Triketone dioxygenase variants for herbicide resistance

Abstract

The present disclosure relates to recombinant DNA molecules and engineered proteins useful for conferring tolerance to β-triketone herbicides, such as mesotrione. The present disclosure also provides herbicide-tolerant transgenic plants, plant parts, cells and seeds containing the recombinant DNA molecules, and methods of using the same.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 307,525, filed February 7, 2022, the entire disclosure of which is incorporated herein by reference.

[0002] The present disclosure relates to the fields of agriculture, plant biotechnology, and plant molecular biology. More specifically, the present disclosure relates to recombinant DNA molecules encoding engineered proteins useful for conferring tolerance to β-triketone herbicides, such as mesotrione, a phytotoxic inhibitor of plant hydroxyphenylpyruvate dioxygenase (HPPD). The present disclosure also provides herbicide-tolerant transgenic plants, plant parts, cells, and seeds comprising the recombinant DNA molecules, as well as methods of using the same.

[0003] Incorporating sequence tables A sequence listing in computer readable form is submitted with this application by electronic submission and is incorporated herein by reference in its entirety. The sequence listing is contained in a file named MONS_523WO_ST26, which is 89.7 kilobytes in size (measured on the MS Windows operating system) and was created on February 1, 2023. [Background technology]

[0004] Agricultural crop production often utilizes transgenic traits created using biotechnology methods. A foreign gene, also known as a transgene, can be introduced into a plant to produce the transgenic trait. Expression of the transgene in the plant confers a trait to the plant, such as herbicide resistance. Examples of transgenic traits that are herbicide resistant include glyphosate resistance, glufosinate resistance, and dicamba resistance. With an increasing number of weed species that are resistant to commonly used herbicides, new herbicide resistance traits are needed in the art. Herbicides of particular interest include herbicides that inhibit hydroxyphenylpyruvate dioxygenase (HPPD), referred to as HPPD inhibitor herbicides. HPPD inhibitor herbicides provide control of a wide variety of herbicide-resistant weeds, making these herbicide resistance traits particularly useful in combination with one or more other herbicide resistance trait(s) in cropping systems. Provided herein are novel engineered enzymes useful for conferring HPPD herbicide tolerance in plants. Summary of the Invention

[0005] Provided herein is a recombinant DNA molecule comprising a nucleic acid sequence encoding a protein having triketone dioxygenase (TDO) activity, the protein having at least 70% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 and 16, the protein comprising at least one amino acid substitution compared to SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 or 16, the protein comprising at least one amino acid substitution at position 230 of SEQ ID NO: 2, threonine (T), alanine (A), leucine (L), valine (V), isoleucine (I), serine (S) or glycine (G), serine (S) at position corresponding to position 301 of SEQ ID NO: 2, arginine (R) at position corresponding to position 332 of SEQ ID NO: 2, isoleucine (I) at position corresponding to position 335 of SEQ ID NO: 2, tyrosine (Y), phenylalanine (F) or glycine (G) at position corresponding to position 141 of SEQ ID NO: 2. The amino acid sequence may include histidine (H), alanine (A), isoleucine (I), leucine (L), serine (S), threonine (T), valine (V) or glycine (G) at a position corresponding to position 229 of SEQ ID NO:2, alanine (A), isoleucine (I), leucine (L) or valine (V) at a position corresponding to position 299 of SEQ ID NO:2, or any combination thereof, and (i) if threonine (T), alanine (A), leucine (L), valine (V), isoleucine (I) or serine (S) is present at a position corresponding to position 230 of SEQ ID NO:2, then glycine (G) must be present at a position corresponding to position 229 of SEQ ID NO:2, and (ii) if alanine (A), leucine (L), valine (V), isoleucine (I), threonine (T) or serine (S) is present at a position corresponding to position 229 of SEQ ID NO:2, then glycine (G) must be present at a position corresponding to position 230 of SEQ ID NO:2.

[0006] In some embodiments, the proteins described herein have at least 70% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 6, 8, 10, 12, 14, and 16, and the protein comprises a threonine (T) at a position corresponding to position 230 of SEQ ID NO:2, a serine (S) at a position corresponding to position 301 of SEQ ID NO:2, an arginine (R) at a position corresponding to position 332 of SEQ ID NO:2, an isoleucine (I) at a position corresponding to position 335 of SEQ ID NO:2, or any combination thereof. In other embodiments, the protein comprises a threonine (T) at a position corresponding to position 230 of SEQ ID NO:2, a serine (S) at a position corresponding to position 301 of SEQ ID NO:2, an arginine (R) at a position corresponding to position 332 of SEQ ID NO:2, and an isoleucine (I) at a position corresponding to position 335 of SEQ ID NO:2. In certain embodiments, the protein has at least 70% sequence identity to SEQ ID NO:2 and contains one or more amino acid substitutions selected from the group consisting of H141F, H141Y, G229A, G229I, G229L, G229S, G229T, G229V, T230A, T230G, T230I, T230L, T230S, T230V, L299A, L299I, L299V, and any combination thereof. In some embodiments, the protein has at least 70% sequence identity to SEQ ID NO:4 and comprises one or more amino acid substitutions selected from the group consisting of H141F, H141Y, A230G, A230I, A230L, A230S, A230T, A230V, G231A, G231I, G231L, G231S, G231T, G231V, F300A, F300I, F300L, F300V, G302S, I333R, and any combination thereof. In other embodiments, the protein comprises one or more amino acid substitutions selected from the group consisting of G231T, G302S, I333R, and any combination thereof. In a further embodiment, the protein further comprises an isoleucine (I) at position 336 of SEQ ID NO:4.

[0007] In additional embodiments, the proteins described herein have at least 70% sequence identity to SEQ ID NO:6 and include one or more amino acid substitutions selected from the group consisting of Q140F, Q140H, Q140Y, G229A, G229I, G229L, G229S, G229T, G229V, P230A, P230G, P230I, P230L, P230S, P230T, P230V, L299A, L299I, L299V, A301S, L332R, V335I, and any combination thereof. In further embodiments, the proteins include one or more amino acid substitutions selected from the group consisting of P230T, A301S, L332R, V335I, and any combination thereof. In some embodiments, the protein has at least 70% sequence identity to SEQ ID NO:8 and comprises one or more amino acid substitutions selected from the group consisting of H144F, H144Y, G233A, G233I, G233L, G233S, G233T, G233V, G234A, G234I, G234L, G234S, G234T, G234V, F303A, F303I, F303L, F303V, G305S, L336R, F339I, and any combination thereof. In certain embodiments, the protein comprises one or more amino acid substitutions selected from the group consisting of G234T, G305S, L336R, F339I, and any combination thereof. In other embodiments, the protein has at least 70% sequence identity to SEQ ID NO: 10 and comprises one or more amino acid substitutions selected from the group consisting of H145F, H145Y, G234A, G234I, G234L, G234S, G234T, G234V, G235A, G235I, G235L, G235S, G235T, G235V, F304A, F304I, F304L, F304V, A306S, V337R, L340I, and any combination thereof. In certain embodiments, the protein comprises one or more amino acid substitutions selected from the group consisting of G235T, A306S, V337R, L340I, and any combination thereof.In some embodiments, the protein has at least 70% sequence identity to SEQ ID NO: 12 and comprises one or more amino acid substitutions selected from the group consisting of Q140F, Q140H, Q140Y, G229A, G229I, G229L, G229S, G229T, G229V, F299A, F299I, F299L, F299V, G230A, G230I, G230L, G230S, G230T, G230V, G301S, I332R, L335I, and any combination thereof. In certain embodiments, the protein comprises one or more amino acid substitutions selected from the group consisting of G230T, G301S, I332R, L335I, and any combination thereof. In other embodiments, the protein has at least 70% sequence identity to SEQ ID NO: 14 and comprises one or more amino acid substitutions selected from the group consisting of H141F, H141Y, A212G, A212I, A212L, A212S, A212T, A212V, G213A, G213I, G213L, G213S, G213T, G213V, F282A, F282I, F282L, F282V, G284S, V315R, V318I, and any combination thereof. In further embodiments, the protein comprises one or more amino acid substitutions selected from the group consisting of G213T, G284S, V315R, V318I, and any combination thereof. In some embodiments, the protein has at least 70% sequence identity to SEQ ID NO: 16 and comprises one or more amino acid substitutions selected from the group consisting of Q140F, Q140H, Q140Y, G229A, G229I, G229L, G229S, G229T, G229V, F299A, F299I, F299L, F299V, G230A, G230I, G230L, G230S, G230T, G230V, G301S, I332R, L335R, and any combination thereof. In certain embodiments, the protein comprises one or more amino acid substitutions selected from the group consisting of G230T, G310S, I332R, L335R, and any combination thereof. In some embodiments, the protein has at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 and 16.In other embodiments, the protein has at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16. In particular embodiments, the protein comprises SEQ ID NO: 48. In further embodiments, the protein further comprises a leucine (L) at a position corresponding to position 11 of SEQ ID NO: 2, a lysine (K) at a position corresponding to position 18 of SEQ ID NO: 2, a glutamine (Q) at a position corresponding to position 32 of SEQ ID NO: 2, a proline (P) at a position corresponding to position 35 of SEQ ID NO: 2, an isoleucine (I) at a position corresponding to position 48 of SEQ ID NO: 2, a glycine (G) at a position corresponding to position 54 of SEQ ID NO: 2, an isoleucine (I) at a position corresponding to position 82 of SEQ ID NO: 2, an aspartic acid (D) at a position corresponding to position 89 of SEQ ID NO: 2, a histidine (H) at a position corresponding to position 116 of SEQ ID NO: 2, a glutamic acid (E) at a position corresponding to position 120 of SEQ ID NO: 2, 2, a valine (V) at a position corresponding to position 133 of SEQ ID NO:2, a histidine (H) at a position corresponding to position 167 of SEQ ID NO:2, a cysteine ​​(C) at a position corresponding to position 173 of SEQ ID NO:2, a phenylalanine (F) at a position corresponding to position 199 of SEQ ID NO:2, a threonine (T) at a position corresponding to position 204 of SEQ ID NO:2, a glycine (G) at a position corresponding to position 230 of SEQ ID NO:2, a glycine (G) at a position corresponding to position 242 of SEQ ID NO:2, a proline (P) at a position corresponding to position 256 of SEQ ID NO:2, a serine (S) at a position corresponding to position 274 of SEQ ID NO:2, a lysine (K) at a position corresponding to position 279 of SEQ ID NO:2, or any combination thereof.

[0008] Also provided herein is a recombinant DNA molecule comprising a nucleic acid sequence encoding a protein having triketone dioxygenase (TDO) activity, the protein having at least 50% identity to SEQ ID NO:2 and containing one or more amino acid substitutions selected from the group consisting of I11L, A18K, K32Q, S35P, L48I, S54G, V82I, S89D, N116H, Q120E, I133V, N167H, T173C, L199F, A204T, T230G, S242G, E256P, C274S, R279K, and any combination thereof. In some embodiments, the protein comprises the following amino acid substitutions: I11L, A18K, K32Q, S35P, L48I, S54G, V82I, S89D, N116H, Q120E, I133V, N167H, T173C, L199F, A204T, T230G, S242G, E256P, C274S, and R279K. In other embodiments, the protein comprises the following amino acid substitutions: I11L, A18K, K32Q, L48I, S54G, V82I, S89D, Q120E, I133V, N167H, T173C, L199F, A204T, T230G, S242G, E256P, C274S, and R279K. In certain embodiments, the protein comprises the following amino acid substitutions: I11L, A18K, K32Q, L48I, S54G, V82I, S89D, N116H, I133V, N167H, T173C, L199F, A204T, T230G, S242G, E256P, C274S, and R279K. In some embodiments, the protein comprises the following amino acid substitutions: I11L, A18K, K32Q, L48I, S54G, V82I, S89D, N116H, Q120E, I133V, N167H, T173C, L199F, A204T, T230G, E256P, C274S, and R279K. In other embodiments, the protein comprises the following amino acid substitutions: I11L, A18K, K32Q, S35P, L48I, S54G, V82I, S89D, Q120E, I133V, N167H, T173C, L199F, A204T, T230G, E256P, C274S, and R279K.In some embodiments, the protein comprises the following amino acid substitutions: I11L, A18K, K32Q, S35P, L48I, S54G, V82I, S89D, N116H, I133V, N167H, T173C, L199F, A204T, T230G, E256P, C274S, and R279K. In other embodiments, the protein comprises the following amino acid substitutions: I11L, A18K, K32Q, S35P, L48I, S54G, V82I, S89D, N116H, Q120E, I133V, N167H, T173C, L199F, A204T, T230G, C274S, and R279K. In further embodiments, the protein comprises the following amino acid substitutions: I11L, A18K, K32Q, S35P, L48I, S54G, V82I, S89D, N116H, Q120E, I133V, N167H, T173C, L199F, A204T, T230G, E256P, and R279K. In particular embodiments, the protein comprises the following amino acid substitutions: I11L, A18K, S35P, S54G, V82I, S89D, N116H, Q120E, N167H, T173C, L199F, A204T, T230G, C274S, and R279K. In other embodiments, the protein comprises I11L, A18K, K32Q, S35P, L48I, S54G, V82I, S89D, N116H, Q120E, I133V, N167H, T173C, A204T, T230G, S242G, E256P, and C274S amino acid substitutions. In some embodiments, the protein further comprises a threonine (T) at a position corresponding to position 230 of SEQ ID NO:2, a serine (S) at a position corresponding to position 301 of SEQ ID NO:2, an arginine (R) at a position corresponding to position 332 of SEQ ID NO:2, and an isoleucine (I) at a position corresponding to position 335 of SEQ ID NO:2. In other embodiments, the protein has at least 80% identity to SEQ ID NO:2. In further embodiments, the protein has at least 90% identity to SEQ ID NO:2. In certain embodiments, the protein comprises SEQ ID NO: 18, 28, 30, 32, 34, 36, 38, 40, 42 or 44.

[0009] Provided herein is a recombinant DNA molecule comprising a nucleic acid sequence encoding a protein having triketone dioxygenase (TDO) activity, the nucleic acid sequence comprising SEQ ID NO:1. In some embodiments, the DNA molecule further comprises a heterologous promoter operably linked to the nucleic acid sequence encoding the protein having triketone dioxygenase (TDO) activity. In other embodiments, the promoter comprises a constitutive promoter. In further embodiments, the heterologous promoter functions in the plant cell. In certain embodiments, the recombinant DNA molecule is comprised within the genome of the plant cell. Also provided are DNA constructs comprising the recombinant DNA molecules and engineered proteins encoded by the recombinant DNA molecules described herein.

