Compositions and methods for targeting cytochrome p450 reductase

Abstract

The present disclosure relates to compositions and methods for targeting cytochrome P450 reductase that can be utilized to control weeds, invasive plants, or other undesired plants.

Description

COMPOSITIONS AND METHODS FOR TARGETING CYTOCHROME P450 REDUCTASECROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to provisional application U.S. Serial No. 63 / 531,116, filed August 7, 2023, which is incorporated herein by reference in its entirety.SEQUENCE LISTING XML

[0002] The instant application contains a sequence listing, which has been submitted in XML file format by electronic submission and is hereby incorporated by reference in its entirety. The XML file, created on August 7, 2024, is named P14870WO00.xml and is 879,376 bytes in size.TECHNICAL FIELD

[0003] The present disclosure relates to the field of biotechnology. More specifically, the present disclosure relates to compositions and methods for control of weeds, invasive plants, and any undesired plant vegetation.BACKGROUND

[0004] Weeds cause economic losses in crop production due to competition for light, water, and nutrients. Weed control can be conducted by labor-intensive hand operations, by mechanical means, and in modern agriculture, most often by efficient and effective herbicides. Herbicides reduce the amount of time, labor, and fuel required to control weeds. Cost-effective and safe herbicides have been discovered and commercialized for nearly every crop and landscape market. However, highly effective herbicides exert substantial selection pressure on weed populations for resistance to the herbicides. Any genetic trait conferring higher survival can be selected and enriched in subsequent generations of the weed species. Herbicide resistance has become a critical problem for many cropping production systems. Many problematic weed species including kochia, Palmer amaranth, waterhemp, ryegrass, blackgrass, barnyardgrass, horseweed, redroot pigweed, and common lambsquarters have evolved resistance to the major herbicides used for their control.SUMMARY

[0005] This disclosure provides polynucleotides and methods for regulating cytochrome P450 reductase (CPR) genes in plants, e.g., by providing a FANA antisense oligonucleotide targeting a transcript of the CPR gene. By targeting cytochrome P450 reductase, herbicide metabolism mediated by cytochrome P450s is diminished.

[0006] Polynucleotides comprising a sequence essentially identical or essentially complementary to at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least24, or at least 25 contiguous nucleotides of a CPR gene of a plant or to a transcript of the CPR gene are provided. In certain embodiments, the polynucleotides are capable of hybridizing under physiological conditions in a cell of the plant to the CPR gene or to the transcript of the CPR gene and reducing expression of the CPR.

[0007] Herbicidal compositions and formulations comprising the polynucleotides of the disclosure are provided. Herbicidal composition and formulations comprising a CPR inhibitor (e.g., diphenyleneiodonium) are also provided.

[0008] Methods of controlling weeds comprising providing to the weed a composition that reduces expression or activity a CPR are provided. In certain embodiments, the methods comprise providing to the weed a polynucleotide or an herbicidal composition or formulation of the disclosure.

[0009] While multiple embodiments are disclosed, still other embodiments of the present disclosure will become apparent based on the detailed description, which shows and describes illustrative embodiments of the disclosure. Accordingly, the figures and detailed description are to be regarded as illustrative in nature and not restrictive.BRIEF DESCRIPTION OF THE FIGURES

[0010] The following drawings form part of the specification and are included to further demonstrate certain embodiments. In some instances, embodiments can be best understood by referring to the accompanying figures in combination with the detailed description presented herein. The description and accompanying figures may highlight a certain specific example, or a certain embodiment. However, one skilled in the art will understand that portions of the example or embodiment may be used in combination with other examples or embodiments.

[0011] FIG. 1 shows the process of electron transfer from NADPH-cytochrome P450 reductase to the catalytic site of cytochrome P450 monooxygenase. Two electrons are sequentially transferred from NADPH to the heme group of P450 through the cofactors FAD and FMN, each transferred in single-electron steps. Cytochrome P450, once activated by the electron transfer, catalyzes the oxidative breakdown of a substrate by inserting one oxygen atom into the chemical compound. Created with Biorender.

[0012] FIG. 2A-B shows the two-step PCR for genotyping mutant lines. FIG. 2A shows the “wild-type PCR” reaction amplifies a region that is present in wild type and heterozygous lines, but it does not amplify in homozygous lines. FIG. 2B shows the “T-DNA PCR” amplifies only if T-DNA insertion is present. LP - left primer, RP - right primer, LBbl.3 - left border primer for T-DNA insertion.

[0013] FIG. 3 is a scheme representing the Arabidopsis crosses to generate transgenic Arabidopsis CYP81A12 with loss of function in atrl or atr2. Fl plants were genotyped for T- DNA insertion and CYP81A12 presence. F2 plants were genotyped for homozygous T-DNA insertion and two copies of CYP81A12 using ddPCR. M - male, F - female. Created with BioRender.

[0014] FIG. 4A-B shows the T-DNA location in^ZRf (FIG. 4A) and ATR2 (FIG. 4B) genes in SALK lines and primers localization for real time qPCR gene expression analysis. The triangle indicates the location of the T-DNA insertion in the genes: atrl -a and atr2-b have T-DNA insertion in the 5’UTR and 4thintron, respectively, and atr2-a and atr2-b have T-DNA insertion in the 3rdand 12thexon, respectively. Boxes indicate exons or untranslated regions (UTR), black line indicates introns. Created with BioRender.

[0015] FIG. 5A-D shows T-DNA insertion genotyping for ATR1 (FIG. 5A, FIG. 5B) and ATR2 gene (FIG. 5C, FIG. 5D) in different Arabidopsis mutant lines. Each includes four sample plants of each line and one wildtype columbia O (Col_0) as a control. Each plant was submitted for two PCR, first reaction was “wildtype PCR” using primer set LP + RP, and second reaction “T-DNA PCR” reaction using primers set LBbl.3 + RP.

[0016] FIG. 6A-J shows relative gene expression oiATRl an A TR2 gene in different mutant and transgenic mutant Arabidopsis lines. FIG. 6A-D shows ATR1 gene expression analyzed in four different locations using different primer sets ATRI A, ATR1 B, ATR1 C and ATR1 D. FIG. 6E-I shows A TR2 gene expression analyzed in five different locations using primer set ATR2 A, ATR2 B, ATR2 C, ATR2 D and ATR2 E. FIG. 6 J shows expression of CYP81A12 with only one primer set. ATR1 and ATR2 gene expression was calculated using Col_0 as control. CYP81A12 gene expression was calculated using transgenic Arabidopsis CYP81A12 as control. * Significant by Dunnett’s Test.

[0017] FIG 7A-D shows different lines of Arabidopsis in response to increasing doses of different herbicides. FIG. 7A shows penoxsulam, FIG. 7B shows chlorsulfuron, FIG. 7C shows mesotrione, and FIG. 7D shows 2,4-D. Col_0, wild-type; CYP81A12, transgenic line expression CYP81A12,' CYP81A12 atrl-a, transgenic Arabidopsis expressing CYP81A12 carrying T-DNA insertion on A TR1 5’UTR; CYP81A12 atrl-b, transgenic Arabidopsis expressing CYP81A12 carrying T-DNA insertion on A TR1 4thintron; CYP81A12 alr2-a. transgenic Arabidopsis expressing CYP81A12 carrying T-DNA insertion on ATR2 3thintron; CYP81A12 atr2-b, transgenic Arabidopsis expressing CYP81A12 carrying T-DNA insertion on ATR2 12thexon.

[0018] FIG. 8A-G shows sensitivity of wild type (Col_0), mutant atrl -a. alrl-b. alr2-a. alr2-b. transgenic Arabidopsis CYP81A12 and transgenic mutant Arabidopsis CYP81A12 atrl-a,CYP81A12 atrl-b, CYP81A12 atr2-a, CYP81A12 atr2-b. Pictures at 14 d-old seedlings growing in Vi MS media containing different herbicide concentrations. FIG. 8A shows the plate map, i MS control untreated and basta (25mg L'1); FIG. 8B-G shows Vi MS plates with different doses of bensulfuron-methyl, chlorsulfuron, penoxsulam, propoxy carb azone, mesotrione, and bentazon.

[0019] FIG. 9A-B shows LC-MS / MS analysis of mesotrione metabolites found in different Arabidopsis lines. Arabidopsis lines analyzed were wild-type (Col_0), transgenic CYP81A12 and transgenic mutants CYP81A12 atrl-a, CYP81A12 atrl-b, CYP81A12 atr2-a, CYP81A12 atr2-b. FIG. 9A shows a MS chromatogram 24 h after herbicide application. The retention time for parental mesotrione and hydroxy-mesotrione is 10.3- and 5.9-min, respectively. Peak at retention time 7.7 min is present in all samples, even in untreated plants. FIG. 9B shows the peak area of mesotrione and hydroxy-mesotrione at 6, 24, 48 and 96 h after mesotrione application on different Arabidopsis lines. * p-value <0.05 by Dunnett’s Test.

[0020] FIG. 10A-B shows LC-MS / MS analysis of chlorsulfuron metabolites found in different Arabidopsis lines. Arabidopsis lines analyzed were wild-type (Col_0), transgenic CYP81A12 and transgenic mutants CYP81A12 atrl-a, CYP81A12 atrl-b, CYP81A12 atr2-a, CYP81A12 atr2-b. FIG. 10A shows a MS chromatogram 192 h after herbicide application. The retention time for parental chlorsulfuron and hydroxy-chlorsulfuron 7.01 and 5.9 min, respectively. Peak at retention time 4.01 min is present in all samples, even in untreated plants. FIG. 10B shows the peak area of chlorsulfuron and hydroxy-chlorsulfuron at different time points after chlorsulfuron application. * p-value <0.05 by Dunnett’s Test.

[0021] FIG. 11 shows a phylogenetic tree of 69 CPR protein sequences from 13 different weed species and others 6 plant species with known CPRs sequences and classification. The phylogenetic tree shows branching of class I and II CPRs. Different CPR were named as “a”, “b” or “c” and the copies were numbered as “.1”, “.2” or “.3” for one, two or three copies, respectively. * Different types of CPR have different copy number in the same species. #CPR, number of cytochrome P450 reductases; #cp, number of copies. Am - Alopecurus myosuroides (blackgrass), At - Arabidopsis thaliana (Arabidopsis), Ap -Amaranthus palmeri (Palmer amaranth), Atu - Amaranthus tuberculatus (waterhemp), Bs - Bassia scoparia (kochia), Bt - Bromus tectorum (downy brome), Cc - Conyza canadensis (horseweed), Cf - Chenopodium formosanum (Djulis), Ec - Echinochloa crus-galli (barnyardgrass), Eo - E. oryzicola (late watergrass), Eco - E. colona (jungle rice), Ei - Eleusine indica (goosegrass), Gh - Gossypium hirsutum (cotton), Gm - Glycine max (soybean), Ip - Ipomoea purpurea (common morning-glory), Mt - Medicago truncatula (Barrel medic), Os - Oryza sativa (rice), Pa - Poa annua (annual bluegrass), Zm - Zea mays (com).DETAILED DESCRIPTION

[0022] So that the present disclosure may be more readily understood, certain terms are first defined. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the disclosure pertain. Many methods and materials similar, modified, or equivalent to those described herein can be used in the practice of the embodiments of the present disclosure without undue experimentation, the preferred materials and methods are described herein. In describing and claiming the embodiments of the present disclosure, the following terminology will be used in accordance with the definitions set out below.

[0023] It is to be understood that all terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting in any manner or scope. For example, as used in this specification and the appended claims, the singular forms “a,” “an” and “the” can include plural referents unless the content clearly indicates otherwise. Thus, for example, reference to “a mutation” includes a single mutation, as well as two or more mutations; reference to “a plant” includes one plant, as well as two or more plants; and so forth. Similarly, the word “or” is intended to include “and” unless the context clearly indicate otherwise. The word “or” means any one member of a particular list and also includes any combination of members of that list. Further, all units, prefixes, and symbols may be denoted in its SI accepted form.

[0024] Numeric ranges recited within the specification are inclusive of the numbers defining the range and include each integer within the defined range. Throughout this disclosure, various embodiments of this disclosure are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges, fractions, and individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6, and decimals and fractions, for example, 1.2, 3.8, lU, and 43 / 4. This applies regardless of the breadth of the range.

[0025] Unless otherwise stated, nucleic acid sequences in the text of this specification are given, when read from left to right, in the 5' to 3' direction. One of skill in the art would be aware that agiven DNA sequence is understood to define a corresponding RNA sequence which is identical to the DNA sequence except for replacement of the thymine (T) nucleotides of the DNA with uracil (U) nucleotides. Thus, providing a specific DNA sequence is understood to define the exact RNA equivalent. A given first polynucleotide sequence, whether DNA or RNA, further defines the sequence of its exact complement (which can be DNA or RNA), a second polynucleotide that hybridizes perfectly to the first polynucleotide by forming Watson-Crick base-pairs. For DNA:DNA duplexes (hybridized strands), base-pairs are adenine:thymine or guanine: cytosine; for DNA:RNA duplexes, base-pairs are adenine:uracil or guanine:cytosine.

[0026] The term “effective amount” of a composition provided herein refers to the amount of the composition capable of performing the specified function for which an effective amount is expressed. The exact amount required can vary from composition to composition and from function to function, depending on recognized variables such as the compositions and processes involved. An effective amount can be delivered in one or more applications. An appropriate “effective amount” can be determined by the skilled artisan via routine experimentation.

[0027] As used herein, the term “foliage” is intended to mean all parts and organs of plants above the ground. Non-limiting examples include leaves, stalks, stems, flowers, fruits, etc.

[0028] As used herein, the term “foliar application”, “foliarly applied”, and variations thereof, is intended to include application to the foliage or above ground portions of the plant, (e.g., the leaves of the plant).

[0029] Generally, the term “herbicide” is used herein to mean an active ingredient that kills, controls or otherwise adversely modifies the growth of plants. The preferred amount or concentration of the herbicide is an “effective amount” or “effective concentration.” By “effective amount” and “effective concentration” is intended an amount and concentration, respectively, that is sufficient to kill or inhibit the growth of a similar, wild-type, plant, plant tissue, plant cell, or host cell, but that said amount does not kill or inhibit as severely the growth of the herbicide-resistant plants, plant tissues, plant cells, and host cells of the present disclosure. Typically, the effective amount of an herbicide is an amount that is routinely used in agricultural production systems to kill weeds of interest. Such an amount for a given herbicide is known to those of ordinary skill in the art. Herbicidal activity is exhibited by herbicides useful for the present disclosure when they are applied directly to the plant or to the locus of the plant at any stage of growth or before planting or emergence. The effect observed depends upon the plant species to be controlled, the stage of growth of the plant, the application parameters of dilution and spray drop size, the particle size of solid components, the environmental conditions at the time of use, the specific compound employed, the specific adjuvants and carriers employed, thesoil type, and the like, as well as the amount of chemical applied. These and other factors can be adjusted as is known in the art to promote non-selective or selective herbicidal action. Generally, the herbicide treatments can be applied PPI (Pre Plant Incorporated), PPSA (Post plant surface applied), PRE- or POST-emergent. Postemergent treatment typically occurs to relatively immature undesirable vegetation to achieve the maximum control of weeds.

[0030] By a “herbicide tolerant” or “herbicide resistant” plant, it is intended that a plant that is tolerant or resistant to at least one herbicide at a level that would normally kill, or inhibit the growth of, a normal or wildtype plant. Levels of herbicide that normally inhibit growth of a non- tolerant plant are known and readily determined by those skilled in the art. Examples include the amounts recommended by manufacturers for application. The maximum rate is an example of an amount of herbicide that would normally inhibit growth of a non-tol erant plant. For the present disclosure, the terms “herbicide tolerant” and “herbicide resistant” are used interchangeably and are intended to have an equivalent meaning and an equivalent scope. Similarly, the terms “herbicide tolerance” and “herbicide resistance” are used interchangeably and are intended to have an equivalent meaning and an equivalent scope. Similarly, the terms “tolerant” and “resistant” are used interchangeably and are intended to have an equivalent meaning and an equivalent scope. As used herein, in regard to an herbicidal composition useful in various embodiments hereof, terms such as herbicides, and the like, refer to those agronomically acceptable herbicide active ingredients (A.I.) recognized in the art.

