Proporphyrinogen oxidase (PPO) resistant plants

Abstract

The present application discloses transgenic plants expressing the fungal protoporphyrinogen oxidase (PPO), and methods of conferring herbicide tolerance (HT) to PPO type inhibitors and controlling weeds by selective protection of transgenic crop crops expressing the fungal PPO enzyme.

Description

[Field of the Invention]

[0001] The present invention relates to transgenic plants and methods for conferring herbicide tolerance (HT) to protoporphyrinogen oxidase (PPO) type inhibitors and controlling weeds.

[0002]

background

[0003] Weeds have been a major cause of crop yield losses since the origins of agriculture.

[0004] Worldwide, weeds compete with crops for space, nutrients, water and light, causing damage estimated to be equivalent to a loss of about one-third of potential crop yield.

[0005] Weeds also compete with pastures, reducing productive land to unusable scrub. In addition, weeds can be toxic, offensive, produce burrs, thorns, or interfere with the use and management of desirable plants by contaminating harvests or interfering with livestock.

[0006] Herbicides are the most commonly used and effective means of controlling weeds. However, herbicide resistance is increasing, and hundreds of weed biotypes have been identified that are resistant to one or more herbicides.

[0007] Protoporphyrinogen oxidase inhibitors (PPO-inhibitors) are a class of herbicides that target the protoporphyrinogen oxidase (PPO) enzyme, which is active in chloroplasts and mitochondria and is involved in the catalysis of the organic compound protoporphyrin IX - a precursor of heme and chlorophyll. Inhibition interferes with the biosynthesis of heme (WSSA group: 14; HRAC group: E) and ultimately leads to the accumulation of peroxidants, which can cause cell membrane rupture. Therefore, PPO inhibitors hinder the growth of plants and are also called cell membrane disruptors.

[0008] Herbicides that act by inhibiting PPO are well known and include the following herbicide families: phenylpyrazoles, pyrimidinediones, oxazolidinediones, diphenyl ethers, oxadiazoles, N-phenylphthalimides, triazinones, thiadiazoles, triazolinones and triazolopyridones, which are commercially known under names such as Star, Goal, Strike and Aurora. Such herbicides include the active compounds fluazifop-butyl, fomesafen, metsulfuron-butyl, foramsulfuron-butyl, sulfentrazone, acifluorfen, lactofen-butyl, flufenacet-butyl, saflufenacil or others.

[0009] PPO inhibitors can be used in the growing process of crops, provided that the crops are resistant / tolerant to the herbicides. Therefore, the treatment of plants that are susceptible to PPO inhibition (such as but not limited to broadleaf plants) is limited. It is particularly difficult to achieve resistance that provides commercial tolerance levels to at least some desirable PPO-type inhibitors. Therefore, new methods are needed to impart tolerance to PPO herbicides to various crops and crop varieties. [Summary of the invention]

[0010] Transgenic plants and methods for conferring protoporphyrin oxidase (PPO) type inhibitor herbicide tolerance (HT) and controlling weeds by selectively protecting transgenic crops expressing fungal derived PPO enzymes are disclosed.

[0011] Advantageously, disclosed are transgenic plants, cells and seeds that have been genetically modified with an exogenous fungal-derived PPO enzyme, variant, mutant, motif, fragment and / or protein fusion thereof, which when expressed in the plant provide resistance or tolerance to PPO-type inhibitor herbicides, thereby conferring on the plant the desirable trait of herbicide tolerance (HT).

[0012] Advantageously and surprisingly, while the structure and sequence of the residues making up the herbicide pocket showed a high degree of similarity, several key structural / geometric differences were identified that may explain the favorable differences in the herbicide tolerance activity of the fungal PPO enzymes of the present invention relative to human PPOX or plant PPO.

[0013] The present invention includes amino acid sequences encoding PPO polypeptides derived from or isolated from fungi and having enzymatic activity and complementing the endogenous PPO enzyme of the plant.

[0014] In some embodiments, the fungus-derived PPO comprises variants, mutants, motifs, fragments and / or protein fusions thereof.

[0015] According to certain aspects, a transgenic plant expressing a protoporphyrinogen oxidase (PPO) from an exogenous fungus is provided, wherein the PPO from the exogenous fungus comprises an amino acid sequence having at least 80% similarity to at least one sequence shown in any one of SEQ ID NOs: 2, 94, 96 to 98, or any combination thereof, wherein the PPO from the fungus comprises no more than The pocket opening size is determined by comparing a simulated 3D structure / fold of a PPO of fungal origin to a crystal structure of human PPOX bound to acifluorfen, wherein the simulated 3D structure / fold of the PPO of fungal origin is structurally aligned with the crystal structure of human PPO and overlaps with acifluorfen, wherein the PPO of fungal origin confers resistance or tolerance to at least one PPO inhibitor herbicide.

[0016] In some embodiments, the exogenous fungus-derived PPO comprises an amino acid sequence having at least 90% similarity to any one of SEQ ID NOs: 2, 94, 96-98, or any combination thereof.

[0017] In some embodiments, the bag opening size of any one of the fungal-derived PPOs comprises at least difference.

[0018] According to some embodiments, the exogenous fungal-derived PPO comprises: a first arginine, which structurally corresponds to the position of arginine at position 97 (human Arg97) in human PPOX, and / or a second arginine, which structurally corresponds to the position of arginine at position 62 (human Arg62) in human PPOX, wherein the first and / or second arginine of the fungal-derived PPO has a geometric orientation different from the geometric orientation of human Arg62 and / or human Arg97, which is determined by comparing the simulated 3D structure / fold of the fungal-derived PPO with the crystal structure of human PPOX bound to trifluorfen, wherein the simulated 3D structure / fold of the fungal-derived PPO is structurally aligned with the crystal structure of human PPO and overlaps with trifluorfen.

[0019] In some embodiments, the different geometric orientations include different distances between the first arginine residue of the fungal-derived PPO and the overlapping trifluorfen, wherein the distances include a first distance (d1) between the guanidinium group of the first arginine residue of the fungal-derived PPO and the C11 hydroxyl group of the overlapping trifluorfen, wherein d1 is greater than

[0020] In some embodiments, the different geometric orientations include different distances between the second arginine residue of the fungal-derived PPO and the overlapping trifluorfen, wherein the distance includes a second distance (d2) between the guanidinium group of the second arginine residue of the fungal-derived PPO and the carbonyl oxygen of C11 of the overlapping trifluorfen, wherein d2 is greater than

[0021] In some embodiments, the sum of the distances d1 and d2 is greater than about

[0022] In some embodiments, the sum of the distances d1 and d2 of the fungal PPO differs from the sum of the distances d1 and d2 of the human PPOX by more than about Wherein d1 and d2 of human PPOX are the distance between the guanidinium group of human Arg97 and the hydroxyl group of C11 of trifluorfen (d1 human PPOX) and the distance between the guanidinium group of human Arg62 and the carbonyl group of C11 of trifluorfen, (d2 human PPOX), which are determined by comparing the simulated 3D structure / fold of fungal-derived PPO with the crystal structure of human PPOX bound to trifluorfen, wherein the simulated 3D structure / fold of the fungal-derived PPO is structurally aligned with the crystal structure of human PPO and overlaps with trifluorfen.

[0023] In some embodiments, the exogenous fungus-derived PPO comprises an amino acid sequence having at least 80% similarity to at least one sequence shown in any one of SEQ ID NOs: 2, 94, 96-98, or any combination thereof.

[0024] In some embodiments, the exogenous fungus-derived PPO comprises an amino acid sequence having at least 90% similarity to at least one sequence shown in any one of SEQ ID NOs: 2, 94, 96-98, or any combination thereof.

[0025] According to some embodiments, the exogenous fungal-derived PPO comprises a phenylalanine at position 404 (F404), which structurally corresponds to a valine at position 347 (V347) of human PPOX, as determined by comparing a simulated 3D structure / fold of the fungal-derived PPO with a crystal structure of human PPOX bound to trifluorfen, wherein the simulated 3D structure / fold of the fungal-derived PPO is structurally aligned with the crystal structure of human PPO and overlaps with trifluorfen.

[0026] According to some embodiments, the exogenous fungal-derived PPO comprises a phenylalanine at position 383 (F383), which structurally corresponds to the phenylalanine at position 331 (F331) of human PPOX, which is determined by comparing the simulated 3D structure / fold of the fungal-derived PPO with the crystal structure of human PPOX bound to trifluorfen, wherein the simulated 3D structure / fold of the fungal-derived PPO is structurally aligned with the crystal structure of human PPO and overlaps with trifluorfen.

[0027] In some embodiments, the exogenous fungal-derived PPO comprises an amino acid sequence having at least 80% similarity to the sequence shown in SEQ ID NO:96.

[0028] In some embodiments, the exogenous fungal-derived PPO comprises an amino acid sequence having at least 90% similarity to the sequence shown in SEQ ID NO:96.

[0029] In some embodiments, the exogenous fungal-derived PPO confers resistance or tolerance to at least one PPO inhibitor herbicide.

[0030] In some embodiments, the one or more PPO inhibitors are selected from the following herbicide families: phenylpyrazoles, pyrimidinediones, oxazolidinediones, diphenyl ethers, oxadiazoles, N-phenylphthalimides, triazinones, thiadiazoles, triazolinones, and triazolopyridones, or any combination thereof.

[0031] In some embodiments, the fungal-derived PPO belongs to a fungal species of the clade Leotiomyceta.

[0032] In some embodiments, the fungal-derived PPO belongs to a fungal species selected from the group consisting of Aspergillus sp., Penicillium sp., Trichoderma sp., Rhodotorula sp., and Hesseltinella sp.

[0033] In some embodiments, the exogenous fungal-derived PPO has less than 40% similarity in amino acid sequence to the endogenous plant PPO enzyme.

[0034] In some embodiments, the exogenous fungal-derived PPO enzyme further comprises a plant chloroplast transit peptide.

[0035] In some embodiments, the plant is selected from the group consisting of: corn, wheat, rice, barley, soybean, cowpea, chickpea, cotton, sorghum, beans, rapeseed, alfalfa, flax, sunflower, safflower, millet, rye, sugarcane, sugar beet, cocoa, tea, Brassica, coffee, sweet potato, flax, peanut, clover; lettuce, tomato, gourd, cassava, potato, carrot, radish, pea, lentil, cabbage, cauliflower, broccoli, Brussels sprouts, pepper, pineapple, citrus, apple, pear, peach, apricot, walnut, avocado, banana, palm, eucalyptus, poplar, pine, coconut, orchid, morning glory, carnation, rose, switch grass, prairie grass, Indian grass, big bluestem grass, camellia, or any combination thereof.

[0036] In some embodiments, the plant is selected from the group consisting of corn, wheat, rice, soybean, cowpea, chickpea, cotton, sorghum, rapeseed, alfalfa, sunflower, sugarcane, sugar beet, camellia, or any combination thereof.

[0037] In some embodiments, the one or more PPO inhibitors are selected from the following herbicide families: phenylpyrazoles, pyrimidinediones, oxazolidinediones, diphenyl ethers, oxadiazoles, N-phenylphthalimides, triazinones, thiadiazoles, triazolinones, and triazolopyridones, or any combination thereof.

[0038] In some embodiments, the plant is a broadleaf plant.

[0039] According to some embodiments, the comparison of the 3D structure / fold of the fungal derived PPO to the crystal structure of human PPO bound to acifluorfen comprises comparing a modeled structure of the fold of the fungal derived PPO or a crystal structure showing the fold of the fungal derived PPO.

[0040] According to certain aspects, a transgenic plant is provided, which expresses an exogenous fungal-derived protoporphyrinogen oxidase (PPO) that confers resistance or tolerance to at least one PPO inhibitor herbicide, wherein the fungal-derived PPO enzyme comprises an amino acid sequence having at least 80% similarity to at least one sequence shown in any one of SEQ ID NOs: 1 to 7 and SEQ ID NOs: 86, 88 to 89, 91 to 99, or any combination thereof.

[0041] In some embodiments, the fungal PPO enzyme comprises an amino acid sequence having at least 90% similarity to at least one of SEQ ID NOs: 1-7 and SEQ ID NOs: 86, 88-89, 91-99, or any combination thereof.

[0042] According to some embodiments, the fungal-derived PPO enzyme comprises no more than The pocket opening size was determined by comparing the simulated 3D structure / fold of the fungal-derived PPO to the crystal structure of human PPOX bound to trifluorfen, wherein the simulated 3D structure / fold of the fungal-derived PPO was structurally aligned with the crystal structure of human PPO and overlapped with trifluorfen.

[0043] According to some embodiments, the exogenous fungal-derived PPO comprises: a first arginine, which structurally corresponds to the position of arginine at position 97 (human Arg97) in human PPOX, and / or a second arginine, which structurally corresponds to the position of arginine at position 62 (human Arg62) in human PPOX, wherein the first and / or second arginine of the fungal-derived PPO has a geometric orientation different from the geometric orientation of human Arg62 and / or human Arg97, which is determined by comparing the simulated 3D structure / fold of the fungal-derived PPO with the crystal structure of human PPOX bound to trifluorfen, wherein the simulated 3D structure / fold of the fungal-derived PPO is structurally aligned with the crystal structure of human PPO and overlaps with trifluorfen.

[0044] According to some embodiments, the exogenous fungal-derived PPO comprises a phenylalanine at position 404 (F404), which structurally corresponds to a valine at position 347 (V347) of human PPOX, as determined by comparing a simulated 3D structure / fold of the fungal-derived PPO with a crystal structure of human PPOX bound to trifluorfen, and wherein the simulated 3D structure / fold of the fungal-derived PPO is structurally aligned with the crystal structure of human PPO and overlaps with trifluorfen.

[0045] According to some embodiments, the exogenous fungal-derived PPO comprises a phenylalanine at position 383 (F383), which structurally corresponds to the phenylalanine at position 331 (F331) of human PPOX, which is determined by comparing the simulated 3D structure / fold of the fungal-derived PPO with the crystal structure of human PPOX bound to trifluorfen, wherein the simulated 3D structure / fold of the fungal-derived PPO is structurally aligned with the crystal structure of human PPO and overlaps with trifluorfen.

[0046] In some embodiments, the fungal-derived PPO comprises an amino acid sequence that has less than 40% similarity to the amino acid sequence of an endogenous plant PPO enzyme.

[0047] In some embodiments, the fungal-derived PPO enzyme further comprises a plant chloroplast transit peptide.

[0048] In some embodiments, the PPO of fungal origin belongs to a fungal species selected from the group consisting of Aspergillus sp., Fusarium sp., Rhizopus sp., Penicillium sp., Trichoderma sp., Rhodotorulasp., Hesseltinella sp., Spizellomyces sp., Rhizophagus sp., Alternaria sp., and Cladosporium sp.

[0049] In some embodiments, the fungal-derived PPO enzyme belongs to a fungal species selected from the group consisting of Aspergillus, Fusarium, Rhizopus, Penicillium, and Trichoderma.

[0050] In some embodiments, the fungal-derived PPO belongs to a fungal species selected from the group consisting of Aspergillus sp., Penicillium sp., Trichoderma sp., Rhodotorula sp., and Hesseltinella sp.

[0051] In some embodiments, the PPO enzyme of fungal origin belongs to a fungal species of the clade Leotiomyceta.

[0052] In some embodiments, the plant is selected from the group consisting of: corn, wheat, rice, barley, soybean, cowpea, chickpea, cotton, sorghum, beans, rapeseed, alfalfa, flax, sunflower, safflower, millet, rye, sugarcane, sugar beet, cocoa, tea, Brassica, coffee, sweet potato, flax, peanut, clover; lettuce, tomato, gourd, cassava, potato, carrot, radish, pea, lentil, cabbage, cauliflower, broccoli, Brussels sprouts, pepper, pineapple, citrus, apple, pear, peach, apricot, walnut, avocado, banana, palm, eucalyptus, poplar, pine, coconut, orchid, morning glory, carnation, rose, switch grass, prairie grass, Indian grass, big bluestem grass, camellia, or any combination thereof.

[0053] In some embodiments, the plant is selected from the group consisting of corn, wheat, rice, soybean, cowpea, chickpea, cotton, sorghum, rapeseed, alfalfa, sunflower, sugarcane, sugar beet, camellia, or any combination thereof.

[0054] In some embodiments, the one or more PPO inhibitors are selected from the following herbicide families: phenylpyrazoles, pyrimidinediones, oxazolidinediones, diphenyl ethers, oxadiazoles, N-phenylphthalimides, triazinones, thiadiazoles, triazolinones, and triazolopyridones, or any combination thereof.

[0055] In some embodiments, the plant is a broadleaf plant.

[0056] In some embodiments, the plant is a cereal plant.

[0057] According to some aspects, there is provided a seed of a transgenic plant according to any of the above embodiments.

[0058] According to some aspects, according to any of the above embodiments, there is provided a product derived from a transgenic plant.

[0059] According to some aspects, a method is provided for controlling weed growth using at least one PPO inhibitor herbicide in a location where crop plants are grown, wherein the crop plants include a transgenic plant according to any one of embodiments 1 to 36, and the method includes applying a weed controlling amount of one or more PPO inhibitor herbicides.

[0060] In some embodiments, the one or more PPO inhibitors are selected from the following herbicide families: phenylpyrazoles, pyrimidinediones, oxazolidinediones, diphenyl ethers, oxadiazoles, N-phenylphthalimides, triazinones, thiadiazoles, triazolinones, and triazolopyridones, or any combination thereof.

[0061] According to certain aspects, a method for conferring resistance or tolerance to one or more PPO inhibitor herbicides in a crop plant is provided, the method comprising genetically modifying the plant to express an exogenous fungal-derived PPO enzyme, wherein the fungal-derived PPO enzyme comprises an amino acid sequence having at least 80% similarity to at least one sequence shown in any one of SEQ ID NOs: 1-7 or SEQ ID NOs: 86, 88-89, 91-99, or any combination thereof.

[0062] According to some embodiments, provided herein are transgenic plants expressing an exogenous fungal-derived protoporphyrinogen oxidase (PPO) that confers resistance or tolerance to at least one PPO inhibitor herbicide.

