Plants modified in protoporphyrinogen oxidase (PPO) with adventitious traits

Abstract

Provided herein are crop plant(s) and crop plant cells genetically modified to express a mutated-plant Protoporphyrinogen Oxidase (PPO). Further provided are methods for their preparation and uses thereof for conferring herbicide resistance and for controlling weeds where the crop plant(s) grow.

Description

[0001] PLANTS MODIFIED IN PROTOPORPHYRINOGEN OXIDASE (PPO) WITH ADVENTITIOUS TRAITS

[0002] FIELD OF THE INVENTION

[0003] The present invention generally pertains to the field of genetically modified plants with beneficial Herbicide Tolerance (HT) traits. Particularly, the invention relates to protoporphyrinogen oxidase (PPO) enzyme diversification.

[0004] BACKGROUND OF THE INVENTION

[0005] Weeds disrupt the cultivation and management of desired crop plants, and generally present complex obstacles to agricultural productivity and sustainability.

[0006] PPO-inhibitors are a class of herbicides that target the enzyme protoporphyrinogen oxidase (PPO) and are useful in controlling a broad spectrum of weeds. Yet, an important role has been assigned to PPO-inhibiting herbicides as an effective tool for controlling broadleaf weeds in soybean and com farming operations, including those weeds resistant to other herbicide classes.

[0007] However, controlling weed growth where, for example, where soybean and corn crop plants are growing, is only possible if the crop plants are resistant / tolerant to the herbicide.

[0008] Indeed, it has been confirmed that the presence of a single dominant allele (or a single gene) (i.e., The PPO enzyme) in soybeans confers increased tolerance to PPO herbicides compared to varieties lacking this genetic trait. Herbicide tolerant soybean plants resistant to various other herbicide groups such as Enlist E3® soybeans, XtendFlex® soybeans, and LibertyLink® GT27 soybeans have been commercialized and are available for use by farmers offering growers options for effective weed management while maintaining crop productivity.

[0009] However, resistance to various herbicides has emerged as a significant concern in weed management, with multiple weed species developing mechanisms to withstand the effects of these herbicides, including by developing mutations in the target site of the herbicide, and non-target site mechanisms, metabolic detoxification processes such as herbicide degradation through conjugation with glutathione and / or glucose, or reduced herbicide uptake into plant cells. In this well-familiar evolutionary-like arms race, providing crop plants with novel mechanisms for resistance to PPO-based inhibition is crucial for gaining competitive advantages in agriculture.

[0010] Therefore, there is an unmet need in providing novel beneficial Herbicide Tolerance traits to crop plants, such as soybean crop plants.

[0011] SUMMARY OF THE INVENTION

[0012] According to some embodiments, there are provided herein advantageous compositions and methods for conferring herbicide tolerance traits to various crop plants, by expressing in the plants modified (mutated) Protoporphyrinogen Oxidase (PPO) protein having one or more mutations in a designated structural pocket of the PPO, in particular, at position(s) structurally corresponding to positions 123, 151, 225, 371, 409 and / or 412 of a reference PPO enzyme. In some embodiments, the reference PPO enzyme has an amino acid sequence as denoted by SEQ ID NO: 2.

[0013] According to some embodiments, further provided herein are modified PPO enzymes, nucleic acids encoding the same, compositions including the same and crop plants or crop plant cells expressing or harboring the same.

[0014] According to one aspect, there is provided a crop plant or a crop plant cell genetically modified to express a plant Protoporphyrinogen Oxidase (PPO) amino acid sequence comprising a mutation(s) / modification at a position(s) structurally corresponding to at least one amino acid position selected from positions 123, 151, 225, 371, 409, and 412 or any combination thereof of a (reference) PPO enzyme having an amino acid sequence as set forth in SEQ ID NO: 2, wherein the mutated-PPO confers resistance or tolerance to at least one PPO-inhibitor herbicide, compared to a non-mutated plant of the same genus.

[0015] Advantageously, in-planta screening of expression libraries containing large collections of randomly mutated PPO mRNA coding sequences, and expression of the herein disclosed mutated-PPO in Arabidopsis thaliana model plants resulted in plants having resistance or tolerance to PPO-inhibitor herbicides, compared to a non-mutated plant of the same genus. Unexpectedly and surprisingly, identification of the mutations uncovered the positions and the type of substitution in the PPO CDS underlying the enhanced herbicide tolerance (HT) trait and revealed that the beneficial and adventitious mutations occurred at amino acid residues that were essential for the interaction with the herbicide rO-RF-SGVYA-V-FGQL- LGTI-Y-I] by forming the enzyme’s herbicide binding pocket.

[0016] In some embodiments, the present disclosure provides novel positions identified as capable of extending plant tolerance to PPO herbicides. In some embodiments, newly discovered substitutions in some of the disclosed positions are capable of extending plant tolerance to PPO herbicides.

[0017] Further advantageously, in some embodiments, comparison of the sequence and the structure of PPO enzymes of different plant species showcased the high conservation between the various PPO enzymes, especially, between positions of structurally corresponding residues essential for the interaction with the herbicide / substrate with the enzyme’s pocket.

[0018] According to other aspects, the disclosure provides a method for controlling weeds growth where crop plants are growing, including the genetically modified plant expressing the mutated-plant Protoporphyrinogen Oxidase (PPO) enzyme, as well as a method for conferring resistance or tolerance to PPO inhibitor herbicides in a crop plant genetically modified to express the mutated-plant Protoporphyrinogen Oxidase (PPO) enzyme. Each possibility is a separate embodiment.

[0019] According to some aspects, there is provided a crop plant or a crop plant cell genetically modified to express a plant Protoporphyrinogen Oxidase (PPO) amino acid sequence comprising a mutation(s) at a position(s) structurally corresponding to at least one amino acid position selected from positions 123, 151, 225, 371, 409, and 412 or any combination thereof of a reference PPO enzyme having an amino acid sequence as set forth in SEQ ID NO: 2 wherein the mutated-PPO confers resistance or tolerance to at least one PPO-inhibitor herbicide compared to a non-mutated plant of the same genus. Each possibility is a separate embodiment.

[0020] According to some embodiments, the mutated-plant Protoporphyrinogen Oxidase (PPO) comprising the mutation(s) at a position(s) structurally corresponding to at least one amino acid position selected from positions 123, 151, 225, 371, 409, and 412 or any combination thereof of a PPO enzyme having an amino acid sequence as set forth in SEQ ID NO: 2, is derived from a WT-plant PPO characterized by a structural pocket formed by amino acid residues Q-RF-SGVYA-V-FGQL-LGTI-Y-I. Each possibility is a separate embodiment. Each possibility is a separate embodiment.

[0021] In some embodiments, the position of the amino acid residues Q-RF-SGVYA-V- FGQL-LGTI-Y-I structurally corresponds to positions 123-150: 151-222:226-371-396:399- 409:412-432-481 of a PPO enzyme having an amino acid sequence as set forth in SEQ ID NO: 2, as shown in Table 1, herein below. Each possibility is a separate embodiment.

[0022] In further related embodiments, the mutated-plant Protoporphyrinogen Oxidase (PPO) amino acid sequence comprising the mutation(s) at a position(s) structurally corresponding to at least one amino acid position selected from positions 123, 151, 225, 371, 409, and 412 or any combination thereof of a PPO enzyme having an amino acid sequence as set forth in SEQ ID NO: 2, is derived from one or more WT-plant PPO enzyme(s) having an amino acids sequence selected from any one of SEQ ID NO: 14-47 listed in Table 3 or Table 4. Each possibility is a separate embodiment.

[0023] According to some embodiments, there is provided a crop plant or a crop plant cell genetically modified to express a mutated plant Protoporphyrinogen Oxidase (PPO) peptide / protein having an amino acid sequence having one or more mutation(s) at position(s) structurally corresponding to at least one amino acid position selected from positions 123, 151, 225, 371, 409, and 412, or any combination thereof, of a PPO enzyme having an amino acid sequence as set forth in SEQ ID NO: 2, wherein the mutated-PPO confers resistance or tolerance to at least one PPO-inhibitor herbicide compared to a non- genetically modified plant of the same genus.

[0024] According to some embodiments, the mutated PPO is derived from a WT-plant PPO characterized by a structural pocket formed by amino acid residues Q-RF-SGVYA-V-FGQL- LGTI-Y-I, such as represented by SEQ ID NO: 73.

[0025] According to some embodiments, the position of the amino acid residues Q-RF- SGVYA-V-FGQL-LGTI-Y-I includes at least one position(s) structurally corresponding to any one of positions 123-150: 151-222:226-371-396:399-409:412-432-481 of a PPO enzyme having an amino acid sequence as set forth in SEQ ID NO: 2. In other embodiments, the mutated-plant Protoporphyrinogen Oxidase (PPO) includes a PPO transgenic to the crop plant and / or PPO endogenous to the crop plant. Each possibility is a separate embodiment.

[0026] In some embodiments, the mutated-plant Protoporphyrinogen Oxidase (PPO) is selected from a mutated-plant Protoporphyrinogen Oxidase 1 (PPO1) and / or a mutated- Protoporphyrinogen Oxidase 2 (PPO2). Each possibility is a separate embodiment.

[0027] In a specific embodiment, the mutated-plant Protoporphyrinogen Oxidase (PPO) comprises a mutated-Protoporphyrinogen Oxidase 1 (PPO1).

[0028] According to some embodiments, the PPO amino acid sequence comprises a mutation(s) at a position(s) structurally corresponding to at least one amino acid position selected from positions 123, 151, 225, and 371, or any combination thereof. Each possibility is a separate embodiment.

[0029] In related embodiments, the PPO amino acid sequence comprises a mutation(s) at a position(s) structurally corresponding to at least one amino acid position selected from positions 123 and 225. Each possibility is a separate embodiment.

[0030] In a specific embodiment, the PPO amino acid sequence comprises a mutation(s) at a position(s) structurally corresponding to amino acid position 225.

[0031] In some embodiments, the mutation(s) at position(s) structurally corresponding to the at least one amino acid position 123 in SEQ ID NO: 2, comprises at least one of a polar uncharged amino acid selected from: Serine (S), Threonine (T), Asparagine (N), Cysteine (C) or Selenocysteine (U), and Tyrosine (Y), or any combination thereof. Each possibility is a separate embodiment.

[0032] In a specific embodiment, the mutation(s) at position(s) structurally corresponding to the at least one amino acid position 123 in SEQ ID NO: 2 comprises Serine (S).

[0033] In some embodiments, the mutation(s) at position(s) structurally corresponding to the at least one amino acid position 151 in SEQ ID NO: 2, comprises at least one of a non-polar amino acid or hydrophobic side chain amino acid selected from: Alanine (A), Valine (V), Leucine (L), Isoleucine (I), Methionine (M), Proline (P), Tyrosine (Y) and Tryptophan (W), or any combination thereof. Each possibility is a separate embodiment. In a specific embodiment, the mutation(s) at position(s) structurally corresponding to the at least one amino acid position 151 in SEQ ID NO: 2 comprises Leucine (L).

[0034] In some embodiments, the mutation(s) at position(s) structurally corresponding to the at least one amino acid position 225 in SEQ ID NO: 2, comprises at least one of a non-polar or positively charged amino acid selected from: Glycine (G), Alanine (A), Arginine (R), and Lysine (K), or any combination thereof. Each possibility is a separate embodiment.

[0035] In related embodiments, the mutation(s) at position(s) structurally corresponding to the at least one amino acid position 225 in SEQ ID NO: 2, comprises at least one of a nonpolar or positively charged amino acid selected from: Arginine (R), Glycine (G), and Lysine (K), or any combination thereof. Each possibility is a separate embodiment.

[0036] In specific embodiments, the mutation(s) at position(s) structurally corresponding to the at least one amino acid position 225 in SEQ ID NO: 2 comprises at least one of a positively charged amino acid selected from: Arginine (R) and / or Lysine (K). Each possibility is a separate embodiment.

[0037] In other embodiments, the mutation(s) at position(s) structurally corresponding to the at least one amino acid position 371 in SEQ ID NO: 2, comprises at least one of a non-polar amino acid or hydrophobic side chain amino acid selected from: Glycine (G) and / or Alanine (A). Each possibility is a separate embodiment.

[0038] In a specific embodiment, the mutation(s) at position(s) structurally corresponding to the at least one amino acid position 371 in SEQ ID NO: 2 comprises Alanine (A).

[0039] In further embodiments, the mutation(s) at position(s) structurally corresponding to the at least one amino acid position selected from 409 and 412 in SEQ ID NO: 2, comprises at least one of a polar uncharged amino acid selected from: Serine (S), Threonine (T), Asparagine (N), Glutamine (Q), Cysteine (C) or Selenocysteine (U), and Tyrosine (Y), or any combination thereof. Each possibility is a separate embodiment.

[0040] In other embodiments, the mutation(s) at position(s) structurally corresponding to the at least one amino acid position selected from 409 and 412 in SEQ ID NO: 2 comprises at least one of a polar uncharged amino acid selected from: Threonine (T) and / or Asparagine (N). Each possibility is a separate embodiment. In other embodiments, the mutation(s) at position(s) structurally corresponding to the at least one amino acid position 409 in SEQ ID NO: 2 comprises Asparagine (N).

[0041] In a specific embodiment, the mutation(s) at position(s) structurally corresponding to the at least one amino acid position 412 in SEQ ID NO: 2 comprises Threonine (T).

[0042] According to another embodiment, the mutated-plant Protoporphyrinogen Oxidase (PPO) amino acid sequence further comprises at least one additional mutation(s) at a position(s) structurally corresponding to any amino acid position(s) in SEQ ID NO: 2.

[0043] According to additional embodiments, the mutated-plant Protoporphyrinogen Oxidase (PPO) amino acid sequence comprises at least one double mutation at a combination of positions structurally corresponding to at least two amino acid positions in SEQ ID NO: 2 selected from positions: (1) 123 S and 15 IL; (2) 123 S and 225R; (3) 123 S and 225G; (4) 123 S and 225K; (5) 123S and 371A; (6) 123S and 409N; (7) 123S and 412T; (8) 151L and 225R; (9) 151L and 225G (10) 151L and 225K; (11) 151L and 371A; (12) 151L and 409N; (13) 151L and 412T; (14) (14) 225R and 371A; (15) 225R and 409N; (16) 225R and 412T; (17); (17) 225G and 371A; (18) 225G and 409N; (19) 225G and 412T; (20) 225K and 371A; (21) 225K and 409N; (22) 225K and 412T; (23) 371 A and 409N; (24) 371 A and 412T; (25) 409N and 412T according to combinations 1-25 as shown in Table 2, or any combination thereof. Each possibility is a separate embodiment.

[0044] According to specific embodiments, the mutated-plant Protoporphyrinogen Oxidase (PPO) comprises at least one double mutation at a combination of positions structurally corresponding to at least two amino acid positions in SEQ ID NO: 2 selected from combinations 1-24 in Table 2, or any combination thereof. Each possibility is a separate embodiment.

