Methods and Compositions for PPO Herbicide Tolerance

Abstract

The present invention relates to biotechnology and provides novel recombinant DNA molecules and engineered proteins for conferring tolerance to protoporphyrinogen oxidase inhibitor herbicides. The invention also provides herbicide-tolerant transgenic plants, seeds, cells, and plant parts containing the recombinant DNA molecules, as well as methods of using the same.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of priority of U.S. Provisional Application No. 62 / 599,386, filed on Dec. 15, 2017, the disclosure of which is incorporated herein by reference in its entirety. Field of the Invention

[0003] The present invention relates to the fields of agriculture, plant biotechnology, and molecular biology. More specifically, the present invention relates to recombinant DNA molecules encoding engineered proteins that provide tolerance to herbicides that inhibit protoporphyrinogen oxidase, and methods of using the same.

[0004] Incorporation of Sequence Listing

[0005] The sequence listing in computer - readable form is submitted electronically together with this application and is incorporated herein by reference in its entirety. The sequence listing is contained in a file named MONS429WO_ST25.txt, which is 296 kilobytes in size (measured in the operating system MS Windows) and was created on Dec. 13, 2018. Background of the Invention

[0007] Crop production often utilizes transgenic traits formed using biotechnological methods. Heterologous genes (also referred to as transgenes) can be introduced into plants to produce transgenic traits. The expression of transgenes in plants confers traits such as herbicide tolerance to the plants. Examples of transgenic herbicide - tolerance traits include glyphosate tolerance, glufosinate tolerance, and dicamba tolerance. As the number of weed species resistant to commonly used herbicides increases, there is a need in the art for new herbicide - tolerance traits. Herbicides of particular interest include those that inhibit protoporphyrinogen oxidase (PPO, EC 1.3.3.4), referred to as PPO herbicides. PPO herbicides provide control of a range of herbicide - resistant weeds, making traits that confer tolerance to these herbicides particularly useful in agricultural systems incorporating one or more other herbicide - tolerance traits. The present invention provides novel engineered herbicide - tolerant protoporphyrinogen oxidases that can be used to provide PPO herbicide tolerance in plants. Summary of the Invention

[0008] In one aspect, the present invention provides a recombinant DNA molecule comprising a heterologous promoter operably linked to a nucleic acid molecule encoding a protein having protoporphyrinogen oxidase activity with herbicide tolerance, wherein the protein has at least about 50% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-23 and comprises at least one first amino acid substitution at positions corresponding to residues 125 to 146 of SEQ ID NO: 1, wherein the substitution is selected from the group consisting of: L125I, L125V, R126A, Y127W, P128A, P128D, P128E, P128K, P128L, P128Q, P128R, P128S, P128T, R129A, R129E, R129G, R129H, R129I, R129K, R129L, R129N, R129Q, R129S, Y130L, R131A, W132A, W132F, W132I, W132K, W132L, W132P, W132R, W132S, W132T, W132V, W132Y, I133A, D134A, D134N, D134Q, D134T, K135A, K135Q, K135R, K135S, K135T, K135V, V136A, M137A, M137C, M137I, M137L, M137S, M137V, I138L, I138M, I138V, Q139A, Q139C, Q139E, Q139G, Q139H, Q139K, Q139L, Q139M, Q139R, Q139S, L140A, L140C, L140F, L140G, L140H, L140I, L140M, L140N, L140Q, L140S, L140T, L140V, L140W, L140Y, I141V, M142L, M142S, M142V, R143A, M144A, T145A, G146A, G146D, G146H, G146K, and G146N.In certain embodiments, the protein has at least about 50% sequence identity, at least about 60% sequence identity, at least about 70% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least about 90% sequence identity, at least about 91% sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, and at least about 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1-23, and comprises at least one first amino acid substitution at positions corresponding to residues 125 to 146 of SEQ ID NO: 1, wherein the substitution is selected from the group consisting of: L125I, L125V, R126A, Y127W, P128A, P128D, P128E, P128K, P128L, P128Q, P128R, P128S, P128T, R129A, R129E, R129G, R129H, R129I, R129K, R129L, R129N, R129Q, R129S, Y130L, R131A, W132A, W132F, W132I, W132K, W132L, W132P, W132R, W132S, W132T, W132V, W132Y, I133A, D134A, D134N, D134Q, D134T, K135A, K135Q, K135R, K135S, K135T, K135V, V136A, M137A, M137C, M137I, M137L, M137S, M137V, I138L, I138M, I138V, Q139A, Q139C, Q139E, Q139G, Q139H, Q139K, Q139L, Q139M, Q139R, Q139S, L140A, L140C, L140F, L140G, L140H, L140I, L140M, L140N, L140Q, L140S, L140T, L140V, L140W, L140Y, I141V, M142L, M142S, M142V, R143A, M144A, T145A, G146A, G146D, G146H, G146K, and G146N. In some embodiments, the protein comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 of the amino acid substitutions. In another embodiment, the protein has at least about 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 24-124 and 249-263.In another embodiment, the protein comprises HemG class protoporphyrinogen oxidase. In another embodiment, at least one first amino acid substitution is located in the long-chain insertion loop of such HemG class protoporphyrinogen oxidase. In another embodiment, the recombinant DNA molecule of the present invention is incorporated into the genome of a plant cell.

[0009] In certain embodiments, a heterologous promoter (e.g., a promoter functional in a plant cell) is operably linked to a nucleic acid molecule encoding a protein having herbicide-tolerant protoporphyrinogen oxidase activity, wherein the protein has at least 50% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-23 and comprises at least one first amino acid substitution at positions corresponding to residues 125 to 146 of SEQ ID NO: 1, wherein the substitution is selected from the group consisting of: L125I, L125V, R126A, Y127W, P128A, P128D, P128E, P128K, P128L, P128Q, P128R, P128S, P128T, R129A, R129E, R129G, R129H, R129I, R129K, R129L, R129N, R129Q, R129S, Y130L, R131A, W132A, W132F, W132I, W132K, W132L, W132P, W132R, W132S, W132T, W132V, W132Y, I133A, D134A, D134N, D134Q, D134T, K135A, K135Q, K135R, K135S, K135T, K135V, V136A, M137A, M137C, M137I, M137L, M137S, M137V, I138L, I138M, I138V, Q139A, Q139C, Q139E, Q139G, Q139H, Q139K, Q139L, Q139M, Q139R, Q139S, L140A, L140C, L140F, L140G, L140H, L140I, L140M, L140N, L140Q, L140S, L140T, L140V, L140W, L140Y, I141V, M142L, M142S, M142V, R143A, M144A, T145A, G146A, G146D, G146H, G146K, and G146N. Such resulting DNA molecules may also comprise a transit sequence whose function is to localize the protein within the cell.

[0010] In another aspect, the present invention provides a DNA construct comprising a recombinant DNA molecule provided herein, such as a recombinant DNA molecule comprising a heterologous promoter operably linked to a nucleic acid molecule encoding a protein having protoporphyrinogen oxidase activity with herbicide tolerance, wherein the protein has at least 50% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1-23 and comprises at least one first amino acid substitution at positions corresponding to residues 125 to 146 of SEQ ID NO: 1, wherein the substitution is selected from the group consisting of: L125I, L125V, R126A, Y127W, P128A, P128D, P128E, P128K, P128L, P128Q, P128R, P128S, P128T, R129A, R129E, R129G, R129H, R129I, R129K, R129L, R129N, R129Q, R129S, Y130L, R131A, W132A, W132F, W132I, W132K, W132L, W132P, W132R, W132S, W132T, W132V, W132Y, I133A, D134A, D134N, D134Q, D134T, K135A, K135Q, K135R, K135S, K135T, K135V, V136A, M137A, M137C, M137I, M137L, M137S, M137V, I138L, I138M, I138V, Q139A, Q139C, Q139E, Q139G, Q139H, Q139K, Q139L, Q139M, Q139R, Q139S, L140A, L140C, L140F, L140G, L140H, L140I, L140M, L140N, L140Q, L140S, L140T, L140V, L140W, L140Y, I141V, M142L, M142S, M142V, R143A, M144A, T145A, G146A, G146D, G146H, G146K, and G146N. In another embodiment, the engineered protein is encoded by the recombinant DNA molecule provided herein.

[0011] In another aspect, the present invention provides a transgenic plant, seed, cell or plant part comprising a recombinant DNA molecule provided herein, such as a recombinant DNA molecule comprising a heterologous promoter operably linked to a nucleic acid molecule encoding a protein having protoporphyrinogen oxidase activity with herbicide tolerance, wherein the protein has at least 50% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1-23 and comprises at least one first amino acid substitution at positions corresponding to residues 125 to 146 of SEQ ID NO: 1, wherein the substitution is selected from the group consisting of: L125I, L125V, R126A, Y127W, P128A, P128D, P128E, P128K, P128L, P128Q, P128R, P128S, P128T, R129A, R129E, R129G, R129H, R129I, R129K, R129L, R129N, R129Q, R129S, Y130L, R131A, W132A, W132F, W132I, W132K, W132L, W132P, W132R, W132S, W132T, W132V, W132Y, I133A, D134A, D134N, D134Q, D134T, K135A, K135Q, K135R, K135S, K135T, K135V, V136A, M137A, M137C, M137I, M137L, M137S, M137V, I138L, I138M, I138V, Q139A, Q139C, Q139E, Q139G, Q139H, Q139K, Q139L, Q139M, Q139R, Q139S, L140A, L140C, L140F, L140G, L140H, L140I, L140M, L140N, L140Q, L140S, L140T, L140V, L140W, L140Y, I141V, M142L, M142S, M142V, R143A, M144A, T145A, G146A, G146D, G146H, G146K, and G146N. In one embodiment, the transgenic plant, seed, cell or plant part is tolerant to at least one PPO herbicide.In another embodiment, the PPO herbicides are selected from the group consisting of acifluorfen, fomesafen, lactofen, fluoroglycofen-ethyl, oxyfluorfen, flumioxazin, azafenidin, carfentrazone-ethyl, sulfentrazone, fluthiacet-methyl, oxadiargyl, oxadiazon, pyraflufen-ethyl, sulfentrazone, and S-3100. In another embodiment, the transgenic plant, seed, cell, or plant part is tolerant to at least one second herbicide.

[0012] In another aspect, the present invention provides a method for conferring PPO herbicide tolerance to a plant, seed, cell, or plant part, the method comprising: heterologously expressing the engineered protein of the present invention in the plant, seed, cell, or plant part. In some embodiments, the herbicide tolerance is to at least one PPO herbicide selected from the group consisting of acifluorfen, fomesafen, lactofen, fluoroglycofen-ethyl, oxyfluorfen, flumioxazin, azafenidin, carfentrazone-ethyl, sulfentrazone, fluthiacet-methyl, oxadiargyl, oxadiazon, pyraflufen-ethyl, sulfentrazone, and S-3100.

[0013] In another aspect, the present invention provides a method for generating a herbicide-tolerant plant, the method comprising the steps of: a) transforming a plant cell with a recombinant DNA molecule provided herein, such as a recombinant DNA molecule comprising a heterologous promoter operably linked to a nucleic acid molecule encoding a protein having herbicide-tolerant protoporphyrinogen oxidase activity, wherein the protein has at least 50% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1-23 and comprises at least one first amino acid substitution at positions corresponding to residues 125 to 146 of SEQ ID NO: 1, wherein the substitution is selected from the group consisting of: L125I, L125V, R126A, Y127W, P128A, P128D, P128E, P128K, P128L, P128Q, P128R, P128S, P128T, R129A, R129E, R129G, R129H, R129I, R129K, R129L, R129N, R129Q, R129S, Y130L, R131A, W132A, W132F, W132I, W132K, W132L, W132P, W132R, W132S, W132T, W132V, W132Y, I133A, D134A, D134N, D134Q, D134T, K135A, K135Q, K135R, K135S, K135T, K135V, V136A, M137A, M137C, M137I, M137L, M137S, M137V, I138L, I138M, I138V, Q139A, Q139C, Q139E, Q139G, Q139H, Q139K, Q139L, Q139M, Q139R, Q139S, L140A, L140C, L140F, L140G, L140H, L140I, L140M, L140N, L140Q, L140S, L140T, L140V, L140W, L140Y, I141V, M142L, M142S, M142V, R143A, M144A, T145A, G146A, G146D, G146H, G146K, and G146N; and b) regenerating a plant from the plant cell comprising the recombinant DNA molecule. In one embodiment, the method further comprises the step of selecting the plant or its progeny for PPO herbicide tolerance. In another embodiment, the method further comprises the step of crossing the regenerated plant with itself or with a second plant to produce progeny.

