID NO: 51) or GXCX3-10(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M) (SEQ ID NO: 52). In certain embodiments, the defensin peptide variants described above (i) have 30 amino acid residues or less; or (ii) comprise, consist essentially of, or consist of 15, 16, or 17-30 amino acid residues.In certain embodiments, the defensin peptide variants described above comprise, consist essentially of, or consist of peptides corresponding to the C-terminus of a defensin peptide comprising the conserved C1, C2, C3 and C4 cysteines (e.g., as contained in the full-length defensin peptides of SEQ ID NOs: 8, 16, 20, 23, 26, 29, 37, 38, 39, 40, 41, 89-122, or SEQ ID NOs: 123-434 and 436-576), where independent tryptophan, tyrosine, phenylalanine, leucine, valine, isoleucine or methionine substitutions of the C2 and C3 residues corresponding to the C2 and C3 residues in the reference C-terminal defensin peptide may result in a defensin variant peptide comprising a modified gamma core variant sequence of SEQ ID NO: 48, 49, 50, 51 or 52, optionally comprising a peptide having exactly two cysteine ​​residues corresponding to the conserved C1 and C4 cysteines of the reference defensin C-terminal peptide.

[0052] In certain embodiments, the defensin peptide variant comprises an amino acid sequence having at least 70%, 75%, 80%, 82%, 85%, 90%, 92%, 94%, 95% or 100% sequence identity over the entire length of SEQ ID NO:3-7, 12-15, 17-20, 22, 23, 25, 26, 28, 29, 31, 32, 42, 578, 579, 581, 582, 584 or 585, wherein the peptide does not include the corresponding full length or C-terminal sequence of a defensin peptide of SEQ ID NO:8, 16, 20, 23, 26, 29, 37, 38, 39, 40, 41, 123-434 or 436-576. In certain embodiments, such defensin peptide variants having at least 70%, 75%, 80%, 82%, 85%, 90%, 92%, 94%, 95% or 100% sequence identity over the entire length of SEQ ID NO:3-7, 12-15, 17-20, 22, 23, 25, 26, 28, 29, 31, 32, 42, 578, 579, 581, 582, 584 or 585 further comprise the gamma-core consensus sequence of GXCX3-9C (SEQ ID NO:9) or GXCX3-10C (SEQ ID NO:10); where X is any amino acid or X is selected from cationic and / or hydrophobic amino acids.In certain embodiments, such defensin peptide variants having at least 70%, 75%, 80%, 82%, 85%, 90%, 92%, 94%, 95% or 100% sequence identity over the entire length of SEQ ID NOs: 3-7, 12-15, 17-20, 22, 23, 25, 26, 28, 29, 31, 32, 42, 578, 579, 581, 582, 584 or 585 include modified gamma core variant sequence GXCX3-8(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO: 33), GXCX3-9(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO: 34), GXCX3-10(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO: 587), GXCX3-12(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO: 588), GXCX3-15(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO: 589), GXCX3-8(F / W / Y) (SEQ ID NO: 43 ...9), GXCX3-10(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO: 589), GXCX3-10(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO: 589), GXCX3-10(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO: 589), GXCX3-10(F / W / Y)(F X3-9(F / W / Y) (SEQ ID NO: 44), GXCX3-8(F / W / Y / L / V / I / M) (SEQ ID NO: 45), GXCX3-10(F / W / Y / L / V / I / M) (SEQ ID NO: 46), GXCX3-8(L / V / I / M)(F / W / Y)(L / V / I / M) (SEQ ID NO: 47), GXCX3-10(L / V / I / M)(F / W / Y)(L / V / I / M) (SEQ ID NO: 48), GXCX3-8(F / W / Y / L / V / I / M)(F / W / Y)(F / W / Y / L / V / I / M) (SEQ ID NO: 49), GXCX3-10(F / W / Y / L / V / I / M)(F / W / Y)(F / W / Y / L / V / I / M) (SEQ ID NO: 50), GXCX3-8(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M) (SEQ ID NO: 51) or GXCX3-10(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M) (SEQ ID NO: 52).In certain embodiments, such defensin peptide variants have at least 70%, 75%, 80%, 82%, 85%, 90%, 92%, 94%, 95% or 100% sequence identity over the entire length of SEQ ID NOs: 3-7, 2-15, 17-20, 22, 23, 25, 26, 28, 29, 31, 32, 42, 578, 579, 581, 582, 584 or 585, and have a net positive charge of at least about 5.8 at neutral pH and / or a hydrophobicity percentage of at least about 15%. In certain embodiments, the first structural characteristic of the defensin peptide variant is a net positive charge at neutral pH. In certain embodiments, the defensin peptide variant has a net positive charge of at least +2, +3, +3.5, +4, +5, +6, +7, +8, +9 or +10 at neutral pH. In certain embodiments, the defensin peptide variants have a net positive charge of at least +3, +3.5, +4, +5, +6, or +7 to about +8, +9, or +10 at neutral pH. In certain embodiments, the hydrophobicity percentage of such defensin peptide variants is at least about 15%-30%, about 16%-19%, or about 28%-30%. In certain embodiments, the defensin peptide variants described above comprise, consist essentially of, or consist of (i) 30 amino acid residues or less; or (ii) 15, 16, or 17 to 30 amino acid residues. In certain embodiments, any of the defensin peptide variants described above includes peptides having exactly two cysteine ​​residues, optionally corresponding to the conserved C1 and C4 cysteines of a reference defensin C-terminal peptide.

[0053] In certain embodiments, the defensin peptide variants contain amino acid substitutions that improve or maintain the net positive charge of the peptide at neutral pH and / or improve or maintain the hydrophobicity of the peptide. Amino acid substitutions in SEQ ID NO:3-7, 12-15, 17-20, 22, 23, 25, 26, 28, 29, 31, 32, 42, 578, 579, 581, 582, 584 or 585 that can maintain a net positive charge of the peptide at neutral pH include substitutions of the lysine, arginine or Dab (diaminobutyric acid) residue in SEQ ID NO:3-7, 12-15, 17-20, 22, 23, 25, 26, 28, 29, 31, 32, 42, 578, 579, 581, 582, 584 or 585 with a different amino acid residue selected from the group consisting of lysine, arginine or Dab (diaminobutyric acid) or other unnatural amino acids that are positively charged at neutral pH. Amino acid substitutions in SEQ ID NO:3-7, 12-15, 17-20, 22, 23, 25, 26, 28, 29, 31, 32, 42, 578, 579, 581, 582, 584 or 585 that may increase the net positive charge at neutral pH include substitution of a polar (e.g., cysteine ​​or threonine) or non-polar (e.g., glycine) residue in SEQ ID NO:3-7, 12, 12-15, 17-20, 22, 23, 25, 26, 28, 29, 31, 32, 42, 578, 579, 581, 582, 584 or 585 with a different amino acid residue selected from the group consisting of lysine, arginine, Dab (diaminobutyric acid) or other unnatural amino acid residues that are positively charged at neutral pH. Amino acid substitutions in SEQ ID NO: 3 to 7, 12 to 15, 17 to 20, 22, 23, 25, 26, 28, 29, 31, 32, 42, 578, 579, 581, 582, 584, or 585 that can maintain the hydrophobicity of the peptide include substitutions of glycine, valine, phenylalanine, or isoleucine residues in SEQ ID NO: 3 to 7, 12 to 15, 17 to 20, 22, 23, 25, 26, 28, 29, 31, 32, 42, 578, 579, 581, 582, 584, or 585 with different amino acid residues selected from the group consisting of glycine, alanine, valine, leucine, phenylalanine, isoleucine, or methionine.Amino acid substitutions in SEQ ID NO: 3-7, 12-15, 17-20, 22, 23, 25, 26, 28, 29, 31, 32, 42, 578, 579, 581, 582, 584 or 585 that may increase the hydrophobicity of the peptide include substitutions of polar (e.g., cysteine ​​or threonine) residues in SEQ ID NO: 3-7, 12-15, 17-20, 22, 23, 25, 26, 28, 29, 31, 32, 42, 578, 579, 581, 582, 584 or 585 with different amino acid residues selected from the group consisting of glycine, alanine, valine, leucine, phenylalanine or isoleucine. In certain embodiments, such substitutions that improve or maintain the net positive charge or hydrophobicity of the peptide include defensin peptide variants having the gamma-core consensus sequence of GXCX3-9C (SEQ ID NO: 9) or GXCX3-10C (SEQ ID NO: 10). In certain embodiments, such substitutions that improve or maintain the net positive charge or hydrophobicity of the peptide are made to the modified gamma core variant sequences GXCX3-8(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO:33) or GXCX3-9(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO:34), GXCX3-10(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO:587), GXCX3-12(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO:588), GXCX3-15(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO:589) GXCX3-8( F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO:33), GXCX3-8(F / W / Y) (SEQ ID NO:43) or GXCX3-9(F / W / Y) (SEQ ID NO:44). In certain embodiments, such substitutions which improve or maintain the net positive charge or hydrophobicity of the peptide include defensin peptide variants having a modified gamma core variant sequence, which may include the modified gamma core variant sequence of SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51 or SEQ ID NO:52.In certain embodiments, the above-mentioned defensin peptide variants comprise, consist essentially of, or consist of (i) 30 amino acid residues or less; or (ii) 15, 16, or 17 to 22, 24, 16, 28, or 30 amino acid residues. In certain embodiments, any of the above-mentioned defensin peptide variants includes a peptide having exactly two cysteine ​​residues, optionally including two cysteine ​​residues corresponding to the conserved C1 and C4 cysteines of a reference defensin C-terminal peptide.

