Methods and compositions to reduce undesirable phenotypic characteristics and / or increase durability of insecticidal proteins in plants

Abstract

NOT VISIBLE DUE TO STATUS OF PATENT

Description

METHODS AND COMPOSITIONS TO REDUCE UNDESIRABLE PHENOTYPIC CHARACTERISTICS AND / OR INCREASE DURABILITY OF INSECTICIDAL PROTEINS IN PLANTSCROSS REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 550,748, filed on February 7, 2024, the disclosure of which is incorporated herein by reference in its entirety.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The official copy of the sequence listing is submitted electronically via Patent Center as an XML formatted sequence listing with a file named “118742-WO-SEC-l Sequence Listing. XML” and is filed concurrently with the specification. The sequence listing contained in this XML formatted document is part of the specification and is herein incorporated by reference in its entirety.FIELD

[0003] This disclosure relates to the field of plant genetics and molecular biology. Provided are novel compositions and methods for mitigating undesirable phenotypic characteristics attributable to the presence of certain transgenic proteins, including insecticidal proteins, when expressed in transgenic plants. Also provided are novel compositions and methods for increasing protein durability and expression levels in transgenic plants. Also provided are novel compositions and methods for modulating the activity of engineered proteins. Novel engineered peptides, polypeptides, and chimeric polypeptides, and methods of producing and using the same, are also contemplated.BACKGROUND

[0004] Biological control of insect pests of agricultural significance using a microbial agent, such as fungi, bacteria or another species of insect affords an environmentally friendly and commercially attractive alternative to synthetic chemical pesticides. Generally speaking, the use of biopesticides presents a lower risk of pollution and environmental hazards and biopesticides provide greater target specificity than is characteristic of traditional broad-spectrum chemicalinsecticides. In addition, biopesticides often cost less to produce and thus improve economic yield for a wide variety of crops.

[0005] Certain species of microorganisms of the genus Bacillus are known to possess pesticidal activity against a range of insect pests including Lepidoptera, Diptera, Coleoptera, Hemiptera and others. Bacillus thuringiensis (Bt) and Bacillus popilliae are among the most successful biocontrol agents discovered to date. Insect pathogenicity has also been attributed to strains of B. larvae, B. lentimorbus, B. sphaericus and B. cereus. Microbial insecticides, particularly those obtained from Bacillus strains, have played an important role in agriculture as alternatives to chemical pest control. Additionally, insecticidal proteins have been discovered from other microorganisms, fungi, and plants that show agricultural promise as transgenic insect control traits.

[0006] Crop plants have been developed with enhanced insect resistance by genetically engineering crop plants to produce pesticidal proteins from Bacillus, Fem plants, and other sources. For example, corn and cotton plants have been genetically engineered to produce pesticidal proteins isolated from strains of Bacillus thuringiensis. These genetically engineered crops are now widely used in agriculture and have provided the farmer with an environmentally friendly alternative to traditional insect-control methods.

[0007] Nevertheless, it is possible for some transgenic plants expressing insecticidal proteins to exhibit undesirable phenotypic responses at different development stages or under different conditions. For example, Milan et al state in U.S. patent application publication number 2011 / 0023194 that expression of Vip2 in cells of plants results in serious developmental pathology and phenotypic alterations to the plant itself. One approach is to engineer constructs and identify transgenic plants with a balance between efficacy and agronomy. However, this can be difficult to achieve with some insecticidal proteins where even very low-level expression has undesirable phenotypic effects.

[0008] Accordingly, there remains a need for new compositions and methods directed to mitigating undesirable phenotypic characteristics or undesirable agronomic phenotypes in certain transgenic plants, for enhancing expression of insecticidal and other transgenic polypeptides of interest in transgenic plants, and for increasing efficacy and durability of insecticidal proteins in transgenic plants.SUMMARY

[0009] In one aspect, compositions and methods for mitigating undesirable phenotypic characteristics attributable to the presence of one or more transgenic polypeptides of interest, such as for example insecticidal polypeptides of interest, in a transgenic plant are provided. Compositions include polypeptides and nucleic acid molecules encoding a multimerization domain, chimeric fusion polypeptides, expression constructs comprising the nucleic acid molecules, and host cells and plants comprising the chimeric fusion polypeptides or expression constructs. Compositions also include multimerization domain peptide sequences, including chimeric polypeptides comprising a multimerization domain peptide, one or more cleavable linkers, and one or more insecticidal polypeptides of interest. Compositions also comprise transformed bacteria, plants, plant cells, tissues and seeds.

[0010] In another aspect, provided is a chimeric polypeptide comprising an insecticidal polypeptide and a heterologous multimerization domain. In one embodiment, provided is a chimeric polypeptide comprising an insecticidal polypeptide and a trimerization domain. In another embodiment, the insecticidal polypeptide of the chimeric polypeptide exhibits an altered activity compared to an insecticidal polypeptide lacking the heterologous multimerization domain. In one non-limiting embodiment, the altered activity is selected from: reduced phytotoxicity in plants, reduced activity in a non-target organism, increased activity in a target organism, and increased expression in a host plant.

[0011] In another aspect, provided are chimeric fusion polypeptides wherein the insecticidal polypeptide and the heterologous multimerization domain are linked by a linker sequence. In one embodiment, the linker sequence is a cleavable linker. In another embodiment, the linker sequence further comprises at least one protease cleavage site. In a non-limiting embodiment, protease cleavage site is specific to a protease present in Lepidopteran gastrointestinal fluid or Coleopteran gastrointestinal fluid.

[0012] Provided are isolated or recombinant nucleic acid molecules capable of encoding a chimeric polypeptide comprising a multimerization domain, wherein the multimerization domain has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more sequence selected from SEQ ID NOs: 1, 2, or 8-16, as well as amino acid substitutions, deletions, insertions, fragments thereof, and combinations thereof. Nucleic acid sequences that areIcomplementary to a nucleic acid sequence of the embodiments or that hybridize to a sequence of the embodiments are also encompassed. In another aspect, provided are chimeric fusion polypeptides comprising a multimerization domain having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more sequence selected from SEQ ID NOs: 1, 2, or 8- 16.

[0013] In another aspect, DNA constructs are encompassed. Also provided are DNA constructs comprising nucleic acid molecules encoding a chimeric polypeptide comprising a multimerization domain and an insecticidal polypeptide, and optionally one or more linkers. In one non-limiting embodiment, provided are DNA constructs comprising nucleic acid molecules encoding a fusion polypeptide comprising a multimerization domain having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more sequence selected from SEQ ID NOs: 1, 2, or 8-16, and optionally one or more linkers having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more sequence selected from any one of SEQ ID NOs: 3-7 or 17, and one or more insecticidal polypeptides of interest. Also, provided are plants, plant cells, and microorganisms comprising the DNA constructs described herein. In another embodiment, provided are plants, plant cells, or microorganisms comprising the DNA constructs described herein, wherein the plants, plant cells, or microorganisms further comprises an additional polynucleotide sequence encoding a different insecticidal protein. In a further embodiment, the additional polynucleotide sequence encodes a chimeric polypeptide comprising an insecticidal polypeptide and a heterologous multimerization domain.

[0014] In another aspect, provided are methods for modifying activity of an insecticidal polypeptide, wherein the method comprises engineering the insecticidal polypeptide to include a heterologous multimerization domain, thereby modifying the activity of the insecticidal polypeptide. In one non-limiting embodiment, the modified activity of an insecticidal polypeptide is selected from: reduced phytotoxicity in plants, reduced activity in a non-target organism, increased activity in a target organism, and increased expression in a host plant. In another non-limiting embodiment, the heterologous multimerization domain and the insecticidal polypeptide are joined or linked by a linker sequence. In one-non-limiting embodiment, the linker sequence comprises at least one protease cleavage site. In another embodiment, the protease cleavage site is specific to a protease present in Lepidopteran gastrointestinal fluid or ColeopteranIgastrointestinal fluid. In one embodiment, the target organism is selected from: coleoptera, lepidoptera, and Hemiptera, and the non-target organism is selected from: non-pest insects of row crops, corn, soybean, and cotton.

[0015] In one aspect, provided are method for modifying an activity of a two-component insecticidal polypeptide system, the method comprising linking a heterologous multimerization domain comprising a first component insecticidal polypeptide of the two-component system to the N-terminus of the heterologous multimerization domain, the method further comprising linking a second component insecticidal polypeptide of the two-component system to the C- terminus of the heterologous multimerization domain, thereby modifying the activity of the two- component insecticidal polypeptide system. In one non-limiting embodiment, the modifying an activity of a two-component insecticidal polypeptide system is selected from: reduced phytotoxicity in plants, reduced activity in a non-target organism, increased activity in a target organism, or increased expression in a host plant. In another non-limiting embodiment, each one of the first component and second component of the two-component insecticidal polypeptide comprising the intervening heterologous multimerization domain by a linker sequence. In one embodiment, the linker sequence further comprises at least one protease cleavage site, wherein the at least one protease cleavage site is specific to a protease present in Lepidopteran gastrointestinal fluid or Coleopteran gastrointestinal fluid.

[0016] In one aspect, provided are methods of increasing efficacy of an insecticidal polypeptide by engineering a chimeric fusion polypeptide comprising a multimerization domain linked to the insecticidal polypeptide. In one embodiment, the method comprises expressing in a plant an engineered chimeric insecticidal polypeptide comprising a heterologous multimerization domain, wherein the level of expression of the engineered chimeric insecticidal polypeptide is increased compared to an insecticidal polypeptide lacking the heterologous multimerization domain, and wherein the increased level of the engineered chimeric insecticidal polypeptide results in increased efficacy against a target pest.

[0017] In another aspect, provided are methods of increasing durability of an insecticidal polypeptide, the method comprising expressing in a plant an engineered chimeric insecticidal polypeptide comprising a heterologous multimerization domain, wherein the level of expression of the engineered chimeric insecticidal polypeptide is increased compared to an insecticidal ipolypeptide lacking the heterologous multimerization domain, and wherein the increased level of the engineered chimeric insecticidal polypeptide increases durability of the insecticidal polypeptide against a target pest.

[0018] In another aspect, provided are methods of designing or engineering a trimerization domain, the method comprising changing the distribution of amino acid residues in a polypeptide sequence that promote trimerization, wherein (i) the inward facing amino acid residues are hydrophobic, allowing formation of a hydrophobic core; and (ii) the outward facing amino acid residues allow for electrostatic interactions with adjacent subunits of the trimer.

[0019] In a non-limiting embodiment, provided are compositions and methods for designing / engineering a chimeric polypeptide comprising a trimerization domain comprising a heptad repeat comprising an amino acid sequence in the order ‘ABCDEF’, wherein every ‘A’ and ‘D’ amino acid residue comprises a hydrophobic side-chain, and wherein every ‘E’ and ‘G’ amino acid residue comprises a charged side-chain. In another embodiment, the ‘A’ and ‘D’ amino acid residue comprises a hydrophobic amino acid selected from Valine or Leucine. In another embodiment, the trimerization domain of the prior embodiment comprises a hydrophobic amino acid in the 1st position, in the 8th and 20th position and wherein every ‘E’ amino acid is a positively charged amino acid and every ‘G’ amino acid is a negatively charged amino acid, wherein the pattern can also be reversed where every 'E’ position is a negatively charged amino acid and every 'G’ is a positively charged amino acid.

[0020] In another aspect the compositions and methods of the embodiments are useful for the production of organisms with enhanced pest resistance or tolerance. These organisms and compositions comprising the organisms are desirable for agricultural purposes.BRIEF DESCRIPTION OF THE FIGURES

[0021] FIG. 1 illustrates the structure of a trimer complex, wherein each monomeric subunit comprises a trimerization (or multimerization) domain fused to Toxin A by a linker and optionally a proteolytic cleavage site that can act as an activation switch. The chimeric fusion protein forms a trimer mediated by the helical trimerization domain. The trimeric structure prevents the toxin from forming an active complex, and the trimeric structure renders the toxin inactive. Cleavage of the proteolytic cleavage site releases the toxin from the trimerization domain to form an active complex. In some embodiments, the proteolytic cleavage site may be endogenously present in Toxin A.

[0022] FIG. 2 illustrates the structure of a trimer complex, wherein each monomeric subunit comprises a bipolar fusion protein comprising two different toxins located at the opposite termini of the intervening trimerization (or a multimerization) domain. ToxinB is located at the N- terminal end and ToxinA is located at the C-terminal end of the fusion protein. Two different cleavable linkers may be present, represented by solid and dotted lines.

[0023] FIG. 3 illustrates the structure of a trimer complex, wherein each monomeric subunit comprises a chimeric fusion protein linked by linkers that prevent adverse steric interactions between the toxin molecules in a trimer complex. The linkers may further comprise a proteolytic cleavage sequence illustrated by a solid line.

[0024] FIG. 4(A). illustrates the structure of a dimer complex, wherein each monomeric subunit comprises a dimerization domain fused to a toxin by a linker and an optional proteolytic cleavage site that can act as an activation switch. (B) Illustrates the structure of a tetrameric complex, wherein each monomeric subunit comprises a tetramerization domain fused to a toxin by a linker and an optional proteolytic cleavage site that can act as an activation switch.

[0025] FIG. 5 illustrates the structure of a dimer complex, wherein each monomeric subunit comprises a bipolar fusion protein containing a dimerization domain. At one end of the fusion protein there is a monomeric toxin (Toxin A). At the opposite end of the protein there is a dimeric protein (ToxinB), where each Toxin A monomer is fused to a multimerization domain that comes together to form a dimer in solution. Each end of the multimerization domain has a cleavable linker. Having the ability to use a multimerization domain that forms a dimer in solution may alleviate possible aggregation that can happen if a trimer-forming multimerization domain isIfused to a dimer-forming toxin. The stoichiometry of the dimeric toxin is preserved with a dimerforming domain. It may also allow delivery of two different toxins within one reading frame. The efficacy of each of the toxins may also be amplified by increasing the local concentration of both toxins in proximity to the site-of-action or cell membrane. Having the toxins close together may also decrease the LC50 or IC50 value of the insecticidal protein compared to an unfused WT insecticidal protein. IC50 measures the growth inhibition concentration affecting 50% of the test larvae. LC50 measures the lethal concentration affecting 50% of the larvae (LC50).

[0026] FIG.6 illustrates the structure of a heterotrimer wherein one multimerization domain that forms a trimer in solution has an amino acid with a large side chain (tryptophan, for example) and the other two helices have smaller amino acid residues at the corresponding position (alanine, for example) to accommodate the large amino acid from the first multimerization domain to form a trimer. This may serve as a lock and key mechanism to allow two different toxins to come together at the same terminal end of the trimer in a 2: 1 ratio. Further contemplated is a heterotrimer or a hetero-multimer comprising at least 3 different toxins.DETAILED DESCRIPTION

[0027] It is to be understood that this disclosure is not limited to the particular methodology, protocols, cell lines, genera, and reagents described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure.

[0028] As used herein the singular forms "a", "and", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the protein" includes reference to one or more proteins and equivalents thereof, and so forth. All technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs unless clearly indicated otherwise.

[0029] As used herein the terms “undesirable phenotypic characteristic(s)”, “undesirable phenotype(s)”, in a plant, or “undesirable plant phenotype(s)” include, but are not limited to, plant phytotoxicity, low protein production in planta, poor plant growth, poor developmental pathology, and undesirable phenotypic alterations.

[0030] As used herein, the terms “fusion polypeptide”, “fusion polynucleotide”, “chimeric polypeptide” or “chimeric fusion polypeptide” include, but are not limited to, engineered polypeptide(s) or polynucleotide(s) encoding the engineered polypeptide(s), wherein the engineered polypeptide(s) comprises at least one multimerization domain and at least one polypeptide of interest. In specific embodiments, the polypeptide of interest may include a polypeptide having insecticidal activity. In some embodiments, the at least one multimerization domain and at least one polypeptide of interest may be linked by an intervening linker sequence. In yet other embodiments, the linker sequence may further comprise a proteolytic cleavage (cut) site.

[0031] As used herein, the terms “fusion”, “fusing”, “joining”, or “linking” of two polypeptides include, but are not limited to, engineering two polynucleotides to encode two polypeptide sequences to be expressed in a translation fusion, where the two polypeptide sequences are either translationally fused directly, or are joined or linked by an intervening linker peptide sequence. In one embodiment, the linker peptide sequence may be a linker peptide and / or a multimerization domain peptide. In another embodiment, the linker peptide sequence may comprise at least one proteolytic cleavage site. In yet another embodiment, three or more polypeptide sequences may be expressed in a translation fusion.

[0032] As used herein, the term “multimerization domain” is used herein to refer to a multimerization domain derived from an organism and may have been modified for use as a component of a chimeric fusion protein contemplated herein. As used herein, the term “engineered multimerization domain” is used herein to refer to a multimerization domain derived from an organism and engineered or modified by substituting, adding, or deleting, one or more amino acid residues to modify its multimerization activity for use as a component of a chimeric fusion protein contemplated herein. The term “heterologous multimerization domain” is used herein to refer to a multimerization domain that is heterologous to the polypeptide of interest in the chimeric fusion protein.

[0033] Some insecticidal proteins showing high efficacy in certain insects may induce undesirable phenotypic effects when expressed in plants under certain conditions. The mechanism of action underpinning the undesirable phenotypic characteristics elicited when insecticidal proteins are recombinantly expressed in planta is unknown and is likely very diverse.Possible mechanisms may include negative epitopes that drive undesirable phenotypic plant responses, such as by blocking an essential plant factor, pore formation, and protein aggregation, and may be unique to each insecticidal protein. In one embodiment, reducing undesirable phenotypic characteristics, including, but not limited to, phytotoxicity, in plants will allow high- dose delivery of certain insecticidal proteins without significantly sacrificing yield and / or durability. Many strategies have been successfully used to inhibit pore-forming toxins including small molecules, synthetic nanoparticles, antibodies, antibody mimetics, and polyvalent inhibitors that have been reviewed in the literature (Omersa, et al. (2019) Toxins (Basel) 11(9):545). These strategies can be difficult to employ with insecticidal traits as they must preserve insecticidal activity while mitigating undesirable phenotypic characteristics and be capable of being recombinantly expressed in planta.

[0034] Creating a universal platform for mitigating undesirable phenotypic characteristics attributable to the presence of certain polypeptides of interest (e.g., insecticidal proteins) is desirable, for example by using a generic multimerization strategy by fusing to a multimerization domain that is independent of the undesirable phenotypic characteristics mechanism. Described herein are compositions and methods for reducing undesirable phenotypic characteristics, compositions and methods for increasing expression, and compositions and methods for increasing durability of insecticidal proteins, by fusing the insecticidal protein to at least one multimerization domain.

