Methods of identifying endophytes, associated compositions, and methods of use

Abstract

This disclosure relates to compositions and methods of producing compositions for improving plant traits. This disclosure provides methods of identifying native endophytes, genetically engineering said endophytes, and recolonizing plant(s) which the endophytes were isolated from. Further, this disclosure provides compositions made by the methods disclosed herein and using the same.

Description

METHODS OF IDENTIFYING ENDOPHYTES, ASSOCIATED COMPOSITIONS, ANDMETHODS OF USERELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 567,475, filed March 20, 2024, incorporated by reference herein for all purposes.REFERENCE TO ELECTRONIC SEQUENCE LISTING

[0002] The application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on March 6. 2025, is named “139891-00801. xml” and is 21,890 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety.FIELD OF THE DISCLOSURE

[0003] The present disclosure relates to methods for identifying native plant endophytes and genetically engineering said endophytes to modify the phenotype of a plant or provide additional beneficial functions. For example, a native endophyte can be selected and genetically engineered to express peptides or polypeptides, which benefit the growth of the plant, the immediate environment surrounding the plant, or can generate byproducts for extraction.BACKGROUND OF THE INVENTION

[0004] Due to an increasing population, it is estimated that global food production needs to increase by 60-70% by 2050. Climate change, global instability, agricultural diseases, and adversarial actions threaten global food security'. Every year, pests and pathogens destroy nearly 10-23% of crops, with another 10-20% lost post-harvest. Shifts in climate exacerbate this, as pathogenic fungi expand their reach from the tropics toward the poles. In the past several years, fungal wheat stem rust infections, which typically infect plants in the tropics, have been reported as far north as Ireland. The geographic spread of these diseases and rising temperatures offers the greater possibility for opportunistic infection, meaning plant pathogens could jump from plants to livestock and humans.

[0005] Agriculture in the United States is especially susceptible to natural and adversarial disruption. Farms are vast and largely unprotected, making them ideal targets for ouradversaries. Further, U.S. agriculture is primarily monoculture; such practices breed treatmentresistant infectious diseases and pests. These agricultural practices provide the perfect storm for a potential fungal outbreak. Fungi are highly resilient, can survive in soil for 40 years, and produce airborne spores. Given the emergence of fungicide-resistant pathogens, the next fungal outbreak could devastate U.S. food production.

[0006] The current state-of-the-art methods combating fungal outbreaks include spraying fungicides or incorporating microbes antagonistic to the pathogens into the soil. However, these methods suffer considerable drawbacks and risk being unable to keep up with future fungal outbreaks. The use of fungicidal sprays is under intense scrutiny, given the potential for adverse effects on human and livestock health as well as driving the development of antifungal- resistant fungi. While inoculating soil with fungal antagonistic microbes is considered safer and has demonstrated efficacy in the lab, field trials fail to show significant protection against pathogenic fungi. Engineering plant to be more pest resistant is time consuming and can potentially take years to develop.

[0007] Thus, there is a need for rapid, scalable, and cost effective methods and compositions for providing probiotics to plants, particularly agricultural plants, to improve growth and resistance to environmental stresses, including pest infection. Provided herein are novel methods for developing genetically engineered endophytes and synthetic endophyte-plant compositions to impart traits of interest to the plant.SUMMARY OF THE INVENTION

[0008] This disclosure provides, at least in part, a method of identifying a genetically tractable microbial endophyte, the method comprising: isolating a plurality of microbial endophytes from one or more tissues of a plant; conjugating the plurality of isolated microbial endophytes with one or more microorganisms comprising a library of broad host range plasmids having at least one selection marker; and selecting one or more conjugated microbial endophytes that have one or more selection markers.

[0009] In some embodiments, the method further comprises transforming the selected microbial endophytes with one or more heterologous nucleic acid sequences encoding one or more heterologous peptides and / or polypeptides.

[0010] In some embodiments, the microbial endophytes comprising one or more heterologous nucleic acid sequences is screened for expression of the one or more heterologous peptides and / or polypeptides.

[0011] In another aspect, the present disclosure provides a method of producing a genetically engineered microbial endophyte, the method comprising: isolating a plurality of microbial endophytes from one or more tissues of a plant; conjugating the plurality of isolated microbial endophytes with one or more microorganisms comprising a library of broad host range plasmids having at least one selection marker and one or more heterologous nucleic acid sequences encoding one or more heterologous peptides and / or polypeptides; selecting one or more conjugated microbial endophytes that have one or more selection markers; culturing the one or more selected microbial endophytes.

[0012] In some embodiments, the selected microbial endophytes is screened for expression of the one or more heterologous peptides and / or polypeptides.

[0013] In some embodiments, the plurality of microbial endophytes comprises one or more bacteria or one or more fungi.

[0014] In some embodiments, the one or more bacteria is of the genus Bacillus, Herbaspirillum, Rhizobium, Bradyrhizobium, Pseudomonas, Ralstonia, Streptomyces, Acidovorax, Rhodococcus. Acetobacteraceae, Acidobacteriaceae, Acidothermaceae, Aerococcaceae, Alcaligenaceae, Alicyclobacillaceae, Alteromonadaceae. Anaerolineaceae, Aurantimonadaceae, Bacillaceae, Bacteriovoracaceae, Bdellovibrionaceae,Bradyrhizobiaceae, Brevibacteriaceae, Brucellaceae, Burkholder iaceae, Carboxydocellaceae, Caulobacleraceae, Cellulomonadaceae, Chilinophagaceae, Chromaliaceae, Chthoniobacteraceae, Chthonomonadaceae, Clostridiaceae. Comamonadaceae, Corynebacteriaceae, Coxiellaceae, Cryomorphaceae, Cyclobacteriaceae, Cytophagaceae, Deinococcaceae, Dermabacteraceae, Dermacoccaceae, Enter obacteriaceae,Enterococcaceae, Erythrobacteraceae, Fibrobacteraceae, Flammeovirgaceae,Flavobacteriaceae, Frankiaceae. Fusobacteriaceae, Gaiellaceae. Gemmatimonadaceae,Geodermatophilaceae, Glycomycetaceae, Haliangiaceae, Halomonadaceae, Holosporaceae, Hyphomicrobiaceae, lamiaceae, Intrcisporangiacecie, Kineosporiacecie, Koribacterciceae, Lachnospiraceae, Lactobacillaceae, Legionellaceae, Leptospiraceae, Leuconostocaceae, Methylobacteriaceae, Methylocystaceae, Methylophilaceae, Microbacteriaceae, Micrococcaceae, Micromonosporaceae, Moraxellaceae, Mycobacter iaceae,Mycoplasmataceae, Myxococcaceae, Nakamurellaceae. Neisseriaceae, Nitrosomonadaceae, Nocardiaceae, Nocar dioidaceae, Oceanospirillaceae, Opitutaceae, Oxalobacteraceae, Paenibacillaceae, Parachlamydiaceae, Pasteurellaceae, Patulibacteraceae,Peptostreptococcaceae, Phyllobacteriaceae, Piscirickettsiaceae, Planctomycetaceae, Planococcaceae, Polyangiaceae, Porphyromonadaceae, Prevotellaceae,Promicromonosporaceae, Pseudomonadaceae, Pseudonocardiaceae, Rhizobiaceae,Rhodobacteraceae, Rhodospirillaceae, Roseiflexaceae, Rubrobacteriaceae. Sandaracinaceae,Sanguibacteraceae. Saprospiraceae, Segniliparaceae, Shewanellaceae, Sinobacteraceae,Solibacteraceae, Solimonadaceae, Solirubrobacteraceae, Sphingobacteriaceae,Sphingomonadaceae, Spiroplasmataceae, Sporichthyaceae, Sporolactobaci llaceae,Staphylococcaceae, Strept coccaceae, Streptomycetaceae, Syntrophobacteraceae,Veillonellaceae, Verrucomicrobiaceae. Weeksellaceae, Xanthobacteraceae, and / orXanthomonadaceae and the one or more fungi is of the genus Epichloe, Neotyphodium, Khuskia, Epicoccum, Curvidaria, Serendipita, Mycosphaerella, Piriformospora, Cladosporium, Fusarium, Colletotrichum, Phomopsis, Beauveria, Talaromyces, Apiospora, Aspergillaceae, Ceratobasidiaceae, Coniochaetaceae, Cordycipitaceae, Corticiaceae, Cystofllobasidiaceae, Davidiellaceae, Debaryomycetaceae, Dothioraceae, Erysiphaceae, Filobasidiaceae, Glomerellaceae, Hydnaceae, Hypocreaceae, Leptosphaeriaceae, Montagnulaceae, Mortierellaceae, Mycosphaerellaceae, Nectriaceae, Orbiliaceae, Phaeosphaeriaceae, Pleosporaceae, Pseudeurotiaceae, Rhizopodaceae, Sclerotiniaceae, Stereaceae. and / or Trichocomaceae .

[0015] In some embodiments, the tissues of the plant are selected from the group consisting of roots, shoots, stems, leaves, rhizomes, trichomes, pneumatophores, inflorescences, pollen and seeds.

[0016] In some embodiments, the library of broad host range plasmids comprises one or more sequences from pCUl , RK2, R300B, R1162, pSa, pR288, pBBRl, PBR322-based plasmids, pUC series plasmids, pSC 101 -derived plasmids, pEpi series plasmids, pL2 series plasmids, pGreen vector series, pH7 vector series, pBI121 and pCAMBIA series, pKl l series, pK13 series, pGWB series plasmids, pUbi vector series, pNovl vectors, pC1301, pC1302, pMDC, pPZP, pVBN, pSHP, pSAG-based vectors, pPha-tl, pGWB, pHygro series, pSL1180 series, pT7-based vectors, pMOL98, RK2, RP4,RP1,R68, PB10, RA3, IncN, IncP-1, IncU, IncPromA, Incl8, RK404, pDSK509, pDSK519, pRK415, RSF1010, IncW, pJC8, pJC24, PBBR1MCS, pK18mobsacB, pLAFRl, pCCIFOS, pWKS30, pUTmmi-TK, pEcoRi, PGEM- T, pGEM. pFOSl, pCCIFOS. pFOS!RK2. pF0S6, pYAC4, pYAC3, pYAC5, pYACl. pBBRIMCS-BAC, pSClOl-based bacterial artificial chromosome (BACs), pFOSBAC, pFosBAC, pK18mobSacB-based BACs, or a combination thereof.

[0017] In some embodiments, the at least one selection marker is resistance to an antibiotic. In some embodiments, the antibiotic is selected from the group consisting of kanamycin,spectinomycin, streptomycin, ampicillin, carbenicillin, bleomycin, erythromycin, polymyxin B, tetracycline, and chloramphenicol.

[0018] In some embodiments, the one or more heterologous nucleic acid sequences are between about 0.01 kb to about 20 kb, about 0.1 kb to about 10 kb, about 0.5 kb to about 15 kb, about 0.1 to about 1 kb, or about 5 kb to about 10 kb.

[0019] In some embodiments, the one or more peptides and / or polypeptides comprise a small molecule, a phytohormone, an enzyme, a transcription factor, and / or a nuclease.

[0020] In some embodiments, the plant is of the genus Zea, Triticum, Oryza, Zizania, Hordeum, Sorghum, Eleusine, Panicum, Pennisetum, Setaria, Avena, Triticosecale, Secale, Psathyrostachys, Bambuseae, Saccharum, Glycine max, Brassica, Gossypium, Medicago, Manihot, Solanum, Solanum, Sporobolus, Odontarrhena, Streptanthus, Pisum, Cicer, Lens, or Linum.

[0021] In another aspect, the present disclosure provides a method of preparing a plant comprising a plurality of genetically engineered microbial endophytes, a method comprising: applying to an exterior surface of the plant the plurality of genetically engineered microbial endophytes, wherein the plurality of genetically engineered microbial endophyte are derived from one or more microbial endophyte species isolated from a species of the plant and comprises one or more heterologous nucleic acid sequences encoding one or more heterologous peptides and / or polypeptides, and wherein the plant comprising the plurality of genetically engineered microbial endophytes has one or more traits of interest.

[0022] In some embodiments, the method further comprises applying to the exterior of the plant one or more additional genetically engineered microbial endophytes, wherein the additional one or more genetically engineered microbial endophy tes are from one or more species isolated from a species of the plant and comprises one or more heterologous nucleic acid sequences encoding one or more heterologous peptides and / or polypeptides.

[0023] In some embodiments, the one or more additional genetically engineered microbial endophytes are of different genera compared to each other.

[0024] In some embodiments, the one or more additional genetically engineered microbial endophytes are of the same genus compared to each other.

[0025] In some embodiments, the trait of interest of the plant is selected from the group consisting of germination rate, emergence rage, drought tolerance, freeze tolerance, shoot biomass, root biomass, seeding root length, yield, metal ion binding, nitrogen fixing, pollutant degrading, anti-fungal, herbicidal, nematicidal, and / or insecticidal.

[0026] In some embodiments, the microbial endophyte is isolated from one or more tissue of the plant. In some embodiments, the one or more tissues of the plant is selected from the group consisting of roots, shoots, stems, leaves, rhizomes, trichomes, pneumatophores, inflorescences, pollen and seeds.

[0027] In some embodiments, the plant is of the genus Zea, Triticum, Oryza, Zizania, Hordeum, Sorghum, Eleusine. Panicum, Pennisetum, Setaria, Avena, Triticosecale, Secale, Psathyrostachys. Bambuseae, Saccharum, Glycine max, Brassica. Gossypium, Medicago, Manihot, Solanum, Solanum, Sporobolus, Odontarrhena, Streptanthus, Pisum, Cicer. Lens, or Linum.

[0028] In some embodiments, the exterior surface of the plant comprises a seed coat, leaves, root, shoots, and / or flowers.

[0029] In some embodiments, the plurality of genetically engineered microbial endophytes comprise 2, 3, 4, 5, 6, 7, 8, 9, or 10 different genetically engineered microbial endophytes each of which comprises different one or more heterologous nucleic acid sequences encoding one or more heterologous peptides and / or polypeptides.

[0030] In another aspect, the present disclosure provides composition comprising a plant and a genetically engineered microbial endophyte, wherein the genetically engineered microbial endophyte is from a genus in symbiosis with the plant and comprises one or more heterologous nucleic acid sequences encoding one or more heterologous peptides and / or polypeptides, wherein the plant is contacted with the genetically engineered microbial endophyte in an amount effective to colonize the plant and to improve one or more traits of interest, and wherein the genetically engineered microbial endophyte is produced by a method comprising: isolating a plurality of microbial endophytes from one or more tissues of a plant; conjugating the plurality of isolated microbial endophytes with one or more microorganisms comprising a library of broad host range plasmids having at least one selection marker and the one or more heterologous nucleic acid sequences encoding the one or more heterologous peptides and / or polypeptides; selecting one or more conjugated microbial endophytes that have one or more selection markers; and culturing the one or more selected microbial endophytes in conditions to induce expression of the more heterologous peptides and / or polypeptides.

[0031] In some embodiments, the traits of interest is selected from the group consisting of germination rate, emergence rage, drought tolerance, freeze tolerance, shoot biomass, root biomass, seeding root length, yield, metal ion binding, pollutant degrading, anti-fungal, herbicidal, nematicidal, and / or insecticidal.

[0032] In some embodiments, the plurality of microbial endophytes comprises one or more bacteria or one or more fungi.

[0033] In some embodiments, the one or more bacteria is of the genus Bacillus, Herbaspirillum, Rhizobium, Bradyrhizobium, Pseudomonas, Ralstonia, Streptomyces, Acidovorax, Rhodococcus, Acetobacteraceae, Acidobacter iaceae, Acidothermaceae, Aerococcaceae, Alcaligenaceae, Alley clobacillaceae, Alteromonadaceae, Anaerolineaceae, Aurantimonadaceae, Bacillaceae, Bacteriovoracaceae, Bdellovibrionaceae, Bradyrhizobiaceae, Brevibacteriaceae, Brucellaceae, Burkholderiaceae, Carboxydocellaceae, Caulobact er aceae, Cellulomonadaceae, Chitinophagaceae, Chromatiaceae,Chthoniobacter aceae, Chthonomonadaceae, Clostridiaceae, Comamonadaceae,Corynebacteriaceae. Coxiellaceae, Cryomorphaceae, Cyclob act er iaceae, Cytophagaceae,Deinococcaceae, Dermabacteraceae. Dermacoccaceae, Enter obact er iaceae,Enterococcaceae, Erythrobacteraceae, Fibrobacteraceae, Flammeovirgaceae,Flavobacteriaceae, Frankiaceae, Fusobacteriaceae, Gaiellaceae, Gemmatimonadaceae,Geodermatophilaceae, Glycomycetaceae, Haliangiaceae, Halomonadaceae. Holosporaceae, Hyphomicrobiaceae, lamiaceae, Intrasporangiaceae, Kineospor iaceae. Koribacteraceae, Lachnospir aceae, Lactobacillaceae, Legionell aceae, Leptospiraceae, Leuconostocaceae, Me thylobact er iaceae, Methylocystaceae, Methylophilaceae, Microbacteriaceae,Micrococcaceae, Micromonosporaceae, Moraxellaceae, Mycobacler iaceae, Mycoplasmataceae, Myxococcaceae, Nakamurellaceae, Neisseriaceae, Nitrosomonadaceae, Nocardiaceae, Nocar dioidaceae, Oceanospirillaceae, Opitutaceae, Oxalobacteraceae, Paenibacillaceae, Parachlamydiaceae, Pasteurellaceae, Patulibacteraceae,Peptostreptococcaceae, Phyllobacteriaceae, Piscirickettsiaceae, Planet omycetaceae,Planococcaceae, Polyangiaceae, Porphyromonadaceae, Prevotellaceae,Promicromonosporaceae, Pseudomonadaceae, Pseudonocardiaceae, Rhizobiaceae, Rhodob act er aceae, Rhodospirillaceae, Roseiflexaceae, Rubrobacteriaceae, Sandaracinaceae, Sanguibacteraceae, Saprospiraceae, Segniliparaceae, Shewanellaceae, Sinobact er aceae, Solibacter aceae, Solimonadaceae, Solirubr obact er aceae, Sphingobacteriaceae,Sphingomonadaceae, Spiroplasmataceae, Sporichthyaceae, Sporolactobacillaceae,Staphylococcaceae, Streptococcaceae, Streptomycetaceae, Syn trophobacteraceae,Veillonellaceae, Verrucomicrobiaceae, Weeksellaceae, Xanthobacter aceae, and / orXanthomonadaceae', and the one or more fungi is of the genus Epi chloe, Neotyphodium, Khuskia, Epicoccum, Curvularia, Serendipita, Mycosphaerella, Piriformospora, Cladosporium, Fusarium, Collet otrichum. Phomopsis, Beauveria, Talaromyces, Apiospora,Aspergillaceae, Ceratobasidiaceae, Coniochaetaceae, Cordycipitaceae, Corticiaceae,Cystofllobasidiaceae, Davidiellaceae, Debaryomycetaceae. Dothioraceae, Erysiphaceae,Filobasidiaceae, Glomerellaceae, Hydnaceae, Hypocreaceae, Leptosphaeriaceae,Montagnulaceae, Mortierellaceae, Mycosphaerellaceae, Nectriaceae, Orbiliaceae,Phaeosphaeriaceae, Pleosporaceae, Pseudeurotiaceae, Rhizopodaceae, Sclerotiniaceae, Stereaceae. and / or Trichocomaceae.

