Antimicrobial root nodule-specific cysteine-rich peptide folded variants

Abstract

Provided are compositions and methods employing antimicrobial root nodule specific cysteine-rich (NCR) peptides and variants thereof, including folded variants of NCR peptides (NCRPFV), including folded variants of antimicrobial NCR peptides and proteins (NCRPFV) that exhibit advantageous antifungal properties and desirable resistance or sensitivity to protease cleavage, the antimicrobial NCRPFV may be applied directly ex vivo to a plant, human or animal, or may be applied in vivo to a plant, human or animal.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 498,059, filed April 25, 2023, pursuant to 35 USC § 119. The entire contents of that provisional patent application are incorporated herein by reference, including but not limited to the description, claims, and abstract, and any figures, tables, appendices, or illustrations therein.

[0003] sequence list

[0004] This application contains a sequence list that has been electronically submitted in XML format, and is incorporated herein by reference in its entirety. The XML copy created on April 19, 2024, is named P13860WO01_SequenceListing.xml and has a size of 333,911 bytes. Technical Field

[0005] This disclosure generally relates to antimicrobial peptides and proteins for controlling pathogenic microorganisms. This document provides compositions and methods employing antimicrobial root nodule-specific cysteine-rich (NCR) peptides and proteins, including folded variants (NCR_PFVs) of antimicrobial NCR peptides and proteins exhibiting favorable antifungal properties and / or desired resistance or sensitivity to protease cleavage. These antimicrobial NCR_PFVs can be applied directly to plants, humans, or animals ex vivo, or can be administered to plants, humans, or animals in vivo. Background Technology

[0006] Both animals and plants are susceptible to microbial infections, which, if left untreated, can lead to significant morbidity and mortality in humans and veterinary patients, and cause losses in food crops, which are particularly vulnerable to contamination and spoilage during storage. Protecting agriculturally vital crops from pathogenic microorganisms is crucial for maximizing crop yields. Fungal infections are a particular problem in humid climates and can be a major issue during crop storage, during which such infections can lead to spoilage and contamination of food or forage products with mycotoxins. Unfortunately, modern planting methods, harvesting, and storage systems can promote plant pathogen infections.

[0007] Controlling pathogens in humans, veterinary animals, and plants is complicated by the need to simultaneously manage multiple genera of pathogenic microorganisms, including fungi, dimorphic fungi, yeasts, molds, and oomycetes. For example, plants are susceptible to infections by a wide variety of microorganisms, including *Alternaria*, *Ascochyta*, *Botrytis*, *Cercospora*, *Colletotrichum*, *Diplodia*, *Erysiphe*, *Fusarium*, *Gaeumanomyces*, *Helminthosporium*, *Macrophomina*, *Magnaporthe*, *Nectria*, *Peronospora*, *Phoma*, and *Phakopsora*. Species from the genera *Phymatotrichum*, *Phytophthora*, *Plasmopara*, *Podosphaera*, *Puccinia*, *Pythium*, *Pyrenophora*, *Pyricularia*, *Rhizoctonia*, *Sclerotium*, *Sclerotinia*, *Septoria*, *Thielaviopsis*, *Uncinula*, *Venturia*, and *Verticillium* are all recognized plant pathogens.

[0008] Therefore, in the presence of multiple pathogens, antimicrobial agents controlling a limited subset of microbial pathogens provide insufficient or ineffective protection. Furthermore, in many cases, plant pathogens have already developed resistance to existing antimicrobial agents.

[0009] Hancock, AAC 43(6) :1317 (1999) and Yourt, PNAS 101(19) 7363 (2004) reviewed antimicrobial peptides. Antimicrobial peptides include root nodule-specific cysteine-rich (NCR) peptides and defensin peptides.

[0010] Defensins are small peptides rich in cysteine, consisting of approximately 45-54 amino acids. These peptides constitute an important component of plant innate immunity (Sathoff, Phytopathology).109 :402 (2019)). PCT patent publication WO 2010 / 146,067 describes antimicrobial root nodule-specific cysteine-rich (NCR) peptides from Medicago truncatula (barrel alfalfa). Montiel et al., Molec. Plant Microb. Inter. 29:210-219 (2016) describe NCR peptides from chickpea (Cicer arietinum) and show that those NCR peptides are involved in the terminal differentiation of endosymbiotic bacteria. WO2020 / 146360 discloses the antimicrobial and antioomytic activities of certain NCR peptides. Some NCR peptides exhibit antimicrobial properties when applied to free-living bacteria and can mediate bacterial cell death and early root nodule senescence. Yang, PNAS 114 :6848-6853 (2017) and Wang, PNAS 114 :6854-6859 (2017). Antimicrobial NCRs (AMPs) are cationic and contain conserved cysteine ​​residues that form intramolecular disulfide bonds. Cysteine ​​substitution or disulfide bond modification can affect the antimicrobial activity of some NCR peptides that have only four conserved cysteine ​​residues. Haag, J. Biol. Chem. 287(14) :10791-8 (2012) and Isozumi, Nature Sci. Rep. 11: 9923 (2021).

[0011] Nodule-specific cysteine-rich (NCR) peptides are commonly expressed in the root nodules of leguminous plants, including chickpeas, alfalfa, *Galega orientalis*, *Medicago sativa*, *Astragalus canadensis*, peas (*Pisum sativum*), *Ononis spinosa*, *Onobrychis viciifolia*, and *Oxytropis lambertii*. These peptides mediate the differentiation of root nodule bacteria into nitrogen-fixing cells, thereby maintaining bacterial survival. (Wang, Mol. Plant-Microbe Int.) 31 (2) :240-8(2018),Van de Velde, Science 327 :1122-1126(2010), Kim, PNAS 112 :15238-15243 (2015), and Horvath, PNAS112 :15232-15237 (2015).

[0012] Despite recent advances in the control and treatment of microbial infections and the recognition of the significant antimicrobial properties and activities of certain antimicrobial peptides, significant unmet needs remain for effective ways to prevent, treat, and control pathogenic microorganisms. Summary of the Invention

[0013] This disclosure is based on the discovery that certain cationic antimicrobial rhizobium-specific cysteine-rich (NCR) peptides and proteins can form alternative disulfide pairs and thus fold into a variety of different conformations, each with unique secondary (e.g., α-helices and β-sheets) and tertiary structures, and that said NCR peptide and protein structural variants possess different antimicrobial activities and resistance to protease cleavage. As disclosed herein, NCR peptide fold variants exhibiting desired levels of antimicrobial activity and protease resistance can be purified from heterogeneous mixtures containing multiple (i.e., two or more) NCR peptide fold variants, and the purified NCR peptide fold variants can be advantageously used in the compositions and methods disclosed herein. Therefore, in various aspects, this disclosure provides rhizobium-specific cysteine-rich NCR peptide fold variant 1 (NCR_PFV1), compositions containing antimicrobial NCR_PFV1, methods for preparing antimicrobial NCR_PFV1 compositions, methods for using such antimicrobial NCR_PFV1 compositions, and devices containing antimicrobial NCR_PFV1, including medical devices.

[0014] In some embodiments, this document provides compositions comprising a cationic 6-Cys nodule-specific cysteine-rich (NCR) peptide fold variant 1 (NCR_PFV1), wherein the NCR peptide comprises, from its N-terminus, a sequential amino acid sequence comprising: (1) a first cysteine ​​pair motif comprising C1 and C2; (2) a first intercalation sequence (IS1); (3) a second cysteine ​​pair motif comprising C3 and C4; (4) a second intercalation sequence (IS2); and (5) a third cysteine ​​pair motif comprising C5 and C6, wherein the NCR peptide is associated with CaNCR7 (SEQ ID NO: 2), CaNCR13 (SEQ ID NO: 1), CaNCR14 (SEQ ID NO: 3), or CaNCR15 (SEQ ID NO: 4). 4) Having less than 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity, wherein the cysteine ​​residues C1, C2, C3, C4, C5, and C6 of NCR_PFV1 form a first group of disulfide bonds, and NCR_PFV1 thereby adopts a first conformation exhibiting greater antimicrobial activity, as compared to root nodule-specific cysteine-rich NCR peptide fold variant 2 (NCR_PFV2), wherein NCR_PFV2 has the same amino acid sequence as NCR_PFV1 but forms a different second group of disulfide bonds between cysteine ​​residues C1, C2, C3, C4, C5, and C6 and adopts a different second conformation, and wherein the composition is substantially free of NCR_PFV2, and optionally wherein at least about 80%, 90%, 95%, 98%, 99%, or 99.5% (by weight) of the total NCR peptide in the composition is NCR_PFV1.

[0015] In related embodiments, this document provides compositions comprising a cationic 4-Cys nodule-specific cysteine-rich (NCR) peptide fold variant 1 (NCR_PFV1), wherein the NCR peptide comprises, from its N-terminus, a sequential amino acid sequence comprising: (1) a first cysteine ​​pair motif comprising C1 and C2; (2) a first intercalation sequence (IS1); and (3) a second cysteine ​​pair motif comprising C3 and C4, wherein the NCR peptide is associated with CaNCR7 (SEQ ID NO: 2), CaNCR13 (SEQ ID NO: 1), CaNCR14 (SEQ ID NO: 3), or CaNCR15 (SEQ ID NO: 4). 4) Having less than 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity, wherein the cysteine ​​residues C1, C2, C3, and C4 of NCR_PFV1 form a first group of disulfide bonds, and NCR_PFV1 thereby adopts a first conformation exhibiting greater antimicrobial activity, as compared to root nodule-specific cysteine-rich NCR peptide fold variant 2 (NCR_PFV2), which has the same amino acid sequence as NCR_PFV1 but forms a different second group of disulfide bonds between cysteine ​​residues C1, C2, C3, and C4 and adopts a different second conformation, and wherein the composition is substantially free of NCR_PFV2, optionally wherein at least about 80%, 90%, 95%, 98%, 99%, or 99.5% (by weight) of the total NCR peptide in the composition is NCR_PFV1.

[0016] In other embodiments, this document provides a method for preparing a composition comprising a cationic antimicrobial 6-Cys or 4-Cys root nodule-specific cysteine-rich (NCR) peptide fold variant 1 (NCR_PFV1), the method comprising: (a) separating a fraction comprising NCR peptide fold variant 1 (NCR_PFV1) from a mixture comprising NCR_PFV1 and NCR_PFV2 or NCR_PFV2 peptide fragments thereof, and one or more fractions comprising NCR peptide fold variant 2 (NCR_PFV2) or NCR_PFV2 peptide fragments thereof; and (b) combining the fraction comprising NCR_PFV1, or a formulation further purified from the NCR_PFV1 fraction, with an agriculturally, pharmaceutically, or veterinarily feasible carrier, diluent, or excipient, wherein the NCR peptide is combined with CaNCR7 (SEQ ID NO: 2), CaNCR13 (SEQ ID NO: 1), CaNCR14 (SEQ ID NO: 3), or CaNCR15 (SEQ ID NO: 2). The composition is prepared by having sequence identity of less than 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% for NO:4.

[0017] In a further embodiment, this document provides a method for preventing or reducing crop damage or post-harvest loss caused by plant pathogenic microorganisms, the method comprising: contacting a plant, plant seed, pre- or post-harvest cereal, pre- or post-harvest melon or fruit, or pre- or post-harvest vegetable with an effective amount of a composition comprising a cationic antimicrobial 6-Cys or 4-Cys root nodule-specific cysteine-rich (NCR) peptide fold variant 1 (NCR_PFV1) under conditions suitable for preventing or reducing crop damage or post-harvest loss, wherein the NCR peptide has a sequence identity of less than 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% with CaNCR7 (SEQ ID NO: 2), CaNCR13 (SEQ ID NO: 1), CaNCR14 (SEQ ID NO: 3), or CaNCR15 (SEQ ID NO: 4).

[0018] In some other embodiments, this document provides a medical device operably combined with a composition comprising a cationic antimicrobial 6-Cys or 4-Cys root nodule-specific cysteine-rich (NCR) peptide fold variant 1 (NCR_PFV1), wherein the medical device comprises at least one surface that is locally coated or impregnated with the composition, and wherein the NCR peptide has a sequence identity of less than 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% with CaNCR7 (SEQ ID NO: 2), CaNCR13 (SEQ ID NO: 1), CaNCR14 (SEQ ID NO: 3), or CaNCR15 (SEQ ID NO: 4).

[0019] In a further embodiment, this document provides a method for treating, preventing, or inhibiting microbial infections in a subject (including plant, human, or non-human animal) in need of such treatment, prevention, or inhibition, the method comprising administering (in vitro or in vivo) to the subject, under conditions and for a duration suitable for treating, preventing, or inhibiting the microbial infection, a therapeutically effective amount of a composition comprising a cationic antimicrobial 6-Cys or 4-Cys root nodule-specific cysteine-rich (NCR) peptide fold variant 1 (NCR_PFV1), wherein the NCR peptide has a sequence identity of less than 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% with CaNCR7 (SEQ ID NO: 2), CaNCR13 (SEQ ID NO: 1), CaNCR14 (SEQ ID NO: 3), or CaNCR15 (SEQ ID NO: 4).

[0020] In related embodiments, this document provides a composition comprising a variant 1 (NCR_PFV1) of a cationic antimicrobial 6-Cys or 4-Cys root nodule-specific cysteine-rich (NCR) peptide fold for use in treating, preventing, or inhibiting microbial infections in a subject with such need, wherein the NCR peptide has a sequence identity of less than 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% with CaNCR7 (SEQ ID NO: 2), CaNCR13 (SEQ ID NO: 1), CaNCR14 (SEQ ID NO: 3), or CaNCR15 (SEQ ID NO: 4).

[0021] In other related embodiments, this document provides plant portions at least partially coated with a composition comprising a cationic antimicrobial 6-Cys or 4-Cys root nodule-specific cysteine-rich (NCR) peptide fold variant 1 (NCR_PFV1), wherein the NCR peptide has a sequence identity of less than 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% with CaNCR7 (SEQ ID NO: 2), CaNCR13 (SEQ ID NO: 1), CaNCR14 (SEQ ID NO: 3), or CaNCR15 (SEQ ID NO: 4).

[0022] These and other aspects of this disclosure will be better understood in conjunction with the following accompanying drawings and detailed description, which illustrate certain aspects of various embodiments. Detailed Implementation

[0023] In some embodiments, this disclosure provides compositions enriched with cationic and antimicrobial 6-Cys and 4-Cys root nodule-specific cysteine-rich (NCR) peptide folded variants (NCR_PFVs) having a defined set of disulfide pairs, employing a unique conformation with a distinctive tertiary structure, exhibiting improved antimicrobial activity, and optionally demonstrating desired resistance or sensitivity to protease cleavage compared to other NCR_PFVs with different disulfide pairs. In some embodiments, this disclosure provides cationic and antimicrobial 6-Cys and 4-Cys root nodule-specific cysteine-rich (NCR) peptide folded variants (NCR_PFVs) having a defined set of disulfide pairs, exhibiting desired resistance or sensitivity to protease cleavage compared to other NCR_PFVs with different disulfide pairs. Cationic and antimicrobial 6-Cys and 4-Cys NCR_PFVs can be purified from heterogeneous mixtures containing multiple (i.e., two or more) NCR folding variants, and the desired purified NCR_PFVs can be advantageously used in the compositions, methods, and apparatus disclosed herein. The antimicrobial 6-Cys and 4-Cys root nodule-specific cysteine-rich (NCR) peptides described above and below as objects of this disclosure are peptides having sequence identity of less than 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% with CaNCR7 (SEQ ID NO: 2), CaNCR13 (SEQ ID NO: 1), CaNCR14 (SEQ ID NO: 3), or CaNCR15 (SEQ ID NO: 4).

[0024] These and other aspects of this disclosure can be better understood by referring to the following non-limiting definitions.

[0025] definition

[0026] As used in this article, the terms “microorganism,” “multi-microorganism,” and “microbial” refer to bacteria, fungi (including yeasts), and oomycetes.

[0027] As used herein, the phrases “susceptible microorganism (or multiple microorganisms)”, “susceptible microorganism infection”, etc., refer to microorganisms that infect plants, humans and non-human animals, or microbial infections thereof, whose growth is inhibited by antimicrobial peptides, including root nodule-specific cysteine-rich (NCR) and defensin peptides, their variants and their polymers.

[0028] As used herein, the phrases “combating microbial damage,” “combating or controlling microbial damage,” or “controlling microbial damage” refer to reducing damage to plants (e.g., ornamental or crop plants) or to ornamental 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 crop plants. Adverse effects of microbial growth are understood to include any type of plant tissue damage or necrosis, any type of reduced plant yield, any reduction in the value of crop plant products, and / or the generation of undesirable microbial metabolites or microbial growth byproducts (including mycotoxins).

[0029] As used herein, the phrases “inhibition of the growth of plant pathogenic microorganisms”, “inhibition of microbial growth”, etc., refer to any method that results in a measurable reduction in microbial growth, including but not limited to any measurable reduction in the number and / or extent of microbial cells, spores, conidia, or mycelia. As used herein, “inhibition of the growth of plant pathogenic microorganisms” should also be understood to include any measurable reduction in the adverse effects of microbial growth in plants. Adverse effects of microbial growth in plants include any type of plant tissue damage or necrosis, any type of reduced plant yield, any reduction in the value of crop plant products, and / or the production of undesirable microbial metabolites or microbial growth byproducts (including but not limited to mycotoxins). As used herein, the phrases “inhibition of microbial growth”, etc., unless otherwise stated, may include inhibition in plants, humans, or animals.

[0030] As used herein, the phrase “antimicrobial peptide” and the term “antimicrobial peptide” as used herein refer to peptides, particularly nodule-specific cysteine-rich (NCR) peptides, which exhibit one or more of the following characteristics: inhibiting the growth of microbial cells, killing microbial cells, disrupting or delaying stages of the microbial life cycle (e.g., spore germination, spore formation, or mating), and / or disrupting microbial cell infection, penetration, or spread within plants or other susceptible subjects (including humans, livestock, poultry, fish, or companion animals (e.g., dogs or cats)).

[0031] As used herein, the phrase “cation-tolerant” refers to an NCR peptide or its variant that exhibits equivalent in vitro antifungal or antimicrobial activity in the presence of 100 mM KCl or 100 mM NaCl, or an in vitro reduction in antifungal or antimicrobial activity of no more than about 1.5, 2, 3, or 4 times in the absence of KCl or NaCl.