[0014] In another aspect, the present invention provides a method for controlling or preventing weed growth in a plant growth area, the method comprising applying an effective amount of at least one PPO herbicide to a plant growth area comprising a transgenic plant or seed as provided herein, such as a transgenic plant or seed comprising a recombinant DNA molecule comprising a heterologous promoter operably linked to a nucleic acid molecule encoding a protein having herbicide-tolerant protoporphyrinogen oxidase activity, wherein the protein has at least 50% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1-23 and comprises at least one first amino acid substitution at positions corresponding to residues 125 to 146 of SEQ ID NO: 1, wherein the substitution is selected from the group consisting of: L125I, L125V, R126A, Y127W, P128A, P128D, P128E, P128K, P128L, P128Q, P128R, P128S, P128T, R129A, R129E, R129G, R129H, R129I, R129K, R129L, R129N, R129Q, R129S, Y130L, R131A, W132A, W132F, W132I, W132K, W132L, W132P, W132R, W132S, W132T, W132V, W132Y, I133A, D134A, D134N, D134Q, D134T, K135A, K135Q, K135R, K135S, K135T, K135V, V136A, M137A, M137C, M137I, M137L, M137S, M137V, I138L, I138M, I138V, Q139A, Q139C, Q139E, Q139G, Q139H, Q139K, Q139L, Q139M, Q139R, Q139S, L140A, L140C, L140F, L140G, L140H, L140I, L140M, L140N, L140Q, L140S, L140T, L140V, L140W, L140Y, I141V, M142L, M142S, M142V, R143A, M144A, T145A, G146A, G146D, G146H, G146K and G146N, wherein the transgenic plant or seed is tolerant to the PPO herbicide. In certain embodiments, the PPO herbicide is selected from the group consisting of: acifluorfen, fomesafen, lactofen, fluoroglycofen-ethyl, oxyfluorfen, flumioxazin, pyraflufen-ethyl, carfentrazone-ethyl, sulfentrazone, fluthiacet-methyl, oxadiazon, pyraclonil, saflufenacil, and S-3100.

[0015] In another aspect, the present invention provides a method for identifying a nucleotide sequence encoding a protein having herbicide-tolerant protoporphyrinogen oxidase activity, the method comprising a) transforming an Escherichia coli (E. coli) strain lacking herbicide-tolerant PPO enzyme activity with a bacterial expression vector comprising a recombinant DNA molecule provided herein, such as a recombinant DNA molecule comprising a heterologous promoter operably linked to a nucleic acid molecule encoding a protein having herbicide-tolerant protoporphyrinogen oxidase activity, wherein the protein has at least 50% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-23 and comprises at least one first amino acid substitution at positions corresponding to residues 125 to 146 of SEQ ID NO: 1, wherein the substitution is selected from the group consisting of: L125I, L125V, R126A, Y127W, P128A, P128D, P128E, P128K, P128L, P128Q, P128R, P128S, P128T, R129A, R129E, R129G, R129H, R129I, R129K, R129L, R129N, R129Q, R129S, Y130L, R131A, W132A, W132F, W132I, W132K, W132L, W132P, W132R, W132S, W132T, W132V, W132Y, I133A, D134A, D134N, D134Q, D134T, K135A, K135Q, K135R, K135S, K135T, K135V, V136A, M137A, M137C, M137I, M137L, M137S, M137V, I138L, I138M, I138V, Q139A, Q139C, Q139E, Q139G, Q139H, Q139K, Q139L, Q139M, Q139R, Q139S, L140A, L140C, L140F, L140G, L140H, L140I, L140M, L140N, L140Q, L140S, L140T, L140V, L140W, L140Y, I141V, M142L, M142S, M142V, R143A, M144A, T145A, G146A, G146D, G146H, G146K, and G146N; and b) growing the transformed E. coli to identify a protein having herbicide-tolerant protoporphyrinogen oxidase activity.

[0016] In another aspect, the present invention provides a method for screening for herbicide tolerance genes, the method comprising a) expressing in a plant cell a recombinant DNA molecule provided herein, such as a recombinant DNA molecule comprising a heterologous promoter operably linked to a nucleic acid molecule encoding a protein having protoporphyrinogen oxidase activity with herbicide tolerance, wherein the protein has at least 50% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-23 and comprises at least one first amino acid substitution at positions corresponding to residues 125 to 146 of SEQ ID NO: 1, wherein the substitution is selected from the group consisting of: L125I, L125V, R126A, Y127W, P128A, P128D, P128E, P128K, P128L, P128Q, P128R, P128S, P128T, R129A, R129E, R129G, R129H, R129I, R129K, R129L, R129N, R129Q, R129S, Y130L, R131A, W132A, W132F, W132I, W132K, W132L, W132P, W132R, W132S, W132T, W132V, W132Y, I133A, D134A, D134N, D134Q, D134T, K135A, K135Q, K135R, K135S, K135T, K135V, V136A, M137A, M137C, M137I, M137L, M137S, M137V, I138L, I138M, I138V, Q139A, Q139C, Q139E, Q139G, Q139H, Q139K, Q139L, Q139M, Q139R, Q139S, L140A, L140C, L140F, L140G, L140H, L140I, L140M, L140N, L140Q, L140S, L140T, L140V, L140W, L140Y, I141V, M142L, M142S, M142V, R143A, M144A, T145A, G146A, G146D, G146H, G146K, and G146N; and b) identifying a plant cell that exhibits tolerance to a PPO herbicide.

[0017] In another aspect, the present invention provides a method for generating a plant that is tolerant to a PPO herbicide and at least one other herbicide, the method comprising a) obtaining a transgenic plant that comprises a recombinant DNA molecule provided herein, such as a recombinant DNA molecule comprising a heterologous promoter operably linked to a nucleic acid molecule encoding a protein having herbicide-tolerant protoporphyrinogen oxidase activity, wherein the protein has at least 50% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-23 and comprises at least one first amino acid substitution at positions corresponding to residues 125 to 146 of SEQ ID NO: 1, wherein the substitution is selected from the group consisting of: L125I, L125V, R126A, Y127W, P128A, P128D, P128E, P128K, P128L, P128Q, P128R, P128S, P128T, R129A, R129E, R129G, R129H, R129I, R129K, R129L, R129N, R129Q, R129S, Y130L, R131A, W132A, W132F, W132I, W132K, W132L, W132P, W132R, W132S, W132T, W132V, W132Y, I133A, D134A, D134N, D134Q, D134T, K135A, K135Q, K135R, K135S, K135T, K135V, V136A, M137A, M137C, M137I, M137L, M137S, M137V, I138L, I138M, I138V, Q139A, Q139C, Q139E, Q139G, Q139H, Q139K, Q139L, Q139M, Q139R, Q139S, L140A, L140C, L140F, L140G, L140H, L140I, L140M, L140N, L140Q, L140S, L140T, L140V, L140W, L140Y, I141V, M142L, M142S, M142V, R143A, M144A, T145A, G146A, G146D, G146H, G146K, and G146N; b) crossing the plant with a second plant that comprises tolerance to at least one other herbicide, and c) selecting progeny plants resulting from the crossing that comprise tolerance to the PPO herbicide and at least one other herbicide.

[0018] In another aspect, the present invention provides a method for reducing the development of herbicide-tolerant weeds, the method comprising a) growing a transgenic plant in a crop growth environment, the transgenic plant comprising a recombinant DNA molecule provided herein, such as a recombinant DNA molecule comprising a heterologous promoter operably linked to a nucleic acid molecule encoding a protein having herbicide-tolerant protoporphyrinogen oxidase activity, wherein the protein has at least 50% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-23 and comprises at least one first amino acid substitution at positions corresponding to residues 125 to 146 of SEQ ID NO: 1, wherein the substitution is selected from the group consisting of: L125I, L125V, R126A, Y127W, P128A, P128D, P128E, P128K, P128L, P128Q, P128R, P128S, P128T, R129A, R129E, R129G, R129H, R129I, R129K, R129L, R129N, R129Q, R129S, Y130L, R131A, W132A, W132F, W132I, W132K, W132L, W132P, W132R, W132S, W132T, W132V, W132Y, I133A, D134A, D134N, D134Q, D134T, K135A, K135Q, K135R, K135S, K135T, K135V, V136A, M137A, M137C, M137I, M137L, M137S, M137V, I138L, I138M, I138V, Q139A, Q139C, Q139E, Q139G, Q139H, Q139K, Q139L, Q139M, Q139R, Q139S, L140A, L140C, L140F, L140G, L140H, L140I, L140M, L140N, L140Q, L140S, L140T, L140V, L140W, L140Y, I141V, M142L, M142S, M142V, R143A, M144A, T145A, G146A, G146D, G146H, G146K, and G146N; and b) applying a PPO herbicide and at least one other herbicide to the crop growth environment, wherein the crop plant is tolerant to the PPO herbicide and at least one other herbicide. In one embodiment, the PPO herbicide is selected from the group consisting of: acifluorfen, fomesafen, lactofen, oxyfluorfen, ethoxyfen, flumioxazin, sulfentrazone, carfentrazone-ethyl, mesotrione, fluthiacet-methyl, oxadiargyl, oxadiazon, pyraflufen-ethyl, saflufenacil, and S-3100.In another embodiment, the at least one other herbicide is selected from the group consisting of: ACC enzyme inhibitors, ALS inhibitors, EPSPS inhibitors, synthetic auxins, photosynthesis inhibitors, glutamine synthetase inhibitors, HPPD inhibitors, PPO inhibitors, and long-chain fatty acid inhibitors. In another embodiment, the ACC enzyme inhibitor is an aryloxyphenoxypropionate or cyclohexanedione; the ALS inhibitor is a sulfonylurea, imidazolinone, triazolopyrimidine, or triazolinone; the EPSPS inhibitor is glyphosate; the synthetic auxin is a phenoxy herbicide, benzoic acid, carboxylic acid, or semicarbazone; the photosynthesis inhibitor is a triazine, triazinone, nitrile, benzothiadiazole, or urea; the glutamine synthetase inhibitor is glufosinate; the HPPD inhibitor is an isoxazole, pyrazolone, or triketone; the PPO inhibitor is a diphenyl ether, N-phenylphthalimide, aryltriazinone, or pyrimidinedione; or the long-chain fatty acid inhibitor is a chloroacetamide, oxyacetamide, or pyrazole. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] This patent or application file contains at least one drawing presented in color. Copies of this patent or patent application publication with one or more color drawings will be provided by the Patent Office upon request and payment of the necessary fees.

[0020] Figure 1A and Figure 1B: Sequence alignment of the long insert loops of a subset of 23 microbial HemG PPO enzymes is shown, with conserved long insert loops highlighted in black. Sequences are arranged in descending overall sequence identity relative to H_N90 (SEQ ID NO:1). HemG001 (SEQ ID NO:11) and HemG003 (SEQ ID NO:13) represent HemG PPO proteins with more than 70% overall sequence identity to H_N90. The next 15 unframed sequences, which are H_N30 (SEQ ID NO:4), H_N40 (SEQ ID NO:5), H_N60 (SEQ ID NO:7), H_N20 (SEQ ID NO:3), H_N70 (SEQ ID NO:8), HemG005 (SEQ ID NO:15), H_N100 (SEQ ID NO:9), HemG002 (SEQ ID NO:12), HemG004 (SEQ ID NO:14), H_N110 (SEQ ID NO:10), H_N10 (SEQ ID NO:2), HemG006 (SEQ ID NO:16), HemG007 (SEQ ID NO:17), H_N50 (SEQ ID NO:6), and HemG013 (SEQ ID NO:23), represent HemG PPO proteins with 50 - 70% overall sequence identity to H_N90. The last 5 framed sequences, which are HemG008 (SEQ ID NO:18), HemG009 (SEQ ID NO:19), HemG011 (SEQ ID NO:21), HemG012 (SEQ ID NO:22), and HemG010 (SEQ ID NO:20), represent HemG PPO proteins with 40 - 50% overall sequence identity to H_N90.

[0021] Figure 2 : A universal genetic code chart is shown that displays all possible mRNA triplet codons (where T in the DNA molecule is replaced by U in the RNA molecule) and the amino acids encoded by each codon.

[0022] Figure 3: A graphical representation showing all the changes found at each residue in the long-chain insertion loop of H_N90 (SEQ ID NO:1) from microbial genome screening. The black box at the top represents the native H_N90 sequence. The boxes below the H_N90 sequence list each of the 20 amino acids. The numbers listed above the H_N90 sequence indicate the relative amino acid positions. Solid grey shading represents amino acid changes identified in the ≥50% sequence identity group. Vertical grey stripe shading represents amino acid changes identified in the 40%-50% sequence identity group. The remaining white unfilled boxes represent amino acid changes not observed in the starting microbial dataset at ≥40% overall sequence identity.

