Herbicide resistant HPPD variants identified via directed evolution

Abstract

Embodiments of the present disclosure pertains to a modified plant or seed that includes a mutated 4-hydroxyphenylpyruvate dioxygenase (HPPD) gene. The mutated HPPD gene expresses an HPPD protein that confers herbicide resistance to the modified plant or seed. Additional embodiments of the present disclosure pertain to methods of producing modified plants or seeds by introducing a mutated HPPD gene to the plant or seed. The mutated HPPD gene may express a protein that includes SEQ ID NO: 1 or a sequence with at least 65% sequence identity to SEQ ID NO: 1. The mutated HPPD gene may also express a protein that includes SEQ ID NO: 2 or a sequence with at least 65% sequence identity to SEQ ID NO: 2.

Description

PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032 TITLE HERBICIDE RESISTANT HPPD VARIANTS IDENTIFIED VIA DIRECTED EVOLUTION CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 541,802, filed on September 30, 2023. The entirety of the aforementioned application is incorporated herein by reference. SEQUENCE DISCLOSURE STATEMENT

[0002] Pursuant to 37 C.F.R. § 1.834, Applicant has submitted a sequence listing in XML format (“Sequence Listing”). The name of the file containing the Sequence Listing is “AF13368.P057WO.xml”. The date of the creation of the Sequence Listing is September 27, 2024. The size of the Sequence Listing is 39,000 bytes. Applicant hereby incorporates by reference the material in the Sequence Listing. BACKGROUND

[0003] A need exists for the development of plants and seeds that have improved resistance to herbicides. Numerous embodiments of the present disclosure aim to address the aforementioned need. SUMMARY

[0004] In some embodiments, the present disclosure pertains to a modified plant or seed. In some embodiments, the modified plant or seed includes a mutated 4-hydroxyphenylpyruvate dioxygenase (HPPD) gene. In some embodiments, the mutated HPPD gene expresses an HPPD protein that confers herbicide resistance to the modified plant or seed.PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032

[0005] Additional embodiments of the present disclosure pertain to methods of producing modified plants or seeds. In some embodiments, the methods of the present disclosure include introducing a mutated 4-hydroxyphenylpyruvate dioxygenase (HPPD) gene to the plant or seed. In some embodiments, the mutated HPPD gene expresses an HPPD protein that confers herbicide resistance to the modified plant or seed.

[0006] In some embodiments, the mutated HPPD gene expresses a protein that includes SEQ ID NO: 1 or a sequence with at least 65% sequence identity to SEQ ID NO: 1. In some embodiments, the mutated HPPD gene expresses a protein that includes SEQ ID NO: 2 or a sequence with at least 65% sequence identity to SEQ ID NO: 2. DESCRIPTION OF THE DRAWINGS

[0007] FIGS.1A-1D show the establishment of a cotton 4-hydroxyphenylpyruvate dioxygenase (HPPD) expression system in E. coli. FIG.1A shows the high accumulation of pigment in the E. coli strain C43 (DE3) with the plasmids pET-30b-HPPD after isopropyl β-d-1- thiogalactopyranoside (IPTG) induction. FIG.1B shows the test of IPTG concentrations for the induction of HPPD on agar plates. FIG.1C shows the effects of IPTG induction on the growth of the colonies. FIG. 1D shows the inhibition of HPPD activity by different concentrations of mesotrione. The LB medium contained 1 mg mL-1 Tyr unless labeled with no Tyr.

[0008] FIG. 2 shows mutations in the cotton HPPD conferring mesotrione resistance. HPPD sequences from Zea mays (Zm), Arabidopsis thaliana (At) and Gossypium hirsutum (Gh) were aligned to show the protein sequence similarity. Identified mutations in cotton HPPD are shown with red arrows.PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032

[0009] FIGS. 3A-3C show the resistance of single and multiple mutations in cotton HPPD to mesotrione. FIG.3A shows mesotrione resistant test on agar plates.1, HPPD wild type (grey).2- 5: HPPD with single mutation (orange); 2, P329S; 3, T331A; 4, K335E; 5, G412S. 6-11: HPPD with double mutations (blue); 6, P329S+T331A; 7, P329S+K335E; 8, T331A+K335E; 9, P329S+G412S; 10, T331A+G412S; 11, K335E+G412S. 12-15: HPPD with triple mutations (green); 12, P329S+T331A+K335E; 13, P329S+T331A+G412S; 14, P329S+K335E+G412S; 15, T331A+K335E+G412S. 16: HPPD with quadruple mutations (yellow), P329S+T331A+ K335E+G412S. FIG.3B shows the inhibition of wild type and HPPD variants by mesotrione in liquid culture. Darker color indicates higher HPPD activity. FIG. 3C shows IC50 of mesotrione on the wild type and 15 HPPD mutants. Different letters above the bars indicate significant differences. Statistical analysis was performed with One-Way ANOVA and Tukey’s post-hoc HSD test (P < 0.05, n ≥ 3).

[0010] FIGS. 4A-4B show genetic complementation of the Arabidopsis athppd mutant with the cotton HPPD variants. FIG. 4A shows the native activity of the wild type and HPPD variants determined in E. coli. Different letters above the bars indicate significant differences. Statistical analysis was performed with One-Way ANOVA and Tukey’s post-hoc HSD test (P < 0.05, n ≥ 3). FIG.4B shows transgenic Arabidopsis athppd homozygous mutant expressing the wild type and mutant HPPD.

[0011] FIGS. 5A-5B show herbicide resistance of the Arabidopsis transgenic plants expressing cotton wild type and variant HPPD. FIG.5A shows a comparison of herbicide resistance of the transgenic seedlings on agar plates supplemented with different concentrations of mesotrione. FIG.5B shows chlorophyll content of seedlings shown in (FIG.5A). Different letters above the bars indicate significant differences. Statistical analysis was performed with One-Way ANOVA and Tukey’s post-hoc HSD test (P < 0.05, n ≥ 3).

[0012] FIG.6 shows the IC50 of mesotrione on the wild type and variants of soybean HPPD (single mutated and multi-mutated variants included).PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032

[0013] FIG.7 shows herbicide resistance of Arabidopsis expressing wild-type and variant soybean HPPD at different concentrations of mesotrione (0-100 nM). (1: soybean WT HPPD; 2: T93A; 3: T385A; 4: N389D; 5: T93A+N389D; 6: N389D+A394V; 7: N389D+K453E; 8: G61E+T93A+T385A; 9: A271V+T385A+K453E; 10: P60S+G61E+N389D; 11: T93A+A271V+T385A+K453E; 12: P60S+G61E+T385A+N389D; 13: P60S+P383S+N389D+A394V).

[0014] FIG.8 shows the chlorophyll content of Arabidopsis seedlings expressing wild-type and variant HPPDs, grown for two weeks on agar medium with specified mesotrione concentrations (0 nM, 30 nM, 50 nM). DETAILED DESCRIPTION

[0015] It is to be understood that both the foregoing general description and the following detailed description are illustrative and explanatory, and are not restrictive of the subject matter, as claimed. In this application, the use of the singular includes the plural, the word “a” or “an” means “at least one”, and the use of “or” means “and / or”, unless specifically stated otherwise. Furthermore, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements or components that includes one unit and elements or components that include more than one unit unless specifically stated otherwise.

[0016] The section headings used herein are for organizational purposes and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated herein by reference in their entirety for any purpose. In the event that one or more of the incorporated literature and similar materials defines a term in a manner that contradicts the definition of that term in this application, this application controls.PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032

[0017] Weeds, as biotic factors, exert significant pressure on crop productivity by competing for essential resources such as nutrients, light, space, and water. Weeds often outpace certain crops during early growth stages, gaining a competitive advantage that can stifle crop growth. Moreover, weeds create an environment conducive to other biotic threats like fungi, insects, and bacteria, directly endangering crops.

[0018] Consequently, weed elimination during crop growth is imperative. Various techniques have been employed for weed management, which includes mechanical, chemical, and biological methods. Labor-intensive manual weeding was the primary method before the mid-20th century, followed by the widespread adoption of synthetic herbicides over the past seven decades, reducing costs and improving weed management efficiency.

[0019] Commercial herbicides operate through diverse mechanisms, including inhibiting biosynthetic enzymes for essential amino acids, such as 5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase), acetolactate synthase, and glutamine synthetase. Some classes of herbicides disrupt photosynthesis by binding to the quinone-binding protein D-1, while others target lipid synthesis in monocotyledonous weeds.

[0020] Additionally, herbicides such as phosphoric amide and dinitroaniline interfere with cell division and cell wall formation by interacting with tubulin. Among these, 4- hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors include a group of commercial herbicides for weed control. HPPD is crucial in the tyrosine metabolic pathway, converting hydroxyphenylpyruvate (HPPA) into homogentisate (HGA), a precursor for α-tocopherol and plastoquinone. Inhibiting HPPD disrupts plastoquinone levels, leading to defects in carotenoid biosynthesis and disruption of photosystem II function. The lack of carotenoids results in oxidative damage and chlorophyll destruction, causing leaf bleaching, impaired photosynthesis, and plant death.PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032

[0021] While broad-spectrum herbicides effectively control weeds, their application time and duration are limited due to crop sensitivity. The advent of herbicide-resistant crops, notably glyphosate-resistant varieties since the mid-1990s, revolutionized weed control by enabling the use of broad-spectrum herbicides without harming crops. The prevalence of herbicide-resistant crops, particularly glyphosate-resistant ones like soybeans, canola, and cotton, has surged due to glyphosate's efficacy and the need for fewer herbicide applications.

[0022] Creating herbicide-resistant crops typically involves three main methods: overexpression of the target enzyme, expression of a resistant target enzyme, and herbicide detoxification. Overexpression of the target enzyme using strong promoters is often inefficient. Thus, expressing a naturally resistant or mutated target gene tolerant to herbicides is preferred. For instance, Monsanto created glyphosate-resistant crops by introducing a gene encoding a microbial enzyme insensitive to glyphosate. Alternatively, crops can be infused with enzymes neutralizing herbicide effects. Examples of such enzymes include phosphinothricin N-acetyltransferase from Streptomyces, which confers tolerance to glufosinate, and nitrilase from Klebsiella ozaenae, which provides resistance to bromoxynil and ioxynil.

[0023] The reliance on glyphosate alone for weed management has become unsustainable due to the proliferation of glyphosate-resistant weeds, necessitating the development of new weed control methods. Cotton, a major producer of natural fiber, is also grappling with issues related to glyphosate-resistant weeds in its cultivation systems. The enzyme HPPD involved in tyrosine catabolism in nearly all aerobic organisms serves as the target for commercial herbicides, with fewer resistant weeds reported so far.PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032

[0024] According to the International Survey of Herbicide Resistant Weeds, resistant weeds to HPPD targeted herbicides have been found in five weed species, including palmer amaranth, tall waterhemp, redroot pigweed, wild radish, and Chinese sprangletop. In the 17 reported mesotrione resistant weeds, almost all of them are also resistant to multiple herbicides with different modes of action, indicating that the resistance is likely caused by nontarget-site mechanisms. In fact, recent reports indicate that metabolic detoxification appears to be the major mechanism causing weed resistance to HPPD target herbicides.