[0054] In certain embodiments, one or more amino acids in any of the above or other variant defensin peptide variant sequences are replaced with another amino acid that is similar in charge and polarity to the original amino acid, i.e., a conservative amino acid substitution. Substitutions for amino acids within a defensin peptide variant or protein or defensin peptide sequence may be selected from other members of the class to which the original amino acid belongs. Amino acids may be divided into four groups: (1) acidic amino acids; (2) basic amino acids; (3) neutral polar amino acids; and (4) neutral nonpolar amino acids. Representative amino acids within these various groups include: (1) acidic (anionic, negatively charged) amino acids, such as aspartic acid and glutamic acid; (2) basic (cationic, positively charged) amino acids, such as arginine, histidine, and lysine; (3) neutral polar amino acids, such as glycine, serine, threonine, cysteine, cystine, tyrosine, asparagine, and glutamine; and (4) neutral nonpolar (hydrophobic) amino acids, such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Conservative amino acid changes within the defensin peptide sequence can be made by substituting an amino acid within one of these groups for another amino acid within the same group. Biologically functional equivalents of defensin peptide variants can have 10 or fewer conservative amino acid changes, 7 or fewer conservative amino acid changes, or 5, 4, 3, 2, or 1 conservative amino acid changes. Thus, the coding nucleotide sequence (e.g., gene, plasmid DNA, cDNA, or synthetic DNA) has corresponding base substitutions that allow for encoding biologically functional equivalent forms of the defensin peptide variants. Certain semi-conservative substitutions in the defensin peptide variants are also provided, including (i) substitutions of neutral polar amino acid residues with neutral non-polar (hydrophobic) amino acid residues; or (ii) substitutions of neutral non-polar (hydrophobic) amino acid residues with neutral polar amino acid residues. In particular, semi-conservative substitutions of neutral polar tyrosine residues with hydrophobic amino acid residues are provided.A biologically functional equivalent of a defensin peptide variant may have no more than 10 semi-conservative amino acid changes, no more than 7 semi-conservative amino acid changes, or 5, 4, 3, 2 or 1 semi-conservative amino acid changes.

[0055] Also provided herein are nucleic acid molecules encoding any of the above-mentioned defensin peptide variants. Also provided herein are recombinant DNA molecules comprising the above-mentioned nucleic acid molecules, particularly recombinant DNA molecules comprising a heterologous promoter operably linked to the above-mentioned nucleic acid molecules.

[0056] The defensin peptide variants provided herein can be operably linked to another defensin peptide variant, defensin or antimicrobial peptide via a spacer peptide sequence that is not susceptible to cleavage by endoproteinases, including plant endoproteinases. Such peptide linker sequences linking peptides in multimeric or multidomain proteins have been disclosed (Argos, 1990; George RA, Heringa (2002)). Examples of suitable peptide sequences from multimeric or multidomain proteins that can be used as spacer domains include the immunoglobulin hinge region from immunoglobulins, the linker between lipoyl and E3 binding domains in pyruvate dehydrogenase (Turner et al., 1993), the linker between the central domain and the C-terminal domain in cysteine ​​proteinases (P9; Mottram et al., 1989) and functional variants thereof. Spacer peptides for use in defensin variant proteins can also be wholly or partially synthetic peptide sequences. Such synthetic spacer peptides are designed to provide a flexible link between at least one defensin peptide variant and another peptide (including a defensin peptide variant or a defensin peptide) and to be resistant to cleavage by endogenous plant endoproteinases or other endoproteinases. In certain embodiments, the length of the synthetic spacer peptide can be between about 3, 4, 8, 10, 12, or 16 to about 20, 24, 28, 30, 40, or 50 amino acid residues in length. In certain embodiments, the synthetic spacer peptide can include a glycine-rich or glycine / serine-containing peptide sequence. The present compositions and peptide designs suitable for flexible linking of protein domains described in the literature (Chen et al., 2013) can be adapted for use as spacer peptides in the defensin variant proteins provided herein.Spacer peptides useful for linking defensin monomers, as described in U.S. Patent Publication Nos. 20190194268 and 20190185877, each of which is incorporated by reference in its entirety herein, may also be used to link the defensin peptide variants disclosed herein to other defensin peptide variants, defensins, antimicrobial peptides, or other peptides.

[0057] The defensin peptide variants provided herein can be operably linked to another defensin peptide variant, defensin, or antimicrobial peptide via a linker peptide sequence that is susceptible to cleavage by an endoproteinase, including a plant endoproteinase. In certain embodiments, the resulting defensin variant protein can be expressed in a cell such that an endoproteinase cleaves the defensin variant protein to provide at least one defensin peptide variant and another peptide (including a defensin peptide variant or a defensin peptide). Such a defensin variant protein can be provided in a cellular compartment (e.g., cytoplasm, mitochondria, plastids, vacuoles, or endoplasmic reticulum) or extracellular space (i.e., to the apoplast) that has an endoproteinase that cleaves the linker peptide. Cleavable linker peptides are disclosed in WO2014078900, Vasivarama and Kirti, 2013a, Franqois et al, Vasivarama and Kirti, 2013b and WO2017127558 and may be used in the defensin variant proteins provided herein.

[0058] Expression cassettes can be constructed that provide expression of defensin peptide variants in monocotyledonous plants, dicotyledonous plants, or both. Such defensin peptide variant expression cassette construction can be accomplished in either a plant expression vector or in the genome of a plant. An expression cassette is a DNA construct in which various promoters, codes (e.g., encoding defensin peptide variants), and polyadenylation sequences are operably linked. In general, an expression cassette generally includes a promoter operably linked to a sequence of interest that is operably linked to a polyadenylation or terminator region. In certain instances, including expression of a recombinant or edited polynucleotide in a monocotyledonous plant, it can also be useful to include an intron sequence. If an intron sequence is included, it is generally placed in the 5' untranslated leader region of the recombinant or edited polynucleotide. In certain instances, it can also be useful to incorporate specific 5' untranslated sequences in the recombinant or edited polynucleotide to promote stability of the transcript or to promote efficient translation of the transcript. Expression cassettes and vectors for expression of other defensin peptides or proteins in plants, including those disclosed in U.S. Patent No. 10,253,328, which is incorporated herein by reference in its entirety, can be adapted for expression of defensin peptide variants in transgenic plants. Any of the defensin peptide variant expression vectors can be introduced into the chromosomes of a host plant via methods such as Agrobacterium-mediated transformation, Rhizobium-mediated transformation, Sinorhizobium-mediated transformation, particle-mediated transformation, DNA transfection, DNA electroporation, or "whiskers"-mediated transformation.The above-mentioned methods of introducing transgenes are described in U.S. Patent Application Publication No. 20050289673 (Agrobacterium-mediated transformation of maize), U.S. Patent No. 7,002,058 (Agrobacterium-mediated transformation of soybean), U.S. Patent No. 6,365,807 (grain-mediated transformation of rice), and U.S. Patent No. 5,004,863 (Agrobacterium-mediated transformation of cotton), each of which is incorporated by reference herein in its entirety.

[0059] In certain embodiments, plants comprising recombinant or edited polynucleotides encoding defensin peptide variants can be obtained by using techniques that provide site-specific insertion of heterologous DNA into the genome of the plant (e.g., by CRISPR, TALEN, or zinc finger nuclease-mediated gene editing). In certain embodiments, at least a DNA fragment encoding the defensin peptide variant is site-specifically integrated into the genome of a plant cell, tissue, part, or whole plant to generate a sequence within its genome that encodes the defensin peptide variant. Examples of methods for inserting foreign DNA at specific sites in the plant genome using site-specific nucleases such as meganucleases or zinc finger nucleases are disclosed at least in Voytas, 2013. Examples of methods for inserting foreign DNA into plant genomes using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (Cas)-guide RNA technology and Cas endonucleases are disclosed at least by Svitashev et al., 2015; Murovec et al., 2017; Kumar and Jain, 2015; and U.S. Patent Publication No. 20150082478, which is specifically incorporated by reference herein in its entirety.

