Compositions and methods for modifying plant characteristic without modifying plant genome

Abstract

To provide a symbiont forming inoculum which is capable of imparting one or more desired traits into a target plant without introducing heterogeneous DNA into the entire plant.SOLUTION: Provided is a symbiont forming inoculum comprising a polynucleotide encoding a phytohormone biosynthetic enzyme and a polynucleotide of interest, the phytohormone biosynthetic enzyme being at least one cytokinin biosynthetic enzyme and / or an auxin biosynthetic enzyme.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Statement regarding electronic filing of sequence listings In lieu of a paper copy, a Sequence Listing in ASCII text format, filed under 37 CFR § 1.821, entitled 1554-3WO_ST25.txt, 126,446 bytes in size, created on September 17, 2020, and submitted via EFS-Web, is provided. This Sequence Listing is hereby incorporated by reference herein for its disclosure.

[0002] Priority statement This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 62 / 903,183, filed September 20, 2019, the entire contents of which are incorporated herein by reference.

[0003] The present invention relates to symbiont-forming inocula and symbionts comprising polynucleotides encoding one or more plant hormone genes and at least one polynucleotide of interest that can be used to modify the characteristics of a host plant without modifying the host plant's genome. [Background technology]

[0004] Bacteria of the genus Agrobacterium have been studied for decades as plant pathogens that cause crown gall disease. This disease results in the formation of plant masses (or galls) that grow at the site of infection by Agrobacterium species on the plant. Galls that develop on mature plants may result in little or no phenotypic response or impact on plant growth, depending on the pathogen and host genotype and the age of the infected host. However, galls on younger plants can have significant deleterious effects on plant growth and other characteristics. Gall formation is induced as a result of the bacteria's ability to enter the plant wound site and transfer a portion of DNA (called T-DNA, or transfer DNA, located on the Ti-plasmid Agrobacterium species plasmid) to neighboring plant cells. After entering the plant cell, the T-DNA is directed to the nucleus, where it is integrated into the plant's genome.

[0005] In the 1940s, before Agrobacterium species were known to transfer DNA into plant genomes, researchers discovered that the bacterium stably altered plant cells, making them "immortal" and allowing them to grow in in vitro culture without the need for plant hormones. It is now known that tumor formation is the result of T-DNA containing genes for plant hormone synthesis that are expressed when inserted into the plant cell genome. The plant hormones subsequently produced cause the plant cells to begin cell division, no longer under the control of plant-produced cell division signals.

[0006] Since the 1980s, Agrobacterium species have been used in research and applications for whole plant transformation due to their ability to insert T-DNA into the genome of target plants. Such T-DNA can be engineered to deliver genes that confer desired traits to target plants. To achieve transformation, "disarmed" strains of Agrobacterium species have been developed that do not form galls, and therefore the resulting plants only realize the direct effect of the delivered gene of interest to produce the desired phenotype.

[0007] However, gene transfer and transformed plants are not always desirable for several reasons. First, plant transformation is a labor-intensive process, and the success rate of transforming plant germline tissues is low. The success of transgenic gene expression in plants can be affected by the expression of neighboring genes and the copy number of the transgene insert within the plant genome. Second, traditional processes of gene transfer do not facilitate real-time responses to environmental stress, pests, or pathogens. Instead, this process is performed in a laboratory setting and therefore cannot be used as a dynamic response to temporal stimuli. Third, transformed genomes containing heterologous DNA may be present in harvested material from plants (e.g., in harvested fruits or vegetables), and most markets require that such transformed DNA be absent from the resulting edible food. Furthermore, there is a demand to prevent pollen from transgenic plants from being present in the environment. Summary of the Invention [Problem to be solved by the invention]

[0008] Therefore, there is a need for methods that can confer one or more desired traits to a target plant without introducing heterologous DNA into the whole plant. [Means for solving the problem]

[0009] One aspect of the present invention provides a symbiont formation inoculum comprising a polynucleotide encoding a plant hormone biosynthetic enzyme and a polynucleotide of interest, wherein the plant hormone biosynthetic enzyme is at least one cytokine biosynthetic enzyme and / or auxin biosynthetic enzyme.

[0010] A second aspect provides a symbiont comprising a plant cell that contains and expresses a polynucleotide encoding a plant hormone biosynthetic enzyme and a polynucleotide of interest, wherein the plant hormone biosynthetic enzyme is at least one cytokinin biosynthetic enzyme and / or auxin biosynthetic enzyme, and the plant cell of the symbiont divides autonomously. In some aspects, the plant cell comprises at least two cells.

[0011] A third aspect of the present invention provides a method for generating a symbiont-forming inoculum, comprising the step of introducing into a cell a polynucleotide encoding a plant hormone biosynthetic enzyme and a polynucleotide of interest, or introducing into a transgenic cell containing the polynucleotide of interest a polynucleotide encoding a plant hormone biosynthetic enzyme, wherein the plant hormone biosynthetic enzyme is at least one cytokinin biosynthetic enzyme and / or auxin biosynthetic enzyme, thereby generating a symbiont-forming inoculum.

[0012] A fourth aspect of the present invention is a method for producing a symbiont formation inoculum, comprising: (a) (i) introducing a polynucleotide encoding a plant hormone biosynthetic enzyme and a polynucleotide sequence of interest into / on at least one site (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) on a plant (or a portion thereof (e.g., an explant)), or transplanting / inoculating plant cells / bacterial cells containing the same (e.g., a polynucleotide encoding a plant hormone biosynthetic enzyme and a polynucleotide sequence of interest) onto at least one site on the plant (or a portion thereof); or (ii) introducing a polynucleotide encoding a plant hormone biosynthetic enzyme into / on at least one site on the plant (or a portion thereof), or transplanting plant cells / bacterial cells containing the same onto at least one site on the plant (or a portion thereof). The method includes the steps of: (ii) transplanting / inoculating the plant (or portion thereof) wherein the plant (or portion thereof) comprises a polynucleotide sequence of interest and the plant hormone biosynthetic enzyme (a) is at least one cytokinin biosynthetic enzyme and / or auxin biosynthetic enzyme, thereby generating a symbiont on the plant (or portion thereof) comprising a polynucleotide encoding the plant hormone biosynthetic enzyme and the polynucleotide sequence of interest; and (b) selecting one or more cells (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more cells) from the symbiont on the plant to provide one or more cells comprising the polynucleotide encoding the plant hormone biosynthetic enzyme and the polynucleotide sequence of interest, thereby generating a symbiont-forming inoculum.

[0013] A fifth aspect of the present invention provides a method for modifying host plant characteristics without modifying the host plant genome, the method comprising the steps of transplanting a symbiont formation inoculum of the present invention or a symbiont of the present invention onto at least one site (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more sites) on a host plant, and culturing the symbiont formation inoculum or symbiont at the at least one site on the host plant to form a symbiont at the at least one site on the host plant, wherein a polynucleotide of interest is expressed in the symbiont on the host plant, and an expression product of the polynucleotide of interest and / or a product made using the expression product of the polynucleotide of interest is transported into the host plant, thereby modifying the host plant characteristics.

[0014] A sixth aspect of the present invention provides a method for producing a biological molecule or a biologically active molecule, the method comprising the steps of providing a symbiont of the present invention in which a polynucleotide of interest encodes a biologically active molecule and collecting the biologically active molecule produced by the symbiont, and / or providing a host plant of the present invention in which a polynucleotide of interest encodes a biologically active molecule and collecting the biologically active molecule produced in the symbiont and the host plant.

[0015] A seventh aspect of the present invention provides a method for delivering a compound of interest to a host plant, the method comprising the steps of transplanting a symbiont-forming inoculum of the present invention or a symbiont of the present invention onto at least one site (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sites) on the host plant, and culturing the symbiont-forming inoculum or symbiont at least one site on the host plant to allow a symbiont to form at least one site on the host plant, wherein a polynucleotide of interest is expressed in the symbiont, and an expression product of the polynucleotide of interest and / or a product made using the expression product of the polynucleotide of interest is transported into the host plant, thereby delivering the compound of interest to the plant.

[0016] An eighth aspect of the present invention provides a method for generating a host plant comprising modified characteristic(s) without modifying the genotype of the host plant, the method comprising the steps of transplanting a symbiont formation inoculum of the present invention or a symbiont of the present invention onto at least one site (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sites) on the host plant, and culturing the symbiont formation inoculum or symbiont at the at least one site on the host plant to allow a symbiont to form at the at least one site on the host plant, wherein a polynucleotide of interest is expressed in the symbiont, and an expression product of the polynucleotide of interest and / or a product made using the expression product of the polynucleotide of interest is transported into the host plant, thereby resulting in a plant comprising a modified phenotype without modifying the genotype.

[0017] Further provided are symbiont-forming inocula, symbionts, host plants, plants and cells and / or protoplasts produced by the methods of the invention, as well as nucleic acids, expression cassettes and vectors comprising same for carrying out the methods.

[0018] These and other aspects of the invention are described in more detail in the description of the invention below. [Brief explanation of the drawings]

[0019] [Figure 1] FIG. 1 demonstrates symbiont formation using co-inoculation and single-strain inoculation (Agrobacterium), as well as a gene gun method for delivering genes encoding plant hormone production (PH) and polynucleotide of interest (POI) into plant cells. [Figure 2] FIG. 1 shows an example of a plasmid map ("pSYM") encoding at least one plant hormone polypeptide (a plant growth regulator (PGR) expression cassette) and a polynucleotide of interest (POI). AscI, XmaI, and SpeI are restriction sites, NosT is the nopaline synthase terminator, and Kan represents the kanamycin selection marker. [Figure 3]

[0023] Figure 1 is an illustration of examples of various routes for generating symbionts. DNA delivery can be performed using any method, e.g., bacteria, irradiation, electroporation, whiskers, protoplast fusion, etc. "Active tissue" is tissue immortalized with plant hormone (PH) genes, "mixed culture" is a collection of cells with a variety of different gene insertions and expression, "symbiont formation inoculum" is inoculum that can be used to form symbionts on plants (e.g., DNA, bacterial cells, plant cells, etc.), and symbiont is plant tissue (e.g., one or more plant cells) optionally located on the plant, that carries both the PH gene(s) and the polynucleotide(s) of interest (POI). [Figure 4] Figure 1 shows symbiont formation on citrus fruits 60 days after inoculation. Panels A and B show symbionts formed using co-inoculation (e.g., multiple Agrobacterium strains). Panels C and D show symbionts formed using single-strain inoculation. [Figure 5] Figure 1 shows examples of inoculation techniques using Agrobacterium species. Panel A shows the use of tweezers for Citrus plants. Panels B and C show examples of two types of needles for tomato plants: a tattoo needle (Panel B) and a hypodermic needle (Panel C). [Figure 6] Figure 1 shows examples of symbionts on various crop species. Panel A, pecan. Panel B, tomato. Panel C, citrus. Panel D, tobacco (Nicotiana benthamiana). [Figure 7] FIG. 1 shows symbiont-forming inoculum (in the form of plant callus tissue) growing on solid medium, showing high levels of mCherry production. [Figure 8] Figure 1 shows examples of various types of symbiont-forming inoculum grown on solid (panels A and B) and liquid (panels C and D) media: tomato (panels A and C) and citrus (panels B and D). [Figure 9]Figures show examples of symbiont transplants on citrus at 1 and 6 weeks (panels A and B) and on tomato at 2 and 6 weeks (panels D and E). Panels C and F illustrate vascularization (C) and green fluorescent protein (GFP) production (panel F) of the transplanted citrus symbiont. Silicone tape (panel A) or parafilm (panel D) is initially used to control humidity at the transplant site. [Figure 10] FIG. 1 shows a plasmid map of an example plasmid carrying multiple (e.g., "stacked") polynucleotides of interest (POIs) encoding a product(s) of interest. [Figure 11] Examples of symbiont stacking and POI stacking are shown. Autofluorescence (Panel A) and GFP fluorescence (Panel B) of multiple individual small GFP pSYMs on a tomato plant. Panels C-E show stacking of two pSYM plasmids with different polynucleotides of interest (POIs) on a single plant: autofluorescence (Panel C), mCherry (Panel D), and GFP (Panel E). Panels F-H show stacking of multiple polynucleotides of interest (POIs) in a single pSYM: autofluorescence (Panel F), mCherry (Panel G), and GFP (Panel H). [Figure 12] Tomato and citrus symbionts expressing high levels of green fluorescent protein (GFP). Panel A. GFP protein accumulation in the tomato symbiont is visible to the naked eye. Panels B and C show cross sections of citrus symbionts established with a single strain inoculation (Agrobacterium sp.). Arrows indicate areas of high GFP accumulation within the symbiont. [Figure 13] Figure 1 shows immunodetection of mCherry produced in symbionts formed using a single-strain inoculation (Agrobacterium spp.) on tomato using a Western blot detection method. mCherry was detectable up to a 10-7 dilution of the original protein extract. [Figure 14]Micrographs of symbionts on tomato plants containing a polynucleotide of interest encoding the mCherry florescent protein. Panel A shows UV autofluorescence of developing plant vascular tissue beginning to extend into the symbiont tissue. Panel B shows production and accumulation of Red mCherry within the symbiont and accumulation in vascular tissue that has grown into the symbiont tissue. Panel C shows developing vascular tissue within the symbiont. Panel D shows mCherry fluorescence detection in stem vascular tissue, demonstrating export of the mCherry protein outside the symbiont. [Figure 15] Figure 1 shows a cross section 1-2 cm above a GFP-expressing symbiont in a tomato stem, illustrating export of the POI product. The arrow indicates GFP accumulation. [Figure 16] Detection of mCherry in various parts of a tomato host plant with two attached symbionts, both of which contain a polynucleotide encoding mCherry protein production: Panel A: symbiont 1, Panel B: stem above symbiont 1, Panel C: symbiont 2, Panel D: stem above symbiont 2, Panel E: below symbiont 1, Panel F: below symbiont 2, Panel G: control. [Figure 17] PCR detection of polynucleotide of interest (GFP, GFP+) in the symbiont ("Sym") versus the stem of a tomato host plant, illustrating that only the symbiont is genetically transformed. "GFP+" is GFP linked to a secretory pathway targeting sequence (endoplasmic reticulum (ER) targeting sequence). [Figure 18] Expression of the citrus FLOWER LOCUS T gene (FT3) in tomato symbionts induces dwarfism of the host plant (panel A) compared with tomato plants inoculated with wild-type Agrobacterium species alone (panel B). [Figure 19]This figure shows citron plants infected with Candidatus Liberibacter asiaticus, the causative agent of citrus greening. Panels A, C, and E illustrate citron plants with symbionts that produce antimicrobial peptides. Panels B, D, and F are citron plants with wild-type Agrobacterium species as controls. [Figure 20] Figure 19 shows the percent relative reduction of Candidatus Liberibacter asiaticus (CLas) in citrus leaves expressing the antimicrobial peptide oncocin operably linked to an ER targeting sequence (oncocin+), compared to citrus inoculated with wild-type Agrobacterium sp., where 4-month-old symbionts were formed on host citrus plants by co-inoculation (see Figure 19). [Figure 21] Figure 1 is a graph illustrating the efficacy of expression of a desired product against Candidatus Liberibacter asiaticus (CLas). The percent relative reduction of CLas is shown in symbiont tissues expressing antimicrobial peptides (oncocin or TMOF) with (+) and without a signal sequence compared to tissues inoculated with wild-type Agrobacterium species. GFP+ is tissue expressing green florescent protein with a signal sequence but without the antimicrobial peptide. TMOF = trypsin-modulating oostatic factor. [Figure 22] Figure 1 shows tobacco (Nicotiana benthamiana) co-inoculated with a symbiont-forming inoculum containing a polynucleotide of interest encoding a bacterial effector protein previously shown to induce effector-triggered immunity in Nictamiana. Panel A: Healthy plant before inoculation. Panel B: One week after inoculation, the arrow below indicates the inoculation site. Panel C: Dead plant two weeks after inoculation. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention will be described hereinafter with reference to the accompanying drawings and examples, which illustrate embodiments of the invention. This description is not intended to be a detailed catalog of all the various ways in which the invention may be practiced or all the features that may be added to the invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be omitted from that embodiment. Thus, in some embodiments of the invention, the invention contemplates the elimination or omission of any feature or combination of features described herein. Furthermore, numerous modifications of and additions to the various embodiments suggested herein will be apparent to those skilled in the art in view of this disclosure and do not depart from the invention. Thus, the following description is intended to illustrate some specific embodiments of the invention, but does not exhaustively specify all permutations, combinations, and variations thereof.

[0021] As used herein, the word "exemplary" means "serving as an example, instance, or illustration." The embodiments described herein are not limiting, but rather exemplary only. It is to be understood that any described embodiment is not necessarily to be construed as preferred or advantageous over other embodiments. Furthermore, the terms "embodiments of the invention," "embodiments," or "invention" do not require that all embodiments of the invention include the described feature, advantage, or mode of operation.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0023] All publications, patent applications, patents, and other references cited herein are incorporated by reference in their entirety for the teachings relevant to the sentence and / or paragraph in which the reference is presented.

[0024] Mention of trade names or products in this publication is for specific information purposes only and does not imply recommendation or endorsement by the U.S. Department of Agriculture.

[0025] Unless the context dictates otherwise, it is specifically contemplated that the various features of the invention described herein can be used in any combination. Furthermore, in some embodiments of the invention, the invention also contemplates that any feature or combination of features described herein can be excluded or omitted. By way of example, if the specification states that a composition includes components A, B, and C, it is specifically contemplated that any one or combination of A, B, or C can be omitted and rejected, either alone or in any combination.

[0026] As used in the description of this invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the content clearly dictates otherwise.

[0027] Also, as used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted as the alternative ("or").

[0028] As used herein, the term "about," when referring to a measurable value such as an amount or concentration, is meant to encompass variations of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified value, as well as the specified value. For example, "about X," where X is a measurable value, means including X and variations of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of X. Ranges provided herein for measurable values ​​can include any other ranges and / or individual values ​​therein.

[0029] Phrases used herein such as "X to Y" and "about X to Y" should be interpreted as including X and Y. Phrases used herein such as "about X to Y" mean "about X to about Y", and phrases such as "about X to Y" mean "about X to about Y".

[0030] The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise stated herein, and each separate value is incorporated into the specification as if it were individually listed herein. For example, if the range 10 to 15 is disclosed, then 11, 12, 13, and 14 are also disclosed.

[0031] As used herein, the terms "comprise," "comprises," and "comprising" specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0032] As used herein, the transitional phrase "consisting essentially of" means that the scope of a claim should be construed to include the specified materials or steps recited in the claim and that do not materially affect the basic and novel feature(s) of the claimed invention. Thus, when used in the claims of the present invention, the term "consisting essentially of" is not intended to be construed as equivalent to "comprising."

[0033] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances in which said event or circumstance occurs and instances in which said event or circumstance does not occur. For example, the phrase "optionally comprising X" means that the composition may or may not contain X.

[0034] As used herein, the terms "increase," "increasing," "increased," "enhance," "enhanced," "enhancing," and "enhancement" (and grammatical variations thereof) describe an increase of at least about 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500%, or more compared to a control. For example, a host plant having an altered trait may exhibit increased tolerance or resistance to a pest, where the increased tolerance or resistance is from about 5% to about 500% compared to a control plant.

[0035] As used herein, the terms "reduce," "reduced," "reducing," "reduction," "diminish," and "decrease" (and grammatical variations thereof) describe, for example, a decrease of at least about 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% compared to a control. In certain embodiments, a decrease may result in no or essentially no detectable activity or amount (i.e., an insignificant amount, e.g., less than about 10%, or even less than 5%).

[0036] As used herein, the terms "express," "expresses," "expressed," or "expression," etc., with respect to nucleic acid molecules and / or nucleotide sequences (e.g., RNA or DNA), indicate that the nucleic acid molecule and / or nucleotide sequence has been transcribed and, optionally, translated. Thus, the nucleic acid molecule and / or nucleotide sequence may express a polypeptide of interest, or, for example, a functional, untranslated RNA.

[0037] A "heterologous" or "recombinant" nucleotide sequence is a nucleotide sequence that is not naturally associated with the host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring nucleotide sequence. Thus, as used herein, the term "heterologous" refers to a nucleotide / polypeptide that is derived from a foreign species, or, if derived from the same species, has been substantially altered in composition and / or genomic locus from its native form by deliberate human intervention. As an example, a heterologous polynucleotide may encode a nucleotide sequence that is native to the organism but is operably linked to a heterologous promoter, thereby providing a heterologous polynucleotide.

[0038] A "native" or "wild-type" nucleic acid, nucleotide sequence, polypeptide, or amino acid sequence refers to a naturally occurring or endogenous nucleic acid, nucleotide sequence, polypeptide, or amino acid sequence.

[0039] As used herein, the terms "nucleic acid," "nucleic acid molecule," "nucleotide sequence," and "polynucleotide" refer to RNA or DNA, whether linear or branched, single-stranded or double-stranded, or a hybrid thereof. This term also encompasses RNA / DNA hybrids. When synthetically producing dsRNA, less common bases such as inosine, 5-methylcytosine, 6-methyladenine, and hypoxanthine can also be used for antisense, dsRNA, and ribozyme pairing. For example, polynucleotides containing C-5 propyne analogs of uridine and cytidine have been shown to bind RNA with high affinity and to be potent antisense inhibitors of gene expression. Other modifications, such as modifications to the phosphodiester backbone or the 2'-hydroxyl in the ribose sugar group of RNA, can also be made.

[0040] As used herein, the term "nucleotide sequence" refers to a heteropolymer of nucleotides or the sequence of these nucleotides from the 5' to 3' end of a nucleic acid molecule, including DNA or RNA molecules, including cDNA, DNA fragments or portions, genomic DNA, synthetic (e.g., chemically synthesized) DNA, plasmid DNA, mRNA, and antisense RNA, any of which may be single-stranded or double-stranded. The terms "nucleotide sequence," "nucleic acid," "nucleic acid molecule," "nucleic acid construct," "oligonucleotide," and "polynucleotide" are also used interchangeably herein to refer to a heteropolymer of nucleotides. The nucleic acid molecules and / or nucleotide sequences provided herein are presented herein from left to right in the 5' to 3' direction and are represented using the standard code for representing nucleotide letters, as set forth in the U.S. Sequencing Rules, 37 CFR 1.821-1.825, and World Intellectual Property Organization (WIPO) Standard ST.25. As used herein, the term "5' region" can refer to the region of a polynucleotide closest to the 5' end of a polynucleotide. Thus, for example, an element in the 5' region of a polynucleotide can be located anywhere from the first nucleotide at the 5' end of the polynucleotide to a nucleotide located in the middle of the polynucleotide. As used herein, the term "3' region" can refer to the region of the polynucleotide closest to the 3' end of the polynucleotide. Thus, for example, an element in the 3' region of a polynucleotide can be located anywhere from the first nucleotide at the 3' end of the polynucleotide to a nucleotide located in the middle of the polynucleotide.

[0041] As used herein, the term "fragment" or "portion" with respect to a nucleic acid refers to a fragment that is reduced in length (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 400, 450, 500, 550, 600, 650, 700, 750, 80 "Nucleic acid fragment" refers to a nucleic acid comprising, consisting essentially of, and / or consisting of a contiguous nucleotide sequence of nucleotides that is identical or nearly identical (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical) to a corresponding portion of a reference nucleic acid. Such nucleic acid fragments may, where appropriate, be included within larger constituent polynucleotides.

[0042] The terms "fragment" or "portion," as used herein with respect to a polypeptide, may refer to a polypeptide that is reduced in length relative to a reference polypeptide and that comprises, consists essentially of, and / or consists of an amino acid sequence of consecutive amino acids that is identical or nearly identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical) to the corresponding portion of the reference polypeptide. Such polypeptide fragments may, where appropriate, be included within a constituent larger polypeptide. In some embodiments, a polypeptide fragment comprises, consists essentially of, or consists of at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 260, 270, 280, 290, or more consecutive amino acids of a reference polypeptide. The present invention will now be described with reference to the following examples. It should be understood that these examples are not intended to limit the scope of the claimed invention, but rather are intended to be illustrative of particular embodiments. Any variations of the exemplified methods that occur to those skilled in the art are intended to be within the scope of the present invention.

[0043] The term "functional fragment," as used herein with respect to nucleic acids, refers to a nucleic acid that encodes a functional fragment of a polypeptide.

[0044] As used herein, the term "gene" refers to a nucleic acid molecule that can be used to produce mRNA, antisense RNA, miRNA, anti-microRNA antisense oligodeoxyribonucleotide (AMO), etc. A gene may or may not be used to produce a functional protein or gene product. A gene can include both coding and non-coding regions (e.g., introns, regulatory elements, promoters, enhancers, termination sequences, and / or 5' and 3' untranslated regions). A gene may be "isolated," which means a nucleic acid that is substantially or essentially free from components normally found associated with the nucleic acid in its natural state. Such components include other cellular material, culture medium from recombinant production, and / or various chemicals used in the chemical synthesis of nucleic acids.

[0045] The term "mutation" refers to point mutations (e.g., missense or nonsense, or single base pair insertion or deletion resulting in a frameshift), insertion, deletion, and / or truncation. When a mutation is a substitution of a residue in an amino acid sequence with another residue, or a deletion or insertion of one or more residues in the sequence, the mutation is typically described by identifying the original residue, followed by the position of the residue in the sequence, and then the identity of the newly substituted residue. Truncation can include truncation at the C-terminus of the polypeptide or the N-terminus of the polypeptide. Polypeptide truncation can be the result of deletion of the corresponding 5' or 3' end of the gene encoding the polypeptide. Frameshift mutations can occur when one or more base pair deletions or insertions are introduced into a gene. Frameshift mutations in a gene can result in the production of a polypeptide that is longer, shorter, or the same length as the wild-type polypeptide, depending on when the first stop codon occurs after the mutated region of the gene. Deletions can also cause mutations in non-coding portions of the gene, such as promoters.

[0046] As used herein, the term "complementary" or "complementarity" refers to the natural binding of polynucleotides by base pairing under permissive salt and temperature conditions. For example, the sequence "AGT" (5' to 3') binds to the complementary sequence "TCA" (3' to 5'). Complementarity between two single-stranded molecules can be "partial," where only a portion of the nucleotides bind, or complete, where complete complementarity exists between the single-stranded molecules. The degree of complementarity between nucleic acid strands has a significant impact on the efficiency and strength of hybridization between nucleic acid strands.

[0047] As used herein, "complementary" can mean 100% complementarity to the comparison nucleotide sequence, or it can mean less than 100% complementarity to the comparison nucleotide sequence (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, etc. complementarity).

[0048] Various nucleic acids or proteins that share homology are referred to herein as "homologues." The term homologue includes homologous sequences from the same species and other species, as well as orthologous sequences from the same species and other species. "Homology" refers to the level of similarity between two or more nucleic acid and / or amino acid sequences in terms of percent positional identity (i.e., sequence similarity or identity). Homology also refers to the concept of similar functional properties between various nucleic acids or proteins. Thus, the compositions and methods of the present invention further include homologues to the nucleotide and polypeptide sequences of the present invention. As used herein, "orthologous" refers to homologous nucleotide and / or amino acid sequences in different species that arose during speciation from a common ancestral gene. Homologues of the nucleotide sequences of the present invention have substantial sequence identity (e.g., at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) to said nucleotide sequences of the present invention.

[0049] As used herein, "sequence identity" refers to the degree to which two optimally aligned polynucleotide or polypeptide sequences are consistent across the entire alignment window of components, such as nucleotides or amino acids. "Identity" can be easily calculated by known methods, including but not limited to those described in Computational Molecular Biology (Lesk, AM, ed.), Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, ed.), Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, AM and Griffin, HG, eds.), Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.), Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.), Stockton Press, New York (1991).

[0050] As used herein, the term "percent sequence identity" or "percent identity" refers to the percentage of identical nucleotides in a linear polynucleotide sequence of a reference ("query") polynucleotide molecule (or its complementary strand) compared to a test ("subject") polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned. In some embodiments, "percent sequence identity" can refer to the percentage of identical amino acids in an amino acid sequence compared to a reference polypeptide.

[0051] The phrases "substantially identical" or "substantial identity," as used herein in the context of two nucleic acid molecules, nucleotide sequences, or polypeptide sequences, refer to two or more sequences or subsequences that have at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% nucleotide or amino acid residue identity when compared and aligned for maximum correspondence as measured using one of the following sequence comparison algorithms or by visual inspection. In some embodiments of the invention, substantial identity exists over a region of contiguous nucleotides of a nucleotide sequence of the invention that is from about 10 nucleotides to about 20 nucleotides, from about 10 nucleotides to about 25 nucleotides, from about 10 nucleotides to about 30 nucleotides, from about 15 nucleotides to about 25 nucleotides, from about 30 nucleotides to about 40 nucleotides, from about 50 nucleotides to about 60 nucleotides, from about 70 nucleotides to about 80 nucleotides, from about 90 nucleotides to about 100 nucleotides, from about 100 nucleotides to about 200 nucleotides, from about 100 nucleotides to about 300 nucleotides, from about 100 nucleotides to about 400 nucleotides, from about 100 nucleotides to about 500 nucleotides, from about 100 nucleotides to about 600 nucleotides, from about 100 nucleotides to about 800 nucleotides, from about 100 nucleotides to about 900 nucleotides, or more, or any range therein up to the full length of the sequence.

[0052] In sequence comparison, typically, one sequence serves as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence(s) relative to the reference sequence based on the designated program parameters.

[0053] Optimal alignment of sequences for aligning a comparison window is well known to those skilled in the art and can be performed by tools such as Smith and Waterman's local homology algorithm, Needleman and Wunsch's homology alignment algorithm, Pearson and Lipman's similarity search method, and optionally by computer implementations of these algorithms, such as GAP, BESTFIT, FASTA, and TFASTA, available as part of the GCG® Wisconsin Package® (Accelrys Inc., San Diego, CA). The "fractional identity" of the aligned segments of a test sequence and a reference sequence is the number of identical components shared by the two aligned sequences divided by the total number of components in the reference sequence segment, for example, the entire reference sequence or a smaller, defined portion of the reference sequence. Percent sequence identity is expressed as the fractional identity multiplied by 100. Comparison of one or more polynucleotide sequences can be to a full-length polynucleotide sequence or a portion thereof, or to a longer polynucleotide sequence. For purposes of the present invention, "percent identity" may also be determined using BLASTX version 2.0 for translated nucleotide sequences and BLASTN version 2.0 for polynucleotide sequences.

[0054] Two nucleotide sequences can also be considered to be substantially complementary if the two sequences hybridize to each other under stringent conditions. In some embodiments, two nucleotide sequences are considered to be substantially complementary if they hybridize to each other under highly stringent conditions.

[0055] "Stringent hybridization conditions" and "stringent hybridization wash conditions" in the context of nucleic acid hybridization experiments such as Southern and Northern hybridization are sequence-dependent and vary under various environmental parameters. An extensive guide to nucleic acid hybridization can be found in Tijssen Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes part I chapter 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays," Elsevier, New York (1993). In general, highly stringent hybridization and wash conditions are those that, for a particular sequence, achieve a thermal melting point (T m ) is chosen to be approximately 5°C lower than

[0056] T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are defined as the temperature T mThe hybridization conditions are chosen to be equal to . An example of stringent hybridization conditions for hybridization of complementary nucleotide sequences with more than 100 complementary residues on a filter in a Southern or Northern blot is 50% formamide with 1 mg heparin at 42°C, with hybridization carried out overnight. An example of highly stringent wash conditions is 0.15 M NaCl at 72°C for approximately 15 minutes. An example of stringent wash conditions is a 0.2x SSC wash at 65°C for 15 minutes (see Sambrook, supra, for a description of SSC buffer). Often, a low stringency wash precedes a high stringency wash to remove background probe signal. For example, an example of a medium stringency wash for a duplex of more than 100 nucleotides is 1x SSC at 45°C for 15 minutes. An example of low stringency washing for duplexes of, for example, more than 100 nucleotides is 4-6×SSC at 40°C for 15 minutes. For short probes (e.g., about 10-50 nucleotides), stringent conditions typically include a salt concentration of less than about 1.0 M Na ion, typically about 0.01-1.0 M Na ion (or other salt), pH 7.0-8.3, and a temperature typically of at least about 30°C. Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. Generally, a signal-to-noise ratio of 2-fold (or higher) than that observed for an unrelated probe in a particular hybridization assay indicates detection of specific hybridization. Nucleotide sequences that do not hybridize to each other under stringent conditions are still substantially identical if the proteins they encode are substantially identical. This can occur, for example, when copies of nucleotide sequences are generated using the maximum codon degeneracy permitted by the genetic code.

[0057] Any polynucleotide and / or recombinant nucleic acid molecule of the present invention can be codon-optimized for expression in any species of interest. Codon optimization is well known in the art and involves modifying nucleotide sequences for codon usage bias using a species-specific codon usage table. The codon usage table is generated based on sequence analysis of the most highly expressed genes for the species of interest. If the nucleotide sequence is expressed in the nucleus, the codon usage table is generated based on sequence analysis of the highly expressed nuclear genes for the species of interest. The modification of the nucleotide sequence is determined by comparing the species-specific codon usage table with the codons present in the native polynucleotide sequence. As is understood in the art, codon optimization of a nucleotide sequence results in a nucleotide sequence that has less than 100% identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, etc.) to a native nucleotide sequence, but still encodes a polypeptide having the same function as that encoded by the original native nucleotide sequence. Thus, in some embodiments of the present invention, polynucleotides of interest and / or polynucleotides encoding plant hormone biosynthetic enzymes and / or nucleic acid constructs comprising the same can be codon optimized for expression in a particular species of interest.

[0058] In some embodiments, recombinant nucleic acid molecules, nucleotide sequences, and polypeptides of the invention are "isolated." An "isolated" nucleic acid molecule, "isolated" nucleotide sequence, or "isolated" polypeptide is a nucleic acid molecule, nucleotide sequence, or polypeptide that exists apart from its native environment by the hand of man and is therefore not a product of nature. An isolated nucleic acid molecule, nucleotide sequence, or polypeptide may exist in a purified form, at least partially separated from at least some of the other components of a naturally occurring organism or virus, e.g., cellular or viral structural components, or other polypeptides or nucleic acids that are typically found in association with the polynucleotide. In some embodiments, an isolated nucleic acid molecule, isolated nucleotide sequence, and / or isolated polypeptide is at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more pure.