[0023] Figure 4 : A graphical representation showing the results obtained from enzyme function analysis. The black box at the top represents the native H_N90 sequence (SEQ ID NO:1). The boxes below the H_N90 sequence list each of the 20 amino acids. The numbers listed above the H_N90 sequence are the relative amino acid positions. Vertical grey stripe shading indicates amino acid modifications that render the enzyme non-functional. Light grey shading with black letters indicates amino acid modifications that impair enzyme function. Dark grey shading with white letters indicates amino acid changes that keep the enzyme fully functional. Black shading represents the native amino acids in the H_N90 sequence. The remaining white unfilled boxes represent amino acid changes not detected in this analysis.

[0024] Figure 5 : A graphical version showing the results obtained from herbicide tolerance analysis. Tolerance is measured relative to H_N90 tolerance. The black box at the top represents the native H_N90 sequence (SEQ ID NO:1). The boxes below the H_N90 sequence list each of the 20 amino acids. The numbers listed above the H_N90 sequence are the relative amino acid positions. Vertical grey stripe shading represents relative tolerance scores of 0 - 24, indicating that amino acid modifications confer little herbicide tolerance. Vertical grey stripe shading represents relative tolerance scores of 25 - 49, indicating that amino acid modifications confer weak herbicide tolerance. Solid light grey shading with black letters represents relative tolerance scores of 50 - 74, indicating that amino acid modifications confer moderate herbicide tolerance. Solid dark grey shading with white letters represents relative tolerance scores of 75 - 100, indicating that amino acid modifications confer good herbicide tolerance. Boxes with dark grey shading and thick black borders represent amino acid modifications showing relative tolerance scores greater than 100, indicating that amino acid modifications confer better herbicide tolerance than H_N90. Black shading represents the native amino acids in the H_N90 sequence. The remaining white unfilled boxes represent amino acid changes not detected in this analysis.

[0025] Brief description of the sequence

[0026] SEQ ID NO:1 is the amino acid sequence of H_N90.

[0027] SEQ ID NOs: 2 to 10 are the amino acid sequences of the microbial HemG PPO enzymes with conserved long-chain insertion loops.

[0028] SEQ ID NOs: 11 to 23 are the amino acid sequences of various HemG PPO enzymes with variable long-chain insertion loops.

[0029] SEQ ID NOs: 24 to 124 and SEQ ID NOs: 249 to 263 are the amino acid sequences of 116 recombinant HemG PPO variants each incorporating mutations into the long-chain insertion loop.

[0030] SEQ ID NO: 125 is the DNA sequence encoding SEQ ID NO: 1.

[0031] SEQ ID NOs: 126 to 147 are the DNA sequences encoding SEQ ID NOs: 2 to 23, respectively.

[0032] SEQ ID NOs: 148 to 248 and SEQ ID NOs: 264 to 278 are the DNA sequences encoding SEQ ID NOs: 24 to 124 and SEQ ID NOs: 249 to 263, respectively. Detailed implementation manners

[0033] The following descriptions and definitions are provided to better define the present invention and guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise specified, terms should be understood according to the conventional usage of those of ordinary skill in the relevant art.

[0034] Protoporphyrinogen oxidase functions in the biosynthetic pathways of chlorophyll and heme, where protoporphyrinogen oxidase converts protoporphyrinogen IX to protoporphyrin IX. Herbicide-tolerant protoporphyrinogen oxidase can be used to generate cells, plants, and seeds that are sensitive to the application of one or more PPO herbicides and can be used in agricultural and weed control methods. The present invention provides novel engineered proteins that are herbicide-tolerant protoporphyrinogen oxidase, recombinant DNA molecules encoding these novel engineered proteins, compositions comprising these novel engineered proteins, and methods of using these novel engineered proteins. For example, in one embodiment, the present invention provides a DNA construct for expression in cells, plants, and seeds, the DNA construct comprising a recombinant DNA molecule encoding an engineered herbicide-tolerant protoporphyrinogen oxidase. In another embodiment, the present invention provides an engineered protein having herbicide-tolerant protoporphyrinogen oxidase activity. In another embodiment, the present invention provides methods and compositions for obtaining and improving herbicide-tolerant protoporphyrinogen oxidase using protein engineering and bioinformatics tools. The present invention also provides methods and compositions for generating cells, plants, and seeds that are tolerant to PPO herbicides, and weed control methods using the cells, plants, and seeds.

[0035] The present invention provides novel engineered proteins and recombinant DNA molecules encoding them. As used herein, the term "engineered" refers to non-natural DNA, proteins, cells, or organisms that do not normally exist in nature but are formed through human intervention. "Engineered protein", "engineered enzyme", or "engineered PPO" refers to a protein, enzyme, or PPO whose amino acid sequence is conceived and formed in the laboratory using one or more techniques in biotechnology, protein design, or protein engineering, such as molecular biology, protein biochemistry, bacterial transformation, plant transformation, site-directed mutagenesis, use of random mutagenesis, genome editing, gene editing, gene cloning, DNA ligation, DNA synthesis, protein synthesis, and DNA shuffling for directed evolution. For example, an engineered protein may have one or more deletions, insertions, or substitutions relative to the coding sequence of a wild-type protein, and each deletion, insertion, or substitution may consist of one or more amino acids. Genetic engineering can be used to form a DNA molecule encoding an engineered protein (e.g., engineered PPO) that is herbicide-tolerant and comprises at least one first amino acid substitution relative to a wild-type PPO protein as described herein.

[0036] Examples of engineered proteins provided herein are herbicide-tolerant PPOs comprising one or more amino acid substitutions selected from: L125I, L125V, R126A, Y127W, P128A, P128D, P128E, P128K, P128L, P128Q, P128R, P128S, P128T, R129A, R129E, R129G, R129H, R129I, R129K, R129L, R129N, R129Q, R129S, Y130L, R131A, W132A, W132F, W132I, W132K, W132L, W132P, W132R, W132S, W132T, W132V, W132Y, I133A, D134A, D134N, D134Q, D134T, K135A, K135Q, K135R, K135S, K135T, K135V, V136A, M137A, M137C, M137I, M137L, M137S, M137V, I138L, I138M, I138V, Q139A, Q139C, Q139E, Q139G, Q139H, Q139K, Q139L, Q139M, Q139R, Q139S, L140A, L140C, L140F, L140G, L140H, L140I, L140M, L140N, L140Q, L140S, L140T, L140V, L140W, L140Y, I141V, M142L, M142S, M142V, R143A, M144A, T145A, G146A, G146D, G146H, G146K, and G146N, including all possible combinations thereof, wherein the position of the one or more amino acid substitutions is relative to the amino acid positions shown in SEQ ID NO:1. In certain embodiments, the engineered proteins provided herein comprise one, two, three, four, five, six, seven, eight, nine, ten, or more of any combination of such substitutions.

[0037] In one embodiment, the engineered protein provided by the present invention has herbicide-tolerant protoporphyrinogen oxidase activity. As used herein, "herbicide-tolerant protoporphyrinogen oxidase" means the ability of protoporphyrinogen oxidase to maintain at least some of its protoporphyrinogen oxidase activity in the presence of one or more PPO herbicides. The term "protoporphyrinogen oxidase activity" means the ability to catalyze the six-electron oxidation (removal of electrons) of protoporphyrinogen IX to form protoporphyrin IX, i.e., the ability to catalyze the dehydrogenation of protoporphyrinogen to form protoporphyrin. The enzymatic activity of protoporphyrinogen oxidase can be measured by any means known in the art, such as by an enzyme assay, wherein the production of the product of protoporphyrinogen oxidase or the consumption of the substrate of protoporphyrinogen oxidase in the presence of one or more PPO herbicides is measured via fluorescence, high performance liquid chromatography (HPLC), or mass spectrometry (MS). Another example of an assay for measuring the enzymatic activity of protoporphyrinogen oxidase is a bacterial assay, such as the assay described herein, whereby recombinant protoporphyrinogen oxidase is expressed in bacterial cells that are otherwise lacking in PPO activity, and the ability of the recombinant protoporphyrinogen oxidase to complement this knockout phenotype is measured. As used herein, a "hemG knockout strain" means an organism (such as Escherichia coli) or a somatic cell of an organism that lacks HemG activity such that it cannot grow on a growth medium lacking heme, or such that growth in the absence of heme is detectably impaired relative to an otherwise isogenic strain that contains functional HemG. A hemG knockout strain of Escherichia coli, for example, can be prepared according to knowledge in the art, such as according to the Escherichia coli HemGPPO sequence (Ecogene accession number EG11485; Sasarman et al., "Nucleotide sequence of the hemG gene involved in the protoporphyrinogen oxidase activity of E. coli K12" Can J Microbiol 39:1155-1161, 1993).

[0038] Engineered proteins can be produced by altering or modifying a wild-type protein sequence to produce a new protein that has one or more improved properties or such as altered V 最大 、K m 、K i 、IC 50, a novel combination of useful protein properties such as substrate specificity, inhibitor / herbicide specificity, substrate selectivity, the ability to interact with other components in the cell (such as chaperone proteins or membranes), and protein stability. The modification can be made at specific amino acid positions in the protein and can be carried out by replacing the typical amino acid found at the same position in nature (i.e., in the wild-type protein) with an alternative amino acid. Amino acid modification can be achieved in the form of a single amino acid substitution in the protein sequence or in combination with one or more other modifications (such as one or more other amino acid substitutions, deletions, or additions). In one embodiment of the invention, the engineered protein has altered protein properties (such as those that result in reduced sensitivity to one or more herbicides) compared to the wild-type protein, or has the ability to confer tolerance to one or more herbicides on a transgenic plant expressing the engineered protein.Thus, in one embodiment, the present invention provides an engineered protein (such as a PPO enzyme) having herbicide-tolerant protoporphyrinogen oxidase activity and a recombinant DNA molecule encoding the same, wherein the engineered protein has one or more amino acid substitutions selected from the group consisting of: L125I, L125V, R126A, Y127W, P128A, P128D, P128E, P128K, P128L, P128Q, P128R, P128S, P128T, R129A, R129E, R129G, R129H, R129I, R129K, R129L, R129N, R129Q, R129S, Y130L, R131A, W132A, W132F, W132I, W132K, W132L, W132P, W132R, W132S, W132T, W132V, W132Y, I133A, D134A, D134N, D134Q, D134T, K135A, K135Q, K135R, K135S, K135T, K135V, V136A, M137A, M137C, M137I, M137L, M137S, M137V, I138L, I138M, I138V, Q139A, Q139C, Q139E, Q139G, Q139H, Q139K, Q139L, Q139M, Q139R, Q139S, L140A, L140C, L140F, L140G, L140H, L140I, L140M, L140N, L140Q, L140S, L140T, L140V, L140W, L140Y, I141V, M142L, M142S, M142V, R143A, M144A, T145A, G146A, G146D, G146H, G146K, and G146N and all combinations thereof, wherein the position of one or more amino acid substitutions is relative to the amino acid positions shown in SEQ ID NO:1. In certain embodiments, the engineered proteins provided herein comprise one, two, three, four, five, six, seven, eight, nine, ten, or more of any combination of such substitutions, wherein the modification is made at a position that is functionally similar to the position of the amino acids provided in SEQ ID NO:1. The amino acid sequences of recombinant or engineered HemG variant PPOs are provided in Table 1.

[0039] Table 1. Amino acid sequences of recombinant or engineered HemG variant PPOs.

[0040]

[0041]

[0042]

[0043] Similar modifications can be made at analogous positions in any PPO enzyme by aligning the amino acid sequence of the PPO enzyme to be mutated with the amino acid sequence of a PPO enzyme having protoporphyrinogen oxidase activity conferring herbicide tolerance. An example of a sequence encoding a PPO enzyme having protoporphyrinogen oxidase activity conferring herbicide tolerance is SEQ ID NO:1. Figure 1A and Figure 1B shows an alignment of H_N90, the PPO enzyme of SEQ ID NO:1, exemplary known PPO enzymes (SEQ ID NOs:2 - 10), and a variety of PPO enzymes (SEQ ID NOs:11 - 23). Making the amino acid modifications described herein in the proteins of SEQ ID NOs:2 - 23, for example, using the sequence identity information as shown in Figure 1A and Figure 1B to produce a PPO enzyme having protoporphyrinogen oxidase activity conferring herbicide tolerance is well within the capabilities of those skilled in the art. The amino acid sequence of the microbial HemG PPO is provided in Table 2.

[0044] Table 2. Amino acid sequence of the microbial HemG PPO.