[0060] The expression of defensin peptide variants in yeast is also specifically contemplated herein. The construction of expression vectors for the production of heterologous proteins in various yeast genera is well established. In general, such expression vectors generally include a promoter operably linked to a sequence of interest operably linked to a polyadenylation or terminator region. Examples of yeast genera that have been successfully used to express heterologous genes include Candida, Kluveromyces, Hansuela, Pichia, Saccharomyces, Schizosaccharomyces, and Yarrowia. A general description of expression vectors and transformation systems for Saccharomyces can be found in Kingsman et al. (1985). Expression vectors and transformation systems useful for yeasts other than Saccharomyces are described in Reiser et al. (1990). Expression cassettes and vectors for expression of other defensin peptides or proteins in yeast, including those disclosed in U.S. Patent No. 10,253,328, which is incorporated by reference in its entirety, can be adapted for expression of defensin peptide variants in yeast.

[0061] The expression of defensin peptide variants in bacteria is also specifically contemplated herein. The construction of expression vectors for the production of heterologous proteins in various bacterial systems has been described. In general, such expression vectors generally include a promoter operably linked to a sequence encoding a peptide sequence of interest (e.g., a signal transit peptide region at the n-terminus of a defensin variant peptide) operably linked to a prokaryotic terminator region. Examples of bacterial genera that have been used successfully to express heterologous genes include Acinetobacter, Alcaligenes, Azotobacter, Bacillus, Escherichia, Lactobacillus, Lactococcus, Streptomyces, and Pseudomonas. E. coli expression systems useful for producing proteins that contain disulfide bonds and that can be adapted for use in expressing the defensin variant peptides provided herein include those described in Kuddus et al. (2017) Biotechnol Prog 233:1520-1528. doi:10.1002 / btpr.Protein Science 2508, Kiedzierska et al. (2008) Protein Expr Purif 60, 82-88, Chang et al. (2015) Amino acids 47, 579-587, Buchko et al. (2018) Protein Science 27, 1611-1623, Marques et al. (2008) J Appl Microbiol 106, 1640-1648, and Pazgier et al. (2006 ... 49, 1-8.Systems for expressing proteins containing disulfide bonds that can be adapted for expression of defensin peptides in E. coli include those disclosed in U.S. Patent Application Publication No. 2020 / 0172915 and Berkmen, M. Protein Expr Purif. 2012;82(1):240-51. doi:10.1016 / j.pep.2011.10.009, which are incorporated by reference in their entireties.

[0062] Other bacterial expression systems useful for producing proteins containing disulfide bonds and that can be adapted for use in expressing the defensin variant peptides provided herein include those described in U.S. Pat. No. 10,604,761, which is incorporated by reference in its entirety.

[0063] Also provided are antimicrobial compositions for agricultural, pharmaceutical or veterinary use, comprising either an antimicrobial plant or antimicrobial human or veterinary, pathogenic microorganism inhibiting amount ("antimicrobial effective amount") of one or more of the present isolated and purified antimicrobial defensin peptide variants or biologically functional equivalents thereof. Such compositions may comprise one or any combination of the defensin peptide variants or proteins disclosed herein and an agriculturally, pharma- ceutical or veterinary practicably acceptable carrier, diluent or excipient. As indicated below, other components relevant in agricultural and therapeutic contexts may also be included in such compositions. Antimicrobial compositions may be used to inhibit the growth of or kill pathogenic microorganisms susceptible to the defensin peptide variants associated with plant, human or animal microbial infections. Such antimicrobial compositions may be formulated for topical administration and may be applied locally to either plants, the plant environment (including soil) or to humans or animals. Such antimicrobial compositions may be formulated for enteral, parenteral and / or intravenous administration of the compositions and administered to a subject in need thereof; such subjects may be humans, livestock, poultry, fish or companion animals. The defensin peptide variants may be formulated alone, in any combination with each other, or may further be formulated in combination with other conventional antimicrobial therapeutic compounds, such as, but not limited to, polyene antimicrobial agents; imidazole, triazole and thiazole antimicrobial agents; allylamines; and echinocandins routinely used in human and veterinary medicine. Administration of a composition comprising a defensin peptide variant to a human or animal subject in need thereof may be accomplished via a variety of routes, including topical application, enteral administration, parenteral administration and / or intravenous administration. The antimicrobial peptides and compositions may be used to control microbial pathogens or contaminants, including: (i) bacterial pathogens of plants or animals, optionally a member of the group of the Enterobacteriaceae, optionally the bacterial pathogen being Salmonella sp., Escherichia sp. or Listeria sp.(ii) Fusarium sp., Alternaria sp., Aphenomyces sp., Verticillium sp., Phytophthora sp., Colletotrichum sp., Botrytis sp., Cercospora sp., Phakopsora sp. Rhizoctonia sp., Sclerotinia sp., Pythium sp., Phoma sp., Leptosphaeria sp., Gaeumannomyces sp., Puccinia sp. Septoria sp., Penicillium sp., Lasiodiplodia sp., Phomopsis sp., Mycosphaerella sp., Golovinomyces sp., Erisyphe sp., Albugo sp., Setosphaeria sp., Cochliobolus sp., Helminthosporium sp., Diplodia sp. or Stenocarpella sp.(iii) Aspergillus, Cryptococcus, Penicillium, Rhizopus, Apophysomyces, Cunninghamella, Saksenaea, Rhizomucor, Syncephalostrum. , Cokeromyces, Actinomucor, Pythium, Fusarium, Histoplasmosis, Coccidiomyces or Blastomyces species; (iv) Candida species (Candida species includes Candida albicans); albicans (C. albicans), C. auris, C. glabrata, C. parapsilosis, C. tropicalis or C. krusei); or (v) the dermatophyte is Trichophyton rubrum, Trichophyton interdigitale, Trichophyton violaceum, Trichophyton tonsurans, Trichophyton soudanense, Trichophyton mentagrophytes, Microsporum flavum. flavum, Epidermophyton floccosum, and Microsporum gypseum.

[0064] Agricultural compositions comprising any of the defensin peptide variant molecules of the present invention alone or in any combination may be formulated as described, for example, in Winnacker-Kuchler (1986) Chemical Technology, Fourth Edition, Volume 7, Hanser Verlag, Munich; van Falkenberg (1972-1973) Pesticide Formulations, Second Edition, Marcel Dekker, NY; and K. Martens (1979) Spray Drying Handbook, Third Edition, G. Goodwin, Ltd., London. Formulation aids, such as carriers, inert materials, surfactants, solvents and other additives, are also well known in the art, for example, Watkins, Handbook of Insecticide Dust Diluents and Carriers, Second Edition, Darland Books, Caldwell, NJ, and Winnacker-Kuchler (1986) Chemical Technology, Fourth Edition, Volume 7, Hanser Verlag, Munich; Verlag, Munich. These formulations can also be used to prepare mixtures of the defensin peptide variants and proteins of the invention with other insecticidal actives, fertilizers and / or growth regulators, etc., in the form of finished formulations or tank mixes.

[0065] Either alone or in combination with other active agents, the antimicrobial defensin peptide variants of the present invention may be applied to a subject or plant at a concentration ranging from about 0.1 pg / ml to about 100 mg / ml or from about 5 pg / ml to about 5 mg / ml at a pH ranging from about 3.0 to about 9.0. Such compositions may be buffered, for example, using phosphate buffer at about 1 mM to 1 M, about 10 mM to about 100 mM, or about 15 mM to about 50 mM. In the case of low buffer concentrations, salts may be added to improve ionic strength. In certain embodiments, sodium salts may be added or provided in compositions comprising defensin peptide variants and proteins, including NaCl in the range of about 1 mM to about 1 M, about 1 mM, 5 mM or 10 mM to about 20 mM, 50 mM, 100 mM, 150 mM or 200 mM, or about 10 mM to about 100 mM. In certain embodiments, a potassium salt comprising KCl in the range of about 1 mM, 5 mM, or 10 mM to about 20 mM, 50 mM, 100 mM, 150 mM, or 200 mM may be added or provided in a composition comprising a defensin peptide variant and a protein. In certain embodiments, a calcium salt comprising CaCl2 in the range of about 0.1 mM, 0.5 mM, or 1 mM to about 2 mM, 5 mM, 10 mM, or 20 mM may be added or provided in a composition comprising a defensin peptide variant.

