Insecticidal proteins

Abstract

Compositions and methods for controlling insect pests are disclosed. In particular, novel insecticidal proteins that are toxic to at least coleopteran insect pests are provided. Nucleic acid molecules encoding the novel insecticidal proteins are also provided. Also disclosed are methods of making the insecticidal proteins and methods of using the insecticidal proteins as well as nucleic acids encoding the insecticidal proteins of the present disclosure, e.g., in transgenic plants to confer protection from insect damage.

Description

[0001] This application is a divisional application of the invention patent application with the application date of October 13, 2020, application number 202080067951.4, and invention name “Insecticide Protein”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 62 / 914,667, filed on October 14, 2019, the contents of which are incorporated herein by reference in their entirety.

[0004] References to sequence listings

[0005] A sequence listing in ASCII text format is provided as an alternative to a paper copy, which is submitted pursuant to 37 CFR §1.821, is entitled "81942-WO-REG-ORG-P-1_ST25.txt", is approximately 113 kilobytes in size, was generated on September 30, 2020, and was submitted via EFS-Web. This sequence listing is hereby incorporated by reference into this specification for its disclosure. Technical Field

[0006] The present invention relates to the fields of protein engineering, plant molecular biology, and pest control. More specifically, the present invention relates to novel proteins and variants thereof having insecticidal activity, nucleic acids (expressed to produce insecticidal proteins), methods for making these insecticidal proteins and corresponding nucleic acids, and methods for using these insecticidal proteins and corresponding nucleic acids to control insects. Background Art

[0007] Insect pests are a major cause of crop losses. In the United States alone, billions of dollars are lost annually due to infestations by various insect species. In addition to losses to field crops, insect pests are a burden to vegetable and fruit growers, to producers of ornamental flowers, and a nuisance to gardeners and homeowners.

[0008] Several species of corn rootworm are considered the most destructive pests of corn. In the United States alone, three species—the western corn rootworm (Diabrotica virgifera virgifera), the northern corn rootworm (D. longicornis barberi), and the southern corn rootworm (D. undecimpunctata howardi)—cause over $1 billion in annual corn losses in the U.S. Corn Belt. A significant corn rootworm pest in the southern United States is the Mexican corn rootworm (Diabrotica virgifera zeae). In South America, the South American rootworm (Diabrotica Speciosa) is considered a significant pest of corn. The western corn rootworm spread to Europe in 1992 and has been causing economic losses throughout major corn-growing regions since 2008. Corn rootworm larvae cause the most substantial plant damage by feeding almost exclusively on corn roots. This damage has been shown to increase plant lodging, reduce grain yield and vegetative yield, and alter the nutrient content of the grain. Larval feeding also indirectly affects corn by opening a pathway into the roots for bacterial and fungal infections that cause root and stem rot. Adult corn rootworms are active in corn fields in late summer, where they feed on ears, silks, and pollen, thereby disrupting normal pollination.

[0009] Corn rootworms are primarily controlled through intensive applications of chemical pesticides, which are active by inhibiting insect growth, preventing insect feeding or reproduction, or causing mortality. This results in good corn rootworm control, but these chemicals can sometimes affect other beneficial organisms. Another problem caused by the widespread use of chemical pesticides is the emergence of resistant insect populations. Yet another problem is the fact that corn rootworm larvae feed underground, making rescue treatments with insecticides difficult. Consequently, most insecticide applications are carried out preventatively at planting time. This practice results in a significant environmental burden. This situation has been partially improved through various farmland management practices, but there is an increasing need for alternative pest control mechanisms.

[0010] Biological pest control agents, such as strains of Bacillus thuringiensis (Bt) expressing insecticidal toxins like delta-endotoxins (Δ-endotoxins; also known as crystalline toxins or Cry proteins), have also been applied to crop plants on a small scale, producing satisfactory results against certain insect pests. These delta-endotoxins are proteins contained within a crystalline matrix that are known to possess insecticidal activity when ingested by certain insects. Such Cry proteins from Bacillus thuringiensis have been expressed in transgenic crop plants and commercially developed to control certain lepidopteran and coleopteran insect pests. For example, starting in 2003, transgenic corn hybrids that control corn rootworms by expressing Cry3Bb1, Cry34Ab1 / Cry35Ab1, or modified Cry3A (mCry3A) or eCry3.1Ab proteins have been commercially available in the United States.

[0011] Although it has been shown that transgenic plants expressing Cry proteins are very effective, insect pests that are resistant to the Cry proteins expressed in certain transgenic plants are now known. Therefore, there is still a need to identify new and effective pest control proteins that provide economic benefits to farmers and are environmentally acceptable. Particularly needed are proteins that are toxic to Diabrotica species (a major corn pest) that have different modes of action compared to the Cry proteins in existing insect control products to mitigate resistance development. In addition, it is desirable to deliver insect control proteins through these products that minimize the environmental burden (such as through transgenic plants). Summary of the Invention

[0012] In view of these needs, in some embodiments, the present disclosure provides insecticidal proteins that are particularly derived from Rhizobium family bacteria, but can also be derived from other types of bacteria. Examples of such insecticidal proteins are illustrated herein and such insecticidal proteins, whether derived from Rhizobium family or other different bacteria, are collectively referred to as Rhizobium family insecticidal proteins (RIPs). The present disclosure also provides variants of RIPs, and proteins substantially identical to RIPs and variants thereof. Examples of the amino acid sequences of RIPs disclosed herein include, but are not limited to, any one of SEQ ID NOs: 1-21. The RIPs disclosed herein are toxic to insect pests. For example, the proteins disclosed herein can be used to control economically important insect pests, including coleopteran insects, such as western corn rootworm (WCR; corn rootworm), northern corn rootworm (NCR; long-horned barbel rootworm), southern corn rootworm (SCR; cucumber rootworm rootfeeding subspecies eleven-spotted) and / or Mexican corn rootworm (MCR; Mexican corn rootworm).

[0013] The present disclosure also provides nucleic acid molecules comprising one or more nucleotide sequences encoding RIP or variant RIP, their complementary sequences, or nucleotide sequences substantially identical to RIP or RIP variants. Examples of nucleotide sequences encoding RIP or variant RIP of the present disclosure include, but are not limited to, any one of SEQ ID NOs: 22-49.

[0014] The present disclosure also provides vectors comprising recombinant nucleic acids encoding the RIPs and / or variant RIPs of the present disclosure; plants or microorganisms comprising and capable of expressing such nucleic acids; plants transformed with such nucleic acids, such as transgenic corn plants; progeny of such plants containing nucleic acids stably incorporated and inherited in a Mendelian manner, and / or seeds of such plants and such progeny. The present disclosure also provides breeding methods for introducing transgenes comprising nucleic acid molecules of the present disclosure into progeny plants and various germplasms.

[0015] The present disclosure also provides compositions and formulations comprising the RIPs and / or variant RIPs of the present disclosure, which are capable of inhibiting the ability of pests to survive, grow and / or reproduce, or to limit insect-associated damage or loss to crops, e.g., by applying the RIPs or RIP variants as part of a composition or formulation to an area or plant infested by insects, or by preventatively treating an area or plant susceptible to insect infestation to confer protection against insect pests.

[0016] The present disclosure further provides methods for making RIPs or variants thereof, and methods for using the nucleic acids, for example, to control insects in microorganisms or to confer protection from insect damage in transgenic plants. Such microorganisms can be, for example, endophytes that colonize maize roots and deliver the RIPs of the present disclosure to the maize rhizosphere, thereby protecting the roots from damage by corn rootworm feeding.

[0017] The RIPs and / or variant RIPs of the present disclosure can be used alone or in combination with other insect control agents and strategies to impart enhanced pest control efficiency against the same insect pests and / or to increase the spectrum of target insects with minimal environmental impact.

[0018] Other aspects and advantages of the present invention will become apparent to those skilled in the art upon study of the following description and non-limiting examples of the invention.

[0019] Brief description of the sequences in the sequence listing

[0020] SEQ ID NO: 1 is the amino acid sequence of Ensif_aridCRW (RIP1Aa). SEQ ID NO: 2 is the amino acid sequence of Sinorhiz_GL28CRW (RIP2Aa). SEQ ID NO: 3 is the amino acid sequence of Rhizo_bactCRW (RIP3Aa). SEQ ID NO: 4 is the amino acid sequence of Rhiz_SPYCRW (RIP4Aa). SEQ ID NO: 5 represents the amino acid sequence of variant RIP3Aa.

[0021] SEQ ID NO: 6 is the amino acid sequence of RIP3Aa-I50L.

[0022] SEQ ID NO: 7 is the amino acid sequence of RIP3Aa-I53L.

[0023] SEQ ID NO: 8 is the amino acid sequence of RIP3Aa-I56L.

[0024] SEQ ID NO: 9 is the amino acid sequence of RIP3Aa-A62C.

[0025] SEQ ID NO: 10 is the amino acid sequence of RIP3Aa-A62L.

[0026] SEQ ID NO: 11 is the amino acid sequence of RIP3Aa-A64C.

[0027] SEQ ID NO: 12 is the amino acid sequence of RIP3Aa-A64L.

[0028] SEQ ID NO: 13 is the amino acid sequence of RIP3Aa-I81L.

[0029] SEQ ID NO: 14 is the amino acid sequence of RIP3Aa-I126L.

[0030] SEQ ID NO: 15 is the amino acid sequence of RIP3Aa-I153L.

[0031] SEQ ID NO: 16 is the amino acid sequence of RIP3Aa-I169L.

[0032] SEQ ID NO: 17 is the amino acid sequence of RIP3Aa-I185L.

[0033] SEQ ID NO: 18 is the amino acid sequence of RIP3Aa-I207L.

[0034] SEQ ID NO: 19 is the amino acid sequence of RIP3Aa-I219L.

[0035] SEQ ID NO: 20 is the amino acid sequence of RIP3Aa-I275L.

[0036] SEQ ID NO: 21 is the amino acid sequence of SUMO-RIP1Aa.

[0037] SEQ ID NO:22 is the nucleotide sequence of Ensif_aridCRW (rip1Aa). SEQ ID NO:23 is the nucleotide sequence of Sinorhiz_GL28CRW (rip2Aa). SEQ ID NO:24 is the nucleotide sequence of Rhizo_bactCRW (rip3Aa). SEQ ID NO:25 is the nucleotide sequence of Rhiz_SPYCRW (rip4Aa). SEQ ID NO:26 is the E. coli-optimized nucleotide sequence of rip1Aa.

[0038] SEQ ID NO: 27 is the E. coli optimized nucleotide sequence of rip2Aa.

[0039] SEQ ID NO: 28 is the nucleotide sequence of rip3Aa optimized for E. coli.

[0040] SEQ ID NO: 29 is the E. coli optimized nucleotide sequence of rip4Aa.

[0041] SEQ ID NO:30 is the nucleotide sequence of rip3Aa-I50L optimized for E. coli. SEQ ID NO:31 is the nucleotide sequence of rip3Aa-I53L optimized for E. coli. SEQ ID NO:32 is the nucleotide sequence of rip3Aa-I56L optimized for E. coli. SEQ ID NO:33 is the nucleotide sequence of rip3Aa-A62C optimized for E. coli. SEQ ID NO:34 is the nucleotide sequence of rip3Aa-A62L optimized for E. coli. SEQ ID NO:35 is the nucleotide sequence of rip3Aa-A64L optimized for E. coli. SEQ ID NO:36 is the nucleotide sequence of rip3Aa-A64C optimized for E. coli. SEQ ID NO:37 is the nucleotide sequence of rip3Aa-I81L optimized for E. coli. SEQ ID NO:38 is the nucleotide sequence of rip3Aa-I126L optimized for E. coli. SEQ ID NO:39 is the nucleotide sequence of rip3Aa-I153L optimized for E. coli. SEQ ID NO:40 is the nucleotide sequence of rip3Aa-I169L optimized for E. coli. SEQ ID NO:41 is the nucleotide sequence of rip3Aa-I185L optimized for E. coli. SEQ ID NO:42 is the nucleotide sequence of rip3Aa-I207L optimized for E. coli. SEQ ID NO:43 is the nucleotide sequence of rip3Aa-I219L optimized for E. coli. SEQ ID NO:44 is the nucleotide sequence of rip3Aa-I275L optimized for E. coli. SEQ ID NO:45 is the nucleotide sequence of SUMO-rip1Aa.

[0042] SEQ ID NO:46 is the maize-optimized nucleotide sequence of rip1Aa.

[0043] SEQ ID NO:47 is the maize-optimized nucleotide sequence of rip2Aa.

[0044] SEQ ID NO:48 is the maize-optimized nucleotide sequence of rip3Aa.

[0045] SEQ ID NO:49 is the maize-optimized nucleotide sequence of rip4Aa.

[0046] SEQ ID NO:50 comprises the RIP1Aa cytotoxin domain.

[0047] SEQ ID NO:51 comprises the RIP2Aa cytotoxin domain.

[0048] SEQ ID NO:52 comprises the RIP3Aa cytotoxin domain.

[0049] SEQ ID NO:53 comprises the RIP4Aa cytotoxin domain. DETAILED DESCRIPTION

[0050] This description is not intended to be an exhaustive list of all the different ways in which the invention may be implemented, or of all the features that may be added to the invention. For example, features described with respect to one embodiment may be incorporated into other embodiments, and features described with respect to a particular embodiment may be deleted from that embodiment. Thus, the present invention contemplates that, in some embodiments of the invention, any feature or combination of features set forth herein may be excluded or omitted. Furthermore, in view of this disclosure, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art without departing from the invention. Therefore, the following description is intended to illustrate some specific embodiments of the invention and is not intended to be an exhaustive description of all permutations, combinations, and variations thereof.

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. The terms used in the description of the invention herein are for the purpose of describing particular embodiments only and are not intended to limit the invention.

[0052] All publications, patent applications, patents, and other references cited herein are incorporated by reference in their entirety for the teachings of the relevant sentence and / or paragraph to which the reference is addressed.

[0053] The nucleotide sequences provided herein are presented from left to right in a 5' to 3' orientation and are represented using the standard code for nucleotide bases as described in 37 CFR §§ 1.821-1.825 and World Intellectual Property Organization (WIPO) Standard ST.25, e.g., adenine (A), cytosine (C), thymine (T), and guanine (G).

[0054] Amino acids are also indicated using WIPO Standard ST.25, for example: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C), glutamine (Gln; Q), glutamic acid (Glu; E), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V). As used herein, "X" or "Xaa" in an amino acid sequence indicates that the amino acid at that position may be any of the 20 known amino acids or may be any of the amino acids listed herein.

[0055] Unless the context indicates otherwise, it is expressly contemplated that the various features of the invention described herein may be used in any combination. Furthermore, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features recited herein may be excluded or omitted. For example, if the specification recites a composition comprising components A, B, and C, it is expressly contemplated that any one or combination of A, B, or C may be omitted or disclaimed, singly or in any combination.

[0056] definition

[0057] As used in accordance with this disclosure, unless otherwise indicated, the following terms shall be understood to have the following meanings:

[0058] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a plant" is a reference to one or more plants and includes equivalents thereof known to those skilled in the art, and so forth.

[0059] As used herein, the term "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

[0060] The term "about" is used herein to mean approximately, roughly, about, or around. When the term "about" is used in conjunction with a numerical range, it defines the range by extending the boundaries above and below the stated values. Typically, the term "about" is used herein to define a numerical value to a variation of 20%, preferably 10% above and below (higher or lower) above and below the stated value. With respect to temperature, the term "about" means ±1°C, preferably ±0.5°C. When the term "about" is used in the context of the present invention (e.g., in combination with a temperature or molecular weight value), the exact value (i.e., without "about") is preferred.

[0061] As used herein, phrases such as “between about X and Y,” “between about X and about Y,” “from X to Y,” and “from about X to about Y” (and similar phrases) should be interpreted to include X and Y unless the context indicates otherwise.

[0062] As used herein, term " amplification " means using at least one nucleic acid molecule as template to construct multiple copies of a nucleic acid molecule or multiple copies complementary to the nucleic acid molecule. Amplification systems include polymerase chain reaction (PCR) systems, ligase chain reaction (LCR) systems, nucleic acid sequence-based amplification (NASBA, Cangene, Mississauga, Ontario), Q-β replicase systems, transcription-based amplification systems (TAS) and strand displacement amplification (SDA). See, for example, Diagnostic Molecular Microbiology: Principles and Applications [diagnostic molecular microbiology: principles and applications], PERSING et al., eds., American Society for Microbiology [American Society for Microbiology], Washington (Washington, DC), (1993). The product of amplification is referred to as "amplicon".

[0063] The "activity" of the insecticidal proteins of the present disclosure means that the insecticidal proteins act as orally active insect control agents, have toxic effects, and / or are able to interfere with or prevent insect feeding, which may or may not cause the death of the insect. When the insecticidal proteins of the present disclosure are delivered to an insect, this result is typically the death of the insect, or the insect does not feed on a source of the insecticidal protein that is available to the insect. "Pesticide" is defined as a toxic biological activity that is able to control harmful organisms (such as insects, nematodes, fungi, bacteria or viruses), preferably by killing or destroying them. "Insecticidal" is defined as a toxic biological activity that is able to control insects, preferably by killing them. A "pesticide" is an agent with pesticidal activity. An "insecticide" is an agent with insecticidal activity.

[0064] As used herein, the term "chimeric construct" or "chimeric gene" or "chimeric polynucleotide" or "chimeric nucleic acid" (or similar terms) refers to a construct or molecule comprising two or more polynucleotides from different sources assembled into a single nucleic acid molecule. The term "chimeric construct," "chimeric gene," "chimeric polynucleotide," or "chimeric nucleic acid" refers to any construct or molecule containing, but not limited to, (1) a polynucleotide (e.g., DNA) comprising a regulatory polynucleotide and a coding polynucleotide that are not found together in nature (i.e., at least one polynucleotide in the construct is heterologous with respect to at least one of its other polynucleotides), or (2) a polynucleotide that encodes a portion of a protein that is not naturally contiguous, or (3) a portion of a promoter that is not naturally contiguous. Additionally, a chimeric construct, chimeric gene, chimeric polynucleotide, or chimeric nucleic acid may comprise a regulatory polynucleotide and a coding polynucleotide derived from different sources, or a regulatory polynucleotide and a coding polynucleotide derived from the same source but arranged in a manner different from that found in nature. In some embodiments of the present disclosure, a chimeric construct, chimeric gene, chimeric polynucleotide, or chimeric nucleic acid comprises an expression cassette comprising a polynucleotide of the present disclosure under the control of a regulatory polynucleotide, particularly a regulatory polynucleotide functional in plants or bacteria.

[0065] A "coding sequence" is a nucleic acid sequence that is transcribed into RNA (such as mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA). Preferably, the RNA is then translated in an organism to produce a protein.

[0066] "Controlling" insects means inhibiting the ability of insect pests to survive, grow, feed, and / or reproduce through toxic effects, or limiting insect-associated damage or losses in crop plants. "Controlling" insects may or may not mean killing the insects, although it preferably means killing the insects.

[0067] As used herein, " codon optimized " sequence means following nucleotide sequence, and wherein these codons are selected to reflect the specific codon preference that host cell or organism can have.This typically is to complete in such a way, and this mode is in order to keep the amino acid sequence of the polypeptide encoded by the nucleotide sequence to be optimized.In certain embodiments, the dna sequence dna of recombinant DNA construct comprises the cell (for example, animal, plant or fungal cell) that has been expressed therein for this construct and has carried out codon optimized sequence.For example, the construct to be expressed in plant cell can make its whole or part of sequence (for example, the first gene suppression element or gene expression element) carry out codon optimized for expression in plant.See for example, U.S. Patent number 6,121,014, incorporated herein by reference.

[0068] The terms “comprises” or “comprising” when used in this specification indicate the presence of stated features, integers, steps, operations, elements, or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, or groups thereof.

[0069] As used herein, the transition phrase "consisting essentially of (and grammatical variations) means that the scope of the claim is to be interpreted to encompass the specified materials or steps recited in the claim and those that do not materially alter one or more of the basic and novel characteristics of the claimed invention. Thus, the term "consisting essentially of" when used in the claims of the present invention is not intended to be interpreted as equivalent to "comprising."

