Antimicrobial polypeptides from plant argonaute proteins

Abstract

This disclosure provides small antimicrobial polypeptides and methods of treating or preventing infection with a bacterial or fungal pathogen using such polypeptides.

Description

PATENT Atorney Docket No. 118380-1544273-261210PC Client Ref. No. UC-2025-670-2ANTIMICROBIAL POLYPEPTIDES FROM PLANT ARGONAUTE PROTEINSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority benefit of U.S. Provisional Application No.63 / 748,953, filed January 23, 2025, which is incorporated by reference for all purposes.BACKGROUND

[0002] Antimicrobial peptides (AMPs) are small cationic and amphiphilic peptides / proteins that contribute to protein-based defenses in host immunity systems in various species including human, animals, and plants. AMPs show potent antimicrobial efficacy against bacteria, fungi, and viruses. They can be applied to treat or prevent various microbial diseases and have antimicrobial mechanisms that differ from those of antibiotics.BRIEF SUMMARY

[0003] This summary features certain aspects of the disclosure and is not intended to be a comprehensive summary of inventive aspects the disclosure.

[0004] In one aspect, the disclosure features a method of treating or preventing a pathogenic bacterial or fungal infection in a plant, the method comprising contacting the plant with an antimicrobial Moirai polypeptide comprising a net charge of at least +3 and a glycine content of at least 25%, wherein the antimicrobial Moirai polypeptide comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 1-99 as set forth in Table 1. In some embodiments, the antimicrobial Moirai polypeptide has at least 80% identity to any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, or 11. In some embodiments, the antimicrobial Moirai polypeptide has at least 90% identity, or at least 95% identity, to any one of SEQ ID NOs: 1-99 as set forth in Table 1. In some embodiments, the antimicrobial Moirai polypeptide has at least 90% identity, or at least 95% identity, to any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, or 11. In some embodiments, the Moirai polypeptide lacks an N-terminal methionine. For example, a sequence of any one of SEQ ID NOs: 1, 2, 5, 8, 9, or 10-29 may lack the N-terminal methionine. In some instances, the antimicrobial Moirai polypeptide comprises any one of SEQ ID NOs: 1-99 as set forth in Table 1. In some instances, the polypeptide lacks the N-terminal methionine shown in Table 1. In somePATENT Atorney Docket No. 118380-1544273-261210PC Client Ref. No. UC-2025-670-2 embodiments, the antimicrobial Moirai polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, or 11. In some instances, the polypeptide lacks the N-terminal methionine shown in Table 1. In some embodiments, the antimicrobial Moirai polypeptide is 15-300 amino acids in length. In some embodiments, the antimicrobial Moirai polypeptide comprises a glycine rich motif GnRGn or comprises a glycine rich motif GnRGnDGnRGY. In some instances, such motifs are repeated 1-5 times. In some instances, the pathogenic bacteria is gram-negative bacteria (e.g., a Liberibacter sp., an Agrobacterium sp., a Pseudomonas sp., a Xanthomonas sp., aXylella sp.) or bacteria that does not have a cell wall. In some embodiments, the pathogenic fungus is aBotrytis sp. or Verticillium sp.

[0005] In another aspect, the disclosure features an isolated antimicrobial Moirai polypeptide having antibacterial and antifungal activity, wherein the Moirai polypeptide comprises a net charge of at least +3 and a glycine content of at least 25%, and comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 1-99, or at least 80% identity to any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, or 11. In some embodiments, the antimicrobial Moirai polypeptide has at least 90% identity, or at least 95% identity, to any one of SEQ ID NOs: 1-99 as set forth in Table 1 or at least 90% identity, or at least 95% identity, to any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, or 11. In some instances, the antimicrobial Moirai polypeptide comprises any one of SEQ ID NOs: 1-99 as set forth in Table 1 or comprises the sequence of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, or 11. In some embodiments, the Moirai polypeptide lacks an N-terminal methionine. For example, in some embodiments a polypeptide comprising SEQ ID NO: 2 can lack the N-terminal methionine. In some embodiments, the antimicrobial Moirai polypeptide is 15-300 amino acids in length. In some embodiments, the antimicrobial Moirai polypeptide comprises a glycine rich motif GnRGn or comprises a glycine rich motif GnRGnDGnRGY. In some instances, such a motif is repeated 1-5 times. In some embodiments, the antimicrobial Moirai polypeptide targets a pathogenic bacterium or fungus. In some instances, the pathogenic bacteria is gram-negative bacteria, (e.g., a. Liberibacter sp., an Agrobacterium sp., a Pseudomonas sp., a Xanthomonas sp., a Xylella sp.) or bacteria that does not have a cell wall. In some embodiments, the pathogenic fungus is aBotrytis sp. or Verticillium sp.

[0006] In a further aspect, the disclosure provides an agricultural composition comprising an antimicrobial Moirai polypeptide as described herein. In some embodiments, thePATENT Atorney Docket No. 118380-1544273-261210PC Client Ref. No. UC-2025-670-2 agricultural composition comprises an additional agent that targets the pathogenic bacteria or fungi.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] FIG. 1 shows results of an antimicrobial activity assay for two plant disease fungi, Verticillium dahlias and Botrytis cinerea treated with 30 |iM Moirai 104 or 30 |iM Moirai 170.

[0008] FIG. 2. shows results of an antimicrobial activity assay for two plant disease fungi, Verticillium dahliae and Botrytis cinerea treated with 50 |iM N1 or 50 |iM N2.

[0009] FIG. 3 shows the results of an antimicrobial activity assay for a suite of gramnegative bacteria and the gram -positive bacteria Bacillus subtilis treated with 10 |iM Moirai 104, 10 |iM Moirai 170, or 100 |lg / ml streptomycin.

[0010] FIG. 4A shows the results of an antimicrobial activity assay for Liberibacter crescens and Xylella fastidosa treated with N1 or N2 polypeptide at the indicated concentrations.

[0011] FIG. 4B shows the results of an antimicrobial activity assay for Pseudomonas syringae pv. Tomato DC 3000 (EV) and Agrobacterium tumefaciens treated with N1 or N2 polypeptide at the indicated concentrations.

[0012] FIG. 5 shows the results of treatment with N1 (30 |lM), N2 (30 |lM), and Moirai 104 (10 |iM) applied to the surface of tomato fruit inoculated with Botrytis cinerea.

[0013] FIG. 6 shows the results of treatment of Arabidopsis leaves with Moirai 104 (10 |lM) and Moirai 170 (10 |iM) polypeptide prior to Botrytis cinerea inoculation.

[0014] FIG. 7 shows the results of root dip pretreatment with Moirai 104 (10 |iM) and Moirai 170 (10 |iM) prior to the inoculation of Arabidopsis with Verticillium dahliae.

[0015] FIG. 8 shows the results of root dip pretreatment with Moirai 104 (10 |iM) and Moirai 170 (10 |iM) prior to the inoculation of tomatoes with Verticillium dahliae.

[0016] FIG. 9 shows the results of two-time trunk injection of Moirai 104 and Moirai 170 into grape plants prior to Xylella fastidosa infection.PATENT Atorney Docket No. 118380-1544273-261210PC Client Ref. No. UC-2025-670-2

[0017] FIG. 10 shows the results of treatment with Moirai 104 and Moirai 170 on Arabidopsis prior to infection with Xanthomonas campestris.