[0063] According to some embodiments, the fungus-derived PPO enzyme comprises an amino acid sequence having at least 70% similarity to the PPO isolated from the fungus.

[0064] According to some embodiments, the fungal-derived PPO enzyme comprises an amino acid sequence having at least 70%, at least 80% or at least 90% similarity to at least one fungal-isolated sequence shown in any one of SEQ ID NOs: 1-7 or 86-100 or any combination thereof. Each possibility is a separate embodiment.

[0065] According to some embodiments, the PPO isolated from fungi comprises any one or more sequences shown in any one of SEQ ID NOs: 1 to 7 or 86 to 100, or any combination thereof. Each possibility is a separate embodiment.

[0066] According to some embodiments, the PPO of fungal origin comprises at least one amino acid motif having at least 85% similarity to any one of SEQ ID NOs: 16 to 85 or any combination thereof. Each possibility is a separate embodiment.

[0067] According to some embodiments, the fungus-derived PPO comprises at least one amino acid motif having at least 95% similarity to at least one sequence shown in any one of SEQ ID NOs: 16 to 85 or any combination thereof. Each possibility is a separate embodiment.

[0068] According to some embodiments, the PPO of fungal origin comprises at least 5 amino acid motifs having at least 85% similarity to the sequence shown in any one of SEQ ID NOs: 16 to 85; and each motif is selected from a different group of the following: SEQ ID NOs: 16 to 22 or SEQ ID NOs: 23 to 29 or SEQ ID NOs: 30 to 36 or SEQ ID NOs: 37 to 43 or SEQ ID NOs: 44 to 50 or SEQ ID NOs: 51 to 57 or SEQ ID NOs: 58 to 64 or SEQ ID NOs: 65 to 71 or SEQ ID NOs: 72 to 78 or SEQ ID NOs: 79 to 85, or any combination thereof. Each possibility is a separate embodiment.

[0069] According to some embodiments, the PPO of fungal origin comprises 10 amino acid motifs having at least 85% similarity to the sequences shown in any one of SEQ ID NOs: 16 to 85 or any combination thereof; and wherein each motif is selected from a different group of the following: SEQ ID NOs: 16 to 22 or SEQ ID NOs: 23 to 29 or SEQ ID NOs: 30 to 36 or SEQ ID NOs: 37 to 43 or SEQ ID NOs: 44 to 50 or SEQ ID NOs: 51 to 57 or SEQ ID NOs: 58 to 64 or SEQ ID NOs: 65 to 71 or SEQ ID NOs: 72 to 78 or SEQ ID NOs: 79 to 85, or any combination thereof. Each possibility is a separate embodiment.

[0070] According to some embodiments, the fungal-derived PPO has less than 50% similarity to the amino acid sequence of the endogenous plant PPO enzyme, in some instances less than 45%, in some instances less than 40%, in some instances less than 35%, in some instances less than 30%, in some instances less than 25%, in some instances less than 20%, in some cases less than 15%, in some cases less than 10%, in some cases less than 5%. Each possibility is a separate embodiment.

[0071] According to some embodiments, the motif of the fungal-derived PPO has less than 50% similarity to the amino acid sequence of the endogenous plant PPO enzyme, in some instances less than 45%, in some instances less than 40%, in some instances less than 35%, in some instances less than 30%, in some instances less than 25%, in some cases less than 20%, in some cases less than 15%, in some cases less than 10%, in some cases less than 5%. Each possibility is a separate embodiment.

[0072] According to some embodiments, the RNA transcript of the fungal-derived PPO comprises introns or lacks introns.

[0073] According to some embodiments, the fungal-derived PPO enzyme further comprises a plant chloroplast transit peptide.

[0074] According to some embodiments, the fungal-derived PPO is selected from the group consisting of: Aspergillus sp., Fusarium sp., Rhizopus sp., Penicillium sp., Trichoderma sp., Rhodotorula sp., Hesseltinella sp., Spizel lomyces sp., Rhizophagus sp., Alternaria sp. and Cladosporium sp., or any combination thereof. Each possibility is a separate embodiment.

[0075] According to some embodiments, the fungal-derived PPO enzyme is isolated from a fungus of a genus selected from the group consisting of Aspergillus, Fusarium, Rhizopus and Penicillium, or any combination thereof. Each possibility is a separate embodiment.

[0076] According to some embodiments, the fungal-derived PPO enzyme is isolated from Aspergillus awamori, Aspergillus oryzae or Aspergillus melleus or any combination thereof. Each possibility is a separate embodiment.

[0077] According to some embodiments, the PPO enzyme of fungal origin is isolated from Fusarium venenatum or Fusarium fujikuroi or any combination thereof. Each possibility is a separate embodiment.

[0078] According to some embodiments, the fungal-derived PPO enzyme is isolated from Rhizopus arrhizus or Rhizopus stolonifer or any combination thereof. Each possibility is a separate embodiment.

[0079] According to some embodiments, the fungal-derived PPO enzyme is isolated from Penicillium oxalicum or Penicillium roqueforti or any combination thereof. Each possibility is a separate embodiment.

[0080] According to some embodiments, the fungal-derived PPO enzyme is isolated from Trichoderma reesei.

[0081] According to some embodiments, the fungal-derived PPO enzyme is isolated from Rhodotorula toruloide.

[0082] According to some embodiments, the fungal-derived PPO enzyme is isolated from Hesseltinel lavesiculosa.

[0083] According to some embodiments, the fungal-derived PPO enzyme is isolated from Spizel lomyces punctatus.

[0084] According to some embodiments, the fungal-derived PPO enzyme is isolated from Rhizophagus irregularis.

[0085] According to some embodiments, the transgenic plant is selected from the group consisting of: corn, wheat, rice, barley, soybean, cowpea, chickpea, cotton, sorghum, beans, rapeseed, alfalfa, flax, sunflower, safflower, millet, rye, sugarcane, sugar beet, cocoa, tea, Brassica, coffee, sweet potato, flax, peanut, clover; lettuce, tomato, gourd, cassava, potato, carrot, radish, pea, lentil, cabbage, cauliflower, broccoli, Brussels sprouts, pepper, pineapple, citrus, apple, pear, peach, apricot, walnut, avocado, banana, palm, eucalyptus, poplar, pine, coconut, orchid, morning glory, carnation, rose, switchgrass, prairie grass, Indian grass, big bluestem grass, camellia, or any combination thereof. Each possibility is a separate embodiment.

[0086] According to some embodiments, the transgenic plant is selected from the group consisting of: corn, wheat, rice, soybean, cowpea, chickpea, cotton, sorghum, rapeseed, alfalfa, sunflower, sugarcane, sugar beet, camellia, or any combination thereof. Each possibility is a separate embodiment.

[0087] According to some embodiments, the transgenic plant is selected from the group consisting of: corn, soybean, cotton, rapeseed, or any combination thereof. Each possibility is a separate embodiment.

[0088] According to some embodiments, the one or more PPO inhibitors are selected from the following herbicide families: phenylpyrazoles, pyrimidinediones, oxazolidinediones, diphenyl ethers, oxadiazoles, N-phenylphthalimides, triazinones, thiadiazoles, triazolinones and triazolopyridones, or any combination thereof. Each possibility is a separate embodiment.

[0089] According to some embodiments, the transgenic plant expressing exogenous fungal-derived protoporphyrinogen oxidase (PPO) is a broadleaf plant.

[0090] According to some embodiments, the transgenic plant expressing exogenous fungal-derived protoporphyrinogen oxidase (PPO) conferring resistance or tolerance to at least one PPO inhibitor herbicide is a seed.

[0091] According to some embodiments, the product derived from the transgenic plant expresses an exogenous fungal-derived protoporphyrinogen oxidase (PPO) that confers resistance or tolerance to at least one PPO inhibitor herbicide.

[0092] According to some embodiments, provided herein is a method of controlling weed growth in growing crops using at least one PPO inhibitor herbicide, wherein the crop plants comprise transgenic plants, the method comprising applying a weed controlling amount of one or more PPO inhibitor herbicides.

[0093] According to some embodiments, the one or more PPO inhibitors are selected from the following herbicide families: phenylpyrazoles, pyrimidinediones, oxazolidinediones, diphenyl ethers, oxadiazoles, N-phenylphthalimides, triazinones, thiadiazoles, triazolinones and triazolopyridones, or any combination thereof.

[0094] According to some embodiments, provided herein is a method of conferring resistance or tolerance to one or more PPO inhibitor herbicides in a crop plant, the method comprising genetically modifying the plant to express an exogenous fungal-derived PPO enzyme.

[0095] According to some embodiments, the fungus-derived PPO enzyme comprises an amino acid sequence having at least 70% similarity to at least one sequence shown in any one of SEQ ID NOs: 1-7.

[0096] Further embodiments, features, advantages and full scope of application of the present invention will be apparent from the detailed description and accompanying drawings given below. However, it should be understood that the detailed description, although showing the preferred embodiments of the present invention, is given only by way of illustration, because various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description.

Brief Description of the Figures

[0098] The invention will now be described in conjunction with certain embodiments and implementations with reference to the following schematic drawings.

[0099] Figure 1A-1B Shown are the results for WT Arabidopsis plants ( Figure 1A ) and transgenic plants expressing PPO isolated from fungi ( Figure 1B ) was used to evaluate the PPO tolerance.

[0100] Figure 1A A graph illustrating the susceptibility of Arabidopsis plants to Strike is shown. WT plants were treated with low concentrations of Strike (flumifenamid 50%, by Adama) at 0.000015% and 0.00003% diluted in water.

[0101] Figure 1B Evaluation of fungal isolated PPO tolerance genes expressed in transgenic plants is shown. Results of two experiments (Trials #1 and 2) using PPO-type inhibitor herbicides Strike (50% fluazifop-butyl, Adama) or Aurora (40% foramsulfuron, FMC) are shown, with application concentrations ranging from 0.01% to 1%, as shown. The tolerance level exhibited by transgenic plants was assessed using a plant resilience score, which is given on a scale of 0 to 5, where "0" represents a highly damaged plant and "5" represents a highly resistant plant. Genes isolated from fungi include PPOs from Aspergillus sp. (SEQ ID NO: 1, SEQ ID NO: 8 and SEQ ID NO: 2, SEQ ID NO: 9), Fusarium sp. (SEQ ID NO: 3, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12), Rhizopus sp. (SEQ ID NO: 6) and Penicillium sp. (SEQ ID NO: 14).

[0102] Figure 2A B shows an image of a transgenic plant expressing a fungal isolated PPO tolerance gene and treated with two types of PPO type inhibitor herbicides, Strike (fluazifop 50%, by Adama) ( Figure 2A ) or Aurora (40% foramsulfuron-butyl, made by FMC) ( Figure 2B ), concentrations ranging from 0.01% to 0.1%, or received no treatment (0%), as indicated. The effects of PPO inhibitors on transgenic plants were compared with control plants transformed with an empty vector.

[0103] Figure 3A Representative graphs are shown, presenting plant resilience scores on a scale of 1 to 5, where "1" represents highly damaged plants and "5" represents highly resistant plants.

[0104] Figure 3B A table showing the results of the in planta screening as a list of the SEQ ID NOs of the fungal isolated PPOs, their fungal species, and the scores they gave in the in planta screening performed in the presence of Strike (based on Figure 3A The list includes PPO genes isolated from fungi without cTP having an amino acid sequence shown in any one of SEQ ID NOs: 1 to 7 and 86 to 100, and their respective chimeric PPO genes with N-terminal cTP, which are transformed into Arabidopsis plants and have an amino acid sequence shown in any one of SEQ ID NOs: 8 to 14 and 104 to 108.

[0105] Figure 3C A table showing the results of the in planta screening is shown, listing the top five SEQ ID NOs of PPO genes isolated from fungi and the scores they gave in the in planta screening performed in the presence of the array of four PPO herbicides of Group 14 (based on Figure 3A The elasticity scale of 100 μg / kg / hectare was used for the analysis of the elasticity scale of 100 μg / kg / hectare. When the concentration increases, the concentration of Strike is 2.5 kg / hectare, the concentration of Star is 1.25 kg / hectare, the concentration of Goal is 1.15 kg / hectare, and the concentration of Aurora is 1.0 kg / hectare. The list includes PPO genes isolated from fungi without cTP having an amino acid sequence shown in any one of SEQ ID NOs: 2, 94, and 96-98, and their respective chimeric PPO genes with N-terminal cTP, which are transformed into Arabidopsis plants and have an amino acid sequence shown in any one of SEQ ID NOs: 9, 112, and 114-116.

[0106] Figure 4A A ribbon diagram showing the 3D structure of soybean (Glycine max) PPO (SEQ ID NO: 102) - a typical PPO protein - presents the structural components of the herbicide binding pocket (yellow). The alpha helices are located on several beta pleated sheets (labeled in yellow) that form the main contact points for the herbicide molecule. Distal to the pocket opening, a small number of residues form a hydrophobic pocket that stabilizes the herbicide portion (outlined by red dots).

[0107] Figure 4BA surface view of the 3D structure of human PPOX (SEQ ID NO: 101) is shown, conformationally presented with the pocket opening facing the viewer. The herbicide acifluorfen is shown in purple at its bonding location.

[0108] Figure 5A A surface view of the 3D structure of human PPOX (SEQ ID NO: 101) is shown, with: (I) a frontal view of the herbicide binding pocket (defined by the yellow line); (II) an alpha helical structure (shown in yellow) located at the pocket opening of each human, plant and fungal PPO, whose amino acid sequences are represented by SEQ ID NOs: 101-103 and SEQ ID NOs: 2, 94, 96-98, even though their amino acid sequences are different. Also shown is a yellow dashed line representing the measured distance of the pocket opening size defined and measured from the same position on the alpha helix, where valine is at position 170 (V170) in human PPOX (or the corresponding position on the structure in SEQ ID NOs: 101-103 and SEQ ID NOs: 2, 94, 96-98), to arginine at position 62 (Arg62) (or SEQ ID NOs: 101-103 and SEQ ID NOs: 2, 94, 96-98), which is located at the widest part of the opening.

[0109] Figure 5B The pocket opening size (yellow dashed line) of human-sensitive PPO (SEQ ID NO: 101), plant-sensitive PPO (SEQ ID NO: 102-103) and fungal-resistant PPO (SEQ ID NO: 2, 94, 96-98) is shown. The pocket opening size is measured as the distance between the amino group of the side chain of human Arg62 or the amino acid at the structurally corresponding position in plant and fungal PPO and V170 of human PPOX or the amino acid at the structurally corresponding position in plant and fungal PPO. Human V170 or the amino acid at the structurally corresponding position in plant and fungal PPO is located at Figure 5A In the α-helical structure shown, human Arg62 or the amino acids at the corresponding positions in the structures of plant and fungal PPO are located at the widest point of the opening.

[0110] Figure 5C A schematic diagram showing the corresponding positions of three amino acids in the structure: valine, isoleucine and threonine, which are involved in the measurement of the opening distance of the herbicide binding pocket because they are located in the α-helix. These three amino acids are either human V170 of the PPO having the amino acid sequence represented by SEQ ID NO: 101, or amino acids located at corresponding positions in any PPO whose amino acid sequences are represented by SEQ ID NO: 102-103 and SEQ ID NO: 2, 94, 96-98 (see Figure 6B; valine, isoleucine, or threonine positions structurally corresponding to human V170 are marked in green). Specific atoms used to measure distances to the same position on the alpha helix are marked with gold asterisks. Despite differences in the side chains, the three indicated atoms occupy the same structurally corresponding position in three-dimensional space, so the measurements can be compared.

[0111] Fig. 6A A schematic diagram showing two arginine residues located within the herbicide binding pocket (e.g., Arg97 (d1) and Arg62 (d2) in human PPOX or other amino acids at corresponding positions based on the structural alignment of fungal PPO) is shown (see Figure 6B ) and the oxygen atoms of the acifluorfen herbicide (crystallized with human PPO) located at the hydroxyl group (distance 1; d1) or carbonyl group (distance 2; d2) of C11, respectively. The yellow dashed lines indicate the measured distances.

[0112] Figure 6B The amino acid residues, their positions and the amino acids at the corresponding positions are shown, which form the herbicide binding pocket of several PPOs that are structurally aligned. The structural alignment of the amino acids associated with the herbicide binding pocket is presented in a tabular form. Residues that affect tolerance are marked in red and green. SEQ ID NO: 101 (Homo sapiens PPOX data is a crystalline form containing trifluorfen) is the reference sequence for the structural alignment / position, including position arginine (Arg62) and position arginine (Arg97) and other amino acids that form the pocket position (i.e., positions 62, 97-98, 168-172, 314, 331-334, 344-347, 368 and / or 419 of SEQ ID NO: 101). The amino acids that structurally correspond to the reference human positions are in SEQ ID NOs: 102-103 and SEQ ID NOs: 2, 94, 96-98. Human PPOX (SEQ ID NO: 101) and soybean (Glycine max) PPO (GmPPO from soybean) (SEQ ID NO: 102) are both considered PPO herbicide sensitive / susceptible proteins.

[0113] Figure 6CThe hydrogen bond distances between the herbicide acifluorfen and Arg62 and Arg97 (or other Args at corresponding positions based on the structural alignment of fungal PPOs) in human PPOX in the herbicide binding domain pocket are shown. The 3D predicted structures of human PPO (SEQ ID NO: 101) in light green were superimposed with fungal PPO proteins (SEQ ID NO: 100 and SEQ ID NOs: 9, 94 and 96-98) and hydrogen bonds were measured and summarized in Table 2. Longer distances are associated with tolerance to the herbicide Star at a concentration of 1.25 kg / hectare. The yellow dashed line indicates the measured distance.

[0114] Figure 7 A 3D diagram is shown showing the geometric orientation of valine V347 in the PPO binding pocket of the susceptible human PPOX represented by SEQ ID NO: 101 compared to the corresponding (by structural alignment) phenylalanine F404 of the tolerant fungal PPO represented by SEQ ID NO: 96 (see Figure 6B ). SEQ ID NO:96 (right side) shows a phenylalanine in a position where most other proteins would have a valine or isoleucine. The added aromatic compound faces the pocket and interferes with the intermolecular bridge connecting the amino acid portions of the herbicide.

