Compositions and Methods for Weed Control

Abstract

This disclosure particularly relates to methods and compositions for weed control, e.g., a method for selectively controlling weeds at a site by applying to the site a pesticidal composition comprising an SDPS-inhibiting herbicide in a weed controlling amount, wherein the crop plants are modified to comprise an SDPS that confers upon the crop plants tolerance to the herbicide that inhibits SPDS. The compositions also particularly include recombinant polynucleotides suitable for use in the methods.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims priority to U.S. Patent Application No. 62 / 850,248, filed May 20, 2019, the entire content of which is incorporated herein by reference.

[0003] Sequence Listing

[0004] This application is accompanied by a sequence listing entitled 81880 - WO - REG - ORG - P - 1_SEQ LIST_ST25.txt, created on March 15, 2020, and having a size of approximately 1,533 kb. This sequence listing is incorporated herein by reference in its entirety. This sequence listing is submitted with this application via EFS - Web and complies with 37 C.F.R.§1.824(a)(2)-(6) and (b). Technical Field

[0005] This disclosure generally relates to compositions and methods for controlling weeds in crops, including compositions and methods for conferring herbicide tolerance to solanesyl diphosphate synthase (SDPS) - inhibitory herbicides. Background Art

[0006] The use of herbicide - tolerant transgenes to engineer crops to be tolerant to herbicides and thereby expand the use of certain herbicides into additional crops has been widely reported. Herbicide tolerance can be conferred simply by overexpression of a gene encoding a herbicide target protein and / or by expression of a transgene encoding an altered and thus herbicide - insensitive target site (e.g., in the case of glyphosate tolerance, glyphosate - insensitive 5 - enolpyruvylshikimate - 3 - phosphate synthase) and / or by expression of an enzyme that metabolizes the herbicide into an inactive form (e.g., in the case of glufosinate tolerance, phosphinothricin N - acetyltransferase). Similarly, in - situ mutagenesis (directed or otherwise) has been used to mutate, for example, the acetolactate synthase (ALS) or acetyl - CoA carboxylase (ACCase) herbicide target genes to produce mutant herbicide - tolerant crop lines. In addition to early examples of tolerance to the non - selective herbicides glyphosate and glufosinate, there are now numerous reports of transgenes and methods for conferring herbicide tolerance to other herbicides, such as those that act by inhibiting: 4 - hydroxyphenylpyruvate dioxygenase (HPPD), protoporphyrinogen oxidase (PPO), and several auxinic herbicides, particularly dicamba and 2,4 - D.

[0007] The applicant still needs additional compositions and methods to confer herbicide tolerance to other herbicides, for example, to provide additional application flexibility for growers and to provide additional tolerance management options. More particularly, the applicant needs compositions and methods to confer herbicide tolerance to a herbicidal compound that exerts its herbicidal effect by inhibiting solanesyl diphosphate synthase.

[0008] In higher plants, solanesyl diphosphate synthase is involved in the biosynthesis of ubiquinone and plastoquinone. The role of these enzymes is to provide solanesyl diphosphate esters, which serve as precursors for the side chains of ubiquinone and plastoquinone. In Arabidopsis thaliana, SDPS1 has been characterized as the enzyme responsible for producing the precursor of the ubiquinone side chain, while the plastids targeting SDPS2 are responsible for producing the precursor of the plastoquinone side chain. The plastoquinone biosynthesis pathway has previously been targeted by herbicides that inhibit HPPD or HST enzymes to produce a characteristic bleaching phenotype. The present disclosure is particularly based on the applicant's work on certain herbicidal compounds that exert their herbicidal effect by inhibiting solanesyl diphosphate synthase. Summary of the Invention

[0009] The present disclosure thus particularly relates to compositions and methods for selectively controlling weeds at a site. The present disclosure further relates to recombinant DNA technology and particularly to the production of transgenic plants that exhibit significant resistance or significant tolerance to herbicides when compared to non-transgenic plants. The present disclosure further relates to DNA editing technology and particularly to the production of DNA-edited plants that exhibit significant resistance or significant tolerance to herbicides when compared to non-transgenic plants.

[0010] Tolerance to solanesyl diphosphate synthase (SDPS)-inhibiting herbicides in plants has not been reported because it has not previously been considered a target site for certain classes of herbicidal compounds. Transgenic plants overexpressing the solanesyl diphosphate synthase gene or expressing or overexpressing its variants are tolerant to the herbicide. Thus, the present invention thus particularly provides an opportunity to utilize solanesyl diphosphate synthase-inhibiting herbicides in a broader agricultural context.

[0011] In one example, the present disclosure includes a method for selectively controlling weeds at a site comprising crop plants and weeds. The method comprises applying to the site a pest control amount of a pesticidal composition comprising a solanesyl diphosphate synthase-inhibiting herbicide, wherein the crop plants are modified to comprise a solanesyl diphosphate synthase that provides the crop plants with tolerance to the solanesyl diphosphate synthase-inhibiting herbicide.

[0012] The crop plants can be modified with a recombinant polynucleotide that provides a solanesyl diphosphate synthase that provides the crop plants with tolerance to a solanesyl diphosphate synthase-inhibiting herbicide.

[0013] In some instances, geranylgeranyl diphosphate synthase can be derived from Arabidopsis thaliana, Triticum aestivum (Wheat), Hordeum vulgare (Barley), Oryza sativa (Rice), Zea mays (Maize), Glycine max (Soybean), Chlamydomonas reinhardtii or Chlorella fusca. In some instances, geranylgeranyl diphosphate synthase can be selected from: SDPSs of SEQ ID NO: 1-18, 45-349 and 663-665; or "modified" SDPSs that have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequences shown in SEQ ID NO: 1-18, 45-349 and 663-665, or SDPSs having a motif selected from the group consisting of 655-662; or SDPSs having at least one mutation at a position corresponding to one of the following amino acid positions in SEQ ID NO: 5: F240L, F227L, F229L, F247L, L120A, L120R, L120W, L123A, L123C, L123D, L123N, L123S, L123W, E127A, E127G, E127K, E127Y, N128L, N128P, V130D, V130K, L131A, L131E, L131M, L131P, A134V, F139D, F139K, F139N, F139R, F139T, P148I, P148L, P148M, P148T, P148V, V151E, V151F, V151I, V151M, V151N, L174F, L174T, A175I, A175P, A175S, E176A, E176D, E176H, E176K, E176N, E176P, E176Y, I177A, I177C, I177F, I177L, I177M, I177S, I177T, I177Y, I178G, I178Q, I178W, E179I, M180I, M180Q, M180S, M180Y, M180W, I181M, I181N, A184G, A184S, A184T, T183C, T183Q, S185A, S185T, S185G, I187E, I187F, I187T, I187V, H188F, H188I, H188L, H188M,H188V, V191A, V191T, I204A, I204F, I204G, I204H, I204K, I204Q, I204R, I204S, I204T, Y208A, Y208D, Y208E, Y208H, Y208I, Y208K, Y208L, Y208M, Y208N, Y208Q, Y208R, Y208S, Y208T, Y208V, G209N, T210Y, R211D, R211E, R211N, R211T, R211V, L215I, L215M, A216T, F219A, M220I, M220C, F221W, A222G, A222M, A222S, Q223A, Q223E, Q223F, Q223G, Q223H, Q223I, Q223K, Q223L, Q223M, Q223R, Q223Y, S224F, S224I, S224M, S224N, S224Q, S224T, S224V, S225C, S225F, S225H, S225I, S225K, S225M, S225N, S225Q, S225T, S225V, S225Y, W226A, W226C, W226E, W226I, W226L, W226Q, W226R, W226T, W226V, F227D, F227L, F227M, F227R, F227V, F227W, L228C, L228I, L228M, L228T, L228V, A229H, A229I, A229L, A229M, A229N, A229T, N230E, N230R, E235G, K238G, K238N, K238S, L239A, L239R, I240A, I240C, I240W, S241A, S241H, S241N, S241T, V243A, V243G, V243N, V243Q, V243S, I244A, I244F, I244G, I244H, I244K, I244L, I244M, I244N, I244P, I244Q, I244S, I244V, I244Y, K245F, K245H, K245M, K245N, K245W, D246E, D246M, D246N, D246Q, D246S, D246T, D246Y, F247E, F247L, F247M, F247N, F247V, A248P, S249A, S249E, S249F, S249G, S249K, S249L, S249N, S249Q, S249T, S249V, S249Y, G250A, I252L, I252M, I252V, K253L, A255TA255W, S256N, T257E, T257G, T257H, T257M, T257Q, T257W, Y274D, Y274G, Y274L, Y274M, Y274Q, T276S, L279F, I280W, I280F, A282G, A282H, A282K, A282N, A282R, S283C, S283F, S283I, S283M, S283T, S283W, R306F, R306H, R306L, R306N, L310G, G309A, G309F, G309M, G309S, L310D, L310E, L310F, L310H, L310N, L310Q, L310W, L310Y, F312C, F312I, F312L, F312M, F312V, Q313A, Q313C, Q313D, Q313S, and Q313T. In some instances, the geranylgeranyl diphosphate synthase can contain a mutation corresponding to the F240L mutation or an equivalent numbering as shown in SEQ ID NO. 3 or 5. In some instances, the geranylgeranyl diphosphate synthase is provided by editing an endogenous geranylgeranyl diphosphate synthase, for example, to achieve any of the above mutations.

[0014] When editing an endogenous SDPS, for example, it can be edited in a variety of ways. For example, the editing can include at least one of the following: (a) the production of one or more alternative spliced transcripts of the polynucleotide encoding the geranylgeranyl diphosphate synthase; (b) the deletion of one or more nucleotides in the polynucleotide encoding the geranylgeranyl diphosphate synthase; (c) a frameshift mutation of one or more exons of the polynucleotide encoding the geranylgeranyl diphosphate synthase; (d) the substitution of one or more nucleotides in the polynucleotide encoding the geranylgeranyl diphosphate synthase; and (e) the deletion or modification of one or more nucleotides of a regulatory element operably linked to the expression of the geranylgeranyl diphosphate synthase, where the regulatory element includes at least one of a promoter, an intron, a 3' UTR, and a terminator. The target of the editing can vary and can include, for example, at least one edit to encode a mutation corresponding to one of the amino acid positions in Table 1.

[0015] A variety of editing constructs can be used according to the present disclosure. For example, constructs that can be used include nucleic acids and optionally at least one guide RNA, the nucleic acid encoding a DNA-modifying enzyme selected from the group consisting of site-specific nucleases, the site-specific nucleases selected from the group consisting of: meganucleases (MN), zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), Cas nucleases (e.g., Cas9 or Cas12), Cpf1 (sometimes also referred to as Cas12) nucleases, dCas9-FokI, dCpf1-FokI, chimeric Cas9-cytidine deaminase, chimeric Cas9-adenine deaminase, chimeric FEN1-FokI and Mega-TAL, nickase Cas9 (nCas9), chimeric dCas9 non-FokI nucleases and dCpf1 non-FokI nucleases; the at least one guide RNA corresponding to a target sequence selected from the sequences encoding the amino acids of Table 1. In many instances, the DNA-modifying enzyme is a site-specific nuclease, the site-specific nuclease selected from the group consisting of: Cas9 nuclease, Cfp1 nuclease, dCas9-FokI, dCpf1-FokI, chimeric Cas9-cytidine deaminase, chimeric Cas9-adenine deaminase, chimeric FEN1-FokI and Mega-TAL, nickase Cas9 (nCas9), chimeric dCas9 non-FokI nucleases and dCpf1 non-FokI nucleases, and will include a guide RNA.

[0016] The target sequence can vary and can include sequences 15-25 nucleotides in length (including sequences encoding the amino acids of Table 1, e.g., sequences of 3 nucleotides).

[0017] In many instances of the crop plants and methods disclosed herein, the crop plants can contain additional recombinant polynucleotides encoding additional herbicide-tolerant enzymes. For example, the additional herbicide-tolerant enzymes can be selected from the group consisting of: 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), HST, glyphosate acetyltransferase (GAT), cytochrome P450, phosphinothricin acetyltransferase (PAT), acetolactate synthase (ALS), protoporphyrinogen oxidase (PPGO), hydroxyphenylpyruvate dioxygenase (HPPD), and dicamba-degrading enzyme.

[0018] In addition, it should be clear that the pesticidal composition can contain additional herbicides. For example, one or more additional herbicides can include glyphosate or its salts, glufosinate or its salts, chloroacetanilide, alachlor, acetochlor, metolachlor, S - metolachlor; photosystem II inhibitors, triazines, ametryn, atrazine, cyanazine, terbuthylazine, triazinon, hexazinone, simazine, ureas, chlorotoluron, diuron, isoproturon, linuron, buthiuron; ALS inhibitors, sulfonylureas, amidosulfuron, chlorsulfuron, flucpyrimiddin - sulfuron, halosulfuron - methyl, nicosulfuron, flupyrsulfuron - methyl, rimsulfuron, tribenuron - methyl, trifloxysulfuron, tritosulfuron, diphenyl ethers, acifluorfen, fomesafen, HPPD - inhibiting herbicides, mesotrione, pyroxasulfone, dicamba, and 2,4 - D.

[0019] This disclosure also pertains to recombinant polynucleotides. In one embodiment, the recombinant polynucleotide comprises (i) a region encoding solanesyl diphosphate synthase operably linked to a plant - operable promoter; and (ii) optionally, at least one additional region operably linked to a plant - operable promoter, the region encoding a herbicide - tolerance enzyme selected from the group consisting of hydroxyphenylpyruvate dioxygenase (HPPD), 5 - enolpyruvylshikimate - 3 - phosphate synthase (EPSPS), glyphosate acetyltransferase (GAT), cytochrome P450, phosphinothricin acetyltransferase (PAT), acetolactate synthase (ALS), protoporphyrinogen oxidase (PPGO), hydroxyphenylpyruvate dioxygenase (HPPD), and dicamba - degrading enzyme.

[0020] The region encoding geranylgeranyl diphosphate synthase can be (a) a nucleic acid sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NO: 20-41, 350-654, and 666-668; or a nucleic acid sequence encoding a polypeptide that comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-18, 45-349, and 663-665; or a nucleic acid sequence encoding a polypeptide that comprises an amino acid sequence selected from the group consisting of: an amino acid sequence that is at least 95% identical to SEQ ID NO: 1-18, 45-349, and 663-665; or a nucleic acid sequence encoding an SDPS that has a motif selected from SEQ ID NO: 655-662; or a nucleic acid sequence encoding an SDPS that has at least one mutation at a position corresponding to one of the following amino acid positions in SEQ ID NO: 5: F240L, F227L, F229L, F247L, L120A, L120R, L120W, L123A, L123C, L123D, L123N, L123S, L123W, E127A, E127G, E127K, E127Y, N128L, N128P, V130D, V130K, L131A, L131E, L131M, L131P, A134V, F139D, F139K, F139N, F139R, F139T, P148I, P148L, P148M, P148T, P148V, V151E, V151F, V151I, V151M, V151N, L174F, L174T, A175I, A175P, A175S, E176A, E176D, E176H, E176K, E176N, E176P, E176Y, I177A, I177C, I177F, I177L, I177M, I177S, I177T, I177Y, I178G, I178Q, I178W, E179I, M180I, M180Q, M180S, M180Y, M180W, I181M, I181N, A184G, A184S, A184T, T183C, T183Q, S185A, S185T, S185G, I187E, I187F, I187T, I187V, H188F, H188I, H188L, H188M, H188V, V191A, V191T, I204A, I204F, I204G, I204H, I204K, I204Q, I204R, I204S, I204T, Y208A, Y208D, Y208E, Y208H, Y208I, Y208K, Y208L, Y208M, Y208N, Y208Q, Y208R, Y208S, Y208T, Y208V, G209N,T210Y, R211D, R211E, R211N, R211T, R211V, L215I, L215M, A216T, F219A, M220I, M220C, F221W, A222G, A222M, A222S, Q223A, Q223E, Q223F, Q223G, Q223H, Q223I, Q223K, Q223L, Q223M, Q223R, Q223Y, S224F, S224I, S224M, S224N, S224Q, S224T, S224V, S225C, S225F, S225H, S225I, S225K, S225M, S225N, S225Q, S225T, S225V, S225Y, W226A, W226C, W226E, W226I, W226L, W226Q, W226R, W226T, W226V, F227D, F227L, F227M, F227R, F227V, F227W, L228C, L228I, L228M, L228T, L228V, A229H, A229I, A229L, A229M, A229N, A229T, N230E, N230R, E235G, K238G, K238N, K238S, L239A, L239R, I240A, I240C, I240W, S241A, S241H, S241N, S241T, V243A, V243G, V243N, V243Q, V243S, I244A, I244F, I244G, I244H, I244K, I244L, I244M, I244N, I244P, I244Q, I244S, I244V, I244Y, K245F, K245H, K245M, K245N, K245W, D246E, D246M, D246N, D246Q, D246S, D246T, D246Y, F247E, F247L, F247M, F247N, F247V, A248P, S249A, S249E, S249F, S249G, S249K, S249L, S249N, S249Q, S249T, S249V, S249Y, G250A, I252L, I252M, I252V, K253L, A255T, A255W, S256N, T257E, T257G, T257H, T257M, T257Q, T257W, Y274D, Y274G, Y274L, Y274M, Y274Q, T276S, L279F, I280W, I280F, A282G, A282H, A282K, A282N, A282R, S283C, S283F, S283I, S283M, S283T,S283W, R306F, R306H, R306L, R306N, L310G, G309A, G309F, G309M, G309S, L310D, L310E, L310F, L310H, L310N, L310Q, L310W, L310Y, F312C, F312I, F312L, F312M, F312V, Q313A, Q313C, Q313D, Q313S, and Q313T.

[0021] This disclosure also relates to plant cells that are tolerant to geranylgeranyl diphosphate synthase inhibitory herbicides. The plant cells can comprise a recombinant polynucleotide as described above.

[0022] This disclosure also relates to nucleic acid molecules that comprise a nucleotide sequence encoding a protein that confers tolerance to geranylgeranyl diphosphate synthase inhibitory herbicides in crop plants, wherein the nucleotide sequence (a) encodes a protein that comprises an amino acid sequence having at least 80% to at least 99% sequence identity to any one of SEQ ID NOs: 1-18, 45-349, and 663-665; (b) is selected from SEQ ID NOs: 20-41, 350-654, and 666-668; (c) is a synthetic sequence of (a) or (b) that has been codon optimized for expression in transgenic organisms; or (d) is SDPS that has a motif selected from SEQ ID NOs: 655-662; or SDPS that has at least one mutation at a position corresponding to one of the amino acid positions of SEQ ID NO: 5, as exemplified above. In certain instances, the protein comprises the amino acid sequence of SEQ ID NO: 3 or 13-18.

[0023] This disclosure also relates to chimeric genes that comprise a heterologous promoter operably linked to a nucleic acid molecule as described above. In typical embodiments, the heterologous promoter is a plant-expressible promoter. Suitable plant-expressible promoters include ubiquitin, Cestrum yellow vein virus, maize TrpA, OsMADS 6, maize histone H3, bacteriophage T3 gene 9 5' UTR, maize sucrose synthase 1, maize alcohol dehydrogenase 1, maize light-harvesting complex, maize heat shock protein, maize mtl, pea small subunit RuBP carboxylase, rice actin, rice cyclophilin, Ti plasmid mannopine synthase, Ti plasmid nopaline synthase, petunia chalcone isomerase, legume glycine-rich protein 1, potato glycoprotein, lectin, CaMV 35S, and S-E9 small subunit RuBP carboxylase promoter.

[0024] This disclosure also relates to plasmids that comprise a chimeric gene as described above, and to plants that comprise a chimeric gene as described above, and to plants that have been edited as described above.

[0025] In other instances, the present disclosure relates to the use of recombinant geranylgeranyl diphosphate synthase in an in vitro screening method for identifying geranylgeranyl diphosphate synthase inhibitory herbicides.

[0026] In other instances, the present disclosure relates to the use of recombinant geranylgeranyl diphosphate synthase in an in vitro screening method for identifying geranylgeranyl diphosphate synthase variants that have increased tolerance to geranylgeranyl diphosphate synthase inhibitory herbicides.

[0027] Thus, according to an embodiment of the present invention, there is provided a method for selectively controlling weeds at a site comprising crop plants and weeds, the method comprising applying to the site a pesticidal composition comprising a SDPS inhibitory herbicide in a weed controlling amount, wherein the crop plants are modified such that they comprise SDPS that provides tolerance to the SDPS inhibitory herbicide for the crop plants.

[0028] Brief Description of the Sequence Listing

[0029] SEQ ID NO:1 is the AA sequence of Arabidopsis thaliana SDPS1.

[0030] SEQ ID NO:2 is the AA sequence of Arabidopsis thaliana SDPS2.

[0031] SEQ ID NO:3 is the AA sequence of Arabidopsis thaliana SDPS2 F240L.

[0032] SEQ ID NO:4 is the AA sequence of Zea mays SDPS1.

[0033] SEQ ID NO:5 is the AA sequence of Zea mays SDPS2.

[0034] SEQ ID NO:6 is the AA sequence of Triticum aestivum SDPS.

[0035] SEQ ID NO:7 is the AA sequence of Hordeum vulgare SDPS.

[0036] SEQ ID NO:8 is the AA sequence of Glycine max SDPS.

[0037] SEQ ID NO:9 is the AA sequence of Oryza sativa (japonica) SDPS.

[0038] SEQ ID NO:10 is the AA sequence of Chlorella vulgaris SDPS.

[0039] SEQ ID NO:11 is the AA sequence of Chlorella vulgaris SDPS F227L.

[0040] SEQ ID NO:12 is the AA sequence of Chlamydomonas reinhardtii SDPS.

[0041] SEQ ID NO:13 is the AA sequence of His-Trunc Arabidopsis thaliana SDPS2.

[0042] SEQ ID NO:14 is the AA sequence of His-Trunc Arabidopsis thaliana SDPS2 F240L.

[0043] SEQ ID NO:15 is the AA sequence of His-Trunc Zea mays SDPS1.

[0044] SEQ ID NO:16 is the AA sequence of His-Trunc Zea mays SDPS2.

[0045] SEQ ID NO:17 is the AA sequence of His-Trunc Zea mays SDPS1 F229L.

[0046] SEQ ID NO:18 is the AA sequence of His-Trunc Zea mays SDPS2 F247L.

[0047] SEQ ID NO:19 is the DNA sequence of the plasmid.

[0048] SEQ ID NO:20 is the DNA sequence of Arabidopsis thaliana SDPS1.

[0049] SEQ ID NO:21 is the DNA sequence of Arabidopsis thaliana SDPS2.

[0050] SEQ ID NO:22 is the DNA sequence of Arabidopsis thaliana SDPS2 F240L.

[0051] SEQ ID NO:23 is the DNA sequence of Zea mays SDPS1.

[0052] SEQ ID NO:24 is the DNA sequence of Zea mays SDPS2.

[0053] SEQ ID NO:25 is the DNA sequence of Triticum aestivum SDPS.

[0054] SEQ ID NO:26 is the DNA sequence of Hordeum vulgare SDPS.

[0055] SEQ ID NO:27 is the DNA sequence of Glycine max SDPS.

[0056] SEQ ID NO:28 is the DNA sequence of Oryza sativa (japonica) SDPS.

[0057] SEQ ID NO:29 is the DNA sequence of Chlorella SDPS.

[0058] SEQ ID NO:30 is the DNA sequence of Chlorella SDPS F227L.

[0059] SEQ ID NO:31 is the DNA sequence of Chlamydomonas reinhardtii SDPS.

[0060] SEQ ID NO:32 is the DNA sequence of E. coli-optimized His-Trunc Arabidopsis SDPS2.

[0061] SEQ ID NO:33 is the DNA sequence of E. coli-optimized His-Trunc Arabidopsis SDPS2 F240L.

[0062] SEQ ID NO:34 is the DNA sequence of E. coli-optimized His-Trunc Zea mays SDPS1.

[0063] SEQ ID NO:35 is the DNA sequence of E. coli-optimized His-Trunc Zea mays SDPS2.

[0064] SEQ ID NO:36 is the DNA sequence of E. coli-optimized His-Trunc Zea mays SDPS1 F229L.

[0065] SEQ ID NO:37 is the DNA sequence of E. coli-optimized His-Trunc Zea mays SDPS2 F247L.

[0066] SEQ ID NO:38 is the DNA sequence of Nicotiana tabacum-optimized Arabidopsis SDPS2.

[0067] SEQ ID NO:39 is the DNA sequence of Nicotiana tabacum-optimized Arabidopsis SDPS2 F240L.

[0068] SEQ ID NO:40 is the DNA sequence of Nicotiana tabacum-optimized Chlorella SDPS.

[0069] SEQ ID NO:41 is the DNA sequence of Nicotiana tabacum-optimized Chlorella SDPS F227L.

[0070] SEQ ID NO:42 is the DNA sequence of SDPS Cpf1-artificial plasmid.

[0071] SEQ ID NO:43 is the DNA sequence of Zea mays, target sequence.

[0072] SEQ ID NO:44 is the DNA sequence of Cpf1 nuclease.

[0073] SEQ ID NO:45 is the protein sequence of His-Trunc maize SDPS2 L120A.

[0074] SEQ ID NO:46 is the protein sequence of His-Trunc maize SDPS2 L120R.

[0075] SEQ ID NO:47 is the protein sequence of His-Trunc maize SDPS2 L120W.

[0076] SEQ ID NO:48 is the protein sequence of His-Trunc maize SDPS2 L123A.

[0077] SEQ ID NO:49 is the protein sequence of His-Trunc maize SDPS2 L123C.

[0078] SEQ ID NO:50 is the protein sequence of His-Trunc maize SDPS2 L123D.

[0079] SEQ ID NO:51 is the protein sequence of His-Trunc maize SDPS2 L123N.

[0080] SEQ ID NO:52 is the protein sequence of His-Trunc maize SDPS2 L123S.