[0066] Many conventional microbial antibiotics and chemical antimicrobials (e.g., antifungals) with which the defensin peptide variants and proteins of the present invention can be combined are described in Worthington and Walker (1983) The Pesticide Manual, Seventh Edition, British Crop Protection Council. These include, for example, polyoxins, nikkomycins, carboxamides, aromatic carbohydrates, carboxins, morpholines, inhibitors of sterol biosynthesis, and organophosphorus compounds. In addition, azole, triazole, and echinocandin fungicides can also be used. Other active ingredients that can be formulated in combination with the antimicrobial peptides and proteins of the present invention include, for example, insecticides, attractants, sterilants, acaricides, nematicides, and herbicides. U.S. Patent No. 5,421,839, incorporated herein by reference in its entirety, contains a comprehensive summary of many active substances with which materials such as the antimicrobial defensin peptide variants and proteins of the present invention can be formulated.

[0067] Agriculturally useful antimicrobial compositions encompassed herein also include those in the form of host cells such as bacteria and microbial cells capable of producing defensin peptide variants and proteins and colonizing plants, including roots, shoots, leaves, or other parts of the plant. The term "plant-colonizing microorganisms" is used herein to refer to microorganisms including those capable of colonizing the plant itself and / or any part of the plant's environment and capable of expressing the defensin variant antimicrobial peptides and proteins of the present invention in the plant and / or plant environment. Plant-colonizing microorganisms may exist in a mutualistic or non-detrimental relationship with the plant in the plant's environment. U.S. Pat. No. 5,229,112, incorporated herein by reference in its entirety, discloses various plant-colonizing microorganisms that can be modified to express antimicrobial peptides and proteins applicable to the defensin variant antimicrobial peptides and proteins disclosed herein and methods of use thereof.Microorganisms that colonize plants expressing the defensin variant antimicrobial peptides and proteins disclosed herein that are useful in inhibiting microbial growth in plants include Bacillus spp., including Bacillus thuringiensis, Bacillus israelensis, and Bacillus subtilis, Candidatus Liberibacter asiaticus, and other Bacillus species. asiaticus; Pseudomonas spp.; Arthrobacter spp., Azospyrillum spp., Clavibacter spp., Escherichia spp.; Agrobacterium spp., e.g. A. radiobacter, Rhizobium spp., Erwinia spp. spp., Azotobacter spp., Azospirillum spp., Klebsiella spp., Alcaligenes spp., Rhizobacterium spp., Xanthomonas spp., Ralstonia spp., and Flavobacterium spp. In certain embodiments, the microorganism is a yeast selected from the group consisting of Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica. In certain embodiments, the microorganism colonizing the plant may be an endophytic bacterium or microorganism.When applying the defensin peptide variant molecules of the invention to the rhizosphere, rhizosphere-colonizing bacteria from the genus Pseudomonas are particularly useful, especially fluorescent pseudomonads such as Pseudomonas fluorescens, which are particularly competitive in the plant rhizosphere and in colonizing the root surface of many plants. Examples of suitable foliar (leaf)-colonizing bacteria are P. putida, P. syringae and Erwinia species.

[0068] Embodiment The following numbered embodiments form part of the present disclosure: 1a. A peptide comprising SEQ ID NO:1, but not including the corresponding full length sequence of a defensin peptide of SEQ ID NO:8, 16, 20, 23, 26, 29, 37, 38, 39, 40 or 41, and optionally comprising a modified gamma-core consensus sequence GXCX3-8(F / W / Y)(F / W / Y)(F / W / Y) as set forth in SEQ ID NO:33, GXCX3-9(F / W / Y)(F / W / Y)(F / W / Y) as set forth in SEQ ID NO:34, GXCX3-8(F / W / Y) (SEQ ID NO:43) or GXCX3-9(F / W / Y) (SEQ ID NO:44); and / or optionally wherein the C-terminal cysteine ​​residue or the C-terminal amino acid residue is amidated.

[0069] 1b. A peptide having at least 50%, 55%, 60%, 68%, 75%, 82% or 94% sequence identity over the entire length of any one of SEQ ID NOs: 3, 4, 5 or 6, but not identical to SEQ ID NO: 8, optionally wherein any amino acid substitutions in said sequence improve or maintain a net positive charge at neutral pH and / or improve or maintain the hydrophobicity of the peptide, and optionally wherein the C-terminal cysteine ​​residue is amidated.

[0070] 1c. A peptide having at least 50%, 55%, 60%, 68%, 75%, 82%, 94%, 95% or 100% sequence identity over the entire length of SEQ ID NO:7, 12, 13, 14, 15, 17-20, 22, 23, 25, 26, 28, 29, 31, 32, 42, 578, 579, 581, 582, 584 or 585, but not identical to SEQ ID NO:8, 16, 20, 23, 26, 29, 37, 38, 39, 40 or 41, and optionally SEQ ID NO:5, SEQ ID NO:13. 578, 579, 581, 582, 584 or 585 of the peptide comprises a disulfide bond between two cysteine ​​residues, optionally any substitution improves or maintains a net positive charge at neutral pH and / or improves or maintains the hydrophobicity of the peptide, and optionally the C-terminal cysteine ​​residue or the C-terminal cysteine ​​residue is amidated.

[0071] 1d. A defensin C-terminal peptide variant comprising conserved C1 and C4 cysteine ​​residues corresponding to the N-terminal and C-terminal cysteines of a reference defensin C-terminal peptide, wherein the conserved C2 and C3 cysteine ​​residues of said reference defensin C-terminal peptide are independently replaced with tryptophan, tyrosine, phenylalanine, leucine, valine, isoleucine, or methionine; optionally, a defensin C-terminal peptide variant having at least 3, 3.5, 4, 5, or 6 net positive charges and a hydrophobic amino acid content of at least 18%.

[0072] 1e. Modified gamma-core consensus sequences GXCX3-8(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO: 33), GXCX3-9(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO: 34), GXCX3-10(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO: 587), GXCX3-12(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO: 588), GXCX3-15(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO: 5 89), GXCX3-8(F / W / Y) (SEQ ID NO: 43), GXCX3-9(F / W / Y) (SEQ ID NO: 44), GXCX3-8(F / W / Y / L / V / I / M) (SEQ ID NO: 45), GXCX3-10(F / W / Y / L / V / I / M) (SEQ ID NO: 46), GXCX3-8(L / V / I / M)(F / W / Y)(L / V / I / M) (SEQ ID NO: 47), GXCX3-10(L / V / I / M)(F / W / Y)(L / V / I / M) (SEQ ID NO: 48), GXCX3-8(F / W / Y / L / V / I / M)(F / W / Y)(F / W / Y / L / V / I / M) (SEQ ID NO: 49), GXCX3-10(F / W / Y / L / V / I / M)(F / W / Y)(F / W / Y / L / V / I / M) (SEQ ID NO: 50), GXCX3-8(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M) (SEQ ID NO: 51) or GXCX3-10(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M) (SEQ ID NO: No. 52), optionally wherein the peptide further comprises a conserved C4 cysteine ​​residue, optionally wherein the cysteine ​​residue in the modified gamma-core consensus sequence forms a disulfide bond with the conserved C4 cysteine ​​residue, and / or optionally has at least 3, 3.5, 4, 5 or 6 net positive charges and a hydrophobic amino acid content of at least 18%.

[0073] 1f. Modified gamma-core consensus sequences GXCX3-8(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO: 33), GXCX3-9(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO: 34), GXCX3-10(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO: 587), GXCX3-12(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO: 588), GXCX3-15(F / W / Y)(F / W / Y)(F / W / Y)((SEQ ID NO:589), GXCX3-8(F / W / Y)(SEQ ID NO:43) or GXCX3-9(F / W / Y)(SEQ ID NO:44), GXCX3-8(F / W / Y / L / V / I / M)(SEQ ID NO:45), GXCX3-10(F / W / Y / L / V / I / M)(SEQ ID NO:46), GXCX3-8(L / V / I / M)(F / W / Y)(L / V / I / M)(SEQ ID NO:47), GXCX3-10(L / V / I / M)(F / W / Y)(L / V / I / M) (SEQ ID NO: 48), GXCX3-8(F / W / Y / L / V / I / M)(F / W / Y)(F / W / Y / L / V / I / M) (SEQ ID NO: 49), GXCX3-10(F / W / Y / L / V / I / M)(F / W / Y)(F / W / Y / L / V / I / M) (SEQ ID NO: 50), GXCX3-8(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M) (SEQ ID NO: 51) or GXCX3-10(F / W / Y / L / V 52), further comprising a second C-terminal cysteine ​​residue located C-terminal to the cysteine ​​residue in the modified gamma-core consensus sequence, having at least 3, 3.5, 4, 5 or 6 net positive charges and a hydrophobic amino acid content of at least 18%, and optionally comprising a disulfide bond between the two cysteine ​​residues.