[0035] The present disclosure is drawn to compositions and methods for mitigating undesirable phenotypic characteristics attributable to the presence of one or more transgenic polypeptides of interest, such as for example insecticidal polypeptides of interest, in a transgenic plant. The methods involve transforming organisms with nucleic acid sequences encoding one or more multimerization domains fused to one or more polypeptides of interest (e.g., insecticidal polypeptides of interest), and optionally one or more linkers. In some embodiments, the linkers may further comprise a proteolytic cleavage site. In particular, the nucleic acid sequences of the embodiments are useful for preparing plants and microorganisms that possess pesticidal activity. Thus, transformed bacteria, plants, plant cells, plant tissues and seeds are provided. The compositions include nucleic acid molecules encoding engineered chimeric fusion polypeptides, expression constructs comprising the nucleic acid molecules, and host cells comprising theconstructs. Compositions also include engineered multimerization peptide sequences, including chimeric fusion polypeptides comprising one or more multimerization domains, one or more linkers, and one or more polypeptides of interest (e.g., insecticidal polypeptides of interest). Compositions also comprise transformed bacteria, plants, plant cells, tissues and seeds. The nucleic acid sequences find use in the construction of expression vectors for subsequent transformation into organisms of interest and for the generation of multimerization domain, or chimeric fusion polypeptides described herein by utilizing aspects of certain methods known in the art, such as site directed mutagenesis, domain swapping, or DNA shuffling.

[0036] The chimeric polypeptides comprising at least one multimerization domain and at least one insecticidal polypeptide of interest find use in controlling or killing Lepidopteran, Coleopteran, Dipteran, fungal, Hemipteran and nematode pest populations and for producing compositions with pesticidal activity. Insect pests of interest include, but are not limited to, Lepidoptera species including but not limited to: Com Earworm (CEW, Helicoverpa zea), European Corn Borer (ECB, Ostrinia nubialis), Fall Armyworm (FAW, Spodoptera frugiperda), Southern Armyworm (SAW, Spodoptera eridania), Soybean looper (SBL, Pseudoplusia includens), diamond-back moth, e.g., Helicoverpa zea Boddie, and velvet bean caterpillar (VBC, Anticarsia gemmatalis Hiibner) and Coleoptera species including but not limited to Western corn rootworm (Diabrotica virgifera) - WCRW, Southern corn rootworm (Diabrotica undecimpunctata howardi) - SCRW, and Northern com rootworm (Diabrotica barberi) - NCRW.

[0037] The present disclosure is drawn to compositions and methods for controlling pests. In one embodiment, the methods involve transforming organisms with nucleic acid sequences encoding Toxin F polypeptide. In particular, the nucleic acid sequences of the embodiments are useful for preparing plants and microorganisms that possess pesticidal activity. Thus, transformed bacteria, plants, plant cells, plant tissues and seeds are provided. The compositions include pesticidal nucleic acids and proteins of plant or bacterial species. In one embodiment the nucleic acid sequences find use in the construction of expression vectors for subsequent transformation into organisms of interest, as probes for the isolation of other homologous (or partially homologous) genes, and for the generation of altered Toxin F polypeptides by utilizing aspects of certain methods known in the art, such as site directed mutagenesis, domain swapping, or DNA shuffling.I I

[0038] In one embodiment the Toxin F polypeptides find use in controlling or killing Lepidopteran, Coleopteran, Dipteran, fungal, Hemipteran and nematode pest populations and for producing compositions with pesticidal activity. Insect pests of interest include, but are not limited to, Lepidoptera species including but not limited to: Com Earworm (CEW; Helicoverpa zea), European Corn Borer (ECB; Ostrinia nubialis), Fall Armyworm (FAW; Spodoptera frugiperda), Soybean looper (SBL; Pseudoplusia includens), diamond-back moth, e.g., Helicoverpa zea Boddie; and velvet bean caterpillar e.g., Anticarsia gemmatalis Htibner and Coleoptera species including but not limited to Western com rootworm (WCRW; Diabrotica virgifera), Southern corn rootworm (SCRW; Diabrotica undecimpunctata howardi), and Northern com rootworm (NCRW; Diabrotica barberi).

[0039] The terms “insecticidal polypeptide” or “insecticidal protein” or “toxin” is used herein to refer to a toxin that has toxic activity against one or more pests. For example, pests may include members of the Lepidoptera, Coleoptera, Diptera, Hemiptera orders or the Nematoda phylum or a protein that has homology to such a protein. Insecticidal proteins of interest have been isolated from organisms including but not limited to, for example, Bacillus sp., Bacillus thurengiensis (“Bt”), Pseudomonas sp., Photorhabdus sp., Xenorhabdus sp., Clostridium bifermentans and Paenibacillus popilliae, as well as from plant species including but not limited to Selaginella, Polystichum, Adiantum, Coniogramme, Davallia, Didymochlaena, Humata, Onoclea, and Tectaria species.Methods of Utilizing Multimerization domain and Chimeric Polypeptides comprising said multimerization domain in Transgenic Plants

[0040] The multimerization domain (MMD), and chimeric polypeptides comprising them, as described herein are useful for reducing undesirable plant phenotypes of transgenic plants expressing polypeptides of interest such as insecticidal proteins of interest. The multimerization domain, and chimeric fusion polypeptides comprising them, as described herein are also useful for improving the expression and efficacy of polypeptides of interest, and also for increasing the durability of certain insecticidal proteins of interest in a plant.

[0041] In one embodiment of a non-limiting hypothesis, the mechanisms causing undesirable phenotypic characteristics, such as but not limited to phytotoxicity, in plants in response to certain fipolypeptides of interest (e g., insecticidal proteins) are complicated and may include, but are not limited to biochemical changes such as lipid peroxidation, enzyme inactivation, cell injury or death, and disruption of membrane and ion homeostasis; molecular changes such as chromosomal aberrations, DNA damage, altered cell division, and gene regulation; and physiological changes such as disturbed membrane, altered stomatai opening, reduced photosynthesis, chlorosis, necrosis, and impacts on reproductive growth.

[0042] In one embodiment, the presentation of undesirable plant phenotypes in response to the expression of one or more polypeptides of interest may be reduced by the association of one or more multimerization domains with the one or more polypeptides of interest, such as insecticidal polypeptides of interest, and may be used in conjunction with one or more linker sequences. The one or more multimerization domains may be the same multimerization domain, or different multimerization domains, and may be used in conjunction with one or more linker sequences, and may further comprise a cleavable site.

[0043] In one embodiment, a first chimeric fusion polypeptides may be expressed or coexpressed in a plant with a different chimeric polypeptide of interest, wherein the first fusion polypeptide comprises a multimerization domain and an insecticidal polypeptide and wherein the second fusion polypeptide comprises a multimerization domain and an insecticidal polypeptide.

[0044] In another embodiment, a multimerization domain may be expressed in a plant as a fusion protein to the transgenic polypeptide of interest, such as an insecticidal polypeptide of interest. In some embodiments, fusion proteins are provided comprising one or more polypeptides of interest fused to a multimerization domain peptide, represented by a formula selected from:R’-L-M1, M’-L-R1, R^M1, M’-R1, R’-L-M^L-R2, R^L-M'-L-R1, R^M^R2, R2-MJ-RJ, M2-Rx- M1

[0045] wherein R1 is a transgenic polypeptide of interest, such as an insecticidal polypeptide of interest, and R2 is a second transgenic polypeptide of interest, such as an insecticidal polypeptide of interest, and Ml is a multimerization domain peptide, and M2 is the same multimerization domain peptide as Ml or alternatively is a second multimerization domain peptide. The R1 polypeptide is fused either directly or through a linker (L) segment to the Ml polypeptide. TheI|term "directly" defines fusions in which the polypeptides are linked without a peptide linker. Thus “L” represents a chemical bound or polypeptide segment to which both R1 and Ml are fused in frame. In one exemplary embodiment, L is a linear peptide to which R1 and Ml are bound by amide bonds linking the carboxy terminus of R1 to the amino terminus of L and carboxy terminus of L to the amino terminus of Ml .

[0046] By "fused in frame" is meant that there is no translation termination or disruption between the reading frames of R1 and Ml. The linking group (L) is generally a polypeptide of between 1 and 500 amino acids in length. The linkers linking the two molecules are preferably designed to (1) allow the two molecules to fold and act independently of each other, (2) not have a propensity for developing an ordered secondary structure which could interfere with the functional domains of the two polypeptides, (3) have minimal hydrophobic or charged characteristic which could interact with the functional polypeptide domains and (4) provide steric separation of R1 and Ml. A linker usually consists of either a flexible loop or flexible residues or loops at the ends and a rigid core region in the middle for separating R1 away from Ml. Typically surface amino acids in flexible protein regions include Gly, Asn and Ser. Virtually any permutation of amino acid sequences containing Gly, Asn and Ser would be expected to satisfy the above criteria for flexible loop. Other neutral amino acids, such as Ala may also be used in flexible loop linker sequence under certain circumstances. The rigid core region in a linker is typically of helical content that can be designed by modem computational tools. Residues that are favored to form a helix include Met, Ala, Leu, Glu and Lys. Additional amino acids may also be included in the linkers due to the addition of unique restriction sites in the linker sequence to facilitate construction of the fusions. In one embodiment, the linker has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence selected from SEQ ID NOs: 3-7. In another embodiment, the linker is selected from SEQ ID NOs: 3-7.

[0047] In some embodiments the linkers or portions of the linkers comprise sequences selected from the group of formulas: (Gly3Ser)n, (Gly4Ser)n, (Gly5Ser)n, (GlynSer)n or (AlaGlySer)n where n is an integer. One example of a highly-flexible linker is the (GlySer)-rich spacer region present within the pill protein of the filamentous bacteriophages, e.g. bacteriophages Ml 3 or fd (Schaller, et al., 1975). Also included are linkers in which an endopeptidase recognition sequence is included, such as for example, Plasmin, Enterokinase, Kallikerin, Urokinase, TissuePlasminogen activator, clostripain, Chymosin, Collagenase, Russell's Viper Venom Protease, Postproline cleavage enzyme, V8 protease, Thrombin and factor Xa. In some embodiments the linker comprises the amino acids from the multi-gene expression vehicle (MGEV)(See International Patent Application Publication No. WO2007 / 137329), which is cleaved by vacuolar proteases. In other embodiments, peptide linker segments from the hinge region of heavy chain immunoglobulins IgG, IgA, IgM, IgD or IgE provide an angular relationship between the attached polypeptides. Especially useful are those hinge regions where the cysteines are replaced with serines. Linkers of the present disclosure include the endogenous recognition sequences of digestive endopeptidases obtained from crop pests, including those insects disclosed herein, and derivatives thereof. Linkers of the present disclosure may include sequences derived from murine IgG gamma 2b hinge region in which the cysteines have been changed to serines. The fusion proteins are not limited by the form, size or number of linker sequences employed and the only requirement of the linker is that functionally it does not interfere adversely with the folding and function of the individual molecules of the fusion.Methods of Evaluating the Effects of Multimerization domain

[0048] Various assays are available for evaluating the mitigation of undesirable plant phenotypes by the inclusion of one or more multimerization domain with the one or more polypeptides of interest, such as one or more insecticidal polypeptides, or the expression of a chimeric fusion polypeptide comprising a multimerization domain peptides, one or more linkers, and one or more insecticidal polypeptides of interest.

[0049] For example, the mitigation of transgenic or insecticidal active-induced undesirable plant phenotypes, such as but not limited to phytotoxicity, may be visually or spectrophotometrically evaluated by growing transgenic plants or transgenic plant cells or tissues that include a multimerization domain peptide fused to one or more transgenic or insecticidal proteins and comparing the growth parameters at certain times with appropriate control plants expressing the same transgene without said multimerization domain peptide, and also non-transgenic null controls. Likewise, decreased plant health may be visually or spectrophotometrically evaluated in a substantially similar manner. Options for screening with the ability to quantify expression, iphytotoxicity, and insect efficacy include but are not limited to maize protoplast assays, bush bean transient assays, and crop transient and stable assays.

[0050] Transgene expression may be evaluated in a similar experimental format, utilizing tools such as Western blot, ELISA, mass spectrometry, Octet, SPR, FRET and other protein quantitation methods to determine the levels of transgene expression.

[0051] Transformation efficiency may be evaluated, for example, using the Quick corn assay method(s) as disclosed in U.S. Patent Application Publication Number US2017 / 0121722, which is herein incorporated by reference in its entirety.Methods of Generating Multimerization domain and Linkers

[0052] A variety of multimerization domain and linkers are contemplated. Multimerization domain peptides may be directly selected from peptide-protein of interest fusion libraries for those enhancing expression of transgenic polypeptides of interest, reducing an undesirable phenotypic response such as a phytotoxic phenotype or for altering (i.e., enhancing) protein accumulation levels in transgenic plants or appropriate surrogate. “Multimerization domain” or “multimerization domain peptide” as used herein refers to a peptide that when expressed as an N- and / or C-terminal fusion partner linked to a transgenic polypeptide of interest, such as for example an insecticidal protein of interest, in planta has at least one of: a mitigating effect on one or more undesirable phenotypic responses of the plant, such as but not limited to phytotoxic phenotypic response to the expression of the transgenic polypeptide of interest, such as for example an insecticidal protein of interest; improves the agronomic phenotype of transgenic plants expressing polypeptides of interest such as insecticidal proteins of interest; increases the expression of one or more heterologous polypeptides of interest in a plant, such as for example an insecticidal protein of interest; increases accumulation of the heterologous polypeptide of interest, such as for example an insecticidal protein of interest in the plant; increases the durability of one or more heterologous polypeptides of interest in a plant, such as for example an insecticidal protein of interest; and / or, alters the accumulation levels of the heterologous polypeptide of interest, such as for example an insecticidal protein of interest, in the plant. The protein of interest can be, for example, a transgenically-expressed protein in a plant cell such asan insecticidal protein. In specific embodiments, a multimerization domain may be a dimerization domain, trimerization domain, or a tetramerization domain.

[0053] Directly screening for reduced undesirable phenotypic characteristics and / or improved expression may occur in plants, plant cells, plant tissues, yeast or other surrogate assays such as those disclosed herein. Peptides of different sequence, position, length, and sequence of linker may be selected or designed using structural modeling information for testing in appropriate assays. Peptides that increase or decrease the stability of specific protein conformations may be isolated. Many insecticidal proteins are known to undergo significant conformational changes upon activation in the target pest digestive tract. Limiting these conformations to those commensurate with a healthy plant phenotype could be desirable.Engineered Multimerization domain, Linker Peptides, and Variants Thereof

[0054] In one embodiment, certain engineered multimerization domain polypeptides are encompassed by the disclosure. In some embodiments, an engineered multimerization domain polypeptide comprises, consists essentially of, or alternatively consists of, an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more sequence selected from SEQ ID NOs: 1, 2, or 8-16, as well as variants thereof and engineered multimerization domain polypeptides having sufficient homology to any one or more of SEQ ID NOs: 1, 2, or 8-16. In one embodiment, certain multimerization domains are encompassed by the disclosure. In some embodiments, a multimerization domain peptide comprises, or alternatively consists of, an amino acid sequence of any one or more of SEQ ID NOs: 1, 2, or 8-16, as well as variants thereof and multimerization domain peptides having sufficient homology to any one or more of SEQ ID NOs: 1, 2, or 8-16. In another embodiment, certain cleavable linker peptides are encompassed by the disclosure. In some embodiments, a linker peptide comprises, or alternatively consists of, an amino acid sequence of any one or more of SEQ ID NOs: 3-7, or 17, as well as variants thereof and linker peptides having sufficient homology to any one or more of SEQ ID NOs: 3-7, or 17.

[0055] “Sufficiently identical” or “sufficiently homologous” are used herein to refer to an amino acid sequence that has at least about 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology compared to a reference sequence using one of the alignment programs described herein using standard parameters. In some embodiments the sequence homology is against the full-length sequence of an engineered multimerization domain peptide, a multimerization domain peptide, or a linker peptide. In some embodiments, the engineered multimerization domain polypeptide, multimerization domain peptide, or linker peptide has at least about 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to any one of SEQ ID NOs: 1, 2, 8-16, or 3-7, 17, or 67, respectively. The term “about” when used herein in context with percent sequence identity means + / - 0.5%. One of skill in the art will recognize that these values can be appropriately adjusted to determine corresponding homology of proteins taking into account amino acid similarity and the like. In some embodiments the sequence identity is calculated using ClustalW algorithm in the ALIGNX® module of the Vector NTI® Program Suite (Invitrogen Corporation, Carlsbad, CA) with all default parameters. In some embodiments the sequence identity is across the entire length of polypeptide calculated using ClustalW algorithm in the ALIGNX® module of the Vector NTI® Program Suite (Invitrogen Corporation, Carlsbad, CA) with all default parameters.

[0056] As used herein, the terms “protein,” “peptide,” “peptide molecule,” or “polypeptide” includes any molecule that comprises five or more amino acids. It is well known in the art that protein, peptide or polypeptide molecules may undergo modification, including post- translational modifications, such as, but not limited to, disulfide bond formation, glycosylation, phosphorylation or oligomerization. Thus, as used herein, the terms “protein,” “peptide,” “peptide molecule” or “polypeptide” includes any protein that is modified by any biological or non-biological process. The terms “amino acid” and “amino acids” refer to all naturally occurring L-amino acids.

[0057] A “recombinant protein” is used herein to refer to a protein that is no longer in its natural environment, for example in vitro or in a recombinant bacterial or plant host cell.

[0058] “Substantially free of cellular material” as used herein refers to a polypeptide including preparations of protein having less than about 30%, 20%, 10% or 5% (by dry weight) of non- pesticidal protein (also referred to herein as a “contaminating protein”).

[0059] “Fragments” or “biologically active portions” include polypeptide fragments comprising amino acid sequences sufficiently identical to a multimerization domain peptide or a linker peptide and that exhibit multimerization activity or linker peptide activity as described above. “Fragments” or “biologically active portions” of multimerization domain, or linker peptides include fragments comprising amino acid sequences sufficiently identical to the amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more sequence selected from SEQ ID NOs: 1, 2, 8-16, or 3-7, or 17 , wherein the multimerization domain peptide or linker peptide has multimerization activity or linker peptide activity, respectively. Such biologically active portions can be prepared by recombinant techniques and evaluated for multimerization activity or linker peptide activity. In some embodiments, the multimerization domain peptide, or linker peptide fragment is an N-terminal and / or a C-terminal truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids from the N-terminus and / or C-terminus relative to any one of SEQ ID NOs: 1, 2, 8- 16, or 3-7, or 17, respectively, e.g., by proteolysis, by insertion of a start codon, by deletion of the codons encoding the deleted amino acids and concomitant insertion of a start codon. In some embodiments, the multimerization domain peptide or linker peptide fragment is an N-terminal truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, amino acids from the N-terminus of any one of SEQ ID NOs: 1, 2, 8-16, or 3-7, or 17, respectively. In some embodiments, the multimerization domain peptide, or linker peptide fragment is an N- terminal and / or a C-terminal truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids from the N-terminus and / or C-terminus relative to any one of SEQ ID NOs: 1, 2, 8-16, or 3-7, or 17, respectively.