[0081] SEQ ID NO:53 is the protein sequence of His-Trunc maize SDPS2 L123W.

[0082] SEQ ID NO:54 is the protein sequence of His-Trunc maize SDPS2 E127A.

[0083] SEQ ID NO:55 is the protein sequence of His-Trunc maize SDPS2 E127G.

[0084] SEQ ID NO:56 is the protein sequence of His-Trunc maize SDPS2 E127K.

[0085] SEQ ID NO:57 is the protein sequence of His-Trunc maize SDPS2 E127Y.

[0086] SEQ ID NO:58 is the protein sequence of His-Trunc maize SDPS2 N128L.

[0087] SEQ ID NO:59 is the protein sequence of His-Trunc maize SDPS2 N128P.

[0088] SEQ ID NO:60 is the protein sequence of His-Trunc maize SDPS2 V130D.

[0089] SEQ ID NO:61 is the protein sequence of His-Trunc maize SDPS2 V130K.

[0090] SEQ ID NO:62 is the protein sequence of His-Trunc maize SDPS2 L131A.

[0091] SEQ ID NO:63 is the protein sequence of His-Trunc maize SDPS2 L131E.

[0092] SEQ ID NO:64 is the protein sequence of His-Trunc maize SDPS2 L131M.

[0093] SEQ ID NO:65 is the protein sequence of His-Trunc maize SDPS2 L131P.

[0094] SEQ ID NO:66 is the protein sequence of His-Trunc maize SDPS2 A134V.

[0095] SEQ ID NO:67 is the protein sequence of His-Trunc maize SDPS2 F139D.

[0096] SEQ ID NO:68 is the protein sequence of His-Trunc maize SDPS2 F139K.

[0097] SEQ ID NO:69 is the protein sequence of His-Trunc maize SDPS2 F139N.

[0098] SEQ ID NO:70 is the protein sequence of His-Trunc maize SDPS2 F139R.

[0099] SEQ ID NO:71 is the protein sequence of His-Trunc maize SDPS2 F139T.

[0100] SEQ ID NO:72 is the protein sequence of His-Trunc maize SDPS2 P148I.

[0101] SEQ ID NO:73 is the protein sequence of His-Trunc maize SDPS2 P148L.

[0102] SEQ ID NO:74 is the protein sequence of His-Trunc maize SDPS2 P148M.

[0103] SEQ ID NO:75 is the protein sequence of His-Trunc maize SDPS2 P148T.

[0104] SEQ ID NO:76 is the protein sequence of His-Trunc maize SDPS2 P148V.

[0105] SEQ ID NO:77 is the protein sequence of His-Trunc maize SDPS2 V151E.

[0106] SEQ ID NO:78 is the protein sequence of His-Trunc maize SDPS2 V151F.

[0107] SEQ ID NO:79 is the protein sequence of His-Trunc maize SDPS2 V151I.

[0108] SEQ ID NO:80 is the protein sequence of His-Trunc maize SDPS2 V151M.

[0109] SEQ ID NO:81 is the protein sequence of His-Trunc maize SDPS2 V151N.

[0110] SEQ ID NO:82 is the protein sequence of His-Trunc maize SDPS2 L174F.

[0111] SEQ ID NO:83 is the protein sequence of His-Trunc maize SDPS2 L174T.

[0112] SEQ ID NO:84 is the protein sequence of His-Trunc maize SDPS2 A175I.

[0113] SEQ ID NO:85 is the protein sequence of His-Trunc maize SDPS2 A175P.

[0114] SEQ ID NO:86 is the protein sequence of His-Trunc maize SDPS2 A175S.

[0115] SEQ ID NO:87 is the protein sequence of His-Trunc maize SDPS2 E176A.

[0116] SEQ ID NO:88 is the protein sequence of His-Trunc maize SDPS2 E176D.

[0117] SEQ ID NO:89 is the protein sequence of His-Trunc maize SDPS2 E176H.

[0118] SEQ ID NO:90 is the protein sequence of His-Trunc maize SDPS2 E176K.

[0119] SEQ ID NO:91 is the protein sequence of His-Trunc maize SDPS2 E176N.

[0120] SEQ ID NO:92 is the protein sequence of His-Trunc maize SDPS2 E176P.

[0121] SEQ ID NO:93 is the protein sequence of His-Trunc maize SDPS2 E176Y.

[0122] SEQ ID NO:94 is the protein sequence of His-Trunc maize SDPS2 I177A.

[0123] SEQ ID NO:95 is the protein sequence of His-Trunc maize SDPS2 I177C.

[0124] SEQ ID NO:96 is the protein sequence of His-Trunc maize SDPS2 I177F.

[0125] SEQ ID NO:97 is the protein sequence of His-Trunc maize SDPS2 I177L.

[0126] SEQ ID NO:98 is the protein sequence of His-Trunc maize SDPS2 I177M.

[0127] SEQ ID NO:99 is the protein sequence of His-Trunc maize SDPS2 I177S.

[0128] SEQ ID NO:100 is the protein sequence of His-Trunc maize SDPS2 I177T.

[0129] SEQ ID NO:101 is the protein sequence of His-Trunc maize SDPS2 I177Y.

[0130] SEQ ID NO:102 is the protein sequence of His-Trunc maize SDPS2 I178G.

[0131] SEQ ID NO:103 is the protein sequence of His-Trunc maize SDPS2 I178Q.

[0132] SEQ ID NO:104 is the protein sequence of His-Trunc maize SDPS2 I178W.

[0133] SEQ ID NO:105 is the protein sequence of His-Trunc maize SDPS2 E179I.

[0134] SEQ ID NO:106 is the protein sequence of His-Trunc maize SDPS2 M180I.

[0135] SEQ ID NO:107 is the protein sequence of His-Trunc maize SDPS2 M180Q.

[0136] SEQ ID NO:108 is the protein sequence of His-Trunc maize SDPS2 M180S.

[0137] SEQ ID NO:109 is the protein sequence of His-Trunc maize SDPS2 M180Y.

[0138] SEQ ID NO:110 is the protein sequence of His-Trunc maize SDPS2 M180W.

[0139] SEQ ID NO:111 is the protein sequence of His-Trunc maize SDPS2 I181M.

[0140] SEQ ID NO:112 is the protein sequence of His-Trunc maize SDPS2 I181N.

[0141] SEQ ID NO:113 is the protein sequence of His-Trunc maize SDPS2 A184G.

[0142] SEQ ID NO:114 is the protein sequence of His-Trunc maize SDPS2 A184S.

[0143] SEQ ID NO:115 is the protein sequence of His-Trunc maize SDPS2 A184T.

[0144] SEQ ID NO:116 is the protein sequence of His-Trunc maize SDPS2 T183C.

[0145] SEQ ID NO:117 is the protein sequence of His-Trunc maize SDPS2 T183Q.

[0146] SEQ ID NO:118 is the protein sequence of His-Trunc maize SDPS2 S185A.

[0147] SEQ ID NO:119 is the protein sequence of His-Trunc maize SDPS2 S185T.

[0148] SEQ ID NO:120 is the protein sequence of His-Trunc maize SDPS2 S185G.

[0149] SEQ ID NO:121 is the protein sequence of His-Trunc maize SDPS2 I187E.

[0150] SEQ ID NO:122 is the protein sequence of His-Trunc maize SDPS2 I187F.

[0151] SEQ ID NO:123 is the protein sequence of His-Trunc maize SDPS2 I187T.

[0152] SEQ ID NO:124 is the protein sequence of His-Trunc maize SDPS2 I187V.

[0153] SEQ ID NO:125 is the protein sequence of His-Trunc maize SDPS2 H188F.

[0154] SEQ ID NO:126 is the protein sequence of His-Trunc maize SDPS2 H188I.

[0155] SEQ ID NO:127 is the protein sequence of His-Trunc maize SDPS2 H188L.

[0156] SEQ ID NO:128 is the protein sequence of His-Trunc maize SDPS2 H188M.

[0157] SEQ ID NO:129 is the protein sequence of His-Trunc maize SDPS2 H188V.

[0158] SEQ ID NO:130 is the protein sequence of His-Trunc maize SDPS2 V191A.

[0159] SEQ ID NO:131 is the protein sequence of His-Trunc maize SDPS2 V191T.

[0160] SEQ ID NO:132 is the protein sequence of His-Trunc maize SDPS2 I204A.

[0161] SEQ ID NO:133 is the protein sequence of His-Trunc maize SDPS2 I204F.

[0162] SEQ ID NO:134 is the protein sequence of His-Trunc maize SDPS2 I204G.

[0163] SEQ ID NO:135 is the protein sequence of His-Trunc maize SDPS2 I204H.

[0164] SEQ ID NO:136 is the protein sequence of His-Trunc maize SDPS2 I204K.

[0165] SEQ ID NO:137 is the protein sequence of His-Trunc maize SDPS2 I204Q.

[0166] SEQ ID NO:138 is the protein sequence of His-Trunc maize SDPS2 I204R.

[0167] SEQ ID NO:139 is the protein sequence of His-Trunc maize SDPS2 I204S.

[0168] SEQ ID NO:140 is the protein sequence of His-Trunc maize SDPS2 I204T.

[0169] SEQ ID NO:141 is the protein sequence of His-Trunc maize SDPS2 Y208A.

[0170] SEQ ID NO:142 is the protein sequence of His-Trunc maize SDPS2 Y208D.

[0171] SEQ ID NO:143 is the protein sequence of His-Trunc maize SDPS2 Y208E.

[0172] SEQ ID NO:144 is the protein sequence of His-Trunc maize SDPS2 Y208H.

[0173] SEQ ID NO:145 is the protein sequence of His-Trunc maize SDPS2 Y208I.

[0174] SEQ ID NO:146 is the protein sequence of His-Trunc maize SDPS2 Y208K.

[0175] SEQ ID NO:147 is the protein sequence of His-Trunc maize SDPS2 Y208L.

[0176] SEQ ID NO:148 is the protein sequence of His-Trunc maize SDPS2 Y208M.

[0177] SEQ ID NO:149 is the protein sequence of His-Trunc maize SDPS2 Y208N.

[0178] SEQ ID NO:150 is the protein sequence of His-Trunc maize SDPS2 Y208Q.

[0179] SEQ ID NO:151 is the protein sequence of His-Trunc maize SDPS2 Y208R.

[0180] SEQ ID NO:152 is the protein sequence of His-Trunc maize SDPS2 Y208S.

[0181] SEQ ID NO:153 is the protein sequence of His-Trunc maize SDPS2 Y208T.

[0182] SEQ ID NO:154 is the protein sequence of His-Trunc maize SDPS2 Y208V.

[0183] SEQ ID NO:155 is the protein sequence of His-Trunc maize SDPS2 G209N.

[0184] SEQ ID NO:156 is the protein sequence of His-Trunc maize SDPS2 T210Y.

[0185] SEQ ID NO:157 is the protein sequence of His-Trunc maize SDPS2 R211D.

[0186] SEQ ID NO:158 is the protein sequence of His-Trunc maize SDPS2 R211E.

[0187] SEQ ID NO:159 is the protein sequence of His-Trunc maize SDPS2 R211N.

[0188] SEQ ID NO:160 is the protein sequence of His-Trunc maize SDPS2 R211T.

[0189] SEQ ID NO:161 is the protein sequence of His-Trunc maize SDPS2 R211V.

[0190] SEQ ID NO:162 is the protein sequence of His-Trunc maize SDPS2 L215I.

[0191] SEQ ID NO:163 is the protein sequence of His-Trunc maize SDPS2 L215M.

[0192] SEQ ID NO:164 is the protein sequence of His-Trunc maize SDPS2 A216T.

[0193] SEQ ID NO:165 is the protein sequence of His-Trunc maize SDPS2 F219A.

[0194] SEQ ID NO:166 is the protein sequence of His-Trunc maize SDPS2 M220I.

[0195] SEQ ID NO:167 is the protein sequence of His-Trunc maize SDPS2 M220C.

[0196] SEQ ID NO:168 is the protein sequence of His-Trunc maize SDPS2 F221W.

[0197] SEQ ID NO:169 is the protein sequence of His-Trunc maize SDPS2 A222G.

[0198] SEQ ID NO:170 is the protein sequence of His-Trunc maize SDPS2 A222M.

[0199] SEQ ID NO:171 is the protein sequence of His-Trunc maize SDPS2 A222S.

[0200] SEQ ID NO:172 is the protein sequence of His-Trunc maize SDPS2 Q223A.

[0201] SEQ ID NO:173 is the protein sequence of His-Trunc maize SDPS2 Q223E.

[0202] SEQ ID NO:174 is the protein sequence of His-Trunc maize SDPS2 Q223F.

[0203] SEQ ID NO:175 is the protein sequence of His-Trunc maize SDPS2 Q223G.

[0204] SEQ ID NO:176 is the protein sequence of His-Trunc maize SDPS2 Q223H.

[0205] SEQ ID NO:177 is the protein sequence of His-Trunc maize SDPS2 Q223I.

[0206] SEQ ID NO:178 is the protein sequence of His-Trunc maize SDPS2 Q223K.

[0207] SEQ ID NO:179 is the protein sequence of His-Trunc maize SDPS2 Q223L.

[0208] SEQ ID NO:180 is the protein sequence of His-Trunc maize SDPS2 Q223M.

[0209] SEQ ID NO:181 is the protein sequence of His-Trunc maize SDPS2 Q223R.

[0210] SEQ ID NO:182 is the protein sequence of His-Trunc maize SDPS2 Q223Y.

[0211] SEQ ID NO:183 is the protein sequence of His-Trunc maize SDPS2 S224F.

[0212] SEQ ID NO:184 is the protein sequence of His-Trunc maize SDPS2 S224I.

[0213] SEQ ID NO:185 is the protein sequence of His-Trunc maize SDPS2 S224M.

[0214] SEQ ID NO:186 is the protein sequence of His-Trunc maize SDPS2 S224N.

[0215] SEQ ID NO:187 is the protein sequence of His-Trunc maize SDPS2 S224Q.

[0216] SEQ ID NO:188 is the protein sequence of His-Trunc maize SDPS2 S224T.

[0217] SEQ ID NO:189 is the protein sequence of His-Trunc maize SDPS2 S224V.

[0218] SEQ ID NO:190 is the protein sequence of His-Trunc maize SDPS2 S225C.

[0219] SEQ ID NO:191 is the protein sequence of His-Trunc maize SDPS2 S225F.

[0220] SEQ ID NO:192 is the protein sequence of His-Trunc maize SDPS2 S225H.

[0221] SEQ ID NO:193 is the protein sequence of His-Trunc maize SDPS2 S225I.

[0222] SEQ ID NO:194 is the protein sequence of His-Trunc maize SDPS2 S225K.

[0223] SEQ ID NO:195 is the protein sequence of His-Trunc maize SDPS2 S225M.

[0224] SEQ ID NO:196 is the protein sequence of His-Trunc maize SDPS2 S225N.

[0225] SEQ ID NO:197 is the protein sequence of His-Trunc maize SDPS2 S225Q.

[0226] SEQ ID NO:198 is the protein sequence of His-Trunc maize SDPS2 S225T.

[0227] SEQ ID NO:199 is the protein sequence of His-Trunc maize SDPS2 S225V.

[0228] SEQ ID NO:200 is the protein sequence of His-Trunc maize SDPS2 S225Y.

[0229] SEQ ID NO:201 is the protein sequence of His-Trunc maize SDPS2 W226A.

[0230] SEQ ID NO:202 is the protein sequence of His-Trunc maize SDPS2 W226C.

[0231] SEQ ID NO:203 is the protein sequence of His-Trunc maize SDPS2 W226E.

[0232] SEQ ID NO:204 is the protein sequence of His-Trunc maize SDPS2 W226I.

[0233] SEQ ID NO:205 is the protein sequence of His-Trunc maize SDPS2 W226L.

[0234] SEQ ID NO:206 is the protein sequence of His-Trunc maize SDPS2 W226Q.

[0235] SEQ ID NO:207 is the protein sequence of His-Trunc maize SDPS2 W226R.

[0236] SEQ ID NO:208 is the protein sequence of His-Trunc maize SDPS2 W226T.

[0237] SEQ ID NO:209 is the protein sequence of His-Trunc maize SDPS2 W226V.

[0238] SEQ ID NO:210 is the protein sequence of His-Trunc maize SDPS2 F227D.

[0239] SEQ ID NO:211 is the protein sequence of His-Trunc maize SDPS2 F227L.

[0240] SEQ ID NO:212 is the protein sequence of His-Trunc maize SDPS2 F227M.

[0241] SEQ ID NO:213 is the protein sequence of His-Trunc maize SDPS2 F227R.

[0242] SEQ ID NO:214 is the protein sequence of His-Trunc maize SDPS2 F227V.

[0243] SEQ ID NO:215 is the protein sequence of His-Trunc maize SDPS2 F227W.

[0244] SEQ ID NO:216 is the protein sequence of His-Trunc maize SDPS2 L228C.

[0245] SEQ ID NO:217 is the protein sequence of His-Trunc maize SDPS2 L228I.

[0246] SEQ ID NO:218 is the protein sequence of His-Trunc maize SDPS2 L228M.

[0247] SEQ ID NO:219 is the protein sequence of His-Trunc maize SDPS2 L228T.

[0248] SEQ ID NO:220 is the protein sequence of His-Trunc maize SDPS2 L228V.

[0249] SEQ ID NO:221 is the protein sequence of His-Trunc maize SDPS2 A229H.

[0250] SEQ ID NO:222 is the protein sequence of His-Trunc maize SDPS2 A229I.

[0251] SEQ ID NO:223 is the protein sequence of His-Trunc maize SDPS2 A229L.

[0252] SEQ ID NO:224 is the protein sequence of His-Trunc maize SDPS2 A229M.

[0253] SEQ ID NO:225 is the protein sequence of His-Trunc maize SDPS2 A229N.

[0254] SEQ ID NO:226 is the protein sequence of His-Trunc maize SDPS2 A229T.

[0255] SEQ ID NO:227 is the protein sequence of His-Trunc maize SDPS2 N230E.

[0256] SEQ ID NO:228 is the protein sequence of His-Trunc maize SDPS2 N230R.

[0257] SEQ ID NO:229 is the protein sequence of His-Trunc maize SDPS2 E235G.

[0258] SEQ ID NO:230 is the protein sequence of His-Trunc maize SDPS2 K238G.

[0259] SEQ ID NO:231 is the protein sequence of His-Trunc maize SDPS2 K238N.

[0260] SEQ ID NO:232 is the protein sequence of His-Trunc maize SDPS2 K238S.

[0261] SEQ ID NO:233 is the protein sequence of His-Trunc maize SDPS2 L239A.

[0262] SEQ ID NO:234 is the protein sequence of His-Trunc maize SDPS2 L239R.

[0263] SEQ ID NO:235 is the protein sequence of His-Trunc maize SDPS2 I240A.

[0264] SEQ ID NO:236 is the protein sequence of His-Trunc maize SDPS2 I240C.

[0265] SEQ ID NO:237 is the protein sequence of His-Trunc maize SDPS2 I240W.

[0266] SEQ ID NO:238 is the protein sequence of His-Trunc maize SDPS2 S241A.

[0267] SEQ ID NO:239 is the protein sequence of His-Trunc maize SDPS2 S241H.

[0268] SEQ ID NO:240 is the protein sequence of His-Trunc maize SDPS2 S241N.

[0269] SEQ ID NO:241 is the protein sequence of His-Trunc maize SDPS2 S241T.

[0270] SEQ ID NO:242 is the protein sequence of His-Trunc maize SDPS2 V243A.

[0271] SEQ ID NO:243 is the protein sequence of His-Trunc maize SDPS2 V243G.

[0272] SEQ ID NO:244 is the protein sequence of His-Trunc maize SDPS2 V243N.

[0273] SEQ ID NO:245 is the protein sequence of His-Trunc maize SDPS2 V243Q.

[0274] SEQ ID NO:246 is the protein sequence of His-Trunc maize SDPS2 V243S.

[0275] SEQ ID NO:247 is the protein sequence of His-Trunc maize SDPS2 I244A.

[0276] SEQ ID NO:248 is the protein sequence of His-Trunc maize SDPS2 I244F.

[0277] SEQ ID NO:249 is the protein sequence of His-Trunc maize SDPS2 I244G.

[0278] SEQ ID NO:250 is the protein sequence of His-Trunc maize SDPS2 I244H.

[0279] SEQ ID NO:251 is the protein sequence of His-Trunc maize SDPS2 I244K.

[0280] SEQ ID NO:252 is the protein sequence of His-Trunc maize SDPS2 I244L.

[0281] SEQ ID NO:253 is the protein sequence of His-Trunc maize SDPS2 I244M.

[0282] SEQ ID NO:254 is the protein sequence of His-Trunc maize SDPS2 I244N.

[0283] SEQ ID NO:255 is the protein sequence of His-Trunc maize SDPS2 I244P.

[0284] SEQ ID NO:256 is the protein sequence of His-Trunc maize SDPS2 I244Q.

[0285] SEQ ID NO:257 is the protein sequence of His-Trunc maize SDPS2 I244S.

[0286] SEQ ID NO:258 is the protein sequence of His-Trunc maize SDPS2 I244V.

[0287] SEQ ID NO:259 is the protein sequence of His-Trunc maize SDPS2 I244Y.

[0288] SEQ ID NO:260 is the protein sequence of His-Trunc maize SDPS2 K245F.

[0289] SEQ ID NO:261 is the protein sequence of His-Trunc maize SDPS2 K245H.

[0290] SEQ ID NO:262 is the protein sequence of His-Trunc maize SDPS2 K245M.

[0291] SEQ ID NO:263 is the protein sequence of His-Trunc maize SDPS2 K245N.

[0292] SEQ ID NO:264 is the protein sequence of His-Trunc maize SDPS2 K245W.

[0293] SEQ ID NO:265 is the protein sequence of His-Trunc maize SDPS2 D246E.

[0294] SEQ ID NO:266 is the protein sequence of His-Trunc maize SDPS2 D246M.

[0295] SEQ ID NO:267 is the protein sequence of His-Trunc maize SDPS2 D246N.

[0296] SEQ ID NO:268 is the protein sequence of His-Trunc maize SDPS2 D246Q.

[0297] SEQ ID NO:269 is the protein sequence of His-Trunc maize SDPS2 D246S.

[0298] SEQ ID NO:270 is the protein sequence of His-Trunc maize SDPS2 D246T.

[0299] SEQ ID NO:271 is the protein sequence of His-Trunc maize SDPS2 D246Y.

[0300] SEQ ID NO:272 is the protein sequence of His-Trunc maize SDPS2 F247E.

[0301] SEQ ID NO:273 is the protein sequence of His-Trunc maize SDPS2 F247L.

[0302] SEQ ID NO:274 is the protein sequence of His-Trunc maize SDPS2 F247M.

[0303] SEQ ID NO:275 is the protein sequence of His-Trunc maize SDPS2 F247N.

[0304] SEQ ID NO:276 is the protein sequence of His-Trunc maize SDPS2 F247V.

[0305] SEQ ID NO:277 is the protein sequence of His-Trunc maize SDPS2 A248P.

[0306] SEQ ID NO:278 is the protein sequence of His-Trunc maize SDPS2 S249A.

[0307] SEQ ID NO:279 is the protein sequence of His-Trunc maize SDPS2 S249E.

[0308] SEQ ID NO:280 is the protein sequence of His-Trunc maize SDPS2 S249F.

[0309] SEQ ID NO:281 is the protein sequence of His-Trunc maize SDPS2 S249G.

[0310] SEQ ID NO:282 is the protein sequence of His-Trunc maize SDPS2 S249K.

[0311] SEQ ID NO:283 is the protein sequence of His-Trunc maize SDPS2 S249L.

[0312] SEQ ID NO:284 is the protein sequence of His-Trunc maize SDPS2 S249N.

[0313] SEQ ID NO:285 is the protein sequence of His-Trunc maize SDPS2 S249Q.

[0314] SEQ ID NO:286 is the protein sequence of His-Trunc maize SDPS2 S249T.

[0315] SEQ ID NO:287 is the protein sequence of His-Trunc maize SDPS2 S249V.

[0316] SEQ ID NO:288 is the protein sequence of His-Trunc maize SDPS2 S249Y.

[0317] SEQ ID NO:289 is the protein sequence of His-Trunc maize SDPS2 G250A.

[0318] SEQ ID NO:290 is the protein sequence of His-Trunc maize SDPS2 I252L.

[0319] SEQ ID NO:291 is the protein sequence of His-Trunc maize SDPS2 I252M.

[0320] SEQ ID NO:292 is the protein sequence of His-Trunc maize SDPS2 I252V.

[0321] SEQ ID NO:293 is the protein sequence of His-Trunc maize SDPS2 K253L.

[0322] SEQ ID NO:294 is the protein sequence of His-Trunc maize SDPS2 A255T.

[0323] SEQ ID NO:295 is the protein sequence of His-Trunc maize SDPS2 A255W.

[0324] SEQ ID NO:296 is the protein sequence of His-Trunc maize SDPS2 S256N.

[0325] SEQ ID NO:297 is the protein sequence of His-Trunc maize SDPS2 T257E.

[0326] SEQ ID NO:298 is the protein sequence of His-Trunc maize SDPS2 T257G.

[0327] SEQ ID NO:299 is the protein sequence of His-Trunc maize SDPS2 T257H.

[0328] SEQ ID NO:300 is the protein sequence of His-Trunc maize SDPS2 T257M.

[0329] SEQ ID NO:301 is the protein sequence of His-Trunc maize SDPS2 T257Q.

[0330] SEQ ID NO:302 is the protein sequence of His-Trunc maize SDPS2 T257W.

[0331] SEQ ID NO:303 is the protein sequence of His-Trunc maize SDPS2 Y274D.

[0332] SEQ ID NO:304 is the protein sequence of His-Trunc maize SDPS2 Y274G.

[0333] SEQ ID NO:305 is the protein sequence of His-Trunc maize SDPS2 Y274L.