[0031] As used herein, the terms “include,” “includes,” and “including” are to be construed as at least having the features to which they refer while not excluding any additional unspecified features.

[0032] As used herein, the term “modified antisense oligonucleotide” refers to antisense oligonucleotides (ASOs) containing a modified sugar. Antisense oligonucleotides are single stranded oligonucleotides that recognize nucleic acid sequences via Watson-Crick base pairing and cause pre- or post-transcriptional gene silencing. The antisense oligonucleotide binds to its target mRNA, and forms a duplex that is recognized by RNase H, which in turn induces the cleavage of the mRNA, the steric blocking of translation machinery, or the prevention of necessary RNA interactions.

[0033] A “naturally occurring nucleotide” or “unmodified nucleotide” contains normally occurring sugars (D-ribose or D-2-deoxyribose) and a phosphodiester backbone that is readily degraded by nucleases. As used herein, an unmodified nucleotide is also referred to as a deoxy rib onucl eoti de .

[0034] The terms “plants” and “vegetation” include, but are not limited to, germinant seeds, emerging seedlings, plants emerging from vegetative propagules, and established vegetation.

[0035] The term “primer” as used herein encompasses any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process, such as PCR. Typically, primers are oligonucleotides from 10 to 30 nucleotides in length, but longer sequences may be used. Primers may be provided in single or double-stranded form. Probes may be used as primers, but are designed to bind to the target DNA or RNA and need not be used in an amplification process.

[0036] The term “weed” refers to all those plants which grow in locations where they are undesired. The terms “undesirable vegetation”, “harmful plants”, “unwanted plants”, “weeds” and “weed species”, as used herein, are synonyms. The weeds of the present disclosure can also include, for example, crop plants that are growing in an undesired location. For example, a volunteer maize plant that is in a field that predominantly comprises soybean plants can be considered a weed, if the maize plant is undesired in the field of soybean plants.Cytochrome P450 Reductase

[0037] Cytochrome P450 reductase (CPR) is a membrane-bound enzyme localized in the endoplasmic reticulum that plays a critical role in the biosynthesis of a wide range of compounds in living organisms, including plants, animals, and bacteria. The structure of P450 reductase is divided in four domains, including TV-terminal flavin mononucleotide (FMN)- binding domain, a connecting domain, an flavin adenine dinucleotide (FAD)-binding domain, and a C-terminal NADPH-binding domain (FIG. 1). CPR enzymes arose from of a fusion of different genes encoding FAD-binding, ferredoxin-NADP+, and an FMN-flavodoxin. Plants require CPR to supply two electrons for P450 enzymes to conduct each monooxygenase reaction with an organic substrate. The CPR reduction includes multiple steps for electron transfer. The first step is reduction from NADPH to FAD, then FMN, and finally to the P450 acceptor (FIG. 1). The transfer of electrons to the P450 active site in the P450 catalytic cycle relies on a robust and intricate CPR:P450 complex in the membrane, making CPR function an essential component of specialized metabolism.

[0038] In contrast to the huge number of P450 genes within plant genomes, for example, 246 in Arabidopsis thaliana (Arabidopsis), 328 in Oryza spp. (rice), 867 in Echinochloa crus-galli (barnyardgrass), 694 in A. colona (jungle rice), and 323 in Conyza canadensis (horseweed), CPRs generally have only two or three copies in plant genomes. There are two CPR genes in Arabidopsis, referred to as Arabidopsis thaliana cytochrome P450 reductase 1 (ATR1 2 (ATR2) and one hypothetical reductase 3 (ATR3) that encodes a diflavin reductase essential forembryogenesis, but that is unable to reduce P450 in vitro. The isoforms are grouped in two distinct groups with different roles. Class I is A TRL which is involved in primary and basal metabolism. Class II is ATR2, which is inducible by environmental stimuli such as wounding, pathogen attack, or high light intensity exposure.

[0039] Compositions and methods of the disclosure are useful for reducing the expression or activity of a cytochrome P450 reductase (CPR) in a plant cell. Examples of nucleotide and amino sequences of CPR genes from various weed species are summarized in Table 1.TABLE 1

[0040] Those skilled in the art may also find CPR genes in additional plant species based on genome synteny and sequence similarity. In certain embodiments, additional CPR genes can be obtained by hybridization or PCR using sequences based on the nucleotide sequences noted above.

[0041] In a PCR approach, oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any plant of interest. Methods for designing PCR primers and PCR cloning are generally known in the art. See, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.). See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New York).

[0042] In hybridization techniques, all or part of a known polynucleotide is used as a probe that selectively hybridizes to other corresponding polynucleotides present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from a chosen organism. The hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with a detectable group such as32P, or any other detectable marker. Methods for preparation of probes for hybridization and for construction of cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

[0043] By “hybridizing to” or “hybridizing specifically to” refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA. “Bind(s) substantially” refers to complementary hybridization between a probe nucleic acid and a targetnucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target nucleic acid sequence.

[0044] “Stringent hybridization conditions” and “stringent hybridization wash conditions” in the context of nucleic acid hybridization experiments such as Southern and Northern hybridizations are sequence dependent, and are different under different environmental parameters. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology -Hybridization with Nucleic Acid Probes part I chapter 2 “Overview of principles of hybridization and the strategy of nucleic acid probe assays” Elsevier, New York. Generally, highly stringent hybridization and wash conditions are selected to be about 5 °C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. Typically, under “stringent conditions” a probe will hybridize to its target subsequence, but to no other sequences.

[0045] The T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the Tmfor a particular probe. An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or northern blot is 50% formamide with 1 mg of heparin at 42 °C, with the hybridization being carried out overnight. An example of highly stringent wash conditions is 0.1 5M NaCl at 72 °C for about 15 minutes. An example of stringent wash conditions is a 0.2* SSC wash at 65 °C for 15 minutes (see, Sambrook, infra, for a description of SSC buffer). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is 1 *SSC at 45 °C for 15 minutes. An example low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4-6*SSC at 40 °C for 15 minutes. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically involve salt concentrations of less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30 °C Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. In general, a signal to noise ratio of 2* (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the proteinsthat they encode are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.

[0046] The following are examples of sets of hybridization / wash conditions that may be used to clone nucleotide sequences that are homologues of reference nucleotide sequences: a reference nucleotide sequence preferably hybridizes to the reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50 °C with washing in 2*SSC, 0.1% SDS at 50 °C, more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50 °C with washing in 1 *SSC, 0.1% SDS at 50 °C, more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50 °C with washing in 0.5xSSC, 0.1% SDS at 50 °C, preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50 °C with washing in 0.1 *SSC, 0.1% SDS at 50 °C, more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50 °C with washing in O.l xSSC, 0.1% SDS at 65 °C.

[0047] In certain embodiments, the CPR gene comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97% at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to at least one of SEQ ID NOs: 1-4, 7-120.

[0048] In certain embodiments, the CPR gene encodes a polypeptide having at least 80%, at least 90%, at least 95%, at least 96%, at least 97% at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to at least one of SEQ ID NOs: 5, 6, 121-177.

[0049] To determine the percent identity of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity=number of identical positions / total number of positions e.g., overlapping positions)x l00). In one embodiment, the two sequences are the same length. In another embodiment, the percent identity is calculated across the entirety of the reference sequence. The percent identity between two sequences can be determined using techniques similar to those described below, with or without allowing gaps. In calculating percent identity, typically exact matches are counted. A gap, i.e. a position in an alignment where a residue is present in one sequence but not in the other, is regarded as a position with non-identical residues.

[0050] The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A nonlimiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Set.USA 90:5873-5877. Such an algorithm is incorporated into the BLASTN and BLASTX programs of Altschul et al. (1990) J. Mol. Biol. 215:403. BLAST nucleotide searches can beperformed with the BLASTN program, score=100, wordlength=12, to obtain nucleotide sequences homologous to target genes of the disclosure. BLAST protein searches can be performed with the BLASTX program, score=50, wordlength=3, to obtain amino acid sequences homologous to proteins encoded by the target genes of the disclosure. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., BLASTX and BLASTN) can be used. Alignment may also be performed manually by inspection.

[0051] Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the ClustalW algorithm (Higgins et al. (1994) Nucleic Acids Res. 22:4673-4680). ClustalW compares sequences and aligns the entirety of the amino acid or DNA sequence, and thus can provide data about the sequence conservation of the entire amino acid sequence. The ClustalW algorithm is used in several commercially available DNA / amino acid analysis software packages, such as the ALIGNX module of the Vector NTI Program Suite (Invitrogen Corporation, Carlsbad, Calif.). After alignment of amino acid sequences with ClustalW, the percent amino acid identity can be assessed. Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller (1988) CABIOS 4: 11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG Wisconsin Genetics Software Package, Version 10 (available from Accelrys, Inc., 9685 Scranton Rd., San Diego, Calif., USA). When utilizing the ALIGN program for comparing amino acid sequences, a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.Polynucleotides

[0052] As used herein, the term “DNA”, “DNA molecule”, “DNA polynucleotide molecule” refers to a single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA) molecule of genomic or synthetic origin, such as, a polymer of deoxyribonucleotide bases or a DNA polynucleotide molecule. As used herein, the term “DNA sequence”, “DNA nucleotide sequence” or “DNA polynucleotide sequence” refers to the nucleotide sequence of a DNA molecule.

[0053] As used herein, the term “RNA”, “RNA molecule”, “RNA polynucleotide molecule” refers to a single-stranded RNA (ssRNA) or double-stranded RNA (dsRNA) molecule of genomic or synthetic origin, such as, a polymer of ribonucleotide bases that comprise single ordouble stranded regions. Unless otherwise stated, nucleotide sequences in the text of this specification are given, when read from left to right, in the 5' to 3' direction and generally represent the plus or sense strand of a double strand polynucleotide. It is recognized that the minus or antisense strand may be the functional moiety of a double strand polynucleotide as described herein although unless indicate the disclosed polynucleotide sequences will represent the plus or sense strand polynucleotide sequence. The nomenclature used herein is that required by Title 37 of the United States Code of Federal Regulations § 1.822 and set forth in the tables in WIPO Standard ST.25 (1998), Appendix 2, Tables 1, 2, and 3.

[0054] As used herein, “polynucleotide” refers to a DNA or RNA molecule containing multiple nucleotides and generally refers both to “oligonucleotides” (a polynucleotide of typically 50 or fewer nucleotides in length) and polynucleotides of 51 or more nucleotides.

[0055] Embodiments include compositions including oligonucleotides having a length of 18-25 nucleotides (18-mers, 19-mers, 20-mers, 21-mers, 22-mers, 23-mers, 24-mers, or 25-mers), for example, oligonucleotides essentially homologous or essentially complementary to a cytochrome P450 reductase, for example, SEQ ID NO: 5, 6, or 121-177 (Table 1) or fragments thereof or medium-length polynucleotides having a length of 26 or more nucleotides (polynucleotides of 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, or about 300 nucleotides), for example, polynucleotides fragments thereof or long polynucleotides having a length greater than about 300 nucleotides (for example, polynucleotides of between about 300 to about 400 nucleotides, between about 400 to about 500 nucleotides, between about 500 to about 600 nucleotides, between about 600 to about 700 nucleotides, between about 700 to about 800 nucleotides, between about 800 to about 900 nucleotides, between about 900 to about 1000 nucleotides, between about 300 to about 500 nucleotides, between about 300 to about 600 nucleotides, between about 300 to about 700 nucleotides, between about 300 to about 800 nucleotides, between about 300 to about 900 nucleotides, or about 1000 nucleotides in length, or even greater than about 1000 nucleotides in length, for example up to the entire length of a target gene including coding or non-coding or both coding and non-coding portions of the cytochrome P450 reductase target gene), for example, polynucleotides of SEQ ID NO: 1-4, or 7- 120, wherein the selected polynucleotides or fragments thereof are homologous or complementary to a segment of SEQ ID NO: 1-4, or 7-120 and suppresses, represses orotherwise delay the expression of the cytochrome P450 reductase gene. Where a polynucleotide is double-stranded, its length can be similarly described in terms of base pairs. A target gene comprises any polynucleotide in a plant cell or fragment thereof for which the modulation of the expression of the target gene is provided by the methods and compositions. A gene has noncoding genetic elements (components) that provide for the function of the gene, these elements are polynucleotides that provide gene expression regulation, such as, a promoter, an enhancer, a 5' untranslated region, intron regions, and a 3' untranslated region. Oligonucleotides and polynucleotides can be made to any of the genetic elements of a gene and to polynucleotides spanning the junction region of a genetic element, such as, an intron and exon, the junction region of a promoter and a transcribed region, the junction region of a 5' leader and a coding sequence, the junction of a 3' untranslated region and a coding sequence.

[0056] Polynucleotide compositions used in the various embodiments include compositions including oligonucleotides or polynucleotides or a mixture of both, including RNA or DNA or RNA / DNA hybrids or chemically modified oligonucleotides or polynucleotides or a mixture thereof. In some embodiments, the polynucleotide may be a combination of ribonucleotides and deoxyribonucleotides, for example, synthetic polynucleotides consisting mainly of ribonucleotides but with one or more terminal deoxyribonucleotides or synthetic polynucleotides consisting mainly of deoxyribonucleotides but with one or more terminal dideoxyribonucleotides. In some embodiments, the polynucleotide includes non-canonical nucleotides such as inosine, thiouridine, or pseudouridine. In some embodiments, the polynucleotide includes chemically or enzymatically modified nucleotides. Examples of chemically modified oligonucleotides or polynucleotides are well known in the art; see, for example, US Patent Publication 20110171287, US Patent Publication 20110171176, and US Patent Publication 20110152353, US Patent Publication, 20110152346, US Patent Publication 20110160082, herein incorporated in its entirety by reference hereto. For example, including but not limited to the naturally occurring phosphodiester backbone of an oligonucleotide or polynucleotide can be partially or completely modified with phosphorothioate, phosphorodithioate, or methylphosphonate intemucleotide linkage modifications, modified nucleoside bases or modified sugars can be used in oligonucleotide or polynucleotide synthesis, and oligonucleotides or polynucleotides can be labeled with a fluorescent moiety (for example, fluorescein or rhodamine) or other label (for example, biotin).

[0057] The polynucleotides can be single- or double-stranded RNA or single- or doublestranded DNA or double-stranded DNA / RNA hybrids or modified analogues thereof, and can be of oligonucleotide lengths or longer. In more specific embodiments, the polynucleotides thatprovide single-stranded RNA in the plant cell are selected from the group of (a) a singlestranded RNA molecule (ssRNA), (b) a single-stranded RNA molecule that self-hybridizes to form a double-stranded RNA molecule, (c) a double-stranded RNA molecule (dsRNA), (d) a single- stranded DNA molecule (ssDNA), (e) a single-stranded DNA molecule that selfhybridizes to form a double-stranded DNA molecule, and (f) a single-stranded DNA molecule including a modified Pol III gene that is transcribed to an RNA molecule, (g) a double-stranded DNA molecule (dsDNA), (h) a double-stranded DNA molecule including a modified Pol III gene that is transcribed to an RNA molecule, (i) a double-stranded, hybridized RNA / DNA molecule, or combinations thereof. In some embodiments, these polynucleotides include chemically modified nucleotides or non-canonical nucleotides. In some embodiments, the oligonucleotides may be blunt-ended or may comprise a 3' overhang of from 1-5 nucleotides of at least one or both of the strands. Other configurations of the oligonucleotide are known in the field and are contemplated herein. In embodiments of the method the polynucleotides include double-stranded DNA formed by intramolecular hybridization, double-stranded DNA formed by intermolecular hybridization, double-stranded RNA formed by intramolecular hybridization, or double-stranded RNA formed by intermolecular hybridization. In one embodiment the polynucleotides include single-stranded DNA or single-stranded RNA that self-hybridizes to form a hairpin structure having an at least partially double-stranded structure including at least one segment that will hybridize to RNA transcribed from the gene targeted for suppression. Not intending to be bound by any mechanism, it is believed that such polynucleotides are or will produce single-stranded RNA with at least one segment that will hybridize to RNA transcribed from the gene targeted for suppression. In certain other embodiments the polynucleotides further include a promoter, generally a promoter functional in a plant, for example, a pol II promoter, a pol III promoter, a pol IV promoter, or a pol V promoter.