[0074] 2. The peptide according to embodiment 1a, 1b, 1c, 1d, 1e or 1f, comprising: (i) an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, 17-20, 22, 23, 25, 26, 28, 29, 31, 32, 42, 578, 579, 581, 582, 584 or 585; optionally, the peptide of SEQ ID NO:5, SEQ ID NO:13, SEQ ID NO:14, 15, 18, 19, 20, 22, 23, 26, 28, 29, 32, 42, 578, 579, 581, 582, 584 or 585 comprises a disulfide bond between two cysteine ​​residues; (ii) SEQ ID NO:7, SEQ ID NO:12, or (iii) a peptide that exhibits antimicrobial activity; or (iv) an amino acid sequence having at least 50%, 55%, 60%, 68%, 75%, 82%, 94%, 95% or 100% sequence identity over the entire length of SEQ ID NO:13, SEQ ID NO:14, 15, 17-20, 22, 23, 25, 26, 28, 29, 31, 32, 18, 19, 20, 22, 23, 26, 28, 29, 32, 42, 578, 579, 581, 582, 584 or 585 (wherein the peptide does not include the corresponding full-length sequence of a defensin peptide of SEQ ID NO:8, 16, 20, 23, 26, 29, 37, 38, 39, 40 or 41; optionally, the peptide comprises a disulfide bond between two cysteine ​​residues); or

[0075] 3. A peptide according to embodiment 1a, 1b, 1c, 1d, 1e, 1f or 2, comprising: (i) 30 amino acid residues or less; (ii) 15, 16 or 17-30 amino acid residues; or (iii) comprising, consisting essentially of or consisting of 15, 16 or 17-30 amino acid residues and containing two cysteine ​​residues.

[0076] 4. A composition comprising a peptide according to any one of embodiments 1a, 1b, 1c, 1d, 1e or 1f-3 and an agriculturally, pharma- ceutical or veterinarily acceptable carrier, diluent or excipient.

[0077] 5. The composition of embodiment 4, wherein the peptide is provided in a concentration of from about 0.1, 0.5, 1.0 or 5 pg / ml to about 1, 5, 20, 50 or 100 mg / ml or from about 0.1, 0.5, 1.0 or 5 pg / gram to about 1, 5, 20, 50 or 100 mg / gram, and optionally comprises a sodium salt at a concentration of at least 100 mM and / or a calcium salt at a concentration of at least 2 mM.

[0078] 6. A method for (i) preventing or reducing crop damage caused by phytopathogenic microorganisms; or (ii) preventing contamination of a plant, a plant part, a seed, livestock feed derived therefrom, or food derived therefrom with undesirable microorganisms, comprising contacting the plant, the plant seed, another part of said plant, livestock feed derived therefrom, or food derived therefrom with an effective amount of the composition of embodiment 4 or embodiment 5.

[0079] 7. The phytopathogenic or undesirable microorganism is (i) a bacterial pathogen of plants or animals, optionally a member of the Enterobacteriaceae family, and optionally a Salmonella sp., Escherichia sp., or Listeria sp.; or (ii) a Fusarium sp., Alternaria sp., Aphenomyces sp., Verticillium sp., Phytophthora sp., Colletotrichum sp., Botrytis sp., Cercospora sp., Phakopsora sp. Rhizoctonia sp., Sclerotinia sp., Pythium sp., Phoma sp., Leptosphaeria sp., Gaeumannomyces sp., Puccinia sp., Septoria sp., Penicillium sp., Lasiodiplodia sp., Phomopsis 7. The method of embodiment 6, wherein the species of the fungus is Mycosphaerella sp., Golovinomyces sp., Erisyphe sp., Albugo sp., Setosphaeria sp., Cochliobolus sp., Helminthosporium sp., Diplodia sp. or Stenocarpella sp.

[0080] 8. A medical device comprising a device and the composition of embodiment 4 or 5, the medical device comprising at least one surface topically coated and / or impregnated with said composition.

[0081] 9. The medical device of embodiment 8, which is a stent, a catheter, a contact lens, a condom, a patch or a septum.

[0082] 10. A method for treating, preventing or inhibiting a microbial infection in a subject in need thereof, comprising administering to the subject an effective amount of the composition of embodiment 4 or 5.

[0083] 11. The method of embodiment 10, wherein said administering comprises topical, enteral, parenteral and / or intravenous introduction of said composition.

[0084] 12. The method of embodiment 10 or 11, wherein the subject is a human, livestock, poultry, fish or companion animal.

[0085] 13. The method of embodiment 12, wherein the microbial infection is an infection of the mucosa, eyes, skin and / or nails, and the composition is applied to the mucosa, eyes, skin and / or nails.

[0086] 14. The method of any one of embodiments 10-13, wherein the microbial infection is caused by a dermatophyte, and the dermatophyte is optionally selected from the group consisting of Trichophyton rubrum, Trichophyton interdigitale, Trichophyton violaceum, Trichophyton tonsurans, Trichophyton soudanense, Trichophyton mentagrophytes, Microsporum flavum, Epidermophyton floccosum, and Microsporum gypseum.

[0087] 15. The microbial infection is (i) optionally a member of the Enterobacteriaceae family, optionally Salmonella sp., Escherichia sp., or Listeria sp.; or (ii) Aspergillus, Cryptococcus, Penicillium, Rhizopus, Apophysomyces, Cunninghamella, The method according to any one of embodiments 10 to 13, wherein the bacterial pathogen of animals is of the species Bacillus subtilis, Bacillus subtilis, Bacillus spp., Bacillus subtilis ...

[0088] 16. The method of any one of embodiments 10 to 13, wherein the microbial infection is caused by a Candida species, and the Candida species is Candida albicans (C. albicans), C. auris, C. glabrata, C. parapsilosis, C. tropicalis, or C. krusei.

[0089] 17. The composition of embodiment 4 or 5, for use in a method for treating, preventing, or inhibiting a microbial infection in a subject in need thereof.

[0090] 18. The composition of embodiment 17, wherein the subject is a human, livestock, poultry, fish or companion animal.

[0091] 19. A plant part at least partially coated with the composition according to embodiment 4 or 5.

[0092] 20. The plant part of embodiment 19, which is a seed, optionally a corn, soybean, wheat, rice, cotton, Brassica sp. or tomato seed.

[0093] 21. The plant part of embodiment 19, which is a fruit, vegetable or flower.

[0094] 22. A recombinant polynucleotide comprising a polynucleotide encoding an antimicrobial peptide according to embodiment 1a, 1b, 1c, 1d, 1e, 1f, 2 or 3, wherein the polynucleotide encoding a first antimicrobial peptide is operably linked to a polynucleotide comprising a promoter heterologous to the polynucleotide encoding the first antimicrobial peptide, and optionally any amino acid substitutions in said sequence improve or maintain a net positive charge and / or improve or maintain the hydrophobicity of the peptide.

[0095] 23. The recombinant polynucleotide of embodiment 22, further comprising a polynucleotide encoding (i) a transport peptide, a vacuole-targeting peptide and / or an endoplasmic reticulum-targeting peptide; (ii) a plastid-targeting peptide; and / or (iii) a polynucleotide encoding a polyadenylation or transcription termination signal, wherein the polynucleotides of (i), (ii) and / or (iii) are operably linked to the polynucleotide encoding the antimicrobial peptide.

[0096] 24. The recombinant polynucleotide of embodiment 22 or 23, wherein the polynucleotide encoding the first antimicrobial peptide is inserted into a heterologous nuclear or plastid genome of a cell and is operably linked to an endogenous promoter located in the heterologous nuclear or plastid genome.

[0097] 25. A plant nuclear or plastid genome comprising a polynucleotide encoding an antimicrobial peptide according to embodiment 1a, 1b, 1c, 1d, 1e, 1f, 2 or 3, wherein said polynucleotide is heterologous to said nuclear or plastid genome, and said polynucleotide is operably linked to an endogenous promoter of said nuclear or plastid genome.

[0098] 26. A cell comprising a recombinant polynucleotide according to embodiment 22 or a genome according to embodiment 25, which is optionally a bacterial, yeast or plant cell.

[0099] 27. A plant comprising a recombinant polynucleotide according to embodiment 22 or a genome according to embodiment 25.

[0100] 28. A plant part of a plant according to embodiment 26, comprising the recombinant polynucleotide or genome, said plant part being a seed, stem, leaf, root, tuber, flower or fruit.

[0101] 29. A method for producing a plant seed that provides a plant with resistance to infection by a plant pathogenic microorganism, comprising the steps of: (i) selfing or crossing a plant of embodiment 22; and (ii) harvesting seeds of said plant from said selfing or crossing, said seeds comprising a recombinant polynucleotide of said plant, thereby producing a plant seed that provides the plant with resistance to infection by a plant pathogenic microorganism.

