WO2022241008A2

WO2022241008A2 - Compositions and methods to increase insect resistance in plants

Info

Publication number: WO2022241008A2
Application number: PCT/US2022/028799
Authority: WO
Inventors: Minviluz G. Stacey; Cuong X. Nguyen; Gary Stacey
Original assignee: The Curators Of The University Of Missouri
Priority date: 2021-05-11
Filing date: 2022-05-11
Publication date: 2022-11-17
Also published as: WO2022241008A3

Abstract

The disclosure relates to CYP72A polypeptides that mediate resistance to insect pests in plants. Also disclosed are plants comprising polynucleotides encoding the CYP72A polypeptides along with related methods of using polynucleotides that encode the CYP72A polypeptides to confer resistance to feeding by insect pests.

Description

1

TITLE: COMPOSITIONS AND METHODS TO INCREASE INSECT

RESISTANCE IN PLANTS

GOVERNMENT SUPPORT

[0001] This invention was made with government support under Grant Number 1444581 awarded by the National Science Foundation. The government has certain rights in the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to provisional application U.S. Serial No. 63/201,745, filed May 11, 2021, which is incorporated herein by reference in its entirety.

SEQUENCE LISTING

[0003] The instant application contains a sequence listing which has been submitted in ASCII format by electronic submission and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 9, 2022, is named P13567WOOO_ST25.txt and is 4,874,784 bytes in size.

TECHNICAL FIELD

[0004] The present disclosure relates to the field of biotechnology. More specifically, the present disclosure relates to compositions and methods for control of insect pests in plants.

BACKGROUND

[0005] Japanese beetles ( Popillia japonica ) feed on the leaves, flowers, and fruits of over 300 plant species of wild and cultivated plants in 79 families. Japanese beetle infestation is widespread across all states east of the Mississippi River, with sporadic infestations reported in California, Iowa, Missouri, and Nebraska. Each year $234 million losses occur due to Japanese beetle infestation. There is an ongoing need to identify new and effective methods for controlling Japanese beetles as well as other insect pests.

SUMMARY

[0006] The present disclosure provides compositions and methods for conferring insect resistance to plants, plant parts, and plant cells.

[0007] Modified plants having resistance to an insect pest are provided. The modified plants comprise increased expression of a polynucleotide encoding a cytochrome P45072A (CYP72A) polypeptide relative to an unmodified plant. In certain embodiments, the CYP72A polypeptide 2 comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 4. In certain embodiments, the modified plants comprise a heterologous polynucleotide encoding the CYP72A polypeptide.

[0008] Progeny, plant parts, and plant cells of the modified plants are also provided.

[0009] Methods for producing a plant with resistance to an insect pest are provided. The methods comprise increasing expression of a polynucleotide encoding a CYP72A polypeptide in the plant, wherein the insect resistance of the plant is increased when compared to a plant that lacks the increased expression are provided. In certain embodiments, the methods comprise introducing to a plant a polynucleotide encoding the CYP72A polypeptide, wherein the polynucleotide is operably linked to a promoter functional in the plant cell.

[0010] Polynucleotides capable of conferring resistance to an insect pest are provided. The polynucleotides encode a CYP72A polypeptide having at least 80%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 4. In certain embodiments, the polynucleotide is operably linked to a heterologous promoter that is operable in a plant cell.

[0011] Expression constructs, vectors, biological samples, plants, plant parts, plant cells comprising the polynucleotides are also provided.

[0012] Methods of reducing insect damage to a plant crop are provided. The methods comprise cultivating a plurality of plants comprising increased expression of a polynucleotide encoding a CYP72A polypeptide, wherein the plurality of plants of the plant crop have resistance to an insect pest, thereby reducing insect damage to the plant crop.

[0013] Commodity plant products prepared from the aforementioned plants, plant parts, and plant cells, wherein the product comprises the CYP72A polypeptide or the polynucleotide encoding the CYP72A polypeptide are provided. Methods for producing a commodity plant product comprising processing the aforementioned plants or plant parts to obtain the product are also provided.

[0014] Methods for identifying a plant that is resistant to an insect pest comprising providing a biological sample from a plant; quantifying expression of a CYP72A gene in the biological sample; and determining that the plant is resistant to the insect pest based on the quantification, wherein the CYP72A gene is differentially expressed in the plant that is resistant to the insect pest compared to a susceptible plant are provided. Kits for identifying an insect-resistant plant are also provided.

[0015] While multiple embodiments are disclosed, still other embodiments of the present disclosure will become apparent based on the detailed description, which shows and describes 3 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

[0016] 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.

[0017] FIG. 1A-C shows soybean fast neutron (FN) mutant line M027 has increased susceptibility to Japanese beetle and soybean looper larvae. FIG. 1A shows photographs of wild type (Williams 82) and M027 mutant plants infested with Japanese beetle grown at the Bradford Research and Experiment Center (BREC), summer 2017. FIG. IB and FIG. 1C show photographs and consumed leaf area, respectively, of detached 2^nd trifoliate leaflet of Williams 82 and M027 leaves infested with soybean looper ( Chrysodeixis includens) larvae (3^rd instar) after 20 hours in a choice assay. Data represent means ± SE, n=18. ***P < 0.001 indicate significant differences (/-test). Scale bar, 2 cm.

[0018] FIG. 2A-B shows identification of GmCYP72A141 as the causative genetic lesion for increased insect susceptibility and expression of GmCYP72A141 in various soybean tissues.

FIG. 2A shows phylogenetic relationships of GmCYP72A141 indicating that it is orthologous to the licorice ( Glycyrrhiza uralensis) GuCYP72A154 gene encoding a P450 monooxygenase involved in saponin biosynthesis. Two proteins, GmCYP72A148 and GmCYP72A120, encoded by genes homologous to GmCYP72A141 are also shown. FIG. 2B shows the expression profile of GmCYP72A141 in different soybean Williams 82 tissues. mRNA expression profile was determined by qRT-PCR. Shown are means ± SD for n = 4, One-way ANOVA with post hoc Tukey HSD test, different letters indicate significant differences between tissues p < 0.01.

[0019] FIG. 3A-C shows that high expression of soybean GmCYP72A141 in transgenic Arabidopsis inhibited cabbage looper ( Trichoplusia ni ) growth. FIG. 3A shows quantitative real-time PCR analysis of the expression of GmCYP72A141 in leaves of three-week-old Arabidopsis transgenic lines 2R-CYP, 5RCYP, and 6RCYPs. Col-OpFGC is negative control transformed with vector only. Relative transcript levels were normalized using SAND as an internal reference. Mean values ± SD for n = 4 are shown. Statistical analysis was done by one way ANOVA followed by a post-hoc Tukey's multiple range test. Different letters denoted 4 significant differences at < 0.01. FIG. 3B shows caterpillar fresh weights at eight (8) days after inoculation of newly-hatched cabbage looper larvaes on three-four week old transgenic Arabidopsis plants (no choice feeding assays). Mean values ± SD for n = 23 are shown. Statistical analysis was done by one-way ANOVA followed by a post-hoc Tukey's multiple range test. Different letters denoted significant differences at < 0.01. FIG. 3C shows representative images of caterpillars cultivated on the different transgenic Arabidopsis plants at eight (8) days after larval inoculation. Bars = 5 mm.

[0020] FIG. 4 shows leaf consumption by cabbage loopers ( Trichoplusia ni ) was reduced on transgenic Arabidopsis over-expressing GmCYP72A141. Shown are representative photographs of detached leaves of Arabidopsis wild-type control (ColO-pFGC) and GmCYP72A141 over expressing (6R-CYP) lines eight (8) hours after inoculation with 2nd instar cabbage loopers (choice assay). In this assay, detached leaves of Col-0 and 6R-CYP were placed in petri dishes then inoculated with cabbage loopers; each petri dish contained one Col-0 and one 6R-CYP leaf. [0021] FIG. 5 shows relative expression level of GmCYP72A141 Glyma.06g238500) in fully expanded first trifoliate leaves of WT (Maverick), CRISPR-45 (CRISPR mutant) and three overexpression lines (K15, K-29 and 0X23) . Mean± SD at p<0.01 with one-way ANOVA follow by Tukey HSD test, n=4. Note line 0X23 and CRISPR lines are homozygous plants at T4 generation. Other overexpression lines are T1 plants. Plants were grown under greenhouse condition.

[0022] FIG. 6A-C shows the soybean CRISPR line mutant with null mutation in GmCYP72A141 (Glyma.06g238500) is more susceptible to chewing insect compared to WT control. FIG. 6A shows representative photographs of 2^nd trifoliate leaflets at 20 hours after feeding with soybean looper. FIG. 6B and FIG. 6C show quantification of consumed leaf area. Data represent means ± SE, n>18. ***P < 0.001 indicate significant differences (/-test). Scale bar is 2 cm

[0023] FIG. 7A-C shows the soybean GmCYP72A141 Glyma.06g238500) overexpression line CYP-OX_23 is less favorable to chewing insect compared to WT control. FIG. 7A shows representative photographs of 2^nd trifoliate leaflets at 20 hours 20 hours after feeding with soybean looper. FIG. 7B and FIG. 7C shows quantification of consumed leaf area. Data represent means ± SE, n=ll. ***P < 0.001 indicate significant differences (/-test). Scale bar 2 cm 5

DETAILED DESCRIPTION

[0024] Soybean CYP72A141 (GmCYP72A141) encodes a cytochrome P450 monooxygense enzyme involved in the biosynthesis of triterpene glycosides, also called saponins. Saponins are widespread throughout the plant kingdom and constitute one of the largest and structurally diverse class of specialized metabolites. Saponins are amphipathic compounds containing hydrophobic triterpenoid agly cones called sapogenin and one or more hydrophilic sugar moieties. Biologically, plant saponins are generally considered defensive compounds against pathogenic microbes and herbivores. Saponins increase mortality levels by lowering food intake and affecting movement of food in the insect gut due to toxicity and less digestibility. Plant- derived saponins have been proven to be effective against important insect pests like aphids, beetles, weevils, leafhoppers, worms, and moths. GmCYP72A141 is orthologous to the Glycyrrhiza uralensis (licorice) GuCYP72A154, an enzyme that catalyzes the oxidation at C-30 of b-amyrin, a precursor of the triterpenoid saponin glycyrrhizin. Studies in yeast showed that GmCYP72A141 catalyzes the oxidation of b-amyrin at C-29 rather than at C-30. Moreover, soybean is not known to produce glycyrrhizin. While not wishing to be bound by any theory, applicants believe that GmCYP72A141 oxidizes the C-29 of b-amyrin to produce a saponin(s) or triterpene(s) that has anti-herbivore properties.

[0025] 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.

[0026] 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. 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. [0027] 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 6 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,

1 ½, and 4 ¾. This applies regardless of the breadth of the range.

[0028] As used herein, the phrase “biological sample” refers to either intact or non-intact ( e.g ., milled seed or plant tissue, chopped plant tissue, lyophilized tissue) plant tissue. It may also be an extract comprising intact or non-intact seed or plant tissue. The biological sample can comprise flour, meal, flakes, syrup, oil, starch, and cereals manufactured in whole or in part to contain crop plant by-products. In certain embodiments, the biological sample is “non- regenerable” (i.e., incapable of being regenerated into a plant or plant part).

[0029] 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.

[0030] The term "introduced" in the context of inserting a nucleic acid into a cell, means "transfection" or "transformation" or "transduction" and includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

[0031] As used herein, “modified”, in the context of plants, seeds, plant components, plant cells, and plant genomes, refers to a state containing changes or variations from their natural or native state. For instance, a “native transcript” of a gene refers to an RNA transcript that is generated from an unmodified gene. Typically, a native transcript is a sense transcript. Modified plants or seeds contain molecular changes in their genetic materials, including either genetic or epigenetic modifications. Typically, modified plants or seeds, or a parental or progenitor line thereof, have been subjected to mutagenesis, genome editing (e.g., without being limiting, via methods using site-specific nucleases), genetic transformation (e.g., without being limiting, via methods of Agrobacterium transformation or microprojectile bombardment), or a combination thereof. In one embodiment, a modified plant provided herein comprises no non-plant genetic material or 7 sequences. In yet another embodiment, a modified plant provided herein comprises no interspecies genetic material or sequences.

[0032] As used herein, the terms “nucleic acid,” “nucleic acid molecule,” “nucleotide sequence” and “polynucleotide” can be used interchangeably and encompass both RNA and DNA, including cDNA, genomic DNA, mRNA, synthetic (e.g., chemically synthesized) DNA or RNA and chimeras of RNA and DNA. The term polynucleotide, nucleotide sequence, or nucleic acid refers to a chain of nucleotides without regard to length of the chain. The nucleic acid can be double-stranded or single-stranded. Where single-stranded, the nucleic acid can be a sense strand or an antisense strand. The nucleic acid can be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such oligonucleotides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases. The present disclosure further provides a nucleic acid that is the complement (which can be either a full complement or a partial complement) of a nucleic acid, nucleotide sequence, or polynucleotide.