[0058] The term “gene” refers to components that comprise chromosomal DNA, plasmid DNA, cDNA, intron and exon DNA, artificial DNA polynucleotide, or other DNA that encodes a peptide, polypeptide, protein, or RNA transcript molecule, and the genetic elements flanking the coding sequence that are involved in the regulation of expression, such as, promoter regions, 5' leader regions, 3' untranslated region that may exist as native genes or transgenes in a plant genome. Any of the components of the gene are potential targets for a polynucleotide of the disclosure.

[0059] The polynucleotides of the disclosure are designed to modulate expression by inducing regulation or suppression of an endogenous cytochrome P450 reductase gene in a plant and are designed to have a nucleotide sequence essentially identical or essentially complementary to thenucleotide sequence of an endogenous cytochrome P450 reductase of a plant or to the sequence of RNA transcribed from an endogenous cytochrome P450 reductase of a plant.

[0060] By “essentially identical” or “essentially complementary” is meant that the polynucleotides (or at least one strand of a double-stranded polynucleotide or portion thereof, or a portion of a single strand polynucleotide) are designed to hybridize to the endogenous gene noncoding sequence or to RNA transcribed (known as messenger RNA or an RNA transcript) from the endogenous gene to effect regulation or suppression of expression of the endogenous gene.

[0061] Embodiments of polynucleotides have sequence complementarity that need not be 100 percent, but is at least sufficient to permit hybridization to RNA transcribed from the target gene or DNA of the target gene to form a duplex to permit a gene silencing mechanism. Thus, in embodiments, a polynucleotide fragment is designed to be essentially identical to, or essentially complementary to, a sequence of 18 or more contiguous nucleotides in either the target cytochrome P450 reductase gene sequence or messenger RNA transcribed from the target gene.

[0062] By “essentially identical” is meant having 100 percent sequence identity or at least about 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity when compared to the sequence of 18 or more contiguous nucleotides in either the target gene or RNA transcribed from the target gene; by “essentially complementary” is meant having 100 percent sequence complementarity or at least about 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence complementarity when compared to the sequence of 18 or more contiguous nucleotides in either the target gene or RNA transcribed from the target gene. In some embodiments, polynucleotides are designed to have 100 percent sequence identity to or complementarity to one allele or one family member of a given target gene (coding or noncoding sequence of a gene); in other embodiments the polynucleotides are designed to have 100 percent sequence identity to or complementarity to multiple alleles or family members of a given target gene in one or more plant species.FANA Antisense Oligonucleotides

[0063] FANA antisense oligonucleotides are modified synthetic single-stranded nucleic acid analogs that can modulate gene expression by enzymatic degradation or steric blocking of an RNA target. FANA antisense oligonucleotides can recognize and bind to specific RNA forms, including mRNA, through complementary base pairing. Contrary to the RNAi pathway that requires the interaction of several enzymes (RISC complex), FANA single-stranded antisense oligonucleotides use RNase H-mediated RNA cleavage. Ribonuclease H (RNase H), an endogenous enzyme present in both prokaryotes and eukaryotes, recognizes the FANA / mRNAduplex and cleaves the RNA within the hybrid. Following cleavage, the fragmented mRNA is further degraded by nucleases, whereas the FANA antisense oligonucleotides are recycled within the cell; the degradation of multiple mRNA copies by a single FANA antisense oligonucleotide increases the silencing efficiency and lowers the dose required. Furthermore, unlike siRNAs that are only processed in the cytoplasm, FANA antisense oligonucleotides can shuttle into the nucleus of eukaryotic cells and silence nuclear RNA. FANA antisense oligonucleotides that do not induce RNase H cleavage can inhibit translation of mRNAs, by binding tightly to the transcript thus preventing ribosomal assembly. FANA antisense oligonucleotides have substantial advantages over a dsRNA system, including higher stability in storage, stability at room temperature, and longer stability and resistance to degradation within the cell.

[0064] The chemistry and construction of FANA antisense oligonucleotides has been described elsewhere in detail (see, e.g., U.S. Pat. Nos. 8,278,103 and 9,902,953, each of which is specifically incorporated herein in their entirety by reference). The FANA antisense oligonucleotides and methods of using them disclosed herein contemplate any FANA chemistries known in the art. In some embodiments, a FANA antisense oligonucleotide comprises an internucleoside linkage comprising a phosphate, thereby being an oligonucleotide. In some embodiments, the sugar-modified nucleosides and / or 2'-deoxynucleosides comprise a phosphate, thereby being sugar-modified nucleotides and / or 2'-deoxynucleotides. In some embodiments, a FANA antisense oligonucleotide comprises an internucleoside linkage comprising a phosphorothioate. In some embodiments, the internucleoside linkage is selected from phosphorothioate, phosphorodithioate, methylphosphorothioate, Rp-phosphorothioate, Sp- phosphorothioate. In some embodiments, a FANA antisense oligonucleotide comprises one or more intemucleotide linkages selected from (a) phosphodiester; (b) phosphotriester; (c) phosphorothioate; (d) phosphorodithioate; (e) Rp-phosphorothioate; (f) Sp-phosphorothioate; (g) boranophosphate; (h) methylene (methylimino) (3 'QU — N(QU) — 05'); (i) 3'-thioformacetal (3'S — QU — 05'); (j) amide (3'QU — C(0)NH-5'); (k) methylphosphonate; (1) phosphoramidate (3'-OP(O2) — N5'); and (m) any combination of (a) to (1).

[0065] In some embodiments, FANA antisense oligonucleotides comprising alternating segments or units of sugar-modified nucleotides (e.g., arabinonucleotide analogues [e.g., FANA]) and 2'-deoxyribonucleotides (DNA) are utilized. In some embodiments, a FANA antisense oligonucleotide disclosed herein comprises at least 2 of each of sugar-modified nucleotide and 2'-deoxynucleotide segments, thereby having at least 4 alternating segments overall. Each alternating segment or unit may independently contain 1 or a plurality ofnucleotides. In some embodiments, each alternating segment or unit may independently contain 1 or 2 nucleotides. In some embodiments, the segments each comprise 1 nucleotide. In some embodiments, the segments each comprise 2 nucleotides. In some embodiments, the plurality of nucleotides may consist of 2, 3, 4, 5, or 6 nucleotides. A FANA antisense oligonucleotide can contain an odd or even number of alternating segments or units and may commence and / or terminate with a segment containing sugar-modified nucleotide residues or DNA residues. Thus, a FANA antisense oligonucleotide can be represented as follows: A1-D1-A2-D2-A3-D3 . . . Az-Dz, where each of Ai, A2, etc. represents a unit of one or more (e.g., 1 or 2) sugar-modified nucleotide residues (e.g., FANA) and each of Di, D2, etc. represents a unit of one or more (e.g., 1 or 2) DNA residues. The number of residues within each unit may be the same or variable from one unit to another. The oligonucleotide may have an odd or an even number of units. The oligonucleotide may start (i.e. at its 5' end) with either a sugar-modified nucleotide-containing unit (e.g., a FANA-containing unit) or a DNA-containing unit. The oligonucleotide may terminate (i.e. at its 3' end) with either a sugar-modified nucleotide-containing unit or a DNA- containing unit. The total number of units may be as few as 4 (i.e. at least 2 of each type).

[0066] In some embodiments, a FANA antisense oligonucleotide disclosed herein comprises alternating segments or units of arabinonucleotides and 2'-deoxynucleotides, wherein the segments or units each independently comprise at least one arabinonucleotide or 2'- deoxynucleotide, respectively. In some embodiments, the segments each independently comprise 1 to 2 arabinonucleotides or 2'-deoxynucleotides. In some embodiments, the segments each independently comprise 2 to 5 or 3 to 4 arabinonucleotides or 2'-deoxynucleotides. In some embodiments, a FANA antisense oligonucleotide disclosed herein comprises alternating segments or units of arabinonucleotides and 2'-deoxynucleotides, wherein the segments or units each comprise one arabinonucleotide or 2'-deoxynucleotide, respectively. In some embodiments, the segments each independently comprise about 3 arabinonucleotides or 2'-deoxynucleotides. In some embodiments, a FANA antisense oligonucleotide disclosed herein comprises alternating segments or units of arabinonucleotides and 2'-deoxynucleotides, wherein the segments or units each comprise one arabinonucleotide or 2'-deoxynucleotide, respectively. In some embodiments, a FANA antisense oligonucleotide disclosed herein comprises alternating segments or units of arabinonucleotides and 2'-deoxynucleotides, wherein said segments or units each comprise two arabinonucleotides or 2'-deoxynucleotides, respectively.

[0067] In some embodiments, a FANA antisense oligonucleotides disclosed herein has a structure selected from:a) (Ax-Dy)n I b) (Dy-Ax)nII c) (Ax-Dy)m-Ax-Dy-AxIII d) (Dy-Ax)m-Dy-Ax-Dy IV wherein each of m, x and y are each independently an integer greater than or equal to 1, n is an integer greater than or equal to 2, A is a sugar-modified nucleotide and D is a 2'- deoxy rib onucl eoti de .

[0068] For example, a FANA antisense oligonucleotide disclosed herein has structure I wherein x=l, y=l and n=10, thereby having a structure: A-D-A-D-A-D-A-D-A-D-A-D-A-D-A-D-A-D-A-D.

[0069] In another example, a FANA antisense oligonucleotide disclosed herein has structure II wherein x=l, y=l and n=10, thereby having a structure:D-A-D-A-D-A-D-A-D-A-D-A-D-A-D-A-D-A-D-A.

[0070] In another example, a FANA antisense oligonucleotide disclosed herein has structure III wherein x=l, y=l and n=9, thereby having a structure:A-D-A-D-A-D-A-D-A-D-A-D-A-D-A-D-A-D-A-D-A.

[0071] In another example, a FANA antisense oligonucleotide disclosed herein has structure IV wherein x=l, y=l and n=9, thereby having a structure:D-A-D-A-D-A-D-A-D-A-D-A-D-A-D-A-D-A-D-A-D.

[0072] In another example, a FANA antisense oligonucleotide disclosed herein has structure I wherein x=2, y=2 and n=5, thereby having a structure:A-A-D-D-A-A-D-D-A-A-D-D-A-A-D-D-A-A-D-D.

[0073] In another example, a FANA antisense oligonucleotide disclosed herein has structure II wherein x=2, y=2 and n=5, thereby having a structure:D-D-A-A-D-D-A-A-D-D-A-A-D-D-A-A-D-D-A-A.

[0074] In another example, a FANA antisense oligonucleotide disclosed herein has structure III wherein x=2, y=2 and m=4, thereby having a structure:A-A-D-D-A-A-D-D-A-A-D-D-A-A-D-D-A-A-D-D-A-A.

[0075] In another example, a FANA antisense oligonucleotide disclosed herein has structure IV wherein x=2, y=2 and m=4, thereby having a structure:D-D-A-A-D-D-A-A-D-D-A-A-D-D-A-A-D-D-A-A-D-D.

[0076] The antisense oligonucleotides of the disclosure include at least one FANA modified nucleotide. The antisense oligonucleotide sequence may include a modified sugar moiety for all or only a portion of the nucleotides in the sequence. In some embodiments, the antisenseoligonucleotides may have all modified sugar moiety nucleotides in the sequence. In some embodiments, the antisense oligonucleotides may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 unmodified nucleotides. In certain embodiments, the antisense oligonucleotide includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 FANA modified nucleotides. In certain other embodiments, the antisense oligonucleotide of the disclosure includes at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 FANA modified nucleotides.

[0077] In some embodiments, at least one unmodified nucleotide is located in the antisense oligonucleotide between strings of nucleotides which have modified sugar moieties. For example, an antisense oligonucleotide may have a string of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more FANA modified nucleotides, followed by a string of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more unmodified nucleotides, followed by another string of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more FANA modified nucleotides. In certain embodiments, when one or more unmodified nucleotides are flanked by FANA modified nucleotides, the unmodified nucleotide section may be referred to as a “nucleotide gap sequence.” The gap sequence may consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 unmodified nucleotides. In some embodiments, the antisense oligonucleotide may have a single gap sequence or may have more than one nucleotide gap sequence in the same molecule. The string of FANA modified nucleotides on each side of the unmodified nucleotide gap sequence can be of the same or of different lengths. For example, the antisense oligonucleotide may have 8 FANA modified nucleotides, followed by 7 unmodified nucleotides, followed by a second string of FANA modified nucleotides that is the same or different in number of FANA modified nucleotides as the first modified string. In certain embodiments the antisense oligonucleotide consists of FANA sugar modified nucleotides sequences flanking a gap sequence of unmodified nucleotides. For example, the antisense oligonucleotide comprises a FANA modified sequence between 1 and 10 nucleotides in length, then an unmodified nucleotide sequence between 1 and 10 nucleotides in length, followed by another FANA modified sequence between 1 and 10 nucleotides in length, with this pattern of modified and unmodified nucleotides optionally repeating.

[0078] Table 2 shows exemplary FANA nucleoside placement within 21-mer FANA antisense oligonucleotide. In some embodiments, FANA modified nucleotides are positioned according to any of Formulas 1-16.TABLE 2

[0079] The formulas shown in Table 2 can be applied to any sequence of the disclosure (e.g., the sequences set forth in SEQ ID NOs: 211-290), wherein X represents an unmodified nucleotide and bolded and underlined nucleotides represent a FANA modified nucleotide.

[0080] In certain embodiments, the antisense oligonucleotides may be between 1 and 60 nucleotides long. Embodiments of this disclosure include antisense oligonucleotides having a length of 18-25 nucleotides (e.g., 18-mers, 19-mers, 20-mers, 21-mers, 22-mers, 23-mers, 24- mers, or 25-mers), or antisense oligonucleotides having a length of 26 or more nucleotides (e.g., 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotides).

[0081] Antisense oligonucleotides as described herein include at least one segment of 18 or more contiguous nucleotides that are complementary to a fragment of equivalent size of the DNA of a target gene or the RNA transcript of the target gene.Herbicidal Compositions

[0082] The herbicidal compositions used in the embodiments disclosed herein comprise at least one polynucleotide (e.g., two or more, such as two, three, four, five, six, seven, eight, nine, ten, etc.). For their use in practice, the polynucleotides of the disclosure can be used alone and can also advantageously be used in formulations in combination or association with one or more other compatible components, which are suitable for the desired use and which are acceptable for use in agriculture. The formulations can be of any type known in the art which are suitable for application onto all types of crops. The compositions disclosed herein may be formulated for various agricultural applications (e.g., seed coating formulations, foliar applications, in-furrow applications, drench applications, etc.). The compositions described herein may be formulated with one or more suitable auxiliaries to achieve a particular purpose (e.g., to coat seeds, for foliar applications, for dilution, etc.). These formulations, which can be prepared in any manner known in the art, also form part of the disclosure.

[0083] In certain embodiments, the formulation comprises at least one polynucleotide of the disclosure and at least one agriculturally suitable auxiliary, e.g., a carrier or a surfactant.