[0070] In the context of the present disclosure, "corresponding to" or "corresponds to" means that when the amino acid sequences of variant or homolog proteins are aligned with each other, the amino acids "corresponding to" certain enumerated positions in the variant or homolog protein are those aligned with these positions in the reference protein, but not necessarily in these exact numerical positions relative to the specific reference amino acid sequence of the present disclosure. For example, if SEQ ID NO: 1 is the reference sequence and is aligned with SEQ ID NO: 2, then the amino acid Leu (L) at position 153 (L153) of SEQ ID NO: 2 "corresponds to" Leu (L) at position 154 (L154) of SEQ ID NO: 1, or, for example, Asn (N) at position 2 (N2) of SEQ ID NO: 2 "corresponds to" Ala (A) A2 of SEQ ID NO: 1.

[0071] "Delivering" a composition or toxic protein means that the composition or toxic protein contacts the insect, which promotes oral uptake of the composition or toxic protein, resulting in a toxic effect and control of the insect. The composition or toxic protein can be delivered in a number of recognized ways, including but not limited to transgenic plant expression, one or more formulated protein compositions, one or more sprayable protein compositions, a bait matrix, or any other art-recognized protein delivery system.

[0072] The term "domain" refers to a group of amino acids that are conserved at specific positions along the alignment of the sequences of evolutionarily related proteins. Although amino acids at other positions may differ between homologs, amino acids that are highly conserved at specific positions indicate amino acids that are likely to be essential in the structure, stability, or function of the protein. They are identified by their high degree of conservation in aligned sequences of a family of protein homologs and can be used as identifiers to determine whether any polypeptide in question belongs to a previously identified group of polypeptides.

[0073] "Insect control effective amount" means a concentration of an insecticidal protein that inhibits the ability of insects to survive, grow, feed and / or reproduce through toxic effects, or limits insect-related damage or crop plant loss. An "effective insect controlling amount" may or may not mean killing the insects, although it preferably means killing the insects.

[0074] As used herein, "expression cassette" means a nucleic acid sequence capable of directing the expression of a specific nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to a target nucleotide sequence, which is operably linked to a termination signal. It also typically comprises the sequence required for the appropriate translation of the nucleotide sequence. The expression cassette comprising the target nucleotide sequence may have at least one of its components that is heterologous relative to at least one of its other components. The expression cassette can also be naturally occurring but has been an expression cassette obtained in a recombinant form useful for heterologous expression. However, typically, the expression cassette is heterologous relative to the host, i.e., the specific nucleic acid sequence of the expression cassette is not naturally present in the host cell and must have been introduced into the host cell or the ancestor of the host cell through a transformation event. The expression of the nucleotide sequence in the expression cassette can be under the control of a constitutive promoter or an inducible promoter, which initiates transcription only when the host cell is exposed to some specific external stimuli. In the case of multicellular organisms (such as plants), the promoter can also be specific for a specific tissue, organ, or developmental stage.

[0075] The expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous to at least one of its other components. The expression cassette may also comprise a native promoter driving its native gene, but has been obtained in a recombinant form useful for heterologous expression. This use of the expression cassette renders it non-naturally occurring in the cell into which it is introduced.

[0076] The expression cassette can also optionally be included in a transcription and / or translation termination region (i.e., terminator) that plays a role in plants. A variety of transcription terminators are available for use in the expression cassette and are responsible for transcription termination and correct mRNA polyadenylation when exceeding the heterologous nucleotide sequence of interest. The terminator can be natural for the transcription initiation region, can be natural for the operably connected nucleotide sequence of interest, can be natural for plant hosts, or can be derived from another source (i.e., external or heterologous for promoter, nucleotide sequence of interest, plant hosts, or any combination thereof). Suitable transcription terminators include, but are not limited to, CAMV 35S terminator, tml terminator, nopaline synthase terminator, and / or pea rbcs E9 terminator. These terminators can be used in both monocotyledons and dicotyledons. In addition, the natural transcription terminator of the coding sequence can be used. Any terminator known to be available for use in plants can be used in the context of this disclosure.

[0077] When used with reference to polynucleotides (such as genes, ORFs or parts thereof, or transgenics of plants), the term "expression" refers to the process by which the genetic information encoded in a gene is converted into RNA (e.g., mRNA, rRNA, tRNA or snRNA) by the "transcription" of a gene (i.e., via the enzymatic action of an RNA polymerase), and, where applicable (e.g., if the gene encodes a protein), into a protein by the "translation" of the mRNA. Gene expression can be regulated at many stages of the process. For example, in the case of an antisense construct or a dsRNA construct, expression can refer only to the transcription of the antisense RNA or only to the transcription of the dsRNA. In an embodiment, "expression" refers to the transcription and stable accumulation of a sense (mRNA) or functional RNA. "Expression" can also refer to the production of a protein.

[0078] " gene " is the limited area that is positioned at genome and comprises coding nucleic acid sequence, and typically also comprises other main regulatory nucleic acids that are responsible for controlling this coding part expression (that is, transcription and translation).Gene can also comprise other 5 ' and 3 ' untranslated sequences and terminator sequences.Other possible element is for example intron.As found in nature, the regulatory nucleic acid sequence of gene may not be operably connected with this associated nucleotide sequence under normal circumstances, and therefore can not be mosaic gene.

[0079] "Gene of interest" refers to any nucleic acid molecule that, when transferred to an organism, such as a bacterium or a plant, confers a desired trait on the bacterium or plant, such as antibiotic resistance, virus resistance, insect resistance, disease resistance, or resistance to other harmful organisms, herbicide tolerance, abiotic stress tolerance, male sterility, modified fatty acid metabolism, modified carbohydrate metabolism, improved nutritional value, improved performance in an industrial process, or altered reproductive capacity. A "gene of interest" can also be a gene transferred to a bacterium or plant for the production of a commercially valuable enzyme or metabolite in the plant.

[0080] "Heterologous" nucleic acid sequence or nucleic acid molecule is a nucleic acid sequence or nucleic acid molecule that is not naturally associated with the host cell into which the nucleic acid sequence is introduced, including multiple copies of non-naturally occurring nucleic acid sequences. Heterologous nucleic acid sequence or nucleic acid molecule can comprise chimeric sequences, such as chimeric expression cassettes, in which the promoter and coding region are derived from multiple source organisms. The promoter sequence can be a constitutive promoter sequence, a tissue-specific promoter sequence, a chemically inducible promoter sequence, a wound-inducible promoter sequence, a stress-inducible promoter sequence, or a developmental stage-specific promoter sequence.

[0081] A "homologous" nucleic acid sequence is a nucleic acid sequence naturally associated with a host cell into which it is introduced.

[0082] "Homologous recombination" is the reciprocal exchange of nucleic acid segments between homologous nucleic acid molecules.

[0083] As used herein, "hypothetical protein" refers to a protein whose existence has been predicted but lacks experimental evidence of its expression in vivo. Genomes or organisms (such as bacteria or plants) are sequenced, and a large number of predicted open reading frames are usually generated, and the functions of these reading frames are not easy to specify. These proteins, whether isolated or conservative hypothetical proteins, account for about 20% to about 40% of the proteins encoded in each newly sequenced genome. Even if there is enough evidence to show that the product of the gene is expressed, it is difficult to specify its function through technologies such as microarrays and mass spectrometry because it lacks identity to the protein sequence with annotated biochemical functions. Usually, most protein sequences are inferred from the computational analysis of genomic DNA sequences. Hypothetical proteins are usually created by gene prediction software during genome analysis. When the bioinformatics tools for gene identification find large open reading frames without characteristic homologs in the protein database, such tools usually return the name "hypothetical protein" as an annotation.

[0084] The term "motif" or "consensus sequence" or "signature" refers to a short conserved region in the sequence of evolutionarily related proteins. A motif is often a highly conserved portion of a domain, but may also comprise only a portion of a domain, or be located outside a conserved domain (if all amino acids of the motif are located outside the defined domain).

[0085] In the context of two nucleic acid or amino acid sequences, the term "identity" or "identical" or "substantially identical" refers to two or more sequences or subsequences that have at least 60%, preferably at least 80%, more preferably 90%, even more preferably 95%, and most preferably at least 99% nucleotide or amino acid residue identity when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. Preferably, substantial identity exists over a region having a length of at least about 50 residues or bases, more preferably over a region of at least about 100 residues or bases, and most preferably, the sequences are substantially identical over at least about 150 residues or bases. In a particularly preferred embodiment, the sequences are substantially identical over the entire length of the coding region. In addition, substantially identical nucleic acid or amino acid sequences perform essentially the same function.

[0086] For sequence comparison, typically, one sequence serves as a reference sequence to which a test sequence is compared. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer (if necessary, subsequence coordinates are specified), and the parameters of the sequence algorithm program are specified. The sequence comparison algorithm then calculates the sequence identity percentages of the test sequence(s) relative to the reference sequence based on the specified program parameters.

[0087] Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search by similarity method of Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Street, Madison, Wisconsin), or by visual inspection (see generally Ausubel et al., infra).

[0088] An example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in the following literature: Altschul et al., J. Mol. Biol. [Molecular Biology] 215: 403-410 (1990). Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (the National Center for Biotechnology Information, U.S. National Library of Medicine, 8600 Rockville Pike, Bethesda, Maryland 20894). This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or meet some positive threshold score T when compared to words of the same length in the database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., 1990). These initial neighborhood word hits serve as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatched residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is stopped when the cumulative alignment score decreases by the amount X from its maximum achieved value; when the cumulative score approaches 0 or below due to the accumulation of one or more negative-scoring residue alignments; or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses a word length (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both chains as defaults. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad Sci. USA 89:10915 (1989)).

[0089] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0090] Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions. The phrase "specifically hybridizes" means that a molecule binds, double-strands, or hybridizes only to a specific nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA. "Substantially binds" refers to complementary hybridization between a probe nucleic acid and a target nucleic acid, and encompasses minor mismatches that can be accommodated by reducing the stringency of the hybridization medium to achieve desired detection of the target nucleic acid sequence.

[0091] In the context of nucleic acid hybridization experiments (such as DNA hybridization and RNA hybridization), "stringent hybridization conditions" and "stringent hybridization wash conditions" are sequence-dependent and are different under different environmental parameters. Longer sequences hybridize specifically at higher temperatures. A broad guide to nucleic acid hybridization 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 higher than the thermal melting point (T for the specific sequence) at a defined ionic strength and pH. m Typically, under "stringent conditions," a probe will hybridize to its target sequence, but to no other sequences.

[0092] T mIt 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 T for a specific probe. m . An example of stringent hybridization conditions for hybridization of complementary nucleic acids (which have more than 100 complementary residues on the filter in a Southern or Northern blot) is 50% formamide with 1 mg of heparin at 42°C, and the hybridization is carried out overnight. An example of a high stringency wash condition is 0.15 M NaCl at 72°C for about 15 minutes. An example of a stringent wash condition is a 0.2×SSC wash at 65°C for 15 minutes (see, Sambrook, below, for a description of SSC buffer). Typically, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example of a medium stringency wash for, for example, a duplex of more than 100 nucleotides is 1×SSC at 45°C for 15 minutes. An example of a low stringency wash for, for example, a duplex of more than 100 nucleotides is 4-6×SSC at 40°C for 15 minutes. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically involve a salt concentration of less than about 1.0 M Na ions, 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 by adding destabilizing agents such as formamide. In general, a signal-to-noise ratio that is 2-fold (or higher) is observed in a particular hybridization assay compared to an unrelated probe, indicating that specific hybridization has been detected. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the proteins they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.

[0093] The following are examples of settings of hybridization / wash conditions that can be used to clone homologous nucleotide sequences that are substantially identical to the reference nucleotide sequences of the present disclosure: a reference nucleotide sequence preferably hybridizes to the reference nucleotide sequence under the following conditions: 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50°C, and washing in 2×SSC, 0.1% SDS at 50°C; more desirably, 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50°C, and washing in 1×SSC, 0.1% SDS at 50°C; still more desirably, 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50°C, and washing in 0.5×SSC, 0.1% SDS at 50°C; preferably, 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50°C, and washing in 0.5×SSC, 0.1% SDS at 50°C. EDTA at 50°C and in 0.1×SSC, 0.1% SDS at 50°C; more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50°C and in 0.1×SSC, 0.1% SDS at 65°C.

[0094] Another indication that two nucleic acid sequences or proteins are substantially identical is that the protein encoded by the first nucleic acid immunologically cross-links with or specifically binds to the protein encoded by the second nucleic acid. Thus, a protein is typically substantially identical to a second protein, e.g., where the two proteins differ only by conservative substitutions.

[0095] The term "isolated" nucleic acid molecule, polynucleotide, or protein is a nucleic acid molecule, polynucleotide, or protein that is no longer present in its natural environment. The isolated nucleic acid molecules, polynucleotides, or proteins disclosed herein can exist in a purified form, or can exist in a recombinant host, such as a transgenic bacterium or a transgenic plant. Thus, claims to "isolated" nucleic acid molecules as recited herein encompass nucleic acid molecules when the nucleic acid molecule is contained within the genome of a transgenic plant.

[0096] A "nucleic acid molecule" or "nucleic acid sequence" is a segment of single-stranded or double-stranded DNA or RNA that can be isolated from any source. In the context of the present disclosure, a nucleic acid molecule is typically a segment of DNA. In some embodiments, the nucleic acid molecules of the present disclosure are isolated nucleic acid molecules.

[0097] "Operably linked" refers to the association of polynucleotides on a single nucleic acid fragment such that the function of one affects the function of the other. For example, a promoter is operably linked to a coding polynucleotide or functional RNA when it is capable of affecting the expression of the coding polynucleotide or functional RNA (i.e., the coding polynucleotide or functional RNA is under the transcriptional control of the promoter). A coding polynucleotide in either the sense or antisense orientation can be operably linked to a regulatory polynucleotide.

[0098] As used herein, "pesticide," "insecticide," and the like refer to the ability of a RIP of the present disclosure to control a pest or the amount of a RIP that can control a pest as defined herein. Thus, a pesticide RIP can kill or inhibit the ability of a pest (e.g., an insect pest) to survive, grow, feed, or reproduce.

[0099] The terms "protein," "peptide," and "polypeptide" are used interchangeably herein.

[0100] A "plant" is any plant at any stage of development, particularly a seed plant. Exemplary plants include, but are not limited to, corn (Zea mays), canola (Brassica napus, Brassica rapa ssp.), alfalfa (Medicago saliva), rice (Oryza sativa, including but not limited to indica and / or japonica), rapeseed (Brassica napus), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), sunflower (Helianthus annuus), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanut (Arachis hypogaea), cotton (Gossypium hirsutum), sweet potato (Ipomoea batatas), cassava (Manihot esculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musas spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia nut (Macadamia integrifolia), almond (Prunus amygdalus), sugar beet (Beta vulgaris), apple (Malus pumila), blackberry (Rubus spp.), strawberry (Fragaria spp.), walnut (Juglans regia), grape (Vitis vinifera), apricot (Prunus armeniaca), cherry (Prunus spp.), peach (Prunus persica). persica), plum (Prunus domestica), pear (Pyrus communis), watermelon (Citrullus vulgaris), duckweed (Lemna spp.), oats (Avena sativa), barley (Hordium vulgare), vegetables, ornamentals, conifers, and turfgrasses (e.g., ornamental, recreational, or forage), as well as biomass grasses (e.g., switchgrass and miscanthus).

[0101] Vegetables include, but are not limited to, Solanum species (e.g., tomatoes, Lycopersicon esculentum), lettuce (e.g., Lactuea sativa), carrots (Caucus carota), cauliflower (Brassica oleracea), celery (Apium graveolens), eggplant (Solanum melongena), asparagus (Asparagus officinalis), okra (Abelmoschus esculentus), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), members of the genus Cucurbita such as C. hubbard, winter squash (C. moschata), zucchini (C. pepo), C. crookneck, C. argyrosperma, C. argyrosperma ssp sororia, C. digitata, C. ecuadorensis, C. foetidissima, C. lundelliana, and C. martinezii, and members of the genus Cucumis such as cucumber (Cucumber, Cucumis sativus), cantaloupe (C. cantalupensis), and muskmelon (C. melo).

[0102] Ornamental plants include, but are not limited to, azaleas (Rhododendron spp.), hydrangeas (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnations (Dianthus caryophyllus), poinsettias (Euphorbia pulcherima), and chrysanthemums.

[0103] Conifers that can be used to practice the present disclosure include, for example, pines such as loblolly pine (Pinustaeda), slash pine (Pinus elliotii), jack pine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii); western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); firs such as silver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as western arborvitae (Thuja sempervirens). plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis).

[0104] Turfgrasses include, but are not limited to, zoysia, bentgrass, fescue, bluegrass, Augustinegrass, Bermudagrass, bufallograsses, ryegrass, and orchardgrass.

[0105] Also included are plants that serve primarily as laboratory models, such as Arabidopsis thaliana.

[0106] A "plant cell" is a structural and physiological unit of a plant, comprising a protoplast and a cell wall. A plant cell can be in the form of an isolated single cell or a cultured cell, or as part of a higher-order organizational unit such as, for example, a plant tissue, a plant organ, or a whole plant.

[0107] "Plant cell culture" means a culture of plant units such as, for example, protoplasts, cell culture cells, cells in plant tissue, pollen, pollen tubes, ovules, embryo sacs, zygotes, and embryos at various stages of development.

[0108] "Plant material" refers to leaves, stems, roots, flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant.

[0109] A "plant organ" is a distinct and clearly structured and differentiated part of a plant, such as a root, stem, leaf, flower bud or embryo.

[0110] As used herein, "plant tissue" means a group of plant cells organized into structural and functional units. This includes any plant tissue in plants or in culture. This term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue cultures, and any group of plant cells organized into structural and / or functional units. The use of this term in conjunction with or alone with any specific type of plant tissue listed above or otherwise encompassed by this definition is not intended to exclude any other type of plant tissue.

[0111] "Polynucleotide" refers to a polymer composed of many nucleotide monomers covalently bonded in a chain. Such "polynucleotides" include DNA, RNA, modified oligonucleotides (for example, oligonucleotides containing bases atypical for biological RNA or DNA, such as 2'-O-methylated oligonucleotides) and the like. In some embodiments, nucleic acids or polynucleotides can be single-stranded, double-stranded, multi-stranded, or a combination thereof. Unless otherwise indicated, in addition to any explicitly indicated polynucleotides, the specific nucleic acids or polynucleotides of the present disclosure optionally further comprise or encode complementary polynucleotides.

[0112] A "polynucleotide of interest" refers to any polynucleotide that, when transferred into an organism (e.g., a plant), confers a desired trait to that organism, such as insect resistance, disease resistance, herbicide tolerance, antibiotic resistance, improved nutritional value, improved performance in an industrial process, production of a commercially valuable enzyme or metabolite, or altered reproductive capacity.

[0113] "Promoter" is an untranslated DNA sequence upstream of a coding region that contains an RNA polymerase binding site and initiates transcription of the DNA. The promoter region may also include other elements that act as regulators of gene expression.

[0114] As used herein, the term "recombination" refers to the following form of a nucleic acid molecule (for example, DNA or RNA) or protein or organism, which is not usually found in nature and is produced by human intervention. As used herein, a "recombinant nucleic acid molecule" is a nucleic acid molecule comprising a combination of polynucleotides, which polynucleotides do not naturally coexist and are the result of human intervention, for example, a nucleic acid molecule composed of a combination of at least two polynucleotides heterologous to each other, or artificially synthesized (for example, using the polynucleotides synthesized by the nucleotide sequence of assembly) and comprising a nucleic acid molecule different from a polynucleotide typically present in nature, or comprising a nucleic acid molecule artificially incorporated into the genomic DNA of a host cell and the transgenic nucleic acid molecule in the relevant flanking DNA of the host cell genome. Another example of a recombinant nucleic acid molecule is a DNA molecule produced by inserting a transgenic into the genomic DNA of a plant, which can ultimately result in the expression of a recombinant RNA / or protein molecule in the organism. As used herein, a "recombinant plant" is a plant that is not usually present in nature, is the result of human intervention, and contains transgenic and / or heterologous nucleic acid molecules incorporated into its genome. Due to this type of genome change, recombinant plants are significantly different from relevant wild-type plants.

