EA040497B1 - NEW CHIMERIC INSECTICIDAL PROTEINS TOXIC OR INHIBITORIC AGAINST LEPIDOPTHER PESTS - Google Patents

Description

Link to related application

This application claims priority over U.S. Provisional Application No. 62/064989, filed Oct. 16, 2014, the contents of which are incorporated herein by reference in their entirety.

Enabling a sequence listing

The sequence listing in machine readable form is filed with this application in electronic format and incorporated herein by reference in its entirety. The sequence listing is contained in a file created on August 23, 2016, with the file name P34230WO00_Seq_PCT_Art34.txt, and is 898,229 bytes in size (as measured in the MSWindows® operating system).

Technical field

The present invention relates generally to the field of proteins having insect inhibitory activity. The present application describes a new class of chimeric insecticidal proteins with inhibitory activity against agricultural pests of cultivated plants and seeds. In particular, the disclosed class of proteins has insecticidal activity against Lepidoptera insect pests. Plants, plant parts, and seeds are provided that contain a recombinant nucleic acid molecule encoding one or more of the described toxic proteins.

State of the art

Improving crop yields, including corn, soybeans, sugar cane, rice, wheat, vegetables and cotton, is becoming increasingly important. In addition to the growing demand for agricultural products to provide food, clothing and energy for a growing population, climate impacts and pressure from a growing population to use non-agricultural land, the amount of available arable land for agriculture is predicted to decrease. These factors have led to less than optimistic food security projections, especially in the absence of significant improvements in plant biotechnology and agronomic practices. In light of these factors, environmentally sustainable improvements in technology, farming practices and pest management are vital tools to expand crop production on the limited amount of arable land available for agriculture.

Insects, especially those within the order Lepidoptera, are considered the main cause of damage to field crops, thereby reducing crop yields in infested areas. Lepidoptera pest species that have a negative impact on agriculture include, without limitation, fall armyworm (Spodoptera frugiperda), little cutworm (Spodoptera exigua), lettuce cutworm (Mamestra configurata), upsilon cutworm (Agrotis ipsilon), cabbage cutworm ( Trichoplusia ni), soybean cutworm (Chrysodeixis includens), velvet bean cutworm (Anticarsia gemmatalis), clover cutworm (Hypena scabra), tobacco leafworm (Heliothis virescens), cotton cutworm (Agrotis subterranea), meadow cutworm (Pseudaletia unipuncta), square cutworm ( Agrotis orthogonia), stem corn borer (Ostrinia nubilalis), moth moth (Amyelois transitella), corn moth (Crambus caliginosellus), meadow borer (Herpetogramma licarsisalis), sunflower moth (Homoeosoma electellum), grain corn borer (Elasmopalpus lignosellus), codling moth apple tree (Cydia pomonella), grape leaflet (Endopiza viteana), oriental peach leaflet (Grapholita molesta), sunflower leafworm (Suleima helianthana), cabbage moth (Plutella xylostella), pink bollworm (Pectinophora gossypiella), pink borer (Sesamia inferens), gypsy moth (Lymantria dispar), American cotton bollworm (Alabama argillacea), leaf roller fruit trees (Archips argyrospila), cut golden leafworm (Archips rosana), Asian stem moth or yellow rice moth (Chilo suppressalis), rice leafworm (Cnaphalocrocis medinalis), corn moth (Crambus caliginosellus), herbal moth (Crambus teterrellus), moth southwestern corn cutworm (Diatraea grandiosella)), sugar cane moth (Diatraea saccharalis), Egyptian cotton cutworm (Earias insulana), spotted cutworm (Earias vittella), American cutworm (Helicoverpa armigera), American corn cutworm, soy cutworm or cotton cutworm ( Helicoverpa zea), meadow moth (Eerpetogramma licarsisalis), grapevine leafworm (L obesia botrana), citrus miner moth (Phyllocnistis citrella), cabbage leaf miner (Pieris brassicae), turnip or turnip moth (Pieris rapae), Asian cotton bollworm or Asian cotton bollworm (Spodoptera litura), and nightshade miner (Tuta absoluta).

Historically, synthetic chemical insecticides have been used extensively as a pest control agent in agriculture. Concerns about the environment and human health, in addition to emerging problems with resistance, have spurred the research and development of biological pesticides. This research has led to the progressive discovery and use of various entomopathogenic microbial species, including bacteria.

The biological control paradigm changed when the potential of entomopathogenic bacteria, especially bacteria belonging to the genus Bacillus, was discovered and applied in the development of agents for biological pest control. Bacillus thuringiensis (Bt) strains have been used as a source of insecticidal proteins since Bt strains have been found to exhibit high toxicity against specific insects. Bt strains are known to produce deltaendotoxins that are localized in the parasporal inclusion crystal body at the onset of sporulation and during the stationary growth phase (eg, Cry proteins) and are also known to produce a secreted insecticidal protein. When ingested by susceptible insects, delta-endotoxins, as well as secreted toxins, exert their effect on the surface of the midgut epithelium, destroying the cell membrane, leading to cell destruction and death. Genes encoding insecticidal proteins have also been identified in bacterial species other than Bt, including other Bacillus and many other bacterial species such as Brevibacillus laterosporus, Lysinibacillus sphaericus (is formerly known as Bacillus sphaericus) and Paenibacillus popilliae.

Crystalline and secreted soluble insecticidal protein toxins are highly specific for their hosts and have gained worldwide acceptance as alternatives to chemical insecticides. For example, insecticidal toxic proteins are used in a variety of agricultural applications to protect valuable plants from insect infestation, reduce the need for chemical pesticides, and increase yields. Insecticidal toxic proteins are used to control agricultural pests of cultivated plants by mechanized methods, such as spraying dispersed microbial preparations containing various strains of bacteria on the surface of plants, and using genetic transformation methods to obtain transgenic plants and seeds expressing the insecticidal toxic protein.

The use of transgenic plants expressing insecticidal proteins is recognized worldwide. For example, in 2012, 26.1 million hectares were planted with transgenic crops expressing Bt toxins (James, C., Global Status of Commercialized Biotech/GM Crops: 2012. ISAAA Report Summary agriculture) No. 44). The global use of transgenic insect-protected crops and the limited number of insecticidal proteins used for these crops has created a selection pressure for existing insect alleles that confer resistance to currently used insecticidal proteins.

The development of resistance in target pests to insecticidal proteins creates an ongoing need to discover and develop new forms of insecticidal proteins that are useful in controlling the increase in insect resistance to transgenic crops expressing insecticidal proteins. New insecticidal proteins with improved efficacy, and which control a wider range of susceptible insect species, reduce the number of surviving insects that can develop resistant alleles. In addition, the use in the same plant of two or more transgenic insecticidal proteins that are toxic to the same pest and exhibit different modes of action reduces the likelihood of resistance in any individual target insect species.

Therefore, there is a strong need to identify additional insecticidal proteins with improved insecticidal properties, such as improved efficacy against a wider range of target insect pest species and different mechanisms of action compared to the toxins currently used in agronomic practices. To meet this need, the present invention describes novel Cry1 chimeric insecticidal proteins that exhibit activity against numerous target Lepidoptera pest species.

Members of the Cry1 crystalline protein family known in the art exhibit biological activity against Lepidoptera pests. The form of the precursor of Cry 1 crystalline proteins consists of two segments of approximately equal size. The carboxy-terminal portion of the precursor protein, known as the protoxin segment, stabilizes crystal formation and exhibits no insecticidal activity. The amino-terminal half of the precursor protein contains the toxin segment of the Cry1 protein and, based on the alignment of conserved or substantially conserved sequences within members of the Cry1 family, can be further divided into three structural domains: domain I, domain II, and domain III. Domain I contains about the first third of the active toxin segment and has been shown to be essential for channel formation. Domains II and III are involved in receptor binding and are specific to insect species, depending on the insect and insecticide being studied.

The likelihood of randomly generating a chimeric protein with improved properties from the assortment of domain structures of numerous native insecticidal proteins known in the art is negligible. This is a result of the complex nature of the protein structure, oligomerization and activation (including the appropriate proteolytic processing of the chimeric precursor, if expressed in such a form) required to release the insecticidal protein segment. Only through careful selection of protoxins and specific targets in each outcome

- 2 040497 functional chimeric insecticidal toxins that exhibit improved insecticidal activity compared to the original proteins from which the chimeras are derived can be created in order to create a chimeric structure. It is known in the art that reassembly of the protoxin and toxin domains I, II, and III of any two or more toxins that differ from each other often results in proteins that exhibit defective crystal formation or that completely lack any detectable insecticidal activity directed at a preferred insect pest species. Trial and error alone is effective in producing insecticidal chimeras, and even so, the skilled artisan is not ultimately certain of a chimera that exhibits insecticidal activity that is equivalent to or improved over any single toxic protein from which it is derived. protoxin or chimera toxin domains. For example, the literature provides numerous examples of the construction or assembly of chimeric proteins from two or more crystalline 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): 1805-1813; Bosch, et al. (US patent No. 6204246); Malvar and Gilmer (U.S. Patent 6,017,534). In each of these examples, many of the resulting chimeras did not exhibit insecticidal or crystal-forming properties that were equivalent to or improved over the precursor proteins from which the chimeric components were derived.

The essence of the invention

Recombinant nucleic acid molecules are proposed that encode chimeric insecticidal proteins that are toxic to Lepidoptera plant pests. Each of the chimeric insecticidal proteins can be used alone or in combination with each other and with other insecticidal proteins and insect inhibitory agents in formulations and in planta conditions; thus providing alternatives to the insecticidal proteins and insecticide chemicals currently used in agricultural systems.

In some embodiments of the invention, this document describes a chimeric insecticidal protein containing the amino acid sequence indicated in any of the sequences of SEQ ID NO: 21, 10, 28, 7, 4, 13, 16, 19, 23, 25, 30, 33, 36, 39, 41, 43, 45, 47, 50, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, or 111. This chimeric insecticidal protein exhibits inhibitory activity against insect species of the order Lepidoptera, such as, without limitation, Anticarsia gemmatalis, Diatraea saccharalis, Elasmopalpus lignosellus, Helicoverpa zea, Heliothis virescens, Chrysodeixis includens, Spodoptera cosmioides, Spodoptera eridania, Spodoptera frugiperda, Spodoptera exigua, Helicoverpa armigera, Spodoptera litura, Pectinophora gossypiella, Diatraea grandiosella, Earias vitella, Helicoverpa gelotopeon, and Rachiplusia nu.

In another embodiment of the invention, a polynucleotide encoding a chimeric insecticidal protein, wherein the polynucleotide is operably linked to a heterologous promoter, and the chimeric insecticidal protein contains the amino acid sequence indicated in any of the sequences of SEQ ID NOs: 21, 10, 28, 7, 4, 13, 16 , 19, 23, 25, 30, 33, 36, 39, 41, 43, 45, 47, 50, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77 , 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, or 111. Also contemplated is a polynucleotide encoding a chimeric insecticidal protein, wherein the polynucleotide contains the nucleotide sequence, which optionally hybridizes under stringent conditions to the reverse complement of the polynucleotide sequence specified in any of SEQ ID NOs: 1, 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 22, 24, 26, 27, 29, 31, 32, 34, 35, 37, 38, 40, 42, 44, 46, 48, 49, 51, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98 100 102 104 106 108 110 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 or 130; or encodes a chimeric insecticidal protein comprising the amino acid sequence shown in any of SEQ ID NOs: 21, 10, 28, 7, 4, 13, 16, 19, 23, 25, 30, 33, 36, 39, 41, 43, 45, 47, 50, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109 or 111.

In other embodiments of the invention, this document describes a host cell containing a polynucleotide specified in any of SEQ ID NO: 1, 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 22, 24, 26, 27, 29, 31, 32, 34, 35, 37, 38, 40, 42, 44, 46, 48, 49, 51, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, or 130, wherein the host cell is selected from the group consisting of a bacterial host cell or plant cell host. Contemplated bacterial host cells include Agrobacterium, Rhizobium, Bacillus, Brevibacillus, Escherichia, Pseudomonas, Klebsiella, and Erwinia; and wherein the Bacillus species is Bacillus cereus or Bacillus thuringiensis, said Brevibacillus is Brevibacillus laterosperous, and said Escherichia is Escherichia coli. In addition, putative plant host cells include monocots or dicots.

Other embodiments of the invention, this document describes compositions with inhibitory activity against insects containing a chimeric insecticidal protein, with

- 3 040497 containing the amino acid sequence specified in any of SEQ ID NOs: 21, 10, 28, 7, 4, 13, 16, 19, 23, 25, 30, 33, 36, 39, 41, 43, 45, 47 , 50, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99 , 101, 103, 105, 107, 109, or 111. In some embodiments, the insect-inhibiting composition may further comprise at least one insect-inhibiting agent other than the chimeric insecticidal protein. Contemplated agents having insect inhibitory activity other than the chimeric insecticidal protein include a protein having insect inhibitory activity, a dsRNA molecule having insect inhibitory activity, and a chemical compound having insect inhibitory activity. These insect inhibitory agents, other than the chimeric insecticidal protein, may be active against one or more pest species from the orders Lepidoptera, Coleoptera, Hemiptera, Homoptera, or Thysanoptera.