[0032] As used herein, the phrase “common sequence” refers to an amino acid sequence derived by comparing two or more homologous sequences and deriving a new sequence that has a conserved set of alternative amino acid residues of the homologous sequence at each position in the peptide sequence.

[0033] As used herein, the term "peptide variant" refers to any peptide with antimicrobial activity that comprises one or more nonconserved amino acid substitutions in a root nodule-specific cysteine-rich (NCR) peptide. In addition to such nonconserved substitutions, NCR peptide variants may also comprise peptides having conserved amino acid substitutions, deletions of 1 to 5 amino acids at the N-terminus, and deletions within one or more amino acid residues as described herein.

[0034] As used herein, “percentage identity” or “sequence identity” refers to the number of identical elements (i.e., amino acids or nucleotides) within a defined length of two protein fragments that produce the maximum number of identical elements in the alignment, and is calculated by dividing the number of identical elements by the total number of elements within the defined length of the aligned fragments and multiplying by 100.

[0035] As used herein, the term "endopeptidase" refers to a peptidase that cleaves the peptide bond between two internal amino acid residues in a peptide sequence. Endopeptidases may also be called "endopeptidases" or "endopeptidases." Therefore, the proteolytic activity of endopeptidases, endoproteases, or endopeptidases differs from that of "exopeptidases," which cleave the peptide bond between the terminal amino acid residues of a peptide.

[0036] As used herein, the terms "heteropeptide" or "heteropeptide" refer to peptides that are not found in cells or organisms in nature, such as nodule-specific cysteine-rich (NCR) peptides. Therefore, heteropeptides include peptides located in subcellular or extracellular locations, peptides expressed in tissues other than subcellular or extracellular locations, or peptides or proteins found in naturally occurring cells or organisms that are expressed in tissues.

[0037] As used herein, the term "amino acid" refers to an organic compound containing an amino (−NH3) and a carboxyl (−CO2) functional group, as well as the side chain (R group) characteristic of each amino acid. In some cases, single-letter amino acid codes are used in this paper to represent peptides, as follows: 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).

[0038] As used herein, the terms “acidic” or “anionic” are used interchangeably and refer to amino acids such as aspartic acid and glutamic acid.

[0039] As used herein, the terms “basic” or “cationic” are used interchangeably to refer to amino acids such as arginine, histidine, and lysine.

[0040] As used herein, “percentage identity” or “sequence identity” refers to the number of identical amino acids within a defined length of two peptide or protein fragments that produce the maximum number of identical elements in the alignment, and is calculated by dividing the number of identical elements by the total number of elements within the defined length of the aligned fragment and multiplying by 100.

[0041] It should be understood that the foregoing definitions will be used in this document when they are inconsistent with those provided in any patent or non-patent reference incorporated herein by reference, any patent or non-patent reference listed herein, or any patent or non-patent reference found elsewhere.

[0042] Unless otherwise expressly stated, the practice of this disclosure will employ conventional methods and techniques commonly used in the fields of microbiology, molecular biology, structural biology, and enzymology, which are well known and readily available to those skilled in the art. Such methods and techniques are described in detail in laboratory manuals and in scientific and patent literature. See, for example, Sambrook et al., “Molecular Cloning: A Laboratory Manual” (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989); Maniatis et al., “Molecular Cloning: A Laboratory Manual” (1982); “DNA Cloning: A Practical Approach, Volumes I and II” (edited by Glover); “Oligonucleotide Synthesis” (edited by Gait, 1984); Ausubel et al. (edited), “Current Protocols in Molecular Biology” (John Wiley & Sons, 1994); “Nucleic Acid Hybridization” (edited by Hames and Higgins, 1985); “Transcription and Translation” (edited by Hames and Higgins, 1984); “Animal Cell Culture” (edited by Freshney, 1986); and Perbal, “A Practical Guide To Molecular Cloning”. (1984). All publications, patents and patent applications cited in this article, whether above or below, are hereby incorporated in their entirety by reference.

[0043] Antimicrobial root nodule-specific cysteine-rich peptide fold variant (NCR_PFV) composition

[0044] In some embodiments, this disclosure provides compositions comprising cationic antimicrobial rhizobium-specific cysteine-rich (NCR) peptide fold variants. The compositions according to these embodiments (1) comprise NCR peptide fold variant 1 (NCR_PFV1) having desired antimicrobial activity and / or resistance / sensitivity to proteolytic degradation, and (2) are substantially free of NCR peptide fold variant 2 (NCR_PFV2) having reduced antimicrobial activity and / or resistance / sensitivity to proteolytic degradation.

[0045] Table 1 lists exemplary cationic antimicrobial 6-Cys root nodule-specific cysteine-rich peptides that can be advantageously used in the compositions disclosed herein, including NCR peptides from plants such as chickpea, alfalfa, goat's bean, alfalfa, astragalus, pea, red clover, donkey bean, and blueberry. The full-length sequences of these NCR peptides are also provided in the sequence listing. Variations of such NCR peptides comprising amino acid additions (e.g., addition of one or more amino acid residues at the N-terminus or C-terminus), insertions, deletions, or substitutions may also be used in the compositions provided herein.

[0046]

[0047] In certain aspects of these compositions, the cationic antimicrobial root nodule-specific cysteine-rich (NCR) peptide is a 6-Cys NCR peptide having (1) a first cysteine ​​pair motif comprising C1 and C2; (2) a first intercalation sequence (IS1); (3) a second cysteine ​​pair motif comprising C3 and C4; (4) a second intercalation sequence (IS2); and (5) a third cysteine ​​pair motif comprising C5 and C6. Typically, the cationic 6-Cys NCR according to these embodiments has an isoelectric point between 8.0 and 12.0 and / or NCR_PFV1 exhibits greater protease resistance than NCR_PFV2. The antimicrobial 6-Cys root nodule-specific cysteine-rich (NCR) peptide described above and below for the purposes of this disclosure is a peptide having less than 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity with CaNCR7 (SEQ ID NO: 2), CaNCR13 (SEQ ID NO: 1), CaNCR14 (SEQ ID NO: 3), or CaNCR15 (SEQ ID NO: 4).

[0048] Based on these aspects, the cysteine ​​residues C1, C2, C3, C4, C5, and C6 of 6-Cys NCR peptide fold variant 1 (NCR_PFV1) form a first set of disulfide bonds, thereby adopting a first conformation exhibiting greater antimicrobial activity and preferred resistance / sensitivity to protease digestion, as compared to NCR peptide fold variant 2 (NCR_PFV2), which has the same amino acid sequence as NCR_PFV1 but forms a different second set of disulfide bonds between cysteine ​​residues C1, C2, C3, C4, C5, and C6 and adopts a different second conformation, which has undesirable reduced antimicrobial activity and / or undesirable resistance / sensitivity to protease digestion.

[0049] This article illustrates 6-Cys NCR peptide folding variant 1 (NCR_PFV1), which forms a first set of disulfide bonds including C1-C4, C2-C5, and C3-C6 disulfide bonds, while 6-Cys NCR peptide folding variant 2 (NCR_PFV1) forms a second set of disulfide bonds in addition to C1-C4 and C2-C5 disulfide bonds, for example, a second set of disulfide bonds including C1-C2, C3-C6, and C4-C5 disulfide bonds.

[0050] In some aspects of these embodiments, the 6-Cys NCR may include a first cysteine ​​pair motif, which is a C1-5AA-C2 motif having five amino acids (A1, A2, A3, A4, and A5) intercalated from its N-terminus with N-terminal C1 and C-terminal C2, wherein A5 may be: an anionic amino acid selected from Asp (D) and Glu (E); or a cationic amino acid selected from His (H), Arg (R), and Lys (K); or an aliphatic amino acid selected from Gly (G), Ala (A), Val (V), Leu (L), Ile (I), and Pro (P); wherein A4 may be: a cationic amino acid selected from His (H), Arg (R), and Lys (K); or an aliphatic amino acid selected from Gly (G), Ala (A), Val (V), Leu (L), Ile (I), and Pro (P); or aliphatic amino acid selected from Asn (N) and Gln. (Q) is an amide amino acid; or a polar amino acid selected from Ser (S) and Thr (T); and wherein A3 can be: a cationic amino acid selected from His (H), Arg (R) and Lys (K); or an anionic amino acid selected from Asp (D) and Glu (E); or a polar amino acid selected from Ser (S) and Thr (T).

[0051] In other aspects of these embodiments, the 6-Cys NCR may include a second cysteine ​​pair motif, which is a C3-5-10AA-C4 motif comprising, from its N-terminus, five to ten amino acids (A6, A7, A8, A9, A10, A20, A30, A40, A50, A60, A70, A80, A90, A1 ... 10 To A 11 A 12 A 13 A 14 A 15 This example is a 6-Cys NCR, in which the C3-5-10AA-C4 motif contains, in a continuous sequence from its N-terminus, seven amino acids (A6, A7, A8, A9, A10AA-C4) flanked by an N-terminal C3 and a C-terminal C4. 10 A 11 and A 12 A6 is an aliphatic amino acid selected from Gly (G), Ala (A), Val (V), Leu (L), Ile (I), and Pro (P); or a cationic amino acid selected from His (H), Arg (R), and Lys (K). 12 It is: anionic amino acids selected from Asp (D) and Glu (E); or cationic amino acids selected from His (H), Arg (R) and Lys (K); or amide amino acids selected from Asn (N) and Gln (Q).

[0052] In a further aspect of these embodiments, the 6-Cys NCR may include a C5-1AA-C6 motif comprising an amino acid (A) side-terminated with an N-terminal C5 and a C-terminal C6. 16 ), where A 16 It is: cationic amino acids selected from His (H), Arg (R) and Lys (K); or aliphatic amino acids selected from Gly (G), Ala (A), Val (V), Leu (L), Ile (I) and Pro (P); or anionic amino acids selected from the group consisting of Asp (D) and Glu (E).

[0053] In related aspects of these embodiments, the 6-Cys NCR may include a first intercalation sequence (IS1) having one to eight amino acids or four to five amino acids and a second intercalation sequence (IS2) having four to six amino acids.

[0054] Representative 6-Cys NCRs according to these embodiments may contain amino acid sequences that have at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% sequence identity with any NCR presented in Table 1 or the corresponding NCR in the sequence listing.

[0055] Depending on the intended precise application, the compositions according to these embodiments may contain agriculturally, pharmaceutically, or veterinarily viable carriers, diluents, or excipients.

[0056] Table 2 lists exemplary cationic antimicrobial 4-Cys root nodule-specific cysteine-rich peptides that can be advantageously used in the compositions disclosed herein, including NCR peptides from plants such as alfalfa, goat's bean, alfalfa, astragalus, pea, and red clover. The full-length sequences of these NCR peptides are also provided in the sequence listing. Variations of such NCR peptides, including amino acid additions (e.g., addition of one or more amino acids at the N-terminus or C-terminus), insertions, deletions, or substitutions, may also be used in the compositions provided herein.

[0057]

[0058] In certain aspects of these compositions, the cationic antimicrobial rhizobium-specific cysteine-rich (NCR) peptide is a 4-Cys NCR peptide comprising (1) a first cysteine ​​pair motif including C1 and C2, (2) a first intercalation sequence (IS1), and (3) a second cysteine ​​pair motif including C3 and C4. Typically, the cationic 4-Cys NCR according to these embodiments has an isoelectric point between 8.0 and 12.0 and / or NCR_PFV1 exhibits greater protease resistance than NCR_PFV2. The antimicrobial 4-Cys rhizobium-specific cysteine-rich (NCR) peptides described above and below as objects of this disclosure are peptides having less than 60% sequence identity with CaNCR7 (SEQ ID NO: 2), CaNCR13 (SEQ ID NO: 1), CaNCR14 (SEQ ID NO: 3), or CaNCR15 (SEQ ID NO: 4).

[0059] According to these embodiments, cysteine ​​residues C1, C2, C3, and C4 of 4-Cys NCR peptide fold variant 1 (NCR_PFV1) form a first set of disulfide bonds, thereby adopting a first conformation. Compared with NCR peptide fold variant 2 (NCR_PFV2), which has the same amino acid sequence as NCR_PFV1 but forms a different second set of disulfide bonds between cysteine ​​residues C1, C2, C3, and C4 and adopts a different second conformation, it exhibits greater antimicrobial activity and preferred resistance / sensitivity to protease digestion. The second conformation has reduced antimicrobial activity and / or undesirable resistance or undesirable sensitivity to protease digestion.

[0060] This article illustrates 4-CysNCR peptide folding variant 1 (NCR_PFV1), wherein the first set of disulfide bonds formed by NCR_PFV1 includes C1-C2 and C3-C4 disulfide bonds, and wherein 4-Cys peptide folding variant 2 (NCR_PFV2) forms a second set of disulfide bonds in addition to C1-C2 and C3-C4 disulfide bonds, for example, a second set of disulfide bonds including C1-C3 and C2-C4 disulfide bonds or a second set of disulfide bonds including C1-C4 and C2-C3 disulfide bonds.

[0061] In some aspects of these embodiments, the 4-Cys NCR may include a first cysteine ​​pair motif, which is a C1-5AA-C2 motif having five amino acids (A1, A2, A3, A4, and A5) in a continuous sequence starting from its N-terminus, flanked by an N-terminal C1 and a C-terminal C2, wherein A5 may be an anionic amino acid selected from Asp (D) and Glu (E); wherein A4 may be a cationic amino acid selected from the group consisting of His (H), Arg (R), and Lys (K); and / or wherein A3 may be an anionic amino acid selected from the group consisting of Asp (D) and Glu (E).

[0062] In other aspects of these embodiments, the 4-Cys NCR may include a second cysteine ​​pair motif, which is a C3-4AA-C4 motif comprising, in a continuous sequence from its N-terminus, four amino acids (A6, A7, A8, and A9) flanked by an N-terminal C3 and a C-terminal C4, wherein A6 is: a cationic amino acid selected from His (H), Arg (R), and Lys (K); or an aliphatic amino acid selected from Gly (G), Ala (A), Val (V), Leu (L), Ile (I), and Pro (P); wherein A7 is: a cationic amino acid selected from His (H), Arg (R), and Lys (K); or an amide amino acid selected from Asn (N) and Gln (Q); or an aliphatic amino acid selected from Gly (G), Ala (A), Val (V), Leu (L), Ile (I), and Pro (P); and / or wherein A8 is: a cationic amino acid selected from Gly (G), Ala (A), Val (V), Leu (L), Ile (I), and Pro (P); and / or wherein A8 is: a cationic amino acid selected from Gly (G), Ala (A), Val (Q), and Leu (L), Ile (I), and Pro (P); Aliphatic amino acids selected from (V), Leu (L), Ile (I) and Pro (P); or amide amino acids selected from Asn (N) and Gln (Q); or cationic amino acids selected from His (H), Arg (R) and Lys (K).

[0063] In related aspects of these embodiments, the 4-Cys NCR may include a first intercalation sequence (IS1) having five to thirteen amino acids or five to eleven amino acids.

[0064] Representative 4-Cys NCRs according to these embodiments may contain an amino acid sequence having at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% sequence identity with any NCR presented in Table 2 (in some embodiments, other than alfalfa NCR169 (SEQ ID NO: 6) or NCR247 (SEQ ID NO: 5)) or the corresponding 4-Cys NCR peptide presented in the sequence listing.

[0065] Depending on the intended precise application, the compositions according to these embodiments may contain agriculturally, pharmaceutically, or veterinarily viable carriers, diluents, or excipients.

[0066] 6-Cys and 4-Cys NCR_PFV1 can be isolated from a mixture containing desired NCR_PFV1 and one or more undesirable NCR_PFV2 to provide a composition enriched with the desired NCR_PFV1. Compositions containing the desired NCR_PFV1 generally exhibit improved unit activity (e.g., units of antimicrobial activity per microgram or micromolar of NCR protein) relative to compositions containing the desired NCR_PFV1 and one or more NCR_PFV2 with reduced activity. Depending on the precise application of the 6-Cys and 4-Cys NCR_PFV1 compositions, it is desirable to employ NCR_PFV1 with increased protease resistance or increased protease sensitivity (e.g., to serine proteases, including trypsin) compared to other undesirable NCR_PFV2.

[0067] Compositions containing desired 6-Cys or 4-Cys NCR_PFV1 may be substantially free of undesirable NCR_PFV2. In some embodiments, such compositions may contain less than about 20%, 10%, 5%, 2%, 1%, or 0.5% by weight of undesirable 6-Cys or 4-Cys NCR_PFV2, and respectively, contain at least about 80%, 90%, 95%, 98%, 99%, or 99.5% by weight of desired 6-Cys or 4-Cys NCR_PFV1. In some embodiments, compositions containing desired NCR_PFV1 and substantially free of undesirable NCR_PFV2 may contain less than about 20%, 10%, 5%, 2%, 1%, or 0.5% by weight of a combined amount of desired NCR_PFV1 and undesirable NCR_PFV2 in the composition as undesirable NCR_PFV2. In some embodiments, a composition comprising desired NCR_PFV1 and substantially free of undesirable NCR_PFV2 may contain at least about 80%, 90%, 95%, 98%, 99%, or 99.5% by weight of total NCR peptides (e.g., total NCR peptides in the form of desired NCR_PFV1 and one or more undesirable NCR_PFV1 and one or more undesirable NCR_PFV2) as desired NCR_PFV1. In some embodiments, less than 20%, 10%, 5%, 2%, 1%, or 0.5% by weight of total NCR peptides (e.g., total NCR peptides in the form of desired NCR_PFV1 and one or more undesirable NCR_PFV2) in the composition is undesirable NCR PFV2, and respectively, at least about 80%, 90%, 95%, 98%, 99%, or 99.5% by weight of total NCR peptides in the composition is desired NCR PFV1.

[0068] It is conceivable that compositions containing or enriched with desired 6-Cys or 4-Cys NCR_PFV1, which exhibits increased protease resistance relative to undesirable 6-Cys or 4-Cys NCR_PFV2, may be useful in certain methods disclosed herein for the desired extension of the shelf life or half-life of NCR_PFV1. Alternatively, it is conceivable that compositions containing or enriched with desired 6-Cys or 4-Cys NCR_PFV1, which exhibits increased protease sensitivity relative to undesirable 6-Cys or 4-Cys NCR_PFV2, may be useful in such methods for the desired shortening of the half-life of NCR_PFV1.