[0034] In some embodiments, the tissues of the plant is selected from the group consisting of roots, shoots, stems, leaves, rhizomes, trichomes, pneumatophores, inflorescences, and seeds.

[0035] In some embodiments, the library of broad host range plasmids comprises one or more sequences from pCUl, RK2, R300B, R1162, pSa, pR288, pBBRl, PBR322-based plasmids, pUC series plasmids, pSC 101 -derived plasmids, pEpi series plasmids, pL2 series plasmids, pGreen vector series, pH7 vector series, pBI121 and pCAMBIA series, pKl l series, pK13 series, pGWB series plasmids, pUbi vector series, pNovl vectors, pC1301, pC1302, pMDC, pPZP, pVBN, pSHP, pSAG-based vectors, pPha-tl, pGWB, pHygro series, pSL1180 series, pT7-based vectors, pMOL98, RK2, RP4,RP1,R68, PB10, RA3, IncN, IncP-1. IncU, IncPromA. Incl8. RK404, pDSK509, pDSK519, pRK415, RSF1010, IncW. pJC8, pJC24. PBBR1MCS, pK18mobsacB, pLAFRl, pCCIFOS, pWKS30, pUTmmi-TK, pEcoRi, PGEM- T, pGEM, pFOSl, pCCIFOS, pFOSlRK2, pF0S6, pYAC4, pYAC3, pYAC5, pYACl, pBBRIMCS-BAC, pSClOl-based bacterial artificial chromosome (BACs), pFOSBAC, pFosBAC, pK18mobSacB-based BACs. or a combination thereof.

[0036] In some embodiments, the at least one selection marker is resistance to an antibiotic. In some embodiments, the antibiotic is selected from the group consisting of kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, bleomycin, erythromycin, polymyxin B, tetracycline, and chloramphenicol.

[0037] In some embodiments, the one or more heterologous peptides and / or polypeptides comprise a peptide, an enzyme, a transcription factor, and / or a nuclease.

[0038] In some embodiments, the plant is of the genus Zea, Triticum, Oryza, Zizania, Hordeum, Sorghum, Eleusine. Panicum, Pennisetum, Setaria, Avena, Triticosecale, Secale, Psathyrostachys. Bambuseae, Saccharum, Glycine max, Brassica. Gossypium, Medicago, Manihot, Solanum, Solanum, Sporobolus, Odontarrhena, Streptanthus, Pisum, Cicer. Lens, or Linum.

[0039] In some embodiments, the genetically engineered microbial endophyte comprises 2, 3, 4, 5, 6. 7, 8. 9, or 10 different genetically engineered microbial endophytes each of whichcomprises different one or more heterologous nucleic acid sequences encoding one or more heterologous peptides and / or polypeptides.

[0040] In another aspect, the present disclosure provides a method of producing a genetically engineered microbial endophyte capable of binding a metal ion, the method comprising: culturing one or more selected microbial endophytes produced by the method according to any one of the methods disclosed herein and conjugating the selected microbial endophytes with one or more heterologous nucleic acid sequences encoding one or more cell-surface or secreted metal ion binding peptides; or culturing one or more selected microbial endophytes produced by the method according to any one of the methods disclosed herein , wherein the one or more heterologous nucleic acid sequences encodes one or more cell-surface metal ion binding peptides.

[0041] In some embodiments, the method further comprises conjugating the genetically engineered microbial endophyte with one or more heterologous nucleic acid sequences encoding one or more plant growth promoting hormones or phytohormones; and / or conjugating the genetically engineered microbial endophyte with an inducible kill switch.

[0042] In some embodiments, the metal ion is selected from the group consisting of gold, silver, palladium, platinum, rhodium, copper, nickel, and lanthanide. In some embodiments, the metal ion is nickel.

[0043] In some embodiments, the plant growth hormone is auxin and / or gibberellic acid.

[0044] In some embodiments, the metal ion binding peptide is a nickel binding peptide (NBP). In some embodiments, the nickel binding peptide is selected from the group consisting of any one of peptide sequences from SEQ ID NOs: 1-17.

[0045] In some embodiments, the plant species is selected from the group consisting of: S'. bicolor, P. virgatum, S. alter niflorus, B. juncea, O. chalcidica, and Streptanthus polygaloides . In some embodiments, the plant species is Streptanthus polygaloides.

[0046] In some embodiments, the metal ion is lanthanide. In some embodiments, the metal ion binding peptide is a lanthanide binding peptide (LBP). In some embodiments, in the lanthanide binding peptide is LanMl.

[0047] In some embodiments, the plant species is Panicum virgatum.

[0048] In another aspect, the present disclosure provides a method of producing a plant capable of accumulating metal ions, the method comprising: applying to an exterior surface of the plant one or more genetically engineered microbial endophytes produced by the method according to any one of the methods disclosed herein, wherein the plant is grown in soil comprising metal ions.

[0049] In another aspect, the present disclosure provides a method of isolating metal ions from the plant produced by the method according to any one of the methods disclosed herein, the method comprising homogenizing the plant tissues; and incubating the homogenized plant tissues in an acid solution.

[0050] In another aspect, the present disclosure provides a method of producing a genetically engineered microbial endophyte capable of producing one or more anti-microbial agents, the method comprising: culturing one or more selected microbial endophytes produced by the method according to any one of the methods disclosed herein and conjugating the selected microbial endophytes with one or more heterologous nucleic acid sequences encoding one or more anti-microbial agents; or culturing one or more selected microbial endophytes produced by the method according to any one of the methods disclosed herein, wherein the one or more heterologous nucleic acid sequences encodes one or more anti-microbial agents.

[0051] In some embodiments, the anti-microbial agent is an anti-fungal agent or an antibacterial agent.

[0052] In some embodiments, the method further comprises conjugating the genetically engineered microbial endophyte with one or more heterologous nucleic acid sequences encoding one or more plant grow th promoting hormones or phytohormones; and / or conjugating the genetically engineered microbial endophyte with an inducible kill switch.

[0053] In some embodiments, the plant is of the genus Zea, Trilicum, Oryza, Zizania, Hordeum, Sorghum, Eleusine. Panicum, Pennisetum, Setaria. Avena. Triticosecale, Secale, Psathyrostachys, Bambuseae, Saccharum, Glycine max. Brassica, Gossypium, Medicago, Manihot, Solarium, Solatium, Sporobolus, Odontarrhena, Streptanthus, Pisum, Cicer, Lens, or Linum. In some embodiments, the plant is Triticum aestivum. In some embodiments, the one or more antifungal agents inhibit Funsarium spp. and / or R. solani.

[0054] In another aspect, the present disclosure provides a method of producing a plant comprising a genetically engineered microbial endophyte capable of producing one or more anti-microbial agents, the method comprising: applying to an exterior surface of the plant one or more genetically engineered microbial endophytes produced by the method according to any one of the methods disclosed herein, wherein the plant is grown in an environment susceptible to microbial infection.

[0055] In another aspect, the present disclosure provides a method of producing a genetically engineered microbial endophyte capable of producing pigment compound, the method comprising: culturing one or more selected microbial endophytes produced by the method according to any one of the methods disclosed herein and conjugating the selected microbialendophytes with one or more heterologous nucleic acid sequences encoding one or more amino acid catalyzing enzymes, wherein the amino acid catalyzing enzymes can catalyze one or more amino acids into one or more pigment compounds; or culturing one or more selected microbial endophytes produced by the method according to any one of the methods disclosed herein, wherein the one or more heterologous nucleic acid sequences encodes one or more amino acid catalyzing enzymes, wherein the amino acid catalyzing enzymes can catalyze one or more amino acids into one or more pigment compounds.

[0056] In another aspect, the present disclosure provides a method of producing a genetically engineered microbial endophyte capable of secreting plant signaling molecule, the method comprising: culturing one or more selected microbial endophytes produced by the method according to any one of the methods disclosed herein and conjugating the selected microbial endophytes with one or more heterologous nucleic acid sequences encoding one or more plant signaling molecules; or culturing one or more selected microbial endophytes produced by the method according to any one of the methods disclosed herein, wherein the one or more heterologous nucleic acid sequences encodes one or more plant signaling molecules.

[0057] In another aspect, the present disclosure provides a method of producing a genetically engineered microbial endophyte for bioremediation, the method comprising: culturing one or more selected microbial endophytes produced by the method according to any one of the methods disclosed herein and conj ugating the selected microbial endophytes with one or more heterologous nucleic acid sequences one or more environmental pollution degrading enzymes; or culturing one or more selected microbial endophytes produced by the method according to any one of the methods disclosed herein , wherein the one or more heterologous nucleic acid sequences encodes one or more environmental pollution degrading enzy mes.

[0058] In another aspect, the present disclosure provides a method of producing a genetically engineered microbial endophyte capable of increasing nitrogen fixation, the method comprising: culturing one or more selected microbial endophytes produced by the method according to any one of the methods disclosed herein and conjugating the selected microbial endophytes with one or more heterologous nucleic acid sequences encoding one or more enzymes capable of fixing nitrogen; or culturing one or more selected microbial endophytes produced by the method according to any one of the methods disclosed herein, wherein the one or more heterologous nucleic acid sequences encodes one or more enzymes capable of fixing nitrogen.BRIEF DESCRIPTION OF THE DRAWINGS

[0059] FIG. 1 depicts a schematic of the workflow to identify and engineer microbial endophytes: extracting microbial endophytes from plant root; endophytes are subjected to a conjugation pipeline using a broad host range plasmid or library of broad host range plasmids; microbes are plated on a selective media.

[0060] FIG. 2A depicts the proof of concept of the disclosed technology: 1) conjugative microbe delivers GFP expressing plasmid into root endophyte isolate; 2) Phragmites australis (Pa) root is cut and incubated with GFP expressing bacteria; 3) endophytic bacteria recolonizes root and fluorescence is imaged. FIG. 2B depicts an exemplary real-world application: endophytes are engineered to express an inducible plasmid; engineered microbes are cultured; engineered microbes are seeded to Pa soil and colonize the Pa root. When desired phenotype is needed, inducer is applied and engineered microbes produce product from within the plant.

[0061] FIGs. 3A-3C show that JS IB is capable of endosymbiotically recolonizing Phragmites australis (Pa) roots. FIG. 3A shows the imaging of JS1B incubated with Pa root clipping for 3 days. FIGs. 3B-3C show that microbes Pseudomonas tremae (FIG. 3B) and Comomonas oxytoca (FIG. 3C) isolated from soil failed to colonizing t? root, suggesting that the identified plant specific microbes are capable of colonizing cells endosymbiotically.

[0062] FIGs. 4A-4B show that endophyte colonization is plant specific. FIG. 4A shows P. australis root cell colonization. FIG. 4B shows switch grass root cell colonization.

[0063] FIG. 5 depicts a schematic of the workflow to engineer genetically tractable endophytes with desired traits of interest: root endophytes are extracted from valuable crops in various environments; a library of microbes is constructed by identifying genetically tractable plant endophytes; microbes are engineered to various functions, including but not limited to delivering RNAi against virus, conferring drought tolerance, and remediating toxic chemicals in the soil.

[0064] FIGs. 6A-6D show endophytes as a targeted biotechnological tool. FIG. 6A depicts that Pseudomonas putida (red) recolonizes P. australis. FIG. 6B depicts that Klebsiella sp. (cyan) recolonizes P. virgatum. FIG. 6C depicts that / < putida colonizes host plant tissue (P. australis while failing to establish itself in P. virgatum. FIG. 6D depicts that Nicotiana benthimiana plants treated with control endophytes (solid line) and endophytes producing plant growth inhibiting proteins (dashed line).

[0065] FIG. 7 demonstrates rapid and modular biosensing in plants, according to an exemplary aspect. Endophytes can be engineered to be responsive to various stimuli and secrete plant signaling molecule when activated. Such engineered endophytes can then be applied to theplant to develop plant biosensors that are responsive to the corresponding stimuli. By applying a pool of endophytes engineered to respond to diverse stimuli, multiple biosensors can be developed on a single plant.

[0066] FIGs. 8A-8B depict the experiment to identify soil-thriving microbe strains for mobilizable biosensor system. FIG. 8A shows that various strains of bacteria isolated from soil were conjugated with fluorescent protein. Bacteria successfully conjugated with fluorescent protein has shown fluorescence under fluorescent microscopy. The bacteria are then incubated in the soil and bacterial growth and survival are analyzed by counting Colony-forming units (CFU) on the agar plate. FIG. 8B shows survival of bacteria over a 2-week period was recorded, and compared with the E. coli control. Based on the results, E. coli growth is below limit of detection, while within all tested strains, A. sandrelli has the highest growth rate after re-introduced to the soil.

[0067] FIGs. 9A-9B show results of mobilizable biosensor system. Bacteria species expressing eBeads sensor system for 2-polyphenol (2-PP) can be selected and isolated in less than a week. FIG. 9A demonstrates selection of four unique species expressing eBeads sensor system. The native eBeads sensors are activated by 200nM 2-PP. FIG. 9B shows the comparison of 2-PP responsive strains cultured under OmM 2-PP and under 200mM 2-PP. The 2-PP responsive strains show7darker color when co-cultured with 200mM 2-PP, indicating activation of the biosensor system.

[0068] FIG. 10 depicts the workflow to identify and engineer endophytes for bioremediation: Root endophytes are extracted from valuable crops in various environments; Conjugate root endophytes and identifying genetically tractable, plant endophytes; Native microbial communities are engineered to deliver pollution degrading enzymes in the soil via root networks.

[0069] FIG. 11 depicts the workflow to identify and engineer root-associated microbes for underground nickel extraction: Task 1) Engineer Ni-binding peptides; Task 2) Display and secrete Ni binding peptides (NBPs) from root-associated microbes to accelerate Ni accumulation and tolerance in a variety of plants, and increase plant biomass through the production of plant growth promoting hormones; Task 3) Extract and quantify nickel.

[0070] FIG. 12 shows genetic constructs designed for NBP surface display in E. coli. The constructs with a carrier protein made of a signal peptide (SP) and a surface anchor (SA), a protein tag to facilitate quantification, and a terminal (top) on intramolecular (bottom) NBP.

[0071] FIG. 13 depicts the workflow to develop endophytic microbes to deliver antifungal peptides directly to the roots of plants to prevent fungal outbreaks: Use generative Al to predictnovel antifungal peptides (AFPs); Select for efficient AFPs; Engineer wheat endophytes to produce AFPs: and apply engineered endophytes to growing plants to treat / prevent fungal infections.

[0072] FIGs. 14A-14B depict the successful production of small molecules in switchgrass roots (Arrow) (FIG. 14A) and proteins in Nicotiana benthamiana leaf (Arrow) (FIG. 14B).

[0073] FIG. 15 demonstrates heterologous indigoidine production by engineered endophytes. Indigoidine BHR plasmid are conjugated to a pool of isolated endophytic microbes. Conjugated microbes selected on agar plate demonstrate heterologous indigoidine production.

[0074] FIG. 16 demonstrates that plant colonizing microbes can be engineered to deliver small molecule or protein effectors directly and continuously to crops. The procedure includes steps: homogenizing root from target crop, selecting pool of microbes, conjugated selected pool of microbes with BHR plasmid pool, selecting conjugated microbes and colonizing plants with the selected genetically engineered microbes.

[0075] FIG. 17 depicts pipeline to identify genetically tractable root endophytes: Endophytes are extracted from the roots of a plant; Endophytes are subjected to a conjugation pipeline using a library of broad host range plasmids; Microbes are plated on a selective media enriching for endophytic microbes: Transgenic microbes are then assayed using fluorescence microscopy for the ability to recolonize root cells; and Selected microbes are applied to the plants. The genetically engineered microbes express Fosmidomycin (and its analogue FR9000098), which is a small molecule that interferes with terpene biosynthesis, which causes bleaching of leaves and external portions of the plant.

[0076] FIGs. 18A-18B demonstrate selection of tractable hosts with a desired phenotype from various environment. FIG. 18A shows examples of marine bacteria. FIG. 18B shows examples of soil bacteria.DETAILED DESCRIPTION OF THE INVENTION

[0077] As used herein and in the appended claims, the singular forms “a”, “an’", and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to a “peptide” is a reference to one or more peptides and equivalents thereof known to those skilled in the art, and so forth.

[0078] The term “about” is used herein to mean within the ty pical ranges of tolerances in the art. For example, “about” can be understood as about 2 standard deviations from the mean. According to certain embodiments, about means +10%. According to certain embodiments,about means +5%. When about is present before a series of numbers or a range, it is understood that "about’ ' can modify each of the numbers in the series or range.

[0079] The term “at least’’ prior to a number or series of numbers (e.g., “at least two”) is understood to include the number adj acent to the term “at least”, and all subsequent numbers or integers that could logically be included, as clear from context. When at least is present before a series of numbers or a range, it is understood that “at least” can modify each of the numbers in the series or range.

