Truncated chimeric insecticidal proteins

Abstract

The present invention relates to nucleotide sequences encoding novel truncated chimeric pesticidal proteins that exhibit Lepidoptera inhibitory activity. Particular embodiments provide compositions and plants, plant parts, and transformed seeds containing recombinant nucleic acid molecules encoding one or more of the chimeric pesticidal proteins.

Description

Technical Field

[0001] The present invention generally relates to the field of insect inhibitory proteins useful in agriculture. This patent application teaches a novel class of chimeric insecticidal proteins that exhibit inhibitory activity against insect pests associated with cultivated plants and seeds. Specifically, this patent application teaches a class of proteins with insecticidal activity against Lepidoptera insect pests. Also taught are plants, plant parts, and seeds containing recombinant nucleic acid molecules encoding one or more insecticidal proteins. Background Art

[0002] For improving the crop productivity of plant (comprising cotton, soybean, corn, sugarcane, rice and wheat etc.) with agricultural significance, there is growing demand.Except the increasing demand for the agricultural products of food, clothing and energy supply for growing population, climate-related impact and the pressure of growing population using land for the purpose beyond agricultural practice tend to reduce the amount of land that can be used for agriculture.These impacts produce larger demand for the productivity and efficiency of agricultural crops.In view of growing demand, agricultural pest management and control technology are extremely important tools that increase the output of the land that per unit can be used for agriculture.

[0003] Insects, especially those belonging to the order Lepidoptera, are the major cause of damage to agricultural crops, reducing the yield of the affected crops. Lepidoptera pest species that negatively impact agriculture include, for example, Agrotis ipsilon, Agrotis subterranea, Agrotis orthogonia, Alabama argillacea, Anticarsia gemmatalis, Amyelois transitella, Archips argyrospila, Archips rosana, Chilo suppressalis, Chrysodeixis includens, Cnaphalocrocism medinalis, Crambus caliginosellus, Crambus teterrellus, Cydia pomonella, Diatraea spp. saccharalis), Southwestern corn borer (Diatraea grandiosella), cotton leaf roller (Earias vitela), Egyptian borer (Earias insulana), emerald borer (Earias vittella), small corn borer (Elasmopalpus lignosellus), grape borer (Endopizaviteana), pear borer (Grapholita molesta), cotton bollworm (Helicoverpa armigera), South American bollworm (Helicoverpa gelotopeon), American bollworm (Helicoverpa zea), green bollworm (Heliothis virescens), rice leaf cutter (Herpetogramma licarsisalis), American sunflower borer (Homoeosoma electellum), green clover moth (Hypena scabra), grape flower roller (Lobesia botrana), gypsy moth (Lymantria dispar), Mamestra configurata, Ostrinianubilalis, Pectinophora gossypiella, Pieris brassicae, Pieris rapae,rapae), Phyllocnistis citrella (Citrus Leafminer), Plutellaxylostella (Diamondback Moth), Pseudaletia unipuncta (Branchworm), Rachiplusia nu (Two-colored Bearded Moth), Sesamia inferens (Seed Borer), Spodoptera cosmioides (South American Bean Moth), Spodoptera exigua (Beet Armyworm), Spodoptera eridania (Southern Gray Armyworm), Spodoptera frugiperda (Fall Armyworm), Spodoptera litura (Spodoptera litura), Suleima helianthana (Sunflower Bud Moth), Trichoplusia ni (Cheese Looper), and Tuta absoluta (Tomato Leafminer).

[0004] Synthetic chemical pesticides have historically been used to control insect pests in agriculture. However, environmental and human health concerns, as well as emerging resistance issues, have spurred research and development of biopesticides. This research effort has led to the increasing observation and use of several entomopathogenic microbial species, particularly bacteria from the genus Bacillus.

[0005] When the potential of entomopathogenic bacteria, especially those belonging to the genus Bacillus, was revealed, the paradigm of biological control was changed. The identification of the entomopathogenic potential of various strains of Bacillus bacteria, especially Bacillus thuringiensis (Bt), represented a major advance in the art of insect pest control. A large family of proteins with high toxicity to specific insects has been identified in several strains of the bacterium Bt. Among them, delta-endotoxins (e.g., Cry proteins) and secreted insecticidal proteins located within the parasporal crystal inclusions during the establishment of sporulation and the stationary growth phase have been identified. After ingestion, the insecticidal proteins of Bt exert their effects on the surface of the insect's midgut epithelium by destroying the cell membrane, leading to cell rupture and death.

[0006] Bt's secreted and crystalline insecticidal proteins are highly specific for their hosts and have been recognized and accepted worldwide as an important alternative to chemical pesticides. For example, insecticidal proteins are used on a variety of agricultural crops to protect crop plants from insect infestation, reducing the need for chemical pesticides and increasing productivity. Bt insecticidal proteins are used to control agriculturally relevant pests of crop plants by applying microbial agricultural compositions to the plants and using genetic transformation techniques to produce transgenic plants and seeds expressing the insecticidal proteins.

[0007] The global use of insect-resistant transgenic plant crops and the limited number of insecticidal proteins used in these crops has created selection pressure for insect-resistance alleles on the insecticidal proteins currently in use.

[0008] The development of resistance to insecticidal proteins in target insect pests has created a continuing need to identify and develop new proteins to control these resistant insect populations. New insecticidal proteins with improved efficacy and alternative activity profiles against different insect species are important in controlling resistant insects. In addition, the use of two or more insecticidal proteins with different modes of action against the same insect pest in the same transgenic plant reduces the potential for resistance in any target insect species.

[0009] Therefore, there is a continuing need to identify new proteins with improved pesticidal properties, such as efficacy, activity against more than one species of insect pests, and a different mode of action compared to toxins currently used in agricultural practice.

[0010] To address this need, the present invention describes novel chimeric insecticidal Cry proteins that exhibit activity against important species of insect pests.

[0011] The Cry protein naming system based on amino acid similarity was originally proposed by Crickmore, N. et al., 1998. Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62, 807–813, and updated by Crickmore, N. et al., 2021. A structure-based nomenclature for Bacillus thuringiensis and other bacteria-derived pesticidal proteins. Journal of Invertebrate Pathology Vol. 186, (107438).

[0012] In recent classifications, Cry-type proteins represent proteins originally isolated from Bacillus thuringiensis crystals, whose precursor forms consist of two similarly sized halves: an amino-terminal active portion, subdivided into three domains based on conserved or substantially conserved sequence alignments; and a carboxyl-terminal portion, which is known to stabilize crystal formation and exhibit no insecticidal activity. Domain I of the active amino-terminal portion comprises approximately one-third of the active toxin segment and has been shown to be essential for channel formation. Domains II and III of the active amino-terminal portion are both involved in receptor binding and insect species specificity, depending on the insect and the insecticidal protein being examined.

[0013] The possibility of arbitrarily generating chimeric proteins with improved properties from the various domain structures of the large number of natural insecticidal proteins known in the art is minimal. This is a result of the complex and unpredictable nature of the protein structure, oligomerization, and activation (including correct proteolytic processing of the chimeric precursor if expressed in this manner) necessary to release the insecticidal protein segments. The generation of functional chimeric insecticidal toxins with improved insecticidal activity compared to the parent protein from which the chimera is derived involves careful selection of subunits, domains, and specific targets within each parent protein.

[0014] It is known in the art that the reassembly of domains I, II, and III of any two or more toxins that are different from each other generally results in a crystal-formed protein structure that shows a complete absence of any detectable insecticidal activity against a preferred target insect pest species. Effective insecticidal chimeras are designed solely by trial and error, and even then, one skilled in the art cannot guarantee that he will obtain a chimera that shows insecticidal activity that is equivalent to or improved compared to the single parent toxin protein from which the constituent protoxin or chimeric toxin domains may have been derived. For example, the literature reports a large number of examples of constructing or assembling chimeric proteins from two or more crystallized protein precursors. See, for example, Jacqueline S. Knight et al. "A Strategy for Shuffling Numerous Bacillus thuringiensis Crystal Protein Domains". J. Economic Entomology, 97(6)(2004): pp. 1805-1813; U.S. Patent No. 6,204,246; U.S. Patent No. 6,017,534; and U.S. Patent No. 10,233,217. In each of these examples, many of the resulting chimeras did not exhibit equivalent or improved insecticidal or crystal-forming properties compared to the precursor proteins from which the chimera components were derived. Summary of the Invention

[0015] The present invention provides novel truncated chimeric Cry proteins formed by recombination of domains I, II, and III of the active amino-terminal portions of the Bacillus thuringiensis (Bt) Cry proteins Cry1Ab, Cry1B, Cry1C, Cry1Da, Cry1E, Cry1Fb, and Cry2Aa, in the absence of a carboxy-terminal protoxin domain. The truncated chimeric proteins of the present invention have inhibitory and toxic activity against Lepidoptera crop insect pests. Furthermore, the present invention also includes nucleic acid molecules encoding the aforementioned truncated chimeric proteins, expression cassettes containing recombinant nucleic acid molecules encoding one or more truncated chimeric proteins, plant cells, plants, plant parts, and seeds.

[0016] Each of the truncated chimeric insecticidal proteins can be used alone or in combination with other insecticidal proteins and insect inhibitors in formulations and for expression in plants. In this way, alternatives to insecticidal proteins and chemical pesticides used in agricultural systems are provided.

[0017] In certain embodiments described herein, the truncated chimeric insecticidal protein comprises (i) domain I of a Cry protein from any one of SEQ ID NO:98, SEQ ID NO:102, SEQ ID NO:106, SEQ ID NO:111, SEQ ID NO:115, SEQ ID NO:119, or SEQ ID NO:123; (ii) domain II of a Cry protein from any one of SEQ ID NO:99, SEQ ID NO:103, SEQ ID NO:107, SEQ ID NO:112, SEQ ID NO:116, SEQ ID NO:120, or SEQ ID NO:124; and (iii) domain III of a Cry protein from any one of SEQ ID NO:100, SEQ ID NO:104, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:113, SEQ ID NO:117, SEQ ID NO:121, or SEQ ID NO:125, and lacks the carboxyl-terminal protoxin domain.

[0018] In preferred embodiments, the truncated chimeric pesticidal protein comprises an amino acid sequence as established in any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48, and lacks the carboxyl-terminal protoxin domain.

[0019] The chimeric insecticidal protein has inhibitory activity against Lepidoptera insect species, such as but not limited to small cutworms, ground butterfly moths, gray cutworms, cotton leaf corrugation moths, velvet bean moths, navel orange borers, fruit tree yellow moths, rose yellow moths, striped stem borers, soybean moths, rice leaf rollers, dark-striped grass moths, meadow web borers, apple moths, small sugarcane borers, southwestern corn borers, cotton leaf rollers, Egyptian borers, emerald borers, small corn borers, grape borers, pear borers The following are the main insects: cotton bollworm, cotton bollworm, South American cotton bollworm, American cotton bollworm, green cotton bollworm, rice leaf cutter borer, American sunflower borer, green clover armyworm, grape flower moth, gypsy moth, collared armyworm, European corn borer, pink bollworm, cabbage butterfly, cabbage butterfly, citrus leafminer, diamondback moth, shaving moth, two-colored bearded moth, large stem borer, South American bean armyworm, beet armyworm, southern gray armyworm, fall armyworm, armyworm, sunflower bud moth, cabbage looper and tomato leafminer.

[0020] In another embodiment, a polynucleotide encoding a truncated chimeric insecticidal protein is provided, wherein the truncated chimeric insecticidal protein comprises (i) a Cry protein domain I of any one of SEQ ID NO: 98, SEQ ID NO: 102, SEQ ID NO: 106, SEQ ID NO: 111, SEQ ID NO: 115, SEQ ID NO: 119 or SEQ ID NO: 123; (ii) domain II of a Cry protein from any one of SEQ ID NO: 99, SEQ ID NO: 103, SEQ ID NO: 107, SEQ ID NO: 112, SEQ ID NO: 116, SEQ ID NO: 120 or SEQ ID NO: 124; and (iii) domain II of a Cry protein from any one of SEQ ID NO: 100, SEQ ID NO: 104, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 113, SEQ ID NO: 117, SEQ ID NO: 121 or SEQ ID NO: NO:125, wherein the protein lacks the carboxyl-terminal protoxin domain. In a preferred embodiment, the chimeric protein pesticide comprises the amino acid sequence of any one of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48, wherein the amino acid sequence lacks the carboxyl-terminal protoxin domain.Also provided are polynucleotides encoding truncated chimeric insecticidal proteins, wherein the polynucleotide comprises a nucleotide sequence that optionally: hybridizes under stringent conditions to the reverse complement of the polynucleotide sequence of any one of SEQ ID NO: 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96, wherein the polynucleotide lacks the region encoding the carboxyl-terminal protoxin domain; or encodes a truncated chimeric insecticidal protein comprising (i) SEQ ID NO: 98, SEQ ID NO: 102, SEQ ID NO: 106, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120 NO:111, SEQ ID NO:115, SEQ ID NO:119 or SEQ ID NO:123; (ii) domain II of a Cry protein from any one of SEQ ID NO:99, SEQ ID NO:103, SEQ ID NO:107, SEQ ID NO:112, SEQ ID NO:116, SEQ ID NO:120 or SEQ ID NO:124; and (iii) domain III of a Cry protein from any one of SEQ ID NO:100, SEQ ID NO:104, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:113, SEQ ID NO:117, SEQ ID NO:121 or SEQ ID NO:125, wherein the protein lacks a carboxyl-terminal protoxin domain. Preferably, the truncated chimeric insecticidal protein comprises the amino acid sequence of any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48, wherein the amino acid sequence lacks the carboxyl-terminal protoxin domain. In another supplementary embodiment, an expression cassette comprising the polynucleotide defined above is provided.