[0010] Also provided are transgenic plants, plant seeds, plant cells or plant parts comprising the recombinant DNA molecules described herein. In some embodiments, the transgenic plants, plant seeds, plant cells or plant parts are tolerant to at least one 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor herbicide. In other embodiments, the HPPD inhibitor herbicide is selected from the group consisting of mesotrione, benzobicyclon (BBC), tembotrione, sulcotrione, tefuryltrione, 2-(2-nitro-4-trifluoromethylbenzoyl)cyclohexane-1,3-dione (NTBC), pyrazolate, benzofenap, pyrazoxyfen, isoxaflutole, pyrazolinate, topramezone, and any combination thereof. In some embodiments, the transgenic plants, plant seeds, plant cells or plant parts are tolerant to at least a second herbicide. In certain embodiments, the second herbicide is selected from the group consisting of ACCase inhibitors, ALS inhibitors, EPSPS inhibitors, synthetic auxins, photosynthesis inhibitors, glutamine synthesis inhibitors, HPPD inhibitors, PPO inhibitors and long-chain fatty acid inhibitors.In further embodiments, the ACCase inhibitor is an aryloxyphenoxypropionate or cyclohexanedione, the ALS inhibitor is a sulfonylurea, an imidazolinone, a triazolopyrimidine or a triazolinone, the EPSPS inhibitor is glyphosate, the synthetic auxin is a phenoxy herbicide, a benzoic acid, a carboxylic acid or a semicarbazone, the photosynthesis inhibitor is a triazine, a triazinone, a nitrile, a benzothiadiazole or a urea, the glutamine synthesis inhibitor is glufosinate, the HPPD inhibitor is an isoxazole, a pyrazolone or a triketone, the PPO inhibitor is a diphenyl ether, an N-phenylphthalimide, an aryltriazinone or a pyrimidinedione, or the long-chain fatty acid inhibitor is a chloroacetamide, an oxyacetamide or a pyrazole. Commercial products containing the recombinant DNA molecules described herein are also provided.

[0011] Further disclosed herein is a method for conferring HPPD inhibitor herbicide tolerance to a plant, plant seed, plant cell or plant part, the method comprising heterologously expressing in the plant, plant seed, plant cell or plant part an engineered protein encoded by a recombinant DNA molecule comprising a nucleic acid sequence encoding a protein having triketone dioxygenase (TDO) activity, the protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 and 16. and wherein the protein contains at least one amino acid substitution compared to SEQ ID NO:2, 4, 6, 8, 10, 12, 14 or 16, and the protein contains at least one amino acid substitution at position 230 of SEQ ID NO:2, threonine (T), alanine (A), leucine (L), valine (V), isoleucine (I), serine (S) or glycine (G), at position corresponding to position 230 of SEQ ID NO:2, serine (S) at position corresponding to position 301 of SEQ ID NO:2, arginine (R) at position corresponding to position 332 of SEQ ID NO:2, arginine (R) at position corresponding to position 335 of SEQ ID NO:2, arginine (R) at position corresponding to position 336 of SEQ ID NO:2, arginine (R) at position corresponding to position 337 of SEQ ID NO:2, arginine (R) at position corresponding to position 339 of SEQ ID NO:2, arginine (R) at position corresponding to position 340 of SEQ ID NO:2, arginine (R) at position corresponding to position 341 of SEQ ID NO:2, arginine (R) at position corresponding to position 342 of SEQ ID NO:2, arginine (R) at position corresponding to position 343 of SEQ ID NO:2, arginine (R) at position corresponding to position 344 of SEQ ID NO:2, arginine (R) at position corresponding to position 345 of SEQ ID NO:2, arginine (R) at position corresponding to position 346 of SEQ ID NO:2, arginine (R) at position corresponding to position 347 of SEQ ID NO:2, arginine (R) at position corresponding to position 348 of SEQ ID NO:2, arginine (R) at position corresponding to position 349 of SEQ ID NO 2; alanine (A), isoleucine (I), leucine (L), serine (S), threonine (T), valine (V) or glycine (G) at a position corresponding to position 229 of SEQ ID NO:2; alanine (A), isoleucine (I), leucine (L) or valine (V) at a position corresponding to position 299 of SEQ ID NO:2, or any combination thereof; (i) a nucleotide sequence of SEQ ID NO:2 It is necessary that (ii) if threonine (T), alanine (A), leucine (L), valine (V), isoleucine (I), or serine (S) is present at the position corresponding to 230, then glycine (G) is present at the position corresponding to 229 of SEQ ID NO:2, and (iii) if alanine (A), leucine (L), valine (V), isoleucine (I), threonine (T), or serine (S) is present at the position corresponding to 229 of SEQ ID NO:2, then glycine (G) is present at the position corresponding to 230 of SEQ ID NO:2.

[0012] Provided herein is a method for producing a herbicide-tolerant plant, the method comprising: (a) transforming a plant cell with a recombinant DNA molecule described herein; and (b) regenerating a plant from the plant cell containing the recombinant DNA molecule. In some embodiments, the method further comprises selecting the plant or its progeny for HPPD inhibitor resistance. In other embodiments, the method comprises crossing the regenerated plant with itself or with a second plant to generate progeny. Also provided herein is a method for producing a plant that is tolerant to an HPPD inhibitor herbicide and at least one other herbicide, the method comprising: (a) crossing a plant provided herein with a second plant that contains resistance to at least one other herbicide; and (b) selecting a progeny plant resulting from the cross and containing resistance to the HPPD inhibitor herbicide and at least one other herbicide.

[0013] Also provided herein is a method for controlling or preventing the growth of weeds in a plant growing area, the method comprising spraying an effective amount of at least one HPPD inhibitor herbicide to a plant growing area containing a transgenic plant or seed provided herein, the transgenic plant or seed being resistant to the HPPD inhibitor herbicide. Also provided herein is a method for screening for herbicide resistance genes, the method comprising (a) expressing a recombinant DNA molecule of the present disclosure in a plant cell, and (b) identifying a plant cell that is resistant to the HPPD inhibitor herbicide. Also provided herein is a method for reducing the occurrence of herbicide resistant weeds, the method comprising (a) growing a plant described in the present disclosure in a crop growing environment, and (b) spraying the crop growing environment with an HPPD inhibitor herbicide and at least one other herbicide, the crop plant being resistant to the HPPD inhibitor herbicide and at least one other herbicide. [Brief description of the drawings]

[0014] [Figure 1]A, B, and C show that constitutive expression of codon-optimized rice TDO (SEQ ID NO:2) in soybean confers superior resistance to mesotrione. A shows images of plants from transgenic event 125 treated with mesotrione at a range of 1x to 16x the commercial use rate. Mesotrione herbicide use rates (application rates) are expressed as grams active ingredient per hectare (g ai / ha), with 1x commercial use rate being 105 g ai / ha. B shows images of wild-type control plants treated with mesotrione at a range of 1x to 16x the commercial use rate. C shows mesotrione-induced injury rates in plants from the TDO transgenic event and the wild-type control. Plants at the V1 growth stage were sprayed with mesotrione (in Syngenta's Callisto® in 1% crop oil) at 1x, 2x, 4x, 8x, and 16x the commercial rate (equivalent to 105, 210, 420, 840, and 1680 g ai / ha, respectively). Crop injury ratings were taken 13 days after mesotrione treatment. Each bar represents the mean + / - standard error (SE). [Diagram 2] Figure 1 shows that expression of Os.TDO in transgenic soybean plants conferred resistance to tembotrione and sulcotrione in addition to mesotrione. Os.TDO transgenic event 125 and wild-type control plants were sprayed with mesotrione (Callisto, 210 g ai / ha), tembotrione (Laudis, 184 g ai / ha), and sulcotrione (Sulcogan, 660 g ai / ha) at twice the commercial use rate. Each bar represents the mean injury rating of eight replicates + / - SE. * indicates significant difference compared to corresponding control at p<0.01. [Diagram 3]A and B show the effect of increasing temperature on mesotrione injury and the correlation with the decrease in TDO protein levels detected by ELISA. Os.TDO transgenic soybean plants and wild type control plants were grown at 80°F until the V2 growth stage, after which half of the plants were transferred to 99°F. After 3 days, leaf samples were harvested to measure TDO protein levels, and then the plants were treated with 2x and 8x the application rate of mesotrione. Crop injury ratings were obtained 11 days after mesotrione treatment. A shows plants expressing Os.TDO transgenic soybean event 125 treated with mesotrione and grown at 99°F (left) or 80°F (center), and wild type plants treated and grown at 80°F (right). B shows data showing the correlation of increased mesotrione injury to decreased TDO protein levels in transgenic soybean plants grown under high temperature conditions. Black bars represent TDO protein levels (ppm) per dry weight. The grey bars represent the percentage of plant injury caused by 2x and 8x mesotrione treatments. [Figure 4A] Figure 1 shows TDO expression levels in transgenic soybean plants. Soybeans were planted under typical greenhouse conditions (85°F daytime, 75°F nighttime) and grown to approximately the V3 growth stage. At that time, half of the plants were transferred to a hotter growth room (99°F daytime, 82°F nighttime, 60% relative humidity) for 3 days prior to collection of leaf samples and spray application of a 4x rate of mesotrione. Mesotrione was applied to both greenhouse and growth room plants. Visual damage ratings were taken 7 days after treatment. Figure 1 shows Os.TDO protein levels (ppm) in transgenic soybean plants with Os.TDO driven by a strong constitutive promoter and two different moderate constitutive promoters (Med A and Medium B). [Figure 4B]Figure 1 shows the corresponding response to mesotrione injury at elevated temperatures. Soybeans were planted under typical greenhouse conditions (85°F daytime, 75°F nighttime) and grown to approximately the V3 growth stage. At that time, half of the plants were transferred to a higher temperature growth room (99°F daytime, 82°F nighttime, 60% relative humidity) for three days prior to collection of leaf samples and spray application of a 4x application rate of mesotrione. Mesotrione was applied to both greenhouse and growth room plants. Visual injury ratings were taken 7 days after treatment. Percent crop injury following mesotrione application is shown. For these transgenic plants, the grey bars represent mesotrione injury ratings at 85°F and the black bars represent mesotrione injury ratings at 99°F. [Figure 5A] Figure 1 shows protein content and percent damage by ELISA for transgenic soybean plants containing Os.TDO variants. Figure 2 shows protein levels (ppm) by ELISA assay for Os.TDO variants with improved enzyme stability at higher temperatures. TDO variants were grown and compared at 85°F (gray) and 99°F (black). Os.TDO served as the control. [Figure 5B] Figure 1 shows protein content and percent injury by ELISA for transgenic soybean plants containing Os.TDO variants. Figure 2 shows injury ratings (%) for soybean transgenic plants expressing Os.TDO and its variants after mesotrione application. TDO variants were grown and compared at 85°F (gray) and 99°F (black). Os.TDO served as the control. [Figure 6] A comparison of the enzyme activity at room temperature of the variants from round 2. The enzyme activity was expressed as Kcat / Km, M-1sec-1. [Figure 7] The active site of TDO is shown, with amino acid residues T230, S301, R332, and I335 (boxed) of Os.TDO that are hypothesized to be activity determinants. The amino acid residues are modeled based on the structure of Arabidopsis anthocyanidin synthase (1GP5), and all other amino acids shown are numbered from 1GP5. Mesotrione (marked Meso) is shown in the predicted three-dimensional structure. [Figure 8] Figure 1 shows the activities of TDO proteins from rice, maize, sorghum, and a sorghum variant (SwM) containing four amino acid substitutions (P230T, A301S, L332R, V335I) at the corresponding positions corresponding to rice TDO. Activities are expressed as nmol / min / mg protein and were the average of three separate assays on the same enzyme sample (technical replicates). Asterisks indicate that the activity was significantly different from the blank (p<0.05). [Figure 9A] Phylogenetic analysis of the active sites of TDO-like proteins is shown. Sequence alignment of 11 TDO sequences from various plant species with Arabidopsis anthocyanidin synthase (also known as At.dioxy or Arabidopsis leucoanthocyanidin dioxygenase) is shown. All residues within 7 Å of DQH (trans-dihydroquercetin) are highlighted in bold and underlined. Residues of interest are marked with numbers that refer to the crystal structure of Arabidopsis anthocyanidin synthase (1GP5). Residues corresponding to amino acids 236, 306, 338, and 341 of At.dioxy (encircled by solid lines) have been experimentally explored, and residues corresponding to amino acids 144 and 304 of At.dioxy (encircled by dashed lines) may also contribute to activity in monocots. Numbers in parentheses after each protein name correspond to its sequence number. [Figure 9B] Phylogenetic analysis of the active sites of TDO-like proteins. The crystal structure of anthocyanidin synthase (1GP5) in complex with DQH (trans-dihydroquercetin, shown in light grey) is shown, with all residues with side chains within 7 Å of DQH highlighted in dark grey. [Figure 9C] FIG. 1 shows a phylogenetic analysis of the active site of TDO-like proteins. The 1GP5 structure is shown with positions of Phe144, Val235, Ser236, and Phe304 highlighted. DQH is shown in black. [Figure 9D]Figure 1 shows a phylogenetic analysis of the active sites of TDO-like proteins. The phylogenetic tree was generated by performing 1000 rounds of bootstrapping on the aligned active site residues using the RAxML program. [Figure 10] Protein sequence alignment of Os.TDO with its homologues in other plant species and the closely related protein At.dioxy. Amino acid residues in solid boxed boxes represent unique amino acid residues T230, S301, R332, and I335 in the active site of Os.TDO. Dashed boxed boxes represent non-unique Os.TDO amino acid residues in the active site. Amino acid residues that are conserved only among TDO protein sequences are indicated by solid triangles, whereas residues that are conserved among all sequences are indicated by hollow triangles. Numbers in parentheses after each protein name correspond to its SEQ ID NO: [Figure 11] 1 shows a universal genetic code table showing all possible mRNA triplet codons (where T in a DNA molecule is replaced by U in an RNA molecule) and the amino acid encoded by each codon. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] A brief description of the sequence SEQ ID NO:1 and SEQ ID NO:2 are the polynucleotide and amino acid sequences of Os.TDO, a TDO from Oryza sativa. The polynucleotide sequence was codon optimized for dicotyledonous expression.