[0102] References Argos, (1990) J Mol Biol. Feb 20;211(4):943-58. Broadley, MR, Bowen, HC, Cotterill, HL, Hammond, JP, Meacham, MC, Mead, A., and White, PJ (2003). Journal of Experimental Botany 54, 1431-1446. Buchko et al. (2018) Protein Science 27, 1611-1623. Chang et al.(2015)Amino Acids 47,579-587. Chen et al.,(2013)Adv Drug Deliv Rev.;65(10):1357-1369. da Silva Conceicao,A.,and Broekaert,WF(1999).Plant Defensins.In Pathogenesis-related proteins in plants,S.Muthukrishnan,ed(New York:CRC Press),pp.247-260. Franqois et al.,Plant Physiology(2002)128:1346-1358. George RA,and Heringa(2002)J Protein Eng.15(11):871-879. Hanks,JN,et al.,(2005).Plant Mol Biol 58,385-399. Kerenga BK,et al.,(2019)Front.Microbiol.10:795.doi:10.3389 / fmicb.2019.00795 Kiedzierska,et al.(2008)Protein Expr Purif 60,82-8. Kingsman SM,et al.,(1985)Biotechnol Genet Eng Rev.3:377-416. Kuddus et al.(2017).Biotechnol Prog 233:1520-1528.doi:10.1002 / btpr.Protein Science 2508. Kumar,V.and Jain,M.(2015)J Exp Bot 66:47-57. In Lacerda et ah,Frontiers in Microbio.(2014)5(116):1-10. Lay,F.T.,and Anderson,M.A.(2005)Curr Protein Pept Sci 6,85-101. Marques et al.(2008)J Appl Microbiol 106,1640-1648. Mottram et al.,FEBS Lett.(1989)258(2):211-215. Murovec et al.,Plant Biotechnol J.2017 Aug;15(8):917-926. Pazgier et al.(2006)Protein Expr Pur 49,1-8. Reiser J,et al.,(1990)Adv Biochem Eng Biotechnol.;43:75-102. Sagaram et al.,(2011)PLOS ONE 6:el8550. Sagaram et al.,(2013)PLoS ONE,8(12):e82485. Shabala,S.,and Pottosin,I.I.(2010)Signal.Commun.Plants 87-110. Svitashev et al.,(2015)Plant Physiology,169(2):931-945. Spelbrink,R.G.,et al.,(2004).Plant Physiol 135,2055-2067. Terras,F.R.,et al.,(1992).J Biol Chem 267,15301-15309. Thomma,B.P.H.J.,Cammue,B.P.A.,and Thevissen,K.(2002).Planta 216,193-202. Turner et al.,(1993)Protein Eng.6(1):101-108. Voytas,D.Annual Review of Plant Biology,Vol.64:327-350,2013. Vasivarama and Kirti(2013a)Plant Cell Tiss Organ Cult,115:309-319. Vasivarama and Kirti(2013b)Funct Integr Genomics 13:435-443. Vriens,K.,et al.Molecules 2014,19,12280-12303;doi:10.3390 / moleculesl90812280. White,P.,and Karley,A.(2010).Potassium.In Cell Biology of Metals and Nutrients;Hell,R.,Mendel,R.R.,Eds.;Springer:Berlin / Heidelberg,Germany.,199-224

Example

[0103] Example 1 Crude peptides (GMA4C-AC, GMA4C_V1, GMA4C_V2 and GMA4C_V3) were chemically synthesized by Biomatik Inc, Canada, GMA4C_V4 and GMA4C_V5 were synthesized by Alan Scientific Inc. (USA), and they were further purified using a linear gradient of acetonitrile / water mixtures on a C-18 reversed-phase HPLC (Agilent Technologies, USA). The HPLC fractions were lyophilized and resuspended in nuclease-free water. The concentration of each peptide was determined using a BCA assay performed according to the manufacturer's protocol (Thermo-Fisher Scientific, USA). Antifungal activity was determined in an assay in SFM medium. SFM medium contained K2HPO4 (2.5 mM), MgSO4 (50 μM), CaCl2 (50 μM), FeSO4 (5 μM), CoCl2 (0.1 μM), CuSO4 (0.1 μM), Na2MoO4 (2 μM), H3BO3 (0.5 μM), KI (0.1 μM), ZnSO4 (0.5 μM), MnSO4 (0.1 μM), glucose (10 g / liter), asparagine (1 g / liter), methionine (20 mg / liter), myo-inositol (2 mg / liter), biotin (0.2 mg / liter), thiamine-HCL (1 mg / liter), and pyridoxine-HCL (0.2 mg / liter), pH 7.0.

[0104] [Table 4]

[0105] [Table 5]

[0106] Antifungal activity in vitro under low cation conditions: GMA4C variants GMA4C_V1A, GMA4C_V3A, GMA4C_V4A and GMA4C_V5A show equivalent antifungal activity to the wild-type GMA4C_AC control in vitro against B. cinerea, F. graminearum, F. oxysporum and P. capsici. GMA4C variant GMA4C_V2A shows 2-fold reduced antifungal activity against B. cinerea and F. graminearum compared to the wild-type GMA4C_AC control in vitro. GMA4C_GMA4C_V6A exhibits a four-fold increase in antifungal activity against B. cinerea compared to the wild-type GMA4C_AC control in vitro.

[0107] Antifungal activity in planta. When applied to detached Nicotiana benthamiana leaves, GMA4C_V1A and GMA4C_V3A are more potent than GMA4C_AC or GMA4C_V2A in reducing symptoms of gray mold caused by B. cinerea, as shown in Figures 1A and 1B. When applied to tomato leaves, GMA4C_V1A is more effective than GMA4C_AC in reducing disease symptoms caused by P. capsici.

[0108] Antifungal activity in vitro in the presence of cations. When tested for antifungal activity against B. cinerea in the presence of 100 mM NaCl, 100 mM KCl or 2 mM CaCl2, all of the peptides, including GMA4C_AC, retain their antifungal activity in the presence of 100 mM NaCl or 100 mM KCl. However, GMA4C_V1A and GMA4C_V4A are two times more active than GMA4C_AC. In the presence of 2 mM CaCl2, only GMA4C_V1A and GMA4C_V4A inhibit fungal growth at 6 μM, while other peptides such as the plant defensins MtDef4 or OeDef1 show little activity at this concentration.

[0109] [Table 6]

[0110] Antifungal activity against human fungal pathogens: GMA4C_V1A and GMA4C_V3A, but not the parent MtDef4 defensin, display antifungal activity against C. auris and C. glabrata in cation-rich RPMI medium.

[0111] [Table 7]

[0112] [Table 8]

[0113] Example 2. Activity of GMA4C-variants against pathogenic microorganisms Antifungal activity was tested using half strength potato dextrose broth for peptides and RPMI for the control antifungal fluconazole and voriconazole. CLSI M27 and M38 methods were used to determine MICs. The minimum inhibitory concentration (MIC) is the concentration of the compound, protein or peptide at which there is no significant growth of the microorganism compared to the growth of the microorganism in growth medium lacking the compound, protein or peptide.

[0114] All studies were performed in RPMI buffered with 0.165M MOPS. The concentration range for peptides in potato dextrose broth was 0.06-2 mcg / ml, for fluconazole was 0.125-64 mcg / ml, and for voriconazole was 0.03-16 mcg / ml. MICs were determined at 24-72 hours.

[0115] [Table 9]

[0116] [Table 10]

[0117] [Table 11]

[0118] [Table 12]

[0119] [Table 13]

[0120] All pathogen isolates used in this assay are resistant to the antifungal drug fluconazole. Antifungal assays were performed in half-strength potato dextrose broth.

[0121] [Table 14]

[0122] [Table 15]

[0123] [Table 16]

[0124] Example 3. Antimicrobial activity of GMA4C_V1A against human bacterial pathogens Salmonella Typhimurium var. Copenhagen, enterotoxigenic E. coli-F4 and Listeria monocytogens (F5244) strains were grown overnight at 37°C on LB agar plates. A small number of bacteria were scraped off the plates, added to Mueller-Hinton (MH) growth medium and grown to log phase. 1–3x10 6 Cells were diluted to CFU / mL and 50ul was added to each well in a polypropylene 96-well plate. The synthesized peptide GMA4C_V1A was diluted in 0.2% BSA, 0.01% acetic acid solution and added to concentrations of 0.2, 0.4, 0.80, 1.6, 3.25, 7.5, 15, 30 and 60 and 120μM. 50μl of each peptide solution was then added to 50μl of bacterial cells. The plates were covered with parafilm and incubated overnight at 37°C. The OD of MH medium alone was 1.0μl. 600 nm values ​​and OD at each concentration compared to that of bacterial growth in MH medium without peptide. 600 Concentration at which bacterial growth inhibition was determined based on nm values. The minimum inhibitory concentration (MIC) is the concentration of peptide at which there is no significant growth of the microorganism compared to the growth of the microorganism in growth medium lacking the peptide.