[0060] In one embodiment, “Fragments” or “biologically active portions” include polypeptide fragments comprising amino acid sequences sufficiently identical to an Toxin F polypeptide and that exhibit insecticidal activity. “Fragments” or “biologically active portions” of Toxin F polypeptides include fragments comprising amino acid sequences sufficiently identical to the amino acid sequence of SEQ ID NO: 67, wherein the Toxin F polypeptide has insecticidalactivity. Such biologically active portions can be prepared by recombinant techniques and evaluated for insecticidal activity. In some embodiments, the Toxin F polypeptide fragment is an N-terminal and / or a C-terminal truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or more amino acids from the N- terminus and / or C-terminus relative to SEQ ID NO: 67, e.g., by proteolysis, by insertion of a start codon, by deletion of the codons encoding the deleted amino acids and concomitant insertion of a start codon, and / or insertion of a stop codon. In some embodiments, the Toxin F polypeptide fragment is an N-terminal truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 amino acids from the N-terminus of SEQ ID NO: 67. In some embodiments, the Toxin F polypeptide fragment is an N-terminal and / or a C-terminal truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or more amino acids from the N-terminus and / or C-terminus relative to SEQ ID NO: 67.

[0061]

[0062] “Variants” as used herein refers to proteins or polypeptides having an amino acid sequence that is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the parental amino acid sequence.

[0063] In some embodiments a multimerization domain peptide comprises an amino acid sequence having at least about 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the full length or a fragment of the amino acid sequence of any one of SEQ ID NOs: 1, 2, 8-16, wherein the multimerization domain peptide has multimerization activity.

[0064] In some embodiments a multimerization domain peptide, or linker peptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more sequence selected from any one or more of SEQ ID NOs: 1, 2, 8-16, or 3-7, or 17, respectively, having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or moreamino acid substitutions compared to the amino acid at the corresponding position of any one or more of the respective SEQ ID NOs: 1, 2, 8-16, or 3-7, or 17.

[0065] In some embodiments the sequence identity is across the entire length of the polypeptide calculated using ClustalW algorithm in the ALIGNX® module of the Vector NTI® Program Suite (Invitrogen Corporation, Carlsbad, CA) with all default parameters.

[0066] Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a multimerization domain peptide, or a linker peptide can be prepared by mutations in the DNA. This may also be accomplished by one of several forms of mutagenesis, such as for example site-specific double strand break technology, and / or in directed evolution. In some aspects, the changes encoded in the amino acid sequence will not substantially affect the function of the protein. Such variants will possess the desired multimerization or linker activity. However, it is understood that the ability of a multimerization domain peptide to confer multimerization activity or other polypeptide physical property may be improved or altered by the use of such techniques upon the compositions of this disclosure.

[0067] Conservative amino acid substitutions may be made at one or more predicted nonessential amino acid residues. A “nonessential” amino acid residue is a residue that can be altered without altering the biological activity. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include: amino acids with basic side chains (e.g., lysine, arginine, histidine); acidic side chains (e.g., aspartic acid, glutamic acid); polar, negatively charged residues and their amides (e.g., aspartic acid, asparagine, glutamic, acid, glutamine; uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine); small aliphatic, nonpolar or slightly polar residues (e.g., Alanine, serine, threonine, proline, glycine); nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); large aliphatic, nonpolar residues (e.g., methionine, leucine, isoleucine, valine, cystine); beta-branched side chains (e.g., threonine, valine, isoleucine); aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine); large aromatic side chains (e g., tyrosine, phenylalanine, tryptophan).11

[0068] Amino acid substitutions may be made in nonconserved regions that retain function. In general, such substitutions would not be made for conserved amino acid residues or for amino acid residues residing within a conserved motif, where such residues are essential for protein activity. Examples of residues that are conserved and that may be essential for protein activity include, for example, residues that are identical between all proteins contained in an alignment of similar or related polypeptides to the sequences of the embodiments (e.g., residues that are identical in an alignment of homologous proteins). Examples of residues that are conserved but that may allow conservative amino acid substitutions and still retain activity include, for example, residues that have only conservative substitutions between all proteins contained in an alignment of similar or related polypeptides to the sequences of the embodiments (e g., residues that have only conservative substitutions between all proteins contained in the alignment homologous proteins). However, one of skill in the art would understand that functional variants may have minor conserved or non-conserved alterations in the conserved residues.

[0069] In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, (1982) J Mol Biol. 157(1): 105-32). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.

[0070] It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, ibid). These are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (_3.5); aspartate (-3.5); asparagine (-3.5); lysine (_3.9) and arginine (-4.5). In making such changes, the substitution of amino acids whose hydropathicindices are within +2 is preferred, those which are within +1 are particularly preferred, and those within +0.5 are even more particularly preferred.

[0071] It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. US Patent Number 4,554,101, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein.

[0072] As detailed in US Patent Number 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0. +0.1); glutamate (+3.0. +0.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (—0.4); proline (-0.5 +0.1); alanine (—0.5); histidine (-0.5); cysteine (—1.0); methionine (—1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).

[0073] Alternatively, alterations may be made to the multimerization domain peptide or linker peptide sequence at the amino or carboxy terminus without substantially affecting activity. This can include insertions, deletions, or alterations introduced by modem molecular methods, such as PCR, including PCR amplifications that alter or extend the protein coding sequence by virtue of inclusion of amino acid encoding sequences in the oligonucleotides utilized in the PCR amplification.

[0074] Variant nucleotide and amino acid sequences of the disclosure also encompass sequences derived from mutagenic and recombinogenic procedures such as DNA shuffling. With such a procedure, one or more different multimerization domain peptide, or linker peptide coding regions can be used to create new engineered multimerization domain, or linker peptides possessing the desired properties. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. For example, using this approach, sequence motifs encoding a domain of interest may be shuffled between a pesticidal gene and other known pesticidal genes to obtain a new gene coding for a protein with an improved property of interest, such as an increased insecticidal activity. Strategies for such DNA shuffling are known in the art. See, for example, Stemmer, (1994) Proc. Natl. Acad. Sci. USA 91 : 10747-10751; Stemmer, (1994) Nature 370:389-391; Crameri, et al., (1997)Nature Biotech. 15:436-438; Moore, et al., (1997) J. Mol. Biol. 272:336-347; Zhang, et al., (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri, et al., (1998) Nature 391 :288-291; and US Patent Numbers 5,605,793 and 5,837,458.

[0075] In some embodiments the engineered multimerization domain peptide, or linker peptide has a modified physical property. As used herein, the term “physical property” refers to any parameter suitable for describing the physical-chemical characteristics of a protein. As used herein, “physical property of interest” and “property of interest” are used interchangeably to refer to physical properties of proteins that are being investigated and / or modified. Examples of physical properties include, but are not limited to, net surface charge and charge distribution on the protein surface, net hydrophobicity and hydrophobic residue distribution on the protein surface, surface charge density, surface hydrophobicity density, total count of surface ionizable groups, surface tension, protein size and its distribution in solution, melting temperature, heat capacity, and second virial coefficient. Examples of physical properties also include, multimerization domain or linker peptides having digestibility of proteolytic fragments in an insect gut. Models for digestion by simulated gastric fluids are known to one skilled in the art (Fuchs, R.L. and J.D. Astwood. Food Technology 50: 83-88, 1996; Astwood, J.D., et al Nature Biotechnology 14: 1269-1273, 1996; Fu TJ et al J. Agric Food Chem. 50: 7154-7160, 2002).

[0076] In some embodiments an engineered multimerization domain peptide, or linker peptide are provided. In one embodiment, the multimerization domain peptide or linker peptide comprises the amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more sequence selected from of any one or more of SEQ ID NOs: 1, 2, 8-16, or 3-7, or 17.

[0077] In some embodiments, chimeric multimerization domain polypeptides are provided comprising regions of at least two different multimerization domains of the disclosure.

[0078] In some embodiments, chimeric polypeptides are provided comprising at least two different insecticidal polypeptides linked to a multimerization domain. In other embodiments, chimeric fusion polypeptides are provided comprising at least one multimerization domain having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a peptide selected from SEQ ID NOs: 1, 2, or 8-16, and at least one linker peptide having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more sequence selected from SEQ ID NOs:3-7, or 17. In a further embodiment, the chimeric fusion polypeptide comprising at least one multimerization domain peptide having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more sequence selected from SEQ ID NOs: 1, 2, or 8-16 and at least one linker peptide having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more sequence selected from any one of SEQ ID NOs: 3-7, or 17 also comprises at least one polypeptide of interest, for example, at least one insecticidal polypeptide of interest.

[0079] In another embodiment fusion proteins are provided that include within its amino acid sequence an amino acid sequence comprising a multimerization domain peptide and linker peptide of the disclosure. In one embodiment, the multimerization domain peptide and linker peptide may comprise an additional fusion to an insecticidal or other polypeptide of interest. Methods for design and construction of fusion proteins (and polynucleotides encoding same) are known to those of skill in the art. Polynucleotides encoding a multimerization domain peptide and optionally a linker peptide may be fused to signal sequences which will direct the localization of the fusion peptide to particular compartments of a prokaryotic or eukaryotic cell and / or direct the secretion of the of the embodiments from a prokaryotic or eukaryotic cell.

[0080] For example, in E. coli, one may wish to direct the expression of the multimerization domain peptide to the periplasmic space through, but are not limited to, the use of a pelB signal sequence like the pelB pectate lyase signal sequence, the maltose binding protein (MBP) signal sequence, MBP, the ompA signal sequence, the signal sequence of the periplasmic E. coli heat- labile enterotoxin B-subunit and the signal sequence of alkaline phosphatase. See also the commercially available pMAL series of vectors (particularly the pMAL-p series) available from New England Biolabs (Ipswich, MA).

[0081] Plant plastid transit peptide / polypeptide fusions are known in the art. Apoplast transit peptides such as rice or barley alpha-amylase secretion signal are also known in the art. The plastid transit peptide is generally fused N-terminally to the polypeptide to be targeted (e.g., the fusion partner). In one embodiment, the fusion protein may comprise, or alternatively consist essentially of, the plastid transit peptide, the multimerization domain peptide to be targeted, optionally a linker peptide, and a polypeptide of interest. In such embodiments, the plastid transit peptide is preferably at the N-terminus of the fusion protein but may include additional amino acid residues N-terminal to the plastid transit peptide. In a specific embodiment, the plastid transitpeptide is in the N-terminal half, N-terminal third or N-terminal quarter of the fusion protein. Most or all of the plastid transit peptide is generally cleaved from the fusion protein upon insertion into the plastid. In one embodiment, the plastid transit peptide cleavage site may be homogenous or alternatively may vary by 1-10 amino acids. The plastid transit peptide can be recombinantly fused to a second protein in one of several ways. For example, a restriction endonuclease recognition site can be introduced into the nucleotide sequence of the transit peptide at a position corresponding to its C-terminal end and the same or a compatible site can be engineered into the nucleotide sequence of the protein to be targeted at its N-terminal end. Care must be taken in designing these sites to ensure that the coding sequences of the transit peptide and the second protein are kept "in frame" to allow the synthesis of the desired fusion protein. In some cases, it may be preferable to remove the initiator methionine of the second protein when the new restriction site is introduced. The introduction of restriction endonuclease recognition sites on both parent molecules and their subsequent joining through recombinant DNA techniques may result in the addition of one or more extra amino acids between the transit peptide and the second protein. This generally does not affect targeting activity as long as the transit peptide cleavage site remains accessible and the function of the second protein is not altered by the addition of these extra amino acids at its N-terminus. Alternatively, one skilled in the art can create a precise cleavage site between the transit peptide and the second protein (with or without its initiator methionine) using gene synthesis (Stemmer, et al., (1995) Gene 164:49- 53) or similar methods. In addition, the transit peptide fusion can intentionally include amino acids downstream of the cleavage site. The amino acids at the N-terminus of the mature protein can affect the ability of the transit peptide to target proteins to plastids and / or the efficiency of cleavage following protein import. This may be dependent on the protein to be targeted. See, e.g., Comai, et al., (1988) J. Biol. Chem. 263(29)45104-9.Nucleic Acid Molecules, and Variants and Fragments Thereof

[0082] Isolated or recombinant nucleic acid molecules comprising nucleic acid sequences encoding multimerization domain polypeptides, or linker peptides or biologically active portions thereof, as well as nucleic acid molecules sufficient for use as hybridization probes to identify nucleic acid molecules encoding proteins with regions of sequence homology are provided. Asused herein, the term “nucleic acid molecule” refers to DNA molecules (e.g., recombinant DNA, cDNA, genomic DNA, plastid DNA, mitochondrial DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.

[0083] An "isolated" nucleic acid molecule (or DNA) is used herein to refer to a nucleic acid sequence (or DNA) that is no longer in its natural environment, for example in an in vitro or in a heterologous recombinant bacterial or plant host cell. In some embodiments, an isolated nucleic acid molecule, or biologically active portion thereof, is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. An isolated nucleic acid is free of sequences (optimally protein encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. A “recombinant” nucleic acid molecule (or DNA) is used herein to refer to a nucleic acid sequence (or DNA) that is in a recombinant bacterial or plant host cell. In some embodiments, an “isolated” or “recombinant” nucleic acid is free of sequences (preferably protein encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For purposes of the disclosure, “isolated” or “recombinant” when used to refer to nucleic acid molecules excludes isolated chromosomes. For example, in various embodiments, the recombinant nucleic acid molecules encoding engineered multimerization domain peptides, and linker peptides can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, 0.1 kb, 0.05 kb, or 0.01 kb of nucleic acid sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.

[0084] In some embodiments an isolated nucleic acid molecule encoding an multimerization domain polypeptide, and a linker peptide has one or more change in the nucleic acid sequence compared to the native or genomic nucleic acid sequence. In some embodiments the change in the native or genomic nucleic acid sequence includes but is not limited to: changes in the nucleic acid sequence due to the degeneracy of the genetic code; changes in the nucleic acid sequence due to the amino acid substitution, insertion, deletion and / or addition compared to the native or genomic sequence; removal of one or more intron; deletion of one or more upstream or2|downstream regulatory regions; and deletion of the 5’ and / or 3’ untranslated region associated with the genomic nucleic acid sequence. In some embodiments the nucleic acid molecule encoding a multimerization domain peptide is a non-genomic sequence.

[0085] A variety of polynucleotides that encode engineered multimerization domain polypeptides and multimerization domain with or without linker peptides, or related proteins are contemplated. Such polynucleotides are useful for production of engineered multimerization domain peptides, for example as multimerization domain peptide fusion proteins, in host cells when operably linked to a suitable promoter, transcription termination and / or polyadenylation sequences. Such polynucleotides are also useful as probes for isolating homologous or substantially homologous polynucleotides that encode related proteins.Polynucleotides Encoding Multimerization domain or Linker Peptides

[0086] One source of polynucleotides that encode multimerization domain peptides, linker peptides, or related proteins is multimerization domain polynucleotides encoding the multimerization domain peptides having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more sequence selected from SEQ ID NOs: 1, 2, 8-16, or 3-7, or 17 . These polynucleotides can be used to express multimerization domain peptides as chimeric polypeptides.

[0087] Polynucleotides encoding multimerization domain polypeptides or fusion polypeptides, including chimeric polypeptides as described herein, can also be synthesized de novo from an engineered multimerization domain polypeptide or chimeric polypeptide sequence. The sequence of the polynucleotide gene can be deduced from a multimerization domain chimeric polypeptide sequence through use of the genetic code. Computer programs such as “BackTranslate” (GCG™ Package, Acclerys, Inc. San Diego, Calif.) can be used to convert a peptide sequence to the corresponding nucleotide sequence encoding the peptide. Examples of multimerization domain polypeptide sequences that can be used to obtain corresponding nucleotide encoding sequences include, but are not limited to the multimerization domain polypeptide of SEQ ID NOs: 1, 2, or 8-16. Furthermore, multimerization domain fusion polynucleotide sequences of the disclosure can be designed so that they will be expressed in plants.

[0088] In some embodiments the nucleic acid molecule encoding a multimerization domain polypeptide is a polynucleotide encoding the polypeptide sequence set forth in any one of SEQ ID NOs: 1, 2, or 8-16, respectively, and variants, fragments and complements thereof. In one embodiment, the nucleic acid molecule encoding a multimerization domain polypeptide is selected from any one of SEQ ID NOs: 89-97.

[0089] “Complement” is used herein to refer to a nucleic acid sequence that is sufficiently complementary to a given nucleic acid sequence such that it can hybridize to the given nucleic acid sequence to thereby form a stable duplex. “Polynucleotide sequence variants” is used herein to refer to a nucleic acid sequence that except for the degeneracy of the genetic code encodes the same polypeptide.

[0090] In some embodiments the nucleic acid molecule encodes a multimerization domain polypeptide or a variant comprising one or more amino acid substitutions to the amino acid sequence of any one of SEQ ID NOs: 1, 2, or 8-16, respectively. In other embodiments the nucleic acid molecule encodes a linker sequence comprising one or more amino acid substitutions to the amino acid sequence of any one of SEQ ID NOs: 3-7, or 17. In one embodiment, the nucleic acid molecule encoding a multimerization domain polypeptide is selected from any one of SEQ ID NOs: 84-88 and 98.

[0091] Also provided are nucleic acid molecules that encode transcription and / or translation products that are subsequently spliced to ultimately produce functional multimerization domain polypeptides or chimeric polypeptides. Splicing can be accomplished in vitro or in vivo, and can involve cis- or trans-splicing. The substrate for splicing can be polynucleotides (e.g., RNA transcripts) or polypeptides. An example of cis-splicing of a polynucleotide is where an intron inserted into a coding sequence is removed and the two flanking exon regions are spliced to generate a multimerization domain polypeptide or fusion polypeptide encoding sequence, for example as a chimeric polypeptide comprising a multimerization domain, linker peptide, and an insecticidal polypeptide of interest. An example of trans-splicing would be where a polynucleotide is encrypted by separating the coding sequence into two or more fragments that can be separately transcribed and then spliced to form the full-length pesticidal encoding sequence. The use of a splicing enhancer sequence, which can be introduced into a construct, can facilitate splicing either in cis or trans-splicing of polypeptides. Thus, in some embodiments thepolynucleotides do not directly encode a full-length multimerization domain polypeptide or fusion polypeptide, but rather encode a fragment or fragments of a multimerization domain polypeptide or chimeric polypeptide. These polynucleotides can be used to express a functional multimerization domain or chimeric polypeptide, such as for example a fusion with an insecticidal polypeptide of interest and a linker peptide, through a mechanism involving splicing, where splicing may occur at the level of polynucleotide (e.g., intron / exon) and / or polypeptide (e.g., intein / extein). This may be useful, for example, in controlling expression of pesticidal activity, since a multimerization domain or chimeric polypeptide comprising the multimerization domain peptide will only be expressed if all required fragments are expressed in an environment that permits splicing processes to generate functional product. In another example, introduction of one or more insertion sequences into a polynucleotide can facilitate recombination with a low homology polynucleotide; use of an intron or intein for the insertion sequence facilitates the removal of the intervening sequence, thereby restoring function of the encoded variant.

