WO2020146949A1 - Plantes ayant une tolérance accrue aux herbicides inhibiteurs de la biosynthèse de la cellulose - Google Patents

Plantes ayant une tolérance accrue aux herbicides inhibiteurs de la biosynthèse de la cellulose Download PDF

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Publication number
WO2020146949A1
WO2020146949A1 PCT/CA2020/050046 CA2020050046W WO2020146949A1 WO 2020146949 A1 WO2020146949 A1 WO 2020146949A1 CA 2020050046 W CA2020050046 W CA 2020050046W WO 2020146949 A1 WO2020146949 A1 WO 2020146949A1
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seq
cesa
plant
polypeptide
nucleic acid
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PCT/CA2020/050046
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English (en)
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Dario Torquato BONETTA
Julian Geoffrey Northey
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Frontier Agri-Science Inc.
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Publication of WO2020146949A1 publication Critical patent/WO2020146949A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C12N9/1059—Cellulose synthases (2.4.1.12; 2.4.1.29)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/60—Isolated nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01012—Cellulose synthase (UDP-forming) (2.4.1.12)

Definitions

  • the present invention relates to methods for conferring on plants agricultural level tolerance to herbicides. More specifically, the present invention relates to methods and plants obtained by mutagenesis and cross-breeding and transformation that have an increased tolerance to herbicides, particularly cellulose-biosynthetic inhibiting (CBI) herbicides.
  • CBI cellulose-biosynthetic inhibiting
  • weeds unwanted plants
  • An ideal treatment is one that can be applied to an entire field, eliminating only the unwanted plants and leaving the crop plants unharmed.
  • One such treatment system involves the use of crop plants that are tolerant to a herbicide. When the herbicide is applied to a field of herbicide-tolerant crop plants, the crop plants continue to thrive while non-herbicide-tolerant weeds are killed or severely damaged.
  • Plant cell walls are complex structures composed of high-molecular-weight polysaccharides, proteins, and lignins.
  • cellulose a hydrogen-bonded p-l,4-linked glucan microfibril
  • cellulose is the main load-bearing wall component and a key precursor for industrial applications.
  • Cellulose is synthesized by large multimeric cellulose synthase (CESA) complexes (E.C.2.4.1.12), tracking along cortical microtubules at the plasma membrane.
  • CESA cellulose synthase
  • the only known components of these complexes are the cellulose synthase proteins.
  • Recent studies have identified tentative interaction partners for the CESAs and have shown that the migratory patterns of the CESA complexes depend on
  • cotton cellulose synthase genes termed CESA1 and CESA2
  • CESA1 and CESA2 were identified in a collection of expressed sequence tag (EST) sequences on the basis of weak sequence similarity to genes for cellulose synthase from bacteria (Richmond and Somerville. Plant Physiology, 2000, Vol. 124, 495-498).
  • EST expressed sequence tag
  • the genes were expressed at high levels in cotton fibers at the onset of secondary wall synthesis and a purified fragment of one of the corresponding proteins was shown to bind UDP-Glc, the proposed substrate for cellulose biosynthesis.
  • CESA genes Ten full-length CESA genes have been sequenced from Arabidopsis, and there is a genome survey sequence that may indicate one additional family member.
  • CESA family which includes RSW1 and IRX3 (AtCESA7), and six families of structurally related genes of unknown function designated as the“cellulose synthase-like" genes (Cs/A, Cs/B, Cs/C, CsID, CsIE, and CsIG).
  • Arabidopsis plant lines containing mutations in CESA 1, CESA 3 and CESA 6 show defects in primary cell wall cellulose accumulation. These defects indicate that CESA1, 3, and 6 are required for primary cell wall production. Furthermore, mutant lines have been used to support that CESA 4,
  • CESA 7, and CESA 8 are required for secondary cell wall synthesis.
  • Null mutants for CESA 1 and CESA 3 are lethal, while CESA 6 null mutants only exhibit subtle growth phenotypes. Therefore, it was proposed that CESA 1 and 3 are both essential to primary cell wall production, while CESA 6 may be redundant. It was found that CESAs 2, 5, and 9 have partial redundancy to CESA 6, and are likely incorporated into the primary cell wall cellulose synthase complex in place of CESA6 at different developmental stages.
  • WO 2013/142968 describes plant cellulose synthase (CESA) alleles identified by mutagenizing plants and screening said plants with a cellulose biosynthetic inhibitor (CBI).
  • CBIs employed in WO 2013/142968 include dichlobenil, chlorthiamid, isoxaben, flupoxam, and quinclorac, particularly isoxaben or flupoxam (named fpxl-1 to fpxl-3 [CESA3], fpx 2-1 to fpx2-3 [CESA1] and ixrl-1 to ixrl-7 [CESA3], ixr2-l to ixr2-2 [CESA6] mutants of Arabidopsis CESA wildtype enzymes).
  • CBIs include, but are not limited to, indaziflam, a member of the alkylazine family that is active at picomolar concentrations and has a long soil residual making it an outstanding pre-emergent herbicide.
  • the alkylazine scaffold has shown to be an excellent lead compound for CBI discovery and optimization.
  • This group includes indaziflam, triaziflam, and AE FI 50944. Additional CBIs are included in WO2017068543 whereby particular fpx and ixr mutants mentioned above confer resistance to these CBIs.
  • a plant or plant part comprising a polynucleotide encoding a mutated CESA polypeptide, the expression of said polynucleotide conferring to the plant or plant part tolerance to at least one CBI herbicide.
  • a seed capable of germination into a plant comprising in at least some of its cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a mutated CESA polypeptide encoded by the polynucleotide, the expression of the mutated CESA polypeptide conferring to the plant tolerance to at least one CBI herbicide.
  • a plant cell of or capable of regenerating a plant comprising in at least some of its cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a mutated CESA polypeptide encoded by the polynucleotide, the expression of the mutated CESA polypeptide conferring to the plant tolerance to at least one CBI herbicide, wherein the plant cell comprises the polynucleotide operably linked to a promoter.
  • an isolated nucleic acid which encodes a mutant CESA polypeptide comprising a mutation corresponding to position S1052 in SEQ ID NO: 1, or a fragment or ortholog thereof encoding the mutant CESA polypeptide wherein the fragment or ortholog comprises the SI 052 mutation, retains the function of the mutant CESA polypeptide and is at least 68% identical to SEQ ID NO: 1.
  • an isolated nucleic acid which encodes a mutant CESA polypeptide comprising a mutation corresponding to position G863 in SEQ ID NO: 5, or a fragment or ortholog thereof encoding the mutant CESA polypeptide wherein the fragment or ortholog comprises the G863 mutation, retains the function of the mutant CESA polypeptide and is at least 75% identical to SEQ ID NO: 5.
  • the isolated nucleic acid sequence may be at least 75% identical to SEQ ID NO: 4 or 8 or may encode a polypeptide which is at least 75% identical to SEQ ID NO: 3 or 7.
  • the mutated CESA polypeptide may comprise the sequence of a CESA orthologue, paralogue, or homologue, wherein the amino acid sequence differs from the wild type amino acid sequence at one or more positions corresponding to at least position SI 052 of SEQ ID NO: 1 or position G863 in SEQ ID NO: 5.
  • the mutated CESA polypeptide may comprise a mutation to a non-polar amino acid, such as a A, V, L, I, F, W or M residue at position S1052.
  • the mutated CESA polypeptide may comprise a S1052F mutation, corresponding to the sequence of SEQ ID NO: 1.
  • the mutated CESA polypeptide may comprise a mutation to a polar amino acid, including a G, Q, N, S, T, Y or C residue at position G863.
  • the mutated CESA polypeptide may comprise a G863S mutation, corresponding to the sequence of SEQ ID NO: 5.
  • the orthologue, paralogue, or homologue may include, without limitation: Arabidopsis thaliana CESA (SEQ ID NO: 9) Capsella Rubella CESA (SEQ ID NO: 10), Brassica rapa FPsc CESA (SEQ ID NO: 11), Brassica oleracea capitata CESA (SEQ ID NO: 12), Gossypium raimondii CESA (SEQ ID NO: 13); Glycine max CESA (SEQ ID NO: 14), Medicago truncatula CESA (SEQ ID NO: 15), Panicum virgatum CESA (SEQ ID NO: 16), Sorghum bicolor CESA (SEQ ID NO: 17), Oryza sativa CESA (SEQ ID NO: 18), Brachypodium distachyon CESA (SEQ ID NO: 19), Zea mays CESA (SEQ ID NO: 20), Physcomitrella patens CESA (SEQ ID
  • the nucleic acid sequence may be 80%, 85%, 90%, 99% or 100% identical to SEQ ID NO: 4 or 8 or encode a polypeptide which is 80%, 85%, 90%, 99% or 100% identical or similar SEQ ID NO: 3 or 7.
  • the present invention may provide a vector, host cell, seed or plant comprising a nucleic acid as defined above.
  • a mutant CESA polypeptide comprising a mutation at a position corresponding to SI 052 in SEQ ID NO: 1, or a fragment or ortholog of the mutant CESA polypeptide comprising the SI 052 mutation, which retains the function of the mutant CESA polypeptide and is at least 68% identical to SEQ ID NO: 1.
  • the mutated CESA polypeptide may comprise a mutation to a non-polar amino acid, such as a A, V, L, I, F, W or M residue at position SI 052.
  • a mutant CESA polypeptide comprising a mutation at a position corresponding to G863 in SEQ ID NO: 5, or a fragment or ortholog of the mutant CESA polypeptide comprising the G863 mutation, which retains the function of the mutant CESA polypeptide and is at least 75% identical to SEQ ID NO: 5.
  • the mutated CESA polypeptide may comprise a mutation to a polar amino acid, such as a G, Q, N, S, T, Y or C residue at position G863.
  • the mutated CESA polypeptide may be a CESA orthologue, paralogue, or homologue, wherein the amino acid sequence differs from the wild type amino acid sequence at one or more positions corresponding to at least position SI 052 of SEQ ID NO: 1.
  • the orthologue, paralogue, or homologue may be: Arabidopsis thaliana CESA (SEQ ID NO: 9) Capsella Rubella CESA (SEQ ID NO: 10), Brassica rapa FPsc CESA (SEQ ID NO: 11), Brassica oleracea capitata CESA (SEQ ID NO: 12), Gossypium raimondii CESA (SEQ ID NO: 13); Glycine max CESA (SEQ ID NO: 14), Medicago truncatula CESA (SEQ ID NO: 15), Panicum virgatum CESA (SEQ ID NO: 16), Sorghum bicolor CESA (SEQ ID NO: 17), Oryza sativa CESA (SEQ ID NO: 18), Brachypodium distachyon CESA (SEQ ID NO: 19), Zea mays CESA (SEQ ID NO: 20), Physcomitrella patens
  • plants or seeds thereof having a genotype characterized by resistance to at least one herbicidal compound, the plant or seed thereof comprising a mutant cesa gene comprising at least one mutation in the cesa sequence corresponding to one or more of positions SI 052 in SEQ ID NO: 1 or G863 in SEQ ID NO: 5.
  • Such plants and seeds may comprise the nucleic acid or encode the mutant CESA polypeptide described above, or else as further described throughout this specification.
  • the plant or seed described above, and throughout this specification, together with at least one CBI herbicidal compound in a method to inhibit growth of one or more undesired plants.
  • the at least one CBI herbicidal compound is flupoxam, dichlobenil, chlorthiamid, isoxaben, indaziflam, triaziflam or a combination thereof.
  • the CBI herbicidal compound is flupoxam.
  • the plant or seed thereof may encode a mutant polypeptide comprising a mutation corresponding to at least one of position SI 052 in SEQ ID NO: 1 or G863 in SEQ ID NO: 5, or a fragment or ortholog of the mutant polypeptide in which the fragment or ortholog comprises the at least one SI 052 or G863 mutation, retains the function of the mutant polypeptide and is at least 68% identical to SEQ ID NO: 1 or at least 75% identical to SEQ ID NO: 5.
  • the mutant polypeptide is as described in further detail above, or else as further described throughout this specification.
  • the nucleic acid sequence may be 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 4 or the polypeptide sequence may be 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:
  • nucleic acid sequence may be 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 8 or the polypeptide sequence may be 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 7 (G863).
  • Also provided herein is a method for controlling weeds at a locus for growth of a plant, the method comprising:
  • the herbicide composition will typically be applied to the weeds and to the plant produced by the seed.
  • the expression of the nucleic acid of the invention in the plant results in the plant's increased resistance to CBI herbicides as compared to a wild type variety of the plant.
  • FIG. 1 is a protein alignment of homologous CESA sequences in plant species. The alignment was made using CLUSTAL 0 (ver. 1.2.4).“Arabidopsis” refers to the CESA sequence in Arabidopsis thaliana (SEQ ID NO: 9).“Capsella” refers to the CESA sequence in Capsella Rubella (SEQ ID NO: 10).“Field” refers to the CESA sequence in Brassica rapa FPsc (Field mustard; SEQ ID NO: 11).“Brassica Oleracea” refers to the CESA sequence in Brassica Oleracea (SEQ ID NO: 12).“Gossypoum” refers to the CESA sequence in
  • Gossypium raimondii (SEQ ID NO: 13).“Glycine” refers to the CESA sequence in Glycine max (SEQ ID NO: 14).“Medicago” refers to the CESA sequence in Medicago truncatula (SEQ ID NO: 15).“Panicum” refers to the CESA sequence in Panicum virgatum (SEQ ID NO: 16).“Sorghum” refers to the CESA sequence in Sorghum bicolor (SEQ ID NO: 17).