In yet another embodiment of the invention, this document describes a seed containing an insect-inhibitory effective amount of a chimeric insecticidal protein containing the amino acid sequence indicated in any of the sequences of SEQ ID NOS: 21, 10, 28, 7, 4, 13, 16, 19, 23, 25, 30, 33, 36, 39, 41, 43, 45, 47, 50, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109 or 111; or a polynucleotide listed in any of SEQ ID NOs: 1, 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 22, 24, 26, 27, 29, 31 , 32, 34, 35, 37, 38, 40, 42, 44, 46, 48, 49, 51, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 , 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 113, 114, 115, 116, 117, 118, 119 , 120, 121, 122, 123, 124, 125, 126, 127, 128, 129 or 130.

Also contemplated are methods for controlling lepidopteran pests comprising the step of contacting the lepidopteran pest with an inhibitory amount of a chimeric insecticidal protein of the present invention.

In another embodiment of the invention, this document describes a transgenic plant cell, plant or plant part containing a chimeric insecticidal protein, and the chimeric insecticidal protein contains any amino acid sequence specified in any of SEQ ID NO: 21, 10, 28, 7, 4, 13, 16, 19, 23, 25, 30, 33, 36, 39, 41, 43, 45, 47, 50, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109 or 111; or the chimeric insecticidal protein contains a protein having at least 94% identity with SEQ ID NO: 21, 10; at least 93% identity with SEQ ID NO: 28, at least 87% identity with SEQ ID NO: 7; at least 90% identity with SEQ ID NO: 4; at least 91% identity with SEQ ID NO: 13; at least 64% identity with SEQ ID NO: 16; at least 66% identity with SEQ ID NO: 19; at least 86% identity with SEQ ID NO: 23; at least 91% identity with SEQ ID NO: 25; at least 94% identity with SEQ ID NO: 30; at least 91% identity with SEQ ID NO: 33; at least 64% identity with SEQ ID NO: 36; at least 66% identity with SEQ ID NO: 39; at least 94% identity with SEQ ID NO: 41; at least 8 4% identity with SEQ ID NO: 43; at least 93% identity with SEQ ID NO: 45; at least 94% identity with SEQ ID NO: 47; at least 91% identity with SEQ ID NO: 50; or at least 93% identity with SEQ ID NO: 53; or at least 87% identity with SEQ ID NO: 85, 93, 105; or at least 85% identity with SEQ ID NOs: 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79; or at least 88% identity with SEQ ID NOs: 91, 87, 89; or at least 89% identity with SEQ ID NO: 107, 111; or at least 90% identity with SEQ ID NO: 97; at least 91% identity with SEQ ID NO: 109; or at least 93% identity with SEQ ID NO: 83; or at least 94% identity with SEQ ID NO: 91 or 103; or at least 95% identity with SEQ ID NO: 95, 101; or at least 98% identity with SEQ ID NO: 99. Methods for controlling lepidoptera pests are also contemplated, comprising the step of exposing the transgenic plant cell, plant, or plant part to the pest, wherein said plant cell, plant, or plant part expresses a Lepidoptera inhibitory amount of the chimeric insecticidal protein.

In other embodiments, this document provides commercial products derived from a plant cell, plant, or plant part, wherein the product contains a detectable amount of a chimeric insecticidal protein. Commercial products under consideration include vegetable biomass, oil, flour, animal feed, flour, flakes, bran, fluff, rinds and processed seeds.

Yet another method disclosed herein is a method for producing a seed containing a chimeric insecticidal protein, comprising the steps of: planting at least one seed containing the chimeric insecticidal protein; growing plants from said seed; and harvesting seed from plants, wherein the harvested seed contains an insecticidal protein.

Recombinant polynucleotide molecules encoding a chimeric insecticidal protein containing a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20 , 22, 24, 26, 27, 29, 31, 32, 34, 35, 37, 38, 40, 42, 44, 46, 48, 49, 51, 52, 54, 56,

- 4 040497

58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112

113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129 or 130; and optionally a polynucleotide sequence encoding an insect inhibitory agent other than the chimeric insecticidal protein.

Another recombinant nucleic acid molecule contemplated herein comprises a heterologous promoter operably linked to a polynucleotide segment encoding chimeric insecticidal proteins, the chimeric insecticidal protein comprising any amino acid sequence shown in any of SEQ ID NOs: 21, 10, 28, 7, 4 , 13, 16, 19, 23, 25, 30, 33, 36, 39, 41, 43, 45, 47, 50, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73 , 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109 or 111; or the chimeric insecticidal protein contains a protein having: at least 94% identity with SEQ ID NO: 21, 10; at least 93% identity with SEQ ID NO: 28; at least 87% identity with SEQ ID NO: 7; at least 90% identity with SEQ ID NO: 4; at least 91% identity with SEQ ID NO: 13; at least 64% identity with SEQ ID NO: 16; at least 66% identity with SEQ ID NO: 19; at least 86% identity with SEQ ID NO: 23; at least 91% identity with SEQ ID NO: 25; at least 94% identity with SEQ ID NO: 30; at least 91% identity with SEQ ID NO: 33; at least 64% identity with SEQ ID NO: 36; at least 66% identity with SEQ ID NO: 39; at least 94% identity with SEQ ID NO: 41; at least 8 4% identity with SEQ ID NO: 43; at least 93% identity with SEQ ID NO: 45; at least 94% identity with SEQ ID NO: 47; at least 91% identity with SEQ ID NO: 50; or at least 93% identity with SEQ ID NO: 53; or at least 87% identity with SEQ ID NO: 85, 93, 105; or at least 85% identity with SEQ ID NOs: 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79; or at least 88% identity with SEQ ID NOs: 91, 87, 89; or at least 89% identity with SEQ ID NO: 107, 111; or at least 90% identity with SEQ ID NO: 97; or at least 91% identity with SEQ ID NO: 109; or at least 93% identity with SEQ ID NO:83; or at least 94% identity with SEQ ID NO: 91, 103; or at least 95% identity with SEQ ID NO: 95, 101; or at least 98% identity with SEQ ID NO: 99; or the polynucleotide segment hybridizes to a polynucleotide having the nucleotide sequence specified in any of SEQ ID NOs: 1, 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 22, 24 , 26, 27, 29, 31, 32, 34, 35, 37, 38, 40, 42, 44, 46, 48, 49, 51, 52, 54, 56, 58, 60, 62, 64, 66, 68 , 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 113, 114, 115 , 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129 or 130.

Other implementations, features and advantages of the present invention will be apparent from the following detailed description, examples and claims.

Brief description of the sequences

SEQ ID NO: 1 is the recombinant DNA sequence encoding TIC1100 used for expression in a bacterial cell.

SEQ ID NO: 2 is a synthetic DNA sequence encoding TIC1100 for expression in a plant cell.

SEQ ID NO: 3 is a synthetic DNA sequence encoding TIC1100 for expression in a plant cell.

SEQ ID NO: 4 is the amino acid sequence of TIC1100.

SEQ ID NO: 5 is the recombinant DNA sequence encoding TIC860 used for expression in a bacterial cell.

SEQ ID NO: 6 is a synthetic DNA sequence encoding TIC860 for expression in a plant cell.

SEQ ID NO: 7 is the amino acid sequence of TIC860.

SEQ ID NO: 8 is a recombinant DNA sequence encoding TIC867 used for expression in a bacterial cell.

SEQ ID NO: 9 is a synthetic DNA sequence encoding TIC867 for expression in a plant cell.

SEQ ID NO: 10 is the amino acid sequence of TIC867.

SEQ ID NO: 11 is the recombinant DNA sequence encoding TIC867_20 used for expression in a bacterial cell.

SEQ ID NO: 12 is a synthetic DNA sequence encoding TIC867_20 for expression in a plant cell.

SEQ ID NO: 13 is the amino acid sequence of TIC867_20.

SEQ ID NO: 14 is the recombinant DNA sequence encoding TIC867_21 used for expression in a bacterial cell.

SEQ ID NO: 15 is a synthetic DNA sequence encoding TIC867_21 for expression in a plant cell.

SEQ ID NO: 16 is the amino acid sequence of TIC867_21.

- 5 040497

SEQ ID NO: 17 is a recombinant DNA sequence encoding

TIC867_22 used for expression in a bacterial cell.

SEQ ID NO: 18 is a synthetic DNA sequence encoding

TIC867_22, for plant cell expression.

SEQ ID NO: 19 is the amino acid sequence of TIC867_22.

SEQ ID NO: 20 is a synthetic DNA sequence encoding TIC867_23 for expression in a plant cell.

SEQ ID NO: 21 is the amino acid sequence of TIC867_23.

SEQ ID NO: 22 is a synthetic DNA sequence encoding TIC867_24 for expression in a plant cell.

SEQ ID NO: 23 is the amino acid sequence of TIC867_24.

SEQ ID NO: 24 is a synthetic DNA sequence encoding TIC867_24 for expression in a plant cell.

SEQ ID NO: 25 is the amino acid sequence of TIC867_25.

SEQ ID NO: 26 is a recombinant DNA sequence encoding TIC868 used for expression in a bacterial cell.

SEQ ID NO: 27 is a synthetic DNA sequence encoding TIC868 for expression in a plant cell.

SEQ ID NO: 28 is the amino acid sequence of TIC868.

SEQ ID NO: 29 is a synthetic DNA sequence encoding TIC868_9 for expression in a plant cell.

SEQ ID NO: 30 is the amino acid sequence of TIC868_9.

SEQ ID NO: 31 is a recombinant DNA sequence encoding TIC868_10 used for expression in a bacterial cell.

SEQ ID NO: 32 is a synthetic DNA sequence encoding a TIC868 variant, TIC868_10, for expression in a plant cell.

SEQ ID NO: 33 is the amino acid sequence of TIC868_10.

SEQ ID NO: 34 is the recombinant DNA sequence encoding TIC868_11 used for expression in a bacterial cell.

SEQ ID NO: 35 is a synthetic DNA sequence encoding TIC868_11 for expression in a plant cell.

SEQ ID NO: 36 is the amino acid sequence of TIC868_11.

SEQ ID NO: 37 is the recombinant DNA sequence encoding TIC868_12 used for expression in a bacterial cell.

SEQ ID NO: 38 is a synthetic DNA sequence encoding TIC868_12 for expression in a plant cell.

SEQ ID NO: 39 is the amino acid sequence of TIC868_12.

SEQ ID NO: 40 is a synthetic DNA sequence encoding TIC868_13 for expression in a plant cell.

SEQ ID NO: 41 is the amino acid sequence of TIC868_13.

SEQ ID NO: 42 is a synthetic DNA sequence encoding TIC868_14 for expression in a plant cell.

SEQ ID NO: 43 is the amino acid sequence of TIC868_14.

SEQ ID NO: 44 is a synthetic DNA sequence encoding TIC868_15 for expression in a plant cell.

SEQ ID NO: 45 is the amino acid sequence of TIC868_15.

SEQ ID NO: 46 is a synthetic DNA sequence encoding TIC868_29 for expression in a plant cell.

SEQ ID NO: 47 is the amino acid sequence of TIC868_29.

SEQ ID NO: 48 is a recombinant DNA sequence encoding TIC869 used for expression in a bacterial cell.

SEQ ID NO: 49 is a synthetic DNA sequence encoding TIC869 for expression in a plant cell.

SEQ ID NO: 50 is the amino acid sequence of TIC869.

SEQ ID NO: 51 is a recombinant DNA sequence encoding TIC836 used for expression in a bacterial cell.

SEQ ID NO: 52 is a synthetic DNA sequence encoding TIC836 for expression in a plant cell.

SEQ ID NO: 53 is the amino acid sequence of TIC836.

SEQ ID NO: 54 is the DNA sequence encoding the chimeric amino acid sequence of TIC713.

SEQ ID NO: 55 is the amino acid sequence of TIC713 translated

- 6 040497 from the open reading frame indicated in SEQ ID NO: 54.

SEQ ID NO: 56 is the DNA sequence encoding the chimeric amino acid sequence of TIC843.

SEQ ID NO: 57 is the amino acid sequence of TIC843 translated from the open reading frame shown in SEQ ID NO: 56.

SEQ ID NO: 58 is the DNA sequence encoding the chimeric amino acid sequence of TIC862.

SEQ ID NO: 59 is the amino acid sequence of TIC862 translated from the open reading frame shown in SEQ ID NO: 58.

SEQ ID NO: 60 is the DNA sequence encoding the chimeric amino acid sequence of TIC1099.

SEQ ID NO: 61 is the amino acid sequence of TIC1099 translated from the open reading frame shown in SEQ ID NO: 60.

SEQ ID NO: 62 is the DNA sequence encoding the chimeric amino acid sequence TIC1099-T507E.

SEQ ID NO: 63 is the amino acid sequence of TIC1099-T507E translated from the open reading frame shown in SEQ ID NO: 62.

SEQ ID NO: 64 is the DNA sequence encoding the chimeric amino acid sequence TIC1099-R522K.

SEQ ID NO: 65 is the amino acid sequence of TIC1099-R522K translated from the open reading frame shown in SEQ ID NO: 64.

SEQ ID NO: 66 is the DNA sequence encoding the chimeric amino acid sequence TIC1099-K490S.

SEQ ID NO: 67 is the amino acid sequence of TIC1099-K490S translated from the open reading frame shown in SEQ ID NO: 66.

SEQ ID NO: 68 is the DNA sequence encoding the chimeric amino acid sequence TIC1099-T562R.

SEQ ID NO: 69 is the amino acid sequence of TIC1099-T562R translated from the open reading frame shown in SEQ ID NO: 68.

SEQ ID NO: 70 is the DNA sequence encoding the chimeric amino acid sequence TIC1099-S553R.

SEQ ID NO: 71 is the amino acid sequence of TIC1099-S553R translated from the open reading frame shown in SEQ ID NO: 70.