[0092] Nucleic acid molecules that are fragments of these nucleic acid sequences encoding multimerization domain polypeptide are also encompassed by the embodiments. “Nucleotide fragment” as used herein refers to a portion of the nucleic acid sequence encoding a multimerization domain peptide. A nucleotide fragment of a nucleic acid sequence may encode a biologically active portion of a multimerization domain peptide or it may be a fragment that can be used as a hybridization probe or PCR primer using methods disclosed below. Nucleic acid molecules that are fragments of a nucleic acid sequence encoding a multimerization domain peptide comprise at least about 21, 24, 27, 30, 33, 36, 39, 45, 60, 75, 90, 120, 150, 180, 210, 240, 270, or 300 contiguous nucleotides or up to the number of nucleotides present in a full-length nucleic acid sequence encoding an engineered multimerization domain peptide disclosed herein, depending upon the intended use. “Contiguous nucleotides” is used herein to refer to nucleotide residues that are immediately adjacent to one another. Fragments of the nucleic acid sequences of the embodiments will encode protein fragments that retain the biological activity of the multimerization domain peptide and, hence, retain multimerization domain activity. “Retains multimerization activity” is used herein to refer to a polypeptide having at least about 10%, at least about 30%, at least about 50%, at least about 70%, 80%, 90%, 95% or higher of the activityof any one of the full-length multimerization domain peptides set forth in SEQ ID NOs: 1, 2, or 8-16.

[0093] "Percent (%) sequence identity" with respect to a reference sequence (subject) is determined as the percentage of amino acid residues or nucleotides in a candidate sequence (query) that are identical with the respective amino acid residues or nucleotides in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any amino acid conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (e.g., percent identity of query sequence = number of identical positions between query and subject sequences / total number of positions of query sequence xlOO).

[0094] In some embodiments, a multimerization domain polynucleotide encodes a multimerization domain polypeptide comprising an amino acid sequence having at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity across the entire length of the amino acid sequence of SEQ ID NO: 1, 2, or 8-16. In some embodiments a multimerization domain polynucleotide encodes a multimerization domain peptide comprising an amino acid sequence having at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity across the entire length of the amino acid sequence of any one of SEQ ID NOs: 1, 2, or 8-16. In some embodiments a linker polynucleotide encodes a linker peptide comprising an amino acid sequence having at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity across the entire length of the amino acid sequence of any one of SEQ ID NOs: 3-7, or 17.11

[0095] In one embodiment is contemplated a polynucleotide sequence having at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity across the entire length of the polynucleotide sequence of any one of SEQ ID NOs: 84-164.

[0096] In another embodiment is contemplated a polypeptide sequence having at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity across the entire length of the polypeptide sequence of any one of SEQ ID NOs: 18-83.

[0097] The embodiments also encompass nucleic acid molecules encoding multimerization domain peptide variants. “Variants” of the multimerization domain polypeptide or multimerization domain-peptide encoding nucleic acid sequences include those sequences that encode the multimerization domain disclosed herein but that differ conservatively because of the degeneracy of the genetic code as well as those that are sufficiently identical as discussed above. Naturally occurring allelic variants can be identified with the use of well-known molecular biology techniques, such as polymerase chain reaction (PCR) and hybridization techniques as outlined below. Variant nucleic acid sequences also include synthetically derived nucleic acid sequences that have been generated, for example, by using site-directed mutagenesis but which still encode the multimerization domain peptide disclosed as discussed below.

[0098] The present disclosure provides isolated or recombinant polynucleotides that encode any of the multimerization domain or linker peptides disclosed herein. Those having ordinary skill in the art will readily appreciate that due to the degeneracy of the genetic code, a multitude of nucleotide sequences encoding multimerization domain or linker peptides of the present disclosure exist.

[0099] The skilled artisan will further appreciate that changes can be introduced by mutation of the nucleic acid sequences, such as for example by introducing one or more nucleotide substitutions, additions and / or deletions into the corresponding nucleic acid sequence disclosed herein, thereby leading to changes in the amino acid sequence of the encoded multimerization domain peptide, without altering the biological activity of the proteins. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated^2mutagenesis. Such variant nucleic acid sequences are also encompassed by the present disclosure.

[0100] Alternatively, variant nucleic acid sequences can be made by introducing mutations randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant multimerization domain peptide mutants can be screened for activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be expressed recombinantly, and the activity of the multimerization domain polypeptide can be determined using assay techniques disclosed herein or known in the art. The polynucleotides of the disclosure and fragments thereof are optionally used as substrates for a variety of recombination and recursive recombination reactions, in addition to standard cloning methods as set forth in, e.g., Ausubel, Berger and Sambrook, i.e., to produce additional multimerization domain polypeptide homologues and fragments thereof with desired properties. Libraries of variant polynucleotides so produced, cells comprising said libraries, and any recombinant polynucleotide produced by such methods are also embodiments of the disclosure. Additionally, such methods optionally comprise selecting a variant polynucleotide from such libraries based on multimerization domain polypeptide activity, as is wherein such recursive recombination is done in vitro or in vivo.

[0101] A variety of diversity generating protocols, including nucleic acid recursive recombination protocols are available and fully described in the art. The procedures can be used separately and / or in combination to produce one or more variants of a nucleic acid or set of nucleic acids, as well as variants of encoded proteins. Individually and collectively, these procedures provide robust, widely applicable ways of generating diversified nucleic acids and sets of nucleic acids (including, e.g., nucleic acid libraries) useful, e g., for the engineering or rapid evolution of nucleic acids, proteins, pathways, cells and / or organisms with new and / or improved characteristics.

[0102] Descriptions of a variety of diversity generating procedures for generating modified nucleic acid sequences, e.g., those coding for polypeptides having engineered multimerization activity, or having pesticidal activity, or fragments thereof, are found in the following publications and the references cited therein: Soong, et al., (2000) Nat Genet 25(4):436-439; Stemmer, et al., (1999) Tumor Targeting 4: 1-4; Ness, et al., (1999) Nat Biotechnol 17:893-896; Chang, et al., (1999) Nat Biotechnol 17:792-697; Minshull and Stemmer, (1999) Curr OpinJ3Chem Biol 3:284-290; Christians, et al., (1999) Nat Biotechnol 17:259-264; Crameri, et al., (1998) Nature 391 :288-291; Crameri, et al., (1997) Nat Biotechnol 15:436-438; Zhang, et al., (1997) PNAS USA 94:4504-4509; Patten, et al., (1997) Curr Opin Biotechnol 8:724-733; Crameri, et al., (1996) Nat Med 2: 100-103; Crameri, et al., (1996) Nat Biotechnol 14:315-319; Gates, et al., (1996) JMol Biol 255:373-386; Stemmer, (1996) “Sexual PCR and Assembly PCR” In: The Encyclopedia of Molecular Biology. VCH Publishers, New York. pp. 447-457; Crameri and Stemmer, (1995) BioTechniques 18: 194-195; Stemmer, et al., (1995) Gene, 164:49-53; Stemmer, (1995) Science 270: 1510; Stemmer, (1995) Bio / Technology 13:549-553; Stemmer, (1994) Nature 370:389-391 and Stemmer, (1994) PNAS USA 91 : 10747-10751.

[0103] Mutational methods of generating diversity include, for example, site-directed mutagenesis (Ling, et al., (1997) Anal Biochem 254(2):157-178; Dale, et al., (1996) Methods Mol Biol 57:369-374; Smith, (1985) Ann Rev Genet 19:423-462; Botstein and Shortle, (1985) Science 229: 1193-1201; Carter, (1986) Biochem J 237: 1-7 and Kunkel, (1987) “The efficiency of oligonucleotide directed mutagenesis” in Nucleic Acids & Molecular Biology (Eckstein and Lilley, eds., Springer Verlag, Berlin)); mutagenesis using uracil containing templates (Kunkel, (1985) PNAS USA 82:488-492; Kunkel, et al., (1987) Methods Enzymol 154:367-382 and Bass, et al., (1988) Science 242:240-245); oligonucleotide-directed mutagenesis (Zoller and Smith, (1983) Methods Enzymol 100:468-500; Zoller and Smith, (1987) Methods Enzymol 154:329- 350 (1987); Zoller and Smith, (1982) Nucleic Acids Res 10:6487-6500), phosphorothioate- modified DNA mutagenesis (Taylor, et al., (1985) Nucl Acids Res 13:8749-8764; Taylor, et al., (1985) Nucl Acids Res 13:8765-8787 (1985); Nakamaye and Eckstein, (1986) Nucl Acids Res 14:9679-9698; Sayers, et al., (1988) Nucl Acids Res 16:791-802 and Sayers, et al., (1988) Nucl Acids Res 16:803-814); mutagenesis using gapped duplex DNA (Kramer, et al., (1984) Nucl Acids Res 12:9441-9456; Kramer and Fritz, (1987) Methods Enzymol 154:350-367; Kramer, et al., (1988) Nucl Acids Res 16:7207 and Fritz, et al., (1988) Nucl Acids Res 16:6987-6999).

[0104] Additional suitable methods include point mismatch repair (Kramer, et al., (1984) Cell 38:879-887), mutagenesis using repair-deficient host strains (Carter, et al., (1985) Nucl Acids Res 13:4431-4443 and Carter, (1987) Methods in Enzymol 154:382-403), deletion mutagenesis (Eghtedarzadeh and Henikoff, (1986) Nucl Acids Res 14:5115), restriction-selection and restriction-purification (Wells, et al., (1986) Phil Trans R SocLond A 317:415-423), mutagenesisby total gene synthesis (Nambiar, et al., (1984) Science 223: 1299-1301; Sakamar and Khorana, (1988) Nucl Acids Res 14:6361-6372; Wells, et al., (1985) Gene 34:315-323 and Grundstrom, et al., (1985) Nucl Acids Res 13:3305-3316), double-strand break repair (Mandecki, (1986) PNAS USA, 83:7177-7181 and Arnold, (1993) Curr Opin Biotech 4:450-455). Additional details on many of the above methods can be found in Methods Enzymol Volume 154, which also describes useful controls for trouble-shooting problems with various mutagenesis methods.

[0105] The nucleotide sequences of the embodiments can also be used to isolate corresponding sequences from other sources that serve as sources of polynucleotides from which multimerization domain polypeptide may be generated, in light of the teachings provided herein. In this manner, methods such as PCR, hybridization, and the like can be used to identify such sequences based on their sequence homology to the sequences set forth herein. Sequences that are selected based on their sequence identity to any of the entire sequences set forth herein or to fragments thereof are encompassed by the embodiments.

[0106] In hybridization methods, all or part of the pesticidal nucleic acid sequence can be used to screen cDNA or genomic libraries. Methods for construction of such cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook and Russell, (2001), supra. The hybridization probes may be genomic DNA fragments, synthetic oligonucleotides, cDNA fragments, RNA fragments or other oligonucleotides and may be labeled with a detectable group such as 32P or any other detectable marker, such as other radioisotopes, a fluorescent compound, an enzyme or an enzyme co-factor. Degenerate primers designed on the basis of conserved nucleotides or amino acid residues in the nucleic acid sequence or encoded amino acid sequence can additionally be used. The probe typically comprises a region of nucleic acid sequence that hybridizes under stringent conditions to at least about 12, at least about 25, at least about 50, 75, 100, 125, 150, 175 or 200 consecutive nucleotides of nucleic acid sequences encoding multimerization domain polypeptide of the disclosure or a fragment or variant thereof. Methods for the preparation of probes for hybridization and stringency conditions are generally known in the art and are disclosed in Sambrook and Russell, (2001), supra, herein incorporated by reference.

[0107] For example, an entire nucleic acid sequence, encoding a multimerization domain polypeptide, disclosed herein or one or more portions thereof may be used as a probe capable ofspecifically hybridizing to corresponding nucleic acid sequences encoding a multimerization domain polypeptide or fusion partner polypeptide-like sequences and messenger RNAs. To achieve specific hybridization under a variety of conditions, such probes include sequences that are unique and are preferably at least about 10 nucleotides in length or at least about 20 nucleotides in length. Such probes may be used to amplify corresponding sequences from a chosen sample source. This technique may be used as a diagnostic assay to determine the presence of coding sequences in a sample of interest. Hybridization techniques include hybridization screening of plated DNA libraries (either plaques or colonies; see, for example, Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y ).

[0108] Hybridization of such sequences may be carried out under stringent conditions. “Stringent conditions” or “stringent hybridization conditions” is used herein to refer to conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in 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, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, preferably less than 500 nucleotides in length.AlphaFold

[0109] In another embodiment, portions of the polypeptides disclosed herein including, but not limited to, domains, structurally significant regions, minimal active core polypeptide, receptor binding domains, active sites, and protease cleavage sites may be identified using the AlphaFold computational program.

[0110] AlphaFold is a computational method that can regularly predict protein structures with atomic accuracy even in cases in which no similar structure is known. The AlphaFold network directly predicts the 3D coordinates of all heavy atoms for a given protein using the primary amino acid sequence and aligned sequences of homologues as inputs. The AlphaFold methodsare scalable to very long proteins with accurate domains and domain-packing, and the model is able to provide precise, per-residue estimates of its reliability that should enable confident use of its structure predictions. (Jumper, J., Evans, R., Pritzel, A. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583-589 (2021).Antibodies

[0111] Antibodies to an multimerization domain polypeptide of the embodiments or to variants or fragments thereof are also encompassed. The antibodies of the disclosure include polyclonal and monoclonal antibodies as well as fragments thereof which retain their ability to bind to a multimerization domain polypeptide. An antibody, monoclonal antibody or fragment thereof is said to be capable of binding a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody, monoclonal antibody or fragment thereof. The term "antibody" (Ab) or "monoclonal antibody" (Mab) is meant to include intact molecules as well as fragments or binding regions or domains thereof (such as, for example, Fab and F(ab).sub.2 fragments) which are capable of binding hapten. Such fragments are typically produced by proteolytic cleavage, such as papain or pepsin. Alternatively, hapten-binding fragments can be produced through the application of recombinant DNA technology or through synthetic chemistry.

[0112] Methods for the preparation of the antibodies of the present disclosure are generally known in the art. For example, see, Antibodies, A Laboratory Manual, Ed Harlow and David Lane (eds.) Cold Spring Harbor Laboratory, N.Y. (1988), as well as the references cited therein. Standard reference works setting forth the general principles of immunology include: Klein, J. Immunology: The Science of Cell-Noncell Discrimination, John Wiley & Sons, N.Y. (1982); Dennett, et al., Monoclonal Antibodies, Hybridoma: A New Dimension in Biological Analyses, Plenum Press, N.Y. (1980) and Campbell, "Monoclonal Antibody Technology," In Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 13, Burdon, et al., (eds.), Elsevier, Amsterdam (1984). Antibodies against or antigen-binding portions thereof can be produced by a variety of techniques, including conventional monoclonal antibody methodology, for example the standard somatic cell hybridization technique of Kohler and Milstein, (1975) Nature 256:495. Other techniques for producing monoclonal antibody can also be employed such as viral orJ 7oncogenic transformation of B lymphocytes. An animal system for preparing hybridomas is a murine system. Immunization protocols and techniques for isolation of immunized splenocytes for fusion are known in the art. Fusion partners (e g., murine myeloma cells) and fusion procedures are also known. The antibody and monoclonal antibodies of the disclosure can be prepared by utilizing multimerization domain polypeptides as antigens.

[0113] A kit for detecting the presence of a multimerization domain polypeptide or detecting the presence of a nucleotide sequence encoding a multimerization domain polypeptide in a sample is provided. In one embodiment, the kit provides antibody-based reagents for detecting the presence of a multimerization domain polypeptide in a tissue sample. In another embodiment, the kit provides labeled nucleic acid probes useful for detecting the presence of one or more polynucleotides encoding a multimerization domain polypeptide. The kit is provided along with appropriate reagents and controls for carrying out a detection method, as well as instructions for use of the kit.Nucleotide Constructs, Expression Cassettes and Vectors

[0114] The use of the term "nucleotide constructs" herein is not intended to limit the embodiments to nucleotide constructs comprising DNA. Those of ordinary skill in the art will recognize that nucleotide constructs, particularly polynucleotides and oligonucleotides composed of ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides, may also be employed in the methods disclosed herein. The nucleotide constructs, nucleic acids, and nucleotide sequences of the embodiments additionally encompass all complementary forms of such constructs, molecules, and sequences. Further, the nucleotide constructs, nucleotide molecules, and nucleotide sequences of the embodiments encompass all nucleotide constructs, molecules, and sequences which can be employed in the methods of the embodiments for transforming plants including, but not limited to, those comprised of deoxyribonucleotides, ribonucleotides, and combinations thereof. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. The nucleotide constructs, nucleic acids, and nucleotide sequences of the embodiments also encompass all forms of nucleotide constructs including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures and the like.

[0115] A further embodiment relates to a transformed organism such as an organism selected from plant and insect cells, bacteria, yeast, baculovirus, protozoa, nematodes and algae. The transformed organism comprises a DNA molecule of the embodiments, an expression cassette comprising the DNA molecule or a vector comprising the expression cassette, which may be stably incorporated into the genome of the transformed organism.

[0116] The sequences of the embodiments are provided in DNA constructs for expression in the organism of interest. The construct will include 5' and 3' regulatory sequences operably linked to a sequence of the embodiments. The term "operably linked" as used herein refers to the association of two or more nucleic acid fragments on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation. Optionally, operably linked may also mean that the nucleic acid sequences being linked are contiguous and where necessary to link two protein coding regions in the same reading frame. The construct may additionally contain at least one additional gene to be co-transformed into the organism. Alternatively, the additional gene(s) can be provided on multiple DNA constructs.

[0117] Such a DNA construct is provided with a plurality of restriction sites for insertion of the multimerization domain polypeptide, linker peptide, Toxin F, or fusion partner, gene sequence(s) of the disclosure to be under the transcriptional regulation of the regulatory regions. The DNA construct may additionally contain selectable marker genes.

