Insecticidal proteins

Abstract

Disclosed are compositions and methods for controlling insect pests. In particular, novel insect inhibitory proteins comprising two different components, both of which are necessary for biological activity against at least a coleopteran insect, are provided. Nucleic acid molecules encoding the novel insecticidal proteins are also provided. Methods of making the insecticidal proteins, and methods of using the insecticidal proteins and nucleic acids encoding the insecticidal proteins of the invention, e.g., to confer protection from insect damage in transgenic plants, are also disclosed.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 951,025, filed December 20, 2019, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the fields of molecular biology and pest control. More particularly, this invention relates to novel insecticidal proteins comprising two distinct components, both of which are essential for maximum biological activity. The invention further relates to expressing nucleic acids that produce these insecticidal proteins, methods for manufacturing these insecticidal proteins and corresponding nucleic acids, and methods for using these insecticidal proteins and corresponding nucleic acids to control insects. Background Technology

[0004] Insect pests are a major cause of crop loss. In the United States alone, billions of dollars are lost annually due to infestations by various genera of insects. In addition to losses in field crops, insect pests are a burden for vegetable and fruit growers, producers of ornamental flowers, and a nuisance for gardeners and homeowners.

[0005] Several species of corn rootworms are considered among the most destructive corn pests. 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 a billion dollars in damage to corn annually in the US 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 leaf beetle (Diabroticaspeciosa) is considered a major corn pest. The western corn rootworm spread to Europe in 1992 and has been causing economic losses throughout major corn-growing regions since 2008. The larvae of the corn rootworm cause the most substantial plant damage by feeding almost exclusively on corn roots. This damage has been shown to increase lodging, reduce grain yield and nutrient yield, and alter the nutrient content of grains. Larval feeding also indirectly affects maize by opening pathways for bacterial and fungal infections that cause root and stem rot to enter the roots. Adult maize rootworms are active in maize fields in late summer, where they feed on ears, silks, and pollen, thereby interfering with normal pollination.

[0006] Corn rootworms are primarily controlled through intensive application of chemical pesticides, which are active by inhibiting insect growth, preventing feeding or reproduction, or causing death. This achieves good control of corn rootworms, 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 arises because corn rootworm larvae feed underground, making remedial treatment with insecticides difficult. Therefore, most insecticide application is done preventatively at planting time. This practice results in a significant environmental burden. Various farm management practices have partially improved this situation, but there is a growing need for alternative pest control mechanisms.

[0007] Biological pest control agents, such as strains of Bacillus thuringiensis (Bt) expressing pest-killing toxins like delta-endotoxins (Δ-endotoxins; also known as crystalline toxins or Cry proteins), have been applied to crop plants on a small scale with satisfactory results against certain insect pests. These delta-endotoxins are proteins housed within a crystalline matrix and 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 have been commercially developed to control certain Lepidoptera and Coleoptera insect pests. For example, transgenic maize hybrids controlling maize rootworms by expressing Cry3Bb1, Cry34Ab1 / Cry35Ab, modified Cry3A (mCry3A), or eCry3.1Ab proteins, began to be commercially available in the United States in 2003.

[0008] Most of the nearly 200 known Bt Cry proteins possess some degree of lepidopteran activity associated with them. The majority of identified Bt insect inhibitory proteins do not exhibit coleopteran control activity. Therefore, identifying additional coleopteran-specific insect inhibitory proteins is particularly important, at least for commercial purposes.

[0009] Although the use of transgenic plants expressing Cry proteins has shown great effectiveness, insect pests resistant to the Cry proteins expressed in some transgenic plants are now known. Therefore, there remains a need to identify novel and effective pest control proteins that offer economic benefits to farmers and are environmentally acceptable. Of particular demand are proteins toxic to species of the root leaf beetle (Diabrotica), a major maize pest, which act differently from Cry proteins in existing insect control products to mitigate resistance development. Furthermore, delivery of insect control proteins through products that minimize environmental burden, such as transgenic plants, is promising. Summary of the Invention

[0010] In view of these needs, the present invention provides novel insecticidal proteins encoded by nucleotide sequences at least recognizable in bacterial genomes, particularly in the orders *Shingobacteriale* and *Rhodobacterale*, but also recognizable in the genomes of bacteria of other orders. Examples of such insecticidal proteins are illustrated herein, and such insecticidal proteins, whether derived from *Shingobacteriale*, *Rhodobacterale*, or different orders, are collectively referred to as sBin insecticidal proteins (sBin-IP), and particularly sBin1-IP and sBin2-IP. The sBin1 and sBin2 proteins of the present invention are two components of a binary toxin exhibiting toxicity to at least beetle insect pests. The present invention also provides variants of the sBin-IP of the present invention, as well as proteins substantially identical to the sBin-IP and its variants of the present invention. Examples of the amino acid sequences of the sBin-IP of the present invention include, but are not limited to, any one of SEQ ID NO:1-6, wherein SEQ ID NO:1, 3, and 5 are the amino acid sequences of the sBin1 protein, and SEQ ID NO:2, 4, and 6 are the amino acid sequences of the sBin2 protein. The sBin-IP of the present invention is toxic to insect pests. For example, the proteins of the present invention can be used for the economical control of important insect pests, including Coleoptera, such as species of the genus *Fireflyus*. Such species include, but are not limited to, the Western Corn Rootworm (WCR), the Northern Corn Rootworm (NCR), the Southern Corn Rootworm (SCR), and the Mexican Corn Rootworm (MCR).

[0011] The present invention further provides a nucleic acid molecule comprising one or more nucleotide sequences encoding sBin-IP or variants of sBin-IP, their complementary sequences, or nucleotide sequences substantially identical to sBin-IP or variants of sBin-IP. Examples of nucleotide sequences encoding sBin-IP or variants of sBin-IP of the present invention include, but are not limited to, any one of SEQ ID NO: 7-24, wherein SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, and 23 are nucleotide sequences encoding sBin1 protein, and SEQ ID NO: 8, 10, 12, 14, 16, 18, 20, 22, and 24 are nucleotide sequences encoding sBin2 protein.

[0012] The present invention also provides vectors comprising recombinant nucleic acids encoding the sBin-IP and / or variants of the present invention; plants or microorganisms comprising and capable of expressing such nucleic acids; plants transformed with such nucleic acids, such as transgenic maize plants; progeny of such plants containing stably incorporated and Mendelian-inherited nucleic acids; and / or seeds of such plants and such progeny. The present invention also provides methods of breeding to introduce transgenic molecules comprising the nucleic acid molecules of the present invention into progeny plants and various germplasms.

[0013] The present invention also provides compositions and formulations containing sBin-IP and / or variants of sBin-IP of the present invention, which are capable of inhibiting the survival, growth and / or reproduction of insect pests, or limiting the ability of insect-related damage or loss to crops, for example, by applying sBin-IP or variants thereof as part of a composition or formulation to insect-infested areas or plants, or by preventatively treating insect-vulnerable areas or plants to provide protection against insect pests.

[0014] The present invention further provides a method for manufacturing sBin-IP or variants thereof, and a method for using these nucleic acids, for example, to control insects in microorganisms or to confer protection against insect damage in transgenic plants. Such microorganisms may be, for example, colonizing maize roots and delivering the sBin-IP of the present invention to endophytes in the rhizosphere of maize, thereby protecting the roots from damage caused by maize rootworms.

[0015] The sBin-IP and / or variants of the present invention 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 increase the spectrum of target insects with minimal environmental impact.

[0016] Other aspects and advantages of the invention will become clear to those skilled in the art from the following description and non-limiting examples.

[0017] A brief description of sequences in a sequence list.

[0018] SEQ ID NO:1 is the amino acid sequence of Seg_korCRW1(sBin1Aa).

[0019] SEQ ID NO:2 is the amino acid sequence of Seg_korCRW2(sBin2Aa).

[0020] SEQ ID NO:3 is the amino acid sequence of Dyad_SG02CRW1(sBin1Ba).

[0021] SEQ ID NO:4 is the amino acid sequence of Dyad_SG02CRW2(sBin2Ab).

[0022] SEQ ID NO:5 is the amino acid sequence of Parac_pantoCRW1(sBin1Ca).

[0023] SEQ ID NO:6 is the amino acid sequence of Parac_pantoCRW2(sBin2Ba).

[0024] SEQ ID NO:7 is the nucleotide sequence of Seg_korCRW1.

[0025] SEQ ID NO:8 is the nucleotide sequence of Seg_korCRW2.

[0026] SEQ ID NO:9 is the nucleotide sequence of Dyad_SG02CRW1.

[0027] SEQ ID NO:10 is the nucleotide sequence of Dyad_SG02CRW2.

[0028] SEQ ID NO:11 is the nucleotide sequence of Parac_pantoCRW1.

[0029] SEQ ID NO:12 is the nucleotide sequence of Parac_pantoCRW2.

[0030] SEQ ID NO:13 is the optimized nucleotide sequence of E. coli Seg_korCRW1.

[0031] SEQ ID NO:14 is an optimized nucleotide sequence of E. coli Seg_korCRW2.

[0032] SEQ ID NO:15 is the optimized nucleotide sequence of E. coli Dyad_SG02CRW1.

[0033] SEQ ID NO:16 is the optimized nucleotide sequence of E. coli Dyad_SG02CRW2.

[0034] SEQ ID NO:17 is the optimized nucleotide sequence of Parac_pantoCRW1 E. coli.

[0035] SEQ ID NO:18 is an optimized nucleotide sequence of E. coli Parac_pantoCRW2.

[0036] SEQ ID NO:19 is the optimized nucleotide sequence of Seg_korCRW1 corn.

[0037] SEQ ID NO20 is the optimized nucleotide sequence of Seg_korCRW2 corn.

[0038] SEQ ID NO:21 is the optimized nucleotide sequence of Dyad_SG02CRW1 corn.

[0039] SEQ ID NO:22 is the optimized nucleotide sequence of Dyad_SG02CRW2 corn.

[0040] SEQ ID NO:23 is the optimized nucleotide sequence of Parac_pantoCRW1 corn.

[0041] SEQ ID NO:24 is the optimized nucleotide sequence of Parac_pantoCRW2 corn. Detailed Implementation

[0042] This description is not intended to be a detailed list of all the different ways in which the invention can be implemented, or of all the features that can be added to the invention. For example, a feature described with respect to one embodiment may be incorporated into other embodiments, and a feature described with respect to a particular embodiment may be removed from that embodiment. Therefore, the invention contemplates that in some embodiments of the invention, any feature or combination of features stated herein may be excluded or omitted. Furthermore, given this disclosure, various variations and additions to the different 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 exhaustive in describing all permutations, combinations, and variations thereof.

[0043] 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 this invention pertains. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0044] All publications, patent applications, patents and other references cited herein, with respect to the teachings of relevant sentences and / or paragraphs, are incorporated in their entirety by reference.

[0045] The nucleotide sequences provided herein are represented from left to right in a 5' to 3' direction and are represented using standard codes representing nucleotide bases, as described in 37 CFR §§ 1.821-1.825 and WIPO Standard ST. 25, such as: adenine (A), cytosine (C), thymine (T), and guanine (G).

[0046] Amino acids are also indicated using WIPO standard ST.25, such as: 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; 1), 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 the amino acid sequence indicates that the amino acid at that position can be any of the 20 known amino acids or any of the amino acids listed herein.

[0047] Unless the context otherwise indicates, it is expressly contemplated that the various features of the invention described herein can be used in any combination. Furthermore, the invention is also contemplated that in some embodiments of the invention, any feature or combination of features stated herein may be excluded or omitted. For example, if this specification states that a composition comprises components A, B, and C, it is expressly contemplated that any one of A, B, or C, or any combination thereof, may be omitted and abandoned individually or in any combination.

[0048] definition

[0049] Unless otherwise specified, the following terms shall be understood to have the following meanings when used in accordance with this disclosure:

[0050] As used herein and in the appended claims, the singular forms “a / an” and “the” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “a plant” refers to one or more plants and includes their equivalents known to those skilled in the art, etc.

[0051] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more associated listed items, together with the absence of a combination when interpreted in terms of alternatives (“or”).

[0052] The term "about" is used herein to mean approximately, roughly, or about. When the term "about" is used in conjunction with a numerical range, it defines the range by extending the boundaries to be higher than and lower than the stated value. Generally, the term "about" is used herein to define a value to be higher than and lower than the specified value by a variation of 20%, preferably around 10% (higher or lower). With respect to temperature, the term "about" means ±1°C, preferably ±0.5°C. When the term "about" is used in the context of this invention (e.g., in combination with temperature or molecular weight values), an exact value (i.e., without "about") is preferred.

[0053] Unless the context otherwise indicates, 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) as used herein shall be interpreted as including both X and Y.

[0054] As used herein, the term "amplified" refers to the construction of multiple copies of a nucleic acid molecule or multiple copies complementary to that nucleic acid molecule using at least one nucleic acid molecule as a template. 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 (TAS) systems, and strand displacement amplification (SDA). See, for example, Diagnostic Molecular Microbiology: Principles and Applications, eds. PERSING et al., American Society for Microbiology, Washington, DC, (1993). The product of amplification is called an "amplifier".

[0055] The "activity" of the insecticidal protein of this invention means that the insecticidal protein functions as an orally active insect control agent, has toxic effects, and / or can interfere with or prevent insect feeding, which may or may not cause the death of the insect. When the insecticidal protein of this invention is delivered to an insect, this result is typically the death of the insect, or the insect not feeding on a source in which the insecticidal protein is available to the insect. "Pesticide" is defined as a toxic biological activity that controls pests (such as insects, nematodes, fungi, bacteria, or viruses), preferably by killing or destroying them. "Insecticidal" is defined as a toxic biological activity that controls insects, preferably by killing them. "Pesticide" is an agent having pesticide activity. "Insecticidal agent" is an agent having insecticide activity.

[0056] As used herein, the terms “chimeric construct” or “chimeric gene” or “chimeric polynucleotide” or “chimeric nucleic acid” (or similar terms) refer to a construct or molecule that comprises two or more polynucleotides from different sources assembled into a single nucleic acid molecule. The terms “chimeric construct,” “chimeric gene,” “chimeric polynucleotide,” or “chimeric nucleic acid” refer to any construct or molecule that contains, but is not limited to, (1) a polynucleotide (e.g., DNA), including regulatory and coding polynucleotides not found together in nature (i.e., at least one polynucleotide in the construct is heterologous relative to at least one of its other polynucleotides), or (2) a polynucleotide encoding a protein portion that is not naturally adjacent, or (3) a promoter portion that is not naturally adjacent. Additionally, a chimeric construct, chimeric gene, chimeric polynucleotide, or chimeric nucleic acid may comprise regulatory and coding polynucleotides derived from different sources, or may comprise regulatory and coding polynucleotides derived from the same source but arranged in a manner different from that found in nature. In some embodiments of the invention, the chimeric construct, chimeric gene, chimeric polynucleotide, or chimeric nucleic acid comprises an expression cassette containing the polynucleotide of the invention under the control of a regulatory polynucleotide, particularly a regulatory polynucleotide that is functional in plants or bacteria.

[0057] 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.

[0058] "Controlling" insects means suppressing the ability of insect pests to survive, grow, feed, and / or reproduce through toxic effects, or limiting insect-related damage or loss in crop plants. "Controlling" insects may or may not mean killing insects, although it preferably means killing insects.

[0059] As used herein, a “codon-optimized” sequence means a nucleotide sequence in which codons are selected to reflect a specific codon preference that a host cell or organism may have. This is typically done in a manner that preserves the amino acid sequence of the polypeptide encoded by the nucleotide sequence to be optimized. In some embodiments, the DNA sequence of the recombinant DNA construct includes a sequence that has been codon-optimized for the cells (e.g., animal, plant, or fungal cells) in which the construct is to be expressed. For example, a construct to be expressed in plant cells may have all or part of its sequence (e.g., a first gene repressor element or gene expression element) codon-optimized for expression in plants. See, for example, U.S. Patent No. 6,121,014, which is incorporated herein by reference.

[0060] The term "comprises" or "comprising" as used in this specification indicates the presence of a described feature, integer, step, operation, element, or component, but does not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, or groups thereof.

[0061] As used herein, the transitional phrase “consistently of…” (and grammatical variations) means that the scope of the claim is to be interpreted as covering the specified materials or steps enumerated in the claim and those that do not substantially alter one or more fundamental and novel features of the claimed invention. Therefore, when used in the claims of this invention, the term “consistently of…” is not intended to be interpreted as equivalent to “comprising.”

[0062] In the context of this invention, "corresponding to" or "corresponds to" means that when the amino acid sequences of variant or homologous proteins are compared with each other, the amino acids "corresponding to" certain enumerated positions in the variant or homologous protein are those compared with those positions in the reference protein, but these precise numerical positions are unnecessary relative to the specific reference amino acid sequence of this invention. For example, if SEQ ID NO:1 is the reference sequence and is compared with SEQ ID NO:3, then the amino acid Phe(F) at position 201 (F201) of SEQ ID NO:3 "corresponds to" Phe(F) at position 200 (F200) of SEQ ID NO:1, or, for example, Thr(T) at position 105 (T105) of SEQ ID NO:2 "corresponds to" Ser(S) at position 105 (S105) of SEQ ID NO:1.

[0063] "Delivery" of a composition or toxic protein means that the composition or toxic protein comes into contact with an insect, which promotes its oral ingestion, resulting in toxicity and control over the insect. The composition or toxic protein can be delivered in many recognized ways, including but not limited to transgenic plant expression, one or more formulated protein compositions, one or more sprayable protein compositions, bait matrix, or any other recognized protein delivery system in the field.

[0064] The term "domain" refers to a group of amino acids conserved at a specific position along the sequence alignment of an evolutionarily related protein. While amino acids at other positions may vary between homologues, highly conserved amino acids at a specific position indicate that they are likely essential for the protein's structure, stability, or function. Identifying a domain by its high conservation in the aligned sequences of a protein homologue family, it can be used as an identifier to determine whether any polypeptide under discussion belongs to a previously identified polypeptide group.

[0065] "Effective insect control dose" refers to the concentration of insecticidal proteins that inhibit the ability of insects to survive, grow, feed, and / or reproduce through toxic effects, or limit insect-related damage or crop loss. "Effective insect control dose" may or may not mean killing insects, although it preferably means killing insects.

[0066] As used herein, an "expression cassette" refers to a nucleic acid sequence capable of directing the expression of a specific nucleotide sequence in a suitable host cell, containing a promoter operatively linked to the target nucleotide sequence, which in turn is operatively linked to a termination signal. It also typically contains the sequence required for proper translation of the nucleotide sequence. An expression cassette containing 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 already obtained in a recombinant form useful for heterologous expression. However, typically, the expression cassette is heterologous relative to the host, meaning that 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 its ancestor through a transformation event. Expression of the nucleotide sequence in the expression cassette can be under the control of a constitutive or inducible promoter, which initiates transcription only when the host cell is exposed to certain external stimuli. In the case of multicellular organisms (such as plants), the promoter can also be tissue- or organ-specific, or developmentally stage-specific.

[0067] Expression cassettes containing the target nucleotide sequence can be chimeric, meaning that at least one of its components is heterologous relative to at least one of its other components. Expression cassettes can also be expression cassettes containing a natural promoter driving their native gene, however, they have been obtained in a recombinant form useful for heterologous expression. This use of the expression cassette makes it not so naturally present in the cell in which it is introduced.

[0068] The expression cassette may optionally include a transcriptional and / or translational termination region (i.e., a termination region) that functions in plants. Various transcription terminators are available for use in the expression cassette and are responsible for transcriptional termination beyond the target heterologous nucleotide sequence and proper mRNA polyadenylation. The termination region may be natural for the transcription initiation region, natural for the target nucleotide sequence to which it is operatively linked, natural for the plant host, or derived from another source (i.e., foreign or heterologous to the promoter, target nucleotide sequence, plant host, or any combination thereof). Suitable transcription terminators include, but are not limited to, the CAMV 35S terminator, the tml terminator, the carmine synthase terminator, and / or the pea rbcs E9 terminator. These terminators can be used in both monocotyledonous and dicotyledonous plants. Furthermore, natural transcription terminators encoding the sequence may be used. Any terminator known to function in plants that is available may be used in the context of this invention.

[0069] When used with reference to polynucleotides (such as plant genes, ORFs or portions thereof, or transgenes), the term "expression" refers to the process by which genetic information encoded in a gene is converted into RNA (e.g., mRNA, rRNA, tRNA, or snRNA) through the "transcription" of the gene (i.e., via the enzymatic action of RNA polymerase), and, where applicable (e.g., if the gene encodes a protein), into a protein through the "translation" of the mRNA. Gene expression can be regulated at many stages of this process. For example, in the case of an antisense construct or a dsRNA construct, expression may refer only to the transcription of that antisense RNA or only to the dsRNA. In embodiments, "expression" refers to the transcription and stable accumulation of sense (mRNA) or functional RNA. "Expression" can also refer to the production of proteins.

[0070] A "gene" is a defined region within the genome that contains a sequence of nucleic acids encoding a protein, and typically also contains other major regulatory nucleic acids responsible for controlling the expression of that coding region (i.e., transcription and translation). Genes may also contain additional 5' and 3' untranslated sequences and termination sequences. Other possible elements include, for example, introns. As found in nature, the regulatory nucleic acid sequences of a gene may not normally be operatively linked to the associated nucleic acid sequence, and therefore are not chimeric genes.

[0071] A "target gene" refers to any nucleic acid molecule that, when transferred to an organism, such as bacteria or a plant, confers a desired trait (such as antibiotic resistance, virus resistance, insect resistance, disease resistance, or resistance to other harmful organisms, herbicide tolerance, tolerance to abiotic stress, male sterility, modified fatty acid metabolism, modified carbohydrate metabolism, improved nutritional value, improved performance in industrial processes, or altered reproductive capacity). A "target gene" can also be a gene transferred to bacteria or a plant for the production of commercially valuable enzymes or metabolites in that plant.

[0072] "Heterologous" nucleic acid sequences or molecules are nucleic acid sequences or molecules that are not naturally associated with the host cell in which the nucleic acid sequence is introduced, including multiple non-natural copies of naturally occurring nucleic acid sequences. Heterologous nucleic acid sequences or molecules may contain chimeric sequences, such as chimeric expression cassettes, in which the promoter and coding region are derived from a multi-source organism. Promoter sequences can be constitutive promoter sequences, tissue-specific promoter sequences, chemically inducible promoter sequences, wound-inducible promoter sequences, stress-inducible promoter sequences, or developmental stage-specific promoter sequences.

[0073] "Homologous" nucleic acid sequences are nucleic acid sequences that are naturally associated with the host cells in which they are introduced.

[0074] Homologous recombination is the exchange of nucleic acid fragments between homologous nucleic acid molecules.

