TOXINAS CRY1CA MODIFICADAS, MÉTODO DE PRODUÇÃO DAS MESMAS, SEQUÊNCIA DE ÁCIDO NUCLEICO, VETOR DE EXPRESSÃO, E MÉTODOS PARA CONTROLE DE PRAGAS DE INSETO DE PLANTAS

Description

1 / 96 Descriptive Report of the Invention Patent for MODIFIED CRY1CA TOXINS, METHOD OF PRODUCING THEM, NUCLEIC ACID SEQUENCE, EXPRESSION VECTOR, AND METHODS FOR CONTROLLING PLANT INSECT PESTS. Separated from BR112017013514-0, filed on December 16, 2015. DESCRIPTION FIELD

[0001] The present invention relates to the modification of a pesticidal toxin from Bacillus thuringiensis, the polynucleotide sequences that encode these toxins, and transgenic plants that produce these toxins. BACKGROUND OF THE DESCRIPTION

[0002] Insects and other pests cost farmers billions of dollars annually in crop losses and expenses to keep these pests under control. In addition to field crop losses, insect pests are also a burden for vegetable and fruit farmers, ornamental flower growers, and home gardeners. Losses caused by insect pests in agricultural production environments include reduced crop yields, reduced crop quality, and increased harvesting costs.

[0003] Insect pests are primarily controlled by intensive applications of chemical pesticides, which are active by inhibiting insect growth, preventing insect feeding or reproduction, or causing death. Good insect control can thus be achieved, but these chemicals can sometimes affect other beneficial insects. Another problem resulting from the widespread use of chemical pesticides is the emergence of resistant insect populations. This has been partially mitigated by various resistance management practices, but there is a growing need. Petition 870240065834, dated 02 / 08 / 2024, page 14 / 210 2 / 96 of alternative pest control agents. Biological pest control agents, such as strains of Bacillus thuringiensis (Bt) expressing pesticidal toxins like delta-endotoxins, have also been applied to cultivated plants with satisfactory results, offering an alternative or complement to chemical pesticides. Genes encoding some of these delta-endotoxins have been isolated, and their expression in heterologous hosts has been shown to provide another tool for controlling economically important insect pests. In particular, the expression of insecticidal toxins, such as Bacillus thuringiensis delta-endotoxins, in transgenic plants has provided efficient protection against selected insect pests, and transgenic plants expressing such toxins have been commercialized, allowing farmers to reduce applications of chemical insect control agents.

[0004] Lepidoptera are an important group of agricultural, horticultural, and domestic pests that cause a great deal of damage each year. This order of insects includes larvae and adults that feed on leaves and roots. Lepidopteran insect pests include, but are not limited to, the following: Achoroia grisella, Acleris gloverana, Acleris variana, Adoxophyes orana, Agrotis ipsilon (black yellowtail BCW), Alabama argillacea, Alsophila pometaria, Amyelois transitella, Anagasta kuehniella, Anarsia lineatella, Anisota senatoria, Antheraea pernyi, Anticarsia gemmatalis (VBC velvety bean caterpillar), Archips sp., Argyrotaenia sp., Athetis mindara, Bombyx mori, Bucculatrix thurberiella, Cadra cautella, Choristoneura sp., Cochylls hospes, Colias eurytheme, Corcyra cephalonica, Cydia latiferreanus, Cydia pomonella, Datana integerrima, Dendrolimus sibericus, Desmia feneralis, Diaphania hyalinata, Diaphania nitidalis, Diatraea grandiosella (southwestern corn borer) Petition 870240065834, dated 02 / 08 / 2024, p. 15 / 210 3 / 96 SWCB), Diatraea saccharalis, Ennomos subsignaria, Eoreuma loftini, Esphestia elutella, Erannis tilaria, Estigmene acrea, Eulia salubricola, Eupocoellia ambiguella, Eupoecilia ambiguella, Euproctis chrysorrhoea, Euxoa messoria, Galleria mellonella, Grapholita molesta, Harrisina americana, Helicoverpa subflexa, Helicoverpa zea (cew corn borer), Heliothis virescens (TBW tobacco borer), Hemileuca oliviae, Homoeosoma electellum, Hyphantia cunea, Keiferia lycopersicella, Lambdina fiscellaria fiscellaria, Lambdina fiscellaria lugubrosa, Leucoma salicis, Lobesia botrana, Loxostege sticticalis, Lymantria dispar, Macalla thyrisalis, Malacosoma sp., Mamestra brassicae, Mamestra configurate, Manduca quinquemaculata, Manduca sexta, Maruca testulalis, Melanchra picta, Operophtera winter, Orgyia sp., Ostrinia nubilalis (European corn borer ECB), Paleacrita vernata, Papiapema nebris (common stem borer), Papilio cresphontes, Pectinophora gossypiella, Phryganidia californica, Phyllonorycter blancardella, Pieris napi, Pieris rapae, Plathypena scabra, Platynota flouendana, Platynota stultana, Platyptilia carduidactyla, Plodia interpunctella, Plutella xylostella (DBM cabbage moth), Pontia protodice, Pseudaletia unipuncta, Pseudoplusia includens (SBL soybean larva), Sabulodes aegrotata, Schizura concinna, Sitotroga cerealella, Spilonta ocellana, Spodoptera eridania (southern SAW caterpillar), Spodoptera frugiperda (FAW fall caterpillar), Spodoptera exigua (beetroot caterpillar BAW), Thaurnstopoea pityocampa, Ensola bisselliella, Trichoplusia ni (cabbage larva CL), Udea rubigalis, Xylomyges curiails, and Yponomeuta padella. Any genus listed above (and others), in general, may also be targeted as a part of the present invention.All additional insects in any of these genera (as targets) are also included within the scope of this invention.

[0005] Bacillus thuringiensis (Bt) is a bacterium that forms Petition 870240065834, dated 02 / 08 / 2024, page 16 / 210 4 / 96 Gram-positive, soil-transmitted spores that produce insecticidal crystalline proteins known as delta endotoxins or Cry proteins (reviewed in Schnepf et al., 1998). New crystalline proteins (Cry) with novel insecticidal properties continue to be discovered at an increasing rate, and more than 440 Cry genes have been reported. Currently, there are more than 450 unique Cry and Cytotoxin (Cyt) proteins classified among 57 levels of primary homology. Cry proteins are named based on the degree of sequence identity, with primary, secondary, and tertiary boundaries occurring at approximately 45%, 78%, and 95% identity, respectively; closely related alleles receive new quaternary designations (Crickmore et al., 1998). An extensive list of delta endotoxins is maintained and regularly updated at http: / / www.lifesci.sussex.ac.uk / home / Neil_Crickmore / Bt / intro.html.Currently, there are over 73 major Cry toxin groups (Cry1-Cry73), with additional Cyt toxins and Vegetative Insecticidal Protein (VIP) toxins and others. Many of each numbered group have capitalized subgroups, and the capitalized subgroups have lowercase sub-subgroups. (Cry1 has AL, and Cry1 A has ai, for example).

[0006] Bt proteins have been used to create insect-resistant transgenic plants that have been successfully registered or deregulated and commercialized to date. These include CrylAb, CrylAc, Cry1F, Vip3A, Cry34Ab1 / Cry35Ab1 and Cry3Bb in maize, CrylAc, Vip3A and Cry2Ab in cotton, and Cry3A in potato. Bt toxins represent more than 90% of the bioinsecticide market and essentially the entire source of genes for transgenic crops that have been developed to provide resistance to insect feeding.

[0007] Cry proteins are oral intoxicants that work Petition 870240065834, dated 02 / 08 / 2024, page 17 / 210 5 / 96 through action on the midgut cells of susceptible insects. The active forms of many Cry proteins comprise three distinct protein domains. The best-studied Bt proteins are members of the three-domain Cry delta endotoxins. These proteins range in size from approximately 70 kDa to 130 kDa. Primary protein sequence analysis reveals five highly conserved sequence blocks and a high degree of sequence variability between conserved blocks three and five (Schnepf et al., 1998).

[0008] Three-dimensional crystal structures have been determined for Cry1Aa1, Cry2Aa1, Cry3Aa1, CrySBbl, Cry4Aa, Cry4Ba, and Cry8Ea1 as examples. These structures are remarkably similar and consist of three distinct domains with the following aspects (reviewed in Maagd et al., 2003). Domain I is a bundle of seven alpha helices where helix five is surrounded by six amphipathic helices. This domain has been implicated in midgut membrane insertion and pore formation. It shares homology with other pore-forming proteins, including hemolysins and colicins. Domain II is composed of three antiparallel beta sheets packaged in a beta prism. This domain shares homology with certain carbohydrate-binding proteins, including vitelline and jacaline. The loops of this domain play important roles in binding insect midgut receptors.In Cry1A proteins, the exposed surface loops at the tips of the beta sheets of domain II are involved in binding to lepidopteran cadherin receptors. Domain III is a beta sandwich structure that interacts with a second class of receptors, examples of which are aminopeptidase and alkaline phosphatase in the case of Cry1A proteins (Piggot and Ellar, 2007). Structurally, this domain is related to... Petition 870240065834, dated 02 / 08 / 2024, page 18 / 210 6 / 96 carbohydrate-binding domains of proteins such as glucanases, galactose oxidase, sialidase, and others. This domain binds certain classes of receptor proteins and may participate in the insertion of an oligomeric toxin pre-pore. The conserved Bt sequence blocks 2 and 3 are arranged near the N-terminus and C-terminus of domain 2, respectively. Therefore, these conserved sequence blocks 2 and 3 are approximate boundary regions between the three functional domains. These conserved DNA regions and protein homology have been exploited for the design of recombinant Bt toxins (US Patent No. 6,090,931, WO 91 / 01087, WO 95 / 06730, WO 1998022595).

[0009] One proposed model for the mode of action of the Cry protein is based on the formation of pores in the midgut membranes of susceptible insects (Knowles and Ellar, 1987). In the current version of this model (Bravo et al., 2007), binding to both cadherin and aminopeptidase receptors in the lepidopteran midgut membranes is required for Cry protein toxicity.According to the pore formation model, Cry protein intoxication involves several steps: 1) proteolytic processing of soluble Cry protoxin into an activated core toxin; 2) binding of Cry protein to cadherin receptors in the insect midgut; 3) further proteolytic cleavage at the N-terminal of the core toxin to remove an α-helical region; 4) oligomerization of the Cry protein to form a prepore; 5) binding of the prepore to second-site membrane receptors (aminopeptidases and alkaline phosphatases); 6) insertion of the prepore into the membrane; and 7) conduction of osmotic cell lysis to midgut rupture and insect death.

[0010] The widespread adoption of insect-resistant transgenic plant technology gives rise to concern of Petition 870240065834, dated 02 / 08 / 2024, pages 19 / 210 7 / 96 that pest populations will develop resistance to the insecticidal proteins produced by these plants. Several strategies have been suggested to preserve the utility of Bt-based insect resistance traits, including implanting proteins at a high dose in combination with a refuge, and alternating or co-implanting different toxins (McGaughey et al. (1998), Bt Resistance Management, Nature Biotechnol. 16:144-146).

[0011] The development of insect resistance to Bt Cry proteins can occur through several mechanisms (Heckel et al., 2007, Piggot and Ellar, 2007). Multiple classes of receptor proteins for Cry proteins have been identified within insects, and there are several examples within each receptor class. Resistance to a particular Cry protein can develop, for example, through a mutation within the toxin-binding portion of a cadherin domain of a receptor protein. Another means of resistance can be mediated through a protoxin-processing protease. Thus, resistance to Cry1A toxins in Lepidoptera species has a complex genetic basis, with at least four distinct major resistance genes. Lepidopteran insects resistant to Cry proteins have been developed in the field by Plutella xylostella (Tabashnik, 1994), Trichoplusia ni (Janmaat and Myers 2003, 2005), Helicoverpa zea (Tabashnik et al., 2008) and Spodoptera frugiperda (Storer, et al., 2010). The development of new high-potency Cry proteins will provide additional tools for the management of lepidopteran insect pests.

[0012] This invention provides Bt insecticidal proteins that are effective in controlling insects that are resistant to CrylAc and Cry1F. These protein toxins can be advantageously used to protect agronomic crops from insect feeding damage. Petition 870240065834, dated 02 / 08 / 2024, pages 20 / 210 8 / 96 The ability to express these insect toxins in such a way that a sufficient quantity of the functionally active protein is present in a culture of interest is also an objective of this invention. BRIEF SUMMARY OF THE INVENTION

[0013] A modified Cry1Ca toxin comprising residues 2 to 68 of SEQ ID NO: 2 wherein amino acid residue 54 is selected from the group consisting of Gly and Ala, amino acid residue 57 is selected from the group consisting of Leu and Met, and amino acid residue 68 is selected from the group consisting of Val, Phe and Ile. A modified Cry1Ca toxin comprising residues 2 to 628 of SEQ ID NO: 10 wherein amino acid residue 54 is selected from the group consisting of Gly and Ala, amino acid residue 57 is selected from the group consisting of Leu and Met, amino acid residue 68 is selected from the group consisting of Val, Phe and Ile, amino acid residue 73 is selected from the group consisting of Trp, Ala and Met, amino acid residue 596 is selected from the group consisting of Phe, Met and Ala and amino acid residue 620 is selected from the group consisting of Leu and Phe.The modified Cry1Ca toxins of the foregoing further comprise a terminal carboxy extension consisting of amino acid residues 629 to 1164 of SEQ ID NO: 36. The modified Cry1Ca toxins of the foregoing further comprise a terminal carboxy extension consisting of amino acid residues 629 to 1164 of SEQ ID NO: 36. The modified Cry1Ca toxins of the foregoing further comprise a terminal amino extension consisting of amino acid residues 1 to 74 of SEQ ID NO: 40. The modified Cry1Ca toxins of the foregoing further comprise a terminal amino extension consisting of amino acid residues 1 to 74 of SEQ ID NO: 40. The modified Cry1Ca toxins of the foregoing. Petition 870240065834, dated 02 / 08 / 2024, page 21 / 210 9 / 96 further comprise an amino-terminal extension consisting of amino acid residues 1 to 74 of SEQ ID NO: 40. The modified CrylCa toxins of the foregoing further comprise an amino-terminal extension consisting of amino acid residues 1 to 74 of SEQ ID NO: 40.

[0014] The DNA encoding the modified CrylCa toxins, the transgenic plants that produce modified CrylCa toxins, and the methods for controlling insect pests using the modified CrylCa toxins are included in the invention.

[0015] The invention in question relates to new materials and methods for controlling arthropod pests that are harmful to plants and agriculture. In a preferred embodiment, the present invention provides materials and methods for controlling Lepidoptera pests.

[0016] The specific Bt Cry proteins (endotoxins, toxins) useful according to the invention include toxins that can be obtained from Bt isolates designated as MR-1206. The present invention also includes the use of mutants of the exemplified Bt isolate and toxins that have improved the active properties of Lepidoptera, which resist protease processing or are expressed at high levels when genes are transformed into a heterologous expression system. The procedures for producing mutants are well known in microbiological art. Ultraviolet light and chemical mutagens such as nitrosoguanidine are widely used for this purpose.

[0017] The toxins of the protein in question can be applied or delivered to come into contact with the target insects in several ways. For example, transgenic plants (in which the protein is produced and present in the plant) can be used and are well known in the art. The expression of the toxin genes also Petition 870240065834, dated 02 / 08 / 2024, page 22 / 210 10 / 96 can be selectively targeted to specific plant tissues, such as roots, leaves, etc. This can be achieved through the use of tissue-specific promoters, for example. Spray applications are another example and are also known in the art. The proteins in question can be appropriately formulated for the desired end use and then sprayed (or otherwise applied) on and / or around the plant and / or in the vicinity of the plant to be protected, before an infestation is discovered, after the target insects are discovered, both before and after, and similarly. The protein in question can also be appropriately formulated and applied to seeds as a seed treatment that allows the protein to be in contact with the root area of ​​the plant to protect it from root-feeding insects. Bait granules, for example, can also be used and are known in the art.

[0018] The proteins in question can be used to protect virtually any type of plant against damage caused by a Lepidopteran insect. Examples of such plants include corn, sunflower, soybean, cotton, canola, rice, sorghum, wheat, barley, vegetables, ornamental plants, peppers (including hot peppers), sugar beet, fruit, and grass, to name just a few. The plants especially preferred are corn, soybean, and cotton. One of the most preferred plants is corn. Another of the most preferred plants is soybean. Another of the most preferred plants is cotton.

[0019] In one embodiment of the present invention, the polynucleotide sequences of the present invention encode toxins of approximately 68 to 71 kDa. These toxins are used to control Lepidopteran pests, especially fall armyworms, diamondback moths, southwestern corn borer, southern corn borer, corn rootworm, and European corn borer. In a preferred embodiment, the Petition 870240065834, dated 02 / 08 / 2024, page 23 / 210 11 / 96 The present invention relates to plant cells transformed with at least one polynucleotide sequence of the present invention such that the transformed plant cells produce and contain pesticidal toxins of the invention in tissues consumed by the target pests.

[0020] Alternatively, the Bt isolate of the invention in question, or recombinant microbes expressing genes encoding the pesticidal toxin proteins described herein, can be used to control insect pests. In this respect, the invention includes the treatment of substantially intact Bt cells and / or recombinant cells containing the toxins of the invention, treated to prolong pesticidal activity when the substantially intact cells are applied to the environment of a target pest. The treated cell acts as a protective coating for the pesticidal toxin. The toxin becomes active after ingestion by a target insect.

[0021] One aspect of the invention relates to isolated nucleic acid molecules comprising nucleotide sequences encoding pesticidal proteins and polypeptides or their biologically active parts, as well as sufficient nucleic acid molecules for use as hybridization probes to identify nucleic acids encoding the claimed toxins. As used herein, the term nucleic acid molecule is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and DNA or RNA analogs generated using nucleotide analogs. The nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA.

[0022] The nucleotide sequences encoding the proteins of the present invention include the sequences presented in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, Petition 870240065834, dated 02 / 08 / 2024, p. 24 / 210 12 / 96 37, 39, and their complements. A complement is a nucleotide sequence that is sufficiently complementary to a given nucleotide sequence such that it can hybridize with that sequence to form a stable duplex (double-stranded) molecule. The corresponding amino acid sequences for pesticide-active modified CrylCa toxins encoded by these nucleotide sequences are presented in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40.