[0033] By “operably linked” or “operably associated,” it is meant that the indicated elements are functionally related to each other, and are also generally physically related. Thus, the term “operably linked” or “operably associated” as used herein, refers to nucleotide sequences on a single nucleic acid molecule that are functionally associated. Therefore, a first nucleotide sequence that is operably linked to a second nucleotide sequence means a situation when the first nucleotide sequence is placed in a functional relationship with the second nucleotide sequence. For instance, a promoter is operably associated with a nucleotide sequence if the promoter effects the transcription or expression of said nucleotide sequence. Those skilled in the art will appreciate that the control sequences (e.g., promoter) need not be contiguous with the nucleotide sequence to which it is operably associated, as long as the control sequences function to direct the expression thereof. Thus, for example, intervening untranslated, yet transcribed, sequences can be present between a promoter and a nucleotide sequence, and the promoter can still be considered “operably linked” to the nucleotide sequence.

[0034] As used herein, “plant” refers to a whole plant, any part thereof, or a cell or tissue culture derived from a plant, comprising any of: whole plants, plant components or organs (e.g., leaves, stems, roots, etc.), plant tissues, seeds, plant cells, and/or progeny of the same. A progeny plant can be from any filial generation, e.g., FI, F2, F3, F4, F5, F6, F7, etc. A plant cell is a biological cell of a plant, taken from a plant or derived through culture from a cell taken from a plant. [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. 8

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] A “promoter” is an untranslated DNA sequence upstream of a coding region that contains the binding site for RNA polymerase and initiates transcription of the DNA. A “promoter region” can also include other elements that act as regulators of gene expression. Promoters can include, for example, constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred and tissue-specific promoters for use in the preparation of recombinant nucleic acid molecules.

[0037] “Regulatory elements” refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory elements may include, but are not limited to, promoters, translation leader sequences, introns, and polyadenylation recognition sequences. Regulatory elements present on a recombinant DNA construct that is introduced into a cell can be endogenous to the cell, or they can be heterologous with respect to the cell. The terms "regulatory element" and "regulatory sequence" are used interchangeably herein.

Cytochrome P45072A

[0038] Cytochrome P45072A (CYP72A) sequences are provided that confer a plant with resistance to an insect pest. Such sequences include the amino acid sequence set forth in SEQ ID NO: 4, and variants thereof. Also provided are polynucleotide sequences encoding such amino acid sequences, including SEQ ID NOs: 1, 2, and 3.

[0039] Several embodiments also relate to the use of CYP72A or variants thereof that confer resistance to insect pests, including coleopteran. “Variants” is intended to mean substantially similar sequences. For polynucleotides, a variant comprises a deletion and/or addition of one or more nucleotides at one or more internal sites within the native polynucleotide and/or a substitution of one or more nucleotides at one or more sites in the native polynucleotide. As used herein, a “native” polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively. For polynucleotides, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode CYP72A polypeptides described above. Naturally occurring allelic variants can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction 9

(PCR) and hybridization techniques as outlined herein. Variant polynucleotides also include synthetically derived polynucleotides, such as those generated, for example, by using site- directed mutagenesis but which still encode a CYP72A polypeptide conferring insect resistance. Generally, variants of a particular polynucleotide will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular polynucleotide.

[0040] In certain embodiments, variants of a polynucleotide include at least one nucleotide substitution, insertion, or deletion so that they do not recite a naturally occurring nucleic acid sequence.

[0041] Variants of a particular polynucleotide encoding a CYP72A that confers insect resistance are encompassed and can be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide. Percent sequence identity between any two polypeptides can be calculated using sequence alignment programs and algorithms described below. Where any given pair of polynucleotides is evaluated by comparison of the percent sequence identity shared by the two polypeptides they encode, the percent sequence identity between the two encoded polypeptides is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

[0042] Methods of alignment of sequences for comparison are well known in the art and can be accomplished using mathematical algorithms such as the algorithm of Myers and Miller (1988) CABIOS 4: 11-17; the local alignment algorithm of Smith et al. (1981) Adv. Appl.

Math. 2:482; the global alignment algorithm of Needleman and Wunsch (1970) J. Mol.

Biol. 48:443-453; and the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci.

USA 872264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873- 5877. Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the GCG Wisconsin Genetics Software Package, Version 10 (available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif., USA).

[0043] Homologs (e.g., orthologs, paralogs) of SEQ ID NO: 4 encompassed by the present disclosure include, but are not limited to, polypeptides comprising the amino acid sequences set forth in SEQ ID NOs: 23-1282. Table 1 provides a summary of soybean homologs of GmCYP72A141 (Glyma.06G238500). 10

[0044] “Orthologs” and “paralogs” encompass evolutionary concepts used to describe the ancestral relationships of genes. Paralogs are genes within the same species that have originated through duplication of an ancestral gene; orthologs are genes from different organisms that have originated through speciation, and are also derived from a common ancestral gene.

TABLE 1

[0045] Additional polynucleotide sequences encoding a CYP72A polypeptide may be identified using methods well known in the art based on their ability to confer resistance to an insect pest. For example, candidate CYP72A genes are expressed in tobacco, Arabidopsis, or other easily transformed plant and the resultant transformant plants assessed for their resistance to insect(s) of interest.

[0046] Those skilled in the art may also find further candidate CYP72A genes based on genome synteny and sequence similarity. In one embodiment, additional gene candidates can be obtained by hybridization or PCR using sequences based on the CYP72A nucleotide sequences noted above.

[0047] 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 11 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).

[0048] 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 as ³²P, 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.).

[0049] 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 target nucleic 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.

[0050] “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 (T_m) 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. 12

[0051] The Tm 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 xSSC at 45 °C for 15 minutes. An example low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4-6xSSC 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 2x (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 proteins that 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.

[0052] 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 NaPCri, 1 mM EDTA at 50 °C with washing in 2xSSC, 0.1% SDS at 50 °C, more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPCri, 1 mM EDTA at 50 °C with washing in lx SSC, 0.1% SDS at 50 °C, more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPOr, 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 NaPCri, 1 mM EDTA at 50 °C with washing in O.lxSSC, 0.1% SDS at 50 °C, more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 MNaPOr, 1 mM EDTA at 50 °C with washing in O.lxSSC, 0.1% SDS at 65 °C.

[0053] The terms “polypeptide” and “protein” are generally used interchangeably and refer to a single polypeptide chain which may or may not be modified by addition of non-amino acid groups. It would be understood that such polypeptide chains may associate with other 13 polypeptides or proteins or other molecules such as co-factors. The terms “proteins” and “polypeptides” as used herein also include variants, mutants, modifications, analogous and/or derivatives of the polypeptides of the disclosure as described herein.

[0054] With regard to a defined polypeptide, it will be appreciated that percent identity figures higher than those provided above will encompass preferred embodiments. Thus, where applicable, in light of the minimum percent identity figures, it is preferred that the CYP72A polypeptide comprises an amino acid sequence which is at least 40%, more preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least

99.9% identical to SEQ ID NO: 4.

[0055] By “variant” polypeptide is intended a polypeptide derived from the protein of SEQ ID NO: 4, by deletion (so-called truncation) or addition of one or more amino acids to the N- terminal and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein. Such variants may result from, for example, genetic polymorphism or from human manipulation. Methods for such manipulations are generally known in the art.

[0056] “Derivatives” of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived. Thus, functional variants and fragments of the CYP72A polypeptides, and nucleic acid molecules encoding them, also are within the scope of the present disclosure, and unless specifically described otherwise, irrespective of the origin of said polypeptide and irrespective of whether it occurs naturally

[0057] In addition, one of ordinary skill in the art will further appreciate that changes can be introduced by mutation into the nucleotide sequences thereby leading to changes in the amino acid sequence of the encoded proteins without altering the biological activity of the proteins. 14

Thus, for example, an isolated polynucleotide molecule encoding a CYP72A polypeptide having an amino acid sequence that differs from that of SEQ ID NO: 4 can be created by introducing one or more nucleotide substitutions, additions, or deletions into the corresponding nucleotide sequence, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site- directed mutagenesis and PCR-mediated mutagenesis. Such variant nucleotide sequences are also encompassed by the present disclosure. For example, preferably, conservative amino acid substitutions may be made at one or more predicted preferably nonessential amino acid residues. A “nonessential” amino acid residue is a residue that can be altered from the wild-type sequence of a protein without altering the biological activity, whereas an “essential” amino acid residue is required for biological activity.

[0058] A deletion refers to removal of one or more amino acids from a protein. An insertion refers to one or more amino acid residues being introduced into a predetermined site in a protein. Insertions may comprise N-terminal and/or C-terminal fusions as well as intra-sequence insertions of single or multiple amino acids. Generally, insertions within the amino acid sequence will be smaller than N- or C-terminal fusions, of the order of about 1 to 10 residues. Examples of N- or C-terminal fusion proteins or peptides include the binding domain or activation domain of a transcriptional activator as used in the yeast two-hybrid system, phage coat proteins, (histidine)-6-tag, glutathione S -transferase-tag, protein A, maltose-binding protein, dihydrofolate reductase, Tag· 100 epitope, c-myc epitope, FLAG^®-epitope, lacZ, CMP (calmodulin-binding peptide), HA epitope, protein C epitope and VSV epitope.

[0059] A substitution refers to replacement of amino acids of the protein with other amino acids having similar properties (such as similar hydrophobicity, hydrophilicity, antigenicity, propensity to form or break a-helical structures or b-sheet structures). Amino acid substitutions are typically of single residues but may be clustered depending upon functional constraints placed upon the polypeptide and may range from 1 to 10 amino acids; insertions will usually be of the order of about 1 to 10 amino acid residues. A conservative amino acid substitution is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, 15 phenylalanine, tryptophan, histidine). Such substitutions would not be made for conserved amino acid residues, or for amino acid residues residing within a conserved motif. Conservative substitution tables are well known in the art (see for example Creighton (1984) Proteins. W.H. Freeman and Company (Eds).

[0060] Amino acid substitutions, deletions and/or insertions may readily be made using peptide synthetic techniques well known in the art, such as solid phase peptide synthesis and the like, or by recombinant DNA manipulation. Methods for the manipulation of DNA sequences to produce substitution, insertion or deletion variants of a protein are well known in the art. For example, techniques for making substitution mutations at predetermined sites in DNA are well known to those skilled in the art and include Ml 3 mutagenesis, T7-Gen in vitro mutagenesis (USB, Cleveland, Ohio), QuickChange Site Directed mutagenesis (Stratagene, San Diego, Calif.), PCR-mediated site-directed mutagenesis or other site-directed mutagenesis protocols. [0061] In certain embodiments, the polypeptides include at least one amino acid substitution, insertion, or deletion so that they do not recite a naturally occurring amino acid sequence.

[0062] Several embodiments relate to increasing expression of a CYP72A gene in a plant. The term “increased expression” or “overexpression” as used herein means any form of expression that is additional to the original wild-type expression level. The original wild-type expression level might also be zero (absence of expression). Methods for increasing expression of genes or gene products are well documented in the art and include, for example, overexpression driven by appropriate promoters, the use of transcription enhancers or translation enhancers. Isolated nucleic acids which serve as promoter or enhancer elements may be introduced in an appropriate position (typically upstream) of a non-heterologous form of a polynucleotide so as to upregulate expression of a nucleic acid encoding the protein of interest. For example, endogenous promoters may be altered in vivo by mutation, deletion, and/or substitution (see, Kmiec, U.S.

Pat. No. 5,565,350; Zarling et al., W09322443), or isolated promoters may be introduced into a plant cell in the proper orientation and distance from a CYP72A gene so as to control the expression of the gene.

[0063] Targeted modification of plant genomes through the use of genome editing methods can be used to increase expression of a CYP72A gene through modification of plant genomic DNA. Genome editing methods can enable targeted insertion of one or more nucleic acids of interest into a plant genome. Genome editing uses engineered nucleases such as RNA guided DNA endonucleases or nucleases composed of sequence specific DNA binding domains fused to a non-specific DNA cleavage module. These engineered nucleases enable efficient and precise genetic modifications by inducing targeted DNA double stranded breaks that stimulate the cell's 16 endogenous cellular DNA repair mechanisms to repair the induced break. Such mechanisms include, for example, error prone non-homologous end joining (NHEJ) and homology directed repair (HDR).

[0064] In the presence of donor plasmid with extended homology arms, HDR can lead to the introduction of single or multiple transgenes to correct or replace existing genes. In the absence of donor plasmid, NHEJ-mediated repair yields small insertion or deletion mutations of the target that cause gene disruption. Engineered nucleases useful in the methods of the present disclosure include zinc finger nucleases (ZFNs), transcription activator-like (TAL) effector nucleases (TALEN) and CRISPR/Cas9 type nucleases.