[0025] HPPD inhibitors can be categorized into five main groups: isoxazole, pyrazolone, β- triketone, isoxazolidinone, and azole carboxamides. Among these, the β-triketone family represents a promising group of herbicides well-suited for agricultural crop management. However, the absence of crops resistant to these herbicides limits their usage and efficiency.

[0026] In sum, herbicides play a crucial role in boosting crop yields. However, the emergence of herbicide-resistant weeds and the susceptibility of crops to herbicides have posed significant challenges to their efficacy. Accordingly, a need exists for the development of plants and seeds that have improved resistance to herbicides. Numerous embodiments of the present disclosure aim to address the aforementioned need.

[0027] In some embodiments, the present disclosure pertains to a modified plant or seed. In some embodiments, the modified plant or seed includes a mutated 4-hydroxyphenylpyruvate dioxygenase (HPPD) gene. In some embodiments, the mutated HPPD gene expresses an HPPD protein that confers herbicide resistance to the modified plant or seed.

[0028] Additional embodiments of the present disclosure pertain to methods of producing modified plants or seeds. In some embodiments, the methods of the present disclosure include introducing a mutated 4-hydroxyphenylpyruvate dioxygenase (HPPD) gene to the plant or seed. In some embodiments, the mutated HPPD gene expresses an HPPD protein that confers herbicide resistance to the modified plant or seed.PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032

[0029] As set forth in more detail herein, the modified plants or seeds of the present disclosure can have numerous embodiments. As also set forth in more detail herein, the methods of the present disclosure can include numerous embodiments.

[0030] Mutated HPPD genes

[0031] The modified plants or seeds of the present disclosure can include various types of mutated HPPD genes. Additionally, the methods of the present disclosure can produce various types of mutated HPPD genes. For instance, in some embodiments, the mutated HPPD gene includes an endogenous HPPD gene. In some embodiments, the mutated HPPD gene includes an exogenous HPPD gene. In some embodiments, the mutated HPPD gene produces a physiologically active HPPD enzyme.

[0032] Various methods may be utilized to introduce mutated HPPD genes to plants or seeds. For instance, in some embodiments, the mutated HPPD gene is introduced by a method that includes, without limitation, chemical mutation, site directed mutagenesis, irradiation, introduction of a gene editing system to the plant or seed, introduction of the mutated HPPD gene as an exogenous gene, alteration of the endogenous HPPD gene, or combinations thereof.

[0033] In some embodiments, the mutated HPPD gene is introduced at a seedling stage of a plant. In some embodiments, the mutated HPPD gene is introduced within somatic embryos of a plant cell prior to somatic embryo development and subsequent germination into seedlings. In some embodiments, the mutated HPPD gene is introduced at an adult stage of a plant.

[0034] In some embodiments, the mutated HPPD gene is introduced as an exogenous gene. In some embodiments, the mutated HPPD gene is introduced by altering an endogenous HPPD gene of the plant or seed.PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032

[0035] In some embodiments, the mutated HPPD gene is introduced by introduction of a gene editing system. In some embodiments, the gene editing system includes a clustered regularly interspaced short palindromic repeats (CRISPR) / Cas nuclease (Cas) system (CRISPR / Cas system) to the plant or seed. In some embodiments, the CRISPR / Cas system includes at least one Cas nuclease and at least one guide RNA. In some embodiments, the Cas nuclease includes, without limitation, class 2 of Cas nucleases, Cas 9, Cas 12, Cas13, Cpf1, or combinations thereof. In some embodiments, the CRISPR / Cas system includes a CRISPR / Cas9 system.

[0036] Herbicide resistance

[0037] Expressed HPPD proteins in the modified plants or seeds of the present disclosure can confer resistance to various herbicides. For instance, in some embodiments, the expressed HPPD protein confers herbicide resistance to herbicides that target HPPD. In some embodiments, the expressed HPPD protein confers herbicide resistance to β-triketone herbicides. In some embodiments, the expressed HPPD protein confers herbicide resistance to mesotrione.

[0038] Plant or seed

[0039] The modified plants or seeds of the present disclosure can include numerous varieties of plants or seeds. For instance, in some embodiments, the plant or seed includes, without limitation, cotton, corn, maize, rice, sorghum, soybean, wheat, N. benthamiana, Arabidopsis, tobacco, tomato, lettuce, common beans, potato, grapes, oilseed rape, sunflower, watermelon, alfalfa, varieties thereof, or combinations thereof. In some embodiments, the plant or seed includes cotton. In some embodiments, the plant or seed includes soybean.

[0040] Expressed HPPD proteins.

[0041] The mutated HPPD genes of the present disclosure can express various HPPD proteins. For instance, in some embodiments, the mutated HPPD gene expresses a protein that includes SEQ ID NO: 1. In some embodiments, SEQ ID NO: 1 includes the following sequence:PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032 MVQTNQSGSGNDFKLVGFSNFVRSNPKSDRFTVKRFHHIEFWCTDATNVARRFSWGLG MQFVAKSDLSTGNLTHASYLLRSRDLNFLFTAPYSPSIAVAQNLSPQSTASIPSFDHSLCR SFAATHGLGVRAIAIEVDDAETAFTTSVTHGALPFCPPTPLGDVATIAEVKLYGDVVLRY VSYTTTINSDHDFLPGFEKIEDTLSYPLDYGLRRLDHAVGNVPELGPAVSYVKSFTGFHE FAEFTAEDVGTSESGLNSVVLANNEEMVLLPMNEPVFGTKRKSQIQTYLEHNEGAGVQ HLALVSEDIFKTLREMRKRSFVGGFEFMPSPX329P X331YYKX335LKQRAGDILSDEQIKECEELGILVDRDDQGTLLQIFTKPVGDRPTIFIEIIQRI GCMVKDEEGKQYQKGGCGGFGKX412NFSELFKSIEEYEKSLEAKQSQNP

[0042] In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 65% sequence identity to SEQ ID NO: 1. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 70% sequence identity to SEQ ID NO: 1. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 75% sequence identity to SEQ ID NO: 1. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 80% sequence identity to SEQ ID NO: 1. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 85% sequence identity to SEQ ID NO: 1. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 90% sequence identity to SEQ ID NO: 1. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 95% sequence identity to SEQ ID NO: 1. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 99% sequence identity to SEQ ID NO: 1.

[0043] In some embodiments, each of X329, X331, X335, and X412 represents an amino acid. In some embodiments, at least one of X329, X331, X335, and X412 in SEQ ID NO: 1 includes a mutation. In some embodiments, the mutation includes at least one of the following: (1) a mutation of X329to Serine (S), (2) a mutation of X331 to Alanine (A), (3) a mutation of X335 to Glutamic Acid (E), (4) a mutation of X412 to Serine (S), or combinations thereof.PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032

[0044] In some embodiments, the mutation includes a mutation of X329from Proline (P) to Serine (S). In some embodiments, the mutation includes a mutation of X329 from Threonine (T) to Serine (S).

[0045] In some embodiments, the mutation includes a mutation of X331from Threonine (T) to Alanine (A). In some embodiments, the mutation includes a mutation of X331 from Aspartic Acid (D) to Alanine (A). In some embodiments, the mutation includes a mutation of X331 from Asparagine (N) to Alanine (A). In some embodiments, the mutation includes a mutation of X331from Serine (S) to Alanine (A).

[0046] In some embodiments, the mutation includes a mutation of X335 from Lysine (K) to Glutamic Acid (E). In some embodiments, the mutation includes a mutation of X335from Asparagine (N) to Glutamic Acid (E). In some embodiments, the mutation includes a mutation of X335 from Glycine (G) to Glutamic Acid (E).

[0047] In some embodiments, the mutation includes a mutation of X412 from Glycine (G) to Serine (S). In some embodiments, the mutation includes a mutation of X412from Aspartic acid (D) to Serine (S).PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032

[0048] In some embodiments, the mutation includes a mutation of X329to Serine (S), and a mutation of X331 to Alanine (A). In some embodiments, the mutation includes a mutation of X329 to Serine (S), and a mutation of X335 to Glutamic Acid (E). In some embodiments, the mutation includes a mutation of X331to Alanine (A), and a mutation of X335to Glutamic Acid (E). In some embodiments, the mutation includes a mutation of X329 to Serine (S), and a mutation of X412 to Serine (S). In some embodiments, the mutation includes a mutation of X331 to Alanine (A), and a mutation of X412to Serine (S). In some embodiments, the mutation includes a mutation of X335 to Glutamic Acid (E), and a mutation of X412 to Serine (S). In some embodiments, the mutation includes a mutation of X329 to Serine (S), a mutation of X331 to Alanine (A), and a mutation of X335to Glutamic Acid (E). In some embodiments, the mutation includes a mutation of X329to Serine (S), a mutation of X331to Alanine (A), and a mutation of X412to Serine (S). In some embodiments, the mutation includes a mutation of X329 to Serine (S), a mutation of X335 to Glutamic Acid (E), and a mutation of X412 to Serine (S). In some embodiments, the mutation includes a mutation of X331to Alanine (A), a mutation of X335to Glutamic Acid (E), and a mutation of X412 to Serine (S). In some embodiments, the mutation includes a mutation of X329 to Serine (S), a mutation of X331 to Alanine (A), a mutation of X335 to Glutamic Acid (E), and a mutation of X412to Serine (S).PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032

[0049] In some embodiments, the mutation includes a mutation of X329to Serine (S) and a mutation of X335 to Glutamic Acid (E). In some embodiments, such a mutated HPPD gene expresses a protein that includes SEQ ID NO: 23. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 65% sequence identity to SEQ ID NO: 23. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 70% sequence identity to SEQ ID NO: 23. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 75% sequence identity to SEQ ID NO: 23. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 80% sequence identity to SEQ ID NO: 23. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 85% sequence identity to SEQ ID NO: 23. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 90% sequence identity to SEQ ID NO: 23. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 95% sequence identity to SEQ ID NO: 23. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 99% sequence identity to SEQ ID NO: 23.PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032

[0050] In some embodiments, the mutation includes a mutation of X329to Serine (S), a mutation of X331 to Alanine (A), and a mutation of X335 to Glutamic Acid (E). In some embodiments, such a mutated HPPD gene expresses a protein that includes SEQ ID NO: 24. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 65% sequence identity to SEQ ID NO: 24. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 70% sequence identity to SEQ ID NO: 24. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 75% sequence identity to SEQ ID NO: 24. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 80% sequence identity to SEQ ID NO: 24. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 85% sequence identity to SEQ ID NO: 24. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 90% sequence identity to SEQ ID NO: 24. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 95% sequence identity to SEQ ID NO: 24. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 99% sequence identity to SEQ ID NO: 24.PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032

[0051] In some embodiments, the mutation includes a mutation of X329to Serine (S), a mutation of X331 to Alanine (A), and a mutation of X412 to Serine (S). In some embodiments, such a mutated HPPD gene expresses a protein that includes SEQ ID NO: 25. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 65% sequence identity to SEQ ID NO: 25. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 70% sequence identity to SEQ ID NO: 25. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 75% sequence identity to SEQ ID NO: 25. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 80% sequence identity to SEQ ID NO: 25. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 85% sequence identity to SEQ ID NO: 25. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 90% sequence identity to SEQ ID NO: 25. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 95% sequence identity to SEQ ID NO: 25. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 99% sequence identity to SEQ ID NO: 25.PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032

