Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
• • 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

Definitions

• the present invention relates to modified protoporphyrinogen oxidase (PPO) enzymes having one or more mutations at specific amino acid positions which increase the resistance of the enzyme to PPO-inhibiting compounds such as herbicides.
• PPO protoporphyrinogen oxidase
• the invention further relates to plants or parts thereof comprising the modified PPO enzymes, methods of making the plants, and methods of controlling undesired vegetation in the vicinity of said plants.
• BACKGROUND The present invention relates to the production of modified PPO enzymes, and plants containing said enzymes, that are resistant to herbicides.
• Protoporphyrinogen oxidase (PPO) enzymes (EC 1.3.3.4), are well conserved eukaryotic enzymes which convert protoporphyrinogen IX to protoporphyrin IX.
• PPO is a flavoprotein located in the inner mitochondrial membrane, which catalyses the removal of six hydrogens (four from methylene bridges and two from ring nitrogens) to form protoporphyrin- IX. This involves a three-step, six-electron flavin adenine dinucleotide–dependent oxidation that consumes molecular oxygen. It is essential to mitochondrial heme functions.
• Herbicides of particular interest in the agricultural field include those that inhibit PPO enzymes, referred to as PPO inhibiting herbicides. Given that heme function is essential to the survival of plants, it has been identified as a herbicidal target. Recently, effective herbicidal compounds which inhibit PPO enzymes have been developed. Upon contact with plants, the herbicides cause cell death and eventually death of the plant. Industrially, herbicides are used in agriculture to remove unwanted vegetation such as weeds from cultivated crops. However, non-specific herbicides which target essential pathways, such as those that target heme synthesis, will affect both crops and the unwanted vegetation, making them difficult to use without destroying the valuable crop.
• Agricultural crop production often utilizes transgenic traits created using the methods of biotechnology.
• a heterologous gene also known as a transgene, can be introduced into a plant to produce a transgenic trait. Expression of the transgene in the plant confers a trait, such as herbicide tolerance, to the plant.
• Herbicide tolerant traits allow a plant to grow, whilst non-tolerant weed species are eradicated by contact with the herbicide.
• transgenic herbicide tolerance traits include glyphosate tolerance, glufosinate tolerance, and dicamba tolerance.
• glyphosate tolerance glyphosate tolerance
• glufosinate tolerance glyphosate tolerance
• dicamba tolerance glyphosate tolerance
• new herbicides and accompanying new herbicide tolerance traits are needed in the field. Given that various herbicides which target PPO have been developed, there is thus a need to develop traits conferring tolerance to PPO-inhibiting herbicides, which will be particularly useful in crops.
• the present invention aims to solve one or more of the above-mentioned problems in the art.
• a modified Protoporphyrinogen Oxidase (PPO) enzyme or functional fragment thereof wherein the modified Protoporphyrinogen Oxidase or functional fragment thereof comprises at least one mutation at one or more amino acid residues selected from residues 362, 365 and/or 479 of SEQ ID NO:1, or residues corresponding thereto.
• PPO Protoporphyrinogen Oxidase
• a modified Protoporphyrinogen Oxidase (PPO) enzyme or functional fragment thereof wherein the modified Protoporphyrinogen Oxidase or functional fragment thereof comprises a mutation at amino acid residues 305 and 426 of SEQ ID NO:1, or residues corresponding thereto, wherein the mutation at amino acid residue 305, or a residue corresponding thereto, is a substitution to leucine (305L) and wherein the mutation at amino acid residue 426, or a residue corresponding thereto, is a substitution to valine (426V).
• the modified PPO enzyme comprises substitutions S305L and Y426V with reference to SEQ ID NO: 1 or residue corresponding thereto.
• the modified PPO enzyme comprises substitutions T305L and Y426V with reference to SEQ ID NO: 2 or residue corresponding thereto. In certain embodiments, the modified PPO enzyme comprises or consists of an amino acid sequence according to SEQ ID NO: 125. In certain embodiments, the modified PPO enzyme comprises or consists of an amino acid sequence according to SEQ ID NO: 139.
• nucleic acid comprising a polynucleotide encoding a modified PPO enzyme or functional fragment thereof according to the first or second aspect.
• a recombinant vector comprising a nucleic acid according to the third aspect.
• a plant or part thereof comprising a modified PPO enzyme or functional fragment thereof according to the first or second aspect, a nucleic acid according to the third aspect, or a recombinant vector according to the fourth aspect.
• a method of producing a plant or part thereof according to the fifth aspect comprising: modifying the plant or part thereof to comprise a PPO enzyme according to the first or second aspect.
• a method of controlling undesired vegetation in the vicinity of a plant or part thereof according to the fifth aspect, or at a locus for growth of a plant according to the fifth aspect comprising applying an effective amount of at least one compound which inhibits a PPO enzyme to the undesired vegetation, the plant or part thereof, and/or the locus, and/or optionally planting a seed at the locus wherein the seed is capable of producing a plant according to the fifth aspect.
• the invention primarily relates to modified Protoporphyrinogen Oxidase (PPO) enzymes that have increased resistance to compounds such as herbicides, in comparison to wild type or control PPO enzymes.
• PPO Protoporphyrinogen Oxidase
• PPO enzyme or “modified PPO enzyme” and the amino acid sequences thereof also refers to and is intended to encompass isolated polynucleotides encoding such an enzyme.
• modified polynucleotide “mutated polynucleotide”, “mutated polypeptide”, or “modified polypeptide” is a polynucleotide or polypeptide that has been altered through human intervention.
• mutated polynucleotide has a sequence that differs from the sequence of the corresponding non-mutated or non-modified polynucleotide or polypeptide (for example a wild type or naturally occurring polynucleotide or polypeptide) by at least one nucleotide or amino acid.
• the mutated polynucleotide or modified polynucleotide comprises an alteration that results from a guide polynucleotide/Cas endonuclease system as disclosed herein.
• Protoporphyrinogen Oxidase refers to a key enzyme in the biosynthesis of protoporphyrin IX. PPO catalyzes the last common step in chlorophyll and heme biosynthesis, which is the oxidation of protoporphyrinogen IX to protoporphyrin IX. (Matringe et al. 1989. Biochem. 1. 260: 231). Inhibition of protoporphyrinogen oxidase is a mechanism of action for several commercial herbicides, including the nitrophenyl ethers acifluorfen and fomesafen and the pyrimidinediones butafenacil and saflufenacil.
• the visible symptoms of treatment are chlorosis and desiccation.
• the damage is caused by an accumulation of protoporphyrin IX in the plant cells by inhibiting protox within the tetrapyrrole biosynthesis pathway.
• PPOX1 and PPOX2 are encoded by two nucleus-localised homologous genes, PPOX1 and PPOX2.
• PPOX1 is targeted exclusively to plastids, providing Proto for heme and chlorophyll synthesis, while PPOX2 has been found in plastid envelope and mitochondria in spinach.
• N. tabacum PPOX2 was shown to be solely a mitochondrial protein.
• the PPO enzyme has been modified to increase activity of the enzyme, suitably within a plant or a part thereof.
• the plant may have been modified to comprise a modified PPO enzyme having increased activity when compared to the unmodified, suitably wildtype PPO enzyme, suitably within the plant or a part thereof.
• the activity of the modified PPO enzyme is increased to a level which provides the plant with increased resistance to a compound that inhibits PPO enzymatic activity relative to an unmodified enzyme or control enzyme.
• the activity of the modified PPO enzyme is increased to a level which provides a plant with increased resistance to a compound that inhibits PPO enzymatic activity relative to an unmodified enzyme or control enzyme.
• the activity of the modified PPO enzyme is increased to a level of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150% greater than the activity thereof in an unmodified or control plant when exposed to a compound that inhibits PPO enzymatic activity.
• an enzyme assay which measures the consumption of a substrate or production of a product over time, suitably in an in vitro environment.
• Such assays may be spectrophotometric, fluorometric, calorimetric, chemiluminescent, light scattering or microscale thermophoresis.
• the modified PPO enzyme comprises one or more modifications, suitably one or more mutations.
• the one or more mutations are a selection from deletions, insertions, substitutions etc.
• mutation it is meant any substitution, deletion or insertion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids.
• the modified PPO enzyme may be a modified PPO1 enzyme.
• the modified PPO enzyme may include at least one amino acid modification at any one of amino acid positions 362, 365 and/or 479 of SEQ ID NO:1, or residues corresponding thereto.
• the modified PPO enzyme may comprise an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity to an amino acid sequence of SEQ ID NO: 1, 2, 4 - 151, 153 -302, or 305 - 336 respectively, or a functional fragment thereof.
• the modified PPO enzyme may comprise an amino acid sequence having at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to an amino acid sequence of SEQ ID NO: 1, 2, 4 - 151, 153 -302, or 305 - 336 respectively, or a functional fragment thereof.
• the modified PPO enzyme may comprise an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity to an amino acid sequence of SEQ ID NO: 37- 39, 58, 59, 97 - 99, 118, 119, 125 - 137, 139 - 151, 188 - 190, 209, 210, 248 - 250, 269, 270, 277 - 288 or 291 - 302 respectively, or a functional fragment thereof.
• the modified PPO enzyme may comprise an amino acid sequence having at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to an amino acid sequence of SEQ ID NO: 37- 39, 58, 59, 97 - 99, 118, 119, 125 - 137, 139 - 151, 188 - 190, 209, 210, 248 - 250, 269, 270, 277 - 288 or 291 - 302 respectively, or a functional fragment thereof.
• the modified PPO enzyme may comprise an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity to an amino acid sequence of SEQ ID NO: 58, 59, 126, 127, 130,
• the modified PPO enzyme may comprise an amino acid sequence having at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to an amino acid sequence of SEQ ID NO: 58, 59, 126, 127, 130, 131,
• the modified PPO enzyme may comprise an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity to an amino acid sequence of SEQ ID NO: 125, 139, 276, 290, 126,131 , 134, 140, 145, 148, 277, 282, 285, 291, 296, or 299 respectively, or a functional fragment thereof.
• the modified PPO enzyme may comprise an amino acid sequence having at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to an amino acid sequence of SEQ ID NO: 125, 139, 276, 290, 126,131, 134, 140, 145, 148, 277, 282, 285, 291, 296, or 299 respectively, or a functional fragment thereof.
• the modified PPO enzyme may comprise an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity to an amino acid sequence of SEQ ID NO: 125, 126, 127, 131, 132, 134, 276, 277, 278, 282, 283, 285, 139, 140, 141, 145, 146, 148, 290, 291, 292, 296, 297, or 299 respectively, or a functional fragment thereof.
• the modified PPO enzyme may comprise an amino acid sequence having at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to an amino acid sequence of SEQ ID NO: 125, 126, 127, 131, 132, 134, 276, 277, 278, 282, 283, 285, 139, 140, 141, 145, 146, 148, 290, 291, 292, 296, 297, or 299 respectively, or a functional fragment thereof.
• the modified PPO enzyme may comprise an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity to an amino acid sequence of SEQ ID NO: 125, 131, 134, 139, 145, 148, 276, 282, 285, 290, 296 or 299 respectively, or a functional fragment thereof.
• the modified PPO enzyme may comprise an amino acid sequence having at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to an amino acid sequence of SEQ ID NO: 125, 131, 134, 139, 145, 148, 276, 282, 285, 290, 296 or 299 respectively, or a functional fragment thereof.
• the modified PPO enzyme may comprise an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity to an amino acid sequence of SEQ ID NO: 125, 139, 276, or 290, respectively, or a functional fragment thereof.
• the modified PPO enzyme may comprise an amino acid sequence having at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to an amino acid sequence of SEQ ID NO: 125, 139, 276, or 290 respectively, or a functional fragment thereof.
• the modified PPO enzyme may comprise an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity to an amino acid sequence of SEQ ID NO: 131, 145, 282, or 296, respectively, or a functional fragment thereof.
• the modified PPO enzyme may comprise an amino acid sequence having at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to an amino acid sequence of SEQ ID NO: 131, 145, 282, or 296 respectively, or a functional fragment thereof.
• the modified PPO enzyme may comprise an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity to an amino acid sequence of SEQ ID NO: 126, 140, 277, or 291, respectively, or a functional fragment thereof.
• the modified PPO enzyme may comprise an amino acid sequence having at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to an amino acid sequence of SEQ ID NO: 126, 140, 277, or 291 respectively, or a functional fragment thereof.
• the modified PPO enzyme may comprise an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity to an amino acid sequence of SEQ ID NO: 134, 148, 285, or 299, respectively, or a functional fragment thereof.
• the modified PPO enzyme may comprise an amino acid sequence having at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to an amino acid sequence of SEQ ID NO: 134, 148, 285, or 299 respectively, or a functional fragment thereof.
• the modified PPO enzyme may comprise an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity to an amino acid sequence of SEQ ID NO: 132, 283, 146 or 297, respectively, or a functional fragment thereof.
• the modified PPO enzyme may comprise an amino acid sequence having at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to an amino acid sequence of SEQ ID NO: 132, 283, 146 or 297 respectively, or a functional fragment thereof.
• the modified PPO enzyme may comprise an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity to an amino acid sequence of SEQ ID NO: 127, 278, 141 or 292, respectively, or a functional fragment thereof.
• the modified PPO enzyme may comprise an amino acid sequence having at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to an amino acid sequence of SEQ ID NO: 127, 278, 141 or 292 respectively, or a functional fragment thereof.
• the modified PPO enzyme may be derived from Arabidopsis thaliana. In one embodiment, the modified PPO enzyme may be derived from Setaria italica.
• a modified PPO enzyme of the invention may comprise an amino acid sequence according to SEQ ID NO:1 (Arabidopsis thaliana PPO) wherein positions 362, 365 and/or 479 or residues corresponding thereto have been modified.
• a modified PPO enzyme of the invention may comprise an amino acid sequence according to SEQ ID NO:2 (Setaria italica PPO) wherein positions 360, 363 and/or 477 or residues corresponding thereto have been modified. Positions 360, 363 and 477 respectively correspond to positions 362, 365 and 479 of SEQ ID NO: 1.
• a modified PPO enzyme of the invention may comprise an amino acid sequence according to SEQ ID NO:1 (Arabidopsis thaliana PPO) wherein positions 305 and/or 426 or residues corresponding thereto have been modified, suitably to be leucine and valine respectively.
• a modified PPO enzyme of the invention may comprise an amino acid sequence according to SEQ ID NO:2 (Setaria italica PPO) wherein positions 303 and/or 424 or residues corresponding thereto have been modified, suitably to be leucine and valine respectively.
• a modified PPO enzyme of the invention may comprise an amino acid sequence according to SEQ ID NO:1 (Arabidopsis thaliana PPO) wherein position 362 has been modified.
• a modified PPO enzyme of the invention may comprise an amino acid sequence according to SEQ ID NO:1 (Arabidopsis thaliana PPO) wherein position 365 has been modified.
• a modified PPO enzyme of the invention may comprise an amino acid sequence according to SEQ ID NO:1 (Arabidopsis thaliana PPO) wherein position 479 has been modified.
• a modified PPO enzyme of the invention may comprise an amino acid sequence according to SEQ ID NO:2 (Setaria italica PPO) wherein position 360 has been modified.
• a modified PPO enzyme of the invention may comprise an amino acid sequence according to SEQ ID NO:2 (Setaria italica PPO) wherein position 363 has been modified.
• a modified PPO enzyme of the invention may comprise an amino acid sequence according to SEQ ID NO:2 (Setaria italica PPO) wherein position 477 has been modified.
• a modified PPO of the invention may comprise an amino acid sequence of at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 98% or at least 99% sequence identity to an amino acid sequence according to SEQ ID NO: 1, wherein at least one of positions 362, 365 and 479 thereof, or corresponding positions thereto, have been modified.
• a modified PPO of the invention may comprise an amino acid sequence of at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 98% or at least 99% sequence identity to an amino acid sequence according to SEQ ID NO: 2, wherein at least one of positions 360, 363 and 477 thereof, or corresponding positions thereto, have been modified.
• a modified PPO of the invention may comprise one or more amino acid sequence motifs as set out in Table 27
• position 362 of SEQ ID NO: 1 or corresponding positions thereto may be substituted with a cysteine. In some examples, position 362 of SEQ ID NO: 1 or corresponding positions thereto may be substituted with a phenylalanine.
• position 360 of SEQ ID NO: 2 or corresponding positions thereto may be substituted with a cysteine. In some examples, position 360 of SEQ ID NO: 2 or corresponding positions thereto may be substituted with a phenylalanine.
• position 365 of SEQ ID NO: 1 or corresponding positions thereto may be substituted with a methionine. In some examples, position 365 of SEQ ID NO: 1 or corresponding positions thereto may be substituted with a leucine.
• position 363 of SEQ ID NO: 2 or corresponding positions thereto may be substituted with a methionine. In some examples, position 363 of SEQ ID NO: 2 or corresponding positions thereto may be substituted with a leucine.
• position 479 of SEQ ID NO: 1 or corresponding positions thereto may be substituted with a methionine. In some examples, position 479 of SEQ ID NO: 1 or corresponding positions thereto may be substituted with an asparagine.
• position 477 of SEQ ID NO: 2 or corresponding positions thereto may be substituted with a methionine. In some examples, position 477 of SEQ ID NO: 2 or corresponding positions thereto may be substituted with an asparagine.
• the modified PPOs of the invention may include one or more additional modifications in addition to those detailed above. For example, modification of any one of the amino acid residues 305, 361, 404, 426, 431 and/or 461 of SEQ ID NO: 1 or residues corresponding thereto.
• residue 305 of SEQ ID NO: 1 or a residue corresponding thereto may be substituted with leucine.
• residue 361 of SEQ ID NO: 1 or a residue corresponding thereto may be substituted with threonine.
• residue 361 of SEQ ID NO: 1 or a residue corresponding thereto may be substituted with cysteine.
• residue 361 of SEQ ID NO: 1 or a residue corresponding thereto may be substituted with aspartic acid.
• residue 361 of SEQ ID NO: 1 or a residue corresponding thereto may be substituted with glutamine.
• residue 404 of SEQ ID NO: 1 or a residue corresponding thereto may be substituted with alanine.
• residue 426 of SEQ ID NO: 1 or a residue corresponding thereto may be substituted with leucine.
• residue 426 of SEQ ID NO: 1 or a residue corresponding thereto may be substituted with cysteine.
• residue 426 of SEQ ID NO: 1 or a residue corresponding thereto may be substituted with threonine.
• residue 426 of SEQ ID NO: 1 or a residue corresponding thereto may be substituted with valine.
• residue 426 of SEQ ID NO: 1 or a residue corresponding thereto may be substituted with methionine.
• residue 431 of SEQ ID NO: 1 or a residue corresponding thereto may be substituted with phenylalanine.
• residue 431 of SEQ ID NO: 1 or a residue corresponding thereto may be substituted with alanine.
• residue 431 of SEQ ID NO: 1 or a residue corresponding thereto may be substituted with arginine.
• residue 461 of SEQ ID NO: 1 or a residue corresponding thereto may be substituted with glutamine.
• residue 303 of SEQ ID NO: 2 or a residue corresponding thereto may be substituted with leucine.
• residue 359 of SEQ ID NO: 2 or a residue corresponding thereto may be substituted with threonine.
• residue 359 of SEQ ID NO: 2 or a residue corresponding thereto may be substituted with cysteine.
• residue 359 of SEQ ID NO: 2 or a residue corresponding thereto may be substituted with aspartic acid.
• residue 359 of SEQ ID NO: 2 or a residue corresponding thereto may be substituted with glutamine.
• residue 402 of SEQ ID NO: 2 or a residue corresponding thereto may be substituted with alanine.
• residue 424 of SEQ ID NO: 2 or a residue corresponding thereto may be substituted with leucine.
• residue 424 of SEQ ID NO: 2 or a residue corresponding thereto may be substituted with cysteine.
• residue 424 of SEQ ID NO: 2 or a residue corresponding thereto may be substituted with threonine.
• residue 424 of SEQ ID NO: 2 or a residue corresponding thereto may be substituted with valine.
• residue 424 of SEQ ID NO: 2 or a residue corresponding thereto may be substituted with methionine.
• residue 429 of SEQ ID NO: 2 or a residue corresponding thereto may be substituted with phenylalanine.
• residue 429 of SEQ ID NO: 2 or a residue corresponding thereto may be substituted with asparagine.
• residue 459 of SEQ ID NO: 2 or a residue corresponding thereto may be substituted with glutamine.
• the modified PPO enzyme may include modifications at amino acid residue positions 365, 426, and 479 of SEQ ID NO: 1 or residues corresponding thereto. In some examples, the modified PPO enzyme may include modifications at amino acid residue positions 305, 404, 426, 431, and 479 of SEQ ID NO: 1 or residues corresponding thereto.
