Pantaphos compounds and methods

Abstract

Transgenic plants and / or plant material comprising a nucleic acid molecule having a constitutive promoter sequence that is functional in plants and a nucleotide sequence operably linked to the constitutive promoter sequence, where the nucleotide sequence encodes an HvrF protein of Pantoea ananatis, thereby conferring pantaphos resistance to the transgenic plant and / or plant material.

Description

[0001] PANTAPHOS COMPOUNDS AND METHODS

[0002] RELATED APPLICATIONS

[0003] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 542,662, filed October 5, 2023, which is incorporated herein by reference.

[0004] GOVERNMENT SUPPORT

[0005] This invention was made with government support under Grant No. 1R01GM127659- 01 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] BACKGROUND OF THE INVENTION

[0007] One of the greatest challenges for modem agriculture is the emerging resistance to current herbicides and the lack of new and effective herbicides. Current pesticide resistance in the agricultural industry results in losses of global crop yield of up to 50 percent and costs the United States alone billions of dollars. Because of the increased resistance to conventional herbicides, alternative strategies to combat the rise of invasive plant species among crops are desperately needed. The emerging weed resistance and the lack of compounds with new modes of action have inspired the investigation into microbial natural products as sources for novel phytotoxins. Fortunately, microbially produced natural products remain an underdeveloped resource for herbicidal compounds, and with the rapid advancements in microbial genomics, the ability to uncover bioactive natural products with new modes of action has become quite robust. About 69% of all newly registered pesticides from 1997 to 2010 were of natural product origin with roughly one third produced directly from microbial sources without any chemical derivatization. Thus, microbially derived compounds continue to be promising next-generation herbicides.

[0008] Pantaphos is a phosphonic acid natural product made by the plant pathogen Pantoea ananatis (Figure 1). This bacterium is a primary cause of onion center rot, a devastating plant disease with major economic impacts. The organism also causes significant disease in other important crops including rice, corn, melon, wheat, and sudangrass. Importantly, P. ananatis strains that lack the ability to produce pantaphos do not cause center rot, while application of pantaphos in the absence of bacteria mimics the disease. Thus, pantaphos is the key virulence factor in P. ananatis-induced onion center rot. Moreover, the phytotoxicity of purified pantaphos is comparable to that of the widely used weed-killers glyphosate and glufosinate, suggesting the molecule has potential as an herbicide. Thus, a detailed understanding of pantaphos biosynthesis could be useful both for controlling plant disease and for herbicide development.

[0009] Accordingly, there is a need to characterize the pantaphos biosynthetic gene cluster to identify the essential components and properties of these enzymes so that effective counter measures are found against pantaphos and Pantoea ananatis infection. The present disclosure satisfies these needs.

[0010] SUMMARY OF THE INVENTION

[0011] The pantaphos biosynthetic gene cluster was identified by comparing the genome sequences of pathogenic and non-pathogenic P. ananatis strains, which revealed a genomic island designated HiVir (for “high virulence”) that was specific to pathogenic strains. Among the genes of the HiVir locus is hvrA (also known as pepM), which is predicted to encode phosphoenolpyruvate phosphonomutase (PEP mutase), the enzyme which catalyzes the first step in all characterized phosphonate biosynthetic pathways. Accordingly, the pathogenicity island was predicted to direct the synthesis of a small molecule phosphonate required for onion pathogenesis, a hypothesis that was validated by showing that hvrA mutants were avirulent. The hvrA gene is encoded within an eleven gene operon that presumably encodes a multi-step pathway for synthesis of the putative phosphonate phytotoxin (Figure 1). A twelfth gene, designated hvrL is located immediately downstream of the operon and may, or may not, be part of the biosynthetic gene cluster. Characterization of mutants with non-polar deletions showed that the first six genes in the operon (hvrA through hvrF) are required for onion necrosis, while mutants in hvrG through hvrJ produce smaller lesions, suggesting they contribute to the phenotype, but are not required. In contrast, hvrK mutants are fully virulent suggesting this gene is not required for synthesis of the toxin. Unfortunately, the ability of these mutants to produce pantaphos was not directly examined, probably because conditions that induce expression of the hvr operon outside the onion host have yet to be found. Nevertheless, the hvr operon is clearly responsible for pantaphos biosynthesis, because genetically engineered strains that express these genes from a tightly regulated, IPTG-inducible promoter make pantaphos only under inducing conditions. When induced, the engineered strains also produce phosphonomethylmaleate (PMME, Figure 1), a probable biosynthetic intermediate, and third phosphonate, which was not identified due to its instability.

[0012] Based on the functions of homologous proteins, we proposed a metabolic scheme for pantaphos production that follows a chemical logic similar to the tricarboxyclic acid (TCA) cycle and leucine biosynthetic pathways. The proposed pathway involves the early biosynthetic intermediate, 2-phosphonomethylmalate (PMM), which is also involved in biosynthesis of the phosphonate natural products FR-900098 and fosfomycin. In organisms that produce these phosphonate antibiotics, PMM is produced from phosphoenolpyruvate by the enzymes PEP mutase and PMM synthase. Homologs of these enzymes are also encoded by the hvr operon (HvrA and HvrC, respectively). In the proposed pantaphos pathway, PMM would be converted to PMME by an aconitase-like dehydration catalyzed by HvrDE, followed by hydroxylation via a two-component monooxygenase comprised of HvrB and HvrK to yield pantaphos.

[0013] While the proposed pantaphos biosynthetic pathway is consistent with the available data, it has yet to be experimentally validated. In the present disclosure, we address this issue by characterizing the in vitro enzymatic activity of selected Hvr proteins using heterologously expressed proteins. Although the predicted functions are broadly consistent with the model, we were surprised to find that the biosynthetic pathway requires the SAM-dependent methyltransferase HvrF, despite the fact that pantaphos has no SAM-derived carbon atoms. This irreversible methyltransferase reaction catalyzed by HvrF provides the thermodynamic driving force needed to shift the equilibrium of preceding unfavorable steps, while producing methylated intermediates that are likely to be less toxic than those predicted in our original scheme. Based on the biochemical results, pantaphos synthesis requires only six of the eleven genes in the hvr operon, a conclusion that was confirmed by heterologous production of the molecule in Escherichia coli hosts expressing these genes.

[0014] Accordingly, this disclosure provides nucleic acid molecules comprising the hvr operon (hvrA-hvrl.) of Pantoea Sp. and engineered cells comprising the same. In some embodiments, the disclosure provides a nucleic acid molecule comprising an hvr operon (hvrA-hvrE) of Pantoea Sp. operably linked to an inducible promotor sequence, wherein induction of the promoter and expression of the genes of the hvr operon causes the production of 2- (hydroxy(phosphono)methyl) maleic acid (pantaphos).

[0015] The dislcsoure also provides for transgenic plants and / or plant parts, or plants derived from the plant parts that comprise a nucleic acid molecule that encodes one or more polypeptides sufficient to confer pantahos resistance to the transgenicn plant or plant part.

[0016] In some embodiments, a nucleic acid molecule that may be used to transform a plant, a plant cell, or a plant part, and may comprises a constitutive promoter sequence that is functional in plants and a nucleotide sequence operably linked to the constitutive promoter sequence, wherein the nucleotide sequence encodes an HvrF protein of Pantoea ananatis comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 11.

[0017] In some embodiments, a pantaphos-resistant plant cell may comprise a recombinant nucleic acid molecule, the recombinant nucleic acid molecule comprising: i) a constitutive promoter sequence that is functional in plant cells; and ii) a nucleotide sequence encoding a polypeptide comprising a 5-adenosylmethione (SAM)-dependent methyltransferase, wherein the (SAM)-dependent methyltransferase comprises an HvrF protein of Pantoea ananatis wherein the constitutive promoter sequence is heterologous with respect to the nucleotide sequence and is configured to cause sufficient expression of the polypeptide in the plant cell comprising the recombinant nucleic acid molecule to confer pantaphos resistance to the plant cells.

[0018] In other embodiments, a method for producing pantaphos-resistant plant cells may comprise: (a) transforming plant cells using a recombinant DNA construct to provide transformed plant cell; and (b) regenerating pantaphos-resistant plants from the transformed plant cells, wherein the recombinant DNA construct comprises: i) a constitutive promoter sequence that is functional in the plant cells; and ii) a coding sequence that encodes a polypeptide comprising an 5-adenosylmethione (SAM)-dependent methyltransferase, wherein the promoter sequence is heterologous with respect to the coding sequence and is configured to cause sufficient expression of the polypeptide in the transformed plant cell in order to confer resistance to pantaphos.

[0019] These and other features and advantages of this invention will be more fully understood from the following detailed description of the invention taken together with the accompanying claims. It is noted that the scope of the claims is defined by the recitations therein and not by the specific discussion of features and advantages set forth in the present description.

[0020] BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The following drawings form part of the specification and are included to further demonstrate certain embodiments or various aspects of the invention. In some instances, embodiments of the invention can be best understood by referring to the accompanying drawings in combination with the detailed description presented herein. The description and accompanying drawings may highlight a certain specific example, or a certain aspect of the invention. However, one skilled in the art will understand that portions of the example or aspect may be used in combination with other examples or aspects of the invention.

[0022] Figure 1. Phosphonate natural products made by Pantoea ananatis. The structures of two previously characterized phosphonate natural products are shown at the top, with the hvr operon responsible for their synthesis shown below.

[0023] Figure 2. Hydratase / dehydratase activity of HvrDE. The reaction catalyzed by HvrDE is shown at the top with31P NMR spectra of reactions containing the indicated substrate and enzyme combinations shown below. The bars indicate the peaks corresponding to PMM and PMME, which shift slightly due to minor variations in pH between reactions.

[0024] Figure 3A-B. Methylation of PMME and pantaphos by HvrF. The reactions with PMME (panel A) and pantaphos (panel B) are shown at the top with the31P-NMR spectra for reactions containing the indicated components below. All reactions also contained S- adenosylmethionine (SAM) and the enzyme 5-adenosylhomocysteine (SAH) nucleosidase. The colored vertical bars indicate the peaks corresponding to each compound, which shift slightly due to minor variations in pH between reactions. Off-pathway rearrangement products from PMME methylation are indicated by asterisks. Figures 9 & 10 show1H-31P HMBC analysis of these off-pathway products. Abbreviations: adenosine (Ade), 5-(5-deoxy-D-ribos- 5-yl)-L-homocysteine (dRibo-Hcy).

[0025] Figure 4. Conversion of PMM to methyl-PMME by the combined action of HvrDE and HvrF. The reactions catalyzed by HvrDE and HvrF are shown at the top with31P NMR spectra of reactions containing the indicated substrate and enzyme combinations shown below. All reactions also contained S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) nucleosidase. The colored vertical bars indicate the peaks corresponding to each compound, which shift slightly due to minor variations in pH between reactions. Off-pathway rearrangement products from PMME methylation are indicated by asterisks.

[0026] Figure 5. Conversion of methyl-PMME to methylpantaphos by HvrB / HvrK. The reaction catalyzed by HvrB / HvrK is shown at the top with31P NMR spectra of reactions containing the indicated components shown below. The colored vertical bars indicate the peaks corresponding to each compound, which shift slightly due to minor variations in pH between reactions. Off-pathway rearrangement products from PMME methylation are indicated by asterisks.

[0027] Figure 6A-C. Heterologous production of pantaphos and pathway intermediates in E. coli. Spent media from E. coli cultures carrying the indicated plasmids grown under inducing and non-inducing conditions were analyzed by31P NMR. Spectra are derived from strains carrying pAP42 (Panel A), pAP40 (Panel B), and pAP44 (Panel C). The genes present in each plasmid are presented in the arrow diagrams between each pair of spectra. The genes present on each plasmid are expressed from the IPTG-inducible Ptac promoter indicated by the arrows. The colored vertical bars indicate the peaks corresponding to each compound, which shift slightly due to minor variations in pH between reactions. The identity of these molecules was validated by1H-31P HMBC analyses. Figure 7. SDS-PAGE analysis of purified Hvr proteins. The indicated Hvr proteins, which, except for HvrD, carried N-terminal 6X-histidine tags, were affinity purified after heterologous expression in E. coli, then separated by SDS-PAGE and visualized by staining with Coomassie Blue. Untagged HvrD was co-expressed with tagged HvrE, which allowed purification of both proteins as a complex. Predicted molecular weights for each protein are: HvrB, 51 kDa; HvrD, 44 kDa; HvrE, 23 kDa; HvrF, 32 kDa; HvrK, 19 kDa; HvrH, 44 kDa, HvrG, 20 kDa: HvrJ, 41 kDa.

[0028] Figure 8. Identification of the HvrK cofactor. The UV-vis absorbance spectrum of the cofactor extracted from HvrK with diagnostic peaks at 373 and 446 nm is shown at the top. A photograph of purified enzyme is shown in the inset. The total ion chromatogram from LC-MS analysis of the extracted cofactor is shown at the bottom. The strong peak at m / z = 457.1 is diagnostic for FMN.

[0029] Figure 9. Requirements for the HvrB / HvrK monooxygenase reaction. Enzyme reactions containing the indicated components were examined by31P NMR. The colored vertical bars indicate the position of the methyl-PMME and methylpantaphos, which shift slightly due to minor variations in pH between reactions. The top two spectra show the requirement for HvrB with NADH of NADPH. The next two spectra show that the reaction proceeds in the absence of HvrK with nicotinamide cofactors. The bottom two spectra show that FMN is required for this reaction. Off-pathway rearrangement products from PMME methylation are indicated by asterisks.

[0030] Figure 10A-B. Phytotoxicity of pantaphos towards ri. thaliana. ( A) Treatment of germinated wild-type A. thaliana seedlings with pantaphos and (B) the corresponding effects on root length and dry weight. The data are Mean± S.E.M. of 12 biological replicates. The asterisks indicate significant differences between untreated (water) and pantaphos treated seedlings using Welch’s t-test: **P::::0.0018; ***P = 0.0006.

[0031] Figure 11A-E. HvrF expression confers pantaphos resistance to A. thaliana. (A) The DNA construct used for HvrF overexpression in 4. thaliana under a constitutive promotor. The bar gene was used for selection of transgenic lines. (B) Pantaphos resistance assay using A. thaliana Col-0 (WT) and independent transgenic lines overexpressing HvrF (#2, #6, #12, and #14). The images were captured nine days post-treatment after treating six-day old seedlings. (C) Pantaphos dose response curves of WT and HvrF overexpression lines. The data are Mean ± S.E.M. of 4-8 biological replicates. The asterisks indicate significant differences between WT and transgenic seed lines using Dunn's Kruskal- Wallis test: *P< 0.05; < 0.01;

[0032] *** / ’ < 0.001; **** / ’ < 0.0001. (D) Immunoblot analysis showing HvrF expression in the transgenic seed lines. The HvrF protein was detected using anti-HA antibodies. (E) Linear relationship of relative HvrF protein expression and 50% lethal dose (LD50) in the transgenic seed lines.

[0033] Figure 12A-H. Growth phenotypes of pantaphos-treated A. thaliana overexpressing HvrF. Growth curves of A. thaliana Col-0 (WT) and independent transgenic lines overexpressing HvrF (#2, #6, #12, and #14) treated with different pantaphos concentrations (0 (A), 1.5 (B), 7.5 (C), 15 (D), 75 (E), 150 (F), 300 (G), 750 (H) pM). Pantaphos was administered to six-day old seedlings and the seedlings were observed for nine days post treatment. Total leaf area was used to determine plant growth. The data are Mean± S.E.M. of 4-8 biological replicates. The asterisks indicate significant differences between WT and transgenic seed lines using Dunn’s Kruskal-Wallis test: *P < 0.05; **P< 0.01; ***P < 0.001; ****P < 0.0001.

[0034] Figure 13A-B. Resistance to Pantoea ananatis infection in A. thaliana overexpressing HvrF. A. thaliana Col-0 wild-type (HvrF-) and transgenic plant line #2 overexpressing HvrF (HvrF 4- ) were inoculated with 6.4 x 106cells / mL of (A) P. ananatis LMG5342 (WT) and (B) P. ananatis LMG5342 Δhvr mutant, which lacks the ability to produce pantaphos. Seedlings were imaged after 2-, 4-, and 7-days post inoculation (DPI). Images are representative of 12 biological replicates per condition.

[0035] DETAILED DESCRIPTION

[0036] Definitions.

[0037] The following definitions are included to provide a clear and consistent understanding of the specification and claims. As used herein, the recited terms have the following meanings. All other terms and phrases used in this specification have their ordinary meanings as one of skill in the art would understand. Such ordinary meanings may be obtained by reference to technical dictionaries, such as Hawley ’s Condensed Chemical Dictionary 14thEdition, by R.J. Lewis, John Wiley & Sons, New York, N.Y., 2001., or Singleton, et al., Dictionary of Microbiology and Molecular Biology, 2d ed., John Wiley and Sons, New York (1994), and Hale & Markham, The Harper Collins Dictionary of Biology. Harper Perennial, N.Y. (1991). General laboratory techniques (DNA extraction, RNA extraction, cloning, PCR amplification, cell culturing, etc.) are known in the art and described, for example, in Molecular Cloning: A Laboratory Manual, J. Sambrook et al., 4th edition, Cold Spring Harbor Laboratory Press, 2012.

[0038] References in the specification to "one embodiment", "an embodiment", etc., indicate that the embodiment described may include a particular aspect, feature, structure, moiety, or characteristic, but not every embodiment necessarily includes that aspect, feature, structure, moiety, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment referred to in other portions of the specification. Further, when a particular aspect, feature, structure, moiety, or characteristic is described in connection with an embodiment, it is within the knowledge of one skilled in the art to affect or connect such aspect, feature, structure, moiety, or characteristic with other embodiments, whether or not explicitly described.

[0039] The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a compound" includes a plurality of such compounds, so that a compound X includes a plurality of compounds X. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for the use of exclusive terminology, such as "solely," "only," and the like, in connection with any element described herein, and / or the recitation of claim elements or use of "negative" limitations.

[0040] The term "and / or" means any one of the items, any combination of the items, or all of the items with which this term is associated. The phrases "one or more" and "at least one" are readily understood by one of skill in the art, particularly when read in context of its usage. For example, the phrase can mean one, two, three, four, five, six, ten, 100, or any upper limit approximately 10, 100, or 1000 times higher than a recited lower limit. For example, one or more substituents on a phenyl ring refers to one to five, or one to four, for example if the phenyl ring is disubstituted.

[0041] As will be understood by the skilled artisan, all numbers, including those expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, are approximations and are understood as being optionally modified in all instances by the term "about." These values can vary depending upon the desired properties sought to be obtained by those skilled in the art utilizing the teachings of the descriptions herein. It is also understood that such values inherently contain variability necessarily resulting from the standard deviations found in their respective testing measurements. When values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value without the modifier "about" also forms a further aspect.

[0042] The terms "about" and "approximately" are used interchangeably. Both terms can refer to a variation of ± 5%, ± 10%, ± 20%, or ± 25% of the value specified. For example, "about 50" percent can in some embodiments carry a variation from 45 to 55 percent, or as otherwise defined by a particular claim. For integer ranges, the term "about" can include one or two integers greater than and / or less than a recited integer at each end of the range. Unless indicated otherwise herein, the terms "about" and "approximately" are intended to include values, e.g., weight percentages, proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, composition, or embodiment. The terms "about" and "approximately" can also modify the endpoints of a recited range as discussed above in this paragraph.

[0043] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges recited herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof, as well as the individual values making up the range, particularly integer values. It is therefore understood that each unit between two particular units are also disclosed. For example, if 10 to 15 is disclosed, then 11, 12, 13, and 14 are also disclosed, individually, and as part of a range. A recited range (e.g., weight percentages or carbon groups) includes each specific value, integer, decimal, or identity within the range. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, or tenths. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art, all language such as "up to", "at least", "greater than", "less than", "more than", "or more", and the like, include the number recited and such terms refer to ranges that can be subsequently broken down into sub-ranges as discussed above. In the same manner, all ratios recited herein also include all sub-ratios falling within the broader ratio. Accordingly, specific values recited for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for radicals and substituents. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

[0044] This disclosure provides ranges, limits, and deviations to variables such as volume, mass, percentages, ratios, etc. It is understood by an ordinary person skilled in the art that a range, such as “numberl” to “number2”, implies a continuous range of numbers that includes the whole numbers and fractional numbers. For example, 1 to 10 means 1, 2, 3, 4, 5, ... 9, 10. It also means 1.0, 1.1, 1.2. 1.3, . . ., 9.8, 9.9, 10.0, and also means 1.01, 1.02, 1.03, and so on. If the variable disclosed is a number less than “numberl 0”, it implies a continuous range that includes whole numbers and fractional numbers less than numberl 0, as discussed above. Similarly, if the variable disclosed is a number greater than “numberl 0”, it implies a continuous range that includes whole numbers and fractional numbers greater than number 10. These ranges can be modified by the term “about”, whose meaning has been described above.

[0045] One skilled in the art will also readily recognize that where members are grouped together in a common manner, such as in a Markush group, the invention encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group. Additionally, for all purposes, the invention encompasses not only the main group, but also the main group absent one or more of the group members. The invention therefore envisages the explicit exclusion of any one or more of members of a recited group. Accordingly, provisos may apply to any of the disclosed categories or embodiments whereby any one or more of the recited elements, species, or embodiments, may be excluded from such categories or embodiments, for example, for use in an explicit negative limitation.

[0046] The term "contacting" refers to the act of touching, making contact, or of bringing to immediate or close proximity, including at the cellular or molecular level, for example, to bring about a physiological reaction, a chemical reaction, or a physical change, e.g., in a solution, in a reaction mixture, in vitro, or in vivo.

[0047] An "effective amount" refers to an amount effective to treat a disease, disorder, and / or condition, or to bring about a recited effect. The disease, disorder, and / or condition is in a living organism such as in an animal, plant, or crop. For example, an effective amount can be an amount effective to reduce the progression or severity of the condition or symptoms being treated. Determination of a therapeutically effective amount is well within the capacity of persons skilled in the art. The term "effective amount" is intended to include an amount of a compound described herein, or an amount of a combination of compounds described herein, e.g., that is effective to treat or prevent a disease or disorder, or to treat the symptoms of the disease or disorder, in a host. Thus, an "effective amount" generally means an amount that provides the desired effect.

[0048] As used herein, the terms “providing”, “administering,” “introducing,” are used interchangeably herein and refer to the placement of a compound of the disclosure into a subj ect by a method or route that results in at least partial localization of the compound to a desired site. The compound can be administered by any appropriate route that results in delivery to a desired location in the subject.