[0118] The DNA construct will generally include in the 5' to 3' direction of transcription: a transcriptional and translational initiation region (i.e., a promoter), a DNA sequence of the embodiments, and a transcriptional and translational termination region (i.e., termination region) functional in the organism serving as a host. The transcriptional initiation region (i.e., the promoter) may be native, analogous, foreign, or heterologous to the host organism and / or to the sequence of the embodiments. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence. The term "foreign" as used herein indicates that the promoter is not found in the native organism into which the promoter is introduced. Where the promoter or any other nucleotide or amino acid sequence is "foreign" or "heterologous" in reference to asequence is a sequence that originates from a foreign species or, if from the same species, is substantially modified from its native form in composition and / or genomic locus by deliberate human intervention. For example, a promoter operably linked to a heterologous polynucleotide is from a species different from the species from which the polynucleotide was derived, or, if from the same / analogous species, one or both are substantially modified from their original form and / or genomic locus, or the promoter is not the native promoter for the operably linked polynucleotide. As used herein, a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence. Where the promoter is a native or natural sequence, the expression of the operably linked sequence is altered from the wild-type expression, which results in an alteration in phenotype.

[0119] In some embodiments the DNA construct comprises a polynucleotide encoding a multimerization domain polypeptide or chimeric polypeptide(s) of the embodiments, and may optionally include a polynucleotide encoding one or more genes of interest. In some embodiments the DNA construct comprises a polynucleotide encoding a chimeric fusion protein comprising a multimerization domain polypeptide, one or more linker peptides, and one or more polypeptides of interest, such as an insecticidal polypeptide of interest, of the embodiments.

[0120] In some embodiments the DNA construct may also include a transcriptional enhancer sequence. As used herein, the term an “enhancer” refers to a DNA sequence which can stimulate promoter activity, and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. Various enhancers are known in the art including for example, introns with gene expression enhancing properties in plants (, the ubiquitin intron (i.e., the maize ubiquitin intron 1 (see, for example, NCBI sequence S94464)), the omega enhancer or the omega prime enhancer (Gallie, et al., (1989) Molecular Biology of RNA ed. Cech (Liss, New York) 237-256 and Gallie, et al., (1987) Gene 60:217-25), the CaMV 35S enhancer (see, e.g., Benfey, et al., (1990) EMBO J. 9: 1685-96) and the enhancers of US Patent Number 7,803,992 may also be used. The above list of transcriptional enhancers is not meant to be limiting. Any appropriate transcriptional enhancer can be used in the embodiments.

[0121] The termination region may be native with the transcriptional initiation region, may be native with the operably linked DNA sequence of interest, may be native with the plant host ormay be derived from another source (i.e., foreign or heterologous to the promoter, the sequence of interest, the plant host or any combination thereof).

[0122] Convenient termination regions are available from the Ti-plasmid of A. turn efaci ens, such as the octopine synthase and nopaline synthase termination regions. See also, Guerineau, et al., (1991) Mol. Gen. Genet. 262: 141-144; Proudfoot, (1991) Cell 64:671-674; Sanfacon, et al., (1991) Genes Dev. 5: 141-149; Mogen, et al., (1990) Plant Cell 2:1261-1272; Munroe, et al., (1990) Gene 91 : 151-158; Ballas, et al., (1989) Nucleic Acids Res. 17:7891-7903 and Joshi, et al., (1987) Nucleic Acid Res. 15:9627-9639.

[0123] Where appropriate, a nucleic acid may be optimized for increased expression in the host organism. Thus, where the host organism is a plant, the synthetic nucleic acids can be synthesized using plant-preferred codons for improved expression. See, for example, Campbell and Gowri, (1990) Plant Physiol. 92: 1-11 for a discussion of host-preferred usage. For example, although nucleic acid sequences of the embodiments may be expressed in both monocotyledonous and dicotyledonous plant species, sequences can be modified to account for the specific preferences and GC content preferences of monocotyledons or dicotyledons as these preferences have been shown to differ (Murray et al. (1989) Nucleic Acids Res. 17:477-498). Thus, the maize-preferred for a particular amino acid may be derived from known gene sequences from maize. Maize usage for 28 genes from maize plants is listed in Table 4 of Murray, et al., supra. Methods are available in the art for synthesizing plant-preferred genes. See, for example, Murray, et al., (1989) Nucleic Acids Res. 17:477-498, and Liu H et al. Mol Bio Rep 37:677-684, 2010, herein incorporated by reference. A Zea maize usage table can be also found at kazusa.or.jp / / cgi- bin / show.cgi?species=4577, which can be accessed using the www prefix. A Glycine max usage table can be found at kazusa.or.jp / / cgi-bin / show.cgi?species=3847&aa=l&style=N, which can be accessed using the www prefix. In some embodiments the recombinant nucleic acid molecule encoding a multimerization domain polypeptide or chimeric polypeptide has maize optimized codons.

[0124] Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exonintron splice site signals, transposon-like repeats, and other well-characterized sequences that may be deleterious to gene expression. The GC content of the sequence may be adjusted to levelsaverage for a given cellular host, as calculated by reference to known genes expressed in the host cell. The term "host cell" as used herein refers to a cell which contains a vector and supports the replication and / or expression of the expression vector is intended. Host cells may be prokaryotic cells such as E. coli or eukaryotic cells such as yeast, insect, amphibian or mammalian cells or monocotyledonous or dicotyledonous plant cells. An example of a monocotyledonous host cell is a maize host cell. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures.

[0125] The expression cassettes may additionally contain 5' leader sequences. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picomavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein, et al., (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie, et al., (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf Mosaic Virus), human immunoglobulin heavy-chain binding protein (BiP) (Macejak, et al., (1991) Nature 353:90-94); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling, et al., (1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie, et al., (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256) and maize chlorotic mottle virus leader (MCMV) (Lommel, et al., (1991) Virology 81 :382-385). See also, Della-Cioppa, et al., (1987) Plant Physiol. 84:965-968. Such constructs may also contain a “signal sequence” or “leader sequence” to facilitate co-translational or post-translational transport of the peptide to certain intracellular structures such as the chloroplast (or other plastid), endoplasmic reticulum or Golgi apparatus.

[0126] “Signal sequence” as used herein refers to a sequence that is known or suspected to result in co-translational or post-translational peptide transport across the cell membrane. In eukaryotes, this typically involves secretion into the Golgi apparatus, with some resulting glycosylation. Insecticidal toxins of bacteria are often synthesized as protoxins, which are proteolytically activated in the gut of the target pest (Chang, (1987) Methods Enzymol. 153:507-516). In some embodiments, the signal sequence is located in the native sequence or may be derived from a sequence of the embodiments. “Leader sequence” as used herein refers to any sequence that when translated, results in an amino acid sequence sufficient to trigger co-translational transport of the peptide chain to a subcellular organelle. Thus, this includes leader sequences targeting transportand / or glycosylation by passage into the endoplasmic reticulum, passage to vacuoles, plastids including chloroplasts, mitochondria, and the like. Research in proteomics of the higher plant chloroplast has identified numerous nuclear-encoded thylakoid lumen proteins (Kieselbach et al. FEBS LETT 480:271-276, 2000; Peltier et al. Plant Cell 12:319-341, 2000; Bricker et al. Biochem. Biophys Acta 1503:350-356, 2001), the lumen targeting signal peptide of which can potentially be used in accordance with the present disclosure. (See Kieselbach et al., Photosynthesis Research, 78:249-264, 2003, in particular, Table 2 of this publication disclosing 85 proteins from the chloroplast lumen, which is incorporated herein by reference).

[0127] Suitable chloroplast transit peptides (CTP) are well known to one skilled in the art also include chimeric CT’s comprising but not limited to, an N-terminal domain, a central domain or a C-terminal domain from a CTP from Oryza sativa 1-decoy-D xylose-5 -Phosphate Synthase Oryza sativa-Superoxide dismutase Oryza sativa-soluble starch synthase Oryza sativa-NADP- dependent Malic acid enzyme Oryza sativa-Phospho-2-dehydro-3 -deoxyheptonate Aldolase 2 Oryza sativa-L- Ascorbate peroxidase 5 Oryza sativa-Phosphoglucan water dikinase, Zea Mays ssRUBISCO, Zea Mays-beta-glucosidase, Zea Mays-Malate dehydrogenase, Zea Mays Thioredoxin M-type (See US Patent Application Publication 2012 / 0304336).

[0128] The recombinant nucleic acid molecule encoding a multimerization domain polypeptide or chimeric polypeptide to be targeted to the chloroplast may be optimized for expression in the chloroplast to account for differences in usage between the plant nucleus and this organelle. In this manner, the nucleic acids of interest may be synthesized using chloroplast-preferred sequences.

[0129] In preparing the expression cassette, the various DNA fragments may be manipulated so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, re-substitutions, e.g., transitions and transversions, may be involved.

[0130] A number of promoters can be used in the practice of the embodiments. The promoters can be selected based on the desired outcome. The nucleic acids can be combined withconstitutive, tissue-preferred, inducible or other promoters for expression in the host organism. Suitable constitutive promoters for use in a plant host cell include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 1999 / 43838 and US Patent Number 6,072,050; the core CaMV 35S promoter (Odell, et al., (1985) Nature 313:810- 812); rice actin (McElroy, et al., (1990) Plant Cell 2: 163-171); ubiquitin (Christensen, et al., (1989) Plant Mol. Biol. 12:619-632 and Christensen, et al., (1992) Plant Mol. Biol. 18:675-689); pEMU (Last, et al., (1991) Theor. Appl. Genet. 81 :581-588); MAS (Velten, et al., (1984) EMBO J. 3:2723-2730); ALS promoter (US Patent Number 5,659,026) and the like. Other constitutive promoters include, for example, those discussed in US Patent Number 6,177,611.

[0131] Depending on the desired outcome, it may be beneficial to express the gene from an inducible promoter. Of particular interest for regulating the expression of the nucleotide sequences of the embodiments in plants are wound-inducible promoters. Such wound-inducible promoters, may respond to damage caused by insect feeding, and include potato proteinase inhibitor (pin II) gene (Ryan, (1990) Ann. Rev. Phytopath. 28:425-449; Duan, et al., (1996) Nature Biotechnology 14:494-498); wunl and wun2; winl and win2 (Stanford, et al., (1989) Mol. Gen. Genet. 215:200-208); systemin (McGurl, et al., (1992) Science 225:1570-1573); WIP1 (Rohmeier, et al., (1993) Plant Mol. Biol. 22:783-792; Eckelkamp, et al., (1993) FEBS Letters 323:73-76); MPI gene (Corderok, et al., (1994) Plant J. 6(2): 141 -150) and the like.

[0132] Additionally, pathogen-inducible promoters may be employed in the methods and nucleotide constructs of the embodiments. Such pathogen-inducible promoters include those from pathogenesis-related proteins (PR proteins), which are induced following infection by a pathogen; e.g., PR proteins, SAR proteins, beta-1, 3-glucanase, chitinase, etc. See, for example, Redolfi, et al., (1983) Neth. J. Plant Pathol. 89:245-254; Uknes, et al., (1992) Plant Cell 4: 645- 656 and Van Loon, (1985) Plant Mol. Virol. 4: 111-116. See also, WO 1999 / 43819.

[0133] Of interest are promoters that are expressed locally at or near the site of pathogen infection. See, for example, Marineau, et al., (1987) Plant Mol. Biol. 9:335-342; Matton, et al., (1989) Molecular Plant-Microbe Interactions 2:325-331; Somsisch, et al., (1986) Proc. Natl. Acad. Sci. USA 83:2427-2430; Somsisch, et al., (1988) Mol. Gen. Genet. 2:93-98 and Yang, (1996) Proc. Natl. Acad. Sci. USA 93: 14972-14977. See also, Chen, et al., (1996) Plant J. 10:955- 966; Zhang, et al., (1994) Proc. Natl. Acad. Sci. USA 91 :2507-2511; Warner, et al., (1993) PlantJ. 3: 191-201; Siebertz, et al., (1989) Plant Cell 1:961-968. Of particular interest is the inducible promoter for the maize PRms gene, whose expression is induced by the pathogen Fusarium moniliforme (see, for example, Cordero, et al., (1992) Physiol. Mol. Plant Path. 41: 189-200).

[0134] Chemical -regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. Depending upon the objective, the promoter may be a chemical-inducible promoter, where application of the chemical induces gene expression or a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical-inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR- la promoter, which is activated by salicylic acid. Other chemical-regulated promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena, et al., (1991) Proc. Natl. Acad. Sci. USA 88: 10421-10425 and McNellis, et al., (1998) Plant J. 14(2):247-257) and tetracyclineinducible and tetracycline-repressible promoters (see, for example, Gatz, et al., (1991) Mol. Gen. Genet. 227:229-237).

[0135] Tissue-preferred promoters can be utilized to target a multimerization domain polypeptide or fusion polypeptide, including chimeric polypeptides comprising a multimerization domain polypeptide, one or more linker peptides, and one or more polypeptides of interest, such as an insecticidal polypeptide of interest, expression within a particular plant tissue. Tissue-preferred promoters include those discussed in Yamamoto, et al., (1997) Plant J. 12(2)255-265; Kawamata, et al., (1997) Plant Cell Physiol. 38(7): 792-803; Hansen, et al., (1997) Mol. Gen Genet. 254(3):337-343; Russell, et al., (1997) Transgenic Res. 6(2): 157-168; Rinehart, et al., (1996) Plant Physiol. 112(3): 1331 - 1341 ; Van Camp, et al., (1996) Plant Physiol. 112(2): 525-535; Canevascini, et al., (1996) Plant Physiol. 112(2):513-524; Yamamoto, et al., (1994) Plant Cell Physiol. 35(5):773-778; Lam, (1994) Results Probl. Cell Differ. 20: 181-196; Orozco, et al., (1993) Plant Mol Biol. 23(6): 1129-1138; Matsuoka, et al., (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590 and Guevara-Garcia, et al., (1993) Plant J. 4(3):495-505. Such promoters can be modified, if necessary, for weak expression.

[0136] Leaf-pref erred promoters are known in the art. See, for example, Yamamoto, et al., (1997) Plant J. 12(2):255-265; Kwon, et al., (1994) Plant Physiol. 105:357-67; Yamamoto, et al., (1994) Plant Cell Physiol. 35(5):773-778; Gotor, et al., (1993) Plant J. 3:509-18; Orozco, et al., (1993) Plant Mol. Biol. 23(6): 1129-1138 and Matsuoka, et al., (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590.

[0137] Root-preferred or root-specific promoters are known and can be selected from the many available from the literature or isolated de novo from various compatible species. See, for example, Hire, et al., (1992) Plant Mol. Biol. 20(2):207-218 (soybean root-specific glutamine synthetase gene); Keller and Baumgartner, (1991) Plant Cell 3(10): 1051-1061 (root-specific control element in the GRP 1.8 gene of French bean); Sanger, et al., (1990) Plant Mol. Biol. 14(3):433-443 (root-specific promoter of the mannopine synthase (MAS) gene of Agrobacterium tumefaciens) and Miao, et al., (1991) Plant Cell 3(1): 11-22 (full-length cDNA clone encoding cytosolic glutamine synthetase (GS), which is expressed in roots and root nodules of soybean). See also, Bogusz, et al., (1990) Plant Cell 2(7):633-641, where two root-specific promoters isolated from hemoglobin genes from the nitrogen-fixing nonlegume Parasponia andersonii and the related non-nitrogen-fixing nonlegume Trema tomentosa are described. The promoters of these genes were linked to a b-glucuronidase reporter gene and introduced into both the nonlegume Nicotiana tabacum and the legume Lotus comiculatus, and in both instances rootspecific promoter activity was preserved. Leach and Aoyagi, (1991) describe their analysis of the promoters of the highly expressed rolC and rolD root-inducing genes of Agrobacterium rhizogenes (see, Plant Science (Limerick) 79(l):69-76). They concluded that enhancer and tissue-preferred DNA determinants are dissociated in those promoters. Teeri, et al., (1989) used gene fusion to lacZ to show that the Agrobacterium T-DNA gene encoding octopine synthase is especially active in the epidermis of the root tip and that the TR2' gene is root specific in the intact plant and stimulated by wounding in leaf tissue, an especially desirable combination of characteristics for use with an insecticidal or larvicidal gene (see, EMBO J. 8(2):343-350). The TRI' gene fused to nptll (neomycin phosphotransferase II) showed similar characteristics. Additional root-preferred promoters include the VfENOD-GRP3 gene promoter (Kuster, et al., (1995) Plant Mol. Biol. 29(4):759-772) and rolB promoter (Capana, et al., (1994) Plant Mol. Biol. 25(4):681-691. See also, US Patent Number 5,401,836; .

[0138] "Seed-preferred" promoters include both "seed-specific" promoters (those promoters active during seed development such as promoters of seed storage proteins) as well as "seedgerminating" promoters (those promoters active during seed germination). See, Thompson, et al., (1989) BioEssays 10: 108. Such seed-preferred promoters include, but are not limited to, Ciml (cytokinin-induced message); cZ19Bl (maize 19 kDa zein); and milps (myo-inositol-1- phosphate synthase) (see, US Patent Number 6,225,529). Gamma-zein and Glb-1 are endospermspecific promoters. For dicots, seed-specific promoters include, but are not limited to, Kunitz trypsin inhibitor 3 (KTi3) (Jofuku and Goldberg, (1989) Plant Cell 1 : 1079-1101), bean b- phaseolin, napin, b-conglycinin, glycinin 1, soybean lectin, cruciferin, and the like. For monocots, seed-specific promoters include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein, waxy, shrunken 1, shrunken 2, globulin 1, etc. See also, WO 2000 / 12733, where seed-preferred promoters from endl and end2 genes are disclosed. In dicots, seed specific promoters include but are not limited to seed coat promoter from Arabidopsis, pBAN; and the early seed promoters from Arabidopsis, p26, p63, and p63tr. A promoter that has “preferred” expression in a particular tissue is expressed in that tissue to a greater degree than in at least one other plant tissue. Some tissue-preferred promoters show expression almost exclusively in the particular tissue.

[0139] Where low level expression is desired, weak promoters will be used. Generally, the term "weak promoter" as used herein refers to a promoter that drives expression of a coding sequence at a low level. By low level expression at levels of between about 1 / 1000 transcripts to about 1 / 100,000 transcripts to about 1 / 500,000 transcripts is intended. Alternatively, it is recognized that the term “weak promoters” also encompasses promoters that drive expression in only a few cells and not in others to give a total low level of expression. Where a promoter drives expression at unacceptably high levels, portions of the promoter sequence can be deleted or modified to decrease expression levels.

[0140] Such weak constitutive promoters include, for example the core promoter of the Rsyn7 promoter (WO 1999 / 43838 and US Patent Number 6,072,050), the core 35S CaMV promoter, and the like. Other constitutive promoters include, for example, those disclosed in US Patent Number 6,177,611.

[0141] The above list of promoters is not meant to be limiting. Any appropriate promoter can be used in the embodiments.