  • “Oryza” refers to the CESA sequence in Oryza sativa (SEQ ID NO: 18).“Brachypodium” refers to the CESA sequence in Brachypodium distachyon (SEQ ID NO: 19).“Zea” refers to the CESA sequence in Zea mays (SEQ ID NO: 20).“Physcomitrella” refers to the CESA sequence in Physcomitrella patens (SEQ ID NO: 21).“Vitis” refers to the CESA sequence in Vitis vinifera (SEQ ID NO: 22).“Phaseolus” refers to the CESA sequence in Phaseolus vulgaris (SEQ ID NO: 23).“Barley” refers to the CESA sequence in Hordeum vulgare (SEQ ID NO: 24).
  • FIG. 2 illustrates the results of analyzing the wheat mutants Tafxrl-1 and Tafxrl-2 root length as a percentage relative to control with an increasing concentration of flupoxam. Roots were measured after seven (7) days of growth.
  • FIG. 3 shows photographs of the growth of the wheat mutants Tafxrl-1 and Tafxrl-2 relative to wild-type seeds on 5 micromolar flupoxam. Wild-type wheat seeds are also shown after growing on water as a control.
  • FIG. 4 shows photographs of the wheat mutants Tafxrl-1 and Tafxrl-2 and Wild-type seeds as senescened plants.
  • nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.
  • sequence Listing is incorporated by reference herein. In the accompanying sequence listing:
  • SEQ ID NO: 1 provides the protein sequence for the Triticum aestivum gene
  • SEQ ID NO: 2 provides the cesa nucleotide sequence that encodes SEQ ID NO: 1.
  • SEQ ID NO: 3 provides the protein sequence for a mutant Triticum aestivum cesa gene Traes_2BS_064B02A89 with a mutation at S1052F, also referred to herein as Tafxrl-1.
  • SEQ ID NO: 4 provides the nucleotide sequence that encodes SEQ ID NO: 3.
  • SEQ ID NO: 5 provides the protein sequence for a mutant Triticum aestivum cesa gene Traes_2DS_C80293002 identified herein as Tafxr2-1 CESA1-2DS.
  • SEQ ID NO: 6 provides the nucleotide sequence that encodes SEQ ID NO: 5.
  • SEQ ID NO: 7 provides the protein sequence for a mutant Triticum aestivum cesa gene Traes_2DS_C80293002 with a mutation at G863S, also referred to herein as Tafxr2-1.
  • SEQ ID NO: 8 provides the nucleotide sequence that encodes SEQ ID NO: 7.
  • SEQ ID NO: 9 provides a protein sequence for a CESA ortholog in Arabidopsis thaliana.
  • SEQ ID NO: 10 provides a protein sequence for a CESA ortholog in Capsella rubella.
  • SEQ ID NO: 11 provides a protein sequence for a CESA ortholog in Field mustard plant.
  • SEQ ID NO: 12 provides a protein sequence for a CESA ortholog in Brassica
  • SEQ ID NO: 13 provides a protein sequence for a CESA ortholog in Gossypium raimondii.
  • SEQ ID NO: 14 provides a protein sequence for a CESA ortholog in Glycine max.
  • SEQ ID NO: 15 provides a protein sequence for a CESA ortholog in Medicago truncatula.
  • SEQ ID NO: 16 provides a protein sequence for a CESA ortholog in Panicum virgatum.
  • SEQ ID NO: 17 provides a protein sequence for a CESA ortholog in Sorghum bicolor.
  • SEQ ID NO: 18 provides a protein sequence for a CESA ortholog in Oryza sativa.
  • SEQ ID NO: 19 provides a protein sequence for a CESA ortholog in Brachypodium distachyon.
  • SEQ ID NO: 20 provides a protein sequence for a CESA ortholog in Zea mays.
  • SEQ ID NO: 21 provides a protein sequence for a CESA ortholog in Physcomitrella patens.
  • SEQ ID NO: 22 provides a protein sequence for a CESA ortholog in Vitis vinifera.
  • SEQ ID NO: 23 provides a protein sequence for a CESA ortholog in Phaseolus vulgaris.
  • SEQ ID NO: 24 provides a protein sequence for a CESA ortholog in Hordeum vulgare.
  • the inventors have found for the first time wheat plants resistant to flupoxam through a mutated variant of the cesa gene. In addition, the inventors have surprisingly found that the mutated variant in the cesa gene also has resistance to additional CBI herbicides.
  • control of undesired vegetation or weeds is to be understood as meaning the killing of weeds and/or otherwise retarding or inhibiting the normal growth of the weeds. Weeds, in the broadest sense, are understood as meaning all those plants which grow in locations where they are undesired.
  • the weeds of the present invention include, for example, dicotyledonous and monocotyledonous weeds.
  • Dicotyledonous weeds include, but are not limited to, weeds of the genera: Sinapis, Lepidium, Galium, Stellaria, Matricaria, Anthemis, Galinsoga, Chenopodium, Ulrica, Senecio, Amaranthus, Portulaca, Xanthium, Convolvulus, Ipomoea, Polygonum, Sesbania, Ambrosia, Cirsium, Carduus, Sonchus, Solanum, Rorippa, Rotala, Lindernia, Lamium, Veronica, Abutilon, Emex, Datura, Viola, Galeopsis, Papaver, Centaurea, Trifolium, Ranunculus, and Taraxacum.
  • Monocotyledonous weeds include, but are not limited to, weeds of the genera: Echinochloa, Setaria, Panicum, Digitaria, Phleum, Poa, Festuca, Eleusine, Brachiaria, Lolium, Bromus, Avena, Cyperus, Sorghum, Agropyron, Cynodon, Monochoria, Fimbristyslis, Sagittaria, Eleocharis, Scirpus, Paspalum, lschaemum, Sphenoclea, Dactyloctenium, Agrostis, Alopecurus, and Apera.
  • the weeds of the present invention can include, for example, crop plants that are growing in an undesired location.
  • a volunteer maize plant that is in a field that predominantly comprises soybean plants can be considered a weed, if the maize plant is undesired in the field of soybean plants.
  • plant is used in its broadest sense as it pertains to organic material and is intended to encompass eukaryotic organisms that are members of the Kingdom Plantae, examples of which include but are not limited to vascular plants, vegetables, grains, flowers, trees, herbs, bushes, grasses, vines, ferns, mosses, fungi and algae, etc., as well as clones, offsets, and parts of plants used for asexual propagation (e.g. cuttings, pipings, shoots, rhizomes, underground stems, clumps, crowns, bulbs, corms, tubers, rhizomes, plants/tissues produced in tissue culture, etc.).
  • asexual propagation e.g. cuttings, pipings, shoots, rhizomes, underground stems, clumps, crowns, bulbs, corms, tubers, rhizomes, plants/tissues produced in tissue culture, etc.
  • plant further encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, leaves, roots (including tubers), flowers, florets, fruits, pedicles, peduncles, stamen, anther, stigma, style, ovary, petal, sepal, carpel, root tip, root cap, root hair, leaf hair, seed hair, pollen grain, microspore, cotyledon, hypocotyl, epicotyl, xylem, phloem, parenchyma, endosperm, a companion cell, a guard cell, and any other known organs, tissues, and cells of a plant, and tissues and organs, wherein each of the aforementioned comprise the gene/nucleic acid of interest.
  • plant also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the aforementioned comprises the gene/nucleic acid of interest.
  • Plants that are particularly useful in the methods of the invention include all plants which belong to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants including fodder or forage legumes, ornamental plants, food crops, trees or shrubs selected from the list comprising Acer spp., Actinidia spp., Abelmoschus spp., Agave sisalana, Agropyron spp., Agrostis stolonifera, Allium spp., Amaranthus spp., Ammophila arenaria, Ananas comosus, Annona spp., Apium graveolens, Arachis spp, Artocarpus spp., Asparagus officinalis, Avena spp.
  • Avena sativa e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida
  • Averrhoa carambola e.g. Bambusa sp.
  • Benincasa hispida Bertholletia excelsea
  • Beta vulgaris Brassica spp.
  • Brassica napus e.g. Brassica napus, Brassica rapa ssp.
  • Lathyrus spp. Lens culinaris, Linum usitatissimum, Litchi chinensis, Lotus spp., Luffa acutangula, Lupinus spp., Luzula sylvatica, Lycopersicon spp. (e.g.
  • Sambucus spp. Secale cereals, Sesamum spp., Sinapis sp., Solanum spp. (e.g. Solanum tuberosum, Solanum integrifolium or Solanum lycopersicum), Sorghum bicolor, Spinacia spp., Syzygium spp., Tagetes spp., Tamarindus indica, Theobroma cacao, Trifolium spp., Tripsacum dactyloides, Triticosecale rimpaui, Triticum spp. (e.g.
  • the plant is a crop plant.
  • crop plants include inter alia soybean, sunflower, canola, alfalfa, rapeseed, cotton, tomato, potato or tobacco.
  • the plant is a
  • the plant is a cereal, such as rice, maize, wheat, barley, millet, rye, sorghum or oats.
  • herbicide is used herein to mean an active ingredient that kills, controls or otherwise adversely modifies the growth of plants.
  • the preferred amount or concentration of the herbicide is an "effective amount” or “effective concentration.”
  • an amount and concentration is intended an amount and concentration, respectively, that is sufficient to kill or inhibit the growth of a similar, wild- type, plant, plant tissue, plant cell, or host cell, but that said amount does not kill or inhibit as severely the growth of the herbicide-resistant plants, plant tissues, plant cells, and host cells of the present invention.
  • the effective amount of an herbicide is an amount that is routinely used in agricultural production systems to kill weeds of interest. Such an amount is known to those of ordinary skill in the art.
  • Herbicidal activity is exhibited by herbicides useful for the present invention when they are applied directly to the plant or to the locus of the plant at any stage of growth or before planting or emergence. The effect observed depends upon the plant species to be controlled, the stage of growth of the plant, the application parameters of dilution and spray drop size, the particle size of solid components, the environmental conditions at the time of use, the specific compound employed, the specific adjuvants and carriers employed, the soil type, and the like, as well as the amount of chemical applied. These and other factors can be adjusted as is known in the art to promote non-selective or selective herbicidal action. Generally, it is preferred to apply the herbicide post-emergence to relatively immature undesirable vegetation to achieve the maximum control of weeds.
  • an “herbicide-tolerant” or “herbicide-resistant” plant it is intended that a plant that is tolerant or resistant to at least one herbicide at a level that would normally kill, or inhibit the growth of, a normal or wild-type plant.
  • levels of herbicide that normally inhibit growth of a non-tolerant plant are known and readily determined by those skilled in the art. Examples include the amounts recommended by manufacturers for application. The maximum rate is an example of an amount of herbicide that would normally inhibit growth of a non-tolerant plant.
  • the terms "herbicide-tolerant” and “herbicide-resistant” are used interchangeably and are intended to have an equivalent meaning and an equivalent scope.
  • CESA- inhibiting herbicides refer to those agronomically acceptable herbicide active ingredients (AT.) recognized in the art.
  • terms such as fungicide, nematicide, pesticide, and the like refer to other agronomically acceptable active ingredients recognized in the art.
  • terms such as herbicide-tolerant and herbicide-tolerance refer to the ability of such enzyme or polypeptide to perform its physiological activity in the presence of an amount of an herbicide A.I.
  • a CESA enzyme when used specifically in regard to a CESA enzyme, it refers specifically to the ability to tolerate a CESA-inhibitor.
  • herbicide-tolerant mutated CESA protein or “herbicide -resistant mutated CESA protein”
  • a CESA protein displays higher CESA activity, relative to the CESA activity of a wild- type CESA protein, when in the presence of at least one herbicide that is known to interfere with CESA activity and at a concentration or level of the herbicide that is known to inhibit the CESA activity of the wild-type CESA protein.
  • CESA activity of such an herbicide-tolerant or herbicide-resistant mutated CESA protein may be referred to herein as “herbicide-tolerant” or “herbicide-resistant” CESA activity.
  • “recombinant” when referring to nucleic acid or polypeptide, indicates that such material has been altered as a result of human application of a recombinant technique, such as by polynucleotide restriction and ligation, by polynucleotide overlap- extension, or by genomic insertion or transformation.
  • a gene sequence open reading frame is recombinant if that nucleotide sequence has been removed from it natural text and cloned into any type of artificial nucleic acid vector.
  • the term recombinant also can refer to an organism having a recombinant material, e.g., a plant that comprises a recombinant nucleic acid can be considered a recombinant plant.
  • transgenic plant refers to a plant that comprises a heterologous polynucleotide.
  • the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations.
  • the heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant expression cassette.
  • Transgenic is used herein to refer to any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been so altered by the presence of heterologous nucleic acid including those transgenic organisms or cells initially so altered, as well as those created by crosses or asexual propagation from the initial transgenic organism or cell.
  • a "recombinant" organism is a "transgenic” organism.
  • transgenic as used herein is not intended to encompass the alteration of the genome (chromosomal or extra- chromosomal) by conventional plant breeding methods (e.g., crosses) or by naturally occurring events such as, e.g., self-fertilization, random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non- recombinant transposition, or spontaneous mutation.
  • mutant refers to an organism or DNA thereof having alteration(s) in the biomolecular sequence of its native genetic material as compared to the sequence of the genetic material of a corresponding wild-type organism or DNA, wherein the alteration(s) in genetic material were induced and/or selected by human action.