[0023] Nucleic acid molecules that are fragments of the nucleotide sequences encoding the claimed toxin are also included in the present invention. By fragment is meant a portion of the nucleotide sequence that encodes a fragment of a claimed modified CrylCa toxin. A fragment of a nucleotide sequence may encode a biologically active portion of a claimed toxin protein, or it may be a fragment that can be used as a hybridization probe or PCR primer using the methods described below. BRIEF DESCRIPTION OF THE SEQUENCES SEQ ID NO: 1 DNA sequence encoding DIG-468 SEQ ID NO: 2 is the sequence number for the DIG-468 protein. SEQ ID NO: 3 DNA sequence encoding DIG-483 SEQ ID NO: 4 is the sequence number for the DIG-483 protein. SEQ ID NO: 5 DNA sequence encoding DIG-485 SEQ ID NO: 6 is the sequence number for the DIG-485 protein. SEQ ID NO: 7 DNA sequence encoding DIG-487 SEQ ID NO: 8 is the sequence number for the DIG-487 protein. SEQ ID NO: 9 DNA sequence encoding DIG-462 SEQ ID NO: 10 is the sequence number for the DIG-462 protein. SEQ ID NO: 11 DNA sequence encoding DIG-463 Petition 870240065834, dated 02 / 08 / 2024, page 25 / 210 13 / 96 SEQ ID NO: 12 is the sequence number for the DIG-463 protein. SEQ ID NO: 13 DNA sequence encoding DIG-464 SEQ ID NO: 14 is the sequence number for the DIG-464 protein. SEQ ID NO: 15 DNA sequence encoding DIG-465 SEQ ID NO: 16 is the sequence number for the DIG-465 protein. SEQ ID NO: 17 DNA sequence encoding DIG-466 SEQ ID NO: 18 is the sequence number for the DIG-466 protein. SEQ ID NO: 19 DNA sequence encoding DIG-467 SEQ ID NO: 20 is the sequence number for the DIG-467 protein. SEQ ID NO: 21 DNA sequence encoding DIG-469 SEQ ID NO: 22 is the sequence number for the DIG-469 protein. SEQ ID NO: 23 DNA sequence encoding DIG-473 SEQ ID NO: 24 is the sequence number for the DIG-473 protein. SEQ ID NO: 25 DNA sequence encoding DIG-474 SEQ ID NO: 26 is the sequence number for the DIG-474 protein. SEQ ID NO: 27 DNA sequence encoding DIG-482 SEQ ID NO: 28 is the sequence number for the DIG-482 protein. SEQ ID NO: 29 DNA sequence encoding the modified Cry1Ca codon optimized for maize (IRDIG544.11) SEQ ID NO: 30 is the toxin sequence of the modified Cry1Ca protein (IRDIG544.11) SEQ ID NO: 31 DNA sequence encoding a modified Cry1Ca, IRDIG544.12, with high GC codon optimization. SEQ ID NO: 32 protein toxin sequence for IRDIG544.12 SEQ ID NO: 33 an optimized dicotyledonous DNA sequence encoding the modified Cry1Ca, IRDIG544.9 SEQ ID NO: 34 modified Cry1Ca protein sequence, IRDIG544.9 SEQ ID NO: 35 an optimized DNA sequence of Petition 870240065834, dated 02 / 08 / 2024, pages 26 / 210 14 / 96 dicotyledons encoding modified CrylCa, IRDIG544.8 SEQ ID NO: 36 protein codon IRDIG544.8 optimized for dicotyledons SEQ ID NO: 37 DNA sequence encoding a modified Cry1Ca toxin fused to the Cry1Ab protoxin segment SEQ ID NO: 38 is the toxin sequence of the protein produced from the DNA of SEQ ID NO: 37 SEQ ID NO: 39 High GC codon-optimized DNA sequence encoding a modified Cry1Ca, IRDIG544.12, fused with TraP12 SEQ ID NO: 40 is the modified Cry1Ca toxin, IRDIG544.12, fused with TraP12. BRIEF DESCRIPTION OF THE FIGURES

[0024] Figure 1 shows the expression levels of DIG-465 by construct 115752 and DIG-473 by construct 115753 in corn leaves T1 tested by leaf perforations.

[0025] Figure 2 is a graph of the amount of leaf damage in maize caused by FAW or Cry1Fa-resistant FAW versus the level of DIG-465 expression.

[0026] Figure 3 is a graph of the amount of leaf damage in maize caused by FAW or Cry1Fa-resistant FAW versus the level of DIG-473 expression. DETAILED DESCRIPTION OF THE INVENTION

[0027] By using the term genetic material in this document, it means to include all genes, nucleic acid, DNA and RNA. These sequences have been altered in such a way as to increase the stability of the protein toxin expressed when the gene is transformed into a plant, specifically maize and dicotyledons. The protein toxins discussed here are typically referred to as insecticides or insecticide-related. By insecticides and insecticide-related Petition 870240065834, dated 02 / 08 / 2024, pages 27 / 210 15 / 96 insecticides, it is understood here that the protein toxins possess a functional activity as further defined herein and are used as insect control agents.

[0028] Functional activity here means that protein toxins function as insect control agents insofar as the proteins are orally active, or have a toxic effect, or are able to interrupt or prevent feeding, which may or may not cause the insect's death. When an insect comes into contact with an effective amount of toxin released through the expression of the transgenic plant, formulated protein compositions, sprayable protein compositions, a bait matrix, or other delivery system, the results are typically the death of the insect, or the insects do not feed on the source that makes the toxins available to the insects.

[0029] By the use of the term oligonucleotides is meant a macromolecule consisting of a short chain of RNA or DNA nucleotides. Such length may be at least one nucleotide, but is typically in the range of about 10 to about 12 nucleotides. Determining the length of the oligonucleotide is well within the ability of a specialist and should not be a limitation in this document. Therefore, oligonucleotides may be shorter than 10 or longer than 12. The invention in question relates not only to polynucleotide sequences encoding these classes of toxins, but also to the use of these polynucleotide sequences to produce recombinant hosts expressing the toxins.

[0030] By using the term toxic or toxicity as used here, it means that the toxins produced by Bacillus thuringiensis have functional activity as defined here.

[0031] Through the use of the term CrylCa toxin(s) Petition 870240065834, dated 02 / 08 / 2024, pages 28 / 210 16 / 96 modified, the intention is to include all protein sequences from the Sequence Listing and all their variants described herein.

[0032] By using the term genetic material in this document, it is intended to include all genes, nucleic acid, DNA and RNA.

[0033] For nucleotide residue designations of polynucleotides, DNA, RNA, oligonucleotides and primers, and for amino acid residue designations of proteins, the standard IUPAC abbreviations are used throughout this document. Nucleic acid sequences are presented in the standard 5' to 3' direction, and protein sequences are presented in the standard amino terminus (N) to carboxy terminus (C) direction.

[0034] The toxins and genes of the present invention can be further defined by their amino acid and nucleotide sequences, and the sequence of single fragments comprised by full-length DNA and amino sequences. The sequences of molecules within each new class can be defined herein in terms of homology to certain exemplified sequences, as well as in terms of the ability to hybridize or be amplified by certain exemplified probes and primers. The classes of toxins provided herein can also be identified based on their immunoreactivity with certain antibodies.

[0035] Toxin structure. The toxin of the present invention can also be characterized in terms of its structure and domain composition. The correlation of protein sequence variability with differences in the bioactivity spectrum led to initial hypotheses that the hypervariable regions between blocks three and five are responsible for the differences in insect specificity among Bt delta-endotoxins.

[0036] When the gene encoding the native full-length CrylCa protein was inserted and expressed in maize cells, Petition 870240065834, dated 02 / 08 / 2024, pages 29 / 210 In 17 / 96, at least 5 detectable proteolytic degradation products were observed. These five polypeptides were determined to have the following amino acid lengths: 1-1164, 1-628, 29-628, 74-628, and 74-596. Of the five CrylCa degradation products detected, two fragments were observed to be inactive against major carrier insect pests. In most cases, these two inactive fragments represented a major part of the CrylCa-related proteins detected in maize cells. The full-length native gene expression for CrylCa in maize resulted in plants having insufficient functional activity against major insect pests such as S. frugiperda.

[0037] When the gene expressing the truncated native CrylCa protein (aa 1-628) was inserted and expressed in maize cells, less proteolytic processing occurred. Most remained unprocessed and functionally active. Thus, expressing the truncated CrylCa gene in maize cells resulted in plants having sufficient functional activity against major insect pests due to reduced proteolysis in maize cells.

[0038] Altering the primary amino acid sequence of CrylCa takes into account the continued biological activity against major insect pests and results in less proteolytic processing of the protein, as measured in vitro using chymotrypsin as the protease enzyme. The less proteolytic processing of the altered CrylCa protein results in greater amounts of functionally active protein accumulating in plants and resulting in greater activity against the target insect pests.

[0039] Protease-sensitive variants. Insect gut proteases generally function in assisting the insect in obtaining necessary amino acids from dietary proteins. Proteases Petition 870240065834, dated 02 / 08 / 2024, page 30 / 210 18 / 96 The best understood digestive proteases in insects are serine proteases, which appear to be the most common type (Englemann and Geraerts, 1980), particularly in Lepidoptera species. Coleoptera insects have guts that are more neutral to acidic than Lepidoptera guts. Most Coleoptera larvae and adults, for example, the Colorado potato beetle, have a slightly acidic midgut, and cysteine ​​proteases provide the main proteolytic activity (Wolfson and Murdock, 1990). More precisely, Thie and Houseman (1990) identified and characterized the cysteine ​​proteases, cathepsin type B and cathepsin type H, and the aspartyl protease, cathepsin type D, in the Colorado potato beetle. Gillikin et al., (1992) characterized the proteolytic activity in the guts of western corn rootworm larvae and found mainly cysteine ​​proteases. US Patent No.7230167 reported that the serine protease, cathepsin G, exists in the western maize rootworm. The diversity and different activity levels of proteases in the insect's gut may influence the insect's sensitivity to a particular Bt toxin.

[0040] In one embodiment, the toxins possess specific alterations in their amino acid sequences that significantly reduce the level of protease processing of the protein expressed by proteases naturally found in maize plants. Changes in amino acids result in higher levels of functional protein activity when expressed in maize. Protease cleavage sites can be introduced at desired locations through chemical gene synthesis or overlap PCR combination (Horton et al., 1989). Serine protease recognition sequences, for example, can optionally be inserted into specific sites in the Cry protein structure to affect protein processing at elimination points. Petition 870240065834, dated 02 / 08 / 2024, p. 31 / 210 19 / 96 desired within the midgut of certain insect pests. Serine proteases from the midgut of lepidopterans such as trypsin or trypsin-like enzymes, chymotrypsin, elastase, etc. (Christeller et al., 1992) can be exploited with respect to the activation of Cry proteins through the design of protease recognition sequences at the desired processing sites. Similarly, serine proteases from Coleoptera such as trypsin, chymotrypsin, and cathepsin-type G protease can be similarly exploited through the design of recognition sequences at the desired processing sites. Furthermore, cysteine ​​proteases from Coleoptera such as cathepsins (B-type, L-type, O-type and K-type proteases) (Koiwa et al., 2000 and Bown et al., 2004), metalloproteases such as ADAM 10 (Ochoa-Campuzano et al., 2007), and aspartic acid proteases such as type D and E cathepsins, pepsin, plasmepsin and chymosin, can be exploited through the design of recognition sequences at the desired processing sites.

[0041] The scope of this invention includes CrylCa variant insecticidal proteins that are produced by manipulating the coding sequence for the insecticidal proteins in question through the introduction or elimination of protease processing sites at appropriate positions to enable or eliminate proteolytic cleavage of a larger variant protein by proteases from insects, plants, or microorganisms. The end result of such manipulation is the generation of toxin molecules having the same activity, or better, as the intact native toxin protein (full length).

[0042] Unlike the high sequence specificity associated with Type II restriction endonucleases in Petition 870240065834, dated 02 / 08 / 2024, p. 32 / 210 20 / 96 recognition and cleavage of their DNA substrates, proteolytic enzymes are more nonspecific in the amino sequence comprising the cleavage recognition site. Some generalities have been discovered regarding the amino acid structures comprising some protease cleavage sites, in particular, cathepsin G compared to cathepsins B, K, L and S (Bown et al., 2004). In the protease cleavage site nomenclature in the illustrations below, amino acid residues upstream (i.e., towards the N-terminal) of the cleavage site are numbered P1, P2, P3, P4, P5, etc., with residue P1 being immediately adjacent to the cleavage site and residue P5 being the fifth most distal to the cleavage site in the N-terminal direction. The amino acid residues downstream (i.e., towards the C-terminus) of the cleavage site are numbered PT, P2', P3', P4', P5', etc.Cathepsin G is known to exhibit preferential cleavage after the P1 residues glutamine, lysine, tryptophan, or phenylalanine, where the P2, P3, P4, P5, etc. and PT, P2', P3', P4', P5', etc. residues can be any of the 20 amino acids normally found in natural proteins. Somewhat improved sequence specificity of the cleavage site is demonstrated by cathepsins B, K, L, and S, where the P2 amino acid side chain fits into a cathepsin S2 substrate binding site. The PT residue is immediately adjacent to the cleavage site, and the P5' residue is the fifth most distal to the cleavage site in the C-terminal direction.The S2 site of these cathepsins preferentially interacts with P2 amino acids having large hydrophobic side chains (e.g., as found in valine, leucine, isoleucine, phenylalanine, tryptophan, and tyrosine) and disfavors interaction with P2 residues having charged side chains (except that cathepsins B and L accept the large hydrophilic charged side chain of arginine at the P2 position). Some. Petition 870240065834, dated 02 / 08 / 2024, p. 33 / 210 21 / 96 Specificity is seen in the amino acid identity at the P3 position. For example, cathepsin L preferentially cleaves after arginine at the P1 position, whereas phenylalanine or arginine occupy the P2 position. The P3 amino acid can be an aromatic type (e.g., phenylalanine, tryptophan, histidine, or tyrosine) or a hydrophobic type (e.g., alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, or tyrosine). The P4, P5, etc. positions and PT, P2', P3', P4', P5', etc. can be any of the 20 amino acids normally found in natural proteins.

[0043] Proteolytic cleavage also depends on the availability of the cleavage sequence in question to the respective protease; sequestration of the potential cleavage site within the three-dimensional structure of the protein can make the protein resistant to cleavage by the particular protease. It is believed that the diversity and different activity levels of insect gut proteases can influence the insect's sensitivity to a Bt toxin. A person versed in biochemical and molecular biology techniques can examine the biochemical characteristics (including, but not limited to, the determination of the amino acid sequences comprising the N-terminal and C-terminal of the polypeptide) of insecticidal protein fragments generated by protease cleavage / activation of larger proteins by gut proteases of susceptible insects.It is also possible to characterize the protease regime of the guts of non-susceptible insects or host plants, and to design, at appropriate locations within the coding sequence for the Bt insecticidal protein, the sequences suitable for cleavage by the intestinal proteases of non-susceptible insects or likely host plants in which the Bt insecticidal protein will be transgenically produced. Such analyses and manipulations of the Bt insecticidal protein in question are understood to be within the scope of this invention. Petition 870240065834, dated 02 / 08 / 2024, page 34 / 210 22 / 96

[0044] In another embodiment, the toxins have specific alterations in their amino acid sequences that significantly increase the level of protein expression when expressed in a variety of different expression systems, including plants and bacteria. The result of increased protein expression is increased functional activity in the expression system. This is advantageous in delivering a high dose of toxin to the insect, which can prevent the occurrence of resistance in insects to the toxins due to the survival of small insect populations that receive a sub-lethal dose of the protein toxin.

[0045] Genes and toxins. The protein molecules of the embodiments in this document comprise amino acid sequences that are homologous to known pesticidal proteins, particularly Bt Cry proteins, more particularly the CrylCa protein (Genbank Accession No. AAA22343). The predicted amino acid sequences encoded by a nucleotide sequence of the embodiments are described as SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 and 40.

[0046] The sequence of toxins of the present invention is provided as SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 and 40. In a preferred embodiment, the toxins of the present invention possess at least one of the following characteristics: (a) said toxin is encoded by a nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, or their complementary sequences, (b) said toxin is immunoreactive with an antibody triggered against a pesticide toxin of approximately 68-71 kDa, or a Petition 870240065834, dated 02 / 08 / 2024, page 35 / 210 23 / 96 fragment thereof, from an isolate of Bacillus thuringiensis, (c) said toxin is encoded by a nucleotide sequence wherein a portion of said nucleotide sequence can be amplified by PCR using a pair of primers to produce a fragment of about 25 to 40 bp, (d) said toxin comprises a pesticidal moiety of the amino acid sequences shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 and 40, (e) said toxin comprises an amino acid sequence that has at least about 90% homology with a pesticidal moiety of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 and 40, (f) said toxin is encoded by a nucleotide sequence that hybridizes under stringent conditions with an insecticidal moiety of a nucleotide sequence selected from the group consisting of DNA encoding the SEQ IDs: 2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 and 40, (g) said toxin is immunoreactive with an antibody to a pesticide toxin of approximately 68 kDa or 130 kDa, or a fragment thereof, from an isolate of Bacillus thuringiensis, MR1206, (h) said toxin comprises an amino acid sequence that has at least about 90% homology with an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 and 40 and the pesticide parts of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 and 40.

[0047] The specific genes exemplified here, variations of these genes, and fragments of these genes may also be Petition 870240065834, dated 02 / 08 / 2024, p. 36 / 210 24 / 96 obtained, for example, through synthetic construction by methods currently practiced by any of the various commercial suppliers (see, for example, US Patent No. 7482119). These genes, or their parts or variants, can also be constructed synthetically, for example, through the use of a gene synthesizer and the methods of, for example, US Patent No. 5,380,831. Alternatively, synthetic or naturally occurring gene variations can be easily constructed using standard molecular biology techniques for the formation of point mutations. Fragments of these genes can also be produced using commercially available exonucleases or endonucleases according to standard procedures. For example, enzymes such as Bal31 or site-directed mutagenesis can be used to systematically cut nucleotides from the ends of these genes.Similarly, gene fragments that encode the active toxin fragments can be obtained using a variety of restriction enzymes.

[0048] The nucleic acid molecules that are fragments of a nucleotide sequence encoding the claimed toxin comprise at least about 15, 20, 30, 40, 50, 60, 75, 100, 200, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 3000, 3500 nucleotides, or up to the number of nucleotides present in a claimed full-length insecticidal toxin-coding nucleotide sequence described herein (e.g., 1878 nucleotides for SEQ ID NO: 1, 3495 nucleotides for SEQ ID NO: 37), depending on the intended use. A fragment of a nucleotide sequence encoding a biologically active portion of a claimed protein of the invention will encode at least about 15, Petition 870240065834, dated 02 / 08 / 2024, p. 37 / 210 25 / 96 25, 30, 40, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1000, 1100 or 1200 contiguous amino acids, or up to the total number of amino acids present in a full-length insecticidal protein of the invention (For example, 625 amino acids for SEQ ID NO: 2 or 1164 amino acids for SEQ ID NO: 38).

[0049] Recombinant hosts. The toxin-coding genes of the invention in question can be introduced into a wide variety of microbial or plant hosts. Expression of the toxin gene results, directly or indirectly, in the intracellular production and maintenance of the pesticidal protein. With suitable microbial hosts, for example, Pseudomonas, the microbes can be applied to the pest environment, where they will proliferate and can be ingested. The result is pest control. Alternatively, the microbe hosting the toxin gene can be treated under conditions that prolong the toxin activity and stabilize the cell. The treated cell, which retains the toxic activity, can then be applied to the target pest environment.

[0050] Where the toxin gene is introduced via a suitable vector into a microbial host, and said host is applied to the environment in a living state, it is essential that certain host microbes be utilized. Host microorganisms are selected which are known to occupy the phytosphere (phyllosphere, rhizosphere and / or rhizoplane) of one or more crops of interest. These microorganisms are selected so as to be able to successfully compete in the particular environment (crops and other insect habitats) with native wild-type microorganisms, provide stable maintenance and expression of the polypeptide pesticide gene and, desirably, provide enhanced protection of the pesticide against Petition 870240065834, dated 02 / 08 / 2024, page 38 / 210 26 / 96 environmental degradation and inactivation.

[0051] Bt spores or recombinant host cells can also be treated before being applied or formulated for application to plants. For example, isolated Bt spores and / or toxin crystals can be chemically treated to prolong insecticidal activity and thus incorporate a treated polypeptide of the invention (US Patent No. 4,695,462 and Gaertner et al., 1993).

