Antimicrobial peptides and their modifications

Abstract

Antimicrobial modified defensin or defensin-like peptides, modified C-terminal fragments of defensins or defensin-like peptides, and nucleic acids encoding them are disclosed. Compositions containing the defensin mutant peptides and methods of using them to control microbial infections in plants and vertebrates, and to control feed and food contamination are also disclosed.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims the benefit of U.S. Provisional Patent Application No. 63 / 603,414, filed November 28, 2023, and U.S. Patent Application No. 63 / 476,488, filed December 21, 2022, each of which is incorporated by reference in its entirety.

[0002] Government support statement This invention was made with government support under National Science Foundation EAGER Award No. 1955461 awarded by the National Science Foundation. The government has certain rights in this invention.

[0003] Sequence Listing The sequence listing contained in the file entitled "P14299WO00", which is 823,641 bytes as measured on a Windows operating system and was created on December 19, 2023, and which was submitted electronically herewith, is incorporated herein by reference in its entirety. [Background technology]

[0004] Protecting agriculturally important crops from pathogenic microorganisms (e.g., fungi or oomycetes) is crucial for improving crop yields. Microbial infections are particularly problematic in humid climates and can be a major concern during crop storage. Such infections can result in spoilage and contamination of food or feed products with microbial toxins. Unfortunately, modern growing methods, harvesting, and storage systems can facilitate the transmission of plant pathogens.

[0005] Suppression of plant pathogens is further complicated by the need to simultaneously suppress multiple microorganisms from different genera. For example, Alternaria, Ascochyta, Aphanomyces, Botrytis, Cercospora, Colletotrichum, Diplodia, Erysipe, Fusarium, Gaeumannomyces, Helminthosporium, Leptosphaeria, Macrohomina, Magnaporthe, Nectria, Peronospora, Phoma, Phakopsora, Phymatotrichum, Phytophthora, Plasmopara, Podosfera, Puccinia, Pythium, Pyrenophora, Pyricularia, Rhizoctonia, Sclerotium, Sclerotinia, Septoria, Thielaviopsis, Uncinula, Venturia, and Verticillium are all recognized plant pathogens.

[0006] Certain microorganisms (e.g., fungi, including molds, yeasts, and dimorphic fungi, or oomycetes) can also be pathogenic to a variety of vertebrates, including humans, livestock, companion animals, fish, etc. Microorganisms including dermatophytes, Aspergillus, Candida, Cryptococcus, Coccidiomyces, Penicillium, Rhizopus, Apophysomyces, Cunninghamella, Succenaea, Rhizomucor, Syncephalastrum, Cocheromyces, Actinomycosis, Pythium, Fusarium, Histoplasma, or Blastomyces are also important vertebrate pathogens.

[0007] A group of proteins known as defensins has been shown to inhibit plant pathogens. Defensins have previously been identified as small, cysteine-rich peptides of approximately 45–54 amino acids that constitute key components of plant innate immunity (Shafee et al., 2016; Thomma et al., 2002; Lay and Anderson, 2005; Vriens et al., 2014). Defensins, widely distributed in plants, vary significantly in their amino acid composition. However, they all possess a compact structure stabilized by four or five intramolecular disulfide bonds. Plant defensins have previously been characterized to contain a conserved gamma-core (i.e., γ-core) peptide containing the conserved GXCX3-9C (X = 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 antibacterial activity of certain defensins correlates with the presence of positively charged amino acid residues within the gamma core peptide (Spelbrink et al., Plant Physiol., 2004; Sagaram et al., 2013).

[0008] Plant defensins have been extensively 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). When expressed in transgenic plants, they confer strong resistance to microbial pathogens (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 pure proteins were added to in vitro antibacterial 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 sativa (Hanks et al., 2005). In vitro experiments demonstrated that the cloned M. truncatula defensin protein MtDef2 has little or no antibacterial activity (Spelbrink et al., 2004). In contrast, the Medicago sativa defensin proteins MtDef4 (U.S. Patent No. 7,825,297, incorporated herein by reference in its entirety) and MtDef5 (WO2014179260 and U.S. Patent Application Publication No. 20160208278, both incorporated herein by reference in their entirety) possess antibacterial activity. The C-terminal 16-amino acid peptide GMA4-C of the MtDef4 defensin protein inhibits Fusarium graminearum in vitro at concentrations as low as 3 μM (Sagaram et al., 2011).

[0010] Plant defensins with potent in vitro antifungal activity often fail to confer effective disease resistance in plants. 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 crossing of the fungal cell envelope via 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 between 100 and 200 mM (Shabala and Pottosin, 2010) and between 10 and 200 mM in the apoplast (White and Karley, 2010). Calcium is an essential secondary micronutrient whose concentration can range from 0.1% to 6% of the plant's dry weight (Broadley et al., 2003). Sodium (Na+) concentrations in plants range 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 when the concentration of monovalent and divalent cations, such as 100 mM KCl or 2 mM CaCl2, is increased. However, the maize (Zea mays) defensin ZmD32, which has a predicted charge of +10.1 at pH 7, exhibits inhibitory activity against Candida spp. and E. coli in the presence of 100 mM NaCl. Similarly, the Nicotiana benthamiana 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

[0012] A peptide comprising the amino acid sequence of a modified defensin or modified defensin-like peptide, wherein the wild-type gamma core consensus peptide GXCX3-9C or GXCX3-22C of the wild-type defensin or wild-type defensin-like peptide is selected from the group consisting of the peptide sequences GXCX3-9(F / W / Y), GXCX3-9(F / W / Y / L / V / I / M), GXCX3-9(F / W / Y)(F / W / Y / M)(F / W / Y), GXCX3-9(F / W / Y / L / V / I / M), / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M), GXCX9-22(F / W / Y), GXCX16-22(F / W / Y), GXCX9-22(F / W / Y / L / V / I / M), GXCX16-22( F / W / Y / L / V / I / M), GXCX9-22(F / W / Y)(F / W / Y / L / V / I / M)(F / W / Y), GXCX16-22(F / W / Y)(F / W / Y / L / V / I / M)(F / W / Y), GXCX9-22(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M), GXCX16-22(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M), GXCX3- 9(F / W / Y)(R / K / H)(F / W / Y), GXCX3-9(F / W / Y / L / V / I / M)(R / K / H)(F / W / Y / L / V / I / M), GXCX9-22(F / W / Y)(R / K / H)(F / W / Y), GXCX16 and a modified gamma core consensus peptide comprising GXCX16-22(F / W / Y)(R / K / H)(F / W / Y), GXCX16-22(F / W / Y / L / V / I / M)(R / K / H)(F / W / Y / L / V / I / M), or GXCX9-22(F / W / Y / L / V / I / M)(R / K / H)(F / W / Y / L / V / I / M), wherein the peptide has a net positive charge of at least 3 and a hydrophobic amino acid content of at least 18%.A peptide comprising the amino acid sequence of a modified defensin peptide fragment, wherein the wild-type gamma core consensus peptide GXCX3-9C or GXCX3-22C of the corresponding wild-type defensin peptide fragment is the peptide sequence GXCX3-9(F / W / Y), GXCX3-9(F / W / Y / L / V / I / M), GXCX3-9(F / W / Y)(F / W / Y / M)(F / W / Y), GXCX3-9(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M), GXCX9-22(F / W / Y)(F / W / Y)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M), Y), GXCX16-22(F / W / Y), GXCX9-22(F / W / Y / L / V / I / M), GXCX16-22(F / W / Y / L / V / I / M), GXCX9-22(F / W / Y)(F / W / Y / L / V / I / M)(F / W / Y), GXCX16-22(F / W / Y)(F / W / Y / L / V / I / M)(F / W / Y), GXCX9-22(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M), GXCX16-22(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M) (F / W / Y / L / V / I / M), GXCX3-9(F / W / Y)(R / K / H)(F / W / Y), GXCX3-9(F / W / Y / L / V / I / M)(R / K / H)(F / W / Y / L / V / I / M), GXCX9-22(F / W / Y)(R / K / H)(F / W / Y) , GXCX16-22(F / W / Y)(R / K / H)(F / W / Y), GXCX16-22(F / W / Y / L / V / I / M)(R / K / H)(F / W / Y / L / V / I / M), or GXCX9-22(F / W / Y / L / V / I / M)(R / K / H)(F / W / Y / L / V / I / M), wherein the peptide further comprises a second C-terminal cysteine ​​residue located C-terminal to the cysteine ​​residue of the modified gamma core consensus sequence, the peptide having a net positive charge of at least 3 and a hydrophobic amino acid content of at least 18%, and optionally the peptide comprises, consists essentially of, or consists of (i) no more than 30 amino acid residues, or (ii) between 15, 16, or 17 and 30 amino acid residues.1. A peptide comprising a C-terminal fragment of a defensin-like peptide, wherein the C-terminal fragment lacks 1 to 35 amino-terminal amino acids of the corresponding wild-type defensin-like peptide, and / or has the peptide sequence GXCX3-9(F / W / Y), GXCX3-9(F / W / Y / L / V / I / M), GXCX3-9(F / W / Y)(F / W / Y / M)(F / W / Y), GXCX3-9(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M), GXCX9-22(F / W / Y), GXCX16-22(F / W / Y), GXCX9-22(F / W / Y / L / V / I / M), GXCX16-22 (F / W / Y / L / V / I / M), GXCX9-22(F / W / Y)(F / W / Y / L / V / I / M)(F / W / Y), GXCX16-22(F / W / Y)(F / W / Y / L / V / I / M)(F / W / Y), G XCX9-22(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M), GXCX16-22(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)( F / W / Y / L / V / I / M), GXCX3-9(F / W / Y)(R / K / H)(F / W / Y), GXCX3-9(F / W / Y / L / V / I / M)(R / K / H)(F / W / Y / L / V / I / M), GXCX9 Peptides are provided, including modified gamma core consensus peptides including GXCX-22(F / W / Y)(R / K / H)(F / W / Y), GXCX16-22(F / W / Y)(R / K / H)(F / W / Y), GXCX16-22(F / W / Y / L / V / I / M)(R / K / H)(F / W / Y / L / V / I / M), or GXCX9-22(F / W / Y / L / V / I / M)(R / K / H)(F / W / Y / L / V / I / M).A peptide comprising, consisting essentially of, or consisting of SEQ ID NOs: 729, 731, 733, 735, 736, 828, 829, 830, 831, 832, or 833, a variant thereof comprising conservative amino acid substitutions of 1 to 2, 3, 4, or 5 amino acid residues, or a variant thereof having at least 90% or 95% sequence identity thereto, wherein the gamma core consensus peptide is conserved in the variant, or the gamma core consensus peptide is the peptide sequence GXCX3-9(F / W / Y), GXCX3-9(F / W / Y / L / V / I / M), GXCX3-9(F / W / Y)(F / W / Y / M)(F / W / Y) , GXCX3-9(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M), GXCX9 -22(F / W / Y), GXCX16-22(F / W / Y), GXCX9-22(F / W / Y / L / V / I / M), GXCX16- 22(F / W / Y / L / V / I / M), GXCX9-22(F / W / Y)(F / W / Y / L / V / I / M)(F / W / Y), GXC X16-22(F / W / Y)(F / W / Y / L / V / I / M)(F / W / Y), GXCX9-22(F / W / Y / L / V / I / M )(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M), GXCX16-22(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M), GXCX3-9(F / W / Y)(R / K / H)(F / W / Y), GX CX3-9(F / W / Y / L / V / I / M)(R / K / H)(F / W / Y / L / V / I / M), GXCX9-22(F / W / Y)( and optionally, the gamma core consensus peptide comprises the peptide sequence GXCX3-9C or GXCX9-16C. In certain embodiments, the peptides exhibit antibacterial activity, which optionally is one or more of antifungal or antibacterial activity.In certain embodiments, the peptide is an isolated peptide. Also provided are compositions comprising any of the aforementioned peptides and an agriculturally, pharmaceutical, or veterinarily acceptable carrier, diluent, or excipient. Also provided are compositions comprising any of the aforementioned 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 caused by plant pathogenic microorganisms or (ii) preventing contamination of plants, plant parts, seeds, feed derived therefrom, or food derived therefrom by undesirable microorganisms, comprising contacting the plant, plant seed, or other part of the plant with an effective amount of any of the aforementioned compositions, 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 aforementioned compositions, which optionally include an agriculturally acceptable carrier, diluent, or excipient.

[0015] Methods are provided for treating, preventing, or inhibiting a microbial infection in a subject in need thereof, comprising administering to said subject an effective amount of the aforementioned composition.

[0016] Also provided is a medical device comprising a device and the aforementioned composition, the device having at least one surface topically coated and / or impregnated with the composition, the composition optionally comprising a pharmaceutically or veterinarily acceptable carrier, diluent, or excipient.

[0017] Also provided are methods for treating, preventing, or inhibiting a microbial infection in a subject in need thereof, comprising administering to the subject an effective amount of any of the aforementioned compositions, wherein the composition optionally comprises a pharmaceutically or veterinarily acceptable carrier, diluent, or excipient. Also provided are uses of any of the aforementioned compositions in methods for treating, preventing, or inhibiting a microbial or yeast infection in a subject in need thereof. Also provided are uses of any of the aforementioned 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.

[0018] Recombinant polynucleotides are provided, including a polynucleotide encoding a peptide comprising any of the aforementioned peptides, wherein the polynucleotide encoding the antimicrobial peptide is operably linked to a polynucleotide comprising a promoter heterologous to the polynucleotide encoding the peptide: (i) a defensin peptide comprising the long gamma core consensus sequence GXCX16-22C, optionally comprising SEQ ID NOs: 578, 608, 612, or a variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NOs: 578, 608, or 612, a variant thereof comprising a deletion of 1 to 10 N-terminal amino acid residues, a variant thereof comprising a deletion of 1 to 10 C-terminal amino acid residues, and / or a variant thereof having conservative amino acid substitutions of 1 to 10 amino acid residues; or (ii) a wild-type gamma core consensus peptide GXCX 3-9 C, GXCX 9-16 C or GXCX 3-22C, optionally selected from the group consisting of SEQ ID NOs: 534, 538, 560, 565, 569, 573, 583, 598, 603, 616, 624, 628, 633, 645, 697, 702, 711, 715, 723, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 5, 736, or SEQ ID NOs: 534, 538, 560, 565, 569, 573, 583, 598, 603, 616, 624, 628, 633, 645, 697, 702, 711, 715, 723, 728, 729, 730, 731, 732, 733, 734, 73 ... A recombinant polynucleotide encoding a peptide comprising a defensin or defensin-like peptide, including variants thereof having 90%, 95%, 98%, or 99% sequence identity with the defensin or defensin-like peptide, variants thereof comprising a deletion of 1 to 10 or 1 to 35 N-terminal amino acid residues, variants thereof comprising a deletion of 1 to 10 C-terminal amino acid residues, and / or variants thereof having conservative amino acid substitutions of 1 to 10 amino acid residues, wherein the polynucleotide encoding the peptide is operably linked to a polynucleotide comprising a promoter heterologous to the polynucleotide encoding the peptide.

[0019] (i) a peptide encoded by any of the foregoing recombinant polynucleotides; (ii) a defensin peptide comprising the long gamma core consensus sequence GXCX16-22C, optionally comprising SEQ ID NO:578, 608, 612, or a variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:578, 608, or 612, a variant thereof comprising a deletion of 1 to 10 N-terminal amino acid residues, a variant thereof comprising a deletion of 1 to 10 C-terminal amino acid residues, and / or a variant thereof having conservative amino acid substitutions of 1 to 10 amino acid residues; or (iii) a defensin or defensin-like peptide comprising the wild-type gamma core consensus peptide GXCX3-9C, GXCX9-16C, or GXCX3-22C, optionally comprising SEQ ID NO:534, 538, 560, 565. , 569, 573, 583, 598, 603, 616, 624, 628, 633, 645, 697, 702, 711, 715, 723, 728, 729, 730, 731, 732, 733, 734, 735, 736, or SEQ ID NOs: 534, 538, 560, 565, 569, 573, 583, 598, 603, 616, 624, 628, 633, 645, 697, 702, 711, 715, 723, 728, 729, 730, 731, 732, 733, 734, 735, 736 4, 735, 736, a variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to 4, 735, 736, a variant thereof comprising a deletion of 1 to 10 N-terminal amino acid residues, a variant thereof comprising a deletion of 1 to 10 C-terminal amino acid residues, and / or a variant thereof having conservative amino acid substitutions of 1 to 10 amino acid residues. Compositions are provided that include the peptides of the foregoing embodiments and an agriculturally, pharmaceutical, or veterinarily acceptable carrier, diluent, or excipient.

[0020] Provided is a nuclear genome or plastid genome of a plant comprising a polynucleotide encoding an antimicrobial peptide, including any of the aforementioned peptides, wherein the polynucleotide is heterologous to the nuclear genome or plastid genome, and the polynucleotide is operably linked to an endogenous promoter of the nuclear genome or plastid genome.

[0021] Also provided are cells, plants, and plant parts, including seeds, that contain the recombinant polynucleotide or genome and / or are at least partially coated with any of the compositions. Also provided are methods for producing plant seeds that provide plants that are resistant to infection by plant pathogenic microorganisms, the methods comprising: (i) selfing or crossing the plants; and (ii) harvesting seeds from the selfing or crossing that contain the plant's recombinant polynucleotide, thereby producing plant seeds that provide plants that are resistant to infection by plant pathogenic microorganisms. Also provided are methods for producing antifungal peptides, the methods comprising: (i) culturing cells that contain the recombinant polynucleotide under conditions in which the peptide, defensin, or defensin-like peptide is expressed by the cells; and (ii) purifying the peptide, defensin, or defensin-like peptide from the culture. [Brief explanation of the drawings]

[0022] [Figure 1] A non-limiting subset of reference wild-type defensin peptides aligned with the conserved C1, C2, C3, C4, C5, C6, C7, and C8 cysteines (bold) is shown. The conserved gamma core peptide is underlined. [Figure 2]A non-limiting subset of reference wild-type defensin peptides is shown aligned with the conserved C1, C2, C3, C4, C5, C6, C7, and C8 cysteines (bold). The conserved gamma-core peptide is underlined. Below each wild-type defensin peptide, non-limiting examples of modified defensin peptides and modified defensin C-terminal fragments are shown aligned with the conserved, unsubstituted C1, C2, C3, C4, C5, and C8 cysteines (bold), in which at least the conserved C6 and C7 cysteine ​​residues are substituted. The substituted residues in the modified defensin peptides and modified defensin C-terminal fragments are double underlined and italicized. The conserved gamma-core peptide is underlined in the wild-type defensin peptide and is not present in the modified gamma-core consensus peptide. The corresponding amino acid residue numbers of the modified defensin peptide present in the modified C-terminal fragment peptide are shown in parentheses. [Figure 3A] 1 shows the preventative effect of antifungal peptide PD122.1.1 (SEQ ID NO: 715 with an N-terminal alanine residue). [Figure 3B] 1 shows the curative effect of antifungal peptide PD122.1.1 (SEQ ID NO: 715 with an N-terminal alanine residue). [Figure 4] 1 shows the curative effect of peptide PD101.1 on leaves of wheat plants inoculated with Z. tritici. DETAILED DESCRIPTION OF THE INVENTION

[0023] definition The term "and / or," as used herein, should be understood as a specific disclosure of each of two particular features or components with or without the other. Thus, the term "and / or" used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to encompass 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 (alone); B (alone); and C (alone).