[0115] "Regulatory elements" refer to sequences involved in controlling the expression of a nucleotide sequence. Regulatory elements include promoters operably linked to a nucleotide sequence of interest and termination signals. They also typically encompass sequences required for proper translation of the nucleotide sequence.

[0116] Rhizobium family insecticidal proteins (RIPs) are proteins encoded by genes found in at least the genome of Rhizobium family bacteria that contain a cytotoxin domain and are active against at least insect pests in the genus Diabrotica. A "variant RIP" is an engineered RIP by replacing and / or deleting at least one amino acid found in a naturally occurring RIP and / or inserting an amino acid into the RIP sequence. As used herein, unless otherwise indicated, when the term "Rhizobium family insecticidal protein" or "RIP" is used alone, it is meant to include both naturally occurring proteins and engineered variant proteins.

[0117] The terms "substitution," "insertion," "addition," and "deletion" are used herein with reference to amino acid or nucleotide sequences. "Substitution" refers to the replacement of one or more nucleotides or amino acids with different nucleotides or amino acids, respectively. "Insertion" or "addition" is a change in a nucleotide or amino acid sequence that results in the addition of one or more nucleotides or amino acid residues, respectively, compared to the naturally occurring sequence. "Deletion" is defined as a change in a nucleotide or amino acid sequence in which one or more nucleotides or amino acid residues, respectively, are absent. Amino acid substitutions are typically single residue substitutions; insertions will generally be on the order of about 1 to 20 amino acids, although significantly larger insertions can be tolerated. Deletions range from about 1 to about 20 residues, although in some cases, deletions can be much larger. Substitutions, deletions, insertions, or any combination thereof can be used to arrive at the final variant polypeptide. Typically, a few amino acids are altered to minimize changes in the molecule. However, in some cases, greater changes can be tolerated. In certain embodiments, an amino acid substitution can be the result of replacing one amino acid with another having similar structural and / or chemical properties, such as replacing isoleucine with valine, i.e., a conservative amino acid substitution. Insertions or deletions can optionally range from 1 to 5 amino acids. In embodiments, substitutions can be made according to known "conservative substitutions." A "conservative substitution" is a substitution of an amino acid from one class with an amino acid from the same class, where the class is defined by common physicochemical amino acid side chain properties and a high frequency of substitution in homologous proteins found in nature. In contrast, in certain embodiments, the substitution is non-conservative. A "non-conservative substitution" is a substitution of an amino acid from one class with an amino acid from another class.

[0118] "Transformation" is a process for introducing heterologous nucleic acid into a host cell or organism. In specific embodiments, "transformation" means the stable integration of a DNA molecule into the genome (nucleus or plastid) of the organism of interest.

[0119] "Transformed / transgenic / recombinant" refers to a host organism, such as a bacterium or plant, into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the host's genome, or the nucleic acid molecule can also exist as an extrachromosomal molecule. Such extrachromosomal molecules are capable of autonomous replication. Transformed cells, tissues, or plants should be understood to encompass not only the end product of the transformation process, but also its transgenic progeny. A "non-transformed," "non-transgenic," or "non-recombinant" host refers to a wild-type organism, such as a bacterium or plant, that does not contain the heterologous nucleic acid molecule.

[0120] The present disclosure provides compositions and methods for controlling harmful insect pests. In particular, the present disclosure relates to insecticidal proteins and variants thereof, referred to herein as Rhizobiaceae insecticidal proteins (RIPs), which are active against at least Coleopteran insects, such as the corn rootworm (Western corn rootworm; WCR), Diabrotica barbatus (Diabrotica barber) (northern corn rootworm; NCR) and / or cucumber rootworm (southern corn rootworm; SCR) and / or other species of Diabrotica (including the Mexican corn rootworm (Mexican corn rootworm; MCR) and the South American rootworm (cucurbit beetle). The inventors of the present invention have discovered that certain proteins described in the art as hypothetical proteins, which are said to be encoded in the genome of at least one Gram-negative bacterium in the order Rhizobiales, are surprisingly insecticidal. More specifically, the coding sequences for these hypothetical proteins are found in the genome of bacteria of the family Rhizobiaceae. The family Rhizobiaceae is a family of the phylum Proteobacteria that includes several subgroups associated with plants, particularly plant roots. More specifically, the coding sequences for hypothetical proteins with insecticidal effects were found in the genomes of bacteria of the family Rhizobiaceae of the genera Sinorhizobium, Xiphium, Rhizobium and related genera. Even more specifically, the hypothetical proteins exemplified herein as insecticidal proteins are found in the genomes of bacteria of the family Rhizobiaceae. The coding sequences of the white protein include, but are not limited to, those in the genomes of Ensiferaridi strain (NCBI: txid1708715), Sinorhizobium sp. GL28 strain (NCBI: txid1358418), unclassified Rhizobiales bacterial strain (NCBI: txid1909294), and Rhizobium sp. SPY-1 strain (NCBI: txid2547961). After synthesizing nucleic acid molecules encoding the above-mentioned proteins and expressing the proteins in transgenic E. coli bacteria, the inventors determined that the proteins described in the art as hypothetical proteins surprisingly have insecticidal activity, particularly against rootworm insect pests. Such insecticidal proteins are generally referred to herein as Rhizobium family insecticidal proteins (RIPs), and those specifically exemplified herein are designated as RIP1Aa (Enf_adiCRW; SEQ ID NO: 166). SEQ ID NO: 1), RIP2Aa (Sinorhiz_GL28CRW; SEQ ID NO: 2), RIP3Aa (Rhizo_bactCRW; SEQ ID NO: 3), and RIP4Aa (Rhiz_SPCRW; SEQ ID NO: 4). One skilled in the art will recognize that, using the teachings of the present disclosure, one skilled in the art can identify sequences related to those described above, including, but not limited to, sequences in bacteria, nucleic acid molecules from environmental samples, and genomic databases, where such sequences may be designated as hypothetical, or they may have some other known function, etc. Such related sequences are contemplated to be encompassed by the present disclosure.The skilled artisan will understand the meaning of the term "related sequences" after reading this disclosure.As described in further detail below, the RIPs of the present disclosure possess unique cytotoxin domains that confer activity against at least coleopteran insect pests.

[0121] The present disclosure also relates to nucleic acids whose expression produces the disclosed RIPs, and to methods of making and using these RIPs to control insect pests. In certain non-limiting embodiments, expression of these nucleic acids produces insecticidal proteins that can be used to control at least coleopteran insects (such as western corn rootworm, northern corn rootworm, and / or southern corn rootworm), particularly when expressed in transgenic plants (such as transgenic corn plants).

[0122] In some non-limiting embodiments, the present disclosure encompasses nucleic acid molecules comprising, consisting essentially of, or consisting of a nucleotide sequence encoding a protein that is toxic to an insect pest (i.e., an insecticidal protein), wherein the nucleotide sequence (a) encodes a protein comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 1-21, or a toxin fragment thereof; (b) encodes a protein comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 1-21, or a toxin fragment thereof; In some embodiments, the insecticidal protein comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NOs: 1-21 or a toxic fragment thereof. In other embodiments, the nucleotide sequence comprises, consists essentially of, or consists of any one of SEQ ID NOs: 22-49 or a toxin encoding fragment thereof.

[0123] In some non-limiting embodiments, the present disclosure encompasses chimeric genes comprising a heterologous promoter operably linked to a nucleic acid molecule comprising, consisting essentially of, or consisting of a nucleotide sequence encoding a protein toxic to an insect pest, wherein the nucleotide sequence (a) encodes a protein comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 1-21, or a toxin fragment thereof; (b) encodes a protein comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 1-21, or a toxin fragment thereof; In some embodiments, the chimeric gene comprises an amino acid sequence of any one of SEQ ID NOs: 1-21 or a toxic fragment thereof. In other embodiments, the nucleotide sequence comprises any one of SEQ ID NOs: 22-49 or a toxin encoding fragment thereof. In some aspects of these embodiments, the chimeric gene is an expression cassette.

[0124] In other non-limiting embodiments, the promoter contained in the chimeric gene or expression cassette of the present disclosure is a plant-expressible promoter. In aspects of these embodiments, the plant-expressible promoter is selected from the group consisting of: ubiquitin, tuberose yellow virus, maize TrpA, OsMADS 6, maize H3 histone, bacteriophage T3 gene 9 5'UTR, maize sucrose synthase 1, maize alcohol dehydrogenase 1, maize light-harvesting complex, maize heat shock protein, maize mtl, pea small subunit RuBP carboxylase, rice actin, rice cyclophilin, Ti plasmid mannopine synthase, Ti plasmid nopaline synthase, petunia chalcone isomerase, legume glycine-rich protein 1, potato glycoprotein, lectin, CaMV 35S, and S-E9 small subunit RuBP carboxylase promoter.

[0125] In some non-limiting embodiments, the insecticidal protein encoded by the nucleic acid molecules of the present disclosure, or the chimeric genes of the present disclosure, or the expression cassettes of the present disclosure is active against coleopteran insect pests. In some aspects of these embodiments, the coleopteran insect pest is in the genus Diabrotica. In other aspects, the Diabrotica insect pest is Diabrotica zeae (Western corn rootworm; WCR), Diabrotica barbatae (Northern corn rootworm; NCR) and / or Diabrotica elevenspinata rootfeeding subspecies (Southern corn rootworm; SCR) and / or other Diabrotica species (including Mexican corn rootworm (Mexican corn rootworm; MCR).

[0126] In some non-limiting embodiments, the chimeric gene or expression cassette of the present disclosure comprises a nucleotide sequence encoding a RIP of the present disclosure, wherein the nucleotide sequence is codon-optimized for expression in a transgenic organism. In some aspects of these embodiments, the transgenic organism is a bacterium or a plant.

[0127] In other non-limiting embodiments, the transgenic bacteria are of the genus Bacillus, Clostridium, Xenorhabdus, Photorhabdus, Pasteurella, Escherichia, Pseudomonas, Erwinia, Serratia, Klebsiella, Salmonella, Pasteurella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Sinorhizobium, Sword Bacteria, Methylophilus, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, Sphingomonas, Burkholderia, Candidatus Glomeribacter, Dai's Bacteria, Grass Spirillum, Bradyrhizobium, Staphylococcus, Methylophilus, Greedy Bacteria, Streptococcus, Sphingobacteraceae (Chitinophaga) or Alcaligenes. In other embodiments, the transgenic bacteria are Escherichia coli. In other embodiments, the nucleotide sequence comprises, consists essentially of, or consists of any one of SEQ ID NOs: 26-45.

[0128] In other non-limiting embodiments, the transgenic plant is a monocot or a dicot. In other embodiments, the dicot is selected from the group consisting of soybean, sunflower, tomato, Brassica crops, cotton, sugar beet, and tobacco. In other aspects, the monocot is selected from the group consisting of barley, maize, oats, rice, sorghum, sugarcane, and wheat. In some aspects, the transgenic plant is a maize plant. In other embodiments, the nucleotide sequence comprises codons optimized for expression in maize. In still other embodiments, the nucleotide sequence comprises, consists essentially of, or consists of any one of SEQ ID NOs: 46-49.

[0129] In some non-limiting embodiments, the present disclosure encompasses proteins, and optionally isolated proteins, that are toxic to insect pests, i.e., insecticidal proteins, wherein the protein or isolated protein comprises, consists essentially of, or consists of: (a) an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 1-21, or a toxin fragment thereof; (b) an amino acid sequence comprising, consisting essentially of, or consisting of: any one of SEQ ID NOs: 1-21, or a toxin fragment thereof; (c) an amino acid sequence encoded by a nucleotide sequence that is identical to any one of SEQ ID NOs: 1-21; NO: 22-49, or a toxin encoding fragment thereof, having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; (d) an amino acid sequence encoded by a nucleotide sequence comprising, consisting essentially of, or consisting of any one of SEQ ID NO: 22-49, or a toxin encoding fragment thereof; or (e) the amino acid sequence of any one of (a)-(d), comprising a cytotoxin domain selected from the group consisting of SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53. One skilled in the art will recognize that modifications may be made to the exemplary RIPs encompassed by the present disclosure. Such modifications and substantially identical nucleic acid or amino acid molecules are encompassed by the present disclosure.

[0130] The present disclosure also includes engineered Rhizobium family insecticidal proteins, which can be described as mutant RIPs or variant RIPs or modified RIPs of the present disclosure. In some embodiments, the modification may include the replacement and / or deletion of one or more amino acids in the naturally occurring RIP sequence and / or the insertion of one or more additional amino acids into the naturally occurring RIP sequence. In other embodiments, the modification may include the replacement and / or deletion and / or insertion of one or more amino acids in the engineered RIP. The replacement and / or insertion may be with naturally occurring amino acids or non-naturally occurring amino acids. In some non-limiting embodiments, the modification comprises, is essentially composed of, or is composed of: the replacement and / or insertion and / or deletion of one or more of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and / or valine at the amino acid position of the RIP amino acid sequence. Such substitutions and / or insertions and / or deletions can be achieved by changing the codons in the nucleotide sequence encoding the RIP, thereby generating a modified rip nucleotide sequence encoding an engineered RIP (which is a mutant RIP or variant RIP or modified RIP of the present disclosure).

[0131] In some non-limiting embodiments, the RIP is modified by the following substitutions and / or insertions: (a) one or more amino acids with aliphatic hydrophobic side chains (e.g., alanine, isoleucine, methionine and / or valine; in embodiments, the amino acid is not alanine); (b) one or more amino acids with aromatic hydrophobic side chains (e.g., phenylalanine, tryptophan and / or tyrosine); (c) one or more amino acids with polar neutral side chains (e.g., asparagine, cysteine, glutamine, serine and / or threonine); (d) one or more amino acids with acidic side chains (e.g., aspartic acid and / or glutamic acid); one or more amino acids with basic side chains (e.g., arginine, histidine and / or lysine); (e) one or more glycine residues; (f) one or more proline residues; or (g) any combination of (a) to (f).

[0132] In other embodiments, amino acids are substituted and / or deleted and / or inserted in any of the amino acid sequences of the RIPs disclosed herein, particularly in any of the amino acid sequences of SEQ ID NOs: 1-4. In other embodiments, amino acids are substituted in SEQ ID NO: 3. In other embodiments, the substituted amino acids in SEQ ID NO: 3 are located at positions 50, 52, 56, 62, 64, 81, 126, 153, 169, 185, 207, 219, and / or 275. In other embodiments, the amino acid at position 50 is substituted with L, the amino acid at position 52 is substituted with L, the amino acid at position 56 is substituted with L, the amino acid at position 62 is substituted with C or L, the amino acid at position 64 is substituted with C or L, the amino acid at position 81 is substituted with L, the amino acid at position 126 is substituted with L, the amino acid at position 153 is substituted with L, the amino acid at position 169 is substituted with L, the amino acid at position 185 is substituted with L, the amino acid at position 207 is substituted with L, the amino acid at position 218 is substituted with L, or the amino acid at position 275 is substituted with L.

[0133] In another embodiment, present disclosure provides a kind of chimeric RIP toxin, it includes the protein fusion tag connected with a complete RIP sequence or a part of RIP sequence (such as cytotoxin domain).The protein fusion tag can be connected to N-terminal (for example, at amino acid 1 or 2 of RIP sequence), or alternatively, the protein fusion tag can be connected to the C-terminal of RIP sequence.The protein fusion tag can be polyhistidine, polyarginine, haloalkane dehalogenase, streptavidin combination, glutathione s-transferase (GST), maltose binding protein (MBP), thioredoxin, small ubiquitin-like modifier (SUMO), N-utilizing substance A (NusA), protein disulfide isomerase I (DsbA), Mistic, ketosteroid isomerase (KSI) or TrpE, c-myc, hemagglutinin antigen (HA), FLAG, 1D4, calmodulin binding peptide, chitin binding domain, cellulose binding domain, S-tag or Softag3 protein fusion tag. These can be used in methods for producing, isolating, or purifying any of the RIP toxins disclosed herein. The disclosure also provides recombinant polynucleotides, such as constructs, encoding fusion tags linked to the RIP toxins disclosed herein. In some embodiments, a SUMO tag is linked to the N-terminus of the RIP1Aa toxin to generate a SUMO-RIP1Aa toxin (SEQ ID NO: 21) encoded by a SUMO-rip1Aa polynucleotide (SEQ ID NO: 45).

[0134] In some non-limiting embodiments, the present disclosure encompasses variant RIPs comprising, consisting essentially of, or consisting of an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a) SEQ ID NO: 1 and further comprising at least one mutation at a position corresponding to amino acid positions 1-345 of SEQ ID NO: 1; or b) SEQ ID NO: 2 NO:2 having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity and further comprising at least one mutation at a position corresponding to amino acid positions 1-344 of SEQ ID NO:2; or c) a mutation with a residue corresponding to amino acid position 1-344 of SEQ ID NO:2; NO:3 having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity and further comprising at least one mutation at a position corresponding to amino acid position 1-351 of SEQ ID NO:3; or d) a mutation at a position corresponding to amino acid position 1-351 of SEQ ID NO:3; NO: 4 has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, and further comprises at least one mutation at a position corresponding to amino acid positions 1-347 of SEQ ID NO: 4. In other non-limiting embodiments, the mutation is at an amino acid position corresponding to amino acid position 50, 52, 56, 62, 64, 81, 126, 153, 169, 185, 207, 219, or 275 of SEQ ID NO: 3, or any combination thereof. In other non-limiting embodiments, the mutation is at position 50, 52, 56, 62, 64, 81, 126, 153, 169, 185, 207, 219, or 275 of SEQ ID NO:3.In other embodiments, the mutation at position 50 is I50L, the mutation at position 52 is I52L, the mutation at position 56 is I56L, the mutation at position 62 is A62C or A62L, the mutation at position 64 is A64C or A64L, the mutation at position 81 is I81L, the mutation at position 126 is I126L, the mutation at position 153 is I153L, the mutation at position 169 is I169L, the mutation at position 185 is I185L, the mutation at position 207 is I207L, the mutation at position 2019 is I219L, or the mutation at position 275 is I275L. In yet other embodiments, the variant RIP comprises, consists essentially of, or consists of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, ID NO:19, or SEQ ID NO:20.

[0135] In some non-limiting embodiments, when tested under the same conditions (e.g., enzyme concentration, protein concentration, pH, temperature and / or time), the variant RIP of the present disclosure has enhanced digestion by a mammalian digestive protease (e.g., pepsin) compared to a suitable control and / or a parent molecule without the modification of the present disclosure. Methods for evaluating protein digestion with pepsin and other digestive proteases are known in the art. For example, digestion using pepsin can be performed at about 37° C. and about pH 1.2, optionally with an enzyme concentration of about 10 units (U) of pepsin / microgram of protein.

[0136] In some embodiments, the RIPs of the present disclosure, including variant RIPs of the present disclosure, are active against insect pests of the order Coleopteran. Coleopteran insects include, but are not limited to, any currently known or later identified Coleopteran insects, including those of the suborders Protocoleoptera, Myxophaga, Carnivora, and Polyphaga, and any combination thereof.

[0137] In some non-limiting embodiments, the RIP toxins and variant RIP toxins of the present disclosure are active against Diabrotica species. Diabrotica is a genus of beetles in the order Coleoptera, commonly known as "corn rootworms" or "cucumber beetles." Exemplary Diabrotica species include, but are not limited to: Diabrotica longicornis barberi (northern corn rootworm), Diabrotica nominata (western corn rootworm), Diabrotica eudicotus (southern corn rootworm), D. balteata (banded cucumber beetle), D. undecimpunctata undecimpunctata (western spotted cucumber beetle), D. significata (3-spotted leaf beetle), D. speciosa (chrysanthemum beetle), Mexican corn rootworm (Mexican corn rootworm), Benni rootworm (D. beetle), Crista rootworm (D. cristata), Curvi rootworm (D. curvipustulata), Dissimilis rootworm (D. dissimilis), Elegantula rootworm (D. elegantula), Emorsitans rootworm (D. graminea), Hispanic rootworm (D. hispanolae), Lemini rootworm (D. graminea), and Lamini rootworm (D. graminea). D. lemniscata, D. linsleyi, D. milleri, D. nummularis, D. occlusa, D. porracea, D. scutellata, D. tibialis, D. trifasciata, and D. viridula; and any combination thereof.