[0075] As used in this article, a "hypothetical protein" refers to a protein whose existence has been predicted but for which experimental evidence of its expression in vivo is lacking. Sequencing genomes or organisms (such as bacteria or plants) typically generates a large number of predicted open reading frames (OPFs), the functions of which are difficult to assign. These proteins, whether isolated or conserved hypothetical proteins, account for approximately 20% to 40% of the proteins encoded in each newly sequenced genome. Even with sufficient evidence that the gene's product is expressed, it is difficult to assign its function using techniques such as microarrays and mass spectrometry because it lacks identity with protein sequences that have been annotated with biochemical functions. Typically, most protein sequences are inferred from computational analysis of genomic DNA sequences. Hypothetical proteins are typically created by gene prediction software during genome analysis. When bioinformatics tools used for gene identification find large ORFs in protein databases without characteristic homologs, such tools typically return the name "hypothetical protein" as an annotation.

[0076] The terms "motif," "shared sequence," or "signature" refer to a short, conserved region in the sequence of an evolution-related protein. A motif is often a highly conserved portion of a domain, but it can also include only a part of the domain or be located outside a conserved domain (if all the amino acids of the motif are located outside the domain it defines).

[0077] In the context of two nucleic acid or amino acid sequences, the terms "identity," "identical," or "substantially identical" refer 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 by one of the following sequence comparison algorithms or by visual inspection. Preferably, substantial identity exists throughout a region having a length of at least about 50 residues or bases, more preferably throughout a region having a length of at least about 100 residues or bases, and most preferably these sequences are substantially identical for at least about 150 residues or bases. In a particularly preferred embodiment, these sequences are substantially identical throughout the length of the coding region. Furthermore, substantially identical nucleic acid or amino acid sequences substantially perform the same function.

[0078] For sequence comparisons, typically, one sequence serves as a reference sequence to be compared with a test sequence. When using a sequence comparison algorithm, the test and reference sequences are input into the computer (with subsequence coordinates specified if necessary), and the parameters of the sequence comparison algorithm program are specified. The sequence comparison algorithm then calculates the percentage of sequence identity between this or these test sequences and the reference sequence based on the specified program parameters.

[0079] The optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. [Advances in Applied Mathematics] 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. [Journal of Molecular Biology] 48:443 (1970), by the similarity method of Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 85:2444 (1988), by the computerized implementation 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 Ausubel et al., below).

[0080] One example of an algorithm suitable for determining sequence identity percentages and sequence similarity is the BLAST algorithm, described in Altschul et al., J. Mol. Biol. [Journal of Molecular Biology] 215:403-410 (1990). Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (US National Library of Medicine, 8600 Rockville Pike, Bethesda, 20894, MD). This algorithm involves first identifying high-scoring sequence pairs (HSPs) in the query sequence by identifying short codewords of length W. These high-scoring sequence pairs match or satisfy a positive threshold score T when compared to codewords of the same length in a database sequence. T is called the neighboring codeword score threshold (Altschul et al., 1990). These initial neighboring codeword hits act as seeds for an initial search to discover longer HSPs containing them. These codeword hits are then extended along each sequence in both directions until the accumulated alignment score can increase. For nucleotide sequences, the accumulated score is calculated using 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 score matrix is ​​used to calculate the accumulated score. Extension of these codeword hits in each direction is stopped when the accumulated alignment score decreases by an amount X from its maximum value; when the accumulated score tends to 0 or below 0 due to the accumulation of one or more negative score residue alignments; or when the end of either sequence is reached. The parameters W, T, and X of the BLAST algorithm determine the alignment sensitivity and speed. The BLASTN program (for nucleotide sequences) uses a word length (W) of 11, an expected value (E) of 10, a cutoff of 100, M = 5, N = -4, and two-strand comparisons as default values. For amino acid sequences, the BLASTP program uses a word length (W) of 3, an expected value (E) of 10, and a BLOSUM62 score matrix as default values ​​(see Henikoff and Henikoff, Proc. Natl. Acad Sci. USA [Proceedings of the National Academy of Sciences] 89:10915 (1989)).

[0081] In addition to calculating the percentage of sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin and Altschul, Proc. Nat'l. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indication of the probability that a match will occur by chance between two nucleotide or amino acid sequences. For example, if the minimum sum probability in a comparison of the test nucleic acid sequence and 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, then the test nucleic acid sequence is considered similar to the reference sequence.

[0082] Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize with each other under stringent conditions. The phrase "specific hybridization" refers to a molecule binding, double-stranding, or hybridizing only with a specific nucleotide sequence under stringent conditions, which occurs when that sequence is present in a complex mixture (e.g., total cellular) of DNA or RNA. "Substantially binding" refers to complementary hybridization between the probe nucleic acid and the target nucleic acid, and encompasses a small number of mismatches that can be adapted by reducing the stringency of the hybridization medium to achieve the desired detection of the target nucleic acid sequence.

[0083] In the context of nucleic acid hybridization experiments (such as DNA and RNA hybridization), "strict hybridization conditions" and "strict hybridization washing conditions" are sequence-dependent and vary under different environmental parameters. Longer sequences hybridize specifically at higher temperatures. Extensive guidance on nucleic acid hybridization can be found in the following literature: Tijssen (1993), Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Chapter 2, Part I, "Overview of principles of hybridization and the strategy of nucleic acid probe assays," Elsevier, New York. Typically, highly stringent hybridization and washing conditions are selected at defined ionic strengths and pH values ​​relative to the melting point (T0) of the specific sequence. m The temperature is approximately 5°C lower. Typically, under “strict conditions,” the probe will hybridize with its target sequence but not with other sequences.

[0084] Tm This is the temperature at which 50% of the target sequence hybridizes with a perfectly matched probe (under specified ionic strength and pH). Very stringent conditions are selected as T equal to the specific probe temperature. m An example of stringent hybridization conditions for hybridization of complementary nucleic acids (which have more than 100 complementary residues on the filter in DNA or RNA blots) is overnight hybridization at 42°C with 50% formamide containing 1 mg heparin. An example of highly stringent wash conditions is 0.15 M NaCl at 72°C for approximately 15 minutes. An example of stringent wash conditions is 0.2×SSC wash at 65°C for 15 minutes (see Sambrook, below, for instructions on SSC buffer). Typically, a low-stringent wash is performed before a high-stringent wash to remove background probe signal. An example of a moderately stringent wash for duplexes of more than 100 nucleotides is 1×SSC at 45°C for 15 minutes. An example of a low-stringent wash for duplexes 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 Na+ salt concentration of less than about 1.0 M, typically about 0.01 to 1.0 M Na+ (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 a destabilizing agent (such as formamide). Generally, a signal-to-noise ratio that is 2-fold (or higher) higher than that of an unrelated probe in a specific hybridization assay indicates that specific hybridization has been detected. If nucleic acids that do not hybridize to each other under stringent conditions encode substantially the same protein, they remain substantially the same. This occurs, for example, when copies of nucleic acids are created using the maximum degree of codon degeneracy allowed by the genetic code.

[0085] The following are examples of hybridization / washing conditions that can be used to clone homologous nucleotide sequences (which are substantially identical to the reference nucleotide sequence of the present invention): the reference nucleotide sequence is preferably hybridized with the reference nucleotide sequence under the following conditions: in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4, 1mM EDTA at 50°C, and washed at 50°C in 2×SSC, 0.1% SDS; more preferably, in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4, 1mM EDTA at 50°C, and washed at 50°C in 1×SSC, 0.1% SDS; still more preferably, in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4, 1mM EDTA at 50°C, and washed at 50°C in 0.5×SSC, 0.1% SDS; preferably in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4, 1mM EDTA, 0.5M NaPO4, 1mM EDTA. Washed at 50°C in EDTA and at 50°C in 0.1×SSC and 0.1% SDS; more preferably washed at 50°C in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4, and 1mM EDTA and at 65°C in 0.1×SSC and 0.1% SDS.

[0086] Another indication that two nucleic acid sequences or proteins are substantially identical is that the protein encoded by the first nucleic acid undergoes an immune cross-linking reaction with or specifically binds to the protein encoded by the second nucleic acid. Thus, the protein is typically substantially identical to the second protein, for example, where the two proteins are distinguished only by conserved substitutions.

[0087] The term "isolated" nucleic acid molecule, polynucleotide, or protein refers to a nucleic acid molecule, polynucleotide, or protein that is no longer present in its natural environment. The isolated nucleic acid molecules, polynucleotides, or proteins of this invention may exist in a purified form or may exist in a recombinant host, such as transgenic bacteria or transgenic plants. Therefore, the claims regarding "isolated" nucleic acid molecules, as enumerated herein, cover nucleic acid molecules when they are contained within the genome of a transgenic plant.

[0088] 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 this invention, a nucleic acid molecule is typically a segment of DNA. In some embodiments, the nucleic acid molecule of this invention is an isolated nucleic acid molecule.

[0089] "Operationally linked" refers to the association of multiple nucleotides on a single nucleic acid fragment, such that the function of one affects the function of the other. For example, when a promoter can influence the expression of a polynucleotide or functional RNA (i.e., the polynucleotide or functional RNA is under the transcriptional control of that promoter), then the promoter and the polynucleotide or functional RNA are operationally linked. Polynucleotides encoded in either the forward or antisense direction can be operationally linked to regulatory polynucleotides.

[0090] As used herein, "pesticide" and "insecticide" refer to the ability of the sBin-IP of the present invention to control pests or the amount of sBin-IP that can control pests as defined herein. Therefore, the pest-killing sBin-IP can kill or inhibit the ability of pests (e.g., insect pests) to survive, grow, feed, or reproduce.

[0091] The terms “protein,” “peptide,” and “polypeptide” are used interchangeably in this article.

[0092] “Plant” means any plant at any stage of development, especially seed plants. Exemplary plants include, but are not limited to, maize (Zea mays), canola (Brassica napus, Brassica rapa ssp.), alfalfa (Medicago saliva), rice (Oryzasativa, including but not limited to indica and / or japonica rice), rapeseed (European rapeseed), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), sunflower (Helianthusannus), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanut (Arachis hypogaea), and cotton (Gossypium). Hirsutum), sweet potato (Ipomoea batatas), cassava (Manihot esculenta), coffee (Coffee subspecies (Cofea spp.)), coconut (Cocosnucifera), pineapple (Ananas comosus), citrus tree (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musaspp.).Avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beet (Beta vulgaris), apple (Malus pumila), blackberry (Rubus), strawberry (Fragaria), walnut (Juglans regia), grape (Vitis vinifera), apricot (Prunus armeniaca), cherry (Prunus), peach (Prunus armeniaca). Fruits include persica, plums (Prunus domestica), pears (Pyrus communis), watermelons (Citrullus vulgaris), duckweed (Lemna spp.), oats (Avena sativa), barley (Hordium vulgare), vegetables, ornamental plants, conifers and turfgrass (e.g., for ornamental, recreational or forage purposes), and biomass grasses (e.g. switchgrass and miscanthus).

[0093] Vegetables include, but are not limited to, species of the genus *Solanum* (e.g., tomato, *Lycopersicon esculentum*), lettuce (e.g., lettuce, *Lactuea sativa*), carrot (*Caucus carota*), cauliflower (*Brassica oleracea*), celery (*Apium graveolens*), eggplant (*Solanum melongena*), asparagus (*Asparagus officinalis*), okra (*Abelmoschus esculentus*), green beans (*Phaseolus vulgaris*), green beans (*Phaseolus limensis*), peas (*Lathyrus spp.*), and squash (*Cucurbita*). Members include Cuban squash (C. hubbard), winter squash (C. moschata), dense zucchini (C. pepo), crooked squash (C. crookneck), C. argyrosperma, C. argyrosperma ssp sororia, C. digitata, C. ecuadorensis, dryland squash (C. foetidissima), C. lundelliana, and C. martinezii, as well as members of the genus Cucumis such as cucumber (Cucumis sativus), cantaloupe (C. cantalupensis), and melon (C. melon).

[0094] Ornamental plants include, but are not limited to, rhododendrons (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.

[0095] The conifers that can be used in the practice disclosed herein include, for example, pine trees such as loblolly pine (Pinustaeda), slash pine (Pinus elliotii), jack pine (Pinus ponderosa), black pine (Pinus contorta), and Monterey pine (Pinus radiata); Douglas fir (Pseudotsuga menziesii); western hemlock (Tsuga canadensis); western spruce (Picea glauca); redwood (Sequoia sempervirens); fir trees such as silver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedar trees such as western cypress (Thuja spp.). plicata)) and Alaska yellow-cedar (Chamaecyparis nootkatensis)).

[0096] Turfgrasses include, but are not limited to, Zoysia japonica, creeping bentgrass, fescue, Kentucky bluegrass, Augustinian grass, Bermuda grass, bufallograsses, ryegrass, and orchardgrass.

[0097] It also includes plants that primarily serve as laboratory models, such as Arabidopsis thaliana.

[0098] A plant cell is the structural and physiological unit of a plant, consisting of a protoplast and a cell wall. Plant cells can exist as isolated single cells or cultured cells, or as part of higher-level tissue units such as, for example, plant tissues, plant organs, or the whole plant.

[0099] "Plant cell culture" refers to a culture of plant units (such as, for example, protoplasts, cell culture cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes, and embryos at different developmental stages).

[0100] "Plant material" means leaves, stems, roots, flowers or parts of flowers, fruits, pollen, egg cells, zygotes, seeds, cuts, cell or tissue cultures, or any other part or product of a plant.

[0101] "Plant organs" are unique and distinct structured and differentiated parts of a plant, such as roots, stems, leaves, flower buds, or embryos.

[0102] As used herein, “plant tissue” means a group of plant cells organized into structural and functional units. This includes any plant tissue in a plant or culture. The term includes, but is not limited to, the whole plant, plant organs, plant seeds, tissue cultures, and any group of plant cells organized into structural and / or functional units. The combined or separate use of this term with any particular type of plant tissue as listed above or otherwise covered by this definition is not intended to exclude any other type of plant tissue.

[0103] "Polynucleotide" refers to a polymer composed of a number of nucleotide monomers covalently bonded together in a chain. Such "polynucleotides" include DNA, RNA, modified oligonucleotides (e.g., oligonucleotides containing bases atypical for biological RNA or DNA, such as 2'-O-methylated oligonucleotides), etc. In some embodiments, the nucleic acid or polynucleotide may be single-stranded, double-stranded, multi-stranded, or a combination thereof. Unless otherwise stated, in addition to any explicitly indicated polynucleotide, the specific nucleic acid or polynucleotide of the present invention optionally also contains or encodes a complementary polynucleotide.

[0104] "Target polynucleotide" refers to any polynucleotide that, when transferred into an organism (e.g., a plant), imparts a desired characteristic to that organism, such as insect resistance, disease resistance, herbicide tolerance, antibiotic resistance, improved nutritional value, improved performance in industrial processes, the production of commercially valuable enzymes or metabolites, or altered reproductive capacity.

[0105] A promoter is the untranslated DNA sequence upstream of a coding region that contains an RNA polymerase binding site and initiates DNA transcription. Promoter regions may also include other elements that act as regulators of gene expression.

[0106] As used herein, the term “recombinant” refers to a nucleic acid molecule (e.g., DNA or RNA) or protein or organism that is not normally found in nature and is therefore produced through human intervention. As used herein, a “recombinant nucleic acid molecule” is a nucleic acid molecule containing a combination of polynucleotides that do not naturally coexist and are the result of human intervention; for example, a nucleic acid molecule consisting of a combination of at least two polynucleotides that are heterologous to each other, or a nucleic acid molecule that is artificially synthesized (e.g., polynucleotides synthesized using assembled nucleotide sequences) and contains polynucleotides that are different from those normally found in nature, or a nucleic acid molecule containing transgenes artificially incorporated into the genomic DNA of a host cell and into the related flanking DNA of that host cell's genome. Another example of a recombinant nucleic acid molecule is a DNA molecule produced by inserting a transgene into the genomic DNA of a plant, which can ultimately lead to the expression of recombinant RNA / or protein molecules in that organism. As used herein, a “recombinant plant” is a plant that does not normally exist in nature, is the result of human intervention, and contains transgenes and / or heterologous nucleic acid molecules incorporated into its genome. Due to such genomic alterations, recombinant plants are significantly different from their corresponding wild-type plants.

[0107] "Regulatory elements" are sequences that participate in controlling the expression of a nucleotide sequence. Regulatory elements include promoters operatively linked to the target nucleotide sequence and termination signals. They also typically encompass the sequences required for proper translation of that nucleotide sequence.

[0108] "sBin insecticidal protein" (sBin-IP) is a protein encoded by genes found in the genomes of bacteria in the orders Sphingomonas and Rhodophytes, and is a binary toxin component active against insect pests in the genus *Lepidoptera*. In some embodiments, the bacterial genome belongs to the genus *Dyadobacter*, the genus *Segetibacter*, or the genus *Paracococcus*. In some embodiments, the *D. alkalitolerans* species are selected from the group consisting of: *D. arcticus*, *D. beijingensis*, *D. crusticola*, *D. endophyticus*, *D. fermentans*, *D. ginsengisoli*, *D. hamtensis*, *D. jejuensis*, *D. koreensis*, *D. psychrophilus*, *D. sediminis*, *D. soli*, and *D. tibetensis*. In some embodiments, the *S. aerophilus* species are selected from the group consisting of: *S. aerophilus* and *S. koreensis*.In some embodiments, the species of *Paracoccus* are selected from the group consisting of: *P. alcaliphilus*, *P. alkenifer*, *P. aminophilus*, *P. aminovorans*, *P. bengalensis*, *P. carotinifaciens*, *P. denitrificans*, *P. ferrooxidans*, *P. haeundaensis*, *P. halotolerans*, *P. homiensis*, *P. kawasakiensis*, *P. kocurii*, *P. kondratievae*, *P. koreensis*, *P. marcusii*, *P. methylutens*, and *P. thalassemia*. Paracoccus pantotrophus, Paracoccus seriniphilus, Paracoccus solventivorans, Paracoccus thiocyanatus, Paracoccus thiophilus, Paracoccus versutus, Paracoccus yeei, and Paracoccus zeaxanthinifaciens.

[0109] Referring to the amino acid or nucleotide sequence, the terms “substitution,” “insertion,” “addition,” and “deletion” are used herein. “Substitution” refers to replacing one or more nucleotides or amino acids, respectively, with a different nucleotide or amino acid. “Insertion” or “addition” is a change in a nucleotide or amino acid sequence that results in the addition of one or more nucleotide 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 nucleotide or amino acid residues are absent, respectively. Amino acid substitution is typically a single residue substitution; insertions are generally 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. Substitution, deletion, insertion, or any combination thereof can be used to obtain the final variant polypeptide. Typically, some amino acids are changed to minimize changes in the molecule. However, in some cases, larger changes can be tolerated. In some embodiments, amino acid substitution can be the result of replacing an amino acid with another amino acid having similar structure and / or chemical properties, such as replacing isoleucine with valine, i.e., a conserved amino acid substitution. Insertions or deletions may optionally range from 1 to 5 amino acids. In the embodiments, substitutions may be made according to known “conserved substitutions.” "Conservative substitution" refers to the substitution of an amino acid in one class by an amino acid from another class, where the class is defined by common physicochemical amino acid side chain characteristics and high substitution frequency in homologous proteins found in nature. Conversely, in some embodiments, the substitution is non-conservative. "Non-conservative substitution" refers to the substitution of an amino acid in one class with an amino acid from another class.

[0110] "Transformation" is a method used to introduce heterologous nucleic acids into a host cell or organism. In a specific embodiment, "transformation" means the stable integration of a DNA molecule into the genome (nucleus or plasmid) of the target organism.

[0111] "Transformed / GMO / Recombinant" refers to a host organism (e.g., bacteria or plants) in which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the host's genome, or it can exist as an extrachromosomal molecule. This extrachromosomal molecule is capable of autonomous replication. Transformed cells, tissues, or plants should be understood to encompass not only the end products of the transformation process but also their transgenic progeny. "Non-transformed," "non-GMO," or "non-recombinant" hosts refer to wild-type organisms, such as bacteria or plants, that do not contain the heterologous nucleic acid molecule.

[0112] This invention provides compositions and methods for controlling harmful insect pests. In particular, this invention relates to insecticidal proteins and variants thereof, referred herein as rhizobium insecticidal proteins (sBin-IP), which are active against at least Coleoptera insects, such as the western maize root beetle (WCR) and the Diabrotica barber. (Northern corn rootworm; NCR) and / or the root-eating subspecies of the cucumber eleven-spotted leaf beetle (Southern corn rootworm; SCR) and / or other root-flying leaf beetle species (including the Mexican corn root-flying leaf beetle (Mexican corn rootworm; MCR) and the South American root-flying leaf beetle (gourd beetle). The inventors of this invention have discovered that certain proteins described in the art as hypothetical proteins are surprisingly components of binary insecticidal toxins, which are allegedly encoded in the genomes of at least Gram-negative bacteria in the orders Sphingomonasales and Rhodophytes. More particularly, coding sequences of hypothetical proteins with insecticidal activity have been found to be present in the genomes of Gram-negative bacteria of the genera *Trebrobacter*, *Pseudomonas*, *Paracoccus*, and related genera. Even more particularly, coding sequences of hypothetical proteins exemplified herein as insecticidal proteins include, but are not limited to, strains of *Trebrobacter* Korea (SEQ ID NO:7 and SEQ ID NO:8), strain SG02 of the genus *Pseudomonas* (SEQ ID NO:9 and SEQ ID NO:10), and strain *Paracoccus pantotrophus* (SEQ ID NO:11 and SEQ ID NO:8). Those sequences in the genome of (SEQ ID NO: 12). After synthesizing nucleic acid molecules encoding the above-mentioned proteins and expressing the proteins in transgenic *E. coli* bacteria, the inventors determined that proteins described in the art as hypothetical proteins surprisingly possess insecticidal activity, particularly against pests of the genus *Lepidoptera*. Such insecticidal proteins are generally designated herein as sBin insecticidal proteins (sBin-IP), and those specifically exemplified herein are designated as sBin1Aa (SEQ ID NO: 1), sBin2Aa (SEQ ID NO: 2), sBin1Ba (SEQ ID NO: 3), sBin2Ab (SEQ ID NO: 4), sBin1Ca (SEQ ID NO: 5), and sBin2Ba (SEQ ID NO: 12). NO:6). Those skilled in the art will recognize that, using the teachings of this invention, they can identify sequences related to those described above, including but not limited to those found in bacteria, nucleic acid molecules from environmental samples, and genomic databases, where such sequences may be hypothetical or may have some other known function, etc. Such related sequences are intended to be encompassed within this invention. Those skilled in the art will understand the meaning of the term "related sequence" upon reading this disclosure.As described in further detail below, the sBin-IP of the present invention is a component of a binary toxin, and together they work to impart activity against pests at least in the order Coleoptera.