[0045] According to further specific embodiments, the mutated-plant Protoporphyrinogen Oxidase (PPO) comprises at least one double mutation at a combination of positions structurally corresponding to at least two amino acid positions in SEQ ID NO: 2 selected from combinations 2-4, 8-10, and 14-22, in Table 2, or any combination thereof, and wherein the at least one double mutation comprises a position that corresponds to position 225 in SEQ ID NO: 2. Each possibility is a separate embodiment. In one embodiment, the mutated-plant Protoporphyrinogen Oxidase (PPO) comprises amino acid sequence having at least 98% sequence identity to the endogenous PPO enzyme of the crop plant.

[0046] In a related embodiment, the mutated-plant PPO comprises amino acid sequence having at least 99% identity to the endogenous PPO enzyme of the crop plant.

[0047] In a further related embodiment, the mutated-plant Protoporphyrinogen Oxidase (PPO) comprises PPO endogenous to the crop plant.

[0048] According to some embodiments, the crop plant comprises at least one crop selected from the group consisting of maize, wheat, rice, barley, soybean, cowpea, chickpea, cotton, sorghum, beans, rapeseed / canola, alfalfa, flax, sunflower, safflower, millet, rye, sugarcane, sugar beet, cocoa, tea, Brassica napus, Brassica rapa, Brassica oleracea, coffee, sweet potato, flax, peanut, clover, lettuce, tomato, cucurbits, cassava, potato, carrot, radish, pea, lentils, cabbage, cauliflower, broccoli, brussels sprouts, peppers, pineapple, citrus, apples, pears, peaches, apricots, walnuts, avocado, banana, palm, eucalyptus, poplar, pine, coconut, orchids, petunia, carnations, roses, Poa species (bluegrasses), Festuca species (fescues), Lolium species (rye grass), Phalaris species (canary grasses), switchgrass, prairie grasses, Indian grasses, big bluestem grass, and camelina, or any combination thereof. Each possibility is a separate embodiment.

[0049] In more specific embodiments, the crop plant is selected from the group consisting of maize, wheat, rice, soybean, cowpea, chickpea, cotton, sorghum, rapeseed / canola, alfalfa, sunflower, sugarcane, sugar beet, camelina, Poa species (bluegrasses), Festuca species (fescues), Lolium species (rye grass), Phalaris species (canary grasses) or any combination thereof. Each possibility is a separate embodiment.

[0050] In some embodiments, the crop plant is a soybean plant.

[0051] According to some embodiments, the mutated-plant Protoporphyrinogen Oxidase (PPO) comprises at least one mutated-PPO enzyme selected from the group consisting of maize PPO, wheat PPO, rice PPO, barley PPO, soybean PPO, cowpea PPO, chickpea PPO, cotton PPO, sorghum PPO, beans PPO, rapeseed / canola PPO, alfalfa PPO, flax PPO, sunflower PPO, safflower PPO, millet PPO, rye PPO, sugarcane PPO, sugar beet PPO, cocoa PPO, tea PPO, Brassica napus PPO, Brassica rapa PPO, Brassica oleracea PPO, coffee PPO, sweet potato PPO, flax PPO, peanut PPO, clover PPO, lettuce PPO, tomato PPO, cucurbits PPO, cassava PPO, potato PPO, carrot PPO, radish PPO, pea PPO, lentils PPO, cabbage PPO, cauliflower PPO, broccoli PPO, brussels sprouts PPO, peppers PPO, pineapple PPO, citrus PPO, apples PPO, pears PPO, peaches PPO, apricots PPO, walnuts PPO, avocado PPO, banana PPO, palm PPO, eucalyptus PPO, poplar PPO, pine PPO, coconut PPO, orchids PPO, petunia PPO, carnations PPO, roses PPO, Poa species (bluegrasses) PPO, Festuca species (fescues) PPO, Lolium species (rye grass) PPO, Phalaris species (canary grasses) PPO, switchgrass PPO, prairie grasses PPO, Indian grasses PPO, big bluestem grass PPO, and camelina PPO, or any combination thereof. Each possibility is a separate embodiment.

[0052] In one embodiment, the mutated-plant Protoporphyrinogen Oxidase (PPO) is a mutated-soybean PPO.

[0053] In some embodiments, the mutated-soybean PPO comprises at least one mutation selected from Q123S, F151L, Y225R, Y225G, Y225K, V371 A, L409N, and I412T as set forth in any one of SEQ ID NOs: 3-10, or any combination thereof. Each possibility is a separate embodiment.

[0054] In some embodiments, the mutated-soybean PPO comprises at least one mutation selected from Q123S, F151L, Y225R, Y225G, Y225K, and V371A as set forth in any one of SEQ ID NOs: 3-8, or any combination thereof. Each possibility is a separate embodiment.

[0055] In some specific embodiments, the mutated-soybean PPO comprises at least one mutation selected from Y225R, Y225G, and Y225K as set forth any one of SEQ ID NOs: 5- 7, or any combination thereof. Each possibility is a separate embodiment.

[0056] According to some embodiments, the mutated-soybean PPO comprises at least one double mutation at a combination of positions structurally corresponding to at least two amino acid positions in SEQ ID NO: 2 selected from any of combinations 1-25 (SEQ ID NOs 48-72, respectively), in Table 2, or any combination thereof.

[0057] According to specific embodiments, the mutated-soybean Protoporphyrinogen Oxidase (PPO) comprises at least one double mutation at a combination of positions structurally corresponding to at least two amino acid positions in SEQ ID NO: 2 selected from any of combinations 2-4 (SEQ ID NOs: 49-51, respectively), 8-10 (SEQ ID NOs: 55-57, respectively), and 14-22 (SEQ ID NOs.: 61-69, respectively), in Table 2, or any combination thereof, and wherein the at least one double mutation comprises a position that corresponds to position 225 in SEQ ID NO: 2. Each possibility is a separate embodiment.

[0058] In some embodiments, the mutated-soybean PPO comprises a soybean PPO1.

[0059] The crop plant or a crop plant cell of any one of claims 1-41, wherein the one or more PPO inhibitors is selected from the group consisting of herbicide families Phenylpyrazoles, Pyrimidinediones, Oxazolidinedione, Diphenylethers, Oxadiazoles, N-phenylphthalimides, Triazinone, Thiadiazoles, Triazolinones, and Triazolopyridinones, or any combination thereof. Each possibility is a separate embodiment.

[0060] In some specific embodiments, the one or more PPO inhibitors is selected from the group consisting of herbicide families Diphenylethers, Oxadiazoles, and Triazolinones. Each possibility is a separate embodiment.

[0061] In one embodiment, the plant is a broadleaf plant.

[0062] In other embodiments, the plant is a cereal and / or grass type plant. Each possibility is a separate embodiment.

[0063] According to an aspect, there is provided a seed of the genetically modified plant of any one of the preceding embodiments.

[0064] According to an aspect, disclosed is a product derived from the genetically modified crop plant or a crop plant cell of any one of the preceding embodiments.

[0065] According to another aspect, there is provided a method of controlling weeds growth where crop plants are growing, using at least one PPO inhibitor herbicide, wherein the crop plants comprise the genetically modified plant according to any one of the preceding embodiments, the method comprises applying a weed-controlling amount of the one or more PPO inhibitor herbicide. Each possibility is a separate embodiment.

[0066] In some embodiments, the one or more PPO-inhibitor herbicides are selected from the group consisting of herbicides families of the groups Phenylpyrazoles, Pyrimidinediones, Oxazolidinedione, Diphenylethers, Oxadiazoles, N-phenylphthalimides, Triazinone, Thiadiazoles, Triazolinones, and Triazolopyridinones, or any combination thereof. Each possibility is a separate embodiment. According to other aspects, disclosed is a method for conferring resistance or tolerance to one or more PPO inhibitor herbicide in a crop plant, the method comprising genetically modifying the plant to express a mutated plant Protoporphyrinogen Oxidase (PPO) enzyme, wherein the plant PPO enzyme have an amino acid sequence comprising mutation(s) at position(s) structurally corresponding to at least one amino acid position selected from positions 123, 151, 225, 371, 409, and 412 or any combination thereof, of a PPO enzyme having an amino acid sequence as set forth in SEQ ID NO: 2. Each possibility is a separate embodiment.

[0067] In some embodiments, the mutated-plant Protoporphyrinogen Oxidase (PPO) comprises PPO transgenic to the crop plant and / or PPO endogenous to the crop plant. Each possibility is a separate embodiment.

[0068] In some embodiments, the mutated-plant Protoporphyrinogen Oxidase (PPO) amino acid sequence comprises a mutation(s) at a position(s) structurally corresponding to at least one amino acid position selected from positions 123, 151, 225 and 371, or any combination thereof. Each possibility is a separate embodiment.

[0069] In another embodiment, the mutated-plant Protoporphyrinogen Oxidase (PPO) amino acid sequence further comprises at least one additional mutation(s) at a position(s) structurally corresponding to any position(s) in SEQ ID NO: 2.

[0070] In some embodiments, the mutated-plant Protoporphyrinogen Oxidase (PPO) comprises at least one double mutation at a combination of positions structurally corresponding to at least two amino acid positions in SEQ ID NO: 2 selected from any of combinations 1-25 in Table 2, or any combination thereof. Each possibility is a separate embodiment.

[0071] In some embodiments, the mutated-plant Protoporphyrinogen Oxidase (PPO) comprises amino acid sequence having at least 98% identity to the endogenous PPO enzyme of the crop plant.

[0072] In one embodiment, the mutated-plant Protoporphyrinogen Oxidase (PPO) comprises a mutated-soybean PPO. According to other aspects, there is provided a crop plant or a crop plant cell genetically modified to express a soybean Protoporphyrinogen Oxidase (PPO) comprising mutation(s) in at least one position(s) selected from Q123, F 151 , Y225, V371, L409, and 1412, or any combination thereof, wherein the mutated-PPO confers resistance or tolerance to at least one PPO-inhibitor herbicide. Each possibility is a separate embodiment.

[0073] According to some embodiments, the mutated-soybean Protoporphyrinogen Oxidase (PPO) comprises at least one mutation selected from Q123S, F151L, Y225R, Y225G, Y225K, V371A, L409N, and I412T, as set forth in any of the mutated-soybean PPO1 amino acid sequences in any one of SEQ ID NOs: 3-10, or any combination thereof, and wherein the mutated-PPO confers resistance or tolerance to at least one PPO-inhibitor herbicide. Each possibility is a separate embodiment.

[0074] According to some embodiments, the mutated-soybean Protoporphyrinogen Oxidase (PPO) comprises at least one mutation selected from Q123S, F151L, Y225R, Y225G, Y225K, and V371A as set forth in the mutated-soybean PPO1 amino acid sequences in any one of SEQ ID NOs: 3-8, or any combination thereof. Each possibility is a separate embodiment.

[0075] In some specific embodiments, the mutated-soybean Protoporphyrinogen Oxidase (PPO) comprises at least one mutation selected from Y225R, Y225G, and Y225K, as set forth in the mutated-soybean PPO amino acid sequences set forth in any one of SEQ ID NOs: 5-7, or any combination thereof. Each possibility is a separate embodiment.

[0076] In some specific embodiments, the mutated-soybean Protoporphyrinogen Oxidase (PPO) comprises at least one mutation selected from Q123S, Y225R, Y225G, and Y225K, as set forth in the mutated-soybean PPO amino acid sequences set forth in any one of SEQ ID NOs: 5-7, or any combination thereof. Each possibility is a separate embodiment.

[0077] In one embodiment, the mutated-soybean Protoporphyrinogen Oxidase (PPO) comprises a mutated-soybean Protoporphyrinogen Oxidase 1 (PPO1).

[0078] Certain embodiments of the present disclosure may include some, all, or none of the above advantages. One or more technical advantages may be readily apparent to those skilled in the art from the figures, descriptions and claims included herein. Moreover, while specific advantages have been enumerated above, various embodiments may include all, some, or none of the enumerated advantages. BRIEF DESCRIPTION OF THE FIGURES

[0079] The invention will now be described in relation to certain examples and embodiments with reference to the following illustrative figures.

[0080] FIG. 1A illustrates the different components of the binary vector backbone (pPA35H) that is used for cloning the native coding sequence of the soybean (Glycine max) Protoporphyrinogen oxidase gene (GmPPOl) downstream to a constitutive CaMV-35S promoter (35S); thereby creating a chimeric 35S-GmPP01 sequence. RB and LB are right and left border recombination sites, respectively. BlpR is a bialaphos resistance cassette that confers resistance to the herbicide bialaphos or Basta (Bayer CropScience) used as a positive selection marker for plant transformation. Ter is the terminator site. The binary pPA35H vector containing the chimeric 35S~GmPP01 sequence is referred to as pPA35S-WT-GmPP01.

[0081] FIG. IB illustrates DNA libraries of mutated GmPPOl CDS. A large collection of approximately 105clones of mutated-GmPPOl CDS were generated using random mutagenesis, cloned into binary vector (pPA35H) downstream to the 35S promoter thereby creating chimeric pool of 35S-mutated GmPPOl sequences which are introduced into Agrobacterium using a high-throughput transformation (floral dip). The Agrobacterium library was then transformed into Arabidopsis thaliana plants for functional screening inplanta. This process resulted in the generation of plants where, on average, each plant harbors a single copy of genetically modified DNA element with one or more alterations in the GmPPOl mRNACDS. The binary pPA35H vector containing the mutated GmPPOl sequence is referred to as pPA35S-mutated-GmPP01.

[0082] FIG. 2A display s representative pictures from an in-planta functional screening assay for PPO inhibitor tolerant plants. T1 transformed plants were initially selected on Basta to identify transgenic plants, followed by functional selection that identified plants with mutations in the PPO1 enzyme that conferred high levels of tolerance to the PPO inhibitors 200ng / ml Carfentrazon-ethyl (Aurora, FMC), 1250ng / ml Oxadiazon (Star, Tapazol), or 120ng / ml Oxyfluorfen (Strike, Adama) (FIG. 2A). Shown are transgenic plants expressing WT- GmPPOl enzyme (SEQ ID NO: 2) compared with high tolerant plants carrying a Y225R mutation, and high tolerant plants carrying a Fl 5 IL mutation.

[0083] FIG. 2B summarizes the relative tolerance exhibited by Arabidopsis plants expressing mutated-soybean PPO enzyme CDS (mutated-GmPPOl) to several PPO-inhibiting herbicides, compared with plants expressing WT-GmPPOl enzyme (SEQ ID NO: 2). The PPO-inhibiting herbicides used were Carfentrazone-ethyl (at a concentration of 200ng / ml; Oxadiazon 1250 ng / ml; and Oxyfluorfen 120 ng / ml). Highly tolerant plants are designed by (+) and susceptible plants by (-). Additionally, the table indicates (v) which of the functionally selected adventitious mutations are characterized by novel unique position (i.e., Q123S, F151L, Y225R, Y225G, Y225K, V371A) and two others by novel substitution at positions previously identified to confer resistance (i.e., L409N and I412T).