[0016] SEQ ID NO:3 and SEQ ID NO:4 are the polynucleotide and amino acid sequences of Zm.TDO, a TDO homologue from Zea mays. The polynucleotide sequence was codon optimized for dicotyledonous expression.

[0017] SEQ ID NO:5 and SEQ ID NO:6 are the polynucleotide and amino acid sequences of Sb.TDO, a TDO homologue from Sorghum bicolor. The polynucleotide sequence was codon optimized for dicotyledonous expression.

[0018] SEQ ID NO:7 and SEQ ID NO:8 are the polynucleotide and amino acid sequences of Si.TDO, a TDO homologue from Setaria italica. The polynucleotide sequence was codon optimized for dicotyledonous expression.

[0019] SEQ ID NO:9 and SEQ ID NO:10 are the polynucleotide and amino acid sequences of Ta.TDO, a TDO homologue from Triticum aestivum. The polynucleotide sequence was codon optimized for dicotyledonous expression.

[0020] SEQ ID NO:11 and SEQ ID NO:12 are the polynucleotide and amino acid sequences of ANDge.TDO, a TDO homologue from Andropogon gerardii. The polynucleotide sequence was codon optimized for dicotyledonous expression.

[0021] SEQ ID NO: 13 and SEQ ID NO: 14 are the polynucleotide and amino acid sequences of Cl.Col.TDO1, a TDO homologue from Coix lacryma-jobi. The polynucleotide sequence was codon-optimized for dicotyledonous expression.

[0022] SEQ ID NO:15 and SEQ ID NO:16 are the polynucleotide and amino acid sequences of Cl.Col.TDO2, a TDO homologue from Coix lacryma-jobi. The polynucleotide sequence was codon-optimized for dicotyledonous expression.

[0023] SEQ ID NO:17 and SEQ ID NO:18 are the polynucleotide and amino acid sequences of Os.TDO variant D08. The polynucleotide sequence was codon optimized for dicotyledonous expression.

[0024] SEQ ID NO:19 and SEQ ID NO:20 are the polynucleotide and amino acid sequences of Os.TDO variant B03. The polynucleotide sequence was codon optimized for dicotyledonous expression.

[0025] SEQ ID NO:21 and SEQ ID NO:22 are the polynucleotide and amino acid sequences of Os.TDO variant F09. The polynucleotide sequence was codon optimized for dicotyledonous expression.

[0026] SEQ ID NO:23 and SEQ ID NO:24 are the polynucleotide and amino acid sequences of Os.TDO variant A04. The polynucleotide sequence was codon optimized for dicotyledonous expression.

[0027] SEQ ID NO:25 and SEQ ID NO:26 are the polynucleotide and amino acid sequences of Os.TDO variant B09. The polynucleotide sequence was codon optimized for dicotyledonous expression.

[0028] SEQ ID NO:27 and SEQ ID NO:28 are the polynucleotide and amino acid sequences of Os.TDO variant TDO_1. The polynucleotide sequence was codon optimized for dicotyledonous expression.

[0029] SEQ ID NO:29 and SEQ ID NO:30 are the polynucleotide and amino acid sequences of Os.TDO variant TDO_2. The polynucleotide sequence was codon optimized for dicot expression.

[0030] SEQ ID NO:31 and SEQ ID NO:32 are the polynucleotide and amino acid sequences of Os.TDO variant TDO_3. The polynucleotide sequence was codon optimized for dicot expression.

[0031] SEQ ID NO: 33 and SEQ ID NO: 34 are the polynucleotide and amino acid sequences of Os.TDO variant TDO_4. The polynucleotide sequence was codon optimized for dicot expression.

[0032] SEQ ID NO:35 and SEQ ID NO:36 are the polynucleotide and amino acid sequences of Os.TDO variant TDO_5. The polynucleotide sequence was codon optimized for dicotyledonous expression.

[0033] SEQ ID NO:37 and SEQ ID NO:38 are the polynucleotide and amino acid sequences of Os.TDO variant TDO_6. The polynucleotide sequence was codon optimized for dicot expression.

[0034] SEQ ID NO:39 and SEQ ID NO:40 are the polynucleotide and amino acid sequences of Os.TDO variant TDO_7. The polynucleotide sequence was codon optimized for dicotyledonous expression.

[0035] SEQ ID NO: 41 and SEQ ID NO: 42 are the polynucleotide and amino acid sequences of Os.TDO variant TDO_8. The polynucleotide sequence was codon optimized for dicot expression.

[0036] SEQ ID NO: 43 and SEQ ID NO: 44 are the polynucleotide and amino acid sequences of Os.TDO variant TDO_9. The polynucleotide sequence was codon optimized for dicot expression.

[0037] SEQ ID NO: 45 is a different codon-optimized polynucleotide sequence of Os.TDO variant TDO_6. The corresponding amino acid sequence is SEQ ID NO: 38.

[0038] SEQ ID NO: 46 is a different codon-optimized polynucleotide sequence of Os.TDO variant TDO_8. The corresponding amino acid sequence is SEQ ID NO: 42.

[0039] SEQ ID NO: 47 and SEQ ID NO: 48 are the polynucleotide and amino acid sequences of Sb.TDO variant SwM. The polynucleotide sequence was codon optimized for dicot expression.

[0040] SEQ ID NO: 49 is the polynucleotide sequence of HIS1, a wild-type TDO from Oryza sativa. The corresponding amino acid sequence is SEQ ID NO: 2.

[0041] SEQ ID NO:50 is the amino acid sequence of leucoanthocyanidin dioxygenase (At.dioxy) from Arabidopsis thaliana. Leucoanthocyanidin dioxygenase is also known as anthocyanidin synthase.

[0042] SEQ ID NO:51 is the amino acid sequence of Gm.TDO, a TDO homologue from soybean.

[0043] SEQ ID NO:52 is the amino acid sequence of At.TDO, a TDO homologue from Arabidopsis thaliana.

[0044] SEQ ID NO:53 is the amino acid sequence of the TDO homologue, Nt.TDO, from Nicotiana tabacum.

[0045] SEQ ID NO:54 is the amino acid sequence of anthocyanidin synthase from Arabidopsis thaliana. It is identical to SEQ ID NO:50, except for the lack of the initial methionine. SEQ ID NO:54 is also referred to herein as 2BRT.

[0046] Detailed Description In plants, hydroxyphenylpyruvate dioxygenase (HPPD) is a key enzyme in catalyzing the biosynthesis of plastoquinone and tocopherol. Plastoquinone is a cofactor for phytoene desaturase, an enzyme important in the production of carotenoids. Carotenoids protect chlorophyll from degradation and are necessary for photosynthesis. Carotenoids and tocopherols are involved in the detoxification of reactive oxygen species and the scavenging of free radicals in plant tissues. Inhibition of HPPD leads to uncoupling of photosynthesis, lack of auxiliary light-harvesting pigments, and most importantly, the destruction (bleaching) of chlorophyll by ultraviolet radiation and reactive oxygen species due to the lack of photoprotection normally provided by carotenoids (Norris et al., 1995). Bleaching of photosynthetically active tissues leads to growth inhibition and death of the plant. Several molecules that inhibit HPPD have proven to be very effective herbicides. The triketone family is one of the most widely used HPPD inhibitor herbicides.

[0047] The present disclosure overcomes the limitations of the prior art by providing novel engineered proteins, herein referred to as TDO proteins, and recombinant DNA molecules encoding them, as well as compositions and methods of using them. TDO proteins are proteins that contain an Fe2OG dioxygenase domain that have the ability to inactivate triketone herbicides. As used herein, inactivating a herbicide means that the herbicide no longer has herbicidal activity on the plant. TDO proteins exhibit novel substrate selectivity, useful enzyme kinetics, and improved enzyme stability at high temperatures. Transgenic plants expressing TDO proteins demonstrate improved tolerance to sprays of triketone herbicides.

[0048] I. Engineered Proteins and Recombinant DNA Molecules Provided herein are novel engineered proteins and recombinant DNA molecules encoding them. As used herein, TDO, "triketone dioxygenase," or a protein having "triketone dioxygenase activity" refers to a protein, specifically a protein that contains an Fe(II) / 2-oxoglutarate-dependent dioxygenase or Fe2OG dioxygenase domain that has the ability to hydroxylate a triketone herbicide. As used herein, the term "engineered" refers to a non-natural DNA, protein, cell, or organism that is not normally found in nature and that has been created by human intervention. An "engineered protein," "engineered enzyme," or "engineered triketone dioxygenase" refers to a protein, enzyme, or triketone dioxygenase whose amino acid sequence has been conceived and created in a laboratory using one or more of the techniques of biotechnology, protein design, or protein engineering, such as molecular biology, protein biochemistry, bacterial transformation, plant transformation, site-directed mutagenesis, directed evolution using random mutagenesis, genome editing, gene editing, gene cloning, DNA ligation, DNA synthesis, protein synthesis, and DNA shuffling. For example, an engineered protein may have one or more deletions, insertions, or substitutions compared 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 create a DNA molecule encoding an engineered protein, such as an engineered TDO protein that includes at least a first amino acid substitution compared to a wild-type TDO protein, as described herein.

[0049] An example of an engineered protein provided herein is an Fe2OG dioxygenase domain-containing protein (referred to herein as a "TDO protein" or "TDO enzyme") having at least 70% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16 and having triketone dioxygenase activity, the protein comprising at least one amino acid substitution compared to SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, or 16, the protein comprising at least one amino acid substitution selected from the group consisting of threonine (T), alanine (A), leucine (L), valine (V), isoleucine (I), serine (S), or glycine (G) at a position corresponding to position 230 of SEQ ID NO: 2, serine (S) at a position corresponding to position 301 of SEQ ID NO: 2, arginine (R) at a position corresponding to position 332 of SEQ ID NO: 2, isoleucine (I) at a position corresponding to position 335 of SEQ ID NO: 2, tyrosine (I) at a position corresponding to position 141 of SEQ ID NO: 2, or glycine (G) at a position corresponding to position 230 ... (Y), phenylalanine (F) or histidine (H), alanine (A), isoleucine (I), leucine (L), serine (S), threonine (T), valine (V) or glycine (G) at a position corresponding to position 229 of SEQ ID NO:2, alanine (A), isoleucine (I), leucine (L) or valine (V) at a position corresponding to position 299 of SEQ ID NO:2, or any combination thereof, and (i) threonine ( 2 at position corresponding to position 229 of SEQ ID NO:2 if (A), leucine (L), valine (V), isoleucine (I), threonine (T), alanine (A), leucine (L), valine (V), isoleucine (I), or serine (S) is present, then a glycine (G) must be present at a position corresponding to position 229 of SEQ ID NO:2, and (ii) a glycine (G) must be present at a position corresponding to position 230 of SEQ ID NO:2 if (A), leucine (L), valine (V), isoleucine (I), threonine (T), or serine (S) is present at a position corresponding to position 229 of SEQ ID NO:2. In certain embodiments, the engineered proteins provided herein include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of any combination of such substitutions.

[0050] The engineered protein is an enzyme with triketone dioxygenase activity. As used herein, "triketone dioxygenase activity" refers to the ability of a protein to inactivate one or more HPPD inhibitor herbicides. The term "triketone dioxygenase activity" refers to the ability to catalyze the hydroxylation of a triketone herbicide, such as mesotrione, thereby inactivating the herbicide. The enzyme activity of triketone dioxygenase can be measured by any means known in the art, for example, by an enzyme assay that measures the second hydroxylation of a triketone, such as the production of oxymesotrione from mesotrione, in the presence of a protein with triketone dioxygenase activity by fluorescence, high performance liquid chromatography (HPLC) or mass spectrometry (MS).

[0051] As used herein, "wild type" means naturally occurring. As used herein, a "wild type DNA molecule", "wild type polypeptide", or "wild type protein" is a naturally occurring DNA molecule, polypeptide, or protein, i.e., a DNA molecule, polypeptide, or protein that exists before in nature. A wild type version of a polypeptide, protein, or DNA molecule may be useful for comparison with an engineered protein or gene. An example of a wild type protein useful for comparison with an engineered protein provided by the present disclosure is the HIS1 enzyme from Oryza sativa. An example of a wild type DNA molecule useful for comparison with a recombinant DNA molecule provided by the present disclosure is the HIS1 gene from Oryza sativa. A wild type version of a protein or DNA molecule may be useful as a control in an experiment.

[0052] "Wild-type plant" refers to a naturally occurring plant. Such wild-type plants can also be useful for comparison with plants that contain recombinant or engineered DNA molecules or proteins. Examples of wild-type plants that are useful for comparison with plants that contain recombinant or engineered DNA molecules or proteins can be the same kind of plant as the plant that contains engineered DNA molecules or proteins, such as the protein that confers herbicide resistance, and thus are genetically distinct from the plant that contains herbicide resistance.

[0053] As used herein, "control" refers to an experimental control designed for comparison purposes.For example, in transgenic plant analysis, the control plant is the same type of plant as the experimental plant (i.e., the plant being tested), but does not contain the transgenic insertion, recombinant DNA molecule, DNA construct, or variant protein or gene of the experimental plant. Examples of control plants useful for comparison with transgenic plants include non-transgenic LH244 maize (ATCC Accession No. PTA-1173) for corn plants, non-transgenic A3555 soybean (ATCC Accession No. PTA-10207) for soybean plants, non-transgenic Coker 130 (Plant Variety Protection (PVP) No. 8900252) for cotton plants, non-transgenic Brassica napus variety 65037 fertility restorer line (Canada Plant Breeders' Rights Application 06-5517) for canola or Brassica napus plants, and non-transgenic wheat variety Samson germplasm (PVP 1994) for wheat plants. As used herein, the term "recombinant" refers to non-natural DNA, proteins, cells, seeds, or organisms that are the result of genetic engineering and are created by human intervention. A "recombinant DNA molecule" is a DNA molecule that contains a DNA sequence that does not occur in nature and is therefore 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 a DNA molecule provided herein that encodes a protein having triketone dioxygenase activity operably linked to a heterologous promoter. A "recombinant protein" is a protein that contains an amino acid sequence that does not occur in nature and is therefore the result of human intervention, such as an engineered protein. A recombinant cell, seed, or organism is a cell, seed, or organism that contains transgenic or heterologous DNA or protein, such as a transgenic plant cell, seed, or plant that contains a DNA construct of the present disclosure or an engineered protein.