[0049] The terms "inhibit", "inhibiting", and "inhibition" refer to the slowing, halting, or reversing the growth or progression of a disease, infection, condition, or group of cells. The inhibition can be greater than about 20%, 40%, 60%, 80%, 90%, 95%, or 99%, for example, compared to the growth or progression that occurs in the absence of the treatment or contacting. An "effective amount" refers to an amount effective to bring about a recited effect, such as an amount necessary to form products in a reaction mixture. Determination of an effective amount is typically within the capacity of persons skilled in the art, especially in light of the detailed disclosure provided herein. The term "effective amount" is intended to include an amount of a compound or reagent described herein, or an amount of a combination of compounds or reagents described herein, e.g., that is effective to form products in a reaction mixture. Thus, an "effective amount" generally means an amount that provides the desired effect.

[0050] The term “substantially” as used herein, is a broad term and is used in its ordinary sense, including, without limitation, being largely but not necessarily wholly that which is specified. For example, the term could refer to a numerical value that may not be 100% the full numerical value. The full numerical value may be less by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, or about 20%.

[0051] Wherever the term “comprising” is used herein, options are contemplated wherein the terms “consisting of’ or “consisting essentially of’ are used instead. As used herein, “comprising” is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, "consisting of excludes any element, step, or ingredient not specified in the aspect element. As used herein, "consisting essentially of' does not exclude materials or steps that do not materially affect the basic and novel characteristics of the aspect. In each instance herein any of the terms "comprising", "consisting essentially of and "consisting of may be replaced with either of the other two terms. The disclosure illustratively described herein may be suitably practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.

[0052] A polynucleotide is "heterologous to" an organism or a second polynucleotide if it originates from a foreign species, or, if from the same species, is modified from its original form. For example, a promoter operably linked to a heterologous coding sequence refers to a coding sequence from a species different from that from which the promoter was derived, or, if from the same species, a coding sequence which is not naturally associated with the promoter (e. g. a genetically engineered coding sequence or an allele from a different ecotype or variety).

[0053] "Transgene", "transgenic" or "recombinant" refers to a polynucleotide manipulated by man or a copy or complement of a polynucleotide manipulated by man. For instance, a transgenic expression cassette comprising a promoter operably linked to a second polynucleotide may include a promoter that is heterologous to the second polynucleotide as the result of manipulation by man (e.g., by methods described in Sambrook et al., Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (1989) or Current Protocols in Molecular Biology Volumes 1 -3, John Wiley & Sons, Inc. (1994-1998)) of an isolated nucleic acid comprising the expression cassette. In another example, a recombinant expression cassette may comprise polynucleotides combined in such a way that the polynucleotides are extremely unlikely to be found in nature. For instance, restriction sites or plasmid vector sequences manipulated by man may flank or separate the promoter from the second polynucleotide. One of skill will recognize that polynucleotides can be manipulated in many ways and are not limited to the examples above.

[0054] A polynucleotide "exogenous to" an individual organism is a polynucleotide which is introduced into the organism by any means other than by a sexual cross.

[0055] As used herein “promoter” includes reference to a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A “plant promoter” is a promoter capable of initiating transcription in plant cells whether or not its origin is a plant cell. Illustrative plant promoters include, but are not limited to, those that are obtained from plants, plant viruses, and bacteria which comprise genes expressed in plant cells such as Agrobacterium or Rhizobium. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, or seeds. Such promoters are referred to as “tissue preferred”. Promoters that initiate transcription only in certain tissue are referred to as “tissue specific”. A “cell type” specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. An “inducible” or “repressible” promoter is a promoter that is under environmental control. Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions or the presence of light. Tissue specific, tissue preferred, cell type specific, and inducible promoters constitute the class of “non-constitutive” promoters. A “constitutive” promoter is a promoter that is active under most environmental conditions.

[0056] As used herein, “recombinant,” when referring to nucleic acid or polypeptide, indicates that such material has been altered as a result of human application of a recombinant technique, such as by polynucleotide restriction and ligation, by polynucleotide overlap-extension, or by genomic insertion or transformation. A gene sequence open reading frame is recombinant if that nucleotide sequence has been removed from its natural context and cloned into any type of artificial nucleic acid vector. The term recombinant also can refer to an organism having a recombinant material, e.g., a plant that comprises a recombinant nucleic acid can be considered a recombinant plant.

[0057] “Regulatory elements” refer to nucleotide sequences located upstream (5' noncoding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory elements may include, but are not limited to, promoters, translation leader sequences, introns, and polyadenylation recognition sequences. Regulatory elements present on a recombinant DNA construct that is introduced into a cell can be endogenous to the cell, or they can be heterologous with respect to the cell. The terms “regulatory element” and “regulatory sequence” are used interchangeably herein.

[0058] The terms "operable linkage" or "operably linked" are generally understood as meaning an arrangement in which a genetic control sequence, e.g., a promoter, enhancer or terminator, is capable of exerting its function with regard to a polynucleotide being operably linked to it, for example a polynucleotide encoding a polypeptide. Function, in this context, may mean for example control of the expression, i.e., transcription and / or translation, of the nucleic acid sequence. Control, in this context, encompasses for example initiating, increasing, governing, or suppressing the expression, i.e., transcription and, if appropriate, translation. Controlling, in turn, may be, for example, tissue- and / or time-specific. It may also be inducible, for example by certain chemicals, stress, pathogens, and the like. Preferably, operable linkage is understood as meaning for example the sequential arrangement of a promoter, of the nucleic acid sequence to be expressed and, if appropriate, further regulatory elements such as, for example, a terminator, in such a way that each of the regulatory elements can fulfill its function when the nucleic acid sequence is expressed. An operably linkage does not necessarily require a direct linkage in the chemical sense. For example, genetic control sequences like enhancer sequences are also capable of exerting their function on the target sequence from positions located at a distance to the polynucleotide, which is operably linked. Preferred arrangements are those in which the nucleic acid sequence to be expressed is positioned after a sequence acting as promoter so that the two sequences are linked covalently to one another. The distance between the promoter and the amino acid sequence encoding polynucleotide in an expression cassette, is preferably less than 200 base pairs, especially preferably less than 100 base pairs, very especially preferably less than 50 base pairs. The skilled worker is familiar with a variety of ways in order to obtain such an expression cassette. However, an expression cassette may also be constructed in such a way that the nucleic acid sequence to be expressed is brought under the control of an endogenous genetic control element, for example an endogenous promoter, for example by means of homologous recombination or else by random insertion. Such constructs are likewise understood as being expression cassettes for the purposes of the invention.

[0059] The terms "express," "expressing," "expressed" and "expression" refer to expression of a gene product (e.g., a biosynthetic enzyme of a gene of a pathway or reaction defined and described in this application) at a level that the resulting enzyme activity of this protein encoded for or the pathway or reaction that it refers to allows metabolic flux through this pathway or reaction in the organism in which this gene / pathway is expressed in. The expression can be done by genetic alteration of the microorganism that is used as a starting organism. In some embodiments, a microorganism can be genetically altered (e.g., genetically engineered) to express a gene product at an increased level relative to that produced by the starting microorganism or in a comparable microorganism which has not been altered. Genetic alteration includes, but is not limited to, altering or modifying regulatory sequences or sites associated with expression of a particular gene (e.g. by adding strong promoters, inducible promoters or multiple promoters or by removing regulatory sequences such that expression is constitutive), modifying the chromosomal location of a particular gene, altering nucleic acid sequences adjacent to a particular gene such as a ribosome binding site or transcription terminator, increasing the copy number of a particular gene, modifying proteins (e.g., regulatory proteins, suppressors, enhancers, transcriptional activators and the like) involved in transcription of a particular gene and / or translation of a particular gene product, or any other conventional means of deregulating expression of a particular gene using routine in the art (including but not limited to use of antisense nucleic acid molecules, for example, to block expression of repressor proteins).

[0060] In some embodiments, a microorganism can be physically or environmentally altered to express a gene product at an increased or lower level relative to level of expression of the gene product unaltered microorganism. For example, a microorganism can be treated with, or cultured in the presence of an agent known, or suspected to increase transcription of a particular gene and / or translation of a particular gene product such that transcription and / or translation are enhanced or increased. Alternatively, a microorganism can be cultured at a temperature selected to increase transcription of a particular gene and / or translation of a particular gene product such that transcription and / or translation are enhanced or increased.

[0061] The term "vector", preferably, encompasses phage, plasmid, fosmid, viral vectors as well as artificial chromosomes, such as bacterial or yeast artificial chromosomes. Moreover, the term also relates to targeting constructs which allow for random or site-directed integration of the targeting construct into genomic DNA. Such target constructs, preferably, comprise DNA of sufficient length for either homologous or heterologous recombination as described in detail below. The vector encompassing the polynucleotide of the present invention, preferably, further comprises selectable markers for propagation and / or selection in a recombinant microorganism. The vector may be incorporated into a recombinant microorganism by various techniques well known in the art. If introduced into a recombinant microorganism, the vector may reside in the cytoplasm or may be incorporated into the genome. In the latter case, it is to be understood that the vector may further comprise nucleic acid sequences which allow for homologous recombination or heterologous insertion. Vectors can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.

[0062] The terms "transformation" and "transfection", conjugation and transduction, as used in the present context, are intended to comprise a multiplicity of prior-art processes for introducing foreign nucleic acid (for example DNA) into a recombinant microorganism, including calcium phosphate, rubidium chloride or calcium chloride co-precipitation, DEAE- dextran-mediated transfection, lipofection, natural competence, carbon-based clusters, chemically mediated transfer, electroporation or particle bombardment. Methods for many species of microorganisms are readily available in the literature.

[0063] Nucleic acid sequences cited herein are written in a 5' to 3' direction unless indicated otherwise. The term "nucleic acid" refers to either DNA or RNA or a modified form thereof comprising the purine or pyrimidine bases present in DNA (adenine "A", cytosine "C", guanine "G", thymine "T") or in RNA (adenine "A", cytosine "C", guanine "G", uracil "U"). Interfering RNAs provided herein may comprise "T" bases, for example at 3' ends, even though "T" bases do not naturally occur in RNA. In some cases, these bases may appear as "dT" to differentiate deoxyribonucleotides present in a chain of ribonucleotides.

[0064] A first nucleic acid or protein sequence displays “substantial identity” or “substantial similarity” to a reference nucleic acid sequence or protein if, when optimally aligned (with appropriate nucleotide or amino acid insertions or deletions totaling less than 20 percent of the reference sequence over the window of comparison) with the other nucleic acid (or its complementary strand) or protein, there is at least about 60% nucleotide sequence equivalence, even better would be 70%, preferably at least about 80% equivalence, more preferably at least about 85% equivalence, and most preferably at least about 90% equivalence over a comparison window of at least 20 nucleotide or amino acid positions, preferably at least 50 nucleotide or amino acid positions, more preferably at least 100 nucleotide or amino acid positions, and most preferably over the entire length of the first nucleic acid or protein. Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm(s), preferably by computerized implementations of these algorithms (which can be found in, for example, Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.). The reference nucleic acid may be a full-length molecule or a portion of a longer molecule. Alternatively, two nucleic acids have substantial identity if one hybridizes to the other under stringent conditions. Appropriate hybridization conditions can be determined empirically, or can be estimated based, for example, on the relative G+C content of the probe and the number of mismatches between the probe and target sequence, if known. Hybridization conditions can be adjusted as desired by varying, for example, the temperature of hybridizing or the salt concentration (Sambrook et al., Molecular Cloning. A Laboratory Manual, 2ndEdition, Cold Spring Harbor Press, 1989).

[0065] Hybridization conditions are dependent on the sequence of the polynucleotide and will be different in different circumstances. As used herein “stringent conditions” are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific polynucleotide at a defined ionic strength and pH. The “thermal melting point” is the temperature (under defined ionic strength and pH) at which 50% of a target molecule hybridizes to a completely complementary molecule. Appropriate stringent conditions which promote DNA hybridization, for example, 6. Ox sodium chloride / sodium citrate (SSC) at about 45° C., followed by a wash of 2. Ox SSC at 50° C., are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, incorporated herein by reference in its entirety. For example, the salt concentration in the wash step can be selected from a low stringent condition of about 2. Ox SSC at 50° C. to a high stringency of about 0.2xSSC at 50° C. In addition, the temperature in the wash step can be increased from low stringent conditions at room temperature, about 22° C., to high stringent conditions at about 65° C. Both temperature and salt concentration may be varied, or either the temperature or the salt concentration may be held constant while the other variable is changed. For the purposes of this disclosure, stringent conditions include at least one wash in 2. Ox SSC at a temperature of at least about 50° C. for 20 minutes, or equivalent conditions.

[0066] As used herein, the term “confer” refers to providing a characteristic or trait, such as herbicide tolerance or resistance and / or other desirable traits to a plant.

[0067] Generally, the term “herbicide” is used herein to mean an active ingredient that kills, controls or otherwise adversely modifies the growth of plants. The preferred amount or concentration of the herbicide is an “effective amount” or “effective concentration.” By “effective amount” and “effective concentration” is intended an amount and concentration, respectively, that is sufficient to kill or inhibit the growth of a similar, wildtype, plant, plant tissue, plant cell, or host cell, but that said amount does not kill or inhibit as severely the growth of the herbicide-resistant plants, plant tissues, plant cells, and host cells of the present disclosure. Typically, the effective amount of an herbicide is an amount that is routinely used in agricultural production systems to kill weeds of interest. Such an amount for a given herbicide is known to those of ordinary skill in the art. Herbicidal activity is exhibited by herbicides useful for the present disclosure when they are applied directly to the plant or to the locus of the plant at any stage of growth or before planting or emergence. The effect observed depends upon the plant species to be controlled, the stage of growth of the plant, the application parameters of dilution and spray drop size, the particle size of solid components, the environmental conditions at the time of use, the specific compound employed, the specific adjuvants and carriers employed, the soil type, and the like, as well as the amount of chemical applied. These and other factors can be adjusted as is known in the art to promote non-selective or selective herbicidal action. Generally, the herbicide treatments can be applied PPI (Pre Plant Incorporated), PPSA (Post plant surface applied), PRE- or POST-emergent. Post emergent treatment typically occurs to relatively immature undesirable vegetation to achieve the maximum control of weeds.

[0068] By a “herbicide-tolerant” or “herbicide-resistant” plant, it is intended that a plant that is tolerant or resistant to at least one herbicide at a level that would normally kill, or inhibit the growth of, a normal or wildtype plant. Levels of herbicide that normally inhibit growth of a non-tolerant plant are known and readily determined by those skilled in the art. Examples include the amounts recommended by manufacturers for application. The maximum rate is an example of an amount of herbicide that would normally inhibit growth of a non-tolerant plant. For the present disclosure, the terms “herbicide-tolerant” and “herbicide-resistant” are used interchangeably and are intended to have an equivalent meaning and an equivalent scope. Similarly, the terms “herbicide-tolerance” and “herbicide-resistance” are used interchangeably and are intended to have an equivalent meaning and an equivalent scope. Similarly, the terms “tolerant” and “resistant” are used interchangeably and are intended to have an equivalent meaning and an equivalent scope. As used herein, in regard to an herbicidal composition useful in various embodiments hereof, terms such as herbicides, and the like, refer to those agronomically acceptable herbicide active ingredients (A.I.) recognized in the art. As used herein, an “herbicide tolerance trait” is a trait imparting improved herbicide tolerance to a plant as compared to the wild-type plant. As used herein, “plant” refers to a whole plant, any part thereof, or a cell or tissue culture derived from a plant, comprising any of: whole plants, plant components or organs (e.g., leaves, stems, roots, etc.), plant tissues, seeds, plant cells, and / or progeny of the same. A progeny plant can be from any filial generation, e.g., Fl, F2, F3, F4, F5, F6, F7, etc. A plant cell is a biological cell of a plant, taken from a plant or derived through culture from a cell taken from a plant. Plant parts include, but are not limited to seeds microspores, pollen, anthers, silk, spike, ovules, ovaries, flowers, pods, cobs, embryos, stems, leaves, roots, and calli.

[0069] Embodiments of the Technology.

[0070] Compositions and methods for conferring herbicide tolerance to plants, plant parts, and plant cells are provided. Modified plants having tolerance to an herbicide, the modified plant, plant part, or plant cell having increased expression of a polynucleotide encoding a HvrF polypeptide of Pantoea ananatis relative to an unmodified plant are provided. In certain embodiments, the modified plants comprise a heterologous polynucleotide encoding the HvrF polypeptide. Progeny, plant seeds, plant parts, and plant cells of the modified plants are also provided.

[0071] Exemplary herbicide resistant plants, plant parts, and plant cells comprise one or more heterologous polynucleotides comprising a promoter sequence operably linked to a nucleotide sequence that encodes one or more polypeptides that, when expressed in the plant, plant part, and plant cell, confers resistance the herbicide. In one preferred embodiment, the herbicide is pantaphos, a phosphonic acid natural product made by the plant pathogen Pantoea ananatis:

[0072] In certain embodiments, the pantaphos is the R isomer of pantaphos, (A)-2- (hydroxy(phosphono)methyl)maleic acid.

[0073] Accordingly, embodiments of nucleic acid molecules that may be used to confer pantaphos resistance to a plant, plant part, or plant cell may comprise a constitutive promoter sequence that is functional in plants and a nucleotide sequence operably linked to the constitutive promoter sequence, wherein the nucleotide sequence encodes an HvrF protein of Pantoea ananatis comprising an amino acid sequence having at least about 50% sequence identity, at least about 60% sequence identity, at least about 70% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least about 90% sequence identity, at least about 91% sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, or at least about 99% sequence identity to SEQ ID NO: 11.

[0074] In some embodiments, a nucleic acid molecule that may be used to confer pantaphos resistance to a plant, plant part, or plant cell comprises a constitutive promoter sequence that is functional in plants and a nucleotide sequence operably linked to the constitutive promoter sequence, wherein the nucleotide sequence encodes the HvrF protein of Pantoea ananatis comprises the amino acid sequence at least 95% identical to SEQ ID NO: 11. In some embodiments, the nucleotide sequence encodes the HvrF protein of Pantoea ananatis comprising an amino acid sequence according to SEQ ID NO: 11.

[0075] In some embodiments, the nucleic acid molecule includes a nucleotide sequence comprises an hvrF gene of Pantoea ananatis having at least about 80% sequence identity, at least about 85% sequence identity, at least about 90% sequence identity, at least about 91% sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, or at least about 99% sequence identity to SEQ ID NO: 12. In some embodiments, the nucleic acid molecule includes a nucleotide sequence comprises a codon optimized hvrF gene of Pantoea ananatis having at least 95% sequence identity to SEQ ID NO: 27. In some embodiments, the nucleotide sequence comprises an hvrF gene of Pantoea ananatis according to SEQ ID NO: 12 or a codon optimized hvrF gene of Pantoea ananatis according to SEQ ID NO: 27. In some embodiments, the nucleic acid molecule includes a nucleotide sequence comprising SEQ ID NO: 26. In some embodiments, the nucleic acid molecule comprises plasmid JA477 (SEQ ID NO: 31).

[0076] Various promoter sequences may be used in the practice of the invention. The promoters can be selected based on the desired outcome. The nucleic acids can be combined with constitutive, tissue-preferred, or other promoters for expression in plants.

[0077] Constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99 / 43838 and U.S. Patent No. 6,072,050; the core CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2: 163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81 :581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Patent No. 5,659,026), and the like. Other constitutive promoters include, for example, U.S. Patent Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.

[0078] Tissue-preferred promoters can be utilized to target enhanced expression within a particular plant tissue. Such tissue-preferred promoters include, but are not limited to, leafpreferred promoters, root-preferred promoters, seed-preferred promoters, and stem-preferred promoters. Some examples of tissue-preferred promoters are described by, e.g., Yamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol. Gen Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2): 157-168; Rinehart et al. (1996) Plant Physiol. 112(3): 1331-1341; Van Camp et al. (1996) Plant Physiol. 112(2): 525-535; Canevascini et al. (1996) Plant Physiol. 112(2):513- 524; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20: 181-196; Orozco ef a / . (1993) Plant Mol Biol. 23(6): 1129-1138; Matsuoka et al. (1993) Voc Natl. Acad. ScL USA 90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J 4(3):495-505. Promoters can be modified, if necessary, for weak expression.

[0079] In some embodiments, the promoter sequence operably linked to the coding sequence comprises a promoter sequence that may include, but are not limited to, p35s, pUbi, pActinl, pNos, pRBCS, pAP3, pGSSP, pPG1090, pPYKIO, pCAB, pRD29A, or pER. In some preferred embodiments, the plant promoter is a constitutive promoter sequence. In some preferred embodiments, the promoter sequence comprises a plant virus promoter sequence.

[0080] Plant viral promoters can also be used, such as the cauliflower mosaic virus 19S and 35S promoters (CaMV 19S and CaMV 35S promoters, respectively) to control gene expression in a plant (U.S. Patent Nos. 5,352,605; 5,530,196 and 5,858,742 for example). Enhancer sequences derived from the CaMV can also be utilized (U.S. Patent Nos. 5,164,316; 5,196,525; 5,322,938; 5,530,196; 5,352,605; 5,359,142; and 5,858,742 for example). Thus, in some embodiments, the promoter sequence comprises a Cauliflower Mosaic Virus 35S (35S) promoter. Optionally, the nucleic acid molecule also may include a Cauliflower Mosaic Virus 35S (35S) terminator sequence. Optionally, the nucleic acid molecule also may include one or more enhancer elements operable in a plant.

[0081] Certain nucleic acid molecules include DNA constructs and / or DNA molecules for use with embodiments of the invention, and may include, for example, expression vectors. Expression vectors include at least one genetic marker operably linked to a regulatory element (for example, a promoter) that allows transformed cells containing the marker to be either recovered by negative selection, i.e., inhibiting growth of cells that do not contain the selectable marker gene, or by positive selection, i.e., screening for the product encoded by the genetic marker. Many commonly used selectable marker genes for plant transformation are well known in the transformation arts, and include, for example, genes that code for enzymes that metabolically detoxify a selective chemical agent which may be an antibiotic or an herbicide, or genes that encode an altered target which is insensitive to the inhibitor. A few positive selection methods are also known in the art.

[0082] One commonly used selectable marker gene for plant transformation is the neomycin phosphotransferase II (nptll) gene which, when under the control of plant regulatory signals, confers resistance to kanamycin. (Fraley et al., Proc. Natl. Acad. Sci. USA, 80:4803 (1983)). Another commonly used selectable marker gene is the hygromycin phosphotransferase gene which confers resistance to the antibiotic hygromycin. (Vanden Elzen et al., Plant Mol. Biol, 5:299 (1985)).