[0059] In some embodiments, an isolated nucleic acid molecule, nucleotide sequence, or polypeptide can exist in a non-native environment, such as, for example, a recombinant host cell. Thus, for example, with respect to a nucleotide sequence, the term "isolated" means that it is separated from the chromosome and / or cell in which it naturally occurs. A polynucleotide is also isolated if it is separated from the chromosome and / or cell in which it naturally occurs and then inserted into a non-naturally occurring genetic context, chromosome, and / or cell (e.g., a different host cell, different regulatory sequences, and / or a different genomic location than that in which it is found in nature). Thus, although recombinant nucleic acid constructs, polynucleotides, and the polypeptides they encode are "isolated" in that they exist away from their native environment by the hand of man and are therefore not products of nature, in some embodiments they can be introduced into and exist in a recombinant host cell.

[0060] In any of the embodiments described herein, the polynucleotides or nucleic acid constructs of the invention may be operably associated with various promoters and / or other regulatory elements for expression in plants and / or plant cells. Thus, in some embodiments, the polynucleotides or nucleic acid constructs of the invention may further comprise one or more promoters, introns, enhancers, and / or terminators operably linked to one or more nucleotide sequences.

[0061] As used herein with reference to a polynucleotide, "operably linked" or "operably associated" means that the indicated elements are functionally related and, generally, physically related to one another. Thus, as used herein, the terms "operably linked" or "operably associated" refer to nucleotide sequences on a single nucleic acid molecule that are functionally related. Thus, a first nucleotide sequence operably linked to a second nucleotide sequence refers to a situation in which the first nucleotide sequence is placed in a functional relationship with the second nucleotide sequence. For example, a promoter is operably associated with a nucleotide sequence if it effects the transcription or expression of the nucleotide sequence. Those skilled in the art will understand that a control sequence (e.g., a promoter) need not necessarily be contiguous with the nucleotide sequence to which it is operably associated, so long as it functions to direct the expression of the nucleotide sequence. Thus, for example, an intervening untranslated but transcribed nucleic acid sequence can be present between the promoter and the nucleotide sequence, and the promoter can still be considered "operably linked" to the nucleotide sequence.

[0062] As used herein, the term "linked" with reference to a polypeptide refers to the attachment of one polypeptide to another. A polypeptide may be linked to another polypeptide directly (e.g., via a peptide bond) or through a linker (at the N-terminus and / or C-terminus). As an example, a polypeptide may be linked to a targeting sequence, optionally at the N-terminus or C-terminus, or both. As used herein, a "linker" may refer to a chemical group or molecule that links two molecules or moieties.

[0063] A "promoter" is a nucleotide sequence that controls or regulates the transcription of a nucleotide sequence (e.g., a coding sequence) operably associated with the promoter. The coding sequence controlled or regulated by a promoter may encode a polypeptide and / or functional RNA. Typically, a "promoter" refers to a nucleotide sequence that contains a binding site for RNA polymerase II and directs the initiation of transcription. In general, promoters are found 5' or upstream to the start of the coding region of the corresponding coding sequence. A promoter may contain other elements that act as regulators of gene expression, such as a promoter region. These include a TATA box consensus sequence and often a CAAT box consensus sequence (Breathnach and Chambon, (1981) Ann. Rev. Biochem. 50:349). In plants, the CAAT box may be replaced by an AGGA box (Messing et al. (1983) in Genetic Engineering of Plants, T. Kosuge, C. Meredith, and A. Hollaender (eds.), Plenum Press, pp. 211-227).

[0064] Promoters useful in the present invention can include, for example, constitutive, inducible, temporally-regulated, developmentally-regulated, and chemically-regulated promoters for use in preparing recombinant nucleic acid molecules, such as "synthetic nucleic acid constructs" or "protein-RNA complexes." These various types of promoters are known in the art.

[0065] The choice of promoter may vary depending on the time and space requirements for expression, and may also vary based on the host cell to be transformed.The promoters for many different organisms are well known in the art.Based on the extensive knowledge existing in the art, suitable promoters can be selected for specific target host organisms.Therefore, for example, much is known about the promoters upstream of the highly constitutively expressed genes in model organisms, and such knowledge can be easily accessed and implemented in other systems as needed.

[0066] In some embodiments, promoters functional in plants can be used in the constructs of the present invention. Non-limiting examples of promoters useful for driving expression in plants include the promoter of RubisCo small subunit gene 1 (PrbcS1), the promoter of actin gene (Pactin), the promoter of nitrate reductase gene (Pnr), and the promoter of duplicated carbonic anhydrase gene 1 (Pdca1) (see Walker et al., Plant Cell Rep., 23:727-735 (2005); Li et al., Gene, 403:132-142 (2007); Li et al., Mol Biol. Rep., 37:1143-1154 (2010)). PrbcS1 and Pactin are constitutive promoters, while Pnr and Pdca1 are inducible promoters. Pnr is induced by nitrate and repressed by ammonium (Li et al., Gene, 403:132-142 (2007)), and Pdca1 is induced by salt (Li et al., Mol. Biol. Rep., 37:1143-1154 (2010)). In some embodiments, promoters useful in the present invention are RNA polymerase II (Pol II) promoters. In some embodiments, the U6 promoter or 7SL promoter from maize (Zea mays) may be useful in the constructs of the present invention. In some embodiments, the U6c promoter and / or 7SL promoter from maize may be useful for driving expression of a guide nucleic acid. In some embodiments, the U6c promoter, U6i promoter, and / or 7SL promoter from soybean (Glycine max) may be useful in the constructs of the present invention. In some embodiments, the U6c promoter, U6i promoter, and / or 7SL promoter from soybean may be useful for driving expression of a guide nucleic acid.

[0067] Examples of constitutive promoters useful in plants include, but are not limited to, the Cestrum virus promoter (cmp) (U.S. Pat. No. 7,166,770), the rice actin 1 promoter (Wang et al. (1992) Mol. Cell. Biol., 12:3399-3406 and U.S. Pat. No. 5,641,876), the CaMV 35S promoter (Odell et al. (1985) Nature, 313:810-812), the CaMV 19S promoter (Lawton et al. (1987) Plant Mol. Biol., 9:315-324), the nos promoter (Ebert et al. (1987) Proc. Natl. Acad. Sci. USA, 84:5745-5749), the Adh promoter (Walker et al. (1987) Proc. Natl. Acad. Sci. USA, 84:6624-6629), the sucrose synthase promoter (Yang and Russell (1990) Proc. Natl. Acad. Sci. USA, 87:4144-4148), and the ubiquitin promoter. Constitutive promoters derived from ubiquitin accumulate in many cell types. Ubiquitin promoters have been cloned from several plant species for use in transgenic plants, such as sunflower (Binet et al., 1991, Plant Science, 79:87-94), maize (Christensen et al., 1989, Plant Molec. Biol., 12:619-632), and Arabidopsis (Norris et al., 1993, Plant Molec. Biol., 21:895-906). The maize ubiquitin promoter (UbiP) has been developed in transgenic monocotyledonous plant systems, and its sequence and vectors constructed for the transformation of monocotyledonous plants are disclosed in Patent Publication EP 0 342 926. The ubiquitin promoter is suitable for expressing the nucleotide sequences of the present invention in transgenic plants, particularly monocotyledonous plants.Additionally, promoter expression cassettes have been described by McElroy et al. (Mol. Gen. Genet. 231:150-160 (1991)) that can be readily modified for expression of the nucleotide sequences of the present invention and are particularly suitable for use in monocotyledonous hosts.

[0068] Additionally, promoters functional in chloroplasts can be used. Non-limiting examples of such promoters include the bacteriophage T3 gene 9 5'UTR and other promoters disclosed in U.S. Patent No. 7,579,516. Other promoters useful in the present invention include, but are not limited to, the S-E9 small subunit RuBP carboxylase promoter and the Kunitz trypsin inhibitor gene promoter (Kti3).

[0069] Additional regulatory elements useful in the present invention include, but are not limited to, introns, enhancers, termination sequences, and / or 5' and 3' untranslated regions.

[0070] Introns useful in the present invention can be introns identified in plants, isolated from them, and then inserted into an expression cassette for use in plant transformation. As will be understood by those skilled in the art, introns can contain sequences necessary for self-excision and are incorporated in-frame into the nucleic acid construct / expression cassette. Introns can be used as spacers to separate multiple protein-coding sequences in a nucleic acid construct, or introns can be used within a single protein-coding sequence, for example, to stabilize mRNA. When used within a protein-coding sequence, they are inserted "in-frame" with an excision site. Introns can also be associated with a promoter to improve or modify expression.

[0071] Non-limiting examples of introns useful in the present invention include introns from the ADHI gene (e.g., Adh1-S introns 1, 2, and 6), the ubiquitin gene (Ubi1), the RuBisCO small subunit (rbcS) gene, the RuBisCO large subunit (rbcL) gene, the actin gene (e.g., the actin-1 intron), the pyruvate dehydrogenase kinase gene (pdk), the nitrate reductase gene (nr), the duplicated carbonic anhydrase gene 1 (Tdca1), the psbA gene, the atpA gene, or any combination thereof.

[0072] In some embodiments, polynucleotides and / or nucleic acid constructs of the present invention can be "expression cassettes" or can be contained within an expression cassette. Expression cassettes and / or vectors can contain one or more polynucleotides and / or nucleic acid constructs of the present invention. When multiple polynucleotides and / or nucleic acid constructs are contained in an expression cassette or vector, the multiple polynucleotides and / or nucleic acid constructs can be considered "stacked" in the expression cassette / nucleic acid construct. In some embodiments, a host plant can be attached with multiple symbionts that deliver expression products of the expression cassette(s) to the host plant in any combination, and can also be considered "stacked." These can include using expression cassettes with one or more polynucleotides and / or nucleic acid constructs used to produce one or more expression products in the host plant in any combination of stacked configuration(s).

[0073] As used herein, "expression cassette" refers to a recombinant nucleic acid molecule (e.g., a nucleic acid construct of the invention (e.g., a synthetic tracr nucleic acid construct, a synthetic CRISPR nucleic acid construct, a synthetic CRISPR array, a chimeric nucleic acid construct, a nucleotide sequence encoding a polypeptide of interest, a nucleotide sequence encoding a cas9 nuclease)) comprising a nucleotide sequence of interest, said nucleotide sequence being operably associated with at least a regulatory sequence (e.g., a promoter). Accordingly, some aspects of the invention provide expression cassettes designed to express the nucleotide sequences of the invention.

[0074] An expression cassette containing a nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous to at least one of its other components. The expression cassette may be naturally occurring but obtained in a recombinant form useful for heterologous expression.

[0075] The expression cassette can also optionally contain a transcriptional and / or translational termination region (i.e., termination region) functional in the selected host cell. A variety of transcription terminators are available for use in the expression cassette and are responsible for terminating transcription across the heterologous nucleotide sequence of interest and for correct mRNA polyadenylation. The termination region can be native to the transcriptional initiation region, native to the operably linked nucleotide sequence of interest, native to the host cell, or derived from another source (i.e., foreign or heterologous to the promoter, nucleotide sequence of interest, host, or any combination thereof).

[0076] The expression cassette can also include a nucleotide sequence for a selectable marker that can be used to select transformed host cells. As used herein, a "selectable marker" refers to a nucleotide sequence that, when expressed, confers a distinct phenotype on host cells expressing the marker, thus allowing such transformed cells to be distinguished from those that do not possess the marker. Such a nucleotide sequence may encode either a selectable or a screenable marker, depending on whether the marker confers a trait that can be selected for by chemical means, such as by using a selection agent (e.g., an antibiotic), or whether the marker is simply a trait that can be identified through observation or testing, such as by screening (e.g., fluorescence). Of course, numerous examples of suitable selectable markers are known in the art and can be used in the expression cassettes described herein.

[0077] In addition to expression cassettes, the nucleic acid molecules and nucleotide sequences described herein can be used in connection with vectors. The term "vector" refers to a composition for transferring, delivering, or introducing a nucleic acid (or multiple nucleic acids) into a cell. A vector includes a nucleic acid molecule that contains the nucleotide sequence(s) to be transferred, delivered, or introduced. Vectors for use in transforming host organisms are well known in the art. Non-limiting examples of general classes of vectors include, but are not limited to, viral vectors, plasmid vectors, phage vectors, phagemid vectors, cosmid vectors, fosmid vectors, bacteriophages, artificial chromosomes, or binary vectors of Agrobacterium species, which may be double-stranded or single-stranded, linear or circular, and may or may not be self-infecting or mobilizable. Vectors, as defined herein, can transform prokaryotic or eukaryotic hosts either by integration into the cellular genome or by being extrachromosomal (e.g., an autonomously replicating plasmid with an origin of replication). Also included is a shuttle vector, which refers to a DNA vehicle capable of replicating, naturally or by design, in two different host organisms, which may be selected from actinomycetes and related species, bacteria, and eukaryotes (e.g., cells of higher plants, mammals, yeast, or fungi). In some representative embodiments, the nucleic acid in the vector is under the control of, and operably linked to, an appropriate promoter or other regulatory element for transcription in the host cell. The vector may be a bifunctional expression vector that functions in multiple hosts. In the case of genomic DNA, it may contain its own promoter or other regulatory element, and in the case of cDNA, it may be under the control of an appropriate promoter or other regulatory element for expression in the host cell. Thus, the nucleic acid molecules and / or expression cassettes of the present invention can be contained in a vector as described herein and known in the art.

[0078] As used herein, "modifying" or "modification" and grammatical variations thereof with reference to a host plant means a change in at least one host plant characteristic without a concomitant change in the host plant genome or genotype.

[0079] As used herein, the terms "inoculate," "inoculating," "inoculated," and grammatical variations thereof refer to the act of contacting a biological entity (i.e., a plant) with a bioactive composition (e.g., a symbiont-forming inoculum). The bioactive composition may be referred to as an inoculum (e.g., a symbiont-forming inoculum).

[0080] As used herein, "contact," "contacting," "contacted," and grammatical variations thereof refer to bringing together the components of a desired reaction under conditions suitable for carrying out the desired reaction (e.g., inoculation, introduction, transformation, transfection, transplantation, etc.).

[0081] In the context of a polynucleotide (e.g., a polynucleotide encoding a plant hormone biosynthetic gene, a polynucleotide of interest), "introducing," "introduce," or "introduced" (and grammatical variations thereof) means presenting the polynucleotide to a host organism or a cell of said organism (e.g., a host cell) in a manner such that the polynucleotide is accessible to the interior of the cell. When multiple polynucleotides are introduced, these polynucleotides can be assembled as part of a single polynucleotide or nucleic acid construct or as separate polynucleotides or nucleic acid constructs, which can be located on the same or different expression constructs or transformation vectors. Thus, these polynucleotides can be introduced into a cell in a single transformation event or in separate transformation / transfection events, or, for example, they can be incorporated into an organism by conventional breeding protocols. Thus, in some embodiments of the present invention, one or more polynucleotides or nucleic acid constructs of the present invention (e.g., a polynucleotide encoding a plant hormone biosynthetic enzyme and / or a polynucleotide of interest) can be introduced into bacterial or plant cells for use as a symbiont formation inoculum to generate a symbiont.

[0082] As used herein, the terms "transplant," "transplanting," or "transplantation" (and grammatical variations thereof) refer to the process of inserting at least one plant cell (e.g., 1, 2, 3, 4, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 1000, 5000, 10,000, 100,000, or more cells) containing one or more polynucleotides encoding at least one plant hormone biosynthetic enzyme and one or more polynucleotides of interest into / onto at least one site on a host plant.

[0083] As used herein, the terms "transformation" or "transfection" refer to the introduction of heterologous nucleic acid into a cell. Cellular transformation can be stable or transient. Thus, in some embodiments, host cells or host organisms are stably transformed with nucleic acid molecules of the present invention. In other embodiments, host cells or host organisms are transiently transformed with recombinant nucleic acid molecules of the present invention.

[0084] "Transient transformation" in the context of polynucleotides means introducing a polynucleotide into a cell without integrating it into the genome of the cell.

[0085] By "stably introducing" or "stably introduced" in the context of a polynucleotide introduced into a cell is intended that the introduced polynucleotide is stably integrated into the genome of the cell, and thus the cell is stably transformed with the polynucleotide.

[0086] As used herein, "stable transformation" or "stably transformed" refers to the introduction of a nucleic acid molecule into a cell and its integration into the cell's genome. The integrated nucleic acid molecule can therefore be inherited by its progeny, more particularly by progeny in multiple successive generations. As used herein, "genome" also includes nuclear and plastid genomes, and thus includes the integration of a nucleic acid into, for example, the genome of a chloroplast or mitochondrion. As used herein, stable transformation can also refer to a transgene that is maintained extrachromosomally, for example, as a minichromosome or plasmid.

[0087] Transient transformation can be detected, for example, by enzyme-linked immunosorbent assay (ELISA) or Western blot, or mass spectrometry, which can detect the presence of peptides or polypeptides encoded by one or more transgenes introduced into an organism. Stable transformation of a cell can be detected, for example, by Southern blot hybridization assay of the cell's genomic DNA using a nucleic acid sequence that specifically hybridizes with the nucleotide sequence of the transgene introduced into an organism (e.g., plant, mammal, insect, archaea, bacteria, etc.). Stable transformation of a cell can be detected, for example, by Northern blot hybridization assay of the cell's RNA using a nucleic acid sequence that specifically hybridizes with the nucleotide sequence of the transgene introduced into a plant or other organism. Stable transformation of a cell can also be detected, for example, by polymerase chain reaction (PCR) or other amplification reactions known in the art using specific primer sequences that hybridize with the target sequence(s) of the transgene, resulting in amplification of the transgene sequence, which can be detected according to standard methods. Transformation can also be detected by direct sequencing and / or hybridization protocols well known in the art.

[0088] As described herein, the polynucleotides, nucleic acid constructs, and expression cassettes of the present invention are stably integrated into the genome of the cells of the symbiont or symbiont-forming inoculum.

[0089] The recombinant nucleic acid molecules / polynucleotides of the present invention can be introduced into cells by any method known to those skilled in the art. The methods of the present invention do not depend on a particular method for introducing one or more nucleotide sequences into an organism, but only on them gaining access to the interior of at least one cell of the organism.

[0090] In some embodiments of the invention, the transformation of the cell comprises nuclear transformation, while in other embodiments, the transformation of the cell comprises plastid transformation (e.g., chloroplast transformation).

[0091] Procedures for transforming both prokaryotic and eukaryotic organisms, including plant and bacterial cells, are well known and routine in the art and are described throughout the literature (see, e.g., Jiang et al., 2013. Nat. Biotechnol., 31:233-239; Ran et al., Nature Protocols, 8:2281-2308 (2013)). Non-limiting examples of transformation methods include bacterial-mediated nucleic acid delivery (e.g., via Agrobacterium), viral-mediated nucleic acid delivery, silicon carbide or nucleic acid whisker-mediated nucleic acid delivery, liposome-mediated nucleic acid delivery, microinjection, microparticle bombardment, calcium phosphate-mediated transformation, cyclodextrin-mediated transformation, electroporation, nanoparticle-mediated transformation, sonication, infiltration, PEG-mediated nucleic acid uptake, and transformation via any other electrical, chemical, physical (mechanical), and / or biological mechanism that results in the introduction of nucleic acid into plant cells, including any combination thereof. General guides to various plant transformation methods known in the art include Miki et al. ("Procedures for Introducing Foreign DNA into Plants," Methods in Plant Molecular Biology and Biotechnology, Glick, B.R. and Thompson, J.E., eds., (CRC Press, Inc., Boca Raton, 1993), pp. 67-88) and Rakowoczy-Trojanowska (Cell. Mol. Biol. Lett. 7:849-858 (2002)). A general guide to yeast transformation is Guthrie and Fink (1991) (Guide to yeast genetics and molecular biology, Methods in Enzymology, (Academic Press, San Diego) 194:1-932), and a guide to methods for bacterial transformation is Aune and Aachmann (Appl. Microbiol. Biotechnol. 85:1301-1313 (2010)).

[0092] When multiple polynucleotides are introduced, they can be assembled as part of a single nucleic acid construct or as separate nucleic acid constructs, and can be located on the same or different nucleic acid constructs. Thus, the nucleotide sequences can be introduced into the target cells in a single transformation event or in separate transformation events, or, where relevant, the nucleotide sequences can be incorporated into the plant as part of a breeding protocol.

[0093] As used herein, the term "T-DNA" refers to transfer DNA, a DNA segment in Agrobacterium species known in the art as being transferred into the genome of (transformed into) a plant infected by Agrobacterium.

[0094] As used herein, the term "single-strain inoculation" refers to the inoculation of plant cells with a single bacterial strain, wherein the polynucleotide encoding a plant hormone biosynthetic enzyme and at least one polynucleotide of interest desired for transforming the plant cell are present in the single bacterial strain.

[0095] As used herein, the term "co-inoculation" refers to the inoculation of plant cells with at least two bacterial strains, one strain carrying a polynucleotide encoding a plant hormone biosynthetic enzyme and another strain carrying at least one polynucleotide of interest necessary to transform the plant cell.

[0096] As used herein, "symbiont formation inoculum" refers to a composition that can be inoculated into a host plant and used to generate the symbionts described herein. In some embodiments, a "symbiont formation inoculum" can include a nucleic acid construct comprising a polynucleotide encoding a plant hormone biosynthetic enzyme described herein and a polynucleotide of interest described herein. In some embodiments, a "symbiont formation inoculum" can include cells (e.g., bacterial cells or plant cells) comprising a polynucleotide encoding a plant hormone biosynthetic enzyme and a polynucleotide of interest. In some embodiments, a "symbiont formation inoculum" can be harvested from a symbiont, and can be a single cell or multiple cells of the symbiont (e.g., a portion of the symbiont, e.g., about 0.005 micrograms to about 1 gram of the symbiont, e.g., about 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 3 0, 35, 40, 45, 50, 100, 200, 300, 400, 500, 1000, 2000, 3000, 4000, 5000 micrograms to about 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 milligrams, or any range or value therein.

[0097] A "polynucleotide encoding a plant hormone biosynthetic enzyme" refers to one or more polynucleotides (e.g., 1, 2, 3, 4, 5, or more) that encode one or more plant hormone biosynthetic enzymes (e.g., 1, 2, 3, 4, 5, or more), where the one or more plant hormone biosynthetic enzymes may be any of the cytokinin biosynthetic enzymes and / or auxin biosynthetic enzymes described herein. In some embodiments, the plant hormone biosynthetic enzyme or the polynucleotide encoding it can be derived from a bacterial species, e.g., a bacterial auxin biosynthetic enzyme or a bacterial cytokinin biosynthetic enzyme (e.g., Agrobacterium species (e.g., A. tumefaciens, A. fabrum, A. rhizogenes, A. vitis), Rhizobium species (e.g., R. tumefaciens, R. skierniewicense, R. lusitanum), or Pseudomonas savastanoi). In some embodiments, the plant hormone biosynthetic enzyme or polynucleotide encoding it can be derived from a plant species, such as a plant auxin biosynthetic enzyme or a plant cytokinin biosynthetic enzyme (e.g., rice (Oryza sativa), maize, Arabidopsis thaliana)). In some embodiments, the plant hormone biosynthetic enzyme or polynucleotide encoding it can be derived from an insect species or can be a plant hormone analog. Examples of polynucleotides encoding plant hormone biosynthetic enzymes include, but are not limited to, any one of the nucleotide sequences of SEQ ID NOs: 1, 3, 5, or 21, or a nucleotide sequence having at least about 80% identity thereto (e.g., 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity).In some embodiments, polynucleotides encoding plant hormone biosynthetic enzymes useful in the present invention encode any one of the amino acid sequences of SEQ ID NOs: 2, 4, 6-20, 22, or 23, or an amino acid sequence having at least about 80% identity thereto (e.g., 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity). Examples of plant hormone biosynthetic polypeptides useful in the present invention include, but are not limited to, any one of the amino acid sequences of SEQ ID NOs: 2, 4, 6-20, 22, or 23, or an amino acid sequence having at least about 80% identity thereto (e.g., 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity). In some embodiments, the plant hormone biosynthetic enzyme is an auxin biosynthetic enzyme. Auxin biosynthetic enzymes useful in the present invention include, but are not limited to, indole-3-acetamide hydrolase (e.g., iaaH, TMS2, AUX2) (EC number: EC 3.5.1.4), amidase 1 (e.g., AtAMI1) (EC 3.5.1.4), tryptophan 2-monooxygenase (e.g., iaaM, TMS1, AUX1) (EC 1.13.12.3), indole-3-lactate synthase (EC 1.1.1.110), L-tryptophan-pyruvate aminotransferase 1 (e.g., In some embodiments, the plant hormone biosynthetic enzyme may include tryptophan aminotransferase-related protein 1 (e.g., TAA1, TIR2, CKRC1, SAV3, WEI8) (EC 2.6.1.99), tryptophan aminotransferase-related protein 1 (e.g., TAR1) (EC 2.6.1.27), indole-3-acetaldehyde oxidase (e.g., IAA oxidase, AO1, Ao-1, AtAO-1, ​​ZmAO1, NtAO1, AtAO1) (EC 1.2.3.7), and / or tryptophan decarboxylase 1 (TDC1) / tryptophan decarboxylase 2 (TDC2) (EC 4.1.1.105). In some embodiments, the plant hormone biosynthetic enzyme is a cytokinin biosynthetic enzyme.Cytokinin biosynthetic enzymes useful in the present invention include, but are not limited to, isopentenyltransferase (Ipt) (also known as adenosine phosphate-isopentenyltransferase, adenylate dimethylallyltransferase, (dimethylallyl)adenosine tRNA methylthiotransferase) (EC number: 2.5.1.27 or 2.5.1.75 or 2.5.1.112) and / or Tzs (also known as dimethyltransferase, isopentenyltransferase, trans-zeatin generating protein, adenylate dimethylallyltransferase) (EC 2.5.1.27). Any combination of plant hormone biosynthetic enzymes capable of initiating autonomous division of plant cells to form the symbiont formation inoculum and symbionts described herein may be used. In some embodiments, combinations of plant hormone biosynthetic enzymes that can be utilized in the present invention include, but are not limited to, SEQ ID NO: 1 / 2 and SEQ ID NO: 3 / 4 and optionally SEQ ID NO: 5 / 6, SEQ ID NO: 8 and SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, and / or SEQ ID NO: 12 and SEQ ID NO: 13. Any combination of polynucleotides encoding auxin plant hormone biosynthetic enzymes and polynucleotides encoding cytokinin plant hormone biosynthetic enzymes that are capable of initiating autonomous replication in plant cells can be used to generate the symbionts and symbiont-forming inocula described herein.

[0098] "Polynucleotide of interest" refers to a polynucleotide that encodes a molecule (e.g., one or more polypeptides, peptides, coding RNA, or non-coding RNA, e.g., a bioactive molecule) for expression in a symbiont and, optionally, transported from the symbiont into a host plant to which the symbiont is anchored at one or more sites. In some embodiments, the polynucleotide of interest may encode a bioactive molecule or may encode a biosynthetic enzyme for a bioactive molecule (e.g., a polypeptide involved in the biosynthesis of a bioactive molecule).

[0099] As used herein, "modifying a host plant characteristic" means that at least one aspect or response of the host plant is altered by the growth of the symbiont of the present invention on the host plant. Such an aspect can include the presence of a biomolecule (produced in the symbiont and transported to the host plant) that is otherwise not found in the host plant or found in reduced amounts in the host plant (e.g., not found in or present in reduced amounts in a symbiont-free host plant), including, but not limited to, insecticidal biomolecules, antimicrobial biomolecules (antibacterial, antifungal), nematicidal biomolecules, antiviral biomolecules, herbicidal biomolecules, biomolecules that confer herbicide resistance / tolerance, biomolecules that confer disease resistance / tolerance, biomolecules that confer abiotic stress resistance / tolerance, biomolecules that modify plant structure and growth / morphology (e.g., nucleic acids encoding polypeptides and other factors (e.g., non-coding nucleic acids) that affect growth / morphology, plant hormones, etc.), biostimulants, RNAs, aptamers, and / or pharmaceuticals. In some embodiments, an "altered host plant characteristic" comprises an increased amount of a biomolecule (e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%) than would normally be found in the host plant. In some embodiments, an "altered host plant characteristic" includes, for example, an altered response to an insect, herbicide, plant pathogen (e.g., plant pathogenic bacteria, fungi, and / or viruses), nematode, environmental factor (e.g., heat, cold, salinity, etc.). In some embodiments, a host plant with an altered characteristic may comprise a symbiont that produces and delivers a herbicide to the host plant, thereby killing the host plant. Thus, in some embodiments, an altered host plant characteristic may be the presence of a herbicidal biomolecule and the death of the host plant. In some embodiments, "modifying host plant characteristics" can include modifying two or more characteristics (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more characteristics) of a host plant.Thus, an altered characteristic of a host plant comprising a symbiont of the invention can be the presence of two or more biomolecules (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) that are otherwise absent (or present in reduced amounts) in a host plant that does not comprise a symbiont of the invention, and / or the altered characteristic of a host plant comprising a symbiont of the invention can include two or more altered or modified responses that are not otherwise observed in a host plant that does not comprise a symbiont of the invention. To obtain two or more altered characteristics (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, or more altered characteristics) in a host plant comprising a symbiont of the invention, the symbiont on the plant can comprise two or more POIs, and / or the symbiont can comprise two or more symbionts (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, or more symbionts), where at least two of the two or more symbionts each comprise at least one POI that is different from a POI contained in another symbiont.

[0100] As used herein, a "symbiont" refers to a plant cell or cells that contain a polynucleotide encoding a plant hormone biosynthetic enzyme (e.g., at least one polynucleotide encoding one or more plant hormone biosynthetic enzymes) and a polynucleotide of interest, where the one or more plant hormone biosynthetic enzymes are cytokinin biosynthetic enzymes and / or auxin biosynthetic enzymes, and the symbiont is growing on a host plant. The cell(s) of the "symbiont" divide autonomously due to expression of the polynucleotide(s) encoding the plant hormone biosynthetic enzymes. "Symbionts" are: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500 , 4000, 4500, 5000, 6000, 7000, 8000, 9000, or 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, or 100,000 or more cells. Thus, in some embodiments, the symbiont can be a single plant cell containing at least one pSYM, a plasmid containing at least one polynucleotide (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more polynucleotides) encoding one or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) plant hormone biosynthetic enzymes, and at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) polynucleotide(s) of interest (POI), or it can include two or more cells, each containing at least one pSYM, a plasmid containing at least one polynucleotide encoding one or more plant hormone biosynthetic enzymes, and at least one polynucleotide (POI). The symbiont cells divide autonomously, which form undifferentiated multicellular structures on the plant.In some embodiments, the undifferentiated multicellular structure (e.g., symbiont) that is formed may visually resemble, for example, a gall, a plant food body, a symbiont home, an extrafloral nectary, a nodule, a plant neoplasm, or a gall, but is biochemically / genetically distinct due to at least the transgene expressed in the symbiont.

[0101] In some embodiments, the symbiont may be removed from the original host plant and cultured in a laboratory setting and / or transplanted onto another plant (e.g., used as symbiont formation inoculum). When the symbiont or at least one cell from the symbiont is cultured, "progeny symbiont material" may be used to refer to new symbiont material that forms over time and grows from the original material removed from the host plant.

[0102] The present invention is directed to host plants that contain at least one modified trait without modifying the host plant's genome. The present invention is further directed to methods and compositions for producing host plants that contain at least one modified trait without modifying the host plant's genome.

[0103] The present invention exploits the understanding that auxin and cytokinin genes, when expressed in plant cells, cause the plant cells to autonomously divide and form undifferentiated multicellular structures. In nature, such structures include, for example, galls initiated by infection of plants with Agrobacterium species. This ability to generate autonomously dividing cells is utilized by the present inventors in conjunction with the expression of a polynucleotide of interest (POI) in the autonomously dividing cells to generate undifferentiated multicellular structures (symbionts) that produce a product through the expression of the POI(s). Understanding that such undifferentiated multicellular structures can be grown on a host plant, the present inventors have now uniquely demonstrated that the POI-expressing undifferentiated multicellular structures (symbionts) of the present invention can be used to deliver products to a host plant and to modify a host plant's characteristic(s) without modifying the host plant's genome. Such plants (e.g., host plants) and related products (symbionts and symbiont-forming inoculants) are produced using various embodiments of the methods and compositions described herein, as well as numerous variations and additions to the various embodiments provided herein that will be apparent to those of skill in the art in light of this disclosure and do not depart from the invention.

[0104] In some embodiments, the present invention provides a symbiont formation inoculum comprising a polynucleotide encoding a plant hormone biosynthetic enzyme and a polynucleotide of interest, wherein the plant hormone biosynthetic enzyme is at least one cytokinin biosynthetic enzyme and / or auxin biosynthetic enzyme.

[0105] In some embodiments, the symbiont formation inoculum can be a nucleic acid composition (e.g., pSYM) comprising a polynucleotide encoding a plant hormone biosynthetic enzyme and a polynucleotide of interest that can be delivered to a host plant to produce a symbiont described herein. In some embodiments, the symbiont formation inoculum can be a cell (e.g., a bacterial cell or a plant cell) comprising a polynucleotide encoding a plant hormone biosynthetic enzyme and a polynucleotide of interest (e.g., comprising pSYM) that can be transplanted onto at least one site of a plant (e.g., a host plant) to produce a symbiont described herein.

[0106] In some embodiments, the nucleic acid construct of the present invention comprises a polynucleotide encoding a plant hormone biosynthetic enzyme and at least one polynucleotide of interest, wherein the plant hormone biosynthetic enzyme is a cytokinin biosynthetic enzyme and / or an auxin biosynthetic enzyme. As described herein, the polynucleotide encoding the plant hormone biosynthetic enzyme may encode one or more plant hormone biosynthetic enzymes. In some embodiments, the one or more plant hormone biosynthetic enzymes may be encoded by multiple polynucleotides. That is, when multiple plant hormone biosynthetic enzymes are contained in a single nucleic acid construct, they may be encoded by the same polynucleotide or separate polynucleotides.