[0045]

[0046]

[0047] As used herein, the term "recombinant" refers to non-naturally occurring DNA, protein, cell, seed, or organism that results from genetic engineering and is formed through human intervention. A "recombinant DNA molecule" is a DNA molecule that contains a DNA sequence that is non-naturally occurring and thus the result of human intervention, such as a DNA molecule that contains at least two DNA molecules that are heterologous to each other. An example of a recombinant DNA molecule is the DNA molecule provided herein encoding a herbicide-tolerant protoporphyrinogen oxidase that is operably linked to a heterologous promoter. A "recombinant protein" is a protein that contains an amino acid sequence that is non-naturally occurring and thus the result of human intervention, such as an engineered protein. A recombinant cell, seed, or organism is a cell, seed, or organism that contains a transgene or heterologous DNA or protein, such as a transgenic plant cell, seed, or plant that contains a DNA construct or engineered protein of the present invention.

[0048] As used herein, "wild-type" means naturally occurring. A "wild-type DNA molecule" or a "wild-type protein" is a naturally occurring form of a DNA molecule or a protein, i.e., a form of a DNA molecule or a protein that pre-exists in nature. The wild-type form of a DNA molecule or a protein can be used for comparison with a recombinant or engineered DNA molecule or protein. An example of a wild-type protein that can be used for comparison with an engineered protein provided by the present invention is the PPO enzyme (H_N90) from Enterobacter cloacae as provided in SEQ ID NO:1.

[0049] A "wild-type plant" is a naturally occurring plant. Such wild-type plants can also be used for comparison with plants containing recombinant or engineered DNA molecules or proteins. An example of a wild-type plant that can be used for comparison with a plant containing a recombinant or engineered DNA molecule or protein can be a plant of the same type as a plant containing an engineered DNA molecule or protein (such as a protein conferring a herbicide tolerance trait), and thus is genetically different from a plant containing a herbicide tolerance trait.

[0050] In certain embodiments, a wild-type plant can also be used as or referred to as a "control plant". As used herein, "control" means an experimental control designed for comparison purposes. For example, a control plant in transgenic plant analysis is a plant of the same type as the experimental plant (i.e., the plant to be tested), but does not contain the transgenic insert, recombinant DNA molecule or DNA construct of the experimental plant. Examples of control plants that can be used for comparison with transgenic plants include: for maize plants, non-transgenic LH244 maize (ATCC deposit number PTA-1173); for comparison with soybean plants: non-transgenic A3555 soybean (ATCC deposit number PTA-10207); for comparison with cotton plants: non-transgenic Coker 130 (Plant Variety Protection (PVP) number 8900252); for comparison with canola or Brassica napus plants: non-transgenic Brassica napus variety 65037 restorer line (Canadian Plant Breeders' Rights Application 06-5517); for comparison with wheat plants: non-transgenic wheat variety Samson germplasm (PVP 1994).

[0051] As used herein, the term "DNA" or "DNA molecule" refers to a double-stranded DNA molecule of genomic or synthetic origin read from the 5′ (upstream) end to the 3′ (downstream) end (i.e., a polymer or polynucleotide molecule of deoxyribonucleotide bases). As used herein, the term "DNA sequence" refers to the nucleotide sequence of a DNA molecule. The nomenclature used herein corresponds to Title 37 of the Code of Federal Regulations §1.822 and is set forth in Tables 1 and 3 of Appendix 2 of WIPO Standard ST.25 (1998).

[0052] The present disclosure provides a nucleic acid molecule encoding a protein having protoporphyrinogen oxidase activity with one or more amino acid substitutions selected from the group consisting of: L125I, L125V, R126A, Y127W, P128A, P128D, P128E, P128K, P128L, P128Q, P128R, P128S, P128T, R129A, R129E, R129G, R129H, R129I, R129K, R129L, R129N, R129Q, R129S, Y130L, R131A, W132A, W132F, W132I, W132K, W132L, W132P, W132R, W132S, W132T, W132V, W132Y, I133A, D134A, D134N, D134Q, D134T, K135A, K135Q, K135R, K135S, K135T, K135V, V136A, M137A, M137C, M137I, M137L, M137S, M137V, I138L, I138M, I138V, Q139A, Q139C, Q139E, Q139G, Q139H, Q139K, Q139L, Q139M, Q139R, Q139S, L140A, L140C, L140F, L140G, L140H, L140I, L140M, L140N, L140Q, L140S, L140T, L140V, L140W, L140Y, I141V, M142L, M142S, M142V, R143A, M144A, T145A, G146A, G146D, G146H, G146K, and G146N and all combinations thereof, wherein the position of the one or more amino acid substitutions is relative to the amino acid positions shown in SEQ ID NO:1.

[0053] As used herein, the term "DNA molecule encoding a protein" refers to a DNA molecule comprising a DNA sequence encoding a protein. As used herein, the term "protein" refers to a chain of amino acids linked by peptide (amide) bonds and includes both polypeptide chains folded or arranged in a biologically functional manner and polypeptide chains not folded or arranged in a biologically functional manner. As used herein, "protein coding sequence" means a DNA sequence encoding a protein. As used herein, "sequence" means an ordered arrangement of nucleotides or amino acids. A "DNA sequence" may refer to a nucleotide sequence or a DNA molecule containing a nucleotide sequence; a "protein sequence" may refer to an amino acid sequence or a protein containing an amino acid sequence. The boundaries of a protein coding sequence are generally determined by a translation initiation codon at the 5' end and a translation termination codon at the 3' end.

[0054] As used herein, the term "isolated" refers to separating a molecule at least in part from other molecules that are normally associated with it in its native state. In one embodiment, the term "isolated" refers to separating a DNA molecule from the nucleic acids that normally flank the DNA molecule in its native state. For example, a DNA molecule encoding a protein that naturally occurs in bacteria will be an isolated DNA molecule if it is not within the DNA of the bacteria in which the DNA molecule encoding the protein is naturally found. Thus, a DNA molecule that is fused to or operably linked to one or more other DNA molecules that are not related in nature, for example by recombinant DNA or plant transformation techniques, is considered isolated herein. Such molecules are considered isolated even when integrated into the chromosome of a host cell together with other DNA molecules or present in a nucleic acid solution.

[0055] Any number of methods well known to those of skill in the art can be used to isolate and manipulate the DNA molecules or fragments thereof disclosed herein. For example, polymerase chain reaction (PCR) techniques can be used to amplify a particular starting DNA molecule or generate variants of the original molecule. DNA molecules or fragments thereof can also be obtained by other techniques, such as by directly synthesizing the fragment by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer.

[0056] Due to the degeneracy of the genetic code, a variety of different DNA sequences can encode a protein, such as the altered or engineered proteins disclosed herein. For example, Figure 2 a universal genetic code chart is provided that shows all possible mRNA triplet codons (where T in the DNA molecule is replaced by U in the RNA molecule) and the amino acids encoded by each codon. Mutations can be introduced into the DNA sequence encoding the wild-type PPO enzyme by using methods known in the art and the information provided in Figure 2 to generate a DNA sequence encoding a PPO enzyme having the amino acid substitutions described herein. Forming alternative DNA sequences encoding the same or substantially the same altered or engineered proteins described herein is well within the ability of those of skill in the art. These variant or alternative DNA sequences are within the scope of the embodiments described herein. As used herein, reference to a "substantially identical" sequence refers to a sequence having amino acid substitutions, deletions, additions, or insertions that do not substantially alter the functional activity of the protein encoded by the DNA molecule of the embodiments described herein. Allelic variants of the nucleotide sequences encoding wild-type or engineered proteins are also included within the scope of the embodiments described herein. Amino acid substitutions other than those specifically listed or naturally occurring in wild-type or engineered PPO enzymes are also contemplated within the scope of the embodiments described herein, provided that the PPO enzyme having the substitution still generally retains the same functional activity described herein.

[0057] The recombinant DNA molecules of the present invention can be synthesized and modified in whole or in part by methods known in the art, wherein sequences useful for DNA manipulation (such as restriction endonuclease recognition sites or recombination-based cloning sites), plant-preferred sequences (such as plant-codon usage or Kozak consensus sequences), or sequences useful for DNA construct design (such as spacer or linker sequences) need to be provided. The present invention includes recombinant DNA molecules and engineered proteins having at least 50% sequence identity, at least 60% sequence identity, at least 70% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, and at least 99% sequence identity with any one of the recombinant DNA molecules or amino acid sequences provided herein and having herbicide-tolerant protoporphyrinogen oxidase activity. As used herein, the term "percent sequence identity" or "sequence identity %" refers to the percentage of identical nucleotides or amino acids in the linear polynucleotide or amino acid sequence of a reference ('query') sequence (or its complementary strand) compared to a test ('target') sequence (or its complementary strand) when the two sequences are optimally aligned (with a total of less than 20% appropriate nucleotide or amino acid insertions, deletions, or gaps in the comparison window) in a reference sequence. The optimal alignment of sequences for the comparison window is well known to those skilled in the art and can be performed by tools such as the local identity algorithm of Smith and Waterman, the identity alignment algorithm of Needleman and Wunsch, the similarity search method of Pearson and Lipman, and computer implementations of these algorithms, such as GAP, BESTFIT, FASTA, and TFASTA, which can be used, for example, with default parameters of Wisconsin (Accelrys Inc., San Diego, CA), part of the sequence analysis software packages of MEGAlign (DNAStar Inc., 1228 S. Park St., Madison, WI 53715) and MUSCLE (version 3.6) (RC Edgar, “MUSCLE: multiple sequence alignment with high accuracy and high throughput” Nucleic Acids Research 32(5):1792-7 (2004)). The “identity score” for the aligned segment of a test sequence with a reference sequence is the number of identical components shared by the two aligned sequences divided by the total number of components in the aligned portion of the reference sequence segment (i.e., the entire reference sequence or a smaller defined portion of the reference sequence). The percent sequence identity is expressed as the identity score multiplied by 100. One or more sequences can be compared to the full-length sequence or a portion thereof or to a longer sequence.

[0058] As used herein, a "DNA construct" is a recombinant DNA molecule that contains two or more heterologous DNA sequences. DNA constructs can be used for transgene expression and can be contained in vectors and plasmids. A DNA construct can be used in a vector for transformation (i.e., introducing heterologous DNA into a host cell) to produce recombinant bacteria or transgenic plants and cells (and can thus also be contained in the plastid DNA or genomic DNA of transgenic plants, seeds, cells, or plant parts). As used herein, a "vector" means any recombinant DNA molecule that can be used for bacterial or plant transformation. The DNA molecules provided by the present invention can be inserted, for example, as part of a DNA construct of a DNA molecule operably linked to a heterologous gene expression element that functions in plants to effect the expression of the engineered protein encoded by the DNA molecule. Methods for preparing and using DNA constructs and vectors are well known in the art and are described in detail, for example, in manuals and protocols including Michael R. Green and Joseph Sambrook, "Molecular Cloning: A Laboratory Manual" (Fourth Edition) ISBN: 978-1-936113-42-2, Cold Spring Harbor Laboratory Press, NY (2012). Components of a DNA construct or a vector containing a DNA construct include one or more gene expression elements operably linked to a transcribable nucleic acid sequence, such as the following: a promoter for expressing the operably linked DNA, an operably linked DNA molecule encoding a protein, and an operably linked 3' untranslated region (UTR). Gene expression elements that can be used to practice the present invention include, but are not limited to, one or more of the following types of elements: promoters, 5'UTRs, enhancers, leaders, cis-acting elements, introns, transit sequences, 3'UTRs, and one or more selectable marker transgenes.

[0059] The term "transgene" refers to a DNA molecule that has been artificially incorporated into the genome of an organism through human intervention, such as by plant transformation methods. As used herein, the term "transgenic" means containing a transgene. For example, a "transgenic plant" refers to a plant that contains a transgene in its genome, and a "transgenic trait" refers to a characteristic or phenotype that is conferred or brought about by the presence of a transgene incorporated into the plant genome. Due to such genomic alterations, transgenic plants are plants that are significantly different from the relevant wild-type plants, and transgenic traits are traits that do not naturally occur in wild-type plants. The transgenic plants of the present invention contain the recombinant DNA molecules and engineered proteins provided by the present invention.