[0083] Additional selectable marker genes of bacterial origin that confer resistance to antibiotics include gentamycin acetyl transferase, streptomycin phosphotransferase and aminoglycoside-3 '-adenyl transferase, the bleomycin resistance determinant (Hayford et al., Plant Physiol., 86: 1216 (1988); Jones et al., Mol. Gen. Genet., 210:86 (1987); Svab et al., Plant Mol. Biol., 14 : 197 (1990); Hille et al., Plant Mol. Biol., 7171 (1986)). Other selectable marker genes confer resistance to herbicides such as glyphosate, glufosinate, or bromoxynil (Comai et al., Nature, 317:741-744 (1985); Gordon-Kamm et al., Plant Cell, 2:603-618 (1990); Stalke et al., Science, 242:419-423 (1988)).

[0084] Selectable marker genes for plant transformation not of bacterial origin include, for example, mouse dihydrofolate reductase, plant 5 -enolpyruvylshikimate-3 -phosphate synthase, and plant acetolactate synthase (Eichholtz et al., Somatic Cell Mol. Genet., 13:67 (1987); Shah et al., Science, 233:478 (1986); Charest et al., Plant Cell Rep., 8:643 (1990)).

[0085] Another class of marker genes for plant transformation requires screening of presumptively transformed plant cells, rather than direct genetic selection of transformed cells, for resistance to a toxic substance such as an antibiotic. These genes are particularly useful to quantify or visualize the spatial pattern of expression of a gene in specific tissues and are frequently referred to as reporter genes because they can be fused to a gene or gene regulatory sequence for the investigation of gene expression. Commonly used genes for screening presumptively transformed cells include P-glucuronidase (GUS), P-galactosidase, luciferase, and chloramphenicol acetyltransferase (Jefferson, R. A., Plant Mol. Biol. Rep. ,5.3X1 (1987); Teeri et al., EMBO J., 8:343 (1989); Koncz et al., Proc. Natl. Acad. Sci. USA, 84: 131 (1987); DeBlock et al., EMBO J., 3: 1681 (1984)). In vivo methods for visualizing GUS activity that do not require destruction of plant tissue are available (Molecular Probes, Publication 2908, IMAGENE GREEN, pp. 1- 4 (1993); Naleway et al., J. Cell Biol. , 115: 151a (1991)). More recently, a gene encoding Green Fluorescent Protein (GFP) has been utilized as a marker for gene expression in prokaryotic and eukaryotic cells (Chalfie et al., Science, 263:802 (1994)). GFP and mutants of GFP may be used as screenable markers.

[0086] Methods for analyzing a gene integration into the plant genome are known to the person skilled in the art and comprise, but are not limited to polymerase chain reaction (PCR), including, inter alia, real time quantitative PCR, multiplex PCR, RT-PCR, nested PCR, analytical PCR and the like, microscopy, including bright and dark field microscopy, dispersion staining, phase contrast, fluorescence, confocal, differential interference contrast, deconvolution, electron microscopy, UV microscopy, IR microscopy, scanning probe microscopy, the analysis of plant or plant cell metabolites, RNA analysis, proteome analysis, functional assays for determining a functional integration, e.g. of a marker gene or a transgene of interest, or of a knock-out, Southern-Blot analysis, sequencing, including next generation sequencing, including deep sequencing or multiplex sequencing and the like, and combinations thereof.

[0087] Where appropriate, nucleic acid sequences may be optimized for increased expression in a transformed plant. For example, coding sequences can be provided that comprise plantpreferred codons for improved expression in a plant. See, for example, Campbell and Gowri (1990) Plant Physiol., 92: 1-11 for a discussion of host-preferred codon usage. Methods also are known in the art for preparing plant-preferred genes. See, for example, U.S. Patent Nos. 5,380,831, and 5,436,391, and Murray et al . (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference. Further, additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious poiyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well-characterized sequences that may be deleterious to gene expression. The G-C content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. Also, if desired, sequences can be readily modified to avoid predicted hairpin secondary mRNA structures. Nucleotide sequences for enhancing gene expression can also be used in the plant expression vectors. These include, for example, the introns of the maize Adhl, intronl gene (Callis et al. Genes and Development 1 : 1183-1200, 1987), and leader sequences, (W -sequence) from the Tobacco Mosaic virus (TMV), Maize Chlorotic Mottle Virus and Alfalfa Mosaic Virus (Gallic et al. Nucleic Acid Res. 15:8693-8711, 1987 and Skuzeski et al. Plant Mol. Biol. 15:65-79, 1990). The first intron from the shrunken- 1 locus of maize, has been shown to increase expression of genes in chimeric gene constructs. U.S. Patent Nos. 5,424,412 and 5,593,874 disclose the use of specific introns in gene expression constructs, and Gallic et al. (Plant Physiol. 106:929-939, 1994) also have shown that introns are useful for regulating gene expression on a tissue specific basis. To further enhance or to optimize gene expression, the plant expression vectors of the invention also may contain DNA sequences containing matrix attachment regions (MARs). Plant cells transformed with such modified expression systems, then, may exhibit overexpression or constitutive expression of a nucleotide sequence of the invention.

[0088] Expression vectors may additionally contain 5' leader sequences in the expression construct. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picomavirus leaders, for example, EMCV leader (Encephalomyo carditis 5 ' noncoding region) (Elroy-Stein et al. (1989) PNAS, 86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Vims) (Gallic et al. (1995) Gene 165(2):233- 238), MDMV leader (Maize Dwarf Mosaic Virus) (Virology 154:9-20), and human immunoglobulin heavy -chain binding protein (BiP) (Macejak et al. (1991 ) Nature 353:90-94); untranslated leader from the coat protein m RN A of alfalfa mosaic vims ( AMV RNA 4) (Jobling et al. (1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallic et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss, New' York), pp. 237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel et al. (1991) Virology 81 :382-385). See also, Della- Cioppa et al. (1987) Plant Physiol. 84:965-968.

[0089] Other methods known to enhance translation also can be utilized, for example, introns, and the like. In preparing an expression vector, the various nucleic acid fragments may be manipulated, so as to provide for the nucleic acid sequences in the proper orientation and, as appropriate, in die proper reading frame. Toward this end, adapters or linkers may be employed to join the nucleic acid fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous nucleic acid, removal of restriction sites, or the Like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved.

[0090] Methods for increasing expression of genes or gene products are well documented in the art and include, for example, overexpression driven by appropriate promoters, the use of transcription enhancers or translation enhancers. Isolated nucleic acids which serve as promoter or enhancer elements may be introduced in an appropriate position (typically upstream) of a non-heterologous form of a polynucleotide so as to upregulate expression of a nucleic acid encoding the protein of interest. Classical enhancers are cis-acting elements that increase gene transcription and can also be included in the expression construct. Classical enhancer elements are known in the art, and include, but are not limited to, the CaMV 35S enhancer element, cytomegalovirus (CMV) early promoter enhancer element, and the SV40 enhancer element. Intron -mediated enhancer elements that enhance gene expression are also known in the art.

[0091] Additionally, expression constructs may optionally contain a transcription termination sequence, a translation termination sequence, a sequence encoding a signal peptide, and / or enhancer elements. Transcription termination regions can typically be obtained from the 3' untranslated region of a eukaryotic or viral gene sequence. Transcription termination sequences can be positioned downstream of a coding sequence to provide for efficient termination.

[0092] The disclosure also provides for pantaphos-resistant plant cells and methods of making the same. In some embodiments, a pantaphos-resistant plant cell comprises a recombinant nucleic acid molecule, the recombinant nucleic acid molecule comprising: i) a constitutive promoter sequence that is functional in plant cells; and ii) a nucleotide sequence encoding a polypeptide comprising a 5-adenosylmethione (SAM)-dependent methyltransferase, wherein the constitutive promoter sequence is heterologous with respect to the nucleotide sequence and is configured to cause sufficient expression of the polypeptide in the plant cell comprising the recombinant nucleic acid molecule to confer pantaphos resistance to the plant cells.

[0093] In some embodiments, the bacterial (SAM)-dependent methyltransferase is derived from Pantoea ananatis. Preferably, the bacterial (SAM)-dependent methyltransferase comprises an HvrF protein of Pantoea ananatis. In some embodiments, a nucleic acid molecule encodes a protein having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 11. In other embodiments, the nucleic acid molecule encodes a protein as set forth in SEQ ID NO: 11.

[0094] In some embodiment of a pantaphos-resistant plant cell, the recombinant nucleic acid molecule includes a nucleotide sequence comprises a hvrF gene of Pantoea ananatis having at least 95% sequence identity to SEQ ID NO: 12 or a codon optimized hvrF gene of Pantoea ananatis having at least 95% sequence identity to SEQ ID NO: 27. In some embodiments, the nucleotide sequence comprises an hvrF gene of Pantoea ananatis according to SEQ ID NO: 12 or a codon optimized hvrF gene of Pantoea ananatis according to SEQ ID NO: 27. In some embodiments, a pantaphos-resistant plant, plant cell, or plant part comprises plasmid JA477 (SEQ ID NO: 31).

[0095] In some embodiments, the promoter sequence comprises one of p35s, pUbi, pActinl, pNos, pRBCS, pAP3, pGSSP, pPG1090, pPYKIO, pCAB, pRD29A, and pER. In some embodiments, the promoter sequence comprises a constitutive promoter sequence comprising a plant virus promoter sequence. In some embodiments, the promoter sequence comprises a Cauliflower Mosaic Virus 35S (35S) promoter. Optionally, the nucleic acid molecule also may include a Cauliflower Mosaic Virus 35S (35S) terminator sequence. Optionally, the nucleic acid molecule also may include one or more enhancer elements operable in a plant.

[0096] In some embodiments, the pantaphos-resistant plant cell is a cell of Agave spp., Alga spp., alfalfa (Medicago sativa), Arabidopsis thaliana. banana (Musa spp. / bamboo (Phyllostachys spp., Bambusa spp., Pseudosasa spp.), barley (Hordeum spp.) Brassica spp., broccoli (Brassica oleracea var. ilalica), cabbage (Brassica oleracea var. capitata), Cannabis spp. (e.g., C. sativa, C. indica, C. ruderalis), carrot (Daucus carota), Carrizo Cane (Arundo donax), cassava (Manihot esculenla), castor (Ricinus communis), cauliflower (Brassica oleracea var. botrytis), celery (Apium graveolens), chickpea (Cicer arietinum), Chinese cabbage (Brassica rapa), coconut (Cocos nucifera), coffee (Coffea arabica; Coffea canephor;), com (Zea mays), clover (Trifolium spp.), cotton (Gossypium sp.), cucumber (Cucumis sativus), Douglas fir (Pseudotsuga menziesii), eggplant (Solanum melongena), eucalyptus (Eucalyptus globulus), field mustard (Brassica rapa), false flax (Camelina spp.), flax (Linum usitatissimum), garlic (Allium sativum), grape (Vitis spp.), hops (Humulus spp,), ice plant (Mesembryanthemum crystallinum), Jatropha (Jatropha curcas), leek (Allium porrum), lettuce (Lactuca sativa), millets (Panicum miliaceum), oat (Avena spp.), olive (Olea spp.), onion (Allium spp.), Opuntia spp., palm (Arecaceae spp.), pasture grass (Holcus spp.), pea (Pisum sativum), peanut (Arachis hypogaea), pepper (Capsicum spp.), potato (Solanum tuberosum), radish (Raphanus sativus), rapeseed / canola (Brassica napus), rice (Oryza spp., Zizania spp., Porteresia spp.), rye (Secale cereale), sorghum (Sorghum bicolor), soybean (Glycine max), spinach (Spinacia oleracea), squash (Cucurbita spp.), strawberry (Fragaria spp.), sugar beet (Beta Vulgaris), sugarcane (Saccharum spp.), sunflower (Helianthus spp.), sweet gum (Liquidambar styracijlua), sweet potato (Ipomoea batatas), switchgrass (Panicum spp.), tea (Camellia spp.), tobacco (Nicotiana spp.), tomato (Solanum lycopersicum), triticale (xTriticosecale), grass (Poaceae spp.), watercress (Nasturtium officinale), watermelon (Citrullus lanatus), and wheat (Triticum spp.).

[0097] In some embodiments a transgenic plant resistant to Pantoea ananatis infection and / or to pantaphos produced by Pantoea ananatis may comprise a nucleic acid molecule comprising a constitutive promoter sequence that is functional in plants and a nucleotide sequence operably linked to the constitutive promoter sequence, wherein the nucleotide sequence encodes an HvrF protein of Pantoea ananatis comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 11.

[0098] The disclosure also provides for transgenic plant parts that are resistant to pantaphos comprising the nucleic acid molecule of claim 1, wherein the plant parts comprise one or more of protoplasts, leaves, stems, roots, root tips, anthers, pistils, seed, grain, embryo, pollen, ovules, cotyledon, hypocotyl, pod, flower, shoot, tissue, petiole, cells, and meristematic cells. In some embodiments, a transgenic plant may be derived from one mor more plant parts. Also provided are progeny of the transgenic plants described herein, wherein the progeny comprises the nucleic acid molecule that confers resistance to pantaphos. Also provided are plant seeds comprising the nucleic acid molecule encoding the HvrF protein, wherein expression of the HvrF protein confers pantaphos resistance to the plant seed.

[0099] The disclosure also provides for methods of producing pantaphos-resistant plant cells. For example, in one embodiment, a method for producing pantaphos-resistant plant cells comprises: (a) transforming plant cells using a recombinant DNA construct to provide transformed plant cell; and (b) regenerating pantaphos-resistant plants from the transformed plant cells, wherein the recombinant DNA construct comprises: i) a constitutive promoter sequence that is functional in the plant cells; and ii) a coding sequence that encodes a polypeptide comprising an 5-adenosylmethione (SAM)-dependent methyltransferase, wherein the promoter sequence is heterologous with respect to the coding sequence and is configured to cause sufficient expression of the polypeptide in the transformed plant cell in order to confer resistance to pantaphos.

[0100] In some embodiments, 5-adenosylmethione (SAM)-dependent methyltransferase comprises the HvrF protein of Pantoea ananatis. In some embodiments, the HvrF protein of Pantoea ananatis comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 11 or is identical to SEQ ID NO: 11.

[0101] In some embodiments, a method of conferring pantaphos resistance to a plant comprises transforming the plant with a nucleic acid molecule comprising a nucleotide sequence operably linked to a promoter sequence, and optionally, one or more enhancer elements, wherein the nucleotide sequence comprises: a) a nucleotide sequence at least 95% identical to one or more of SEQ ID NO: 10, SEQ ID NO: 26, and SEQ ID NO: 27; b) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO: 11; c) a nucleotide sequence as set forth in any one of SEQ ID NO: 10, SEQ ID NO: 26, and SEQ ID NO: 27; or d) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 11. In other embodiments, a method of producing a transgenic plant comprising transforming a plant with the nucleic acid molecule, thereby producing the transgenic plant, wherein the nucleic acid molecule comprising a constitutive promoter sequence that is functional in plants and a nucleotide sequence operably linked to the constitutive promoter sequence, wherein the nucleotide sequence encodes an HvrF protein of Pantoea ananatis comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 11. In some embodiments, the method further comprises producing a progeny plant comprising the nucleic acid molecule by crossing the transgenic plant with: a) itself; b) a second plant from the same plant line; c) a wild type plant; or d) a second plant from a different plant line; to produce a seed, growing the seed to produce a progeny plant; and selecting a progeny plant with increased pantaphos resistance as compared to a control plant.

[0102] Recombinant DNA molecules of the instant invention can be transformed into a many commercially useful plants. In a preferred embodiment of the invention, a transgenic plant expressing the desired protein is to be produced. In this case the desired protein may be operably linked to particular promoter to control expression of the encoded polypeptide. Various methods for the introduction of a desired polynucleotide sequence encoding the desired protein into plant cells are available and known to those of skill in the art and include, but are not limited to: (1) physical methods such as microinjection, electroporation, and microprojectile mediated delivery (biolistics or gene gun technology); (2) virus mediated delivery methods; and (3) Agrobacterium-mediated transformation methods.

[0103] The most commonly used methods for transformation of plant cells are the Agrobacterium-mediated DNA transfer process and the biolistics or microprojectile bombardment mediated process (i.e., the gene gun). Typically, nuclear transformation is desired but where it is desirable to specifically transform plastids, such as chloroplasts or amyloplasts, plant plastids may be transformed utilizing a microprojectile mediated delivery of the desired polynucleotide.

[0104] Agrobacterium-mediated transformation is achieved through the use of a genetically engineered soil bacterium belonging to the genus Agrobacterium. A number of wild-type and disarmed strains of Agrobacterium tumefaciens and Agrobacterium rhizogenes harboring Ti or Ri plasmids can be used for gene transfer into plants. Gene transfer is done via the transfer of a specific DNA known as “T-DNA” that can be genetically engineered to carry any desired piece of DNA into many plant species.

[0105] Agrobacterium-meddated genetic transformation of plants involves several steps. The first step, in which the virulent Agrobacterium and plant cells are first brought into contact with each other, is generally called “inoculation”. Following the inoculation, the Agrobacterium and plant cells / tissues are permitted to be grown together for a period of several hours to several days or more under conditions suitable for growth and T-DNA transfer. This step is termed “co-culture”. Following co-culture and T-DNA delivery, the plant cells are treated with bactericidal or bacteriostatic agents to kill the Agrobacterium remaining in contact with the explant and / or in the vessel containing the explant. If this is done in the absence of any selective agents to promote preferential growth of transgenic versus non-transgenic plant cells, then this is typically referred to as the “delay” step. If done in the presence of selective pressure favoring transgenic plant cells, then it is referred to as a “selection” step. When a “delay” is used, it is typically followed by one or more “selection” steps.

[0106] With respect to microprojectile bombardment (U.S. Patent No. 5,550,318; U.S. Patent No. 5,538,880; U.S. Patent No. 5,610,042; and PCT Publication WO 95 / 06128;), particles are coated with nucleic acids and delivered into cells by a propelling force. Exemplary particles include those comprised of tungsten, platinum, and preferably, gold.

[0107] An illustrative embodiment of a method for delivering DNA into plant cells by acceleration is the Biolistics Particle Delivery System (BioRad, Hercules, Calif.), which can be used to propel particles coated with DNA or cells through a screen, such as a stainless steel or Nytex screen, onto a filter surface covered with monocot plant cells cultured in suspension.

[0108] Microprojectile bombardment techniques are widely applicable and may be used to transform virtually any plant species. Examples of species that have been transformed by microprojectile bombardment are illustrated in in U.S. Patent No. 5,015,580 (soybean); U.S. Patent No. 5,550,318 (corn); U.S. Patent No. 5,538,880 (com); U.S. Patent No. 5,914,451 (soybean); U.S. Patent No. 6,160,208 (corn); U.S. Patent No. 6,399,861 (com); U.S. Patent No. 6,153,812 (wheat) and U.S. Patent No. 6,365,807 (rice) and Agrobacterium-mediated transformation is described in U.S. Patent No. 5,159,135 (cotton); U.S. Patent No. 5,824,877 (soybean); U.S. Patent No. 5,463,174 (canola); U.S. Patent No. 5,591,616 (com); U.S. Patent No. 5,846,797 (cotton); U.S. Patent No. 6,384,301 (soybean), U.S. Patent No. 7,026,528 (wheat) U.S. Patent No. and 6,329,571 (rice), US Patent Publication 2004 / 0087030 (cotton), and US Patent Publication 2001 / 0042257 (sugar beet), for enabling the production of transgenic plants.

[0109] The regeneration, development, and cultivation of plants from various transformed explants is well documented in the art. This regeneration and growth process typically includes the steps of selecting transformed cells and culturing those individualized cells through the usual stages of embryonic development through the rooted plantlet stage. Transgenic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are thereafter planted in an appropriate plant growth medium such as soil. Cells that survive the exposure to the selective agent, or cells that have been scored positive in a screening assay, may be cultured in media that supports regeneration of plants. Developing plantlets are transferred to soil less plant growth mix, and hardened off, prior to transfer to a greenhouse or growth chamber for maturation.

[0110] The present invention can be used with any transformable cell or tissue. By transformable as used herein is meant a cell or tissue that is capable of further propagation to give rise to a plant. Those of skill in the art recognize that a number of plant cells or tissues are transformable in which after insertion of exogenous DNA and appropriate culture conditions the plant cells or tissues can form into a differentiated plant. Tissue suitable for these purposes can include but is not limited to immature embryos, scutellar tissue, suspension cell cultures, immature inflorescence, shoot meristem, nodal explants, callus tissue, hypocotyl tissue, cotyledons, roots, and leaves.

[0111] Any suitable plant culture medium can be used. Examples of suitable media would include but are not limited to MS-based media (Murashige and Skoog, Physiol. Plant, 15:473- 497, 1962) or N6-based media (Chu et al., Scientia Sinica 18:659, 1975) supplemented with additional plant growth regulators including but not limited to auxins, cytokinins, ABA, and gibberellins. Those of skill in the art are familiar with the variety of tissue culture media, which when supplemented appropriately, support plant tissue growth and development and are suitable for plant transformation and regeneration. These tissue culture media can either be purchased as a commercial preparation, or custom prepared and modified. Those of skill in the art are aware that media and media supplements such as nutrients and growth regulators for use in transformation and regeneration and other culture conditions such as light intensity during incubation, pH, and incubation temperatures that can be optimized for the particular variety of interest.