[0107] Plant hormone biosynthetic enzymes useful in the symbiont formation inoculants of the present invention can be any auxin or cytokinin biosynthetic enzyme that can be expressed in plant cells to produce autonomously dividing or replicating plant cells, and optionally to produce callus cultures, suspension cultures, and / or undifferentiated multicellular structures. In some embodiments, the plant hormone biosynthetic enzyme or the polynucleotide encoding it can be derived from a bacterial species, e.g., a bacterial auxin biosynthetic enzyme or a bacterial cytokinin biosynthetic enzyme. In some embodiments, the plant hormone biosynthetic enzyme or the polynucleotide encoding it can be derived from a plant species, e.g., a plant auxin biosynthetic enzyme or a plant cytokinin biosynthetic enzyme. Examples of polynucleotides encoding plant hormone biosynthetic enzymes useful in the present invention include, but are not limited to, any one of the nucleotide sequences of SEQ ID NOs: 1, 3, 5, or 21, or a nucleotide sequence having at least about 80% identity thereto (e.g., 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity). In some embodiments, polynucleotides encoding plant hormone biosynthetic enzymes useful in the invention encode any one of the amino acid sequences of SEQ ID NOs: 2, 4, 6-20, 22, or 23, or an amino acid sequence having at least about 80% identity thereto (e.g., 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity). Examples of plant hormone biosynthetic polypeptides useful in the present invention include, but are not limited to, any one of the amino acid sequences of SEQ ID NOs: 2, 4, 6-20, 22, or 23, or an amino acid sequence having at least about 80% identity thereto (e.g., 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity). In some embodiments, the plant hormone biosynthetic enzyme is an auxin biosynthetic enzyme.Auxin biosynthetic enzymes useful in the present invention include, but are not limited to, indole-3-acetamide hydrolase (iaaH) (EC number: EC 3.5.1.4), amidase 1 (EC 3.5.1.4), tryptophan 2-monooxygenase (IaaM) (EC 1.13.12.3), indole-3-lactic acid synthase (EC 1.1.1.110), L-tryptophan-pyruvate aminotransferase 1 (EC 2.6.1.99), tryptophan aminotransferase-related protein 1 (EC 2.6.1.27), indole-3-acetaldehyde oxidase (EC 1.2.3.7), and / or tryptophan decarboxylase 1 / tryptophan decarboxylase 2 (EC 4.1.1.105). In some embodiments, the plant hormone biosynthetic enzyme is a cytokinin biosynthetic enzyme. Cytokinin biosynthetic enzymes useful in the present invention include, but are not limited to, isopentenyltransferase (Ipt) (also known as adenosine phosphate-isopentenyltransferase, adenylate dimethylallyltransferase, (dimethylallyl)adenosine tRNA methylthiotransferase) (EC number: 2.5.1.27 or 2.5.1.75 or 2.5.1.112), and / or Tzs (also known as dimethyltransferase, isopentenyltransferase, trans-zeatin generating protein, adenylate dimethylallyltransferase) (EC 2.5.1.27).

[0108] In some embodiments, polynucleotides (EC number: EC 3.5.1.4) encoding indole-3-acetamide hydrolases (e.g., iaaH, Aux2, Tms2) include, but are not limited to, nucleotide sequences having at least 80% identity to SEQ ID NO: 1. In some embodiments, indole-3-acetamide hydrolase polynucleotides useful in the invention may encode an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 2, 7, 9, 11, or 13. In some embodiments, an indole-3-acetamide hydrolase may comprise an amino acid sequence having at least 80% identity to any one of the amino acid sequences of SEQ ID NOs: 2, 7, 9, 11, or 13. Additional exemplary indole-3-acetamide hydrolases (and polynucleotides encoding same) useful in embodiments of the present invention have accession numbers (UniProt / NCBI) including, but not limited to, P06618, AAD30488.1, WP_010974823.1, WP_172691448.1, WP_172690897.1, WP_10891462.1, WP _172691118.1, NSZ87871.1, BAA76345.1, CAA39649.1, WP_070167543.1, P25016.1, WP_156536347.1, NSY72470.1, WP_156536347.1, WP_156638711.1, WP_045231698.1, WP_174183178.1, and / or AAB41868.1.

[0109] In some embodiments, amidase 1 (e.g., AMI1, AtAMI1) (EC 3.5.1.4) can comprise an amino acid sequence (At1GO8980) having at least 80% identity to the amino acid sequence of SEQ ID NO: 14. In some embodiments, amidase 1 polynucleotides useful in the invention encode an amino acid sequence having at least 80% identity to SEQ ID NO: 14.

[0110] In some embodiments, polynucleotides encoding tryptophan 2-monooxygenases (e.g., IaaM, Tms1, Aux1) (EC 1.13.12.3) include, but are not limited to, the nucleotide sequence of SEQ ID NO: 3 or a nucleotide sequence having at least 80% identity to SEQ ID NO: 3. In some embodiments, tryptophan 2-monooxygenase polynucleotides useful in the invention may encode an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 4, 8, 10, or 12. In some embodiments, tryptophan 2-monooxygenases useful in the invention may comprise an amino acid sequence having at least 80% identity to any one of the amino acid sequences of SEQ ID NOs: 4, 8, 10, or 12. Additional exemplary tryptophan 2-monooxygenase (and polynucleotides encoding same) accession numbers (UniProt / NCBI) include, but are not limited to, P25017, AAD30489.1, BAA76346.1, AYM09598.1, AYM14954.1, AYM61129.1 CAB44640.1, CUX71287.1, WP_040132230.1, AAF77123.1, WP_104680323.1, P25017.1, P0A3V2.1, MBB3947410.1, WP_162163087.1, NSY99416.1, AKC10880.1, AVH45197.1, and / or AYDO4913.1.

[0111] In some embodiments, an indole-3-lactic acid synthase (EC 1.1.1.110) can comprise an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, an indole-3-lactic acid synthase polynucleotide useful in the invention can be the nucleotide sequence of SEQ ID NO: 5 or a nucleotide sequence having at least 80% identity to SEQ ID NO: 5. In some embodiments, an indole-3-lactic acid synthase polynucleotide encodes an amino acid sequence having at least 80% identity to SEQ ID NO: 6. Additional exemplary indole-3-lactic acid synthase accession numbers (UniProt) useful in the invention include, but are not limited to, WP_052675630.1, WP_083212579.1, WP_172691447.1, and / or WP_010891463.1.

[0112] In some embodiments, an L-tryptophan-pyruvate aminotransferase 1 (e.g., TAA1, TIR2, CKRC1, SAV3, WEI8) (EC 2.6.1.99) useful in the present invention can comprise an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 15 (UniProt Q927N2). In some embodiments, an L-tryptophan-pyruvate aminotransferase 1 polynucleotide encodes an amino acid sequence having at least 80% identity to SEQ ID NO: 15.

[0113] In some embodiments, a tryptophan aminotransferase-related protein 1 (e.g., TAR1) (EC 2.6.1.27) useful in the invention can comprise an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 16 (UniProt Q9LR29). In some embodiments, a tryptophan aminotransferase-related protein 1 polynucleotide encodes an amino acid sequence having at least 80% identity to SEQ ID NO: 16.

[0114] In some embodiments, an indole-3-acetaldehyde oxidase (e.g., IAA oxidase, AO-1, ​​AO1, zmAO1, NtAO1, AtAO1, AtAO-1) useful in the invention (EC 1.2.3.7) can comprise an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 17 (UniProt O23887) and / or SEQ ID NO: 18 (UniProt Q7G193). In some embodiments, an indole-3-acetaldehyde oxidase polynucleotide encodes an amino acid sequence having at least 80% identity to SEQ ID NO: 17 and / or SEQ ID NO: 18.

[0115] In some embodiments, tryptophan decarboxylase 1 (e.g., TDC1) and / or tryptophan decarboxylase 2 (e.g., TDC2) (EC 4.1.1.105) may be used in the present invention to initiate autonomous cell division in plant cells. Tryptophan decarboxylase 1 useful in the present invention includes, but is not limited to, an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 19 (UniProt Q6ZJK7). In some embodiments, a tryptophan decarboxylase 1 polynucleotide encodes an amino acid sequence having at least 80% identity to SEQ ID NO: 19. In some embodiments, tryptophan decarboxylase 2 (e.g., TDC2) (EC 4.1.1.105) includes, but is not limited to, an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 20 (UniProt Q7XHL3). In some embodiments, a tryptophan decarboxylase 2 polynucleotide encodes an amino acid sequence having at least 80% identity to SEQ ID NO: 20.

[0116] Cytokinin biosynthetic enzymes useful for initiating autonomous cell division in plant cells include, but are not limited to, the cytokinin biosynthetic enzyme called isopentenyltransferase (Ipt). Other names for the Ipt enzyme include adenosine phosphate-isopentenyltransferase, adenylate dimethylallyltransferase, and (dimethylallyl)adenosine tRNA methylthiotransferase (EC Nos. 2.5.1.27, 2.5.1.75, or 2.5.1.112). In some embodiments, a polynucleotide encoding Ipt comprises the nucleotide sequence of SEQ ID NO:21 or a nucleotide sequence having at least 80% identity to SEQ ID NO:21. In some embodiments, an Ipt polynucleotide useful in the present invention may encode an amino acid sequence having at least 80% identity to any one of SEQ ID NOs:22. In some embodiments, an Ipt useful in the present invention may comprise an amino acid sequence having at least 80% identity to SEQ ID NO:22. Additional exemplary Ipt polypeptide accession numbers (UniProt / NCBI) include, but are not limited to, WP_010891460.1, NZ87873.1, WP_172690592.1, CAB44641.1, WP_172690722.1, BAA76344.1, WP_156638720.1, WP_104680324.1, NTA56762.1, WP_010892365.1, AAB41870.1, WP_032488312.1, WP_156 536348.1, WP_065657522.1, WP_0324488268.1, AAZ50399.1, WP_080830665.1, AYM20353.1, WP_174005331.1, WP_173994930.1, WP_111221726.1, WP032489582.1, WP_174156215.1, WP_17404522.5.1, WP_070167542.1, WP_172691205.1, and / or CAA54540.1.

[0117] Additional cytokinin biosynthetic enzymes include adenylate dimethylallyltransferase enzymes (e.g., tzs) (EC 2.5.1.27). Alternative names for Tzs enzymes include dimethyltransferase, isopentenyltransferase, trans-zeatin generating protein, and adenylate dimethylallyltransferase. Tzs polypeptides useful in the present invention include, but are not limited to, those comprising an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO:23 (UniProt P14011). In some embodiments, a Tzs polynucleotide encodes an amino acid sequence at least 80% identical to SEQ ID NO:23.

[0118] Any combination of auxin and cytokinin biosynthetic enzymes and / or polynucleotides encoding auxin and cytokinin biosynthetic enzymes, such as those described herein, can be used to generate symbionts and / or symbiont-forming inoculants. In some embodiments, the plant hormone biosynthetic enzymes encoded in the nucleic acid constructs of the present invention can be indole-3-acetamide hydrolase (iaaH), tryptophan 2-monooxygenase (IaaM), and / or isopentenyltransferase (Ipt). In some embodiments, the nucleic acid constructs of the present invention can further comprise a polynucleotide encoding a plant hormone biosynthetic enzyme that is indole-3-lactic acid synthase.

[0119] The present inventors have shown that expression of polynucleotides encoding plant hormone biosynthetic enzymes in plant cells as described herein can induce undifferentiated cell growth and symbiont formation on plants such as pecan, citrus, potato, tomato, and Nicotiana benthamiana. From literature related to crown gall disease and other similar disorders, it is understood that increased levels of cytokinin and auxin result from the integration of T-DNA containing plant hormone biosynthetic enzymes (e.g., IaaH, IaaM, and Ipt) into the plant genome, and that increased production of auxin and cytokinin by T-DNA-transformed cells promotes cell division. The present inventors have leveraged this knowledge to develop the present invention, thus generating symbiont and symbiont-forming inoculum of the present invention and host plants with modified characteristics without modifications in their genomes. Undifferentiated callus growth is the result of both increased auxin and cytokinin levels and the maintenance of a relatively high cytokinin-to-auxin ratio. The elevation of cytokinin and auxin is typically 2-fold to over 100-fold higher than that observed in non-tumorigenic tissues. For example, in tobacco cells, the observed cytokinin-to-auxin ratio is approximately 40:1. Generally, the cytokinin-to-auxin ratio ranges from about 5:1 to about 50:1 for the initiation of autonomous division and the formation of undifferentiated growth. As is known in the art, the cytokinin-to-auxin ratio required to generate undifferentiated growth can vary based on the plant species and the analytical method used to detect various plant hormone levels.

[0120] In some embodiments, a plant cell containing a nucleic acid construct of the present invention that includes a polynucleotide encoding a plant hormone biosynthetic enzyme (wherein the plant hormone biosynthetic enzyme is a cytokinin biosynthetic enzyme and an auxin biosynthetic enzyme) but does not include a polynucleotide of interest described herein may be referred to as an "active cell." Therefore, as used herein, "active cell" refers to an autonomously replicating plant cell that includes a polynucleotide encoding a plant hormone biosynthetic enzyme, where the plant hormone biosynthetic enzyme is a cytokinin biosynthetic enzyme and an auxin biosynthetic enzyme. Such active cells may be used to generate "active tissue." After introducing a polynucleotide of interest into the cells of the active cell or active tissue, the active plant cell or tissue may be referred to as a symbiont-formed inoculum. A symbiont is generated by transplanting the symbiont-formed inoculum onto at least one site on a host plant. Because cells (e.g., one or more cells (e.g., tissues)) can be harvested from the symbiont for various purposes, these cells (or tissues) may be referred to as the symbiont itself, or, when transplanted onto at least one site on a host plant, may be considered a symbiont-formed inoculum.

[0121] In some embodiments, cells from naturally formed galls, nodal galls, plant vegetative bodies, symbiont harborages, extrafloral nectaries, nodules, plant neoplasms, and / or autonomously replicating endosperm can be used to generate symbiont formation inoculum. Cells from structures such as these that naturally contain polynucleotides encoding plant hormone biosynthetic enzymes replicate autonomously. Such cells can be used to generate symbiont formation inoculum by transforming a cell(s) with at least one POI, resulting in the generation of symbiont formation inoculum containing cells harboring the polynucleotides encoding plant hormone biosynthetic enzymes and at least one POI. Like other symbiont formation inoculum, symbiont formation inoculum generated in this manner can also be used to generate symbionts on host plants.

[0122] A polynucleotide of interest useful in a symbiont formation inoculum of the invention refers to a polynucleotide that encodes a molecule described herein (e.g., one or more polypeptides, peptides, coding RNA, or non-coding RNA, e.g., a bioactive molecule) for expression in the symbiont, optionally transported from the symbiont into a host plant to which the symbiont is anchored at one or more sites, and optionally, when transported into the host plant, the molecule can confer new characteristics to the host plant without altering the genotype or genome of the host plant. In some embodiments, the polynucleotide of interest may encode a biomolecule and / or bioactive molecule, as described herein, and / or may encode a biosynthetic enzyme for a biomolecule and / or bioactive molecule (e.g., a polypeptide involved in the biosynthesis of a biomolecule and / or bioactive molecule). The "polynucleotide of interest" included in the symbiont formation inoculum can be one polynucleotide of interest, or it can be two or more polynucleotides of interest. When two or more polynucleotides of interest are included in the symbiont formation inoculum, the symbiont formation inoculum can be referred to as a "stacked" symbiont formation inoculum. The stacked symbiont formation inoculum can be used to form one or more stacked symbionts on a host plant. As a further example of stacking, when the symbiont formation inoculum comprises bacterial cells, the bacterial cells can contain at least two different POIs on one plasmid or at least two different plasmids.

[0123] In some embodiments, the nucleic acid constructs of the present invention may further comprise a polynucleotide encoding a plastid polypeptide (e.g., a plasticity polypeptide). A plastid polypeptide useful in the present invention can be any now known or later discovered plastid polypeptide that can benefit the morphology and structure of the symbiont formed using the nucleic acid constructs of the present invention (see, e.g., Leon Otten, Curr Topics Microbiol Immunol, 418:375-419 (2018)). Examples of plastid polypeptides useful in the nucleic acid constructs of the present invention include, but are not limited to, those provided in Table 1. In some embodiments, the plastid polypeptide can be 6b, rolB, rolC, and / or orf13. In some embodiments, multiple polynucleotides encoding plastid polypeptides can be included in the nucleic acid constructs of the present invention.

[0124] In some embodiments, the polynucleotide encoding a plant hormone biosynthetic enzyme and / or the polynucleotide of interest of the symbiont formation inoculum may be operably linked to regulatory elements including, but not limited to, a promoter sequence, a terminator sequence, and / or an intron. In some embodiments, when the polynucleotide encoding a plant hormone biosynthetic enzyme and / or the polynucleotide of interest are both operably linked to a promoter, they may each be operably linked to the same promoter or separate promoters in any combination. In some embodiments, when the polynucleotide encoding a plant hormone biosynthetic enzyme and / or the polynucleotide of interest are both operably linked to a terminator sequence, they may each be operably linked to the same terminator or separate terminators in any combination.

[0125] The nucleic acid construct of the present invention comprising a polynucleotide encoding a plant hormone biosynthetic enzyme may encode more than one plant hormone biosynthetic enzyme. In some embodiments, the encoded multiple plant hormone biosynthetic enzymes may be operably linked to a single promoter or separate promoters in any combination. For example, if the polynucleotide encoding the plant hormone biosynthetic enzyme encodes a polynucleotide encoding indole-3-acetamide hydrolase (iaaH), a polynucleotide encoding tryptophan 2-monooxygenase (IaaM), and a polynucleotide encoding isopentenyltransferase (Ipt), the polynucleotide encoding iaaH, the polynucleotide encoding IaaM, the polynucleotide encoding Ipt (and / or the polynucleotide encoding indole-3-lactic acid synthase), and the polynucleotide of interest may each be operably linked to a single promoter or at least two separate promoters in any combination. In some embodiments, the polynucleotides encoding iaaH, IaaM, and Ipt (and / or indole-3-lactate synthase) may be operably linked to a single promoter, and at least one polynucleotide of interest may be operably linked to a separate promoter. In some embodiments, the polynucleotide encoding indole-3-lactate synthase may be operably linked to the same promoter as or a separate promoter from the polynucleotides operably linked to any other plant hormone biosynthetic enzymes (e.g., polynucleotides encoding iaaH, IaaM, and / or Ipt).

[0126] In some embodiments, the nucleic acid construct of the present invention may further comprise a polynucleotide encoding a plast polypeptide, which may be operably linked to a promoter, which may be the same promoter as or a separate promoter from the polynucleotide operably linked to the plant hormone biosynthetic enzyme (e.g., a polynucleotide encoding iaaH, IaaM, and / or Ipt, or a polynucleotide encoding indole-3-lactic acid synthase). As will be appreciated by those of skill in the art, any of the polynucleotide combinations described herein may be under the control of (operably linked to) one or more regulatory elements (any combination of separate or the same regulatory elements), including, but not limited to, a promoter and / or a terminator.

[0127] In some embodiments, a regulatory element (e.g., promoter, terminator, intron) can be endogenous to the polynucleotide or cell(s) of the symbiont or symbiont formation inoculum to which it is operably linked. In some embodiments, a regulatory element (e.g., promoter, terminator, intron) can be heterologous (e.g., recombinant, chimeric) to the polynucleotide or cell(s) of the symbiont or symbiont formation inoculum to which it is operably linked.

[0128] Any promoter functional in plants that provides the desired expression level and location in plant cells can be used in the present invention. Thus, for example, the promoter can be a constitutive promoter. In some embodiments, the promoter can be an inducible promoter. In some embodiments, an inducible promoter can be inducible for programmed cell death. Examples of promoters include, but are not limited to, the CaMV35s promoter or the plant ubiquitin promoter (Ubi, e.g., Ubi-1). Additional promoters are disclosed above.

[0129] In some embodiments, for use in a symbiont formation inoculum, a polynucleotide of interest may encode a polypeptide operably linked to a targeting sequence such that the polypeptide, upon expression, can translocate out of the symbiont and enter the host plant and / or be located in a desired part of the host plant. The choice of targeting sequence depends on the desired location of the polypeptide encoded by the polynucleotide of interest. In some embodiments, a targeting sequence may be used to target a protein to a membrane, an intracellular location, or an extracellular location. In some embodiments, the targeting sequence is an endoplasmic reticulum targeting sequence, a mitochondrial targeting sequence, a chloroplast targeting sequence, a nuclear targeting (nuclear transport) sequence, a vacuolar targeting sequence, a peroxisome targeting sequence, a lysosome targeting sequence, a membrane targeting sequence, or a plant viral transport protein. In some embodiments, a polynucleotide of interest may encode a polypeptide operably linked to multiple (e.g., 1, 2, 3, 4, 5, or more) targeting sequences such that the polypeptide, upon expression, can translocate out of the symbiont and / or be located in multiple locations, e.g., in the host plant.

[0130] In some embodiments, the polynucleotide encoding the plant hormone biosynthetic enzyme and the polynucleotide of interest are contained in one or more nucleic acid constructs (e.g., one or more expression cassettes), in any combination, together or separately. In some embodiments, the polynucleotide encoding at least one plastopolypeptide may be contained in a nucleic acid construct, and optionally, the polynucleotide encoding at least one plastopolypeptide may be contained in the same or a separate nucleic acid construct (e.g., expression cassette) as the polynucleotide encoding the plant hormone biosynthetic enzyme and / or the polynucleotide of interest. The nucleic acid construct of the present invention may be contained in or can be an expression cassette. In some embodiments, the expression cassette of the present invention may be contained in a vector. Any vector suitable for introducing the nucleic acid construct into a cell may be used. By way of example, the vector may include, but is not limited to, a plasmid, T-DNA, bacterial artificial chromosome, viral vector, or binary bacterial artificial chromosome.

[0131] In some embodiments, the nucleic acid constructs of the invention and / or expression cassettes and / or vectors comprising same may be contained in cells, optionally plant or bacterial cells. Thus, the symbiont formation inoculum of the invention may comprise a polynucleotide encoding a plant hormone biosynthetic enzyme and a polynucleotide of interest in a cell, where the plant hormone biosynthetic enzymes include at least one cytokinin biosynthetic enzyme and at least one auxin biosynthetic enzyme, and optionally the cell is a plant or bacterial cell.

[0132] In some embodiments, the symbiont formation inoculum comprises cells comprising a polynucleotide encoding a plant hormone biosynthetic enzyme and a polynucleotide of interest, and the cells can be bacterial cells, optionally comprising a type IV secretion system (T4SS, e.g., a T4ASS (e.g., a VirB / D4 system), a T4BSS) or a type III secretion system (T3SS). In some embodiments, the bacterial cell can be a cell of an Agrobacterium species, a Rhizobium species, a Mesorhizobium species, a Sinorhizobium species, a Bradyrhizobium species, a Phyllobacterium species, an Ochrobactrum species, an Azobacter species, a Closterium species, a Klebsiella species, a Rhodospirillum species, or a Xanthomonas species. In some embodiments, the Agrobacterium species cell can be an A. tumefaciens (e.g., biovar 1), A. rhizogenes (e.g., biovar 2), A. vitis (e.g., biovar 3), or A. fablum (e.g., strain C58) cell. In some embodiments, the Pseudomonas species cell can be a P. savastanoi pv. Savastanoi cell.

[0133] The ability of bacteria to transfer DNA into plant cells is well known, both in natural settings (e.g., crown gall disease) and artificially (plant transformation). Researchers worldwide are exploiting the natural ability of Agrobacterium species to transfer DNA into plant cells and using this to expand the bacteria's natural host range far beyond its native range. As known to those skilled in the fields of plant disease and plant DNA transfer, the natural host range of Agrobacterium species is very broad. However, since at least the early 1980s, through human intervention, the ability of these bacteria to transfer DNA into plants has been further extended to many other species that are not natural hosts. An exemplary list of plants that are natural hosts for Agrobacterium species, and many that have been shown to be capable of being transformed using Agrobacterium species, is provided in Table 2. These and other plant genera and species can be used as host plants or to generate the symbiont-forming inocula described herein. In some embodiments, plant genera and species that may be used as host plants, and from which symbiont-forming inocula may be made, include, but are not limited to, those provided in Table 4 or in the plant list provided in the preceding paragraph of the Examples section below.

[0134] In some embodiments, the symbiont formation inoculum comprises cells comprising a polynucleotide encoding a plant hormone biosynthetic enzyme and a polynucleotide of interest, and the cells may be plant cells, and optionally the plant cells may be from any plant, including, but not limited to, angiosperms (e.g., dicotyledons or monocotyledons), gymnosperms, algae (e.g., macroalgae, e.g., Rhodophyta (red algae), Phaeophyta (brown algae), and Chlorophyta (green algae), Chrysophyceae (chrysophyceae)), bryophytes, ferns, and / or fern ally (i.e., ferns).

[0135] The symbiont-forming inoculum containing the polynucleotide encoding a plant hormone biosynthetic enzyme and the polynucleotide of interest, when contained in plant cells, can be in the form of a plant callus, callus culture, or suspension culture.

[0136] The present invention further provides a symbiont comprising a plant cell that contains and expresses a polynucleotide encoding a plant hormone biosynthetic enzyme and a polynucleotide of interest, wherein the plant hormone biosynthetic enzyme is at least one cytokinin biosynthetic enzyme and / or auxin biosynthetic enzyme, and the plant cell of the symbiont divides autonomously. In some embodiments, the plant cell comprises at least two plant cells (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more cells). Symbionts comprising multiple cells can form plant calli, callus cultures, or suspension cultures. Symbionts comprising multiple plant cells can form undifferentiated multicellular structures.

[0137] A polynucleotide of interest useful in the symbionts of the invention refers to a polynucleotide that encodes a molecule described herein (e.g., one or more polypeptides, peptides, coding RNA, or non-coding RNA, e.g., biomolecule, bioactive molecule) for expression in the symbiont, optionally transported from the symbiont into a host plant to which the symbiont is anchored at one or more sites, and optionally, where the molecule, when transported into the host plant, can confer new characteristics to the host plant without altering the genotype or genome of the host plant. In some embodiments, the polynucleotide of interest may encode a biomolecule and / or bioactive molecule, as described herein, and / or may encode a biosynthetic enzyme for a biomolecule and / or bioactive molecule (e.g., a polypeptide involved in the biosynthesis of a bioactive molecule). The "polynucleotide of interest" contained in a symbiont can be one polynucleotide of interest, or it can be two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more) polynucleotides of interest. When two or more polynucleotides of interest are contained in a single symbiont, the symbionts can be referred to as "stacked" symbionts. Furthermore, one or more symbionts formed on a host plant, where at least two of the symbionts contain different POIs, can be referred to as "stacked symbionts." Stacking can also include the formation of one or more symbionts on a host plant, where all symbionts contain the same POI(s).

[0138] In some embodiments, the polynucleotide encoding a plant hormone biosynthetic enzyme contained in a symbiont may encode one or more plant hormone biosynthetic enzymes. In some embodiments, one or more plant hormone biosynthetic enzymes may be encoded by one or more polynucleotides. That is, when a symbiont contains a polynucleotide encoding multiple plant hormone biosynthetic enzymes, the multiple plant hormone biosynthetic enzymes may be encoded in any combination on the same polynucleotide as another plant hormone biosynthetic enzyme or on separate polynucleotides.

[0139] Plant hormone biosynthetic enzymes useful in the symbionts of the present invention can be any auxin or cytokinin biosynthetic enzyme that can be expressed in a plant cell to produce an autonomously dividing or replicating plant cell, and optionally to produce an undifferentiated multicellular structure. These are described in detail above and include auxin biosynthetic enzymes, including, but not limited to, indole-3-acetamide hydrolase (iaaH) (EC number: EC 3.5.1.4), amidase 1 (EC 3.5.1.4), tryptophan 2-monooxygenase (IaaM) (EC 1.13.12.3), indole-3-lactate synthase (EC 1.1.1.110), L-tryptophan-pyruvate aminotransferase 1 (EC 2.6.1.99), tryptophan aminotransferase-related protein 1 (EC 2.6.1.27), indole-3-acetaldehyde oxidase (EC 1.2.3.7), and / or tryptophan decarboxylase 1 / tryptophan decarboxylase 2 (EC 4.1.1.105). In some embodiments, the plant hormone biosynthetic enzyme is a cytokinin biosynthetic enzyme, including, but not limited to, isopentenyltransferase (Ipt) (also known as adenosine phosphate-isopentenyltransferase, adenylate dimethylallyltransferase, (dimethylallyl)adenosine tRNA methylthiotransferase) (EC Numbers: 2.5.1.27 or 2.5.1.75 or 2.5.1.112), and / or Tzs (also known as dimethyltransferase, isopentenyltransferase, trans-zeatin generating protein, adenylate dimethylallyltransferase) (EC 2.5.1.27). In some embodiments, the plant hormone biosynthetic enzyme can be indole-3-acetamide hydrolase (iaaH), tryptophan 2-monooxygenase (IaaM), and / or isopentenyltransferase (Ipt). In some embodiments, the symbiont of the present invention may further comprise a polynucleotide encoding a plant hormone biosynthetic enzyme that is indole-3-lactic acid synthase.

[0140] In some embodiments, the symbionts of the invention may further comprise a polynucleotide encoding a plast polypeptide (e.g., a plasticity polypeptide). A plast polypeptide useful in the invention can be any now known or later discovered plast polypeptide that can benefit the structure of a symbiont formed using a nucleic acid construct of the invention. Examples of plast polypeptides useful in the symbionts of the invention include, but are not limited to, those provided in Table 1. In some embodiments, the plast polypeptide can be 6b, rolB, rolC, and / or orf13. In some embodiments, multiple polynucleotides encoding plast polypeptides can be included in the symbionts of the invention.

[0141] For expression in the cells of the symbiont, the polynucleotide encoding the plant hormone biosynthetic enzyme and / or the polynucleotide of interest may be operably linked to regulatory elements, including, but not limited to, a promoter sequence, a terminator sequence, and / or an intron. In some embodiments, when the polynucleotide encoding the plant hormone biosynthetic enzyme and / or the polynucleotide of interest are both operably linked to a promoter, they may each be operably linked to the same promoter or separate promoters in any combination. In some embodiments, when the polynucleotide encoding the plant hormone biosynthetic enzyme and / or the polynucleotide of interest are both operably linked to a terminator sequence, they may each be operably linked to the same terminator or separate terminators in any combination.

[0142] For example, if the polynucleotide encoding a plant hormone biosynthetic enzyme encodes a polynucleotide encoding indole-3-acetamide hydrolase (iaaH), a polynucleotide encoding tryptophan 2-monooxygenase (IaaM), and a polynucleotide encoding isopentenyltransferase (Ipt), the polynucleotide encoding iaaH, the polynucleotide encoding IaaM, the polynucleotide encoding Ipt, and the polynucleotide of interest may be operably linked to a single promoter or at least two separate promoters in any combination. In some embodiments, the polynucleotide encoding iaaH, the polynucleotide encoding IaaM, and the polynucleotide encoding Ipt may be operably linked to a single promoter, and at least one polynucleotide of interest may be operably linked to a separate promoter. In some embodiments, the polynucleotide encoding an indole-3-lactic acid synthase may be operably linked to a promoter that may be the same as or a separate promoter from any other polynucleotide operably linked to a plant hormone biosynthetic enzyme (e.g., a polynucleotide encoding iaaH, IaaM, and / or Ipt). In some embodiments, the polynucleotide encoding a plast polypeptide may be operably linked to a promoter that may be the same as or a separate promoter from the polynucleotide operably linked to a plant hormone biosynthetic enzyme (e.g., a polynucleotide encoding iaaH, IaaM, and / or Ipt, or a polynucleotide encoding an indole-3-lactic acid synthase). As will be appreciated by those of skill in the art, any of the polynucleotide combinations described herein may be under the control of (operably linked to) one or more regulatory elements (any combination of separate or the same regulatory elements), including, but not limited to, a promoter and / or a terminator.

[0143] In some embodiments, a regulatory element (e.g., promoter, terminator, intron) can be endogenous or heterologous (e.g., recombinant) to the polynucleotide to which it is operably linked or to one or more plant cells of the symbiont.

[0144] Any promoter functional in plants that provides the desired expression level and location in plant cells can be used in the present invention. Thus, for example, the promoter can be a constitutive promoter. In some embodiments, the promoter can be an inducible promoter. In some embodiments, an inducible promoter can be inducible for programmed cell death. Examples of promoters include, but are not limited to, the CaMV35s promoter or the plant ubiquitin promoter (Ubi, e.g., Ubi-1). Additional regulatory elements comprising the promoter are as disclosed above.

[0145] In some embodiments, the polynucleotide encoding the plant hormone biosynthetic enzyme and the polynucleotide of interest are contained together or separately in one or more nucleic acid constructs (e.g., one or more expression cassettes) in any combination. In some embodiments, the polynucleotide encoding at least one plastopolypeptide may be contained in a nucleic acid construct, and optionally, the polynucleotide encoding at least one plastopolypeptide may be contained in the same or a separate nucleic acid construct (e.g., expression cassette) as the polynucleotide encoding the plant hormone biosynthetic enzyme and / or the polynucleotide of interest. The nucleic acid construct of the present invention may be contained in or can be an expression cassette. In some embodiments, the expression cassette of the present invention may be contained in a vector. Any vector suitable for introducing a nucleic acid construct into a cell may be used. By way of example, the vector may include, but is not limited to, a plasmid, T-DNA, bacterial artificial chromosome, viral vector, or binary bacterial artificial chromosome.

[0146] In some embodiments, the polynucleotide of interest may encode a polypeptide operably linked to a targeting sequence such that the polypeptide can be localized to a desired part of the host plant upon expression in the symbiont. The selection of the targeting sequence depends on the desired location of the polypeptide encoded by the polynucleotide of interest. In some embodiments, the targeting sequence may be used to target a protein to a membrane, an intracellular location, or an extracellular location. In some embodiments, the targeting sequence is an endoplasmic reticulum targeting sequence, a mitochondrial targeting sequence, a chloroplast targeting sequence, a nuclear targeting (nuclear transport) sequence, a vacuolar targeting sequence, a peroxisome targeting sequence, a lysosome targeting sequence, or a plant viral movement protein. In some embodiments, the polynucleotide of interest may encode a polypeptide operably linked to multiple (e.g., 1, 2, 3, 4, 5, or more) targeting sequences such that the polypeptide can be localized to multiple desired parts of the host plant upon expression. In some embodiments, the polypeptide can be operably linked to multiple targeting sequences to sequentially target multiple locations. For example, a polypeptide operably linked to a chloroplast targeting sequence and a membrane targeting sequence may be targeted first to the chloroplast and then to the membrane. In some embodiments, a polynucleotide encoding a plant hormone biosynthetic enzyme (e.g., a polynucleotide encoding iaaH, a polynucleotide encoding IaaM, and / or a polynucleotide encoding Ipt, and / or an indole-3-lactic acid synthase) and / or a polynucleotide encoding at least one plastid polypeptide may be operably linked to a nuclear targeting sequence.

[0147] In some embodiments, the symbiont comprising a polynucleotide encoding a plant hormone biosynthetic enzyme (e.g., a polynucleotide encoding iaaH, a polynucleotide encoding IaaM, and / or a polynucleotide encoding Ipt, and / or an indole-3-lactic acid synthase), and / or a polynucleotide encoding at least one plastid polypeptide, the plant hormone biosynthetic enzyme, and / or the polynucleotide encoding the plastid polypeptide is operably linked to a nuclear targeting sequence.