[0024] As used herein, the terms "corresponding," "corresponding to," and the like, when used in the context of amino acid positions, mutations, and / or substitutions in any given 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), refer to amino acid residues in the given peptide sequence that have the same positions in the given peptide as residues in the reference amino acid sequence when the given peptide is aligned to the reference sequence. In certain embodiments, the alignment is of the four conserved cysteine ​​residues of the defensin C-terminal peptide of the defensin variant peptide with the reference defensin C-terminal peptide sequence (e.g., as shown in Figures 2 and 3).

[0025] As used herein, the terms "comprises," "including," and "comprising" should be interpreted as having at least the features they refer to, while not excluding additional unspecified features.

[0026] Where a term is provided in the singular, other embodiments are also provided that are described by the plural of that term.

[0027] As used herein, the phrase "antimicrobial peptide" refers to a peptide that exhibits one or more of the following characteristics: inhibiting the growth of microbial cells; killing microbial cells; disrupting or delaying a stage of the microbial life cycle, such as spore germination, sporulation, or conjugation; and / or disrupting the infection, penetration, or spread of microbial cells in plants or other susceptible subjects, including humans, livestock, poultry, fish, or companion animals (e.g., dogs or cats).

[0028] As used herein, the terms "acidic" or "anionic" are used interchangeably to refer to amino acids such as aspartic acid and glutamic acid.

[0029] As used herein, the term "amino acid" refers to an organic compound containing an amino (-NH3) functional group and a carboxylate (-CO2) functional group, along with a side chain (R group) characteristic of each amino acid. Amino acid residues in polypeptides are referred to herein, in certain cases, by the single-letter amino acid code, such as 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).

[0030] As used herein, the terms "basic" and "cationic" are used interchangeably to refer to amino acids such as arginine, histidine, and lysine.

[0031] As used herein, the phrase "cation-tolerant" refers to a defensin peptide or modified defensin or defensin-like peptide that exhibits equivalent in vitro antifungal or antibacterial activity in the presence of 100 mM KCl or 100 mM NaCl, or that exhibits no more than about a 1.5-fold, 2-fold, 3-fold, or 4-fold decrease in in vitro antifungal or antibacterial activity, compared to the antifungal activity of the defensin peptide or modified defensin or defensin-like peptide in the absence of KCl or NaCl.

[0032] As used herein, the phrase "consensus sequence" refers to an amino acid sequence, DNA sequence, or RNA sequence created by aligning two or more homologous sequences and deriving a new sequence that, at each position in the created sequence, has the conserved amino acid, deoxyribonucleic acid, or ribonucleic acid residue of the homologous sequences, or has an alternative set of amino acid, deoxyribonucleic acid, or ribonucleic acid residues.

[0033] As used herein, the phrases "preventing crop damage" and "reducing crop damage" refer to preventing or reducing damage to crop plants or crop plant products due to infection by microbial pathogens. More generally, these phrases refer to reducing the adverse effects caused by the presence of pathogenic microorganisms in crops. Adverse effects from 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 product, and / or the production of undesirable microbial metabolic products or microbial growth by-products, including mycotoxins.

[0034] As used herein, the term "defensin peptide" refers to a peptide containing a conserved gamma core peptide and two additional cysteine ​​residues located C-terminal to the C-terminal cysteine ​​residue of the conserved gamma core peptide. Plant defensins have previously been characterized as containing the conserved GXCX3-9C gamma core peptide sequence (where X is any amino acid residue) (Lacerda et al.) or the conserved GXCX3-10C variant gamma core peptide sequence (where X is any amino acid residue other than cysteine). In certain embodiments, the defensin peptides disclosed herein may also contain non-canonical defensin gamma core peptides, including GXCX3-12C, GXCX3-15C, GXCX3-22C, or GXCX16-22C. Thus, as used herein, a plant defensin, or a C-terminal peptide comprising a defensin or a fragment thereof, can comprise the conserved GXCX3-9C, GXCX3-12C, GXCX3-15C, GXCX3-22C, or GXCX16-22C gamma core peptide sequence, where X is any amino acid residue except cysteine. Defensin peptides include proteins that are antimicrobial, capable of permeabilizing cell membranes, capable of binding phospholipids, capable of binding sphingolipids, or exhibit any combination of these properties. Defensin peptides can be naturally occurring or non-naturally occurring (e.g., synthetic and / or chimeric).

[0035] As used herein, the phrase "defensin-like peptide" refers to a peptide that contains a conserved gamma core peptide but lacks at least one of two additional cysteine ​​residues located C-terminal to the C-terminal cysteine ​​residue of the conserved gamma core peptide. In certain embodiments, a defensin-like peptide may therefore contain two or three disulfide bonds (as opposed to the four in classical C8 defensins as depicted in Figures 1 and 2) and may contain a cysteine-stabilized α / β fold. In certain embodiments, the defensin-like peptides disclosed herein may also include defensin gamma core peptides containing the GXCX3-12C, GXCX3-15C, GXCX9-16C, GXCX3-22C, or GXCX16-22C peptide sequence. Examples of defensin-like peptides disclosed herein include peptides of SEQ ID NOs: 728, 730, 732, 734, and variants thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, and 99% identity thereto.

[0036] As used herein, the word "exemplary" refers to an example, instance, or illustration and, unless otherwise stated, does not refer to a most preferred embodiment.

[0037] As used herein, the phrases "modified defensin peptide," "modified defensin-like peptide," or "modified defensin or defensin-like peptide" are used to describe mutant defensin or defensin-like peptides that include (i) the conserved gamma-core peptide of GXCX3-12C, GXCX3-15C, GXCX3-22C, GXCX9-22C, or GXCX16-22C and at least one amino acid substitution in the corresponding wild-type defensin or defensin-like peptide, (ii) a C-terminal fragment of a wild-type defensin or defensin-like peptide that includes the conserved gamma-core peptide of GXCX3-12C, GXCX3-15C, GXCX3-22C, GXCX9-22C, or GXCX16-22C and lacks at least one amino acid residue at the N-terminus of the conserved gamma-core peptide, or (iii) a modified gamma-core variant sequence. In certain embodiments, the modified defensin or defensin-like peptides provided herein have the wild-type gamma core consensus peptide GXCX of the wild-type defensin or wild-type defensin-like peptide. 3-9 C or GXCX 3-22C is a modified gamma core consensus peptide (e.g., peptide sequence GXCX3-9(F / W / Y), GXCX3-9(F / W / Y / L / V / I / M), GXCX3-9(F / W / Y)(F / W / Y / M)(F / W / Y), GXCX3-9(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M), GXCX9-22(F / W / Y), GXCX16- 22(F / W / Y), GXCX9-22(F / W / Y / L / V / I / M), GXCX16-22(F / W / Y / L / V / I / M), GXCX9-22(F / W / Y)(F / W / Y / L / V / I / M )(F / W / Y), GXCX16-22(F / W / Y)(F / W / Y / L / V / I / M)(F / W / Y), GXCX9-22(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M), GXCX16-22(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M), GXCX3-9(F / W / Y)(R / K / H )(F / W / Y), GXCX3-9(F / W / Y / L / V / I / M)(R / K / H)(F / W / Y / L / V / I / M), GXCX9-22(F / W / Y)(R / K / H)(F / W / Y), GXCX and variants of full-length defensin peptides in which X is replaced by any amino acid other than cysteine ​​(GXCX16-22(F / W / Y)(R / K / H)(F / W / Y), GXCX16-22(F / W / Y / L / V / I / M)(R / K / H)(F / W / Y / L / V / I / M), or GXCX9-22(F / W / Y / L / V / I / M)(R / K / H)(F / W / Y / L / V / I / M), where X is any amino acid except cysteine. In certain embodiments, the modified defensin or defensin-like peptides provided herein are less than a full-length defensin or defensin-like peptide (e.g., a C-terminal fragment of a defensin or defensin-like peptide comprising a gamma core peptide sequence or a modified gamma core sequence (e.g., a peptide) that comprises, consists essentially of, or consists of: (i) 30 amino acid residues or less, or (ii) 15, 16, or 17 to 30 amino acid residues).

[0038] As used herein, the term "modified defensin C-terminal fragment" refers to a fragment of a defensin protein in which at least one amino acid residue has been deleted from the N-terminus and the wild-type consensus gamma core sequence has been replaced with a modified gamma core peptide. In certain embodiments, the modified defensin C-terminal fragment comprises a substitution of at least the conserved C6 cysteine ​​or at least the conserved C6 and C7 cysteines of a wild-type defensin with a phenylalanine, tryptophan, tyrosine, leucine, valine, isoleucine, or methionine residue. In certain embodiments, the modified defensin C-terminal fragment is a modified gamma core peptide GXCX3-9(F / W / Y), GXCX3-9(F / W / Y / L / V / I / M), GXCX3-9(F / W / Y)(F / W / Y / M)(F / W / Y), GXCX3-9(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M), GXCX9-22(F / W / Y), GXCX16-22(F / W / Y), GXCX9-22(F / W / Y / L / V / I / M), GXCX16-22(F / W / Y / L / V / I / M), GXCX9-22(F / W / Y )(F / W / Y / L / V / I / M)(F / W / Y), GXCX16-22(F / W / Y)(F / W / Y / L / V / I / M)(F / W / Y), GXCX9-22(F / W / Y / L / V / I / M )(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M), GXCX16-22(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M ), GXCX3-9(F / W / Y)(R / K / H)(F / W / Y), GXCX3-9(F / W / Y / L / V / I / M)(R / K / H)(F / W / Y / L / V / I / M), GXCX9-22( F / W / Y)(R / K / H)(F / W / Y), GXCX16-22(F / W / Y)(R / K / H)(F / W / Y), GXCX16-22(F / W / Y / L / V / I / M)(R / K / H)(F / W / Y / L / V / I / M), or GXCX9-22(F / W / Y / L / V / I / M)(R / K / H)(F / W / Y / L / V / I / M) (where X is any amino acid other than cysteine).

[0039] As used herein, the phrase "reference defensin peptide" or "wild-type defensin" refers to a peptide having the conserved GXCX 3-9 C or GXCX 3-22 "C" refers to a full-length defensin peptide containing the C gamma-core sequence and two additional conserved cysteine ​​residues located at the C-terminus of the gamma-core peptide sequence, where the cysteine ​​located closest to the N-terminus of the reference defensin C-terminal peptide corresponds to the cysteine ​​located closest to the N-terminus of the gamma-core sequence. In certain embodiments, the reference defensin C-terminal peptide thus contains eight conserved cysteine ​​residues, designated herein as C1 (most N-terminal in both the peptide and the gamma-core peptide sequence), C2 (C-terminal to C1), C3 (C-terminal to C2), C4 (C-terminal to C3), and C5 (C-terminal to C4 and located in the gamma-core peptide sequence). An alignment of a non-limiting subset of reference defensin C-terminal peptides containing the C1, C2, C3, C4, C5, C6, C7, and C8 cysteines with the conserved gamma-core peptide sequence is shown in Figure 1. An alignment of a non-limiting subset of wild-type defensin peptides of SEQ ID NOs: 366, 743, 746, 749, and 752, which contain the conserved gamma core peptide sequence and the C1, C2, C3, C4, C5, C6, C7, and C8 cysteines, with exemplary modified defensin peptides and exemplary modified defensin C-terminal fragments in which at least the conserved C6 and C7 cysteines have been replaced with the amino acids shown is shown in Figure 2. Other wild-type defensin peptides include those contained in the full-length defensin peptides of SEQ ID NOs: 11-41, 81 to 533, 534, 538, 542, 546, 551, 555, 560, 565, 569, 573, 578, 583, 588, 593, 598, 603, 608, 612, 616, 620, 624, 628, 633, 637, 641, 645, 649, 653, 657, 662, 666, 670, 674, 679, 683, 688, 692, 697, 702, 706, 711, 715, 719, 723, 743, 746, 749, or 752.

[0040] As used herein, the terms "editing," "editing," "edited," and the like refer to the process or product by which insertions, deletions, and / or nucleotide substitutions are introduced into a genome. Such processes include methods of inducing homologous sequence-dependent repair and / or non-homologous end joining at one or more sites within the genome.

[0041] As used herein, the phrase "gene-edited plant" or "edited plant" refers to a plant that contains one or more nucleotide insertions, deletions, substitutions, or any combination thereof, in the plant's genomic DNA. Such gene-edited plants can be constructed by techniques including CRISPR / Cas endonuclease-mediated editing, meganuclease-mediated editing, artificial zinc finger endonuclease-mediated editing, etc.

[0042] As used herein, the term "heterologous," when used in the context of 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 sequence(s) have been modified from their / their original form; (iii) a second polynucleotide that is disposed in an order and / or orientation, or genomic location or environment relative to the first polynucleotide, that is different from the order and / or orientation, or genomic location or environment of the first and second polynucleotides in a naturally occurring cell; or (iv) a second polynucleotide that is not present in a naturally occurring cell that contains the first polynucleotide. Heterologous polynucleotides include polynucleotides that promote transcription (e.g., promoter and enhancer elements), polynucleotides that increase the amount of transcripts (e.g., introns, 5'UTRs, and 3'UTRs), polynucleotides that promote translation, or combinations thereof, as well as polynucleotides that encode modified defensins or defensin-like or defensin peptides, spacer peptides, or localization peptides. In certain embodiments, the nuclear genome or plastid genome may contain a first polynucleotide, where the second polynucleotide is heterologous to the nuclear genome or plastid genome. "Heterologous" polynucleotides that promote transcription, transcript amount, translation, or combinations thereof, and polynucleotides that encode modified defensins or defensin-like or defensin peptides, spacer peptides, or localization peptides may be autologous to the cell, but are arranged in an order and / or orientation, or genomic location or environment, that differs from the order and / or orientation, or genomic location or environment, in a naturally occurring cell.Polynucleotides that promote transcription, transcript abundance, translation, or a combination thereof, as well as polynucleotides that encode modified defensin or defensin-like peptides or defensin peptides, spacer peptides, or localization can be heterologous to another polynucleotide if the polynucleotides are not operably linked to each other in naturally occurring cells. A heterologous peptide or protein includes a peptide or protein that is not present in a cell or organism when the cell or organism is in its natural state. Thus, a heterologous peptide or protein includes a peptide or protein that is localized to an intracellular location, an extracellular location, or is expressed in a tissue different from the intracellular location, extracellular location, or tissue in which the peptide is found in the cell or organism in nature. A heterologous polynucleotide includes a polynucleotide that is not present in a cell or organism when the cell or organism is in its natural state.

[0043] As used herein, phrases such as "inhibiting the growth of plant pathogenic microorganisms," "inhibiting microbial growth," and the like refer to methods that result in any measurable reduction in microbial growth, where microbial growth includes any measurable reduction in the number and / or extent of microbial cells, spores, conidia, or hyphae. As used herein, "inhibiting the growth of plant pathogenic microorganisms" is understood to also 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 plant yield reduction, any reduction in the value of the crop product, and / or the production of undesirable microbial metabolic products or microbial growth by-products, including mycotoxins. As used herein, phrases such as "inhibiting microbial growth" can include inhibition in plants, humans, or animals, unless otherwise specified.

[0044] As used herein, the phrase "percent identity" or "sequence identity" refers to the number of elements (i.e., amino acids or nucleotides) in sequences that are identical within a specified length of two DNA, RNA segments in an alignment that results in the maximum number of identical elements, which is calculated by dividing the number of identical elements by the total number of elements in the specified length of the aligned segments and multiplying by 100.

[0045] The term "transgenic" refers to an organism or its progeny, in which the DNA of the nuclear or organelle genome of the organism or its progeny contains an inserted foreign DNA molecule of 10 nucleotides or more in length. A "transgenic plant" refers to a plant or its progeny, in which the DNA of the nuclear or plastid genome of the plant or its progeny contains an introduced foreign DNA molecule of 10 nucleotides or more in length. Such introduced foreign DNA molecules can be naturally occurring, non-naturally occurring (e.g., synthetic and / or chimeric), from heterologous sources, or from autologous sources.

[0046] In the event that any of the above definitions conflicts with a definition provided in any patent or non-patent literature incorporated by reference herein, any patent or non-patent literature cited herein, or any patent or non-patent literature found elsewhere, it will be understood that the above definition shall be used herein.

[0047] Further explanation Provided herein are antimicrobial peptides called modified defensin or defensin-like peptides. The antimicrobial peptides and proteins can be applied directly to plants, feed, or food, or can be applied to plants in the form of microorganisms that produce the modified defensin or defensin-like peptides or proteins, or the plants can be genetically edited to produce the modified defensin or defensin-like peptides or proteins. The present disclosure also relates to recombinant or edited polynucleotides, microorganisms and plants transformed with recombinant or edited polynucleotides, plants containing genetically edited nuclear or plastid genomes encoding the modified defensin or defensin-like peptides and proteins, and compositions containing the modified defensin or defensin-like peptides and proteins that are useful for suppressing pathogenic microorganisms, including plant pathogenic microorganisms. In certain embodiments, defensin mutant proteins comprising two modified defensin or defensin-like peptides, or a modified defensin or defensin-like peptide and another peptide (including a modified defensin or defensin-like peptide or defensin peptide), can provide improved inhibition of microbial growth when compared to proteins comprising only one of the antimicrobial peptides found in the defensin mutant protein. In certain embodiments, the modified defensins or defensin-like peptides provided herein are cation-tolerant. Such cation-tolerant defensins may be more effective than cation-sensitive defensins in providing effective suppression of plant pathogenic microorganisms in transgenic crops. The cation-tolerant defensins provided herein can 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 can 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).Also provided herein is a recombinant polynucleotide comprising a polynucleotide encoding a modified defensin, modified defensin C-terminal fragment, defensin-like molecule, modified defensin-like molecule, or peptide encoding a defensin comprising a long C16-C22 gamma core consensus peptide, operably linked to a polynucleotide comprising a promoter heterologous to the polynucleotide encoding the peptide. In certain embodiments, the peptide is a modified defensin, modified defensin C-terminal fragment, or defensin-like peptide.In certain embodiments, the modified defensin and defensin-like peptides are selected from the group consisting of SEQ ID NOs: 535, 536, 537, 539, 540, 541, 543, 544, 545, 547, 548, 549, 550, 552, 553, 554, 556, 557, 558, 559, 561, 562, 563, 564, 566, 567, 568, 570, 571, 572, 574, 575, 576, 577, 579, 580, 581, 582, 584 , 585, 586, 587, 589, 590, 591, 592, 594, 595, 596, 597, 599, 600, 601, 602, 604, 605, 606, 607, 609, 610, 611, 613, 614, 615, 617, 618, 619, 621, 622, 623, 625, 626, 627, 629, 630, 631, 632, 634, 635, 636, 638, 639, 640, 642, 643, 644, 646, 647, 648 8, 650, 651, 652, 654, 655, 656, 658, 659, 660, 661, 663, 664, 665, 667, 668, 669, 671, 672, 673, 675, 676, 677, 678, 680, 681, 682, 684, 685, 686, 687, 689, 690, 691, 693, 694, 695, 696, 698, 699, 700, 701, 703, 704, 705, 707, 708, 709, 710, 712, 7 13, 714, 716, 717, 718, 720, 721, 722, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 744, 745, 747, 748, 750, 751, 753, 754, 755, 756 peptides, and variants thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, and 99% sequence identity thereto.