[0142] Generally, the expression cassette will comprise a selectable marker gene for the selection of transformed cells. Selectable marker genes are utilized for the selection of transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones and 2,4-dichlorophenoxyacetate (2,4-D). Additional examples of suitable selectable marker genes include, but are not limited to, genes encoding resistance to chloramphenicol (Herrera Estrella, et al., (1983) EMBO J. 2:987-992); methotrexate (Herrera Estrella, et al., (1983) Nature 303:209-213 and Meijer, et al., (1991) Plant Mol. Biol. 16:807- 820); streptomycin (Jones, et al., (1987) Mol. Gen. Genet. 210:86-91); spectinomycin (Bretagne- Sagnard, et al., (1996) Transgenic Res. 5: 131-137); bleomycin (Hille, et al., (1990) Plant Mol. Biol. 7: 171-176); sulfonamide (Guerineau, et al., (1990) Plant Mol. Biol. 15: 127-136); bromoxynil (Stalker, et al., (1988) Science 242:419-423); glyphosate (Shaw, et al., (1986) Science 233:478-481 and US Patent Application Serial Numbers 10 / 004,357 and 10 / 427,692); phosphinothricin (DeBlock, et al., (1987) EMBO J. 6:2513-2518). See generally, Yarranton, (1992) Curr. Opin. Biotech. 3:506-511; Christopherson, et al., (1992) Proc. Natl. Acad. Sci. USA 89:6314-6318; Yao, et al., (1992) Cell 71 :63-72; Reznikoff, (1992) Mol. Microbiol. 6:2419- 2422; Barkley, et al., (1980) in The Operon, pp. 177-220; Hu, et al., (1987) Cell 48:555-566; Brown, et al., (1987) Cell 49:603-612; Figge, et al., (1988) Cell 52:713-722; Deuschle, et al., (1989) Proc. Natl. Acad. Sci. USA 86:5400-5404; Fuerst, et al., (1989) Proc. Natl. Acad. Sci. USA 86:2549-2553; Deuschle, et al., (1990) Science 248:480-483; Gossen, (1993) Ph.D. Thesis, University of Heidelberg; Reines, et al., (1993) Proc. Natl. Acad. Sci. USA 90: 1917-1921; Labow, et al., (1990) Mol. Cell. Biol. 10:3343-3356; Zambretti, et al., (1992) Proc. Natl. Acad. Sci. USA 89:3952-3956; Bairn, et al., (1991) Proc. Natl. Acad. Sci. USA 88:5072-5076; Wyborski, et al., (1991) Nucleic Acids Res. 19:4647-4653; Hillenand-Wissman, (1989) Topics Mol. Struc. Biol. 10: 143-162; Degenkolb, et al., (1991) Antimicrob. Agents Chemother. 35: 1591-1595; Kleinschnidt, et al., (1988) Biochemistry 27: 1094-1104; Bonin, (1993) Ph.D. Thesis, University of Heidelberg; Gossen, et al., (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Oliva, et al., (1992) Antimicrob. Agents Chemother. 36:913-919; Hlavka, et al., (1985) Handbook of Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin) and Gill, et al., (1988) Nature 334:721-724.

[0143] The above list of selectable marker genes is not meant to be limiting. Any selectable marker gene can be used in the embodiments.Plant Transformation

[0144] The methods of the embodiments involve introducing a multimerization domain polypeptide or fusion polypeptide or polynucleotide, including chimeric fusion polypeptides comprising a multimerization domain polypeptide, one or more linker peptides, and one or more polypeptides of interest, such as an insecticidal polypeptide of interest, into a plant. "Introducing" as used herein means presenting to the plant the polynucleotide or polypeptide in such a manner that the sequence gains access to the interior of a cell of the plant. The methods of the embodiments do not depend on a particular method for introducing a polynucleotide or polypeptide into a plant, only that the polynucleotide(s) or polypeptide(s) gains access to the interior of at least one cell of the plant. Methods for introducing polynucleotide(s) or polypeptide(s) into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.

[0145] "Stable transformation" is a transformation in which the polynucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by the progeny thereof. "Transient transformation" as used herein means that a polynucleotide is introduced into the plant and does not integrate into the genome of the plant. “Plant” as used herein generically includes whole plants, plant organs, plant tissues, seeds, plant cells, seeds and progeny of the same. The plant is a monocot or dicot. Plant cells include, without limitation, cells from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen and microspores. A “plant element" is intended to reference either a whole plant or a plant component, which may comprise differentiated and / or undifferentiated tissues, for example but not limited to plant tissues, parts, and cell types. In one embodiment, a plant element is one of the following: whole plant, seedling, meristematic tissue, ground tissue, vascular tissue, dermal tissue, seed, leaf, root, shoot, stem, flower, fruit, stolon,bulb, tuber, corm, keiki, shoot, bud, tumor tissue, and various forms of cells and culture (e.g., single cells, protoplasts, embryos, callus tissue). It should be noted that a protoplast is not technically "intact" plant cell (as naturally found with all components), as protoplasts lack a cell wall, plant organ" refers to plant tissue or a group of tissues that constitute a morphologically and functionally distinct part of a plant. A plant element "is synonymous to a portion" of a plant, and refers to any part of the plant, and can include distinct tissues and / or organs, and may be used interchangeably with tissue" throughout. Similarly, a plant reproductive element" is intended to generically reference any part of a plant that can initiate other plants via either sexual or asexual reproduction of that plant, for example but not limited to: seed, seedling, root, shoot, cutting, scion, graft, stolon, bulb, tuber, corm, keiki, or bud. The plant element may be in plant or in a plant organ, tissue culture, or cell culture.

[0146] Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection (Crossway, et al., (1986) Biotechniques 4:320-334), electroporation (Riggs, et al., (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606), Agrobacterium-mediated transformation (US Patent Numbers 5,563,055 and 5,981,840), direct gene transfer (Paszkowski, et al., (1984) EMBO J. 3:2717-2722) and ballistic particle acceleration (see, for example, US Patent Numbers 5,879,918; 5,886,244 and 5,932,782; Tomes, et al., (1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips, (Springer-Verlag, Berlin) and McCabe, et al., (1988) Biotechnology 6:923-926) and Led transformation (WO 00 / 28058). For potato transformation see, Tu, et al., (1998) Plant Molecular Biology 37:829-838 and Chong, et al., (2000) Transgenic Research 9:71- 78. Additional transformation procedures can be found in Weissinger, et al., (1988) Ann. Rev. Genet. 22:421-477; Sanford, et al., (1987) Particulate Science and Technology 5:27-37 (onion); Christou, et al., (1988) Plant Physiol. 87:671-674 (soybean); McCabe, et al., (1988) Bio / Technology 6:923-926 (soybean); Finer and McMullen, (1991) In Vitro Cell Dev. Biol. 27P:175-182 (soybean); Singh, et al., (1998) Theor. Appl. Genet. 96:319-324 (soybean); Datta, et al., (1990) Biotechnology 8:736-740 (rice); Klein, et al., (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein, et al., (1988) Biotechnology 6:559-563 (maize); US PatentNumbers 5,240,855; 5,322,783 and 5,324,646; Klein, et al., (1988) Plant Physiol. 91 :440-444 (maize); Fromm, et al., (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren, et al., (1984) Nature (London) 311 :763-764; US Patent Number 5,736,369 (cereals); Bytebier, et al., (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet, et al., (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman, et al., (Longman, New York), pp. 197-209 (pollen); Kaeppler, et al., (1990) Plant Cell Reports 9:415-418 and Kaeppler, et al., (1992) Theor. AppL Genet. 84:560-566 (whisker-mediated transformation); D'Halluin, et al., (1992) Plant Cell 4: 1495-1505 (electroporation); Li, et al., (1993) Plant Cell Reports 12:250-255 and Christou and Ford, (1995) Annals of Botany 75:407-413 (rice); Osjoda, et al., (1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens).

[0147] In specific embodiments, the sequences of the embodiments can be provided to a plant using a variety of transient transformation methods. Such transient transformation methods include, but are not limited to, the introduction of the fusion polynucleotide or variants and fragments thereof directly into the plant or the introduction of the transcript into the plant or the introduction of the fusion polypeptide transcript into the plant. Such methods include, for example, microinjection or particle bombardment. See, for example, Crossway, et al., (1986) Mol Gen. Genet. 202: 179-185; Nomura, et al., (1986) Plant Sci. 44:53-58; Hepler, et al., (1994) Proc. Natl. Acad. Sci. 91 :2176-2180 and Hush, et al., (1994) The Journal of Cell Science 107:775-784. Alternatively, the polynucleotide(s) can be transiently transformed into the plant using techniques known in the art. Such techniques include viral vector system and the precipitation of the polynucleotide in a manner that precludes subsequent release of the DNA. Thus, transcription from the particle-bound DNA can occur, but the frequency with which it is released to become integrated into the genome is greatly reduced. Such methods include the use of particles coated with poly ethylimine (PEI; Sigma #P3143).

[0148] Methods are known in the art for the targeted insertion of a polynucleotide at a specific location in the plant genome. In one embodiment, the insertion of the polynucleotide at a desired genomic location is achieved using a site-specific recombination system. See, for example, WO 1999 / 25821, WO 1999 / 25854, WO 1999 / 25840, WO 1999 / 25855 and WO 1999 / 25853. Briefly, the polynucleotide of the embodiments can be contained in transfer cassette flanked by two nonidentical recombination sites. The transfer cassette is introduced into a plant have stablyincorporated into its genome a target site which is flanked by two non-identical recombination sites that correspond to the sites of the transfer cassette. An appropriate recombinase is provided and the transfer cassette is integrated at the target site. The polynucleotide of interest is thereby integrated at a specific chromosomal position in the plant genome.

[0149] Plant transformation vectors may be comprised of one or more DNA vectors needed for achieving plant transformation. For example, it is a common practice in the art to utilize plant transformation vectors that are comprised of more than one contiguous DNA segment. These vectors are often referred to in the art as “binary vectors”. Binary vectors as well as vectors with helper plasmids are most often used for Agrobacterium-mediated transformation, where the size and complexity of DNA segments needed to achieve efficient transformation is quite large, and it is advantageous to separate functions onto separate DNA molecules. Binary vectors typically contain a plasmid vector that contains the cis-acting sequences required for T-DNA transfer (such as left border and right border), a selectable marker that is engineered to be capable of expression in a plant cell, and a “gene of interest” (a gene engineered to be capable of expression in a plant cell for which generation of transgenic plants is desired). Also present on this plasmid vector are sequences required for bacterial replication. The cis-acting sequences are arranged in a fashion to allow efficient transfer into plant cells and expression therein. For example, the selectable marker gene and the pesticidal gene are located between the left and right borders. Often a second plasmid vector contains the trans-acting factors that mediate T-DNA transfer from Agrobacterium to plant cells. This plasmid often contains the virulence functions (Vir genes) that allow infection of plant cells by Agrobacterium, and transfer of DNA by cleavage at border sequences and vir-mediated DNA transfer, as is understood in the art (Hellens and Mullineaux, (2000) Trends in Plant Science 5:446-451). Several types of Agrobacterium strains (e.g. LBA4404, GV3101, EHA101, EHA105, etc.) can be used for plant transformation. The second plasmid vector is not necessary for transforming the plants by other methods such as microprojection, microinjection, electroporation, polyethylene glycol, etc.

[0150] In general, plant transformation methods involve transferring heterologous DNA into target plant cells (e g., immature or mature embryos, suspension cultures, undifferentiated callus, protoplasts, etc.), followed by applying a maximum threshold level of appropriate selection (depending on the selectable marker gene) to recover the transformed plant cells from a group ofuntransformed cell mass. Following integration of heterologous foreign DNA into plant cells, one then applies a maximum threshold level of appropriate selection in the medium to kill the untransformed cells and separate and proliferate the putatively transformed cells that survive from this selection treatment by transferring regularly to a fresh medium. By continuous passage and challenge with appropriate selection, one identifies and proliferates the cells that are transformed with the plasmid vector. Molecular and biochemical methods can then be used to confirm the presence of the integrated heterologous gene of interest into the genome of the transgenic plant.

[0151] Explants are typically transferred to a fresh supply of the same medium and cultured routinely. Subsequently, the transformed cells are differentiated into shoots after placing on regeneration medium supplemented with a maximum threshold level of selecting agent. The shoots are then transferred to a selective rooting medium for recovering rooted shoot or plantlet. The transgenic plantlet then grows into a mature plant and produces fertile seeds (e.g., Hiei, et al., (1994) The Plant Journal 6:271-282; Ishida, et al., (1996) Nature Biotechnology 14:745-750). Explants are typically transferred to a fresh supply of the same medium and cultured routinely. A general description of the techniques and methods for generating transgenic plants are found in Ayres and Park, (1994) Critical Reviews in Plant Science 13:219-239 and Bommineni and Jauhar, (1997) Maydica 42: 107-120. Since the transformed material contains many cells; both transformed and non-transformed cells are present in any piece of subjected target callus or tissue or group of cells. The ability to kill non-transformed cells and allow transformed cells to proliferate results in transformed plant cultures. Often, the ability to remove non-transformed cells is a limitation to rapid recovery of transformed plant cells and successful generation of transgenic plants.

[0152] The cells that have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick, et al., (1986) Plant Cell Reports 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having constitutive or inducible expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited andthen seeds harvested to ensure that expression of the desired phenotypic characteristic has been achieved.

[0153] The nucleotide sequences of the embodiments may be provided to the plant by contacting the plant with a virus or viral nucleic acids. Generally, such methods involve incorporating the nucleotide construct of interest within a viral DNA or RNA molecule. It is recognized that the recombinant proteins of the embodiments may be initially synthesized as part of a viral polyprotein, which later may be processed by proteolysis in vivo or in vitro to produce the desired multimerization domain polypeptide or fusion chimeric polypeptide. It is also recognized that such a viral polyprotein, comprising at least a portion of the amino acid sequence of a multimerization domain polypeptide or chimeric polypeptide of the embodiments, may have the desired activity, such as pesticidal activity. Such viral polyproteins and the nucleotide sequences that encode for them are encompassed by the embodiments. Methods for providing plants with nucleotide constructs and producing the encoded proteins in the plants, which involve viral DNA or RNA molecules, are known in the art.

[0154] Methods for transformation of chloroplasts are known in the art. See, for example, Svab, et al., (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530; Svab and Maliga, (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab and Maliga, (1993) EMBO J. 12:601-606. The method relies on particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination. Additionally, plastid transformation can be accomplished by transactivation of a silent plastid-borne transgene by tissue-preferred expression of a nuclear-encoded and plastid-directed RNA polymerase. Such a system has been reported in McBride, et al., (1994) Proc. Natl. Acad. Sci. USA 91 :7301-7305.

[0155] The embodiments further relate to plant-propagating material of a transformed plant of the embodiments including, but not limited to, seeds, tubers, corms, bulbs, leaves and cuttings of roots and shoots.

[0156] The embodiments may be used for transformation of any plant species, including, but not limited to, monocots and dicots. Examples of plants of interest include, but are not limited to, com (Zea mays), Brassica sp. (e g., B. napus, B. rapa, B. juncea), particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereal e), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum">4glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Elelianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp.), sugarcane (Saccharum spp.), oats, barley, and vegetables.

[0157] Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo). Plants of the embodiments include crop plants (for example, corn, alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, etc.), such as corn and soybean plants.

[0158] Turf grasses include, but are not limited to: annual bluegrass (Poa annua); annual ryegrass (Lolium multiflorum); Canada bluegrass (Poa compressa); Chewing’s fescue (Festuca rubra); colonial bentgrass (Agrostis tenuis); creeping bentgrass (Agrostis palustris); crested wheatgrass (Agropyron desertorum); fairway wheatgrass (Agropyron cristatum); hard fescue (Festuca longifolia); Kentucky bluegrass (Poa pratensis); orchardgrass (Dactylis glomerata); perennial ryegrass (Lolium perenne); red fescue (Festuca rubra); redtop (Agrostis alba); rough bluegrass (Poa trivialis); sheep fescue (Festuca ovina); smooth bromegrass (Bromus inermis); tall fescue (Festuca arundinacea); timothy (Phleum pratense); velvet bentgrass (Agrostis canina); weeping alkaligrass (Puccinellia distans); western wheatgrass (Agropyron smithii); Bermuda grass (Cynodon spp.); St. Augustine grass (Stenotaphrum secundatum); zoysia grass (Zoysia spp.); Bahia grass (Paspalum notatum); carpet grass (Axonopus affinis); centipede grass (Eremochloa ophiuroides); kikuyu grass (Pennisetum clandesinum); seashore paspalum (Paspalum vaginatum); blue gramma (Bouteloua gracilis); buffalo grass (Buchloe dactyloids); sideoats gramma (Bouteloua curtipendula).

[0159] Plants of interest include grain plants that provide seeds of interest, oil-seed plants, and leguminous plants. Seeds of interest include grain seeds, such as com, wheat, barley, rice, sorghum, rye, millet, etc. Oil-seed plants include cotton, soybean, safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, flax, castor, olive, etc. Leguminous plants include beans and peas.Beans include guar, locust bean, fenugreek, soybean, garden beans, cowpea, mung bean, lima bean, fava bean, lentils, chickpea, etc.

[0160] Following introduction of heterologous foreign DNA into plant cells, the transformation or integration of heterologous gene in the plant genome is confirmed by various methods such as analysis of nucleic acids, proteins and metabolites associated with the integrated gene.

[0161] PCR analysis is a rapid method to screen transformed cells, tissue or shoots for the presence of incorporated gene at the earlier stage before transplanting into the soil (Sambrook and Russell, (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). PCR is carried out using oligonucleotide primers specific to the gene of interest or Agrobacterium vector background, etc.

[0162] Plant transformation may be confirmed by Southern blot analysis of genomic DNA (Sambrook and Russell, (2001) supra). In Northern blot analysis, RNA is isolated from specific tissues of transformant, fractionated in a formaldehyde agarose gel, and blotted onto a nylon filter according to standard procedures that are routinely used in the art (Sambrook and Russell, (2001) supra). Expression of RNA encoded by the fusion polynucleotide or pesticidal gene is then tested by hybridizing the filter to a radioactive probe derived from a fusion polynucleotide, by methods known in the art (Sambrook and Russell, (2001) supra). Western blot, biochemical assays and the like may be carried out on the transgenic plants to confirm the presence of protein encoded by the pesticidal gene by standard procedures (Sambrook and Russell, 2001, supra) using antibodies that bind to one or more epitopes present on the multimerization domain polypeptide or fusion polypeptides.Methods to Introduce Genome Editing Technologies into Plants

[0163] In some embodiments, the disclosed fusion polynucleotide compositions can be introduced into the genome of a plant using genome editing technologies, or previously introduced fusion polynucleotides in the genome of a plant may be edited using genome editing technologies. For example, the disclosed polynucleotides can be introduced into a desired location in the genome of a plant through the use of double-stranded break technologies such as TALENs, meganucleases, zinc finger nucleases, CRISPR-Cas, and the like. For example, the disclosed polynucleotides can be introduced into a desired location in a genome using a CRISPR- i6Cas system, for the purpose of site-specific insertion. The desired location in a plant genome can be any desired target site for insertion, such as a genomic region amenable for breeding or may be a target site located in a genomic window with an existing trait of interest. Existing traits of interest could be either an endogenous trait or a previously introduced trait.