[0054] As used herein, the term "DNA" or "DNA molecule" refers to a double-stranded DNA molecule (i.e., a polymer of deoxyribonucleotide bases or polynucleotide molecules) of genomic or synthetic origin read from the 5' (upstream) end to the 3' (downstream) end. As used herein, the term "DNA sequence" refers to the nucleotide sequence of a DNA molecule. The nomenclature used herein corresponds to that of Title 37, United States Code of Federal Regulations § 1.822 and is set forth in the tables in WIPO Standard ST.25 (1998), Appendix 2, Tables 1 and 3.

[0055] The present disclosure provides a nucleic acid molecule encoding a protein comprising at least 70% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16 and having triketone dioxygenase activity, the protein comprising at least one amino acid substitution compared to SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, or 16, the protein comprising at least one amino acid substitution at position 230 of SEQ ID NO: 2, threonine (T), alanine (A), leucine (L), valine (V), isoleucine (I), serine (S), or glycine (G) at position 301 of SEQ ID NO: 2, threonine (T), alanine (A), leucine (L), valine (V), isoleucine (I), serine (S), or glycine (G) at position 302 of SEQ ID NO: 2, or at position 303 of SEQ ID NO: 2, 2; serine (S) at a position corresponding to position 332 of SEQ ID NO:2; arginine (R) at a position corresponding to position 335 of SEQ ID NO:2; tyrosine (Y), phenylalanine (F) or histidine (H) at a position corresponding to position 141 of SEQ ID NO:2; alanine (A), isoleucine (I), leucine (L), serine (S), threonine (T), valine (V) or glycine (G) at a position corresponding to position 229 of SEQ ID NO:2; alanine (A), isoleucine (I), leucine (L) or valine (V) at a position corresponding to position 299 of SEQ ID NO:2, or any combination thereof. However, (i) if threonine (T), alanine (A), leucine (L), valine (V), isoleucine (I) or serine (S) is present at the position corresponding to position 230 of SEQ ID NO:2, then glycine (G) must be present at the position corresponding to position 229 of SEQ ID NO:2, and (ii) if alanine (A), leucine (L), valine (V), isoleucine (I), threonine (T) or serine (S) is present at the position corresponding to position 229 of SEQ ID NO:2, then glycine (G) must be present at the position corresponding to position 230 of SEQ ID NO:2.

[0056] As used herein, the term "protein-coding DNA molecule" refers to a DNA molecule that comprises a DNA sequence that encodes 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 that are folded or arranged in a biologically functional manner and those that are not. As used herein, "protein coding sequence" refers to a DNA sequence that encodes a protein. As used herein, "sequence" refers to a sequential arrangement of nucleotides or amino acids. A "DNA sequence" may refer to a series of nucleotides or a DNA molecule that comprises a series of nucleotides, and a "protein sequence" may refer to a series of amino acids or a protein that comprises a series of amino acids. The boundaries of a protein coding sequence are usually determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3'-terminus.

[0057] The engineered proteins include, among others, modified V max , K m , K i ,I C 50These may be produced by altering or modifying the wild-type protein sequence to produce new proteins with modified property(s) or novel combinations of useful protein properties, such as substrate specificity, inhibitor / herbicide specificity, substrate selectivity, ability to interact with partner proteins or other components within the cell such as membranes, and protein stability. Modifications may be made at specific amino acid positions in the protein, or by substituting an alternative amino acid for a typical amino acid found at that same position in nature (i.e., in the wild-type protein). Amino acid modifications may be made as single amino acid substitutions in the protein sequence, or in combination with one or more other modifications, such as one or more other amino acid substitution(s), deletions, or additions. In some embodiments, the engineered protein has an altered protein property, such as a property that results in reduced sensitivity to one or more herbicides compared to the wild-type protein, or a property that results in the ability to confer resistance to one or more herbicides to a transgenic plant expressing the engineered protein. Thus, in other embodiments, the disclosure provides engineered proteins, such as TDO proteins, and recombinant DNA molecules encoding same, having one or more amino acid substitution(s) selected from the group consisting of H141F, H141Y, G229A, G229I, G229L, G229S, G229T, G229V, T230A, T230G, T230I, T230L, T230S, T230V, L299A, L299I, L299V, and all combinations thereof, where the position of the amino acid substitution(s) is relative to the amino acid position set forth in SEQ ID NO: 2. In certain embodiments, the engineered proteins provided herein include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of any combination of such substitutions, where the modifications are made at positions relative to functionally equivalent positions to positions in the amino acid sequence provided as SEQ ID NO: 2.

[0058] Similar modifications can be made at analogous positions in any protein containing an Fe2OG dioxygenase domain by aligning the amino acid sequence of the Fe2OG dioxygenase domain-containing protein to be mutated with the amino acid sequence of the Fe2OG dioxygenase domain-containing protein having triketone dioxygenase activity. An example of a sequence encoding an Fe(II) / 2-oxoglutarate-dependent dioxygenase with triketone dioxygenase activity that can be used for alignment is SEQ ID NO:2. FIG. 10 shows an alignment of Os.TDO, the TDO protein of SEQ ID NO:2, and homologs of Os.TDO (SEQ ID NOs:4, 6, 8, 10, 12, 14, and 16). As shown in FIG. 10, it is well within the capabilities of one of ordinary skill in the art to use sequence homology information to make the amino acid modifications described herein in, for example, the protein of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, or 16 to generate a TDO protein.

[0059] As used herein, the term "isolated" refers to at least partially separating a molecule from other molecules that are typically associated with it in its natural state. As used herein, the term "isolated" refers to a DNA molecule that is separated from the nucleic acids that normally flank the DNA molecule in its natural state. For example, a DNA molecule that encodes a protein that naturally occurs in bacteria would be an isolated DNA molecule if it were not present within the DNA of the bacteria in which the DNA molecule encoding the protein is naturally found. Thus, a DNA molecule that is fused or operably linked to one or more other DNA molecule(s) with which it would not be associated in nature, for example as a result of recombinant DNA or plant transformation techniques, is considered isolated herein. Such molecules are considered isolated even if they are integrated into the chromosome of a host cell or are present in a nucleic acid solution with other DNA molecules.

[0060] A number of methods well known in the art can be used to isolate and manipulate DNA molecules, or fragments thereof, as disclosed herein. For example, polymerase chain reaction (PCR) techniques can be used to amplify a particular starting DNA molecule or to produce variants of the original molecule. DNA molecules, or fragments thereof, can also be obtained by other techniques, such as by directly synthesizing the fragments by chemical means, as is commonly accomplished by using automated oligonucleotide synthesizers.

[0061] Due to the degeneracy of the genetic code, a variety of different DNA sequences may encode proteins such as the modified or engineered proteins disclosed herein. For example, FIG. 11 provides a universal genetic code table showing 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. DNA sequences encoding TDO proteins having the amino acid substitutions described herein can be generated by introducing mutations into a DNA sequence encoding a wild-type TDO protein using methods known in the art and the information provided in FIG. 11. It is well within the capabilities of one of ordinary skill in the art to create alternative DNA sequences that encode the same or essentially the same modified or engineered proteins as those described herein. These variants or alternative DNA sequences are within the scope of the embodiments described herein. As used herein, reference to "essentially the same" sequence refers to sequences that encode amino acid substitutions, deletions, additions, or insertions that do not significantly alter the functional activity of the protein encoded by the DNA molecules of the embodiments described herein. Allelic variants of the nucleotide sequences encoding wild-type or engineered proteins are also encompassed within the scope of the embodiments described herein. Amino acid substitutions other than those specifically exemplified or naturally occurring in wild-type or engineered TDO proteins are also contemplated within the scope of the embodiments described herein, so long as the TDO protein having such substitutions still retains substantially the same functional activity as described herein.

[0062] Where it is desirable to provide sequences useful for DNA manipulation (such as restriction enzyme recognition sites or recombination-based cloning sites), sequences preferred in plants (such as plant codon usage or Kozak consensus sequences), or sequences useful for designing DNA constructs (such as spacer or linker sequences), the recombinant DNA molecules provided herein may be synthesized and modified, either in whole or in part, by methods known in the art. The disclosure 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 to any of the recombinant DNA molecules or amino acid sequences provided herein and have triketone dioxygenase activity. As used herein, the term "percent sequence identity" or "% sequence identity" refers to the percentage of identical nucleotides or amino acids in a linear polynucleotide or amino acid sequence of a reference ("query") sequence (or its complement) compared to a test ("subject") sequence (or its complement) when the two sequences are optimally aligned (with appropriate nucleotide or amino acid insertions, deletions, or gaps totaling less than 20 percent of the reference sequence over the comparison window).Optimal alignment of sequences to align a comparison window is well known to those of skill in the art and may be performed by tools such as the Smith and Waterman local homology algorithm, the Needleman and Wunsch homology alignment algorithm, the Pearson and Lipman similarity search method, as well as by computer implementations of these algorithms, such as GAP, BESTFIT, FASTA, and TFASTA available as part of the sequence analysis software package GCG® Wisconsin Package® (Accelrys Inc., San Diego, CA), 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)), for example using default parameters. The "identity fraction" for an aligned segment of a test sequence and a reference sequence is the number of identical elements shared by the two aligned sequences in the portion of the segment of the reference sequence that is being aligned, i.e., the entire reference sequence or a smaller defined portion of the reference sequence, divided by the total number of elements. Percent sequence identity is expressed as the percent identity multiplied by 100. Comparison of one or more sequences may be to the full length sequence or a portion thereof, or to a longer sequence.

[0063] II. Expression Constructs As used herein, a "DNA construct" is a recombinant DNA molecule that contains two or more heterologous DNA sequences. The DNA construct is useful for expressing transgenes and may be included in vectors and plasmids. The DNA construct may be used in a vector for transformation (i.e., introduction of heterologous DNA into a host cell) to produce recombinant bacteria or transgenic plants and cells (and as such may be included in the plasmid DNA or genomic DNA of a transgenic plant, seed, cell, or plant part). As used herein, a "vector" refers to any recombinant DNA molecule that can be used for bacterial or plant transformation. The DNA molecules provided herein can be inserted into a vector as part of a DNA construct, for example, having the DNA molecule operably linked to a heterologous gene expression element that functions to affect expression of an engineered protein encoded by the DNA molecule in a plant. Methods for making and using DNA constructs and vectors are well known in the art and are described in detail in handbooks and laboratory manuals, including, for example, 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 for 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, examples of which include a promoter for expressing an operably linked DNA, an operably linked protein-coding DNA molecule, and an operably linked 3' untranslated region (UTR), as follows. Useful gene expression elements include one or more of the above types of elements, such as promoters, 5' UTRs, enhancers, leaders, cis-acting elements, introns, transport sequences, 3' UTRs, and one or more selectable marker transgenes, but are not limited to these.

[0064] The term "transgene" refers to a DNA molecule that is artificially integrated into the genome of an organism as a result of human intervention, such as by plant transformation techniques. As used herein, the term "transgenic" is meant to include transgenes, e.g., 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 conveyed or conferred by the presence of a transgene integrated into the plant genome. As a result of such genome modification, a transgenic plant is distinct from an associated wild-type plant, and a transgenic trait is a trait that is not naturally found in a wild-type plant. Transgenic plants provided herein include the recombinant DNA molecules and engineered proteins of the present disclosure.

[0065] As used herein, the term "heterologous" refers to the relationship between two or more things that are not normally associated in nature, e.g., come from different sources, or are not normally found together in nature in any other manner. For example, a DNA molecule or protein can be heterologous to another DNA molecule, protein, cell, plant, seed, or organism when they are not normally found together or in the same association in nature. In certain embodiments, a first DNA molecule is heterologous to a second DNA molecule when these two DNA molecules are not normally found together in nature in the same association. For example, a protein-encoding recombinant DNA molecule is heterologous to an operably linked promoter when such a combination is not normally found in nature. Similarly, a protein is heterologous to an operably linked second protein, such as a transport peptide, when such a combination is not normally found in nature. In another embodiment, a recombinant DNA molecule encoding a TDO protein is heterologous to an operably linked promoter that is functional in a plant cell when such a combination is not normally found in nature. A recombinant DNA molecule can also be heterologous to the cell, seed, or organism into which it is inserted if it would not naturally occur in that cell, seed, or organism.

[0066] "Heterologous protein" refers to a protein that exists in a plant, seed, cell, tissue, or organism that does not naturally exist, or that is operably linked to a protein that is not naturally associated with it.An example of a heterologous protein is the engineered TDO protein that contains at least the first amino acid substitution described herein, which is 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 transport peptide, that is not naturally associated with it, or that is introduced into a plant cell that does not naturally exist, using genetic engineering techniques.

[0067] As used herein, "operably linked" refers to two or more DNA molecules or two or more proteins linked in such a manner that one can affect the function of the other. The operably linked DNA molecules or operably linked proteins may be part of a single contiguous molecule and may or may not be contiguous. For example, a promoter is operably linked to a protein-encoding DNA molecule in a DNA construct when the two DNA molecules are positioned such that the promoter can affect expression of the transgene.

[0068] The DNA construct of the present disclosure may comprise a promoter operably linked to the protein-encoding DNA molecule provided herein, so that the promoter drives the expression of the engineered protein. Promoters useful in the implementation of the contemplated embodiments include promoters that function in cells to express the operably linked DNA molecule, such as bacterial or plant promoters. Plant promoters are diverse and well known in the art, including, for example, inducible, viral, synthetic, constitutive, temporally regulated, spatially regulated, or spatio-temporally regulated promoters.

[0069] In some embodiments, the DNA constructs provided herein include a DNA sequence encoding a trafficking sequence operably linked to a heterologous DNA sequence encoding a TDO protein, such that the trafficking sequence facilitates intracellular localization of the protein molecule. Trafficking sequences are known in the art as signal sequences, targeting peptides, targeting sequences, localization sequences, and transit peptides. Examples of trafficking sequences are chloroplast transit peptides (CTPs), mitochondrial transit sequences (MTSs), or chloroplast-mitochondria dual transit peptides. By facilitating intracellular localization of the protein, the trafficking sequence may increase the accumulation of the recombinant protein, protect the protein from proteolysis, or improve the level of herbicide resistance, thereby reducing the level of damage to the cell, seed, or organism after application of the herbicide. CTPs and other targeting molecules that may be used in connection with the present disclosure are well known in the art.