[0065] A zinc finger nuclease (ZFN) comprises a DNA-binding domain and a DNA-cleavage domain, wherein the DNA binding domain is comprised of at least one zinc finger and is operatively linked to a DNA-cleavage domain. The zinc finger DNA-binding domain is at the N- terminus of the protein and the DNA-cleavage domain is located at the C-terminus of said protein.

[0066] A ZFN must have at least one zinc finger. In a preferred embodiment, a ZFN would have at least three zinc fingers in order to have sufficient specificity to be useful for targeted genetic recombination in a host cell or organism. Typically, a ZFN having more than three zinc fingers would have progressively greater specificity with each additional zinc finger.

[0067] The zinc finger domain can be derived from any class or type of zinc finger. In a particular embodiment, the zinc finger domain comprises the Cis2His2 type of zinc finger that is very generally represented, for example, by the zinc finger transcription factors TFIIIA or Spl.

In a preferred embodiment, the zinc finger domain comprises three Cis2His2 type zinc fingers. The DNA recognition and/or the binding specificity of a ZFN can be altered in order to accomplish targeted genetic recombination at any chosen site in cellular DNA. Such modification can be accomplished using known molecular biology and/or chemical synthesis techniques (see, for example, Bibikova et ak, 2002).

[0068] The ZFN DNA-cleavage domain is derived from a class of non-specific DNA cleavage domains, for example the DNA-cleavage domain of a Type II restriction enzyme such as Fold (Kim et ak, 1996). Other useful endonucleases may include, for example, Hhal, Hindlll, Nod, BbvCI, EcoRI, Bgll, and AlwI.

[0069] A transcription activator-like (TAL) effector nuclease (TALEN) comprises a TAL effector DNA binding domain and an endonuclease domain. TAL effectors are proteins of plant pathogenic bacteria that are injected by the pathogen into the plant cell, where they travel to the nucleus and function as transcription factors to turn on specific plant genes. The primary amino 17 acid sequence of a TAL effector dictates the nucleotide sequence to which it binds. Thus, target sites can be predicted for TAL effectors, and TAL effectors can be engineered and generated for the purpose of binding to particular nucleotide sequences.

[0070] Fused to the TAL effector-encoding nucleic acid sequences are sequences encoding a nuclease or a portion of a nuclease, typically a nonspecific cleavage domain from a type II restriction endonuclease such as Fokl (Kim et al., 1996). Other useful endonucleases may include, for example, Hhal, Hindlll, Nod, BbvCI, EcoRI, Bgll, and Ahvl. The fact that some endonucleases (e.g., Fokl) only function as dimers can be capitalized upon to enhance the target specificity of the TAL effector. For example, in some cases each Fokl monomer can be fused to a TAL effector sequence that recognizes a different DNA target sequence, and only when the two recognition sites are in close proximity do the inactive monomers come together to create a functional enzyme. By requiring DNA binding to activate the nuclease, a highly site-specific restriction enzyme can be created.

[0071] A sequence-specific TALEN can recognize a particular sequence within a preselected target nucleotide sequence present in a cell. Thus, in some embodiments, a target nucleotide sequence can be scanned for nuclease recognition sites, and a particular nuclease can be selected based on the target sequence. In other cases, a TALEN can be engineered to target a particular cellular sequence.

[0072] Distinct from the site-specific nucleases described above, the clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas system provides an alternative to ZFNs and TALENs for inducing targeted genetic alterations, via RNA-guided DNA cleavage.

[0073] CRISPR systems rely on CRISPR RNA (crRNA) and transactivating chimeric RNA (tracrRNA) for sequence-specific cleavage of DNA. Three types of CRISPR/Cas systems exist: in type II systems, Cas9 serves as an RNA-guided DNA endonuclease that cleaves DNA upon crRNA-tracrRNA target recognition. CRISPR RNA base pairs with tracrRNA to form a two- RNA structure that guides the Cas9 endonuclease to complementary DNA sites for cleavage. [0074] The CRISPR system can be portable to plant cells by co-delivery of plasmids expressing the Cas endonuclease and the necessary crRNA components. The Cas endonuclease may be converted into a nickase to provide additional control over the mechanism of DNA repair (Cong et al., 2013).

[0075] CRISPRs are typically short partially palindromic sequences of 24-40 bp containing inner and terminal inverted repeats of up to 11 bp. Although isolated elements have been detected, they are generally arranged in clusters (up to about 20 or more per genome) of repeated units spaced by unique intervening 20-58 bp sequences. CRISPRs are generally homogenous 18 within a given genome with most of them being identical. However, there are examples of heterogeneity in, for example, the Archaea (Mojica et al., 2000).

Expression Constructs

[0076] CYP71 A polynucleotides as described herein can be provided in an expression construct. Expression constructs generally include regulatory elements that are functional in the intended host cell in which the expression construct is to be expressed. Thus, a person of ordinary skill in the art can select regulatory elements for use in bacterial host cells, yeast host cells, plant host cells, insect host cells, mammalian host cells, and human host cells. Regulatory elements include promoters, transcription termination sequences, translation termination sequences, enhancers, and polyadenylation elements. As used herein, the term “expression construct” refers to a combination of nucleic acid sequences that provides for transcription of an operably linked nucleic acid sequence.

[0077] An expression construct can comprise a promoter sequence operably linked to a polynucleotide sequence encoding a CYP72A polypeptide as described herein. Promoters can be incorporated into a polynucleotide using standard techniques known in the art. Multiple copies of promoters or multiple promoters can be used in an expression construct as described herein.

In some embodiments, a promoter can be positioned about the same distance from the transcription start site in the expression construct as it is from the transcription start site in its natural genetic environment. Some variation in this distance is permitted without substantial decrease in promoter activity. A transcription start site is typically included in the expression construct.

[0078] The choice of promoter will vary depending on the temporal and spatial requirements for expression, and also depending on the host cell to be transformed. Thus, for example, expression of the nucleotide sequences can be in any plant and/or plant part, (e.g., in leaves, in stems, in inflorescences, in roots, seeds and/or seedlings, and the like). In many cases, however, expression in multiple tissues is desirable. Although many promoters from dicotyledons have been shown to be operational in monocotyledons and vice versa, a dicotyledonous promoter may be selected for expression in dicotyledons, and a monocotyledonous promoter for expression in monocotyledons. However, there is no restriction to the provenance of selected promoters; it is sufficient that they are operational in driving the expression of the nucleotide sequences in the desired cell.

[0079] If the expression construct is to be provided in or introduced into a plant cell, then plant viral promoters, such as, for example, a cauliflower mosaic virus (CaMV) 35S (including the 19 enhanced CaMV 35S promoter (see, for example U.S. Pat. No. 5,106,739)) or a CaMV 19S promoter or a cassava vein mosaic can be used. Other promoters that can be used for expression constructs in plants include, for example, zein promoters including maize zein promoters, prolifera promoter, Ap3 promoter, heat shock promoters, T-DNA G- or 2'-promoter of A. tumefaciens, polygalacturonase promoter, chalcone synthase A (CHS-A) promoter from petunia, tobacco PR-la promoter, ubiquitin promoter, actin promoter, alcA gene promoter, pin2 promoter (Xu et al., 1993), maize Wipl promoter, maize trpA gene promoter (U.S. Pat. No. 5,625,136), maize CDPKgene promoter, and RUBISCO SSU promoter (U.S. Pat. No. 5,034,322) can also be used. Constitutive promoters (such as the CaMV, ubiquitin, actin, or NOS promoter), developmentally-regulated promoters, and inducible promoters (such as those promoters than can be induced by heat, light, hormones, or chemicals) are also contemplated for use with polynucleotide expression constructs described herein. These various types of promoters are known in the art.

[0080] Examples of constitutive promoters include, but are not limited to, cestrum virus promoter (cmp) (U.S. Pat. No. 7,166,770), the rice actin 1 promoter (Wang et al. (1992) Mol. Cell. Biol. 12:3399-3406; as well as U.S. Pat. No. 5,641,876), CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812), CaMV 19S promoter (Lawton et al. (1987) Plan! Mol. Biol. 9:315- 324), nos promoter (Ebert et al. (1987) Proc. Natl. Acad. Sci USA 84:5745-5749), Adh promoter (Walker et al. (1987) Proc. Natl. Acad. Sci. USA 84:6624-6629), sucrose synthase promoter (Yang & Russell (1990) Proc. Natl. Acad. Sci. USA 87:4144-4148), and the ubiquitin promoter. The constitutive promoter derived from ubiquitin accumulates in many cell types. Ubiquitin promoters have been cloned from several plant species for use in transgenic plants, for example, sunflower (Binet et al., 1991. Plant Science 79: 87-94), maize (Christensen et al., 1989. Plant Molec. Biol. 12: 619-632), and arabidopsis (Norris et al. 1993. Plant Molec. Biol. 21:895-906). The maize ubiquitin promoter (UbiP) has been developed in transgenic monocot systems and its sequence and vectors constructed for monocot transformation are disclosed in the patent publication EP 0342 926. The ubiquitin promoter is suitable for the expression of the nucleotide sequences in transgenic plants, especially monocotyledons. Further, the promoter expression cassettes described by McElroy et al. {Mol. Gen. Genet. 231: 150-160 (1991)) can be easily modified for the expression of the nucleotide sequences and are particularly suitable for use in monocotyledonous hosts.

[0081] In some embodiments, tissue specific/tissue preferred promoters can be used. Tissue specific or preferred expression patterns include, but are not limited to, green tissue specific or preferred, root specific or preferred, stem specific or preferred, and flower specific or preferred. 20

Promoters suitable for expression in green tissue include many that regulate genes involved in photosynthesis and many of these have been cloned from both monocotyledons and dicotyledons. Non-limiting examples of tissue-specific promoters include those associated with genes encoding the seed storage proteins (such as b-conglycinin, cruciferin, napin and phaseolin), zein or oil body proteins (such as oleosin), or proteins involved in fatty acid biosynthesis (including acyl carrier protein, stearoyl-ACP desaturase and fatty acid desaturases (fad 2-1)), and other nucleic acids expressed during embryo development (such as Bce4, see, e.g., Kridl et al. (1991) Seed Sci. Res. 1:209-219; as well as EP Patent No. 255378). Tissue- specific or tissue-preferential promoters useful for the expression of the nucleotide sequences in plants, particularly maize, include but are not limited to those that direct expression in root, pith, leaf or pollen. Such promoters are disclosed, for example, in WO 93/07278, herein incorporated by reference in its entirety. Other non-limiting examples of tissue specific or tissue preferred promoters include the cotton rubisco promoter disclosed in U.S. Pat. No. 6,040,504; the rice sucrose synthase promoter disclosed in U.S. Pat. No. 5,604,121; the root specific promoter described by de Framond (FEBS 290:103-106 (1991); EP 0452269 to Ciba-Geigy); the stem specific promoter described in U.S. Pat. No. 5,625,136 (to Ciba-Geigy) and which drives expression of the maize trpA gene; and the cestrum yellow leaf curling virus promoter disclosed in WO 01/73087, all incorporated by reference.

[0082] Additional examples of tissue-specific/tissue preferred promoters include, but are not limited to, the root-specific promoters RCc3 (Jeong et al. Plant Physiol. 153:185-197 (2010)) and RB7 (U.S. Pat. No. 5,459,252), the lectin promoter (Lindstrom et al. (1990) Der.

Genet. 11:160-167; and Vodkin (1983) Prog. Clin. Biol. Res. 138:87-98), com alcohol dehydrogenase 1 promoter (Dennis et al. (1984) Nucleic Acids Res. 12:3983-4000), S-adenosyl- L-methionine synthetase (SAMS) (Vander Mijnsbrugge et al. (1996) Plant and Cell Physiology, 37(8): 1108-1115), com light harvesting complex promoter (Bansal et al.

(1992) Proc. Natl. Acad. Sci. USA 89:3654-3658), com heat shock protein promoter (O'Dell et al. (1985) EMBOJ. 5:451-458; and Rochester et al. (1986) EMBOJ. 5:451-458), pea small subunit RuBP carboxylase promoter (Cashmore, “Nuclear genes encoding the small subunit of ribulose-l,5-bisphosphate carboxylase” pp. 29-39 In: Genetic Engineering of Plants (Hollaender ed., Plenum Press 1983; and Poulsen et al. (1986 )Mol. Gen. Genet. 205:193-200), Ti plasmid mannopine synthase promoter (Langridge et al. (1989) Proc. Natl. Acad. Sci. USA 86:3219- 3223), Ti plasmid nopaline synthase promoter (Langridge et al. (1989), supra), petunia chalcone isomerase promoter (van Tunen et al. (1988) EMBO J. 7:1257-1263), bean glycine rich protein 1 promoter (Keller et al. (1989) Genes Dev. 3:1639-1646), truncated CaMV 35S promoter (O'Dell 21 et al. (1985) Nature 313:810-812), potato patatin promoter (Wenzler et al. (1989) Plant Mol. Biol. 13:347-354), root cell promoter (Yamamoto et al. (1990) Nucleic Acids Res. 18:7449), maize zein promoter (Kriz et al. (1987 )Mol. Gen. Genet. 207:90-98; Langridge et al.