SEQ ID NO: 72 is the DNA sequence encoding the chimeric amino acid sequence TIC1099-G498D.

SEQ ID NO: 73 is the amino acid sequence of TIC1099-G498D translated from the open reading frame shown in SEQ ID NO: 72.

SEQ ID NO: 74 is the DNA sequence encoding the chimeric amino acid sequence TIC1099-K490A.

SEQ ID NO: 75 is the amino acid sequence of TIC1099-K490A translated from the open reading frame shown in SEQ ID NO: 74.

SEQ ID NO: 76 is the DNA sequence encoding the chimeric amino acid sequence TIC1099-E564A.

SEQ ID NO: 77 is the amino acid sequence of TIC1099-E564A translated from the open reading frame shown in SEQ ID NO: 76.

SEQ ID NO: 78 is the DNA sequence encoding the chimeric amino acid sequence of TIC1103.

SEQ ID NO: 79 is the amino acid sequence of TIC1103 translated from the open reading frame shown in SEQ ID NO: 78.

SEQ ID NO: 80 is the DNA sequence encoding the chimeric amino acid sequence TIC1101.

SEQ ID NO: 81 is the amino acid sequence of TIC1101 translated from the open reading frame shown in SEQ ID NO: 80.

SEQ ID NO: 82 is the DNA sequence encoding the chimeric amino acid sequence of TIC845.

SEQ ID NO: 83 is the amino acid sequence of TIC845 translated from the open reading frame shown in SEQ ID NO: 82.

SEQ ID NO: 84 is the DNA sequence encoding the chimeric amino acid sequence of TIC846.

SEQ ID NO: 85 is the amino acid sequence of TIC846 translated from the open reading frame shown in SEQ ID NO: 84.

SEQ ID NO: 86 is the DNA sequence encoding the chimeric amino acid sequence of TIC858.

SEQ ID NO: 87 is the amino acid sequence of TIC858 translated from the open reading frame shown in SEQ ID NO: 86.

SEQ ID NO: 88 is the DNA sequence encoding the chimeric amino acid sequence of TIC865.

SEQ ID NO: 89 is the amino acid sequence of TIC865 translated from the open reading frame shown in SEQ ID NO: 88.

SEQ ID NO: 90 is the DNA sequence encoding the chimeric amino acid sequence of TIC866.

SEQ ID NO: 91 is the amino acid sequence of TIC866 translated from the open reading frame shown in SEQ ID NO: 90.

SEQ ID NO: 92 is the DNA sequence encoding the chimeric amino acid sequence of TIC838.

SEQ ID NO: 93 is the amino acid sequence of TIC838 translated from the open reading frame shown in SEQ ID NO: 92.

SEQ ID NO: 94 is the DNA sequence encoding the chimeric amino acid sequence of TIC839.

SEQ ID NO: 95 is the amino acid sequence of TIC839 translated from the open reading frame shown in SEQ ID NO: 94.

SEQ ID NO: 96 is the DNA sequence encoding the chimeric amino acid sequence of TIC841.

SEQ ID NO: 97 is the amino acid sequence of TIC841 translated from the open reading frame shown in SEQ ID NO: 96.

SEQ ID NO: 98 is the DNA sequence encoding the chimeric amino acid sequence of TIC842.

SEQ ID NO: 99 is the amino acid sequence of TIC842 translated from the open reading frame shown in SEQ ID NO: 98.

SEQ ID NO: 100 is the DNA sequence encoding the chimeric amino acid sequence of TIC850.

SEQ ID NO: 101 is the amino acid sequence of TIC850 translated from the open reading frame shown in SEQ ID NO: 100.

SEQ ID NO: 102 is the DNA sequence encoding the chimeric amino acid sequence of TIC859.

SEQ ID NO: 103 is the amino acid sequence of TIC859 translated from the open reading frame shown in SEQ ID NO: 102.

SEQ ID NO: 104 is the DNA sequence encoding the chimeric amino acid sequence of TIC861.

SEQ ID NO: 105 is the amino acid sequence of TIC861 translated from the open reading frame shown in SEQ ID NO: 104.

SEQ ID NO: 106 is the DNA sequence encoding the chimeric amino acid sequence of TIC848.

SEQ ID NO: 107 is the amino acid sequence of TIC848 translated from the open reading frame shown in SEQ ID NO: 106.

SEQ ID NO: 108 is the DNA sequence encoding the chimeric amino acid sequence of TIC849.

SEQ ID NO: 109 is the amino acid sequence of TIC849 translated from the open reading frame shown in SEQ ID NO: 108.

SEQ ID NO: 110 is the DNA sequence encoding the chimeric amino acid sequence of TIC847.

SEQ ID NO: 111 is the amino acid sequence of TIC847 translated from the open reading frame shown in SEQ ID NO: 110.

SEQ ID NO: 112 is a plant cell encoding TIC713.

SEQ ID NO: 113 is a plant cell encoding TIC713.

SEQ ID NO: 114 is a plant cell encoding TIC843.

SEQ ID NO: 115 is a plant cell encoding TIC862.

SEQ ID NO: 116 is a plant cell encoding TIC1099.

SEQ ID NO: 117 is synthetic synthetic synthetic synthetic synthetic synthetic sequence sequence sequence sequence sequence sequence sequence

DNA

DNA

DNA

DNA

DNA

DNA for for for for for for expression expression expression expression expression expression

- 8 040497 plant cell encoding TIC1103.

SEQ ID NO: 118 is a synthetic plant cell DNA sequence encoding TIC845.

SEQ ID NO: 119 is a synthetic plant cell DNA sequence encoding TIC846.

SEQ ID NO: 120 is a synthetic plant cell DNA sequence encoding TIC858.

SEQ ID NO: 121 is a synthetic plant cell DNA sequence encoding TIC866.

SEQ ID NO: 122 is a synthetic plant cell DNA sequence encoding TIC838.

SEQ ID NO: 123 is a synthetic plant cell DNA sequence encoding TIC841.

SEQ ID NO: 124 is a synthetic plant cell DNA sequence encoding TIC842.

SEQ ID NO: 125 is a synthetic plant cell DNA sequence encoding TIC850.

SEQ ID NO: 126 is a synthetic plant cell DNA sequence encoding TIC859.

SEQ ID NO: 127 is a synthetic plant cell DNA sequence encoding TIC861.

SEQ ID NO: 128 is a synthetic plant cell DNA sequence encoding TIC848.

SEQ ID NO: 129 is a synthetic plant cell DNA sequence encoding TIC849.

SEQ ID NO: 130 is a synthetic plant cell DNA sequence encoding TIC847.

Detailed description of the present invention

A problem in the field of pest control can be for expression in for expression in for expression in for expression in for expression in for expression in for expression in for expression in for expression in for expression in for expression in for expression in for expression in is characterized as the need for novel insecticidal proteins that are effective against target pests, exhibit broad spectrum toxicity to target pest species, are able to be expressed in plants without causing undesirable agronomic problems, and provide an alternative mode of action compared to currently available toxins that use in plants for commercial purposes. This document describes novel chimeric insecticidal proteins and addresses each of these needs, in particular for a wide range of Lepidoptera insect pests.

In order to avoid the development of insect resistance to currently used insecticidal proteins, eliminating their shortcomings, new insecticidal proteins with different mechanisms of action (MA), as well as with a wide spectrum of action and effectiveness for the control of Lepidoptera, are needed. One way to solve this problem is to find new insecticidal proteins from various biological sources, preferably from bacteria, fungi or plants. Another approach is to shuffle segments between different Bt proteins that show structural similarity to create new chimeric Bt proteins with insect inhibitory properties. The likelihood of creating a chimeric protein with improved properties by recombining the domain structures of numerous native insecticidal crystal proteins known in the art is negligible. See, for example, Jacqueline S. Knight, et al. A Strategy for Shuffling Numerous Bacillus thuringiensis Crystal Protein Domains. J. Economic Entomology, 97 (6) (2004): 18051813.

This document describes the sequence of recombinant nucleic acid molecules that encode new chimeric insecticidal proteins. These insecticidal proteins address the current need in the art for the development of additional toxic insecticidal proteins with improved insecticidal properties, such as increased efficacy against a wider range of target insect pest species and different mechanisms of action. Members of this group of proteins, including the exemplary proteins provided herein, exhibit insecticidal activity against Lepidoptera pest species.

The term segment or fragment is used herein to describe continuous amino acid or nucleic acid sequences that are shorter than the full amino acid or nucleotide sequence describing the disclosed chimeric insecticidal protein. A segment or fragment exhibiting insect inhibitory activity is also disclosed herein if alignment of such segment or fragment with the corresponding region of the chimeric insecticidal protein results in amino acid sequence identity of any percentage of a fraction from about 65% to about 100% between the segment or fragment and the corresponding

- 9 040497 plot of chimeric insecticidal protein.

In the context of this application, the terms active or activity, pesticidal activity or pesticidal or insecticidal activity, insect inhibitor or insecticidal refer to the effectiveness of a toxic agent, such as an insecticidal protein, in inhibition (inhibition of growth, nutrition, fertility or viability), suppression (inhibition of growth, feeding, fecundity or viability), control (control of pest infestation, control of feeding intensity of insect pests on a particular crop containing an effective amount of an insecticidal protein), or eradication (causing morbidity, mortality or reduced fertility) of the pest. These terms encompass the result of providing a pesticidally effective amount of an insecticidal protein to a pest, wherein exposure of the insecticidal protein to the pest results in morbidity, mortality, reduced fertility, or stunted growth. The terms also include repelling a pest from a plant, plant tissue, plant part, seed, plant cell, or specific geographic location, which plant can grow by providing a pesticidally effective amount of insecticidal protein to or on the plant. In general, pesticidal activity refers to the ability of a toxic agent to be effective in inhibiting growth, development, viability, foraging behavior, mating behavior, fertility, or any measurable reduction caused by adverse effects, in connection with the provision of a given protein, protein fragment, segment protein or polynucleotide, a specific target pest, including, but not limited to, insects of the order Lepidoptera. The insecticidal protein may be produced by the plant or may be applied to the plant or to the environment where the plant is located. The terms bioactivity, effective, efficient, or variations thereof are also terms used interchangeably throughout this application to describe the effect of the chimeric insecticidal proteins of the present invention on target insect pests.

A pesticidally effective amount of the toxic agent provided in the diet of the target pest exhibits pesticidal activity upon contact of the toxic agent with the pest. The toxic agent may be an insecticidal protein, or one or more chemical agents known in the art. The insecticidal chemical agents and insecticidal proteinaceous agents may be used alone or in combination with each other. Chemical agents include, without limitation, dsRNA molecules targeting specific genes for suppression in the target pest, organochlorides, organophosphates, carbamates, pyrethroids, neonicotinoids, and ryanoids. Insecticidal proteinaceous agents include the chimeric insecticidal proteins provided herein, as well as other proteinaceous toxic agents, including those that target Lepidoptera pest species, as well as proteinaceous toxins that are used to control other plant pests, such as Cry- proteins available in the art for use in the control of Coleoptera, Thysanoptera, Hemiptera and Homoptera species.

Reference to a pest, especially a crop pest, is intended to mean crop pests, especially those Lepidoptera pests controlled by the chimeric insecticidal proteins described. However, reference to insect pest may also include insect pests Coleoptera, Hemiptera and Homoptera, as well as nematodes and fungi, under conditions where the toxic agents that target these pests are colocalized or present with a chimeric insecticidal protein or a protein that has about 65 to about 100% identity with the chimeric insecticidal protein.

The chimeric insecticidal proteins described herein exhibit insecticidal activity against Lepidoptera insect pests, including adults, pupae, larvae, and neonates, as well as members of the order Hemiptera, including adults and nymphs. Insects of the order Lepidoptera include, but are not limited to, prairie armyworm, armyworm, leafworm, and armyworm from the family Noctuidae, e.g. Agrotis ipsilon), cabbage cutworm (Trichoplusia ni), soy cutworm (Pseudoplusia includens), velvet bean cutworm (Anticarsia gemmatalis), clover cutworm (Hypena scabra), tobacco leafworm (Heliothis virescens), cotton cutworm (Agrotis subterranea), meadow cutworm ( Pseudaletia unipuncta), rectangular scoop (Agrotis orthogonia); borers, sheath-bearers, butterflies, smoky leafworms, caterpillars of cabbage butterflies and pyromorphids from the family Pyralidae, for example, stem corn borer (Ostrinia nubilalis), moth moth (Amyelois transitella), corn moth (Crambus caliginosellus), meadow borer (Herpetogramma licarsisalis), sunflower moth (Homoeosoma electellum), corn grinder (Elasmopalpus lignosellus); leafworm, cone moth, codling moth, and fruit leafworms from the Tortricidae family, for example, apple codling moth (Cydia pomonella), grape leafworm (Endopiza viteana), eastern peach leafworm (Grapholita molesta), sunflower leafworm (Suleima helianthana); and many other economically important members of the order Lepidoptera, such as the cabbage moth (Plutella xylostella), the pink bollworm (Pectinophora gossypiella) and the gypsy moth (Lymantria dispar). Other insect pests of the order Lepi

- 10 040497 doptera includes, for example, Alabama argillacea (American cotton bollworm), Archips argyrospila (fruit tree leafworm), Archips rosana (golden leafworm) and other Archips species, Chilo suppressalis (Asian stem moth or yellow rice moth), Cnaphalocrocis medinalis (rice leafworm), Crambus caliginosellus (corn moth), Crambus teterrellus (grass moth), Diatraea grandiosella (southwestern corn moth), Diatraea saccharalis (sugarcane moth), Earias insulana (Egyptian cotton bollworm), Earias vittella (spotted armyworm ), Helicoverpa armigera (American cutworm), Helicoverpa zea (cotton cutworm or American corn cutworm), Heliothis virescens (tobacco leafworm), Herpetogramma licarsisalis (meadow moth), Lobesia botrana (cluster leafworm), Phyllocnistis citrella (citrus passer moth), Pieris brassicae (cabbage white), Pieris rapae (turnip or turnip white), Plutell a xylostella (cabbage moth), Spodoptera exigua (small cutworm), Spodoptera litura (Asian cotton bollworm, Asian cotton bollworm) and Tuta absoluta (nightshade miner).