[0060] As used herein, the term "heterologous" refers to a relationship between two or more things that are not normally associated in nature, such as a relationship derived from different sources or not normally found together in nature in any other way. For example, a DNA molecule or protein can be heterologous relative to another DNA molecule, protein, cell, plant, seed, or organism if they are not normally found together in nature or in the same context. In certain embodiments, a first DNA molecule is heterologous to a second DNA molecule if the two DNA molecules are not normally found together in nature in the same context. For example, a recombinant DNA molecule encoding a protein is heterologous relative to an operably linked promoter if the combination of the recombinant DNA molecule encoding the protein and the operably linked promoter is not normally found in nature. Similarly, a protein is heterologous relative to a second operably linked protein (such as a transit peptide) if the combination of the protein and the second operably linked protein (such as a transit peptide) is not normally found in nature. In another embodiment, a recombinant DNA molecule encoding a PPO enzyme is heterologous relative to an operably linked promoter functional in a plant cell if the combination of the recombinant DNA molecule encoding the PPO enzyme and the operably linked promoter functional in a plant cell is not normally found in nature. A recombinant DNA molecule can also be heterologous relative to a cell, seed, or organism into which it is inserted if it does not naturally occur in the cell, seed, or organism.

[0061] A "heterologous protein" is a protein that is present in a plant, seed, cell, tissue, or organism in which it does not naturally occur or is operably linked to a protein to which it is not naturally linked. Examples of heterologous proteins are engineered PPO enzymes that contain at least one first amino acid substitution described herein and are expressed in any plant, seed, cell, tissue, or organism. Another example is a protein that is operably linked to a second protein (such as a transit peptide or a herbicide tolerance protein) to which it is not naturally linked or a protein that is introduced into a plant cell using genetic engineering techniques in which it does not naturally occur.

[0062] As used herein, "operably linked" means that two or more DNA molecules or two or more proteins are linked in such a way that one enables the function of the other. The operably linked DNA molecules or operably linked proteins can be part of a single continuous molecule and may or may not be adjacent. For example, in a DNA construct, a promoter and a DNA molecule encoding a protein are operably linked, where the two DNA molecules are arranged such that the promoter enables the expression of the transgene.

[0063] The DNA constructs of the present invention may include a promoter operably linked to a DNA molecule encoding a protein provided by the present invention, whereby the promoter drives the expression of the engineered protein. Promoters useful in practicing the present invention include those that function in a cell to cause the expression of an operably linked DNA molecule, such as bacterial or plant promoters. Plant promoters vary and are well known in the art and include, for example, inducible, viral, synthetic, constitutive, temporally regulated, spatially regulated, or spatio-temporally regulated promoters.

[0064] In one embodiment of the present invention, the DNA constructs provided herein include a DNA sequence encoding a transit sequence, which is operably linked to a heterologous DNA sequence encoding a PPO enzyme, whereby the transit sequence aids in the intracellular localization of the protein molecule. Transit sequences are known in the art as signal sequences, targeting peptides, targeting sequences, localization sequences, and transit peptides. Examples of transit sequences are chloroplast transit peptides (CTP), mitochondrial transit sequences (MTS), or dual chloroplast and mitochondrial transit peptides. By facilitating the intracellular localization of the protein, the transit sequence can increase the accumulation of the recombinant protein, prevent protein degradation, or increase the level of herbicide tolerance, and thereby reduce the level of damage in cells, seeds, or organisms after herbicide application. CTPs and other targeting molecules that can be used in combination with the present invention are well known in the art. The DNA sequence encoding the transit sequence can be operably linked to the DNA sequence encoding the PPO enzyme as provided herein. Such an operable linkage may involve removing the initiating methionine codon (ATG) at the 5' end of the PPO sequence, although this is not necessarily done, and the transit sequence will aid in the intracellular localization of the protein molecule with or without removal of the initiating methionine codon.

[0065] As used herein, "transgene expression", "causing transgene expression", "protein expression", and "causing protein expression" mean the production of a protein by the process of transcribing a DNA molecule into messenger RNA (mRNA) and translating the mRNA into a polypeptide chain that ultimately folds into a protein. The DNA molecule encoding the protein can be operably linked to a heterologous promoter in a DNA construct for expressing the protein in a cell transformed with the recombinant DNA molecule.

[0066] In one aspect, the present invention provides cells, tissues, plants, and seeds comprising the recombinant DNA molecules or engineered proteins of the present invention. These cells, tissues, plants, and seeds comprising the recombinant DNA molecules or engineered proteins exhibit tolerance to one or more PPO herbicides.

[0067] One method of generating such cells, tissues, plants, and seeds is by plant transformation. Methods suitable for transforming host plant cells for use in the present invention include any method by which DNA can be introduced into the cell (e.g., where a recombinant DNA construct is stably integrated into the plant chromosome) and are well known in the art. Two effective and widely used methods for cell transformation are Agrobacterium-mediated transformation and microprojectile bombardment-mediated transformation. For example, the microprojectile bombardment method is illustrated in U.S. Patent Nos. US 5,550,318; US 5,538,880; US 6,160,208; and US 6,399,861. The Agrobacterium-mediated transformation method is described in U.S. Patent No. US 5,591,616, which is incorporated herein by reference in its entirety. Before or after regenerating such cells into plants, cells having the recombinant DNA molecule or engineered protein of the present invention can be selected for the presence of the recombinant DNA molecule or engineered protein, e.g., by the enzyme activity it encodes.

[0068] Another method of generating the cells, plants, and seeds of the present invention is by genome modification using site-specific integration or genome editing. Targeted modification of the plant genome can be utilized to form improved plant lines by modification of plant genomic DNA through the use of genome editing methods. As used herein, "site-directed integration" is a genome editing method that enables the targeted insertion of one or more nucleic acids of interest into the plant genome. Methods suitable for altering a wild-type DNA sequence or a pre-existing transgenic sequence or for inserting DNA into the plant genome at a predetermined chromosomal locus include any method known in the art. Exemplary methods include the use of sequence-specific nucleases such as zinc finger nucleases, engineered or native meganucleases, TALE-endonucleases, or RNA-guided endonucleases (e.g., the clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 system, CRISPR / Cpf1 system, CRISPR / CasX system, CRISPR / CasY system, CRISPR / cascade system). Several embodiments relate to genome editing methods for introducing precise base pair modifications into the plant genome by using single-stranded oligonucleotides as described by Sauer et al., Plant Physiology 170(4):1917–1928 (2016). Genome editing methods for modifying, deleting, or inserting nucleic acid sequences into genomic DNA are known in the art.

[0069] In certain embodiments, the present invention provides for the modification or replacement of an existing coding sequence (such as a PPO coding sequence or another existing transgenic insert) within the plant genome with a sequence encoding an engineered protein (such as an engineered PPO coding sequence of the present invention) or an expression cassette comprising such an engineered protein. Several embodiments relate to the use of known genome editing methods, such as zinc finger nucleases, engineered or native meganucleases, TALE-endonucleases or RNA-guided endonucleases (e.g., clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 system, CRISPR / Cpf1 system, CRISPR / CasX system, CRISPR / CasY system, CRISPR / cascade system).

[0070] Accordingly, several embodiments may relate to a recombinant DNA construct comprising one or more expression cassettes encoding a site-specific nuclease and optionally one or more any associated proteins to effect genome modification. These nuclease expression cassettes may be present in the same molecule or vector (in cis) or on separate molecules or vectors (in trans) with a donor template for templated editing or an expression cassette comprising a nucleic acid sequence encoding a PPO protein as described herein. Several methods for site-directed integration are known in the art that involve different sequence-specific nucleases (or a complex of a protein or guide RNA or both) that cleave genomic DNA to create a double-strand break (DSB) or a nick at a desired genomic locus or site. As is understood in the art, during the process of repairing the DSB or nick introduced by the nuclease, the donor template DNA, transgene or expression cassette may become integrated into the genome at the DSB or nick site. The presence of one or more homologous arms in the DNA to be integrated may facilitate the acceptance of the inserted sequence and its targeting to the plant genome by homologous recombination, although the insertion event may proceed by non-homologous end joining (NHEJ).

[0071] As used herein, the term "double-strand break inducer" refers to any agent that can induce a double-strand break (DSB) in a DNA molecule. In some embodiments, the double-strand break inducer is a site-specific genome modification enzyme.

[0072] As used herein, the term "site-specific genome modifying enzyme" refers to any enzyme that can modify a nucleotide sequence in a sequence-specific manner. In some embodiments, the site-specific genome modifying enzyme modifies the genome by inducing a single-strand break. In some embodiments, the site-specific genome modifying enzyme modifies the genome by inducing a double-strand break. In some embodiments, the site-specific genome modifying enzyme comprises a cytidine deaminase. In some embodiments, the site-specific genome modifying enzyme comprises an adenine deaminase. In the present disclosure, site-specific genome modifying enzymes include endonucleases, recombinases, transposases, deaminases, helicases, and any combination thereof. In some embodiments, the site-specific genome modifying enzyme is a sequence-specific nuclease.

[0073] In one aspect, the endonuclease is selected from the group consisting of meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), Argonaute (non-limiting examples of Argonaute proteins include Thermus thermophilus Argonaute (TtAgo), Pyrococcus furiosus Argonaute (PfAgo), Natronobacterium gregoryi Argonaute (NgAgo), RNA-guided nucleases such as CRISPR-associated nucleases, non-limiting examples of CRISPR-associated nucleases include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, CasX, CasY, their homologs or their modified forms).

[0074] In some embodiments, the site-specific genome modification enzyme is a recombinase. Non-limiting examples of recombinases include tyrosine recombinases linked to DNA recognition motifs, selected from the group consisting of Cre recombinase, Gin recombinase, Flp recombinase, and Tnp1 recombinase provided herein. In one aspect, the Cre recombinase or Gin recombinase provided herein is tethered to a zinc finger DNA binding domain or a TALE DNA binding domain or a Cas9 nuclease. In another aspect, the serine recombinases provided herein that are linked to DNA recognition motifs are selected from the group consisting of PhiC31 integrase, R4 integrase, and TP-901 integrase. In another aspect, the DNA transposases provided herein that are linked to DNA binding domains are selected from the group consisting of TALE-piggyBac and TALE-Mutator.

[0075] Any of the DNAs of interest provided herein can be integrated into a target site of a chromosomal sequence by introducing the DNA of interest and the provided site-specific genome modification enzyme. Any of the methods provided herein can use any of the site-specific genome modification enzymes provided herein.

[0076] In one aspect, the present invention provides cells, plants, and seeds that are tolerant to PPO inhibitor herbicides. Such cells, plants, and seeds can be used in agricultural methods such as weed control and crop production.

[0077] As used herein, "herbicide" is any molecule used to control, inhibit, or prevent the growth of one or more plants. Exemplary herbicides include acetyl-CoA carboxylase (ACC enzyme) inhibitors (e.g., aryloxyphenoxypropionates and cyclohexanediones); acetolactate synthase (ALS) inhibitors (e.g., sulfonylureas, imidazolinones, triazolopyrimidines, and triazolinones); 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitors (e.g., glyphosate), synthetic auxins (e.g., phenoxys, benzoics, carboxylic acids, semicarbazones), photosynthesis (photosystem II) inhibitors (e.g., triazines, triazinones, nitriles, benzothiadiazoles, and ureas), glutamine synthetase (GS) inhibitors (e.g., glufosinate and bialaphos), 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors (e.g., isoxazoles, pyrazolinones, and triketones), protoporphyrinogen oxidase (PPO) inhibitors (e.g., diphenyl ethers, N-phenylphthalimides, aryltriazinones, and pyrimidine diones), very long chain fatty acid inhibitors (e.g., chloroacetamides, oxyacetamides, and pyrazoles), cellulose biosynthesis inhibitors (e.g., indaziflam), photosystem I inhibitors (e.g., paraquat), microtubule assembly inhibitors (e.g., pendimethalin), and phytoene desaturase (PDS) inhibitors (e.g., norflurazone), among others.

[0078] As used herein, a "PPO herbicide" is a chemical that targets protoporphyrinogen oxidase (PPO) and inhibits its enzymatic activity. Protoporphyrinogen oxidase catalyzes the dehydrogenation of protoporphyrinogen IX to form protoporphyrin IX, which is a precursor to heme and chlorophyll. Inhibition of protoporphyrinogen oxidase results in the formation of reactive oxygen species, which causes cell membrane rupture and ultimately leads to the death of susceptible cells. PPO herbicides are well known in the art and are commercially available. Examples of PPO herbicides include, but are not limited to, diphenyl ethers (such as acifluorfen, its salts and esters, aclonifen, bifenox, its salts and esters, ethoxyfen, its salts and esters, fluoronitrofen, furyloxyfen, halosafen, chlomethoxyfen, fluoroglycofen, its salts and esters, lactofen, its salts and esters, oxyfluorfen, and fomesafen, its salts and esters); thiadiazoles (such as fluthiacet-methyl and thidiazimin); pyrimidine diones or phenyluracils (such as benzfendizone, butafenacil, ethyl [3-(2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetate (CAS Registry No. 353292-31-6 and referred to herein as S-3100), flupropacil, sulfentrazone, and tiafenacil); phenylpyrazoles (such as fluazolate, pyraflufen-ethyl, and pyraflufen); oxadiazoles (such as oxadiargyl and oxadiazon); triazolinones (such as azafenidin, bencarbazone, carfentrazone, its salts and esters, and mesotrione); oxazolidinediones (such as pentoxazone); N-phenylphthalimides (such as cinidon-ethyl, flumiclorac, flumiclorac pentyl, and flumioxazin); benzoxazinone derivatives (such as 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3,4-dihydro-3-oxo-4-prop-2-ynyl-2H-1,4-benzoxazin-6-yl)-1,3,5-triazinan-2,4-dione); flufenpyr and flufenpyr-ethyl; pyraclonil; and profluazol).The protoporphyrinogen oxidase and cells, seeds, plants, and plant parts provided by the present invention exhibit herbicide tolerance to one or more PPO herbicides.