[0164] In some embodiments, where the disclosed fusion polynucleotide has previously been introduced into a genome, genome editing technologies may be used to alter or modify the introduced polynucleotide sequence. Site specific modifications that can be introduced into the disclosed fusion polynucleotide compositions include those produced using any method for introducing site specific modification, including, but not limited to, through the use of gene repair oligonucleotides, or through the use of double-stranded break technologies such as TALENs, meganucleases, zinc finger nucleases, CRISPR-Cas, and the like. Such technologies can be used to modify the previously introduced polynucleotide through the insertion, deletion or substitution of nucleotides within the introduced polynucleotide. Alternatively, double-stranded break technologies can be used to add additional nucleotide sequences to the introduced polynucleotide. Additional sequences that may be added include, additional expression elements, such as enhancer and promoter sequences. In another embodiment, genome editing technologies may be used to position additional insecticidally-active proteins in close proximity to the disclosed fusion polynucleotide compositions disclosed herein within the genome of a plant, in order to generate molecular stacks of proteins of interest such as insecticidally-active proteins.

[0165] An “altered target site,” “altered target sequence.” “modified target site,” and “modified target sequence” are used interchangeably herein and refer to a target sequence as disclosed herein that comprises at least one alteration when compared to non-altered target sequence. Such "alterations" include, for example: (i) replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, or (iv) any combination of (i) - (iii).Stacking of traits in transgenic plant

[0166] Transgenic plants may comprise a stack of one or more fusion polynucleotides disclosed herein with one or more additional polynucleotides resulting in the production or suppression of multiple polypeptide sequences. Transgenic plants comprising stacks of polynucleotidesequences can be obtained by either or both of traditional breeding methods or through genetic engineering methods. These methods include, but are not limited to, breeding individual lines each comprising a polynucleotide of interest, transforming a transgenic plant comprising a gene disclosed herein with a subsequent gene and co- transformation of genes into a single plant cell. As used herein, the term “stacked” includes having the multiple traits present in the same plant (i.e., both traits are incorporated into the nuclear genome, one trait is incorporated into the nuclear genome and one trait is incorporated into the genome of a plastid or both traits are incorporated into the genome of a plastid). In one non-limiting example, “stacked traits” comprise a molecular stack where the sequences are physically adjacent to each other. A trait, as used herein, refers to the phenotype derived from a particular sequence or groups of sequences. Co-transformation of genes can be carried out using single transformation vectors comprising multiple genes or genes carried separately on multiple vectors. If the sequences are stacked by genetically transforming the plants, the polynucleotide sequences of interest can be combined at any time and in any order. The traits can be introduced simultaneously in a co-transformation protocol with the polynucleotides of interest provided by any combination of transformation cassettes. For example, if two sequences will be introduced, the two sequences can be contained in separate transformation cassettes (trans) or contained on the same transformation cassette (cis). Expression of the sequences can be driven by the same promoter or by different promoters. In certain cases, it may be desirable to introduce a transformation cassette that will suppress the expression of the polynucleotide of interest. This may be combined with any combination of other suppression cassettes or overexpression cassettes to generate the desired combination of traits in the plant. It is further recognized that polynucleotide sequences can be stacked at a desired genomic location using a site-specific recombination system. See, for example, WO 1999 / 25821, WO 1999 / 25854, WO 1999 / 25840, WO 1999 / 25855 and WO 1999 / 25853, all of which are herein incorporated by reference.

[0167] In some embodiments, one or more of the polynucleotides encoding the multimerization domain polypeptide or fusion polypeptide(s) disclosed herein, including fusion polynucleotides encoding chimeric polypeptides comprising one or more multimerization domain polypeptide, one or more linker peptides, and one or more polypeptides of interest, such as an insecticidal polypeptide of interest, alone or stacked with one or more additional insect resistance traits canbe stacked with one or more additional input traits (e.g., herbicide resistance, fungal resistance, virus resistance, stress tolerance, disease resistance, male sterility, stalk strength, and the like) or output traits (e.g., increased yield, modified starches, improved oil profile, balanced amino acids, high lysine or methionine, increased digestibility, improved fiber quality, drought resistance, and the like). Thus, the polynucleotide embodiments can be used to provide a complete agronomic package of improved crop quality with the ability to flexibly and cost effectively control any number of agronomic pests.

[0168] Transgenes useful for stacking include but are not limited to: transgenes that confer resistance to an herbicide; transgenes that confer or contribute to an altered grain characteristic; genes that control male-sterility; genes that create a site for site specific DNA integration; genes that affect abiotic stress resistance; genes that confer increased yield, genes that confer plant digestibility; and transgenes that confer resistance to insects or disease.

[0169] Examples of transgenes that confer resistance to insects include genes encoding a Bacillus thuringiensis protein, a derivative thereof or a synthetic polypeptide modeled thereon. See, for example, Geiser, et al., (1986) Gene 48: 109, who disclose the cloning and nucleotide sequence of a Bt delta-endotoxin gene. Moreover, DNA molecules encoding delta-endotoxin genes can be purchased from American Type Culture Collection (Rockville, Md.), for example, under ATCC® Accession Numbers 40098, 67136, 31995 and 31998. Other non-limiting examples of Bacillus thuringiensis transgenes being genetically engineered are given in the following patents and patent applications: US Patent Numbers 5,188,960; 5,689,052; 5,880,275; 5,986,177;6,023,013, 6,060,594, 6,063,597, 6,077,824, 6,620,988, 6,642,030, 6,713,259, 6,893,826,7,105,332; 7,179,965, 7,208,474; 7,227,056, 7,288,643, 7,323,556, 7,329,736, 7,449,552,7,468,278, 7,510,878, 7,521,235, 7,544,862, 7,605,304, 7,696,412, 7,629,504, 7,705,216,7,772,465, 7,790,846, 7,858,849 and WO 1991 / 14778; WO 1999 / 31248; WO 2001 / 12731; WO 1999 / 24581 and WO 1997 / 40162.

[0170] Genes encoding pesticidal proteins may also be stacked including but are not limited to: insecticidal proteins from Pseudomonas sp. such as PSEEN3174 (Monalysin, (2011) PLoS Pathogens, 7: 1-13), from Pseudomonas protegens strain CHAO and Pf-5 (previously fluorescens) (Pechy-Tarr, (2008) Environmental Microbiology 10:2368-2386: GenBank Accession No. EU400157); from Pseudomonas taiwanensis (Liu, et al., (2010) J. Agric. Food Chem. 58: 12343-12349) and from Pseudomonas pseudoalcaligenes (Zhang, et al., (2009) Annals of Microbiology 59:45-50 and Li, et al., (2007) Plant Cell Tiss. Organ Cult. 89: 159-168); insecticidal proteins from Photorhabdus sp. and Xenorhabdus sp. (Hinchliffe, et al., (2010) The Open Toxinology Journal 3: 101-118 and Morgan, et al., (2001) Applied and Envir. Micro. 67:2062-2069), US Patent Number 6,048,838, and US Patent Number 6,379,946; a PIP-1 polypeptide of US Patent Number 9,688,730; an AffP-lA and / or AffP-lB polypeptide of US Patent Number 9,475,847; a PIP -47 polypeptide of US Patent Number 10,006,045; an IPD045 polypeptide, an IPD064 polypeptide, an IPD074 polypeptide, an IPD075 polypeptide, and an IPD077 polypeptide of PCT Publication Number WO 2016 / 114973; an IPD080 polypeptide of International Patent Application Publication Number W02018 / 075350; an IPD078 polypeptide, an IPD084 polypeptide, an IPD085 polypeptide, an IPD086 polypeptide, an IPD087 polypeptide, an IPD088 polypeptide, and an IPD089 polypeptide of International Patent Application Publication Number WO2018 / 084936; PIP-72 polypeptide of US Patent Publication Number US20160366891; a PtIP-50 polypeptide and a PtIP-65 polypeptide of US Patent Application Publication Number US20170166921; an IPD098 polypeptide, an IPD059 polypeptide, an IPD108 polypeptide, an IPD109 polypeptide of International Patent Application Publication Number WO2018 / 232072; a PtIP-83 polypeptide of US Publication Number US20160347799; a PtIP-96 polypeptide of US Publication Number US20170233440; an IPD079 polypeptide of PCT Publication Number WO2017 / 23486; an IPD082 polypeptide of International Patent Application Publication Number WO 2017 / 105987, an IPD090 polypeptide of International Patent Application Publication Number WO2017 / 192560, an IPD093 polypeptide of International Patent Application Publication Number WO2018 / 111551; an IPD103 polypeptide of International Patent Application Publication Number W02018 / 005411; an IPD101 polypeptide of International Patent Application Publication Number WO2018 / 118811; an IPD121 polypeptide of International Patent Application Publication Number WO2018 / 208882, and 8-endotoxins including, but not limited to, the Cryl, Cry2, Cry3, Cry4, Cry 5, Cry6, Cry7, Cry 8, Cry9, Cry 10, Cryl 1, Cryl2, Cryl3, Cryl4, Cryl5, Cryl6, Cryl7, Cryl8, Cryl9, Cry20, Cry21, Cry22, Cry23, Cry 24, Cry25, Cry26, Cry27, Cry 28, Cry 29, Cry 30, Cry31, Cry32, Cry33, Cry34, Cry35,Cry36, Cry37, Cry38, Cry39, Cry40, Cry41, Cry42, Cry43, Cry44, Cry45, Cry 46, Cry47, Cry49, Cry50, Cry51, Cry52, Cry53, Cry 54, Cry55, Cry56, Cry57, Cry58, Cry59, Cry60, Cry61, Cry62, Cry63,Cry64, Cry65, Cry66, Cry67, Cry68, Cry69, Cry 70, Cry71, and Cry 72 classes of 8-endotoxin genes and the B. thuringiensis cytolytic Cytl and Cyt2 genes. Members of these classes of B. thuringiensis insecticidal proteins well known to one skilled in the art (see, Crickmore, et al., "Bacillus thuringiensis toxin nomenclature" (2011), at lifesci.sussex.ac.uk / home / Neil_Crickmore / Bt / which can be accessed on the world-wide web using the "www" prefix).

[0171] Examples of 6-endotoxins also include but are not limited to CrylA proteins of US Patent Numbers 5,880,275 and 7,858,849; a DIG-3 or DIG-11 toxin (N-terminal deletion of a-helix 1 and / or a-helix 2 variants of Cry proteins such as CrylA) of US Patent Numbers 8,304,604 and 8.304,605, CrylB of US Patent Application Serial Number 10 / 525,318, US Patent Application Publication Number US20160194364, and US Patent Numbers 9,404,121 and 8,772,577; CrylB variants of PCT Publication Number WO2016 / 61197 and Serial Number PCT / US 17 / 27160; CrylC of US Patent Number 6,033,874; Cry ID protein of US20170233759; a Cry IE protein of PCT Publication Number WO2018 / 075197; Cry IF of US Patent Numbers 5,188,960, 6,218,188; CrylA / F chimeras of US Patent Numbers 7,070,982; 6,962,705 and 6,713,063; a CrylJ variant of US Publication US20170240603; a Cry2 protein such as Cry2Ab protein of US Patent Number 7,064,249; a Cry3A protein including but not limited to an engineered hybrid insecticidal protein (eHIP) created by fusing unique combinations of variable regions and conserved blocks of at least two different Cry proteins (US Patent Application Publication Number 2010 / 0017914); a Cry4 protein; a Cry5 protein; a Cry6 protein; Cry8 proteins of US Patent Numbers 7,329,736, 7,449,552, 7,803,943, 7,476,781, 7,105,332, 7,378,499 and 7,462,760; a Cry9 protein such as such as members of the Cry9A, Cry9B, Cry9C, Cry9D, Cry9E, and Cry9F families; a Cry 15 protein of Naimov, et al., (2008) Applied and Environmental Microbiology 74:7145-7151; a Cry22, a Cry34Abl protein of US Patent Numbers 6,127,180, 6,624,145 and 6,340,593; a CryET33 and CryET34 protein of US Patent Numbers 6,248,535, 6,326,351, 6,399,330, 6,949,626, 7,385,107 and 7,504,229; a CryET33 and CryET34 homologs of US Patent Publication Number 2006 / 0191034, 2012 / 0278954, and PCT Publication Number WO 2012 / 139004; a Cry35Abl protein of US Patent Numbers 6,083,499, 6,548,291 and 6,340,593; a Cry46 protein of US Patent Number 9,403,881, a Cry 51 protein, a Cry binary toxin; a TIC901 or related toxin; TIC807 of US 2008 / 0295207; ET29, ET37, TIC809, TIC810, TIC812, TIC127,TIC128 of PCT US 2006 / 033867; engineered Hemipteran toxic proteins of US Patent Application Publication Number US20160150795, AXMI-027, AXMI-036, and AXMI-038 of US Patent Number 8,236,757; AXMI-031, AXMI-039, AXMI-040, AXMI-049 of US Patent Number 7,923,602; AXMI-018, AXMI-020 and AXMI-021 of WO 2006 / 083891; AXMI-010 of WO 2005 / 038032; AXMI-003 of WO 2005 / 021585; AXMI-008 of US Patent Application Publication Number 2004 / 0250311; AXMI-006 of US Patent Application Publication Number 2004 / 0216186; AXMI-007 of US Patent Application Publication Number 2004 / 0210965; AXMI-009 of US Patent Application Number 2004 / 0210964; AXMI-014 of US Patent Application Publication Number 2004 / 0197917; AXMI-004 of US Patent Application Publication Number 2004 / 0197916; AXMI-028 and AXMI-029 of WO 2006 / 119457; AXMI- 007, AXMI-008, AXMI-0080rf2, AXMI-009, AXMI-014 and AXMI-004 of WO 2004 / 074462; AXMI-150 of US Patent Number 8,084,416; AXMI-205 of US Patent Application Publication Number 2011 / 0023184; AXMI-011, AXMI-012, AXMI-013, AXMI-015, AXMI-019, AXMI- 044, AXMI-037, AXMI-043, AXMI-033, AXMI-034, AXMI-022, AXMI-023, AXMI-041, AXMI-063 and AXMI-064 of US Patent Application Publication Number 2011 / 0263488; AXMI046, AXMI048, AXMI050, AXMI051, AXMI052, AXMI053, AXMI054, AXMI055, AXMI056, AXMI057, AXMI058, AXMI059, AXMI060, AXMI061, AXMI067, AXMI069, AXMI071, AXMI072, AXMI073, AXMI074, AXMI075, AXMI087, AXMI088, AXMI093, AXMI070, AXMI080, AXMI081, AXMI082, AXMI091, AXMI092, AXMI096, AXMI097, AXMI098, AXMI099, AXMI100, AXMI101, AXMI102, AXMI103, AXMI104, AXMI107, AXMI108, AXMI109, AXMI110, AXMI111, AXMI112, AXMI114, AXMI116, AXMI117, AXMI118, AXMI119, AXMI120, AXMI121, AXMI122, AXMI123, AXMI124, AXMI125, AXMI126, AXMI127, AXMI129, AXMI151, AXMI161, AXMI164, AXMI183, AXMI132, AXMI137, AXMI138 of US Patent US8461421 and US8,461,422; AXMI-R1 and related proteins of US Patent Application Publication Number 2010 / 0197592; AXMI221Z, AXMI222z, AXMI223z, AXMI224z and AXMI225z of WO 2011 / 103248; AXMI218, AXMI219, AXMI220, AXMI226, AXMI227, AXMI228, AXMI229, AXMI230 and AXMI231 of WO 2011 / 103247; AXMI-115, AXMI-113, AXMI-005, AXMI-163 and AXMI-184 of US Patent Number 8,334,431; AXMI-001, AXMI-002, AXMI-030, AXMI-035 and AXMI-045 of US Patent Application Publication Number 2010 / 0298211; AXMI-066 and AXMI-076 of US PatentApplication Publication Number 2009 / 0144852; AXMI128, AXMI130, AXMI131, AXMI133, AXMI140, AXMI141, AXMI142, AXMI143, AXMI144, AXMI146, AXMI148, AXMI149, AXMI152, AXMI153, AXMI154, AXMI155, AXMI156, AXMI157, AXMI158, AXMI162, AXMI165, AXMI166, AXMI167, AXMI168, AXMI169, AXMI170, AXMI171, AXMI172,AXMI173, AXMI174, AXMI175, AXMI176, AXMI177, AXMI178, AXMI179, AXMI180, AXMI181, AXMI182, AXMI185, AXMI186, AXMI187, AXMI188, AXMI189 of US Patent Number 8,318,900; AXMI079, AXMI080, AXMI081, AXMI082, AXMI091, AXMI092,AXMI096, AXMI097, AXMI098, AXMI099, AXMI100, AXMI101, AXMI102, AXMI103,AXMI104, AXMI107, AXMI108, AXMI109, AXMI110, dsAXMIl l l, AXMI112, AXMI114,AXMI116, AXMI117, AXMI118, AXMI119, AXMI120, AXMI121, AXMI122, AXMI123,AXMI124, AXMI1257, AXMI1268, AXMI127, AXMI129, AXMI164, AXMI151, AXMI161,AXMI183, AXMI132, AXMI138, AXMI137 of US Patent US 8461421; AXMI192 of US PatentUS8,461,415; AXMI281 of US Patent Application Publication Number US20160177332; AXMI422 of US Patent Number US8,252,872; and Cry proteins such as CrylA and Cry3A having modified proteolytic sites of US Patent Number 8,319,019; and a Cry 1 Ac, Cry2Aa and CrylCa toxin protein from Bacillus thuringiensis strain VBTS 2528 of US Patent Application Publication Number 2011 / 0064710. The Cry proteins MP032, MP049, MP051, MP066, MP068, MP070, MP091S, MP109S, MP114, MP121, MP134S, MP183S, MP185S, MP186S, MP195S,MP197S, MP208S, MP209S, MP212S, MP214S, MP217S, MP222S, MP234S, MP235S, MP237S, MP242S, MP243, MP248, MP249S, MP251M, MP252S, MP253, MP259S, MP287S, MP288S, MP295S, MP296S, MP297S, MP300S, MP304S, MP306S, MP310S, MP312S,MP314S, MP319S, MP325S, MP326S, MP327S, MP328S, MP334S, MP337S, MP342S,MP349S, MP356S, MP359S, MP360S, MP437S, MP451S, MP452S, MP466S, MP468S,MP476S, MP482S, MP522S, MP529S, MP548S, MP552S, MP562S, MP564S, MP566S,MP567S, MP569S, MP573S, MP574S, MP575S, MP581S, MP590, MP594S, MP596S, MP597, MP599S, MP600S, MP601S, MP602S, MP604S, MP626S, MP629S, MP630S, MP631S,MP632S, MP633S, MP634S, MP635S, MP639S, MP640S, MP644S, MP649S, MP651S,MP652S, MP653S, MP661S, MP666S, MP672S, MP696S, MP704S, MP724S, MP729S,MP739S, MP755S, MP773S, MP799S, MP800S, MP801S, MP802S, MP803S, MP805S,MP809S, MP815S, MP828S, MP831S, MP844S, MP852, MP865S, MP879S, MP887S,MP891S, MP896S, MP898S, MP935S, MP968, MP989, MP993, MP997, MP1049, MP1066, MP1067, MP1080, MP1081, MP1200, MP1206, MP1233, and MP1311 of US Patent 11,492,639. Other Cry proteins are well known to one skilled in the art (see, Crickmore, et al., Microbiology and Molecular Biology Reviews (1998) Vol 62: 807-813; and Crickmore, et al., "Bacillus thuringiensis toxin nomenclature" (2016), at btnomenclature.info / which can be accessed on the world-wide web using the "www" prefix). The insecticidal activity of Cry proteins is well known to one skilled in the art (for review, see, van Frannkenhuyzen, (2009) J. Invert. Path. 101 : 1-16). The use of Cry proteins as transgenic plant traits is well known to one skilled in the art and Cry-transgenic plants including but not limited to Cry 1 Ac, CrylAc+Cry2Ab, CrylAb, CrylA.105, CrylF, CrylFa2, CrylF+CrylAc, Cry2Ab, Cry3A, mCry3A, Cry3Bbl, Cry34Abl, Cry35Abl, Vip3A, mCry3A, Cry9c and CBI-Bt have received regulatory approval (see, Sanahuja, (2011) Plant Biotech Journal 9:283-300 and the CERA (2010) GM Crop Database Center for Environmental Risk Assessment (CERA), ILSI Research Foundation, Washington D.C. at cera-gmc.org / index. php?action=gm_crop_database which can be accessed on the world-wide web using the “www” prefix). More than one pesticidal proteins well known to one skilled in the art can also be expressed in plants such as Vip3Ab & CrylFa (US2012 / 0317682); CrylBE & CrylF (US2012 / 0311746); CrylCA & CrylAB(US2012 / 0311745); CrylF & CryCa (US2012 / 0317681); CrylDA & CrylBE (US2012 / 0331590); CrylDA & CrylFa (US2012 / 0331589); CrylAB & CrylBE(US2012 / 0324606); Cr lFa & Cry2Aa and Cry II & CrylE (US2012 / 0324605); Cry34Ab / 35Ab & Cry6Aa (US20130167269); Cry34Ab / VCry35Ab & Cry3Aa (US20130167268); CrylDa & CrylCa (US 9796982); Cry3Aa & Cry6Aa (US 9798963); and Cry3A & CrylAb or Vip3Aa (US9,045,766). Pesticidal proteins also include insecticidal lipases including lipid acyl hydrolases of US Patent Number 7,491,869, and cholesterol oxidases such as from Streptomyces (Purcell et al. (1993) Biochem Biophys Res Commun 15:1406-1413). Pesticidal proteins also include VIP (vegetative insecticidal proteins) toxins of US Patent Numbers 5,877,012, 6, 107,279, 6,137,033, 7,244,820, 7,615,686, and 8,237,020, and the like. Other VIP proteins are well known to one skilled in the art (see, lifesci.sussex.ac.uk / home / Neil_Crickmore / Bt / vip.html which can be accessed on the world-wide web using the "www" prefix). Pesticidal proteins also include MP467omplex (TC) proteins, obtainable from organisms such as Xenorhabdus, Photorhabdusand Paenibacillus (see, US Patent Numbers 7,491,698 and 8,084,418). Some TC proteins have “stand alone” insecticidal activity and other TC proteins enhance the activity of the stand-alone toxins produced by the same given organism. The toxicity of a “stand-alone” TC protein (from Photorhabdus, Xenorhabdus or Paenibacillus, for example) can be enhanced by one or more TC protein “potentiators” derived from a source organism of a different genus. There are three main types of TC proteins. As referred to herein, Class A proteins (“Protein A”) are stand-alone toxins. Class B proteins (“Protein B”) and Class C proteins (“Protein C”) enhance the toxicity of Class A proteins. Examples of Class A proteins are TcbA, TcdA, XptAl and XptA2. Examples of Class B proteins are TcaC, TcdB, XptBIXb and XptCIWi. Examples of Class C proteins are TccC, XptClXb and XptBIWi. Pesticidal proteins also include spider, snake and scorpion venom proteins. Examples of spider venom peptides include but are not limited to lycotoxin-1 peptides and mutants thereof (US Patent Number 8,334,366).