In the context of this application, the term isolated DNA molecule or an equivalent term or phrase means that a DNA molecule is a DNA molecule that is present alone or in combination with other compositions, but not in its natural environment. For example, nucleic acid elements such as a coding sequence, an intron sequence, an untranslated leader sequence, a promoter sequence, a transcription termination sequence, and the like, which are naturally found in the DNA of an organism's genome, are not considered isolated as long as the element is in the organism's genome and at the location in the genome where it occurs naturally. However, each of these elements and portions thereof would be isolated within the scope of this disclosure provided that the element is within the organism's genome and at the location in the genome where it naturally occurs. Similarly, a nucleotide sequence encoding an insecticidal protein, or any naturally occurring insecticidal variant of that protein, will be an isolated nucleotide sequence if the nucleotide sequence is not found in the DNA of a bacterium that naturally contains the sequence encoding the protein. A synthetic nucleotide sequence encoding the amino acid sequence of a natural insecticidal protein will be considered isolated for the purposes of this disclosure. For the purposes of this disclosure, any transgenic nucleotide sequence, i. a DNA nucleotide sequence inserted into the cell genome of a plant or bacterium or present in an extrachromosomal vector is considered an isolated nucleotide sequence, whether it is present within a plasmid or similar structure used for cell transformation, in the genome of a plant or bacterium, or present in detectable amounts in tissues, progeny, biological samples or commercial products derived from a plant or bacterium.

As described in the examples below, approximately eight hundred and forty-four (844) nucleotide sequences were constructed from the protoxin and toxin domains of known insecticidal toxins (referred to herein as parent proteins) by chimerogenesis, which encode chimeric insecticidal proteins, and were expressed and tested in a bioassay for activity against Lepidoptera. A small number of engineered chimeric insecticidal proteins have shown increased activity against Lepidoptera or increased spectrum against Lepidoptera compared to the original proteins from which its toxin components were derived.

These new chimeric insecticidal proteins with increased activity against Lepidoptera or enhanced spectrum against Lepidoptera were constructed from the following protoxin and toxin domains of the original insecticidal proteins: Cry1Ah (domain I), Cry1Bb1 (domains I and II), Cry1Be2 (domains I and II) , Cry1Ja1 (domains I and II), Cry1Fa1 (domains I and II), Cry1Ac (domain II and protoxin), Cry1Ca (domain III and protoxin), Cry1Ka (domain III and protoxin), Cry1Jx (domain III), Cry1Ab (domain III), Cry1Ab3 (protoxin), Cry1Da1 (protoxin), Cry4 (protoxin), Cry9 (protoxin), Cry1Be (protoxin), and Cry1Ka (protoxin).

In particular, novel chimeric insecticidal proteins of the present invention with increased activity against Lepidoptera or enhanced spectrum of Lepidoptera include the following combinations of protoxins and domains: TIC1100/SEQ ID NO: 4 (domain I-Cry1Ah, domain II-Cry1Ac, domain III-Cry1Ca, protoxin-Cry1Ac), TIC860/SEQ ID NO: 7 (domain I-Cry1Bb1, domain II-Cry1BB1, domain III-Cry1Ca, protoxin-Cry1Ac), TIC867/SEQ ID NO: 10 (domain I-Cry1Be2, domain II-Cry1Be2 , domain IIICry1Ka, protoxin-Cry1Ab3), TIC868/SEQ ID NO: 28 (domain I-Cry1Be2, domain II-Cry1Be2 and domain IIICry1Ca, protoxin-Cry1Ab3), TIC869/SEQ ID NO: 50 (domain I-Cry1Ja1, domain II -Cry1Ja1, domain IIICry1Jx, protoxin-Cry1Ab3) and TIC836/SEQ ID NO: 53 (domain I-Cry1Fa1, domain II-Cry1Fa1, domain IIICry1Ab, protoxin-Oy1Ac).

Variants in which amino acid substitutions or alternative protoxin domains have been introduced have also been constructed for the TIC867 and TIC868 chimeric insecticidal proteins. In particular, these variants of TIC867 and TIC868 include the following amino acid substitutions or alternative protoxin domains: TIC867_20/SEQ ID NO: 13 (Cry1Da1 alternative protoxin domain),

- 11 040497

TIC867_21/SEQ ID NO: 16 (Cry4 alternative protoxin domain), TIC867_22/SEQ ID NO: 19 (Cry9 alternative protoxin domain), TIC867_23/SEQ ID NO: 21 (Cry1Be alternative protoxin domain), TIC867_24/SEQ ID NO: 23 ( Cry1Ka alternative protoxin domain), TIC867_25/SEQ ID NO: 25 (Cry1Ka alternative protoxin domain), TIC868_9/SEQ ID NO: 30 (N240S_Y343Q_N349T amino acid modification), TIC868_10/SEQ ID NO: 33 (Cry1Da1 alternative protoxin domain), TIC868_11 ID NO: 36 (alternative Cry4 protoxin domain), TIC868_12/SEQ ID NO: 39 (alternative protoxin Cry 9 domain), TIC868_13/SEQ ID NO: 41 (alternative Cry1Be protoxin domain), TIC868_14/SEQ ID NO: 43 (alternative protoxin Cry1Ka domain), TIC868_15/SEQ ID NO: 45 (alternative Cry1Ca protoxin domain) and TIC868_29/SEQ ID NO: 47 (amino acid modification Q136Y_Y343Q_N349T).

As demonstrated in the examples, each of these variants of TIC867 and TIC868 has an altered lepidoptera activity and/or reduced lepidoptera activity spectrum of the parent chimeric insecticidal protein, thus indicating that the alternative protoxin domain and amino acid substitutions had a direct effect on insecticidal activity and spectrum of chimeric insecticidal proteins TIC867 and TIC868.

Many of the chimeric insecticidal proteins exhibit insecticidal activity against numerous species of Lepidoptera insect pests. In particular, the new chimeric insecticidal proteins disclosed in this application exhibit activity against one or more of the following lepidopteran insect pests: velvet bean cutworms (VBC, Anticarsia gemmatalis), sugar cane moths (SCB, Diatraea saccharalis), corn borer (LSCB, Elasmopalpus lignosellus), American corn armyworm (CEW, Helicoverpa zea), soybean armyworm (SPW, Helicoverpa zea), cotton bollworm CBW, Helicoverpa zea), tobacco leafworm (TBW, Heliothis virescens), soybean armyworm (SBL, Chrysodeixis includens ), Black armyworm (BLAW, Spodoptera cosmioides), Southern armyworm (SAW, Spodoptera eridania), Corn armyworm (FAW, Spodoptera frugiperda), Little armyworm (BAW, Spodoptera exigua), American armyworm (OBW, Helicoverpa armigera), Asian cotton armyworm cutworm (OLW, Spodoptera litura), pink bollworm (PBW, Pectinophora gossypiella), southwestern corn moth (SWCB, Diatraea grandiosella), spiny northern bark sideworm (SBW, Earias vitella), South American armyworm (SABW, Helicoverpa gelotopeon), and sunflower moth (SFL, Rachiplusia nu). Thus, the exemplary proteins described herein share a common function and exhibit insecticidal activity against insect pest species of the order Lepidoptera, including adults, larvae, and pupae.

Proteins that resemble chimeric insecticidal proteins can be identified by comparison with each other using various computer algorithms known in the art. For example, amino acid sequence identity of proteins associated with chimeric insecticidal proteins can be analyzed using Clustal W alignment using these default parameters: comparison matrix: weights, alignment deletion penalty: 10.0, deletion extension penalty: 0 .05, hydrophilic deletions: on, hydrophilic residues: GPSNDQERK, residue specific deletion penalty: on (Thompson, et al (1994) Nucleic Acids Research, 22:4673-4680). Percent amino acid identity is further calculated as: 100% times (amino acid identity/claimed protein length). Other alignment algorithms also available in the art provide similar results to those obtained using Clustal W alignment and are discussed in this application.

It is assumed that the target protein exhibiting insect inhibitory activity is disclosed in this application if the alignment of such target protein with the test chimeric insecticidal proteins indicated in SEQ ID NO: 4, 7, 10, 13, 16, 19, 21, 23 , 25, 28, 30, 33, 36, 39, 41, 43, 45, 47, 50, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79 , 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109 and 111 results in at least about 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, 96, 97, 98, 99, or about 100% amino acid sequence identity (or any percentage within this range) between the protein of interest and the protein of interest.

As described further in the examples of this application, synthetic or artificial sequences encoding chimeric insecticidal proteins have been designed for use in plants. Illustrative synthetic nucleotide sequences that have been designed for use in plants are shown in SEQ ID NO: 2 and 3 (TIC1100), SEQ ID NO: 6 (TIC860), SEQ ID NO: 9 (TIC867), SEQ ID NO: 12 ( TIC867_20), SEQ ID NO: 15 (TIC867_21), SEQ ID NO: 18 (TIC867_22), SEQ ID NO: 20 (TIC867_23), SEQ ID NO: 22 (TIC867_24), SEQ ID NO: 24 (TIC867_25), SEQ ID NO: 27 (TIC868), SEQ ID NO: 29 (TIC868_9), SEQ ID NO: 32 (TIC868_10), SEQ ID NO: 35 (TIC868_11), SEQ ID NO: 38 (TIC868_12), SEQ ID NO: 40 (TIC868_13) ), SEQ ID NO: 42 (TIC868_14), SEQ ID NO: 44 (TIC868_15), SEQ ID NO: 46 (TIC868_29), SEQ ID NO: 49 (TIC869) and SEQ ID NO: 52 (TIC836), SEQ ID NO : 112 and 113 (TIC713), SEQ ID NO: 114 (TIC843), SEQ ID NO: 115 (TIC862), SEQ ID NO: 116 (TIC1099), SEQ ID NO: 117 (TIC1103), SEQ ID NO: 118 ( TIC845), SEQ ID NO: 119 (TIC846), SEQ ID NO: 120

- 12 040497 (TIC858), SEQ ID NO: 121 (TIC866), SEQ ID NO: 122 (TIC838), SEQ ID NO: 123 (TIC841), SEQ ID NO:

124 (TIC842), SEQ ID NO: 125 (TIC850), SEQ ID NO: 126 (TIC859), SEQ ID NO: 127 (TIC861), SEQ ID

NO: 128 (TIC848), SEQ ID NO: 129 (TIC849), and SEQ ID NO: 130 (TIC847).

For expression in plant cells, the chimeric insecticidal proteins can be expressed so that they reside in the cytosol or are targeted to different plant cell organelles. For example, targeting a protein to a chloroplast can increase the levels of expressed protein in a transgenic plant, preventing the appearance of other phenotype traits. Targeting can also lead to increased pest resistance in the transgenic event. The target peptide or transit peptide is a short peptide chain (3-70 amino acids long) that directs protein transport to a specific region of the cell, including the nucleus, mitochondria, endoplasmic reticulum (ER), chloroplast, apoplast, peroxisome, and plasma membrane. Some signal peptides are cleaved from the protein by signal peptidase after protein transport. To target the chloroplast, proteins contain transit peptides that contain about 40-50 amino acids. For a description of the use of chloroplast transit peptides, see US Pat. Examples of such isolated chloroplast proteins include, without limitation, those associated with the small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase, ferredoxin, ferredoxin oxidoreductase, protein I and protein II of the light harvesting complex, thioredoxin F, enolpyruvate shikimate phosphate synthase ( EPSP) and the transit peptides described in US Pat. Incorporation of a suitable chloroplast transit peptide such as Arabidopsis thaliana TPX EPSP (TPX2) (see Klee et al., Mol. Gen. Genet. 210:437-442, 1987) or Petunia hybrida EPSP TPX (TPX4) (see della - Cioppa et al., Proc. Natl. Acad. Sci. USA83: 6873-6877, 1986) have been shown to target heterologous EPSP protein sequences to chloroplasts in transgenic plants (see US Pat. Nos. 5,627,061, 5,633,435 and 5,312,910 and EP 0218571, EP 189707, EP 508909 and EP 924299). To target chimeric insecticidal proteins to the chloroplast, the chloroplast transit peptide coding sequence is 5' operably linked and in frame to a synthetic coding sequence encoding a chimeric insecticidal protein that has been engineered for optimal expression in plant cells.