[0052] A large number of microorganisms are known to inhabit the phylloplane (the surface of plant leaves) and / or the rhizosphere (the soil surrounding plant roots) of a wide variety of important crops. These microorganisms include bacteria, algae, and fungi. Of particular interest are microorganisms such as bacteria, for example, genera Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium, Sinorhizobium, Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes; and fungi, particularly yeast, for example, genera Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium.Of particular interest are phytosphere bacterial species such as Pseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum, Agrobacterium tumefaciens, Agrobacterium radiobacter, Rhodopseudomonas spheroides, Xanthomonas campestris, Sinorhizobium meliloti (formerly Rhizobium meliloti), Alcaligenes eutrophus and Azotobacter vinelandii; and phytosphere yeast species such as Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S. odorus, Kluyveromyces veronae, and Aureobasidium pollulans. Privately. Petition 870240065834, dated 02 / 08 / 2024, pages 39 / 210 27 / 96 of interest are the pigmented microorganisms.

[0053] A preferred embodiment of the present invention is the transformation of plants with genes encoding the insecticidal protein in question or its variants. The transformed plants are resistant to attack by a target insect pest by virtue of the presence of controlling amounts of the insecticidal protein in question or its variants in the transformed plant cells. By incorporating genetic material encoding the insecticidal properties of Bt insecticidal toxins into the genome of a plant devoured by a particular insect pest, the adult or larvae would die after consuming the food plant. Numerous members of the monocotyledonous and dicotyledonous classifications have been transformed. Transgenic agronomic crops as well as fruits and vegetables are of commercial interest. Such crops include, but are not limited to, maize, rice, soybeans, canola, sunflower, alfalfa, sorghum, wheat, cotton, peanuts, tomatoes, potatoes, and the like.Several techniques exist for introducing foreign genetic material into monocotyledonous or dicotyledonous plant cells, and for obtaining fertile plants that stably maintain and express the introduced gene. Such techniques include accelerating the delivery of genetic material coated in microparticles directly into the cells (US Patents 4945050 and 5141131). Plants can be transformed using Agrobacterium technology, see US Patent 5177010, US Patent 5104310, European Patent Application 0131624B1, European Patent Application 120516, European Patent Application 159418B, European Patent Application 176112, US Patent 5149645, US Patent 5469976, US Patent 5464763, US Patent 4940838, US Patent 4693976, European Patent Application 116718, European Patent Application 290799, European Patent Application 320500, European Patent Application 604662. Petition 870240065834, dated 02 / 08 / 2024, page 40 / 210 28 / 96 European Patent Application 627752, European Patent Application 0267159, European Patent Application 0292435, US Patent 5231019, US Patent 5463174, US Patent 4762785, US Patent 5004863, and US Patent 5159135. Other transformation technologies include WHISKERS™ technology, see US Patent 5302523 and US Patent 5464765. Electroporation technology has also been used to transform plants, see WO 87 / 06614, US Patent 5472869, US Patent 5384253, WO 9209696, and WO 9321335. All these patents and transformation publications are incorporated by reference. In addition to numerous technologies for plant transformation, the type of tissue that comes into contact with the foreign genes can also vary. Such tissue would include, but not be limited to, embryogenic tissue, type I and II callus tissue, hypocotyl, meristem, and the like.Almost all plant tissues can be transformed during differentiation using appropriate techniques within the skill of a specialist.

[0054] The genes encoding modified CrylCa insecticidal toxins and variants can be inserted into plant cells using a variety of techniques that are well known in the specialty as described above. For example, a large number of cloning vectors comprising a marker that allows the selection of transformed microbial cells and a functional replication system in E. coli are available for the preparation and modification of foreign genes for insertion into higher plants. Such manipulations may include, for example, the insertion of mutations, truncations, additions, deletions, or substitutions as desired for the intended use. The vectors comprise, for example, pBR322, pUC series, M13mp series, pACYC184, etc. Consequently, the sequence encoding the Cry protein or variants can be inserted into the vector at a restriction site. Petition 870240065834, dated 02 / 08 / 2024, page 41 / 210 29 / 96 suitable. The resulting plasmid is used for the transformation of E. coli, whose cells are cultured in a suitable nutrient medium, then harvested and subjected to lysis so that viable quantities of the plasmid are recovered. Sequence analysis, restriction fragment analysis, electrophoresis, and other molecular biochemical-biological methods are generally performed as analytical methods. After each manipulation, the DNA sequence used can be cleaved and joined to the next DNA sequence. Each manipulated DNA sequence can be cloned into the same or other plasmids.

[0055] Depending on the plant transformation method, auxiliary DNA sequences may be required. If, for example, a Ti or Ri plasmid is used for plant cell transformation, then at least one right-edge repeat of the TDNA, but often both the right-edge repeat and the left-edge repeat of the Ti or Ri plasmid, will be joined as the flanking region of the desired genes to be inserted into the plant cell. The use of T-DNA-containing vectors for plant cell transformation has been intensively researched and sufficiently described in EP 120516; Lee and Gelvin (2008), Fraley et al., (1986), and An et al., (1985), and is well established in the field.

[0056] Once the inserted DNA has been integrated into the plant genome, it is relatively stable across subsequent generations. The vector used to transform the plant cell typically contains a selectable marker gene that encodes a protein that confers tolerance of transformed plant cells to a herbicide or antibiotic, such as bialafos, kanamycin, G418, bleomycin, or hygromycin, inter alia. The individually employed selectable marker gene must, therefore, allow for the selection of transformed cells while preventing the growth of cells that do not contain it. Petition 870240065834, dated 02 / 08 / 2024, page 42 / 210 30 / 96 the inserted DNA is suppressed by the selective compound.

[0057] A large number of techniques are available for inserting DNA into a plant host cell. These techniques include transformation with T-DNA released by Agrobacterium tumefaciens or Agrobacterium rhizogenes as the transforming agent. Additionally, fusion of plant protoplasts with liposomes containing the DNA to be released, direct DNA injection, biolistic transformation (microparticle bombardment), or electroporation, as well as other possible methods, can be employed. A person versed in the field of plant transformation will understand that multiple methodologies are available for the production of transformed plants, and they can be modified and specialized to accommodate the biological differences between various host plant species.

[0058] If Agrobacterium strains are used for transformation, the DNA to be inserted will be cloned into special plasmids, namely, an intermediate (transport) vector or a binary vector. Intermediate vectors can be integrated into the Ti or Ri plasmid or their derivatives through homologous recombination due to sequences that are homologous between the Ti or Ri plasmid and the intermediate plasmid. The Ti or Ri plasmid also comprises the vir region containing vir genes necessary for T-DNA transfer. Intermediate vectors cannot replicate in Agrobacteria. The intermediate vector can be transferred to Agrobacterium tumefaciens via a helper plasmid (through bacterial conjugation), through electroporation, via direct DNA transfer, chemically mediated transformation, or through other methodologies. Binary vectors can replicate autonomously in both E. coli and Agrobacterium cells.They comprise sequences, structured by the repeating regions of... Petition 870240065834, dated 02 / 08 / 2024, page 43 / 210 31 / 96 right and left edges of the T-DNA, which may include a functional selectable marker gene for the selection of transformed plant cells, a cloning linker, cloning polylinker, or other sequence that may function as an introduction site for genes intended for plant cell transformation. They may be transformed directly into Agrobacterium cells (Holsters et al., (1978)) by electroporation, or by direct DNA, chemically mediated transformation, or introduced by bacterial conjugation, or through other methodologies. The Agrobacterium used as a host cell is to comprise a plasmid carrying a vir region. The vir region is necessary for the transfer of the T-DNA into the plant cell. The additional additive T-DNA regions to that containing the gene encoding the Bt insecticidal toxin protein or its variants may be present in the Agrobacterium host cell.Bacterial cells thus transformed are used for the transformation of plant cells. Plant explants (e.g., leaf fragments, stem segments, roots, but also protoplasts or cells grown in suspension) can be advantageously cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes for the transfer of DNA into the plant cell. Whole plants can then be regenerated from the infected plant material after placement in suitable growth conditions and culture medium, which may contain antibiotics or herbicides for the selection of transformed plant cells. The plants thus obtained can then be tested for the presence of the inserted DNA.

[0059] The transformed cells grow within plants in the usual way. They can form germ cells and transmit the transformed characteristics to the offspring plants. Such plants can be grown in the normal way and crossed with plants that Petition 870240065834, dated 02 / 08 / 2024, page 44 / 210 32 / 96 possess the same transformed hereditary factors or other hereditary factors. The resulting hybrid individuals possess the corresponding phenotypic properties, for example, the ability to control the feeding of insect plant pests.

[0060] No special requirements are made for the construction of plasmids in the case of those used for injection and electroporation. It is possible to use common plasmids, such as, for example, those derived from puC, suitably modified to contain all the desired genes to be transferred to plant cells.

[0061] The activity of recombinant polynucleotides inserted into plant cells may be dependent on the influence of endogenous plant DNA adjacent to the insertion. Thus, another option is to take advantage of events that are known to be excellent locations in a plant genome for insertions. See, for example, WO 2005 / 103266 A1, regarding the Cry1F and CrylAc cotton events; the Bt insecticidal toxin gene in question can be substituted at those genomic loci instead of Cry1F or CrylAc insertions. Targeted homologous recombination, for example, can be used according to the present invention. This type of technology is the subject, for example, of WO 03 / 080809 and the corresponding US published application (USPA 20030232410), regarding the use of zinc linkers for targeted recombination. The use of recombinases (cre-lox and flp-frt, for example) is also known in the art.

[0062] In a preferred embodiment of the present invention, plants will be transformed with genes in which the codon usage of the protein coding region has been optimized for plants. See, for example, U.S. Patent No. 5,380,831, which is incorporated herein by reference. Also advantageously, plants encoding a truncated toxin will be used. The truncated toxin typically Petition 870240065834, dated 02 / 08 / 2024, page 45 / 210 33 / 96 will encode approximately 55% to approximately 80% of the total length toxin. Methods for creating synthetic Bt genes for use in plants are known in the art (Stewart, 2007).

[0063] Another variable is the choice of a selectable marker. The preference for a particular marker is at the discretion of the specialist, but any of the following selectable markers may be used together with any other gene not listed in this document that may function as a selectable marker. Such selectable markers include, but are not limited to, the aminoglycoside phosphotransferase gene of the Tn5 transposon (Aph II) which encodes resistance to the antibiotics kanamycin, neomycin and G418, as well as those genes which encode tolerance to glyphosate; hygromycin; methotrexate; phosphinothricin (bialaphos); imidazolinones, sulfonylureas and triazolopyrimidine herbicides such as chlorsulfuron; bromoxynil, dalapon and the like. Examples of such genes are provided in Merlo, (2002), which is incorporated herein by reference.

[0064] In addition to a selectable marker, it may be desirable to use a reporter gene. In some cases, a reporter gene can be used without a selectable marker. Reporter genes are genes that typically do not provide a growth advantage to the recipient organism or tissue. The reporter gene typically encodes a protein that provides some phenotypic changes or enzymatic properties. A preferred reporter gene is the glucuronidase gene (GUS). Other examples of reporter genes are provided in Merlo (2002).

[0065] Regardless of the transformation technique, the gene is preferably incorporated into a gene transfer vector adapted to express the Bt insecticidal toxin genes and variants in the plant cell through the inclusion of a promoter in the vector. Petition 870240065834, dated 02 / 08 / 2024, p. 46 / 210 34 / 96 plant. In addition to plant promoters, promoters from a variety of sources can be efficiently used in plant cells to express foreign genes. For example, promoters of bacterial origin, such as the octopin synthase promoter, the nopalin synthase promoter, the mannopin synthase promoter; promoters of viral origin such as the 35S and 19S promoters of cauliflower mosaic virus, and others, can be used. Plant promoters include, but are not limited to, ribulose-1,6-bisphosphate (RUBP) small subunit carboxylase (ssu), beta-conglycinin promoter, phaseolin promoter, ADH (alcohol dehydrogenase) promoter, heat shock promoters, ADF (actin depolymerizing factor) promoter, and tissue-specific promoters. Promoters may also contain certain enhancer sequence elements that can improve transcription efficiency.Typical enhancers include, but are not limited to, ADH1-intron 1 and ADH1-intron 6. Constitutive promoters can be used. Constitutive promoters direct continuous gene expression in almost all cell types and at almost all times (e.g., actin, ubiquitin, CaMV 35S). Tissue-specific promoters are responsible for gene expression in specific cell or tissue types, such as leaves or seeds (e.g., zein, oleosin, napin, ACP (Acyl Carrier Protein)), and these promoters can also be used. Promoters that are active during a certain stage of plant development, as well as those active in specific plant tissues and organs, can also be used.Examples of such promoters include, but are not limited to, promoters that are specific to the root, specific to pollen, specific to the embryo, specific to corn silks, specific to cotton fiber, specific to the seed endosperm. Petition 870240065834, dated 02 / 08 / 2024, page 47 / 210 35 / 96 specific to the phloem, and similar.

[0066] Under certain circumstances it may be desirable to use an inducible promoter. An inducible promoter is responsible for gene expression in response to a specific signal, such as: physical stimulus (e.g., heat shock genes); light (e.g., RUBP carboxylase); hormone (e.g., glucocorticoid); antibiotic (e.g., tetracycline); metabolites; and stress (e.g., drought). Other desirable transcription and translation elements that function in plants can be used, such as untranslated 5' leader sequences, RNA transcription termination sequences, and polyadenylate addition signal sequences. Numerous plant-specific gene transfer vectors are known in the art.

[0067] The present invention includes plant cells that are not totipotent, plant cells that are not propagation materials (e.g., leaf cells in some embodiments, seed cells are excluded from some embodiments) and are incapable of differentiating into whole plants. The present invention includes plant cells that have a different use for regeneration into a whole plant. For example, said plant cells can be used to produce a protein (such as a DIG-465 protein of the present invention). Thus, the plant cells of the present invention include those that have a different use from totipotent cells (i.e., some cells of the present invention are not regenerable into a whole plant). However, some embodiments include seed cells and plant cells that can be regenerated into a whole plant.

[0068] Another method for identifying the toxins and genes of the present invention is through the use of oligonucleotide probes. These probes are detectable nucleotide sequences. These Petition 870240065834, dated 02 / 08 / 2024, page 48 / 210 36 / 96 sequences can become detectable by virtue of an appropriate radioactive label or can be prepared inherently fluorescent as described in US Patent No. 6268132. As is well known in the art, if the probe molecule and the nucleic acid sample hybridize through the formation of strong base-pairing bonds between the two molecules, it can be reasonably assumed that the probe and the sample possess substantial sequence homology. Preferably, hybridization is conducted under stringent conditions by techniques well known in the art, as described, for example, in Keller and Manak (1993). Probe detection provides a means to determine in a known manner whether hybridization has occurred. Such probe analysis provides a rapid method for identifying toxin-coding genes of the present invention.The nucleotide segments used as probes according to the invention can be synthesized using a DNA synthesizer and standard procedures. These nucleotide sequences can also be used as PCR primers to amplify the genes of the present invention.

[0069] As used herein, the terms stringent conditions or stringent hybridization conditions are intended to refer to the conditions under which a probe will hybridize (anneale) with its target sequence to a detectably greater degree than with other sequences (e.g., at least 2 times over formation). Stringent conditions are sequence-dependent and will differ under different circumstances. By controlling the stringency of the hybridization and / or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, the severity conditions can be adjusted to allow for some inequalities in Petition 870240065834, dated 02 / 08 / 2024, p. 49 / 210 37 / 96 sequences, so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is smaller than about 1000 nucleotides in length, preferably less than 500 nucleotides in length.

[0070] Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to pH 8.3, and the temperature is at least around 30°C for short probes (e.g., 10 to 50 nucleotides) and at least around 60°C for long probes (e.g., greater than 50 nucleotides). Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. Low rigor conditions include hybridization with a 30% to 35% formamide buffer solution, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37°C and a wash in 1x to 2x SSC (20x SSC = 3.0 M NaCl / 0.3 M trisodium citrate) at 50°C to 55°C. Exemplary moderate rigor conditions include hybridization in 40% to 45% formamide, 1.0 M NaCl, 1% SDS at 37°C and a wash in 0.5x to 1x SSC at 55°C to 60°C.Exemplary high rigor conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37°C and a wash in 0.1xSSC at 60°C to 65°C. Optionally, the wash buffers may comprise from about 0.1% to about 1% SDS. The hybridization duration is generally less than about 24 hours, usually from about 4 to about 12 hours.

[0071] Specificity is typically a function of post-hybridization washes, the critical factors being ionic strength and the temperature of the final wash solution. For DNA / DNA hybrids, the thermal melting point (Tm) is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes with Petition 870240065834, dated 02 / 08 / 2024, p. 50 / 210 38 / 96 a perfectly matched probe. Tm is reduced by approximately 1°C for each 1% mismatch; thus, Tm, hybridization conditions, and / or washing conditions can be adjusted to facilitate annealing of sequences of the desired identity. For example, if sequences with >90% identity are sought, Tm can be decreased by 10°C. Generally, stringent conditions are selected to be approximately 5°C lower than Tm for the specific sequence and its complement at a defined ionic strength and pH. However, highly stringent conditions may utilize hybridization and / or washing at 1°C, 2°C, 3°C, or 4°C lower than Tm; Moderately stringent conditions may use hybridization and / or washing at 6°C, 7°C, 8°C, 9°C or 10°C lower than Tm, and low stringency conditions may use hybridization and / or washing at 11°C, 12°C, 13°C, 14°C, 15°C or 20°C lower than Tm.

[0072] Tm (in °C) can be determined experimentally or approximated by calculation. For DNA-DNA hybrids, Tm can be approximated by the Meinkoth and Wahl equation (1984): Tm(°C) = 81.5°C + 16.6(log M) + 0.41(%GC) - 0.61(% formamide) - 500 / L; where M is the molarity of monovalent cations, %GC is the percentage of guanosine and cytosine nucleotides in the DNA, % formamide is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs.

[0073] Alternatively, Tm is described by the following formula (Beltz et al., 1983). Tm(°C) = 81.5°C + 16.6(log[Na+]) + 0.41(%GC) - 0.61(% formamide) - 600 / L where [Na+] is the molarity of sodium ions, %GC is the percentage of guanosine and cytosine nucleotides in the DNA, and % formamide is the Petition 870240065834, dated 02 / 08 / 2024, page 51 / 210 39 / 96 is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in the base pairs.

[0074] Using the equations, the hybridization and washing compositions, and the desired Tm, those with practical skill will understand that variations in the rigidity of the hybridization and / or washing solutions are inherently described. If the desired degree of miscombination results in a Tm lower than 45°C (aqueous solution) or 32°C (formamide solution), it is preferable to increase the SSC concentration so that a higher temperature can be used. An extensive guide to nucleic acid hybridization is found in Tijssen (1993) and Ausubel et al., (1995). See also Sambrook et al., (1989).

[0075] Hybridization of DNA immobilized on Southern blots with radioactively labeled gene-specific probes can be performed by conventional methods (Sambrook et al., Supra). Radioactive isotopes used to label polynucleotide probes may include 32P, 33P, 14C, or 3H. Incorporation of radioactive isotopes into polynucleotide probe molecules can be done by any of several well-known methods used by those versed in the field of molecular biology. (See, for example, Sambrook et al., Supra). In general, hybridization and subsequent washes can be performed under stringent conditions that take into account the detection of target sequences with homology to the claimed toxin-coding genes.For double-stranded DNA gene probes, hybridization can be performed overnight at 20 to 25°C below the Tm of the DNA hybrid in SSPE 6X, Denhardt Solution 5X, 0.1% SDS, 0.1 mg / mL denatured DNA [20X SSPE is 3M NaCl, 0.2M NaHPO4 and 0.02M EDTA (sodium salt of ethylenediaminetetraacetic acid); Denhardt Solution 100X is 20 g / L polyvinylpyrrolidone, 20 g / L Ficoll type 400 and 20 g / L albumin]. Petition 870240065834, dated 02 / 08 / 2024, p. 52 / 210 40 / 96 bovine serum (fraction V)].