[0138] Other non-limiting examples of coleopteran insect pests according to the present disclosure include Leptinotarsa ​​spp., such as the potato beetle (Colorado potato beetle); Chrysomelas spp., such as C. scripta (cottonwood leaf beetle); Hypothenemus spp., such as H. hampei (coffee berry borer); Sitophilus spp., such as S. zeamais (maize weevil); Epitrix spp., such as E. hirtipennis (tobacco flea beetle); (tobacco flea beetle) and E. cucumeris (potato flea beetle): Phyllotreta spp., such as P. cruciferae (crucifer flea beetle) and P. pusilla (western black flea beetle); Anthonomus spp., such as A. eugenii (pepper weevil); Hemicrepidus spp., such as H. memnonius (wireworms); Melanotus spp., such as M. communis (wireworms); Ceutorhychus spp. spp., such as C. assimilis (cabbage seedpod weevil); Aeolus spp., such as A. mellillus (wireworm); Aeolus spp., such as A. mancus (wheatwireworm); Horistonotus spp., such as H. uhlerii (sandwireworm); Sphenophorus spp., such as S. maidis (maize billbug), S. zeae (timothy billbug), billbug), S. parvulus (bluegrass billbug), and S. callosus (southern corn billbug); Phyllophaga spp. (white grubs); Chaetocnema spp., such as C. pulicaria (corn flea beetle); Popillia spp., such as P.japonica (Japanese beetle); Epilaxna spp., such as E. varivestis (Mexican bean beetle); Cerotoma spp., such as C. trifurcate (Bean leaf beetle); Epicauta spp., such as E. pestifera and E. lemniscata (lister beetles); and any combination of the foregoing.

[0139] The RIPs of the present disclosure may also be active against Lepidoptera. Such Lepidoptera include, but are not limited to, any insect classified as such, now known or later identified, including those species within the suborders Hypogyroptera, Proboscis, and Heterotrichum, and any combination thereof. Exemplary Lepidoptera include, but are not limited to, Ostrinia species, such as O. nubilalis (European corn borer); Plutella species, such as P. xylostella (diamondback moth); Spodoptera species, such as S. frugiperda (fall armyworm), S. ornithogalli (yellowstriped armyworm), S. praefica (western yellowstriped armyworm), S. eridania (southern armyworm), and S. exigua (beet armyworm); Spodoptera species, such as A. ipsilon (black cutworm), A. segetum (common cutworm), A. gladiaria (clayback cutworm), and A. cutworm and A. orthogonia (palewestern cutworm); Striacosta spp., such as S. albicosta (western bean cutworm); Helicoverpa spp., such as H. zea (corn earworm), H. punctigera (native budworm), S. littoralis (Egyptian cotton leafworm), and H. armigera (cotton bollworm); Heliothis spp., such as Heliothis virescens (tobacco budworm); Diatraea spp., such as D. grandiosella (southwestern corn borer); borer and D. saccharalis (sugarcane borer); Trichoplusia spp., such as T.ni, cabbage looper; Sesamia spp., such as S. nonagroides (Mediterranean corn borer); Pectinophora spp., such as P. gossypiella (pink bollworm); Cochylis spp., such as C. hospes (banded sunflower moth); Manduca spp., such as M. sexta (tobacco hornworm) and M. quinquemaculata (tomato hornworm); Elasmopalpus spp., such as E. lignosellus (lesser cornstalk borer); borer); Pseudoplusia spp., such as P. includens (soybean moth); Anticarsia spp., such as Spodoptera velvet bean caterpillar; Plathypena spp., such as P. scabra (green cloverworm); Pieris spp., such as P. brassicae (cabbage butterfly); Papaipema spp., such as P. nebris (stalk borer); Pseudaletia spp., such as P. unipuncta (common armyworm); Peridromas spp., such as P. saucia (variegated cutworm); cutworm; Keiferia spp., such as K. lycopersicella (tomato pinworm); Artogeia spp., such as A. rapae (imported cabbageworm); Phthorimaea spp., such as P. operculella (potato tuberworm); Crymodes spp., such as C.devastator, glassy cutworm; Feltia spp., such as F. ducens, dingy cutworm; and any combination of the foregoing.

[0140] The RIPs of the disclosure may also be active against Hemiptera, Diptera, Lygus species, and / or other piercing-sucking insects (e.g., piercing-sucking insects of the Order Orthoptera or Thysanoptera). Insects of the order Diptera include, but are not limited to, any Diptera insect now known or hereafter identified, including, but not limited to, Liriomyza spp., such as L. trifolii (leaf miner) and L. sativae (vegetable leaf miner); Scrobipalpula spp., such as S. absoluta (tomato leaf miner); Delia spp., such as D. platura (seedcorn maggot), D. brassicae (cabbage maggot), and D. radicum (cabbage root fly); Psilia spp., such as P. rosae (carrot rust fly); Tetanops spp., such as P. rosae (carrot rust fly); spp.), such as T. myopaeformis (sugarbeet root maggot); and any combination of the foregoing.

[0141] Insects of the order Orthoptera include, but are not limited to, any Orthoptera insect now known or later identified, including, but not limited to, Melanoplus spp., such as M. differentialis (Differential grasshopper), M. femurrubrum (Redlegged grasshopper), M. bivittatus (Twostriped grasshopper); and any combination thereof.

[0142] Insects of the order Thysanoptera include, but are not limited to, any Thysanoptera insect now known or later identified, including, but not limited to, Frankliniella spp., such as F. occidentalis (western flower thrips) and F. fusca (tobacco thrips); and Thrips spp., such as T. tabaci (onion thrips), T. palmi (melon thrips); and any combinations of the foregoing.

[0143] The RIPs of the present disclosure may also be active against nematodes. As used herein, the term "nematode" encompasses any currently known or later identified organism classified as belonging to the phylum Nematoda of the animal kingdom, including but not limited to nematodes in the class Adenophora (including, for example, the orders Rhynchoides, Isopharyngea, Monodontia, Lanceolata, Trichodesmata, Chondriomorpha, Muspasianales, Lactophagus, Chromophora, Taenelena, Catenellata, and Monogyria) and / or the class Tubulonema (including, for example, the orders Rhabditis, Strongylida, Ascaris, Spirulina, Camelida, Digastricales, Sphaeroides, and Aphelenchus).

[0144] Nematodes include, but are not limited to, parasitic nematodes, such as root-knot nematodes, cyst nematodes, and / or decay nematodes. Exemplary genera of nematodes according to the present disclosure include, but are not limited to, root-knot nematodes (root-knot nematodes), Heterodera (cyst nematodes), Globodera (cyst nematodes), Penetrating nematodes (piercing nematodes), Renoides (kidney-shaped kidney-shaped nematodes), Pratylenchus (rot nematodes), Aphelenchus (leaf nematodes), Heliconema (heliconema), Nematode (lance nematodes), Pseudocercosus (short and thick root nematodes), Long-nematode nematodes, Pearl Nematode (pseudo-root-knot nematodes), Subanguina, Stinging nematodes, Microcircle nematodes (microcircle nematodes), nematodes), Ring Nematodes (ring nematodes), Stem Nematodes (stem nematodes), Trypanoidea (tie nematodes), Semirothrix (semirothrix nematodes), Sheath Nematodes (sheath nematodes), Subroot Nematodes (subroot nematodes), Root-knot Nematodes (Hypsoperine), Ditylenchus (large stem nematodes), Melinius spp., Punctodera, Quinisulcius, Shield Nematodes (shield nematodes), Xiphinema (dagger nematodes), Dwarf Nematodes (dwarf nematodes), Penelope nematodes (puncture nematodes), Bursaphelenchus (roundworms), and any combination thereof.

[0145] Exemplary plant parasitic nematodes according to the present disclosure include, but are not limited to, Belonolaimus gracilis, Belonolaimus longicaudatus, Bursaphelenchus xylophilus (pine wood nematode), Criconemoides ornata, Ditylenchus destructor (potato rot nematode), Ditylenchus dipsaci (stem and bulb nematode), Globodera pallida (potato cyst nematode), Globodera rostochiensis (golden nematode), Heterodera glycines (soybean cyst nematode), Heteroderas schachtii (sugar beet cyst nematode), and Ditylenchus schachtii (potato cyst nematode). nematode); Heterodera zeae, corn cystnematode; Heterodera avenae,cereal cyst nematode), Heterodera carotae, Heterodera trifolii, Hoplolaimus columbus, Hoplolaimus galeatus, Hoplolaimus magnistylus, Longidorus breviannulatus, Meloidogynearenaria, Meloidogyne chitwoodi, Meloidogyne hapla, Meloidogyne incognita, Meloidogyne javanica, Mesocriconema xenoplax, Nacobbus aberrans, Naccobus dorsalis, Paratrichodorus christiei, Paratrichodorus minor), Pratylenchus brachyurus, Pratylenchus crenatus, Pratylenchus hexincisus, Pratylenchus negletus, Pratylenchus penetrans, Pratylenchus projectus, Pratylenchus scribneri, Pratylenchus tenuicaudatus, Pratylenchus thornei, Pratylenchus zeae, Punctodera chaccoensis, Quinisulcius acutus, Radopholus similis, Rotylenchulus reniformis, Tylenchorhynchus dubius), Tylenchulus semipenetrans, Siphinema americanum, X. Mediterraneum, and any combination of the foregoing.

[0146] The present disclosure also encompasses recombinant vectors and / or recombinant constructs, and these recombinant vectors or constructs may also be referred to as vectors or constructs, which comprise expression cassettes and / or nucleic acid molecules of the present disclosure. In this type of vector, these nucleic acids are preferably in expression cassettes, and these expression cassettes comprise regulatory elements for expressing nucleotide molecules in host cells that can express nucleotide molecules. This type of regulatory element generally comprises a promoter and a termination signal and preferably also comprises elements, and these elements allow effective translation of the polypeptide coded by the nucleic acid of the present disclosure. The vector comprising nucleic acid can replicate (preferably as an extrachromosomal molecule) in specific host cells and therefore can be used to increase the nucleic acid of the present disclosure in these host cells.

[0147] The present disclosure also encompasses host cells comprising recombinant vectors, expression cassettes or nucleic acid molecules of the present disclosure. In other embodiments, such vectors are viral vectors and are used to replicate nucleotide sequences in specific host cells (e.g., insect cells or plant cells). Recombinant vectors are also used to transform the nucleic acid molecules of the present disclosure into host cells, whereby these nucleic acid molecules are stably integrated into the DNA of a transgenic host. In certain embodiments, the host cell is a bacterial cell or a plant cell. In some aspects of these embodiments, the bacterial cell is in the genus Bacillus, Clostridium, Xenorhabdus, Photorhabdus, Pasteurella, Escherichia, Pseudomonas, Erwinia, Serratia, Klebsiella, Salmonella, Pasteurella, Xanthomonas, Streptomyces, Rhizobium, Sinorhizobium, Sword Pseudomonas, Rhodopseudomonas, Methylophilus, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, Sphingomonas, Burkholderia, Candidatus Glomeribacter, Dai Shi, Grass Spirillum, Bradyrhizobium, Staphylococcus, Methylophilus, Greedy Bacteria, Streptococcus, Sphingobacteraceae or Alcaligenes. In other aspects of these embodiments, the host cell for such recombinant vectors is an endophyte or epiphyte. In some other aspects of these embodiments, the host cell is a plant cell, such as a dicot cell or a monocot cell. In other aspects, the dicot cell is selected from the group consisting of a soybean cell, a sunflower cell, a tomato cell, a Brassica plant cell, a cotton cell, a sugar beet cell, and a tobacco cell. In still other aspects, the monocot cell is selected from the group consisting of a barley cell, a maize cell, an oat cell, a rice cell, a sorghum cell, a sugar cane cell, and a wheat cell.

[0148] In some non-limiting embodiments of the present disclosure, at least one of the nucleic acid molecules of the present disclosure is inserted into an appropriate expression cassette (comprising a promoter and a termination signal). Expression of the nucleic acid can be constitutive, or an inducible promoter that initiates transcription in response to various types of stimuli can be used. In another embodiment, the cell in which the insecticidal protein of the present disclosure is expressed is a microorganism, such as a virus, bacteria, or fungus. In yet another embodiment, a virus (such as a baculovirus) contains a nucleic acid of the present disclosure in its genome and, after infecting an appropriate eukaryotic cell (suitable for viral replication and expression of the nucleic acid), expresses a large amount of the corresponding insecticidal protein. The insecticidal protein thus produced is used as an insecticide. Alternatively, a baculovirus engineered to contain the nucleic acid is used to infect insects in vivo and kill them through expression of the insecticidal toxin or through a combination of viral infection and expression of the insecticidal toxin. In another embodiment, the present disclosure also encompasses a method for producing a polypeptide having insecticidal activity, comprising culturing a host cell under conditions in which a nucleic acid molecule encoding the polypeptide is expressed.

[0149] Bacterial cells are also hosts for expressing the nucleic acid of the present disclosure. In one embodiment, non-pathogenic symbiotic bacteria (so-called endophytes) that can live and replicate in plant tissues are used, or non-pathogenic symbiotic bacteria (so-called epiphytes) that can be settled in phyllosphere or rhizosphere are used. Such bacteria include the bacterium of the following genus: Agrobacterium, Alcaligenes, Azospirillum, Azotobacter, Bacillus, Corynebacterium, Enterobacter, Erwinia, Flavobacterium, Klebsiella, Pseudomonas, Rhizobium, Sinorhizobium, Sword Pseudomonas, Serratia, Streptomyces, Sphingomonas, Burkholderia, Candidatus Glomeribacter, Dai Shi Pseudomonas, Grass Spirillum, Bradyrhizobium, Staphylococcus, Methylophilus, Greedy Phage, Streptococcus, Sphingobacteraceae and Xanthomonas. Symbiotic fungi such as Trichoderma and Glucocladium are also possible hosts for expressing the nucleic acids of the invention for the same purpose.

[0150] These gene manipulation techniques are specific for different available hosts and are known in the art. For example, expression vectors pKK223-3 and pKK223-2 can be used to express heterologous genes after tac or trc promoters in Escherichia coli (in transcription or translation fusion). In order to express the operon encoding multiple ORFs, the simplest method is to insert the operon into a vector (such as pKK223-3) in transcriptional fusion, allowing the homologous ribosome binding site of the heterologous gene to be utilized. Overexpression techniques in gram-positive species (such as Bacillus) are also known in the art, and can be used in the context of the present disclosure (Quax et al., in: Industrial Microorganisms: Basic and Applied Molecular Genetics [industrial microorganisms: basic and applied molecular genetics], editor Baltz et al., American Society for Microbiology [American Society for Microbiology], Washington (1993)). Alternative systems for overexpression rely on, for example, yeast vectors and include the use of Pichia, Saccharomyces, and Kluyveromyces (Sreekrishna, In: Industrial microorganisms: basic and applied molecular genetics, Baltz, Hegeman, and Skatrud, eds., American Society for Microbiology, Washington (1993); Dequin and Barre, Biotechnology L2: 173-177 (1994); van den Berg et al., Biotechnology 8: 135-139 (1990)).

[0151] In yet other embodiments, the present disclosure encompasses methods for controlling insect pests comprising delivering an insecticide protein of the present disclosure in an insect control-effective amount to the insect pest. In some aspects of these embodiments, the insecticide protein is delivered via a transgenic plant or by topical application of an insecticidal composition comprising the insecticide protein. In other aspects, the transgenic plant or the insecticidal composition comprises a second insecticide different from the RIP of the present disclosure. In still other aspects, the second insecticide is a protein, dsRNA, or a chemical. In still other aspects, the protein is selected from the group consisting of: Cry protein, VIP toxin, potato glycoprotein, protease, protease inhibitor, urease, α-amylase inhibitor, pore-forming protein, lectin, engineered antibody or antibody fragment, or chitinase; or the chemical is a carbamate, pyrethroid, organophosphate, friprole, neonicotinoid, organochloride, nereistoxin or a combination thereof; or the chemical comprises an active ingredient selected from the group consisting of: carbofuran, carbaryl, methomyl, cypermethrin, tefluthrin, permethrin, cyfluthrin, λ-cyfluthrin, cypermethrin, deltamethrin, chlorpyrifos, chloroxyfos, dimethoate, anthiophanate, malathion, methyl parathion, phorate, terbufos, tert-butylpyrimidophos, fipronil, acetamiprid, imidacloprid, thiacloprid, thiamethoxam, endosulfan, sulfamethoxam and a combination thereof.

[0152] In some embodiments of the present disclosure, at least one of the RIP toxins disclosed herein is expressed in a higher organism, such as a plant. Transgenic plants expressing an insecticide protein in an insect-control-effective amount protect themselves from damage by insect pests. When an insect pest begins to feed on the transgenic plant, it also ingests the expressed insecticide protein. This can prevent the insect from further feeding on plant tissue and / or can even injure or kill the insect. The nucleic acid molecules disclosed herein are inserted into an expression cassette, which can then be stably integrated into the plant's genome. In other embodiments, the nucleic acid molecules are included in non-pathogenic, self-replicating viruses. Plants transformed according to the present disclosure can be monocots or dicots and include, but are not limited to, corn, wheat, oats, turf grass, pasture grass, flax, barley, rye, sweet potato, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, pepper, celery, squash, pumpkin, hemp, zucchini, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tomato, sorghum, sugarcane, sugar beet, sunflower, rapeseed, clover, tobacco, carrot, cotton, alfalfa, rice, potato, eggplant, cucumber, Arabidopsis species, and woody plants, such as coniferous and deciduous trees.

[0153] In some embodiments, the present disclosure encompasses methods of producing a protein that is toxic to an insect pest, i.e., an insecticidal protein, comprising: (a) obtaining a host cell comprising a gene that itself comprises an expression cassette and / or nucleic acid molecule of the present disclosure; and (b) growing the host cell or a transgenic host comprising the host cell under conditions whereby the transgenic host cell produces the protein that is toxic to an insect pest.

[0154] In other embodiments, the present disclosure encompasses methods for producing transgenic plants or plant parts having enhanced insect resistance compared to control plants or plant parts, the methods comprising: (a) introducing a chimeric gene or expression cassette or vector comprising a nucleic acid molecule encoding an insecticidal protein of the present disclosure into a plant or plant part, wherein the insecticidal protein is expressed in the plant or plant part, thereby producing a plant or plant part having enhanced insect resistance. In other embodiments, the chimeric gene, expression cassette or vector can encode a RIP toxin of the present disclosure comprising, consisting essentially of, or consisting of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical or similar to any one of SEQ ID NOs: 1-21. "Enhanced" insect resistance can be measured by any toxic effect of the transgenic plant on insect pests that feed on the transgenic plant. The enhanced insect resistance can be 0%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000% greater insecticidal activity than a control plant not expressing the insecticidal protein. Plants or plant parts having enhanced insect resistance compared to control plants or plant parts can be produced by plant transformation, plant tissue culture, or breeding methods. Plants or plant parts can be produced by sexual or asexual propagation methods. Any suitable control plant or plant part can be used, for example, a plant having the same or similar genetic background grown in the same environment. In an embodiment, the control plant or plant part has the same genetic background and is grown in the same environment as the described plant, but does not contain the molecules of the disclosure, while the described plant contains the nucleic acid molecules of the disclosure.