Expression cassettes and vectors containing these synthetic or artificial nucleotide sequences were constructed and introduced into corn, cotton and soybean plant cells according to transformation methods and techniques known in the art. The transformed cells were regenerated into transformed plants which were found to express the chimeric insecticidal protein. To test pesticidal activity, biotesting was carried out in the presence of larvae of lepidopteran pests using plant leaf disks obtained from transformed plants. Compositions of recombinant nucleic acid molecules that encode chimeric insecticidal proteins are considered. For example, chimeric insecticidal proteins can be expressed by recombinant DNA constructs in which the ORF polynucleotide molecule encoding the chimeric insecticidal protein is operably linked to genetic expression elements such as a promoter and any other regulatory element necessary for expression in the system for which the construct is intended. . Non-limiting examples include a plant functional promoter operably linked to a synthetic chimeric insecticidal protein coding sequence to express the chimeric insecticidal protein in plants, or a Bt functional promoter operably linked to a chimeric insecticidal protein coding sequence to express the protein in a Bt bacterium or other bacillus. Other elements may be operably linked to sequences encoding chimeric insecticidal proteins, including, but not limited to, enhancers, introns, untranslated leaders, encoded protein immobilization tags (histidine tag), translocation peptides (i.e., plastid transit peptides, signal peptides), polypeptide sequences for post-translational modifying enzymes, ribosome binding sites and RNAi target sites.

Examples of recombinant polynucleotide molecules provided herein include, without limitation, a heterologous promoter operably linked to a polynucleotide such as SEQ ID NO: 1, 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17 , 18, 20, 22, 24, 26, 27, 29, 31, 32, 34, 35, 37, 38, 40, 42, 44, 46, 48, 49, 51, 52, 54, 56, 58, 60 , 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110 . in SEQ ID NO: 4 (TIC1100), 7 (TIC860), 10 (TIC867), 13 (TIC867_20), 16 (TIC867_21), 19 (TIC867_22), 21 (TIC867_23), 23 (TIC867_24), 25 (TIC867_25), 28 (TIC868), 30 (TIC868_9), 33

- 13 040497 (TIC868_10), 36 (TIC868_11), 39 (TIC867_12), 41 (TIC867_13), 43 (TIC867_14), 45 (TIC867_15), 47 (TIC867_29), 50 (TIC869), 53 (TIC836), 55 (TIC711) ), 57 (TIC843), 59 (TIC862), 61 (TIC1099), 63 (TIC1099T507E), 65 (TIC1099-R522K), 67 (TIC1099-K490S), 69 (TIC1099-T562R), 71 (TIC1099-S533R), 73 (TIC1099-G498D), 75 (TIC1099-K490A), 77 (TIC1099-E564A), 79 (TIC1103), 81 (TIC1101), 83 (TIC845), 85 (TIC846), 87 (TIC858), 89 (TIC865) , 91 (TIC866), 93 (TIC838), 95 (TIC839), 97 (TIC841), 99 (TIC842), 101 (TIC850), 103 (TIC859), 105 (TIC861), 107 (TIC848), 109 (TIC849) and 111 (TIC847). The heterologous promoter can also be operably linked to synthetic DNA coding sequences encoding a plastid-targeted chimeric insecticidal protein and a non-targeted chimeric insecticidal protein. It is contemplated that the codons of the recombinant nucleic acid molecule encoding the chimeric insecticidal protein described herein may be replaced by synonymous codons (known in the art as silent substitutions).

A recombinant DNA molecule or construct containing a sequence encoding a chimeric insecticidal protein may further comprise a region of DNA that encodes one or more toxic agents that can be engineered to be co-expressed or co-expressed with a DNA sequence encoding a chimeric insecticidal protein other than the chimeric one. an insecticidal protein, a dsRNA molecule having insect inhibitory activity, or an accessory protein. Accessory proteins include, without limitation, cofactors, enzymes, binding partners, or other agents that enhance the effectiveness of an insect inhibitory agent, for example, by promoting its expression, affecting its stability in plants, optimizing free energy for oligomerization, increasing its toxicity and increasing its spectrum of activity. The accessory protein may facilitate the uptake of one or more insect inhibitory agents, for example, or enhance the toxic effect of the toxic agent.

The recombinant DNA molecule or construct can be assembled such that all proteins or dsRNA molecules are expressed from a single promoter, or each protein or dsRNA molecule is expressed under the control of a single promoter, or in some combination thereof. The proteins of the present invention can be expressed from a multigene expression system in which the chimeric insecticidal protein is expressed from a common nucleotide segment that also contains other open reading frames and promoters, depending on the type of expression system chosen. For example, a bacterial multigene expression system may use a single promoter to drive the expression of multiple/tandem open reading frames from a single operon (ie, polycistronic expression). In another example, a plant multigene expression system may use multiply unlinked expression cassettes, each of which expresses a different protein or other toxic agent, such as one or more dsRNA molecules.

Recombinant nucleic acid molecules or recombinant DNA constructs containing a sequence encoding a chimeric insecticidal protein can be delivered to host cells by vectors such as a plasmid, baculovirus, synthetic chromosome, virion, cosmid, phagemid, phage, or viral vector. Such vectors can be used to achieve stable or transient expression of the chimeric insecticidal protein coding sequence in the host cell, or later expression of the encoded polypeptide. An exogenous recombinant polynucleotide or recombinant DNA construct that contains a sequence encoding a chimeric insecticidal protein and that is introduced into a host cell, referred to herein as a transgene.

Provided herein are transgenic bacteria, transgenic plant cells, transgenic plants, and transgenic plant parts that contain a polynucleotide that encodes any one or more chimeric insecticidal proteins. The term bacterial cell or bacterium may include, without limitation, an Agrobacterium, Bacillus, Escherichia, Salmonella, Pseudomonas, or Rhizobium cell. The term plant cell or plant may include, without limitation, a dicot cell or a monocot cell. Contemplated plants and plant cells include, without limitation, a cell or plant of alfalfa, banana, barley, bean, broccoli, cabbage, carrot, cassava, castor bean, cauliflower, celery, chickpea, bok choy, citrus, coconut, clover, cotton, pumpkin, cucumber, Douglas fir, eggplant, eucalyptus, flax, garlic, grapes, hops, leek, lettuce, Loblolly pine, millet, melon, walnut, oats, peas, peanuts, pepper, pigeon pea, pine, potato, poplar, pumpkin, radish pine, radish, canola, rice, cereal, rye, American saffron, shrub, sorghum, southern pine, soybean, spinach, squash, strawberry, sugar beet, sugar cane, sunflower, sweet corn, amberwood, sweet potatoes, millet, tea, tobacco, tomato, triticale, turf grass, watermelon, wheat. In some embodiments, transgenic plants and parts of transgenic plants that have been regenerated from a transgenic plant cell are provided. In some embodiments of the invention, transgenic plants can be obtained from a transgenic seed by cutting, picking, grinding, or otherwise separating a part from a plant. In certain embodiments, the plant part may be a seed, pod, leaf, flower, stem, root, or any part thereof or a non-reproducible part of a transgenic plant part. As used herein, a non-regenerating portion of a transgenic plant part is a portion that cannot be induced to form a whole plant or that cannot be induced to form a whole plant capable of sexual and/or asexual reproduction. In some embodiments, the non-regenerated portion of the selected plant part is a portion of a transgenic seed, bolus, leaf, flower, stem, or root.

Methods for obtaining transgenic plants that contain an amount of chimeric insecticidal proteins that inhibit Lepidoptera insects are presented. Such plants can be obtained by introducing a polynucleotide that encodes the chimeric insecticidal proteins provided herein into a plant cell and selecting a plant derived from said plant cell that expresses an insect or Lepidoptera inhibitory amount of the insecticidal substance. Plants can be obtained from plant cells by regenerating seeds, pollen or meristems obtained by transformation methods. Methods for transforming plants are known in the art. For example, Agrobacterium mediated transformation is described in US Patent Application Publications 2009/0138985 A1 (soybean), 2008/0280361 A1 (soybean), 2009/0142837 A1 (corn), 2008/0282432 (cotton) and 2008/0256667 (cotton) .

Plants expressing chimeric insecticidal proteins 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 resistance genes, yield or stress tolerance genes, and so on. or features can be combined into one vector so that all features are related.

Processed plant products, wherein the processed product contains a detectable amount of a chimeric insecticidal protein, segments and fragments having insect inhibitory activity, or a fragment or any distinctive part thereof, are also disclosed in this application. In some embodiments, the treated product is selected from the group consisting of plant parts, plant biomass, oil, flour, sugar, animal feed, flour, flakes, bran, fiber, treated seed and seed husks. In certain embodiments of the invention, the processed product is non-recyclable. The plant product may include commercial or other commercial products derived from the transgenic plant or part of the transgenic plants, wherein the commercial or other products can be traced by detecting nucleotide segments or expressed RNA or proteins that encode or contain distinct portions of the chimeric insecticidal protein.

Also disclosed in this application are methods for controlling insects, in particular infestations of cultivated plants with lepidopteran insects using chimeric insecticidal proteins. Such methods may include growing a plant containing a Lepidoptera insect-inhibiting amount of the chimeric insecticidal protein. In some embodiments of the invention, such methods may further include any one or more of: (i) applying any composition containing or encoding a chimeric insecticidal protein to a plant or seed that gives rise to a plant; and (ii) transforming a plant or plant cell that gives rise to a plant with a polynucleotide encoding a chimeric insecticidal protein. In general, it is contemplated that the chimeric insecticidal protein can be provided in a composition present in a microorganism or present in a transgenic plant to provide inhibitory activity against lepidopteran insects.

In some embodiments, the chimeric insecticidal protein is an insecticidally active ingredient in a composition having insect inhibitory activity obtained by culturing a recombinant Bacillus or any other recombinant bacterial cell transformed to express the chimeric insecticidal protein under conditions suitable for expression. Such a composition can be obtained by drying, lyophilizing, homogenizing, extracting, filtering, centrifuging, sedimenting or concentrating a culture of such recombinant cells expressing/producing the chimeric insecticidal protein. Such a process may result in the formation of a cell extract, cell suspension, cell homogenate, cell lysate, cell supernatant, cell filtrate, or cell precipitate from Bacillus or other entomopathogenic bacteria. Having obtained the engineered insecticidal protein in this way, a composition that contains the engineered insecticidal protein can contain bacterial cells, bacterial spores, as well as parasporal bodies and can be supplied for various applications, including spray products having inhibitory activity against agricultural insects or preparations of fodder biomass with inhibitory activity against insects in fodder bioassays.

The above compound or preparation may further contain an agriculturally acceptable carrier such as bait, powder, dust, tablets, granules, sprays, emulsion, colloidal suspension, aqueous solution, Bacillus spores or crystalline preparations or processed seeds.

- 15 040497 exchange. The compound or preparation may also further comprise a recombinant plant cell, plant tissue, seed, or plant transformed to express one or more proteins; or bacteria transformed to express one or more proteins. Depending on the level of insect or insecticidal inhibition inherent in the recombinant polypeptide and the level of the compound or preparation to be used in the plant or diet assay, the compound or preparation may contain varying weight amounts of the recombinant polypeptide, for example, from 0.0001 to 0.001 , up to 0.01, up to 1, up to 99% by weight of the recombinant polypeptide.

In an embodiment of the invention, in order to reduce the likelihood of developing resistance, the composition having inhibitory activity against insects, or a transgenic plant containing a chimeric insecticidal protein, may further contain at least one additional toxic agent that exhibits inhibitory activity against the same species of Lepidoptera insects, but which is different from the chimeric insecticidal protein. Possible additional toxic agents for such a composition comprise a protein having inhibitory activity against insects and a dsRNA molecule having inhibitory activity against insects. One example of the use of such ribonucleotide sequences for insect pest control is described in Baum, et al. (US Patent Publication 2006/0021087 A1). Such additional Lepidoptera pest control polypeptide(s) may be selected from the group consisting of a protein having insect inhibitory activity such as, but not limited to, Cry1A (US Pat. No. 5,880,275), Cry1Ab, Cry1Ac, Cry1A.105, Cry1Ae, Cry1B (U.S. Patent Publication 10/525,318), Cry1C (U.S. Patent No. 6,033,874), Cry1D, Cry1E, Cry1F, and Cry1A/F Chimeras (U.S. Patents 7,070,982, 6,962,705, and 6,713,063), Cry1G, Cry1H, Cry1I, Cry1J, Cry1K, Cry1L, Cry2A, Cry2Ab (U.S. Patent No. 7064249), Cry2Ae, Cry4B, Cry6, Cry7, Cry8, Cry9, Cryl5, Cry43A, Cry43B, Cry51Aal, ET66, TIC400, TIC800, TIC834, TIC1415, Vip3A, VIP3Ab, VIP-3B, AXMI 001, AXMI-002, AXMI-030, AXMI-035 and AXMI-045 (U.S. Patent Publication 2013-0117884 A1), AXMI-52, AXMI-58, AXMI-88, AXMI-97, AXMI-102, AXMI-112 , AXMI-117, AXMI-100 (US Patent Publication 2013-0310543 A1), AXMI-115, AXMI-113, AXMI-005 (US Patent Publication 2013-0104259 A1), AXMI-134 (US Patent Publication 2013-0167264 A1 ), AXMI-150 (patent publication US 2010-0160231 A1), AXMI-184 (US Patent Publication 2010-0004176 A1), AXMI-196, AXMI-204, AXMI-207, AXMI-209 (US Patent Publication 2011-0030096 A1), AXMI-218, AXMI -220 (US Patent Publication 2014-0245491 A1), AXMI221z, AXMI-222z, AXMI-223z, AXMI-224z, AXMI-225z (US Patent Publication 2014-0196175 A1), AXMI-238 (US Patent Publication 2014-0033363 A1 ), AXMI-270 (U.S. Patent Pub 20140223598 A1), AXMI-345 (U.S. Patent Pub 2014-0373195 A1), DIG-3 (U.S. Patent Pub 2013-0219570 Al), DIG-5 (U.S. Patent Pub 2010-0317569 A1 ), DIG-11 (US Patent Publication 2010-0319093 A1), AfIP-1A and its derivatives (US Patent Application Publication 2014-0033361 A1), AfIP-1B and its derivatives (US Patent Publication 2014-0033361 A1), PIP- 1APIP-1B (U.S. Patent Pub 2014-0007292 A1), PSEEN3174 (U.S. Patent Pub 2014-0007292 A1), AECFG592740 (Patent Pub 2014-0007292 A1), Pput_1063 (U.S. Patent Pub 2014-0007292 A1), Pput_1064 (U.S. Patent Pub 2014-0007292 A1), 2014 -0007292 A1), GS-135 and its derivatives (U.S. Patent Pub 2012-0233726 A1), GS153 and its derivatives (U.S. Patent Pub 2012-0192310 A1), GS154 and its derivatives (U.S. Patent Pub 2012-0192310 A1), GS155 and its derivatives (Patent Publication 2012-0192310 A1), SEQ ID NO: 2 and its derivatives as described in US Patent Publication 2012-0167259 A1, SEQ ID NO: 2 and its derivatives as described in US Patent Publication 20120047606 A1, SEQ ID NO: 2 and its derivatives as described in US Patent Publication 2011-0154536 A1, SEQ ID NO: 2 and its derivatives as described in US Patent Publication 2011-0112013 A1, SEQ ID NO: 2 and 4, and its derivatives as described in US Patent Publication 2010-0192256 A1, SEQ ID NO: 2 and derivatives thereof as described in US Patent Publication 2010-0077507 A1, SEQ ID NO: 2 and derivatives thereof as described in US Patent Publication 2010- 0077508 A1, SEQ ID NO: 2 and derivatives thereof, as described in US Patent Publication 2009-0313721 A1, SEQ ID NO: 2 or 4, and derivatives thereof, as described in US Patent Publication 2010-0269221 A1, SEQ ID NO: 2 and its derivatives as described in US Pat. their derivatives as described in US2008-0172762 A1, US2011-10055968 A1 and US2012-0117690 A1; SEQ ID NO: 2 and its derivatives, as described in US7510878 (B2), SEQ ID NO: 2 and its derivatives, as described in US patent No. 7812129 (B1); etc.