[0113] The present invention also relates to nucleic acids (the expression of which produces the sBin-IP of the present invention), and to methods for manufacturing and using these sBin-IPs to control insect pests. In some non-limiting embodiments, the expression of these nucleic acids produces insecticidal proteins that can be used to control at least Coleoptera insects (such as western maize rootworm, northern maize rootworm, and / or southern maize rootworm), particularly when expressed in transgenic plants (such as transgenic maize plants).

[0114] In some non-limiting embodiments, the present invention covers nucleic acid molecules comprising a nucleotide sequence encoding a protein toxic to insect pests, wherein the nucleotide sequence (a) encodes a protein comprising an amino acid sequence having at least 80% to at least 99% sequence identity with any of SEQ ID NO:1-6 or its toxin-coding fragment; or (b) has at least 80% to at least 99% sequence identity with any of SEQ ID NO:7-12 or its toxin-coding fragment; or (c) is a synthetic sequence of (a) or (b) that has been codon-optimized for expression in transgenic organisms. In other embodiments, the insecticidal protein comprises, substantially comprises, or comprises the amino acid sequence of any of SEQ ID NO:1-6 or its toxin-coding fragment. In other embodiments, the nucleotide sequence comprises, substantially comprises, or comprises the toxin-coding fragment of any of SEQ ID NO:7-12. In still other embodiments, the synthetic nucleotide sequence comprises, substantially comprises, or comprises the toxin-coding fragment of any of SEQ ID NO:13-24.

[0115] In some non-limiting embodiments, the present invention covers chimeric genes comprising a heterologous promoter operatively linked to a nucleic acid molecule comprising, substantially comprising, or comprising: a nucleotide sequence encoding a protein toxic to insect pests, 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 having 100% sequence identity with any of SEQ ID NO:1-6 or its toxin fragment; (b) 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 96%, at least 97%, at least 98%, at least 99%, or having 100% sequence identity with any of SEQ ID NO:1-6 or its toxin fragment; Any one of SEQ ID NO: 7-24 or its toxin-coding fragment 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; or (c) is a synthetic sequence of (a) or (b) that has been codon-optimized for expression in a transgenic organism. In other embodiments, the insecticidal protein comprises the amino acid sequence of any one of SEQ ID NO: 1-6 or its toxin-coding fragment. In other embodiments, the nucleotide sequence comprises any one of SEQ ID NO: 7-24 or its toxin-coding fragment. In some aspects of these embodiments, the chimeric gene is an expression cassette.

[0116] In other non-limiting embodiments, the promoters included in the chimeric genes or expression cassettes of the present invention are plant-expressible promoters. In aspects of these embodiments, the plant-expressible promoters are selected from promoters of the group consisting of: ubiquitin, *Cestrum nocturnum* flavivirus, maize TrpA, OsMADS 6, maize H3 histone, 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 mannitol synthase, Ti plasmid annatone synthase, petunia chalcone isomerase, soybean glycine-rich protein 1, potato glycoprotein, lectin, CaMV 35S, and S-E9 small subunit RuBP carboxylase promoters.

[0117] In some non-limiting embodiments, the insecticidal protein encoded by the nucleic acid molecule of the present invention, the chimeric gene of the present invention, or the expression cassette of the present invention is active against Coleoptera insect pests. In some aspects of these embodiments, the Coleoptera insect pests are in the genus *Radiata*. In other aspects, *Radiata* insect pests are *Radiata maize* (Western maize rootworm; WCR), *Radiata barba* (Northern maize rootworm; NCR), and / or *Radiata cucumeroides* (Southern maize rootworm; SCR) and / or other *Radiata* species (including *Radiata mexicanica* (Mexican maize rootworm; MCR)).

[0118] In some non-limiting embodiments, the chimeric gene or expression cassette of the present invention includes a nucleotide sequence encoding the sBin-IP of the present invention, 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.

[0119] In other non-limiting embodiments, the transgenic bacteria are from the following genera: *Bacillus*, *Clostridium*, *Pathogenic Bacillus*, *Luminobacter*, *Pasteurella*, *Escherichia*, *Pseudomonas*, *Erwinia*, *Serratia*, *Klebsiella*, *Salmonella*, *Pasteurella*, *Xanthomonas*, *Streptomyces*, *Rhizobium*, *Rhodopseudomonas*, *Sinobacterium*, *Cyclophorus*, *Methylophilus*, *Agrobacterium*, *Acetobacter*, *Lactobacillus*, *Arthrobacter*, *Azotobacter*, *Leuconostoc*, *Sphingomonas*, *Burkholderia*, *Candidatus Glomeribacter*, *Dystrophus*, *Streptococcus*, *Chitinophaga*, or *Alcaligenes*. In other embodiments, the transgenic bacteria are *Escherichia coli*. In other embodiments, the nucleotide sequence comprises, is substantially composed of, or is composed of any one of SEQ ID NO:13-18.

[0120] In other non-limiting embodiments, the transgenic plant is a monocotyledonous or dicotyledonous plant. In still other embodiments, the dicotyledonous plant is selected from the group consisting of: soybean, sunflower, tomato, brassica crops, cotton, sugar beet, and tobacco. In another aspect, the monocotyledonous plant is selected from the group consisting of: barley, corn, oats, rice, sorghum, sugarcane, and wheat. In some aspects, the transgenic plant is a corn plant. In other embodiments, the nucleotide sequence contains codons optimized for expression in corn. In still other embodiments, the nucleotide sequence comprises, is substantially composed of, or is composed of any one of SEQ ID NO:19-24.

[0121] In some non-limiting embodiments, the present invention covers proteins toxic to insect pests, and optionally isolated proteins, i.e., insecticidal proteins, wherein the protein or isolated protein comprises, is substantially composed of, or is composed 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 having 100% sequence identity with any one of SEQ ID NO:1-6 or its toxic fragment; (b) an amino acid sequence comprising, is substantially composed of, or is composed of: any one of SEQ ID NO:1-6 or its toxic fragment; (c) an amino acid sequence encoded by a nucleotide sequence that is identical to SEQ ID NO:1-6. Any one of SEQ ID NO: 7-24 or its toxin-coding fragment 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 has 100% sequence identity; (d) an amino acid sequence encoded by a nucleotide sequence comprising, substantially comprising, or comprising any one of SEQ ID NO: 7-24 or its toxin-coding fragment. Those skilled in the art will recognize that the exemplary sBin-IP covered by this invention can be modified. Such modifications and substantially identical nucleic acid or amino acid molecules are covered by this invention.

[0122] This invention also covers engineered sBin insecticidal proteins, which can be described as mutant sBin-IPs or variant sBin-IPs or modified sBin-IPs of this invention. In some embodiments, modification may comprise substitution and / or deletion of one or more amino acids in the naturally occurring sBin-IP sequence and / or insertion of one or more additional amino acids into the naturally occurring sBin-IP sequence. In other embodiments, modification may comprise substitution and / or deletion and / or insertion of one or more amino acids in engineered sBin-IPs. Substitution and / or insertion may be with naturally occurring or non-naturally occurring amino acids. In some non-limiting embodiments, modification comprises, substantially comprises, or comprises the following: substitution and / or insertion and / or deletion of one or more of the following amino acids at the amino acid positions of the sBin-IP amino acid sequence: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and / or valine. Such substitutions and / or insertions and / or deletions can be achieved by altering the codons in the nucleotide sequence encoding sBin-IP, thereby producing a modified sBin-IP nucleotide sequence encoding engineered sBin-IP (which is the mutant sBin-IP or variant sBin-IP or modified sBin-IP of this invention).

[0123] In some non-limiting embodiments, sBin-IP is modified by the following substitutions and / or insertions: (a) one or more amino acids having an aliphatic hydrophobic side chain (e.g., alanine, isoleucine, methionine, and / or valine; in the embodiments, the amino acid is not alanine); (b) one or more amino acids having an aromatic hydrophobic side chain (e.g., phenylalanine, tryptophan, and / or tyrosine); (c) one or more amino acids having a polar neutral side chain (e.g., asparagine, cysteine, glutamine, serine, and / or threonine); (d) one or more amino acids having an acidic side chain (e.g., aspartic acid and / or glutamic acid); one or more amino acids having a basic side chain (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).

[0124] In other embodiments, amino acids in any amino acid sequence of the sBin-IP of the present invention, particularly any amino acid sequence of SEQ ID NO:1-6, are substituted and / or deleted and / or inserted. In other embodiments, an amino acid in SEQ ID NO:1 is substituted. In still other embodiments, the substituted amino acid in SEQ ID NO:1 is located at positions 52, 102, 151, and / or 297. In other embodiments, the amino acid at position 52 is substituted with A, the amino acid at position 102 is substituted with T, the amino acid at position 151 is substituted with A or K, or the amino acid at position 297 is substituted with S or T.

[0125] In another embodiment, the present invention provides a chimeric sBin-IP protein comprising a protein fusion tag linked to the complete sBin-IP sequence or a portion thereof (e.g., a cytotoxic domain). The protein fusion tag may be attached to the N-terminus (e.g., at amino acid 1 or 2 of the sBin-IP sequence), or alternatively, the protein fusion tag may be attached to the C-terminus of the sBin-IP sequence. Protein fusion tags can be polyhistidine, polyarginine, haloalkanes dehalogenase, streptavidin-binding enzyme, 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 tags. These can be used in methods for producing, isolating, or purifying any sBin-IP toxin of the present invention. The present invention also provides recombinant polynucleotides, such as constructs, that encode a fusion tag linked to the sBin-IP toxin of the present invention.

[0126] In some non-limiting embodiments, the present invention covers an insecticidal composition comprising a first component and a second component acting together as an insecticidal toxin, wherein the first component is a peptide selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5, and the second component is a peptide selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6, and wherein the insecticidal toxin is active against at least one genus of root leaf beetle pests.

[0127] In other embodiments, the first component of the insecticidal composition comprises SEQ ID NO:1 and the second component comprises SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6; or the first component comprises SEQ ID NO:3 and the second component comprises SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6; or the first component comprises SEQ ID NO:5 and the second component comprises SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6; or the first component comprises SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5 and the second component comprises SEQ ID NO:2; or the first component comprises SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5 and the second component comprises SEQ ID NO:4; or the first component comprises SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5 and the second component comprises SEQ ID NO:6. In other embodiments of the insecticidal composition, the first component comprises SEQ ID NO:1 and the second component comprises SEQ ID NO:2. In still other embodiments of the insecticidal composition, the first component comprises SEQ ID NO:3 and the second component comprises SEQ ID NO:4. In a further embodiment of the insecticidal composition, the first component comprises SEQ ID NO:5 and the second component comprises SEQ ID NO:6.

[0128] In other embodiments, the insecticidal composition is active against pests of the genus *Fireflyus*. In still other embodiments, the pests of *Fireflyus* are selected from *Fireflyus* species selected from the group consisting of: *Fireflyus maize*, *Fireflyus bark*, *Fireflyus cucumeris*, and *Fireflyus maize*.

[0129] In other embodiments, the insecticidal composition of the present invention further comprises a second pesticide. In other embodiments, the second pesticide is a biological agent or a chemical agent. In other embodiments, the biological agent is or is derived from insecticidal proteins of Bacillus thuringiensis, Bacillus cereus, pathogenic bacteria, luminescent bacteria, Bacillus retrosporum, Bacillus spherulites, Chromobacterium, Yersinia pestis, Paenibacillus popiliae, or Clostridium species. In other embodiments, the biological agent is or is derived from dsRNA, Cry protein, Vip protein, potato glycoprotein, protease, protease inhibitor, urease, α-amylase inhibitor, porogen, lectin, engineered antibody or antibody fragment, or chitinase. In other embodiments, the chemical agent is a carbamate, pyrethroid, organophosphate, neonicotinoid, organochloride, nereistoxin, or a combination thereof; or (d) the chemical agent comprises an active ingredient selected from the group consisting of: carbofuran, methamidophos, methomyl, bifenthrin, heptafluthrin, permethrin, cypermethrin, lambda-cyhalothrin, lambda-cyhalothrin, deltamethrin, chlorpyrifos, oxychloride, dimethoate, ethoprophos, malathion, methyl parathion, phorate, terbufos, terbufos, fipronil, acetamiprid, imidacloprid, thiamethoxam, endosulfan, sulfadiazine, and combinations thereof.

[0130] In some non-limiting embodiments, the invention covers transgenic plants that produce the insecticidal compositions of the invention. In other embodiments, the transgenic plant is a transgenic corn plant that produces the binary toxins of the invention.

[0131] In some embodiments, the present invention covers isolated and purified antibodies that specifically bind to the sBin-IP peptide, its immunologically detectable variants, and the epitope described herein, the sBin-IP peptide being selected from the group consisting of peptides: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, said antibodies being produced by the immune system of a vertebrate in response to exposure of all or part of the peptide to the animal's immune system.

[0132] In some embodiments, the present invention covers a method for detecting the presence of a peptide in a sample, the method comprising obtaining a solution suspected of containing the peptide, probing the solution with an antibody as claimed in claim 29, and detecting the binding of the antibody to the peptide; wherein the peptide is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, and immunologically detectable variants thereof.

[0133] In some embodiments, the present invention covers a kit for detecting the presence of a peptide in a sample, the kit comprising, in a suitable container device, an antibody that binds to the peptide, reagents necessary for mixing the peptide and the antibody in solution, providing the antibody, a control antibody, a control antigen, and at least a first immunoassay reagent of these reagents, and instructions for detecting the binding; wherein the peptide is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, and immunologically detectable variants thereof.

[0134] In some embodiments, the sBin-IP of the present invention, including variants of the sBin-IP, is active against beetle pests. Beetles include, but are not limited to, any beetles currently known or subsequently identified, including those from the suborders Protocoryptales, Myxoboloids, Carnivorous, and Polyphagous, and any combinations thereof.

[0135] In some non-limiting embodiments, the binary toxin of the present invention is active against species of the genus *Radiata*. *Radiata* is a genus of beetles in the order Coleoptera, commonly known as "corn rootworm" or "cucumber beetle". Exemplary species of the genus *Diabrotica* include, but are not limited to: *Diabrotica longicornis barberi* (Northern Maize Rootworm), *Diabrotica longicornis barberi* (Western Maize Rootworm), *Diabrotica balteata* (Banded Cucumber Beetle), *Diabrotica undecimpunctata* (Western Spotted Cucumber Beetle), *Diabrotica significata* (3-Spotted Leaf Beetle), *Diabrotica speciosa* (Chrysanthemum Beetle), *Diabrotica beniensis* (Mexican Maize Rootworm), *Diabrotica beniensis* (Benevolent Leafworm), *Diabrotica crispata* (Crestorant Leafworm), and *Diabrotica kowaigen* (Benevolent Leafworm). *D. curvipustulata*, *D. dissimilis*, *D. elegantula*, *D. emorsitans*, *D. graminea*, *D. hispanolae*, *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.

[0136] Other non-limiting examples of pests of the order Coleoptera according to the invention include species of the genus *Leptinotarsa*, such as the potato leaf beetle (Colorado potato beetle); species of the genus *Chrysomelaspp.*, such as the cottonwood leaf beetle (*C. scripta*); species of the genus *Hypothenemus*, such as the coffee berry borer (*H. hampei*); species of the genus *Sitophilus*, such as the maize weevil (*S. zeamais*); and species of the genus *Epitrix*, such as the tobacco flea beetle (*E. hirtipennis*). (Tobacco flea beetle) and (Potato flea beetle (E. cucumeris, potato flea beetle)); species of the genus *Phyllotreta*, such as *P. cruciferae* (cruciferflea beetle) and *P. pushilla* (western black flea beetle); species of the genus *Anthonomus*, such as the pepper weevil (A. eugenii); species of the genus *Hemicrepidus*, such as the wireworm (H. memnonius); species of the genus *Melanotus*, such as the common wireworm (M. communis); species of the genus *Ceutorhychus*. Species of the genus *Aeolus*, such as *C. assimilis* (cabbage seedpod weevil); species of the genus *Aeolus*, such as *A. mancus* (wheat wireworm); species of the genus *Horistonotus*, such as *H. uhlerii* (sand wireworm); species of the genus *Sphenophorus*, such as *S. maidis* (maize billbug), *S. zeae* (timothybillbug), and *S. zeae* (timothybillbug). *Parvulus* (bluegrass billbug) and *S. callosus* (southern corn billbug); species of the genus *Phyllophaga* (white grub); species of the genus *Chaetocnema*, such as *C. pulicaria* (corn flea beetle); species of the genus *Popillia*, such as the Japanese ...Japanese beetle (Epilachna spp.); species of the genus *Epilachna*, such as *E. variantstis* (Mexican bean beetle); species of the genus *Cerotoma*, such as *C. trifurcate* (bean leaf beetle); species of the genus *Epicauta*, such as *E. pestifera* and *E. lemniscata* (lister beetles); and any combination of the foregoing.

[0137] The binary toxin or sBin-IP of this invention is also active against lepidopteran insects. Such lepidopteran insects include, but are not limited to, any insects currently known or subsequently identified that are classified as lepidopteran insects. Exemplary Lepidoptera insects include, but are not limited to: species of the genus *O. nubilalis*, such as the European corn borer; species of the genus *P. xylostella*, such as the diamondback moth; species of the genus *Spodopteras*, such as the fall armyworm (*S. frugiperda*), the yellow-striped armyworm (*S. ornithogalli*), the western yellow-striped armyworm (*S. praefica*), the southern armyworm (*S. eridania*), and the beet armyworm (*S. exigua*); species of the genus *A. ipsilon*, the black cutworm (*A. segetum*), the common cutworm (*A. gladiaria*), and the pale western gray cutworm (*A. orthogonia*). Species of the genus *Striacostas*, such as *S. albicosta* (western bean cutworm); species of the genus *Helicoverpa*, such as *H. zea* (corn earworm), *H. punctigera* (native budworm), *S. littoralis* (Egyptian cotton leafworm), and *H. armigera* (cotton bollworm); species of the genus *Heliothis*, such as *H. virescens* (tobacco budworm); and species of the genus *Diatraea*, such as *D. grandiosella* (southwestern corn borer). (cornborer) and small sugarcane borer (D. saccharalis) (sugarcane borer); species of the genus Trichoplusia spp., such as the white-spotted cutworm (T.).* *ni* (cabbage looper); *Sesamia* spp., such as the Mediterranean corn borer (S. nonagroides); *Pectinophora* spp., such as the pink bollworm (P. gossypiella); *Cochylis* spp., such as the banded sunflower moth (C. hospes); *Manduca* spp., such as the tobacco hornworm (M. sexta) and the tomato hornworm (M. quinquemaculata); *Elasmopalpus* spp., such as the South American corn borer (E. lignosellus); and the lesser cornstalk borer (*E. lignosellus*). Species of the genus *Pseudoplusia*, such as the soybean looper (*P. includens*); species of the genus *Anticarsia*, such as the bean caterpillar (*P. velutina*); species of the genus *Plathypena*, such as the alfalfa green cloverworm (*P. scabra*); species of the genus *Pieris*, such as the cabbage butterfly (*P. brassicae*); species of the genus *Papaipema*, such as the stalk borer (*P. nebris*); species of the genus *Pseudaletia*, such as the common armyworm (*P. unipuncta*); species of the genus *Peridroma*, such as the variegated cutworm (*P. saucia*). Cutworm); Species of the genus *Keiferia*, such as the tomato pinworm (*K. lycopersicella*); Species of the genus *Artogeia*, such as the cabbage worm (*A. rapae*); Species of the genus *Phthorimaea*, such as the potato tuberworm (*P. operculella*); Species of the genus *Crymodes*, such as *C.*devastator, glassy cutworm; species of the genus *Feltia*, such as *F. ducens*, dingy cutworm; and any combination thereof.

[0138] The binary toxin of the present invention, sBin-IP, is also active against hemiptera, dipterans, *Hemiptera* species and / or other piercing-sucking insects (e.g., orthoptera or tsioptera). Diptera insects include, but are not limited to, any currently known or subsequently identified Diptera insects, including, but not limited to, species of the genus *Liriomyza*, such as *L. trifolii* (leafminer) and *L. sativa* (vegetable leafminer); species of the genus *Scrobipalpula*, such as *S. absoluteuta* (tomato leafminer); species of the genus *Delia*, such as *D. platura* (seedcorn maggot), *D. brassicae* (cabbage maggot), and *D. radicum* (cabbage root fly); species of the genus *Psilia*, such as *P. rosae* (carrot rust fly); and species of the genus *Tetanops*. (spp.), such as the beetroot maggot (T. myopaeformis) (beetroot maggot); and any combination thereof.

[0139] Orthoptera include, but are not limited to, any orthoptera species currently known or subsequently identified, including, but not limited to, species of the genus Melanoplus spp., such as M. differentialis (Differential grasshopper), M. femurrubrum (Redlegged grasshopper), and M. bivittatus (Twostriped grasshopper); and any combination thereof.

[0140] Thysanoptera include, but are not limited to, any thysanoptera insects currently known or subsequently identified, including, but not limited to, species of the genus *Frankliniella* such as *F. occidentalis* (western flower thrips) and *F. fusca* (tobacco thrips); and species of the genus *Thrips* such as *T. tabaci* (onion thrips) and *T. palmi* (melon thrips); and any combination thereof.

[0141] The binary toxin of the present invention or the sBin-IP of the present invention may also be active against nematodes. As used herein, the term "nematode" encompasses any organism currently known or subsequently identified that is classified into the phylum Nematoda of the animal kingdom, including but not limited to nematodes in the class Adenocarcinales (including, for example, Sphenodontia, Isophora, Monodentaria, Lyacodontia, Trichodontia, Cordyceps, Msipa, Phyllodontia, Chrysodontia, Trichodontia, Lepidodendron, and Monogony) and / or nematodes in the class Cytoneonephrales (including, for example, Rhizoctonia, Strigiformes, Ascaridia, Cyclocera, Cameloidea, Digastricia, Padnaeida, and Scalycero).

[0142] Nematodes include, but are not limited to, parasitic nematodes, such as root-knot nematodes, cyst nematodes, and / or carrion nematodes. Exemplary genera of nematodes according to the present invention include, but are not limited to: *Globodera* (root-knot nematodes), *Globodera* (cyst nematodes), *Globodera* (cyst nematodes), *Globodera* (perforating nematodes), *Globodera* (kidney-shaped nematodes), *Globodera* (carrion nematodes), *Globodera* (leaf nematodes), *Globodera* (spiral nematodes), *Globodera* (spear nematodes), *Globodera* (short-thick root nematodes), *Globodera* (long-needle nematodes), *Globodera* (false root-knot nematodes), *Globodera* (false root-knot nematodes), *Globodera* (subanguina), *Globodera* (stinging nematodes), and *Globodera* (small ring nematodes). Nematodes, including the genera *Cyclophora*, *Steel Nematode*, *Tridentella*, *Half-rotor Nematode*, *Sheath Nematode*, *Root-knot Nematode*, *Hypsoperine*, *Large-stem Nematode*, *Melinius*, *Punctodera*, *Quinisulcius*, *Shield Nematode*, *Dagger Nematode*, *Dwarf Nematode*, *Piercing Nematode*, *Ascaris*, and any combination thereof.