[0076] Washing can typically be carried out as follows: (1) Twice at room temperature for 15 minutes in SSPE 1X, 0.1% SDS (low stiffness wash). (2) Once at Tm -20°C for 15 minutes in SSPE 0.2X, SDS at 0.1% (moderate rigidity wash).

[0077] For oligonucleotide probes, hybridization can be performed overnight at 10 to 20°C below the Tm of the hybrid in SSPE 6X, Denhardt solution 5X, 0.1% SDS, 0.1 mg / mL of denatured DNA. The Tm for oligonucleotide probes can be determined by the following formula (Suggs et al., 1981). Tm(°C) = 2(number of T / A base pairs) + 4(number of G / C base pairs)

[0078] Washing can typically be carried out as follows: (1) Twice at room temperature for 15 minutes SSP 1X, SDS at 0.1% (low stiffness wash). (2) Once at hybridization temperature for 15 minutes in SSPE 1X, SDS at 0.1% (moderate wash).

[0079] A professional versed in the technique will realize that the molecules of the hybridization probe and the hybrid molecules formed between probe and target molecules can become detectable by means other than radioactive labeling.

[0080] Variant toxins. The useful genes and toxins according to the present invention include not only the truncated sequences described, but also full-length sequences, fragments of these sequences, variants, mutants, and fusion proteins that retain the characteristic pesticidal activity of the toxins specifically exemplified herein. As used herein, the terms Petition 870240065834, dated 02 / 08 / 2024, p. 53 / 210 41 / 96 variants or variations of genes refer to nucleotide sequences that encode the same toxins or that encode equivalent toxins having pesticidal activity. As used herein, the term equivalent toxins refers to toxins having the same or essentially the same biological activity against the target pests as the claimed toxins. Thus, the variant or variations of the claimed toxins will have at least about 30%, preferably at least about 50%, more preferably at least about 70%, and even more preferably at least about 80% of the activity of the claimed toxins. Methods for measuring pesticidal activity are well known in the art and are exemplified herein.Variants here are intended to include proteins or polypeptides that have an amino acid sequence that is at least about 60%, 65%, preferably about 70%, 75%, more preferably about 80%, 85%, most preferably about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of the SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 and 40. Variants also include polypeptides encoded by a nucleic acid molecule that hybridizes with the nucleic acid molecule of the SEQ ID NOs. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 or 39, or a complement thereof, under stringent conditions. Such variants will generally maintain the claimed activity. The variants include polypeptides that differ in amino acid sequence due to mutagenesis. The variant proteins included by the present invention are insecticidally active.

[0081] Variant proteins can also be designed to differ at the primary amino acid sequence level while retaining the same or similar overall essential three-dimensional structure, surface charge distribution, and more. Petition 870240065834, dated 02 / 08 / 2024, p. 54 / 210 42 / 96 See, for example, US Patent No. 7,058,515; Larson et al., (2002); Crameri et al., (1997); Stemmer, W.P.C. (1994a); Stemmer, W.P.C. (1994b) Stemmer, W. P. C. (1995); Crameri et al., (1996a) and Crameri et al., (1996b).

[0082] Certain toxins of the present invention have been specifically exemplified in this document. Since these toxins are only exemplary of the toxins of the present invention, it should be readily apparent that the present invention comprises variants or equivalent toxins (and nucleotide sequences encoding equivalent toxins) having the same or similar pesticidal activity as the exemplified toxin. The equivalent toxins will have amino acid homology with an exemplified toxin. The amino acid identity will typically be greater than 60%, preferably greater than 75%, more preferably greater than 80%, most preferably greater than 90%, and may be greater than 95%. The amino acid homology will be highest in the critical regions of the toxin that are responsible for the biological activity or are involved in determining the three-dimensional configuration that is ultimately responsible for the biological activity.In this regard, certain amino acid substitutions are acceptable and can be anticipated if these substitutions are in regions that are not critical to activity or are conservative amino acid substitutions that do not affect the three-dimensional configuration of the molecule. For example, amino acids can be placed into the following classes: nonpolar, polar uncharged, basic, and acidic. Conservative substitutions whereby an amino acid of one class is replaced by another amino acid of the same type fall within the scope of the present invention, provided that the substitution does not materially alter the biological activity of the compound. Table 1 provides a list of examples of amino acids belonging to each class. Petition 870240065834, dated 02 / 08 / 2024, p. 55 / 210 43 / 96 Table 1 Amino Acid Classes and Examples. Amino Acid Class Examples of Amino Acids Nonpolar Side Chains Ala, Vai, Leu, Ie, Pro, Met, Phe, Trp Polar Uncharged Side Chains Gly, Ser, Thr, Cys, Tyr, Asn, Gin Acidic Side Chains Asp, Glu Basic Side Chains Lys, Arg, His Beta-branched Side Chains Thr, Vai, Ie Aromatic Side Chains Tyr, Phe, Trp, His

[0083] In some cases, non-conservative substitutions can also be made. The critical factor is that these substitutions must not significantly impair the biological activity of the toxin.

[0084] The preferred insecticidal toxin proteins of the present invention are encoded by a nucleotide sequence sufficiently identical to the nucleotide sequences of the SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 or 39. By sufficiently identical is meant an amino acid or nucleotide sequence having at least about 60% or 65% sequence identity, preferably about 70% or 75% sequence identity, more preferably about 80% or 85% sequence identity, most preferably about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity when compared to a reference sequence as analyzed by one of the alignment programs described herein, employing standard parameters.A person skilled in the technique will recognize that these values ​​can be appropriately adjusted to determine the corresponding identity of proteins encoded by two nucleotide sequences, taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like.

[0085] To determine the percentage identity of two Petition 870240065834, dated 02 / 08 / 2024, page 56 / 210 44 / 96 amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical positions / total number of positions (e.g., overlapping positions) x 100). In one embodiment, the two sequences are of the same length. The percent identity between two sequences can be determined using techniques similar to those described below, with or without allowing gaps. When calculating percent identity, typically exact matches are counted.

[0086] Determining the percent identity between two sequences can be performed using a mathematical algorithm. A non-limiting example of a mathematical algorithm used for comparing two sequences is the Karlin and Altschul (1990) algorithm, modified as in Karlin and Altschul (1993). Such an algorithm is incorporated into the BLASTN and BLASTX programs of Altschul et al. (1990). BLAST searches can be conveniently used to identify homologous (similar) sequences to a query sequence in nucleic or protein databases. Nucleotide searches by BLAST can be performed with the BLASTN program, score = 100, word length = 12, to identify nucleotide sequences having homology with the claimed nucleic acid molecules of the invention.BLAST protein searches can be performed using the BLASTX program, score = 50, word length = 3, to identify amino acid sequences having homology with the claimed insecticidal protein molecules of the invention.

[0087] To obtain open alignments for comparison purposes, Gapped BLAST can be used as described in Petition 870240065834, dated 02 / 08 / 2024, p. 57 / 210 45 / 96 Altschul et al., (1997). Alternatively, PSI-Blast can be used to perform a repeated search that detects distant connections between molecules Altschul et al., (1997). When using the BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., BLASTX and BLASTN) can be used. See www.ncbi.nlm.nih.gov. Alignment can also be performed manually via inspection.

[0088] A non-limiting example of a mathematical algorithm used for sequence comparison is the ClustalW algorithm (Thompson et al., (1994)). ClustalW compares sequences and aligns the entire amino acid or DNA sequence and therefore can provide data on the conservation of the amino acid sequence or the entire nucleotide sequence. The ClustalW algorithm is used in several commercially available DNA / amino acid analysis software packages, such as the ALIGNX module of the Vector NTI Program Suite (Invitrogen, Inc., Carlsbad, CA). When aligning amino acid sequences with ALIGNX, one can conveniently use the default settings with an open Gap penalty of 10, an extension Gap penalty of 0.1, and the blosum63mt2 comparison matrix. sequences can be evaluated.When aligning two DNA sequences with ALIGNX, one can conveniently use the default settings with an open Gap penalty of 15, an extended Gap penalty of 6.6, and the swgapdnamt comparison matrix. After aligning two DNA sequences with ALIGNX, the percent identity between the two sequences can be evaluated.

[0089] A second, non-limiting example of a useful software program for analyzing ClustalW alignments is GeneDoc™. Petition 870240065834, dated 02 / 08 / 2024, page 58 / 210 46 / 96 (developed by Karl Nicholas, http: / / iubio.bio.indiana.edu / soft / nnolbio / ibmpc / genedoc-readme.html). GeneDoc™ allows the assessment of amino acid (or DNA) similarity and identity between multiple proteins.

[0090] Another non-limiting example of a mathematical algorithm used for sequence comparison is the Myers and Miller algorithm (1988). This algorithm is incorporated into the wSTRETCHER program, which is part of the wEMBOSS sequence alignment software package (available at http: / / emboss.sourceforge.net / ). STRETCHER calculates an ideal global alignment of two sequences using a modification of the classic dynamic programming algorithm that uses linear space. The result is a standard alignment file. The substitution matrix, open insertion penalty, and open extension penalties used to calculate the alignment can be specified. When using the STRETCHER program to compare nucleotide sequences, an open Gap penalty of 16 and an extension Gap penalty of 4 can be used. The scoring matrix file for comparing DNA sequences is EDNAFULL.When used to compare amino acid sequences, an open Gap penalty of 12 and an extension Gap penalty of 2 can be used. The scoring matrix file for comparing protein sequences is EBLOSUM62.

[0091] Another non-limiting example of a mathematical algorithm used for sequence comparison is the Needleman and Wunsch algorithm (1970), which is incorporated into the GAP Version 10 and wNEEDLE sequence alignment software packages (http: / / emboss.sourceforge.net / ). The GAP 10 version can be used to determine sequence identity or similarity using the following parameters: for a nucleotide sequence, % of Petition 870240065834, dated 02 / 08 / 2024, pages 59 / 210 47 / 96 Identity and % similarity are found using a GAP Weight of 50 and a Length Weight of 3, and the nwsgapdna.cmp scoring matrix. For amino acid sequence comparison, % identity or % similarity were determined using a GAP weight of 8 and a length weight of 2, and the BLOSUM62 scoring program. wNEEDLE reads two input sequences, finds the optimal alignment (including gaps) along their entire length, and records its optimal global sequence alignment to the file. The algorithm uses a dynamic programming method to ensure that the alignment is optimal, by exploring all possible alignments and choosing the best one. A scoring matrix is ​​read that contains values ​​for each possible residue or nucleotide match.wNEEDLE finds the alignment with the highest possible score where the score of an alignment is equal to the sum of the matches obtained from the scoring matrix, minus the penalties resulting from opening and widening gaps in the aligned sequences. The replacement matrix and the gap opening and widening penalties are specified by the user. When amino acid sequences are compared, a standard Open Gap penalty of 10, a Gap widening penalty of 0.5, and the EBLOSUM62 comparison matrix are used. When DNA sequences are compared using wNEEDLE, an Open Gap penalty of 10, a Gap widening penalty of 0.5, and the EDNAFULL comparison matrix are used.

[0092] Equivalent programs may also be used. By equivalent program is meant any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide residue matches and sequence identity. Petition 870240065834, dated 02 / 08 / 2024, pages 60 / 210 48 / 96 identical percentage when compared to the corresponding alignment generated by ALIGNX, wNEEDLE, or wSTRETCHER. The % identity is the percentage of identical matches between the two sequences over the reported aligned region (including any gaps in length), and the % similarity is the percentage of matches between the two sequences over the reported aligned region (including any gaps in length).

[0093] Toxin fragments and equivalents. Fragments and equivalents that retain the pesticidal activity of the exemplified toxins shall be within the scope of the present invention. Similarly, because of the redundancy of the genetic code, a variety of different DNA sequences may encode the amino acid sequences described herein. It is well within the ability of a person trained in the art to create such alternative DNA sequences that encode them, or essentially the same toxins. Such variant DNA sequences are within the scope of the present invention. As used herein, the reference to essentially the same sequence refers to sequences that have amino acid substitutions, deletions, additions, or insertions that do not materially affect the pesticidal activity. Fragments that retain the pesticidal activity are also included in this definition.

[0094] Alterations can be made to the amino or carboxy terminus of the insecticidal proteins and variants of the invention resulting in polypeptides that retain biological activity. Biologically active fragments or parts include polypeptide fragments comprising amino acid sequences sufficiently identical to the amino acid sequence shown in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 and 40. A Petition 870240065834, dated 02 / 08 / 2024, p. 61 / 210 49 / 96 The biologically active part of a delta endotoxin protein can be a polypeptide that is, for example, 10, 25, 50, 100 or more amino acids in length. Such biologically active parts can be prepared through recombinant protein engineering techniques well known in the art and evaluated for insecticidal activity. The methods for measuring pesticidal activity are well known in the art. As used herein, a fragment encompasses at least 8 contiguous amino acids of the SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 and 40.The invention also covers other fragments, however, such as any fragment in the protein larger than approximately 10, 20, 30, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150 or 1200 amino acids, up to the total length of the insecticidal proteins or variant proteins of the following SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 and 40.

[0095] Fragments with improved biological activity, pest spectrum, or the ability to control resistant insect populations are also provided in the present invention. Modifications can be made to Cry proteins to produce fragments with improved pore formation and thus pesticidal activity. In the case of three-domain Cry proteins, domain 1 is composed of seven α-helices involved in pore formation in the midgut of susceptible insects. Modified DIG proteins with improved activity can be designed to have N-terminal deletions in regions with putative secondary structure homology to α-helix 1 and α-helix 2 of domain 1.

[0096] Proteases can be used to directly obtain active fragments of these toxins. A fragment of a claimed insecticidal toxin will comprise at least about 15, 25, 30, Petition 870240065834, dated 02 / 08 / 2024, page 62 / 210 50 / 96, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1000, 1100 or 1200 contiguous amino acids, or up to the total number of amino acids present in a full-length insecticidal toxin of the invention (for example, 625 amino acids for SEQ ID NO: 2, or 625 amino acids for SEQ ID NO: 4).

[0097] Core toxin and protoxin chimeras. Most crystalline protein molecules of Bacillus thuringiensis delta-endotoxin consist of two functional segments. The protease-resistant core toxin is the first segment and corresponds to approximately the first half of the protein molecule. The approximately C-terminal half of the molecule is the second segment. For the purposes of this application, this second segment will be referred to here as the protoxin segment. The protoxin segment is believed to participate in the formation of toxin crystals (Arvidson et al., (1989)). The complete 130 kDa toxin molecule is rapidly processed in the resistant core segment by protease in the insect gut.The protoxin segment can thus transmit a partial insect specificity with respect to the toxin by limiting the accessibility of the nucleus to the insect by reducing protease processing of the toxin molecule (Haider et al., (1986)) or by reducing the solubility of the toxin (Aronson et al., (1991)).

[0098] Advantageously linked chimeric proteins within the toxin domains of CrylFa and CrylAb have been reported (US Patent No. 5,527,883). Other successes in the area have been reported in the literature. For example, the construction of hybrid delta-endotoxins is reported in the following related technique. Inti. Pat. Appl. Publ. No. WO 95 / 30753 discloses the construction of hybrid B. thuringiensis delta-endotoxins for production in Pseudomonas fluorescens wherein the fragment Petition 870240065834, dated 02 / 08 / 2024, page 63 / 210 The non-toxic protoxin segment 51 / 96 of Cry1F has been replaced by the non-toxic protoxin fragment of CrylAc / CrylAb described in US Patent No. 25,128,130. This patent also discloses the construction of hybrid B. thuringiensis delta-endotoxins for production in P. fluorescens wherein a portion of the non-toxic protoxin segment of CrylAc is replaced with the corresponding non-toxic protoxin fragment of CrylAb. US Patent No. 25,055,294 discloses the construction of a specific hybrid delta-endotoxin between CrylAc (amino acid residues 1-466) and CrylAb (amino acid residues 466-1155) for production in P. fluorescens. Although the aforementioned patent discloses the construction of a hybrid toxin within the active toxin segment, no specification is given regarding the insecticidal activity of the hybrid toxin. International Patent Application Publication No. WO 95 / 30752 discloses the construction of delta endotoxins from B.Hybrid delta-endotoxins for production in P. fluorescens, in which the non-toxic protoxin segment of Cry1C is replaced by the non-toxic protoxin segment of CrylAb. The aforementioned application further discloses that the activity against Spodoptera exigua for the hybrid delta-endotoxin is improved compared to that of the original active toxin, Cry1C. International Patent Application Publication No. WO 95 / 06730 discloses the construction of a hybrid B. thuringiensis delta-endotoxin consisting of domains 1 and 2 of CrylE coupled to domain 3 and the non-toxic protoxin segment of Cry1C. Insect bioassays performed against Manduca sexta (sensitive to Cry1C and CrylE), Spodoptera exigua (sensitive to Cry1C), and Mamestra brassicae (sensitive to Cry1C) show that the Cry1E / Cry1C hybrid toxin is active against M. sexta, S. exigua, and M. brassicae. Bioassay results were expressed as EC50 values ​​(toxin concentration that provides a reduction in growth of...). Petition 870240065834, dated 02 / 08 / 2024, page 64 / 210 52 / 96 50%) instead of LC50 values ​​(toxin concentration that provides 50% mortality). Although the delta-endotoxins used for the bioassay were produced in B. thuringiensis, only the artificially generated active segments of the delta-endotoxins were used, and not the naturally produced crystals typically produced by B. thuringiensis that are present in commercial formulations of B. thuringiensis. The bioassay results indicated that the LC50 values ​​for the CrylE / Cry1C hybrid crystal against S. frugiperda were 1.5 to 1.7 times lower (i.e., more active) than for native Cry1C. This technique also discloses the construction of a B. thuringiensis delta endotoxin hybrid between CrylAb (domains 1 and 2) and Cry1C (domain 3 and the non-toxic protoxin segment), although no data is provided on the activity or utility of the hybrid toxin.

[0099] Lee et al., (1995) report the construction of B. thuringiensis hybrid delta-endotoxins between CrylAc and CrylAa within the active toxin segment. The artificially generated active segments of the hybrid toxins were used to examine protein interactions in brush border membrane vesicles of susceptible insects (BBMV). The bioactivity of the hybrid toxins was not reported. Honee et al., (1991) report the construction of hybrid delta-endotoxins between CrylC (domain 1) and CrylAb (domains 2 and 3) and the reciprocal hybrid between CrylAb (domain 1) and CrylC (domains 2 and 3). These hybrids did not show a significant increase in activity against susceptible insects. Furthermore, the CrylC (domain 1) / CrylAb (domains 2 and 3) hybrid toxin was observed to be hypersensitive to protease degradation. A report by Schnepf et al., (1990) discloses the construction of a CrylAc hybrid toxin in which a small portion of domain 2 was replaced by the corresponding CrylAa region, although no significant increase in activity against the larvae of. Petition 870240065834, dated 02 / 08 / 2024, page 65 / 210 53 / 96 susceptible insects were observed.

[00100] The chimeric toxins of the present invention comprise a complete core N-terminal toxin portion of a Bt toxin and, at some point after the end of the toxin portion, the protein has a transition to a heterologous protoxin sequence. The transition to the heterologous protoxin segment may occur approximately at the native toxin / protoxin junction or a portion of the native protoxin (extending beyond the toxin portion) may be retained with the downstream transition to the heterologous protoxin. For example, a chimeric toxin of the present invention may have the complete toxin portion of a modified CrylCa toxin such as amino acids 1-628 of DIG-473 or DIG-465 and a heterologous protoxin segment (amino acids 629 to the C-terminal). In a preferred embodiment, the heterologous protoxin segment portion is removed from CrylAb.