• the modified PPO enzyme may include modifications at amino acid residue positions 305 and 365 of SEQ ID NO: 1 or residues corresponding thereto.
• the modified PPO enzyme may include modifications at amino acid residue positions 305, 362 and 404 of SEQ ID NO: 1 or residues corresponding thereto. In some examples, the modified PPO enzyme may include modifications at amino acid residue positions 305, 361 , 365, 431 and 479 of SEQ ID NO: 1 or residues corresponding thereto.
• the modified PPO enzyme may include modifications at amino acid residue positions 305, 426 and 479 of SEQ ID NO: 1 or residues corresponding thereto. In some examples, the modified PPO enzyme may include modifications at amino acid residue positions 426, 461 and 479 of SEQ ID NO: 1 or residues corresponding thereto. In some examples, the modified PPO enzyme may include modifications at amino acid residue positions 305, 361 and 365 of SEQ ID NO: 1 or residues corresponding thereto. In some examples, the modified PPO enzyme may include modifications at amino acid residue positions 305, 361 , 362, 426 and 479 of SEQ ID NO: 1 or residues corresponding thereto.
• the modified PPO enzyme may include modifications at amino acid residue positions 361 and 365 of SEQ ID NO: 1 or residues corresponding thereto.
• the modified PPO enzyme may include modifications at amino acid residue positions 365, 404, and 479 of SEQ ID NO: 1 or residues corresponding thereto. In some examples, the modified PPO enzyme may include modifications at amino acid residue positions 305, 404, and 479 of SEQ ID NO: 1 or residues corresponding thereto. In some examples, the modified PPO enzyme may include modifications at amino acid residues positions 305 and 426 of SEQ ID NO: 1 or residues corresponding thereto.
• the modified PPO enzyme may include modifications at amino acid residue positions 363, 424, and 477 of SEQ ID NO: 2 or residues corresponding thereto. In some examples, the modified PPO enzyme may include modifications at amino acid residue positions 303, 402, 424, 429, and 477 of SEQ ID NO: 2 or residues corresponding thereto.
• the modified PPO enzyme may include modifications at amino acid residue positions 303 and 363 of SEQ ID NO: 2 or residues corresponding thereto.
• the modified PPO enzyme may include modifications at amino acid residue positions 303, 360 and 402 of SEQ ID NO: 2 or residues corresponding thereto. In some examples, the modified PPO enzyme may include modifications at amino acid residue positions 303, 359, 363, 429 and 477 of SEQ ID NO: 2 or residues corresponding thereto.
• the modified PPO enzyme may include modifications at amino acid residue positions 303, 424 and 477 of SEQ ID NO: 2 or residues corresponding thereto. In some examples, the modified PPO enzyme may include modifications at amino acid residue positions 424, 459 and 477 of SEQ ID NO: 2 or residues corresponding thereto. In some examples, the modified PPO enzyme may include modifications at amino acid residue positions 303, 359 and 363 of SEQ ID NO: 2 or residues corresponding thereto. In some examples, the modified PPO enzyme may include modifications at amino acid residue positions 303, 359, 360, 424 and 477 of SEQ ID NO: 2 or residues corresponding thereto.
• the modified PPO enzyme may include modifications at amino acid residue positions 359 and 363 of SEQ ID NO: 2 or residues corresponding thereto.
• the modified PPO enzyme may include modifications at amino acid residue positions 363, 402, and 477 of SEQ ID NO: 2 or residues corresponding thereto. In some examples, the modified PPO enzyme may include modifications at amino acid residue positions 303, 402, and 477 of SEQ ID NO: 2 or residues corresponding thereto. In some examples, the modified PPO enzyme may include modifications at amino acid residues positions 303 and 424 of SEQ ID NO: 2 or residues corresponding thereto. Suitably, the modified PPO may have an amino acid sequence having at least 30% sequence identity to SEQ ID NO: 37 (wherein the amino acid sequence includes the substitution Y362F compared to SEQ ID NO:1).
• the modified PPO may have an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 37.
• the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 37.
• the modified PPO may comprise an amino acid sequence according to SEQ ID NO:
• the modified PPO may essentially consist of an amino acid sequence according to SEQ ID NO: 37.
• the modified PPO may have an amino acid sequence having at least 30% sequence identity to SEQ ID NO: 38 (wherein the amino acid sequence includes the substitution V365L compared to SEQ ID NO:1).
• the modified PPO may have an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 38.
• the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 38.
• the modified PPO may comprise an amino acid sequence according to SEQ ID NO:
• the modified PPO may essentially consist of an amino acid sequence according to SEQ ID NO: 38.
• the modified PPO may have an amino acid sequence having at least 30% sequence identity to SEQ ID NO: 39 (wherein the amino acid sequence includes the substitution V365M compared to SEQ ID NO:1).
• the modified PPO may have an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 39.
• the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 39.
• the modified PPO may comprise an amino acid sequence according to SEQ ID NO:
• the modified PPO may essentially consist of an amino acid sequence according to SEQ ID NO: 39.
• the modified PPO may have an amino acid sequence having at least 30% sequence identity to SEQ ID NO: 58 (wherein the amino acid sequence includes the substitution L479M compared to SEQ ID NO:1).
• the modified PPO may have an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 58.
• the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 58.
• the modified PPO may comprise an amino acid sequence according to SEQ ID NO:
• the modified PPO may essentially consist of an amino acid sequence according to SEQ ID NO: 58.
• the modified PPO may have an amino acid sequence having at least 30% sequence identity to SEQ ID NO: 59 (wherein the amino acid sequence includes the substitution L479N compared to SEQ ID NO:1).
• the modified PPO may have an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 59.
• the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 59.
• the modified PPO may comprise an amino acid sequence according to SEQ ID NO:
• the modified PPO may essentially consist of an amino acid sequence according to SEQ ID NO: 59.
• the modified PPO may have an amino acid sequence having at least 30% sequence identity to SEQ ID NO: 97 (wherein the amino acid sequence includes the substitution Y360F compared to SEQ ID NO:2).
• the modified PPO may have an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 97.
• the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 97.
• the modified PPO may comprise an amino acid sequence according to SEQ ID NO:
• the modified PPO may essentially consist of an amino acid sequence according to SEQ ID NO: 97.
• the modified PPO may have an amino acid sequence having at least 30% sequence identity to SEQ ID NO: 98 (wherein the amino acid sequence includes the substitution V363L compared to SEQ ID NO:2).
• the modified PPO may have an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 98.
• the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 98.
• the modified PPO may comprise an amino acid sequence according to SEQ ID NO:
• the modified PPO may essentially consist of an amino acid sequence according to SEQ ID NO: 98.
• the modified PPO may have an amino acid sequence having at least 30% sequence identity to SEQ ID NO: 99 (wherein the amino acid sequence includes the substitution V363M compared to SEQ ID NO:2).
• the modified PPO may have an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 99.
• the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 99.
• the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 99.
• the modified PPO may essentially consist of an amino acid sequence according to SEQ ID NO: 99.
• the modified PPO may have an amino acid sequence having at least 30% sequence identity to SEQ ID NO: 118 (wherein the amino acid sequence includes the substitution V477M compared to SEQ ID NO:2).
• the modified PPO may have an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 118.
• the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 118.
• the modified PPO may comprise an amino acid sequence according to SEQ ID NO:
• the modified PPO may essentially consist of an amino acid sequence according to SEQ ID NO: 118.
• the modified PPO may have an amino acid sequence having at least 30% sequence identity to SEQ ID NO: 119 (wherein the amino acid sequence includes the substitution V477N compared to SEQ ID NO:2).
• the modified PPO may have an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 119.
• the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 119.
• the modified PPO may comprise an amino acid sequence according to SEQ ID NO:
• the modified PPO may essentially consist of an amino acid sequence according to SEQ ID NO: 119.
• the modified PPO may have an amino acid sequence having at least 30% sequence identity to SEQ ID NO: 125 (wherein the amino acid sequence includes the substitutions S305L and Y426V compared to SEQ ID NO:1).
• the modified PPO may have an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 125.
• the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 125.
• the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 125.
• the modified PPO may essentially consist of an amino acid sequence according to SEQ ID NO: 125.
• the modified PPO may have an amino acid sequence having at least 30% sequence identity to SEQ ID NO: 126 (wherein the amino acid sequence includes the substitutions V365L, Y426V, and L479N compared to SEQ ID NO:1).
• the modified PPO may have an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 126.
• the modified PPO may have an amino acid sequence having at least 85% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 126.
• the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 126.
• the modified PPO may essentially consist of an amino acid sequence according to SEQ ID NO: 126.
• the modified PPO may have an amino acid sequence having at least 30% sequence identity to SEQ ID NO: 127 (wherein the amino acid sequence includes the substitution S305L, G404A, Y426V, T431 R, and L479N compared to SEQ ID NO:1).
• the modified PPO may have an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 127.
• the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 127.
• the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 127.
• the modified PPO may essentially consist of an amino acid sequence according to SEQ ID NO: 127.
• the modified PPO may have an amino acid sequence having at least 30% sequence identity to SEQ ID NO: 128 (wherein the amino acid sequence includes the substitution S305L and V365M compared to SEQ ID NO:1).
• the modified PPO may have an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 128.
• the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 128.
• the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 128.
• the modified PPO may essentially consist of an amino acid sequence according to SEQ ID NO: 128.
• the modified PPO may have an amino acid sequence having at least 30% sequence identity to SEQ ID NO: 129 (wherein the amino acid sequence includes the substitution S305L, Y362F, and G404A compared to SEQ ID NO:1).
• the modified PPO may have an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 129.
• the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 129.
• the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 129.
• the modified PPO may essentially consist of an amino acid sequence according to SEQ ID NO: 129.
• the modified PPO may have an amino acid sequence having at least 30% sequence identity to SEQ ID NO: 130 (wherein the amino acid sequence includes the substitution S305L, Y361T, V365L, T431 R, and L479M compared to SEQ ID NO:1).
• the modified PPO may have an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 130.
• the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 130.
• the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 130.
• the modified PPO may essentially consist of an amino acid sequence according to SEQ ID NO: 130.
• the modified PPO may have an amino acid sequence having at least 30% sequence identity to SEQ ID NO: 131 (wherein the amino acid sequence includes the substitution S305L, Y426V, and L479M compared to SEQ ID NO:1).
• the modified PPO may have an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 131.
• the modified PPO may have an amino acid sequence having at least 85% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 131.
• the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 131.
• the modified PPO may essentially consist of an amino acid sequence according to SEQ ID NO: 131.
• the modified PPO may have an amino acid sequence having at least 30% sequence identity to SEQ ID NO: 132 (wherein the amino acid sequence includes the substitution Y426M, T461Q, and L479M compared to SEQ ID NO:1).
• the modified PPO may have an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 132.
• the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 132.
• the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 132.
• the modified PPO may essentially consist of an amino acid sequence according to SEQ ID NO: 132.
• the modified PPO may have an amino acid sequence having at least 30% sequence identity to SEQ ID NO: 133 (wherein the amino acid sequence includes the substitution S305L, Y361D, and V365M compared to SEQ ID NO:1).
• the modified PPO may have an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 133.
• the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 133.
• the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 133.
• the modified PPO may essentially consist of an amino acid sequence according to SEQ ID NO: 133.
• the modified PPO may have an amino acid sequence having at least 30% sequence identity to SEQ ID NO: 134 (wherein the amino acid sequence includes the substitution S305L, Y361C, Y362F, Y426M, and L479M compared to SEQ ID NO:1).
• the modified PPO may have an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 134.
• the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 134.
• the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 134.
• the modified PPO may essentially consist of an amino acid sequence according to SEQ ID NO: 134.
• the modified PPO may have an amino acid sequence having at least 30% sequence identity to SEQ ID NO: 135 (wherein the amino acid sequence includes the substitution Y361 D and V365L compared to SEQ ID NO:1).
• the modified PPO may have an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 135.
• the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 135.
• the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 135.
• the modified PPO may essentially consist of an amino acid sequence according to SEQ ID NO: 135.
• the modified PPO may have an amino acid sequence having at least 30% sequence identity to SEQ ID NO: 136 (wherein the amino acid sequence includes the substitution V365L, G404A, and L479N compared to SEQ ID NO:1).
• the modified PPO may have an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 136.
• the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 136.
• the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 136.
• the modified PPO may essentially consist of an amino acid sequence according to SEQ ID NO: 136.
• the modified PPO may have an amino acid sequence having at least 30% sequence identity to SEQ ID NO: 137 (wherein the amino acid sequence includes the substitution S305L, G404A, and L479N compared to SEQ ID NO:1).
• the modified PPO may have an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 137.
• the modified PPO may have an amino acid sequence having at least 85% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 137.
• the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 137.
• the modified PPO may essentially consist of an amino acid sequence according to SEQ ID NO: 137.
• the modified PPO may have an amino acid sequence having at least 30% sequence identity to SEQ ID NO: 138 (wherein the amino acid sequence includes the substitution T303L and Y424M compared to SEQ ID NO:2).
• the modified PPO may have an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 138.
• the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 138.
• the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 138.
• the modified PPO may essentially consist of an amino acid sequence according to SEQ ID NO: 138.
• the modified PPO may have an amino acid sequence having at least 30% sequence identity to SEQ ID NO: 139 (wherein the amino acid sequence includes the substitution T303L and Y424V compared to SEQ ID NO:2).
• the modified PPO may have an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 139.
• the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 139.
• the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 139.
• the modified PPO may essentially consist of an amino acid sequence according to SEQ ID NO: 139.
• the modified PPO may have an amino acid sequence having at least 30% sequence identity to SEQ ID NO: 140 (wherein the amino acid sequence includes the substitution V363L, Y424V, and L477N compared to SEQ ID NO:2).
• the modified PPO may have an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 140.
• the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 140.
• the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 140.
• the modified PPO may essentially consist of an amino acid sequence according to SEQ ID NO: 140.
• the modified PPO may have an amino acid sequence having at least 30% sequence identity to SEQ ID NO: 141 (wherein the amino acid sequence includes the substitution T303L, G402A, Y424V, T429R, and L477N compared to SEQ ID NO:2).
• the modified PPO may have an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 141.
• the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 141.
• the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 141.
• the modified PPO may essentially consist of an amino acid sequence according to SEQ ID NO: 141.
• the modified PPO may have an amino acid sequence having at least 30% sequence identity to SEQ ID NO: 142 (wherein the amino acid sequence includes the substitution T303L and V363M compared to SEQ ID NO:2).
• the modified PPO may have an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 142.
• the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 142.
• the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 142.
• the modified PPO may essentially consist of an amino acid sequence according to SEQ ID NO: 142.
• the modified PPO may have an amino acid sequence having at least 30% sequence identity to SEQ ID NO: 143 (wherein the amino acid sequence includes the substitution T303L, Y360F and G402A compared to SEQ ID NO:2).
• the modified PPO may have an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 143.
• the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 143.
• the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 143.
• the modified PPO may essentially consist of an amino acid sequence according to SEQ ID NO: 143.
• the modified PPO may have an amino acid sequence having at least 30% sequence identity to SEQ ID NO: 144 (wherein the amino acid sequence includes the substitution T303L, Y359T, V363L, T429R and L477M compared to SEQ ID NO:2).
• the modified PPO may have an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 144.
• the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 144.
• the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 144.
• the modified PPO may essentially consist of an amino acid sequence according to SEQ ID NO: 144.
• the modified PPO may have an amino acid sequence having at least 30% sequence identity to SEQ ID NO: 145 (wherein the amino acid sequence includes the substitution T303L, Y424V and L477M compared to SEQ ID NO:2).
• the modified PPO may have an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 145.
• the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 145.
• the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 145.
• the modified PPO may essentially consist of an amino acid sequence according to SEQ ID NO: 145.
• the modified PPO may have an amino acid sequence having at least 30% sequence identity to SEQ ID NO: 146 (wherein the amino acid sequence includes the substitution Y424M V459Q and L477M compared to SEQ ID NO:2).
• the modified PPO may have an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 146.
• the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 146.
• the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 146.
• the modified PPO may essentially consist of an amino acid sequence according to SEQ ID NO: 146.
• the modified PPO may have an amino acid sequence having at least 30% sequence identity to SEQ ID NO: 147 (wherein the amino acid sequence includes the substitution T303L, Y359D and V363M compared to SEQ ID NO:2).
• the modified PPO may have an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 147.
• the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 147.
• the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 147.
• the modified PPO may essentially consist of an amino acid sequence according to SEQ ID NO: 147.
• the modified PPO may have an amino acid sequence having at least 30% sequence identity to SEQ ID NO: 148 (wherein the amino acid sequence includes the substitution T303L, Y359C, Y360F, Y424M and L477M compared to SEQ ID NO:2).
• the modified PPO may have an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 148.
• the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 148.
• the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 148.
• the modified PPO may essentially consist of an amino acid sequence according to SEQ ID NO: 148.
• the modified PPO may have an amino acid sequence having at least 30% sequence identity to SEQ ID NO: 149 (wherein the amino acid sequence includes the substitution Y359D and V363L compared to SEQ ID NO:2).
• the modified PPO may have an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 149.
• the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 149.
• the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 149.
• the modified PPO may essentially consist of an amino acid sequence according to SEQ ID NO: 149.
• the modified PPO may have an amino acid sequence having at least 30% sequence identity to SEQ ID NO: 150 (wherein the amino acid sequence includes the substitution V363L, G402A and L477N compared to SEQ ID NO:2).
• the modified PPO may have an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 150.
• the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 150.
• the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 150.
• the modified PPO may essentially consist of an amino acid sequence according to SEQ ID NO: 150.
• the modified PPO may have an amino acid sequence having at least 30% sequence identity to SEQ ID NO: 151 (wherein the amino acid sequence includes the substitution T303L, G402A and L477N compared to SEQ ID NO:2).
• the modified PPO may have an amino acid sequence having at least 50% sequence identity to SEQ ID NO: 151.
• the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 151.
• the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 151.
• the modified PPO may essentially consist of an amino acid sequence according to SEQ ID NO: 151.
• a PPO enzyme which has resistance to a compound which inhibits PPO enzymatic activity, whether partial or complete may comprise an amino acid which has at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%
• a modified PPO enzyme which has resistance to a compound which inhibits PPO enzymatic activity may be a homologue of any of SEQ ID NOs: 1, 2, 4 - 151, 153 -302, or 305 - 336.
• “homologue” refers to a protein that is functionally equivalent, i.e. has the same enzymatic activity as an enzyme having an amino acid sequence according to SEQ ID NO 1, 2, 4 - 151 , 153 -302, or 305 - 336 (i.e. acts as a PPO enzyme as defined herein), but may have a limited number of amino acid substitutions, deletions, insertions or additions in the amino acid sequence.
• Homologues need not display the same level of enzymatic activity. Homologues may have lower sequences identities, for example, at least 20%, at least 25%, at least 30%, at least 35% or at least 40% or more sequence identity to a PPO enzyme identified herein, but are capable of carrying out the same enzymatic reaction.
• the invention therefore, includes any isoforms of PPO enzymes and their mutations as defined herein.
• Identity refers to the degree of sequence variation between two given nucleic acid or amino acid sequences.
• sequence comparison typically one sequence acts as a reference sequence to which test sequences are compared.
• sequence comparison algorithm When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
• Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math.2: 482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol.
• HSPs high scoring sequence pairs
• Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0).
• M forward score for a pair of matching residues
• N penalty score for mismatching residues; always ⁇ 0.
• a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached.
• the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
• W wordlength
• E expectation
• W wordlength
• E expectation
• BLOSUM62 scoring matrix see, Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)
• the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci.
• test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0.1. In one embodiment less than about 0.01, and in one embodiment less than about 0.001.
• EMBOSS Needle is available, e.g., from EMBL-EBI such as at the following website: ebi.ac.uk/Tools/psa/emboss_needle/ and as described in the following publication: “The EMBL-EBI search and sequence analysis tools APIs in 2019.” Madeira et al. Nucleic Acids Research, June 2019, 47(W1):W636-W641.
• the term “equivalent program” as used herein refers to any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by EMBOSS Needle
• a PPO enzyme encoded by a nucleic acid or a PPO enzyme of the invention may be a functional fragment of a PPO enzyme as described herein.
• a "functional fragment” refers to a protein fragment that retains protein function.
• a functional fragment of a PPO enzyme is a fragment, portion or part of a PPO protein that is still capable of catalysing the 6-electron oxidation of protoporphyrinogen-IX to form protoporphyrin-IX.
• the mutations defined herein may be located at a position ‘corresponding to’ an amino acid position listed in another PPO enzyme. It is possible to compare PPO polypeptides by sequence comparison and locating conserved regions that correspond to the amino acid positions listed.
• the term “equivalent amino acids” or “corresponding amino acids” refers to amino acids in a sequence of interest, which correspond to those amino acids of an identified reference sequence, typically herein the reference sequence is SEQ ID NO:1 or 2 for PPO enzymes.