[0143] Exemplary plant parasitic nematodes according to the present invention 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), and *Heteroderaschachtii* (sugar beet cyst nematode). (Heterodera zeae, corn cyst nematode); Heterodera avenae, corn cyst nematodeThe following nematodes are listed: cereal cyst nematode, carrot heteroderma (Heterodera carotae), red clover heteroderma (Heterodera trifolii), Columbus nematode (Hoplolaimus columbus), cap-shaped nematode (Hoplolaimus galeatus), Hoplolaimus magnistylus, Longidorus breviannulatus, peanut root-knot nematode (Meloidogynearenaria), Colombian root-knot nematode (Meloidogyne chitwoodi), northern root-knot nematode (Meloidogynehapla), southern root-knot nematode (Meloidogyne incognita), Javan root-knot nematode (Meloidogynejavanica), Mesocriconema xenoplax, abnormal pearl nematode (Nacobbus aberrans), Naccobus dorsalis, Paratrichodorus christiei, and Paratrichodorus microscopic hairy nematode. The following nematodes are listed: minor, shortest-tailed short-bodied nematode (Pratylenchus brachyurus), notched short-bodied nematode (Pratylenchus crenatus), hexincisus, rejected short-bodied nematode (Pratylenchus neglectus), piercing short-bodied nematode (Pratylenchus penetrans), projectus, scribneri, tenuicaudatus, thornei, maize short-bodied nematode (Pratylenchus zeae), Punctodera chaccoensis, Quinisulciusacutus, banana perforating nematode (Radopholus similis), kidney-shaped rotifer (Rotylenchulus reniformis), and cis- and reversible dwarf nematode (Tylenchorhynchus). *Dubius*, *Tylenchulus semipenetrans*, *Siphinema americanum*, *X. mediterraneum*, and any combination thereof.

[0144] This invention also covers recombinant vectors and / or recombinant constructs, which may also be referred to as vectors or constructs, containing the expression cassettes and / or nucleic acid molecules of this invention. In such vectors, the nucleic acids are preferably contained in expression cassettes that contain regulatory elements for expressing nucleotide molecules in host cells capable of expressing nucleotide molecules. These regulatory elements typically contain promoters and termination signals and preferably also contain elements that allow efficient translation of the polypeptide encoded by the nucleic acids of this invention. Vectors containing nucleic acids are capable of replicating in specific host cells (preferably as extrachromosomal molecules) and can therefore be used to amplify the nucleic acids of this invention in these host cells.

[0145] The present invention also covers host cells containing the recombinant vectors, expression cassettes, or nucleic acid molecules of the present invention. In other embodiments, such vectors are viral vectors and are used to replicate nucleotide sequences in specific host cells (e.g., insect or plant cells). Recombinant vectors are also used to transform the nucleic acid molecules of the present invention into host cells, whereby these nucleic acid molecules are stably integrated into the DNA of the transgenic host. In some embodiments, the host cell is a bacterial cell or a plant cell. In some aspects of these embodiments, the bacterial cells are from the genera *Bacillus*, *Clostridium*, *Pathogenic Bacillus*, *Luminobacter*, *Pasteurella*, *Escherichia*, *Pseudomonas*, *Erwinia*, *Serratia*, *Klebsiella*, *Salmonella*, *Pasteurella*, *Xanthomonas*, *Streptomyces*, *Rhizobium*, *Sinobacterium sinense*, *Streptococcus*, *Rhizobium sinense*, *Rhizobium*, *Methylophilus*, *Agrobacterium*, *Acetobacter*, *Lactobacillus*, *Arthrobacter*, *Azotobacter*, *Leuconostoc*, *Sphingomonas*, *Burkholderia*, *Candidatus Glomeribacter*, *Dystrophus*, *Streptococcus*, *Sphingobacterium*, or *Alcaligenes*. In other aspects of these embodiments, the host cells used for such recombinant vectors are endophytes or epiphytes. In some other aspects of these embodiments, the host cell is a plant cell, such as a dicotyledonous plant cell or a monocotyledonous plant cell. In other aspects, the dicotyledonous plant cell is selected from the group consisting of: soybean cells, sunflower cells, tomato cells, brassica crop cells, cotton cells, sugar beet cells, and tobacco cells. In still other aspects, the monocotyledonous plant cell is selected from the group consisting of: barley cells, corn cells, oat cells, rice cells, sorghum cells, sugarcane cells, and wheat cells.

[0146] In some non-limiting embodiments of the invention, at least one of the nucleic acid molecules of the invention is inserted into a suitable expression cassette (containing a promoter and a termination signal). Expression of the nucleic acid can be constitutive or can use an inducible promoter that responds to various types of stimuli to initiate transcription. In another embodiment, the cell expressing the insecticidal protein of the invention is a microorganism, such as a virus, bacteria, or fungus. In yet another embodiment, a virus (such as a baculovirus) contains the nucleic acid of the invention in its genome and expresses a large amount of the corresponding insecticidal protein after infecting a suitable eukaryotic cell (suitable for viral replication and expression of the nucleic acid). The resulting insecticidal protein is used as an insecticide. Alternatively, a baculovirus engineered to include the nucleic acid is used to infect insects in vivo and kill them by expression of the insecticidal toxin or by a combination of viral infection and expression of the insecticidal toxin. In another embodiment, the invention also covers a method for producing a polypeptide with insecticidal activity, the method comprising culturing host cells under conditions in which a nucleic acid molecule encoding the polypeptide is expressed.

[0147] Bacterial cells are also hosts used to express the nucleic acids of the present invention. In one embodiment, non-pathogenic symbiotic bacteria (so-called endophytes) capable of living and replicating within plant tissues, or non-pathogenic symbiotic bacteria (so-called epiphytes) capable of settling in the leaf or rhizosphere, are used. Such bacteria include bacteria from the following genera: Agrobacterium, Alcaligenes, Azotobacter, Azotobacter, Bacillus, Corynebacterium, Enterobacter, Erwinia, Flavobacterium, Klebsiella, Pseudomonas, Rhizobium, Rhizobium sinense, Rhizobium, Serratia, Streptomyces, Sphingomyelinus, Burkholderia, Candidatus Glomeribacter, Diplodia, Spirilaria, Rhizobium stoloniferum, Staphylococcus, Methylophilus, Gluconobacterium, Streptococcus, Sphingosporidiaceae, and Xanthomonas. Symbiotic fungi (such as Trichoderma and Gynostemma) are also possible hosts for expressing the nucleic acids of the present invention for the same purpose.

[0148] These gene manipulation techniques are specific to the different available hosts and are known in the art. For example, expression vectors pKK223-3 and pKK223-2 can be used in *E. coli* to express heterologous genes after a tac or trc promoter (in transcriptional or translational fusion). The simplest method for expressing operons encoding multiple ORFs is to insert the operon into a vector (such as pKK223-3) during transcriptional fusion, allowing access to the homologous ribosome binding site of the heterologous gene. Overexpression techniques in Gram-positive species (such as *Bacillus*) are also known in the art and can be used in the context of this invention (Quax et al., in: *Industrial Microorganisms: Basic and Applied Molecular Genetics*, edited by Baltz et al., American Society for Microbiology, Washington (1993)). Alternative systems for overexpression rely on, for example, yeast vectors and include the use of *Pichia pastoris*, *Saccharomyces*, and *Kluyveromyces* (Sreekrishna, in: *Industrial microorganisms: basic and applied molecular genetics*, edited by Baltz, Hegeman, and Skautrud, American Society for Microbiology, Washington (1993); Dequin and Barre, *Biotechnology* L2:173-177 (1994); van den Berg et al., *Biotechnology* 8:135-139 (1990)).

[0149] In other embodiments, the present invention covers a method for controlling insect pests, the method comprising delivering an insect-controlling effective amount of the insecticidal protein of the present invention to the insect pest. In some aspects of these embodiments, the insecticidal protein is delivered via transgenic plants or via topical application of an insecticidal composition comprising the insecticidal protein. In other aspects, the transgenic plants or insecticidal compositions comprise a second insecticide different from the sBin-IP of the present invention. In still other aspects, the second insecticide is a protein, dsRNA, or a chemical. In other respects, the protein is selected from the group consisting of: Cry protein, VIP toxin, potato glycoprotein, protease, protease inhibitor, urease, α-amylase inhibitor, porogen, 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 contains an active ingredient selected from the group consisting of: carbofuran, methamidophos, methomyl, bifenthrin, heptafluthrin, permethrin, cypermethrin, lambda-cyhalothrin, cypermethrin, deltamethrin, chlorpyrifos, oxychloride, dimethoate, ethoprophos, malathion, methyl parathion, phorate, terbufos, terbufos, fipronil, acetamiprid, imidacloprid, thiamethoxam, endosulfan, sulfadiazine or chlorpyrifos or a combination thereof.

[0150] In some embodiments of the invention, at least one of the sBin-IP toxins of the invention is expressed in a higher organism, such as a plant. Transgenic plants expressing insect-effective amounts of an insecticidal protein protect themselves from insect pests. When an insect pest begins to feed on this transgenic plant, it also ingests the expressed insecticidal protein. This can prevent the insect from further biting into plant tissue and / or even harm or kill the insect. The nucleic acid molecule of the invention is inserted into an expression cassette, and then the nucleic acid can be stably integrated into the genome of the plant. In other embodiments, the nucleic acid molecule comprises a non-pathogenic, self-replicating virus. The plants transformed according to the present invention can be monocotyledonous or dicotyledonous plants, and include, but are not limited to: corn, wheat, oats, turfgrass, pasture grass, flax, barley, rye, sweet potato, beans, peas, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, pepper, celery, squash, pumpkin, hemp, dense zucchini, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tomato, sorghum, sugarcane, beet, sunflower, rapeseed, clover, tobacco, carrot, cotton, alfalfa, rice, potato, eggplant, cucumber, Arabidopsis species, and woody plants such as conifers and deciduous trees.

[0151] In some embodiments, the present invention covers a method for producing a protein toxic to insect pests, i.e., an insecticidal protein, the method comprising: (a) obtaining a host cell containing a gene that itself contains an expression cassette and / or nucleic acid molecule of the present invention; and (b) growing the transgenic host cell or a transgenic host containing the host cell under conditions in which the transgenic host cell produces a protein toxic to insect pests.

[0152] In other embodiments, the present invention covers a method for producing transgenic plants or plant parts with enhanced insect resistance compared to control plants or plant parts, the method comprising: (a) introducing a chimeric gene, expression cassette, or vector containing a nucleic acid molecule encoding an insecticidal protein of the present invention 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 with enhanced insect resistance. In other embodiments, the chimeric gene, expression cassette, or vector may encode the sBin-IP toxin of the present invention, the toxin comprising, substantially comprising, or comprising of: 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% of the same or similar amino acid sequence as any one of SEQ ID NO: 1-6. The “enhanced” insect resistance can be measured by any toxic effects of the transgenic plant on insect pests that feed on the transgenic plant. Compared to control plants that do not express insecticidal proteins, the enhanced insecticidal activity 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% higher. Plants or plant parts with enhanced insecticidal 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 reproduction methods. Any suitable control plant or plant part can be used, such as a plant grown in the same environment with the same or similar genetic background. In the embodiments, the control plant or plant part has the same genetic background as the described plant and is grown in the same environment, but does not contain the molecules of the present invention, while the described plant contains the nucleic acid molecules of the present invention.

[0153] In other embodiments, the present invention covers a method for enhancing insect resistance in a plant or plant part compared to a control plant or plant part, the method comprising expressing a nucleic acid molecule or expression cassette of the present invention in the plant or plant part, wherein expression of a heterologous nucleic acid of the expression cassette results in enhanced insect resistance in the plant or plant part compared to a control plant or plant part. In some embodiments, the expression cassette or nucleic acid molecule comprises a promoter operatively linked to a heterologous nucleic acid molecule containing a nucleotide sequence comprising, substantially comprising, or comprising the following: (a) a nucleotide sequence of any one of SEQ ID NO: 7-24; (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 the nucleotide sequence of any one of SEQ ID NO: 7-24; (c) a nucleotide sequence encoding a protein, wherein the amino acid sequence of the protein comprises, substantially comprises, or comprises the following: any one of SEQ ID NO: 1-6; (d) a nucleotide sequence encoding a protein, wherein the amino acid sequence of the protein is identical to the nucleotide sequence of any one of SEQ ID NO: 7-24. NO: The amino acid sequence of any one of NOs 1-6 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 has been 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. Nucleic acid molecules or expression cassettes may be introduced into plants. In some embodiments, nucleic acid molecules or expression cassettes may be introduced into a plant portion, and a plant containing the nucleic acid molecules or expression cassette may be generated from that plant portion.

[0154] In some embodiments, the invention covers a method for producing plants with enhanced insect resistance compared to control plants, the method comprising detecting a heterologous nucleic acid comprising the nucleic acid molecule or expression cassette of the invention in a plant part, and producing a plant from the plant part thereby producing a plant with enhanced insect resistance compared to a control plant. In another embodiment, the invention covers a method for identifying plants or plant parts with enhanced insect resistance compared to control plants or plant parts, the method comprising detecting the nucleic acid molecule or expression cassette of the invention in the plant or plant part thereby identifying plants or plant parts with enhanced insect resistance. In another embodiment, the expression cassette or a diagnostic fragment thereof is detected in the amplification product of a nucleic acid sample from the plant or plant part. The diagnostic fragment may be a nucleic acid molecule at least 10 consecutive nucleotides long, which is unique to the expression cassette of the invention.

[0155] In other embodiments, the present invention covers a method for producing plants with enhanced insect resistance compared to control plants or plant parts, the method comprising hybridizing a first parent plant with a second parent plant, wherein at least the first parent plant contains a heterologous nucleic acid containing the nucleic acid molecule or expression cassette of the present invention in its genome; and producing offspring, wherein the offspring comprises at least one plant having the heterologous nucleic acid in its genome and exhibiting enhanced insect resistance compared to control plants.

[0156] In some aspects of the above embodiments, the method of the present invention confers enhanced insect resistance to beetle pests on plants or plant parts. Examples demonstrate insect control against beetle pests. In other aspects, the method of the present invention confers enhanced insect resistance to species of the genus *Radiata* (including *Radiata maize*, *Radiata barba*, *Radiata cucumberis* subspecies *Rhizophora cucumeroides*, *Radiata mexicanata*, and / or *Radiata septemloba*) and / or related species. In further embodiments, the method of the present invention confers enhanced insect resistance to *Radiata western maize*, *Radiata barba*, and / or *Radiata cucumberis* subspecies *Rhizophora cucumeroides*.

[0157] In some embodiments, the present invention covers transgenic plants comprising the heterologous nucleic acid molecule or expression cassette of the present invention, which confers enhanced insect resistance upon transcription or translation. In some aspects of these embodiments, the heterologous nucleic acid molecule or expression cassette comprises 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 nucleotide sequences to any one of SEQ ID NO:7-24. In other embodiments, the transgenic plant is a dicotyledonous or monocotyledonous plant. In other areas, genetically modified plants include alfalfa, apples, apricots, artichokes, arugula, asparagus, avocados, bananas, beans, beets, blackberries, blueberries, broccoli, Brussels sprouts, cabbage, canola, Roman melons, carrots, cassava, cauliflower, celery, cherries, coriander, citrus fruits, Clementi oranges, coffee beans, corn, cotton, cucumbers, Douglas fir, eggplants, endive, mustard greens, eucalyptus, fennel, figs, gourds, grapes, grapefruits, cantaloupes, jicama, kiwifruit, lettuce, leeks, and lemons. Lemon, lime, sago palm, mango, melon, mushroom, nuts, okra, onion, orange, ornamental plants, papaya, parsley, pea, peach, peanut, pear, pepper, persimmon, pine, pineapple, plantain, plum, pomegranate, poplar, potato, pumpkin, pineapple, radish pine, red chicory, radish, raspberry, rice, rye, sorghum, southern pine, soybean, spinach, small pumpkin, strawberry, beet, sunflower, sweet potato, sweetgum, citrus, tea, tobacco, tomato, turf, vine, watermelon, potato, or densely growing zucchini. In other respects, genetically modified plants include millet, switchgrass, corn, sorghum, wheat, oats, turfgrass, pasture grass, flax, rice, sugarcane, rapeseed, or barley. In other embodiments, the transgenic plant is a transgenic maize (corn) plant containing an sBin-IP coding sequence (where codon optimization is used for expression in maize), such as any one of SEQ ID NO:19-24.

[0158] In some embodiments, the present invention covers nucleic acid molecules encoding the insecticidal protein of the present invention, which are modified and optimized for expression in transgenic plants. Although genes from microbial organisms can be expressed at high levels in plants without modification in many cases, low expression in transgenic plants may 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 invention can be altered to conform to plant preferences while maintaining the encoded amino acids, or certain amino acid alterations can be made to the encoded insecticidal protein. Furthermore, high expression in plants is preferably achieved by 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 be poorly expressed in plants due to the presence of the ATTTA motif, which can destabilize information, and the AATAAA motif, which may cause inappropriate polyadenylation. In embodiments, sequences can be modified to cater to specific codon preferences and GC content preferences of monocotyledonous or dicotyledonous plants, as these preferences have been shown to be distinct (Murray et al. Nucl. Acids Res. [Nucleic Acid Research] 17:477-498 (1989)). Furthermore, these nucleic acids are screened for the presence of inappropriate splicing sites that could cause information shortening. Well-known site-directed mutagenesis, PCR, and synthetic gene construction techniques can be used, for example, using the methods described in published patent applications EP 0385 962, EP 0 359 472, and WO93 / 07278, to modify all necessary changes (such as those described above) within these nucleic acids.

[0159] In some embodiments of the invention, the coding sequence of the insecticidal protein of the invention is produced according to the procedure disclosed in U.S. Patent 5,625,136 (incorporated herein by reference). In this procedure, maize-preferred codons are used, i.e., single codons that most frequently encode amino acids in maize. Maize-preferred codons for specific amino acids may, for example, be derived from known gene sequences of maize. Maize codons for 28 genes from the maize plant are used as found in Murray et al., Nucleic Acids Research [Nucleic Acid Sequences] 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 understood that all or any part of the gene sequence can be optimized or synthetic. That is, synthetic or partially optimized sequences can also be used.

[0160] For more efficient translation initiation, the sequence adjacent to the starting methionine can be modified. For example, they can be modified by including sequences known to be effective in plants. Joshi has proposed suitable common sequences for plants (NAR 15:6643-6653 (1987)), and Clontech has proposed another common translation initiator (1993 / 1994 catalog, page 210). These common sequences are suitable for use with the nucleic acids of the present invention. In embodiments, these sequences are incorporated into constructs containing nucleic acids to achieve and include ATG (without modification of the second amino acid), or alternatively to achieve and include GTC after ATG (with the possibility of modifying the second amino acid of the transgene).

[0161] In transgenic plants, the expression of these nucleic acids is driven by promoters that function in the plant. The choice of promoter will vary depending on the temporal and spatial needs of expression, and also on the target species. Therefore, expression of the nucleic acids of the present invention in leaves, stalks or stems, spikes, inflorescences (e.g., spikes, panicles, rachis, etc.), roots, and / or seedlings is preferred. However, in many cases, protection against more than one type of insect pest is sought, and therefore expression in multiple tissues is desirable. Although many promoters from dicotyledons have been shown to be operable in monocotyledons and vice versa, it is ideal to select dicotyledonous promoters for expression in dicotyledons and monocotyledonous promoters for expression in monocotyledons. However, there are no restrictions on the origin of the selected promoters; it is sufficient as long as they can effectively drive the expression of nucleic acids in the desired cells.

[0162] In some embodiments, promoters constitutively expressed are used, including actin or ubiquitin or cmp promoters, or CaMV35S and 19S promoters. The nucleic acids of the present invention can also be expressed under the regulation of promoters regulated by chemical methods. Preferred techniques for chemical induction of gene expression are detailed in published application EP 0 332 104 (Ciba-Geigy) and U.S. Patent 5,614,395. A preferred promoter for chemical induction is the tobacco PR-1a promoter.

[0163] In other embodiments, a class of wound-inducing promoters may be used. Numerous promoters expressed at wound sites and also at sites of plant pathogen infection have been described. Ideally, such promoters should be locally active only at the site of infection, and in this way, the insecticidal proteins of the present invention accumulate only in cells that require the synthesis of these proteins to kill invading insect pests. Preferred promoters of this type include those described in the following literature: Stanford et al. Mol. Gen. Genet. [Molecular Genetics] 215:200-208 (1989), Xu et al. Plant Molec. Biol. [Plant Molecular Biology] 22:573-588 (1993), Logemann et al. Plant Cell [Plant Cell] 1:151-158 (1989), Rohrmeier and Lehle, Plant Molec. Biol. [Plant Molecular Biology] 22:783-792 (1993), Firek et al. Plant Molec. Biol. [Plant Molecular Biology] 22:129-142 (1993), and Warner et al. Plant J. [Plant Journal] 3:191-201 (1993).

[0164] Tissue-specific or tissue-preferred promoters for expressing genes encoding the insecticidal proteins of the present invention in plants (particularly maize) are those promoters that are expressed directly in roots, pith, leaves, or pollen (particularly roots). Such promoters, for example 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.

[0165] In addition, promoters that function in plastids can be used. Non-limiting examples of such promoters include the phage T3 gene 9 5'UTR and other promoters disclosed in U.S. Patent No. 7,579,516. Other promoters suitable for use in this invention include, but are not limited to, the S-E9 small subunit RuBP carboxylase promoter and the Kunitz trypsin inhibitor gene promoter (Kti3).

[0166] In some embodiments of the present invention, inducible promoters may be used. Therefore, for example, chemically regulated promoters may be used to regulate the expression of the nucleotide sequence of the present invention by applying exogenous chemical regulators. The regulation of the expression of the nucleotide sequence of the present invention via a chemically regulated promoter ensures that the polypeptide of the present invention can be synthesized only when the crop plant is treated with the induced chemical. Depending on the purpose, the promoter may be a chemically inducible promoter when the expression of the nucleotide sequence of the present invention is induced by the application of chemicals, or a chemically repressive promoter when the expression of the nucleotide sequence of the present invention is inhibited by the application of chemicals.

[0167] Chemically inducible promoters are known in the art and include, but are not limited to, the corn In2-2 promoter (activated by a benzenesulfonamide herbicide safener), the corn GST promoter (activated by a hydrophobic electrophilic compound used as a pre-germination herbicide), and the tobacco PR-1a promoter (activated by salicylic acid) (e.g., the PR1a system), steroid-responsive promoters (see, for example, Schena et al. (1991) Proc. Natl. Acad. Sci. [Proceedings of the National Academy of Sciences] USA 88, 10421-10425 and McNellis et al. (1998) Plant Glucocorticoid-inducible promoters in J. [Plant Journal] 14, 247-257, as well as tetracycline-inducible and tetracycline-repressor 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.