[0080] As used herein, “up to” as in “up to 10” is understood as up to and including 10, i.e., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0081] Ranges provided herein are understood to include all individual integer values and all subranges within the ranges.

[0082] The terms “composition” and “formulation” are used interchangeably herein to refer to a product of the described invention that comprises all active and inert ingredients.

[0083] The term “decrease” and its various grammatical forms is used herein to refer to a diminution, a reduction, an attenuation or abatement of the degree, intensity, extent, size, amount, density or number of occurrences, events or characteristics.

[0084] The term “reduce” and its various grammatical forms is used herein to refer to a diminution, a decrease, an attenuation or abatement of the degree, intensity, extent, size, amount, density or number of occurrences, events or characteristics.

[0085] The term “improves” is used to convey that the present invention changes either the appearance, form, characteristics and / or the physical attributes of the subject, organ, tissue or cell to which it is being provided, applied or administered. For example, and without limitation, the change in form may be demonstrated by any of the following alone or in combination: a decrease in one or more infection pests, e.g., fungus, infecting the plant; increase growth and biomass of the plant; and reduce the necessity for external fertilizers.

[0086] The term “expression” as used herein generally refers to the action of a gene in the production of a protein or phenotype. More specifically, it refers to the process by which a polynucleotide is transcribed from a DNA template (such as into an mRNA or other RNA transcript, e.g., RNAi or miRNA) and / or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referred to as “gene product.” If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. Expression may also refer to the post-translational modification of a polypeptide or protein.

[0087] The term “gene’' as used herein refers to a region of DNA that controls a discrete hereditary characteristic, usually corresponding to a single protein or RNA. This definition includes the entire functional unit, encompassing coding DNA sequences, noncoding regulatory DNA sequences, and introns.

[0088] As used herein, each of the terms "peptide,"’ “polypeptide" and "protein" refer to two or more amino acids covalently linked by an amide bond or non-amide equivalent. A peptide generally is considered an amino acid polymer of 40 or fewer amino acids. A polypeptide generally is considered an amino acid polymer containing more than 40 amino acids or more. The amino acid sequence also can include modifications typically associated with post- translational processing of proteins, for example, cyclization (e.g., disulfide or amide bond), phosphorylation, glycosylation, carboxylation, ubiquitination, myristylation, or lipidation.

[0089] As used herein, the term “tissue” refers to a collection of similar cells and the intercellular substances surrounding them. For example, tissues of a plant can include, but are not limited to, root tissue, stem tissue, leaf tissue, or flower tissue.

[0090] The term “transcription factor” as used herein refers to proteins that bind to DNA- regulatory sequences (enhancers and silencers), usually localized in the 5 ’-upstream region of target genes, to modulate the rate of gene transcription. This may result in increased or decreased gene transcription, protein synthesis, and subsequent altered cellular function.Embodiments

[0091] According to one aspect, the present disclosure provides a method of identifying a genetically tractable microbial endophyte, the method comprising: isolating a plurality' of microbial endophytes from one or more tissues of a plant; conjugating the plurality of isolated microbial endophytes with one or more microorganisms comprising a library’ of broad host range plasmids having at least one selection marker; and selecting one or more conjugated microbial endophytes that have one or more selection markers.

[0092] According to some embodiments, “genetically tractable” means that a biological organism can be easily manipulated at the genetic level, including but not limited to, introducing, modifying or deleting genes within the biological organism.

[0093] According to some embodiments, an “endophyte” is an organism that lives within a plant or is otherwise associated therewith, and does not cause disease or harm the plant otherwise. Endophytes can occupy the intracellular or extracellular spaces of plant tissue, including the leaves, stems, flowers, fruits, seeds, or roots. An endophyte can be for example abacterial or fungal organism, and can confer a beneficial property to the host plant such as an increase in yield, biomass, resistance, or fitness.

[0094] According to some embodiments, the term “capable of’ living inside or on the surface of a plant means that the endophyte has the appropriate features permitting it to live inside or on the surface of a plant. For example, the endophyte may produce the necessary7substances to avoid rejection by the plant, and be able to use the nutrients provided by the plant to live.

[0095] According to some embodiments, as used herein, the term “bacterium” or “bacteria” refers in general to any prokaryotic organism, and may' reference an organism from either Kingdom Eubacteria (Bacteria), Kingdom Archaebacteria (Archae), or both.

[0096] According to some embodiments, “plurality of microbial endophytes” refer to one or more endophytes that are genetically identical or genetically diverse. For example, a plurality of microbial endophytes can be isolated from a plant, e.g., from the roots, stem, leaves, and / or flowers, and pooled together where the plurality7can include bacterial cells that are genetically identical or genetically diverse, and / or fungal cells that are genetically identical or genetically diverse.

[0097] According to some embodiments, “conjugating” or “conjugation” or grammatical variation thereof refers to the process where one microbial cell directly transfers genetic materials to another bacterial cell through physical contact, with the donor cell forming a connection with the recipient cell.

[0098] According to some embodiments, the term “isolated” is intended to specifically reference an organism, cell, tissue, polynucleotide, or polypeptide that is removed from its original source and purified from additional components with which it was originally associated. For example, an endophyte may be considered isolated from a plant, e g., root cells, if it is removed from that plant source and purified so that it is isolated from any additional components with which it was originally associated. Similarly, an endophyte may be removed and purified from a plant or plant element so that it is isolated and no longer associated with its source plant or plant element. In some cases, the term “isolated” is used to describe a bacterium or an endophyte that has been removed from its host plant.

[0099] According to some embodiments, a “host plant” can includes any plant, particularly an agricultural plant, which an endophyte can colonize. As used herein, an endophyte is said to “colonize” a plant or seed when it can be stably detected within the plant or seed over a period time, such as one or more days, weeks, months or years, in other words, a colonizing endophyte is not transiently associated with the plant or seed. In some embodiments, such host plants are agricultural plants.

[0100] According to some embodiments, the terms “plant element” or “tissue of a plant” can be used interchangeably, and are intended to generically reference either a whole plant or a plant component, including but not limited to plant tissues, parts, and cell types. A plant element or a tissue of a plant may be one of the following: whole plant, seedling, meristematic tissue, ground tissue, vascular tissue, dermal tissue, seed, leaf, root, shoot, stem, flower, fruit, stolon, bulb, tuber, corm, keikis, shoot, bud.

[0101] According to some embodiments, the method further comprises transforming the selected microbial endophytes with one or more heterologous nucleic acid sequences encoding one or more heterologous peptides and / or polypeptides.

[0102] According to some embodiments, the term "heterologous" refers to two entities not naturally being associated with each other. For example, as used herein, “heterologous nucleic acid sequence” is a nucleic acid sequence not naturally associated with a host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring nucleic acid sequence. For another example, as used herein, “heterologous peptide or polypeptide” refers to an amino acid sequence of a peptide or polypeptide or protein that is not naturally occurring in the host cell.

[0103] According to some embodiments, the term "nucleic acid" refers to at least two nucleotide monomers linked together.

[0104] According to some embodiments, “nucleotide” refers to a phosphate ester of a nucleoside, as a monomer unit or within a nucleic acid, “nucleotide 5'-triphosphate” refers to a nucleotide with a triphosphate ester group at the 5' position, and are sometimes denoted as “NTP”, or “dNTP” and “ddNTP” to particularly point out the structural features of the ribose sugar. The triphosphate ester group can include sulfur substitutions for the various oxygens, e.g. alpha-thio-nucleotide 5'-triphosphates.

[0105] According to some embodiments, peptide is a short chain of amino acids linked by peptide bonds, with 2-50 amino acids long. According to some embodiments, polypeptide is along chain of amino acids linked by peptide bonds, w ith more than 50 amino acids long.

[0106] According to some embodiments, the microbial endophytes comprise one or more heterologous nucleic acid sequences is screened for expression of the one or more heterologous peptides and / or polypeptides.

[0107] According to some embodiments, a broad host range plasmid is a plasmid that can transfer to, replicate in and persist in a wide range of host microorganisms.

[0108] According to some embodiments, a selection marker is a gene introduced into cells, which confers one or more traits suitable for artificial selection. A selection marker can bepositive, such as antibiotic selection, conferring selective advantage to the host organism, or negative, such as thymidine kinase, eliminating or inhibiting growth of the host organism upon selection.

[0109] According to one aspect, the present disclosure also provides a method of producing a genetically engineered microbial endophyte, the method comprising: isolating a plurality of microbial endophytes from one or more tissues of a plant; conjugating the plurality of isolated microbial endophytes with one or more microorganisms comprising a library of broad host range plasmids having at least one selection marker and one or more heterologous nucleic acid sequences encoding one or more heterologous peptides and / or polypeptides; selecting one or more conjugated microbial endophytes that have one or more selection markers; and culturing the one or more selected microbial endophytes.

[0110] According to some embodiments, the selected microbial endophytes is screened for expression of the one or more heterologous peptides and / or polypeptides.

[0111] According to some embodiments, the plurality of microbial endophytes comprises one or more bacteria or one or more fungi.

[0112] According to some embodiments, the one or more bacteria is of the genus Bacillus, Herbaspirillum. Rhizobium, Bradyrhizobium. Pseudomonas, Ralstonia, Streptomyces, Acidovorax, Rhodococcus, Acetobacteraceae, Acidobacteriaceae, Acidothermaceae, Aerococcaceae, Alcaligenaceae, Alicyclobacillaceae, Alteromonadaceae, Anaerolineaceae, Aurantimonadaceae, Bacillaceae, Bader iovoracaceae, Bdellovibnonaceae,Bradyrhizobiaceae, Brevibacteriaceae, Brucellaceae, Burkholderiaceae, Carboxydocellaceae, Caulobacteraceae, Cellulomonadaceae, Chitinophagaceae, Chromatiaceae, Chthoniobacteraceae, Chthonomonadaceae, Clostridiaceae, Comamonadaceae, Corynebacteriaceae, Coxiellaceae, Cryomorphaceae, Cyclobacteriaceae, Cytophagaceae, Deinococcaceae, Dermabacteraceae, Dermacoccaceae, Enter oba eriaceae,Enterococcaceae, Erythrobacteraceae, Fibrobaderaceae, Flammeovirgaceae,Flavobacteriaceae, Frankiaceae, Fusobaderiaceae, Gaiellaceae, Gemmatimonadaceae, Geodermatophilaceae, Glycomycetaceae, Haliangiaceae, Halomonadaceae, Holosporaceae, Hyphomicrobiaceae, lamiaceae, Intrasporangiaceae. Kineosporiaceae, Koribaderaceae, Lachnospiraceae, Lactobacillaceae, Legionellaceae, Leptospiraceae, Leuconostocaceae, Methylobaderiaceae, Methylocystaceae, Methylophilaceae, Microbaderiaceae, Micrococcaceae, Micromonosporaceae, Moraxellaceae, Mycobaderiaceae, Mycoplasmataceae, Myxococcaceae, Nakamurellaceae, Neisseriaceae, Nitrosomonadaceae, Nocardiaceae, Nocar dioidaceae, Oceanospirillaceae, Opitutaceae, Oxalobaderaceae,Paenibacillaceae, Parachlamydiaceae, Pasteurellaceae, Patulibacteraceae,Peptostreptococcaceae, Phyllobacteriaceae, Piscirickettsiaceae, Planctomycetaceae,Planococcaceae, Polyangiaceae, Porphyromonadaceae, Prevotellaceae,Promicromonosporaceae, Pseudomonadaceae, Pseudonocardiaceae, Rhizobiaceae, Rhodobacteraceae, Rhodospirillaceae, Roseiflexaceae, Rubrobacteriaceae, Sandaracinaceae, Sanguibacteraceae, Saprospiraceae, Segniliparaceae, Shewanellaceae, Sinobacteraceae, Solibacteraceae, Solimonadaceae, Solirubrobacteraceae, Sphingobacteriaceae,Sphingomonadaceae, Spiroplasmataceae, Sporichthyaceae, Sporolactobaci llaceae,Staphylococcaceae, Streptococcaceae, Streptomycetaceae, Syntrophobacteraceae,Veillonellaceae, Verrucomicrobiaceae, Weeksellaceae, Xanthobacteraceae, and / orXanthomonadaceae .

[0113] According to some embodiments, the one or more fungi is of the genus Epichloe,Neotyphodium, Khuskia, Epicoccum, Curvularia, Serendipitci, Mycosphaerella,Piriformospora, Cladosporium, Fusarium, Colletotrichum, Phomopsis, Beauveria,Talaromvces. Apiospora, Aspergillaceae. Ceratobasidiaceae, Coniochaetaceae,Cordycipitaceae. Corticiaceae, Cystofllobasidiaceae, Davidie llaceae, Debaryomycetaceae, Dothioraceae, Erysiphaceae, Filobasidiaceae, Glomerellaceae, Hydnaceae, Hypocreaceae, Leptosphaeriaceae, Montagnulaceae, Mor tier ellaceae, Mycosphaerellaceae, Nectriaceae, Orbiliaceae, Phaeosphaeriaceae, Pleosporaceae, Pseudeurotiaceae, Rhizopodaceae, Sclerotiniaceae, Stereaceae, and / or Trichocomaceae .

[0114] According to some embodiments, the tissues of the plant is selected from the group consisting of roots, shoots, stems, leaves, rhizomes, trichomes, pneumatophores, inflorescences, and seeds.

[0115] According to some embodiments, the library of broad host range plasmids comprises one or more of origin of replication (oriV) sequences from the following plasmids or incompatibility groups pCUl, RK2, R300B, R1162, pSa, pR288, pBBRl, PBR322-based plasmids, pUC series plasmids, pSClOl-derived plasmids, pEpi series plasmids, pL2 series plasmids, pGreen vector series, pH7 vector series, pBI121 and pCAMBIA series. pKl 1 series, pK13 series. pGWB series plasmids. pUbi vector series, pNovl vectors. pC1301. pC1302. pMDC, pPZP, pVBN, pSHP, pS AG-based vectors, pPha-tl, pGWB, pHygro series, pSLl 180 series, pT7-based vectors, pMOL98, RK2, RP4,RP1,R68, PB10, RA3, IncN, IncP-1, IncU, IncPromA, Incl8, RK404, pDSK509, pDSK519, pRK415, RSF1010, IncW, cosmids (e.g., pJC8, pJC24, PBBR1MCS. pK18mobsacB, pLAFRl. pCCIFOS, pWKS30. pUTmini-TK, pEcoRi, PGEM-T, pGEM), fosmids (e.g., pFOSl, pCCIFOS, pFOSlRK2, pF0S6), yeastartificial chromosomes (YACs) (e.g., pYAC4, pYAC3, pYAC5, pYACl), and / or Bacterial artificial chromosomes (BACs) (e.g., pBBRIMCS-BAC, pSClOl-based BACs, pFOSBAC, pFosBAC, pK18mobSacB based BACs).

[0116] According to some embodiments, at least one selection marker is resistance to an antibiotic. According to some embodiments, the antibiotic is selected from the group consisting of kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, bleomycin, erythromycin, polymyxin B. tetracycline, and chloramphenicol.

[0117] According to some embodiments, the one or more heterologous nucleic acid sequences are at least about 0.01 kb, about 0. 1 kb, about 0.5 kb, about 1 kb, about 2 kb, about 3 kb, about 4 kb, about 5 kb, about 6 kb, about 7 kb, about 8 kb, about 9 kb, about 10 kb, about 11 kb, about 12 kb, about 13 kb, about 14 kb, about 15 kb, about 16 kb, about 17 kb, about 18 kb, about 19 kb, about 20 kb. According to some embodiments, the one or more heterologous nucleic acid sequences are at least about between about 0.01 kb to about 20 kb, about 0. 1 kb to about 10 kb, about 0.5 kb to about 15 kb, about 0.1 to about 1 kb, or about 5 kb to about 10 kb.

[0118] According to some embodiments, the one or more peptides and / or polypeptides comprise a small molecule, a phytohormone, an enzyme, a transcnption factor, or a nuclease.

[0119] According to some embodiments, a small molecule or micromolecule is a low molecular weight (equals or smaller than 1000 Daltons) organic or inorganic compound that may regulate a biological process, with a size on the order of 1 nm.

[0120] According to some embodiments, a phytohormone refers to a polypeptide hormone, which control all aspects of plant growth and development, including embryogenesis, the regulation of organ size, pathogen defense, stress tolerance and reproductive development. Each plant cell is capable of producing phytohormones. According to some embodiments, the terms "plant growth promoting hormones” and "phytohormones" can be used interchangeably, both refer to, chemical substances that promote plant growth, development and stress responses.

[0121] According to some embodiments, an enzyme refers to proteins that act as biological catalysts by accelerating chemical reactions.

[0122] According to some embodiments, a transcription factor refers to proteins that bind to DNA-regulatory sequences (enhancers and silencers), usually localized in the 5 ’-upstream region of target genes, to modulate the rate of gene transcription. This may result in increased or decreased gene transcription, protein synthesis, and subsequent altered cellular function.

[0123] According to some embodiments, nuclease is an enzyme which possesses catalytic activity for DNA cleavage.

[0124] According to some embodiments, the plant is of the genus Zea, Triticum, Oryza, Zizania, Hordeum. Sorghum, Eleusine, Panicum, Pennisetum, Setaria, Avena, Triticosecale, Secale, Psathyrostachys, Bambuseae, Saccharum, Glycine max, Brassica, Gossypium, Medicago, Manihot, Solanum, Solanum, Sporobolus, Odontarrhena, Streptanthus, Pisum, Cicer, Lens, or Linum.

[0125] According to some embodiments, the plant species is selected from the group consisting of: S. bicolor. P. virgatum, S. alterniflorus, B. juncea, O. chalcidica. and Streptanthus polygaloides. According to some embodiments, the plant species is Streptanthus polygaloides. According to some embodiments, the plant species is Panicum virgatum. According to some embodiments, the plant species is Triticum aestivum.

[0126] According to one aspect, the present disclosure also provides a method of preparing a plant comprising a plurality of genetically engineered microbial endophytes, a method comprising: applying to an exterior surface of the plant the plurality of genetically engineered microbial endophytes, wherein the plurality of genetically engineered microbial endophyte are derived from one or more microbial endophyte species isolated from a species of the plant and comprises one or more heterologous nucleic acid sequences encoding one or more heterologous peptides and / or polypeptides, and wherein the plant comprising the plurality of genetically engineered microbial endophytes has one or more traits of interest.