[0069] Suitable 6-Cys or 4-Cys NCR peptides for generating the NCR_PFV1 peptide may include 6-Cys NCR peptides comprising the amino acid sequences listed in Table 1 or 4-Cys NCR peptides comprising the amino acid sequences listed in Table 2. Variations of these 6-Cys and 4-Cys NCR peptides are contemplated, wherein, for example, one or more hydrophobic, basic, and / or acidic amino acid residues are substituted with alternative hydrophobic, basic, and / or acidic amino acid residues. In the embodiments provided herein, the 6-Cys and 4-Cys NCR peptide variants comprise an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity over the entire length of one or more NCR peptides presented in Tables 1 and 2 and / or listed as SEQ ID NO: 8 to 371, but having less than 60% sequence identity with CaNCR7 (SEQ ID NO: 2), CaNCR13 (SEQ ID NO: 1), CaNCR14 (SEQ ID NO: 3), or CaNCR15 (SEQ ID NO: 4). Suitable 6-Cys or 4-Cys NCR peptides used in the compositions and methods provided herein do not include any 6-Cys NCR peptides or variants thereof that have more than 60% sequence identity with CaNCR7 (SEQ ID NO: 2), CaNCR13 (SEQ ID NO: 1), CaNCR14 (SEQ ID NO: 3), or CaNCR15 (SEQ ID NO: 4), which are disclosed in U.S. Patent Publication No. US 2022 / 0061333, which is incorporated herein by reference in its entirety.

[0070] The conserved cysteine ​​residues in the 6-Cys NCR peptide can be identified as C1, C2, C3, C4, C5, and C6 as shown in Table 1, and the conserved cysteine ​​residues in the 4-Cys NCR peptide can be identified as C1, C2, C3, and C4 as shown in Table 2. For the 6-Cys NCR peptide, the conserved cysteine ​​residue closest to the N-terminus is C1, and the conserved cysteine ​​residue closest to the C-terminus is C6, as shown in Table 1. For the 4-Cys NCR peptide, the conserved cysteine ​​residue closest to the N-terminus is C1, and the conserved cysteine ​​residue closest to the C-terminus is C4, as shown in Table 2.

[0071] In some aspects of these embodiments, 6-Cys or 4-Cys NCR peptides that may employ alternative disulfide pairs and constitute NCR peptide fold variants may have a net positive charge at neutral pH. For example, some NCR peptides have a net positive charge of at least +4, +5, +6, +7, +8, +9, or +10 to +12, +13, +14, or +15 at neutral pH, or alternatively, at least +5, +6, +7, +8, +9, or +10. It should be understood that such a net positive charge in 6-Cys or 4-Cys NCR peptides may be achieved by methods including: (i) maintaining or replacing the cationic (basic) amino acid residues found in the NCR peptides (including the NCR peptides presented in Tables 1 and 2, respectively) with another cationic amino acid residue; (ii) replacing the anionic or polar amino acid residues found in the NCR peptides (including the NCR peptides presented in Tables 1 and 2, respectively) with basic amino acid residues; or a combination of (i) and (ii). Such a net positive charge in 6-Cys or 4-Cys NCR peptides can be achieved by preferentially selecting or substituting cationic amino acid residues at variable positions corresponding to the variable positions of the NCR peptides.

[0072] In other aspects of these embodiments, 6-Cys or 4-Cys NCR peptides that use alternative disulfide pairs and constitute NCR peptide fold variants may include a significant percentage of hydrophobic amino acid residues. For example, 6-Cys or 4-Cys NCR peptides may contain at least about 25%, 26%, 28%, 30%, 32%, 34%, or 36% to 37%, 38%, 40%, 42%, or 45% hydrophobic amino acid residues, or at least about 25%, 26%, 28%, 30%, 32%, 34%, 36%, 37%, or 38% hydrophobic amino acid residues. It should be understood that such a percentage of hydrophobic amino acids in a 6-Cys or 4-Cys NCR peptide can be achieved by methods including: (i) maintaining the hydrophobic amino acid residues found in the 6-Cys or 4-Cys NCR peptide (including the NCR peptides presented in Tables 1 and 2, respectively) or replacing such residues with another hydrophobic amino acid residue or a neutral polar amino acid residue; (ii) replacing the polar amino acid residues found in the 6-Cys or 4-Cys NCR peptide (including the NCR peptides presented in Tables 1 and 2, respectively) with hydrophobic amino acid residues; (iii) replacing neutral polar amino acids with hydrophobic amino acids; or a combination of (i), (ii), and (iii). Such a percentage of hydrophobic amino acids in a 6-Cys or 4-Cys NCR peptide can be achieved by preferentially selecting or replacing hydrophobic amino acid residues at variable positions corresponding to variable positions in the 6-Cys or 4-Cys NCR peptide.

[0073] In a further aspect of these embodiments, the 6-Cys or 4-Cys NCR peptide comprises 4, 5, or 6 conserved C1, C2, C3, C4, C5, and C6 cysteine ​​residues listed in Tables 1 and 2, respectively. The 6-Cys NCR_PFV may contain disulfide bonds corresponding to the C1-C4, C2-C5, and C3-C6 disulfide bonds in the 6-Cys NCR peptide presented in Table 1, while the 4-Cys NCR_PFV may contain disulfide bonds corresponding to the C1-C2 and C3-C4 disulfide bonds in the 4-Cys NCR peptide presented in Table 2.

[0074] The 6-Cys NCR peptide may also contain four of the six conserved cysteine ​​residues: C1, C2, C3, C4, C5, and C6. For example, one or two cysteyl residues in the 6-Cys NCR peptide may be substituted or deleted by different amino acid residues to provide an NCR peptide having only four of the six conserved cysteine ​​residues: C1, C2, C3, C4, C5, and C6. Alternatively, one or two cysteine ​​residues in the 6-Cys NCR peptide may be substituted by another amino acid residue, including glycine, serine, threonine, cysteine, cystine, tyrosine, asparagine, or glutamine residues, to provide an NCR peptide having only four of the six conserved cysteine ​​residues: C1, C2, C3, C4, C5, and C6. For example, it is desirable to replace one or two conserved cysteine ​​residues with serine residues to provide an NCR peptide having only four of the six conserved C1, C2, C3, C4, C5, and C6 cysteine ​​residues. Such an NCR peptide having only four of the six conserved cysteine ​​residues may include deletions or substitutions of C1 and C6 cysteine ​​residues, and thus will contain C2, C3, C4, and C5 cysteine ​​residues. Alternatively, an NCR peptide having only four of the six conserved cysteine ​​residues may include deletions or substitutions of C2 or C6 cysteine ​​residues in an NCR peptide containing only C2, C3, C4, C5, and C6 cysteine ​​residues, and thus will contain either C3, C4, C5, and C6 cysteine ​​residues or C2, C3, C4, and C5 cysteine ​​residues. In some embodiments, the conserved cysteine ​​residues corresponding to C1 and C4 cysteine ​​residues are substituted with another amino acid residue (e.g., serine) to provide an NCR peptide having only 4 of the 6 conserved C1, C2, C3, C4, C5, and C6 cysteine ​​residues. In some embodiments, the conserved cysteine ​​residues corresponding to C2 and C5 cysteine ​​residues are substituted with another amino acid residue (e.g., serine) to provide an NCR peptide having only 4 of the 6 conserved C1, C2, C3, C4, C5, and C6 cysteine ​​residues.

[0075] In a further aspect of these embodiments, the 6-Cys or 4-Cys NCR peptide comprises a common core sequence GX1CKCVX2R or a variant thereof, said variant comprising the insertion, deletion, and / or substitution of one or more amino acid residues. For example, X1 may be F or Y, X2 may be absent or V, or X1 may be F, Y, W, I, V, A, or M and / or X2 may be absent or F, Y, W, I, V, A, or M. Alternatively, the NCR peptide may also comprise a variant of the common core GX1CKCVX2R, wherein one or both cationic residues (K and / or R) may be substituted by another cationic amino acid residue (K, R, or H). Such NCR peptides may also comprise a variant of the common core GX1CKCVX2R, wherein one or both conserved tyrosine or valine amino acid residues may be substituted by a hydrophobic amino acid (F, W, I, A, or M) or a tyrosine residue (Y).

[0076] Suitable NCR peptides for preparing compositions according to these embodiments may comprise an amino acid sequence having at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with one or more 6-Cys NCR peptide sequences presented in Table 1 or one or more 4-Cys NCR peptide sequences presented in Table 2, wherein one or more hydrophobic, basic, and / or acidic amino acid residues are respectively substituted by other hydrophobic, basic, and / or acidic amino acid residues.

[0077] In any 6-Cys or 4-Cys NCR peptide presented in Tables 1 and 2, or in variant 6-Cys or 4-Cys NCR peptides, one or more amino acids may be substituted with other amino acids whose charge and polarity are similar to the original amino acid, i.e., conserved amino acid substitutions. The substitutions for amino acids within the 6-Cys or 4-Cys NCR peptide sequence may be selected from other members of the class to which the original amino acid belongs. Amino acids can be classified 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 different groups include, but are not limited to: (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. Conserved amino acid alterations within NCR peptide sequences can be achieved by replacing one amino acid in one of these groups with another amino acid from the same group.

[0078] Biologically equivalent NCR peptides can have 10 or fewer conserved amino acid alterations, 7 or fewer conserved amino acid alterations, or 5, 4, 3, 2, or 1 conserved amino acid alterations. The encoding nucleotide sequence (e.g., gene, plasmid DNA, cDNA, or synthetic DNA) will therefore have corresponding base substitutions, enabling it to encode the biologically equivalent form of the NCR peptide.

[0079] Certain semi-conserved substitutions in NCR peptides include: (i) substitution of a neutral polar amino acid residue with a neutral nonpolar (hydrophobic) amino acid residue; or (ii) substitution of a neutral nonpolar (hydrophobic) amino acid residue with a neutral polar amino acid residue. In particular, semi-conserved substitutions of neutral polar tyrosine residues with hydrophobic amino acid residues are provided. Semi-conserved substitutions of hydrophobic amino acid residues with tyrosine residues are also provided. Biologically equivalent NCR peptides may have 10 or fewer semi-conserved amino acid changes, 7 or fewer semi-conserved amino acid changes, or 5, 4, 3, 2, or 1 semi-conserved amino acid changes.

[0080] Any functional fragment of a 6-Cys or 4-Cys NCR disclosed herein may include an N-terminal deletion, a C-terminal deletion, an internal deletion, or any combination thereof, which retains the conserved cysteine ​​residues of the 6-Cys or 4-Cys NCR intact or contains at least four or five of the six conserved C1, C2, C3, C4, C5, and C6 cysteine ​​residues of the 6-Cys NCR. The functional fragment may contain deletions of at least one, two, three, four, five, six, or seven or more amino acid residues from the N-terminus, C-terminus, internal region, or any combination thereof. For example, an antimicrobial fragment of a 6-Cys or 4-Cys NCR peptide may contain at least about 10, 14, 15, 18, or 20 about 22, 24, 25, 26, 27, or 28 amino acid residues from the C-terminus of the NCR peptide. Chimeric NCR peptides containing portions of any 6-Cys or 4-Cys NCRs disclosed herein, or containing variants or fragments of those 6-Cys or 4-Cys NCRs, may be used alone or in the NCR proteins provided herein.

[0081] Methods for preparing, testing, and using antimicrobial NCR_PFV compositions

[0082] Within the relevant embodiments, this disclosure provides methods for preparing, testing, and using antimicrobial NCR_PFV compositions comprising cationic antimicrobial rhizonospecific cysteine-rich (NCR) peptide fold variants (PFVs), said compositions (1) comprising NCR peptide fold variant 1 (NCR_PFV1) having desired antimicrobial activity and / or resistance / sensitivity to proteolysis, and (2) excluding NCR peptide fold variant 2 (NCR_PFV2) having reduced antimicrobial activity and / or undesirable resistance or sensitivity to proteolysis.

[0083] 6-Cys or 4-Cys NCR_PFV1 may contain an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the amino acid sequence presented in Table 1 or Table 2, respectively, wherein one or more hydrophobic, basic, and / or acidic amino acid residues are substituted with hydrophobic, basic, and / or acidic amino acid residues, respectively; a variant of the amino acid sequence presented in Table 1 or Table 2, wherein one or more neutral polar amino acid (e.g., tyrosine) residues are substituted with hydrophobic amino acid residues; and / or wherein one or more hydrophobic amino acid residues are substituted with neutral polar amino acid residues (e.g., tyrosine); its functional fragments; and its chimeras.

[0084] 6-Cys or 4-Cys NCR peptides or proteins can be synthesized de novo from the NCR peptide sequences disclosed herein, or expressed from the nucleotide sequences encoding 6-Cys or 4-Cys NCR peptides or proteins. The sequence encoding the nucleotide sequence of the peptide or protein can be deduced from the NCR peptide sequence using a reference genetic code. Computer programs such as BackTranslate (GCG™ software package, Acclerys, San Diego, CA) can be used to convert peptide sequences into the corresponding nucleotide sequences encoding that peptide.

[0085] This paper specifically envisions the expression of 6-Cys and 4-Cys NCR peptides in yeasts and filamentous fungi to generate NCR_PFV1. The construction of expression vectors for generating heterologous proteins in various yeast genera is well-established. Typically, such expression vectors contain a promoter operatively linked to the sequence of interest, which is operatively linked to a polyadenylated or terminator region. Examples of yeast genera that have been successfully used for heterologous gene expression include *Candida*, *Kluveromyces*, *Hansuela*, *Pichia*, *Saccharomyces*, *Schizosaccharomyces*, and *Yarrowia*. A general description of yeast expression vectors and transformation systems can be found in Kingsman et al. (1985) *Biotechnol Genet Eng Rev. 3:377-416*. Reiser et al. (1990) described expression vectors and transformation systems that can be used outside of the yeast genus in Adv Biochem Eng Biotechnol.;43:75-102.Other examples of fungal systems suitable for expressing NCR peptides include filamentous fungal systems, such as *Acremonium*, *Aspergillus*, *Aureobasidium*, *Bjerkandera*, *Ceriporiopsis*, *Chrysosporium*, *Coprinus*, *Coriolus*, *Cryptococcus*, *Filibasidium*, *Fusarium*, *Humicola*, *Magnaporthe*, *Mucor*, *Myceliophthora*, and *Neoca*. The system of genera including *Illimastix*, *Neurospora*, *Paecilomyces*, *Penicillium*, *Phanerochaete*, *Phlebia*, *Piromyces*, *Pleurotus*, *Schizophyllum*, *Talaromyces*, *Thermoascus*, *Thielavia*, *Tolypocladium*, *Trametes*, and *Trichoderma* (e.g., U.S. Patent Nos. 11,046,736 and 11,180,767, each of which is incorporated herein by reference in its entirety). Other systems applicable to the expression of NCR peptides include the Chrysosporium lucknowense system (e.g., U.S. Patent Nos. 8,871,493 and 9,175,296, each of which is incorporated herein by reference in its entirety).

[0086] This document specifically envisions the expression of NCR peptides to generate NCR_PFV in bacterial cells including those of the genus *Escherichia coli* (e.g., *E. coli*). Systems for expressing proteins containing disulfide bonds can be adapted to express defensin peptides in *E. coli*, including those disclosed in: U.S. Patent Publication No. US2020 / 0172915 (which is incorporated herein by reference in its entirety) and Berkmen, M. Protein Expr Purif. 82(1):240-51 (2012). Other systems that can be used to express proteins containing disulfide bonds, applicable to the expression of NCR peptides in E. coli, include those disclosed in: Kuddus, Biotechnol Prog 233 :1520-1528 (2017); Kiedzierska, Protein ExprPurif 60 :82-88(2008); Chang, Amino Acids 47: 579–587(2015); Buchko, ProteinScience 27 :1611-1623(2018); Marques, J Appl Microbiol 106 :1640-1648 (2008); and Pazgier, Protein Expr Pur 49 :1-8(2006).

[0087] Typically, promoters and polyadenylated regions are selected based on their operability in a given bacterial, yeast, or fungal host. For example, the AOX1 or AOX2 promoter of *Pichia pastoris* can be used in combination with the AOX1, AOX2, p40, or p76 polyadenylated sequences of *Pichia pastoris* to express heterologous proteins, such as NCR peptides. The AOX1 and AOX2 promoters are particularly useful in *Pichia pastoris* because both promoters provide abundant expression of the linked heterologous gene when induced by the addition of methanol to the growth medium. The use of these *Pichia pastoris* promoters and polyadenylated sequences is described in U.S. Patent No. 4,855,231, the entire contents of which are incorporated herein by reference. Similarly, the MOX, DHAS, or FMDH promoters of *Hansenula polymorpha* can be used to express heterologous proteins, such as NCRs in *Hansenula polymorpha*. The MOX, DHAS, or FMDH promoters are particularly useful in *Hansenula polymorpha* because these promoters provide abundant expression of the linked heterologous gene when induced by the addition of methanol to the growth medium. The use of MOX and DHAS promoters in *Hansenula polymorpha* is described in U.S. Patent No. 5,741,672, and the use of the FMDH promoter in *Hansenula polymorpha* is described in U.S. Patent No. 5,389,525, each of which is incorporated herein by reference in its entirety. For *Kluyveromyces kusnezoffii*, a lactase promoter and a polyadenylated sequence can be used to express heterologous genes, such as NCRs. Expression of heterologous genes operatively linked to a lactase promoter and a polyadenylated sequence can be achieved by growing *Kluyveromyces kusnezoffii* in the presence of galactose. The use of a lactase promoter and a polyadenylated sequence in *Kluyveromyces kusnezoffii* is described in its entirety in U.S. Patent No. 6,602,682, the entire contents of which are expressly incorporated herein by reference.

[0088] It is also envisioned to provide heterologous proteins (such as NCRs) secreted into growth media by transformed yeast or fungi via yeast, bacterial, or fungal expression vectors. The secretion of mature NCR peptides is typically achieved by operatively linking a signal peptide sequence, or a signal peptide and a propeptide sequence, to a mature NCR protein or peptide-coding sequence. Examples of useful signal peptides for secreting heterologous proteins in yeast include, but are not limited to, α-factor signal peptides, invertase signal peptides, and PHOl signal peptides, all of which are derived from yeast. α-factor signal peptides are typically derived from *Saccharomyces*, *Kluyveromyces*, or *Candida*, while PHOl signal peptides are derived from *Pichia pastoris*.