[0021] In another embodiment, provided herein is a host cell comprising the polynucleotide described above, wherein the host cell is selected from bacterial host cells or plant host cells.Bacterial host cells include Agrobacterium (Agrobacterium), Rhizobium (Rhizobium), Bacillus (Bacillus), Brevibacillus (Brevibacillus), Escherichia (Escherichia), Pseudomonas (Pseudomonas), Klebsiella (Klebsiella), Erwinia (Erwinia), wherein the Bacillus species is Bacillus cereus or Bacillus thuringiensis, Brevibacillus is preferably Brevibacillus laterosporus, and Escherichia is preferably Escherichia coli.The plant cell considered includes monocots and dicots.

[0022] Other embodiments provided herein include insect inhibitor compositions comprising truncated chimeric insecticidal proteins comprising (i) domain I of a Cry protein of any one of SEQ ID NO: 98, SEQ ID NO: 102, SEQ ID NO: 106, SEQ ID NO: 111, SEQ ID NO: 115, SEQ ID NO: 119, or SEQ ID NO: 123; (ii) domain II of a Cry protein from any one of SEQ ID NO: 99, SEQ ID NO: 103, SEQ ID NO: 107, SEQ ID NO: 112, SEQ ID NO: 116, SEQ ID NO: 120, or SEQ ID NO: 124; and (iii) domain II of a Cry protein from any one of SEQ ID NO: 100, SEQ ID NO: 104, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 113, SEQ ID NO: 117, SEQ ID NO: 121, or SEQ ID NO: 123. NO:125, wherein the protein lacks the carboxyl-terminal protoxin domain; preferably, the truncated chimeric protein comprises the amino acid sequence of any one of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48, wherein the amino acid sequence lacks the carboxyl-terminal protoxin domain. In certain embodiments, the insect inhibitor composition further comprises at least one insect inhibitor different from the chimeric insecticidal protein. Insect inhibitors contemplated in addition to chimeric insecticidal proteins include insect inhibitor proteins, insect inhibitor dsRNA molecules and insect inhibitor chemicals. These insect inhibitors, except for the chimeric insecticidal proteins, may exhibit activity against one or more pest species of the orders Lepidoptera, Coleoptera, Hemiptera, Homoptera, or Thysanoptera.

[0023] In another embodiment disclosed herein, the seed comprises an effective insect-inhibiting amount of: a chimeric insecticidal protein comprising (i) domain I of a Cry protein of any one of SEQ ID NO: 98, SEQ ID NO: 102, SEQ ID NO: 106, SEQ ID NO: 111, SEQ ID NO: 115, SEQ ID NO: 119, or SEQ ID NO: 123; (ii) domain II of a Cry protein from any one of SEQ ID NO: 99, SEQ ID NO: 103, SEQ ID NO: 107, SEQ ID NO: 112, SEQ ID NO: 116, SEQ ID NO: 120, or SEQ ID NO: 124; and (iii) domain II of a Cry protein from any one of SEQ ID NO: 100, SEQ ID NO: 104, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 113, SEQ ID NO: 117, SEQ ID NO: 121, or SEQ ID NO: 123. NO:125 any one of the Cry protein domain III, wherein the protein lacks the carboxyl terminal protoxin domain; preferably, the truncated chimeric protein comprises SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48 any one of the amino acid sequence, wherein the amino acid sequence lacks the carboxyl terminal protoxin domain; or a polynucleotide, which comprises SEQ ID NO:49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 or 96, wherein the polynucleotide lacks the region encoding the carboxyl-terminal protoxin domain.

[0024] The method of controlling lepidopteran pests further comprises contacting the lepidopteran pests with an inhibitory amount of a chimeric pesticidal protein of the present invention.

[0025] In another embodiment, a plant genome is provided, comprising a nucleic acid molecule encoding a truncated chimeric insecticidal protein according to the present invention or an expression cassette containing the nucleic acid molecule.

[0026] The present invention further provides a plant cell, plant or part of a transgenic plant comprising a chimeric insecticidal protein or a nucleic acid molecule encoding the same, wherein: the truncated chimeric insecticidal protein comprises (i) a Cry protein domain I of any one of SEQ ID NO: 98, SEQ ID NO: 102, SEQ ID NO: 106, SEQ ID NO: 111, SEQ ID NO: 115, SEQ ID NO: 119 or SEQ ID NO: 123; (ii) domain II of a Cry protein from any one of SEQ ID NO: 99, SEQ ID NO: 103, SEQ ID NO: 107, SEQ ID NO: 112, SEQ ID NO: 116, SEQ ID NO: 120 or SEQ ID NO: 124; and (iii) domain II of a Cry protein from any one of SEQ ID NO: 100, SEQ ID NO: 104, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 113, SEQ ID NO: 117, SEQ ID NO: 121 or SEQ ID NO: 123. NO:125, wherein the protein lacks the carboxyl-terminal protoxin domain; preferably, the truncated chimeric protein comprises SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48, wherein the amino acid sequence lacks the carboxyl-terminal protoxin domain.

[0027] In another embodiment, a chimeric insecticidal protein is provided which: has at least 85% identity to SEQ ID NO: 1, at least 80% identity to SEQ ID NO: 2, at least 79% identity to SEQ ID NO: 3, at least 82% identity to SEQ ID NO: 4, at least 79% identity to SEQ ID NO: 5, at least 87% identity to SEQ ID NO: 6, at least 91% identity to SEQ ID NO: 7, at least 77% identity to SEQ ID NO: 8, at least 81% identity to SEQ ID NO: 9, at least 82% identity to SEQ ID NO: 10, at least 76% identity to SEQ ID NO: 11, at least 91% identity to SEQ ID NO: 12, at least 92% identity to SEQ ID NO: 13, at least 86% identity to SEQ ID NO: 14, at least 82% identity to SEQ ID NO: 15, at least 88% identity to SEQ ID NO: 16, at least 89% identity to SEQ ID NO: 17, at least 91% identity to SEQ ID NO: 18, at least 92% identity to SEQ ID NO: 19, at least 93% identity to SEQ ID NO: 20 SEQ ID NO: 17 is at least 97% identical to SEQ ID NO: 18, at least 81% identical to SEQ ID NO: 19, at least 86% identical to SEQ ID NO: 20, at least 76% identical to SEQ ID NO: 21, at least 78% identical to SEQ ID NO: 22, at least 78% identical to SEQ ID NO: 23, at least 86% identical to SEQ ID NO: 24, at least 85% identical to SEQ ID NO: 25, at least 83% identical to SEQ ID NO: 26, at least 83% identical to SEQ ID NO: 27, at least 85% identical to SEQ ID NO: 28, at least 79% identical to SEQ ID NO: 29, at least 85% identical to SEQ ID NO: 30, at least 80% identical to SEQ ID NO: 31, at least 84% identical to SEQ ID NO: 33, at least 86% identical to SEQ ID NO: 34, at least 87% identical to SEQ ID NO: 35 NO:33 is at least 78% identical to SEQ ID NO:33, is at least 85% identical to SEQ ID NO:34, is at least 80% identical to SEQ ID NO:35, is at least 87% identical to SEQ ID NO:36, is at least 93% identical to SEQ ID NO:37, is at least 84% identical to SEQ ID NO:38, is at least 81% identical to SEQ ID NO:39, is at least 80% identical to SEQ ID NO:40, is at least 84% identical to SEQ ID NO:41,At least 93% identical to SEQ ID NO: 42, at least 86% identical to SEQ ID NO: 43, at least 70% identical to SEQ ID NO: 44, at least 75% identical to SEQ ID NO: 45, at least 73% identical to SEQ ID NO: 46, at least 50% identical to SEQ ID NO: 47, or at least 50% identical to SEQ ID NO: 48, wherein the amino acid sequence lacks the carboxyl-terminal protoxin domain. Also provided are nucleic acid molecules encoding the aforementioned proteins, expression cassettes containing recombinant nucleic acid molecules encoding one or more chimeric proteins provided herein, plant cells, plants, plant parts, and seeds.

[0028] Also provided are methods of controlling a lepidopteran pest comprising exposing the pest to a truncated chimeric protein described herein, a plant cell, a plant, or a part of a transgenic plant, wherein the plant cell, plant, or part of the plant expresses the truncated chimeric protein described herein.

[0029] In other embodiments described herein, plant products obtained from plant cells, plants, or parts of plants are provided, wherein the product comprises a detectable amount of a chimeric insecticidal protein. Plant products include plant biomass, oils, bran, animal feed, flour, chips, hulls, and processed seeds.

[0030] Another method described herein is a method of producing seeds comprising a chimeric pesticidal protein, the method comprising: planting at least one seed comprising a chimeric pesticidal protein; growing a plant from the seed; and harvesting seeds from the plant, wherein the harvested seeds comprise the chimeric pesticidal protein.

[0031] A recombinant polynucleotide molecule encoding a truncated chimeric insecticidal protein comprises a nucleotide sequence of any one of SEQ ID NO: 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 or 96, wherein the polynucleotide lacks the region encoding the carboxyl-terminal protoxin domain; and optionally, a polynucleotide sequence encoding an insect inhibitor other than the chimeric insecticidal protein is also contemplated herein.

[0032] Another recombinant nucleic acid molecule contemplated herein comprises a heterologous promoter operably linked to a polynucleotide segment encoding a chimeric insecticidal protein, wherein: the truncated chimeric insecticidal protein comprises (i) a Cry protein domain I of any one of SEQ ID NO: 98, SEQ ID NO: 102, SEQ ID NO: 106, SEQ ID NO: 111, SEQ ID NO: 115, SEQ ID NO: 119, or SEQ ID NO: 123; (ii) domain II of a Cry protein from any one of SEQ ID NO: 99, SEQ ID NO: 103, SEQ ID NO: 107, SEQ ID NO: 112, SEQ ID NO: 116, SEQ ID NO: 120, or SEQ ID NO: 124; and (iii) domain II of a Cry protein from any one of SEQ ID NO: 100, SEQ ID NO: 104, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 113, SEQ ID NO: 117, SEQ ID NO: 121, or SEQ ID NO: 123. NO:125, wherein the protein lacks the carboxyl-terminal protoxin domain; or a truncated chimeric insecticidal protein comprising a protein having at least 85% identity to SEQ ID NO: 1, at least 80% identity to SEQ ID NO: 2, at least 79% identity to SEQ ID NO: 3, at least 82% identity to SEQ ID NO: 4, at least 79% identity to SEQ ID NO: 5, at least 87% identity to SEQ ID NO: 6, at least 91% identity to SEQ ID NO: 7, at least 77% identity to SEQ ID NO: 8, at least 81% identity to SEQ ID NO: 9, at least 82% identity to SEQ ID NO: 10, at least 76% identity to SEQ ID NO: 11, at least 91% identity to SEQ ID NO: 12, at least 92% identity to SEQ ID NO: 13, at least 86% identity to SEQ ID NO: 14, at least 87% identity to SEQ ID NO: 15, at least 88% identity to SEQ ID NO: 16, at least 89% identity to SEQ ID NO: 17, at least 91% identity to SEQ ID NO: 18, at least 92% identity to SEQ ID NO: 19, at least 93% identity to SEQ ID NO: 20, at least 94% identity to SEQ ID NO: 21, at least 95% identity to SEQ ID NO: 22 SEQ ID NO: 15 is at least 82% identical to SEQ ID NO: 16, at least 88% identical to SEQ ID NO: 17, at least 97% identical to SEQ ID NO: 17, at least 81% identical to SEQ ID NO: 18, at least 86% identical to SEQ ID NO: 19, at least 76% identical to SEQ ID NO: 20, at least 78% identical to SEQ ID NO: 21, at least 78% identical to SEQ ID NO: 22,at least 78% identical to SEQ ID NO:23, at least 86% identical to SEQ ID NO:24, at least 85% identical to SEQ ID NO:25, at least 83% identical to SEQ ID NO:26, at least 83% identical to SEQ ID NO:27, at least 85% identical to SEQ ID NO:28, at least 79% identical to SEQ ID NO:29, at least 85% identical to SEQ ID NO:30, at least 80% identical to SEQ ID NO:31, at least 84% identical to SEQ ID NO:33, at least 78% identical to SEQ ID NO:33, at least 85% identical to SEQ ID NO:34, at least 80% identical to SEQ ID NO:35, at least 87% identical to SEQ ID NO:36, at least 93% identical to SEQ ID NO:37, at least 84% identical to SEQ ID NO:38, at least 86% identical to SEQ ID NO:39. NO:39 is at least 81% identical to SEQ ID NO:39, at least 80% identical to SEQ ID NO:40, at least 84% identical to SEQ ID NO:41, at least 93% identical to SEQ ID NO:42, at least 86% identical to SEQ ID NO:43, at least 70% identical to SEQ ID NO:44, at least 75% identical to SEQ ID NO:45, at least 73% identical to SEQ ID NO:46, at least 50% identical to SEQ ID NO:47 or at least 50% identical to SEQ ID NO:48; or the polynucleotide segment is at least 50% identical to SEQ ID NO:49; 94, 95, or 96, wherein the polynucleotide lacks the region encoding the carboxyl-terminal protoxin domain.