[0127] According to some embodiments, the method further comprises applying to the exterior of the plant one or more additional genetically engineered microbial endophytes, wherein the additional one or more genetically engineered microbial endophytes are from one or more species isolated from a species of the plant and comprises one or more heterologous nucleic acid sequences encoding one or more heterologous peptides and / or polypeptides.

[0128] According to some embodiments, the one or more additional genetically engineered microbial endophytes are of different genera compared to each other. According to some embodiments, the one or more additional genetically engineered microbial endophytes are of the same genus compared to each other.

[0129] According to some embodiments, the trait of interest of the plant is selected from the group consisting of germination rate, emergence rage, drought tolerance, freeze tolerance, shoot biomass, root biomass, seeding root length, yield, metal ion binding, nitrogen fixing, pollutant degrading, anti-fungal, herbicidal, nematicidal, and / or insecticidal.

[0130] According to some embodiments, the microbial endophyte is isolated from one or more tissue of the plant. According to some embodiments, the exterior surface of the plant comprises a seed coat, leaves, root, shoots, and / or flowers.

[0131] According to some embodiments, the plurality of genetically engineered microbial endophytes comprise 2, 3, 4, 5. 6, 7. 8, 9, or 10 different genetically engineered microbial endophytes each of which comprises different one or more heterologous nucleic acid sequences encoding one or more heterologous peptides and / or polypeptides.

[0132] According to one aspect, the present disclosure also provides a composition comprising a plant and a genetically engineered microbial endophyte, wherein the genetically engineered microbial endophyte is from a genus in symbiosis with the plant and comprises one or more heterologous nucleic acid sequences encoding one or more heterologous peptides and / or polypeptides, wherein the plant is contacted with the genetically engineered microbial endophyte in an amount effective to colonize the plant and to improve one or more traits of interest, and wherein the genetically engineered microbial endophyte is produced by a method comprising: isolating a plurality of microbial endophytes from one or more tissues of a plant; conjugating the plurality' of isolated microbial endophytes with one or more microorganisms comprising a library of broad host range plasmids having at least one selection marker and the one or more heterologous nucleic acid sequences encoding the one or more heterologous peptides and / or polypeptides; selecting one or more conjugated microbial endophytes that have one or more selection markers; and culturing the one or more selected microbial endophytes in conditions to induce expression of the more heterologous peptides and / or polypeptides.

[0133] According to one aspect, the present disclosure also provides a method of producing a genetically engineered microbial endophyte capable of binding a metal ion. the method comprising: culturing one or more selected microbial endophytes produced by the method disclosed herein and conjugating the selected microbial endophytes with one or more heterologous nucleic acid sequences encoding one or more cell-surface metal ion binding peptides; or culturing one or more selected microbial endophytes produced by the method disclosed herein, wherein the one or more heterologous nucleic acid sequences encodes one or more cell-surface metal ion binding peptides.

[0134] According to some embodiments, the method further comprising: conjugating the genetically engineered microbial endophyte with one or more heterologous nucleic acid sequences encoding one or more plant growth promoting hormones or phytohormones; and / or conjugating the genetically engineered microbial endophyte with an inducible kill switch.

[0135] According to some embodiments, the metal ion is selected from the group consisting of gold, silver, palladium, platinum, rhodium, copper, nickel, and lanthanide. According to some embodiments, the metal ion is nickel. According to some embodiments, the metal ion is lanthanide.

[0136] According to some embodiments, the plant grow th hormone is auxin, gibberellic acid, cytokinin, ethylene and / or abscisic acid.

[0137] According to some embodiments, the metal ion binding peptide is a nickel binding peptide (NBP). According to some embodiments, the nickel binding peptide is selected from the group consisting of any one of peptide sequences from SEQ ID NO: 1-17.

[0138] According to some embodiments, the metal ion binding peptide is a lanthanide binding peptide (LBP). According to some embodiments, the lanthanide binding peptide is LanMl.

[0139] According to one aspect, the present disclosure also provides a method of producing a plant capable of accumulating metal ions, the method comprising: applying to an exterior surface of the plant one or more genetically engineered microbial endophytes. According to some embodiments, the engineered microbial endophytes are produced by the method disclosed herein. According to some embodiments, the plant is grown in soil comprising metal ions.

[0140] According to one aspect, the present disclosure also provides a method of isolating metal ions from the plant produced by the method disclosed herein, the method comprising: homogenizing the plant tissues; and incubating the homogenized plant tissues in an acid solution.

[0141] According to one aspect, the present disclosure also provides a method of producing a genetically engineered microbial endophyte capable of producing one or more anti-microbial agents, the method comprising: culturing one or more selected microbial endophytes produced by the method disclosed herein and conjugating the selected microbial endophytes with one or more heterologous nucleic acid sequences. According to some embodiments, the method further comprises conjugating the genetically engineered microbial endophyte with one or more heterologous nucleic acid sequences encoding one or more plant growth promoting hormones or phytohormones; and / or conjugating the genetically engineered microbial endophyte with an inducible kill switch. According to some embodiments, the one or more heterologous nucleic acid sequences encodes one or more anti-microbial agents.

[0142] According to some embodiments, the anti-microbial agent is an anti-fungal agent or an anti-bacterial agent. According to some embodiments, the one or more antifungal agents inhibit Funsarium spp. and / or R. solani.

[0143] According to one aspect, the present disclosure also provides a method of producing a plant comprising a genetically engineered microbial endophyte capable of producing one or more anti-microbial agents, the method comprising: applying to an exterior surface of the plant one or more genetically engineered microbial endophytes produced by the method disclosedherein. According to some embodiments, the plant is grown in an environment susceptible to microbial infection.

[0144] According to one aspect, the present disclosure also provides a method of producing a genetically engineered microbial endophyte capable of producing pigment compound, the method comprising: culturing one or more selected microbial endophytes produced by the method disclosed herein and conjugating the selected microbial endophytes with one or more heterologous nucleic acid sequences encoding one or more amino acid catalyzing enzymes, wherein the amino acid catalyzing enzymes can catalyze one or more amino acids into one or more pigment compounds; or culturing one or more selected microbial endophytes produced by the method disclosed herein. According to some embodiments, the one or more heterologous nucleic acid sequences encodes one or more amino acid catalyzing enzymes. According to some embodiments, the amino acid catalyzing enzymes can catalyze one or more amino acids into one or more pigment compounds.

[0145] According to one aspect, the present disclosure also provides a method of producing a genetically engineered microbial endophyte capable of secreting plant signaling molecule, the method comprising: cultunng one or more selected microbial endophytes produced by the method disclosed herein and conjugating the selected microbial endophytes with one or more heterologous nucleic acid sequences encoding one or more plant signaling molecules. According to some embodiments, the one or more heterologous nucleic acid sequences encodes one or more plant signaling molecules.

[0146] According to one aspect, the present disclosure also provides a method of producing a genetically engineered microbial endophyte for bioremediation, the method comprising: culturing one or more selected microbial endophytes produced by the method disclosed herein and conjugating the selected microbial endophytes with one or more heterologous nucleic acid sequences one or more environmental pollution degrading enzymes. According to some embodiments, the one or more heterologous nucleic acid sequences encodes one or more environmental pollution degrading enzymes.

[0147] According to one aspect, the present disclosure also provides a method of producing a genetically engineered microbial endophyte capable of increasing nitrogen fixation, the method comprising: culturing one or more selected microbial endophytes produced by the method disclosed herein and conjugating the selected microbial endophytes with one or more heterologous nucleic acid sequences encoding one or more enzymes capable of fixing nitrogen. According to some embodiments, the one or more heterologous nucleic acid sequences encodes one or more enzy mes capable of fixing nitrogen.EXAMPLESEXAMPLE 1: Identifying genetically tractable plant endophytes

[0148] Root endophytes were extracted from Phragmites australis (Pa) roots. Samples were grown up in try ptic soy broth (TSB) and shuttled through the conjugation pipeline. Library' of Broad Host Range (BHR) plasmids in E. coli was conjugated with isolated root endophytes. Conjugated bacteria were plated on diaminopimelic acid (DAP) minus Burke’s media with Acetate. Colored colonies were sub-cultured in Burke’s acetate and Marine Broth. 16s PCR sequencing was performed on the sub-cultures and sent for sequencing. All sub cultured colonies were frozen in -80°C. (FIG. 1).

[0149] For proof of concept, conjugative microbes delivered GFP expressing plasmid into the root endophyte isolate. The isolated and GFP+ endophyte was incubated with P. australis plants, specifically, the P. australis root was cut to allow- for re-colonization by the GFP+ endophyte. Once the GFP+ endophyte has re-colonized the P. australis cells, the root was imaged for fluorescence under a microscope. (FIG. 2A). For real world application, the genetically engineered endophytes include an expression vector including an heterologous gene encoding a gene of interest. Once the genetically7engineered endophytes are cultured and expanded, the engineered endophytes are seeded into P. australis soil. Genetically engineered endophytes colonize the P. australis root. When the desired phenotype is needed, an inducer is applied and bacteria produce the product from within the plant. (FIG. 2B).

[0150] Genetically engineered endophytes are shown to re-colonize a plant. Pseudomonas (JS1B) were isolated from P. australis plant clippings, and Pseudomonas tremae w as isolated from the soil. Each strain was conjugated w ith a GFP expressing plasmid, cultured, and allowed to re-colonize P. australis. JS1B was able to recolonize P. australis as shown in FIGs. 3A and 4A, however P. tremae (GFP+), as shown in FIG. 3B, was not able to recolonize P. australis. JS5E (GFP+), isolated from switch grass, was shown to recolonize switchgrass, however JS1B (GFP+) was unable to recolonize switchgrass. (FIG. 4B). Lastly, Comomonas oxytoca was also isolated and conjugated with the GFP expressing vector, however, C. oxytoca (GFP+) was unable to re-colonize P. australis. (FIG. 3C)EXAMPLE 2: Pipeline to genetically engineering plant endophytes to express a gene of interest

[0151] Root endophytes are extracted from valuable crops in various environments. The endophytes are conjugated w ith a library7of broad host range plasmids with a selection marker.Individual endophytes can be selected based on the selection marker and engineered further with a vector including a gene(s) which encode a beneficial nucleic acid sequence, e.g.. RNAi, or a peptide and / or polypeptide, e.g., growth hormone or anti-microbial agent.

[0152] The genetically engineered endophytes can also be engineered by isolating endophytes from a specific plant, and conjugating the endophytes with a library of broad host range plasmids, which include the gene(s) of interest. This method can increase throughput to more rapidly identify the genus and species of endophytes that naturally are found in the plant and simultaneously engineer the endophytes to express the gene of interest, e.g., plant growth inducing proteins or plant growth inhibiting proteins. The gene of interest can improve the traits of the plant, e.g., drought resistance or increase the plants defense against microbial infection. The gene of interest can also be a peptide or polypeptide that can extract molecules or ions from the soil, e.g., metal ions or pollutants. The gene of interest can also be a peptide or polypeptide that allow-s the plant to produce a byproduct which the plant would not normally produce in nature or would only produce said byproduct at very low' levels. (FIG. 5).EXAMPLE 3: Identify genetically tractable, lanthanide-tolerant endophytes

[0153] The BIOMSE pipeline w as developed to enable endophyte identification, modification, and reintroduction into the host. BIOMSE utilizes a broad host range conjugation library that enables the identification of genetically tractable root endophytes. The plasmids from the library are housed in E. coll and carry antibiotic and fluorescent markers as well as the genes needed for conjugation. Following conjugation, endophytes that receive the plasmids are selected and tested for the ability' to recolonize roots. Genetically tractable endophytes targeted to Phragmites australis and Panicum virgatum are shown in FIGs. 6A-6C.

[0154] P. virgatum will be collected from soils containing elevated levels of rare earth elements (REEs) and the BIOMSE pipeline will be used to identify genetically tractable endophytes. To ensure that genetically tractable microbes are lanthanide-tolerant, the isolated microbes will be assessed in growth medium containing high concentrations of REEs. Microbes with high REE tolerance will be screened for the ability to recolonize host plants.

[0155] Many synthetic and naturally-occurring REE-binding peptides and proteins have been identified, either through phage display, rational design, proteomics, or homology modeling

[0018] , Plant endophytes were engineered to alter plant metabolism (FIG. 6D) and the same approach can be used to turn any plant into an REE hyperaccumulator. LanMl, an eighteen amino acid motif found in lanmodulin. a highly-selective LBP identified in the model methylotroph, Methylobacterium extorquens [16,19-20], The root endophytes will beengineered to express cell-surface displayed and secreted LBPs

[0021] , The secretion of LBPs will serve to chelate REEs and promote the translocation to the leaves, while cell surface display on root endophytes will improve the REE tolerance and enable them to continue producing LBPs for dissemination in the plant [22-24],

[0156] To provide baseline performance of the LBPs, will first display tagged LBPs on the surface of E. coll, leveraging the many genetic tools available for surface display, before moving them into the identified endophytes. The display of heterologous peptides in microbial hosts will be accomplished by genetically linking them to a native carrier protein, consisting of a signal peptide, SpyTag and a cell surface anchor

[0025] , The features will mediate transport across the cell envelope and attachment to the bacterial cell surface [22-24] . The proper surface display will be confirmed through microscopy by staining the genetically modified strains with SpyCatcher-GFP, leveraging the highly specific covalent binding between the SpyTag and the SpyCatcher

[0025] , Following this, the ability of modified bacteria to bind free metal ions in solution will be tested. Upon confirmation of metal binding, constructs will be generated for secreted LBPs lacking the cell surface anchor to enable dissemination to the plant.

[0157] A robust biocontainment strategy will be developed, as containment is essential for developing deployable microbe-based phytomining strategies. The benefit of using endophytes is that endophytes are housed in, or closely associated with, roots or other plant tissues. Therefore, plant-based cues can be used to ensure that microbes do not escape the plant hosts. An inducible kill switch using CRISPR Cas3 will be engineered to ensure microbial killing and DNA destruction [26-27], Specifically, bacterial promoters and transcription factors responsive to plant molecular cues will be identified, like auxin [28-29], The molecular circuitry that represses kill switch activation when endophytes are associated with the plant host will be used. If microbes escape into the environment where the plant cue is no longer present, the kill switch will be activated. Kill switch molecular circuitry will be incorporated into the broad host range plasmids. Since the plasmids can be shared with other microbes through conjugation, incorporating the kill switch in the plasmid is the only w ay to ensure plasmid containment.

[0158] To further ensure containment, methionine synthase will be knocked out in the identified endophytes to generate auxotrophies. Constructs containing the kill switches described above will also contain a cassette that elicits expression of methionine synthase in the presence of auxin. If modified microbes are not associated with plants, endophytes will not be able to make methionine and will die. This will provide an additional line of defense and will further ensure that modified microbes do not escape into the environment.

[0159] P. virgatum will be inoculated with engineered endophytes, for example by watering. Upon inoculation, the presence of engineered endophytes will be confirmed and plants containing engineered endophytes will be assessed for the ability to grow in REE enriched soils, consistent with soil levels found in the Appalachian coal basin. Specifically, survivability, root elongation, and lanthanide concentrations in the roots and leaves using ICP- MS will be assessed.References

[0160] 1. Index of / pubs / periodicals / mcs2024. www. pubs. usgs.gov / periodicals / mcs2024 / mcs2024-rare-earths. pdf.

[0161] 2. Dinh, T., Dobo, Z. & Kovacs, H. Phytomining of rare earth elements - A review; Chemosphere 297. 134259 (2022).

[0162] 3. Lopez-Santos, P. West Virginia to Treat Acid Mine Drainage and Recover Rare Earth Oxides. The Environmental Council of the States (ECOS) (2019).

[0163] 4. Rabbani, M., Taqi Rabbani, M., Muthoni, F., Sun. Y. & Vahidi, E. Advancing phytomining: Harnessing plant potential for sustainable rare earth element extraction. Bioresour. Technol. 401. 130751 (2024).

[0164] 5. Vuppaladadiyam, S. S. V. et al. Can e-waste recycling provide a solution to the scarcity of rare earth metals? An overview of e-waste recycling methods. Sci. Total Environ. 924, 171453 (2024).

[0165] 6. Joradon. P. et al. Phytoremediation technology for recovery of Ni by Acacia plants in association with Bacillus amyloliquefaciens isolated from E-waste contaminated site. Int. J. Phytoremediation 1-10 (2023)

[0166] 7. Dodbiba, G. & Fujita, T. Trends in Extraction of Rare Earth Elements from Coal Ashes: A Review. Recycling 8, 17 (2023).

[0167] 8. Lai, Y., Wang, Q., Yang, L. & Huang, B. Subcellular distribution of rare earth elements and characterization of their binding species in a newly discovered hyperaccumulator Pronephrium simplex. Taianta 70, 26-31 (2006).

[0168] 9. Kikis, C., Thalassinos, G. & Antoniadis, V. Soil Phytomining: Recent Developments — A Review; Soil Syst. 8. 8 (2024).

[0169] 10. Skuza, L., Szucko-Kociuba, I., Filip, E. & Bozek, I. Natural Molecular Mechanisms of Plant Hyperaccumulation and Hypertolerance towards Heavy Metals. Int. J. Mol. Sci. 23, 9335 (2022).

[0170] 11. WVU Today | WVU receives $3M from DoD to boost rare earth extraction capacity. (2023).

[0171] 12. Dance, A. Microbial miners take on rare-earth metals. Nature 623, 876-878 (2023).

[0172] 13. Roszczenko-Jasihska, P. et al. Gene products and processes contributing to lanthanide homeostasis and methanol metabolism in Methylorubrum extorquens AMI. Sci. Rep. 10, 12663 (2020).

[0173] 14. Native Plant Profile: Switchgrass (Panicum virgatum).