[0115] Figure 8 A 3D diagram is shown showing the geometric orientation of phenylalanine F331 in the PPO binding pocket of the susceptible human PPOX represented by SEQ ID NO: 101 compared to the corresponding (by structural alignment) phenylalanine F383 of the resistant fungal PPO represented by SEQ ID NO: 96 (see Figure 6B ). F331 together with leucine at position 334 ( Figure 6B ) stabilizes the herbicide molecule in the human PPOX pocket. Differently, SEQ ID NO: 96 contains a phenylalanine in the corresponding position F383, which is perpendicular to the direction of human PPOX, so the herbicide molecule is unstable in the pocket, which helps its tolerance.

[0116]

Detailed description

[0117] In the following description, various aspects of the disclosure will be described. For ease of explanation, specific configurations and details are specified herein so that different aspects of the disclosure can be thoroughly understood. However, it is also obvious to those skilled in the art that the disclosure can be implemented without providing specific details herein. In addition, well-known features may be omitted or simplified in order not to obscure the disclosure.

[0118] In order to facilitate the understanding of the present invention, a number of terms and phrases are defined below. It should be understood that these terms and phrases are used for descriptive purposes rather than for limiting purposes, and therefore, the terms or phrases of this specification should be interpreted by a skilled craftsman based on the teachings and guidance provided herein, combined with the knowledge of a person of ordinary skill in the art.

[0119] According to certain aspects, the present invention provides transgenic plants expressing an exogenous fungal-derived protoporphyrinogen oxidase (PPO) enzyme that confers resistance or tolerance to at least one PPO inhibitor herbicide, wherein the fungal-derived PPO enzyme comprises an amino acid sequence having at least 80% similarity to a PPO isolated from a fungus.

[0120] According to certain aspects, the present invention discloses transgenic plants, cells and seeds that have been genetically modified with a PPO enzyme of exogenous fungal origin, variants, mutants, motifs, fragments and / or protein fusions thereof, which when expressed in plants provide resistance or tolerance to PPO-type inhibitor herbicides, thereby conferring the desirable trait of herbicide tolerance (HT) to the plant. Each possibility is a separate embodiment.

[0121] In some embodiments, the amino acid sequence encoding the PPO polypeptide derived from or isolated from fungi is included, and has an enzyme activity that complements the endogenous PPO enzyme of the plant. In some embodiments, the fungal-derived PPO also includes variants, mutants, motifs, fragments and protein fusions thereof. Each possibility is a separate embodiment.

[0122] In some embodiments, methods for controlling weeds in agricultural fields by selectively protecting transgenic crops expressing fungal-derived PPOs are also provided.

[0123] In a specific embodiment, the method comprises introducing into the plant at least one expression cassette comprising a promoter operably linked to a nucleotide sequence encoding a PPO polypeptide of the present invention, variant, mutant, motif, fragment and / or protein fusion thereof.

[0124] As used herein, the singular forms "a", "an" and "the" shall also include the plural forms unless the context clearly indicates otherwise. "A" and "an" are used herein to refer to one or more (i.e., at least one) of the objects described. For example, "anucleic acid" refers to one or more nucleic acids.

[0125] As used herein, "optional" or "optional" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not occur.

[0126] As used herein, the term "about" when referring to a measurable value (such as an amount, a time duration, etc.) is meant to include variations of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1%, as these variations are suitable for performing the disclosed methods.

[0127] As used herein, the terms "PPO inhibitor," "PPO-type inhibitor herbicide," or "PPO herbicide" refer to substances that target protoporphyrinogen oxidase (PPO) and inhibit its catalytic activity, thereby preventing the enzyme from participating in the catalysis of the organic compound protoporphyrin IX - a precursor of heme and chlorophyll. These terms can be used interchangeably.

[0128] Protoporphyrinogen oxidase inhibitors (PPO inhibitors) are a class of herbicides that inhibit plant growth by interfering with the biosynthesis of heme, thereby preventing oxygen from being carried into chloroplasts. Inhibition of PPO oxidase activity not only weakens photosynthesis by hindering the production of heme and chlorophyll, but also leads to the formation of highly reactive molecules that attack and damage lipid and protein membranes, causing cells and organelles to leak and rapidly decompose.

[0129] Non-limiting examples of PPO-type inhibitor herbicides include the following herbicide families: phenylpyrazoles, pyrimidinediones, oxazolidinediones, diphenyl ethers, oxadiazoles, N-phenylphthalimides, triazinones, thiadiazoles, triazolinones and triazolopyridones, which are commercially known under the names Star, Goal, Strike and Aurora, for example. Such herbicides include the active compounds fluazifop-butyl, fomesafen, flufenacet-butyl, mesotrione, trifloxystrobin, lactofen, flufenacet-butyl, bensulfuron-butyl and the like.

[0130] As used herein, the term "conferring" means providing a property or trait to plants, such as herbicide tolerance or resistance, such that the plants grow and develop in the presence of the herbicide with little or no herbicidal effect.

[0131] As used herein, a plant that is substantially "tolerant" to an herbicide exhibits a rightward shift in the dose / response curve when treated with the herbicide relative to the same dose / response exhibited by an intolerant plant (i.e., a tolerant plant requires a higher concentration of herbicide to exhibit the same herbicidal effect as an intolerant plant). Tolerant plants typically require at least twice as much herbicide as intolerant plants to produce a given herbicidal effect. Plants that are substantially "resistant" to an herbicide exhibit few symptoms, including browning (necrosis), lysis, greening, or other lesions of the tissue within 1 to 2 hours after exposure, or at least no symptoms that significantly affect the growth and development of the plant when the herbicide is used at concentrations and rates that are commonly employed by the agricultural community to suppress or kill weeds in fields. The terms "tolerance" and "resistance" may be used interchangeably.

[0132] As used herein, the terms "tolerance," "resistance," "improved resistance," "improved herbicide tolerance," or "improved tolerance to PPO inhibitors" may be used interchangeably and may refer to the improved ability of a fungal PPO (derived or isolated), variant, motif, mutant, or fragment thereof, or protein fusion thereof, including but not limited to a chloroplast transit peptide (cTP), to be exogenously expressed in a transgenic plant, relative to an unmodified (WT) plant or a plant that does not express a fungal PPO-derived gene but expresses an "empty vector" as a control.

[0133] Alternatively, the term "tolerance" may refer to the ability of a transgenic plant to resist the herbicidal activity of a PPO inhibitor due to the expression of an exogenous fungal PPO, variant, motif, mutant or fragment thereof or protein fusion thereof.

[0134] In some embodiments, the term "conferring tolerance" may refer to or include the improved ability of a fungal PPO to confer enhanced / increased / elevated tolerance; or the ability of a transgenic plant to resist the herbicidal activity of a PPO inhibitor; relative to a plant that is not modified (WT) or does not express a fungal PPO-derived gene but expresses an "empty vector" as a control. Each possibility is a different embodiment.

[0135] In some embodiments, the improved ability of the fungal PPO to confer enhanced / increased / elevated tolerance; or the ability of the transgenic plant to resist the herbicidal activity of the PPO inhibitor is determined by giving the plant a resilience score on a scale of 1 to 5, where "1" represents a highly damaged plant and "5" represents a highly resistant plant; if the score is at least 2, or preferably at least 3, even more preferably at least 4, even more preferably 5, then the fungal PPO confers enhanced / increased / elevated tolerance to the plant, and the plant is resistant, relative to a WT plant or a plant expressing an "empty vector" with a score of 1. Each possibility is a different embodiment.

[0136] In some embodiments, the term "granting a tolerance" includes at least 2 points, or preferably at least 3 points, even more preferably at least 4 points, even more preferably 5 points. Each possibility is a different embodiment.

[0137] In this regard, it is best to refer to Figure 3A , but may also refer to Figure 1B , where a scale of 0 to 5 is used, where "0" represents highly damaged plants (no tolerance) and "5" represents highly resistant plants.

[0138] The term "plant" as used herein refers to a whole plant, any part thereof, or a culture thereof, at any stage of development, such as plant seeds, plant cells, plant tissues, and plant organs. Examples of plant parts include, but are not limited to, meristems, roots, stems, leaves, flowers, fruits, and seeds, at any stage of development, in plants, in vitro, or in culture.

[0139] The present invention is applicable to any plant or crop plant species, including but not limited to monocots and dicots. Examples of crop species include, but are not limited to, corn (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularly 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., Pennisetum glaucum, Panicum meliaceous, Setaria italica, Eleusine coracana), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum, peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potatoes (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp.), grapes (Vitis vinifera), palm trees from the palm family (Arecaceae) (e.g., coconut (Cocos nucifera), palm (Elaeis guineensis), date palm (Phoenix spp.), pineapple (Ananascomosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), bananas (Musa spp.), avocado (Persea americana), fig (Ficus carica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Caricapapaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), eucalyptus (Eucalyptus sp.), pine (Pinus spp.), sugar beet (Betavulgaris), sugar cane (Saccharum ssp.), oats, barley, plants, ornamental plants, lawn grasses (Poaceae) and conifers. Each possibility is a separate embodiment. .

[0140] Other examples of crop species include, but are not limited to: cowpea, chickpea, bean, rapeseed, clover; lettuce, tomato, gourd, carrot, radish, pea, lentil, cabbage, cauliflower, broccoli, Brussels sprouts, pepper, apple, pear, peach, apricot, walnut, poplar, pine, orchid, morning glory, carnation, rose, switchgrass, prairie grass, Indian grass, big bluestem grass, camellia.

[0141] As used herein, the term "fungal origin" refers to protoporphyrinogen oxidase (PPO) genes and gene products or sequences thereof, and variants, mutants, motifs or fragments thereof, and includes synthetic or cloned PPO or protein fusions thereof, which are expressed in cells or represented in libraries. The terms "fungal origin" and "PPO origin" can be used interchangeably.

[0142] According to some embodiments, the fungal-derived PPO confers resistance or tolerance to at least one PPO inhibitor herbicide.

[0143] According to some embodiments, the fungus-derived PPO has an amino acid sequence as shown in any one of SEQ ID NOs: 1-100.

[0144] According to some embodiments, the fungus-derived PPO is a PPO isolated from a fungus, and its amino acid sequence is as described in any one of SEQ ID NOs: 1 to 7 or 86 to 100. Each possibility is a separate embodiment.

[0145] According to some embodiments, the fungus-derived PPO has an amino acid sequence of a PPO-derived motif as set forth in any one of SEQ ID NOs: 16-85.

[0146] According to some embodiments, the fungus-derived PPO has an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to the PPO isolated from the fungus.

[0147] According to some embodiments, the fungus-derived PPO has an amino acid sequence comprising at least 95% similarity to the PPO isolated from the fungus.

[0148] As used herein, the term "fungal isolate" refers to native full-length PPO genes and gene products, or sequences thereof, originally isolated or extracted from fungi as genomic DNA or RNA (i.e., then processed into cDNA), including synthetic or cloned PPO genes and gene products, or sequences thereof, or protein fusions thereof, expressed in cells, or represented in libraries.

[0149] According to some embodiments, the fungus-isolated PPO or fungus-derived PPO may be modified to include a signal peptide or a targeting peptide, including but not limited to, for example, but not limited to, a chloroplast transit peptide (cTP).

[0150] In some embodiments, the fungal isolated PPO or protein fusion of the fungal derived PPO comprises a fusion with a chloroplast transit peptide (cTP).

[0151] According to some embodiments, fungal isolated PPOs may be modified to optimize their function, including but not limited to by optimizing genetic codon usage for optimal expression in plant cells.

[0152] According to some embodiments, the fungal isolated PPO or fungal derived PPO of the present invention, variants, mutants, motifs and / or fragments thereof, belongs to the clade Leotiomyceta.

[0153] According to some embodiments, the fungus-derived or fungus-isolated PPO of the present invention belongs to the following genera: Aspergillus, Fusarium, Rhizopus, Penicillium, Trichoderma, Alternaria and Cladosporium.

[0154] According to some embodiments, the PPO isolated from fungi has an amino acid sequence as set forth in any one of SEQ ID NOs: 1 to 7. Each possibility is a separate embodiment.

[0155] According to some embodiments, the fungus-derived PPO enzyme comprises an amino acid sequence having at least 70% similarity to a PPO isolated from a fungus, at least 75% similarity to a PPO isolated from a fungus, at least 80% similarity to a PPO isolated from a fungus, at least 85% similarity to a PPO isolated from a fungus, at least 90% similarity to a PPO isolated from a fungus, at least 95% similarity to a PPO isolated from a fungus, at least 96% similarity to a PPO isolated from a fungus, at least 97% similarity to a PPO isolated from a fungus, at least 98% similarity to a PPO isolated from a fungus, at least 99% similarity to a PPO isolated from a fungus, at least 99.9% similarity to a PPO isolated from a fungus, or more. Each possibility is a separate embodiment.

[0156] According to some embodiments, the fungus-derived PPO comprises fungus-isolated PPO.

[0157] According to some embodiments, the PPO sequences of fungal origin share at least 60% similarity with each other, in some cases at least 65% similarity, in some cases at least 70% similarity, in some cases at least 75% similarity, in some cases at least 80% similarity, in some cases at least 85% similarity, in some cases at least 90% similarity, in some cases at least 95% similarity, in some cases at least 96%, in some cases at least 97%, in some cases at least 98%, or in some cases at least 99% similarity.

[0158] According to some embodiments, a fungal-derived nucleic acid template is used to amplify PPO; in some embodiments, the fungal-derived nucleic acid template used to amplify PPO is a transcript of a fungal genomic DNA template or mRNA template; in some embodiments, the fungal-derived nucleic acid template used to amplify PPO is a synthetic or cloned PPO expressed in a cell or represented by a library. Each possibility is a separate embodiment.

[0159] According to some embodiments, the RNA transcript of the fungus-derived PPO sequence of the present invention contains introns; according to some embodiments, the RNA transcript of the fungus-derived PPO sequence of the present invention completely lacks or partially lacks introns; according to some embodiments, the RNA transcript of the fungus-derived PPO sequence of the present invention includes at least one intron, or at least 2 introns, or at least 5 introns, or at least half of the intron full-length natural transcript. Each possibility is a separate embodiment.

[0160] According to some embodiments, the RNA transcripts of the fungus-derived PPO polypeptides of the present invention are expressed as fully mature intron-free RNA products; according to some embodiments, the RNA transcripts of the fungus-derived PPO polypeptides of the present invention are expressed as unspliced ​​or partially spliced ​​premature RNA products. Each possibility is a separate embodiment.

[0161] The terms "similarity" and "sequence similarity" are used interchangeably and refer to the level of "identity" or "positivity" between two sequences when they are compared by alignment using an alignment tool. As used herein, similarity is with respect to a directly compared PPO of fungal origin or a PPO isolated from a fungus, or with respect to any other PPO gene or sequence directly referenced, including but not limited to genes from plants, humans or other organisms.

[0162] As used herein, the term "identity" means that two aligned residues are identical. As used herein, the term "positive" means that two aligned residues are not identical, but they have chemical, physical and / or structural properties that make them nearly substitutable. For example, lysine and arginine are both positively charged amino acids and are therefore considered "positive" to each other, while aspartic acid and glutamic acid are negatively charged amino acids and are therefore also considered "positive" to each other.

[0163] In some embodiments, the amino acid sequence of the fungal-derived PPO comprises % similarity to the amino acid sequence of the PPO isolated from fungi. In some embodiments, the amino acid sequence of the fungal-derived PPO comprises % identity to the amino acid sequence of the PPO isolated from fungi.

[0164] In some embodiments, the % similarity of the amino acid sequence of the PPO of fungal origin comprises at least 60%, at least 65%, at least 70%, at least 75%, 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 88%, at least 89%, 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%, at least 99.9% or more similarity to the amino acid sequence of the PPO isolated from the fungus. Each possibility is a separate embodiment.

[0165] In some embodiments, the % identity of the amino acid sequence of the PPO of fungal origin comprises at least 30%, at least 35%, at least 40%, at least 45%, 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 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 80% identity or more to the amino acid sequence of the PPO isolated from the fungus. Each possibility is a separate embodiment.

[0166] As used herein, when comparing two amino acid sequences, the level of similarity may refer to the degree of similarity between the residues in the aligned sequences.

[0167] In some embodiments, the similarity level between two amino acid sequences is the degree of identity (total %) between the residues of the aligned sequences (total percentage of the complete sequences). In some embodiments, the degree of similarity between two amino acid sequences is the degree of identity (total %) between the residues of the aligned sequences (total percentage of the complete sequences).

[0168] Thus, sequence similarity refers to the best match of residues (i.e., sequence similarity can be determined by multiple sequence alignment (MSA) or the like. The best match is determined by an algorithm that finds the fewest number of editing operations (insertions, deletions, and substitutions) to align one sequence to another. Sequence alignment tools (e.g., CLUSTALW) can be used to accurately compare, analyze, and identify the level of similarity between two or more fragments, variants, mutants, motifs, fragments, and / or homologous sequences of a protein or gene with statistical confidence. According to some embodiments, the best match can also be determined by an algorithm that finds spatially overlapping residues based on the structure or model of the translated protein. According to some embodiments, the best match for two residues can also be determined based on the common portion of the catalytic machinery of the two proteins.

[0169] As described above, a multiple sequence alignment (MSA) can be used for sequence similarity analysis (calculation of % similarity or % identity) based on the best matching of residues.

[0170] The present invention also encompasses the terms "variants," "mutants," "motifs," and "fragments" of the disclosed nucleotide sequences and the amino acid sequences of the proteins encoded thereby, and refers to similar sequences.

[0171] According to some embodiments, the present invention includes amino acid sequences isolated from fungi or derived from PPO, including variants, mutants, motifs and fragments thereof, which may have at least 60% similarity to each other, in some instances at least 65% similarity, in some instances at least 70% similarity, in some instances at least 75% similarity, in some instances at least 80% similarity, in some cases at least 85% similarity, in some cases at least 90% similarity, in some cases at least 95% similarity, in some cases at least 96%, in some cases at least 97%, in some cases at least 98%, or in some cases at least 99% similarity. Each possibility is a separate embodiment.