[0125] [Table 17]

[0126] Example 4. Antimicrobial activity of defensin peptide fragments Crude chemically synthesized defensin-derived peptides of 80-85% purity were obtained from Biomatik Inc, Canada or Alan Scientific Inc., USA. Each peptide was further purified using C-18 reversed-phase HPLC (Agilent, Singapore). HPLC fractions containing the peptides were lyophilized and resuspended in nuclease-free water. For their accurate quantification, concentrations were determined using the BCA assay using the manufacturer's protocol (Thermo-Fisher Scientific).

[0127] Fungal strains of Botrytis cinerea T-4, Alternaria alternata, Cercospora sojina and Colletotrichum gloeosporioides were cultured in their respective regular growth media as shown in Table 16. Fungal spores were harvested by flooding the fungal growth plates with sterile water. The spore suspension was filtered through two layers of Miracloth, centrifuged at 13,600 rpm for 1 min, washed and resuspended in low-salt synthetic fungal medium (SFM) (U.S. Patent No. 6,316,407). The spore suspension was adjusted to the desired spore count using a hemocytometer.

[0128] [Table 18]

[0129] The antifungal activity of truncated defensin-derived peptides and their variants against the fungal pathogens B. cinerea, A. alternata, C. sojina, and C. gloeosporioides was determined spectrophotometrically using a 96-well plate assay (Sagaram et al., (2011) PLoS ONE 6:e18550. doi:10.1371 / journal.pone.0018550). 45 μL (approximately 10 5 Forty-five microliters of peptides at concentrations of 0.375, 0.75, 1.5, 3, and 12 μM were added to each well of a microtiter plate containing a spore suspension (B. cinerea spores / ml). After 48 h, quantitative fungal growth inhibition was determined by measuring absorbance at 595 nm using a microplate reader (Tecan Infinite M200 ProTecan Systems Inc., San Jose, CA). A resazurin cell viability assay was used to determine fungal cell viability (Chadha and Kale, (2015) Lett Appl Microbiol 61, 238-244, Li et al., (2019) Mol Plant Microbe Interact 32, 1649-1664). After incubation of the pathogen / peptide mixture for 48 h, 10 μl of 0.1% resazurin solution was added to each well and reincubated overnight. A color change of resazurin from blue to pink or colorless indicated the presence of viable fungal cells. The MIC value of each peptide was determined as the lowest concentration of peptide at which no blue color change was observed. MIC values ​​of defensin-derived peptides and their variants were also determined in SFM and SFM supplemented with 100 mM NaCl, 100 mM or 2 mM CaCl2 as described above.

[0130] The semi-in planta antifungal activity of each defensin-derived peptide and its variants against B. cinerea was determined using detached leaves of N. benthamiana Nb1 as previously described (Li et al., (2019) Mol Plant Microbe Interact 32, 1649-1664; Velivelli et al., (2020) Proceedings of the National Academy of Sciences. 117, 16043-16054). Each peptide was tested at concentrations of 1.5 μM, 3 μM and 6 μM. After incubation of each peptide / fungal spore mixture at room temperature for 48 h, photographs of the leaves were taken under white light. High-resolution fluorescence images were also taken using CropReporter (PhenoVation, Wageningen, Netherlands). These images were compared to the F of diseased areas affected by B. cinerea infection. V / F M The calculated values ​​are shown for the maximum quantum yield of photosystem II. The colors in the images represent five different classes ranging from Class I to Class V (0.000-0.700) that indicate the varying degree of tissue damage. The green color in each image corresponds to Class V, ranging from 0.600 to ≥ 0.700, which indicates healthy areas of the leaf surface, while the red color corresponds to Class I, ranging from 0.000-0.160, which indicates severely damaged or diseased leaf surfaces.

[0131] The primary amino acid sequence, length, net charge and percentage of hydrophobic amino acids of the defensin-derived peptides are shown in Table 17. These peptides are derived from the plant defensins OeDef1, MtDef4, MsDef1 and MtDef5A. Amino acid substitutions in the wild-type sequence of each peptide were made to improve the net charge and hydrophobicity. In addition, disulfide bonds were introduced into specific variants to make them pseudocyclic. Peptides capable of forming a single disulfide bond and pseudocyclic peptides are indicated as "+" in the "Disulfide Bond" column of Table 17. All peptides also possess a carboxy-terminal amide group.

[0132] [Table 19]

[0133] The minimum inhibitory concentration (MIC) values ​​of each defensin-derived peptide and its variants were determined (Table 18). It is hypothesized that the presence of cations significantly weakens the electrostatic interaction between positively charged defensins and negatively charged fungal membranes (Chu et al., (2013) Antimicrobial Agents and Chemotherapy 57:4050-4052). Therefore, we determined the antifungal activity of each peptide against B. cinerea in SFM supplemented with 100 mM NaCl, 100 mM KCl or 2 mM CaCl2 (Table 18).

[0134] [Table 20]

[0135] The MIC values ​​of the defensin-derived peptides were also tested against Alternaria alternata, Cercospora sojina, and Colletotrichum gloeosporioides (Table 19).

[0136] [Table 21]

[0137] The semi-in planta antifungal activity of GMA4C_V9, GMA4C_V10, GMAOe1C_WT, GMAOe1C_V3, GMAOe1C_V4, GMA1C_V1 and GMA1C_V2 peptides against B. cinerea was determined by using detached leaves of N. benthamiana. Each peptide at concentrations of 1.5 μM, 3 μM and 6 μM was applied as a drop to the leaves and freshly prepared conidial inoculum was immediately applied to each drop of the peptide. Leaves were assessed for reduction of Botrytis symptoms 48 hours after inoculation by measuring the lesion size compared to the control without peptide. Both GMA4C_V9 and GMA4C_V10 peptides are effective in reducing Botrytis symptoms. However, at the low concentrations of 1.5 μM and 3 μM, GMA4C_V10 was more effective than GMA4C_V9 in reducing symptoms of Botrytis cinerea (FIG. 5).

[0138] Drop inoculation assays were also performed to test the antifungal activity of GMAOe1C_WT, GMAOe1C_V1, and GMAOe1C_V2. The results revealed that GMAOe1C_V1 and GMAOe1C_V2 at concentrations of 3 and 6 μM completely suppressed the symptoms of Botrytis cinerea, while GMAOe1C_WT was only effective at 6 μM. At a concentration of 1.5 μM, GMAOe1C_V3 was more effective than GMAOe1C_V4 or GMAOe1C_WT (Figure 6).

[0139] Drop inoculation assays were also performed to test the antifungal activity of GMA1C_V1 and GMA1C_V2 (Figure 6). GMA1C_V2 at 3 μM and 6 μM completely suppressed the symptoms of Botrytis cinerea. GMA1C_V1 failed to reduce disease symptoms at these concentrations. At a concentration of 1.5 μM, GMA1C_V2 was more effective than GMA1C_V1 (Figure 7).

[0140] The results above indicate that a panel of modifications (eg, amino acid substitutions) introduced into these truncated defensin-derived peptides confer greater antifungal activity than the wild-type truncated peptides.

[0141] The breadth and scope of the present disclosure should not be limited by any of the above examples.

Claims

1. A peptide comprising the following amino acid sequence (a) A modified gamma-core consensus sequence GXCX3-10(F / W / Y / L / V / I / M)(F / W / Y)(F / W / Y / L / V / I / M) (SEQ ID NO: 50), GXCX3-10(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO: 587), GXCX3-8(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO: 33), GXCX3-9(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO: 34), GXCX3-8(F / W / Y) (SEQ ID NO: 43), GXCX3-9(F / W / Y) (SEQ ID NO: 44), GXCX3-8(F / W / Y / L / V / I / M) (SEQ ID NO: 45), GXCX3-10(F / W / Y / L / V / I / M) (SEQ ID NO: 46), GXCX3-8(L / V / I / M)(F / W / Y)(L / V / I / M) (SEQ ID NO: 47), GXCX3-10(L / V / I / M)(F / W / Y)(L / V / I / M) (SEQ ID NO: 48), GXCX3-8(F / W / Y / L / V / I / M)(F / W / Y)(F / W / Y / L / V / I / M) (SEQ ID NO: 49), GXCX3-8(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M) (SEQ ID NO: 51) or GXCX3-10(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M) (SEQ ID NO: 52) (the peptide further comprises a second C-terminal cysteine residue located at the C-terminus relative to the cysteine residue in the modified gamma-core consensus sequence, the peptide has at least 3 net positive charges, and the hydrophobic amino acid content is at least 18%); or (b) SEQ ID NO: 1 (the peptide does not include the corresponding full-length sequences of the defensin peptides of SEQ ID NOs: 8, 16, 20, 23, 26, 29, 37, 38, 39, 40 or 41; optionally, the peptide comprises the modified gamma-core consensus sequence GXCX3-8(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO: 33), GXCX3-9(F / W / Y)(F / W / Y)(F / W / Y) (SEQ ID NO: 34), GXCX3-8(F / W / Y) (SEQ ID NO: 43) or GXCX3-9(F / W / Y) (SEQ ID NO: 44); and / or optionally the C-terminal cysteine residue or C-terminal amino acid residue is amidated).