[0174] 15. Shrestha. P., Belliturk, K. & Gorres, J. H. Phytoremediation of Heavy Metal- Contaminated Soil by Switchgrass: A Comparative Study Utilizing Different Composts and Coir Fiber on Pollution Remediation, Plant Productivity, and Nutrient Leaching. Int. J. Environ. Res. Public. Health 16, 1261 (2019).

[0175] 16. Cotruvo, J. A. Jr., Featherston, E. R., Mattocks, J. A., Ho, J. V. & Laremore, T. N. Lanmodulin: A Highly Selective Lanthanide-Binding Protein from a Lanthanide-Utilizing Bacterium. J. Am. Chem. Soc. 140, 15056-15061 (2018).

[0176] 17. Zytnick, A. M. et al. Identification and characterization of a small-molecule metallophore involved in lanthanide metabolism. Proc. Natl. Acad. Sci. 121, e2322096121 (2024).

[0177] 18. Ye. Q., Wang, D. & Wei. N. Engineering biomaterials for the recover}' of rare earth elements. Trends Biotechnol. 42, 575-590 (2024).

[0178] 19. Verma, G. et al. Investigation of Rare Earth Element Binding to a Surface-Bound Affinity Peptide Derived from EF-Hand Loop I of Lanmodulin. ACS Appl. Mater. Interfaces 16. 16912-16926 (2024).

[0179] 20. Gutenthaler, S. M. et al. Lanmodulin peptides - unravelling the binding of the EF- Hand loop sequences stripped from the structural corset. Inorg. Chem. Front. 9, 4009-4021.

[0180] 21. Wang, Y ., Selvamani, V., Yoo, I.-K., Kim, T. W. & Hong, S. H. A Novel Strategy for the Microbial Removal of Heavy Metals: Cell-surface Display of Peptides. Biotechnol. Bioprocess Eng. 26, 1-9 (2021).

[0181] 22. van Bloois, E., Winter, R. T., Kolmar, H. & Fraaije, M. W. Decorating microbes: surface display of proteins on Escherichia coli. Trends Biotechnol. 29, 79-86 (2011).

[0182] 23. Tozakidis, I. E. P., Luken, L. M., tiffing, A., Meyers, A. & Jose, J. Improving the autotransporter-based surface display of enzymes in Pseudomonas putida KT2440. Microb. Biotechnol. 13, 176-184 (2020).

[0183] 24. Dvorak, P., Bayer, E. A. & de Lorenzo, V. Surface Display of Designer Protein Scaffolds on Genome-Reduced Strains of Pseudomonas putida. ACS Synth. Biol. 9, 2749-2764 (2020).

[0184] 25. Molinari, S. et al. A de novo matrix for macroscopic living materials from bacteria. Nat. Commun. 13, 5544 (2022).

[0185] 26. Rottinghaus, A. G., Ferreiro, A., Fishbein, S. R. S., Dantas, G. & Moon, T. S. Genetically stable CRISPR-based kill switches for engineered microbes. Nat. Commun. 13, 672 (2022).

[0186] 27. Caliando. B. J. & Voigt, C. A. Targeted DNA degradation using a CRISPR device stably carried in the host genome. Nat. Commun. 6, 6989 (2015).

[0187] 28. Conway, J. M. et al. Diverse MarR bacterial regulators of auxin catabolism in the plant microbiome. Nat. Microbiol. 7, 1817-1833 (2022).

[0188] 29. Lavy, M. & Estelle, M. Mechanisms of auxin signaling. Dev. Camb. Engl. 143, 3226-3229 (2016).EXAMPLE 4: Modular biosensing in plants

[0189] Engineering a plant to respond to a specific signal takes a lot of time and effort, whereas engineering microbes is relatively fast and straightforward. A single sensor plant that responds to a specific microbially secreted signal will be engineered. A library of stimuli responsive endophytes that secrete the plant signaling molecule when activated will also be designed and engineered. (FIG. 7).

[0190] Soil isolates were conjugated with GFP expressing vectors. Example of colonies in bright field verses fluorescent channel. (FIG. 8A). A. calconceticus , C. donisae. A. sandrelli, K. cryocrescens, and K. oxytocu were compared to E. coli for survival in soil over 2 weeks. A. sandrelli showed a significant increase in growth in soil while the other isolates did not grow as readily. (FIG. 8B).

[0191] eBead sensors are an engineered encapsulated biosensor, which was developed to hold E. coli capable of detecting 2-phenylphenol (2-PP), a known fungicide and, in high amounts, is an environmental hazard. See Luisi et al., ACS Sens. 2022, 7, 9, 2589-2596. Use of the eBead system is shown in FIGs. 9A and 9B. 2-PP is activated the biosensor in the eBead and a colonies produce a darker color compared to no 2-PP control. A. sandrelli will be engineered into an eBead system.EXAMPLE 5: Ground pollution remediating endophytes

[0192] Engineer genetically tractable plant endophytes will be generated to degrade pollutants in the soil and water table, according to the methods as described herein.

[0193] Despite being incredibly promising, most plant-microbe bioremediation systems fail. Engineering non-model plants is incredibly time consuming (years). Engineered microbes cannot establish an ecological niche in the outside environment. The technology allows us to engineer the endophytes from native plants bypassing plant genetics and working within a controllable niche (plant root).

[0194] Root endophytes will be extracted from valuable crops in various environments. The endophytes will be conjugated with the broad host range plasmid library, as described herein, and isolated based on genetically tractability. Native microbes will be genetically engineered with genes which encode enzymes involved in the breakdown of pollutants in the soil or water. If the pathway to breakdown the pollutants requires more than one enzyme, multiple endophytes can be engineered to express one or more enzymes needed for the pathway. (FIG. 10).Example 6: Genetically engineered endophytes for underground nickel extraction

[0195] As the United States transitions to a green energy economy, valuable metals like nickel (Ni) will have an outsized role due to the critical need in batteries. Although soils contain varying amounts of Ni, the Ni is too dilute to mine traditionally. Phytomining, using plants to extract valuable metals, offers a potential solution to extract Ni from soil. Genetically engineered plant-microbe interactions can result in the extraction of 250kg Ni / hectare / year. Endophytes of native plants will be used to enhance Ni accumulation. First, root-associated microbes will be identified and engineered to 1 ) display and secrete Ni binding peptides (NBPs) from root-associated microbes to accelerate Ni accumulation and tolerance in a variety' of plants and 2) increase plant biomass through the production of plant growth promoting hormones (FIG. 11). The genetically engineered endophyte derived from a native endophyte can turn any plant into a Ni hyperaccumulator.

[0196] To leverage plant-microbe symbiosis, synthetic biology will be used to rapidly develop Ni hyperaccumulation in plants to provide a low-cost solution enabling high-efficiency Ni extraction from soil. Microbial peptide surface display will be used to develop engineered root endophytes that bind Ni on the cell surface and secret Ni-binding peptides. Genetically tractable root endophytes will be identified from multiple plants species, including the model Ni hyperaccumulating (HA) plant, Streptanthus polygaloides . Endophytes will be engineered to increase the biomass and the Ni storage capacity of the plants. Additionally, eco-friendly approaches for Ni extraction will be developed.Nickel and Nickel Binding Peptides (NBPs)

[0197] Nickel is an essential micronutrient in plants due to its role as a cofactor of several enzymes, including the nitrogen recycling urease enzyme5. Deficiencies in Ni result in various plant defects such as reduced growth, chlorosis, metabolic dysregulation, and induction of senescence. However, excessive amounts of Ni result in growth reduction, decreases in biomass, and developmental defects6. A critical component of Ni homeostasis in plants is the production of phytochelatins. short metal-binding compounds that sequester toxic concentrations of heavy metals and deliver them to the vacuole7. In the Ni HA plant Alyssum murale, phytochelators and NBPs serve to bind Ni and prevent toxicity8.

[0198] The use of NBTs in practical applications has garnered interested in recent years. Engineered NBPS have been identified with high affinity and specificity, making them the ideal candidates for industrial applications, including bioremediation and phytomining9. Previously, engineered NBPs displayed on the surface of yeast and bacteria have been used for bioremediation bacteria10,11. The engineered microbes display a significant increase in Ni bioadsorption, specifically on the microbial cell surface12. Plant endophytes will be engineered to display and secrete NBPs to enable microbial and plant Ni tolerance and accumulation.Endophytes for modulating plant Ni uptake and storage

[0199] The plant microbiome can be divided into two parts, external and internal. Both consist of a plant associated microorganisms, consisting of bacteria, archaea, and fungi, and both play a role in disease suppression, enhancing nutrient uptake, and stress resistance. Given the functions, microbiome engineering offers an approach to modulating many aspects of plant growth, development, and fitness. Efforts to date, have primarily focused on soil microbiome engineering; however, most of the attempts have fail when deployed beyond the lab, as soil composition is highly dynamic and influenced by many extrinsic factors13. Endophytes live in plants, either in cells or interstitial spaces. Given that endophytes are resident in plant tissues, endophytes represent a more stable target for microbiome engineering; however, few endophytes have been modified to date14.

[0200] Recently, BIOMSE protocols have been developed for endophyte modification and reintroduction to the host (FIG. 1). BIOMSE utilizes a broad host range conjugation library, which is used to identify genetically tractable root endophytes. The plasmids from this library are housed in E. coli and carry antibiotic and fluorescent markers and the genes needed for conjugation. Following selection for endophytes that received the plasmid, roots can be easily recolonized with the modified microbes. This approach has several advantages over existing approaches. Endophytes are resident in plant tissue, a more stable and less dynamic environment than microbes that reside in the soil microbiome. Native endophytes are welladapted to the plant host and are less susceptible to local environmental changes. Additionally, the technologies disclosed herein are plant agnostic and will provide a path to turn any plant into an Ni HA.

[0201] Endophytes will be engineered to display NBPs on the cell surfaces and secret NBPs to the plant. This will increase the Ni tolerance in the microbe and Ni accumulation and tolerance in the plant. In addition to using endophytes to improve Ni uptake and tolerance, endophytes will be engineered to produce plant growth hormones, such as auxin or gibberellic acid, increasing the biomass available for Ni accumulation and storages.Nickel extraction and quantification

[0202] Current methods for extracting metals from plants involves either acid leaching or incineration, which result in the conversion of plant matter into ash concentrated with the metal of interest, such as nickel (Ni). The methods rely on harsh chemicals and add to greenhouse gas emissions worldwide. Environmentally friendly Ni extraction methods will be established to minimize the use of caustic chemicals, and the impact on global Ni supply and greenhouse emissions can be assessed.

[0203] The genetically engineered endophytes capable of extracting Nickel from the soil can result in extracting greater than 30 mg Ni / gdb. Focusing too much on a single Ni HA plant risks restricting operating capabilities to a particular region and soil type. Engineering specific plants with genes involved in Ni accumulation is promising but incredibly time-consuming. This work demonstrates an approach that could be highly tailored and broadly deployable, ensuring a flexible, robust, and rapid approach to phytomining. Specifically engineering native endophytes can be done rapidly according to the method described herein, compared to traditional methods in engineering root microbiomes which tend to fail outside a laboratory setting13. To this end, genetically tractable native plant endophytes can be utilized to broadly turn any plant into a Ni hyperaccumulator and further enhance the Ni accumulating capability of known HA plants.Methods

[0204] Secreted high-affinity Ni-binding peptides (NBPs) will be engineered and will be introduced into root endophytes identified from diverse plant species, including Sorghum bicolor (sorghum), Pcmicum virgatum (switchgrass), Sporobolus alterniflorus (spartina), and the Ni hyperaccumulators, Brassica juncea (mustard), Odontarrhena chalcidica, and Streptanthus polygaloides (milkwort). The identified endophytes will be engineered to secrete high-affinity NBPs, which will function as a Ni buffer in the xylem, increasing Ni tolerance. Additionally, endophytes will be engineered to express growth factors to promote plant growth,increasing the amount of plant biomass available for Ni accumulation and storage. This approach will allow us to rapidly modify endophytes native to any plant, turning that plant into aNi HA.Nickel binding peptide engineering (NBP)

[0205] Nickel hyperaccumulators have many adaptations that enable them to tolerate and accumulate high levels of Ni. One of the adaptations is the ability to bind accumulated Ni in complexes that reduce its toxicity. For example, in the vasculature of Alyssum murale. a well- studied Ni HA, histidine, malate, and other low molecular weight organic acids function to chelate Ni and limit its toxicity8. Synthetic and naturally-occurring high-affinity Ni-binding peptides have been identified. The peptides, which reversibly bind Ni and function as Ni chelators, represent ideal candidates for broadly bolstering plant Ni tolerance.

[0206] Saccharomyces cerevisiae was previously engineered for cell-surface display of NBPs identified from a phage display library. Display of the peptides, which were highly selective for Ni, increased the Ni-binding capacity of the yeast by nearly 80% while having little impact on the absorption of other metals. Root endophytes will be engineered to express cell-surface displayed and secreted NBPs. The secretion of the NBPs will serve to chelate Ni in the plant, while cell surface display on root endophytes improves Ni tolerance, enabling continued endophyte survival and NBP production for dissemination to the plant.

[0207] While numerous NBPs have been identified, comparing binding affinities is challenging, given the use of varying assays to determine effectiveness of the NBPs. Additionally, not all of the peptides have been tested in the context of a larger fusion protein. The Molinari Lab has extensive experience in protein engineering, particularly for cell surface display. Secreted and displayed versions of the peptides in Table 1 can be expressed in Escherichia coli and the Ni binding specificities and affinities can be determined. Candidates with the highest affinity and specificity will then be optimized for expression in root endophytes. The endophyte-produced peptides will be assessed for the ability to bind Ni before introduction into plants.Table 1: Nickel Binding PeptidesEndophyte engineering to enhance Ni tolerance and accumulation

[0208] The root microbiome comprises bacteria, fungi, and archaea that promote nutrient uptake, stress tolerance, disease tolerance, and plant growth. Given the benefits, numerous researchers have sought to genetically modify plant microbiomes to influence plant growth via various routes; however, most of the attempts have failed outside of a laboratory setting. Competition with resident microbes, soil heterogeneity, temperature fluctuations, and runoff all unpredictably influence microbial community composition. Because of this, engineered communities often fail13. Engineered endophytes offer a potential solution to this problem; however, until recently, there have been few tools to enable genetic modification of the organisms.

[0209] If only the host’s function is important rather than its specific identify, a host-agnostic strategy can be adopted. This approach involves screening a large pool of microbes simultaneously for genetic tractabilify using one or more broad host range (BHR) plasmids. By doing so, the chances of identifying a host organism that is both genetically tractable and capable of performing the desired function are greatly increased compared to testing individual strains one at a time. Furthermore, advances in genetic toolkits have enabled the delivery of plasmids to non-model microbes by incorporating diverse origins of replication, antibiotic selection markers. The toolkits are often housed in specialized conjugative donor strains to facilitate plasmid transfer. Previously, root tissue from Phragmites australis was extracted anda library of BHR plasmids was used to identify genetically accessible endophytes (FIGs. 3C and 4A). Upon identification, endophytes carrying a fluorescent reporter were reinoculated into the host and shown to colonize roots upon reinoculation. BIOMSE (Building Inter- Organismal Mutualisms for Synthetic Engineering) refers to methods of endophyte modification as described herein.

[0210] The method disclosure herein can be used to turn any plant into a Ni HA and promote plant biomass production to provide a sink for Ni accumulation. The approach as disclosure herein will be utilized to identify' root endophytes from diverse plant species, including Ni HA and non-HA species. Endophytes from the plants will be modified to express cell surface- linked and secreted Ni-binding peptides to increase endophyte and plant Ni tolerance and accumulation. Previous work has demonstrated that treating Ni HA plants with the auxin increases in biomass 2-fold and Ni extraction 30-40%.31To promote biomass production and Ni accumulation, endophytes will be engineered to produce key phytohormones, including auxin and gibberellic acid. The grow th factors promote root elongation, which would expand underground plant surface area, provide more biomass for endophytes to occupy, and, ultimately. Ni access. Together, the modifications are projected to accumulate more than 250 kg of Ni per hectare per year in harvestable plant biomass.Ni extraction and supply chain impact

[0211] While methods exist for extracting metals from plant material, the methods could be more environmentally friendly. The predominant methods include plant material incineration for producing Ni-concentrated plant ash or using acid-based solutions to leach the Ni from plant material

[0056] , While the generation of plant ash provides a concentrated Ni source, incineration of this material contributes to global greenhouse gas emissions. Acid-leaching is viewed as more environmentally friendly; however, developing more environmentally friendly approaches may be necessary [32,33],

[0212] Non-standard methods for Ni extraction from plant and plant-associated microbial material will be used. Because secreted and surface-displayed NBPs contain SpyTags, the feasibility’ of using anti-Spy tag antibodies to separate NBPs from plant material will be accessed. While this approach would still require acid extraction to obtain Ni, the concentration of Ni-bound NBPs would dramatically reduce the amount of caustic chemicals required for Ni extraction. Spy Tag containing high-affinity NBPs have the potential to provide a strategy for an eco-friendlier approach to Ni extraction than existing approaches.

[0213] As described herein, broad host range plasmids can be used to capture genetically tractable microbes from soil and plant tissue. Engineering tractable endophytes to secrete high-affinity NBPs to improve plant Ni accumulation may result in toxicity to the plant and / or endophyte. Engineered endophytes will be assessed to identify those with the highest Ni tolerance and engineer them to improve Ni tolerance further. The engineering strategy to endophytes will be used for multiple plant species, including known Ni HAs. Lastly, NBPs will be selected with the highest affinity and specificity7for Ni; however, the peptides will bind other metals to some extent.Identification and optimization of NBPs for endophyte secretion and display (UMD)

[0214] Displaying foreign metal-binding proteins or peptides on the cell surface using microbial display systems enhances the ability7of microorganisms to bind to heavy metals, increasing the endophytes resistance to the metals and the capacity of the endophyte to absorb the metals. This strategy has been employed for the remediation of waters and soils contaminated with heavy metals

[0034] , Secretion of the same peptides could be used to increase Ni resistance and enhance Ni accumulation in plants. In this task, Ni-binding peptides will be identified with high Ni affinity and specificity.