[0048] Modified defensin peptides and modified defensin C-terminal fragments include peptides comprising, consisting essentially of, or consisting of a sequence in which the gamma-core consensus peptide of a defensin or defensin C-terminal fragment is replaced with a modified gamma-core peptide. In certain embodiments, such replacement of a gamma-core consensus peptide (e.g., GXCX3-9C or GXCX3-22C) with a modified gamma-core peptide can be achieved by substituting one or more amino acids in the gamma-core consensus peptide with one or more amino acids defined in the modified gamma-core peptide. In certain embodiments, the substitution can include substituting the C-terminal cysteine ​​residue in the wild-type gamma-core consensus peptide of a defensin peptide (e.g., the cysteine ​​corresponding to C6 in Figures 1 and 2) with (i) a phenylalanine, tryptophan, or tyrosine residue, or (ii) a phenylalanine, tryptophan, tyrosine, leucine, valine, isoleucine, or methionine residue. In certain embodiments, the substitution can include replacing both the C-terminal cysteine ​​residue of the wild-type gamma core consensus peptide (e.g., the cysteine ​​corresponding to C6 in Figures 1 and 2) and the closest C-terminal cysteine ​​of the defensin peptide or defensin C-terminal peptide fragment (e.g., the cysteine ​​corresponding to C7 in Figures 1 and 2) with (i) a phenylalanine, tryptophan, or tyrosine residue, or (ii) a phenylalanine, tryptophan, tyrosine, leucine, valine, isoleucine, or methionine residue.In certain embodiments, the substitutions include substitutions of the C-terminal cysteine ​​residue of the wild-type gamma core consensus peptide (e.g., the cysteine ​​corresponding to C6 in Figures 1 and 2) and the closest C-terminal cysteine ​​of the defensin peptide or defensin C-terminal peptide fragment (e.g., the cysteine ​​corresponding to C7 in Figures 1 and 2) with (i) a phenylalanine, tryptophan, or tyrosine residue, or (ii) a phenylalanine, tryptophan, tyrosine, leucine, valine, isoleucine, or methionine residue, and additional substitutions of the amino acid located between the two cysteines (e.g., the amino acid residue between the cysteines corresponding to the C6 defensin and the C7 defensin in Figures 1 and 2) with (i) a phenylalanine, tryptophan, or tyrosine residue, (ii) a phenylalanine, tryptophan, tyrosine, leucine, valine, isoleucine, or methionine residue, or (iii) a lysine, arginine, or histidine residue. In certain embodiments, one or more additional amino acids located between the N-terminal cysteine ​​and the C-terminal cysteine ​​of wild-type gamma core peptide are substituted (e.g., substitution of a residue corresponding to X3-22 in GXCX3-22C gamma core peptide). Substitution of a residue corresponding to X3-22 in GXCX3-22C gamma core peptide may include an amino acid substitution that increases or maintains the peptide's net positive charge at neutral pH and / or increases or maintains the peptide's hydrophobicity. Thus, in certain embodiments, substitution of a residue corresponding to X3-22 in GXCX3-22C gamma core peptide may include substitution of one or more anionic aspartic acid or glutamic acid residues with cationic amino acids such as arginine, histidine, and lysine, and / or with neutral nonpolar (hydrophobic) amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine.Wild-type defensins containing wild-type gamma core peptides that can be substituted or replaced as described above and elsewhere herein to obtain antimicrobial peptides include those of SEQ ID NOS: 11-41, 81 to 533, 534, 538, 542, 546, 551, 555, 560, 565, 569, 573, 578, 583, 588, 593, 598, 603, 608, 612, 616, 620, 624, 628, 63 3, 637, 641, 645, 649, 653, 657, 662, 666, 670, 674, 679, 683, 688, 692, 697, 702, 706, 711, 715, 719, 723, 743, 746, 749, or 752, and gamma core peptide-containing variants thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, and 99% identity thereto. SEQ ID NOs: 11 to 41, 81 to 533, 534, 538, 542, 546, 551, 555, 560, 565, 569, 573, 578, 583, 588, 593, 598, 603, 608, 612, 616, 620, 624, 628, 633, 637, 641, 645, 649, 653, 657, 662, 666, 670, 674, 679, 683, 688, 690, 700, 702, 704, 706, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 76 and modified C-terminal fragments containing deletions of modified gamma-core peptides of wild-type defensins, including 92, 697, 702, 706, 711, 715, 719, 723, 743, 746, 749, or 752, and gamma-core peptide-containing variants thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, and 99% identity thereto. Modified defensin peptides and modified C-terminal variants thereof also include those listed in Table 1 below. Table 1 lists wild-type defensins and modified defensin peptides or modified C-terminal fragments containing modified gamma-core peptides with amino acid substitutions in the wild-type defensin peptide, in order. For example, SEQ ID NOs: 535 and 536 contain variants of the wild-type defensin of SEQ ID NO: 534, while SEQ ID NO: 537 contains a modified C-terminal fragment of SEQ ID NO: 534.In certain embodiments, the modified defensin peptides disclosed above are selected from the group consisting of SEQ ID NOs: 536, 540, 544, 549, 553, 558, 563, 567, 571, 576, 581, 586, 591, 596, 601, 606, 610, 614, 618, 622, 626, 631, 635, 639, 643, 647, 651, 655, 660, 664, 668, 672, 677, 681, 686, 690, 695, 700, 704, 709, 717, 721, 726, 745, 748, 751, 754, or 756. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0049] In certain embodiments, the modified defensin or defensin-like peptides of the present disclosure are characterized by comprising a modified defensin gamma-core peptide that is involved in the antifungal activity of plant defensins. Gamma-core peptides or modified gamma-core peptides typically contain a net positive charge and at least one hydrophobic amino acid. In certain embodiments, the modified defensin or defensin-like peptide can comprise a gamma-core consensus sequence of GXCX3-9C or GXCX3-22C, where X is any amino acid other than cysteine, and 3 to 9 or 3 to 22 non-cysteine ​​amino acid residues are located between the two cysteine ​​residues of the gamma-core consensus peptide. In certain embodiments, the modified defensin or defensin-like peptide comprises a modified gamma core peptide comprising the peptide sequence GXCX3-9(F / W / Y), GXCX3-9(F / W / Y / L / V / I / M), GXCX9-22(F / W / Y), GXCX16-22(F / W / Y), GXCX9-22(F / W / Y / L / V / I / M), GXCX16-22(F / W / Y / L / V / I / M), and includes 3 to 9, 9 to 22, or 16 to 22 amino acid residues other than cysteine ​​followed by a C-terminal phenylalanine, tryptophan, tyrosine, leucine, valine, isoleucine, or methionine residue.In certain embodiments, the modified defensin or modified defensin-like peptide has the peptide sequence GXCX3-9(F / W / Y)(F / W / Y / M)(F / W / Y), GXCX3-9(F / W / Y / L / V / I / M), (F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M), GXCX9-22(F / W / Y)(F / W / Y / L / V / I / M)(F / W / Y), GXCX16-22(F / W / Y)(F / W / Y / L / V / I / M)(F / W / Y), GXCX9- 22(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M), or GXCX16-22(F / W / Y / L / V / and (I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M), wherein the C-terminus of 3 to 9, 9 to 22, or 16 to 22 amino acid residues other than cysteine ​​is followed at the C-terminus by (i) a phenylalanine, tryptophan, tyrosine, leucine, valine, isoleucine, or methionine residue, wherein the C-terminus of (i) is followed at the C-terminus by (ii) a phenylalanine, tryptophan, tyrosine, leucine, valine, isoleucine, or methionine residue, and the C-terminus of (ii) is followed at the C-terminus by (iii) a phenylalanine, tryptophan, tyrosine, leucine, valine, isoleucine, or methionine residue.In certain embodiments, the modified defensin or modified defensin-like peptide has the peptide sequence GXCX3-9(F / W / Y)(R / K / H)(F / W / Y), GXCX3-9(F / W / Y / L / V / I / M)(R / K / H)(F / W / Y / L / V / I / M), GXCX9-22(F / W / Y)(R / K / H)(F / W / Y), GXCX16-22(F / W / Y)(R / K / H)(F / W / Y), GXCX16-22(F / W / Y / L / V / I / M)(R / K / H)(F / W / Y / L / V / I / M), or GXCX9-22(F / W / Y / L / V and a modified gamma core peptide comprising 3 to 9, 9 to 22, or 16 to 22 amino acid residues other than cysteine, followed at the C-terminus by (i) a phenylalanine, tryptophan, tyrosine, leucine, valine, isoleucine, or methionine residue, wherein (i) is followed at the C-terminus by (ii) an arginine, lysine, or histidine residue, and (ii) is followed at the C-terminus by (iii) a phenylalanine, tryptophan, tyrosine, leucine, valine, isoleucine, or methionine residue. In certain embodiments, the modified defensin or defensin-like peptide comprises any of the aforementioned modified gamma core sequences, wherein X is preferentially selected from cationic and / or hydrophobic amino acids, and any variable (X) amino acid residue of the modified gamma core is selected from the group consisting of SEQ ID NOS: 11-41, 81 to 533, 534, 538, 542, 546, 551, 555, 560, 565, 569, 573, 578, 583, 588, 593, 598, 603, 608, 612, 616, 620, 624, 628, 633, 637, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709 5, 649, 653, 657, 662, 666, 670, 674, 679, 683, 688, 692, 697, 702, 706, 711, 715, 719, 723, 743, 746, 749, or 752, or in a wild-type defensin-like peptide, including a polypeptide of SEQ ID NO:728, SEQ ID NO:730, SEQ ID NO:732, or SEQ ID NO:734.While the gamma core peptide is involved in phospholipid and / or sphingolipid binding, certain amino acids outside the gamma core motif are also thought to be involved in phospholipid and sphingolipid binding. In certain embodiments, the X3-22 amino acid sequence between the cysteines corresponding to C6 and C7 in the corresponding region of the modified defensin or defensin-like peptide also contributes to antibacterial activity.In certain embodiments, modified defensin and defensin-like peptides having one or more of any of the foregoing modified gamma core sequences include those listed in SEQ ID NOs: 535, 536, 537, 539, 540, 541, 543, 544, 545, 547, 548, 549, 550, 552, 553, 554, 556, 557, 558, 559, 561, 562, 563, 564, 566, 567, 568, 570, 571, 572, 574, 575, 576, 577, , 579, 580, 581, 582, 584, 585, 586, 587, 589, 590, 591, 592, 594, 595, 596, 597, 599, 600, 601, 602, 604, 605, 606, 607, 609, 610, 611, 613, 614, 615, 617, 618, 619, 621, 622, 623, 625, 626, 627, 629, 630, 631, 632, 634, 635, 636, 638, 639, 640, 642, 643, 644 4, 646, 647, 648, 650, 651, 652, 654, 655, 656, 658, 659, 660, 661, 663, 664, 665, 667, 668, 669, 671, 672, 673, 675, 676, 677, 678, 680, 681, 682, 684, 685, 686, 687, 689, 690, 691, 693, 694, 695, 696, 698, 699, 700, 701, 703, 704, 705, 707, 708, 709, 710, 712, 713, 714, 716, 717, 718, 720, 721, 722, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 744, 745, 747, 748, 750, 751, 753, 754, 755, 756, and variants thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, and 99% sequence identity thereto.

[0050] Also provided herein are defensin-like peptides comprising modified defensins or modified C-terminal fragments thereof. In certain embodiments, the modified defensin or defensin-like C-terminal fragment comprises, consists essentially of, or consists of (i) 30 or fewer amino acid residues, or (ii) 15, 16, or 17 to 30 amino acid residues. In certain embodiments, the C-terminal fragment has a net positive charge of at least 3 and a hydrophobic amino acid content of at least 18%. In certain embodiments, the modified defensin or defensin-like protein comprising the C-terminal fragment comprises a total of two cysteine ​​peptides, and optionally comprises a disulfide bond between the two cysteine ​​residues in the modified C-terminal fragment. In certain embodiments, the C-terminal fragment of the aforementioned modified defensin peptide comprises, consists essentially of, or consists of a peptide corresponding to the C-terminus of the defensin peptide including the conserved cysteines at C5 and C8, wherein tryptophan, tyrosine, or phenylalanine substitutions of residues corresponding to residues C6 to C7 result in a defensin C-terminal fragment comprising the modified gamma core variant sequence GXCX3-9(F / W / Y)(F / W / Y)(F / W / Y), GXCX3-22(F / W / Y)(F / W / Y)(F / W / Y), GXCX16-22(F / W / Y)(F / W / Y)(F / W / Y), GXCX3-9(F / W / Y), GXCX16-22(F / W / Y), or GXCX3-22(F / W / Y).In certain embodiments, the modified defensin peptide C-terminal fragment comprises, consists essentially of, or consists of a peptide corresponding to the C-terminus of a defensin peptide including the conserved cysteines at C5 and C8, wherein tryptophan, tyrosine, phenylalanine, leucine, valine, isoleucine, or methionine substitutions of residues corresponding to residues C6 through C7 result in a modified gamma core mutation. Defensin C-terminal fragments containing the target sequence GXCX3-9(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M), GXCX16-22(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M), or GXCX3-22(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) can be obtained. In certain embodiments, the modified defensin or defensin-like C-terminal fragment comprises GXCX3-9(F / W / Y)(R / K / H)(F / W / Y), GXCX3-9(F / W / Y / L / V / I / M)(R / K / H)(F / W / Y / L / V / I / M), GXCX9-22(F / W / Y)(R / K / H)(F / W / Y), GXCX16-22(F / W / Y)(R / K / H)(F / W / Y), GXCX16-22(F / W / Y / L / V / I / M)(R / K / H)(F / W / Y / L / V / I / M), or GXCX9-22(F / W / Y / L / V / I / M)(R / K / H)(F / W / Y / L / V / I / M). In certain embodiments, the defensin or defensin-like C-terminal fragment comprises any of the aforementioned modified gamma core consensus sequences, where X is not cysteine ​​and is preferentially selected from cationic amino acids (e.g., R, K, or H) and / or hydrophobic amino acids (e.g., F / W / Y / L / V / I / M). In certain embodiments, the defensin or defensin-like protein C-terminal fragment can comprise a peptide having only two (2) cysteine ​​residues, where the two (2) cysteine ​​residues optionally correspond to the conserved C5 and C8 cysteines of a reference or wild-type defensin peptide (e.g., as shown in Figure 2).In certain embodiments, the aforementioned C-terminal fragments are selected from the group consisting of SEQ ID NOs: 11-41, 81 to 533, 534, 538, 542, 546, 551, 555, 560, 565, 569, 573, 578, 583, 588, 593, 598, 603, 608, 612, 616, 620, 624, 628, 633, 637, 641, 645, 649, 653, 657, 662, 666, 670, 674, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 746, 747, In certain embodiments, the C-terminal fragments may comprise an N-terminal deletion of about 1 to 35 amino acids and amino acid substitutions in at least the gamma core peptide of a wild-type defensin polypeptide of SEQ ID NO: 728, SEQ ID NO: 730, SEQ ID NO: 732, SEQ ID NO: 734, or a variant thereof having at least 70%, 75%, 80%, 90%, or 95% sequence identity thereto. Exemplary modified defensin C-terminal fragments include those comprising, consisting essentially of, or consisting of those set forth in Table 1, as well as those comprising, consisting essentially of, or consisting of peptides corresponding to amino acid residues 32-47 of SEQ ID NO: 742, amino acid residues 32-53 of SEQ ID NO: 745, amino acid residues 31-45 of SEQ ID NO: 748, amino acid residues 33-50 of SEQ ID NO: 751, and amino acid residues 34-49 of SEQ ID NO: 754 (e.g., as depicted in Figure 2). Exemplary modified defensin C-terminal fragments comprise, consist essentially of, or consist of peptides of SEQ ID NOs: 828, 829, 830, 831, 832, or 833. Variants of any of the foregoing C-terminal fragments containing conservative amino acid substitutions of one to two, three, four, or five amino acid residues are also provided.

[0051] In certain embodiments, optionally isolated defensin-like peptides and defensin-like C-terminal fragments are provided. In certain embodiments, such defensin-like peptides include peptides of SEQ ID NOs: 728, 730, 732, 734, or variants thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity thereto. In certain embodiments, defensin-like C-terminal fragments are provided that include a deletion of 1 to about 35 N-terminal amino acid residues of SEQ ID NOs: 729, 731, 733, 735, 736, or variants thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity thereto.

[0052] In certain embodiments, the modified defensins, modified defensin-like peptides, defensin-like peptides, and C-terminal fragments thereof provided herein have a net positive charge of at least about 5.8 at neutral pH and / or a hydrophobicity ratio of at least about 15%. In certain embodiments, the first structural feature of the modified defensin or defensin-like peptide is a net positive charge at neutral pH. In certain embodiments, the modified defensin or defensin-like peptide 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 modified defensin or defensin-like peptide has 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 of such modified defensin or defensin-like peptides is at least about 15% to 30%, about 16% to 19%, or about 28% to 30%. In certain embodiments, the modified defensin or defensin-like peptides comprise, consist essentially of, or consist of (i) 30 or fewer amino acid residues, or (ii) 15, 16, or 17 to 30 amino acid residues. In certain embodiments, any of the modified defensin or defensin-like peptides includes peptides having only two (2) cysteine ​​residues, and optionally includes two cysteine ​​residues corresponding to the conserved C5 and C8 cysteines of the reference defensin C-terminal peptide.In certain embodiments, modified defensin and defensin-like peptides having any one or more of the foregoing net positive charge and / or hydrophobicity ratios include those listed in SEQ ID NOs: 535, 536, 537, 539, 540, 541, 543, 544, 545, 547, 548, 549, 550, 552, 553, 554, 556, 557, 558, 559, 561, 562, 563, 564, 566, 567, 568, 570, 571, 572, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 63 76, 577, 579, 580, 581, 582, 584, 585, 586, 587, 589, 590, 591, 592, 594, 595, 596, 597, 599, 600, 601, 602, 604, 605, 606, 607, 609, 610, 611, 613, 614, 615, 617, 618, 619, 621, 622, 623, 625, 626, 627, 629, 630, 631, 632, 634, 635, 636, 638, 639, 640, 642, 643 3, 644, 646, 647, 648, 650, 651, 652, 654, 655, 656, 658, 659, 660, 661, 663, 664, 665, 667, 668, 669, 671, 672, 673, 675, 676, 677, 678, 680, 681, 682, 684, 685, 686, 687, 689, 690, 691, 693, 694, 695, 696, 698, 699, 700, 701, 703, 704, 705, 707, 708, 709, 71 70%, 75%, 80%, 85%, 90%, 95%, 98%, and 99% sequence identity thereto.