[0084] A carrier is a solid or liquid, natural or synthetic, organic or inorganic substance that is generally inert. The carrier generally improves the application of the polynucleotide, for instance, to plants, plants parts or seeds. Examples of suitable solid carriers include, but are not limited to, ammonium salts, in particular ammonium sulfates, ammonium phosphates and ammonium nitrates, natural rock flours, such as kaolins, clays, talc, chalk, quartz, attapulgite, montmorillonite and diatomaceous earth, silica gel and synthetic rock flours, such as finely divided silica, alumina and silicates. Examples of typically useful solid carriers for preparing granules include, but are not limited to crushed and fractionated natural rocks such as calcite, marble, pumice, sepiolite and dolomite, synthetic granules of inorganic and organic flours and granules of organic material such as paper, sawdust, coconut shells, maize cobs and tobacco stalks. Examples of suitable liquid carriers include, but are not limited to, water, organic solvents and combinations thereof. Examples of suitable solvents include polar and nonpolar organic chemical liquids, for example from the classes of aromatic and nonaromatic hydrocarbons (such as cyclohexane, paraffins, alkylbenzenes, xylene, toluene, tetrahydronaphthalene, alkylnaphthalenes, chlorinated aromatics or chlorinated aliphatic hydrocarbons such as chlorobenzenes, chloroethylenes or methylene chloride), alcohols andpolyols (which may optionally also be substituted, etherified and / or esterified, such as ethanol, propanol, butanol, benzyl alcohol, cyclohexanol or glycol), ketones (such as acetone, methyl ethyl ketone, methyl isobutyl ketone, acetophenone, or cyclohexanone), esters (including fats and oils) and (poly)ethers, unsubstituted and substituted amines, amides (such as dimethylformamide or fatty acid amides) and esters thereof, lactams (such as N- alkylpyrrolidones, in particular N-methylpyrrolidone) and lactones, sulfones and sulfoxides (such as dimethyl sulfoxide), oils of vegetable or animal origin, nitriles (alkyl nitriles such as acetonitrile, propionotrilie, butyronitrile, or aromatic nitriles, such as benzonitrile), carbonic acid esters (cyclic carbonic acid esters, such as ethylene carbonate, propylene carbonate, butylene carbonate, or dialkyl carbonic acid esters, such as dimethyl carbonate, diethyl carbonate, dipropyl carbonate, dibutyl carbonate, dioctyl carbonate). The carrier may also be a liquefied gaseous extender, i.e. liquid which is gaseous at standard temperature and under standard pressure, for example aerosol propellants such as halohydrocarbons, butane, propane, nitrogen and carbon dioxide.

[0085] In certain embodiments, the amount of carrier ranges from 1% to 99.9%, from 5% to 99%, from 10% to 95%, or from 20% to 90% by weight of the composition.

[0086] Surfactants are well known in the art and any combination of suitable surfactants or surfactant systems may be used with the compositions described herein. The following includes non-limiting examples of surfactants which may be suitable for use with the compositions described herein.

[0087] The compositions described herein may comprise one or more anionic surfactants. The anionic surfactant(s) may be either water soluble anionic surfactants, water insoluble anionic surfactants, or a combination of water soluble anionic surfactants and water insoluble anionic surfactants. Mixtures of anionic and nonionic surfactants may also be used in the compositions.

[0088] Anionic surfactants are surfactants having a hydrophilic moiety in an anionic or negatively charged state in aqueous solution. Non-limiting examples of anionic surfactants include sulfonic acids, sulfuric acid esters, carboxylic acids, and salts thereof. Non-limiting examples of water soluble anionic surfactants include alkyl sulfates, alkyl ether sulfates, alkyl amido ether sulfates, alkyl aryl polyether sulfates, alkyl aryl sulfates, alkyl aryl sulfonates, monoglyceride sulfates, alkyl sulfonates, alkyl amide sulfonates, alkyl aryl sulfonates, benzene sulfonates, toluene sulfonates, xylene sulfonates, cumene sulfonates, alkyl benzene sulfonates, alkyl diphenyloxide sulfonate, alpha-olefin sulfonates, alkyl naphthalene sulfonates, paraffin sulfonates, lignin sulfonates, alkyl sulfosuccinates, ethoxylated sulfosuccinates, alkyl ether sulfosuccinates, alkylamide sulfosuccinates, alkyl sulfosuccinamate, alkyl sulfoacetates, alkylphosphates, phosphate ester, alkyl ether phosphates, acyl sarconsinates, acyl isethionates, N-acyl taurates, N-acyl-N-alkyltaurates, alkyl carboxylates, or a combination thereof.

[0089] The compositions described herein may comprise one or more nonionic surfactants. The nonionic surfactant may be either water soluble nonionic surfactants, water insoluble nonionic surfactants, or a combination of water soluble nonionic surfactants and water insoluble nonionic surfactants.

[0090] Non-limiting examples of water insoluble nonionic surfactants include alkyl and aryl: glycerol ethers, glycol ethers, ethanolamides, sulfoanylamides, alcohols, amides, alcohol ethoxylates, glycerol esters, glycol esters, ethoxylates of glycerol ester and glycol esters, sugar- based alkyl polyglycosides, polyoxyethylenated fatty acids, alkanolamine condensates, alkanolamides, tertiary acetylenic glycols, polyoxyethylenated mercaptans, carboxylic acid esters, polyoxyethylenated polyoxyproylene glycols, sorbitan fatty esters, or combinations thereof. Also included are EO / PO block copolymers (EO is ethylene oxide, PO is propylene oxide), EO polymers and copolymers, polyamines, and polyvinylpynolidones. Commercially available water insoluble nonionic surfactants that may be suitable for the compositions described herein include TOMADOL® 91-2.5, TOMADOL® 23-1, TOMADOL® 23-3, SPAN™ 20, SPAN™ 40, SPAN™ 60, SPAN™ 65, SPAN™ 80, SPAN™ 85, ARLATONE® TV, ATLAS® G-1086, ATLAS® G-1096, ATLOX® 1045 A, CIRRASOL® G-1086, CIRRASOL® G- 1096, and combinations thereof.

[0091] Non-limiting examples of water soluble nonionic surfactants include sorbitan fatty acid alcohol ethoxylates and sorbitan fatty acid ester ethoxylates. Commercially available water soluble nonionic surfactants that may be suitable for the compositions described herein include TOMADOL® 9-11, TOMADOL® 23-7, TOMADOL® 91-6, TWEEN® 20, TWEEN® 21, TWEEN® 40, TWEEN® 60, TWEEN® 80, Surfonic L24-4, and combinations thereof.

[0092] In certain embodiments, the amount of surfactant ranges from 0.01% to 10%, from 0.05% to 5%, or from 0.1 to 1% by weight of the composition.

[0093] The compositions described herein may also comprise organosilicone surfactants, silicone-based antifoams used as surfactants in silicone-based and mineral-oil based antifoams. In certain embodiments, the compositions described herein may also comprise alkali metal salts of fatty acids (e.g., water soluble alkali metal salts of fatty acids and / or water insoluble alkali metal salts of fatty acids).

[0094] Further examples of suitable auxiliaries include water repellents, drying agents, binders (adhesive, tackifier, fixing agent, such as carboxymethylcellulose, natural and synthetic polymers in the form of powders, granules or latices, such as gum arabic, polyvinyl alcohol andpolyvinyl acetate, natural phospholipids such as cephalins and lecithins and synthetic phospholipids, polyvinylpyrrolidone and tylose), thickeners and secondary thickeners (such as cellulose ethers, acrylic acid derivatives, xanthan gum, modified clays, e.g. , the products available under the name Bentone, and finely divided silica), stabilizers (e.g., cold stabilizers, preservatives (e.g., di chlorophene, benzyl alcohol hemiformal, l,2-Benzisothiazolin-3-on, 2- methyl-4-isothiazolin-3-one), antioxidants, light stabilizers, in particular UV stabilizers, or other agents which improve chemical and / or physical stability), dyes or pigments (such as inorganic pigments, e.g., iron oxide, titanium oxide and Prussian Blue; organic dyes, e.g., alizarin, azo and metal phthalocyanine dyes), antifoams (e.g., silicone antifoams and magnesium stearate), antifreezes, stickers, gibberellins and processing auxiliaries, mineral and vegetable oils, perfumes, waxes, nutrients (including trace nutrients, such as salts of iron, manganese, boron, copper, cobalt, molybdenum and zinc), protective colloids, thixotropic substances, penetrants, sequestering agents and complex formers.

[0095] The compositions may be in any customary formulation type, such as solutions (e.g., aqueous solutions), emulsions, water- and oil-based suspensions, powders (e.g., wettable powders, soluble powders), dusts, pastes, granules (e.g., soluble granules, granules for broadcasting), suspoemulsion concentrates, natural or synthetic products impregnated with the polynucleotides, fertilizers and also microencapsulations in polymeric substances. The polynucleotides may be present in a suspended, emulsified or dissolved form. Examples of particular suitable formulation types are solutions, water-soluble concentrates, dispersible concentrates, suspensions and suspension concentrates, emulsifiable concentrates, emulsions, capsules, pastes, wettable powders or dusts, pressings, granules, as well as gel formulations for the treatment of plant propagation materials such as seeds.

[0096] Compositions of the disclosure can include polynucleotides designed to target multiple genes, or multiple segments of one or more genes. The target can include multiple consecutive segments of a target gene, multiple non-consecutive segments of a target gene, multiple alleles of a target gene, or multiple target genes from one or more species.

[0097] In certain embodiments, the target sequence may be specific to a single species. In certain embodiments, the targeted sequence may have activity across all species in a genus or family. Control of multiple species within a single application may be achieved by use of polynucleotides that match targeted transcripts across all species, or by combination of multiple polynucleotides targeting the transcript of one gene with sequence specificity for each respective species.Nanoparticles

[0098] The compositions of the disclosure may include a nanoparticle to improve delivery and uptake into the plant.

[0099] As used herein, the term “nanoparticle” refers to particles having at least one cross- sectional dimension of less than about 1 micron. A nanoparticle can also be referred to as a “nanostructure.” A nanoparticle can have at least one cross-sectional dimension of less than about 500 nm, less than about 250 nm, less than about 100 nm, less than about 75 nm, less than about 50 nm, less than about 25 nm, less than about 10 nm, or, in some cases, less than about 1 nm. Examples of nanoparticles include nanotubes (e.g., carbon nanotubes), nanowires (e.g., carbon nanowires), graphene, star poly cations, and quantum dots (e.g., carbon dots), among others.

[0100] A variety of nanoparticles can be used. Sometimes a nanoparticle can be a carbon-based nanoparticle. As used herein, a “carbon-based nanoparticle” can include a fused network of aromatic rings wherein the nanoparticle includes primarily carbon atoms. In some instances, a nanoparticle can have a cylindrical, pseudo-cylindrical, or horn shape. A carbon-based nanoparticle can include a fused network of at least about 10, at least about 50, at least about 100, at least about 1000, at least about 10,000, or, in some cases, at least about 100,000 aromatic rings. A carbon-based nanoparticle may be substantially planar or substantially non-planar, or may include a planar or non-planar portion. A carbon-based nanoparticle may optionally include a border at which the fused network terminates. For example, a sheet of graphene includes a planar carbon-containing molecule including a border at which the fused network terminates, while a carbon nanotube includes a non-planar carbon-based nanoparticle with borders at either end. In some cases, the border may be substituted with hydrogen atoms. In some cases, the border may be substituted with groups comprising oxygen atoms (e.g., hydroxyl).

[0101] In some embodiments, a nanoparticle can include or be a nanotube. The term “nanotube” is given its ordinary meaning in the art and can refer to a substantially cylindrical molecule or nanoparticle including a fused network of primarily six-membered rings (e.g., six-membered aromatic rings). In some cases, a nanotube can resemble a sheet of graphite formed into a seamless cylindrical structure. It should be understood that a nanotube may also include rings or lattice structures other than six-membered rings. Typically, at least one end of the nanotube may be capped, i.e., with a curved or non-planar aromatic group. A nanotube may have a diameter of the order of nanometers and a length on the order of microns, tens of microns, hundreds of microns, or millimeters, resulting in an aspect ratio greater than about 100, about 1000, about 10,000, or greater. In some embodiments, a nanotube can have a diameter of less than about 1micron, less than about 500 nm, less than about 250 nm, less than about 100 nm, less than about 75 nm, less than about 50 nm, less than about 25 nm, less than about 10 nm, or, in some cases, less than about 1 nm.

[0102] In some embodiments, a nanotube may include a carbon nanotube. The term “carbon nanotube” can refer to a nanotube including primarily carbon atoms. Examples of carbon nanotubes can include single-walled carbon nanotubes (SWNTs), double-walled carbon nanotubes (DWNTs), multi -walled carbon nanotubes (MWNTs) (e.g., concentric carbon nanotubes), inorganic derivatives thereof, and the like. In some embodiments, a carbon nanotube can be a single-walled carbon nanotube. In some cases, a carbon nanotube can be a multi-walled carbon nanotube (e.g., a double-walled carbon nanotube).

[0103] The term “quantum dot” is given its normal meaning in the art. Examples of materials from which quantum dots can be made include PbS, PbSe, CdS, CdSe, ZnS, and ZnSe, among others. Carbon dots are quantum sized carbon nanoparticles which have emerged as being nanoparticles with potential to replace heavy metal containing toxic quantum dots. In certain embodiments, the nanoparticle comprises or is a carbon dot.Herbicides

[0104] The compositions of the disclosure may be used to reduce the expression of a cytochrome P450 reductase gene in weeds and restore utility of herbicides at originally labeled use rates on herbicide resistant weeds.

[0105] In certain embodiments, the compositions described herein may comprise one or more herbicides. Alternatively, the one or more herbicides may be applied either simultaneously or applied sequentially, with the compositions disclosed herein. In certain embodiments, the herbicide may be a pre-emergent herbicide, a post-emergent herbicide, or a combination thereof.

[0106] Non-limiting examples of suitable types of herbicides including, for example, HPPD inhibitor herbicides, acetolactate synthase (ALS) inhibitor herbicides, auxin herbicides, protoporphyrinogen oxidase (PPO) inhibitor herbicides, acetyl-CoA carboxylase (ACCase) inhibitor herbicides, photosystem II (PSII) inhibitor herbicides, or phytoene desaturase (PDS) inhibitor herbicides.

[0107] HPPD inhibitor herbicides include triketones, isoxazoles, and pyrazolinates.

[0108] The triketones include, but are not limited to, sulcotrione [i.e. 2-[2-chloro-4- (methylsulfonyl)benzoyl]-l,3-cyclohexanedione], mesotrione [i.e. 2-[4-(methylsulfonyl)-2- nitrobenzoyl]-l,3-cyclohexanedione]; tembotrione [i.e. 2-[2-chloro-4-(methylsulfonyl)-3- [(2,2,2,-tri-fluoroethoxy)methyl]benzoyl]-l,3-cyclo-hexanedione]; tefuryltrione [i.e. 2-[2- chloro-4-(methylsulfonyl)-3-[[(tetrahydro-2-furanyl)methoxy]methyl]benzoyl]-l,3cyclohexanedione]]; bicyclopyrone [i.e. 4-hydroxy-3-[[2-[(2-methoxyethoxy) methyl]-6- (trifluoromethyl)-3-pyridinyl]carbonyl]bicyclo [3.2.1]oct-3-en-2-one]; benzobicyclone [i.e. 3-(2- chloro-4-mesylbenzoyl)-2-phenylthiobicyclo [3.2.1]oct-2-en-4-one];

[0109] The isoxazoles include, but are not limited to isoxaflutole [i.e. (5-cyclopropyl-4- isoxazolyl)[2-(methylsulfonyl)-4-(trifluoromethyl)phenyl]methanone] or corresponding diketonitriles.

[0110] The pyrazolinones include, but are not limited to, topramezone [i.e. [3-(4,5-dihydro-3- isoxazolyl)-2-methyl-4-(methylsulfonyl)phenyl](5-hydroxy-l -methyl- IH-pyrazol -4- yl)m ethanone], pyrasulfotole [(5-hydroxy-l,3-dimethylpyrazol-4-yl(2-mesyl- 4trifluaromethylphenyl) methanone]; pyrazoxyfen [2-[4-(2,4-dichlorobenzoyl)-l,3- dimethy Ipy razol - 5 -y 1 oxy ] acetophenone] .[OHl] Further compound classes of HPPD inhibitor herbicides are the N-(tetrazol-5-yl)- and N- (triazol-5-yl)arylcarboxamides as disclosed in PCT / EP2011 / 064820 and the N-(1,2,5-Oxadiazol- 3-yl)benzamides as disclosed in WO2011 / 035874.