[0052] In some embodiments, the mutation includes a mutation of X331to Alanine (A), and a mutation of X335 to Glutamic Acid (E). In some embodiments, such a mutated HPPD gene expresses a protein that includes SEQ ID NO: 26. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 65% sequence identity to SEQ ID NO: 26. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 70% sequence identity to SEQ ID NO: 26. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 75% sequence identity to SEQ ID NO: 26. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 80% sequence identity to SEQ ID NO: 26. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 85% sequence identity to SEQ ID NO: 26. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 90% sequence identity to SEQ ID NO: 26. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 95% sequence identity to SEQ ID NO: 26. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 99% sequence identity to SEQ ID NO: 26.PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032

[0053] In some embodiments, the mutation includes a mutation of X335to Glutamic Acid (E) and a mutation of X412 to Serine (S). In some embodiments, such a mutated HPPD gene expresses a protein that includes SEQ ID NO: 27. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 65% sequence identity to SEQ ID NO: 27. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 70% sequence identity to SEQ ID NO: 27. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 75% sequence identity to SEQ ID NO: 27. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 80% sequence identity to SEQ ID NO: 27. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 85% sequence identity to SEQ ID NO: 27. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 90% sequence identity to SEQ ID NO: 27. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 95% sequence identity to SEQ ID NO: 27. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 99% sequence identity to SEQ ID NO: 27.PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032

[0054] In some embodiments, the mutation includes a mutation of X331to Alanine (A). In some embodiments, such a mutated HPPD gene expresses a protein that includes SEQ ID NO: 28. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 65% sequence identity to SEQ ID NO: 28. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 70% sequence identity to SEQ ID NO: 28. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 75% sequence identity to SEQ ID NO: 28. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 80% sequence identity to SEQ ID NO: 28. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 85% sequence identity to SEQ ID NO: 28. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 90% sequence identity to SEQ ID NO: 28. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 95% sequence identity to SEQ ID NO: 28. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 99% sequence identity to SEQ ID NO: 28.PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032

[0055] In some embodiments, the mutation includes a mutation of X329to Serine (S) and a mutation of X331 to Alanine (A). In some embodiments, such a mutated HPPD gene expresses a protein that includes SEQ ID NO: 29. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 65% sequence identity to SEQ ID NO: 29. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 70% sequence identity to SEQ ID NO: 29. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 75% sequence identity to SEQ ID NO: 29. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 80% sequence identity to SEQ ID NO: 29. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 85% sequence identity to SEQ ID NO: 29. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 90% sequence identity to SEQ ID NO: 29. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 95% sequence identity to SEQ ID NO: 29. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 99% sequence identity to SEQ ID NO: 29.PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032

[0056] In some embodiments, the mutation includes a mutation of X329to Serine (S), a mutation of X335 to Glutamic Acid (E), and a mutation of X412 to Serine (S). In some embodiments, such a mutated HPPD gene expresses a protein that includes SEQ ID NO: 30. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 65% sequence identity to SEQ ID NO: 30. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 70% sequence identity to SEQ ID NO: 30. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 75% sequence identity to SEQ ID NO: 30. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 80% sequence identity to SEQ ID NO: 30. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 85% sequence identity to SEQ ID NO: 30. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 90% sequence identity to SEQ ID NO: 30. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 95% sequence identity to SEQ ID NO: 30. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 99% sequence identity to SEQ ID NO: 30.PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032

[0057] In some embodiments, the mutation includes a mutation of X331to Alanine (A), a mutation of X335 to Glutamic Acid (E), and a mutation of X412 to Serine (S). In some embodiments, such a mutated HPPD gene expresses a protein that includes SEQ ID NO: 31. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 65% sequence identity to SEQ ID NO: 31. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 70% sequence identity to SEQ ID NO: 31. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 75% sequence identity to SEQ ID NO: 31. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 80% sequence identity to SEQ ID NO: 31. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 85% sequence identity to SEQ ID NO: 31. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 90% sequence identity to SEQ ID NO: 31. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 95% sequence identity to SEQ ID NO: 31. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 99% sequence identity to SEQ ID NO: 31.PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032

[0058] In some embodiments, the mutation includes a mutation of X329to Serine (S), a mutation of X331 to Alanine (A), a mutation of X335 to Glutamic Acid (E), and a mutation of X412 to Serine (S). In some embodiments, such a mutated HPPD gene expresses a protein that includes SEQ ID NO: 32. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 65% sequence identity to SEQ ID NO: 32. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 70% sequence identity to SEQ ID NO: 32. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 75% sequence identity to SEQ ID NO: 32. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 80% sequence identity to SEQ ID NO: 32. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 85% sequence identity to SEQ ID NO: 32. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 90% sequence identity to SEQ ID NO: 32. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 95% sequence identity to SEQ ID NO: 32. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 99% sequence identity to SEQ ID NO: 32.

[0059] In some embodiments, the mutated HPPD gene expresses a protein that is analogous to SEQ ID NO: 1. In some embodiments, the analogous gene corresponds to any plant HPPD gene. In some embodiments, the mutated gene expresses a mutated HPPD protein with amino acid changes that correspond to X329, X331, X335, and X412in SEQ ID NO: 1.

[0060] In some embodiments, the HPPD gene expresses a protein that includes SEQ ID NO: 2. In some embodiments, SEQ ID NO: 2 includes the following sequence:PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032 MPMYTPSLSAPSSNHIQPSVTLPLYITTTKLNLKQQHHTTPMPIPMCNEIQAQAQAQAQ X60X61FKLVGFKNFVRTNPKSDRFQVNRFHHIEFWCX93DATNASRRFSWGLGMPIVAKS DLSTGNQIHASYLLRSGDLSFLFSAPYSPSLSAGSSAASSASIPSFDAATCLAFAAKHGFG VRAIALEVADAEAAFSASVAKGAEPASPPVLVDDRTGFAEVRLYGDVVLRYVSYKDAA PQAPHADPSRWFLPGFEAAASSSSFPELDYGIRRLDHX271VGNVPELAPAVRYLKGFSGF HEFAEFTAEDVGTSESGLNSVVLANNSETVLLPLNEPVYGTKRKSQIETYLEHNEGAGV QHLALVTHDIFTTLREMRKRSFLGGFEFMPSPX383PX385YYAX389LHNRX394ADVLTVDQI KQCEELGILVDRDDQGTLLQIFTKPVGDRPTIFIEIIQRIGCMVEDEEGX453VYQKGACGG FGKGNFSELFKSIEEYEKTLEAKRTA

[0061] In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 65% sequence identity to SEQ ID NO: 2. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 70% sequence identity to SEQ ID NO: 2. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 75% sequence identity to SEQ ID NO: 2. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 80% sequence identity to SEQ ID NO: 2. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 85% sequence identity to SEQ ID NO: 2. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 90% sequence identity to SEQ ID NO: 2. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 95% sequence identity to SEQ ID NO: 2. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 99% sequence identity to SEQ ID NO: 2.PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032

[0062] In some embodiments, each of X60, X61, X93, X271, X383, X385, X389, X394, and X453represents an amino acid. In some embodiments, at least one of X60, X61, X93, X271, X383, X385, X389, X394, and X453 in SEQ ID NO: 2 includes a mutation. In some embodiments, the mutation includes at least one of the following: (1) a mutation of X60to Serine (S), (2) a mutation of X61to Glutamic Acid (E), (3) a mutation of X93 to Alanine (A), (4) a mutation of X271 to Valine (V), (5) a mutation of X383 to Serine (S), (6) a mutation of X385 to Alanine (A), (7) a mutation of X389 to Aspartic Acid (D), (8) a mutation of X394to Valine (V), (9) a mutation of X453to Glutamic Acid (E), or combinations thereof.

[0063] In some embodiments, the mutation includes a mutation of X60 from Proline (P) to Serine (S). In some embodiments, the mutation includes a mutation of X61from Glycine (G) to Glutamic Acid (E). In some embodiments, the mutation includes a mutation of X93from Threonine (T) to Alanine (A). In some embodiments, the mutation includes a mutation of X271 from Alanine (A) to Valine (V). In some embodiments, the mutation includes a mutation of X383 from Proline (P) to Serine (S). In some embodiments, the mutation includes a mutation of X385from Threonine (T) to Alanine (A). In some embodiments, the mutation includes a mutation of X389 from Asparagine (N) to Aspartic Acid (D). In some embodiments, the mutation includes a mutation of X394 from Alanine (A) to Valine (V). In some embodiments, the mutation includes a mutation of X453from Lysine (K) to Glutamic Acid (E).PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032

[0064] In some embodiments, the mutation includes a mutation of X60from Asparagine (N) to Serine (S). In some embodiments, the mutation includes a mutation of X60 from Alanine (A) to Serine (S). In some embodiments, the mutation includes a mutation of X61 from Aspartic acid (D) to Glutamic Acid (E). In some embodiments, the mutation includes a mutation of X93from Glycine (G) to Alanine (A). In some embodiments, the mutation includes a mutation of X271 from Isoleucine (I) to Valine (V). In some embodiments, the mutation includes a mutation of X385 from Asparagine (N) to Alanine (A). In some embodiments, the mutation includes a mutation of X385from Serine (S) to Alanine (A). In some embodiments, the mutation includes a mutation of X389 from Lysine (K) to Aspartic Acid (D). In some embodiments, the mutation includes a mutation of X389from Glycine (G) to Aspartic Acid (D). In some embodiments, the mutation includes a mutation of X453from Glutamine (Q) to Glutamic Acid (E). In some embodiments, the mutation includes a mutation of X453 from Arginine (R) to Glutamic Acid (E).PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032

[0065] In some embodiments, the mutation includes a mutation of X61to Glutamic Acid (E), and a mutation of X385 to Alanine (A). In some embodiments, the mutation includes a mutation of X271 to Valine (V), and a mutation of X385 to Alanine (A). In some embodiments, the mutation includes a mutation of X385to Alanine (A), and a mutation of X453to Glutamic Acid (E). In some embodiments, the mutation includes a mutation of X61 to Glutamic Acid (E), and a mutation of X389 to Aspartic Acid (D). In some embodiments, the mutation includes a mutation of X93 to Alanine (A), and a mutation of X389to Aspartic Acid (D). In some embodiments, the mutation includes a mutation of X389 to Aspartic Acid (D), and a mutation of X394 to Valine (V). In some embodiments, the mutation includes a mutation of X389 to Aspartic Acid (D), and a mutation of X453to Glutamic Acid (E). In some embodiments, the mutation includes a mutation of X61to Glutamic Acid (E), a mutation of X93to Alanine (A), and a mutation of X385to Alanine (A). In some embodiments, the mutation includes a mutation of X271 to Valine (V), a mutation of X385 to Alanine (A), and a mutation of X453 to Glutamic Acid (E). In some embodiments, the mutation includes a mutation of X60to Serine (S), a mutation of X61to Glutamic Acid (E), and a mutation of X389 to Aspartic Acid (D). In some embodiments, the mutation includes a mutation of X61 to Glutamic Acid (E), a mutation of X93 to Alanine (A), a mutation of X385 to Alanine (A), and a mutation of X394to Valine (V). In some embodiments, the mutation includes a mutation of X93to Alanine (A), a mutation of X271 to Valine (V), a mutation of X385 to Alanine (A), and a mutation of X453 to Glutamic Acid (E). In some embodiments, the mutation includes a mutation of X60 to Serine (S), a mutation of X61to Glutamic Acid (E), a mutation of X385to Alanine (A), and a mutation of X389to Aspartic Acid (D). In some embodiments, the mutation includes a mutation of X60 to Serine (S), a mutation of X383 to Serine (S), a mutation of X389 to Aspartic Acid (D), and a mutation of X394 to Valine (V). In some embodiments, the mutations include a combination of the mutations of X60,X61, X93, X271, X383, X385, X389, X394, and X453.PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032