In other embodiments of the invention, the composition or transgenic plant having insect inhibitory activity may further comprise at least one additional toxic agent that exhibits inhibitory activity against insect pests that are not inhibited by the chimeric insecticidal proteins of the present invention (such insects pests such as Coleoptera, Hemiptera and Homoptera) in order to expand the spectrum of inhibitory activity against insects.

Such an additional toxic agent for the control of beetle pests

- 16 040497 may be selected from the group consisting of a protein having inhibitory activity against insects, such as, without limitation, Cry3Bb (US patent No. 6501009), Cry1C variants, Cry3a variants, Cry3, Cry3B, Cry34/35, 5307, AXMI134 (U.S. Patent Pub 2013-0167264 A1) AXMI-184 (U.S. Patent Pub 2010-0004176 A1), AXMI-205 (U.S. Patent Pub 20140298538 A1), axmi207 (U.S. Patent Pub 2013-0303440 A1), AXMI-218, AXMI -220 (U.S. Patent Pub 20140245491 A1), AXMI-221z, AXMI-223z (U.S. Patent Pub 2014-0196175 A1), AXMI-279 (U.S. Patent Pub 2014-0223599 A1), AXMI-R1 and variants thereof (U.S. Patent Pub. 2010-0197592 A1, TIC407, TIC417, TIC431, TIC807, TIC853, TIC901, TIC1201, TIC3131, DIG-10 (US Patent Publication 2010-0319092 A1), eHIPs (US Patent Application Publication 2010/0017914) US Pat. No. 2012-0210462 A1) and et-hexatoxin-Hv1a (Patent Application Publication US2014-0366227 A1).

Such additional hemipteran pest control toxic agent may be selected from the group consisting of proteins active against hemipterans such as, without limitation, TIC1415 (U.S. Patent Publication 2013-0097735 A1), TIC807 (U.S. Patent No. 8609936), TIC834 (US Patent Publication 2013-0269060 A1), AXMI-036 (US Patent Publication 20100137216 A1), and AXMI-171 (US Patent Publication 2013-0055469 A1). Additional polypeptides for the control of Coleoptera, Lepidoptera and Homoptera insect pests can be found on the Bacillus thuringiensis toxin nomenclature site maintained by Neil Creekmore (on the World Wide Web at btnomenclature.info).

Sequences encoding chimeric insecticidal proteins and sequences having a significant percentage identity with 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 proteins and can be used to screen for other proteins that are closely related.

In addition, nucleotide sequences encoding chimeric insecticidal proteins can be used as probes and primers for screening and identifying other members of the class using thermal cycling or isothermal amplification and hybridization techniques. For example, oligonucleotides derived from the sequences set forth in SEQ ID NO: 2 can be used to determine the presence or absence of a chimeric insecticidal transgene in a deoxyribonucleic acid sample obtained from a commercial product. Given the sensitivity of certain nucleic acid detection methods that use oligonucleotides, it is contemplated that oligonucleotides derived from the sequences set forth in any of SEQ ID NO: 2 can be used to detect the corresponding chimeric insecticidal protein in commercial products derived from pooled sources, where only a portion of the commercial product is derived from a transgenic plant containing any of SEQ ID NO: 2.

Examples.

In view of the foregoing, those skilled in the art will appreciate that the following disclosed embodiments are only representative of the present invention and may be embodied in various forms. Thus, the specific structural and functional details disclosed herein should not be interpreted as limiting.

Example 1

Creation and cloning of sequences encoding new chimeric insecticidal proteins active against Lepidoptera.

This example illustrates the creation of new chimeric insecticidal proteins and the cloning and expression of chimeric insecticidal proteins.

Recombinant nucleic acid sequences were constructed from known Cry protein genes to produce polynucleotide sequences encoding novel chimeric insecticidal proteins. The resulting polynucleotide sequences were cloned into a Bacillus thuringiensis (Bt) plasmid expression vector. After confirming the polynucleotide sequence, the expression plasmid was transformed into Bt and expressed. Preparations of new chimeric proteins were analyzed for activity against various Lepidoptera pests.

Many polynucleotide sequences encoding chimeric insecticidal proteins have been generated and tested in a bioassay. Not all chimeric insecticidal proteins showed activity. Only a few of the chimeric insecticidal proteins were selected based on their activity against specific Lepidoptera demonstrated in a bioassay. Amino acid variants in which amino acid substitutions or alternative protoxin domains have been introduced have also been derived from the original TIC867 and TIC868 chimeric insecticidal proteins. Components of chimeric insecticidal proteins (domains I, II and III and protoxin) according to the present invention are presented in table. 1. Also presented are amino acid substitutions in TIC868 variants relative to

- 17 040497 of the original TIC868 protein sequence.

Table 1. New chimeric pesticidal proteins and their components

Toxin ORT SEQ ID NO: Domi Dom2 Dom3 Protoxin Amino acid modifications * TIC1100 4 CrylAh CrylAc CrylCa CrylAc TIC860 7 CrylBbl CrylBbl CrylCa CrylAc TIC867 10 CrylBe2 CrylBe2 CrylKa CrylAb3 Т1С867_20 13 CrylBe2 CrylBe2 CrylKa CrylDal Т1С867_21 16 CrylBe2 CrylBe2 CrylKa Cry4 Т1С867_22 19 CrylBe2 CrylBe2 CrylKa Cry9 Т1С867_23 21 CrylBe2 CrylBe2 CrylKa CrylBe Т1С867_24 23 CrylBe2 CrylBe2 CrylKa CrylKa Т1С867_25 25 CrylBe2 CrylBe2 CrylKa CrylCa TIC868 28 CrylBe2 CrylBe2 CrylCa CrylAb3 Т1С868_9 thirty CrylBe2 CrylBe2 CrylCa CrylAb3 N240S_Y343Q_N3 49T Т1С868_10 33 CrylBe2 CrylBe2 CrylCa CrylDal Т1С868_11 36 CrylBe2 CrylBe2 CrylCa Cry4 Т1С868_12 39 CrylBe2 CrylBe2 CrylCa Cry9 Т1С868_13 41 CrylBe2 CrylBe2 CrylCa CrylBe Т1С868_14 43 CrylBe2 CrylBe2 CrylCa CrylKa Т1С868_15 45 CrylBe2 CrylBe2 CrylCa CrylCa Т1С868_29 47 CrylBe2 CrylBe2 CrylCa CrylAb3 Q136Y_Y343Q_N3 49T TIC869 50 CrylJal CrylJal CrylJx CrylAb3 TIC836 53 CrylFal CrylFal CrylAb CrylAc

* amino acid mutations are identified using the standard IUPAC amino acid code. See IUPAC-IUB Joint Commission on Biochemical Nomenclature on Biochemical Nomenclature. Nomenclature and notation for amino acids and peptides. Eur. J Biochem. 138:9-37(1984). The first abbreviation of the first amino acid sequence denotes the original amino acid in the scaffold protein, the number represents the position of the amino acid, and the second abbreviation of the amino acid sequence denotes the amino acid placed at that position in the improved protein.

Example 2

New chimeric insecticidal proteins show activity against Lepidoptera pests.

This example illustrates testing of the chimeric insecticidal proteins described in Example 1 and the Lepidoptera activity observed for the chimeric insecticidal proteins.

Polynucleotide sequences encoding the chimeric insecticidal proteins were expressed in Bt.

The expressed chimeric insecticidal proteins were then analyzed against a variety of Lepidoptera known to be pests of corn, sugarcane, soybean and cotton, as well as other crops. In particular, insecticidal proteins were analyzed for activity against velvet cutworm (VBC, Anticarsia gemmatalis), sugar cane moth (SCB, Diatraea saccharalis), corn borer (LSCB, Elasmopalpus lignosellus), cotton bollworm (CEW, Helicoverpa zea), tobacco leafworm (TBW, Heliothis virescens), soybean cutworm (SBL, Chrysodeixis includens), black cutworm (BLAW, Spodoptera cosmioides), southern cutworm (SAW, Spodoptera eridania), fall armyworm (FAW, Spodoptera frugiperda), little cutworm (BAW, Spodoptera exigua), American cutworm (OBW, Helicoverpa armigera), Asian cotton cutworm (OLW, Spodoptera litura), pink bollworm ((PBW, Pectinophora gossypiella), upsilon cutworm (BCW, Agrotis ipsilon), southwestern corn moth (SWCB, Diatraea grandiosella), northern spiny bollworm (SBW, Earias vitella), and stem corn borer (ECB, Ostrinia nubilalis). called the bollworm (SPW) and cotton bollworm (CBW). Activity was determined using a combination of mortality and stunting scores and an MIC50 score (minimum inhibitory concentration for 50%). The MIC50 refers to the moult-inhibiting concentration, with both dead larvae and L1 larvae (larvae that do not molt until their second instars) counted in the assessment. In table. 2 shows the activity of each chimeric insecticidal protein. The '+' sign indicates the activity observed in a particular pest.

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Table 2. Biological activity against selected Lepidoptera

Insect

Toxin PRT SEQ ID NO: VBC SCB LSCB CEWSPWCBW BLAW TBW SBL SAW FAW BAW OBW OLW PBW BCW SWCB ECB SBW TIC1100 4 + + + + + + + TIC860 7 + + + + + + + + + + + + + TIC867 10 + + + + + + + + + Т1С867_ 20 13 Т1С867_ 21 16 + Т1С867_ 22 19 + + TIC868 28 + + + + + + + + + + Т1С868_ 10 33 + Т1С868_ eleven 36 + Т1С868_ 12 39 + TIC869 50 + + + + + TIC836 53 + + + + +

As can be seen from Table. 2 above, most of the chimeric insecticidal proteins exhibited activity against one or more Lepidoptera pest species.

Example 3

Synthesis of genes encoding chimeric insecticidal proteins for expression in plants.

This Example illustrates the synthesis of polynucleotides encoding chimeric insecticidal proteins for expression in plants.

Synthetic coding sequences have been designed for use in the expression of chimeric insecticidal proteins in plants. Synthetic sequences were designed and synthesized according to methods generally described in US Pat. No. 5,500,365, avoiding some inappropriate problematic sequences such as plant polyadenylation sequences enriched in ATTTA and A/T while maintaining the amino acid sequence of the chimeric insecticidal protein. The nucleotide sequences for these genes encoding chimeric insecticidal proteins for expression in plants are listed in Table 1. 3.

Table 3. Polynucleotide sequences encoding chimeric insecticidal proteins for use in plants

insecticidal protein DNA SEQ ID NO: ORT SEQ ID NO: TIC1100 2 4 TIC1100 3 4 TIC860 6 7 TIC867 9 10 TIC867_20 12 13 TIC867_21 15 16 TIC867_22 18 19 TIC867_23 20 21 TIC867_24 22 23 TIC867_25 24 25 TIC868 27 28 TIC868_9 29 thirty TIC868_10 32 33 TIC868_11 35 36 TIC868_12 38 39 TIC868_13 40 41 TIC868_14 42 43 TIC868-15 44 45 TIC868_29 46 47 TIC869 49 50 TIC836 52 53

- 19040497

Example 4

Expression cassettes for the expression of chimeric insecticidal proteins in plants

This example illustrates the construction of expression cassettes containing polynucleotide sequences for use in plants that encode chimeric insecticidal proteins.