2. The peptide according to claim 1, comprising: (i) conserved C1 and C4 cysteine residues corresponding to the N-terminal and C-terminal cysteines of the reference defensin C-terminal peptide, wherein amino acid residues 16 and 18 corresponding to the conserved C2 and C3 cysteine residues of the reference defensin C-terminal peptide are independently substituted with tryptophan, tyrosine, phenylalanine, leucine, valine, isoleucine or methionine; optionally, the defensin peptide variant has at least 3 net positive charges and a hydrophobic amino acid content of at least 18%; or (ii) an amino acid sequence having at least 50%, 55%, 60%, 68%, 75%, 82% or 94% sequence identity over the full length of SEQ ID NO: 7, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, 15, 17-20, 22, 23, 25, 26, 28, 29, 31, 32, 42, 578, 579, 581, 582, 584 or 585 (wherein the peptide does not include the corresponding full-length sequence of the defensin peptide of SEQ ID NO: 8, 16, 20, 23, 26, 29, 37, 38, 39, 40 or 41); optionally, the peptide of (i) or (ii) includes a disulfide bond between the two cysteine residues.

3. The peptide according to claim 1 or 2, comprising: (i) 30 amino acid residues or less; or (ii) comprising, consisting essentially of, or consisting of 15, 16 or 17-30 amino acid residues.

4. A composition comprising the peptide according to claim 1 or 2 and an agriculturally acceptable, pharmaceutically acceptable or veterinarily acceptable carrier, diluent or excipient.

5. The composition according to claim 4, wherein the peptide is provided at a concentration of about 0.1, 0.5, 1.0 or 5 pg / ml to about 1, 5, 20, 50 or 100 mg / ml or at a concentration of about 0.1, 0.5, 1.0 or 5 pg / gram to about 1, 5, 20, 50 or 100 mg / gram, and optionally includes a sodium salt at a concentration of at least 100 mM and / or a calcium salt at a concentration of at least 2 mM.

6. A method for preventing or reducing crop damage caused by phytopathogenic microorganisms, comprising contacting a plant, plant seed or other part of the plant with an effective amount of the composition according to claim 4.

7. The method according to claim 6, wherein the phytopathogenic microorganism is Fusarium sp., Alternaria sp., Verticillium sp., Phytophthora sp., Colletotrichum sp., Botrytis sp., Cercospora sp., Phakopsora sp., Rhizoctonia sp., Sclerotinia sp., Pythium sp., Phoma sp., Leptosphaeria sp., Gaeumannomyces sp., Puccinia sp., Septoria sp., Penicillium sp., Diplodia sp., Phomopsis sp., Mycosphaerella sp., Golovinomyces sp., Erysiphe sp., Albugo sp., Setosphaeria sp., Cochliobolus sp., Helminthosporium sp., Diplodia sp. or Stenocarpella sp.

8. A medical device comprising the device and the composition according to claim 4, wherein the device comprises at least one surface that is locally coated with and / or impregnated with the composition.

9. The medical device according to claim 8, which is a stent, catheter, contact lens, condom, patch or septum.

10. Use of the peptide according to claim 1 or 2 for the preparation of a composition for treating, preventing or inhibiting a microbial infection in a subject in need thereof, wherein the composition may further comprise an agriculturally acceptable, pharmaceutically acceptable or veterinarily acceptable carrier, diluent or excipient. Use according to claim 10, wherein the composition is formulated for topical, enteral, parenteral and / or intravenous administration.

12. Use according to claim 10, wherein the subject is a human, livestock, poultry, fish or companion animal.

13. Use according to claim 10, wherein the microbial infection is an infection of the mucosa, eye, skin and / or nail, and the composition is applied to the mucosa, eye, skin and / or nail.

14. Use according to claim 10, wherein the microbial infection is caused by dermatophytes, and the dermatophytes are optionally selected from the group consisting of Trichophyton rubrum, Trichophyton interdigitale, Trichophyton violaceum, Trichophyton tonsurans, Trichophyton soudanense, Trichophyton mentagrophytes, Microsporum flavum, Epidermophyton floccosum and Microsporum gypseum.

15. Use according to claim 10, wherein the microbial infection is caused by species of Aspergillus, Cryptococcus, Penicillium, Rhizopus, Apophysomyces, Cunninghamella, Saksenaea, Rhizomucor, Syncephalastrum, Cokeromyces, Actinomucor, Pythium, Fusarium, Histoplasmosis or Blastomyces.

16. The microbial infection is caused by a Candida species, and the Candida species is Candida albicans (C. albicans), C. auris, C. glabrata, C. parapsilosis, C. tropicalis or C. krusei, the use according to claim 10.

17. The composition according to claim 4, further comprising an antifungal agent.

18. The composition according to claim 17, further comprising an insecticide or a nematicide.

19. A method for producing a plant part for agricultural use, comprising applying the composition according to claim 4 to a plant part to obtain a plant part at least partially coated with the composition.

20. The method according to claim 19, wherein the plant part is a seed, and the seed is optionally a corn, soybean, wheat, rice, cotton, Brassica sp. or tomato seed.

21. The method according to claim 19, which is a fruit, vegetable or flower.

22. A recombinant polynucleotide comprising a polynucleotide encoding a peptide comprising the peptide according to claim 1 or 2, wherein the polynucleotide encoding the first antimicrobial peptide is operably linked to a promoter that is heterologous to the polynucleotide encoding the first antimicrobial peptide, and optionally any amino acid substitution in the sequence improves or maintains the net positive charge and / or improves or maintains the hydrophobicity of the peptide.

23. The recombinant polynucleotide according to claim 22, further comprising (i) a transit peptide, a vacuolar targeting peptide and / or an endoplasmic reticulum targeting peptide; (ii) a plastid targeting peptide; and / or (iii) a polynucleotide encoding a polyadenylation or transcription termination signal, and the polynucleotide of (i), (ii) and / or (iii) is operably linked to the polynucleotide encoding the antimicrobial peptide.

24. The polynucleotide encoding the first antimicrobial peptide is inserted into the heterologous nucleus or plastid genome of a cell and is operably linked to an endogenous promoter located in the heterologous nucleus or plastid genome, the recombinant polynucleotide according to claim 22.

25. A plant comprising a polynucleotide encoding a peptide comprising the peptide according to claim 1 or 2, wherein the polynucleotide is heterologous to the plant and the polynucleotide is operably linked to a promoter.

26. A cell comprising the recombinant polynucleotide according to claim 22, optionally a bacterial, yeast or plant cell.

27. The cell is a yeast cell, and the yeast cell is a Candida, Kluyveromyces, Hansuela, Pichia, Saccharomyces, Schizosaccharomyces or Yarrowia cell, the cell according to claim 26.

28. The cell is a bacterial cell, and the bacterial cell is an Acinetobacter, Alcaligenes, Azotobacter, Bacillus, Escherichia, Lactobacillus, Lactococcus, Streptomyces or Pseudomonas cell, the cell according to claim 26.

29. (i) self-pollinating or crossing the plant according to claim 25; (ii) harvesting seeds containing the recombinant polynucleotide of the plant from the self-pollination or the crossing, thereby producing plant seeds that confer resistance to infection by phytopathogenic microorganisms. A method for producing plant seeds that confer resistance to infection by phytopathogenic microorganisms, comprising the steps of:

30. A method for preparing an agricultural composition comprising formulating the peptide according to claim 1 with a carrier, an inert substance, a surfactant or a solvent.

31. The method according to claim 30, wherein the composition is formulated with other insecticidal active substances, fertilizers and / or growth regulating substances.

32. A method for producing the peptide according to claim 1, comprising expressing the peptide in a yeast or bacterial cell containing an expression vector encoding the peptide according to claim 1.

33. A plant part at least partially coated with the composition according to claim 4.

34. The plant part according to claim 33, wherein the plant part is a seed, and the seed is optionally a corn, soybean, wheat, rice, cotton, Brassica sp. or tomato seed.

Images