[0033] Other embodiments, features, and advantages of the present invention will become apparent from the following detailed description, examples, and claims.

[0034] Brief description of sequence

[0035] SEQ ID NO: 1 is the amino acid sequence of the truncated chimeric protein EMS_Q1.

[0036] SEQ ID NO: 2 is the amino acid sequence of the truncated chimeric protein EMS_Q2.

[0037] SEQ ID NO: 3 is the amino acid sequence of the truncated chimeric protein EMS_Q3.

[0038] SEQ ID NO: 4 is the amino acid sequence of the truncated chimeric protein EMS_Q4.

[0039] SEQ ID NO: 5 is the amino acid sequence of the truncated chimeric protein EMS_Q5.

[0040] SEQ ID NO: 6 is the amino acid sequence of the truncated chimeric protein EMS_Q6.

[0041] SEQ ID NO: 7 is the amino acid sequence of the truncated chimeric protein EMS_Q7.

[0042] SEQ ID NO: 8 is the amino acid sequence of the truncated chimeric protein EMS_Q8.

[0043] SEQ ID NO: 9 is the amino acid sequence of the truncated chimeric protein EMS_Q9.

[0044] SEQ ID NO: 10 is the amino acid sequence of the truncated chimeric protein EMS_Q10.

[0045] SEQ ID NO: 11 is the amino acid sequence of the truncated chimeric protein EMS_Q11.

[0046] SEQ ID NO: 12 is the amino acid sequence of the truncated chimeric protein EMS_Q12.

[0047] SEQ ID NO: 13 is the amino acid sequence of the truncated chimeric protein EMS_Q13.

[0048] SEQ ID NO: 14 is the amino acid sequence of the truncated chimeric protein EMS_Q14.

[0049] SEQ ID NO: 15 is the amino acid sequence of the truncated chimeric protein EMS_Q15.

[0050] SEQ ID NO: 16 is the amino acid sequence of the truncated chimeric protein EMS_Q16.

[0051] SEQ ID NO: 17 is the amino acid sequence of the truncated chimeric protein EMS_Q17.

[0052] SEQ ID NO: 18 is the amino acid sequence of the truncated chimeric protein EMS_Q18.

[0053] SEQ ID NO: 19 is the amino acid sequence of the truncated chimeric protein EMS_Q19.

[0054] SEQ ID NO: 20 is the amino acid sequence of the truncated chimeric protein EMS_Q20.

[0055] SEQ ID NO: 21 is the amino acid sequence of the truncated chimeric protein EMS_Q21.

[0056] SEQ ID NO: 22 is the amino acid sequence of the truncated chimeric protein EMS_Q22.

[0057] SEQ ID NO: 23 is the amino acid sequence of the truncated chimeric protein EMS_Q23.

[0058] SEQ ID NO: 24 is the amino acid sequence of the truncated chimeric protein EMS_Q24.

[0059] SEQ ID NO: 25 is the amino acid sequence of the truncated chimeric protein EMS_Q25.

[0060] SEQ ID NO: 26 is the amino acid sequence of the truncated chimeric protein EMS_Q26.

[0061] SEQ ID NO: 27 is the amino acid sequence of the truncated chimeric protein EMS_Q27.

[0062] SEQ ID NO: 28 is the amino acid sequence of the truncated chimeric protein EMS_Q28.

[0063] SEQ ID NO: 29 is the amino acid sequence of the truncated chimeric protein EMS_Q29.

[0064] SEQ ID NO: 30 is the amino acid sequence of the truncated chimeric protein EMS_Q30.

[0065] SEQ ID NO: 31 is the amino acid sequence of the truncated chimeric protein EMS_Q31.

[0066] SEQ ID NO: 32 is the amino acid sequence of the truncated chimeric protein EMS_Q32.

[0067] SEQ ID NO: 33 is the amino acid sequence of the truncated chimeric protein EMS_Q33.

[0068] SEQ ID NO: 34 is the amino acid sequence of the truncated chimeric protein EMS_Q34.

[0069] SEQ ID NO: 35 is the amino acid sequence of the truncated chimeric protein EMS_Q35.

[0070] SEQ ID NO: 36 is the amino acid sequence of the truncated chimeric protein EMS_Q36.

[0071] SEQ ID NO: 37 is the amino acid sequence of the truncated chimeric protein EMS_Q37.

[0072] SEQ ID NO: 38 is the amino acid sequence of the truncated chimeric protein EMS_Q38.

[0073] SEQ ID NO: 39 is the amino acid sequence of the truncated chimeric protein EMS_Q39.

[0074] SEQ ID NO:40 is the amino acid sequence of the truncated chimeric protein EMS_Q40.

[0075] SEQ ID NO:41 is the amino acid sequence of the truncated chimeric protein EMS_Q41.

[0076] SEQ ID NO:42 is the amino acid sequence of the truncated chimeric protein EMS_Q42.

[0077] SEQ ID NO:43 is the amino acid sequence of the truncated chimeric protein EMS_Q43.

[0078] SEQ ID NO:44 is the amino acid sequence of the truncated chimeric protein EMS_Q44.

[0079] SEQ ID NO:45 is the amino acid sequence of the truncated chimeric protein EMS_Q45.

[0080] SEQ ID NO:46 is the amino acid sequence of the truncated chimeric protein EMS_Q46.

[0081] SEQ ID NO: 47 is the amino acid sequence of the truncated chimeric protein EMS_Q47.

[0082] SEQ ID NO:48 is the amino acid sequence of the truncated chimeric protein EMS_Q48.

[0083] SEQ ID NO: 49 is the nucleotide sequence encoding the truncated chimeric protein EMS_1.

[0084] SEQ ID NO: 50 is the nucleotide sequence encoding the truncated chimeric protein EMS_2.

[0085] SEQ ID NO: 51 is the nucleotide sequence encoding the truncated chimeric protein EMS_3.

[0086] SEQ ID NO: 52 is the nucleotide sequence encoding the truncated chimeric protein EMS_4.

[0087] SEQ ID NO: 53 is the nucleotide sequence encoding the truncated chimeric protein EMS_5.

[0088] SEQ ID NO: 54 is the nucleotide sequence encoding the truncated chimeric protein EMS_6.

[0089] SEQ ID NO: 55 is the nucleotide sequence encoding the truncated chimeric protein EMS_7.

[0090] SEQ ID NO: 56 is the nucleotide sequence encoding the truncated chimeric protein EMS_8.

[0091] SEQ ID NO: 57 is the nucleotide sequence encoding the truncated chimeric protein EMS_9.

[0092] SEQ ID NO: 58 is the nucleotide sequence encoding the truncated chimeric protein EMS_10.

[0093] SEQ ID NO: 59 is the nucleotide sequence encoding the truncated chimeric protein EMS_11.

[0094] SEQ ID NO: 60 is the nucleotide sequence encoding the truncated chimeric protein EMS_12.

[0095] SEQ ID NO: 61 is the nucleotide sequence encoding the truncated chimeric protein EMS_13.

[0096] SEQ ID NO: 62 is the nucleotide sequence encoding the truncated chimeric protein EMS_14.

[0097] SEQ ID NO: 63 is the nucleotide sequence encoding the truncated chimeric protein EMS_15.

[0098] SEQ ID NO: 64 is the nucleotide sequence encoding the truncated chimeric protein EMS_16.

[0099] SEQ ID NO: 65 is the nucleotide sequence encoding the truncated chimeric protein EMS_17.

[0100] SEQ ID NO: 66 is the nucleotide sequence encoding the truncated chimeric protein EMS_18.

[0101] SEQ ID NO: 67 is the nucleotide sequence encoding the truncated chimeric protein EMS_19.

[0102] SEQ ID NO: 68 is the nucleotide sequence encoding the truncated chimeric protein EMS_20.

[0103] SEQ ID NO: 69 is the nucleotide sequence encoding the truncated chimeric protein EMS_21.

[0104] SEQ ID NO: 70 is the nucleotide sequence encoding the truncated chimeric protein EMS_22.

[0105] SEQ ID NO: 71 is the nucleotide sequence encoding the truncated chimeric protein EMS_23.

[0106] SEQ ID NO: 72 is the nucleotide sequence encoding the truncated chimeric protein EMS_24.

[0107] SEQ ID NO: 73 is the nucleotide sequence encoding the truncated chimeric protein EMS_25.

[0108] SEQ ID NO: 74 is the nucleotide sequence encoding the truncated chimeric protein EMS_26.

[0109] SEQ ID NO: 75 is the nucleotide sequence encoding the truncated chimeric protein EMS_27.

[0110] SEQ ID NO: 76 is the nucleotide sequence encoding the truncated chimeric protein EMS_28.

[0111] SEQ ID NO: 77 is the nucleotide sequence encoding the truncated chimeric protein EMS_29.

[0112] SEQ ID NO: 78 is the nucleotide sequence encoding the truncated chimeric protein EMS_30.

[0113] SEQ ID NO: 79 is the nucleotide sequence encoding the truncated chimeric protein EMS_31.

[0114] SEQ ID NO: 80 is the nucleotide sequence encoding the truncated chimeric protein EMS_32.

[0115] SEQ ID NO: 81 is the nucleotide sequence encoding the truncated chimeric protein EMS_33.

[0116] SEQ ID NO: 82 is the nucleotide sequence EMS_34 encoding the truncated chimeric protein.

[0117] SEQ ID NO: 83 is the nucleotide sequence encoding the truncated chimeric protein EMS_35.

[0118] SEQ ID NO: 84 is the nucleotide sequence EMS_36 encoding the truncated chimeric protein.

[0119] SEQ ID NO: 85 is the nucleotide sequence encoding the truncated chimeric protein EMS_37.

[0120] SEQ ID NO: 86 is the nucleotide sequence encoding the truncated chimeric protein EMS_38.

[0121] SEQ ID NO: 87 is the nucleotide sequence encoding the truncated chimeric protein EMS_39.

[0122] SEQ ID NO: 88 is the nucleotide sequence encoding the truncated chimeric protein EMS_40.

[0123] SEQ ID NO: 89 is the nucleotide sequence encoding the truncated chimeric protein EMS_41.

[0124] SEQ ID NO:90 is the nucleotide sequence encoding the truncated chimeric protein EMS_42.

[0125] SEQ ID NO:91 is the nucleotide sequence encoding the truncated chimeric protein EMS_43.

[0126] SEQ ID NO:92 is the nucleotide sequence encoding the truncated chimeric protein EMS_44.

[0127] SEQ ID NO:93 is the nucleotide sequence encoding the truncated chimeric protein EMS_45.

[0128] SEQ ID NO:94 is the nucleotide sequence EMS_46 encoding the truncated chimeric protein.

[0129] SEQ ID NO:95 is the nucleotide sequence encoding the truncated chimeric protein EMS_47.