[0112] The regeneration, development and cultivation of plants from single plant protoplast transformants or from various transformed explants is well known in the art. See generally, Maliga et al., Methods in Plant Molecular Biology, Cold Spring Harbor Press (1995); Weissbach and Weissbach, In: Methods for Plant Molecular Biology, Academic Press, San Diego, Calif. (1988). Plants of the present invention can be part of or generated from a breeding program and may also be reproduced using apomixis. Methods for the production of apomictic plants are known in the art. See, e.g., U.S. Patent No. 5,811,636. In addition to direct transformation of a plant material with a recombinant DNA, a transgenic plant cell nucleus can be prepared by crossing a first plant having cells with a transgenic nucleus with recombinant DNA with a second plant lacking the transgenic nucleus. For example, recombinant DNA can be introduced into a nucleus from a first plant line that is amenable to transformation to transgenic nucleus in cells that are grown into a transgenic plant which can be crossed with a second plant line to introduce the recombinant DNA into the second plant line. A transgenic plant with recombinant DNA providing an enhanced trait, e.g., enhanced yield, can be crossed with transgenic plant line having other recombinant DNA that confers another trait, for example herbicide resistance or pest resistance, to produce progeny plants having recombinant DNA that confers both traits. Typically, in such breeding for combining traits the transgenic plant donating the additional trait is a male line and the transgenic plant carrying the base traits is the female line. The progeny of this cross will segregate such that some of the plants will carry the DNA for both parental traits and some will carry DNA for one parental trait; such plants can be identified by markers associated with parental recombinant DNA, e.g. marker identification by analysis for recombinant DNA or, in the case where a selectable marker is linked to the recombinant, by application of the selecting agent such as a herbicide for use with a herbicide tolerance marker, or by selection for the enhanced trait. Progeny plants carrying DNA for both parental traits can be crossed back into the female parent line multiple times, for example usually 6 to 8 generations, to produce a progeny plant with substantially the same genotype as one original transgenic parental line but for the recombinant DNA of the other transgenic parental line.

[0113] In certain embodiments of the invention the recombinant DNA insertion is “targeted” in order to achieve site-specific integration, for example to replace an existing gene in the genome, to use an existing promoter in the plant genome, or to insert a recombinant polynucleotide at a predetermined site known to be active for gene expression. Several sitespecific recombination systems exist which are known to function implants include cre-lox as disclosed in U.S. Patent No. 4,959,317 andFLP-FRT as disclosed in U.S. Patent No. 5,527,695, both incorporated herein by reference. Other methods of creating transgenic plants are described, for example, in U.S. Patent Nos. 4,940,835 and 11,700,809, and U.S. Patent Publication Nos. 2022 / 0162631, 2016 / 0264980, and 2023 / 0128674.

[0114] Additionally, this disclosure provides nucleic acid molecules comprising the hvr operon (hvrA-hvrE) of Pantoea Sp. In some embodiments, the disclosure provides a nucleic acid molecule comprising an hvr operon (hvrA-hvrl.) of Pantoea Sp. operably linked to an inducible promotor sequence, wherein induction of the promoter and expression of the genes of the hvr operon causes the production of 2-(hydroxy(phosphono)methyl) maleic acid (pantaphos).

[0115] In some embodiments, a nucleic acid molecule comprises a nucleotide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or 100% sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, or SEQ ID NO: 24.

[0116] In some embodiments, a nucleic acid molecule comprising an inducible promoter sequence operably linked to a nucleotide sequence encoding one or more polypeptides having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or 100% sequence identity to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, or SEQ ID NO: 23.

[0117] In other embodiments, the nucleotide sequence comprises hvr A, hvrB, hvrC, hvrD, hvrE, hvrF and hvrl of Pantoea Sp. In another embodiment, the hvr operon comprises hvr A, hvrB, hvrC, hvrl). hvrE, hvrF, and hvrl of Pantoea ananatis.

[0118] Suitable inducible promoters for use with various embodiments include, but are not limited to, bacteriophage promoters (e.g. Plslcon, T3, T7, SP6, PL) and bacterial promoters (e.g. Pbad, PmgrB, Ptrc2, Plac / ara, Ptac, Pm), or hybrids thereof (e.g. PLlacO, PLtetO). Examples of bacterial promoters for use in accordance with the present disclosure include, without limitation, positively regulated E. coh promoters such as positively regulated σ70 promoters (e.g., inducible pBad / araC promoter, Lux cassette right promoter, modified lamdba Pirn promote, plac Or2-62 (positive), pBad / AraC with extra REN sites, pBad, P(Las) TetO, P(Las) CIO, P(Rhl), Pu, Fee A, pRE, cadC, hns, pLas, pLux), σspromoters (e.g., Pdps), σ32promoters (e.g., heat shock) and σ54 promoters (e.g., glnAp2); negatively regulated E. coh promoters such as negatively regulated σ70promoters (e.g., Promoter (PRM+), modified lamdba Prm promoter, TetR-TetR-4C P(Las) TetO, P(Las) CIO, P(Lac) IQ, RecA_DlexO_DLacOl, dapAp, FecA, Pspac-hy, pci, plux-cL plux-lac, CinR, CinL, glucose controlled, modifed Pr, modifed Prm+, FecA, Pcya, rec A (SOS), Rec A (SOS), EmrR_regulated, Betl_regulated, pLacJux, pTet_Lac, pLac / Mnt, pTet / Mnt, LsrA / cI, pLux / cI, LacIQ, pLacIQl, pLas / cI, pLas / Lux, pLux / Las, pRecA with LexA binding site, reverse BBa R00I 1 , pLacI / ara-1, pLacIq, rrnB Pl, cadC, hns, PfhuA, pBad / araC, nhaA, OmpF, RcnR), c8promoters (e g., Lutz-Bujard LacO with alternative sigma factor σ38), a32promoters (e.g., Lutz-Bujard LacO with alternative sigma factor3and cP4promoters (e.g., glnAp2); negatively regulated B. subtilis promoters such as repressible B. subtilis σApromoters (e.g.. Gram-positive IPTG-inducible, Xyl, hyper-spank) and σBpromoters. Other inducible microbial promoters and / or bacterial promoters may be used in accordance with the present disclosure In some embodiments, the inducible promoter is a lac or a tac promoter. In some embodiments, the inducible promoter is a tac promoter.

[0119] In other embodiments, the disclosure provides for a recombinant cell for producing pantaphos or a salt thereof, wherein the recombinant cell comprises a nucleic acid molecule comprising an hvr operon (hvrA-hvrl.) of Pantoea Sp. operably linked to an inducible promotor sequence; or the hvr operon comprises hvr A, hvrB, hvrC, hvrl), hvrE, hvrF and hvrl of Pantoea Sp. , or the hvr operon comprises hvr A, hvrB, hvrC, hvrD, hvrE, hvrF, and hvrl of Pantoea ananatis.

[0120] In some embodiments, the cell is a cell selected from a genus selected from the group consisting of Pantoea, Clostridium, Zymomonas, Escherichia, Salmonella, Serratia, Erwinia, Klebsiella, Shigella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Lactococcus, Enterococcus, Alcaligenes, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Schizosaccharomyces, Kluveromyces, Yarrowia, Pichia, Zygosaccharomyces, Debaryomyces, Candida, Brettanomyces, Pachysolen, Hansenula, Issatchenkia, Trichosporon, Yamadazyma, and Saccharomyces. In some embodiments, the cell is a cell of one of the genus Pantoea, Escherichia, or Saccharomyces . In some embodiments, the cell is Pantoea ananatis cell, Escherichia coli cell, or Saccharomyces cerevisiae cell.

[0121] The disclosure also provides for a recombinant bacterial cell for producing phosphomethylmaleate (PMME) or a salt thereof, wherein the recombinant bacterial cell comprises an hvr A gene, an hvrC gene, an hvrD gene, an hvrE gene, an hvrF gene, and an hvrl gene, wherein each of the genes are derived from Pantoea ananatis. In some embodiments, the genes are operably linked to an inducible promoter sequence. In some embodiments, the recombinant bacterial cell is Pantoea ananatis ox Escherichia coli.

[0122] Methods of making recombinant cells and nucleic acid molecules are known in the art and are described herein and, for example, in International Patent Publication Nos. WO / 2020 / 090940 and WO2014129898; and U.S. Patent Publication Nos. 2019 / 0256838 and 2014 / 0134689.

[0123] The following Examples are intended to illustrate the above invention and should not be construed as to narrow its scope. One skilled in the art will readily recognize that the Examples suggest many other ways in which the invention could be practiced. It should be understood that numerous variations and modifications may be made while remaining within the scope of the invention.

[0124] EXAMPLES

[0125] Example 1. Characterization of Pantaphos biosynthesis pathway.

[0126] Hydratase / dehydratase activity of HvrDE. HvrDE is a member of the aconitase superfamily whose closest characterized homolog is the heterodimeric leucine biosynthetic enzyme LeuCD, which converts 2-isopropylmalate to 3-isopropylmalate via a dehydrated intermediate (Figure 1). Based on the structural similarity of the substrates and the mechanism of LeuCD, we predicted HvrDE would dehydrate PMM to form PMME. To test this, we purified HvrD and HvrE after heterologous expression in E. coli. Although the 6X-histidine affinity tag was present only on HvrE, the two proteins purified as a complex when coexpressed in the same strain (Figure 7). Thus, like LeuCD, HvrD and HvrE comprise a heterodimer. When HvrDE was incubated with PMM, followed by31P NMR analysis to characterize the phosphorus compounds present in the assay, no new products were formed (Figure 2). However, when incubated with PMME, a small amount of PMM was observed. Thus, as purified HvrDE catalyzes hydration of PMME, albeit poorly. Because members of the aconitase superfamily are known to require a labile [4Fe / 4S] cluster that is easily damaged during purification under aerobic conditions, we repeated the experiment after in vitro reconstitution of HvrDE with ferrous iron and dithiothreitol. Although the reconstituted enzyme still showed no detectable activity with PMM, it completely converted PMME to PMM, consistent with the idea that chemical equilibrium of the hydration / dehydration reaction strongly favors the hydrated substrate PMM, as it does for several other members of this protein family.

[0127] The unfavorable dehydration of PMM is driven by methylation of PMME. Assuming the HvrDE reaction favors the substrate PMM, subsequent consumption of PMME (or another downstream product) should be highly favorable to drive pantaphos production. Initially, we suspected that hydroxylation of PMME to form pantaphos would fulfill this function; however, the monooxygenase that catalyzes this reaction (HvrB) does not use PMME as a substrate (see below). The putative 5-adenosylmethionine (SAM)-dependent methyltransferase HvrF was also a reasonable candidate because such reactions are typically irreversible due to favorable thermodynamics and the rapid degradation of the product S- adenosylhomocysteine (SAH) by abundant cellular enzymes such as SAH nucleosidase. Accordingly, we examined whether HvrF could methylate PMME using enzyme purified after expression in E. coli. Incubation of PMME with SAM, HvrF and SAH nucleosidase led to the appearance of several new peaks in31P NMR (Figure 3). The major peak at -16.6 ppm was tentatively identified as methyl-PMME based on the chemical shifts of protons identified via1H-31P HMBC NMR. Additional results reported below strongly support this assignment. The minor phosphorus species observed in these reactions are likely to be chemically rearranged derivatives of methyl-PMME based on changes observed in the reaction products over time. Thus, after 24 hours incubation in the absence of enzyme, the integration values for the vinyl protons linked to the major phosphorus peak decreased, while those coupled to the minor phosphorus peaks increased, suggesting that methyl-PMME undergoes spontaneous rearrangements around the carbon-carbon double bond. Due to the continued changes in signal intensity over time, we were unable to collect the NMR data needed to confidently assign a structure to these products.

[0128] To test whether PMME methylation was sufficient to drive the unfavorable dehydration reaction, we incubated PMM with reconstituted HvrDE in the presence or absence of HvrF, SAM and SAH nucleosidase (Figure 4). As previously observed, HvrDE was unable to dehydrate PMM; however, when HvrF, SAM and SAH nucleosidase were added to the reaction, a new peak at -16.3 ppm was observed. Mixing this reaction with the products of the PMME methylation reaction showed that the new peak was identical to the product of the HvrF reaction, namely methyl-PMME. Thus, HvrDE can dehydrate PMM to PMME, but SAM- dependent methylation of the dehydrated product is required to drive the reaction forward.

[0129] SAM-dependent methylation of pantaphos by HvrF. Given the structural similarity of PMME and pantaphos, we also asked whether HvrF could methylate the latter. Indeed, incubation of pantaphos, HvrF, SAM and SAH nucleosidase led to complete conversion of pantaphos to a new phosphonate without any side-products (dp -14.3 ppm; Figure 3). This finding suggests that the hydroxyl moiety, which differentiates pantaphos and PMME, prevents rearrangement of the methylated product. After extended incubations in aqueous solution in the absence of enzyme, the putative methylated product was converted back to pantaphos. Thus, the ester linkage in methylpantaphos is chemically labile. Resuspension in pure methanol stabilized the molecule allowing full structural elucidation via a series of NMR experiments. These data show that the product, designated methylpantaphos, has a single methyl-ester modification of the carboxylate moiety proximal to the phosphonate.

[0130] HvrB catalyzes the hydroxylation of methyl-PMME with the aid of HvrK. The evidence presented above show that methyl-PMME is an intermediate in pantaphos biosynthesis. Subsequent conversion of methyl-PMME to pantaphos would then require hydroxylation and demethylation, presumably in that order to prevent rehydration of PMME by HvrDE. We predicted a two-component monooxygenase system comprised of HvrB and HvrK would be responsible for the hydroxylation reaction based on the homology of these proteins to the well-characterized nitrilotriacetate monooxygenase from Chelatobacter heintzii. As the name implies, members of this enzyme family require a monooxygenase (HvrB), which catalyzes flavin-dependent hydroxylation using molecular oxygen as the co-substrate, as well as an NAD(P)H-dependent flavin-reductase (HvrK) to produce the reduced flavin used in the reaction. Consistent with this idea, purified HvrK contains tightly bound flavin mononucleotide (FMN), based on the UV-visible and high-resolution mass spectra of cofactor released from the enzyme by heat denaturation (Figure 8). Purified HvrB was colorless, suggesting that it does not bind flavins tightly.

[0131] Although we predicted that methyl-PMME, rather than PMME, would be the hydroxylation substrate, we assessed HvrB / HvrK activity with both substrates using FMN and either NADH or NADPH as electron carriers. As expected, PMME was not consumed in these reactions, showing that it is not a monooxygenase substrate and that it cannot be the coupling enzyme used to drive the HvrDE-dependent desaturation of PMM as described above. In contrast, reactions containing methyl-PMME and either NADH or NADPH produced a new phosphorus compound (dp ~13 ppm), which was shown to be methylpantaphos, by spiking with an authentic standard (Figure 5). Significantly, production of methylpantaphos from methyl-PMME shows that both molecules carry the methyl-ester on the same carbon. Thus, the tentative methyl-PMME structure shown above is correct.

[0132] Additional experiments show that hydroxylation of methyl-PMME requires HvrB, FMN and either NADH or NADPH, but surprisingly not HvrK (Figure 9). This finding suggests that HvrB can reduce FMN using nicotinamide cofactors in the absence of HvrK. To provide additional insight into this phenomenon, we characterized the flavin reductase activity of both HvrK and HvrB using a standard spectrophotometric assay (Table 1). HvrK is a highly active flavin reductase, capable of reducing both FMN and FAD, with marginally higher activity with the former. The enzyme has a strong preference for NADH, with rates 60 to 100- fold higher than observed with NADPH. HvrB was also able to reduce both flavins with either NADH or NADPH at rates that were significantly higher than the chemical rate observed in no-enzyme controls, but much lower than observed with HvrK. Thus, although HvrB can slowly convert methyl-PMME to pantaphos in the absence of HvrK in vitro, it is probable that a partner flavin reductase would increase the rate of pantaphos synthesis in vivo. Table 1: Flavin reductase activitiesaof HvrB and HvrK.

[0133] “Enzyme activity reported as the average activity (N =3) in nmol / min / mg of enzyme.

[0134] '’Rates attributed to chemical reduction by NAD(P)H were subtracted from the observed enzyme rates to yield the final enzyme activity.

[0135] Conversion of methylpantaphos to pantaphos. Although our in vitro data suggest that methylpantaphos is slowly converted to pantaphos in the absence of enzymes, it seems likely that this reaction is enzymatically catalyzed in vivo. A reasonable candidate for this reaction is HvrJ, which has no characterized homologs in Genbank (Figure 1). To test whether HvrJ has methylesterase activity, we incubated methylpantaphos with and without purified HvrJ; however, after 12-hour incubations at room temperature, no pantaphos was observed. Thus, assuming that HvrJ was purified in an active form, it is not a methylpantaphos methylesterase.

[0136] Production of pantaphos and PMME in E. coli. Our biochemical data suggest that the first six genes in the hvr operon, hvrA through hvrF, are sufficient for methylpantaphos biosynthesis, with the possibility that hvrK contributes to the efficiency of the monooxygenase reaction. To test whether these results hold true in vivo, we constructed plasmids that express these genes in various combinations from a tightly regulated, IPTG-inducible promoter. Each plasmid also carried the gene for the putative MFS-family transporter HvrJ, to allow export of pathway products into the medium. After transformation of E. coli (a close relative of P. ananatis), spent media from cultures grown in the presence and absence of the inducer were examined by31P NMR to assess whether phosphonic acids were produced (Figure 6). When grown under non-inducing conditions, none of the strains produced detectable levels of phosphonic acids; however, a variety of phosphonates were produced and secreted when the transformants were grown under inducing conditions. Strains expressing hvrA, hvrC, hvrD, hvrE, hvrF and hvrl mainly produced PMME, with a small amount of PMM also being observed. Strains carrying a similar plasmid that also included the hvrB gene, produced the same products, as well as a small amount of pantaphos. Thus, despite the poor conversion of PMME to pantaphos in this strain, HvrA, HvrB, HvrC, HvrD, HvrE, HvrF and Hvrl are clearly sufficient for in vivo production of pantaphos. A possible explanation for the poor conversion of PMME to pantaphos in this strain lies in the absence of the putative HvrB partner protein HvrK. However, the phosphonate production profile of strains carrying plasmids that included hvrK was essentially the same as that observed in strains without this gene, suggesting this is not the case.

[0137] Bioactivity of pathway intermediates. The bioactivity of PMME was not previously examined due to insufficient material, a problem that was remedied during this work. Accordingly, we assessed the bioactivity of PMME against plants, bacteria and fungi. Despite the structural similarity of PMME and pantaphos, the former was inactive when tested against Arabidopsis (Table 2). Thus, the hydroxyl moiety of pantaphos is critical for its phytotoxicity. Like pantaphos, PMME was completely non-toxic for a variety of fungi and bacteria, including E. coli strains engineered to allow inducible expression of a broad substrate specificity phosphonate transporter. Due to the instability of the methyl ester intermediates we were unable to test the bioactivity of methyl-PMME and methylpantaphos.

[0138] Table 2: Bioactivity of PMME against representative organisms.

[0139] “Minimum inhibitory concentration (MIC) determined based on CLSI guidelines.

[0140] '’Not tested.

[0141] The results presented here are broadly consistent with a previously proposed model for pantaphos biosynthesis, with the significant difference being the involvement of methylated intermediates formed by the SAM-dependent methyltransferase HvrF (Scheme I).

[0142]

[0143] Scheme I. The revised pathway for pantaphos biosynthesis. Biosynthetic steps confirmed by in vitro data are indicated by solid-lines. Steps proposed based on homology to previously characterized enzymes are indicated by dotted-lines. Abbreviations: PEP, phosphoenolpyruvate; PnPy, phosphonopyruvate; Ac-CoA, acetyl-coenzyme A; CoASH, coenzyme A; PMM, phosphonomethylmalate; PMME, phosphonomethylmaleate; SAM, S- adenosylmethionine; SAH, S-adenosylhomocysteine; FMN, flavin mononucleotide; NAD(P)H, nicotinamide adenine dinucleotide (phosphate).

[0144] The absolute requirement for the methylation reaction derives from the unfavorable thermodynamics of the preceding dehydration step and highlights the unusual role for HvrF in pantaphos biosynthesis. As noted in Krebs early studies of the TCA cycle, the chemical equilibrium of dehydration reactions, such as those catalyzed by fumarase and aconitase, strongly favors the hydrated products. Subsequent studies have reproduced these findings for other dehydratase reactions. Thus, it is not surprising that the equilibrium of the homologous HvrDE-catalyzed reaction favors PMM. In pathways like the TCA cycle and leucine biosynthesis, the unfavorable equilibrium of the dehydration reaction is overcome by consumption of the product in highly favorable decarboxylation-coupled dehydrogenase reactions (isocitrate dehydrogenase and isopropylmalate dehydrogenase, respectively). In the pantaphos biosynthetic pathway, an irreversible SAM-dependent methyltransferase reaction serves to pull the unfavorable dehydration of PMM, which, to our knowledge, is a unique function for this class of enzyme. An additional benefit of this reaction would be detoxification of intra-cellular pantaphos, which based on structural similarity to isocitrate and isopropylmalate, has been suggested to inhibit central metabolism in the producing host. Support for this idea comes from analysis of a close relative of HvrF, the trans- aconitate methyltransferase of yeast, which is known to detoxify an inhibitory by-product of the TCA cycle. Intriguingly, it seems that the preferred substrate for this yeast enzyme is actually the leucine biosynthetic intermediate 3-isopropylmalate, the product of another HvrDE homolog isopropylmalate dehydratase. The biological role for methylated 3-isopropylmalate is currently a matter of speculation, but the results presented here suggest the intriguing possibility that a SAM-dependent methyltransferase may play an auxiliary role in driving an unfavorable reaction in leucine biosynthesis, and by extension in other dehydration-dependent pathways as well.

[0145] The revised pathway for production of pantaphos requires only the first six genes in the hvr operon (hvrA-hvrE), a finding that was validated by production of pantaphos in recombinant E. coli hosts. The putative export protein Hvrl is likely to be required in vivo, although we did not conduct heterologous expression experiments in clones lacking this gene. Significantly, the same six genes (hvrA-hvrF) are the only ones in the hvr operon that have strong virulence defects when mutated in P. ananatis, consistent with previous results showing that pantaphos is the key virulence factor in onion center rot. These results beg the question of how methylpantaphos, the in vitro product made by the six required enzymes, is converted to pantaphos, which is the product observed in spent media of producing strains. While our in vitro stability studies clearly indicate that spontaneous hydrolysis is possible, it almost certainly occurs at a rate that is too slow to account for in vivo production of pantaphos. We suspected that HvrJ, the only protein encoded by the hvr locus without a putative function, might play this role, but were unable to show methylesterase activity with the purified protein. Further, heterologous production of the unmethylated phosphonates pantaphos and PMME in vivo did not require additional genes beyond the putative transporter Hvrl, suggesting the possibility is that hydrolysis occurs during, or after, export of the methylated products. The accumulation of PMME in spent media supports this idea, because if PMME were produced in the cytoplasm it would either be rapidly hydrated to form PMM, or remethylated by HvrF in a metabolically expensive futile cycle that would consume large amounts of SAM and ATP. An alternate explanation is that promiscuous extracellular enzymes are responsible for conversion of methyl-PMME and methylpantaphos to PMME and pantaphos, respectively.