[0155] In other embodiments, the present disclosure encompasses a method of increasing insect resistance in a plant or plant part compared to a control plant or plant part, the method comprising expressing in the plant or plant part a nucleic acid molecule or expression cassette of the present disclosure, wherein expression of the heterologous nucleic acid of the expression cassette results in the plant or plant part having increased insect resistance compared to the control plant or plant part. In some embodiments, the expression cassette or nucleic acid molecule comprises a promoter operably linked to a heterologous nucleic acid molecule comprising a nucleotide sequence comprising, consisting essentially of, or consisting of: (a) a nucleotide sequence of any one of SEQ ID NOs: 22-49; (b) a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a nucleotide sequence of any one of SEQ ID NOs: 22-49; (c) a nucleotide sequence encoding a protein, wherein the amino acid sequence of the protein comprises, consists essentially of, or consists of: any one of SEQ ID NOs: 1-21; (d) a nucleotide sequence encoding a protein, wherein the amino acid sequence of the protein is identical to any one of SEQ ID NOs: 1-22. ID NO: 1-21 any one of the amino acid sequence is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical; (e) the nucleotide sequence of any one of (a) to (d) above, which is codon-optimized for expression in a transgenic host organism; or (f) a nucleotide sequence complementary to the nucleotide sequence of any one of (a) to (e) above. The nucleic acid molecule or expression cassette can be introduced into a plant. In some embodiments, the nucleic acid molecule or expression cassette can be introduced into a plant part, and a plant comprising the nucleic acid molecule or expression cassette can be produced from the plant part.

[0156] In certain embodiments, present disclosure encompasses methods for producing plants having enhanced insect resistance compared to control plants, the method comprising detecting a heterologous nucleic acid comprising a nucleic acid molecule or expression cassette of the present disclosure in a plant part, and producing a plant from the plant part, thereby producing a plant having enhanced insect resistance compared to a control plant. In a further embodiment, present disclosure encompasses methods for identifying a plant or plant part having enhanced insect resistance compared to a control plant or plant part, the method comprising detecting a nucleic acid molecule or expression cassette of the present disclosure in the plant or plant part, thereby identifying a plant or plant part having enhanced insect resistance. In a further embodiment, the expression cassette or its diagnostic fragment is detected in an amplified product from a nucleic acid sample of the plant or plant part. The diagnostic fragment can be a nucleic acid molecule of at least 10 consecutive nucleotides long that is unique to the expression cassette of the present disclosure.

[0157] In other embodiments, the present disclosure encompasses methods of producing plants having enhanced insect resistance compared to a control plant or plant part, the method comprising crossing a first parent plant with a second parent plant, wherein at least the first parent plant comprises in its genome a heterologous nucleic acid comprising a nucleic acid molecule or expression cassette of the present disclosure; and producing progeny generations, wherein the progeny generations comprise at least one plant having in its genome the heterologous nucleic acid and exhibiting enhanced insect resistance compared to a control plant.

[0158] In some aspects of the above-described embodiments, the method of the present disclosure gives plants or plant parts the insect resistance of the enhancement for coleopteran insect pests. In an example, insect control of coleopteran insect pests has been demonstrated. In a further aspect, the method of the present disclosure gives plants or plant parts the insect resistance of the enhancement for Diabrotica species (including corn rootworms, Barker's rootworms, cucumber eleven-star leaf beetle root-feeding subspecies, Mexican corn rootworms and / or South American leaf beetles (Diabrotica peciosa)) and / or related species. In a further embodiment, the method of the present disclosure gives plants or plant parts the insect resistance of the enhancement for western corn rootworms, Barker's rootworms and / or cucumber eleven-star leaf beetle root-feeding subspecies.

[0159] In some embodiments, the present disclosure encompasses transgenic plants comprising a heterologous nucleic acid molecule or expression cassette of the present disclosure that, when transcribed or translated, confers enhanced insect resistance to the transgenic plant. In some aspects of these embodiments, the heterologous nucleic acid molecule or expression cassette comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 21-49. In other embodiments, the transgenic plant is a dicot or a monocot. In another aspect, the transgenic plant is alfalfa, apple, apricot, artichoke, arugula, asparagus, avocado, banana, bean, beet, blackberry, blueberry, broccoli, Brussels sprouts, cabbage, canola, cantaloupe, carrot, cassava, cauliflower, celery, cherry, cilantro, citrus, clementine, coffee bean, corn, cotton, cucumber, Douglas fir, eggplant, endive, kohlrabi, eucalyptus, fennel, fig, gourd, grape, grapefruit, honeydew melon, jicama, kiwi, lettuce, leek, In some embodiments, the transgenic plant is a transgenic plant selected from the group consisting of lemon, sour orange, loblolly pine, mango, muskmelon, mushroom, nut, okra, onion, orange, ornamental plant, papaya, parsley, pea, peach, peanut, pear, pepper, persimmon, pine, pine, plantain, plum, pomegranate, poplar, potato, pumpkin, quince, radiata pine, red chicory, radish, raspberry, rice, rye, sorghum, southern pine, soybean, spinach, small pumpkin, strawberry, beet, sunflower, sweet potato, liquidambar, citrus, tea, tobacco, tomato, turf, vine, watermelon, potato or green zucchini. In other aspects, the transgenic plant is millet, switchgrass, maize, sorghum, wheat, oat, lawn grass, pasture grass, flax, rice, sugarcane, rape or barley. In other embodiments, the transgenic plant is a transgenic maize (corn) plant comprising a rip coding sequence (wherein codon optimized for expression in maize), such as any one of SEQ ID NO:46-49.

[0160] In some embodiments, the present disclosure encompasses nucleic acid molecules encoding the insecticidal proteins of the present disclosure that have been modified and optimized for expression in transgenic plants. While genes from microbial organisms can in many cases be expressed at high levels in plants without modification, low expression in transgenic plants can be due to microbial nucleic acids having codons that are not preferred in plants. It is known in the art that all organisms have specific preferences for codon usage, and the codons of the nucleic acids described in the present disclosure can be altered to conform to plant preferences while maintaining the amino acids encoded thereby, or by making certain amino acid changes to the encoded insecticidal protein. Furthermore, high expression in plants is best achieved with coding sequences having a GC content of at least about 35%, preferably greater than about 45%, more preferably greater than about 50%, and most preferably greater than about 60%. Microbial nucleic acids with low GC content may express poorly in plants due to the presence of ATTTA motifs, which can destabilize the message, and AATAAA motifs, which can cause inappropriate polyadenylation. In embodiments, the sequences can be modified to accommodate the specific codon preferences and GC content preferences of monocots or dicots, as these preferences have been shown to be different (Murray et al. Nucl. Acids Res. 17:477-498 (1989)). In addition, the nucleic acids are screened for the presence of illogical splice sites that may cause message shortening. All desired changes within the nucleic acids, such as those described above, can be altered using well-known techniques of site-directed mutagenesis, PCR, and synthetic gene construction, for example, using the methods described in published patent applications EP 0 385 962, EP 0 359 472, and WO 93 / 07278.

[0161] In some embodiments of the present disclosure, the coding sequence of the insecticide protein of the present disclosure is manufactured according to the program disclosed in U.S. Patent No. 5,625,136 (incorporated herein by reference). In this program, the preferred codons of maize, i.e., the single codons that most frequently encode the amino acid in maize, have been used. The preferred codons of maize for specific amino acids can, for example, be derived from the known gene sequences of maize. The maize codons for 28 genes from maize plants are used to be found in Murray et al., Nucleic Acids Research [nucleotide sequence] 17:477-498 (1989), the disclosure of which is incorporated herein by reference. In this way, these nucleotide sequences can be optimized for expression in any plant. It is recognized that all or any part of this gene sequence can be optimized or synthetic. That is, synthetic or partially optimized sequences can also be used.

[0162] For more effective translation initiation, the sequence adjacent to the start methionine can be modified. For example, they can be modified by comprising sequences known to be effective in plants. Joshi has proposed a suitable consensus sequence for plants (NAR 15:6643-6653 (1987)), and Clontech has proposed another consensus translation initiator (1993 / 1994 catalogue, page 210). These consensus sequences are suitable for use with the nucleic acids disclosed herein. In an embodiment, these sequences are incorporated into the construct comprising the nucleic acid, reaching and including ATG (and not modifying the second amino acid), or alternatively reaching and including the GTC after the ATG (with the possibility of modifying the second amino acid of the transgenic).

[0163] In transgenic plants, the expression of these nucleic acids is driven by promoters that play a role in plants. The selection of promoters will vary according to the time and space required for expression, and also according to the target species. Therefore, the expression of the nucleic acids disclosed herein in leaves, stalks or stems, spikes, inflorescences (such as spikes, panicles, cobs, etc.), roots, and / or seedlings is preferred. However, in many cases, protection is sought for more than one type of insect pest, and therefore expression in multiple tissues is desirable. Although it has been shown that many promoters from dicots are operable in monocots and vice versa, it is ideal to select dicot promoters for expression in dicots, and to select monocot promoters for expression in monocots. However, there is no restriction on the origin of the selected promoter; as long as they can effectively drive nucleic acid expression in the desired cell, it is enough.

[0164] In certain embodiments, a promoter for constitutive expression has been used, including actin or ubiquitin or cmp promoters, or CaMV35S and 19S promoters. The nucleic acid of this disclosure can also be expressed under the regulation of a promoter that is regulated by a chemical method. The preferred technology for chemical induction of genetic expression is described in detail in open application EP 0 332 104 (Ciba-Geigy) and United States Patent (USP) 5,614,395. The preferred promoter for chemical induction is the tobacco PR-1a promoter.

[0165] In other embodiments, a class of wound-inducible promoters can be used. Numerous promoters have been described that express at the site of wounding and also at the site of infection by plant pathogens. Ideally, such promoters should be locally active only at the site of infection, and in this way, the insecticidal proteins of the present disclosure accumulate only in the cells that need to synthesize these proteins to kill the invading insect pests. Such preferred promoters include 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), and Warner et al. Plant J. 3:191-201 (1993).

[0166] Tissue-specific or tissue-preferred promoters for expressing genes encoding the insecticidal proteins of the present disclosure in plants, particularly corn, are those that express directly in roots, pith, leaves, or pollen, particularly roots. Such promoters, such as those isolated from PEPC or trpA, are disclosed in U.S. Patent No. 5,625,136, or those isolated from MTL, are disclosed in U.S. Patent No. 5,466,785. Both U.S. patents are incorporated herein by reference in their entirety.

[0167] In addition, promoters that function in plastids can be used. Non-limiting examples of such promoters include the bacteriophage T3 gene 9 5'UTR and other promoters disclosed in U.S. Patent No. 7,579,516. Other promoters suitable for use in the present disclosure include, but are not limited to, the S-E9 small subunit RuBP carboxylase promoter and the Kunitz trypsin inhibitor gene promoter (Kti3).

[0168] In some embodiments of the present disclosure, an inducible promoter can be used. Thus, for example, a chemically regulated promoter can be used to regulate the expression of the nucleotide sequence of the present disclosure by applying an exogenous chemical regulator. The expression of the nucleotide sequence of the present disclosure is regulated by a chemically regulated promoter so that the polypeptide of the present disclosure can be synthesized only when the crop plants are treated with inducing chemicals. Depending on the purpose, when applying chemicals to induce the expression of the nucleotide sequence of the present disclosure, the promoter can be a chemically inducible promoter, or when applying chemicals to suppress the expression of the nucleotide sequence of the present disclosure, the promoter can be a chemically repressible promoter.

[0169] Chemically 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 used as pre-emergence herbicides), and the tobacco PR-1a promoter (which is activated by salicylic acid) (e.g., the PR1a system), steroid-responsive promoters (see, e.g., Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88, 10421-10425 and McNellis et al. (1998) Plant Genetics, 1999; 10421-10425). J. [Plant Journal] 14, 247-257) as well as tetracycline-inducible promoters and tetracycline-repressible promoters (see, for example, Gatz et al. (1991) Mol. Gen. Genet. [Molecular Genetics] 227, 229-237 and U.S. Patent Nos. 5,814,618 and 5,789,156), Lac repressor system promoters, copper-inducible system promoters, salicylic acid-inducible system promoters (e.g., PR1a system), glucocorticoid-inducible promoters (Aoyama et al. (1997) Plant J. [Plant Journal] 11: 605-612) and ecdysone-inducible system promoters.

[0170] Other non-limiting examples of inducible promoters include ABA-inducible and cell swelling-inducible promoters, the auxin-binding protein gene promoter (Schwob et al. (1993) Plant J. 4:423-432), the UDP-glucose flavonoid glycosyltransferase 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 J. 119:185-197). 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 benzenesulfonamide-inducible (U.S. Pat. No. 5,364,780) and ethanol-inducible (International Patent Application Publication Nos. WO 97 / 06269 and WO 97 / 06268) systems and glutathione S-transferase promoters. Similarly, 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 may be used. Other chemically inducible promoters suitable for directing expression of the disclosed nucleotide sequences in plants are disclosed in U.S. Pat. No. 5,614,395, which is incorporated herein by reference in its entirety. Chemical induction of gene expression is also described in detail in published application EP 0 332 104 (granted to Ciba-Geigy) and U.S. Pat. No. 5,614,395. In some embodiments, the promoter for chemical induction may be the tobacco PR-1a promoter.

[0171] In a further embodiment, the nucleotide sequences of the present disclosure can be operably associated with a promoter that is inducible by wounding or infection by a pest or pathogen (e.g., an insect or nematode plant pest). Numerous promoters have been described that express at the site of wounding and / or at the site of pest attack (e.g., insect / nematode feeding) or infection by a plant pathogen. Ideally, such a promoter would be locally active only at or near the site of attack, and in this way, expression of the nucleotide sequences of the present disclosure would be concentrated in the cells that have been invaded or fed. 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. Patent No. 5,750,386; U.S. Patent No. 5,955,646; U.S. Patent No. 6,262,344; U.S. Patent No. 6,395,963; U.S. Patent No. 6,703,541; U.S. Patent No. 7,078,589; U.S. Patent No. 7,196,247; U.S. Patent No. 7,223,901; and U.S. Patent Application Publication No. 2010043102.

[0172] In some embodiments of the present disclosure, a "minimal promoter" or "basic promoter" is used. The minimal promoter can recruit and bind to RNA polymerase II complex and its auxiliary proteins to allow transcription initiation and extension. In certain embodiments, the minimal promoter is constructed to comprise only the nucleotides / nucleotide sequences of the selected promoter necessary for the binding of transcription factors and the transcription of the target nucleotide sequence, and this target nucleotide sequence is operably associated with a minimal promoter including but not limited to a TATA box sequence. In other embodiments, the minimal promoter lacks the cis sequence for recruiting and binding transcription factors, and these transcription factors regulate (e.g., enhance, repress, confer tissue specificity, confer induction or repressibility) transcription. The minimal promoter is typically placed upstream (i.e., 5') of the nucleotide sequence to be expressed. Therefore, the nucleotides / nucleotide sequences from any promoter available with the present disclosure can be selected to be used as the minimal promoter.

[0173] Numerous other sequences can be incorporated into the expression cassettes described herein. These sequences include sequences that have been shown to enhance expression, such as intron sequences (e.g., from Adhl and bronzel) and viral leader sequences (e.g., from TMV, MCMV, and AMV).

[0174] The nucleic acid of this disclosure may be more preferably targeted for expression in plants at different cellular locations. In some cases, the location in the cytosol may be desirable, and in other cases, the location in a certain subcellular organelle may be preferred. Use technology well known in the art to carry out subcellular localization of the transgenic encoding enzymes. Typically, DNA encoding a target peptide from a gene product of known organelle targeting is operated and fused to the upstream of the nucleic acid. Many such target sequences for chloroplasts are known and have demonstrated their function in heterologous constructs. The expression of the nucleic acid of this disclosure is also targeted to the endoplasmic reticulum or vacuole of the host cell. The technology for realizing it is well known in the art.

[0175] The carrier that is suitable for plant transformation is described in other places in this specification sheets.For agrobacterium-mediated transformation, binary vectors or the carrier carrying at least one T-DNA border sequence are suitable, and for direct gene transfer, any carrier is all suitable, and the linear DNA containing only the purpose construct is perhaps preferred.In the case of direct gene transfer, it is possible to use conversion or co-transformation (Schocher et al., Biotechnology [biological technology] 4:1093-1096 (1986)) with a single DNA species. For direct gene transfer and agrobacterium-mediated transformation, conversion is usually (but not necessarily) carried out with a selective marker, and this selective marker can provide resistance to antibiotics (kanamycin, hygromycin or methotrexate) or herbicides (basta). The plant transformation vector comprising the nucleic acid molecules of this disclosure can also include following genes (such as phosphomannose isomerase; PMI), and these genes provide the positive selection of transgenic plants, as disclosed in United States Patents 5,767,378 and 5,994,629 (incorporated herein by reference). However, the choice of selectable marker is not critical to the present disclosure.

[0176] In certain embodiments, nucleic acid can be transformed into nuclear genome. In another embodiment, the nucleic acid of the present disclosure is directly transformed into plastid genome. The main advantage of plastid transformation is that plastids are generally able to express bacterial genes without substantial codon optimization, and plastids can express multiple open reading frames under the control of a single promoter. In U.S. Patent Nos. 5,451,513, 5,545,817 and 5,545,818, in PCT application No. WO 95 / 16783, and in McBride et al., (1994), Proc. Nati. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 91, 7301-7305, plastid transformation technology is widely described. Basic chloroplast transformation technology involves, for example, using biolistic or protoplast transformation (e.g., calcium chloride or PEG-mediated transformation), the cloned plastid DNA region flanking the selective marker is introduced into a suitable target tissue together with the gene of interest. These 1 to 1.5 kb flanking regions (designated targeting sequences) promote homologous recombination with the plastid genome and thus allow replacement or modification of specific regions of the plastid. Initially, point mutations in the chloroplast 16S rRNA and rps12 genes (conferring resistance to spectinomycin and / or streptomycin) were used as selective markers for transformation (Svab, Z., Hajdukiewicz, P., and Maliga, P. (1990) Proc. Nati. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 87, 8526-8530; Staub, J. M., and Maliga, P. (1992) Plant Cell [Plant Cell] 4, 39-45). This produces stable homoplasmic transformants at a frequency of approximately 1 per 100 target leaf bombardments. The presence of a cloning site between these markers allows the creation of plastid targeting vectors for the introduction of foreign genes (Staub, JM and Maliga, P. (1993) EMBO J. 12, 601-606). A substantial increase in transformation frequency was achieved by replacing a recessive rRNA or r-protein antibiotic resistance gene with a dominant selectable marker (the bacterial aadA gene, which encodes the spectinomycin-detoxifying enzyme aminoglycoside-3'-adenylyltransferase) (Svab, Z. and Maliga, P. (1993) Proc. Natl. Acad. Sci. USA 90, 913-917).Previously, this marker has been successfully used for high-frequency transformation of the plastid genome of the green alga Chlamydomonas reinhardtii (Goldschmidt-Clermont, M. (1991) Nucl. Acids Res. [Nucleic Acids Research] 19: 4083-4089). Other selective markers useful for plastid transformation are known in the art and are included within the scope of the present disclosure. Typically, approximately 15-20 cell division cycles are required after transformation to achieve a homoplasmic state. Plastid expression (wherein a gene is inserted into all of the thousands of copies of the circular plastid genome present in each plant cell by homologous recombination) takes advantage of the large copy number over nuclear-expressed genes to allow expression levels that can easily exceed 10% of total soluble plant protein. In a preferred embodiment, the nucleic acids of the present disclosure are inserted into a plastid-targeted vector and transformed into the plastid genome of the desired plant host. Plants homozygous for a plastid genome comprising a nucleic acid of the present disclosure are obtained, and these plants are preferentially capable of high expression of the nucleic acid.

[0177] In other embodiments, the transgenic plant of the present disclosure may comprise a heterologous nucleic acid molecule encoding at least one other desired proterties. Other proterties may be encoded on a heterologous nucleic acid molecule identical to the nucleic acid molecule of the present disclosure, or may be encoded on a second heterologous nucleic acid molecule. Other desired proterties may confer insect resistance to a second insect pest, insect resistance to the same insect pest, abiotic stress tolerance, male sterility, herbicide resistance, bacterial disease resistance, fungal disease resistance, viral disease resistance, nematode resistance, modified fatty acid metabolism, modified carbohydrate metabolism, improved nutritional value, improved performance in an industrial process, or the reproductive capacity of a change. Other desired proterties may also induce commercially valuable enzymes or metabolites to produce in the plant.