(1983) Cell 34:1015-1022; Reina et al. (1990) Nucleic Acids Res. 18:6425; Reina et al.

(1990) Nucleic Acids Res. 18:7449; and Wandelt et al. (1989) Nucleic Acids Res. 17:2354), globulin-1 promoter (Belanger et al. (1991) Genetics 129:863-872), a-tubulin cab promoter (Sullivan et al. (1989 )Mol. Gen. Genet. 215:431-440), PEPCase promoter (Hudspeth & Grula (1989) Plant Mol. Biol. 12:579-589), R gene complex-associated promoters (Chandler et al. (1989) Plant Cell 1:1175-1183), and chalcone synthase promoters (Franken et al. (1991) EMBO J. 10:2605-2612). In some particular embodiments, the nucleotide sequences are operatively associated with a root-preferred promoter.

[0083] Promoters useful for seed-specific expression include the pea vicilin promoter (Czako et al. (1992) o/. Gen. Genet. 235:33-40; as well as the seed-specific promoters disclosed in U.S. Pat. No. 5,625,136. Useful promoters for expression in mature leaves are those that are switched on at the onset of senescence, such as the SAG promoter from Arabidopsis (Gan et al.

(1995) Science 270:1986-1988).

[0084] In addition, promoters functional in plastids can be used. Non-limiting examples of such promoters include the bacteriophage T3 gene 95' UTR and other promoters disclosed in U.S. Pat. No. 7,579,516. Other promoters include but are not limited to the S-E9 small subunit RuBP carboxylase promoter and the Kunitz trypsin inhibitor gene promoter (Kti3).

[0085] In some embodiments, inducible promoters can be used. Thus, for example, chemical- regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. Regulation of the expression of nucleotide sequences via promoters that are chemically regulated enables the polypeptides to be synthesized only when the crop plants are treated with the inducing chemicals. Depending upon the objective, the promoter may be a chemical-inducible promoter, where application of a chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression.

[0086] Chemical inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR- la promoter, which is activated by salicylic acid ( e.g ., the PRla system), steroid steroid-responsive promoters (see, e.g., the glucocorticoid- inducible promoter in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88, 10421-10425 and 22

McNellis et al. (1998) Plant J. 14, 247-257) and tetracycline-inducible and tetracycline- repressible promoters (see, e.g., Gatz et al. (1991)Mo/. Gen. Genet. 227, 229-237, and U.S. Pat. Nos. 5,814,618 and 5,789,156, Lac repressor system promoters, copper-inducible system promoters, salicylate-inducible system promoters (e.g., the PRla system), glucocorticoid- inducible promoters (Aoyama et al. (1997) Plant J. 11:605-612), and ecdysone-inducible system promoters.

[0087] Other non-limiting examples of inducible promoters include ABA- and turgor-inducible promoters, the auxin-binding protein gene promoter (Schwob et al. (1993) Plant J. 4:423-432), the UDP glucose flavonoid glycosyl-transferase promoter (Ralston et al.

(1988) Genetics 119:185-197), the MPI proteinase inhibitor promoter (Cordero et al.

(1994) Plant J. 6:141-150), and the glyceraldehyde-3-phosphate dehydrogenase promoter (Kohler et al. (1995) Plant Mol. Biol. 29: 1293-1298; Martinez et al. (1989) J. Mol.

Biol. 208:551-565; and Quigley et al. (1989) J. Mol. Evol. 29:412-421). Also included are the benzene sulphonamide-inducible (U.S. Pat. No. 5,364,780) and alcohol-inducible (Int'l Patent Application Publication Nos. WO 97/06269 and WO 97/06268) systems and glutathione 5- transferase promoters. Likewise, one can use any of the inducible promoters described in Gatz (1996) Current Opinion Biotechnol. 7:168-172 and Gatz (1997) Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:89-108. Other chemically inducible promoters useful for directing the expression of the nucleotide sequences in plants are disclosed in U.S. Pat. No. 5,614,395 herein incorporated by reference in its entirety. Chemical induction of gene expression is also detailed in the published application EP 0 332 104 (to Ciba-Geigy) and U.S. Pat. No. 5,614,395. In some embodiments, a promoter for chemical induction can be the tobacco PR- la promoter.

[0088] In further embodiments, the nucleotide sequences can be operatively associated with a promoter that is wound inducible or inducible by pest or pathogen infection (e.g., an insect pest). Numerous promoters have been described which are expressed at wound sites and/or at the sites of pest attack (e.g., insect feeding) or phytopathogen infection. Ideally, such a promoter should be active only locally at or adjacent to the sites of attack, and in this way expression of the nucleotide sequences will be focused in the cells that are being invaded. Such promoters include, but are not limited to, those described by Stanford et al., Mol. Gen. Genet. 215:200-208 (1989), Xu et al. Plant Molec. Biol. 22:573-588 (1993), Logemann et al. Plant Cell 1:151-158 (1989), Rohrmeier and Lehle, Plant Molec. Biol. 22:783-792 (1993), Firek et al. Plant Molec.

Biol. 22:129-142 (1993), Warner et al. Plant J. 3:191-201 (1993), U.S. Pat. Nos. 5,750,386, 5,955,646, 6,262,344, 6,395,963, 6,703,541, 7,078,589, 7,196,247, 7,223,901, and U.S. Patent Application Publication 2010043102. 23

[0089] A number of non-translated leader sequences derived from viruses are known to enhance gene expression. Specifically, leader sequences from Tobacco Mosaic Virus (TMV, the “co sequence”), Maize Chlorotic Mottle Virus (MCMV) and Alfalfa Mosaic Virus (AMV) have been shown to be effective in enhancing expression (Gallie et al. (1987) Nucleic Acids Res. 15:8693-8711; and Skuzeski et al. (1990) Plant Mol. Biol. 15:65-79). Other leader sequences known in the art include, but are not limited to, picomavirus leaders such as an encephalomyocarditis (EMCV) 5' noncoding region leader (Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130); potyvirus leaders such as a Tobacco Etch Virus (TEV) leader (Allison et al. (1986) Virology 154:9-20); Maize Dwarf Mosaic Virus (MDMV) leader (Allison et al. (1986), supra); human immunoglobulin heavy -chain binding protein (BiP) leader (Macejak & Samow (1991) Nature 353:90-94); untranslated leader from the coat protein mRNA of AMV (AMV RNA 4; Jobling & Gehrke (1987) Nature 325:622-625); tobacco mosaic TMV leader (Gallie et al. (1989) Molecular Biology of RNA 237-256); and MCMV leader (Lommel et al. (1991) Virology 81:382-385). See also, Della-Cioppa et al. (1987) Plant Physiol. 84:965-968. [0090] Expression constructs may optionally contain a transcription termination sequence, a translation termination sequence, a sequence encoding a signal peptide, and/or enhancer elements. Transcription termination regions can typically be obtained from the 3' untranslated region of a eukaryotic or viral gene sequence. Transcription termination sequences can be positioned downstream of a coding sequence to provide for efficient termination. A signal peptide sequence is a short amino acid sequence typically present at the amino terminus of a protein that is responsible for the relocation of an operably linked mature polypeptide to a wide range of post-translational cellular destinations, ranging from a specific organelle compartment to sites of protein action and the extracellular environment. Targeting gene products to an intended cellular and/or extracellular destination through the use of an operably linked signal peptide sequence is contemplated for use with the polypeptides described herein. Classical enhancers are cis-acting elements that increase gene transcription and can also be included in the expression construct. Classical enhancer elements are known in the art, and include, but are not limited to, the CaMV 35 S enhancer element, cytomegalovirus (CMV) early promoter enhancer element, and the SV40 enhancer element. Intron-mediated enhancer elements that enhance gene expression are also known in the art. These elements must be present within the transcribed region and are orientation dependent. Examples include the maize shrunken- 1 enhancer element (Clancy and Hannah, 2002).

[0091] An expression construct can include a nucleotide sequence for a selectable marker, which can be used to select a transformed plant, plant part and/or plant cell. As used herein, 24

“selectable marker” means a nucleotide sequence that when expressed imparts a distinct phenotype to the plant, plant part and/or plant cell expressing the marker and thus allows such transformed plants, plant parts and/or plant cells to be distinguished from those that do not have the marker. Such a nucleotide sequence may encode either a selectable or screenable marker, depending on whether the marker confers a trait that can be selected for by chemical means, such as by using a selective agent (e.g., an antibiotic, herbicide, or the like), or on whether the marker is simply a trait that one can identify through observation or testing, such as by screening. Of course, many examples of suitable selectable markers are known in the art and can be used in the expression cassettes described herein.

[0092] Examples of selectable markers include, but are not limited to, a nucleotide sequence encoding neo or nptll, which confers resistance to kanamycin, G418, and the like (Potrykus et al. (1985) o/. Gen. Genet. 199:183-188); anucleotide sequence encoding bar, which confers resistance to phosphinothricin; a nucleotide sequence encoding an altered 5- enolpyruvylshikimate-3-phosphate (EPSP) synthase, which confers resistance to glyphosate (Hinchee et al. (1988) Biotech. 6:915-922); anucleotide sequence encoding anitrilase such as bxn from Klebsiella ozaenae that confers resistance to bromoxynil (Stalker et al.

(1988) Science 242:419-423); anucleotide sequence encoding an altered acetolactate synthase (ALS) that confers resistance to imidazolinone, sulfonylurea or other ALS -inhibiting chemicals (EP Patent Application No. 154204); a nucleotide sequence encoding a methotrexate-resistant dihydrofolate reductase (DHFR) (Thillet et al. (1988) J. Biol. Chem. 263:12500-12508); a nucleotide sequence encoding a dalapon dehalogenase that confers resistance to dalapon; a nucleotide sequence encoding a mannose-6-phosphate isomerase (also referred to as phosphomannose isomerase (PMI)) that confers an ability to metabolize mannose (U.S. Pat.

Nos. 5,767,378 and 5,994,629); anucleotide sequence encoding an altered anthranilate synthase that confers resistance to 5-methyl tryptophan; and/or a nucleotide sequence encoding hph that confers resistance to hygromycin. One of skill in the art is capable of choosing a suitable selectable marker for use in an expression cassette.

[0093] Additional selectable markers include, but are not limited to, a nucleotide sequence encoding b-glucuronidase or uidA (GUS) that encodes an enzyme for which various chromogenic substrates are known; an R-locus nucleotide sequence that encodes a product that regulates the production of anthocyanin pigments (red color) in plant tissues (Dellaporta et al., “Molecular cloning of the maize R-nj allele by transposon-tagging with Ac,” pp. 263-282 In: Chromosome Structure and Function: Impact of New Concepts, 18th Stadler Genetics Symposium (Gustafson & Appels eds., Plenum Press 1988)); anucleotide sequence encoding b- 25 lactamase, an enzyme for which various chromogenic substrates are known (e.g., PAD AC, a chromogenic cephalosporin) (Sutcliffe (1978) Proc. Natl. Acad. Sci. USA 75:3737-3741); a nucleotide sequence encoding xylE that encodes a catechol dioxygenase (Zukowsky et al.

(1983) Proc. Natl. Acad. Sci. USA 80:1101-1105); a nucleotide sequence encoding tyrosinase, an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone, which in turn condenses to form melanin (Katz et al. (1983) J. Gen. Microbiol. 129:2703-2714); a nucleotide sequence encoding b-galactosidase, an enzyme for which there are chromogenic substrates; a nucleotide sequence encoding luciferase (lux) that allows for bioluminescence detection (Ow et al.

(1986) Science 234:856-859); a nucleotide sequence encoding aequorin, which may be employed in calcium-sensitive bioluminescence detection (Prasher et al. (1985) Biochem. Biophys. Res. Comm. 126:1259-1268); or a nucleotide sequence encoding green fluorescent protein (Niedz et al. (1995) Plant Cell Reports 14:403-406). One of skill in the art is capable of choosing a suitable selectable marker for use in an expression cassette.

[0094] Optionally the gene encoding the CYP72A polypeptide is codon optimized to remove features inimical to expression and codon usage is optimized for expression in the particular crop (see, for example, U.S. Pat. No. 6,051,760; EP 0359472; EP 80385962; EP 0431829; and Perlak et al. (1991) PNAS USA 88:3324-3328; all of which are herein incorporated by reference).