[0070] As used herein, "transgene expression," "expressing a transgene," "protein expression," and "expressing a protein" refer to the production of a protein through the process of transcribing a DNA molecule into messenger RNA (mRNA), translating the mRNA into a polypeptide chain that is ultimately folded into a protein. A protein-encoding DNA molecule may be operably linked in a DNA construct to a heterologous promoter for use in expressing the protein in a cell transformed with the recombinant DNA molecule.

[0071] III. Transgenic plants In one aspect, the present disclosure provides cells, tissues, plants and seeds comprising the recombinant DNA molecules or engineered proteins described herein. These cells, tissues, plants and seeds comprising the recombinant DNA molecules or engineered proteins exhibit tolerance to one or more HPPD inhibitor herbicide(s).

[0072] One method of producing such cells, tissues, plants and seeds is by plant transformation. Methods for transformation of host plant cells suitable for use with the present disclosure include any method by which DNA can be introduced into the cell (e.g., recombinant DNA constructs are stably integrated into plant chromosomes) and are well known in the art. Two effective and widely used methods for cell transformation are Agrobacterium-mediated transformation and biolistic-mediated transformation. Biolistic methods are exemplified, for example, in U.S. Pat. Nos. 5,550,318, 5,538,880, 6,160,208, and 6,399,861. Agrobacterium-mediated transformation methods are described, for example, in U.S. Pat. No. 5,591,616. Cells carrying the recombinant DNA molecule or engineered protein of the present disclosure can be selected for the presence of the recombinant DNA molecule or engineered protein, for example, by its encoded enzyme activity, before or after regenerating such cells into plants.

[0073] According to some embodiments, another method of producing cells, plants and seeds is by genome modification using site-specific integration or genome editing. Targeted modification of plant genome through the use of genome editing methods can be used to create improved plant lines through modification of plant genome DNA. As used herein, "site-specific integration" refers to genome editing methods that allow targeted insertion of one or more nucleic acids of interest into plant genome. Suitable methods for modifying wild-type DNA sequences or existing transgenic sequences or inserting DNA into plant genome at a predefined chromosomal site include any method known in the art. Exemplary methods include the use of sequence-specific nucleases such as zinc finger nucleases, engineered or natural meganucleases, TALE-endonucleases, or RNA-guided endonucleases (e.g., Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) / Cas9 systems, CRISPR / Cpf1 systems, CRISPR / CasX systems, CRISPR / CasY systems, CRISPR / Cascade systems). Some embodiments relate to methods of genome editing by introducing precise base pair modifications in plant genomes 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.

[0074] In some embodiments, modification or replacement of an existing coding sequence, such as a TDO coding sequence or another existing transgenic insertion, in a plant genome with a sequence encoding an engineered protein, such as the engineered TDO coding sequence provided herein, or with an expression cassette encoding such an engineered protein, is provided. Some embodiments involve the use of known genome editing methods, such as zinc finger nucleases, engineered or natural meganucleases, TALE-endonucleases, or RNA-guided endonucleases (e.g., Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) / Cas9 system, CRISPR / Cpf1 system, CRISPR / CasX system, CRISPR / CasY system, CRISPR / Cascade system).

[0075] Thus, some embodiments may relate to recombinant DNA constructs that include expression cassette(s) encoding site-specific nucleases and optionally any associated protein(s) for genome modification. These nuclease expression cassette(s) may be present in the same molecule or vector (in cis) as the donor template for templated editing or an expression cassette that includes a nucleic acid sequence encoding a TDO protein as described herein, or on a separate molecule or vector (in trans). Several methods for site-specific integration are known in the art that involve different sequence-specific nucleases (or protein complexes or guide RNAs or both) that cleave genomic DNA to generate double-strand breaks (DSBs) or nicks at desired genomic sites or loci. As understood in the art, during the process of repairing the DSB or nicks introduced by the nuclease enzyme, the donor template DNA, transgene, or expression cassette may become integrated into the genome at the site of the DSB or nick. The presence of homologous arm(s) in the DNA to be integrated may facilitate recruitment and targeting of the insertion sequence into the plant genome by homologous recombination during the repair process, although the insertion event may also occur through non-homologous end joining (NHEJ).

[0076] As used herein, the term "double-strand break inducer" refers to any agent capable of inducing a double-strand break (DSB) in a DNA molecule. In some embodiments, the double-strand break inducer is a site-specific genome modifying enzyme.

[0077] As used herein, the term "site-specific genome modifying enzyme" refers to any enzyme capable of modifying 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, the site-specific genome modifying enzyme includes an endonuclease, a recombinase, a transposase, a deaminase, a helicase, and any combination thereof. In some embodiments, the site-specific genome modifying enzyme is a sequence-specific nuclease.

[0078] In one aspect, the endonuclease is a meganuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), an Argonaute (non-limiting examples of Argonaute proteins include Thermus thermophilus Argonaute (TtAgo), Pyrococcus furiosus Argonaute (PfAgo), and Natronobacterium gregoryi Argonaute (NgAgo)), an RNA-guided nuclease, such as a CRISPR-associated nuclease (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, Cs c2, 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, homologs thereof, or modified versions thereof).

[0079] In some embodiments, the site-specific genome modifying enzyme is a recombinase. Non-limiting examples of recombinases include tyrosine recombinases linked to DNA recognition motifs provided herein, selected from the group consisting of Cre recombinase, Gin recombinase, Flp recombinase, and Tnp1 recombinase. 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 recombinase linked to a DNA recognition motif provided herein is selected from the group consisting of PhiC31 integrase, R4 integrase, and TP-901 integrase. In another aspect, the DNA transposase linked to a DNA binding domain provided herein is selected from the group consisting of TALE-piggyBac and TALE-Mutator.

[0080] Any of the DNA of interest provided herein can be integrated into a target site in a chromosomal sequence by introducing the DNA of interest and a site-specific genome modification enzyme provided. Any of the methods provided herein can utilize any of the site-specific genome modification enzymes provided herein.

[0081] IV. Herbicide-resistant plants Provided herein are cells, plants and seeds that are tolerant to HPPD inhibitor herbicides. Such cells, plants and seeds are useful in agricultural methods such as weed control and crop production.

[0082] As used herein, a "herbicide" is any molecule used to control, inhibit, or interfere with the growth of one or more plants. Exemplary herbicides include, among others, acetyl-CoA carboxylase (ACCase) 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., phenoxy, benzoic acid, carboxylic acid, semicarbazones), photosynthesis (photosystem II) inhibitors (e.g., triazines, triazinones, nitriles, benzothiadiazoles, and ureas), glutamine synthetase (GS) inhibitors (e.g., glutamine synthetase inhibitors ... These include, for example, benzophenone, benzoyl perfluorooctanoate, benzotriazole, benzocaine, benzoyl perfluorooctanoate ...

[0083] As used herein, "HPPD herbicides" are chemicals that target and inhibit the enzymatic activity of 4-hydroxyphenyl-pyruvate-dioxygenase (HPPD), an Fe-containing enzyme that catalyzes the second reaction in the catabolism of tyrosine, the conversion of 4-hydroxyphenylpyruvate to homogentisic acid. Inhibition of 4-hydroxyphenyl-pyruvate-dioxygenase prevents the formation of plant carotenoid pigments, which in turn leads to the degradation of chlorophyll and ultimately to lipid peroxidation of cell membranes. HPPD inhibitor herbicides are well known in the art and are commercially available. Examples of HPPD inhibitor herbicides include, but are not limited to, aclonifen, amitrole, beflubutamid, benzofenap, clomazone, diflufenican, fluridone, flurochloridone, flurtamone, isoxaclorthol, isoxaflutole, mesotrione, norflurazone, picolinafen, pyrazolinate, pyrazoxyfen, sulcotrione, tembotrione, tolpyralate, topramezone and tefuryltrione salts and esters thereof, and mixtures thereof.Proteins having triketone dioxygenase activity, as well as cells, seeds, plants and plant parts provided by the present disclosure, exhibit herbicide resistance to one or more HPPD inhibitor herbicides(es).

[0084] As used herein, "herbicide-tolerant" or "herbicide-tolerance" refers to the ability to be completely or partially unaffected by the presence or application of one or more herbicides, e.g., to resist the toxic effects of the herbicide when applied. A cell or organism is "herbicide-tolerant" if it is capable of maintaining at least some normal growth or phenotype in the presence of one or more herbicides. A trait is a herbicide-tolerant trait if its presence can confer improved resistance to the herbicide to a cell, plant, or seed compared to a wild-type or control cell, plant, or seed. A crop containing the herbicide-tolerant 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 in the presence of the herbicide compared to a wild-type or control enzyme. Herbicide tolerance can be complete or partial insusceptibility to a particular herbicide and can be expressed as a percent (%) of tolerance or insusceptibility to a particular herbicide.

[0085] Contemplated plants that can be produced using the herbicide-tolerance traits provided herein can include any plant, including crop plants such as soybean (Glycine max), corn (Zea mays), cotton (Gossypium spp.), and Brassica plants, among others.

[0086] Herbicides may be applied to the plant growing area including the plants and seeds provided by the present disclosure as a method for controlling weeds. Provided herein are plants and seeds that include herbicide-tolerant traits and are therefore tolerant to the application of one or more HPPD inhibitor herbicides. The application of the herbicide may be at the recommended commercial use rate (1X) or any fraction or multiple thereof, for example, twice the recommended commercial use rate (2X). The application rate of the herbicide may be expressed as pounds acid equivalent per acre (lb ae / acre) or grams acid equivalent per hectare (g ae / ha), or as pounds active ingredient per acre (lb ai / acre) or grams active ingredient per hectare (g ai / ha), depending on the herbicide and formulation. The application of the herbicide includes at least one HPPD inhibitor herbicide. The plant growing area may or may not include weed plants at the time of the application of the herbicide. The herbicidally effective dose of HPPD inhibitor herbicide(s) for use in an area to control weeds may range from about 0.1X to about 30X the label rate(s) over the growing season. One acre equals 2.47105 hectares and one pound equals 453.592 grams. Herbicide rates can be converted between English and metric systems as follows: (lb ai / ac) multiplied by 1.12 = (kg ai / ha), (kg ai / ha) multiplied by 0.89 = (lb ai / ac).

[0087] The herbicide applications may be sequential or tank-mixed with one HPPD inhibitor herbicide, two HPPD inhibitor herbicides, or a combination of several HPPD inhibitor herbicides or any other compatible herbicide. Multiple applications of one herbicide or two or more herbicides (in combination or alone), 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), may be used throughout the growing season on areas containing the transgenic plants of the present disclosure for control of a wide variety of dicotyledonous weeds, monocotyledonous weeds, or both.

[0088] As used herein, a "weed" is any undesirable plant. The plant may be generally considered undesirable for agricultural or horticultural purposes (e.g., Amaranthus species) or may be considered undesirable under certain circumstances (e.g., one species of crop plant in a field of a different species, aka a volunteer plant).

[0089] The transgenic plants, progeny, seeds, plant cells, and plant parts of the present disclosure may also contain one or more additional traits. The additional traits may be introduced by crossing a plant containing a transgene comprising a recombinant DNA molecule provided by the present disclosure with another plant containing one or more additional trait(s). As used herein, "crossing" refers to the propagation of two individual plants to produce a progeny plant. The two plants may thus be crossed to produce progeny containing desirable traits from each parent. As used herein, "progeny" refers to the descendants of any generation of a parent plant, and the transgenic progeny contains the DNA construct provided by the present disclosure and inherited from at least one parent plant.

[0090] The additional trait(s) may also be introduced by co-transforming a DNA construct for the additional transgenic trait(s) with a DNA construct comprising a recombinant DNA molecule provided by the present disclosure (e.g., all DNA constructs present as part of the same vector used for plant transformation), or by inserting the additional trait(s) into a transgenic plant comprising a DNA construct provided herein or vice versa (e.g., by using any of the plant transformation or genome editing methods 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 may include transgenic or non-transgenic tolerance to one or more herbicides, such as ACCase inhibitors (e.g., aryloxyphenoxypropionates and cyclohexanediones), ALS inhibitors (e.g., sulfonylureas, imidazolinones, triazolopyrimidines, and triazolinones), EPSPS inhibitors (e.g., glyphosate), synthetic auxins (e.g., phenoxy, benzoic acid, carboxylic acid, semicarbazones), photosynthesis inhibitors (e.g., triazines, triazinones, nitriles, benzothiadiazoles, and ureas), glutamine synthesis inhibitors (e.g., glufosinate), HPPD inhibitors (e.g., isoxazoles, pyrazolones, and triketones), PPO inhibitors (e.g., diphenyl ethers, N-phenylphthalimides, aryltriazinones, and pyrimidinediones), and long chain fatty acid inhibitors (e.g., chloroacetamindes, oxyacetamides, and pyrazoles), among others.Examples of herbicide resistance proteins useful for generating additional herbicide resistance traits are known in the art and include glyphosate-tolerant 5-enolpyruvylshikimate 3-phosphate synthase (e.g., CP4 EPSPS, 2mEPSPS), glyphosate oxidoreductase (GOX), glyphosate N-acetyltransferase (GAT), herbicide-resistant acetolactate synthase (ALS) / acetohydroxyacid synthase (AHAS), herbicide-resistant 4-hydroxyphenylpyruvate dioxygenase (HPPD), dicamba monooxygenase (DMO), phosphinothricin acetyltransferase (PAT), herbicide-resistant glutamine synthetase (GS), 2,4-dichlorophenoxypropionic acid dioxygenase (TfdA), R-2,4-dichlorophenoxypropionic acid dioxygenase (RdpA), S-2,4-dichlorophenoxypropionic acid dioxygenase (SdpA), herbicide-resistant protoporphyrinogen oxidase (PPO), and cytochrome P450 monooxygenase. Exemplary insect resistance traits may include resistance to one or more insect members within one or more of the orders Lepidoptera, Coleoptera, Hemiptera, Thysanoptera, Diptera, Hymenoptera, and Orthoptera, among others. Such additional traits are well known to those of skill in the art, and for example, a list of such transgenic traits is provided by the Animal and Plant Health Inspection Service (APHIS) of the United States Department of Agriculture (USDA).