[0018] FIG. 11 A shows the antimicrobial activity of Moirai polypeptides of Lactuca sativa AGO2, Brassica napus AGO2, and Sorghum bicolor AGO2 on Botrytis cinerea and Verticillium dahliae (Table 2).

[0019] FIG. 11B shows the antimicrobial activity of Moirai polypeptides of Oryza sativa AGO2, Zea mays AGO2, and Lactuca sativa AGO2-Like on Botrytis cinerea and Verticillium dahliae (Table 2).

[0020] FIG. 12A shows antimicrobial activity of Brassica napus, Lactuca sativa, Oryza sativa, Sorghum bicolor, and Zea mays on Pseudomonas syringae pv. tomato DC 300, Xanthomonas campestris pv. Campestris, and Agrobacterium tumefaciens.

[0021] FIG. 12B shows antimicrobial activity of Brassica napus, Lactuca sativa, Oryza sativa, Sorghum bicolor, and Zea mays on Liberibacter crescens and Xylella fastidiosa.

[0022] FIG. 13 shows a phylogenetic tree.

[0023] FIG. 14 shows the results of treatment with Moirai 104 and two Moirai polypeptides of Lactuca sativa on lettuce leaves and vegetables prior to infection with Botrytis cinerea.

[0024] FIG. 15 shows the antimicrobial activity of Moirai polypeptides of Moirai 104, Moirai 170, Lactuca sativa AGO2, Lactuca sativa AGO2-Like, Sorghum bicolor and Oryza sativa on Fusarium oxysporum and Fusarium verticilioides.

[0025] FIG. 16 shows the antimicrobial activity of Moirai polypeptides of Moirai 104, Moirai 170, Lactuca sativa AGO2, Lactuca sativa AGO2-Like, Sorghum bicolor and Oryza sativa on Pseudomonas aeruginosa, Klebsiella pnuemoniae, and Salmonella typhimurium.DETAILED DESCRIPTIONIntroduction

[0026] Unlike animal ARGON AUTE (AGO) proteins, some plant AGOs have an N-terminal region preceding the conserved domain characteristic of AGO proteins. This region varies among plant AGO proteins in amino acid length and function. The AGO3 protein of Arabidopsis thaliana is particularly distinct in structure. It possesses a long RG-rich N-terminal region. The disclosure is based, at least in part, on our findings that the ArabidopsisPATENT Atorney Docket No. 118380-1544273-261210PC Client Ref. No. UC-2025-670-2 AGO3 protein undergoes cleavage, resulting in a C-terminal truncated AGO and a short cationic N-terminal protein. This 170 amino acid long AGO3 N-terminal region was divided into two distinct regions based on polypeptide repeats. The repeat region, spanning from the 1stto 104thamino acid (aa), is predominantly composed of five 17 or 19 aa repeats. The remaining region (amino acids 105-170) shows an AGO3-speciftc sequence. Antibacterial and antifungal assays determined that these two cationic proteins (referred to in the experimental section as “Moirai 104” and “Moirai 170”) exhibited strong antimicrobial activity against both bacteria and fungi. These polypeptides have a positive charge and possess a glycine-rich region, which may contain various similar or identical glycine-rich repeats. We also identified similar glycine-rich motifs in the N-terminal region of other plant AGO proteins, repeating 1-8 times in the N-terminal region. These antimicrobial polypeptides (AMPs) also demonstrated efficacy in protecting plants such as Arabidopsis, tomato, and grape from fungal and bacterial infection.

[0027] The disclosure thus provides AMPs that are administered to prevent or treat a disease caused by a bacterial (e.g., gram-negative bacteria) or fungal pathogen.Terminology

[0028] As used herein, the term “antimicrobial polypeptides / proteins” or “AMPs” refers to small cationic amphiphilic polypeptides or short polypeptides having antimicrobial activity (e.g., antibacterial and antifungal activity that results in death of the target microbe). In the context of the present disclosure, AMPs are cationic amphiphilic polypeptides from the N-terminal region of various plant ARGONAUTE (AGO) proteins. For purposes of this disclosure AMPs from the N-terminal regions of plant AGO proteins are also referred to as “Moirai.” In some instances, Moirai polypeptides are less than 300 amino acids in length, have a high positive net charge of +3 or greater (e.g., +3 to 13.5) or greater than 13.5, and in some instances, are Gly-rich (c.g, have a glycine content of 25% or greater). Indicated charges are calculated using the “Antimicrobial Peptide Calculator and Predictor” from the APD3 database available at aps.unmc.edu (Wang, G., Li, X. and Wang, Z. (2016) APD3: the antimicrobial peptide database as a tool for research and education. Nucleic Acids Research 44, D1087-D1093; Wang, G., Li, X. and Wang, Z. (2009) APD2: the updated antimicrobial peptide database and its application in peptide design. Nucleic Acids Research 37, D933-D937; Wang, Z. and Wang, G. (2004) APD: the antimicrobial peptide database. Nucleic Acids Research 32, D590-D592). In some embodiments, a Moirai polypeptide comprises atPATENT Atorney Docket No. 118380-1544273-261210PC Client Ref. No. UC-2025-670-2 least one Gly-rich motif. In typical embodiments, a Moirai polypeptide having antimicrobial activity comprises repeating Gly-rich sequences. The Gly-rich motif is in some instances GnRGnDGnRGY. In some instances, this motif is repeated 1-5 times. Some Moirai sequences have more conserved sequences with this motif whereas some Moirai sequences have partial conservation: GnRGn. In some instances, this motif is repeated 1-5 times. An AMP peptide with a single repeat typically has a charge of +3, whereas a polypeptide having two or more repeating motifs have a positive charge greater than 5. A single repeat is typically 20 amino acids or fewer in length (e.g., 18 or 19 amino acids in length). In most instances, stronger antimicrobial activity is observed with Moirai containing more than one repeat. The 3D structure determined using Alpha fold 3 indicates that Moirai polypeptides with more repeats tend to form a unique helix-like (spiral) structure. The traditional a-helix is arranged in a right-handed helical structure where each amino acid residue corresponds to a 100° turn in the helix, and a translation of 1.5 A (0.15 nm) along the helical axis. In the Moirai polypeptide structure, the polypeptides with a greater number of repeats tend to form a large spring-like spiral structure. However, the diameter of each turn is not same, as it depends on the number of amino acids in each repeat.

[0029] As used herein, the term “treat” or “treating” in the context of treating a disease refers to the reduction, delay, or eradication of at least one symptom of a disease of a plant or animal resulting from pathogen infection (e.g., a fungal pathogen or a gram-negative bacterial pathogen or a bacterial pathogen lacking a cell wall). “Treating” a disease does not necessarily result in eradication or cure of the disease but reduces at least one symptom caused by the disease.

[0030] As used herein, the term “prevent” or “preventing” a disease refers to protecting a plant (or animal in instances where the disease is, for example, a mammal) at risk for the disease from developing the disease, or decreasing the risk that a subject plant (or animal) may develop the disease.