[0334] SEQ ID NO:306 is the protein sequence of His-Trunc maize SDPS2 Y274M.

[0335] SEQ ID NO:307 is the protein sequence of His-Trunc maize SDPS2 Y274Q.

[0336] SEQ ID NO:308 is the protein sequence of His-Trunc maize SDPS2 T276S.

[0337] SEQ ID NO:309 is the protein sequence of His-Trunc maize SDPS2 L279F.

[0338] SEQ ID NO:310 is the protein sequence of His-Trunc maize SDPS2 I280W.

[0339] SEQ ID NO:311 is the protein sequence of His-Trunc maize SDPS2 I280F.

[0340] SEQ ID NO:312 is the protein sequence of His-Trunc maize SDPS2 A282G.

[0341] SEQ ID NO:313 is the protein sequence of His-Trunc maize SDPS2 A282H.

[0342] SEQ ID NO:314 is the protein sequence of His-Trunc maize SDPS2 A282K.

[0343] SEQ ID NO:315 is the protein sequence of His-Trunc maize SDPS2 A282N.

[0344] SEQ ID NO:316 is the protein sequence of His-Trunc maize SDPS2 A282R.

[0345] SEQ ID NO:317 is the protein sequence of His-Trunc maize SDPS2 S283C.

[0346] SEQ ID NO:318 is the protein sequence of His-Trunc maize SDPS2 S283F.

[0347] SEQ ID NO:319 is the protein sequence of His-Trunc maize SDPS2 S283I.

[0348] SEQ ID NO:320 is the protein sequence of His-Trunc maize SDPS2 S283M.

[0349] SEQ ID NO:321 is the protein sequence of His-Trunc maize SDPS2 S283T.

[0350] SEQ ID NO:322 is the protein sequence of His-Trunc maize SDPS2 S283W.

[0351] SEQ ID NO:323 is the protein sequence of His-Trunc maize SDPS2 R306F.

[0352] SEQ ID NO:324 is the protein sequence of His-Trunc maize SDPS2 R306H.

[0353] SEQ ID NO:325 is the protein sequence of His-Trunc maize SDPS2 R306L.

[0354] SEQ ID NO:326 is the protein sequence of His-Trunc maize SDPS2 R306N.

[0355] SEQ ID NO:327 is the protein sequence of His-Trunc maize SDPS2 L310G.

[0356] SEQ ID NO:328 is the protein sequence of His-Trunc maize SDPS2 G309A.

[0357] SEQ ID NO:329 is the protein sequence of His-Trunc maize SDPS2 G309F.

[0358] SEQ ID NO:330 is the protein sequence of His-Trunc maize SDPS2 G309M.

[0359] SEQ ID NO:331 is the protein sequence of His-Trunc maize SDPS2 G309S.

[0360] SEQ ID NO:332 is the protein sequence of His-Trunc maize SDPS2 L310D.

[0361] SEQ ID NO:333 is the protein sequence of His-Trunc maize SDPS2 L310E.

[0362] SEQ ID NO:334 is the protein sequence of His-Trunc maize SDPS2 L310F.

[0363] SEQ ID NO:335 is the protein sequence of His-Trunc maize SDPS2 L310H.

[0364] SEQ ID NO:336 is the protein sequence of His-Trunc maize SDPS2 L310N.

[0365] SEQ ID NO:337 is the protein sequence of His-Trunc maize SDPS2 L310Q.

[0366] SEQ ID NO:338 is the protein sequence of His-Trunc maize SDPS2 L310W.

[0367] SEQ ID NO:339 is the protein sequence of His-Trunc maize SDPS2 L310Y.

[0368] SEQ ID NO:340 is the protein sequence of His-Trunc maize SDPS2 F312C.

[0369] SEQ ID NO:341 is the protein sequence of His-Trunc maize SDPS2 F312I.

[0370] SEQ ID NO:342 is the protein sequence of His-Trunc maize SDPS2 F312L.

[0371] SEQ ID NO:343 is the protein sequence of His-Trunc maize SDPS2 F312M.

[0372] SEQ ID NO:344 is the protein sequence of His-Trunc maize SDPS2 F312V.

[0373] SEQ ID NO:345 is the protein sequence of His-Trunc maize SDPS2 Q313A.

[0374] SEQ ID NO:346 is the protein sequence of His-Trunc maize SDPS2 Q313C.

[0375] SEQ ID NO:347 is the protein sequence of His-Trunc maize SDPS2 Q313D.

[0376] SEQ ID NO:348 is the protein sequence of His-Trunc maize SDPS2 Q313S.

[0377] SEQ ID NO:349 is the protein sequence of His-Trunc maize SDPS2 Q313T.

[0378] SEQ ID NO:350 is the DNA sequence of E. coli-optimized His-Trunc maize SDPS2 L120A.

[0379] SEQ ID NO:351 is the DNA sequence of E. coli-optimized His-Trunc maize SDPS2 L120R.

[0380] SEQ ID NO:352 is the DNA sequence of E. coli-optimized His-Trunc maize SDPS2 L120W.

[0381] SEQ ID NO:353 is the DNA sequence of His-Trunc maize SDPS2 L123A optimized for E. coli.

[0382] SEQ ID NO:354 is the DNA sequence of His-Trunc maize SDPS2 L123C optimized for E. coli.

[0383] SEQ ID NO:355 is the DNA sequence of His-Trunc maize SDPS2 L123D optimized for E. coli.

[0384] SEQ ID NO:356 is the DNA sequence of His-Trunc maize SDPS2 L123N optimized for E. coli.

[0385] SEQ ID NO:357 is the DNA sequence of His-Trunc maize SDPS2 L123S optimized for E. coli.

[0386] SEQ ID NO:358 is the DNA sequence of His-Trunc maize SDPS2 L123W optimized for E. coli.

[0387] SEQ ID NO:359 is the DNA sequence of His-Trunc maize SDPS2 E127A optimized for E. coli.

[0388] SEQ ID NO:360 is the DNA sequence of His-Trunc maize SDPS2 E127G optimized for E. coli.

[0389] SEQ ID NO:361 is the DNA sequence of His-Trunc maize SDPS2 E127K optimized for E. coli.

[0390] SEQ ID NO:362 is the DNA sequence of His-Trunc maize SDPS2 E127Y optimized for E. coli.

[0391] SEQ ID NO:363 is the DNA sequence of His-Trunc maize SDPS2 N128L optimized for E. coli.

[0392] SEQ ID NO:364 is the DNA sequence of His-Trunc maize SDPS2 N128P optimized for E. coli.

[0393] SEQ ID NO:365 is the DNA sequence of His-Trunc maize SDPS2 V130D optimized for E. coli.

[0394] SEQ ID NO:366 is the DNA sequence of His-Trunc maize SDPS2 V130K optimized for E. coli.

[0395] SEQ ID NO:367 is the DNA sequence of His-Trunc maize SDPS2 L131A optimized for E. coli.

[0396] SEQ ID NO:368 is the DNA sequence of His-Trunc maize SDPS2 L131E optimized for E. coli.

[0397] SEQ ID NO:369 is the DNA sequence of His-Trunc maize SDPS2 L131M optimized for E. coli.

[0398] SEQ ID NO:370 is the DNA sequence of His-Trunc maize SDPS2 L131P optimized for E. coli.

[0399] SEQ ID NO:371 is the DNA sequence of His-Trunc maize SDPS2 A134V optimized for E. coli.

[0400] SEQ ID NO:372 is the DNA sequence of His-Trunc maize SDPS2 F139D optimized for E. coli.

[0401] SEQ ID NO:373 is the DNA sequence of His-Trunc maize SDPS2 F139K optimized for E. coli.

[0402] SEQ ID NO:374 is the DNA sequence of His-Trunc maize SDPS2 F139N optimized for E. coli.

[0403] SEQ ID NO:375 is the DNA sequence of His-Trunc maize SDPS2 F139R optimized for E. coli.

[0404] SEQ ID NO:376 is the DNA sequence of His-Trunc maize SDPS2 F139T optimized for E. coli.

[0405] SEQ ID NO:377 is the DNA sequence of His-Trunc maize SDPS2 P148I optimized for E. coli.

[0406] SEQ ID NO:378 is the DNA sequence of His-Trunc maize SDPS2 P148L optimized for E. coli.

[0407] SEQ ID NO:379 is the DNA sequence of His-Trunc maize SDPS2 P148M optimized for E. coli.

[0408] SEQ ID NO:380 is the DNA sequence of His-Trunc maize SDPS2 P148T optimized for E. coli.

[0409] SEQ ID NO:381 is the DNA sequence of His-Trunc maize SDPS2 P148V optimized for E. coli.

[0410] SEQ ID NO:382 is the DNA sequence of His-Trunc maize SDPS2 V151E optimized for E. coli.

[0411] SEQ ID NO:383 is the DNA sequence of His-Trunc maize SDPS2 V151F optimized for E. coli.

[0412] SEQ ID NO:384 is the DNA sequence of His-Trunc maize SDPS2 V151I optimized for E. coli.

[0413] SEQ ID NO:385 is the DNA sequence of His-Trunc maize SDPS2 V151M optimized for E. coli.

[0414] SEQ ID NO:386 is the DNA sequence of His-Trunc maize SDPS2 V151N optimized for E. coli.

[0415] SEQ ID NO:387 is the DNA sequence of His-Trunc maize SDPS2 L174F optimized for E. coli.

[0416] SEQ ID NO:388 is the DNA sequence of His-Trunc maize SDPS2 L174T optimized for E. coli.

[0417] SEQ ID NO:389 is the DNA sequence of His-Trunc maize SDPS2 A175I optimized for E. coli.

[0418] SEQ ID NO:390 is the DNA sequence of His-Trunc maize SDPS2 A175P optimized for E. coli.

[0419] SEQ ID NO:391 is the DNA sequence of His-Trunc maize SDPS2 A175S optimized for E. coli.

[0420] SEQ ID NO:392 is the DNA sequence of His-Trunc maize SDPS2 E176A optimized for E. coli.

[0421] SEQ ID NO:393 is the DNA sequence of His-Trunc maize SDPS2 E176D optimized for E. coli.

[0422] SEQ ID NO:394 is the DNA sequence of His-Trunc maize SDPS2 E176H optimized for E. coli.

[0423] SEQ ID NO:395 is the DNA sequence of His-Trunc maize SDPS2 E176K optimized for E. coli.

[0424] SEQ ID NO:396 is the DNA sequence of His-Trunc maize SDPS2 E176N optimized for E. coli.

[0425] SEQ ID NO:397 is the DNA sequence of His-Trunc maize SDPS2 E176P optimized for E. coli.

[0426] SEQ ID NO:398 is the DNA sequence of His-Trunc maize SDPS2 E176Y optimized for E. coli.

[0427] SEQ ID NO:399 is the DNA sequence of His-Trunc maize SDPS2 I177A optimized for E. coli.

[0428] SEQ ID NO:400 is the DNA sequence of His-Trunc maize SDPS2 I177C optimized for E. coli.

[0429] SEQ ID NO:401 is the DNA sequence of His-Trunc maize SDPS2 I177F optimized for E. coli.

[0430] SEQ ID NO:402 is the DNA sequence of His-Trunc maize SDPS2 I177L optimized for E. coli.

[0431] SEQ ID NO:403 is the DNA sequence of His-Trunc maize SDPS2 I177M optimized for E. coli.

[0432] SEQ ID NO:404 is the DNA sequence of His-Trunc maize SDPS2 I177S optimized for E. coli.

[0433] SEQ ID NO:405 is the DNA sequence of His-Trunc maize SDPS2 I177T optimized for E. coli.

[0434] SEQ ID NO:406 is the DNA sequence of His-Trunc maize SDPS2 I177Y optimized for E. coli.

[0435] SEQ ID NO:407 is the DNA sequence of His-Trunc maize SDPS2 I178G optimized for E. coli.

[0436] SEQ ID NO:408 is the DNA sequence of His-Trunc maize SDPS2 I178Q optimized for E. coli.

[0437] SEQ ID NO:409 is the DNA sequence of His-Trunc maize SDPS2 I178W optimized for E. coli.

[0438] SEQ ID NO:410 is the DNA sequence of His-Trunc maize SDPS2 E179I optimized for E. coli.

[0439] SEQ ID NO:411 is the DNA sequence of His-Trunc maize SDPS2 M180I optimized for E. coli.

[0440] SEQ ID NO:412 is the DNA sequence of His-Trunc maize SDPS2 M180Q optimized for E. coli.

[0441] SEQ ID NO:413 is the DNA sequence of His-Trunc maize SDPS2 M180S optimized for E. coli.

[0442] SEQ ID NO:414 is the DNA sequence of His-Trunc maize SDPS2 M180Y optimized for E. coli.

[0443] SEQ ID NO:415 is the DNA sequence of His-Trunc maize SDPS2 M180W optimized for E. coli.

[0444] SEQ ID NO:416 is the DNA sequence of His-Trunc maize SDPS2 I181M optimized for E. coli.

[0445] SEQ ID NO:417 is the DNA sequence of His-Trunc maize SDPS2 I181N optimized for E. coli.

[0446] SEQ ID NO:418 is the DNA sequence of His-Trunc maize SDPS2 A184G optimized for E. coli.

[0447] SEQ ID NO:419 is the DNA sequence of His-Trunc maize SDPS2 A184S optimized for E. coli.

[0448] SEQ ID NO:420 is the DNA sequence of His-Trunc maize SDPS2 A184T optimized for E. coli.

[0449] SEQ ID NO:421 is the DNA sequence of His-Trunc maize SDPS2 T183C optimized for E. coli.

[0450] SEQ ID NO:422 is the DNA sequence of His-Trunc maize SDPS2 T183Q optimized for E. coli.

[0451] SEQ ID NO:423 is the DNA sequence of His-Trunc maize SDPS2 S185A optimized for E. coli.

[0452] SEQ ID NO:424 is the DNA sequence of His-Trunc maize SDPS2 S185T optimized for E. coli.

[0453] SEQ ID NO:425 is the DNA sequence of His-Trunc maize SDPS2 S185G optimized for E. coli.

[0454] SEQ ID NO:426 is the DNA sequence of His-Trunc maize SDPS2 H188F optimized for E. coli.

[0455] SEQ ID NO:427 is the DNA sequence of His-Trunc maize SDPS2 H188I optimized for E. coli.

[0456] SEQ ID NO:428 is the DNA sequence of His-Trunc maize SDPS2 H188L optimized for E. coli.

[0457] SEQ ID NO:429 is the DNA sequence of His-Trunc maize SDPS2 H188M optimized for E. coli.

[0458] SEQ ID NO:430 is the DNA sequence of His-Trunc maize SDPS2 H188V optimized for E. coli.

[0459] SEQ ID NO:431 is the DNA sequence of His-Trunc maize SDPS2 I187E optimized for E. coli.

[0460] SEQ ID NO:432 is the DNA sequence of His-Trunc maize SDPS2 I187F optimized for E. coli.

[0461] SEQ ID NO:433 is the DNA sequence of His-Trunc maize SDPS2 I187T optimized for E. coli.

[0462] SEQ ID NO:434 is the DNA sequence of His-Trunc maize SDPS2 I187V optimized for E. coli.

[0463] SEQ ID NO:435 is the DNA sequence of His-Trunc maize SDPS2 V191A optimized for E. coli.

[0464] SEQ ID NO:436 is the DNA sequence of His-Trunc maize SDPS2 V191T optimized for E. coli.

[0465] SEQ ID NO:437 is the DNA sequence of His-Trunc maize SDPS2 I204A optimized for E. coli.

[0466] SEQ ID NO:438 is the DNA sequence of His-Trunc maize SDPS2 I204F optimized for E. coli.

[0467] SEQ ID NO:439 is the DNA sequence of His-Trunc maize SDPS2 I204G optimized for E. coli.

[0468] SEQ ID NO:440 is the DNA sequence of His-Trunc maize SDPS2 I204H optimized for E. coli.

[0469] SEQ ID NO:441 is the DNA sequence of His-Trunc maize SDPS2 I204K optimized for E. coli.

[0470] SEQ ID NO:442 is the DNA sequence of His-Trunc maize SDPS2 I204Q optimized for E. coli.

[0471] SEQ ID NO:443 is the DNA sequence of His-Trunc maize SDPS2 I204R optimized for E. coli.

[0472] SEQ ID NO:444 is the DNA sequence of His-Trunc maize SDPS2 I204S optimized for E. coli.

[0473] SEQ ID NO:445 is the DNA sequence of His-Trunc maize SDPS2 I204T optimized for E. coli.

[0474] SEQ ID NO:446 is the DNA sequence of His-Trunc maize SDPS2 Y208A optimized for E. coli.

[0475] SEQ ID NO:447 is the DNA sequence of His-Trunc maize SDPS2 Y208D optimized for E. coli.

[0476] SEQ ID NO:448 is the DNA sequence of His-Trunc maize SDPS2 Y208E optimized for E. coli.

[0477] SEQ ID NO:449 is the DNA sequence of His-Trunc maize SDPS2 Y208H optimized for E. coli.

[0478] SEQ ID NO:450 is the DNA sequence of His-Trunc maize SDPS2 Y208I optimized for E. coli.

[0479] SEQ ID NO:451 is the DNA sequence of His-Trunc maize SDPS2 Y208K optimized for E. coli.

[0480] SEQ ID NO:452 is the DNA sequence of His-Trunc maize SDPS2 Y208L optimized for E. coli.

[0481] SEQ ID NO:453 is the DNA sequence of His-Trunc maize SDPS2 Y208M optimized for E. coli.

[0482] SEQ ID NO:454 is the DNA sequence of His-Trunc maize SDPS2 Y208N optimized for E. coli.

[0483] SEQ ID NO:455 is the DNA sequence of His-Trunc maize SDPS2 Y208Q optimized for E. coli.

[0484] SEQ ID NO:456 is the DNA sequence of His-Trunc maize SDPS2 Y208R optimized for E. coli.

[0485] SEQ ID NO:457 is the DNA sequence of His-Trunc maize SDPS2 Y208S optimized for E. coli.

[0486] SEQ ID NO:458 is the DNA sequence of His-Trunc maize SDPS2 Y208T optimized for E. coli.

[0487] SEQ ID NO:459 is the DNA sequence of His-Trunc maize SDPS2 Y208V optimized for E. coli.

[0488] SEQ ID NO:460 is the DNA sequence of His-Trunc maize SDPS2 G209N optimized for E. coli.

[0489] SEQ ID NO:461 is the DNA sequence of His-Trunc maize SDPS2 T210Y optimized for E. coli.

[0490] SEQ ID NO:462 is the DNA sequence of His-Trunc maize SDPS2 R211D optimized for E. coli.

[0491] SEQ ID NO:463 is the DNA sequence of His-Trunc maize SDPS2 R211E optimized for E. coli.

[0492] SEQ ID NO:464 is the DNA sequence of His-Trunc maize SDPS2 R211N optimized for E. coli.

[0493] SEQ ID NO:465 is the DNA sequence of His-Trunc maize SDPS2 R211T optimized for E. coli.

[0494] SEQ ID NO:466 is the DNA sequence of His-Trunc maize SDPS2 R211V optimized for E. coli.

[0495] SEQ ID NO:467 is the DNA sequence of His-Trunc maize SDPS2 L215I optimized for E. coli.

[0496] SEQ ID NO:468 is the DNA sequence of His-Trunc maize SDPS2 L215M optimized for E. coli.

[0497] SEQ ID NO:469 is the DNA sequence of His-Trunc maize SDPS2 A216T optimized for E. coli.

[0498] SEQ ID NO:470 is the DNA sequence of His-Trunc maize SDPS2 F219A optimized for Escherichia coli.

[0499] SEQ ID NO:471 is the DNA sequence of His-Trunc maize SDPS2 M220I optimized for Escherichia coli.

[0500] SEQ ID NO:472 is the DNA sequence of His-Trunc maize SDPS2 M220C optimized for Escherichia coli.

[0501] SEQ ID NO:473 is the DNA sequence of His-Trunc maize SDPS2 F221W optimized for Escherichia coli.

[0502] SEQ ID NO:474 is the DNA sequence of His-Trunc maize SDPS2 A222G optimized for Escherichia coli.

[0503] SEQ ID NO:475 is the DNA sequence of His-Trunc maize SDPS2 A222M optimized for Escherichia coli.

[0504] SEQ ID NO:476 is the DNA sequence of His-Trunc maize SDPS2 A222S optimized for Escherichia coli.

[0505] SEQ ID NO:477 is the DNA sequence of His-Trunc maize SDPS2 Q223A optimized for Escherichia coli.

[0506] SEQ ID NO:478 is the DNA sequence of His-Trunc maize SDPS2 Q223E optimized for Escherichia coli.

[0507] SEQ ID NO:479 is the DNA sequence of His-Trunc maize SDPS2 Q223F optimized for Escherichia coli.

[0508] SEQ ID NO:480 is the DNA sequence of His-Trunc maize SDPS2 Q223G optimized for Escherichia coli.

[0509] SEQ ID NO:481 is the DNA sequence of His-Trunc maize SDPS2 Q223H optimized for Escherichia coli.

[0510] SEQ ID NO:482 is the DNA sequence of His-Trunc maize SDPS2 Q223I optimized for Escherichia coli.

[0511] SEQ ID NO:483 is the DNA sequence of His-Trunc maize SDPS2 Q223K optimized for E. coli.

[0512] SEQ ID NO:484 is the DNA sequence of His-Trunc maize SDPS2 Q223L optimized for E. coli.

[0513] SEQ ID NO:485 is the DNA sequence of His-Trunc maize SDPS2 Q223M optimized for E. coli.

[0514] SEQ ID NO:486 is the DNA sequence of His-Trunc maize SDPS2 Q223R optimized for E. coli.

[0515] SEQ ID NO:487 is the DNA sequence of His-Trunc maize SDPS2 Q223Y optimized for E. coli.

[0516] SEQ ID NO:488 is the DNA sequence of His-Trunc maize SDPS2 S224F optimized for E. coli.

[0517] SEQ ID NO:489 is the DNA sequence of His-Trunc maize SDPS2 S224I optimized for E. coli.

[0518] SEQ ID NO:490 is the DNA sequence of His-Trunc maize SDPS2 S224M optimized for E. coli.

[0519] SEQ ID NO:491 is the DNA sequence of His-Trunc maize SDPS2 S224N optimized for E. coli.

[0520] SEQ ID NO:492 is the DNA sequence of His-Trunc maize SDPS2 S224Q optimized for E. coli.

[0521] SEQ ID NO:493 is the DNA sequence of His-Trunc maize SDPS2 S224T optimized for E. coli.

[0522] SEQ ID NO:494 is the DNA sequence of His-Trunc maize SDPS2 S224V optimized for E. coli.

[0523] SEQ ID NO:495 is the DNA sequence of His-Trunc maize SDPS2 S225C optimized for E. coli.

[0524] SEQ ID NO:496 is the DNA sequence of His-Trunc maize SDPS2 S225F optimized for Escherichia coli.

[0525] SEQ ID NO:497 is the DNA sequence of His-Trunc maize SDPS2 S225H optimized for Escherichia coli.

[0526] SEQ ID NO:498 is the DNA sequence of His-Trunc maize SDPS2 S225I optimized for Escherichia coli.

[0527] SEQ ID NO:499 is the DNA sequence of His-Trunc maize SDPS2 S225K optimized for Escherichia coli.

[0528] SEQ ID NO:500 is the DNA sequence of His-Trunc maize SDPS2 S225M optimized for Escherichia coli.

[0529] SEQ ID NO:501 is the DNA sequence of His-Trunc maize SDPS2 S225N optimized for Escherichia coli.

[0530] SEQ ID NO:502 is the DNA sequence of His-Trunc maize SDPS2 S225Q optimized for Escherichia coli.

[0531] SEQ ID NO:503 is the DNA sequence of His-Trunc maize SDPS2 S225T optimized for Escherichia coli.

[0532] SEQ ID NO:504 is the DNA sequence of His-Trunc maize SDPS2 S225V optimized for Escherichia coli.

[0533] SEQ ID NO:505 is the DNA sequence of His-Trunc maize SDPS2 S225Y optimized for Escherichia coli.

[0534] SEQ ID NO:506 is the DNA sequence of His-Trunc maize SDPS2 W226A optimized for Escherichia coli.

[0535] SEQ ID NO:507 is the DNA sequence of His-Trunc maize SDPS2 W226C optimized for Escherichia coli.

[0536] SEQ ID NO:508 is the DNA sequence of His-Trunc maize SDPS2 W226E optimized for Escherichia coli.

[0537] SEQ ID NO:509 is the DNA sequence of E. coli - optimized His - Trunc maize SDPS2 W226I.

[0538] SEQ ID NO:510 is the DNA sequence of E. coli - optimized His - Trunc maize SDPS2 W226L.

[0539] SEQ ID NO:511 is the DNA sequence of E. coli - optimized His - Trunc maize SDPS2 W226Q.

[0540] SEQ ID NO:512 is the DNA sequence of E. coli - optimized His - Trunc maize SDPS2 W226R.

[0541] SEQ ID NO:513 is the DNA sequence of E. coli - optimized His - Trunc maize SDPS2 W226T.

[0542] SEQ ID NO:514 is the DNA sequence of E. coli - optimized His - Trunc maize SDPS2 W226V.

[0543] SEQ ID NO:515 is the DNA sequence of E. coli - optimized His - Trunc maize SDPS2 F227D.

[0544] SEQ ID NO:516 is the DNA sequence of E. coli - optimized His - Trunc maize SDPS2 F227L.

[0545] SEQ ID NO:517 is the DNA sequence of E. coli - optimized His - Trunc maize SDPS2 F227M.

[0546] SEQ ID NO:518 is the DNA sequence of E. coli - optimized His - Trunc maize SDPS2 F227R.

[0547] SEQ ID NO:519 is the DNA sequence of E. coli - optimized His - Trunc maize SDPS2 F227V.

[0548] SEQ ID NO:520 is the DNA sequence of E. coli - optimized His - Trunc maize SDPS2 F227W.