Transformation Methods

[0095] Suitable methods for transformation of host plant cells include virtually any method by which DNA or RNA can be introduced into a cell (for example, where a recombinant DNA construct is stably integrated into a plant chromosome or where a recombinant DNA construct or an RNA is transiently provided to a plant cell) and are well known in the art. Two effective methods for cell transformation are Agrobaclerium-mediaied transformation and microprojectile bombardment-mediated transformation. Microprojectile bombardment methods are illustrated, for example, in U.S. Pat. Nos. 5,550,318; 5,538,880; 6,160,208; and 6,399,861. Agrobacterium- mediated transformation methods are described, for example in U.S. Pat. No. 5,591,616, which is incorporated herein by reference in its entirety. Transformation of plant material is practiced in tissue culture on nutrient media, for example a mixture of nutrients that allow cells to grow in vitro. Recipient cell targets include, but are not limited to, meristem cells, shoot tips, hypocotyls, calli, immature or mature embryos, and gametic cells such as microspores and pollen. Callus can be initiated from tissue sources including, but not limited to, immature or mature embryos, 26 hypocotyls, seedling apical meristems, microspores and the like. Cells containing a transgenic nucleus are grown into transgenic plants.

[0096] In transformation, DNA is typically introduced into only a small percentage of target plant cells in any one transformation experiment. Marker genes are used to provide an efficient system for identification of those cells that are stably transformed by receiving and integrating a recombinant DNA molecule into their genomes. Preferred marker genes provide selective markers which confer resistance to a selective agent, such as an antibiotic or an herbicide. Potentially transformed cells are exposed to the selective agent. In the population of surviving cells are those cells where, generally, the resistance-conferring gene is integrated and expressed at sufficient levels to permit cell survival. Cells can be tested further to confirm stable integration of the exogenous DNA. Commonly used selective marker genes include those conferring resistance to antibiotics such as kanamycin and paromomycin (nptll), hygromycin B (aph IV), spectinomycin (aadA) and gentamycin (aac3 and aacC4) or resistance to herbicides such as glufosinate (bar or pat), dicamba (DMO) and glyphosate (aroA or EPSPS). Examples of such selectable markers are illustrated in U.S. Pat. Nos. 5,550,318; 5,633,435; 5,780,708 and 6,118,047. Markers which provide an ability to visually screen transformants can also be employed, for example, a gene expressing a colored or fluorescent protein such as a luciferase or green fluorescent protein (GFP) or a gene expressing a beta-glucuronidase or uidA gene (GUS) for which various chromogenic substrates are known.

[0097] Transformation of a cell may be stable or transient. Thus, in some embodiments, a plant cell is stably transformed with a nucleic acid molecule. In other embodiments, a plant is transiently transformed with a nucleic acid molecule. “Transient transformation” in the context of a polynucleotide means that a polynucleotide is introduced into the cell and does not integrate into the genome of the cell. By “stably introducing” or “stably introduced” in the context of a polynucleotide introduced into a cell is intended the introduced polynucleotide is stably incorporated into the genome of the cell, and thus the cell is stably transformed with the polynucleotide.

[0098] “Stable transformation” or “stably transformed” as used herein means that a nucleic acid is introduced into a cell and integrates into the genome of the cell. As such, the integrated nucleic acid is capable of being inherited by the progeny thereof, more particularly, by the progeny of multiple successive generations. “Genome” as used herein also includes the nuclear and the plastid genome, and therefore includes integration of the nucleic acid into, for example, the chloroplast genome. Stable transformation as used herein can also refer to a transgene that is maintained extrachromasomally, for example, as a minichromosome. 27

[0099] Transient transformation may be detected by, for example, an enzyme-linked immunosorbent assay (ELISA) or Western blot, which can detect the presence of a peptide or polypeptide encoded by one or more transgene introduced into an organism. Stable transformation of a cell can be detected by, for example, a Southern blot hybridization assay of genomic DNA of the cell with nucleic acid sequences which specifically hybridize with a nucleotide sequence of a transgene introduced into an organism (e.g., a plant). Stable transformation of a cell can be detected by, for example, a Northern blot hybridization assay of RNA of the cell with nucleic acid sequences which specifically hybridize with a nucleotide sequence of a transgene introduced into a plant or other organism. Stable transformation of a cell can also be detected by, e.g., a polymerase chain reaction (PCR) or other amplification reactions as are well known in the art, employing specific primer sequences that hybridize with target sequence(s) of a transgene, resulting in amplification of the transgene sequence, which can be detected according to standard methods Transformation can also be detected by direct sequencing and/or hybridization protocols well known in the art.

[0100] A nucleic acid (e.g., SEQ ID NOs: 1-3, or a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 4) can be introduced into a cell by any method known to those of skill in the art.

[0101] In some embodiments, transformation of a cell comprises nuclear transformation. In other embodiments, transformation of a cell comprises plastid transformation (e.g., chloroplast transformation).

[0102] Procedures for transforming plants are well known and routine in the art and are described throughout the literature. Non-limiting examples of methods for transformation of plants include transformation via bacterial-mediated nucleic acid delivery (e.g., via Agrobacteria), viral-mediated nucleic acid delivery, silicon carbide or nucleic acid whisker- mediated nucleic acid delivery, liposome mediated nucleic acid delivery, microinjection, microparticle bombardment, calcium-phosphate-mediated transformation, cyclodextrin- mediated transformation, electroporation, nanoparticle-mediated transformation, sonication, infiltration, PEG-mediated nucleic acid uptake, as well as any other electrical, chemical, physical (mechanical) and/or biological mechanism that results in the introduction of nucleic acid into the plant cell, including any combination thereof. General guides to various plant transformation methods known in the art include Miki et al. (“Procedures for Introducing Foreign DNA into Plants” in Methods in Plant Molecular Biology and Biotechnology, Glick, B. R. and Thompson, J. E., Eds. (CRC Press, Inc., Boca Raton, 1993), pages 67-88) and Rakowoczy-Trojanowska (Cell. Mol. Biol. Lett. 7:849-858 (2002)). 28

[0103] Agrobacterium- mediated transformation is a commonly used method for transforming plants, in particular, dicot plants, because of its high efficiency of transformation and because of its broad utility with many different species. Agrobacterium- mediated transformation typically involves transfer of the binary vector carrying the foreign DNA of interest to an appropriat Q Agrobacterium strain that may depend on the complement of vir genes carried by the host Agrobacterium strain either on a co-resident Ti plasmid or chromosomally (Uknes et al. (1993) Plant Cell 5: 159-169). The transfer of the recombinant binary vector to Agrobacterium can be accomplished by a triparental mating procedure using Escherichia coli carrying the recombinant binary vector, a helper E. coli strain that carries a plasmid that is able to mobilize the recombinant binary vector to the target Agrobacterium strain. Alternatively, the recombinant binary vector can be transferred to Agrobacterium by nucleic acid transformation (Hofgen & Willmitzer (1988) Nucleic Acids Res. 16:9877).

[0104] Transformation of a plant by recombinant Agrobacterium usually involves co-cultivation of the Agrobacterium with explants from the plant and follows methods well known in the art. Transformed tissue is regenerated on selection medium carrying an antibiotic or herbicide resistance marker between the binary plasmid T-DNA borders.

[0105] Another method for transforming plants, plant parts and/or plant cells involves propelling inert or biologically active particles at plant tissues and cells. See, e.g., U.S. Pat. Nos. 4,945,050; 5,036,006 and 5,100,792. Generally, this method involves propelling inert or biologically active particles at the plant cells under conditions effective to penetrate the outer surface of the cell and afford incorporation within the interior thereof. When inert particles are utilized, the vector can be introduced into the cell by coating the particles with the vector containing the nucleic acid of interest. Alternatively, a cell or cells can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle. Biologically active particles (e.g., dried yeast cells, dried bacterium or a bacteriophage, each containing one or more nucleic acids sought to be introduced) also can be propelled into plant tissue.

[0106] Thus, in particular embodiments, a plant cell can be transformed by any method known in the art and as described herein and intact plants can be regenerated from these transformed cells using any of a variety of known techniques. Plant regeneration from plant cells, plant tissue culture and/or cultured protoplasts is described, for example, in Evans et al. (Handbook of Plant Cell Cultures, Vol. 1, MacMilan Publishing Co. New York (1983)); and Vasil I. R. (ed.) (Cell Culture and Somatic Cell Genetics of Plants, Acad. Press, Orlando, Vol. I (1984), and Vol. II (1986)). Methods of selecting for transformed transgenic plants, plant cells and/or plant tissue culture are routine in the art and can be employed in the methods provided herein. 29

[0107] Likewise, the genetic properties engineered into the transgenic seeds and plants, plant parts, and/or plant cells described above can be passed on by sexual reproduction or vegetative growth and therefore can be maintained and propagated in progeny plants. Generally, maintenance and propagation make use of known agricultural methods developed to fit specific purposes such as harvesting, sowing or tilling.

[0108] A nucleotide sequence therefore can be introduced into the plant, plant part and/or plant cell in any number of ways that are well known in the art. The methods do not depend on a particular method for introducing one or more nucleotide sequences into a plant, only that they gain access to the interior of at least one cell of the plant. Where more than one nucleotide sequence is to be introduced, they can be assembled as part of a single nucleic acid construct, or as separate nucleic acid constructs, and can be located on the same or different nucleic acid constructs. Accordingly, the nucleotide sequences can be introduced into the cell of interest in a single transformation event, in separate transformation events, or, for example, in plants, as part of a breeding protocol.

Plants with Resistance to an Insect Pest

[0109] Several embodiments relate to plant cells, plant tissues, plants, and seeds that comprise a polynucleotide encoding a CYP72A polypeptide, wherein expression of the polynucleotide confers resistance to an insect. Plants may be monocots or dicots, and may include, for example, rice, wheat, barley, oats, rye, sorghum, maize, grape, tomato, potato, lettuce, broccoli, cucumber, peanut, melon, pepper, carrot, squash, onion, soybean, alfalfa, sunflower, cotton, canola, and sugar beet plants.

[0110] Plants that are useful in the methods of the present disclosure include all plants which belong to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants including fodder or forage legumes, ornamental plants, food crops, trees or shrubs selected from the list comprising Acer spp ,Actinidia spp., Abelmoschus spp Agave sisalana, Agropyron spp ,Agrostis stolonifera, Allium spp.,Amaranthus spp.,Ammophila arenaria, Ananas comosus, Annona spp.,Apium graveolens, Arachis spp, Artocarpus spp Asparagus officinalis, Avena spp. (e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida), Averrhoa carambola, Bambusa sp., Benincasa hispida, Bertholletia excelsea, Beta vulgaris, Brassica spp. (e.g. Brassica napus, Brassica rapa ssp. [canola, oilseed rape, turnip rape]), Cadaba farinosa, Camellia sinensis, Canna indica, Cannabis sativa, Capsicum spp., Carex elata, Carica papaya, Carissa macrocarpa, Carya spp., Carthamus tinctorius, Castanea spp., Ceiba pentandra, Cichorium endivia, Cinnamomum spp., Citrullus lanatus, 30

Citrus spp., Cocos spp., Coffea spp., Colocasia esculenta, Cola spp., Corchorus sp.,

Coriandrum sativum, Corylus spp., Crataegus spp., Crocus sativus, Cucurbita spp., Cucumis spp., Cynara spp., Daucus carota, Desmodium spp., Dimocarpus longan, Dioscorea spp., Diospyros spp., Echinochloa spp., Elaeis (e.g. Elaeis guineensis, Elaeis oleifera), Eleusine coracana, Eragrostis tef, Erianthus sp., Eriobotrya japonica, Eucalyptus sp., Eugenia uniflora, Fagopyrum spp., Fagus spp., Festuca arundinacea, Ficus carica, Fortunella spp., Fragaria spp., Ginkgo biloba, Glycine spp. (e.g. Glycine max, Soja hispida or Soja max), Gossypium hirsutum, Helianthus spp. (e.g. Helianthus annuus), Hemerocallis fulva, Hibiscus spp., Hordeum spp. (e.g. Hordeum vulgar e ), Ipomoea batatas, Juglans spp., Lactuca sativa, Lathyrus spp., Lens culinaris, Linum usitatissimum, Litchi chinensis, Lotus spp., Luffa acutangula, Lupinus spp., Luzula sylvatica, Lycopersicon spp. (e.g. Lycopersicon esculentum, Lycopersicon lycopersicum, Lycopersicon pyriforme), Macrotyloma spp ,Malus spp.. Malpighia emarginata, Mammea americana, Mangifera indica, Manihot spp.,Manilkara zapota, Medicago sativa, Melilotus spp., Mentha spp.,Miscanthus sinensis, Momordica spp ,Morus nigra, Musa spp., Nicotiana spp., Olea spp., Opuntia spp., Ornithopus spp., Oryza spp. (e.g. Oryza sativa, Oryza latifolia), Panicum miliaceum, Panicum virgatum, Passiflora edulis, Pastinaca sativa, Pennisetum sp.,