• a region of equivalent or corresponding amino acids may be determined by aligning the amino acid sequences of the proteins from the different species, using an alignment program such as BLAST® or ClustalW. Note that the corresponding positions in a sequence of interest should be determined by comparison with a like for like reference sequence.
• the reference PPO sequence should also lack a targeting peptide.
• the reference sequence used herein may be SEQ ID NO: 1 , 2 (with transit peptides), 153 or 154 (without transit peptides). Any amino acid positions listed herein in relation to a PPO enzyme sequence comprising a targeting peptide still apply to a PPO enzyme sequence from the same organism without a targeting peptide.
• a “corresponding” amino acid position to a given SEQ ID NO is determined using Geneious as a global alignment with free end gaps having the following parameters: cost matrix Blossum 62, gap open penalty 12, gap extension penalty 3, refinement iterations 2; or an equivalent program thereof.
• a “corresponding” amino acid position to a given SEQ ID NO is determined using EMBOSS Needle default parameters: BLOSUM62; Gap Open 10, GAP EXTEND 0.5; END GAP OPEN 10 and END GAP EXTEND 0.5, or an equivalent program thereof.
• EMBOSS Needle default parameters BLOSUM62; Gap Open 10, GAP EXTEND 0.5; END GAP OPEN 10 and END GAP EXTEND 0.5, or an equivalent program thereof.
• the term “equivalent program” as used herein refers to any sequence comparison program that, for any two sequences in question, generates an alignment having identical corresponding nucleotide or amino acid residue matches when compared to the corresponding alignment generated by the program provided above.
• Mutations may include deletions or substitutions or combinations thereof.
• the mutations may be conservative or non-conservative amino acid substitutions.
• Constant amino acid substitutions refer to the interchangeability of residues having similar side chains, and thus typically involves substitution of an amino acid in a polypeptide with amino acids within the same or similar defined class of amino acids.
• an amino acid with an aliphatic side chain may be substituted with another aliphatic amino acid, e.g., alanine, valine, leucine, and isoleucine
• an amino acid with a hydroxyl side chain may be substituted with another amino acid with a hydroxyl side chain, e.g., serine and threonine
• amino acids having aromatic side chains may be substituted with another amino acid having an aromatic side chain, e.g., phenylalanine, tyrosine, tryptophan, and histidine
• an amino acid with a basic side chain may be substituted with another amino acid with a basic side chain, e.g., lysine and arginine
• an amino acid with an acidic side chain may be substituted with another amino acid with another
• Non-conservative substitution refers to substitution of an amino acid in a polypeptide with an amino acid with significantly differing side chain properties. Non-conservative substitutions may use amino acids between, rather than within, the defined groups and may affect (a) the structure of the peptide backbone in the area of the substitution (e.g., proline for glycine) (b) the charge or hydrophobicity, or (c) the bulk of the side chain.
• an exemplary non-conservative substitution can be an acidic amino acid substituted with a basic or aliphatic amino acid; an aromatic amino acid substituted with a small amino acid; and a hydrophilic amino acid substituted with a hydrophobic amino acid.
• “Deletion” refers to modification of a polypeptide by removal of one or more amino acids in comparison to a wild-type or control polypeptide.
• Deletions can comprise removal of 1 or more amino acids, 2 or more amino acids, or 3 or more amino acids of the polypeptide while retaining enzymatic activity.
• Deletions can comprise a continuous segment or can be discontinuous.
• the modified PPG enzyme may further comprise a transit peptide, suitably a mitochondrial and/or chloroplast transit peptide, suitably at the N or C terminus thereof. Suitably, at the N-terminus thereof.
• the transit peptide is a mitochondrial transit peptide.
• the transit peptide is a chloroplast transit peptide.
• the transit peptide comprises a chloroplast transit peptide and a mitochondrial transit peptide.
• mitochondrial or chloroplast transit peptides are present in wild type PPO enzymes.
• the modified PPO enzyme may comprise a native, endogenous mitochondrial or chloroplast transit peptide.
• the endogenous mitochondrial or chloroplast transit peptide may be replaced with a heterologous mitochondrial or chloroplast transit peptide, derived from an enzyme or a different PPO enzyme.
• the modified PPO enzyme may further be modified by the addition of a sequence.
• a sequence Suitably by the addition of a sequence to its N or C terminus.
• the sequence is a heterologous transit peptide.
• the heterologous transit peptide is added to the C terminus of the PPO enzyme.
• the heterologous transit peptide is added to the N terminus of the PPO enzyme
• the modified PPO enzyme comprises a heterologous transit peptide, suitably a heterologous mitochondrial or chloroplast transit peptide, suitably at the N terminus thereof.
• the transit peptide is a heterologous mitochondrial transit peptide.
• the transit peptide is a heterologous chloroplast transit peptide.
• the transit peptide is a heterologous dual targeting chloroplast/mitochondrial transit peptide.
• the heterologous transit peptide is added to the modified PPO enzyme, suitably such that the modified PPO enzyme is produced as a fusion protein with the heterologous transit peptide.
• the heterologous mitochondrial or chloroplast transit peptide is a plant mitochondrial or chloroplast transit peptide.
• the heterologous mitochondrial or chloroplast transit peptide may be derived from a heterologous PPO enzyme, suitably from a wild type heterologous PPO enzyme.
• the heterologous mitochondrial or chloroplast transit peptide may be derived from a heterologous plant PPO enzyme.
• transit peptides include those set forth in WO2017198859, US10745712 and US10563220, each of which in incorporated by reference in their entirety.
• Suitable transit peptides may be selected from: MELSLLRPTTQSLLPSFSKPNLRLNVYKPLRLRC (SEQ ID NO: 303); or MVAAAMATAPSAGVPPLRGTRGPARFRIRGVSVRC (SEQ ID NO: 304).
• the modified PPO enzyme may comprise a sequence according to any one of SEQ ID NOs: 153 to 302 or 321 to 336.
• the modified PPO enzyme may comprise a sequence having at least 30% sequence identity to a sequence according to any one of SEQ ID NOs: 153 to 302 or 321 to 336.
• such a modified PPO enzyme comprising a sequence according to any one of SEQ ID NOs: 153 to 302 or 321 to 336 may further comprise a transit peptide at the C or N terminus thereof, suitably which may be added thereto.
• a transit peptide at the C or N terminus thereof, suitably which may be added thereto.
• a mitochondrial transit peptide and/or a chloroplastic transit peptide may be added thereto.
• the modified PPO enzyme may comprise both a mitochondrial transit peptide and chloroplastic transit peptide.
• a chloroplastic and mitochondrial transit peptide as described herein.
• positions 362, 365, and 479 of SEQ ID NO: 1 (wild type Arabidopsis thaliana PPO including naturally occurring transit peptide) would respectively be denoted as positions 328, 331, and 445 of SEQ ID NO: 153 (wild type Arabidopsis thaliana PPO without transit peptide).
• positions 360, 363, and 477 of SEQ ID NO: 2 (wild type Setaria Italica PPO including naturally occurring transit peptide) would respectively be denoted as positions 325, 328 and 442 of SEQ ID NO: 154 (wild type Setaria Italica PPO without transit peptide).
• the modified PPO enzyme may comprise one or more additional elements, regions or domains that act to improve or help purification, expression, and/or stability of the modified PPO enzyme.
• the modified PPO enzymes provided herein may include one or more purification tags, detections tags and/or cleavage sites.
• a detection tag may be amino acid sequence that can be bound by a specific binding partner or emits a signal to allow the modified PPO enzyme to be detected or visualised.
• detection tags include V5 tag, Xpress tag, myc tag, 6XHis, GST, BioEase tag, capTEV tag, florescent tags (such as GFP), Lumio tag, HA tag, and FLAG tag.
• a purification tag may be an amino acid sequence that can be used to help purify the modified PPO enzymes when produced by recombinant methods.
• purification tags may allow for binding of the modified PPO enzyme to a resin or bead via the purification tag.
• purification tags include, 6XHis, GST, BioEase tag, capTEV tag.
• the modified PPO enzymes provided herein may include a 6XHis tag (i.e. a tag including 6 histidine residues).
• the modified PPO enzymes provided herein may include a 6XHis tag according to SEQ ID NO: 375.
• a cleavage sit refers to an amino acid sequence that may be cleaved or cut by a specific cleavage enzyme such as a protease.
• Cleavage sites may allow for simplified removal of tags that may be included. Cleavage may be carried our after purification and before transfer of a recombinant protein into a host.
• a cleavage site may be placed adjacent to a tag allowing cleavage of the tag using a protease enzyme.
• cleavage sites include Tobacco Etch Virus (TEV) site, enterokinase (EK) cleavage site, HRV3C site, and Factor Xa site.
• the modified PPO enzymes provided herein may include a TEV site.
• TEV site for example as TEV site according to SEQ ID NO: 376.
• the modified PPO enzymes provided herein include a His-Tag and TEV site as defined in SEQ ID NO: 377 or 378.
• the plants or parts thereof of the invention may further be modified to comprise an additional trait of interest.
• the additional trait of interest increases resistance to a different compound which inhibits a different plant metabolic process.
• the plants or parts thereof of the invention may further be modified to comprise increased resistance to a compound which inhibits a plant metabolic process other than the those involving a PPO enzyme.
• this may be regarded as ‘stacking’ of resistance.
• resistance to a compound which inhibits PPO enzymes may be stacked with resistance to another compound which inhibits a different enzyme or metabolic pathway, in the plants of the invention.
• the plant or part thereof exhibits a second compound-resistant trait.
• the plants or parts thereof of the invention may further be modified to comprise increased resistance to a compound which inhibits a different plant enzyme to PPO.
• the plants or parts thereof of the invention may further be modified to comprise increased resistance to a compound which inhibits an essential plant metabolic process. By inhibiting a plant metabolic process, this may mean inhibiting one or more enzymes of a plant metabolic process.
• the plants or parts thereof of the invention may further be modified to comprise increased resistance to a compound which is not a compound that targets PPO enzymes, but which targets a different essential plant enzyme and metabolic process.
• the plant or part thereof may comprise an additional modified enzyme, suitably which has been modified to increase its resistance to the compound, which inhibits a different plant enzyme to PPO.
• such compounds are herbicides.
• the plant or part thereof may comprise increased resistance to a herbicide which inhibits PPO enzymatic activity and increased resistance to another herbicide, suitably which inhibits a different plant metabolic process.
• the plant or part thereof exhibits a second herbicide-resistant trait.
• modified PPO enzymes and variants thereof provided herein can therefore be stacked with one or more additional modified enzymes which confers a desirable trait such as, for example, insect, disease or herbicide resistance or other desirable agronomic traits of interest including, but not limited to, traits associated with high oil content; traits associated with increase protein content, increased digestibility; balanced amino acid content; improved drought resistance, modified maturity and/or flowering time, and high energy content.
• a desirable trait such as, for example, insect, disease or herbicide resistance or other desirable agronomic traits of interest including, but not limited to, traits associated with high oil content; traits associated with increase protein content, increased digestibility; balanced amino acid content; improved drought resistance, modified maturity and/or flowering time, and high energy content.
• traits may refer to properties of both seed and non-seed plant tissues, or to food or feed prepared from plants or seeds having such traits.
• gene or trait “stacking” comprises combining desired genes or traits into one transgenic plant line.
• Stacking can include the introduction of transgenic traits of interest, genome edited traits of interest or native traits of interest.
• the additional polynucleotide can be introduced by a variety of approaches, as described herein in relation to polynucleotides encoding the modified PPO enzymes, including by transgenic means, by breeding, targeted integration via genome editing system, or by genome editing.
• plant breeders stack transgenic traits by making crosses between parents that each have a desired trait and then identifying offspring that have both of these desired traits (so-called “breeding stacks”).
• Another way to stack genes is by transferring two or more genes into the cell nucleus of a plant at the same time during transformation.
• the two or more genes may be transferred via distinct expression cassettes or via a common expression cassette.
• Another way to stack genes is by re-transforming a transgenic plant comprising a desired trait with another gene of interest conferring another desired trait to thereby provide a progeny transgenic plant comprising the combination of traits.
• Such methods can include, for example, random integration techniques or targeted integration via a gene editing system such as Crispr or meganucleases.
• gene stacking can be used to combine two different insect resistance traits, two different herbicide resistance traits, two different agronomic performance traits, an insect resistance trait with a disease resistance trait, a herbicide resistance trait (such as, for example, Bt11 ), or an agronomic performance trait, etc.
• a selectable marker in addition to a gene of interest would also be considered gene stacking.
• the offspring or progeny plant having the desired combination of traits is identified through the use of genetic markers or molecular markers including but not limited to SNPs, QTLs, primers or probes directed to desired trait- associated genes or transgenes, promoters, microRNAs, siRNAs, mRNAs, ds RNAs, transcriptional profiles, and methylation patterns.
• genetic markers or molecular markers including but not limited to SNPs, QTLs, primers or probes directed to desired trait- associated genes or transgenes, promoters, microRNAs, siRNAs, mRNAs, ds RNAs, transcriptional profiles, and methylation patterns.
• a polynucleotide or vector described herein can include an additional coding sequence for one or more polypeptides or double stranded RNA molecules (dsRNA) of interest for agronomic traits that primarily are of benefit to a seed company, grower or grain processor.
• dsRNA double stranded RNA molecules
• a polypeptide of interest can be any polypeptide encoded by a nucleotide sequence of interest, such as an enzyme.
• Non-limiting examples of polypeptides of interest that are suitable for production in plants include those resulting in agronomically important traits such as herbicide resistance (also sometimes referred to as “herbicide tolerance”), disease resistance, virus resistance, bacterial pathogen resistance, insect resistance, nematode resistance, or fungal resistance, such as modified enzymes conferring these traits.
• herbicide resistance also sometimes referred to as “herbicide tolerance”
• disease resistance also sometimes referred to as “herbicide tolerance”
• virus resistance also sometimes referred to as “herbicide tolerance”
• bacterial pathogen resistance e.g., insect resistance, nematode resistance
• fungal resistance e.g., U.S. Patent Nos. 5,569,823; 5,304,730; 5,495,071 ; 6,329,504; and 6,337,431.
• the polypeptide also can be one that increases plant vigor or yield (including traits that allow a plant to grow at different temperatures, soil conditions and levels of sunlight and precipitation), or one that allows identification of a plant exhibiting a trait of interest (e.g., a selectable marker, seed coat color, relative maturity group, etc.).
• a trait of interest e.g., a selectable marker, seed coat color, relative maturity group, etc.
• the additional polypeptide of interest to be stacked with the modified PPO enzyme provided herein may comprise any known polypeptide of interest or a modified enzyme in the art that has been modified to increase resistance to a known compound which inhibits a plant metabolic process.
• such compounds are herbicides.
• the additional polypeptide of interest or modified enzyme may provide additional herbicide resistance trait, suitably by providing increased herbicide resistance.
• the plant or part thereof may comprise a modified PPO enzyme which provides increased resistance to a herbicide which inhibits PPO and an additional enzyme which provides increased resistance to another herbicide, suitably which inhibits a different plant metabolic process.
• the polypeptides of interest stacked with the modified PPO enzymes provide herein confer resistance/tolerance to a herbicide that inhibits the growing point or meristem, such as an imidazalinone or a sulfonylurea may be suitable in some embodiments.
• a herbicide that inhibits the growing point or meristem such as an imidazalinone or a sulfonylurea
• Exemplary polynucleotides in this category code for mutant ALS and AHAS enzymes as described, e.g., in U.S. Patent Nos. 5,767,366 and 5,928,937.
• U.S. Patent Nos. 4,761 ,373 and 5,013,659 are directed to plants resistant to various imidazalinone or sulfonamide herbicides.
• 4,975,374 relates to plant cells and plants containing a nucleic acid encoding a modified glutamine synthetase (GS) enzyme resistant to inhibition by herbicides that are known to inhibit GS, e.g., phosphinothricin and methionine sulfoximine.
• GS glutamine synthetase
• U.S. Patent No. 5,162,602 discloses plants resistant to inhibition by cyclohexanedione and aryloxyphenoxypropanoic acid herbicides. The resistance is conferred by a modified acetyl coenzyme A carboxylase (ACCase) enzyme. Additional herbicide tolerant traits that confer tolerance to ACCase inhibitors can be found in WO2014144951, WO2017138986, WO2018205995, WG2021088601, WO2011028836, and WO2011028833.
• the polypeptide of interest stacked with the modified PPO enzymes provided herein confer resistance to glyphosate are also suitable for the disclosure. See, e.g., U.S. Patent No. 4,940,835 and U.S. Patent No. 4,769,061.
• U.S. Patent No. 5,554,798 discloses transgenic glyphosate resistant maize plants, which resistance is conferred by a modified 5-enolpyruvyl-3-phosphoshikimate (EPSP) synthase gene.
• EPP 5-enolpyruvyl-3-phosphoshikimate
• Polynucleotides coding for resistance to phosphono compounds such as glufosinate ammonium or phosphinothricin, and pyridinoxy or phenoxy propionic acids and cyclohexones are also suitable. See, European Patent Application No. 0242 246. See also, U.S. Patent Nos. 5,879,903, 5,276,268 and 5,561 ,236.
• Additional herbicide tolerant traits that can be stacked with the modified PPOs disclosed herein include, PPO tolerant traits including, for example, one or more PPO trait set forth in US20190062777, US10370677, US11124803, WO2017217793, WO2020251313, US10392630, US10378023 , WO2016099153, WO2019117579, WO2019117578, and US10100329, each of which is herein incorporated by reference in their entirety.
• HPPD tolerant traits include: W02009144079, US8642748, EP2453012, W02013026740, US9078446, US10793872, US10508089, US10400249, US10597674, WO2018119364 , WO2018119361, US11180770, US20200157086, US20210147866, US11279944, US202000331866, WO2019227036, WO2019227028, WO2022115296, WO2011068567, and those disclosed in Maeda et al. (2019) Science, vol 365, issue 6451, pp 393-396, each of which is herein incorporated by reference in their entirety.
• ACCase tolerant traits include: US20120284812, US20120284853, US20160108423, US20160244780, US20160264990, US20170275645, US20210153448, US10696975B2, US10370678, CN109082416 , US10694694, US20170265469, US20170231225, each of which is herein incorporated by reference.
• Dicamba tolerant traits include, for example, RE45048 or US7884262.
• Various traits the confer tolerance to AOPP herbicides, phenoxy acid herbicides and/or pyridinyloxy acid herbicides include, for example, US10174337, US8278505, W005107437,
• WO1 1022469, US10023874, and US2019241903 (and other traits therein), each of which is herein incorporated by reference.
• Additional herbicides tolerant traits of interest for stacking include glucosyl transferase polypeptides as set forth in 2018213022 or Solanesyl Diphosphate Synthase polypeptides as set forth in W02020236790, or a BIO3-BIO1 and/or BioA enzyme as described in European patent application EP23154964.3, each of which is herein incorporated by reference in their entirety.
• suitable polynucleotides include those which confer resistance to herbicides that inhibit photosynthesis, such as a triazine and a benzonitrile (nitrilase) See, U.S. Patent No. 4,810,648.
• Additional suitable polynucleotides coding for herbicide resistance include those coding for resistance to 2,2-dichloropropionic acid, sethoxydim, haloxyfop, imidazolinone herbicides, sulfonylurea herbicides, triazolopyrimidine herbicides, s-triazine herbicides and bromoxynil.
• adverse environmental conditions abiotic stresses
• alterations in plant architecture or development including changes in developmental timing. See, e.g., U.S. Patent Publication No. 2001/0016956 and U.S. Patent No. 6,084,155.
• Disease resistance proteins such as enzymes that increase resistance to various plant disease including rust, include, but are not limited to, one or more of the various resistance genes set forth in: W02019103918; WO202100878; WO2021022022; WO2021260673; WO2022173659; WO2022159341 ; WO2021154632A1, W02021022026, W02021022101, US20220135997; US10842097; or WO2022140257; each of which is incorporated by reference in their entirety.
• the modified PPO enzymes described herein or active variant or fragments thereof is stacked with a native trait that confers disease resistance.
• the various intervals, locus or resistance genes as set forth in W02009079729, US9091681, W02010009404, WO2017222827, W02021000878, W02021022026, WO2021022101 , WO2021154632, WO2022173659, (each of which is incorporated by reference in their entirety) can used to introduce a trait of interest.
• Disease resistance proteins and/or native traits that increase resistance to various plant diseases including NCLB include, for example, US8921646 , US2021000059, US10858668, US20200199610, W02022/013268, W02022/013268, US9040772, US10897862, EP3839073, each of which is herein incorporated by reference.
• Additional suitable polynucleotides include those coding for insecticidal polypeptides such as insecticidal enzymes. These polypeptides may be produced in amounts sufficient to control, for example, insect pests (i.e. , insect controlling amounts). It is recognized that the amount of production of an insecticidal polypeptide in a plant necessary to control insects or other pests may vary depending upon the cultivar, type of pest, environmental factors and the like. Polynucleotides useful for additional insect or pest resistance include, for example, those that encode toxins identified in Bacillus organisms.