[0549] SEQ ID NO:521 is the DNA sequence of E. coli - optimized His - Trunc maize SDPS2 L228C.

[0550] SEQ ID NO:522 is the DNA sequence of His-Trunc maize SDPS2 L228I optimized for E. coli.

[0551] SEQ ID NO:523 is the DNA sequence of His-Trunc maize SDPS2 L228M optimized for E. coli.

[0552] SEQ ID NO:524 is the DNA sequence of His-Trunc maize SDPS2 L228T optimized for E. coli.

[0553] SEQ ID NO:525 is the DNA sequence of His-Trunc maize SDPS2 L228V optimized for E. coli.

[0554] SEQ ID NO:526 is the DNA sequence of His-Trunc maize SDPS2 A229H optimized for E. coli.

[0555] SEQ ID NO:527 is the DNA sequence of His-Trunc maize SDPS2 A229I optimized for E. coli.

[0556] SEQ ID NO:528 is the DNA sequence of His-Trunc maize SDPS2 A229L optimized for E. coli.

[0557] SEQ ID NO:529 is the DNA sequence of His-Trunc maize SDPS2 A229M optimized for E. coli.

[0558] SEQ ID NO:530 is the DNA sequence of His-Trunc maize SDPS2 A229N optimized for E. coli.

[0559] SEQ ID NO:531 is the DNA sequence of His-Trunc maize SDPS2 A229T optimized for E. coli.

[0560] SEQ ID NO:532 is the DNA sequence of His-Trunc maize SDPS2 N230E optimized for E. coli.

[0561] SEQ ID NO:533 is the DNA sequence of His-Trunc maize SDPS2 N230R optimized for E. coli.

[0562] SEQ ID NO:534 is the DNA sequence of His-Trunc maize SDPS2 E235G optimized for E. coli.

[0563] SEQ ID NO:535 is the DNA sequence of His-Trunc maize SDPS2 K238G optimized for Escherichia coli.

[0564] SEQ ID NO:536 is the DNA sequence of His-Trunc maize SDPS2 K238N optimized for Escherichia coli.

[0565] SEQ ID NO:537 is the DNA sequence of His-Trunc maize SDPS2 K238S optimized for Escherichia coli.

[0566] SEQ ID NO:538 is the DNA sequence of His-Trunc maize SDPS2 L239A optimized for Escherichia coli.

[0567] SEQ ID NO:539 is the DNA sequence of His-Trunc maize SDPS2 L239R optimized for Escherichia coli.

[0568] SEQ ID NO:540 is the DNA sequence of His-Trunc maize SDPS2 I240A optimized for Escherichia coli.

[0569] SEQ ID NO:541 is the DNA sequence of His-Trunc maize SDPS2 I240C optimized for Escherichia coli.

[0570] SEQ ID NO:542 is the DNA sequence of His-Trunc maize SDPS2 I240W optimized for Escherichia coli.

[0571] SEQ ID NO:543 is the DNA sequence of His-Trunc maize SDPS2 S241A optimized for Escherichia coli.

[0572] SEQ ID NO:544 is the DNA sequence of His-Trunc maize SDPS2 S241H optimized for Escherichia coli.

[0573] SEQ ID NO:545 is the DNA sequence of His-Trunc maize SDPS2 S241N optimized for Escherichia coli.

[0574] SEQ ID NO:546 is the DNA sequence of His-Trunc maize SDPS2 S241T optimized for Escherichia coli.

[0575] SEQ ID NO:547 is the DNA sequence of His-Trunc maize SDPS2 V243A optimized for Escherichia coli.

[0576] SEQ ID NO:548 is the DNA sequence of His-Trunc maize SDPS2 V243G optimized for Escherichia coli.

[0577] SEQ ID NO:549 is the DNA sequence of His-Trunc maize SDPS2 V243N optimized for Escherichia coli.

[0578] SEQ ID NO:550 is the DNA sequence of His-Trunc maize SDPS2 V243Q optimized for Escherichia coli.

[0579] SEQ ID NO:551 is the DNA sequence of His-Trunc maize SDPS2 V243S optimized for Escherichia coli.

[0580] SEQ ID NO:552 is the DNA sequence of His-Trunc maize SDPS2 I244A optimized for Escherichia coli.

[0581] SEQ ID NO:553 is the DNA sequence of His-Trunc maize SDPS2 I244F optimized for Escherichia coli.

[0582] SEQ ID NO:554 is the DNA sequence of His-Trunc maize SDPS2 I244G optimized for Escherichia coli.

[0583] SEQ ID NO:555 is the DNA sequence of His-Trunc maize SDPS2 I244H optimized for Escherichia coli.

[0584] SEQ ID NO:556 is the DNA sequence of His-Trunc maize SDPS2 I244K optimized for Escherichia coli.

[0585] SEQ ID NO:557 is the DNA sequence of His-Trunc maize SDPS2 I244L optimized for Escherichia coli.

[0586] SEQ ID NO:558 is the DNA sequence of His-Trunc maize SDPS2 I244M optimized for Escherichia coli.

[0587] SEQ ID NO:559 is the DNA sequence of His-Trunc maize SDPS2 I244N optimized for Escherichia coli.

[0588] SEQ ID NO:560 is the DNA sequence of His-Trunc maize SDPS2 I244P optimized for Escherichia coli.

[0589] SEQ ID NO:561 is the DNA sequence of His-Trunc maize SDPS2 I244Q optimized for Escherichia coli.

[0590] SEQ ID NO:562 is the DNA sequence of His-Trunc maize SDPS2 I244S optimized for Escherichia coli.

[0591] SEQ ID NO:563 is the DNA sequence of His-Trunc maize SDPS2 I244V optimized for Escherichia coli.

[0592] SEQ ID NO:564 is the DNA sequence of His-Trunc maize SDPS2 I244Y optimized for Escherichia coli.

[0593] SEQ ID NO:565 is the DNA sequence of His-Trunc maize SDPS2 K245F optimized for Escherichia coli.

[0594] SEQ ID NO:566 is the DNA sequence of His-Trunc maize SDPS2 K245H optimized for Escherichia coli.

[0595] SEQ ID NO:567 is the DNA sequence of His-Trunc maize SDPS2 K245M optimized for Escherichia coli.

[0596] SEQ ID NO:568 is the DNA sequence of His-Trunc maize SDPS2 K245N optimized for Escherichia coli.

[0597] SEQ ID NO:569 is the DNA sequence of His-Trunc maize SDPS2 K245W optimized for Escherichia coli.

[0598] SEQ ID NO:570 is the DNA sequence of His-Trunc maize SDPS2 D246E optimized for Escherichia coli.

[0599] SEQ ID NO:571 is the DNA sequence of His-Trunc maize SDPS2 D246M optimized for Escherichia coli.

[0600] SEQ ID NO:572 is the DNA sequence of His-Trunc maize SDPS2 D246N optimized for Escherichia coli.

[0601] SEQ ID NO:573 is the DNA sequence of His-Trunc maize SDPS2 D246Q optimized for Escherichia coli.

[0602] SEQ ID NO:574 is the DNA sequence of His-Trunc maize SDPS2 D246S optimized for E. coli.

[0603] SEQ ID NO:575 is the DNA sequence of His-Trunc maize SDPS2 D246T optimized for E. coli.

[0604] SEQ ID NO:576 is the DNA sequence of His-Trunc maize SDPS2 D246Y optimized for E. coli.

[0605] SEQ ID NO:577 is the DNA sequence of His-Trunc maize SDPS2 F247E optimized for E. coli.

[0606] SEQ ID NO:578 is the DNA sequence of His-Trunc maize SDPS2 F247L optimized for E. coli.

[0607] SEQ ID NO:579 is the DNA sequence of His-Trunc maize SDPS2 F247M optimized for E. coli.

[0608] SEQ ID NO:580 is the DNA sequence of His-Trunc maize SDPS2 F247N optimized for E. coli.

[0609] SEQ ID NO:581 is the DNA sequence of His-Trunc maize SDPS2 F247V optimized for E. coli.

[0610] SEQ ID NO:582 is the DNA sequence of His-Trunc maize SDPS2 A248P optimized for E. coli.

[0611] SEQ ID NO:583 is the DNA sequence of His-Trunc maize SDPS2 S249A optimized for E. coli.

[0612] SEQ ID NO:584 is the DNA sequence of His-Trunc maize SDPS2 S249E optimized for E. coli.

[0613] SEQ ID NO:585 is the DNA sequence of His-Trunc maize SDPS2 S249F optimized for E. coli.

[0614] SEQ ID NO:586 is the DNA sequence of His-Trunc maize SDPS2 S249G optimized for E. coli.

[0615] SEQ ID NO:587 is the DNA sequence of His-Trunc maize SDPS2 S249K optimized for Escherichia coli.

[0616] SEQ ID NO:588 is the DNA sequence of His-Trunc maize SDPS2 S249L optimized for Escherichia coli.

[0617] SEQ ID NO:589 is the DNA sequence of His-Trunc maize SDPS2 S249N optimized for Escherichia coli.

[0618] SEQ ID NO:590 is the DNA sequence of His-Trunc maize SDPS2 S249Q optimized for Escherichia coli.

[0619] SEQ ID NO:591 is the DNA sequence of His-Trunc maize SDPS2 S249T optimized for Escherichia coli.

[0620] SEQ ID NO:592 is the DNA sequence of His-Trunc maize SDPS2 S249V optimized for Escherichia coli.

[0621] SEQ ID NO:593 is the DNA sequence of His-Trunc maize SDPS2 S249Y optimized for Escherichia coli.

[0622] SEQ ID NO:594 is the DNA sequence of His-Trunc maize SDPS2 G250A optimized for Escherichia coli.

[0623] SEQ ID NO:595 is the DNA sequence of His-Trunc maize SDPS2 I252L optimized for Escherichia coli.

[0624] SEQ ID NO:596 is the DNA sequence of His-Trunc maize SDPS2 I252M optimized for Escherichia coli.

[0625] SEQ ID NO:597 is the DNA sequence of His-Trunc maize SDPS2 I252V optimized for Escherichia coli.

[0626] SEQ ID NO:598 is the DNA sequence of His-Trunc maize SDPS2 K253L optimized for Escherichia coli.

[0627] SEQ ID NO:599 is the DNA sequence of His-Trunc maize SDPS2 A255T optimized for Escherichia coli.

[0628] SEQ ID NO:600 is the DNA sequence of His-Trunc maize SDPS2 A255W optimized for E. coli.

[0629] SEQ ID NO:601 is the DNA sequence of His-Trunc maize SDPS2 S256N optimized for E. coli.

[0630] SEQ ID NO:602 is the DNA sequence of His-Trunc maize SDPS2 T257E optimized for E. coli.

[0631] SEQ ID NO:603 is the DNA sequence of His-Trunc maize SDPS2 T257G optimized for E. coli.

[0632] SEQ ID NO:604 is the DNA sequence of His-Trunc maize SDPS2 T257H optimized for E. coli.

[0633] SEQ ID NO:605 is the DNA sequence of His-Trunc maize SDPS2 T257M optimized for E. coli.

[0634] SEQ ID NO:606 is the DNA sequence of His-Trunc maize SDPS2 T257Q optimized for E. coli.

[0635] SEQ ID NO:607 is the DNA sequence of His-Trunc maize SDPS2 T257W optimized for E. coli.

[0636] SEQ ID NO:608 is the DNA sequence of His-Trunc maize SDPS2 Y274D optimized for E. coli.

[0637] SEQ ID NO:609 is the DNA sequence of His-Trunc maize SDPS2 Y274G optimized for E. coli.

[0638] SEQ ID NO:610 is the DNA sequence of His-Trunc maize SDPS2 Y274L optimized for E. coli.

[0639] SEQ ID NO:611 is the DNA sequence of His-Trunc maize SDPS2 Y274M optimized for E. coli.

[0640] SEQ ID NO:612 is the DNA sequence of His-Trunc maize SDPS2 Y274Q optimized for E. coli.

[0641] SEQ ID NO:613 is the DNA sequence of His-Truncated maize SDPS2 T276S optimized for E. coli.

[0642] SEQ ID NO:614 is the DNA sequence of His-Truncated maize SDPS2 L279F optimized for E. coli.

[0643] SEQ ID NO:615 is the DNA sequence of His-Truncated maize SDPS2 I280W optimized for E. coli.

[0644] SEQ ID NO:616 is the DNA sequence of His-Truncated maize SDPS2 I280F optimized for E. coli.

[0645] SEQ ID NO:617 is the DNA sequence of His-Truncated maize SDPS2 A282G optimized for E. coli.

[0646] SEQ ID NO:618 is the DNA sequence of His-Truncated maize SDPS2 A282H optimized for E. coli.

[0647] SEQ ID NO:619 is the DNA sequence of His-Truncated maize SDPS2 A282K optimized for E. coli.

[0648] SEQ ID NO:620 is the DNA sequence of His-Truncated maize SDPS2 A282N optimized for E. coli.

[0649] SEQ ID NO:621 is the DNA sequence of His-Truncated maize SDPS2 A282R optimized for E. coli.

[0650] SEQ ID NO:622 is the DNA sequence of His-Truncated maize SDPS2 S283C optimized for E. coli.

[0651] SEQ ID NO:623 is the DNA sequence of His-Truncated maize SDPS2 S283F optimized for E. coli.

[0652] SEQ ID NO:624 is the DNA sequence of His-Truncated maize SDPS2 S283I optimized for E. coli.

[0653] SEQ ID NO:625 is the DNA sequence of His-Truncated maize SDPS2 S283M optimized for E. coli.

[0654] SEQ ID NO:626 is the DNA sequence of His-Trunc maize SDPS2 S283T optimized for E. coli.

[0655] SEQ ID NO:627 is the DNA sequence of His-Trunc maize SDPS2 S283W optimized for E. coli.

[0656] SEQ ID NO:628 is the DNA sequence of His-Trunc maize SDPS2 R306F optimized for E. coli.

[0657] SEQ ID NO:629 is the DNA sequence of His-Trunc maize SDPS2 R306H optimized for E. coli.

[0658] SEQ ID NO:630 is the DNA sequence of His-Trunc maize SDPS2 R306L optimized for E. coli.

[0659] SEQ ID NO:631 is the DNA sequence of His-Trunc maize SDPS2 R306N optimized for E. coli.

[0660] SEQ ID NO:632 is the DNA sequence of His-Trunc maize SDPS2 L310G optimized for E. coli.

[0661] SEQ ID NO:633 is the DNA sequence of His-Trunc maize SDPS2 G309A optimized for E. coli.

[0662] SEQ ID NO:634 is the DNA sequence of His-Trunc maize SDPS2 G309F optimized for E. coli.

[0663] SEQ ID NO:635 is the DNA sequence of His-Trunc maize SDPS2 G309M optimized for E. coli.

[0664] SEQ ID NO:636 is the DNA sequence of His-Trunc maize SDPS2 G309S optimized for E. coli.

[0665] SEQ ID NO:637 is the DNA sequence of His-Trunc maize SDPS2 L310D optimized for E. coli.

[0666] SEQ ID NO:638 is the DNA sequence of His-Trunc maize SDPS2 L310E optimized for E. coli.

[0667] SEQ ID NO:639 is the DNA sequence of His-Trunc maize SDPS2 L310F optimized for E. coli.

[0668] SEQ ID NO:640 is the DNA sequence of His-Trunc maize SDPS2 L310H optimized for E. coli.

[0669] SEQ ID NO:641 is the DNA sequence of His-Trunc maize SDPS2 L310N optimized for E. coli.

[0670] SEQ ID NO:642 is the DNA sequence of His-Trunc maize SDPS2 L310Q optimized for E. coli.

[0671] SEQ ID NO:643 is the DNA sequence of His-Trunc maize SDPS2 L310W optimized for E. coli.

[0672] SEQ ID NO:644 is the DNA sequence of His-Trunc maize SDPS2 L310Y optimized for E. coli.

[0673] SEQ ID NO:645 is the DNA sequence of His-Trunc maize SDPS2 F312C optimized for E. coli.

[0674] SEQ ID NO:646 is the DNA sequence of His-Trunc maize SDPS2 F312I optimized for E. coli.

[0675] SEQ ID NO:647 is the DNA sequence of His-Trunc maize SDPS2 F312L optimized for E. coli.

[0676] SEQ ID NO:648 is the DNA sequence of His-Trunc maize SDPS2 F312M optimized for E. coli.

[0677] SEQ ID NO:649 is the DNA sequence of His-Trunc maize SDPS2 F312V optimized for E. coli.

[0678] SEQ ID NO:650 is the DNA sequence of His-Trunc maize SDPS2 Q313A optimized for E. coli.

[0679] SEQ ID NO:651 is the DNA sequence of His-Trunc maize SDPS2 Q313C optimized for E. coli.

[0680] SEQ ID NO:652 is the DNA sequence of His-Truncated maize SDPS2 Q313D optimized for Escherichia coli.

[0681] SEQ ID NO:653 is the DNA sequence of His-Truncated maize SDPS2 Q313S optimized for Escherichia coli.

[0682] SEQ ID NO:654 is the DNA sequence of His-Truncated maize SDPS2 Q313T optimized for Escherichia coli.

[0683] SEQ ID NO:655 is the SDPS protein sequence motif of N, X1, N, X2, X3, X4, X5, X6, G, X7, X8, X9, P, X10, X11, X12, X13, A, X14, X15, Q, I, X16, X17, A, G, G, K.

[0684] SEQ ID NO:656 is the SDPS protein sequence motif of K, X1, X2, R, X3, X4, X5, X6, F, L.

[0685] SEQ ID NO:657 is the SDPS protein sequence motif of H, X1, R, X2, X3, X4, X5, X6, 7, X8, X9, H, X10, X11, X12, L, X13, X14, D, D, X15, X16, D.

[0686] SEQ ID NO:658 is the SDPS protein sequence motif of G, X1, X2, T, X3, X4, X5, X6, X7, X8, X9, X10, X11, A, V, X12, X13, G, D, X14.

[0687] SEQ ID NO:659 is the SDPS protein sequence motif of X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, L, E.

[0688] SEQ ID NO:660 is the SDPS protein sequence motif of N, X1, X2, V, I, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, X14, X15, E, X16, X17, Q, X18, X19, X20.

[0689] SEQ ID NO:661 is the SDPS protein sequence motif of S, X1, X2, K, X3, A, S, X4, X5, A, X6, X7, X8.

[0690] SEQ ID NO:662 is the SDPS protein sequence motif of G, X1, X2, L, X3, X4, X5, X6, X7, V, V.

[0691] SEQ ID NO:663 is the protein sequence of His-Truncated Zea mays SDPS2 N128Y.

[0692] SEQ ID NO:664 is the protein sequence of His-Truncated Zea mays SDPS2 T183G.

[0693] SEQ ID NO:665 is the protein sequence of His-Truncated Zea mays SDPS2 A184C.

[0694] SEQ ID NO:666 is the DNA sequence of E. coli-optimized His-Truncated Zea mays SDPS2 N128Y.

[0695] SEQ ID NO:667 is the DNA sequence of E. coli-optimized His-Truncated Zea mays SDPS2 T183C.

[0696] SEQ ID NO:668 is the DNA sequence of E. coli-optimized His-Truncated Zea mays SDPS2 A184C. BRIEF DESCRIPTION OF THE DRAWINGS

[0697] Figure 1 Shows a graphical representation of vector F240L for transformation and gene insertion.

[0698] Figure 2 Shows a graphical representation of a vector for transformation and gene editing.

[0699] Figure 3 Is a table showing the damage scores of 4 plant populations expressing various SDPS genes. DETAILED DESCRIPTION

[0700] This description is not intended to be an exhaustive catalog of all the different ways in which the invention can be implemented or all the features that can be added to the invention. For example, features illustrated with respect to one embodiment can be incorporated into other embodiments, and features illustrated with respect to a particular embodiment can be deleted from that embodiment. Thus, the invention contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. Additionally, in light of this disclosure, numerous variations and additional embodiments of the different embodiments presented herein will be apparent to those skilled in the art, and this does not depart from the invention. Accordingly, the following description is intended to illustrate some particular embodiments of the invention and does not exhaustively recite all of its permutations, combinations, and variations.

[0701] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terms used in the description of the invention herein are for the purpose of describing particular embodiments only and are not intended to limit the invention.

[0702] Definitions

[0703] As used herein and in the appended claims, the singular forms "a / an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a plant" refers to one or more plants and includes equivalents thereof known to those skilled in the art, and the like.

[0704] As used herein, the phrase "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, together with the absence of combinations when interpreted in an alternative ("or") sense.

[0705] The term "about" is used herein to mean approximately, roughly, around or in the vicinity of. When the term "about" is used in conjunction with a numerical range, it delimits that range by extending the boundaries above and below the stated numerical value. In general, the term "about" is used herein to limit a numerical value to a variation of 20%, preferably plus or minus 10% (higher or lower), above and below the specified value. With regard to temperature, the term "about" means ±1 °C, preferably ±0.5 °C. When the term "about" is used in the context of the present invention (e.g., in combination with temperature or molecular weight values), the exact value (i.e., without "about") is preferred.

[0706] As used herein, the term "amplified" means the construction of multiple copies of a nucleic acid molecule or multiple copies complementary to the nucleic acid molecule using at least one nucleic acid molecule as a template. Amplification systems include polymerase chain reaction (PCR) systems, ligase chain reaction (LCR) systems, nucleic acid sequence-based amplification (NASBA, Cangene, Mississauga, Ontario), Q-β replicase systems, transcription-based amplification systems (TAS), and strand displacement amplification (SDA). See, e.g., Diagnostic Molecular Microbiology: Principles and Applications, edited by PERSING et al., American Society for Microbiology, Washington, D.C., (1993). The amplified product is referred to as an "amplicon".

[0707] "Assembled sequence", "assembled polynucleotide", "assembled nucleotide sequence", etc. according to the present invention are synthetic polynucleotides prepared by aligning overlapping sequences of polynucleotides or portions of sequenced polynucleotides (i.e., k-mers, all possible subsequences of length k of the reads obtained by DNA sequencing), which are determined from genomic DNA using DNA sequencing techniques. Assembled sequences typically contain base-calling errors, which may be misdetermined bases, insertions, and / or deletions compared to the native DNA sequence contained in the genome from which the genomic DNA was obtained. Thus, for example, an "assembled polynucleotide" may encode a protein, and according to the present invention, neither the polynucleotide nor the protein is a natural product but exists only through human action.

[0708] As used herein, the terms "chimeric construct" or "chimeric gene" or "chimeric polynucleotide" or "chimeric nucleic acid" (or like terms) refer to a construct or molecule that comprises two or more polynucleotides of different origin assembled into a single nucleic acid molecule. The terms "chimeric construct", "chimeric gene", "chimeric polynucleotide" or "chimeric nucleic acid" refer to any construct or molecule that contains, but is not limited to, (1) a polynucleotide (e.g., DNA) that includes regulatory and coding polynucleotides that are not found together in nature (i.e., at least one polynucleotide in the construct is heterologous relative to at least one of its other polynucleotides), or (2) a polynucleotide that encodes protein moieties that are not naturally contiguous, or (3) promoter portions that are not naturally contiguous. Additionally, a chimeric construct, chimeric gene, chimeric polynucleotide or chimeric nucleic acid can comprise regulatory and coding polynucleotides derived from different sources, or can comprise regulatory and coding polynucleotides derived from the same source but arranged in a manner different from that found in nature. In some embodiments of the invention, the chimeric construct, chimeric gene, chimeric polynucleotide or chimeric nucleic acid comprises an expression cassette that contains a polynucleotide of the invention under the control of a regulatory polynucleotide that is functional, particularly in a plant or a bacterium, under the control of a regulatory polynucleotide.

[0709] A "coding sequence" is a nucleic acid sequence that is transcribed into RNA (such as mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA). Preferably, the RNA is then translated in an organism to produce a protein.

[0710] As used herein, a "codon-optimized" sequence means a nucleotide sequence in which the codons are selected to reflect the particular codon preference that a host cell or organism may have. This is typically done in such a way as to maintain the amino acid sequence of the polypeptide encoded by the nucleotide sequence to be optimized. In certain embodiments, the DNA sequence of a recombinant DNA construct includes a sequence that has been codon-optimized for the cell (e.g., an animal, plant, or fungal cell) in which the construct is to be expressed. For example, a construct to be expressed in a plant cell can have all or part of its sequence (e.g., a first gene silencing element or gene expression element) codon-optimized for expression in plants. See, e.g., U.S. Patent No. 6,121,014, which is incorporated herein by reference.

[0711] The term "comprises" or "comprising", as used in this specification, indicates the presence of the stated feature, integer, step, operation, element, or component, but does not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, or groups thereof.

[0712] As used herein, the transitional phrase "consisting essentially of" (and grammatical variations thereof) means that the scope of the claim is to be interpreted to cover the specified materials or steps recited in the claim and those that do not materially affect one or more of the basic and novel features of the claimed invention. Thus, when used in the claims of the present invention, the term "consisting essentially of" is not intended to be construed as equivalent to "comprising".

[0713] In the context of the present invention, "corresponding to" means that when referring to the amino acid sequence of a variant or homolog of SDPS, the amino acids "corresponding to" certain enumerated positions in that variant or homolog protein are those that align with these positions in the reference protein, but are not necessarily in these exact numerical positions relative to the specific reference amino acid sequence of the present invention.

[0714] The term "domain" refers to a group of amino acids that are conserved at specific positions along the alignment of the sequences of evolutionarily related proteins. While the amino acids at other positions may vary among homologs, the amino acids that are highly conserved at specific positions indicate amino acids that are likely to be essential for the structure, stability, or function of the protein. Identified by its high conservation in the aligned sequences of a family of protein homologs, it can be used as an identifier to determine whether any polypeptide under discussion belongs to a previously identified group of polypeptides.