[0168] Other non-restricted examples of inducible promoters include ABA-inducible and cell swelling-inducible promoters, promoters of auxin-binding protein genes (Schwob et al. (1993) Plant J. [Plant Journal] 4:423-432), promoters of UDP-glucose flavonoid glycosyltransferases (Ralston et al. (1988) Genetics [Genetics] 119:185-197), promoters of MPI protease inhibitors (Cordero et al. (1994) Plant J. [Plant Journal] 6:141-150), and promoters of glyceraldehyde-3-phosphate dehydrogenases (Kohler et al. (1995) Plant Mol. Biol. [Plant Molecular Biology] 29:1293-1298; Martinez et al. (1989) J. Mol. Biol. [Journal of Molecular Biology] 208:551-565; and Quigley et al. (1989) J. Mol. Evol. [Journal of Molecular Evolution] 29:412-421). Also included are benzylsulfonamide-inducible (US Patent 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 the following literature may be used: Gast (1996) Current Opinion Biotechnol. [New Insights in Biotechnology] 7:168-172 and Gast (1997) Annu. Rev. Plant Physiol. Plant Mol. Biol. [Annual Review of Plant Physiology and Plant Molecular Biology] 48:89-108. Other chemically inducible promoters suitable for guiding the expression of the nucleotide sequences of the present invention in plants are disclosed in U.S. Patent 5,614,395, which is incorporated herein by reference in its entirety. Chemical induction of gene expression is also detailed in Publication EP 0332 104 (granted to Ciba-Geigy) and U.S. Patent 5,614,395. In some embodiments, the promoter used for chemical induction may be the tobacco PR-1a promoter.

[0169] In a further embodiment, the nucleotide sequence of the present invention can be operatively associated with a promoter that is wound-inducible or inducible by infection by a pest or pathogen (e.g., insects or nematodes). Numerous promoters expressed at wound sites and / or at sites of pest attack (e.g., insect / nematode feeding) or plant pathogen infection have been described. Ideally, such promoters should be locally active only at or near the site of attack, and in this way, the expression of the nucleotide sequence of the present invention will be concentrated in the invaded or fed cells. Such promoters include, but are not limited to, those described below: Stanford et al., Mol. Gen. Genet. [Molecular and General Genetics] 215:200-208 (1989); Xu et al., Plant Molec. Biol. [Plant Molecular Biology] 22:573-588 (1993); Logemann et al., Plant Cell [Plant Cell] 1:151-158 (1989); Rohrmeier and Lehle, Plant Molec. Biol. [Plant Molecular Biology] 22:783-792 (1993); Firek et al., Plant Molec. Biol. [Plant Molecular Biology] 22:129-142 (1993); Warner et al., Plant J. [Plant Journal] 3:191-201 (1993); U.S. Patent Nos. 5,750,386; 5,955,646; 6,262,344; 6,395,963; 6,703,541; 7,078,589; 7,196,247; 7,223,901; and U.S. Patent Application Publication 2010043102.

[0170] In some embodiments of the invention, a “minimal promoter” or “basic promoter” is used. A minimal promoter is capable of recruiting and binding the RNA polymerase II complex and its accessory proteins to allow transcription initiation and elongation. In some embodiments, the minimal promoter is constructed to contain only a nucleotide / nucleotide sequence from a selected promoter necessary for the binding of transcription factors and transcription of a target nucleotide sequence, which is operatively associated with a minimal promoter including, but not limited to, a TATA box sequence. In other embodiments, the minimal promoter lacks a cis sequence for recruiting and binding transcription factors that regulate (e.g., enhance, repress, tissue-specific, inducible, or repressive) transcription. The minimal promoter is typically placed upstream (i.e., 5') of the nucleotide sequence to be expressed. Therefore, a nucleotide / nucleotide sequence from any promoter available with this invention can be selected as the minimal promoter.

[0171] Numerous other sequences can be incorporated into the expression cassette described in this invention. These sequences include sequences that have already shown enhanced expression, such as intron sequences (e.g., from Adhl and Bronzel) and viral leader sequences (e.g., from TMV, MCMV, and AMV).

[0172] Targeted expression of the nucleic acid of the present invention in plants for different cellular locations may be more preferred. In some cases, localization in the cytosol may be desirable, while in others, localization in a specific subcellular organelle may be preferred. Subcellular localization of transgenes encoding enzymes is performed using techniques well known in the art. Typically, DNA encoding a target peptide from a gene product known to target a cell organelle is manipulated and fused upstream of the nucleic acid. Many such target sequences for chloroplasts are known and their function in heterologous constructs has been demonstrated. The expression of the nucleic acid of the present invention is also targeted to the endoplasmic reticulum or vacuoles of the host cell. Techniques for achieving this are well known in the art.

[0173] Vectors suitable for plant transformation are described elsewhere in this specification. For Agrobacterium-mediated transformation, binary vectors or vectors carrying at least one T-DNA boundary sequence are suitable, while for direct gene transfer, any vector is suitable, and containing only the linear DNA of the target construct may be preferred. In the case of direct gene transfer, transformation or co-transformation with a single DNA species can be used (Schocher et al., Biotechnology 4:1093-1096 (1986)). For both direct gene transfer and Agrobacterium-mediated transformation, transformation is usually (but not necessary) performed with a selective marker that can provide resistance to antibiotics (kanamycin, hygromycin, or methotrexate) or herbicides (basta). Plant transformation vectors containing the nucleic acid molecules of the present invention may also contain genes such as phosphomannose isomerase; PMI, which provide positive selection for transgenic plants, as disclosed in U.S. Patents 5,767,378 and 5,994,629 (incorporated herein by reference). However, the choice of selective marker is not essential to the present invention.

[0174] In some embodiments, nucleic acids can be converted into a nuclear genome. In another embodiment, the nucleic acids of the present invention are directly converted into a plastid genome. The main advantage of plastid conversion is that plastids can generally express bacterial genes without substantial codon optimization, and that plastids can express multiple open reading frames under the control of a single promoter. Plastid conversion techniques are extensively described 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] 91,7301-7305. Basic chloroplast conversion techniques involve, for example, using bio-projectiles or protoplast conversion (e.g., calcium chloride or PEG-mediated conversion) to introduce a cloned plastid DNA region flanking a selective marker, along with the target gene, into a suitable target tissue. These 1- to 1.5 kb flanking regions (referred to as target sequences) facilitate homologous recombination with the plastome genome and thus allow for the substitution or modification of specific regions of the plastome. Initially, point mutations in the chloroplast 16S rRNA and rps12 genes (conferring resistance to spectinomycin and / or streptomycin) were used as selectivity markers for transformation (Svab, Z., Hajdukiewicz, P., and Maliga, P. (1990) Proc. Nati. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 87, 8526-8530; Staub, JM, and Maliga, P. (1992) Plant Cell [Plant Cell] 4, 39-45). This produced stable homologous transformants at a frequency of approximately one per 100 target leaf bombardments. The presence of cloning sites between these markers allows for the creation of plasmid-targeted vectors for the introduction of foreign genes (Staub, JM and Maliga, P. (1993) EMBO J. [European Journal of Molecular Biology] 12, 601-606). A substantial increase in transformation frequency is achieved by replacing recessive rRNA or r-protein antibiotic resistance genes with a dominant-selective marker (bacterial aadA gene, which encodes the spectinomycin-detoxifying enzyme aminoglycoside-3'-adenosyltransferase) (Svab, Z. and Maliga, P. (1993) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 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 Acid Research] 19:4083-4089). Other selective markers for plastid transformation are known in the art and are covered within the scope of this invention. Typically, approximately 15-20 cell division cycles are required after transformation to achieve a homogeneous state. Plastid expression (where genes are inserted via homologous recombination into all the thousands of copies of the circular plastid genome present in each plant cell) takes advantage of the large copy number of genes beyond nuclear expression, allowing expression levels exceeding 10% of total soluble plant proteins to be easily achieved. In a preferred embodiment, the nucleic acid of the invention is inserted into a plastid-targeted vector and transformed into the plastid genome of the desired plant host. Plants homologous to the plastid genome containing the nucleic acid of the invention are obtained, and these plants are able to preferentially overexpress the nucleic acid.

[0175] In other embodiments, the transgenic plant of the present invention may comprise a heterologous nucleic acid molecule encoding at least one additional desired trait. The additional trait may be encoded on the same heterologous nucleic acid molecule as the nucleic acid molecule of the present invention, or on a second heterologous nucleic acid molecule. The additional desired trait may confer insect resistance to a second insect pest, insect resistance to the same insect pest, tolerance to abiotic stress, 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 industrial processes, or altered reproductive capacity. The additional desired trait may also induce the production of commercially valuable enzymes or metabolites within the plant.

[0176] In some embodiments, the desired additional property is a secondary pest control agent. The secondary pest control agent may be active against any plant pest, including insects, nematodes, fungi, viruses, or bacteria. Examples of insect-plant pests include, but are not limited to: species of the genus *Nilaparvata* (e.g., *N. lugens*, also known as the brown planthopper); species of the genus *Laodelphax* (e.g., *L. striatellus*, also known as the small brown planthopper); species of the genus *Nephotettix* (e.g., *N. virescens*, *N. cincticeps*, or *N. nigropictus*, also known as the rice leafhopper); species of the genus *Sogatella* (e.g., *S. furcifera*, also known as the white-backed planthopper); and species of the genus *Blissus*. Species of the genus *Scotinophora* (e.g., *B. leucopterus* leucopterus) (chinch bug); Species of the genus *Scotinophora* (e.g., *S. vermidulate*, rice blackbug); Species of the genus *Acrosternum* (e.g., *A. hilare*, green stink bug); Species of the genus *Parnara* (e.g., *P. guttata*, rice skipper); Species of the genus *Chilo* (e.g., *C. suppressalis*, rice striped stem borer), and *C. auricilius*, golden-fringed stem borer. (e.g., *C. polychrysus*, or ...*Inferens* (pink rice borer); *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); *D. grandiosella* (sugarcane borer, or southwestern corn borer). * *Narnaga* species (e.g., *N. aenescens*, green rice caterpillar); *Xanthodes* species (e.g., *X. transversa*, green caterpillar); *S. praefica* species (e.g., fall armyworm, beet armyworm, climbing cutworm, or western yellowstriped armyworm); *Mythimna* species (e.g., armyworm, *Mythmna seperata*, *Pseudaletia seperata*); *C. spp.* species (e.g., corn earworm); *C. brunnea* species (e.g., grapecolaspis); *Lissorhoptrus* species. spp. (e.g., rice water weevil (L. oryzophilus); rice weevil species (e.g., rice scale weevil (E. squamos)); rice plant weevil species (D. armigera, rice hispa); rice weevil species (Oulema spp.)(e.g., rice leaf beetle (O. oryzae); rice weevil species (e.g., rice weevil (S. oryzae)); rice gall midge species (e.g., rice gall midge (P. oryzae)); dwarf fly species (e.g., small rice leafminer (H. griseola) or rice stem maggot (H. sasakii)); yellow leaf miner species (e.g., stem fly (C. oryzae)). Species of the genus *Magngot* (e.g., *Magngot* nominate subspecies *Magngot*, *Magngot* barry's leaf beetle (northern corn rootworm), *Magngot* cucumber root-eating subspecies *Magngot* cucumber, *Magngot* Mexican corn rootworm); *Magngot* cucumber stripe leaf beetle (ribbed cucumber beetle); species of the genus *O. nubilalis* (European corn borer); species of the genus *Noctuid* (e.g., black cutworm); species of the genus *Elasmopalpus* (e.g., *E. Lignosellus* (lesser cornstalk borer)); species of the genus *Hornet* (horseman worm); species of the genus *Cyclocephala* (e.g., northern masked rhinoceros beetle). chafer) or Southern masked chafer (C. immaculata); species of the genus *Popillia* (e.g., Japanese masked chafer); species of the genus *C.* (e.g., corn flea beetle); species of the genus *C.* (e.g., corn weevil); species of the genus *Rhopalosiphum* (e.g., corn aphid (R. maidis)); species of the genus *Anuraphis* (e.g., corn root aphid (A. maidiradicis)); species of the genus *Melanoplus* (e.g., red-legged locust, long-fronted locust, or blood locust).*Sanguinipes* (migratory grasshopper); *Hylemya* species (e.g., *H. platura*, seedcorn maggot); *Anaphothrips* species (e.g., grass thrips); *Solenopsis* species (e.g., leaf-stealing ant *S. milesta*); *Tetranychus* species (e.g., two-spotted spider mite *T. urticae*, carmine spider mite *T. cinnabarinus*); *Pectinophora* species (e.g., cotton bollworm *H. armigera*, American bollworm *H. armigera*); Species of the genus *P. gossypiella* (e.g., pink bollworm); species of the genus *Earias* (e.g., spotted bollworm); species of the genus *Heliothis* (e.g., tobacco shoot moth); species of the genus *A. grandis* (boll weevil); species of the genus *Pseudatomoscelis* (e.g., cotton fleahopper); species of the genus *Trialeurodes* (e.g., banded-winged whitefly *T. abutiloneus*, greenhouse whitefly *T. vaporariorum*); species of the genus *Bemisia*. spp. (e.g., silverleaf whitefly (B. argentifolii)); Aphis spp. (e.g., cotton aphid (A. gossypii)); Lygus spp. (e.g., tarnished plantbug (L. lineolaris) or bean pod grass bug (L.*Hesperus* (western tarnished plant bug); *Euschistus* spp. (e.g., *E. conspersus*, consterse stink bug); *Chlorochroa* spp. (e.g., *C. sayi*, Say stink bug); *Nezara* spp. (e.g., *N. viridula*, southern green stink bug); *Thrips* spp. (e.g., tobacco thrips); *Flower thrips* spp. (e.g., tobacco thrips or western flower thrips); *Leptinotarsa* spp. (e.g., *L. decemlineata*, Colorado potato beetle). Species of the genus *Lema* (e.g., *L. trilineata*, *L. texana*, *E. cucumeris*, *E. hirtipennis*, *E. tuberis*, *E. tuberis*, *E. vittata*, *Phaedon* (e.g., *P. cochleariae*, *P. cochleariae*, *P. cochleariae*, *P. cochleariae*, *P. cochleariae*, *P. cochleariae*, *P. cochleariae*, *P. tardina ... Leaf beetle; Species of the genus *Acheta* (e.g., house cricket); Species of the genus *Empoasca* (e.g., potato leafhopper); Species of the genus *Myzus* (e.g., peach aphid).* *persicae* (green peach aphid); *Paratrioza* spp. (e.g., *P. cockerelli*); *Conoderus* spp. (e.g., *C. falli*, southern potato wireworm, or *C. vespertinus*, tobacco wireworm); *P. operculella* (potatotuberworm); *Macrosiphum* spp. (e.g., *M. euphorbiae*, potato aphid); *Thyanta* spp. (e.g., *T. pallidovirens*, red-shouldered stink bug). stinkbug); species of the genus *P. operculella* (e.g., potato tuberworm); species of the genus *P. operculella* (e.g., tomato fruitworm); species of the genus *P. operculella* (e.g., tomato pinworm); species of the genus *Limonius* (hornworm); species of the genus *Manduca* (e.g., tobacco hornworm, *M. sexta* and tomato hornworm, *M. quinquemaculata*); species of the genus *Leymus chinensis* (e.g., American serpentine leafminer, trifolli, or huidobrensis leafminer); species of the genus *Drosophila* (e.g., Drosophila melanogaster). * *D. melanogaster*, *D. yakuba*, *D. pseudoobscura*, or *D. simulans*; species of *Carabus* (e.g., *C. granulatus*); species of *Chironomus* (e.g., *C. tentanus*); species of *C. felis* (cat flea); species of *Diaprepes* (e.g., *D. abbreviatus* root weevil); species of *Ips* (e.g., *I. pini* (pine engraver)); species of *Tribolium*.(e.g., *T. castaneum*, red floor beetle); *Glossina* species (e.g., *G. Morstinans*, tsetse fly); *Anopheles* species (e.g., *A. gambiae*, malaria mosquito); *H. armigera* species (e.g., African bollworm); *Acyrthosiphon* species (e.g., pea aphid); *Apis* species (e.g., Italian bee *A. melifera*, honeybee); *Homalodisca* species (e.g., glassy-winged sharpshooter); *Aedes* species. spp. (e.g., Aedes aegypti (yellow fever mosquito))); spp. of the genus *Bombyx* (e.g., *B. mori* (silkworm))); spp. of the genus *Locusta* (e.g., *L. miigratoria* (migratory locust))); spp. of the genus *Boophilus* (e.g., *B. microplus* (cattle tick))); spp. of the genus *Acanthoscurria* (e.g., *A. Gomesiana* (red-haired chocolate-colored bird eater))); spp. of the genus *Diploptera* (e.g., *D. punctata* (pacific beetle cockroach)); spp. of the genus *Heliconius* (e.g., *H. erato* (red passion flower butterfly)). Butterfly (H. melopomene) or Red-banded Butterfly (Postman Butterfly); Curculio species (e.g., Oak Weevil (C. glandium)); Diamondback moth species (e.g., Diamondback Moth (P. xylostella)); Amblyomma species.(e.g., cattle tick (A. variegatum)); species of the genus *Anteraea* (e.g., silkworm (A. Yamamai)); and species of the genus *Armigeres* (e.g., harassing mosquito (A. subalbatus)).

[0177] The binary toxin of this invention can be used in combination with other pest control agents to increase the target range of pests. Furthermore, the binary toxin of this invention, when used in combination with a second insecticide (which has a different mode of action or targets different receptors in the insect gut), has specific uses for preventing and / or managing insect resistance. In some embodiments, the binary toxin of the present invention is combined with a second insecticidal protein selected from the group consisting of: Cry1A, Cry1Ab, Cry1Ac, Cry1A.105, Cry1Ae, Cry1B, Cry1C, Cry1C variant, Cry1D, Cry1E, Cry1F, Cry1A / F chimera, Cry1G, Cry1H, Cry1I, Cry1J, Cry1K, Cry1L, Cry2A, Cry2Ab, Cry2Ae, Cry3, Cry3A variant, Cry3B, Cry4B, Cry6, Cry7, Cry8, Cry9, Cry15, Cry34, Cry35, Cry43A, Cry43B, Cry46A, Cry51Aa1, PtIP-96, PtIP-83. PHI-4, MP467, MP81, PS149B1, DIG-3, DIG-5, DIG-10, DIG-11, DIG-17, DIG-657, IRDIG28688.1, IRDIG28688.1, IRDIG28684.1, IRDIG28682.1, 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 trophic insecticidal proteins, mCry3A, eCry3.1Ab, AXMI-001, AXMI-002, AXMI-030, AXMI-035, AXMI-036, AXMI-045, AXMI52, AXMI58, AXMI88, AXMI97, AXMI102, AXMI112, AXMI113, AXMI115, AXMI117, AXMI100, AXMI-115, AXMI-113, and AXMI-005, AXMI134, AXMI-150, 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, ET33, ET34, ET35, ET66, ET70, TIC400, TIC407, TIC417, TIC431, TIC800, TIC807, TIC834, TIC836, TIC844, TIC853, TIC860 or its variants, TIC867 or its variants, TIC868 or its variants, 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 aforementioned insecticidal proteins. The second insecticide may also be an agent selected from the group consisting of: α-amylase, peroxidase, cholesterol oxidase, potato glycoprotein, protease, protease inhibitor, urease, α-amylase inhibitor, porin, chitinase, lectin, engineered antibody or antibody fragment, Bacillus cereus insecticidal protein, pathogenic bacillus species (such as X. nematophila or X. bovienii) insecticidal protein, luminescent bacillus species (such as P. luminescens or P. asymobiotica) insecticidal protein, and brevispera species (such as B. lateralis).Laterosporous insecticidal proteins, Lysinibacillus spp. (e.g., L. Sphearicus) insecticidal proteins, Chromobacterium spp. (e.g., C. subtsugae or C. piscinae) insecticidal proteins, Yersinia spp. (e.g., Y. entomophaga) insecticidal proteins, Bacillus spp. (e.g., P. propylaea) insecticidal proteins, Clostridium spp. (e.g., C. bifermentans) insecticidal proteins, and lignin. In other embodiments, the second agent may be at least one derivative of a suicide insect toxin complex (Tc) (the complex being derived from the genera *Luminobacterium*, *Xenorhabus*, *Serratia*, or *Yersinia*). In other embodiments, the second insecticidal protein may be a binary toxin derived from insect-killing bacteria (such as ISP1A and ISP2A from *Bacillus laterosporus* or BinA and BinB from *Bacillus spheroidosa*). The combination of the sBin-IP of the present invention and the second pest-killing agent can be achieved through the expression of both in transgenic plants. In some embodiments, the transgenic plant is a transgenic maize plant. In other embodiments, the combination in the transgenic maize plant is the sBin-IP of the present invention and mCry3A and / or eCry3.1Ab and / or Cry3Bb1 and / or Cry34 / Cry35.

[0178] In some embodiments, the transgenic plants of the present invention may contain at least one non-protein secondary pest killer. In a preferred embodiment, the secondary pest killer is an interfering RNA molecule. Interfering RNA molecules typically contain at least one RNA fragment targeting a target gene, a spacer sequence, and a second RNA fragment complementary to the first RNA fragment, thereby forming a double-stranded RNA structure. RNA interference (RNAi) occurs when an organism recognizes double-stranded RNA (dsRNA) molecules and hydrolyzes them. The resulting hydrolysis products are small RNA fragments of about 19-24 nucleotides in length, referred to as small interfering RNAs (siRNAs). These siRNAs then diffuse or are carried throughout the organism, including across the cell membrane, where they hybridize with mRNA (or other RNAs) and cause RNA hydrolysis. The interfering RNA is recognized by the RNA interference silencing complex (RISC), in which the effector strand (or “guide strand”) of the RNA resides. This guide strand acts as a template for recognizing and disrupting the double-stranded sequence. This process is repeated each time the siRNA hybridizes with its complementary RNA target, effectively preventing those mRNAs from being translated and thus “silencing” the expression of specific genes transcribed from the mRNA. Interfering RNAs (RNAs) are known in the art for use in insect control (see, for example, disclosure WO 2013 / 192256, which is incorporated herein by reference). Interfering RNAs designed for insect control produce non-naturally occurring double-stranded RNAs that utilize the natural RNAi pathway in insects to trigger the downregulation of target genes, which may lead to cessation of feeding and / or growth and may cause the death of insect pests. Interfering RNA molecules may confer insect resistance to the same target pests as the proteins of the present invention or may target different pests. Target insect plant pests may feed on them by chewing, sucking, or piercing. Interfering RNAs are known in the art for use in insect control. In other embodiments, interfering RNAs may confer resistance to non-insect plant pests, such as nematode pests or viral pests.