  • Examples of human action that can be used to produce a mutagenized organism or DNA include, but are not limited to, as illustrated in regard to herbicide tolerance: tissue culture of plant cells (e.g., calli) and selection thereof with herbicides (e.g., CBI herbicides), treatment of plant cells with a chemical mutagen such as EMS and subsequent selection with herbicide(s); or by treatment of plant cells with x-rays and subsequent selection with herbicide(s). Any method known in the art can be used to induce mutations. Methods of inducing mutations can induce mutations in random positions in the genetic material or can induce mutations in specific locations in the genetic material (i.e., can be directed mutagenesis techniques), such as by use of a genoplasty technique.
  • herbicide tolerance tissue culture of plant cells (e.g., calli) and selection thereof with herbicides (e.g., CBI herbicides), treatment of plant cells with a chemical mutagen such as EMS and subsequent selection with herbicide(s); or by treatment of plant cells with x
  • a "genetically modified organism” is an organism whose genetic characteristics contain alteration(s) that were produced by human effort causing transfection that results in transformation of a target organism with genetic material from another or “source” organism, or with synthetic or modified-native genetic material, or an organism that is a descendant thereof that retains the inserted genetic material.
  • the source organism can be of a different type of organism (e.g., a GMO plant can contain bacterial genetic material) or from the same type of organism (e.g., a GMO plant can contain genetic material from another plant).
  • mutagenized is used to refer to a plant or other organism, or the DNA thereof, in which no such transgenic material is present, but in which the native genetic material has become mutated so as to differ from a
  • wild-type or corresponding wild-type plant means the typical form of an organism or its genetic material, as it normally occurs, as distinguished from, e.g., mutagenized and/or recombinant forms.
  • control cell or “similar, wild-type, plant, plant tissue, plant cell or host cell” is intended a plant, plant tissue, plant cell, or host cell, respectively, that lacks the herbicide-resistance characteristics and/or particular polynucleotide of the invention that are disclosed herein.
  • descendant refers to any generation plant.
  • a descendant is a first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth generation plant.
  • seed refers to a first generation plant.
  • seed comprises seeds of all types, such as, for example, true seeds, caryopses, achenes, fruits, tubers, seedlings and similar forms.
  • seed refers to true seed(s) unless otherwise specified.
  • the seed can be seed of transgenic plants or plants obtained by traditional breeding methods. Examples of traditional breeding methods can include cross-breeding, selfing, back-crossing, embryo rescue, in crossing, out-crossing, inbreeding, selection, asexual propagation, and other traditional techniques as are known in the art.
  • CBI herbicide-tolerant plant lines described as useful herein can be employed in weed control methods either directly or indirectly, i.e. either as crops for herbicide treatment or as CBI herbicides-tolerance trait donor lines for development, as by traditional plant breeding, to produce other varietal and/or hybrid crops containing such trait or traits. All such resulting variety or hybrids crops, containing the ancestral CBI herbicide-tolerance trait or traits can be referred to herein as progeny or descendant of the ancestral, CESA- inhibiting herbicide-tolerant line(s).
  • the present invention provides a plant or plant part comprising a polynucleotide encoding a mutated CESA polypeptide, the expression of said polynucleotide confers to the plant or plant part tolerance to CBI herbicides.
  • the plant has been previously produced by a process comprising in situ mutagenizing plant cells or seeds, to obtain plant cells or plants which express a mutated CESA.
  • the polynucleotide encoding the mutated CESA polypeptide comprises the nucleic acid sequence set forth in SEQ ID NO: 4 or 8 or a variant or derivative thereof.
  • the mutated CESA polypeptide for use according to the present invention is a functional variant having, over the full-length of the variant, at least about 68%, illustratively, at least about 70%, 75%, 80%, 90%, 95%, 98%, 99% or more amino acid sequence identity to SEQ ID NO: 3 or 7.
  • the mutated CESA polypeptide for use according to the present invention is a functional fragment of a polypeptide having the amino acid sequence set forth at least in part in SEQ ID NOS: 3 and 7.
  • CESA polynucleotide molecules and CESA polypeptides of the invention encompass polynucleotide molecules and polypeptides comprising a nucleotide or an amino acid sequence that is sufficiently identical to nucleotide sequence set forth in SEQ ID NO: 4 or 8, or to the amino acid sequence set forth in SEQ ID NO: 3 or 7.
  • the term "sufficiently identical" is used herein to refer to a first amino acid or nucleotide sequence that contains a sufficient or minimum number of identical or equivalent (e.g., with a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have a common structural domain and/or common functional activity.
  • sequence identity refers to the extent to which two optimally aligned DNA or amino acid sequences are invariant throughout a window of alignment of
  • an “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components that are shared by the two aligned sequences divided by the total number of components in reference sequence segment, i.e., the entire reference sequence or a smaller defined part of the reference sequence. "Percent identity” is the identity fraction times 100.
  • Optimal alignment of sequences for aligning a comparison window are well known to those skilled in the art and may be conducted by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search for similarity method of Pearson and Lipman, and preferably by computerized implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA available as part of the GCG. Wisconsin Package. (Accelrys Inc. Burlington, Mass.)
  • an “isolated polynucleotide” or“isolated nucleic acid”, including DNA, RNA, or a combination of these, single or double stranded, in the sense or antisense orientation or a combination of both, dsRNA or otherwise we mean a polynucleotide which is at least partially separated from the polynucleotide sequences with which it is associated or linked in its native state.
  • the isolated polynucleotide is at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturally associated.
  • an isolated polynucleotide can be an exogenous polynucleotide present in, for example, a transgenic organism which does not naturally comprise the polynucleotide.
  • polynucleotide(s) used interchangeably herein and refer to nucleotides, either ribonucleotides or deoxyribonucleotides or a combination of both, in a polymeric unbranched form of any length.
  • mutated Cesa nucleic acid refers to a Cesa nucleic acid having a sequence that is mutated from a wild-type Cesa nucleic acid and that confers increased CBI herbicide tolerance to a plant in which it is expressed.
  • mutated CESA refers to the replacement of an amino acid of the wild-type primary sequences of SEQ ID NO: 1 or 5, or a variant, a derivative, a homologue, an orthologue, or paralogue thereof, with another amino acid.
  • the expression “mutated amino acid” will be used below to designate the amino acid which is replaced by another amino acid, thereby designating the site of the mutation in the primary sequence of the protein.
  • the CESA nucleotide sequence encoding a mutated CESA comprises the sequence of SEQ ID NO: 4 or 8, or a variant or derivative thereof.
  • the CESA nucleotide sequences encompass homologues, paralogues and orthologues of SEQ ID NO: 4 or 8 as defined hereinafter.
  • the term "variant" with respect to a sequence is intended to mean substantially similar sequences.
  • variants include those sequences that, because of the degeneracy of the genetic code, encode the identical amino acid sequence of the native protein.
  • Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques.
  • variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis and for open reading frames, encode the native protein comprising the sequence of SEQ ID NO:, as well as those that encode a polypeptide having amino acid substitutions relative to the native protein, e.g.
  • nucleotide sequence variants of the invention will have at least 30, 40, 50, 60, to 70%, e.g., preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, generally at least 80%, e.g., 81%-84%, at least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98% and 99% nucleotide "sequence identity" to the nucleotide sequence of SEQ ID NO: 4 or 8.
  • the query sequence is at least 45 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 45 nucleotides.
  • the query sequence is at least 150 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 150 nucleotides. More preferably, the query sequence is at least 300 nucleotides in length and the GAP analysis aligns the two sequences over a region of at least 300 nucleotides. Even more preferably, the GAP analysis aligns the two sequences over their entire length.
  • polypeptides [0079] By “substantially purified polypeptide” or “purified” a polypeptide is meant that has been separated from one or more lipids, nucleic acids, other polypeptides, or other
  • the substantially purified polypeptide is at least 60% free, more preferably at least 75% free, and more preferably at least 90% free from other components with which it is naturally associated.
  • the purified polypeptide can be a recombinantly produced polypeptide.
  • polypeptide and protein are generally used
  • polypeptide chains may associate with other polypeptides or proteins or other molecules such as co-factors.
  • proteins and “polypeptides” as used herein also include variants, mutants, modifications, analogous and/or derivatives of the polypeptides of the invention as described herein.
  • the query sequence is at least 25 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 25 amino acids. More preferably, the query sequence is at least 50 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 50 amino acids. More preferably, the query sequence is at least 100 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 100 amino acids. Even more preferably, the query sequence is at least 250 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 250 amino acids. Even more preferably, the GAP analysis aligns the two sequences over their entire length.
  • polypeptide of the invention comprises an amino acid sequence which is at least 65%, more preferably at least 70%, more preferably at least75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to SEQ ID NO: 3 or 7.
  • variant polypeptide is intended a polypeptide derived from the protein of SEQ ID NO: 3 or 7, by deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and/or C- terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein.
  • variants may result from, for example, genetic polymorphism or from human manipulation. Methods for such manipulations are generally known in the art.
  • Derivatives of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived.
  • functional variants and fragments of the CESA polypeptides, and nucleic acid molecules encoding them also are within the scope of the present invention, and unless specifically described otherwise, irrespective of the origin of said polypeptide and irrespective of whether it occurs naturally.
  • Various assays for functionality of a CESA polypeptide can be employed.
  • a functional variant or fragment of the CESA polypeptide can be assayed to determine its ability to confer CBI herbicide tolerance.
  • CBI herbicide tolerance can be defined as insensitivity to CESA inhibiting herbicides sufficient to provide a determinable increase in tolerance to CBI herbicides in a plant or plant part comprising a recombinant polynucleotide encoding the variant or fragment of the CESA polypeptide, wherein the plant or plant part expresses the variant or fragment at up to about 0.5%, illustratively, about 0.05 to about 0.5%, about 0.1 to about 0.4%, and about 0.2 to about 0.3%, of the total cellular protein relative to a similarly treated control plant that does not express the variant or fragment.
  • the mutated CESA polypeptide is a functional variant or fragment of a CESA having the amino acid sequence set forth in SEQ ID NOS: 3, 5, 7, 9, 11 wherein the functional variant or fragment has at least about 80% amino acid sequence identity to SEQ ID NO: 1.
  • the functional variant or fragment further has CBI herbicide tolerance defined as insensitivity to CESA inhibiting herbicides sufficient to provide a determinable increase in tolerance to CBI herbicides in a plant or plant part comprising a recombinant polynucleotide encoding the variant or fragment, wherein the plant or plant part expresses the variant or fragment at up to about 0.5% of the total cellular protein to a similarly treated control plant that does not express the variant or fragment.
  • CBI herbicide tolerance defined as insensitivity to CESA inhibiting herbicides sufficient to provide a determinable increase in tolerance to CBI herbicides in a plant or plant part comprising a recombinant polynucleotide encoding the variant or fragment, wherein the plant or plant part expresses the variant or fragment at up to about 0.5% of the total cellular protein to a similarly treated control plant that does not express the variant or fragment.
  • Homologues of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived.
  • an isolated polynucleotide molecule encoding a mutated CESA polypeptide having an amino acid sequence that differs from that of SEQ ID NO: 3 or 7 can be created by introducing one or more nucleotide substitutions, additions, or deletions into the corresponding nucleotide sequence, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.
  • Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR- mediated mutagenesis. Such variant nucleotide sequences are also encompassed by the present invention. For example, preferably, conservative amino acid substitutions may be made at one or more predicted preferably nonessential amino acid residues.
  • a "nonessential" amino acid residue is a residue that can be altered from the wild-type sequence of a protein without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity.
  • a deletion refers to removal of one or more amino acids from a protein.
  • An insertion refers to one or more amino acid residues being introduced into a predetermined site in a protein. Insertions may comprise N-terminal and/or C-terminal fusions as well as intra sequence insertions of single or multiple amino acids. Generally, insertions within the amino acid sequence will be smaller than N- or C-terminal fusions, of the order of about 1 to 10 residues.
  • N- or C-terminal fusion proteins or peptides include the binding domain or activation domain of a transcriptional activator as used in the yeast two-hybrid system, phage coat proteins, (histidine)-6-tag, glutathione 5- transferase-tag, protein A, maltose-binding protein, dihydrofolate reductase, Tag.100 epitope, c-myc epitope, FLAG®- epitope, lacZ, CMP (calmodulin-binding peptide), HA epitope, protein C epitope and VSV epitope.
  • a transcriptional activator as used in the yeast two-hybrid system
  • phage coat proteins phage coat proteins
  • glutathione 5- transferase-tag protein A
  • maltose-binding protein dihydrofolate reductase
  • Tag.100 epitope c-myc epitope
  • FLAG®- epitope FLAG®- epitope
  • lacZ
  • a substitution refers to replacement of amino acids of the protein with other amino acids having similar properties (such as similar hydrophobicity, hydrophilicity, antigenicity, propensity to form or break a-helical structures or 6-sheet structures).
  • substitutions are typically of single residues, but may be clustered depending upon functional constraints placed upon the polypeptide and may range from 1 to 10 amino acids; insertions will usually be of the order of about 1 to 10 amino acid residues.
  • a conservative amino acid substitution is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar sidechains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • substitutions would not be made for conserved amino acid residues, or for amino acid residues residing within a conserved motif.
  • Conservative substitution tables are well known in the art (see for example Creighton (1984) Proteins. W.H. Freeman and Company (Eds).