• Bt insecticidal proteins include the Cry proteins such as CrylAa, CrylAb, CrylAc, Cry1B, Cry1C, Cry1D, Cryl Ea, Cryl Fa, Cry3A, Cry9A, Cry9B, Cry9C, and the like, as well as vegetative insecticidal proteins such as Vip1 , Vip2, Vip3, and the like.
• an additional polypeptide is an insecticidal polypeptide, such as an enzyme, derived from a non-Bt source, including without limitation, an alpha-amylase, a peroxidase, a cholesterol oxidase, a patatin, a protease, a protease inhibitor, a urease, an alpha-amylase inhibitor, a pore-forming protein, a chitinase, a lectin, an engineered antibody or antibody fragment, a Bacillus cereus insecticidal protein, a Xenorhabdus spp. (such as X. nematophila orX. bovienii) insecticidal protein, a Photorhabdus spp.
• an enzyme derived from a non-Bt source, including without limitation, an alpha-amylase, a peroxidase, a cholesterol oxidase, a patatin, a protease, a protease inhibitor, a
• insecticidal protein such as P. luminescens or P. asymobiotica
• Brevibacillus spp. such as B. laterosporous insecticidal protein
• Lysinibacillus spp. such as L. sphearicus
• Chromobacterium spp. such as C. subtsugae or C. foundedae
• Yersinia spp. such as Y. entomophaga
• insecticidal protein such as P. propylaea
• Clostridium spp. such as C. bifermentans
• insecticidal protein such as C. bifermentans
• Pseudomonas spp. such as P. fluorescens
• lignin such as P. fluorescens
• the additional polypeptide or enzyme is a resistance protein such as an enzyme conferring enhanced pathogen resistance, such as enhanced resistance to any one of the following pathogens: soy cyst nematode, bacterial pustule, root knot nematode, frog eye leaf spot, phytopthora, brown stem rot, nematode, Asian Soybean Rust, smut, Golovinomyces cichoracearum, Erysiphe cichoracearum, Blumeria graminis, Podosphaera xanthii, Sphaerotheca fuliginea, Pythium ultimum, Uncinula necator, Mycosphaerella pinodes, Magnaporthe grisea, Bipolaris oryzae, Magnaporthe grisea, Rhizoctonia solani, Phytophthora sojae, Schizaphis graminum, Bemisia tabaci, Rhopalosiphum maidis, Derocera
• Exemplary polynucleotides encoding proteins that confer increased pathogen resistance that may be stacked with the modified PPO enzymes of the invention include polynucleotides encoding proteins such as enzymes that confer increased ASR resistance as described in US Patent publication Nos. US 20200354739 and PCT Publications Nos. W02019103918, WO2021154632A1, W02021022022, W02021022026, W02021022101, WO2021260673, and WO2021263249, each of which is incorporated by reference in its entirety.
• Polypeptides that are suitable for production in plants further include those that improve or otherwise facilitate the conversion of harvested plants or plant parts into a commercially useful product, including, for example, increased or altered carbohydrate content or distribution, improved fermentation properties, increased oil content, increased protein content, improved digestibility, and increased nutraceutical content, e.g., increased phytosterol content, increased tocopherol content, increased stand content or increased vitamin content.
• Polypeptides of interest also include, for example, those resulting in or contributing to a reduced content of an unwanted component in a harvested crop, e.g., phytic acid, or sugar degrading enzymes.
• the polypeptide of interest can directly or indirectly contribute to the existence of a trait of interest (e.g., increasing cellulose degradation by the use of a heterologous cellulase enzyme). Any such polypeptides may be stacked with the modified PPO enzymes of the invention.
• the polypeptide contributes to improved digestibility for food or feed.
• Xylanases are hemicellulolytic enzymes that improve the breakdown of plant cell walls, which leads to better utilization of the plant nutrients by an animal. This leads to improved growth rate and feed conversion. Also, the viscosity of the feeds containing xylan can be reduced.
• Heterologous production of xylanases in plant cells also can facilitate lignocellulosic conversion to fermentable sugars in industrial processing.
• Numerous xylanases from fungal and bacterial microorganisms have been identified and characterized (see, e.g., U.S. Patent No. 5,437,992; Coughlin et al. (1993) “Proceedings of the Second TRICEL Symposium on Trichoderma reesei Cellulases and Other Hydrolases” Espoo; Souminen and Reinikainen, eds. (1993) Foundation for Biotechnical and Industrial Fermentation Research 8:125-135; U.S. Patent Publication No. 2005/0208178; and PCT Publication No.
• WO 03/16654 WO 03/16654
• three specific xylanases XYL-I, XYL-II, and XYL-III
• T. reesei Teenkanen et al. (1992) Enzyme Microb. Technol. 14:566; Torronen et al. (1992) Bio/Technology 10:1461; and Xu et al. (1998) Appl. Microbiol. Biotechnol. 49:718).
• Any such polypeptides may be stacked with the modified PPO enzymes of the invention.
• a polypeptide useful for the disclosure can be a polysaccharide degrading enzyme. Plants of this disclosure producing such an enzyme may be useful for generating, for example, fermentation feedstocks for bioprocessing.
• enzymes useful for a fermentation process include alpha amylases, proteases, pullulanases, isoamylases, cellulases, hemicellulases, xylanases, cyclodextrin glycotransferases, lipases, phytases, laccases, oxidases, esterases, cutinases, granular starch hydrolyzing enzyme and other glucoamylases.
• Polysaccharide-degrading enzymes include: starch degrading enzymes such as a-amylases (EC 3.2.1.1), glucuronidases (E.C. 3.2.1.131); exo-1,4-a-D glucanases such as amyloglucosidases and glucoamylase (EC 3.2.1.3), p-amylases (EC 3.2.1.2), a-glucosidases (EC 3.2.1.20), and other exo-amylases; starch debranching enzymes, such as a) isoamylase (EC 3.2.1.68), pullulanase (EC 3.2.1.41), and the like; b) cellulases such as exo-1,4-3-cellobiohydrolase (EC 3.2.1.91), exo-1,3-p-D-glucanase (EC 3.2.1.39), p-glucosidase (EC 3.2.1.21); c) L-arabinases,
• the a-amylase is the synthetic a-amylase, Amy797E, described is US Patent No. 8,093,453, herein incorporated by reference in its entirety. Any such polypeptides may be stacked with the modified PPO enzymes of the invention.
• proteases such as fungal and bacterial proteases.
• Fungal proteases include, but are not limited to, those obtained from Aspergillus, Trichoderma, Mucor and Rhizopus, such as A. niger, A. awamori, A. oryzae and M. miehei.
• the polypeptides of this disclosure can be cellobiohydrolase (CBH) enzymes (EC 3.2.1.91).
• the cellobiohydrolase enzyme can be CBH1 or CBH2. Any such polypeptides may be stacked with the modified PPO enzymes of the invention.
• hemicellulases such as mannases and arabinofuranosidases (EC 3.2.1.55); ligninases; lipases (e.g., E.C. 3.1.1.3), glucose oxidases, pectinases, xylanases, transglucosidases, alpha 1 ,6 glucosidases (e.g., E.C. 3.2.1.20); esterases such as ferulic acid esterase (EC 3.1.1.73) and acetyl xylan esterases (EC 3.1.1.72); and cutinases (e.g. E.C. 3.1.1.74). Any such polypeptides may be stacked with the modified PPO enzymes of the invention.
• the modified PPO enzymes described herein may be stacked with polynucleotides that encode polypeptides which increase protein content, and/or alter seed composition and/or fatty acid content.
• sequences include, but are not limited to, sequences disclosed in PCT Appl. No. PCT/CN2022/075977 and PCT Appl. No.
• the modified PPO enzymes described herein are stacked with a modified BIO3-BIO1 and/or BioA enzyme and/or a modified biotin synthesis pathway.
• the plant or part thereof described herein comprises modified BIO3-BIO1 and/or BioA enzyme.
• the additional modified enzyme is a modified BIO3-BIO1 and/or BioA enzyme.
• the plant or part thereof may further be modified to comprise a BIO3-BIO1 and/or BioA enzyme that provides the plant or part thereof with increased resistance to a compound which inhibits a BIO3-BIO1 and/or BioA enzyme and/or the biotin synthesis pathway relative to an unmodified plant.
• the plant or part thereof may be modified to comprise a BIO3-BIO1 and/or BioA enzyme having one or more modifications which provide the plant or part thereof with a BIO3-BIO1 and/or BioA resistance trait, suitably by providing increased resistance to a compound which inhibits a biotin synthesis and ergo BIO3-BIO1 and/or BioA enzyme relative to an unmodified plant.
• the plant or part thereof may comprise a recombinant polynucleotide encoding a BIO3-BIO1 and/or BioA enzyme having one or more modifications.
• the or each modification provides the plant with an increased resistance to a compound which inhibits the BIO3-BIO1 and/or BioA enzyme relative to an unmodified plant.
• the or each modification provides the plant with an increased resistance to a herbicide which inhibits the BIO3-BIO1 and/or BioA enzymes and the biotin synthesis pathway.
• the plant or part thereof may further be modified to comprise a BIO3-BIO1 and/or BioA enzyme as described in European patent application EP23154964.3, which is incorporated herein by reference.
• the plants of the present invention include both non-transgenic plants and transgenic plants.
• non-transgenic plant is intended to mean a plant lacking recombinant DNA in its genome, but containing a mutant nucleic acid molecule in the plant cell genome which has been mutated through human intervention using mutagenic techniques, such as chemical mutagenesis, gene editing or by those methods provided herein.
• Non-transgenic plants may encompass those plants having mutant or modified sequences as a result of natural processes, such as plants including spontaneous PPO enzymes that provide the desired resistance to compounds that inhibit PPO enzymatic activity or by the use of gene editing techniques.
• the non-transgenic plant comprises a modified PPO enzyme that has been altered through gene editing to comprise at least one or more of the modifications disclosed herein. Such gene editing modifications will increase the resistance of the plant to the herbicide of interest.
• transgenic plant is intended to mean a plant comprising recombinant DNA in its genome.
• recombinant when referring to nucleic acid or polypeptide, indicates that such material has been conceived of and created in the laboratory using one or more of the techniques of biotechnology, protein design, or protein engineering, such as molecular biology, protein biochemistry, bacterial transformation, plant transformation, site- directed mutagenesis, directed evolution using random mutagenesis, genome editing, gene editing, gene cloning, DNA ligation, DNA synthesis, protein synthesis, and DNA shuffling.
• a recombinant technique such as by polynucleotide restriction and ligation, by polynucleotide overlapextension, or by genomic insertion or transformation.
• a gene sequence open reading frame is recombinant if that nucleotide sequence has been removed from its 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.
• Such a transgenic plant can be produced by introducing recombinant DNA into the genome of the plant.
• progeny of the plant can also comprise the recombinant DNA.
• a progeny plant that comprises at least a portion of the recombinant DNA of at least one progenitor transgenic plant is also a transgenic plant.
• heterologous in reference to a polypeptide or polynucleotide sequence is a sequence that originates, for example, from a cell or an organism from a foreign species. Alternatively, if the sequence originates from the same species, it is derived from a cell or organism having a different genetic background; or if from the same genetic background, it is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. As such, heterologous sequences are in a configuration not found in nature.
• spontaneous mutant refers to mutants or variants that arise from the parent strain without the intentional use of mutagens i.e. they are considered as not genetically modified (non-GMO). Spontaneous mutants in respect of plants may also be known as sports, breaks, or chimeras.
• the plant or part thereof of the invention is transgenic.
• the plant or plant part thereof of the invention is non-transgenic and comprises a gene edit that increases the plant’s or plant part’s tolerance to a herbicide of interest.
• the plant or part thereof comprises a recombinant polynucleotide encoding a modified PPO enzyme.
• the plant or part thereof comprises a polynucleotide encoding a modified or mutated PPO enzyme.
• the polynucleotide encoding the modified PPO enzyme may comprise one or more modifications.
• the polynucleotide may be operable to express a modified PPO enzyme having one or more modifications or mutations.
• the expression of said polynucleotide provides or confers to the plant or part thereof increased resistance to a compound which inhibits PPO enzymatic activity.
• Suitable PPO modifications are defined herein and can include any one of SEQ ID NO: 1, 2, 4 -151 , 153-302 and 305-336 or active variants thereof as described elsewhere herein.
• PPO modifications are defined herein and can include any one of SEQ ID NO: 37- 39, 58, 59, 97 - 99, 118, 119, 125 - 137, 139 - 151, 188 - 190, 209, 210, 248 - 250, 269, 270, 277 - 288 or 291 - 302 or active variants thereof as described elsewhere herein.
• the modified or mutated PPO enzyme is encoded by a recombinant polynucleotide stably integrated into the plant's genome.
• the modified or mutated PPO enzyme is encoded by a polynucleotide, suitably a gene, comprising a non-transgenic modification, such as an edit, within the genome of the plant.
• the plant or part thereof may be modified to comprise a modified PPO enzyme wherein the modified PPO enzyme is overexpressed and wherein the modified PPO enzyme comprises one or more mutations provided herein which provide the plant or part thereof with increased resistance to a compound which inhibits PPO enzymatic activity relative to an unmodified or control PPO enzyme or plant.
• Suitable modified PPOs can include any one of SEQ ID NO: 1 , 2, 4 -151, 153-302 and 305-336.
• modified PPOs can include any one of SEQ ID NO: 37- 39, 58, 59, 97 - 99, 118, 119, 125 - 137, 139 - 151 , 188 - 190, 209, 210, 248 - 250, 269, 270, 277 - 288 or 291 - 302 or active variants thereof as described elsewhere herein.
• the plant has been transformed with said recombinant polynucleotide. Suitable means of transformation are described hereinbelow.
• the transformed parts of plants, transformed plant cells or a transformed plant protoplasts as described herein may be regenerated to produce a modified plant as described herein.
• the cells can be cultured, then regenerated into whole plants.
• “Regeneration” refers to the process of growing a plant from a plant cell (for example, plant protoplast or explant). Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker that has been introduced together with the desired nucleotide sequences. Choice of methodology for the regeneration step is not critical.
• the present invention may be for use with any plant species and the progeny thereof, including, but not limited to, monocots and dicots.
• plant species of interest include, but are not limited to, corn or maize (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea), including 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 annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum, T.
• Brassica sp. e.g., B. napus, B. rapa, B. juncea
• Turgidum ssp. durum soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solarium tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea 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
• plants of the present invention are crop plants (for example, sunflower, Brassica sp., cotton, sugar, beet, soybean, peanut, alfalfa, safflower, tobacco, corn, rice, wheat, rye, barley triticale, sorghum, millet, etc.).
• crop plants for example, sunflower, Brassica sp., cotton, sugar, beet, soybean, peanut, alfalfa, safflower, tobacco, corn, rice, wheat, rye, barley triticale, sorghum, millet, etc.
• plants of the present invention may also include various types of cover crop plants.
• cover crop plants include, but are not limited to, Brassica sp. (e.g., B. carinata, B. napus, B. rapa, B. hirta, B. Juncea, B. nigra), radish (Raphanus sativus), Camelina sp. (e.g., C. sativa), pennycress (Thlaspi arvense), clover (Trifolium sp., e.g., T. incarnatum, T. pratense, T. repens, T. subterraneum), field peas (Pisum sativum), Vicia sp.
• Brassica sp. e.g., B. carinata, B. napus, B. rapa, B. hirta, B. Juncea, B. nigra
• radish Raphanus sativus
• progeny and “progeny plant” refer to a plant generated from a vegetative or sexual reproduction from one or more parent plants.
• a progeny plant may be obtained by cloning or selfing a single parent plant, or by crossing two parental plants.
• plant is intended to mean a plant at any developmental stage, as well as any part or parts of a plant that may be attached to or separate from a whole intact plant.
• parts of a plant include, but are not limited to, seed, organs, tissues, and cells of a plant including, plant calli, plant clumps, plant protoplasts and plant cell tissue cultures from which plants can be regenerated.
• Examples of particular plant parts include a stem, a leaf, a root, an inflorescence, a flower, a floret, a fruit, a pedicle, a peduncle, a stamen, an anther, a stigma, a style, an ovary, a petal, a sepal, a carpel, a root tip, a root cap, a root hair, a leaf hair, a seed hair, a pollen grain, a microspore, an embryos, an ovule, a cotyledon, a hypocotyl, an epicotyl, xylem, phloem, parenchyma, endosperm, a companion cell, a guard cell, and any other known organs, tissues, and cells of a plant.
• a seed is a plant part.
• a "plant cell” is a structural and physiological unit of a plant, comprising a protoplast and a cell wall.
• the plant cell may be in the form of an isolated single cell or a cultured cell, or as a part of a higher organized unit such as, for example, plant tissue, a plant organ, or a whole plant.
• a "plant part” is a distinct and visibly structured and differentiated part of a plant such as a root, stem, leaf, flower bud, or embryo.
• the plants, progeny thereof or parts thereof of the invention express at least one of a modified PPO enzyme as disclosed herein.
• Expression of the enzymes, polynucleotides encoding said enzymes, or expression vectors of the invention provides a plant that is at least partially resistant to compounds which inhibit PPO enzymatic activity, such as those herbicides described herein.
• the plants, progeny thereof or parts thereof of the invention have increased resistance to a herbicide which inhibits PPO enzymatic activity.
• the increase in resistance may be determined by comparison to a wild-type or control plant as described herein.
• a plant that has not been modified to include or express the modified PPO enzymes, polynucleotides encoding them, or expression vectors of the invention may be determined by comparison to a wild-type or control plant as described herein.
• Such methods may include (i) a method of producing a modified plant or part thereof having an increased resistance to a compound which inhibits PPO enzymatic activity, and (ii) a method of increasing the resistance of a plant or part thereof to a compound which inhibits PPO enzymatic activity.
• either method comprises a step of modifying the plant or part thereof to comprise a modified PPO enzyme that provides the increased resistance.
• Suitably modifying the plant may comprise providing the plant or part thereof with a modified PPO enzyme having one or more modifications, wherein the or each modification provides the increased resistance.
• Such steps may comprise providing the plant or part thereof with a recombinant polynucleotide encoding a modified PPO enzyme as described herein.
• Suitably providing may comprise introducing the recombinant polynucleotide encoding a modified PPO enzyme as provided herein into the plant or part thereof, or introducing the modified PPO protein into the plant or part thereof.
• the modified PPO enzyme may be introduced into a plant or part thereof by introducing a polynucleotide of the invention which encodes a modified PPO enzyme.
• plants of the invention may be referred to as modified or transgenic plants.
• introducing the modified PPO enzyme into a plant or part thereof may be carried out by transforming the plant or part thereof with a recombinant polynucleotide encoding a modified PPO enzyme.
• the recombinant polynucleotide may further encode a transit peptide, such as a mitochondrial or chloroplast transit peptide.
• a transit peptide such as a mitochondrial or chloroplast transit peptide.
• the recombinant polynucleotide may comprise a chimeric polynucleotide encoding a modified PPO as described above and a transit peptide operably linked thereto, suitably a mitochondrial or chloroplast transit peptide operably linked thereto. Suitable transit peptides are described elsewhere herein.
• the recombinant polynucleotide may be part of an expression construct, or comprised in an expression vector.
• the expression construct or vector may comprise one or more expression elements such as a promoter, as is described elsewhere herein.
• the methods may comprise providing, introducing or transforming the plant or part thereof with an expression construct or expression vector comprising a polynucleotide encoding a modified PPO enzyme, suitably which may be a recombinant polynucleotide.
• the methods may further comprise a step of inducing expression of the recombinant polynucleotide to produce the modified PPO enzyme provided herein in the plant or part thereof.
• inducing expression of the polynucleotide may comprise contacting the plant or part thereof with an inducer.
• the polynucleotide encoding the modified PPO enzyme may be under the control of an inducible promoter, suitably therefore the expression construct or vector may comprise an inducible promoter operably linked to the polynucleotide encoding the modified PPO enzyme. Suitable inducible promoters and inducers are well known in the art.
• a plant may be modified by in situ editing of the endogenous genetic material in order to provide a gene that expresses a modified PPO enzyme which provides increased resistance to compounds which inhibit PPO enzymatic activity.
• a plant may be provided with the components of a gene editing system for modifying an endogenous gene sequence of the plant encoding PPO enzyme at one or more positions to produce a modified gene sequence encoding a modified PPO enzyme which provides increased resistance to a compound which PPO enzymatic activity.
• the plant may be transformed with one or more polynucleotides encoding a gene editing system for modifying an endogenous gene sequence of the plant encoding PPO enzyme at one or more positions to produce a modified gene sequence encoding modified PPO enzyme which provides increased resistance to a compound which inhibits PPO enzymatic activity.