[00101] A person skilled in this art will observe that Bt toxins, even within a certain class, will vary to some extent in length and in the precise location of the transition from the toxin portion of the core to the protoxin portion. The transition from the toxin portion of the core to the protoxin portion will typically occur between about 50% and about 60% of the total toxin length. The chimeric toxin of the present invention includes the full extent of this N-terminal toxin portion of the core, such as the total length of 628 amino acids of the insecticidal toxin protein IRDIG544.12. SEQ ID NO: 15 discloses the 1887-nucleotide DNA sequence encoding DIG-465, of which the 1887 5'-terminal nucleotides comprise the coding region for the CrylCa core toxin segment with an L57A mutation (leucine at amino acid position 57 substituted for alanine), an embodiment of the present invention. SEQ ID NO: 16 discloses the Petition 870240065834, dated 02 / 08 / 2024, page 66 / 210 SEQ ID NO: 24 discloses the amino acid sequence 628 of the full-length DIG-465 polypeptide, comprising the N-terminal core portion of CrylCa with the aforementioned amino acid substitutions. SEQ ID NO: 23 discloses the 1887 nucleotide sequence of the DNA encoding DIG-473, which comprises the coding region for the toxin segment of the CrylCa core with an F596M mutation (phenylalanine at amino acid position 596, substituted for methionine), another subject of the invention. SEQ ID NO: 24 discloses the amino acid sequence 628 of the full-length DIG-473 polypeptide, comprising the CrylCa portion with the aforementioned amino acid substitutions.

[00102] With respect to the protoxin portion, the total extent of the native CrylAb protoxin portion extends from the toxin end of the full-length CrylAb protein to the C-terminal of the molecule. Attention is drawn to the final approximately 100 to 150 amino acids of this protoxin, which are most critical to include in the chimeric toxin of the present invention.

[00103] Because Cry proteins have selective insecticidal activity, most are active against a limited range of target pests. Therefore, there is a need to further improve the biological activity attributes of Cry proteins. Cry proteins with unique binding characteristics and modes of action are useful in strategies to expand the range of insect pests controlled or against the development of Bt resistance.

[00104] Modifications of Domain III. As described herein, the Bt insecticidal toxins of the present invention are 3-domain type toxins, comprising Domain I, Domain II, and Domain III. Domain III binds certain classes of receptor proteins and may participate in the insertion of an oligomeric toxin pre-pore. Specific hybrid toxins that included substitutions of Domain III Petition 870240065834, dated 02 / 08 / 2024, page 67 / 210 55 / 96 showed superior toxicity against Spodoptera exigua (de Maagd et al., 1996) and there is guidance on the design of Cry toxin domain switching (Knight et al., 2004).

[00105] Domain I Modifications. Numerous studies using biochemical and molecular approaches have provided information on the determinants of Cry protein binding and insertion into insect midgut membranes (reviewed in Piggot and Ellar, 2007). Domain I of Cry1A and Cry3A proteins has been studied in relation to its ability to insert and form pores in membranes. The α4 and α5 helices of the domain play fundamental roles in membrane insertion and pore formation (Walters et al., 1993; Gazit et al., 1998; Nunez-Valdez et al., 2001), with the other helices proposed to come into contact with the membrane surface like the striations of an umbrella (Gazit et al., 1998).

[00106] The alpha 3 helix appears in some cases to be necessary for the formation and toxicity of oligomeric pre-pores. Some α-helix 3 mutants are able to bind receptors but do not form oligomers and are not toxic to Manduca sexta (reviewed in Jimenez-Juarez et al., 2008). However, proteolytically activated forms of Cry3Aa1 lack α 1, 2, and 3 helices (Carroll et al., 1997).

[00107] The alpha 1 helix is ​​removed after receptor binding. Gomez et al. (2002) found that CrylAb oligomers formed after binding to the BBMV receptor lacked the 1 portion of the α-helix of domain I. Furthermore, Soberon et al. (2007) showed that N-terminal deletion mutants of CrylAb and Cry 1 Ac lacking approximately 60 amino acids that comprise the α 1 helix in the three-dimensional Cry structure are able to assemble molecular weight monomers around 60 kDa in pre-pores in Petition 870240065834, dated 02 / 08 / 2024, pages 68 / 210 56 / 96 absence of cadherin linkage. These N-terminal deletion mutants have been reported to be active against Cry-resistant insect larvae. Furthermore, Diaz-Mendoza et al. (2007) described 43 kDa and 46 kDa CrylAb fragments that maintained activity against the Mediterranean corn borer (Sesamia nonagrioides). These fragments were shown to include amino acid residues 116 to 423; however, the precise amino acid sequences have not been elucidated, and the mechanism of activity of these proteolytic fragments is unknown. The results of Gomez et al. (2002), Soberon et al. (2007), and Diaz-Mendoza et al. (2007) contrast with those of Hofte et al. (1986), who reported that deletion of 36 amino acids from the N-terminal of CrylAb resulted in loss of insecticidal activity.

[00108] Anti-toxin antibodies. The equivalent toxins and / or the genes encoding these equivalent toxins can be derived from Bacillus thuringiensis isolates and / or DNA libraries using the teachings provided herein. There are several methods for obtaining the pesticide toxins of the present invention. For example, immunoreactive antibodies to the pesticide toxins described and claimed herein can be used to identify and isolate other toxins from a protein mixture. Specifically, antibodies can be boosted for the portions of the toxins that are more constant and more distinct from other Bt toxins. These antibodies can then be used to specifically identify the equivalent toxins with characteristic activity, for example, by immunoprecipitation, enzyme-linked immunosorbent assay (ELISA) or immunoblotting (western blotting).Antibodies to the toxins described here, or to equivalent toxins, or fragments of these toxins, can be easily prepared using standard procedures in this technique. The genes. Petition 870240065834, dated 02 / 08 / 2024, pages 69 / 210 57 / 96, which encode these toxins, can then be obtained from the microorganisms that produce the toxins.

[00109] Once the Bt insecticidal toxin has been isolated, specific antibodies to the toxin can be created by conventional methods that are well known in the art. Repeated injections into a host of choice over a period of weeks or months will elicit an immune response and result in significant anti-Bt toxin serum titers. Preferred hosts are mammalian species, and the most highly preferred species are rabbits, goats, sheep, and mice. Blood drawn from such immunized animals can be processed by established methods to obtain antiserum (polyclonal antibodies) reactive with the Bt insecticidal toxin. The antiserum can then be purified by affinity through adsorption onto the toxin according to techniques known in the art.Affinity-purified antiserum can be further purified by isolating the immunoglobulin fraction within the antiserum using procedures known in the art. The resulting material will be a heterogeneous population of immunoglobulins reactive with the Bt insecticidal toxin.

[00110] Anti-Bt toxin antibodies can also be generated by preparing a semi-synthetic immunogen consisting of a synthetic peptide fragment of the Bt insecticidal toxin conjugated with an immunogenic carrier. Numerous schemes and useful instruments for the production of peptide fragments are well known in the art. Many suitable immunogenic carriers such as bovine serum albumin (BSA) or limpet hemocyanin are also well known in the art, as are the techniques for coupling the immunogen and carrier proteins. Once the semi-synthetic immunogen has been constructed, the procedure for producing antibodies specific to the toxin fragment Petition 870240065834, dated 02 / 08 / 2024, pages 70 / 210 The Bt insecticide 58 / 96 is identical to that used to produce antibodies reactive with natural Bt toxin.

[00111] Anti-Bt toxin monoclonal antibodies (MAbs) are readily prepared using purified Bt insecticidal toxin. Methods for producing MAbs have been practiced for over 15 years and are well known to those skilled in the art. Repeated intraperitoneal or subcutaneous injections of purified Bt insecticidal toxin in adjuvant will elicit an immune response in most animals. Hyperimmunized B lymphocytes are removed from the animal and fused with a suitable fusion-pair cell line capable of indefinite culture. Preferred animals whose B lymphocytes can be hyperimmunized and used in MAb production are mammals. The most preferred animals are rats and mice, and the most preferred is the BALB / c mouse breed.

[00112] Numerous mammalian cell lines are suitable fusion pairs for hybridoma production. Many of these lines are available from the American Type Culture Collection (ATCC, Manassas, VA) and commercial suppliers. Preferred fusion pair cell lines are derived from mouse myelomas, and the HL-1® Friendly myeloma-653 cell line (Ventrex, Portland, ME) is most preferred. Once fused, the resulting hybridomas are cultured in a selective growth medium for one to two weeks. Two well-known selection systems are available to eliminate unfused myeloma cells, or fusions between myeloma cells, from the mixed hybridoma culture. The choice of selection system depends on the immunized mouse strain and the myeloma fusion pairs used. The AAT selection system, described by Taggart and Samloff (1983), can be used; however, the HAT (hypoxanthine, Petition 870240065834, dated 02 / 08 / 2024, pp. 71 / 210 The 59 / 96 aminopterin, thymidine) assay, described by Littlefield (1964), is preferable because of its compatibility with the preferred mouse strain and the fusion pairs mentioned above. The spent growth medium is then filtered for immune-specific MAb secretion. Enzyme-linked immunosorbent assay (ELISA) procedures are most suitable for this purpose; however, radioimmunoassays adapted for high-volume screening are also acceptable. Several screenings designed to consecutively reduce the considerable number of irrelevant or less desirable cultures can be performed. Cultures secreting MAbs reactive with the Bt insecticidal toxin can be screened for cross-reactivity with Bt insecticidal toxins. MAbs that preferentially bind to the preferred Bt insecticidal toxin can be isotyped using commercially available assays.Preferred MAbs are of the IgG class, and the most highly preferred MAbs are of the lgG1 and lgG2a subisotypes.

[00113] Hybridoma cultures that secrete preferred MAbs can be subcloned multiple times to establish monoclonal capability and stability. Well-known methods for subcloning non-adherent eukaryotic cell cultures include limiting dilution techniques, soft agarose, and fluorescence-activated cell sorting. After each subcloning, the resulting cultures are preferably reanalyzed for antibody secretion and isotype to ensure that a stable preferred MAb-secreting culture has been established.

[00114] Anti-Bt toxin antibodies are useful in various methods of detecting the claimed Bt insecticidal toxin of the present invention, and its variants or fragments. It is well known that antibodies labeled with a clearance group can be used to identify the presence of antigens in a variety of Petition 870240065834, dated 02 / 08 / 2024, p. 72 / 210 60 / 96 environments. Radioisotope-labeled antibodies have been used for decades in radioimmunoassays to identify, with high precision and sensitivity, the presence of antigens in a variety of biological fluids. More recently, enzyme-labeled antibodies have been used as a substitute for radiolabeled antibodies in the ELISA assay. Furthermore, the immunoreactive antibodies to the Bt insecticidal toxin of the present invention can be linked to an immobilizing substance such as a reservoir or polystyrene particle and used in immunoassays to determine if the Bt toxin is present in a test sample.

[00115] In a preferred embodiment, insecticidal proteins or a variant are released orally via a transgenic plant comprising a nucleic acid sequence expressing a toxin of the present invention. The present invention provides a method of producing an insect-resistant transgenic plant comprising introducing a nucleic acid molecule of the invention into the plant wherein the toxin is expressed in the transgenic plant in an amount effective to control an insect. In a non-limiting example, a basic cloning strategy may be to subclone the full-length or modified Cry coding sequences (CDS) into a plant expression plasmid at the Ncol and Saci restriction sites.The resulting plant expression cassettes containing the appropriate Cry coding region under the control of plant expression elements (e.g., plant-expressed promoters, 3'-terminal transcription termination and polyadenylate addition determinants, and others) are subcloned into a binary vector plasmid using, for example, Gateway® technology or standard restriction enzyme fragment cloning procedures. LR Clonase™ (Invitrogen), by. Petition 870240065834, dated 02 / 08 / 2024, page 73 / 210 For example, 61 / 96 can be used to recombine full-length and modified plant gene expression cassettes into a binary plant transformation plasmid if Gateway® technology is used. It is convenient to employ a binary plant transformation vector harboring a bacterial gene that confers resistance to the antibiotic spectinomycin when the plasmid is present in E. coli and Agrobacterium cells. It is also convenient to employ a binary vector plasmid containing a selectable expressed plant marker gene that is functional in the desired host plants.Examples of selectable expressed plant marker genes include, but are not limited to, the Tn5 transposon aminoglycoside phosphotransferase gene (Aph II) which encodes resistance to the antibiotics kanamycin, neomycin and G418, as well as those genes which encode tolerance to glyphosate; hygromycin; methotrexate; phosphinothricin (bialaphos), imidazolinones, sulfonylureas and triazolopyrimidine herbicides such as chlorsulfuron, bromoxynil, dalapon, and the like.

[00116] Alternatively, the structure of the binary plant transformation vector plasmid containing the DIG465, DIG-473, DIG-468, DIG-483, DIG-462, DIG-463, DIG-464, DIG466, DIG-467, DIG-469, DIG-474, DIG-482, DIG-485, DIG-487, IRDIG544.8, IRDIG544.9, IRDIG544.11 or IRDIG544.12 gene insertion is performed by fingerprinting the restriction digestion of plasmid DNA prepared from candidate Agrobacterium isolates by standard molecular biology methods well known to those versed in the technique of Agrobacterium manipulation.

[00117] Those versed in the technique of obtaining transformed plants through Agrobacterium-mediated transformation methods will understand that other Agrobacterium strains besides Z707S can be used and the choice of strain may Petition 870240065834, dated 02 / 08 / 2024, p. 74 / 210 62 / 96 depends on the identity of the host plant species to be transformed.

[00118] The following are examples illustrating procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixing ratios are by volume, unless otherwise stated. All patents, patent applications, provisional applications and publications referenced or cited herein are incorporated by reference in their entirety, to the extent that they are not inconsistent with the explicit teachings of this descriptive report. Unless specifically indicated or implied, the terms a, an, or at least as used herein mean at least one. EXAMPLE 1 Design of an optimized plant version of the coding sequence for Bt insecticidal proteins.

[00119] A DNA sequence having a plant codon influence was designed and synthesized to produce insecticidal proteins in transgenic monocotyledonous and dicotyledonous plants. A codon usage table for maize (Zea mays L.) was calculated from 706 protein coding sequences (CDS) obtained from sequences deposited in GenBank. Codon usage tables for tobacco (Nicotiana tabacum, 1268 CDS), canola (Brassica napus, 530 CDS), cotton (Gossypium hirsutum, 197 CDS), and soybean (Glycine max, 1000 CDS) were obtained by downloading data from the website http: / / www.kazusa.or.jp / codon / . A set of influenced codons comprising highly used codons common to both maize and dicotyledon datasets, in appropriate weighted average relative amounts, was calculated after omitting any redundant codons used less than about 10%. Petition 870240065834, dated 02 / 08 / 2024, pages 75 / 210 63 / 96 of the total codon uses for this amino acid in any plant type. To derive an optimized plant sequence encoding the insecticidal protein, codon substitutions for the insecticidal protein DNA sequences were performed such that the resulting DNA sequence had the total codon composition of the plant-optimized codon influence table. Further sequence refinements were made to eliminate undesirable restriction enzyme recognition sites, potential plant intron binding sites, long A / T or C / G residue series, and other motifs that might interfere with the stability, transcription, or translation of the coding region RNA in plant cells. Other changes were made to introduce the desired restriction enzyme recognition sites and to eliminate long internal open reading frames (structures other than +1).These changes were all made within the constraints of partial codon-influenced composition retention. To complete the design, a sequence encoding translational stop codons in all 6 open reading frames was added to the 3' end of the coding regions, and appropriate restriction recognition sites were added to the 5' and 3' ends of the sequences. Synthesis of the designed sequence was performed by a commercial supplier (DNA2.0, Menlo Park, CA). Further guidance regarding the production of synthetic genes can be found, for example, in WO 97 / 13402 and US Patent No. 25,380,831.

[00120] The optimized plant DNA sequences encoding the DIG proteins of the present invention (SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 and 40) are described as SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and 39. DNA molecules comprising the Petition 870240065834, dated 02 / 08 / 2024, p. 76 / 210 64 / 96 sequences described in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and 39 were synthetically assembled by a commercial entity (DNA2.0). EXAMPLE 2 Construction of expression plasmids encoding insecticidal toxins and expression in bacterial hosts.

[00121] Standard cloning methods were used in the construction of Pseudomonas fluorescens (Pf) expression plasmids, designed to produce proteins DIG-465 (SEQ ID NO: 16), DIG-473 (SEQ ID NO: 24), DIG-468 (SEQ ID NO: 2), DIG-483 (SEQ ID NO: 4), DIG-462 (SEQ ID NO: 10), DIG-463 (SEQ ID NO: 12), DIG-464 (SEQ ID NO: 14), DIG-466 (SEQ ID NO: 18), DIG-467 (SEQ ID NO: 20), DIG-469 (SEQ ID NO: 22), DIG-474 (SEQ ID NO: 26), DIG-482 (SEQ ID NO: 28), DIG-485 (SEQ ID NO: 6) and DIG-487 (SEQ ID NO: 8) encoded by plant-optimized coding regions. Restriction endonucleases were obtained from New England BioLabs (NEB, Ipswich, MA) and T4 DNA Ligase (Invitrogen Corporation, Carlsbad, CA) was used for DNA ligation.

[00122] The basic cloning strategy involved subcloning the toxin coding sequence DIG-465, DIG-473, DIG-468, DIG-483, DIG-462, DIG-463, DIG-464, DIG-466, DIG-467, DIG-469, DIG-474, DIG-482, DIG-485 or DIG-487 (CDS) into pDOW1169 at restriction sites such as Spel and Xhol, whereby it was placed under the expression control of the Ptac promoter and the rrnBT1T2 terminator of the pKK223-3 plasmid (PL Pharmacia, Milwaukee, Wl). pDOW1169 is a low-copy plasmid with the origin of replication of RSF1010, a pyrF gene, and a ribosome-binding site preceding restriction enzyme recognition sites into which DNA fragments containing protein-coding regions can be introduced (US Patent Application). Petition 870240065834, dated 02 / 08 / 2024, p. 77 / 210 65 / 96 (US20080193974). The expression plasmid was transformed by electroporation into DC454 (a near-wild-type P. fluorescens strain having βpyrF and lsc::laclQI mutations), or its derivatives, recovered in SOC-Soy hydrolysate medium and coated on selective medium (uracil-deficient M9 glucose agar, Sambrook et al., Supra). Details of the microbiological manipulations are available in Squires, CH et al., (2004), in US Patent Application 20060008877, in US Patent Application 20080193974 and in US Patent Application 20080058262, incorporated herein by reference. The strains were validated by restriction digestion of the miniprep plasmid DNA.

[00123] Growth and Expression Analysis in Shaker Flasks. The production of DIG-465, DIG-473, DIG-468, DIG-483, DIG-462, DIG-463, DIG-464, DIG-466, DIG-467, DIG-469, DIG-474, DIG-482, DIG-485 or DIG-487 toxins for insect characterization and bioassay was performed using P. strains.fluorescens cultures were grown in shake flasks housing the expression constructs. Seed cultures grown in M9 medium supplemented with 1% glucose and trace elements were used to inoculate 50 ml of defined minimum medium with 5% glycerol (Teknova Cat. # 3D7426, Hollister, CA). Expression of the insecticidal protein toxin gene via the Ptac promoter was induced by the addition of isopropyl-pD-1 thiogalactopyranoside (IPTG) after an initial 24-hour incubation at 30°C with shaking. Cultures were analyzed at the time of induction and at various time points after induction. Cell density was measured by optical density at 600 nm (OD60o). Other culture media suitable for the growth of Pseudomonas fluorescens may also be used, for example, as described in Huang et al., 2007 and in US Patent Application 20060008877.