[0178] In some embodiments, the desired additional trait is a second pesticide.The second pesticide can be active against any plant pest, including insects, nematodes, fungi, viruses, or bacteria. Examples of insect plant pests include, but are not limited to, Nilaparvata spp. (e.g., N. lugens (brown planthopper)); Laodelphax spp. (e.g., L. striatellus (small brown planthopper)); Nephotettix spp. (e.g., N. virescens or N. cincticeps (green leafhopper), or N. nigropictus (rice leafhopper)); Sogatella spp. (e.g., S. furcifera (white-backed planthopper); Blissus spp. (e.g., S. cincticeps (green leafhopper), or N. nigropictus (rice leafhopper)); spp.) (e.g., B. leucopterus (chinch bug)); Scotinophora spp. (e.g., S. vermidulate (rice blackbug)); Acrosternum spp. (e.g., A. hilare (green stink bug)); Parnara spp. (e.g., P. guttata (riceskipper)); Chilo spp. (e.g., C. suppressalis (rice striped stem borer), C. auricilius (gold-fringed rice borer); stemborer), or C. polychrysus (dark-headed stem borer); Chilotraea spp. (e.g., C. polychrysa (rice stalk borer); Sesamia spp. (e.g., S. inferens (pink riceborer); Tryporyza spp.) (e.g., T. innotata (white rice borer), or T. incertulas (yellow rice borer)); Cnaphalocrocis spp. (e.g., C. medinalis (rice leafroller); Agromyza spp. (e.g., A. oryzae (leafminer), or A. parvicornis (corn blot leafminer)); Cnaphalocrocis spp. (e.g., T. innotata (white rice borer), or T. incertulas (yellow rice borer)); Cnaphalocrocis spp. (e.g., C. medinalis (rice leafroller); Agromyza spp. (e.g., A. oryzae (leafminer), or A. parvicornis (corn blot leafminer)); Cnaphalocrocis spp. (e.g., D. grandiosella (southwestern corn borer); Narnaga spp. (e.g., N. aenescens (green rice borer)); caterpillar); Xanthodes spp. (e.g., X. transversa, green caterpillar); Spodoptera spp. (e.g., S. frugiperda (fall armyworm), S. exigua, beet armyworm, S. littoralis, climbing cutworm, or S. praefica, western yellowstriped armyworm); Mythimna spp. (e.g., Mythmnaseperata, Pseudaletia seperata); Spodoptera spp. (e.g., corn earworm (corn moth)); Colaspis spp. (e.g., C. brunnea, grape colaspis); Lissorhoptrus spp. (e.g., L. oryzophilus (rice water weevil); Echinocnemus spp. (e.g., E. squamos (rice plant weevil)); Diclodispa spp. (e.g., D. armigera, rice hispa); Oulema spp. (e.g., O.oryzae (leaf beetle); Sitophilus spp. (e.g., S. oryzae, rice weevil); Pachydiplosis spp. (e.g., P. oryzae, rice gall midge); Hydrellia spp. (e.g., H. griseola (small rice leaf miner), or H. sasakii, rice stem maggot); Chlorops spp. (e.g., C. oryzae, stem maggot); Diabrotica species (e.g., O. nubilalis (Western corn rootworm), O. barnettii (Northern corn rootworm), O. elevenspotted (Southern corn rootworm), O. mexicana (Mexican corn rootworm); Diabrotica spp. (Banded cucumber beetle); Ostrinia species (e.g., O. nubilalis (European corn borer); Spodoptera species (e.g., Agrotissima cutworm (black cutworm)); Elasmopalpus species (e.g., E. lignosellus (lesser cornstalk borer)); Cyclocephalus species (wireworms); Cyclocephala species (e.g., C. borealis (northern masked rhinoceros beetle)); chafer or southern masked chafer (C. immaculata); Popillia spp. (e.g., P. japonica, Japanese beetle); Chaetocnema spp. (e.g., C. pulicaria, corn flea beetle); Sphenophorus spp. (e.g., S. maidis, maize billbug); Rhopalosiphum spp. (e.g., R. maidis (corn leaf aphid); Anuraphis spp. (e.g., A.maidiradicis (corn root aphid); Melanoplus spp. (e.g., M. femurrubrum (redlegged grasshopper), M. differentialis (differential grasshopper), or M. sanguinipes (migratory grasshopper); Hylemya spp. (e.g., H. platura (seedcorn maggot); Anaphothrips spp. (e.g., A. obscrurus (grassthrips)); Solenopsis spp. (e.g., S. milesta (thief ant)); Tetranychus spp. (e.g., T. urticae (twospotted spider mite)); mite), T. cinnabarinus (carmine spider mite); Heliothis spp. (e.g., corn earworm (cotton bollworm), or H. armigera (American bollworm); Pectinophora spp. (e.g., P. gossypiella (pink bollworm); Earias spp. (e.g., E. vittella (spotted bollworm); Heliothis spp. (e.g., H. virescens (tobacco budworm); Heliothis spp. (e.g., A. grandis (bollworm); weevil); Pseudatomoscelis spp. (e.g., P. seriatus (cotton fleahopper); Trialeurodes spp. (e.g., T. abutiloneus (banded-winged whitefly), T. vaporariorum (greenhouse whitefly); Bemisia spp. (e.g., B.argentifolii (silverleaf whitefly); Aphis spp. (e.g., A. gossypii (cotton aphid); Lygus spp. (e.g., L. lineolaris (tarnished plant bug) or L. hesperus (western tarnished plant bug)); Euschistus spp. (e.g., E. conspersus (consperse stinkbug)); Chlorochroa spp. (e.g., C. sayi (Say stinkbug)); Green stink bug spp. (e.g., Nezara spp.) (e.g., N. viridula (southern green stink bug)); Thrips species (e.g., T. tabaci (onion thrips)); Frankliniella species (e.g., F. fusca (tobacco thrips) or F. occidentalis (western flower thrips); Leptinotarsa ​​species (e.g., L. decemlineata (Colorado potato beetle), L. juncta (false potato beetle) or L. texana (Texan false potato beetle)). potato beetle); Lema species (e.g., three-lined potato beetle); Epitrix spp. (e.g., potato flea beetle (E. cucumeris), tobacco flea beetle (E. hirtipennis), or tuber flea beetle (E. tuberis); Meloidogyne species (e.g., E.vittata (striped blister beetle); Phaedon spp. (e.g., P. cochleariae (mustard leaf beetle); Epilachna spp. (e.g., E. varivetis (Mexican bean beetle); Acheta spp. (e.g., A. domesticus (house cricket); Empoasca spp. (e.g., E. fabae (potato leafhopper); Myzus spp. (e.g., M. persicae (green peach aphid); Paratrioza spp. spp.) (e.g., P. cockerelli (psyllid)); Conoderus spp. (e.g., C. falli, southern potato wireworm or C. vespertinus, tobacco wireworm); Conoderus spp. (e.g., P. operculella, potato tuberworm); Macrosiphum spp. (e.g., M. euphorbiae (potato aphid); Thyanta spp. (e.g., T. Pallidovirens (redshouldered stinkbug)); Conoderus spp. (e.g., P. operculella, potato tuberworm); Spodoptera spp. (e.g., P. corn earworm (tomato bollworm)); fruitworm); Lycopodiella species (e.g., Codling moth (tomato pinworm)); Limonius spp. (wireworms); Manduca spp., such as M. sexta (tobacco hornworm) and M. quinquemaculata (tomato hornworm); Liriomyza spp. (e.g., L. sativae, L. trifoliata, L. quinquemacul ...trifolli or L. huidobrensis (leaf miner); Drosophilla spp. (e.g., Drosophila melanogaster, D. yakuba, D. pseudoobscura, or D. simulans); Carabus spp. (e.g., C. granulatus); Chironomus spp. (e.g., C. tentanus); Ctenocephalides spp. (e.g., C. felis, cat flea); Diaprepes spp. (e.g., D. abbreviation, root weevil); Ips spp. (e.g., I. pini, pine engraver); Tribolium spp. (e.g., T. innotata, red floor beetle). beetle); Glossina spp. (e.g., G. Morsitans (tsetse fly); Anopheles spp. (e.g., A. gambiae (malaria mosquito)); Helicoverpa spp. (e.g., H. armigera (African Bollworm); Acyrthosiphon spp. (e.g., A. pisum (pea aphid)); Apis spp. (e.g., A. melifera (honeybee)); Homalodisca spp. (e.g., H. coagulate (glassy-winged sharpshooter)); Aedes spp. (e.g., Ae. aegypti (yellow fever mosquito) fever mosquito); Bombyx spp. (e.g., B. mori (silkworm); Locusta spp. (e.g., L. migratoria (migratory locust); Boophilus spp. (e.g., B. microplus (cattle tick); Acanthoscurria spp. (e.g., A.Gomesiana (red-haired chololate bird eater); Diploptera spp. (e.g., D. punctata, pacific beetle cockroach); Heliconius spp. (e.g., H. erato (red passion flower butterfly) or H. melpomene (postman butterfly); Curculio spp. (e.g., C. glandium, acorn weevil); Plutella xylostella (e.g., P. xylostella, diamondback moth); Amblyomma spp. (e.g., A. variegatum, cattle tick); tick); Anteraea species (e.g., A. yamamai (silkmoth); and Armige species (e.g., A. subalbatus).

[0179] The RIP toxins of the present disclosure can be used in combination with other insecticides to increase the target range of pests. In addition, the RIP toxins of the present disclosure used in combination with a second insecticide (which has a different mode of action or targets a different receptor in the insect gut) has specific utility for preventing and / or managing insect resistance. In some embodiments, the RIP toxins of the present disclosure are combined with a second insecticidal protein selected from the group consisting of Cry1A, Cry1Ab, Cry1Ac, Cry1A.105, Cry1Ae, Cry1B, Cry1B variants, Cry1C, Cry1C variants, Cry1D, Cry1D variants, Cry1E, Cry1F, Cry1A / F chimeras, Cry1G, Cry1H, Cry1I, Cry1J, Cry1K, Cry1L, Cry2A, Cry2Ab, Cry2Ae, Cry3, Cry3A variants body, Cry3B, Cry4B, Cry6, Cry7, Cry8, Cry9, Cry15, Cry34, Cry35, Cry43A, Cry43B, Cry46A, Cry51Aa1, PtIP-96, PtIP-83, PHI-4, M P467, MP81, PS149B1, DIG-3, DIG-5, DIG-10, DIG-11, DIG-17, DIG-657, IRDIG28688.1, IRDIG28688.1, IRDIG28684.1, IRDIG28 682.1, IRDIG28680.1, IRDIG28674.1, IRDIG28672.1, IRDIG27642, IRDIG28688.1, IRDIG28686.1, IRDIG28684.1, IRDIG28682 .1, IRDIG28680.1, IRDIG28674.1, IRDIG28672.1, IRDIG27642, IRDIG28678.2, IRDIG28678.1, IRDIG31125.1, IRDIG28696.1, IRDIG29781.1, IRDIG29779.1, IRDIG30844.1, IRDIG30850.1, IRDIG30852.1, IRDIG30854.1, IRDIG30856.1, IRDIG30858.1, IRDIG30862.1, IRDIG30860.1, IRDIG30848.1, RETIRE2021, VIP3A, VIP3B, VIP3Ab, binary VIP1 and VIP2, or other vegetative insecticidal proteins, mCry3A, eCry3.1Ab, AXMI-001, AXMI-002, AXMI-030, AXMI-035, AXMI-036, AXMI-045, AXMI52, AXMI58, AXMI88, AXMI97, AX MI102, AXMI112, AXMI113, AXMI115, AXMI117, AXMI100, AXMI-115, AXMI-113, and AXMI-005, AXMI134, AXMI-15 0, AXMI171, AXMI-184, AXMI196, AXMI204, AXMI207, AXMI209, AXMI205, AXMI218, AXMI220, AXMI221z, AXMI222z, AXMI223z, AXMI224z and AXMI225z, AXMI238, AXMI270, AXMI279, AXMI345, AXMI-R1 and its variants, IP3 and its variants, ET29, E T33, ET34, ET35, ET66, ET70, TIC400, TIC407, TIC417, TIC431, TIC800, TIC807, TIC834, TIC836, TIC844, TIC853, TIC860 or variants thereof, TIC867 or variants thereof, TIC868 or variants thereof, TIC869, TIC900 or related proteins, TIC901, TIC1100, TIC1201, TIC1362, TIC1414, TIC1415, TIC1422, TIC1497, TIC1498, TIC1885, TIC1886, TIC1922, TIC1925, TIC1974, TIC2032, TIC2120, TIC2160, TIC3131, TIC3244, TIC6757, TIC7243, TIC7472, and TIC7473 proteins, or hybrid proteins or chimeras made from any of the foregoing insecticidal proteins. The second insecticide can also be an agent selected from the group comprising: alpha amylase, peroxidase, cholesterol oxidase, potato glycoprotein, protease, protease inhibitor, urease, alpha-amylase inhibitor, pore-forming protein, chitinase, lectin, engineered antibody or antibody fragment, Bacillus cereus insecticidal protein, Xenorhabdus species (such as X. nematophila or Xenorhabdus bovienii) insecticidal protein, Photorhabdus species (such as P. luminescens or P. asymobiotica) insecticidal protein, Brevibacillus species (such as B. laterosporous) insecticidal protein, Lysinibacillus spp.In some embodiments, the second agent may be an insecticidal protein derived from at least one insecticide toxin complex (Tc) derived from Photorhabdus, Xenorhabus, Serratia, or Yersinia. In still other embodiments, the second insecticidal protein may be a binary toxin derived from an insecticide bacterium, such as ISP1A and ISP2A from Brevibacillus laterosporus or BinA and BinB from Bacillus sphaericus. The combination of the disclosed RIP and a second pesticide can be expressed in a transgenic plant. In some embodiments, the transgenic plant is a transgenic corn plant. In other embodiments, the combination in the transgenic corn plant is a disclosed RIP and mCry3A and / or eCry3.1Ab and / or Cry3Bb1 and / or Cry34 / Cry35.

[0180] In some embodiments, the transgenic plants disclosed herein may include at least one non-protein second pesticidal agent. In a preferred embodiment, the second pesticidal agent is an interfering RNA molecule. Interfering RNA molecules typically include at least one RNA fragment for the target gene, a spacer sequence, and a second RNA fragment complementary to the first RNA fragment, thereby forming a double-stranded RNA structure. When an organism recognizes double-stranded RNA (dsRNA) molecules and hydrolyzes them, RNA interference (RNAi) occurs. The resulting hydrolyzate is a small RNA fragment of about 19-24 nucleotides in length, which is referred to as small interfering RNA (siRNA). These siRNAs then diffuse or are carried throughout the organism, including across the cell membrane, where they hybridize with mRNA (or other RNA) and cause the hydrolysis of RNA. Interfering RNA is recognized by the RNA interference silencing complex (RISC), in which the effector strand (or "guide strand") of RNA is located. This guide strand serves as a template for the recognition and destruction of duplex sequences. Each time siRNA hybridizes with its complementary RNA target, this process is repeated, effectively preventing those mRNAs from being translated, and thus "silencing" the expression of the specific gene from which the mRNA is transcribed. Interfering RNA is known in the art to be useful for insect control (see, for example, publication WO 2013 / 192256, which is incorporated herein by reference). Interfering RNA designed for insect control produces non-naturally occurring double-stranded RNA that utilizes the natural RNAi pathway in insects to trigger the downregulation of target genes, which may result in cessation of feeding and / or growth and may cause the death of insect pests. Interfering RNA molecules can confer insect resistance to target pests identical to the proteins disclosed herein or can target different pests. Target insect plant pests can be fed by chewing, sucking, or piercing. Interfering RNA is known in the art to be useful for insect control. In other embodiments, interfering RNA can confer resistance to non-insect plant pests (such as nematode pests or viral pests).

[0181] The co-expression of more than one pesticide in the same transgenic plant can be achieved by making a single recombinant vector (comprising the coding sequence of more than one pesticide in a so-called molecular stack) and genetically engineering the plant so that all the pesticides are contained and expressed in the transgenic plant. Such molecular stacking can also be prepared using mini-chromosomes, as described in, for example, U.S. Patent No. 7,235,716. Alternatively, a transgenic plant comprising a nucleic acid encoding a first pesticide can be retransformed with a different nucleic acid encoding a second pesticide, etc. Alternatively, a plant (parent 1) can be genetically engineered for expression of the genes disclosed herein. A second plant (parent 2) can be genetically engineered for expression of a second pesticide. By hybridizing parent 1 with parent 2, a progeny plant expressing all the genes introduced into parent 1 and parent 2 is obtained.

[0182] Transgenic plants or seeds comprising the insecticidal proteins of the present disclosure may also be treated with insecticides or insecticidal seed coatings, as described in U.S. Patent Nos. 5,849,320 and 5,876,739 (incorporated herein by reference). Where the insecticides or insecticidal seed coatings of the present disclosure and the transgenic plants or seeds are active against the same target insects (e.g., coleopteran pests or rootworm target pests), the combination (i) is useful in methods for further enhancing the activity of the compositions of the present disclosure against the target insects and (ii) in methods for preventing resistance to the compositions of the present disclosure by providing yet another mechanism of action against the target insects. Thus, the present disclosure provides methods for enhancing control of rootworm insect populations, comprising providing a transgenic plant or seed of the present disclosure and applying an insecticide or insecticidal seed coating of the present disclosure to the plant or seed.

[0183] Even where the insecticidal seed coating is active against different insects, the insecticidal seed coating is useful for expanding the spectrum of insect control, for example, by adding an insecticidal seed coating active against Lepidoptera insects to transgenic seeds of the present disclosure (which in some embodiments are active against Coleoptera and some Lepidoptera), the resulting coated transgenic seeds control both Lepidoptera and Coleoptera pests.

[0184] Examples of such insecticides and / or insecticidal seed coatings include, but are not limited to, carbamates, pyrethroids, organophosphates, friprole, neonicotinoids, organochlorides, nereistoxins, or combinations thereof. In another embodiment, the insecticide or insecticidal seed coating is selected from the group consisting of carbofuran, carbaryl, methomyl, bifenthrin, tefluthrin, permethrin, cyfluthrin, lambda-cyhalothrin, cypermethrin, deltamethrin, chlorpyrifos, chloroxyphos, dimethoate, ethoprophos, malathion, methyl parathion, phorate, terbufos, tebupirimiphos, fipronil, acetamiprid, imidacloprid, thiacloprid, thiamethoxam, endosulfan, sulfamic acid, and combinations thereof. Commercial products comprising such insecticides and insecticidal seed coatings include, but are not limited to (Carbofuran), (Methomyl, Methomyl, Nanaid), (carbaryl), (Bifenthrin), (tefluthrin), (Cypermethrin), (Cypermethrin), Delta (Deltamethrin), (λ-cyhalothrin), (Permethrin), (Permethrin), (Bifenthrin), (Bifenthrin), (tefluthrin)), (lambda cyhalothrin), (chlorpyrifos), (Chlorine Oxyphosphorus), (Methoate), (Phorate), (phorate, flucythinate), (Phorate), (terbufos), (Dimethoate), isochlorophos, (fipronil)), (Thiamethoxam), (Imidacloprid), (Imidacloprid), (thiamethoxam) and (Cyfluthrin, Pyrifos).

[0185] In some embodiments, the present disclosure also encompasses a composition comprising an insecticide protein of the present disclosure in an effective amount for controlling insects. In a further embodiment, the composition comprises a suitable agricultural carrier and the RIP of the present disclosure. The agricultural carrier may include adjuvants, mixtures, enhancers, etc. that are beneficial to the application of the active ingredient (such as the protein of the present disclosure, including a protein comprising, consisting essentially of, or consisting of: an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to any one of SEQ ID NOs: 1-21). Suitable carriers should not be phytotoxic to valuable crops (particularly at the concentrations used when the composition is applied in the presence of crops) and should not chemically react with the compounds of the active ingredients herein (i.e., the polypeptides or other composition ingredients of the present disclosure). Such mixtures can be designed for direct application to crops, or can be concentrates or formulations that are typically diluted with additional carriers and adjuvants before application. They may include inert or active ingredients and may be solid (such as, for example, dusts, powders, granules, water-dispersible granules or wettable powders) or liquid (such as emulsifiable concentrates, solutions, emulsions or suspensions). Suitable agricultural vehicles may include liquid vehicles such as water, toluene, xylene, naphtha, crop oils, acetone, methyl ethyl ketone, cyclohexanone, trichloroethylene, perchloroethylene, ethyl acetate, amyl acetate, butyl acetate, propylene glycol monomethyl ether and diethylene glycol methyl ether, methanol, ethanol, isopropyl alcohol, amyl alcohol, ethylene glycol, propylene glycol, glycerol, and the like. Water is generally the vehicle of choice for diluting concentrates. Suitable solid vehicles may include talc, pyrophyllite clay, silica, attapulgite clay, kieselguhr, chalk, diatomaxeous earth, lime, calcium carbonate, bentonite, Fuller's earth, cottonseed hulls, wheat flour, soy flour, pumice, wood flour, walnut shell flour, lignin, and the like. In other embodiments, the proteins of the present disclosure can be encapsulated in a synthetic matrix (such as a polymer) and applied to the surface of a host (such as a plant). Insect uptake of host cells allows the insect control agent to be delivered to the insect and results in a toxic effect on the insect pest.