[0084] FIGs. 3A-3D: Presents sequence alignment of PPO1 proteins from different plant species. The amino acid sequences of 35 different PPO1 proteins were analyzed using Clustal Omega with default settings to determine their level of conservation and variation. The results were displayed in Jalview using Blosum62 coloring. In this visualization, blue represents conserved residues, light blue indicates not conserved residues but with a positive Blosum matrix score, and white signifies residues with a negative Blosum matrix score. The residues of the herbicide binding pocket are marked by black arrows. Novel positions identified to extend plant tolerance to PPO herbicides are marked as underlined bold (green), while newly discovered residues / mutations in previously published positions are marked as underlined italics (highlighted in red). Amino acids forming the pocket: Q-RF-SGVYA-V-FGQL-LGTI- Y-I are marked with rectangles, showing the high level of conservation (100% sequence identity) of the pocket formed by these amino acids which was common to all 34 different plant PPO CDSs. Abbreviation in the left hand side of the figure corresponds with the Abbreviation in Table 3.

[0085] DETAILED DESCRIPTION

[0086] In the following description, various aspects of the disclosure will be described. For the purpose of explanation, specific configurations and details are set forth in order to provide a thorough understanding of the different aspects of the disclosure. However, it will also be apparent to one skilled in the art that the disclosure may be practiced without specific details being presented herein. Furthermore, well-known features may be omitted or simplified in order not to obscure the disclosure.

[0087] To facilitate an understanding of the present invention, a number of terms and phrases are defined below. It is to be understood that these terms and phrases are for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art.

[0088] Definitions

[0089] As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise, “a” and “an” are used herein to refer to one or more than one (i.e., to at least one) of the stated object, unless the context clearly dictates otherwise. By way of example, “a crop plant” means one or more crop plants.

[0090] The term “may” refer to an optional or possible approach / possibility, but not a requirement. The term “can” refer to a permissible or plausible approach / possibility, but not a requirement.

[0091] As used herein, "optional" or "optionally" means that the subsequently described event or circumstance does or does not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0092] As used herein, the term "about" when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass deviations / variations of ±20% or in some embodiments ±10%, or in some embodiments ±5%, or in some embodiments ±1%, or in some embodiments ±0.1% from the specified value, as such deviations are appropriate to perform the disclosed methods.

[0093] As used herein, the term “comprising” is synonymous with the terms "including," "containing," or "characterized by," and is inclusive or open-ended i.e. does not exclude additional, unrecited elements. According to some embodiments, the term comprising may be replaced with the term with the term “consisting of’ which excludes any element, step, or ingredient not specified in the claim. According to some embodiments, the term comprising may be replaced with the term “consisting essentially of’ which limits the scope of a claim to the specified materials or steps "and those that do not materially affect the basic and novel characteristics" of the claimed invention.

[0094] As used herein, the terms “prevent”, “reduce”, “attenuate”, “ameliorate”, “alleviate”, and “inhibit” are used interchangeably. As used herein, the terms “enhanced”, “increased”, “elevated”, “improved” are used interchangeably.

[0095] The disclosed invention addresses the concern of Herbicide Tolerance (HT) crops by evaluating a plant expression library that included tens of thousands of altered mRNA sequences that were generated using random mutagenesis and code for mutated-plant Protoporphyrinogen Oxidase (PPO) coding sequence (CDS), namely of a soybean PPO enzyme, and which were functionally screened and selected for their ability to confer improved Herbicide Tolerance (HT) trait, in some embodiments.

[0096] Reference is made to Examples 1-2, FIGs 1A-1B demonstrating / illustrating the construction of the plant expression library of mutated-PPO CDSs.

[0097] Expression of the mutated-PPO in Arabidopsis thaliana model plants resulted with some plants having resistance or tolerance to PPO-inhibitor herbicides, compared to a nonmutated plant of the same genus, in some embodiments.

[0098] Identification of the mutations uncovered the positions and the type of substitution in the PPO CDS underlying the enhanced HT trait, and revealed that the beneficial and adventitious mutations were of amino acid residues that interacted with the herbicide / substrate F Q-RF - SGVYA- V-F GQL-LGTI- Y-I1 and are located within the enzyme’s pocket, in some embodiments.

[0099] Reference is made to Example 3-5, FIGs. 2A-2B and Tables 1-2, demonstrating the resistance / Herbicide Tolerance (HT) and the identification of the underlying genetic changes in the PPO1 CDS elements.

[0100] Comparison of the sequence and the structure of PPO enzymes of different plant species showcased the high conservation between the various PPO enzymes, especially, between positions of structurally corresponding residues essential for herbicide interaction / binding in the enzyme’s pocket, in some embodiments.

[0101] Reference is made to Example 5-6, FIGs. 3A-3F and Table 3, demonstrating sequence and structure analyses of PPOs of different organism and species, indicative of the wide applicability of the invention. The invention, therefore, discloses a crop plant, or a crop plant cell, genetically modified to express a plant Protoporphyrinogen Oxidase (PPO) amino acid sequence that includes the mutation(s) at a position(s) structurally corresponding to certain disclosed amino acid position(s) of the soybean (Glycine max) PPO amino acid sequence, in some embodiments.

[0102] The invention further discloses a method for controlling weeds growth where crop plants are growing including the genetically modified plant, as well as a method for conferring resistance or tolerance to PPO inhibitor herbicides in a crop plant genetically modified to express the mutated-plant Protoporphyrinogen Oxidase (PPO) enzyme, in some embodiments.

[0103] According to one aspect, there is provided a crop plant or a crop plant cell genetically modified to express a plant Protoporphyrinogen Oxidase (PPO) amino acid sequence comprising a mutation(s) at a position(s) structurally corresponding to at least one amino acid position selected from positions 123, 151, 225, 371, 409, and 412 or any combination thereof of a PPO enzyme having an amino acid sequence as set forth in SEQ ID NO: 2 wherein the mutated-PPO confers resistance or tolerance to at least one PPO-inhibitor herbicide compared to a non-mutated plant of the same genus. Each possibility is a separate embodiment.

[0104] In specific embodiments, the PPO amino acid sequence includes a mutation(s) at a position(s) structurally corresponding to at least one amino acid position selected from positions 123, 151, 225, and 371, or any combination thereof. Each possibility is a separate embodiment.

[0105] In additional specific embodiments, the PPO amino acid sequence includes a mutation(s) at a position(s) structurally corresponding to at least one amino acid position selected from positions 123 and / or 225. Each possibility is a separate embodiment.

[0106] In a further specific embodiment, the PPO amino acid sequence comprises a mutation(s) at a position(s) structurally corresponding to amino acid position 225.

[0107] In some embodiments, the mutated-plant Protoporphyrinogen Oxidase (PPO) is selected from a mutated-plant Protoporphyrinogen Oxidase 1 (PPO1) and / or a mutated- Protoporphyrinogen Oxidase 2 (PPO2). Each possibility is a separate embodiment. In some specific embodiments, the mutated-plant Protoporphyrinogen Oxidase (PPO) comprises a mutated-Protoporphyrinogen Oxidase 1 (PPO1). In some specific embodiments, the mutated-plant Protoporphyrinogen Oxidase (PPO) comprises a mutated- Protoporphyrinogen Oxidase 2 (PPO2).

[0108] As used herein, the terms “PPO-inhibitor”, “PPO-type inhibitor herbicide” or “PPO-herbicide” may be interchangeably used to refer to a class of herbicides / substances that target the enzyme Protoporphyrinogen Oxidase (PPO) which is active in chloroplast and mitochondria and participates in the catalysis of the organic compound Protoporphyrin IX - a precursor of heme and chlorophyll.

[0109] PPO-inhibitors thus inhibit plant growth. Inhibition of PPO oxidation enzymatic activity not only attenuates photosynthesis due to blockage of the production of heme and chlorophyll but also causes the protoporphyrinogen (protogen) precursor to leak out into the cytoplasm, where it is converted to the highly photodynamic protoporphyrin IX (proto). In the presence of light, proto generates a burst of reactive oxygen species (ROS) including lipid peroxides that destroys lipids and protein membranes, causing cells and organelles to become leaky and disintegrate rapidly. For this reason, PPO inhibitors are also called cell membrane disruptors.

[0110] Non-limiting examples of PPO-type inhibitor herbicides include the following herbicide families, the Phenylpyrazoles, Pyrimidinediones, Oxazolidinedione, Diphenylethers, Oxadiazoles, N-phenylphthalimides, Triazinone, Thiadiazoles, Triazolinones, and Triazolopyridinones, or any combination thereof. Each possibility is a separate embodiment.

[0111] The commercial names of some PPO-inhibitors are, for example, Star, Goal, Strike and Aurora. Such herbicides include the active compounds flumioxazin, fomesafen, fluthiacet, carfentrazone-ethyl, sulfentrazone, acifluorfen, lactofen, flumiclorac, saflufenacil, oxadiazon, and oxyfluorfen, or others.

[0112] As used herein, the term “Herbicide Tolerance” (HT) refers to a genetic trait, that when manifested in a plant provides an enhanced / improved tolerance or resistance against PPO-inhibitors. The enhancement / improvement is of the genetically modified plant that expresses the mutated-PPO conferring HT trait in comparison to a non-mutated plant of the same genus that is completely unmodified (WT) or to a non-mutated plant of the same genus that carries a transgene that expresses a WT PPO. In one example, but not limited to, the trait may be conferred by the herein disclosed mutated-PPO amino acid sequences denoted by any one of SEQ ID NOs: 3-10,

[0113] As used herein, in some embodiments, a non-mutated plant includes an unmodified (WT) plant and / or a genetically modified plant that carries a transgene that expresses a WT- PPO as control, such as, for example, but not limited to, a WT-soybean PPO as the one denoted by SEQ ID NO: 2. Each possibility is a separate embodiment.

[0114] Herbicide tolerant plants grow and develop without or with minor herbicidal effect in the presence of the herbicide, as can be seen in representative FIG. 2A.

[0115] The “tolerance” can be characterized by a dose / response curve which is shifted to the right with respect to the same dose / response exhibited by non-tolerant plants (i.e., a tolerant plant will require using higher concentrations of herbicide to show the same herbicidal effect exhibited by the non-tolerant plant). Tolerant plants will typically require at least twice as much herbicide as non-tolerant plants in order to produce a given herbicidal effect. Plants that are substantially “resistant” to the herbicide exhibit few, if any of the symptoms that can occur within 1 to 3 days after exposure and include browning (necrosis) of the tissue, lytic, chlorotic or other lesions or, at least, none that impact significantly on the growth and development of the plant, when subjected to the herbicide at concentrations and rates which are typically employed by the agricultural community to inhibit or kill weeds in the field.

[0116] The terms “tolerance” and “resistance” may be interchangeably used.

[0117] According to some embodiments, the mutated-plant Protoporphyrinogen Oxidase (PPO) comprises PPO transgenic to the crop plant and / or PPO endogenous to the crop plant. Each possibility is a separate embodiment.

[0118] The term “endogenous to the crop plant” used herein, refers to “gene-edited” plants, or cells thereof, wherein the native PPO gene was “edited” using gene editing tools, including any method known in the art commonly used to direct and introduce a specific change in a specific locus to nucleic acid sequence, for example, using engineered nucleases such as meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALEN), or clustered regularly interspaced short palindromic repeats (CRISPR). Each possibility is a separate embodiment. The term “transgenic” used herein, refers to transgenic plants or cells thereof transformed with a nucleic acid of foreign / exog enous origin that has been introduced into their genome by transformation with Agrobacterium, biolistics, protoplasts, viral expression (transient) etc. as known in the art, in order to achieve transient or stable expression of the transgene, that according to some embodiments may include the PPO gene.

[0119] The term “genetically modified” used herein, may refer to a “gene-edited” plant or “transgenic” plant.

[0120] As used herein, the term “plant” refers to the whole plant, any parts thereof, or cultures thereof, such as a plant seed, a plant cell, a plant tissue, and a plant organ, at any stage of development. Examples of parts of a plant include but are not limited to meristems, roots, stems, leaves, flowers, fruits, and seeds, at any stage of development, in-planta, in-vitro, or in culture.

[0121] As used herein, the term “model plant” may refer to any plant that typically is from an extensively studied plant species chosen for the ease of investigating herbicide tolerance. The model plants provide biological insights relevant to other plants such as crop plants. In some embodiments, the model plant includes Arabidopsis thaliana.

[0122] In some embodiments, the plant includes a crop plant.

[0123] As used herein, the term “crop” refers to any plant that can be grown and harvested extensively for profit or subsistence. The disclosed invention can be applied to any plant or crop plant species, including, but not limited to, monocots and dicots.

[0124] Examples of crop plant species include, but are not limited to: maize, wheat, rice, barley, soybean, cowpea, chickpea, cotton, sorghum, beans, rapeseed / canola, alfalfa, flax, sunflower, safflower, millet, rye, sugarcane, sugar beet, cocoa, tea, Brassica napus, Brassica rapa, Brassica oleracea, coffee, sweet potato, flax, peanut, clover, lettuce, tomato, cucurbits, cassava, potato, carrot, radish, pea, lentils, cabbage, cauliflower, broccoli, brussels sprouts, peppers, pineapple, citrus, apples, pears, peaches, apricots, walnuts, avocado, banana, palm, eucalyptus, poplar, pine, coconut, orchids, petunia, carnations, roses, Poa species (bluegrasses), Festuca species (fescues), Lolium species (rye grass), Phalaris species (canary grasses), switchgrass, prairie grasses, Indian grasses, big bluestem grass, and camelina, or any combination thereof. Each possibility is a separate embodiment. As used herein the term “structurally corresponding” refers to an amino acid position(s) in a PPO enzyme coding sequence (CDS), that corresponds to a position of an amino acid in a reference PPO CDS, as determined by structural alignment / simulation of the 3D structure / folding. In addition, by performing structural alignment the level of structural homology / similarity between the PPO enzyme and the reference protein may be determined.

[0125] In the present invention, the structural homology / similarity between PPO1 proteins from different organisms was measured. Specifically, the level of structural homology between two fungal PPO1 proteins (SEQ ID NOs: 11-12), a human PPO1 protein (SEQ ID NO: 13) and 34 different plant PPO CDSs denoted by any of SEQ ID NOs: 14-47, and the reference CDS of WT soybean PPO1 denoted by SEQ ID NO: 2 were compared and determined.

[0126] A well-known measure of “Structural homology” or “Structural similarity” between two amino acid sequences is the RMSD (A).

[0127] The analysis of the structural simulation resulted with a characteristic (RMSD (A)) value for each comparison of the PPO1 proteins from the different organisms and plants and soybean PPO1. The level of sequence identity (%) was also determined.

[0128] Reference is now made to Example 5, FIGs 3A-3D and Table 3 which exemplifies the structural alignment and presents the level of structural homology (RMSD (A)) and sequence homology (identity %) between the PPO1 proteins from the different organisms and plants and soybean PPO1. Moreover, the analysis showcased which amino acid positions in any one of the CDS denoted by SEQ ID NOs: 14-47 structurally corresponds to which amino acid position in WT soybean (Glycine max) PPO1 CDS denoted by SEQ ID NO: 2.