[0146] The ability of HvrB to catalyze a flavin dependent monooxygenase reaction in the absence of HvrK was also somewhat surprising, but consistent with the observation that the hvrK gene is absent from many organisms that encode homologs of the hvr gene cluster. Based on protein homology, HvrB is clearly a member of the two-component monooxygenase family, which require a partner protein to generate the reduced flavin required for such reactions. While some of the monooxygenase proteins from this family are able to use reduced flavins produced by other cellular reductases, we are not aware of any that possess inherent NAD(P)H-dependent flavin reductase activity. Thus, HvrB appears unique in this regard, a finding that is even more puzzling given the lack of NAD(P)H binding domains within the protein. Nevertheless, the inherent flavin reductase activity of HvrB is rather low relative to that of HvrK, thus it seems likely that a partner protein is helpful in vivo. Interestingly, the preference of HvrB for NADPH seems to complement the preference of HvrK for NADH. Thus, the endogenous flavin reductase activity of HvrB may have evolved to compensate for the cofactor preference of HvrK during growth conditions when NADPH is limiting. Importantly, the predominant production of PMME in heterologous hosts, regardless of whether HvrK is present, shows that the monooxygenase reaction can be a bottleneck in pantaphos biosynthesis. Production of PMME is also seen in the native host under some growth conditions, reinforcing this conclusion. Thus, resolving these unanswered questions has significant implications for understanding pantaphos biosynthesis.

[0147] Finally, the revised pantaphos biosynthetic pathway serves as a starting point for future studies aimed at reducing crop losses due to P. ananatis infections and the potential development of pantaphos as a cost-effective herbicide. Accordingly, because pantaphos production is required for P. ananatis virulence, it should be possible to develop inhibitors of the enzymes characterized here that could be used as crop protection agents. The estimated $60 million dollar per years losses attributed to onion center rot in the United States alone provides a strong motivation for such an effort. Crop losses due to weed infestations are also a major problem, which is compounded by the accelerating occurrence of herbicide resistant strains. Thus, there is an urgent need for development of novel herbicides, especially ones with novel biological targets. Whether pantaphos is useful in this regard remains to be seen, but a key issue in answering this question lies in the cost of production. The ability to overproduce this natural product in engineered hosts offers one path to lowering cost of production, while avoiding the chemical waste issues inherent to chemical synthesis.

[0148] Example 2. Methods and Materials.

[0149] Bacteria, plasmids, and growth conditions. E. coli strains carrying plasmids were typically grown at 37°C in LB medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl) supplemented with the appropriate antibiotic. Antibiotic concentrations of 50 mg / mL filter- sterilized kanamycin (Kan), 100 mg / mL filter-sterilized ampicillin (Amp), or 20 mg / mL filter- sterilized chloramphenicol (Chi) were used. Phosphonate induction media (PIM) was prepared according to Polidore et al., mBio. 2021 Feb 2;12(l):e03402-20. Chemicals. Pantaphos and PMME were obtained by bacterial fermentation, extraction, and purification as previously described in Polidore et al. General chemical and molecular biology reagents were purchased from MilliporeSigma, QIAGEN, New England Biolabs, and Thermo Fisher Scientific.

[0150] NMR specifications. The31P NMR spectra were recorded on an Agilent DD2600 MHz spectrometer (243 MHz for31P). The1H and13C spectra were recorded on a Bruke Avance Illr 500 MHz spectrometer (500 MHz for 'H and 125 MHz for13C) equipped with a BBFO CryoProbe and SampleXpress autosampler. Samples were prepared in either D2O or CD3OD as the locking solvent. NMR spectra were analyzed using MestReNova version 11.0.1-17801.

[0151] Sanger sequencing of hvr genes. Based on the P. ananatis LMG5342 draft genome sequence (GenBank Accession number GCF 000283875.1), the hvrB, hvrE, hvrl, and hvrJ genes were predicted to contain frameshift mutations; however, we previously showed that the strain is fully virulent in onion pathogenesis assays and is able to make pantaphos, casting doubt on the accuracy of the submitted sequence. Accordingly, we resequenced the problematic regions of the hvr operon using Sanger sequencing. P. ananatis LMG5342 genomic DNA was prepared using Qiagen DNeasy® UltraClean Microbial Kit and gene regions containing the predicted frameshift mutation were amplified using NEB® Q5 DNA Polymerase reactions with forward and reverse primer sets. PCR products were cleaned using Qiagen® QIAquick PCR Purification Kit and submitted for Single Pass DNA Sequencing at ACGT, Inc. (Germantown, MD).

[0152] Plasmid construction. Digested plasmids were assembled using NEBuilder® HiFi DNA Assembly Master Mix with PCR-amplified hvr genes from P. ananatis LMG5342 genomic DNA that was prepared using Qiagen DNeasy® UltraClean Microbial Kit. Following successful transformation into DH5a / lpir, or WM4778 for heterologous production plasmids, plasmid clones were confirmed by Single Pass DNA Sequencing at ACGT, Inc. (Germantown, MD) or Plasmidsaurus (Eugene, OR). Final plasmid constructs were transformed into E. coli BL21 (DE3) or PK11466 or BW19719 for protein expression.

[0153] Purification of 6X-histidine tagged proteins after expression in E. coli. A starter culture of the appropriate expression strain was grown overnight at 37°C in LB medium supplemented with the appropriate antibiotic(s). The following day, 6 L of LB, supplemented with the appropriate antibiotic, was inoculated with a 2% inoculum from the starter culture and grown at 37°C. The culture was monitored until ODeoo reached 0.6-0.8, then 1 mM isopropyl β-d-1 -thiogalactopyranoside (IPTG) was added and the culture was shifted to 16°C incubation overnight. The next day, the cells were pelleted using centrifugation and the cell pellet saved in -80 °C or used directly for the next steps. The cells were lysed using a French Press at 16000 psi, and the resulting cell lysate centrifuged at 10,000 x g for 1 hour at 4 °C to remove insoluble material. The clarified cell lysate was added to a Ni-NTA Agarose column equilibrated with three column volumes with Binding Buffer (20 mM Tris-HCl pH 7.8, 10 mM imidazole, 300 mM NaCl, 10% glycerol). The column was then washed with three column volumes of Binding Buffer, followed by three column volumes of Wash Buffer (20 mM Tris-HCl pH 7.8, 50 mM imidazole, 300 mM NaCl, 10% glycerol). Finally, the protein on the column was eluted with Elution Buffer (20 mM Tris-HCl pH 7.8, 250 mM imidazole, 300 mM NaCl, 10% glycerol). The protein elution was concentrated using either an Amicon 10 KDa or 30 KDa cutoff spin column depending on protein size, and the imidazole removed using a Sephadex™ G-25 M column pre-washed and eluted with Desalting Buffer (20 mM Tris-HCl pH 7.8, 300 mM NaCl, 10% glycerol). The resulting sample was then aliquoted and saved in -80°C. Protein size and purity were checked on SDS-PAGE (Figure 7) and quantified using Bradford reagent (BioRad).

[0154] HvrDE activity assays. The activity of HvrDE with 2-phosphonom ethylmalate and 2- phosphonomethylmaleate substrates was tested with and without iron-sulfur cluster reconstitution. The initial reaction solution consisted of 100 mM Tris-HCl pH 8.0, 5 mM MgCh, 1 mM dithiothreitol (DTT), 5 mM KC1, and 3 mM HvrDE. For enzyme reconstitution, 5 mM DTT and 1 mM ammonium iron(II) sulfate were added to the enzyme mixture and incubated for 20 minutes at room temperature. For the coupled reaction with HvrF, 5 mM S-adenosyl-L- methionine (SAM), 3 mM MtnN (SAH nucleosidase), and 5 mM HvrF were added following enzyme reconstitution. The reaction was initiated by addition of either 400 mM 2- phosphonomethylmalate (PMME) or 1.25 mM 2-phosphonomethylmaleate (PMM) and incubated in the dark at room temperature overnight. The following day, to separate out any iron precipitates, the solution was centrifuged max speed at 4°C, and the supernatant transferred to fresh tube. The proteins were removed from the supernatant via centrifugation in an Amicon 10 KDa cutoff spin column from which the flowthrough, containing the reaction products, was collected and dried via lyophilization. The freeze-dried solution was reconstituted in D2O plus 5 mM EDTA pH 8.0 and analyzed by31P NMR.

[0155] HvrF activity assays and purification of methyl-phosphonates. HvrF methyltransferase enzymatic assays were adapted from Lin et al., J Biol Chem 287, 37010- 37020 (2012). The activity of HvrF with 2-phosphonomethylmalate, 2- phosphonomethylmaleate, or pantaphos substrates was tested either using purified SAM or a one-pot reaction using MetK (E. coli SAM synthetase). Typical reactions consisted of 100 mM Tris-HCl pH 8.0, 5 mM MgCh, 5 mM KC1, 3 mM MtnN (E. coli 5’-methylthioadenosine / S- adenosyl-L-homocysteine nucleosidase), and 5 mM HvrF. For most experiments, a one-pot reaction was used where SAM was made in situ via MetK synthesis. For these reactions, 7.5 mM MetK, 4 mM L-methionine, and 5 mM ATP were included in the reaction mixture. In some cases, as indicated, reactions used purified SAM as described below. The reaction was initiated by addition of 1 mM 2-phosphonomethylmaleate or 1 mM pantaphos and incubated in the dark at room temperature overnight. The following day, the proteins were removed via centrifugation in an Amicon 10 KDa cutoff spin column from which the flowthrough, containing the reaction products, was collected and dried via lyophilization. The freeze-dried solution was reconstituted in D2O plus 5 mM EDTA at pH 8.0, then analyzed via31P NMR. For purification of the methylated intermediates produced from HvrF reactions, freeze-dried samples were reconstituted in methanol, incubated on ice for ten minutes, then centrifuged at max speed at 4°C to remove insoluble material. The methanol soluble fraction was screened for methylated phosphonates using31P NMR with deuterated methanol as the locking solvent. Methylated phosphonate intermediates were saved in -80°C until later use.

[0156] HvrB and HvrK cofactor identification and activity assays. For co-factor extraction,

[0157] 4.5 mg of HvrK was concentrated using an Amicon 10 KDa cutoff spin column. The spin column was washed six times with filtered water, and the protein was re-extracted from the filter membrane using IX phosphate-buffered saline. The re-extracted protein sample was boiled at 99°C for 5 minutes followed by 50°C for 5 minutes. The boiled protein sample was then centrifuged through an Amicon 10 KDa cutoff spin column, washed three times with filtered water, and the flowthrough was flash frozen and dried to completion via lyophilization. The dried sample was reconstituted in 200 mL of water and analyzed using LC-MS. The extracted co-factor sample (10 mL) was injected onto a Synergi 4u Fusion-RP column (150 x

[0158] 4.6 mm, 4 mm) connected to a Thermo Q-Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer system monitoring for masses corresponding to FAD (m / z = 786.2) and FMN (m / z = 457.1) in positive mode. The column was first equilibrated with ten column volumes of 5% Buffer B (95% acetonitrile with 0.1% formic acid) / 95% Buffer A (5% acetonitrile with 0.1% formic acid) and the masses monitored using a linear gradient from 5% to 95% Buffer B. The activity of HvrB and HvrK was tested with 2-phosphonomethylmaleate or methyl-2- phosphonomethylmaleate using FMN, NADH, and NADPH. Typical reactions consisted of 100 mM Tris-HCl pH 8.0, 5 mM MgCh, 5 mM KC1, 2.5 mM NAD(P)H, 50 mM FMN, 2.5 pM HvrB and 2.5 pM HvrK. The reactions were initiated by addition of 1.15 mM 2- phosphonomethylmaleate or methyl-2-phosphonomethylmaleate. Preparation of S-adenosyl-L-methionine (SAM). The enzymatic synthesis of SAM was adapted from Lin et al., J Biol Chem 287, 37010-37020 (2012) and Park et al., Bioorg Med Chem 4, 2179-2185 (1996). The reactions consisted of 100 mM Tris-HCl pH 8, 13 mM KC1, 13 mM ATP, 13 mM MgCh, 1 mM EDTA, 9.5 mM MetK, 10 mM L-methionine, 8% b- mercaptoethanol, 0.5 U / mL pyrophosphatase, and water up to 2 mL total reaction volume. The reaction mixture was incubated in the dark for about 16 h, then proteins were removed via centrifugation in an Amicon 10 KDa cutoff spin column from which the flowthrough, containing the reaction products, was collected. Next, SAM was precipitated from the flowthrough using acetone (90% final concentration) and incubated on ice for at least ten minutes. The precipitate was collected via centrifugation at max speed at 4°C and the precipitate was washed three times with 100% acetone. The precipitated pellet was air dried then reconstituted in water, aliquoted into PCR tubes, and dried to completion via lyophilization. The dried SAM was saved in -80°C until use. The concentration of SAM was determined using a Varian Cary 4000 spectrophotometer by absorbance at 260 nm using an extinction coefficient of 14,700 M'1cm'1. SAM sample purity was assessed using an HPLC method. Samples were injected onto a Zorbax SB-C18 column (3.0 x 150 mm, 3.5 mm) equilibrated with ten column volumes of 5% Buffer B (acetonitrile with 10 mM ammonium acetate at pH 4) / 95% Buffer A (10 mM ammonium acetate at pH 4) using a linear gradient from 5% to 95% Buffer B over 20 minutes at a flow rate of 0.2 mL / min. The samples were monitored for ATP, SAM, and the degradation products, S-adenosyl-L-homocysteine (SAH) and 5 ’-methylthioadenosine (MTA) at 260 nm.

[0159] Flavin reductase activity assays with HvrB and HvrK. Flavin-dependent cytochrome c reduction assays were adapted from Imlay et al., J Biol Chem 266, 6957-6965 (1991) and McCord et al., J Biol Chem 244, 6049-6055 (1969). In brief, 20 mM cytochrome c, 0.5 mM NADPH, and 50 mM FMN were sequentially added in that order to an initial mixture of enzyme buffer (100 mM Tris-HCl pH 8, 5 mM KC1, and 5 mM MgCh), then 5 mM HvrB and / or 5 mM HvrK were sequentially added in that order to the mixture. Absorbance readings were carried out using an Agilent Cary 4000 UV-Vis Spectrophotometer zeroed with enzyme buffer. Continuous absorbance measurements were recorded for at least 2 minutes in between additions. The increase in absorbance at 550 nm indicated reduction of cytochrome c. Three independent experiments were performed for each reaction condition. Enzyme activity was determined using the linear portion of absorbance increase over time and the extinction coefficients of 12,000 M-1crn-1for FMN and 11,500 M^cm'1for FAD. Data were compiled and analyzed using GraphPad Prism 8.0.2.263. Bioactivity assays. Bioactivity assays were performed as previously described in Polidore et al. 2021. The rich medium conditions included Murashige and Skoog (MS) agar medium for plants, yeast-peptone-dextrose (YPD) medium for fungi, and Luria-Bertani (LB) agar for bacteria. Bioactivity in minimal medium was determined using MOPS-glucose and MOPS-acetate for bacteria, and yeast-minimal-media (YMM) medium for fungi.

[0160] In vivo production of pantaphos and PMME. A single colony of E. coli BW19719 carrying the corresponding heterologous production plasmid was inoculated into a starter culture of 5 mL of phosphonate induction media (PIM) and grown at 37 °C for 48 hrs. Two 500 mL shake flasks were prepared with 100 mL PIM medium each and inoculated with 2% bacteria from the starter culture. Flasks were incubated with vigorous shaking at 37 °C with monitoring of the optical density at 600 nm (ODeoo). Once culture ODeoo reached 0.5, 1 mM IPTG was added to one of the flasks and incubated at 37°C. After 48 hours, bacterial cells were removed via centrifugation and the spent media was dried via freeze-drying. The dried material was reconstituted in 0.5 mL 100% D2O with 10 mM EDTA and analyzed for production of phosphonates by31P NMR.

[0161] Structure elucidation of methylpantaphos. Methylpantaphos was produced via an in vitro enzyme reaction between pantaphos and HvrF and partially purified using a methanol precipitation to remove contaminants as described in Methods. The supernatant was then dried and reconstituted in 100% CD3OH prior to proton, carbon, and phosphorus NMR analyses. Because the pantaphos carbon assignments have already been established and due to the contamination of other methanol soluble components from the reaction mixture, we relied only on two-dimensional carbon spectra for carbon assignments of methylpantaphos. The1H-31P HMBC spectrum for methylpantaphos revealed the characteristic signals for pantaphos protons at dn 6.54 (d, J=6.00 Hz) and 4.73 ppm (d, J=18.0 Hz). The large coupling constant J of 18.0 Hz is typical for protons bound to the adjacent carbon to a phosphorus atom in phosphonic acids. These protons were also correlated to the phosphorus atom of the compound at dp 14.58 ppm in the indicating close proximity (within 3-bond distance) to P. The downfield signal at dn 6.54 indicates the alkene structure of pantaphos is maintained in methylpantaphos.

[0162] Proton-carbon HSQC experiments revealed the coupling of protons at dn 4.73 ppm to a carbon signal at de 70.12 ppm. This carbon chemical shift and the coupling of the protons that are adjacent to phosphorus closely resemble position 1 of pantaphos. Therefore, this signal at de 70.12 ppm is assigned as carbon position 1. Proton-carbon HMBC experiments revealed the coupling of protons at dn 4.73 ppm to other carbons at de 131.51, 135.63, and 168.34 ppm supporting the assignment of these carbons at positions adjacent to or nearby carbon 1. The carbon signal at de 135.63 ppm aligns with the chemical shifts for the vinyl carbon at position 2 of pantaphos and 2-phosphonomethylmaleate supporting a position 2 assignment. Based on proton-carbon HSQC and the vinyl carbon assignments established for pantaphos, the carbon signal at de 131.51 ppm correlates with the vinyl carbon proton signal at dn 6.54 ppm, which fully supports the assignment of this carbon to position 4. The carbon signals at de 168.34 and 174.91 ppm align with the chemical shift ranges predicted for carboxylic acids suggesting assignment to carbon positions 3 or 5. The proton-carbon HMBC revealed that the carbon signal at de 168.34 correlates with the protons on both carbon 1 (de 70.12 ppm) and carbon 4 (de 131.51 ppm) suggesting this carbon is less than a four-bond distance away. However, since the carbon signal at de 174.91 ppm does not correlate with the protons on both carbon 1 (de 70.12 ppm) and carbon 4 (de 131.51 ppm), it must be greater than or equal to a four-bond distance away. These proton-carbon HMBC data support the assignment of carbons at de 168.34 and 174.91 ppm to positions 3 and 5 respectively. Lastly, proton-carbon HMBC revealed a correlation between carbon 3 (de 168.34 ppm) and a new proton signal at dn 3.76 ppm. The proton signal at dH 3.76 ppm correlates to the carbon signal at de 51.12 ppm (assigned as position 6), which both align with the chemical shift ranges for a methyl ester. Since the methyl protons (dH3.r16 ppm) do not show a correlation to carbon 5 (de 174.91 ppm) and only exhibit a correlation to carbon 3 (de 168.34 ppm), the methyl ester carbon (de 51.12 ppm) must be located adjacent to carbon position 3. Based on these NMR assignments the compound structure is identified as (2E)-4-hydroxy-3-(methoxycarbonyl)-4-phosphonobut-2-enoic acid.

[0163] Tabulated values for1H and13C NMR:

[0164] 'H NMR (CD3OH, 600 MHz): 3 3.76 (s, 3H, methyl), 4.73 (d, 1H, J = 18.0 Hz), 6.54 (d, 1H, J = 6.00 Hz). The five non-solvent exchangeable protons agree with the proposed structure.

[0165] 13C NMR (CD3OH, 150 MHz) : 3 51.12, 70.12, 131.51, 135.63, 168.34, 174.91. The six unique carbon signals agree with the proposed structure.

[0166] Example 3. Plant resistance towards the potent phytotoxin pantaphos by an O- methyltransferase.

[0167] Pantaphos, a small molecule phosphonate natural product, has shown promise as a nextgeneration herbicide. Pantaphos was recently discovered as a virulence factor produced from pathogenic strains of Pantoea ananatis. P. ananatis is an economically important plant pathogen and the cause of onion center rot as well as disease in a variety of food crops including maize, rice, pineapple, and wheat. Strains of P. ananatis lacking the ability to produce pantaphos do not cause disease in onion, moreover, treatment with pantaphos results in severe onion rot as well as necrosis in mustard. Thus, pantaphos may have agricultural usefulness like that of the widely used phosphonate herbicides glyphosate (Roundup®) and phosphinothricin (Basta®).

[0168] During pantaphos biosynthesis, a S-adenosylmethionine-dependent O- methyltransferase, HvrF is required to drive an unfavorable dehydration reaction to convert phosphonomethylmalate to phosphonomethylmaleate (PMME) through the formation of a PMME methyl ester intermediate. Interestingly, it was found that HvrF also methylates pantaphos, which is not too surprising given the structural similarity between PMME and pantaphos which differ by a lone hydroxyl group. However, due to the hydrolysis of the methyl ester in vitro, bioactivity of methylpantaphos was unable to be assessed. Given the lability of the methyl ester and that HvrF catalyzes the complete conversion of pantaphos to methylpantaphos, it is likely that there is a biological necessity to prevent free-floating pantaphos during biosynthesis. The exact reason for this remains to be seen. However, based on the structural similarity of pantaphos to aconitate and isocitrate, it has been proposed that pantaphos may inhibit one or more steps of the TCA cycle.

[0169] Because bioactive phosphonates tend to inhibit essential pathways shared among the producing-organisms and their target hosts, the producing-organism must maintain a mechanism to prevent self-targeting. As demonstrated by some antimicrobial phosphonates including rhizocticin, plumbemycin, phosalacine, and dehydrophos, a common strategy involves chemically modifying the toxic phosphonate molecule into an inactive form that can still be taken up and processed by the target host. In the case of dehydrophos, the modification of the phosphonate with a glycine-leucine dipeptide linkage prevents release of the toxic phosphonate inside the cells of the producing-organism and still maintains toxicity towards target hosts. Thus, HvrF may be needed to detoxify intracellular pantaphos in vivo.

[0170] Here we show that in vivo expression of HvrF is sufficient to neutralize pantaphos toxicity, and that HvrF-expressing plants show tolerance to pantaphos treatment and pantaphos-mediated infection by P. ananatis. The use of the HvrF methyltransferase for the development of pantaphos- and infection-resistant plants elevates pantaphos as an herbicidal candidate and points to a novel strategy for combating Pantoea virulence among essential food crops.