A variety of plant expression cassettes were constructed using the polynucleotide sequences encoding the chimeric insecticidal proteins shown in Table 1. 3 and intended for expression in plants. Such expression cassettes are useful for transient expression in plant protoplasts or plant cell transformation. Exemplary expression cassettes have been designed taking into account the possible placement of the protein within the cell. One set of expression cassettes was designed so that the protein was translated and stored in the cytosol. Another set of expression cassettes was designed to contain a transit peptide adjacent to the toxin protein to allow targeting of cell organelles such as the chloroplast or plastid. All expression cassettes were designed to start at the 5' end with a promoter, which may consist of several promoter elements, enhancer elements, or other expression elements known to those skilled in the art, operably linked to increase expression of the transgene. The promoter sequence is typically followed continuously by one or more leader sequences at the 3' end of the promoter. The intron sequence is typically located at the 3' end of the leader sequence to improve expression of the transgene. The toxin or transit peptide coding sequence and the toxin coding sequence are typically located at the 3' end of the operably linked promoter, leader and intron. The 3'-UTR sequence is typically located at the 3' end of the coding sequence to facilitate termination of transcription and provide a sequence important for polyadenylation of the resulting transcript. All of the elements described above were operably linked and arranged sequentially, often with additional sequences used to construct the expression cassette.

Example 5

Lepidoptera activity of a chimeric insecticidal protein in stably transformed maize.

This example illustrates the inhibitory activity exhibited by chimeric insecticidal proteins against lepidopteran pests expressed by corn plants and offered as food for the corresponding corn pests.

Maize variety LH244 was transformed into the binary transformation vectors described in Example 4 using the Agrobacterium mediated transformation method.

Transformed cells were induced to form plants using methods known in the art. Bioassay using leaf disks of the plant was performed in analogy to those described in US patent No. 8344207. Tissue obtained from untransformed plants of variety LH244 was used as a negative control. Multiple transformation targets for each binary vector were evaluated against Fall Armyworm (CEW, Helicoverpa zea), Fall Armyworm (FAW, Spodoptera frugiperda), Fall Armyworm (BCW, Agrotis ipsilon) and Southwestern Corn Both (SWCB, Diatraea grandiosella).

Biotesting using leaf discs was performed on transgenic plants of R0 and F ₁ generations. In addition, the degree of leaf damage was assessed for whole transgenic Fj plants expressing certain chimeric insecticidal proteins infested with lepidoptera pests. Fj transgenic events expressing TIC860 and TIC868 were also evaluated for activity in the field against FAW, CEW, and SWCB. The results of the analysis are given in table. 4. The '+' sign indicates the activity observed in a particular pest. As can be seen from Table. 4, most of the chimeric insecticidal proteins and many of the chimeric insecticidal protein variants have shown activity against one or more Lepidoptera pest species.

- 20 040497

Table 4. Bioassay of activity of chimeric insecticidal protein from stably transformed maize leaf tissue

Toxin PRT SEQ ID NO: VBC SCB LSCB CEWSPWCBW BLAW TBW SBL SAW FAW BAW OBW OLW PBW BCW SWCB ERU SBW TIC1100 4 + + + + + + + TIC860 7 + + + + + + + + + + + + + TIC867 10 + + + + + + + + + TIC86720 13 nt nt nt nt nt nt nt nt nt nt nt nt nt NT nt TIC86721 16 nt nt nt + nt nt nt nt nt nt nt nt nt nt nt nt TIC86722 19 nt nt nt + nt nt nt nt + nt nt nt nt nt nt nt nt TIC868 28 + + + + + + + + + + + TIC86810 nt nt nt + nt nt nt nt + nt nt nt nt nt nt nt nt TIC86811 36 nt nt nt + nt nt nt nt + nt nt nt nt nt nt nt nt TIC86812 39 nt nt nt + nt nt nt nt + nt nt nt nt nt nt nt nt TIC869 50 + + + + + + TIC836 53 + + + + +

Example 6

Lepidoptera activity of chimeric insecticidal protein in stably transformed soybeans.

This example illustrates the inhibitory activity exhibited by a chimeric insecticidal protein against lepidopteran pests expressed by soybean plants and offered as food for the respective insect pests.

The coding sequences for the selected chimeric insecticidal protein were altered for expression in plants, cloned into a binary plant transformation vector, and used to transform soybean plant cells. Plant transformation vectors contained a first transgenic cassette for the expression of a chimeric insecticidal protein as described in Example 4 and a second transgenic cassette for selection of transformed plant cells using spectinomycin selection. In some cases, for example, in the case of TIC1100, TIC860 and TIC836, the chloroplast transit peptide coding sequence was operably linked to the chimeric insecticidal protein coding sequence. Analyzes were performed using TIC1100, TIC860 and TIC836 targeted and untargeted to the plastid. In table. 5 below shows the chimeric insecticidal protein and the TIC867 chimeric insecticidal protein variant and associated coding sequences used for expression in stably transformed soybeans.

Soybean plant cells were transformed into the binary transformation vectors described above using the Agrobacterium mediated transformation method. As a result, transformed plant cells were induced to form soybean plants. Leaf tissues were harvested and used in bioassay as described in Example 5, or alternatively, lyophilized tissues were used as insect food for bioassay. Biotesting was performed against FAW, southern armyworm (SAW, Spodoptera eridania), soybean cutworm (SBL, Chrysodeixis includens), bollworm (SPW, Helicoverpa zea), velvet bean cutworm (VBC, Anticarsia gemmatalis), tobacco leafworm (TBW, Heliothis virescens) , black armyworm (BLAW, Spodoptera cosmioides), grain corn grinder (LSCB, Elasmopalpus lignosellus) and American cutworm (OBW, Helicoverpa armigera).

In table. 5 shows activity against selected Lepidoptera species for each insecticidal protein in R ₀ generation plants, with '+' indicating activity. As can be seen from Table. 5, each of the chimeric insecticidal proteins expressed in stably transformed soybean showed activity against several Lepidoptera species. It should be noted separately that the TIC867 variant, TIC867_23, showed activity against SPW.

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Table 5. Bioassay of activity of chimeric insecticidal protein from leaf tissue of stably transformed _R0 soybean

insecticidal protein FAW SAW SBL SPW VBC TBW BLAW LSCB OBW TIC1100 + + + + + + + TIC860 + + + + + TIC867 + + + + + + Т1С867_20 + + Т1С867_21 + + Т1С867_22 + + Т1С867_23 + + + + Т1С867_24 + + Т1С867_25 + + TIC868 + + + + + TIC869 + + + + TIC836 + + + + + + +

The selected transformed objects were allowed to self-pollinate and the resulting grains were grown. Leaf tissues were collected from R1 generation plants and used in a feed bioassay. R1 plants expressing TIC1100, TIC860, TIC867, TIC868, TIC869 and TIC836 were tested for activity against SAW, SBL, SPW and VBC. In table. 6 shows the activity observed in these tests. The '+' sign indicates the activity observed in a particular pest. As shown in Table. 6, the most expressed chimeric insecticidal protein from R ₁ generation plants showed activity against one or more Lepidoptera species.

Table 6. Bioassay of activity of chimeric insecticidal protein from leaf tissue of stably transformed R1 soybean

Toxin SAW SBL SPW VBC TIC1100 + + + TIC860 + + + TIC867 + TIC868 + + + TIC869 + + + TIC836 + + +

In table. 7 shows the results of a greenhouse field trial using stably transformed R1 generation soybean plants expressing TIC1100, TIC860, and TIC836. Species used to infect plants in the greenhouse include SAW, SBL and SPW. Resistance was defined as defoliation in soybean plants that is less than or equal to fifteen percent. The resistance observed in the experimental cell corresponds to the resistance observed in the leaf tissue of the R ₁ generation of soybean plants, the analysis presented in table. 6. The '+' sign indicates the activity observed in a particular pest.

Table 7. Activity profile of TIC1100, TIC860 and

TIC836 expressed in generation R1 soybeans investigated in greenhouse field trials

Toxin SAW SBL SPW TIC1100 + + TIC860 + + TIC836 + +

Greenhouse field trials using stably transformed R1 generation soybean plants expressing TIC867 and TIC869 were also conducted at two different sites in Argentina, Acevedo and Fontezuela. Species used to infect plants in greenhouses include South American armyworm (SABW, Helicoverpa gelotopeon), VBC, BLAW, and sunflower moth (SFL, Rachiplusia pi). Resistance was defined as defoliation in soybean plants that is less than or equal to fifteen percent. In table. 8 below shows the observed resistance. The '+' sign indicates the activity observed in a particular pest. As shown in Table. 8, transgenic soybean plants expressing TIC867 showed resistance to BLAW and VBC. Transgenic soybean plants expressing TIC869 have shown resistance to SABW, SFL, BLAW, and VBC.

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Table 8. Activity profile of TIC867 and TIC869 expressed in generation R1 soybeans tested in greenhouse field trials

Acevedo Fontesuela Toxin SABW SFL VBC SABW BLAW VBC TIC867 + + + TIC869 + + + + +

Example 7

Lepidoptera activity of a chimeric insecticidal protein in stably transformed cotton.

This example shows the inhibitory activity exhibited by a chimeric insecticidal protein against lepidoptera pests when expressed in cotton plants and is proposed as a diet for the respective pests.

The coding sequences for the selected chimeric insecticidal protein, modified for expression in plants, were cloned into a binary plant transformation vector and used to transform cotton plant cells. The resulting binary vectors were similar to those described in Example 4 and were used to express plastid- and non-targeted TIC860 (coding sequence: SEQ ID NO: 6; protein sequence: SEQ ID NO: 7), TIC867 ( coding sequence: SEQ ID NO: 9; protein sequence: SEQ ID NO: 10), TIC868 (coding sequence: SEQ ID NO: 27; protein sequence: SEQ ID NO: 28) and TIC867_23 (coding sequence: SEQ ID NO: 20 ; protein sequence: SEQ ID NO: 23).

Cotton plant cells were transformed using the Agrobacterium mediated transformation method. Transformed cotton cells were induced to form whole plants. Cotton leaf tissue was used in biotesting as described in Example 5 against cotton bollworm (CBW, Helicoverpa zea), FAW, TBW and SBL. In table. 9 shows the activity observed against these Lepidoptera species for TIC860, TIC867, and TIC868 in stably transformed R ₀ generation cotton, with '+' indicating activity. As can be seen from Table. 9, TIC860, TIC867, and TIC868 showed activity against two or more Lepidoptera pests in stably transformed R ₀ generation cotton.

Table 9. Activity Bioassay

TIC860, TIC867 and TIC868 from stably transformed ________ R generation cotton sheet fabric ₀ ________

Toxin CBW FAW TBW SBL TIC860 + + TIC867 + + + NT TIC868 + +

The selected transformed objects were used to generate R1 seeds. R1 plants expressing TIC860, TIC867, and TIC868 were tested for resistance to CBW, FAW, TBW, and SBL. Leaf, bud and boll tissues were used in the analysis. In table. 10 shows the activity observed in these tests. The '+' sign indicates the activity observed in a particular pest. As shown in Table. 10, TIC860 showed anti-FAW activity in leaf tissue. In addition, the chimeric insecticidal protein TIC867 showed activity against CBW and FAW in leaf, bud and boll tissue, and against TBW and SBL in leaf. The chimeric insecticidal protein TIC868 demonstrated activity against FAW in leaf, bud, and boll tissues, as well as TBW and SBL in leaf.

Table 10. Bioassay of chimeric insecticidal protein activity from leaf tissue is stable

Example 8

Cloning of sequences encoding novel chimeric insecticidal proteins active against Lepidoptera.

Recombinant nucleic acid sequences were constructed from known

- 23 040497 Cry protein genes for obtaining coding sequences encoding new chimeric insecticidal proteins. The resulting coding sequences were cloned into a Bacillus thuringiensis (Bt) plasmid expression vector. After confirming the cloned sequence, the expression plasmid was transformed into Bt and expressed. Preparations of new chimeric Cry proteins have been investigated for activity against various Lepidoptera pests. Many coding sequences encoding chimeric insecticidal proteins have been generated and tested in bioassays. Not all chimeric insecticidal proteins showed activity. Only a few of the chimeric insecticidal proteins have been selected based on their activity against specific Lepidoptera and are shown in Table 1. 11 below.

Table 11. Novel chimeric pesticidal proteins and their respective bacterial and protein coding sequences

pesticide protein Bacterial DNA SEQ ID NO: Protein SEQID NO: TIC713 54 55 TIC843 56 57 TIC862 58 59 TIC1099 60 61 TIC1103 78 79 TIC1101 80 81 TIC845 82 83 TIC846 84 85 TIC858 86 87 TIC865 88 89 TIC866 90 91 TIC838 92 93 TIC839 94 95 TIC841 96 97 TIC842 98 99 TIC850 100 101 TIC859 102 103 TIC861 104 105 TIC848 106 107 TIC849 108 109 TIC847 110 111

Example 9

New chimeric insecticidal proteins show activity against Lepidoptera pests.