[0179] Co-expression of more than one biocidal agent in the same transgenic plant can be achieved by creating a single recombinant vector (containing the coding sequences of more than one biocidal agent in a so-called molecular stack) and genetically engineering the plant to contain and express all the biocidal agents in the transgenic plant. Such molecular stacks can also be created using miniature chromosomes, as described, for example, in U.S. Patent 7,235,716. Alternatively, the transgenic plant containing nucleic acid encoding the first biocidal agent can be re-transformed with different nucleic acids encoding a second biocidal agent, etc. Alternatively, a plant (parent 1) can be genetically engineered for the expression of the genes of the present invention. A second plant (parent 2) can be genetically engineered for the expression of a second biocidal agent. By crossing parent 1 with parent 2, progeny plants expressing all the genes introduced into parents 1 and 2 are obtained.

[0180] Transgenic plants or seeds including the insecticidal protein of the present invention can 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). In cases where the insecticides or insecticidal seed coatings of the present invention, along with the transgenic plants or seeds, are effective against the same target insect (e.g., Coleoptera pests or root leaf beetle target pests), this combination is useful (i) in a method for further enhancing the activity of the composition of the present invention against the target insect and (ii) in a method for preventing resistance to the composition of the present invention by providing yet another mechanism of action against the target insect. Therefore, the present invention provides a method for enhancing the control of root leaf beetle populations, comprising providing the transgenic plants or seeds of the present invention and applying the insecticides or insecticidal seed coatings of the present invention to the plants or seeds.

[0181] Even when the insecticidal seed coating is active against different insects, the insecticidal seed coating is useful for expanding the scope of insect control. For example, by adding an insecticidal seed coating that is active against lepidopteran insects to the transgenic seeds of the present invention (which are active against coleopteran and some lepidopteran insects in some embodiments), the resulting coated transgenic seeds control both lepidopteran and coleopteran insect pests.

[0182] Examples of such insecticides and / or insecticidal seed coatings include, but are not limited to, carbamates, pyrethroids, organophosphates, neonicotinoids, organochlorides, neurotoxins, or combinations thereof. In another embodiment, the insecticide or insecticidal seed coating is selected from the group consisting of: carbofuran, carbaryl, methomyl, bifenthrin, cypermethrin, deltamethrin, cypermethrin, deltamethrin, chlorpyrifos, oxychloride, dimethoate, ethoprophos, malathion, methyl parathion, phorate, terbufos, terbufos, pyrimethanil, fipronil, acetamiprid, imidacloprid, thiamethoxam, endosulfan, chlorpyrifos, and combinations thereof. Commercial products containing such insecticides and insecticidal seed coatings include, but are not limited to, [list of products]. (Carbofuran) (Methodoxime, Methodoxime, Nanade) (Aminonaphthalene) (bifenthrin) (heptafluthrin), (Cypermethrin) (Cypermethrin), Delta (deltamethrin) (λ-cyhalothrin), (permethrin) (permethrin) (bifenthrin) (bifenthrin) (heptafluthrin) (λ cyhalothrin), (chlorpyrifos) (phosphorus oxychloride) (Meconium) (Methyl phorate) (Phraphoscine, flucythinate) (Methyl phorate) (tert-butylphosphide), (Dimethoate), isochloride, (fipronil) (thiamethoxam) (Imidacloprid) (Imidacloprid) (Thiamethoxam) and (Cypermethrin, Pyrimethanil).

[0183] In some embodiments, the invention also covers a composition comprising an insecticidal amount of the insecticidal protein of the invention. In further embodiments, the composition comprises a suitable agricultural carrier and a binary toxin of the invention. 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 invention, including proteins comprising, substantially comprising, 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 NO: 1-6). A suitable carrier should not be phytotoxic to valuable crops (particularly at the concentration used when applying the composition in the presence of the crop) and should not chemically react with compounds of the active ingredient herein (i.e., peptides or other compositional components of the invention). Such mixtures may be designed for direct application to crops or may be concentrates or formulations that are typically diluted with additional carriers and adjuvants prior to application. These can include inert or active components and can be solids (e.g., dusts, powders, granules, water-dispersible granules, or wettable powders) or liquids (e.g., emulsifiable concentrates, solutions, emulsions, or suspensions). Suitable agricultural carriers can include liquid carriers such as water, toluene, xylene, naphtha, crop oil, acetone, methyl ethyl ketone, cyclohexanone, trichloroethylene, perchloroethylene, ethyl acetate, amyl acetate, butyl acetate, propylene glycol monomethyl ether and diethylene glycol methyl ether, methanol, ethanol, isopropanol, pentanol, ethylene glycol, propylene glycol, glycerin, etc. Water is often the preferred carrier for diluting concentrates. Suitable solid carriers can include talc, pyrophyllite clay, silica, clay, kieselguhr, chalk, diatomaceous earth, lime, calcium carbonate, bentonite, bleaching clay, cottonseed hulls, wheat flour, soybean flour, pumice, wood flour, walnut shell powder, lignin, etc. In other embodiments, the proteins of the present invention may 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 for the delivery of insect control agents to the insect and results in toxic effects on insect pests.

[0184] In further embodiments, the compositions of the present invention may be powders, dusts, pills, granules, sprays, emulsions, colloids, or solutions. The compositions of the present invention may be prepared by dehydration, freeze-drying, homogenization, extraction, filtration, centrifugation, sedimentation, or concentration of bacterial cell cultures. The compositions of the present invention may 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 invention by weight. The compositions of the present invention may contain at least a second pesticide (which may be insecticidal, nematode-killing, fungicidal, or bactericidal). The at least second pesticide may have insecticidal activity against the same insect as or different insects as the polypeptide of the present invention. The second pesticide may be a polypeptide. The pesticide may be interfering RNA. Secondary biocides can be microorganisms (such as bacteria) containing nucleic acid molecules encoding the biocides and / or containing the biocides (such as peptides or interfering RNA). The microorganisms may be attenuated, heat-inactivated, or freeze-dried. The microorganisms may die or be unable to reproduce. Secondary biocides can be insecticides, such as carbofuran, acetamiprid, methomyl, bifenthrin, heptafluthrin, permethrin, deltamethrin, lambda-cyhalothrin, cypermethrin, deltamethrin, chlorpyrifos, oxychloride, dimethoate, ethoprophos, malathion, methyl parathion, phorate, terbufos, tebufenozide, fipronil, acetamiprid, imidacloprid, thiamethoxam, endosulfan, chlorpyrifos, or combinations thereof, or commercial products containing insecticides and insecticidal seed coatings as described above.

[0185] The compositions of the present invention (e.g., compositions comprising the proteins of the present invention and agriculturally acceptable carriers) can be used in conventional agricultural methods. Agriculturally acceptable carriers are formulations that can be used to apply compositions comprising the polypeptides of the present invention to plants or seeds. For example, the compositions of the present invention can be mixed with water and / or fertilizer and can be applied to desired locations before and / or after emergence by any means, such as aircraft spray cans, irrigation equipment, direct injection sprayers, backpack spray cans, livestock dipping tanks, farm equipment used for ground spraying (e.g., nozzle sprayers, manual sprayers), etc. The desired location can be soil, plants, etc.

[0186] The compositions of the present invention can be applied to seeds or plant propagules in any physiological state at any time: between seed harvesting and sowing; during or after sowing; and / or after germination. Preferably, the seeds or plant propagules are in a sufficiently robust state to cause little or no damage during treatment, including physical or biological damage. The formulations can be applied to seeds or plant propagules using conventional coating techniques and machinery such as fluidized bed technology, drum milling methods, rotostatic seed processors, and rotary drum coaters.

[0187] In some embodiments, the present invention further includes a method for controlling a population of Coleoptera pests, the method comprising contacting the pest population with an insect-controlling effective amount of the present invention's binary toxin, wherein the binary toxin comprises, substantially comprises, or comprises at least two amino acid sequences identical in 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% of any one of SEQ ID NO: 1-6. Contact includes members of the pest population ingesting or taking in an insecticidal protein. The insecticidal protein may be incorporated into the insect's diet or may be expressed or present in plant tissue subsequently ingested by the insect population. In a further embodiment, controlling a population of Coleoptera pests comprises killing the insects by contacting them with an insect-controlling effective amount of the present invention's insecticidal protein.

[0188] The present invention also includes a method for increasing plant yield, the method comprising growing in a field a plant or its seeds containing a nucleic acid molecule that has been stably incorporated into the expression cassette of the present invention in its genome, wherein the field is infested with a pest, and the polypeptide has insecticidal activity against the pest.

[0189] Once the desired nucleic acid is transformed into a specific plant species, it can be propagated in that species or transferred to other varieties of the same species (especially commercial varieties) using traditional breeding techniques.

[0190] In some embodiments, the present invention covers a method for providing maize growers with means of controlling populations of root leaf beetles in maize crops, the method comprising (a) selling or providing to the grower transgenic maize seeds containing nucleic acid molecules, expression cassettes, vectors or chimeric genes of the present invention; and (b) advertising to the grower that the transgenic maize seeds produce transgenic maize plants that control populations of root leaf beetles.

[0191] In some embodiments, the present invention also covers a method for identifying an insecticidal protein comprising, substantially comprising, or comprising 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 having 100% sequence identity with any of SEQ ID NO:1-6 or its toxin fragment, the method comprising the steps of: (a) generating a primer pair that amplifies SEQ ID NO:1-6 from a nucleic acid sample. (a) A polynucleotide or its complementary sequence of any one of NO:7-12 is selected, (b) an orthologous polynucleotide is amplified from the nucleic acid sample, (c) the nucleic acid sequence of the orthologous polynucleotide is identified, (d) a protein encoded by the orthologous polynucleotide is generated, and (e) the protein of step (d) is determined to have insecticidal activity against insect pests.

[0192] Example

[0193] Embodiments of the present invention can be better understood by referring to the following examples. The foregoing and following descriptions of embodiments of the invention, as well as various embodiments, are not intended to limit the claims, but are illustrative. Therefore, it should be understood that the claims are not intended to be limited to the specific details of these examples. Those skilled in the art will understand that other embodiments of the invention can be practiced without departing from the spirit and scope of this disclosure, which is defined by the appended claims. Recombinant DNA and molecular cloning techniques generally accepted in this field can be found in the following publications, such as J. Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd edition, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press (2001); T.J. Silhavy, M.L. Berman, and L.W. Wenquist, *Experiments with Gene Fusions*, Cold Spring Harbor Laboratory, NY (1984); Ausubel, FM et al., *Current Protocols in Molecular Biology*, John Wiley and Sons Inc., NY (1988); Reiter et al., *Methods in Arabidopsis Research*, World Scientific Press (1992); and Schultz et al., *Plant Molecular Biology Manual*, Kluwer Academic. Publishers [Krugwert Academic Publishers] (1998).

[0194] Example 1: Identification of the genomic sequence encoding an insecticidal protein in bacteria.

[0195] 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 *Sphingosporobacteria*, *Pseudomonas*, and related genera within the order *Sphingosporobacteria*. Four candidate sequences were selected for expression and testing against insect pests. Four candidate nucleotide sequences were identified in the genomes of *Sphingosporobacteria* strains from Korea (SEQ ID NO:1 and SEQ ID NO:2) and *Pseudomonas* species SG02 (SEQ ID NO:3 and SEQ ID NO:4). Codon-optimized versions of the *E. coli* encoding each candidate sequence were generated, SEQ ID NO:13-16, and individually introduced into the pET29a bacterial expression vector, designated pET29a-13, pET29a-14, pET29a-15, and pET29a-16, respectively, to produce the proteins. Each pET29a expression vector was transformed into *E. coli* BL21*(DE3) and lysates were prepared from isopropyl β-D-1-thiogalactopyranoside (IPTG)-induced cultures (producing protein overnight at approximately 18°C). The insecticidal activity of the lysates against the western maize rootworm (WCR) was assessed in a feed incorporation bioassay. Briefly, *E. coli* lysates were mixed with an equal volume of heated artificial insect feed (Bioserv, Frenchtown, NJ)) in 1.5 mL centrifuge tubes and then applied to small culture dishes. After the diet-sample mixture cooled and solidified, WCR larvae were added to each plate. The plates were sealed and maintained under ambient laboratory conditions for temperature, light, and relative humidity. Buffer without lysates, lysates from *E. coli* BL21*(DE3) cultures (containing empty pET29a vectors), and artificial insect feed alone served as negative controls. Mortality percentages and growth inhibition observations were obtained at 4 and 6 days post-infection, designated as s = small larvae, m = medium larvae, and l = large larvae.

[0196] The results shown in Table 1 indicate that lysates from *E. coli* cultures (expressing proteins encoded in the genomes of *Sphingosporales*) surprisingly possess insecticidal activity against pests of the *Leymus* genus, particularly when combined. These sBin insecticidal protein (sBin-IP) combinations were identified as binary toxins. The binary toxin containing *Bacillus* proteins was designated as the Seg_kor binary toxin and contains sBin1Aa (SEQ ID NO: 1) and sBin2Aa (SEQ ID NO: 2). The binary toxin containing paired *Bacillus* proteins was designated as the Dyad-SG02 binary toxin and contains sBin1Ba (SEQ ID NO: 3) and sBin2Ab (SEQ ID NO: 4). The sBin1 protein is approximately 32–33 kDa and, based on sequence comparison search, is an ortholog of ETX-MTX2. The sBin2 protein is approximately 12 kDa and shows no similarity to any known protein family. However, they can function as small beta-sheets.

[0197] Table 1. Insecticidal activity of lysates containing sBin protein against WCR.

[0198]

[0199] Using the bioassay method described above, the Dyad_SG02 binary toxin containing the sBin1Ba and sBin2Ab protein components was tested against the western maize rootworm and the northern maize rootworm at different concentrations. The results are shown in Table 2, indicating that sBin-IP is active against both species of the root leaf beetle genus.

[0200] Table 2. Insecticidal activity of lysates containing sBin1Ba and sBin2Ab against maize rootworm.

[0201]

[0202]

[0203] Example 2. The purified sBin-IP protein is active against the genus *Rhizophora*.

[0204] The insecticidal properties of the Dyad-SG02 binary toxin were further characterized. Two liters of *E. coli* BL21*(DE3) cells carrying either a pET vector encoding sBin1Ba or a pET vector encoding sBin2Ab were grown in LB medium at 37°C. When the OD reached 0.8–1.0, IPTG (1 mM) was added to the culture, and the culture was then transferred to 18°C ​​and incubated for 18 h. The cell pellet was harvested and resuspended in 20 mM Tris (pH 8.5) containing 10% glycerol. Cells were lysed using a Freund's cell crusher, and the lysate was then centrifuged at 100 kJ / g. 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 binds sBin proteins; each protein was eluted from the column using a linear NaCl gradient. The high-salt buffer consisted of 20 mM Tris (pH 8.5) and 0.5 M NaCl containing 10% glycerol. The purest fractions were combined and concentrated to approximately 2 ml. The protein was loaded onto a Sephadex 200 gel filter column pre-equilibrated in 1X PBS. The purity of the fractions from the Sephadex 200 column was analyzed by SDS-PAGE. The purest fractions were combined and concentrated to approximately 7 mg / ml, then stored at -80°C. The purified protein was then tested against WCR larvae using a dietary incorporation method substantially as described in Example 1. As shown in Table 3, the components sBin1Ba and sBin2Ab of the Dyad-SG02 binary toxin were inactive individually, but when combined, they resulted in 100% mortality. At concentrations tested against WCR and NCR, the sBin protein showed no activity against the southern maize rootworm.

[0205] Table 3. Insecticidal activity of sBin1Ba and sBin2Ab against corn rootworm.

[0206]

[0207] Example 3. Identification of the genomic sequence encoding an insecticidal protein in Paracoccus species.

[0208] 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 genus *Paragonimus* and related genera within the order Rhodobacteria. Two candidate sequences (SEQ ID NO:5 and SEQ ID NO:6) were identified in pantrophic *Paragonimus* strains. Codon-optimized versions of each candidate coding sequence were generated from *E. coli*, SEQ ID NO:17 and SEQ ID NO:18, and were individually introduced into pET29a bacterial expression vectors, designated pET29a-17 and pET29a-18, respectively, to produce proteins. Each pET29a expression vector was transformed into *E. coli* BL21*(DE3), and lysates were produced from isopropyl β-D-1-thiogalactopyranoside (IPTG)-induced cultures (producing proteins overnight at approximately 18°C). In a feed incorporation bioassay, the insecticidal activity of the lysates against the western maize rootworm (WCR) was assessed. In short, *E. coli* lysates were mixed with an equal volume of heated artificial insect feed (Bioserv, Frenchtown, NJ) in 1.5 mL centrifuge tubes and then applied to small petri dishes. 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 for temperature, light, and relative humidity. Buffer without lysates, lysates from *E. coli* BL21*(DE3) cultures (containing empty pET29a vectors), and artificial insect feed alone served as negative controls. Mortality percentages and growth inhibition observations were obtained at 4 and 6 days post-infection, designated as s = small larvae, m = medium larvae, and l = large larvae.

[0209] The results shown in Table 4 indicate that the lysates from *E. coli* cultures (expressing proteins encoded in the genomes of Rhodophytes) surprisingly possessed insecticidal activity against root leaf beetle pests, particularly *Hemiberlesia sapiens* and *Hemiberlesia sapiens*. These sBin insecticidal protein (sBin-IP) combinations were designated as binary toxins. The binary toxin containing *Bacillus sapiens* proteins was designated as the Seg_kor binary toxin and contained sBin1Aa (SEQ ID NO:1) and sBin2Aa (SEQ ID NO:2). The binary toxin containing paired *Bacillus sapiens* proteins was designated as the Dyad-SG02 binary toxin and contained sBin1Ba (SEQ ID NO:3) and sBin2Ab (SEQ ID NO:4). At concentrations tested for WCR and NCR, the sBin proteins showed no activity against *Hemiberlesia sapiens*.

[0210] Table 4. Insecticidal activity of lysates containing sBin protein against WCR.

[0211]

[0212] Unlike the aforementioned binary toxins, the ETX-MTX2-like protein in the Parac-panto binary toxin sBin1Ca possesses some insecticidal activity. However, the insecticidal activity of the Parac-panto binary toxin sBin1Ca+sBin2Ba is higher than that of either component alone. Therefore, the maximum insecticidal activity requires both components, sBin1Ca+sBin2Ba. Example 4. Characterization of sBin protein function.

[0213] This example describes replacing the sBin2 protein of one binary toxin of the present invention with the sBin2 protein of a different binary toxin of the present invention. The smaller protein component sBin2Aa of the Seg-kor binary toxin was combined with the larger protein component sBin1Ba of the Dyad_SG02 binary toxin for testing to determine whether the binary toxin containing the heterologous protein was still effective against the corn rootworm. The resulting heterologous binary toxin was tested at two different concentrations in the bioassay described above.

[0214] The results shown in Table 5 indicate that the components in the different binary toxins of the present invention are cross-functional.

[0215] Table 5. Insecticidal activity of sBin mixture against WCR

[0216]

[0217] Example 5. Sequence relationship of sBin-IP.

[0218] Tables 6 and 7 show the sBin-IP activity alignment and sequence identity comparison for *Betula* species. The sBin1 fraction (Table 6) showed low identity across its full sequence length. The sBin2 fractions from the Seg_kor and Dyad_SG02 binary toxins showed 84% identity, while the sBin2 fraction from the Parac_panto binary toxin showed only about 56%–59% identity with sBin2 fractions from other binary toxins (Table 7).

[0219] Table 6. Comparison of alignment and identity percentage of sBin1 protein.

[0220]

[0221] Under the amino acid label, "." indicates the same amino acid.

[0222] Table 7. Comparison of alignment and identity percentage of sBin2 protein.

[0223]

[0224] Example 6. Insecticidal activity of sBin toxin against Cry-resistant WCR.

[0225] To determine whether the sBin toxin activity occurs through a different mode of action than Cry3-related proteins, SUMO-labeled sBin1Ba and sBin2Ab were purified as described above, and their efficacy against Cry3Bb-resistant WCR strains, against strains resistant to modified Cry3A (mCry3A) protein (mCry3A-R), and against strains resistant to eCry3.1Ab protein (eCry3.1Ab-R) was tested. Feed incorporation assays were performed essentially as described above, and mortality and growth inhibition were observed at 4 and / or 6 days post-infection, where s = small larvae, m = medium larvae, and l = large larvae. A negative control was 1x PBS. Wild-type WCR strains without resistance to Cry proteins were used as positive controls. As shown in Table 8, the binary toxins of this invention exhibit insecticidal activity against Cry-resistant WCR strains, indicating that these proteins have a unique mode of action compared to Cry proteins from Bacillus thuringiensis. Therefore, the combination of the binary toxin of the present invention (e.g., Dyad-SG02 binary toxin containing sBin1Ba + sBin2Ab) and Cry protein will effectively mitigate the development of resistance to Cry protein or to the binary toxin.

[0226] Table 8. Activity of binary toxins against Cry-resistant WCRs.

[0227]

[0228] Example 7. Convert corn using sBin-IP encoded sequences.

[0229] Nucleotide sequences encoding the sBin1Aa, ​​sBin2Aa, sBin1Ba, sBin2Ab, sBin1Ca, and sBin2Ba of the present invention, or variants thereof, such as any one of SEQ ID NO: 1-6, or maize-optimized nucleotide sequences, such as any one of SEQ ID NO: 19-24 generated as described, for example, in U.S. Patent No. 6,051,760, may be converted into maize to control maize rootworms.

[0230] Two or three plant expression cassettes were constructed to introduce the sBin-IP coding sequence into maize. The first cassette contains a plant-expressible promoter operatively linked to an sBin1 coding sequence (e.g., SEQ ID NO: 19, 21, or 23), which is operatively linked to a terminator that functions in maize. The second cassette contains a plant-expressible promoter operatively linked to an sBin2 coding sequence (e.g., SEQ ID NO: 20, 22, or 24), which is operatively linked to a terminator that functions in maize. The third cassette contains a plant-expressible promoter operatively linked to a pmi coding sequence encoding a selectively labeled phosphomannose isomerase (PMI), which is operatively linked to a terminator that functions in maize. Optionally, a first expression cassette can be constructed that operatively links a plant-expressible promoter to a nucleotide sequence containing functionally fused sBin1 and sBin2 coding sequences, which are operatively linked to a terminator that functions in maize. A second expression cassette then contains a selective marker. A recombinant plant transformation binary vector containing the two or three expression cassettes is generated for use in maize transformation experiments.

[0231] The binary vector was transformed into Agrobacterium tumefaciens using standard molecular biology techniques. To prepare Agrobacterium for transformation, the cells were cultured overnight in liquid YPC medium at 28°C and 220 rpm.