[0715] As used herein, an "expression cassette" means a nucleic acid molecule capable of directing the expression of at least one polynucleotide of interest (e.g., a polynucleotide encoding SDPS) in a suitable host cell, the nucleic acid molecule comprising a promoter operably linked to the polynucleotide of interest (which is operably linked to a termination signal). An "expression cassette" also typically comprises additional polynucleotides required for the correct translation of the polynucleotide of interest. The expression cassette may also comprise other polynucleotides that are not necessary for the direct expression of the polynucleotide of interest but are present due to convenient restriction sites for removing the expression cassette from an expression vector. An expression cassette comprising one or more polynucleotides of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression cassette may also be a naturally occurring expression cassette that has been obtained in a recombinant form useful for heterologous expression. However, typically, the expression cassette is heterologous to the host, i.e., the polynucleotide of interest in the expression cassette is not naturally present in the host cell and must have been introduced into the host cell or an ancestor of the host cell by a transformation process or a breeding process. The expression of one or more polynucleotides of interest in the expression cassette is generally under the control of a promoter. In the case of a multicellular organism (such as a plant), the promoter may also be specific or preferential for a particular tissue, or organ, or developmental stage. When transformed into a plant, the expression cassette or a fragment thereof may also be referred to as an "inserted polynucleotide" or an "insert polynucleotide".

[0716] As used herein, in reference to a particular polynucleotide, a "full-length sequence" means a complete nucleic acid sequence having the native or mutant SDPS sequence. A "native sequence" is intended to mean an endogenous sequence, i.e., a non-engineered sequence found in the genome of an organism.

[0717] Thus, a fragment of a nucleotide sequence of the invention may encode a biologically active portion of an SDPS polypeptide, or it may be a fragment that can be used as a hybridization probe, etc. or a PCR primer (using the methods disclosed below). A biologically active portion of a mutant SDPS polypeptide can be prepared by isolating a portion of one of the nucleotide sequences of the invention, expressing the portion of the encoded mutant SDPS protein (e.g., by in vitro recombinant expression), and assaying the activity of the portion of the encoded mutant SDPS protein. A nucleic acid molecule (a fragment of a nucleotide sequence of the invention) comprises at least 15, 20, 50, 75, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, or 1300 consecutive nucleotides, or up to the number of nucleotides present in the full-length nucleotide sequences disclosed herein.

[0718] "Variant" is intended to mean substantially similar sequences. For polynucleotides, variants include the deletion and / or addition of one or more nucleotides at one or more internal sites within a reference polynucleotide and / or the substitution of one or more nucleotides at one or more sites in a mutant SDPS polynucleotide. As used herein, a "reference" polynucleotide or polypeptide comprises the SDPS nucleotide sequence or amino acid sequence, respectively. As used herein, a "native" polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively. Those of ordinary skill in the art will recognize that variants of these nucleic acids of the invention will be constructed such that the open reading frame is maintained. For polynucleotides, conservative variants include those sequences that encode the amino acid sequence of a mutant SDPS polypeptide of the invention (due to the degeneracy of the genetic code). Such naturally occurring allelic variants can be identified using well-known molecular biology techniques such as the polymerase chain reaction (PCR) and hybridization techniques outlined below. Variant polynucleotides also include synthetically derived polynucleotides, such as those generated by site-directed mutagenesis but still encoding a mutant SDPS protein of the invention. Generally, variants of a particular polynucleotide of the invention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with that particular polynucleotide, as determined by the sequence alignment programs and parameters described elsewhere herein.

[0719] Variants of a particular polynucleotide of the invention (i.e., the reference polynucleotide) can also be evaluated by comparing the percent sequence identity between the polypeptide encoded by the variant polynucleotide and the polypeptide encoded by the reference polynucleotide. The percent sequence identity between any two polypeptides can be calculated using the sequence alignment programs and parameters described elsewhere herein. Where any given pair of polynucleotides of the invention is evaluated by comparing the percent sequence identity shared by the two polypeptides they encode, the percent sequence identity between the two encoded polypeptides is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more across the entirety of the glucosyltransferase sequence described herein.

[0720] A "variant" protein is intended to mean a protein derived from a reference protein by deletion or addition of one or more amino acids at one or more internal sites in the SDPS protein and / or substitution of one or more amino acids at one or more sites in the SDPS protein. Variant proteins encompassed by the present invention are biologically active, i.e., they continue to possess the desired biological activity of the SDPS protein, i.e., the SDPS enzyme activity as described herein. Such variants can arise, for example, from genetic polymorphism or from human manipulation. A biologically active variant of the mutant SDPS protein of the present invention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity across the entire amino acid sequence of the mutant SDPS protein, as determined by the sequence alignment procedures and parameters described elsewhere herein. A biologically active variant of the protein of the present invention may differ from the protein by as few as 1 - 15 amino acid residues, as few as 1 - 10 (such as 6 - 10), as few as 5 (such as 4, 3, 2, or even 1) amino acid residues. For example, a variant amino acid sequence described as F227L will include substitution of the phenylalanine residue at position 227 with a leucine residue.

[0721] "Gene" is defined herein as a genetic unit that contains one or more polynucleotides, that occupies a specific position on a chromosome or plasmid, and that contains the genetic instructions for a specific characteristic or trait in an organism.

[0722] When referring to the use of a gene or polynucleotide or polypeptide, the term "heterologous" means that the gene or polynucleotide or polypeptide is not in its natural environment or is part of an environment in which it does not naturally occur (i.e., has been artificially altered). For example, a heterologous gene can include a polynucleotide introduced from one species into another species. A heterologous gene can also include a polynucleotide that is native to an organism but has been altered in some way (e.g., by mutation; addition of multiple copies; ligation to a non-native promoter or enhancer polynucleotide, etc.). A heterologous gene can further comprise a plant gene polynucleotide that includes the cDNA form of a plant gene; these cDNAs can be expressed in the sense orientation (to produce mRNA) or in the antisense orientation (to produce an antisense RNA transcript that is complementary to the mRNA transcript). In one aspect of the invention, a heterologous gene is distinguished from an endogenous plant gene in that the heterologous gene polynucleotide is typically ligated to a polynucleotide that includes regulatory elements such as a promoter, and these polynucleotides are not found to be naturally associated with the gene encoding the protein encoded by the heterologous gene or with the plant gene polynucleotide in the chromosome, or with a portion of the chromosome not found in nature (e.g., a gene expressed at a locus where the gene is not normally expressed). Additionally, a "heterologous" polynucleotide is a polynucleotide that is not naturally associated with the host cell into which the polynucleotide is introduced, including non-naturally occurring multiple copies of a naturally occurring polynucleotide.

[0723] "Homologous recombination" is the exchange ("crossing over") of DNA segments between two DNA molecules or chromatids of paired chromosomes in regions of the same polynucleotide. A "recombination event" is understood herein to mean meiotic crossing over.

[0724] When a nucleic acid sequence encodes a polypeptide that has the same amino acid sequence as the polypeptide encoded by a reference nucleic acid sequence, such nucleotide sequence "coencodes" with such reference nucleic acid sequence.

[0725] The term "isolated" nucleic acid molecule, polynucleotide or protein is a nucleic acid molecule, polynucleotide or protein that is no longer in its natural environment. The isolated nucleic acid molecules, polynucleotides or proteins of the invention can exist in a purified form or can exist in a recombinant host, such as a transgenic bacterium or a transgenic plant. Thus, the requirements for an "isolated" nucleic acid molecule as recited herein encompass a nucleic acid molecule when it is contained within the genome of a transgenic plant.

[0726] A "nucleic acid molecule" is a single-stranded or double-stranded DNA or RNA that can be isolated from any source or can be synthetically prepared. In the context of the present invention, a nucleic acid molecule is typically a segment of DNA.

[0727] "Operably linked" means the association of polynucleotides on a single nucleic acid fragment such that the function of one affects the function of the other. For example, a promoter is operably linked to a coding polynucleotide or functional RNA when it is capable of affecting the expression of the coding polynucleotide or functional RNA (i.e., the coding polynucleotide or functional RNA is under the transcriptional control of the promoter). A coding polynucleotide in the sense or antisense orientation can be operably linked to a regulatory polynucleotide.

[0728] "Plant" means any plant at any stage of development, particularly seed plants.

[0729] "Plant cell" is the structural and physiological unit of a plant, comprising a protoplast and a cell wall. Plant cells can be in the form of isolated single cells or cultured cells, or as part of a higher organizational unit such as, for example, a plant tissue, a plant organ, or a whole plant.

[0730] "Plant cell culture" means a culture of plant units such as, for example, protoplasts, cultured cells of a cell culture, cells in a plant tissue, pollen, pollen tubes, ovules, embryo sacs, zygotes, and embryos at various stages of development.

[0731] "Plant material" means leaves, stems, roots, flowers or parts of flowers, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant.

[0732] "Plant organ" is a distinct and visibly structured and differentiated part of a plant, such as a root, stem, leaf, bud, or embryo.

[0733] As used herein, "plant tissue" means a group of plant cells organized into structural and functional units. It includes any plant tissue in a plant or in culture. This term includes, but is not limited to: whole plants, plant organs, plant seeds, tissue cultures, and any group of plant cells organized into a structural or functional unit. The combined or separate application of this term with any specific type of plant tissue listed above or otherwise covered by this definition is not intended to exclude any other type of plant tissue.

[0734] "Polynucleotide" refers to a polymer composed of many nucleotide monomers covalently bonded in a chain. Such "polynucleotides" include DNA, RNA, modified oligonucleotides (e.g., oligonucleotides containing bases atypical of biological RNA or DNA, such as 2'-O-methylated oligonucleotides), and the like. In some embodiments, a nucleic acid or polynucleotide can be single-stranded, double-stranded, multi-stranded, or a combination thereof. Unless otherwise indicated, a particular nucleic acid or polynucleotide of the present invention optionally contains or encodes a complementary polynucleotide in addition to any polynucleotide specifically indicated.

[0735] "Target polynucleotide" refers to any polynucleotide that, when transferred into an organism (e.g., a plant), confers a desired trait to that organism, such as insect resistance, disease resistance, herbicide tolerance, antibiotic resistance, improved nutritional value, improved performance in industrial processes, production of commercially valuable enzymes or metabolites, or altered reproductive capacity.

[0736] The term "promoter" refers to a polynucleotide, typically upstream (5') of its coding polynucleotide, that controls the expression of the coding polynucleotide by providing recognition for the RNA polymerase and other factors required for correct transcription.

[0737] "Protoplast" is an isolated plant cell that lacks a cell wall or has only a partial cell wall.

[0738] As used herein, the term "recombinant" refers to a form of a nucleic acid molecule (e.g., DNA or RNA), protein, or organism that is not normally found in nature and is thus produced through human intervention. As used herein, a "recombinant nucleic acid molecule" is a nucleic acid molecule that includes a combination of polynucleotides that do not naturally occur together and are the result of human intervention, e.g., a nucleic acid molecule composed of a combination of at least two polynucleotides that are heterologous to each other, or a nucleic acid molecule that is artificially synthesized (e.g., a polynucleotide synthesized using assembled nucleotide sequences) and contains a polynucleotide that deviates from the polynucleotides normally found in nature, or a nucleic acid molecule that contains a transgene artificially incorporated into the genomic DNA of a host cell and the genomic flanking DNA associated therewith. Another example of a recombinant nucleic acid molecule is a DNA molecule produced by inserting a transgene into the genomic DNA of a plant, which can ultimately result in the expression of a recombinant RNA / or protein molecule in the organism. As used herein, a "recombinant plant" is a plant that is not normally found in nature, is the result of human intervention, and contains a transgene and / or heterologous nucleic acid molecule incorporated into its genome. Due to such genomic alterations, a recombinant plant is significantly different from the related wild-type plant.

[0739] "Regulatory element" refers to a sequence that participates in controlling the expression of a nucleotide sequence. Regulatory elements include a promoter and a termination signal operably linked to a nucleotide sequence of interest. They also typically encompass sequences required for proper translation of the nucleotide sequence, e.g., introns, 3' UTRs, and terminators.

[0740] In the context of two nucleic acid or amino acid sequences, the terms "identity" or "identical" or "substantially identical" refer to two or more sequences or subsequences having at least 60%, preferably at least 80%, more preferably 90%, even more preferably 95%, and most preferably at least 99% nucleotide or amino acid residue identity when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. Preferably, substantial identity exists over a region of the sequences that has a length of at least about 50 residues or bases, more preferably over a region of at least about 100 residues or bases, and most preferably the sequences are substantially identical over at least about 150 residues or bases. In particularly preferred embodiments, the sequences are substantially identical over the entire length of the coding region. Additionally, substantially identical nucleic acid or amino acid sequences perform substantially the same function.

[0741] For sequence comparison, typically, one sequence acts as a reference sequence to which the test sequence is compared. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer (specifying subsequence coordinates if necessary), and the parameters of the sequence algorithm program are designated. The sequence comparison algorithm then calculates the percentage of sequence identity of one or more test sequences relative to the reference sequence based on the designated program parameters.

[0742] Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by the computerized implementation of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin), or by visual inspection (generally see Ausubel et al., infra).

[0743] An example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in the following references: Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information, U.S. National Library of Medicine, 8600 Rockville Pike, Bethesda, MD 20894, USA. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., 1990). These initial neighborhood word hits act as seeds for initiating a search to find longer HSPs containing them. Then, these word hits are extended in both directions along each sequence until the cumulative alignment score can no longer increase. For nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for a mismatch residue; always <0) are used to calculate the cumulative score. For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction is stopped when the cumulative alignment score drops from its maximum achieved value by an amount X; when the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments; or when the end of either sequence is reached. The parameters W, T, and X of the BLAST algorithm determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M = 5, N = -4, and comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad Sci. USA 89:10915 (1989)).

[0744] In addition to calculating the percent sequence identity, the BLAST algorithm performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Nat'l Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the sum probability (P(N)), which gives an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, if the sum probability is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001 in a comparison of a test nucleic acid sequence to a reference nucleic acid sequence, the test nucleic acid sequence is considered similar to the reference sequence.

[0745] Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions. The phrase "specific hybridization" refers to the binding, duplexing, or hybridization of a molecule only to a particular nucleotide sequence under stringent conditions, as occurs when the sequence is present in a complex mixture (e.g., total cellular) DNA or RNA. "Substantially bound" refers to the complementary hybridization between a probe nucleic acid and a target nucleic acid and encompasses minor mismatches that can be accommodated by decreasing the stringency of the hybridization medium to achieve the desired detection of the target nucleic acid sequence.

[0746] In the context of nucleic acid hybridization experiments such as DNA and RNA hybridization, "stringent hybridization conditions" and "stringent hybridization wash conditions" are sequence-dependent and are different under different environmental parameters. Longer sequences hybridize specifically at higher temperatures. Extensive guidance for nucleic acid hybridization is found in the following: Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Acid Probes, Chapter 2, Part I, "Overview of principles of hybridization and the strategy of nucleic acid probe assays," Elsevier, New York. Typically, for a specific sequence at a defined ionic strength and pH, high stringency hybridization and wash conditions are selected to be approximately 5°C lower than the thermal melting point (Tm). Typically, under "stringent conditions," a probe will hybridize to its target sequence but not to other sequences.

[0747] Tm is the temperature at which 50% of the target sequence hybridizes to a perfectly matched probe (at a defined ionic strength and pH). Very stringent conditions are selected to be equal to the Tm for a particular probe. Examples of stringent hybridization conditions for the hybridization of complementary nucleic acids (which have over 100 complementary residues on a filter in a DNA or RNA blot) are hybridization overnight at 42 °C in 50% formamide with 1 mg heparin. Examples of high stringency wash conditions are 0.15 M NaCl at 72 °C for approximately 15 minutes. Examples of stringent wash conditions are 0.2×SSC wash at 65 °C for 15 minutes (see, Sambrook, infra, for description of SSC buffer). Typically, a low stringency wash is performed prior to a high stringency wash to remove background probe signal. Examples of moderate stringency wash for a duplex of, e.g., over 100 nucleotides are 1×SSC at 45 °C for 15 minutes. Examples of low stringency wash for a duplex of, e.g., over 100 nucleotides are 4 - 6×SSC at 40 °C for 15 minutes. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically involve a salt concentration of less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30 °C. Stringent conditions can also be achieved by adding destabilizing agents such as formamide. In general, a signal-to-noise ratio of 2-fold (or higher) above that observed for an unrelated probe in a particular hybridization assay indicates specific hybridization is detected. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially the same if the proteins they encode are substantially the same. For example, this occurs when copies of a nucleic acid are created using the maximum codon degeneracy permitted by the genetic code.

[0748] The following are examples of the settings of hybridization / washing conditions that can be used to clone homologous nucleotide sequences that are substantially identical to the reference nucleotide sequence of the present invention: The reference nucleotide sequence preferably hybridizes to the reference nucleotide sequence under the following conditions: in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50 °C, and washed in 2x SSC, 0.1% SDS at 50 °C; more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50 °C, and washed in 1x SSC, 0.1% SDS at 50 °C; still more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50 °C, and washed in 0.5x SSC, 0.1% SDS at 50 °C; preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50 °C, and washed in 0.1x SSC, 0.1% SDS at 50 °C; more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50 °C, and washed in 0.1x SSC, 0.1% SDS at 65 °C.

[0749] Another indication that two nucleic acid sequences or proteins are substantially identical is that the protein encoded by the first nucleic acid undergoes an immunocrosslinking reaction with or binds specifically to the protein encoded by the second nucleic acid. Thus, the proteins are typically substantially identical to the second protein, for example where the two proteins differ only by conservative substitutions.

[0750] As used herein, "synthetic polynucleotide" refers to a polynucleotide that contains bases or structural features not present in naturally occurring polynucleotides.

[0751] Plants that are substantially "tolerant" to a herbicide (when they are subjected to the herbicide) provide a dose / response curve that is shifted to the right compared to that provided by similarly treated non-tolerant-like plants. Such dose / response curves plot "dose" on the x-axis and "percent kill", "weed control efficacy", etc. on the y-axis. To achieve a given weed control efficacy, tolerant plants typically require at least about twice as much herbicide as non-tolerant-like plants. Plants that are substantially "resistant" to a herbicide show little (if any) necrosis, lysis, chlorosis, or other lesions when subjected to the concentrations and rates of herbicide typically used in the agricultural community to kill weeds in the field.

[0752] "Transformation" is a method for introducing heterologous nucleic acid into a host cell or organism. In particular, "transformation" means that a DNA molecule is stably integrated into the genome of the target organism.

[0753] "Transformed / Transgenic / Recombinant" refers to a host organism, such as a bacterium or a plant, into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome of the host, or the nucleic acid molecule can also exist as an extrachromosomal molecule. Such an extrachromosomal molecule is capable of autonomous replication. Transformed cells, tissues or plants should be understood to cover not only the end products of the transformation process, but also their transgenic progeny. "Non-transformed", "non-transgenic", or "non-recombinant" hosts refer to wild-type organisms, such as bacteria or plants, that do not contain the heterologous nucleic acid molecule.

[0754] For example, once the SDPS polynucleotide conferring herbicide resistance has been cloned into an expression system, either alone or in combination with one or more additional nucleic acid molecules encoding polypeptides conferring desired traits, it has been transformed into a plant cell. The receptor and target expression cassettes of the present invention can be introduced into plant cells in a manner recognized by a variety of techniques. The term "introduced" in the context of a polynucleotide (e.g., a nucleotide construct of interest) is meant to refer to delivering the polynucleotide into a plant in such a way that the polynucleotide gains access to the interior of the plant cell. Where more than one polynucleotide is to be introduced, the polynucleotides can be assembled as part of a single nucleotide construct, or as separate nucleotide constructs, and can be located on the same or different transformation vectors. Thus, or as part of a breeding program, these polynucleotides can be introduced into the target host cell in a single transformation event, in separate transformation events, for example, in a plant. The methods of the present invention do not depend on the particular method used to introduce one or more polynucleotides into a plant, only on gaining access to the interior of at least one cell of the plant for one or more polynucleotides. Methods known in the art for introducing multiple polynucleotides into plants include, but are not limited to, transient transformation methods, stable transformation methods, and virus-mediated methods.

[0755] In the context of a polynucleotide, "transient transformation" is intended to mean that the polynucleotide is introduced into the plant and it does not integrate into the genome of the plant.

[0756] In the context of a polynucleotide introduced into a plant, "stably introducing" or "stably introduced" means that the introduced polynucleotide is stably incorporated into the plant genome, and thus the plant has been stably transformed with the polynucleotide.

[0757] "Stable transformation" or "stably transformed" is intended to mean that a polynucleotide (e.g., a nucleotide construct as described herein) introduced into a plant integrates into the genome of the plant and is capable of being inherited by its progeny, and more particularly, is inherited through multiple successive generations of progeny.

[0758] For those of ordinary skill in the art of plant transformation, numerous transformation vectors are available and known for plant transformation, and the genes relevant to the present invention can be used in conjunction with any such vector. The choice of vector will depend on the preferred transformation technique and the target species to be transformed. For certain target species, different antibiotic or herbicide selectable markers may be preferred. Selectable markers commonly used in transformation include the nptII gene that confers resistance to kanamycin and related antibiotics (Messing and Vierra Gene 19:259-268 (1982); Bevan et al., Nature 304:184-187 (1983)), the pat and bar genes that confer resistance to the herbicide glufosinate (also known as phosphinothricin; see White et al., Nucl. Acids Res 18:1062 (1990), Spencer et al. Theor. Appl. Genet 79:625-631 (1990) and U.S. Patent Nos. 5,561,236 and 5,276,268), the hph gene that confers resistance to the antibiotic hygromycin (Blochinger and Diggelmann, Mol. Cell Biol. 4:2929-2931), and the dhfr gene that confers resistance to methotrexate (Bourouis et al., EMBO J. 2(7):1099-1104 (1983)), the EPSPS gene that confers resistance to glyphosate (U.S. Patent Nos. 4,940,935 and 5,188,642), the glyphosate N-acetyltransferase (GAT) gene that also confers resistance to glyphosate (Castle et al. (2004) Science, 304:1151-1154; U.S. Patent Application Publication Nos. 20070004912, 20050246798 and 20050060767), and the 6-phosphomannose isomerase gene that provides the ability to metabolize mannose (U.S. Patent Nos. 5,767,378 and 5,994,629). Alternatively, and in a preferred embodiment, the SDPS gene of the present invention is used in combination with the use of a suitable substrate PSII herbicide as a selection agent, which itself is used as a selectable marker.

[0759] Methods for regenerating plants are also well known in the art. For example, Ti plasmid vectors have been used for delivering foreign DNA, as well as direct DNA uptake, liposomes, electroporation, microinjection, and microprojectiles. In addition, bacteria from the genus Agrobacterium can be used to transform plant cells. The following are representative techniques for transforming both dicotyledonous and monocotyledonous plants along with descriptions of representative plastid transformation techniques.

[0760] For transformation using Agrobacterium tumefaciens, many vectors are available. These vectors typically carry at least one T-DNA border sequence and include vectors such as pBIN19 (Bevan, Nucl. Acids Res. [Nucleic Acids Research] (1984)). For the construction of vectors useful in Agrobacterium transformation, see, for example, U.S. Patent Application Publication No. 2006 / 0260011, incorporated herein by reference.

[0761] Transformation without using Agrobacterium tumefaciens avoids the need for T-DNA sequences in the selected transformation vector and thus vectors lacking these sequences can be utilized in addition to those containing T-DNA sequences as described above, for example. Agrobacterium-independent transformation techniques include transformation via particle bombardment, protoplast uptake (e.g., PEG and electroporation), and microinjection. The choice of vector depends largely on the preference for the species to be transformed. For the construction of these vectors, see, for example, U.S. Application No. 20060260011, incorporated herein by reference.

[0762] Transformation techniques for dicotyledonous plants are also well known in the art and include Agrobacterium-based techniques as well as techniques that do not require Agrobacterium. Agrobacterium-independent techniques involve the direct uptake of foreign genetic material through protoplasts or cells. This can be accomplished by PEG or electroporation-mediated uptake, particle bombardment-mediated delivery, or microinjection. Examples of these techniques are described by Paszkowski et al., EMBO J. [European Molecular Biology Organization Journal] 3:2717-2722 (1984), Potrykus et al., Mol. Gen. Genet. [Molecular Genetics and General Genetics] 199:169-177 (1985), Reich et al., Biotechnology [Biotechnology] 4:1001-1004 (1986), and Klein et al., Nature [Nature] 327:70-73 (1987). In each case, these transformed cells are regenerated into whole plants using standard techniques known in the art.

[0763] For the transformation of dicotyledonous plants, Agrobacterium-mediated transformation is the preferred technique because of its high transformation efficiency and its broad utility for many different species. Agrobacterium transformation typically involves transferring a binary vector carrying the foreign DNA of interest (e.g., pCIB200 or pCIB2001) into a suitable Agrobacterium strain, which can rely on the complement of vir genes carried by the host Agrobacterium strain (e.g., CIB542 strain for pCIB200 and pCIB2001 (Uknes et al. Plant Cell 5:159-169 (1993))) or on a cointegrated Ti plasmid or on the chromosome. Transfer of the recombinant binary vector into Agrobacterium is achieved by a triparental mating method using Escherichia coli carrying the recombinant binary vector and a helper Escherichia coli strain carrying a plasmid (e.g., pRK2013) and capable of mobilizing the recombinant binary vector into the target Agrobacterium strain. Alternatively, the recombinant binary vector can be transferred into Agrobacterium by DNA transformation (Hofgen and Willmitzer, Nucl. Acids Res. 16:9877 (1988)).

[0764] Transformation of the target plant species by recombinant Agrobacterium typically involves co-cultivation of the Agrobacterium with an explant from the plant and follows protocols well known in the art. The transformed tissue is regenerated on a selective medium carrying an antibiotic marker or a herbicide resistance marker between the T-DNA borders of the binary plasmid.