[0171] In vivo expression of HvrF in plants confers pantaphos resistance. Previous research has shown pantaphos kills both onion and mustard, but it has not been tested against Arabidopsis. To test whether pantaphos causes potent phytotoxicity in Arabidopsis, we treated germinated A. thaliana Col-0 wild-type seedlings with pantaphos and observed complete killing of the plants (Fig. 10). To address whether in vivo activity of HvrF is sufficient to neutralize pantaphos toxicity, we constructed transgenic plants using an Agrobacterium- mediated transformation of A. thaliana Col-0 with a gene construct containing HvrF under constitutive expression from the Cauliflower Mosaic Virus promotor (35S P) (Fig. 11 A). Four independent HvrF+ seeds lines (#2, #6, #12, and #14) were isolated and subjected to increasing concentrations of pantaphos (Fig. 1 IB). At pantaphos concentrations of 15 mM and above, A. thaliana Col-0 (WT) showed a severe growth defect and significantly lower total leaf area compared to the HvrF overexpression lines (Figs. 11C and 12). In contrast to the significant growth inhibition of Arabidopsis WT, the transgenic plant lines showed complete tolerance up to 150 mM pantaphos (Fig. 12). Although all transgenic plant lines showed resistance to pantaphos, transgenic plant #2 exhibited the highest tolerance with an LDso (50% lethal dose) of 109 mM compared to 5 mM for Arabidopsis WT and 83, 85, and 98 mM for transgenic lines #14, #6, and #12, respectively (Table 3). To identify if the differences in pantaphos resistance profiles of the transgenic plant lines was due to different levels of HvrF protein expression, we analyzed HvrF expression against measured LDso and found that a lower relative HvrF protein expression led to a lower LDso (Fig. 11D-E). These data show that plants expressing HvrF are resistance to pantaphos treatment, and that constitutive expression in vivo is sufficient to detoxify pantaphos.

[0172] Table 3. Pantaphos resistance parameters for A. thaliana overexpressing HvrF.

[0173] Summary of the parameters estimated from a log-logistic model fitted for total leaf area to different pantaphos concentrations (in μM) against A. thaliana Col-0 (WT) and independent transgenic lines overexpressing HvrF (#2, #6, #12, and #14). C, lower limit; D, upper limit; b. slope of curves; LDso, 50% lethal dose; LD90, 90% lethal dose; R2, coefficient of determination. The values are the Mean ± S.E.M. of parameters estimated by the fitted model. Plants expressing HvrF show tolerance to P. ananatis pathogenicity. Because Arabidopsis expressing HvrF are resistant to pantaphos and since pantaphos is a key virulence factor produced during pathogenic P. ananatis infection, we tested whether the expression of HvrF in Arabidopsis can attenuate P. ananatis virulence during infection. Wild-type A. thaliana (HvrF-) inoculated with the pantaphos-producing strain P. ananatis LMG5342 resulted in total plant death four days post inoculation (Fig. 13 A). This effect was abolished in the plants inoculated with the mutant lacking the pantaphos biosynthetic gene cluster (DAvr), suggesting the plant death associated with P. ananatis infection is due to the production of pantaphos by the bacteria (Fig. 13B). In contrast to the plant death observed in the wild-type plants, plant line #2 (HvrF+) showed significant reduction in virulence during P. ananatis LMG5342 infection (Fig. 13 A). Minor yellowing of plant leaves was observed in P. ananatis WT and Pfhvr treatments suggesting there are some negative effects on plant health attributed to the bacterial burden itself. However, the lack of growth inhibition and death in the HvrF+ plants infected with the pantaphos-production strain suggests that HvrF expression in plants serves as a mechanism of resistance to pantaphos-mediated pathogenicity.

[0174] The use of herbicides in agriculture relies on the ability to target only the weeds leaving crops unharmed. Thus, crops must exhibit insensitivity to herbicide treatment. This typically involves either expression of a resistant target protein, altered penetration or translocation of the herbicide, and / or detoxification of the herbicide through chemical modification. For example, an A -acetyl transferase, corresponding to the bar gene from S. hygroscopicus or the homologous pat gene from S. viridochromogenes Tu494, encoded by these phosphinothricin- producing strains confers resistance to the widely used herbicide phosphinothricin (Basta®) when expressed in plants. In addition, a novel phosphinothricin A -acetyl transferase (MAT) from Nocardia sp. strain AB2253 was shown to detoxify phosphinothricin and facilitate herbicide-resistant plants. Thus, the use of hvrF as a marker for pantaphos tolerance could be very useful to construct “Pantaphos Ready” crops that are resistant to pantaphos treatment in the field.

[0175] In addition, the construction of HvrF-expressing crops could also be very useful for alleviating pantaphos-mediated plant disease. For example, onions that express HvrF may show decreased sensitivity to onion center rot caused by P. ananatis infection.

[0176] Bacteria, plasmids, and growth conditions. E. coli strains carrying plasmids were grown at 37°C in sterile Luria-Bertani medium (LB; 10 g / L tryptone, 10 g / L NaCl, 5 g / L yeast extract) or M9 minimal medium (M9; Cold Spring Harbor Protocols) with the appropriate antibiotic. Antibiotic concentrations of 50 mg / mL filter-sterilized kanamycin (Kan) and 50 mg / mL filter-sterilized spectinomycin (Spec) were used where appropriate.

[0177] Chemicals. Pantaphos was obtained through bacterial fermentation and purification. General molecular biology consumables and reagents were purchased from Millipore-Sigma, New England Biolabs, QIAGEN, Thermo Fisher Scientific. Glufosinate ammonium (phosphinothricin) was purchased from GoldBio.

[0178] Arabidopsis thaliana growth conditions. Arabidopsis thaliana Columbia (Col-0) seeds were purchased from the Arabidopsis Biological Resource Center (Columbus, OH, USA). Plants were germinated and grown using Murashige and Skoog medium (MS; Millipore- Sigma) with 0.7% (w / v) agar under the following day / light cycle conditions; 16 h light at 23°C with 100 mmol m2s ' light intensity and 8 h dark at 18°C (long day conditions).

[0179] Expression and purification of HvrF from Pantoea ananatis LMG5342. The pAP13 plasmid containing the gene encoding for 6X-histidine-LVPRGS-HvrF was transformed into E. coli Rosetta (DE3) cells. A starter culture inoculated with a single His-HvrF E. coli colony was inoculated into 100 mL of LB-Kan and incubated in a shaking incubator overnight at 37°C. The following day, 6 L of M9-Kan was inoculated with a 2% inoculum from the starter culture and grown at 37°C. When culture ODeoo reached 0.6-0.8, the cultures were cooled to 18°C for one hour in a shaking incubator and supplemented with 100 mg / mL of lysine, phenylalanine, and threonine, and 50 mg / mL of leucine, isoleucine, and valine. Cultures were incubated for 15 min, then a freshly made stock of selenomethionine (TCI Chemicals) was added for a final concentration of 0.05 g / L and the cultures incubated an additional 15 min. Finally, 1 mM of isopropyl P-D-l -thiogalactopyranoside (IPTG; GoldBio) was added to each flask and the cultures were incubated in a shaking incubator for 18 hrs at 18°C. Cells were harvested by centrifugation at 5000 x g for 20 minutes and resuspended in 500 mM NaCl, 20 mM Tris-HCl pH 8.0, 10% (v / v) glycerol buffer before being subjected to lysis via sonication. The resultant lysate was clarified by centrifugation at 14,000 x g for 45 min at 4°C and the supernatant was loaded onto a 5 mL HisTrap HP affinity column (Cytiva) which had previously been equilibrated with Buffer A (30 mM imidazole, 20 mM Tris-HCl pH 8, 1 M NaCl). An additional wash of the loaded column was performed using Buffer A prior to eluting the protein by employing an AKTAprime plus FPLC system set to a linear gradient of 0-100% Buffer B (250 mM imidazole, 20 mM Tris-HCl pH 8, 1 M NaCl) over 40 mL at a flow rate of 2 mL / min. Fractions were analyzed via SDS-PAGE gel to ensure the presence of the protein prior to being pooled. These pooled fractions were then subjected to dialysis against a buffer of 20 mM Tris- HCl pH 7.5, 300 mM NaCl, and 10% (v / v) glycerol. Simultaneously during the dialysis, the protein was subjected to digestion via thrombin cleavage (Thrombin Bovine from Bovine Plasma Activity; Thermo Fisher Scientific) to remove the 6X-histidine affinity tag. The resultant solution was then again loaded onto a 5 mL HisTrap HP affinity column (Cytiva) equilibrated with Buffer A. The loaded column was washed with Buffer A and then eluted in one step using Buffer B and fractions analyzed by SDS-PAGE. The flowthrough, which contained the untagged protein, was further purified by size exclusion chromatography using a 120 mL Superdex® 200 10 / 300 GL column (GE Healthcare) with 20 mM Tris-HCl pH 7.5 and 300 mM NaCl as the running buffer. The resultant fractions were analyzed via SDS-PAGE to confirm the presence of the protein and purity. Fractions containing selenomethionineincorporated pure protein were pooled and frozen at -80°C for future use.

[0180] Construction of HvrF overexpression plant lines in Arabidopsis thaliana. A GenSmart™ codon optimized hemagglutinin-tagged (HA) HvrF sequence from Pantoea ananatis LMG5342 was assembled into a binary vector by Loop assembly with Cauliflower Mosaic Virus 35S (35S) promoter and terminator, to which a phosphinothricin resistance gene (bar) and nopaline synthase (NOS) promoter and terminator were added to select for bacteria transformants (Pollak et al., New Phytol 222, 628-640 (2019)). The assembled construct, JV477, was confirmed by whole plasmid sequencing (MGH CCIB DNA Core; Cambridge, MA) and transformed via electroporation into Agrobacterium tumefaciens GV3101 with selection on LB-Spec. Stable transformation of the JV477 expression construct into Arabidopsis thaliana Columbia (Col-0) was performed via an A. tumefaciens GV3101 mediated floral dip method (Clough etal., Plant J 16, 735-743 (1998); Zhang etal., NatProtoc 1, 641-646 (2006)). Four independent transformation events were selected on MS-0.7% agar with 15 mg / L phosphinothricin and used for immunoblot analysis using HA Epitope Tag Polyclonal Antibody DyLight™ 800 (Thermo Fisher Scientific) to confirm protein expression. Transgenic seed lines expressing HvrF were used for pantaphos resistance assays and pathogen infection assays.

[0181] Pantaphos resistance assay using Arabidopsis thaliana. Seeds of Arabidopsis thaliana Col-0 wild-type (WT) and HvrF overexpressors (HvrF+) were surface sterilized by soaking in 70% (v / v) ethanol for 5 min, then in 50% (v / v) bleach for 15 min followed by rinsing five times with sterile water. Sterilized seeds were covered with foil and placed in 4°C for two days. Cold-treated seeds were transferred to MS-0.7% agar medium under long day conditions for six days until the cotyledons fully opened. Addition of 100 mM phosphinothricin was included in the medium for HvrF+ seeds. Healthy seedlings with fully opened cotyledons were then transplanted into 24-well Falcon® plates (Corning) containing 1 mL of MS-0.7% agar per well. Then, 20 mL of various concentrations of pantaphos were applied to each well directly onto the seedling. The plates were monitored for nine days until the seedlings reached the principal growth stage 1.04 according to Boyes et al., Plant Cell 13, 1499-1510 (2001). Total leaf area of the seedlings was used to determine plant growth responding to pantaphos and measured with ImageJ software (Rasband W.S., Image! U. S. National Institutes of Health, Bethesda, Maryland, USA, (1997-2018).) . The relationship between the total leaf area of each genotype and pantaphos concentration was analyzed using log-logistic regression. The fitted model was: where C = the mean response at the highest herbicide dose (lower limit), D = the mean response of the untreated control (upper limit), b = the slope of the line at LD50, and LD50= 50% lethal dose. Parameters estimated from fitting models are listed in Table 3. The curve fitting analysis was performed using OriginPro® 2023 software. Statistical analysis for biological replicates was performed in R (v 4.3.0) using a Dunn’s Kruskal-Wallis test with adjusted comparisons.

[0182] Pathogen infection and bioactivity assays. Arabidopsis thaliana Col-0 wild-type (WT) and HvrF overexpression (HvrF+) seeds were surface sterilized by soaking with 10% (v / v) bleach for 5 minutes, rinsing three times with sterile H2O, then soaking in 70% (v / v) ethanol for 5 minutes followed by rinsing three times with sterile H2O. Sterilized seeds were transferred to a petri dish (Avantor) containing 25 mL of MS-0.7% agar. Addition of 100 mM phosphinothricin was included in the medium for HvrF+ seeds. Plates were incubated at 4°C for 18-24 hrs then transferred to a growth room with long day conditions for 5-6 days until the cotyledons fully opened. Healthy seedlings with fully opened cotyledons were transferred to sterile 24-well Falcon® plates (Coming) containing 1.5 mL of MS-0.7% agar per well and incubated in a growth room with long day conditions for five days. For pantaphos bioactivity assays, 0.5 mM purified pantaphos or water was spotted onto the seedlings and incubated in a growth room with long day conditions for five days. For pathogen infection assays, seedlings were first incubated in a growth room with long day conditions for five days. A single colony from a LB-agar plate streaked with P. ananatis incubated 18-24 hrs was resuspended in 0.5 mL of IX phosphate-buffered saline. This corresponded to an ODeoo of around 0.03 or 6.4xl06cells / mL of bacteria confirmed via plate counting. Then, 10 mL of this bacterial suspension was inoculated directly onto the center of the 5-day old seedlings. The inoculated seedlings were incubated at ambient temperature in the dark for 16 hrs then transferred to a growth room with long day conditions where plates were imaged daily.

[0183] Example 4. Sequences

[0184] (HvrA)

[0185] MIKKLIAEKGTLIFIEAHNPLSALIASKAEQTNSEGRIVKFDGIWSSSLTDSASRGIPDNETLALS SRLENIADIRNVTDMPIIMDADTGGKPEHFSYYVKRMINNGVNGVIIEDKTGLKKNSLFGTEV EQTLADINDFSEKIKRGKSAVYIDDFMIIARLESLIAGFDVEHALERADAYVEAGADGIMIHSC KKTPDEVFLFSTKFRKKYPSVPLICVPTTYSATSNRELSEAGFNVIIYANHMLRAAYKAMENV SKEILRYGRTAEIEKSCMSVKEIISLIP (SEQ ID NO: 1)

[0186] (HvrA) atgatcaaaaaacttattgcagaaaagggtactctgatttttattgaggcccataatccgctctccgcattaattgcgtctaaagcagaacaaactaattc agaaggccgtattgtcaaatttgacggtatatggtcaagctcgttaacggactcagcatctcgcggtattcccgataacgaaacactggcattaagca gcaggttagaaaatattgctgatatccgaaatgtgacagacatgcccatcatcatggatgctgatacggggggaaaaccagaacattttagttattac gtaaaaagaatgattaacaacggtgtaaatggcgtcatcatcgaagataaaacaggattaaagaaaaattctttgttcggcactgaagtagaacagac tctcgcagatattaatgatttttcagagaagattaaaagaggaaaatctgcagtttatattgatgattttatgatcatagccagacttgaaagtcttattgca gggttcgacgtagaacatgcactcgaacgtgccgacgcatacgtcgaagccggggcagacggaattatgattcatagttgtaagaagactccgga tgaggttttcttattcagtacgaaatttcggaaaaaatatccatcagtaccattaatttgtgttcctactacttattctgcaaccagcaacagagaactcagt gaagcgggttttaacgtgatcatttatgcaaaccatatgctcagggctgcttataaagcaatggaaaatgtttcaaaagaaatattgagatatggcagg acggcagagatagaaaaatcttgcatgagtgtaaaggaaattatttcactgattccttaa (SEQ ID NO: 2)

[0187] (HvrB)

[0188] MHECKGNYFTDSLKRMTMKKKEMIIGAYISYGTGHHPASWRESGVNAAAALDIDTYANLAR VCEKGLADLLFLADTPSVFQDNMDGYGSRVSVLEPLSLLSYLASQTQNIGLVATASTTYKHPY NIAREFASLDYISKGRAGWNLVTSSKSDAAKNFGLAAHPEHSKRYDMAWEAWQVISGLWDS WEDNALVRNKTSGQFFVKDKYREINFEGEYFNVKGPLNIARPPQGYPVIVQAGSSEEGKELA AKTADIVFTAQNNIEDAKKFYDDLKGRMEKYGRSKSELLILPGLSFYIASDESKARKKLNDLN ALIPQSFGLSMLSDLLGGVDLKNNDPEGPLPDLPKSNGNQSRQKIIIDLARKEKLSIKQLYEKIII SRGHYTFTGSYQDLADEMIKWVENEACDGFNIMPPLMPESLINLFDHVIPLIQARGWYKKSY STGTLREKLGLKRPTNKLFNQ (SEQ ID NO:3)

[0189] (HvrB) atgataataggtgcctatatatcgtatggaacaggacatcatcctgcttcatggcgcgaaagtggcgtaaatgcagcggcagcgcttgatattgatac gtatgccaatcttgcaagagtatgtgagaaaggattagctgatttactttttcttgcagatacgccttctgtatttcaggacaatatggacggttatggca gcagggtatctgtactggagccattatcattattatcgtatttagcatctcaaacacaaaatattgggctggttgcgacggcttcaacgacgtacaaaca cccttacaacattgcgagggaatttgcttcgctggattacattagcaaaggaagggcagggtggaatctggtgacatcctcaaagtcggatgcggct aaaaactttggccttgccgctcatccagaacattcaaagcgttatgatatggcctgggaagcatggcaggttatcagtggtttgtgggacagctggga agacaatgctttagtaaggaataaaaccagtggacaattcttcgttaaggataaataccgggaaataaattttgagggtgaatattttaatgtaaaaggc ccattaaacatagctcgccctcctcagggctatcccgtcatcgtccaggcgggttcctctgaggaaggaaaagagttagctgcaaaaacagcagat attgtttttactgcacaaaacaatattgaagatgcaaaaaaattctatgatgatcttaaagggcgaatggaaaaatacggaaggtcaaagagtgaactt cttattcttcccggattaagcttttatattgcaagtgacgaatctaaagcccgtaaaaagcttaatgatctcaacgcactgatccctcaatcatttggctta agtatgttatcagatttactgggtggggttgatttaaaaaacaatgatcctgaaggaccattaccagatttaccgaaatctaatggtaatcagagtagac aaaaaattattatcgatttagctcgaaaagaaaaactatctatcaagcaactttatgaaaaaataatcatttcaagaggacattatacatttacaggttcct atcaagatttagcagatgagatgattaagtgggttgaaaatgaagcatgtgatggtttcaacattatgcctcctctcatgcctgaatctcttattaatctttt cgatcatgtcattccacttattcaggcaagaggatggtataaaaaatcatattctactggaacattaagagaaaaactggggcttaaaagacctactaa taaattgtttaatcaataa (SEQ ID NO:4)

[0190] (HvrC)

[0191] MLNKNLILEDTTLRDGEQAPGVAFTPEQKVEIFYLLANMGVKWIEAGIPAMKGDEVKALSEM LERKNEINIIAWNRGVLEDIEYSISLGFKAVHIGLPTSAIHLEKSVKKDKSWLVKTASDLVKFAK DKGMFVSISAEDIGRTDIGFLQEYAQVVAEAGADRLRLSDTIGILSPAQYKEKVSLLNKNVNID LQCHCHNDFGFAVANTLAGIEAGARYFHVCVNGIGERAGMPDLAQVAMALHFFHGVDLGLD LTKLIALSETVARYSHQKISPWQPIVGDNVFAHESGIHANGMLKDSSTFEPFDPATVGGERRLV VGKHSGRAIIKHFLEESGVKAADDKALDRCLERVRSHAVRHPGGIPPHVLVDLYTAG (SEQ ID NO: 5)

[0192] (HvrC) atgcttaataaaaatctgattcttgaagataccactttacgtgatggtgagcaggcgccaggtgttgcatttacaccagagcaaaaagtagaaatttttt atctacttgcaaatatgggcgttaaatggatcgaagccggaatacctgcgatgaagggtgatgaagtaaaggctctgtcggaaatgttagagagaa aaaatgaaattaacatcatcgcgtggaaccgaggcgtgcttgaagacattgagtacagtatctcactggattcaaagcggtgcatatcgggctacc gactcagctatccatttagagaaaagcgttaagaaagataagtcctggcttgtaaagacggcttcagatttagttaagtttgccaaagacaaagggat gtttgtttctatcagcgcagaagatataggccgaacagatatggatctacaggagtatgcacaggtagttgctgaggctggagccgatcgtcttcg cctctctgacacgatggtatctttctcctgcacaatacaaagaaaaagtctctttgttaaataagaacgtcaacatcgattgcagtgccatgccaca atgattttggttttgcagtgctaacacgctggcaggcatgaagcaggagcacgctactttcatgtctgcgtcaatggcattggtgaaagggctggaa tgccagacctggcacaagttgctatggcattgcactttttccacggggttgatttagggctcgatttaacaaaattaatcgcgtgagtgaaacggtcg ccaggtacagccatcaaaaaatcagtccatggcagccgatcgtaggcgataacgtttttgcacatgaatcgggcatcacgcaaatggtatgctcaa agacagcagtacttttgaaccctcgacccagctacggtgggaggagaacgacgtctggtcgtgggtaaacattccggtcgcgccattatcaaacat tttctcgaagaatcaggcgtgaaagctgccgacgataaggctcttgatcgctgtttagaacgcgtgagaagtcatgccgtgcgccaccccggtggg atccctccacatgtatagttgatctgtataccgcggggtaa (SEQ ID NO:6)

[0193] (HvrD)

[0194] MKSNQPIVNQIIASHSGRGQVSAGELITVDVDYVYVQDGNSPTVAKLFQDYHLSEVLKPDKIG FFFDHSVLVPDKTMAKRVNEAMEFAKKLGINIYSRGEGISHVIALESKIFKPGNIVLGADSHTC TGGAVQSLALGMGASDILVAMLTGQTWLKVPQTVHLCIKGKTGKDVRAKDVMLALLNKYG QTPFLYKSIEVSGEWAEELTLDEAASFASMAVELGAKCIFMPDGQGRPEGLLKADASVADSVI NFSVSELMPHIAPPHSPLYAKPANDFEGLKFDYIFIGSCTNSRLEDIKEVAEIVAGKTIHPDIHCL LTPGSKSVYLKALQAGYIDTLIRSGIIVTPPGCGACVGTQGTIPADGEKVLSTMNRNFKGRMG NAEADIFLCSPRTAAMVALNGTVPHFEGESAYE (SEQ ID NO: 7)