[0089] Particularly useful signal peptide sequences, or signal peptide and propeptide sequences, for protein secretion in yeast are derived from the *Saccharomyces cerevisiae* α-factor and are described in U.S. Patent Nos. 4,546,082, 4,588,684, 4,870,008, and 5,602,034, all of which are incorporated herein by reference in their entirety. The *Saccharomyces cerevisiae* α-factor signal peptide and propeptide sequences consist of amino acids 1-83 of the primary, unprocessed translation product of the *Saccharomyces cerevisiae* α-matching factor gene (GenBank accession number: P01149). In some embodiments, the α-matching factor signal peptide sequence comprising amino acids 1 to 19-23 of the α-matching factor proprotein can be directly ligated to the N-terminus of a mature NCR protein to provide secretion of the mature NCR protein. In this case, the signal peptide is cleaved from the mature NCR protein during secretion. Alternatively, the α-matching factor signal peptide and propeptide can be operatively ligated to a mature NCR coding sequence via a cleavage site sequence. This cleavage site sequence can comprise a variety of sequences that provide a leader sequence and proteolytic processing of the gene of interest.

[0090] In the native *Saccharomyces cerevisiae* α-matching factor gene, the cleavage site sequence corresponds to amino acid residues 84-89 and is represented by the sequence Lys84-Arg85-Glu86-Ala87-Glu88-Ala89 (SEQ ID NO: 7). The Lys-Arg sequence corresponds to the KEX2 protease recognition site, while the Glu-Ala-Glu-Ala sequence corresponds to the repeating dipeptidyl aminopeptidase or STE13 recognition site. In some embodiments, a DNA fragment encoding the 89-amino acid *Saccharomyces cerevisiae* α-factor signal peptide, the propeptide coding region, and the entire natural spacer region (i.e., the N-terminal 89 amino acid residues of the α-matching factor precursor protein contained in the Lys-Arg KEX2 protease cleavage site at residues 84 and 85 and the Glu-Ala-Glu-Ala dipeptidyl aminopeptidase or STE13 recognition site at residues 86-89) is operatively ligated to a sequence encoding the mature NCR protein.

[0091] When the N-terminal 89 amino acids of the α-matching factor precursor protein are fused to the N-terminus of a foreign protein (e.g., NCR), the propeptide sequence is typically dissociated from the foreign protein by cleavage at the KEX2 or STE13 recognition site by an endogenous yeast protease. In other embodiments, a DNA fragment encoding a smaller 85-amino acid Saccharomyces cerevisiae α-factor signal peptide, propeptide, and KEX2 spacer element (i.e., only the N-terminal 85 amino acid residues of the α-matching factor precursor protein contained at the Lys-Arg KEX2 protease cleavage site at residues 84 and 85) is operatively ligated to the sequence encoding the mature NCR protein. When the N-terminal 85 amino acids of the α-matching factor precursor protein are fused to the N-terminus of a foreign protein (e.g., NCR), the propeptide sequence is typically dissociated from the foreign protein by cleavage at the KEX2 recognition site by an endogenous yeast protease. Therefore, the NCR protein can be expressed without the glu-ala repeat sequence.

[0092] To obtain transformant yeast strains expressing NCR peptides, yeast NCR expression cassettes (e.g., yeast promoter, yeast signal peptide coding sequence, mature NCR protein sequence, and polyadenylated sequence) are typically combined with other sequences that provide selection for transformant yeast strains. Examples of useful alternative marker genes include genes encoding ADE, HIS5, HIS4, LEU2, URA3, ARG4, TRP1, LYS2, proteins conferring resistance to bleomycin or fulvic acid, proteins conferring resistance to chloramphenicol, proteins conferring resistance to G418 or genimycin, proteins conferring resistance to hygromycin, proteins conferring resistance to methotrexate, AR04-OFP, and FZF1-4 proteins. Similar alternative marker cassettes for conferring antibiotic resistance or rescuing auxotrophic traits are used in bacterial or fungal systems.

[0093] DNA molecules containing a yeast NCR expression cassette and optional marker genes are introduced into yeast cells via techniques such as yeast protoplast transfection or electroporation. In some embodiments, the DNA molecule containing the yeast NCR expression cassette and optional marker genes is introduced as a linear DNA fragment and integrated into the genome of a transformed yeast host cell. Integration can occur at random sites in the yeast host cell genome or at specific sites in the yeast host cell genome. Integration at specific sites in the yeast host cell genome is typically accomplished through homologous recombination between sequences contained in the expression vector and sequences in the yeast host cell genome. Homologous recombination is typically accomplished by linearizing the expression vector within homologous sequences (e.g., within the AOX1 promoter sequence of the Pichia pastoris expression vector when the expression vector is integrated into the endogenous AOX1 gene of the Pichia pastoris host cell). In other embodiments, the yeast expression cassette may also contain additional sequences, such as autonomously replicating sequences (ARS), which provide for the replication of the DNA containing the expression cassette as an extrachromosomal (non-integrating) element. Such extrachromosomal elements are typically maintained in yeast cells by the presence of continuously selected optional marker genes. Yeast artificial chromosomes (YACs) containing sequences for replication and mitosis transmission are another type of vector that can be used to maintain DNA constructs in yeast hosts.

[0094] Yeast, bacterial, or fungal cells transformed with a yeast or fungal NCR expression cassette can be used to produce a mixture of NCR_PFVs, which can be used to obtain formulations enriched with the desired NCR_PFV. The desired NCR_PFV can be used as an antimicrobial agent to produce antimicrobial compositions that can be applied to plants or food, or to produce antimicrobial compositions that can be applied to human or non-human subjects. Methods for producing NCR_PFV mixtures typically begin by culturing yeast, bacterial, or fungal cells transformed with an NCR expression cassette under conditions where mature NCR molecules are expressed in yeast cells. Typically, the conditions for expressing mature NCR molecules in yeast, bacterial, or fungal cells are conditions that allow or specifically induce expression of the yeast promoter in the yeast expression cassette that is operatively linked to the NCR coding sequence. When the yeast is Pichia pastoris and the signal peptide / MD gene is controlled by the AOX1 or AOX2 promoter, the addition of methanol to the growth medium will induce expression of the mature NCR protein. Similarly, when the yeast is *Hansenula polymorpha* and the signal peptide / MD gene is under the control of the MOX, DHAS, or FMDH promoter, adding methanol to the growth medium will provide expression of the mature NCR protein. Alternatively, when the yeast is *Kluyveromyces var. xylooligosacchari* and the signal peptide / De / 5 gene is under the control of the lactase promoter, adding galactose to the growth medium will provide expression of the mature NCR protein.

[0095] Once the transformed yeast, bacterial, or fungal culture is incubated for a sufficiently long time under culture conditions that provide mature NCR peptide expression, the desired NCR PFV can be separated from any undesired NCR PFV as a mixture from the culture. Sufficient incubation time can be determined by periodically harvesting portions or aliquots of the culture and measuring the presence of desired and / or undesired NCR PFV. For example, HPLC analysis can be used to monitor the production of both desired and undesired NCR PFV. In some embodiments, the incubation period can be optimized to produce the desired NCR PFV, such that the culture is enriched with the desired NCR PFV.

[0096] The methods according to these embodiments include (a) separating a fraction containing 6-Cys or 4-Cys NCR peptide fold variant 1 (NCR_PFV1) from a mixture containing NCR_PFV1 and NCR_PFV2 or NCR_PFV2 peptide fragments thereof, and one or more fractions containing 6-Cys or 4-Cys NCR peptide fold variant 2 (NCR_PFV2) or NCR_PFV2 peptide fragments thereof; and (b) combining the fraction containing NCR_PFV1, or a formulation further purified from the NCR_PFV1 fraction, with an agriculturally, pharmaceutically, or veterinarily feasible carrier, diluent, or excipient, thereby preparing the composition.

[0097] A mixture comprising NCR_PFV1 and NCR_PFV2 or a fragment of NCR_PFV2 thereof may be prepared by: (a) culturing microorganisms expressing recombinant polynucleotides in a fermentation broth, the recombinant polynucleotides comprising a transcription promoter operatively linked to a polynucleotide encoding a signal peptide co-located and upstream of the polynucleotide encoding NCR_PFV1 or a variant thereof; (b) isolating the microorganisms from the fermentation broth (e.g., by size exclusion chromatography), the fermentation broth containing the mixture; (c) lysing the microorganisms to obtain cell lysates; and (d) separating insoluble fragments in the cell lysates from an aqueous fraction containing the mixture.

[0098] The isolation of the desired NCR_PFV from the culture can be partial or complete. For NCR expression vectors in which the signal peptide is operatively linked to a sequence encoding a mature NCR protein, a mixture containing the desired NCR_PFV and any raNCR_PFV or other undesired NCR_PFV (e.g., increasing or decreasing protease resistance) can be recovered from yeast cell culture medium, bacterial periplasm, or fungal cell culture medium. In some embodiments, yeast or fungal cell culture medium containing mature NCR protein present in both the desired and undesired NCR_PFV forms can be separated from the yeast or fungal cells by centrifugation or filtration, thereby providing a composition containing a mixture of the desired and undesired NCR_PFV. In some embodiments, formulations containing bacterial periplasmic proteins of mature NCR proteins existing in both desired and undesirable NCR_PFV forms can be obtained by methods including physical force, pressure, osmotic shock, or chemical treatment (e.g., EDTA + heat or detergent) to provide compositions containing a mixture of desired and undesirable NCR_PFV.

[0099] Such methods for isolating bacterial periplasmic proteins can be adapted from methods for isolating other periplasmic proteins (e.g., Schimek et al., (2020) Biotechnology Progress, 36(5), e2999, doi.org / 10.1002 / btpr.2999). Yeast or fungal cell culture media containing mature NCR proteins, or formulations of bacterial periplasmic proteins, can be further processed by any combination of dialysis and / or concentration techniques (e.g., precipitation, lyophilization, filtration) to produce a composition containing at least one NCR_PFV (e.g., a mixture of desired and undesired NCR_PFVs). In some embodiments, size exclusion membranes can be used to enrich fractions containing the mixture of desired and undesired NCR_PFVs.

[0100] A combination of various separation techniques can also be used to produce a mixture of NCR_PFV. For example, cell culture medium can be separated from cells by centrifugation and then dialyzed or adjusted. In some embodiments, the buffer used for dialysis or adjustment is a 25 mM sodium acetate buffer at approximately pH 4.5–6.0. ​​The dialysate is then subjected to ion exchange chromatography. For example, a cation exchange resin equilibrated with a 25 mM sodium acetate buffer at approximately pH 6.0, such as CM-Sephadex C-25, can be used. The NCR protein bound to the cation exchange resin is washed and then eluted. For example, the column described above is washed with a 25 mM sodium acetate buffer at approximately pH 6.0 and then eluted with 1 M NaCl, 50 mM Tris, pH 7.6. The fraction containing the NCR protein is identified by assay or UV absorption and then concentrated through a size-cutoff filter membrane. The concentrated NCR protein is then dialyzed to obtain substantially or substantially pure NCR protein in a buffer. The buffer includes buffers such as 10 mM Tris, pH 7.6.

[0101] Compositions containing or enriched with desired NCR_PFVs can be obtained by methods that provide separation of the desired NCR_PFV from undesired NCR_PFV(s). In some embodiments, such methods may include standard protein separation techniques for enriching the desired NCR_PFV based on different chemical properties other than mass (e.g., hydrodynamic radii of different NCR_PFVs and / or charge properties of different NCR_PFVs). In some embodiments, chromatographic techniques such as sedimentation, size exclusion chromatography (SEC), ion exchange chromatography, and / or affinity chromatography may be used to produce compositions enriched with desired NCR_PFVs. Disclosed methods for separating proteins with different conformations by SEC can be applied to the separation of desired NCR_PFV(s) from one or more undesired NCR_PFV(s) (La Verde et al., Bio Protoc. 20 April 2017;7(8):e2230. doi:10.21769 / BioProtoc.2230).

[0102] In some embodiments, the method may provide a composition comprising a desired NCR_PFV, wherein the composition comprises less than 5%, 2%, 1%, 0.5%, 0.25%, or 0.1% by weight of one or more undesired NCR_PFV(s). In some embodiments, the method may provide a composition comprising a desired NCR_PFV, wherein less than 5%, 2%, 1%, or 0.5% by weight of the total combined amount of the desired NCR_PFV and one or more undesired NCR_PFV(s) in the composition is / is an undesired NCR_PFV(s). In some embodiments, the method may provide a composition comprising a desired NCR_PFV, wherein at least about 80%, 90%, 95%, 98%, 99%, or 99.5% by weight or molar of the total NCR peptide in the composition is the desired NCR_PFV.

[0103] In some embodiments, the method may provide a composition in which less than 20%, 10%, 5%, 2%, 1%, or 0.5% by weight of total NCR peptides are undesired NCR_PFVs, or in which at least about 80%, 90%, 95%, 98%, 99%, or 99.5% by weight of total NCR peptides are desired NCR_PFVs. The total NCR peptides in the composition by weight can be determined by quantifying the combined total amount of desired and undesired NCR_PFVs in the composition. Quantification of total NCR peptides and / or isolated NCR_PFVs can be performed by a variety of methods, including immunoassays (e.g., ELISA, RIA, where a reference curve is established using purified NCR peptides) or mass spectrometry (Zhang et al. Methods Mol Biol. 2010;673:211-222.doi:10.1007 / 978-1-60761-842-3_13).

[0104] In some embodiments, compositions rich in the desired NCR_PFV can be obtained by utilizing the relative sensitivity of the desired NCR_PFV to proteases (e.g., serine proteases such as trypsin). In embodiments where the desired NCR_PFV exhibits increased resistance to proteases (e.g., at a given protease concentration), a mixture containing both desired and undesired NCR_PFV can be treated with a protease at a concentration capable of cleaving the undesired NCR_PFV while keeping the desired NCR_PFV intact. The intact NCR_PFV can then be separated from the resulting fragments of NCR peptides produced by cleaving the undesired NCR_PFV by methods including size exclusion membrane filtration, SEC, and / or IEC. Suitable proteases for such methods can include serine proteases from the trypsin family. In some embodiments, the serine protease cleaves the undesired NCR_PFV at the carboxyl terminus of an arginine or lysine residue in the undesired NCR_PFV under non-denaturing conditions. In some embodiments, the trypsin family serine proteases are recombinant bovine, porcine, human, or microbial trypsin, optionally wherein the microbial trypsin is Streptomyces trypsin or a variant thereof. In some embodiments, about 6.25, 10, or 12.5 to about 15, 20, or 25 BAEE units / mL of trypsin (e.g., Sigma-Aldrich catalog number T1426, St. Louis, Missouri) can be incubated with NCR PFV at 37°C in 60 mM monobasic phosphate buffer at pH 7.5 for about 1, 2, or 3 to about 4, 5, or 6 hours to cleave undesirable NCR PFV while leaving the desired NCR PFV intact. One BAEE (N α Benzoyl-L-arginine ethyl ester hydrochloride) trypsin substrate units will produce 0.001 ΔA253 (absorbance change at 253 nm) per minute with BAEE as substrate at pH 7.6, 25°C, and a reaction volume of 3.2 mL (1 cm optical path).

[0105] In some aspects of these methods, a mixture comprising NCR_PFV1 and NCR_PFV2, or a NCR_PFV2 peptide fragment thereof, is treated with a protease under non-denaturing conditions sufficient to produce an NCR_PFV2 peptide fragment but insufficient to produce an NCR_PFV1 peptide fragment. Suitable proteases include serine proteases of the trypsin family that cleave the NCR peptide at the carboxyl terminus of the Arg(R) or Lys(K) of the NCR peptide. Serine proteases of the trypsin family include recombinant bovine, porcine, human, or microbial trypsin, and optionally, wherein the microbial trypsin is streptomycin or a variant thereof.

[0106] The 6-Cys or 4-Cys NCR_PFV1 according to this embodiment can exhibit increased phospholipid binding compared to the corresponding 6-Cys or 4-Cys NCR_PFV2. The desired 6-Cys or 4-Cys NCR_PFV1 provided herein can exhibit lower IC50 values ​​against one or more microbial pathogens, improved phospholipid binding, or any combination thereof compared to any of the following: (i) a mixture comprising the desired 6-Cys or 4-Cys NCR_PFV1 and one or more corresponding undesired 6-Cys or 4-Cys NCR_PFV2, or (ii) 6-Cys or 4-Cys NCR_PFV2 alone. The desired 6-Cys or 4-Cys NCR_PFV1 can be identified and / or selected by choosing a 6-Cys or 4-Cys NCR_PFV1 that provides improved phospholipid binding compared to either of the following, in order to obtain a lower IC50 value against one or more microbial pathogens: (i) a mixture comprising the desired 6-Cys or 4-Cys NCR_PFV1 and one or more corresponding undesired 6-Cys or 4-Cys NCR_PFV2, or (ii) 6-Cys or 4-Cys NCR_PFV2 alone.

[0107] Suitable assays for determining improved phospholipids include protein-lipid coverage assays (e.g., Dowler et al., 2002, Sci STKE. April 23, 2002; 2002(129):16), surface plasmon resonance assays (e.g., Baron and Pauron, 2014, Bio-protocol 4(18): e1237), biotin-capture lipid affinity assays (e.g., Davidson et al., 2006, Lipid Research, 47, 440-449), titration calorimetry assays (e.g., Miller and Cistola, 1993 Molecular and Cellular Biochemistry, 123(1): 29-37), etc.

[0108] Typically, the permeability of microbial plasma membranes treated with 6-Cys or 4-Cys NCR_PFV1 as described herein can be increased compared to that treated with the corresponding 6-Cys or 4-Cys NCR_PFV2. Membrane permeability can be measured using a variety of techniques that include dye uptake. Convenient dye uptake assays that can be used to assess changes in membrane permeability include uptake assays of Hoechst 33342 (H0342), Rhodamine 123, SYTOX™ Green, etc. These dyes enter microbial cells only when the plasma membrane of the microbial cell is permeated by NCR peptides, defensins, or other membrane permeabilizers. Without being bound by theory, in some embodiments, it is considered that NCR proteins comprising an NCR peptide and another antifungal peptide linked by a spacer peptide can provide improved microbial inhibition by increasing the permeability of the treated microbial membrane compared to that treated with the NCR peptide.