[0066] In some embodiments, the mutation includes a mutation of X389to Aspartic Acid (D). In some embodiments, such a mutated HPPD gene expresses a protein that includes SEQ ID NO: 33. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 65% sequence identity to SEQ ID NO: 33. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 70% sequence identity to SEQ ID NO: 33. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 75% sequence identity to SEQ ID NO: 33. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 80% sequence identity to SEQ ID NO: 33. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 85% sequence identity to SEQ ID NO: 33. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 90% sequence identity to SEQ ID NO: 33. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 95% sequence identity to SEQ ID NO: 33. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 99% sequence identity to SEQ ID NO: 33.PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032

[0067] In some embodiments, the mutation includes a mutation of X389to Aspartic Acid (D) and a mutation of X453 to Glutamic Acid (E). In some embodiments, such a mutated HPPD gene expresses a protein that includes SEQ ID NO: 34. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 65% sequence identity to SEQ ID NO: 34. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 70% sequence identity to SEQ ID NO: 34. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 75% sequence identity to SEQ ID NO: 34. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 80% sequence identity to SEQ ID NO: 34. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 85% sequence identity to SEQ ID NO: 34. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 90% sequence identity to SEQ ID NO: 34. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 95% sequence identity to SEQ ID NO: 34. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 99% sequence identity to SEQ ID NO: 34.PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032

[0068] In some embodiments, the mutation includes a mutation of X60to Serine (S), a mutation of X61 to Glutamic Acid (E), and mutation of X389 to Aspartic Acid (D). In some embodiments, such a mutated HPPD gene expresses a protein that includes SEQ ID NO: 35. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 65% sequence identity to SEQ ID NO: 35. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 70% sequence identity to SEQ ID NO: 35. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 75% sequence identity to SEQ ID NO: 35. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 80% sequence identity to SEQ ID NO: 35. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 85% sequence identity to SEQ ID NO: 35. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 90% sequence identity to SEQ ID NO: 35. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 95% sequence identity to SEQ ID NO: 35. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 99% sequence identity to SEQ ID NO: 35.PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032

[0069] In some embodiments, the mutation includes a mutation of X60to Serine (S), a mutation of X383 to Serine (S), a mutation of X389 to Aspartic Acid (D), and a mutation of X394 to Valine (V). In some embodiments, such a mutated HPPD gene expresses a protein that includes SEQ ID NO: 36. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 65% sequence identity to SEQ ID NO: 36. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 70% sequence identity to SEQ ID NO: 36. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 75% sequence identity to SEQ ID NO: 36. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 80% sequence identity to SEQ ID NO: 36. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 85% sequence identity to SEQ ID NO: 36. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 90% sequence identity to SEQ ID NO: 36. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 95% sequence identity to SEQ ID NO: 36. In some embodiments, the mutated HPPD gene expresses a protein that includes a sequence with at least 99% sequence identity to SEQ ID NO: 36.

[0070] In some embodiments, the mutated HPPD gene expresses a protein that is analogous to SEQ ID NO: 2. In some embodiments, the analogous gene corresponds to any plant HPPD gene. In some embodiments, the mutated gene expresses a mutated HPPD protein with amino acid changes that correspond to X60, X61, X93, X271, X383, X385, X389, X394, and X453in SEQ ID NO: 2.

[0071] Additional Embodiments

[0072] Reference will now be made to more specific embodiments of the present disclosure and experimental results that provide support for such embodiments. However, Applicant notes that the disclosure below is for illustrative purposes only and is not intended to limit the scope of the claimed subject matter in any way.

[0073] Example 1. Herbicide resistant HPPD variants identified via directed evolutionPCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032

[0074] In this Example, by using directed evolution, four single nucleotide changes in the cotton HPPD gene conferring herbicide resistance were identified. These single mutations and their combinations enhanced herbicide resistance without significantly impairing its native activity in plants. Applicant aimed to identify mutations within the cotton HPPD gene that confer resistance to mesotrione, a widely used triketone herbicide.

[0075] Through the establishment of a high-throughput mutant screening system in E. coli, Applicant identified four single nucleotide changes leading to amino acid substitutions in HPPD, resulting in mesotrione resistance while preserving native enzymatic activity. Various combinations of these mutations displayed synergistic effects on herbicide resistance. Additionally, the HPPD variants were able to complement the Arabidopsis athppd mutant, indicating their retention of sufficient native activity crucial for plant growth and development. Expression of these cotton HPPD variants in Arabidopsis resulted in heightened herbicide resistance. These findings offer critical insights into the target amino acids of HPPD for gene editing, indicating the ability to develop herbicide-resistant cotton.

[0076] Example 1.1. Construction of the vector with recombinant HPPD

[0077] The cotton HPPD gene (Gohir.A07G080400) was identified based on the Arabidopsis HPPD protein sequence. Total RNA extracted from cotton (Coker 312) leaves was used for cDNA synthesis by using reverse transcription polymerase chain reaction (RT-PCR). The HPPD coding sequence (CDS) (1311bp) was amplified from cDNAs by PCR with the designed primers (F 5’- CGCGGATCCAATGGTCCAGACAAACCAGTCTG-3’ (SEQ ID NO: 9) and R 5’- ACGCGTCGACTCATGGATTCTGAGATTGTTTGGC-3’ (SEQ ID NO: 10)).

[0078] The PCR product of HPPD CDS was then digested with BamHI and SalI restriction enzymes and ligated to pETBlue-2 vector. After sequencing and confirming the HPPD CDS sequence, the HPPD CDS fragment was excised from pETBlue-2-HPPD vector and ligated with pET-30b to create pET-30b-HPPD plasmid.

[0079] Example 1.2. HPPD expression and inhibition by herbicide mesotrione on agar platesPCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032

[0080] The plasmid pET-30b-HPPD was transformed into C43 (DE3), and the transformed bacterial cells were spread on the LB agar plate containing 0.2 mM IPTG, 50 g mL-1 Kanamycin and 1 mg mL-1 Tyr. The expression of HPPD was assessed by the color change of the colonies harboring pET-30b-HPPD. When Tyr and IPTG were not added, the colonies were white. However, when Tyr and IPTG were supplemented, the color of colonies changed to brownish, owing to the production of melanin-like pigment formed from HGA, the product of HPPD enzyme. The inhibition of HPPD activity by mesotrione was determined by supplementing mesotrione to the medium and the reduction of brownish color in the colonies.

[0081] Example 1.3. Determination of the IC50 of mesotrione on HPPD inhibition

[0082] The bacteria C43 (DE3) with pET-30b-HPPDs were cultured overnight in 5 mL LB liquid media with 1 mg mL-1 Tyr and 50 g mL-1 Kanamycin. The bacterial cultures were then diluted at a 1:100 ratio with new LB liquid medium supplemented with Kan and Tyr. After culture for 2 hours in a shaker, the OD600 was determined and adjusted to around 0.6. Equal volume of the culture was dispensed to different tubes, then IPTG was added to a final concentration of 0.2 mM, and mesotrione was supplemented with different concentrations.

[0083] After about 14 hours culture in a shaking incubator, different levels of brownish color in the cultures under the treatment with various concentrations of mesotrione were observed. The cultures were centrifuged, the supernatants were collected, and the content of soluble brownish pigment was measured with a spectrophotometer at 400 nm. The IC50 of mesotrione on HPPD inhibition was calculated by using the online tool (https: / / www.aatbio.com / tools / ic50-calculator).

[0084] Example 1.4. Random mutagenesis and screening for herbicide-resistant variants of HPPDPCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032

[0085] The mutator E. coli strain XL1-red (endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac mutD5 mutS mutT Tn10 (Tet r)) was used to generate random point mutations. This mutator strain lacks certain functional genes (mutS, mutT, mutD) involved in mismatch repair, resulting in defects in DNA repair and thus increased replicative mutations in E. coli. (Muteeb and Sen, 2010). The pET- 30b-HPPD plasmid was transformed into XL1-red and the transformants were divided and cultured on 10 agar plates. 10 independent HPPD mutagenized libraries were prepared by collecting all the colonies from each plate, culturing in a shaker for 14 hours, and isolating plasmids separately. The plasmids from each pool were transformed to C43 (DE3), and the transformed C43 (DE3) cells were spread on LB agar plates with 0.2 mM IPTG, 50 mg mL-1 Kanamycin, 1 mg mL-1 Tyr and 20 μM mesotrione. The herbicide-resistant HPPD variants were isolated based on the increased brownish color compared with most of other colonies on the plates due to less inhibition of HPPD by mesotrione.

[0086] Example 1.5. Site-directed mutagenesis to combine identified mutations in the HPPD gene

[0087] The combination of single mutations to create double, triple, and quadruple mutants of HPPD was carried out by using site-directed mutagenesis. The primers for the introduction of point mutations in the vector pET-30b-HPPD are listed in Table 1.PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032 Combined mutations Base mutation Forward primer Reverse primer P329S+T331A T331A TTCATGCCGTCGCCGTCGC TATAATAAGCCGGCGACGGC CGGCTTATTATA GACGGCATGAA P329S+T331A+K335E T331A+K335E(SEQ ID NO: 15)(SEQ ID NO: 16)P329S+K335E P329S CCGACTTATTATAAAGAAT CCCTTTGCTTCAATTCTTTAT TGAAGCAAAGGG AATAAGTCGG (SEQ ID NO: 17) (SEQ ID NO: 18) T331A+K335E T331A CCGGCTTATTATAAAGAAT CCCTTTGCTTCAATTCTTTAT TGAAGCAAAGGG AATAAGCCGG (SEQ ID NO: 19) (SEQ ID NO: 20) P329S+G412S P329S T331A+G412S T331A K335E + G412S K335E GGTGGTTTTGGGAAAAGC GCTCAGAAAAGTTGCTTTTC P329S+T331A+G412S P329S+T331A AACTTTTCTGAGC CCAAAACCACC P329S+K335E+G412S P329S+K335E (SEQ ID NO: 21) (SEQ ID NO: 22) T331A+K335E+G412S T331A+K335E P329S+T331A+K335E P329S+T331A+ +G412S K335E Letters underlined indicate mutated nucleotides, and italicized bold letters indicate nucleotides to be mutated. Table 1. Primers used for site-directed mutagenesis to generate the combinations of the four mutations.PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032

[0088] As the first step, double mutations including P329S+T331A, P329S+K335E, T331A+K335E, P329S+G412S, T331A+G412S and K335E + G412S were made based on vectors pET-30b-HPPD with single mutations. Then triple mutations including P329S+T331A+K335E, P329S+T331A+G412S, P329S+K335E+G412S and T331A+K335E+G412S were constructed based on vectors pET-30b-HPPD with double mutations. Finally, quadruple mutations P329S+T331A+K335E+G412S was constructed based on vector with triple mutations P329S+T331A+K335E. Totally, 11 mutants with multiple mutations in HPPD were constructed in pET-30b.