[0031] As used herein, the term “disease resistance” refers to the ability of a plant to not be affected by infection by a pathogenic microbe, such as a pathogenic bacteria or pathogenic fungus.

[0032] As used herein, the term “disease tolerance” refers to the ability of a plant to continuously grow and survive despite being infected by a pathogenic microbe, such as a pathogenic bacteria or pathogenic fungus.PATENT Atorney Docket No. 118380-1544273-261210PC Client Ref. No. UC-2025-670-2

[0033] As used herein, the term "plant" includes whole plants, shoot vegetative organs / structures (e.g., leaves, stems and tubers), roots, flowers and floral organs / structures (e.g., bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (e.g., vascular tissue, ground tissue, and the like) and cells (e.g, guard cells, egg cells, and the like), and progeny of same. The term “plant” also includes naturally occurring mutants and genetically modified plants. A “genetically modified plant” refers to a plant having a genome that has been manipulated so that it is different than a wild-type plant of the same species, variety or cultivar, e.g., to add a gene or genetic element, remove a gene or genetic element, mutate a gene or genetic element, change chromatin structure, change gene or protein expression levels, or processes in a cell. In the context of the present disclosure, genetically modified plants may include genetic modifications in an AGO gene encoding an AMP, for example to introduce a cleavage site in the N-terminal region.

[0034] An "expression cassette" refers to a nucleic acid construct that, when introduced into a host cell, results in transcription and / or translation of an RNA or polypeptide, respectively.

[0035] As used herein, the term “polynucleotide” refers to an oligonucleotide, or nucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin, which may be single- or double-stranded, and represent the sense or anti-sense strand. A single polynucleotide is translated into a single polypeptide.

[0036] As used herein, the terms “protein” and “polypeptide” are used interchangeably and describe a polymer in which the monomers are amino acid residues which are joined together through amide bonds. A polypeptide is intended to encompass any amino acid sequence, either naturally occurring, recombinant, or synthetically produced. In some instances, “peptide” is used to reference a relatively short protein. However, “peptide” as used herein does not limit the length of the polymer of amino acids.

[0037] As used herein, the term "substantial identity" or "substantially identical," used in the context of nucleic acids or polypeptides, refers to a sequence that has at least 50% sequence identity with a reference sequence. Alternatively, percent identity can be any integer from 50% to 100%. In some embodiments, a sequence is substantially identical to a reference sequence (e.g., any one of the sequences set forth in Table 1) if the sequence has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%PATENT Atorney Docket No. 118380-1544273-261210PC Client Ref. No. UC-2025-670-2 sequence identity to the reference sequence as determined using the methods described herein; preferably BLASTP using standard parameter. In some embodiments, a sequence is substantially identical to a reference sequence (e.g., any one of the sequences set forth in Table 1) if the sequence has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the reference sequence as determined using the methods described herein; preferably BLASTP using standard parameters, as described below.

[0038] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0039] A comparison window includes reference to a segment of any one of a number of contiguous positions, e.g., a segment of at least 10 residues. In some embodiments, the comparison window has from 10 to 600 residues, e.g., about 10 to about 30 residues, about 10 to about 20 residues, about 50 to about 200 residues, or about 100 to about 150 residues, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.

[0040] Algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotidePATENT Atorney Docket No. 118380-1544273-261210PC Client Ref. No. UC-2025-670-2 sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=l, N=-2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Set. USA 89:10915 (1989)). For purposes of this disclosure, percent identity of amino acids sequence use the BLASTP program with default parameters.

[0041] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'L Acad. Set. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, an amino acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test amino acid sequence to the reference amino acid sequence is less than about 0.01, more preferably less than about 10'5, and most preferably less than about IO'20.Antimicrobial Polypeptides

[0042] The disclosure provides Moirai polypeptides that have antimicrobial effects, e.g., cell killing effect, on bacteria (e.g., gram negative bacteria or bacteria lacking cell walls) and fungi. Such Moirai polypeptides may be used for the treatment or prevention of microbial disease caused by bacterial or fungal pathogens. In some instances, Moirai polypeptides of the present disclosure are employed to treat or prevent disease in plants. In other instances, Moirai polypeptides are used for the treatment of animals (e.g., including humans).

[0043] In some embodiments, a Moirai polypeptide of the present disclosure that has antimicrobial activity towards a bacterial or fungal pathogen (e.g., a gram-negative bacterial pathogen or a fungal pathogen), is a small polypeptide (e.g., from 15-300 amino acid in length), has a net positive charge of 3 or greater and a glycine content of at least 25% or atPATENT Atorney Docket No. 118380-1544273-261210PC Client Ref. No. UC-2025-670-2 least 30%. In some instances, such a Moirai polypeptide comprises a partially conserved repeat comprising a sequence GnRGn. In some instances, the Moirai polypeptide comprises a repeat sequence GnRGnDGnRGY. In some embodiments, such repeat sequences are repeated 1-5 times.

[0044] In some instances, a Moirai polypeptide of the present disclosure has a net positive charge of 3 or greater and a glycine content of at least 25% or at least 30% and comprises a sequence that has at least 80% or 85% identity to any one of the sequence set forth in Table 1. In some embodiments, the Moirai polypeptide comprises a sequence having at least 90%, 91%, 92%, 93%, or 94% sequence identity to any one of the sequences set forth in Table 1. In some embodiments, the Moirai polypeptide comprises a sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the sequences set forth in Table 1. In some instances, the Moirai polypeptide comprises 2 or more repeats. In some instances, each repeat is 18-20 amino acids in length. In some embodiments, the Moirai polypeptide comprises the amino acid sequence of one of the Moirai polypeptide sequences provided in Table 1. In some instances, the amino acid sequence of the Moirai polypeptide does not comprise the N-terminal methionine shown for a polypeptide sequence in Table 1.

[0045] In some embodiments, a Moirai polypeptide of the present disclosure comprises a sequence having at least one amino acid substitution (e.g., 2, 3, 4, 5, or 6; or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions for any sequence 150 or greater in length) relative to a sequence of any one of the sequences set forth in Table 1 and conserves a glycine content of at least 25% and a net positive charge of at least +3 for a Moirai polypeptide not containing repeats and at least +5 for a Moirai polypeptide containing two or more repeats.

[0046] In some embodiments, a Moirai polypeptide of the present disclosure has a net charge of +3 or higher and glycine content of at least 25%, often at least 30%, and comprises an amino acid sequence having at least 80% identity or at least 85% identity to any one of the polypeptide sequences shown in Table 1. In some embodiments, the Moirai polypeptide has a net charge of +3 or higher and glycine content of at least 30% and comprises an amino acid sequence having at least 90% identity to any one of the polypeptide sequences shown in Table 1. In some embodiments, the Moirai polypeptide has a net charge of +3 or higher and glycine content of at least 30% and comprises an amino acid sequence having at least 95% identity to any one of the polypeptide sequences shown in Table 1. In some embodiments, aPATENT Atorney Docket No. 118380-1544273-261210PC Client Ref. No. UC-2025-670-2 Moirai polypeptide having at least 80%, 85%, 90% or 95% identity to a polypeptide sequence set forth in Table 1 comprises a Gly-rich motif GnRGnDGnRGY. In some embodiments, a Moirai polypeptide having at least 80%, 85%, 90% or 95% identity to a polypeptide sequence set forth in Table 1 comprises a Gly-rich motif GnRGnDGnRGY: GnRGn. In some embodiments, such repeat sequences are repeated 1-5 times.