[0172] The PPO-derived variants, mutants, motifs or fragments of the fungal-derived polypeptides of the present invention may be encoded to retain the biological activity of the full-length fungal isolated PPO protein, or they may have improved activity, or have increased or decreased activity relative to the fungal isolated PPO or other PPO-derived polypeptides (including specific sequences derived from it, including but not limited to other sequences from plants, humans or other organisms).

[0173] As used herein, the terms "variant" or "mutant" refer to similar sequences. These terms are used for sequences of fungal origin or any other PPO gene or sequence directly referenced, including but not limited to PPO genes from plants, humans or other organisms.

[0174] When directly mentioned, variant may refer to naturally occurring allelic variation (ie, genetic diversity, polymorphism), or when directly mentioned, it may refer to genetic engineering (ie, mutation or altered form). Otherwise, the terms variant and mutant as used herein may be used interchangeably.

[0175] Polynucleotide variants or mutants or polypeptide variants or mutants, including deletions, substitutions and / or additions of one or more residues at one or more positions of the disclosed PPO-derived sequence. One of skill in the art will recognize that variants or mutants of nucleic acids can be constructed to maintain an open reading frame, so that variants or mutants of nucleotide sequences can encode polypeptides or fragments thereof that retain the biological activity of the full-length fungal isolated or PPO-derived protein, have improved activity, or have increased or decreased activity relative to fungal isolated PPO or other PPO-derived polypeptides. For polynucleotides, conservative variants include those sequences that encode the amino acid sequence of a fungal-derived PPO polypeptide due to the degeneracy of the genetic code. Variants or mutants can be identified using well-known molecular biology techniques, for example, using next-generation sequencing (NGS), polymerase chain reaction (PCR) and hybridization techniques. For example, variant or mutant polynucleotides can be generated using site-directed mutagenesis or solid phase synthesis. According to some embodiments, variants or mutants of the nucleotide sequence encoding the functional fungus-derived protein will have at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence similarity with the PPO isolated from fungi or other PPO-derived polypeptides (including specific amino acids derived therefrom), as determined by sequence alignment tools. Each possibility is a separate embodiment.

[0176] As used herein, the term "motif" refers to a consecutive sequence of amino acids in a polypeptide, which may include a specific pattern of amino acid residues and / or a structure that characterizes it.

[0177] According to some embodiments, the motif in the polypeptide isolated from fungi or derived from PPO may have a corresponding amino acid sequence in the PPO polypeptide of plants, humans or other species. For example, such correspondence is determined by performing optimal matching using multiple sequence alignment (MSA).

[0178] According to some embodiments, the motif of the present invention can be a PPO polypeptide of fungal origin or a polypeptide isolated from a fungus, which has similarity in at least Aspergillus, Fusarium, Rhizopus, Penicillium, Trichoderma, Alternaria and Cladosporium.

[0179] According to some embodiments, the motif of the present invention may be a PPO polypeptide of fungal origin or a polypeptide isolated from fungi, which has similarity at least in Aspergillus, Fusarium, Rhizopus, and Penicillium.

[0180] According to some embodiments, the PPO polypeptide of fungal origin or the polypeptide isolated from fungi has similarity between at least Aspergillus, Fusarium, Rhizopus, Penicillium, Trichoderma, Alternaria and Cladosporium.

[0181] According to some embodiments, the PPO polypeptide derived from fungi or isolated from fungi has similarities at least among Aspergillus, Fusarium, Rhizopus, and Penicillium.

[0182] According to some embodiments, the motif of the present invention can be any sequence that shares at least about 60%, 65%, 70%, 75%, 80%, 85%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence similarity with any one of the amino acid sequences listed in SEQ ID NOs: 16-85.

[0183] According to some embodiments, the motif of the present invention can retain the biological activity of the full-length fungal isolated PPO protein, or it can have improved, enhanced or reduced activity relative to the fungal isolated PPO or other PPO-derived polypeptides (including specific sequences derived therefrom).

[0184] According to some embodiments, a motif of the invention includes at least about 2 amino acids, at least about 4 amino acids, at least about 10 amino acids, at least about 15 amino acids, at least about 20 amino acids, at least about 25 amino acids, at least about 30 amino acids, at least about 40 amino acids, or at least 45 amino acids. Each possibility is a different embodiment.

[0185] According to some embodiments, a motif of the invention comprises about 2 to about 50 amino acids, about 4 to about 45 amino acids, about 10 to about 40 amino acids, about 15 to about 35 amino acids, or about 15 to about 30 amino acids. Each possibility is a different embodiment.

[0186] According to some embodiments, the fungus-derived PPO polypeptide disclosed herein includes 10 motifs. According to some embodiments, motif 1 comprises any one of the amino acid sequences shown in SEQ ID NOs: 16 to 22; motif 2 comprises any one of the amino acid sequences shown in SEQ ID NOs: 23 to 29; motif 3 comprises any one of the amino acid sequences shown in SEQ ID NOs: 30 to 36; motif 4 comprises any one of the amino acid sequences shown in SEQ ID NOs: 37 to 43; motif 5 comprises any one of the amino acid sequences shown in SEQ ID NOs: 44 to 50; motif 6 comprises any one of the amino acid sequences shown in SEQ ID NOs: 51 to 57; motif 7 comprises any one of the amino acid sequences shown in SEQ ID NOs: 58 to 64; motif 8 comprises any one of the amino acid sequences shown in SEQ ID NOs: 65 to 71; motif 9 comprises any one of the amino acid sequences shown in SEQ ID NOs: 72 to 78; motif 10 comprises any one of the amino acid sequences shown in SEQ ID NOs: 79 to 85. Each possibility is a different embodiment.

[0187] According to some embodiments, the fungus-derived PPO polypeptide disclosed herein comprises 10 motifs. According to some embodiments, motif 1 comprises any one of the amino acid sequences shown in SEQ ID NOs: 16 to 22; motif 2 comprises any one of the amino acid sequences shown in SEQ ID NOs: 23 to 29; motif 3 comprises any one of the amino acid sequences shown in SEQ ID NOs: 30 to 36; motif 4 comprises any one of the amino acid sequences shown in SEQ ID NOs: 37 to 43; motif 5 comprises any one of the amino acid sequences shown in SEQ ID NOs: 44 to 50; motif 6 comprises any one of the amino acid sequences shown in SEQ ID NOs: 51 to 57; motif 7 comprises any one of the amino acid sequences shown in SEQ ID NOs: 58 to 64; motif 8 comprises any one of the amino acid sequences shown in SEQ ID NOs: 65 to 71; motif 9 comprises any one of the amino acid sequences shown in SEQ ID NOs: 72 to 78; motif 10 comprises any one of the amino acid sequences shown in SEQ ID NOs: 79 to 85. Each possibility is a different embodiment.

[0188] The term "fragment" as used herein refers to a portion of a nucleotide sequence or a portion of an amino acid sequence. According to some embodiments, the nucleotide sequence fragment encoding the functional fungal separation or PPO-derived protein of the present invention will encode at least 2, 4, 7, 10, 15, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 150, 180, 200, 250, 300, 350 consecutive amino acids, or up to the total number of amino acids present in the full-length polypeptide of the present invention. Each possibility is a different embodiment.

[0189] Those skilled in the art will recognize that the nucleic acid fragments of the present invention can be constructed to maintain an open reading frame, so that variants, mutants, motifs or fragments of the fungal isolated or PPO-derived polypeptides of the present invention can be encoded to retain the biological activity of the full-length fungal-derived PPO protein, or they may have improved activity, or have increased or decreased activity relative to the fungal isolated PPO or other PPO-derived polypeptides (including specific sequences derived therefrom).

[0190] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical analogue of the corresponding natural amino acid, as well as to naturally occurring amino acid polymers. The polypeptides of the present invention can be produced from the nucleic acids disclosed herein, or by using standard molecular biology techniques. For example, the PPO polypeptides, variants, mutants, motifs and / or fragments thereof of the present invention can be produced by expressing a recombinant nucleic acid of the present invention in an appropriate host cell, or by chemical synthesis or by a combination of ex vivo procedures (e.g., protease digestion and purification).

[0191] As used herein, "nucleic acid" includes deoxyribonucleotide or ribonucleotide polymers in single- or double-stranded form and, unless otherwise limited, includes known analogs that have the essential properties of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides.

[0192] As used herein, the term "encoding" or "encoding" when used in the context of a specific nucleic acid means that the nucleic acid contains the necessary information for translating the nucleotide sequence directly into a specific protein. The information for protein coding is specified by using codons. Nucleic acids encoding proteins of the present invention may contain non-translated sequences (e.g., introns) within the translated region of the nucleic acid, or may lack or partially lack such intermediate non-translated sequences (e.g., such as cDNA).

[0193] As used herein, the term "transgenic" refers to a plant or cell thereof transformed with a nucleic acid of exogenous / exogenous origin that has been introduced into its genome by transformation with Agrobacterium, biomass, protoplasts, viral expression (transient), etc., as known in the art, to achieve transient or stable expression of the transgene. The terms "transgenic" and "transgenic" can be used interchangeably.

[0194] According to some embodiments, the expression cassette comprises a promoter operably linked to a nucleotide sequence encoding a fungus-derived PPO polypeptide of the present invention, variant, mutant, motif and / or fragment thereof. Typically, the expression cassette will comprise a selectable marker gene for selecting transformed cells.

[0195] According to some embodiments, the fungal-derived PPO sequence is fused to a chloroplast transit peptide (cTP).

[0196] According to some embodiments, the fungal-derived PPO sequence confers high tolerance to the PPO inhibitor herbicides Strike and / or Aurora to the transgenic plants. Each possibility is a separate embodiment.

[0197] According to some embodiments, some fungal-derived PPO sequences confer high-level tolerance to the PPO inhibitor herbicide Strike, but not to Aurora, on transgenic plants. According to some embodiments, some fungal-derived PPO sequences confer high-level tolerance to the PPO inhibitor herbicide Aurora, but not to Strike, on transgenic plants.

[0198] According to some embodiments, a high level of tolerance means that the tolerance of the transgenic plant is about 10% higher than that of the WT control plant. 4 times (4 orders of magnitude) or more.

[0199] According to some embodiments, very high tolerance levels mean that the tolerance to the treatment is about 10 times higher than that of WT or other plants expressing the empty cassette without the fungal-derived gene and serving as control plants. 5 (5 orders of magnitude).

[0200] According to some embodiments, transgenic plants expressing a fungal-derived PPO gene exhibited favorable improved growth over all concentration ranges tested (0.01% to 0.5% for Aurora and 0.01% to 1% for Strike), and thus increased tolerance to both PPO inhibitor herbicides when applied in a dose-dependent manner.

[0201] According to some embodiments, all of the proteins derived from Aspergillus sp. (SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 2, SEQ ID NO: 9 and SEQ ID NO: 86-88), Fusarium sp. (SEQ ID NO: 3, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 89 and 90), Rhizopus sp. (SEQ ID NO: 6 and SEQ ID NO: 91-93), Penicillium sp. (SEQ ID NO: 14, SEQ ID NO: 94 and SEQ ID NO: 95), Trichoderma sp. (SEQ ID NO: 96), Rhodotorula sp. (SEQ ID NO: 97), Hesseltinella sp. (SEQ ID NO: 98), Spizellomyces sp. (SEQ ID NO: 99), NO: 99) or Rhizophagus sp. (SEQ ID NO: 100) conferring high tolerance to the PPO inhibitor herbicides Strike and / or Aurora to the transgenic plants. Each possibility is a separate embodiment.

[0202] According to some embodiments, transgenic plants expressing Aspergillus sp. genes (SEQ ID NO: 1, SEQ ID NO: 8 and SEQ ID NO: 2, SEQ ID NO: 9), Fusarium sp. genes (e.g., SEQ ID NO: 10 and SEQ ID NO: 12) and Rhizopus sp. genes (SEQ ID NO: 6) exhibit very high levels of resistance to Strike. Each possibility is a separate embodiment.

[0203] According to some embodiments, transgenic plants expressing genes from Aspergillus sp. (e.g., SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 2, SEQ ID NO: 9) and genes from Rhizopus sp. (SEQ ID NO: 6) and Penicillium sp. (SEQ ID NO: 14) exhibit very high levels of resistance to Aurora compared to control plants. Each possibility is a separate embodiment.

[0204] According to some embodiments, the Penicillium gene (SEQ ID NO: 14) confers very high resistance to Aurora, but not to Strike, to the transgenic plant.

[0205] According to some embodiments, the Fusarium gene (eg, SEQ ID NO: 10 and SEQ ID NO: 12) confers high-level resistance to Strike, but not Aurora, to the transgenic plant.

[0206] According to some embodiments, transgenic plants expressing a PPO gene isolated from a fungus exhibited favorable improved growth over all concentration ranges tested (0.01% to 0.5% for Aurora and 0.01% to 1% for Strike), and thus increased tolerance to both PPO inhibitor herbicides when applied in a dose-dependent manner.

[0207] According to some embodiments, 10 motifs (motifs 1-10) are identified in PPO of fungal origin. According to some embodiments, the motifs are about 4 to about 30 amino acids in length.

[0208] According to some embodiments, motifs of fungal isolated PPO sequences may share more common amino acid sequences and / or structural features between enzymes of Aspergillus, Fusarium, Rhizopus and Penicillium than the corresponding sequences of plant derived PPOs between Arabidopsis, tobacco and soybean enzymes, for example, motifs 1, 2, 3, 4, 6, 7 and 10. Each possibility is a separate embodiment.

[0209] According to some embodiments, the plant-derived PPO sequences may share more common amino acid sequences and / or structural features between them than motifs shared between enzymes of the plant in comparison to the corresponding sequences.

[0210] According to some embodiments, a total of 70 motif sequences were identified in PPO enzymes isolated from fungi of the genera Aspergillus sp., Fusarium sp., Rhizopus sp., and Penicillium sp., and are considered distinct and distinguishable with respect to their corresponding sequences in plants.

[0211] According to some embodiments, the 70 motifs have amino acid sequences as shown in SEQ ID NOs: 16-85.

[0212] According to some embodiments, the PPO polypeptide of fungal origin contains 10 motifs that are considered to be distinct and distinguishable relative to their corresponding sequences in the PPO enzyme in plants.

[0213] According to certain aspects, a transgenic plant is provided, which expresses a protoporphyrinogen oxidase (PPO) of exogenous fungal origin, wherein the enzyme comprises an amino acid sequence having at least 80% similarity to a PPO sequence isolated from a fungus as shown in any one of SEQ ID NOs: 2, 94, 96 to 98, or any combination thereof. Each possibility is a separate embodiment.

[0214] In some embodiments, the exogenous fungal-derived PPO comprises an amino acid sequence having at least 90% similarity to the PPO sequence isolated from a fungus as set forth in any one of SEQ ID NOs: 2, 94, 96-98, or any combination thereof. Each possibility is a separate embodiment.

[0215] In some embodiments, the protoporphyrinogen oxidase (PPO) of fungal origin comprises an amino acid sequence having at least 80% similarity, 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 88%, at least 89%, 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%, at least 99.9% or more similarity to the amino acid sequence of PPO isolated from fungi. Each possibility is a separate embodiment.

[0216] In some embodiments, the amino acid sequence of the PPO isolated from the fungus comprises SEQ ID NO: 2, 94, 96 to 98. Each possibility is a separate embodiment.

[0217] Reference now Figures 3A to 3C .

[0218] According to some embodiments, the fungal-derived PPO comprises less than The bag opening size.

[0219] According to some embodiments, the fungal-derived PPO comprises less than about Less than about Less than about Less than about Less than about Less than about Less than about Less than about Less than about or less than about Each possibility is a separate embodiment.

[0220] According to some embodiments, the fungal-derived PPO comprises about about about about Each possibility is a separate embodiment.

[0221] In some embodiments, the bag opening size of any one of the fungal-derived PPOs comprises at least difference / reduction.

[0222] In some embodiments, the bag opening size of any one of the fungal-derived PPOs comprises at least about 1:1 relative to the bag opening size of human PPOX. At least about At least about At least about At least At least At least or at least or greater. Each possibility is a separate implementation.

[0223] In some embodiments, the bag opening size of any one of the fungus-derived PPOs comprises about about about about Each possibility is a separate implementation.

[0224] As used herein, the term "pocket opening size" refers to the space on the surface of the PPO enzyme through which the herbicide enters its binding site and is measured as the distance between the amino group of the side chain of human Arg62, or the structurally corresponding amino acid in plant and fungal PPOs, to V170 of human PPOX or the structurally corresponding amino acid in fungal or plant PPOs. Human V170 or the structurally corresponding amino acid in fungal or plant PPOs is located Figure 5A In the α-helical structure shown, the amino acid at the structurally corresponding position of human Arg62 or fungal or plant PPO is located at the widest point of the opening, such as Figure 5A and Figure 5B shown.

[0225] In some embodiments, in which the amino acid at the position structurally corresponding to human Arg62 in plant and fungal PPO is not arginine (ie, see Figure 6B: Q123 (SEQ ID NO: 102), T70 (SEQ ID NO: 103) or S102 (SEQ ID NO: 96)), the distance is measured from the same position on the alpha helix to the terminal amino group of the glutamine side chain in SEQ ID NO: 102, or the methyl group at the terminal end of the threonine side chain in SEQ ID NO: 103, or the hydroxyl group at the terminal end of the serine side chain in SEQ ID NO: 96. Although these three amino acids have different chemical groups, they occupy the same position in the structure and form the inner edge of the pocket opening, so the measured distance is the smallest, so that a specific atom is always located at the end of the side chain.

[0226] In some embodiments, the amino acid sequence of the PPO isolated from the fungus comprises one or more of SEQ ID NOs: 2, 94, 96-98, or any combination thereof. Each possibility is a separate embodiment.

[0227] In some embodiments, the amino acid sequence of the PPO isolated from the fungus comprises one or more of SEQ ID NOs: 2, 94, 97-98, or any combination thereof. Each possibility is a separate embodiment.