[0195] (HvrD) atgaaaagtaatcagccgattgttaaccagatcattgcgtcacacagtggcagaggtcaggtttcagcaggtgaactgatcacggtagatgtgacta cgtctatgttcaggatggaaattcaccgaccgtggcaaaactgtttcaggattatcatctgtctgaggtgctaaaacccgataaaatcgggttcttctc gaccattcagttctggtacctgataaaaccatggctaaacgtgtcaacgaggccatggaatttgcaaaaaaacttggaataaacatctattcacgagg ggagggaatagtcacgtcattgccctggagagtaaaatatttaaacccggcaatatagtgctgggcgcagattcccatactgtacagggggggc cgtacagtctttagcgctgggaatgggggctcggatattctggttgctatgttaacaggacaaacgtggttgaaagtccctcaaacagtccatttgtgt attaagggtaaaacgggaaaagatgtgcgggcaaaagatgtcatgttggcacttttaaataagtacggacaaacaccatttctttataaatcgatcgaa gtttcaggggaatgggcagaagagctaacgcttgacgaagctgcaagttttgcaagtatggctgtgagttaggagccaaatgcatatttatgccag atgggcaaggcaggcctgaggggctattgaaggcggatgcctcagtggcagacagtgtcatcaatttttctgtatcagaattaatgccccatatcgc accacctcacagtccttgtatgctaaacctgcaaatgactttgagggtctgaaatttgattatattttcattggaagctgtactaacagcagacttgaaga tatcaaagaggtggccgaaatgtgctggtaaaacaatacatcccgatattcactgccttctgacgccaggatcgaaaagtgtttatctaaaagctctc caggcgggatatatcgatacgctatccgctcgggcattattgtcaccccaccgggttgtggagcttgtgtgggtacccaaggaaccattcctgcgg atggggagaaagtattaagtacgatgaaccgcaattttaagggaagaatggggaatgctgaggcagacatctttttatgttctccacgaactgcagc gatggttgcattgaacggcactgttccacattttgagggagagtccgcatatgagtaa (SEQ ID NO: 8)

[0196] (HvrE)

[0197] MFHILRESPHMSKTAENYRVRRVEGNISTDDIIPARYKHMYTEPAQLAPHLFESRFPGFRETLSI NDVLVCDQIFGIGSSREQAVTTLLACGVKYVFSPSFGRIFFRNSWNLGLHAIEVDTSELADLSEI KIELTGGVIYTENNQINFFPPSSQMTAIVSAGGIIPYTINKIMEKKGDILRGYSNEK (SEQ ID NO: 9)

[0198] (HvrE) ctgtccacattttgagggagagtccgcatatgagtaaaactgctgaaaattatcgcgtcagacgcgtggaagggaacatctcgaccgacgatattat acctgcgcgctataaacatatgtataccgagccggcccagctggcaccgcatctttttgagagccgttttcccggatttagggaaacgctcagtatca atgatgtgctgtatgtgatcaaatatcggtatagggagttcgcgggagcaggcagtaacaactctgttggcatgtggtgttaaatatgtattttcccct tcttcgggaggattttttttaggaactcttggaatttgggtttacatgcgattgaggtcgatacgagtgaacttgcagatttaagtgaaattaaaatagaa ctgactggaggggtaatttatacagaaaataatcaaataaattttttccctcccagctcgcagatgacggcaattgtcagtgcaggtggcataataccc tacaccataaataaaattatggaaaaaaaaggtgatattttaagaggtatagcaatgaaaagtga (SEQ ID NO: 10)

[0199] (HvrF)

[0200] MKSEKFDGLADNYDKYRPRYPAILFKEIHDWMQPSAKNIYDIGAGTGIAIEGMTRVTGKHYD FTAIDISEDMIKKGREKLPGTTWVKGKAEDILSDKSRIDVIMAAQSFQWMDRAKTLEVSIKSL NKGGVFAVLQNNRDYRNNEMLNKYEGLLEKFSPGYSRHYRDYDYENEITNVFKLPIANFKK VVTGWTMEMISEDFFGFISSSTQVQRAIENDRNGFWKEIEILIDEHSVGGKISIDYISELFIAKK RDDS (SEQ ID NO: 11)

[0201] (HvrF) atgaaaagtgaaaagttgatggttggctgataactatgataaatatcgtccccgtatcctgcaatccttttcaaggaaatccatgactggatgcagc cgtctgccaaaaatatatacgatatggcgcaggcacaggtattgctatgaaggtatgacacgtgtcactggaaaacactatgatttcacggcgata gatatttctgaagatatgataaaaaaaggaagggaaaaactgcctggtacgacttgggttaaaggaaaagcggaagatatctttctgataaaagcc gtattgacgtcattatggcggcacagtcctccaatggatggatagagctaaaacattagaagtttcgataaaatctttaaataagggcggggtttttgc agttttgcaaaacaatcgagattacagaaataatgaaatgcttaacaaatatgaaggtttgctagagaaatttagtccaggttacagcagacatatcgc gactatgactatgaaaatgaaatcaccaatgtttttaaatgcctattgctaactttaagaaagtcgtcacagggtggactatggaaatgatttcagaaga tttttttggattcatttcttcctcaacccaggtacaaagagctatgagaacgatcgtaatggattctggaaggagatgaaatctgatgacgaacactc agttggtggaaaaatagcatagattatataagtgagttatttatagctaagaagcgtgatgatcatag (SEQ ID NO: 12)

[0202] (HvrG)

[0203] MIHSMTIQDISIEQASYEDAKLLRKALEKVYEPYTLNFSPTALQFTENIIAQESSKWLVAKYKS DIVGAVRYELYDIYLDFHFLCVTPPFRKMGVGNELFHKLKKIAYEKRKDFMKIVLRDSLSYNR RYFESKGFYFYHKYQTNMHSVFILKLNGEKP (SEQ ID NO: 13)

[0204] (HvrG) atgacgattcaggacatcagcattgaacaagcttcgtatgaagatgcgaagctgttaagaaaggctttagaaaaagtttacgaaccttatacactgaat ttctcacctaccgctttgcagttactgaaaatatcatgctcaggaatcctcgaagtggctagtcgcgaaatacaagtcagacatgtgggggcagtg agatatgaacttatgatatttatctggactccattttctctgcgtcacaccaccatcagaaaaatgggagtagggaatgaatatttcataaactgaag aaaatagcgtatgaaaaaagaaaggattttatgaagatcgttttgcgagattcgctaagctataaccgacgctattttgaaagtaaaggattttacttttat cataaataccagacaaatatgcatagtgtatttattttaaaattaaacggtgaaaagccatga (SEQ ID NO: 14)

[0205] (HvrH)

[0206] MNKKALVIGLKSNMERVIKGLNEIEFIIIDRGTLDNESIDYIINLSDDLMHKKSFSYVIASSEDFI ALAGLLRNRYSLYGEKYYKSTIATNKFLMRNFCSGFLSCPKFWLSGEIINSENLLLSSQKDYIV KPLTGSSAKHVETVTQEDLNQYLNENNKLMLIEEKVLMRDEYHLDCIIKDGNILFSTLSIYDR PILEAKSKNRASINLPDGTRLHEDALILATNLQSHFEMTNGVFHIEMYHTQDGFILGEFGIRPP GAGVTDMYYMYRGVDFWEAFIYSQIDKEFILPLNHKSDKYCAAIGICSSFPVDDIRSTSKVSV DKYVNLRENAAKPAVPSSTSFNHMIYVSSSSLEEIRNFLHDISDRQE (SEQ ID NO: 15)

[0207] (HvrH) atgaataaaaaagcattggtattgggtgaaaagtaatatggaaagagtcatcaaagggctcaatgagatagaatttatattattgacagaggaaca ctggataacgaaagtatagatatatcataatctttcagatgatttaatgcataaaaaatcattcagtatgttatagccagttctgaggattttatcgctttg gctgggtatgcgtaatcgtattcactttatggtgaaaaatatataaaagtacaatgcaaccaataaattcctgatgcgtaatttttgctcaggtttttta tcctgtccaaagttctggctatcaggtgagatcatcaatcagagaatcttttactgtcatcccagaaagatacatcgtaaaacctcttacaggtagttct gcaaaacatgtcgaaacagtcacacaggaagacttaaaccaatatcttaatgaaaataacaaattgatgttaattgaagaaaaggttttaatgagagat gaatatcatctgatgtataatcaaagacggtaatatcctcttttcaacactgtcaatttacgacaggcctatactcgaggcaaaaagtaaaaacagag ccagtatcaactacccgatggcacacggcttcatgaagatgccttaatacttgccacgaatttgcaaagtcattttgaaatgactaatggcgtttttcat atgaaatgtatcatactcaggatggatttatactcggtgagtttggtatccgccctcccggagcgggtgtgaccgatatgtatatatgtatagaggcg tcgatttctgggaggcatttatttattctcagatgataaagaatttattttacccctaaatcataagtccgataaatatgtgctgctatgggatatgttcttc tttcccggtcgatgatatcagaagtacgtcaaaagtatccgtggataaatatgtaaatttgcgtgagaatgctgctaaacccgcagttcccagtcaac gtcttttaatcatatgatttatgtttcttcatcatcctagaagaaataaggaattttttgcatgatatttctgacagacaagagtaa (SEQ ID NO: 16) (Hvrl)

[0208] MLKYETKKNAFFQIYNFLSCSAEGIQTVIFLWLIYHETHSPMLVSLTIVSSYLPSAVLGFFFLKK ADASSPGKQLFISNVSLSAISLIVYFILMRNEGFELITLSIFYLAQAVLSVVKMFNKTSQNRIIRT AFSNSDAIKALQLASSGMQAAQVFGAAIGGWAISTGYYMQALMLTCCIYLLNIYISTLFEKGN PDNKDAEVIVQIHSVTKKNESFTYLFRSKDFLLPLIFTVPSSGALQFLNTSLPSLSSLYGNSEKIY PVLNMTLQCAVIISGIAAALNMLSLKSSLRFSLCISGICLILMCLSSRNYYAVYFFLFLTSFFVSW HMISIKVLTNQMPDIENIGKFTMMRNSVASGVKIVFSISSGAFLTFYSITTTYLILAILLIFFNVL WVCQSRSFNYEDLGDVKFQK (SEQ ID NO: 17)

[0209] (Hvrl) atgttaaaatatgaaaccaaaaaaaacgctttcttccagatatacaattttctttcatgctcggctgaagggattcagacggttatattttgtggctatata tcatgaaactcatagtccgatgctgtcagtctgactatagtttcttcatatcttccatcagccgtataggattctttttcctcaaaaaagctgatgcgagc agtcctggaaaacagttatttatcagtaatgtttctctagtgccatatcgctaatgtctattttattttaatgagaaatgagggttttgaactaatcacgctc agtatcttttatttggcacaggccgtatatctgtgtcaaaatgtttaataagacatcccaaaatcgcattatcagaacagcattcagtaatagtgatgca ataaaagcactcagctgcctcctctggcatgcaggccgctcaggtttttggcgcggccataggaggatgggctatcagtacgggtactacatgc aagcgctcatgctgacctgctgcatctaccttttgaatatttatatttcgacactttttgagaaagggaatcctgataataaagacgctgaggttatgtgc agatcactctgtcactaagaaaaatgaatccttacatatctttttcgcagtaaggactttttgtaccctgatttttactgttccatcaagtggagctttac aatttttaaacacatcccttcctcatatctctctatatggtaatagcgagaaaattatccagtgctgaatatgactctacaatgtgctgtcatatctcgg ggatagcagcagcactcaatatgctttctctaaagtcttcattacggttttctctgtgtatttctggcatctgtctgattttgatgtgttatcatcgagaaat atatgccgtatacttttttttatttctcactctttttttgtttcatggcatatgatttctataaggtactcacgaatcaaatgcctgatatagaaaatatggta aatcacaatgatgagaaactcagtcgcttctggtgtgaaaatgtcttttctatttcgtctggtgcttttttgactttttacagcataactacaactatctaat actgcgatactgcttatttttttcaatgtactatgggtttgtcagagtcgcagttttaactatgaggatttaggtgatgttaaatttcaaaagtga (SEQ ID NO: 18)

[0210] (HvrJ)

[0211] MLNFKSDFSEFITGFYLKQFTHLNTQEREHVLETLGVTPSAINEFITSEDIYITLPHASMNVFFP RAGVSRYVYDLQNSNKNAFHLRFFLTHTNFSDLNWRPYAWWFNNGGKIDKLTFFTRNKKKK HNIVYSLKPNEMHSASVDRRLRHDFDTSMKFKRISLSFIYMTAVQEVNSGFAHKGRTLYLPLD AFVAFIIHQAKNDVISFNFLEAFLSQAQCRRLNGEELSFTSEWRDAFIFDNFTNIALLNFFQPAA FVGGEKMDNYWHQVIEKWKMALPNQSEVEFALPTNLVMPAVQEYIYPYKPSSDIAEQLIKNN IPYSLTMAIQEHDLFSKKE (SEQ ID NO: 19)

[0212] (HvrJ) atgttaaattcaaaagtgatttttctgaatttatactggttttatcttaaacagtttactcatttaaatacgcaggaaagagaacatgtgcttgaaacgctt ggcgtcacacccagcgcaatcaatgagtttataacatcagaagatatttatataccttacctcatgcaagtatgaatgttttttttccaagagcgggggt ctcacgttatgtctacgatctacaaaatagtaacaaaaacgcgtccatttaaggtttttctgacccataccaactttagcgatctgaactggcgcccta tgcatggtggtttaataacggcgggaaaatagataaacttacctttttcacacgaaataaaaaaaagaaacataatatcgtttactcgctaaaaccaaat gaaatgcactctgcctctgtagatcgacgtctgcgtcatgacttgatacaagtatgaagtttaaacgtatttctctagctcatctatatgacagccgttc aagaggtcaatagcggtttgcacataaaggccgtaccctttatctcccactgacgcatttgtagcttttattattcatcaggcaaaaaacgatgttatat cttttaattttttagaagcttttttaagccaggcacaatgcagaagactgaatggcgaagaattgagttttaccagtgaatggcgcgatgcatttatctttg ataattttacaaacattgcgctgttaaatttttttcagcctgcagcatttgtgggaggcgaaaaaatggataactactggcatcaggtgattgaaaaatgg aaaatggcataccaaatcaatctgaagtgagtttgcgtacccactaatctggtcatgcctgccgttcaagagtatatttacccttataaaccatcaag cgatatagcagaacagcttattaaaaataacatcccatacagtcttaccatggctattcaggaacatgatcttttttcaaagaaagaataa (SEQ ID NO: 20)

[0213] (HvrK)

[0214] MLDKSAFRHAMSHLPTAVTIVTSSGSCGAAACTVSSVCSVTDDPPTLLVCINRASNNNSVIRN NGSLCVSILSGEQSNIAMQCANHQVSVEERLALFDADVLVTGSPAVVDSVCCLDCRVDNIVES GTHSVFFCQVLSSKAFVGKDALVYYCRNYHRVS (SEQ ID NO: 21)

[0215] (HvrK) atgctagataaaagtgcttttcgccacgcaatgtcgcatcttcctactgcagtcactattgtaacatcaagcggctcctgcggggctgcagcatgtaca gtttcatccgtgtgttctgtcaccgacgacccacccactttactcgtatgtataaatcgtgcctccaataacaatagtgttataagaaataatggctctttgt gtgtcagcattctttcaggagagcaaagcaacattgcaatgcaatgtgctaatcatcaggtttcagttgaggagcgcttagcattattcgacgcagac gttctggtcacggggtctcctgctgtagttgattccgtttgctgtttagattgtcgtgtagataatattgtggaatcaggaacccactctgtcttcttttgtca ggttctttcttctaaagcttttgtggggaaagatgcgctggtttattattgccggaattatcatcgtgtatcataa (SEQ ID NO: 22)

[0216] (HvrL)

[0217] MMKSLIITSHFNSDLSSMACDEFGLKRSLIFYTPKINELSLGLKYEYYDKIDSYSVRLMELGNE IDFIWSTSGSEINSFAELKAVELAGLRNIYLGMNENAFSTAAYKSITQELMAFLGVSIPKGLQCN TKKEIHDFLEYNGGTIVCKANNGAGGVNQFYCQRFDDIYKLPHEVTDWYVEQFLKGLEFSV NAYMLNGFYIASPIMFKGETDIHSGHAMDKFRYISKLKNKSLNEKINAILSKISNTNIFNGWIE VEFIKTHQDLVVIEINARYNGTIRATGYACNENLYQLDLESKIYNKFSSQLNHENEVIEMPIHLK LETGLKEFGFVQKMKSRKTNTGRATIWGEDQCELLERIKNTELEIYSERIIHGINESKELFEKYI (SEQ ID NO: 23)

[0218] (HvrL) atgatgaaaagtttaattattacgtcgcattttaatagtgatttatcctctatggcatgcgatgagtttggattaaaaagatcactaattttctatactccaaa aataaatgaacttagcttaggtttaaaatatgagtactacgataagatagattcttactctgtaagattaatggagcttgggaatgaaattgattttatatgg agcacttccggttctgaaatcaactcgttcgctgaattaaaagctgttgagttggcaggattaagaaacatctatctaggcatgaatgaaaatgctttttc tacagctgcttataaaagcatcacgcaagagttaatggcatttttgggtgttagtattcccaaagggcttcaatgtaacacaaaaaaagaaatacatgat ttccttgaatataatggtggaactatagtctgtaaagctaacaatggagcaggtggtgtaaatcaattttattgccaaagatttgatgacatatataaactc cctcatgaggtaactgattggtatgtagaacagtttctgaaaggacttgagttttctgtaaacgcttatatgctaaatgggttttatatagcctccccgata atgtttaaaggcgaaactgatattcatagcgggcacgcaatggacaaattcaggtatattagcaagctaaaaaataaaagcttaaatgaaaaaataaa tgccatattaagtaaaattagtaataccaatatctttaatggatggatagaggttgagtttataaaaacacatcaggatttggtagttatagaaattaatgc gaggtataatggaacaatcagagctactggatatgcttgtaatgaaaatttatatcaattggatttggaaagtaaaatttataataaattttcttcacaatta aatcatgaaaacgaagtcattgagatgccaattcatttgaaacttgaaacaggtttaaaagaatttggtttcgtccaaaaaatgaaatctcgaaaaacca acacaggaagggccacaatatggggtgaagatcaatgtgaactattagagaggatcaaaaatactgaattagaaatatactcagaaagaattattcat ggcataaatgagagtaaggagttattcgaaaaatatatttaa (SEQ ID NO: 24)

[0219] HA-Tag_HvrF_AT_codon-optimized (HA-tag in bold and the 5 ’ and 3 ’ extensions for Loop assembly underlined) cactctgtggtctcaaatgtatccgtacgacgtcccggactacgcgtacccttacgacgtaccggactacgcatacccgtatgatgttccggat tatgcgatgaagtctgaaaagtttgatggtctggctgataattatgacaagtatcgaccaagatatccggcgatactatttaaggagatccatgattgg atgcaacccagcgcaaaaaatatctatgacattggggccggaacgggtattgccatagaaggaatgactagggttaccggcaagcactatgatttta ctgctattgatatctccgaggatatgatcaagaagggaagagagaagcttcctggaacaacatgggtgaaaggaaaagctgaagatattttgtcgga taaaagtaggatcgatgttatcatggctgctcagagttttcaatggatggatcgagctaaaactttagaagtatctattaaatctctcaacaagggcgga gtctttgcagtccttcaaaacaaccgcgactatagaaacaatgagatgcttaacaaatacgaaggtttgttggagaaattctcacctgggtacagccgt cattacagagattacgattatgagaatgaaatcacgaatgtgtttaagctcccaatagcgaacttcaaaaaagtagttaccggttggacaatggaaatg atcagtgaggacttcttcggttttatatcaagctcaactcaggttcagcgggcaattgaaaatgatagaaatggtttctggaaagaaatagagattttaat agatgaacattctgtgggcgggaagatttcgattgactacatttccgagctgttcattgcaaagaaacgtgacgattcttgagctttgagaccacgaag tg (SEQ ID NO: 25)

[0220] HA-Tag HvrF AT codon-optimized (HA-tag in bold) tatccgtacgacgtcccggactacgcgtacccttacgacgtaccggactacgcatacccgtatgatgttccggattatgcgatgaagtctgaa aagtttgatggtctggctgataattatgacaagtatcgaccaagatatccggcgatactatttaaggagatccatgattggatgcaacccagcgcaaa aaatatctatgacattggggccggaacgggtattgccatagaaggaatgactagggttaccggcaagcactatgattttactgctattgatatctccga ggatatgatcaagaagggaagagagaagcttcctggaacaacatgggtgaaaggaaaagctgaagatattttgtcggataaaagtaggatcgatgt tatcatggctgctcagagttttcaatggatggatcgagctaaaactttagaagtatctattaaatctctcaacaagggcggagtctttgcagtccttcaaa acaaccgcgactatagaaacaatgagatgcttaacaaatacgaaggtttgttggagaaattctcacctgggtacagccgtcattacagagattacgat tatgagaatgaaatcacgaatgtgtttaagctcccaatagcgaacttcaaaaaagtagttaccggttggacaatggaaatgatcagtgaggacttcttc ggttttatatcaagctcaactcaggttcagcgggcaattgaaaatgatagaaatggtttctggaaagaaatagagattttaatagatgaacattctgtgg gcgggaagatttcgattgactacatttccgagctgttcattgcaaagaaacgtgacgattcttga (SEQ ID NO: 26)