[0129] The structural alignment revealed the high level of conservation (100% sequence identity) of the pocket formed by amino acids Q-RF-SGVYA-V-FGQL-LGTI-Y-I which was common to all 34 different plant PPO CDSs denoted by any of SEQ ID NOs: 14-47, and the reference CDS of WT soybean PPO1 denoted by SEQ ID NO: 2 (FIGs. 3A-3D; positions marked with rectangles). As used therein, the sign when introduced between two designated amino acids (for example, “Q-R”) refers to an intervening stretch / sequence of amino acids, and may include any number of intervening amino acids, present between the designated amino acids. The number and / or identity of intervening amino acids may be similar or different between different species. In some embodiments, the plant Protoporphyrinogen Oxidase (PPO) amino acid sequence comprising the mutation(s) at a position(s) structurally corresponding to at least one amino acid position selected from positions 123, 151, 225, 371, 409, and 412 or any combination thereof of a PPO enzyme having an amino acid sequence as set forth in SEQ ID NO: 2, is characterized by a structural pocket formed by an amino acid Q-RF-SGVYA-V- FGQL-LGTI-Y-I.

[0130] In some embodiments, the amino acid Q-RF-SGVYA-V-FGQL-LGTI-Y-I includes positions structurally corresponding to positions 123-150: 151-222:226-371-396:399- 409:412-432-481 of a PPO enzyme having an amino acid sequence as set forth in SEQ ID NO: 2, according to Table 1.

[0131] In some embodiments, the amino acid pocket stretch may include the following number of intervening amino acids, between the designated amino acid: For example, the number of intervening amino acids between Q and RF may be 26. For example, the number of intervening amino acids between F (of RF) and S (of SGVYA) may be 70. For example, the number of intervening amino acids between A (of SGVYA) and V may be 144. For example, the number of intervening amino acids between V and F (of FGQL) may be 24. For example, the number of intervening amino acids between F (of FGQL) and L (of LGTI) may be 9. For example, the number of intervening amino acids between I (of LGTI) and Y may be 19. For example, the number of intervening amino acids between Y and I may be 48.

[0132] In some embodiments, the amino acid pocket stretch may be represented by the sequence: QX26RFX70SGVYAX144VX24FGQLX9LGTIX19YX48I (SEQ ID NO: 73), wherein X is any amino acid, and n is the number of the X amino acids.

[0133] In some embodiments, the plant Protoporphyrinogen Oxidase (PPO) amino acid sequence comprising the mutation(s) at a position(s) structurally corresponding to at least one amino acid position selected from positions 151, 225, 371, 409, and 412 or any combination thereof of a PPO enzyme having an amino acid sequence as set forth in SEQ ID NO: 2, is characterized by a structural pocket formed by an amino acid RF - SGVYA- V-FGQL-LGTI-Y- I. In some embodiments, such a structural pocket may be represented by the sequence: RFX70SGVYAX144VX24FGQLX9LGTIX19YX48I (SEQ ID NO: 74), wherein X is any amino acid, and n is the number of the X amino acids. In some embodiments, the plant Protoporphyrinogen Oxidase (PPO) amino acid sequence includes mutation(s) at a position(s) structurally corresponding to at least one amino acid position selected from positions 123, 151, 225, 371, or any combination thereof of a PPO enzyme having an amino acid sequence as set forth in SEQ ID NO: 2, is characterized by a structural pocket formed by an amino acid Q-RF-SGVYA-V-FGQL. In some embodiments, such a structural pocket may be represented by the sequence: QX26RFX70SGVYAX144VX24FGQL (SEQ ID NO: 75), wherein X is any amino acid, and n is the number of the X amino acids.

[0134] In some embodiments, the plant Protoporphyrinogen Oxidase (PPO) amino acid sequence includes mutation(s) at a position(s) structurally corresponding to at least one amino acid position selected from positions 151, 225, 371, or any combination thereof of a PPO enzyme having an amino acid sequence as set forth in SEQ ID NO: 2, is characterized by a structural pocket formed by an amino acid RF-SGVYA-V-FGQL. In some embodiments, such a structural pocket may be represented by the sequence: RFX70SGVYAX144VX24FGQL (SEQ ID NO: 76).

[0135] In some embodiments, the amino acid pocket stretch may be represented by the sequence: QX24-28RFX68.72SGVYAX142-146VX22-2622-26FGQLX7.11LGTIX17-21YX46-50I, wherein X is any amino acid, and the range n is the number of the X amino acids.

[0136] In some embodiments, the plant Protoporphyrinogen Oxidase (PPO) amino acid sequence comprising the mutation(s) at a position(s) structurally corresponding to at least one amino acid position selected from positions 123, 151, 225, 371, 409, and 412 or any combination thereof of a PPO enzyme having an amino acid sequence as set forth in SEQ ID NO: 2, comprises one or more PPO enzyme selected from any one of SEQ ID NO: 14-47 listed in Table 3 or Table 4.

[0137] According to some embodiments, the mutation(s) in the PPO amino acid sequence at position(s) structurally corresponding to the at least one amino acid position 123 in SEQ ID NO: 2, includes at least one of a polar uncharged amino acid selected from: Serine (S), Threonine (T), Asparagine (N), Cysteine (C) or Selenocysteine (U), and Tyrosine (Y), or any combination thereof. Each possibility is a separate embodiment. According to some embodiments, the mutation(s) in the PPO amino acid sequence at position(s) structurally corresponding to the at least one amino acid position 123 in SEQ ID NO: 2 includes Serine (S).

[0138] According to some embodiments, the mutation(s) in the PPO amino acid sequence at position(s) structurally corresponding to the at least one amino acid position 151 in SEQ ID NO: 2, includes at least one of a non-polar amino acid or hydrophobic side chain amino acid selected from: Alanine (A), Valine (V), Leucine (L), Isoleucine (I), Methionine (M), Proline (P), Tyrosine (Y) and Tryptophan (W), or any combination thereof. Each possibility is a separate embodiment.

[0139] According to some embodiments, the mutation(s) in the PPO amino acid sequence at position(s) structurally corresponding to the at least one amino acid position 151 in SEQ ID NO: 2 includes Leucine (L).

[0140] According to some embodiments, the mutation(s) in the PPO amino acid sequence at position(s) structurally corresponding to the at least one amino acid position 225 in SEQ ID NO: 2, includes at least one of a non-polar or positively charged amino acid selected from: Glycine (G), Alanine (A), Arginine (R), and Lysine (K), or any combination thereof. Each possibility is a separate embodiment.

[0141] According to some embodiments, the mutation(s) in the PPO amino acid sequence at position(s) structurally corresponding to the at least one amino acid position 225 in SEQ ID NO: 2, includes at least one of a non-polar or positively charged amino acid selected from: Arginine (R), Glycine (G), and Lysine (K), or any combination thereof. Each possibility is a separate embodiment.

[0142] According to some embodiments, the mutation(s) in the PPO amino acid sequence at position(s) structurally corresponding to the at least one amino acid position 225 in SEQ ID NO: 2 includes at least one of a positively charged amino acid selected from: Arginine (R) and / or Lysine (K). Each possibility is a separate embodiment.

[0143] The crop plant or crop plant cell of any one of claims 1-15, wherein the mutation(s) at position(s) structurally corresponding to the at least one amino acid position 225 in SEQ ID NO: 2 is selected from: Arginine (R) and / or Glycine (G). Each possibility is a separate embodiment. According to some embodiments, the mutation(s) in the PPO amino acid sequence at position(s) structurally corresponding to the at least one amino acid position 371 in SEQ ID NO: 2, includes at least one of a non-polar amino acid or hydrophobic side chain amino acid selected from: Glycine (G) and / or Alanine (A). Each possibility is a separate embodiment.

[0144] According to some embodiments, the mutation(s) in the PPO amino acid sequence at position(s) structurally corresponding to the at least one amino acid position 371 in SEQ ID NO: 2 includes Alanine (A).

[0145] According to some embodiments, the mutation(s) in the PPO amino acid sequence at position(s) structurally corresponding to the at least one amino acid position selected from 409 and 412 in SEQ ID NO: 2, includes at least one of a polar uncharged amino acid selected from: Serine (S), Threonine (T), Asparagine (N), Glutamine (Q), Cysteine (C) or Selenocysteine (U), and Tyrosine (Y), or any combination thereof. Each possibility is a separate embodiment.

[0146] According to some embodiments, the mutation(s) in the PPO amino acid sequence at position(s) structurally corresponding to the at least one amino acid position selected from 409 and 412 in SEQ ID NO: 2 includes at least one of a polar uncharged amino acid selected from: Threonine (T) and / or Asparagine (N). Each possibility is a separate embodiment.

[0147] According to some embodiments, the mutation(s) in the PPO amino acid sequence at position(s) structurally corresponding to the at least one amino acid position 409 in SEQ ID NO: 2 includes Asparagine (N).

[0148] According to some embodiments, the mutation(s) in the PPO amino acid sequence at position(s) structurally corresponding to the at least one amino acid position 412 in SEQ ID NO: 2 includes Threonine (T).

[0149] Reference is now made to Example 3 and Table 1.

[0150] In some embodiments, the mutated-plant Protoporphyrinogen Oxidase (PPO) amino acid sequence includes at least one additional mutation(s) at a position(s) structurally corresponding to any amino acid position(s) in SEQ ID NO: 2.

[0151] In some related embodiments, the at least one additional mutation(s) includes the at least one double mutation. In some embodiments, the mutated-plant Protoporphyrinogen Oxidase (PPO) amino acid sequence includes at least one double mutation at a combination of positions structurally corresponding to at least two amino acid positions in SEQ ID NO: 2 selected from positions: (1) 123S and 151L; (2) 123S and 225R; (3) 123S and 225G; (4) 123S and 225K; (5) 123S and 371A; (6) 123S and 409N; (7) 123S and 412T; (8) 151L and 225R; (9) 151L and 225G (10) 151L and 225K; (11) 151L and 371A; (12) 151L and 409N; (13) 151L and 412T; (14) (14) 225R and 371A; (15) 225R and 409N; (16) 225R and 412T; (17); (17) 225G and 371A; (18) 225G and 409N; (19) 225G and 412T; (20) 225K and 371 A; (21) 225K and 409N; (22) 225K and 412T; (23) 371A and 409N; (24) 371A and 412T; (25) 409N and 412T according to combinations 1-25 in Table 2, or any combination thereof.

[0152] In some embodiment, combinations 1-25 shown in Table 2, include combinations of two commonly known positions found to have novel mutations / substitutions identified as capable of extending plant tolerance to PPO herbicides.

[0153] According to a specific embodiment, the mutated-plant Protoporphyrinogen Oxidase (PPO) includes at least one double mutation at a combination of positions structurally corresponding to at least two amino acid positions in SEQ ID NO: 2 selected from combinations 1-25 in Table 2, or any combination thereof.

[0154] In some embodiments, combinations 1-24 include at least one novel / unique position identified as capable of extending plant tolerance to PPO herbicides.

[0155] According to another specific embodiment, the mutated-plant Protoporphyrinogen Oxidase (PPO) comprises at least one double mutation at a combination of positions structurally corresponding to at least two amino acid positions in SEQ ID NO: 2 selected from combinations 1-10, and 14-22 in Table 2, or any combination thereof, and wherein the at least one double mutation includes a position that corresponds to positions 225 and / or 123 in SEQ ID NO: 2. In some embodiment, combinations 1-10 and 14-22 include position 225.

[0156] According to another specific embodiment, the mutated-plant Protoporphyrinogen Oxidase (PPO) comprises at least one double mutation at a combination of positions structurally corresponding to at least two amino acid positions in SEQ ID NO: 2 selected from combinations 2-4, 8-10, and 14-22 in Table 2, or any combination thereof, and wherein the at least one double mutation includes a position that corresponds to position 225 in SEQ ID NO: 2. In some embodiment, combinations 2-4, 8-10 include position 225. Reference is now made to Example 4 and Table 2.

[0157] According to some embodiments, the identified genetic change(s) are introduced into the PPO CDS of a crop plant that expresses a PPO enzyme having amino acid sequence at least 98% identity to the GmPPOl enzyme utilizing gene-editing methods, in order to generate genetically edited / modified crop plants possessing Herbicide Tolerance HT / resistance.

[0158] According to some additional embodiments, the mutated-plant Protoporphyrinogen Oxidase (PPO) comprises amino acid sequence having at least 98% sequence identity to the endogenous PPO enzyme of the crop plant.

[0159] According to some additional embodiments, the mutated-plant PPO comprises amino acid sequence having at least 99% identity to the endogenous PPO enzyme of the crop plant.

[0160] According to some embodiments, the mutated-plant Protoporphyrinogen Oxidase (PPO) comprises PPO endogenous to the crop plant.

[0161] According to some additional embodiments, the mutated-plant Protoporphyrinogen Oxidase (PPO) comprises amino acid sequence having at least 98% sequence identity to the endogenous PPO enzyme of the crop plant, and wherein the mutated-plant Protoporphyrinogen Oxidase (PPO) comprises PPO endogenous to the crop plant.

[0162] According to some embodiments, the crop plant comprises at least one crop selected from maize, wheat, rice, barley, soybean, cowpea, chickpea, cotton, sorghum, beans, rapeseed / canola, alfalfa, flax, sunflower, safflower, millet, rye, sugarcane, sugar beet, cocoa, tea, Brassica napus, Brassica rapa, Brassica oleracea. coffee, sweet potato, flax, peanut, clover, lettuce, tomato, cucurbits, cassava, potato, carrot, radish, pea, lentils, cabbage, cauliflower, broccoli, brussels sprouts, peppers, pineapple, citrus, apples, pears, peaches, apricots, walnuts, avocado, banana, palm, eucalyptus, poplar, pine, coconut, orchids, petunia, carnations, roses, Poa species (bluegrasses), Festuca species (fescues), Lolium species (rye grass), Phalaris species (canary grasses), switchgrass, prairie grasses, Indian grasses, big bluestem grass, and camelina, or any combination thereof. Each possibility is a separate embodiment.

[0163] In some specific embodiments, the crop plant is selected from the group consisting of maize, wheat, rice, soybean, cowpea, chickpea, cotton, sorghum, rapeseed / canola, alfalfa, sunflower, sugarcane, sugar beet, camelina, Poa species (bluegrasses), Festuca species (fescues), Lolium species (rye grass), Phalaris species (canary grasses) or any combination thereof. Each possibility is a separate embodiment.

[0164] In even more specific embodiments, the crop plant is a soybean plant.

[0165] In one embodiment, the crop plant comprises a broadleaf plant.

[0166] In other embodiments, the crop plant comprises a cereal and / or grass type plant. Each possibility is a separate embodiment.

[0167] The term “cereal” is used herein to refer to a grass species cultivated for seeds or for propagation. Non limiting examples of cereal crop plants include: corn, wheat, rye, barley, oat, rice, millet, sorghum and triticale.