[0228] In some embodiments, the amino acid sequence of the PPO isolated from the fungus comprises one or more of SEQ ID NOs: 2 and 98. Each possibility is a separate embodiment.

[0229] Reference now Figures 5A to 5C , Figure 6B and Table 1.

[0230] In some embodiments, the fungal-derived PPO confers resistance or tolerance to at least one PPO inhibitor herbicide.

[0231] In some embodiments, the fungal-derived PPO confers resistance or tolerance to at least one PPO inhibitor herbicide, at least 2, at least 3, at least 5, at least 7, or at least 10 PPO inhibitor herbicides. Each possibility is a separate embodiment.

[0232] In some embodiments, the pocket opening size of the fungal derived PPO is determined by comparing the modeled 3D structure / fold of the fungal derived PPO to the crystal structure of human PPO bound to acifluorfen (provided by PDB identifier: 4IVO).

[0233] In some embodiments, the simulated 3D structure / fold of the fungal-derived PPO is structurally aligned with the crystal structure of human PPO and superimposed with acifluorfen.

[0234] As used herein, the term "simulated 3D structure / fold", "simulated structure" or "structural alignment" or "structure-based alignment" refers to the calculation / modeling of 3D structure / fold using structural alignment / structural simulation / structural homology, including the superposition of a target sequence (e.g., a fungal PPO) on a solved crystal structure to calculate the 3D structure / fold of the target sequence (i.e., simulated structure) to determine the structural similarity between the simulated structure and the crystal structure, and to determine the structural similarity between the simulated structure and the crystal structure using RMSD (root mean square deviation) measurement. The simulation may include, for example, the fungal PPO sequence is structurally aligned and superimposed to the crystal structure of human PPOX bound to trifluorfen (provided by PDB identifier: 4IVO) to simulate the 3D structure / fold of the fungal PPO. In some embodiments thereof, the simulated 3D structure / fold of the fungal-derived PPO is superimposed with trifluorfen. In some preferred embodiments, the structural simulation is performed according to the description of the method in preferred embodiment 5, but other embodiments may also refer to the description of embodiment 3.

[0235] The terms human PPO and human PPOX are used interchangeably.

[0236] The term "comparative simulated 3D structure / fold" as used herein may refer to, but is not limited to, a comparison between a simulated 3D structure / fold of a fungal-derived PPO superimposed with acifluorfen and a crystal structure of human PPOX bound to acifluorfen (provided by PDB identifier: 4IVO); and may include, but is not limited to, for example: any structural similarities / differences that can be identified / measured (e.g., pocket opening sizes, distances, and geometric orientations), and that may be related to the identities of amino acids at corresponding positions on the structure, differences in geometric orientations of amino acids at corresponding positions on the structure, differences in geometric orientations of structural elements (e.g., secondary and tertiary structures), measurements of distances or angles between different amino acids, and distances or angles between amino acids and herbicide / acifluorfen.

[0237] In some embodiments, the geometric orientation includes direction, angular position and / or distance, or any combination thereof. Each possibility is a separate embodiment. In some embodiments, the geometric orientation includes distance.

[0238] As used herein, the term "geometric orientation", when referring to structural comparisons between amino acid molecules / polymers located in 3D space, can be interpreted in the broad sense accepted in the art, including reference to parameters including, but not limited to, directions, angular positions, distances, and overall arrangement / identity of atoms and molecules in 3D space, etc. When structurally aligned and superimposed - two corresponding atoms / molecules / polymers can be characterized by differences in the non-limiting parameters of geometric orientation described herein, and as a direct result of these differences between them - there may be other differences, such as, but not limited to: distances from a reference point ("third party") that can be characterized / measured and compared. As an example: when the sequence of fungal PPO is structurally aligned and superimposed based on the human PPO crystal structure, and two structures are compared - human Arg97 or human Arg62, each structure has a different geometric orientation relative to the amino acid at the corresponding position in the fungal PPO, and as a direct result of their difference in geometric orientation - human Arg97 or human Arg62, for example, the measured distance between acifluorfen bound to a reference point ("third party") and their corresponding amino acid positions to the same reference point, for example, superimposed acifluorfen ("third party").

[0239] In some embodiments, comparing the 3D structure / fold of the fungus-derived PPO to the crystal structure of human PPO bound to acifluorfen comprises comparing a simulated structure of the fold of the fungus-derived PPO.

[0240] In some embodiments, the comparison of the 3D structure / fold of the fungus-derived PPO to the crystal structure of human PPO bound to acifluorfen comprises comparing the crystal structure of the fungus-derived PPO.

[0241] According to some embodiments, the exogenous fungal-derived PPO comprises: a first arginine, which structurally corresponds to the position of arginine at position 97 in human PPOX (human Arg97), and / or a second arginine, which structurally corresponds to the position of arginine at position 62 in human PPOX (human Arg62). Each possibility is a separate embodiment.

[0242] In some embodiments, the first and / or second arginine of the fungal-derived PPO has a different geometric orientation compared to the geometric orientation of human Arg62 and / or human Arg97. Each possibility is a separate embodiment.

[0243] In some embodiments, the position of the fungal-derived PPO corresponds structurally to the position of Arg62 and / or Arg97, and their different geometric orientations are determined by comparing the simulated 3D structure / fold of the fungal-derived PPO with the crystal structure of human PPOX bound to acifluorfen (provided with PDB identifier: 4IVO). Each possibility is a separate embodiment.

[0244] In some embodiments, the simulated 3D structure / fold of the fungal-derived PPO is structurally aligned with the crystal structure of human PPO and superimposed with acifluorfen.

[0245] As used herein, the terms "structurally corresponding", "structurally corresponding positions" or "structurally corresponding positions" are used interchangeably and may refer to amino acid positions, such as fungal PPO, that are structurally homologous as determined by structural alignment / mimicry / folding of the fungal PPO. For example, corresponding amino acid positions may be composed according to Figure 6B The enzyme binding site of the herbicide (e.g., SEQ ID NOs: 2, 94, 96-98 and 100, SEQ ID NOs: 102 and 103), and may correspond in structure to the herbicide binding site according to Figure 6B The human enzyme herbicide binding site of human PPOX is any of the following positions, namely, position 62, 97-98, 168-172, 314, 331-334, 344-347, 368 and / or 419 of SEQ ID NO: 101.

[0246] According to some embodiments, the different geometric orientations include different distances between the first arginine residue of the fungus-derived PPO and the overlapping trifluorfen, wherein the distances include a first distance (d1) between the guanidinium group of the first arginine residue of the fungus-derived PPO and the hydroxyl group of C11 of the overlapping trifluorfen, wherein d1 is greater than

[0247] In some embodiments, the distance (d1) is greater than Greater than Greater than Greater than Greater than Greater than Each possibility is a separate implementation.

[0248] In some embodiments, the distance (d1) is and between and Each possibility is a separate implementation.

[0249] According to some embodiments, the different geometric orientations include different distances between the second arginine residue of the fungal-derived PPO and the overlapping trifluorfen, wherein the distance includes a second distance (d2) between the guanidinium group of the second arginine residue of the fungal-derived PPO and the carbonyl oxygen of C11 of the overlapping trifluorfen, wherein d2 is greater than

[0250] In some embodiments, the distance (d2) is greater than Greater than about Greater than about Greater than about Greater than about Greater than about Greater than about Greater than about Greater than about Greater than about Each possibility is a separate implementation.

[0251] In some embodiments, the distance (d2) is Peace Between, about Peace Between, about Peace Each possibility is a separate implementation.

[0252] According to some embodiments, the sum of the distances d1 and d2 is greater than about

[0253] In some embodiments, the sum of the distances d1 and d2 is greater than about Greater than about Greater than about Greater than about Greater than about Greater than about Each possibility is a separate implementation.

[0254] In some embodiments, the sum of the distances d1 and d2 is about Peace between, or about Peace Each possibility is a separate implementation.

[0255] According to some embodiments, the sum of the distances d1 and d2 of the fungal-derived PPO differs from the sum of the distances d1 and d2 of the human PPOX by more than about Wherein d1 and d2 of human PPOX are the distance between the guanidinium group of human Arg97 and the hydroxyl group of C11 of acifluorfen (d1 of human PPOX) and the distance between the guanidinium group of human Arg62 and the carbonyl group of C11 of acifluorfen (d2 of human PPOX).

[0256] In some embodiments, different geometric orientations comprising d1 and d2 are determined by comparing the modeled 3D structure / fold of the fungus-derived PPO to the crystal structure of human PPOX bound to acifluorfen (provided with PDB identifier: 4IVO).

[0257] In some embodiments, the simulated 3D structure / fold of the fungal-derived PPO is structurally aligned with the crystal structure of human PPO and superimposed with acifluorfen.

[0258] Reference now Figures 6A to 6C and Table 2.

[0259] According to some embodiments, the exogenous fungal-derived PPO comprises an amino acid sequence having at least 80% similarity to the PPO sequence isolated from fungi as shown in any one of SEQ ID NOs: 2, 94, 96-98, or any combination thereof. Each possibility is a separate embodiment.

[0260] According to some embodiments, the exogenous fungal-derived PPO comprises an amino acid sequence having at least 90% similarity to a PPO isolated from a fungus as shown in any one of SEQ ID NOs: 2, 94, 96-98, or any combination thereof. Each possibility is a separate embodiment.

[0261] In some embodiments, the fungal-derived protoporphyrinogen oxidase (PPO) comprises an amino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or more similarity to the amino acid sequence of the fungal-isolated PPO. Each possibility is a separate embodiment.

[0262] In some embodiments, the amino acid sequence of the PPO isolated from the fungus comprises a sequence represented by any one of SEQ ID NOs: 2, 94, 96 to 98 or any combination thereof. Each possibility is a separate embodiment.

[0263] According to some embodiments, the exogenous fungus-derived PPO comprises a phenylalanine at position 404 (F404), which structurally corresponds to a valine at position 347 (V347) of human PPOX.

[0264] In some embodiments, the corresponding phenylalanine position of the fungal derived PPO is determined by comparing the modeled 3D structure / fold of the fungal derived PPO to the crystal structure of human PPO bound to acifluorfen (provided by PDB identifier: 4IVO).

[0265] In some embodiments, the simulated 3D structure / fold of the fungal-derived PPO is structurally aligned with the crystal structure of human PPO and superimposed with acifluorfen.

[0266] Reference now Figure 6B and Figure 7 .

[0267] According to some embodiments, the exogenous fungus-derived PPO comprises a phenylalanine at position 383 (F383), which structurally corresponds to the phenylalanine at position 331 (F331) of human PPOX.

[0268] In some embodiments, the corresponding phenylalanine position of the fungal derived PPO is determined by comparing the modeled 3D structure / fold of the fungal derived PPO to the crystal structure of human PPO bound to acifluorfen (provided by PDB identifier: 4IVO).

[0269] In some embodiments, the simulated 3D structure / fold of the fungal-derived PPO is structurally aligned with the crystal structure of human PPO and superimposed with acifluorfen.

[0270] In some embodiments, the exogenous fungal-derived PPO comprises an amino acid sequence having at least 80% similarity to the fungal-isolated PPO sequence shown in SEQ ID NO:96.

[0271] In some embodiments, the exogenous fungal-derived PPO comprises an amino acid sequence that is at least 90% similar to the fungal-isolated PPO of the sequence shown in SEQ ID NO:96.

[0272] According to some embodiments, the exogenous fungal-derived PPO confers resistance or tolerance to at least one PPO inhibitor herbicide.

[0273] In some embodiments, the one or more PPO inhibitors are selected from the following herbicide families: phenylpyrazoles, pyrimidinediones, oxazolidinediones, diphenyl ethers, oxadiazoles, N-phenylphthalimides, triazinones, thiadiazoles, triazolinones, and triazolopyridones, or any combination thereof.

[0274] In some embodiments, the protoporphyrinogen oxidase (PPO) of fungal origin comprises an amino acid sequence having at least 80% similarity, 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%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9% or more similarity to the amino acid sequence of PPO isolated from fungi. Each possibility is a separate embodiment.

[0275] Reference now Figure 6B and Figure 8 .

[0276] In some embodiments, the mode / mechanism of obtaining PPO herbicide tolerance in the fungal-derived or fungal-isolated PPO includes at least one of the following:

[0277] (i) The size of the bag opening is reduced, affecting the entry of the herbicide into the binding site (reducing the opening size);

[0278] (ii) disrupting the stability of hydrogen bonds at positions corresponding to human positions Arg62 and Arg97 (by structural alignment), affecting binding to the herbicide (i.e., increasing the distance);

[0279] (iii) phenylalanine at position 404 (F404), which corresponds structurally to valine at position 347 (V347) of human PPOX, affects binding to the herbicide (i.e., steric hindrance); and

[0280] (iv) phenylalanine at position 383 (F383), which corresponds structurally to phenylalanine at position 331 (F331) of human PPOX, affecting binding to the herbicide (i.e., unstable conformation); or

[0281] Any combination of these. Each possibility is a separate implementation.

[0282] In some embodiments, the amino acid sequence of the PPO isolated from the fungus comprises one or more sequences represented by any one of SEQ ID NOs: 2, 94, 96-98, or any combination thereof. Each possibility is a separate embodiment.

[0283] In some embodiments, the amino acid sequence of the PPO isolated from the fungus comprises one or more of SEQ ID NOs: 2, 94, 97-98, or any combination thereof. Each possibility is a separate embodiment.

[0284] In some embodiments, the amino acid sequence of the PPO isolated from the fungus comprises SEQ ID NO: 2 and / or 98. Each possibility is a separate embodiment.

[0285] In some embodiments, the amino acid sequence of the PPO isolated from the fungus comprises SEQ ID NO: 96. Each possibility is a separate embodiment.

[0286] Reference is now made to Table 3 which summarizes the modes / mechanisms of acquisition of PPO herbicide tolerance in fungal isolated PPOs.

[0287] In some embodiments, the fungal-derived PPO is isolated from a fungal species of the clade Leotiomyceta.

[0288] In some embodiments, the fungal-derived PPO is isolated from a fungal species selected from the group consisting of Aspergillus sp., Penicillium sp., Trichoderma sp., Rhodotorula sp., and Hesseltinella sp.

[0289] In some embodiments, the exogenous fungal-derived PPO has less than 40% similarity in amino acid sequence to the endogenous plant PPO enzyme.

[0290] In some embodiments, the exogenous fungal-derived PPO enzyme further comprises a plant chloroplast transit peptide.

[0291] In some embodiments, the plant is selected from the group consisting of: corn, wheat, rice, barley, soybean, cowpea, chickpea, cotton, sorghum, beans, rapeseed, alfalfa, flax, sunflower, safflower, millet, rye, sugarcane, sugar beet, cocoa, tea, Brassica, coffee, sweet potato, flax, peanut, clover; lettuce, tomato, gourd, cassava, potato, carrot, radish, pea, lentil, cabbage, cauliflower, broccoli, Brussels sprouts, pepper, pineapple, citrus, apple, pear, peach, apricot, walnut, avocado, banana, palm, eucalyptus, poplar, pine, coconut, orchid, morning glory, carnation, rose, switchgrass, prairie grass, Indian grass, big bluestem grass, camellia, and any combination thereof. Each possibility is a separate embodiment.

[0292] In some embodiments, the plant is selected from the group consisting of corn, wheat, rice, soybean, cowpea, chickpea, cotton, sorghum, rapeseed, alfalfa, sunflower, sugarcane, sugar beet, camellia, or any combination thereof. Each possibility is a separate embodiment.

[0293] In some embodiments, the one or more PPO inhibitors are selected from the following herbicide families: phenylpyrazoles, pyrimidinediones, oxazolidinediones, diphenyl ethers, oxadiazoles, N-phenylphthalimides, triazinones, thiadiazoles, triazolinones, and triazolopyridones, or any combination thereof. Each possibility is a separate embodiment.

[0294] In some embodiments, the plant is a broadleaf plant.

[0295] In some embodiments, the comparison of the 3D structure / fold of the fungal-derived PPO to the crystal structure of human PPO bound to acifluorfen comprises comparing a modeled structure of the fungal-derived PPO fold or a crystal structure showing the folded fungal-derived PPO.

[0296] According to another aspect, a transgenic plant is provided, which expresses an exogenous fungal-derived protoporphyrinogen oxidase (PPO) that confers resistance or tolerance to at least one PPO inhibitor herbicide, wherein the fungal-derived PPO enzyme comprises an amino acid sequence having at least 80% similarity to a fungal isolated PPO sequence as shown in any one of SEQ ID NOs: 1 to 7 and SEQ ID NOs: 86, 88 to 89, 91 to 99, or any combination thereof. Each possibility is a separate embodiment.

[0297] In some embodiments, the fungal PPO enzyme comprises an amino acid sequence having at least 90% similarity to the fungal isolated PPO sequence shown in any one of SEQ ID NOs: 1-7 and SEQ ID NOs: 86, 88-89, 91-99, or any combination thereof.

[0298] In some embodiments, the protoporphyrinogen oxidase (PPO) of fungal origin comprises an amino acid sequence having at least 80% similarity, 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 88%, at least 89%, 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%, at least 99.9% or more similarity to the amino acid sequence of PPO isolated from fungi. Each possibility is a separate embodiment.

[0299] In some embodiments, the amino acid sequence of the PPO isolated from the fungus comprises any one of SEQ ID NOs: 1 to 7 and SEQ ID NOs: 86, 88 to 89, 91 to 99 or any combination thereof. Each possibility is a separate embodiment.

[0300] In some embodiments, the fungal-derived PPO enzyme comprises no more than The bag opening size.

[0301] In some embodiments, the exogenous fungal-derived PPO comprises: a first arginine that structurally corresponds to the position of arginine at position 97 in human PPOX (human Arg97), and / or a second arginine that structurally corresponds to the position of arginine at position 62 in human PPOX (human Arg62), wherein the first and / or second arginine of the fungal-derived PPO has a geometric orientation that is different from the geometric orientation of human Arg62 and / or human Arg97, wherein the positions of the fungal-derived PPO structurally correspond to Arg62 and / or Arg97, and their different geometric orientations are determined by comparing a simulated 3D structure / fold of the fungal-derived PPO with a crystal structure of human PPOX bound to trifluorfen (provided with PDB identifier: 4IVO), wherein the simulated 3D structure / fold of the fungal-derived PPO is structurally aligned with the crystal structure of human PPO and overlaps with trifluorfen.