[0053] In certain embodiments, the modified defensins, modified defensin-like peptides, or defensin-like peptides provided herein (e.g., Table 1, Figure 2, and the Sequence Listing) and variants thereof can include amino acid substitutions that increase or maintain the net positive charge of the peptide at neutral pH and / or increase or maintain the hydrophobicity of the peptide. Amino acid substitutions that can maintain the net positive charge of the modified defensin, modified defensin-like, or defensin-like peptide at neutral pH include replacing a lysine, arginine, or histidine residue in the modified defensin, modified defensin-like, or defensin-like peptide with a different amino acid residue selected from the group consisting of lysine, arginine, Dab (diaminobutyric acid), or other non-naturally occurring amino acids that are positively charged at neutral pH. Amino acid substitutions in modified defensins, modified defensin-like, or defensin-like peptides that can increase the net positive charge at neutral pH include replacing a polar (e.g., cysteine ​​or threonine) or non-polar (e.g., glycine) residue in the modified defensin, modified defensin-like, or defensin-like peptide with a different amino acid residue selected from the group consisting of lysine, arginine, Dab (diaminobutyric acid), or other non-naturally occurring amino acid residues that are positively charged at neutral pH. Amino acid substitutions in modified defensins, modified defensin-like, or defensin-like peptides that can maintain the hydrophobicity of the peptide include replacing a glycine, valine, phenylalanine, or isoleucine residue in the modified defensin, modified defensin-like, or defensin-like peptide with a different amino acid residue selected from the group consisting of glycine, alanine, valine, leucine, phenylalanine, isoleucine, or methionine. Amino acid substitutions in the modified defensin, modified defensin-like, or defensin-like peptide that can increase the hydrophobicity of the peptide include replacing polar (e.g., cysteine ​​or threonine) residues in the modified defensin, modified defensin-like, or defensin-like peptide with glycine, alanine, valine, leucine, phenylalanine,or isoleucine. In certain embodiments, such substitutions that increase or maintain the net positive charge or hydrophobicity of the peptide include modified defensin or defensin-like peptides having the gamma core consensus sequence of GXCX3-9C or GXCX9-22C. In certain embodiments, such substitutions that increase or maintain the net positive charge or hydrophobicity of the peptide include modified defensin or defensin-like peptides having the modified gamma core variant sequences provided herein. In certain embodiments, such substitutions that increase or maintain the net positive charge or hydrophobicity of the peptide include GXCX3-9(F / W / Y), GXCX3-9(F / W / Y / L / V / I / M), GXCX3-9(F / W / Y)(F / W / Y / M)(F / W / Y), GXCX3-9(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M), GXCX9-22(F / W / Y), GXCX16-22(F / W / Y), GXCX9-22(F / W / Y / L / V / I / M), GXCX16-22(F / W / Y / L / V / I / M), GXCX9-22(F / W / Y)(F / W / Y / L / V / I / M)(F / W / Y), GXCX16-22(F / W / Y)(F / W / Y / L / V / I / M)(F / W / Y), GXCX9-22(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M), GXCX16-22(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M), GXCX3-9(F / W / Y)(R / K / H)(F / W / Y), GXCX3-9(F / W / Y / L / V / I / M)(R / K / H)(F / W / Y / L / V / I / M), GXCX9-22(F / W / Y)(R / K / H)(F In certain embodiments, the modified defensin or defensin-like peptide comprises a modified gamma core mutant sequence of GXCX16-22(F / W / Y)(R / K / H)(F / W / Y), GXCX16-22(F / W / Y / L / V / I / M)(R / K / H)(F / W / Y / L / V / I / M), or GXCX9-22(F / W / Y / L / V / I / M)(R / K / H)(F / W / Y / L / V / I / M).The aforementioned modified defensin or defensin-like peptides comprise, consist essentially of, or consist of (i) 30 or fewer amino acid residues, or (ii) 15, 16, or 17 to 22, 24, 16, 28, or 30 amino acid residues. In certain embodiments, any of the aforementioned modified defensin or defensin-like peptides includes peptides having only two (2) cysteine ​​residues, and optionally, two cysteine ​​residues corresponding to the conserved C5 and C8 cysteines of the reference defensin C-terminal peptide. In certain embodiments, modified defensin and defensin-like peptides that can be substituted as described above or elsewhere herein include those listed in SEQ ID NOs: 535, 536, 537, 539, 540, 541, 543, 544, 545, 547, 548, 549, 550, 552, 553, 554, 556, 557, 558, 559, 561, 562, 563, 564, 566, 567, 568, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 6 71, 572, 574, 575, 576, 577, 579, 580, 581, 582, 584, 585, 586, 587, 589, 590, 591, 592, 594, 595, 596, 597, 599, 600, 601, 602, 604, 605, 606, 607, 609, 610, 611, 613, 614, 615, 617, 618, 619, 621, 622, 623, 625, 626, 627, 629, 630 , 631, 632, 634, 635, 636, 638, 639, 640, 642, 643, 644, 646, 647, 648, 650, 651, 652, 654, 655, 656, 658, 659, 660, 661, 663, 664, 665, 667, 668, 669, 671, 672, 673, 675, 676, 677, 678, 680, 681, 682, 684, 685, 686, 687, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 646, 647 91, 693, 694, 695, 696, 698, 699, 700, 701, 703, 704, 705, 707, 708, 709, 710, 712, 713, 714, 716, 717, 718, 720, 721, 722, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 744, 745, 747, 748, 750, 751, 753, 754, 755,In certain embodiments, modified defensin peptides that can be substituted as described above or elsewhere herein include peptides of SEQ ID NOS: 11-41, 81 to 533, 534, 538, 542, 546, 551, 555, 560, 565, 569, 573, 578, 583, 588, 593, 598, 603, 608, 612, 616, 620, 624, 628, 633, 637, 641, 645, 649, 653, 657, 662, 666, 670, 674, 679, 683, 688, 692, 697, 702, 706, , 711, 715, 719, 723, 743, 746, 749, or 752, wherein the wild-type gamma core consensus peptide GXCX3-9C or GXCX3-22C of the wild-type defensin or wild-type defensin-like peptide is selected from the group consisting of peptide sequences GXCX3-9(F / W / Y), GXCX3-9(F / W / Y / L / V / I / M), GXCX3-9(F / W / Y)(F / W / Y / M)(F / W / Y), GXCX3-9(F / W / Y / L / V / I / M)(F / W / Y), and GXCX3-9(F / W / Y / L / V / I / M)(F / W / Y). W / Y / L / V / I / M), GXCX9-22(F / W / Y), GXCX16-22(F / W / Y), GXCX9-22(F / W / Y / L / V / I / M), GXCX16-22(F / W / Y / L / V / I / M), GXCX9-22(F / W / Y)(F / W / Y / L / V / I / M)(F / W / Y), GXCX16-22(F / W / Y)(F / W / Y / L / V / I / M)(F / W / Y), GXCX9-22(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M), GXCX1 6-22(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M), GXCX3-9( F / W / Y)(R / K / H)(F / W / Y), GXCX3-9(F / W / Y / L / V / I / M)(R / K / H)(F / W / Y / L / V / I / M), GXCX9-22(F / W / Y)(R / K / H)(F / W / Y), GXCX16-22(F / W / Y)(R / K / H)(F / W / Y), GXCX16-22(F / W / Y / L / V / I / M)(R / K / H)(F / W / Y / L / V / I / M),or a modified gamma core consensus peptide comprising GXCX9-22(F / W / Y / L / V / I / M)(R / K / H)(F / W / Y / L / V / I / M), optionally with the N-terminal residues 1-35 deleted.

[0054] In certain embodiments, one or more amino acids in any of the foregoing or other modified defensin or defensin-like peptide sequences are substituted with another amino acid(s) that is similar in charge and polarity to the original amino acid, i.e., a conservative amino acid substitution. Substitutes for amino acids in a modified defensin or defensin-like peptide or protein, or defensin peptide sequence, can be selected from other members of the class to which the originally occurring amino acid belongs. Amino acids are 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 one amino acid within one of these groups for another amino acid within the same group. Biologically functional equivalents of modified defensins or defensin-like peptides can have no more than 10 conservative amino acid changes, no more than 7 conservative amino acid changes, or 5, 4, 3, 2, or 1 conservative amino acid change. Thus, the encoding nucleotide sequence (e.g., gene, plasmid DNA, cDNA, or synthetic DNA) can have the corresponding base substitutions to encode a biologically functional equivalent form of the modified defensin or defensin-like peptide. Specific semi-conservative substitutions in the modified defensin or defensin-like peptide are also provided, including (i) the substitution of a neutral polar amino acid residue with a neutral non-polar (hydrophobic) amino acid residue, or (ii) the substitution of a neutral non-polar (hydrophobic) amino acid residue with a neutral polar amino acid residue. In particular,Semi-conservative substitutions of neutral polar tyrosine residues with hydrophobic amino acid residues are provided. Biologically functional equivalents of modified defensins or defensin-like peptides can have 10 or fewer semi-conservative amino acid changes, 7 or fewer semi-conservative amino acid changes, or 5, 4, 3, 2, or 1 semi-conservative amino acid changes. In certain embodiments, as described above or elsewhere herein, modified defensin and defensin-like peptides that may be conservatively or semi-conservatively substituted include those of SEQ ID NOs: 535, 536, 537, 539, 540, 541, 543, 544, 545, 547, 548, 549, 550, 552, 553, 554, 556, 557, 558, 559, 561, 562, 563, 564, 566, 567, 568, 570, 571, 572, 574, 575, 576, 577, 579, 580, 581, 582, 584, 585, 586, 587, 589, 590, 591, 592, 594, 595, 596, 597, 599, 600, 601, 602, 604, 605, 606, 607, 609, 610, 611, 613, 614, 615, 617, 618, 619, 621, 622, 623, 625, 626, 627, 629, 630, 631 1, 632, 634, 635, 636, 638, 639, 640, 642, 643, 644, 646, 647, 648, 650, 651, 652, 654, 655, 656, 658, 659, 660, 661, 663, 664, 665, 667, 668, 669, 671, 672, 673, 675, 676, 677, 678, 680, 681, 682, 684, 685, 686, 687, 689, 690, 691, 693, 694, 750, 751, 753, 754, 755, and 756. In certain embodiments, modified defensin peptides that may be conservatively or semi-conservatively substituted, as described above or elsewhere herein, include peptides set forth in SEQ ID NOS: 11-41,81 to 533, 534, 538, 542, 546, 551, 555, 560, 565, 569, 573, 578, 583, 588, 593, 598, 603, 608, 612, 616, 620, 624, 628, 633, 637, 641, 645, 649, 653, 657, 662, 666, 670, 674, 679, 683, 688, 692, 697, 702, 706, 711, 715, 719, 723, 743, 746, 749, or 752, and are wild-type defensin or wild-type defensin-like peptides. The wild-type gamma core consensus peptide GXCX3-9C or GXCX3-22C is expressed as the peptide sequence GXCX3-9(F / W / Y), GXCX3-9(F / W / Y / L / V / I / M), GXCX3-9(F / W / Y)(F / W / Y / M)(F / W / Y), GXCX3-9(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M), GXCX9-22(F / W / Y), GXCX16-22(F / W / Y), GXCX9-22(F / W / Y / L / V / I / M), GXCX16-22(F / W / Y ... Y / L / V / I / M), GXCX9-22(F / W / Y)(F / W / Y / L / V / I / M)(F / W / Y), GXCX16-22(F / W / Y)(F / W / Y / L / V / I / M)(F / W / Y), GXCX9-22(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M), GXCX16-22(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M )(F / W / Y / L / V / I / M), GXCX3-9(F / W / Y)(R / K / H)(F / W / Y), GXCX3-9(F / W / Y / L or GXCX9-22(F / W / Y / L / V / I / M)(R / K / H)(F / W / Y), GXCX16-22(F / W / Y)(R / K / H)(F / W / Y), GXCX16-22(F / W / Y / L / V / I / M)(R / K / H)(F / W / Y / L / V / I / M), or GXCX9-22(F / W / Y / L / V / I / M)(R / K / H)(F / W / Y / L / V / I / M), optionally with the N-terminal residues 1 to 35 deleted.

[0055] Also provided herein are nucleic acid molecules encoding any of the aforementioned modified defensin or defensin-like peptides. Also provided herein are recombinant DNA molecules comprising the aforementioned nucleic acid molecules, particularly recombinant DNA molecules comprising a heterologous promoter operably linked to the aforementioned nucleic acid molecule.

[0056] The modified defensin or defensin-like peptides provided herein can be operably linked to another modified defensin or defensin-like peptide, defensin, or antimicrobial peptide via a spacer peptide sequence that is resistant to cleavage by endoproteases, including plant endoproteases. Such peptide linker sequences for joining peptides within 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 the lipoyl domain and E3-binding domain of pyruvate dehydrogenase (Turner et al., 1993), the linker between the central domain and C-terminal domain of a cysteine ​​proteinase (P9; Mottram et al., 1989), and functional variants thereof. Spacer peptides for use in defensin mutant proteins can also be wholly or partially synthetic peptide sequences. Such synthetic spacer peptides are designed to provide a flexible linkage between at least one modified defensin or defensin-like peptide and another peptide (including a modified defensin or defensin-like peptide or defensin peptide) and to be resistant to cleavage by endogenous plant or other endoproteases. In certain embodiments, the length of the synthetic spacer peptide can be between about 3, 4, 8, 10, 12, or 16 and about 20, 24, 28, 30, 40, or 50 amino acid residues. In certain embodiments, the synthetic spacer peptide can comprise a glycine-rich or glycine / serine-containing peptide sequence. The composition and design of peptides suitable for flexible linkage of protein domains described in the literature (Chen et al., 2013) can be adapted for use as spacer peptides in the defensin mutant proteins provided herein.Spacer peptides useful for joining defensin monomers, as described in U.S. Patent Application Publications US20190194268 and US20190185877, each of which is incorporated herein by reference in its entirety, can also be used to join the modified defensin or defensin-like peptides disclosed herein to other modified defensin or defensin-like peptides, defensins, antimicrobial peptides, or other peptides.

[0057] The modified defensin or defensin-like peptides provided herein can be operably linked to another modified defensin or defensin-like peptide, defensin, or antimicrobial peptide via a linker peptide sequence that is susceptible to cleavage by an endoprotease, including a plant endoprotease. In certain embodiments, the resulting defensin mutant protein can be expressed intracellularly such that the endoprotease cleaves the defensin mutant protein to provide at least one modified defensin or defensin-like peptide and another peptide (including a modified defensin or defensin-like peptide or defensin peptide). Such defensin mutant proteins can be provided in an intracellular compartment (e.g., cytoplasm, mitochondria, plastids, vacuoles, or endoplasmic reticulum) or in the extracellular space (i.e., apoplast) that contains an endoprotease 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 can be used in the defensin mutant proteins provided herein.

[0058] Expression cassettes can be constructed that provide expression of modified defensins or defensin-like peptides in monocotyledonous plants, dicotyledonous plants, or both. Such modified defensin or defensin-like peptide expression cassettes can be constructed either in a plant expression vector or in the plant genome. Expression cassettes are DNA constructs that operably link various promoters, coding sequences (e.g., sequences encoding modified defensins or defensin-like peptides), and polyadenylation sequences. In general, expression cassettes typically contain a promoter operably linked to a sequence of interest operably linked to a polyadenylation or terminator region. In certain cases, including expression of recombinant or edited polynucleotides in monocotyledonous plants, it may also be useful to include intron sequences. When an intron sequence is included, it is typically located in the 5' untranslated leader region of the recombinant or edited polynucleotide. In some cases, it may also be useful to incorporate specific 5' untranslated sequences into the recombinant or edited polynucleotide to increase the stability of the transcript or 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 modified defensin or defensin-like peptides in transgenic plants. Any modified defensin or defensin-like peptide expression vector 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 "whisker"-mediated transformation.Methods for introducing the aforementioned 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 (particle-mediated transformation of rice), and U.S. Patent No. 5,004,863 (Agrobacterium-mediated transformation of cotton), each of which is incorporated herein by reference in its entirety.

[0059] In certain embodiments, plants containing a recombinant or edited polynucleotide encoding a modified defensin or defensin-like peptide can be obtained by using techniques that provide site-specific insertion of heterologous DNA into the plant genome (e.g., by CRISPR, TALEN, or zinc finger nuclease-mediated gene editing). In certain embodiments, a DNA fragment encoding at least the modified defensin or defensin-like peptide is site-specifically integrated into the genome of a plant cell, tissue, part, or pre-plant to create a sequence encoding the modified defensin or defensin-like peptide within the genome. Exemplary methods for inserting foreign DNA into specific sites in a 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 Application Publication No. 20150082478, the entire contents of which are incorporated herein by reference.

[0060] The expression of modified defensins or defensin-like peptides 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 usually contain a promoter operably linked to a sequence of interest, which is operably linked to a polyadenylation or terminator region. Examples of yeast genera in which heterologous genes have been successfully expressed include Candida, Kluyveromyces, Hansuella, Pichia, Saccharomyces, Schizosaccharomyces, and Yarrowia. A review of expression vectors and transformation systems for Saccharomyces is provided by Kingsman et al. (1985). Expression vectors and transformation systems useful for yeasts other than Saccharomyces are described by Reiser et al. (1990). Expression cassettes and vectors for the expression of other defensin peptides or proteins in yeast, including those disclosed in U.S. Pat. No. 10,253,328, the entire contents of which are incorporated herein by reference, can be adapted for the expression of modified defensin or defensin-like peptides in yeast.

[0061] The expression of modified defensins or defensin-like peptides in bacteria is also specifically discussed herein. The construction of expression vectors for the production of heterologous proteins in various bacterial systems is described. In general, such expression vectors usually contain a promoter operably linked to a sequence encoding the desired peptide sequence(s) (e.g., the N-terminal signaling peptide region of a defensin mutant peptide) operably linked to a prokaryotic terminator region. Examples of bacterial genera in which heterologous genes have been successfully expressed include Acinetobacter, Alcaligenes, Azotobacter, Bacillus, Escherichia coli, Lactobacillus, Lactococcus, Streptomyces, and Pseudomonas. E. coli 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 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) Protein Expr Purif 49, 1-8. Systems for expressing proteins containing disulfide bonds include those disclosed in U.S. Patent Application Publication No. 2020 / 0172915, which can be adapted for expression of defensin peptides in E. coli, and Berkmen, M. Protein Expr Purif. 2012;82(1):240-51. doi:10.1016 / j.pep.2011.10.009, which is incorporated by reference in its entirety.