  • Amino acid substitutions, deletions and/or insertions may readily be made using peptide synthetic techniques well known in the art, such as solid phase peptide synthesis and the like, or by recombinant DNA manipulation. Methods for the manipulation of DNA sequences to produce substitution, insertion or deletion variants of a protein are well known in the art. For example, techniques for making substitution mutations at predetermined sites in DNA are well known to those skilled in the art and include M13 mutagenesis, T7-Gen in vitro mutagenesis (USB,
  • “Derivatives” further include peptides, oligopeptides, polypeptides which may, compared to the amino acid sequence of the naturally-occurring form of the protein, such as the protein of interest, comprise substitutions of amino acids with non-naturally occurring amino acid residues, or additions of non-naturally occurring amino acid residues.
  • Derivatives of a protein also encompass peptides, oligopeptides, polypeptides which comprise naturally occurring altered (glycosylated, acylated, prenylated, phosphorylated, myristoylated, sulphated etc.) or non-naturally altered amino acid residues compared to the amino acid sequence of a naturally-occurring form of the polypeptide.
  • a derivative may also comprise one or more non-amino acid substituents or additions compared to the amino acid sequence from which it is derived, for example a reporter molecule or other ligand, covalently or non-covalently bound to the amino acid sequence, such as a reporter molecule which is bound to facilitate its detection, and non-naturally occurring amino acid residues relative to the amino acid sequence of a naturally-occurring protein.
  • a reporter molecule or other ligand covalently or non-covalently bound to the amino acid sequence, such as a reporter molecule which is bound to facilitate its detection, and non-naturally occurring amino acid residues relative to the amino acid sequence of a naturally-occurring protein.
  • “derivatives” also include fusions of the naturally-occurring form of the protein with tagging peptides such as FLAG, HI36 or thioredoxin (for a review of tagging peptides, see Terpe, Appl. Microbiol. Biotechnol. 60, 523-533, 2003).
  • “Orthologues” and “paralogues” encompass evolutionary concepts used to describe the ancestral relationships of genes. Paralogues are genes within the same species that have originated through duplication of an ancestral gene; orthologues are genes from different organisms that have originated through speciation, and are also derived from a common ancestral gene.
  • SEQ ID NOS 1 and 13 - 24 A non-limiting list of examples of homologues of CESA from different plants are listed as SEQ ID NOS 1 and 13 - 24. It will be understood by the person skilled in the art that the sequences of SEQ ID NOS: 1 and 13 - 24, represent orthologues and paralogues to SEQ ID NO: 1. An alignment of these sequences can be found in Figure 1. A summary of the identities and positive matches from the alignment can be found below in Table 1.
  • Table 1 Protein alignment of homologous CESA sequences in plant species relative to
  • CESA in wheat SEQ ID NO: 1. The alignment was made using CLUSTAL 0 (ver. 1.2.4).
  • amino acids at other positions can vary between homologues, amino acids that are highly conserved at specific positions indicate amino acids that are likely essential in the structure, stability or function of a protein. Identified by their high degree of conservation in aligned sequences of a family of protein homologues, they can be used as identifiers to determine if any polypeptide in question belongs to a previously identified polypeptide family.
  • motif 1 or "consensus sequence” refers to a short conserved region in the sequence of evolutionarily related proteins. Motifs are frequently highly conserved parts of domains, but may also include only part of the domain, or be located outside of conserved domain (if all of the amino acids of the motif fall outside of a defined domain). [0092] Specialist databases exist for the identification of domains, for example, SMART
  • GAP uses the algorithm of Needleman and Wunsch ((1970) J Mob Biol 48: 443-453) to find the global (i.e. spanning the complete sequences) alignment of two sequences that maximizes the number of matches and minimizes the number of gaps.
  • the BLAST algorithm (Altschul et al. (1990) J Mob Biol 215: 403-10) calculates percent sequence identity and performs a statistical analysis of the similarity between the two sequences.
  • the software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information (NCB!).
  • Homologues may readily be identified using, for example, the ClustalW multiple sequence alignment algorithm (version 1.83), with the default pairwise alignment parameters, and a scoring method in percentage (See Figure 1). Global percentages of similarity and identity may also be determined using one of the methods available in the MatGAT software package (Campanella et al., BMC Bioinformatics. 2003 Jul 10;4:29. MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences.). Minor manual editing may be performed to optimize alignment between conserved motifs, as would be apparent to a person skilled in the art. Furthermore, instead of using full-length sequences for the identification of homologues, specific domains may also be used.
  • sequence identity values may be determined over the entire nucleic acid or amino acid sequence or over selected domains or conserved motif(s), using the programs mentioned above using the default parameters.
  • Smith-Waterman algorithm is particularly useful (Smith TF, Waterman MS (1981) J. Mol. Biol 147(1); 195-7).
  • the proteins of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (1985) PNAS, 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S. Patent No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein.
  • variant nucleotide sequences can be made by introducing mutations randomly along all or part of a coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened to identify mutants that encode proteins that retain activity. For example, following mutagenesis, the encoded protein can be expressed recombinantly, and the activity of the protein can be determined using standard assay techniques.
  • mutated CESA are those that increase the herbicide tolerance of the plant, but leave the biological activity of the CESA polypeptide substantially unaffected. Examples of these mutant nucleic acid sequences can be found in SEQ ID NOS: 4 and 8. Examples of the peptide sequences can be found in SEQ ID NOS: 3 and 7.
  • a mutant CESA polypeptide having a mutation at SI 052 or G863, including that comprising the sequence of SEQ ID NO: 3 or 7, may also include a variant, derivative, orthologue, paralogue or homologue thereof, the key amino acid residues of which being substituted by any other amino acid. It will be understood by the person skilled in the art that amino acids located in a close proximity to the positions of amino acids mentioned below may also be substituted.
  • a variant, derivative, orthologue, paralogue or homologue thereof comprises a mutated CESA, wherein an amino acid ⁇ 3, ⁇ 2 or ⁇ 1 amino acid positions from a key amino acid is substituted by any other amino acid.
  • Herbicide resistance or tolerance can be determined by generating a transgenic plant or host cell, preferably a plant cell, comprising a nucleic acid sequence of the library of step a) and comparing the transgenic plant with a control plant or host cell, preferably a plant cell.
  • Embodiments of the present invention may relate to an isolated and or recombinantly produced and/or synthetic nucleic acid encoding a mutated CESA as disclosed, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 4 or 8, or a variant or derivative thereof.
  • nucleic acid sequence comprising the sequence of SEQ ID NO: 4 or 8, or a homolog thereof, or its derivatives or parts thereof; or
  • (c) (a) and (b); are not found in their natural, genetic environment or have been modified by genetic engineering methods, wherein the modification may by way of example be a substitution, addition, deletion, inversion or insertion of one or more nucleotide residues.
  • Natural genetic environment means the natural genomic or chromosomal locus in the organism of origin or inside the host organism or presence in a genomic library.
  • the natural genetic environment of the nucleic acid sequence is preferably retained at least in part.
  • the environment borders the nucleic acid sequence at least on one side and has a sequence length of at least 50 bp, preferably at least 500 bp, particularly preferably at least 1,000 bp, most particularly preferably at least 5,000 bp.
  • a naturally occurring expression cassette - for example the naturally occurring combination of the natural promoter of the nucleic acid sequence according to the invention with the corresponding gene - turns into a transgenic expression cassette when the latter is modified by unnatural, synthetic ("artificial") methods such as by way of example a mutagenation.
  • the present invention encompasses a progeny or a descendant of an CBI herbicides-tolerant plant of the present invention as well as seeds derived from the CBI herbicides-tolerant plants of the invention and cells derived from the CBI herbicides- tolerant plants of the invention.
  • the present invention provides a progeny or descendant plant derived from a plant comprising in at least some of its cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a mutated CESA polypeptide encoded by the polynucleotide, wherein the progeny or descendant plant comprises in at least some of its cells the recombinant polynucleotide operably linked to the promoter, the expression of the mutated CESA polypeptide conferring to the progeny or descendant plant tolerance to the CBI herbicides.
  • seeds of the present invention preferably comprise the
  • a seed is capable of germination into a plant comprising in at least some of its cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a mutated CESA polypeptide encoded by the polynucleotide, the expression of the mutated CESA polypeptide conferring to the progeny or descendant plant tolerance to the CBI herbicides.
  • plant cells of the present invention are capable of regenerating a plant or plant part. In other embodiments, plant cells are not capable of regenerating a plant or plant part. Examples of cells not capable of regenerating a plant include, but are not limited to, endosperm, seed coat (testa & pericarp), and root cap.
  • the present invention provides a plant cell of or capable of regenerating a plant comprising in at least some of its cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a mutated CESA polypeptide encoded by the polynucleotide, the expression of the mutated CESA polypeptide conferring to the plant tolerance to the CBI herbicides, wherein the plant cell comprises the recombinant polynucleotide operably linked to a promoter.
  • the present invention provides a plant cell comprising a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a mutated CESA polypeptide encoded by the polynucleotide, the expression of the mutated CESA polypeptide conferring to the cell tolerance to the CBI herbicides.
  • the invention refers to a plant cell transformed by a nucleic acid encoding a mutated CESA polypeptide according to the present invention or to a plant cell which has been mutated to obtain a plant expressing a nucleic acid encoding a mutated CESA polypeptide according to the present invention, wherein expression of the nucleic acid in the plant cell results in increased resistance or tolerance to a CBI herbicide as compared to a wild type variety of the plant cell.
  • the mutated CESA polypeptide encoding nucleic acid comprises a polynucleotide sequence selected from the group consisting of: a) a polynucleotide as shown in SEQ ID NO: 4 or 8, or a variant or derivative thereof; b) a polynucleotide encoding a polypeptide as shown in SEQ ID NO: 3 or 7, or a variant or derivative thereof; c) a polynucleotide comprising at least 60 consecutive nucleotides of any of a) or b); and d) a polynucleotide complementary to the polynucleotide of any of a) through c).
  • CESA- inhibiting herbicide-tolerant plants hereof examples include, without limitation, grain, oil, and meal.
  • a plant product is plant grain (e.g., grain suitable for use as feed or for processing), plant oil (e.g., oil suitable for use as food or biodiesel), or plant meal (e.g., meal suitable for use as feed).
  • a plant product prepared from a plant or plant part comprising in at least some of its cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a mutated CESA polypeptide encoded by the polynucleotide, the expression of the mutated CESA polypeptide conferring to the a plant or plant part tolerance to the CBI herbicides.
  • the invention refers to a method of producing a transgenic plant cell with an increased resistance to a CBI herbicide as compared to a wild type variety of the plant cell comprising, transforming the plant cell with an expression cassette comprising a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a mutated CESA polypeptide encoded by the polynucleotide.
  • the invention refers to a method of producing a transgenic plant comprising, (a) transforming a plant cell with an expression cassette comprising a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a mutated CESA polypeptide encoded by the polynucleotide, and (b) generating a plant with an increased resistance to CBI herbicide from the plant cell.
  • the present invention provides a method for producing a CBI herbicide-tolerant plant.
  • the method comprises: regenerating a plant from a plant cell transformed with a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a mutated CESA polypeptide encoded by the polynucleotide, the expression of the mutated CESA polypeptide conferring to the plant tolerance to the CBI herbicides.
  • transcription of a specific gene or specific genes or specific genetic construct means the transcription of a gene or genes or genetic construct into structural RNA (rRNA, tRNA) or mRNA with or without subsequent translation of the latter into a protein.
  • expression or “gene expression” in particular means the transcription of a gene or genes or genetic construct into structural RNA (rRNA, tRNA) or mRNA with or without subsequent translation of the latter into a protein.
  • the process includes transcription of DNA and processing of the resulting mRNA product.
  • the at least one nucleic acid is "over-expressed” by methods and means known to the person skilled in the art.
  • the term "increased expression” or “overexpression” as used herein means any form of expression that is additional to the original wild-type expression level. Methods for increasing expression of genes or gene products are well documented in the art and include, for example, overexpression driven by appropriate promoters, the use of transcription enhancers or translation enhancers.
  • Isolated nucleic acids which serve as promoter or enhancer elements may be introduced in an appropriate position (typically upstream) of a non- heterologous form of a polynucleotide so as to upregulate expression of a nucleic acid encoding the polypeptide of interest.
  • endogenous promoters may be altered m vivo by mutation, deletion, and/or substitution (see, Kmiec, US 5,565,350; Zarling et al., W09322443), or isolated promoters may be introduced into a plant cell in the proper orientation and distance from a gene of the present invention so as to control the expression of the gene.
  • polypeptide expression it is generally desirable to include a
  • the polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from T- DNA.
  • the 3' end sequence to be added may be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene.
  • An intron sequence may also be added to the 5' untranslated region (UTR) or the coding sequence of the partial coding sequence to increase the amount of the mature message that accumulates in the cytosol.
  • nucleic acid sequences may be optimized for increased expression in a transformed plant.
  • coding sequences that comprise plant-preferred codons for improved expression in a plant can be provided. See, for example, Campbell and Gowni (1990) Plant Physiol., 92: 1-11 fora discussion of host-preferred codon usage. Methods also are known in the art for preparing plant-preferred genes. See, for example, U.S. Patent Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference.
  • mutated cesa nucleic acids of the invention are provided in expression cassettes for expression in the plant of interest.