[0047] In some embodiments, a Moirai polypeptide having antibacterial and / or antifungal activity comprises at least one and preferably two repeats of a repeated sequence shown in Table 1. In some embodiments, the repeats are from the same N-terminal AGO polypeptide. In other embodiments, the repeats from the N-terminal AGO region are from different AGO polypeptides.

[0048] In some embodiments, a Moirai polypeptide for the treatment or prevention of an infection by a microbial pathogen (e.g., a bacterial or fungal pathogen) has a net positive charge of 3 or greater and a glycine content of at least 30% and comprises a sequence that has at least 80% or 85% identity to any one of the sequences of a polypeptide listed in Table 2. In some embodiments, the Moirai polypeptide comprises a sequence having at least 90%, 91%, 92%, 93%, or 94% sequence identity to any one of the sequences of a polypeptide listed in Table 2. In some embodiments, the Moirai polypeptide comprises a sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the sequences of a polypeptide listed in Table 2. In some instances, the Moirai polypeptide comprises 2 or more repeats. In some instances, each repeat is 18-20 amino acids in length. In some embodiments, the Moirai polypeptide comprises the amino acid sequence of one of the Moirai polypeptides listed in Table 2. In some instances, the amino acid sequence of the Moirai polypeptide does not comprise the N-terminal methionine of the sequence shown in Table 1. Sequences of the polypeptides listed in Table 2 are provided in Table 1.

[0049] In some embodiments, a Moirai polypeptide of the present disclosure comprises a sequence having at least one amino acid substitution (e.g., 2, 3, 4, 5, or 6; or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions for any sequence 150 or greater in length) relative to a sequence of any one of the polypeptides set forth in Table 2 and conserves a glycine content of at least 25% and a net positive charge of at least +3 for a polypeptide Moirai not containing repeats and at least +5 for a Moirai polypeptide containing two or more repeats. Sequences of the polypeptides listed in Table 2 are provided in Table 1.PATENT Atorney Docket No. 118380-1544273-261210PC Client Ref. No. UC-2025-670-2

[0050] In some embodiments, a Moirai polypeptide of the present disclosure has a net charge of +3 or higher and glycine content of at least 25%, often at least 30%, and comprises an amino acid sequence having at least 80% identity or at least 85% identity to any one of the polypeptides shown in Table 2. In some embodiments, the Moirai polypeptide has a net charge of +3 or higher and glycine content of at least 30% and comprises an amino acid sequence having at least 90% identity to any one of the polypeptides shown in Table 2. In some embodiments, the Moirai polypeptide has a net charge of +3 or higher and glycine content of at least 30% and comprises an amino acid sequence having at least 95% identity to any one of the polypeptides shown in Table 2. In some embodiments, a Moirai polypeptide has at least 80%, 85%, 90% or 95% identity to a polypeptide sequence of a polypeptide set forth in Table 2 and comprises a Gly-rich motif GnRGnDGnRGY. In some embodiments, the motif is repeated 1-5 times. In some embodiments, a Moirai polypeptide has at least 80%, 85%, 90% or 95% identity to a sequence of a polypeptide set forth in Table 2 and comprises a Gly-rich motif GnRGnDGnRGY: GnRGn. In some embodiments, the motif is repeated 1-5 times. Sequences of the polypeptides listed in Table 2 are provided in Table 1.

[0051] In some embodiments, a Moirai polypeptide having antibacterial and / or antifungal activity comprises at least one and preferably two repeats of a repeated sequence of a polypeptide shown in Table 2. In typical embodiments, the repeats are from the same N-terminal AGO polypeptide. In other embodiments, the repeats from the N-terminal AGO region of different AGO polypeptides. Sequences of the polypeptides listed in Table 2 are provided in Table 1.

[0052] AGO-derived Moirai polypeptides can be tested for inhibitory activity, typically cell killing against one or more pathogens of interest (e.g., the suite of microbes employed in the technical section) including gram-negative bacteria such as plant disease oc-proteobacteria (e.g., Liberibacter crescens and Agrobacterium tumefaciens) and y-proteobacteria (e.g., Pseudomonas syringae, Xanthomonas campestris pv campeslris. and Xylella fastidiosa). Candidate Moirai polypeptides can also be tested for antifungal activity (e.g., activity against Botrytis, e.g., Botrytis cinerea and / or Verticillium, e.g., Verticillium dahliae) using an assay demonstrated in the technical section. Thus, for example, bacteria can be grown in suitable medium and culturing conditions and activity assess by determining the proportion of cells that die using a staining assay that distinguishes between living and dead cells.PATENT Atorney Docket No. 118380-1544273-261210PC Client Ref. No. UC-2025-670-2 Table 1. Illustrative Moirai polypeptides.PATENT Atorney Docket No. 118380-1544273-261210PC Client Ref. No. UC-2025-670-2PATENT Atorney Docket No. 118380-1544273-261210PC Client Ref. No. UC-2025-670-2PATENT Atorney Docket No. 118380-1544273-261210PC Client Ref. No. UC-2025-670-2PATENT Atorney Docket No. 118380-1544273-261210PC Client Ref. No. UC-2025-670-2PATENT Atorney Docket No. 118380-1544273-261210PC Client Ref. No. UC-2025-670-2PATENT Atorney Docket No. 118380-1544273-261210PC Client Ref. No. UC-2025-670-2PATENT Atorney Docket No. 118380-1544273-261210PC Client Ref. No. UC-2025-670-2PATENT Atorney Docket No. 118380-1544273-261210PC Client Ref. No. UC-2025-670-2PATENT Atorney Docket No. 118380-1544273-261210PC Client Ref. No. UC-2025-670-2PATENT Atorney Docket No. 118380-1544273-261210PC Client Ref. No. UC-2025-670-2PATENT Atorney Docket No. 118380-1544273-261210PC Client Ref. No. UC-2025-670-2PATENT Atorney Docket No. 118380-1544273-261210PC Client Ref. No. UC-2025-670-2PATENT Atorney Docket No. 118380-1544273-261210PC Client Ref. No. UC-2025-670-2PATENT Atorney Docket No. 118380-1544273-261210PC Client Ref. No. UC-2025-670-2PATENT Atorney Docket No. 118380-1544273-261210PC Client Ref. No. UC-2025-670-2PATENT Atorney Docket No. 118380-1544273-261210PC Client Ref. No. UC-2025-670-2PATENT Atorney Docket No. 118380-1544273-261210PC Client Ref. No. UC-2025-670-2 Table 2. Antimicrobial activity of illustrative Moirai polypeptides.PATENT Atorney Docket No. 118380-1544273-261210PC Client Ref. No. UC-2025-670-2PATENT Atorney Docket No. 118380-1544273-261210PC Client Ref. No. UC-2025-670-2 Delivery of Moirai polypeptides to plants

[0053] A Moirai polypeptide of the present disclosure can be provided to a plant in an agricultural composition using any number of methods, including for example injecting spraying, soaking, dusting, coating, and the like. As used herein, the term “agricultural composition” refers to a composition formulated for application to a plant or plant part (e.g., seed, cutting, shoots, etc.). An agricultural composition is typically in liquid form (e.g., for application by spraying or soaking) but can be in the form of a powder for rehydration or application (e.g., dusting or dry coating) or gaseous (e.g., for enclosed environments). The agricultural composition can be concentrated (e.g., for dilution in water or other solvent). An agricultural composition can also include more than one active ingredient (e.g., a Moirai polypeptide) in combination with a fungicide, herbicide, fertilizer, etc.