[0130] SEQ ID NO:96 is the nucleotide sequence encoding the truncated chimeric protein EMS_48.

[0131] SEQ ID NO: 97 is the amino acid sequence of the parent protein Cry1C.

[0132] SEQ ID NO:98 is the amino acid sequence of domain 1 of Cry1C.

[0133] SEQ ID NO: 99 is the amino acid sequence of domain II of Cry1C.

[0134] SEQ ID NO: 100 is the amino acid sequence of domain III of Cry1C.

[0135] SEQ ID NO: 101 is the amino acid sequence of the parent protein Cry1B.

[0136] SEQ ID NO: 102 is the amino acid sequence of domain I of Cry1B.

[0137] SEQ ID NO: 103 is the amino acid sequence of domain II of Cry1B.

[0138] SEQ ID NO: 104 is the amino acid sequence of domain III of Cry1B.

[0139] SEQ ID NO: 105 is the amino acid sequence of the parent protein Cry1Ab.

[0140] SEQ ID NO: 106 is the amino acid sequence of domain 1 of Cry1Ab.

[0141] SEQ ID NO: 107 is the amino acid sequence of domain II of CrylAb.

[0142] SEQ ID NO: 108 is the amino acid sequence of the III_a domain of Cry1Ab.

[0143] SEQ ID NO: 109 is the amino acid sequence of the III_b domain of Cry1Ab.

[0144] SEQ ID NO: 110 is the amino acid sequence of the parent protein Cry1E.

[0145] SEQ ID NO: 111 is the amino acid sequence of domain I of Cry1E.

[0146] SEQ ID NO: 112 is the amino acid sequence of domain II of Cry1E.

[0147] SEQ ID NO: 113 is the amino acid sequence of domain III of Cry1E.

[0148] SEQ ID NO: 114 is the amino acid sequence of the parent protein Cry1Da.

[0149] SEQ ID NO: 115 is the amino acid sequence of domain I of Cry1Da.

[0150] SEQ ID NO: 116 is the amino acid sequence of domain II of Cry1Da.

[0151] SEQ ID NO: 117 is the amino acid sequence of domain III of Cry1Da.

[0152] SEQ ID NO: 118 is the amino acid sequence of the parent protein Cry1Fb.

[0153] SEQ ID NO: 119 is the amino acid sequence of domain 1 of Cry1Fb.

[0154] SEQ ID NO: 120 is the amino acid sequence of domain II of Cry1Fb.

[0155] SEQ ID NO: 121 is the amino acid sequence of domain III of Cry1Fb.

[0156] SEQ ID NO: 122 is the amino acid sequence of the parent protein Cry2Aa.

[0157] SEQ ID NO: 123 is the amino acid sequence of domain 1 of Cry2Aa.

[0158] SEQ ID NO: 124 is the amino acid sequence of domain II of Cry2Aa.

[0159] SEQ ID NO: 125 is the amino acid sequence of domain III of Cry2Aa. BRIEF DESCRIPTION OF THE DRAWINGS

[0160] Figure 1-4 The test results reported in Example 5 are shown.

[0161] Figure 1 : Graphical results of the mean injury scores (Davis Scale) across three evaluations (7, 14, and 21 days after infestation) in a greenhouse assay manually infested with Spodoptera frugiperda in 44 pots of maize events containing the chimeric protein coding sequence EMS_Q6 (SEQ ID NO: 6). Non-transgenic maize L3 was added as a negative control (C-), and commercial transgenic maize resistant to Spodoptera frugiperda was added as a positive control (C+).

[0162] Figure 2 : Graphical results of the mean injury scores (Davis Scale) between three evaluations (7, 14, and 21 days after infestation) of 22 events (Ev1 to Ev22) of corn containing the chimeric protein coding sequence EMS_Q6 (SEQ ID NO: 6) in a field trial artificially infested with Spodoptera frugiperda. Two non-transgenic corn (C-) and two commercial transgenic corn (C+) were evaluated as negative and positive controls, respectively.

[0163] Figure 3: Representative results of field trials with artificial and natural infestations of Spodoptera frugiperda in (A) transgenic maize expressing the chimeric protein EMS_Q6 (SEQ ID NO: 6) and (B) non-transgenic maize controls. Note that the transgenic maize plants showed no damage, while conventional maize plants showed leaf damage caused by attack by Spodoptera frugiperda.

[0164] Figure 4 Results of a laboratory bioassay using corn leaves 5 days after infestation with neonate larvae of Spodoptera frugiperda. (A) Transgenic corn expressing the chimeric protein EMS_Q6 (SEQ ID NO: 6) and (B) non-transgenic control corn (C-). Note that in the corn leaves expressing the EMS_Q6 protein, all caterpillars died and there was no leaf damage. In the control corn, feeding damage was evident, and live, well-developed caterpillars were present. DETAILED DESCRIPTION

[0165] A problem in agricultural insect pest control technology can be identified as the need for new insecticidal proteins that are effective against target insect pests, exhibit a broad spectrum of toxicity against target insect pest species, have the ability to be expressed in plants without causing adverse agronomic problems, and provide an alternative mode of action compared to proteins currently used in agriculture. The truncated chimeric insecticidal proteins disclosed herein address each of these needs, particularly against a broad spectrum of lepidopteran insect pests.

[0166] In order to prevent the development of insect resistance to currently used insecticidal proteins or to avoid insect resistance to currently used insecticidal proteins, novel insecticidal proteins with different modes of action and broad spectrum and efficacy are necessary for controlling Lepidoptera. One way to address this demand is to exchange segments between several Bt proteins that show structural similarity to produce novel Bt proteins with insect inhibitory properties. However, the possibility of producing chimeric proteins with improved properties from the various domain structures of a large number of natural insecticidal proteins known in the art is extremely low. See, for example, Jacqueline S. Knight et al. "A Strategy for Shuffling Numerous Bacillus thuringiensis Crystal Protein Domains". J. Economic Entomology, 97 (6) (2004): pp. 1805 to 1813.

[0167] Disclosed herein are sequences of recombinant nucleic acid molecules encoding novel chimeric insecticidal proteins. These insecticidal proteins address the ongoing need in the art for additional toxic insecticidal proteins with improved insecticidal properties, such as increased efficacy, activity against a broader spectrum of target insect pest species, and a different mode of action. Members of this group of proteins, including those exemplified and provided herein, exhibit insecticidal activity against Lepidoptera insect pest species.

[0168] The terms "segment" or "fragment" are used herein to describe a contiguous amino acid or nucleic acid sequence that is shorter than the full-length amino acid or nucleic acid sequence describing a disclosed chimeric insecticidal protein. If alignment of a segment or fragment that exhibits insect inhibitory activity with the corresponding portion of the chimeric insecticidal protein results in any fractional percentage of amino acid sequence identity between the segment or fragment and the corresponding portion of the chimeric insecticidal protein from about 50% to about 100%, then such segment or fragment is also disclosed herein.

[0169] References to the terms "active" or "activity"; "pesticidal activity" or "pesticide" or "insecticidal activity," "insect inhibitor," "insecticide" in this application refer to the ability of a toxic agent, such as a pesticidal protein, to inhibit (inhibit growth, feeding, egg production, or viability), prevent (in a specific crop containing an effective amount of the pesticidal protein, prevent pest infestation, prevent pest feeding activity), or kill (cause disease, mortality, or reduced egg production) a pest. These terms are intended to include the results of providing an effective amount of the pesticidal protein to the pest, wherein exposure of the pest to the pesticidal protein results in disease, mortality, reduced egg production, or growth retardation. These terms also include the repulsion of plant pests from plant tissues, plant parts, seeds, plant cells, or a specific geographic location where the plant can be cultivated due to the provision of an effective amount of the pesticidal protein as a pesticide in or on the plant. Generally speaking, insecticidal activity refers to the ability of an insecticidal protein to effectively inhibit the growth, development, viability, feeding behavior, mating behavior, egg-laying capacity, or any measurable reduction in side effects caused by the insects feeding on the protein, protein fragment, protein segment, or polynucleotide of a specific target pest (including but not limited to insects of the order Lepidoptera). The insecticidal protein can be produced by a plant, or it can be provided to the plant or the environment within the location in which the plant is located. The terms "biological activity," "efficacy," or variations thereof are also terms used interchangeably in this application to describe the effects of the truncated chimeric insecticidal proteins of the present invention on target insect pests.

[0170] The term "truncated" refers to an amino acid sequence of a protein that lacks the carboxyl terminal portion of the protoxin present in the native and / or parent Cry1 and Cry2 proteins compared to the corresponding native and / or parent sequence. The term "truncated" also refers to a nucleotide sequence that encodes a truncated protein and thus lacks the region encoding the carboxyl terminal protoxin domain.

[0171] When provided in the diet of target pests, the toxicant of insecticidal effective amount shows insecticidal activity when the toxicant contacts the pest. The toxicant can be an insecticidal protein or one or more chemicals known in the art. Insecticide chemicals and insecticidal protein agents can be used alone or in combination with each other. Chemical agents include but are not limited to dsRNA molecules, organochlorides, organophosphates, carbamates, pyrethroids, neonicotinoids and ryanodine classes of target-specific genes for suppressing target pests. Insecticide protein agents include chimeric insecticidal proteins provided by the application, and other toxic protein agents, including those of target lepidopteran pest species, and protein toxins for controlling other plant pests, such as Cry proteins of species that can be used to control Coleoptera, Thysanoptera, Hemiptera and Homoptera in this area.

[0172] Reference to pests, particularly agricultural pests, refers to crop insect pests, particularly Lepidoptera pests, which are controlled by the truncated chimeric insecticidal proteins disclosed herein. However, reference to pests may also include plant pests of the orders Coleoptera, Hemiptera, and Homoptera, as well as nematodes, when a toxicant for these pests is combined or present with the truncated chimeric insecticidal protein or a protein having 50% to about 100% identity to the truncated chimeric insecticidal protein.

[0173] The truncated chimeric insecticidal proteins described herein show insecticidal activity against insect pests from Lepidoptera species (including adults, pupae, larvae and neonates) and Hemiptera species (including adults and nymphs). Lepidoptera insects include, but are not limited to, cutworms, ground moths, gray cutworms, cotton leaf moths, velvet bean moths, navel orange borers, fruit tree yellow moths, rose yellow moths, striped stem borers, soybean moths, rice leaf rollers, dark-striped grass borers, meadow web borers, codling moths, small sugarcane borers, southwestern corn borers, cotton leaf rollers, Egyptian borers, green borers, small corn borers, grape borers, pear borers, cotton bollworms, South American cotton borers, bollworm, American bollworm, green bollworm, rice leaf cutter borer, American sunflower borer, green clover armyworm, grape flower roller, gypsy moth, collared leafworm, European corn borer, pink bollworm, large cabbage butterfly, cabbage butterfly, citrus leafminer, diamondback moth, shaving moth, two-colored bearded moth, large stem borer, South American bean armyworm, beet armyworm, southern armyworm, fall armyworm, armyworm, sunflower bud moth, cabbage looper and tomato leafminer.

[0174] Reference to an "isolated DNA molecule" or equivalent terms or phrases in this application means that the DNA molecule exists alone or in combination with other compositions, but is not in its natural environment. For example, nucleic acid elements naturally present in the DNA of the genome of an organism, such as coding sequences, intron sequences, untranslated primary sequences, promoter sequences, transcription termination sequences, etc., are not considered "isolated" because the elements are within the genome of the organism and in their naturally occurring location within the genome. However, each of these elements, as well as sub-portions of these elements, will be "isolated" within the scope of this document so long as the elements are not within the genome of the organism and in their naturally occurring location within the genome. Similarly, a nucleotide sequence encoding an insecticidal protein or any naturally occurring insecticidal variant of the protein is an isolated nucleotide sequence, provided that the nucleotide sequence is not within the DNA of the bacterium in which the sequence encoding the protein is naturally present. For the purposes of this document, a sequence of synthetic nucleotides encoding the amino acid sequence of a naturally occurring insecticidal protein will be considered isolated. For the purposes herein, any recombinant nucleotide sequence, such as a DNA nucleotide sequence inserted into the genome of a cell of a plant or bacterium or present in an extrachromosomal vector, is considered an isolated nucleotide sequence if it is present within a plasmid or similar construct used to transform a cell, is present within the genome of a plant or bacterium, or is present in detectable amounts in a tissue, progeny, biological sample, or consumable product obtained from the plant or bacterium.