[0089] Example 1.6. Construction of the vectors for expressing HPPD in plants

[0090] Wild type HPPD, four single mutants of HPPD and 11 mutants with multiple mutations in HPPD were introduced into the plant expression vector pCAMBIA 99-1. The wild type and variant HPPD CDSs in pET-30b were double digested with BamHI and SalI restriction enzymes and the HPPD fragments were ligated to the vector pCAMBIA 99-1 respectively, creating the plant expression cassette of HPPD under the control of 35S promoter. After sequencing the constructed plasmids, the vectors were used for Arabidopsis transformation.

[0091] Example 1.7. Transformation of cotton wild type and variant HPPDs into the Arabidopsis T-DNA insertion mutant

[0092] The Arabidopsis T-DNA insertion mutant SALK_076732 was obtained from ABRC. The annotated position of T-DNA insertion at TAIR is C / 2012859-2013258 in chromosome 1. For genotyping of the T-DNA insertion, the mutant seeds were germinated on ½ Murashige and Skoog (MS) agar medium, and the seedlings were then transferred to soil for further growth. Before bolting of the plants, several leaves were collected from each plant and genomic DNA was extracted via the sucrose extraction method.PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032

[0093] The extracted DNA was used for genotyping by PCR with designed primers (F1 5’- TCAGCGATCGTAACTGCTTC-3’ (SEQ ID NO: 11) and R1 5’- GCTCAACGAAACACACAGTTC-3’ (SEQ ID NO: 12); F2 5’- ACATAAGTTAAAGCCGGACC-3’ (SEQ ID NO: 13) and LBb1.3 5’- ATTTTGCCGATTTCGGAAC-3’ (SEQ ID NO: 14)). The F1 and R1 primer pair was designed to amplify the wild type genomic region, while the F2 and LBb1.3 primer pair was for the detection of T-DNA insertion. Since the HPPD gene is known as an essential gene, PCR-based genotyping only identified wild type and heterozygous, and the heterozygous progeny was used for transformation with the cotton HPPD.

[0094] The heterozygous seeds were geminated on ½ MS agar medium, and the seedlings were then transferred to soil. The healthy Arabidopsis plants starting to flower were used for transformation with the Agrobacterium GV3101 harboring the plasmids 99-1-HPPDs by using the floral dip method. The seeds from transformed plants were screened for positive transgenic plants by using ½ MS medium with 25 ug mL-1 Hygromycin. The hygromycin resistant seedlings were then transferred to soil. When the plants started bolting, leaf materials were collected from each plant and DNA was extracted. The extracted DNA was used for genotyping of the T-DNA insertion and verification of heterozygous mutant, homozygous mutant or wild type in the transgenic plants.

[0095] Example 1.8. Complementation and herbicide-resistant assay of the transgenic Arabidopsis with the cotton HPPD

[0096] T2 seeds were collected from the T1 transgenic lines in the background of heterozygous or homozygous mutation of the T-DNA insertion. The T2 seeds were grown on ½ MS plates containing hygromycin to select the hygromycin resistant seedlings. For herbicide-resistant assay, the T2 seedlings were transferred to new ½ MS plates containing different concentrations of mesotrione. The photos showing growth phenotypes were taken at one week and two weeks of seedling growth. The chlorophyll content was measured after treatment for two weeks. The chlorophyll was extracted with 100% acetone and the chlorophyll content was measured according to the previous described method.PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032

[0097] For genetic complementation assay, individual T2 transgenic plants harboring different cotton HPPD variants were genotyped by PCR for the T-DNA insertion. The homozygous mutants of the Arabidopsis athppd expressing the cotton HPPD variants were used to determine whether the cotton HPPD variants could complement the Arabidopsis HPPD gene by observing the growth and development of the plants.

[0098] Example 1.9. Complementation and herbicide-resistant assay of the transgenic Arabidopsis with the cotton HPPD

[0099] T2 seeds were collected from the T1 transgenic lines in the background of heterozygous or homozygous mutation of the T-DNA insertion. The T2 seeds were grown on ½ MS plates containing hygromycin to select the hygromycin resistant seedlings. For herbicide-resistant assay, the T2 seedlings were transferred to new ½ MS plates containing different concentrations of mesotrione. The photos showing growth phenotypes were taken at one week and two weeks of seedling growth. The chlorophyll content was measured after treatment for two weeks. The chlorophyll was extracted with 100% acetone and the chlorophyll content was measured according to the previous described method.

[0100] For genetic complementation assay, individual T2 transgenic plants harboring different cotton HPPD variants were genotyped by PCR for the T-DNA insertion. The homozygous mutants of the Arabidopsis athppd expressing the cotton HPPD variants were used to determine whether the cotton HPPD variants could complement the Arabidopsis HPPD gene by observing the growth and development of the plants.

[0101] Example 1.10. Establishment of a high-throughput screening for herbicide-resistant HPPD variantsPCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032

[0102] Applicant first attempted to use a constitutive expression system for HPPD by using the vector pETBlue-2 and the bacterial strain BL21 (DE3). After testing the HPPD protein expression and enzyme inhibition by herbicide mesotrione, Applicant found that the pigment accumulated in the bacterial cells was not dark enough for discriminating color changes in colonies, which may be due to low expression of HPPD in bacteria.

[0103] Applicant then switched to the IPTG-inducible vector pET-30b and found that increasing concentrations of IPTG clearly inhibited bacterial growth, which suggested that the product of HPPD is somewhat toxic to E. coli. Applicant thus selected a BL21 (DE3) derived strain named C43 (DE3) known to tolerate high expression of toxic proteins. In fact, C43 (DE3) strain allowed high expression of HPPD, which was indicated by the dark color of the pigment (FIG.1A).

[0104] Condition optimization for HPPD induction suggested that 0.2 mM IPTG was an appropriate concentration (FIGS. 1A-1C). To determine the herbicide concentration for the screening of herbicide-resistant HPPD variants, Applicant tested six different concentrations of mesotrione on HPPD activity in E. coli C43 (DE3) transformed with pET-30b-HPPD and grown on LB agar medium containing 0.2 mM IPTG, 50 g mL-1 Kanamycin and 1 mg mL-1 Tyr. The concentration of 20 uM was found to be an appropriate concentration of mesotrione, resulting in slightly less than half brownish color of the colonies comparing with the colonies without herbicide (FIG.1D), and thus 20 uM mesotrione was chosen for large scale screening of the HPPD mutants.

[0105] Example 1.11. Identification of HPPD mutations conferring mesotrione resistancePCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032

[0106] To screen for herbicide-resistant HPPD variants, Applicant generated HPPD mutant libraries by introducing the plasmid pET-30b-HPPD into the mutator strain E. coli XL1-red. The mutation rate was determined by sequencing the HPPD gene in 20 plasmids, and two random mutations in the HPPD gene were found, which suggested a mutation rate of approximately 1 / 15000. The mutated plasmids were transformed into C43 (DE3) and the transformed bacterial cells were grown on the LB agar medium supplemented with 0.2 mM IPTG and 20 uM mesotrione. The colonies displayed darker color compared with most of the colonies were selected and considered as putative herbicide-resistant HPPD variants.

[0107] After multiple rounds of screening using the 10 HPPD mutant libraries, in total about a hundred colonies with darker brownish color were isolated from approximately 300,000 colonies. The isolated colonies were subjected to a rescreening by streaking the mutant colonies side by side with the wild type pET-30b-HPPD in C43 (DE3) on LB agar plates supplemented with 20 uM mesotrione.

[0108] After rescreening, 39 colonies showing clear increase in brownish color compared to the colonies with the wild type HPPD were chosen, and the plasmids were isolated. After sequencing the HPPD gene, a total of four different mutations in the HPPD gene were identified. Among the four mutations, a C to T transition, resulting in an amino acid change from Proline (P) to Serine (S) at the position 329 of the protein, was found to be the most identified mutation from the screening. Out of 39 confirmed colonies, 28 harbored this mutated HPPD. The second most identified mutation was an A to G transition, which causes an amino acid change from Threonine (T) to Alanine (A) at position 331. This mutation was recovered from six confirmed colonies. The third identified mutation was also an A to G transition, which was identified from two confirmed colonies, resulting in a change of Lysine (K) to Glutamic Acid (E) at position 335. The fourth identified mutation was a G to A transition, which results in a change of Glycine (G) to Serine (S) at position 412. The fourth mutation was recovered from three confirmed colonies. The four mutations in HPPD protein conferring herbicide resistance are shown in FIG.2.

[0109] Example 1.12. The effects of combined HPPD mutations on herbicide resistancePCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032

[0110] To test whether the combinations of identified mutations could provide higher herbicide resistance, Applicant created double, triple, and quadruple mutations of these four single mutations by using site-directed mutagenesis. Double mutations include P329S+T331A, P329S+K335E, T331A+K335E, P329S+G412S, T331A+G412S and K335E + G412S. Triple mutations include P329S+T331A+K335E, P329S+T331A+G412S, P329S+K335E+G412S and T331A+K335E+G412S. Quadruple mutations include P329S+T331A+K335E+G412S.

[0111] Applicant spread E. coli C43 (DE3) with single and multiple mutations of HPPD on agar plates with 0.2 mM IPTG, 50 g mL-1 Kanamycin, 1 mg mL-1 Tyr and different concentrations of herbicide mesotrione. The results showed that, on agar medium, all mutation combinations of HPPD exhibited similar enzyme activity compared with the wild type HPPD without herbicide mesotrione, which was indicated by the similar levels of pigment accumulation in the cells.

[0112] Nevertheless, starting from 60 uM herbicide mesotrione, the wild type and single mutation of HPPD showed significantly decreased activity compared with the double, triple and quadruple mutations of HPPD. At the concentration of 140 uM herbicide mesotrione, the colonies with wild type HPPD showed nearly no brown color, while combined mutations conferred higher resistance to herbicide mesotrione, especially the triple and quadruple mutations of HPPD (FIG. 3A). In liquid culture, most of the combined mutations also showed higher resistance to mesotrione than the single mutations, which was shown by darker color indicative of higher accumulation of the pigment (FIG.3B).

[0113] To quantitatively compare the different HPPD variants, the IC50of mesotrione on the 15 HPPD variants and wild type were determined. The result showed that all the 15 HPPD variants exhibited higher IC50 than wild type, ranging from about 3- to 40-fold higher IC50, and all triple mutants and the quadruple mutant showed significantly higher IC50 than the single mutants (FIG. 3C).