• An endogenous PPO encoding gene sequence may be edited in situ by way of gene editing techniques in order to provide a modified PPO enzyme that is at least partially resistant to compound that inhibits PPO enzymatic activity, such as those described herein and as such a modified plant as described herein.
• Such genome editing and/or mutagenesis technologies are well known in the art.
• introduction may be accomplished by any manner known in the art, including: introgression, transgenic, or site-directed nucleases (SDN).
• SDN site-directed nucleases
• the modification to the gene sequence is introduced by way of site- directed nuclease (SDN).
• the SDN is selected from: meganuclease, zinc finger, transcription activator- like effector nucleases system (TALEN) or Clustered Regularly Interspaced Short Palindromic Repeats system (CRISPR) system.
• SDN is also referred to as “genome editing”, or genome editing with engineered nucleases (GEEN).
• GEEN genome editing with engineered nucleases
• SDN may comprises techniques such as: Meganucleases, Zinc finger nucleases (ZFNs), Transcription Activator-Like Effector-based Nucleases (TALEN) (Feng et al. 2013 Cell Res. 23, 1229-1232, Sander & Joung Nat. Biotechnol. 32, 347-3552014), and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-Cas) system.
• CRISPR-Cas Clustered Regularly Interspaced Short Palindromic Repeats
• Gene editing may also be achieved by SDN-2.
• SDN-2 is similar to SDN, but also provides a small nucleotide template complementary to the area of the break. The template contains one or more sequences modifications to the genomic DNA which are incorporated to create the mutation to the target gene.
• the gene editing system may include a CRISPR-Cas system.
• guide RNA generally refers to an RNA molecule (or a group of RNA molecules collectively) that can bind to a CRISPR system effector, such as a Cas or a Cpf 1 protein, and aid in targeting the Cas or Cpfl protein to a specific location within a target polynucleotide (e.g., a DNA).
• a guide RNA of the invention can be an engineered, single RNA molecule (sgRNA), where for example the sgRNA comprises a crRNA segment and optionally a tracrRNA segment.
• a guide RNA of the invention can also be a dual-guide system, where the crRNA and tracrRNA molecules are physically distinct molecules which then interact to form a duplex for recruitment of a CRISPR system effector, such as Cas9, and for targeting of that protein to the target polynucleotide.
• a CRISPR system effector such as Cas9
• crRNA refers to an RNA molecule or to a portion of an RNA molecule that includes a polynucleotide targeting guide sequence, a stem sequence involved in protein-binding, and, optionally, a 3'-overhang sequence.
• the polynucleotide targeting guide sequence is a nucleic acid sequence that is complementary to a sequence in a target DNA (for example a gene encoding a PPG enzyme). This polynucleotide targeting guide sequence is also referred to as the “protospacer”.
• the polynucleotide targeting guide sequence of a crRNA molecule interacts with a target DNA in a sequence-specific manner via hybridization (i.e., base pairing).
• the nucleotide sequence of the polynucleotide targeting guide sequence of the crRNA molecule may vary and determines the location within the target DNA that the guide RNA and the target DNA will interact.
• the polynucleotide targeting guide sequence of a crRNA molecule can be modified (e.g., by genetic engineering) to hybridize to any desired sequence within a target DNA.
• the polynucleotide targeting guide sequence of a crRNA molecule of the invention can have a length from about 12 nucleotides to about 100 nucleotides.
• the polynucleotide targeting guide sequence of a crRNA can have a length of from about 12 nucleotides (nt) to about 80 nt, from about 12 nt to about 50 nt, from about 12 nt to about 40 nt, from about 12 nt to about 30 nt, from about 12 nt to about 25 nt, from about 12 nt to about 20 nt, or from about 12 nt to about 19 nt.
• the polynucleotide targeting guide sequence of a crRNA can have a length of from about 17 nt to about 27 nts.
• the polynucleotide targeting guide sequence of a crRNA can have a length of from about 19 nt to about 20 nt, from about 19 nt to about 25 nt, from about 19 nt to about 30 nt, from about 19 nt to about 35 nt, from about 19 nt to about 40 nt, from about 19 nt to about 45 nt, from about 19 nt to about 50 nt, from about 19 nt to about 60 nt, from about 19 nt to about 70 nt, from about 19 nt to about 80 nt, from about 19 nt to about 90 nt, from about 19 nt to about 100 nt, from about 20 nt to about 25 nt, from about 20 nt to about 30 nt, from about 20 nt to about 35 nt, from about 20 nt to about 40 nt, from about 20 nt to about 45 nt, from about 20 nt to about 50
• the nucleotide sequence of the polynucleotide targeting guide sequence of a crRNA can have a length at least about 12 nt. In some embodiments, the polynucleotide targeting guide sequence of a crRNA is 20 nucleotides in length. In some embodiments, the polynucleotide targeting guide sequence of a crRNA is 19 nucleotides in length.
• the present invention also provides a guide RNA comprising an engineered crRNA, wherein the crRNA comprises a bait RNA segment capable of hybridizing to a genomic target sequence.
• This engineered crRNA may be a physically distinct molecule, as in a dual-guide system.
• tracrRNA refers to an RNA molecule or portion thereof that includes a protein-binding segment (e.g., the protein-binding segment is capable of interacting with a CRISPR-associated protein, such as a Cas9).
• the present invention also provides a guide RNA comprising an engineered tracrRNA, wherein the tracrRNA further comprises a bait RNA segment that is capable of binding to a donor DNA molecule.
• the engineered tracrRNA may be a physically distinct molecule, as in a dualguide system, or may be a segment of a sgRNA molecule.
• the guide RNA does not contain a tracrRNA, as it is known in the art that some CRISPR-associated nucleases, such as Cpfl (also known as Casl2a), do not require a tracrRNA for its RNA-mediated endonuclease activity (Qi et al., (2013), Cell, 152: 1173-1183; Zetsche et al., (2015), Cell 163: 759-771).
• Cpfl also known as Casl2a
• Such a guide RNA of the invention may comprise a crRNA with the bait RNA operably linked at the 5’ or 3’ end of the crRNA.
• Cpfl also has RNase activity on its cognate pre-crRNA (Fonfara et al., (2016), Nature, doi.org/10.1038/naturel7945).
• a guide RNA of the invention may comprise multiple crRNAs which the Cpfl possesses to mature crRNAs. Each of these crRNAs may be operably linked to a bait RNA. At least one of these crRNAs may be operably linked to a bait RNA.
• the bait RNA may be specific to a sequence of interest (SOI), or it may be a “universal” bait, which has a corresponding “universal” prey sequence on the donor DNA molecule.
• the present invention also provides a polynucleotide comprising a sequence encoding a guide RNA of the invention.
• the polynucleotide may be a DNA or an RNA molecule.
• the polynucleotide molecule may be circularized or linear.
• the polynucleotide may be single stranded, partially double-stranded, or double-stranded.
• the polynucleotide may be complexed with at least one polypeptide.
• the polypeptide may have a nucleic acid recognition or nucleic acid binding domain.
• the polypeptide may be a shuttle for mediating delivery of, for example, a polynucleotide of the invention, a nuclease, and optionally a donor molecule.
• the polypeptide may be a Feldan Shuttle (U.S. Patent Publication No. 20160298078, herein incorporated by reference).
• the polynucleotide may comprise an expression cassette capable of driving the expression of the polynucleotide.
• the polynucleotide may further comprise additional expression cassettes, capable of expressing, for example, a nuclease such as a CRISPR-associated nuclease.
• the plant or part thereof may be provided with, specifically transformed with, a Cas enzyme, or one or more polynucleotides encoding a Cas enzyme, and a polynucleotide sequence encoding a guide RNA.
• the guide RNA is complementary to a PPO gene or regulatory sequences thereof, in the plant or part thereof.
• the guide RNA is operable to target the Cas enzyme to edit the PPO gene and provide the plant or part thereof with increased resistance to a compound which inhibits PPO enzymatic activity pathway relative to an unmodified plant.
• a plant may be modified by providing the plant or part thereof with one or more regulatory RNA sequences operable to target a gene that encodes a PPO enzyme or a regulatory sequence thereof.
• the plant or part thereof may be transformed with one or more regulatory RNA sequences operable to target a gene that encodes a PPO enzyme or a regulatory sequence thereof.
• the regulatory RNA sequence may be complementary to, and bind to, a gene that encodes a PPO enzyme or a regulatory sequence thereof in the plant or part thereof, and act to provide increased resistance to compounds which inhibit PPO enzymatic activity.
• Transformation refers to a process of introducing an exogenous nucleic acid molecule (for example, a recombinant polynucleotide) into a cell or protoplast and that exogenous nucleic acid molecule is incorporated into a host cell genome or an organelle genome (for example, chloroplast or mitochondria) or is capable of autonomous replication.
• Transformed or “transgenic” refers to a cell, tissue, organ, or organism into which a foreign nucleic acid, such as an expression vector or recombinant nucleic acid molecule has been introduced.
• the nucleic acid molecule can be stably integrated into the genome of the host or the nucleic acid molecule can also be present as an extrachromosomal molecule. Such an extrachromosomal molecule can be auto-replicating.
• the nucleic acid molecule can also be introduced into the genome of the chloroplast or the mitochondria of a plant cell.
• Methods of transformation of plant cells or tissues include, but are not limited to, Agrobacterium mediated transformation method and the Biolistics or particle-gun mediated transformation method.
• Suitable plant transformation vectors for the purpose of Agrobacterium mediated transformation include-those elements derived from a tumor inducing (Ti) plasmid of Agrobacterium tumefaciens, for example, right border (RB) regions and left border (LB) regions, and others disclosed by Herrera-Estrella et al., Nature 303:209 (1983); Bevan, Nucleic Acids Res. 12:8711-8721 (1984); Klee et al., Bio-Technology 3(7):637-642 (1985).
• Ti tumor inducing
• nucleic acid molecules of this invention can be used to insert the nucleic acid molecules of this invention into plant cells. Such methods may involve, but are not limited to, for example, the use of liposomes, electroporation, chemicals that increase free DNA uptake, free DNA delivery via microprojectile bombardment, and transformation using viruses or pollen.
• a “transgenic” or “transformed” cell or plant also includes progeny of the cell or plant and progeny produced from a breeding program employing such a “transgenic” plant as a parent in a cross and exhibiting an altered phenotype resulting from the presence of the foreign nucleic acid molecule.
• the transgenic plants may be homozygous for the polynucleotide encoding a modified PPO enzyme described herein (i.e. those that contain two added genes encoding the enzyme at the same position on each chromosome of the chromosome pair). Homozygous transgenic plants may be obtained by crossing (self-pollinating) independent transgenic plant isolates containing a single added gene, germinating some of the resulting seeds, and transforming the resulting plant with the target gene.
• transgenic plants containing a nucleic acid molecule that encodes recombinant modified PPO enzymes of the invention are well known in the art.
• the regenerated plants may be self-pollinated to provide homozygous transgenic plants, as discussed above. Otherwise, pollen obtained from the regenerated plants is crossed to seed-grown plants of agronomically important lines. Conversely, pollen from plants of these important lines is used to pollinate regenerated plants.
• the at least partially resistant plants and progeny of such plants described herein can be used in methods for preparing at least partially resistant plants, plants having increased tolerance to compounds which inhibit PPO enzymatic activity, and seeds of such plants.
• the plants exemplified herein may be used in breeding programs to develop additional at least partially herbicide resistant plants, such as commercial varieties of such plants.
• a first parent plant may be used in crosses with a second parent plant, where at least one of the first or second parent plants contains a modified PPO enzyme as described herein.
• One application of the process is in the production of F1 hybrid plants.
• Another aspect of this process is that the process can be used for the development of novel parent, dihaploid or inbred lines.
• a plant line as described herein could be crossed to any second plant, and the resulting hybrid progeny each selfed and/or sibbed for about 5 to 7 or more generations, thereby providing a large number of distinct, parent lines.
• These parent lines could then be crossed with other lines and the resulting hybrid progeny analyzed for beneficial characteristics. In this way, novel lines conferring desirable characteristics could be identified.
• Various breeding methods may be used in the methods, including haploidy, pedigree breeding, single-seed descent, modified single seed descent, recurrent selection, and backcrossing.
• the plants and progeny thereof may display a synergistic effect rather than additive effect of tolerance to compounds which inhibit PPO enzymatic activity, whereby the level of tolerance in the plants and the progeny thereof comprising multiple mutations is greater than the combined tolerance of plants comprising a single modified PPO enzyme.
• Plant lines containing the modified PPO enzymes of the present invention can be crossed by either natural or mechanical techniques. Mechanical pollination can be effected either by controlling the types of pollen that can be transferred onto the stigma or by pollinating by hand.
• any breeding method may be used in the methods of the present invention.
• the resistant plants of the present invention may be bred using a haploid method.
• parents having the genetic basis for the desired complement of characteristics are crossed in a simple or complex cross.
• Crossing refers to the transfer of pollen from one plant to a different plant. Progeny of the cross are grown and microspores (immature pollen grains) are separated and filtered, using techniques known to those skilled in the art [(e.g. Swanson, E. B.
• microspore culture in Brassica napus L.
• Swanson, E. B. (1990) Microspore culture in Brassica, pp. 159-169 in Methods in Molecular Biology, vol. 6, Plant Cell and Tissue Culture, Humana Press] These microspores exhibit segregation of genes.
• the microspores are cultured in the presence of an appropriate AHAS-inhibitor herbicide, such as imazethapyr (e.g. PURSUITTM) or imazamox (e.g.
• pedigree breeding may be used for the improvement of largely self-pollinating crops such as Brassica and canola.
• Pedigree breeding starts with the crossing of two genotypes, each of which may have one or more desirable characteristics that is lacking in the other or which complements the other. If the two original parents do not provide all of the desired characteristics, additional parents can be included in the crossing plan. These parents may be crossed in a simple or complex manner to produce a simple or complex F1.
• An F2 population is produced from the F1 by selfing one or several F1 plants, or by intercrossing two FTs (i.e. , sib mating).
• the best individuals may begin in the F2 generation, and beginning in the F3 the best families, and the best individuals within the best families are selected. Replicated testing of families can begin in the F4 generation to improve the effectiveness of selection for traits with low heritability.
• F6 and F7 the best lines or mixtures of phenotypically similar lines may be tested for potential release as new cultivars.
• the pedigree method is more time-consuming than the haploidy method for developing improved plants which are at least partially resistant to PPO enzyme inhibiting compounds, because the plants exhibit segregation for multiple generations, and the recovery of desirable traits is relatively low.
• the single seed descent (SSD) procedure may also be used to breed improved varieties.
• the SSD procedure in the strict sense refers to planting a segregating population, harvesting a sample of one seed per plant, and using the population of single seeds to plant the next generation.
• the plants from which lines are derived will each trace to different F2 individuals.
• the number of plants in a population declines each generation due to failure of some seeds to germinate or some plants to produce at least one seed. As a result, not all of the plants originally sampled in the F2 population will be represented by a progeny when generation advance is completed.
• canola breeders commonly harvest one or more pods from each plant in a population and thresh them together to form a bulk. Part of the bulk is used to plant the next generation and part is put in reserve.
• the procedure has been referred to as modified single-seed descent or the pod-bulk technique.
• the multiple-seed procedure has been used to save labour at harvest. It is considerably faster to thresh pods with a machine than to remove one seed from each by hand for the single-seed procedure.
• the multiple-seed procedure also makes it possible to plant the same number of seeds of a population each generation of inbreeding. Enough seeds are harvested to make up for those plants that did not germinate or produce seed.
• Backcross breeding can be used to transfer a gene or genes for a simply inherited, highly heritable trait from a source variety or line (the donor parent) into another desirable cultivar or inbred line (the recurrent parent).
• the donor parent a source variety or line
• the recurrent parent a desirable cultivar or inbred line
• individuals possessing the phenotype of the donor parent are selected and are repeatedly crossed (backcrossed) to the recurrent parent.
• backcrossing is complete, the resulting plant is expected to have the attributes of the recurrent parent and the desirable trait transferred from the donor parent.
• Improved varieties may also be developed through recurrent selection. In this method, genetically variable population of heterozygous individuals is either identified or created by intercrossing several different parents. The best plants are selected based on individual superiority, outstanding progeny, or excellent combining ability.
• At least partially resistant plants can be produced by cross-pollinating a first plant with a second plant and allowing the pollen acceptor plant (can be either the first or second plant) to produce seed from this cross pollination. Seeds and progeny plants generated therefrom can have the mutation crossed into the genome of the seed and/or progeny plants.
• the pollen-acceptor plant can be either the first or second plant.
• the first plant comprises a nucleic acid encoding a modified PPO enzyme as disclosed herein.
• the second plant can be any compatible plant and may comprise a second same or different modified PPO enzyme.
• the first and second enzymes may comprise a nucleic acid encoding the same or different amino acid substitution(s) relative to a wild-type modified PPO enzyme. Seeds or progeny plants arising from the cross which comprise one or two nucleic acids encoding modified PPO enzymes can be selected.
• each of the resulting progeny plants comprises one copy of each of the first and second nucleic acid molecules and the selection step can be omitted.
• progeny plants comprising both nucleic acid molecules can be selected, for example, by analyzing the DNA of progeny plants to identify progeny plants comprising both the first and second nucleic acid molecules or by testing the progeny plants for increased herbicide tolerance.
• Descendent and/or progeny plants may be evaluated for the nucleic acid molecules of the present invention by any method to determine the presence of a specific modified PPO nucleic acid or enzyme.
• a plant or part thereof that includes a modified PPO enzyme of the invention, or a nucleic acid or expression vector encoding it by exposing the plant or part thereof to an effective amount of a compound which inhibits a PPO enzymatic activity sufficient to prevent or reduce the growth of a plant that does not include at least a modified PPO enzyme of the invention, or a nucleic acid or expression vector encoding it. It may then be determined by the methods described herein whether the plant has been affected (e.g. has reduced growth or reduced damage) by the compound. Plants that are unaffected by the compound may then be selected.
• Methods of determining whether a plant includes the modified PPO enzyme of the invention, or a nucleic acid or expression vector encoding it and/or is affected by a compound which inhibits PPO enzymatic activity include phenotypic evaluations, genotypic evaluations, or combinations thereof.
• the progeny plants may be evaluated in subsequent generations for resistance to the compound, and other desirable traits.
• Resistance to compounds which inhibit PPO enzymatic activity may be evaluated by exposing plants to one or more appropriate compounds and evaluating injury. Some traits, such as lodging resistance and plant height, may be evaluated through visual inspection of the plants, while earliness of maturity may be evaluated by a visual inspection of seeds within pods (siliques). Other traits, such as oil percentage, protein percentage, and total glucosinolates of seeds may be evaluated using techniques such as Near Infrared Spectroscopy and/or liquid chromatography and/or gas chromatography.
• Plants of the present invention can also be identified using any genotypic analysis method. Genotypic evaluation of the plants includes using techniques such as Isozyme Electrophoresis, Restriction Fragment Length Polymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), Allele-specific PCR (AS-PCR), DNA Amplification Fingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs), Amplified Fragment Length Polymorphisms (AFLPs), Simple Sequence Repeats (SSRs) which are also referred to as "Microsatellites”.
• RFLPs Restriction Fragment Length Polymorphisms
• RAPDs Randomly Amplified Polymorphic DNAs
• AP-PCR Arbitrarily Primed Polymerase Chain Reaction
• AS-PCR Allele-specific PCR
• DAF Sequence Characterized Amplified Regions
• compositions and methods for analyzing the genotype of the plants include those methods disclosed in U.S. Publication No. 2004/0171027, U.S. Publication No. 2005/02080506, and U.S. Publication No. 2005/0283858, the entireties of which are hereby incorporated by reference. Evaluation and manipulation (through exposure to one or more appropriate compounds which inhibit PPO enzymatic activity) may occur over several generations. The performance of the new lines may be evaluated using objective criteria in comparison to check varieties. Lines showing the desired combinations of traits are either crossed to another line or selfpollinated to produce seed.
• “Sequencing DNA” refers to determining the nucleic acid sequence of a piece of DNA, e.g. of a gene. Standard methods and commercial services are known in the art. Basic methods for DNA sequencing include the Maxam-Gilbert method and the chain termination method. High-throughput techniques have also been developed and may be used in the method of the present invention. These high-throughput techniques include, but are not limited to, Massively parallel signature sequencing (MPSS), Polony sequencing, 454 pyrosequencing, Illumina (Solexa) sequencing, Combinatorial probe anchor synthesis (cPAS), SOLiD sequencing, Ion Torrent semiconductor sequencing, DNA nanoball sequencing, Heliscope single molecule sequencing, Single molecule real time (SMRT) sequencing and Nanopore DNA sequencing.