[0112] Examples of ALS inhibitor herbicides include, but are not limited to, sulfonylureas (such as bensulfuron, chlorsulfuron, halosulfuron, nicosulfuron, rimsulfuron, sulfometuron, and triflusulfuron); imidazolinones (such as imazamox, imazethapyr, imazapic, imazamethabenz- methyl, and imazaquin); triazolopyrimidines (such as flumetsulam, diclosulam, florasulam, chloransulam-methyl, and metosulam); and triazolinones (such as flucarbazone, thiencarbazone- methyl, and propoxycarbazone).

[0113] Examples of auxin herbicides include benzoic acids, phenoxy carboxylic acids, pyridine carboxylic acids, quinoline carboxylic acids, semi-carbasones, diflufenzopyr, 2,4-D, 2,4-DB, MCPA, MCPB, mecoprop, dicamba, clopyralid, fluroxypyr, picloram, triclopyr, aminopyralid, aminocyclopyrachlor, and quinclorac.

[0114] Examples of ACCase inhibitor herbicides include aryloxyphenoxypropionates (such as clodinafop, cyhalofop, diclofop, fenoxaprop, fluazifop, haloxyfop, propaquizafop, and quizalofop); cyclohexanediones (such as alloxydim, butroxydim, clethodim, cycloxydim, profoxydim, sethoxydim, tepraloxydim, and tralkoxydim), and phenylpyrazolines (such as pinoxaden).

[0115] Examples of PPO inhibitor herbicides include, but are not limited to, diphenylethers (such as acifluorfen, aclonifen, bifenox, ethoxyfen, fluoronitrofen, furyloxyfen, halosafen, chlomethoxyfen, fluoroglycofen, lactofen, oxyfluorfen, and fomesafen); thiadiazoles (such as fluthiacet-methyl and thidiazimin); pyrimidinediones or phenyluracils (such as benzfendizone, butafenacil, ethyl [3-2-chloro-4-fluoro-5-(l-methyl-6-trifluoromethyl-2,4-dioxo-l,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetate, flupropacil, saflufenacil, and tiafenacil); phenylpyrazoles (such as fluazolate, pyraflufen and pyraflufen-ethyl); oxadiazoles (such as oxadiargyl and oxadiazon); triazolinones (such as azafenidin, bencarbazone, carfentrazone, and sulfentrazone); oxazolidinediones (such as pentoxazone); N- phenylphthalimides (such as cinidon-ethyl, flumiclorac, flumiclorac-pentyl, and flumioxazin); benzoxazinone derivatives (such as l,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3,4-dihydro-3-oxo- 4-prop-2-ynyl-2H-l,4-benzoxazin-6-yl)-l, 3, 5-triazinane-2, 4-dione); flufenpyr and flufenpyr- ethyl; pyraclonil; and profluazol.

[0116] Examples of PSII inhibitor herbicides include, but are not limited to, bentazon, linuron, hexazinone, metribuzin, atrazine, diuron, isoproturon, monolinuron, desmedipham, metamitron, propanil, amicarbzone, fluometuron, phenmedipham, pyridate, ametryn, cynazine, dimefuron, fluometuron, methibenzuron, metoxuron, prometryn, simazine, simetryn, terbacil, terbuthylazine, chlorotoluron, and trietazine.

[0117] Examples of PDS inhibitor herbicides include, but are not limited to, norflurazon, diflufenican, fluorochloridone, flurtamone, picolinafen, and fluridone.

[0118] Commercial products containing each of these compounds are readily available. Herbicide concentration in the composition will generally correspond to the labeled use rate for a particular herbicide.

[0119] Herbicide applications may be sequentially or tank mixed with one, two, or a combination of several herbicides or any other compatible herbicide. Multiple applications of one herbicide or of two or more herbicides, in combination or alone, may be used over a growing season to areas comprising plants for the control of a broad spectrum of dicot weeds, monocot weeds, or both, for example, two applications (such as a pre-planting application and a post-emergence application or a pre-emergence application and a post-emergence application) or three applications (such as a pre-planting application, a pre-emergence application, and a postemergence application or a pre-emergence application and two post-emergence applications).Application to Plants

[0120] All plants and plant parts can be treated in accordance with the disclosure. Here, plants are to be understood to mean all plants and plant parts such as wanted and unwanted wild plants or crop plants (including naturally occurring crop plants), for example cereals (wheat, rice, triticale, barley, rye, oats), maize, soybean, potato, sugar beet, sugar cane, tomatoes, pepper, cucumber, melon, carrot, watermelon, onion, lettuce, spinach, leek, beans, Brassica oleracea (e.g., cabbage) and other vegetable species, cotton, tobacco, oilseed rape, and also fruit plants (with the fruits apples, pears, citrus fruits and grapevines). Crop plants can be plantswhich can be obtained by conventional breeding and optimization methods or by biotechnological and genetic engineering methods or combinations of these methods. Plants should be understood to mean all developmental stages, such as seeds, seedlings, young (immature) plants up to mature plants. Plant parts should be understood to mean all parts and organs of the plants above and below ground, such as shoot, leaf, flower and root, examples given being leaves, needles, stalks, stems, flowers, fruit bodies, fruits and seeds, and also tubers, roots and rhizomes. Parts of plants also include harvested plants or harvested plant parts and vegetative and generative propagation material, for example seedlings, tubers, rhizomes, cuttings and seeds.

[0121] Methods of using the compositions of the disclosure to control one or more weed species are provided. Examples of weed species controlled using the methods described herein include, but are not limited to, Italian ryegrass (Lolium mulliflorum), kochia (Kochia scoparia), redroot pigweed (Amaranthus relroflexiis). Palmer amaranth (Amaranthu palmeri), bamyardgrass (Echinochloa crus-galli), Common lambsquarters (Chenopodium album), Russian thistle (Salsola iberica), Common waterhemp (Amaranthus tuberculatus, syn. Amaranthus rudis), smooth pigweed (Amaranthus hybridus), powell amaranth (Amaranthus powellii), spiny amaranth (Amaranthus spinosus), black-grass (Alopecurus myosuroides), annual ryegrass (Lolium rigidum), perennial ryegrass (Lolium perenne), sourgrass (Digitaria insularis), junglerice (Echinochloa colona), Echinochloa phyllopogon, wild oats (Avena fatua), feral rye (Secale cereale), downy brome (Bromus tectorum), ripgut brome (Bromus diandrus), jointed goatgrass (Aegilops cylindrica), silky bentgrass (Apera spica-venti), green foxtail (Setaria viridis), yellow foxtail (Setaria pumila), eastern black nightshade (Solanum ptychanthum), horseweed (Conyza canadensis), hairy fleabane (Conyza bonariensis), tall fleabane (Conyza sumatrensis), broadleaf signalgrass (Urochloa platyphylla), giant foxtail (Setaria faberi), dandelion (Taraxacum officinale), blue mustard (Chorispora tenella), flixweed (Descurainia sophia), shepherd’ s-purse (Capsella bursa-pastoris), Sicklepod (Senna obtusifolia), hemp sesbania (Sesbania herbacea), velvetleaf (Abutilon theophrasti), johnsongrass (Sorghum halepense), giant ragweed (Ambrosia trifida), common ragweed (Ambrosia artemisiifolia), prickly sida (Sida spinosa), pitted momingglory, (Ipomoea lacunose), and puncturevine (Tribulus terrestris).

[0122] The treatment of the plants and plant parts with the compositions of the disclosure is carried out directly or by action on their surroundings or habitat using customary treatment methods, for example by dipping, spraying, atomizing, irrigating, evaporating, dusting, fogging, broadcasting, foaming, painting, spreading-on, injecting, watering (drenching), drip irrigatingand, in the case of propagation material, in particular in the case of seed, furthermore as a powder for dry seed treatment, a solution for liquid seed treatment, a water-soluble powder for slurry treatment, by incrusting, by coating with one or more coats, etc.

[0123] A direct treatment of the plants is foliar application, i.e. the compositions of the disclosure are applied to the foliage. The compositions of the disclosure also access the plants via the root system. The plants are then treated by the action of the compositions of the disclosure on the habitat of the plant. This may be done, for example, by drenching, or by mixing into the soil or the nutrient solution, i.e. the locus of the plant (e.g., soil) is impregnated with a liquid form of the compositions of the disclosure, or by soil application, i.e. the compositions of the disclosure are introduced in solid form (e.g., in the form of granules) into the locus of the plants, or by drip application (often also referred to as “chemigation”), i.e. the liquid application of the compositions of the disclosure from surface or sub-surface driplines over a certain period of time together with varying amounts of water at defined locations in the vicinity of the plants.

[0124] Compositions of the disclosure can be applied to weeds, crops, soil, and any other desired target using any delivery methodology known to those of skill in the art. For example, compositions can be applied to the desired locale via methods and forms including, but not limited to, shank injection, sprays, granules, flood / furrow methods, sprinklers, fumigation, root soaking and drip irrigation. In embodiments of the disclosure where the compositions are sprayed onto a desired locale, the compositions can be delivered as a liquid suspension, emulsion, microemulsion or powder. In other embodiments, granules or microcapsules can be used to deliver the compositions of the disclosure.

[0125] The compositions of the disclosure can be applied to plants or crops by any convenient method, for example, by using a fixed application system such as a center pivot irrigation system. In certain embodiments, application to fields of plants or crops is made by air spraying, i.e., from an airplane or helicopter, or by land spraying. For example, land spraying may be carried out by using a high flotation applicator equipped with a boom, by a back-pack sprayer or by nurse trucks or tanks. One of skill in the art will recognize that these application methodologies are provided by way of example and that any applicable methods known in the art or developed in the future can be utilized.

[0126] In certain embodiments, a method for controlling one or more weeds comprises directly contacting a weed with one or more of the compositions described herein. Non-limiting examples of contacting the weed include spraying the weed, drenching the weed, dripping onto the weed, or dusting the weed with one or more of the compositions described herein. In oneembodiment, the contacting step is repeated (e.g., more than once, as in the treating step is repeated twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, etc.). The contacting step can occur at any time during the growth of the weed. In one embodiment, contacting a weed with one or more of the compositions described herein occurs before the weed begins to grow. In another embodiment, contacting a weed with one or more of the compositions described herein occurs after the weed has started to grow.

[0127] In certain embodiments, a method for controlling one or more weeds comprises contacting a crop plant or plant part with one or more of the compositions described herein. Non-limiting examples of contacting the crop plant or plant part include spraying the crop plant or plant part, drenching the crop plant or plant part, dripping onto the crop plant or plant part, or dusting the crop plant or plant part with one or more of the compositions described herein. In one embodiment, the contacting step is repeated (e.g., more than once, as in the treating step is repeated twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, etc.). The contacting step can occur at any time during the growth of the crop plant or plant part. In one embodiment, contacting the crop plant or plant part with one or more of the polynucleotides described herein occurs before the crop plant begins to grow. In another embodiment, contacting the crop plant or plant part with one or more of the compositions described herein occurs after the crop plant has started to grow.

[0128] In certain embodiments, a method for controlling one or more weeds comprises treating a soil with one or more of the compositions described herein. Without being bound by theory, it is believed the one or more weeds will come into contact with the compositions when in contact with a treated soil. Non -limiting examples of treating the soil include spraying the soil, drenching the soil, dripping onto the soil, and / or dusting the soil with one or more of the compositions described herein.

[0129] In another embodiment, the method further comprises the step of planting a plant or plant part in the soil. The planting step can occur before, after, or during the treatment of the soil with one or more of the compositions described herein. In one embodiment, the planting step occurs before the soil is treated with one or more of the compositions described herein. In another embodiment, the planting step occurs during the treatment of the soil with one or more of the compositions described herein (e.g., the planting step occurs substantially simultaneously with the treating step, etc.). In still another embodiment, the planting step occurs after the soil is treated with one or more of the compositions described herein.

[0130] In certain embodiments, seeds are coated with one or more of the compositions described herein. Seeds may be treated with the compositions described herein in several ways includingvia spraying or dripping. Spray and drip treatment may be conducted by formulating compositions described herein and spraying or dripping the compositions onto seed via a continuous treating system (which is calibrated to apply treatment at a predefined rate in proportion to the continuous flow of seed), such as a drum-type of treater. Batch systems, in which a predetermined batch size of seed and compositions as described herein are delivered into a mixer, may also be employed. Systems and apparatuses for performing these processes are commercially available from numerous suppliers. In another embodiment, the treatment entails coating seeds. One such process involves coating the inside wall of a round container with the compositions described herein, adding seeds, then rotating the container to cause the seeds to contact the wall and the compositions, a process known in the art as container coating. Seeds can be coated by combinations of coating methods. Soaking typically entails using liquid forms of the compositions described. For example, seeds can be soaked for about 1 minute to about 24 hours (e.g., for at least 1 min, 5 min, 10 min, 20 min, 40 min, 80 min, 3 hours, 6 hours, 12 hours, 24 hours).Embodiments

[0131] The following numbered embodiments also form part of the present disclosure:

[0132] 1. A polynucleotide comprising a sequence essentially identical or essentially complementary to at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of a cytochrome P450 reductase (CPR) gene of a plant or to a transcript of the CPR gene.

[0133] 2 The polynucleotide of embodiment 1, wherein the polynucleotide is capable of hybridizing under physiological conditions in a cell of the plant to the CPR gene or to the transcript of the CPR gene and reducing expression of the CPR.

[0134] 3 The polynucleotide of embodiment 1 or embodiment 2, wherein the polynucleotide is a single-stranded RNA, a double-stranded RNA, a single-stranded DNA, a double-stranded DNA, or a double-stranded DNA / RNA hybrid.

[0135] 4. The polynucleotide of any one of embodiments 1-3, wherein the polynucleotide comprises at least one FANA-modified nucleotide.

[0136] 5. The polynucleotide of any one of embodiments 1-4, wherein the polynucleotide comprises a sequence essentially identical or essentially complementary to at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 1-4 or 7-120.

[0137] 6. The polynucleotide of any one of embodiments 1-5, wherein the polynucleotide consists of a sequence essentially identical or essentially complementary to 21 contiguous nucleotides of SEQ ID NO: 1-4 or 7-120.

[0138] 7 The polynucleotide of any one of embodiments 1-6, wherein the polynucleotide comprises or consists of the sequence of at least one of SEQ ID NOs: 211-290.

[0139] 8. The polynucleotide of any one of embodiments 1-7, wherein the CPR gene has at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1-4 or 7-120.

[0140] 9. The polynucleotide of any one of embodiments 1-8, wherein the CPR gene encodes a polypeptide having at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 5, 6, or 121-177.

[0141] 10. An herbicidal composition comprising the polynucleotide of any one of embodiments 1-9; and a carrier.

[0142] 11. The herbicidal composition of embodiment 10, wherein the composition further comprises an herbicide.

[0143] 12. The herbicidal composition of embodiment 11, wherein the herbicide comprises a 4- hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor herbicide, an acetolactate synthase (ALS) inhibitor herbicide, an auxin herbicide, a protoporphyrinogen oxidase (PPO) inhibitor herbicide, an acetyl-CoA carboxylase (ACCase) inhibitor herbicide, a photosystem II (PSII) inhibitor herbicide, or a phytoene desaturase (PDS) inhibitor herbicide.

[0144] 13. The herbicidal composition of any one of embodiments 10-12, wherein the composition further comprises a surfactant.

[0145] 14. The herbicidal composition of any one of embodiments 10-13, wherein the composition further comprises a nanoparticle.

[0146] 15. The herbicidal composition of embodiment 10, wherein the composition is in the form of a liquid, a powder, a granule, a paste, a pellet, or a gel.

[0147] 16. A plant or plant cell comprising the polynucleotide of any one of embodiments 1-9.

[0148] 17. A plant seed coated with a composition comprising the polynucleotide of any one of embodiments 1-9.

[0149] 18. An herbicidal composition comprising a cytochrome P450 reductase (CPR) inhibitor; and a carrier.

[0150] 19. The herbicidal composition of embodiment 18, wherein the CPR inhibitor comprises diphenyl enei odonium .

[0151] 20. The herbicidal composition of embodiment 18, wherein the composition further comprises an herbicide.