[0114] Example 1.13. Complementation of the Arabidopsis athppd mutant with the cotton HPPD variantsPCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032

[0115] Since HPPD is an essential gene in plants, the native activities of the HPPD variants are critical for their applications in creating herbicide-resistant crops by gene editing. To determine the native activity of the HPPD variants, the bacteria with HPPD wild type and variants were incubated in the liquid culture with 0.2 mM IPTG, 50 g mL-1 Kanamycin and 1 mg mL-1 Tyr without mesotrione. The OD400 and OD600 were measured after culture for 14 hours respectively.

[0116] The results showed that all 15 HPPD variants had similar or slightly decreased OD400 values when compared to the wild type HPPD at 14 hours (FIG.4A). This result indicated that, although these mutations conferred resistance to mesotrione, these variants still maintained their native activities comparable to the wild type.

[0117] Whether the herbicide-resistant HPPD variants could sustain normal plant growth was further tested in Arabidopsis by using the Arabidopsis athppd mutant. The Arabidopsis T-DNA insertion mutant (Salk_076732) of HPPD was PCR verified, and as expected, homozygous mutant could not be recovered because of the essential role of HPPD in plants. Therefore, Applicant used the heterozygous athppd mutant for transformation with the cotton HPPD variants. At the T2 generation, homozygous athppd mutant lines with the cotton HPPD transgene were identified in several transgenic lines, indicating that the cotton HPPD variants could functionally complement the Arabidopsis HPPD gene (FIG. 4B). This further supports that the cotton HPPD variants, despite conferring herbicide resistance, maintained native activity sufficient for plant growth and development.

[0118] However, among the complemented lines, the line with K335E mutation showed white spots on leaves and young shoots and produced fewer seeds, and the line with K335E+G412S displayed yellowish leaves and shoots and produced less seeds. Applicant failed to identify homozygous T-DNA mutant lines with the cotton HPPD variants G412S, P329S+G412S, P329S+K335E+G412S and P329S+T331A+K335E+G412S even at the T3 generation, suggesting that these four variants may not maintain sufficient native activities for plant growth.

[0119] Example 1.14. Herbicide resistance of the cotton HPPD variants in ArabidopsisPCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032

[0120] To determine whether expression of the cotton HPPD variants confers tolerance to herbicide mesotrione in plants, Applicant tested the response of the Arabidopsis plants expressing the cotton wild type HPPD and the variant HPPD to mesotrione. The tolerance assay was carried out on agar plates.

[0121] Determination of appropriate concentrations of mesotrione for tolerance assay on agar plates suggested that the concentrations range from 1-7 μM were too high because all seedlings were bleached in one to two weeks, while concentrations range from 2-30 nM may be too low for the assay because all seedlings grew normal and green after one to two weeks treatment.

[0122] Applicant then decided to use the concentrations 0, 25, 50, 100, and 150 nM for the tolerance assay on agar plates. One-week-old T2 transgenic seedling selected from hygromycin containing agar medium were transferred onto ½ MS agar plates supplemented with mesotrione. One week after transfer, the transgenic lines with the HPPD variants T331A, P329S+T331A, P329S+K335E, T331A+K335E, K335E + G412S, P329S+T331A+K335E, P329S+T331A+G412S, and P329S+K335E+G412S showed clear better growth and greener leaves than the transgenic line with the cotton wild type HPPD. The transgenic lines with the HPPD variants P329S, K335E, G412S, P329S+G412S, T331A+G412S, T331A+K335E+G412S, and P329S+T331A+K335E+G412S were only slightly better or similar to that with the wild type HPPD (FIG. 5A). Chlorophyll content assay indicated that the lines T331A, P329S+T331A, P329S+K335E, T331A+K335E, K335E+G412S, P329S+T331A+K335E, and P329S+T331A+G412S showed strong resistance to mesotrione, among which P329S+K335E, P329S+T331A+K335E and P329S+T331A+G412S were the strongest ones for herbicide resistance (FIG.5B).

[0123] Example 1.15. Herbicide resistance of the soybean HPPD variants in ArabidopsisPCT Application Attorney Docket No. AF13368.P057WO Texas Tech No. 2023-032

[0124] To determine whether expression of the soybean HPPD variants confers tolerance to herbicide mesotrione in plants, Applicant tested the response of the Arabidopsis plants expressing the soybean wild type HPPD and the variant HPPD to mesotrione. The tolerance assay was carried out on agar plates. Table 2 lists the mutations of the soybean HPPD protein variants. The full sequence is listed in SEQ ID NO: 2. Table 2. The mutations of the soybean HPPD protein variants. The full sequence is listed in SEQ ID NO: 2

[0125] FIG. 6 shows the IC50 of mesotrione on the wild type and variants of soybean HPPD (single mutated and multi-mutated variants included). The IC50test was conducted under different concentrations of mesotrione ranging from 0 uM to 200 uM. The results indicate that these mutant variants exhibit a stronger resistance to herbicides compared to the wild type, ranging from 1.5- to 15-fold increases. Furthermore, the combination of multiple single mutants demonstrates an enhanced herbicide resistance compared to individual single mutants, with most of the quadruple mutant displaying about 15-fold increase in herbicide resistance compared to the wild type.PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032

[0126] To assess the herbicide tolerance of soybean HPPD variants in plants, Applicant evaluated the response of Arabidopsis plants expressing both wild-type soybean HPPD and its variants to mesotrione. The results are summarized in FIG. 7, where agar plates were supplemented with mesotrione concentrations ranging from 0 to 100 nM. One-week-old T2 transgenic seedlings, pre- selected on hygromycin B-containing agar, were transferred to ½ MS agar plates containing mesotrione at 0, 10, 20, 30, 40, 50, and 100 nM.

[0127] Chlorophyll content was analyzed post-treatment for two weeks to quantify herbicide tolerance. The results are summarized in FIG.8. After two weeks, the transgenic lines with the HPPD variants N389D, N389D+K453E, and P60S+G61E+N389D showed lightly better growth compared to WT HPPD. Additionally, P60S+P383S+N389D+A394V showed significantly better growth compared to WT HPPD. Chlorophyll content assay after two weeks also confirmed the results above. For P60S+P383S+N389D+A394V, the chlorophyll content had no apparent difference under 0 nM, 30 nM, 50 nM compared to WT HPPD at 0 nM, which means P60S+P383S+N389D+A394V can grow well on 100 nM mesotrione plate as good as on plate without mesotrione.

[0128] Example 1.16. Discussion

[0129] In this Example, Applicant identified distinct nucleotide mutations in cotton and soybean HPPD, resulting in corresponding amino acid changes that significantly enhanced resistance to the herbicide mesotrione. Combining these mutations further bolstered mesotrione resistance. This indicates that the high throughput mutant screening system employed in this Example is highly effective on identifying single nucleotide mutations for herbicide resistant HPPD variants, which provides basis for creating additional resistant variants by testing the combinations of the identified mutations.PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032

[0130] Since HPPD is a conserved protein in plants, Applicant expects that this screening system can be applied to the HPPD genes from other crops, and the identified mutations could also be useful for creating mesotrione resistant HPPD variants in other crops. Although the screening system in E. coli was effective, Applicant noticed that some of the identified mutations and their combinations behaved differently in E. coli and plants.

[0131] In particular, the P329S in cotton HPPD was the most identified mutation in the E. coli screening and showed significantly elevated resistance to mesotrione in E. coli. However, this mutation displayed little effect on mesotrione resistance in the transgenic Arabidopsis. This discrepancy suggests that the HPPD enzyme might act somewhat differently in bacteria and plants, which requires attention in the screening for other herbicide resistant HPPD variants.

[0132] Among the four identified mutations in cotton HPPD, one mutation (G412S) occurs on helix11, while three others (P329S, T331A, K335E) are located at the surrounding area that interacts with helix11 in HPPD. Helix 11 is a structural component of the HPPD C-terminal, which has been implicated in herbicide resistance. This helix may act as a "gate" that can either shield the active site or allow solvent access.

[0133] Without being bound by theory, mutations within or near helix 11 may alter the binding site conformation, affecting herbicide accessibility. For instance, maize exhibits a higher resistance to herbicide mesotrione compared to Arabidopsis, possibly due to the preference of AtHPPD for an open helix11 state, while ZmHPPD favors a closed helix 11 state. The P329S and G412S mutations may result in the formation of additional hydrogen bonds between the helix 11 and the surrounding area, making the helix 11 in more closed conformation and thus preventing mesotrione access to the active site. The T331A substitution could disrupt the hydrogen bond formed by T331 with other regions and alter the accessibility of the active site pocket. The K335E change may affect the association between the helix 11 and the surrounding area by forming salt bridge with the lysine residues in the helix 11.PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032

[0134] Herbicide-resistant crops targeting HPPD offer promising weed management strategies. By disrupting pigment and plastoquinone synthesis, these herbicides effectively control a broad spectrum of weeds, including resistant strains. Incorporating HPPD-resistant variants into crops reduces reliance on traditional herbicides, aligning with sustainable agriculture goals.

[0135] In sum, Applicant aimed to identify mutations in cotton and soybean HPPD conferring herbicide resistance and introduce them via gene editing to create herbicide-resistant cotton. Through a high-throughput screening system in E. coli, Applicant identified four mutations conferring mesotrione resistance while maintaining native activities. These cotton HPPD variants, either individually or in combination, complemented the Arabidopsis athppd mutant and significantly enhanced herbicide resistance in plants.

[0136] Prior approaches utilized transgenic crops with herbicide resistant variant genes from other sources, such as bacterial genes. The approach in this Example is more advantageous for various reasons. First, the amino acid changes identified in this Example were not previously known. Second, plants that incorporate these mutations through gene editing methods can be herbicide resistant plants without being labeled as transgenic crops. Thus, the crops produced by the methods in this Example can create novel crops for better weed management.

[0137] Without further elaboration, it is believed that one skilled in the art can, using the description herein, utilize the present disclosure to its fullest extent. The embodiments described herein are to be construed as illustrative and not as constraining the remainder of the disclosure in any way whatsoever. While the embodiments have been shown and described, many variations and modifications thereof can be made by one skilled in the art without departing from the spirit and teachings of the invention. Accordingly, the scope of protection is not limited by the description set out above, but is only limited by the claims, including all equivalents of the subject matter of the claims. The disclosures of all patents, patent applications and publications cited herein are hereby incorporated herein by reference, to the extent that they provide procedural or other details consistent with and supplementary to those set forth herein.

Claims

PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032 WHAT IS CLAIMED IS:

1. A modified plant or seed, wherein the modified plant or seed comprises a mutated 4- hydroxyphenylpyruvate dioxygenase (HPPD) gene, wherein the mutated HPPD gene expresses an HPPD protein that confers herbicide resistance to the modified plant or seed.

2. The modified plant or seed of claim 1, wherein the mutated HPPD gene comprises an endogenous HPPD gene.

3. The modified plant or seed of claim 1, wherein the mutated HPPD gene comprises an exogenous HPPD gene.

4. The modified plant or seed of claim 1, wherein the plant or seed is selected from the group consisting of cotton, corn, maize, rice, sorghum, soybean, wheat, N. benthamiana, Arabidopsis, tobacco, tomato, lettuce, common beans, potato, grapes, oilseed rape, sunflower, watermelon, alfalfa, varieties thereof, or combinations thereof.