[0054] In some embodiments, an agricultural composition comprising one or more polypeptides described herein can also include one or more of: a surface-active agent, an inert carrier, a preservative, a humectant, a feeding stimulant, an attractant, an encapsulating agent, a binder, an emulsifier, a dye, a UV protective, a buffer, a flow agent, a fertilizer, a nitrogen fixation agent, micronutrient donors, or other preparations that influence plant growth. The agricultural composition can also include one or more agrochemicals including: herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides, acaracides, plant growth regulators, harvest aids, and fertilizers, which can also be combined with carriers, surfactants or adjuvants as appropriate for the agrochemical. Suitable earners and adjuvants can be solid or liquid and correspond to the substances ordinarily employed in formulation technology (e.g.. natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, binders, or fertilizers). The active ingredients of the present disclosure are normally applied in the form of compositions and can be applied to the crop area, plant, or seed to be treated. For example, the compositions of the present disclosure can be applied during growth, seeding, or storage.

[0055] Surface-active agents that can be used with the presently described polypeptides include anionic compounds such as a carboxylate of, for example, a metal; carboxylate of a long chain fatty acid; an N-acylsarcosinate; mono- or di-esters of phosphoric acid with fatty alcohol ethoxylates or salts of such esters; fatty alcohol sulfates such as sodium dodecyl sulfate, sodium octadecyl sulfate, or sodium acetyl sulfate; ethoxylated fatty alcohol sulfates; ethoxylated alkylphenol sulfates; lignin sulfonates; petroleum sulfonates; alkyl arylPATENT Atorney Docket No. 118380-1544273-261210PC Client Ref. No. UC-2025-670-2 sulfonates such as alkyl-benzene sulfonates or lower alkyl naphthalene sulfonates (e.g., butylnaphthalene sulfonate); salts of sulfonated naphthalene-formaldehyde condensates; salts of sulfonated phenol-formaldehyde condensates; more complex sulfonates such as the amide sulfonates, e.g., the sulfonated condensation product of oleic acid and N-methyl taurine; or the dialkyl sulfosuccinates (e.g., the sodium sulfonate or dioctyl succinate). Non-ionic agents include condensation products of fatty acid esters, fatty alcohols, fatty acid amides or fatty-alkyl- or alkenyl-substituted phenols with ethylene oxide, fatty esters of polyhydric alcohol ethers (e.g., sorbitan fatty acid esters), condensation products of such esters with ethylene oxide (e.g., polyoxyethylene sorbitan fatty acid esters), block copolymers of ethylene oxide and propylene oxide, acetylenic glycols (e.g., 2,4,7,9-tetraethyl-5-decyn-4,7-diol), or ethoxylated acetylenic glycols. Examples of a cationic surface-active agent include, for instance, an aliphatic mono-, di-, or polyamine such as an acetate, naphthenate or oleate; oxygen-containing amine such as an amine oxide of polyoxyethylene alkylamine; an amide-linked amine prepared by the condensation of a carboxylic acid with a di- or polyamine; or a quaternary ammonium salt.

[0056] Examples of inert materials or inert carriers that can be used include, but are not limited to, inorganic minerals such as kaolin, phyllosilicates, carbonates, sulfates, phosphates, or botanical materials such as cork, powdered corncobs, peanut hulls, rice hulls, and walnut shells.

[0057] Herbicides that can be used with the presently described polypeptides include compounds that kill or inhibit growth or replication of undesired plants, typically a subset of plants that is distinct from the desired plant or crop. There are several modes of action:ACCase inhibition, carotenoid biosynthesis inhibition, cell wall synthesis inhibition, ALS inhibition, ESP synthase inhibition, glutamine synthase inhibition, HPPD inhibition, microtubule assembly inhibition, PPO inhibition, etc. Examples of commercially available herbicides include One-Time®, MSMA, Corvus®, Volunteer®, Escalade®, Q4®, RaptorC®, Acumen®, Seneor®, Bullet®, TopNotch®, Valor®, PastureGard®, glyphosate (Roundup®), DSMA, Break-Up®, Hyvar®, Barricade®, etc. Herbicides can be mixed with “herbicide safeners” to reduce general toxicity of the herbicide, as described in Riechers et al. (2010) Plant Physiol. 153:3.

[0058] Pesticides (e.g., nematicides, molluscicides, insecticides, miticide, and acaricides) can be used in combination with the presently disclosed polypeptides to kill or reduce thePATENT Atorney Docket No. 118380-1544273-261210PC Client Ref. No. UC-2025-670-2 population of undesirable pests affecting the plant. Pesticides can also be used with repellants or pheromones to disrupt mating behavior. Insecticides are directed to insects, and include those of botanical origin (e.g., allicin, nicotine, oxymatrine, jasmolin I and II, quassia, rhodojaponin HI, and limonene), carbamate insecticides (e.g., carbaryl, carbofuran, carbosulfan, oxamyl, CPMC, EMPC, and fenobucarb), fluorine insecticides, formamidine insecticides, fumigants (e.g., ethylene oxide, methyl bromide, and carbon disulfide), chitin synthesis inhibitors, macrocyclic lactone insecticides, neonicotinoid insecticides, organophosphate insecticides, urea and thiourea insecticides, etc. Nematicides affect nematodes and include organophosphorus nematicides (e.g., diamidafos, fosthiazate, heterophos, phsphamidon, and triazophos), fumigant nematicides (e.g., carbon disulfide, methyl bromide, and methyl iodide), abamectin, carvacrol, carbamate nematicides (e.g., benomyl, and oxamyl), etc. Molluscicides are directed to slugs and snails and include allicin, bromoacetamide, thiocarb, trifenmorph, fen tin, copper sulfate, etc. Many pesticides target more than one type of pest, so that one or two can be selected to target insects, mollusks, nematodes, mitogens, etc.

[0059] Fertilizers typically provide macro- and micronutrients in a form that can be utilized by the plant or a plant-associated organism. These include nitrogen, phosphorus, potassium, sulfur, calcium, potassium, boron, chlorine, copper, iron, manganese, molybdenum, zinc, nickel, and selenium. Fertilizers are often tailored to specific soil conditions or for particular crops or plants. Fertilizers that can be used include naturally-occurring (e.g., manure, bone meal, compost, fish meal, wood chips, etc.), modified, concentrated and / or chemically synthesized materials (e.g., UAN, anhydrous ammonium nitrate, urea, potash, etc.). Suppliers include Scott®, SureCrop®, BCF®, RVR®, Gardenline®, and many others known in the art.