[0148] In some embodiments, the symbiont may include a polynucleotide encoding a plant hormone biosynthetic enzyme (e.g., a polynucleotide encoding iaaH, a polynucleotide encoding IaaM, a polynucleotide encoding Ipt) and a polynucleotide of interest, where the polynucleotide encoding the plant hormone biosynthetic enzyme and the polynucleotide of interest are operably linked to a single promoter or to at least two separate promoters, in any combination. In some embodiments, when the polynucleotide encoding the plant hormone biosynthetic enzyme encodes iaaH, IaaM, and Ipt, the polynucleotide(s) encoding iaaH, IaaM, and Ipt are operably linked to a single promoter, and the polynucleotide of interest is operably linked to a separate promoter.

[0149] In some embodiments, the symbiont may include a polynucleotide encoding at least one plastopolypeptide operably linked to a promoter, and optionally, the polynucleotide encoding the at least one plastopolypeptide is operably linked to the same promoter as or a separate promoter from the polynucleotide encoding the plant hormone biosynthetic enzyme and / or the polynucleotide of interest. In some embodiments, the promoter, single promoter, separate promoters, and / or two or more separate promoters are endogenous to the cells of the symbiont. In some embodiments, the promoter, single promoter, separate promoters, and / or two or more separate promoters are heterologous to the cells of the symbiont. In some embodiments, one or more of the promoter, single promoter, separate promoters, and / or two or more separate promoters may be endogenous to the cells of the symbiont, while at least one of the promoter, single promoter, separate promoters, and / or two or more separate promoters is heterologous to the cells of the symbiont. In some embodiments, the polynucleotide encoding the plant hormone biosynthetic enzyme may be heterologous to the plant cells of the symbiont. In some embodiments, the polynucleotide encoding the plant hormone biosynthetic enzyme may be endogenous to the plant cell of the symbiont, hi some embodiments, the polynucleotide encoding the plant hormone biosynthetic enzyme may be operably linked to a heterologous promoter (e.g., heterologous to the polynucleotide encoding the plant hormone biosynthetic enzyme and / or the plant cell of the symbiont) or an endogenous promoter (e.g., endogenous to the polynucleotide encoding the plant hormone biosynthetic enzyme or the plant cell symbiont).

[0150] Plant cells for use as symbionts of the present invention (e.g., plant cells comprising a polynucleotide encoding a plant hormone biosynthetic enzyme and a polynucleotide of interest) can be any plant cell, including, but not limited to, angiosperm cells (e.g., dicotyledonous or monocotyledonous plants), gymnosperm cells, algal cells (e.g., macroalgae, e.g., Rhodophyta (red algae), Phaeophyta (brown algae), and Chlorophyta (green algae), Chrysophyceae (chrysophyceae)), bryophyte cells, fern and / or fern-like plant cells (i.e., ferns). In some embodiments, plant cells useful in the present invention include, but are not limited to, those listed in Table 2 or Table 4, or in the plant list provided in the preceding paragraph of the Examples section below. In some embodiments, plant cells include, but are not limited to, citrus cells, tomato cells, corn cells, pecan cells, and tobacco cells.

[0151] The symbiont can be transplanted into a plant (e.g., a host plant) at one or more locations on the plant. Thus, the present invention further provides a host plant comprising at least one symbiont of the present invention, wherein the symbiont is located at at least one site (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sites) on the plant. A plant (e.g., a host plant) of the present invention can include multiple symbionts located at various sites on the plant or host plant. As used herein, a "site" on a plant can be any location on the plant or any part of the plant for growing a symbiont. Examples of sites or locations for symbionts include, but are not limited to, explants, embryos, leaves, shoots, stems, branches, grains, ears, cobs, husks, stalks, epidermal tissue, apical meristems, floral tissue (e.g., pollen, pistils, ovules, anthers, stamens, corollas, sepals, petals, receptacles, filaments, styles, stigmas, etc.), fruits, seeds, pods, capsules, cotyledons, hypocotyls, petioles, tubers, corms, roots, root tips, symbionts, galls, plant vegetative bodies, symbiont harborages, extrafloral nectaries, nodules, plant neoplasms, or galls.

[0152] In some embodiments, when the symbiont is contained on at least one site on the host plant, the polynucleotide of interest contained in the symbiont is expressed in the symbiont, and an expression product of the polynucleotide of interest and / or a product made using the expression product of the polynucleotide of interest is transported into the host plant. The host plant can be a wild-type plant of any age or size (e.g., a seedling, a young plant, or a mature plant). Host plants include, but are not limited to, angiosperms (e.g., dicotyledonous or monocotyledonous plants), gymnosperms, macroalgae (e.g., Rhodophyta (red algae), Phaeophyta (brown algae), and Chlorophyta (green algae), Chrysophyceae (chrysophyceae)), bryophytes, and / or ferns and / or fern-like plants (i.e., ferns), as described herein. In some embodiments, plants useful in the present invention include, but are not limited to, those listed in Table 2 and / or Table 4 and / or the plant list provided in the preceding paragraph of the Examples section below. In some embodiments, exemplary plants useful in the present invention include citrus plants (eg, grapefruit, oranges, lemons, limes, etc.), tomato plants, corn plants, pecan plants, and tobacco plants.

[0153] In some embodiments, the symbiont may be harvested from the host plant, and a product comprising the biomolecule(s) and / or bioactive molecule(s) may be isolated / collected from the harvested symbiont. Any biomolecule or bioactive molecule, such as those described herein, may be produced in and collected / isolated from the symbiont of the present invention. The products collected from the symbiont and symbiont-containing host plant may be used for any purpose for which the product is suitable. Non-limiting examples of such uses include specialty chemicals, pharmaceuticals, cosmetics, lubricants, dyes / pigments, fuels, foods, and / or nutritional products.

[0154] The present invention further provides methods for producing compositions of the present invention, including symbiont-forming inocula, symbionts, and host plants containing symbionts of the present invention. Symbiont-forming inocula of the present invention can be compositions containing one or more nucleic acid constructs (e.g., 1, 2, 3, 4, 5, or more) comprising at least one polynucleotide of interest and at least one plant hormone biosynthetic enzyme (e.g., one or more polynucleotides (e.g., 1, 2, 3, 4, 5, or more) encoding one or more plant hormone biosynthetic enzymes (e.g., 1, 2, 3, 4, 5, or more)), wherein the biosynthetic enzyme(s) comprise an auxin biosynthetic enzyme and / or a cytokinin biosynthetic enzyme. In some embodiments, a symbiont formation inoculum of the invention can include one or more cells (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, or more cells) comprising one or more nucleic acid constructs comprising a polynucleotide encoding a plant hormone biosynthetic enzyme, where the biosynthetic enzyme comprises an auxin biosynthetic enzyme and / or a cytokinin biosynthetic enzyme, and a polynucleotide of interest. In some embodiments, the cells are plant cells. In some embodiments, the cells are bacterial cells.

[0155] Thus, there is provided a method for generating a symbiont formation inoculum, comprising introducing into a cell a polynucleotide encoding a plant hormone biosynthetic enzyme and a polynucleotide of interest, or introducing into a transgenic cell containing the polynucleotide of interest a polynucleotide encoding a plant hormone biosynthetic enzyme, wherein the plant hormone biosynthetic enzyme is at least one cytokinin biosynthetic enzyme and / or auxin biosynthetic enzyme, thereby generating a symbiont formation inoculum. In some embodiments, the method for generating a symbiont formation inoculum further comprises culturing the cells to generate a population of cells comprising the polynucleotide encoding the plant hormone biosynthetic enzyme and the polynucleotide of interest.

[0156] The present invention provides a method for producing a symbiont formation inoculum, comprising the steps of: (a) (i) introducing into / onto at least one site (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sites) a polynucleotide encoding a plant hormone biosynthetic enzyme and a polynucleotide sequence of interest, or transplanting plant cells (e.g., viable plant cells comprising at least one polynucleotide encoding a plant hormone-encoding enzyme) containing the same, or inoculating onto at least one site on the plant (or portion thereof, e.g., explant, stem, etc.) a bacterial cell (e.g., at least one polynucleotide encoding a plant hormone-encoding enzyme) containing the same, or (ii) introducing into / onto at least one site (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sites) a plant (or portion thereof, e.g., explant, stem, etc.) a polynucleotide encoding a plant hormone biosynthetic enzyme, or transplanting plant cells containing the same, comprising the polynucleotide sequence of interest. (b) inoculating one or more bacterial cells (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 75, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, selecting, from the symbiont on the plant, 70, 80, 90, 100, 200, 300, 400, 500, 1000, 2500, 5000, 10,000, 50,000, 100,000, or more cells, e.g., a portion of tissue from the symbiont, e.g., about 0.005 μg to about 1 g or more), to provide one or more cells comprising a polynucleotide encoding a plant hormone biosynthetic enzyme and a polynucleotide sequence of interest, thereby producing a symbiont-forming inoculum.In some embodiments, when the method for generating a symbiont-formed inoculum first includes generating the symbiont on at least one site on the plant, the at least one site on the plant is in an above-ground portion of the plant. In some embodiments, the at least one site on the plant is in an underground portion of the plant. In some embodiments, the method for generating a symbiont-formed inoculum may further include (c) culturing one or more cells from (b) to generate a population of plant cells (e.g., a callus, callus culture, and / or suspension culture) comprising a polynucleotide encoding a plant hormone biosynthetic enzyme and a polynucleotide sequence of interest.

[0157] When plant cells are used to generate symbiont formation inoculum by transplanting the cells onto a plant or part thereof, or when bacterial cells are used to generate symbiont formation inoculum by inoculating the bacterial cells onto a plant or part thereof, the cells can be a single cell or can be two or more cells (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10 cells to about 100,000 cells or more). When the cells are plant cells, and the plant cells used to generate symbiont formation inoculum contain at least one polynucleotide encoding a plant hormone enzyme (e.g., a polynucleotide encoding an auxin biosynthetic enzyme and a polynucleotide encoding a cytokinin biosynthetic enzyme), but do not contain a polynucleotide of interest for use in modifying a host plant characteristic without modifying the host plant genome, the plant cells can be referred to as "active" plant cells. The active plant cells are modified with at least one polynucleotide encoding a plant hormone enzyme that allows the cells to reproduce autonomously, thereby allowing the active cells to form undifferentiated structures (gall-like structures) when transplanted onto a plant or part thereof. When the active plant cells divide autonomously to form tissue, the tissue can be called "active tissue." Symbiotic inoculum is produced from active plant cells or active tissue only if the cells or tissue contain a polynucleotide of interest for use in modifying host plant characteristics without modifying the host plant genome.

[0158] Polynucleotides of interest useful in the methods of the invention for producing compositions of the invention, including the symbiont-forming inocula, symbionts, and host plants containing symbionts of the invention, include any polynucleotides of interest that may be useful for modifying host plant characteristics or that may be useful in the production of a biomolecule in / by the symbiont and / or host plant containing at least one symbiont. A biomolecule is any molecule produced by a living organism and / or part thereof (e.g., a cell or a cell-free system).

[0159] The polynucleotide of interest may encode any molecule described herein (e.g., one or more polypeptides, peptides, coding RNA, or non-coding RNA, e.g., bioactive molecules), which may be expressed in the symbiont and, optionally, transferred from the symbiont into a host plant to which the symbiont is anchored at one or more sites, and optionally, when transferred into the host plant, may confer new characteristics to the host plant without altering the genotype or genome of the host plant. In some embodiments, the polynucleotide of interest may encode a biomolecule and / or bioactive molecule, as described herein, and / or may encode a biosynthetic enzyme for a biomolecule and / or bioactive molecule (e.g., a polypeptide involved in the biosynthesis of a bioactive molecule). As described herein, a "polynucleotide of interest" for use in generating the symbiont formation inoculum described herein can be one polynucleotide of interest, or it can be two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more) polynucleotides of interest. When two or more polynucleotides of interest are included in the symbiont formation inoculum, the symbiont formation inoculum can be referred to as a "stacked" symbiont formation inoculum. A stacked symbiont formation inoculum can be used to form one or more symbionts on a host plant, which can be referred to as stacked symbiont(s). As a further example of stacking, when the symbiont formation inoculum includes bacterial cells, the bacterial cells can include at least two different POIs on one plasmid or at least two different plasmids.

[0160] As described herein, any auxin or cytokinin biosynthetic enzyme that can be expressed in a plant cell as described herein to produce an autonomously dividing or replicating plant cell can be used to create a symbiont formation inoculum. Exemplary auxin and cytokinin biosynthetic enzymes and the polynucleotides encoding them are described in detail above and include, but are not limited to, auxin biosynthetic enzymes including indole-3-acetamide hydrolase (iaaH) (EC number: EC 3.5.1.4), amidase 1 (EC 3.5.1.4), tryptophan 2-monooxygenase (IaaM) (EC 1.13.12.3), indole-3-lactic acid synthase (EC 1.1.1.110), L-tryptophan-pyruvate aminotransferase 1 (EC 2.6.1.99), tryptophan aminotransferase-related protein 1 (EC 2.6.1.27), indole-3-acetaldehyde oxidase (EC 1.2.3.7), and / or tryptophan decarboxylase 1 / tryptophan decarboxylase 2 (EC 4.1.1.105). In some embodiments, the plant hormone biosynthetic enzyme is a cytokinin biosynthetic enzyme. Cytokinin biosynthetic enzymes useful in the present invention include, but are not limited to, isopentenyltransferase (Ipt) (also known as adenosine phosphate-isopentenyltransferase, adenylate dimethylallyltransferase, (dimethylallyl)adenosine tRNA methylthiotransferase) (EC number: 2.5.1.27 or 2.5.1.75 or 2.5.1.112) and / or Tzs (also known as dimethyltransferase, isopentenyltransferase, trans-zeatin generating protein, adenylate dimethylallyltransferase) (EC 2.5.1.27). In some embodiments, the plant hormone biosynthetic enzyme may be indole-3-acetamide hydrolase (iaaH), tryptophan 2-monooxygenase (IaaM), and / or isopentenyltransferase (Ipt), and optionally, indole-3-lactic acid synthase, and any combination thereof.

[0161] In some embodiments, the method of generating a symbiont formation inoculum may further include introducing into at least one site on a cell or on a plant a polynucleotide encoding at least one plastopolypeptide (e.g., a plasticity polypeptide), optionally wherein the plastopolypeptide includes, but is not limited to, a plastopolypeptide provided in Table 1. In some embodiments, the plastopolypeptide is 6b, rolB, rolC, and / or orf13.

[0162] The polynucleotides encoding the plant hormone biosynthetic enzymes and the polynucleotide of interest may be included together or separately in one or more nucleic acid constructs (e.g., one or more expression cassettes and / or vectors) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more constructs) for introduction into the cell. In some embodiments, the polynucleotides encoding plastid polypeptides (e.g., at least one plastid polypeptide, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more constructs) may be included in one or more nucleic acid constructs (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more constructs), optionally where the polynucleotide encoding at least one plastid polypeptide is in the same or a separate nucleic acid construct (e.g., expression cassette) as the polynucleotides encoding the plant hormone biosynthetic enzymes and / or the polynucleotide of interest. In some embodiments, nucleic acid constructs comprising a polynucleotide encoding a plant hormone biosynthetic enzyme, a polynucleotide of interest, and / or a polynucleotide encoding a plastid polypeptide may be included in an expression cassette, which may be the same or separate expression cassettes. In some embodiments, one or more nucleic acid constructs (or expression cassettes comprising them) may be included in one or more vectors (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more). Any vector useful for transferring polynucleotides into cells may be used in the nucleic acid constructs of the present invention. In some embodiments, the vector may be a plasmid, T-DNA, bacterial artificial chromosome, viral vector, or binary bacterial artificial chromosome, or any combination thereof, for use with the polynucleotides, nucleic acid constructs, and / or expression cassettes of the present invention.

[0163] In some embodiments, the polynucleotide of interest introduced into a cell according to the method of the present invention can encode a polypeptide operably linked to a targeting sequence. In some embodiments, the targeting sequence positions the protein at a membrane, an intracellular location, or an extracellular location. In some embodiments, the targeting sequence can be, but is not limited to, a membrane targeting sequence, an endoplasmic reticulum targeting sequence, a mitochondrial targeting sequence, a chloroplast targeting sequence, or a plant virus movement protein.

[0164] In some embodiments, the polynucleotide can be targeted to the nucleus. Thus, the polynucleotide encoding the plant hormone biosynthetic enzyme described herein and / or the polynucleotide encoding the plastid polypeptide described herein can be operably linked to a nuclear localization sequence to target the polynucleotide to the nucleus of the cell.

[0165] In some embodiments, the polynucleotide encoding a plant hormone biosynthesis gene and / or the polynucleotide of interest may be operably linked to regulatory elements, including, but not limited to, a promoter sequence, a terminator sequence, and / or an intron. In some embodiments, when the polynucleotide encoding a plant hormone biosynthesis gene and / or the polynucleotide of interest are both operably linked to a promoter, they may each be operably linked to the same promoter or to separate promoters in any combination. In some embodiments, when the polynucleotide encoding a plant hormone biosynthesis gene and / or the polynucleotide of interest are both operably linked to a terminator sequence, they may each be operably linked to the same terminator or to separate terminators in any combination. In some embodiments, the polynucleotide encoding a plant hormone biosynthesis enzyme and the polynucleotide of interest are each operably linked to a single promoter. In some embodiments, the polynucleotide encoding a plant hormone biosynthesis enzyme and the polynucleotide of interest are operably linked to at least two separate promoters in any combination. In some embodiments, when the polynucleotide encoding the plant hormone biosynthetic enzyme encodes two or more plant hormone biosynthetic enzymes (e.g., iaaH, IaaM, and Ipt), the polynucleotide(s) encoding the two or more plant hormone biosynthetic enzymes are operably linked to a single promoter and the polynucleotide of interest is operably linked to a separate promoter.

[0166] In some embodiments, when polynucleotides encoding multiple plant hormone biosynthetic enzymes are introduced (e.g., a polynucleotide encoding iaaH, a polynucleotide encoding IaaM, a polynucleotide encoding Ipt, and / or a polynucleotide encoding indole-3-lactic acid synthase), the polynucleotides encoding the multiple plant hormone biosynthetic enzymes may be operably linked to the same or separate promoters, which may be the same or separate promoters as the promoter operably linked to the polynucleotide of interest. In some embodiments, the polynucleotides encoding iaaH, the polynucleotides encoding IaaM, and the polynucleotides encoding Ipt are operably linked to a single promoter, and the polynucleotides of interest are operably linked to separate promoters. In some embodiments, the polynucleotides encoding plant hormone biosynthetic enzymes (e.g., iaaH, IaaM, and Ipt, and / or indole-3-lactic acid synthase) are operably linked to a single promoter, and the polynucleotides of interest are operably linked to the same promoter.

[0167] In some embodiments, the polynucleotide encoding at least one plast polypeptide may be operably linked to a promoter. In some embodiments, the polynucleotide encoding at least one plast polypeptide is operably linked to the same promoter as the polynucleotide encoding the plant hormone biosynthetic enzyme and / or the polynucleotide of interest. In some embodiments, the polynucleotide encoding at least one plast polypeptide is operably linked to a promoter that is separate from the promoter operably linked to the polynucleotide encoding the plant hormone biosynthetic enzyme and / or the polynucleotide of interest.

[0168] Any promoter that allows the polynucleotide encoding the plant hormone enzyme and / or the polynucleotide of interest to be expressed can be used. As described herein, the selection of the promoter can vary depending on the temporal and spatial requirements for expression and can also vary based on the host cell to be transformed. Promoters with different expression patterns for many different organisms are well known in the art. Promoters useful in generating symbiont formation inoculum can be endogenous to one or more cells of the symbiont formation inoculum, heterologous to one or more cells of the symbiont formation inoculum, or any combination thereof. In some embodiments, the promoter can be endogenous or heterologous to the polynucleotide to which it is operably linked.

[0169] In some embodiments, promoters useful in generating symbiont formation inoculum are constitutive promoters, hi some embodiments, promoters useful in generating symbiont formation inoculum are inducible promoters.

[0170] Bacterial cells useful for generating symbiont formation inocula can be any bacterial cell that contains a type IV secretion system (T4SS, e.g., a T4ASS (e.g., a VirB / D4 system), a T4BSS), or a type III secretion system (T3SS). Such bacterial systems are well known in the art and include, but are not limited to, species of the genus Agrobacterium (e.g., A. tumefaciens (e.g., biovar 1), A. rhizogenes (e.g., biovar 2), A. vitis (e.g., biovar 3), A. fablum (e.g., strain C58), Rhizobium spp., Mesorhizobium spp., Sinorhizobium spp., Bradyrhizobium spp., Pseudomonas spp. (e.g., P. savastanoi pv. savastanoi), Phyllobacterium spp., Ochrobactrum spp., Azobacter spp., Closterium spp., Klebsiella spp., Rhodospirillum spp., or Xanthomonas spp.

[0171] Any plant cell that can subsequently be used to form symbionts on plants can be used to generate the symbiont formation inoculum. Such plant cells include, but are not limited to, those derived from angiosperms (e.g., dicotyledons or monocotyledons), gymnosperms, algae (e.g., macroalgae, e.g., Rhodophyta (red algae), Phaeophyta (brown algae), and Chlorophyta (green algae), Chrysophyceae (chrysophyceae)), bryophytes, ferns, and / or fern-like plants (i.e., ferns). Cells can be derived from wild-type or transgenic plants (e.g., seedlings, young plants, or mature plants) of any age or size. In some embodiments, plant cells useful in the present invention include, but are not limited to, those listed in Table 2, Table 4, or in the plant list provided in the preceding paragraph of the Examples section below. In some embodiments, exemplary plant cells useful in the present invention include citrus cells (eg, grapefruit, orange, lemon, lime, etc.), tomato cells, corn cells, pecan cells, and tobacco cells.

[0172] Plant cells useful for generating symbiont-forming inoculum can be derived from any plant part, including, but not limited to, plant cell culture (callus, callus culture, or suspension culture), protoplast, seedling, explant, embryo, leaf, shoot, stem, branch, grain, ear, cob, husk, stalk, epidermal tissue, apical meristem, floral tissue (e.g., pollen, pistil, ovule, anther, stamen, corolla, sepals, petals, receptacle, filament, style, stigma, etc.), fruit, seed, pod, capsule, cotyledon, hypocotyl, petiole, tuber, corm, root, root tip, symbiont, gall, phytotroph, symbiont harborage, extrafloral nectary, nodule, gall, or plant neoplasm.

[0173] As described herein, in some embodiments, when plant cells are used to generate symbiont formation inoculum, the at least one site on the plant can be any site on the plant, including, but not limited to, an explant, embryo, leaf, shoot, stem, branch, grain, ear, cob, husk, stalk, epidermal tissue, apical meristem, floral tissue (e.g., pollen, pistil, ovule, anther, stamen, corolla, sepals, petals, receptacle, filament, style, stigma, etc.), fruit, seed, pod, capsule, cotyledon, hypocotyl, petiole, tuber, corm, root, root tip, symbiont, nodal gall, plant vegetative body, symbiont harborage, extrafloral nectary, nodule, plant neoplasm, or gall.

[0174] Nucleic acid constructs (e.g., polynucleotides, expression cassettes, and / or vectors) can be introduced into cells via any known method. Procedures for transforming both prokaryotic and eukaryotic organisms, including plants, are well known and routine in the art and are described throughout the literature. In some embodiments, nucleic acid constructs of the invention (e.g., polynucleotides encoding plant hormone biosynthetic enzymes, polynucleotides of interest, and / or expression cassettes and / or vectors comprising same) can be introduced into cells via methods including, but not limited to, bacterial-mediated transformation, agroinfiltration, viral-mediated transformation, particle bombardment (biolistics), electroporation, microinjection, lipofection (liposome-mediated transformation), sonication, silicon fiber-mediated transformation, chemically stimulated DNA uptake (e.g., polyfection, e.g., polyethylene glycol (PEG)-mediated transformation), and / or laser microbeam (UV)-induced transformation.

[0175] In some embodiments, when (i) a polynucleotide encoding a plant hormone biosynthetic enzyme and a polynucleotide sequence of interest or (ii) a polynucleotide encoding a plant hormone biosynthetic enzyme are comprised in at least one plant cell, the at least one plant cell may be transplanted onto at least one site (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sites) on the plant. In some embodiments, one or more cells (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more cells) transplanted into the at least one site are cultured at the site to generate a population of plant cells comprising the polynucleotide encoding the plant hormone biosynthetic enzyme and the polynucleotide sequence of interest, forming a symbiont, wherein one or more cells from the symbiont on the plant are selected to provide one or more cells comprising the polynucleotide encoding the plant hormone biosynthetic enzyme and the polynucleotide sequence of interest, thereby generating a symbiont-forming inoculum.

[0176] In some embodiments, when a plant is used to generate a symbiont formation inoculum, at least one site on the plant can be wounded at the inoculation site before, simultaneously with, or after the step of introducing the symbiont into at least one site on the plant (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sites). Similarly, when a symbiont is transplanted onto at least one site on a host plant, at least one site on the host plant can be wounded before, simultaneously with, or after the step of transplanting. Wounding for introduction or transplantation can be performed in any manner that results in cracking the outer surface (epidermis, cuticle, bark) of the plant or part thereof at the site where introduction or transplantation is to occur. Such tools can include, but are not limited to, tweezers or forceps, a knife, a needle (e.g., hypodermic, dissecting, tattooing, sewing, etc.), a toothpick, and / or a syringe. Additionally, any standard transplantation tool can be used for introduction or transplantation as described herein.

[0177] In some embodiments, introducing a polynucleotide of the invention (e.g., a polynucleotide encoding a plant hormone biosynthetic enzyme (e.g., at least one polynucleotide encoding at least one plant hormone biosynthetic enzyme), a polynucleotide of interest, an expression cassette(s) or vector(s) comprising the same) into a plant cell, plant, or part thereof is carried out via bacterial-mediated transformation and comprises co-culturing the plant cell or plant (or part thereof, e.g., an explant) with cells of at least one bacterial species or strain (e.g., 1, 2, 3, 4, 5, or more), wherein the bacterial cells comprise one or more of the polynucleotide encoding the plant hormone biosynthetic enzyme, the polynucleotide of interest, and / or at least one polynucleotide encoding at least one plastid polypeptide. In some embodiments, the plant (or part thereof, e.g., an explant) may be wounded at the inoculation site prior to or during co-cultivation with cells of the at least one bacterial strain. In some embodiments, the cells of at least one bacterial species or strain include cells of at least two bacterial species or strains, and the polynucleotide encoding the plant hormone enzyme is contained in a bacterial strain that is separate from the bacterial strain containing the at least one polynucleotide of interest (e.g., double bacterial transformation). As described herein, bacterial cells useful for generating symbiont formation inoculum can be any bacterial cell that contains a type IV secretion system (T4SS, e.g., T4ASS (e.g., VirB / D4 system), T4BSS) or a type III secretion system (T3SS), including, but not limited to, species of Agrobacterium (e.g., A. tumefaciens (e.g., biovar 1), A. rhizogenes (e.g., biovar 2), A. tumefaciens (e.g., biovar 3), A. rhizogenes (e.g., biovar 4), A. tumefaciens (e.g., biovar 5), A. tumefaciens (e.g., biovar 6), A. tumefaciens (e.g., biovar 7), A. tumefaciens (e.g., biovar 8), A. tumefaciens (e.g., biovar 9), A. tumefaciens (e.g., biovar 10), A. tumefaciens (e.g., biovar 11), A. tumefaciens (e.g., biovar 12), A. tumefaciens (e.g., biovar 13), A. tumefaciens (e.g., biovar 14), A. tumefaciens (e.g., biovar 15), A. tumefaciens (e.g., biovar 16), A. tumefaciens (e.g., biovar 17), A. tumefaciens (e.g., biovar 2), A. bitis (e.g., biovar 3), A. fabrum (e.g., strain C58), Rhizobium species, Mesorhizobium species, Sinorhizobium species, Bradyrhizobium species, Pseudomonas species, Phyllobacterium species, Ochrobactrum species, Azobacter species, Closterium species, Klebsiella species, Rhodospirillum species, or Xanthomonas species.In some embodiments, the bacterial cell is a Pseudomonas species (e.g., P. savastanoi pv. savastanoi). In some embodiments, the bacterial cell can be a Pseudomonas savastanoi pv. savastanoi cell. The plant species to which this method can be applied are not limited. As noted above, since at least the early 1980s, the ability of bacteria to transfer DNA into plants through human intervention has expanded to numerous species beyond those naturally infected by bacteria. Table 2 provides non-limiting examples of plants that are natural hosts for Agrobacterium species, as well as some plants that are not natural hosts but have been shown to be capable of being transformed using Agrobacterium species. As would be readily apparent to one of skill in the art, the plant genera and species listed in Table 2, as well as any other plant genera and species, can be used as host plants or to generate the symbiont formation inoculum described herein. In some embodiments, plant genera and species that may be used as host plants, and from which symbiont-forming inocula may be made, include, but are not limited to, those provided in Table 4 or in the plant list provided in the preceding paragraph of the Examples section below.

[0178] In some embodiments, the method for generating a symbiont formation inoculum may further include editing at least one nucleic acid in at least one cell of the symbiont formation inoculum to generate at least one edited nucleic acid in the symbiont formation inoculum. Any known gene editing technology may be used, including but not limited to nuclease-based editing systems, including but not limited to CRISPR-Cas technology, zinc finger nuclease (ZFN) technology, transcription activator-like effector nuclease (TALEN) technology, and engineered meganuclease technology. In some embodiments, the at least one edited nucleic acid has modified expression. In some embodiments, modified expression comprises increased expression compared to the same nucleic acid without the same modification. In some embodiments, modified expression comprises decreased expression compared to the same nucleic acid without the same modification.

[0179] The present invention further provides a symbiont formation inoculum produced by the methods of the present invention. In some embodiments, the symbiont formation inoculum is a bacterial culture that includes polynucleotides encoding plant hormone biosynthetic enzymes (e.g., one or more (e.g., 1, 2, 3, 4, 5, or more) polynucleotides encoding one or more (e.g., 1, 2, 3, 4, 5, or more) plant hormone biosynthetic enzymes) and a polynucleotide of interest (e.g., at least one polynucleotide of interest (e.g., 1, 2, 3, 4, 5, or more)). In some embodiments, the symbiont formation inoculum includes two or more cells in the form of a plant cell culture (e.g., a callus or a cell suspension) that include polynucleotides encoding plant hormone biosynthetic enzymes (e.g., one or more polynucleotides encoding one or more plant hormone biosynthetic enzymes) and a polynucleotide of interest (e.g., at least one polynucleotide of interest).

[0180] In some embodiments, the invention provides cells or protoplasts derived from the symbiont-forming inoculum of the invention, wherein the cells or protoplasts comprise a polynucleotide encoding a plant hormone biosynthetic enzyme and a polynucleotide of interest.

[0181] Further provided is a method for modifying a host plant characteristic without modifying the plant genome, comprising the steps of transplanting a symbiont formation inoculum of the invention or a symbiont of the invention into at least one site (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sites) on a host plant, and culturing the symbiont formation inoculum at least one site on the host plant to form a symbiont at the at least one site on the host plant, wherein a polynucleotide of interest is expressed in the symbiont on the host plant, and an expression product of the polynucleotide of interest and / or a product made using the expression product of the polynucleotide of interest is delivered into the host plant, thereby modifying the host plant characteristic. "Modifying a host plant characteristic without modifying the plant genome" refers to a change in the morphology, metabolism, biochemistry, and / or physiology of the host plant that does not change the genotype of the host plant.

[0182] Polynucleotides of interest useful in symbionts of the invention for plant host characterization can include polynucleotides encoding molecules described herein (e.g., one or more polypeptides, peptides, coding RNAs, or non-coding RNAs, e.g., biomolecules, bioactive molecules) for expression in the symbiont that is anchored on the host plant at one or more sites, such that when the molecule is delivered into the host plant, the molecule can confer new characteristics to the host plant without altering the genotype or genome of the host plant. In some embodiments, a polynucleotide of interest can encode a biomolecule or bioactive molecule, as described herein, or can encode a biosynthetic enzyme for a biomolecule and / or bioactive molecule (e.g., a polypeptide involved in the biosynthesis of a biomolecule or bioactive molecule). As described herein, a "polynucleotide of interest" contained in a symbiont formed on a host plant can be one polynucleotide of interest, or can be two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more) polynucleotides of interest. When two or more polynucleotides of interest are contained in a symbiont, the symbiont may be referred to as a "stacked" symbiont. Furthermore, one or more symbionts formed on a host plant, where at least two of the symbionts contain different POIs, may be referred to as a "stacked symbiont." Stacking may also include the formation of one or more symbionts on a host plant, where all symbionts contain the same POI(s).

[0183] In some embodiments, the polynucleotide encoding a plant hormone biosynthetic enzyme contained in a symbiont used to confer an altered host plant characteristic may encode one or more plant hormone biosynthetic enzymes. In some embodiments, one or more plant hormone biosynthetic enzymes may be encoded by one or more polynucleotides. That is, when a symbiont contains polynucleotides encoding multiple plant hormone biosynthetic enzymes, the multiple plant hormone biosynthetic enzymes may be encoded in any combination on the same polynucleotide as another plant hormone biosynthetic enzyme or on separate polynucleotides.

[0184] The plant hormone biosynthetic enzymes expressed in the symbionts of the present invention can be any auxin or cytokinin biosynthetic enzymes that can be expressed in plant cells to produce autonomously dividing or replicating plant cells, and optionally to produce undifferentiated multicellular structures. As described herein, any auxin or cytokinin biosynthetic enzymes that can be expressed in plant cells to produce autonomously dividing or replicating plant cells can be used to create the symbiont formation inoculum. Exemplary auxin and cytokinin biosynthetic enzymes and the polynucleotides encoding them are described in detail above and include, but are not limited to, auxin biosynthetic enzymes including indole-3-acetamide hydrolase (iaaH) (EC number: EC 3.5.1.4), amidase 1 (EC 3.5.1.4), tryptophan 2-monooxygenase (IaaM) (EC 1.13.12.3), indole-3-lactic acid synthase (EC 1.1.1.110), L-tryptophan-pyruvate aminotransferase 1 (EC 2.6.1.99), tryptophan aminotransferase-related protein 1 (EC 2.6.1.27), indole-3-acetaldehyde oxidase (EC 1.2.3.7), and / or tryptophan decarboxylase 1 / tryptophan decarboxylase 2 (EC 4.1.1.105). In some embodiments, the plant hormone biosynthetic enzyme is a cytokinin biosynthetic enzyme. Cytokinin biosynthetic enzymes useful in the present invention include, but are not limited to, isopentenyltransferase (Ipt) (also known as adenosine phosphate-isopentenyltransferase, adenylate dimethylallyltransferase, (dimethylallyl)adenosine tRNA methylthiotransferase) (EC number: 2.5.1.27 or 2.5.1.75 or 2.5.1.112) and / or Tzs (also known as dimethyltransferase, isopentenyltransferase, trans-zeatin generating protein, adenylate dimethylallyltransferase) (EC 2.5.1.27).In some embodiments, the plant hormone biosynthetic enzymes may be indole-3-acetamide hydrolase (iaaH), tryptophan 2-monooxygenase (IaaM), and / or isopentenyltransferase (Ipt), and optionally, indole-3-lactic acid synthase, and any combination thereof. In some embodiments, the plant hormone biosynthetic enzymes may be indole-3-acetamide hydrolase (iaaH), tryptophan 2-monooxygenase (IaaM), and / or isopentenyltransferase (Ipt), in any combination. In some embodiments, the symbiont of the present invention may further comprise a polynucleotide encoding a plant hormone biosynthetic enzyme that is indole-3-lactic acid synthase.