Per sea spp., Petroselinum crispum, Phalaris arundinacea, Phaseolus spp., Phleum pratense, Phoenix spp., Phragmites australis, Physalis spp., Pinus spp., Pistacia vera, Pisum spp., Poa spp., Populus spp., Prosopis spp., Prunus spp., Psidium spp., Punica granatum, Pyrus communis, Quercus spp., Raphanus sativus, Rheum rhabarbarum, Ribes spp., Ricinus communis, Rubus spp., Saccharum spp., Salix sp., Sambucus spp., Secale cereale, Sesamum spp., Sinapis sp., Solanum spp. (e.g. Solanum tuberosum, Solanum integrifolium or Solanum lycopersicum ), Sorghum bicolor, Spinacia spp., Syzygium spp., Tagetes spp., Tamarindus indica, Theobroma cacao, Trifolium spp., Tripsacum dactyloides, Triticosecale rimpaui, Triticum spp. (e.g. Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum, Triticum monococcum or Triticum vulgare), Tropaeolum minus, Tropaeolum majus, Vaccinium spp., Vicia spp., Vigna spp., Viola odorata, Vitis spp., Zea mays, Zizania palustris, Ziziphus spp., amaranth, artichoke, asparagus, broccoli, Brussels sprouts, cabbage, canola, carrot, cauliflower, celery, collard greens, flax, kale, lentil, oilseed rape, okra, onion, potato, rice, soybean, strawberry, sugar beet, sugar cane, sunflower, tomato, squash, tea and algae, amongst others. In certain embodiments, the plant is a crop plant. Examples of crop plants include inter alia soybean, sunflower, canola, alfalfa, rapeseed, cotton, tomato, potato, or tobacco. 31

[0111] Certain embodiments encompass a progeny or a descendant of an insect-resistant plant as well as seeds derived from the insect-resistant plants and cells derived from the insect-resistant plants as described herein.

[0112] In some embodiments, the present disclosure provides a progeny or descendant plant derived from a plant comprising in at least some of its cells a polynucleotide operably linked to a promoter functional in a plant cell, the promoter capable of expressing a CYP72A polypeptide encoded by the polynucleotide, wherein the progeny or descendant plant comprises in at least some of its cells the recombinant polynucleotide operably linked to the promoter, the expression of the CYP72A polypeptide conferring to the progeny or descendant plant resistance to the insect.

[0113] In one embodiment, seeds of the present disclosure preferably comprise the insect resistance characteristics of the plant. In other embodiments, a seed is capable of germination into a plant comprising in at least some of its cells a polynucleotide operably linked to a promoter functional in a plant cell, the promoter capable of expressing a CYP72A polypeptide encoded by the polynucleotide, the expression of the CYP72A polypeptide conferring to the progeny or descendant plant resistance to the insects.

[0114] In some embodiments, plant cells of the present disclosure are capable of regenerating a plant or plant part. In other embodiments, plant cells are not capable of regenerating a plant or plant part. Examples of cells not capable of regenerating a plant include, but are not limited to, endosperm, seed coat (testa and pericarp), and root cap.

[0115] In another embodiment, the disclosure refers to a plant cell transformed by a nucleic acid encoding a CYP72A polypeptide as described herein, wherein expression of the nucleic acid in the plant cell results in increased resistance to an insect as compared to a wild type variety of the plant cell.

[0116] Several embodiments provide a commodity plant product prepared from the insect- resistant plants. In some embodiments, examples of plant products include, without limitation, grain, oil, and meal. In one embodiment, a commodity plant product is plant grain (e.g., grain suitable for use as feed or for processing), plant oil (e.g., oil suitable for use as food or biodiesel), or plant meal (e.g., meal suitable for use as feed). A preferred commodity plant product is fodder, seed meal, oil, or seed-treatment-coated seeds. In certain embodiments, the meal and/or oil comprise the CYP72A polynucleotide or CYP72A protein.

[0117] In certain embodiments, a commodity plant product prepared from a plant or plant part is provided, wherein the plant or plant part comprises in at least some of its cells a polynucleotide operably linked to a promoter functional in plant cells, the promoter capable of expressing a 32

CYP72A polypeptide encoded by the polynucleotide, the expression of the CYP72A polypeptide conferring to the plant or plant part resistance to the insect.

[0118] The product may be produced at the site where the plant has been grown, the plants and/or parts thereof may be removed from the site where the plants have been grown to produce the product. Typically, the plant is grown, the desired harvestable parts are removed from the plant, if feasible in repeated cycles, and the product made from the harvestable parts of the plant. The step of growing the plant may be performed only once each time the method is performed, while allowing repeated times the steps of product production e.g. by repeated removal of harvestable parts of the plants of the disclosure and if necessary further processing of these parts to arrive at the product. It is also possible that the step of growing the plants is repeated and plants or harvestable parts are stored until the production of the product is then performed once for the accumulated plants or plant parts. Also, the steps of growing the plants and producing the product may be performed with an overlap in time, even simultaneously to a large extend or sequentially. Generally, the plants are grown for some time before the product is produced. [0119] The composition and methods of the present disclosure find use in producing plants with resistance to an insect pest. Insect pests include insects selected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthoptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularly Coleoptera and Lepidoptera. Insect pests of the disclosure for the major crops include, but are not limited to:

[0120] Maize: Ostrinia nubilalis, European com borer; Agrotis ipsilon, black cutworm; Helicoverpa zea, com earworm; Spodoptera frugiperda, fall army worm; Diatraea grandiosella, southwestern com borer; Elasmopalpus lignosellus, lesser cornstalk borer; Diatraea saccharalis, surgarcane borer; Diabrotica virgifera, western com rootworm; Diabrotica longicomis barberi, northern com rootworm; Diabrotica undecimpunctata howardi, southern com rootw orm; Melanolus spp., wireworms; Cyclocephala borealis, northern masked chafer (white grub); Cyclocephala immaculata, southern masked chafer (white gmb); Popillia japonica, Japanese beetle; Chaetocnema pulicaria, com flea beetle; Sphenophorus maidis, maize billbug; Rhopalosiphum maidis, com leaf aphid; Anuraphis maidiradicis, com root aphid; Blissus leucopterus leucopterus, chinch bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplus sanguinipes, migratory grasshopper; Hylemya platura, seedcom maggot; Agromyza parvicornis, com blot leafminer; Anaphothrips obscrurus, grass thrips; Solenopsis milesta, thief ant; Tetranychus urticae, two-spotted spider mite; 33

[0121] Sorghum: Chilo partellus, sorghum borer; Spodoptera frugiperda, fall army worm; Helicoverpa zea, com earworm; Elasmopalpus lignosellus, lesser cornstalk borer; Feltia subterranea, granulate cutworm; Phyllophaga crinita, white grub; Eleodes, Conoderus, and Aeolus spp., wireworms; Oulema melanopus, cereal leaf beetle; Chaetocnema pulicaria, com flea beetle; Sphenophorus maidis, maize billbug; Rhopalosiphum maidis com leaf aphid; Sipha flava, yellow sugarcane aphid; Blissus leucopterus leucopterus, chinch bug; Contarinia sorghicola, sorghum midge; Tetranychus cinnabarinus , carmine spider mite; Tetranychus urticae, twospotted spider mite;

[0122] Wheat: Pseudaletia unipunctata, army worm; Spodoptera frugiperda, fall army worm; Elasmopalpus lignosellus, lesser cornstalk borer; Agrotis orthogonia, western cutworm; Elasmopalpus lignosellus, lesser cornstalk borer; Oulema melanopus, cereal leaf beetle; Hypera punctata, clover leaf weevil; Diabrotica undecimpunctata howardi, southern com rootworm; Russian wheat aphid; Schizaphis graminum, greenbug; Macrosiphum avenae, English grain aphid; Melanoplus femurrubrum, redlegged grasshopper; Melanoplus differentialis , differential grasshopper; Melanoplus sanguinipes, migratory grasshopper; Mayetiola destructor, Hessian fly; Sitodiplosis mosellana, wheat midge; Meromyza americana, wheat stem maggot; Hylemya coarctata, wheat bulb fly; Frankliniella fusca, tobacco thrips; Cephus cinctus, wheat stem sawfly; Aceria tulipae, wheat curl mite;

[0123] Sunflower: Suleima helianthana, sunflower bud moth; Homoeosoma electellum, sunflower moth; zygogramma exclamationis, sunflower beetle; Bothyrus gibbosus, carrot beetle; Neolasioptera murtfeldtiana, sunflower seed midge;

[0124] Cotton: Heliothis virescens, cotton budworm; Helicoverpa zea, cotton bollworm; Spodoptera exigua, beet army worm; Pectinophora gossypiella, pink bollworm; Anthonomus grandis grandis, boll weevil; Aphis gossypii, cotton aphid; Pseudatomoscelis seriatus, cotton fleahopper; Trialeurodes abutilonea, banded-winged whitefly; Lygus lineolaris, tarnished plant bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplus differentialis, differential grasshopper; Thrips tabaci, onion thrips; Franklinkiella fusca, tobacco thrips; Tetranychus cinnabarinus, carmine spider mite; Tetranychus urticae, two-spotted spider mite;

[0125] Rice: Diatraea saccharalis, sugarcane borer; Spodoptera frugiperda, fall army worm; Helicoverpa zea, com earworm; Colaspis brunnea, grape colaspis; Lissorhoptrus oryzophilus, rice water weevil; Sitophilus oryzae, rice weevil; Nephotettix nigropictus, rice leafhopper; Blissus leucopterus leucopterus, chinch bug; Acrostemum hilare, green stink bug; 34

[0126] Soybean: Pseudoplusia includens, soybean looper; Anticarsia gemmatalis, velvetbean caterpillar; Plathypena scabra, green cloverworm; Ostrinia nubilalis, European com borer; Agrotis ipsilon, black cutworm; Spodoptera exigua, beet armyworm; Heliothis virescens, cotton budworm; Helicoverpa zea, cotton bollworm; Epilachna varivestis, Mexican bean beetle; Myzus persicae, green peach aphid; Empoasca fabae, potato leafhopper; Acrosternum hilare, green stink bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplus differentialis , differential grasshopper; Hylemya platura, seedcom maggot; Sericothrips variabilis, soybean thrips; Thrips tabaci, onion thrips; Tetranychus turkestani, strawberry spider mite; Tetranychus urticae, two-spotted spider mite;

[0127] Barley: Ostrinia nubilalis, European com borer; Agrotis ipsilon, black cutworm; Schizaphis graminum, greenbug; Blissus leucopterus leucopterus, chinch bug; Acrosternum hilare, green stink bug; Euschistus servus, brown stink bug; Delia platura, seedcom maggot; Mayetiola destructor, Hessian fly; Petrobia latens, brown wheat mite;

[0128] Oil Seed Rape: Brevicoryne brassicae, cabbage aphid; Phyllotreta cruciferae, Flea beetle; Mamestra configurata, Bertha armyworm; Plutella xylostella, Diamond-back moth; Delia ssp., Root maggots.

[0129] The plants of the disclosure may be used in a plant breeding program. The goal of plant breeding is to combine, in a single variety or hybrid, various desirable traits. For field crops, these traits may include, for example, resistance to diseases and insects, tolerance to heat and drought, tolerance to chilling or freezing, reduced time to crop maturity, greater yield and better agronomic quality. With mechanical harvesting of many crops, uniformity of plant characteristics such as germination and stand establishment, growth rate, maturity, and plant height is desirable. Traditional plant breeding is an important tool in developing new and improved commercial crops. This disclosure encompasses methods for producing a plant by crossing a first parent plant with a second parent plant wherein one or both of the parent plants is a plant displaying a phenotype as described herein.

[0130] Plant breeding techniques known in the art and used in a plant breeding program include, but are not limited to, recurrent selection, bulk selection, mass selection, backcrossing, pedigree breeding, open pollination breeding, restriction fragment length polymorphism enhanced selection, genetic marker enhanced selection, doubled haploids and transformation. Often combinations of these techniques are used.

[0131] The development of hybrids in a plant breeding program requires, in general, the development of homozygous inbred lines, the crossing of these lines and the evaluation of the crosses. There are many analytical methods available to evaluate the result of a cross. The oldest 35 and most traditional method of analysis is the observation of phenotypic traits. Alternatively, the genotype of a plant can be examined.

[0132] A genetic trait which has been engineered into a particular plant using transformation techniques can be moved into another line using traditional breeding techniques that are well known in the plant breeding arts. For example, a backcrossing approach is commonly used to move a transgene from a transformed plant to an elite inbred line and the resulting progeny would then comprise the transgene(s). Also, if an inbred line was used for the transformation, then the transgenic plants could be crossed to a different inbred in order to produce a transgenic hybrid plant. As used herein, "crossing" can refer to a simple X by Y cross or the process of backcrossing, depending on the context.