[0168] According to some embodiments, the mutated-plant Protoporphyrinogen Oxidase (PPO) includes at least one mutated-PPO enzyme selected from the group consisting of maize PPO, wheat PPO, rice PPO, barley PPO, soybean PPO, cowpea PPO, chickpea PPO, cotton PPO, sorghum PPO, beans PPO, rapeseed / canola PPO, alfalfa PPO, flax PPO, sunflower PPO, safflower PPO, millet PPO, rye PPO, sugarcane PPO, sugar beet PPO, cocoa PPO, tea PPO, Brassica napus PPO, Brassica rapa PPO, Brassica oleracea PPO, coffee PPO, sweet potato PPO, flax PPO, peanut PPO, clover PPO, lettuce PPO, tomato PPO, cucurbits PPO, cassava PPO, potato PPO, carrot PPO, radish PPO, pea PPO, lentils PPO, cabbage PPO, cauliflower PPO, broccoli PPO, brussels sprouts PPO, peppers PPO, pineapple PPO, citrus PPO, apples PPO, pears PPO, peaches PPO, apricots PPO, walnuts PPO, avocado PPO, banana PPO, palm PPO, eucalyptus PPO, poplar PPO, pine PPO, coconut PPO, orchids PPO, petunia PPO, carnations PPO, roses PPO, Poa species (bluegrasses) PPO, Festuca species (fescues) PPO, Lolium species (rye grass) PPO, Phalaris species (canary grasses) PPO, switchgrass PPO, prairie grasses PPO, Indian grasses PPO, big bluestem grass PPO, and camelina PPO, or any combination thereof.

[0169] In some specific embodiments, the mutated-plant Protoporphyrinogen Oxidase (PPO) includes a mutated-soybean PPO.

[0170] In some specific embodiments, the mutated-plant Protoporphyrinogen Oxidase (PPO) includes a mutated-soybean PPO1. In some embodiments, the mutated- soybean PPO includes at least one mutation selected from Q123S, F151L, Y225R, Y225G, Y225K, V371A, L409N, and I412T, as set forth in any one of the mutated-soybean PPO amino acid sequences represented by SEQ ID NOs: 3-10, or any combination thereof. Each possibility is a separate embodiment.

[0171] In specific embodiments, the mutated-soybean PPO includes at least one mutation selected from Q123S, F151L, Y225R, Y225G, Y225K, and V371A, as set forth in any one of the mutated-soybean PPO amino acid sequences represented by SEQ ID NOs: 3-8, or any combination thereof. Each possibility is a separate embodiment.

[0172] In further specific embodiments, the mutated-soybean PPO includes at least one mutation selected from Y225R, Y225G, and Y225K, as set forth in any one of the mutated- soybean PPO amino acid sequences represented by SEQ ID NOs: 5-7, or any combination thereof. Each possibility is a separate embodiment.

[0173] In some embodiments, the mutated-soybean PPO includes mutation(s) at least at one position(s) selected from Q123, F151, Y225, V371, L409, and 1412, or any combination thereof. Each possibility is a separate embodiment.

[0174] In some embodiments, the mutated-soybean PPO includes mutation(s) at least at one position(s) selected from Q123X1, F151X2, Y225X3, V371 X4, L409X5, and I412X5, or any combination thereof. Each possibility is a separate embodiment.

[0175] In some embodiments, the mutation in Xi includes amino acid residues selected from: Serine (S), Threonine (T), Asparagine (N), Cysteine (C) or Selenocysteine (U), and Tyrosine (Y), or any combination thereof. Each possibility is a separate embodiment.

[0176] In some embodiments, the mutation X2includes amino acid residues selected from: Alanine (A), Valine (V), Leucine (L), Isoleucine (I), Methionine (M), Proline (P), Tyrosine (Y) and Tryptophan (W), or any combination thereof. Each possibility is a separate embodiment.

[0177] In some embodiments, the mutation X3includes amino acid residues selected from: Glycine (G), Alanine (A), Arginine (R), and Lysine (K), or any combination thereof. Each possibility is a separate embodiment. In some embodiments, the mutation X4 includes amino acid residues selected from: Glycine (G) and / or Alanine (A), or any combinations thereof. Each possibility is a separate embodiment.

[0178] In some embodiments, the mutation X5 includes amino acid residues selected from: Serine (S), Threonine (T), Asparagine (N), Glutamine (Q), Cysteine (C) or Selenocysteine (U), and Tyrosine (Y), or any combination thereof. Each possibility is a separate embodiment.

[0179] In specific embodiments, the mutated- soybean PPO includes mutation(s) at least at one position(s) selected from Q123X1, F151 X2, Y225X3, and V371X4, or any combination thereof. Each possibility is a separate embodiment.

[0180] In a further specific embodiment, the mutated-soybean PPO includes mutation(s) at least at position Y225X3.

[0181] In some embodiments, the mutated-soybean PPO includes at least one double mutation at a combination of positions structurally corresponding to at least two amino acid positions in SEQ ID NO: 2 selected from any of combinations 1-25 in Table 2, or any combination thereof. Each possibility is a separate embodiment.

[0182] In some embodiments, the mutated-soybean Protoporphyrinogen Oxidase (PPO) includes at least one double mutation at a combination of positions structurally corresponding to at least two amino acid positions in SEQ ID NO: 2 selected from any of combinations 1-25 in Table 2, or any combination thereof, and wherein the at least one double mutation comprises a position that corresponds to position 225 in SEQ ID NO: 2. Each possibility is a separate embodiment.

[0183] In some specific embodiments, the mutated-plant Protoporphyrinogen Oxidase (PPO) comprises a mutated-soybean PPO1.

[0184] According to some embodiments, the one or more PPO inhibitors is selected from the group consisting of herbicide families Phenylpyrazoles, Pyrimidinediones, Oxazolidinedione, Diphenylethers, Oxadiazoles, N-phenylphthalimides, Triazinone, Thiadiazoles, Triazolinones, and Triazolopyridinones, or any combination thereof. Each possibility is a separate embodiment. According to specific embodiments, the one or more PPO inhibitors is selected from the group consisting of herbicide families Diphenylethers, Oxadiazoles, and Triazolinones. Each possibility is a separate embodiment.

[0185] According to specific embodiments, the one or more PPO inhibitors is selected from the group consisting of herbicide families Diphenylethers, Oxadiazoles, and Triazolinones, including the active ingredients Oxyfluorfen, Oxadiazon and Carfentrazone-ethyl, respectively. Each possibility is a separate embodiment.

[0186] In one embodiment, the plant comprises a broadleaf plant.

[0187] In other embodiments, the plant comprises a cereal and / or grass type plant. Each possibility is a separate embodiment.

[0188] According to some embodiments, there is provided a seed of the genetically modified plant of any one of the aforementioned embodiments.

[0189] According to some embodiments, there is provided a product derived from the genetically modified of any one of the aforementioned embodiments.

[0190] According to another aspect of the disclosed invention, there is provided a method of controlling weeds growth where crop plants are growing, using at least one PPO inhibitor herbicide, wherein the crop plants include the genetically modified plant according to any one of the aforementioned embodiments, the method include applying a weed-controlling amount of the one or more PPO inhibitor herbicide.

[0191] In some embodiments, the one or more PPO-inhibitor herbicides are selected from the group consisting of herbicides families of the groups Phenylpyrazoles, Pyrimidinediones, Oxazolidinedione, Diphenylethers, Oxadiazoles, N-phenylphthalimides, Triazinone, Thiadiazoles, Triazolinones, and Triazolopyridinones, or any combination thereof. Each possibility is a separate embodiment.

[0192] According to yet another aspect of the disclosed invention, there is provided method for conferring resistance or tolerance to one or more PPO inhibitor herbicide in a crop plant, the method includes genetically modifying the plant to express a mutated plant Protoporphyrinogen Oxidase (PPO) enzyme, wherein the plant PPO enzyme have an amino acid sequence comprising mutation(s) at position(s) structurally corresponding to at least one amino acid position selected from positions 123, 151, 225, 371, 409, and 412 or any combination thereof, of a PPO enzyme having an amino acid sequence as set forth in SEQ ID NO: 2. Each possibility is a separate embodiment.

[0193] In some embodiments, the mutated-plant Protoporphyrinogen Oxidase (PPO) comprises transgenic PPO and / or the endogenous PPO of the crop plant. Each possibility is a separate embodiment.

[0194] In some embodiments, the mutated-plant Protoporphyrinogen Oxidase (PPO) is endogenous to the crop plant; and wherein the crop plant is genetically modified using endogenous PPO utilizing gene editing tools. Each possibility is a separate embodiment.

[0195] In some embodiments, the mutated-plant Protoporphyrinogen Oxidase (PPO) amino acid sequence comprises a mutation(s) at a position(s) structurally corresponding to at least one amino acid position selected from positions 123, 151, 225 and 371, or any combination thereof. Each possibility is a separate embodiment.

[0196] The crop plant or crop plant cell of any one of claims 1-7, wherein the PPO amino acid sequence comprises a mutation(s) at a position(s) structurally corresponding to at least one amino acid position selected from positions 123 and 225.

[0197] In some embodiments, the mutated-plant Protoporphyrinogen Oxidase (PPO) amino acid sequence comprises a mutation(s) at a position structurally corresponding to amino acid position 225.

[0198] In some embodiments, the mutated-plant Protoporphyrinogen Oxidase (PPO) amino acid sequence further comprises at least one additional mutation(s) at a position(s) structurally corresponding to any position(s) in SEQ ID NO: 2.

[0199] In some embodiments, the mutated-plant Protoporphyrinogen Oxidase (PPO) comprises at least one double mutation at a combination of positions structurally corresponding to at least two amino acid positions in SEQ ID NO: 2 selected from any of combinations 1-25 in Table 2, or any combination thereof. Each possibility is a separate embodiment. In some embodiments, the mutated-plant Protoporphyrinogen Oxidase (PPO) comprises amino acid sequence having at least 98% identity to the endogenous PPO enzyme of the crop plant.

[0200] In some embodiments, the mutated-plant Protoporphyrinogen Oxidase (PPO) comprises a mutated- soybean PPO.

[0201] In some embodiments, the mutated- soybean Protoporphyrinogen Oxidase (PPO) comprises at least one mutation selected from Q123S, F151L, Y225R, Y225G, Y225K, V371A, L409N, and I412T, as set forth in any of the mutated-soybean PPO1 amino acid sequences in any one of SEQ ID NOs: 3-10, or any combination thereof, and wherein the mutated-PPO confers resistance or tolerance to at least one PPO-inhibitor herbicide. Each possibility is a separate embodiment.

[0202] In some embodiments, the mutated-soybean Protoporphyrinogen Oxidase (PPO) comprises at least one mutation selected from Q123S, F151L, Y225R, Y225G, Y225K, and 371 A as set forth in the mutated-soybean PPO1 amino acid sequences in any one of SEQ ID NOs: 3-8, or any combination thereof. Each possibility is a separate embodiment.

[0203] In some embodiments, the mutated-soybean Protoporphyrinogen Oxidase (PPO) comprises at least one mutation selected from Y225R, Y225G, and Y225K, as set forth in the mutated-soybean PPO amino acid sequences set forth in any one of SEQ ID NOs: 5-7, or any combination thereof. Each possibility is a separate embodiment.

[0204] In some embodiments, the mutated-soybean Protoporphyrinogen Oxidase (PPO) comprises a mutated-soybean Protoporphyrinogen Oxidase 1 (PPO1).

[0205] According to some embodiments, there is provided a modified Protoporphyrinogen Oxidase (PPO) protein, said modified PPO includes one or more amino acid replacements at a position(s) structurally corresponding to at least one amino acid position selected from positions 123, 151, 225, 371, 409, and 412, or any combination thereof, of a reference PPO protein having an amino acid sequence as set forth in SEQ ID NO: 2.

[0206] According to some embodiments, when the modified PPO is expressed in a crop plant, the modified PPO confers resistance or tolerance to at least one PPO-inhibitor herbicide, compared to a plant of the same genus, not expressing the modified PPO. According to some embodiments, there is provided a nucleic acid encoding for the modified PPO protein. In some embodiments, there is provided a vector (such as an expression vector) including the nucleic acid encoding for the modified PPO protein.

[0207] According to some embodiments, there is provided a plant cell harboring the modified PPO and / or a nucleic acid including the same.

[0208] The following examples are presented in order to more fully illustrate some embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

[0209] EXAMPLES

[0210] Materials and Methods

[0211] To generate a crop(s) plant(s), for example, a soybean crop plant, resistant or tolerant to PPO inhibitor-type herbicides, experiments were set to identify resistant / tolerant conferring mutation(s) in a soybean (Glycine max) PPO enzyme coding sequence (CDS) utilizing Arabidopsis thaliana model plants.

[0212] Initially, a soybean PPO1 mRNA coding sequence was cloned, and one or two genetic alterations were randomly introduced in multiple copies of the PPO1 CDS resulting in genetic expression library containing numerous mutated PPO1 coding sequences (CDS)

[0213] Next, the genetic library was introduced into Arabidopsis model plants utilizing high throughput transformation, such that on average each model plant receives a single altered CDS element. A high-throughput in-planta screening for PPO inhibitor-type herbicide tolerance (HT) was performed comparing tolerance of transgenic plants expressing the native GmPPOl enzyme CDS having the amino acid sequence as set forth in SEQ ID NO: 2 with the tolerance of plants expressing the mutated-GmPPOl enzyme CDS.

[0214] In the following step, transgenic plants exhibiting improved / enhanced Herbicide Tolerance (HT) were selected and one or more of the genetic changes in the GmPPOl CDS that conferred the tolerance were identified. These mutated-GmPPOl enzyme CDS have the amino acid sequence as set forth in SEQ ID NOs: 3-10.

[0215] Finally, a crop(s) plant(s), such as soybean crop plants, are genetically modified to express the mutated GmPPOl CDS, thereby conferring on these plants a resistance or tolerance to herbicides of the PPO inhibitor type, namely flumioxazin, oxyfluorfen, oxadiazon and carfentrazone-ethyl, which are the active ingredients in Strike, Goal, Star, and Aurora, respectfully.

[0216] Example 1: Cloning of the soybean (Glycine max) PPO1 coding sequence (CDS), for transformation and determination of the selection cut-off for enhanced natural Herbicide Tolerance (HT)

[0217] A soybean (Glycine max) Protoporphyrinogen Oxidase 1 (PPO1) mRNA coding sequence CDS coding for a native PPO1 enzyme (GmPPOl) was cloned into the backbone of the pPA35H binary vector, commonly used for libraries construction, downstream to a constitutive 35S promoter; thereby creating a 35S(WT)GmPP01 chimeric sequence (FIG. 1A).