The coding sequences encoding the chimeric insecticidal proteins were expressed in Bt. The expressed proteins were then analyzed against various Lepidoptera known to be pests of corn, sugarcane, soybeans and cotton, as well as other crops. The insecticidal proteins were analyzed for activity against velvet cutworm (VBC, Anticarsia gemmatalis), sugarcane moth (SCB, Diatraea saccharalis), corn borer (LSCB, Elasmopalpus lignosellus), bollworm (CEW, Helicoverpa zea), tobacco leafworm (TBW, Heliothis virescens), soybean cutworm (SBL, Chrysodeixis includens), black cutworm (BLAW, Spodoptera cosmioides), southern cutworm (SAW, Spodoptera eridania), fall armyworm (FAW, Spodoptera frugiperda), little cutworm (BAW, Spodoptera exigua), American cutworm (OBW, Helicoverpa armigera), Asian cotton cutworm (OLW, Spodoptera litura), pink bollworm (PBW, Pectinophora gossypiella), upsilon cutworm (BCW, Agrotis ipsilon), southwestern corn moth (SWCB, Diatraea grandiosella) and stem corn borer (ESB, Ostrinia nubilalis). The American bollworm (CEW, Helicoverpa zea) is also referred to as the bollworm (SPW) and cotton bollworm (CBW). Activity was determined using a combination of mortality and stunting scores as well as MIC50 scores. The MIC50 refers to the moult-inhibiting concentration, with both dead larvae and L1 larvae (larvae that do not molt until their second instars) counted in the assessment. In table. 12 below shows the activity of each chimeric insecticidal protein. The '+' sign indicates the activity observed in a particular pest.

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Table 12. Biological activity against selected Lepidoptera

Insect ORT SEQ ID NO: VBC SCB LSCB CEW SPW CBW TBW SBL in L.A.W. SAW FAW BAW OBW OLW PBW BCW SWCB ECB 55 + + + + + + + + + + + + + + 57 + + + + + + + + + + + 59 + + + + + + + + + 61 + + + + + + + + + + + + 79 + + + + + + + + + + + + + 81 + + + + 83 + + + + + + + 85 + + + + + 87 + + + + + + + + 89 + + + + + + + + + 91 + + + + + + 93 + + + + + 95 + + + + + + 97 + + + + + + 99 + + + + + + + + + + + + + 101 + + + + + + + + + 103 + + + + + + + + + + + 105 + + + + + 107 + + + + + + + + + + 109 + + + + + + + + + + 111 + + + + + + + + +

As can be seen from Table. 12 above, all chimeric insecticidal proteins are active against several species of Lepidoptera insects.

Example 10

Amino acid variants of TIC 1099 show activity against Lepidoptera.

The coding sequence for TIC 1099 (SEQ ID NO: 60) was modified by methods known in the art to change specific amino acids from the original TIC 1099 protein sequence (SEQ ID NO: 61). The resulting variants were tested for activity against selected Lepidoptera. In table. 13 below shows TIC 1099 variants and corresponding DNA and protein SEQ ID NOs.

Table 13. TIC 1099 options

Variant TIC1099 DNA SEQ ID NO: ORT SEQ ID NO: TIC1099-T507E 62 63 TIC1099-R522K 64 65 TIC1099-K490S 66 67 TIC1099-T562R 68 69 TIC1099-S553R 70 71 TIC1099-G498D 72 73 TIC1099-K490A 74 75 TIC1099-E564A 76 77

For each variant, the mortality and growth retardation of Fall Armyworm (FAW, Spodoptera frugiperda), American Fall Armyworm (CEW, Helicoverpa zea), Fall Armyworm (BCW, Agrotis ipsilon), Soybean Fall Armyworm (SBL, Chrysodeixis includens), and Fall Armyworm (SBL, Chrysodeixis includens) and -western (SWCB, Diatraea grandiosella). In table. 14 below shows the activity against each lepidopteran pest. Activity was assessed from + to ++++ depending on the percentage of mortality.

Table 14 Activity of TIC1099 variants against Lepidoptera

Option TIC1099 ORT SEQ ID NO: FAW CEW BCW SBL SWCB TIC1099 61 + + + + + + + + + + + + + + + +++ TIC1099-T507E 63 + + + + + + + + + + + + + + + + +++ + TIC1099-R522K 65 + + + + + + + + +++ + + + + +++ + TIC1099-K490S 67 + + + + + + + + + + + ++ + TIC1099-T562R 69 + + + + + + + + + + + + + + + + +++ + TIC1099-S553R 71 + + + + + + + + +++ + + + + + +++ + TIC1099-G498D 73 + + + + + + + + + + + + + + + + +++ + TIC1099-K490A 75 + + + + + + + + + + + + + + + +++ TIC1099-E564A 77 + + + ++ +++ + +

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All variants of TIC1099 demonstrated activity against the five species tested

Lepidoptera. Several variants showed lower activity than others for specific pests.

Example 11.

Chimeric insecticidal proteins expressed in stably transformed maize show activity against lepidoptera pests.

Synthetic coding sequences were designed to express the encoded protein in plants, cloned into a binary plant vector for transformation, and used to transform maize plant cells. Plant transformation vectors contain a first transgenic cassette for the expression of a chimeric insecticidal protein, which contains a constitutive promoter operably linked 5' to a leader operably linked 5' to an intron operably linked 5' to a sequence encoding the chimeric insecticidal protein, which in turn is operably linked connected 5' with 3'-UTR and; a second transgenic cassette for selection of transformed plant cells using glyphosate selection. Below in table. 15 shows chimeric insecticidal protein coding sequences for use in maize expression corresponding to SEQ ID NO.

Table 15. Sequences encoding insecticidal proteins and corresponding SEQ ID NO

Maize cultivar LH244 was transformed with the binary transformation vectors described above using the Agrobacterium mediated transformation method. Transformed cells were induced to form plants using methods known in the art. Bioassay using leaf disks of the plant was performed in analogy to those described in US patent No. 8344207. Tissue obtained from untransformed plants of variety LH244 was used as a negative control. Multiple transformation targets for each binary vector were evaluated against Fall Armyworm (CEW, Helicoverpa zea), Fall Armyworm (FAW, Spodoptera frugiperda), Upsilon Cutworm (BCW, Agrotis ipsilon) and Southwestern Corn Both (SWCB, Diatraea grandiosella) . The results of the analysis are shown in table. 16, where + denotes activity.

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Table 16. Bioassay for activity of chimeric insecticidal protein from stably transformed corn leaf tissue

insecticidal protein CEW FAW BCW SWCB TIC713 + + + TIC843 + + + TIC862 + + + TIC1099 + + + + TIC1103 + + + TIC845 + + + TIC846 + + + TIC858 + + + TIC866 + + + TIC838 + + TIC841 + + + TIC842 + + + + TIC850 + + + TIC859 + + + TIC861 + + + TIC848 + + + TIC849 + + + TIC847 + +

As can be seen in Table. 16 above, all chimeric insecticidal proteins showed activity against two or more Lepidoptera species.

Example 12.

Chimeric insecticidal proteins expressed in stably transformed soybean exhibit activity against lepidopteran pests.

The coding sequences for the selected chimeric insecticidal protein were altered for expression in plants, cloned into a binary plant transformation vector, and used to transform soybean plant cells. Plant transformation vectors contain a first transgenic cassette for the expression of a chimeric insecticidal protein that contains a constitutive promoter operably linked 5' to a leader operably linked 5' to the chimeric insecticidal protein coding sequence, which in turn is operably linked 5' to 3' NTO and; a second transgenic cassette for selection of transformed plant cells using spectinomycin selection. In table. 17 below shows chimeric insecticidal protein coding sequences for use in soybean expression corresponding to SEQ ID NO.

Table 17. Sequences encoding insecticidal proteins and corresponding SEQ ID NO

insecticidal protein DNA SEQ ID NO: ORT SEQ ID NO: TIC1103 117 79 TIC866 121 91 TIC842 124 99 TIC849 129 109

Soybean plant cells were transformed with the binary transformation vectors described above using the Agrobacterium mediated transformation method. As a result, transformed plant cells were induced to form soybean plants. Leaf tissues were harvested and used in bioassay as described in Example 11, or alternatively, freeze-dried tissues were used as insect food for bioassay. Biotesting was performed against Fall Armyworm (FAW, Spodoptera frugiperda), Southern Fall Armyworm (SAW, Spodoptera eridania), Soybean Armyworm (SBL, Chrysodeixis includens), Bollworm (SPW, Helicoverpa zea), Velvet Bean Armyworm (VBC, Anticarsia gemmatalis), soybean cutworm (SBL, Chrysodeixis includens), tobacco leafworm (TBW, Heliothis virescens), black cutworm (BLAW, Spodoptera cosmioides), southern cutworm (SAW, Spodoptera eridania), grain corn grinder (LSCB, Elasmopalpus lignosellus) and American cutworm

Claims (18)

Kanskaya (OBW, Helicoverpa armigera). Below in table. 18 shows activity against selected Lepidoptera species for each insecticidal protein in plants of the Ro generation. Table 18. Bioassay of activity of chimeric insecticidal protein from leaf tissue of stably transformed R ₀ soybean insecticidal protein TIC1103 FAW SAW + SBL + SPW + VBC + TBW + BLAW SAW LSCB + OBW + TIC866 + + + + + TIC842 + + + + + + + + + TIC849 + + + + + + As can be seen from Table. 18, each of the chimeric insecticidal proteins expressed in stably transformed soybean showed activity against several Lepidoptera species. The selected transformed objects were allowed to self-pollinate and the resulting grains were grown. Leaf tissues were collected from R1 generation plants and used in feed biotesting. In table. 19 shows the activity of insecticidal proteins expressed in R1 soybean leaf tissue observed in some species of Lepidoptera. Table 19. Bioassay of chimeric insecticidal protein activity from leaf tissue is stable proteins from R ₁ generation plants have shown activity against two or more Lepidoptera species. All compositions disclosed and claimed herein can be made and performed without undue experimentation in light of the present disclosure. While the compositions of the present invention have been described in terms of the above illustrative embodiments, those skilled in the art will appreciate that variations, variations, modifications, and rearrangements may be applied to the composition described herein without departing from the concept, spirit, and scope of the present inventions. More specifically, it will be apparent that certain agents that are similar both chemically and physiologically can be used instead of the agents described herein, achieving the same or similar results. All such similar substitutions and modifications obvious to those skilled in the art are considered to be within the spirit, scope and concept of the present invention as defined in the appended claims. All publications and published patent documents cited in this specification are incorporated herein by reference to the same extent as if each individual publication or patent application were specifically and individually indicated for inclusion by reference. CLAIM 1. A chimeric insecticidal protein comprising an amino acid sequence having at least 94% identity with the amino acid sequence shown in SEQ ID NO: 21, wherein the chimeric insecticidal protein exhibits inhibitory activity against insect species of the order Lepidoptera. 2. Chimeric insecticidal protein according to claim 1, characterized in that the insect species is selected from the group consisting of: Anticarsia gemmatalis, Diatraea saccharalis, Elasmopalpus lignosellus, Helicoverpa zea, Heliothis virescens, Chrysodeixis includens, Spodoptera cosmioides, Spodoptera eridania, Spodoptera frugiperda, Spodoptera exigua, Spodoptera litura, Pectinophora gossypiella, Diatraea grandiosella, Earias vitella, Helicoverpa gelotopeon and Rachiplusia nu. 3. A polynucleotide encoding a chimeric insecticidal protein according to claim 1. 4. A polynucleotide according to claim 3, characterized in that it is operably linked to a heterologous promoter. 5. A host cell comprising a polynucleotide according to claim 3, wherein the host cell is selected from the group consisting of a bacterial host cell and a plant host cell. - 28 040497 6. The host cell according to claim 5, wherein the bacterial host cell is selected from the group consisting of Agrobacterium, Rhizobium, Bacillus, Brevibacillus, Escherichia, Pseudomonas, Klebsiella, and Erwinia. 7. The host cell according to claim 5, wherein the plant host cell is selected from the group consisting of a monocot cell and a dicot cell. 8. Composition having inhibitory activity against insects, containing a chimeric insecticidal protein at π. 1. 9. The insect-inhibiting composition of claim 8, further comprising at least one insect-inhibiting agent other than the chimeric insecticidal protein selected from the group consisting of a protein having insect-inhibiting activity. insects, and a dsRNA molecule having inhibitory activity against insects. 10. An insect-inhibiting composition according to claim 9, characterized in that at least one other insecticidal agent is active against one or more pest species of the orders Lepidoptera, Coleoptera, Hemiptera, Homoptera or Thysanoptera. 11. Seed showing inhibitory activity against insect species of the order Lepidoptera, containing: A. chimeric insecticidal protein at π. 1; or b. a polynucleotide encoding a chimeric insecticidal protein according to claim 1. 12. A method for controlling a lepidoptera pest, comprising contacting the lepidoptera pest with an inhibitory amount of a chimeric insecticidal protein at π. 1. 13. A transgenic plant, plant cell, or plant part containing a chimeric insecticidal protein according to claim 1, wherein the transgenic plant, plant cell, or plant part exhibits inhibitory activity against insect species of the order Lepidoptera. 14. A method for controlling a lepidoptera pest, comprising exposing the pest to the transgenic plant, plant cell, or plant part of claim 13, said plant, plant cell, or plant part expressing a Lepidoptera-inhibitory amount of the chimeric insecticidal protein. 15. A method for obtaining a seed exhibiting inhibitory activity against insect species of the order Lepidoptera, including: A. planting a transgenic plant according to claim 13; And b. collecting progeny seed from said transgenic plant, wherein the collected progeny seed contains the chimeric insecticidal protein at π. 1. 16. A processed plant product containing a polynucleotide encoding a chimeric insecticidal protein according to claim 1, further defined as selected from the group consisting of: plant biomass, oil, meal, animal feed, flour, flakes, bran, fiber, husk and processed seed. 17. Chimeric insecticidal protein according to claim 1, characterized in that it contains the amino acid sequence represented by SEQ ID NO: 21. 18. The processed plant product of claim 16, comprising the chimeric insecticidal protein of claim 1. Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky per., 2