[0109] The identification of transgenic MsDefl-expressing potato plants resistant to Verticillium dahliae using a combination of microbial disease severity assays and colony formation assays with expression assays has been described (US Patent Nos. 6,916,970 and Gao et al., 2000, Nature Biotechnology 18, 1307-1310). Similarly, it is conceivable that plants treated with 6-Cys or 4-Cys NCR_PFV1 could be identified by scoring the treated plants to determine their resistance to the microbial pathogens that infect them, thus identifying plants resistant to or controlling various microbial pathogens. Examples of microbial resistance conferred by NCR_PFV1, which can be determined by observing a reduction in disease symptoms or microbial growth, include: resistance of treated maize to *Fusarium verticillioides*, *Fusarium moniliforme*, *Colletotrichum graminicola*, *Stenocarpella maydis*, and / or *Cercospora zeae-maydis*; resistance of treated wheat to *Fusarium graminearum*, powdery mildew (*Erysiphegraminis f. sp. tritici*), stripe rust, stem rust, or leaf rust (*Puccinia tritici*); and resistance of treated cotton to *Fusarium oxysporum*. Resistance to *Verticillium dahlia* and *Oxysporum*; resistance of treated rice to *Magnaporthe oryzae* and *Rhizoctonia solani*; and resistance of treated soybeans to Asian soybean rust (*Phakopsora pachyrhizi*), *Phytophthora* root rot (*Phytophthora* sp.), white mold (*Sclerotinia* sp.), sudden death syndrome (*Fusarium virguliforme*), and / or brown stem rot (*Phialophora gregata*).

[0110] The desired amount of 6-Cys or 4-Cys NCR_PFV1 inhibitory against pathogenic microorganisms in a given plant can also be identified by measuring the reduction in adverse effects caused by microbial growth in such plants. This reduction can be determined by comparing the degree of adverse effects in NCR_PFV1-treated plants with control plants of other aspects identical to those treated with a control composition lacking NCR_PFV1 or containing the corresponding NCR_PFV2. Measurable adverse effects of microbial growth in plants include any type of plant tissue damage or necrosis, any type of reduced plant yield, any reduction in the value of crop plant products, and / or the production of undesirable microbial metabolites or byproducts of microbial growth, including but not limited to mycotoxins. Mycotoxins comprise a variety of toxic molecules produced by microbial species, including but not limited to polyketides (including aflatoxin, demethylazopyrrolizin, O-methylazopyrrolizin, etc.), fumonisins, alperisins (e.g., A1s A2, B1s B2), sphingomyelins (A, B, C, and D), trichothecenes, fumifungins, etc. Methods for quantifying mycotoxin levels have been extensively documented. Furthermore, commercial kits are available for measuring mycotoxins such as aflatoxin, fumonisin, deoxynivalenol, and zearalenone (VICAM, Watertown, Massachusetts, USA).

[0111] Infection of certain plants by specific plant pathogens can have significant and easily observable effects on plant growth. Therefore, plants treated with the desired 6-Cys or 4-Cys NCR_PFV1 can be distinguished from controls treated with NCR peptides lacking the peptide or containing the corresponding raNCR_PFV by attacking such plants with the pathogenic plant microorganism and observing a reduction in symptoms typically associated with such infections. Such observations can be facilitated by co-infecting control plants treated with NCR peptides lacking the peptide or containing the corresponding undesirable 6-Cys or 4-Cys NCR_PFV2 with other control plants of similar characteristics. Identification of treated plants that control or resist microbial infection can be based on observed reductions in disease symptoms, determination of reduced microbial growth in infected plants (e.g., by determining the number of colony-forming units per gram of infected tissue), and / or determination of the amount of mycotoxins present in the infected plant tissue.

[0112] Plants of interest include food crop plants and biofuel or energy crop plants. The plants, parts thereof, and harvested parts thereof (e.g., grains, fruits, and vegetables) to which the methods and compositions disclosed herein may be applied may refer to directly edible plants, parts thereof, and harvested parts thereof, or plants that produce edible products (e.g., commonly used to feed humans directly or indirectly through animals). The plants, parts thereof, and harvested parts used in this article include cereal crops (e.g., wheat, rice, corn, barley, oats, sorghum, rye, and millet), legumes (e.g., peanuts, chickpeas, lentils, kidney beans, soybeans, and lima beans); root or tuber crops (e.g., potatoes, sweet potatoes, and cassava); oilseed crops (e.g., rapeseed, wheat, peanuts, palm, coconut, safflower, cottonseed, sunflower, flax, and olives); sugar crops (e.g., sugarcane and sugar beets); fruit crops (e.g., bananas, oranges, apples, pears, breadfruit, pineapples, strawberries, grapes, and cherries); vegetable and tuber crops (e.g., tomatoes, lettuce, carrots, melons, asparagus, and brassica); nut crops (e.g., cashews, peanuts, walnuts, pistachios, and almonds); forage and turfgrass; forage legumes (e.g., alfalfa and clover); and medicinal crops (e.g., cannabis). sp.), coffee, cocoa, kola nut, poppy; spice and flavoring crops (e.g., vanilla, sage, thyme, fennel, saffron, menthol, peppermint, spearmint, coriander), fiber crops (e.g., cotton or hemp), and biofuel crops (e.g., castor bean, flaxseed, switchgrass, Miscanthus and Jatropha), plants, parts thereof, and harvested parts thereof.

[0113] Analysis of different NCR_PFVs for the allocation of specific disulfide bonds can be performed using mass spectrometry-based methods (Weinfurtner, Oxidative Folding of Proteins: Basic Principles, Cellular Regulation and Engineering, 2018, pp. 81-98, DOI: 10.1039 / 9781788013253-00081; Tang and Speicher, Current protocols in protein science, Vol. 96, No. 1 (2019):e86. doi:10.1002 / cpps.86).

[0114] Antimicrobial compositions for agricultural, pharmaceutical, or veterinary use are also provided, comprising one or more isolated, purified, antimicrobial modified NCR_PFV peptides of the present invention, or their biological equivalents, at an inhibitory amount (“antimicrobial effective amount”) against an antimicrobial plant or antimicrobial human or veterinary pathogenic microorganism. Such compositions may comprise one or any combination of NCR peptides disclosed herein, along with agriculturally, pharmaceutically, or veterinarily feasible carriers, diluents, or excipients. Other components relevant in agricultural and therapeutic contexts may also be included in such compositions, as shown below. The antimicrobial compositions can be used to inhibit or kill NCR protein or peptide-sensitive pathogenic microorganisms associated with microbial infections in plants, humans, or animals. Such antimicrobial compositions can be formulated for topical application and applied topically to plants, plant environments (including soil), or humans or animals.

[0115] Agricultural compositions comprising any NCR molecule of the present invention, alone or in any combination, can be formulated according to the methods described in the following literature: for example, Winnacker-Kuchler (1986) Chemical Technology, 4th edition, Vol. 7, Hanser Verlag, Munich; van Falkenberg (1972-1973) Pesticide Formulations, 2nd edition, Marcel Dekker, NY; and K. Martens (1979) Spray Drying Handbook, 3rd edition, GG. Goodwin, Ltd., London. Formulation aids (such as carriers, inert materials, 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, 2nd edition, Darland Books, Caldwell, NJ; and Winnacker-Kuchler (1986) Chemical Technology, 4th edition, Vol. 7, Hanser Verlag, Munich. These formulations can also be used to prepare compositions in which the desired NCR_PFV is combined with other pesticide actives, fertilizers and / or growth regulators (e.g., finished formulations or canned mixtures).

[0116] Whether used alone or in combination with other active agents, the antimicrobial NCR_PFV peptide of the present invention can be administered at concentrations ranging from about 0.1 μg / ml to about 100 mg / ml, or from about 5 μg / ml to about 5 mg / ml, at a pH range of about 3.0 to about 9.0. Such compositions can be buffered, for example, with phosphate buffers ranging from about 1 mM to 1 M, from about 10 mM to about 100 mM, or from about 15 mM to about 50 mM. In cases of low buffer concentrations, salts can be added to increase ionic strength. In some embodiments, NaCl in the range of about 1 mM to about 1 M or from about 10 mM to about 100 mM can be added.

[0117] Many conventional microbial antibiotics and chemical fungicides that can be combined with the NCR_PFV of the present invention are described in Worthington and Walker (1983), The Pesticide Manual, 7th Edition, British Crop Protection Council. These include, for example, polyoxins, scintillans, carboxamides, aromatic carbohydrates, carboxylamines, morpholine, sterol biosynthesis inhibitors, and organophosphate compounds. Additionally, azoles, triazoles, and echinococcins can be used as fungicides. Other active ingredients that can be formulated in combination with the antimicrobial peptides and proteins of the present invention include, for example, insecticides, attractants, sterilizers, acaricides, nematicides, and herbicides. U.S. Patent No. 5,421,839 (which is incorporated herein by reference in its entirety) contains a comprehensive summary of many active agents that can be formulated with substances such as the antimicrobial NCR peptides and proteins of the present invention.

[0118] The compositions provided herein can be applied in an antimicrobial effective amount, which will vary depending on a variety of factors, such as the specific fungal pathogen to be controlled, the specific plant (and plant parts or soil) to be treated, and the method of applying the composition containing the desired NCR_PFV.

[0119] The desired NCR_PFV and its biofunctional equivalents, and compositions comprising them (e.g., in the examples numbered below), provided herein can be used to inhibit the growth of a variety of susceptible microorganisms in plants. In some embodiments, the growth of microorganisms of the following genera or species can be inhibited: *Alternaria* (e.g., *Alternaria brassicicola*; *Alternaria solani*)); *Ascochyta* (e.g., *Ascochytapisi*)); *Aspergillus* (e.g., *Aspergillus flavus*; *Aspergillus fumigatus*)); *Botrytis* (e.g., *Botrytis cinerea*)); *Cercospora* (e.g., *Cercospora kikuchii*; *Cercospora maize*). * *Zeae-maydis*); *Colletotrichum* (e.g., *Colletotrichum lindemuthianum*); *Diplodia* (e.g., *Diplodia maydis*); *Erysiphe* (e.g., *Erysiphe graminis* f.sp. graminis; *Erysiphe graminis* f.sp. hordei); *Fusarium* (e.g., *Fusarium nivale*; *Fusarium oxysporum*; *Fusarium graminearum*; *Fusarium culmorum*; *Fusarium solani*; *Fusarium moniliforme*; *Fusarium roseum*. roseum); Gaeumanomyces (e.g., wheat graminis).*Helminthosporium* (e.g., *Helminthosporium turcicum*, *Helminthosporium carbonum*, *Helminthosporium maydis*); *Macrophomina* (e.g., *Macrophomina phaseolina*, *Magnaporthe grisea*); *Nectria* (e.g., *Nectria heamatococca*); *Peronospora* (e.g., *Peronospora manshurica*, *Peronospora tabacina*); *Phakopsora* (e.g., *Phakopsora pachyrhizi*); *Phoma* (e.g., *Phoma pachyrhizi*). betae); Phymatotrichum (e.g., Phymatotrichum omnivorum); Phytophthora (e.g., Phytophthora cinnamomi; Phytophthora cactorum; Phytophthora phaseoli; Phytophthora parasitica; Phytophthora citrophthora; Phytophthora sojae; Phytophthora pathogenicum). *Plasmopara* (e.g., *Plasmoparaviticola*); *Podosphaera* (e.g., *Podosphaeraleucotricha*); *Puccinia* (e.g., *Puccinia sorghi*, *Puccinia striiformis*, *Puccinia graminis* f.sp.); *Puccinia sorghi* (e.g., *Puccinia sorghi*, *Puccinia striiformis*, *Puccinia graminis* f.sp.); *Puccinia graminis* f.sp. (*Puccinia sorghi*, *Puccinia striiformis*, *Puccinia graminis* f.sp.).*Puccinia tritici*; *Puccinia asparagi*; *Puccinia recondita*; *Puccinia arachidis*; *Pythium* (e.g., *Pythium aphanidermatum*; *Pythium ultimum*); *Pyrenophora* (e.g., *Pyrenophoratritici-repentens*); *Pyricularia* (e.g., *Pyricularia oryzae*); *Rhizoctonia* (e.g., *Rhizoctonia solani*; *Rhizoctonia graminifolia*). cerealis); Sclerotium (e.g., Sclerotium rolfsii); Sclerotinia (e.g., Sclerotinia sclerotiorum); Septoria (e.g., Septoria lycopersici; Septoria aglycines; Septoria nodorum; Septoria tritici); Thieviopsis (e.g., Thieviopsis basicola); Uncinula (e.g., Uncinula necator); Venturia (e.g., Venturia aubergina). (inaequalis); and Verticillium (e.g., Verticillium dahliae; Verticillium albo-atrum).

[0120] Also provided are pharmaceutical or veterinary compositions comprising a desired antimicrobially effective amount of NCR_PFV and a pharmaceutically acceptable or veterinary-feasible carrier. Such pharmaceutical or veterinary compositions can be used to inhibit the growth of susceptible pathogenic microorganisms infecting humans or animals, or to kill them, i.e., to treat such fungal infections by administering such compositions to patients or other subjects in need. In some embodiments, compositions comprising NCR_PFV can be formulated by means of methods described in Remington: The Science and Practice of Pharmacy (2005), 21st edition, University of the Science of Philadelphia, Lippincott Williams & Wilkins. In some embodiments, the composition may comprise NCR_PFV at concentrations ranging from about 0.1 μg / mL to about 100 mg / mL, or from about 5 μg / mL to about 5 mg / mL, at a pH ranging from about 3.0 to about 9.0. Such compositions can be buffered, for example, with phosphate-buffered saline at concentrations of about 1 mM to about 1 M, about 10 mM to about 100 mM, or about 15 mM to 50 mM. When the buffer concentration is low, salt can be added to increase the ionic strength. In some embodiments, NaCl in the range of about 1 mM to about 1 M, or about 10 mM to about 100 mM, can be added.

[0121] NCR_PFV can be formulated alone or in combination with other conventional antimicrobial therapeutic compounds, such as, but not limited to, polyene antimicrobial agents; imidazole, triazole and thiazole antimicrobial agents; allylamines; and echinocandins.

[0122] The administration of a composition containing NCR_PFV to human or animal subjects in need can be accomplished through a variety of routes, including topical application, enteral, parenteral, and / or intravenous administration.

[0123] In a further embodiment, this disclosure provides a method for preventing or reducing crop damage or post-harvest loss caused by plant pathogenic microorganisms, the method comprising contacting a plant, plant seed, pre- or post-harvest cereal, pre- or post-harvest melon or fruit, or pre- or post-harvest vegetable with an effective amount of a composition comprising a cationic antimicrobial 6-Cys or 4-Cys root nodule-specific cysteine-rich (NCR) peptide fold variant 1 (NCR_PFV1) under conditions suitable for preventing or reducing crop damage or post-harvest loss.

[0124] In other embodiments, this disclosure provides a medical device operably combined with a composition comprising a cationic antimicrobial 6-Cys or 4-Cys root nodule-specific cysteine-rich (NCR) peptide fold variant 1 (NCR_PFV1), wherein the medical device comprises at least one surface that is locally coated or impregnated with the composition.

[0125] In a further embodiment, this document provides a method for treating, preventing, or inhibiting microbial infection in a subject (including plant, human, or non-human animal) in need, the method comprising administering (in vitro or in vivo) to the subject, under conditions and for a duration suitable for treating, preventing, or inhibiting the microbial infection, a therapeutically effective amount of a composition comprising a cationic antimicrobial 6-Cys or 4-Cys root nodule-specific cysteine-rich (NCR) peptide fold variant 1 (NCR_PFV1).

[0126] In related embodiments, this document provides a method for treating, preventing, or inhibiting microbial infections in subjects who require the use of a composition comprising a cationic antimicrobial 6-Cys or 4-Cys root nodule-specific cysteine-rich (NCR) peptide fold variant 1 (NCR_PFV1).

[0127] In other related embodiments, this document provides plant portions at least partially coated with a composition comprising a cationic antimicrobial 6-Cys or 4-Cys root nodule-specific cysteine-rich (NCR) peptide fold variant 1 (NCR_PFV1).

[0128] Example

[0129] The following numbered embodiments form part of this disclosure:

[0130] 1a. A composition comprising a root nodule-specific cysteine-rich NCR peptide fold variant 1 (NCR_PFV1), wherein the NCR peptide is a cationic 6-Cys NCR peptide comprising, from its N-terminus, a sequential amino acid sequence of: (1) a first cysteine ​​pair motif comprising C1 and C2; (2) a first intercalation sequence (IS1); (3) a second cysteine ​​pair motif comprising C3 and C4; (4) a second intercalation sequence (IS2); and (5) a third cysteine ​​pair motif comprising C5 and C6, wherein the NCR peptide is associated with CaNCR7 (SEQ ID NO: 2), CaNCR13 (SEQ ID NO: 1), CaNCR14 (SEQ ID NO: 3), or CaNCR15 (SEQ ID NO: 4). 4) Having less than 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity, wherein the cysteine ​​residues C1, C2, C3, C4, C5, and C6 of said NCR_PFV1 form a first group of disulfide bonds, and said NCR_PFV1 thereby adopts a first conformation exhibiting greater antimicrobial activity, as compared to root nodule-specific cysteine-rich NCR peptide fold variant 2 (NCR_PFV2), said NCR_PFV2 having the same amino acid sequence as said NCR_PFV1 but forming a different second group of disulfide bonds between cysteine ​​residues C1, C2, C3, C4, C5, and C6 and adopting a different second conformation, and wherein said composition is substantially free of NCR_PFV2, optionally wherein at least about 80%, 90%, 95%, 98%, 99%, or 99.5% (by weight) of said total NCR peptide in said composition is DEF_PFV1.

[0131] 1b. A composition comprising a root nodule-specific cysteine-rich NCR peptide fold variant 1 (NCR_PFV1), wherein the NCR peptide is a cationic 6-Cys NCR peptide comprising, from its N-terminus, a sequential amino acid sequence of: (1) a first cysteine ​​pair motif comprising C1 and C2; (2) a first intercalation sequence (IS1); (3) a second cysteine ​​pair motif comprising C3 and C4; (4) a second intercalation sequence (IS2); and (5) a third cysteine ​​pair motif comprising C5 and C6, wherein the NCR peptide is associated with CaNCR7 (SEQ ID NO: 2), CaNCR13 (SEQ ID NO: 1), CaNCR14 (SEQ ID NO: 3), or CaNCR15 (SEQ ID NO: 4). 4) Possessing less than 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity, wherein the cysteine ​​residues C1, C2, C3, C4, C5, and C6 of NCR_PFV1 form the first group of disulfide bonds, and NCR_PFV1 thereby adopts a first conformation exhibiting greater antimicrobial activity, increased protease resistance, or increased protease sensitivity, such as the cysteine-rich NCR peptide fold variant 2 specific to root nodules. Compared to (NCR_PFV2), said NCR_PFV2 has the same amino acid sequence as NCR_PFV1 but forms a different second set of disulfide bonds and adopts a different second conformation between cysteine ​​residues C1, C2, C3, C4, C5 and C6, and said composition is substantially free of NCR_PFV2, and optionally, said composition comprises at least about 80%, 90%, 95%, 98%, 99% or 99.5% (by weight) of total NCR peptides, which is DEF_PFV1.