[0175] As further described in the Examples, through chimeric sequence development work, approximately forty-eight (48) nucleotide sequences encoding truncated chimeric insecticidal proteins were constructed from the toxin domains of known insecticidal proteins (referred to herein as "parent proteins"), expressed, and tested for activity against Lepidoptera in bioassays. The truncated chimeric proteins constructed according to the teachings herein exhibited superior activity in controlling Lepidoptera compared to the ancestral proteins from which their toxin components were derived.

[0176] These chimeric insecticidal proteins with enhanced lepidopteran activity or improved lepidopteran spectrum were constructed from the following amino-terminal toxin domains of the Bt parent proteins Cry1Ab, Cry1B, Cry1C, Cry1Da, Cry1E, Cry1Fb, and Cry2Aa. In particular, the novel chimeric insecticidal proteins of the present invention with enhanced lepidopteran activity or improved lepidopteran spectrum comprise the following domain combinations: Cry1C Domain I (SEQ ID NO: 98), Cry1B (SEQ ID NO: 102), Cry1Ab (SEQ ID NO: 106), Cry1E (SEQ ID NO: 111), Cry1Da (SEQ ID NO: 115), Cry1Fb (SEQ ID NO: 119), Cry2Aa (SEQ ID NO: 123); Cry1C Domain II (SEQ ID NO: 99), Cry1B (SEQ ID NO: 103), Cry1Ab (SEQ ID NO: 107), Cry1E (SEQ ID NO: 112), Cry1Da (SEQ ID NO: 116), Cry1Fb (SEQ ID NO: 120), Cry2Aa (SEQ ID NO: 124); and Cry1C Domain III (SEQ ID NO: 98), Cry1B (SEQ ID NO: 102), Cry1Ab (SEQ ID NO: 106), Cry1E (SEQ ID NO: 111), Cry1Da (SEQ ID NO: 115), Cry1Fb (SEQ ID NO: 119), Cry2Aa (SEQ ID NO: 123). NO:100), Cry1B (SEQ ID NO:104), Cry1Ab_a (SEQ ID NO:108), Cry1Ab_b (SEQ ID NO:109), Cry1E (SEQ ID NO:113), Cry1Da (SEQ ID NO:117), Cry1Fb (SEQ ID NO:121), Cry2Aa (SEQ ID NO:125).

[0177] Exemplary truncated chimeric proteins according to the present invention are referred to as EMS_Q1 (SEQ ID NO:1), EMS_Q2 (SEQ ID NO:2), EMS_Q3 (SEQ ID NO:3), EMS_Q4 (SEQ ID NO:4), EMS_Q5 (SEQ ID NO:5), EMS_Q6 (SEQ ID NO:6), EMS_Q7 (SEQ ID NO:7), EMS_Q8 (SEQ ID NO:8), EMS_Q9 (SEQ ID NO:9), EMS_Q10 (SEQ ID NO:10), EMS_Q11 (SEQ ID NO:11), EMS_Q12 (SEQ ID NO:12), EMS_Q13 (SEQ ID NO:13), EMS_Q14 (SEQ ID NO:14), EMS_Q15 (SEQ ID NO:15), EMS_Q16 (SEQ ID NO:16), EMS_Q17 (SEQ ID NO:17), EMS_Q18 (SEQ ID NO:18), EMS_Q19 (SEQ ID NO:19), EMS_Q20 (SEQ ID NO:20), EMS_Q21 (SEQ ID NO:21), EMS_Q22 (SEQ ID NO:22), EMS_Q23 (SEQ ID NO:23), EMS_Q24 (SEQ ID NO:24), EMS_Q25 (SEQ ID NO:25), EMS_Q26 (SEQ ID NO:26), EMS_Q27 (SEQ ID NO:27), EMS_Q28 (SEQ ID NO:28), EMS_Q29 (SEQ ID NO:29), EMS_Q30 (SEQ ID NO:30), EMS_Q31 (SEQ ID NO:31), EMS_Q32 (SEQ ID NO:32), EMS_Q33 (SEQ ID NO:33), EMS_Q34 (SEQ ID NO:34), EMS_Q35 (SEQ ID NO:35), EMS_Q36 (SEQ ID NO:36), EMS_Q37 (SEQ ID NO:37), EMS_Q38 (SEQ ID NO:38), EMS_Q39 (SEQ ID NO:39), EMS_Q40 (SEQ ID NO:40), EMS_Q41 (SEQ ID NO:41), EMS_Q42 (SEQ ID NO:42), EMS_Q43 (SEQ ID NO:43), EMS_Q44 (SEQ ID NO:44), EMS_Q45 (SEQ IDNO:45), EMS_Q46 (SEQ ID NO:46), EMS_Q47 (SEQ ID NO:47) and EMS_Q48 (SEQ ID NO:48).

[0178] Many truncated chimeric insecticidal proteins exhibit insecticidal activity against multiple species of Lepidoptera insect pests. In particular, the truncated chimeric insecticidal proteins described in this application exhibit activity against Spodoptera frugiperda. Thus, the exemplary proteins described in this application relate to common functions and exhibit insecticidal activity against insect pests from Lepidoptera species, including adults, larvae, and pupae.

[0179] Chimeric insecticide proteins can be identified by comparing them to each other using various computer-based algorithms known in the art. For example, the amino acid sequence identity of a chimeric insecticidal protein involving a truncation can be analyzed using Clustal W alignment with these standard parameters: weight matrix: blosum, gap open penalty: 10.0, gap extension penalty: 0.05, hydrophilic gap: bonded, hydrophilic residue: GPSNDQERK, specific residue gap penalty: associated (Thompson et al. (1994) Nucleic Acids Research, 22: 4,673 to 4,680). The amino acid identity percentage is further calculated by multiplying the product of (number of identical amino acids / length of the protein in question) by 100%. Other alignment algorithms can also be used in the art to provide results similar to those obtained using Clustal W alignments and are considered in this application.

[0180] If an interrogation protein exhibiting insect inhibitory activity is aligned with a truncated chimeric insecticidal protein established in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48 and results in at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59% between the interrogation protein and the substance 94%, 95%, 96%, 97%, 98%, 99%, or about 100% amino acid sequence identity (or any fractional percentage within that range) to the query protein.

[0181] As further described in the Examples of this application, synthetic or artificial sequences encoding chimeric insecticidal proteins have been designed for use in plants. Exemplary synthetic nucleotide sequences that have been designed for use in plants established in the field are EMS_Q1 (SEQ ID NO:49), EMS_Q2 (SEQ ID NO:50), EMS_Q3 (SEQ ID NO:51), EMS_Q4 (SEQ ID NO:52), EMS_Q5 (SEQ ID NO:53), EMS_Q6 (SEQ ID NO:54), EMS_Q7 (SEQ ID NO:55), EMS_Q8 (SEQ ID NO:56), EMS_Q9 (SEQ ID NO:57), EMS_Q10 (SEQ ID NO:58), EMS_Q11 (SEQ ID NO:59), EMS_Q12 (SEQ ID NO:60), EMS_Q13 (SEQ ID NO:61), EMS_Q14 (SEQ ID NO:62), EMS_Q15 (SEQ ID NO:63), EMS_Q16 (SEQ ID NO:64), EMS_Q17 (SEQ ID NO:65), NO:65), EMS_Q18 (SEQ ID NO:66), EMS_Q19 (SEQ ID NO:67), EMS_Q20 (SEQ ID NO:68), EMS_Q21 (SEQ ID NO:69), EMS_Q22 (SEQ ID NO:70), EMS_Q23 (SEQ ID NO:71), EMS_Q24 (SEQ ID NO:72), EMS_Q25 (SEQ ID NO:73), EMS_Q26 (SEQ ID NO:74), EMS_Q27 (SEQ ID NO:75), EMS_Q28 (SEQ ID NO:76), EMS_Q29 (SEQ ID NO:77), EMS_Q30 (SEQ ID NO:78), EMS_Q31 (SEQ ID NO:79), EMS_Q32 (SEQ ID NO:80), EMS_Q33 (SEQ ID NO:81), EMS_Q34 (SEQ ID NO:82), EMS_Q35 (SEQ ID NO:83), EMS_Q36 (SEQ ID NO:84), EMS_Q37 (SEQ ID NO:85), EMS_Q38 (SEQ ID NO:86), EMS_Q39 (SEQ ID NO:87), EMS_Q40 (SEQ ID NO:88), EMS_Q41 (SEQ ID NO:89), EMS_Q42 (SEQ ID NO:90), EMS_Q43 (SEQID NO: 91), EMS_Q44 (SEQ ID NO: 92), EMS_Q45 (SEQ ID NO: 93), EMS_Q46 (SEQ ID NO: 94), EMS_Q47 (SEQ ID NO: 95) and EMS_Q48 (SEQ ID NO: 96).

[0182] For expression in plant cells, chimeric insecticidal proteins can be expressed to be present in the cytosol or directed to various organelles of plant cells. For example, protein targeting to chloroplasts can result in an increase in the protein level expressed in transgenic plants, preventing the occurrence of different phenotypes. Targeting can also result in an increase in the effectiveness of pest resistance in transgenic events. Targeting peptides or transport peptides are short peptide chains (3 to 70 amino acid lengths) that direct protein transport to specific regions of the cell, including the nucleus, mitochondria, endoplasmic reticulum (ER), chloroplasts, apoplasts, peroxisomes, and plasma membranes. Some transport peptides are cut off from the protein by peptidase after protein transport. For targeting to chloroplasts, the protein contains a transport peptide with about 40 to 50 amino acids. The use of chloroplast transport peptides is described in, for example, WO2013116758, US 5,188,642, US 5,728,925, and US 9,150,625. Many proteins located in chloroplasts are expressed from nuclear genes as precursors and are directed to chloroplasts by chloroplast transport peptides (CTPs). Examples of such isolated chloroplast proteins include, but are not limited to, those associated with the small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase, ferredoxin, ferredoxin oxidoreductase, light-harvesting complex I and protein II, thioredoxin F, and enolpyruvylshikimate phosphate synthase (EPSPS). Proteins that have been shown in vivo and in vitro to be not expressed in chloroplasts can be directed to chloroplasts using protein fusions with heterologous CTPs, and CTPs are sufficient to target proteins to chloroplasts. It has been shown that incorporation of a suitable chloroplast transport peptide, such as the EPSPS CTP from Arabidopsis thaliana (CTP2) (see Klee et al., Mol. Gen. Genet. 210: 437-442, 1987) or the EPSPS CTP from Petunia hybrida (CTP4) (see Della-Cioppa et al., Proc. Natl. Acad. Sci. USA 83: 6,873-6,877, 1986), directs the protein sequence of heterologous EPSPS to the chloroplasts in transgenic plants (see US 5,627,061; US 5,633,435; and US 5,312,910; and EP 0 218 571; EP 189 707; EP 508 909; and EP 924 299). To target the chimeric insecticidal protein to chloroplasts, a sequence encoding a chloroplast transit peptide is operably placed 5' to a synthetic coding sequence encoding a chimeric insecticidal protein designed to be expressed in plant cells.

[0183] According to the method and setting of transformation program known in the art, expression cassettes and vectors containing these synthetic or artificial nucleotide sequences are constructed and introduced into the plant cells of Escherichia coli and corn (Zea mays). The transformed corn cells are regenerated into plants expressing the transformed chimeric insecticidal protein of truncation. In order to test insecticidal activity, bioassays are carried out in the presence of newborn larvae (1 day old) of lepidopteran pests. Compositions of recombinant nucleic acid molecules encoding chimeric insecticidal proteins have been considered. For example, chimeric insecticidal proteins can be expressed with recombinant DNA constructs, wherein the polynucleotide molecules encoding chimeric insecticidal proteins with ORFs are operably connected to gene expression elements, such as promoters and any other regulatory elements necessary for construct expression in the system. Non-limiting examples include functional plant promoters that are operably connected to the synthetic chimeric insecticidal protein of the sequence encoding the chimeric insecticidal protein for expressing the chimeric insecticidal protein in plants, or Bt functional promoters that are operably connected to the sequence encoding the chimeric insecticidal protein for expressing the protein in other species of Bt bacteria or Bacillus. Other elements can be operably linked to the chimeric pesticidal protein, including but not limited to enhancers, introns, untranslated leader sequences, encoded protein anchoring peptides (HIS-tags), translocation peptides (i.e., plastid transport peptides, signal peptides), polypeptide sequences for post-translational modification enzymes, ribosome binding sites, and RNAi targeting sites.