[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, the entire contents of which are incorporated herein by reference.

[0063] Also provided are antimicrobial compositions for agricultural, pharmaceutical, or veterinary use, comprising an antimicrobial plant, or antimicrobial human or veterinary, pathogenic microorganism-inhibiting amount (an "antimicrobially effective amount") of one or more of the isolated, purified antimicrobial modified defensin or defensin-like peptides of the present invention, or biologically functional equivalents thereof. Such compositions can comprise one of the modified defensin or defensin-like peptides or proteins disclosed herein, or any combination thereof, and an agriculturally, pharmaceutically, or veterinarily acceptable carrier, diluent, or excipient. Other ingredients relevant to agricultural and therapeutic situations can also be included in such compositions, as described below. The antimicrobial compositions can be used to inhibit the growth of or kill modified defensin- or defensin-like peptide-susceptible pathogenic microorganisms associated with microbial infections of plants, humans, or animals. Such antimicrobial compositions can be formulated for topical administration and applied topically to either plants, the plant's environment (including soil), or humans or animals. Such antimicrobial compositions can be formulated for enteral, parenteral, and / or intravenous administration of the compositions and can be administered to a subject in need thereof, which may be a human, livestock, poultry, fish, or companion animal. The modified defensins or defensin-like peptides can be formulated alone or in any combination with each other, any of which can further be combined with other conventional antimicrobial therapeutic compounds, such as, by way of non-limiting example, polyene antimicrobials, imidazole, triazole, and thiazole antimicrobials, allylamines, and echinocandins, which are routinely used in human and veterinary medicine. Administration of compositions comprising modified defensins or defensin-like peptides to a human or animal subject in need thereof can be achieved via a variety of routes, including topical application, enteral administration, parenteral administration, and / or intravenous administration.The antimicrobial peptides and compositions are useful for treating (i) bacterial pathogens of plants or animals, where the bacterial pathogen is optionally a member of the Enterobacteriaceae family, and optionally the bacterial pathogen is Salmonella, Escherichia, or Listeria; (ii) bacterial pathogens of the genus Fusarium, Alternaria, Aphenomyces, Verticillium, Phytophthora, Colletotrichum, Botrytis, Cercospora, Phakopsora, Rhizoctonia, Sclerotinia, and the like; (iii) Aspergillus, Cryptococcus, Penicillium, Rhizopus, or Rhizopus; (iv) Aspergillus, Cryptococcus, Penicillium, Rhizopus, or Rhizopus; (v) Aspergillus, Cryptococcus, Penicillium, Rhizopus, or Rhizopus; (vi ... (iv) a fungus of the genus Candida, such as Candida albicans (C. albicans), C. auris, C. glabrata, C. parapsilosis, C. tropicalis, or the like; It can be used to inhibit microbial pathogens or contaminants, including Candida species, such as C. krusei, or (v) optionally, dermatophytes 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.

[0064] Agricultural compositions containing any of the modified defensin or defensin-like peptide molecules of the present invention, alone or in any combination, can 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 substances, surfactants, solvents, and other additives are also well known in the art and are described, for example, in 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. These formulations can also be used to prepare mixtures of the modified defensins or defensin-like peptides or proteins of the present invention with other insecticidal active substances, fertilizers, and / or growth regulators, etc., in the form of finished formulations or tank mixes.

[0065] The antimicrobial modified defensin or defensin-like peptides of the present invention, whether used alone or in combination with other active agents, can be applied to a subject or plant at a concentration of about 0.1 pg / ml to about 100 mg / ml, or about 5 pg / ml to about 5 mg / ml, at a pH of about 3.0 to about 9.0. Such compositions can be buffered, for example, with a phosphate buffer of about 1 mM to 1 M, about 10 mM to about 100 mM, or about 15 mM to about 50 mM. If the buffer concentration is low, salt can be added to increase the ionic strength. In certain embodiments, sodium salts, including NaCl, can be added or provided to compositions containing modified defensin or defensin-like peptides and proteins 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, compositions comprising modified defensin or defensin-like peptides and proteins may be supplemented or provided with a potassium salt, including 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. In certain embodiments, compositions comprising modified defensin or defensin-like peptides may be supplemented or provided with a calcium salt, including CaCl, 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.

[0066] Numerous conventional microbial antibiotics and chemical antimicrobial agents (e.g., fungicides) that can be combined with the modified defensin or defensin-like peptides and proteins of the present invention 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, sterol biosynthesis inhibitors, and organophosphorus compounds. Additionally, 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, fungicides, miticides, nematicides, and herbicides. U.S. Patent No. 5,421,839, incorporated herein by reference in its entirety, provides a comprehensive overview of many active ingredients that can be formulated with substances such as the antimicrobial modified defensin or defensin-like peptides and proteins of the present invention.

[0067] Agriculturally useful antimicrobial compositions encompassed herein also include host cell forms, such as bacterial and microbial cells, capable of producing modified defensin or defensin-like peptides and proteins and colonizing plants, including plant roots, shoots, leaves, or other parts. The term "plant-colonizing microorganism" is used herein to refer to a microorganism capable of colonizing the plant itself and / or any part of the plant's environment and expressing the defensin variant antimicrobial peptides and proteins of the present invention in the plant and / or plant environment. Plant-colonizing microorganisms are microorganisms that can exist in a symbiotic or non-harmful relationship with the plant in the plant's environment. U.S. Patent No. 5,229,112, incorporated herein by reference in its entirety, discloses various plant-colonizing microorganisms that can be engineered to express antimicrobial peptides and proteins, as well as methods of use thereof that are applicable to the defensin variant antimicrobial peptides and proteins disclosed herein. Plant-colonizing microorganisms expressing the defensin variant antimicrobial peptides and proteins disclosed herein that are useful for inhibiting microbial growth in plants include bacteria selected from the group consisting of Bacillus species, including Bacillus thuringiensis, Bacillus israelensis, and Bacillus subtilis, Candidatus, Liberibacter asiaticus, Pseudomonas species, Arthrobacter species, Azospirillum species, Clavibacter species, Escherichia coli species, Agrobacterium species, including A. radiobacter, Rhizobium species, Erwinia species, Azotobacter species, Azospirillum species, Klebsiella species, Alcaligenes species, Rhizobacterium species, Xanthomonas species, Ralstonia species, and Flavobacterium species. 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 plant-colonizing microorganism can be an endophytic bacterium or a microorganism.When applying the modified defensin or defensin-like peptide molecules of the present invention to the rhizosphere, rhizosphere-colonizing bacteria of the genus Pseudomonas are particularly useful, and in particular fluorescent Pseudomonas, such as Pseudomonas fluorescens, are particularly competitive in the plant rhizosphere and form large colonies on the surface of plant roots. Examples of suitable foliar (leaf)-colonizing bacteria include P. putida, P. syringae, and Erwinia species.

[0068] Embodiment The following numbered embodiments form part of this disclosure:

[0069] 1. A peptide comprising the amino acid sequence of a modified defensin or modified defensin-like peptide, wherein the wild-type gamma core consensus peptide GXCX3-9C or GXCX3-22C of the wild-type defensin or wild-type defensin-like peptide is selected from the group consisting of the peptide sequences GXCX3-9(F / W / Y), GXCX3-9(F / W / Y / L / V / I / M), GXCX3-9(F / W / Y)(F / W / Y / M)(F / W / Y), GXCX3-9(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M), GXCX9-22(F / W / Y), GXCX16-22(F / W / Y), GXCX9-22(F / W / Y / L / V / I / M), GXCX16- 22(F / W / Y / L / V / I / M), GXCX9-22(F / W / Y)(F / W / Y / L / V / I / M)(F / W / Y), GXCX16-22(F / W / Y)(F / W / Y / L / V / I / M)(F / W / Y), GXCX9-22 (F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M), GXCX16-22(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M), GX CX3-9(F / W / Y)(R / K / H)(F / W / Y), GXCX3-9(F / W / Y / L / V / I / M)(R / K / H)(F / W / Y / L / V / I / M), GXCX9-22(F / W / Y)(R / K / H)(F / W / Y), G A peptide having a modified gamma core consensus peptide comprising XCX16-22(F / W / Y)(R / K / H)(F / W / Y), GXCX16-22(F / W / Y / L / V / I / M)(R / K / H)(F / W / Y / L / V / I / M), or GXCX9-22(F / W / Y / L / V / I / M)(R / K / H)(F / W / Y / L / V / I / M), wherein the peptide has a net positive charge of at least 3 and a hydrophobic amino acid content of at least 18%.

[0070] 2. (i) The wild-type defensin is a polynucleotide selected from the group consisting of SEQ ID NOs: 11-41, 81 to 533, 534, 538, 542, 546, 551, 555, 560, 565, 569, 573, 578, 583, 588, 593, 598, 603, 608, 612, 616, 620, 624, 628, 633, 637, 641, 645, 649, 653, 657, 662, 666, 670, 674, 679, 683, 688, 692, 697, 702, 706, 711, 715, 719, 723, 743, 746, 749, and 752. or (ii) the wild-type defensin-like peptide comprises the polypeptide of SEQ ID NO: 728, SEQ ID NO: 730, SEQ ID NO: 732, SEQ ID NO: 734, or a variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% sequence identity thereto; or (iii) the wild-type defensin-like peptide comprises the polypeptide of SEQ ID NO: 728, SEQ ID NO: 730, SEQ ID NO: 732, SEQ ID NO: 734, or a variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% sequence identity thereto.

[0071] 3. The modified defensin peptide is selected from the group consisting of SEQ ID NOs: 536, 540, 544, 549, 553, 558, 563, 567, 571, 576, 581, 586, 591, 596, 601, 606, 610, 614, 618, 622, 626, 631, 635, 639, 643, 647, 651, 655, 660, 664, 668, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 720, 721, 722, 726, 727, 728, 730, 731, 732, 733, 734, 735, 736, 737, 738, 740, 741, 742, 743, 744, 745, 74 3. The peptide of embodiment 1 or 2, having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% sequence identity to 72, 677, 681, 686, 690, 695, 700, 704, 709, 717, 721, 726, 745, 748, 751, 754, or 756.

[0072] 4. The modified defensin peptide or modified defensin-like peptide is selected from the group consisting of SEQ ID NOs: 535, 536, 537, 539, 540, 541, 543, 544, 545, 547, 548, 549, 550, 552, 553, 554, 556, 557, 558, 559, 561, 562, 563, 564, 566, 567, 568, 570, 571, 572, 574, 575, 576, 577, 579, 580, 581, 582, 584, 585, 586, 587, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642 90, 591, 592, 594, 595, 596, 597, 599, 600, 601, 602, 604, 605, 606, 607, 609, 610, 611, 613, 614, 615, 617, 618, 619, 621, 622, 623, 625, 626, 627, 629, 630, 631, 632, 634, 635, 636, 638, 639, 640, 642, 643, 644, 646, 647, 648, 650, 651, 652, 654, 655, 656, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 7 59, 660, 661, 663, 664, 665, 667, 668, 669, 671, 672, 673, 675, 676, 677, 678, 680, 681, 682, 684, 685, 686, 687, 689, 690, 691, 693, 694, 695, 696, 698, 699, 700, 701, 703, 704, 705, 707, 708, 709, 710, 712, 713, 714, 716, 717, 718, 720, 721, 722, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 7 4. The peptide of embodiment 3, comprising 27, 728, 729, 730, 731, 732, 733, 734, 735, 736, 744, 745, 747, 748, 750, 751, 753, 754, 755, 756, or a variant thereof comprising a deletion of 1 to 10 N-terminal amino acid residues, a deletion of 1 to 10 C-terminal amino acid residues, and / or conservative amino acid substitutions of 1 to 10 amino acid residues, wherein the modified gamma core consensus peptide is preserved in the variant.

[0073] 5. A peptide comprising the amino acid sequence of a modified defensin peptide fragment, wherein the wild-type gamma core consensus peptide GXCX3-9C or GXCX3-22C of the corresponding wild-type defensin peptide fragment is selected from the group consisting of the peptide sequences GXCX3-9(F / W / Y), GXCX3-9(F / W / Y / L / V / I / M), GXCX3-9(F / W / Y)(F / W / Y / M)(F / W / Y), GXCX3-9(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M), GXCX9-22(F / W / Y), GXCX16-22(F / W / Y), GXCX9-22(F / W / Y / L / V / I / M), GXCX16-22(F / W / Y / L / V / I / M), GXCX9-22(F / W / Y)(F / W / Y / L / V / I / M)(F / W / Y), GXCX16-22 (F / W / Y)(F / W / Y / L / V / I / M)(F / W / Y), GXCX9-22(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M), GXCX16-22(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M), GXCX3-9(F / W / Y)(R / K / H)(F / W / Y), GXCX3-9(F / W / Y / L / V / I / M)(R / K / H)(F / W / Y / L / V / I / M), GXCX9-22(F / W / Y)(R / K / H)(F / W / Y), GXCX16-22(F / W / Y)(R / K / H)(F / W / Y), GXCX16-22(F / W / Y / L / V / I / M )(R / K / H)(F / W / Y / L / V / I / M), or GXCX9-22(F / W / Y / L / V / I / M)(R / K / H)(F / and wherein the amino acid sequence of the modified gamma core consensus peptide is replaced with a modified gamma core consensus peptide comprising a C-terminal amino acid sequence (W / Y / L / V / I / M), the peptide further comprising a second C-terminal cysteine ​​residue located C-terminal to the cysteine ​​residue of the modified gamma core consensus sequence, the peptide having a net positive charge of at least 3 and a hydrophobic amino acid content of at least 18%, and optionally the peptide comprises, consists essentially of, or consists of (i) 30 or fewer amino acid residues, or (ii) 15, 16, or 17 to 30 amino acid residues.

[0074] 6. The peptide of embodiment 5, wherein the peptide comprises a modified C-terminal fragment of a wild-type defensin peptide, the modification comprising replacing the wild-type gamma core consensus peptide GXCX3-9C or GXCX3-22C of the wild-type defensin C-terminal fragment with a modified gamma core consensus peptide, optionally wherein the peptide comprises one additional cysteine ​​residue at the C-terminus of the modified gamma core consensus peptide sequence, and optionally wherein the peptide comprises a disulfide bond between two cysteine ​​residues in the modified C-terminal fragment having the one additional cysteine ​​residue.

[0075] 7. The peptide of embodiment 6, wherein the peptide comprises, consists essentially of, or consists of SEQ ID NOs: 537, 541, 545, 550, 554, 559, 564, 568, 572, 577, 582, 587, 592, 597, 602, 607, 611, 615, 619, 623, 627, 632, 636, 640, 644, 648, 652, 656, 661, 665, 669, 673, 678, 682, 687, 691, 696, 701, 705, 710, 714, 718, 722, 727, or a variant thereof comprising conservative amino acid substitutions of 1 to 2, 3, 4, or 5 amino acid residues, and wherein the modified gamma core consensus peptide is conserved in the variant.

[0076] 8. A peptide comprising a C-terminal fragment of a defensin-like peptide, wherein the C-terminal fragment lacks 1 to 35 amino-terminal amino acids of the corresponding wild-type defensin-like peptide, and / or has the peptide sequence GXCX3-9(F / W / Y), GXCX3-9(F / W / Y / L / V / I / M), GXCX3-9(F / W / Y)(F / W / Y / M)(F / W / Y), GXCX3-9(F / W / Y / L / V / I / M)( F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M), GXCX9-22(F / W / Y), GXCX16-22(F / W / Y), GXCX9-22(F / W / Y / L / V / I / M), GXCX16 -22(F / W / Y / L / V / I / M), GXCX9-22(F / W / Y)(F / W / Y / L / V / I / M)(F / W / Y), GXCX16-22(F / W / Y)(F / W / Y / L / V / I / M)(F / W / Y), GXCX9-22(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M), GXCX16-22(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M), GXCX3-9(F / W / Y)(R / K / H)(F / W / Y), GXCX3-9(F / W / Y / L / V / I / M)(R / K / H)(F / W / Y / L / V / I / M), Peptides comprising modified gamma core consensus peptides including GXCX9-22(F / W / Y)(R / K / H)(F / W / Y), GXCX16-22(F / W / Y)(R / K / H)(F / W / Y), GXCX16-22(F / W / Y / L / V / I / M)(R / K / H)(F / W / Y / L / V / I / M), or GXCX9-22(F / W / Y / L / V / I / M)(R / K / H)(F / W / Y / L / V / I / M).

[0077] 9. A peptide comprising, consisting essentially of, or consisting of SEQ ID NO: 729, 731, 733, 735, 736, 828, 829, 830, 831, 832, or 833, or a variant thereof containing conservative amino acid substitutions of 1 to 2, 3, 4, or 5 amino acid residues, or a variant thereof having at least 90% or 95% sequence identity thereto, wherein the gamma core consensus peptide is conserved in the variant or the gamma core consensus peptide is the peptide sequence GXCX3 -9(F / W / Y), GXCX3-9(F / W / Y / L / V / I / M), GXCX3-9(F / W / Y)(F / W / Y / M)(F / W / Y), GXCX3-9(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M), GXCX9-22(F / W / Y), GXCX16-22(F / W / Y), GXCX9-22(F / W / Y / L / V / I / M), GX CX16-22(F / W / Y / L / V / I / M), GXCX9-22(F / W / Y)(F / W / Y / L / V / I / M)(F / W / Y ), GXCX16-22(F / W / Y)(F / W / Y / L / V / I / M)(F / W / Y), GXCX9-22(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M), GXCX16-22(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M), GXCX3-9(F / W / Y)(R / K / H)(F / W / Y), GXCX3-9(F / W / Y / L / V / I / M)(R / K / H)(F / W / Y / L / V / I / M), GXCX9-22(F or GXCX9-22(F / W / Y / L / V / I / M)(R / K / H)(F / W / Y), and optionally the gamma core consensus peptide comprises the peptide sequence GXCX3-9C or GXCX9-16C.

[0078] 10. A composition comprising a peptide according to any one of embodiments 1 to 9 and an agriculturally, pharmaceutically or veterinarily acceptable carrier, diluent or excipient.

[0079] 11. The composition of embodiment 10, 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 about 0.1, 0.5, 1.0, or 5 pg / gram to about 1, 5, 20, 50, or 100 mg / gram, and optionally, wherein the composition 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.

[0080] 12. A method for preventing or reducing crop damage caused by plant pathogenic microorganisms, comprising contacting a plant, a plant seed, or other part of said plant with an effective amount of the composition of embodiment 10.

[0081] 13. The method of embodiment 12, wherein the plant pathogenic microorganism is Fusarium, Alternaria, Verticillium, Phytophthora, Colletotrichum, Botrytis, Cercospora, Phakopsora, Rhizoctonia, Sclerotinia, Pythium, Phoma, Leptosphaeria, Gaeumannomyces, Puccinia, Septoria, Penicillium, Lasiodiplodia, Phomopsis, Mycosphaerella, Golobinomyces, Erysiphe, Albugo, Cetosphaeria, Cochliobolus, Helminthosporium, Diplodia, Magnaporthe, or Stenocarpella.