[0185] In some embodiments, the symbionts of the invention may further comprise and express a polynucleotide encoding a plast polypeptide (e.g., a plasticity polypeptide). A plast polypeptide useful in the invention can be any now known or later discovered plast polypeptide that can benefit the structure of a symbiont formed using a nucleic acid construct of the invention. Examples of plast polypeptides useful in the symbionts of the invention include, but are not limited to, those provided in Table 1. In some embodiments, the plast polypeptide can be 6b, rolB, rolC, and / or orf13. In some embodiments, multiple polynucleotides encoding plast polypeptides can be included in the symbionts of the invention.

[0186] In some embodiments, culturing the symbiont-forming inoculum, when contained within bacterial cells on a host plant, can further include culturing in the presence of acetosyringone at a concentration ranging from about 10 μM to about 200 μM, or any range or value therein (e.g., about 10, 15, 20, 25, 30, 350, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 μM, or any range or value therein) (e.g., about 50 μM to about 150 μM, about 75 μM to about 125 μM, about 85 μM to about 100 μM). In some embodiments, when cultured in the presence of acetosyringone, the acetosyringone is present at a concentration of about 100 μM.

[0187] In some embodiments, a symbiont formation inoculum comprising bacterial cells can be used to modify host plant characteristics without modifying the host plant genome. In some embodiments, a symbiont formation inoculum containing an Agrobacterium species can be delivered to, for example, a first plant. The Agrobacterium species can be in the form of one or more strains, at least one strain containing a nucleic acid (which can be provided, for example, in a T-DNA) encoding at least one plant hormone biosynthetic enzyme that induces symbiont formation, and at least one strain containing a nucleic acid (which can be provided, for example, in a T-DNA) comprising a polynucleotide of interest that encodes a desired trait to impart to the host plant. Thus, delivery of the inoculum may cause one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) symbionts to form on the first plant, and the symbionts may express the nucleic acid delivered by the Agrobacterium species. The symbionts have increased vascularization in the symbiont tissue, which supports rapid growth, faster metabolism, and efficient export pathways, and ultimately systemic movement of the desired molecule throughout the plant. In some embodiments, the symbionts may then be removed from the first plant and added / transplanted onto a second plant (e.g., a host plant) to functionally communicate with the host plant, thus providing the host plant with desired traits, but without transforming or altering the host plant's genome or introducing heterologous or xenobiotic DNA into the host plant, to form plant tissue. In some embodiments, prior to transplantation into a host plant, the removed symbionts become symbiont-formed inoculum, which may be cultured without the use of Agrobacterium species to form bacteria-free symbiont-formed inoculum, which may then be transplanted into a host plant.

[0188] Regarding the choice of strain(s) of Agrobacterium species for use in the present invention, various single strains or combinations thereof can be used to achieve the desired results. According to one embodiment, the inoculum contains at least two strains in a host plant, where at least one strain is an "active strain" (e.g., a wild-type reference strain) containing a polynucleotide encoding at least one plant hormone biosynthetic enzyme, and at least one other strain is not an active strain (e.g., a "disarmed" or "transducible" strain) but contains a nucleic acid (e.g., T-DNA) that confers a desired trait (a polynucleotide of interest). Because wild-type Agrobacterium species are known to form galls, the active strain may be isolated from nature, such as strain FL-F54 described herein. The desired trait can be, for example, having antimicrobial or anti-insect properties, altering plant physiology, or other. The trait can be expressed or achieved by one or more molecules (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more molecules), such as molecules encoded by nucleic acids (e.g., T-DNA) in the transforming Agrobacterium species. These molecules can be small molecules, large molecules, proteins, polymers, or other molecules, as desired. Multiple active strains and / or multiple transforming strains can be used, as desired for a particular application. Alternatively, a single strain can be used that induces symbiont formation and also induces the desired trait in the host plant to which the symbiont is attached, without modifying the host plant genome. There is no limit to the plant species to which this method can be applied. Today, it is routine to transfer DNA into plants using Agrobacterium and other bacterial species. Non-limiting examples of plants that are natural hosts for Agrobacterium species, as well as some plants that are not natural hosts but have been shown to be capable of being transformed using Agrobacterium species, are provided in Table 2. The plants listed in this table are from many different plant families, including both dicotyledonous and monocotyledonous plants, demonstrating that the types of plants on which this method can be used are not limited.In some embodiments, plant genera and species that may be used in this method include, but are not limited to, those provided in Table 4 or in the plant list provided in the preceding paragraph of the Examples section below.

[0189] In addition to a carrier, the inoculum may contain one or more strain(s) of Agrobacterium species described above (e.g., 1, 2, 3, 4, 5, or more strains), and other components as desired. When multiple strains are used, various strain ratios may be used as desired, e.g., a ratio of 1:10 active strain to transforming strain. Agrobacterium species delivery inocula are well known in the art, and an appropriate one can be selected based on the desired results in a particular application. For example, the inoculum may contain an aqueous buffer such as MES (2-ethanesulfonic acid), Tris (tris(hydroxymethyl)aminomethane), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), or a salt-based buffer such as PBS (phosphate-buffered saline), one or more salts such as magnesium chloride, a transformation enhancer such as acetosyringone or other phenols that can enhance virulence, and / or an adjuvant including a wetting agent / penetration-enhancing surfactant, including but not limited to anionic, cationic, and nonionic surfactants. Delivery of the inoculum may be accomplished by any known method, such as via the use of a needle, puncture wound, or other direct delivery system, i.e., drill or air blast, and may be automated or manual.

[0190] Symbiont formation can be observed visually, and symbiont size can optionally be controlled through known means, such as chemical control (i.e., GALLEX® (AgBioChem Inc., Los Molinos, CA)). Symbiont formation can take various periods of time depending on the host plant species and plant age used. For example, sufficient symbiont formation can take from several days to several months to develop. In some embodiments, symbionts or symbiont tissue can be collected from a first plant and then cultured for volumetric or storage purposes. In some embodiments, symbionts can be transferred directly from a first plant to a second plant (e.g., a host plant) without culture. However, it may be desirable to first culture the symbiont-formed inoculum to (a) remove residual bacteria, such as by natural attrition or active sterilization, or (b) determine that the symbiont-formed inoculum expresses the desired trait(s). Removal of residual Agrobacterium species can occur over time by natural attenuation, such as by feeding a culture that does not support the bacteria, thus causing it to die off, or by active means, such as sterilization with the application of bleach and / or antibiotics or other methods that actively kill the bacterial culture. Determining whether the symbiont-informing inoculum or symbiont expresses the desired trait can be achieved by simple observation if the trait is phenotypically visible (such as color), or by analyzing the culture medium / host plant for the target compound(s) produced by the symbiont or symbiont-forming inoculum, or by any other known means.

[0191] The symbiont can be removed from the plant by any known and applicable means and used as a symbiont formation inoculum for attachment (transplantation) to a second plant (e.g., a host plant). Note that the entire removed symbiont for use as a symbiont formation inoculum may not necessarily achieve the desired results. For example, only a portion of stable material from the symbiont (e.g., one or more cells) can be removed and used for attachment / transplantation to a host plant. In such a method, cells of a single removed and cultured symbiont can be expanded to provide material for transplantation onto multiple host plants (e.g., as a symbiont formation inoculum). Techniques for transplanting one plant or plant part, such as a symbiont or symbiont formation inoculum, onto another plant (e.g., a host plant) are well known in the art and can be used in the present invention. Preferentially, the symbiont formation inoculum can be added to the host plant such that the formed symbiont is in functional communication with the vascular system of the host plant or such that functional communication with the vascular system of the host plant is achievable. Transplantation methods may allow the symbiont tissue to develop the necessary vascular connections after transplantation, even if these connections are not established simultaneously with transplantation. In this way, the desired traits / compounds produced by the symbiont can travel through the vascular system of the second plant. In some embodiments, the desired traits / compounds produced by the symbiont can be transported to the host plant via the apoplast and / or symplast. In some embodiments, the desired traits / compounds produced by the symbiont can be transported to the host plant via the apoplast, symplast, or the vascular system that develops between the host plant and the symbiont, or any combination thereof.

[0192] The first (the original plant on which the symbiont grows or develops) and second plant (e.g., the host plant onto which the symbiont-forming inoculum can be grafted) can be of the same species or different species, depending on the specific interoperability (i.e., graft compatibility) of the plant materials between the different species.

[0193] In some embodiments, viable cells / tissues may be formed by inoculating a plant with at least one viable strain of Agrobacterium sp. as described above, and the viable cells / tissues may be removed from the first plant and then cultured in a solution containing at least one transforming strain of Agrobacterium sp. After sufficient uptake of nucleic acid (e.g., T-DNA) from the transforming strain, plant cells containing both the polynucleotide(s) encoding the plant hormone biosynthetic enzyme and the transforming nucleic acid (POI) (e.g., symbiont-forming inoculum) may be present in the culture. These cells (e.g., symbiont-forming cells or inoculum) may be selected by known methods and then used as desired. For example, cells may be selected, removed, and cultured to generate more symbiont-forming inoculum (e.g., bacterial cell masses containing two or more cells, callus tissue, and / or suspension cultures) having the desired trait. Alternatively, or in addition, selected cells of the symbiont-forming inoculum can then be used as described above (i.e., sterilized and transplanted onto a second plant (such as a host plant).

[0194] In some embodiments, bacteria (e.g., cells of one or more Agrobacterium species containing at least one pSYM) containing at least one pSYM (e.g., a polynucleotide encoding at least one plant hormone biosynthetic enzyme and at least one POI) can be delivered directly to (inoculated onto) a host plant. In some embodiments, the symbiont formation inoculum can include one Agrobacterium species strain or multiple Agrobacterium species strains as described above (e.g., one strain containing one or more polynucleotides encoding at least one plant hormone biosynthetic enzyme and one strain containing a POI, or a single strain containing both one or more polynucleotides encoding at least one plant hormone biosynthetic enzyme and a POI). In this way, the resulting symbiont tissue formed on the host plant will serve as a beneficial biofactory for or on the host plant for the desired molecule(s) without the need to transform the host plant. In some embodiments, some or all of the inoculated strains may be engineered to have low vigor, so that after a useful symbiont (i.e., a symbiont that has vascular overgrowth and produces the desired molecules for the desired trait) is formed on the plant, the bacteria die off and are no longer present in the symbiont.

[0195] In some embodiments, culturing the symbiont formation inoculum on the host plant can further include culturing under conditions that increase humidity. For example, the site on the host plant that is contacted with the symbiont formation inoculum or onto which the symbiont is transplanted can be covered to increase humidity in the area adjacent to the symbiont or symbiont formation inoculum. Any type of cover that maintains humidity in the area surrounding the symbiont or symbiont formation inoculum transplanted onto the host plant can be used. As an example, the symbiont or symbiont formation inoculum located at the site on the host plant can be wrapped or covered with a film to maintain humidity. In some embodiments, the film can include, but is not limited to, plastic film, silicone tape, and / or parafilm. In some embodiments, to increase humidity in the area of ​​the symbiont or symbiont-formed inoculum, the symbiont or symbiont-formed inoculum on the host plant can be covered after transplantation (e.g., immediately or within about 15 minutes to 5 hours) for about 1 hour to about 72 hours or more, about 1 hour to about 48 hours or more, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours to about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 hours or more, or any range or value therein. In some embodiments, the symbiont or symbiont-formed inoculum on the host plant may be covered for about 10 to about 30 hours (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 hours), optionally for about 24 hours, after transplantation (host plant) or inoculation (inoculum formation) to increase humidity in the area of ​​the symbiont or symbiont-formed inoculum.

[0196] In some embodiments, the host plant can be wounded at at least one site prior to or simultaneously with transplantation of the at least one site on the host plant. Wounding can be performed in any manner using any tool useful for cracking the outer surface (epidermis, cuticle, bark) of the plant or plant part at the site where the symbiont or symbiont-forming inoculum will be transplanted. Such tools can include, but are not limited to, tweezers or forceps, a knife, a needle (e.g., hypodermic, dissecting, tattooing, sewing, etc.), a toothpick, and / or a syringe. Additionally, any standard transplantation tool can be used for introducing or transplanting as described herein.

[0197] In some embodiments, the at least one site on the host plant can be on an above-ground portion of the host plant and / or on an below-ground portion of the host plant.

[0198] In some embodiments, the symbiont is transplanted onto the host plant at least twice. In some embodiments, the symbiont formation inoculum is transplanted onto the host plant at least twice. In some embodiments, the symbiont and / or symbiont formation inoculum is transplanted onto at least two sites on the host plant.

[0199] In some embodiments, the expression product of a polynucleotide of interest can be a transcription product or a translation product, or a variant thereof. As an example, the expression product of a polynucleotide of interest can be methylation of a transcription product. In some embodiments, the expression product of a polynucleotide of interest can be, for example, glycosylation of a translation product. The translation product can be a protein (polypeptide) or a peptide. The transcription product is ribonucleic acid (RNA). In some embodiments, the RNA is coding RNA (e.g., mRNA). In some embodiments, the RNA is non-coding RNA, including, but not limited to, transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), piwi-interacting RNA (piRNA), microRNA (miRNA), long non-coding RNA (lncRNA), and / or small interfering RNA (siRNA).

[0200] In some embodiments, the expression product of a polynucleotide of interest may be a biosynthetic enzyme that may be used to make another product, which may include, but is not limited to, a chemical, a protein (polypeptide / peptide), or a polynucleotide.

[0201] Modified host plant characteristics can include modifications of any plant characteristic, including, but not limited to, alterations in the host plant's metabolism, alterations in the host plant's structure (e.g., morphology), and / or alterations in the host plant's metabolism, biochemistry, and / or physiology. In some embodiments, the modified host plant characteristic can be, for example, an altered plant response to a disease-causing organism, such as, for example, a fungus, bacterium, virus, and / or protozoan. Thus, in some embodiments, the modified host plant characteristic can result in increased tolerance / resistance to a disease-causing organism compared to a plant without the symbiont. Disease-causing organisms can include, but are not limited to, a fungus, bacterium, virus, and / or protozoan. In some embodiments, the modified host plant characteristic is increased (induced) expression of a plant defense gene, thereby resulting in a host plant with increased disease resistance. In some embodiments, plant defense genes that can be increased include "W-box" defense genes. W-box defense genes can include, but are not limited to, CAD1, NPR1, and / or PR2. In some embodiments, plant defense genes can be increased in the host plant through the production of a chemical in the symbiont, such as a chemical that is delivered to the host plant via the symbiont and stimulates a systemic acquired resistance response in the host plant. In some embodiments, plant defense genes can be increased in the host plant through the production of a chemical in the host plant that stimulates a systemic acquired resistance response in the host plant, where the biochemical pathway that produces the chemical in the host plant has been modified by the product of the polynucleotide of interest in the symbiont, which is delivered to the host plant.

[0202] In some embodiments, the modified host plant characteristic can be, for example, a change in the plant's response to insects or nematodes. Thus, in some embodiments, the modified host plant characteristic is increased insect tolerance / resistance compared to a plant without the symbiont. Insects against which tolerance or resistance can be increased include, but are not limited to, insects of the orders Lepidoptera, Coleoptera, Hemiptera, Thysanoptera, and Diptera. In some embodiments, the modified host plant characteristic is increased nematode tolerance / resistance compared to a plant without the symbiont. Nematodes to which tolerance or resistance can be increased include, but are not limited to, root-knot nematodes (Meloidogyne spp.), cyst nematodes (Heterodera spp. and Globodera spp.), root-lesion nematodes (Pratylenchus spp.), burrowing nematodes (Radopholus similis), reniform nematodes (Rotylenchulus reniformis), grape nematodes (Xiphinema index), and citrus nematodes (Tylenchulus semipenetrans).

[0203] In some embodiments, the modified host plant characteristic can be a change in the plant's response to a bacterial pest. Thus, in some embodiments, the modified host plant characteristic is increased tolerance / resistance to the bacterial pest compared to a plant without the symbiont. The present invention relates to a method for the treatment of bacterial flora, including, but not limited to, bacterial flora, such as Xantomonas axonopodis, Xanthomonas campestris, Erwinia amylovora, Erwinia carotovora, Candidatus Liberibacter asiaticus, Candidatus Liberibacter solanacearum, Pseudomonas syringae, Xylella fastidiosa, Dickeya solani, Dickeya dadantii, Pectobacterium carotovorum, and / or Ralstonia wilt. The present invention provides methods and compositions that can increase resistance or tolerance to a number of bacterial pests, including Bacillus solanacearum.

[0204] In some embodiments, the modified host plant characteristic can be a change in the plant's response to a herbicide. Thus, in some embodiments, the modified host plant characteristic is increased herbicide tolerance / resistance compared to a plant without the symbiont. Examples of herbicides to which host plant characteristics may be modified to tolerate or tolerate include, but are not limited to, glyphosate, triazines, dicamba, 2,4-D, clopyralid, flumioxazin, carfentrozone-ethyl, sulfentrozon, lactofen, fomesafen, acifluorfen, mesotrione, sulcotrione, tembotrione, topramezone, picolinafen, clomazone, isoxaflutole, mefenacet, flufenacet, imazamox, imazapyr, imazethapyr, rimsulfuron, tribenuron-methyl, triasulfuron, nicosulfuron, sulfosulfuron, sulfometuron-methyl, mesosulfuron-methyl, azimsulfuron, amidosulfuron, cyclosulfamuron, flumetsulam, metosulam, florasulam, diclosulam, and / or thiencarbazone-methyl. Thus, in some embodiments, the modified host plant characteristic is increased herbicide tolerance / resistance compared to a plant without the symbiont. The increased herbicide tolerance / resistance in the host plant can be to one herbicide, or can be to two or more different herbicides.

[0205] In some embodiments, the modified host plant characteristic can be a change in the plant's response to abiotic stress. In some embodiments, the modified host plant characteristic is tolerance to abiotic stress compared to a plant not comprising the symbiont of the present invention. In some embodiments, the host plant may exhibit increased tolerance to multiple abiotic stresses (e.g., 1, 2, 3, 4, 5, or more abiotic stresses). As used herein, the term "abiotic stress" refers to an external, non-living factor that can cause deleterious effects on a plant. Thus, abiotic stress as used herein includes, but is not limited to, low temperature resulting in freezing, chilling, heat, or high temperature, drought, excess water, high light intensity, low light intensity, high UV radiation, salinity, ozone, and / or combinations thereof. Parameters of abiotic stress factors are species-specific and even variety-specific and therefore vary widely according to the species / variety exposed to the abiotic stress. Thus, one species may be severely affected by temperatures as high as 23°C, while another may not be affected until temperatures reach at least 30°C. Temperatures above 30°C dramatically reduce the yield of most important crops. This is due to a decline in photosynthesis, which begins at approximately 20–25°C, and the increased carbohydrate demand of crops grown at higher temperatures. Critical temperatures are not absolute and vary depending on factors such as the crop's acclimation to prevailing environmental conditions. Furthermore, because most crops are exposed to multiple abiotic stresses at once, interactions between stresses affect plant responses. For example, damage from excess light occurs at lower intensities as temperatures rise beyond the photosynthetic optimum. Water-stressed plants are less able to cool overheated tissues due to reduced transpiration, further exacerbating the effects of excess (high) heat and / or excessive (high) light intensity. Therefore, the specific parameters that affect crop productivity, such as high / low temperature, light intensity, and drought, vary with species, variety, degree of acclimation, and exposure to a combination of environmental conditions.

[0206] As used herein, "increased tolerance to abiotic stress" refers to the ability of a plant or portion thereof comprising a symbiont of the present invention that is exposed to an abiotic stress to tolerate a given abiotic stress better than a control plant or portion thereof (i.e., a plant or portion thereof that is exposed to the same abiotic stress and does not contain the symbiont). Increased tolerance to abiotic stress can be measured using various parameters, including, but not limited to, the size and number of plants or portions thereof (e.g., the number and size of fruits), the level or amount of cell division, the amount of flower stunting, the amount of sunburn damage, crop yield, etc. Thus, in some embodiments of the present invention, a plant or portion thereof comprising a symbiont of the present invention and having increased tolerance to abiotic stress will have, for example, reduced flower stunting compared to a plant or portion thereof that is exposed to the same stress but does not contain the symbiont. Thus, in some embodiments, expression of a polynucleotide of interest in a symbiont can confer increased abiotic stress tolerance to a host plant. In some embodiments, the presence of biomolecules and / or bioactive molecules produced by the symbiont and transported to the host plant can confer increased abiotic stress tolerance to the host plant, thereby modifying host plant characteristics.

[0207] In some embodiments, the modified host plant characteristic is modified host plant morphology. The symbionts described herein containing and expressing a polynucleotide of interest can be used to alter any plant structure, including but not limited to, leaves, stems, flowers, roots, buds, seeds, meristems, fruits, tubers, etc. In some embodiments, modified morphology includes, but is not limited to, shortened internodes, increased lateral branching, and / or increased flowering compared to a plant without the symbiont.

[0208] In some embodiments, the modified host plant characteristic is the presence of a biomolecule, a bioactive molecule, and / or a polypeptide involved in the biomolecule and / or bioactive molecule biosynthesis, encoded by or resulting from the expression of a polynucleotide of interest (e.g., the polynucleotide of interest encodes a polypeptide or regulatory nucleic acid that affects the production of a bioactive molecule in the plant), which can then be transported into the host plant, thereby modifying the host plant characteristic; the modified host characteristic can include and / or be the result of the presence of the biomolecule and / or bioactive molecule. As described herein, the symbiont formed on the plant develops a vascular system that connects with that of the host plant. In some embodiments, the biomolecule and / or bioactive molecule produced in the symbiont (e.g., expressed by the polynucleotide of interest) can be transported to the host plant via the connected vascular system or tissue. In some embodiments, transport of biomolecules and / or bioactive molecules from the symbiont to the host plant can be systemic transport. In some embodiments, biomolecules and / or bioactive molecules produced in the symbiont can be transported to the host plant via the apoplast and / or symplast of the connected tissues between the symbiont and the host plant. In some embodiments, transport of biomolecules and / or bioactive molecules from the symbiont to the host plant can be via any combination of the connected vascular systems of the symbiont and the host plant, the apoplastic pathway, and / or the symplast pathway.

[0209] Thus, in some embodiments, a polynucleotide of interest encoding a biological molecule and / or biologically active molecule is contained in a symbiont of the present invention that is transplanted onto a host plant, the polynucleotide of interest is expressed in the symbiont, and the biological molecule and / or biologically active molecule is transported to the host plant.

[0210] In some embodiments, biomolecules and / or bioactive molecules may include, but are not limited to, pharmaceuticals, biostimulants, biofungicides, bioherbicides, insecticidal proteins / peptides, trypsin-modulating oostatic factors (TMOFs), Bacillus thuringiensis toxins, plant insecticidal proteins (VIPs), nutrients, plant growth regulators, RNAi, plant antibodies, stylet sheath inhibitor proteins, ribozymes, bacteriocins, plant lipids, plant fatty acids, plant oils, antimicrobial peptides, aptamers, CRISPR-Cas system polypeptides and corresponding CRISPR guide nucleic acids, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and / or engineered meganucleases. A "biomolecule" is any molecule produced by a living organism and / or part thereof (e.g., a cell or a cell-free system). Thus, a biomolecule includes any molecule produced by a symbiont that is derived directly or indirectly from a polynucleotide of interest contained and expressed in the symbiont and that is optionally transported to a host plant to which the symbiont is attached or attached. A biomolecule can also refer to a biomolecule (e.g., a second biomolecule) produced in a host plant as a result of transport into the host plant of a different biomolecule (e.g., a first biomolecule) expressed in the symbiont from a POI (e.g., the POI may encode an enzyme involved in the biosynthesis of the biomolecule, which is then produced in the host plant using the biosynthetic enzyme). A "biomolecule" includes, but is not limited to, a "biologically active molecule." A biologically active molecule includes any biomolecule that comprises a biological activity, numerous non-limiting examples of which are described herein.

[0211] As used herein, a "pharmaceutical agent" includes, but is not limited to, a therapeutic protein, a therapeutic polynucleotide, and / or a therapeutic chemical. In some embodiments, a pharmaceutical agent may include, but is not limited to, a vaccine, an antibody, a recombinant antibody, an antibody fragment, a fusion protein, an antibody fusion protein, human serum albumin, gastric lipase, insulin, glucocerebrosidase, a growth factor, a cytokine, hepatitis B surface antigen (HBsAg), Apo-A1, alpha-galactosidase (PRX-102), alpha-galactosidase (PRX-102), acetylcholinesterase (PRX-105), anti-tumor necrosis factor (Pr-anti-TNF), IgG, interferon-alpha, plasmin, lactoferrin, lysozyme, and / or collagen.

[0212] Examples of bacteriocins that may be encoded in a polynucleotide of interest include, but are not limited to, acidocin, actagardine, agrocin, alveycin, aureocin, aureocin A53, aureocin A70, vicine, carnosine, carnocycline, caseicin, cerein, circularin A, colicin, curvaticin, divercin, duramycin, enterocin, enterolysin, epidermin / gallidermin, erwiniocin, gardimicin, gassericin A, glycinecin, halocin, haloduracin, klebicin, lactocin S, lactococcin, lacticin, leucine ... Mention may be made of leucoccin, lysostaphin, macedocin, mersacidin, mesentericin, microbisporicin, microcin S, mutacin, nisin, paenibacillin, planosporicin, pediocin, pentosin, planosporicin, pneumocyclicin, pyocin, reutericin 6, sakacin, salivaricin, subrancin, subtilin, sulfolobicin, tasmancin, thuricin 17, trifolitoxin, variacin, vibriocin, warnericin, and / or warnerin.

[0213] Additional antimicrobial peptides useful in the present invention that may be encoded by a polynucleotide of interest include gramicidin (AVGALAVVVWLWLWLW SEQ ID NO: 35), magainin 2 (GIGKFLHSAKKFGKAFVGEIMNS SEQ ID NO: 36), LL-37 (cathelicidin) (LGDFFRKSKEKIGKEFKRIVQRIKFLRNLVPRTES SEQ ID NO: 37), pyrophocolicin (PrAMP) (VDKGSYLPRPTPPRPIYNRN SEQ ID NO: 38), nisin A (lantibiotic) (ITSISLCTPGCKTGALMGCNMKTATCHCSIHVSK SEQ ID NO: 39), HNP1 (α-defensin) (ACYCRIPACIAGERRYGTCIYQGRLWAFCC SEQ ID NO: 40), TAP (β-defensin) NPVSCVRNK (GICVPIRCPGSMKQIGTCVGRAVKCCRKK SEQ ID NO: 41), plectasin (GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY SEQ ID NO: 42), colistin (XTXXKLLXXT SEQ ID NO: 43) (X = 2,4-diaminobutanoic acid), daptomycin (WNDTGKDADGSEY SEQ ID NO: 44), microcin J25 (VGIGTPIFSYGGGAGHVPEYF SEQ ID NO: 45), alamethicin (peptaibol) (PBABAQBVBGLBPVBBEQ SEQ ID NO: 46) (B = α-aminoisobutyric acid), gramicidin (SVKLFPVKLFP SEQ ID NO: 47), subtilosin A (NKGCATCSIGAACLVDGPIPDFEIAGATGLFGLWG SEQ ID NO: 48), Kalata B1 (cyclotide) (GLPVCGETCVGGTCNTPGCTCSWPVCTRN SEQ ID NO: 49), and rhesus theta-defensin 1 (RTD-1) (GFCRCLCRRGVCRCICTR SEQ ID NO: 50).

[0214] Examples of bioinsecticides that may be encoded by a polynucleotide of interest include Javretox (e.g., SEQ ID NO: 24, polypeptide SEQ ID NO: 25), trypsin-modulating oostatic factor (TMOF) (e.g., SEQ ID NO: 26, polypeptide SEQ ID NOs: 27, 28), Bacillus thuringiensis toxins (e.g., delta-endotoxins, e.g., Cry (crystal) toxins, Cyt (cytotoxic) toxins) (e.g., SEQ ID NO: 33, polypeptide SEQ ID NO: 34), stylet sheath inhibitor proteins (e.g., ficin (e.g., SEQ ID NO: 51), bromelain), and / or plant insecticidal proteins (Vips). These are well-known polypeptides. Bacillus thuringiensis toxins include, for example, Cry (crystal) toxins (e.g., Cry I, Cry II, Cry III, Cry IV), Cyt (cytotoxic) toxins, plant insecticidal proteins (Vips) (which are classified into four families, Vip1, Vip2, Vip3, and Vip4, according to their degree of amino acid similarity), and secreted insecticidal protein (Sip) toxins. These proteins include toxins with varying spectrums of toxicity, which can be broad or narrow (e.g., toxic only to certain insect groups).

[0215] In some embodiments, the biologically active molecule encoded by the polynucleotide of interest is Jabretox (peptide JBTX), trypsin-modulating oostatic factor (TMOF), B. thuringiensis delta-endotoxin, Cry toxin, Cyt toxin, leghemoglobin, nitrogenase, ficin, bromelain, bacteriocin, nisin, oncocin, and / or an oncocin analog (e.g., SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32).

[0216] In some embodiments, the altered host plant characteristic is the presence of a bioactive molecule (e.g., a biocidal molecule) and increased resistance / tolerance to a plant pathogen compared to a plant without the symbiont, and the presence of a bioactive molecule transported into the plant from the symbiont. In some embodiments, the biocide is a bacteriocin or antimicrobial peptide and the plant pathogen is a bacterium. In some embodiments, the bacteriocin or antimicrobial peptide is oncocin and / or nisin.

[0217] In some embodiments, the altered host plant characteristics are the presence of an insecticidal protein (e.g., a bioinsecticide) and increased insect tolerance or resistance compared to a symbiont-free plant, as well as the presence of an insecticidal protein transported into the plant from the symbiont. In some embodiments, the insecticidal protein is Jabretox, trypsin-modulating oostatic factor (TMOF), Bacillus thuringiensis toxin (e.g., delta-endotoxin), optionally a Cry (crystal) toxin, a Cyt (cytotoxic) toxin, a plant insecticidal protein (Vip) or a secreted insecticidal protein (Sip) toxin, and / or a stylet sheath inhibitor protein, optionally ficin and / or bromelain.

[0218] In some embodiments, a stylet sheath inhibitory protein can be expressed by a polynucleotide of interest in a symbiont of the present invention. Such inhibitory peptides are known, as exemplified in U.S. Patent Application Publication No. 2018 / 0199577. Examples of stylet sheath inhibitory peptides useful for expression in a symbiont include, but are not limited to, those listed in Table 3.

[0219] Table 4 provides an illustrative list of examples of plants and diseases or pests (e.g., insect and / or nematode pests) to which the plants are vulnerable. In some embodiments, the present invention may be used to provide increased tolerance / resistance in plants to these diseases and pests.

[0220] In some embodiments, the altered host plant characteristic is the presence or increase or decrease of plant lipid, plant fatty acid, and / or plant oil production.

[0221] In some embodiments, the altered host plant characteristic is the presence or increased or decreased production of a plant growth regulator (e.g., auxin, cytokinin, gibberellin, ethylene, growth inhibitor / retardant) and altered growth. In some embodiments, the altered growth can be increased or decreased growth of the host plant and / or increased or decreased growth of a part of the host plant as a result of the transport of a growth regulator from the symbiont into the host plant or the transport of a bioactive molecule from the symbiont into the host plant, resulting in increased or decreased production of a growth regulator (e.g., a plant hormone biosynthetic enzyme) in the host plant. The increased or decreased production of the plant growth regulator and the altered growth is relative to a control plant (e.g., a plant without the symbiont and the presence of the plant growth regulator and / or an increased or decreased production of the plant growth regulator).

[0222] In some embodiments, the modified host plant characteristics are the presence or increase in the production of RNA and the increase / decrease in the production of polynucleotides, peptides, or polypeptides. The RNA useful in the present invention can be any RNA that can be used to modify plant characteristics, such as any RNA used in RNA interference (RNAi). In some embodiments, the RNA can include, but is not limited to, siRNA, dsRNA, miRNA, and / or shRNA. Exemplary RNAs include dvsnf7, ccomt, dCS, asn1, phL, RI, PGAS, and / or ppo5.

[0223] The present invention further provides host plants having altered characteristics produced by the methods of the present invention.

[0224] Also provided herein are methods for producing a biomolecule or bioactive molecule, the methods comprising the steps of providing a symbiont of the invention in which a polynucleotide of interest encodes the biomolecule and / or bioactive molecule and collecting the biomolecule and / or bioactive molecule produced in the symbiont or symbiont-formed inoculum, and / or providing a host plant of the invention in which a polynucleotide of interest encodes the biomolecule and / or bioactive molecule and collecting the biomolecule and / or bioactive molecule produced in the symbiont-formed inoculum and / or the symbiont and / or the host plant.

[0225] Further provided is a method for delivering a compound of interest to a host plant, the method comprising the steps of transplanting a symbiont-forming inoculum of the present invention or a symbiont of the present invention onto at least one site (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sites) on a host plant, and culturing the symbiont-forming inoculum or symbiont at least one site on the host plant to allow a symbiont to form at least one site on the host plant, wherein a polynucleotide of interest is expressed in the symbiont, and an expression product of the polynucleotide of interest and / or a product produced using the expression product of the polynucleotide of interest is transported into the host plant, thereby delivering the compound of interest to the plant.