  • the cassette will include regulatory sequences operably linked to a mutated cesa nucleic acid sequence of the invention.
  • regulatory element refers to a polynucleotide that is capable of regulating the transcription of an operably linked polynucleotide. It includes, but not limited to, promoters, enhancers, introns, 5' UTRs, and 3' UTRs.
  • operably linked is intended a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence.
  • operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame.
  • the cassette may additionally contain at least one additional gene to be co-transformed into the organism.
  • the additional gene(s) can be provided on multiple expression cassettes.
  • Such an expression cassette is provided with a plurality of restriction sites for insertion of the mutated cesa nucleic acid sequence to be under the transcriptional regulation of the regulatory regions.
  • the expression cassette may additionally contain selectable marker genes.
  • the expression cassette of the present invention will include in the 5'-3' direction of transcription, a transcriptional and translational initiation region (i.e., a promoter), a mutated CESA encoding nucleic acid sequence of the invention, and a transcriptional and translational termination region (i.e., termination region) functional in plants.
  • the promoter may be native or analogous, or foreign or heterologous, to the plant host and/or to the mutated cesa nucleic acid sequence of the invention. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence. Where the promoter is "foreign" or "heterologous" to the plant host, it is intended that the promoter is not found in the native plant into which the promoter is introduced.
  • a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence. While it may be preferable to express the mutated cesa nucleic acids of the invention using heterologous promoters, the native promoter sequences may be used. Such constructs would change expression levels of the mutated CESA protein in the plant or plant cell. Thus, the phenotype of the plant or plant cell is altered.
  • the termination region may be native with the transcriptional initiation region, may be native with the operably linked mutated CESA sequence of interest, may be native with the plant host, or may be derived from another source (i.e., foreign or heterologous to the promoter, the mutated cesa nucleic acid sequence of interest, such as cesa , the plant host, or any combination thereof).
  • Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262: 141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5: 141-149; Mogen et al. (1990) Plant Cell 2: 1261-1272; Munroe et al. (1990) Gene 91 : 151-158; Belles t al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al.
  • the gene(s) may be optimized for increased expression in the transformed plant. That is, the genes can be synthesized using plant-preferred codons for improved expression. See, for example, Campbell and Gown (1990) Plant Physiol. 92: 1-11 for a discussion of host-preferred codon usage. Methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Patent Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference.
  • the expression cassettes of the invention can include another selectable marker gene for the selection of transformed cells.
  • Selectable marker genes including those of the present invention, are utilized for the selection of transformed cells or tissues.
  • Marker genes include, but are not limited to, genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase 11 (NEO) and hygromycinphosphotransferase (H PT), as well as genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones, and 2,4- dichlorophenoxyacetate (2,4-D).
  • selectable marker genes are not meant to be limiting. Any selectable marker gene can be used in the present invention. Further, additional sequence modifications are known to enhance gene expression in acellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well - characterized sequences that may be deleterious to gene expression.
  • the G-C content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. Also, if desired, sequences can be readily modified to avoid predicted hairpin secondary mRNA structures.
  • Nucleotide sequences for enhancing gene expression can also be used in the plant expression vectors. These include, for example, introns of the maize Adh gene Adhl-S intron 1,2, and 6 (Callis et al. Genes and Development 1 : 1183-1200, 1987), and leader sequences, (W- sequence) from the Tobacco Mosaic virus (TMV), Maize Chlorotic Mottle Virus and AlfalfaMosaic Virus (Gallie et al. Nucleic Acid Res.
  • the first intron from the shrunken-1 locus of maize has been shown to increase expression of genes in chimeric gene constructs.
  • U.S. Pat. Nos. 5,424,412 and 5,593,874 disclose the use of specific introns in gene expression constructs, and Gallie et al. (Plant Physiol. 106:929-939, 1994) also have shown that introns are useful for regulating gene expression on a tissue specific basis.
  • the plant expression vectors of the invention also may contain DNA sequences containing matrix attachment regions (MARs). Plant cells transformed with such modified expression systems, then, may exhibit overexpression or constitutive expression of a nucleotide sequence of the invention.
  • MARs matrix attachment regions
  • an isolated recombinant expression vector comprising the expression cassette containing a mutated cesa nucleic acid nucleic acid as described above, wherein expression of the vector in a host cell results in increased tolerance to a CBI herbicide as compared to a wild type variety of the host cell.
  • the term "vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.
  • vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • Other vectors e.g., non-episomal mammalian vectors
  • certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as
  • expression vectors In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions.
  • the recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operably linked to the nucleic acid sequence to be expressed. Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells or under certain conditions. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of polypeptide desired, etc.
  • the expression vectors of the invention can be introduced into host cells to thereby produce polypeptides or peptides, including fusion polypeptides or peptides, encoded by nucleic acids as described herein (e.g., mutated CESA polypeptides, fusion polypeptides, etc.)
  • Expression vectors may additionally contain 5' leader sequences in the expression construct. Such leader sequences can act to enhance translation.
  • Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyo carditis 5' noncoding region) (Elroy-Stein et al.
  • TEV leader tobacco Etch Virus
  • MDMV leader Maize Dwarf Mosaic Virus
  • CiP human immunoglobulin heavy-chain binding protein
  • AMV RNA 4 untranslated leader from the coat protein mRNA of alfalfa mosaic virus
  • TMV tobacco mosaic virus leader
  • RNA sequences in the proper orientation and, as appropriate, in the proper reading frame.
  • MCMV chlorotic mottle virus leader
  • Other methods known to enhance translation also can be utilized, for example, introns, and the like.
  • the various nucleic acid fragments may be manipulated, so as to provide for the nucleic acid sequences in the proper orientation and, as appropriate, in the proper reading frame.
  • adapters or linkers may be employed to join the nucleic acid fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous nucleic acid, removal of restriction sites, or the like.
  • in vitro mutagenesis primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved.
  • a number of promoters can be used in the practice of the invention. The promoters can be selected based on the desired outcome.
  • the nucleic acids can be combined with constitutive, tissue-preferred, or other promoters for expression in plants.
  • Constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99/43838 and U.S. Patent No. 6,072,050; the core CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2: 163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619- 632 and Christensen et al. (1992) Plant Mol. Biol.
  • pEMU Last et al. (1991) Theor. Appl. Genet. 81 :581- 588); MAS (Velten et al. (1984) EMBO J. 3:2723- 2730); ALS promoter (U.S. Patent No. 5,659,026), and the like.
  • Other constitutive promoters include, for example, U.S. Patent Nos. 5,608,149; 5,608, 144; 5,604,121 ;
  • tissue-preferred promoters can be utilized to target enhanced expression within a particular plant tissue.
  • tissue-preferred promoters include, but are not limited to, leaf- preferred promoters, root-preferred promoters, seed- preferred promoters, and stem- preferred promoters.
  • tissue-preferred promoters are described by, e.g., Yamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol. Gen Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2): 157-168; Rinehart et al.
  • the nucleic acids of interest can be targeted to the chloroplast for expression.
  • the expression vector will additionally contain a chloroplast- targeting sequence comprising a nucleotide sequence that encodes a chloroplast transit peptide to direct the gene product of interest to the chloroplasts.
  • a chloroplast-targeting sequence comprising a nucleotide sequence that encodes a chloroplast transit peptide to direct the gene product of interest to the chloroplasts.
  • transit peptides are known in the art.
  • "operably linked" means that the nucleic acid sequence encoding a transit peptide (i.e., the chloroplast-targeting sequence) is linked to the desired coding sequence of the invention such that the two sequences are contiguous and in the same reading frame.
  • chloroplast transit peptide known in the art can be fused to the amino acid sequence of a
  • CESA polypeptide of the invention by operably linking a chloroplast-targeting sequence to the 5'- end of a nucleotide sequence encoding the CESA polypeptide.
  • Chloroplast targeting sequences are known in the art and include the chloroplast small subunit of ribulose-I,5-bisphosphate carboxylase (Rubisco) (de Castro Silva Filho et al.(1996) Plant Mol. Biol. 30:769-780; Schnell et al. (1991) J Biol. Chem.
  • the method relies on particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination. Additionally, plastid transformation can be accomplished by transactivation of a silent plastid-borne transgene by tissue-preferred expression of a nuclear-encoded and plastid-directed RNA polymerase. Such a system has been reported in McBride et al. (1994) Proc. Natl. Acad. Sci.
  • the nucleic acids of interest to be targeted to the chloroplast may be optimized for expression in the chloroplast to account for differences in codon usage between the plant nucleus and this organelle. In this manner, the nucleic acids of interest may be synthesized using chloroplast-preferred codons. See, for example, U.S. Patent No. 5,380,831, herein incorporated by reference. Numerous plant transformation vectors and methods for transforming plants are available. See, for example, An, G. et al. (1986) Plant PysioL, 81 :301- 305; Fry, I, et al. ( 1987) Plant Cell Rep. 6:321-325; Block, M. (1988) Theor. Appl. Genet .16: 161 -11 A; Hinchee, et al. (1990) Stadler. Genet. Symp.2032 ⁇ 2.203-2 ⁇ 2; Cousins, et al.
  • the methods of the invention involve introducing a polynucleotide construct into a plant.
  • introducing is intended presenting to the plant the polynucleotide construct in such a manner that the construct gains access to the interior of a cell of the plant.
  • the methods of the invention do not depend on a particular method for introducing a polynucleotide construct to a plant, only that the polynucleotide construct gains access to the interior of at least one cell of the plant.
  • Methods for introducing polynucleotide constructs into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods. The term
  • introduction or “transformation” as referred to herein further means the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer.
  • Plant tissue capable of subsequent clonal propagation may be transformed with a genetic construct of the present invention and a whole plant regenerated there from. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed.
  • tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, mega gametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem).
  • the polynucleotide may be transiently or stably introduced into a host cell and may be maintained non-integrated, for example, as a plasmid. Alternatively, it may be integrated into the host genome.
  • the resulting transformed plant cell may then be used to regenerate a transformed plant in a manner known to persons skilled in the art.
  • stable transformation is intended that the polynucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by descendent thereof.
  • transient transformation is intended that a polynucleotide construct introduced into a plant does not integrate into the genome of the plant.
  • the nucleotide sequences of the invention are inserted using standard techniques into any vector known in the art that is suitable for expression of the nucleotide sequences in a plant or plant cell. The selection of the vector depends on the preferred transformation technique and the target plant species to be transformed.
  • the encoding nucleotide sequence is operably linked to a plant promoter, e.g.
  • a promoter known in the art for high-level expression in a plant cell is then introduced into a plant cell that is susceptible to CBI herbicides; and a transformed plant is regenerated.
  • the transformed plant is tolerant to exposure to a level of CBI herbicides that would kill or significantly injure a plant regenerated from an untransformed cell.
  • This method can be applied to any plant species or crops.
  • Methodologies for constructing plant expression vectors and introducing foreign nucleic acids into plants are generally known in the art. For example, foreign DNA can be introduced into plants, using tumor-inducing (Ti) plasmid vectors.
  • Ti tumor-inducing
  • Agrobacterium tumefaciens each of which is herein incorporated by reference.
  • Transgenic plants are preferably produced via Agrobacterium-mediated transformation.
  • An advantageous transformation method is the transformation in planta.
  • Methods for Agrobacterium-mediated transformation of rice include well known methods for rice transformation, such as those described in any of the following: European patent application EP 1198985 Al, Aldemita and Hodges (Planta 199: 612-617, 1996); Chan et al. (Plant Mol Biol 22 (3): 491-506, 1993), Hiei et al. (Plant J 6 (2): 271-282, 1994), which disclosures are incorporated by reference herein as if fully set forth.
  • the preferred method is as described in either Ishida et al. (Nat. Biotechnol 14(6): 745-50, 1996) or Frame et al.
  • the nucleic acids or the construct to be expressed is preferably cloned into a vector, which is suitable for transforming Agrobacterium tumefaciens, for example pBinl9 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711).
  • Agrobacteria transformed by such a vector can then be used in known manner for the transformation of plants, such as plants used as a model, like Arabidopsis (Arabidopsis thaliana is within the scope of the present invention not considered as a crop plant), or crop plants such as, by way of example, tobacco plants, for example by immersing bruised leaves or chopped leaves in an agrobacterial solution and then culturing them in suitable media.
  • Agrobacterium mediated plant transformation can be performed using for example the GV3101(pMP90) (Koncz and Schell, 1986, Mol. Gen. Genet. 204:383-396) or LBA4404 (Clontech) Agrobacterium tumefaciens strain. Transformation can be performed by standard transformation and regeneration techniques (Deblaere et al., 1994, Nucl. Acids. Res. 13:4777-4788; Gelvin, Stanton B.
  • rapeseed can be transformed via cotyledon or hypocotyl transformation (Moloney et al., 1989, Plant Cell Report 8:238-242; De Block et al., 1989, Plant Physiol. 91 :694-701).
  • Use of antibiotics for Agrobacterium and plant selection depends on the binary vector and the Agrobacterium strain used for transformation. Rapeseed selection is normally performed using kanamycin as selectable plant marker.
  • Agrobacterium mediated gene transfer to flax can be performed using, for example, a technique described by Mlynarova et al., 1994, Plant Cell Report 13:282-285.