[0215] The initial phase of this proj ect involves selecting the most suitable NBP for modulating Ni accumulation in plants and endophyte survival. Although numerous peptides have been identified for the capacity to bind Ni metal, comparing binding efficiency is challenging due to the use of various assays to describe their effectiveness. As an effective comparison, previously identified NBPs (Table 1) will be displayed on the surface of Escherichia coli. leveraging the many genetic tools available for surface display.

[0216] The display of heterologous peptides in microbial hosts involves genetically linking them to a native carrier protein, comprising a signal peptide and a surface anchor

[0035] , This will mediate the export across the cell envelope and its attachment to the bacterial cell surface. Common carriers used for this purpose are proteins whose structure is made of transmembrane B-strands connected at the periplasmic side w ith short turns and at the outside with long surface- accessible loops. Heterologous peptides are inserted into extracellular loops for surface display. This strategy is preferable, especially in non-model organisms, because it only requires the insertion of a small peptide in an unstructured region, likely not disrupting the tertiary structure or the functionality of the protein. However, many of the identified NBPs are isolated from the N-terminus of larger proteins, suggesting that NBPs are more effective in terminal positions. The library in Table 1 will be screened as terminal and intramolecular insertion of a carrier protein and characterize nickel binding activity in both contexts (FIG. 12).

[0217] The proper surface display will be confirmed through microscopy by staining the genetically modified strains with SpyCatcher-GFP, leveraging the highly specific covalentbinding between the SpyTag and the SpyCatcher

[0036] , The ability of each genetically engineered E. coli strain to bind nickel will be tested by using Ni-NTA HisSorb Strips, as described by Dong et al.

[0037] , Briefly, induced strains will be incubated with Ni-NTA HisSorb Strips and allowed to bind. Ni-NTA HisSorb Strips will be then washed, and attached cells eluted by vortexing. Eluted cells will be diluted and plated on agar plates for colony-forming unit (CFU) count. A higher number of CFU correlates with a higher ability to bind nickel.

[0218] To assess the capability of the bacterial clones to bind free metal ions in solution, a competitive assay will be employed to evaluate the nickel-binding capacity of the chosen NBPs. Induced bacteria will be allowed to bind to nickel-immobilized well, and then eluted to estimate the number of bacterial colonies. This assay will be also adopted to determine the specificity of nickel-binding compared to other heavy metals possibly present in soils such as, copper, cadmium, lead, and zinc. This assay will provide a relative characterization of NBPs to facilitate nickel absorption when displayed on the surface of bacteria. For absolute quantification, immunoblotting of membrane-bound proteins using anti-SpyTag antibodies will be performed. Three NBPs with the highest binding affinity and specificity for secretion to plants and display the surface of endophytes will be selected.Optimize NBPs for display on endophytes

[0219] The top three NBPs will be optimized for secretion from and display on genetically tractable root bacterial endophytes identified by the method described herein. For Gramnegative bacteria, the best-performing E. coli NBP fusion proteins will be cloned into the broad host range plasmids and protein function will be accessed as described herein. The E. coli signal peptide and surface anchor sequences may not be adequate for NBP secretion and cell surface anchoring in Gram-negative endophytes. If so, signal peptides and surface anchor proteins from the endophyte host will also be identified and constructs will be modified to include those motifs. Gram-negative hosts for peptide display and secretion will be used primarily, as those constructs will likely require fewer modifications than those designed for Gram-positive bacteria.

[0220] As with E. coli, peptides can be displayed on the outer membrane of Gram-negative species using a short transmembrane B-barrel structure, including the common soil bacterium Pseudomonas putida [38,39], Since Gram-positive organisms lack an outer membrane, peptides are displayed by using C-terminal fusions to a peptidoglycan binding motifs

[0040] , Previously identified signal sequences and peptidoglycan binding domains will be used to engineer NBP surface displayed proteins in Gram positive organisms. If secretion and displayare inadequate, signal peptides and carbohydrate binding motifs for the native endophyte host will be identified and incorporated those motifs into the NBP constructs.Endophyte identification and modulation of Ni accumulation

[0221] BIOMSE technology will be used to extract and identify genetically tractable root- associated microorganisms from five diverse plant species, including S', bicolor, P. virgatum, S. alterniflorus, and the Ni hyperaccumulators, B. juncea, O. chalcidica, and S. polygaloides . Plants will be collected from the field at various stages of growth. For known Ni HA plants, samples will be collected from soils known to contain high concentrations of Ni and Ni- depleted soils. Soil samples will be collected to confirm Ni concentration. A representative sample of the colonizing microbes will be collected as described herein.

[0222] Upon field collection, roots from the plants will be homogenized and the endophytes will be extracted. The extracted endophytes will be co-cultured with E. coli containing the BHR plasmid library for conjugation. Following conjugation, microbes will be plated on selective Burke’s medium, w hich is a nitrogen-depleted medium. Organisms capable of growing on this medium can fix nitrogen, and nitrogen-fixing microbes are commonly found in mutualistic relationships with plants, decreasing the likelihood of obtaining plant pathogens. Microbes that grow on selective medium will be screened for the presence of the fluorescent marker included in the plasmid, and 16S sequencing can be used to identify those microbes. The identified microbes will be screened for 1) ability to colonize the roots of the host plant using fluorescence microscopy; and 2) Ni tolerance.Engineer endophytes to produce phytohormones

[0223] The overall goal is to maximize Ni extract and per-plant Ni yield. One way to accomplish this is to increase plant biomass. It is well documented that microbes produce key phytohormones like auxin and gibberellic acid (GA), increasing biomass and root elongation in the plant hosts [41-43], Increasing root elongation would expand the underground surface area occupied by plants, providing additional sites for endophytes to occupy and increased access to Ni stores. Identified organisms will be tested to determine level of auxin or gibberellic acid production. Auxin and gibberellic biosynthetic pathways will be incorporated into the BHR plasmid or BHR plasmid library for delivery to identified endophytes which do not produce auxin or gibberellic. Endophytes containing the plasmids will be reintroduced to the host plant, and plant growth and Ni accumulation will be assessed.Biocontainment engineering

[0224] Biocontainment is essential for developing deployable microbe-based phytomining improvement strategies. The benefit of using endophytes is that endophytes are housed in orclosely associated with roots. Therefore, plant-based cues can be used to ensure that microbes do not escape the plant hosts. An inducible kill switch can be engineered using CRISPR Cas3 to ensure microbial killing and DNA destruction [44,45], Specifically, bacterial promoters and transcription factors responsive to plant molecular cues will be identified. The bacterial promoters and transcription factors can be used as the basis for molecular circuitry that can repress activation of the kill switch when endophytes are associated with the plant host. If microbes escape into the environment where the plant cue is no longer present, the kill switch will be activated. Kill switch molecular circuitry will be incorporated into the broad host range plasmids. Since the plasmids can be shared with other microbes through conjugation, incorporating the kill switch in the plasmid is the only way to ensure plasmid containment.Ni extraction from plant tissue and supply chain impacts

[0225] Existing methods for metal extraction from plants include acid leaching or incineration, which turns plant material into Ni-concentrated ash. The processes involve using caustic chemicals and contribute to global greenhouse gas emissions. Existing Ni extraction protocols will be evaluated and eco-friendly protocols will be developed to obtain SpyTag-NBP bound Ni.

[0226] Plants provide an effective strategy for concentrating Ni found in Ni-rich soils; however, existing strategies for extracting Ni from plants grown in those soils present challenges. Existing methods are not eco-friendly and require significant energy input and caustic chemicals. Specifically, metals can be leached from the plant using an acid solution. Three Ni recovery approaches will be used: Cyanex 272, oxidative precipitation, and organic chelators

[0032] ,

[0227] In addition to employing traditional means of Ni extraction, plant and root material can be homogenized to generate a concentrated plant slurry. Since the NBPs contain SpyTags, the NBP-bound Ni can be immuneprecipitated to create an NBP-bound Ni concentrate. The Ni can be extracted using one of the above methods.

[0228] Finally, factors that impact Ni uptake and accumulation will be identified. The impact of planting density on metal accumulation may be accessed, as both overplanting and underplanting could have diminishing return on plant Ni uptake and accumulation. Soil parameters, like pH and the presence of competing ions, may influence Ni uptake and accumulation.References

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[0274] 46. Althaf, S. & Babbitt, C. W. Disruption risks to material supply chains in the electronics sector. Resour. Conserv. Recycl. 167, 105248 (2021).Example 7: Genetically engineering endophytes to secrete anti-microbial agents

[0275] Genetically tractable root colonizing microbes will be generated to produce novel antifungal peptides to protect wheat from fungicide-resistant plant pathogens. An predictive Al model will be developed to generate novel antifungal peptides (AFPs) and other anti-microbial agents. Genetically tractable Trilicum aeslivum (common wheat) endophytes will be identified and engineered to produce novel AFPs.

[0276] Pathogenic fungi blighting wheat fields pose a particular threat to the food supply. The plant disease-causing fungi, like Fusarium spp. and Rhizoctonia solani, have been reported to9 10 reduce wheat yield by 70-80% in affected fields . At a global scale, fungal infections are thought to be responsible for 100-200 billion dollars in crop loss per year11. With climate change driving the spread of fungi and monoculture practices leading to increases in fungicidal resistance, a rapid and robust system to combat the spreading threat of fungal plant pathogens , . , , 3,12 must be developed

[0277] Biocontrol probiotics using endophytic microbes as vectors to deliver antifungal peptides directly to the roots of wheat plants to prevent Fusarium spp. and . solani outbreaks will be developed (FIG. 13). Endophytes are symbiotic microbes that live within the cells or tissues of plants, where endophytes are protected from the environment and can deliver therapeutics directly to the plant. The current state-of-the-art methods combating fungal outbreaks include spraying fungicides or incorporating microbes antagonistic to the pathogensinto the soil. However, such methods suffer considerable drawbacks and risk being unable to keep up with future fungal outbreaks. The use of fungicidal sprays is under intense scrutiny, given the potential for adverse effects on human and livestock health as well as driving the13 development of antifungal-resistant fungi . While inoculating soil with fungal antagonistic microbes is considered safer and has demonstrated efficacy in the lab, field trials fail to show significant protection against pathogenic fungi. This lapse in protection is thought to be caused by the inability of fungal antagonistic microbes to establish an ecological niche in a field 14 setting . Native wheat endophytes capable of long-term environmental persistence to express such novel antifungal peptides may be engineered.Preliminary AI / ML approach to discover novel candidate antifungal peptidesAntifungal peptides (AFPs) are short, naturally occurring or synthetic sequences of about 10 - 50 amino acids that inhibit the growth of fungi through various mechanisms. Many AFPs can function broadly by forming pores in the cell membrane. Such AFPs not only target fungi but can also kill bacteria and archaea. Such AFPs are not of interest to BioSPIDER (platform adapted from BIOSME), as such AFPs may harm beneficial members of the plant microbiome. Instead, classes of AFPs that specifically target fungi will be used. One class of AFPs specifically targets fungal cell wall synthesis. The fungal cell wall is heavily enriched in p~ glucan and chitin, and AFPs that disrupt fungal cell wall synthesis do so by inhibiting the enzymes involved in fungal cell wail synthesis, 0-(l,3)-glucan synthase and chitin synthase.15Hie mechanisms of action for other AFPs is less well understood. For example, plants produce a class of small cationic AFPs known as defensins. Such AFPs are taken up by fungal pathogens where the AFPs initiate lysis from the cytoplasm of the pathogen16. Since AFPs are a part of the17 plant's innate immune system, overexpressing them in plants is considered safe and effective . Like any antimicrobial, the development of resistance is still an issue. The next generation of AFPs must be produced rapidly and continuously as fungi develop resistance to provide plants18 with an advantage over fungal pathogens and ensure the robustness of U.S. agriculture . Engineered endophytes will provide a platform for the delivery of the next generation AFPs in the absence of engineered plants.Developing a pipeline to identify genetically tractable root endophytes

[0278] The plant microbiome consists of plant-associated bacteria, archaea, and fungi that reside in and on plant tissue. Constituents of the plant microbiome play an essential role indisease suppression, nutrient uptake, and stress resistance. Given these crucial functions, microbiome engineering offers a powerful approach to modulating many aspects of plant growth, development, and fitness. Efforts to date have primarily focused on engineering the soil microbiome. Most of these attempts have failed when deployed beyond the lab, as soil 23 composition is highly dynamic and influenced by many extrinsic factors . Endophytes are constituents of the plant microbiome that live in plants, either in the plant cells or interstitial spaces. Given that endophytes are resident in plant tissues, endophytes represent a more stable 24 target for microbiome engineering; however, few endophytes have been modified to date .

[0279] As disclosed herein, a pipeline for endophyte modification and reintroduction to the host plant, e.g., BIOMSE (Building Inter-Organismal Mutualisms for Synthetic Engineering). BIOMSE utilizes broad host range plasmids to identify genetically tractable root endophytes25’26. The plasmids from this library are housed in E. coli and carry antibiotic and fluorescent markers with the genes needed for conjugation. Following selection for endophytes that received the plasmid, roots can be recolonized with the modified microbes. This approach has several advantages over existing methods. Endophytes are resident in plant tissue, a more stable and less dynamic environment than microbes in the soil microbiome. Native endophytes are already well adapted to the plant host and be less susceptible to local environmental changes. Endophytes will be engineered to secret AFPs into the T. aestivum roots. The production of such AFPs will serve as a way to make wheat crop resistant to infection and serve as a method to treat diseased crops. Additionally, the technologies disclosed herein are plant agnostic. This approach can be adapted for use in many other relevant crops and has already been demonstrated on a variety of plants, including grasses and nightshades.

[0280] Genetically engineering environmental microbes has traditionally been a challenge. A critical step to engineering an organism is delivering the novel genetic material into the cell. This is typically done using a circular, self- replicating unit of DNA known as a plasmid, however most plasmids used in the lab have been designed for well-studied organisms like Escherichia coli and Bacillus subtilis, and are not compatible with the vast majority of microbes in the environment. Broad Host Range (BHR) plasmids like RSF1010 typically encode replication machinery and can replicate within a much greater diversity of organisms. Previously, root tissue was extracted from Phragmites australis and used a library of BHR plasmids to identify genetically accessible endophytes (FIGs. 3A-3C). Upon identification, endophytes carrying a fluorescent reporter were reinoculated into the host and shown tocolonize roots upon reinoculation. Genetically tractable root endophytes for three different plants, including Nicotiana benthamiana, Phragmites australis, and Panicum vtrgaium. have been identified.Methods:

[0281] The BioSPIDER project will adapt the BIOSME pipelines into a singular platform for rapidly producing biocontrol probiotics capable of countering essentially any existing or emerging agricultural biothreat. As a proof-of-concept, wheat and two of its fungal pathogens, Fusarium spp. and R. solani. will be used. A large number of AFPs will be initially down- selected to only those with validated in vitro activity against the fungal pathogens. Validated peptides will be further down-selected based on the ability to be successfully expressed in wheat colonizing host chassis strains identified using the BIOSME Wheat Pipeline. Such rudimentary biocontrol probiotic strains will be further engineered to have enhanced genomic stability and AFP expression with an integrated kill switch that prevents the unwanted spread in the environment. The platform’s output will be a library of optimized biocontrol probiotics that will be evaluated in wheat for efficacy, longevity, and any potential adverse effects on crop health or surrounding environment.In vitro testing of predicted hits

[0282] To identify the best peptides to include in the engineered probiotics, approximately 40 peptides per pathogen will be ordered for synthesis. Each peptide will be screened for antifungal activity against Fusarium spp. and R. solani with a broth microdilution assay. Peptides with reasonably low minimal inhibitory concentrations (MICs) may be further screened for undesirable activities, including hemolytic activity, cytotoxicity in plants, and activity against commensal organisms. The five most effective and species-specific peptides will be prioritized for downstream tasking.Expected Outcomes:

[0283] The initial step of the pipeline will generate approximately 10,000 candidate antifungal peptides that were initially down-selected based on criteria like charge and % hydrophobicity7. Five down-selected peptides were synthesized and tested against Fusarium, of which two demonstrated inhibitory activity. At least 35 predicted antifungal peptides per pathogen will be examined. At least five peptides with in vitro activity against each pathogen are expected.Adapt BIOSME pipeline for wheat colonizing host chassis isolation

[0284] The BIOSME pipeline to isolate wheat-colonizing microbes that can be engineered to deliver antifungals directly to the root environment. The pipeline will select microbes that are 1) naturally genetically tractable and 2) native to the wheat rhizosphere, emphasizing endophytic microbes. This approach will allow us to readily engineer isolates to produce the antifungal and give engineered probiotic strains a significant advantage in environmental persistence over non-native or lab adapted organisms like E. coli.BIOSME-Wheat pipeline development

[0285] The wheat rhizosphere will be characterized by performing shotgun metagenomic sequencing of the roots of wheat crops at various stages of growth purchased from local farmers. Additional plasmid donor strains will be developed from the highly abundant genera in samples that contain many root colonizing or endophytic species, which will bias the hits37 from the pipeline toward those organisms as disclosed herein . Selected donor species will38 be engineered to have a selectable autotrophy, like diaminopimelic acid , to enable removal of the engineered microbes from the environmental microbe population following conjugation. Metagenomic datasets will also be analyzed using computational tools like 39PlasmidHostFinder to identify abundant plasmids in the wheat rhizosphere, which will be added to the existing plasmid library to further bias the platform tow ard target organisms.Wheat Colonizing Host Chassis Isolation and Characterization

[0286] The w heat root-associated microbiome will be extracted from homogenized root tissue used for sequencing. To assess genetic tractability, extracted root-associated microbes will be co-cultured with a donor microbe containing the plasmid library for conjugation.

[0287] Following conjugation, microbes will be plated on selective Burke’s medium, which is a nitrogen-depleted medium. Organisms capable of growing on this medium can fix nitrogen, and nitrogen-fixing microbes are commonly found in mutualistic relationships with plants, decreasing the likelihood of obtaining plant pathogens. Microbes that grow on selective medium, indicating plasmid uptake, will be screened for the fluorescent marker included in the plasmid, and 16S rDNA sequencing will be used to identify those microbes. The identified microbes will be screened for the ability to colonize the roots of the host plant using fluorescence microscopy.