[0079] As used herein, "herbicide tolerant" or "herbicide tolerance" means the ability to be completely or partially unaffected by the presence or application of one or more herbicides (e.g., resist the toxic effects of herbicides upon application). A cell or organism is "herbicide tolerant" if it can maintain at least some normal growth or phenotype in the presence of one or more herbicides. A trait is a herbicide tolerance trait if the presence of the trait results in improved herbicide tolerance of a cell, plant, or seed as compared to a wild-type or control cell, plant, or seed. A crop plant containing a herbicide tolerance trait can continue to grow and is minimally affected by the presence of the herbicide. A target enzyme is "herbicide tolerant" if it exhibits improved enzyme activity relative to a wild-type or control enzyme in the presence of a herbicide. Herbicide tolerance can be complete or partial insensitivity to a particular herbicide and can be expressed as a percentage (%) of tolerance or insensitivity to a particular herbicide.

[0080] Plants that can be produced and that are covered by the present invention having the herbicide tolerance trait can include, for example, any plant, including crop plants such as soybean (Glycine max), corn (Zea mays), cotton (Gossypium sp.), and Brassica plants, among others.

[0081] Herbicides can be applied to a plant growth area containing plants and seeds provided by the present invention as a method of controlling weeds. The plants and seeds provided by the present invention contain a herbicide tolerance trait and are thus tolerant to the application of one or more PPO herbicides. The herbicide application can be at the recommended commercial rate (1X) or any fraction or multiple thereof, such as twice the recommended commercial rate (2X). The herbicide rate can be expressed in terms of herbicide and partitioning as pounds of acid equivalent per acre (lb ae / acre) or grams of acid equivalent per hectare (g ae / ha) or as pounds of active ingredient per acre (lb ai / acre) or grams of active ingredient per hectare (g ai / ha). The herbicide application comprises at least one PPO herbicide. The plant growth area may or may not contain weed plants when the herbicide is applied. The herbicidally effective dose of one or more PPO herbicides for controlling weeds in an area can consist of a range from about 0.1X to about 30X the labeled rate during one growing season. The 1X labeled rates of some exemplary PPO herbicides are provided in Table 3. One (1) acre is equal to 2.47105 hectares and one (1) pound is equal to 453.592 grams. The herbicide rate can be converted between English and metric as follows: (lb ai / ac) multiplied by 1.12 = (kg ai / ha) and (kg ai / ha) multiplied by 0.89 = (lb ai / ac).

[0082] Table 3. Exemplary PPO Herbicides

[0083]

[0084]

[0085] The herbicide application can be sequential or tank-mixed with one, two, or a combination of several PPO herbicides or any other compatible herbicide. One herbicide or a combination of two or more herbicides can be applied multiple times, either alone or in combination, during the growing season to an area containing the transgenic plants of the present invention to control a broad spectrum of dicotyledonous weeds, monocotyledonous weeds, or both, for example, two applications (such as a pre-plant application and a post-emergence application or a pre-emergence application and a post-emergence application) or three applications (such as a pre-plant application, a pre-emergence application, and a post-emergence application or a pre-emergence application and two post-emergence applications).

[0086] As used herein, "weed" is any unwanted plant. A plant can be generally considered unwanted for agricultural or horticultural purposes (e.g., Amaranthus species) or can be considered unwanted in a particular situation (e.g., a crop plant of one species within a different species field, also known as a volunteer plant).

[0087] The transgenic plants, progeny, seeds, plant cells, and plant parts of the present invention may also contain one or more additional traits. The additional traits can be introduced by crossing a plant containing a transgene comprising the recombinant DNA molecule provided by the present invention with another plant containing one or more additional traits. As used herein, "crossing" means breeding two individual plants to produce a progeny plant. Thus, two plants can be crossed to produce a progeny containing the desired traits from each parent. As used herein, "progeny" means any generation of offspring of a parental plant, and transgenic progeny contain the DNA construct provided by the present invention and inherited from at least one parental plant.

[0088] One or more additional traits can also be introduced by co-transforming a DNA construct with a DNA construct comprising the recombinant DNA molecule provided by the present invention (e.g., all DNA constructs present as part of the same vector used for plant transformation) against said one or more additional transgenic traits or by inserting one or more additional traits into a transgenic plant comprising the DNA construct provided by the present invention or vice versa (e.g., by using any of the methods of plant transformation or genome editing on a transgenic plant or plant cell). Such additional traits include, but are not limited to, increased insect resistance, increased water use efficiency, increased yield performance, increased drought resistance, increased seed quality, improved nutritional quality, hybrid seed production, and herbicide tolerance, where the traits are measured relative to wild-type plants. Exemplary additional herbicide tolerance traits can include transgenic or non-transgenic tolerance to one or more herbicides such as: ACC enzyme inhibitors (e.g., aryloxyphenoxypropionates and cyclohexanediones), ALS inhibitors (e.g., sulfonylureas, imidazolinones, triazolopyrimidines, and triazolinones), EPSPS inhibitors (e.g., glyphosate), synthetic auxins (e.g., phenoxys, benzoics, carboxylic acids, semicarbazones), photosynthesis inhibitors (e.g., triazines, triazinones, nitriles, benzothiadiazoles, and ureas), glutamine synthetase inhibitors (e.g., glufosinate), HPPD inhibitors (e.g., isoxazoles, pyrazolinones, and triketones), PPO inhibitors (e.g., diphenyl ethers, N-phenylphthalimides, aryltriazinones, and pyrimidinediones), and long-chain fatty acid inhibitors (e.g., chloroacetamides, oxyacetamides, and pyrazoles). Examples of herbicide tolerance proteins that can be used to generate additional herbicide tolerance traits are well known in the art and include, but are not limited to, glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthase (e.g., CP4 EPSPS, 2m EPSPS), glyphosate oxidoreductase (GOX), glyphosate N-acetyltransferase (GAT), herbicide-tolerant acetolactate synthase (ALS) / acetohydroxyacid synthase (AHAS), herbicide-tolerant 4-hydroxyphenylpyruvate dioxygenase (HPPD), dicamba monooxygenase (DMO), phosphinothricin acetyltransferase (PAT), herbicide-tolerant glutamine synthetase (GS), 2,4-dichlorophenoxypropionate dioxygenase (TfdA), R-2,4-dichlorophenoxypropionate dioxygenase (RdpA), S-2,4-dichlorophenoxypropionate dioxygenase (SdpA), herbicide-tolerant protoporphyrinogen oxidase (PPO), and cytochrome P450 monooxygenase. Exemplary insect resistance traits can include resistance to one or more insect members within one or more of the orders such as Lepidoptera, Coleoptera, Hemiptera, Thysanoptera, Diptera, Hymenoptera, and Orthoptera.Such additional traits are well known to those skilled in the art; for example, the United States Department of Agriculture (USDA) Animal and Plant Health Inspection Service (APHIS) provides a list of such transgenic traits.

[0089] Transgenic plants and progeny that are tolerant to PPO herbicides can be used in any breeding method known in the art. In plant lines containing two or more traits, the traits can be independently segregating, linked, or a combination of both in plant lines containing three or more transgenic traits. As is common in vegetative propagation, backcrossing to the parental plant and outcrossing to non-transgenic plants are also encompassed. Descriptions of breeding methods commonly used for different traits and crops are well known to those skilled in the art. To confirm the presence of the one or more transgenes in a particular plant or seed, a variety of assays can be performed. Such assays include, for example, molecular biology assays such as Southern and Northern blot analyses, PCR, and DNA sequencing; biochemical assays such as detecting the presence of a protein product, e.g., by immunological means (ELISA and Western blot) or by enzyme function; plant part assays such as leaf or root assays; and by analyzing the phenotype of the whole plant.

[0090] The introgression of transgenic traits into the plant genotype is achieved through the backcross conversion process. A plant genotype into which transgenic traits have been introgressed can be referred to as a backcross-converted genotype, line, inbred, or hybrid. Similarly, a plant genotype lacking the desired transgenic trait can be referred to as an unconverted genotype, line, inbred, or hybrid.

[0091] As used herein, the term "comprising" means "including but not limited to".

[0092] Having thus described the invention in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing from the scope of the invention as defined in the appended claims. In addition, it should be understood that the examples in this disclosure are provided as non-limiting examples.

[0093] Examples

[0094] Example 1: Sequence Diversity within the Long Insertion Loop of the Microbial HemG Protein

[0095] Collections of different HemG PPO enzymes were examined for diversity in the long insertion loop of the protein. The long insertion loop is a defining feature of the microbial HemG PPO enzyme and is approximately 25 residues in length in the presence of major conserved residues (Boynton et al., Biochemistry (2009) 48:6705 - 6711).

[0096] Genomic analysis was performed using an initial set of 1,013 HemG PPO sequences from various microorganisms. Algorithms were designed to capture the overall sequence diversity of the proteins and the diversity within the long insert loop. Sequences were then sorted into multiple groups based on overall sequence similarity within the initial set.

[0097] To form the first group, all sequences in the initial set with ≥70% sequence identity to HemG PPO H_N90 (SEQ ID NO:1) with herbicide tolerance protoporphyrinogen oxidase activity were identified. The analysis was then repeated to identify sequences with ≥70% sequence identity to any of the sequences identified in the first analysis. Finally, the search was repeated a third time to identify sequences with ≥70% sequence identity to any of the sequences identified in the second analysis. The results of the three analyses were pooled together to form the first group, which represents sequences from three iterations of the ≥70% sequence identity analysis, a total of 273 HemG PPO sequences.

[0098] The second group was formed by focusing on the remaining ungrouped sequences from the initial set. All sequences with 50%-70% sequence identity to HemG PPO H_N90 were identified. The analysis was then repeated to identify sequences with ≥70% sequence identity to any of the sequences identified in the first analysis. Finally, the search was repeated a third time to identify sequences with ≥70% sequence identity to any of the sequences identified in the second analysis. The results of the three analyses were pooled together to form the second group, which represents sequences from all three iterations, a total of 278 HemG PPO sequences.

[0099] The third group was formed by focusing on the still remaining ungrouped sequences from the initial set. All sequences with 40%-50% sequence identity to HemG PPO H_N90 were identified. The analysis was then repeated to identify sequences with ≥70% sequence identity to any of the sequences identified in the first analysis. A third search was performed to identify sequences with ≥70% sequence identity to any of the sequences identified in the second analysis, but this last iteration did not capture any additional sequences from the initial set. The results of the two analyses were pooled together to form the third group, which represents sequences from two iterations, a total of 66 HemG PPO sequences.

[0100] The three groups of sequences were then used to analyze the changes present at the 25th amino acid in the long insert loop. The long insert loop sequences of each PPO were identified and compiled. Unexpectedly, it was found that for the first and second groups, the sequence changes in this domain were similar, even though the sequences of the two groups had up to 50% total sequence variation when combined and represented 551 different sequences. Figure 3Provide a review of the changes found in the long-chain insertion loop in 617 sequences from three groups. Among the 25 amino acid positions in the long-chain insertion loop, 211 different amino acids were identified from 617 HemG PPO sequences. Figure 1A and Figure 1B Shows the sequence alignment of the long-chain insertion loop (highlighted in black) of a subset of 23 microbial HemG PPO sequences.

[0101] A set of 17 HemG PPO sequences was selected to represent the changes found in the long-chain insertion loop. When compared using pairwise sequence alignment, the protein sequences of the 17 different HemG PPO enzymes had identity percentages in the range of approximately 15% to 98% over the full-length sequences. These 17 different HemG PPO enzymes were tested for protoporphyrinogen oxidase activity and herbicide tolerance.