[0091] The transgenic plants and progeny that are tolerant to HPPD inhibitor herbicides may be used by any breeding method known in the art. In plant lines containing two or more traits, the traits may be independently segregated, linked, or in plant lines containing three or more transgenic traits, a combination of both. Backcrossing to the parent plant and outcrossing with non-transgenic plants are also contemplated, as is vegetative propagation. Descriptions of breeding methods commonly used for different traits and crops are well known to those skilled in the art. Various assays may be performed to confirm the presence of the transgene(s) in a particular plant or seed. Such assays include, for example, molecular biology assays such as Southern and Northern blotting, PCR, and DNA sequencing, biochemical assays such as detection of the presence of protein products by immunological means (ELISA and Western blot) or by enzyme function, plant part assays such as leaf or root assays, and also by analysis of the whole plant phenotype.

[0092] The introgression of a transgenic trait into a plant genotype is achieved as a result of a backcross conversion process. The plant genotype into which the transgenic trait has been introgressed may be referred to as a backcross conversion genotype, line, inbred, or hybrid. Similarly, the plant genotype lacking the desired transgenic trait may be referred to as an unconverted genotype, line, inbred, or hybrid.

[0093] As used herein, the term "including" means "including, but not limited to."

[0094] Although the present disclosure has been described in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing from the spirit and scope of the present disclosure as further defined in the appended claims. Moreover, it should be recognized that all examples in the present disclosure, including those below, are provided as non-limiting examples. EXAMPLES

[0095] Example 1: Ectopic expression of TDO codon-optimized proteins in soybean confers superior mesotrione tolerance Mesotrione, a β-triketone herbicide, is a highly potent competitive inhibitor of 4-hydroxyphenylpyruvate dioxygenase (HPPD) and has been developed as a selective herbicide for many crops, including corn. Mesotrione provides pre- and post-emergence control of many important broadleaf weeds, as well as some annual weeds, in corn fields (Mitchell et al., 2001). However, it cannot be used to control weeds in soybean fields due to the sensitivity of soybean to mesotrione (Mitchell et al., 2001). The development of soybean plants that can tolerate twice (2X) the herbicide application rates recommended for commercial use of mesotrione, resulting in less than 10% herbicide injury, provides soybean farmers with an additional tool for weed control.

[0096] The rice HPPD inhibitor susceptibility 1 (HIS1) gene was found to confer tolerance to β-triketone herbicides, including mesotrione, but was not evaluated to determine whether the tolerance level was sufficient for commercial use (Maeda et al., 2019). HIS1 encodes an Fe(II) / 2-oxoglutarate-dependent dioxygenase that catalyzes the hydroxylation of multiple β-triketone herbicides, including benzobicyclon, tefuryltrione, sulcotrione, mesotrione, and tembotrione, resulting in their inactivation. To test whether ectopic expression of the rice HIS1 gene in soybean could confer sufficient mesotrione tolerance for trait development, the rice HIS1 gene sequence was codon-optimized for expression in soybean and designated as Os.TDO (SEQ ID NO:2).

[0097] The Os.TDO sequence was cloned into a series of binary vectors using standard cloning methods known in the art, and its expression is driven by various constitutive promoters and various 3'UTRs. Transgenic soybean plants containing these constructs were developed using the Agrobacterium-mediated transformation system as described in Larue et al. (2020). The copy number of the transgene was determined by Taqman assay, and events with a single copy of the T-DNA insertion were selected for evaluation of mesotrione resistance. Briefly, homozygous lines of R2 and subsequent generations of three soybean transgenic events derived from construct 32 were selected for mesotrione resistance greenhouse assays. Construct 32 contained two expression cassettes between the T-DNA borders; one for expression of Os.TDO and one for expression of a selectable marker. The Os.TDO cassette utilizes the cauliflower mosaic virus (CaMV) 35S promoter and the 5'UTR of petunia HSP70 to drive expression of Os.TDO and subsequently direct polyadenylation of the mRNA. i The 3'UTR of the nopaline synthase (nos) gene from the T-DNA region of the plasmid was utilized. The selectable marker cassette, which confers resistance to spectinomycin and streptomycin, contains the promoter, intron, and leader sequence of the Arabidopsis thaliana actin 7 gene, the transit peptide region of Arabidopsis thaliana EPSPS, the coding region of the E. coli Tn7 adenylyltransferase (AAD(3")) gene, and the 3' untranslated region of the Agrobacterium tumefaciens nopaline synthase gene.

[0098] Plants were grown in 3.5 inch pots with soybean seedling medium in a growth room at 28°C / 24°C (light / dark) and a 16-h light / 8-h dark photoperiod at 700 μE and fertilized with Peters general purpose fertilizer. Eight plants at the V1 growth stage were sprayed with mesotrione (in Syngenta's Callisto® in 1% crop oil) at 1x, 2x, 4x, 8x, and 16x the commercial rate (equivalent to 105, 210, 420, 840, and 1680 g active ingredient / ha, respectively). Crop damage ratings were taken 13 days after mesotrione treatment.

[0099] As shown in Figure 1A and Figure 1C, the three transgenic events showed excellent resistance to up to 16 times the commercial spray use rate of mesotrione (1X = 105 g ai / ha) without off-type, whereas wild-type control plants were severely damaged by mesotrione at all applied use rates (Figure 1B and Figure 1C). When treated with 8 times or less the use rate of mesotrione, the damage rate of all TDO transgenic events was less than 10%. When treated with extremely high doses of mesotrione (16X), the damage rate in the events remained less than 15% (Figure 1C). Furthermore, expression of Os.TDO in soybean provided resistance to two other triketone HPPD inhibitors, tembotrione and sulcotrione, although the level of resistance was not as strong as that observed with mesotrione (Figure 2).

[0100] Example 2: Identification and screening of TDO homologs for mesotrione resistance To investigate whether HIS1 from other species can also confer resistance to HPPD inhibitor herbicides, homologs were identified from various plant species using bioinformatics methods known in the art. Plant expression vectors containing TDO homologous sequences from Zea mays, Sorghum bicolor, foxtail millet, bread wheat, Andropogon gerardii, Job's tears, and Oryza sativa were constructed and verified using standard molecular cloning techniques. These homologous sequences were optimized for enhanced expression in dicotyledonous plants using an in-house sequence optimization program. Nucleotides downstream of the ATG of the wheat TDO sequence (nucleotide positions +4 to +11) were identified for enhanced protein expression in plants. [ka] The sequence was codon-optimized as (Sawant et al., 2001), resulting in an alanine to serine substitution at the fourth amino acid position from the N-terminus (A4S). Table 1 shows information on sequences identified in other plant species. [Table 1]

[0101] Plant expression vectors containing the selected TDO homologues under the control of one of two constitutive promoters were transformed into soybean by Agrobacterium tumefaciens-mediated methods. After Agrobacterium transformation, R0 transgenic soybean plants containing a single copy of the transgene were identified by molecular characterization and segregation analysis. Transgenic and wild-type (control) plants were grown in 6-inch pots filled with BM7 soilless mix in a growth room under conditions of 28°C / 23°C (light / dark) and a 16-h light / 8-h dark photoperiod. Plants were fertilized with 15-5-15 liquid fertilizer and treated with 210 g ai / ha mesotrione (1X=105 g ai / ha; Syngenta's Callisto® in 1% crop oil) at the V3-V4 growth stage. Plants were visually scored on a 0% to 100% scale (0% = no damage, 100% = plant death) compared to wild-type controls 7–14 days after treatment. Results summarized in Table 2 represent the average of all events (10–15) tested for each construct. [Table 2]

[0102] The results in Table 2 show that transgenic plants expressing Os.TDO showed minimal damage by mesotrione. Homologues from maize, sorghum, and foxtail millet showed reduced but still significant damage, whereas homologues from wheat, big bluestem, and Job's tears did not appear to offer any protection against mesotrione.

[0103] Example 3: Improved mesotrione resistance by enhanced expression Crop plants are often grown in different locations around the world and may be exposed to a wide range of climatic conditions. One of these climatic variables includes a wide range of temperatures. Previous studies on herbicide resistance trait development have shown that building robust resistance traits often requires that the enzymes conferring these traits are stable at higher temperatures (e.g., field conditions).

[0104] As shown in the previous example, expression of Os.TDO in transgenic soybean plants confers tolerance to mesotrione. However, when plants were grown in a growth chamber experiment with elevated temperatures to mimic a hot field environment, trait performance was reduced. This reduced performance was associated with reduced TDO protein expression (Figure 3).

[0105] One approach taken to address the instability of the TDO protein at higher temperatures was to improve the expression of the TDO protein through the selection of expression elements, particularly promoters, within the plant transformation constructs. Transgenic soybean plants expressing Os.TDO under the control of a strong constitutive promoter (construct 32), a moderate constitutive promoter A (construct 9), and a different moderate constitutive promoter B (construct 1) were generated at 85 °C. ° F or 99 ° A growth chamber experiment was conducted at F. At the V3-V4 growth stage, the plants were treated with 4X mesotrione (in Syngenta's Callisto® in 1% crop oil) (420 g ai / ha, 1X = 105 g ai / ha). Plants were visually scored on a 0% to 100% scale (0% = no damage, 100% = plant death) 7-14 days after treatment compared to untreated transgenic and wild-type controls.

[0106] As shown in Figure 4A, when the temperature is 85 ° F to 99 ° When elevated to 85°F, Os.TDO protein in transgenic plants was significantly reduced regardless of the promoter used. However, plants with a strong constitutive promoter still produced / retained higher Os.TDO content compared to plants with a moderate constitutive promoter. Higher Os.TDO protein content by the strong constitutive promoter was also significantly reduced at normal plant cultivation temperatures (85°F). ° F) and high temperatures (99 °F) both resulted in reduced plant damage (Figure 4B).

[0107] Example 4: Optimization of TDO protein for temperature stabilization to improve mesotrione resistance A rational design approach based on the ortholog analysis described above was employed to design a set of TDO variants with improved temperature stability along with desirable enzymatic activity. In the first round of screened variants, five variants passed high-throughput screening and were further characterized. [Table 3]

[0108] As shown in Table 3, all five variants showed superior thermostability (defined as the temperature at which 50% of the enzyme is denatured) compared to Os.TDO. However, all five also showed reduced enzyme activity in vitro. Enzyme activity for the hydroxylation of mesotrione was measured at room temperature and K cat / K m ,M -1 sec -1 Four of these variants were tested in soybean plants. All of the variants tested in transgenic soybean plants grew at normal growing temperatures (85 ° F) and high temperatures (99 ° Leaf samples collected from plants grown at Os.TDO (F) showed that enzyme stability tended to be significantly improved or was improved, as shown by ELISA (Figure 5A). However, when transgenic soybean plants were assayed for resistance to mesotrione sprays, three of the four variants showed damage ratings that tended to be significantly higher or were often higher than the Os.TDO control, especially at normal growing temperatures. Only one variant, called D08 (construct 10), had damage ratings similar to the Os.TDO control at normal temperatures and a possible trend toward lower damage compared to the Os.TDO control at elevated temperatures (Figure 5B).

[0109] Further testing of the best variant, D08, in field conditions confirmed that this variant provided good resistance to mesotrione spray treatments but was not significantly different from control plants expressing Os.TDO driven by a strong constitutive promoter. Because this trial was conducted in the field, the temperature range to which the plants were exposed was not controlled.

[0110] Based on these observations, additional optimization rounds focused on improving enzyme activity while maintaining improved temperature stability to further reduce herbicide damage to mesotrione sprays in plants at both normal and higher temperatures. Variant D08 was used as the base variant for all round 2 optimizations. Rational design, taking into account the results of round 1 screening, was used to engineer variants in round 2. Much of the rational design work focused on selected point amino acid changes (relative to wild type) made in round 1 in D08 that could be restored to the wild type amino acid (or similar). Here the overall approach was to identify amino acid positions that were important for activity, and amino acid positions that were important for stability. The goal was to find amino acid positions that were independent of each other, allowing for improvement of both stability and activity without adversely affecting the opposing improvement goals, to build variants with improved properties for both objectives. In principle, improving either the stability or activity of an enzyme often has an opposite effect on the other property. For example, improving stability can result in an enzyme that is highly resistant to denaturation at higher temperatures, but at the cost of reduced overall enzyme activity. Efforts have focused on improving both properties simultaneously, but this is often a much greater operational challenge.

[0111] In the second round of optimization, approximately 300 variants were designed based on the D08 template, exploring different amino acid positions that could be reverted back to the wild-type amino acid in the majority. Of these, approximately 250 were successfully cloned and screened in high-throughput screening, and a subset was selected for more detailed characterization. Eight candidates were ultimately selected after completing several rounds of enzyme characterization. These candidates underwent detailed in vitro enzyme characterization and were also tested in planta assays. All of these eight candidates showed improved enzyme stability compared to the starting point of Os.TDO (data not shown). However, only two of the candidates (TDO_6 and TDO_8) had significantly improved enzyme activity over the D08 variants, and none of the enzymes had better enzyme activity than Os.TDO at room temperature (Figure 6).

[0112] The next set of experiments focused on balancing the improvement of enzyme stability with the reduced activity observed at the optimum temperature of soybean plants. Potentially, improved stability could result in more active enzymes being available in the plant cells across a reasonable temperature range, and thus improved tolerance even if the activity of each enzyme was somewhat reduced. To test this hypothesis, eight variants from the second round of biochemical analysis described in the previous paragraph were transformed into soybean by Agrobacterium-mediated transformation, along with three controls. One of the controls (TDO_9) was a variant from the high-throughput screen with weak activity. The other two controls were the D08 variant and Os.TDO. R0 transgenic soybean plants were sprayed with mesotrione herbicide and screened for herbicide tolerance. Due to some technical issues in the plant transformation process unrelated to the transgene itself, the three variants did not produce any transgenic plants for testing. These included the Os.TDО control, the D08 variant, and one round 2 variant. Plants were scored for percent damage as described in the previous example. All variants showed damage ratings in the low to mid 30s, demonstrating the resistance of these plants compared to the wild-type transformed germplasm controls, which averaged 45% damage (Table 4). [Table 4]

[0113] As shown in Table 4, the wild type (WT) control had the highest damage among the variants tested. Although the Os.TDO control failed to transform in this experiment, a typical damage rating for R0 soybean (using the same expression element) would have been in the high 20% to low 30%. This suggests that the variants had damage similar to that expected for Os.TDO under optimal growing conditions. Results from the second round of variants provided insight into sites / amino acid changes that may help stabilize the TDO protein, especially for crops that may be exposed to higher temperatures. Furthermore, these amino acid changes are candidates for gene editing of native traits in rice or similar crops to improve those native traits, or for editing the transgene itself in crops such as soybean.