[0133] The development of a hybrid in a plant breeding program involves three steps: (1) the selection of plants from various germplasm pools for initial breeding crosses; (2) the selfing of the selected plants from the breeding crosses for several generations to produce a series of inbred lines, which, while different from each other, breed true and are highly homozygous and (3) crossing the selected inbred lines with different inbred lines to produce the hybrids. During the inbreeding process, the vigor of the lines decreases. Vigor is restored when two different inbred lines are crossed to produce the hybrid. An important consequence of the homozygosity and homogeneity of the inbred lines is that the hybrid created by crossing a defined pair of inbreds will always be the same. Once the inbreds that give a superior hybrid have been identified, the hybrid seed can be reproduced indefinitely as long as the homogeneity of the inbred parents is maintained.

[0134] Plants of the present disclosure may be used to produce, e.g., a single cross hybrid, a three-way hybrid or a double cross hybrid. A single cross hybrid is produced when two inbred lines are crossed to produce the FI progeny. A double cross hybrid is produced from four inbred lines crossed in pairs (A x B and C x D) and then the two FI hybrids are crossed again (A x B) times (C x D). A three-way cross hybrid is produced from three inbred lines where two of the inbred lines are crossed (A x B) and then the resulting FI hybrid is crossed with the third inbred (A x B) x C. Much of the hybrid vigor and uniformity exhibited by FI hybrids is lost in the next generation (F2). Consequently, seed produced by hybrids is consumed rather than planted.

Detection Tools

[0135] Several embodiments provide a method for identifying an insect resistant plant, or cells or tissues thereof. In some embodiments, the method includes using primers or probes which specifically recognize a portion of the sequence of the gene. In an embodiment, the method is 36 based on identifying the expression level of a CYP72A gene in the plant. In some embodiments, a PCR-based technique is used to quantify the expression of a CYP72A gene that is differentially expressed in resistant plants compared to susceptible plants prior to treatment. In other words, basal expression levels are heightened in resistant plants compared to susceptible plants prior to exposure to insect feeding.

[0136] In some embodiments, the identification is performed using polymerase chain reaction. The method may also include providing a detectable marker specific to the CYP72A gene. In embodiments, the detection is performed using an Enzyme-Linked Immunosorbent Assay (ELISA), a quantitative real-time polymerase chain reaction (qPCR), or an RNA-hybridization technique.

[0137] Several embodiments provide kits for identifying insect-resistant plants, the kits comprising at least two primers or probes that specifically recognize the CYP72A gene. For example, primers have been developed to amplify and/or quantify the expression of the CYP72A gene associated with SEQ ID NO: 1. By evaluating the expression level of the gene, one skilled in the art is able to determine whether a plant sample comes from an insect resistant plant. In certain embodiments, the primers comprise SEQ ID NOs: 5 or 6. In an embodiment, the kit includes more than one primer pair. The kit may also include one or more positive or negative controls.

[0138] In some embodiments, the kits include a specific probe having a sequence which corresponds to or is complementary to a sequence having between about 80% and about 100% sequence identity with a specific region of the CYP72A gene. In some embodiments, the kit includes a specific probe which corresponds to or is complementary to a sequence having between about 90% and about 100% sequence identity with a specific region of the CYP72A gene.

[0139] The methods, kits, and primers can be used for different purposes including, but not limited to the following: identifying the presence or absence of insect resistance in plants, including plant material such as seeds or cuttings; and tailoring an insecticide regime to effectively and economically manage insect pests affecting agricultural crops.

Embodiments

[0140] The following numbered embodiments also form part of the present disclosure:

[0141] 1. A modified plant, or a progeny, a plant part, or a plant cell thereof, having resistance to an insect pest, the modified plant comprising increased expression of a polynucleotide 37 encoding a cytochrome P45072A (CYP72A) polypeptide relative to a corresponding unmodified plant.

[0142] 2. The modified plant of embodiment 1, wherein the CYP72A polypeptide comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 99% sequence identity to any of SEQ ID NOs: 4 or 23-33.

[0143] 3. The modified plant of embodiment 1 or embodiment 2, wherein the plant comprises a heterologous polynucleotide encoding the CYP72A polypeptide.

[0144] 4. The modified plant of any one of embodiments 1-3, wherein the polynucleotide encoding the CYP72A polypeptide is operably linked to a heterologous promoter functional in a plant cell.

[0145] 5. The modified plant of embodiment 4, wherein the promoter is a constitutive promoter, a tissue-specific promoter, or an inducible promoter.

[0146] 6. The modified plant of any one of embodiments 1-5, wherein the CYP72A polypeptide comprises the amino acid sequence of any of SEQ ID NOs: 4 or 23-33.

[0147] 7. The modified plant of any one of embodiments 1-6, wherein the CYP72A polypeptide is soybean CYP72A141 (GmCYP72A141).

[0148] 8. The modified plant of any one of embodiments 1-7, wherein the polynucleotide encoding the CYP72A polypeptide comprises a nucleotide sequence having at least 80%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 1, 2, or 3.

[0149] 9. The modified plant of any one of embodiments 1-8, wherein the polynucleotide encoding the CYP72A polypeptide comprises the nucleotide sequence of SEQ ID NO: 1, 2, or 3. [0150] 10. The modified plant of any one of embodiments 1-9, wherein the plant is a maize, rice, wheat, barley, oat, rye, millet, sorghum, tobacco, tomato, potato, soybean, brassica, alfalfa, sunflower, or cotton plant.

[0151] 11. The modified plant of embodiment 10, wherein the plant is a soybean plant.

[0152] 12. The modified plant of any one of embodiments 1-11, wherein the insect pest is a coleopteran, a hemipteran, or a lepidopteran.

[0153] 13. A seed or an asexual propagate of the modified plant of any one of embodiments 1- 12

[0154] 14. A method for producing a plant with resistance to an insect pest, the method comprising: increasing expression of a polynucleotide encoding a CYP72A polypeptide in the plant, wherein the insect resistance of the plant is increased when compared to a plant that lacks the increased expression. 38

[0155] 15. The method of embodiment 14, wherein the CYP72A polypeptide comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 99% sequence identity to any of SEQ ID NOs: 4 or 23-33.

[0156] 16. The method of embodiment 14 or embodiment 15 comprising introducing a polynucleotide encoding the CYP72A polypeptide in a plant cell; and regenerating a plant from the plant cell.

[0157] 17. The method any one of embodiments 14-16, wherein the polynucleotide encoding the CYP72A polypeptide is operably linked to a heterologous promoter functional in a plant cell. [0158] 18. The method of embodiment 17, wherein the promoter is a constitutive promoter, a tissue-specific promoter, or an inducible promoter.

[0159] 19. The method of any one of embodiments 14-18, wherein the CYP72A polypeptide comprises the amino acid sequence of any of SEQ ID NOs: 4 or 23-33.

[0160] 20. The method of any one of embodiments 14-19, wherein the CYP72A polypeptide is GmCYP72A141.

[0161] 21. The method of any one of embodiments 14-20, wherein the polynucleotide encoding the CYP72A polypeptide comprises a nucleotide sequence having at least 80%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 1, 2, or 3.

[0162] 22. The method of any one of embodiments 14-21, wherein the polynucleotide encoding the CYP72A polypeptide comprises the nucleotide sequence of SEQ ID NO: 1, 2, or 3.

[0163] 23. The method of any one of embodiments 14-22, wherein the plant is a maize, rice, wheat, barley, oat, rye, millet, sorghum, tobacco, tomato, potato, soybean, brassica, alfalfa, sunflower, or cotton plant.

[0164] 23. The method of embodiment 23, wherein the plant is a soybean plant.

[0165] 24. The method of any one of embodiments 14-23, wherein the insect pest is a coleopteran, a hemipteran, or a lepidopteran.

[0166] 25. A nucleic acid construct comprising a polynucleotide encoding a CYP72A polypeptide, wherein the CYP72A polypeptide has at least 80%, at least 90%, at least 95%, or at least 99% sequence identity to any of SEQ ID NOs: 4 or 23-33, and wherein the polynucleotide is operably linked to a heterologous promoter that is operable in a plant cell.

[0167] 26. The nucleic acid construct of embodiment 25, wherein the polynucleotide encoding the CYP72A polypeptide comprises a nucleotide sequence having at least 80%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 1, 2, or 3.

[0168] 27. The nucleic acid construct of embodiment 25 or embodiment 26, wherein the CYP72A polypeptide comprises the amino acid sequence of any of SEQ ID NOs: 4 or 23-33. 39

[0169] 28. The nucleic acid construct of any one of embodiments 25-27, wherein the polynucleotide encoding the CYP72A polypeptide comprises the nucleotide sequence of SEQ ID NO: 1, 2, or 3.

[0170] 29. The nucleic acid construct of any one of embodiments 25-29, wherein the CYP72A polypeptide is GmCYP72A141.

[0171] 30. The nucleic acid construct of any one of embodiments 25-30, wherein the promoter is a constitutive promoter, a tissue-specific promoter, or an inducible promoter.

[0172] 31. A vector comprising the nucleic acid construct of any one of embodiments 25-30. [0173] 32. A plant or plant cell comprising the nucleic acid construct of any one of embodiments 25-30 or the vector of embodiment 31.

[0174] 33. A method of producing a plant having resistance to infestation by an insect pest, the method comprising: (a) crossing the plant of any one of embodiments 1-12 with itself or another plant to produce seed; and (b) growing a progeny plant from the seed to produce a plant having resistance to infestation by the insect pest.

[0175] 34. The method of embodiment 33, further comprising: (c) crossing the progeny plant with itself or another plant; and (d) repeating steps (b) and (c) for an additional 0-7 generations to produce a plant having increased resistance to infestation by the insect.

[0176] 35. A crop comprising a plurality of the plants of any one of embodiments 1-12 planted together in an agricultural field.

[0177] 36. A method of reducing insect damage to a plant crop, the method comprising: cultivating a plurality of the plants of any one of embodiments 1-12 as a plant crop, wherein the plurality of plants of the plant crop have resistance to an insect pest, thereby reducing insect damage to the plant crop.

[0178] 37. A commodity plant product prepared from the plant, plant part, or plant cell of any one of embodiments 1-12, wherein the product comprises the CYP72A polypeptide or the polynucleotide encoding the CYP72A polypeptide.

[0179] 38. The commodity plant product of embodiment 37, wherein the product is fodder, seed meal, oil, or seed-treatment-coated seed.

[0180] 39. A method for producing a commodity plant product, the method comprising processing the plant or plant part of any one of embodiments 1-12 to obtain the product, wherein the product comprises the CYP72A polypeptide or the polynucleotide encoding the CYP72A polypeptide.

[0181] 40. The method of embodiment 39, wherein the product is fodder, seed meal, oil, or seed-treatment-coated seeds. 40

[0182] 41. A method for identifying a plant that is resistant to an insect pest, the method comprising: providing a biological sample from a plant; quantifying expression of a CYP72A gene in the biological sample; and determining that the plant is resistant to the insect pest based on the quantification, wherein the CYP72A gene is differentially expressed in the plant that is resistant to the insect pest compared to a susceptible plant of the same species.

[0183] 42. The method of embodiment 41, wherein the biological sample is from a soybean plant.

[0184] 43. The method of embodiment 41 or embodiment 42, wherein the quantifying expression of the CYP72A gene comprises quantifying CYP72A mRNA or CYP72A protein. [0185] 44. The method of any one of embodiments 41-43, wherein the CYP72A gene comprises a nucleotide sequence having at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1, 2, or 3.

[0186] 45. The method of any one of embodiments 41-44, wherein quantifying expression comprises amplifying a nucleic acid using at least two primers.

[0187] 46. The method of embodiment 45, wherein the at least two primers comprise SEQ ID NO: 5 or SEQ ID NO: 6.

[0188] 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.

[0189] 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 claims.

[0190] The following examples are offered by way of illustration and not by way of limitation.

EXAMPLES

Example 1: Fast neutron-induced deletions of Glyma.06g238500 (GmCYP72A141)

[0191] While growing fast neutron-mutagenized plants in the field, a mutant line, M027, was identified that showed increased susceptibility to Japanese beetles compared to the unmodified parental Williams 82 cultivar (FIG. 1A). Back-crosses were performed by pollinating emasculated flowers of the parental cultivar Williams 82 with pollen from mutant plants. Phenotypic evaluation of BC1F2 and BC1F3 plants derived from M027 showed that the 41 phenotype segregated at 3 wild-type: 1 susceptible ratio, indicating that the trait is due to a single recessive allele.