[0186] In other embodiments, the composition of the present disclosure can be a powder, dust, pill, granule, spray, emulsion, colloid or solution. The composition of the present disclosure can be prepared by dehydrating, freeze-drying, homogenizing, extracting, filtering, centrifuging, settling or concentrating a culture of bacterial cells. The composition of the present disclosure can contain at least 1%, about 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or at least 99% of the polypeptide of the present disclosure by weight. The composition of the present disclosure can contain at least a second pesticide (which can be insecticidal, nematocidal, fungicidal, bactericidal). At least the second pesticide can have an insecticidal effect on the same insect as the polypeptide of the present disclosure or a different insect. The second pesticide can be a polypeptide. The pesticide can be an interfering RNA. The second pesticide can be a microorganism (such as a bacterium) that comprises a nucleic acid molecule encoding a pesticide and / or comprises a pesticide (such as a polypeptide or interfering RNA). The microorganism can be attenuated, heat-inactivated or freeze-dried. The microorganism may die or cannot reproduce. The second pesticide can be an insecticide, for example, carbofuran, carbaryl, methomyl, bifenthrin, tefluthrin, permethrin, cyfluthrin, lambda-cyhalothrin, cypermethrin, deltamethrin, chlorpyrifos, chloroxyphos, dimethoate, ethoprophos, malathion, methyl parathion, phorate, terbufos, tert-butyl pyrimidinphos (tebupirimiphos), fipronil, acetamiprid, imidacloprid, thiacloprid, thiamethoxam, endosulfan, sulfamic acid, and combinations thereof, or commercial products containing pesticides as described above and insecticide seed coatings.

[0187] The composition of the present disclosure (for example, a composition comprising an albumen of the present disclosure and an agriculturally acceptable carrier) can be used in traditional agricultural methods. An agriculturally acceptable carrier is a formulation that can be used to apply a composition comprising a polypeptide of the present disclosure to a plant or seed. For example, the composition of the present disclosure can be mixed with water and / or fertilizer, and can be applied to a desired place before and / or after emergence by any means, such as an airplane spray barrel, irrigation equipment, direct injection spray equipment, backpack spray barrel, livestock dip tank, the farm equipment used in ground spraying (for example, a nozzle sprayer, a hand sprayer), etc. The desired place can be soil, plant, etc.

[0188] The compositions of the present disclosure can be applied to seeds or plant propagules in any physiological state at the following time: any time between seed harvest and sowing; or during sowing or after sowing; and / or after germination. Preferably, the seeds or plant propagules are in a sufficiently durable state so that no damage is caused or minimal damage is caused during the treatment process, including physical damage or biological damage. The formulation can be applied to the seeds or plant propagules using conventional coating techniques and machines (such as fluidized bed technology, drum grinding method, static rotation (rotostatic) seed processor and drum coater).

[0189] In some embodiments, the present disclosure also includes methods of controlling a coleopteran pest population comprising contacting the pest population with an insect control-effective amount of a RIP of the present disclosure, wherein the protein comprises, consists essentially of, or consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 1-21. Contacting comprises ingestion or uptake of the insecticidal protein by members of the pest population. The insecticidal protein can be incorporated into the insect's dietary food, or can be expressed or present in plant tissue that the insect population then ingests. In further embodiments, controlling a coleopteran pest population comprises killing the insect by contacting the insect with an insect control-effective amount of an insecticidal protein of the present disclosure.

[0190] The present disclosure further encompasses a method for increasing plant yield, the method comprising growing a plant or its seeds in a field having stably incorporated into its genome a nucleic acid molecule of the expression cassette of the present disclosure, and wherein the field is infested with a pest against which the polypeptide has insecticidal activity.

[0191] Once a desired nucleic acid has been transformed into a particular plant species, it can be propagated within that species or moved into other varieties of the same species (particularly including commercial varieties) using conventional breeding techniques.

[0192] In some embodiments, the present disclosure encompasses a method of providing a corn grower with a means of controlling a rootworm pest population in a corn crop, the method comprising (a) selling or providing to the grower a transgenic corn seed comprising a nucleic acid molecule, expression cassette, vector, or chimeric gene of the present disclosure; and (b) advertising to the grower that the transgenic corn seed produces a transgenic corn plant that controls a rootworm pest population.

[0193] In some embodiments, the present disclosure also encompasses methods of identifying an insecticidal protein comprising, consisting essentially of, or consisting of a nucleotide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 1-21, or a toxin fragment thereof, the method comprising the steps of: (a) generating a primer pair that will amplify the nucleotide sequence of SEQ ID NOs: 1-21 from a nucleic acid sample; NO:22-25 or its complementary sequence, (b) amplifying the orthologous polynucleotide from the nucleic acid sample, (c) identifying the nucleotide sequence of the orthologous polynucleotide, (d) producing a protein encoded by the orthologous polynucleotide, and (e) determining that the protein of step (d) has insecticidal activity against insect pests.

[0194] Examples

[0195] Embodiments of the present invention may be better understood by reference to the following examples. The foregoing and following embodiments of the present invention and the description of various embodiments are not intended to limit the claims, but are illustrative thereof. Therefore, it should be understood that the claims are not intended to be limited to the specific details of these examples. It should be understood by those skilled in the art that other embodiments of the present invention may be practiced without departing from the spirit and scope of the present disclosure, and the scope of the present disclosure is defined by the appended claims. Art-recognized recombinant DNA and molecular cloning techniques can be found in, for example, J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press (2001); T. J. Silhavy, M. L. Berman, and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984), and Ausubel, F. M. et al., Current Protocols in Molecular Biology, New York, John Wiley and Sons Inc., (1988), Reiter et al., Methods in Arabidopsis Research, World Scientific Publishing Company, Inc. World Scientific Press (1992), and Schultz et al., Plant Molecular Biology Manual, Kluwer Academic Publishers (1998).

[0196] Example 1: Identification of sequences encoding insecticidal proteins from the Rhizobiaceae family.

[0197] Based on a proprietary algorithm, candidate nucleotide sequences encoding proteins described in the art as hypothetical proteins were identified in the genomes of Gram-negative bacteria belonging to the genera Sinorhizobium, Xiphium, Rhizobium, and related genera belonging to the Rhizobiaceae family. Four candidate sequences were selected for expression and testing against insect pests. Four candidate nucleotide sequences were identified in the genomes of Xiphium aridi strain (NCBI: txid1708715) (SEQ ID NO: 22), Sinorhizobium species GL28 strain (NCBI: txid1358418) (SEQ ID NO: 23), unclassified Rhizobiales bacterial strain (NCBI: txid1909294) (SEQ ID NO: 24), and Rhizobium species strain SPY-1 (NCBI: txid2547961) (SEQ ID NO: 25). Codon-optimized versions of each candidate coding sequence were generated for E. coli, SEQ ID NOs: 26-29, and introduced individually into the pET29a bacterial expression vector, designated pET29a-26, pET29a-27, pET29a-28, and pET29a-29, to produce the protein. Each pET29a expression vector was transformed into E. coli BL21*(DE3) and lysates were prepared from isopropyl β-D-1-thiogalactopyranoside (IPTG)-induced cultures (protein production overnight at approximately 18°C). In a feed incorporation bioassay, the insecticidal activity of the lysates against western corn rootworm (WCR) was tested. Briefly, the E. coli lysates were mixed with an equal volume of heated artificial insect diet (Bioserv, Frenchtown, New Jersey) in a 1.5 mL centrifuge tube and then applied to a small culture dish. After the diet-sample mixture cooled and solidified, 12 WCR larvae were added to each plate. The plates were sealed and maintained under ambient laboratory conditions with respect to temperature, light, and relative humidity. Buffer without lysate, lysate from an E. coli BL21*(DE3) culture (containing an empty pET29a vector), and artificial insect diet alone served as negative controls. Percent mortality and growth inhibition observations were obtained at 4 and 6 days post-infestation and designated as s = small larvae, m = medium larvae, and l = large larvae.

[0198] The results shown in Table 1 indicate that lysates from E. coli cultures expressing proteins encoded in the genome of Rhizobiaceae bacteria surprisingly possess insecticidal activity against rootworm insect pests. These Rhizobiaceae insecticidal proteins (RIPs) were designated Enf_adiCRW (RIP1Aa; SEQ ID NO: 1), expressed from the pET29a-26 vector; Sinorhiz_GL28CRW (RIP2Aa; SEQ ID NO: 2), expressed from the pET29a-27 vector; Rhizo_bactCRW (RIP3Aa; SEQ ID NO: 3), expressed from the pET29a-28 vector; and Rhiz_SPYCRW (RIP4Aa; SEQ ID NO: 4), expressed from the pET29a-29 vector.

[0199] Table 1: Insecticidal activity of RIPs against WCR

[0200]

[0201] Table 2 shows the alignment and sequence identity comparison of active RIPs against WCR. (National Institutes of Health) gene sequence database (U.S. National Library of Medicine) searches indicate that the disclosed RIPs are not significantly identical to any other sequences. However, the disclosed RIPs appear to have cytotoxin domains. The Enf_adiCRW protein (RIP1Aa; SEQ ID NO: 1) has 345 amino acids, is 38 kDa, and contains a cytotoxin domain from about amino acid position 40 to about amino acid position 234. The Sinorhiz_GL28CRW protein (RIP2Aa; SEQ ID NO: 2) has 344 amino acids, is 37.7 kDa, and contains a cytotoxin domain from about amino acid position 39 to about amino acid position 233. The Rhizo_bactCRW protein (RIP3Aa; SEQ ID NO: 3) has 351 amino acids, is 38.5 kDa, and contains a cytotoxin domain from about amino acid position 46 to about amino acid position 240. The Rhiz_SPYCRW protein (RIP4Aa; SEQ ID NO: 4) has 347 amino acids, is 38.2 kDa, and contains a cytotoxin domain from about amino acid 40 to about amino acid 234. Other bacteria, such as Bacillus thuringiensis, are known to produce proteins with cytotoxin domains that are primarily active against Diptera insects and not Coleopterans, particularly rootworm pests. However, the cytotoxin domains of the RIPs of the present disclosure appear to be unique, and without being bound by theory, it is believed that these unique cytotoxin domains of the RIPs of the present disclosure are responsible for their unexpected activity against coleopteran insect pests, particularly rootworm pests.

[0202] Table 2. Alignment and percent identity comparison of WCR-active RIPs.

[0203]

[0204]

[0205] "." under amino acids indicates identical amino acids

[0206] Table 3. Alignment of RIP cytotoxic domains.

[0207]

[0208]

[0209] Example 2. Efficacy of RIP against corn rootworms.

[0210] To determine the efficacy of the RIPs of the present disclosure against western corn rootworm (WCR; corn rootworm), lysates containing the RIPs of the present disclosure were tested against WCR larvae over a range of concentrations in a diet incorporation assay essentially as described in Example 1. Twelve newborn larvae were tested at each concentration. Mortality and percent growth were determined on days 4 and 6 for RIP2Aa and on days 3 and 6 for RIP3Aa.

[0211] As shown in Table 4, lysates containing RIP2Aa (SEQ ID NO: 2) were effective against WCR even at a 1:100 dilution, producing at least 25% mortality by day 6, compared to no mortality in the control treatment. RIP3Aa was active at the lowest concentration of 1:64, producing 100% mortality by day 6.

[0212] Table 4. Efficacy of RIP lysates against western corn rootworm.

[0213]

[0214]

[0215] The insecticidal properties of the RIP2Aa protein were further characterized. Two liters of E. coli BL21*(DE3) cells carrying pET-rip2Aa were grown in LB medium at 37°C. IPTG (1 mM) was added to the culture when the OD reached 0.8-1.0, and the culture was moved to 18°C ​​and cultured for 18 hours. The cell pellet was harvested and resuspended in 20 mM Tris (pH 8.5) containing 10% glycerol. The cells were lysed using a French press; the lysate was then spun at 100k xg in an ultracentrifuge. The supernatant was collected, filtered, and then loaded onto a HiPrepQ anion exchange column, which was pre-equilibrated in 20 mM Tris (pH 8.5) containing 10% glycerol. The HiPrepQ column efficiently bound RIP2Aa; the protein was eluted from the column using a linear NaCl gradient. High salt buffer consisted of 20 mM Tris (pH 8.5), 0.5 M NaCl containing 10% glycerol. The purest fractions were pooled and concentrated to approximately 2 mL. The RIP2Aa protein was loaded onto a Sephadex 200 gel filtration column that had been pre-equilibrated in 1X PBS. The purity of the fractions from the Sephadex 200 column was analyzed by SDS-PAGE (RIP2Aa (SEQ ID NO: 2) has a predicted molecular weight of 37.7 kDa). The purest fractions were pooled and concentrated to approximately 7 mg / mL and then stored at -80°C. A series of concentrations of pure RIP2Aa protein were then tested against 12 WCR larvae in a diet incorporation assay essentially as described above. As shown in Table 5, RIP2Aa was effective against WCR; 75 μg / mL of RIP2Aa produced at least 75% mortality on day 6.

[0216] Table 5. Activity of RIP2Aa against western corn rootworm.

[0217]

[0218] Example 3. Biological activity of RIP variants.

[0219] Variant RIPs are generated by making single or double amino acid substitutions in the native RIP3Aa sequence (SEQ ID NO: 3). The variants made included the following: the I50 residue was changed to L (RIP3Aa-I150L; Xaa50 of SEQ ID NO:5 is L; SEQ ID NO:6), the I53 residue was changed to L (RIP3Aa-I53L; Xaa53 of SEQ ID NO:5 is L; SEQ ID NO:7), the I56 residue was changed to L (RIP3Aa-I56L; Xaa56 of SEQ ID NO:5 is L; SEQ ID NO:8), the A62 residue was changed to C or L (RIP3Aa-A62C or RIP3Aa-A62L; Xaa62 of SEQ ID NO:5 is C or L; SEQ ID NO:9 or SEQ ID NO:10), the A64 residue was changed to C or L (RIP3Aa-A64C or RIP3Aa-A64L; Xaa64 of SEQ ID NO:5 is C or L; SEQ ID NO:11). NO:11 or SEQ ID NO:12), the I81 residue was changed to L (RIP3Aa-I81L; Xaa81 of SEQ ID NO:5 is L; SEQ ID NO:13), the I126 residue was changed to L (RIP3Aa-I126L; Xaa126 of SEQ ID NO:5 is L; SEQ ID NO:14), the I153 residue was changed to L (RIP3Aa-I153L; Xaa153 of SEQ ID NO:5 is L; SEQ ID NO:15), the I169 residue was changed to L (RIP3Aa-I169L; Xaa169 of SEQ ID NO:5 is L; SEQ ID NO:16), the I185 residue was changed to L (RIP3Aa-I185L; Xaa185 of SEQ ID NO:5 is L; SEQ ID NO:17). NO:17), residue I207 was changed to L (RIP3Aa-I207L; Xaa207 of SEQ ID NO:5 is L; SEQ ID NO:18), residue I219 was changed to L (RIP3Aa-I219L; Xaa219 of SEQ ID NO:5 is L; SEQ ID NO:19), and residue I275 was changed to L (RIP3Aa-I275L; Xaa275 of SEQ ID NO:5 is L; SEQ ID NO:20).

[0220] Each variant was tested in a diet incorporation assay against newborn WCR larvae essentially as described in Example 1. The results are shown in Table 6. The results indicate that certain amino acid substitutions can be made in RIP and still retain insecticidal activity against Diabrotica spp. Certain mutations do appear to delay full toxicity, suggesting that these positions may be important in the mode of action of Rhizobiaceae insecticidal proteins.

[0221] Table 6. Insecticidal activity of variant RIPs.

[0222]

[0223] Example 4. Transformation of maize with a RIP coding sequence.

[0224] Nucleotide sequences encoding RIP1Aa, RIP2Aa, RIP3Aa, RIP4Aa, or variants thereof of the present disclosure, such as any one of SEQ ID NOs: 1-5, or maize-optimized nucleotide sequences, such as any one of SEQ ID NOs: 42-45, which can be generated as described, for example, in U.S. Patent No. 6,051,760, are transformed into corn to control corn rootworms.

[0225] Two plant expression cassettes were constructed to introduce the RIP coding sequence into maize. The first cassette contained the maize ubiquitin 1 (Ubil) promoter operably linked to the rip coding sequence, which was operably linked to the maize Ubi361 terminator. The second cassette contained the maize Ubil promoter operably linked to the pmi coding sequence encoding the selectable marker phosphomannose isomerase (PMI), which was operably linked to the maize Ubil terminator. A recombinant plant transformation binary vector containing the two expression cassettes was generated and used in maize transformation experiments.

[0226] The binary vector was transformed into Agrobacterium tumefaciens using standard molecular biology techniques.To prepare Agrobacterium for transformation, cells were grown overnight in liquid YPC medium at 28°C and 220 rpm.

[0227] Agrobacterium transformation of immature maize embryos was performed essentially as described in Negrotto et al., 2000, Plant Cell Reports 19:798-803. For this example, all culture medium components were essentially as described in Negrotto et al. (as above). However, various culture medium components known in the art may be substituted.

[0228] Briefly, Agrobacterium strain LBA4404 (pSB1) containing a binary vector plant transformation vector was grown on YEP (yeast extract (5 g / L), peptone (10 g / L), NaCl (5 g / L), 15 g / l agar, pH 6.8) solid medium at 28°C for 2-4 days. Approximately 0.8 × 10 9 Agrobacterium was suspended in LS-inf medium (Negrotto et al., supra) supplemented with 100 μM As. The bacteria were pre-induced in this medium for 30 to 60 minutes.

[0229] The immature embryos from the genotype that is suitable for are excised among the liquid LS-inf+100 μM As from the big ear of 8-12 days. Rinse these embryos with fresh infection culture medium. Add agrobacterium solution then, and these embryos are vortexed 30 seconds and allowed to settle 5 minutes together with bacterium. These embryo scutels are upwardly transferred to the LSA culture medium then, and cultivated two to three days in the dark. Subsequently, 20 and 25 embryos of every petri plate (petri plate) are transferred to the LSDc culture medium that is supplemented with cefotaxime (250mg / l) and silver nitrate (1.6mg / l), and cultivated 10 days at 28 ℃ in the dark.

[0230] The immature embryos that will produce embryonic callus are transferred to the LSD1M0.5S culture medium. On this culture medium, culture is continued the selection of about 6 weeks, has the passage culture step of about 3 weeks. Survival callus is transferred to the Reg1 culture medium that is supplemented with mannose. Afterwards in illumination (16 hours light / 8 hours dark scheme) cultivation, green tissue is transferred to the Reg2 culture medium that does not have growth regulator, hatches about 1 to 2 weeks. These plantlets are transferred to the Magenta GA-7 box (Magenta Corp (Magenta Corp), Chicago, Illinois) that contains Reg3 culture medium and are grown in illumination.

[0231] After transformation, selection and regeneration, use The presence of the pmi gene and the rip maize codon-optimized coding sequence in the plants was assayed. The presence of the vector backbone in the plants was also tested. Plants that were negative for the vector backbone and contained a copy of the transgenic vector from the binary vector were transferred to a greenhouse and tested for insecticidal activity against WCR.

[0232] Example 5. Rhizobium insecticidal protein in combination with a second insecticide.