[0132] 2. The composition according to Example 1a or 1b, wherein the first group of disulfide bonds comprises C1-C4 disulfide bonds, C2-C5 disulfide bonds and C3-C6 disulfide bonds.

[0133] 3. The composition according to Example 2, wherein the second group of disulfide bonds comprises disulfide bonds other than C1-C4 disulfide bonds and C2-C5 disulfide bonds.

[0134] 4. The composition according to Example 3, wherein the second group of disulfide bonds comprises C1-C2 disulfide bonds, C3-C6 disulfide bonds and C4-C5 disulfide bonds.

[0135] 5. The composition according to any one of Examples 1a or 1b to 4, wherein the cationic 6-Cys NCR has an isoelectric point between 8.0 and 12.0.

[0136] 6. The composition according to any one of Examples 1a or 1b to 5, wherein the NCR_PFV1 exhibits greater protease resistance than the NCR_PFV2.

[0137] 7. The composition according to any one of Examples 1a or 1b to 6, wherein the first cysteine ​​pair motif is a C1-5AA-C2 motif, the motif comprising, from its N-terminus, five amino acids (A1, A2, A3, A4, and A5) side-attached to an N-terminal C1 and a C-terminal C2 in a continuous sequence.

[0138] 8. The composition according to any one of Examples 1a or 1b to 6, wherein the A5 is: an anionic amino acid selected from the group consisting of Asp (D) and Glu (E); or a cationic amino acid selected from the group consisting of His (H), Arg (R) and Lys (K); or an aliphatic amino acid selected from the group consisting of Gly (G), Ala (A), Val (V), Leu (L), Ile (I) and Pro (P).

[0139] 9. The composition according to any one of Examples 1a or 1b to 6, wherein the A4 is: a cationic amino acid selected from the group consisting of His (H), Arg (R) and Lys (K); or an aliphatic amino acid selected from the group consisting of Gly (G), Ala (A), Val (V), Leu (L), Ile (I) and Pro (P); or an amide amino acid selected from the group consisting of Asn (N) and Gln (Q); or a polar amino acid selected from the group consisting of Ser (S) and Thr (T).

[0140] 10. The composition according to any one of Examples 1a or 1b to 6, wherein the A3 is: a cationic amino acid selected from the group consisting of His (H), Arg (R) and Lys (K); or an anionic amino acid selected from the group consisting of Asp (D) and Glu (E); or a polar amino acid selected from the group consisting of Ser (S) and Thr (T).

[0141] 11. The composition according to any one of Examples 1a or 1b to 6, wherein A5 is an anionic amino acid selected from the group consisting of Asp (D) and Glu (E), wherein A4 is a cationic amino acid selected from the group consisting of His (H), Arg (R) and Lys (K), and wherein A3 is an anionic amino acid selected from the group consisting of Asp (D) and Glu (E).

[0142] 12. The composition according to any one of Examples 1a or 1b to 6, wherein the second cysteine ​​pair motif is a C3-5-10AA-C4 motif, wherein the composition comprises, in a continuous sequence from its N-terminus, five to ten amino acids (A6, A7, A8, A9, A10, A20, A30, A40, A2 ... 10 To A 11 A 12 A 13 A 14 A 15 ).

[0143] 13. The composition according to any one of Examples 1a or 1b to 6, wherein the second cysteine ​​pair motif is a C3-5-10AA-C4 motif, the motif comprising, from its N-terminus, in a continuous sequence of seven amino acids (A6, A7, A8, A9, A10AA, A20AA, A30AA, A40AA, A2 ... 10 A 11 and A 12 ).

[0144] 14. The composition according to any one of Examples 1a or 1b to 6, wherein the A6 is an aliphatic amino acid selected from the group consisting of Gly (G), Ala (A), Val (V), Leu (L), Ile (I) and Pro (P); or a cationic amino acid selected from the group consisting of His (H), Arg (R) and Lys (K).

[0145] 15. The composition according to any one of Examples 1a or 1b to 6, wherein A 12 It is anionic amino acid selected from the group consisting of Asp (D) and Glu (E); or cationic amino acid selected from the group consisting of His (H), Arg (R) and Lys (K); or amide amino acid selected from the group consisting of Asn (N) and Gln (Q).

[0146] 16. The composition according to any one of Examples 1a or 1b to 6, wherein A6 and A 12 These are cationic amino acids selected from the group consisting of His (H), Arg (R), and Lys (K).

[0147] 17. The composition according to any one of Examples 1a or 1b to 6, wherein the third cysteine ​​pair motif is a C5-1AA-C6 motif, the motif comprising an amino acid (A) side-terminated with an N-terminal C5 and a C-terminal C6. 16 ).

[0148] 18. The composition according to any one of Examples 1a or 1b to 6 ，Wherein A 16 It is: cationic amino acids selected from the group consisting of His (H), Arg (R) and Lys (K); aliphatic amino acids selected from the group consisting of Gly (G), Ala (A), Val (V), Leu (L), Ile (I) and Pro (P); or anionic amino acids selected from the group consisting of Asp (D) and Glu (E).

[0149] 19. The composition according to any one of Examples 1a or 1b to 6, wherein A 16 These are cationic amino acids selected from the group consisting of His (H), Arg (R), and Lys (K).

[0150] 20. The composition according to any one of Examples 1a or 1b to 6, wherein the IS1 comprises one to eight amino acids or four to five amino acids.

[0151] 21. The composition according to any one of Examples 1a or 1b to 6, wherein the IS2 comprises four to six amino acids.

[0152] 22. The composition according to any one of Examples 1a or 1b to 6, wherein each of IS1 and IS2 is composed of four amino acids.

[0153] 23. The composition according to any one of Examples 1 to 22, wherein the NCR_PFV1 comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% sequence identity with any one of SEQ ID NO: 8 to 371.

[0154] 24. The composition according to any one of Examples 1 to 23, wherein the composition further comprises an agriculturally, pharmaceutically, or veterinarily feasible carrier, diluent, or excipient.

[0155] 25a. A composition comprising a nodule-specific cysteine-rich NCR peptide fold variant 1 (NCR_PFV1), wherein the NCR peptide is a cationic 4-Cys NCR comprising, from its N-terminus, a sequential amino acid sequence of: (1) a first cysteine ​​pair motif comprising C1 and C2; (2) a first intercalation sequence (IS1); and (3) a second cysteine ​​pair motif comprising C3 and C4, wherein the NCR peptide is associated with CaNCR7 (SEQ ID NO: 2), CaNCR13 (SEQ ID NO: 1), CaNCR14 (SEQ ID NO: 3), or CaNCR15 (SEQ ID NO: 4). 4) Having less than 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity, wherein the cysteine ​​residues C1, C2, C3, and C4 of NCR_PFV1 form a first group of disulfide bonds, and NCR_PFV1 thereby adopts a first conformation exhibiting greater antimicrobial activity, as compared to root nodule-specific cysteine-rich NCR peptide fold variant 2 (NCR_PFV2), wherein NCR_PFV2 has the same amino acid sequence as NCR_PFV1 but forms a different second group of disulfide bonds between cysteine ​​residues C1, C2, C3, C4, C5, and C6 and adopts a different second conformation, and wherein the composition is substantially free of NCR_PFV2, and optionally wherein at least about 80%, 90%, 95%, 98%, 99%, or 99.5% (by weight) of the total NCR peptide in the composition is NCR_PFV1.

[0156] 25b. A composition comprising a root nodule-specific cysteine-rich NCR peptide fold variant 1 (NCR_PFV1), wherein the NCR peptide is a cationic 4-Cys NCR comprising, from its N-terminus, a sequential amino acid sequence of: (1) a first cysteine ​​pair motif comprising C1 and C2; (2) a first intercalation sequence (IS1); and (3) a second cysteine ​​pair motif comprising C3 and C4, wherein the NCR peptide is associated with CaNCR7 (SEQ ID NO: 2), CaNCR13 (SEQ ID NO: 1), CaNCR14 (SEQ ID NO: 3), or CaNCR15 (SEQ ID NO: 4). 4) Having less than 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity, wherein cysteine ​​residues C1, C2, C3, and C4 of NCR_PFV1 form a first group of disulfide bonds, and NCR_PFV1 thereby adopts a first conformation exhibiting greater antimicrobial activity, increased protease resistance, or increased protease sensitivity, as compared to root nodule-specific cysteine-rich NCR peptide fold variant 2 (NCR_PFV2), wherein NCR_PFV2 has the same amino acid sequence as NCR_PFV1 but forms a different second group of disulfide bonds between cysteine ​​residues C1, C2, C3, C4, C5, and C6 and adopts a different second conformation, and wherein the composition is substantially free of NCR_PFV2, optionally wherein at least about 80%, 90%, 95%, 98%, 99%, or 99.5% (by weight) of the total NCR peptide in the composition is NCR_PFV1.

[0157] 26. The composition according to Example 25a or 25b, wherein the first group of disulfide bonds comprises C1-C2 disulfide bonds and C3-C4 disulfide bonds.

[0158] 27. The composition according to Example 26, wherein the second group of disulfide bonds comprises disulfide bonds other than C1-C2 disulfide bonds and C3-C4 disulfide bonds.

[0159] 28. The composition according to Example 27, wherein the second group of disulfide bonds comprises disulfide bonds other than C1-C3 disulfide bonds and C2-C4 disulfide bonds.

[0160] 29. The composition according to Example 25, wherein the first group of disulfide bonds comprises C1-C3 disulfide bonds and C2-C4 disulfide bonds.

[0161] 30. The composition according to Example 29, wherein the second group of disulfide bonds comprises disulfide bonds other than C1-C3 disulfide bonds and C2-C4 disulfide bonds.

[0162] 31. The composition according to Example 25a or 25b, wherein the first group of disulfide bonds comprises C1-C4 disulfide bonds and C2-C3 disulfide bonds.

[0163] 32. The composition according to Example 31, wherein the second group of disulfide bonds comprises disulfide bonds other than C1-C4 disulfide bonds and C2-C3 disulfide bonds.

[0164] 33. The composition according to any one of Examples 25a or 25b to 32, wherein the cationic 4-Cys NCR has an isoelectric point of 8.0 to 12.0.

[0165] 34. The composition according to any one of Examples 25a or 25b to 33, wherein the NCR_PFV1 exhibits greater protease resistance compared to the NCR_PFV2.

[0166] 35. The composition according to any one of Examples 25a or 25b to 33, wherein the first cysteine ​​pair motif is a C1-5AA-C2 motif, the motif comprising, from its N-terminus, five amino acids (A1, A2, A3, A4, and A5) side-attached to an N-terminal C1 and a C-terminal C2 in a continuous sequence.

[0167] 36. The composition according to any one of Examples 25a or 25b to 33, wherein the second cysteine ​​pair motif is a C3-4AA-C4 motif, and wherein it comprises, in a continuous sequence from its N-terminus, five amino acids (A6, A7, A8, and A9) side-attached with an N-terminal C3 and a C-terminal C4, and

[0168] 37. The composition according to any one of Examples 25a or 25b to 33, wherein the IS1 comprises five to thirteen amino acids or five to eleven amino acids.

[0169] 38. The composition according to any one of Examples 25a or 25b to 33, wherein the A5 is an anionic amino acid selected from the group consisting of Asp (D) and Glu (E).

[0170] 39. The composition according to any one of Examples 25a or 25b to 33, wherein the A4 is a cationic amino acid selected from the group consisting of His (H), Arg (R) and Lys (K).

[0171] 40. The composition according to any one of Examples 25a or 25b to 33, wherein the A3 is an anionic amino acid selected from the group consisting of Asp (D) and Glu (E).

[0172] 41. The composition according to any one of Examples 25a or 25b to 33 ， The A6 therein is: a cationic amino acid selected from the group consisting of His (H), Arg (R) and Lys (K); or an aliphatic amino acid selected from the group consisting of Gly (G), Ala (A), Val (V), Leu (L), Ile (I) and Pro (P).

[0173] 42. The composition according to any one of Examples 25a or 25b to 33, wherein the A7 is: a cationic amino acid selected from the group consisting of His (H), Arg (R) and Lys (K); or an amide amino acid selected from the group consisting of Asn (N) and Gln (Q); or an aliphatic amino acid selected from the group consisting of Gly (G), Ala (A), Val (V), Leu (L), Ile (I) and Pro (P).

[0174] 43. The composition according to any one of Examples 25a or 25b to 33, wherein the A8 is: an aliphatic amino acid selected from the group consisting of Gly (G), Ala (A), Val (V), Leu (L), Ile (I) and Pro (P); or an amide amino acid selected from the group consisting of Asn (N) and Gln (Q); or a cationic amino acid selected from the group consisting of His (H), Arg (R) and Lys (K).

[0175] 44. The composition according to any one of Examples 25a or 25b to 33, wherein the NCR_PFV1 comprises an amino acid sequence having at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% sequence identity with any one of SEQ ID NO: 8 to 371, optionally wherein the NCR_PFV1 does not contain the amino acid sequence of NCR169 (SEQ ID NO: 6) or NCR247 (SEQ ID NO: 5).

[0176] 45. The composition according to any one of Examples 25a or 25b to 33, wherein the composition further comprises an agriculturally, pharmaceutically, or veterinarily feasible carrier, diluent, or excipient.

[0177] 46. ​​A method for preparing a composition according to any one of Examples 1a or 1b to 4 or 25a or 25b to 45, the method comprising: (a) separating a fraction containing NCR peptide fold variant 1 (NCR_PFV1) from a mixture comprising NCR_PFV1 and NCR_PFV2 or NCR_PFV2 peptide fragments thereof, and one or more fractions comprising NCR peptide fold variant 2 (NCR_PFV2) or NCR_PFV2 peptide fragments thereof; and (b) combining the fraction containing NCR_PFV1, or a formulation further purified from the NCR_PFV1 fraction, with an agriculturally, pharmaceutically, or veterinarily feasible carrier, diluent, or excipient, thereby preparing the composition.

[0178] 47. The method according to Example 46, the method further comprising obtaining the mixture comprising NCR_PFV1 and NCR_PFV2 or a NCR_PFV2 peptide fragment thereof by: (a) culturing a microorganism expressing a recombinant polynucleotide in a fermentation broth, the recombinant polynucleotide comprising a transcription promoter operatively linked to a polynucleotide encoding a signal peptide co-located with and upstream of the polynucleotide encoding NCR_PFV1 or a variant thereof; and (b) separating the microorganism from the fermentation broth containing the mixture, wherein the microorganism is optionally selected from the group consisting of Candida, Kluyveromyces, Hansenula, Pichia pastoris, Saccharomyces, Schizomyces, or Yersinia, Trichoderma, and Escherichia.

[0179] 48. The method according to Example 46, the method further comprising obtaining the mixture comprising NCR_PFV1 and NCR_PFV2 or a NCR_PFV2 peptide fragment thereof by: (a) culturing a microorganism expressing a recombinant polynucleotide in a fermentation broth, the recombinant polynucleotide comprising a transcription promoter operatively linked to encoding NCR_PFV1 or a variant thereof; (b) separating the microorganism from the fermentation broth, the microorganism comprising the mixture; (c) lysing the microorganism to obtain cell lysates; and (d) separating insoluble fragments in the cell lysates from an aqueous fraction comprising the mixture, wherein the microorganism is optionally selected from the group consisting of Candida, Kluyveromyces, Hansenula, Pichia pastoris, Saccharomyces, Schizosoma, or Yersinia, Trichoderma, and Escherichia.

[0180] 49. The method according to Example 47 or 48, the method further comprising: treating the mixture containing NCR_PFV1 and NCR_PFV2 with a protease under non-denaturing conditions sufficient to produce an NCR_PFV2 peptide fragment but insufficient to produce an NCR_PFV1 peptide fragment.

[0181] 50. The method according to Example 49, wherein the protease is a trypsin family serine protease that cleaves the Arg(R) or Lys(K) end of NCR_PFV2.

[0182] 51. The method according to Example 50, wherein the trypsin family serine protease is a recombinant bovine, porcine, human, or microbial trypsin, and optionally, wherein the microbial trypsin is streptomycin trypsin or a variant thereof.

[0183] 52. The method according to Example 49, the method further comprising separating NCR_PFV1 from the NCR_PFV2 peptide fragment by size exclusion chromatography.

[0184] 53. A method for preventing or reducing crop damage or post-harvest loss caused by plant pathogenic microorganisms, the method comprising: contacting a plant, plant seeds, pre- or post-harvest grains, pre- or post-harvest fruits or vegetables with an effective amount of the composition according to any one of Examples 1a or 1b to 24 or 25a or 25b to 44, and the contact being under conditions suitable for preventing or reducing crop damage or post-harvest loss.

[0185] 54. The method according to Example 53, wherein the plant pathogenic microorganism is selected from the group consisting of Fusarium, Alternaria, Verticillium, Phytophthora, Anthracnose, Botrytis, Cercospora, Laminaria, Rhizoctonia, Sclerotium, Pythium, Stem-point mold, Micrococcus, Top-shell mold, Styloides, Sclerotium, Spp., Penicillium, Trichoderma, Pseudomonas, Erythromyces, Erythromyces, White rust, Trichoderma, Spiralocystis, Helicobacter, Monosporus, or Stenoptera.

[0186] 55. The method according to Example 53, wherein the plant, plant seed, pre- or post-harvest grain, pre- or post-harvest fruit or vegetable is selected from the group consisting of cereal crops, legume crops, root or tuber crops, oil crops, fruit crops, vegetable crops, nut crops, forage or turfgrass crops, forage legumes, medicinal crops, spice or flavoring crops, fiber crops and biofuel crops, plant seed, pre- or post-harvest grain, pre- or post-harvest fruit or vegetable.

[0187] 56. The method according to Example 53, wherein the plant, plant seed, pre- or post-harvest grain, pre- or post-harvest fruit or vegetable is selected from the group consisting of wheat, rice, corn, barley, oats, sorghum, rye, millet, peanut, chickpea, pigeon pea, lentil, kidney bean, soybean, lima bean, potato, sweet potato, cassava, rapeseed, peanut, palm, coconut, safflower, cotton, sunflower, flax, olive, sugarcane, beet, banana, orange, apple, pear, breadfruit, pineapple, strawberry, grape, cherry, coffee, cocoa, tomato, lettuce, carrot, melon, asparagus, Brassica sp., cashew, walnut, pistachio, almond, alfalfa, clover, castor bean, flaxseed, iris, switchgrass, Miscanthus and Jatropha, plant seed, pre- or post-harvest grain, pre- or post-harvest fruit or vegetable.