  • transformation of soybean can be performed using for example a technique described in European Patent No. 0424 047, U.S. Patent No. 5,322,783, European Patent No. 0397 687, Ei.S. Patent No. 5,376,543, or Ei.S. Patent No. 5,169,770. Transformation of maize can be achieved by particle bombardment, polyethylene glycol mediated DNA uptake, or via the silicon carbide fiber technique. (See, for example, Freeling and Walbot "The maize handbook" Springer Verlag: New York (1993) ISBN 3-540-97826-7). A specific example of maize transformation is found in U.S. Patent No. 5,990,387, and a specific example of wheat transformation can be found in PCT Application No. WO
  • polynucleotides of the present invention may be introduced into plants by contacting plants with a virus or viral nucleic acids. Generally, such methods involve incorporating a polynucleotide construct of the invention within a viral DNA or RNA molecule. It is recognized that the polypeptides of the invention may be initially synthesized as part of a viral polyprotein, which later may be processed by proteolysis in vivo or in vitro to produce the desired recombinant polypeptide. Further, it is recognized that promoters of the invention also encompass promoters utilized for transcription by viral RNA polymerases.
  • the present invention may be used for transformation of any plant species, including, but not limited to, monocots and dicots.
  • plant species of interest include, but are not limited to, com or maize (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B.
  • juncea particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annu ), safflower (Carthamus tinctorius), wheat (Triticum aestivum, T. Turgidum ssp.
  • soybean Glycine max
  • tobacco Natural (Nicotiana tabacum), potato (Solarium tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffee spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (P
  • plants of the present invention are crop plants (for example, sunflower, Brassicasp., cotton, sugar, beet, soybean, peanut, alfalfa, safflower, tobacco, corn, rice, wheat, rye, barley triticale, sorghum, millet, etc.).
  • crop plants for example, sunflower, Brassicasp., cotton, sugar, beet, soybean, peanut, alfalfa, safflower, tobacco, corn, rice, wheat, rye, barley triticale, sorghum, millet, etc.
  • the transformation of the chloroplast genome is generally achieved by a process which has been schematically displayed in Klaus et al., 2004 [Nature Biotechnology 22 (2), 225-229] Briefly the sequences to be transformed are cloned together with a selectable marker gene between flanking sequences homologous to the chloroplast genome. These homologous flanking sequences direct site specific integration into the plastome.
  • plant cells or cell groupings are selected for the presence of one or more markers which are encoded by plant-expressible genes co-transferred with the gene of interest, following which the transformed material is regenerated into a whole plant.
  • the plant material obtained in the transformation is, as a rule, subjected to selective conditions so that transformed plants can be distinguished from untransformed plants.
  • the seeds obtained in the above-described manner can be planted and, after an initial growing period, subjected to a suitable selection by spraying.
  • a further possibility consists in growing the seeds, if appropriate after sterilization, on agar plates using a suitable selection agent so that only the transformed seeds can grow into plants.
  • the transformed plants are screened for the presence of a selectable marker such as the ones described above.
  • putatively transformed plants may also be evaluated, for instance using Southern analysis, for the presence of the gene of interest, copy number and/or genomic organization.
  • expression levels of the newly introduced DNA may be monitored using Northern and/or Western analysis, both techniques being well known to persons having ordinary skill in the art.
  • the generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or Tl) transformed plant may be selfed and homozygous second-generation (or T2) transformants selected, and the T2 plants may then further be propagated through classical breeding techniques.
  • the generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and non-transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion).
  • the expression of the nucleic acid in the plant results in the plant's increased tolerance to CBI herbicide as compared to a wild type variety of the plant.
  • the invention refers to a plant, comprising a plant cell according to the present invention, wherein expression of the nucleic acid in the plant results in the plant's increased resistance to CBI herbicide as compared to a wild type variety of the plant.
  • transgenic means with regard to, for example, a nucleic acid sequence, an expression cassette, gene construct or a vector comprising the nucleic acid sequence or an organism transformed with the nucleic acid sequences, expression cassettes or vectors according to the invention, all those constructions brought about by recombinant methods in which either:
  • the natural genetic environment is understood as meaning the natural genomic or
  • the natural genetic environment of the nucleic acid sequence is preferably retained, at least in part.
  • the environment flanks the nucleic acid sequence at least on one side and has a sequence length of at least 50 bp, preferably at least 500 bp, especially preferably at least 1000 bp, most preferably at least 5000 bp.
  • a transgenic plant as described herein is thus understood as meaning, as above, that the nucleic acids of the invention are not at their natural locus in the genome of said plant, it being possible for the nucleic acids to be expressed homologously or
  • transgenic also means that, while the nucleic acids according to the invention or used in the inventive method are at their natural position in the genome of a plant, the sequence has been modified with regard to the natural sequence, and/or that the regulatory sequences of the natural sequences have been modified.
  • Transgenic is preferably understood as meaning the expression of the nucleic acids according to the invention at an unnatural locus in the genome, i.e. homologous or, preferably, heterologous expression of the nucleic acids takes place.
  • Preferred transgenic plants are mentioned herein.
  • the term “transgenic” refers to any plant, plant cell, callus, plant tissue, or plant part that contains all or part of at least one recombinant polynucleotide.
  • recombinant polynucleotide refers to a polynucleotide that has been altered, rearranged, or modified by genetic engineering. Examples include any cloned polynucleotide, or polynucleotides, that are linked or joined to heterologous sequences.
  • recombinant does not refer to alterations of polynucleotides that result from naturally occurring events, such as spontaneous mutations, or from non-spontaneous mutagenesis followed by selective breeding.
  • Non-transgenic plants Plants containing mutations arising due to non-spontaneous mutagenesis and selective breeding are referred to herein as non-transgenic plants and are included in the present invention.
  • the nucleic acids can be derived from different genomes or from the same genome.
  • the nucleic acids are located on different genomes or on the same genome.
  • the present invention involves herbicide-resistant plants that are produced by mutation breeding.
  • Such plants comprise a polynucleotide encoding a mutated cesa and are tolerant to one or more CBI herbicides.
  • Such methods can involve, for example, exposing the plants or seeds to a mutagen, particularly a chemical mutagen such as, for example, ethyl methanesulfonate (EMS) and selecting for plants that have enhanced tolerance to at least one or more CBI herbicide
  • EMS ethyl methanesulfonate
  • the present invention is not limited to herbicide-tolerant plants that are produced by a mutagenesis method involving the chemical mutagen EMS. Any mutagenesis method known in the art may be used to produce the herbicide-resistant plants of the present invention.
  • Such mutagenesis methods can involve, for example, the use of any one or more of the following mutagens: radiation, such as X-rays, Gamma rays (e.g., cobalt 60 or cesium 137), neutrons, (e.g., product of nuclear fission by uranium 2in an atomic reactor), Beta radiation (e.g., emitted from radioisotopes such as phosphorus 32 or carbon 14), and ultraviolet radiation (preferably from 250 to 290 nm), and chemical mutagens such as base analogues (e.g., 5-bromo-uracil), related compounds (e.g., 8-ethoxy caffeine), antibiotics (e.g., streptonigrin), alkylating agents (e.g., sulfur mustards, nitrogen mustards, epoxides, ethylenamines, sulfates, sulfonates, sulfones, lactones), azide, hydroxylamine, nitro
  • Herbicide-resistant plants can also be produced by using tissue culture methods to select for plant cells comprising herbicide- resistance mutations and then regenerating herbicide-resistant plants therefrom. See, for example, U.S. Patent Nos. 5,773,702 and 5,859,348, both of which are herein incorporated in their entirety by reference. Further details of mutation breeding can be found in "Principals of Cultivar Development” Fehr, 1993 Macmillan Publishing Company the disclosure of which is incorporated herein by reference.
  • herbicide-resistant plants according to the present invention can also be produced by using genome editing methods to select for plant cells comprising herbicide-resistance mutations and then regenerating herbicide-resistant plants therefrom.
  • Gene Editing refers to a type of genetic engineering in which DNA is inserted, deleted or replaced in the genome of an organism using engineered nucleases. These nucleases are known to the skilled artisan to create site-specific double-strand breaks at desired locations in the genome. The induced double-strand breaks are repaired through non-homologous end joining or homologous recombination, resulting in targeted mutations.
  • the plant of the present invention comprises at least one mutated cesa nucleic acid and has increased tolerance to a CBI herbicide as compared to a wild-type variety of the plant. It is possible for the plants of the present invention to have multiple mutated cesa nucleic acids from different genomes since these plants can contain more than one genome. For example, a plant contains two genomes, usually referred to as the A and B genomes. Because CESA is a required metabolic enzyme, it is assumed that each genome has at least one gene coding for the CESA enzyme (i.e. at least one cesa gene).
  • the term "cesa gene locus” refers to the position of a cesa gene on a genome
  • the terms " cesa gene” and " cesa nucleic acid” refer to a nucleic acid encoding the CESA enzyme.
  • the cesa nucleic acid on each genome differs in its nucleotide sequence from a Cesa nucleic acid on another genome.
  • One of skill in the art can determine the genome of origin of each Cesa nucleic acid through genetic crossing and/or either sequencing methods or exonuclease digestion methods known to those of skill in the art.
  • the present invention includes plants comprising one, two, three, or more mutated Cesa alleles, wherein the plant has increased tolerance to a CBI herbicide as compared to a wild-type variety of the plant.
  • the mutated Cesa alleles can comprise a nucleotide sequence as defined in SEQ ID NO: 4 or 8, or a variant or derivative thereof, a polynucleotide encoding a polypeptide as defined in SEQ ID NO: 3 or 7, or a variant or derivative, homologue, orthologue, paralogue thereof, a polynucleotide comprising at least 60 consecutive nucleotides of any of the aforementioned polynucleotides; and a polynucleotide complementary to any of the aforementioned polynucleotides.
  • Allelic variants are alternative forms of a given gene, located at the same chromosomal position. Allelic variants encompass Single Nucleotide
  • SNPs Polymorphisms
  • INDELs Small Insertion/Deletion Polymorphisms
  • the size of INDELs is usually less than 100 bp. SNPs and INDELs form the largest set of sequence variants in naturally occurring polymorphic strains of most organisms.
  • cultivar refers to a group of plants within a species defined by the sharing of a common set of characteristics or traits accepted by those skilled in the art as sufficient to distinguish one cultivar or variety from another cultivar or variety. There is no implication in either term that all plants of any given cultivar or variety will be genetically identical at either the whole gene or molecular level or that any given plant will be homozygous at all loci. A cultivar or variety is considered "true breeding" for a particular trait if, when the true- breeding cultivar or variety is self-pollinated, all of the progeny contain the trait.
  • breeding line or “line” refer to a group of plants within a cultivar defined by the sharing of a common set of characteristics or traits accepted by those skilled in the art as sufficient to distinguish one breeding line or line from another breeding line or line. There is no implication in either term that all plants of any given breeding line or line will be genetically identical at either the whole gene or molecular level or that any given plant will be homozygous at all loci.
  • a breeding line or line is considered “true breeding" for a particular trait if, when the true-breeding line or breeding line is self-pollinated, all of the progeny contain the trait. In the present invention, the trait arises from a mutation in a Cesa gene of the plant or seed.
  • the herbicide-resistant plants of the invention that comprise polynucleotides encoding mutated CESA polypeptides also find use in methods for increasing the herbicide- resistance of a plant through conventional plant breeding involving sexual reproduction.
  • the methods comprise crossing a first plant that is an herbicide-resistant plant of the invention to a second plant that may or may not be resistant to the same herbicide or herbicides as the first plant or may be resistant to different herbicide or herbicides than the first plant.
  • the second plant can be any plant that is capable of producing viable progeny plants (i.e., seeds) when crossed with the first plant.
  • the first and second plants are of the same species.
  • the methods can optionally involve selecting for progeny plants that comprise the mutated CESA polypeptides of the first plant and the herbicide resistance characteristics of the second plant.
  • the progeny plants produced by this method of the present invention have increased resistance to an herbicide when compared to either the first or second plant or both. When the first and second plants are resistant to different herbicides, the progeny plants will have the combined herbicide tolerance characteristics of the first and second plants.
  • the methods of the invention can further involve one or more generations of backcrossing the progeny plants of the first cross to a plant of the same line or genotype as either the first or second plant. Alternatively, the progeny of the first cross or any subsequent cross can be crossed to a third plant that is of a different line or genotype than either the first or second plant.
  • the present invention also provides plants, plant organs, plant tissues, plant cells, seeds, and non-human host cells that are transformed with the at least one
  • transformed plants, plant organs, plant tissues, plant cells, seeds, and non-human host cells have enhanced tolerance or resistance to at least one herbicide, at levels of the herbicide that kill or inhibit the growth of an untransformed plant, plant tissue, plant cell, or non-human host cell, respectively.
  • the transformed plants, plant tissues, plant cells, and seeds of the invention are Arabidopsis thaliana and crop plants.
  • the plant of the present invention can comprise a wild type Cesa nucleic acid in addition to a mutated Cesa nucleic acid. It is contemplated that the CBI herbicide tolerant lines may contain a mutation in only one of multiple CESA isoenzymes. Therefore, the present invention includes a plant comprising one or more mutated Cesa nucleic acids in addition to one or more wild type Cesa nucleic acids. In another embodiment, the invention refers to a seed produced by a transgenic plant comprising a plant cell of the present invention, wherein the seed is true breeding for an increased resistance to a CBI herbicide as compared to a wild type variety of the seed.