[0765] Another method for transforming plant cells with genes involves propelling inert or biologically active particles onto plant tissues and cells. This technique is disclosed in U.S. Patent Nos. 4,945,050, 5,036,006, and 5,100,792 (all to Sanford et al.). Generally, this method involves propelling inert or biologically active particles onto cells under conditions effective to penetrate the outer surface of the cell and provide incorporation within it. When using inert particles, the particles can be coated with a vector containing the desired gene to introduce the vector into the cell. Alternatively, the target cell can be surrounded by the vector such that the vector is brought into the cell by the bombardment of the particles. Biologically active particles (e.g., dry yeast cells, dry bacteria, or bacteriophages, each containing the DNA to be introduced) can also be propelled into plant cell tissue.

[0766] The transformation of most monocotyledonous plant species has now also become routine. Preferred techniques include direct gene transfer into protoplasts using PEG or electroporation techniques, and particle bombardment into callus. Patent applications EP0 292 435, EP 0 392 225, and WO 93 / 07278 describe techniques for preparing callus and protoplasts from elite inbred lines of maize, transforming protoplasts using PEG or electroporation, and regenerating maize plants from transformed protoplasts. Gordon-Kamm et al. (Plant Cell 2:603-618 (1990)) and Fromm et al. (Biotechnology 8:833-839 (1990)) have disclosed techniques for using particle bombardment to transform maize lines derived from A188. In addition, WO 93 / 07278 and Koziel et al. (Biotechnology 11:194-200 (1993)) describe techniques for transforming elite inbred lines of maize by particle bombardment. This technique utilizes immature maize embryos 1.5-2.5 mm in length excised from maize ears 14-15 days after pollination and a PDS-1000He Biolistics device for bombardment.

[0767] The transformation of rice can also be carried out by direct gene transfer techniques using protoplasts or particle bombardment. Protoplast-mediated transformation has been described for Japonica and Indica types (Zhang et al. Plant Cell Rep 7:379-384 (1988); Shimamoto et al. Nature 338:274-277 (1989); Datta et al. Biotechnology 8:736-740 (1990)). Both types can also be routinely transformed using particle bombardment (Christou et al. Biotechnology 9:957-962 (1991)). In addition, WO 93 / 21335 describes a technique for transforming rice via electroporation.

[0768] The transformation of monocotyledonous plants using Agrobacterium has also been described. See WO 94 / 00977 and U.S. Patent No. 5,591,616, both of which are incorporated herein by reference. See also Negrotto et al., Plant Cell Reports 19:798-803 (2000), incorporated herein by reference.

[0769] Plants obtained by transformation with the target nucleic acid sequence of the present invention can be any of a variety of plant species, including those of monocotyledonous and dicotyledonous plants; however, the plants used in the method of the present invention are preferably selected from the list of agronomically important target crops shown elsewhere herein. The expression of the gene of the present invention combined with other characteristics important for yield and quality can be incorporated into plant lines by breeding. Methods and techniques of breeding are known in the art. See, for example, Welsh J.R., Fundamentals of Plant Genetics and Breeding, John Wiley & Sons, New York (1981); Crop Breeding, Wood D.R. (ed.), American Society of Agronomy, Madison, Wis. (1983); Mayo O., The Theory of Plant Breeding, 2nd ed., Clarendon Press, Oxford (1987); Singh, D.P., Breeding for Resistance to Diseases and Insect Pests, Springer-Verlag, New York (1986); and Wricke and Weber, Quantitative Genetics and Selection Plant Breeding, Walter de Gruyter and Co., Berlin (1986).

[0770] Nucleotides are represented herein by the following standard abbreviations: adenine (A), cytosine (C), thymine (T), and guanine (G). Amino acids are also represented by the following standard abbreviations: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C), glutamine (Gln; Q), glutamic acid (Glu; E), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0771] The present disclosure particularly provides compositions and methods for selectively controlling weeds at a locus. The present invention further relates to recombinant DNA technology and particularly to the production of transgenic plants that exhibit significant resistance or significant tolerance to herbicides as compared to non-transgenic plants. Plants that are substantially "tolerant" to an herbicide (when they are subjected to the herbicide) provide a dose / response curve that is shifted to the right as compared to that provided by similarly treated non-tolerant plants. Such dose / response curves plot "dose" on the x-axis and "percent kill", "weed control efficacy", etc. on the y-axis. To achieve a given weed control efficacy, tolerant plants typically require at least about twice the amount of herbicide as non-tolerant plants. Plants that are substantially "resistant" to an herbicide show little (if any) necrosis, lysis, chlorosis, or other lesions when subjected to the concentrations and rates of herbicides typically used in the agricultural community to kill weeds in the field.

[0772] Tolerance to solanesyl diphosphate synthase (SDPS)-inhibiting herbicides in plants has not been reported because it has not previously been considered a target site for certain classes of herbicidal compounds. Transgenic plants overexpressing the solanesyl diphosphate synthase gene or variants thereof are tolerant to the herbicide. Additionally, the native SDPS gene can be edited in plants to confer tolerance to the herbicide. Thus, the present disclosure particularly provides an opportunity to utilize SDPS-inhibiting herbicides in a broader agricultural environment.

[0773] Accordingly, in one aspect of the present disclosure, there is provided a method for selectively controlling weeds at a locus comprising crop plants and weeds, the method comprising applying to the locus a pesticidal composition comprising a SDPS-inhibiting herbicide in a weed controlling amount, wherein the crop plants are modified such that they comprise SDPS that provides tolerance to the SDPS-inhibiting herbicide for the crop plants.

[0774] For the purposes of the present invention, a SDPS-inhibiting herbicide is an herbicide that inhibits Arabidopsis thaliana SDPS (i.e., exhibits an IC50 of less than 10 μM, preferably 5 μM in the assay method as described herein).

[0775] In one such embodiment, the SDPS-inhibiting herbicide is a compound having the formula (I): In one such embodiment, the SDPS-inhibiting herbicide is a compound having the formula (I):

[0776]

[0777] wherein

[0778] R 1 is selected from the group consisting of C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkynyl, and C2-C6 alkenyl;

[0779] R 2 is a C1-C2 alkyl group;

[0780] R 3 is selected from the group consisting of: C1-C5 alkyl, C1-C5 haloalkyl, halogen, CN, -CH2OMe, substituted aryl, substituted 5-6 membered heteroaryl, and 4-6 membered heterocyclic group;

[0781] R 4 to R 8 are independently selected from the group consisting of: H, Me, CF3, halogen, CF3, and CN, where 4 to R 8 at least two of which are not hydrogen and 4 to R 8 at most three of which are not hydrogen.

[0782] Exemplary compounds having formula (I) include Herbicide Compound Example 1 and Herbicide Compound Example 2 shown below:

[0783] Herbicide Compound Example 1:

[0784]

[0785] Herbicide Compound Example 2:

[0786]

[0787] In another embodiment of the present invention, the SDPS-inhibiting herbicide is a compound having formula (1) shown directly below

[0788]

[0789] As disclosed in WO 2015 / 089003, the entire content of which is incorporated herein by reference.

[0790] In another embodiment of the present invention, the SDPS-inhibiting herbicide is a compound disclosed in WO 2015 / 108779. Thus, in this embodiment, the SDPS-inhibiting herbicide is a compound having formula (1):

[0791]

[0792] where

[0793] Q is a 5- or 6-membered aromatic heterocycle bonded to the remainder of formula 1 through a carbon atom and optionally substituted with 1 to 4 R 1 substituents;

[0794] Z is O or S;

[0795] Each R 1 is independently a halogen, cyano, nitro, SF5, CHO, C(=O)NH2, C(=S)NH2, SO2NH2, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C2-C4 haloalkenyl, C2-C4 haloalkynyl, C3-C6 cycloalkyl, C3-C6 halocycloalkyl, C4-C8 alkylcycloalkyl, C4-C8 cycloalkylalkyl, C2-C8 alkylcarbonyl, C2-C6 haloalkylcarbonyl, C2-C6 alkoxycarbonyl, C3-C7 cycloalkylcarbonyl, C2-C6 alkylaminocarbonyl, C3-C 10 dialkylaminocarbonyl, C1-C4 alkoxy, C3-C4 alkenyloxy, C3-C4 alkynyloxy, C1-C4 haloalkoxy, C3-C4 haloalkenyloxy, C3-C4 haloalkynyloxy, C3-C6 cycloalkoxy, C3-C6 halocycloalkoxy, C4-C8 cycloalkylalkoxy, C2-C6 alkoxyalkyl, C2-C6 haloalkoxyalkyl, C2-C6 alkoxyhaloalkyl, C2-C6 alkoxyalkoxy, C2-C4 alkylcarbonyloxy, C2-C6 cyanoalkyl, C2-C6 cyanoalkoxy, C1-C4 hydroxyalkyl, C2-C4 alkylthioalkyl, SOnR 1A , Si(CH3)3 or B(-OC(R 1B ))2C(R 1B ))2O-); or optionally a benzene ring substituted by up to 5 substituents independently selected from R 1C ; or a 5- or 6-membered heteroaromatic ring containing ring members selected from carbon atoms and up to 4 heteroatoms independently selected from up to 2 O, up to 2 S and up to 4 N atoms, each ring optionally substituted by up to 3 substituents independently selected from R 1C on the carbon atom ring members and R 1D on the nitrogen atom ring members;

[0796] R 2 is a halogen, cyano, nitro, C1-C4 alkoxy, C1-C4 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, SOnR 2A , C1-C4 haloalkyl or C3-C6 cycloalkyl;

[0797] Each R 3Independently is halogen, cyano, hydroxyl, nitro, amino, CHO, C(=O)NH2, C(=S)NH2, SO2NH2, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C2-C4 haloalkenyl, C2-C4 haloalkynyl, C3-C6 cycloalkyl, C3-C6 halocycloalkyl, C4-C8 alkylcycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 alkylcarbonyl, C2-C6 haloalkylcarbonyl, C2-C6 alkoxycarbonyl, C3-C7 cycloalkylcarbonyl, C1-C4 alkoxy, C3-C4 alkenyloxy, C3-C4 alkynyloxy, C1-C4 haloalkoxy, C3-C4 haloalkenyloxy, C3-C4 haloalkynyloxy, C3-C6 cycloalkoxy, C3-C6 halocycloalkoxy, C4-C8 cycloalkylalkoxy, C2-C6 alkoxyalkyl, C2-C6 haloalkoxyalkyl, C2-C6 alkoxyhaloalkyl, C2-C6 alkoxyalkoxy, C2-C4 alkylcarbonyloxy, C2-C6 cyanoalkyl, C2-C6 cyanoalkoxy, C2-C4 alkylthioalkyl, Si(CH3)3, C≡CSi(CH3)3, C(=O)N(R 3A )(R 3B ), C(=NOR 3C ), C(=N R3D ), SOnR 3E ; or optionally a benzene ring substituted with up to 5 substituents independently selected from R 3F ; or a 5- or 6-membered heteroaromatic ring containing ring members selected from carbon atoms and up to 4 heteroatoms independently selected from up to 2 O, up to 2 S, and up to 4 N atoms, each ring optionally substituted with up to 3 substituents independently selected from R 3F on carbon atom ring members and R 3G on nitrogen atom ring members; or pyrimidyloxy;

[0798] m is 0, 1, 2 or 3;

[0799] Each n is independently 0, 1 or 2;

[0800] Each R 1A , R 2A and R 3E are independently C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 alkylamino or C2-C6 dialkylamino;

[0801] Each R 1B is independently H or C1-C4 alkyl;

[0802] Each R 1Cindependently is hydroxy, halogen, cyano, nitro, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy or C1-C6 haloalkoxy;

[0803] each R 1D independently is cyano, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy or C2-C6 alkylcarbonyl;

[0804] each R 3A independently is C1-C4 alkyl or C1-C4 haloalkyl;

[0805] each R 3B independently is H, C1-C4 alkyl or C1-C4 haloalkyl;

[0806] each R 3C independently is H or C1-C4 alkyl;

[0807] each R 3D independently is H, amino, C1-C4 alkyl or C1-C4 alkylamino;

[0808] each R 3F independently is hydroxy, halogen, cyano, nitro, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy or C1-C6 haloalkoxy; and

[0809] each R 3G independently is cyano, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy or C2-C6 alkylcarbonyl.

[0810] In this embodiment, the compound having the formula (1) is preferably selected from the group consisting of: 5-[2-chloro-6-(5-chloropyrimidin-2-yl)oxy-phenyl]-3-(difluoromethyl)isoxazole, 5-[2-bromo-6-(5-chloropyrimidin-2-yl)oxy-phenyl]-3-(difluoromethyl)isoxazole and 3-(5-chloropyrimidin-2-yl)oxy-2-[3-(difluoromethyl)isoxazol-5-yl]benzonitrile. More preferably, the compound is 5-[2-chloro-6-(5-chloropyrimidin-2-yl)oxy-phenyl]-3-(difluoromethyl)isoxazole.

[0811] In another embodiment of the present invention, the SDPS inhibitory herbicide is a compound having the formula (1) shown immediately below

[0812]

[0813] As disclosed in WO 2016 / 010731, the entire content of which is incorporated herein by reference.

[0814] In another embodiment of the present invention, the SDPS-inhibiting herbicide is a compound of the formula shown directly below

[0815]

[0816] As disclosed in WO 2016 / 014814, the entire content of which is incorporated herein by reference.

[0817] In another embodiment of the present invention, the SDPS-inhibiting herbicide is a compound of formula (1) shown directly below

[0818]

[0819] As disclosed in WO 2016 / 149315, the entire content of which is incorporated herein by reference.

[0820] In another embodiment of the present invention, the SDPS-inhibiting herbicide is a compound of formula (1) disclosed in WO 2016 / 196606. Thus, in this embodiment, the SDPS-inhibiting herbicide is a compound of formula (I) (including all geometric and stereoisomers), its N-oxides and salts:

[0821]

[0822] wherein

[0823] A is:

[0824]

[0825] B is O or S;

[0826] R1 is H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C2-C6 haloalkenyl, C2-C6 haloalkynyl, C3-C6 cycloalkyl, C3-C6 halocycloalkyl, C3-C6 halocycloalkylalkyl, C4-C6 alkylcycloalkyl, C4-C6 cycloalkylalkyl, C1-C6 alkylamino, C1-C6 haloalkylamino, C2-C10 dialkylamino, C2-C10 halodialkylamino, C3-C6 cycloamino, C1-C6 alkoxy, C3-C6 alkenyloxy, C3-C6 alkynyloxy, C1-C6 haloalkoxy, C3-C6 haloalkenyloxy, C3-C6 haloalkynyloxy, C3-C6 cycloalkoxy, C3-C6 halocycloalkoxy, C4-C6 cycloalkylalkoxy, C4-C6 halocycloalkylalkoxy, C2-C6 alkoxyalkyl, C2-C6 haloalkoxyalkyl, C2-C6 alkoxyhaloalkyl, C2-C6 alkoxyalkoxy, C2-C6 cyanoalkyl, C2-C6 cyanoalkoxy, C3-C7 cyanoalkoxyalkyl, C1-C6 hydroxyalkyl, C1-C6 nitroalkyl, C1-C6 alkylthio, C1-C6 haloalkylthio, C3-C8 cycloalkylthio, C1-C6 alkenylthio, C1-C6 alkylsulfinyl, C1-C6 haloalkylsulfonyl, C3-C8 cycloalkylsulfonyl, C2-C6 alkylthioalkyl, C2-C6 haloalkylthioalkyl, benzyl, -N(R7)(OR8), -ON(R9a)(R9b) or -N(R7)N(R9a)(R9b);

[0827] Z is O or S;

[0828] R2 is halogen, cyano, nitro, C1-C6 alkoxy, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C3-C6 cycloalkyl or -SOnR10;

[0829] Each R3 is independently a halogen, cyano, nitro, CHO, C(=O)NH2, C(=S)NH2, SO2NH2, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C2-C4 haloalkenyl, C2-C4 haloalkynyl, C3-C6 cycloalkyl, C3-C6 halocycloalkyl, C4-C6 alkylcycloalkyl, C4-C6 cycloalkylalkyl, C2-C6 alkylcarbonyl, C2-C6 haloalkylcarbonyl, C2-C6 alkoxycarbonyl, C3-C7 cycloalkylcarbonyl, C2-C4 alkoxy, C3-C4 alkenyloxy, C3-C4 alkynyloxy, C1-C4 haloalkoxy, C3-C6 cycloalkoxy, C3-C6 halocycloalkoxy, C4-C6 cycloalkylalkoxy, C2-C6 alkoxyalkyl, C2-C6 haloalkoxyalkyl, C2-C6 alkoxyhaloalkyl, C2-C6 alkoxyalkoxy, C2-C4 alkylcarbonyloxy, C2-C6 cyanoalkyl, C2-C6 cyanoalkoxy, C2-C4 alkylthioalkyl, -C(=O)N(Rlla)(Rllb), -C(=NOR12)H, -C(=N(R13))H or -SOnRl4;

[0830] m is 0, 1, 2 or 3;

[0831] Each n is independently 0, 1 or 2;

[0832] R4 is H, C1-C6 alkyl or C1-C6 haloalkyl;

[0833] R5 is H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C2-C6 haloalkenyl, C2-C6 haloalkynyl, C3-C6 cycloalkyl, C3-C6 halocycloalkyl, C4-C6 alkylcycloalkyl, C4-C6 cycloalkylalkyl, C2-C6 alkoxyalkyl, C2-C6 haloalkoxyalkyl, C2-C6 alkoxyhaloalkyl, C2-C6 cyanoalkyl, C3-C7 cyanoalkoxyalkyl, C1-C6 hydroxyalkyl, C1-C6 nitroalkyl, C2-C6 alkylthioalkyl, C2-C6 haloalkylthioalkyl or benzyl;

[0834] Each R6a and R6b is independently H, C1-C6 alkyl or C1-C6 haloalkyl;

[0835] R7 is H, C1-C6 alkyl or C1-C6 haloalkyl;

[0836] R8 is H, C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkoxyalkyl, C2-C6 haloalkoxyalkyl or C2-C6 cyanoalkyl;

[0837] Each R9a and R9b is independently H, C1-C6 alkyl, or C1-C6 haloalkyl;

[0838] R10 is independently C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkylamino, or C2-C10 dialkylamino;

[0839] Each Rlla is independently C1-C4 alkyl or C1-C4 haloalkyl;

[0840] Each Rllb is independently H, C1-C4 alkyl, or C1-C4 haloalkyl;

[0841] Each R12 is independently H or C1-C4 alkyl;

[0842] Each R13 is independently H, amino, C1-C4 alkyl, or C1-C4 alkylamino;

[0843] Each R14 is independently C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkylamino, or C2-C10 dialkylamino; and R15 is H or C1-C6 alkyl.

[0844] In this embodiment, the compound having formula (1) is preferably selected from the group consisting of: 5-chloro-2-[3-chloro-2-(5,5,5-trifluoropentyl)phenoxy]pyrimidine, 2-[3-bromo-2-(5,5,5-trifluoropentyl)phenoxy]-5-chloro-pyrimidine, 3-(5-chloropyrimidin-2-yl)oxy-2-(5,5,5-trifluoropentyl)benzonitrile, 1-[2-chloro-6-(5-chloropyrimidin-2-yl)oxy-phenyl]-4,4,4-trifluoro-butan-1-one, 1-[2-bromo-6-(5-chloropyrimidin-2-yl)oxy-phenyl]-4,4,4-trifluoro-butan-1-one, and 3-(5-chloropyrimidin-2-yl)oxy-2-(4,4,4-trifluorobutyryl)benzonitrile. More preferably, the compound is 1-[2-chloro-6-(5-chloropyrimidin-2-yl)oxy-phenyl]-4,4,4-trifluoro-butan-1-one.

[0845] In another embodiment of the present invention, the SDPS-inhibiting herbicide is a compound having formula (1) shown immediately below

[0846]

[0847] As disclosed in WO 2017 / 011288, the entire content of which is incorporated herein by reference.

[0848] In another embodiment of the present invention, the SDPS-inhibiting herbicide is a compound having formula (1) shown immediately below

[0849]

[0850] As disclosed in WO 2018 / 204164, the entire content of which is incorporated herein by reference.

[0851] In another embodiment of the present invention, the SDPS-inhibiting herbicide is a compound having formula (I), as disclosed in International Patent Application PCT / EP 2019 / 079971. Thus, in this embodiment, the SDPS-inhibiting herbicide is a compound having formula (I)

[0852]

[0853] or an agriculturally acceptable salt thereof,

[0854] wherein

[0855] Q is a 5-membered aromatic heterocycle optionally substituted with 1 or 2 R3 substituents independently selected from the group consisting of: C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, cyclopropyl, C1-C4 haloalkyl, C1-C2 alkoxy-, C1-C2 haloalkoxy-, halogen, -C(O)C1-C4 alkyl, NO2, -CH2CN, -CN, and -S(O)pC1-C4 alkyl;

[0856] Each R1 is independently selected from the group consisting of: halogen, -CN, nitro, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1-C4 alkoxy-, C1-C4 haloalkoxy-, and -S(O)pC1-C4 alkyl;

[0857] Each R2 is independently selected from the group consisting of: halogen, -CN, NO2, C1-C4 alkyl, C1-C4 haloalkyl, C3-C6 cycloalkyl, C2-C4 alkenyl, C2-C4 alkynyl, -S(O)pC1-C4 alkyl, C1-C4 alkoxy, -C(O)C1-C4 alkyl, -C(O)OC1-C4 alkyl, and C1-C4 haloalkoxy;

[0858] m = 0, 1 or 2;

[0859] n = 0, 1 or 2; and

[0860] p = 0, 1 or 2.

[0861] In this embodiment, the compound having the formula (I) is preferably selected from the group consisting of: 5-[2-[(5-chloro-3-fluoro-2-pyridinyl)oxy]-6-fluoro-phenyl]-3-(difluoromethyl)isoxazole, 5-chloro-3-fluoro-2-[2-[4-(trifluoromethyl)pyrazol-1-yl]phenoxy]pyridine, and 5-chloro-2-[2-[3-(difluoromethyl)isoxazol-5-yl]-3-fluoro-phenoxy]pyridine-3-carbonitrile.

[0862] In another embodiment of the present invention, the SDPS-inhibiting herbicide is a compound having the formula shown immediately below

[0863]

[0864] As disclosed in WO 2019 / 016066, the entire content of which is incorporated herein by reference.

[0865] In another embodiment of the present invention, the SDPS-inhibiting herbicide is a compound having the formula (I) shown immediately below

[0866]

[0867] As disclosed in WO 2020 / 002089, the entire content of which is incorporated herein by reference.

[0868] In another embodiment of the present invention, the SDPS-inhibiting herbicide is a compound having the formula (I) shown immediately below

[0869]

[0870] As disclosed in WO 2020 / 002087, the entire content of which is incorporated herein by reference.

[0871] In another embodiment of the present invention, the SDPS-inhibiting herbicide is a compound having the formula (I) shown immediately below

[0872]

[0873] As disclosed in WO 2020 / 002085, the entire content of which is incorporated herein by reference.

[0874] In another embodiment of the present invention, the SDPS-inhibiting herbicide is a compound having the formula (I) shown immediately below

[0875]

[0876] As disclosed in EP 0061913 A2, the entire content of which is incorporated herein by reference.

[0877] Where applicable, the SDPS inhibitory herbicide may be present as a racemate or as a single enantiomer (or an enantiomer mixture enriched in enantiomers).