[0288] BIOSME isolates will be down-selected based on the ability' to produce the down- selected peptides that have been identified, as differences in metabolism or susceptibility maymake some antifungals incompatible with a given isolate. Because each BIOSME isolate will have a known plasmid that can be easily engineered, the Gibson Assembly technique can be used to rapidly clone the sequence of the identified antifungal peptide into the corresponding plasmid, then insert the peptide-containing plasmid into the isolate and confirm expression via mass spectrometry7.Expected Outcomes:

[0289] Previous implementations of the BIOSME pipeline have produced multiple hits in under one week. Adapting the pipeline specifically for the wheat rhizosphere by adding at least three donor strains and five novel plasmids will expand the number and diversity of genetically^ tractable wheat root colonizing microbes that can be isolated from this environment. More than ten genetically tractable wheat root-colonizing symbionts capable of producing at least one of the identified antifungals are expected.Biocontrol probiotic optimization

[0290] However, down-selected strains capable of colonizing wheat roots and producing identified AFPs will also require additional optimization for deployment in the field. For example, expressing the peptide from the plasmid, while easier, involves using a selectable marker, like an antibiotic, to maintain the plasmid in the isolate, which is feasible for use in the field. Using more advanced genetic engineering techniques, antifungal genes can be integrated into the genomes of the isolates so the antifungal genes are stable without needing a selectable marker. Expression and secretion of the antifungal peptides will be fine-tune to ensure that sufficient concentrations are produced without affecting the fitness and environmental persistence of the host chassis isolate. Finally, isolates will be engineered with a genetically encoded kill switch so the isolates cannot spread unchecked in the environment.Antifungal expression optimization

[0291] Expression of the antifungals will be optimized by testing constitutive promoter libraries with different characterized levels of gene expression40and taxonomic origins41.42Peptide antifungals may require the addition of a secretion tag , which targets the protein or 43 peptide for export via the cell’s secretion machinery . Antifungal expression and secretion optimization will be performed using the plasmid-based antifungal expression strains, as plasmids can be engineered much faster than genome-integrated strains. Secretion of the antifungal w ill be quantified by growing the strain in a nutrient-poor media like VL55 or R2B, ty pes of medium optimized to simulate the conditions microbes encounter in the soil, thenremoving the cells and analyzing the concentration of compound secreted into the supernatant via mass spectrometry. Because plasmids and the genes can exist as multiple copies within a cell, higher titers are expected of the antifungal in plasmid-based antifungal expression strains than what will be produced in the final genome-integrated strains. The artificially high production will be normalized by quantifying the plasmid copy number with qPCR and projecting the amount of compound expected when only one copy of the antifungal gene is present.

[0292] Once the optimal promoter and secretion tag configurations are determined for antifungal production in the specific host chassis strain, the genes will be integrated into the44 genome using allelic exchange , which uses homologous recombination to add or remove DNA at specific locations within the chromosome. This method is ideal for the diverse collection of non-model organisms generated by the methods disclosed herein, as it only requires that a strain can take up a plasmid and express a suitable selectable marker, which, by design, all BIOSME isolates can. The isolate genome will be sequenced to identify a suitable integration site, then a plasmid will be constructed containing the homologous DNA sequences to that integration site, the optimized antifungal expression genes, and an antibiotic resistance gene that to select mutants that have successfully integrated the new genes into the chromosome of the endophyte by plating on a nutrient media containing that antibiotic. The final step will be to remove the antibiotic selection marker using the FLP-FRT recombination. ,4.45.46 methodBiocontainment engineering

[0293] Biocontainment is essential for deploying genetically engineered microbes into the environment. One benefit of using root-associated microbes is that the microbes are housed in or on the roots. Therefore, plant-based cues can be used to ensure that microbes do not escape the plant hosts. An inducible kill switch can be engineered using CRISPR / Cas3 to 47 48 ensure microbial killing by DNA destruction ’ . Specifically, bacterial promoters and transcription factors responsive to plant molecular cues can be identified, such as the49 ethanolamine-responsive PipR transcriptional regulator . Because many of the plant molecular cues with reported microbial responsive systems are not specific to wheat, this strategy would require that the biocontrol probiotics have a narrow endosymbiotic host range for the target crop. This will be tested by looking for biocontrol probiotic colonization in a variety of related and unrelated plants. A temperature dependent riboswitch50may be used.The temperature dependent riboswitch would sterilize biocontrol probiotics at the end of a growing season, and / or a chemically induced system that could be sprayed as needed. Molecular circuitry that can repress the kill switch activation when endophytes are associated with the plant host will be designed. If microbes escape into the environment where the plant cue is no longer present, the kill switch will be activated. Kill switch molecular circuitry will be incorporated into the genome of the engineered probiotic strains, as described herein. The efficacy of the biocontainment kill switch will be evaluated in the whole-plant wheat model.Expected Outcomes

[0294] The optimization of biocontrol probiotics is expected to yield a library of at least five field-deployable strains capable of competing with the native microbiome and persisting in the environment for months while continuously delivering antifungals to the root environment. By leveraging plant-based cues to control the inducible kill switch, a robust biocontainment system that activates upon microbial escape from the plant host can be developed.In plant validation

[0295] For this to be an attractive technology to farmers, the biocontrol probiotics’ efficacy and safety must be demonstrated. Assays will be developed to evaluate the biocontrol probiotics in a laboratory whole-plant wheat model with an intact rhizosphere. Efficacy of the engineered endophytes will be assess in reducing a fungal pathogen burden when delivered as a prophylactic before fungal challenge and after fungal challenge as a post-challenge treatment. Any adverse effects will be measured by quantifying wheat yield and biomass of biocontrol probiotic-treated versus untreated plants. As the surrounding rhizosphere is important for crop health, changes in the surrounding microbial community will also be determined.Wheat and fungi model development

[0296] Wheat is typically planted in the fall, experiences dormancy in the winter, and is harvested in the summer. Developmentally, this timeline is split into eleven stages, beginning 52 with planting and culminating with harvestable grain . Wheat may be grown in a chamber with temperatures ranging between 21 °C to 24°C with a sixteen-hour photopenod. While growth occurs across multiple seasons, two stages are most susceptible to infection by fungal5T pathogens, seedlings and post-anthesis . The seedling stage will be used, because the plants can germinate and infect seedlings over two weeks instead of the several months needed to grow plants to the post-anthesis stage51'53. This approach will enable rapid screening ofendophyte and peptide combinations for efficacy. Further, it is reasonable to assume that crops treated with the endophytes early in development will be protected from fungal pathogens until harvest.

[0297] Because competition from the native microbiome will likely be a major source of failure, performing the validation experiments in sterile soil will not produce results similar to those in plants in a crop environment. The plant model will be developed with an intact rhizosphere by inoculating the agriculturally conditioned soil with frozen stocks of the same wheat root homogenate and surrounding soil used for sequencing.

[0298] Fusarium spp. and R. solani will be maintained on potato dextrose agar plates and synthetic nutrient agar in preparation for infection studies’1’53. Wheat seeds will be sterilized before being planted. Upon sterilization, seeds will be planted in the microbially inoculated, agriculturally conditioned soil and allowed to germinate. Two weeks post-germination, seedlings will be removed from the soil, dipped into a solution containing the fungus, and then re-planted. Efficacy assays will be performed two weeks post-infection.

[0299] Plant roots have been inoculated with endophytes at the seedling stage. For a week, biocontrol probiotics will be introduced to the seedlings by pipetting the probiotics directly at the base of the hypocotyl. This method is reasonably robust and results in microbial colonization of in-lab experiments with other plant systems. Throughout the project, microbes will be added pre- and post-fungal infection to identify a delivery timeline that is most effective in protecting wheal.Efficacy and risk assessment

[0300] Each biocontrol probiotic strain will be evaluated for efficacy and adverse effects and compared to a state-of-the-art fungicide treatment like epoxiconazole. Experiments will be performed with replicates that receive only water, fungicide treatment, or treatment with the probiotic strains. This approach will allow us to establish a healthy baseline and identify any adverse effects of fungicide or probiotic treatment. Following two weeks of fungal challenge, biomass will be measured to assess plant health. Biocontrol efficacy will be determined by measuring fungal pathogen abundance in the rhizosphere after the challenge period using qPCR targeting the fungal pathogen normalized to homogenized root tissue. Similarly, persistence of the biocontrol probiotic will be measured using qPCR targeting the antifungal gene integrated into the genome of the host chassis strain. Adverse effects on the wheat rhizosphere will be evaluated through metagenomic sequencing.Example 8: Genetically engineered soil microbes

[0301] Samples of soil were taken from the environment of interest. The soil samples were resuspended in media and the microbes were extracted from the soil. The microbes were then subjected to the trans-conjugation protocol, as described herein, and plated on enriched selective media (LB / TSA).

[0302] Engineered microbial colonies that formed on the media were grow n up independently and then inoculated back into the soil.

[0303] Immediately after inoculation, a sample of soil was taken, washed and plated on selective media. This serves as timepoint 0, to determine the approximate starting concentration of microbes in the soil.

[0304] After 14 days the soils were sampled again and plated on selective media. Genetically engineered microbes which continued to grow and thrive in the soil after the initial inoculation grew on the selective media.

[0305] Genetically engineered soil microbes can be inoculated into the soil surrounding a plant of interest. A genetically engineered soil microbe and genetically engineered endophyte can be concurrently inoculated into the soil surrounding the plant of interest and the plant of interest.Example 9: Genetically engineering endophytes to produce a byproduct or small molecule

[0306] Bioproduction: FIG. 15 shows indigoidine production by engineered endophytes. Indigoidine BHR plasmid are conjugated to selected microbe. Conjugated microbes selected on agar plate demonstrate heterologous indigoidine production.

[0307] Small Molecule: FIG. 16 show s a schematic of engineering a plant colonizing microbe to deliver small molecule or protein effectors directly and continuously to crops. The procedure includes steps: homogenizing root from target crop, selecting pool of microbes, conjugated selected pool of microbes with BHR plasmid pool, selecting conjugated microbes and colonizing plants with the selected genetically engineered microbes. FIG. 17 depicts pipeline to identify genetically tractable root endophytes: Endophytes are extracted from the roots of a plant; Endophytes are subjected to a conjugation pipeline using a library of broad host range plasmids; Microbes are plated on a selective media enriching for endophytic microbes; Transgenic microbes are then assayed using fluorescence microscopy for the ability to recolonize root cells; and Selected microbes are applied to the plants. The geneticallyengineered microbes express Fosmidomycin (and its analogue FR9000098), which is a small molecule that interferes with terpene biosynthesis, which causes bleaching of leaves and external portions of the plant.INCORPORATION BY REFERENCE

[0308] The entire disclosure of each of the patent documents, including patent application documents, scientific articles, governmental reports, websites, and other references referred to herein is incorporated by reference herein in its entirety for all purposes. In case of a conflict in terminology, the present specification controls. All sequence listings, or Seq. ID. Numbers, disclosed herein are incorporated herein in their entirety.

[0309] Although illustrative embodiments of the present invention have been described herein, it should be understood that the invention is not limited to those described, and that various other changes or modifications may be made by one skilled in the art without departing from the scope or spirit of the invention.

Claims

CLAIMSWhat is claimed is:

1. A method of identifying a genetically tractable microbial endophyte, the method comprising: isolating a plurality of microbial endophytes from one or more tissues of a plant; conjugating the plurality of isolated microbial endophytes with one or more microorganisms comprising a library of broad host range plasmids having at least one selection marker; and selecting one or more conjugated microbial endophytes that have one or more selection markers.

2. The method of claim 1, further transforming the selected microbial endophytes with one or more heterologous nucleic acid sequences encoding one or more heterologous peptides and / or polypeptides.

3. The method of claim 2, wherein the microbial endophytes comprising one or more heterologous nucleic acid sequences is screened for expression of the one or more heterologous peptides and / or polypeptides.

4. A method of producing a genetically engineered microbial endophyte, the method comprising: isolating a plurality of microbial endophytes from one or more tissues of a plant; conjugating the plurality of isolated microbial endophytes with one or more microorganisms comprising a library of broad host range plasmids having at least one selection marker and one or more heterologous nucleic acid sequences encoding one or more heterologous peptides and / or polypeptides; selecting one or more conjugated microbial endophytes that have one or more selection markers; and culturing the one or more selected microbial endophytes.

5. The method of claim 4, wherein the selected microbial endophytes is screened for expression of the one or more heterologous peptides and / or polypeptides.

6. The method of claim 1 or 4, wherein the plurality of microbial endophytes comprises one or more bacteria or one or more fungi.

7. The method of claim 6, wherein the one or more bacteria is of the genus Bacillus, Herbaspirillum, Rhizobium, Bradyrhizobium, Pseudomonas, Ralstonia, Streptomyces , Acidovorax, Rhodococcus, Acetobacteraceae, Acidobacteriaceae, Acidothermaceae, Aerococcaceae, Alcaligenaceae, Alicyclobacillaceae, Alteromonadaceae, Anaerolineaceae, Aurantimonadaceae, Bacillaceae, Bacteriovoracaceae, Bdellovibrionaceae, Bradyrhizobiaceae, Brevibacteriaceae, Brucellaceae, Burkholder iaceae, Carboxydocellaceae, Caulobacteraceae, Cellulomonadaceae, Chitinophagaceae, Chromatiaceae, Chthoniobacteraceae, Chthonomonadaceae, Clostridiaceae, Comamonadaceae, Corynebact er iaceae, Coxiellaceae. Cryomorphaceae, Cyclobacteriaceae, Cytophagaceae, Deinococcaceae, Dermabacteraceae, Dermacoccaceae, Enterobacteriaceae, Enter ococcaceae, Erythrobacteraceae, Fibrobacteraceae, Flammeovirgaceae, Flavobacteriaceae, Frankiaceae, Fusobacteriaceae, Gaiellaceae, Gemmatimonadaceae, Geodermatophi Iaceae, Glycomycetaceae, Haliangiaceae, Hcdomonadaceae, Holosporaceae, Hyphomicrobiaceae, lamiaceae, Intrasporangiaceae, Kineosporiaceae, Koribacteraceae, Lachnospiraceae, Lactobacillaceae, Legionellaceae, Leplospiraceae, Leuconoslocaceae. Me thylobacler iaceae, Methylocystaceae, Methylophilaceae, Microbacteriaceae, Mier ococcaceae, Micromonosporaceae, Moraxellaceae, Mycobacteriaceae, Mycoplasmataceae, Myxococcaceae, Nakamurellaceae, Neisser iaceae, Nitrosomonadaceae, Nocardiaceae, Nocardioidaceae, Oceanospirillaceae, Opitutaceae, Oxalobacteraceae, Paenibacillaceae, Parachlamydiaceae, Pasteurellaceae, Patulibacteraceae, Peptostreptococcaceae, Phyllobacteriaceae, Piscirickettsiaceae, Planet omycetaceae, Planococcaceae, Polyangiaceae, Porphyromonadaceae, Prevotellaceae, Promicromonosporaceae, Pseudomonadaceae, Pseudonocardiaceae, Rhizobiaceae, Rhodobacteraceae, Rhodospirillaceae, Roseiflexaceae, Rubrobacteriaceae, Bandar acinaceae, Sanguibacteraceae, Saprospiraceae, Segniliparaceae, Shewanellaceae, Sinobacteraceae. Solibacteraceae, Solimonadaceae, Solirubrobacteraceae.Sphingobacter iaceae, Sphingomonadaceae, Spiroplasmataceae, Sporichthyaceae, Sporolactobacillaceae, Staphylococcaceae, Streptococcaceae, Streptomycetaceae, Syntrophobacteraceae, Veillonellaceae, Verrucomicrobiaceae, Weeksellaceae, Xanthobacteraceae, and / or Xanthomonadaceae andthe one or more fungi is of the genus Epichloe, Neotyphodium, Khuskia, Epicoccum, Curvularia, Serendipita, Mycosphaerella, Piriformospora, Cladosporium, Fusarium. Colletotrichum. Phomopsis. Beauveria. Talaromyces, Apiospora, Aspergillaceae. Ceratobasidiaceae. Coniochaetacecie, Cordycipitaceae, Corticiaceae, Cystofilobasidiaceae, Davidiellaceae, Debaryomycetaceae, Dothioraceae, Erysiphaceae, Filobasidiaceae, Glomerellaceae, Hydnaceae. Hypocreaceae, Leptosphaeriaceae, Montagnulaceae. Mor tier ellaceae, Mycosphaerellaceae. Nectriaceae. Orbiliaceae, Phaeosphaeriaceae, Pleosporaceae, Pseudeurotiaceae, Rhizopodaceae, Sclerotiniaceae. Stereaceae, and / or Trichocomaceae .

8. The method of any one of claims 1-7, wherein the tissues of the plant is selected from the group consisting of roots, shoots, stems, leaves, rhizomes, trichomes, pneumatophores, inflorescences, pollen and seeds.

9. The method of any one of claims 1-8, wherein the library of broad host range plasmids comprises one or more sequences from pCUl, RK2, R300B, R1162, pSa, pR288, pBBRl, PBR322-based plasmids, pUC series plasmids, pSC 101 -derived plasmids, pEpi series plasmids, pL2 series plasmids, pGreen vector series, pH7 vector series, pBI121 and pCAMBIA series, pKl l series, pK13 series, pGWB series plasmids, pUbi vector series, pNovl vectors. pC1301. pC1302, pMDC. pPZP, pVBN, pSHP. pSAG-based vectors, pPha- tl, pGWB, pHygro series, pSLl 180 series, pT7-based vectors, pMOL98, RK2, RP4,RP1,R68, PB10, RA3, IncN, IncP-1, IncU, IncPromA, Incl8, RK404, pDSK509, pDSK519, pRK415, RSF1010, IncW, pJC8. pJC24, PBBR1MCS, pK18mobsacB, pLAFRl, pCCIFOS. pWKS30, pUTmini-TK. pEcoRi, PGEM-T. pGEM. pFOSl, pCCIFOS, PFOS1RK2, pF0S6, pYAC4, pYAC3, pYAC5, pYACl, pBBRIMCS-BAC, pSClOl-based bacterial artificial chromosome (BACs), pFOSBAC, pFosBAC, pK18mobSacB-based BACs, or a combination thereof.