[0218] The sequence of the constitutive 35S promoter is denoted by nucleic acid sequence of SEQ ID NO: 1 : ttaattaagagctcgcatgcctgcaggtccccagattagccttttcaatttcagaaagaatgctaacccacagatggttagag aggcttacgcagcaggtctcatcaagacgatctacccgagcaataatctccaggaaatcaaataccttcccaagaaggttaaagatgc agtcaaaagattcaggactaactgcatcaagaacacagagaaagatatatttctcaagatcagaagtactattccagtatggacgattc aaggcttgcttcacaaaccaaggcaagtaatagagattggagtctctaaaaaggtagttcccactgaatcaaaggccatggagtcaaa gattcaaatagaggacctaacagaactcgccgtaaagactggcgaacagttcatacagagtctcttacgactcaatgacaagaagaa aatcttcgtcaacatggtggagcacgacacacttgtctactccaaaaatatcaaagatacagtctcagaagaccaaagggcaattgag acttttcaacaaagggtaatatccggaaacctcctcggattccattgcccagctatctgtcactttattgtgaagatagtggaaaaggaa ggtggctcctacaaatgccatcattgcgataaaggaaaggccatcgttgaagatgcctctgccgacagtggtcccaaagatggaccc ccacccacgaggagcatcgtggaaaaagaagacgttccaaccacgtcttcaaagcaagtggattgatgtgatatctccactgacgta agggatgacgcacaatcccactatccttcgcaagacccttcctctatataaggaagttcatttcatttggagagaacacgggggac

[0219] The amino acid sequence of the native soybean PPO1 enzyme (GmPPOl) is denoted by SEQ ID NO: 2: MVSVFNEILFPPNQTLLRPSLHSPTSFFTSPTRKFPRSRPNPILRCSIAEESTASPPKTRDSAPVDC VVVGGGVSGLCIAQALATKHANANVVVTEARDRVGGNITTMERDGYLWEEGPNSFQPSDPMLTMVVD SGLKDELVLGDPDAPRFVLWNRKLRPVPGKLTDLPFFDLMSIGGKIRAGFGALGIRPPPPGHEESVE EFVRRNLGDEVFERLIEPFCSGVYAGDPSKLSMKAAFGKVWKLEKNGGSIIGGTFKAIQERNGASKP PRDPRLPKPKGQTVGSFRKGLTMLPDAISARLGNKVKLSWKLSSISKLDSGEYSLTYETPEGVVSLQ CKTVVLTIPSYVASTLLRPLSAAAADALSKFYYPPVAAVSISYPKEAIRSECLIDGELKGFGQLHPR SQGVETLGTIYSSSLFPNRAPPGRVLLLNYIGGATNTGILSKTDSELVETVDRDLRKILINPNAQDP FVVGVRLWPQAIPQFLVGHLDLLDVAKASIRNTGFEGLFLGGNYVSGVALGRCVEGAYEVAAEVNDF LTNRVYK

[0220] Pre-determined cut-off concentration - The pPA35H vector containing the 35S-WT- GmPPOl chimeric sequence (pPA35S-(WT)GmPP01 construct) was used to transform Arabidopsis model plants, creating stable transgenic T2 events. These events were subsequently utilized to calibrate a PPO inhibitor herbicide cut-off concentration. This predetermined concentration was used in the subsequent stage of plant library screening and selection.

[0221] The process of generating Arabidopsis seeds with T2 generation stable events commenced with the introduction of the binary vector pPA35S-GmPP01 containing the 35S- (WT)GmPPOl chimeric sequence into Agrobacterium. Subsequently, the Agrobacterium was employed to genetically modify Arabidopsis plants. The transformed Arabidopsis seeds underwent germination and were subjected to a selective herbicide to identify the transformed plants. Following this, seeds were harvested, and T2 Arabidopsis seeds underwent another round of germination and treatment with a selective herbicide to confirm the presence of stable transgenic T2 events.

[0222] The selection cut-off value was calibrated to functionally screen the transformed model plants for Herbicide Tolerance (HT) trait, specifically the resistant activity of the GmPPOl enzyme. T2 Arabidopsis plants carrying the pPA35S-(WT)GmPP01 construct were exposed to a concentration gradient of Flumioxazin, a PPO inhibitor-type herbicide, to establish the cut-off concentration. Stable T2 plants showing the highest tolerance to the herbicide were chosen based on set criteria (damage to leaves and growth inhibition). The determined cut-off concentration was used to select transgenic plant libraries containing one or more genetic changes in the GmPPOl CDS that conferred the tolerance.

[0223] Example 2 - Generation of an in-planta genetic library of a large collection of altered soybean PPO1 CDS elements for high-throughput transformation and screening of Herbicide Tolerance (HT) in transgenic Arabidopsis plants A mutagenized genetic library was created by introducing random genetic alterations into the soybean PPO1 CDS sequence, resulting in diverse modifications across various regions of the enzyme's structure.

[0224] The CDS was amplified from the soybean mRNA coding sequence previously cloned into the pPA35H binary vector as described in Example 1. The PCR fragments were then subjected to random mutagenesis using Error Prone PCR methodology according to manufacture instructions. The mutation rate was increased by the changes in the Mn2+and Mg2+ion concentrations along with unbalancing the available nucleotides pools. The manipulation of mutagenesis was monitored by adjusting the number of amplification cycles allowing choice of mutation degree.

[0225] The resulting population of DNA molecules carrying random point mutations coding for mutated GmPPOl mRNA were cloned into the binary vector (pPA35H) downstream to the 35S promoter, generating large pools of 105clones representing mutated PPO1 enzymes (pPA35 S -mutated GmPPOl). This is illustrated in FIG. IB.

[0226] Next, quality of the GmPPOl genetic variant libraries were validated through random sequencing of the library, followed by a statistical analysis of number, distribution, and type of mutations.

[0227] Following preparation and quality validation of a GmPPOl CDS variant library, the obtained plurality of pPA35S-mutated-GmPP01 (or pPA35S-WT-GmPP01) nucleic acid vectors were introduced to Agrobacterium and transformed into Arabidopsis plants utilizing a high-throughput transformation method (floral dip), creating in-planta CDS libraries each containing a collection of approximately 103-105transgenic pPA35S-mutated-GmPP01 (or pPA35S-WT-GmPP01) plants, in which, on average each plant carries a single genetically altered CDS element with at least one genetic changes in the GmPPOl mRNA sequence.

[0228] The evaluation and selection of Herbicide Tolerance (HT) trait in model plants was performed by a high-throughput in-planta functional screening assay using PPO inhibitor herbicide spraying to easily identify low-frequency mutants. First, transgenic pPA35S- mutated-GmPPOl (or pPA35S-WT-GmPP01) T1 seedlings were subjected to Glufosinate ammonium to select the transformed T1 events. Thereafter, 14 days old seedlings were subjected to lxlO'3% 1250 ng / ml Oxadiazon (Star) treatment, in accordance with the predetermined cut-off concentration (examplel). The level of tolerance was evaluated based on the criteria of damage to leaves and growth inhibition. Plants exhibiting significant tolerance to the specified cut-off levels of Oxadiazon (Star) were chosen based on their potential resistance to the herbicide.

[0229] Example 3 - selection of transgenic Arabidopsis plants exhibiting resistance / Herbicide Tolerance (HT) and identification of the underlying genetic changes in the PPO1 CDS elements

[0230] Next, the herbicide-tolerant transgenes in selected plants were amplified using PCR and then sequenced to identify the positions and types of mutations in GmPPOl CDS (the WT sequence is represented by SEQ ID NO: 2) that exhibited Herbicide Tolerance (HT).

[0231] Advantageously, at least 8 adventitious mutated sequences of GmPPOl were identified by sequencing of the transgenes. The amino acid sequences of these mutated-GmPPOl CDS are denoted by any one of SEQ ID NOs: 3-10.

[0232] Surprisingly, the mutations that provide resistance or tolerance to PPO inhibitor herbicide(s) are located within the enzyme's herbicide binding pocket, and are involved in substrate / herbicides interaction.

[0233] The mutations and their positions with respect to WT GmPPOl amino acid CDS denoted by SEQ ID NO: 2, are summarized herein below in Table 1. A minimum of 8 distinct mutations were observed at 6 unique positions within 26 resistant events, identified through the functional herbicide selection assay. The analysis revealed that these 26 resistant events represent the following mutated positions in the GmPPOl protein -

[0123] ,

[0151] ,

[0225] ,

[0371] ,

[0409] and

[0412] - which were all identified as essential to herbicide interaction within the enzyme pocket.

[0234] Surprisingly, all 8 resistant mutations Q123S, F151L, Y225R, Y225G, Y225K, V371A, L409N and I412T represent newly discovered residues / mutations in the soybean PPO1 enzyme CDS and are represented by the amino acid sequence denoted in SEQ ID NOs: 3-10, respectively. Two positions, namely

[0409] and

[0412] , represented by two of the mutations, L409N and I412T, are previously published positions, while the other four positions, namely

[0123] ,

[0151] ,

[0225] and

[0371] , represented by the remaining 6 mutations Q123S, F151L, Y225R, Y225G, Y225K and V371A are newly identified positions uniquely disclosed herein to extend plant tolerance to PPO herbicides. Further surprising, is that out of the 26 events, 18 events shared an identical position

[0225] , suggesting that these six positions, and most particularly position

[0225] may be highly advantageous potent spots that when mutated confer enhanced / improved Herbicide Tolerance (HT) trait.

[0235] Table 1: Soybean PPO1 mutations within the enzyme’s herbicide binding pocket. Shown are soybean PPO1 -specific residues essential for herbicide interact! on / binding in the enzyme’s pocket 1 Q-RF-SGVYA- V-F GQL-LGTI- Y-I1 (gray lower row highlighted in bold) and their positions (the gray upper row). The lower rows of the table illustrate the corresponding mutations found to confer resistance or tolerance to PPO-inhibitor herbicide(s). Novel positions identified to extend plant tolerance to PPO herbicides are those carrying the mutations Q123S, F151L, Y225R, Y225G, Y225K, and V371A (highlighted in bold underlined (originally green)), while newly discovered residues / mutations in previously published positions, are L409N and I412T (highlighted in Italics underlined (originally red).

[0236] To evaluate whether the herein disclosed mutated positions in GmPPOl CDS can confer resistance or tolerance to other PPO-inhibiting herbicides besides Oxadiazon, further experiment with Arabidopsis T2 seeds were performed in the presence of: 1250ng / ml Oxadiazon, 120 ng / ml Oxyfluorfen, and 200ng / ml Carfentrazone-ethyl.

[0237] Advantageously and surprisingly, the identified mutations conferred tolerance to all three PPO-inhibiting herbicides. This is demonstrated and summarized in FIGs 2A-2B. FIG. 2A demonstrates the herbicide tolerance of two representative events containing ‘beneficial mutation’ (i.e., that generated resistance to the herbicides or HT) in the PPO CDS, compared to transgenic plants expressing WT-GmPPO CDS.

[0238] The results are summarized in FIG. 2B, showing that soybean GmPPOl CDS bearing adventitious mutations at positions

[0123] ,

[0151] ,

[0225] ,

[0371] ,

[0409] and

[0412] and expressed in Arabidopsis model plants conferred resistance or tolerance to at least 3 different PPO- inhibiting herbicides including Oxadiazon, Oxyfluorfen, and Carfentrazone-ethyl, relative to Arabidopsis plants expressing WT soybean GmPPOl CDS.

[0239] The amino acid sequences of the mutated- GmPPOl CDS are denoted by any one of SEQ ID NOs: 3-10 below:

[0240] The amino acid sequence of the mutated soybean PPO1 enzyme (GmPPOl) carrying a Q123S mutation is denoted by SEQ ID NO: 3:

[0241] MVSVFNEILFPPNQTLLRPSLHSPTSFFTSPTRKFPRSRPNPILRCSIAEESTASPPKTRDSAPVDC VVVGGG VS GLC I AQ AL AT KH AN AN VVVT E ARDRVGGN I T TME RDG YL WE EG PN S F S P S D PMLTMVVD SGLKDELVLGDPDAPRFVLWNRKLRPVPGKLTDLPFFDLMSIGGKIRAGFGALGIRPPPPGHEESVE EFVRRNLGDEVFERLIEPFCSGVYAGDPSKLSMKAAFGKVWKLEKNGGSIIGGTFKAIQERNGASKP PRDPRLPKPKGQTVGSFRKGLTMLPDAISARLGNKVKLSWKLSSISKLDSGEYSLTYETPEGVVSLQ CKTVVLTIPSYVASTLLRPLSAAAADALSKFYYPPVAAVSISYPKEAIRSECLIDGELKGFGQLHPR SQGVETLGTIYSSSLFPNRAPPGRVLLLNYIGGATNTGILSKTDSELVETVDRDLRKILINPNAQDP FVVGVRLWPQAIPQFLVGHLDLLDVAKASIRNTGFEGLFLGGNYVSGVALGRCVEGAYEVAAEVNDF LTNRVYK

[0242] The amino acid sequence of the mutated soybean PPO1 enzyme (GmPPOl) carrying a F151L mutation is denoted by SEQ ID NO: 4:

[0243] MVSVFNEILFPPNQTLLRPSLHSPTSFFTSPTRKFPRSRPNPILRCSIAEESTASPPKTRDSAPVDC VVVGGGVSGLCIAQALATKHANANVVVTEARDRVGGNITTMERDGYLWEEGPNSFQPSDPMLTMVVD SGLKDELVLGDPDAPRLVLWNRKLRPVPGKLTDLPFFDLMSIGGKIRAGFGALGIRPPPPGHEESVE EFVRRNLGDEVFERLIEPFCSGVYAGDPSKLSMKAAFGKVWKLEKNGGSIIGGTFKAIQERNGASKP PRDPRLPKPKGQTVGSFRKGLTMLPDAISARLGNKVKLSWKLSSISKLDSGEYSLTYETPEGVVSLQ CKTVVLTIPSYVASTLLRPLSAAAADALSKFYYPPVAAVSISYPKEAIRSECLIDGELKGFGQLHPR SQGVETLGTIYSSSLFPNRAPPGRVLLLNYIGGATNTGILSKTDSELVETVDRDLRKILINPNAQDP FVVGVRLWPQAIPQFLVGHLDLLDVAKASIRNTGFEGLFLGGNYVSGVALGRCVEGAYEVAAEVNDF LTNRVYK

[0244] The amino acid sequence of the mutated soybean PPO1 enzyme (GmPPOl) carrying a Y225R mutation is denoted by SEQ ID NO: 5:

[0245] MVSVFNEILFPPNQTLLRPSLHSPTSFFTSPTRKFPRSRPNPILRCSIAEESTASPPKTRDSAPVDC

[0246] VVVGGGVSGLCIAQALATKHANANVVVTEARDRVGGNITTMERDGYLWEEGPNSFQPSDPMLTMVVD

[0247] SGLKDELVLGDPDAPRFVLWNRKLRPVPGKLTDLPFFDLMSIGGKIRAGFGALGIRPPPPGHEESVE EFVRRNLGDEVFERLIEPFCSGVRAGDPSKLSMKAAFGKVWKLEKNGGSIIGGTFKAIQERNGASKP

[0248] PRDPRLPKPKGQTVGSFRKGLTMLPDAISARLGNKVKLSWKLSSISKLDSGEYSLTYETPEGVVSLQ

[0249] CKTVVLTIPSYVASTLLRPLSAAAADALSKFYYPPVAAVSISYPKEAIRSECLIDGELKGFGQLHPR

[0250] SQGVETLGTIYSSSLFPNRAPPGRVLLLNYIGGATNTGILSKTDSELVETVDRDLRKILINPNAQDP

[0251] FVVGVRLWPQAIPQFLVGHLDLLDVAKASIRNTGFEGLFLGGNYVSGVALGRCVEGAYEVAAEVNDF LTNRVYK

[0252] The amino acid sequence of the mutated soybean PPO1 enzyme (GmPPOl) carrying a Y225G mutation is denoted by SEQ ID NO: 6:

[0253] MVSVFNEILFPPNQTLLRPSLHSPTSFFTSPTRKFPRSRPNPILRCSIAEESTASPPKTRDSAPVDC VVVGGGVSGLCIAQALATKHANANVVVTEARDRVGGNITTMERDGYLWEEGPNSFQPSDPMLTMVVD SGLKDELVLGDPDAPRFVLWNRKLRPVPGKLTDLPFFDLMSIGGKIRAGFGALGIRPPPPGHEESVE