  • CBI herbicide-tolerant plants of the present invention can be employed as CBI herbicide-tolerance trait donor lines for development, as by traditional plant breeding, to produce other varietal and/or hybrid crops containing such traitor traits. All such resulting variety or hybrids crops, containing the ancestral CBI herbicides-tolerance trait or traits can be referred to herein as progeny or descendant of the ancestral, CBI herbicides- tolerant line(s).
  • the present invention provides a method for producing a CBI herbicide-tolerant plant.
  • the method comprises: crossing a first CBI herbicide-tolerant plant with a second plant to produce a CBI herbicide- tolerant progeny plant, wherein the first plant and the progeny plant comprise in at least some of their cells a polynucleotide operably linked to a promoter operable in plant cells, the recombinant polynucleotide being effective in the cells of the first plant to express a mutated CESA polypeptide encoded by the
  • the present invention provides a method for producing a CBI herbicide-tolerant progeny plant, the method comprising: crossing a parent plant with a CBI herbicide-tolerant plant to introduce the CBI herbicide tolerance characteristics of the CBI herbicide-tolerant plant into the germplasm of the progeny plant, wherein the progeny plant has increased tolerance to the CBI herbicides relative to the parent plant.
  • the method further comprises the step of introgressing the CBI herbicides- tolerance characteristics through traditional plant breeding techniques to obtain a descendent plant having the CBI herbicides-tolerance characteristics.
  • plants of the invention include those plants which, in addition to being CBI herbicide-tolerant, have been subjected to further genetic modifications by breeding, mutagenesis or genetic engineering, e.g. have been rendered tolerant to applications of specific other classes of herbicides, such as AHAS inhibitors; auxinic herbicides; bleaching herbicides such as hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors or phytoene desaturase (PDS) inhibitors; EPSPS inhibitors such as glyphosate; glutamine synthetase (GS) inhibitors such as glufosinate; lipid biosynthesis inhibitors such as acetyl CoA carboxylase (ACCase) inhibitors; or oxynil (i.e.
  • CBI herbicide-tolerant plants of the invention can be made resistant to multiple classes of herbicides through multiple genetic modifications, such as resistance to both glyphosate and glufosinate or to both glyphosate and a herbicide from another class such as HPPD inhibitors, AHAS inhibitors, or ACCase inhibitors.
  • herbicide resistance technologies are, for example, described in Pest Management Science (at volume, year, page): 61, 2005, 246; 61, 2005, 258; 61, 2005, 277; 61, 2005, 269; 61, 2005, 286; 64, 2008, 326; 64, 2008, 332; Weed Science 57, 2009,
  • CBI herbicide-tolerant plants of the invention may be tolerant to ACCase inhibitors, such as "dims” (e.g., cycloxydim, sethoxydim, clethodim, or tepraloxydim), "fops” (e.g., clodinafop, diclofop, fluazifop, haloxyfop, or quizalofop), and "dens” (such as pinoxaden); to auxinic herbicides, such as dicamba; to EPSPS inhibitors, such as glyphosate; to other CESA inhibitors; and to GS inhibitors, such as glufosinate.
  • ACCase inhibitors such as "dims” (e.g., cycloxydim, sethoxydim, clethodim, or tepraloxydim), "fops” (e.g., clodinafop, diclofop, fluaz
  • CBI herbicide-tolerant plants of the invention may also be tolerant to herbicides having other modes of action, for example, chlorophyll/carotenoid pigment inhibitors, cell membrane disrupters, photosynthesis inhibitors, cell division inhibitors, root inhibitors, shoot inhibitors, and combinations thereof.
  • tolerance traits may be expressed, e.g.
  • mutant or wildtype HPPD proteins as mutant or wildtype PPO proteins, as mutant AHASL proteins, mutant ACCase proteins, mutant EPSPS proteins, or mutant glutamine synthetase proteins; or as mutant native, inbred, or transgenic aryloxyalkanoate dioxygenase (AAD or DHT), haloarylnitrilase (BXN), 2,2- dichloropropionic acid dehalogenase (DEH), glyphosate-N- acetyltransferase (GAT), glyphosate decarboxylase (G DC), glyphosate oxidoreductase (GOX), glutathione-S- transferase (GST), phosphinothricin acetyltransferase (PAT or bar), or CYP450s proteins having an herbicide-degrading activity.
  • AAD or DHT transgenic aryloxyalkanoate dioxygenase
  • BXN 2,2-
  • CBI herbicide-tolerant plants hereof can also be stacked with other traits including, but not limited to, pesticidal traits such as Bt Cry and other proteins having pesticidal activity toward coleopteran, lepidopteran, nematode, or other pests; nutrition or nutraceutical traits such as modified oil content or oil profile traits, high protein or high amino acid concentration traits, and other trait types known in the art.
  • pesticidal traits such as Bt Cry and other proteins having pesticidal activity toward coleopteran, lepidopteran, nematode, or other pests
  • nutrition or nutraceutical traits such as modified oil content or oil profile traits, high protein or high amino acid concentration traits, and other trait types known in the art.
  • CBI herbicide-tolerant plants are also covered which are, by the use of recombinant DNA techniques and/or by breeding and/or otherwise selected for such characteristics, rendered able to synthesize one or more insecticidal proteins, especially those known from the bacterial genus Bacillus, particularly from Bacillus thuringiensis, such as [delta] -endotoxins, e.g. CrylA(b), CrylA(c), CrylF, Cryl F(a2), CryllA(b), CrylllA, CrylllB(bl) or Cry9c; vegetative insecticidal proteins (VIP), e.g.
  • VIP vegetative insecticidal proteins
  • VIPl insecticidal proteins of bacteria colonizing nematodes, e.g. Photorhabdus spp. or Xenorhabdus spp.
  • toxins produced by animals such as scorpion toxins, arachnid toxins, wasp toxins, or other insect-specific neurotoxins
  • toxins produced by fungi such streptomycete toxins
  • plant lectins such as pea or barley lectins
  • agglutinins proteinase inhibitors, such as trypsin inhibitors, serine protease inhibitors, patatin, cystatin or papain inhibitors
  • ribosome-inactivating proteins (RIP) such as ricin, maize-RIP, abrin, luffin, saporin or bryodin
  • steroid metabolism enzymes such as 3 -hydroxy- steroid oxidase, ecdysteroid-IDP-glycosyl-transferase,
  • insecticidal proteins or toxins are to be understood expressly also as pre- toxins, hybrid proteins, truncated or otherwise modified proteins.
  • Hybrid proteins are characterized by a new combination of protein domains, (see, e.g. WO 02/015701).
  • Such toxins or genetically modified plants capable of synthesizing such toxins are disclosed, e.g., in EP-A 374 753, WO 93/007278, WO 95/34656, EP-A 427 529, EP-A 451 878, WO 03/18810 und WO 03/52073.
  • the methods for producing such genetically modified plants are generally known to the person skilled in the art and are described, e.g. in the publications mentioned above.
  • insecticidal proteins contained in the genetically modified plants impart to the plants producing these proteins tolerance to harmful pests from all taxonomic groups of arthropods, especially to beetles (Coeloptera), two-winged insects (Diptera), and moths (Lepidoptera) and to nematodes (Nematoda).
  • expression of one or more protein toxins in the CBI herbicide-tolerant plants is effective for controlling organisms that include, for example, members of the classes and orders: Coleoptera such as the American bean weevil Acanthoscelides obtectus; the leaf beetle Agelastica alni; click beetles (Agriotes lineatus, Agriotes obscurus, Agriotes bicolor); the grain beetle Ahasverus advena; the summer schafer Amphimallon solstitialis; the furniture beetle Anobium punctatum; Anthonomus spp.
  • Coleoptera such as the American bean weevil Acanthoscelides obtectus
  • the leaf beetle Agelastica alni click beetles (Agriotes lineatus, Agriotes obscurus, Agriotes bicolor)
  • weevils the Pygmy mangold beetle Atomaria linearis; carpet beetles(Anthrenus spp., Attagenus spp.); the cowpea weevil Callosobruchus maculates; the fried fruit beetle Carpophilus hemipterus; the cabbage seedpod weevil Ceutorhynchus assimilis; the rape winter stem weevil Ceutorhynchus picitarsis; the wireworms Conoderus vespertinus and Conoderus falli; the banana weevil Cosmopolites sordidus; the New Zealand grass grub Costelytra zealandica; the June beetle Cotinis nitida; the sunflower stem weevil Cylindrocopturus adspersus; the larder beetle Dermestes lardarius; the corn root worms Diabrotica virgifera, Diabrotica virgifera virgifera, and Dia
  • Lasiodermaserricome the Colorado potato beetle Leptinotarsa decemlineata; Lyctus beetles ⁇ Lyctus spp. , the pollen beetle Meligethes aeneus; the common cockshafer Melolontha melolontha; the American spider beetle Mezium americanum; the golden spider beetle Niptus hololeuc s; the grain beetles Oryzaephilus surinamensis and Oryzaephilus Mercator; the black vine weevil Otiorhynchus sulcatus; the mustard beetle Phaedon cochleariae, the crucifer flea beetle Phyllotreta cruciferae; the striped flea beetle Phyllotreta striolata; the cabbage steam flea beetle Psylliodes chrysocephala; P
  • Drosophila spp. Drosophila spp.
  • lsoptera lsoptera (termites) including species from the familes Hodotermitidae, Kalotermitidae, Mastotermitidae, Rhinotermitidae, Serritermitidae, Termitidae, Termopsidae; the tarnished plant bug Lygus lineolaris; the black bean aphid Aphis fabae; the cotton or melon aphid Aphis gossypii; the green apple aphid Aphis pomi; the citrus spiny whitefly Aleurocanthus spiniferus; the sweet potato whitefly Bemesia tabaci; the cabbage aphid Brevicoryne brassicae; the pear psylla Cacopsylla pyricola; the currant aphid Cryptomyzus ribis; the grape phylloxera D
  • Bucculatrixthurberiella (cotton leaf perforator); Bupalus piniarius (pine looper); Carpocapsa pomonella (codling moth); Chilo suppressalis (striped rice borer); Choristoneura fumiferana (eastern spruce budworm); Cochylis hospes (banded sunflower moth); Diatraea grandiosella (southwestern corn borer); Eupoecilia ambiguella (European grape berry moth); Helicoverpa armigera (cotton bollworm); Helicoverpa zea (cotton bollworm); Heliothis virescens (tobacco budworm), Homeosoma electellum (sunflower moth); Homona magnanima (oriental tea tree tortrix moth); Lithocolletis blancardella (spotted tentiform leafminer); Lymantria dispar (gypsy moth); Malacosoma neustria (tent caterpillar); Mamestra brassicae
  • expression of one or more protein toxins in the CBI herbicide-tolerant plants is effective for controlling flea beetles, i.e. members of the flea beetle tribe of family Chrysomelidae, preferably against Phyllotreta spp., such as Phyllotreta cruciferae and/or Phyllotreta triolata.
  • Phyllotreta spp. such as Phyllotreta cruciferae and/or Phyllotreta triolata.
  • expression of one or more protein toxins in the CBI herbicide-tolerant plants is effective for controlling cabbage seedpod weevil, the Bertha armyworm, Lygus bugs, or the diamondback moth.
  • CBI herbicide-tolerant plants are also covered which are, e.g. by the use of recombinant DNA techniques and/or by breeding and/or otherwise selected for such traits, rendered able to synthesize one or more proteins to increase the resistance or tolerance of those plants to bacterial, viral or fungal pathogens.
  • the methods for producing such genetically modified plants are generally known to the person skilled in the art.
  • CBI herbicide-tolerant plants are also covered which are, e.g. by the use of recombinant DNA techniques and/or by breeding and/or otherwise selected for such traits, rendered able to synthesize one or more proteins to increase the productivity (e.g. oil content), tolerance to drought, salinity or other growth- limiting environmental factors or tolerance to pests and fungal, bacterial or viral pathogens of those plants.
  • CBI herbicides-tolerant plants are also covered which are, e.g. by the use of recombinant DNA techniques and/or by breeding and/or otherwise selected for such traits, altered to contain a modified amount of one or more substances or new substances, for example, to improve human or animal nutrition, e.g. oil crops that produce health-promoting long-chain omega-3 fatty acids or unsaturated omega-9 fatty acids (e.g. Nexera(R) rape, Dow Agro Sciences, Canada).
  • CBI herbicide-tolerant plants are also covered which are, e.g. by the use of recombinant DNA techniques and/or by breeding and/or otherwise selected for such traits, altered to contain increased amounts of vitamins and/or minerals, and/or improved profiles of nutraceutical compounds.
  • a method for treating a plant of the present invention comprises contacting the plant with an agronomically acceptable composition.
  • the agronomically acceptable composition comprises a CESA inhibiting herbicide active ingredient (A.I.), such as an azine as described herein.
  • the present invention provides a method for preparing a descendent seed. The method comprises planting a seed of or capable of producing a plant of the present invention. In one embodiment, the method further comprises growing a descendent plant from the seed; and harvesting a descendant seed from the descendent plant. In other embodiments, the method further comprises applying a CBI herbicide herbicidal composition to the descendent plant.
  • the invention refers to harvestable parts of the plant according to the present invention.
  • the harvestable parts comprise the Cesa nucleic acid or CESA protein of the present invention.
  • the harvestable parts may be seeds, roots, leaves and/or flowers comprising the Cesa nucleic acid or CESA protein or parts thereof.
  • Preferred parts of soy plants are soy beans comprising the Cesa nucleic acid or CESA protein.
  • the invention refers to products derived from a plant according to the present invention, parts thereof or harvestable parts thereof.