[00124] Cell Fractionation and SDS-PAGE Analysis of Samples in the Shake Bottle. At each sampling time, Petition 870240065834, dated 02 / 08 / 2024, pp. 78 / 210 66 / 96 0.5 ml aliquots were centrifuged at 14000 x g for five minutes. The cell beads were frozen at -80°C. The soluble and insoluble fractions of the frozen cell bead samples in the shaker flask were generated using BugBuster Master Mix (EMDMillipore® Darmstadt, Germany). Each cell bead was resuspended in 0.5 ml of BundBuster Master Mix™ solution and incubated with shaking at room temperature for 30 minutes. The samples were lysed using a globule shaker with 0.1 mm glass beads for 3 minutes. The lysate was centrifuged at 14000 rpm for 5 minutes and the supernatant was recovered as the soluble fraction. The granule (insoluble fraction) was then resuspended in an equal volume of extraction buffer (8 M urea, 0.5 M NaCl, 25 mM NaPO4, pH 10.4).

[00125] Samples were mixed 1:1 with 2X NuPAGE Tris Glycine SDS Sample Buffer (Invitrogen, Carlsbad, CA) containing dithiothreitol (DTT) and boiled for 5 minutes before loading onto Novex 4-20% Tris Glycine SDS polyacrylamide gel (Invitrogen, Carlsbad, CA). Electrophoresis was performed in the recommended TrisGlycine buffer. Gels were stained with Bio-Safe Coomassie Stain according to the manufacturer's protocol (Bio-Rad Inc., Hercules, CA) and imaged using the GE Typhooon Series Imaging system (Pittsburgh, PA).

[00126] Inclusion body preparation. Cry (IB) protein inclusion body preparations were performed in P. fluorescens fermentation cells that produced the insoluble Bt insecticidal protein, as demonstrated by SDS-PAGE and MALDI-MS (Matrix Assisted Laser Desorption / Ionization Mass Spectrometry). P. fluorescens fermentation pellets were thawed in a water bath at 37 °C. The cells were then placed back in Petition 870240065834, dated 02 / 08 / 2024, pp. 79 / 210 67 / 96 cells were suspended in 25% w / v lysis buffer (50 mM Tris, pH 7.5, 200 mM NaCl, 20 mM disodium EDTA salt (ethylenediaminetetraacetic acid), 1% Triton X-100 and 5 mM Dithiothreitol (DTT); 5 mL / L of bacterial protease inhibitor cocktail (P8465 SigmaAldrich, St. Louis, MO) was added just before use). The cells were suspended using a manual homogenizer on the lowest setting (Tissue Tearer, BioSpec Products, Inc., Bartlesville, OK). Lysozyme (25 mg of SigmaAldrich L7651, from chicken egg white) was added to the cell suspension by mixing with a metal spatula, and the suspension was incubated at room temperature for one hour. The suspension was cooled on ice for 15 minutes, then subjected to ultrasonic vibration using a Branson Sonifier 250 (two 1-minute sessions, 50% duty cycle, 30% productivity). Cell lysis was verified by microscopy.An additional 25 mg of lysozyme was added if necessary, and incubation and ultrasonic vibration were repeated. When cell lysis was confirmed by microscopy, the lysate was centrifuged at 11,500 x g for 25 minutes (4°C) to form the IB pellet, and the supernatant was discarded. The IB pellet was suspended in 100 mL of lysis buffer, homogenized with a hand mixer, and centrifuged as above. The IB pellet was repeatedly washed by suspension (in 50 mL of lysis buffer), homogenization, ultrasonic vibration, and centrifugation until the supernatant became colorless and the IB pellet became firm and whitish in color. For the final wash, the IB pellet was suspended in sterile-filtered distilled water (0.22 µm) containing 2 mM EDTA and centrifuged. The final granule was suspended in sterile, filtered distilled water containing 2 mM EDTA and stored in 1 ml aliquots at -80 °C. Petition 870240065834, dated 02 / 08 / 2024, pp. 80 / 210 68 / 96

[00127] SDS-PAGE analysis and protein quantification in IB preparations were performed by thawing a 1 ml aliquot of IB granule and diluting it 1:20 with sterile-filtered distilled water. The diluted sample was then boiled with 4X reducing sample buffer [250 mM Tris, pH 6.8, 40% glycerol (v / v), 0.4% Bromophenol Blue (w / v), 8% SDS (w / v) and 8% βMercaptoethanol (v / v)] and loaded onto a Novex® 4-20% TrisGlycine, 12+2 reservoir gel (Invitrogen) activated with 1X Tris / Glycine / SDS buffer (BioRad). The gel was run for 60 minutes at 200 volts, then stained with Coomassie Blue (50% G-250 / 50% R-250 in 45% methanol, 10% acetic acid), and decolorized with 7% acetic acid, 5% methanol in distilled water. Target band quantification was performed by comparing densitometric values ​​for the bands to bovine serum albumin (BSA) samples activated in the same gel to generate a standard curve.

[00128] Solubilization of Inclusion Bodies. Six ml of inclusion body suspension from the Pf clone containing the protein DIG-465, DIG-473, DIG-468, DIG-483, DIG-462, DIG-463, DIG-464, DIG-466, DIG-467, DIG-469, DIG-474, DIG-482, DIG-485, or DIG-487 were centrifuged at the highest setting of an Eppendorf model 5415C microcentrifuge (approximately 14000 xg) to sediment the inclusions. The storage buffer supernatant was removed and replaced with 25 ml of 100 mM sodium carbonate buffer, pH 11, in a 50 ml conical tube. The inclusions were resuspended using a pipette and vortexed to mix thoroughly. The tube was placed on a gentle shaking platform at 4°C overnight to extract the target protein. The extract was centrifuged at 30,000 x g for 30 min at 4°C, and the resulting supernatant was concentrated. Petition 870240065834, dated 02 / 08 / 2024, pages 81 / 210 69 / 96 times using an Amicon Ultra-15 regenerated cellulose centrifugal filter device (30000 Molecular Weight Cutoff; Millipore). The sample buffer was then changed to 10 mM CAPS [3-(cyclohexamino)1-propanesulfonic acid] pH 10, using disposable PD-10 columns (GE Healthcare, Piscataway, NJ).

[00129] Gel electrophoresis. The concentrated extract was prepared for electrophoresis by dilution 1:50 in NuPAGE® LDS sample buffer (Invitrogen) containing 5 mM dithiothreitol as a reducing agent and heated at 95°C for 4 minutes. The sample was loaded in duplicate lanes of a 4-12% NuPAGE® gel alongside five BSA standards ranging from 0.2 to 2 pg / lane (for standard curve generation). Voltage was applied at 200V using MOPS SDS running buffer (Invitrogen) until the tracer dye reached the bottom of the gel. The gel was stained with 0.2% Coomassie Blue G-250 in 45% methanol, 10% acetic acid, and decolorized, first briefly with 45% methanol, 10% acetic acid, and then at length with 7% acetic acid, 5% methanol until the background was clear. After decolorization, the gel was scanned with a Biorad Fluor-S Multifilmer. The instrument's Quantity One v.4.5.2. Software was used to obtain volumes subtracted from the formation of colored protein bands and generate the BSA standard curve, which was used to calculate the protein concentration of DIG-465, DIG-473, DIG-468, DIG-483, DIG-462, DIG-463, DIG-464, DIG-466, DIG-467, DIG-469, DIG-474, DIG-482, DIG-485, or DIG-487 in the stock solution.

[00130] The expression level of DIG-465, DIG-473, DIG-468, DIG-483, DIG-463, DIG-464, DIG-466, DIG-467, DIG-469, DIG-474, DIG-482, DIG-485, and DIG-487 was compared with the expression level of truncated CrylCa (DIG-462) when expressed in Pseudomonas fluorescens bacterial cells. Truncated CrylCa (DIG-462) expresses Petition 870240065834, dated 02 / 08 / 2024, page 82 / 210 70 / 96 is expressed at approximately 1 g / L, while DIG-473 is expressed at approximately 0.5 g / L. DIG-465 is expressed at approximately 5 times higher levels than truncated CrylCa, at 4.9 g / L. These in vitro results show that the L57A mutation results in higher expression of truncated CrylCa protein. EXAMPLE 3 Insecticidal activity of DIG proteins produced in Pseudomonas fluorescens

[00131] The insecticidal toxins of Bt DIG-462, DIG-463, DIG-464, DIG-465, DIG-466, DIG-467, DIG-468, DIG-469, DIG-470, DIG-473 and DIG-474 have been shown to be active against Lepidoptera species, including the diamondback moth (DBM, Plutella xylostella (Linnaeus)) and the fall armyworm (FAW, Spodoptera frugiperda (Smith)).

[00132] Sample preparation and bioassays. The embedding body preparations in 10 mM CAPS pH 10 were appropriately diluted in 10 mM CAPS, pH 10, and all bioassays contained a control treatment consisting of this buffer, which served as a formation check for mortality or growth inhibition.

[00133] Protein concentrations in the bioassay buffer were estimated by gel electrophoresis using BSA to create a standard curve for gel densitometry, which was measured using a BioRad imaging system (Fluor-S Multiimager with Quantitative One software version 4.5.2). Proteins in the gel matrix were stained with Coomassie-based dye and decolorized before reading.

[00134] The purified proteins were tested for insecticidal activity in bioassays conducted with neonate Lepidoptera larvae on an artificial insect diet. DBM and FAW larvae were hatched from eggs obtained from a colony maintained by a Petition 870240065834, dated 02 / 08 / 2024, page 83 / 210 71 / 96 commercial insectarium (Benzon Research Inc., Carlisle, PA). rFAW larvae were hatched from eggs collected from a proprietary colony (Dow AgroSciences LLC, Indianapolis, IN).

[00135] These bioassays were conducted in 128-reservoir plastic trays specifically designed for insect bioassays (CD International, Pitman, NJ). Each reservoir contained 1.0 ml of multi-species Lepidoptera diet (Southland Products, Lake Village, AR). A 40 µl aliquot of protein sample was pipetted onto the 1.5 cm² surface area of ​​the diet in each reservoir (26.7 μL / cm²). Cry protein concentrations were calculated as the amount (ng) of DIG protein per square centimeter (cm²) of surface area in the reservoir. The treated trays were kept in a fume hood until the liquid on the diet surface evaporated or was absorbed into the diet.

[00136] Within a few hours of hatching, individual larvae were collected with a moistened camel hair brush and placed on the treated diet, one larva per reservoir. The infested reservoirs were then sealed with transparent plastic adhesive sheets, vented to allow gas exchange (CD International, Pitman, NJ). Bioassay trays were maintained under controlled environmental conditions (28°C, ~60% relative humidity, 16:8 [Light:Dark]) for 5 days, after which the total number of insects exposed to each protein sample, the number of dead insects, and the weight of surviving insects were recorded. The percentage mortality and percentage growth inhibition were calculated for each treatment. Growth inhibition (Gl) was calculated as follows: Gl = [1 - (TWIT / TNIT) / (TWIBC / TNIBC)] where TWIT is the total weight of insects in the treatment, TNIT is the total number of insects in the treatment. Petition 870240065834, dated 02 / 08 / 2024, page 84 / 210 72 / 96 TWIBC is the total weight of insects in the formation check (buffer control), and TNIBC is the total number of insects in the formation check (buffer control).

[00137] In the DBM bioassay, 10 and 300 ng / cm2 of DIG-462, DIG-463, DIG-464, DIG-465, DIG-466, DIG-467, DIG-468, DIG-469, DIG-470, DIG-471, DIG-472, DIG-473 and DIG-474 were tested against the insect sp. FAW was tested with the inclusion body preparation of DIG462, DIG-465, DIG-473 at dilution rates of 1X and 5X. The results of percentage mortality and growth inhibition were compared.

[00138] Mortality was 100% at 300 ng / cm2 for treatments with DIG-462, DIG-463, DIG-464, DIG-465, DIG-466, DIG-468, DIG-469, DIG-473, and DIG-474 (Table 2 and Table 3). Growth inhibition was 70 to 90% at 10 ng / cm2 and 100% at 300 ng / cm2 for treatments with DIG-465 and DIG-473 (Table 2). Table 2 Results of bioassay tests of the proteins DIG-462, DIG-465, and DIG-473 in DBM, measuring both mortality and growth inhibition. Protein Mortality Growth inhibition DIG-462 +++ ++++ DIG-465 ++ ++++ DIG-473 +++ ++++ For Mortality ++ = 0 to 20% at 10 ng / cm2 and 100% at 300 ng / cm2, +++ = 30 to 60% at 10 ng / cm2 and 100% at 300 ng / cm2. For Growth Inhibition ++++ = 70 to 90% growth inhibition at 10 ng / cm2 and 100% inhibition at 300 ng / cm2. Table 3 Bioassay results of protein mutants tested against Petition 870240065834, dated 02 / 08 / 2024, page 85 / 210 73 / 96 DBM at concentrations of 10 ng / cm2 and 300 ng / cm2. Protein % Mortality at 10 ng / cm2 % Mortality at 300 ng / cm2 DIG-462 38 100 DIG-463 63 100 DIG-464 38 100 DIG-465 0 100 DIG-466 38 100 DIG-467 38 88 DIG-468 13 100 DIG-469 0 100 DIG-470 0 50 DIG-471 0 0 DIG-472 0 0 DIG-473 38 100 DIG-474 0 100 BSA 0 0

[00139] The growth inhibition of Cry1Ca core toxin (DIG-462), DIG-465 protein, and DIG-473 for FAW larvae was determined to be >40% for all treatments (Table 4). The proteins were tested at full strength and diluted 5 times with buffer (10 mM CAPS, pH 10). Table 4 Percentage inhibition of DIG-462, DIG-465, and DIG-473 growth for FAW Protein Dilution % Growth Inhibition DIG-462 1X 43 DIG-462 5X 47 DIG-465 1X 81 DIG-465 5X 58 DIG-473 1X 56 DIG-473 5X 48 Buffer 1X 0

[00140] The DBM activity and susceptibility of the purified protein to digestion by chymotrypsin were evaluated. An unexpected and surprising finding was that DIG-473 was resistant to chymotrypsin cleavage while simultaneously having the same potency against DBM as DIG-462. This contrasts with the Cry1Ca core (DIG-462) and DIG-465 proteins, which were Petition 870240065834, dated 02 / 08 / 2024, p. 86 / 210 74 / 96 susceptible to chymotrypsin cleavage in vitro (Table 5). Table 5 Proteins with activity against DBM (DIG-462 is the standard) and Protein resistance to chymotrypsin cleavage. DIG# Mutation Type Activity in DBM Chymotrypsin Resistant 462 Truncated ++++ No | 463 | G54A | ++++ | No | 464 L57M ++++ No | 465 | L57A | ++++ | No | 466 V68F ++++ No | 467 | V68I | ++++ | No | 468 AGPS +++ No | 469 | W73A +++ | Partial | 473 F596M ++++ Yes | 474 | F596A | ++++ | No | 482 G54A / W73M +++ Yes | 483 | G54A / AGPS +++ | Yes | 485 L57A / AGPS +++ No | 487 | L57M / AGPS +++ | No | EXAMPLE 4 Bioassays with European Corn Borer (ECB), Southwestern Corn Borer (SWCB) and Southern Corn Larva (SAW)

[00141] Bioassays were conducted in 32-pool test trays. Approximately 5 ml of a 2% water-agar solution was applied to each pool and the agar was allowed to solidify completely.

[00142] The plants were approximately 3 weeks old and tested in the T^ generation. Three replicates of T1 leaf material were completed. One leaf was cut (T' x 0.5 rectangular) and placed in a single reservoir of the tray. Each reservoir was infested with 10 individual insect larvae (generally less than 24 hours old) of ECB, CrylFa rECB, or SWCB. For SAW, 5 individual insect larvae were infested per reservoir. Seed-based plants derived from B104 congenital lines and plants transformed with Yellow Fluorescent Protein (YFP) served as negative controls.

[00143] The infested reservoirs were then sealed with Petition 870240065834, dated 02 / 08 / 2024, page 87 / 210 75 / 96 transparent plastic adhesive sheets, vented to allow gas exchange (CD International, Pitman, NJ). The trays were placed in a Conviron incubator and maintained at 28°C (16:8h light:dark, 60% RH) for 3 days, after which the total amount of damage to each sheet (0, 5, 10, 15, 25, 50, 75% damage, etc., up to 100%) was recorded.