[0254] EFVRRNLGDEVFERLIEPFCSGVGAGDPSKLSMKAAFGKVWKLEKNGGSIIGGTFKAIQERNGASKP PRDPRLPKPKGQTVGSFRKGLTMLPDAISARLGNKVKLSWKLSSISKLDSGEYSLTYETPEGVVSLQ CKTVVLTIPSYVASTLLRPLSAAAADALSKFYYPPVAAVSISYPKEAIRSECLIDGELKGFGQLHPR SQGVETLGTIYSSSLFPNRAPPGRVLLLNYIGGATNTGILSKTDSELVETVDRDLRKILINPNAQDP

[0255] FVVGVRLWPQAIPQFLVGHLDLLDVAKASIRNTGFEGLFLGGNYVSGVALGRCVEGAYEVAAEVNDF LTNRVYK

[0256] The amino acid sequence of the mutated soybean PPO1 enzyme (GmPPOl) carrying a Y225K mutation is denoted by SEQ ID NO: 7:

[0257] MVSVFNEILFPPNQTLLRPSLHSPTSFFTSPTRKFPRSRPNPILRCSIAEESTASPPKTRDSAPVDC

[0258] VVVGGGVSGLCIAQALATKHANANVVVTEARDRVGGNITTMERDGYLWEEGPNSFQPSDPMLTMVVD

[0259] SGLKDELVLGDPDAPRFVLWNRKLRPVPGKLTDLPFFDLMSIGGKIRAGFGALGIRPPPPGHEESVE

[0260] EFVRRNLGDEVFERLIEPFCSGVKAGDPSKLSMKAAFGKVWKLEKNGGSIIGGTFKAIQERNGASKP

[0261] PRDPRLPKPKGQTVGSFRKGLTMLPDAISARLGNKVKLSWKLSSISKLDSGEYSLTYETPEGVVSLQ

[0262] CKTVVLTIPSYVASTLLRPLSAAAADALSKFYYPPVAAVSISYPKEAIRSECLIDGELKGFGQLHPR

[0263] SQGVETLGTIYSSSLFPNRAPPGRVLLLNYIGGATNTGILSKTDSELVETVDRDLRKILINPNAQDP

[0264] FVVGVRLWPQAIPQFLVGHLDLLDVAKASIRNTGFEGLFLGGNYVSGVALGRCVEGAYEVAAEVNDF LTNRVYK

[0265] The amino acid sequence of the mutated soybean PPO1 enzyme (GmPPOl) carrying a V371 A mutation is denoted by SEQ ID NO: 8:

[0266] MVSVFNEILFPPNQTLLRPSLHSPTSFFTSPTRKFPRSRPNPILRCSIAEESTASPPKTRDSAPVDC

[0267] VVVGGGVSGLCIAQALATKHANANVVVTEARDRVGGNITTMERDGYLWEEGPNSFQPSDPMLTMVVD

[0268] SGLKDELVLGDPDAPRFVLWNRKLRPVPGKLTDLPFFDLMSIGGKIRAGFGALGIRPPPPGHEESVE

[0269] EFVRRNLGDEVFERLIEPFCSGVYAGDPSKLSMKAAFGKVWKLEKNGGSIIGGTFKAIQERNGASKP

[0270] PRDPRLPKPKGQTVGSFRKGLTMLPDAISARLGNKVKLSWKLSSISKLDSGEYSLTYETPEGVVSLQ

[0271] CKTVVLTIPSYVASTLLRPLSAAAADALSKFYYPPAAAVSISYPKEAIRSECLIDGELKGFGQLHPR

[0272] SQGVETLGTIYSSSLFPNRAPPGRVLLLNYIGGATNTGILSKTDSELVETVDRDLRKILINPNAQDP

[0273] FVVGVRLWPQAIPQFLVGHLDLLDVAKASIRNTGFEGLFLGGNYVSGVALGRCVEGAYEVAAEVNDF LTNRVYK The amino acid sequence of the mutated soybean PPO1 enzyme (GmPPOl) carrying a L409N mutation is denoted by SEQ ID NO: 9:

[0274] MVSVFNEILFPPNQTLLRPSLHSPTSFFTSPTRKFPRSRPNPILRCSIAEESTASPPKTRDSAPVDC VVVGGGVSGLCIAQALATKHANANVVVTEARDRVGGNITTMERDGYLWEEGPNSFQPSDPMLTMVVD SGLKDELVLGDPDAPRFVLWNRKLRPVPGKLTDLPFFDLMSIGGKIRAGFGALGIRPPPPGHEESVE EFVRRNLGDEVFERLIEPFCSGVYAGDPSKLSMKAAFGKVWKLEKNGGSIIGGTFKAIQERNGASKP PRDPRLPKPKGQTVGSFRKGLTMLPDAISARLGNKVKLSWKLSSISKLDSGEYSLTYETPEGVVSLQ CKTVVLTIPSYVASTLLRPLSAAAADALSKFYYPPVAAVSISYPKEAIRSECLIDGELKGFGQLHPR SQGVETNGTIYSSSLFPNRAPPGRVLLLNYIGGATNTGILSKTDSELVETVDRDLRKILINPNAQDP FVVGVRLWPQAIPQFLVGHLDLLDVAKASIRNTGFEGLFLGGNYVSGVALGRCVEGAYEVAAEVNDF LTNRVYK

[0275] The amino acid sequence of the mutated soybean PPO1 enzyme (GmPPOl) carrying a I412T mutation is denoted by SEQ ID NO: 10:

[0276] MVSVFNEILFPPNQTLLRPSLHSPTSFFTSPTRKFPRSRPNPILRCSIAEESTASPPKTRDSAPVDC VVVGGGVSGLCIAQALATKHANANVVVTEARDRVGGNITTMERDGYLWEEGPNSFQPSDPMLTMVVD SGLKDELVLGDPDAPRFVLWNRKLRPVPGKLTDLPFFDLMSIGGKIRAGFGALGIRPPPPGHEESVE EFVRRNLGDEVFERLIEPFCSGVYAGDPSKLSMKAAFGKVWKLEKNGGSIIGGTFKAIQERNGASKP PRDPRLPKPKGQTVGSFRKGLTMLPDAISARLGNKVKLSWKLSSISKLDSGEYSLTYETPEGVVSLQ CKTVVLTIPSYVASTLLRPLSAAAADALSKFYYPPVAAVSISYPKEAIRSECLIDGELKGFGQLHPR SQGVETLGTTYSSSLFPNRAPPGRVLLLNYIGGATNTGILSKTDSELVETVDRDLRKILINPNAQDP FVVGVRLWPQAIPQFLVGHLDLLDVAKASIRNTGFEGLFLGGNYVSGVALGRCVEGAYEVAAEVNDF LTNRVYK

[0277] Example 4 - Generation of double mutants

[0278] Double mutants are generated within the enzyme’s herbicide binding pocket based on the established resistance conferred by the single mutation described in Table 1.

[0279] Table 2 hereinbelow summarizes combinations of soybean PPO1 double positions / mutations. Table 2: Soybean PPO1 double positions / mutations within the enzyme’s herbicide binding pocket. The table displays combinations of mutations based on the crossing position of the upper row and left column. Each combination is numbered. Combinations 1-24 represent double mutations wherein at least one of the two positions is novel, and combination 25 represents a double mutation of two commonly known positions found to have novel mutations / substitutions, as detailed in Table 1.

[0280] In some specific examples, with respect of SEQ ID NO:2, combinations 1-25 are represented by amino acid sequences as denoted by SEQ ID NOs: 48-72, respectively.

[0281] Such that: Combination 1 is represented by SEQ ID NO: 48; Combination 2 is represented by SEQ ID NO: 49; Combination 3 is represented by SEQ ID NO: 50; Combination 4 is represented by SEQ ID NO: 51; Combination 5 is represented by SEQ ID NO: 52; Combination 6 is represented by SEQ ID NO: 53; Combination 7 is represented by SEQ ID NO: 54; Combination 8 is represented by SEQ ID NO: 55; Combination 9 is represented by SEQ ID NO: 56; Combination 10 is represented by SEQ ID NO: 57; Combination 11 is represented by SEQ ID NO: 58; Combination 12 is represented by SEQ ID NO: 59; Combination 13 is represented by SEQ ID NO: 60; Combination 14 is represented by SEQ ID NO: 61; Combination 15 is represented by SEQ ID NO: 62; Combination 16 is represented by SEQ ID NO: 63; Combination 17 is represented by SEQ ID NO: 64; Combination 18 is represented by SEQ ID NO: 65; Combination 19 is represented by SEQ ID NO: 66; Combination 20 is represented by SEQ ID NO: 67; Combination 21 is represented by SEQ ID NO: 68; Combination 22 is represented by SEQ ID NO: 69; Combination 23 is represented by SEQ ID NO: 70; Combination 24 is represented by SEQ ID NO: 71; Combination 24 is represented by SEQ ID NO: 72.

[0282] Example 5 - Alignment of PPO1 proteins from different plant species

[0283] To evaluate the applicability of the findings in soybean PPO1 to other plant crop species both sequence alignment (i.e., percent identity) and structural alignment (RMSD (A)) were performed comparing between 33 different plant crop species, with rapeseed and Camelina having 2 homologs each, including soybean PPO1 as a reference.

[0284] The purpose of the alignments was to evaluate the homology / similarity of different PPO1 enzymes, and particularly to evaluate the extent to which the novel positions (Table 1; bold underlined (originally green)) and the positions of the novel mutations / substitutions (Table 1 ; underlined italics (originally red)), found within the soybean PPO1 enzyme’s herbicide binding pocket are conserved and commonly shared between various plant crop species, thereby allowing extrapolation of the herein disclosed findings / conclusions from soybean to other crop species.

[0285] Table 3 The results of the sequence alignment and calculated identity are presented in FIGs. 3A-3D and Table 3, and the RMSD values of the structural alignment are presented in Table 3. The abbreviations for the plant names referenced in FIGs. 3A-3D can be found in Table 3.

[0286] Table 3 shows Sequence (identity (%)) and structural similarity (RMSD (A)) of PPO1 proteins. The sequence identity between each PPO protein from various organisms (SEQ ID NO: 11-47) and soybean PPO (SEQ ID NO: 2) was assessed using Clustal Omega with default settings to determine their level of conservation and variation.

[0287] Structural alignment / similarity was assessed using the “Align” tool in PyMOL (The PyMOL Molecular Graphics System, Version 2.0 Schrodinger, LLC). Structural similarity was assessed by calculating the root mean squared deviation (RMSD) between homologous residues of each protein. The RMSD value gives the average deviation between the corresponding atoms of two proteins and is expressed in angstroms(A). A smaller RMSD indicates a higher similarity between the two structures. A value of 0 means identical tertiary structure.

[0288] The comparative analysis depicted in FIGs. 3A-3D and Table 3, reveals a notable degree of sequence identity / conservation among the PPO1 CDS from various plant species, characterized by low variation, (between 72.4% and 93.19% sequence identity including the CTP which has lower identity, Table 3).

[0289] Table 3 shows that the PPO1 CDS from various plant species exhibit a notable degree of structural similarity and conservation, with minimal variability indicated by RMSD (A) values falling between 0.39 and 1.137 (SEQ ID NO: 14-47), in some embodiments. This is in stark contrast to the PPO1 CDS from Aspergillus, Myxococcus, and Human (SEQ ID NO: 11- 13), which display significantly higher RMSD (A) values, displaying greater structural differences ranging from 3.109 to 6.724, in some embodiments.

[0290] Table 4 presented below lists the corresponding amino acid position of the amino acids of the pocket rO-RF-SGVYA-V-FGQL-LGTI-Y-11, of the different organisms

[0291] Overall, these results indicate that any one of the genetic changes identified in GmPPOl enzyme in Arabidopsis model plants (presented, for example in Table 1 and / or in Table 2) can be introduced to any one of the 33 different PPO1 CDS of the crop plant presented in FIGs. 3A-3D, Table 3 and / or Table 4, for example utilizing gene-editing methods, to obtain a desired level of resistance / tolerance to that seen for the GmPPOl enzyme in Arabidopsis model plants, as presented above (FIGs. 2A-2B and Example 2).

[0292] Example 6 - Generation of an Herbicide Tolerance (HT) genetically modified gene- edited / transgenic crop plants.

[0293] The genetic change(s) identified in model plants (Table 1 and / or Table 2) exhibiting the desired level of resistance / tolerance are introduced into the PPO1 CDS of Glycine max crop plant utilizing gene-editing methods, to generate genetically edited / modified plants possessing Herbicide Tolerance (HT) / resistance.

[0294] The following exemplary crop plants were used: Soybean, Sunflower (Helianthus annuus), Wheat (Triticum aestivum), Barley (Hordeum vulgare), Pearl Milet (Pennisetum glaucum (L.) R. Br), Rice (Oryza sativa), Sorghum (Sorghum hicolor), Tomato (Solanum lycopersicum). Potato (Solanum tuberosum), Sugar beet (Beta vulgaris).

[0295] The identified genetic change(s) are introduced into the PPO CDS of any other crop plant that expresses a PPO enzyme having amino acid sequence at least 98% identity to the GmPPOl enzyme utilizing gene-editing methods, in order to generate genetically edited / modified crop plants possessing Herbicide Tolerance HT / resistance.

[0296] In addition, the genetic change(s) identified in model plants exhibiting the desired level of resistance / tolerance are introduced into a Glycine max crop plant or into any other crop plant by expressing the mutated PPO1 transgene utilizing transgenic engineering methods, in order to generate genetically edited / modified soybeans plants possessing Herbicide Tolerance (HT) / resistance.

[0297] Example 7 - Generation of a Herbicide Tolerance (HT) genetically modified gene- edited / transgenic crop plant(s)

[0298] The following crop plants were used: Soybean, Sunflower (Helianthus annuus), Wheat (Triticum aestivum), Barley (Hordeum vulgare), Pearl Milet (Pennisetum glaucum (L.) R. Br), Rice (Oryza saliva), Sorghum (Sorghum hicolor), Tomato (Solarium lycopersicum). Potato (Solarium tuberosum), Sugar beet (Beta vulgaris).

[0299] The corresponding PPO1 gene of the above species is cloned by extracting RNA and performing RT-PCR using specific primers. The CDS of the gene is cloned into pPA35H binary vector for expression in plants. The endogenous PPO gene (i.e., “reference PPO”), expressed in Arabidopsis, is used as a base line control for calibration of the plant screening using PPO inhibiting herbicides. The PCR fragment of the above reference gene is used as template for creating a mutagenized library, by methods known in the art, for example, using Error Prone PCR (EP -PCR).

[0300] Thus, the obtained PCR fragments are subjected to random mutagenesis using EP -PCR. The mutation rate is controlled using unbalanced DNTPs in the PCR mix. The mutation rate is monitored. The population of the gene’s DNA molecules carrying random point mutations is cloned into the binary vector pPA35H, generating a large pool of 105- 106clones representing mutated gene amplicons. Transformation into E.coli is performed and selection is facilitated using Kanamycin containing plates for the transformed E.coli, which is then used for large volume plasmids preparation (DNALib).