• MPSS Massively parallel signature sequencing
• Polony sequencing Polony sequencing
• 454 pyrosequencing Illumina (Solexa) sequencing
• cPAS Combinatorial probe anchor synthesis
• SOLiD sequencing Ion Torrent semiconductor sequencing
• Sequencing may be carried out using primers that are capable of binding to an isolated polynucleotide of the invention.
• primers that are complimentary to at least a portion of an isolated polynucleotide of the invention are preferred.
• primer refers to an oligonucleotide which is capable of annealing to a polynucleotide target and serving as a point of initiation of DNA synthesis when placed under conditions in which synthesis of a primer extension product is induced (e.g., in the presence of nucleotides and an agent for polymerization such as DNA polymerase and at a suitable temperature and pH).
• a primer in some examples an extension primer and in some examples an amplification primer
• the primer may be an oligodeoxyribonucleotide.
• a primer is typically sufficiently long to prime the synthesis of extension and/or amplification products in the presence of the agent for polymerization.
• the minimum length of the primer can depend on many factors, including, but not limited to temperature and composition (A/T vs. G/C content) of the primer.
• amplification primers these are typically provided as a pair of bi-directional primers consisting of one forward and one reverse primer or provided as a pair of forward primers as commonly used in the art of DNA amplification such as in PGR amplification.
• a “primer” can refer to more than one primer, particularly in the case where there is some ambiguity in the information regarding the terminal sequence(s) of the target region to be amplified.
• a “primer” can include a collection of primer oligonucleotides containing sequences representing the possible variations in the sequence or includes nucleotides which allow a typical base pairing.
• Primers can be prepared by any suitable method known in the art. Methods for preparing oligonucleotides of specific sequence are known in the art, and include, for example, cloning and restriction of appropriate sequences and direct chemical synthesis. Chemical synthesis methods can include, for example, the phospho di- or tri-ester method, the diethylphosphoramidate method and the solid support method disclosed in U.S. Patent No. 4,458,066.
• Primers can be labelled, if desired, by incorporating detectable moieties by for instance spectroscopic, fluorescence, photochemical, biochemical, immunochemical, or chemical moieties.
• Primers diagnostic i.e. able to identify or select based on presence of modified PPO encoding nucleic acids and the modified PPO enzymes thereof as described herein
• Primers diagnostic i.e. able to identify or select based on presence of modified PPO encoding nucleic acids and the modified PPO enzymes thereof as described herein
• the PCR method is well described in handbooks and known to the skilled person.
• target polynucleotides can be detected by hybridization with a probe polynucleotide, which forms a stable hybrid with the target sequence under stringent to moderately stringent hybridization and wash conditions. If it is expected that the probes are essentially completely complementary (i.e., about 99% or greater) to the target sequence, stringent conditions can be used.
• the stringency of hybridization can be reduced.
• conditions are chosen to rule out non-specific/adventitious binding. Conditions that affect hybridization, and that select against non-specific binding are known in the art, and are described in, for example, Sambrook & Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, United States of America. Generally, lower salt concentration and higher temperature hybridization and/or washes increase the stringency of hybridization conditions.
• seeds that are capable of producing a plant or part thereof of the invention.
• seeds that comprise a modified PPO enzyme provided herein, or a polynucleotide or expression vector encoding a modified PPO enzyme provided herein, which provides increased resistance to a compound which inhibits PPO enzymatic activity relative to an unmodified plant.
• seed embraces seeds and plant propagules of all kinds including but not limited to true seeds, seed pieces, suckers, corms, bulbs, fruit, tubers, grains, cuttings, cut shoots and the like.
• Seeds may be treated or untreated seeds.
• the seeds can be treated to improve germination, for example, by priming the seeds, or by disinfection to protect against seed-borne pathogens.
• seeds can be coated with any available coating to improve, for example, plantability, seed emergence, and protection against seed-borne pathogens.
• Seed coating can be any form of seed coating including, but not limited to pelleting, film coating, and encrustments.
• the seed may be germinated and used to produce or grow a plant or part thereof of the invention. That is a plant or part thereof including a modified PPO enzyme which provides increased resistance to a compound which inhibits PPO enzymatic activity relative to an unmodified plant.
• a container including seeds of the invention may contain any number, weight or volume of seeds.
• a container can contain at least, or greater than, about 10, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more seeds.
• the container can contain at least, or greater than, about 1 ounce, 5 ounces, 10, ounces, 1 pound, 2 pounds, 3 pounds, 4 pounds, 5 pounds or more seeds.
• Containers of plant seeds may be any container available in the art.
• a container may be a box, a bag, a packet, a pouch, a tape roll, a pail, a foil, or a tube.
• Seeds contained in a containers may be treated or untreated seeds.
• the seeds can be treated to improve germination, for example, by priming the seeds, or by disinfection to protect against seed-borne pathogens.
• seeds can be coated with any available coating to improve, for example, plantability, seed emergence, and protection against seed-borne pathogens.
• Seed coating can be any form of seed coating including, but not limited to pelleting, film coating, and encrustments.
• At least 10% of seeds within a container may be seeds of the invention.
• at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% of the seeds in the container may be seeds of the invention.
• the seeds of the invention may be hybrid seeds produced by a method including crossing a first plant according to the invention, with a second plant; and obtaining seeds. For example, crossing a plant including modified PPO enzyme, or a polynucleotide or expression vector encoding such a modified PPO enzyme, which provides increased resistance to a compound which inhibits PPO enzymatic activity with another plant.
• hybrid seed refers to a seed produced by cross-pollinating two plants. Plants grown from hybrid seeds may have improved agricultural characteristics, such as better yield, greater uniformity, and/or disease resistance. Hybrid seeds do not breed true, i.e. , the seed produced by self-fertilizing a hybrid plant (the plant grown from a hybrid seed) does not reliably result the next generation in an identical hybrid plant. Therefore, new hybrid seeds must be produced from the parent plant lines for each planting. Since most crop plants have both male and female organs, hybrid seeds can only be produced by preventing self- pollination of the female parent and allowing or facilitating pollination with the desired pollen.
• Hybrid seeds may be the result of a single cross (e.g., a first generation cross between two inbred lines), a modified single cross (e.g., a first generation cross between two inbred lines, one or other of which may have been modified slightly by the use of closely related crossing), a double cross (e.g., a first generation of a cross between two single crosses), a three-way cross (e.g., a first generation of a cross between a single cross and an inbred line), a top cross (e.g., the first generation of a cross between an inbred line and an open-pollinated variety, or the first generation of a cross between a singlecross and an open-pollinated variety), or an open pollinated variety (e.g., a population of plants selected to a standard which may show variation but has characteristics by which a variety can be differentiated from other varieties).
• a single cross e.g., a first generation cross between two inbred lines
• a modified single cross
• cross refers to the fusion of gametes via pollination to produce progeny (e.g., cells, seeds or plants).
• progeny e.g., cells, seeds or plants.
• the term encompasses both sexual crosses (the pollination of one plant by another) and selfing (self-pollination, e.g., when the pollen and ovule are from the same plant).
• crossing refers to the act of fusing gametes via pollination to produce progeny.
• the invention further provides isolated polynucleotides that encode a modified PPO enzyme or fragment thereof as defined in any aspect or embodiment herein, suitably which may encode a modified PPO enzyme or fragment thereof as defined in any of the sequences listed herein, suitably which may encode any of SEQ ID NOs: 1 , 2, 4 - 151 , 153 -302, or 305 - 336. Suitably which may encode any of SEQ ID NOs: 37- 39, 58, 59, 97 - 99, 118, 119, 125 - 137, 139 - 151 , 188 - 190, 209, 210, 248 - 250, 269, 270, 277 - 288 or 291 - 302.
• modified PPO enzymes or functional fragments thereof that may be expressed from such isolated polynucleotides.
• An “isolated” polynucleotide is substantially separated away from other polynucleotide sequences with which the polynucleotide is normally associated, such as, from the chromosomal or extrachromosomal DNA of a cell in which the polynucleotide naturally occurs.
• a polynucleotide may be an isolated polynucleotide when it comprises a transgene or part of a transgene present in the genome of another organism.
• the term also embraces polynucleotides that are biochemically purified so as to substantially remove contaminating polynucleotides and other cellular components.
• Isolated polynucleotides are substantially free of sequences (such as protein encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide is derived.
• the isolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the polynucleotide in genomic DNA of the cell from which the polynucleotide is derived.
• the isolated polynucleotide may be flanked by its native genomic sequences that control its expression in the cell, for example, the native promoter, or native 3 ' untranslated region.
• the expression constructs and vectors, which include at least one polynucleotide of the present invention inserted therein may be any construct or vector capable of delivering the polynucleotide into a host or host cell and allowing expression of the polynucleotide to provide a functional modified PPO enzyme as described herein or fragment thereof.
• constructs or vectors may contain heterologous polynucleotide sequences, that is polynucleotide sequences that are not naturally found adjacent to polynucleotides of the present invention and that may be derived from a species other than the species from which the polynucleotide molecule(s) are derived.
• the construct or vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically the vector is a virus or a plasmid.
• a number of vectors suitable for stable transfection of plant cells or for the establishment of transgenic plants have been described in, e.g., Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp.
• plant expression vectors include, for example, one or more cloned plant genes under the transcriptional control of 5' and 3' regulatory sequences and a dominant selectable marker.
• the vector may be pBIN 19 (Bevan, Nucl. Acids Res. (1984)).
• the expression vector of the invention may include one or more regulatory sequences.
• the expression vectors can contain a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally- regulated, or cell- or tissue-specific expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.
• a promoter regulatory region e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally- regulated, or cell- or tissue-specific expression
• a transcription initiation start site e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally- regulated, or cell- or tissue-specific expression
• a transcription initiation start site e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally- regulated, or cell- or tissue-specific expression
• a transcription initiation start site e.g., a
• “Expression construct” as used herein means a nucleic acid sequence capable of directing expression of a particular nucleic acid sequence in an appropriate host cell, comprising a promoter operably linked to the polynucleotide of interest which is operably linked to termination signal sequences. It also typically comprises sequences required for proper translation of the polynucleotide sequence.
• the expression construct comprising the polynucleotide of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components.
• the expression construct may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. Typically, however, the expression construct is heterologous with respect to the host, i.e.
• the particular polynucleotide of the expression cassette does not occur naturally in the host cell and must have been introduced into the host cell or an ancestor of the host cell by a transformation event.
• the expression of the polynucleotide sequence in the expression construct may be under the control of, for example, a constitutive promoter or of an inducible promoter that initiates transcription only when the host cell is exposed to some particular external stimulus.
• the promoter can also be specific to a particular tissue, or organ, or stage of development.
• regulatory element refers to a nucleic acid that is capable of regulating the transcription and/or translation of an operably linked polynucleotide. Regulatory elements include, but are not limited to, promoters, enhancers, introns, 5' UTRs, and 3' UTRs.
• Expression cassettes may include in the 5 3' direction of transcription, a transcriptional and translational initiation region (e.g., a promoter), a polynucleotide sequence encoding modified PPO enzyme of the invention, and a transcriptional and translational termination region (e.g., termination region) functional in plants.
• a transcriptional and translational initiation region e.g., a promoter
• a polynucleotide sequence encoding modified PPO enzyme of the invention e.g., a transcriptional and translational termination region
• any promoter can be used in the production of the expression construct and vectors including such expression constructs as described herein.
• the promoter may be native or analogous, or foreign or heterologous, to the plant host and/or to the polynucleotide sequences encoding the modified PPO of the invention. Additionally, the promoter may be a 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.
• promoter is “foreign” or “heterologous” to the polynucleotide encoding the modified PPO of the invention, it is intended that the promoter is not the native or naturally occurring promoter for the operably linked polynucleotide of the invention.
• the native promoter sequences may be used in the preparation of the expression constructs.
• Such expression constructs may change expression levels of the modified PPO enzyme in the plant or plant cell. Thus, the phenotype of the plant or plant cell is altered.
• Any promoter can be used in the preparation of expression constructs to control the expression of the polynucleotide encoding modified PPO, such as promoters providing for constitutive, tissue-preferred, inducible, 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 99/43 838 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.
• Tissue-preferred promoters can be utilized to direct expression of the modified PPO enzymes of the invention within a particular plant tissue.
• tissue-preferred promoters include, but are not limited to, leaf-preferred promoters, root-preferred promoters, seedpreferred promoters, and stem- preferred promoters.
• Tissue-preferred promoters include those described in 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 expression constructs may also comprise transcription termination regions. Where transcription terminations regions are used, any termination region may be used in the preparation of the expression cassettes.
• the termination region may be native to the transcriptional initiation region, may be native to the operably linked polynucleotide of interest, may be native to the plant host, or may be derived from another source (i.e. , foreign or heterologous to the promoter, the polynucleotide of interest encoding modified PPO enzyme, the plant host, or any combination thereof).
• termination regions that are available for use in the expression constructs and vectors of the present invention include those 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; 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; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acid Res. 15:9627-9639.
• the expression construct may comprise a Tomato Mosaic Virus (TMV) omega 5’ leader and a modified PPG enzyme encoding gene of interest is excised using Xhol/Kpnl and cloned into pBIN 19 behind a double enhanced 35S promoter and ahead of a NOS 3’ transcription terminator.
• TMV Tomato Mosaic Virus
• a suitable exemplary vector comprising such an expression construct is provided herein as SEQ ID NO: 3.
• the polynucleotides may be optimized for increased expression in a transformed plant. That is, the polynucleotides encoding the modified PPO enzymes can be synthesized using plantpreferred codons for improved expression. See, for example, Campbell and Gowri (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.
• sequence modifications can be made to the polynucleotides of the invention.
• additional sequence modifications that are known to enhance gene expression in a cellular 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 also be adjusted to levels average for a target cellular host, as calculated by reference to known genes expressed in the host cell.
• the sequence can be modified to avoid predicted hairpin secondary mRNA structures.
• polynucleotide sequences may also be used in the preparation of the expression constructs of the present invention, for example to enhance the expression of the modified PPO encoding polynucleotide sequence.
• Such polynucleotide sequences include the introns of the maize Adhl, intron I gene (Callis et al. (1987) Genes and Development 1 :1183- 1200), and leader sequences, (W-sequence) from the Tobacco Mosaic virus (TMV), Maize Chlorotic Mottle Virus and Alfalfa Mosaic Virus (Gallie et al (1987) Nucleic Acid Res. 15:8693-8711, and Skuzeski et al. (1990) Plant Mol. Biol.
• 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. ((1994) Plant Physiol. 106:929-939) also have shown that introns are useful for regulating gene expression on a tissue specific basis. Plant cells transformed with such modified expression constructs or vectors, then, may exhibit overexpression or constitutive expression of a polynucleotide of the invention.
• Expression constructs may additionally contain 5' leader sequences. Such leader sequences can act to enhance translation.
• Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein et al. (1989) Proc. Natl. Acad. ScL USA 86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie et al. (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf Mosaic Virus) (Virology 154:9-20), and human immunoglobulin heavy-chain binding protein (BiP) (Macejak et al.
• EMCV leader Engelphalomyocarditis 5' noncoding region
• potyvirus leaders for example, TEV leader (Tobacco Etch Virus) (Gallie et al. (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf Mosaic Virus) (Virology 154:9-20), and human immunoglob
• the various polynucleotides may be manipulated, so as to provide for the polynucleotides in the proper orientation and, as appropriate, in the proper reading frame.
• adapters or linkers may be employed to join the nucleic acid molecules or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous polynucleotides, removal of restriction sites, or the like.
• in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions may be involved.
• Expression vectors may include additional features.
• gRNA promoters to regulate expression of the at least one gRNA e.g. prOsU3-01, which is the Rice U3 promoter for pol III dependent transcription of non-coding.
• Vectors may similarly include additional features such as selectable markers, e.g. Phosphomannose Isomerase (PMI), and antibiotic resistance genes that can be used to aid recovery of stably transformed plants.
• selectable markers e.g. Phosphomannose Isomerase (PMI)
• antibiotic resistance genes e.g. Phosphomannose Isomerase (PMI)
• operably linked or “operably associated” as used herein, it is meant that the indicated elements are functionally related to each other, and are also generally physically related.
• the term “operably linked” or “operably associated” as used herein refers to polynucleotides on a single nucleic acid molecule that are functionally associated.
• a first polynucleotide sequence or nucleic acid molecule that is operably linked to a second polynucleotide sequence or nucleic acid molecule means a situation when the first polynucleotide sequence or nucleic acid molecule is placed in a functional relationship with the second polynucleotide sequence or nucleic acid molecule.
• a promoter is operably associated with a polynucleotide sequence or nucleic acid molecule if the promoter effects the transcription or expression of said polynucleotide sequence or nucleic acid molecule.
• control sequences e.g., promoter
• the control sequences need not be contiguous with the polynucleotide sequence or nucleic acid molecule to which it is operably associated, as long as the control sequences function to direct the expression thereof.
• intervening untranslated, yet transcribed, sequences can be present between a promoter and a polynucleotide sequence or nucleic acid molecule, and the promoter can still be considered “operably linked” to or “operatively associated” with the polynucleotide sequence or nucleic acid molecule.
• the plants or parts thereof according to the invention are at least partially resistant to inhibition by a compound which inhibits a PPO enzyme (i.e. PPO enzymatic activity).
• PPO enzyme i.e. PPO enzymatic activity.
• the present invention further relates to a method of controlling the growth of undesired vegetation at a locus comprising plants having the modified PPO enzymes provided herein in the vicinity thereof, wherein the method comprises applying an effective amount of at least one PPO-inhibiting herbicide.
• locus includes soil, seeds, seedlings, field, greenhouse, an area of cultivation, as well as, established vegetation.
• the compound which inhibits a PPO enzyme is a herbicide.
• the plants of the invention are resistant to a herbicide which inhibits PPO enzymatic activity (referred to herein as a PPO-inhibiting herbicide), and therefore such plants can be used in methods where these herbicides are applied.
• Herbicides can be applied pre-emergence or post-emergence of the crop plant or the undesired vegetation.
• a plant having increased resistance to a compound which inhibits PPO enzymatic activity may be referred to as an "herbicide-tolerant" or “herbicideresistant” plant.
• Such plants are tolerant or at least partially resistant to at least one compound which inhibits PPO enzymatic activity at a level that would normally kill, or inhibit the growth of a normal, control or wild-type plant lacking the modified PPO enzymes, polynucleotides encoding said enzymes or expression vectors of the invention.
• the term "herbicide” is used herein to mean an active ingredient that kills, controls or otherwise adversely modifies the growth of plants and is typically used to control undesired vegetation.
• plants of the invention may have at least a 2-fold increase in resistance to a compound which inhibits PPO enzymatic activity, such as the inhibiting herbicides described herein.
• plants of the invention may have at least a 2-fold, 3-fold, 4-fold, 5-fold 6-fold, 7-fold, 8-fold, 9-fold, 10-fold increase in resistance.
• the plants of the invention comprising at least one of the modified PPO enzymes disclosed herein may have at least a 2-fold increase in resistance to any of compounds A to E described herein, compared to an unmodified plant.
• the plants of the invention comprising at least one of the modified PPO enzymes disclosed herein may have at least a 2-fold, 3- fold, 4-fold, 5-fold 6-fold, 7-fold, 8-fold, 9-fold, 10-fold increase in resistance to any of compounds A to E described herein, compared to an unmodified plant. In one embodiment, the plants of the invention comprising at least one of the modified PPO enzymes disclosed herein may have at least a 2-fold increase in resistance to any of compounds A to E described herein, compared to a control plant.
• the plants of the invention comprising at least one of the modified PPO enzymes disclosed herein may have at least a 2-fold, 3-fold, 4-fold, 5-fold 6-fold, 7-fold, 8-fold, 9-fold, 10-fold increase in resistance to any of compounds A to E described herein, compared to a control plant.
• a control plant may be a plant comprising an alternative modified PPO enzyme to those of the invention.
• Resistance to compounds which inhibit PPO enzymatic activity may be determined by any known methods for comparing the growth, damage or other properties of two plants after application of a compound which inhibits PPO enzymatic activity to a plant.
• the resistance of a plant of the invention may be determined by comparing the percentage of damage caused to the plant in comparison to a wild-type or control plant after application of a compound which inhibits PPO enzymatic activity, such as the herbicides described herein.
• Increased resistance to a compound which inhibits PPO enzymatic activity refers in the context of the invention to a plant which has been modified to comprise a modified PPO enzyme that has improved or increased resistance to a compound which inhibits PPO enzymatic activity (the PPO-inhibiting herbicide), relative to an unmodified plant (e.g. wild type) or control plant (e.g. a plant comprising a PPO enzyme according to SEQ ID NO: 124).
• the increased resistance may be due to an increased activity of the modified PPO enzyme in the plant, compared to the activity of such enzymes in an unmodified plant, when in the presence of at least one compound that is known to interfere with PPO enzyme activity in plants at a concentration or level that is to known to inhibit the activity of the wild-type PPO protein.