[0232] The transformation of immature corn embryos with Agrobacterium was basically performed as described by Negrotto et al., 2000, Plant Cell Reports 19:798-803. For this example, all the culture medium components were essentially as described by Negrotto et al. (above). However, many culture medium components known in the art can be substituted.

[0233] In short, Agrobacterium strain LBA4404 (pSB1) containing the binary plant transformation vector was grown at 28°C on YEP (yeast extract (5 g / L), peptone (10 g / L), NaCl (5 g / L), 15 g / L agar, pH 6.8) solid medium for 2-4 days. Approximately 0.8 x 10⁻⁶ cells / day were grown. 9 Agrobacterium was suspended in LS-inf medium supplemented with 100 μM As (Negrotto et al., ibid.). The bacteria were pre-induced in this medium for 30 to 60 minutes.

[0234] Immature embryos of suitable genotypes were excised from 8-12 day old ears and placed in liquid LS-inf + 100 μM saturates. These embryos were rinsed with fresh infection medium. Agrobacterium solution was then added, and the embryos were vortexed for 30 seconds and allowed to settle with the bacteria for 5 minutes. The embryos were then transferred scutellum-upward to LSA medium and incubated in the dark for two to three days. Subsequently, embryos with 20 to 25 embryos per petri plate were transferred to LSDc medium supplemented with cefotaxime (250 mg / L) and silver nitrate (1.6 mg / L) and incubated in the dark at 28°C for 10 days.

[0235] Immature embryos producing embryogenic callus were transferred to LSD1M0.5S medium. Cultures were selected on this medium for approximately 6 weeks, followed by a subculturing phase of approximately 3 weeks. Surviving callus tissue was transferred to Reg1 medium supplemented with mannose. After culturing under light (16-hour light / 8-hour dark), the green tissue was transferred to Reg2 medium without growth regulators and incubated for approximately 1 to 2 weeks. These plantlets were then transferred to Magenta GA-7 boxes (Magenta Corp., Chicago, Illinois) containing Reg3 medium and allowed to grow under light.

[0236] After transformation, selection, and regeneration, use The analysis was used to determine the presence of the pmi gene and the codon-optimized coding sequence for the sBin corn cob in the plants. The presence of the vector backbone in the plants was also tested. Plants negative for the vector backbone and containing a one-copy transgene from the binary vector were transferred to a greenhouse and their insecticidal activity against WCR was tested.

[0237] Example 8. The binary toxin of the present invention in combination with a second insecticide.

[0238] The components of the binary toxin of the present invention, as described above, were purified as in Example 2. Cry protein and dsRNA, known to have insecticidal activity against the essential targets, were prepared. In a non-limiting example, the dsRNA may target genes encoding vacuole ATP synthase, β-tubulin, 26S proteosomal subunit p28 protein, EF1α48D, troponin I, transmembrane tetraprotein, clathrin heavy chain, γ-excapsule, β-excapsule, and / or juvenile hormone epoxide hydrolase (PCT patent applications PCT / US17 / 044825; PCT / US17 / 044831; PCT / US17 / 044832; US Patent No. 7,812,219; each incorporated herein by reference). The efficacy of the Cry protein and / or dsRNA and the purified binary toxin components against WCR was tested in a dietary incorporation assay performed substantially as described in Example 1.

[0239] Example 9. In situ editing of the genome in plant cells to generate modified sBin-IP.

[0240] The following examples illustrate genome editing using in situ plant cell genomes to incorporate mutations into the coding sequence of natural sBin-IP (e.g., sBin1Aa (SEQ ID NO:1)).

[0241] Targeted genome modification (also known as genome editing) can be used to introduce mutations into specific DNA sequences. These genome editing technologies, including zinc finger nucleases (ZNF), transcription activator-like effector nucleases (TALEN), large-scale nucleases, and clustered regularly spaced short palindromic repeats (CRISPR), have been successfully applied to more than 50 different organisms, including crop plants. See, for example, Belhaj, K., et al., Plant Methods 9, 39 (2013); Jiang, W., et al., Nucleic Acids Research, 41, e188 (2013). The CRISPR / Cas system used for genome editing is based on the transient expression of the Cas9 nuclease and an engineered single guide RNA (sgRNA) targeting a specific polynucleotide sequence.

[0242] Cas9 is a large, monomeric DNA nuclease that is guided to its DNA target sequence via a complex of two 20-nucleotide (nt) non-coding RNAs: CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). Functionally, it can be acquired as a single synthetic RNA chimera. The Cas9 protein contains two nuclease domains homologous to RuvC and HNH nucleases. The HNH nuclease domain cleaves the complementary DNA strand, while the RuvC-like domain cleaves the non-complementary strand, thus introducing a blunt nick in the target DNA.

[0243] When Cas9 and sgRNA are transiently expressed in living corn cells, specific double-strand breaks (DSBs) are generated in the target DNA in transgenic corn cells. Mutations at the break sites are introduced through non-homologous end joining and homologous directed DNA repair pathways.

[0244] A specific mutation is introduced into the coding sequence of the natural sBin1Aa component protein (SEQ ID NO:1) using a recombinant plasmid expressing the Cas9 nuclease and an sgRNA target (codon-optimized for the sBin1Aa sequence, e.g., SEQ ID NO:19) in transgenic corn. This method is implemented via Agrobacterium tumefaciens infiltration, where Agrobacterium carries a binary plasmid containing the specified target sequence. Upon binding of the sgRNA to the target sBin1Aa coding sequence, the Cas9 nuclease specifically cleaves the coding sequence, introducing one or more desired mutations during DNA repair. Thus, the now mutated sBin1Aa coding sequence encodes a modified variant sBin1Aa protein, for example, where the mutation at T52 residue is changed to A, V102 residue to T, V151 residue to A or K, or K297 residue to S or T, or any combination thereof.

[0245] Plant cells containing genome-edited sBin-IP coding sequences were screened by PCR and sequencing. Callus with genome-edited mutations in the sBin-IP or modified sBin-IP coding sequences was induced and regenerated plants were used for phenotypic evaluation. The insecticidal activity of expressed sBin-IP against the following insects was assessed: western maize rootworm (maize root firefly beetle), northern maize rootworm (Barth's root firefly beetle), southern maize rootworm (cucumber eleven-spotted leaf beetle subspecies), and / or Mexican maize rootworm (Mexican maize root firefly beetle).

[0246] Example 10. Testing the insecticidal activity of sBin-IP against lepidopteran pests.

[0247] The Dyad_SG02 binary toxin was tested against lepidopteran insects. The labeled protein was expressed using the pET-6His-SUMO construct containing sBin1Ba (SEQ ID NO:15) and the construct containing sBin-IP1Aa (SEQ ID NO:16). The constructs were transformed into *E. coli* BL21*(DE3) for protein production. The bioactivity of lysates from bacterial cultures expressing the sBin protein against a group of lepidopteran insect pests, including the corn ear moth (CEW), fall armyworm (FAW), black cutworm (BCW), and European corn borer (ECB), was tested using a diet-overlay bioassay. Positive controls consisted of larvae exposed to lysates of *E. coli* BL21* expressing the Cry protein, known to be active against all four species. Buffer-only and lysates from BL21*(DE3) bacterial cultures carrying the empty pET29 vector were used as negative controls. Mortality was assessed on day 7. Bioassay results showed that the labeled sBin-IP1Aa was active against WCR, but not against any of the Lepidoptera pests tested.

[0248] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and different modifications or variations thereof will be apparent to those skilled in the art and will be included within the spirit and scope of this application and the appended claims.