  • a preferred plant product is fodder, seed meal, oil, or seed-treatment-coated seeds.
  • the meal and/or oil comprise the Cesa nucleic acids or CESA proteins.
  • the invention refers to a method for the production of a product, which method comprises: a) growing the plants of the invention or obtainable by the methods of invention and b) producing said product from or by the plants of the invention and/or parts, e.g. seeds, of these plants.
  • the method comprises the steps: a) growing the plants of the invention, b) removing the harvestable parts as defined above from the plants and c) producing said product from or by the harvestable parts of the invention.
  • the product may be produced at the site where the plant has been grown, the plants and/or parts thereof may be removed from the site where the plants have been grown to produce the product.
  • the plant is grown, the desired harvestable parts are removed from the plant, if feasible in repeated cycles, and the product made from the harvestable parts of the plant.
  • the step of growing the plant may be performed only once each time the methods of the invention is performed, while allowing repeated times the steps of product production e.g. by repeated removal of harvestable parts of the plants of the invention and if necessary further processing of these parts to arrive at the product. It is also possible that the step of growing the plants of the invention is repeated and plants or harvestable parts are stored until the production of the product is then performed once for the accumulated plants or plant parts.
  • the steps of growing the plants and producing the product may be performed with an overlap in time, even simultaneously to a large extend or sequentially. Generally the plants are grown for some time before the product is produced.
  • the products produced by said methods of the invention are plant products such as, but not limited to, a foodstuff, feedstuff, a food supplement, feed supplement, fiber, cosmetic and/or pharmaceutical.
  • Foodstuffs are regarded as compositions used for nutrition and/or for supplementing nutrition.
  • Animal feedstuffs and animal feed supplements, in particular, are regarded as foodstuffs.
  • the inventive methods for the production are used to make agricultural products such as, but not limited to, plant extracts, proteins, amino acids, carbohydrates, fats, oils, polymers, vitamins, and the like. It is possible that a plant product consists of one or more agricultural products to a large extent.
  • the present invention provides a method for controlling weeds at a locus for growth of a plant or plant part thereof, the method comprising: applying a composition comprising CBI herbicides to the locus.
  • the present invention provides a method for controlling weeds at a locus for growth of a plant, the method comprising: applying an herbicide composition comprising CBI herbicides to the locus; wherein said locus is: (a) a locus that contains: a plant or a seed capable of producing said plant; or (b) a locus that is to be after said applying is made to contain the plant or the seed; wherein the plant or the seed comprises in at least some of its cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a mutated CESA polypeptide encoded by the polynucleotide, the expression of the mutated CESA polypeptide conferring to the plant tolerance to C
  • the mutated CESA polypeptide may comprise a mutation corresponding to S1052F (SEQ ID NO: 3), or a fragment of said polypeptide comprising said S1052F mutation; or may comprise a mutation corresponding to G863S (SEQ ID NO: 7), or a fragment of said polypeptide comprising said G863S mutation.
  • the seed may comprise in its cells the wild type or mutated CESA polypeptide, where the polypeptide is a functional variant having, over the full-length of the variant, at least about 65%, more particularly, at least about 80%, 90%, 95%, 98%, 99% or more amino acid sequence identity to SEQ ID NO: 3 or 7.
  • the seed may be planted at the locus, either before, during or after herbicidal treatment.
  • the herbicide composition may be applied to the weeds and to the plant produced by the seed.
  • a plant or seed may be used having a genotype characterized by resistance to a CBI herbicide.
  • the plant or seed may comprise a mutant cesa gene comprising a mutation in the cesa sequence corresponding to SEQ ID NO: 4 or 8, respectively.
  • the plant or seed may be used with a CBI herbicide to inhibit growth of one or more undesired plants.
  • the present invention provides a method for controlling weeds at a locus for growth of a plant, the method comprising: applying a herbicide composition comprising a CBI herbicide to the locus; and planting a seed at the locus, wherein the seed is capable of producing a plant that comprises in at least some of its cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a wild type or mutated CESA polypeptide encoded by the polynucleotide, the expression of the wild type or mutated CESA polypeptide conferring to the plant tolerance to the CBI herbicide.
  • the mutated CESA polypeptide may comprise a mutation corresponding to S1052F in SEQ ID NO: 3, or a fragment or ortholog of said polypeptide comprising said S1052F mutation.
  • the mutated CESA polypeptide may comprise a mutation corresponding to G863S in SEQ ID NO: 7, or a fragment or ortholog of said polypeptide comprising said G863S mutation.
  • the wild type or mutated CESA polypeptide is a functional variant having, over the full-length of the variant, at least about 65%, more particularly, at least about 80%, 90%, 95%, 98%, 99% or more amino acid sequence identity to SEQ ID NO: 3 or 7.
  • a CBI herbicide may be applied to the weeds and to the plant produced by the seed.
  • Herbicide compositions hereof can be applied, e.g., as foliar treatments, soil treatments, seed treatments, or soil drenches. Application can be made, e.g., by spraying, dusting, broadcasting, or any other mode known useful in the art.
  • herbicides can be used to control the growth of weeds that may be found growing in the vicinity of the herbicide-tolerant plants invention.
  • an herbicide can be applied to a plot in which herbicide-tolerant plants of the invention are growing in vicinity to weeds.
  • An herbicide to which the herbicide-tolerant plant of the invention is tolerant can then be applied to the plot at a concentration sufficient to kill or inhibit the growth of the weed. Concentrations of herbicide sufficient to kill or inhibit the growth of weeds are known in the art and are disclosed above.
  • the present invention provides a method for controlling weeds in the vicinity of a CBI herbicide-tolerant plant of the invention.
  • the method comprises applying an effective amount of a CBI herbicide to the weeds and to the herbicide-tolerant plant, wherein the plant has increased tolerance to CBI herbicide when compared to a wild- type plant.
  • the CBI herbicide-tolerant plants of the invention are preferably crop plants, including, but not limited to, sunflower, alfalfa, Brassica sp., soybean, cotton, safflower, peanut, tobacco, tomato, potato, wheat, rice, maize, sorghum, barley, rye, millet, and sorghum.
  • herbicide(s) e.g., CBI herbicides
  • an effective concentration or an effective amount of herbicide(s), or a composition comprising an effective concentration or an effective amount of herbicide(s) can be applied directly to the seeds prior to or during the sowing of the seeds.
  • Seed Treatment formulations may additionally comprise binders and optionally colorants.
  • Binders can be added to improve the adhesion of the active materials on the seeds after treatment.
  • suitable binders are block copolymers EO/PO surfactants but also polyvinylalcoholsl, polyvinylpyrrolidones, polyacrylates,
  • colorants can be included in the formulation. Suitable colorants or dyes for seed treatment formulations are Rhodamin B, C.I. Pigment Red 112, C.I.
  • Solvent Red 1 pigment blue 15:4, pigment blue 15:3, pigment blue 15:2, pigment blue 15: 1, pigment blue 80, pigment yellow 1 , pigment yellow 13, pigment red 112, pigment red 48:2, pigment red 48: 1, pigment red 57: 1 , pigment red 53: 1 , pigment orange 43, pigment orange 34, pigment orange 5, pigment green 36, pigment green 7, pigment white 6, pigment brown 25, basic violet 10, basic violet 49, acid red 51, acid red 52, acid red 14, acid blue 9, acid yellow 23, basic red 10, basic red 108.
  • seed treatment comprises all suitable seed treatment techniques known in the art, such as seed dressing, seed coating, seed dusting, seed soaking, and seed pelleting.
  • the present invention provides a method of treating soil by the application, in particular into the seed drill: either of a granular formulation containing the Indaziflam or auxinic herbicides as a composition/formulation (e.g., a granular formulation), with optionally one or more solid or liquid, agriculturally acceptable carriers and/or optionally with one or more agriculturally acceptable surfactants. This method is advantageously employed, for example, in seedbeds of cereals, maize, cotton, and sunflower.
  • the present invention also comprises seeds coated with or containing with a seed treatment formulation comprising CBI herbicides and at least one other herbicide such as, e.g. , an AHAS-inhibitor selected from the group consisting of amidosulfuron, azimsulfuron, bensulfuron, chlorimuron, chlorsulfuron, cinosulfuron, cyclosulfamuron, ethametsulfuron, ethoxysulfuron, flazasulfuron, flupyrsulfuron, foramsulfuron, halosulfuron, imazosulfuron, iodosulfuron, mesosulfuron, metsulfuron, nicosulfuron, oxasulfuron, primisulfuron, prosulfuron, pyrazosulfuron, rimsulfuron, sulfometuron, sulfosulfuron, thifensulfuron, tria
  • the term "coated with and/or containing” generally signifies that the active ingredient is for the most part on the surface of the propagation product at the time of application, although a greater or lesser part of the ingredient may penetrate into the propagation product, depending on the method of application.
  • the seed treatment application with CBI herbicides or with a formulation comprising the CBI herbicides is carried out by spraying or dusting the seeds before sowing of the plants and before emergence of the plants.
  • the corresponding formulations are applied by treating the seeds with an effective amount of CBI herbicides or a formulation comprising the CBI herbicides.
  • the present invention provides a method for combating undesired vegetation or controlling weeds comprising contacting the seeds of the CBI herbicides-tolerant plants of the present invention before sowing and/or after pregermination with CBI herbicides.
  • the method can further comprise sowing the seeds, for example, in soil in a field or in a potting medium in greenhouse.
  • the method finds particular use in combating undesired vegetation or controlling weeds in the immediate vicinity of the seed.
  • the control of undesired vegetation is understood as the killing of weeds and/or otherwise retarding or inhibiting the normal growth of the weeds.
  • Weeds, in the broadest sense, are understood as meaning all those plants which grow in locations where they are undesired.
  • the weeds may include, for example, dicotyledonous and monocotyledonous weeds.
  • Dicotyledonous weeds include, but are not limited to, weeds of the genera: Sinapis, Lepiclium, Galium, Stellaria, Matricaria, Anthemis, Galinsoga, Chenopodium, Ulrica,
  • Senecio Amaranthus, Portulaca, Xanthium, Convolvulus, lpomoea, Polygonum, Sesbania, Ambrosia, Cirsium, Carduus, Sonchus, Solarium, Rorippa, Rotala, Lindernia, Lamium, Veronica, Abutilon, Emex, Datura, Viola, Galeopsis, Papaver,Centaurea, Trifolium, Ranunculus, and Taraxacum.
  • Monocotyledonous weeds include, but are not limited to, weeds of the genera: Echinochloa, Setaria, Panicum, Digitaria, Phleum, Poa, Festuca, Eleusine, Brachiaria, Lolium, Bromus, Avena, Cyperus, Sorghum, Agropyron, Cynodon, Monochoria, Fimbristyslis, Sagittaria, Eleocharis, Scirpus, Paspalum, lschaemum, Sphenoclea,
  • the weeds may include, for example, crop plants that are growing in an undesired location.
  • a volunteer maize plant that is in a field that predominantly comprises soybean plants can be considered a weed, if the maize plant is undesired in the field of soybean plants.
  • treatment of loci, plants, plant parts, or seeds of the present invention comprises application of an agronomically acceptable composition that does not contain an AT.
  • the treatment comprises application of an agronomically acceptable composition that does not contain a CBI herbicide A.I.
  • the treatment comprises application of an agronomically acceptable composition that does not contain a CBI herbicide A.L, wherein the composition comprises one or more of agronomically-acceptable carriers, diluents, excipients, plant growth regulators, and the like.
  • the treatment comprises application of an agronomically acceptable composition that does not contain a CBI herbicide A.L, wherein the composition comprises an adjuvant.
  • the adjuvant is a surfactant, a spreader, a sticker, a penetrant, a drift-control agent, a crop oil, an emulsifier, a compatibility agent, or combinations thereof.
  • FIG 2 results from analyzing wheat mutants Tafxrl-1 and Tafxrl-2 root length are shown as a percentage relative to control with an increasing concentration of flupoxam. After seven days, pictures of the seedlings were taken and root length was measured using magnification. Measurements are shown in Percent Control (%), which is the percent of the mean root length of each line under control conditions.
  • FIG 3 shows photographs of the growth of the wheat mutants Tafxrl-1 and
  • FIG 4 shows photographs of the wheat mutants Tafxrl-1 and Tafxrl-2 and Wild-type seeds as senescened plants.

Abstract

L'invention concerne des plantes exprimant un polypeptide de synthase de cellulose mutante (CESA) conférant une tolérance aux herbicides inhibiteurs de la biosynthèse de la cellulose (CBI), tels que le flupoxam. Le polypeptide CESA mutant comprend une substitution d'acide aminé à une position correspondant À SI 052 représentée dans SEQ ID NO : 1 ou G863 représentée dans SEQ ID NO : 5; ledit polypeptide ayant au moins 75 % d'identité avec cette dernière; et conférant une tolérance à un herbicide CBI dans une plante. L'invention concerne également des procédés utilisant des mutants CESA comportant une substitution d'acides aminés à une position correspondant à S1052F représentée dans SEQ ID NO : 1 ou G863S représentée dans SEQ ID NO : 5, et des plantes exprimant ledit polypeptide CESA mutant pour une utilisation dans la lutte contre les mauvaises herbes.
PCT/CA2020/050046 2019-01-18 2020-01-17 Plantes ayant une tolérance accrue aux herbicides inhibiteurs de la biosynthèse de la cellulose WO2020146949A1 (fr)

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