[0878] It should be understood that in the above methods, the herbicide composition can be applied to the site before crop emergence and / or after crop emergence (so-called "excessive" application). In a preferred embodiment, the herbicide composition is applied before crop emergence. Single or in fact multiple applications can be made as needed to obtain the desired weed control. The SDPS-inhibiting herbicide can be applied to the site at any suitable rate. Typically, the genome-edited or transgenic plants of the present invention exhibit resistance or tolerance to an SDPS-inhibiting herbicide applied at an amount of from about 5 to about 2,000 grams of active ingredient per hectare (g / ha), which amounts include, for example, about 5 g / ha, about 10 g / ha, about 15 g / ha, about 20 g / ha, about 25 g / ha, about 30 g / ha, about 35 g / ha, about 40 g / ha, about 45 g / ha, about 50 g / ha, about 55 g / ha, about 60 g / ha, about 65 g / ha, about 70 g / ha, about 75 g / ha, about 80 g / ha, about 85 g / ha, about 90 g / ha, about 95 g / ha, about 100 g / ha, about 110 g / ha, about 120 g / ha, about 130 g / ha, about 140 g / ha, about 150 g / ha, about 160 g / ha, about 170 g / ha, about 180 g / ha, about 190 g / ha, about 200 g / ha, about 210 g / ha, about 220 g / ha, about 230 g / ha, about 240 g / ha, about 250 g / ha, about 260 g / ha, about 270 g / ha, about 280 g / ha, about 290 g / ha, about 300 g / ha, about 310 g / ha, about 320 g / ha, about 330 g / ha, about 340 g / ha, about 350 g / ha, about 360 g / ha, about 370 g / ha, about 380 g / ha, about 390 g / ha, about 400 g / ha, about 410 g / ha, about 420 g / ha, about 430 g / ha, about 440 g / ha, about 450 g / ha, about 460 g / ha, about 470 g / ha, about 480 g / ha, about 490 g / ha, about 500 g / ha, about 510 g / ha, about 520 g / ha, about 530 g / ha, about 540 g / ha, about 550 g / ha, about 560 g / ha, about 570 g / ha, about 580 g / ha, about 590 g / ha, about 600 g / ha, about 610 g / ha, about 620 g / ha, about 630 g / ha, about 640 g / ha, about 650 g / ha, about 660 g / ha, about 670 g / ha, about 680 g / ha, about 690 g / ha, about 700 g / ha, about 710 g / ha, about 720 g / ha, about 730 g / ha, about 740 g / ha, about 750 g / ha, about 760 g / ha, about 770 g / ha, about 780 g / ha, about 790 g / ha,Approximately 800 g / ha, approximately 810 g / ha, approximately 820 g / ha, approximately 830 g / ha, approximately 840 g / ha, approximately 850 g / ha, approximately 860 g / ha, approximately 870 g / ha, approximately 880 g / ha, approximately 890 g / ha, approximately 900 g / ha, approximately 910 g / ha, approximately 920 g / ha, approximately 930 g / ha, approximately 940 g / ha, approximately 950 g / ha, approximately 960 g / ha, approximately 970 g / ha, approximately 980 g / ha, approximately 990 g / ha, approximately 1,000 g / ha, approximately 1,010 g / ha, approximately 1,020 g / ha, approximately 1,030 g / ha, approximately 1,040 g / ha, approximately 1,050 g / ha, approximately 1,060 g / ha, approximately 1,070 g / ha, approximately 1,080 g / ha, approximately 1,090 g / ha, approximately 1,100 g / ha, approximately 1,110 g / ha, approximately 1,120 g / ha, approximately 1,130 g / ha, approximately 1,140 g / ha, approximately 1,150 g / ha, approximately 1,160 g / ha, approximately 1,170 g / ha, approximately 1,180 g / ha, approximately 1,190 g / ha, approximately 1,200 g / ha, approximately 1,210 g / ha, approximately 1,220 g / ha, approximately 1,230 g / ha, approximately 1,240 g / ha, approximately 1,250 g / ha, approximately 1,260 g / ha, approximately 1,270 g / ha, approximately 1,280 g / ha, approximately 1,290 g / ha, approximately 1,300 g / ha, approximately 1,310 g / ha, approximately 1,320 g / ha, approximately 1,330 g / ha, approximately 1,340 g / ha, approximately 1,350 g / ha, approximately 360 g / ha, approximately 1,370 g / ha, approximately 1,380 g / ha, approximately 1,390 g / ha, approximately 1,400 g / ha, approximately 1,410 g / ha, approximately 1,420 g / ha, approximately 1,430 g / ha, approximately 1,440 g / ha, approximately 1,450 g / ha, approximately 1,460 g / ha, approximately 1,470 g / ha, approximately 1,480 g / ha, approximately 1,490 g / ha, approximately 1,500 g / ha, approximately 1,510 g / ha, approximately 1,520 g / ha, approximately 1,530 g / ha, approximately 1,540 g / ha, approximately 1,550 g / ha, approximately 1,560 g / ha, approximately 1,570 g / ha, approximately 1,580 g / ha, approximately 1,590 g / ha, approximately 1,600 g / ha, approximately 1,610 g / ha, approximately 1,620 g / ha, approximately 1,630 g / ha, approximately 1,640 g / ha, approximately 1,650 g / ha, approximately 1,660 g / ha, approximately 1,670 g / ha, approximately 1,680 g / ha, approximately 1,690 g / ha, approximately 1,700 g / ha, approximately 1,710 g / ha, approximately 1,720 g / ha, approximately 1,730 g / haAbout 1,740 g / ha, about 1,750 g / ha, about 1,760 g / ha, about 1,770 g / ha, about 1,780 g / ha, about 1,790 g / ha, about 1,800 g / ha, about 1,810 g / ha, about 1,820 g / ha, about 1,830 g / ha, about 1,840 g / ha, about 1,850 g / ha, about 1,860 g / ha, about 1,870 g / ha, about 1,880 g / ha, about 1,890 g / ha, about 1,900 g / ha, about 1,910 g / ha, about 1,920 g / ha, about 1,930 g / ha, about 1,940 g / ha, about 1,950 g / ha, about 1,960 g / ha, about 1,970 g / ha, about 1,980 g / ha, about 1,990 g / ha, or about 2,000 g / ha.

[0879] The term “weed” refers to any unwanted vegetation and includes, for example, residual or “rogue” or “volunteer” crop plants.

[0880] In one aspect of the invention, the crop plant is modified with a recombinant polynucleotide that provides SDPS, which confers tolerance to an SDPS-inhibiting herbicide on the crop plant.

[0881] Typically, the recombinant polynucleotide will comprise (i) a plant-operable promoter operably linked (ii) a region encoding SDPS and (iii) a transcription terminator. Typically, the recombinant polynucleotide will further comprise a region encoding a polypeptide that can target SDPS to sub-organelles such as chloroplasts. The recombinant polynucleotide may further comprise, for example, a transcriptional enhancer. Additionally, the region encoding SDPS may be “codon optimized” according to the plant host in which expression of SDPS is desired. Those skilled in the art are well aware of plant-operable promoters, transcription terminators, chloroplast transit peptides, enhancers, etc. useful in the context of the present invention.

[0882] The SDPS can be a “wild-type” enzyme or it can be an enzyme that has been modified to provide preferential kinetic properties (with respect to providing herbicide-tolerant plants).

[0883] Examples of suitable SDPS are, but are not limited to, those derived from: Arabidopsis thaliana, common wheat (Triticum aestivum), barley (Hordeum vulgare), rice (Oryza sativa), maize (Zea mays), soybean (Glycine max), Chlorella and Chlamydomonas reinhardtii.

[0884] It should be understood that the modified SDPS can be introduced into plants by recombinant polynucleotide means. Figure 1 An example of a plasmid (SEQ ID NO:19) that can be used to introduce the modified SDPS into plants is illustrated. The transformation method is as described above and is known in the art, but briefly, Figure 1 the plasmid in Figure 1 includes a recombinant polynucleotide that contains (i) a region encoding solanesyl diphosphate synthase, which is operably linked to a plant-operable promoter.

[0885] For Figure 1 the vector components in Figure 1 (also relative to SEQ ID NO:19), cNPT2-01-04 (start: 592 end: 1567) encodes neomycin phosphotransferase; cNPT3-01-01 (start: 5869 end: 6660) encodes a phosphotransferase that confers kanamycin resistance; cTETR-01-01 (start: 10273 end: 10923) is the gene for tet R; SDPS2 F240L (start: 12305 end: 13564) encodes the modified SDPS; bNLB-01-01 (start: 2746 end: 2893) shows similarity to the left border of T-DNA; bNRB-01-03 (start: 11297 end: 11458) is the right border of T-DNA; pNOS-01-01 (start: 284 end: 590) is the Nos promoter; p35S-07-01 (start: 11484 end: 11810) is the CaMV 35s promoter; p35S-10-01 (start: 11811 end: 12227) is the CaMV35s promoter; the origin of replication features include oRK2-01-01 (start: 8317 end: 8934) and oCOLE-03-01 (start: 9724 end: 10039); tNOS-01-01 (start: 17 end: 271) is the terminator of nopaline synthase; tNOS-01-01 (start: 1786 end: 2040) is the terminator of nopaline synthase.

[0886] Alternatively, endogenous SDPS can be edited in situ by means of gene editing techniques to provide SDPS that is tolerant to SDPS-inhibiting herbicides. Such genome editing and / or mutagenesis techniques are well known in the art. Similarly, the introduction can be accomplished by any means known in the art, including: introgression, transgenesis, or site-directed nuclease (SDN). In particular, modifications to the nucleic acid sequence are introduced by means of site-directed nuclease (SDN). More particularly, SDN is selected from: meganucleases, zinc fingers, transcription activator-like effector nuclease systems (TALEN), or clustered regularly interspaced short palindromic repeat systems (CRISPR). SDN is also referred to as "genome editing", or genome editing with engineered nucleases (GEEN). This is a type of genetic engineering in which engineered nucleases that create site-specific double-strand breaks (DSBs) at desired loci in the genome are used to insert, delete, or replace DNA in the genome of an organism. The induced double-strand breaks are repaired by non-homologous end joining (NHEJ) or homologous recombination (HR), resulting in targeted mutations ('editing'). In particular, SDN can include techniques such as: meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) (Feng et al. 2013 Cell Res. [Cell Research] 23, 1229-1232, Sander and Joung Nat. Biotechnol. [Nature Biotechnology] 32, 347-355 2014), and clustered regularly interspaced short palindromic repeats (CRISPR-Cas) systems.

[0887] Accordingly, the present disclosure also relates to plasmids that can be used for editing. The plasmids include nucleic acids encoding DNA-modifying enzymes such as site-directed nucleases, for example, Cas9 nuclease, Cfp1 nuclease, dCas9-FokI, dCpf1-FokI, chimeric Cas9-cytidine deaminase, chimeric Cas9-adenine deaminase, chimeric FEN1-FokI, and Mega-TAL, nickase Cas9 (nCas9), chimeric dCas9 non-FokI nucleases, and dCpf1 non-FokI nucleases. The plasmids also include at least one guide RNA. The plasmids can also include additional components, for example, they can include a gRNA promoter, such as prOsU3-01 (which is a rice U3 promoter for pol III-dependent transcription of non-coding RNA) to regulate the expression of at least one gRNA. The vectors can similarly include additional features such as, a selectable marker, for example, phosphomannose isomerase (PMI) and can be used in conjunction with mannose selection to recover stably transformed plants. Additional features include regulatory sequences, for example, promoters and terminators for regulating the expression of the selectable marker.

[0888] The vector may further comprise additional features that assist in transformation, such as features that facilitate Agrobacterium-mediated transformation as described above.

[0889] The target sequence can vary and can include sequences that are 15 - 25 nucleotides in length (including sequences encoding the amino acids of Table 1, such as sequences of 3 nucleotides).

[0890] Figure 2 An example of a plasmid (SEQ ID NO:42) that can be used for editing is illustrated. In this example, the plasmid is a binary CRISPR construct that contains a Cpf1 endonuclease targeting a maize gene region. The ubiquitin promoter drives the expression of the endonuclease and the gRNA. The vector also contains PMI as a selectable marker.

[0891] For Figure 2The vector components shown in (where the positions are relative to SEQ ID NO: 42), cLbCpf1-02 (starting at 2049 and ending at 5831) is an RNA-guided endonuclease of the class II CRISPR / Cas system and is a rice codon-optimized version from the bacterium Lachnospiraceae bacterium ND2006; cPMI-09 (starting at 10334 and ending at 11512) is a synthetic phosphomannose isomerase gene; cSpec-03 (starting at 12240 and ending at 13028) is a gene encoding the enzyme aminoglycoside 3'-adenyltransferase, conferring resistance to spectinomycin and streptomycin for maintaining the vector in Escherichia coli and Agrobacterium; cVirG-09 (starting at 13328 and ending at 13960) is a non-functional VirG gene for cloning purposes; cRepA-01 (starting at 13990 and ending at 15063) encodes a replication protein; bNRB-04 (starting at 4 and ending at 143) is the right border region of the T-DNA of the Agrobacterium tumefaciens nopaline ti plasmid; bNRB-01-01 (starting at 101 and ending at 125) is a right border repeat; bNLB-03 (starting at 11831 and ending at 11960) is the left border region of the T-DNA of the Agrobacterium tumefaciens nopaline ti plasmid; prSoUbi4-02 (starting at 229 and ending at 2030) is a constitutive sugarcane ubi4 promoter; prSoUbi4-02 (starting at 6099 and ending at 7900) is a constitutive sugarcane ubi4 promoter; prUbi1-04 (starting at 8326 and ending at 10317) is a Zea mays polyubiquitin 1 promoter; oVS1-02 (starting at 15106 and ending at 15510) is the origin of replication and partitioning region of the plasmid pVS1 from Pseudomonas and is used as an origin of replication in Agrobacterium tumefaciens hosts; oCOLE-06 (starting at 16188 and ending at 16994) is a ColE1 origin of replication that functions in Escherichia coli; rHH-01 (starting at 7908 and ending at 7950) is a conserved sequence motif of satellite RNAs from certain viruses, including Tobacco ringspot virus, responsible for self-cleavage; rLbCrRNA-01 (starting at 7951 and ending at 7971) is the scaffold crRNA of LbCpf1, also known as the direct repeat (DR) of the guide RNA; rLbgRNACpf1ZmLHT1-01 (starting at 7951 and ending at 7971) is a CRISPR / Cpf1 guide RNA that includes the direct repeat of LbCrRNA from Lachnospiraceae bacterium ND2006 (targeting sequences in the Zea mays genome); rHDV-01 (starting at 7995 and ending at 8062) encodes the sequence of a self-cleaving ribozyme from Hepatitis delta virus (HDV). It should be clear that Figure 2 the examples in are provided for illustration only, and other technicians in the art will be able to easily design vectors according to the present invention.

[0892] It should be further understood that the crop plants used in the methods may further comprise additional recombinant polynucleotides encoding additional herbicide tolerance enzymes. Examples of additional herbicide tolerance enzymes include, for example, herbicide tolerance enzymes selected from the group consisting of: 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), HST, glyphosate acetyltransferase (GAT), cytochrome P450, phosphinothricin acetyltransferase (PAT), acetolactate synthase (ALS), protoporphyrinogen oxidase (PPGO), hydroxyphenylpyruvate dioxygenase (HPPD), phytoene desaturase (PD), dicamba degrading enzymes (such as those taught in WO02 / 068607), and aryloxyphenoxy herbicide degrading enzymes (such as those taught in WO 2007 / 053482 and WO 2005 / 107437).

[0893] The pesticidal composition may further comprise one or more additional pesticidal ingredients. The additional pesticidal ingredients may include, for example, herbicides (as discussed), but may also include fungicides and / or insecticides. Preferably, the pesticidal composition used in the above methods may further comprise one or more additional herbicides to which the crop plants are naturally tolerant or to which they are resistant via the expression of one or more additional transgenes as mentioned herein. In a preferred embodiment, the one or more additional herbicides are selected from the group consisting of: glyphosate (including its agrochemically acceptable salts); glufosinate (including its agrochemically acceptable salts); chloroacetanilides, for example, alachlor, acetochlor, S-metolachlor, S-isometolachlor; photosystem II inhibitors, for example, triazines (such as ametryn, atrazine, cyanazine, and terbuthylazine), triazinones (such as hexazinone and secbumeton), ureas (such as chlorotoluron, diuron, isoproturon, linuron, and buthiuron); ALS-inhibitors, for example, sulfonylureas, such as amidosulfuron, chlorsulfuron, flupyrsulfuron, halosulfuron, nicosulfuron, flazasulfuron, prosulfuron, rimsulfuron, tribenuron-methyl, trifloxysulfuron, and tritosulfuron; diphenyl ethers, for example, acifluorfen and fomesafen; herbicides that inhibit HPPD, such as, mesotrione and pyraflufen-ethyl; dicamba (including its agrochemically acceptable salts) and 2,4-D (including its agrochemically acceptable salts).

[0894] The present invention further provides a recombinant polynucleotide comprising (i) a region encoding SDPS operably linked to a plant- operable promoter and (ii) at least one additional heterologous polynucleotide comprising a region encoding an additional herbicide-tolerant enzyme operably linked to a plant- operable promoter. The additional herbicide-tolerant enzyme is, for example, selected from the group consisting of hydroxyphenylpyruvate dioxygenase (HPPD), 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), glyphosate acetyltransferase (GAT), cytochrome P450, phosphinothricin acetyltransferase (PAT), acetolactate synthase (ALS), protoporphyrinogen oxidase (PPGO), phytoene desaturase (PD), and a dicamba-degrading enzyme (as taught in WO02 / 068607).

[0895] Preferably, the recombinant polynucleotide comprises (i) a region encoding SDPS operably linked to a plant- operable promoter and (ii) a region encoding HPPD operably linked to a plant- operable promoter. The recombinant polynucleotide may further comprise at least two, three or more additional regions, each region encoding, for example, a herbicide-tolerant enzyme as previously defined. Thus, in another preferred embodiment, the recombinant polynucleotide comprises (i) a region encoding SDPS, (ii) a region encoding an HPPD enzyme, and (iii) a region encoding a glyphosate-tolerant enzyme.

[0896] The present invention further provides a vector comprising the recombinant polynucleotide according to the present invention.

[0897] The present invention further relates to a transformed plant overexpressing the SDPS enzyme, which shows significant resistance or significant tolerance to SDPS-inhibiting herbicides as compared to an untransformed similar plant.

[0898] Accordingly, the present invention further provides a plant cell which shows significant resistance or significant tolerance to an SDPS-inhibiting herbicide when compared to an untransformed similar plant cell - said plant cell comprising the recombinant polynucleotide of the present invention as described herein. It should be understood that the region encoding SDPS and any region encoding one or more additional herbicide-tolerant enzymes may be provided on the same ("linked") or actually separate transformed recombinant polynucleotide molecules.

[0899] The plant cell may further comprise additional transgenic traits, such as a heterologous polynucleotide providing resistance to insects, fungi, and / or nematodes.

[0900] The present invention further provides a morphologically normal fertile SDPS inhibitor-tolerant plant, plant cell, tissue, and seed comprising the plant cell according to the present invention.

[0901] The transformed plants or plant cells include, but are not limited to, field crops, fruits and vegetables, such as canola, sunflower, tobacco, sugar beet, cotton, maize, wheat, barley, rice, sorghum, tomato, mango, peach, apple, pear, strawberry, banana, melon, mangelworzel, potato, carrot, lettuce, cabbage, onion, etc. Particularly preferred transgenic plants are soybean, sugarcane, pea, broad bean, poplar, grape, citrus, alfalfa, rye, oat, turfgrass and forage grass, flax and rape, and nut-producing plants not specifically mentioned yet. In a particularly preferred embodiment of the method, the plant is a dicotyledonous plant, preferably selected from the group consisting of: canola, sunflower, tobacco, sugar beet, soybean, cotton, sorghum, tomato, mango, peach, apple, pear, strawberry, banana, melon, potato, carrot, lettuce, cabbage, onion, and particularly preferably soybean. In another preferred embodiment, the plant is maize or rice. Preferably, the plants of the present invention are soybean, rice or maize. The present invention also includes the progeny of the aforementioned plants, as well as the seeds or other propagation materials of such plants and progeny.

[0902] The present invention further provides a method for providing transgenic plants that are tolerant to SDPS-inhibiting herbicides, the method comprising transforming plant material with one or more recombinant polynucleotides comprising a region encoding an SDPS enzyme, selecting the transformed plant material using an SDPS-inhibiting herbicide and regenerating the material into morphologically normal fertile plants.

[0903] The present invention further relates to a method for using a polynucleotide comprising a region encoding SDPS (as a selectable marker) in plant transformation and to the use of a polynucleotide comprising a region encoding SDPS in the production of plants that are tolerant to herbicides that act, in whole or in part, by inhibiting SDPS.

[0904] The present invention further relates to the use of an SDPS inhibitor as a selection agent in plant transformation and to the use of recombinant SDPS in an in vitro screening method for identifying SDPS-inhibiting herbicides.

[0905] This disclosure also relates to various methods for identifying solanesyl diphosphate synthase-inhibiting herbicides. In a typical embodiment, the method comprises expressing SDPS in a plant; and

[0906] Expose a plant to a herbicide, wherein reduced damage in the plant as compared to a control plant that does not express SDPS indicates that the compound is a solanesyl diphosphate synthase-inhibiting herbicide. The SDPS can be selected from (a) SDPSs of SEQ ID NO: 1-18, 45-349, and 663-665; or (b) "modified" SDPSs having an amino acid sequence that is at least 80% identical to the sequences shown in SEQ ID NO: 1-18, 45-349, and 663-665; or (c) "modified" SDPSs having an amino acid sequence that is at least 90% identical to the sequences shown in SEQ ID NO: 1-18, 45-349, and 663-665; or (d) "modified" SDPSs having an amino acid sequence that is at least 95% identical to the sequences shown in SEQ ID NO: 1-18 and 45-349; or (e) SDPSs having a motif selected from SEQ ID NO: 655-662, or (f) SDPSs having at least one mutation at a position corresponding to one of the following amino acid positions of SEQ ID NO: 5: F240L, F227L, F229L, F247L, L120A, L120R, L120W, L123A, L123C, L123D, L123N, L123S, L123W, E127A, E127G, E127K, E127Y, N128L, N128P, V130D, V130K, L131A, L131E, L131M, L131P, A134V, F139D, F139K, F139N, F139R, F139T, P148I, P148L, P148M, P148T, P148V, V151E, V151F, V151I, V151M, V151N, L174F, L174T, A175I, A175P, A175S, E176A, E176D, E176H, E176K, E176N, E176P, E176Y, I177A, I177C, I177F, I177L, I177M, I177S, I177T, I177Y, I178G, I178Q, I178W, E179I, M180I, M180Q, M180S, M180Y, M180W, I181M, I181N, A184G, A184S, A184T, T183C, T183Q, S185A, S185T, S185G, I187E, I187F, I187T, I187V, H188F, H188I, H188L, H188M, H188V, V191A, V191T, I204A, I204F, I204G, I204H, I204K, I204Q, I204R, I204S, I204T, Y208A, Y208D,Y208E, Y208H, Y208I, Y208K, Y208L, Y208M, Y208N, Y208Q, Y208R, Y208S, Y208T, Y208V, G209N, T210Y, R211D, R211E, R211N, R211T, R211V, L215I, L215M, A216T, F219A, M220I, M220C, F221W, A222G, A222M, A222S, Q223A, Q223E, Q223F, Q223G, Q223H, Q223I, Q223K, Q223L, Q223M, Q223R, Q223Y, S224F, S224I, S224M, S224N, S224Q, S224T, S224V, S225C, S225F, S225H, S225I, S225K, S225M, S225N, S225Q, S225T, S225V, S225Y, W226A, W226C, W226E, W226I, W226L, W226Q, W226R, W226T, W226V, F227D, F227L, F227M, F227R, F227V, F227W, L228C, L228I, L228M, L228T, L228V, A229H, A229I, A229L, A229M, A229N, A229T, N230E, N230R, E235G, K238G, K238N, K238S, L239A, L239R, I240A, I240C, I240W, S241A, S241H, S241N, S241T, V243A, V243G, V243N, V243Q, V243S, I244A, I244F, I244G, I244H, I244K, I244L, I244M, I244N, I244P, I244Q, I244S, I244V, I244Y, K245F, K245H, K245M, K245N, K245W, D246E, D246M, D246N, D246Q, D246S, D246T, D246Y, F247E, F247L, F247M, F247N, F247V, A248P, S249A, S249E, S249F, S249G, S249K, S249L, S249N, S249Q, S249T, S249V, S249Y, G250A, I252L, I252M, I252V, K253L, A255T, A255W, S256N, T257E, T257G, T257H, T257M, T257Q, T257W, Y274D, Y274G, Y274L, Y274M, Y274Q, T276SL279F, I280W, I280F, A282G, A282H, A282K, A282N, A282R, S283C, S283F, S283I, S283M, S283T, S283W, R306F, R306H, R306L, R306N, L310G, G309A, G309F, G309M, G309S, L310D, L310E, L310F, L310H, L310N, L310Q, L310W, L310Y, F312C, F312I, F312L, F312M, F312V, Q313A, Q313C, Q313D, Q313S, and Q313T. In many instances, the SDPS variants can include 2, 3, 4, 5, 6, or 7 of the above mutations. The damage observed will typically include bleaching. The exposure ratios can vary and can include exposure in the range of 2.5 ppm to 20 ppm.

[0907] This disclosure also relates to various methods for identifying solanesyl diphosphate synthase variants that have increased tolerance to a reference solanesyl diphosphate synthase-inhibiting herbicide (reference herbicide). The reference herbicide can be, for example, a commercial herbicide or a novel herbicide with an unknown mode of action. The methods typically include obtaining a first plant expressing a first SDPS variant and a second plant expressing a second SDPS variant that contains at least one amino acid different from the first variant; and exposing the first plant and the second plant to the reference herbicide, wherein reduced damage of one plant relative to the other indicates the presence of a variant with increased tolerance to the reference herbicide. Any amino acid in the variant can be altered, and in many embodiments, the variants will differ by two, three, four, five, or more amino acids. Exemplary amino acids for generating variants include those in Table 1. The SDPS can include (a) the SDPS of SEQ ID NOs: 1-18, 45-349, and 663-665; or (b) a "modified" SDPS that has an amino acid sequence that is at least 80% identical to the sequences shown in SEQ ID NOs: 1-18, 45-349, and 663-665; or (c) a "modified" SDPS that has an amino acid sequence that is at least 90% identical to the sequences shown in SEQ ID NOs: 1-18, 45-349, and 663-665; or (d) a "modified" SDPS that has an amino acid sequence that is at least 95% identical to the sequences shown in SEQ ID NOs: 1-18, 45-349, and 663-665; or (e) an SDPS having a motif selected from SEQ ID NOs: 655-662; or (f) an SDPS having at least one mutation at a position corresponding to one of the amino acid positions in SEQ ID NO: 5, as exemplified above. The exposure ratio can vary, for example, within the range of 2.5 ppm to 20 ppm.

[0908] Example 1. Expression of SDPS Enzyme

[0909] The DNA sequence encoding the N-terminal his-tagged SDPS gene (Seq ID 13-18) optimized for E. coli codon usage was synthesized by Genewiz (South Plainfield, USA) to include 5’ NdeI and 3’ NotI restriction sites. These were cloned into the E. coli expression plasmid pET24a (Novagen) via the NdeI and NotI restriction sites, and the resulting plasmid was transformed into E. coli BL21(DE3) and then maintained with 50 μg / ml kanamycin. E. coli BL21(DE3) competent cells from Agilent were transformed according to the manufacturer's instructions. Briefly, 100 μl aliquots of competent cells were thawed, pre-mixed with 1.7 μl of β-mercaptoethanol on ice and then incubated with 1-50 ng of DNA on ice for 30 min with gentle vortexing. Each transformation reaction was briefly (45 s) heated to 42 °C before returning to ice and then mixed with 0.9 ml of SOC medium pre-warmed to 42 °C. The cell suspension was then incubated at 37 °C for 1 h with shaking at 250 rpm, and then 5 and 50 μl aliquots were plated onto LB agar plates containing 50 μg / ml kanamycin. Transformed colonies were picked after overnight growth. After pre-growth in an initial seed culture, the transformed cells were transferred to Formedium auto-induction medium (which has a Terrific broth matrix and includes trace elements (catalog number: AIMTB0210)), and the culture was grown at 37 °C in a 2.5 L flask at 200 rpm for 3 h, followed by overnight growth at 20 °C, 200 rpm. Cells were harvested at 4 °C at 6,000 rpm for 20 min. After biomass harvest, approximately 20 g wet weight of cell paste was resuspended in 100 ml of lysis buffer, which was PBS (phosphate buffered saline) pH 7.4, 10% glycerol, 20 mM imidazole. The cells were stirred for approximately 30 min to resuspend and then lysed using a Constant Systems cell disruptor at a pressure of 20,000 psi. The cell lysate was clarified by centrifugation at 50,000 x g at 4 °C for 30 min in a Beckman JA 25.5 rotor. All subsequent purification steps were carried out at 4 °C. The clarified lysate was then applied to a 5 ml HisTrap FF column, which was equilibrated in PBS (pH 7.4, 10% glycerol, 20 mM imidazole). The column was washed with 20 column volumes of this buffer and then the bound protein was eluted in 3.5 column volumes of PBS (pH 7.4, 10% glycerol, 500 mM imidazole).The eluted protein was then further purified and the buffer was exchanged by flowing it down a GE 26 / 60 S200 SEC column into 150 mM MOPS (pH 8.0, 50 mM NaCl, 10% glycerol). The elution fractions containing the target protein were combined, and the sample was bead formed in liquid nitrogen and subsequently stored at -80 °C. The protein concentration was determined using a Nanodrop ME52070. Based on SDS PAGE stained with Coomassie blue, the protein obtained typically ran as a single major band (corresponding to an expected molecular weight of approximately 44 kDa) (e.g., for the N-terminal his-tagged SEQ ID NO:13), and was typically judged to be > approximately 90% pure based on gel densitometry.