[0114] The selected TDO variants were tested in cotton, as cotton is a target crop that is often exposed to high temperature growing conditions. Three TDO variants, namely D08, TDO_6 and TDO_8, were transformed into cotton (along with Os.TDO control) using similar expression elements in single cassette vectors or as part of multiple cassette vectors. R2 transgenic cotton plants were then screened for resistance to mesotrione. Sixteen plants from two events per construct, at the V3 stage under normal (80°F) and high temperature (100°F) growing conditions, were sprayed with mesotrione at two application rates (2x and 8x). Additionally, the effect of heat treatment on TDO accumulation in cotton plants was measured by ELISA. Cotton plants were planted under normal greenhouse conditions (80°F day / 70°F night) and grown to the V3 growth stage. Half of the plants were then transferred to a hotter growth room (100°F daytime, 82°F nighttime) for heat treatment, while the other half of the plants remained in the same greenhouse (80°F daytime, 70°F nighttime). Leaf samples were collected for TDO protein measurement on the third day after the plants were placed under the various temperature conditions. Plants in both the 100°F growth room and the 80°F greenhouse were sprayed with 2X mesotrione (210 g ai / ha). Leaf samples were collected again 7 days after mesotrione treatment (DAT: days after treatment) for protein assay. Table 5 compares the protein levels of TDO variants in transgenic cotton plants grown under the two test conditions. Protein levels of TDO variants were measured for plants at three time points: 1) immediately before entering the high temperature treatment, 2) 3 days after placement in the control (normal growth temperature) or high temperature (100°F), and 3) 7 days after 2X mesotrione spray. [Table 5]

[0115] In this experiment, Os.TDO plants showed decreased TDO protein levels at high temperature treatment compared to normal conditions at both 3 and 7 days after treatment. However, all three optimized TDO variants did not show significant changes in TDO protein levels between normal and high temperature cultivation conditions, confirming the excellent in planta thermostability of the enzyme. Table 6 shows the results of mesotrione treatment on cotton plants expressing these selected TDO variants. Although all TDO-expressing cotton events showed excellent resistance, two variants, namely D08 and TDO_6, showed injury rates that still tended to be lower than the Os.TDO control at higher temperatures. This suggests that improved thermostability of the enzyme leads to improved herbicide tolerance in cotton. In contrast, wild-type control cotton plants showed high injury (weak resistance) to all mesotrione application. [Table 6]

[0116] Example 5: Structural and functional characterization of triketone dioxygenase from Oryza sativa Among the TDO genes tested from various species, the rice gene (Os.TDO) was unique in its ability to provide plants with strong protection against mesotrione. The only other TDO that provided protection was the maize TDO (Zm.TDO), albeit with a much lower efficacy, as shown in Example 2. The amino acid sequences of various proteins with TDO activity, as well as two closely related proteins, namely BRT and At.dioxy, were aligned to that of Os.TDO. 2BRT and At.dioxy are anthocyanidin synthases from Arabidopsis thaliana. Both are involved in the biosynthesis of anthocyanins and protoanthocyanidins by catalyzing the oxidation of leucoanthocyanidins to anthocyanidins.

[0117] Based on this sequence alignment, the amino acids were divided into several categories, including 1) amino acids conserved throughout all proteins (shown as hollow triangles in Figure 10), 2) amino acids conserved throughout all TDO-type proteins (shown as solid triangles in Figure 10), 3) amino acids near the mesotrione binding site and conserved among many TDO proteins (shown as dashed boxes in Figure 10), and 4) amino acids near the mesotrione binding site but found only in rice TDO or rice and / or maize TDO, as shown as solid boxes in Figure 10. The 2BRT protein is not shown in Figure 10 because it differs from the At.dioxy protein only by the lack of a methionine at the N-terminus. From the latter category, Thr from rice TDO was 230 , Ser 301 , Arg 332 and Ile 335 Four amino acids were identified (Figure 10). The first three of these (Thr 230 , Ser 301 and Arg 332 ) was only found in rice TDO, whereas the fourth Ile 335 were found in both rice and maize TDO. These four amino acids were mapped onto the structure of the rice TDO protein as shown in Figure 7 and were hypothesized to play an important role in conferring resistance to mesotrione.

[0118] To test this hypothesis, four amino acids in sorghum TDO corresponding to amino acids 230, 301, 332, and 335 in rice TDO were replaced with those of rice TDO. The maize, rice, sorghum, and modified sorghum TDO genes were cloned with an N-terminal His6 tag into the pET28 vector for E. coli expression. The proteins were then expressed and purified by affinity chromatography. BL21-Codon Plus (DE3)-RIPL E. coli cells (Stratagene) were used for expression. Cells transformed with the expression vectors were cultured at 100 μg mL -1Kanamycin and 34 μg mL -1 The cells were cultured in autoinduction medium (AIM) supplemented with chloramphenicol.

[0119] The TDO protein used in this study was expressed in AIM cells using an autoinduction system as described by Studier (2014). Briefly, recombinant cultures were grown at 37 °C for 3 h and then grown at 20 °C for 1 or 2 days. Cells were harvested by centrifugation and frozen at -80 °C. The pellet was resuspended in lysis buffer (Bper and Yper [Pierce], ratio 3:1, 25 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, pH 8.0). 150 mM ferric ammonium citrate (1000X) was added to the lysate to a final concentration of 150 μM and incubated on ice for 20 min. The lysate was separated by centrifugation to clarify the supernatant. The clear supernatant was loaded onto a 5 mL Ni column connected to an AKTA Express (GE Healthcare). The column was washed with wash buffer (25 mM Tris-HCl, 300 mM NaCl, 30 mM imidazole, pH 8.0). Protein was eluted with 25 mM Tris-HCl, 300 mM NaCl, 150 mM imidazole, pH 8.0. Protein from the Ni column was concentrated and subjected to size exclusion chromatography using a Superdex 200 column connected to an AKTA Explorer (GE Healthcare) equilibrated with 25 mM Tris, 150 mM NaCl and 1 mM DTT, pH 8.0. Fractions containing pure TDO were pooled. All purification steps were performed at 4°C. Protein concentration was determined spectrophotometrically based on the calculated molar extinction coefficient at 280 nm.

[0120] TDO activity was measured by the second hydroxylation of mesotrione. For routine activity measurements of TDO and for determining the kinetic characteristics of the second hydroxylation, A specific for the second hydroxylation and subsequent nonenzymatic water removal (i.e., conversion of hydroxy-mesotrione to oxy-mesotrione) was used. 330Continuous changes in A were monitored. The reaction mixture (100 μL) contained 50 mM MOPS (pH 7.2), 2.5 mM oxoglutarate, 50 μM iron(II) sulfate heptahydrate, 150 mM KCl, 150 μM sodium ascorbate, 100–600 μM mesotrione, and 50–250 nM TDO protein. The reaction proceeded with a maximum A 330 The reaction was allowed to proceed until K was obtained (typically 20-120 min, depending on the mesotrione concentration), and the linear portion of the curve was determined by visual inspection. The starting concentration of mesotrione was used as an estimate of the concentration of hydroxy-mesotrione; since less than 100% of mesotrione would have been converted by the onset of the linear phase of the second reaction, the starting concentration of hydroxy-mesotrione was therefore K m was always slightly underestimated.

[0121] For activity calculations, aliquots of the reaction were removed at various times during the reaction and quenched by heating at 95°C. The reaction mixtures were analyzed by HPLC and mass spectrometry. Product conversion, followed by peak integration in HPLC, gave a linear plot, and the extinction coefficient delta between the substrate (mesotrione) and the second product (oxy-mesotrione) was calculated using the formula ΔA 330 =(ε p -ε s ) × bΔp, where ΔA 330 is the change in absorbance at 330 nm and b is the light path.

[0122] The specific activities of the TDO variants for the second hydroxylation are shown in Figure 8. Consistent with the plant data, rice TDO (Os.TDO) had the highest activity, followed by maize TDO, which was about 30% less active than rice TDO. Sorghum TDO was not significantly different from the blank, but the sorghum TDO variant, designated SwM, was about 17% more active than the rice TDO protein. The SwM variant contains a combination of substitutions P230T-A301S-L332R-V335I, in which the wild-type sorghum residues at positions 230, 310, 332, and 335 are replaced by those of rice TDO.

[0123] In addition to the full-length alignment of the TDO protein, the amino acid sequences flanking the active site of the TDO homologs were aligned (Figure 9A) and superimposed with the 1GP5 structure of anthocyanidin synthase (ANS) complexed with one of its substrates, DQH (trans-dihydroquercetin). π-stacking between the enzyme and the compound may be important for retaining the compound in the active site. DQH is located at the Phe of 1GP5. 144 When this is mapped to TDO (Figure 9B and Figure 9C), mesotrione is π-stacked with His at the position of Os.TDO. 141 This suggests that the enzyme may have some degree of π-stacking with Glycine 144 (alignment in Figure 9A). In this regard, it is interesting to note that monocots with glutamine at the position corresponding to position 144 of 1GP5 are not active. Extending this further, the corresponding position (Phe in Gm.TDO) may be involved in the synthesis of 1GP5. 141 , Zm.TDO His 141 ) may confer enhanced activity due to the aromaticity and potential π-stacking of the amino acid (Figure 9A).

[0124] The left side of the binding pocket (Ser 236 1GP5), dicotyledonous plants have greater rigidity in this area. Proteins containing two glycine residues in this region may allow enough flexibility in the binding pocket for the reaction to occur, whereas proteins without glycine may not be flexible enough.

[0125] Both active monocot TDO proteins have one glycine and a residue with less backbone flexibility. Thus, a glycine can replace the serine corresponding to position 236 in 1GP5 (see Figure 9A; Thr in Os.TDO). 230 , Gly of Gm.TDO 230 , Gly of Zm.TDO 231 ), and in addition, they can replace the hydrophobic residues (A, L, V, I, T, S) corresponding to position 235 of 1GP5 (see Figure 9A; Gly of Os.TDO).229 , Gly of Gm.TDO 229 , Zm.TDO Ala 230 ). In addition, Val of 1GP5 235 The position corresponding to Gly in Os.TDO (Figure 9A) 229 , Gly of Gm.TDO 229 , Zm.TDO Ala 230 ) to Ser 236 equivalents to threonine, alanine, leucine, valine, isoleucine, or serine, and may be changed to glycine.

[0126] In addition, the leucine, alanine, valine, or isoleucine at position 299 of Os.TDO (corresponding to position F304 of 1GP5) may allow more space for the non-aromatic ring of mesotrione to bind. In the case of DQH, π-stacking exists between Phe and DQH, which is common in dicotyledonous plants such as soybean (Gm.TDO F299). This also explains the difference in the Phe of maize TDO (Zm.TDO) compared to rice TDO. 300 ) may also explain the reduced activity of rice TDO. However, investigating other TDOs based on similarity to the active site of rice TDO, rather than on whole protein similarity, may provide a useful strategy to identify naturally active or other variants.

Claims

1. A recombinant DNA molecule comprising a nucleic acid sequence encoding a protein having triketone dioxygenase (TDO) activity, said nucleic acid sequence comprising SEQ ID NO:

1.

2. 1. A recombinant DNA molecule comprising a nucleic acid sequence encoding a protein having triketone dioxygenase (TDO) activity, said protein having at least 70% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16, said protein comprising at least one amino acid substitution compared to SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, or 16, said protein comprising: threonine (T), alanine (A), leucine (L), valine (V), isoleucine (I), serine (S), or glycine (G) at the position corresponding to position 230 of SEQ ID NO: 2; serine (S) at the position corresponding to position 301 of SEQ ID NO: 2; arginine (R) at the position corresponding to position 332 of SEQ ID NO:2; isoleucine (I) at a position corresponding to position 335 of SEQ ID NO:2; tyrosine (Y), phenylalanine (F) or histidine (H) at the position corresponding to position 141 of SEQ ID NO: 2; alanine (A), isoleucine (I), leucine (L), serine (S), threonine (T), valine (V), or glycine (G) at the position corresponding to position 229 of SEQ ID NO: 2; alanine (A), isoleucine (I), leucine (L), or valine (V) at the position corresponding to position 299 of SEQ ID NO:2, or including any combination thereof, The recombinant DNA molecule of any one of the following requirements: (i) if threonine (T), alanine (A), leucine (L), valine (V), isoleucine (I), or serine (S) is present at the position corresponding to position 230 of SEQ ID NO:2, then glycine (G) must be present at the position corresponding to position 229 of SEQ ID NO:2; and (ii) if alanine (A), leucine (L), valine (V), isoleucine (I), threonine (T), or serine (S) is present at the position corresponding to position 229 of SEQ ID NO:2, then glycine (G) must be present at the position corresponding to position 230 of SEQ ID NO:

2.

3. The protein has at least 70% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 6, 8, 10, 12, 14 and 16, and the protein comprises: threonine (T) at the position corresponding to position 230 of SEQ ID NO:2; serine (S) at the position corresponding to position 301 of SEQ ID NO: 2; arginine (R) at the position corresponding to position 332 of SEQ ID NO:2; Isoleucine (I) at a position corresponding to position 335 of SEQ ID NO:2, or The recombinant DNA molecule of claim 2, comprising any combination thereof.

4. The protein is threonine (T) at the position corresponding to position 230 of SEQ ID NO:2; serine (S) at the position corresponding to position 301 of SEQ ID NO: 2; Arginine (R) at a position corresponding to position 332 of SEQ ID NO: 2, and 4. The recombinant DNA molecule of claim 3, which comprises an isoleucine (I) at a position corresponding to position 335 of SEQ ID NO:

2.

5. 3. The recombinant DNA molecule of claim 2, wherein the protein has at least 70% sequence identity to SEQ ID NO:2 and comprises one or more amino acid substitutions selected from the group consisting of H141F, H141Y, G229A, G229I, G229L, G229S, G229T, G229V, T230A, T230G, T230I, T230L, T230S, T230V, L299A, L299I, L299V, and any combination thereof.