[00144] Reduced feeding damage caused by ECB and CrylFa-resistant ECB (rECB) was observed when insect larvae were exposed to plants containing truncated CrylCa modified protein. When tested in a diet bioassay, where purified full-length CrylCa is placed on top of an artificial insect diet and individual insects are allowed to feed on the diet containing the toxin, the modified CrylCa is observed to be inactive against ECB and rECB. However, when expressed in maize at concentrations >120 ng / cm2, plant CrylCa expression provides unexpected protection against feeding damage caused by ECB and especially rECB. Table 6 Results of the IRDIG544.12 T1 maize bioassay when fed the European Corn Borer (ECB) and CrylFa-resistant ECB (rECB) Plant Name Description Average ECB Damage Average rECB Damage Toxin ng / cm2 Negative control with YFP control 100 88.3 0 Negative control with YFP control 100 93.3 0 Negative control with YFP control 97.7 98.3 0 Negative control with YFP control 99.3 98 0 Negative control with YFP control 100 92.7 0 114269[1]-021.001 AJ.025 IRDIG544.12 w / TraP12 96 85 41 114269[1]-021.001 AJ.018 IRDIG544.12 w / TraP12 94.3 90 42 114269[1]-021.001 AJ.017 IRDIG544.12wZTraP12 99.3 95 34 114269[1]-021.001 AJ.023 IRDIG544.12wZTraP12 99.3 50 36 114269[1]-021.001 AJ.016 IRDIG544.12wZTraP12 99.3 80 33 114260[1]-021 .AJ001.023 IRDIG544.12 66.7 11.7 210 114260(11-021 .AJ001.029 IRDIG544.12 69.7 13.3 230 114260[1]-021 .AJ001.021 IRDIG544.12 66.7 16.7 210 114260[1]-021 .AJ001.016 IRDIG544.12 50 18.3 230 Petition 870240065834, dated 02 / 08 / 2024, pages 88 / 210 76 / 96 Plant Name Description Average ECB Damage Average rECB Damage Toxin ng / cm2 114260[1]-021.AJ001.019 IRDIG544.12 50 18.3 210 114259[1]-009.AJ001.018 IRDIG544.12 95 85 100 114259[1]-009.AJ001.022 IRDIG544.12 86 61.7 130 114259[1]-009.AJ001.021 IRDIG544.12 93.3 75 140 114259[1]-009.AJ001.026 IRDIG544.12 80 95 67 114259[1]-009.AJ001.027 IRDIG544.12 100 61.7 92 114260[1]-010.001AJ.054 IRDIG544.12 68.3 25 180 114260[1]-010.001AJ.048 IRDIG544.12 71.7 18.3 180 114260[1]-010.001AJ.047 IRDIG544.12 66.7 50 140 114260[1]-010.001AJ.052 IRDIG544.12 71.7 20 220 114260[1]-010.001AJ.046 IRDIG544.12 86.7 16.7 200 114269[1]-029.001AJ.027 IRDIG544.12 w / TraP12 91 66.7 39 114269[1]-029.001AJ.028 IRDIG544.12 w / TraP12 88.3 66.7 30 114269[1]-029.001AJ.023 IRDIG544.12 w / TraP12 96.7 88.3 41 114269[1]-029.001AJ.026 IRDIG544.12 w / TraP12 96 76.7 37 114269[1]-029.001AJ.019 IRDIG544.12 w / TraP12 96.7 95 41 114267[1]-009.001AJ.044 IRDIG544.12 w / TraP12 95 70 36 114267[1]-009.001AJ.034 IRDIG544.12 w / TraP12 88.3 56.7 44 114267[1]-009.001AJ.032 IRDIG544.12 w / TraP12 96.7 86.7 41 114267[1]-009.001AJ.037 IRDIG544.12 w / TraP12 96.7 88.3 43 114267[1]-009.001AJ.030 IRDIG544.12 w / TraP12 91.7 80 43 114259[1]-006.001AJ.015 IRDIG544.12 91.7 90 38 114259[1]-006.001AJ.014 IRDIG544.12 98,3 85 41 114259[1]-006.001AJ.005 IRDIG544.12 95 80 40 114259[1]-006.001AJ.010 IRDIG544.12 81,7 86 39 114259[1]-006.001AJ.013 IRDIG544.12 85 88,3 47 114270[1]-027.AJ001.029 IRDIG544.12 w / TraP12 98,7 96,7 170 114270[1]-027.AJ001.030 IRDIG544.12 w / TraP12 91,7 99,3 150 114270[1]-027.AJ001.023 IRDIG544.12 w / TraP12 93,3 95 170 114270[1]-027.AJ001.028 IRDIG544.12 w / TraP12 96,7 85 160 114270[1]-027.AJ001.027 IRDIG544.12 w / TraP12 98,3 86,7 150 114257[1]-016.AJ001.030 IRDIG544.12 100 86,7 100 114257[1]-016.AJ001.024 IRDIG544.12 68,3 97 140 114257[1]-016.AJ001.021 IRDIG544.12 100 99.3 130 114257[1]-016.AJ001.027 IRDIG544.12 100 71.7 130 114257[1]-016.AJ001.022 IRDIG544.12 100 81.7 120 114267[1]-021.AJ001.039 IRDIG544.12 w / TraP12 91,7 61,7 230 114267[1]-021.AJ001.034 IRDIG544.12 w / TraP12 95 80 180 114267[1]-021.AJ001.044 IRDIG544.12 w / TraP12 83,3 75 210 114268[1]-023.AJ001.036 IRDIG544.12 w / TraP12 81 99 320 114268[1]-023.AJ001.041 IRDIG544.12 w / TraP12 100 83,3 410 114268[1]-023.AJ001.034 IRDIG544.12 w / TraP12 93.3 55 520 114268[1]-023.AJ001.039 IRDIG544.12 w / TraP12 91.7 80 620 114268[1]-023.AJ001.026 IRDIG544.12 w / TraP12 98.3 96 440 114268[1]-026.AJ001.053 IRDIG544.12 w / TraP12 90 73.3 390 114268[1]-026.AJ001.046 IRDIG544.12 w / TraP12 90 84.3 500 114268[1]-026.AJ001.037 IRDIG544.12 w / TraP12 99.3 71.7 320 114268[1]-026.AJ001.038 IRDIG544.12 w / TraP12 91.7 65 320 114268[1]-026.AJ001.052 IRDIG544.12 w / TraP12 97.7 66.7 360 114271[1]-011.001AJ.031 IRDIG544.12 w / TraP12 100 95 4. Petição 870240065834, de 02 / 08 / 2024, pág. 89 / 210 77 / 96 Plant Name Description Average ECB Damage Average rECB Damage Toxin ng / cm2 114271 [1]-011.001 AJ.042 IRDIG544.12w / TraP12 100 95 4 114271 [1]-011.001 AJ.043 IRDIG544.12 w / TraP12 96 91.7 4 114271 [1]-011.001 AJ.047 IRDIG544.12w / TraP12 100 97.7 3 114271 [1]-011.001 AJ.046 IRDIG544.12w / TraP12 100 98 3 114270[1]-023.001 AJ.050 IRDIG544.12w / TraP12 90 80 210 114270[1]-023.001 AJ.055 IRDIG544.12w / TraP12 99.3 73.3 260 114270[1]-023.001 AJ.044 IRDIG544.12w / TraP12 100 92 250 114270[1]-023.001 AJ.054 IRDIG544.12w / TraP12 100 88.3 210 114270[1]-023.001 AJ.058 IRDIG544.12w / TraP12 100 82.7 140 B104 control 100 100 0 B104 control 100 100 0 B104 control 100 100 0 B104 control 100 99.3 0 B104 control 100 86 0

[00145] Reduced feeding damage caused by the southwestern corn borer (SWCB) and southern corn larvae (SAW) was observed when insect larvae were exposed to plants containing truncated CrylCa modified protein, in a protein expression range of 140 to 340 ng / cm2 (Table 7). The mean expression was 210 ng / cm2 with a standard deviation of 35. Table 7 Bioassay of IRDIG544.12 Ti maize plants fed on the southwestern corn borer (SWCB) and southern corn larva (SAW) Plant Name Average Damage of SWCB Average Damage of SAW 112726[1]-015.AJ001.047 2 3.0 112726[1]-015.AJ001.030 4 3.0 112726[1]-015.AJ001.019 1 1.3 112726[1]-015.AJ001.034 2 2.0 Negative control with YFP 98.3 94.3 Negative control with YFP 93.3 62.5 Negative control with YFP 100 27.5 Negative control with YFP 92.7 67.5 Negative control with YFP 97.7 30.0 Negative control with YFP 100 45.0 B104 98.3 94.3 B104 100 91.7 B104 100 97.0 B104 65 70.0 B104 100 86.7

[00146] Field trials on corn borers were conducted at two locations: one in Indiana (IN), United States and Petition 870240065834, dated 02 / 08 / 2024, pages 90 / 210 78 / 96 another in Mississippi (MS), United States. Multiple constructs and events were tested for each treatment. Cry1Ab and Cry1F served as positive controls in the ECB tests. The null served as a negative control.

[00147] To evaluate the effectiveness with ECB, each plant received ten second-instar larvae of ECB in the whorl of plants at the V6-V7 stage. In MS, second-instar larvae of the southwestern corn borer (SWCB) were also artificially infested in the whorls of V9 corn (22 larvae per plant). The ECB and SWCB used were obtained from the Benzon laboratory. In both ECB tests, plants were evaluated 2 weeks after infestation regarding foliar damage (Guthrie scale 1 to 9) (Guthrie et al., 1960), where 1 is no visible lesion and 9 is most leaves with long lesions (Table 8). In the MS SWCB tests, plants were examined 4 to 5 days after whorl classification regarding stem damage and live insects. The data collected included the number of tunnels per stem, tunnel length, and live larvae / pupae per stem. Table 8 Scoring criteria for corn borer damage (whorl damage). Scoring Criteria 1 No visible leaf lesions or small number of shot-hole type lesions on some leaves. 2 Small number of shot-hole type lesions on some leaves. 3 Common shot-hole lesion on several leaves. 4 Several leaves with shot holes and prolonged lesions. 5 Several leaves with prolonged lesions. 6 Several leaves with prolonged lesions (approx. 1 inch). 7 Common long lesions on half of the leaves. 8 Common long lesions on about 2 / 3 of the leaves. 9 Most leaves with long lesions.

[00148] ECB field tests. ECB whorl damage was measured relative to Cry1Ca activity and showed protection. Petition 870240065834, dated 02 / 08 / 2024, pages 91 / 210 79 / 96 of whorl significantly better when compared to null. The activities of the Cry1Ca event were not statistically equivalent to the whorl protection provided by Cry1Ab and Cry1F.

[00149] The data generated in MS further reinforced the unexpectedly high level of plant protection with respect to Cry1Ca. High feeding pressure was established in this study. Significant control was measured for Cry1Ca when compared to leaf and stem damage over null. Very few live insects were found surviving on stems with Cry1Ca. Significant whorl and stem protection was measured for events with Cry1Ab and Cry1F when compared to null. Petition 870240065834, dated 02 / 08 / 2024, pages 92 / 210 80 / 96 Table 9 Leaf Whorl Data with ECB, IN (Average Across Several Events) Gene Events Average Whorl Damage Classification (ranking from 1 to 9) Average Range Cry1Ca 4 2.31 B 2.00-2.65 Cry1Ab 8 1.00 A 1.00 Cry1F 12 1.03 A 1.00-1.20 Null 1 4.65 C Means followed by different letters are significantly different (P < 0.05). Table 10 Leaf Whorl and Stem Data with ECB, MS (Average Through Various Events Toxin Gene Number of Events Average Damage Classification in the Whorl (1-9) Average Number of Tunnels per Stem Average Tunnel Length (cm) Average Number of Larvae + Pupae per Stem Cry1Ca 3 2.74 B 0.53 B 1.43 B 0.25 B Cry1Ab 8 1.66 D 0.00 C 0.00 C 0.00 C Cry1F 8 1.85 C 0.00 C 0.00 C 0.00 C Null 1 6.77 A 1.93 A 9.15 A 1.89 A For all data columns, all genetic events were significantly different from the null values ​​(P < 0.05). Within each column, means followed by different letters are significantly different (P < 0.05).

[00150] In the SWCB test, only 2 events per Bt were evaluated. High feeding pressure was established in this study. Statistically equivalent stem protection and the number of larvae and pupae per stem were measured for the Cry1Ab, Cry1F, and Cry1Ca events. Table 11 Leaf Whorl and Stem Data with SWCB, MS Toxin Gene Number of Events Average Damage Classification in the Whorl (1-9) Average Number of Tunnels per Stem Average Tunnel Length (cm) Average Number of Larvae + Pupae per Stem Cry1Ca 2 1.90 C 0.07 B 0.20 B 0.02 B Cry1Ab 2 1.91 C 0.00 B 0.00 B 0.00 B Cry1F 2 2.18 B 0.07 B 0.54 B 0.03 B Null 1 7.17 A 3.53 A 28.62 A 3.06 A Within each column, the means followed by different letters are significantly different (P < 0.05).

[00151] The active form of Cry1Ca is composed of 29 amino acids Petition 870240065834, dated 02 / 08 / 2024, pp. 93 / 210 81 / 96 628. The full length (1-1164) or the cleaved forms (1-628 and 29628) are active when presented to insects, as they are processed in the 29-628 form. EXAMPLE 5 Field tests with the corn moth larva.

[00152] Field trials on maize traction larvae were conducted in Fowler, IN with multiple constructs and events (SEQ ID NO: 31). The null served as a negative control. Each plant received five first larval stage larvae in the green silks of the maize ears. CEWs were obtained from the Benzon Laboratory. Ten maize ears per plot per event were evaluated to assess the level of grain damage in maize ears infested with CEWs. All transgenic events provided significantly lower levels of grain damage when compared to the null. There was significant suppression of larval feeding in plants with CrylCa (Table 12). Table 12 Grain Consumption Data by CEW, IN Gene Inputs Average area (cm2) of grains consumed* % reduction in grains consumed per area (compared to null) CrylCa 4 1.72 B 63.2 Cry1F 12 1.86 B 60.3 Null 1 4.68 A EXAMPLE 6 Transformation of Agrobacterium.

[00153] Standard cloning methods were used in the construction of binary plasmids for plant transformation and expression. Agrobacterium binary plasmids containing crylCa expression cassettes were engineered using Gateway® Technology (Invitrogen, Carlsbad, CA) and used in Agrobacterium-mediated plant transformation. Restriction endonucleases were obtained from New England BioLabs. Petition 870240065834, dated 02 / 08 / 2024, pages 94 / 210 82 / 96 (NEB, Ipswich, MA) and T4 DNA Ligase (Invitrogen) were used for DNA ligation. Gateway reactions were performed using Gateway® LR Clonase® enzyme mixture (Invitrogen). Plasmid preparations were performed using the NucleoSpin® plasmid preparation kit or the NucleoBond® AX Xtra Midi kit (both from Macherey-Nagel), following the manufacturers' instructions. DNA fragments were purified using the QIAquick PCR Purification Kit or the QIAEX II Gel Extraction Kit (both from Qiagen) after isolation from the gel.

[00154] DNA fragments comprising the nucleotide sequences encoding insecticidal proteins or fragments thereof were synthesized by a commercial supplier (e.g., DNA2.0, Menlo Park, CA) and provided as cloned fragments in standard plasmid vectors or were obtained by standard molecular biology manipulation of other constructs containing appropriate nucleotide sequences. Unique restriction sites internal to each gene were identified, and one fragment from each gene was synthesized, each containing a specific deletion or insertion. The modified Cry fragments were subcloned into other Cry fragments at an appropriate restriction site to obtain a region encoding the desired full-length protein, fused proteins, or deleted variant proteins.

[00155] Electrocompetent cells of the Agrobacterium tumefaciens Z707S strain (a streptomycin-resistant derivative of Z707, Hepburn et al., 1985) were prepared and transformed using electroporation (Weigel and Glazebrook, 2002). After electroporation, 1 ml of YEP broth (gm / L: yeast extract, 10, peptone, 10, NaCl, 5) was added to the cuvette and the cell-YEP suspension was transferred to a 15 ml culture tube for incubation at 28°C in a water bath with constant agitation for 4 hours. The cells Petition 870240065834, dated 02 / 08 / 2024, pages 95 / 210 Samples 83 / 96 were laminated onto YEP-supplemented agar (25 g / L) with spectinomycin (200 pg / ml) and streptomycin (250 pg / ml), and the plates were incubated for 2 to 4 days at 28°C. Well-separated individual colonies were selected and streaked onto fresh YEP + agar plates with spectinomycin and streptomycin as before, and incubated at 28°C for 1 to 3 days.

[00156] The presence of the insecticidal protein gene insertion in the binary plant transformation vector was determined by PCR analysis using vector-specific primers with template plasmid DNA prepared from selected Agrobacterium colonies. A 4 ml aliquot of a 15 ml overnight culture grown in YEP with spectinomycin and streptomycin as before was extracted using Qiagen Spin Mini Preps, performed according to the manufacturer's instructions. Plasmid DNA from the binary vector used in the electroporation transformation of Agrobacterium was included as a control. The PCR reaction was completed using Invitrogen Taq DNA polymerase according to the manufacturer's instructions at concentrations of 0.5x.PCR reactions were performed in an MJ Research Peltier Thermal Cycler programmed with the following conditions: Step 1) 94°C for 3 minutes; Step 2) 94°C for 45 seconds; Step 3) 55°C for 30 seconds; Step 4) 72°C for 1 minute per kb of expected product length; Step 5) 29 times Step 2; Step 6) 72°C for 10 minutes. The reaction was maintained at 4°C after cycling. Amplification products were analyzed by agarose gel electrophoresis (e.g., 0.7% to 1% agarose, w / v) and visualized by ethidium bromide staining. A colony was selected whose PCR product was identical to the plasmid control. Petition 870240065834, dated 02 / 08 / 2024, pages 96 / 210 84 / 96 Table 13 Description of the plasmids for the expression of DIG-465 and DIG-473 in maize. Plasmid Description pDAB 115752 ZmUbil / DIG-465 / ZmPer5::SCBV(MAM)v2 / AAD-1v3 / ZmLip pDAB 115753 ZmUbi1 / DIG-473 / ZmPer5::SCBV(MAM)v2 / AAD-1v3 / ZmLip pDAB 112725 ZmUbi1 / Cry1Ca (Zm) / ZmPer5::SCBV(MAM) / AAD-1v3 / ZmLip pDAB 112726 ZmUbil / CrylCa (HGC) / ZmPer5::SCBV(MAM) / AAD-1v3 / ZmLip EXAMPLE 7 Production of Bt DIG-465 and DIG473 insecticidal proteins and variants in monocotyledonous plants.

[00157] Agrobacterium-mediated transformation of maize. Seeds of a High II Ft cross (Armstrong et al., 1991) were planted in 5-gallon pots containing a mixture of 95% Metro-Mix 360 soilless growing medium (Sun Grober Horticulture, Bellevue, WA) and 5% clay / soil. Plants were grown in a greenhouse using a combination of high-pressure sodium and metal halide lamps with a 16:8 hour Light:Dark photoperiod. To obtain immature F2 embryos for transformation, controlled parent pollinations were performed. Immature embryos were isolated 8–10 days post-pollination when the embryos were approximately 1.0–2.0 mm in size.

[00158] Infection and co-culture. Corn cobs were surface-sterilized by scrubbing with liquid soap, immersing in 70% ethanol for 2 minutes, and then immersing in 20% commercial bleach (0.1% sodium hypochlorite) for 30 minutes before rinsing with sterile water. An Agrobacterium cell suspension containing a super-binary vector was prepared by transferring 1 to 2 groups of bacteria cultured in YEP solid medium containing 100 mg / L spectinomycin, 10 mg / L tetracycline, and 250 mg / L streptomycin. Petition 870240065834, dated 02 / 08 / 2024, pages 97 / 210 85 / 96 at 28°C for 2 to 3 days in 5 ml of liquid infection medium (LS Basal Medium (Linsmaier and Skoog, 1965), vitamin N6 (Chu et al., 1975), 1.5 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D), 68.5 g / L sucrose, 36.0 g / L glucose, 6 mM L-proline, pH 5.2) containing 100 μM acetosyringone. The solution was vortexed until a uniform suspension was achieved, and the concentration was adjusted to a final density of 200 Klett units using a Klett-Summerson colorimeter with a purple filter. Immature embryos were isolated directly into a microcentrifuge tube containing 2 ml of the infection medium.The medium was removed and replaced with 1 ml of Agrobacterium solution with a density of 200 Klett units, and the Agrobacterium solution and embryo were incubated for 5 minutes at room temperature and then transferred to co-culture medium (LS Basal Medium, N6 vitamins 1.5 mg / L of 2,4-D, 30.0 g / L of sucrose, L-proline 6 mM, 0.85 mg / L of AgNO3, 100 μM of Acetosyringone, 3.0 g / L of Gellan gum (PhytoTechnology Laboratories, Lenexa, KS), pH 5.8) for 5 days at 25°C under dark conditions.

[00159] After co-culture, the embryos were transferred to the selective medium, after which transformed isolates were obtained over approximately 8 weeks. For the selection of maize tissues transformed with a superbinary plasmid containing a selectable plant-expressible pat or bar marker gene, an LS-based medium (LS Basal medium, vitamins N6, 1.5 mg / L 2,4-D, 0.5 g / L MES (2-(N-morpholino)ethanesulfonic acid monohydrate; PhytoTechnologies Labr.), 30.0 g / L sucrose, 6 mM L-proline, 1.0 mg / L AgNO3, 250 mg / L cefotaxime, 2.5 g / L Gellan gum, pH 5.7) was used with Bialaphos (Gold BioTechnology). The embryos were transferred to selection media containing 3 mg / L of Bialaphos until embryogenic isolates were obtained. Petition 870240065834, dated 02 / 08 / 2024, pages 98 / 210 86 / 96 recovered isolates were resized by transfer to a new selection medium at 2-week intervals for regeneration and subsequent analysis.

[00160] Seed regeneration and production. For regeneration, cultures were transferred to induction medium 28 (MS salts and vitamins, 30 g / L sucrose, 5 mg / L benzylaminopurine, 0.25 mg / L 2,4-D, 3 mg / L Bialaphos, 250 mg / L cefotaxime, 2.5 g / L Gellan gum, pH 5.7) for 1 week under low light conditions (14 pEm-2s-1) then 1 week under high light conditions (approximately 89 pEm-2s-1). The tissues were subsequently transferred to regeneration medium 36 (identically to the induction medium except that it lacks plant growth regulators). When the seedlings grew to 3 to 5 cm in length, they were transferred to glass culture tubes containing SHGA medium (Schenk and Hildebrandt salts and vitamins (1972); PhytoTechnologies Labr.), 1.0 g / L myo-inositol, 10 g / L sucrose and 2.0 g / L gellan gum, pH 5.8) considering further growth and development of the shoot and roots.The plants were transplanted into the same soil mixture as described earlier in this document and grown until flowering in the greenhouse. Controlled pollinations for seed production were conducted.