[0188] 57. A medical device having antimicrobial properties, the medical device comprising a medical device operatively combinable with a composition according to any one of Examples 1a or 1b to 24 or 25a or 25b to 44, wherein the medical device comprises at least one surface that is locally coated or impregnated with the composition.

[0189] 58. The medical device according to embodiment 57, wherein the medical device is selected from the group consisting of a stent, a catheter, a contact lens, a condom, a patch, and a diaphragm.

[0190] 59. A method for treating, preventing or inhibiting a microbial infection in a subject who requires such treatment, the method comprising: administering to the subject a therapeutically effective amount of the composition according to any one of Examples 1a or 1b to 4 or 25a or 25b to 44, under conditions and for a duration suitable for treating, preventing or inhibiting the microbial infection.

[0191] 60. The method according to Example 59, wherein the administration comprises topically, intravenously, parenterally, or intravenously introducing the composition to the subject.

[0192] 61. The method according to Example 59, wherein the subjects are selected from a group consisting of humans, livestock, poultry, fish and companion animals.

[0193] 62. The method according to Example 59, wherein the microbial infection is a microbial infection of mucous membranes, eyes, skin or nails, and the composition is applied to the mucous membranes, eyes, skin or nails.

[0194] 63. The method according to Example 59, wherein the microbial infection is caused by dermatophytes, and wherein the dermatophytes are optionally selected from the group consisting of: Trichophyton rubrum, Trichophyton interdigitale, Trichophyton violaceum, Trichophyton tonsurans, Trichophyton sulphureus, Trichophyton mentagrophytes, Microsporum xanthosporum, Epidermophyton floccosum, and Microsporum gypseum.

[0195] 64. The method according to Example 59, wherein the microbial infection is caused by microorganisms selected from the group consisting of Aspergillus, Cryptococcus, Penicillium, Rhizopus, Squamata, Cladosporium, Hypocreosporium, Rhizopus, Coccidioides, Mucor, Pythium, Fusarium, Histoplasmosis, and Blastomyces.

[0196] 65. The method according to Example 64, wherein the microbial infection is caused by a microorganism selected from the group consisting of the genus Candida, wherein the genus Candida is selected from the group consisting of Candida albicans, Candida auris, Candida glabrata, Candida parapsilosis, Candida tropicalis, and Candida krusei.

[0197] 66. The composition according to Examples 1a or 1b to 24 or 25a or 25b to 44, in a method of treating, preventing or inhibiting microbial infections in a subject with such need.

[0198] 67. The composition according to Example 66, wherein the subject is selected from the group consisting of humans, livestock, poultry, fish and companion animals.

[0199] 68. A plant part, said plant part being at least partially coated with a composition according to any one of Examples 1a or 1b to 24 or 25a or 25b to 44.

[0200] 69. The plant portion according to Example 68, wherein the plant portion is selected from the group consisting of seeds and pre- or post-harvest grains.

[0201] 70. The plant portion according to Example 68, wherein the plant portion is selected from the group consisting of pre- or post-harvest fruits, pre- or post-harvest vegetables, and pre- or post-harvest flowers.

[0202] 71. The plant portion according to Example 68, wherein the plant portion is selected from the group consisting of cereal crops, legume crops, root or tuber crops, oil crops, melon and fruit crops, vegetable crops, nut crops, forage or turfgrass crops, forage legumes, medicinal crops, spice or flavoring crops, fiber crops and biofuel crops.

[0203] 72. The plant portion according to Example 68, wherein the plant portion is selected from the group consisting of wheat, rice, corn, barley, oats, sorghum, rye, millet, peanut, chickpea, pigeon pea, lentil, kidney bean, soybean, lima bean, potato, sweet potato, cassava, rapeseed, peanut, palm, coconut, safflower, cotton, sunflower, flax, olive, sugarcane, beet, banana, orange, apple, pear, breadfruit, pineapple, strawberry, grape, cherry, coffee, cocoa, tomato, lettuce, carrot, melon, asparagus, brassica, cashew, walnut, pistachio, almond, alfalfa, clover, castor bean, flaxseed, iris, switchgrass, Miscanthus, and Jatropha.

[0204] * * * * *

[0205] The term “and / or” as used herein should be understood to explicitly disclose each of two specified features or components, with or without the other. Therefore, the term “and / or” as used 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” as used in phrases such as “A, B, and / or C” is intended to cover 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).

[0206] Where terms are provided in the singular form, other embodiments described by the plural form of the term are also provided. As used herein, the terms “include,” “includes,” and “including” should be interpreted as having at least the features they refer to, without excluding any additional unspecified features. It should be understood that, unless otherwise stated, terms intended to be “open” (e.g., the term “includes” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “comprising” should be interpreted as “including but not limited to,” etc.). Phrases such as “at least one” and “one or more,” and terms such as “a” or “an” include both singular and plural forms.

[0207] It should be further understood that when a feature or aspect of this disclosure is described in the form of a Markush group, this disclosure is also intended to be described in the form of any single member or subgroup of members of the Markush group. Similarly, all scopes disclosed herein also cover all possible subscopes and combinations of subscopes, and languages ​​such as “between,” “at most,” “at least,” “greater than,” and “less than” include numbers referenced in the scope and include each individual member.

[0208] All references cited in this article, whether above or below, including but not limited to patents, patent applications and patent publications (whether U.S., PCT or foreign patents outside the U.S.), and all technical and / or scientific publications, are incorporated herein by reference in their entirety.

[0209] Example

[0210] While various embodiments have been disclosed herein, other embodiments will be apparent to those skilled in the art. The disclosed aspects and embodiments are for illustrative purposes and are not intended to be limiting, and the true scope and spirit are indicated by the claims. This disclosure may be further described with reference to the following embodiments, which are intended to illustrate certain embodiments and are not intended to limit the scope of this disclosure or the claimed subject matter.

[0211] Example 1: Heterologous expression and purification of NCR_PFV1 and NCR_PFV2

[0212] Synthetic NCR genes encoding 6-Cys or 4-Cys NCR peptides (see Tables 1 and 2) were cloned into a linearized pPICZα-A integration vector, co-located with an α-matching factor secretion signal sequence containing a KEX2 cleavage site but lacking a Glu-Ala repeat sequence, and expressed in Pichia pastoris.

[0213] 6-Cys or 4-Cys NCR peptides were purified using CM-Sephadex C-25 cation exchange chromatography and C18 reversed-phase HPLC with slight modifications, such as those used in Sagaram and PLoS One. 6(4) As described in :e18550 (2011). Cells were harvested by centrifugation at 6,000 rpm for 20 min at 4 °C, and the pH of the supernatant was adjusted to 6.0. A cation exchange resin (CM-Sephadex C-25, Sigma, catalog number: C25120) equilibrated with binding buffer (25 mM anhydrous sodium acetate, pH 6.0) was added to the supernatant and incubated overnight at 110 rpm at 4 °C. After collection and washing of the resin with binding buffer, bound proteins were eluted using AKTA FPLC with elution buffer (1 M NaCl, 50 mM Tris, pH 7.6). FPLC fractions containing 6-Cys or 4-Cys NCR peptides were concentrated using an Amicon Ultra-15 centrifuge and filtration system. The concentrated fractions were dialyzed with 10 mM Tris, pH 7.6 and further purified by reversed-phase C18-HPLC.

[0214] Observe the peaks and collect fractions containing NCR_PFV1 (peak 1) and NCR_PFV2 (peak 2) using the methods provided in the Agilent HPLC System Manual (Agilent Technologies Part No. G1380-90000 (1999)). Lyophilize the HPLC fractions containing NCR_PFV1 (peak 1) and NCR_PFV2 (peak 2) peptides and resuspend them in nuclease-free water. Determine the concentrations of NCR_PFV1 and NCR_PFV2 using the BCA method according to the manufacturer's protocol (Thermo-Fisher Scientific, Inc.® "Protein Assay Manual"). Verify the purity and size of NCR_PFV1 and NCR_PFV2 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Perform NMR analysis or protease treatment and LC-MS on the NCR_PFV1 and NCR_PFV2 peptides to determine the disulfide pairs present in each peptide.

[0215] Example 2: Fungal cultures and spore suspensions

[0216] The fungal strain of *Botrytis cinerea* T-4 was cultured in 20% V8 growth medium. *Alternaria* was grown on potato dextrose agar. Fungal spores were harvested by immersing the fungal growth plates in sterile water. The spore suspension was filtered through two layers of Magic Cloth, centrifuged at 13,600 rpm for 1 min, washed, and resuspended in low-salt synthetic fungal medium (SFM) (see U.S. Patent No. 6,916,970). The spore suspension was adjusted to an equivalent spore density using a hemocytometer.

[0217] In the presence of 5 ml of sterile water, conidia of *Cercospora zumi* and *Alternaria alternata* were harvested from fully grown culture plates using an L-shaped spreader. To remove mycelial debris, the spore suspension was filtered through a triple-layered filter cloth and washed twice with sterile water at 13,000 rpm for 2 minutes each time. The spore concentration was adjusted to 1 x 10⁻⁶ using a hemocytometer. 5 Spores / ml.

[0218] Sclerotinia sclerotiorum 555 fungal culture was cultured at room temperature on potato dextrose agar (PDA) medium (BDDiagnostics, Sparks, MD "Difco"). TM It grows on the BBLTM manual, second edition.

[0219] Example 3: Determination of the minimum inhibitory concentration (MIC) of 6-Cys or 4-Cys NCR_PFV1 and NCR_PFV2 in the absence and presence of cations.

[0220] The antifungal activity of 6-Cys or 4-Cys NCR_PFV1 and NCR_PFV2 was evaluated at different concentrations using a 2-fold dilution series of each peptide. The antifungal activity of each peptide was determined spectrophotometrically using a 96-well plate assay (Sagaram, PLoS One 6(4):e18550 (2011) and Sagaram, PLoS One 8(12):e82485 (2013)). Approximately 45 µL of each peptide at different concentrations was added to approximately 10 μL of each well plate containing 45 µL of each peptide. 5 1 spore / ml spore suspension was added to each well of a microtiter plate. After 48 hours, quantitative fungal growth inhibition was determined by measuring absorbance at 595 nm using a microplate reader (Tecan Infinite® M200 Pro, Tecan Systems Inc., San Jose, California). Fungal cell viability was determined by the resplenoid cell viability assay (see Li, MPMI). 32 :1649–1664 (2019) and Velivelli, PNAS 117(27) :16043 (2020)).

[0221] After incubating the pathogen / peptide mixture for 48 hours, 10 µl of 0.1% resazurin solution was added to each well. After incubation overnight, the resazurin dye changed color from blue to pink or colorless, indicating the presence of viable fungal cells. The MIC for each peptide is the lowest concentration at which the blue color does not change. Using this protocol, the MIC values ​​for 6-Cys or 4-Cys NCR_PFV1 and NCR_PFV2 were determined in the presence of 100 mM NaCl and 2 mM CaCl2.

[0222] The MICs of 6-Cys or 4-Cys NCR_PFV1 and NCR_PFV2 against *Botrytis cinerea* were determined in 2X SFM to ascertain antifungal activity. It is hypothesized that antifungal peptides significantly lose their antifungal activity in the presence of cations because the electrostatic interaction between the positively charged peptide and the negatively charged fungal membrane is significantly weakened in the presence of cations. Therefore, the antifungal activity of 6-Cys or 4-Cys NCR_PFV1 and NCR_PFV2 can be determined in SFM supplemented with 100 mM NaCl or 2 mM CaCl2. The loss of antifungal activity in the presence of NaCl or CaCl2 indicates that the antifungal activity is salt-sensitive.

[0223] Example 4: In vivo antifungal activity of 6-Cys or 4-Cys NCR_PFV against Sclerotinia sclerotiorum 555

[0224] For the semi-in vivo antifungal assay, a 1 mm plug was taken from the leading edge of a fungal colony on freshly cultured 2-day-old PDA medium and placed on isolated soybean leaves and pods (6 weeks old) at approximately 3–4 weeks of age. Immediately afterward, 40 μL of various concentrations of NCR_PFV solution or water was applied to the plug, and the plant was incubated at high humidity for 2–3 days prior to evaluation. Lesion severity on each leaf was assessed using the CropReporter system, as described in Li et al., Mol Plant Microbe Interact. Dec 2019;32(12):1649-1664. doi: 10.1094 / MPMI-08-19-0224-R. Epub Oct 2019. PMID: 31425003. High-resolution fluorescence images could be captured using the CropReporter (PhenoVation, Wageningen, Netherlands).

[0225] Example 5: Antifungal activity of 6-Cys or 4-Cys NCR_PFV1 and PFV2 against Botrytis cinerea infection in tomato fruits

[0226] Antifungal activity was determined on commercially available tomato fruits. Tomato fruits were washed with 0.01% bleach and rinsed three times with sterile distilled water. Corundum powder (Fisher Chemical, USA) was gently rubbed onto the fruit surface to create micro-damage. 20 µl of Botrytis cinerea conidia (approximately 1 x 10⁻⁶) were inoculated by drip inoculation. 5 Conidia (one conidia / ml) were inoculated onto the fruit. Conidia of *Botrytis cinerea* were prepared as described above. After drip inoculation, the fruit was incubated in a Ziploc weatherproof box containing damp paper towels to maintain high humidity. 12 hours post-inoculation (hpi), 20 µl of a peptide solution (0.75 µM 6-Cys or 4-Cys NCR_PFV1 or NCR_PFV2) was applied to the drip-inoculated conidia suspension and incubated for 5 days. Fungal growth on the fruit was observed daily.

[0227] Example 6: NCR169 expression in Pichia pastoris produced only one disulfide bond variant with no antifungal activity.

[0228] A codon-optimized synthetic gene for NCR169 (SEQ ID NO: 6), cloned into the pPICZalphaA vector, was received from GenScript. The encoded peptide has an additional alanine residue at its N-terminus (i.e., the polypeptide containing SEQ ID NO: 6 has an additional alanine residue at its N-terminus). The recombinant plasmid was linearized with SacI, and single colonies were inoculated into 50 mL of YPD broth for transformation of Pichia pastoris X-33 and cultured overnight on a rotary shaker at 30°C and 225 rpm. The Pichia pastoris culture was then inoculated into 500 mL of buffered basal glycerol medium and cultured overnight on a rotary shaker at 30°C and 225 rpm. After 48 hours of growth, cells were pelleted by centrifugation at 3,500 rpm for 20 min at room temperature (RT) and resuspended in 1,000 mL of buffered methanol compound medium. The cultures were grown at 25°C for 5 days, with 5 mL of methanol added every 24 hours to maintain gene expression induction. After induction, cells were centrifuged at 3,000 rpm for 10 minutes to precipitate them, and the supernatant was retained. The pH of the supernatant was adjusted to 6.0 using 10 M NaOH, and CM-Sephadex C-25 cation exchange resin was added to bind proteins. The culture was incubated for 2 days at 4°C and 110 rpm in a shaker. The slurry was poured through a Miracloth® into a Buchner funnel, and the resin was collected and loaded into an FPLC (Rapid Protein Liquid Chromatography) column. The resin was thoroughly washed with binding buffer, and then the bound proteins were eluted in 1 M NaCl and 50 mM Tris (pH 7.6). The FPLC fraction was collected and concentrated at 4°C using an Amicon stirred cell concentrator (Cole-Parmer, Vernon Hills, IL) with a molecular weight cutoff (MWCO) of 3,000. The concentrated fraction was dialyzed in the tube with 10 mM Tris (pH 7.6). The dialysis buffer was used for HPLC analysis with a retention time of 32 minutes. The HPLC eluent was lyophilized. The lyophilized protein was resuspended in nuclease-free water, and the protein concentration was determined using a Nanodrop (NanoDrop™ 2000 / 2000c spectrophotometer). HPLC analysis of NCR169 expressed in Pichia pastoris showed only one peak. The purified peptide at a concentration of 12 µM was found to have no antifungal activity against Botrytis cinerea.

Claims

1. A composition comprising a root nodule-specific cysteine-rich NCR peptide fold variant 1 (NCR_PFV1), The NCR peptide is a cationic 6-Cys NCR peptide comprising, from its N-terminus, a continuous amino acid sequence of: (1) a first cysteine ​​pair motif comprising C1 and C2; (2) a first intercalation sequence (IS1); (3) a second cysteine ​​pair motif comprising C3 and C4; (4) a second intercalation sequence (IS2); and (5) a third cysteine ​​pair motif comprising C5 and C6, wherein the NCR peptide has less than 60% sequence identity with CaNCR7 (SEQ ID NO: 2), CaNCR13 (SEQ ID NO: 1), CaNCR14 (SEQ ID NO: 3), or CaNCR15 (SEQ ID NO: 4). In NCR_PFV1, cysteine ​​residues C1, C2, C3, C4, C5, and C6 form a first set of disulfide bonds, and NCR_PFV1 thus adopts a first conformation exhibiting greater antimicrobial activity, as compared to root nodule-specific cysteine-rich NCR peptide fold variant 2 (NCR_PFV2). NCR_PFV2 has the same amino acid sequence as NCR_PFV1 but forms a different second set of disulfide bonds between cysteine ​​residues C1, C2, C3, C4, C5, and C6 and adopts a different second conformation. The composition therein is substantially free of NCR_PFV2.

2. The composition according to claim 1, wherein the first group of disulfide bonds comprises C1-C4 disulfide bonds, C2-C5 disulfide bonds, and C3-C6 disulfide bonds.

3. The composition according to claim 2, wherein the second group of disulfide bonds comprises disulfide bonds other than C1-C4 disulfide bonds and C2-C5 disulfide bonds.

4. The composition according to claim 3, wherein the second group of disulfide bonds comprises C1-C2 disulfide bonds, C3-C6 disulfide bonds, and C4-C5 disulfide bonds.

5. The composition according to any one of claims 1 to 4, wherein the cationic 6-Cys NCR has an isoelectric point between 8.0 and 12.

0.

6. The composition according to any one of claims 1 to 4, wherein the NCR_PFV1 exhibits greater protease resistance than the NCR_PFV2.