[0152] 21. The herbicidal composition of embodiment 20, wherein the herbicide comprises a 4- hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor herbicide, an acetolactate synthase (ALS) inhibitor herbicide, an auxin herbicide, a protoporphyrinogen oxidase (PPO) inhibitor herbicide, an acetyl-CoA carboxylase (ACCase) inhibitor herbicide, a photosystem II (PSII) inhibitor herbicide, or a phytoene desaturase (PDS) inhibitor herbicide.

[0153] 22. A method of controlling weeds, reducing non-target site resistance (NT SR) of a weed, or improving effectiveness of an herbicide on a weed, the method comprising: providing to the weed a composition that reduces expression or activity a cytochrome P450 reductase (CPR) of the weed.

[0154] 23. The method of embodiment 22, wherein the composition comprises a polynucleotide comprising a sequence essentially identical or essentially complementary to at least 18, at least19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of a CPR gene of the weed or to a transcript of the CPR gene.

[0155] 24. The method of embodiment 22 or embodiment 23, wherein the polynucleotide is capable of hybridizing under physiological conditions in a cell of the weed to the CPR gene or to the transcript of the CPR gene and reducing expression of the CPR.

[0156] 25. The method of any one of embodiments 22-24, wherein the polynucleotide is a single- stranded RNA, a double-stranded RNA, a single-stranded DNA, a double-stranded DNA, or a double-stranded DNA / RNA hybrid.

[0157] 26. The method of any one of embodiments 22-25, wherein the polynucleotide comprises at least one FANA-modified nucleotide.

[0158] 27. The method of any one of embodiments 22-26, wherein the polynucleotide comprises a sequence essentially identical or essentially complementary to at least 18, at least 19, at least20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 1-4 or 7-120.

[0159] 28. The method of any one of embodiments 22-27, wherein the polynucleotide consists of a sequence essentially identical or essentially complementary to 21 contiguous nucleotides of SEQ ID NO: 1-4 or 7-120.

[0160] 29. The method of any one of embodiments 22-28, wherein the polynucleotide comprises or consists of the sequence of at least one of SEQ ID NOs: 211-290.

[0161] 30. The method of any one of embodiments 22-29, wherein the CPR gene has at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1-4 or 7-120.

[0162] 31. The method of any one of embodiments 22-30, wherein the CPR gene encodes a polypeptide having at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 5, 6, or 121-177.

[0163] 32. The method of any one of embodiments 22-31, wherein the composition comprises two or more polynucleotides, wherein the two or more polynucleotides target different segments of the same CPR gene or wherein the two or more polynucleotides target multiple CPR genes.

[0164] 33. The method of any one of embodiments 22-32, wherein the composition comprises a CPR inhibitor.

[0165] 34. The method of embodiment 33, wherein the CPR inhibitor comprises diphenyl enei odonium .

[0166] 35. The method of any one of embodiments 22-34, wherein the weed is growing in a field of crop plants.

[0167] 36. The method of embodiment 35, wherein the crop plant is com, soybean, or wheat.

[0168] 37. The method of any one of embodiments 22-36, wherein the weed is a monocot or a dicot.

[0169] 38. The method of any one of embodiments 22-37, wherein the weed is Alopecurus myosuroides (blackgrass), Bassia scoparia (kochia), Bromus tectorum (downy brome), Conyza canadensis (horseweed), Chenopodium formosanum (Djulis), Echinochloa crus-galli (barnyardgrass), E. oryzicola (late watergrass), E. colona (jungle rice), Eleusine indica (goosegrass), Ipomoea purpurea (common morning glory), Medicago truncatula (Barrel medic), or Poa annua (annual bluegrass).

[0170] 39. The method of any one of embodiments 22-38, wherein the weed is herbicide resistant.

[0171] 40. The method of any one of embodiments 22-39, wherein the composition further comprises a nanoparticle.

[0172] 41. The method of any one of embodiments 22-40, wherein the composition further comprises a carrier, a surfactant, or an herbicide.

[0173] 42. The method of any one of embodiments 22-41, wherein the composition is in the form of a liquid, a powder, a granule, a paste, a pellet, or a gel.

[0174] 43. The method of any one of embodiments 22-42, wherein the composition is provided as a foliar application, in-furrow application, or a soil drench application.

[0175] 44. A kit comprising the polynucleotide of any one of embodiments 1-9; and an herbicide.

[0176] All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0177] Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended embodiments.

[0178] The following examples are offered by way of illustration and not by way of limitation.EXAMPLES

[0179] There are two CPR genes in Arabidopsis, referred to s ATRl an A TR2 and one hypothetical ^ TR3 that encodes a diflavin reductase essential for embryogenesis, but that is unable to reduce P450 in vitro. The isoforms are grouped in two distinct groups with different roles. Class I is A TRL which is involved in primary and basal metabolism. Class II is ATR2, which is inducible by environmental stimuli such as wounding, pathogen attack, or high light intensity exposure.

[0180] Examples 1-8 explored iiATRl an A TR2 are the main source of electrons for P450 enzymes and, without being bound by any theory, hypothesized that silencing these genes would decrease P450s that are involved in herbicide metabolism. These examples evaluated the function oiATRl an A TR2 in transgenic Arabidopsis over-expressing the P450 CYP81A12.Example 1: Exemplary Materials and Methods

[0181] The instant example provides exemplary materials and methods utilized for the present disclosure.Plant material

[0182] Mutant SALK lines of Arabidopsis were ordered from the Arabidopsis Biological Resource Center (ABRC). The lines SALK l 11558, SALK 208483C have a T-DNA insertion in Arabidopsis cytochrome P450 reductase 1 (ATR1 - at4g24520), hereafter named as atrl-a and atrl-b, respectively. The lines SALK 026053 and SALK l 52766 have T-DNA insertion in Arabidopsis cytochrome P450 reductase 2 (ATR2 - at4g30210), hereafter named as atr2-a and atr2-b.

[0183] Seeds from mutants and wild-type Columbia O (Col_0) were sown in soil and kept in a growth chamber at 22 / 18°C with a light intensity of 120 pE nr2s'1for 16 h. When seedlings hadfour true leaves, they were transplanted into individual pots and kept in the growth chamber in the same conditions. All the following experiments used the same methods to grow the Arabidopsis plants.Characterization and confirmation of T-DNA insertion

[0184] PCR assays were performed to identify and confirm the presence of T-DNA insertion in AXQ A TRI arA A TR2 genes in the mutant lines and to verify homozygosity. To amplify the wild type (WT) version of ATR1 or ATR2 genes, different primer sets named LP (forward) and RP (reverse) spanning the T-DNA insertion specific for each gene and line were used. The primers to identify the T-DNA insertion for all the mutant lines were RP primers, specific for each gene and line, with LBb 1.3 (FIG. 2). The primer LBb 1.3 is a primer designed in the right border of the T-DNA sequence. Primer sequences of LBbl.3, LP and RP for alrl-a. atrl-b and atr2-a were provided by SIGnAL (http: / / signal.salk.edu / ). Primer sets for atr2-b were designed using Primer3plus. The primer sequences are described in Table 3.TABLE 3. Primers and their characteristics used to genotype by PCRATR1 and ATR2 genes with the T-DNA insertion and CYP81A12 in Fl or F2 plants.LP - left border, RP - right border, LBbl .3 - primer designed in the right border of the T-DNA sequence.

[0185] Leaf tissue was collected from individual plants at rosette stage and incubated in 1.2 mL tubes. Around 20 plants for each mutant line were genotyped. The tissues were frozen at -20°C until further analyses. Frozen materials were ground using a Qiagen Tissuelyzer and DNA wasextracted using a modified hexadecyltrimethylammonium bromide (CTAB) method. DNA quality and concentration were measured using Nanodrop2000.

[0186] PCR reactions were performed with Econotaq Plus 2 X Master Mix. PCR mixtures consisted of 12.5 pL of Econotaq Master Mix, 6.5 pL nuclease-free water, and 2.5 pL of forward and reverse primers (10 pM). Each sample was submitted to two different reactions, called “WT PCR” and “T-DNA PCR”. The former consisted of LP and RP primers to amplify the WT form or heterozygous and the latter was composed of RP and LBbl.3 primers to amplify the T-DNA insertion, if present. PCR cycling conditions were composed by initial denaturation of 94°C for 2 min, followed by 35 cycles consisting of denaturation at 94°C for 30 s, annealing at 55°C for “WT PCR” and 48°C for “T-DNA PCR” for 30 s, followed by extension at 72°C for 70 s, with a final extension time at 72°C for 5 min. The DNA amplification was confirmed by running in 1% agarose gel electrophoresis and PCR products were sent for Sanger sequencing.Generating transgenic herbicide-resistant Arabidopsis with mutant atrl or atr2 genes and double knockout lines

[0187] Aiming to generate transgenic Arabidopsis lines with a loss of function in ATR1 or ATR2, a transgenic Arabidopsis line carrying the P450 CYP81A12 from Echinochloa phyllopogon (synonym E. oryzicola, late watergrass) was used. The transgenic Arabidopsis metabolizes five different herbicide modes of action including acetolactate synthase (ALS) inhibitors (bensulfuron-methyl, pyrazosulfuron-ethyl, chlorsulfuron, azimsulfuron, imazamox, propoxycarbazone-sodium and penoxsulam), 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor (mesotrione), protoporphyrinogen oxidase (PPO) inhibitor (pyraclonil), deoxyxylulose phosphate synthase (DXS) inhibitor (clomazone), and photosystem II (PSII) inhibitor (bentazon). This population hereafter is called Arabidopsis CYP81A12. This line was crossed with mutant lines alrl-a. alrl-b. air 2 -a. and atr2-b to generate transgenic CYP81A12 carrying mutant atrl or atr2, hereafter are named as CYP81A12 atrl -a, CYP81A12 atrl-b, CYP81A12 atr2-a, and CYP81A12 atr2-b.

[0188] Transgenic Arabidopsis CYP81A12 and mutant lines alrl-a. alrl-b. air 2 -a. and atr2-b were grown with the same conditions as indicated before. When plants were in the flowering stage, the mutant lines were crossed with the transgenic Arabidopsis CYP81A12. The cross was repeated three additional times with new flowers within an interval of a week. The crossed flowers were labeled, and the seeds were collected when siliques turned brown. In total, four crosses were performed, cross A - transgenic CYP81A12 X alrl-a, cross B - transgenic CYP81A12 alrl-b. cross C - transgenic CYP81A12 Xatr2-a, and cross D - transgenic CYP81A12 X atr2-b (FIG. 3).

[0189] Fl plants were grown, and tissue collected two weeks after transplanting for DNA extraction. Extracted DNA was used to genotype for T-DNA insertion and presence of CYP81A12 gene. T-DNA insertion was genotyped as indicated before, using WT PCR and T- DNA PCR reactions. PCR amplification for the whole CYP81A12 was performed to genotype for the presence of the gene using genomic DNA. Plants that were heterozygous for T-DNA insertion and had the presence of CYP81A12 were selected and self-pollinated. F2 plants were grown with the same conditions as described before, and tissue collected for genotyping. Genomic DNA was extracted, and T-DNA insertion was genotyped using the same two PCR reactions as indicated before. It was expected that 25% of the F2 population were homozygous for T-DNA insertion. These plants were selected to genotype for the presence of CYP81A12 and those plants that had the gene were selected. These plants were genotyped for copy number analysis of CYP81A12 using QX200 Droplet Digital PCR system (ddPCR) due to its precision and sensitivity for detection of low copy numbers. ddPCR consisted of a master mix composed of 10 pL of 2 XQX200 EVAGREEN® Supermix, 2.5 pL of each primer (5 pM), and 5 pL of genomic DNA (5 ng pL'1).

[0190] Selected samples were processed for droplet generation using QX200™ droplet generator, followed by PCR reaction with initial cycle of 5 min at 95°C followed by a sequence of 40 cycles starting at 95°C for 30 s and 60°C for 1 min. Droplet reading of PCR products was conducted in the QX200 Droplet Reader. The ALS gene was used as a reference gene containing only two copies (one copy per haplotype). Primer sequences for ACS' and CYP81A12 used for ddPCR are listed in Table 4. The samples were analyzed by QuantaSoft AP Software, which measures the number of positive and negative droplets for each gene in each well. Droplets are assigned as positive or negative by thresholding based on their fluorescence amplitude. The software calculates the starting concentration of each target DNA molecule by modeling a Poisson distribution; the formula used for Poisson modeling is: Copies per droplet = -ln(l-p) where p= fraction of positive droplets. Concentrations are provided in units of copies per microliter of input sample with 95% confidence intervals. The ratio of the number of copies per droplet of ALS and CYP81A12 was calculated.TABLE 4. Primers and their characteristics used for gene expression analysis of A TRL ATR2, and CYP81A12.

[0191] To obtain an Arabidopsis line with a double knockout oiATRl and ATR2, mutant lines atrl-C atrl-b, atr2-a, and atr2-b were cultivated under the same conditions as previously indicated. Upon reaching the flowering stage, crosses were performed as described earlier. Fl plants were subjected to genotyping to determine homozygosity for ATR1 an A TR2 using the same PCR reactions as previously mentioned. Heterozygous plants were selected for self- pollination. F2 seeds were then sown in soil, and subsequent plants were genotyped for atrl and atr2 mutations.Gene expression analysis in atrl and atr2 mutant lines

[0192] Mutant and transgenic mutant lines were grown in a growth chamber with the same conditions as specified before. Approximately 100 mg of leaf tissue were collected from plants in the rosette stage. Sampling consisted of cutting the youngest fully expanded leaf for each biological replication, placing in 2 mL tubes, and incubating immediately in liquid nitrogen and afterwards kept at -80°C. Plants of lines Col_0, transgenic CYP81A12, and transgenic mutants CYP81A12 atrl-a, CYP81A12 atrl-b, CYP81A12 atr2-a, and CYP81A12 atr2-b were kept for fresh mass measurement at 28 d after transplant to analyze any fitness cost in the mutant lines.

[0193] RNA extraction was conducted using DIRECT-ZOL™ RNA miniprep from Zymo Research following manufacturer instructions. DNase I treatment was conducted during the RNA extraction protocol. Quantification and quality control were performed using Nanodrop 2000. cDNA synthesis was conducted using ISCRIPT™ from Zymo Research using 1 pg of RNA. Gene expression was analyzed on CFX96 Real Time System using SSOADVANCED™ Universal SYBR® Green Supermix. The reactions consisted of 5 pL of cDNA from samples diluted in Nuclease-Free Water in 1 :20, 2.5 pL of each primer at 5 pM, and 5 pL of SSOADVANCED™ universal SYBR® Green supermix (2 X). Thermal cycler conditions consisted of an initial cycle of 30 s at 95°C, followed by a sequence of 35 cycles starting at 95°C for 10 s and 72°C for 30 s. Melt-curve analysis was performed by adding steps incrementing temperature 65-95°C by 0.5°C.