[0249] All disclosures and patent applications mentioned in this specification indicate the skill level of a person skilled in the art to which this invention pertains. All disclosures and patent applications are incorporated herein by reference to the extent that each individual disclosure or patent application is expressly and individually indicated to be incorporated by reference. sequence list <110> Syngenta Crop Protection AG <120> Insecticidal protein <130> 81986‑WO‑REG‑ORG‑P‑1 <150> US 62 / 951,025 <151> 2019-12-20 <160> twenty four <170> PatentIn version 3.5 <210> 1 <211> 302 <212> PRT <213> Segetibacter koreensis (Korea) <400> 1 Met Ser Leu Gln Tyr Leu Asp Lys Arg Leu Ile Gly Ala Ile Ile Val 1 5 10 15 Asp Ala Trp Gln Asn His Phe Asn Thr Glu Met Asp Pro Ala Asp Gly 20 25 30 Gln Trp Phe Thr Asn Gly Pro Gly Asn Asn Gly Gly Leu Tyr Gly Ile 35 40 45 Leu Thr Asp Thr Leu Ala Ser Glu Leu Lys Phe Leu Pro Asp Gln Gln 50 55 60 Val Phe Thr Met Asn Lys Ile Ala Ala Ala Thr Ser Thr Ala Asp Asn 65 70 75 80 Arg Asn Gly Leu Thr Pro His Gln Thr Val Ser Leu Thr Tyr Glu Tyr 85 90 95 Gln Asn Ser Thr Thr Val Thr His Ser Thr Thr Asn Thr Ile Thr Val 100 105 110 Gly Thr Gly Val Glu Ile Lys Ser Ser Ala Glu Phe Leu Gly Thr Gly 115 120 125 Ala Glu Val Thr Val Ser Phe Asn Thr Glu Tyr Ser Tyr Ser Trp Thr 130 135 140 Glu Glu Arg Ser Glu Ser Val Ser Glu Thr Lys Thr Phe Gly Gln Glu 145 150 155 160 Val Ser Thr Asp Ile Pro Ser Gly Leu Val Tyr Gln Val Thr Leu Leu 165 170 175 Ala Asp Lys Ala Asn Ile Arg Val Pro Phe Tyr Ala Asp Ile Ile Leu 180 185 190 Thr Gly Gln Ser Val Ala Asn Phe Ala Ser Pro Val Asn Gly Gln Lys 195 200 205 Thr Trp Ala Ile Asp Ala Gly Thr Leu Cys Glu Trp Ile Asn Gln Tyr 210 215 220 Gly Ser Ala Gly Asn Glu Ser Tyr Arg Tyr Met Lys Asp Ala Ser Asn 225 230 235 240 Pro Lys Gln Gly Phe Ile Arg Leu Glu Gly Asn Leu Thr Ala Thr Gln 245 250 255 Thr Leu Asn Phe Thr Ala Leu Thr Ser Asp Ile Thr Asp Ser Phe Thr 260 265 270 Ala Thr Ala Gln Pro Ala Leu Met Arg Glu Leu Asn Asn Ser Glu Leu 275 280 285 Glu Lys Leu Glu Ser Lys Ala Ile Lys Lys Val Ala Leu Gly 290 295 300 <210> 2 <211> 109 <212> PRT <213> Segetibacter koreensis <400> 2 Met Pro Leu Gln Gln Ile Gly Lys Met Ser Leu Lys Asn Ser Gly Gly 1 5 10 15 Phe Val Ala Arg Ile Gln Phe Ser Tyr Leu Asp Glu Asn Gly Glu Lys 20 25 30 Lys Leu Thr Gly Gln Ser Gly Asp Val Leu Leu Gly Gln Thr Lys Thr 35 40 45 Leu Asp Pro Gly Glu Met Gly Val Pro Asp Gly Ser Met Thr Tyr Met 50 55 60 Tyr Val Ser Val Val Trp Gly Arg Asp Asn Glu Ala Thr Arg Ala Phe 65 70 75 80 Leu Tyr Gln Lys Gly Asn Val Ser Thr Ala His Tyr Leu Ile Ser Gly 85 90 95 Thr Thr Leu Asn Asn Asp Leu Gly Leu Ile Glu Ile Ser 100 105 <210> 3 [[ID=D34]]<211> 305 <212> PRT <213> Dyadobacter species SG02 <400> 3 Met Ser Leu Lys Phe Leu Asp Lys Gln Arg Ile Gly Ala Ile Ile Val 1 5 10 15 Asp Ala Phe Gln Glu Gln Leu Asn Thr Glu Met Asp Pro Ala Asn Gly 20 25 30 Gln Trp Phe Thr Asn Gly Pro Gly Asn Asn Gly Gly Leu Tyr Gly Ser 35 40 45 Leu Val Asp Ala Leu Ala Ser Ser Leu Val Phe Leu Pro Asn Glu Leu 50 55 60 Val Leu Thr Pro His Lys Leu Ala Ala Asp Thr Ala Ile Ile Asp Asn 65 70 75 80 Arg Asn Gly Leu Thr Pro Lys Ser Ser Ile Thr Leu Ser Tyr Ser Thr 85 90 95 Thr Glu Thr Thr Thr Thr Thr His Thr Val Ser Asn Ala Leu Lys Val 100 105 110 Gly Ile Gly Val Asp Ile Lys Ala Ser Ala Lys Phe Phe Gly Ser Gly 115 120 125 Val Asp Ile Thr Thr Lys Ile Ser Thr Asp Tyr Thr Tyr Ser Trp Ser 130 135 140 Asp Ala Val Ser Lys Ala Ala Ser Glu Thr Lys Gln Phe Ser Gln Thr 145 150 155 160 Val Pro Val Glu Val Pro Thr Gly Arg Val Tyr Gln Val Val Leu Thr 165 170 175 Cys Asp Lys Thr Asp Leu Asn Ala Pro Tyr Tyr Ala Asp Val Thr Leu 180 185 190 Thr Gly Thr Ser Thr Ala Asn Phe Ala Asn Asn Val Asn Gly Lys Asn 195 200 205 Thr Trp Val Leu Asp Ala Gly Thr Leu Cys Glu Trp Ile Asn Arg Ser 210 215 220 Gly Ser Ala Gly Gly Glu Ser His Met Tyr Leu Arg Asp Pro Gln Val 225 230 235 240 Thr Gly Gln Gly Leu Ile Arg Met Arg Gly Ser Leu Thr Ser Ser Ile 245 250 255 Thr Ala Asn Phe Val Val Asn Thr Tyr Asp Ile Thr Asp Thr Tyr Asn 260 265 270 Ala Thr Gly Lys Ala Ala Ile Glu Asn Asp His Leu Phe Ala Ala Ser 275 280 285 Glu Leu Ala Ser Leu Asn Ser Lys Leu Val Ser Glu Lys Val Ile Gly 290 295 300 Lys 305 <210> 4 <211> 109 <212> PRT <213> Dyadobacter species SG02 <400> 4 Met Pro Leu Gln Lys Ile Gly Lys Met Ser Leu Lys Asn Ser Gly Gly 1 5 10 15 Phe Val Ala Arg Val Gln Phe Ser Tyr Leu Asp Asp Asn Gly Glu Lys 20 25 30 Lys Leu Thr Gly Gln Ser Gly Asp Ile Thr Leu Gly Phe Thr Lys Thr 35 40 45 Tyr Asp Pro Gly Glu Met Gly Val Pro Asp Gly Ser Met Val Tyr Met 50 55 60 His Val Phe Val Val Trp Gly Thr Asp Asn Glu Ala Lys Arg Ala Phe 65 70 75 80 Leu Tyr Glu Lys Gly Asn Val Ser Val Ala His Tyr Asn Ile Ser Gly 85 90 95 Thr Thr Leu Asn Asn Asp Leu Gly Leu Ser Asp Ile Ser 100 105 <210> 5 <211> 304 <212> PRT <213> Paracoccus pantotrophus <400> 5 Met Thr Leu Gln Tyr Leu Asp Thr Leu Gly Leu Gly Ala Ile Ile Val 1 5 10 15 Asp Ala Trp Gln Asn His Leu Glu Thr Glu Lys Asp Pro Ala Gly Gly 20 25 30 Gln Trp Phe Ala Asn Gly Pro Gly Asn Asn Gly Gly Leu Phe Gly Lys 35 40 45 Leu Thr Asp Thr Leu Ala Ser Glu Leu Val Leu Asp Val Pro Ala Gln 50 55 60 Thr Phe Ser Val Tyr Gln Ser Ala Ala Ala Thr Gly Ile Val Asp Asn 65 70 75 80 Arg Asn Gly Leu Thr Pro Glu Gln Thr Val Gly Leu Ser Cys Thr Phe 85 90 95 Gln Asp Thr Val Thr Thr Thr His Ser Val Ser Lys Ala Val Lys Thr 100 105 110 Gly Thr Thr Val Ser Ile Lys Gly Thr Ile Asp Ala Lys Val Val Lys 115 120 125 Lys Glu Phe Gly Ile Ser Phe Thr Ala Glu Tyr Ser His Ser Trp Thr 130 135 140 Asp Ala Thr Ala Val Ser Lys Ser Glu Ser Arg Ser Phe Ser Val Ser 145 150 155 160 Val Pro Val Arg Asn Val Pro Ala Gly Arg Val Trp Gln Val Val Leu 165 170 175 Met Ala Asn Lys Lys Glu Leu Ser Met Pro Tyr Arg Ala Asp Ile Ile 180 185 190 Leu Lys Gly Ser Thr Val Ala Asn Phe Leu Ser Pro Ile Arg Gly Gln 195 200 205 Arg Ile Trp Gln Ala Asp Ala Gly Thr Leu Cys Glu Trp Ile Asn Arg 210 215 220 His Gly Ser Ala Gly Asp Glu Ser Trp Ser Tyr Gly Arg Asp Pro Ala 225 230 235 240 Asp Pro Thr Gln Gly Arg Ile Ser Leu Leu Gly Thr Leu Lys Ala Val 245 250 255 His Thr Val Asn Phe Thr Val Arg Thr Leu Asp Val Thr Glu Ser Phe 260 265 270 Arg Pro Asp Gly Asp Gly Gly Leu Val Leu Ala Thr Asn Ala Gly Ser 275 280 285 Glu Ala Pro Val Val Asp Glu Val Leu Val Thr Glu Leu Ala Ala Ala 290 295 300 <210> 6 <211> 110 <212> PRT <213> Paracoccus pantotrophus <400> 6 Met Ser Leu Gln Lys Val Gly Asn Phe Ser Leu His Asn Gly Gly Gly 1 5 10 15 Phe Val Ala Arg Met Lys Phe Ala Tyr Ile Asp Asp Glu Gly Gln Lys 20 25 30 Lys Thr Arg Glu Thr Gly Asp Ile Leu Leu Gly Gln Thr Lys Thr 35 40 45 Ala Lys Leu Glu Glu Phe Asp Ile Pro Asp Gly Ala Leu Val Tyr Leu 50 55 60 His Val Asp Val Val Trp Gly Lys Asp Asn Glu Ala Ala Arg Ala Phe 65 70 75 80 Tyr Glu Arg Gly and Thr Cys Thr Ala Ala Tyr Ile Thr Gly 85 90 95 Thr Thr Leu Ser Asn Thr Leu Gly Leu Ile Asp Val Asn Cys 100 105 110 <210> 7 <211> 909 <212> DNA <213> Segetibacter koreensis (Segetibacter koreensis) <400> 7 atgagccttc atatttaga caagcgactt atcggcgcca taattgttga tgcgtggcaa 60 aaccacttta accagagat ggaccctgcc gatggacagt gtttacgaa tggaccgggg 120 aacaacggag gtctttatgg tatattaca gatacgctcg catctgaatt aaagttctta 180 cctgatcaac aagttttac aatgaatag atagcggctg ctaccagcac agccgataat 240 cgaaatggcc taacgcctca ccagactgtt tcactaacat atgaatatca aaattctacc 300 acagtaacac actctactac caacactata actgtaggaa caggtgtaga aattaaaagc 360 tcggcggaat ttttaggaac aggcgcagaa gttacggtta gttttaatac tgaatacagc 420 tactcctgga ctgaagagag aagtgagtcg gtctcagaaa ctaaaacgtt tggacaagag 480 gtatctaccg acattccctc tggcctggtt taccaggtaa cactgcttgc cgacaaagca 540 aatattagag tgccctttta tgctgatata attcttactg gacagtctgt tgcaaacttt 600 gcaagccctg ttaatggaca aaaaacctgg gctattgatg caggtacttt atgcgaatgg 660 attaatcaat atggatcagc aggtaacgag tcgtatagat acatgaagga tgcgagtaac 720 cccaagcaag gatttattcg gctggagggt aacctgaccg ctactcaaac tttgaatttc 780 accgcattaa caagtgatat tacagattca ttcactgcga cagctcagcc agctcttatg 840 cgtgagttga acaactctga gctagaaaaa ttagaatcta aggctattaa gaaagtagcc 900 ctgggttga 909 <210> 8 <211> 330 <212> DNA <213> Segetibacter koreensis <400> 8 atgccactac aacaaatcgg aaaaatgagc ctcaagaatt ccggaggctt cgtcgccagg 60 atccaattca gctatttaga tgaaaatggc gaaaaaaaac ttacgggcca aagtggagac 120 gtattattag gtcaaacaaa aacattagat ccgggagaaa tgggggtacc ggatggctct 180 atgacttata tgtacgtatc cgttgtctgg ggaagagaca acgaggcgac ccgtgcattc 240 ctttatcaaa aaggaaatgt tagtactgcc cactacctca ttagtggcac gacccttaat 300[[ID=!3]] aacgacctag gattgattga gattagttaa 330 <210> 9 <211> 918 <212> DNA <213> Dyadobacter sp. SG02 <400> 9 atgagtctga aattcttaga taaacaaaga attggtgcca ttatcgtcga tgcttttcag 60 gaacagctca ataccgagat ggaccctgcg aacggacaat ggtttaccaa cggtccgggc 120 aacaatggcg gattgtatgg tagcctggtc gacgcgctgg cctcgtcgct ggtttttctg 180 ccaaacgaac tcgttctgac gccccataaa ctcgctgcgg atacggccat tatagataac 240 cgtaacggac tgactccgaa atcgtcgatc acattgtcat attcgaccac cgaaacgacg 300 acaacaaccc ataccgtatc gaacgcctta aaagtcggga tcggcgtgga catcaaggct 360 tcggcaaaat tcttcgggag cggtgtcgac atcacaacca agatcagcac ggattacacg 420 tacagttgga gcgacgcggt ctccaaagcg gcgtcggaaa cgaagcagtt ttcccagacg 480 gttccggtgg aagtgccgac gggccgggtg tatcaggtgg tactcacctg cgacaaaacc 540 gacctgaacg caccttacta tgcggacgtg acgcttaccg gcacatcaac cgccaatttc 600 gcgaacaatg taaacgggaa gaacacctgg gtactggatg cgggcacatt gtgcgaatgg 660 atcaaccggt cggggtctgc cggcggagaa tctcatatgt acctgcgcga tcctcaggtt 720 acaggccagg gccttatccg catgcgcggc agcctgacct cctccattac cgcgaatttc 780 gtcgtgaaca cctacgatat cacggatact tataacgcaa ctggcaaagc cgccattgaa 840 aacgaccacc tctttgccgc cagcgagctg gcttcgctga atagtaaact tgttagcgaa 900 aaagtgatcg ggaagtaa 918 <210> 10 <211> 330 <212> DNA <213> Dyadobacter species SG02 <400> 10 atgcctttac agaaaatcgg taaaatgagc cttaaaaatt cgggcggctt cgtagcccgc 60 gttcagttta gctatctgga cgacaatggt gaaaaaaaac tgaccggcca aagcggcgac 120 atcacgctcg gtttcaccaa aacgtatgat ccgggtgaaa tgggcgtacc ggacggctca 180 atggtttaca tgcacgtatt cgtcgtttgg ggcaccgaca acgaggcaaa acgtgcattt 240 ctttacgaaa agggcaatgt atccgttgct cactacaata tcagcggcac cacgttgaac 300 aacgacctgg ggctttctga tatcagttaa 330 <210> 11 <211> 915 <212> DNA <213> Paracoccus pantotrophus <400> 11 atgacgctgc aatatctcga cacgcttggg cttggcgcca tcatcgtgga tgcctggcaa 60 aatcaccttg aaaccgagaa ggacccggcc ggcggccaat ggttcgccaa cggtcccggc 120 aacaacggcg ggctgttcgg caagctgacg gacacgctgg catccgaact ggtcttagac 180 gtgccggcgc agaccttcag cgtctaccag tcggcggcgg cgaccgggat cgtggacaac 240 cgcaacggcc tgacgccga gcagacggtc ggcctgtcct gcaccttcca ggacacggtg 300 acgacgacgc attcggtcag caaggcggtc aagacgggca ccacggtctc gatcaagggc 360 accatcgacg ccaaggtggt gaaaggaa ttcggcatca gcttcaccgc cgaatattcg 420 cattcctgga cggatgcgac cgccgtgtcg aaatccgagt cgcgcagctt cagcgtctcg 480 gtgccggtgc gcaacgtgcc cgccggccgg gtctggcagg tcgtgctgat ggccaacaag 540 aaagactca gcatgcccta tcgcgccgac atcatcctga agggcagcac ggtggcgaat 600 ttcctgtcgc cgatccgtgg ccagaggatc tggcaggccg atgccggcac gctgtgcgaa 660 tggatcaacc gccatggctc ggcgggcgac gagtcctgga gctatggccg cgatcccgcc 720 gatcccacgc aggggcgcat ttccctgctg gggacgctga aggcggtcca tacggtcaat 780 ttcaccgtgc ggacgctgga tgtgaccgaa agcttccgac ccgatggcga cggcggtctg 840 gtccttgcga ccaacgccgg gtcggaagcg cctgttgttg acgaggttct ggttaccgag 900 cttgccgctg cctga 915 <210> 12 <211> 333 <212> DNA <213> Paracoccus pantotrophus <400> 12 atgtctctgc agaaagtcgg caatttcagc ctgcacaatg gcggcggctt cgtcgcccgg 60 atgaagttcg cctatatcga cgacgagggc cagaagaaga gcacgcggga aaccggcgac 120 atccttctgg gccagaccaa gaccgcgaaa ctggaagagt tcgacatccc ggacggcgcg 180 ctcgtctatc tgcatgtcga cgtggtctgg ggcaaggaca acgaggccgc gcgcgccttc 240 acctacgagc gcggcaatac ctgcacggcg gcctatacga tcaccggcac gacgctgtcg 300 aacacgctgg ggctgatcga cgtgaactgc tga 333 <210> 13 <211> 909 <212> DNA <213 Artificial sequence <220> <223> Synthetic polynucleotide <400> 13 atgtccctgc agtacttaga taaacgtctt atcggcgcga ttattgttga cgcgtggcag 60 aatcatttta atactgaaat ggatccggcg gatgggcaat ggtttacaaa cggtccaggc 120 aataacggtg gcctctacgg tattttgacc gacaccctcg cctctgagtt aaaatttctg 180 ccggatcaac aggtgttcac catgaataag atcgctgccg cgacttcgac cgcggacaac 240 cgcaatgggc tcaccccgca ccagaccgtt tcgttgacat acgaatatca gaacagcacc 300 actgtgacac acagcacaac gaataccatc acagtgggga ccggcgtcga aattaagtcg 360 agcgccgaat ttctgggtac cggtgcagag gtgactgtgt cctttaatac ggaatattcc 420 tactcatgga cggaggagcg cagcgaatca gtgtccgaaa cgaagacctt tggccaggaa 480 gtctcaaccg acatccctag cggcttggtt taccaggtta cgttactcgc ggataaagct 540 aatatccgg tgccgttcta cgcagacatt atcttaaccg ggcagtcagt tgcgaatttc 600 gcctcaccgg ttaacggcca aaaaacctgg gctatcgatg cgggcaccct gtgcgaatgg 660 atcaaccagt atggtagtgc cggcaatgag agttaccggt atatgaaaga cgccagcaat 720 cccaaacaag gctttattcg tctggagggc aacttaacag cgacacagac cttaacttt 780 acggcattga cgagtgatat cacggattcg tttaccgcta ccgcccaacc ggcgctcatg 840 cgcgaactga ataattcgga actggaaaaa cttgaaagca aagcaattaa aaaagtggcg 900 ctgggctaa 909 <210> 14 <211> 330 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide <400> 14 atgccgcttc agcaaattgg caaaatgagc cttaaaaatt caggcgggtt tgtcgctcgt 60 attcaattca gttacctgga cgaaaatggc gagaaaaaac tgaccgggca atctggcgat 120 gtactgttag gccagaccaa aacgttagat ccaggcgaaa tgggcgtccc agacggtagc 180 atgacgtaca tgtatgtgag tgtggtgtgg gggcgtgata atgaagccac ccgtgctttt 240 ctctaccaga aaggcaacgt atccactgcg cactacctga ttagcggtac tacgttgaat 300 aacgatctgg gtctgattga gatttcgtaa 330 <210> 15 <211> 918 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide <400> 15 atgtcactga aatttttgga taaacaacgc atcggcgcga taatcgttga cgcctttcaa 60 gaacagctta atacggaaat ggatccggcc aatgggcagt ggtttaccaa tgggccaggg 120 aacaatgggg gcttatacgg ctctttagta gacgccttgg catctagtct tgtattcctg 180 ccaaacgaac tggttctac gccgcacaa cttgcagctg atacggctat tattgacaac 240 agaaacgggc tgacgcccaa gtcttcaata acacttctt acagcacgac cgagacacg 300 accacaacc acacagtcag taacgcctta aaagtgggga taggtgtcga tattaagca 360 agtgcgaagt tctttggttc gggtgttgac ataactacga agatatcac agattacacc 420 tattcttgga gcgatgcagt cagtaggcc gcttcggaa ccaacaatt ctcccaacc 480 gtccctgtcg aagtgcccac agggagtc tatcaagtgg ttttgacatg cgacaagacg 540 gacttaacg cgccgtacta tgctgacgtg actcttacag gaacttcgac tgcgaacttt 600 gctaacaacg taaatggga gatacctgg gtactggatg ctggaacatt gtgtgaatgg 660 atcaccgtt cagggagtgc gggaggagaa tcgcacatgt atctgagaga tccacaagtg 720 acaggccaag ggttaatccg gatgagaggt agcttaacaa gctcgattac tgccacttt 780 gttgtaaata cctacgatat aacggacacg tacaatgcca cgggtaaagc tgccattgaa 840 aatgatcacc ttttgccgc gtcggaattg gcaagcctta atagcaaatt ggtcagcgaa 900 aaggtcatag gtaaataa 918 <210> 16 <211> 330 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> 16 atgcctcttc aaaagatcgg caagatgtca ttgaagaatt caggggggctt cgtggccaga 60 gtgcaattta gctatttaga tgataatggc gagaagaaat taaccggaca atctggggac 120 atcactctgg gtttcaccaa aacctacgat ccgggcgaaa tggggggtccc cgacggaagt 180 atggtttata tgcacgtgtt cgttgtttgg ggcactgaca acgaggctaa gcgtgcgttt 240 ttgtacgaaa agggcaatgt gtcagtcgcg cactataaca tttctggtac aaccttaaat 300 aatgatctgg gattgtcaga catcagctaa 330 <210> 17 <211> 915 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> 17 atgaccttac agtaccttga cacgctgggg cttggggcta tcattgtcga tgcgtggcag 60 aatcatttag agacggaaaa agatcccgct ggtgggcagt ggtttgctaa cgggcctggc 120 aacaacggag gacttttcgg taaacttaca gatactttag catctgaatt ggtcttagac 180 gtcccagcac agactttctc agtgtaccag agtgcggcag ccacagggat agtcgataac 240 agaaatgggt taactccaga gcagaccgtt gggttatcat gcacttttca agataccgta 300 actacgaccc attccgtgtc aaaggctgtg aagaccggca caactgtctc catcaagggt 360 acaattgatg ctaaagttgt taaaaaggaa tttggaatct cttttaccgc tgagtactct 420 catagttgga ctgatgctac ggctgttagc aagtcggagt cgcggtcatt ctctgtaagt 480 gtgcccgtta gaaatgttcc agcaggtcgc gtctggcaag tggttcttat ggcgaataag 540 aaagaattaa gtatgccgta cagagcggat atcatactta aagggtctac agtcgccaat 600 tttttgtcgc caatccgcgg gcagagaata tggcaagctg atgccgggac actttgtgaa 660 tggataaata gacacgggag tgctggagac gaatcgtgga gctatggccg cgacccggca 720 gatcccagc aaggccggat aagccttttg gggactttaa aagcagtgca tacggtaaac 780 ttcaccgtcc gtacattgga cgtcaccgag agtttccgcc cggacggcga cggcggactg 840 gtacttgcca ctaacgcggg atccgaggca cccgtggttg acgaagtttt agtaacagaa 900 ttggccgctg cgtaa 915 <210> 18 <211> 333 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide <400> 18 atgtcactgc agaaggtggg caatttcagc cttcataatg gaggtggttt cgtggcccgc 60 atgaagtttg cgtacataga cgatgaaggg caaaaaaaat ccaccagaga gaccggcgac 120 atattgttgg gacagaccaa gactgccaag ttagaagaat ttgatattcc cgacggggct 180 ttggtttact tgcatgtcga cgtggtgtgg ggcaaggata acgaggccgc tagagccttt 240 acctacgaac ggggaaacac ttgtaccgca gcttatacaa tcaccggaac gacgctgagt 300 aatacactgg gtctgataga tgtaaactgc taa 333 <210> 19 <211> 909 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide <400> 19 atgtccctgc agtacctgga caagaggctg atcggcgcca tcatcgtgga cgcctggcag 60 aaccacttca acaccgagat ggacccggcc gacggccagt ggttcaccaa cggcccgggc 120 aacaacggcg gcctgtacgg catcctgacc gacaccctgg cctccgagct gaagttcctg 180 ccggaccagc aggtgttcac catgaacaag atcgccgccg ccacctccac cgccgacaac 240 aggaacggcc tgaccccgca ccagaccgtg tccctgacct acgagtacca gaactccacc 300 accgtgaccc actccaccac caacaccatc accgtgggca ccggcgtgga gatcaagtcc 360 tccgccgagt tcctgggcac cggcgccgag gtgaccgtgt ccttcaacac cgagtactcc 420 tactcctgga ccgaggagag gtccgagtcc gtgtccgaga ccaagacctt cggccaggag 480 gtgtccaccg acatcccgtc cggcctggtg taccaggtga ccctgctggc cgacaaggcc 540 aacatcaggg tgccgttcta cgccgacatc atcctgaccg gccagtccgt ggccaacttc 600 gcctccccgg tgaacggcca gaagacctgg gccatcgacg ccggcaccct gtgcgagtgg 660 atcaaccagt acggctccgc cggcaacgag tcctacaggt acatgaagga cgcctccaac 720 ccgaagcagg gcttcatcag gctggagggc aacctgaccg ccacccagac cctgaacttc 780 accgccctga cctccgacat caccgactcc ttcaccgcca ccgcccagcc ggccctgatg 840 agggagctga acaactccga gctggagaag ctggagtcca aggccatcaa gaaggtggcc 900 ctgggctga 909 <210> 20 <211> 330 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide <400> 20 atgccgctgc agcagatcgg caagatgtcc ctgaagaact ccggcggctt cgtggccagg 60 atccagttct cctacctgga cgagaacggc gagaagaagc tgaccggcca gtccggcgac 120 gtgctgctgg gccagaccaa gaccctggac ccgggcgaga tgggcgtgcc ggacggctcc 180 atgacctaca tgtacgtgtc cgtggtgtgg ggcagggaca acgaggccac cagggccttc 240 ctgtaccaga agggcaacgt gtccaccgcc cactacctga tctccggcac caccctgaac 300 aacgacctgg gcctgatcga gatctcctaa �30 <210> 21 <&11> 917 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide <400> 21 atgtccctga agttcctgga caagcagagg atcggcgcca tcatcgtgga cgccttccag 60 gagcagctga acaccgagat ggacccggcc aacggccagt ggttcaccaa cggcccgggc 120 aacaacggcg gcctgtacgg ctccctggtg gacgccctgg cctcctccct ggtgttcctg 180 ccgaacgagc tggtgctgac cccgcacaag ctggccgccg acaccgccat catcgacaac 240 aggaacggcc tgaccccgaa gtcctccatc accctgtcct actccaccac cgagaccacc 300 accaccaccc acaccgtgtc caacgccctg aaggtgggca tcggcgtgga catcaaggct 360 ccgccaagtt cttcggctcc ggcgtggaca tcaccaccaa gatctccacc gactacacct 420 actcctggtc cgacgccgtg tccaaggccg cctccgagac caagcagttc tcccagaccg 480 tgccggtgga ggtgccgacc ggcagggtgt accaggtggt gctgacctgc gacaagaccg 540 acctgaacgc cccgtactac gccgacgtga ccctgaccgg cacctccacc gccaacttcg 600 ccaacaacgt gaacggcaag aacacctggg tgctggacgc cggcaccctg tgcgagtgga 660 tcaacaggtc cggctccgcc ggcggcgagt cccacatgta cctgagggac ccgcaggtga 720 ccggccaggg cctgatcagg atgaggggct ccctgacctc ctccatcacc gccaacttcg 780 tggtgaacac ctacgacatc accgacacct acaacgccac cggcaaggcc gccatcgaga 840 acgaccacct gttcgccgcc tccgagctgg cctccctgaa ctccaagctg gtgtccgaga 900 aggtgatcgg caagtaa 917 <210> 22 <211> 330 [[ID=1​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​<223> Synthetic polynucleotide <400> 23 atgaccctgc agtacctgga caccctgggc ctgggcgcca tcatcgtgga cgcctggcag 60 aaccacctgg agaccgagaa ggacccggcc ggcggccagt ggttcgccaa cggcccgggc 120 aacaacggcg gcctgttcgg caagctgacc gacaccctgg cctccgagct ggtgctggac 180 gtgccggccc agaccttctc cgtgtaccag tccgccgccg ccaccggcat cgtggacaac 240 aggaacggcc tgaccccgga gcagaccgtg ggcctgtcct gcaccttcca ggacaccgtg 300 accaccaccc actccgtgtc caaggccgtg aagaccggca ccaccgtgtc catcaagggc 360 accatcgacg ccaaggtggt gaagaaggag ttcggcatct ccttcaccgc cgagtactcc 420 cactcctgga ccgacgccac cgccgtgtcc aagtccgagt ccaggtcctt ctccgtgtcc 480 gtgccggtga ggaacgtgcc ggccggcagg gtgtggcagg tggtgctgat ggccaacaag 540 aaggagctgt ccatgccgta cagggccgac atcatcctga agggctccac cgtggccaac 600 ttcctgtccc cgatcagggg ccagaggatc tggcaggccg acgccggcac cctgtgcgag 660 tggatcaaca ggcacggctc cgccggcgac gagtcctggt cctacggcag ggacccggcc 720 gacccgaccc agggcaggat ctccctgctg ggcaccctga aggccgtgca caccgtgaac 780 ttcaccgtga ggaccctgga cgtgaccgag tccttcaggc cggacggcga cggcggcctg 840 gtgctggcca ccaacgccgg ctccgaggcc ccggtggtgg acgaggtgct ggtgaccgag 900 ctggccgccg cctga 915 <210> 24 <211> 333 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Polynucleotide <400> 24 atgtccctgc agaaggtggg caacttctcc ctgcacaacg gcggcggctt cgtggccagg 60 atgaagttcg cctacatcga cgacgagggc cagaagaagt ccaccaggga gaccggcgac 120 atcctgctgg gccagaccaa gaccgccaag ctggaggagt tcgacatccc ggacggcgcc 180 ctggtgtacc tgcacgtgga cgtggtgtgg ggcaaggaca acgaggccgc cagggccttc 240 acctacgaga ggggcaacac ctgcaccgcc gcctacacca tcaccggcac caccctgtcc 300 aacaccctgg gcctgatcga cgtgaactgc tga 333

Claims

1. An insecticidal composition comprising a first component and a second component acting together as an insecticidal toxin, wherein the first component is a peptide composed of SEQ ID NO: 1, and the second component is a peptide composed of SEQ ID NO: 2, and wherein the insecticidal toxin is active against the corn root leaf beetle.

2. An insecticidal composition comprising a first component and a second component acting together as an insecticidal toxin, wherein the first component is a peptide composed of SEQ ID NO: 3, and the second component is a peptide composed of SEQ ID NO: 4, and wherein the insecticidal toxin is active against the corn root leaf beetle.

3. An insecticidal composition comprising a first component and a second component acting together as an insecticidal toxin, wherein the first component is a peptide composed of SEQ ID NO: 3, and the second component is a peptide composed of SEQ ID NO: 2, and wherein the insecticidal toxin is active against the corn root leaf beetle.

4. An insecticidal composition comprising a first component and a second component acting together as an insecticidal toxin, wherein the first component is a peptide composed of SEQ ID NO: 5, and the second component is a peptide composed of SEQ ID NO: 6, and wherein the insecticidal toxin is active against the corn root leaf beetle.

5. The insecticidal composition of claim 2, wherein the insecticidal toxin is active against the longhorn bark leaf beetle.

6. A nucleic acid molecule comprising a nucleotide sequence encoding a first component and a second component as described in any one of claims 1-5.

7. A nucleic acid molecule comprising a nucleotide sequence encoding a first component and a second component as described in any one of claims 1-5 and a heteropromoter, wherein the heteropromoter is operatively linked to the nucleotide sequence encoding the first component and the second component.

8. A nucleic acid molecule comprising a nucleotide sequence encoding a first component and a second component as described in any one of claims 1-5, a first heteropromoter, and a second heteropromoter, wherein the first heteropromoter is operatively linked to the nucleotide sequence encoding the first component and the second heteropromoter is operatively linked to the nucleotide sequence encoding the second component.

9. The nucleic acid molecule of claim 6, wherein the nucleotide sequence comprises one of the following combinations: SEQ ID NO: 7 and 8; SEQ ID NO: 9 and 10; SEQ ID NO: 11 and 12; or SEQ ID NO: 8 and 9.

10. The nucleic acid molecule of claim 6, wherein the nucleotide sequence is a synthetic nucleotide sequence consisting of any one of the following combinations: SEQ ID NO: 13 and 14; SEQ ID NO: 19 and 20; SEQ ID NO: 15 and 16; SEQ ID NO: 17 and 18; SEQ ID NO: 23 and 24; or SEQ ID NO: 14 and 15.

11. The nucleic acid molecule of claim 7 or 8, wherein the heterologous promoter is a plant-expressible promoter.

12. The nucleic acid molecule of claim 11, wherein the plant-expressible promoter is selected from the group of promoters consisting of: ubiquitin, night-blooming jasmine virus, maize TrpA, OsMADS 6, maize H3 histone, 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 mannitol synthase, Ti plasmid annatine synthase, petunia chalcone isomerase, soybean glycine-rich protein 1, potato glycoprotein, lectin, CaMV 35S, and S-E9 small subunit RuBP carboxylase promoter.

13. A recombinant vector comprising the nucleic acid molecule as described in claim 6, 7 or 8.

14. A host cell comprising the recombinant vector as described in claim 13, wherein the host cell is a bacterial cell.

15. The host cell of claim 14, wherein the bacterial cell is from the genera *Bacillus*, *Clostridium*, *Pathogenic Bacillus*, *Luminobacter*, *Pasteurella*, *Escherichia*, *Pseudomonas*, *Erwinia*, *Serratia*, *Klebsiella*, *Salmonella*, *Pasteurella*, *Xanthomonas*, *Streptomyces*, *Rhizobium*, *Sinobacterium sinense*, *Streptococcus*, *Rhizobium sinense*, *Rhizobium*, *Methylophilus*, *Agrobacterium*, *Acetobacter*, *Lactobacillus*, *Arthrobacter*, *Azotobacter*, *Leuconostoc*, or *Alcaligenes*.

16. The host cell of claim 15, wherein the bacterial cell is a Bacillus thuringiensis cell.

17. A method for producing an insecticidal protein, the method comprising culturing a host cell or an organism containing the host cell under conditions in which the host cell produces the insecticidal protein as described in claim 14.

18. A method for producing a transgenic plant or plant part with enhanced insect resistance compared to a control plant or plant part, the method comprising: (a) Introducing into a plant or plant part The nucleic acid molecule of claim 7 or 8, wherein the insecticidal composition of any one of claims 1-5 is expressed in the plant or plant part to produce a plant or plant part with enhanced insect resistance, wherein the enhanced insect resistance is against the maize root firefly beetle or the longhorn bark firefly beetle.

19. The method of claim 18, wherein the introduction step is achieved by: (a) transforming the plant or a plant part; or (b) hybridizing a first plant containing the nucleic acid molecule of claim 7 or 8 with a different second plant.

20. A method for controlling maize root leaf beetle or longhorn bark leaf beetle insect pests, the method comprising delivering an effective amount of the insecticidal composition as described in any one of claims 1-5 to said insect pests or their environment.

21. The method of claim 20, wherein the insecticidal composition of any one of claims 1-5 is delivered via a genetically modified plant or via topical application of a composition comprising the insecticidal composition.

22. The method of claim 21, wherein the transgenic plant or the composition comprises a second insecticide different from the insecticidal composition of any one of claims 1-5.

23. The method of claim 22, wherein the second insecticide is a protein, dsRNA, or a chemical.

24. The method of claim 23, wherein (a) the protein is selected from the group consisting of: Cry protein, Vip protein, potato glycoprotein, protease, protease inhibitor, urease, α-amylase inhibitor, porogen, 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, methamidophos, methomyl, bifenthrin, heptafluthrin, permethrin, deltamethrin, lambda-cyhalothrin, cypermethrin, deltamethrin, chlorpyrifos, oxychloride, dimethoate, ethoprophos, malathion, methyl parathion, phorate, terbufos, terbufos, fipronil, acetamiprid, imidacloprid, thiamethoxam, thiamethoxam, endosulfan, sulfadiazine, and combinations thereof.

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