7. The composition according to any one of claims 1 to 4, wherein the first cysteine ​​pair motif is a C1-5AA-C2 motif, the motif comprising, from its N-terminus, five amino acids (A1, A2, A3, A4, and A5) side-attached to an N-terminal C1 and a C-terminal C2 in a continuous sequence.

8. The composition according to any one of claims 1 to 4, wherein the A5 is: an anionic amino acid selected from the group consisting of Asp (D) and Glu (E); or a cationic amino acid selected from the group consisting of His (H), Arg (R) and Lys (K); or an aliphatic amino acid selected from the group consisting of Gly (G), Ala (A), Val (V), Leu (L), Ile (I) and Pro (P).

9. The composition according to any one of claims 1 to 4, wherein the A4 is: a cationic amino acid selected from the group consisting of His (H), Arg (R) and Lys (K); or an aliphatic amino acid selected from the group consisting of Gly (G), Ala (A), Val (V), Leu (L), Ile (I) and Pro (P); or an amide amino acid selected from the group consisting of Asn (N) and Gln (Q); or a polar amino acid selected from the group consisting of Ser (S) and Thr (T).

10. The composition according to any one of claims 1 to 4, wherein the A3 is: a cationic amino acid selected from the group consisting of His (H), Arg (R) and Lys (K); or an anionic amino acid selected from the group consisting of Asp (D) and Glu (E); or a polar amino acid selected from the group consisting of Ser (S) and Thr (T).

11. The composition according to any one of claims 1 to 4, wherein A5 is an anionic amino acid selected from the group consisting of Asp (D) and Glu (E), wherein A4 is a cationic amino acid selected from the group consisting of His (H), Arg (R) and Lys (K), and wherein A3 is an anionic amino acid selected from the group consisting of Asp (D) and Glu (E).

12. The composition according to any one of claims 1 to 4, wherein the second cysteine ​​pair motif is a C3-5-10AA-C4 motif, the motif comprising, from its N-terminus, five to ten amino acids (A6, A7, A8, A9, A10, A20, A30, A40, A2 ... 10 To A 11 A 12 A 13 A 14 A 15 ).

13. The composition according to any one of claims 1 to 4, wherein the second cysteine ​​pair motif is a C3-5-10AA-C4 motif, the motif comprising, from its N-terminus, a continuous sequence of seven amino acids (A6, A7, A8, A9, A10, A20, A30, A40, A2 ... 10 A 11 and A 12 ).

14. The composition according to any one of claims 1 to 4, wherein the A6 is: an aliphatic amino acid selected from the group consisting of Gly (G), Ala (A), Val (V), Leu (L), Ile (I) and Pro (P); or a cationic amino acid selected from the group consisting of His (H), Arg (R) and Lys (K).

15. The composition according to any one of claims 1 to 4, wherein the A 12 It is: anionic amino acids selected from the group consisting of Asp (D) and Glu (E); or cationic amino acids selected from the group consisting of His (H), Arg (R) and Lys (K); or amide amino acids selected from the group consisting of Asn (N) and Gln (Q).

16. The composition according to any one of claims 1 to 4, wherein the A6 and the A 12 These are cationic amino acids selected from the group consisting of His (H), Arg (R), and Lys (K).

17. The composition according to any one of claims 1 to 4, wherein the third cysteine ​​pair motif is a C5-1AA-C6 motif, the motif comprising an amino acid (A) side-attached to an N-terminal C5 and a C-terminal C6. 16 ).

18. The composition according to any one of claims 1 to 4, wherein the A 16 It is: cationic amino acids selected from the group consisting of His (H), Arg (R) and Lys (K); aliphatic amino acids selected from the group consisting of Gly (G), Ala (A), Val (V), Leu (L), Ile (I) and Pro (P); or anionic amino acids selected from the group consisting of Asp (D) and Glu (E).

19. The composition according to any one of claims 1 to 4, wherein the A 16 These are cationic amino acids selected from the group consisting of His (H), Arg (R), and Lys (K).

20. The composition according to any one of claims 1 to 4, wherein the IS1 comprises one to eight amino acids or four to five amino acids.

21. The composition according to any one of claims 1 to 4, wherein the IS2 comprises four to six amino acids.

22. The composition according to any one of claims 1 to 4, wherein each of IS1 and IS2 is composed of four amino acids.

23. The composition according to any one of claims 1 to 4, wherein the NCR_PFV1 comprises an amino acid sequence having at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% sequence identity with any one of SEQ ID NO: 8 to 371.

24. The composition according to any one of claims 1 to 4, wherein the composition further comprises an agriculturally, pharmaceutically, or veterinarily feasible carrier, diluent, or excipient.

25. A composition comprising a root nodule-specific cysteine-rich NCR peptide fold variant 1 (NCR_PFV1), The NCR peptide is a cationic 4-Cys NCR, which comprises, from its N-terminus, a continuous amino acid sequence comprising: (1) a first cysteine ​​pair motif including C1 and C2; (2) a first intercalation sequence (IS1); and (3) a second cysteine ​​pair motif including C3 and C4, and wherein the NCR peptide has less than 60% sequence identity with CaNCR7 (SEQ ID NO: 2), CaNCR13 (SEQ ID NO: 1), CaNCR14 (SEQ ID NO: 3), or CaNCR15 (SEQ ID NO: 4). In NCR_PFV1, cysteine ​​residues C1, C2, C3, and C4 form a first set of disulfide bonds, and NCR_PFV1 thus adopts a first conformation exhibiting greater antimicrobial activity, as compared to root nodule-specific cysteine-rich NCR peptide fold variant 2 (NCR_PFV2). NCR_PFV2 has the same amino acid sequence as NCR_PFV1 but forms a different second set of disulfide bonds between cysteine ​​residues C1, C2, C3, C4, C5, and C6, and adopts a different second conformation. The composition therein is substantially free of NCR_PFV2.

26. The composition of claim 25, wherein the first group of disulfide bonds comprises C1-C2 disulfide bonds and C3-C4 disulfide bonds.

27. The composition of claim 26, wherein the second group of disulfide bonds comprises disulfide bonds other than C1-C2 and C3-C4 disulfide bonds.

28. The composition of claim 27, wherein the second group of disulfide bonds comprises C1-C3 disulfide bonds and C2-C4 disulfide bonds.

29. The composition of claim 25, wherein the first group of disulfide bonds comprises C1-C3 disulfide bonds and C2-C4 disulfide bonds.

30. The composition of claim 29, wherein the second group of disulfide bonds comprises disulfide bonds other than C1-C3 and C2-C4 disulfide bonds.

31. The composition of claim 25, wherein the first group of disulfide bonds comprises C1-C4 disulfide bonds and C2-C3 disulfide bonds.

32. The composition of claim 31, wherein the second group of disulfide bonds comprises disulfide bonds other than C1-C4 disulfide bonds and C2-C3 disulfide bonds.

33. The composition according to any one of claims 25 to 32, wherein the cationic 4-Cys NCR has an isoelectric point of 8.0 to 12.

0.

34. The composition according to any one of claims 25 to 32, wherein the NCR_PFV1 exhibits greater protease resistance compared to the NCR_PFV2.

35. The composition according to any one of claims 25 to 32, wherein the first cysteine ​​pair motif is a C1-5AA-C2 motif, the motif comprising, from its N-terminus, in a continuous sequence of five amino acids (A1, A2, A3, A4, and A5) side-attached to an N-terminal C1 and a C-terminal C2.

36. The composition according to any one of claims 25 to 32, wherein the second cysteine ​​pair motif is a C3-4AA-C4 motif, the motif comprising, from its N-terminus, in a continuous sequence of four amino acids (A6, A7, A8, and A9) side-attached to an N-terminal C3 and a C-terminal C4, and.

37. The composition according to any one of claims 25 to 32, wherein the IS1 comprises five to thirteen amino acids or five to eleven amino acids.

38. The composition according to any one of claims 25 to 32, wherein the A5 is an anionic amino acid selected from the group consisting of Asp (D) and Glu (E).

39. The composition according to any one of claims 25 to 32, wherein the A4 is a cationic amino acid selected from the group consisting of His (H), Arg (R) and Lys (K).

40. The composition according to any one of claims 25 to 32, wherein the A3 is an anionic amino acid selected from the group consisting of Asp (D) and Glu (E).

41. The composition according to any one of claims 25 to 32, wherein the A6 is: a cationic amino acid selected from the group consisting of His (H), Arg (R) and Lys (K); or an aliphatic amino acid selected from the group consisting of Gly (G), Ala (A), Val (V), Leu (L), Ile (I) and Pro (P).

42. The composition according to any one of claims 25 to 32, wherein the A7 is: a cationic amino acid selected from the group consisting of His (H), Arg (R) and Lys (K); or an amide amino acid selected from the group consisting of Asn (N) and Gln (Q); or an aliphatic amino acid selected from the group consisting of Gly (G), Ala (A), Val (V), Leu (L), Ile (I) and Pro (P).

43. The composition according to any one of claims 25 to 32, wherein the A8 is: an aliphatic amino acid selected from the group consisting of Gly (G), Ala (A), Val (V), Leu (L), Ile (I) and Pro (P); or an amide amino acid selected from the group consisting of Asn (N) and Gln (Q); or a cationic amino acid selected from the group consisting of His (H), Arg (R) and Lys (K).

44. The composition according to any one of claims 25 to 32, wherein the NCR_PFV1 comprises an amino acid sequence having at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% sequence identity with any one of SEQ ID NO: 8 to 371, optionally wherein the NCR_PFV1 does not contain the amino acid sequence of NCR169 (SEQ ID NO: 6) or NCR247 (SEQ ID NO: 5).

45. The composition according to any one of claims 25 to 32, wherein the composition further comprises an agriculturally, pharmaceutically, or veterinarily feasible carrier, diluent, or excipient.

46. ​​A method for preparing a composition according to any one of claims 1 to 4 or 25 to 32, the method comprising: (a) Separating a fraction containing NCR_PFV1 from a mixture containing NCR peptide fold variant 1 (NCR_PFV1) and NCR peptide fold variant 2 (NCR_PFV2) or a fragment of NCR_PFV2 thereof; and (b) The composition is prepared by combining the fraction containing NCR_PFV1, or a formulation further purified from the NCR_PFV1 fraction, with an agriculturally, pharmaceutically, or veterinarily feasible carrier, diluent, or excipient.

47. The method of claim 46, further comprising obtaining the mixture comprising NCR_PFV1 and NCR_PFV2 or an NCR_PFV2 peptide fragment thereof by: (a) Culturing microorganisms expressing recombinant polynucleotides in fermented broth, the recombinant polynucleotides comprising a transcription promoter operatively linked to a polynucleotide encoding a signal peptide, the signal peptide being co-located with and upstream of a polynucleotide encoding NCR_PFV1 or a variant thereof, and (b) Separating the microorganisms from the fermented broth, the fermented broth containing the mixture. The microorganisms mentioned therein are optionally selected from the group consisting of Candida, Kluyveromyces, Hansenula, Pichia pastoris, yeast, fissilla, or Yersinia, Trichoderma and Escherichia.

48. The method of claim 46, further comprising obtaining the mixture comprising NCR_PFV1 and NCR_PFV2 or an NCR_PFV2 peptide fragment thereof by: (a) Culturing microorganisms expressing recombinant polynucleotides in fermented broth, said recombinant polynucleotides comprising a transcription promoter operatively linked to a polynucleotide or a variant thereof encoding NCR_PFV1, and (b) Separating the microorganisms from the fermented broth, the microorganisms comprising the mixture. (c) Disrupt the microorganisms to obtain cell lysates, and (d) Separate the insoluble fragments from the cell lysate from the aqueous fraction containing the mixture. The microorganisms mentioned therein are optionally selected from the group consisting of Candida, Kluyveromyces, Hansenula, Pichia pastoris, yeast, fissilla, or Yersinia, Trichoderma and Escherichia.

49. The method according to claim 47 or 48, wherein the method further comprises: The mixture containing NCR_PFV1 and NCR_PFV2 was treated with a protease under non-denaturing conditions sufficient to produce NCR_PFV2 peptide fragments but insufficient to produce NCR_PFV1 peptide fragments.

50. The method of claim 49, wherein the protease is a trypsin family serine protease that cleaves the NCR_PFV2 at the carboxyl terminus of the Arg(R) or Lys(K) of the NCR_PFV2.

51. The method of claim 50, wherein the trypsin family serine protease is a recombinant bovine, porcine, human, or microbial trypsin, and optionally, wherein the microbial trypsin is streptomycin trypsin or a variant thereof.

52. The method of claim 49, further comprising separating NCR_PFV1 and NCR_PFV2 peptide fragments by size exclusion chromatography.

53. A method for preventing or reducing crop damage or post-harvest losses caused by plant pathogenic microorganisms, the method comprising: The plant, plant seeds, pre- or post-harvest grains, pre- or post-harvest fruits, or pre- or post-harvest vegetables are brought into contact with an effective amount of the composition according to any one of claims 1 to 4 or 25 to 32, and the contact is made under conditions suitable for preventing or reducing crop damage or post-harvest loss.

54. The method according to claim 53, wherein the plant pathogenic microorganism is selected from the group consisting of Fusarium, Alternaria, Verticillium, Phytophthora, Anthracnose, Botrytis, Cercospora, Laminaria, Rhizoctonia, Sclerotium, Pythium, Stem-point mold, Micrococcus, Capillaris, Styloides, Sclerotium, Spp., Penicillium, Trichoderma, Pseudomonas, Powdery mildew, Erythrinae, Erythrinae, White rust, Trichoderma, Spiralocystis, Helicobacter, Monosporus, or Stenoptera.

55. The method of claim 53, wherein the plant, plant seed, pre- or post-harvest cereal, pre- or post-harvest fruit, or pre- or post-harvest vegetable is selected from the group consisting of cereal crops, legume crops, root or tuber crops, oil crops, fruit crops, vegetable crops, nut crops, forage or turfgrass crops, forage legumes, medicinal crops, spice or flavoring crops, fiber crops, and biofuel crops.

56. The method according to claim 53, wherein the plant, plant seed, pre- or post-harvest grain, pre- or post-harvest fruit, or pre- or post-harvest vegetable is selected from the group consisting of wheat, rice, corn, barley, oats, sorghum, rye, millet, peanut, chickpea, pigeon pea, lentil, kidney bean, soybean, lima bean, potato, sweet potato, cassava, rapeseed, peanut, palm, coconut, safflower, cotton, sunflower, flax, olive, sugarcane, beet, banana, orange, apple, pear, breadfruit, pineapple, strawberry, grape, cherry, coffee, cocoa, tomato, lettuce, carrot, melon, asparagus, Brassica sp., cashew, walnut, pistachio, almond, alfalfa, clover, castor bean, flaxseed, iris, switchgrass, Miscanthus and Jatropha, plant seed, pre- or post-harvest grain, pre- or post-harvest fruit, or pre- or post-harvest vegetable.

57. A medical device having antimicrobial properties, the medical device comprising a medical device operatively combinable with a composition according to any one of claims 1 to 4 or 25 to 32, wherein the medical device comprises at least one surface that is locally coated or impregnated with the composition.

58. The medical device of claim 57, wherein the medical device is selected from the group consisting of a stent, a catheter, a contact lens, a condom, a patch, and a diaphragm.

59. A method for treating, preventing, or inhibiting a microbial infection in a subject with such need, the method comprising: Under conditions and for a duration suitable for treating, preventing or inhibiting the microbial infection, the subject is given a therapeutically effective amount of the composition according to any one of claims 1 to 4 or 25 to 32.

60. The method of claim 59, wherein the administration comprises topically, intravenously, parenterally, or intravenously introducing the composition into the subject.

61. The method of claim 59, wherein the subject is selected from the group consisting of humans, livestock, poultry, fish and companion animals.

62. The method of claim 59, wherein the microbial infection is a microbial infection of mucous membranes, eyes, skin, or nails, and the composition is applied to the mucous membranes, eyes, skin, or nails.

63. The method of claim 59, wherein the microbial infection is caused by dermatophytes, and wherein the dermatophytes are optionally selected from the group consisting of: Trichophyton rubrum, Trichophyton interdigitale, Trichophyton violaceum, Trichophyton tonsurans, Trichophyton sulphureus, Trichophyton mentagrophytes, Microsporum xanthosporum, Epidermophyton floccosum, and Microsporum gypseum.

64. The method according to claim 59, wherein the microbial infection is caused by a microorganism selected from the group consisting of Aspergillus, Cryptococcus, Penicillium, Rhizopus, Squamata, Cladosporium, Hypocreosporium, Rhizopus, Coccidioides, Mucor, Pythium, Fusarium, Histoplasmosis, and Blastomyces.

65. The method of claim 64, wherein the microbial infection is caused by a microorganism selected from the group consisting of the genus Candida, wherein the genus Candida is selected from the group consisting of Candida albicans, Candida auris, Candida glabrata, Candida parapsilosis, Candida tropicalis, and Candida krusei.

66. The composition according to any one of claims 1 to 4 or 25 to 32, in a method of treating, preventing or inhibiting a microbial infection in a subject with such need.

67. The composition of claim 66, wherein the subject is selected from the group consisting of humans, livestock, poultry, fish and companion animals.

68. A plant part, said plant part being at least partially coated with the composition according to any one of claims 1 to 4 or 25 to 32.

69. The plant portion according to claim 68, wherein the plant portion is selected from the group consisting of seeds and pre- or post-harvest grains.

70. The plant portion according to claim 68, wherein the plant portion is selected from the group consisting of pre- or post-harvest fruits, pre- or post-harvest vegetables, and pre- or post-harvest flowers.

71. The plant portion according to claim 68, wherein the plant portion is selected from the group consisting of cereal crops, legume crops, root or tuber crops, oil crops, fruit crops, vegetable crops, nut crops, forage or turfgrass crops, forage legumes, medicinal crops, spice or flavoring crops, fiber crops and biofuel crops.

72. The plant portion according to claim 68, wherein the plant portion is selected from the group consisting of wheat, rice, corn, barley, oats, sorghum, rye, millet, peanut, chickpea, pigeon pea, lentil, kidney bean, soybean, lima bean, potato, sweet potato, cassava, rapeseed, peanut, palm, coconut, safflower, cotton, sunflower, flax, olive, sugarcane, beet, banana, orange, apple, pear, breadfruit, pineapple, strawberry, grape, cherry, coffee, cocoa, tomato, lettuce, carrot, melon, asparagus, Brassica, cashew, walnut, pistachio, almond, alfalfa, clover, castor bean, flaxseed, iris, switchgrass, Miscanthus, and Jatropha.

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