[0060] Fungicides are compounds that can kill fungi or inhibit fungal growth or replication. Fungicides that can be used with the presently disclosed polypeptides include contact, translaminar, and systemic fungicides. Examples include sulfur, neem oil, rosemary oil, jojoba, tea tree oil, Bacillus subtihs, Uiocladium, cinnamaldehyde, etc.

[0061] The agricultural compositions of the disclosure can be in a suitable form for direct application or as a concentrate of primary composition that requires dilution with a suitable quantity of water or other diluent before application. The concentration of the polypeptide will vary depending upon the nature of the particular formulation, specifically, whether it is aPATENT Atorney Docket No. 118380-1544273-261210PC Client Ref. No. UC-2025-670-2 concentrate or to be used directly, the type of plant, and in some cases, the infectious agent targeted by the Moirai polypeptide.

[0062] In some embodiments, a Moirai polypeptide is exogenous to the plant ( / .< ., the polypeptide does not occur naturally in the plant by expression of a native gene, without human intervention). Embodiments of an “exogenous” polypeptide include a polypeptide that is produced by in vitro fermentation or by production in cells or tissues or by synthesis and that is then provided to the plant (e.g., in an agricultural composition delivered by injection, dipping, spraying, soaking, irrigation), or a polypeptide expressed in ceils (e.g., bacterial cells) or tissue (e.g., transgenic or genome-edited plant tissue) that is then provided to the plant (e.g., by extraction and delivery in an agricultural composition, or by grafting), or by providing to the plant a recombinant construct that expresses the exogenous polypeptide in the plant, or by modifying the plant’s genome (e.g, by genome editing methods such as CRISPR / Cas, TALENs, base editing, or prime editing) resulting in the plant’s expression of the exogenous polypeptide.

[0063] Any plant can be provided with a Moirai polypeptide to treat or prevent a bacterial infection (e.g, by gram-negative bacteria or bacteria without a cell wall, or other bacteria with a surface membrane / wall structure that can be penetrated by a Moirai polypeptide) or a fungal infection. Plants that can be treated include monocots and dicots. Illustrative examples of plants, include, but are not limited to: citrus, tomato, grape, potato, cucumber, melons, squash, beans, peas, carrots, radish, turnip yam, sweat potato, Brassica, corn, rice, rye, sorghum, flax, alfalfa, millet, wheat, soybean, tobacco, cotton sugar cane, oats, barley, sugar beets, coffee, coconut, pineapple, citrus, banana, cocoa, peach, pear, avocado, olive, mango, cherry, apple, apricot, plum, pistachio, walnut, almond, pecan, and fruit plants such as strawberry, blueberry, raspberry and the like.

[0064] A Moirai polypeptide as described herein used to treat or prevent a plant disease include, but are not limited to, those caused by bacteria that are members of the families Xantomonadaceae, Pseudomonadaceae, and Enterobacteriaceae. Thus, plant disease arising from bacteria of the following genera can be treated or prevent by providing a Moirai polypeptide to a plant: Dickeya, Liberibacter, Erwinia, P ectobacterium, Candidatus, Pantoea, Agrobacterium, Pseudomonas, Ralstonia, Burkholderia, Acidovorax, Xanthomonas, Xylella, Spiroplasma, Phytoplasma, Brenner ia, Lonsdalea, or Xy lophilus .PATENT Atorney Docket No. 118380-1544273-261210PC Client Ref. No. UC-2025-670-2

[0065] In some instances, a Moirai polypeptide can be employed to treat or prevent an infection by a bacterial plant pathogen, including, but not limited to, one of the following: Ralstonia solanacearum, Xanthomonas oryzae pathovars, Xanthomonas campestris pathovars, Xanthomonas axonopodis pathovars, Xanthomonas euvesicatoria pathovars, Xanthomonas hostorum pathovars, Pseudomonas syringae, Pseudomonas viridiflava, Pseudomonas savastonoi, Candidatus liberibacter asiaticus, Candidatus liberibacter solanacearum, Liberibacter ere sc ens, Acidovorax citrulli, Acidovorax avenae, P ectobacterium atrosepticum, P ectobacterium carotovorum, P ectobacterium sp., Agrobacterium tumefaciens, Dickeya (dadantii and solani), Erwinia amylovora, Clavibacter michiganensis (michiganensis and sepedonicus), Xylella fastidiosa, P ectobacterium (carotovorum and atrosepticum), Phytoplasma sp., Spiroplasma sp., Pseudomonas cichorii; Xanthomonas campestris pv. Rhodococcus fascians, xanthomonas campestris pv. Campestris, Ralstonia solanacearum, Xanthomonas axonopodis, Xanthomonas hortorum, orP ectobacterium carotovorum.

[0066] As detailed herein, fungal plant pathogens can also be targeted with a Moirai polypeptide. In some embodiments, the fungal or fungal-like pathogen is a. Botrytis spp., Cercospora spp., Alternaria spp., Didymella spp., Fusarium spp., Erysiphe spp., Colletotrichum spp., Monilinia spp., Mycosphaerella spp., Plasmopara spp., Peronospora spp., Pythium spp., Phytophthora spp., Phomop sis spp., Phakopsora spp., Podosphaera spp., Rhizoctonia spp., Sclerotinia spp., Uncinula spp., Venturia spp., Verticillium sp., Wilsonomyces spp., and Plasmodiophora spp. In some embodiments, the fungal pathogen can be, but is not limited to one of the following: Aspergillus flavus, Albugo occidentalis, Alternaria solani, Alternaria spp., Apiognomonia errabunda, Apiognomonia veneta, Armillaria mellea, Bipolaris maydis, Botrytis cinerea, Botrytis squamosa, Botryosphaeria dothidea, Blumeriella jaapii, Bremia lactucae, Cladosporium carpophilum, Cladosporium caryigenum, Colletotrichum acutatum, Colletotrichum graminicola, Colletotrichum cereale, Colletotrichum gloeosporiodes, Colletotrichum sublineolum, Cochliobolus heterostrophus, Corynespora cassiicola, Discula fraxinea, Cercospora sojina, Cercospora beticola, Blumeria graminis, Didymella bryoniae, Elsinoe fawcetti, Erysiphe necator, Erysiphe lager str oemiae, Erysiphe cichoracearum, Eutypa lata, Eutypa spp., Fusarium graminearum, Fusarium solani, Fusarium oxysporum, Fusarium graminicola, Golovinomyces cichoracearum, Gibberella zeae, Gloeodes pomigena, Gymnosporangium juniperi-virginianae, Hemileia vastatrix, Leveillula taurica, Mycosphaerella fijiensis, Mycosphaerella citri, MycosphaerellaPATENT Atorney Docket No. 118380-1544273-261210PC Client Ref. No. UC-2025-670-2 pomi, Macrophomina spp., Monosporascus cannonballus, Monilinia fructicola, Monilinia laxa, Monilinia fructigena, Neofabraea spp., Podosphaera xanthii, Podosphaera leucotricha, Phomopsis viticola, Phakopsora meibomiae, Phakopsora pachyrhizi, Phytophthora infestans, Puccinia triticina, Puccinia recondita, Puccinia striiformis, Puccinia graminis, Pyrenophora trici-repentis, Rhizoctonia solani, Tilletia barclayena,, Uncinula necator, Uromyces betas, Sclerotium rolfsii, Sclerotinia minor, Sclerotinia sclerotiorium, , Schizothyrium pomi, Septoria glycines, Sphaerotheca pannosa, Sphaerotheca macularis, Venturia inaequalis, Verticillium dahlias, or Wilsonomyces carpophilus,.