[00161] The expression level of DIG-465 by construct 115752 and the expression level of DIG-473 by construct 115753 are shown in Figure 1. Both expressed similar levels of their respective proteins, at approximately 70 to 80 ng / cm2 measured in leaves using a leaf perforation to obtain the tissue sample.

[00162] The SDS-PAGE of the extract was taken from maize expressing the gene encoding the full-length CrylCa protein (MR Petition 870240065834, dated 02 / 08 / 2024, pages 99 / 210 87 / 96 1206) (pm 130 kDa). At least five protein products were detected by immunoblotting using a polyclonal antibody directed against Cry1Ca. The full-length protein (130 kDa), as encoded by the gene inserted into maize, was detected. The other bands represent proteolytic products of this protein. A protein fragment composed of amino acid sequences 1628, representing the core toxin, was determined to have a molecular weight of 70 kDa. A 68 kDa band represented a protein composed of amino acids 29-628, where the first 28 N-terminal amino acids were excluded. The first three bands were functionally active against S. frugiperda and other lepidopteran insects. A fourth band represented a cleaved protein composed of amino acids 74-628 (pm 62 kDa), and a fifth band represented the Cry1Ca protein, which was further processed with amino acids 74-596 (pm 59 kDa).The 62 kDa and 59 kDa bands were not functionally active against S. frugiperda and other lepidopteran insects, but they represent the main protein products. EXAMPLE 8. Bioassay of transgenic corn.

[00163] The bioactivity of the DIG-465 and DIG-473 proteins and variants produced in plant cells has been demonstrated by methods known to those skilled in the art (see, for example, Huang et al., 2006). Efficacy can be demonstrated by feeding various plant tissues or tissue fragments derived from a plant that produces the DIG-465 or DIG-473 protein or variants to target insects in a controlled feeding environment. Alternatively, protein extracts can be prepared from various plant tissues derived from a plant that produces the DIG-465 or DIG-473 protein or variants and incorporated into an artificial diet bioassay as previously described herein. It should be understood that the Petition 870240065834, dated 02 / 08 / 2024, pages 100 / 210 88 / 96 results from such feeding assays should be compared with similarly conducted bioassays employing appropriate control tissues from host plants that do not produce the DIG-465 or DIG-473 protein or variants, or with other control samples.

[00164] The biological activity of several events produced in maize from construct 115752 (DIG-465) was tested to prevent leaf damage caused by feeding activity of FAW or Cry1Fa-resistant FAW (rFAW). The results show that events expressing the DIG-465 protein exhibited less feeding damage than plants not expressing the protein, and that the effect was dose-dependent, with higher DIG-465 expression resulting in less feeding damage caused by FAW or rFAW, with the effect apparently greater against rFAW (Table 14 and Figure 2).

[00165] Similarly, the biological activity of several events produced in maize from construct 115753 (DIG-473) was tested to prevent leaf damage caused by feeding activity of FAW or Cry1Fa-resistant FAW (rFAW). The results show that events expressing the DIG473 protein showed less feeding damage than plants that do not express the protein, and that the effect was dose-dependent, with higher expression of DIG-473 resulting in less feeding damage caused by FAW or rFAW, with the effect apparently greater against rFAW (Table 14 and Figure 3). Table 14 Bioassay data with FAW when exposed to DIG-465, DIG-473, or controls. Plant Name DIG # Average Damage by FAW Average Damage by rFAW Accumulated Toxin (ng / cm2) 115752[1]-001.001 DIG-465 65 20 24 115752[1]-002.001 DIG-465 20 4.375 46 115752[1]-003.001 DIG-465 32.5 3.75 75 Petition 870240065834, dated 02 / 08 / 2024, pp. 101 / 210 89 / 96 Nome da Planta DIG # Dano Médio por FAW Dano Médio por rFAW Toxina acumulada (ng / cm2) 115752[1]-004.001 DIG-465 100 98.75 0.7 115752[1]-005.001 DIG-465 30 8.75 110 115752[1]-006.001 DIG-465 40 11.25 44 115752[1]-007.001 DIG-465 22.5 5 110 115752[1]-009.001 DIG-465 3.125 3.125 56 115752[1]-010.001 DIG-465 17.5 11.25 53 115752[1]-011.001 DIG-465 3.125 1.75 170 115752[1]-012.001 DIG-465 27.5 5 16 115752[1]-013.001 DIG-465 20 1 61 115752[1]-014.001 DIG-465 25 3.75 97 115752[1]-016.001 DIG-465 15 3.75 110 115752[1]-017.001 DIG-465 75 45 2 115752[1]-018.001 DIG-465 25 4.375 72 115752[1]-019.001 DIG-465 10 2.125 37 115752[1]-020.001 DIG-465 25 1 120 115752[1]-021.001 DIG-465 20 1.75 65 115752[1]-022.001 DIG-465 12.5 7.5 110 115752[1]-023.001 DIG-465 35 11.25 84 115752[1]-024.001 DIG-465 47.5 10 67 115752[1]-025.001 DIG-465 20 5 120 115752[1]-026.001 DIG-465 4,375 8 130.00 115752[1]-027.001 DIG-465 5 3,125 56 115752[1]-028.001 DIG-465 10 3,75 96 115752[1]-029.001 DIG-465 4.375 2.5 80 115752[1]-030.001 DIG-465 8.75 10.625 21 115752[1]-031.001 DIG-465 100 100 0 115753[1]-001.001 DIG-473 100 100 0 115753[1]-002.001 DIG-473 12.5 8.75 130 115753[1]-003.001 DIG-473 20 7.5 78 115753[1]-004.001 DIG-473 20 3.125 110 115753[1]-005.001 DIG-473 25 11.875 24 115753[1]-006.001 DIG-473 12.5 1.75 96 115753[1]-007.001 DIG-473 8.75 3.75 89 115753[1]-008.001 DIG-473 25 3.375 130 115753[1]-010.001 DIG-473 20 6.25 89 115753[1]-011.001 DIG-473 15 4.375 100 115753[1]-012.001 DIG-473 13.75 2.125 79 115753[1]-013.001 DIG-473 17.5 2.75 54 115753[1]-014.001 DIG-473 10 12.5 65 115753[1]-015.001 DIG-473 20 3.75 130 115753[1]-016.001 DIG-473 7.5 3.125 49 115753[1]-017.001 DIG-473 2.125 3.125 110 115753[1]-018.001 DIG-473 100 100 0.7 115753[1]-019.001 DIG-473 100 100 0 115753[1]-020.001 DIG-473 11.875 1.75 90 115753[1]-021.001 DIG-473 10 3.75 130 115753[1]-022.001 DIG-473 40 5 120 115753[1]-023.001 DIG-473 7.5 3.75 110 115753[1]-025.001 DIG-473 13.75 3.375 72 115753[1]-026.001 DIG-473 12.5 4.375 150 115753[1]-027.001 DIG-473 5.625 2.5 93 115753[1]-028.001 DIG-473 5 11.875 48 115753[1]-029.001 DIG-473 3.125 3.125 56 115753[1]-030.001 DIG-473 7.5 3.125 120.00 B104 100 100 N / A B104 100 100 N / A B104 100 100 N / A negative control with YFP 100 100 N / A negative control with YFP 100 100 N / A negative control with YFP 100 100 N / A B104 100 100 N / A. Petition 870240065834, dated 02 / 08 / 2024, pages 102 / 210 90 / 96 Plant Name DIG# Average Damage by FAW Average Damage by rFAW Accumulated Toxin (ng / cm2) B104 100 100 N / A B104 100 100 N / A Negative control with YFP 100 100 N / A Negative control with YFP 100 100 N / A Negative control with YFP 100 100 N / A EXAMPLE 9 Production of Bt insecticidal proteins and variants in dicotyledonous plants.

[00166] Arabidopsis Transformation. Arabidopsis thaliana Col-01 was transformed using the floral immersion method (Weigel and Glazebrook, 2002). The selected Agrobacterium colony was used to inoculate 1 ml to 15 ml of YEP broth cultures containing appropriate antibiotics for selection. The culture was incubated overnight at 28°C with constant shaking at 220 rpm. Each culture was used to inoculate two 500 ml cultures of YEP broth containing appropriate antibiotics for selection, and the new cultures were incubated overnight at 28°C with constant shaking. The cells were centrifuged at approximately 8700 xg for 10 minutes at room temperature, and the resulting supernatant was discarded. The cell granule was gently resuspended in 500 ml of infiltration medium containing: 1 / 2x Murashige and Skoog salts (Sigma-Aldrich) / Gamborg vitamin B5 (Gold BioTechnology, St.The substrate consisted of: a 10% (w / v) sucrose, 0.044 μM benzylaminopurine (10 μL / L from 1 mg / mL stock in DMSO), and 300 μL / L Silwet L-77. Plants approximately 1 month old were immersed in the medium for 15 seconds, taking care to ensure the submersion of the most recent inflorescence. The plants were then placed on their sides and covered (transparent or opaque) for 24 hours, washed with water, and placed upright. The plants were grown at 22°C with a light:dark photoperiod of 16:8. Approximately 4 weeks after immersion, the seeds were harvested. Petition 870240065834, dated 02 / 08 / 2024, pages 103 / 210 91 / 96

[00167] Growth and Selection of Arabidopsis. Freshly harvested T1 seed was left to dry for at least 7 days at room temperature in the presence of a desiccant. The seed was suspended in a 0.1% agar / water solution (Sigma-Aldrich) and then stratified at 4°C for 2 days. To prepare for planting, Sunshine Mix LP5 (Sun Gro Horticulture Inc., Bellevue, WA) in 10.5-inch x 21-inch germination trays (TO Plastics Inc., Clearwater, MN) was covered with fine vermiculite, sub-irrigated with Hoagland's solution (Hoagland and Arnon, 1950) until wet, then allowed to drain for 24 hours. Stratified seed was sown on the vermiculite and covered with moisture domes (KORD Products, Bramalea, Ontario, Canada) for 7 days.The seeds were germinated and the plants were grown in a Conviron (Models CMP4030 or CMP3244; Controlled Environments Limited, Winnipeg, Manitoba, Canada) under long-day conditions (16:8 light:dark photoperiod) with a light intensity of 120 to 150 μmol / m²sec at constant temperature (22°C) and humidity (40 to 50%). The plants were initially watered with Hoagland's solution and subsequently with deionized water to keep the soil moist but not wet.

[00168] The domes were removed 5 to 6 days after sowing and the plants were sprayed with a chemical selection agent to kill germinated plants from untransformed seeds. For example, if the expressible selectable marker gene of the plant provided by the binary plant transformation vector was a pat or bar gene (Wehrmann et al., 1996), transformed plants could be selected by spraying with a 1000X solution of Finale (5.78% glufosinate ammonium, Farnam Companies Inc., Phoenix, AZ). Two subsequent sprays were performed at 5 to 7 day intervals. The survivors (plants that grow Petition 870240065834, dated 02 / 08 / 2024, pages 104 / 210 92 / 96 actively) were identified 7 to 10 days after the final spraying and were transplanted into pots prepared with Sunshine Mix LP5. The transplanted plants were covered with a humidity dome for 3-4 days and placed in a Conviron incubator under the above-mentioned growing conditions.

[00169] Those versed in the technique of dicotyledonous plant transformation will understand that other methods of selection of transformed plants are available when other selectable expressible plant marker genes (e.g., herbicide tolerance genes) are used. EXAMPLE 10 Transchemical glycine max comprising the DIG protein

[00170] Ten to twenty transgenic Tode Glycine max plants harboring expression vectors for nucleic acids comprising the CrylCa protein were generated as is known in the art, including, for example, Agrobacterium-mediated transformation. Mature soybean seeds (Glycine max) were sterilized overnight with chlorine gas for sixteen hours. After chlorine gas sterilization, the seeds were placed in an open container in a LAMINAR™ flow hood to dissipate the chlorine gas. Immediately afterwards, the sterilized seeds were soaked with sterile H2O for sixteen hours in the dark using a black box at 24°C.

[00171] Preparation of split soybean seed. The split soybean seed comprising part of an embryonic axis protocol required the preparation of soybean seed material that was longitudinally cut, using a #10 blade attached to a scalpel, along the seed entry organ to separate and remove the seed coat, and split the seed into two cotyledon sections. Careful attention was given to removing Petition 870240065834, dated 02 / 08 / 2024, pp. 105 / 210 93 / 96 partially the embryonic axis, in which about 1 / 2 to 1 / 3 of the embryonic axis remained attached to the nodal end of the cotyledon.

[00172] Inoculation. Split soybean seeds comprising a partial portion of the embryonic axis were then immersed for approximately 30 minutes in a solution of Agrobacterium tumefaciens (e.g., strain EHA 101 or EHA 105) containing a binary plasmid comprising the DIG protein. The Agrobacterium tumefaciens solution was diluted to a final concentration of λ = 0.6 OD650 before immersing the cotyledons comprising the embryonic axis.

[00173] Co-culture. After inoculation, the split soybean seed was co-cultured with the Agrobacterium tumefaciens strain for 5 days in co-culture medium (Wang, Kan. Agrobacterium Protocols. 2. 1. New Jersey: Humana Press, 2006. Print.) in a Petri dish covered with a piece of filter paper.

[00174] Sprout induction. After 5 days of co-cultivation, the split soybean seeds were washed in liquid Sprout Injection (SI) medium consisting of B5 salts, vitamin B5, 28 mg / L ferrous, 38 mg / L Na2EDTA, 30 g / L sucrose, 0.6 g / L MES, 1.11 mg / L BAP, 100 mg / L TIMENTIN™, 200 mg / L cefotaxime and 50 mg / L vancomycin (pH 5.7). The split soybean seeds were then cultured in Sprout Induction Medium I (SI I) consisting of B5 salts, vitamin B5, 7 g / L Noble agar, 28 mg / L Ferrous, 38 mg / L Na2EDTA, 30 g / L sucrose, 0.6 g / L MES, 1.11 mg / L BAP, 50 mg / L TIMENTIN™, 200 mg / L cefotaxime, 50 mg / L vancomycin (pH 5.7), with the flat side of the cotyledon facing upwards and the nodal end of the cotyledon inserted into the medium. After 2 weeks of culture, the explants of the transformed split soybean seed were transferred to Sprout Induction Medium II (SI II) containing SI I medium supplemented with 6 mg / L glufosinate (LIBERTY®). Petition 870240065834, dated 02 / 08 / 2024, pages 106 / 210 94 / 96

[00175] Shoot elongation. After 2 weeks of culture in SI II medium, the cotyledons were removed from the explants and a leveled shoot block containing the embryonic axis was cut by making a cut at the base of the cotyledon. The shoot block isolated from the cotyledon was transferred to shoot elongation medium (SE). The SE medium consisted of MS salts, 28 mg / L ferrous, 38 mg / L Na2EDTA, 30 g / L sucrose and 0.6 g / L MES, 50 mg / L asparagine, 100 mg / L L-pyroglutamic acid, 0.1 mg / L IAA, 0.5 mg / L GA3, 1 mg / L zeatin riboside, 50 mg / L TIMENTIN™, 200 mg / L cefotaxime, 50 mg / L vancomycin, 6 mg / L glufosinate, 7 g / L Noble agar (pH 5.7). Cultures were transferred to fresh SE medium every 2 weeks. The cultures were grown in a CONVIRON™ culture chamber at 24°C with an 18-hour photoperiod and a light intensity of 80 to 90 μmol / m2sec.

[00176] Rooting. The elongated shoots that developed from the cotyledon shoot block were isolated by cutting the elongated shoot with 1 mg / L of IBA (indole-3-butyric acid) for 1 to 3 minutes to promote rooting. Immediately afterwards, the elongated shoots were transferred to rooting medium (MS salts, vitamin B5, 28 mg / L ferrous, 38 mg / L Na2EDTA, 20 g / L sucrose and 0.59 g / L MES, 50 mg / L asparagine, 100 mg / L L-pyroglutamic acid, 7 g / L Noble agar, pH 5.6) in phytin trays.

[00177] Cultivation. After cultivation in a CONVIRON™ grow chamber at 24°C, 18-hour photoperiod, for 1 to 2 weeks, rooted sprouts were transferred to a soil mix in a covered sundae cup and placed in a CONVIRON™ grow chamber (models CMP4030 and CMP3244, Controlled Environments Limited, Winnipeg, Manitoba, Canada) under long-day conditions (16 hours of light / 8 hours of darkness) with an intensity Petition 870240065834, dated 02 / 08 / 2024, pages 107 / 210 95 / 96 light of 120 to 150 μmol / m2sec under constant temperature (22°C) and humidity (40 to 50%) for the acclimatization of seedlings. Rooted seedlings were acclimatized in sundae cups for several weeks before being transferred to the greenhouse for further acclimatization and establishment of robust transgenic soybean plants.

[00178] The development and morphological characteristics of the transgenic lines were compared with non-transformed plants. The characteristics of the root, shoot, foliage, and reproduction of the plant were compared. There was no observable difference in root length and growth patterns of transgenic and non-transformed plants. The plant shoot characteristics such as height, number and size of leaves, flowering time, size, and floral appearance were similar. In general, there were no observable morphological differences between the transgenic lines and those without DIG protein expression when grown in vitro and in soil in the greenhouse. EXAMPLE 11 Transformation of Additional Crop Species

[00179] Cotton is transformed with Bt proteins (with or without a chloroplast transit peptide) to provide control of lepidopteran insects by means of a method known to those skilled in the art, for example, substantially the same techniques previously described in EXAMPLE 9 of U.S. Patent 7,838,733, or Example 12 of International Patent Publication PCT No. WO 2007 / 053482.

[00180] It should be understood that the examples and modalities described here are for illustrative purposes only and that various modifications or alterations in their understanding will be suggested to those skilled in the art and should be incorporated within the spirit Petition 870240065834, dated 02 / 08 / 2024, pages 108 / 210 96 / 96 and the domain of this application and the scope of the appended claims. With the teachings provided herein, a person skilled in the art will be able to easily produce and utilize the various toxins and polynucleotide sequences described herein. Petition 870240065834, dated 02 / 08 / 2024, pp. 109 / 210

Claims

1 / 1 CLAIMS 1. Modified CrylCa toxin, characterized in that it conforms to SEQ ID NO:

24.

2. Nucleic acid sequence, characterized by the fact that it conforms to the SEQ ID NO:

23.

3. A method for controlling plant insect pests, characterized in that it comprises cultivating transgenic plants expressing one or more modified Cry1Ca toxins encoded by a polynucleotide as defined in claim 2, and allowing susceptible pests to feed on said transgenic plants.

4. Method according to claim 3, characterized in that one or more modified Cry1Ca toxins comprise one or more additional modified Cry1Ca toxins.

5. A method for controlling insect pests of plants that have developed resistance to other Cry toxins, characterized in that it comprises cultivating transgenic plants that express the modified Cry1Ca toxin encoded by a polynucleotide as defined in claim 2, and allowing susceptible pests to feed on said transgenic plants.

6. Expression vector, characterized in that it comprises a heterologous promoter in operable binding to a nucleic acid sequence as defined in claim 2 that encodes any modified Cry1Ca toxins as defined in claim 1. Petition 870260028393, dated 03 / 26 / 2026, p. 10 / 12

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