[0226] Also provided are methods for producing a plant comprising an altered characteristic without altering the plant's genotype, comprising transplanting a symbiont-forming inoculum of the invention or a symbiont of the invention onto at least one site (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sites) on a host plant, and culturing the symbiont-forming inoculum or symbiont at least one site on the host plant to allow a symbiont to form at least one site on the host plant, wherein a polynucleotide of interest is expressed in the symbiont, and an expression product of the polynucleotide of interest and / or a product made using the expression product of the polynucleotide of interest is delivered into the host plant, thereby resulting in a plant comprising an altered phenotype without altering the plant's genotype. Also provided are plants produced by the methods of the invention.

[0227] As described herein, a polypeptide encoded by a polynucleotide of the present invention (e.g., a polypeptide encoded by a polynucleotide of interest, a plant hormone biosynthetic enzyme) may be operably linked to a targeting sequence. In some embodiments, a polypeptide may be linked to a targeting sequence at its N-terminus or its C-terminus, or both. A targeting sequence useful in the present invention may be any targeting sequence that can direct / locate a polypeptide or peptide to a particular organelle or plant part. The targeting sequence may be operably linked at the N- or C-terminus of the polynucleotide or nucleic acid molecule, and optionally, the polynucleotide or nucleic acid molecule is heterologous to the targeting sequence.Targeting (or signal) sequences or targeting peptides (and the nucleotide sequences encoding them) are well known in the art and can be found in the Signal Peptide Website: An Information Platform for Signal Sequences and Signal Peptides (www.signalpeptide.de), the Signal Peptide Database (proline.bic.nus.edu.sg / spdb / index) (Choo et al., BMC Bioinformatics, 6:249 (2005) (available at biomedcentral.com / 1471-2105 / 6 / 249 / abstract), ChloroP (cbs.dtu.dk / services / ChloroP / , which predicts the presence of chloroplast transit peptides (cTPs) in protein sequences and the location of potential cTP cleavage sites), LipoP (cbs.dtu.dk / services / LipoP / , which predicts lipoproteins and signal peptides in Gram-negative bacteria), MITOPROT (ihg2.helmholtz-muenchen.de / ihg / mitoprot, which predicts mitochondrial targeting sequences), PlasMit (gecco.org.chemie.uni-frankfurt.de / plasmit / index, which predicts mitochondrial targeting sequences in Plasmodium falciparum) falciparum), Predotar (urgi.versailles.inra.fr / predotar / predotar.html, which predicts mitochondrial and plastid targeting sequences), PTS1 (mendel.imp.ac.at / mendeljsp / sat / pts1 / PTS1predictor.jsp, which predicts peroxisome targeting signal 1-containing proteins), and SignalP (cbs.dtu.dk / services / SignalP / , which predicts the presence and location of signal peptide cleavage sites in amino acid sequences from various organisms, i.e., gram-positive prokaryotes, gram-negative prokaryotes, and eukaryotes).The SignalP method incorporates cleavage site prediction and signal / non-signal peptide prediction based on a combination of several artificial neural networks and hidden Markov models, while TargetP (cbs.dtu.dk / services / TargetP / ) predicts the subcellular location of eukaryotic proteins, with location assignment based on the predicted presence of an N-terminal presequence, i.e., a chloroplast transit peptide (cTP), a mitochondrial targeting peptide (mTP), or any of the secretory pathway signal peptides (SPs). (von Heijne, G., Eur J Biochem, 133(1)17-21(1983); Martoglio et al., Trends Cell Biol, 8(10):410-5(1998); Hegde et al., Trends Biochem Sci, 31(10):563-71(2006); Dultz et al., J Biol Chem, 283(15):9966-76(2008); Emanuelsson et al., Nature Protocols, 2(4)953-971(2007); Zuegge et al., 280(1-2):19-26(2001); Neuberger et al., J Mol Biol., 328(3):567-79(2003); and Neuberger et al., J Mol Biol., 328(3):581-92 (2003). Examples of targeting sequences useful for targeting polypeptides as described herein include, but are not limited to, those provided in Table 5. In some embodiments, the polypeptide encoded by the POI may be operably linked to a sequence that targets the secretory system (e.g., to the endoplasmic reticulum (ER), e.g., an ER targeting sequence).

[0228] As described herein, plants, plant parts, or plant cells useful in embodiments of the present invention may be or be derived from any plant, including, but not limited to, angiosperms (e.g., dicotyledons or monocotyledons), gymnosperms, algae (e.g., macroalgae, e.g., Rhodophyta (red algae), Phaeophyta (brown algae), and Chlorophyta (green algae), Chrysophyceae (chrysophyceae)), bryophytes, ferns, and / or fern-like plants (i.e., ferns).

[0229] Plants useful in the present invention (e.g., for the symbiont-forming inoculants, symbionts, plants, or host plants described herein) include, but are not limited to, Abelia species (abelia), Abelmoschus species (okra), Abies species (fir), Acacia species (acacia), Acalypha species (chenille), Acca species (feijoa, pineapple guava, guavasteen), Acer species (maple), Sawberry, ... Achillea species (yarrow), Achlys species (barberry), Acmella species (holland amaranth), Acoelorrhaphe species (palm), Acorus species (calamus), Acronychia species (aspen), Acrostichum species (fern), Acrotriche species (currant), Actinidia species (kiwi fruit), Baobab species nsonia species (baobab), Adiantum species (capillary fern), Adonidia species (palm), Aechmea species (bromeliad), Aegle species (bell tree), Aesculus species (horse chestnut, horse chestnut), Aframomum species (false cardamom), Agapanthus species (agapanthus), Agaricus species (mushroom), Agastache species (anise) ), Agathosma species (bukko tree), Agave species (agave), Ageratum species (whiteweed), Aglaonema species (green bamboo), Agrimonia species (agrimony), Ailanthus species (ailanthus), Ajuga species (ajuga), Albizia species (silk tree), Alchemilla species (alchemilla),Aleurites species (Kukui), Allamanda species (Allamanda), Allium species (Chive, Garlic, Leek, Onion, Shallot), Alnus species (Alder), Alocasia species (Taro), Aloe species (Aloe), Aloysia species (Lemon Verbena), Alpinia species (Alpinia japonica), Alternanthera species (Joiweed) (Gard), Althaea species (Marshmallow), Amaranthus species (Amaranth), Amelanchier species (Juneberry, Serviceberry), Amomum species (Cardamom), Amphitecna species (Black Calabash), Anacardium species (Cashew), Ananas species (Pineapple), Anaphalis species (Andrographis), Andrographis Andrographis species (false water willow), Andromeda species (rhododendron), Anethum species (dill), Angelica species (angelica), Angelonia species (angolonia), Angostura species (angostura), Annona species (cherimoya, sweetsop, sugar-apple, soursop), Anogeissus species ) seeds (Axelwood), Anthemis seeds (Chamomile), Anthoxanthum seeds (Grass), Anthriscus seeds (Chervil), Anthurium seeds (Tailflower), Antidesma seeds (Bignay), Antigonon seeds (Morning Vine), Antirrhinum seeds (Snapdragon), Apium seeds (Celery),Aquilegia species (Aquilegia), Arabidopsis species (Arabidopsis, Mouse-ear Cress), Aralia species (Walking Stick, Aralia), Araucaria species (Pine), Arbutus species (Madrone, Strawberry), Arctium species (Burdock), Arctostaphylos species (Bearberry, Rhododendron), Ardisia species (Ardisia), Armeria species (Sea Thrush), Armoracia species (Horseradish), Aronia species (Ardisia), ia species (Begonia), Arracacia species (Arracacha), Artemisia species (Wormwood), Artocarpus species (Breadfruit, Jackfruit, Monkey Fruit), Aruncus species (Kernel), Arundinaria species (Bamboo), Asarum species (Ginger), Asclepias species (Milkweed), Ascophyllum species (Feamainn Bhui), Rockweed, Norway Kelp, Knotted Kelp, Knotted Wrack, Egg Wrack wrack), Asimina species (pawpaw), Aspalathus species (rooibos), Asparagus species (asparagus, linden), Aspidistra species (asparagus), Aspidosperma species (quebranch), Asplenium species (nest fern), Aster species seeds (aster), Astragalus seeds (vetch), Asystasia seeds (ascystasia), Athyrium seeds (fern), Atriplex seeds (mountain spinach), Auriculari seeds (edible fungus), Avena seeds (oats), Averrhoa seeds (star fruit),Baccaurea species (Lotkon), Baccharis species (Saltbush), Backhousia species (Ironwood), Bactris species (Peach palm), Balanites species (Torchwood), Baleria species (Violets), Bambusa species (Bamboo), Baptisia species (Indigo), Barbarea species (Caramel) China), Basella species (spinach), Bauhinia species (philippines), Beaucarnea species (palm), Begonia species (begonia), Belamcanda species (lilies), Benincasa species (winter), Berberis species (barberry), Bertholletia species (Brazil nuts), Beta species (beets), Birch species (Betu la) species (birch), Bidens species (Beggarticks), Billardiera species (appleberry), Bischofia species (bishopwood), Bismarckia species (palm), Bixa species (annatto), Blighia species (ackee), Boesenbergia species (fingerroot), Borago species (borage), Palmyra palm (B orassus seeds (palm), Borojoa seeds (borojo), Borrichia seeds (sea oxeye), Boscia seeds (Hanza), Boswellia seeds (frankincense), Bouea seeds (plum mango), Brahea seeds (palm), Brassica seeds (broccoli, chard, cabbage, cauliflower, Chinese cabbage, kale, mustard, mustard greens, rapeseed, rutabaga, Brussels sprouts),Breynia species (snowbush), Brosimum species (breadnuts), Browallia species (amethyst flower), Brunfelsia species (American bush), Buchanania species (chilaurinuts), Bucida species (rosewood), Bumelia species (chittamwood), Bunchosia species (peanut butter fruit), Bursera species Limbo, Butia species (Brazilian palm), Buxus species (Boxwood), Byrsonima species (Locustberry), Caesalpinia species (Caesalpinia), Cajanus species (Pigeonpea), Caladium species (Caladium), Calamagrostis species (Reeds, Smallweed), Calathea species (Calatea, Fritillary, Relen), Calendula dula species (marigold), Calliandra species (fairy duster), Callicarpa species (purple bark), Callistemon species (bottle bottle), Calocedrus species (cypress), Calophyllum species (calico), Calycanthus species (black laurel), Calyptranthes species (lidflower) -, spicewood, mountain bay), Camassia species (bark hyacinth, wild hyacinth), Camelina species (false flax, camellia), Campanula species (bulb bellflower), Campomanesia species (guaviloba), Campsis species (trumpet creeper, hummingbird vine), Canarium species (Pacific almond), Canavalia species (jack bean),Canella species (cinnamon bark), Canna species (canna lily, canna), Cannabis species (cannabis), Capparis species (capers), Capsella species (shepherd's purse), Capsicum species (bell pepper, cayenne pepper, chili, jalapeño, Pepper), Carex species (Sedges), Carica species (Papaya), Carissa species (Carissa numnum), Carnegiea species (Saguaro), Carpentaria species (Carpentaria palm), Carpinus species (Horn), Carpobrotus species (Pigface, Ice plant, Sour fig, Bakuyage), Carthamus species (Safflower) , Carum species (Carum carum), Carya species (Hickory nut, pecan), Caryocar species (Pequy, Suari nut), Caryota species (Caryota), Casasia species (Casasia, 7-year apple), Cassimiroa species (Sapote), Cassia species (Cassia), Castanea species (Chestnut, Chinquapin), Casuarina species (Casuarina), Casuarina Casuarinaceae species (Casuarinaceae), Catalpa species (Catalpa, Catawba), Catharanthus species (Periwinkle), Ceanothus species (Solicha, Buckbrush, Soapbush), Cedrus species (Cedar), Ceiba species (Botanical cotton), Celosia species (Celosia, Woolflower), Celtis species (Celtis berry, Celtis tree), Celastrus species (Celtis japonica, Celtis tree), ntaurium species (Centella), Centella species (Centella asiatica), Centratherum species (Brazilian button flower, Lark daisy), Cephalanthus species (American ragwort), Cerastium species (Southern ragwort), Ceratonia species (Carob), Cercidiphyllum species (Katsura), Cercis species (Redbud),Chaenomeles species (quince), Chaerophyllum species (chervil), Chamaecyparis species (false cypress), Chamaedorea species (bamboo palm, table palm), Chamaemelum species (chamomile), Chamaerops species (European fan palm), Chelidonium species (celandine), Chenopodium species (pigweed), Chilopsis species (Desert Willow), Chimaphila species (Giant Willow), Chimonobambusa species (Bamboo), Chiococca species (Milkberry, Snowdrop), Chionanthus species (Chrysanthemum), Chrysanthemum species (Chrysanthemum), Chrysobalanus species (Squid), Chrysophyllum species (Kainit, Satinleaf), Cicer species (chickpeas), Cichorium species (chicory, endive, arkroot), Cinchona species (cinchona), Cinnamomum species (cinnamon, camphor, cassia), Cirsium species (thistles), Citharexylum species (vervain, zitterwood), Citrillus species (watermelon), Citrus species (citrus, grapefruit, lemon) Lime, Orange, Pummelos, Tangerines), Cladrastis species (Yellowwood), Clarkia species (Godetia), Clausena species (Wampi), Claytonia species (Portulaca), Cleome species (Spider Plant, Bee Plant, Cat Whisker), Clerodendron species (Honeywort, Honeysuckle, Bleeding Heart), Clinopodium species (Calamint),Clusia rosea species (Clusia, Pitch apple), Coccoloba species (Sea buckthorn), Coccothrinax species (Silver palm), Cocos species (Coconut), Coffea species (Coffee), Coleus species (Coleus), Colocasia species (Taro), Colubrinus species (Colu brina species (naked wood, snakewood, mung bean, hog plum), Combretum species (bush willow), Commiphora species (myrrh), Conocarpus species (sycamore), Conradina species (false rosemary), Conringia species (hare's ear mustard) Mustard), Convallaria species (Lily of the valley), Copaifera species (Copaiba), Coptis species (Coptis), Corchorus species (Jute), Cordia species (Mangel, Bocote), Cordyline species (Ti plant, Palm lily), Coreopsis species (Peacock, Tickseed), Coriandrum species (Coriander, Cilantro), Cornus species (Dogwood), Coronilla species (Coro nilla species (Thornberry), Corydalis species (Cypress), Corylus species (Hazel, Hazelnut), Cosmos species (Cosmos, Mexican Aster, Kennequil), Costus species (Spiral Ginger), Cotinus species (Cotinus cotinus), Crambe species (Crambe), Crassocephalum species (Raguleaf, Thickhead, Bologi, Ebolo), Crassula species (Crassula,Pygmyweed), Crataegus species (hawthorn, crataegus, datura, may tree, howberry), Crescentia species (gourd, huingo, krabasi, kalebas), Crinum species (crinum, sea laurel), Crocus species (saffron), Crotalaria species (chipilin), Cryptotaenia species (Mitsuba, Japanese cedar), Cucumis species (cantaloupe, cucumber, melon, gherkin, muskmelon, honeydew), Cucurbita species (pumpkin, summer squash, winter squash), Cuminum species (cumin), Cunninghamia species (Chinese fir, Chinese fir), Cupaniopsis species (Tuckeroo, Soapberry), Cuphea species (Cuphea, cigar plant, heather), Cupressoca yparis seeds (Leyland Cypress, Leyland Cypress), Cupressus seeds (Cypress), Curcuma seeds (Turmeric), Cyamopsis seeds (Guar), Cycas seeds (Cycad), Cyclopia seeds (Honeybush), Cydonia seeds (Quinces), Cymbopogon seeds (Lemongrass), Cynara seeds (Cardoon, Artichoke, Thistle), Chayote Cyperus species (Cyperus officinalis, Cyperus serrata, Cyperus serrata, Nutsedge, Umbrella-sedge, Galingale), Dahlia species (Dahlia), Dalbergia species (Kingwood, Indian Rosewood, African Blackwood, Liriodendron), Daucus species (Carrot), Davidsonia species (Ooray), Delonix species (Delonix regia),Dendranthema species (Chrysanthemum), Dendrocalamus species (Bamboo), Deparia species (Fern), Dermatophyllum species (Mescal bean), Deschampsia species (Tussock grass), Dialium species (Tamarind), Dianthus species (Carnation, Pink, American Dianthus, Dianthus), Dicentra species ntra) species (Bleeding Hearts), Dictyophora species (Stinkhorns), Dietes species (Wood Iris, Fortnite Lily, African Iris, Japanese Iris, Butterfly Iris), Dimocarpus species (Longan), Dioscorea species (Yams), Diospyros species (Persimmons, Black Sapote), Diplazium species (Ferns), Diplotaxis species (Robow) Mustard), Dizygotheca species (Pseudoaralia, Dizygotheca), Dodonaea species (Sugar), Doellingeria species (Cham-chwi), Dombeya species (Dombeya, Dikbas, Pinkball), Dovyalis species (Gooseberry, Kay apple), Dracaena species (Dragon tree, Dracaena), Dryopteris species eris species (ferns), Durio species (durian), Dypsis species (areca palm), Dyschoriste species (snake herb), Dysphania species (foxtail), Echinacea species (flowering cornflower), Echium species (purple berry), Ecklonia species (earberry), Elaeagnus species (silver berry, walnut),Elaeocarpus species (Ceylon olive), Elettaria species (cardamom), Elwendia species (black cumin), Elymus species (quackgrass, barley, wheatgrass), Epilobium species (red herb), Epimedium species (epimedium), Epipremnum species (pothos, Centipede, tongavine, pothos, devil's ivy), Eremocitrus species (desert lime), Erigeron species (flea grass, mugwort), Eriobotrya species (loquat), Eriodictyon species (Santa grass), Ernodea species (Beech creeper, coughbush), Eruca species (arugula), Eryngium species ( Eryngium species (Eryngium, Eryngium, Culanthro), Erythrina species (Deigo, Brassica campestris, Bucale, Kafferboom), Eucalyptus species (Gum, Eucalyptus, Marie), Eucharis species (Amazon lily), Eucommia species (Chinese rubber tree), Eugenia species (Dune myrtle) myrtle, rainforest plum, wild cherry, pitanga, araza), Euodia species (Euodia), Eupatorium species (Eupatorium, Eupatorium, Snakeroot), Euphorbia species (Spurge), Euryops species (Euryops), Eustoma species (Lisianthus, Prairie Gentian), Euterpe species (Assai palm), Exacum species (Persian violet), Fagopyrum species (Buckwheat), Fagus ) species (beech tree), Fatshedera species (tree ivy, Aralia ivy), Ferula species (fennel, musk wood, pepper), Festuca species (fescue), Ficaria species (celandine), Ficus species (fig), Filipendula species (spirit of staghorn), Firmiana species (branchia), Flacourtia species (battocoplum), Flammulina species (enokitake mushroom),Foeniculum species (fennel), Forestiera species (swamp privet), Fortunella species (kumquat), Fothergilla species (alder), Fragaria species (strawberry), Frangula species (cascara), Franklinia species (Franklin tree), Fr axinus species (ashweed), Fritillaria species (fritillary), Fucus species (rockweed), Fumaria species (fritillary), Gaillardia species (gaillardia), Galium species (woodworm), Ganoderma species (reishi mushroom), Garberia species (Garberia, Garber's scrub) starts), Garcinia species (mangosteen, sap tree, garcinia), Gardenia species (gardenia), Gaultheria species (wintergreen, bayberry, snowbell, sharon), Gaylussacia species (huckleberry), Gazania species (gazania, trailing gazania, clumping gazania) ng) Gazania), Geijera species (Geijera, Wilga, Oilbush, Sheepbush), Genipa species (Genip), Gentiana species (Gentiana), Geranium species (Geranium, Cranesbill), Gigantochloa species (Bamboo), Ginkgo species (Ginkgo, Maidenhair tree), Glebionis species (Chrysanthemum, Garland daisy, Garland daisy), Gleditsia species (Honeylocust), Glinus species (Sweet juice), Glycine species (Soybean),Gomphrena species (globe globe), Goodyera species (jade orchid, screw pine), Gordonia species (loblolly-bay), Gossypium species (cottonseed), Grevillea species (grevillea, spider flower, Chinese holly, toothbrush plant) plant), Grewia species (Falsa), Grifola species (Maitake mushroom), Grindelia species (Grindelia), Guaiacum species (Guaiac), Guizotia species (Niger seeds), Gymnema species (Gymnema), Gymnocarpium species (Rabbit fern), Gymnocladus species (Coffee tree, Soap tree), Hakonechloa species (Hakone grass, Halesia), Halesia species (Silver bell, Snowdrop tree) -), Hamamelis species (witch hazel), Hamelia species (firebush, hummingbird bush, scarlet bush, redhead), Hancornia species (mangaba), Harpephyllum species (kaffir plum), Hedychium species (shrimp, ginger lily, cauliflower), Helianthus species (sunflower, Jerusalem artichoke), Helichrysum species (curry plant), Heliconia species (lobster claw, toucan beak) beak), wild plantain, false bird-of-paradise), Helictotrichon species (blue oat grass), Hemerocallis species (daisy), Heracleum species (hogweed), Hericium species (pom pom,Edible mushrooms), Hesperis species (spider radish), Heuchera species (coral bell, alumroot), Hibiscus species (hibiscus, hibiscus, rose of Sharon), Hierochloe species (grass), Hippeastrum species (amaryllis), Hippophae species (sea buckthorn), Holodiscus species (ocean spray, cream bush), Hordeum species (barley), Hosta species (hosta, hosta, Plantain lily), Houttuynia species (Houttuynia), Hovenia species (Hovenia), Howea species (Large-leaved Kentia palm, Mountain palm, Belmore hoea), Hoya species (Wax plant, Wax vine, Wax flower, Hoya), Hybrid species (Astilbe), Hydrangea species (Hydrangea, Western hydrangea), Hydrophyllum species (Goby) Hylocereus species (dragon fruit, pitahaya), Hymenaea species (couril burr), Hymenocallis species (lily of the valley), Hypericum species (St. John's wort, goat weed), Hyphaene species (dome palm), Hypsizygus species (bunashimeji mushroom), Hyssopus species (herb hyssop), Ilex species seeds (holly, ilex), Illicium species (star anise, anise tree), Impatiens species (impatiens, jewelweed, balsam, snapweed, patience, balsam, African balsam), Imperata species (imperata grass), Indigofera species (indigo), Inga species (inga), Ipomoea species (sweet potato, morning glory, water spinach,Kangkung, bindweed, night-glory, Iris species (iris), Irvingia species (dika), Iva species (sambucus), Ixora species (West Indian jasmine, viruchi, rangan, kheme, ponna, chantane, techi, pan, siantan, jarum-jarum, jejarum, jungle flame, jungle geranium, cruz de malta Malta), Jacaranda species (Jacaranda), Jasminum species (Jasmine), Jatropha species (Jatropha curcas, Nettlespurge), Jubaea species (Palm), Juglans species (Walnut), Juncus species (Rush), Juniperus species (Juniper), Justicia species (Foxglove, Spirea, Malabar nut), Kalanchoe species (Kalanchoe, Panda plant, Mother of a thousand) thousands), felt plant), Kalimeris species (Indian aster, Kalimeris aster), Kalmia species (Sheep-laurel, lamb-kill, calf-kill, kill-kid, sheep-poison, spoonwood), Kalopanax species (Caster araria, tree araria, prickly castor oil tree), Kniphofia species (Tritoma, red hot poker, torch lily, knoffler, poker plant), Koelreuteria species (Scutellaria, flame gold, Chinese flame tree), Kunzea species (Kunzea, Kanuka, Leptospermum, Muntry),Lablab seeds (hyacinth bean, bataw, Indian bean), Laburnum seeds (sweet lily, laburnum, laburnum), Lactuca seeds (lettuce, celtuce), Lagenaria seeds (gourd tree, gourd fruit), Lagerstroemia species (crape myrtle), Laminaria species (kelp, kelp), Lansium species (lansones, lansat), Latania species (rattan palm, ratania palm), Launaea species (launea), Laurus species (laurel, sweet bay), Lavandula species (lavender), Lecythis species (paradise nut, monkey pot, cream nut, sapcay) Annatto, Leea species (Udonoki, Talyantan), Lens species (Lentil), Lentinula species (Shiitake), Leonurus species (Motherwort), Lepidium species (Peppercress, Peppergrass, Pepperwort, Tumbleweed), Lepista species (Purple moss, Mushroom-forming fungi), Lespedeza species (Thorn, Lesquercus), Lesquerella species (Lesq uerella species (Gaslight Bladderpod), Lessertia species (Balloon Pea), Leucaena species (Leucaena), Leucanthemum species (Max chrysanthemum, American sea cucumber, Oxeye daisy, Shasta daisy), Leucothoe species (Iwananthya, Sweetbell, Dog hobble, Black laurel), Leucothrinax species (Palm), Levisticum species (Rabbit's (Lewisia), Liatris species (Blazing Star), Licania species (Gopher Apple, Sansapote, Merecure, Oisitica), Ligustrum species (Private Tree), Lilium species (True Lily, Lilium), Limnanthes species (Meadowfoam), Limnophila species (Marshweed), Limonia species (Wood Apple),Limonium species (Siberian pine, statice, caspia, marsh rosemary), Lindera species (spicewood, spicebush, benjamin bush), Linnaea species (beauty bush, Linum), Lippia species (Lippia, Mexican oregano, licorice, verbena), Liquidambar species (American storax, satin walnut, red gum, sweetgum, star gum), Liriodendron Liriodendron species (tulip tree, tulip tree, yellow poplar), Liriope species (liriope, monkey grass, spider grass), Litchi species (lychee), Livistona species (bush laurel), Lobelia species (lobelia), Lobularia species (sweet almond), Lonicera species (honeysuckle), Loropetalum species (witch hazel, Chinese fringeflower) Lotus species (lotus, deervetch, lotus grass, trifoliate), Luffa species (gourd, loofah), Lunaria species (ginseng), Lupinus species (lupine), Lychnis species (clamshell), Lycium species (goji berry, wolfberry, desert thorn), Lycopersicon species (tomato, wild tomato), Lycopus species (gypsy wattle) Lyonia species (Staggerbush, Poor-grub, Mailberry, Heehuckleberry, Hurrahbush), Lysichiton species (Skunk cabbage, Swamp plantain), Lysiloma species (False tamarind, Sabik), Lysimachia species (Loosestrife), Maackia species (Maackia), Macadamia species (Macadamia),Maclura species (cocksparthorn, osage orange, Dyer's mulberry, mandarin melonberry), Macrocystis species (giant kelp, giant bladder kelp), Magnolia species (magnolia), Mahonia species (mahonia, Fremont mahonia, agarita, chaparral berry), Maianthemum species (sasa), Malcolmia species (sea lavender, African mustard), Mallotonia species (sea lavender), Malpighia species (acerola, Barbados cherry, dwarf holly), Malus species (Apple, Crabapple, Crabtree, Wild Apple), Mammea species (Mummy Apple, Tropical Apricot, Sarapee), Mandevilla species (Rock Trumpet, Allamanda), Mangifera species (Mango, White Mango, Jack, Puffin, Paho), Manihot species (Cassava), Manilkara species (Sapodilla, Massaranzaba, Chicle, Sapota), Maranta species (Fritillary, Strawberry, Maranta), Marlierea species (Belquillo), Marrubium vulgare vulgare (horehound), Matisia species (molinillo, chupa-chupa), Matricaria species (German chamomile, false chamomile), Matteuccia species (feather cycad, fern, shuttlecock fern), Matthiola species (stock, stock), Medicago species (alfalfa, medic, medic), Melaleuca species (paperbark, reed, tea tree), Melampodium species (blackfoot), Melia species (chinaberry,Persian ash tree, Melicoccus species (Melicoccus spp., Motoyoe, Quenepa), Melilotus species (Chinese bush clover, sweet clover, spider moth), Melissa species (Lemon balm), Mentha species (Mint), Merrillia species (Flower mellilia, katinga, Malay lemon), Mesembryanthemum species (Ice plant), Mespilus species (Medlar), Metasequoia species a) species (Akebonosugi), Michelia species (Megasus, White Champaka, Goldenrod, Dandy, Togatama), Microcitrus species (Lime), Micromeria species (Yerba buena, White Micromeria, Whiteleaf Savory, Micromeria), Milicia species (Iroko, African Teak, Odum), Millettia species (Pongamia), Mimulus species (Miscanthus), Miscanthus species (Silvergrass, Maiden grass), Mitchella species (bitter melon), Momordica species (bitter melon, southern gourd, spine gourd, cantola, momordica), Monarda species (mountain mint, horse mint, honeysuckle, bergamot), Monstera species (capsicum, shingle plant, five holes plant) plant), Monstera), Montia species (miner's lettuce, chickweed, winter purslane), Morchella species (morels), Morinda species (noni, Indian mulberry, sweet morinda, redgal, yawweed, cheese shrub),Morus species (mulberry), Mucuna species (deer's eye peas, donkey's eye peas, bull's eye peas, hamburger beans), Muhlenbergia species (Muhly), Muntingia species (Jamaica cherry), Murraya species (curry tree, orange jasmine, China box), Musa species (banana, plantain), Myrcianthes species (Lucumillo, twinberry, arrayan, guabiyu), Myrciaria species (jabuticaba, guavaberry, hiva berry) puru, sabara, ybapuru), Myrica species (bayberry, bayberry, candleberry, bay willow, wax myrtle), Myristica species (nutmeg, mace, kumpang, Macassar nutmeg, silver nutmeg), Myroxylon species (balsam), Myrrhis species (sissary, myrrh, sweet chervil), Myrsine species (colicwood, korea, machipo), Myrtus species (myrtle), Nandina species (nandina, heavenly) bamboo, sacred bamboo), Narcissus species (daffodil, narcissus, narcissus), Nasturtium species (water lily, water lily), Nastus species (bamboo), Nelumbo species (lotus), Neomarica species (American iris, apostle's iris, neomarica), Nepeta species (catnip, catmint, catswort), Nephelium species (rambutan, koran, prasan), Nephrolepis species (macho fern, ball fern), Nerium species (oleander,Oleander), Nicotiana species (tobacco), Nigella species (black caraway, nigella, devil-in-a-bush, love-in-a-mist), Noronhia species (Madagascar olive), Nymphaea species (sweet, Water lily, Nyssa species (Nyssa, Water Dogwood), Ochrosia species (Elliptic Yellowwood, Bloodhorn, Coptis, Kauai Yellowwood, Southern Ochrosia), Ocimum species (Basil, Lemon Basil, Sweet Basil, Holy Basil), Odontonema species (Thoothed Threads), Oenocarpus species (Turu Palm, Palma Milpessoa) milpesos, bakaba, patawa), Oenothera species (primrose, evening primrose, suncup, sundrop), Olea species (olive, black ironwood, ironwood, East African olive, Ergon's teak), Onobrychis species (sainfoin), Oplopanax species (spinach, Alaskan ginseng), Opuntia species (prickly pear, tuna cactus, nopal cactus), Origanum species (oregano, marjoram, Cretan dittany, Bible hyssop) hyssop), Oryza seeds (rice, Zizania latifolia, African rice, longstamen rice, red rice, Asian rice), Osmanthus seeds (osmanthus, holly), Osmunda seeds (regalis osmanthus, flowering osmanthus) fern), Osmundastrum species (Osmundastrum), Ostrya species (Oxalis), Oxalis species (Oxalis, Oxalis, False Shamrock, Sour Grass, Oxalise), Oxydendrum species (Sourwood, Sour Tree), Pachira species (Guiana Chestnut, Money Tree, Malabar Chestnut, French Peanut, Provision Tree, Saba Nut, Monguba, Pochote), Pachyrhizus species (Jicama, Yam Bean, Nup, Ahipa),Pachystachys species (cardinals guard, lollipop plant, golden shrimp plant), Paeonia species (peony, Polish rose), Panax species (Korean ginseng, Chinese ginseng, Panax notoginseng, mountain plant, Himalayan ginseng), Pandanus species (pandan, screw palm, Pandanus tree, Nicobar breadfruit, kalka), Pandorea species (Wonga vine, Bower of beauty), Pandora vine, Boat vine), Panicum species (panic grass, foxtail millet, common millet, tumbleweed, maiden cane), Papaver species (poppy), Parkia species (bitter bean, African carob), Parkinsonia species (palo verde, brea, verde olivo), Parrotia species (Persian ironwood, Chinese ironwood), Parthenocissus species (Virginia ivy, sweet pea, seven-leaf creeper, Boston ivy), Passiflora species (passion fruit, passion flower, passionflower vine, invertebrate passionflower, passionflower kudzu), and Parsiflora species (Passion fruit, passion flower, passionflower vine, passionflower kudzu), tinaca seeds (parsnip), Paullinia seeds (guarana, yoko), Paulownia seeds (princess tree, kiri, Korean kiri, dragon tree), Paxistima seeds (Oregon boxleaf, Camby's Mountain Love), Pelargonium seeds (geranium, Dutch geranium, pelargonium), Peltophorum species (Weeping Wattle, Copperpoda, Yellow Flamboyant, Red Poinciana, Yellow Poinciana), Pennisetum species (Fountain Grass, Pearl Millet, Kikuyu Grass, Feathertop Grass), Penstemon species (Rockberry), Pentalinon species (Wild Allamanda),Pentas species (Pentas, Penta), Peperomia species (Radiator Plant, Peperomia), Perilla species (Perilla, Shiso), Persea species (Avocado, Bay, Koyo, Red Bay, Swamp Bay), Persicaria species (Willow Polygonum, Polygonum, Smartweed, Hot Mint), Petasites species (butterbur, coltsfoot), Petroselinum species (parsley), Petunia species (petunia), Peucedanum species (honeydew), Peumus species (Bordeaux), Phaseolus species (bean, wildbee) Phellodendron species (cork oak, Phellodendron bark), Philadelphus species (Philodendron, rascagarganta, vilevine, treelover), Phlox species (phlox, wild sweet William), Phoenix species (date palm, date), Pholiota species (nameko, mushroom), Photinia species (Japanese hawthorn), Phyllanthus species (gooseberry, leaf flower, scrubby spurge) spurge, redroot floater, sand reverchonia, gripeweed, shatterstone), Phyllostachys species (golden bamboo, fishpole bamboo, yellow groove bamboo, Madake bamboo, timber bamboo, moso bamboo), Physalis species (ground cherry, grape ground cherry, husk tomato, Inca berry, poha berry, golden berry, cape gooseberry), Physocarpus species (ninebark), Picea species (spruce),Pilea species (morning glory, rice plant, silver sprinkle, friendship plant, creeping charlie), Pimenta species (allspice, balsam, ciliment), Pimpinella species (anise, aniseed, honeysuckle, chamnamul, saxifrage), Pinckneya species (Georgia bark, pinkneya), Pinus species (pine), Piper species (pepper, parpaloba, Mexican pepper) Pepper leaf, betel vine), Pipturus species (mamaki), Pistacia species (pistachio, mastic), Pisum species (pea), Pithecellobium species (Madras thorn), Pittosporum species (pittosporum, oil nut, cheesewood), Plantago species (plantain), Platanus species (plane tree, sycamore), Platonia species (baki) Bacury), Platycladus species (Biota), Platycodon species (Banana), Plectranthus species (Sparflower), Pleurotus species (Oyster mushroom), Plinia species (Brazilian grape tree, Jaboticaba, Cambuca), Plumbago species (Leadwort), Plumeria ) species (Plumeria, Indian Jasmine), Podocarpus species (Yellowwood, Pine, Illawara Plum), Polygonatum species (Polygonatum), Polypodium species (Siberian laurel, Rock Cap Fern), Polyscias species (Mingararia, 'ohe), Polystichum species (Fern), Poncirus species (Polycrest orange), Pontederia species (Latin Monochaete),Populus species (poplar, aspen, cottonwood), Porophyllum species (coriander), Portulaca species (purslane), Potentilla species (cinquefoil, bellflower, strawberry), Pouteria species (aphid, canistel, lucuma, sapote), Primula species (primrose), Probosc idea seeds (hornbeam), Prosopis seeds (mesquite), Prostanthera seeds (mint bush), Prunella seeds (medicinal herbs), Prunus seeds (almonds, apricots, cherries, chokecherries, nectarines, peaches, plums, plumcots, prunes, sloes), Pseudanamomis seeds (Monos plums) plum), Pseudolarix species (Golden Larch), Pseudotsuga species (Dog Fir), Psidium species (Guava), Psychotria species (Wild Coffee), Ptelea species (Hop Trees), Pteridium species (Ferns), Pterocarpus species (Saunders), Pterocarya species (Japanese Walnut), Pterostyrax species (Large-leaved Pistachio), Ptychosperma sperma seeds (cabbage palm), Pueraria seeds (kudzu), Punica seeds (pomegranate), Pycnanthemum seeds (mountain mint), Pyrostegia seeds (fire vine), Pyrus seeds (European pear), Quararibea seeds (Guayabillo), Quassia seeds (Amargo), Quercus seeds (Oak), Quillaja seeds (Soapwood), Randia seeds (Indigoberry),Raphanus species (radish), Raphia species (chilean palm), Ravenala species (travel tree), Rehmannia species (rehmannia), Rhamnus species (buckthorn), Rhapis species (palm bamboo), Rheum species (rhubarb), Horse chestnut, Rhizophora species (true mangroves), Rhododendron species (azalea, labradoria, rhododendron), Rhus species (sumac), Ribes species (currant, gooseberry, jostaberry), Ricinodendron species (African nuts), Ricinus species (castor oil), Robinia species (black locust), Rorippa species (mustard), Rosa species (rose) , Rosemarinus species (rosemary), Roystonea species (rhubarb palm), Rubus species (raspberry, blackberry, cloudberry, tayberry, young berry), Rudbeckia species (cornflower, cornflower), Ruellia species (wild petunia), Rumex species (sorrel, silverleaf), Ruta species (rue), Sabal species (palmetto, Sabal, Sagittaria species (arrowroot), Salacca species (salac palm), Salix species (willow), Salvia species (sage, salvia, rosemary, chia), Sambucus species (elderberry, elderberry), Sandoricum species (santol), Sanguisorba species (burnt wood), Santalum species (santal), Santalum species (sanvitamin), alia species (Japonica), Sargassum species (Sargassum, Eryngium), Sassafras species (Sassafras), Satureja species (Savory), Savia species (Savia), Scabiosa species (Scabiosa), Scaevola species (Scabiosa, Fanflower, Halfflower, Naupaka, Gullfeed), Schinus species (Pepperberry),Schinziophyton species (Mongongo), Schisandra species (Magnolia Berry), Schizonepeta species (Schisandra), Scolymus species (Scolymus), Scorzonera species (Scorzonera), Secale species (Rye), Sechium species (Chayote), Sedum species (Stonecrop), Senecio species (Senecio) (Gum arabic, Groundsel), Senegalia species (Gum arabic, Catechu), Senna species (Candlebush, Avarum), Sequoia species (Sequoia), Sequoiadendron species (Sequoia), Serenoa species (Saw palmetto), Sesamum species (Sesame), Sesuvium species (Centella asiatica), Shepherdia species (Bush (Fallon's Worm), Sidalcea species (Checkermallow), Silybum species (Milk Thistle), Simarouba species (Simalba), Simmondsia species (Jojoba), Sinapis species (Brassica napus), Sisyrinchium species (Siberian ginseng), Sium species (Siberian ginseng, Water parsnip), Solanum species (Tomato, Potato, Coconut, Sunberry, Pepino) , naranjilla, garden huckleberry, eggplant), Solidago species (goldenrod), Sophora species (honeybee), Sorbus species (rowan, serviceberry), Sorghastrum species (Indian grass), Sorghum species (sorghum), Spartina species (cordgrass), Spathiphyllum species (spath, peace lily),Spathodea species (tree ferns), Sphaeropteris species (tree ferns), Spinacia species (spinach), Spiraea species (spiadonia), Spondias species (mombin), Stachys species (hedge nettle), Stachytarpheta species (long-legged nettle), Stenochlaena species (staghorn fern) laena species (ferns), Sterculia species (tropical chestnuts), Stevia species (stevia), Stewartia species (summer camellias), Stokesia species (stokesia), Strelitzia species (birds of paradise), Stropharia species (mushrooms), Struthiopteris species (deer ferns) fern), Styphnolobium species (necklacepod), Styrax species (snowbell), Suriana species (bay cedar), Sutera species (stella), Swietenia species (mahogany), Syagrus species (overtop palm, lily palm, dung palm), Symphoricarpos species (snowberry), Synsepalum species (miracle fruit), Syringa species (lily of the valley) jack), Syzygium species (brush cherry, waterberry, clove), Tabebuia species (mulberry), Tabernaemontana species (milkwood), Tagetes species (marigold), Talinum species (creeping zinnia), Tamarindus species (tamarind), Tanacetum species (tansy), Taraxacum species (dandelion), Tasmannia species (pepperbush),Taxodium species (Baldcypress, Pondcypress), Taxus species (Yew), Tecoma species (Trumpetbush), Tellima species (Fringecup), Terminalia species (Terminalia, Kakadu Plum), Ternstroemia species (Tember), Tetragonai species (Spinach), Tetrazygia species (Clawfoot), Bur Ash), Teucrium species (Bordeum), Theobroma species (Cacao), Thlaspi species (Shepherd's purse), Thuja species (Thuja occidentalis, Arborvitae, Cedar), Thymus species (Thyme), Thyrsostachys species (Bamboo), Tiarella species (Foam Flower), Tibouchina species (Linden), Tilia species (Linden), Tollumiea species Tolmiea species (Tormiera), Toona species (Red Cedar), Torreya species (Nutmeg, Torreya), Trachycarpus species (Palm), Trachyspermum species (Ajowan), Tradescantia species (Tradescantia), Tragopogon species (Tremella), Tremella species (Fungus), Triadica species (Talcum) Ze), Tribulus species (Tributaria terrestris), Tricholoma species (Fungus), Trientalis species (Starflower), Trifolium species (Clover), Trigonella species (Fenugreek), Trillium species (Trillium), Triticum species (Wheat), Tropaeolum species (Nasturtium), Tsuga species (Hemlock tree),Tuber species (French tuber), Turbinaria species (disc coral, scroll coral, cup coral, vase coral, pagoda coral, ruffled ridge coral), Turnera species (Damiana), Typha species (cattail, staghorn cattail, reed, punk, typha), Uapaca species (sugar plum), Ugni species (Ugni), Ulmus species (elm), Uncaria species (cat's crest) Rho, Gambir), Ungnadia species (Mexican buckeye), Uniola species (Sea oats), Urtica species (Nettles), Vaccinium species (Blueberries, Cranberries, Huckleberries, Cowberries), Valerianella species (Vancouveria), Vancouveria species (Inside-Out Flower), Vangueria species (Spanish Tamarind), Vanilla (vanilla), Vasconcellea species (mountain papaya, babaco), Verbascum species (mullein), Verbena species (verbena), Vernonia species (ironweed), Veronica species (stag beetle), Viburnum species (cranberry, viburnum), Vicia species (vetch), Vigna species (bean), Viola species (pansy) , violets), Vitex species (plums, hollyhocks), Vitis species (grapes), Volvariella species (mushrooms), Washingtonia species (palms), Wedelia species (American columbine), Wisteria species (Wisteria), Withania species (Ashwagandha), Xanthoceras species (yellow horn), Xanthosoma species (American taro),Ximenia species (Tallowwood), Xylopia species (Grain of Selim), Yucca species (Yucca), Zamia species (Cycad), Zanthoxylum species (Pepper), Zea species (Corn, Teosinthe), Zelkova species (Zelkova), Zephyranthes species (Zelkova, Mention may be made of any plant from the genus Zizania (Zizania), Zizania (Zizania), and / or Ziziphus (Zijube).