10. The method of any one of claims 1-9, wherein the at least one selection marker is resistance to an antibiotic.

11. The method of claim 10, wherein the antibiotic is selected from the group consisting of kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, bleomycin, erythromycin, polymyxin B, tetracycline, and chloramphenicol.

12. The method of any one of claims 4-10, wherein the one or more heterologous nucleic acid sequences are between about 0.01 kb to about 20 kb, about 0.1 kb to about 10 kb, about 0.5 kb to about 15 kb, about 0.1 to about 1 kb, or about 5 kb to about 10 kb.

13. The method of any one of claim 4-11, wherein the one or more peptides and / or polypeptides comprise a small molecule, a phytohormone, an enzyme, a transcription factor, and / or a nuclease.

14. The method of any one of claims 1-13, wherein the plant is of the genus Zea, Triticum. Oryza, Zizania, Hordeum, Sorghum. Eleusine. Panicum, Pennisetum. Setaria, Avena, Triticosecale, Secale, Psathyrostachys, Bambuseae, Saccharum, Glycine max. Brassica, Gossypium, Medicago, Manihot, Solanum, Solcinum, Sporobolus, Odontarrhena, Streptanthus, Pisum, Cicer, Lens, or Linum.

15. A method of preparing a plant comprising a plurality of genetically engineered microbial endophytes, a method comprising: applying to an exterior surface of the plant the plurality of genetically engineered microbial endophytes, wherein the plurality of genetically engineered microbial endophyte are derived from one or more microbial endophyte species isolated from a species of the plant and comprises one or more heterologous nucleic acid sequences encoding one or more heterologous peptides and / or polypeptides, and wherein the plant comprising the plurality of genetically engineered microbial endophytes has one or more traits of interest.

16. The method of claim 15, further applying to the exterior of the plant one or more additional genetically engineered microbial endophytes, wherein the additional one or more genetically engineered microbial endophytes are from one or more species isolated from a species of the plant and comprises one or more heterologous nucleic acid sequences encoding one or more heterologous peptides and / or polypeptides.

17. The method of claim 16, wherein the one or more additional genetically engineered microbial endophytes are of different genera compared to each other.

18. The method of claim 1 , wherein the one or more additional genetically engineered microbial endophytes are of the same genus compared to each other.

19. The method of claim 15, wherein the trait of interest of the plant is selected from the group consisting of germination rate, emergence rage, drought tolerance, freeze tolerance, shoot biomass, root biomass, seeding root length, yield, metal ion binding, nitrogen fixing, pollutant degrading, anti-fungal, herbicidal, nematicidal, and / or insecticidal.

20. The method of claim 15 or 19, wherein the microbial endophyte is isolated from one or more tissue of the plant.

21. The method of claim 20, wherein the one or more tissues of the plant is selected from the group consisting of roots, shoots, stems, leaves, rhizomes, trichomes, pneumatophores, inflorescences, pollen and seeds.

22. The method of any one of claims 15-21. wherein the plant is of the genus Zea, Triticum, Oryza, Zizania, Hordeum, Sorghum, Eleusine, Panicum, Pennisetum, Setaria, Avena, Triticosecale, Secale, Psathyrostachys, Bambuseae, Saccharum, Glycine max, Brassica, Gossypium, Medicago, Manihot, Solarium, Solanum, Sporobolus, Odonlarrhena, Streptanthus. Pisum, Cicer, Lens, or Linum.

23. The method of any one of claims 15-22, wherein the exterior surface of the plant comprises a seed coat, leaves, root, shoots, and / or flowers.

24. The method of any one of claims 15-23, wherein the plurality of genetically engineered microbial endophytes comprise 2, 3, 4, 5, 6, 7, 8, 9, or 10 different genetically engineered microbial endophytes each of which comprises different one or more heterologous nucleic acid sequences encoding one or more heterologous peptides and / or polypeptides.

25. A composition comprising a plant and a genetically engineered microbial endophyte, wherein the genetically engineered microbial endophyte is from a genus in symbiosis with the plant and comprises one or more heterologous nucleic acid sequences encoding one or more heterologous peptides and / or polypeptides, wherein the plant is contacted with thegenetically engineered microbial endophyte in an amount effective to colonize the plant and to improve one or more traits of interest, and wherein the genetically engineered microbial endophyte is produced by a method comprising: isolating a plurality of microbial endophytes from one or more tissues of a plant; conjugating the plurality of isolated microbial endophytes with one or more microorganisms comprising a library’ of broad host range plasmids having at least one selection marker and the one or more heterologous nucleic acid sequences encoding the one or more heterologous peptides and / or polypeptides; selecting one or more conjugated microbial endophytes that have one or more selection markers; and culturing the one or more selected microbial endophytes in conditions to induce expression of the more heterologous peptides and / or polypeptides.

26. The composition of claim 25, wherein the traits of interest is selected from the group consisting of germination rate, emergence rage, drought tolerance, freeze tolerance, shoot biomass, root biomass, seeding root length, yield, metal ion binding, nitrogen fixing, pollutant degrading, anti-fungal, herbicidal, nematicidal, and / or insecticidal.

27. The composition of claim 25, wherein the plurality of microbial endophytes comprises one or more bacteria or one or more fungi.

28. The composition of claim 27, wherein the one or more bacteria is of the genus Bacillus, Herbaspirillum, Rhizobium, Bradyrhizobium, Pseudomonas, Ralstonia, Streptomyces , Acidovorax, Rhodococcus, Acetobacteraceae, Acidobacteriaceae, Acidothermaceae, Aerococcaceae, Alcaligenaceae, Alicyclobacillaceae, Alteromonadaceae, Anaerolineaceae, Aurantimonadaceae, Bacillaceae, Bacteriovoracaceae, Bdellovibrionaceae, Bradyrhizobiaceae, Brevibacteriaceae, Brucellaceae, Burkholder iaceae, Carboxydocellaceae, Caulobacteraceae, Cellulomonadaceae, Chitinophagaceae, Chromatiaceae, Chthoniobacteraceae, Chthonomonadaceae, Clostridiaceae, Comamonadaceae, Corynebacteriaceae, Coxiellaceae, Cryomorphaceae, Cyclobacteriaceae, Cytophagaceae, Deinococcaceae, Dermabacteraceae, Dermacoccaceae, Enterobacteriaceae, Enterococcaceae, Erythrobacteraceae, Fibrobacteraceae, Flammeovirgaceae, Flavobacteriaceae, Frankiaceae, Fusobacteriaceae, Gaiellaceae, Gemmatimonadaceae, Geodermatophilaceae, Glycomycetaceae.Haliangiaceae, Halomonadaceae, Holosporaceae, Hyphomicrobiaceae, lamiaceae, Intrasporangiaceae, Kineosporiaceae. Koribacteraceae, Lachnospiraceae, Lactobacillaceae, Legionellaceae, Leptospiraceae, Leuconostocaceae, Methylobacteriaceae, Methylocystaceae. Methylophilaceae, Microbacteriaceae, Micrococcaceae, Micromonosporaceae, Moraxellaceae, Mycobacteriaceae, Mycoplasmataceae, Myxococcaceae, Nakamurellaceae, Neisseriaceae, Nitrosomonadaceae, Nocardiaceae, Nocardioidaceae, Oceanospirillaceae, Opitutaceae, Oxalobacteraceae, Paenibacillaceae, Parachlamydiaceae, Pasteurellaceae, Patulibacteraceae, Peptostreptococcaceae, Phyllobacteriaceae, Piscirickettsiaceae, Planet omycetaceae, Planococcaceae, Polyangiaceae, Porphyromonadaceae, Prevotellaceae, Promicromonosporaceae, Pseudomonadaceae, Pseudonocardiaceae, Rhizobiaceae, Rhodobacteraceae. Rhodospirillaceae, Roseiflexaceae, Rubrobacteriaceae, Sandaracinaceae. Sanguibacteraceae, Saprospiraceae, Segniliparaceae, Shewanellaceae, Sinobacteraceae, Solibacteraceae, Solimonadaceae, Solirubrobacteraceae, Sphingobacteriaceae, Sphingomonadaceae, Spiroplasmataceae, Sporichthyaceae, Sporolactobacillaceae. Staphylococcaceae, Streptococcaceae, Str ept omycetaceae, Syntrophobacteraceae. Veillonellaceae, Verrucomicrobiaceae, Weeksellaceae.Xanthobacteraceae, and / or Xanthomonadaceae.' and the one or more fungi is of the genus Epichloe, Neotyphodium, Khuskia, Epicoccum, Curvularla, Serendipila, Mycosphaerella, Piriformospora, Cladosporium, Fusarium. Colletotrichum, Phomopsis, Beauveria, Talaromyces, Apiospora, Aspergillaceae, Ceratobasidiaceae. Coniochaetaceae, Cordycipitaceae, Corticiaceae, Cystofdobasidiaceae, Davidiellaceae, Debaryomycetaceae, Dothioraceae, Erysiphaceae, Filobasidiaceae, Glomerellaceae, Hydnaceae. Hypocreaceae, Leptosphaeriaceae, Montagnulaceae, Mor tier ellaceae, Mycosphaerellaceae. Nectriaceae, Orbiliaceae, Phaeosphaeriaceae, Pleosporaceae, Pseudeurotiaceae, Rhizopodaceae, Sclerotiniaceae, Stereaceae, and / or Trichocomaceae .

29. The composition of any one of claims 25-28, wherein the tissues of the plant is selected from the group consisting of roots, shoots, stems, leaves, rhizomes, trichomes, pneumatophores, inflorescences, and seeds.

30. The composition of any one of claims 25-29, wherein the library of broad host range plasmids comprises one or more sequences from pCUl. RK2, R300B, R1162, pSa, pR288, pBBRl, PBR322-based plasmids, pUC series plasmids, pSClOl-derived plasmids, pEpiseries plasmids, pL2 series plasmids, pGreen vector series, pH7 vector series, pBI121 and pC AMBIA series, pKl 1 series, pK13 series, pGWB series plasmids, pUbi vector series, pNovl vectors, pC 13Ol, pC1302, pMDC, pPZP, pVBN, pSHP, pSAG-based vectors, pPha- tl, pGWB, pHygro series, pSLl 180 series, pT7-based vectors, pMOL98, RK2, RP4,RP1,R68, PB10, RA3, IncN, IncP-1, IncU, IncPromA, Incl8, RK404, pDSK509, pDSK519, pRK415, RSF1010, IncW, pJC8. pJC24. PBBR1MCS, pK18mobsacB, pLAFRl, pCCIFOS. pWKS30, pUTmini-TK. pEcoRi, PGEM-T. pGEM. pFOSl, pCCIFOS, pFOSlRK2, pF0S6, pYAC4, pYAC3, pYAC5, pYACl, pBBRIMCS-BAC, pSClOl-based bacterial artificial chromosome (BACs), pFOSBAC, pFosBAC, pK18mobSacB-based BACs, or a combination thereof.

31. The composition of any one of claims 25-30, wherein the at least one selection marker is resistance to an antibiotic.

32. The composition of claim 31, wherein the antibiotic is selected from the group consisting of kanamycin, spectinomycin, streptomycin, ampicillin, carbenicilhn, bleomycin, erythromycin, polymyxin B, tetracycline, and chloramphenicol.

33. The composition of any one of claims 25-32, wherein the one or more heterologous peptides and / or polypeptides comprise a peptide, an enzyme, a transcription factor, and / or a nuclease.

34. The composition of any one of claims 25-33, wherein the plant is of the genus Zea, Triticum, Oryza, Zizania, Hordeum, Sorghum, Eleusine. Panicum, Pennisetum, Setaria, Avena, Triticosecale, Secale, Psathyrostachys, Bambuseae, Saccharum, Glycine max, Brassica, Gossypium, Medicago, Manihot, Solanum, Solanum, Sporoboliis, Odontarrhena, Streptanthus, Pisum, Cicer, Lens, or Linum.

35. The composition of any one of claims 25-34, wherein the genetically engineered microbial endophyte comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 different genetically engineered microbial endophytes each of which comprises different one or more heterologous nucleic acid sequences encoding one or more heterologous peptides and / or polypeptides.

36. A method of producing a genetically engineered microbial endophyte capable of binding a metal ion, the method comprising: culturing one or more selected microbial endophytes produced by the method according to claim 1 and conjugating the selected microbial endophytes with one or more heterologous nucleic acid sequences encoding one or more cell-surface or secreted metal ion binding peptides; or culturing one or more selected microbial endophytes produced by the method according to claim 2, wherein the one or more heterologous nucleic acid sequences encodes one or more cell-surface metal ion binding peptides.

37. The method of claim 36, further comprising: conjugating the genetically engineered microbial endophyte with one or more heterologous nucleic acid sequences encoding one or more plant growth promoting hormones or phytohormones; and / or conjugating the genetically engineered microbial endophyte with an inducible kill switch.

38. The method of claim 36 or 37, wherein the metal ion is selected from the group consisting of gold, silver, palladium, platinum, rhodium, copper, nickel, and lanthanide.

39. The method of claim 38, wherein the metal ion is nickel.

40. The method of claim 37, wherein the plant growth hormone is auxin and / or gibberellic acid.

41. The method of claim 36, wherein the metal ion binding peptide is a nickel binding peptide (NBP).

42. The method of claim 19, wherein the nickel binding peptide is selected from the group consisting of any one of peptide sequences from SEQ ID NO: 1-17.

43. The method of claim 21, wherein the plant species is selected from the group consisting of: S. bicolor, P. virgatum, S. alterni floras, B. juncea, O. chalcidica, and Streptanthus polygaloides .

44. The method of claim 43. wherein the plant species is Streptanthus polygaloides.

45. The method of claim 38, wherein the metal ion is lanthanide.

46. The method of claim 36, wherein the metal ion binding peptide is a lanthanide binding peptide (LBP).

47. The method of claim 46, wherein the lanthanide binding peptide is LanMl .

48. The method of any one of claims 45-47, wherein the plant species is Panicum virgatum.

49. A method of producing a plant capable of accumulating metal ions, the method comprising: applying to an exterior surface of the plant one or more genetically engineered microbial endophytes produced by the method according to any one of claims 36-48. wherein the plant is grown in soil comprising metal ions.

50. A method of isolating metal ions from the plant produced by the method according to claim 48. the method comprising homogenizing the plant tissues; and incubating the homogenized plant tissues in an acid solution.

51. A method of producing a genetically engineered microbial endophyte capable of producing one or more anti-microbial agents, the method comprising: culturing one or more selected microbial endophytes produced by the method according to claim 1 and conjugating the selected microbial endophytes with one or more heterologous nucleic acid sequences encoding one or more anti-microbial agents; or culturing one or more selected microbial endophytes produced by the method according to claim 4, wherein the one or more heterologous nucleic acid sequences encodes one or more anti-microbial agents.

52. The method of claim 51, wherein the anti-microbial agent is an anti-fungal agent or an anti-bacterial agent.

53. The method of claim 51, further comprising:conjugating the genetically engineered microbial endophyte with one or more heterologous nucleic acid sequences encoding one or more plant growth promoting hormones or phytohormones; and / or conjugating the genetically engineered microbial endophyte with an inducible kill switch.

54. The method of any one of 36-53, wherein the plant is of the genus Zea, Triticum, Oryza, Zizania, Hordeum, Sorghum, Eleusine, Panicum, Pennisetum, Setaria, Avena, Triticosecale, Secale, Psathyrostachys, Bambuseae, Saccharum, Glycine max, Brassica, Gossypium, Medicago, Manihot, Solarium, Solanum, Sporobolus, Odontarrhena, Streptanthus, Pisum, Cicer, Lens, or Linum.

55. The method of claim 54, wherein the plant is Triticum aestivum.

56. The method of any one of 51-55, wherein the one or more antifungal agents inhibit Funsarium spp. and / or / ?, solani.

57. A method of producing a plant comprising a genetically engineered microbial endophyte capable of producing one or more anti-microbial agents, the method comprising: applying to an exterior surface of the plant one or more genetically engineered microbial endophytes produced by the method according to any one of claims 51-56, wherein the plant is grown in an environment susceptible to microbial infection.

58. A method of producing a genetically engineered microbial endophyte capable of producing pigment compound, the method comprising: culturing one or more selected microbial endophytes produced by the method according to claim 1 and conjugating the selected microbial endophytes with one or more heterologous nucleic acid sequences encoding one or more amino acid catalyzing enzymes, wherein the amino acid catalyzing enzymes can catalyze one or more amino acids into one or more pigment compounds; or culturing one or more selected microbial endophytes produced by the method according to claim 4, wherein the one or more heterologous nucleic acid sequences encodes one or more amino acid catalyzing enzymes, wherein the amino acid catalyzing enzy mes can catalyze one or more amino acids into one or more pigment compounds.

59. A method of producing a genetically engineered microbial endophyte capable of secreting plant signaling molecule, the method comprising: culturing one or more selected microbial endophytes produced by the method according to claim 1 and conjugating the selected microbial endophytes with one or more heterologous nucleic acid sequences encoding one or more plant signaling molecules; or culturing one or more selected microbial endophytes produced by the method according to claim 4. wherein the one or more heterologous nucleic acid sequences encodes one or more plant signaling molecules.

60. A method of producing a genetically engineered microbial endophyte for bioremediation, the method comprising: culturing one or more selected microbial endophytes produced by the method according to claim 1 and conjugating the selected microbial endophytes with one or more heterologous nucleic acid sequences one or more environmental pollution degrading enzymes: or culturing one or more selected microbial endophytes produced by the method according to claim 4, wherein the one or more heterologous nucleic acid sequences encodes one or more environmental pollution degrading enzymes.

61. A method of producing a genetically engineered microbial endophyte capable of increasing nitrogen fixation, the method comprising: culturing one or more selected microbial endophytes produced by the method according to claim 1 and conjugating the selected microbial endophytes with one or more heterologous nucleic acid sequences encoding one or more enzymes capable of fixing nitrogen; or culturing one or more selected microbial endophytes produced by the method according to claim 4, wherein the one or more heterologous nucleic acid sequences encodes one or more enzymes capable of fixing nitrogen.

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