[0301] The DNALib is transformed into Agrobacterium and grown in LB with Kanamycin + Gentamycin + Rifampicin as selection markers of the transformed bacteria. Agrobacterium is harvested and resuspended in Arabidopsis thaliana (At) transformation buffer of 50gr sucrose +300pl Silwet L-77 per IL. Arabidopsis plants are transformed using a high-throughput transformation method (floral dip), creating in-planta CDS libraries each containing a collection of approximately 104-105transgenic pPA35S-mutated-gene plants, in which, on average each plant carries 1-10 genetically altered CDS element. Plants are grown to maturation and then harvested for the TO seeds. The seeds are seeded densely in trays and are applied with BASTA (glufosinate ammonium, is a non-selective herbicide), as a BAR gene selector to distinguish between the WT and the transformed seedlings. Seedlings that show resistance to Glufosinate ammonium are applied with a second selection of PPO herbicides (Group 14). The tolerant T1 plants are scored, documented and grown to seeds. The mutated PPO gene within the tolerant plants is sequenced and re-evaluated for their resistance / tolerance, for a variety of PPO herbicides (Group 14). The mutations are then used for gene editing in the corresponding species, to confer HT to these plants.

[0302] While certain embodiments of the invention have been illustrated and described, it will be clear that the invention is not limited to the embodiments described herein. Numerous 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 by the claims which follow.

Claims

CLAIMS:

1. A crop plant or a crop plant cell genetically modified to express a mutated plant Protoporphyrinogen Oxidase (PPO) peptide / protein having an amino acid sequence comprising a mutation(s) at a position(s) structurally corresponding to at least one amino acid position selected from positions 123, 151, 225, 371, 409, and 412 or any combination thereof, of a PPO enzyme having an amino acid sequence as set forth in SEQ ID NO: 2, wherein the mutated-PPO confers resistance or tolerance to at least one PPO-inhibitor herbicide compared to a non- genetically modified plant of the same genus.

2. The crop plant or crop plant cell of claim 1, wherein the mutated PPO is derived from a WT- plant PPO characterized by a structural pocket formed by amino acid residues Q-RF-SGVYA- V-FGQL-LGTI-Y-I, represented by SEQ ID NO: 73.

3. The crop plant or crop plant cell of claim 2, wherein the position of the amino acid residues Q-RF-SGVYA-V-FGQL-LGTI-Y-I comprises at least one position(s) structurally corresponding to any one of positions 123-150: 151-222:226-371-396:399-409:412-432-481 of a PPO enzyme having an amino acid sequence as set forth in SEQ ID NO: 2.

4. The crop plant or crop plant cell of any one of claims 1-3, wherein the mutated-plant Protoporphyrinogen Oxidase (PPO) comprises PPO transgenic to the crop plant and / or PPO endogenous to the crop plant.

5. The crop plant or crop plant cell of any one of claims 1-4, wherein the mutated-plant Protoporphyrinogen Oxidase (PPO) is selected from a mutated-plant Protoporphyrinogen Oxidase 1 (PPO1) and / or a mutated-Protoporphyrinogen Oxidase 2 (PPO2).

6. The crop plant or crop plant cell of any one of claims 1-5, wherein the PPO amino acid sequence comprises a mutation(s) at a position(s) structurally corresponding to at least one amino acid position selected from positions 123, 151, 225, and 371 of SEQ ID NO: 2, or any combination thereof.

7. The crop plant or crop plant cell of any one of claims 1-6, wherein the PPO amino acid sequence comprises a mutation(s) at a position(s) structurally corresponding to at least one amino acid position selected from positions 123 and 225 of SEQ ID NO: 2.

8. The crop plant or crop plant cell of any one of claims 1-7, wherein the PPO amino acid sequence comprises a mutation(s) at a position(s) structurally corresponding to amino acid position 225 of SEQ ID NO: 2.

9. The crop plant or crop plant cell of any one of claims 1-8, wherein the mutation(s) at position(s) structurally corresponding to the at least one amino acid position 123 in SEQ ID NO: 2, comprises at least one of a polar uncharged amino acid selected from: Serine (S), Threonine (T), Asparagine (N), Cysteine (C) or Selenocysteine (U), and Tyrosine (Y), or any combination thereof.

10. The crop plant or crop plant cell of any one of claims 1-9, wherein the mutation(s) at position(s) structurally corresponding to the at least one amino acid position 123 in SEQ ID NO: 2 comprises Serine (S).

11. The crop plant or crop plant cell of any one of claims 1-10, wherein the mutation(s) at position(s) structurally corresponding to the at least one amino acid position 151 in SEQ ID NO: 2, comprises at least one of a non-polar amino acid or hydrophobic side chain amino acid selected from: Alanine (A), Valine (V), Leucine (L), Isoleucine (I), Methionine (M), Proline (P), Tyrosine (Y) and Tryptophan (W), or any combination thereof.

12. The crop plant or crop plant cell of any one of claims 1-11, wherein the mutation(s) at position(s) structurally corresponding to the at least one amino acid position 151 in SEQ ID NO: 2 comprises Leucine (L).

13. The crop plant or crop plant cell of any one of claims 1-12, wherein the mutation(s) at position(s) structurally corresponding to the at least one amino acid position 225 in SEQ ID NO: 2, comprises at least one of a non-polar or positively charged amino acid selected from: Glycine (G), Alanine (A), Arginine (R), and Lysine (K), or any combination thereof.

14. The crop plant or crop plant cell of any one of claims 1-13, wherein the mutation(s) at position(s) structurally corresponding to the at least one amino acid position 371 in SEQ ID NO: 2, comprises at least one of a non-polar amino acid or hydrophobic side chain amino acid selected from: Glycine (G) and / or Alanine (A).

15. The crop plant or crop plant cell of any one of claims 1-14, wherein the mutation(s) at position(s) structurally corresponding to the at least one amino acid position 371 in SEQ ID NO: 2 comprises Alanine (A).

16. The crop plant or crop plant cell of any one of claims 1-15, wherein the mutation(s) at position(s) structurally corresponding to the at least one amino acid position selected from 409 and 412 in SEQ ID NO: 2 comprises at least one of a polar uncharged amino acid selected from: Threonine (T) and / or Asparagine (N).

17. The crop plant or crop plant cell of any one of claims 1-16, wherein the mutated-plant Protoporphyrinogen Oxidase (PPO) amino acid sequence further comprises at least one additional mutation(s) at a position(s) structurally corresponding to any amino acid position(s) in SEQ ID NO: 2.

18. The crop plant or crop plant cell of any one of claims 1-17, wherein the mutated-plant Protoporphyrinogen Oxidase (PPO) amino acid sequence comprises at least one double mutation at a combination of positions structurally corresponding to at least two amino acid positions in SEQ ID NO: 2 selected from positions: (1) 123 S and 15 IL; (2) 123 S and 225R; (3) 123S and 225G; (4) 123S and 225K; (5) 123S and 371A; (6) 123S and 409N; (7) 123S and 412T; (8) 151L and 225R; (9) 151L and 225G (10) 151L and 225K; (11) 151L and 371A; (12) 151L and 409N; (13) 151L and 412T; (14) (14) 225R and 371A; (15) 225R and 409N; (16) 225R and 412T; (17); (17) 225G and 371A; (18) 225G and 409N; (19) 225G and 412T; (20) 225K and 371 A; (21) 225K and 409N; (22) 225K and 412T; (23) 371 A and 409N; (24) 371 A and 412T; (25) 409N and 412T.

19. The crop plant or crop plant cell of any one of claims 1-18, wherein the mutated-plant Protoporphyrinogen Oxidase (PPO) amino acid sequence is derived from one or more WT- plant PPO enzyme(s) having an amino acids sequence selected from any one of SEQ ID NO: 2 and 14-47.

20. The crop plant or crop plant cell of any one of claims 1-19, wherein the mutated-plant Protoporphyrinogen Oxidase (PPO) comprises amino acid sequence having at least 98% sequence identity to the endogenous PPO enzyme of the crop plant.

21. The crop plant or crop plant cell of any one of claims 1-20, wherein the crop plant comprises at least one crop selected from the group consisting of maize, wheat, rice, barley, soybean,cowpea, chickpea, cotton, sorghum, beans, rapeseed / canola, alfalfa, flax, sunflower, safflower, millet, rye, sugarcane, sugar beet, cocoa, tea, Brassica napus, Brassica rapa, Brassica oleracea, coffee, sweet potato, flax, peanut, clover, lettuce, tomato, cucurbits, cassava, potato, carrot, radish, pea, lentils, cabbage, cauliflower, broccoli, brussels sprouts, peppers, pineapple, citrus, apples, pears, peaches, apricots, walnuts, avocado, banana, palm, eucalyptus, poplar, pine, coconut, orchids, petunia, carnations, roses, Poa species (bluegrasses), Festuca species (fescues), Lolium species (rye grass), Phalaris species (canary grasses), switchgrass, prairie grasses, Indian grasses, big bluestem grass, and camelina, or any combination thereof.

22. The crop plant or crop plant cell of claim 21, wherein the crop plant is selected from the group consisting of maize, wheat, rice, soybean, cowpea, chickpea, cotton, sorghum, rapeseed / canola, alfalfa, sunflower, sugarcane, sugar beet, camelina, Poa species (bluegrasses), Festuca species (fescues), Lolium species (rye grass), Phalaris species (canary grasses) or any combination thereof.

23. The crop plant or crop plant cell of claim 22, wherein the crop plant is a soybean plant.

24. The crop plant or crop plant cell of any one of claims 1-23, wherein the mutated-plant Protoporphyrinogen Oxidase (PPO) is a mutated-soybean PPO.

25. The crop plant or crop plant cell of claim 24, wherein the mutated-soybean PPO comprises at least one mutation selected from Q123S, F151L, Y225R, Y225G, Y225K, V371A, L409N, and I412T as set forth in any one of SEQ ID NOs: 3-10, respectively.

26. The crop plant or crop plant cell of any one of claims 24-25, wherein the mutated-soybean PPO comprises at least one double mutation at a combination of positions structurally corresponding to at least two amino acid positions in SEQ ID NO: 2 selected from any one of SEQ ID NOs 48-72.

27. The crop plant or crop plant cell of any one of claims 24-26, wherein the mutated-soybean Protoporphyrinogen Oxidase (PPO) comprises at least one double mutation at a combination of positions structurally corresponding to at least two amino acid positions in SEQ ID NO: 2 selected from any of SEQ ID NOs: 49-51, 55-57, and 61-69, or any combination thereof, and wherein the at least one double mutation comprises a position that corresponds to position 225 in SEQ ID NO: 2.

28. The crop plant or a crop plant cell of any one of claims 1-27, wherein the one or more PPO inhibitors is selected from the group consisting of herbicide families Phenylpyrazoles, Pyrimidinediones, Oxazolidinedione, Diphenylethers, Oxadiazoles, N-phenylphthalimides, Triazinone, Thiadiazoles, Triazolinones, and Triazolopyridinones, or any combination thereof.

29. The crop plant or a crop plant cell of claim 28, wherein the one or more PPO inhibitors is selected from the group consisting of herbicide families Diphenylethers, Oxadiazoles, and Triazolinones.

30. The crop plant or a crop plant cell of any one of claims 1-29, wherein the plant is a broadleaf plant.

31. The crop plant or a crop plant cell of any one of claims 1-30, wherein the plant is a cereal and / or grass type plant.

32. A seed of the genetically modified plant of any one of claims 1-31.

33. A product derived from the genetically modified crop plant or a crop plant cell of any one of claims 1-32.

34. A method of controlling weeds growth where crop plants are growing, using at least one PPO inhibitor herbicide, wherein the crop plants comprise the genetically modified plant according to any one of claims 1-33, the method comprises applying a weed-controlling amount of the one or more PPO inhibitor herbicide.

35. The method of claim 34, wherein the one or more PPO-inhibitor herbicides are selected from the group consisting of herbicides families of the groups Phenylpyrazoles, Pyrimidinediones, Oxazolidinedione, Diphenylethers, Oxadiazoles, N-phenylphthalimides, Triazinone, Thiadiazoles, Triazolinones, and Triazolopyridinones, or any combination thereof.

36. A method for conferring resistance or tolerance to one or more PPO inhibitor herbicide in a crop plant, the method comprising genetically modifying the plant to express a mutated plant Protoporphyrinogen Oxidase (PPO) enzyme, wherein the plant PPO enzyme have an amino acid sequence comprising mutation(s) at position(s) structurally corresponding to at least one amino acid position selected from positions 123, 151, 225, 371, 409, and 412 or anycombination thereof, of a PPO enzyme having an amino acid sequence as set forth in SEQ ID NO: 2.

37. The method of claim 36, wherein the mutated-plant Protoporphyrinogen Oxidase (PPO) comprises PPO transgenic to the crop plant and / or PPO endogenous to the crop plant.

38. The method of claims 36 or 37, wherein the mutated-plant Protoporphyrinogen Oxidase (PPO) amino acid sequence comprises a mutation(s) at a position(s) structurally corresponding to at least one amino acid position selected from positions 123, 151, 225, 371, or any combination thereof, of SEQ ID NO: 2.

39. The method of any one of claims 36-38, wherein the mutated-plant Protoporphyrinogen Oxidase (PPO) comprises at least one double mutation at a combination of positions structurally corresponding to at least two amino acid positions in SEQ ID NO: 2 selected from any of combinations 1-25 in Table 2, or any combination thereof.

40. The method of any one of claims 36-38, wherein the mutated-plant Protoporphyrinogen Oxidase (PPO) comprises amino acid sequence having at least 98% identity to the endogenous PPO enzyme of the crop plant.

41. The method of any one of claims 36-40, wherein the mutated-plant Protoporphyrinogen Oxidase (PPO) comprises a mutated- soybean PPO.

42. A crop plant or a crop plant cell genetically modified to express a soybean Protoporphyrinogen Oxidase (PPO) comprising mutation(s) in at least one position(s) selected from QI 23, F 151, Y225, V371, L409, and 1412, or any combination thereof, wherein the mutated-PPO confers resistance or tolerance to at least one PPO-inhibitor herbicide.

43. The crop plant or crop plant cell of claim 42, wherein the mutated-soybean Protoporphyrinogen Oxidase (PPO) comprises at least one mutation selected from Q123S, F151L, Y225R, Y225G, Y225K, V371A, L409N, and I412T, as set forth in any one of SEQ ID NOs: 3-10, and wherein the mutated-PPO confers resistance or tolerance to at least one PPO-inhibitor herbicide.

44. The crop plant or crop plant cell of claims 42 or 43, wherein the mutated-soybean Protoporphyrinogen Oxidase (PPO) comprises at least one mutation selected from Q123S, F151L, Y225R, Y225G, Y225K, and V371A as set forth in in any one of SEQ ID NOs: 3-8.

5. The crop plant or crop plant cell of any one of claims 42-44, wherein the mutated- soybean Protoporphyrinogen Oxidase (PPO) comprises at least one mutation selected from Y225R, Y225G, and Y225K, as set forth in any one of SEQ ID NOs: 5-7.

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