[0230] In some embodiments, plants useful in the present invention include, but are not limited to, those listed in Table 2 or Table 4, or in the lists provided in the paragraphs above. In some embodiments, examples of plants useful in the present invention include citrus plants (e.g., grapefruit, oranges, lemons, limes, etc.), tomato plants, corn plants, pecan plants, and tobacco plants.

[0231] [Table 1] JPEG2025170254000003.jpg59153

[0232] [Table 2] JPEG2025170254000005.jpg227153JPEG2025170254000006.jpg231153JPEG2025170254000007.jpg63153

[0233] [Table 3]

[0234] [Table 4] JPEG2025170254000010.jpg197153JPEG2025170254000011.jpg196153JPEG2025170254000012.jpg199153JPEG2025170254 000013.jpg199153JPEG2025170254000014.jpg198153JPEG2025170254000015.jpg199153JPEG2025170254000016.jpg19915 3JPEG2025170254000017.jpg192153JPEG2025170254000018.jpg195153JPEG2025170254000019.jpg197153JPEG2025170254 000020.jpg197153JPEG2025170254000021.jpg198153JPEG2025170254000022.jpg200153JPEG2025170254000023.jpg17153

[0235] [Table 5]

[0236] The present invention will now be described with reference to the following examples. It should be understood that these examples are not intended to limit the scope of the claimed invention, but rather are intended to be illustrative of particular embodiments. Any variations of the exemplified methods that occur to those skilled in the art are intended to be within the scope of the present invention. [Example]

[0237] Symbiont formation inoculum and symbiont inoculation / creation Symbiont formation inocula and symbionts can be generated using several different methods, including i) co-inoculation, ii) single inoculation, and iii) direct DNA inoculation, as illustrated in Figure 1.

[0238] i. The co-inoculation method uses two strains of Agrobacterium species. One strain is a disarmed Agrobacterium species containing a binary vector (e.g., A. tumefaciens strain EHA105) used to express a polynucleotide of interest (POI), and a second wild-type (WT) Agrobacterium strain is used to transfer a plant hormone gene (PHG) into plant cells. Plant cells co-inoculated in this manner, containing both the POI and PH gene (PHG), can be referred to as symbiont-forming inoculum or symbionts, depending on their intended use. In some cases, the cells can be used as symbiont-forming inoculum to form symbionts on a host plant, or when cells located on a plant (or part thereof) are inoculated with bacterial cells in this manner, they can directly form symbionts on the plant.

[0239] Disarmed Agrobacterium strains carrying the binary vector, in this example the A. tumefaciens EHA105 strain, and the WT strain were grown using procedures common in the art, and then each strain was centrifuged to collect bacterial pellets, which were then resuspended in inoculation buffer (10 mM MgCl, 10 mM MES [pH 5.6], 100 μM acetosyringone) and incubated at OD . 600 These were then kept at room temperature for 1-3 hours, and then mixed together and inoculated onto plant tissue, resulting in the formation of the symbiont-forming inoculum or symbionts.

[0240] ii. In the single-inoculation method, only a single Agrobacterium species is used to inoculate plant cells or plants (e.g., host plants). In this example, a disarmed Agrobacterium tumefaciens strain EHA105 carrying a binary vector (e.g., pSYM plasmid, see Figure 2) containing both the POI and PHG was used to inoculate plant cells. The pSYM plasmid contains a cassette of approximately 7.5 kb of plant growth regulators (indole-3-acetamide hydrolase, tryptophan 2-monooxygenase, isopentenyltransferase, and indole-3-lactate synthase) and the POI operably linked to a constitutive or inducible promoter. The pSYM plasmid also contained a selectable marker gene (kanamycin) to enable selection of Agrobacterium species cells carrying the pSYM plasmid. Plant tissue was inoculated with a suspension of Agrobacterium species containing pSYM to form a symbiont-forming inoculum or symbionts.

[0241] iii. Direct DNA inoculation can use a biolistic delivery system to deliver DNA into plant cells or tissues. This is done using metal particles coated with the POI and PHG genes, which are propelled directly into host plant cells without using Agrobacterium species as gene(s) vectors. The cells then incorporate the POI and PHG genes into their genomes, after which the plant tissue can form either a symbiont-forming inoculum or a symbiont. Numerous other methods of direct DNA delivery are known and can be used in place of biolistic bombardment with similar success, including, for example, electroporation, microinjection, lipofection (liposome-mediated transformation), sonication, silicon fiber-mediated transformation, chemically stimulated DNA uptake (e.g., polyfection, e.g., polyethylene glycol (PEG)-mediated transformation), and / or laser microbeam (UV)-induced transformation.

[0242] After the DNA is delivered into the host plant cell genome, expression of the PHG induces and stimulates plant tissue to grow a mixed-culture symbiont (see Figure 3) containing a collection of cells with various gene inserts and expression levels of the POI and PHG. The mixed-culture symbiont can grow autonomously and connect with the host plant via vascular neoplasts by connecting with one or both of the phloem and xylem, where the POI product can be transported to the host plant. The POI product produced by the symbiont can be translocated via the symbiont-derived apoplast and / or symplast and / or through the phloem and / or xylem for dispersal throughout the host plant.

[0243] The mixed culture symbionts can be subsequently excised and grown in hormone-free culture, where cells with desired traits and expression levels can be selected, allowing for the isolation of homogeneous symbiont-forming inoculum(s). Selection of pure culture symbiont-forming inoculum(s) can include, but is not limited to, the use of antibiotic selection (e.g., the use of an antibiotic resistance marker, POI, that allows growth of only transformed cells (i.e., cells that have the POI and PHG)), serial dilution / splitting of the culture, or conversion to protoplasts and single protoplast cells that can be isolated and grown into pure culture. The symbiont-forming inoculum(s) can be selected for expressing desirable traits in addition to expression of the POI and PHG.

[0244] Additionally, when Agrobacterium is used in the symbiosis process, the process of using antibiotics can also be used to eliminate Agrobacterium cells from the mixed culture symbiosis inoculum.

[0245] The final step involves transplanting the selected symbiont-forming inoculum(s) onto a host plant for dispersal within and / or throughout the plant, where it can attach and provide a POI expression product or a product of the POI expression product (e.g., the POI expression product can be an enzyme involved in the biosynthesis of a product in the symbiont, and it is this product that exits the symbiont and is transported into the host plant). After the symbiont-forming inoculum(s) attaches to the plant host, it forms what are called symbiont(s). An example of a symbiont is shown in Figure 4, where panels A and B show citrus symbionts formed 60 days after inoculation using the simultaneous inoculation method. Panels C and D show symbionts formed on citrus using the single-strain inoculation method (e.g., see Figure 1 for a graphical representation of simultaneous and single-strain inoculation).

[0246] In this example, Agrobacterium species carrying pSYM were used to inoculate plant hosts and induce symbiont formation. To do this, Agrobacterium species were grown overnight at 28°C in 10 mL of Luria-Bertani broth supplemented with the appropriate antibiotic (50 μg kanamycin). Both strains were centrifuged to collect a pellet of bacterial cells, which were then resuspended in inoculation buffer (as previously described). Various techniques can be used to inoculate host plants. For example, woody plants such as citrus fruits, which have a hard surface structure on their stems, require a method to pierce the woody stem tissue and penetrate into the plant. Here, citrus toothed tweezers (see Figure 5, panel A) dipped in the Agrobacterium species inoculation solution can be used to pierce citrus bark tissue and deliver the solution to the plant. Herbaceous plants such as tomato (Figure 5, Panels B and C) with flexible stems were inoculated in this example using a tattoo needle (Figure 5, Panel B) or a syringe needle (Figure 5, Panel C) to inject or pass the Agrobacterium seed solution into or through the plant tissue by simply dipping the needle into the Agrobacterium seed solution and piercing the tissue.

[0247] Symbiont tissues can be grown on a range of host plant types. Figure 6 illustrates the formation and growth of symbionts on pecan (Figure 6, panel A), tomato (Figure 6, panel B), citrus (Figure 6, panel C), and Nicotiana benthamiana (Figure 6, panel D). These symbionts were formed by inoculation using one of the methods described above. [Example]

[0248] In vitro culture of symbiont-forming inoculum A symbiont-formed inoculum (e.g., Figure 7) can be generated and used to inoculate additional host plants. This example describes a process for cleaning microbial contamination from symbiont tissue, including removal of Agrobacterium species or other bacteria used to generate the symbionts, and any microbial impurities that may contaminate the agar or liquid medium (e.g., Figure 8). This process allows for the generation and maintenance of in vitro cultures of the symbiont-formed inoculum.

[0249] After the symbionts develop on the host plant, they can be used to create symbiont formation inoculum (Figures 7 and 8). For this purpose, the symbiont tissue was removed from the host plant and rinsed with running tap water for approximately 30 minutes. The rinsed tissue was then washed with ethanol. Subsequently, the tissue was washed with a 10% bleach solution and then with a solution of sterile water. The sterilization step was performed in a laminar flow hood under aseptic conditions using aseptic techniques to avoid external bacterial or fungal contamination.

[0250] After the sterilization step, the tissues were placed on sterile paper to dry (e.g., sterile filter paper) and then placed on solid agar media based on Murashige and Skoog (MS) for both tomato and citrus fruits (Figure 8, panels A and B) or in liquid agar media for both tomato and citrus fruits (Figure 8, panels C and D).

[0251] Growth medium containing antibiotics was used in cell culture to eliminate Agrobacterium species cells and provide only the symbiont-forming inoculum cells. After multiple tissue culture divisions and passages on the medium, homogenous expression of the POI was obtained, as shown in Figure 7. Figure 7 shows the symbiont-forming inoculum expressing mCherry on selective medium, with high expression of the fluorescent marker as shown under UV light and an mCherry filter. [Example]

[0252] Transplantation of symbiont-forming inoculum onto host plants Symbiont tissues (Figure 6) were isolated from various crops and grown on selective agar media, followed by bacterial removal and the generation of symbiont-formed inocula on culture media as described in Example 2. Symbiont-formed inocula transformed with mCherry (Figure 7) or green fluorescent protein (GFP) were used to optimize tissue selection by screening for fluorescence intensity using an mCherry / GFP filter with a UV lamp and by multiple transfers of only fluorescent cells to new selective agar media (as described in Example 2). Symbiont-formed inocula from tomato and citrus fruits were grown under selective solid agar conditions (Figure 8, panel A (tomato) and Figure 8, panel B (citrus)) and selective liquid agar conditions (Figure 8, panel C (tomato) and Figure 8, panel D (citrus)).

[0253] The symbiont-formed inoculum tissue ready for transplantation was removed from the culture medium and then washed in a transplantation solution containing plant hormones (sterile distilled water containing auxin and cytokinin). The transplantation solution is used to support the transplantation efficacy of the symbiont-formed inoculum and host-plant interaction. After washing, the symbiont-formed inoculum tissue from citrus fruits in the transplantation solution was applied to the stem of a citrus plant in a position where the stem epidermal layer had previously been removed. To ensure adhesion of the symbiont-formed inoculum to form symbionts, silicone tape was firmly attached around the symbiont-formed inoculum / symbiont tissue and stem (Figure 9, panel A). As will be appreciated, other methods for holding the symbiont-formed inoculum / symbiont tissue in position on the host plant may be used instead of silicone tape. After approximately 6 weeks, the silicone tape was removed from the symbiont (Figure 9, panel B), and the tissue was excised to evaluate adhesion, vascularization (Figure 9, panel C), and GFP expression (Figure 9, panel F).

[0254] For tomato, symbiont-formed inoculum tissue prepared from tomato was first washed with a transplanting solution containing plant hormones (auxin and cytokinin). The tomato-derived symbiont-formed inoculum tissue was applied to the stems of tomato plants after removing the stem epidermal layer. As with the citrus example, to ensure adhesion of the symbiont tissue to the stem, plastic wrap (e.g., Parafilm® M) was applied to help maintain humidity and contact between the symbiont-formed inoculum and the stem (Figure 9, panel D). After 6 weeks, the symbiont tissue had integrated with the tomato host plant and increased in size (Figure 9, panel E), demonstrating successful transplantation. [Example]

[0255] The versatility of symbionts Symbiont cells can express one or more POIs introduced using one or more vectors / expression cassettes, which can be provided to the cells in one or more steps (e.g., one or more inoculations (e.g., one or more strains of Agrobacterium), one or more introductions using any system known for delivering DNA). Such a method is illustrated in Figure 1. In addition to transplanting various symbiont types onto a single host plant (i.e., one symbiont with one type of POI and one or more additional symbionts containing one or more different POIs), it is also possible to create pSYM plasmids that carry multiple polynucleotides of interest on the same vector / expression cassette / T-DNA region, effectively "stacking" multiple POIs onto a single pSYM plasmid that delivers the desired POIs. It is also possible to "gene stack" the symbionts to form symbionts (as previously described in Examples 1-3). Such POIs can each be regulated by a specific promoter (Figure 10), or by separate promoters, which can be the same or different promoters. It is also possible to use different Agrobacterium species (or other viable bacterial systems), each carrying a unique pSYM with only one POI (Figure 2). pSYMs with multiple POIs are an example of "gene stacking" (Figure 10) and can also be used. The use of different symbiont formation inocula (with the same or different POIs) on the same host plant is an example of "symbiont stacking" to confer the benefits of multiple POIs per plant.

[0256] In this example, we used Agrobacterium-mediated transformation with simultaneous inoculation of separate Agrobacterium symbiont inocula, one carrying a unique pSYM plasmid encoding GFP and the other carrying a pSYM plasmid encoding mCherry. Detection of GFP and mCherry accumulation was performed by fluorescence microscopy (Figure 11). Live symbiont cells were used to track protein localization and dynamics, and symbiont sections were analyzed under a microscope to detect cells expressing GFP, cells expressing mCherry, and cells with both GFP and mCherry expression. This example demonstrates the versatility of symbiont cells that express unique POIs in different cells (Figure 11, panels B–E) or multiple POIs in the same cell (Figure 11, panels G–H), as well as their ability to be supported on the same host plant. [Example]

[0257] POI Product Generation and Export Symbionts can produce and accumulate large amounts of desired POI products (e.g., proteins, Figure 12). Preliminary evaluation suggests that up to 30% of the symbiont tissue may be POI product (Figure 13). Using a single inoculation (e.g., a single strain) of Agrobacterium species, GFP-expressing symbionts were generated on tomato and citrus host plants (Figure 12). The combination of both GFP and mCherry allowed for visualization and quantification of gene expression by measuring fluorescence intensity and protein accumulation using Western blot (Figure 13). To extract total protein from the symbionts, 1 g of symbiont material was converted to a powder by freezing with liquid nitrogen and pulverizing the tissue. This was then suspended in 100 mL of protein extraction buffer (e.g., 150 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% IGEPAL® CA-630, and 1 tablet of 1% (vol / vol) protease inhibitor mixture). For extraction, buffer was added at 2 mL / 1 g of tissue powder. Samples were clarified by centrifugation at 4°C for 20 min. The supernatant was collected and several dilutions were performed to obtain 10 mL of input. -7 Dilutions were made and analyzed under reducing conditions on SDS-PAGE gels. Samples were then blotted onto nitrocellulose membranes and incubated with antibodies according to the manufacturer's protocol (ThermoFisher®). The membranes were incubated with a chemiluminescent substrate, and imaging and data were acquired (Figure 13).

[0258] The symbiont can induce the formation of a sophisticated vascular network with the host plant, consisting of aqueducts and assimilate-transporting sieve elements (Figure 14, panels A and B). Symbiont cells are tightly connected by functional plasmodesmata. Because cells found in the phloem have only primary cell walls, while cells found in the xylem have both primary and secondary cell walls, toluidine blue was used to distinguish between phloem and xylem cells (Figure 14, panel C). The high level of POI expression in symbiont cells, coupled with the high amount of vascular neoplasia, facilitates the transfer of POI into the host plant's vascular tissue. Using the fluorescent proteins GFP and mCherry, we detected and monitored protein accumulation and transfer from symbiont cells to the plant's vascular system using fluorescence microscopy in tomato plants. Host plant tissue 1–2 cm above the symbiont was collected in longitudinal sections (Figure 14, panel D) and transverse sections (Figure 15) to confirm GFP / mCherry transfer (by fluorescence microscopy). Western blot techniques were also used to detect and analyze the accumulation of proteins in the symbionts and in the stems of the plant host, confirming the results from the microscopy analysis (Figure 16).

[0259] Just as symbiont products are transported out of the symbiont and into the host plant, solutes enter the symbiont through the vascular tissue, which is connected to that of the host plant and consists of the phloem for the transport of assimilates and the xylem for water and minerals. While products can move from symbiont cells to the host plant, no genetic material moves from the symbiont to host plant cells. We confirmed that the POI DNA is restricted to the symbiont by PCR detection using specific primers for the POI, and examined the symbiont and neighboring stem sections. PCR analysis of the symbiont and host plant tissues showed that only the symbiont cells were genetically transformed with the POI (Figure 17), indicating that the host plant was not transformed with the POI. This provides the host plant with new traits but does not result in genetic modification. [Example]

[0260] Effects of POI on host plants Symbiont tissue is highly versatile and can be adapted for many different functions or activities. For example, the FLOWERING LOCUS T (FT3) protein is synthesized in leaves and translocates through the graft junction to control flowering in plants, and its overexpression is often associated with plant dwarfism. We generated symbionts on tomato plants using Agrobacterium tumefaciens harboring pSYM to deliver the PHG and FT3 products to plants (Figure 18, panel A). We also generated symbionts on tomato plants using wild-type Agrobacterium tumefaciens (i.e., lacking pSYM) as a control (Figure 18, panel B).

[0261] Tomato plants with FT3-expressing symbionts were bushier, with more branches and leafy structures than controls (compare Figure 18, panels A and B). FT3-expressing symbionts had an increased number of branches (Figure 18, panel A) and altered tomato phyllotaxis, where only one leaf was present at each node and the central stem of the plant was dominant over the lateral stems, as seen in control tomato plants (Figure 18, panel B).

[0262] Symbionts can also be used to modify and modulate plant phenotypes, enhancing resistance to specific pathogens to improve defense mechanisms and increase plant fitness. Figure 19 shows an example of enhancing plant resistance to a specific pathogen. Here, Agrobacterium tumefaciens containing pSYM carrying PHG and oncocin (an antimicrobial peptide) was used to generate symbionts on citrus plants, thereby generating symbionts containing PHG and oncocin (Figure 19, panel A). As a control, wild-type A. tumefaciens was used to generate symbionts on citrus plants without oncocin (Figure 19, panel B). The oncocin-producing symbiont was engineered to translocate oncocin to treat / kill Candidatus Liberibacter asiaticus (CLas). CLas is the causative agent of Huanglong disease (also known as citrus greening), which causes devastating yield losses in citrus fruits worldwide. To date, there is no established cure for this disease. We leveraged the highly vascularized structure of the symbiont to produce and deliver this antimicrobial peptide to the host plant and to the CLas bacteria. To improve peptide export, we fused it to a signal / target sequence peptide (SS or +). Signal sequences are found, for example, in proteins targeted to the endoplasmic reticulum and ultimately destined for extracellular secretion. Both "Oncocin" and "Oncocin+"-expressing symbionts reduced CLas titers over time as determined by qPCR (Figure 20) and also improved plant health (Figure 19, panels C and E), as indicated by a reduction in typical HLB plant symptoms, including reduced bruise-like spots compared to controls (Figure 19, panels D and F).

[0263] As shown, symbionts can be used to express and translocate products that directly interfere with infection or kill pathogens present in the host plant. Oncocin and oncocin+-expressing symbionts have previously been identified as improving citrus health and reducing the titer of CLas bacteria (Figures 19 and 20, as described). To further investigate this, we studied CLas-positive citrus host plants with various symbionts expressing various POIs (GFP+, TMOF, TMOF+, oncocin, and oncocin+) and monitored the CLas titer and efficacy of these various POIs by qPCR analysis of CLas titer effects (Figure 21). Symbionts expressing "GFP+" (GFP with a signal sequence) were used as a control. Results showed that TMOF, TMOF+, oncocin, and oncocin+ all had antibacterial effects against CLas by reducing its titer (Figure 21).

[0264] As shown, the versatility of the symbionts of the present invention provides the ability to improve host plant characteristics and control plant pests. As another example, the symbionts can be used to produce deleterious plant effects, such as being used as herbicides by triggering plant hypersensitive responses and cell death. As an example, Nicotiana benthamiana was injected with a symbiont-forming inoculum carrying a POI of a C. labraxans-derived effector protein recognized by plant nucleotide-binding leucine-rich repeat (NLR) immune receptors, resulting in the overproduction of reactive oxygen species (ROS), which leads to the activation of the cell death process and kills the host plant (Figure 22).

[0265] The foregoing is illustrative of the present invention and should not be construed as limiting thereof. Further variations of the above-described embodiments will be understood by those skilled in the art. Accordingly, the above-described embodiments should be considered illustrative rather than limiting. Therefore, variations to these embodiments can be made by those skilled in the art without departing from the scope of the present invention as defined by the following claims, and it will be understood that equivalents of the claims are encompassed therein.

Claims

1. A symbiont-forming inoculum comprising a polynucleotide encoding a plant hormone biosynthetic enzyme and a target polynucleotide, wherein the plant hormone biosynthetic enzyme is at least one cytokinin biosynthetic enzyme and / or auxin biosynthetic enzyme.

2. The symbiont-forming inoculum of claim 1 , wherein the polynucleotide encoding the plant hormone biosynthetic enzyme and the polynucleotide of interest are contained in a cell, and optionally the cell is a plant cell or a bacterial cell.

3. 3. The symbiont-forming inoculum of claim 1 or claim 2, wherein the plant hormone biosynthetic enzymes are derived from bacterial species and / or plant species.

4. 4. The symbiont-forming inoculum of any one of claims 1 to 3, wherein the plant hormone biosynthetic enzyme is indole-3-acetamide hydrolase (iaaH) (E.C. number: EC 3.5.1.4), amidase 1 (EC 3.5.1.4), tryptophan 2-monooxygenase (IaaM) (EC 1.13.12.3), indole-3-lactic acid synthase (EC 1.1.1.110), L-tryptophan-pyruvate aminotransferase 1 (EC 2.6.1.99), tryptophan aminotransferase-related protein 1 (EC 2.6.1.27), indole-3-acetaldehyde oxidase (EC 1.2.3.7), tryptophan decarboxylase 1 / tryptophan decarboxylase 2 (EC 4.1.1.105), isopentenyltransferase (Ipt), and / or Tzs (EC 2.5.1.27).

5. 5. The symbiont-forming inoculum of any one of claims 1 to 4, wherein the plant hormone biosynthetic enzyme is indole-3-acetamide hydrolase (iaaH), tryptophan 2-monooxygenase (IaaM), and / or isopentenyl transferase (Ipt).

6. The symbiont-forming inoculum according to any one of claims 1 to 5, wherein the plant hormone biosynthetic enzyme is indole-3-lactic acid synthase.

7. 7. The symbiont-forming inoculum of any one of claims 1 to 6, further comprising a polynucleotide encoding a plastid polypeptide (e.g., a plasticity polypeptide), optionally the plastid polypeptide being 6b, rolB, rolC, and / or orf13.

8. A symbiont-forming inoculum described in any one of claims 1 to 7, wherein the polynucleotide encoding the plant hormone biosynthetic enzyme and the target polynucleotide are contained in a single nucleic acid construct or in two or more nucleic acid constructs (e.g., one or more expression cassettes).

9. 9. A symbiont-forming inoculum as described in claim 7 or claim 8, wherein the polynucleotide encoding the plast polypeptide is contained in a single nucleic acid construct, and optionally the polynucleotide encoding the plast polypeptide is in the same or a separate nucleic acid construct (e.g., expression cassette) as the polynucleotide encoding the plant hormone biosynthetic enzyme and / or the polynucleotide of interest.

10. 10. The symbiont-forming inoculum of claim 8 or claim 9, wherein the one or more nucleic acid constructs are contained in one or more vectors.

11. 11. The symbiont-forming inoculum of claim 10, wherein the one or more vectors are a plasmid, a T-DNA, a bacterial artificial chromosome, a viral vector, or a binary bacterial artificial chromosome.

12. 12. A symbiont-forming inoculum according to any one of claims 1 to 11, wherein the target polynucleotide encodes a biomolecule, a bioactive molecule, and / or a polypeptide involved in the biosynthesis of a bioactive molecule.

13. The symbiont-forming inoculum of claim 12, wherein expression of the target polynucleotide confers increased abiotic stress tolerance (e.g., high salt tolerance, high heat tolerance, heavy metal tolerance, cold tolerance, drought tolerance, excess water tolerance, UV radiation tolerance), resistance or increased tolerance to pathogens (e.g., viruses, fungi, bacteria) or pests (e.g., insects, nematodes), or increased tolerance to herbicides.

14. The bioactive molecule may be a biostimulant, a biofungicide, a bioherbicide, an insecticidal protein / peptide (e.g., a bioinsecticide, e.g., Jabretox (peptide JBTX, a bioinsecticide derived from the seeds of jack bean (Canavalia ensiformis))), an insecticidal peptide derived from spider venom (e.g., from Hadronyche versuta), a trypsin-modulating oostatic factor (TMOF), a Bacillus thuringiensis (B. thuringiensis toxins (delta-endotoxins, e.g., Cry toxins, Cyt toxins), plant insecticidal proteins (Vip)), nutrients (e.g., nitrogen, e.g., leghemoglobin, nitrogenase), plant growth regulators (auxins, cytokinins, gibberellins, ethylene, growth inhibitors / retardants), plant lipids, plant fatty acids, plant oils, RNA (e.g., siRNA, dsRNA, miRNA, shRNA), plant antibodies, stylet sheath inhibitory proteins (e.g., ficin, bromelain), ribozymes, bacteriocins, antimicrobial peptides (e.g., oncocin), aptamers, nucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and / or engineered meganucleases.