[0102] The protoporphyrinogen oxidase bacterial screening system was used to test the protoporphyrinogen oxidase activity of the proteins and thus confirm that they are functional PPO enzymes. This screening system uses a functional rescue assay in an Escherichia coli strain that contains a gene knockout of the Escherichia coli HemG PPO enzyme (referred to herein as H_N10 and corresponding to SEQ ID NO: 2). The hemG knockout E. coli strain was transformed with bacterial expression vectors each containing an expression cassette for one of the PPO enzymes and cultured on LB medium. The hemG knockout E. coli strain shows very little growth on classical bacterial media (such as LB medium), but can resume normal growth when the bacterial medium is supplemented with free heme or when a functional protoporphyrinogen oxidase is expressed in the cells. Two controls were used for comparison: green fluorescent protein (GFP) and untransformed cells. Two of the HemG PPO enzymes (HemG014 and HemG015) could not rescue the hemG knockout E. coli strain (no protoporphyrinogen oxidase activity), two showed partial rescue (intermediate) with slower growth, and the remaining 13 showed a complete rescue phenotype (functional). The results are shown in Table 4.

[0103] Design protoplast herbicide tolerance assays to test the herbicide tolerance of 17 different HemG PPO enzymes in plant cells. Recombinant DNA molecules encoding 17 different HemG PPO enzymes (codon-optimized for dicotyledonous plant expression) were synthesized and cloned into plant transformation vectors. The expression constructs contained a recombinant DNA molecule encoding one of the 17 different HemG PPO enzymes operably linked to a plant promoter, a chloroplast transit peptide, and a 3' untranslated region. Soybean protoplasts were transformed with the plant transformation vectors using standard methods. The transformed protoplasts were grown in the presence of 1.0 μM concentration of the PPO herbicide S-3100 or mock treatment (negative control). The protoplasts were then assayed for PPO herbicide tolerance, normalized relative to the score of HemG PPO enzyme H_N90 set at 100. The assays were repeated four times in two batches. The relative tolerance scores for each were averaged and the standard error (SE) was calculated. The tolerance score for the GFP control assay was 0, demonstrating that soybean protoplasts are intolerant to the PPO herbicide in the absence of herbicide tolerance proteins. Two of the various HemG PPO enzymes (HemG014 and HemG015) did not confer tolerance, while the other 14 conferred tolerance scores in the range of 24 to 89 relative to H_N90. The results are shown in Table 4.

[0104] Fifteen of the various HemG PPO enzymes were then expressed in transgenic plants, and the PPO herbicide tolerance of the transgenic plants was analyzed. Recombinant DNA molecules encoding 15 different HemG PPO enzymes (codon-optimized for dicotyledonous or monocotyledonous plant expression) were synthesized and cloned into plant transformation vectors. The expression constructs contained a recombinant DNA molecule encoding one of the 15 different HemG PPO enzymes operably linked to a plant promoter, a chloroplast transit peptide, and a 3' untranslated region.

[0105] Maize cells were transformed with these vectors using Agrobacterium tumefaciens and standard methods known in the art. The regenerated R 0 transgenic seedlings were grown in the greenhouse. At approximately V 2 to V 4During the growth period, plants were sprayed with the PPO herbicide S3100 at a rate of 80 g / ha to evaluate tolerance. Plants were evaluated for damage 1 - 14 days after treatment and damage scores were recorded. For each of the various HemG PPO enzymes in all plants, the percentage of plants with a visual damage score of 20% or less was calculated. Any construct in which 25% or more of the individual plants showed good tolerance (visual damage score of 20% or less) was considered to effectively confer herbicide tolerance. Eight of the various HemG PPO enzymes (HemG001, HemG002, HemG003, HemG004, HemG005, HemG006, HemG011, and HemG012) provided a large number of maize plants that exhibited tolerance to the PPO herbicide (20% or less damage after treatment). The results are shown in Table 4.

[0106] Soybean cells were transformed with these vectors using Agrobacterium tumefaciens and standard methods known in the art. The regenerated R 0 transgenic seedlings were grown in the greenhouse. At approximately V 2 to V 4 During the growth period, plants were sprayed with the PPO herbicide S3100 at a rate of 20 g / ha to evaluate tolerance. Plants were evaluated for damage 1 - 14 days after treatment and damage scores were recorded. For each of the various HemG PPO enzymes in all plants, the percentage of plants with a visual damage score of 20% or less was calculated. Any construct in which 25% or more of the individual plants showed good tolerance (visual damage score of 20% or less) was considered to effectively confer herbicide tolerance. Ten of the various HemG PPO enzymes (HemG001, HemG002, HemG003, HemG004, HemG005, HemG006, HemG007, HemG009, HemG011, and HemG013) provided a large number of soybean plants that exhibited tolerance to the PPO herbicide (20% or less damage after treatment). The results are shown in Table 4.

[0107] Table 4. Testing of various HemG PPO enzymes

[0108]

[0109]

[0110] Of the 15 different HemG PPO enzymes tested in stably transformed maize and soybean (or both), 10 were found to effectively confer herbicide tolerance (producing plants with visual injury scores of 20% or lower for more than 25%). The sequences of these HemG PPO enzymes that effectively confer herbicide tolerance to plants are provided below: HemG001 (SEQ ID NO:11), HemG002 (SEQ ID NO:12), HemG003 (SEQ ID NO:13), HemG004 (SEQ ID NO:14), HemG005 (SEQ ID NO:15), HemG006 (SEQ ID NO:16), HemG007 (SEQ ID NO:17), HemG009 (SEQ ID NO:19), HemG011 (SEQ ID NO:21), and HemG013 (SEQ ID NO:23).

[0111] Example 2: Functional Characterization of Long Chain Insertion Loop Variants

[0112] The long chain insertion loop of the HemG protein has been described as essential for PPO enzyme function and many residues have been reported to be highly conserved (Boynton et al., Biochemistry (2009) 48:6705 - 6711). Recombinant HemG PPO enzymes were formed that introduced amino acid changes within the long chain insertion loop and then the changes in enzyme function were analyzed in a bacterial assay.

[0113] The protoporphyrinogen oxidase activity of the variant proteins was tested using the protoporphyrinogen oxidase bacterial screening system described in Example 2. This assay provided a means to rapidly and easily analyze the protoporphyrinogen oxidase activity of the variant proteins.

[0114] Recombinant HemG PPO enzymes incorporating mutations into the long chain insertion loop were designed as follows. Each amino acid of the long chain insertion loop was considered independently and ranked based on the amount of change identified at that position and how many of those changes were found in which sequence group. Based on this assessment, 21 of the 25 amino acids in the long chain insertion loop were selected for mutagenesis. Mutations were formed in the H_N90 sequence to represent the changes observed at each of these 21 positions, resulting in 109 single amino acid variants. In addition, alanine scanning mutagenesis was performed using the H_N90 sequence to produce mutants having alanine at each position in the long chain insertion loop that was not already alanine in H_N90, resulting in 10 additional variants. Then the total of 119 single amino acid variants were used for screening.

[0115] Recombinant DNA molecules encoding 119 variants were then synthesized and cloned into an expression construct in a bacterial transformation vector. For each variant, the entire nucleotide sequence remained the same as the H_N90 nucleotide sequence, except for the codons for the mutant amino acids. The expression construct contained each of the recombinant DNA molecules encoding 119 variants operably linked to a plant promoter, the APG6 chloroplast transit peptide, and a 3' untranslated region. The positive control consisted of an expression construct containing the H_N90 coding sequence operably linked to a plant promoter, the APG6 chloroplast transit peptide, and a 3' untranslated region. Each vector was individually transformed into a hemG knockout Escherichia coli strain. As a negative control, mock transformations (absence of vector) and a vector for expressing green fluorescent protein (GFP) were individually transformed into the hemG knockout Escherichia coli strain.

[0116] Each of the 119 variants was screened for the ability to restore normal growth of the hemG knockout Escherichia coli strain on LB plates. All plates were scored blindly and independently by three individuals: no growth (variant did not complement), slow growth (variant had reduced enzyme function), or normal growth (variant had full enzyme function and provided full complementation). Growth was measured based on colony size on the plates rather than colony number, and the three individuals agreed on all ratings. Table 5 shows the results of the analysis. In this analysis, 105 of the 119 variants restored normal growth, indicating that these variants had full PPO function; 6 of the 119 variants showed colony growth with a significantly slower growth rate, indicating that these variants had reduced PPO function; and 8 of the 119 variants did not show colony growth, indicating that these variants had no PPO function. As expected, all positive controls showed complementation, although the H_N10 construct grew more slowly than the H_N90 construct. Figure 4 A graphical representation of the results of this analysis is shown.

[0117] Table 5. HemG Variant Complementation Analysis

[0118]

[0119]

[0120]

[0121]

[0122] The results of this analysis indicate that while the long insert loop is highly conserved in the HemG PPO protein, many residues within the loop are flexible in maintaining enzyme function. This is unexpected based on published reports demonstrating that changes in residues within the long insert loop result in loss of enzyme function (Zwerschke, D., Karrie, S., Jahn, D., and Jahn, M. (2014) Biosci. Rep. 34(4), art:e00124. doi:10.1042 / BSR20140081). In this analysis, altered enzyme function was found, particularly in the case of mutations formed at positions G123, L125, Y127, I138, L140, I141, M142, and G147, indicating that changes at these positions are important for altering enzyme function.

[0123] Example 3: Herbicidal Tolerance Characterization of Long Insert Loop Variants

[0124] Recombinant HemG PPO enzymes were formed that introduced amino acid changes within the long insert loop and the changes in herbicide sensitivity in plants were analyzed. A protoplast herbicidal tolerance assay was designed to determine whether the variants could confer tolerance to PPO herbicides in plant cells.

[0125] Soybean protoplasts were transformed using standard methods with the same expression constructs described in Example 2 but using a plant transformation vector. The transformed protoplasts were grown in the presence of 1.0 μM concentration of the PPO herbicide S-3100 or mock treatment (negative control). The protoplasts were then analyzed for PPO herbicidal tolerance, expressed relative to the GFP control and H_N90 (allowing for relative tolerance scores to enable comparison between experiments). The analysis was repeated four times in two batches. The relative tolerance scores for each were averaged and the standard error (SE) was calculated. The tolerance score for the GFP control analysis was 0, demonstrating that soybean protoplasts are intolerant to the PPO herbicide in the absence of the herbicide tolerance protein. The tolerance score for the N-N90 analysis was 100. Table 6 shows the results of the analysis. Thirteen variants conferred little tolerance (similar to the untransformed control or GFP control), six variants conferred weak tolerance (similar to the H_N90 control without CTP), nine variants conferred marginal tolerance (similar to the H_N10 control), and 79 variants conferred good tolerance (similar to the H_N90 control with CTP). Twelve variants had relative tolerance scores greater than 100 (superior to the H_N90 control with CTP). The amino acid changes in these 12 variants were located at 7 residue positions, 4 of which had more than 1 variant that tended to be higher than 100. This indicates that these 7 amino acid sites are of particular interest for improved herbicidal tolerance. Figure 5A graphical representation showing the results of this analysis.

[0126] Table 6. Results of HemG variant protoplast analysis - S3100

[0127]

[0128]

[0129]

[0130]

[0131] A subset of 39 variants (plus controls) was selected for further analysis. In an analysis similar to the S-3100 tolerance analysis described above, these variants were tested for tolerance to three additional PPO herbicides, flumioxazin, sulfentrazone, and lactofen. The transformed protoplasts were treated with flumioxazin (5 nM), sulfentrazone (1 μM), and lactofen (1 μM). Table 7 shows the results of the analysis. Of the 39 variants tested, 30 showed good tolerance to flumioxazin, sulfentrazone, or lactofen and 9 had poor tolerance (tolerance score below 50, indicated as "PT"). Of the 30 variants showing good tolerance, 8 showed a greater change in tolerance to one or more herbicides than the experimental change relative to S-3100. Of these 8 variants, 4 variants conferred higher tolerance to one or more of the herbicides, indicated as "higher" in Table 7 below, while 4 variants conferred lower tolerance to one or more of the herbicides, indicated as "lower" in Table 7 below. Variants indicated as "NSD" had tolerance scores with differences less than the standard error of the known data points.

[0132] Table 7. Results of HemG variant protoplast analysis - flumioxazin, sulfentrazone, and lactofen

[0133]

[0134]

[0135]

[0136] Among the 8 variants in which the tolerance scores for flumioxazin, sulfentrazone or lactofen showed significant differences compared to the tolerance scores for S-3100, 6 variants were located at the hydrophobic residues M137, L140 and M142. The region spanning residues M137 to M142 contains a large number of hydrophobic residues (especially I, L, V, M, A). Analysis of this hydrophobic region indicates that these residues are uniquely important in regulating the functionality of the enzyme variants. In addition, these residues are uniquely important in regulating tolerance to different PPO inhibitor herbicides.