[0082] 14. A medical device comprising a device and the composition of embodiment 4, wherein the device comprises at least one surface topically coated and / or impregnated with the composition.

[0083] 15. The medical device of embodiment 14, wherein the device is a stent, a catheter, a contact lens, a condom, a patch, or a diaphragm.

[0084] 16. 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 the composition of embodiment 10.

[0085] 17. The method of embodiment 16, wherein said administering comprises topical, enteral, parenteral, and / or intravenous introduction of the composition.

[0086] 18. The method of embodiment 16, wherein the subject is a human, livestock, poultry, fish, or companion animal.

[0087] 19. The method of embodiment 16, wherein the microbial infection is an infection of the mucous membranes, eyes, skin, and / or nails, and the composition is applied to the mucous membranes, eyes, skin, and / or nails.

[0088] 20. The method of any one of embodiments 16 to 19, wherein the microbial infection is caused by a dermatophyte, and the dermatophyte is optionally selected from the group consisting of Trichophyton rubrum, Trichophyton interdigital, Trichophyton violaceum, Trichophyton tonsurans, Trichophyton soudanense, Trichophyton mentagrophytes, Microsporum flavum, Epidermophyton floccosum, and Microsporum gypseum.

[0089] 21. The method of any one of embodiments 16 to 19, wherein the microbial infection is caused by Aspergillus, Cryptococcus, Penicillium, Rhizopus, Apophysomyces, Cunninghamella, Succenea, Rhizomucor, Syncephalastrum, Cocheromyces, Actinomycor, Pythium, Fusarium, Histoplasma, or Blastomyces.

[0090] 22. The method of any one of embodiments 16 to 19, wherein the microbial infection is caused by a Candida fungus, and the Candida fungus is Candida albicans (C. albicans), C. auris, C. glabrata, C. parapsilosis, C. tropicalis, or C. krusei.

[0091] 23. The composition of embodiment 10, for use in a method for treating, preventing, or inhibiting a microbial infection in a subject in need thereof.

[0092] 24. The composition of embodiment 23, wherein the subject is a human, livestock, poultry, fish, or companion animal.

[0093] 25. A plant part at least partially coated with the composition of embodiment 10.

[0094] 26. The plant part of embodiment 25, wherein the part is a seed, and the seed is optionally a corn, soybean, wheat, rice, cotton, cruciferous, or tomato seed.

[0095] 27. The plant part of embodiment 25, wherein the plant part is a leaf, stem, fruit, vegetable, root, tuber, or flower.

[0096] 28. A recombinant polynucleotide comprising a polynucleotide encoding a peptide comprising the peptide of any one of embodiments 1 to 9, wherein the polynucleotide encoding the peptide is operably linked to a polynucleotide comprising a promoter heterologous to the polynucleotide encoding the peptide.

[0097] 29. (i) A defensin peptide comprising the long gamma core consensus sequence GXCX16-22C, optionally comprising SEQ ID NO: 578, 608, 612, or a variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 578, 608, or 612, a variant thereof comprising a deletion of 1 to 10 N-terminal amino acid residues, a variant thereof comprising a deletion of 1 to 10 C-terminal amino acid residues, and / or a variant thereof comprising a deletion of 1 to 10 N-terminal amino acid residues. or (ii) a defensin or defensin-like peptide comprising the wild-type gamma core consensus peptide GXCX3-9C, GXCX9-16C or GXCX3-22C, optionally selected from the group consisting of SEQ ID NOs: 534, 538, 560, 565, 569, 573, 583, 598, 603, 616, 624, 628, 633, 645, 697, 702, 711, 715, 723, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 800, 801, 802, 803, 804, 805, 806, 807, 30, 731, 732, 733, 734, 735, 736, or variants thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NOs: 534, 538, 560, 565, 569, 573, 583, 598, 603, 616, 624, 628, 633, 645, 697, 702, 711, 715, 723, 728, 729, 7 ... A recombinant polynucleotide encoding a peptide comprising a defensin or defensin-like peptide, including variants thereof including deletions, variants thereof including deletions of 1 to 10 C-terminal amino acid residues, and / or variants thereof having conservative amino acid substitutions of 1 to 10 amino acid residues, wherein the polynucleotide encoding the peptide is operably linked to a polynucleotide comprising a promoter heterologous to the polynucleotide encoding the peptide.

[0098] 30. The recombinant polynucleotide of embodiment 28 or 29, wherein the recombinant polynucleotide further comprises a polynucleotide encoding (i) a transit peptide, a vacuolar targeting peptide, and / or an endoplasmic reticulum targeting peptide, (ii) a plastid targeting peptide, and / or (iii) a polyadenylation or transcription termination signal, wherein the polynucleotide of (i), (ii), and / or (iii) is operably linked to the polynucleotide encoding the antimicrobial peptide.

[0099] 31. The recombinant polynucleotide of embodiment 28, 29, or 30, wherein the polynucleotide encoding the peptide is inserted into the heterologous nuclear genome or plastid genome of the cell and operably linked to an endogenous promoter located within the heterologous nuclear genome or plastid genome.

[0100] 32. A plant nuclear genome or plastid genome comprising a polynucleotide encoding a peptide comprising the peptide of embodiments 1 to 9, wherein the polynucleotide is heterologous to the nuclear genome or plastid genome, and the polynucleotide is operably linked to an endogenous promoter of the nuclear genome or plastid genome.

[0101] 33. A cell comprising a recombinant polynucleotide according to any one of embodiments 28, 29, 30, or 31, wherein the cell is optionally a bacterial cell, a yeast cell, or a plant cell.

[0102] 34. A plant comprising a recombinant polynucleotide according to any one of embodiments 28, 29, 30, or 31.

[0103] 35. A plant part of a plant according to embodiment 34, wherein the plant part comprises a recombinant polynucleotide, and optionally the plant part is a seed, stem, leaf, root, tuber, flower, vegetable, or fruit.

[0104] 36. A method for producing a plant seed that provides a plant that is resistant to infection by a plant pathogenic microorganism, the method comprising: (i) selfing or crossing a plant described in embodiment 34; and (ii) harvesting seeds from the selfing or crossing that comprise a recombinant polynucleotide of the plant, thereby producing a plant seed that provides a plant that is resistant to infection by a plant pathogenic microorganism.

[0105] 37. A method for producing an antifungal peptide, comprising: (i) culturing a cell described in embodiment 33 under conditions in which the peptide, defensin, or defensin-like peptide is expressed by the cell; and (ii) purifying the peptide, defensin peptide, or defensin-like peptide from the culture.

[0106] 38. The method of embodiment 37, wherein the cell is a yeast cell, and optionally the recombinant polynucleotide comprises a polynucleotide encoding a transport peptide operably linked to a polynucleotide encoding a peptide, defensin peptide, or defensin-like peptide, and optionally the peptide, defensin peptide, or defensin-like peptide is purified from the culture supernatant.

[0107] 39. The method of embodiment 38, wherein the yeast cell is a Candida cell, a Kluyveromyces cell, a Hansella cell, a Pichia cell, a Saccharomyces cell, a Schizosaccharomyces cell, or a Yarrowia cell.

[0108] 40. (i) A defensin peptide comprising the long gamma core consensus sequence GXCX16-22C, optionally SEQ ID NO: 578, 608, 612, or a variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 578, 608, or 612; a variant thereof comprising a deletion of 1 to 10 N-terminal amino acid residues; a deletion of 1 to 10 C-terminal amino acids; or (ii) a defensin or defensin-like peptide comprising the wild-type gamma core consensus peptide GXCX3-9C, GXCX9-16C or GXCX3-22C, optionally selected from SEQ ID NOs: 534, 538, 560, 565, 569, 573, 583, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683 , 603, 616, 624, 628, 633, 645, 697, 702, 711, 715, 723, 728, 729, 730, 731, 732, 733, 734, 735, 736, or SEQ ID NOs: 534, 538, 560, 565, 569, 573, 583, 598, 603, 616, 624, 628, 633, 645, 697, 702, 711, 715, 723, 728, 729, 730, 731, 732, 733, 734, 735, 736, its variants having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to 736, its variants comprising a deletion of 1 to 10 N-terminal amino acid residues, its variants comprising a deletion of 1 to 10 C-terminal amino acid residues, and / or its variants having conservative amino acid substitutions of 1 to 10 amino acid residues.

[0109] 41. A composition comprising a peptide according to embodiment 40 and an agriculturally, pharmaceutically or veterinarily acceptable carrier, diluent or excipient.

[0110] 42. A method for preventing or reducing crop damage caused by plant pathogenic microorganisms, comprising contacting a plant, a plant seed, or other part of said plant with an effective amount of the composition of embodiment 41.

[0111] 43. The method of embodiment 42, wherein the plant pathogenic microorganism is Fusarium, Alternaria, Verticillium, Phytophthora, Colletotrichum, Botrytis, Cercospora, Phakopsora, Rhizoctonia, Sclerotinia, Pythium, Phoma, Leptosphaeria, Gaeumannomyces, Puccinia, Septoria, Penicillium, Lasiodiplodia, Phomopsis, Mycosphaerella, Golobinomyces, Erysiphe, Albugo, Setosphaeria, Cochliobolus, Helminthosporium, Diplodia, Magnaporthe, Stenocarpella, or Zymoseptoria.

[0112] 44. A plant or plant part at least partially coated with the composition according to embodiment 41.

[0113] 45. The plant or plant part of embodiment 44, wherein the plant part is a leaf, seed, or fruit.

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[0147] Vriens, K., et al.Molecules 2014,19,12280-12303;doi:10.3390 / moleculesl90812280.

[0148] White, P., and Karley, A. (2010). Potassium. In Cell Biology of Metals and Nutrients; Hell, R., Mendel, RR, Eds.; Springer: Berlin / Heidelberg, Germany., 199-224 [Example]

[0149] 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 further purified by C-18 reverse-phase HPLC (Agilent Technologies, USA) using a linear gradient of acetonitrile / water mixtures. 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 measured in SFM medium. SFM medium contains 2.5 mM KHPO, 50 μM MgSO, 50 μM CaCl, 50 μM FeSO, 5 μM CoCl, 0.1 μM CuSO, 0.1 μM NaMoO, 2 μM HBO, 0.5 μM KI, 0.1 μM ZnSO, 0.5 μM MnSO, ​​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 pyridoxine-HCl, and 0.2 mg / liter pyridoxine-HCl at pH 7.0. [Table 2] 1 The C-terminus is amidated. [Table 3]

[0150] In vitro antifungal activity under low cation conditions: GMA4C variants GMA4C_V1A, GMA4C_V3A, GMA4C_V4A, and GMA4C_V5A exhibit comparable 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 exhibits a two-fold decrease in antifungal activity against B. cinerea and F. graminearum compared to the wild-type GMA4C_AC control in vitro. GMA4C variant GMA4C_V6A exhibits a four-fold increase in antifungal activity against B. cinerea in vitro compared to the wild-type GMA4C_AC control.

[0151] In planta antifungal activity. As shown in Figures 1A and 1B, 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. When applied to tomato leaves, GMA4C_V1A is more effective than GMA4C_AC in reducing symptoms caused by P. capsici.

[0152] In vitro antifungal activity in the presence of cations. Antifungal activity against B. cinerea was tested in the presence of 100 mM NaCl, 100 mM KCl, or 2 mM CaCl2. All peptides, including GMA4C_AC, retained their antifungal activity in the presence of 100 mM NaCl or 100 mM KCl. However, GMA4C_V1A and GMA4C_V4A were twice as active as GMA4C_AC. In the presence of 2 mM CaCl2, only GMA4C_V1A and GMA4C_V4A inhibited fungal growth at 6 μM, whereas other peptides, such as the plant defensins MtDef4 and OeDef1, showed little or no activity at this concentration. [Table 4] 1 The minimum inhibitory concentration (MIC) is the concentration of a compound, protein, or peptide at which there is no significant growth of a microorganism compared to the growth of the microorganism in a growth medium lacking the compound, protein, or peptide.

[0153] Antifungal activity against human fungal pathogens: GMA4C_V1A and GMA4C_V3A, but not the parent MtDef4 defensin, exhibit antifungal activity against C. auris and C. glabrata in cation-rich RPMI medium. [Table 5] [Table 6] 1 The minimum inhibitory concentration (MIC) is the concentration of a protein or peptide at which there is no significant growth of a microorganism compared to growth of the microorganism in a growth medium lacking the compound, protein, or peptide.

[0154] Example 2 Activity of GMA4C mutants against pathogenic microorganisms Antifungal activity testing used half-strength potato dextrose broth for peptides and RPMI for the comparator antifungals fluconazole and voriconazole. CLSI M27 and M38 methodologies were used to determine MICs. The minimum inhibitory concentration (MIC) is the concentration of a compound, protein, or peptide at which no significant growth of a microorganism occurs compared to growth of the microorganism in a growth medium lacking the compound, protein, or peptide.

[0155] All studies were performed in RPMI buffered with 0.165 M MOPS. Peptide concentrations ranged from 0.06 to 2 mcg / ml, fluconazole concentrations ranged from 0.125 to 64 mcg / ml, and voriconazole concentrations ranged from 0.03 to 16 mcg / ml in potato dextrose broth. MICs were determined after 24 to 72 hours. [Table 7] [Table 8] [Table 9] [Table 10] [Table 11] 1 The full-length MtDef4 protein is described in US Pat. No. 7,825,297.

[0156] All pathogen isolates used in this assay are resistant to the antifungal drug fluconazole. Antifungal assays were performed in half-strength potato dextrose broth. [Table 12] 1 FICI stands for fractional inhibitory concentration index. It is the MIC A Combination / MIC alone + MIC BCombination / MIC B Calculated separately, MIC A MIC and MIC of drug A when combined A ("Alone" is the MIC for drug A alone). In the top table, drug A is a peptide (GMAC_V3A) and drug B is fluconazole. In the bottom table, drug A is the full-length MtDef4 protein and drug B is fluconazole. 2 The full-length MtDef4 protein is described in US Pat. No. 7,825,297, which is incorporated herein by reference in its entirety. [Table 13] 1 The full-length MtDef4 protein is described in US Pat. No. 7,825,297. 2 The full-length OeDef1 defensin protein is described in WO2020 / 146373. [Table 14]

[0157] Example 3 Antibacterial activity of GMA4C_V1A against human bacterial pathogens Copenhagen-type Salmonella typhimurium, enterotoxigenic E. coli F4, and Listeria monocytogenes (F5244) were cultured overnight on LB agar plates at 37°C. A small amount of bacteria was scraped from the plate, added to Mueller-Hinton (MH) growth medium, and cultured until logarithmic phase. 1–3 x 10 cells were cultured. 6 The peptides were diluted to CFU / mL and added in 50 μl portions to each well of a 96-well polypropylene 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, 60, and 120 μM. Next, 50 μl of each peptide solution was added to 50 μl of bacterial cells. The plate was covered with parafilm and incubated for 37 min. o The bacterial growth inhibitory concentration was calculated by the OD of MH medium alone.600 nm values ​​and OD during bacterial growth in peptide-free MH medium 600 OD at each concentration versus nm value 600 The minimum inhibitory concentration (MIC) was determined based on nm values. The minimum inhibitory concentration (MIC) is the concentration of peptide at which no significant growth of the microorganism occurs compared to the growth of the microorganism in growth medium lacking the peptide. [Table 15]

[0158] Example 4 Antibacterial activity of defensin peptide fragments Crude chemically synthesized defensin-derived peptides with 80-85% purity were obtained from Biomatik Inc., Canada, or Alan Scientific Inc., USA. Each peptide was further purified using a C-18 reverse-phase HPLC (Agilent, Singapore). HPLC fractions containing the peptides were lyophilized and resuspended in nuclease-free water. Concentrations were determined using a BCA assay using the manufacturer's protocol (Thermo-Fisher Scientific) for accurate quantification.

[0159] Strains of Botrytis cinerea T-4, Alternaria alternata, Cercospora sojina, and Colletotrichum gloeosporioides were cultured in their respective conventional growth media, as shown in Table 16. Fungal spores were harvested by pouring sterile water onto fungal growth plates. The spore suspension was filtered through two layers of Miracloth, centrifuged at 13,600 rpm for 1 minute, 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. [Table 16]

[0160] The antifungal activity of truncated defensin-derived peptides and their mutants against the fungal pathogens B. cinerea, A. alternata, C. sojina, and C. gloeosporioides was measured spectrophotometrically using a 96-well plate assay (Sagaram et al., (2011) PLoS ONE 6:e18550. doi:10.1371 / journal.pone.0018550). Forty-five microliters of peptide at concentrations of 0.375, 0.75, 1.5, 3, and 12 μM were added to 45 μL (approximately 10 μM) of 1000-kJ / ml PBS. 5 A spore suspension containing 100 B. cinerea spores / ml was added to each well of a microtiter plate. Quantitative fungal growth inhibition was determined after 48 hours by measuring absorbance at 595 nm using a microplate reader (Tecan Infinite M200 ProTecan Systems Inc., San Jose, CA). Fungal cell viability was measured using a resazurin cell viability assay (Chadha and Kale, (2015) Lett Appl Microbiol 61, 238-244; Li et al., (2019) Mol Plant Microbe Interact 32, 1649-1664). After 48 hours of incubation, the pathogen / peptide mixture was incubated with 10 μl of 0.1% resazurin solution, which was then added to each well and reincubated overnight. A color change 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 mutants were also determined as above in SFM and SFM supplemented with 100 mM NaCl, 100 mM, or 2 mM CaCl2.

[0161] The in vivo antifungal activity of each defensin-derived peptide and its mutants against B. cinerea was measured using detached leaves of N. benthamiana Nb1 as previously described (Li et al., (2019) Mol Plant Microbe Interact 32, 1649-1664; Velibelli 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 incubating each peptide / fungal spore mixture at room temperature for 48 h, the leaves were photographed under white light. High-resolution fluorescence images were also taken using CropReporter (PhenoVation, Wageningen, The Netherlands). These images were used to calculate the F of the affected area due to B. cinerea infection. V / F M The maximum quantum yield of photosystem II (PQY) values ​​are shown. The colors in the images represent five different classes, from Class I to Class V (0.000 to 0.700), indicating various degrees of tissue damage. Green in each image represents Class V, ranging from 0.600 to ≥ 0.700, which indicates healthy areas of the leaf surface. In contrast, red represents Class I, ranging from 0.000 to 0.160, which indicates severe damage or disease on the leaf surface.