[0192] Deleted genomic regions in chromosomes 2 and 6 of M027 were identified by comparative genome hybridization (CGH) (Table 2).

TABLE 2

[0193] Co-segregation analysis indicated that the susceptible phenotype co-segregated with the two linked fast neutron-induced deletions in Chr.06 of M027. The two deletions encode a total of 44 genes and based on co-segregation results, one of these soybean genes is therefore responsible for the increased susceptibility trait (Table 3). Evaluation of the functional annotation and expression of the 44 candidate genes lead to the hypothesis that the genetic basis of the susceptibility trait is the deletion of Glyma.06g238500 (GmCYP72A141). Phylogenetic relationships of GmCYP72A141 indicate that it is orthologous to the licorice ( Glycyrrhiza uralensis) GuCYP72A154 gene encoding a P450 monooxygenase involved in saponin biosynthesis (FIG. 2A).

TABLE 3

42

[0194] To further show that M027 harboring a deletion in GmCYP72A141 has increased susceptibility to insect infestation, choice feeding assays were performed with soybean loopers (3rd instar stage) on 2nd trifoliate soybean leaflets. Soybean wild-type and mutant line M027 were sown and grown in a growth chamber maintained at 26°C with a 18h/6h day -night cycle. Fully expanded 2^nd trifoliate leaflet from 4-week-old soybean plants were detached and used for feeding experiments. Leaflets from WT and M027 were put on moisturized paper in the petri dish (15cm in diameter). A larva (3rd instar) soybean looper ( Chrysodeixis includens) was put in the petri dish and allowed to feed on these detached leaflets. Twenty petri dishes were used. The petri dishes were in a growth chamber maintained at 22°C with a 18h/6 h day -night. Twenty- hours after feeding, leaflets were scanned with Epson perfection v500 Scanner, and the consumer area was measured with the ImageJ software. Soybean loopers preferred to feed on M027 leaves compared to wild-type Williams 82 leaves (FIG. 1B-C). These results are consistent with data on the increased susceptibility of M027 to Japanese beetles.

Example 2: GmCYP72A141 overexpression in Arabidopsis

[0195] To determine if GmCYP72A141 is indeed involved in insect resistance, GmCYP72A141 was expressed from the CAMV 35S promoter in Arabidopsis Col-0. To construct the pFGC- GmCYP72A141 vector, GmCYP72A141 CDS from Williams 82 was amplified by PCR using primer pairs (Table 4). The PCR products were separated by 1.5% agarose gel electrophoresis, the DNA band of interest was purified from the gel and ligated to pGEMT^® Easy Vector Systems (Promega, Madison, WI, USA) for sequencing. The pGEMT-GmCYP72A141 and the vector backbone pFGC5941 (GenBank accession no. AY310901) were digested with Ascl - BamHI followed by ligation to produce the pFGC-GmCYP72A141 construct. The positive plasmid was introduced into Agrobacterium tumefaciens strain GV3101 by electroporation and used for subsequent Arabidopsis stable transformations using floral dip approach. The stable transgenic lines were confirmed by basta resistance and qPCR using the appropriate specific primer set (Table 4). T3 transgenic lines were used for insect assays.

[0196] No-choice assays were performed with newly hatched cabbage looper neonate larvae to determine if overexpression of GmCYP72A141 has deleterious effect on looper growth. No choice insect feeding assay was performed using cabbage looper ( Trichoplusia ni ) caterpillars on individual 4-week-old Arabidopsis plants. Newly-hatched cabbage looper caterpillars (n>23) were allowed to feed the Arabidopsis plants lines for eight days in a growth chamber maintained at 21°C with a 10 h/14 h day-night cycle in sealed clear plastic cages. Feeding was done on leaves from Col-0 (wildtype) and three GmCYP72A141 overexpression lines, after which larvae 43 were harvested and weighed. Growth of cabbage loopers on the overexpression lines 2R-CYP and SR-CYP was comparable to growth on Col-0. However, compared to Col-0, a significant decrease in weight was observed for loopers that fed on the overexpression line 6R-CYP. To determine if the different results obtained in the overexpression lines can be explained by the differences in the levels of GmCYP72A141 expression, qRT-PCR was performed on leaves. Indeed, line 6R-CYP that showed inhibitory effect on looper growth had the highest expression of GmCYP72A141.

[0197] Choice assays were performed with 2^nd instar stage cabbage looper on detached leaves from 4-week-old Arabidopsis Col-0 and 6R-CYP plants. Leaves from control and overexpression plants were put on moisturized paper in the petri dish (100mm in diameter). A larva 2^nd instar cabbage looper was put in the petri dish and allowed to feed on these detached leaves. Twenty petri dishes were used. The petri dishes were kept in a growth chamber maintained at 22°C with a 18h/6 h day -night. Eight-hours after feeding, leaves were recorded. The cabbage loopers preferred to feed on wildtype Arabidopsis Col-0 compared to the 6R-CYP line overexpressing GmCYP72A141.

TABLE 4

44

Example 3: CRISPR/Cas9-induced GmCYP72A141 mutant in soybean

Two independent CRISPR lines were generated in soybean, CRISPR-45 (bi-allelic mutant) and CRISPR-Kl 1 (FIG. 5). The soybean CRISPR line mutant with null mutation in GmCYP72A141 Glyma.06g238500) was more susceptible to chewing insects compared to the WT control (FIG. 6A-C). The increased insect susceptibility of CRISPR-45 was similar to that of the fast neutron mutant M027, confirming the function of GmCYP72A141 in promoting insect resistance in soybean.

Example 4: GmCYP72A141 overexpression in soybean

A soybean GmCYP72A141 Glyma.06g238500) overexpression line, CYP-OX_23, was generated (FIG. 5). The CYP-OX_23 was less favorable to chewing insect compared to the WT control (FIG. 7A-C). The increased insect resistance of CYP-OX_23 to insect feeding was similar to that of Arabidopsis overexpression lines, confirming the utility of GmCYP72A141 in promoting insect resistance in soybean.

Claims

45 What is claimed is:

1. A modified plant, or a progeny, a plant part, or a plant cell thereof, having resistance to an insect pest, the modified plant comprising increased expression of a polynucleotide encoding a cytochrome P45072A (CYP72A) polypeptide relative to a corresponding unmodified plant.

2. The modified plant of claim 1, wherein the CYP72A polypeptide comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 4.

3. The modified plant of claim 1, wherein the plant comprises a heterologous polynucleotide encoding the CYP72A polypeptide.

4. The modified plant of claim 1, wherein the polynucleotide encoding the CYP72A polypeptide is operably linked to a heterologous promoter functional in a plant cell.

5. The modified plant of claim 4, wherein the promoter is a constitutive promoter, a tissue- specific promoter, or an inducible promoter.

6. The modified plant of claim 1, wherein the CYP72A polypeptide comprises the amino acid sequence of SEQ ID NO: 4.

7. The modified plant of claim 1, wherein the CYP72A polypeptide is soybean CYP72A141 (GmCYP72A141).

8. The modified plant of claim 1, wherein the polynucleotide encoding the CYP72A polypeptide comprises a nucleotide sequence having at least 80%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 1, 2, or 3.

9. The modified plant of claim 1, wherein the polynucleotide encoding the CYP72A polypeptide comprises the nucleotide sequence of SEQ ID NO: 1, 2, or 3.

10. The modified plant of claim 1, wherein the plant is a maize, rice, wheat, barley, oat, rye, millet, sorghum, tobacco, tomato, potato, soybean, brassica, alfalfa, sunflower, or cotton plant. 46

11. The modified plant of claim 1, wherein the plant is a soybean plant.

12. The modified plant of claim 1, wherein the insect pest is a coleopteran, a hemipteran, or a lepidopteran.

13. A seed or an asexual propagate of the modified plant of any one of claims 1-12.

14. A method for producing a plant with resistance to an insect pest, the method comprising: increasing expression of a polynucleotide encoding a CYP72A polypeptide in the plant, wherein the insect resistance of the plant is increased when compared to a plant that lacks the increased expression.

15. The method of claim 14, wherein the CYP72A polypeptide comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 4.

16. The method of claim 14 comprising introducing a polynucleotide encoding the CYP72A polypeptide in a plant cell; and regenerating a plant from the plant cell.

17. The method of claim 14, wherein the polynucleotide encoding the CYP72A polypeptide is operably linked to a heterologous promoter functional in a plant cell.

18. The method of claim 17, wherein the promoter is a constitutive promoter, a tissue- specific promoter, or an inducible promoter.

19. The method of claim 14, wherein the CYP72A polypeptide comprises the amino acid sequence of SEQ ID NO: 4.

20. The method of claim 14, wherein the CYP72A polypeptide is GmCYP72A141.

21. The method of claim 14, wherein the polynucleotide encoding the CYP72A polypeptide comprises a nucleotide sequence having at least 80%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 1, 2, or 3. 47

22. The method of claim 14, wherein the polynucleotide encoding the CYP72A polypeptide comprises the nucleotide sequence of SEQ ID NO: 1, 2, or 3.

23. The method of claim 14, wherein the plant is a maize, rice, wheat, barley, oat, rye, millet, sorghum, tobacco, tomato, potato, soybean, brassica, alfalfa, sunflower, or cotton plant.

23. The method of claim 14, wherein the plant is a soybean plant.

24. The method of claim 14, wherein the insect pest is a coleopteran, a hemipteran, or a lepidopteran.

25. A nucleic acid construct comprising a polynucleotide encoding a CYP72A polypeptide, wherein the CYP72A polypeptide has at least 80%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 4, and wherein the polynucleotide is operably linked to a heterologous promoter that is operable in a plant cell.

26. The nucleic acid construct of claim 25, wherein the polynucleotide encoding the CYP72A polypeptide comprises a nucleotide sequence having at least 80%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 1, 2, or 3.

27. The nucleic acid construct of claim 25, wherein the CYP72A polypeptide comprises the amino acid sequence of SEQ ID NO: 4.

28. The nucleic acid construct of claim 25, wherein the polynucleotide encoding the CYP72A polypeptide comprises the nucleotide sequence of SEQ ID NO: 1, 2, or 3.

29. The nucleic acid construct of claim 25, wherein the CYP72A polypeptide is GmCYP72A141.

30. The nucleic acid construct of claim 25, wherein the promoter is a constitutive promoter, a tissue-specific promoter, or an inducible promoter.

31. A vector comprising the nucleic acid construct of any one of claims 25-30. 48

32. A plant or plant cell comprising the nucleic acid construct of any one of claims 25-30.

33. A method of producing a plant having resistance to infestation by an insect pest, the method comprising:

(a) crossing the plant of any one of claims 1-12 with itself or another plant to produce seed; and

(b) growing a progeny plant from the seed to produce a plant having resistance to infestation by the insect pest.

34. The method of claim 33, further comprising:

(c) crossing the progeny plant with itself or another plant; and

(d) repeating steps (b) and (c) for an additional 0-7 generations to produce a plant having increased resistance to infestation by the insect.

35. A crop comprising a plurality of the plants of any one of claims 1-12 planted together in an agricultural field.

36. A method of reducing insect damage to a plant crop, the method comprising: cultivating a plurality of the plants of any one of claims 1-12 as a plant crop, wherein the plurality of plants of the plant crop have resistance to an insect pest, thereby reducing insect damage to the plant crop.

37. A commodity plant product prepared from the plant, plant part, or plant cell of any one of claims 1-12, wherein the product comprises the CYP72A polypeptide or the polynucleotide encoding the CYP72A polypeptide.

38. The commodity plant product of claim 37, wherein the product is fodder, seed meal, oil, or seed-treatment-coated seed.

39. A method for producing a commodity plant product, the method comprising processing the plant or plant part of any one of claims 1-12 to obtain the product, wherein the product comprises the CYP72A polypeptide or the polynucleotide encoding the CYP72A polypeptide.

40. The method of claim 39, wherein the commodity plant product is fodder, seed meal, oil, or seed-treatment-coated seeds. 49

41. A method for identifying a plant that is resistant to an insect pest, the method comprising: providing a biological sample from a plant; quantifying expression of a CYP72A gene in the biological sample; and determining that the plant is resistant to the insect pest based on the quantification, wherein the CYP72A gene is differentially expressed in the plant that is resistant to the insect pest compared to a susceptible plant of the same species.

42. The method of claim 41, wherein the biological sample is from a soybean plant.

43. The method of claim 41, wherein the quantifying expression of the CYP72A gene comprises quantifying CYP72A mRNA or CYP72A protein.

44. The method of claim 41, wherein the CYP72A gene comprises a nucleotide sequence having at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1, 2, or 3.

45. The method of claim 41, wherein quantifying expression comprises amplifying a nucleic acid using at least two primers.

46. The method of claim 45, wherein the at least two primers comprise SEQ ID NO: 5 or SEQ ID NO: 6.