6. 5. The recombinant DNA molecule of any one of claims 2 to 4, wherein the protein has at least 70% sequence identity to SEQ ID NO:4 and comprises one or more amino acid substitutions selected from the group consisting of H141F, H141Y, A230G, A230I, A230L, A230S, A230T, A230V, G231A, G231I, G231L, G231S, G231T, G231V, F300A, F300I, F300L, F300V, G302S, I333R, and any combination thereof.

7. 7. The recombinant DNA molecule of claim 6, wherein the protein comprises one or more amino acid substitutions selected from the group consisting of G231T, G302S, I333R, and any combination thereof.

8. 7. The recombinant DNA molecule of claim 6, wherein the protein further comprises an isoleucine (I) at position 336 of SEQ ID NO:

4.

9. 5. The recombinant DNA molecule of any one of claims 2 to 4, wherein the protein has at least 70% sequence identity to SEQ ID NO:6 and comprises one or more amino acid substitutions selected from the group consisting of Q140F, Q140H, Q140Y, G229A, G229I, G229L, G229S, G229T, G229V, P230A, P230G, P230I, P230L, P230S, P230T, P230V, L299A, L299I, L299V, A301S, L332R, V335I, and any combination thereof.

10. 10. The recombinant DNA molecule of claim 9, wherein the protein comprises one or more amino acid substitutions selected from the group consisting of P230T, A301S, L332R, V335I, and any combination thereof.

11. 5. The recombinant DNA molecule of claim 2, wherein the protein has at least 70% sequence identity to SEQ ID NO:8 and comprises one or more amino acid substitutions selected from the group consisting of H144F, H144Y, G233A, G233I, G233L, G233S, G233T, G233V, G234A, G234I, G234L, G234S, G234T, G234V, F303A, F303I, F303L, F303V, G305S, L336R, F339I, and any combination thereof.

12. 12. The recombinant DNA molecule of claim 11, wherein the protein comprises one or more amino acid substitutions selected from the group consisting of G234T, G305S, L336R, F339I, and any combination thereof.

13. 5. The recombinant DNA molecule of any one of claims 2 to 4, wherein the protein has at least 70% sequence identity to SEQ ID NO: 10 and comprises one or more amino acid substitutions selected from the group consisting of H145F, H145Y, G234A, G234I, G234L, G234S, G234T, G234V, G235A, G235I, G235L, G235S, G235T, G235V, F304A, F304I, F304L, F304V, A306S, V337R, L340I, and any combination thereof.

14. 14. The recombinant DNA molecule of claim 13, wherein the protein comprises one or more amino acid substitutions selected from the group consisting of G235T, A306S, V337R, L340I, and any combination thereof.

15. 5. The recombinant DNA molecule of any one of claims 2 to 4, wherein the protein has at least 70% sequence identity to SEQ ID NO: 12 and comprises one or more amino acid substitutions selected from the group consisting of Q140F, Q140H, Q140Y, G229A, G229I, G229L, G229S, G229T, G229V, F299A, F299I, F299L, F299V, G230A, G230I, G230L, G230S, G230T, G230V, G301S, I332R, L335I, and any combination thereof.

16. 16. The recombinant DNA molecule of claim 15, wherein the protein comprises one or more amino acid substitutions selected from the group consisting of G230T, G301S, I332R, L335I, and any combination thereof.

17. 5. The recombinant DNA molecule of any one of claims 2 to 4, wherein the protein has at least 70% sequence identity to SEQ ID NO: 14 and comprises one or more amino acid substitutions selected from the group consisting of H141F, H141Y, A212G, A212I, A212L, A212S, A212T, A212V, G213A, G213I, G213L, G213S, G213T, G213V, F282A, F282I, F282L, F282V, G284S, V315R, V318I, and any combination thereof.

18. 18. The recombinant DNA molecule of claim 17, wherein the protein comprises one or more amino acid substitutions selected from the group consisting of G213T, G284S, V315R, V318I, and any combination thereof.

19. 5. The recombinant DNA molecule of any one of claims 2 to 4, wherein the protein has at least 70% sequence identity to SEQ ID NO: 16 and comprises one or more amino acid substitutions selected from the group consisting of Q140F, Q140H, Q140Y, G229A, G229I, G229L, G229S, G229T, G229V, F299A, F299I, F299L, F299V, G230A, G230I, G230L, G230S, G230T, G230V, G301S, I332R, L335R, and any combination thereof.

20. 20. The recombinant DNA molecule of claim 19, wherein the protein comprises one or more amino acid substitutions selected from the group consisting of G230T, G310S, I332R, L335R, and any combination thereof.

21. The recombinant DNA molecule of any one of claims 2 to 4, wherein the protein has at least 80% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 and 16.

22. The recombinant DNA molecule of any one of claims 2 to 4, wherein the protein has at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 and 16.

23. The recombinant DNA molecule of any one of claims 2 to 4, wherein the protein comprises SEQ ID NO:

48.

24. The protein further comprises: leucine (L) at the position corresponding to position 11 of SEQ ID NO:2; Lysine (K) at the position corresponding to position 18 of SEQ ID NO:2; glutamine (Q) at the position corresponding to position 32 of SEQ ID NO: 2; Proline (P) at the position corresponding to position 35 of SEQ ID NO: 2; isoleucine (I) at the position corresponding to position 48 of SEQ ID NO:2; glycine (G) at the position corresponding to position 54 of SEQ ID NO:2; isoleucine (I) at the position corresponding to position 82 of SEQ ID NO:2; aspartic acid (D) at the position corresponding to position 89 of SEQ ID NO:2; histidine (H) at the position corresponding to position 116 of SEQ ID NO:2; glutamic acid (E) at the position corresponding to position 120 of SEQ ID NO:2; valine (V) at the position corresponding to position 133 of SEQ ID NO:2; histidine (H) at the position corresponding to position 167 of SEQ ID NO:2; a cysteine ​​(C) at a position corresponding to position 173 of SEQ ID NO:2; phenylalanine (F) at the position corresponding to position 199 of SEQ ID NO:2; threonine (T) at the position corresponding to position 204 of SEQ ID NO: 2; glycine (G) at the position corresponding to position 230 of SEQ ID NO:2; glycine (G) at the position corresponding to position 242 of SEQ ID NO:2; Proline (P) at the position corresponding to position 256 of SEQ ID NO: 2; serine (S) at the position corresponding to position 274 of SEQ ID NO: 2; Lysine (K) at the position corresponding to position 279 of SEQ ID NO:2, or The recombinant DNA molecule according to any one of claims 2 to 4, comprising any combination thereof.

25. 1. A recombinant DNA molecule comprising a nucleic acid sequence encoding a protein having triketone dioxygenase (TDO) activity, said protein having at least 50% identity to SEQ ID NO:2 and comprising one or more amino acid substitutions selected from the group consisting of I11L, A18K, K32Q, S35P, L48I, S54G, V82I, S89D, N116H, Q120E, I133V, N167H, T173C, L199F, A204T, T230G, S242G, E256P, C274S, R279K, and any combination thereof.

26. 26. The recombinant DNA molecule of claim 25, wherein the protein comprises the following amino acid substitutions: I11L, A18K, K32Q, S35P, L48I, S54G, V82I, S89D, N116H, Q120E, I133V, N167H, T173C, L199F, A204T, T230G, S242G, E256P, C274S, and R279K.

27. 26. The recombinant DNA molecule of claim 25, wherein the protein comprises the following amino acid substitutions: I11L, A18K, K32Q, L48I, S54G, V82I, S89D, Q120E, I133V, N167H, T173C, L199F, A204T, T230G, S242G, E256P, C274S, and R279K.

28. 26. The recombinant DNA molecule of claim 25, wherein the protein comprises the following amino acid substitutions: I11L, A18K, K32Q, L48I, S54G, V82I, S89D, N116H, I133V, N167H, T173C, L199F, A204T, T230G, S242G, E256P, C274S, and R279K.

29. 26. The recombinant DNA molecule of claim 25, wherein the protein comprises the following amino acid substitutions: I11L, A18K, K32Q, L48I, S54G, V82I, S89D, N116H, Q120E, I133V, N167H, T173C, L199F, A204T, T230G, E256P, C274S, and R279K.

30. 26. The recombinant DNA molecule of claim 25, wherein the protein comprises the following amino acid substitutions: I11L, A18K, K32Q, S35P, L48I, S54G, V82I, S89D, Q120E, I133V, N167H, T173C, L199F, A204T, T230G, E256P, C274S, and R279K.

31. 26. The recombinant DNA molecule of claim 25, wherein the protein comprises the following amino acid substitutions: I11L, A18K, K32Q, S35P, L48I, S54G, V82I, S89D, N116H, I133V, N167H, T173C, L199F, A204T, T230G, E256P, C274S, and R279K.

32. 26. The recombinant DNA molecule of claim 25, wherein the protein comprises the following amino acid substitutions: I11L, A18K, K32Q, S35P, L48I, S54G, V82I, S89D, N116H, Q120E, I133V, N167H, T173C, L199F, A204T, T230G, C274S, and R279K.

33. 26. The recombinant DNA molecule of claim 25, wherein the protein comprises the following amino acid substitutions: I11L, A18K, K32Q, S35P, L48I, S54G, V82I, S89D, N116H, Q120E, I133V, N167H, T173C, L199F, A204T, T230G, E256P, and R279K.

34. 26. The recombinant DNA molecule of claim 25, wherein the protein comprises the following amino acid substitutions: I11L, A18K, S35P, S54G, V82I, S89D, N116H, Q120E, N167H, T173C, L199F, A204T, T230G, C274S, and R279K.

35. 26. The recombinant DNA molecule of claim 25, wherein the protein comprises the following amino acid substitutions: I11L, A18K, K32Q, S35P, L48I, S54G, V82I, S89D, N116H, Q120E, I133V, N167H, T173C, A204T, T230G, S242G, E256P, and C274S.

36. The protein further comprises: threonine (T) at the position corresponding to position 230 of SEQ ID NO:2; serine (S) at the position corresponding to position 301 of SEQ ID NO: 2; Arginine (R) at a position corresponding to position 332 of SEQ ID NO: 2, and 36. The recombinant DNA molecule of any one of claims 25 to 35, comprising an isoleucine (I) at a position corresponding to position 335 of SEQ ID NO:

2.

37. The recombinant DNA molecule of any one of claims 25 to 35, wherein the protein has at least 80% identity with SEQ ID NO:

2.

38. The recombinant DNA molecule of any one of claims 25 to 35, wherein the protein has at least 90% identity with SEQ ID NO:

2.

39. 26. The recombinant DNA molecule of claim 25, wherein the protein comprises SEQ ID NO: 18, 28, 30, 32, 34, 36, 38, 40, 42 or 44.

40. 2. The recombinant DNA molecule of claim 1, wherein the DNA molecule further comprises a heterologous promoter operably linked to the nucleic acid sequence encoding a protein having triketone dioxygenase (TDO) activity.

41. 41. The recombinant DNA molecule of claim 40, wherein the promoter comprises a constitutive promoter.

42. 42. The recombinant DNA molecule of claim 40 or 41, wherein the heterologous promoter functions in a plant cell.

43. The recombinant DNA molecule of claim 1, wherein the recombinant DNA molecule is contained within the genome of a plant cell.

44. A DNA construct comprising the recombinant DNA molecule of claim 1.

45. 10. An engineered protein encoded by the recombinant DNA molecule of claim 1.

46. A transgenic plant, plant seed, plant cell or plant part comprising the recombinant DNA molecule of claim 1.

47. 47. The transgenic plant, plant seed, plant cell or plant part of claim 46, wherein the transgenic plant, plant seed, plant cell or plant part is tolerant to at least one 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor herbicide.

48. 48. The transgenic plant, plant seed, plant cell or plant part of claim 47, wherein the HPPD inhibitor herbicide is selected from the group consisting of mesotrione, benzobicyclon (BBC), tembotrione, sulcotrione, tefuryltrione, 2-(2-nitro-4-trifluoromethylbenzoyl)cyclohexane-1,3-dione (NTBC), pyrazolate, benzofenap, pyrazoxyfen, isoxaflutole, pyrazolinate, topramezone, and any combination thereof.

49. 49. The transgenic plant, plant seed, plant cell or plant part of any one of claims 46 to 48, wherein the transgenic plant, plant seed, plant cell or plant part is tolerant to at least a second herbicide.

50. 49. The transgenic plant, plant seed, plant cell, or plant part of any one of claims 46 to 48, wherein the second herbicide is selected from the group consisting of an ACCase inhibitor, an ALS inhibitor, an EPSPS inhibitor, a synthetic auxin, a photosynthesis inhibitor, a glutamine synthesis inhibitor, an HPPD inhibitor, a PPO inhibitor, and a long-chain fatty acid inhibitor.

51. 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 synthesis 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 chloroacetamide, oxyacetamide, or pyrazole.

52. 10. A commercial product comprising the recombinant DNA molecule of claim 1.

53. 46. ​​A method of conferring HPPD inhibitor herbicide tolerance to a plant, plant seed, plant cell or plant part, the method comprising heterologously expressing in said plant, plant seed, plant cell or plant part an engineered protein of claim 45.

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

55. 55. The method of claim 54, further comprising selecting said plant or its progeny for HPPD inhibitor tolerance.

56. 56. The method of claim 54 or 55, further comprising crossing the regenerated plant with itself or with a second plant to produce progeny.

57. 47. A method for controlling or preventing the growth of weeds in a plant growing area, comprising spraying an effective amount of at least one HPPD inhibitor herbicide to a plant growing area containing the transgenic plant or seed of claim 46, wherein the transgenic plant or seed is tolerant to the HPPD inhibitor herbicide.

58. A method for screening for herbicide resistance genes, comprising: (a) expressing the recombinant DNA molecule of claim 1 in a plant cell; and (b) identifying a plant cell that is resistant to the HPPD inhibitor herbicide.

59. 1. A method for producing a plant that is tolerant to an HPPD inhibitor herbicide and at least one other herbicide, comprising: (a) crossing the plant of claim 46 with a second plant that comprises tolerance to said at least one other herbicide; and (b) selecting a progeny plant resulting from said cross and comprising tolerance to the HPPD inhibitor herbicide and said at least one other herbicide.

60. 1. A method for reducing the occurrence of herbicide-resistant weeds, comprising: (a) growing the plant of claim 46 in a crop growing environment; and (b) spraying an HPPD inhibitor herbicide and at least one other herbicide into the crop growing environment, wherein the crop plants are tolerant to the HPPD inhibitor herbicide and the at least one other herbicide.

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