• Improved resistance means that the plant comprising the enzyme, and the modified PPO enzyme itself, is at least partially resistant to a compound which inhibits PPO enzymatic activity (the PPO-inhibiting herbicide).
• Partially resistant plants of the invention may still have some decreased PPO enzymatic activity when exposed to a compound which inhibits PPO enzymatic activity, such as at most a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, decrease in enzymatic activity.
• the modified PPO enzymes used in the invention may be partially resistant, and may still have some decrease in enzymatic activity when exposed to a compound which inhibits PPO enzymatic activity.
• the plants modified to comprise the modified PPO enzymes may have total or near total resistance to a compound which inhibits PPO enzymatic activity (the PPO- inhibiting herbicide).
• the plants may have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% resistance to a compound which inhibits PPO enzymatic activity.
• the modified PPO enzymes used in the invention may have no substantial decrease in enzymatic activity when exposed to a compound which inhibits PPO enzymatic activity.
• any decrease in activity is less than a decrease in activity relative to the activity of a wild-type enzyme, when in the presence of at least one compound that is known to interfere with PPO enzymatic activity and at a concentration or level of the compound that is to known to inhibit the activity of the wild-type PPO protein.
• a decrease in activity seen for a partially resistant modified PPO enzyme may be a decrease in activity that does not have a negative effect on the growth, propagation or development of a plant comprising a partially resistant modified PPO enzyme.
• the activity of such a partially resistant modified PPO enzyme may be referred to herein as "herbicide-tolerant" or "herbicideresistant” enzymes.
• Plants which are at least partially "resistant" to at least one compound that inhibits PPO enzymatic activity exhibit few, if any, necrotic, lytic, chlorotic or other lesions when subjected to the compound at concentrations and rates which are typically employed by the agricultural community to kill unwanted vegetation in the vicinity of the plant such as a field.
• modified PPO enzymes described herein may be compared to a reference, unmodified, or wild-type enzyme, which otherwise may be termed a control enzyme or a plant comprising such an enzyme.
• wild-type is used to refer to a nucleic acid molecule or protein that can be found in nature as distinct from being artificially produced or mutated by man.
• a reference or unmodified PPO enzyme may be a PPO enzyme derived from the same source species as the modified enzyme, that does not include any modifications of the invention as described herein.
• the reference enzyme may be a wild type enzyme.
• unmodified or “reference” is not intended to necessarily imply that a plant, plant tissue, plant cell, or other host cell lacks any recombinant DNA in its genome, and/or does not possess herbicide resistant characteristics that are different from those disclosed herein.
• Reference, or unmodified PPO enzymes as referred to herein may well include other mutations or modifications that do not affect resistance to compounds which inhibit PPO enzymatic activity.
• a reference enzyme may include mutations or modifications to improve or alter expression, translation or targeting of the control enzyme to specific tissues, organs or cells.
• control enzyme or plant including said enzyme
• the reference to control enzyme may be a PPO enzyme that includes alternative modifications that increase resistance to compounds that inhibit PPO enzymatic activity to those of the invention.
• a wild-type, reference unmodified PPO enzyme may be an enzyme encoded by SEQ ID NO: 1.
• a wild-type, reference unmodified PPO enzyme may be a PPO enzyme encoded by SEQ ID NO: 2.
• a reference or control PPO enzyme may be an enzyme encoded by SEQ ID NO: 124.
• Many herbicides which inhibit PPO enzymatic activity are known in the art and include a structurally diverse range of chemistry. Accordingly, the skilled person will appreciate that a broad range of PPO inhibiting herbicides have utility in the present invention.
• the PPO-inhibiting herbicide is selected from the group consisting of butafenacil, carfentrazone-ethyl, cyclopyranil, epyrifenacil (Herbicide A), flufenoximacil, flumioxazin, fomesafen, oxyfluorfen, pyraflufen-ethyl, saflufenacil (Herbicide E), sulfentrazone, tiafenacil, trifludimoxazin (Herbicide B), compounds disclosed in WO2016/095768 for example 3-(2-chloro-4-fluoro-5-(3-methyl-2,6-dioxo-4-trifluoromethyl-
• the compound which inhibits PPO enzymatic activity may be any combination of one or more such inhibitory compounds, suitably one or more of the compounds listed above. For example, 1 , 2, 3, 4, 5 or more of the inhibitory compounds as described herein.
• the plants of the invention may be used in methods of controlling undesired vegetation in the vicinity of the plant.
• the methods may include applying an effective amount of at least one compound, such as those listed above, which inhibits PPO enzymatic activity to the undesired vegetation and the plant.
• plants of the invention that include a modified PPO enzyme of the invention may be used in methods of enhancing plant growth by controlling undesired vegetation in the vicinity of the plant.
• the methods may include applying an effective amount at least one compound, such as those listed above, which inhibits PPO enzymatic activity to the undesired vegetation and the plant.
• certain modified PPO enzymes may perform better when used with certain compounds which inhibit PPO enzymatic activity.
• certain modified PPO enzymes may provide better resistance to certain compounds which inhibit PPO enzymatic activity compared to other compounds which inhibit PPO enzymatic activity.
• certain combinations of PPO modifications and compounds may be optimal for use in the methods of the invention.
• the plant of the invention may comprise a PPO enzyme having a mutation at position 362 of SEQ ID NO: 1 or a residue corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound D.
• the plant of the invention may comprise a PPO enzyme having a mutation at position 362 of SEQ ID NO: 1 or residue corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound B.
• the plant of the invention may comprise a PPO enzyme having a mutation at position 365 of SEQ ID NO: 1 or residue corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound B.
• the plant of the invention may comprise a PPO enzyme having a mutation at position 365 of SEQ ID NO: 1 or residue corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound E.
• the plant of the invention may comprise a PPO enzyme having a mutation at position 479 of SEQ ID NO: 1 or residue corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound B.
• the plant of the invention may comprise a PPO enzyme having a mutation at position 479 of SEQ ID NO: 1 or residue corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound D.
• the plant of the invention may comprise a PPO enzyme having a mutation at position 479 of SEQ ID NO: 1 or residue corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound E.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 305 and 426 of SEQ ID NO: 1 or residues corresponding thereto wherein the mutation at position 305 is S305L and position 426 is Y426V or residues corresponding thereto (i.e. SEQ ID NO 125 or 139) and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound A.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 305 and 426 of SEQ ID NO: 1 or residues corresponding thereto wherein the mutation at position 305 is S305L and position 426 is Y426V or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound C.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 305 and 426 of SEQ ID NO: 1 or residues corresponding thereto wherein the mutation at position 305 is S305L and position 426 is Y426V or residues corresponding thereto and is for use with any PPO- inhibiting herbicide disclosed herein, including for example compound D.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 305 and 426 of SEQ ID NO: 1 or residues corresponding thereto wherein the mutation at position 305 is S305L and position 426 is Y426V or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound E.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 365, 426 and 479 of SEQ ID NO: 1 or residues corresponding thereto (e.g. SEQ ID NO 126) and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound A.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 365, 426 and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound C.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 365, 426 and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound D.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 365, 426 and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound E.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 404, 426, 431, and 479 of SEQ ID NO: 1 or residues corresponding thereto (e.g. SEQ ID NO 127) and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound A.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 404, 426, 431 , and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound C.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 404, 426, 431, and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound D.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 404, 426, 431, and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound E.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 305 and 365 of SEQ ID NO: 1 or residues corresponding thereto (e.g. SEQ ID NO 128) and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound C.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 305 and 365 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO- inhibiting herbicide disclosed herein, including for example compound D.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 305 and 365 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound E.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 362 and 404 of SEQ ID NO: 1 or residues corresponding thereto (e.g. SEQ ID NO 129) and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound A.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 362 and 404 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound B.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 362 and 404 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound C.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 362 and 404 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound D.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 362 and 404 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound E.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 361, 365, 431 and 479 of SEQ ID NO: 1 or residues corresponding thereto (e.g. SEQ ID NO 130) and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound A.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 361, 365, 431 and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound B.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 361, 365, 431 and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound D.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 361, 365, 431 and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound E.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 426, and 479 of SEQ ID NO: 1 or residues corresponding thereto (e.g. SEQ ID NO 131) and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound A.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 426, and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound B.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 426, and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound C.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 426, and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound D.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 426, and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound E.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 426, 461 and 479 of SEQ ID NO: 1 or residues corresponding thereto (e.g. SEQ ID NO 132) and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound A.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 426, 461 and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound B.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 426, 461 and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound C.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 426, 461 and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound E.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 361, and 365 of SEQ ID NO: 1 or residues corresponding thereto (e.g. SEQ ID NO 133) and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound A.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 361, and 365 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound B.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 361, and 365 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound C.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 361, and 365 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound D.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 361, and 365 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound E.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 361, 362, 426 and 479 of SEQ ID NO: 1 or residues corresponding thereto (e.g. SEQ ID NO 134) and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound A.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 361, 362, 426 and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound C.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 361, 362, 426 and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound D.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 361, 362, 426 and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound E.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 361 and 365 of SEQ ID NO: 1 or residues corresponding thereto (e.g. SEQ ID NO 135) and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound C.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 361 and 365 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO- inhibiting herbicide disclosed herein, including for example compound B.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 361 and 365 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound D.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 361 and 365 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound E.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 365, 404 and 479 of SEQ ID NO: 1 or residues corresponding thereto (e.g. SEQ ID NO 136) and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound B.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 365, 404 and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound C.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 365, 404 and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound D.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 365, 404 and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound E.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 404 and 479 of SEQ ID NO: 1 or residues corresponding thereto (e.g. SEQ ID NO 137) and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound A.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 365, 404 and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound B.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 365, 404 and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound C.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 365, 404 and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound D.
• the plant of the invention may comprise a PPO enzyme having a mutation at positions 365, 404 and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound E.
• Undesired vegetation may include, for example, dicotyledonous and monocotyledonous weeds.
• Dicotyledonous weeds include, but are not limited to, weeds of the genera: Sinapis, Lepidium, Galium, Slellaria, Matricaria, Anthemis, Galinsoga, Chenopodium, Urtica, 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, Ischaemum, Sphenoclea, Dactyloctenium, Agrostis, Alopecurus, and Apera.
• undesired vegetation 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.
• an “effective amount” or “effective concentration” refers to an amount and concentration, respectively, of a compound that inhibits PPO enzymatic activity (a PPO-inhibiting herbicide), that is sufficient to kill or inhibit the growth of a wild-type plant and/or undesired plant, plant tissue, plant cell, microspore, or host cell, but that said amount does not kill or inhibit as severely the growth of the at least partially resistant plants, parts thereof, plant tissues, plant cells, and seeds having the modified PPOs described herein.
• the effective amount is an amount that is routinely used in agricultural production systems to kill unwanted vegetation of interest. Such an amount is known to those of ordinary skill in the art, or can be easily determined using methods known in the art.
• the effective amount in an agricultural production system might be substantially different than an effective amount for a plant culture system such as, for example, the microspore culture system.
• the amount may be small enough to simply retard or suppress the growth or development of a given weed or undesired vegetation, or the amount may be large enough to irreversibly destroy a given weed or the undesired vegetation. Further, the amount may be any amount there-between.
• An effective amount may be at least 1 gram of active compound per hectare (g ai/ha).
• a compound that inhibits PPO enzymatic activity may be applied at a concentration of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265,
• the compound may be applied to the vicinity of the plant pre-emergence of the crop and/or post-emergence of the crop - a so-called “over-the-top” application.
• Preemergent refers to a compound which is applied to the vicinity of an at least partially resistant plant of the invention (e.g., a field or area of cultivation) before the plant emerges visibly from the soil and/or before germination of a seed.
• Postemergent refers to an compound which is applied to the vicinity of an at least partially resistant plant of the invention after a plant emerges visibly from the soil.
• preemergent and postemergent are used with reference to a weed or undesired vegetation in the vicinity of an at least partially resistant plant of the invention, and in some instances these terms are used with reference to a crop plant in the vicinity of an at least partially resistant plant of the invention.
• weed or undesired vegetation When used with reference to a weed or undesired vegetation, these terms may apply to only a particular type of weed or species of weed or undesired vegetation that is present or believed to be present in the area of interest. While any compound which inhibits PPO enzymatic activity may be applied in a preemergent and/or postemergent treatment, some such compounds are known to be more effective in controlling a weed or weeds or undesired plants when applied either preemergence or postemergence. The compound may be applied "preplant incorporation" which involves the incorporation of the compound into the soil prior to planting.
• the rates of application of a compound which inhibits PPO enzymatic activity may vary within wide limits and depend on the nature of the soil, the method of application (preemergence; post-emergence; application to the seed furrow; no tillage application etc.), the plant, the undesired vegetation to be controlled, the prevailing climatic conditions, and other factors governed by the method of application, the time of application and the target plant.
• the compound which inhibits PPO enzymatic activity may be applied at a rate of at least 1 L/ha.
• the compound may be applied at a rate of 200L/ha.
• the application is generally made by spraying the compound, typically by tractor mounted sprayer for large areas, but other methods such as dusting (for powders), drip or drench can also be used.
• the plants and plant parts and seeds are provided having stably incorporated into their genome a modified PPO provided herein, as well as, an additional polynucleotide conferring tolerance to at least one additional herbicide, including for example, herbicides comprising glyphosate, glufosinate, HPPD herbicides, and others.
• additional herbicide including for example, herbicides comprising glyphosate, glufosinate, HPPD herbicides, and others.
• the application of an effective amount of a PPO-inhibiting herbicide can be combined with the application of other herbicides to which the crop is naturally tolerant or to which the crop is resistant via expression of a one or more modified herbicide resistance genes.
• the one or more other herbicides that are applied in combination with the PPO inhibiting herbicide can be applied sequentially (e.g., in succession) or simultaneously without affecting the yield or growth of the herbicide resistant plant. This includes applying the one or more other herbicides in accordance with a schedule based on the application of the PPO inhibiting herbicide. When applied simultaneously, an effective amount of each herbicide is applied to the plant at the same time (e.g., at the same stage of plant growth, and/or as constituents of a herbicide composition comprising the one or more of herbicides and the HPPD inhibiting herbicide). When applied sequentially, an effective amount of each herbicide is applied to the plant in succession.
• enhancing plant growth of a plant means an improvement in plant vigour, an improvement in plant quality, improved tolerance to stress factors, and/or improved input use efficiency.
• the invention described herein also relates to a kit comprising a container and instructions for use, the container comprising a compound which inhibits PPO enzymatic activity, and the instructions comprising a direction to apply the compound to a plant modified to comprise a modified PPO enzyme that provides the plant or part thereof with increased resistance to said compound.
• the compound may be any one of those as defined above.
• the direction to apply the compound may comprise direction to apply the compound in an effective amount as defined above.
• the direction to apply the compound may comprise direction to apply the compound at a particular rate as described above.
• the direction to apply the compound may comprise direction to apply the compound in a particular method such as by spraying as defined above.
• the direction to apply the compound may comprise direction to apply the compound at a particular time such as pre-emergence of the crop and/or post-emergence of the crop, or during a particular season or month.
• the instructions may further comprise direction to prepare the compound such that it can be applied in the desired effective amount and by the desired method.
• directions may include direction to dilute the compound, suitably in a solvent such as water, suitably to an effective concentration.
• compositions comprising a modified PPO enzyme provided herein (such as any of SEQ I D NOs: 1 , 2, 4 - 151 , 153 -302, or 305 - 336 or SEQ I D Nos: 37- 39, 58, 59, 97 - 99, 118, 119, 125 - 137, 139 - 151, 188 - 190, 209, 210, 248 - 250, 269, 270, 277 - 288 or 291 - 302 or active variant thereof) and a PPO inhibiting herbicide, including but not limited to the herbicides disclosed herein as herbicide A, B, C, D, or E.
• a modified PPO enzyme provided herein (such as any of SEQ I D NOs: 1 , 2, 4 - 151 , 153 -302, or 305 - 336 or SEQ I D Nos: 37- 39, 58, 59, 97 - 99, 118, 119, 125 - 137,
• a method of selecting a plant or organism including, for example, a prokaryotic organism such as a bacteria having an increased resistance to a compound which inhibits PPO enzymatic activity relative to an unmodified plant or organism, comprising: (a) providing a plant or organism; (b) optionally mutagenizing the plant or organism; (c) exposing the plant or organism to an effective amount of a compound which inhibits PPO enzymatic activity; and (d) selecting the plant or organism if the plant or organism displays resistance to the compound.
• a plant or organism including, for example, a prokaryotic organism such as a bacteria
• the plant may be a plant according to the first aspect of the invention, and may comprise any of the features defined in relation to such a plant recited herein.
• step (b) may comprise mutagenizing the plant or organism by any known means such as EMS or other chemical treatment, or by X-ray or another radiation treatment.
• EMS electrospray
• to induce modifications, suitably one or more mutations, in the PPO gene may be mutagenizing the plant or organism by any known means such as EMS or other chemical treatment, or by X-ray or another radiation treatment.
• modifications suitably one or more mutations, in the PPO gene.
• the plant or organism comprises one or more modifications to the endogenous or a heterologous PPO gene, suitably one or more mutations to the endogenous or the heterologous PPO gene.
• the modified PPO gene may encode a modified PPO enzyme, suitably as described herein.
• the plant or organism may comprise a modified PPO enzyme as described herein, suitably having one or more mutations as described herein.
• step (c) comprises exposing the plant or organism to an effective amount of a compound as described herein above.
• step (b) comprises exposing the plant or organism to an effective amount of a compound as described herein above.
• step (b) comprises exposing the plant or organism to an effective amount of a compound as described herein above.
• Suitably selecting the plant or organism if the plant or organism displays resistance to the compound may comprise, for example, for plants selecting those plants that do not display any signs of PPO deficiency. Suitably such signs may include stunted growth, wilting, necrosis, discolouration, and the like. For organisms, such as bacteria, resistance to the compound may comprise the ability to grow in the presence of the herbicide.
• a method of identifying a modified PPO enzyme which comprises an increased resistance to a compound which inhibits PPO enzymatic activity comprising: (a) generating a library of modified PPO encoding polynucleotides; (b) screening a population of the resulting modified PPO encoding polynucleotides by expressing each of said polynucleotides in a bacteria, a plant or a plant part and exposing the bacteria, plant or part thereof to an effective amount of a compound which inhibits PPO enzymatic activity; (c) selecting the modified PPO encoding polynucleotides which provide the bacteria, plant or plant part thereof with increased resistance to said compound compared to a reference bacteria, plant or plant part thereof containing an unmodified or control PPO encoding polynucleotide.
• Suitably generating a library of modified PPO encoding polynucleotides may be carried out by any known technique to generate a library of genes comprising different modifications, suitably mutations throughout the gene sequence.
• these methods may be random or directed.
• Such methods may include a step of exposing the PPO encoding polynucleotides to a mutagen, such as a chemical or radiation, or carrying out error prone PCR on the PPO encoding polynucleotides for example.
• Other molecular techniques may include performing DNA shuffling or staggered extension processes on PPO encoding polynucleotides.
• Suitably expressing the modified polynucleotides in a bacteria, a plant or a plant part, such that the modified PPO enzymes are expressed comprises transforming the bacteria, a plant or a plant part with each of the modified polynucleotides. Suitable means of transformation are described elsewhere herein, as are suitable constructs for expression of a PPO encoding polynucleotide.
• exposing the bacteria, plant or part thereof to an effective amount of a compound which inhibits PPO enzymatic activity comprises using an effective amount as described hereinabove, of a suitable compound as described hereinabove.
• Suitably selecting the modified PPO encoding polynucleotides which provide the bacteria, plant or plant part thereof with increased resistance to said compound comprises selecting those host bacteria, plants or plant parts thereof that display resistance to the compound, suitably selecting those that do not display any signs of PPO deficiency.
• Such signs in plants may include stunted growth, wilting, necrosis, discolouration, and the like.
• such signs in bacteria may include stunted growth or death.
• the sign is reduced growth compared to an unmodified or control bacteria or unmodified or control plant.
• Also provided is a method of identifying a compound which inhibits PPO enzymatic activity comprising: (a) generating a modified plant or part as described herein, (b) applying a test compound to the plant or part thereof of step (a) and to an unmodified reference plant; (c) selecting the test compounds which confer reduced growth to the unmodified reference plant as compared to the growth of the modified plant or part thereof.
• the modified plant or plant part is a plant having increased resistance to compounds which inhibit PPO enzymatic activity, as defined according to the first aspect of the invention, and may comprise any of the features defined in relation to such a plant recited herein.
• test compound may be any compound which may have, or which is expected to have an inhibitory effect on a PPO enzyme of the plant.