5. The modified plant or seed of claim 1, wherein the plant or seed comprises cotton.

6. The modified plant or seed of claim 1, wherein the plant or seed comprises soybean.

7. The modified plant or seed of claim 1, wherein the mutated HPPD gene expresses a protein comprising SEQ ID NO: 1, or a sequence with at least 65% sequence identity to SEQ ID NO: 1, wherein SEQ ID NO: 1 comprises: 47 4823-4406-5785v.313368-42PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032 MVQTNQSGSGNDFKLVGFSNFVRSNPKSDRFTVKRFHHIEFWCTDATNVARRFSWGLG MQFVAKSDLSTGNLTHASYLLRSRDLNFLFTAPYSPSIAVAQNLSPQSTASIPSFDHSLCR SFAATHGLGVRAIAIEVDDAETAFTTSVTHGALPFCPPTPLGDVATIAEVKLYGDVVLRY VSYTTTINSDHDFLPGFEKIEDTLSYPLDYGLRRLDHAVGNVPELGPAVSYVKSFTGFHE FAEFTAEDVGTSESGLNSVVLANNEEMVLLPMNEPVFGTKRKSQIQTYLEHNEGAGVQ HLALVSEDIFKTLREMRKRSFVGGFEFMPSPX329P X331YYKX335LKQRAGDILSDEQIKECEELGILVDRDDQGTLLQIFTKPVGDRPTIFIEIIQRI GCMVKDEEGKQYQKGGCGGFGKX412NFSELFKSIEEYEKSLEAKQSQNP, wherein each of X329, X331, X335, and X412represents an amino acid, wherein at least one of X329, X331, X335, and X412 comprises a mutation, wherein the mutation comprises at least one of the following: a mutation of X329 to Serine (S), a mutation of X331 to Alanine (A), a mutation of X335to Glutamic Acid (E), a mutation of X412 to Serine (S), or combinations thereof.

8. The modified plant or seed of claim 7, wherein the mutation comprises at least one of the following: 48 4823-4406-5785v.313368-42PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032 a mutation of X329from Proline (P) to Serine (S), or a mutation of X329from Threonine (T) to Serine (S); a mutation of X331from Threonine (T) to Alanine (A), or a mutation of X331from Aspartic Acid (D) to Alanine (A); a mutation of X335from Lysine (K) to Glutamic Acid (E), a mutation of X335from Asparagine (N) to Glutamic Acid (E), or a mutation of X335 from Glycine (G) to Glutamic Acid (E); a mutation of X412from Glycine (G) to Serine (S); or combinations thereof.

9. The modified plant or seed of claim 7, wherein the mutation comprises at least one of the following: a mutation of X335 to Glutamic Acid (E), and a mutation of X412 to Serine (S); a mutation of X329to Serine (S), a mutation of X331to Alanine (A), and a mutation of X335to Glutamic Acid (E); a mutation of X329 to Serine (S), a mutation of X331 to Alanine (A), and a mutation of X412 to Serine (S); a mutation of X329 to Serine (S), a mutation of X335 to Glutamic Acid (E), and a mutation of X412to Serine (S); 49 4823-4406-5785v.313368-42PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032 a mutation of X331to Alanine (A), a mutation of X335to Glutamic Acid (E), and a mutation of X412 to Serine (S); or a mutation of X329to Serine (S), a mutation of X331to Alanine (A), a mutation of X335to Glutamic Acid (E), and a mutation of X412 to Serine (S).

10. The modified plant or seed of claim 7, wherein the mutation comprises at least one of the following: a mutation of X329 to Serine (S) and a mutation of X335 to Glutamic Acid (E), and wherein the mutated HPPD gene expresses a protein comprising SEQ ID NO: 23 or a sequence with at least 65% sequence identity to SEQ ID NO: 23; a mutation of X329 to Serine (S), a mutation of X331 to Alanine (A), and a mutation of X335 to Glutamic Acid (E), and wherein the mutated HPPD gene expresses a protein comprising SEQ ID NO: 24 or a sequence with at least 65% sequence identity to SEQ ID NO: 24; a mutation of X329 to Serine (S), a mutation of X331 to Alanine (A), and a mutation of X412to Serine (S), and wherein the mutated HPPD gene expresses a protein comprising SEQ ID NO: 25 or a sequence with at least 65% sequence identity to SEQ ID NO: 25; a mutation of X331to Alanine (A), and a mutation of X335to Glutamic Acid (E), and wherein the mutated HPPD gene expresses a protein comprising SEQ ID NO: 26 or a sequence with at least 65% sequence identity to SEQ ID NO: 26; a mutation of X335to Glutamic Acid (E) and a mutation of X412to Serine (S), and wherein the mutated HPPD gene expresses a protein comprising SEQ ID NO: 27 or a sequence with at least 65% sequence identity to SEQ ID NO: 27; 50 4823-4406-5785v.313368-42PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032 a mutation of X331to Alanine (A), and wherein the mutated HPPD gene expresses a protein comprising SEQ ID NO: 28 or a sequence with at least 65% sequence identity to SEQ ID NO: 28; a mutation of X329 to Serine (S) and a mutation of X331 to Alanine (A), and wherein the mutated HPPD gene expresses a protein comprising SEQ ID NO: 29 or a sequence with at least 65% sequence identity to SEQ ID NO: 29; a mutation of X329 to Serine (S), a mutation of X335 to Glutamic Acid (E), and a mutation of X412to Serine (S), and wherein the mutated HPPD gene expresses a protein comprising SEQ ID NO: 30 or a sequence with at least 65% sequence identity to SEQ ID NO: 30; a mutation of X331to Alanine (A), a mutation of X335to Glutamic Acid (E), and a mutation of X412 to Serine (S), and wherein the mutated HPPD gene expresses a protein comprising SEQ ID NO: 31 or a sequence with at least 65% sequence identity to SEQ ID NO: 31; or a mutation of X329 to Serine (S), a mutation of X331 to Alanine (A), a mutation of X335 to Glutamic Acid (E), and a mutation of X412to Serine (S), and wherein the mutated HPPD gene expresses a protein comprising SEQ ID NO: 32 or a sequence with at least 65% sequence identity to SEQ ID NO:

32.

11. The modified plant or seed of claim 1, wherein the HPPD gene expresses a protein comprising SEQ ID NO: 2 or a sequence with at least 65% sequence identity to SEQ ID NO: 2, wherein SEQ ID NO: 2 comprises: 51 4823-4406-5785v.313368-42PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032 MPMYTPSLSAPSSNHIQPSVTLPLYITTTKLNLKQQHHTTPMPIPMCNEIQAQAQAQAQ X60X61FKLVGFKNFVRTNPKSDRFQVNRFHHIEFWCX93DATNASRRFSWGLGMPIVAKS DLSTGNQIHASYLLRSGDLSFLFSAPYSPSLSAGSSAASSASIPSFDAATCLAFAAKHGFG VRAIALEVADAEAAFSASVAKGAEPASPPVLVDDRTGFAEVRLYGDVVLRYVSYKDAA PQAPHADPSRWFLPGFEAAASSSSFPELDYGIRRLDHX271VGNVPELAPAVRYLKGFSGF HEFAEFTAEDVGTSESGLNSVVLANNSETVLLPLNEPVYGTKRKSQIETYLEHNEGAGV QHLALVTHDIFTTLREMRKRSFLGGFEFMPSPX383PX385YYAX389LHNRX394ADVLTVDQI KQCEELGILVDRDDQGTLLQIFTKPVGDRPTIFIEIIQRIGCMVEDEEGX453VYQKGACGG FGKGNFSELFKSIEEYEKTLEAKRTA wherein each of X60, X61, X93, X271, X383, X385, X389, X394, and X453represents an amino acid, wherein at least one of X60, X61, X93, X271, X383, X385, X389, X394, and X453comprises a mutation, wherein the mutation comprises at least one of the following: a mutation of X60 to Serine (S), a mutation of X61to Glutamic Acid (E), a mutation of X93 to Alanine (A), a mutation of X271 to Valine (V), a mutation of X383to Serine (S), 52 4823-4406-5785v.313368-42PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032 a mutation of X385to Alanine (A), a mutation of X389 to Aspartic Acid (D), a mutation of X394 to Valine (V), a mutation of X453to Glutamic Acid (E), or combinations thereof.

12. The modified plant or seed of claim 11, wherein the mutation comprises at least one of the following: a mutation of X60from Proline (P) to Serine (S); a mutation of X61 from Glycine (G) to Glutamic Acid (E); a mutation of X93 from Threonine (T) to Alanine (A); a mutation of X271from Alanine (A) to Valine (V); a mutation of X383 from Proline (P) to Serine (S); a mutation of X385from Threonine (T) to Alanine (A); a mutation of X389 from Asparagine (N) to Aspartic Acid (D); a mutation of X394 from Alanine (A) to Valine (V); or 53 4823-4406-5785v.313368-42PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032 a mutation of X453 from Lysine (K) to Glutamic Acid (E).

13. The modified plant or seed of claim 11, wherein the mutation comprises at least one of the following: a mutation of X389 to Aspartic Acid (D), and wherein the mutated HPPD gene expresses a protein comprising SEQ ID NO: 33 or a sequence with at least 65% sequence identity to SEQ ID NO: 33; a mutation of X389 to Aspartic Acid (D) and a mutation of X453 to Glutamic Acid (E), and wherein the mutated HPPD gene expresses a protein comprising SEQ ID NO: 34 or a sequence with at least 65% sequence identity to SEQ ID NO: 34; a mutation of X60 to Serine (S), a mutation of X61 to Glutamic Acid (E), and mutation of X389 to Aspartic Acid (D), and wherein the mutated HPPD gene expresses a protein comprising SEQ ID NO: 35 or a sequence with at least 65% sequence identity to SEQ ID NO: 35; or a mutation of X60 to Serine (S), a mutation of X383 to Serine (S), a mutation of X389 to Aspartic Acid (D), and a mutation of X394 to Valine (V), and wherein the mutated HPPD gene expresses a protein comprising SEQ ID NO: 36 or a sequence with at least 65% sequence identity to SEQ ID NO:

36.

14. A method of producing a modified plant or seed, wherein the method comprises introducing a mutated 4-hydroxyphenylpyruvate dioxygenase (HPPD) gene to the plant or seed, wherein the mutated HPPD gene expresses an HPPD protein that confers herbicide resistance to the modified plant or seed.

15. The method of claim 14, wherein the mutated HPPD gene is introduced by a method selected from the group consisting of chemical mutation, site directed mutagenesis, irradiation, introduction of a gene editing system to the plant or seed, introduction of the mutated HPPD gene as an exogenous gene, alteration of the endogenous HPPD gene, or combinations thereof. 54 4823-4406-5785v.313368-42PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032 16. The method of claim 14, wherein the mutated HPPD gene is introduced at a seedling stage of a plant.

17. The method of claim 14, wherein the mutated HPPD gene is introduced within somatic embryos of a plant cell prior to somatic embryo development and subsequent germination into seedlings.