[0162] The primary amino acid sequences, lengths, net charges, and percentages 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 increase the net charge and hydrophobicity. Additionally, disulfide bonds were introduced into certain mutants to make them pseudocyclic. Peptides capable of forming a single disulfide bond and pseudocyclic peptides are indicated by a "+" in the "Disulfide Bond" column of Table 17. All peptides also have a carboxy-terminal amide group. [Table 17]

[0163] The minimum inhibitory concentration (MIC) values ​​of each defensin-derived peptide and its mutants were measured (Table 18). It has been 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, the antifungal activity of each peptide against B. cinerea was measured in SFM supplemented with 100 mM NaCl, 100 mM KCl, or 2 mM CaCl (Table 18). [Table 18] 1 The ND is yet to be determined.

[0164] The MIC values ​​of defensin-derived peptides were also tested against Alternaria alternata, Cercospora sojina, and Colletotrichum gloeosporioides (Table 19). [Table 19]

[0165] The in vivo antifungal activity of the GMA4C_V9, GMA4C_V10, GMAOe1C_WT, GMAOe1C_V3, GMAOe1C_V4, GMA1C_V1, and GMA1C_V2 peptides against B. cinerea was measured using detached leaves of N. benthamiana. Concentrations of 1.5 μM, 3 μM, and 6 μM of each peptide were applied as drops to the leaves, and freshly prepared conidial inoculum was applied directly to each drop of peptide. Reduction of Botrytis cinerea symptoms on the leaves 48 h after inoculation was assessed by measuring lesion size compared with the control without peptide. Both the GMA4C_V9 and GMA4C_V10 peptides were effective in reducing Botrytis cinerea symptoms. However, at lower concentrations of 1.5 μM and 3 μM, GMA4C_V10 was more effective than GMA4C_V9 in reducing Botrytis cinerea symptoms.

[0166] Droplet inoculation assays were also performed to test the antifungal activity of GMAOe1C_WT, GMAOe1C_V1, and GMAOe1C_V2. Results revealed that GMAOe1C_V1 and GMAOe1C_V2 completely suppressed gray mold symptoms at concentrations of 3 and 6 μM, whereas 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.

[0167] A drop inoculation assay was also performed to test the antifungal activity of GMA1C_V1 and GMA1C_V2. GMA1C_V2 at 3 μM and 6 μM completely suppressed Botrytis cinerea symptoms. GMA1C_V1 failed to reduce symptoms at these concentrations. At a concentration of 1.5 μM, GMA1C_V2 was more effective than GMA1C_V1.

[0168] The above results demonstrate that a series of modifications (eg, amino acid substitutions) introduced into these truncated defensin-derived peptides confer greater antifungal activity than the wild-type truncated peptides.

[0169] Example 5 Recombinant Expression and Purification of Defensins or Defensin-Like Peptides Codon-optimized synthetic genes encoding defensin or defensin-like peptides were custom synthesized using GenScript (Piscataway, NJ). The synthetic genes were cloned in frame with the α-factor secretion signal sequence between the XhoI and XbaI sites of the pPICZαA vector and expressed in Pichia pastoris X33. An alanine was added to the N-terminus of the defensin or defensin-like sequence to ensure efficient cleavage by the KEX2 cleavage site. Synthetic genes comprising a 5' to 3' XhoI site, three codons including an N-terminal alanine codon, and DNA encoding the wild-type defensins of SEQ ID NOs: 534, 538, 542, 546, 551, 555, 560, 565, 569, 573, 578, 583, 588, 593, 598, 603, 608, 612, 616, 620, 624, 628, 633, 637, 641, 645, 649, 653, 657, 662, 666, 670, 674, 679, 683, 688, 692, 697, 702, 706, 711, 715, 719, and 723 include SEQ ID NOs: 780-823, respectively. Derivatives of the synthetic DNAs of SEQ ID NOs:780-823 that encode the modified defensins and modified C-terminal fragments described in Table 1 or elsewhere herein are constructed by deleting and / or mutating the corresponding codons in SEQ ID NOs:780-823, or by de novo synthesis. Synthetic genes containing a 5' to 3' XhoI site, three codons including an N-terminal alanine codon, and DNA encoding the wild-type defensin-like proteins of SEQ ID NOs:728, 730, 732, and 734 comprise SEQ ID NOs:824, 825, 826, and 827, respectively. Derivatives of the synthetic DNAs of SEQ ID NOs:824, 825, 826, and 827 that encode the modified defensin-like proteins and modified C-terminal fragments thereof described in Table 1 or elsewhere herein are constructed by deleting and / or mutating the corresponding codons in SEQ ID NOs:824, 825, 826, and 827, or by de novo synthesis.

[0170] The pPICZαA vector containing each defensin or defensin-like sequence was linearized by digestion with SacI or PmeI restriction enzyme and transformed into P. pastoris X33 using electroporation. Transformant candidates were selected on yeast extract-peptone-dextrose (YPD) agar plates containing 150 μg / mL Zeocin® and then inoculated into 100 μl of YPD broth containing 500 μg / mL Zeocin®. Transformants that survived at higher Zeocin® concentrations and exhibited higher optical density (OD600) readings were selected and used for small-scale expression studies of defensins or defensin-like peptides. Selected transformants were grown in 2 ml of buffered minimal glycerol (BMG, Invitrogen) medium for 2 days at 30°C in a shaker at 225 rpm. Cells were harvested by centrifugation at 4,000 rpm for 10 minutes and resuspended in 2 ml of buffered minimal methanol (BMM, Invitrogen) medium to induce peptide expression. Cultures were grown at 30°C for 4 days, with 1% (v / v) methanol added every 24 hours to maintain induction. After 4 days, cells were harvested, and 20 μl of supernatant was taken and run on an SDS-PAGE gel for recombinant peptide expression. High-expressing transformants were selected based on the intensity of the peptide band.

[0171] Defensin or defensin-like peptides were purified using CM Sephadex C-25 cation exchange chromatography. Briefly, P. pastoris X33 transformants containing defensins or defensin-like peptides were grown in 100 ml of buffered minimal glycerol (BMG, Invitrogen) medium for 2–3 days in a shaker at 30°C and 225 rpm. Cells were harvested by centrifugation at 4,000 rpm for 10 min at room temperature (RT) and resuspended in 100 ml of buffered minimal methanol (BMM, Invitrogen) medium to induce peptide expression. Cultures were grown at 30°C and 225 rpm for 4 days, with 1% (v / v) methanol added every 24 h to maintain induction. After induction, cells were harvested by centrifugation at 4,000 rpm for 10 min at 4°C. The supernatant was then filtered through a 0.22 μm filter to further remove cellular debris. Cation exchange resin (CM-Sephadex C-25, catalog number: C25120, Sigma) pre-equilibrated with binding buffer (25 mM anhydrous sodium acetate, pH 5.2 / 6.0) was added to the supernatant and incubated overnight at 4°C and 100 rpm. The sample was loaded onto a gravity column (Poly-Prep® Chromatography Columns, catalog number: 7311550, Bio-Rad). After washing the resin with binding buffer, bound proteins were eluted with elution buffer (1 M NaCl, 50 mM Tris, pH 7.6). Fractions containing each defensin or defensin-like peptide were concentrated and dialyzed against 10 mM Tris at pH 7.6 using 1 kD / 3 kD centrifugal filter units. The purity and size of each peptide were determined by SDS-PAGE gel electrophoresis, and peptide concentration was determined by BCA assay.

[0172] Example 6 Antifungal Assay The antifungal activity of defensins and defensin-like peptides is measured in an in vitro assay using 96-well microtiter plates with an Opentron liquid handler. Briefly, 45 µL (approximately 10 µL) of fungal spore suspension (e.g., F. graminearum, B. cinerea, and Z. tritici) is added to the wells of the plate.5 Forty-five microliters of each protein dilution (0, 0.375, 0.75, 1.5, 3, 6, and 12 μM) is added to each well of a microtiter plate containing 100 spores / ml. The plate is incubated at room temperature, and quantitative fungal growth inhibition is estimated by measuring absorbance at 595 nm using a Tecan Infinite M200 Pro (Tecan Systems Inc., San Jose, CA) microplate reader at 48 hours. Fungal cell viability is measured by a resazurin cell viability assay. After 48 hours of incubation, 0.1% resazurin solution is added to each well, and the plate is reincubated overnight. A color change from blue to pink indicates a reduction in resazurin and cell viability.

[0173] Example 7 Peptide Expression, Purification, and In Vitro Antifungal Assay Expression and purification of defensins and defensin-like peptides was performed essentially as described in Example 5. Selected transformants were grown in 2 ml of buffered minimal glycerol medium (BMG, Invitrogen) at 28-30°C in a shaker at 225 rpm for 2 days. Cells were harvested by centrifugation at 4,000 rpm for 10 minutes and resuspended in 2 ml of buffered minimal methanol (BMM, Invitrogen) medium to induce peptide expression. Cultures were grown at 28-30°C for 4 days, with 1% (v / v) methanol added every 24 hours to maintain induction. 100 ml cultures were grown at 28-30°C at 225 rpm for 4 days, with 1% (v / v) methanol added every 24 hours to maintain induction.

[0174] The Pichia pastoris X33 expression system was effectively used to express and purify defensin and defensin-like peptides. Yields of these peptides varied considerably, ranging from no detectable expression to a maximum of 4.47 mg per 100 ml of culture. A summary of purification yields is shown in Table 20 for all peptides. [Table 20-1] [Table 20-2] 1 SEQ ID NO: of defensin peptide lacking N-terminal alanine

[0175] The antifungal activity of defensins and defensin-like peptides was measured in an in vitro assay using 96-well microtiter plates with an Opentron liquid handler. Briefly, 45 µL (approximately 10 µL) of fungal spore suspension (e.g., Fusarium graminearum PH-1, Botrytis cinerea T4, and Zymoseptoria tritici IPO323) was added to the 96-well microtiter plate. 5 Forty-five microliters of each protein dilution (0, 0.187, 0.375, 0.75, 1.5, 3, 6, or 12 μM) was added to each well of a microtiter plate containing 100 spores / ml of spores. The plates were incubated at room temperature, and quantitative fungal growth inhibition was estimated by measuring absorbance at 595 nm after 48 hours for F. graminearum and B. cinerea, and after 72 hours for Z. tritici. Fungal cell viability was measured using a resazurin cell viability assay. After 48 hours of incubation, 0.05% resazurin solution was added to each well of F. graminearum and B. cinerea. For Z. tritici IPO323, 0.025% resazurin solution was added at 72 hours. The plates were then reincubated overnight for F. graminearum and B. cinerea, and up to 14 days for Z. tritici. The change from blue to pink / colorless represents a decrease in resazurin and indicates metabolically active fungal spores.

[0176] In vitro antifungal assays revealed that the peptides inhibited the growth of F. graminearum PH-1, B. cinerea T4, and Z. tritici IPO323. The MIC values ​​of the in vitro antifungal activity of each peptide against these pathogens are shown in Tables 21 and 22. [Table 21-1] [Table 21-2] 1 SEQ ID NO: of defensin peptide lacking N-terminal alanine [Table 22-1] [Table 22-2] 1 SEQ ID NO: of defensin peptide lacking N-terminal alanine

[0177] In the case of F. graminearum, PD87.1.1, PD95.1.1, PD96.1.1, PD99.1.1, and PD106.1.1 showed potent in vitro antifungal activity against F. graminearum at submicromolar concentrations, particularly at 0.75 μM, with some peptides showing activity in the range of 1 μM to over 12 μM. The positive control, amphotericin B, showed an MIC of 6 μM against F. graminearum.

[0178] In the case of Botrytis cinerea, PD87.1.1, PD88.1.1, PD90.1.1, PD95.1.1, PD99.1.1, PD122.1.1, and PD124.1.1 showed potent in vitro antifungal activity against B. cinerea at submicromolar concentrations (0.375 to 0.75 μM). PD81.1.1 and PD106.1.1 showed in vitro antifungal activity against B. cinerea at 1 μM, with some peptides showing activity in the range of 1.5 to 12 μM or greater. The positive control, amphotericin B, showed an MIC of 3 μM against B. cinerea.

[0179] For Z. tritici, MIC values ​​were evaluated on days 1, 7, and 14. Stability was indirectly assessed by observing changes in MIC values ​​over time. PD97.1.1 and PD101.1.1 showed initial MIC values ​​of 0.75 μM on day 1. Subsequent measurements on days 7 and 14 revealed MIC values ​​of 1.5 μM, suggesting that the antifungal activity was relatively stable throughout the evaluation period. PD81.1.1, PD82.1.1, PD87.1.1, PD90.1.1, PD92.1.1, PD102.1.1, PD103.1.1, PD106.1.1, PD118.1.1, and PD122.1.1 showed MIC values ​​ranging from 2 to 3.5 μM on day 14. Some peptides showed MIC values ​​between 4 and 12 μM on day 14, while others showed values ​​above 12 μM, suggesting that the stability of antifungal activity varies among different peptides. The positive control, amphotericin B, showed MIC values ​​against Z. tritici of 0.84 μM on day 1, 5.44 μM on day 7, and 8.92 μM on day 14.

[0180] Example 8: Antifungal activity in plants Pepper - Botrytis cinerea in planta assay Pepper ( Capsicum annuum, California Wonder) plants were grown for 4 weeks in a greenhouse with a 14-h / 10-h light / dark cycle, a daytime temperature of 28°C, a nighttime temperature of 22°C, and a relative humidity of 50%.

[0181] To evaluate preventive activity, a 24 μM solution of PD122.1.1 (SEQ ID NO: 715 with an N-terminal alanine) containing 0.02% Tween was sprayed onto treated pepper plants at a volume of 2 mL / plant. Control plants were prepared similarly, but without peptide. After 24 hours, B. cinerea spores (approximately 7 x 10 spores) suspended in 0.5x SFM were sprayed onto the treated pepper plants. 4 The treated and control pepper plants were sprayed with 1 mL of the compound per plant. The plants were kept in a humid environment at room temperature. Symptoms were observed at 96 HPI.

[0182] For evaluation of curative activity, B. cinerea spores (approximately 7 x 10 ) suspended in 0.5x SFM were used. 4The treated and control pepper plants were sprayed with 1 mL of PD122.1.1 per plant. 24 hours later, 24 μM PD122.1.1 solution containing 0.02% Tween was sprayed on the treated pepper plants at 2 mL per plant. Control plants were prepared similarly, but without the peptide. The plants were kept in a humid environment at room temperature. Symptoms were observed 5 days after the onset of infection.

[0183] The potential of PD122.1.1 to provide both preventative and curative protection against B. cinerea (gray mold) was evaluated by spraying 4-week-old pepper plants with 24 μM PD122.1. Application of PD122.1.1 reduced gray mold symptoms compared to control plants inoculated with the pathogen alone (shown in Figures 3A and 3B). These results demonstrate that spraying PD122.1.1 effectively protects pepper plants from gray mold, providing preventative protection at 96 hours post-inoculation (hpi) (Figure 3A) and curative protection at 5 days post-inoculation (dpi) (Figure 3B).

[0184] In planta assay for wheat - Zymoseptoria trisiti Wheat (bread wheat, bobwhite) plants were grown for 2 weeks in a controlled growth chamber with a 16-h / 8-h light / dark cycle under conditions of 20°C temperature, 200 μMol light intensity, and 70% relative humidity.

[0185] For evaluation of curative activity, Z. tritici spores (1 × 10 ) suspended in sterile water containing 0.1% Tween were used. 6 Both treated and control wheat plants were sprayed with 2 mL of PD101.1.1 (SEQ ID NO: 624 with an N-terminal alanine) at 2 mL / plant. 24 hours later, treated wheat plants were sprayed with 2 mL / plant of a 48 μM solution of PD101.1.1 (SEQ ID NO: 624 with an N-terminal alanine) containing 0.02% Tween. Control plants were prepared similarly, but without the peptide. All plants were placed in a humid environment with a 16-h / 8-h light / dark cycle, a day / night temperature of 20°C, a light intensity of 200 μM, and a relative humidity of 80-95%. Disease symptoms were observed at 21 DPI.

[0186] The potential of PD101.1.1 to provide curative protection against Z. tritici (Septoria tritici leaf spot (STB)) was evaluated by spraying 2-week-old wheat plants with 48 μM PD101.1. Application of PD101.1.1 reduced STB disease symptoms compared to control plants inoculated with the pathogen alone (shown in Figure 4). These results demonstrate that application of PD101.1.1 effectively protects wheat plants from STB disease and provides curative protection 21 days post-inoculation (dpi).

[0187] The breadth and scope of the present disclosure should not be limited by any of the above examples.

Claims

1. A peptide comprising the amino acid sequence of a modified defensin or modified defensin-like peptide, wherein the wild-type gamma core consensus peptide GXCX3-9C or GXCX3-22C of the wild-type defensin or wild-type defensin-like peptide has the peptide sequence GXCX3-9(F / W / Y), GXCX3-9(F / W / Y / L / V / I / M), GXCX3-9(F / W / Y)(F / W / Y / M) ... V / I / M) (F / W / Y / L / V / I / M) (F / W / Y / L / V / I / M), GXCX9-22 (F / W / Y), GXCX16-22 (F / W / Y), GXCX9-22 (F / W / Y / L / V / I / M), GXCX16-22 (F / W / Y / L / V / I / M), GXCX9-22 (F / W / Y) (F / W / Y / L / V / I / M) (F / W / Y), GXCX16-22 (F / W / Y) (F / W / Y / L / V / I / M) (F / W / Y), GXCX9-22 (F / W / Y) / W / Y / L / V / I / M) (F / W / Y / L / V / I / M) (F / W / Y / L / V / I / M), GXCX16-22 (F / W / Y / L / V / I / M) (F / W / Y / L / V / I / M) (F / W / Y / L / V / I / M), GXCX 3-9 (F / W / Y) (R / K / H) (F / W / Y), GXCX3-9 (F / W / Y / L / V / I / M) (R / K / H) (F / W / Y / L / V / I / M), GXCX9-22 (F / W / Y) (R / K / H) (F / W / Y), GXC X16-22(F / W / Y)(R / K / H)(F / W / Y), GXCX16-22(F / W / Y / L / V / I / M)(R / K / H)(F / W / Y / L / V / I / M), or GXCX9-22(F / W / Y / L / V / I / M)(R / K / H)(F / W / Y / L / V / I / M), wherein the peptide has a net positive charge of at least 3 and a hydrophobic amino acid content of at least 18%.

2. The wild-type defensin is selected from the group consisting of SEQ ID NOs: 11 to 41, 81 to 533, 534, 538, 542, 546, 551, 555, 560, 565, 569, 573, 578, 583, 588, 593, 598, 603, 608, 612, 616, 620, 624, 628, 633, 637, 641, 645, 649, 653, 657, 662, 666, 670 , 674, 679, 683, 688, 692, 697, 702, 706, 711, 715, 719, 723, 743, 746, 749, or 752, or the wild-type defensin-like peptide comprises a polypeptide of SEQ ID NO: 728, SEQ ID NO: 730, SEQ ID NO: 732, or SEQ ID NO:

734.