[0194] Primer sets were specifically designed to target various regions of the genes, both upstream and downstream of the T-DNA insertion site. This systematic design allowed for accurate analysis of how the gene was affected by the T-DNA insertion. YorATRl gene, four sets of primers were designed, called ATRI A with forward primers in the 5’UTR and reverse primers in the 1stexon (spanning the T-DNA insertion position in atrl-a line), ATR1 B with forward and reverse primers in the 1stexon, ATR1 C with the forward primer in exon 4thand reverse primer in exon 5th(spanning the T-DNA insertion position in atrl-b line), and ATR1 D with forward in the 8thexon and reverse in the 9thexon (FIG. 4). For ATR2 gene, five sets of primers were designed, ATR2 A with forward and reverse primers localized in the 1stexon, ATR2 B with forward primer in the 3thexon and reverse primer in the 4thexon (spanning the T- DNA insertion position in atr2-a line), ATR2 C with forward primer localized in the 9thand reverse in the 10thexon, ATR2 D with forward primer in the 12thexon and reverse in the 13th(spanning the T-DNA insertion position in atr2-b line), and ATR2 E with forward and reverse primers designed in the 14thexon. The primer locations in ^ZRY and ATR2 are represented inFIG. 4A and FIG. 4B, respectively. Primer sequences for gene expression analysis and their characteristics are listed in Table 4.Herbicide sensitivity of Arabidopsis lines in % MS medium

[0195] Plant growth of different lines of Arabidopsis were evaluated on ’ Murashige and Skoog (MS) solid medium containing different doses of bensulfuron, chlorsulfuron, penoxsulam, propoxycarbazone, mesotrione and bentazon. Seeds of Arabidopsis wild type (Col_0), transgenic Arabidopsis CYP81A12, mutants and transgenic mutant CYP81A12 atrl-a, CYP81A12 alrl-b. CYP81A12 air 2 -CL and CYP81A12 atr2-b were sterilized by chloride gas for 7 h by mixing 100 mL of bleach with 3% hydrochloric acid. Technical grade herbicides were diluted in ethanol (bensulfuron, penoxsulam, propoxycarbazone, bentazon) or methanol (chlorsulfuron, mesotrione) to mix with A MS medium before it solidified. Petri dishes containing A MS with different herbicides doses were divided into 10 sections, where six seeds of each Arabidopsis line were placed. The plates were incubated in a growth chamber at 25°C, 14-h photoperiod, and 110 pE m'2s'1light intensity until the day of evaluation. The number of green leaves were counted 14 d after plating, and the data were normalized to percentage of untreated control.Whole-plant dose response

[0196] Arabidopsis plants of Col_0, transgenic CYP81A12, and transgenic mutant CYP81A12 air 1 -C CYP81A12 atrl-b, CYP81A12 atr2-CL and CYP81A12 atr2-b were grown with the same conditions as specified before. Plants in rosette stage (14 d after transplanting) were sprayed with increasing doses of chlorsulfuron (TELAR® XP, 750 g Kg-1), penoxsulam (Ricer, 217 g a.i. kg'1), mesotrione (Callisto 480g a.i. L'1), and 2,4-D (Shredder 2,4-D LV4, 455g a.e. L'1). The herbicides were applied in an automated spray chamber (Greenhouse Spray Chamber, model Generation IV) using a TJ8002E nozzle, calibrated to deliver 200 L ha'1at a pressure of 280 kPa and speed of 1.2 m s'1. The experimental design was completely randomized in 6 X 11 with four replications. Factor A was Arabidopsis lines. Factor B was increasing herbicides dose for each herbicide. Chlorsulfuron and penoxsulam doses were in range from 0 to 51.2 g a.i. ha'1; mesotrione doses were from 0 to 400 g a.i. ha'1; and 2,4-D doses were from 0 to 128 g a.e. ha'1. The shoot fresh mass was measured at 28 d after herbicide treatment.Herbicide metabolism analysis

[0197] Seedlings of Arabidopsis lines Col_0, transgenic CYP81A12, and transgenic mutant CYP81A12 atr-a, CYP81A12 atr-b, CYP81A12 atr2-a, and CYP81A12 atr2-b were transplanted into pots filled with potting soil and grown under the same conditions as previously described. At rosette stage, mesotrione or chlorsulfuron were applied were applied at 100 and 50 g ha'1,respectively. The doses were chosen based on the whole-plant dose-response curve experiment described above. After herbicide application the plants were kept in the growth chamber at same conditions and whole plants were collected at 6, 12, 24, 48, and 96 h for mesotrione and adding 144 h and 196 h for plants treated with chlorsulfuron. At each time of tissue collection, plant samples were weighed, washed with 80% acetone three times, dried with towel paper, placed in 15 mL Falcon tubes, and kept at -80°C ultra-freezer until the extraction. The extraction was conducted by grinding the tissue with a pestle, then added 10X of plant mass with 80% methanol and 0.1% formic acid. The solution was homogenized for 30 s using PowerGen 125 (Fisher Scientific) and centrifuged for 10 min at 4,700 Xg (Sorvall Legend X1R, Thermo Scientific, Waltham, MA). The supernatant was filtered with Econofltr Nylon 13 mm 0.2 pm (Agilent Technologies, Santa Clara, CA) and injected in the LC-MS / MS.

[0198] LC-MS / MS system consisted of a Nexera X2 UPLC with 2 LC-30AD pumps, a SIL- 30 AC MP autosampler, a DGU-20A5 Prominence degasser, a CTO-30 A column oven, and SPD-M30A diode array detector coupled to an 8040 quadrupole mass-spectrometer (Shimadzu Scientific Instruments, Columbia, MD). For mesotrione, the MS was in positive mode [M+H]+with MRM optimized for mesotrione 340>227.95 and set for 100 ms dwell time with a QI prebias of -16.0V, a collision energy of -18.0V and a Q3 pre-bias of -16.0V and for hydroxymesotrione 356>55>228 and set for 100 ms dwell time with a QI pre-bias of -16V, a collision energy of -18.0V and a Q3 pre-bias of -16.0V. For chlorsulfuron, the MS was in positive mode with MRM optimized for chlorsulfuron 357.9>141.1>167.05 and set for 100 ms dwell time with a QI pre-bias of -25.0V, a collision energy of -20.0V and a Q3 pre-bias of -15.0V and for hydroxy-chlorsulfuron 374.0>127.0>182.0 and set for a collision energy of -35.0. The samples were chromatographed on a 100 X4.6 mm Kinetex 2.6 pm biphenyl column (Phenomenex, Torrance, CA) maintained at 40°C. For both herbicides, solvent A consisted of water with 0.1% formic acid and solvent B was methanol with 0.1% formic acid. The solvent program started at 50% B and increased to 95% B in 5 min and maintained at 95% for 4 min. The solvent was returned to 50% B and maintained there for 3 min before the next injection. The flow rate was set at 0.4 mL min'1and each sample were analyzed with 5 pL injection volumes.Identification and phylogenetic analysis of CPRs in different weeds

[0199] The presence of CPR genes and copy number was searched in several important weed species. The genomes of Alopecurus myosuroides, Chenopodium formosanum, Bassia scoparia, Eleusine indica, Bromus tectorum, Echinochloa crus-galli, E. oryzicola, E. colona, Ipomoea purpurea, Poa annua, and Conyza canadensis were accessible through WeedPedia (weedpedia.weedgenomics.org). CPRs were identified with a blast search using ATR1(AT4G24520) or ATR2 (AT4G30210) against the genomes or utilized the InterPro ID for NADPH-cytochrome P450 reductase (IPR023208). Additionally, the genomes of Amaranthus palmeri and A. tuberculatus were available, and the COGE web browser (genomevolution.org / coge) was used for blasting purposes. The CPR genes in Amaranthus species were identified using a comparative analysis approach. Firstly, ATR1 and ATR2 sequences were subjected to a sequence similarity search against the Amaranthus hypochondriacus (grain amaranth) genome, which is accessible through the Phytozome database. Subsequently, the identified sequences were further compared against the Palmer amaranth and common waterhemp genomes. Other already known plant CPR genes were extracted from GeneBank NCBI, such as corn (Zea mays, CPR2a: NP 001159331.1, CPR2b: NP_001146345.2), rice (Oryza sativa, CPR2a: XP_015651232.1, CPR2b: XP_015650780.1), soybean (Glycine max, CPR1 : NP_001236742.2, CPR2: XP_003549436.1), cotton (Gossypium hirsutum, CPR1: ACN54323.1, CPR2: NP_001314398.2), and Barrel medic (Medicago truncatula, CPR1 : XP 003602898.1, CPR2: XP 003610109.1). Multiple protein alignment was performed using ClustalO. Tree construction with Neighbor-joining method was performed using GENEIOUS PRIME® 2023.0.1. The N-terminal, transmembrane, FMN binding, FAD binding, and NADP+binding domains were identified. The transmembrane was identified using DeepTMHMM tool (dtu.biolib.com / DeepTMHMM).Statistical analysis

[0200] Gene expression analysis oiATRl arA A TR2 genes in mutant plants was performed using ALS as a normalization gene. Expression analyses were performed utilizing four biological and two technical replicates for each line. The mean Ct values and the standard deviation were calculated by treatment. A melt curve analysis confirmed the presence of a single amplified product for each reaction. Relative transcript abundance was calculated using 2('AACt)method for each gene and biotype. The reference population used for the calculation was Col_0. Dunnett’s test (p < 0.05) was used to compare the relative expression between Col_0 and mutant lines.

[0201] Number of green leaves at 14 d after seeds were planted and shoot fresh weight (% of untreated control) at 28 d after herbicide application (DAA) were used as response variable for the Vi MS medium and whole-plant dose-response experiment, respectively. Data were converted into percentages relative to untreated treatment. The drc package from statistical software R v.3.5.3 was used for data analysis. The data were tested for the best nonlinear model by comparing different models by ANOVA() and using function modelFit() testing for lack-of- fit. Based on the analysis, the data were adjusted using the three-parameter log-logistic model with the function modelFit(). For the whole-plant dose response experiment, the herbicide dosescausing 50% reduction in shoot fresh weight (GR50) were determined using the function summaryO and the resistance index (RI) was calculated by the function EDcomp() by dividing the GR50 from transgenic CYP81A12 or transgenic CYP81A12 mutants (atrl or air 2) by the GR50 of Col_0. For the dose-response in Yi MS medium the calculation was the same, using the GR50 obtained by the variable green leaves. The x and j’ data from the dose-response curves of both experiments were extracted from R using the function write. csv() and plotted with the treatment means by GraphPad Prism version 9 for Windows (San Diego, California).

[0202] The metabolism data obtained by LC-MS / MS were submitted to analysis of variance (ANOVA) and the means were compared using Dunnett’s test (p < 0.05). The peak area of the herbicides and metabolites of all the different Arabidopsis lines were compared to the Col_0 line.Example 2: Arabidopsis mutants

[0203] SALK lines atrl-a, atrl-b, and atr2-a were segregating for T-DNA insertion and heterozygous plants were selected to self and seed kept for seed production. atr2-b was homozygous for T-DNA insertion and four plants were self-pollinated to produce seeds. Progeny plants from atrl-a, atrl-b, and atr2-a were genotyped again and individuals homozygous for the T-DNA insertion were selected to self and seeds were produced. Homozygous T-DNA insertion in ATR1 in atrl-a and atrl-b lines and homozygous T-DNA insertion in ATR2 in atr2-a and atr2-b lines were confirmed by PCR. Primers LP and RP for both genes amplified only when Col_0 DNA was in the reaction. No SALK line amplified the WT version of the respective gene and only amplified the T-DNA reaction, confirming the homozygosity for T-DNA insertion. The amplifications were confirmed by electrophoresis in 1% agarose gel (FIG. 5), followed by Sanger sequencing. Based on Sanger sequencing results, the insertion of T-DNA was in the 5’ UTR and in the 4thintron in atrl-a and atrl-b mutant lines, respectively, for ATR1, and in the 4thintron and the 12thexon in atr2-a and atr2-b mutant lines, respectively, for ATR2 (FIG. 5).Example 3: Transgenic Arabidopsis CYP81A12 with atrl and atr2 knockdown

[0204] Fl plants from crosses were genotyped for T-DNA insertion. Around 20 plants were genotyped from each cross and around 90% of the plants were heterozygous for ATR1 T-DNA insertion for cross A and B and heterozygous for ATR2 T-DNA insertion in cross C and D, indicating success in the crosses. These plants were genotyped for CYP81A12 presence by PCR and plants that had the gene were selected for self-pollination to generate F2. F2 plants segregated 1 :2: 1 for ATR1 T-DNA insertion for cross A and B and for ATR2 T-DNA insertion for cross C and D (Table 5). Homozygous mutants were submitted for PCR amplification ofCYP81A12 and when confirmed, samples were submitted for ddPCR. Four, one, three, and five plants out of 15, 12, 9 and 15 homozygous had CYP81A12. These plants were analyzed by ddPCR. When only one gene copy was identified in comparison to ALS gene, the plants were self-pollinated, and their progenies were analyzed again using ddPCR.TABLE 5. PCR genotyping for T-DNA insertion in F2 segregating plants derived from crosses between atrl or atr2 mutants with transgenic Arabidopsis CYP81A12.

[0205] The ddPCR results indicated the same copy number of CYP81A12 and ALS for the parental transgenic line CYP81A12 (Table 6). Plants selected based on previous genotyping (as indicated above from crosses A, B, C, and D) had a ratio of ALS and CYP81A12 from 0.9 to 1.1, suggesting two copies of each gene, one copy per haploid genome. These plants were selected and used for further experiments.TABLE 6. ddPCR results of parental transgenic Arabidopsis CYP81A12 and four selected F3 plants from crosses A (CYP81A12 I atrl -a), B (CYP81A12 I atrl-b\ C (CYP81A12 I atr2-a and D (CYP81A12 I alr2-h .Example 4: atrl and atr2 double knockouts

[0206] Genotyping analysis of F2 plants obtained from various crosses between atrl and atr2 mutants demonstrated an absence of double knockout genotypes (Table 7). A total of 416 F2 plants were genotyped, revealing seven out of the nine possible genotypes. Among the genotyped plants, 46.2% exhibited heterozygosity for both genes, 5% displayed heterozygosity for one gene and wildtype for the other, 6.2% exhibited homozygous mutation in one gene and heterozygosity in the other, while 42.6% exhibited homozygous mutation in one gene and wildtype in the other. Notably, double wildtype and double mutant genotypes were not observed. The segregation pattern observed in the F2 generation deviated from the expected Mendelian segregation for two independent genes, indicating the presence of linked genes.Further investigation revealed that the atrl and atr2 genes are localized on chromosome four of Arabidopsis and are approximately two megabases (MBp) apart, providing supporting evidence that the two genes may be linked.TABLE 7. Genotype analysis of F2 segregating populations derived from crosses between atrl and atr2 Arabidopsis mutants.Example 5: ATR1 and ATR2 gene expression in mutant and transgenic mutant lines

[0207] Mutant atrl -a showed a reduced transcription of ATR1 when the primer set used was spanning the T-DNA insertion (FIG. 6A); however, normal expression of ATR1 gene was observed when primer set used was downstream of the T-DNA insertion (FIG. 6B-D), indicating that the T-DNA insertion in 5’ UTR did not decrease ATR1 expression in atrl -a line. Mutant line alrl-b. which had T-DNA insertion in the 4thintron, had significant reduction in ATR1 transcripts. This transcript disruption was confirmed in fnoATRl transcript when the primer set used spanned the T-DNA insertion and a section of downstream sequence (FIG. 6B- D). Interestingly, this line had increased transcript abundance when the primer set was upstream of the T-DNA (FIG. 6A). These results indicate a possible compensation in the gene expression by the plant for the disrupted gene.

[0208] Mutants for ATR2 showed similar results. Mutant atr2-a had an upregulation of transcript upstream of the T-DNA insertion in this line (FIG. 6E); however, this line had significant reduction of A / R2 expression downstream of the T-DNA insertion, especially whenthe set of primers used was spanning the T-DNA insertion (FIG. 6F-I). Mutant atr2-b had T- DNA insertion in the 12thexon and had a reduction in the transcription upstream and downstream of the T-DNA insertion (FIG. 6E-I). T-DNA insertion in the ATR1 mutant atrl-b and in the ATR2 mutants atr2-a and atr2-b had knockdown of expression of their respective mRNA. CYP81A12 expression was analyzed, and all the transgenic mutant lines had the same transcription production as the transgenic CYP81A12 (FIG. 6J).