[0221] Codon optimized hvrF gene atgaagtctgaaaagtttgatggtctggctgataattatgacaagtatcgaccaagatatccggcgatactatttaaggagatccatgattggatgcaa cccagcgcaaaaaatatctatgacattggggccggaacgggtattgccatagaaggaatgactagggttaccggcaagcactatgattttactgcta ttgatatctccgaggatatgatcaagaagggaagagagaagcttcctggaacaacatgggtgaaaggaaaagctgaagatattttgtcggataaaa gtaggatcgatgttatcatggctgctcagagttttcaatggatggatcgagctaaaactttagaagtatctattaaatctctcaacaagggcggagtcttt gcagtccttcaaaacaaccgcgactatagaaacaatgagatgcttaacaaatacgaaggtttgttggagaaattctcacctgggtacagccgtcatta cagagattacgattatgagaatgaaatcacgaatgtgtttaagctcccaatagcgaacttcaaaaaagtagttaccggttggacaatggaaatgatca gtgaggacttcttcggttttatatcaagctcaactcaggttcagcgggcaattgaaaatgatagaaatggtttctggaaagaaatagagattttaataga tgaacattctgtgggcgggaagatttcgattgactacatttccgagctgttcattgcaaagaaacgtgacgattcttga (SEQ ID NO: 27) pTAC promoter gcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccgggttttctttgcctcacgatcgcccccaaaaca cataaccaattgtatttattgaaaaataaatagatacaactcactaaacatagcaattcagatccggtccagtaatgacctcagaactccatctggatttg ttcagaacgctcggttgccgccgggcgttttttattggtgagaatcgcagcaacttgtcgcgccaatcgagccatgtcgtcgtcaacgaccccccatt caagaacagcaagcagcattgagaactttggaatccagtccctcttccacctgctgatccggatcgatcccgttatcgactgcacggtgcaccaatg cttctggcgtcaggcagccatcggaagctgtggtatggctgtgcaggtcgtaaatcactgcataattcgtgtcgctcaaggcgcactcccgttctgga taatgttttttgcgccgacatcataacggttctggcaaatattctgaaatgagctgttgacaattaatcatcggctcgtataatgtgtggaattgtgagcg gataacaatttcacacaggaaacagaattgatccggccaagcttgtaagaaggagatatacat (SEQ ID NO: 28)

[0222] CaMV 35 S promoter VI ggatcctctagagtcccccgtgttctctccaaatgaaatgaacttccttatatagaggaagggtcttgcgaaggatagtgggattgtgcgtcatccctta cgtcagtggagattccagataggcctaacgcttgtccaagatctattcaggatatcacatcaatccacttgctttgaagacgtggttggaacgtcttcttt ttccacgatgctcctcgtgggtgggggtccatctttgggaccactgtcggcagaggcatcttcaacgatggcctttcctttatcgcaatgatggcatttg taggagccaccttccttttccactatcttcacaataaagtgacagatagctgggcaatggaatccgaggaggtttccggatattaccctttgttgaaaag tctcaattgccctttggtcttctgagactgtatctttgatatttttggagtagacaagtgtgtcgtgctccaccatgttgacga (SEQ ID NO: 29)

[0223] CaMV 35 S Promoter V2 ggatcctctagagtcccccgtgttctctccaaatgaaatgaacttccttatatagaggaagggtcttgcgaaggatagtgggattgtgcgtcatccctta cgtcagtggagatatcacatcaatccacttgctttgaagacgtggttggaacgtcttctttttccacgatgctcctcgtgggtgggggtccatctttggg accactgtcggcagaggcatcttcaacgatggcctttcctttatcgcaatgatggcatttgtaggagccaccttccttttccactatcttcacaataaagt gacagatagctgggcaatggaatccgaggaggtttccggatattaccctttgttgaaaagtctcaattgccctttggtcttctgagactgtatctttgata tttttggagtagacaagtgtgtcgtgctccaccatgttgacga (SEQ ID NO: 30)

[0224] Plasmid JV477 atacttgagaccggatcctgacaggatatattggcgggtaaacctaagagaaaagagcgtttattagaataatcggatatttaaaagggcgtgaaaa ggtttatccgttcgtccatttgtatgtgcatgccaaccacagggttcccctcgggatcaaagtactttgatccaacccctccgctgctatagtgcagtcg gcttctgacgttcagtgcagccgtcatctgaaaacgacatgtcgcacaagtcctaagttacgcgacaggctgccgccctgcccttttcctggcgttttc ttgtcgcgtgttttagtcgcataaagtagaatacttgcgactagaaccggagacattacgccatgaacaagagcgccgccgctggcctgctgggcta tgcccgcgtcagcaccgacgaccaggacttgaccaaccaacgggccgaactgcacgcggccggctgcaccaagctgttttccgagaagatcac cggcaccaggcgcgaccgcccggagctggccaggatgcttgaccacctacgccctggcgacgttgtgacagtgaccaggctagaccgcctggc ccgcagcacccgcgacctactggacattgccgagcgcatccaggaggccggcgcgggcctgcgtagcctggcagagccgtgggccgacacc accacgccggccggccgcatggtgttgaccgtgttcgccggcattgccgagttcgagcgttccctaatcatcgaccgcacccggagcgggcgcg aggccgccaaggcccgaggcgtgaagtttggcccccgccctaccctcaccccggcacagatcgcgcacgcccgcgagctgatcgaccaggaa ggccgcaccgtgaaagaggcggctgcactgcttggcgtgcatcgctcgaccctgtaccgcgcacttgagcgcagcgaggaagtgacgcccacc gaggccaggcggcgcggtgccttccgtgaggacgcattgaccgaggccgacgccctggcggccgccgagaatgaacgccaagaggaacaa gcatgaaaccgcaccaggacggccaggacgaaccgtttttcattaccgaagagatcgaggcggagatgatcgcggccgggtacgtgttcgagcc gcccgcgcacctctcaaccgtgcggctgcatgaaatcctggccggtttgtctgatgccaagctggcggcctggccggccagcttggccgctgaag aaaccgagcgccgccgtctaaaaaggtgatgtgtatttgagtaaaacagcttgcgtcatgcggtcgctgcgtatatgatgcgatgagtaaataaaca aatacgcaaggggaacgcatgaaggttatcgctgtacttaaccagaaaggcgggtcaggcaagacgaccatcgcaacccatctagcccgcgccc tgcaactcgccggggccgatgttctgttagtcgattccgatccccagggcagtgcccgcgattgggcggccgtgcgggaagatcaaccgctaacc gttgtcggcatcgaccgcccgacgattgaccgcgacgtgaaggccatcggccggcgcgacttcgtagtgatcgacggagcgccccaggcggc ggactggctgtgtccgcgatcaaggcagccgactcgtgctgatccggtgcagccaagcccttacgacatatgggccaccgccgacctggtgg agctggttaagcagcgcattgaggtcacggatggaaggctacaagcggcctttgtcgtgtcgcgggcgatcaaaggcacgcgcatcggcggtga ggttgccgaggcgctggccgggtacgagctgcccattcttgagtcccgtatcacgcagcgcgtgagctacccaggcactgccgccgccggcaca accgttctgaatcagaacccgagggcgacgctgcccgcgaggtccaggcgctggccgctgaaattaaatcaaaactcatttgagtaatgaggta aagagaaaatgagcaaaagcacaaacacgctaagtgccggccgtccgagcgcacgcagcagcaaggctgcaacgttggccagcctggcagac acgccagccatgaagcgggtcaactttcagttgccggcggaggatcacaccaagctgaagatgtacgcggtacgccaaggcaagaccataccg agctgctatctgaatacatcgcgcagctaccagagtaaatgagcaaatgaataaatgagtagatgaattttagcggctaaaggaggcggcatggaa aatcaagaacaaccaggcaccgacgccgtggaatgccccatgtgtggaggaacgggcggttggccaggcgtaagcggctgggttgcctgccg gccctgcaatggcactggaacccccaagcccgaggaatcggcgtgagcggtcgcaaaccatccggcccggtacaaatcggcgcggcgctggg tgatgacctggtggagaagtgaaggccgcgcaggccgcccagcggcaacgcatcgaggcagaagcacgccccggtgaatcgtggcaagcg gccgctgatcgaatccgcaaagaatcccggcaaccgccggcagccggtgcgccgtcgataggaagccgcccaagggcgacgagcaaccag attttttcgttccgatgctctatgacgtgggcacccgcgatagtcgcagcatcatggacgtggccgttttccgtctgtcgaagcgtgaccgacgagct ggcgaggtgatccgctacgagcttccagacgggcacgtagaggtttccgcagggccggccggcatggccagtgtgtgggattacgacctggtac tgatggcggtttcccatctaaccgaatccatgaaccgataccgggaagggaagggagacaagcccggccgcgtgttccgtccacacgttgcgga cgtactcaagttctgccggcgagccgatggcggaaagcagaaagacgacctggtagaaacctgcattcggttaaacaccacgcacgttgccatgc agcgtacgaagaaggccaagaacggccgcctggtgacggtatccgagggtgaagcctgattagccgctacaagatcgtaaagagcgaaaccg ggcggccggagtacatcgagatcgagctagctgattggatgtaccgcgagatcacagaaggcaagaacccggacgtgctgacggttcaccccg atactttttgatcgatcccggcatcggccgttttctctaccgcctggcacgccgcgccgcaggcaaggcagaagccagatggttgttcaagacgat ctacgaacgcagtggcagcgccggagagtcaagaagtctgtttcaccgtgcgcaagctgatcgggtcaaatgacctgccggagtacgatttgaa ggaggaggcggggcaggctggcccgatcctagtcatgcgctaccgcaacctgatcgagggcgaagcatccgccggttcctaatgtacggagca gatgctagggcaaattgccctagcaggggaaaaaggtcgaaaaggactctttcctgtggatagcacgtacattgggaacccaaagccgtacattg ggaaccggaacccgtacattgggaacccaaagccgtacattgggaaccggtcacacatgtaagtgactgatataaaagagaaaaaaggcgattttt ccgcctaaaactctttaaaactataaaactcttaaaacccgcctggcctgtgcataactgtctggccagcgcacagccgaagtgctgcaaaaagcg cctacccttcggtcgctgcgctccctacgccccgccgctcgcgtcggcctatcgcggccgctggccgctcaaaaatggctggcctacggccagg caatctaccagggcgcggacaagccgcgccgtcgccactcgaccgccggcgcccacatcaaggcaccctgcctcgcgcgtttcggtgatgacg gtgaaaacctctgacacatgcagctcccggtgacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagc gggtgtggcgggtgtcggggcgcagccatgacccagtcacgtagcgatagcggagtgtatactggctaactatgcggcatcagagcagatgt actgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgctcatccgctcctcgctcactgactc gctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaag aacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatc acaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcct gttccgaccctgccgctaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagtcggtgt aggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtctgagtccaacccggt aagacacgacttatcgccactggcagcagccactggtaacaggatagcagagcgaggtatgtaggcggtgctacagagtctgaagtggtggc ctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaa caaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacgggg tctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgcattctaggtgatatttgccgactacctggtgatctcgcctttcacgtagt ggacaaattcttccaactgatctgcgcgcgaggccaagcgatcttcttctgtccaagataagcctgtctagcttcaagtatgacgggctgatactgg gccggcaggcgctccatgcccagtcggcagcgacatccttcggcgcgattttgccggtactgcgctgtaccaaatgcgggacaacgtaagcac tacatttcgctcatcaccagcccagtcgggcggcgagtccatagcgttaaggtttcatttagcgcctcaaatagatcctgttcaggaaccggatcaaa gagttcctccgccgctggacctaccaaggcaacgctatgtctctgcttttgtcagcaagatagccagatcaatgtcgatcgtggctggctcgaaga tacctgcaagaatgtcatgcgctgccatctccaaattgcagttcgcgcttagctggataacgccacggaatgatgtcgtcgtgcacaacaatggtg actctacagcgcggagaatctcgctctctccaggggaagccgaagtttccaaaaggtcgtgatcaaagctcgccgcgttgtttcatcaagccttac ggtcaccgtaaccagcaaatcaatatcactgtgtggcttcaggccgccatccactgcggagccgtacaaatgtacggccagcaacgtcggttcgag atggcgctcgatgacgccaactacctctgatagtgagtcgatacttcggcgatcaccgctccctcataatgtttaactttgttttagggcgactgccct gctgcgtaacatcgttgctgctccataacatcaaacatcgacccacggcgtaacgcgcttgctgctggatgcccgaggcatagactgtaccccaaa aaaacagtcataacaagccatgaaaaccgccactgcgccgttaccaccgctgcgttcggtcaaggttctggaccagttgcgtgagcgcatacgcta ctgcatacagctacgaaccgaacaggctatgtccactgggtcgtgcctcatccgtttccacggtgtgcgtcacccggcaaccttgggtagca gcgaagtcgaggcatttctgtcctggctggaacagaactattatttcctcctcttttctacagtatttaaagataccccaagaagctaattataacaaga cgaactccaatcactgtccttgcattctaaaaccttaaataccagaaaacagctttttcaaagttgttttcaaagtggcgtataacatagtatcgacgg agccgattttgaaaccgcggtgatcacaggcagcaacgctctgtcatcgttacaatcaacatgctaccctccgcgagatcatccgtgtttcaaacccg gcagctagtgccgttcttccgaatagcatcggtaacatgagcaaagtctgccgcctacaacggctctcccgctgacgccgtcccggactgatgg gctgcctgtatcgagtggtgattttgtgccgagctgccggtcggggagctgttggctggctggtggcaggatatattgtggtgtaaacataacggatc cggtctcaggagaatgaggagagcggagaattaagggagtcacgttatgacccccgccgatgacgcgggacaagccgttttacgtttggaactga cagaaccgcaacgttgaaggagccactcagccgcgggtttctggagtttaatgagctaagcacatacgtcagaaaccattattgcgcgttcaaaagt cgcctaaggtcactatcagctagcaaatatttcttgtcaaaaatgctccactgacgttccataaattcccctcggtatccaattagagtctcatattcactc tcatactgtatttttacaacaattaccaacaacaacaaacaacaaacaacattacaattactatttacaattacaatgagcccagaacgacgcccggcc gacatccgccgtgccaccgaggcggacatgccggcggtctgcaccatcgtcaaccactacatcgagacaagcacggtcaacttccgtaccgagc cgcaggaaccgcaggagtggacggacgacctcgtccgtctgcgggagcgctatccctggctcgtcgccgaggtggacggcgaggtcgccggc atcgcctacgcgggcccctggaaggcacgcaacgcctacgactggacggccgagtcaaccgtgtacgtatccccccgccaccagcggacggg actgggctccacgctctacacccacctgctgaagtccctggaggcacagggcttcaagagcgtggtcgctgtcatcgggctgcccaacgacccga gcgtgcgcatgcacgaggcgctcggatatgccccccgcggcatgctgcgggcggccggcttcaagcacgggaactggcatgacgtgggtttct ggcagctggacttcagcctgccggtaccgccccgtccggtcctgcccgtcaccgagatttgagcttgtcaagcagatcgttcaaacatttggcaata aagtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattacgttaagcatgtaataattaacatgtaatgcatgacgtt atttatgagatgggtttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcgcaaactaggataaattatcgcgc gcggtgtcatctatgttactagatcgacgctagcaaggaggtcaacatggtggagcacgacactctggtctactccaaaaatgtcaaagatacagtct cagaagatcaaagggctattgagacttttcaacaaaggataatttcgggaaacctcctcggattccattgcccagctatctgtcacttcatcgaaagga cagtagaaaaggaaggtggctcctacaaatgccatcattgcgataaaggaaaggctatcattcaagatctctctgccgacagtggtcccaaagatg gacccccacccacgaggagcatcgtggaaaaagaagaggttccaaccacgtctacaaagcaagtggattgatgtgacatctccactgacgtaagg gatgacgcacaatcccactatccttcgcaagacccttcctctatataaggaagttcatttcatttggagaggacacgcaatgtatccgtacgacgtccc ggactacgcgtacccttacgacgtaccggactacgcatacccgtatgatgttccggattatgcgatgaagtctgaaaagtttgatggtctggctgata attatgacaagtatcgaccaagatatccggcgatactatttaaggagatccatgattggatgcaacccagcgcaaaaaatatctatgacattggggcc ggaacgggtattgccatagaaggaatgactagggttaccggcaagcactatgattttactgctattgatatctccgaggatatgatcaagaagggaa gagagaagcttcctggaacaacatgggtgaaaggaaaagctgaagatattttgtcggataaaagtaggatcgatgttatcatggctgctcagagtttt caatggatggatcgagctaaaactttagaagtatctattaaatctctcaacaagggcggagtctttgcagtccttcaaaacaaccgcgactatagaaac aatgagatgcttaacaaatacgaaggtttgttggagaaattctcacctgggtacagccgtcattacagagattacgattatgagaatgaaatcacgaat gtgtttaagctcccaatagcgaacttcaaaaaagtagttaccggttggacaatggaaatgatcagtgaggacttcttcggttttatatcaagctcaactc aggttcagcgggcaattgaaaatgatagaaatggtttctggaaagaaatagagattttaatagatgaacattctgtgggcgggaagatttcgattgact acatttccgagctgttcattgcaaagaaacgtgacgattcttgagcttctctagctagagtcgatcgacaagctcgagtttctccataataatgtgtgagt agttcccagataagggaattagggttcctatagggtttcgctcatgtgttgagcatataagaaacccttagtatgtatttgtatttgtaaaatacttctatca ataaaatttctaattcctaaaaccaaaatccagtactaaaatccagatcgctatacaggagcaggttcggattattattgttagtatgggtgttaagaaaa gccggagaggtatctgtcaccatactagctgcaggacgtgataaagaaactcgtgtagagcccggatgcaaccatctgtcccaaccattacattcca agaagatgtagagtccacgtggctgttctgtacgcgtgaactattcttgtatctctttctctgcgctaggt (SEQ ID NO: 31)

[0225] All publications, patents, and patent documents cited herein are incorporated by reference as though individually incorporated by reference, and in particular, PCT Patent

[0226] Publication No. WO 20233 / 051527 to Metcalf et al. No limitations inconsistent with this disclosure are to be understood therefrom. The invention has been described with reference to various specific and preferred embodiments and techniques. However, many variations and modifications may be made while remaining within the spirit and scope of the invention.

[0227] While specific embodiments have been described above with reference to the disclosed embodiments and examples, such embodiments are only illustrative and do not limit the scope of the invention. Changes and modifications can be made in accordance with ordinary skill in the art without departing from the invention in its broader aspects as defined in the following claims.

Claims

What is claimed is:

1. A nucleic acid molecule comprising a constitutive promoter sequence that is functional in plants and a nucleotide sequence operably linked to the constitutive promoter sequence, wherein the nucleotide sequence encodes an HvrF protein of Pantoea ananatis comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 11.

2. The nucleic acid molecule of claim 1, wherein the HvrF protein of Pantoea ananatis comprises the amino acid sequence of SEQ ID NO: 11.

3. The nucleic acid molecule of claim 1, wherein the nucleotide sequence comprises an hvrF gene of Pantoea ananatis having at least 95% sequence identity to SEQ ID NO: 12; or a codon optimized hvrF gene of Pantoea ananatis having at least 95% sequence identity to SEQ ID NO: 27.

4. The nucleic acid molecule of claim 3, wherein the nucleotide sequence comprises an hvrF gene of Pantoea ananatis according to SEQ ID NO: 12; or a codon optimized hvrF gene of Pantoea ananatis according to SEQ ID NO: 27.

5. The nucleic acid molecule of claim 1, wherein the nucleotide sequence comprises SEQ ID NO: 26.

6. The nucleic acid molecule of claim 1, wherein the constitutive promoter sequence comprises a plant virus promoter sequence.

7. The nucleic acid molecule of claim 6, wherein the constitutive promoter sequence comprises a Cauliflower Mosaic Virus 35S (35S) promoter.

8. The nucleic acid molecule of claim 1, wherein the promoter sequence comprises one of p35s, pUbi, pActinl, pNos, pRBCS, pAP3, pGSSP, pPG1090, pPYKIO, pCAB, pRD29A, and pER.

9. The nucleic acid molecule of claim 1, further comprising one or more enhancer elements operable in a plant.

10. A transgenic plant resistant to Pantoea ananatis infection comprising the nucleic acid molecule of claim 1.

11. A pantaphos-resistant plant cell comprising a recombinant nucleic acid molecule, the recombinant nucleic acid molecule comprising: i) a constitutive promoter sequence that is functional in plant cells; and ii) a nucleotide sequence encoding a polypeptide comprising a 5-adenosylmethione (SAM)-dependent methyltransferase, wherein the (SAM)-dependent methyltransferase comprises an HvrF protein of Pantoea ananatis wherein the constitutive promoter sequence is heterologous with respect to the nucleotide sequence and is configured to cause sufficient expression of the polypeptide in the plant cell comprising the recombinant nucleic acid molecule to confer pantaphos resistance to the plant cells.

12. The pantaphos-resistant plant cell of claim 11, wherein the HvrF protein of Pantoea ananatis comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 11.

13. The pantaphos-resistant plant cell of claim 11, wherein the nucleotide sequence comprises a hvrF gene of Pantoea ananatis having at least 95% sequence identity to SEQ ID NO: 12; or a codon optimized hvrF gene of Pantoea ananatis having at least 95% sequence identity to SEQ ID NO: 27.

14. The pantaphos-resistant plant cell of claim 11, wherein the constitutive promoter sequence comprises a plant virus promoter sequence.

15. The pantaphos-resistant plant cell of claim 14, wherein the promoter sequence comprises a Cauliflower Mosaic Virus 35S (35S) promoter.

16. The pantaphos-resistant plant cell of claim 11, wherein the plant cell is a cell of alfalfa, Arabidopsis, banana, barley, bean, broccoli, cabbage, cannabis, carrot, cassava, castor, cauliflower, celery, chickpea, Chinese cabbage, coconut, coffee, com, clover, cotton, cucumber, Douglas fir, eggplant, eucalyptus, flax, garlic, grape, hops, leek, lettuce, millets,oat, olive, onion, palm, pasture grass, pea, peanut, pepper, potato, radish, rapeseed, rice, rye, sorghum, soybean, spinach, squash, strawberry, sugar beet, sugarcane, sunflower, sweet corn, sweet gum, sweet potato, switchgrass, tea, tobacco, tomato, triticale, grass, watermelon, or wheat.

17. A method for producing pantaphos-resistant plant cells comprising:(a) transforming plant cells using a recombinant DNA construct to provide transformed plant cell; and(b) regenerating pantaphos-resistant plants from the transformed plant cells, wherein the recombinant DNA construct comprises: i) a constitutive promoter sequence that is functional in the plant cells; and ii) a coding sequence that encodes a polypeptide comprising an 5-adenosylmethione (SAM)-dependent methyltransferase, wherein the promoter sequence is heterologous with respect to the coding sequence and is configured to cause sufficient expression of the polypeptide in the transformed plant cell in order to confer resistance to pantaphos.

18. The method of claim 17, wherein the wherein the (SAM)-dependent methyltransferase comprises an HvrF protein of Pantoea ananatis.

19. The method of claim 18, wherein the HvrF protein of Pantoea ananatis comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 11; or wherein the HvrF protein of Pantoea ananatis comprises an amino acid sequence as set forth in SEQ ID NO: 11.

20. A transgenic plant produced according to the method of claim 17.

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