[0067] In some instances, a Moirai polypeptide of the present disclosure can be used to treat or prevent a bacterial infection (e.g., gram-negative bacterial infection) or a fungal infection in any animal, including mammals, avians, reptiles and the like. In some instances, the animal is a chicken, turkey, goose, or other fowl. In some instances, the animal is a mammal (e.g., rat, mouse, cat, dog, pig, guinea pig, hamster, goat, sheep, or horse, among others). In some instances, the mammal is a primate, such as a rhesus macaque or chimpanzee. In some instances, the primate is a human. A Moirai polypeptide may be administered in any suitable pharmaceutical formulation and may be administered topically, intravenously, parenterally, or by any other known route.EXAMPLES

[0068] Unlike animal ARGONAUTE (AGO) proteins, some plant AGOs have an N-terminal region preceding the conserved domain characteristic of AGO proteins. This region varies among plant AGO proteins in amino acid length and function. The AGO3 protein of Arabidopsis thaliana is particularly distinct in structure. It possesses a long RG-rich N-terminal region. The disclosure is based, at least in part, on our findings that the Arabidopsis AGO3 protein undergoes cleavage, resulting in a C-terminal truncated AGO and a short cationic N-terminal protein. In brief, the 170 amino acid long AGO3 N-terminal region was divided into two distinct regions based on polypeptide repeats. An important repeat region spanning from the start codon to the 104thamino acid was identified. This region has five repeats of 18- or 19-amino acids in length that are rich in glycine and arginine, giving the region a positive charge (+13.5). The cleavage site (D170) was identified at the 170thamino acid, which includes the first 104-amino acid repeats and has a net positive charge (+12.75).

[0069] These two polypeptide types from the AGO3 N-terminal region were synthesized and purified. Antibacterial and antifungal assays determined that these cationic proteinsPATENT Atorney Docket No. 118380-1544273-261210PC Client Ref. No. UC-2025-670-2 exhibit strong antimicrobial activity against both bacteria and fungi. Based on the characterization analysis of these antimicrobial proteins, we found that they carry a positive charge and possess a glycine-rich region, which may contain various similar or identical glycine-rich repeats. In AGO proteins from other plant species, a similar glycine-rich motif is found, repeating 1-8 times in the N-terminal region. Various cationic proteins were purified and confirmed to be effective in defending against bacteria and fungi. These antimicrobial peptides (AMPs) have also demonstrated efficacy in protecting plants such as Arabidopsis, tomato, and grape from fungal and bacterial infectionsEXAMPLE 1 : Illustrative techniquesPreparation of AMPs:

[0070] We analyzed the most N-terminal regions of plant AGOs and identified several cationic polypeptides from these regions. These polypeptides share similar characteristics: they are glycine-rich, positively charged, and contain repeating motifs. The sequences and key characteristics of these polypeptides are listed in Table 1. We synthesized the polypeptides and small proteins or purified them using the E. coli expression system.

[0071] A phylogenetic tree (FIG. 13) was constructed for AGO N-terminal regions listed in Table 1. The tree reveals that AGO proteins from various plants (e.g., Brassica plants, such as Brassica rapa, Brassica oleracea var. ole race a. and Brassica napus, Lactuca saliva. Raphanus saliv s, primus dulcis. and beta vulgaris subsp. vulgaris possess a highly conserved N-terminal sequence when compared to AtAGO3 in dicotyledonous plants.Interestingly, certain monocotyledonous plants, such as wheat (e.g., Triticum aestivum), rice (e.g., Oryza saliva), sorghum (e.g., Sorghum bicolor), corn (e.g., Zea mays) and barley (e.g., Hordeum vulgare) appear to be more close evolutionary descent to AtAGO3 based on their N-terminal sequence. Upon analyzing these AGO protein N-terminal sequences, it is evident that some contain multiple repeat sequences. Although these repeat sequences may not be identical to the repeat sequence found in AtAGO3, they share a similar characteristic of having numerous glycine residues. The corresponding sequences and additional information are also listed in Table 1.

[0072] AMPS were assayed for antibacterial and antifungal activity. The results are shown in FIGS. 1-12B and 14-16.PATENT Atorney Docket No. 118380-1544273-261210PC Client Ref. No. UC-2025-670-2 EXAMPLE 2: Antimicrobial assay on plant disease fungiDerived from At AGO 3:

[0073] To evaluate whether these polypeptides exhibit antimicrobial properties against fungi, we selected Moirai 104, Moirai 107, and two shorter polypeptides, N1 and N2, which contain one or two repeats, respectively.

[0074] Moirai 104 and Moirai 170 showed antimicrobial activity on two plant disease fungi: Verticillium dahliae and Botrytis cinerea. Antimicrobial assays were done to demonstrate whether Moirai 104 or Moirai 170 can affect the growth of fungi. The germination of spores was simultaneously observed to be retarded by the application of Moirai 104 or Moirai 170. In this experiment, spores of fungi at a concentration of 10'5were cultured for several hours until the hyphae germinated. 30 |iM Moirai 104 or Moirai 170 was then introduced into the medium. Over time, it was observed that Moirai 104 or Moirai 170 damaged the membrane and penetrated the fungus hyphae. Consequently, the hyphae ceased to grow and eventually died. However, the N1 and N2 polypeptides also show antimicrobial activity against B. cinerea and Verticillium dahliae at high concentrations (50 |iM).EXAMPLE 3 : Antimicrobial assay on pathogenic bacteria

[0075] These polypeptides also exhibited antimicrobial properties against a wide range of bacteria. In this study, we treated the gram-positive bacteria, Bacillus subtilis, and a suite of gram-negative bacteria, including a-proteobacteria Liberibacter crescens and Agrobaclerium tumefaciens and y-proteobacteria Pseudomonas syringae, Xanthomonas campestris pv. campestris and ylella fastidiosa. All of bacteria were grown using their standard medium and culturing conditions. Bacteria were resuspended in 150 mM NaCl to O.D. 0.2. The resuspended bacteria were treated with candidate polypeptides added to provide a final O.D. of 0.1. EthD-III and DMAO dyes were used to stain the bacteria. Bacteria with intact cell membranes stain fluorescent green, whereas red indicated damaged or dead bacteria.Bacteria were incubated with 10 |iM Moirai 104 or Moirai 170 for one hour. However, for N1 and N2, the 10 |iM application did not show obvious antimicrobial activity until the concentration was increased to 50 |iM. Though gram-positive Bacillus was resistant to polypeptide treatment, all tested gram-negative bacteria were highly susceptible to 10 |iM Moirai 104 or Moirai 170 compared to 100 ug / mL of the antibiotic streptomycin. However, the N1 and N2 polypeptides only showed high antimicrobial activity against LiberibacterPATENT Atorney Docket No. 118380-1544273-261210PC Client Ref. No. UC-2025-670-2 crescens, Agrobacterium tumefaciens, P. syringae. and Xylella fastidiosa at a high concentration (50 |iM). These results thus demonstrated the antimicrobial activity of these AGO3 N-terminal fragments against bacteria.EXAMPLE 4: Plant protection assay

[0076] The promising antimicrobial properties of the AGO3-derived polypeptide led us to explore its potential application as an antifungal and antibacterial treatment for plants. To assess their efficacy in post-harvest products, we conducted experiments in which tomato fruits were treated with polypeptides prior to inoculation with Botrytis.