[0302] In some embodiments, the exogenous fungal-derived PPO comprises a phenylalanine at position 404 (F404), which structurally corresponds to a valine at position 347 (V347) of human PPOX, wherein the corresponding phenylalanine position of the fungal-derived PPO is determined by comparing a simulated 3D structure / fold of the fungal-derived PPO with a crystal structure of human PPOX bound to trifluorfen (provided with PDB identifier: 4IVO), wherein the simulated 3D structure / fold of the fungal-derived PPO is structurally aligned with the crystal structure of human PPO and overlaps with trifluorfen.

[0303] In some embodiments, the exogenous fungal-derived PPO comprises a phenylalanine at position 383 (F383) that structurally corresponds to a phenylalanine at position 331 (F331) of human PPOX, wherein the corresponding phenylalanine position of the fungal-derived PPO is determined by comparing a simulated 3D structure / fold of the fungal-derived PPO with a crystal structure of human PPOX bound to trifluorfen (provided with PDB identifier: 4IVO), wherein the simulated 3D structure / fold of the fungal-derived PPO is structurally aligned with the crystal structure of human PPO and overlaps with trifluorfen.

[0304] In some embodiments, the fungal-derived PPO comprises an amino acid sequence that has less than 40% similarity to the amino acid sequence of an endogenous plant PPO enzyme.

[0305] In some embodiments, the fungal-derived PPO comprises an amino acid sequence with less than about 55% similarity, less than about 50% similarity, less than about 45% similarity, less than about 40% similarity, less than about 35% similarity, less than about 30% similarity, less than about 25% similarity, less than about 20% similarity to an endogenous plant PPO enzyme. Each possibility is a separate embodiment.

[0306] In some embodiments, the fungal-derived PPO enzyme further comprises a plant chloroplast transit peptide.

[0307] According to some embodiments, the fungal-derived PPO is isolated from a fungal species selected from the group consisting of Aspergillus sp., Fusarium sp., Rhizopus sp., Penicillium sp., Trichoderma sp., Rhodotorulasp., Hesseltinella sp., Spizellomyces sp., Rhizophagus sp., Alternaria sp. and Cladosporium sp., or any combination thereof. Each possibility is a separate embodiment.

[0308] In some embodiments, the fungal PPO enzyme is isolated from a fungal species selected from the group consisting of Aspergillus, Fusarium, Rhizopus, Penicillium and Trichoderma, or any combination thereof. Each possibility is a separate embodiment.

[0309] In some embodiments, the fungal-derived PPO is isolated from a fungal species selected from the group consisting of Aspergillus sp., Penicillium sp., Trichoderma sp., Rhodotorula sp., Hesseltinella sp., or any combination thereof. Each possibility is a separate embodiment.

[0310] In some embodiments, the fungal-derived PPO enzyme is isolated from a fungal species of the clade Leotiomyceta.

[0311] In some embodiments, the plant is selected from the group consisting of: corn, wheat, rice, barley, soybean, cowpea, chickpea, cotton, sorghum, beans, rapeseed, alfalfa, flax, sunflower, safflower, millet, rye, sugarcane, sugar beet, cocoa, tea, Brassica, coffee, sweet potato, flax, peanut, clover; lettuce, tomato, gourd, cassava, potato, carrot, radish, pea, lentil, cabbage, cauliflower, broccoli, Brussels sprouts, pepper, pineapple, citrus, apple, pear, peach, apricot, walnut, avocado, banana, palm, eucalyptus, poplar, pine, coconut, orchid, morning glory, carnation, rose, switchgrass, prairie grass, Indian grass, big bluestem grass, camellia, and any combination thereof. Each possibility is a separate embodiment.

[0312] In some embodiments, the plant is selected from the group consisting of corn, wheat, rice, soybean, cowpea, chickpea, cotton, sorghum, rapeseed, alfalfa, sunflower, sugarcane, sugar beet, camellia, or any combination thereof. Each possibility is a separate embodiment.

[0313] In some embodiments, the one or more PPO inhibitors are selected from the following herbicide families: phenylpyrazoles, pyrimidinediones, oxazolidinediones, diphenyl ethers, oxadiazoles, N-phenylphthalimides, triazinones, thiadiazoles, triazolinones, and triazolopyridones, or any combination thereof.

[0314] In some embodiments, the plant is a broadleaf plant.

[0315] In some embodiments, the plant is a cereal plant.

[0316] According to certain aspects, according to any of the above embodiments, there is provided a seed of a transgenic plant.

[0317] According to some aspects, a product derived from the transgenic plant of any one of the above embodiments is provided.

[0318] According to certain aspects, a method for controlling weed growth using at least one PPO inhibitor herbicide in growing crops is provided, wherein the crops include the transgenic plants according to any of the above embodiments, and the method includes applying a weed controlling amount of one or more PPO inhibitor herbicides.

[0319] In some embodiments, the one or more PPO inhibitors are selected from the following herbicide families: phenylpyrazoles, pyrimidinediones, oxazolidinediones, diphenyl ethers, oxadiazoles, N-phenylphthalimides, triazinones, thiadiazoles, triazolinones, and triazolopyridones, or any combination thereof.

[0320] According to certain aspects, a method for conferring resistance or tolerance to one or more PPO inhibitor herbicides in a crop plant is provided, the method comprising genetically modifying the plant to express an exogenous fungal-derived PPO enzyme, wherein the fungal-derived PPO enzyme comprises an amino acid sequence having at least 80% similarity to a PPO sequence isolated from a fungus as shown in any one of SEQ ID NOs: 1 to 7 or SEQ ID NOs: 86, 88 to 89, 91 to 99, or any combination thereof.

[0321]

sequence

[0322] According to some embodiments, the PPO polypeptide isolated from natural fungi has an amino acid sequence shown in any one of SEQ ID NOs: 1 to 7 and SEQ ID NOs: 86 to 100:

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331] According to some embodiments, the PPO polypeptide isolated from the chimeric fungus is fused to a chloroplast transient peptide (cTP) and has an amino acid sequence shown in any one of SEQ ID NOs: 8 to 14 and SEQ ID NOs: 104 to 118:

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340] According to some embodiments, human PPO has the amino acid sequence shown in SEQ ID NO: 101:

[0341]

[0342] According to some embodiments, the plant PPO has the amino acid sequence shown in any one of SEQ ID NOs: 102-103 and SEQ ID NOs: 119-121:

[0343]

[0344]

[0345]

[0346] According to some embodiments, the chloroplast transient peptide (cTP) has the amino acid sequence shown in SEQ ID NO: 15:

[0347]

[0348] In order to more fully illustrate some embodiments of the present invention, the following embodiments are proposed. However, they should never be interpreted as limiting the broad scope of the invention. Those skilled in the art can easily design many changes and modifications of the principles disclosed herein without departing from the scope of the present invention. [Example]

[0349] [Example 1: Screening of PPO tolerance genes in plants and identification of PPO tolerance in fungi isolated]

[0350] In plants, transgenic plants are screened to identify transgenes that confer resistance to PPO-type inhibitor herbicides. Transgenic plants that exhibit PPO herbicide tolerance are sampled, and PPO herbicide tolerance genes are isolated and annotated. Fungal genes are identified as conferring the tolerance exhibited. Nucleic acid sequencing is used to identify the fungal source of each gene. Each tolerance-conferring natural gene is amplified from the genomic DNA (gDNA) of the source fungus with a specific primer set complementary to the coding sequence (cds) or untranslated region (UTR) of the gene.

[0351] The amino acid sequence of the isolated fungal PPO gene is as follows:

[0352] SEQ ID NO: 1 (PPO from Aspergillus sp.);

[0353] SEQ ID NO: 2 (PPO from Aspergillus sp.);

[0354] SEQ ID NO: 3 (PPO from Fusarium sp.);

[0355] SEQ ID NO: 4 (PPO from Fusarium sp.);

[0356] SEQ ID NO: 5 (PPO from Fusarium sp.);

[0357] SEQ ID NO: 6 (PPO from Rhizopus sp.);

[0358] SEQ ID NO: 7 (PPO from Penicillium sp.).

[0359] [Example 2: Evaluation of tolerance of PPO genes isolated from fungi in transgenic plants]

[0360]

method

[0361]

Cloning and transformation of fungal PPO genes

[0362] The nucleotide sequence encoding the PPO protein was cloned into a binary plant expression vector under the control of the 35S constitutive promoter and the HSP terminator.

[0363] These vectors are used to transform Arabidopsis plants using Agrobacterium according to the floral dip method.

[0364] Generation of the cTP chimeric PPO gene

[0365] The sequence of the N-terminal chloroplast transient peptide (cTP) (AtPPO1) (amino acids 1 to 38 (AtcTP_38)) of the Arabidopsis chloroplast PPO was added to the sequence of the fungal PPO tolerance gene to enhance the performance of its polypeptide as a PPO inhibitor resistance gene (SEQ ID NO: 8 is cloned from SEQ ID NO: 1, SEQ ID NO: 9 is cloned from SEQ ID NO: 2, SEQ ID NO: 10 is cloned from SEQ ID NO: 3, SEQ ID NO: 11 is cloned from SEQ ID NO: 4, SEQ ID NO: 12 is cloned from SEQ ID NO: 5, SEQ ID NO: 13 is cloned from SEQ ID NO: 6, and SEQ ID NO: 14 is cloned from SEQ ID NO: 7).

[0366]

Evaluation of tolerance of PPO genes isolated from fungi in transgenic plants

[0367] T1 or T2 generation transformed seeds were germinated on Basta selection (Bayer) according to the manufacturer's instructions (~30-35 plants per pot, 4 pots per gene). When two rosette leaves were grown, they were treated with PPO inhibitor herbicides, foliar application of Strike (flumizone 50%, Adama) or Aurora (40% foramsulfuron, FMC), in concentrations ranging from 0.000015% to 1%, diluted in water as shown below.

[0368]

result

[0369] PPO tolerance was evaluated in WT Arabidopsis plants and transgenic plants expressing fungal isolated PPOs having the amino acid sequences represented by SEQ ID NOs: 1-3, 6, and N-terminal cTP fusions represented by SEQ ID NOs: 8-12 and 14 ( Figure 1A-1B ).

[0370] To assess the sensitivity level of Arabidopsis plants to Strike, a preliminary calibration aimed to set the natural tolerance limit of WT plants. To this end, WT Arabidopsis plants were treated with low concentrations of Strike at 0.000015% and 0.00003% diluted in water. Figure 1A WT plants were shown to be susceptible to these low amounts of Strike.

[0371] To evaluate the tolerance level displayed by transgenic Arabidopsis plants expressing PPO genes isolated from different fungi, three experiments were conducted in transgenic plants. In experiment #1, T2 transformed seeds were germinated and plants were treated with 0.01%, 0.1% and 0.5% of Strike. In experiment #2, T2 transformed seeds were germinated in two separate tables, one for each PPO inhibitor selection, and then plants were treated with 0.01%, 0.1%, 0.5% and 1% of Strike and in the second table with 0.01%, 0.1% and 0.5% of Aurora.

[0372] Figure 1B The results of the three experiments described above are summarized. The level of tolerance exhibited by the transgenic plants was assessed using a four data point plant resilience score given on a scale of 0 to 5, where "0" represents a highly compromised plant and "5" represents a highly tolerant / resistant plant.

[0373] Advantageously and unexpectedly, all of the PPO genes were derived from Aspergillus sp. encoding the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 2 and SEQ ID NO: 9, Fusarium sp. encoding the amino acid sequences set forth in SEQ ID NO: 3, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, Rhizopus sp. encoding the amino acid sequence set forth in SEQ ID NO: 6, and Penicillium sp. encoding the amino acid sequence set forth in SEQ ID NO: 14, conferring high tolerance (i.e., tolerance greater than about 10 4 times (4 orders of magnitude) WT).

[0374] Of note, particularly advantageous are transgenic plants expressing the Aspergillus sp. PPO protein (SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 2 and SEQ ID NO: 9), the Fusarium sp. gene (e.g., SEQ ID NO: 10 and SEQ ID NO: 12), and the Rhizopus sp. gene (SEQ ID NO: 6) that exhibit very high levels (i.e., resistance to Strike of about 10 5 (5 orders of magnitude) (compared to control plants)

[0375] Also advantageous are transgenic plants expressing Aspergillus sp. PPO proteins (e.g., SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 2 and SEQ ID NO: 9) and genes from Rhizopus sp. (SEQ ID NO: 6) and Penicillium sp. (SEQ ID NO: 14), which exhibit very high resistance to Aurora compared to control plants.

[0376] It is also noteworthy that the Penicillium PPO protein (SEQ ID NO: 14) confers very high resistance to Aurora but not to Strike in transgenic plants, while on the other hand, the Fusarium proteins (e.g., SEQ ID NO: 10 and SEQ ID NO: 12) confers high levels of resistance to Strike but not to Aurora in transgenic plants. Figure 2A B shows comparative images between transgenic plants from experiments #1 and #2 described above, with plants transformed with an empty vector as a control. These figures show the growth of transgenic plants that received no treatment (0%) or that received increasing concentrations of Strike ( Figure 2A ) or Aurora( Figure 2B ) treatment on transgenic plants expressing the PPO gene isolated from fungi.

[0377] The figures show that transgenic plants expressing the fungal isolated PPO gene showed favorable growth improvements in all concentration ranges tested (0.01% to 0.5% for Aurora and 0.01% to 1% for Strike), and thus increased tolerance to both PPO inhibitor herbicides when applied in a dose-dependent manner.

[0378] [Example 3: Analysis of putative motifs in PPO tolerance genes isolated from fungi]

[0379]

method

[0380]

Multiple sequence alignment (MSA) combined with structural alignment of fungal isolation and plant PPO genes

[0381] The SWISS-MODEL server predicted 3D models of PPO herbicide tolerance genes from each fungal species (Waterhouse, A., 2018). A representative model was selected from each species: SEQ ID NO: 2 (Aspergillus); SEQ ID NO: 3 (Fusarium); SEQ ID NO: 6 (Rhizopus); SEQ ID NO: 7 (Penicillium). Model #1 template is the protoporphyrinogen oxidase structure of human protoporphyrinogen IX oxidase (HsPPO) (PDB: 3NKS.1.A; Qin X., 2011) with an amino acid sequence represented by SEQ ID NO: 101. Using the SWISS-MODEL server, the model was aligned on 3NKS and 1SEZ (N. tabacum mitochondrial PPO2; Koch M., 2004 and PDB) (based on structural alignment).

[0382] Other PPO herbicide tolerance genes were aligned individually to their species representatives and the alignments were combined with the structure-based alignment: SEQ ID NO: 1 to SEQ ID NO: 2 (Aspergillus); SEQ ID NO: 4 and SEQ ID NO: 5 to SEQ ID NO: 3 (Fusarium). The sequences of plant PPOs were combined with the structure-based alignment in the same way: the following amino acid sequences were aligned with the sequence of the mitochondrial PPO of tobacco (N. tabacum) (NtPPO2) having the amino acid sequence represented by SEQ ID NO: 103 using the T-coffee server (Notredame, C., 2000): the mitochondrial PPO from Arabidopsis thaliana (AtPPO2) having the amino acid sequence represented by SEQ ID NO: 119, the chloroplast PPO from tobacco (N. tabacum) (NtPPO1) having the amino acid sequence represented by SEQ ID NO: 120, the Arabidopsis thaliana PPO (AtPPO1) having the amino acid sequence represented by SEQ ID NO: 121, and the soybean PPO (GmPPO1) having the amino acid sequence represented by SEQ ID NO: 102. Based on the sequence of NtPPO2, the alignment was combined with the structure-based alignment.

[0383]

result

[0384] The amino acid sequences of fungal PPO enzymes were analyzed and compared with those of plant and human PPOs to identify putative fungal motifs that might be responsible for the improved tolerance exhibited by the transgenic plants.

[0385] According to some embodiments, 10 motifs (motifs 1 to 10) are identified in PPO isolated from fungi. The motif length is about 4 to 30 amino acids. According to some embodiments, the motifs identified in PPO isolated from fungi advantageously share unique patterns of amino acid sequence and / or structure. According to some embodiments, the motifs (e.g., motifs 1, 2, 3, 4, 6, 7, and 10) of PPO sequences of fungal origin may share more common amino acid sequence and / or structural features between enzymes of Aspergillus, Fusarium, Rhizopus, and Penicillium than the corresponding sequences of PPOs of plant origin shared between enzymes of Arabidopsis, tobacco, and soybean.

[0386] Advantageously, according to some embodiments, a total of 70 motif sequences identified in PPO enzymes isolated from fungi of the genera Aspergillus sp., Fusarium sp., Rhizopus sp., and Penicillium sp. are considered to be different and distinguishable from their corresponding sequences in plant PPO enzymes.

[0387] According to some embodiments, the 70 motifs have amino acid sequences as shown in SEQ ID NOs: 16 to 85. [Example 4: Screening for PPO tolerance genes in plants, identifying PPO tolerance of fungal isolates, and evaluating their tolerance in transgenic plants]

[0388]

Screening of PPO tolerance genes in plants and identification of PPO tolerance genes isolated from fungi

[0389] In addition to the PPO tolerance genes demonstrated in Examples 1-2 above, other PPO genes were screened for tolerance.

[0390] By sequencing and annotating its corresponding nucleic acid transgene, a PPO gene having an amino acid sequence represented by any one of SEQ ID NOs: 86 to 100 was identified as a PPO gene isolated from fungi: After plant screening was performed to identify potential PPO tolerance genes to PPO-type inhibitor herbicides, similar to the above experiment described in Example 1.