[0184] Examples of recombinant polynucleotide molecules provided herein include, but are not limited to, a heterologous promoter operably linked to a polynucleotide such as SEQ ID NO: 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 encoding a polypeptide having a motif of SEQ ID NO: NO:1(EMS_Q1), 2(EMS_Q2), 3(EMS_Q3), 4(EMS_Q4), 5(EMS_Q5), 6(EMS_Q6), 7(EMS_Q7) , 8(EMS_Q8), 9(EMS_Q9), 10(EMS_Q10), 11(EMS_Q11), 12(EMS_Q12), 13(EMS_Q13), 14( EMS_Q14), 15(EMS_Q15), 16(EMS_Q16), 17(EMS_Q17), 18(EMS_Q18), 19(EMS_Q19), 20( EMS_Q20), 21(EMS_Q21), 22(EMS_Q22), 23(EMS_Q23), 24(EMS_Q24), 25(EMS_Q25), 26( 48 (EMS_Q44), 45 (EMS_Q45), 46 (EMS_Q46), 47 (EMS_Q47) and 48 (EMS_Q48). Heterologous promoters can also be operably linked to the coding sequences of synthetic DNA encoding plastid-targeted truncated chimeric insecticidal proteins and non-targeted truncated chimeric insecticidal proteins. It is envisioned that the codons of the recombinant nucleic acid molecules encoding the chimeric insecticidal proteins disclosed herein can be replaced by synonymous codons (referred to in the art as silent substitutions). Methods for optimizing codons for gene expression in host cells of species of interest (including bacteria and plants, such as dicots and monocots) are known in the art.For example, one skilled in the art knows how to use the Optimizer software (http: / / genomes.urv.es / OPTIMIZER / Form.php) (Puigbo, P., Guzmen, E., Romeu, A. and Garcia-Vallve, S. 2007 OPTIMIZER: A web server for optimizing the codon usage of DNA sequences. Nucleic Acids Research, 35: W126-W131) to select parameters for the host cell of interest.

[0185] The recombinant DNA molecule, construct, or expression cassette encoding the truncated chimeric insecticidal protein may further comprise a DNA region encoding one or more toxicants, proteins distinct from the chimeric insecticidal protein, insect-inhibitory dsRNA molecules, or supplemental proteins that can be configured to simultaneously express or co-express the DNA sequence encoding the chimeric insecticidal protein. Supplemental proteins include, but are not limited to, cofactors, enzymes, binding partners, or other agents that aid in the efficacy of the insect inhibitor, such as those that aid expression, influence stability in plants, optimize oligomerization free energy, increase toxicity, or broaden its spectrum of activity. Supplemental proteins may, for example, promote absorption of one or more insect inhibitors or enhance the toxic effects of the toxicant.

[0186] Recombinant DNA molecules, constructs, or expression cassettes can be assembled so that all proteins or dsRNA molecules are expressed from the same promoter, or each protein or dsRNA molecule is under the control of a separate promoter, or some combination thereof. The proteins of the present invention can be expressed from a multigene expression system, wherein the chimeric insecticidal proteins are expressed from a common nucleotide segment, which also contains other open reading frames and promoters, depending on the type of expression system selected. In another example, a plant multigene expression system can use multiple unlinked expression cassettes, each of which expresses a different protein or another toxic agent, such as one or more dsRNA molecules.

[0187] A recombinant nucleic acid molecule or recombinant DNA construct comprising a sequence encoding a chimeric insecticidal protein can be administered to a host cell via a vector, such as a plasmid, baculovirus, synthetic chromosome, virion, cosmid, phagemid, bacteriophage, or viral vector. Such vectors can be used to achieve stable or transient expression of the chimeric insecticidal protein-encoding sequence in the host cell, or subsequent expression of the encoded polypeptide. An exogenous recombinant polynucleotide or recombinant DNA construct comprising a chimeric insecticidal protein sequence encoding sequence and introduced into a host cell is also referred to herein as a "transgene."

[0188] Provided herein are transgenic bacteria, transgenic plant cells, transgenic plants, and parts of transgenic plants containing polynucleotides encoding any one or more chimeric insecticidal proteins. The term "bacterial cell" or "bacteria" may include, but are not limited to, cells of the genus Agrobacterium, Bacillus, Escherichia, Salmonella, Pseudomonas, or Rhizobium. The term "plant cell" or "plant" may include, but are not limited to, dicotyledonous plant cells or monocotyledonous plant cells. The plants and plant cells envisioned include, but are not limited to, beets, watercress, lettuce, alfalfa, cotton, chicory, alstroemeria, peanuts, rice, oats, potatoes, snapdragons, brachiaria, broccoli, coffee, sugarcane, grasses, carrots, rye, barley, chicory, coconut, kale, cauliflower, chrysanthemum, peace lily, spinach, stevia, beans, tobacco, gerbera, baby's breath, lisianthus, castor beans, cassava, passion fruit, foxtail, corn, mustard, forage grasses, pepper, sweet pepper, cabbage, rose, rocket, rubber tree, soybean, sorghum, tomato, wheat, triticale, fruits and vegetables. In certain embodiments, transgenic plants and transgenic plant parts regenerated from transgenic plant cells are provided. In certain embodiments, transgenic plants can be obtained from transgenic seeds or seedlings of transgenic plants. In certain embodiments, the parts of the plant can be seeds, capsules, leaves, flowers, stems, roots or any part thereof, or the non-regenerative parts of transgenic plant parts. In the context of this paper, a "non-regenerable" portion of a transgenic plant part is a portion that cannot be induced to form a complete plant or cannot be induced to form a complete plant capable of sexual or asexual reproduction. In certain embodiments, the non-regenerable portion of a plant part is a portion of a seed, capsule, leaf, flower, stem, or transgenic root.

[0189] Also provided herein is a method for producing transgenic plants comprising a chimeric insecticidal protein comprising a truncated amount of a lepidopteran inhibitory agent. Such plants can be produced by introducing a polynucleotide encoding the chimeric insecticidal protein provided herein into a plant cell, and selecting a plant expressing the chimeric insecticidal protein expressing an insect or lepidopteran inhibitory agent obtained from the plant cell. Plants can be obtained from plant cells by regeneration, seed, pollen, or meristem transformation techniques. Methods for transforming plants are known in the art. For example, Agrobacterium-mediated transformation is described in, for example, US 8,404,930 (corn), US2009 / 0142837 (corn), WO2011095460, US 2009 / 0138985 (soybean), US2008 / 0280361 (soybean), WO2000071733 (cotton), and US2008 / 0256667 (cotton).

[0190] Plants expressing the chimeric insecticidal protein can be crossed by breeding with transgenic events expressing other insecticidal proteins and / or expressing other transgenic traits, such as other insect control traits, herbicide tolerance genes, genes conferring yield or stress tolerance traits, etc., or such traits can be combined in a single vector so that the traits are all linked.

[0191] The present application also discloses processed plant products, wherein the processed product comprises a detectable amount of a truncated chimeric insecticidal protein. In certain embodiments, the processed product is selected from plant parts, plant biomass, oil, flour, sugar, animal feed, bran, chips, hulls, processed seeds, and seeds. In certain embodiments, the processed product is non-renewable. The plant product may comprise a commercial or other consumer product obtained from the transgenic plant or transgenic plant part, wherein the consumer or other product can be tracked through trade by detecting a nucleotide or RNA segment or expressed protein that encodes or comprises a unique portion of the chimeric insecticidal protein.

[0192] This application also discloses methods for controlling insect infestations, particularly of lepidopteran crops, using chimeric insecticidal proteins. Such methods may include cultivating plants comprising an insect-inhibiting amount of the chimeric insecticidal protein. In certain embodiments, such methods may further include any one or more of the following steps: (i) applying any composition comprising or encoding the chimeric insecticidal protein to a plant or a seed that produces a plant; and (ii) transforming the plant or a plant cell that produces a plant with a polynucleotide encoding the chimeric insecticidal protein. In general, it is contemplated that the chimeric insecticidal protein may be provided in a composition, provided in a microorganism, or provided in a transgenic plant to confer insect-inhibiting activity against lepidopteran insects.

[0193] In certain embodiments, the chimeric insecticidal protein is used as the active ingredient in an insecticide composition prepared by culturing recombinant Bacillus or any other recombinant bacterial cells that have been transformed to express a truncated chimeric insecticidal protein under conditions suitable for expression. Such compositions can be prepared by dehydrating, freeze-drying, homogenizing, extracting, filtering, centrifuging, precipitating, or concentrating a culture of such recombinant cells that express / produce the chimeric insecticidal protein. Such methods can produce a Bacillus cell extract or another entomopathogenic bacterial cell extract, a cell suspension, a cell homogenate, a cell lysate, a cell supernatant, a cell filtrate, or a cell pellet. By obtaining the chimeric insecticidal protein thus produced, compositions comprising the chimeric insecticidal protein can include bacterial cells, bacterial spores, and parasporal inclusion bodies and can be formulated for a variety of uses, including as agricultural insecticide spray products or as insecticide formulations in dietary bioassays.

[0194] The above-mentioned composition or formulation may also include an agriculturally acceptable vehicle, such as a bait, powder, dust, pellet, granule, spray, emulsion, colloidal suspension, aqueous solution, spore or crystal preparation of Bacillus, or seed treatment. The composition or formulation may also include recombinant plant cells, plant tissues, seeds or plants transformed to express one or more of the proteins; or bacteria transformed to express one or more of the proteins. Depending on the inherent insecticidal inhibition or insect inhibition level of the recombinant polypeptide and the level of compound or formulation applied to the plant or diet assay, the composition or formulation may include various amounts of the recombinant polypeptide by weight, for example, 0.0001% to 0.001% to 0.01% to 1% to 99% by weight of the recombinant polypeptide.

[0195] In one embodiment, to reduce the likelihood of resistance development, an insect or transgenic plant inhibitory composition comprising a chimeric insecticidal protein may further comprise at least one additional toxicant that exhibits insect inhibitory activity against the same Lepidoptera insect species, but is different from the chimeric insecticidal protein. Additional possible toxicants for such compositions include insect inhibitory proteins and insect inhibitory dsRNA molecules. Examples of such ribonucleotide sequences for use in controlling insect pests are described in US 2006 / 0021087. Such additional polypeptides for controlling lepidopteran pests may be selected from insect inhibitor proteins such as, but not limited to, Cry1A (US 5,880,275), Cry1Ab, Cry1Ac, Cry1A.105, Cry1Ae, Cry1B (US 10 / 525,318), Cry1C (US 6,033,874), chimeric Cry1D, Cry1E, Cry1F and Cry1A / F (US 7,070,982; US 6,962,705; and US 6,713,063), Cry1G, Cry1H, Cry1I, Cry1J, Cry1K, Cry1L, Cry2A, Cry2Ab (US 10 / 525,318), Cry1C (US 6,033,874), chimeric Cry1D, Cry1E, Cry1F and Cry1A / F (US 7,070,982; US 6,962,705; and US 6,713,063), Cry1G, Cry1H, Cry1I, Cry1J, Cry1K, Cry1L, Cry2A, Cry2Ab (US 7,064,249), Cry2Ae, Cry4B, Cry6, Cry7, Cry8, Cry9, Cry15, Cry43A, Cry43B, Cry51Aa1, ET66, TIC400, TIC800, TIC834, TIC1415, Vip3A, VIP3Ab, VIP3B, AXMI-001, AXMI-002, AXMI-030, AXMI-035, and AXMI-045 (US2013 / 0117884), AXMI-52, AXMI-58, AXMI-88, AXMI-97, AXMI-102, AXMI-112, AXMI-117, AXMI-100 (US2013 / 0 310543), AXMI-115, AXMI-113, AXMI-005 (US2013 / 0104259), AXMI-134 (US2013 / 0167264), AXMI-150 (US2010 / 0160231), AXMI-184 (US2010 / 0004176), AXMI-1 96. AXMI-204, AXMI-207, AXMI-209 (US2011 / 0030096), AXMI-218, AXMI-220 (US2014 / 0245491), AXMI-221z, AXMI-222z, AXMI-223z, AXMI-224z, AXMI-225z (US 2014 / 0196175), AXMI-238 (US2014 / 0033363), AXMI-270 (US2014 / 0223598), AXMI-345 (US2014 / 0373195), DIG-3 (US 2013 / 0219570), DIG-5 (US2010 / 0317569), DIG-11 (US 2010 / 0319093), AfIP-1A and its derivatives (US2014 / 0033361), AfIP-1B and its derivatives (US2014 / 0033361), PIP-1APIP-1B (US2014 / 0007292), PSEEN3174 (US2014 / 0007292), AECFG-592740 (US2014 / 0007292), Pput_1063 (US2014 / 0007292), Pput_1064 (US2014 / 0007292), GS-135 and its derivatives (US2012 / 0233726), GS153 and its derivatives (US 2012 / 0192310), GS154 and its derivatives (US2012 / 0192310), GS155 and its derivatives (US2012 / 0192310), SEQ ID NO: 2 and its derivatives described in US2012 / 0167259, SEQ ID NO: 2 and its derivatives described in US2012 / 0047606, SEQ ID NO: 2 and its derivatives described in US 2011 / 0154536, SEQ ID NO: 2 and its derivatives described in US2011 / 0112013, SEQ ID NO: 2 and 4 and its derivatives described in US2010 / 0192256, SEQ ID NO: 2 and its derivatives described in US2010 / 0077507, SEQ ID NO: 2 and its derivatives described in US2010 / 0077508, SEQ ID NO: 2 and its derivatives described in US2009 / 0313721. NO: 2 and its derivatives, SEQ ID NO: 2 or 4 and its derivatives described in US 2010 / 0269221, SEQ ID NO: 2 and its derivatives described in US 7,772,465, CF161_0085 and its derivatives described in document WO2014 / 008054, toxic lepidopteran proteins and their derivatives described in US2008 / 0172762, US2011 / 0055968 and US2012 / 0117690; SEQ ID NO: 2 and its derivatives described in US document 7,510,878, SEQ ID NO: 2 or 4 and its derivatives described in US 7,812,129;NO: 2 and its derivatives; TIC1100, TIC860, TIC867, TIC868, TIC869, TIC836, TIC713, TIC843, TIC862, TIC1099, TIC1103, TIC845, TIC846, TIC858, TIC866, TIC838, TIC841, TIC842, TIC850, TIC859, TIC861, TIC848, TIC849 and TIC847 described in document WO2016 / 061391, and the like.