[0077] We applied Moirai 104, Nl, and N2 at the indicated concentrations (10 |iM, 30 |iM, 30 |iM) onto the surface of the tomato fruits. After two days, we observed remarkable results: the polypeptides nearly completely eradicated the fungal infection. This outcome suggests the potential of these polypeptides as effective treatments against fungal pathogens in postharvest produce, indicating their possible use for extending the shelflife and quality of fruits and vegetables.

[0078] Treatment of Arabidopsis leaves with Moirai 104 (10 |iM) polypeptide prior to B. cinerea inoculation suppressed the lesions to an unnoticeable degree, suggesting a potent antifungal activity of the polypeptide at the plant surface. Indeed, we found that root dip treatment of Arabidopsis with the Moirai 104 (10 |iM) polypeptide before Verticillium dahlias inoculation could protect the plant from fungal infection. The 3 -week Arabidopsis roots were dipped with 10 |iM Moirai 104 for 10 minutes and then were inoculated by 10'5Verticillium dahlias for 5 minutes. The treated seedlings were transferred into clean and fresh soil to observe the phenotype. Similarly, root dipping treatment of tomato plants with Moirai 104 (10 |iM) polypeptide prior to Verticillium dahlias inoculation drastically reduced the wilting symptoms.

[0079] Besides protecting plants from fungi, two-time trunk injection with Moirai 104 (20 |lM) enhanced grape plant resistance to Xylella fastidiosa infection. Pretreatment with the Moirai 104 polypeptide also attenuated Xanthomonas campestris pv. Campestris infection in Arabidopsis.

[0080] Compared to traditional antibiotics, Moirai 104 exhibited more efficacious antibacterial and antifungal activity. Activity was observed even using low concentrations.PATENT Atorney Docket No. 118380-1544273-261210PC Client Ref. No. UC-2025-670-2

[0081] From Table 1 and the phylogenetic tree, we observed that not all of these N-terminal regions have the similar repeats. We analyzed and selected this conserved region with or without repeats from various dicot and monocots including Brassica napus (60 aa), Lactuca sativa (68 aa), Zea mays (140 aa), Oryza sativa (140 aa) and Sorghum bicolor (210 aa). These sequences are provided in Table 1. We purified all candidate polypeptides by bacterial expression system. The purified polypeptides showed similar antibacterial and antifungal effects at high concentration (30 |iM).

[0082] Table 2 shows the polypeptides tested in the present illustrative techniques for antimicrobial activity against bacteria and fungi. Some polypeptides exhibited strong antimicrobial activity, while others showed weaker effects.

Claims

PATENT Atorney Docket No. 118380-1544273-261210PC Client Ref. No. UC-2025-670-2 WHAT IS CLAIMED IS:

1. A method of treating or preventing a pathogenic bacterial or fungal infection in a plant, the method comprising contacting the plant with an antimicrobial Moirai polypeptide comprising a net charge of at least +3 and a glycine content of at least 25%, wherein the antimicrobial Moirai polypeptide comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 1-99 or at least 80% identity to any one of SEQ IDNOs: 1, 2, 3, 4, 5, 6, 7, 8, or 11.

2. The method of claim 1, wherein the antimicrobial Moirai polypeptide has at least 90% identity to any one of SEQ ID NOs: 1-99 or at least 90% identity to any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, or 11.

3. The method of claim 1, wherein the antimicrobial Moirai polypeptide has at least 95% identity to any one of SEQ ID NOs: 1-99 or at least 95% identity to any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, or 11.

4. The method of claim 1, wherein the antimicrobial Moirai polypeptide comprises any one of SEQ ID NOs: 1-99 or any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, or 11, optionally wherein the sequence does not comprise an N-terminal methionine.

5. The method of any one of claims 1-4, wherein the antimicrobial Moirai polypeptide is 15-300 amino acids in length.

6. The method of any one of claims 1-5, wherein the antimicrobial Moirai polypeptide comprises a glycine rich motif GnRGn, optionally wherein the motif is repeated 1-5 times.

7. The method of any one of claims 1-5, wherein the antimicrobial Moirai polypeptide comprises a glycine rich motif GnRGnDGnRGY, optionally wherein the motif is repeated 1-5 times.

8. The method of any one of claims 1-7, wherein the pathogenic bacteria is gram-negative bacteria or bacteria that does not have a cell wall.PATENT Atorney Docket No. 118380-1544273-261210PC Client Ref. No. UC-2025-670-2 9. The method of claim 8, wherein the gram-negative bacteria is a Liberibacter sp., an Agrobacterium sp., a Pseudomonas sp., a Xanthomonas sp., or Xylella sp.

10. The method of any one of claims 1-7, wherein the pathogenic fungus is a Botrytis sp. or Verticillium sp.

11. An isolated antimicrobial Moirai polypeptide having antibacterial and antifungal activity, wherein the Moirai polypeptide comprises a net charge of at least +3 and a glycine content of at least 25% and comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 1-99 or at least 80% identity to any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, or 11.

12. The isolated antimicrobial Moirai polypeptide of claim 11, wherein the Moirai polypeptide has at least 90% identity to any one of SEQ ID NOs: 1-99 or at least 90% identity to any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, or 11.

13. The isolated antimicrobial Moirai polypeptide of claim 11, wherein the Moirai polypeptide has at least 95% identity to any one of SEQ ID NOs: 1-99 or comprises any one of SEQ ID NOs: 1-99; or has at least 95% identity to any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, or 11 or comprises any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, or 11; optionally wherein the sequence does not comprise an N-terminal methionine.

14. The isolated antimicrobial polypeptide of any one of claims 11-13, wherein the Moirai polypeptide is 15-300 amino acids in length.

15. The isolated antimicrobial polypeptide of any one of claims 11-14, wherein the antimicrobial Moirai polypeptide comprises a glycine rich motif GnRGn, optionally wherein the motif is repeated 1-5 times.

16. The isolated antimicrobial polypeptide of any one of claims 11-14, wherein the antimicrobial Moirai polypeptide comprises a glycine rich motif GnRGnDGnRGY, optionally wherein the motif is repeated 1-5 times.

17. The isolated antimicrobial polypeptide of any one of claims 11-16, wherein the antimicrobial Moirai polypeptide targets pathogenic bacteria or fungi.PATENT Atorney Docket No. 118380-1544273-261210PC Client Ref. No. UC-2025-670-2 18. The isolated antimicrobial polypeptide of claim 17, wherein the pathogenic bacteria is gram-negative bacteria.

19. An agricultural composition comprising the antimicrobial Moirai polypeptide of any one of claims 11-18.

20. The agricultural composition of claim 19, wherein the composition comprises an additional agent that targets the pathogenic bacteria or fungi.

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