3. The modified defensin peptide is selected from the group consisting of SEQ ID NOs: 536, 540, 544, 549, 553, 558, 563, 567, 571, 576, 581, 586, 591, 596, 601, 606, 610, 614, 618, 622, 626, 631, 635, 639, 643, 647, 651, 655, 660, 664, 668 , 672, 677, 681, 686, 690, 695, 700, 704, 709, 717, 721, 726, 745, 748, 751, 754, or 756. The peptide of claim 1, having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% sequence identity to

4. The modified defensin peptide or modified defensin-like peptide is selected from the group consisting of SEQ ID NOs: 535, 536, 537, 539, 540, 541, 543, 544, 545, 547, 548, 549, 550, 552, 553, 554, 556, 557, 558, 559, 561, 562, 563, 564, 566, 567, 568, 570, 571, 572, 574, 575, 576, 577, 579, 580, 581, 582, 584, 585, 586, 587, 589, 59 0, 591, 592, 594, 595, 596, 597, 599, 600, 601, 602, 604, 605, 606, 607, 609, 610, 611, 613, 614, 615, 617, 618, 619, 621, 622, 623, 625, 626, 627, 629, 630, 631, 632, 634, 635, 636, 638, 639, 640, 642, 643, 644, 646, 647, 648, 650, 651, 652, 654, 655, 656, 658, 659 , 660, 661, 663, 664, 665, 667, 668, 669, 671, 672, 673, 675, 676, 677, 678, 680, 681, 682, 684, 685, 686, 687, 689, 690, 691, 693, 694, 695, 696, 698, 699, 700, 701, 703, 704, 705, 707, 708, 709, 710, 712, 713, 714, 716, 717, 718, 720, 721, 722, 724, 725, 726, 727 728, 729, 730, 731, 732, 733, 734, 735, 736, 744, 745, 747, 748, 750, 751, 753, 754, 755, 756, or a variant thereof comprising a deletion of 1 to 10 N-terminal amino acid residues, a deletion of 1 to 10 C-terminal amino acid residues, and / or conservative amino acid substitutions of 1 to 10 amino acid residues, wherein the modified gamma core consensus peptide is conserved in the variant.

5. A peptide comprising the amino acid sequence of a modified defensin peptide fragment, wherein the wild-type gamma core consensus peptide GXCX3-9C or GXCX3-22C of the corresponding wild-type defensin peptide fragment is selected from the group consisting of peptide sequences GXCX3-9(F / W / Y), GXCX3-9(F / W / Y / L / V / I / M), GXCX3-9(F / W / Y)(F / W / Y / M)(F / W / Y), GXCX3-9(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M), GXCX9-22(F / W / Y), GXCX16-22 (F / W / Y), GXCX9-22 (F / W / Y / L / V / I / M), GXCX16-22 (F / W / Y / L / V / I / M), GXCX9-22 (F / W / Y) (F / W / Y / L / V / I / M) (F / W / Y), GXCX16-22 (F / W / Y) (F / W / Y / L / V / I / M) (F / W / Y), GXCX9-22 (F / W / Y / L / V / I / M) (F / W / Y / L / V / I / M) (F / W / Y / L / V / I / M), GXCX16-22 (F / W / Y / L / V / I / M) (F / W / Y / L / V / I / M ) (F / W / Y / L / V / I / M), GXCX3-9 (F / W / Y) (R / K / H) (F / W / Y), GXCX3-9 (F / W / Y / L / V / I / M) (R / K / H) (F / W / Y / L / V / I / M), GXCX9-22 (F / W / Y) (R / K / H) (F / W / Y ), GXCX16-22 (F / W / Y) (R / K / H) (F / W / Y), GXCX16-22 (F / W / Y / L / V / I / M) (R / K / H) (F / W / Y / L / V / I / M), or GXCX9-22 (F / W / Y / L / V / I / M) (R / K / H) (F / W / Y / and wherein the amino acid sequence of the modified gamma core consensus peptide is replaced with a modified gamma core consensus peptide comprising a C-terminal cysteine ​​residue (L / V / I / M), said peptide further comprising a second C-terminal cysteine ​​residue located C-terminal to the cysteine ​​residue of said modified gamma core consensus sequence, said peptide having a net positive charge of at least 3 and a hydrophobic amino acid content of at least 18%, and optionally said peptide comprising, consisting essentially of, or consisting of (i) no more than 30 amino acid residues, or (ii) between 15, 16, or 17 and 30 amino acid residues.

6. The peptide of claim 5, wherein the peptide comprises a modified C-terminal fragment of a wild-type defensin peptide, the modification comprising replacing the wild-type gamma core consensus peptide GXCX3-9C or GXCX3-22C of the wild-type defensin C-terminal fragment with the modified gamma core consensus peptide, optionally the peptide comprises one additional cysteine ​​residue at the C-terminus of the modified gamma core consensus peptide sequence, and optionally the peptide comprises a disulfide bond between the two cysteine ​​residues in the modified C-terminal fragment having the one additional cysteine ​​residue.

7. 7. The peptide of claim 6, wherein the peptide comprises, consists essentially of, or consists of SEQ ID NOs: 537, 541, 545, 550, 554, 559, 564, 568, 572, 577, 582, 587, 592, 597, 602, 607, 611, 615, 619, 623, 627, 632, 636, 640, 644, 648, 652, 656, 661, 665, 669, 673, 678, 682, 687, 691, 696, 701, 705, 710, 714, 718, 722, 727, or a variant thereof comprising conservative amino acid substitutions of 1 to 2, 3, 4, or 5 amino acid residues, and wherein the modified gamma core consensus peptide is conserved in the variant.

8. 1. A peptide comprising a C-terminal fragment of a defensin-like peptide, wherein the C-terminal fragment lacks 1 to 35 amino terminal amino acids of the corresponding wild-type defensin-like peptide, and / or has the peptide sequence GXCX3-9(F / W / Y), GXCX3-9(F / W / Y / L / V / I / M), GXCX3-9(F / W / Y)(F / W / Y / M)(F / W / Y), GXCX3-9(F / W / Y / L / V / I / M)(F / W / Y / L / V / I / M) (F / W / Y / L / V / I / M), GXCX9-22 (F / W / Y), GXCX16-22 (F / W / Y), GXCX9-22 (F / W / Y / L / V / I / M), GXCX16- 22 (F / W / Y / L / V / I / M), GXCX9-22 (F / W / Y) (F / W / Y / L / V / I / M) (F / W / Y), GXCX16-22 (F / W / Y) (F / W / Y / L / V / I / M) (F / W / Y ), GXCX9-22 (F / W / Y / L / V / I / M) (F / W / Y / L / V / I / M) (F / W / Y / L / V / I / M), GXCX16-22 (F / W / Y / L / V / I / M) (F / W / Y / L / V / I / M) (F / W / Y / L / V / I / M), GXCX3-9 (F / W / Y) (R / K / H) (F / W / Y), GXCX3-9 (F / W / Y / L / V / I / M) (R / K / H) (F / W / Y / L / V / I / M), Peptides comprising modified gamma core consensus peptides including GXCX9-22(F / W / Y)(R / K / H)(F / W / Y), GXCX16-22(F / W / Y)(R / K / H)(F / W / Y), GXCX16-22(F / W / Y / L / V / I / M)(R / K / H)(F / W / Y / L / V / I / M), or GXCX9-22(F / W / Y / L / V / I / M)(R / K / H)(F / W / Y / L / V / I / M).

9. 1. A peptide comprising, consisting essentially of, or consisting of SEQ ID NOs: 729, 731, 733, 735, 736, 828, 829, 830, 831, 832, or 833, or a variant thereof comprising conservative amino acid substitutions of 1 to 2, 3, 4, or 5 amino acid residues, or a variant thereof having at least 90% or 95% sequence identity thereto, wherein the gamma core consensus peptide is conserved in said variant, or said gamma core consensus peptide has the peptide sequence GXCX 3-9 (F / W / Y), GXCX3-9 (F / W / Y / L / V / I / M), GXCX3-9 (F / W / Y) (F / W / Y / M) (F / W / Y), GXCX3-9 (F / W / Y / L / V / I / M) (F / W / Y / L / V / I / M) (F / W / Y / L / V / I / M), GXCX9-22 (F / W / Y), GXCX16-22 (F / W / Y), GXCX9-22 (F / W / Y / L / V / I / M), GX CX16-22 (F / W / Y / L / V / I / M), GXCX9-22 (F / W / Y) (F / W / Y / L / V / I / M) (F / W / Y ), GXCX16-22 (F / W / Y) (F / W / Y / L / V / I / M) (F / W / Y), GXCX9-22 (F / W / Y / L / V / I / M) (F / W / Y / L / V / I / M) (F / W / Y / L / V / I / M), GXCX16-22 (F / W / Y / L / V / I / M) (F / W / Y / L / V / I / M) (F / W / Y / L / V / I / M), GXCX3-9 (F / W / Y) (R / K / H) (F / W / Y), GXCX3-9 (F / W / Y / L / V / I / M) (R / K / H) (F / W / Y / L / V / I / M), GXCX9-22 (F / and optionally said gamma core consensus peptide comprises the peptide sequence GXCX3-9C or GXCX9-16C.

10. 10. A composition comprising a peptide according to any one of claims 1 to 9 and an agriculturally, pharmaceutically or veterinarily acceptable carrier, diluent or excipient.

11. 11. The composition of claim 10, 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 about 0.1, 0.5, 1.0, or 5 pg / gram to about 1, 5, 20, 50, or 100 mg / gram, and optionally, the composition 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.

12. 11. A method for preventing or reducing crop damage caused by plant pathogenic microorganisms, comprising contacting a plant, plant seed, or other part of said plant with an effective amount of the composition of claim 10.

13. 13. The method of claim 12, wherein the plant pathogenic microorganism is a fungus of the genus Fusarium, Alternaria, Verticillium, Phytophthora, Colletotrichum, Botrytis, Cercospora, Phakopsora, Rhizoctonia, Sclerotinia, Pythium, Phoma, Leptosphaeria, Gaeumannomyces, Puccinia, Septoria, Penicillium, Rasiodiplodia, Phomopsis, Mycosphaerella, Golobinomyces, Erysiphe, Albugo, Setosphaeria, Cochliobolus, Helminthosporium, Diplodia, Magnaporthe, or Stenocarpella.

14. 10. A medical device comprising a device and the composition of claim 4, wherein said device comprises at least one surface topically coated and / or impregnated with said composition.

15. 15. The medical device of claim 14, wherein the device is a stent, a catheter, a contact lens, a condom, a patch, or a diaphragm.

16. 11. 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 claim 10.

17. 17. The method of claim 16, wherein said administering comprises topical, enteral, parenteral, and / or intravenous introduction of said composition.

18. 17. The method of claim 16, wherein the subject is a human, livestock, poultry, fish, or companion animal.

19. 17. The method of claim 16, wherein the microbial infection is an infection of the mucous membrane, eye, skin, and / or nails, and the composition is applied to the mucous membrane, eye, skin, and / or nails.

20. 17. The method of claim 16, wherein the microbial infection is caused by a dermatophyte, and the dermatophyte is optionally selected from the group consisting of Trichophyton rubrum, Trichophyton interdigital, Trichophyton violaceum, Trichophyton tonsurans, Trichophyton soudanense, Trichophyton mentagrophytes, Microsporum flavum, Epidermophyton floccosum, and Microsporum gypseum.

21. 17. The method of claim 16, wherein the microbial infection is caused by Aspergillus, Cryptococcus, Penicillium, Rhizopus, Apophysomyces, Cunninghamella, Succenea, Rhizomucor, Syncephalastrum, Cocheromyces, Actinomycosis, Pythium, Fusarium, Histoplasma, or Blastomyces.

22. 17. The method of claim 16, wherein the microbial infection is caused by a Candida fungus, and the Candida fungus is C. albicans, C. auris, C. glabrata, C. parapsilosis, C. tropicalis, or C. krusei.

23. 11. The composition of claim 10 for use in a method for treating, preventing, or inhibiting a microbial infection in a subject in need thereof.

24. 24. The composition of claim 23, wherein the subject is a human, livestock, poultry, fish, or companion animal.

25. A plant part at least partially coated with the composition of claim 10.

26. 26. The plant part of claim 25, wherein the part is a seed, and the seed is optionally a corn, soybean, wheat, rice, cotton, crucifer, or tomato seed.

27. 26. The plant part of claim 25, wherein the plant part is a leaf, stem, fruit, vegetable, root, tuber, or flower.

28. 10. A recombinant polynucleotide comprising a polynucleotide encoding a peptide comprising the peptide of any one of claims 1 to 9, wherein the polynucleotide encoding the peptide is operably linked to a polynucleotide comprising a promoter heterologous to the polynucleotide encoding the peptide.

29. (i) a defensin peptide comprising the long gamma core consensus sequence GXCX16-22C, optionally comprising SEQ ID NO:578, 608, 612, or a variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:578, 608, or 612, a variant thereof comprising a deletion of 1 to 10 N-terminal amino acid residues, a variant thereof comprising a deletion of 1 to 10 C-terminal amino acid residues, and / or a variant thereof having conservative amino acid substitutions of 1 to 10 amino acid residues, or (ii) a wild-type gamma core consensus peptide GXCX. ３－９ C, GXCX ９－１６ C or GXCX ３－２２ C, optionally selected from the group consisting of SEQ ID NOs: 534, 538, 560, 565, 569, 573, 583, 598, 603, 616, 624, 628, 633, 645, 697, 702, 711, 715, 723, 728, 729, 730, 731, 732, 733, 734, 735, 736, or SEQ ID NOs: 534, 538, 560, 565, 569, 573, 583, 598, 603, 616, 624, 628, 633, 645, 697, 702, 711, 715, 723, 728, 729, 730, 731, 732, 733, 734, 735, 736 A recombinant polynucleotide encoding a peptide comprising a defensin or defensin-like peptide, including variants thereof having 0%, 85%, 90%, 95%, 98%, or 99% sequence identity, variants thereof comprising a deletion of 1 to 10 N-terminal amino acid residues, variants thereof comprising a deletion of 1 to 10 C-terminal amino acid residues, and / or variants thereof having conservative amino acid substitutions of 1 to 10 amino acid residues, wherein the polynucleotide encoding the peptide is operably linked to a polynucleotide comprising a promoter heterologous to the polynucleotide encoding the peptide.

30. 30. The recombinant polynucleotide of claim 28 or 29, wherein the recombinant polynucleotide further comprises a polynucleotide encoding (i) a transit peptide, a vacuolar targeting peptide, and / or an endoplasmic reticulum targeting peptide, (ii) a plastid targeting peptide, and / or (iii) 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.

31. 30. The recombinant polynucleotide of claim 28 or 29, wherein the polynucleotide encoding the peptide is inserted into the heterologous nuclear genome or plastid genome of a cell and is operably linked to an endogenous promoter located within the heterologous nuclear genome or plastid genome.

32. 10. A plant nuclear genome or plastid genome comprising a polynucleotide encoding a peptide comprising the peptide of claims 1 to 9, wherein the polynucleotide is heterologous to the nuclear genome or plastid genome, and the polynucleotide is operably linked to an endogenous promoter of the nuclear genome or plastid genome.

33. 30. A cell comprising a recombinant polynucleotide according to claim 28 or 29, wherein the cell is optionally a bacterial cell, a yeast cell, or a plant cell.

34. 30. A plant comprising the recombinant polynucleotide of claim 28 or 29.

35. 35. A plant part of the plant of claim 34, wherein the plant part comprises the recombinant polynucleotide, and optionally the plant part is a seed, stem, leaf, root, tuber, flower, vegetable, or fruit.

36. 35. A method for producing plant seeds that provide plants that are resistant to infection by plant pathogenic microorganisms, the method comprising: (i) selfing or crossing the plant of claim 34; and (ii) harvesting seeds from the selfing or crossing that comprise the recombinant polynucleotide of the plant, thereby producing plant seeds that provide plants that are resistant to infection by plant pathogenic microorganisms.

37. A method for producing an antifungal peptide, comprising: (i) culturing a cell described in claim 33 under conditions in which the peptide, defensin, or defensin-like peptide is expressed by the cell; and (ii) purifying the peptide, defensin peptide, or defensin-like peptide from the culture.

38. The method of claim 37, wherein the cell is a yeast cell, and optionally the recombinant polynucleotide comprises a polynucleotide encoding a transport peptide operably linked to the polynucleotide encoding the peptide, defensin peptide, or defensin-like peptide, and optionally the peptide, defensin peptide, or defensin-like peptide is purified from the culture supernatant.

39. 39. The method of claim 38, wherein the yeast cell is a Candida cell, a Kluyveromyces cell, a Hansella cell, a Pichia cell, a Saccharomyces cell, a Schizosaccharomyces cell, or a Yarrowia cell.

40. (i) a defensin peptide comprising the long gamma core consensus sequence GXCX16-22C, optionally SEQ ID NO: 578, 608, 612, or a variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 578, 608, or 612; a variant thereof comprising a deletion of 1 to 10 N-terminal amino acid residues; a deletion of 1 to 10 C-terminal amino acid residues; or (ii) a defensin or defensin-like peptide comprising the wild-type gamma core consensus peptide GXCX3-9C, GXCX9-16C or GXCX3-22C, optionally selected from the group consisting of SEQ ID NOs: 534, 538, 560, 565, 569, 573, 583, 598, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 03, 616, 624, 628, 633, 645, 697, 702, 711, 715, 723, 728, 729, 730, 731, 732, 733, 734, 735, 736, or SEQ ID NOs: 534, 538, 560, 565, 569, 573, 583, 598, 603, 616, 624, 628, 633, 645, 697, 702, 711, 715, 723, 728, 729, 730, 731, 732, 733, 734, 735, 7 36, its variants having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to 36, its variants comprising a deletion of 1 to 10 N-terminal amino acid residues, its variants comprising a deletion of 1 to 10 C-terminal amino acid residues, and / or its variants having conservative amino acid substitutions of 1 to 10 amino acid residues.

41. 41. A composition comprising the peptide of claim 40 and an agriculturally, pharmaceutically, or veterinarily acceptable carrier, diluent, or excipient.

42. 42. A method for preventing or reducing crop damage caused by plant pathogenic microorganisms, comprising contacting a plant, plant seed, or other part of said plant with an effective amount of the composition of claim 41.

43. 43. The method of claim 42, wherein the plant pathogenic microorganism is a fungus of the genus Fusarium, Alternaria, Verticillium, Phytophthora, Colletotrichum, Botrytis, Cercospora, Phakopsora, Rhizoctonia, Sclerotinia, Pythium, Phoma, Leptosphaeria, Gaeumannomyces, Puccinia, Septoria, Penicillium, Rasiudiplodia, Phomopsis, Mycosphaerella, Golobinomyces, Erysiphe, Albugo, Setosphaeria, Cochliobolus, Helminthosporium, Diplodia, Magnaporthe, Stenocarpella, or Zymoseptoria.

44. 42. A plant or plant part at least partially coated with the composition of claim 41.

45. 45. The plant or plant part of claim 44, wherein the plant part is a leaf, seed, or fruit.

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