[0391] The amino acid sequence of the isolated fungal PPO gene is as follows:

[0392] SEQ ID NO: 86 (PPO from Trichoderma sp.);

[0393] SEQ ID NO: 87 (PPO from Aspergillus sp.);

[0394] SEQ ID NO: 88 (PPO from Aspergillus sp.);

[0395] SEQ ID NO: 89 (PPO from Fusarium sp.);

[0396] SEQ ID NO: 90 (PPO from Fusarium sp.);

[0397] SEQ ID NO: 91 (PPO from Rhizopus sp.);

[0398] SEQ ID NO: 92 (PPO from Rhizopus sp.).

[0399] SEQ ID NO: 93 (PPO from Rhizopus sp.).

[0400] SEQ ID NO: 94 (PPO from Penicillium sp.).

[0401] SEQ ID NO: 95 (PPO from Penicillium sp.);

[0402] SEQ ID NO: 96 (PPO from Trichoderma sp.);

[0403] SEQ ID NO: 97 (PPO from Rhodotorula sp.);

[0404] SEQ ID NO: 98 (PPO from Hesseltinella sp.);

[0405] SEQ ID NO: 99 (PPO from Spizellomyces sp.);

[0406] SEQ ID NO: 100 (PPO from Rhizophagus sp.).

[0407]

Evaluation of tolerance of PPO genes isolated from fungi in transgenic plants

[0408]

method

[0409]

Cloning and transformation of fungal PPO genes

[0410] The nucleotide sequence encoding the PPO protein was cloned into a binary plant expression vector under the control of the 35S constitutive promoter and the HSP terminator.

[0411] These vectors are used to transform Arabidopsis plants using Agrobacterium according to the floral dip method.

[0412] Generation of the cTP chimeric PPO gene

[0413] Chimeric PPO genes having chloroplast transient peptide (cTP) (SEQ ID NO: 15) represented by the amino acid sequence of SEQ ID NO: 104-118 (N-terminal amino acids 1-38 are cTP from Arabidopsis thaliana) were generated from their respective fungal PPO tolerance genes (represented by SEQ ID NO: 86-100) to enhance the performance of their polypeptides as PPO inhibitor resistance genes in subsequent in planta experiments, i.e., SEQ ID NO: 104 was cloned from SEQ ID NO: 86, SEQ ID NO: 105 was cloned from SEQ ID NO: 87, SEQ ID NO: 106 was cloned from SEQ ID NO: 88, SEQ ID NO: 107 was cloned from SEQ ID NO: 89, SEQ ID NO: 108 was cloned from SEQ ID NO: 90, SEQ ID NO: 109 was cloned from SEQ ID NO: 91, SEQ ID NO: 110 was cloned from SEQ ID NO: 92, SEQ ID NO: 111 was cloned from SEQ ID NO: 93, SEQ ID NO: 112 was cloned from SEQ ID NO: 94, SEQ ID NO: 113 was cloned from SEQ ID NO: 95, SEQ ID NO: 114 was cloned from SEQ ID NO: 96 SEQ ID NO:112 was cloned from SEQ ID NO:94, SEQ ID NO:113 was cloned from SEQ ID NO:95, SEQ ID NO:114 was cloned from SEQ ID NO:96, SEQ ID NO:115 was cloned from SEQ ID NO:97, SEQ ID NO:116 was cloned from SEQ ID NO:98, SEQ ID NO:117 was cloned from SEQ ID NO:99, and SEQ ID NO:118 was cloned from SEQ ID NO:100.

[0414]

Evaluation of tolerance of PPO genes isolated from fungi in transgenic plants

[0415] T2 transformed seeds were germinated on Basta selection (Bayer) according to the manufacturer's instructions (~30-35 plants per pot, four pots per gene). When two rosette leaves were grown, they were treated with a PPO inhibitor Group 14 herbicide, foliar sprays of Strike (flumizone, Adama), Star (oxadiazon, Tapazol), Goal (oxyfluorfen, Corteva) or Aurora (foramsulfuron, FMC), diluted in water, at concentrations of 1.0 kg / ha, 1.15 kg / ha, 1.25 kg / ha or 2.5 kg / ha.

[0416]

result

[0417] Evaluation of tolerance to PPO herbicides (Group 14 herbicides) in Arabidopsis thaliana of WT plants, transgenic plants transformed with an "empty" vector, and transgenic plants expressing the genes described in SEQ ID NOs: 8-14 and SEQ ID NOs: 104-118 and ( Figures 3A to 3C ) Transgenic plants expressing the fungal isolated PPO fused to cTP (at the N-terminus of the protein) as shown.

[0418] To evaluate the relative survival / resilience exhibited by transgenic plants in the experiments described below, a representative resilience scale, including 5 levels of resilience / survival (scored 1–5), where “1” represents highly damaged plants and “5” represents highly resistant plants, was used as a reference for scoring plant survival / resilience, e.g. Figure 3A shown.

[0419] First, if Figure 3B As shown, all transgenic plants carrying / expressing the PPO gene isolated from fungi had an amino acid sequence as represented by any one of SEQ ID NOs: 8 to 14 and SEQ ID NOs: 104 to 118, and tolerance to Strike was determined at a concentration of 2.5 kg / hectare.

[0420] Advantageously, transgenic plants corresponding to SEQ ID NOs: 8-14, 104, 106-107 and 109-117 (and their respective SEQ ID NOs: 1-7, 86, 88-89, 91-99) showed a certain degree of tolerance in the presence of the herbicide (corresponding to a resilience score of 2, 3, 4 or 5) growth. This was compared to control plants (WT and plants transformed with an empty vector), which showed acute growth inhibition and leaf whitening, as indicated by their very low tolerance score of 1.

[0421] Transgenic plants corresponding to SEQ ID NOs: 8-12, 14, 109-111, 114-115 showed higher tolerance levels (corresponding to resilience scores of 3, 4 or 5) in the presence of the herbicide Strike at a concentration of 2.5 kg / hectare.

[0422] Transgenic plants corresponding to SEQ ID NOs: 8-12, 14, 114-115 showed higher tolerance levels (corresponding to resilience scores of 4 or 5) in the presence of the herbicide Strike at a concentration of 2.5 kg / hectare.

[0423] Next, PPO tolerance was assessed in the presence of a wider range of Group 14 PPO herbicides: Strike at 2.5 kg / ha, Star at 1.25 kg / ha, Goal at 1.15 kg / ha and Aurora at 1.0 kg / ha.

[0424] The results of the survival test in the presence of Strike, Star, Goal and Aurora are shown in Figure 3C Shown are only those plants expressing the fungal-isolated PPO gene that exhibited some degree of tolerance to all four herbicides tested (corresponding to resilience scores of 2, 3, 4, or 5), whereas control plants (WT and plants transformed with an empty vector) exhibited acute growth inhibition and leaf bleaching, as indicated by their very low tolerance score of 1.

[0425] Advantageously, the best five fungal isolated PPOs have an amino acid sequence represented by any one of SEQ ID NOs: 9, 112, 114 to 116 and belong to the genera Aspergillus, Penicillium, Trichoderma, Rhodotorula and Hesseltinella.

[0426] The PPO genes isolated from the first three fungi grown in the presence of Strike, Star, Goal and Aurora herbicides had amino acid sequences represented by SEQ ID NOs: 9, 114 and 115 and belonged to the genera Aspergillus, Trichoderma and Rhodotorula, respectively.

[0427] The PPO genes isolated from the first two fungi grown in the presence of Strike, Star, Goal and Aurora herbicides had amino acid sequences represented by SEQ ID NOs: 9 and 115 and belonged to the genera Aspergillus and Rhodotorula, respectively.

[0428] The best performing fungal isolate PPO gene that grew in the presence of Strike, Star, Goal and Aurora herbicides had an amino acid sequence represented by SEQ ID NO: 9 and belonged to the genus Aspergillus.

[0429] Plants expressing the genes represented by SEQ ID NOs: 112 and 116 and belonging to the genera Penicillium and Hesseltinella, respectively, showed tolerance only to Goal and Star herbicides ( Figure 3C ).

[0430] all in all, Figures 3B to 3C It was shown that transgenic plants expressing the PPO gene isolated from fungi belonging to any of the genera Aspergillus, Penicillium, Trichoderma, Rhodotorula and Hesseltinella exhibited favorable improved growth in all concentration ranges tested.

[0431] [Example 5: Structural / geometric analysis of putative amino acid positions in PPO tolerance genes isolated from fungi]

[0432]

method

[0433] The secondary and tertiary structures of the proteins represented by SEQ ID NOs: 1 to 7 and SEQ ID NOs: 86 to 100 were predicted as described in ColabFold V1.5.2 (Mirdita, M., Schütze, K., Moriwaki, Y. et al. ColabFold: making protein folding accessible to all. Nature Methods 19, 679-682 (2022)) (https: / / doi.org / 10.1038 / s41592-022-01488-1). These structures were then compared to crystallized proteins as references, including human protoporphyrinogen IX oxidase (PDB identifier: 4IVO) - SEQ ID NO: 101, crystallized with the herbicide acifluorfen (5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoic acid), and tobacco (Nicotiana tabacum) protoporphyrinogen IX oxidase (PDB identifier: 1SEZ) - SEQ ID NO: 103) to identify their relevant amino acids in their domains and positions / positions of the active sites.

[0434] Proteins were mapped to Pymol (PyMOL molecular graphics system, version 2.0, LLC.) Structural alignments were performed using the RMSD (root mean square deviation) measurement to calculate structural similarity between proteins and colored accordingly.

[0435] The PPO genes isolated for the discovered novel fungi were compared in sequence and structural composition to the active and binding sites of the herbicides acifluorfen (5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoic acid) and FAD (flavin adenine dinucleotide). Using proteins found in the PDB as references for structural homology, the residues involved in substrate and FAD binding were identified, labeled, and compared.

[0436]

result

[0437] The 3D structural features of soybean (Glycine max) PPO (SEQ ID NO: 102) and human PPOX (SEQ ID NO: 101) binding to the herbicide trifluorfen are shown in Figure 4A and Figure 4B .

[0438] It was found that the active site of the substrate protoporphyrinogen IX and the FAD binding site of the PPO protein (having the amino acid sequences represented by SEQ ID NOs: 1 to 7 (corresponding to SEQ ID NOs: 8 to 14, respectively) and SEQ ID NOs: 86 to 100 (corresponding to SEQ ID NOs: 104 to 118, respectively) are mainly similar in structure and function. It was also observed that the PPO protein having the amino acid sequence represented by SEQ ID NO: 2 (corresponding to SEQ ID NO: 9) has the same pocket structure as the herbicide acifluorfen when superimposed on SEQ ID NO: 101.

[0439] While the structure and sequence of the residues that make up the herbicide pocket show a high degree of similarity, several key structural / geometric differences were identified that may explain the favorable differences in herbicide tolerance activity of fungal isolates relative to human PPOX or plant PPOs from soybean (Glycine max), Arabidopsis (Arabidopsis) or tobacco. These include 4 modes for the desired resistance / tolerance:

[0440] (i) the size of the opening of the herbicide bag is reduced;

[0441] (ii) destabilization of hydrogen bonds at positions corresponding (by structural alignment) to human positions Arg62 and Arg97;

[0442] (iii) steric hindrance at a position corresponding (by structural alignment) to human position V347 affects binding to the herbicide; and

[0443] (iv) disrupting conformational stability at a position corresponding (by structural alignment) to human position F331, which could affect binding to the herbicide.

[0444] 【1. The opening size of the herbicide bag is reduced】

[0445] like Figure 5A and Figure 5B (Schematic representation of the structure and binding pocket of human PPOX (SEQ ID NO: 101)), the size of the herbicide binding pocket opening was measured (the distance is represented by the yellow dashed line).

[0446] The size of the herbicide bag opening is measured by measuring the Figure 5A (II) shown in yellow) to the widest point of the bag opening, such as Figure 5B As shown by the yellow dotted line in .

[0447] This distance is measured as

[0448] The α-helical structure is oriented in the same direction in human and plant PPOs having the amino acid sequence represented by SEQ ID NOs: 101 to 103 (susceptible to PPO herbicides), as well as in fungal PPOs having the amino acid sequence represented by any one of SEQ ID NOs: 2, 94, 96 to 98 (showing tolerance to PPO herbicides), and thus serves as a reference point for measuring and comparing bag opening sizes, e.g. Figure 5B shown.

[0449] The size of the pocket opening was measured as the distance between the same position on the α-helix occupied by human V170 or the amino acid (valine, isoleucine, or threonine) at the structurally corresponding position in the plant and fungal PPO and the amino group of the side chain of human Arg62 or the amino acid at the structurally corresponding position in the plant and fungal PPO, representing the widest point of the pocket opening, e.g. Figure 5B and Figure 5C shown.

[0450] However, in cases where the amino acid at the position structurally corresponding to human Arg62 in plant and fungal PPO is not arginine (i.e., see Figure 6B : Q123 (SEQ ID NO: 102), T70 (SEQ ID NO: 103) or S102 (SEQ ID NO: 96)), the distances are measured from the same position occupied by the amino group at the end of the glutamine side chain in SEQ ID NO: 102, the methyl group at the end of the threonine side chain in SEQ ID NO: 103, and the hydroxyl group at the end of the serine side chain in SEQ ID NO: 96. Although these three amino acids have different chemical groups, they occupy the same position in the structure and form the inner edge of the pocket opening, so the measured distance is the smallest, so a specific atom is always located at the end of the side chain.

[0451] The dimensions of the bag openings of the above human, plant and fungal PPOs are shown in Table 1 below.

[0452] [Table 1: Bag opening dimensions of PPO for humans, plants and fungi]

[0453]

[0454] Advantageously and surprisingly, Figure 5B As can be seen from the measured distances shown in and Table 1 , significant differences were found in the size of the herbicide binding pocket opening between the human and plant sensitive PPOs and the fungus tolerant PPOs.

[0455] The PPO proteins of human (SEQ ID NO: 101), soybean (Glycine max) (SEQ ID NO: 102) and tobacco (Nicotiana tabacum) (SEQ ID NO: 103) are sensitive to Group 14 PPO herbicides / inhibitors and have at least In contrast, the PPO protein isolated from a tolerant fungus having an amino acid sequence represented by any one of SEQ ID NOs: 2, 94, 96, 97 and 98 is characterized in that the opening of SEQ ID NO: 96 is much smaller, not exceeding

[0456] The difference in the size of the opening of the herbicide binding pocket may explain the resistance of the fungal isolated PPO having the amino acid sequence represented by any one of SEQ ID NOs: 2, 94, 96, 97 and 98 (or the corresponding cTP fusion having the amino acid sequence represented by any one of SEQ ID NOs: 9, 112, 114 to 116), because the reduction in the measured distance of the fungal PPO relative to that of human or plant PPO may prevent / block / hinder the herbicide from entering the binding pocket, thereby conferring tolerance to the herbicide.

[0457] [2. The structurally unstable hydrogen bonds corresponding to the human positions Arg62 and Arg97]

[0458] Of particular importance is the geometric orientation of the amino acids at the positions corresponding to the two arginine residues, arginine at position 62 (Arg62) and arginine at position 97 (Arg97) located within the herbicide binding pocket of human PPOX.

[0459] like Fig. 6A As shown, the diagram illustrates the hydrogen bonds formed between the guanidinium groups of two arginine residues Arg97 (distance 1) and Arg62 (distance 2) located in the herbicide binding pocket of human PPOX and the oxygen atoms of the trifluorfenapyr herbicide (the hydroxyl group (distance 1) or carbonyl group (distance 2) located at C11, respectively).

[0460] Particularly advantageous and surprising is the discovery that at least one of the two arginine residues (Arg62 and Arg97) identified in Homo sapiens PPOX (SEQ ID NO: 101) is conserved in each of the fungal isolated PPOs having an amino acid sequence represented by any one of SEQ ID NOs: 2, 94, 96 to 98 (corresponding to cTPs containing SEQ ID NOs: 9, 112, 114 to 116).

[0461] Arg62 and Arg97 are conserved in each PPO isolated from a fungus having an amino acid sequence represented by any one of SEQ ID NOs: 2, 94, 97 to 98 (corresponding to cTPs containing SEQ ID NOs: 9, 112, 115 to 116, respectively); whereas only Arg97 is conserved in the PPO isolated from a fungus having an amino acid sequence represented by SEQ ID NO: 96 (corresponding to cTP containing SEQ ID NO: 114).

[0462] The positions corresponding to human Arg62 and human Arg97 are conserved as Figure 6B As shown, the amino acid residues forming the herbicide binding pocket and their positions are shown, structurally aligned, and presented in a table format. SEQ ID NO: 101 (Homo sapiens PPOX data is a crystalline form containing trifluorfen) is the reference sequence for structural alignment, including Arg62 and Arg97, and together with SEQ ID NO: 102 (GmPPO, from soybean (Glycine max)) is considered to be a PPO herbicide sensitive / sensitive protein.

[0463] However, even if the fungal PPO positions corresponding to human Arg62 and human Arg97 (except position S102 of SEQ ID NO:96 or SEQ ID NO:114 which corresponds to human Arg62 which is not conserved) are conserved, their conformational orientations may be affected by protein folding, potentially reducing the ability of these amino acids to effectively bind herbicides, thus promoting / inducing PPO herbicide tolerance (residues that contribute to tolerance in Figure 6B marked in red).

[0464] exist Figure 6C The distances measured in and summarized in Table 2 below are the distances between two arginine residues located within the herbicide binding pocket of human PPOX (human Arg62 and human Arg97) or the guanidinium group of residues at structurally corresponding positions in fungal PPO and the oxygen atom of the trifluorfen herbicide (the hydroxyl group (distance 1; d1) or carbonyl group (distance 2; d2) located at C11, respectively).

[0465] like Figure 6C As shown and summarized in Table 2 below, distance 1 (d1), distance 2 (d2) and the sum of distances are greater in the tolerant fungal PPOs having the amino acid sequences represented by SEQ ID NOs: 2, 94, 96 to 98 relative to the sensitive human PPOX having the amino acid sequence represented by SEQ ID NO: 100 or the sensitive plant PPO having the amino acid sequence represented by SEQ ID NO: 101.