18. The method of claim 14, wherein the mutated HPPD gene is introduced at an adult stage of a plant.

19. The method of claim 14, wherein the mutated HPPD gene is introduced as an exogenous gene.

20. The method of claim 14, wherein the mutated HPPD gene is introduced by altering an endogenous HPPD gene of the plant or seed.

21. The method of claim 14, wherein the mutated HPPD gene is introduced by introduction of a gene editing system.

22. The method of claim 21, wherein the gene editing system comprises a clustered regularly interspaced short palindromic repeats (CRISPR) / Cas nuclease (Cas) system (CRISPR / Cas system) to the plant or seed, wherein the CRISPR / Cas system comprises at least one Cas nuclease and at least one guide RNA.

23. The method of claim 14, wherein the plant or seed is selected from the group consisting of cotton, corn, maize, rice, sorghum, soybean, wheat, N. benthamiana, Arabidopsis, tobacco, tomato, lettuce, common beans, potato, grapes, oilseed rape, sunflower, watermelon, alfalfa, varieties thereof, or combinations thereof. 55 4823-4406-5785v.313368-42PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032 24. The method of claim 14, wherein the plant or seed comprises cotton.

25. The method of claim 14, wherein the plant or seed comprises soybean.

26. The method of claim 14, wherein the mutated HPPD gene expresses a protein comprising SEQ ID NO: 1, or a sequence with at least 65% sequence identity to SEQ ID NO: 1, wherein SEQ ID NO: 1 comprises: MVQTNQSGSGNDFKLVGFSNFVRSNPKSDRFTVKRFHHIEFWCTDATNVARRFSWGLG MQFVAKSDLSTGNLTHASYLLRSRDLNFLFTAPYSPSIAVAQNLSPQSTASIPSFDHSLCR SFAATHGLGVRAIAIEVDDAETAFTTSVTHGALPFCPPTPLGDVATIAEVKLYGDVVLRY VSYTTTINSDHDFLPGFEKIEDTLSYPLDYGLRRLDHAVGNVPELGPAVSYVKSFTGFHE FAEFTAEDVGTSESGLNSVVLANNEEMVLLPMNEPVFGTKRKSQIQTYLEHNEGAGVQ HLALVSEDIFKTLREMRKRSFVGGFEFMPSPX329P X331YYKX335LKQRAGDILSDEQIKECEELGILVDRDDQGTLLQIFTKPVGDRPTIFIEIIQRI GCMVKDEEGKQYQKGGCGGFGKX412NFSELFKSIEEYEKSLEAKQSQNP, wherein each of X329, X331, X335, and X412 represents an amino acid, wherein at least one of X329, X331, X335, and X412comprises a mutation, wherein the mutation comprises at least one of the following: a mutation of X329 to Serine (S), a mutation of X331to Alanine (A), 56 4823-4406-5785v.313368-42PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032 a mutation of X335to Glutamic Acid (E), a mutation of X412 to Serine (S), or combinations thereof.

27. The method of claim 26, wherein the mutation comprises at least one of the following: a mutation of X329 from Proline (P) to Serine (S), or a mutation of X329 from Threonine (T) to Serine (S); a mutation of X331 from Threonine (T) to Alanine (A), or a mutation of X331 from Aspartic Acid (D) to Alanine (A); a mutation of X335 from Lysine (K) to Glutamic Acid (E), a mutation of X335 from Asparagine (N) to Glutamic Acid (E), or a mutation of X335 from Glycine (G) to Glutamic Acid (E); a mutation of X412 from Glycine (G) to Serine (S); or combinations thereof.

28. The method of claim 26, wherein the mutation comprises at least one of the following: a mutation of X335 to Glutamic Acid (E), and a mutation of X412 to Serine (S); a mutation of X329to Serine (S), a mutation of X331to Alanine (A), and a mutation of X335to Glutamic Acid (E); 57 4823-4406-5785v.313368-42PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032 a mutation of X329 to Serine (S), a mutation of X331 to Alanine (A), and a mutation of X412 to Serine (S); a mutation of X329 to Serine (S), a mutation of X335 to Glutamic Acid (E), and a mutation of X412 to Serine (S); a mutation of X331 to Alanine (A), a mutation of X335 to Glutamic Acid (E), and a mutation of X412 to Serine (S); or a mutation of X329to Serine (S), a mutation of X331to Alanine (A), a mutation of X335to Glutamic Acid (E), and a mutation of X412 to Serine (S).

29. The method of claim 26, wherein the mutation comprises at least one of the following: a mutation of X329 to Serine (S) and a mutation of X335 to Glutamic Acid (E), and wherein the mutated HPPD gene expresses a protein comprising SEQ ID NO: 23 or a sequence with at least 65% sequence identity to SEQ ID NO: 23; a mutation of X329 to Serine (S), a mutation of X331 to Alanine (A), and a mutation of X335 to Glutamic Acid (E), and wherein the mutated HPPD gene expresses a protein comprising SEQ ID NO: 24 or a sequence with at least 65% sequence identity to SEQ ID NO: 24; a mutation of X329 to Serine (S), a mutation of X331 to Alanine (A), and a mutation of X412to Serine (S), and wherein the mutated HPPD gene expresses a protein comprising SEQ ID NO: 25 or a sequence with at least 65% sequence identity to SEQ ID NO: 25; 58 4823-4406-5785v.313368-42PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032 a mutation of X331to Alanine (A), and a mutation of X335to Glutamic Acid (E), and wherein the mutated HPPD gene expresses a protein comprising SEQ ID NO: 26 or a sequence with at least 65% sequence identity to SEQ ID NO: 26; a mutation of X335 to Glutamic Acid (E) and a mutation of X412 to Serine (S), and wherein the mutated HPPD gene expresses a protein comprising SEQ ID NO: 27 or a sequence with at least 65% sequence identity to SEQ ID NO: 27; a mutation of X331 to Alanine (A), and wherein the mutated HPPD gene expresses a protein comprising SEQ ID NO: 28 or a sequence with at least 65% sequence identity to SEQ ID NO: 28; a mutation of X329 to Serine (S) and a mutation of X331 to Alanine (A), and wherein the mutated HPPD gene expresses a protein comprising SEQ ID NO: 29 or a sequence with at least 65% sequence identity to SEQ ID NO: 29; a mutation of X329to Serine (S), a mutation of X335to Glutamic Acid (E), and a mutation of X412 to Serine (S), and wherein the mutated HPPD gene expresses a protein comprising SEQ ID NO: 30 or a sequence with at least 65% sequence identity to SEQ ID NO: 30; a mutation of X331 to Alanine (A), a mutation of X335 to Glutamic Acid (E), and a mutation of X412 to Serine (S), and wherein the mutated HPPD gene expresses a protein comprising SEQ ID NO: 31 or a sequence with at least 65% sequence identity to SEQ ID NO: 31; or 59 4823-4406-5785v.313368-42PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032 a mutation of X329to Serine (S), a mutation of X331to Alanine (A), a mutation of X335to Glutamic Acid (E), and a mutation of X412 to Serine (S), and wherein the mutated HPPD gene expresses a protein comprising SEQ ID NO: 32 or a sequence with at least 65% sequence identity to SEQ ID NO:

32.

30. The method of claim 14, wherein the HPPD gene expresses a protein comprising SEQ ID NO: 2 or a sequence with at least 65% sequence identity to SEQ ID NO: 2, wherein SEQ ID NO: 2 comprises: MPMYTPSLSAPSSNHIQPSVTLPLYITTTKLNLKQQHHTTPMPIPMCNEIQAQAQAQAQ X60X61FKLVGFKNFVRTNPKSDRFQVNRFHHIEFWCX93DATNASRRFSWGLGMPIVAKS DLSTGNQIHASYLLRSGDLSFLFSAPYSPSLSAGSSAASSASIPSFDAATCLAFAAKHGFG VRAIALEVADAEAAFSASVAKGAEPASPPVLVDDRTGFAEVRLYGDVVLRYVSYKDAA PQAPHADPSRWFLPGFEAAASSSSFPELDYGIRRLDHX271VGNVPELAPAVRYLKGFSGF HEFAEFTAEDVGTSESGLNSVVLANNSETVLLPLNEPVYGTKRKSQIETYLEHNEGAGV QHLALVTHDIFTTLREMRKRSFLGGFEFMPSPX383PX385YYAX389LHNRX394ADVLTVDQI KQCEELGILVDRDDQGTLLQIFTKPVGDRPTIFIEIIQRIGCMVEDEEGX453VYQKGACGG FGKGNFSELFKSIEEYEKTLEAKRTA wherein each of X60, X61, X93, X271, X383, X385, X389, X394, and X453represents an amino acid, wherein at least one of X60, X61, X93, X271, X383, X385, X389, X394, and X453 comprises a mutation, wherein the mutation comprises at least one of the following: a mutation of X60 to Serine (S), 60 4823-4406-5785v.313368-42PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032 a mutation of X61 to Glutamic Acid (E), a mutation of X93to Alanine (A), a mutation of X271 to Valine (V), a mutation of X383 to Serine (S), a mutation of X385to Alanine (A), a mutation of X389 to Aspartic Acid (D), a mutation of X394to Valine (V), a mutation of X453 to Glutamic Acid (E), or combinations thereof.

31. The method of claim 30, wherein the mutation comprises at least one of the following: a mutation of X60 from Proline (P) to Serine (S); a mutation of X61from Glycine (G) to Glutamic Acid (E); a mutation of X93 from Threonine (T) to Alanine (A); 61 4823-4406-5785v.313368-42PCT Application Attorney Docket No. AF13368.P057WO Texas Tech No.2023-032 a mutation of X271from Alanine (A) to Valine (V); a mutation of X383 from Proline (P) to Serine (S); a mutation of X385 from Threonine (T) to Alanine (A); a mutation of X389from Asparagine (N) to Aspartic Acid (D); a mutation of X394 from Alanine (A) to Valine (V); or a mutation of X453from Lysine (K) to Glutamic Acid (E).

32. The method of claim 30, wherein the mutation comprises at least one of the following: a mutation of X389to Aspartic Acid (D), and wherein the mutated HPPD gene expresses a protein comprising SEQ ID NO: 33 or a sequence with at least 65% sequence identity to SEQ ID NO: 33; a mutation of X389to Aspartic Acid (D) and a mutation of X453to Glutamic Acid (E), and wherein the mutated HPPD gene expresses a protein comprising SEQ ID NO: 34 or a sequence with at least 65% sequence identity to SEQ ID NO: 34; a mutation of X60to Serine (S), a mutation of X61to Glutamic Acid (E), and mutation of X389to Aspartic Acid (D), and wherein the mutated HPPD gene expresses a protein comprising SEQ ID NO: 35 or a sequence with at least 65% sequence identity to SEQ ID NO: 35; or a mutation of X60 to Serine (S), a mutation of X383 to Serine (S), a mutation of X389 to Aspartic Acid (D), and a mutation of X394to Valine (V), and wherein the mutated HPPD gene expresses a protein comprising SEQ ID NO: 36 or a sequence with at least 65% sequence identity to SEQ ID NO:

36. 62 4823-4406-5785v.313368-42

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