[0137] The transformed protoplasts can be assailed with other PPO herbicides, such as diphenyl ethers (such as acifluorfen, its salts and esters, oxyfluorfen, nitrofen, its salts and esters, fluorodifen, its salts and esters, fluroglycofen, fomesafen, fluazinam, chlomethoxyfen, ethyl fluoroglycofen, its salts and esters, salts and esters of lactofen, ethoxyfen and fomesafen, its salts and esters); thiadiazoles (such as fluthiacet-methyl and thidiazimin); pyrimidinediones or phenyluracils (such as bispyribac-sodium, flupropacil, [3-2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetic acid ethyl ester (CAS registration number 353292-31-6 and referred to herein as S-3100), flupropacil, pyriftalid and fluthiacet); phenylpyrazoles (such as propisochlor, pyraflufen-ethyl and pyraflufen); oxadiazoles (such as oxadiargyl and oxadiazon); triazolinones (such as carfentrazone-ethyl, sulfentrazone and carfentrazone, its salts and esters); oxazolidinediones (such as pentoxazone); N-phenylphthalimides (such as cinidon-ethyl, flumiclorac-pentyl and flumiclorac); benzoxazinone derivatives (such as 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3,4-dihydro-3-oxo-4-prop-2-ynyl-2H-1,4-benzoxazin-6-yl)-1,3,5-triazinan-2,4-dione); flufenpyr-ethyl and flufenpyr; bipyrazon; and fluzolate. A mock treatment can be used as a negative control.

[0138] Example 4: Characterization of the Function and Herbicide Tolerance of Combinatorial Variants

[0139] The variants were designed to contain two or more amino acid modifications within the long loop insert in the HemG PPO sequence. These combinatorial variant HemG PPO enzymes were then analyzed to determine PPO activity. The combinatorial variant HemG PPO DNA sequences were synthesized and cloned into an expression cassette. A bacterial transformation vector containing the expression cassette was transformed into a hemG knockout Escherichia coli strain for an initial high-throughput bacterial rescue screen as described in Example 2. The combinatorial variants were screened for the ability to restore normal growth of the hemG knockout Escherichia coli strain.

[0140] The combinatorial variant HemG PPO enzymes were also analyzed for the ability to confer tolerance to PPO herbicides on plant cells. An expression cassette containing the combinatorial variant HemG PPO DNA sequence was used with a plant transformation vector to transform soybean protoplasts. Protoplast tolerance assays were performed as described in Example 3, and the combinatorial variants were screened for the ability to confer herbicide tolerance on plant cells.

[0141] Example 5: Expression and testing of variant HemG PPO enzymes in plants

[0142] The microbial HemG PPO variants described in the above examples can be expressed in stably transformed plants, and the PPO herbicide tolerance of these plants can be analyzed.

[0143] Twenty-five of the microbial HemG PPO variants were tested for herbicide tolerance in stably transformed maize or soybean (or both). A plant transformation vector was constructed containing a recombinant DNA molecule encoding a variant HemG PPO enzyme operably linked to a plant promoter, a transit sequence, and a 3' UTR (where the protein coding sequence was optimized for expression in monocotyledonous or dicotyledonous plants).

[0144] In maize, maize cells were transformed with the plant transformation vector using Agrobacterium tumefaciens and standard methods known in the art. The regenerated R 0 transgenic seedlings were grown in a greenhouse. At approximately V 2 to V 4 growth stage, the R 0 plants were sprayed with S3100 at a rate of 80 g / ha. The plants were then evaluated for damage 1 - 14 days after treatment and the damage scores were recorded. Transgenic plants with a single copy of the transgenic DNA insert (i.e., single-event plants) were identified, and the R 0 plants containing only a single copy and passing the herbicide spray test were self-pollinated to produce R 1 seeds.

[0145] In soybeans, excised embryos were transformed with Agrobacterium tumefaciens and a plant transformation vector using standard methods known in the art. The regenerated R 0 transgenic seedlings were grown in the greenhouse. At approximately V 2 to V 4 the vegetative growth stage, the R 0 plants were sprayed with S3100 at a rate of 20 g / ha. The plants were then evaluated for injury and the injury scores were recorded 1 - 14 days after treatment. Transgenic plants with a single copy of the transgenic DNA insert (i.e., single event plants) were identified, and only those R 0 plants containing only a single copy and passing the herbicide spray test were self - pollinated to produce R 1 seeds. For some variant HemG PPO enzymes, the R 1 plants were grown in the greenhouse and sprayed with S3100 at a rate of 60 g / ha at approximately V 2 to V 4 the vegetative growth stage. The plants were then evaluated for injury and the injury scores were recorded 1 - 14 days after treatment.

[0146] Transgenic soybean and corn plants with a visual injury score of 20% or less were scored as passing the herbicide tolerance screen and thus showed tolerance to the PPO herbicide. The percentage of all plants passing the herbicide tolerance screen was calculated for each variant HemG PPO enzyme. Any construct in which 25% or more of the individual plants showed good tolerance (visual injury score of 20% or less) was considered to effectively confer herbicide tolerance.

[0147] The tests confirmed that the results obtained from the protoplast analysis (performed as described above) were consistent with the results obtained in whole plants, thus validating the use of protoplast analysis as a screening tool. Of the 25 microbial HemG PPO variants tested in stably transformed corn or soybeans (or both), 20 were found to effectively confer herbicide tolerance (producing more than 25% of plants with a visual injury score of 20% or less). Of these twenty, 14 had efficacy results higher than the positive control H_N90. The results are provided in Table 8.

[0148] Table 8. Test results of HemG variants in soybean and corn plants.

[0149]

[0150]

[0151] In cotton, excised embryos (Coker 130) were transformed with these vectors using Agrobacterium tumefaciens and standard methods known in the art. The regenerated R 0The transgenic seedlings are grown in the greenhouse and tested as described above.

[0152] In the case of transgenic plants, other herbicides can be tested for tolerance. This can be done, for example, by growing multiple transgenic plants for each HemG PPO and dividing the plants into groups. Each group is sprayed with one or more PPO herbicides (one PPO herbicide per group) to evaluate tolerance. For example, at the 2-4 true leaf stage, the transgenic plants are sprayed with lactofen at about 220 gai / ha or 440 gai / ha or flumioxazin at about 210 g / ha or 420 g / ha. Then, 1-14 days after treatment, the plants are evaluated for damage and the damage score is recorded. The unsprayed transgenic plants are used for phenotypic comparison with unsprayed non-transgenic plants.

Claims

1. A recombinant DNA molecule, said recombinant DNA molecule comprising a nucleic acid molecule encoding a genetically engineered protein having herbicide-tolerant protoporphyrinogen oxidase activity and a heterologous promoter operably linked to said nucleic acid molecule, wherein said protein comprises a HemG-like protoporphyrinogen oxidase, and wherein the amino acid sequence of said protein is as shown in SEQ ID NO:1 and is engineered to contain an amino acid substitution at positions corresponding to residues 125 to 146 of SEQ ID NO:1, wherein said substitution is selected from the group consisting of: L125I, Y127W, P128A, P128S, P128T, R129A, R129E, R129K, R129L, R129N, R129Q, Y130L, W132I, K135R, M137A, M137C, M137I, M137L, M137S, M137V, I138L, Q139K, Q139S, L140A, L140C, L140F, L140G, L140H, L140I, L140M, L140T, I141V, M142L, M142S, M142V, R143A, G146A, and G146D.

2. The recombinant DNA molecule according to claim 1, wherein the amino acid sequence of said protein is as shown in the group consisting of SEQ ID NO:24 - 124 and 249 - 263.

3. The recombinant DNA molecule according to claim 1, wherein said amino acid substitution is located in a long-chain insertion loop in said enzyme.

4. The recombinant DNA molecule according to claim 1, wherein said heterologous promoter is functional in a plant cell.

5. The recombinant DNA molecule according to claim 4, wherein said nucleic acid molecule is operably linked to a DNA molecule encoding a transit sequence for targeting said protein intracellularly.

6. The recombinant DNA molecule according to claim 1, wherein said recombinant DNA molecule is contained in the genome of a plant cell.

7. A DNA construct, said DNA construct comprising the recombinant DNA molecule according to claim 1.

8. A genetically engineered protein, said genetically engineered protein encoded by the recombinant DNA molecule according to claim 1.

9. A method for conferring PPO herbicide tolerance to a plant, seed, cell, or plant part, said method comprising: heterologously expressing the genetically engineered protein according to claim 8 in said plant, seed, cell, or plant part.

10. The method according to claim 9, wherein the herbicide tolerance is against at least one PPO herbicide selected from the group consisting of acifluorfen, fomesafen, lactofen, fluoroglycofen-ethyl, oxyfluorfen, flumioxazin, pyraflufen-ethyl, carfentrazone-ethyl, sulfentrazone, fluthiacet-methyl, oxadiargyl, oxadiazon, pyraclonil, sulfentrazone, fluthiacet-methyl, fluoroxaprop-p-ethyl, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3,4-dihydro-3-oxo-4-prop-2-ynyl-2H-1,4-benzoxazin-6-yl)-1,3,5-triazinan-2,4-dione, and S-3100.

11. A method for producing a herbicide-tolerant plant, the method comprising the steps of: a) transforming a plant cell with the recombinant DNA molecule according to claim 1; and b) regenerating a plant from the plant cell containing the recombinant DNA molecule.

12. The method according to claim 11, further comprising the step of selecting the plant or its progeny for PPO herbicide tolerance.

13. The method according to claim 11, further comprising the step of crossing the regenerated plant with itself or with a second plant to produce progeny.

14. A method for controlling or preventing weed growth in a plant growth area, the method comprising applying an effective amount of at least one PPO herbicide to a plant growth area containing a transgenic plant or seed containing the recombinant DNA molecule according to claim 1, wherein the transgenic plant or seed is tolerant to the PPO herbicide.

15. The method according to claim 14, wherein the PPO herbicide is selected from the group consisting of acifluorfen, fomesafen, lactofen, fluoroglycofen-ethyl, oxyfluorfen, flumioxazin, pyraflufen-ethyl, carfentrazone-ethyl, sulfentrazone, fluthiacet-methyl, oxadiargyl, oxadiazon, pyraclonil, sulfentrazone, fluthiacet-methyl, fluoroxaprop-p-ethyl, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3,4-dihydro-3-oxo-4-prop-2-ynyl-2H-1,4-benzoxazin-6-yl)-1,3,5-triazinan-2,4-dione, and S-3100.

16. A method for identifying a nucleotide sequence encoding a protein having herbicide-tolerant protoporphyrinogen oxidase activity, the method comprising: a) transforming an Escherichia coli strain lacking herbicide-tolerant PPO enzyme activity with a bacterial expression vector containing the recombinant DNA molecule according to claim 1; and b) growing the transformed Escherichia coli to identify a protein having herbicide-tolerant protoporphyrinogen oxidase activity.

17. A method for screening herbicide tolerance genes, the method comprising: a) expressing the recombinant DNA molecule according to claim 1 in a plant cell; and b) identifying plant cells that exhibit tolerance to a PPO herbicide.

18. A method for producing a plant that is tolerant to a PPO herbicide and at least one other herbicide, the method comprising: a) Obtaining a plant comprising the recombinant DNA molecule as claimed in claim 1; b) Crossing said plant with a second plant comprising tolerance to said at least one other herbicide, and c) Selecting progeny plants resulting from said crossing and comprising tolerance to a PPO herbicide and said at least one other herbicide.

19. A method for reducing the development of herbicide-tolerant weeds, said method comprising: a) Growing a plant comprising the recombinant DNA molecule as claimed in claim 1 in a crop growth environment; and b) Applying a PPO herbicide to said crop growth environment, wherein said crop plant is tolerant to said PPO herbicide.

20. The method as claimed in claim 19, wherein said PPO herbicide is selected from the group consisting of acifluorfen, fomesafen, lactofen, fluoroglycofen-ethyl, oxyfluorfen, flumioxazin, pyraclonil, carfentrazone-ethyl, sulfentrazone, fluthiacet-methyl, oxadiargyl, oxadiazon, pyraflufen-ethyl, sulfentrazone, fluthiacet-methyl, oxadiargyl, oxadiazon, pyraflufen-ethyl, fluthiamide, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3,4-dihydro-3-oxo-4-prop-2-ynyl-2H-1,4-benzoxazin-6-yl)-1,3,5-triazinan-2,4-dione and S-3100.

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