[0910] Example 2. Determination of Geranylgeranyl Diphosphate Synthase Activity

[0911] Polyisoprenyltransferase activity is determined by measuring the phosphate produced by incorporating isopentenyl pyrophosphate (IPP) into geranylgeranyl pyrophosphate (GGPP) that releases pyrophosphate. The assay is coupled with inorganic pyrophosphatase (IPPase) to release inorganic phosphate (quantified using the malachite green reagent). The assay is run in a 96-well microtiter plate. The herbicide is dissolved in dimethyl sulfoxide (DMSO) at a high enough concentration as a stock solution. 2 μl of a diluted herbicide at an appropriate concentration is transferred to a 96-well clear microtiter plate. The enzyme (typically at a stock solution concentration of about 10 mg / ml) is diluted to 128 nM in 100 mM Tricine buffer (pH 8.0, containing 641 mM IPP, 12.8 mM MgCl2, 6.41 mg / ml bovine serum albumin (BSA), 0.26% Tween 20, and 1.28 units / ml IPPase from Escherichia coli). A 78 μl aliquot of this mixture is added to each well of the plate containing the herbicide. The plate is incubated at 25 °C for 60 min, followed by starting the assay with 250 mM GGPP in 20 μl of 100 mM Tricine pH 8.0. Typically, the assay is carried out by incubating at 25 °C for 10 - 60 min, followed by stopping with 200 μl of malachite green reagent. To prepare the malachite green reagent, 600 ml of 1 mM malachite green oxalate is mixed with 200 ml of 33 mM ammonium molybdate in 3.92 M HCl. The color change is allowed to develop for 15 minutes before reading the absorbance at 630 nm in a Tecan M200 microplate reader. A phosphate standard curve is run with each set of assays. A reagent blank control (MAX) is run with DMSO instead of the test herbicide and a full inhibition control (MIN) assay is run with a high enough concentration of the test herbicide to determine the percentage of inhibition in each well using the following expression: percentage of inhibition = 100 x (1 - (X - MIN) / (MAX - MIN)), where X is the absorbance at 630 nm and MIN and MAX are the average absorbances of the controls. The percentage of inhibition values are fitted to a four-parameter logistic regression to determine the half-maximal inhibitory concentration (IC 50 ).

[0912] In an alternative method for determining polyisoprenyltransferase activity, phosphate was quantified using a Thermo Fisher EnzChek assay kit according to the manufacturer's instructions. This method couples the production of inorganic phosphate with the enzymatic conversion of 2-amino-6-mercapto-7-methylpurine riboside (MESG) to ribose 1-phosphate and 2-amino-6-mercapto-7-methylpurine by purine nucleoside phosphorylase (PNP). The herbicide was dissolved in DMSO at a high enough concentration as a stock solution. 4 μl of a diluted herbicide at an appropriate concentration was transferred to a 96-well clear microtiter plate. The enzyme (typically at a stock solution concentration of about 10 mg / ml) was diluted to 227 nM in 100 mM Tricine buffer (pH 8.0, containing 570 mM IPP, 11.4 mM MgCl2, 5.7 mg / ml BSA, 0.23% Tween 20, 1.14 units / ml IPPase from E. coli, 1.14 units / ml PNP, 5% reaction buffer provided by the manufacturer, and 0.23 mM MESG). A 176 μl aliquot of this mixture was added to each well of the plate containing the herbicide. The plate was incubated at 25 °C for 60 min, followed by starting the assay with 500 mM GGPP in 20 μl of 100 mM Tricine pH 8.0. The assay at 25 °C was measured by reading the absorbance at 360 nm (typically for 10 - 60 min) in a Tecan M200 microplate reader. The comparison rate was calculated from the rate of change of absorbance in the linear region of the assay by comparison with a phosphate standard curve. A reagent blank control (MAX) was run with DMSO instead of the test herbicide and a complete inhibition control (MIN) assay was run with a high enough concentration of the test herbicide to determine the percentage of inhibition in each well using the following expression: Percentage of inhibition = 100 x (1 - (X - MIN) / (MAX - MIN)), where X is the rate of change of absorbance at 360 nm and MIN and MAX are the average ratios of the controls. The percentage of inhibition values were fitted to a four-parameter logistic regression to determine the half-maximal inhibitory concentration (IC 50 ).

[0913] Example 3. Identification of Herbicide Tolerance Variants of SDPS Enzyme

[0914] Use the assays described in Example 2 to identify SDPS variants with increased herbicide tolerance to herbicide compounds Examples 1 and 2. The SDPS sequence (such as SEQ ID NO. 16) was codon-optimized for E. coli expression and a single-site saturation library was created at the desired amino acid positions (Twist Bioscience, USA), resulting in a pool of all possible amino acid substitutions at the selected amino acid positions. Table 1 shows the selected amino acid positions that were varied, assayed, and sequenced (numbered based on SEQ ID NO. 5). Table 1A shows the amino acid motifs of the exemplary SDPS motifs of the present invention.

[0915] Table 1.

[0916] L120 I138 E179 D189 A213 Q223 K238 A248 L258 I280 L123 F139 M180 D190 V214 S224 L239 S249 K271 A281 V124 R147 I181 V191 L215 S225 I240 G250 S272 A282 G125 P148 H182 I192 A216 W226 S241 E251 Y273 S283 A126 V151 T183 I204 G217 F227 Q242 I252 Y274 Y302 E127 L174 A184 Y208 D218 L228 V243 K253 K275 R306 N128 A175 S185 G209 F219 A229 I244 Q254 T276 G309 V130 E176 L186 T210 M220 N230 K245 A255 A277 L310 L131 I177 I187 R211 F221 V236 D246 S256 S278 F312 A134 I178 H188 V212 A222 I237 F247 T257 L279 Q313

[0917] Table 1A

[0918]

[0919]

[0920]

[0921] Table 2 shows SDPS variants that demonstrated higher tolerance to herbicides X and Y than the parental sequence SEQ ID NO: 16 in the assays described in Example 2.

[0922]

[0923]

[0924]

[0925]

[0926]

[0927]

[0928]

[0929]

[0930] It is clear that some mutations provide increased tolerance to one or in some cases both of the selected herbicides. In many cases, the same mutation was recovered multiple times (for each library) and demonstrated a persistent tolerance pattern to the herbicide. Data for a single instance of each targeted mutation are shown.

[0931] From this list of variants, further select those that have strong tolerance to one of the herbicides or those that are tolerant to both herbicides. As described in Example 2, these variants were assayed to determine the IC50 values. Table 3 shows the IC50 data for each variant. The data clearly show that the selected examples have significantly increased IC50 values for one or more of the target herbicides.

[0932] Table 3.

[0933]

[0934]

[0935]

[0936]

[0937] Example 4. SDPS Sequences and Expressions in Plants

[0938] Arabidopsis SDPS or its orthologs (see full-length SDPS sequences, including the chloroplast transit peptide), such as SEQ ID No. 1-12 expressed in transgenic tobacco. The DNA sequences encoding these polypeptides (optimized for tobacco or optionally codon-optimized according to the target crop such as soybean) were prepared synthetically. Each sequence was designed to include a 5' fusion with the TMVΩ 5' leader sequence and was flanked by XhoI at the 5' end and KpnI at the 3' end to facilitate direct cloning into a binary vector suitable for Agrobacterium-based plant transformation.

[0939] In one instance, the expression cassette (containing the TMVΩ 5' leader sequence and the SDPS-encoding gene of interest) was cut with XhoI / KpnI and cloned into similarly digested pBIN 19 (Bevan, Nucl. Acids Res. [Nucleic Acids Research] (1984)) (after a double-enhanced 35S promoter and before a NOS 3' transcriptional terminator) and then transformed into Escherichia coli DH5α competent cells. The DNA recovered from this E. coli was used to transform Agrobacterium tumefaciens LBA4404, and the transformed bacteria were selected on medium containing rifampicin and kanamycin. Tobacco tissues were subjected to Agrobacterium-mediated transformation using methods well described in the art or as described herein. For example, a master plate of Agrobacterium tumefaciens containing the binary vector expressing SDPS was used to inoculate 10 ml of LB (L broth) containing 100 mg / l rifampicin plus 50 mg / l kanamycin (using a single colony). This culture was incubated overnight at 28 °C with shaking at 200 rpm. This entire overnight culture was used to inoculate 50 ml volumes of LB containing the same antibiotics. Again, this culture was grown overnight at 28 °C with shaking at 200 rpm. The Agrobacterium cells were pelleted by centrifugation at 3000 rpm for 15 minutes and then resuspended in MS (Murashige and Skoog) medium containing 30 g / l sucrose, pH 5.9, to an OD(600 nM) = 0.6. This suspension was aliquoted into Petri dishes at 25 ml aliquots.

[0940] Clonally micropropagated tobacco shoot cultures were used to excise young leaves (not fully expanded). The midribs and outer leaf margins were removed and discarded, and the remaining leaves were cut into 1 cm² pieces. These leaves were transferred to the Agrobacterium suspension for 20 minutes. The explants were then removed, blotted on sterile filter paper to remove excess suspension, and then transferred to solid NBM medium (MS medium containing 30 g / l sucrose, 1 mg / l BAP (benzylaminopurine), and 0.1 mg / l NAA (naphthaleneacetic acid) at pH 5.9 and solidified with 8 g / l phytagel), with the abaxial surface of each explant in contact with the medium. Approximately 7 explants were transformed per plate, and then the plates were sealed and maintained in a light incubator at 25 °C with a 16-hour light cycle for 3 days.

[0941] The explants were then transferred to NBM medium containing 100 mg / l kanamycin plus antibiotics to further arrest the growth of Agrobacterium (200 mg / l ticarcillin with 250 mg / l carbenicillin). Further subculturing was then performed on this same medium every 2 weeks.

[0942] When shoots began to regenerate from the callus leaf explants, these were transferred to shoot elongation medium (MS medium, 30 g / l sucrose, 8 g / l plant agar, 100 mg / l kanamycin, 200 mg / l ticarcillin, 250 mg / l carbenicillin, pH 5.9). These stable transgenic plants rooted readily within 2 weeks. To provide multiple plants for each event, ultimately allowing more than one herbicide test per transgenic plant, all rooted shoots were micropropagated to produce 3 or more rooted clones.

[0943] Putative transgenic plants that were rooting on medium containing kanamycin and showing vigorous shoot growth were analyzed by PCR using primers that amplified a 500 bp fragment specific for the SDPS transgene of interest. Evaluation of this same primer set on untransformed tobacco conclusively showed that these primers did not amplify any sequences from the native tobacco genome.

[0944] The transformed shoots were divided into 2 or 3 clones and regenerated from kanamycin-resistant callus. These shoots rooted on MS agar containing kanamycin. The surviving rooted explants were regenerated to provide approximately 40 - 50 kanamycin-resistant and PCR-positive events from each event.

[0945] Once rooted, the plantlets were transferred from the agar and potted into 3-inch round pots with 50% peat, 50% John Innes Soil No. 3 with slow-release fertilizer, and watered regularly to establish in the greenhouse for 8 to 12 days. Greenhouse conditions were approximately 24 °C - 27 °C during the day; 18 °C - 21 °C at night and a photoperiod of approximately 14 h. Humidity was regulated to approximately 65% and the light level used was up to 2000 μmol / m at bench level. 2 。

[0946] Thus, a transgenic population of about forty tobacco plants containing genes encoding full-length SDPS genes (such as Seq ID No 2) was generated. Plants were selected from each population based on the same size and subjected to ELISA or Mass Western tests to monitor the protein transgenic SDPS expression levels. The highest expressing T0 lines were selfed in the normal manner and T1 seeds and T2 lines and seeds were generated. Seeds from the highest expressing lines were tested for germination on agar plates containing a series of concentrations of the SDPS inhibitory herbicide as taught in the examples herein, and resistant plant lines were selected as having the least damage to root growth and morphology at the highest concentration of the herbicide. The resistant plant lines showed a dose response to herbicidal damage by the SDPS inhibitor that was shifted to the right compared to similarly grown and treated wild-type and null segregant plants.

[0947] Example 5: Determination of Herbicide Tolerance in Transgenic Tobacco

[0948] Transgenic tobacco populations were generated as described in Example 4, each plant transformation construct containing 20 - 30 transgenic events. These lines were cloned and propagated, and one clone / event was sprayed with 1000 g / ha of the herbicide compound 1. Visual assessments of herbicidal damage were made on the entire population, and herbicide damage scores were given at 7 and 14 days. A score of 1 indicates no visible damage or developmental retardation, while a score of 100 indicates complete plant death.

[0949] Figure 3 The results in show the damage scores for 4 plant populations expressing: the Arabidopsis SDPS2 gene (SEQID NO.2), the Arabidopsis SDPS2 F240L mutant gene (SEQ ID NO.3), the Chlamydomonas SDPS gene (SEQ ID NO.10), or the Chlamydomonas SDPS F227L mutant gene (SEQ ID NO.11). The herbicide damage of a wild-type Samsun tobacco control population was also evaluated. The average damage scores for the entire transgenic plant population for each construct are given in Table 4. It is clear that overexpression of the Chlamydomonas SDPS (SEQ ID NO.10) gene does not increase tolerance to the herbicide compound Example 1, however, the mutant version of the gene carrying the F227L mutation (SEQ ID NO.11) does show increased tolerance. The Arabidopsis gene (SEQ ID NO.2) performs better than the Chlamydomonas wild-type gene in terms of herbicide tolerance, and this tolerance was again improved by addition of the F240L mutation (SEQ ID NO.3).

[0950] Table 4.

[0951] Construct Average Damage (%) WT Control 23.125 pBin TMV CfSDPS 25 pBin TMV CfSDPS_F227L 10.05 pBin TMV AraSDPS2 13.25 pBin TMV AraSDPS2_F240L 5

[0952] Another set of transgenic tobacco plants overexpressing the Arabidopsis thaliana SDPS F240L gene (SEQ ID NO.3) was generated as described above. These plants were sprayed with acifluorfen at 75 g / ha or herbicide Z at 25 g / ha. Herbicidal injury to the plants was evaluated 7 days after treatment and compared to wild-type tobacco plants. Herbicidal injury scores are shown in Table 5.

[0953] Table 5:

[0954]

[0955] In contrast to the injury seen on control plants, many transgenic tobacco events expressing the Arabidopsis thaliana SDPS2 F240L gene (SEQ ID NO.3) showed no injury from herbicide treatment.

[0956] The average herbicidal injury scores of the transgenic tobacco population relative to control wild-type plants are shown in Table 6.

[0957] Table 6:

[0958] Aclonifen 75 g / ha Herbicide Z 25 g / ha pBin AraSDPS2 F240L 16.16666667 4.833333333 Wild Type 40 21.875

[0959] Results showed a significant increase in herbicide tolerance in transgenic lines expressing the Arabidopsis thaliana SDPS2 F240L gene (SEQ ID NO.3).

[0960] Example 6: Tolerance Analysis of Transgenic Tobacco T1 Lines in Liquid Medium

[0961] Seed sterilization:

[0962] Tobacco (Nicotiana tabacum) Samsun wild-type and transgenic lines were sterilized by placing them in a 30 ml Universal container containing approximately 15 ml of freshly prepared 2% Virkon and rotating on a roller for 15 min. The Virkon was removed using an extended fine-tip micropipette and the seeds were washed 4 times with 1% PPM (Plant Preservative Mixture, P820, 250 ml, Apollo Scientific Ltd.).

[0963] Seed germination:

[0964] The seeds were germinated in 90 mm Petri dishes containing 1 / 3 MS + 0.8% agarose (SPI, Duchefa #DU 0463), pH 5.7 (for WT) and 1 / 3 MS + 0.8% agarose + 100 ug / ml kanamycin (for transgenic). Approximately 100 seeds were scattered on each Petri dish. Note: The transgenic seeds are not homozygous, so they are inoculated on kanamycin for selection. (MS = Murashige and Skoog MS medium, #M0221, Melford Laboratories).

[0965] The plates were placed in a transparent plastic "incubator" (covered under the plate) and incubated in a controlled environmental chamber with the following conditions:

[0966] Temperature: 25 °C during the day / 25 °C at night

[0967] Light settings: Photoperiod 16 hours, light level approximately 50 μmol / m 2 / s

[0968] Dose-response test:

[0969] Seven-day-old tobacco seedlings (3 seedlings per well) were aseptically transferred to 24-well plates (flat-bottom cell culture multi-well plates with lids) (Corning Costar #3524) containing 2 ml of 1 / 2 MS + 30 mM sucrose (Fisher #10638403), pH 5.7 per well. Herbicide 2 was serially diluted two-fold starting from 10 ppm in DMSO and added to the medium. Only DMSO was added to the zero-compound control wells. Note: The final concentration of DMSO does not exceed 0.5% as it has an inhibitory effect on the seedlings.

[0970] The plates were placed in a transparent plastic "incubator" (covered under the plate) and incubated in a controlled environmental chamber for 2 weeks with the following conditions:

[0971] Temperature: 24 °C during the day / 18 °C at night

[0972] Light settings: Photoperiod 16 hours, light level 600 μmol / m 2 / s

[0973] Evaluation:

[0974] After 7 days, the bleaching symptoms of the seedlings were visually evaluated and a damage score was given for each well. Each plate was repeated and two damage scores were given in Table 7.

[0975] Table 7:

[0976] Tobacco Lines 10 ppm 5 ppm 2.5 ppm 1.25 ppm 0.75 ppm DMSO WT (Samsun) 95 / 100 95 / 90 50 / 50 0 / 0 0 / 0 0 / 0 ARA SDPS1 100 / 100 100 / 100 50 / 50 0 / 0 0 / 0 0 / 0 ARA SDPS1 100 / 100 90 / 80 50 / 25 0 / 0 0 / 0 0 / 0 ARA SDPS1 100 / 100 80 / 95 25 / 25 0 / 0 0 / 0 0 / 0

[0977]

[0978]

[0979] The presence of the F240L mutation reduced damage under 5 ppm herbicide treatment, although no significant advantage was seen at 10 ppm.

[0980] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and various modifications or variations according to their specifications will suggest to those skilled in the art and will be included within the spirit and scope of this application and within the scope of the appended claims.

[0981] All publications and patent applications mentioned in this specification indicate the level of skill of those skilled in the art to which the present invention pertains. All publications and patent applications are hereby incorporated by reference in their entirety, to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. A method for selectively controlling weeds at a site comprising crop plants and weeds, the method comprising: applying to the site a pest control amount of a pesticidal composition comprising acifluorfen, wherein the crop plants comprise a recombinant polynucleotide encoding a modified solanesyl diphosphate synthase (SDPS) protein, the modified SDPS protein providing tolerance to acifluorfen for the crop plants when expressed in the plants, wherein the modified SDPS protein consists of the amino acid sequence of SEQ ID NO:

3.

2. The method according to claim 1, wherein the solanesyl diphosphate synthase is provided by editing an endogenous solanesyl diphosphate synthase.

3. The method according to claim 2, wherein the editing is introduced by using a nucleic acid and optionally at least one guide RNA, the nucleic acid encoding a DNA modifying enzyme selected from the group consisting of site-specific nucleases, the site-specific nucleases selected from the group consisting of: meganuclease (MN), zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), Cas9 nuclease, Cas12, dCas9-FokI, dCpf1-FokI, chimeric FEN1-FokI and Mega-TAL, nickase Cas9 (nCas9), chimeric dCas9 non-FokI nuclease and dCpf1 non-FokI nuclease; the at least one guide RNA corresponding to the amino acid substitution involved in SEQ ID NO:

3.

4. The method according to claim 3, wherein the DNA modifying enzyme is a site-specific nuclease, the site-specific nuclease selected from the group consisting of: Cas9 nuclease, Cas12 nuclease, dCas9-FokI, dCpf1-FokI, chimeric FEN1-FokI and Mega-TAL, nickase Cas9 (nCas9), chimeric dCas9 non-FokI nuclease and dCpf1 non-FokI nuclease; and comprises the guide RNA.

5. The method according to any one of claims 1-4, wherein the crop plants further comprise another recombinant polynucleotide encoding an additional herbicide tolerance enzyme, wherein the additional herbicide tolerance enzyme is selected from the group consisting of: 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), HST, glyphosate acetyltransferase (GAT), cytochrome P450, phosphinothricin acetyltransferase (PAT), acetolactate synthase (ALS), protoporphyrinogen oxidase (PPGO), hydroxyphenylpyruvate dioxygenase (HPPD) and dicamba degrading enzyme.

6. The method according to any one of claims 1-4, wherein the pesticidal composition comprises one or more additional herbicides.

7. The method according to claim 6, wherein said one or more additional herbicides are selected from the group consisting of: glyphosate or its salts, glufosinate or its salts, alachlor, acetochlor, metolachlor, S - metolachlor, ametryn, atrazine, cyanazine, terbuthylazine, triazinon, hexazinone, simazine, chlorotoluron, diuron, isoproturon, linuron, buthiuron, amidosulfuron, chlorsulfuron, flupyrsulfuron, halosulfuron, nicosulfuron, flazasulfuron, rimsulfuron, tribenuron - methyl, tritosulfuron, acifluorfen, fomesafen, mesotrione, pyroxasulfone, dicamba, and 2,4 - D.

8. A recombinant polynucleotide comprising: a nucleic acid sequence encoding a modified solanesyl - diphosphate synthase (SDPS), said modified SDPS consisting of the amino acid sequence of SEQ ID NO:3; and a plant - operable promoter operably coupled to said nucleic acid sequence, wherein expression of said modified SDPS in a plant confers upon said plant increased tolerance to the SDPS - inhibitory herbicide oxyfluorfen.

9. The recombinant polynucleotide according to claim 8, wherein said plant - operable promoter is heterologous to the nucleic acid sequence encoding the modified SDPS.

10. A recombinant nucleic acid molecule comprising a nucleotide sequence encoding a modified solanesyl - diphosphate synthase (SDPS) protein, the expression of said modified SDPS protein in a crop plant providing tolerance to a solanesyl - diphosphate synthase - inhibitory herbicide, wherein said modified SDPS protein consists of the amino acid sequence of SEQ ID NO:

3.

11. A chimeric gene comprising a heterologous promoter and the recombinant nucleic acid molecule according to claim 10 operably linked to said heterologous promoter.

12. The chimeric gene according to claim 11, wherein said heterologous promoter is a plant - expressible promoter.

13. The chimeric gene according to claim 12, wherein the plant-expressible promoter is selected from the group consisting of the following promoters: Ubiquitin, maize TrpA, OsMADS 6, maize histone H3, bacteriophage T3 gene 9 5'UTR, maize sucrose synthase 1, maize alcohol dehydrogenase 1, maize light - harvesting complex, maize heat - shock protein, maize mtl, pea small subunit RuBP carboxylase, rice actin, rice cyclophilin, Ti plasmid mannopine synthase, Ti plasmid nopaline synthase, petunia chalcone isomerase, legume glycine - rich protein 1, potato glycoprotein, lectin, CaMV 35S, and S - E9 small subunit RuBP carboxylase promoter.

14. A vector comprising the chimeric gene according to any one of claims 11 - 13.

15. A method for identifying a solanesyl - diphosphate synthase (SDPS) - inhibitory herbicide, said method comprising: expressing a modified SDPS in a plant, wherein said modified SDPS consists of the following amino acid sequence: a) SEQ ID NO:3; or b) SEQ ID NO:10 with only the amino acid substitution F227L; or c) SEQ ID NO:5 with only the amino acid substitution F247L; and exposing the plant to a compound, wherein reduced damage in the plant relative to a control plant that does not express the SDPS indicates that the compound is a solanesyl diphosphate synthase inhibitory herbicide.

16. The method according to claim 15, wherein the exposure is in the range of 2.5 ppm to 20 ppm.

17. A method of producing a plant having increased acifluorfen tolerance, the method comprising: introducing into a recipient plant cell a nucleic acid molecule comprising a polynucleotide encoding a modified SDPS protein consisting of the amino acid sequence of SEQ ID NO:3, wherein the nucleic acid molecule is incorporated into the genome of the recipient plant cell; and regenerating the recipient plant cell into a transgenic plant having increased acifluorfen tolerance.

18. The method according to claim 17, wherein the nucleic acid molecule further comprises a heterologous regulatory element operably coupled to the polynucleotide.

19. The method according to claim 18, wherein the heterologous regulatory element is a plant-expressible promoter.

20. The method according to any one of claims 17-19, wherein introducing the nucleic acid molecule into the recipient plant cell is achieved by transforming the recipient plant cell with the nucleic acid molecule.

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