[0233] The RIP of the present disclosure as described above was purified as described in Example 2. dsRNAs were prepared against essential targets and known to have insecticidal activity. In non-limiting examples, dsRNAs can target genes encoding vacuolar ATP synthase, β-tubulin, 26S proteosome subunit p28 protein, EF1α48D, troponin I, tetraspanin, clathrin heavy chain, γ-coat body, β-coat body and / or juvenile hormone epoxide hydrolase (PCT Patent Application Nos. PCT / US17 / 044825; PCT / US17 / 044831; PCT / US17 / 044832; U.S. Patent No. 7,812,219; each of which is incorporated herein by reference). The efficacy of the dsRNA and purified protein against WCR was tested in a dietary incorporation assay performed essentially as described in Example 1.

[0234] Example 6. In situ genome editing in plant cells to generate modified RIPs.

[0235] The following examples illustrate the use of genome editing of plant cell genomes in situ to incorporate mutations described herein, including but not limited to the mutations described in Example 3, into the coding sequence of native RIPs, including RIP3Aa (SEQ ID NO: 3), or into the coding sequence of RIP3Aa proteins that have been modified.

[0236] Directed genome modification (also known as genome editing) can be used to introduce mutations in specific DNA sequences. These genome editing technologies, including zinc finger nucleases (ZNFs), transcription activator-like effector nucleases (TALENs), meganucleases, and clustered regularly interspaced short palindromic repeats (CRISPRs), have been successfully applied to more than 50 different organisms, including crop plants. See, for example, Belhaj, K., et al., Plant Methods [Plant Methods] 9, 39 (2013); Jiang, W., et al., Nucleic Acids Res [Nucleic Acids Research], 41, e188 (2013). The CRISPR / Cas system for genome editing is based on the transient expression of an engineered single guide RNA (sgRNA) of the Cas9 nuclease and a specified target polynucleotide sequence.

[0237] Cas9 is a large, monomeric DNA nuclease that is guided to a DNA target sequence by a complex of two 20-nucleotide (nt) noncoding RNAs: CRIPSR RNA (crRNA) and a transactivating crRNA (tracrRNA), which are functionally accessible as a single synthetic RNA chimera. The Cas9 protein contains two nuclease domains with homology to RuvC and HNH nucleases. The HNH nuclease domain cleaves the complementary DNA strand, while the RuvC-like domain cleaves the non-complementary strand, thereby introducing a blunt cut in the target DNA.

[0238] When Cas9 and sgRNA are transiently expressed in living maize cells, double-strand breaks (DSBs) are generated in specific target DNA in transgenic maize cells. Mutations at the break sites are introduced through non-homologous end joining and homology-directed DNA repair pathways.

[0239] By using a recombinant plasmid expressing the Cas9 nuclease and sgRNA target (maize codon optimized for rip3Aa or mutant rip3Aa sequences) in transgenic maize, specific mutations (such as those described in Example 3 above) are introduced into the coding sequence of the native RIP3Aa insecticidal protein (SEQ ID NO: 3) or mutant RIP3Aa protein. The method is implemented by agroinfiltration of Agrobacterium tumefaciens carrying a binary plasmid containing the specified target sequence of interest. After the sgRNA binds to the target rip3Aa or mutant rip3Aa coding sequence, the Cas9 nuclease specifically cuts the coding sequence and introduces the desired mutation or mutations during the DNA repair process. Thus, the now mutated rip3Aa coding sequence will encode a modified variant RIP3Aa protein, such as the variant described in Example 3, for example, wherein the mutation at residue I50 is changed to L, residue I53 is changed to L, residue I56 is changed to L, residue A62 is changed to C or L, residue A64 is changed to C or L, residue I81 is changed to L, residue I126 is changed to L, residue I153 is changed to L, residue I169 is changed to L, residue I185 is changed to L, residue I207 is changed to L, residue I219 is changed to L, residue I275 is changed to L, or any combination thereof.

[0240] Plant cells containing the genome-edited rip coding sequence were screened by PCR and sequencing. Calli containing genome-edited mutations in the rip or modified rip coding sequence were induced and regenerated into plants for phenotypic evaluation. The expressed RIPs were evaluated for insecticidal activity against western corn rootworm (Diplodia zeae), northern corn rootworm (Diplodia barnettii), southern corn rootworm (Cercidia elevenspottedii rootfeeding subspecies), and / or Mexican corn rootworm (Diplodia mexicana).

[0241] Example 7. Testing of RIPs for Insecticidal Activity Against Lepidoptera Pests.

[0242] The pET-6His-SUMO construct containing rip1Aa (SEQ ID NO: 21) was made to express tagged RIP1Aa (SEQ ID NO: 49). The pET-6His-SUMO-rip1Aa construct was transformed into E. coli BL21* (DE3) for protein production. Lysates from bacterial cultures expressing 6his-SUMO-RIP1Aa were tested for bioactivity against a panel of lepidopteran insect pests including corn ear moth (CEW), fall armyworm (FAW) and soybean looper (SBL) using a diet overlay bioassay. For each experiment, newborn WCR larvae were tested using lysate from a BL21* bacterial culture containing the gene encoding SUMO-RIP1Aa (SEQ ID NO: 1) containing a hexa-histidine tag at the N-terminus. The positive control consisted of larvae exposed to E. coli BL21* lysate expressing the Vip3 protein. Buffer alone and lysate from a BL21*(DE3) bacterial culture carrying an empty pET29 vector were used as negative controls. Mortality was assessed on day 7. The results of the bioassay showed that the labeled RIP1Aa was active against WCR but not against any of the lepidopteran pests tested.

[0243] As described above, bacterial lysates containing RIP2Aa (SEQ ID NO: 2) were also tested against lepidopteran pests including corn moth, European corn borer, black cutworm, and fall armyworm. The results of the bioassays showed that RIP2Aa had no insecticidal activity against any lepidopteran pest insects at the concentrations tested.

[0244] It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or variations from the descriptions thereof will be suggested to those skilled in the art and are intended to be included within the spirit and purview of this application and the scope of the appended claims.

[0245] All publications and patent applications mentioned in this specification are indicative of the levels of skill of those skilled in the art to which this invention 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.

Claims

1. A nucleic acid molecule comprising a nucleotide sequence encoding a protein that is toxic to an insect pest, wherein the nucleotide sequence (a) encodes a protein comprising an amino acid sequence having at least 80% to at least 99% sequence identity to any one of SEQ ID NOs: 1-21, or a toxin fragment thereof; or (b) has at least 80% to at least 99% sequence identity to any one of SEQ ID NOs: 22-49, or a toxin-encoding fragment thereof; or (c) is a synthetic sequence of (a) or (b) that has been codon-optimized for expression in a transgenic organism.

2. The nucleic acid molecule of claim 1, wherein the insecticidal protein comprises the amino acid sequence of any one of SEQ ID NOs: 1-21 or a toxic fragment thereof.

3. The nucleic acid molecule of claim 1, wherein the nucleotide sequence comprises any one of SEQ ID NOs: 22-49 or a toxin encoding fragment thereof.

4. The nucleic acid molecule of claim 1, wherein the synthetic nucleotide sequence comprises any one of SEQ ID NOs: 26-49 or a toxin encoding fragment thereof.

5. A chimeric gene comprising a heterologous promoter operably linked to the nucleic acid molecule of any one of claims 1 to 4. The chimeric gene of claim 5 , wherein the heterologous promoter is a plant-expressible promoter.

7. The chimeric gene of claim 6, wherein the plant-expressible promoter is selected from the group consisting of: ubiquitin, tuberose yellow virus, maize TrpA, OsMADS 6, maize H3 histone, bacteriophage T3 gene 9 5'UTR, maize sucrose synthase 1, maize alcohol dehydrogenase 1, maize light-harvesting complex, maize heat shock protein, maize mtl, pea small subunit RuBP carboxylase, rice actin, rice cyclophilin, Ti plasmid mannopine synthase, Ti plasmid nopaline synthase, petunia chalcone isomerase, legume glycine-rich protein 1, potato glycoprotein, lectin, CaMV 35S, and S-E9 small subunit RuBP carboxylase promoter.

8. The chimeric gene of claim 5, wherein the insect pest is a coleopteran insect pest.

9. The chimeric gene of claim 8, wherein the coleopteran insect pest is a Diabrotica insect pest.

10. The chimeric gene of claim 9, wherein the insect pest of the genus Diabrotica is selected from the group consisting of: corn rootworm, barnett's rootworm, cucumber rootworm subsp. undecemlineata, and maize rootworm.

11. The chimeric gene of claim 5, wherein the transgenic organism is a bacterium or a plant.

12. The chimeric gene of claim 11, wherein the transgenic bacteria is selected from the group consisting of: Bacillus, Clostridium, Xenorhabdus, Photorhabdus, Pasteurella, Escherichia, Pseudomonas, Erwinia, Serratia, Klebsiella, Salmonella, Pasteurella, Xanthomonas, Streptomyces, Rhizobium, Sinorhizobium, Xiphium, Rhodopseudomonas, Methylophilus, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes.

13. The chimeric gene of claim 11, wherein the plant is a maize plant.

14. A protein that is toxic to insect pests, wherein the protein comprises (a) an amino acid sequence having at least 80% to at least 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1-21, or a toxin fragment thereof; or (b) an amino acid sequence comprising any one of SEQ ID NOs: 1-21, or a toxin fragment thereof; or (c) an amino acid sequence encoded by a nucleotide sequence having at least 80% to at least 99% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 21-49, or a toxin-encoding fragment thereof; or (d) an amino acid sequence encoded by a nucleotide sequence comprising any one of SEQ ID NOs: 21-49, or a toxin-encoding fragment thereof; or (e) an amino acid sequence of (a)-(d), which comprises a cytotoxin domain selected from the group consisting of SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO:

53.

15. The protein of claim 14, wherein the insect pest is a coleopteran insect pest.

16. The protein of claim 15, wherein the coleopteran insect pest is a Diabrotica insect pest.

17. The protein of claim 16, wherein the rootworm insect pest is selected from the group consisting of: corn rootworm, barnett's rootworm, cucumber rootworm subsp. undecemlineata, and maize rootworm. A recombinant vector comprising the chimeric gene according to claim 5 . A host cell comprising the recombinant vector according to claim 18 , wherein the host cell is a bacterial cell or a plant cell.

20. The transgenic bacterial cell of claim 19, wherein the bacterial cell is in the genus Bacillus, Clostridium, Xenorhabdus, Photorhabdus, Pasteurella, Escherichia, Pseudomonas, Erwinia, Serratia, Klebsiella, Salmonella, Pasteurella, Xanthomonas, Streptomyces, Rhizobium, Sinorhizobium, Xiphium, Rhodopseudomonas, Methylophilus, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, or Alcaligenes.

21. The transgenic Bacillus cell of claim 20, wherein the Bacillus cell is a Bacillus thuringiensis cell.

22. The transgenic plant cell of claim 19, wherein the plant cell is a dicotyledonous plant cell or a monocotyledonous plant cell.

23. The transgenic plant cell of claim 22, wherein (a) the dicot cell is selected from the group consisting of a soybean cell, a sunflower cell, a tomato cell, a Brassica cell, a cotton cell, a sugar beet cell, and a tobacco cell; or (b) the monocot cell is selected from the group consisting of a barley cell, a maize cell, an oat cell, a rice cell, a sorghum cell, a sugar cane cell, and a wheat cell.

24. A transgenic plant or plant part comprising the transgenic plant cell of claim 23.

25. The transgenic plant or plant part of claim 24, which is a transgenic maize plant or plant part.

26. An engineered insecticidal protein comprising an amino acid sequence having at least 80% to at least 99% sequence identity to a) SEQ ID NO: 1 and further comprising at least one mutation at a position corresponding to any one or any combination of amino acid positions 1-345 of SEQ ID NO: 1; or having at least 80% to at least 99% sequence identity to b) SEQ ID NO: 2 and further comprising at least one mutation at a position corresponding to any one or any combination of amino acid positions 1-344 of SEQ ID NO: 2; or having at least 80% to at least 99% sequence identity to c) SEQ ID NO: 3 and further comprising at least one mutation at a position corresponding to any one or any combination of amino acid positions 1-351 of SEQ ID NO: 3; or having at least 80% to at least 99% sequence identity to d) SEQ ID NO: 4 and further comprising at least one mutation at a position corresponding to any one or any combination of amino acid positions 1-347 of SEQ ID NO:

4.

27. The engineered insecticidal protein of claim 26, wherein the mutation is at an amino acid position corresponding to amino acid position 50, 52, 56, 62, 64, 81, 126, 153, 169, 185, 207, 219, or 275 of SEQ ID NO: 3, or any combination thereof.

28. The engineered insecticidal protein of claim 27, wherein the mutation is at position 50, 52, 56, 62, 64, 81, 126, 153, 169, 185, 207, 219, or 275 of SEQ ID NO:

3.

29. The engineered insecticidal protein of claim 28, wherein the mutation at position 50 is I50L, the mutation at position 52 is I52L, the mutation at position 56 is I56L, the mutation at position 62 is A62C or A62L, the mutation at position 64 is A64C or A64L, the mutation at position 81 is I81L, the mutation at position 126 is I126L, the mutation at position 153 is I153L, the mutation at position 169 is I169L, the mutation at position 185 is I185L, the mutation at position 207 is I207L, the mutation at position 219 is I219L, or the mutation at position 275 is I275L.

30. The engineered insecticidal protein of claim 29, wherein the protein comprises the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO:

20.

31. The engineered insecticidal protein of any one of claims 26-30, wherein the protein is active against at least insect pests of the order Coleopteran.

32. The engineered insecticidal protein of claim 31 , wherein the coleopteran insect pest is a Diabrotica insect pest.

33. The engineered insecticidal protein of claim 32, wherein the Diabrotica insect pest is selected from the group consisting of: corn rootworm, barker's rootworm, cucumber rootworm subsp. undecemlineata, and maize rootworm.

34. An insecticidal composition comprising the protein of claim 14 and an agriculturally acceptable carrier.

35. The composition of claim 34, wherein the agriculturally acceptable carrier is selected from the group consisting of dusts, pellets, granules, sprays, emulsions, colloids, and solutions.

36. The composition of claim 34, wherein the composition is prepared by dehydrating, freeze-drying, homogenizing, extracting, filtering, centrifuging, sedimenting, or concentrating a culture of a Bacillus thuringiensis strain.

37. The composition of claim 34 comprising from about 1% to about 99% by weight of the insecticidal protein.

38. The composition of any one of claims 34-37, wherein the composition further comprises a second pesticide.

39. The composition of claim 38, wherein the second pesticide is a biological agent or a chemical agent.

40. The composition of claim 39, wherein (a) the biological agent is or is derived from a Bacillus thuringiensis insecticidal protein, a Bacillus cereus insecticidal protein, a Xenorhabdus species insecticidal protein, a Photorhabdus species insecticidal protein, a Brevibacillus laterosporus insecticidal protein, a Lysinibacillus sphaericus insecticidal protein, a Chromobacterium species insecticidal protein, a Yersinia entomophage insecticidal protein, a Paenibacillus popiliae insecticidal protein, or a Clostridium species insecticidal protein; (b) the biological agent is or is derived from a dsRNA, a Cry protein, a Vip protein, a potato glycoprotein, a protease, a protease inhibitor, a urease, an α-amylase inhibitor, a pore-forming protein, a lectin, an engineered antibody or antibody fragment, or a chitinase; (c) the chemical agent is a carbamate, a pyrethroid, an organophosphate, a friprole, a neonicotinoid, an organochloride, Nereis toxins or a combination thereof; or (d) the chemical agent comprises an active ingredient selected from the group consisting of carbofuran, carbaryl, methomyl, cypermethrin, tefluthrin, permethrin, cyfluthrin, λ-cyfluthrin, cypermethrin, deltamethrin, chlorpyrifos, chloroxyphos, dimethoate, ethoprophos, malathion, methyl parathion, phorate, terbufos, tert-butylpyrimidophos, fipronil, acetamiprid, imidacloprid, thiacloprid, thiamethoxam, endosulfan, sulfamethoxam and a combination thereof.

41. A method for producing an insecticidal protein, the method comprising culturing the host cell of claim 17 or an organism comprising the host cell under conditions whereby the host cell produces the insecticidal protein.

42. A method of producing a transgenic plant or plant part having enhanced insect resistance compared to a control plant or plant part, the method comprising: (a) introducing into a plant or plant part The chimeric gene of claim 5, wherein the insecticidal protein is expressed in the plant or plant part, thereby producing a plant or plant part with enhanced insect resistance.

43. The method of claim 42, wherein the introducing step is accomplished by: (a) transforming the plant or plant part; or (b) crossing a first plant comprising the chimeric gene with a different second plant.

44. The method of claim 43, wherein the chimeric gene encodes an insecticidal protein having the amino acid sequence of any one of SEQ ID NOs: 1-21.

45. A method of controlling an insect pest comprising delivering an effective amount of the insecticidal protein of claim 12 to the insect pest or its environment.

46. ​​The method of claim 45, wherein the insecticidal protein is delivered via a transgenic plant or by topical application of an insecticidal composition comprising the insecticidal protein.

47. The method of claim 46, wherein the transgenic plant or the insecticidal composition comprises a second insecticide different from the insecticidal protein.

48. The method of claim 47, wherein the second insecticide is a protein, a dsRNA, or a chemical.

49. The method of claim 48, wherein (a) the protein is selected from the group consisting of: Cry protein, Vip protein, potato glycoprotein, protease, protease inhibitor, urease, α-amylase inhibitor, pore-forming protein, lectin, engineered antibody or antibody fragment, or chitinase; (b) the chemical is a carbamate, pyrethroid, organophosphate, friprole, neonicotinoid, organochloride, nereistoxin, or a combination thereof; or (c) the chemical comprises an active ingredient selected from the group consisting of: carbofuran, carbaryl, methomyl, bifenthrin, tefluthrin, permethrin, cyfluthrin, lambda-cyhalothrin, cypermethrin, deltamethrin, chlorpyrifos, chloroxyfos, dimethoate, ethoprophos, malathion, methyl parathion, phorate, terbufos, terbufos, fipronil, acetamiprid, imidacloprid, thiacloprid, thiamethoxam, endosulfan, thiosulfuron, and a combination thereof.

50. The method of any one of claims 45-49, wherein the insect pest is a coleopteran insect pest.

51. The method of claim 50, wherein the coleopteran insect pest is a Diabrotica species.

52. The method of claim 51, wherein the species of Diabrotica is selected from the group consisting of: Diabrotica zea, Diabrotica barbatus, Diabrotica euphorbiae, and Diabrotica zea.

53. A method of reducing the development of resistance in a root beetle population to the insecticidal protein of claim 5, the method comprising expressing the insecticidal protein and an interfering RNA molecule in a transgenic plant ingested by the root beetle population, the interfering RNA molecule inhibiting expression of a target gene in larval and adult root beetles, thereby reducing the development of resistance in the root beetle population compared to a root beetle population exposed only to the insecticidal protein.

54. A method of providing a corn grower with a means of controlling a rootworm pest population in a corn crop, the method comprising (a) selling or providing to the grower transgenic corn seeds comprising the nucleic acid molecule of claim 1; and (b) advertising to the grower that the transgenic corn seeds produce transgenic corn plants that control a rootworm pest population.

55. A method of identifying an insecticidal protein comprising a nucleotide sequence having at least 80% to at least 99% sequence identity to any one of SEQ ID NOs: 22-25, the method comprising the steps of: (a) generating a primer pair that will amplify a polynucleotide of any one of SEQ ID NOs: 21-25, or a complementary sequence thereof, from a nucleic acid sample, (b) amplifying an orthologous gene from the nucleic acid sample, (c) identifying the polynucleotide sequence of the orthologous gene, (d) producing a protein encoded by the orthologous gene, and (e) determining that the protein of step (d) has insecticidal activity against an insect pest.

56. A variant insecticidal protein comprising an amino acid substitution corresponding to an amino acid substitution in SEQ ID NO: 3 selected from the group consisting of I50L, I53L, A62C or L, A64C or L, I81L, I126L, I153L, I169L, I185L, I207L, I219L, and I275L, wherein the variant has at least 90% identity to SEQ ID NO: 3 and is active against insect pests of the genus Diabrotica.

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