• test compound may be related to, derived from, or synthesised from a compound as defined herein, which may be a known compound which inhibits PPO enzymatic activity, such as a known herbicidal compound.
• test compound may be applied in a test amount, suitably the test amount may be the same as any known effective amount for such herbicidal compounds, suitable amounts are defined hereinabove.
• Figure 1 shows a plasmid map of pBin TMV AtPPOl transformation vector.
• Example 1 Expression and assay of an Arabidopsis thaliana PPO1 site saturation variant library in E. coli
• This plasmid was used as the basis of a Site Saturation Variant Library (SSVL) that substituted the codons at 352 amino acid positions for codons that encoded one of the other 19 amino acids, or removed the codon altogether, resulting in a translation product with a deletion at that position.
• SSVL Site Saturation Variant Library
• the SSVL was batch-transformed by electroporation into an E. coli BL21(DE3) hemG strain with the hemG gene replaced by CmR conferring resistance to chloramphenicol and spread onto LA supplemented with 50 pg/mL kanamycin, 25 pg/mL chloramphenicol and 20 pg/mL hematin.
• Each batch consisted of all the plasmids that contained variants at two sites. Colonies from batch transformations were inoculated into 500 pL of Terrific broth supplemented with 50 pg/mL kanamycin, 25 pg/mL chloramphenicol and 20 pg/mL hematin in 96-deepwell blocks.
• Herbicides A to E are as described above.
• Table 2 AtPPOl variants that were identified as being resistant to one or more of herbicides B, D or E.
• the table shows the percentage growth inhibition in the presence of herbicides B, D or E compared to growth in DMSO.
• Example 3 A subset of identified herbicide tolerance mutations from Example 1 were selected to be incorporated into a combinatorial variant library (produced by Twist Bioscience). The selected mutations and designed frequency within the combinatorial library are shown in Table 3.
• Table 3 AtPPOl mutations selected for inclusion in a combinatorial library. A percentage frequency of each mutation within the library is shown.
• the combinatorial library was transformed into E. coli BL21(DE3) hemG and selected for increased tolerance to herbicides as described in Example 1 except for the herbicide concentrations being increased to 25ppm (herbicides B and D) and 50ppm (herbicide E). Plasmids were isolated from herbicide-tolerant strains and sequenced to identify the causal mutation in the AtPPOl gene carried by the pET24 plasmid in each strain. All herbicide- tolerant variants identified are in Table 4.
• Table 4 AtPPOl variants with multiple mutations selected on the basis of strong growth in the presence of herbicide.
• Example 3 Expression and assay of selected Arabidopsis thaliana PPO1 variants and percent-inhibition values versus herbicides A to E.
• This plasmid was expressed in E. coli BL21 (DE3) hemG with 50 pg/mL kanamycin and 25 pg/mL chloramphenicol selection.
• Overnight cultures grown at 37 °C were used to inoculate 150ml AIM (AIM-Terrific Broth Base including Trace elements, Formedium) in 500ml shake flasks at a ratio of 1 :100.
• AIM AIM-Terrific Broth Base including Trace elements, Formedium
• the lysates were centrifuged at 13,00rpm for 10min at 4°C.
• the crude extracts i.e, supernatants
• Protein concentration was measured and calculated using the absorbance at 280 nm. Extracts were diluted 1 in 4 and 10 uL was analysed by SDS PAGE.
• Variant crude extracts were assayed in a fluorescent Protoporphyrinogen Oxidase assay in black 384 well plates with a clear bottom.
• the total volume of the reaction mixture was 100ul and contained 100mM K2HPO4 pH7.2, 1mM Na2EDTA, 5mM DTT, 20% glycerol (v/v), 0.025% Tween 20 (v/v).
• Reactions were started with addition of 1uM protoporphyrinogen IX final assay concentration. Fluorescence was monitored over 30min using a Tecan plate reader with an excitation wavelength of 405 nm, emission wavelength of 635 nm, gain of 90 and 15 flashes.
• the percentage inhibition was calculated as the activity in the presence of one of four concentrations of each herbicide compound A to E (note that the compounds labelled as A to E are identified hereinabove in the relevant section of the description) as a percentage of the enzyme activity in DMSO (Table 5).
• the selected variants showed improved tolerance to at least one of the PPO inhibiting herbicides A to E compared to the parental gene AtPPOl (SEQ ID NO:1) or previously known PPO1 variant (SEQ ID NO:2).
• AtPPOl S305L Y426V
• SEQ ID NO: 125 the improvement in herbicide tolerance was seen across all herbicides tested.
• differential tolerances are seen to the tested herbicides, such as with AtPPOl Y361D V365L (SEQ ID NO: 135) where the improvement over SEQ ID NO: 124 was seen for some of the herbicides (i.e. B, C, D and E) but not for others (i.e. herbicide A).
• AtPPOl SEQ ID NO: 370
• AtPPOl L479M SEQ ID NO: 371
• AtPPOl L479N SEQ ID NO: 372
• AtPPOl S305L Y426V SEQ ID NO: 374
• AtPPOl S305L Y426V L479M SEQ ID NO: 373
• This plasmid was expressed in E. coli BL21 (DE3) with 50 pg/mL kanamycin selection. 10 mL of overnight cultures grown at 37 °C were used to inoculate 1 L AIM (AIM-Terrific Broth Base including Trace elements, Formedium) in 2 L shake flasks.
• AIM AIM-Terrific Broth Base including Trace elements, Formedium
• lysates were centrifuged at 20,000 rpm for 25 min at 4°C. Cleared lysates were loaded on His GraviTrap (Cytiva) columns, washed with 20 mL of lysis buffer and eluted in elution buffer (20 mM Tris pH8, 500 mM sodium chloride, 10% glycerol, 200 mM imidazole, 0.03 % (v/v) Triton X-100). The eluate was desalted on a PD10 column and 2.5-3.2 mL was collected. Protein concentration was measured and calculated using the absorbance at 280 nm. Extracts were diluted 1 in 4 and 10 uL was analysed by SDS PAGE.
• Protoporphyrinogen Oxidase enzymes were assayed for activity using fluorescence adapted from the method of Shepherd & Dailey (Anal Biochem. 2005 344(1) 115-121).
• the assay was performed in black 384 well plates with a clear bottom and the total volume of the reaction mixture was 91 ul.
• the final assay mixture contained 100mM K2HPO4 pH7.2, 1mM Na2EDTA, 5mM DTT, 20% glycerol (v/v), 0.05% Tween 20 (v/v), 1.1% DMSO (v/v), 1uM protoporphyrinogen IX and 1.5nM enzyme.
• AtPPOl V002 SEQ ID NO: 373
• AtPPOl V030 SEQ ID NO: 374
• AtPPOl V002 (SEQ ID NO: 373) has an additional L479M substitution that AtPPO V030 (SEQ ID NO: 374) lacks.
• One mechanism by which substitutions at L479 may enhance tolerance is by increasing the PPO1 enzyme rate of activity (Table 7).
• Table 6 IC50 (nM) of Protoporphyrinogen Oxidase variants treated with different PPO inhibitors. ⁇ 1.0 is below detection limit of assay.
• Table 7 Activity rates of Protoporphyrinogen Oxidase variants in in vitro assay.
• Arabidopsis thaliana PPO1 or orthologues of this for example SEQ ID NOs: 1 and 124 to 137 or Setaria italica PPO1 or orthologues of this, for example SEQ ID NOs: 2 and 138-151 are expressed in transgenic tobacco.
• DNA sequences that encode these polypeptides are prepared synthetically. Each sequence is designed to include a 5’ fusion with TMV omega 5’ leader sequence and such that they are flanked at the 5’ end with Xhoi and at the 3’ end with Kpni to facilitate direct cloning into a suitable binary vector for Agrobacterium-based plant transformation.
• the expression cassette comprising the TMV omega 5’ leader and a PPO1 encoding gene of interest is excised using Xhol/ Kpni and cloned into similarly digested pBIN 19 (Bevan, Nucleic Acids Res. 12:8711-8721 (1984) to create a binary vector, e.g. the vector of Figure 1, (SEQ ID NO: 152) behind a double enhanced 35S promoter ahead of a NOS 3’ transcription terminator and then transformed into E. coli DH5 alpha competent cells.
• DNA recovered from the E. coli is used to transform Agrobacterium tumefaciens LBA4404, and the transformed bacteria are selected on media contain rifampicin and kanamycin.
• Tobacco tissue is subjected to Agrobacterium-mediated transformation using methods well described in the art or as described herein.
• a master plate of Agrobacterium tumefaciens containing the PPO1 expressing binary vector is used to inoculate 10 ml LB (L broth) containing 100 mg/l rifampicin plus 50 mg/l kanamycin using a single bacterial colony. This is incubated overnight at 28 °C shaking at 200 rpm. This entire overnight culture is used to inoculate a 50 ml volume of LB containing the same antibiotics. Again, this is cultured overnight at 28 °C shaking at 200 rpm.
• Explants are then removed, dabbed on sterile filter paper to remove excess suspension, then transferred onto solid NBM medium (MS medium containing 30 g/l sucrose, 1 mg/l BAP (benzylaminopurine) and 0.1 mg/l NAA (napthalene acetic acid) at pH 5.9 and solidified with 8 g/l Plantagar), with the abaxial surface of each explant in contact with the medium. Approximately 7 explants are transferred per plate, which are then sealed and maintained in a lit incubator at 25 °C for a 16 hour photoperiod for 3 days.
• MS medium MS medium containing 30 g/l sucrose, 1 mg/l BAP (benzylaminopurine) and 0.1 mg/l NAA (napthalene acetic acid) at pH 5.9 and solidified with 8 g/l Plantagar
• Explants are then transferred onto NBM medium containing 100 mg/l kanamycin plus antibiotics to prevent further growth of Agrobacterium (200 mg/l timentin with 250 mg/l carbenicillin). Further subculture onto this same medium was then performed every 2 weeks.
• Transformed shoots are divided into 2 or 3 clones and regenerated from kanamycin resistant callus.
• Shoots are rooted on MS agar containing kanamycin.
• Surviving rooted explants are re-rooted to provide approximately 40 - 50 kanamycin resistant and PCR positive events from each event.
• Transgenic populations of about forty tobacco plants that comprise a gene encoding a full length Arabidopsis thaliana PPO1 gene (SEQ ID NO: 1) or variants (SEQ ID NO:1 and 124 to 137) or encoding a full length Setaria italica PPO1 gene (SEQ ID NO: 2) or variants (SEQ ID NO: 138-151) are thus produced. Plants are selected on the basis of similar size from each population and ELISA or Mass Western tests are carried out to monitor protein transgenic PPO expression levels. The highest expressing T0 lines are selected to be taken forward to self and to generate T1 seed and T2 lines and seed in the normal way.
• Seeds from the highest expressing lines are tested for germination on agar plates containing a range of concentrations of PPO-inhibiting herbicides as taught for example herein and resistant plant lines selected as showing the least damage to root or leaf growth and morphology at the highest concentrations of herbicides.
• Resistant plant lines exhibit a dose response in respect of herbicidal damage by PPO1 inhibitors that is shifted to the right in comparison with similarly grown and treated wild type and null segregant plants.
• Example 5 Assay of herbicide tolerance in transgenic tobacco plants expressing heterologous AtPPO1 variants or heterologous SiPPO1 variants.
• the Setaria italica PPO1s (SEQ ID NO: 2 or 138-151) or any one of SEQ ID NO: 1, 2, 4 – 151, 155-302, or 305 – 336 confer resistance to PPO inhibiting compounds when expressed in plants
• GM tobacco lines are produced and tested with PPO inhibiting compounds.
• Transgenic tobacco plants expressing the Arabidopsis thaliana PPO1 gene and variants of (SEQ ID NOs 1 and 124-137) are produced and transgenic tobacco plants expressing the Setaria italica PPO1 gene and variants of (SEQ ID NOs 2 and 138-151) are produced.
• Populations of transgenic tobacco comprising 20-30 transgenic events per plant transformation constructs are generated as described in Example 4.
• Example 5A Assay of herbicide tolerance in transgenic tobacco plants expressing heterologous AtPPO1 variants
• Data for herbicide tolerance in transgenic tobacco plants was generated according to Example 5 above and is shown in Tables 11-24.
• Herbicidal damage was visually assessed across the population and a herbicide damage score given at 7 and 14 days. A score of 0 indicates no visible damage or stunting whereas a score of 100 indicates a complete death of the plant.
• 20 transgenic events i.e. individual transgenic plants
• WT tobacco is included as a control.
• Table 8 shows the treatment regimens used. Table 8: Treatment regimens for testing PPO inhibitor resistance
• Table 9 Plants transformed with Arabidopsis PPO gene variants.
• Table 11 Results of percentage of damage to tobacco plants containing pBin TMV AtPPOI .
• Transgenic corn lines expressing AtPPOl and variants (SEQ ID NO:1 and 124-137) and SiPPOl and variants (SEQ ID NO:2 and 138-151) are created using methods known in the art.
• Agrobacterium strain LBA4404 comprising an expression vector expressing the disclosed AtPPOl and variants (SEQ ID NO: 1 and 124-137) and SiPPOl and variants (SEQ ID NO: 2 and 138- 151) is grown on YEP (yeast extract (5 g/L), peptone (1 Og/L), NaCI (5g/L), 15g/l agar, pH 6.8) solid medium for 2- 4 days at 28°C. Approximately 0.8X 10 9 Agrobacterium cells are suspended in LS-inf media supplemented with 100 pM As. Bacteria are pre-induced in this medium for approximately 30-60 minutes.
• Immature embryos from an inbred maize line are excised from 8-12 day old ears into liquid LS-inf + 100 pM As. Embryos are rinsed once with fresh infection medium. Agrobacterium solution is then added, and embryos are vortexed for 30 seconds and allowed to settle with the bacteria for 5 minutes. The embryos are then transferred scutellum side up to LSAs medium and cultured in the dark for two to three days. Subsequently, between approximately 20 and 25 embryos per petri plate are transferred to LSDc medium supplemented with cefotaxime (250 mg/l) and silver nitrate (1.6 mg/l) and cultured in the dark at approximately 28°C for 10 days.
• Immature embryos, producing embryogenic callus are transferred to LSD1M0.5S medium. The cultures are selected on this medium for approximately 6 weeks with a subculture step at about 3 weeks. Surviving calli are transferred to Reg1 medium supplemented with mannose. Following culturing in the light (16 hour light/ 8 hour dark regiment), green tissues are then transferred to Reg2 medium without growth regulators and incubated for about 1-2 weeks. Plantlets are transferred to Magenta GA-7 boxes (Magenta Corp, Chicago III.) containing Reg3 medium and grown in the light. After about 2-3 weeks, plants are tested for the presence of the disclosed PPO genes by PCR. Positive plants from the PCR assay are transferred to a greenhouse for further evaluation.
• Plants are treated with PPO inhibiting herbicides to confirm the herbicide resistance.
• Example 7 Transformation of Soybean with AtPPOl or SiPPOl variants.
• Soybean plant material can be suitably transformed and fertile plants regenerated by many methods which are well known to one of skill in the art.
• fertile morphologically normal transgenic soybean plants may be obtained by: 1) production of somatic embryogenic tissue from, e.g., immature cotyledon, hypocotyl or other suitable tissue; 2) transformation by particle bombardment or infection with Agrobacterium; and 3) regeneration of plants.
• somatic embryogenic tissue from, e.g., immature cotyledon, hypocotyl or other suitable tissue
• transformation by particle bombardment or infection with Agrobacterium and 3) regeneration of plants.
• cotyledon tissue is excised from immature embryos of soybean, optionally with the embryonic axis removed, and cultured on hormone-containing medium so as to form somatic embryogenic plant material.
• This material is transformed using, for example, direct DNA methods, DNA coated microprojectile bombardment or infection with Agrobacterium, cultured on a suitable selection medium and regenerated, optionally also in the continued presence of selecting agent, into fertile transgenic soybean plants.
• Selection agents may be antibiotics such as kanamycin, hygromycin, or herbicides or, alternatively, selection may be based upon expression of a visualisable marker gene such as GUS.
• Target tissues for transformation include meristematic tissue, somaclonal embryogenic tissue, and flower or flower-forming tissue.
• Other examples of soybean transformation include physical DNA delivery methods, such as particle bombardment (see e.g., Finer & McMullen, In Vitro Cell Dev.
• Soybean transgenic plants can be generated with a binary vector containing AtPPOl and variants (SEQ ID NO:1 and 124-137) or SiPPOl and variants (SEQ ID NO:2 and 138-151) using any available transformation method.
• the PPO gene can provide the means of selection and identification of transgenic tissue.
• a vector is used to transform immature seed targets as described to generate transgenic PPO soybean plants directly using PPO inhibitor, as selection agent.
• a PPO gene can be present in the polynucleotide alongside other sequences which provide additional means of selection/ identification of transformed tissue including, for example, the known genes which provide resistance to kanamycin, hygromycin, phosphinothricin, butafenacil, or glyphosate.
• different binary vectors containing PAT or EPSPS selectable marker genes are known in the art (see e.g., U.S. Patent Application Publication No. 20080229447).
• selectable marker sequences may be present on separate polynucleotides and a process of, for example, cotransformation and co-selection is used.
• a scorable marker gene such as GUS may also be used to identify transformed tissue.
• TO plants are taken from tissue culture to the greenhouse where they are transplanted into water- saturated soil (REDI-EARTH® Plug and Seedling Mix, Sun Gro Horticulture, Bellevue, WA, or Fafard Germinating Mix) mixed with 1 % granular MARATHON® (Olympic Horticultural Products, Co., venue, PA) at 5-10 g/gal soil in 2" square pots.
• the plants are covered with humidity domes and placed in a Conviron chamber (Pembina, ND) with the following environmental conditions: 24°C day; 20°C night; 16-23 hours light- 1-8 hours dark photoperiod; 80% relative humidity.
• plants are sampled and tested for the presence of desired transgene by TAQMAN® analysis using appropriate probes for the PPG genes or promoters. Positive plants are transplanted into 4" square pots containing Fafard #3 soil. Sierra 17-6-12 slow release fertilizer is incorporated into the soil at the recommended rate. The plants are then relocated into a standard greenhouse to acclimatize ( ⁇ 1 week). The environmental conditions are: 27°C day; 21°C night; 14 hour photoperiod (with supplemental light); ambient humidity. After acclimatizing ( ⁇ 1 week), the plants are sampled and tested in detail for the presence and copy number of inserted transgenes. Transgenic soybean plants are grown to maturity for TI seed production.
• Tl plants are grown up, and are tested for the presence of the disclosed PPO genes by PCR. Positive plants from the PCR assay are transferred to a greenhouse for further evaluation. After TAQMAN® analysis, homozygous plants were grown for seed production. Transgenic seeds and progeny plants are used to further evaluate their herbicide tolerance performance and molecular characteristics
• T 1 screening For each event, 36-54 seeds per event were sown. At 10 days after sowing, leaf tissue of all seeds that successfully germinated was sampled for TAQMAN® as noted in Example 7 and zygosity determined. Chi-square analyses were performed on all zygosity data to determine segregation pattern of each event.
• Arabidopsis thaliana PPO1 (with transit peptide) (SEQ ID NO:1)
• Arabidopsis thaliana PPO1 V127I (SEQ ID NO:8) MELSLLRPTTQSLLPSFSKPNLRLNVYKPLRLRCSVAGGPTVGSSKIEGGGGTTITTDCVIV
• Arabidopsis thaliana PPO1 Y219S (SEQ ID NO:19) MELSLLRPTTQSLLPSFSKPNLRLNVYKPLRLRCSVAGGPTVGSSKIEGGGGTTITTDCVIV
• Arabidopsis thaliana PPO1 A220C (SEQ ID NO:20)
• Arabidopsis thaliana PPO1 L360C (SEQ ID NQ:30) MELSLLRPTTQSLLPSFSKPNLRLNVYKPLRLRCSVAGGPTVGSSKIEGGGGTTITTDCVIV GGGISGLCIAQALATKHPDAAPNLIVTEAKDRVGGNIITREENGFLWEEGPNSFQPSDPMLT MVVDSGLKDDLVLGDPTAPRFVLWNGKLRPVPSKLTDLPFFDLMSIGGKIRAGFGALGIRPS
• Arabidopsis thaliana PPO1 Y361 D (SEQ ID NO:31)
• Arabidopsis thaliana PPO1 Y361 F (SEQ ID NO:32)
• Arabidopsis thaliana PPO1 Y361 P (SEQ ID NO:33)
• Arabidopsis thaliana PPO1 Y361Q (SEQ ID NO:34)
• Arabidopsis thaliana PPO1 Y361 R (SEQ ID NO:35)
• Arabidopsis thaliana PPO1 Y361T (SEQ ID NO:36)
• Arabidopsis thaliana PPO1 Y362F (SEQ ID NO:37)
• Arabidopsis thaliana PPO1 Y365L (SEQ ID NO:38)

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