[0196] In other embodiments, to broaden the spectrum of insect inhibition achieved, the insect inhibitor composition or transgenic plant may further comprise at least one additional toxicant that exhibits insect inhibitory activity against insect pests not inhibited by the chimeric insecticidal protein of the present invention (e.g., coleopteran, hemipteran, and homopteran pests).

[0197] Such additional toxicants for controlling coleopteran pests may be selected from insect inhibitor proteins such as, but not limited to, Cry3Bb (US 6,501,009), Cry1C variants, Cry3A variants, Cry3, Cry3B, Cry34 / 35, 5307, AXMI-134 (US 2013 / 0167264), AXMI-184 (US 2010 / 0004176), AXMI-205 (US 2014 / 0298538), AXMI207 (US 2013 / 0303440), AXMI-218, AXMI-220 (US 2014 / 0245491), AXMI-221z, AXMI-223z (US2014-0196175), AXMI-279 (US2014 / 0223599), AXMI-R1 and its variants (US2010 / 0197592, TIC407, TIC417, TIC431, TIC807, TIC853, TIC901, TIC1201, TIC3131, DIG-10 (US2010 / 0319092), eHIPs (US2010 / 0017914), IP3 and its variants (US2012 / 0210462) and ω-hexatoxin-Hv1a (US2014 / 0366227).

[0198] Such additional toxicants for controlling hemipteran pests can be selected from hemipteran active proteins such as, but not limited to, TIC1415 (US2013 / 0097735), TIC807 (US 8,609,936), TIC834 (US2013 / 0269060), AXMI-036 (US 2010 / 0137216), and AXMI-171 (US2013 / 0055469). Additional polypeptides for controlling coleopteran, lepidopteran, and hemipteran insects can be found on the Bacillus thuringiensis toxin nomenclature webpage (http: / / www.lifesci.sussex.ac.uk / home / Neil_Crickmore / Bt / and https: / / www.bpprc.org / ).

[0199] Chimeric insecticidal protein coding sequences and sequences having a substantial percentage of identity to chimeric insecticidal proteins can be identified using methods known to those skilled in the art, such as polymerase chain reaction (PCR), thermal amplification, and hybridization. For example, chimeric insecticidal proteins can be used to generate antibodies that specifically bind to the related protein, and they can be used to study and discover other closely related proteins.

[0200] In addition, nucleotide sequences encoding chimeric pesticidal proteins can be used as probes and primers for screening to identify other members of this class using hybridization and thermal amplification or thermal cycling methods. For example, oligonucleotides derived from the sequence of any one of SEQ ID NOs: 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 can be used to determine the presence or absence of a chimeric pesticidal transgene in a sample of DNA derived from a consumer product. In view of the sensitivity of certain nucleic acid detection methods using oligonucleotides, it is expected that oligonucleotides derived from the sequence provided in any one of SEQ ID NOs:49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 or 96 can be used to detect nucleic acids encoding truncated chimeric pesticidal proteins in plant products from transgenic plants.

[0201] Example

[0202] In view of the above, those skilled in the art will appreciate that the following embodiments described are merely representative of the present invention, which can be implemented in various ways. Therefore, the specific structural and functional details disclosed herein should not be interpreted as limiting.

[0203] Example 1

[0204] Generation of coding sequences for truncated chimeric insecticidal proteins active against Lepidoptera

[0205] This example demonstrates the production of a truncated chimeric insecticidal protein.

[0206] Recombinant nucleic acid sequences were constructed from Cry genes obtained from sequencing of Bacillus thuringiensis strains. First, the C-terminal protoxin domain of each Cry protein was removed to produce truncated parent Cry proteins Cry1C (SEQ ID NO: 97), Cry1B (SEQ ID NO: 101), Cry1Ab (SEQ ID NO: 105), Cry1E (SEQ ID NO: 110), Cry1Da (SEQ ID NO: 114), Cry1Fb (SEQ ID NO: 118) and Cry2Aa (SEQ ID NO: 122). Next, domains I, II and III were identified by aligning the protein sequence with a protein domain database (Pfam or NCBI-BlastP). Table 1 details the identification of each truncated Cry protein and their respective domains. The truncated chimeric proteins were assembled by combining the domains described in Table 2. The resulting polynucleotide sequence was synthesized using codon optimization for expression in Escherichia coli and cloned into a plasmid expression vector and transformed into Escherichia coli. Preparations of proteins expressed in E. coli were used in bioassays against various lepidopteran pest caterpillars to verify insecticidal activity and growth inhibition.

[0207] Table 1: Truncated chimeric insecticidal proteins and their components.

[0208]

[0209]

[0210] Table 2: Truncated chimeric insecticidal proteins and their components.

[0211]

[0212]

[0213]

[0214] Example 2

[0215] Activity testing of truncated chimeric proteins against Spodoptera frugiperda

[0216] This example illustrates the testing of the insecticidal activity of the truncated chimeric protein described in Example 1 against Spodoptera frugiperda.

[0217] The polynucleotide sequences encoding the truncated chimeric proteins were expressed in Escherichia coli and used in bioassays with neonatal caterpillars of Spodoptera frugiperda. Activity was determined based on mortality scores or evaluation of larval growth inhibition during 5 to 7 days of feeding on an artificial diet containing the recombinant chimeric protein preparations. Table 3 illustrates the insecticidal activity results of each chimeric protein against Spodoptera frugiperda. Insecticidal activity is indicated by a "+" sign. A "-" sign indicates that no insecticidal activity was observed, and NT means that the protein was not tested. Distilled water and E. coli containing an empty expression vector were used as negative controls. The mortality rate of the controls was less than 15%.

[0218] Table 3: Insecticidal activity (mortality or growth inhibition) against Spodoptera frugiperda from truncated chimeric proteins expressed in E. coli

[0219]

[0220]

[0221]

[0222] Example 3

[0223] Synthesis of genes encoding truncated chimeric insecticidal proteins for expression in plants

[0224] This example illustrates the synthesis of a polynucleotide encoding a chimeric insecticidal protein for expression in plants.

[0225] The modification of gene codons was carried out with the aid of Optimizer (http: / / genomes.urv.es / OPTIMIZER / Form.php) software (Puigbo P., Guzmen E., Romeu A. and Garcia-Vallve S.2007 OPTIMIZER: A web server for optimizing the codon usage of DNA sequences. Nucleic Acids Research, 35: W126-W131), with the goal being a codon more compatible with corn (Zea mays), and the sequence was sent digitally for commercial synthesis. The optimized gene was synthesized in a pBS plasmid and cloned into a binary vector p7i2x-UibZm (https: / / dna-cloning.com / binaries / ). The synthesized sequence was confirmed by sequencing according to standard techniques.

[0226] Table 4: Polynucleotide sequences encoding novel chimeric proteins for expression in plants.

[0227]

[0228]

[0229] Example 4

[0230] Expression cassettes for expressing chimeric insecticidal proteins in plants

[0231] This example illustrates the construction of an expression cassette for use in plants comprising a polynucleotide sequence encoding a truncated chimeric insecticidal protein.

[0232] Various plant expression cassettes were constructed using the polynucleotide sequences listed in Table 4. The optimized genes were inserted into the binary vector p7i2x-UibZm (https: / / dna-cloning.com / binaries / ), which contains the promoter of the maize ubiquitin gene (ubi) and the terminator of the nopaline synthase gene (nos). This generated a series of plasmids designed to allow protein translation and retention in the plant cytosol. The resulting plasmids containing the genes encoding the chimeric proteins were used to transform Agrobacterium tumefaciens EHA101 bacteria by electroporation (BioRad / MicroPulser). Transformed bacteria containing the expression cassettes were used for genetic transformation of maize.

[0233] Example 5

[0234] Lepidopteran activity of chimeric insecticidal proteins in stably transformed maize

[0235] This example illustrates the testing of the novel chimeric protein described in Example 1 expressed in maize for insecticidal activity against lepidopteran pests in plants.

[0236] The vector containing the polynucleotide sequence SEQ ID NO: 54 was selected from the transformation vector constructed and described in Example 4 for transformation of HiII corn varieties (Armstrong CL, Grenn CE, Phillips RL (1991). Development and availability of germplasm with high type II culture formation response. Maize Genet. Coop. Newsletter. 65: 92-93). The transformation protocol was that described by Frame et al., Agrobacterium tumefaciens-mediated transformation of corn embryos using a standard binary vector system. Plant Physiol. 2002 May; 129 (1): 13-22, with minor modifications. In brief, for the transformation of this genotype, immature embryos 1.8–2.0 mm in length were collected (10-12 days after pollination). The ears for embryo collection were immersed in a 1: 1 solution of commercial bleach (2.5% sodium hypochlorite) and distilled water containing 1-2 drops of Tween 20 for 20 minutes. Then rinse them with sterile distilled water for 5 minutes twice.

[0237] Immature embryos were collected from the surface of the grains by means of a spatula. For the transfer of gene constructs to maize, Agrobacterium tumefaciens EHA101 was used. From a stock culture of Agrobacterium tumefaciens containing the gene construct of interest maintained in glycerol at -80°C, the embryos were grown in a 5% culture medium containing the necessary antibiotics (spectinomycin 100 mg.L-1 and kanamycin 50 mg.L-1). -1 ) of YEP medium (5 g.L -1 Yeast extract; 10g.L -1 Peptone; 5g.L -1 NaCl; 15g.L -1 The plates were streaked onto bacto agar and incubated at 28°C for 2 to 3 days (mother plates).

[0238] For gene transformation, Agrobacterium was streaked using colonies isolated from the mother plate in YEP medium containing the necessary antibiotics. The plates were incubated at 19°C for 2 to 5 days. Agrobacterium was then resuspended in infection medium (4.0 g L) supplemented with 100 μM acetosyringone. -1 N6 salt; 68.4gL -1 Sucrose; 36.0gL -1 Glucose: 0.7 g / L -1Proline; 1.5mg.L-12,4-D; 1.0mL.L -1 N6 Vitamin (1000X = 1.0gL -1 Thiamine HCl; 0.5 g / L -1 Pyridoxine HCl; 0.5 g / L -1 Nicotinic acid); pH 5.2) to reach OD550 = 0.3-0.4 and incubate on a shaker at approximately 150 rpm, 23°C for 2 hours.