14. 14. The symbiont-forming inoculum of any one of claims 1 to 13, wherein the polynucleotide of interest encodes a polypeptide operably linked to a targeting sequence, and optionally the targeting sequence targets the protein to a membrane, an intracellular location, or an extracellular location.

15. 15. The symbiont-forming inoculum of claim 14, wherein the targeting sequence is a membrane targeting sequence, an endoplasmic reticulum targeting sequence, a mitochondrial targeting sequence, a chloroplast targeting sequence, a nuclear targeting sequence, a vacuolar targeting sequence, a peroxisome targeting sequence, a lysosome targeting sequence, or a plant viral movement protein.

16. 16. The symbiont-forming inoculum of any one of claims 1 to 15, wherein the polynucleotide encoding a plant hormone biosynthetic enzyme (e.g., a polynucleotide encoding iaaH, a polynucleotide encoding IaaM, and / or a polynucleotide encoding isopentenyltransferase (Ipt), and / or a polynucleotide encoding indole-3-lactic acid synthase) and / or the polynucleotide encoding a plast polypeptide is operably linked to a nuclear targeting (nuclear localization) sequence.

17. A symbiont-forming inoculum described in any one of claims 1 to 16, wherein the polynucleotide encoding the plant hormone biosynthetic enzyme and the target polynucleotide are each operably linked to a single promoter or to at least two separate promoters in any combination.

18. 18. The symbiont-forming inoculum of any one of claims 1 to 17, wherein when the polynucleotides encoding plant hormone biosynthetic enzymes encode iaaH, IaaM, and Ipt, the polynucleotide(s) encoding iaaH, IaaM, and Ipt are operably linked to a single promoter, and the polynucleotides of interest are operably linked to a single promoter or separate promoters.

19. 19. A symbiont-forming inoculum according to any one of claims 7 to 18, wherein the polynucleotide encoding the plast polypeptide is operably linked to a promoter, and optionally the polynucleotide encoding the plast polypeptide is operably linked to the same promoter or a separate promoter as that operably linked to the polynucleotide encoding the plant hormone biosynthetic enzyme and / or the polynucleotide of interest.

20. 20. The symbiont-forming inoculum of any one of claims 17 to 19, wherein the single promoter, separate promoters, and / or two or more separate promoters are each constitutive or inducible promoters in any combination.

21. 21. The symbiont-forming inoculum of claim 1, wherein the polynucleotide of interest is expressed in the symbiont-forming inoculum.

22. 22. The symbiont-forming inoculum of any one of claims 2 to 21, wherein the bacterial cells comprise a type IV secretion system (T4SS, e.g., T4ASS (e.g., VirB / D4 system), T4BSS) or a type III secretion system (T3SS).

23. The bacterial cells may be from a species of the genus Agrobacterium (e.g., A. tumefaciens (e.g., biovar 1), A. rhizogenes (e.g., biovar 2), A. vitis (e.g., biovar 3), A. fabrum (e.g., C58 strains), Rhizobium species, Mesorhizobium species, Sinorhizobium species, Bradyrhizobium species, Pseudomonas species (e.g., P. savastanoi pv. savastanoi 23. The symbiont-forming inoculum of any one of claims 2 to 22, derived from a bacterial genus of the genus Pseudomonas pv. Savastanoi), Phyllobacterium spp., Ochrobactrum spp., Azobacter spp., Closterium spp., Klebsiella spp., Rhodospirillum spp., or Xanthomonas spp.

24. 22. The symbiont formation inoculum of any one of claims 2 to 21, wherein the symbiont formation inoculum contained in cells comprises two or more cells in the form of a cell culture, a plant callus, a callus culture, and / or a suspension culture.

25. 25. The symbiont-forming inoculum of any one of claims 2 to 21 or 24, wherein the plant cells are derived from a macroalgae, angiosperm, gymnosperm, or pteridophyte.

26. A symbiotic organism comprising a plant cell that contains and expresses a polynucleotide encoding a plant hormone biosynthetic enzyme and a target polynucleotide, wherein the plant hormone biosynthetic enzyme is at least one cytokinin biosynthetic enzyme and / or at least one auxin biosynthetic enzyme, and the plant cell of the symbiont divides autonomously.

27. 27. The symbiont of claim 26, wherein the plant cell comprises a plurality of plant cells and forms an undifferentiated multicellular structure when transplanted onto a plant or part thereof.

28. 28. A symbiont according to claim 26 or claim 27, wherein the plant hormone biosynthetic enzymes are derived from bacterial and / or plant species.

29. 29. The symbiont of any one of claims 26 to 28, wherein the plant hormone biosynthetic enzyme is indole-3-acetamide hydrolase (iaaH) (EC number: EC 3.5.1.4), amidase 1 (EC 3.5.1.4), tryptophan 2-monooxygenase (IaaM) (EC 1.13.12.3), indole-3-lactic acid synthase (EC 1.1.1.110), L-tryptophan-pyruvate aminotransferase 1 (EC 2.6.1.99), tryptophan aminotransferase-related protein 1 (EC 2.6.1.27), indole-3-acetaldehyde oxidase (EC 1.2.3.7), tryptophan decarboxylase 1 / tryptophan decarboxylase 2 (EC 4.1.1.105), isopentenyltransferase (Ipt), and / or Tzs (EC 2.5.1.27).

30. 30. The symbiont of any one of claims 26 to 29, wherein the plant hormone biosynthetic enzymes comprise indole-3-acetamide hydrolase (iaaH), tryptophan 2-monooxygenase (IaaM), and / or isopentenyltransferase (Ipt), and optionally, the plant hormone biosynthetic enzymes comprise indole-3-lactic acid synthase.

31. 31. A symbiont described in any one of claims 26 to 30, further comprising a polynucleotide encoding a plastid polypeptide (e.g., a plasticity polypeptide), optionally wherein the plastid polypeptide is 6b, rolB, rolC, and / or orf13.

32. A symbiont described in any one of claims 26 to 31, wherein the polynucleotide encoding the plant hormone biosynthetic enzyme and the target polynucleotide are contained in a single nucleic acid construct or in two or more nucleic acid constructs (e.g., one or more expression cassettes).

33. A symbiont described in claim 31 or claim 32, wherein the polynucleotide encoding the plast polypeptide is contained in one nucleic acid construct, and optionally the polynucleotide encoding the plast polypeptide is in the same or a separate nucleic acid construct as the polynucleotide encoding the plant hormone biosynthetic enzyme and / or the polynucleotide of interest.

34. A symbiont described in any one of claims 26 to 33, wherein the target polynucleotide encodes a biomolecule, a bioactive molecule, and / or a polypeptide involved in the biosynthesis of a bioactive molecule.

35. The symbiont of claim 34, wherein expression of the polynucleotide of interest confers increased abiotic stress tolerance (e.g., high salt tolerance, high heat tolerance, heavy metal tolerance, cold tolerance, drought tolerance, excess water tolerance, UV radiation tolerance), resistance or increased tolerance to pathogens (e.g., viruses, fungi, bacteria) or pests (e.g., insects, nematodes), or increased tolerance to herbicides.

36. Bioactive molecules include pharmaceuticals, biostimulants, biofungicides, bioherbicides, insecticidal proteins / peptides (e.g., bioinsecticides, e.g., Jabretox (peptide JBTX), insecticidal peptides from spider venom (e.g., from Hadronike versta), trypsin-modulating oostatic factors (TMOF), Bacillus thuringiensis toxins (delta-endotoxins, e.g., Cry toxins, Cyt toxins), plant insecticidal proteins (Vip)), nutrients (e.g., nitrogen, e.g., leghemoglobin, nitrogenase), plant growth regulators (auxins, cytokinins, diamine nucleotides, etc.), and the like.

36. The symbiont of claim 34 or claim 35, wherein the symbiont is a plant endothelial cell, a plant stem cell, a plant endothelial cell (e.g., ectoplasmic reticulum, ectoplasmic reticulum, ectoplasmic reticulum), a plant endothelial cell (e.g. ...

37. 37. The symbiont of any one of claims 26 to 36, wherein the polynucleotide of interest encodes a polypeptide operably linked to a targeting sequence, and optionally the targeting sequence positions the polypeptide at a membrane, an intracellular location, or an extracellular location.

38. The symbiont of claim 37, wherein the targeting sequence is a membrane targeting sequence, an endoplasmic reticulum targeting sequence, a mitochondrial targeting sequence, a chloroplast targeting sequence, or a plant viral movement protein.

39. 39. The symbiont of any one of claims 26 to 38, wherein a polynucleotide encoding a plant hormone biosynthetic enzyme (e.g., a polynucleotide encoding iaaH, a polynucleotide encoding IaaM, a polynucleotide encoding isopentenyltransferase (Ipt), and / or a polynucleotide encoding indole-3-lactic acid synthase) and / or a polynucleotide encoding a plastid polypeptide is operably linked to a nuclear targeting sequence.

40. 40. A symbiont described in any one of claims 26 to 39, wherein the polynucleotide encoding the plant hormone biosynthetic enzyme and the polynucleotide of interest are operably linked to a single promoter or to at least two separate promoters in any combination.

41. 41. A symbiont described in any one of claims 26 to 40, wherein when the polynucleotide encoding the plant hormone biosynthetic enzyme encodes iaaH, IaaM, and Ipt, the polynucleotide(s) encoding iaaH, IaaM, and Ipt are operably linked to a single promoter, and the polynucleotide of interest is operably linked to the same promoter or a separate promoter.

42. 42. A symbiont described in any one of claims 26 to 41, wherein the polynucleotide encoding the plast polypeptide is operably linked to a promoter, and optionally the polynucleotide encoding the plast polypeptide is operably linked to the same promoter or a separate promoter as the polynucleotide encoding the plant hormone biosynthetic enzyme and / or the polynucleotide of interest.

42. 43. The symbiont of any one of claims 26 to 42, wherein the plant cell is derived from a macroalgae, angiosperm, gymnosperm, or pteridophyte.

43. 43. A host plant comprising at least one symbiont according to any one of claims 26 to 42, wherein the at least one symbiont is located on at least one site on the host plant.

44. 44. The host plant of claim 43, wherein at least one part of the host plant is on an explant, embryo, leaf, shoot, stem, branch, grain, panicle, cobion, husk, stalk, epidermal tissue, apical meristem, floral tissue (e.g., pollen, pistil, ovule, anther, stamen, corolla, sepals, petals, receptacle, filament, style, stigma, etc.), fruit, seed, pod, capsule, cotyledon, hypocotyl, petiole, tuber, corm, root, root tip, symbiont, nodal gall, phytotroph, symbiont harborage, extrafloral nectary, nodule, plant neoplasm, or gall.

45. A host plant described in any one of claims 43 or 44, wherein a polynucleotide of interest is expressed in a symbiont and an expression product of the polynucleotide of interest and / or a product made using the expression product of the polynucleotide of interest is transported into the host plant.

46. 46. ​​The host plant of any one of claims 43 to 45, wherein the host plant is derived from a wild-type plant of any age or size (e.g., a seedling, a young plant, or a mature plant).

47. 47. The host plant of any one of claims 43 to 46, wherein the host plant is derived from a macroalgae, angiosperm, gymnosperm, or pteridophyte.

48. A method for generating a symbiont-forming inoculum, comprising the step of introducing into a cell a polynucleotide encoding a plant hormone biosynthetic enzyme and a polynucleotide of interest, or introducing into a transgenic cell containing the polynucleotide of interest a polynucleotide encoding a plant hormone biosynthetic enzyme, wherein the plant hormone biosynthetic enzyme is at least one cytokinin biosynthetic enzyme and / or auxin biosynthetic enzyme, thereby generating a symbiont-forming inoculum, and optionally wherein the cell is a plant cell or a bacterial cell.

49. 49. The method of claim 48, further comprising culturing the cells to produce a population of cells comprising a polynucleotide encoding a plant hormone biosynthetic enzyme and a polynucleotide of interest.

50. (a)(i) introducing a polynucleotide encoding a plant hormone biosynthetic enzyme and a polynucleotide sequence of interest into / onto at least one site on a plant (or a part thereof (e.g., an explant)), or transplanting a plant cell containing the same, or inoculating a bacterial cell containing the same onto at least one site on a plant (or a part thereof); (ii) introducing a polynucleotide encoding a plant hormone biosynthetic enzyme into, or transplanting a plant cell containing, or inoculating a bacterial cell containing the polynucleotide into / on at least one site on the plant (or a part thereof), wherein the plant (or part thereof) of (ii) contains the polynucleotide sequence of interest; The plant hormone biosynthetic enzyme is at least one cytokinin biosynthetic enzyme and / or auxin biosynthetic enzyme, thereby generating a symbiont on the plant (or part thereof) comprising a polynucleotide encoding the plant hormone biosynthetic enzyme and a polynucleotide sequence of interest; (b) selecting one or more cells from the symbiont on the plant to provide one or more cells comprising a polynucleotide encoding a plant hormone biosynthetic enzyme and a polynucleotide sequence of interest, thereby producing a symbiont-forming inoculum; A method for producing a symbiont-forming inoculum, comprising:

51. 51. The method of claim 50, further comprising the step of (c) culturing one or more cells from (b) to produce a plant cell population (e.g., a callus culture and / or a suspension culture) comprising a polynucleotide encoding a plant hormone biosynthetic enzyme and a polynucleotide sequence of interest.

52. 52. The method of claim 50 or claim 51, wherein the plant hormone biosynthetic enzymes are derived from bacterial and / or plant species.

53. 53. The method of any one of claims 50 to 52, wherein the plant hormone biosynthetic enzyme is indole-3-acetamide hydrolase (iaaH) (E.C. number: EC 3.5.1.4), amidase 1 (EC 3.5.1.4), tryptophan 2-monooxygenase (IaaM) (EC 1.13.12.3), indole-3-lactic acid synthase (EC 1.1.1.110), L-tryptophan-pyruvate aminotransferase 1 (EC 2.6.1.99), tryptophan aminotransferase-related protein 1 (EC 2.6.1.27), indole-3-acetaldehyde oxidase (EC 1.2.3.7), tryptophan decarboxylase 1 / tryptophan decarboxylase 2 (EC 4.1.1.105), isopentenyltransferase (Ipt), and / or Tzs (EC 2.5.1.27).

54. 54. The method of any one of claims 50 to 53, wherein the plant hormone biosynthetic enzymes comprise indole-3-acetamide hydrolase (iaaH), tryptophan 2-monooxygenase (IaaM), and / or isopentenyltransferase (Ipt), and / or optionally indole-3-lactic acid synthase.

55. 55. The method of any one of claims 50 to 54, wherein at least one site on the plant is in the above-ground part of the plant and / or in the underground part of the plant.

56. 56. The method of any one of claims 50 to 55, further comprising the step of introducing into at least one site within the cell or on the plant a polynucleotide encoding a plastid polypeptide (e.g., a plasticity polypeptide), optionally the plastid polypeptide being 6b, rolB, rolC, and / or orf13.

57. 57. The method of any one of claims 50 to 56, wherein the polynucleotide encoding the plant hormone biosynthetic enzyme and the polynucleotide of interest are introduced separately in a single nucleic acid construct or in two or more nucleic acid constructs (e.g., one or more expression cassettes).

58. 58. The method of claim 56 or 57, wherein the polynucleotide encoding the plast polypeptide is contained in a single nucleic acid construct, and optionally the polynucleotide encoding the plast polypeptide is introduced in the same or a separate nucleic acid construct as the polynucleotide encoding the plant hormone biosynthetic enzyme and / or the polynucleotide of interest.

59. 59. The method of claim 57 or claim 58, wherein the two or more nucleic acid constructs are contained in one or more vectors, and optionally the vectors are plasmids, T-DNA, bacterial artificial chromosomes, viral vectors, or binary bacterial artificial chromosomes.

60. 60. The method of any one of claims 50 to 59, wherein the polynucleotide of interest encodes a biomolecule, a bioactive molecule, and / or a polypeptide involved in the biosynthesis of a bioactive molecule.

61. The method of claim 60, wherein expression of the target polynucleotide confers increased abiotic stress tolerance (e.g., high salt tolerance, high heat tolerance, heavy metal tolerance, cold tolerance, drought tolerance, excess water tolerance, UV radiation tolerance), resistance or increased tolerance to pathogens (e.g., viruses, fungi, bacteria) or pests (e.g., insects, nematodes), and / or increased tolerance to herbicides.

62. The bioactive molecule may be a biostimulant, a biofungicide, a bioherbicide, an insecticidal protein / peptide (e.g., a bioinsecticide, e.g., Jabretox (peptide JBTX, a bioinsecticide derived from Jack bean seeds), an insecticidal peptide derived from spider venom (e.g., from Hadronike versta), a trypsin-modulating oostatic factor (TMOF), a Bacillus thuringiensis toxin (delta-endotoxin, e.g., Cry toxin, Cyt toxin), a plant insecticidal protein (Vip)), a nutrient (e.g., nitrogen, e.g., leghemoglobin, nitrogenase), a plant growth regulator (oak, 62. The method of claim 60 or 61, wherein the target compound is selected from the group consisting of: cytokinins, gibberellins, ethylene, growth inhibitors / retardants), RNA (e.g., siRNA, dsRNA, miRNA, shRNA), plant antibodies, stylet sheath inhibitory proteins (e.g., ficin, bromelain), ribozymes, bacteriocins, antimicrobial peptides (e.g., oncocin), plant lipids, plant fatty acids, plant oils, aptamers, nucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and / or engineered meganucleases.

63. 63. The method of any one of claims 50 to 62, wherein the polynucleotide of interest encodes a polypeptide operably linked to a targeting sequence, optionally the targeting sequence localizes the protein to a membrane, an intracellular location, or an extracellular location, and optionally the targeting sequence is a membrane targeting sequence, an endoplasmic reticulum targeting sequence, a mitochondrial targeting sequence, a chloroplast targeting sequence, or a plant viral movement protein.

64. 64. The method of any one of claims 50 to 63, wherein the polynucleotide encoding the plant hormone biosynthetic enzyme and / or the polynucleotide encoding the plastid polypeptide is operably linked to a nuclear targeting sequence.

65. 65. The method of any one of claims 50 to 64, wherein the polynucleotide encoding the plant hormone biosynthetic enzyme and the polynucleotide of interest are each operably linked to a single promoter or to at least two separate promoters in any combination.

66. 66. The method of any one of claims 50 to 65, wherein when the polynucleotide encoding the plant hormone biosynthetic enzyme encodes iaaH, IaaM, and Ipt, the polynucleotide(s) encoding iaaH, IaaM, and Ipt are operably linked to a single promoter, and the polynucleotides of interest are operably linked to a single promoter or separate promoters.

67. 67. The method of any one of claims 50 to 66, wherein when the polynucleotide encoding a plant hormone biosynthetic enzyme comprises a polynucleotide encoding iaaH, a polynucleotide encoding IaaM, and a polynucleotide encoding Ipt, the polynucleotide encoding iaaH, the polynucleotide encoding IaaM, and the polynucleotide encoding Ipt are each operably linked to at least two separate promoters, and the polynucleotide of interest is operably linked to a promoter separate from the at least two separate promoters or is linked to at least one of the at least two separate promoters.

68. 68. The method of any one of claims 56 to 67, wherein the polynucleotide encoding the plast polypeptide is operably linked to a promoter, and optionally the polynucleotide encoding the plast polypeptide is operably linked to the same promoter or a separate promoter as the polynucleotide encoding the plant hormone biosynthetic enzyme and / or the polynucleotide of interest.

69. 69. The method of any one of claims 65 to 68, wherein the promoter, the single promoter, and / or the two or more separate promoters are constitutive or inducible promoters.

70. 70. The method of any one of claims 48-54 or 56-69, wherein the bacterial cell comprises a type IV secretion system (T4SS, e.g., T4ASS (e.g., VirB / D4 system), T4BSS) or a type III secretion system (T3SS).

71. 70. The method of any one of claims 48-54 or 56-69, wherein the bacterial cell is from the bacterial genus Agrobacterium spp. (e.g., A. tumefaciens (e.g., biovar 1), A. rhizogenes (e.g., biovar 2), A. vitis (e.g., biovar 3), A. fablum (e.g., strain C58), Rhizobium spp., Mesorhizobium spp., Sinorhizobium spp., Bradyrhizobium spp., Pseudomonas spp. (e.g., P. savastanoi pv. savastanoi), Phyllobacterium spp., Ochrobactrum spp., Azobacter spp., Closterium spp., Klebsiella spp., Rhodospirillum spp., or Xanthomonas spp.

72. 70. The method of any one of claims 50 to 69, wherein the plant, plant part, or plant cell is derived from a wild-type or transgenic plant of any age or size (e.g., a seedling, young plant, or mature plant).

73. 73. The method of any one of claims 50 to 69 or 72, wherein the plant, plant part, or plant cell is derived from a macroalgae, angiosperm, gymnosperm, or pteridophyte.

74. 74. The method of any one of claims 50 to 69, 72, or 73, wherein the plant cell is derived from a plant cell culture (callus culture or suspension culture), protoplast, seedling, explant, embryo, leaf, shoot, stem, branch, grain, ear, cob, husk, stalk, epidermal tissue, apical meristem, floral tissue (e.g., pollen, pistil, ovule, anther, stamen, corolla, sepals, petals, receptacle, filament, style, stigma, etc.), fruit, seed, pod, capsule, cotyledon, hypocotyl, petiole, tuber, corm, root, root tip, symbiont, nodal gall, vegetative body, symbiont harborage, extrafloral nectary, nodule, gall, or plant neoplasm.

75. 75. The method of any one of claims 50-69 or 72-74, wherein the at least one site on the plant is an explant, embryo, leaf, shoot, stem, branch, grain, ear, cob, husk, stalk, epidermal tissue, apical meristem, floral tissue (e.g., pollen, pistil, ovule, anther, stamen, corolla, sepals, petals, receptacle, filament, style, stigma, etc.), fruit, seed, pod, capsule, cotyledon, hypocotyl, petiole, tuber, corm, root, root tip, symbiont, nodal gall, plant vegetative body, symbiont harborage, extrafloral nectary, nodule, plant neoplasm, or gall.

76. 76. The method of any one of claims 50 to 75, wherein the introduction is via bacterial-mediated transformation, agroinfiltration, viral-mediated transformation, particle bombardment (microprojectile bombardment), electroporation, microinjection, lipofection (liposome-mediated transformation), sonication, silicon fiber-mediated transformation, chemically stimulated DNA uptake (e.g. polyfection, e.g. polyethylene glycol (PEG)-mediated transformation) and / or laser microbeam (UV)-induced transformation.

77. 77. The method of any one of claims 50 to 69 or 72 to 76, wherein (i) a polynucleotide encoding a plant hormone biosynthetic enzyme and a polynucleotide sequence of interest or (ii) a polynucleotide encoding a plant hormone biosynthetic enzyme are contained in at least one plant cell, and the at least one plant cell is transplanted onto at least one site on the plant.

78. 78. The method of claim 77, wherein the plant is wounded at least at one location prior to or during transplantation of the at least one plant cell at the at least one location on the plant.

79. 77. The method of claim 76, wherein the introduction is via bacterially-mediated transformation and comprises co-culturing the plant cell or plant (or part thereof, e.g., an explant) with cells of at least one bacterial species or strain, wherein the bacterial cells comprise one or more of a polynucleotide encoding a plant hormone biosynthetic enzyme, a polynucleotide of interest, and / or at least one polynucleotide encoding at least one plastid polypeptide.

80. 80. The method of claim 79, wherein the plant (or part thereof, e.g., an explant) is wounded at least at one site prior to or during co-cultivation with cells of the at least one bacterial strain.

81. 81. The method of claim 79 or claim 80, wherein the cells of at least one bacterial species or strain include cells of at least two bacterial species or strains, and the polynucleotide encoding at least one plant hormone enzyme is contained in a bacterial strain separate from the bacterial strain containing the at least one polynucleotide of interest (e.g., double bacterial transformation).

82. 82. The method of any one of claims 79 to 81, wherein at least one bacterial strain or species and / or at least two bacterial strains or species are bacterial cells comprising a type IV secretion system (T4SS, e.g., T4ASS (e.g., VirB / D4 system), T4BSS) or a type III secretion system (T3SS).

83. 83. The method of claim 82, wherein the bacterial cell is an Agrobacterium species cell (e.g., A. tumefaciens (e.g., biovar 1), A. rhizogenes (e.g., biovar 2), A. vitis (e.g., biovar 3), A. fabrum (e.g., strain C58), Rhizobium species cell, Mesorhizobium species cell, Sinorhizobium species cell, Bradyrhizobium species cell, Pseudomonas species cell (e.g., P. savastanoi pv. savastanoi), Phyllobacterium species cell, Ochrobactrum species cell, Azobacter species cell, Closterium species cell, Klebsiella species cell, Rhodospirillum species cell, or Xanthomonas species cell.

84. 84. The method of any one of Claims 48-83, further comprising editing at least one nucleic acid in at least one cell of the symbiont-forming inoculum to produce at least one edited nucleic acid, optionally wherein the editing is performed with a gene-editing nuclease.

85. 85. The method of Claim 84, wherein at least one edited nucleic acid has altered expression (e.g., increased or decreased expression compared to the same nucleic acid that has not been so altered).

86. 86. A symbiont-forming inoculum produced by the method of any one of claims 48 to 85.

87. 87. The symbiont formation inoculum of claim 86, wherein the inoculum comprises bacterial cells, a bacterial culture, a plant cell, or a plant cell culture (e.g., a callus or cell suspension).

88. 88. A cell or protoplast derived from the symbiont-forming inoculum of claim 86 or claim 87, wherein the cell or protoplast comprises a polynucleotide encoding a plant hormone biosynthetic enzyme and a polynucleotide of interest.

89. Transplanting the symbiont-forming inoculum of claim 86 or claim 87, or the cell of claim 88, or the symbiont of any one of claims 26 to 42 into at least one site on a host plant; culturing the symbiont formation inoculum or symbiont at least one site on the host plant to form a symbiont at least one site on the host plant, wherein the polynucleotide of interest is expressed in the symbiont on the host plant, and an expression product of the polynucleotide of interest and / or a product made using the expression product of the polynucleotide of interest is delivered into the host plant, thereby producing a host plant with modified characteristics; A method for modifying host plant characteristics without modifying the plant genome, comprising:

90. 90. The method of claim 89, wherein the polynucleotide of interest encodes a biomolecule, a bioactive molecule, and / or a polypeptide involved in the biosynthesis of a bioactive molecule.

91. The method of claim 90, wherein expression of the target polynucleotide confers increased abiotic stress tolerance (e.g., high salt tolerance, high heat tolerance, heavy metal tolerance, cold tolerance, drought tolerance, excess water tolerance, UV radiation tolerance), resistance or increased tolerance to pathogens (e.g., viruses, fungi, bacteria) or pests (e.g., insects, nematodes), or increased tolerance to herbicides.

92. The biologically active molecule may be a pharmaceutical (e.g., a therapeutic protein, a therapeutic polynucleotide, a therapeutic chemical, e.g., a vaccine, an antibody, a recombinant antibody, an antibody fragment, a fusion protein, an antibody fusion protein, human serum albumin, gastric lipase, insulin, glucocerebrosidase, a growth factor, a cytokine, hepatitis B surface antigen (HBsAg)), Apo-A1, alpha-galactosidase (PRX-102), acetylcholinesterase (PRX-105), anti-tumor necrosis factor (Pr-anti-TNF), IgG, interferon-alpha, plasmin, lactoferrin, lysozyme, and collagen), a biostimulant, a biofungicide, a bioherbicide, an insecticidal protein / peptide (e.g., a bioinsecticide, e.g., Jabretox (peptide JBTX), a bioinsecticide derived from Jack bean (Jack bean seed), an insecticidal peptide derived from spider venom (e.g., 92. The method of claim 90 or 91, wherein the toxin is selected from the group consisting of a plant soluble toxin (e.g., from Hadronike versta), a trypsin-modulating oostatic factor (TMOF), a Bacillus thuringiensis toxin (delta-endotoxin, e.g., Cry toxin, Cyt toxin), a plant insecticidal protein (Vip), a nutrient (e.g., nitrogen, e.g., leghemoglobin, nitrogenase), a plant growth regulator (auxin, cytokinin, gibberellin, ethylene, growth inhibitor / retardant), RNA (e.g., siRNA, dsRNA, miRNA, shRNA), a plant antibody, a plant lipid, a plant fatty acid, a plant oil, a stylet sheath inhibitor protein (e.g., ficin, bromelain), a ribozyme, a bacteriocin, an antimicrobial peptide (e.g., oncocin), an aptamer, a nuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), and / or an engineered meganuclease.

93. 93. The method of claims 89 to 92, wherein at least one site on the host plant is on an above-ground portion of the host plant and / or on an underground portion of the host plant.

94. 92. The method of any one of claims 89 to 91, wherein the symbiont or symbiont-forming inoculum is transplanted onto the host plant at least twice and / or at at least two sites on the host plant.

95. 95. The method of any one of claims 89 to 94, wherein the expression product is a transcription product or translation product, or a variant thereof.

96. 96. The method of any one of claims 89 to 95, wherein the product made using the expression product of the polynucleotide is a chemical, a protein (polypeptide / peptide), or a polynucleotide.

97. 97. The method of any one of claims 89 to 96, wherein the modified host plant characteristic is increased tolerance / resistance to a disease-causing organism (e.g., a fungus, bacterium, virus).

98. 98. The method of any one of claims 89 to 97, wherein the modified host plant characteristic is increased (induced) expression of a plant defense gene.

99. 99. The method of any one of claims 89 to 98, wherein the modified host plant characteristic is increased insect tolerance / resistance.

100. 100. The method of any one of claims 89 to 99, wherein the modified host plant characteristic is increased nematode tolerance / resistance.

101. 101. The method of any one of claims 89 to 100, wherein the modified host plant characteristic is modified morphology.

102. 102. The method of claim 101, wherein the altered morphology comprises shortened internodes, increased lateral branching, and / or increased flowering.

103. 103. The method of any one of claims 89 to 102, wherein the modified host plant characteristic is the presence of a biomolecule, a bioactive molecule, and / or a polypeptide involved in the biosynthesis of a bioactive molecule.

104. 104. The method of claim 103, wherein the bioactive molecule is a pharmaceutical (e.g., a therapeutic protein, a therapeutic polynucleotide, a therapeutic chemical), a biostimulant, a biofungicide, a bioherbicide, an insecticidal protein / peptide, a trypsin-modulating oostatic factor (TMOF), a Bacillus thuringiensis toxin, a plant insecticidal protein (Vip), a nutrient, a plant growth regulator, RNA, a plant antibody, a stylet sheath inhibitory protein, a ribozyme, a bacteriocin, an antimicrobial peptide, a plant lipid, a plant fatty acid, a plant oil, an aptamer, a nuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), and / or an engineered meganuclease.

105. 105. The method of claim 103 or 104, wherein the biologically active molecule is Jabretox (peptide JBTX), delta-endotoxin, Cry toxin, Cyt toxin, leghemoglobin, nitrogenase, ficin, bromelain, bacteriocin, nisin, and / or oncocin.

106. 106. The method of any one of claims 89 to 105, wherein the modified host plant characteristics are the presence of bioactive molecules (e.g. biocidal molecules) in the host plant and increased resistance / tolerance to plant pathogens.

107. 107. The method of claim 106, wherein the bioactive molecule is a bacteriocin or an antimicrobial peptide and the plant pathogen is a bacterium.

108. 108. The method of claim 107, wherein the bacteriocin or antimicrobial peptide is oncocin and / or nisin.

109. 106. The method of any one of claims 89 to 105, wherein the altered host plant characteristics are the presence of insecticidal proteins (e.g., bioinsecticides) and increased insect tolerance or resistance.

110. 110. The method of claim 109, wherein the insecticidal protein is Jabretox, trypsin-modulating oostatic factor (TMOF), Bacillus thuringiensis toxin (e.g., delta-endotoxin, e.g., Cry toxin, Cyt toxin), stylet sheath inhibitor protein (e.g., ficin, bromelain), and / or botanical insecticidal protein (Vip).

111. 105. The method of any one of claims 89 to 104, wherein the modified host plant characteristic is the presence or increase or decrease of production of a plant growth regulator (e.g., auxin, cytokinin, gibberellin, ethylene, growth inhibitor / retardant) and modified growth.

112. 105. The method of any one of claims 89 to 104, wherein the altered host plant characteristic is the presence or increase in the production of RNA and the increase / decrease in the production of polynucleotides, peptides, or polypeptides.

113. 113. The method of claim 112, wherein the RNA is siRNA, dsRNA, miRNA, or shRNA, optionally dvsnf7, ccomt, dCS, asn1, phL, RI, PGAS, and / or ppo5.

114. 114. A host plant having modified characteristics produced by the method of any one of claims 89 to 113.

115. Providing a symbiont according to any one of claims 26 to 42, wherein the polynucleotide of interest encodes a biomolecule and / or a bioactive molecule, and collecting the biomolecule and / or bioactive molecule produced in the symbiont; and / or providing a host plant according to any one of claims 43 to 47, wherein the polynucleotide of interest encodes a biomolecule and / or a bioactive molecule, and collecting the biomolecule and / or bioactive molecule produced in the symbiont and the host plant.

1. A method for producing a biomolecule and / or a bioactive molecule, comprising:

116. Transplanting the symbiont-forming inoculum of any one of claims 1 to 25, 86, or 87, or the cell of claim 88, or the symbiont of any one of claims 26 to 42 onto at least one site on a host plant; culturing the symbiont-forming inoculum or the symbiont at least one site on the host plant to form a symbiont at least one site on the host plant, wherein the polynucleotide of interest is expressed in the symbiont, and an expression product of the polynucleotide of interest and / or a product made using the expression product of the polynucleotide of interest is transported into the host plant, thereby delivering the compound of interest to the plant; A method for delivering a compound of interest to a host plant, comprising:

117. Transplanting the symbiont-forming inoculum of any one of claims 1 to 25, 86, or 87, or the cell of claim 88, or the symbiont of any one of claims 26 to 42 onto at least one site on a host plant; culturing the symbiont formation inoculum or symbiont at least one site on the host plant to allow a symbiont to form at least one site on the host plant, wherein the polynucleotide of interest is expressed in the symbiont, and an expression product of the polynucleotide of interest and / or a product made using the expression product of the polynucleotide of interest is delivered into the host plant, thereby producing a plant that does not have the modified genotype and includes the modified trait; 20. A method for producing a plant containing an altered trait without altering the genotype of the plant, comprising:

118. 118. A plant produced by the method of claim 117.

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