[0209] Knocking down 47 / ? / or ATR2 did not cause obvious effects on plant growth. Transgenic CYP81A12 had a lower fresh mass with a smaller height of the plants in comparison to Col_0.Example 6: atrl mutant increases herbicide sensitivity in CYP81A12 line

[0210] Based on whole-plant dose response, transgenic Arabidopsis CYP81A12 carrying atrl and atr2 were more sensitive to all herbicides tested, including penoxsulam, chlorsulfuron, mesotrione, and 2,4-D (FIG. 7). The transgenic Arabidopsis CYP81A12 had a resistance index of 6.8, 23.6, and 20.9 for penoxsulam, mesotrione, and 2,4-D, respectively (Table 8). When ATR1 was knocked out (CYP81A12 atrl-b), the resistance index (RI) decreased to 1.0, 5.7, and 4.2, respectively to penoxsulam, mesotrione, and 2,4-D. The chlorsulfuron doses used in this study were not able to control the transgenic Arabidopsis CYP81A12 and CYP81A12 atrl-a (FIG. 7). The resistance to chlorsulfuron of these lines was very high. The lines CYP81A12 atr2-a and CYP81A12 atr2-b showed that the highest doses imparted a slight plant control, and the model was fitted to the three-parameter model. These lines had a RI of 97.7 and 86.7 for chlorsulfuron (Table 8). The line CYP81A12 atrl-b reduced the RI to 3.7 (Table 8). CYP81A12 atrl-b had the highest decrease in the RI to all herbicides. ATR2 knockdown lines (CYP81A12 atr2-a and CYP81A12 atr2-b) also reduced the RI, but not with the same magnitude as atrl-b (Table 8).TABLE 8. Parameters of the logistic equation and resistance index (RI) for shoot fresh weight (% of untreated control) of wild-type (Col_0), transgenic Arabidopsis CYP81A12, transgenic Arabidopsis mutants CYP81A12 atrl-a, CYP81A12 atrl-b, CYP81A12 atr2-a and CYP81A12 atr2-b treated with different doses of penoxsulam, chlorsulfuron, mesotrione and 2,4-D.

[0211] The control of different herbicide doses on Arabidopsis lines grown in i MS medium yielded similar results (FIG. 8 and Table 9). Among the mutants, the CYP81A12 atrl-b line exhibited the most significant reversal of resistance to bensulfuron, penoxsulam, chlorsulfuron, propoxycarbazone, mesotrione, and bentazon, as evidenced by the highest magnitude of response (FIG. 8). Notably, CYP81A12 atrl-b displayed symptoms similar to the Col_0 line at lower herbicide doses (FIG. 8). In contrast, CYP81A12 atrl-a, CYP81A12 atr2-a, and CYP81A12 atr2-b did not exhibit reduced herbicide resistance compared to CYP81A12.Specifically, CYP81A12 displayed resistance indices (RI) of 111.0, 35.7, 2.1, 4.2, 4.6, and 107.3 for bensulfuron, chlorsulfuron, penoxsulam, propoxycarbazone, mesotrione, and bentazon, respectively, while CYP81A12 atrl-a demonstrated reduced RIs of 22.3, 10, 0.9, 1.2, 0.9, and 0.9 for the same herbicides, reflecting similar GR50 values as Col_0 for penoxsulam, propoxycarbazone, mesotrione, and bentazon (Table 9).TABLE 9. Parameters of the logistic equation and resistance index (RI) for leaf number of Arabidopsis seedlings (% of untreated control) of wild-type, atrl-a, atrl-b, atr2-a, atr2-b, and Arabidopsis transgenic CYP81A12, Arabidopsis transgenic mutants CYP81A12 atrl-a, CYP81A12 atrl-b, CYP81A12 atr2-a, CYP81A12 atr2-b growing in MS medium supplemented with different doses of herbicides.Example 7: Reduction of herbicide metabolism

[0212] Transgenic Arabidopsis line CYP81A12 had a smaller peak area for the parental mesotrione than Col_0 in all time points after herbicide application, indicating faster herbicide metabolism (FIG. 9A-B). The transgenic lines CYP81A12 atrl-a, CYP81A12 atr2-a and CYP81A12 atr2-b had similar profiles, with smaller peak area of parental mesotrione than Col_0. The only Arabidopsis line that showed the same peak area as Col_0 was CYP81A12 atrl- b. Hydroxylation of mesotrione was higher than Col_0 for all lines in all times points, except for CYP81A12 atrl-b (FIG. 9A-B), which had a very small amount of hydroxy -mesotri one, highlighting that knockdown oiATRl prevents mesotrione metabolism in CYP81A12 atrl-b. Similar results were observed for chlorsulfuron metabolism (FIG. 10). Transgenic CYP81A12 had a significantly smaller peak area of parental chlorsulfuron than Col_0, inferring faster herbicide metabolism. The transgenic mutant line CYP81A12 atrl-a was not different in areapeak of parental chlorsulfuron and hydroxy-chlorsulfuron when compared to Col_0, indicating a loss of the metabolism capacity.Example 8: NADPH-cytochrome P450 reductase in weeds

[0213] Thirteen different weed species were analyzed, and their corresponding CPRs (cytochrome P450 reductase) were identified (FIG. 11). The number of CPR copies varied within each weed species, ranging from two to three copies. Through the construction of a phylogenetic tree, CPRs can be categorized into two distinct classes, namely CPR I and CPR II (FIG. 11). These findings align with previous research, which suggests that CPR Class I is exclusive to dicot species, while Class II is present in both monocots and dicots.

[0214] To differentiate between the CPRs within weed species, they were denoted as "a", "b", or "c" and individual copies were numbered as ".1", ".2", or ".3" to represent one, two, or three copies, respectively, in the present study. Kochia (Bassia scoparia), common waterhemp, common morningglory (Ipomea purpurea), and Djulis (Chenopodium formosanum) have both CPR 1 and CPR 2 (FIG. 11). Common morning glory, a tetrapioid, possesses two copies of CPR 1 and one copy of CPR 2, whereas the hexapioid Djulis has three copies of CPR 1 and three copies of CPR 2. The diploid kochia hasone copy of each CPR class. On the other hand, the diploids common waterhemp and Palmer amaranth possess one copy of CPR1 and two copies of CPR2 and horseweed possess three copies of only CPR class II. Class II Palmer amaranth CPRs have 93% identity, while the CPRs of horseweed (Class II) had a 74-76% identity among their three copies. Djulis, a hexapioid species with 27 chromosomes, exhibits three copies of each CPR class. Notably, each copy is localized within a different subgenome (B, C, and D).

[0215] Monocot plants exhibit diverse CPR compositions within CPR Class II, forming a distinct branch. Although their classification into specific classes is not documented in the literature, various monocot species possess CPR variants within Class II and we propose a division of CPR variants in grasses into CPRa, CPRb and CPRc class II (FIG. 11). For instance, blackgrass and goosegrass each possess three different CPR II variants: Am CPRa, Am CPRb, and Am CPRc for blackgrass, and Ei CPRa, Ei CPRb, and Ei CPRc for goosegrass. These CPR variants are categorized into separate branches based on phylogenetic analysis. Similarly, annual bluegrass, a tetrapioid species, possesses three CPR variants, with two copies of each CPR variant present in subgenomes A or B. Similarly, late watergrass, an allotetraploid species, exhibits two copies of each of the three CPR variants, distributed across its subgenomes. Jungle rice, an allohexaploid species, possesses nine CPR variants, with copies of each CPR variant located in subgenomes D, E, or F. On the other hand, bamyardgrass, also an allohexaploid species, diverges from jungle rice as it possesses eight CPR variants. Among these variants, twoCPRs (CPRa and CPRc) have three copies each, while one CPR (CRPb) has two copies. It is noteworthy that the diploid downy brome is the sole monocot weed species analyzed in this study with only two CPR variants, whereas the remaining monocot weed species all possess three distinct CPR variants with different copy numbers.

[0216] All CPR genes had a well conserved domain. Among the identified CPRs in weed genomes, three CPRs, namely Eco CPRc. l, Ec_CPRc.3, and Eo CPRc.l, stand out with significantly larger protein sizes compared to the others. These three CPRs have 1520, 1905, and 1948 amino acids, respectively, whereas the remaining CPRs range from 689 to 823 amino acids. Eo CPRc.l, Ec_CPRc.3 had a bigger TV-terminal and additionally Eco CPRc.l had a long C-terminal extensions matching phosphatidylinositol 3 -kinase, which could be an assembly error. Additionally, these three CPRs exhibit a similarity ranging from 87% to 94% among themselves. Additionally, there is close genetic relationship between subgenome D of jungle rice and subgenome C of barnyardgrass. This suggests a common diploid ancestor, explaining the high similarity observed in CPRs among these three species.Example 9: FANA ASO in vitro binding efficiency

[0217] FANA ASOs designed to target Arabidopsis ATRs, Waterhemp (Amaranthus tuberculatus) CPRs, and Palmer amaranth (Amaranthus palmeri) CPRs were synthesized. Sequences of the FANA ASOs are shown in Tables 10, 11, and 12.TABLE 10. FANA ASOs targeting Arabidopsis ATRs.TABLE 11. FANA ASOs targeting waterhemp (Amaranthus tuberculatus) CPRs.TABLE 12. FANA ASOs targeting Palmer amaranth (Amaranthus palmeri) CPRs.

[0218] 1 j_ig of total extracted RNA from Arabidopsis and waterhemp (Amaranlhus tuberculatus) was treated with different concentrations of the FANA-ASOs (1, 5, and 10 pM) and water (control). Afterward, the samples were incubated at room temperature for 20 minutes. Subsequently, cDNA was synthesized and the efficiency of F ANA ASOs binding to the target genes were evaluated using qPCR.

[0219] The results of F ANA ASO in vitro binding efficiency for Arabidopsis ATR (ATR1 and ATR2) are shown in Tables 13 and 14. The FANA ASOs highlighted in bold were the best FANAs with high binding efficiency to the target gene. They decreased the expression of the ATR2 and ATR1 genes by roughly 100% and 80%, respectively. Therefore, these FANAs were used for further experiments.TABLE 13TABLE 14

[0220] The results of F ANA ASOs in vitro binding efficiency for waterhemp CPRs (CPR1, CPR2 and AltCPR2) are shown in Tables 15-17. The FANA ASOs highlighted in bold were the most effective FANAs with high binding efficiency to the target gene. They caused a roughly 100% decrease in the expression of the CPR1, CPR2, and AltCPR2 genes. As a result, these FANAs were used for further experiments.TABLE 15TABLE 16TABLE 17Example 10: FANA ASO petri dish assay

[0221] Tables 18 and 19 show the results of targeting Arabidopsis ATR (ATR1 and ATR2) with the FANA ASOs in a petri dish assay. After a week of application, samples were collected, followed by RNA extraction and qPCR.TABLE 18Bensulfuron (BEN), 100 nM, Mesotrione(MES), 250 nMTABLE 19Bensulfuron (BEN), 100 nM, Mesotrione(MES), 250 nMReferences

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Claims

What is claimed is:

1. A polynucleotide comprising a sequence essentially identical or essentially complementary to at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of a cytochrome P450 reductase (CPR) gene of a plant or to a transcript of the CPR gene.

2. The polynucleotide of claim 1, wherein the polynucleotide is capable of hybridizing under physiological conditions in a cell of the plant to the CPR gene or to the transcript of the CPR gene and reducing expression of the CPR.

3. The polynucleotide of claim 1, wherein the polynucleotide is a single-stranded RNA, a double-stranded RNA, a single-stranded DNA, a double-stranded DNA, or a double-stranded DNA / RNA hybrid.

4. The polynucleotide of claim 1, wherein the polynucleotide comprises at least one FANA- modified nucleotide.

5. The polynucleotide of claim 1, wherein the polynucleotide comprises a sequence essentially identical or essentially complementary to at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 1-4 or 7-120.

6. The polynucleotide of claim 1, wherein the polynucleotide consists of a sequence essentially identical or essentially complementary to 21 contiguous nucleotides of SEQ ID NO: 1-4 or 7-120.

7. The polynucleotide of claim 1, wherein the polynucleotide comprises or consists of the sequence of at least one of SEQ ID NOs: 211-290.

8. The polynucleotide of claim 1, wherein the CPR gene has at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1-4 or 7-120.

9. The polynucleotide of claim 1, wherein the CPR gene encodes a polypeptide having at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 5, 6, or 121-177.

10. An herbicidal composition comprising the polynucleotide of claim 1; and a carrier.

11. The herbicidal composition of claim 10, wherein the composition further comprises an herbicide.

12. The herbicidal composition of claim 11, wherein the herbicide comprises a 4- hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor herbicide, an acetolactate synthase (ALS) inhibitor herbicide, an auxin herbicide, a protoporphyrinogen oxidase (PPO) inhibitor herbicide, an acetyl-CoA carboxylase (ACCase) inhibitor herbicide, a photosystem II (PSII) inhibitor herbicide, or a phytoene desaturase (PDS) inhibitor herbicide.

13. The herbicidal composition of claim 10, wherein the composition further comprises a surfactant.

14. The herbicidal composition of claim 10, wherein the composition further comprises a nanoparticle.

15. The herbicidal composition of claim 10, wherein the composition is in the form of a liquid, a powder, a granule, a paste, a pellet, or a gel.

16. A plant or plant cell comprising the polynucleotide of claim 1.

17. A plant seed coated with a composition comprising the polynucleotide of claim 1.

18. An herbicidal composition comprising a cytochrome P450 reductase (CPR) inhibitor; and a carrier.

19. The herbicidal composition of claim 18, wherein the CPR inhibitor comprises diphenyl enei odonium .

20. The herbicidal composition of claim 18, wherein the composition further comprises an herbicide.

21. The herbicidal composition of claim 20, wherein the herbicide comprises a 4- hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor herbicide, an acetolactate synthase (ALS) inhibitor herbicide, an auxin herbicide, a protoporphyrinogen oxidase (PPO) inhibitor herbicide, an acetyl-CoA carboxylase (ACCase) inhibitor herbicide, a photosystem II (PSII) inhibitor herbicide, or a phytoene desaturase (PDS) inhibitor herbicide.

22. A method of controlling weeds, the method comprising: providing to the weed a composition that reduces expression or activity a cytochrome P450 reductase (CPR) of the weed.

23. The method of claim 22, wherein the composition comprises a polynucleotide comprising a sequence essentially identical or essentially complementary to at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of a CPR gene of the weed or to a transcript of the CPR gene.

24. The method of claim 22, wherein the polynucleotide is capable of hybridizing under physiological conditions in a cell of the weed to the CPR gene or to the transcript of the CPR gene and reducing expression of the CPR.

25. The method of claim 22, wherein the polynucleotide is a single-stranded RNA, a doublestranded RNA, a single-stranded DNA, a double-stranded DNA, or a double-stranded DNA / RNA hybrid.

26. The method of claim 22, wherein the polynucleotide comprises at least one FANA- modified nucleotide.

27. The method of claim 22, wherein the polynucleotide comprises a sequence essentially identical or essentially complementary to at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 1-4 or 7-120.

28. The method of claim 22, wherein the polynucleotide consists of a sequence essentially identical or essentially complementary to 21 contiguous nucleotides of SEQ ID NO: 1-4 or 7- 120.

29. The method of claim 22, wherein the polynucleotide comprises or consists of the sequence of at least one of SEQ ID NOs: 211-290.

30. The method of claim 22, wherein the CPR gene has at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1-4 or 7-120.

31. The method of claim 22, wherein the CPR gene encodes a polypeptide having at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 5, 6, or 121-177.

32. The method of claim 22, wherein the composition comprises two or more polynucleotides, wherein the two or more polynucleotides target different segments of the same CPR gene or wherein the two or more polynucleotides target multiple CPR genes.

33. The method of claim 22, wherein the composition comprises a CPR inhibitor.

34. The method of claim 33, wherein the CPR inhibitor comprises diphenyleneiodonium.

35. The method of claim 22, wherein the weed is growing in a field of crop plants.

36. The method of claim 35, wherein the crop plant is corn, soybean, or wheat.

37. The method of claim 22, wherein the weed is a monocot or a dicot.

38. The method of claim 22, wherein the weed is Alopecurus myosuroides (blackgrass), Bassia scoparia (kochia), Bromus tectorum (downy brome), Conyza canadensis (horseweed), Chenopodium formosanum (Djulis), Echinochloa crus-galli (bamyardgrass), E. oryzicola (late watergrass), E. colona (jungle rice), Eleusine indica (goosegrass), Ipomoea purpurea (common morning glory), Medicago truncatula (Barrel medic), or Poa annua (annual bluegrass).

39. The method of claim 22, wherein the weed is herbicide resistant.

40. The method of claim 22, wherein the composition further comprises a nanoparticle.

41. The method of claim 22, wherein the composition further comprises a carrier, a surfactant, or an herbicide.

42. The method of claim 22, wherein the composition is in the form of a liquid, a powder, a granule, a paste, a pellet, or a gel.

43. The method of claim 22, wherein the composition is provided as a foliar application, infurrow application, or a soil drench application.

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