[0466] [Table 2: Measured distances between residues at corresponding positions in the structure of human Arg62 and human Arg97 or fungal PPO and the oxygen atom of trifluorfen herbicide]

[0467]

[0468] For example, in a tolerant fungus PPO having an amino acid sequence represented by any one of SEQ ID NOs: 2, 94, 96 to 98, the distance (d1) is greater than In the case of the sensitive human PPOX having the amino acid sequence represented by SEQ ID NO: 100 or the sensitive plant PPO having the amino acid sequence represented by SEQ ID NO: 101, d1 does not exceed Furthermore, in the tolerant fungus PPO having an amino acid sequence represented by any one of SEQ ID NOs: 2, 94, 96 to 98, the distance (d2) is at least In the case of the sensitive human PPOX represented by SEQ ID NO: 100 or the sensitive plant PPO represented by SEQ ID NO: 101d1, the distance (d2) is no more than Likewise, the sum of the distances was at least 13.8 in the tolerant fungus PPOs and did not exceed 10.1 in the sensitive PPOs.

[0469] [3. The steric hindrance of the position corresponding to human position V347 affects the size of the binding pocket]

[0470] like Figure 7 As shown, the geometric orientation of valine V347 in the PPO binding pocket of the susceptible human PPOX represented by SEQ ID NO: 101 is compared with the corresponding (by structural alignment) geometric orientation of phenylalanine F404 of the resistant fungal PPO represented by SEQ ID NO: 96 (see Figure 6B ). SEQ ID NO:96 exhibits a phenylalanine at a position where most other proteins would have a valine or isoleucine. The added aromatic compound faces the pocket and interferes with the intermolecular bridge connecting the amino acid portions of the herbicide.

[0471] [4. Unstable conformation corresponding to human position F331]

[0472] like Figure 8 As shown, the geometric orientation of phenylalanine F331 in the PPO binding pocket of the susceptible human PPOX represented by SEQ ID NO: 101 is compared with the corresponding (by structural alignment) geometric orientation of phenylalanine F383 of the resistant fungal PPO represented by SEQ ID NO: 96 (see Figure 6B ). F331 together with leucine at position 334 ( Figure 6B) stabilizes the herbicide molecule in the human PPOX pocket. In contrast, SEQ ID NO: 96 has a phenylalanine at the corresponding position F383 (oriented perpendicularly to human PPOX), so the herbicide molecule is not stable in the pocket, contributing to its tolerance.

[0473] In summary, although the structure and sequence of the residues constituting the herbicide pocket show a high degree of similarity, structural / geometric differences were found between sensitive / susceptible PPOs (i.e., human PPOX or plant PPOs such as from soybean (Glycine max), Arabidopsis (Arabidopsis) or tobacco) and resistant / tolerant PPOs (i.e., fungal isolates). These advantageous and surprising tolerance differences / mechanisms are summarized in Table 3 below.

[0474] Table 3: Summary of modes / mechanisms of PPO herbicide tolerance acquired in fungal PPO isolates

[0475]

[0476] Although certain embodiments of the present invention have been illustrated and described, it is apparent that the present invention is not limited to the embodiments described herein. Many modifications, changes, variations, substitutions and equivalents will be apparent to those skilled in the art without departing from the spirit and scope of the present invention, as described in the following claims.

Claims

1. A transgenic plant expressing a protoporphyrinogen oxidase (PPO) of exogenous fungal origin, wherein the PPO of fungal origin comprises an amino acid sequence having at least 80% similarity to at least one sequence shown in any one of SEQ ID NOs: 2, 94, 96 to 98, or any combination thereof, in, The fungal origin PPO contains no more than The size of the pocket opening was determined by comparing the modeled 3D structure / folding of the fungal-derived PPO with the crystal structure of human PPOX bound to acifluorfen, and wherein the simulated 3D structure / fold of the fungal-derived PPO is structurally aligned with the crystal structure of human PPO and overlaps with trifluorfen, and Wherein, the fungal-derived PPO confers resistance or tolerance to at least one PPO inhibitor herbicide.

2. The transgenic plant according to claim 1, wherein The PPO derived from exogenous fungi comprises an amino acid sequence having at least 90% similarity to at least one sequence shown in any one of SEQ ID NOs: 2, 94, 96 to 98 or any combination thereof.

3. The transgenic plant according to claim 1 or 2, wherein The bag opening size of any of the fungus-derived PPOs comprises at least difference.

4. The transgenic plant according to any one of claims 1 to 3, in, The exogenous fungus-derived PPO comprises: a first arginine which structurally corresponds to the position of arginine at position 97 in human PPOX (human Arg97), and / or a second arginine, which structurally corresponds to the position of arginine at position 62 in human PPOX (human Arg62), wherein the first and / or second arginine of the fungus-derived PPO has a geometric orientation different from the geometric orientation of human Arg62 and / or human Arg97, as determined by comparing a simulated 3D structure / fold of the fungus-derived PPO with a crystal structure of human PPOX bound to acifluorfen, and Therein, the simulated 3D structure / folding of the fungus-derived PPO is structurally aligned with the crystal structure of human PPO and overlaps with trifluorfen.

5. The transgenic plant according to claim 4, in, The different geometric orientations include different distances between the first arginine of the fungus-derived PPO and the overlapping acifluorfen, and wherein the distance includes a first distance (d1) between the guanidinium group of the first arginine residue of the fungus-derived PPO and the overlapping C11 hydroxyl group of acifluorfen, and Where d1 is greater than 6. The transgenic plant according to claim 4 or 5, in, The different geometric orientations include different distances between the second arginine of the fungus-derived PPO and the overlapping acifluorfen, and wherein the distance includes a second distance (d2) between the guanidinium group of the second arginine residue of the fungal-derived PPO and the overlapping carbonyl oxygen of C11 of acifluorfen, and Where d2 is greater than 7. The transgenic plant according to any one of claims 4 to 6, wherein the sum of the distances d1 and d2 is greater than about 8. The transgenic plant according to any one of claims 7, characterized in that in, The sum of the distances d1 and d2 of the fungal PPO differs from the sum of the distances d1 and d2 of human PPOX by more than and Among them, d1 and d2 of human PPOX are: The distance between the guanidinium group of human Arg97 and the C11 hydroxyl group of acifluorfen (d1 human PPOX), and The distance between the guanidinium group of human Arg62 and the carbonyl group of C11 of trifluorfen (d2 human PPOX), This was determined by comparing the modeled 3D structure / fold of the fungal-derived PPO with the crystal structure of human PPOX bound to acifluorfen, and Therein, the simulated 3D structure / folding of the fungus-derived PPO is structurally aligned with the crystal structure of human PPO and overlaps with trifluorfen.

9. The transgenic plant according to any one of claims 5 to 8, wherein The PPO derived from exogenous fungi comprises an amino acid sequence having at least 80% similarity to at least one sequence shown in any one of SEQ ID NOs: 2, 94, 96 to 98 or any combination thereof.

10. The transgenic plant according to claim 9, wherein The PPO derived from exogenous fungi comprises an amino acid sequence having at least 90% similarity to at least one sequence shown in any one of SEQ ID NOs: 2, 94, 96 to 98 or any combination thereof.

11. The transgenic plant according to any one of claims 1 to 10, in, The exogenous fungus-derived PPO comprises a phenylalanine at position 404 (F404), which structurally corresponds to a valine at position 347 (V347) of human PPOX, as determined by comparing a simulated 3D structure / fold of the fungus-derived PPO with a crystal structure of human PPOX bound to acifluorfen, and Therein, the simulated 3D structure / folding of the fungus-derived PPO is structurally aligned with the crystal structure of human PPO and overlaps with trifluorfen.

12. The transgenic plant according to any one of claims 1 to 11, in, The exogenous fungus-derived PPO comprises a phenylalanine at position 383 (F383), which structurally corresponds to the phenylalanine at position 331 (F331) of human PPOX, as determined by comparing a simulated 3D structure / fold of the fungus-derived PPO with a crystal structure of human PPOX bound to acifluorfen, and Therein, the simulated 3D structure / folding of the fungus-derived PPO is structurally aligned with the crystal structure of human PPO and overlaps with trifluorfen.

13. The transgenic plant according to any one of claims 11 or 12, wherein The exogenous fungus-derived PPO comprises an amino acid sequence having at least 80% similarity to the sequence shown in SEQ ID NO:

96.

14. The transgenic plant according to claim 13, wherein The exogenous fungus-derived PPO comprises an amino acid sequence having at least 90% similarity to the sequence shown in SEQ ID NO:

96.

15. The transgenic plant according to any one of claims 1 to 14, wherein The exogenous fungal-derived PPO confers resistance or tolerance to at least one PPO inhibitor herbicide.

16. The transgenic plant according to any one of claims 1 to 15, wherein The one or more PPO inhibitors are selected from the following herbicide families: phenylpyrazoles, pyrimidinediones, oxazolidinediones, diphenyl ethers, oxadiazoles, N-phenylphthalimides, triazinones, thiadiazoles, triazolinones and triazolopyridones, or any combination thereof.

17. The transgenic plant according to any one of claims 1 to 16, wherein The fungal-derived PPO belongs to a fungal species of the clade Leotiomyceta.

18. The transgenic plant according to any one of claims 1 to 17, wherein The fungal-derived PPO belongs to a fungal species selected from the group consisting of Aspergillus sp., Penicillium sp., Trichoderma sp., Rhodotorula sp. or Hesseltinella sp.

19. The transgenic plant according to any one of claims 1 to 18, wherein The exogenous fungus-derived PPO has less than 40% similarity in amino acid sequence with the endogenous plant PPO enzyme.

20. The transgenic plant according to any one of claims 1 to 19, wherein The exogenous fungus-derived PPO enzyme further comprises a plant chloroplast transit peptide.

21. The transgenic plant according to any one of claims 1 to 20, wherein The plant is selected from the group consisting of corn, wheat, rice, barley, soybean, cowpea, chickpea, cotton, sorghum, beans, rapeseed, alfalfa, flax, sunflower, safflower, millet, rye, sugarcane, sugar beet, cocoa, tea, Brassica, coffee, sweet potato, flax, peanut, clover; lettuce, tomato, gourd, cassava, potato, carrot, radish, pea, lentil, cabbage, cauliflower, broccoli, Brussels sprouts, pepper, pineapple, citrus, apple, pear, peach, apricot, walnut, avocado, banana, palm, eucalyptus, poplar, pine, coconut, orchid, morning glory, carnation, rose, switchgrass, prairie grass, Indian grass, big bluestem grass, camellia, or any combination thereof.

22. The transgenic plant according to any one of claims 21, wherein The plant is selected from the group consisting of corn, wheat, rice, soybean, cowpea, chickpea, cotton, sorghum, rapeseed, alfalfa, sunflower, sugarcane, sugar beet, camellia, or any combination thereof.

23. The transgenic plant according to any one of claims 1 to 22, wherein The one or more PPO inhibitors are selected from the following herbicide families: phenylpyrazoles, pyrimidinediones, oxazolidinediones, diphenyl ethers, oxadiazoles, N-phenylphthalimides, triazinones, thiadiazoles, triazolinones and triazolopyridones, or any combination thereof.

24. The transgenic plant according to any one of claims 1 to 23, wherein The plant is a broadleaf plant.

25. The transgenic plant according to any one of claims 1 to 24, wherein The comparison of the 3D structure / fold of the fungal derived PPO to the crystal structure of human PPO bound to acifluorfen comprises comparing a modeled structure of the fold of the fungal derived PPO or a crystal structure showing the fold of the fungal derived PPO.

26. A transgenic plant expressing a protoporphyrinogen oxidase (PPO) of exogenous fungal origin, said enzyme conferring resistance or tolerance to at least one PPO inhibitor herbicide, wherein: The fungus-derived PPO enzyme comprises an amino acid sequence having at least 80% similarity to at least one sequence shown in any one of SEQ ID NOs: 1-7 and SEQ ID NOs: 86, 88-89, 91-99 or any combination thereof.

27. The transgenic plant according to claim 26, wherein The fungus-derived PPO enzyme comprises an amino acid sequence having at least 90% similarity to at least one sequence shown in any one of SEQ ID NOs: 1 to 7 and SEQ ID NOs: 86, 88 to 89, 91 to 99 or any combination thereof.

28. The transgenic plant according to claim 26 or 27, in, The fungal PPO enzyme contains no more than The size of the pocket opening was determined by comparing the modeled 3D structure / folding of the fungal-derived PPO with the crystal structure of human PPOX bound to acifluorfen, and Therein, the simulated 3D structure / folding of the fungus-derived PPO is structurally aligned with the crystal structure of human PPO and overlaps with trifluorfen.

29. The transgenic plant according to any one of claims 26 to 28, in, The exogenous fungus-derived PPO comprises: a first arginine which structurally corresponds to the position of arginine at position 97 in human PPOX (human Arg97), and / or a second arginine, which structurally corresponds to the position of arginine at position 62 in human PPOX (human Arg62), wherein the first and / or second arginine of the fungus-derived PPO has a geometric orientation different from the geometric orientation of human Arg62 and / or human Arg97, as determined by comparing a simulated 3D structure / fold of the fungus-derived PPO with a crystal structure of human PPOX bound to acifluorfen, and Therein, the simulated 3D structure / folding of the fungus-derived PPO is structurally aligned with the crystal structure of human PPO and overlaps with trifluorfen.

30. The transgenic plant according to any one of claims 26 to 29, in, The exogenous fungus-derived PPO comprises a phenylalanine at position 404 (F404), which structurally corresponds to a valine at position 347 (V347) of human PPOX, as determined by comparing a simulated 3D structure / fold of the fungus-derived PPO with a crystal structure of human PPOX bound to acifluorfen, and Therein, the simulated 3D structure / folding of the fungus-derived PPO is structurally aligned with the crystal structure of human PPO and overlaps with trifluorfen.

31. The transgenic plant according to any one of claims 26 to 30, in, The exogenous fungus-derived PPO comprises a phenylalanine at position 383 (F383), which structurally corresponds to the phenylalanine at position 331 (F331) of human PPOX, as determined by comparing a simulated 3D structure / fold of the fungus-derived PPO with a crystal structure of human PPOX bound to acifluorfen, and Therein, the simulated 3D structure / folding of the fungus-derived PPO is structurally aligned with the crystal structure of human PPO and overlaps with trifluorfen.

32. The transgenic plant according to any one of claims 26 to 31, wherein The fungus-derived PPO comprises an amino acid sequence having less than 40% similarity to the amino acid sequence of an endogenous plant PPO enzyme.

33. The transgenic plant according to any one of claims 26 to 32, wherein The fungal-derived PPO enzyme also comprises a plant chloroplast transit peptide.

34. The transgenic plant according to any one of claims 26 to 33, wherein The fungal-derived PPO belongs to a fungal species selected from the group consisting of Aspergillus sp., Fusarium sp., Rhizopus sp., Penicillium sp., Trichoderma sp., Rhodotorula sp., Hesseltinella sp., Spizellomyces sp., Rhizophagus sp., Alternaria sp. and Cladosporium sp.

35. The transgenic plant according to claim 34, wherein The fungal-derived PPO enzyme belongs to a fungal species selected from the group consisting of Aspergillus, Fusarium, Rhizopus, Penicillium and Trichoderma.

36. The transgenic plant according to claim 34, wherein The fungal-derived PPO belongs to a fungal species selected from the group consisting of Aspergillus sp., Penicillium sp., Trichoderma sp., Rhodotorula sp. or Hesseltinella sp.

37. The transgenic plant according to any one of claims 26 to 36, wherein The fungal-derived PPO enzyme belongs to a fungal species of the clade Leotiomyceta.

38. The transgenic plant according to any one of claims 26 to 36, wherein The plant is selected from the group consisting of corn, wheat, rice, barley, soybean, cowpea, chickpea, cotton, sorghum, beans, rapeseed, alfalfa, flax, sunflower, safflower, millet, rye, sugarcane, sugar beet, cocoa, tea, Brassica, coffee, sweet potato, flax, peanut, clover; lettuce, tomato, gourd, cassava, potato, carrot, radish, pea, lentil, cabbage, cauliflower, broccoli, Brussels sprouts, pepper, pineapple, citrus, apple, pear, peach, apricot, walnut, avocado, banana, palm, eucalyptus, poplar, pine, coconut, orchid, morning glory, carnation, rose, switchgrass, prairie grass, Indian grass, big bluestem grass, camellia, or any combination thereof.

39. The transgenic plant according to any one of claims 38, wherein The plant is selected from the group consisting of corn, wheat, rice, soybean, cowpea, chickpea, cotton, sorghum, rapeseed, alfalfa, sunflower, sugarcane, sugar beet, camellia, or any combination thereof.

40. The transgenic plant according to any one of claims 26 to 39, wherein The one or more PPO inhibitors are selected from the following herbicide families: phenylpyrazoles, pyrimidinediones, oxazolidinediones, diphenyl ethers, oxadiazoles, N-phenylphthalimides, triazinones, thiadiazoles, triazolinones and triazolopyridones, or any combination thereof.

41. The transgenic plant according to any one of claims 26 to 40, wherein The plant is a broadleaf plant.

42. The transgenic plant according to any one of claims 26 to 41, wherein The plant is a cereal plant.

43. A seed of the transgenic plant according to any one of claims 1 to 42.

44. A product derived from a transgenic plant according to any one of claims 1 to 43.

45. A method for controlling weed growth in a crop growing area using at least one PPO inhibitor herbicide, wherein: The crop plant comprises a transgenic plant according to any one of claims 1 to 36, and the method comprises applying a weed controlling amount of one or more PPO inhibitor herbicides.

46. ​​The method of claim 40, wherein: The one or more PPO inhibitors are selected from the following herbicide families: phenylpyrazoles, pyrimidinediones, oxazolidinediones, diphenyl ethers, oxadiazoles, N-phenylphthalimides, triazinones, thiadiazoles, triazolinones and triazolopyridones, or any combination thereof.

47. A method for conferring resistance or tolerance to one or more PPO inhibitor herbicides in a crop plant, the method comprising genetically modifying the plant to express a PPO enzyme of exogenous fungal origin, wherein: The fungus-derived PPO enzyme comprises an amino acid sequence having at least 80% similarity to at least one sequence shown in any one of SEQ ID NOs: 1-7 or SEQ ID NOs: 86, 88-89, 91-99, or any combination thereof.

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