[0239] For infection of immature corn embryos, 50 to 100 embryos were collected in 1 mL of infection medium supplemented with acetosyringone. After collection, the embryos were rinsed twice, 1 mL of bacterial culture was added, and the suspension was incubated at 23°C for 5 minutes. After infection, the embryos were transferred to a co-culture medium (4.0 g L -1 N6 salt; 1.5mg.L -1 2,4-D; 30.0gL -1 Sucrose; 0.7gL-1 proline; 1.0mL.L -1 N6 vitamin (1000X); 0.85mg.L -1 AgNO3; 100μM acetosyringone; 300mg.L -1 L-cysteine; 3.0gL -1 phytagel; pH 5.8) with the embryo cotyledon side facing up. The plates were incubated at 20°C in the dark for 3 to 5 days. After co-cultivation, the embryos were transferred to a resting medium (4.0 g L-1 N6 salts; 1.5 mg L-1) at 28°C (dark). -1 2,4-D; 30.0gL -1 Sucrose; 0.5gL -1 MES; 0.7gL -1 Proline; 1.0mL.L-1N6 Vitamin (1000X); 0.85mg.L -1 AgNO3; 100mg.L -1 Tioxin; 3.0gL -1 phytagel; pH 5.8) for 7 to 15 days. The embryos were then transferred to selection medium (4.0 g L -1 N6 salt; 1.5mg.L -1 2,4-D; 30.0gL -1 Sucrose; 0.5gL -1 MES; 0.7gL-1 proline; 1.0mL.L -1 N6 vitamin (1000X); 0.85mg.L -1 AgNO3; 100mg.L -1Tioxin; 1.5 and 3.0 mg / L bialaphos; 3.0 g / L -1 Phytagel; pH 5.8) (25 embryos / plate). These embryos were subcultured every 15 days in selection medium until actively growing calli were selected.

[0240] The selected callus was transferred to regeneration medium (4.62 g L -1 MS salt; 60.0gL -1 Sucrose; 100mg.L -1 Inositol; 1.0mL.L -1 MS vitamins (1000X); 1.5 mg / L bialaphos; 4.0 gL-1 phytagel; pH 5.8) and incubated at 26 ± 2°C (in the dark) for 15 to 21 days. Calli ready for germination with a dry appearance and opaque white color were transferred to germination medium (4.62 gL-1 MS salts; 30.0 gL-1 -1 Sucrose; 100mg.L -1 Inositol; 1.0mL.L -1 MS vitamin (1000X = 0.5gL -1 Thiamine HCl; 0.5 g / L -1 Pyridoxine HCl; 0.05 g / L -1 Niacin); 3.0gL -1 Phytagel; pH 5.8) (12 calli / plate), 25°C, 80-100 μE / m2 / sec light intensity, 16-hour photoperiod). Seedlings with leaves and roots were transferred to a greenhouse within 14 to 20 days.

[0241] When roots were fully developed and leaf structures measured approximately 5 cm in length, seedlings were transplanted into pots containing a mixture of soil and organic matter (2 / 3 soil and 1 / 3 commercially produced organic matter (TDP 30 / 15)) in the greenhouse.

[0242] Genetically modified maize plants containing a gene encoding a chimeric protein (SEQ ID NO: 6) were used in infestation assays in the field and in the greenhouse, as well as in bioassays where caterpillars were fed on fallen leaves.

[0243] In a greenhouse assay, 44 pots of maize events containing the coding sequence for the chimeric protein EMS_Q6 (SEQ ID NO: 6) were used. Non-transgenic maize L3 plants were added as a negative control (C-), and commercial transgenic maize plants resistant to Spodoptera frugiperda were added as a positive control (C+). Maize plants were manually infested with newborn larvae of Spodoptera frugiperda at the V4-V6 stages of development, and plants were evaluated 13 days after infestation using a damage score based on the Davis scale (Davis et al., 1992), where a score of 0 represents no visible damage and a score of 9 represents complete plant destruction. Figure 1 The results of damage scoring are illustrated, where a series of events expressing the chimeric protein EMS_Q6 showed a damage score of 0, similar to the commercial positive control container, while the negative control showed a damage score ranging from 8 to 9.

[0244] In a field trial, 22 events (Ev1 to Ev22) of corn containing a sequence encoding the chimeric protein EMS_Q6 (SEQ ID NO: 6), two positive controls (commercial transgenic corn resistant to fall armyworm) and two negative controls (non-transgenic corn) were artificially infested with newborn larvae of fall armyworm at the V4-V6 stages of corn development, and the plants were evaluated by injury scores based on the Davis scale (Davis, FM; NG, S.; Williams, WP 1992. Visual rating scales for screening whole-stage corn resistance to fall armyworm. Mississippi: Mississippi State University, Technical Bulletin, v.186.9p) 7, 14 and 21 days after infestation, where a score of 0 represents no visible damage and a score of 9 represents complete destruction of the plant. Figure 2 The injury score results represented by the arithmetic mean of 3 evaluations are shown. It should be noted that most of the transgenic events containing the protein EMS_Q6 received a lower average score than the negative control, and some of them received a score lower than the evaluated commercial transgenic products. Figure 3 Representative photographs of damage caused by Spodoptera frugiperda in control plants compared to transgenic plants expressing the chimeric protein EMS_Q6 (SEQ ID NO: 6) are illustrated. Note that the transgenic corn plants did not provide damage, whereas conventional corn plants provided significant leaf damage caused by attack by Spodoptera frugiperda.

[0245] Bioassays were also performed in the laboratory using leaves separated from one of the maize events containing the coding sequence of the chimeric protein EMS_Q6 (SEQ ID NO: 6) and a non-transgenic maize control. To perform this assay, intact leaves of approximately 15 cm in length from one of the events containing the chimeric protein EMS_Q6 (SEQ ID NO: 6) and a negative control (C-) grown in a greenhouse were used. The leaves were rolled up and placed in 50 ml transparent plastic cups. 15 newborn caterpillars of the frugiperda (Spodoptera frugiperda) were placed on each leaf, and the cups were placed in a climate-controlled room with a constant temperature of 25°C, 70% humidity, and a photoperiod of 16 hours light and 8 hours dark. After 5 days, the number of live caterpillars present in each cup was evaluated. Figure 4 The results of the bioassay are shown. It should be noted that in corn leaves expressing the EMS_Q6 protein, all caterpillars died and there was no leaf damage. In the control corn, there was obvious feeding damage and there were alive and well-developed caterpillars.

[0246] In light of the present disclosure, all compositions disclosed and claimed herein may be prepared and performed without undue experimentation. Although the compositions of the present invention have been described in terms of the foregoing illustrative embodiments, it will be apparent to those skilled in the art that changes, variations, modifications, and alterations may be applied to the compositions described herein without departing from the true concept, essence, and scope of the present disclosure. More specifically, it will be apparent that certain reagents related to chemistry and physics may replace the reagents described herein while obtaining the same or similar results. All such substitutions and modifications apparent to those skilled in the art are considered to be within the essence, scope, and concept of the invention as defined in the appended claims.

[0247] All publications and patent documents disclosed in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. A truncated chimeric insecticidal protein comprising: (i) domain I of a Cry protein from any one of SEQ ID NO: 98, SEQ ID NO: 102, SEQ ID NO: 106, SEQ ID NO: 111, SEQ ID NO: 115, SEQ ID NO: 119 or SEQ ID NO: 123; (ii) domain II of a Cry protein from any one of SEQ ID NO: 99, SEQ ID NO: 103, SEQ ID NO: 107, SEQ ID NO: 112, SEQ ID NO: 116, SEQ ID NO: 120, or SEQ ID NO: 124; and (iii) domain III of a Cry protein from any one of SEQ ID NO: 100, SEQ ID NO: 104, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 113, SEQ ID NO: 117, SEQ ID NO: 121 or SEQ ID NO: 125, The protein lacks the carboxy-terminal protoxin domain.

2. A truncated chimeric insecticidal protein comprising a sequence having at least 85% identity to SEQ ID NO: 1, at least 80% identity to SEQ ID NO: 2, at least 79% identity to SEQ ID NO: 3, at least 82% identity to SEQ ID NO: 4, at least 79% identity to SEQ ID NO: 5, at least 87% identity to SEQ ID NO: 6, at least 91% identity to SEQ ID NO: 7, at least 77% identity to SEQ ID NO: 8, at least 81% identity to SEQ ID NO: 9, at least 82% identity to SEQ ID NO: 10, at least 76% identity to SEQ ID NO: 11, at least 91% identity to SEQ ID NO: 12, at least 92% identity to SEQ ID NO: 13, at least 86% identity to SEQ ID NO: 14, at least 82% identity to SEQ ID NO: 15, at least 83% identity to SEQ ID NO: 16, at least 84% identity to SEQ ID NO:

17. NO:16 is at least 88% identical to SEQ ID NO:17, at least 97% identical to SEQ ID NO:18, at least 81% identical to SEQ ID NO:19, at least 86% identical to SEQ ID NO:19, at least 76% identical to SEQ ID NO:20, at least 78% identical to SEQ ID NO:21, at least 78% identical to SEQ ID NO:22, at least 78% identical to SEQ ID NO:23, at least 86% identical to SEQ ID NO:24, at least 85% identical to SEQ ID NO:25, at least 83% identical to SEQ ID NO:26, at least 83% identical to SEQ ID NO:27, at least 85% identical to SEQ ID NO:28, at least 79% identical to SEQ ID NO:29, at least 85% identical to SEQ ID NO:30, at least 80% identical to SEQ ID NO:31, at least 82% identical to SEQ ID NO:

32. NO:33 is at least 84% identical to SEQ ID NO:33, is at least 78% identical to SEQ ID NO:33, is at least 85% identical to SEQ ID NO:34, is at least 80% identical to SEQ ID NO:35, is at least 87% identical to SEQ ID NO:36, is at least 93% identical to SEQ ID NO:37, is at least 84% identical to SEQ ID NO:38, is at least 81% identical to SEQ ID NO:39, is at least 80% identical to SEQ ID NO:40, is at least 84% identical to SEQ ID NO:41,An amino acid sequence that is at least 93% identical to SEQ ID NO:42, at least 86% identical to SEQ ID NO:43, at least 70% identical to SEQ ID NO:44, at least 75% identical to SEQ ID NO:45, at least 73% identical to SEQ ID NO:46, at least 50% identical to SEQ ID NO:47, or at least 50% identical to SEQ ID NO:

48.

3. The truncated chimeric insecticidal protein of claim 1 or 2, wherein the insecticidal protein has inhibitory activity against insect species of the order Lepidoptera.

4. The truncated chimeric insecticidal protein of claim 1 or 2, comprising the amino acid sequence of any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48.

5. A recombinant nucleic acid molecule, wherein the recombinant nucleic acid molecule encodes a truncated chimeric insecticidal protein as defined in claim 1 or 2.

6. The recombinant nucleic acid molecule of claim 5, wherein the recombinant nucleic acid molecule comprises a functional plant promoter operably linked to the coding sequence of the truncated chimeric insecticidal protein.

7. The recombinant nucleic acid molecule of claim 5, wherein the recombinant nucleic acid molecule comprises the nucleotide sequence of any one of SEQ ID NO: 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96, wherein the polynucleotide lacks the region encoding the carboxyl-terminal protoxin domain. A host cell comprising the nucleic acid molecule according to claim 5 .

9. An insect inhibitory composition comprising the truncated chimeric insecticidal protein of claim 1, the recombinant nucleic acid molecule of claim 5, or the cell of claim 7.

10. A plant genome comprising the nucleic acid molecule from claim 5.

11. A plant or plant seed comprising the truncated chimeric insecticidal protein as defined in claim 1 or the nucleic acid molecule according to claim 5.

12. The plant or plant seed of claim 11, wherein the plant is beet, watercress, lettuce, alfalfa, cotton, chicory, alstroemeria, peanut, rice, oat, potato, snapdragon, brachiaria, broccoli, coffee, sugarcane, grass, carrot, rye, barley, chicory, coconut, kale, cauliflower, chrysanthemum, peace lily, spinach, stevia, bean, tobacco, gerbera, baby's breath, lisianthus, castor bean, cassava, passion fruit, foxtail, corn, mustard, grass, pepper, bell pepper, cabbage, rose, arugula, rubber tree, soybean, sorghum, tomato, wheat, triticale, fruit and vegetable plants.

13. A method for controlling lepidopteran pests comprising contacting the lepidopteran pests with an inhibitory amount of the truncated chimeric insecticidal protein of claim 1.

14. A method of controlling a lepidopteran pest comprising exposing the pest to a transgenic plant cell, plant, or plant part, wherein the plant cell, plant, or plant part expresses the recombinant nucleic acid molecule of claim 5.

15. A plant product, characterized in that it comprises a detectable amount of the chimeric insecticidal protein according to claim 1 or 2, the recombinant nucleic acid molecule according to claim 5 or the cell according to claim 8.

16. Use of a plant cell, plant, plant part or seed expressing a truncated chimeric insecticidal protein according to claim 1 or 2, said plant cell, plant, plant part or seed comprising a nucleic acid molecule as defined in claim 5, characterized in that it is used for crossing with a second plant, regenerating a transgenic plant, planting or growing a transgenic plant in situ, producing a plant product or producing a consumer product.

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