[0172] Any of the pesticidal proteins listed above and disclosed herein may be an insecticidal polypeptide of interest according to the compositions and methods of the invention.Gene Silencing

[0173] In some embodiments, the stacked trait may be in the form of silencing of one or more polynucleotides of interest resulting in suppression of one or more target pest polypeptides. In some embodiments, the silencing is achieved using a suppression DNA construct.

[0174] In some embodiments, one or more polynucleotide encoding the polypeptides of the multimerization domain polypeptide or chimeric polypeptides comprising one or more multimerization domain polypeptide, one or more linker peptides, and one or more polypeptides of interest, such as an insecticidal polypeptide of interest, or fragments or variants thereof may be stacked with one or more polynucleotides encoding one or more polypeptides having insecticidal activity or agronomic traits as set forth supra and optionally may further include one or more polynucleotides providing for gene silencing of one or more target polynucleotides as discussed infra.

[0175] Further transgenes that confer resistance to insects may relate to down-regulation of expression of target genes in insect pest species by interfering ribonucleic acid (RNA) molecules through RNA interference. RNA interference refers to the process of sequence-specific post-6transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs) (Fire, et al., (1998) Nature 391 :806). RNAi transgenes may include but are not limited to expression of dsRNA, siRNA, miRNA, iRNA, antisense RNA, or sense RNA molecules that down-regulate expression of target genes in insect pests. PCT Publication WO 2007 / 074405 describes methods of inhibiting expression of target genes in invertebrate pests including Colorado potato beetle. PCT Publication WO 2005 / 110068 describes methods of inhibiting expression of target genes in invertebrate pests including in particular Western corn rootworm as a means to control insect infestation. Furthermore, PCT Publication WO 2009 / 091864 describes compositions and methods for the suppression of target genes from insect pest species including pests from the Lygus genus.

[0176] RNAi transgenes are provided for targeting the vacuolar ATPase H subunit, useful for controlling a coleopteran pest population and infestation as described in US Patent Application Publication 2012 / 0198586. PCT Publication WO 2012 / 055982 describes ribonucleic acid (RNA or double stranded RNA) that inhibits or down regulates the expression of a target gene that encodes: an insect ribosomal protein such as the ribosomal protein LI 9, the ribosomal protein L40 or the ribosomal protein S27A; an insect proteasome subunit such as the Rpn6 protein, the Pros 25, the Rpn2 protein, the proteasome beta 1 subunit protein or the Pros beta 2 protein; an insect P-coatomer of the COPI vesicle, the y-coatomer of the COPI vesicle, the P'- coatomer protein or the ^-coatomer of the COPI vesicle; an insect Tetraspanine 2 A protein which is a putative transmembrane domain protein; an insect protein belonging to the actin family such as Actin 5C; an insect ubiquitin-5E protein; an insect Sec23 protein which is a GTPase activator involved in intracellular protein transport; an insect crinkled protein which is an unconventional myosin which is involved in motor activity; an insect crooked neck protein which is involved in the regulation of nuclear alternative mRNA splicing; an insect vacuolar H+-ATPase G-subunit protein and an insect Tbp-1 such as Tat-binding protein. PCT publication WO 2007 / 035650 describes ribonucleic acid (RNA or double stranded RNA) that inhibits or down regulates the expression of a target gene that encodes Snf7. US Patent Application publication 2011 / 0054007 describes polynucleotide silencing elements targeting RPS 10. PCT publication WO 2016 / 205445 describes polynucleotide silencing elements that reduce fecundity, with target polynucleotides, including NCLB, MAEL, BOULE, and VgR. US Patent Application publication 2014 / 0275208and US2015 / 0257389 describes polynucleotide silencing elements targeting RyanR (DvSSJl) and PAT3. PCT publications WO / 2016 / 138106, WO 2016 / 060911, WO 2016 / 060912, WO 2016 / 060913, and WO 2016 / 060914 describe polynucleotide silencing elements targeting COPI coatomer subunit nucleic acid molecules that confer resistance to Coleopteran and Hemipteran pests. US Patent Application Publications 2012 / 029750, US 20120297501, and 2012 / 0322660 describe interfering ribonucleic acids (RNA or double stranded RNA) that functions upon uptake by an insect pest species to down-regulate expression of a target gene in said insect pest, wherein the RNA comprises at least one silencing element wherein the silencing element is a region of double-stranded RNA comprising annealed complementary strands, one strand of which comprises or consists of a sequence of nucleotides which is at least partially complementary to a target nucleotide sequence within the target gene. US Patent Application Publication 2012 / 0164205 describe potential targets for interfering double stranded ribonucleic acids for inhibiting invertebrate pests including: a Chd3 Homologous Sequence, a Beta-Tubulin Homologous Sequence, a 40 kDa V-ATPase Homologous Sequence, a EFla Homologous Sequence, a 26S Proteosome Subunit p28 Homologous Sequence, a Juvenile Hormone Epoxide Hydrolase Homologous Sequence, a Swelling Dependent Chloride Channel Protein Homologous Sequence, a Glucose-6-Phosphate 1 -Dehydrogenase Protein Homologous Sequence, an Act42A Protein Homologous Sequence, a ADP-Ribosylation Factor 1 Homologous Sequence, a Transcription Factor IIB Protein Homologous Sequence, a Chitinase Homologous Sequences, a Ubiquitin Conjugating Enzyme Homologous Sequence, a Glyceraldehyde-3 -Phosphate Dehydrogenase Homologous Sequence, an Ubiquitin B Homologous Sequence, a Juvenile Hormone Esterase Homolog, and an Alpha Tubuliln Homologous Sequence.Use in Pesticidal Control

[0177] General methods for employing strains comprising a nucleic acid sequence of the embodiments or a variant thereof, in pesticide control or in engineering other organisms as pesticidal agents are known in the art.

[0178] Microorganism hosts that are known to occupy the "phytosphere" (phylloplane, phyllosphere, rhizosphere, and / or rhizoplana) of one or more crops of interest may be selected. These microorganisms are selected so as to be capable of successfully competing in the particularenvironment with the wild-type microorganisms, provide for stable maintenance and expression of the gene(s) expressing one or more of the multimerization domain polypeptide or chimeric fusion polypeptides comprising one or more multimerization domain polypeptide, one or more linker peptides, and one or more polypeptides of interest, such as an insecticidal polypeptide of interest and desirably provide for improved protection of the pesticide from environmental degradation and inactivation.

[0179] Alternatively, the multimerization domain polypeptide or chimeric polypeptides comprising one or more multimerization domain polypeptide, one or more linker peptides, and one or more polypeptides of interest, such as an insecticidal polypeptide of interest are produced by introducing a heterologous gene into a cellular host. Expression of the heterologous gene results, directly or indirectly, in the intracellular production and maintenance of the pesticide. These cells are then treated under conditions that prolong the activity of the toxin produced in the cell when the cell is applied to the environment of target pest(s). The resulting product retains the toxicity of the toxin. These naturally encapsulated multimerization domain polypeptide or chimeric polypeptides comprising one or more multimerization domain polypeptide, one or more linker peptides, and one or more polypeptides of interest, such as an insecticidal polypeptide of interest may then be formulated in accordance with conventional techniques for application to the environment hosting a target pest, e.g., soil, water, and foliage of plants. See, for example EPA 0192319, and the references cited therein.Pesticidal Compositions

[0180] In some embodiments the active ingredients such as insecticidal polypeptides can be applied in the form of compositions and can be applied to the crop area or plant to be treated, simultaneously or in succession, with other compounds. These compounds can be fertilizers, weed killers, Cryoprotectants, surfactants, detergents, pesticidal soaps, dormant oils, polymers, and / or time-release or biodegradable carrier formulations that permit long-term dosing of a target area following a single application of the formulation. They can also be selective herbicides, chemical insecticides, virucides, microbicides, amoebicides, pesticides, fungicides, bacteriocides, nematocides, molluscicides or mixtures of several of these preparations, if desired, together with further agriculturally acceptable carriers, surfactants or application-promotingadjuvants customarily employed in the art of formulation. Suitable carriers and adjuvants can be solid or liquid and correspond to the substances ordinarily employed in formulation technology, e.g. natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, binders or fertilizers. Likewise, the formulations may be prepared into edible “baits” or fashioned into pest “traps” to permit feeding or ingestion by a target pest of the pesticidal formulation.

[0181] Methods of applying an active ingredient or an agrochemical composition that contains chimeric polypeptides comprising one or more multimerization domain, one or more linker peptides, and one or more polypeptides of interest, such as an insecticidal polypeptide of interest and / or of the Toxin F polypeptide(s) include leaf application, seed coating and soil application. The number of applications and the rate of application depend on the intensity of infestation by the corresponding pest.

[0182] The composition may be formulated as a powder, dust, pellet, granule, spray, emulsion, colloid, solution, or such like, and may be prepared by such conventional means as desiccation, lyophilization, homogenation, extraction, filtration, centrifugation, sedimentation or concentration of a culture of cells comprising the polypeptide. In all such compositions that contain at least one such pesticidal polypeptide, the polypeptide may be present in a concentration of from about 1% to about 99% by weight.

[0183] Lepidopteran, Dipteran, Heteropteran, nematode, Hemiptera or Coleopteran pests may be killed or reduced in numbers in a given area by the methods of the disclosure or may be prophylactically applied to an environmental area to prevent infestation by a susceptible pest. Preferably the pest ingests or is contacted with, a pesticidally-effective amount of the polypeptide. “Pesticidally-effective amount” as used herein refers to an amount of the pesticide that can bring about death to at least one pest or to noticeably reduce pest growth, feeding or normal physiological development. This amount will vary depending on such factors as, for example, the specific target pests to be controlled, the specific environment, location, plant, crop or agricultural site to be treated, the environmental conditions and the method, rate, concentration, stability, and quantity of application of the pesticidally-effective polypeptide composition. The formulations may also vary with respect to climatic conditions, environmental considerations, and / or frequency of application and / or severity of pest infestation.

[0184] The pesticide compositions described may be made by formulating either the bacterial cell, Crystal and / or spore suspension or isolated protein component with the desired agriculturally-acceptable carrier. The compositions may be formulated prior to administration in an appropriate means such as lyophilized, freeze-dried, desiccated or in an aqueous carrier, medium or suitable diluent, such as saline or other buffer. The formulated compositions may be in the form of a dust or granular material or a suspension in oil (vegetable or mineral) or water or oil / water emulsions or as a wettable powder or in combination with any other carrier material suitable for agricultural application. Suitable agricultural carriers can be solid or liquid and are well known in the art. The term “agriculturally-acceptable carrier” covers all adjuvants, inert components, dispersants, surfactants, tackifiers, binders, etc. that are ordinarily used in pesticide formulation technology; these are well known to those skilled in pesticide formulation. The formulations may be mixed with one or more solid or liquid adjuvants and prepared by various means, e.g., by homogeneously mixing, blending and / or grinding the pesticidal composition with suitable adjuvants using conventional formulation techniques. Suitable formulations and application methods are known. The plants can also be treated with one or more chemical compositions, including one or more herbicide, insecticides or fungicides. Exemplary chemical compositions include: Cereals Herbicides: Isoproturon, Bromoxynil, Ioxynil, Phenoxies, Chlorsulfuron, Clodinafop, Diclofop, Diflufenican, Fenoxaprop, Florasulam, Fluoroxypyr, Metsulfuron, Triasulfuron, Flucarbazone, lodosulfuron, Propoxy carb azone, Picolinafen, Mesosulfuron, Beflubutamid, Pinoxaden, Amidosulfuron, Thifensulfuron Methyl, Tribenuron, Flupyrsulfuron, Sulfosulfuron, Pyrasulfotole, Pyroxsulam, Flufenacet, Tralkoxydim, Pyroxasulfon; Cereals Fungicides: Carbendazim, Chlorothalonil, Azoxystrobin, Cy proconazole, Cyprodinil, Fenpropimorph, Epoxiconazole, Kresoxim-methyl, Quinoxyfen, Tebuconazole, Trifloxystrobin, Simeconazole, Picoxystrobin, Pyraclostrobin, Dimoxystrobin, Prothioconazole, Fluoxastrobin; Cereals Insecticides: Dimethoate, Lambda-cyhalthrin, Del tarn ethrin, alpha- Cypermethrin, P-cyfluthrin, Bifenthrin, Imidacloprid, Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid, Dinetofuran, Clorphyriphos, Metamidophos, Oxidemethon-methyl, Pirimicarb, Methiocarb; Maize Herbicides: Atrazine, Alachlor, Bromoxynil, Acetochlor, Dicamba, Clopyralid, (S-) Dimethenamid, Glufosinate, Glyphosate, Isoxaflutole, (S-)Metolachlor, Mesotrione, Nicosulfuron, Primisulfuron, Rimsulfuron, Sulcotrione, Foramsulfuron,Topramezone, Tembotrione, Saflufenacil, Thiencarb azone, Flufenacet, Pyroxasulfon; Maize Insecticides: Carbofuran, Chlorpyrifos, Bifenthrin, Fipronil, Imidacloprid, Lambda-Cyhalothrin, Tefluthrin, Terbufos, Thiamethoxam, Clothianidin, Spiromesifen, Flubendiamide, Triflumuron, Rynaxypyr, Deltamethrin, Thiodicarb, P-Cyfluthrin, Cypermethrin, Bifenthrin, Lufenuron, Triflumoron, Tefluthrin, Tebupirimphos, Ethiprole, Cyazypyr, Thiacloprid, Acetamiprid, Dinetofuran, Avermectin, Methiocarb, Spirodi cl ofen, Spirotetramat; Maize Fungicides: Fenitropan, Thiram, Prothioconazole, Tebuconazole, Trifloxystrobin; Cotton Herbicides: Diuron, Fluometuron, MSMA, Oxyfluorfen, Prometryn, Trifluralin, Carfentrazone, Clethodim, Fluazifop-butyl, Glyphosate, Norflurazon, Pendimethalin, Pyrithiobac-sodium, Trifloxysulfuron, Tepraloxydim, Glufosinate, Flumioxazin, Thidiazuron; Cotton Insecticides: Acephate, Aldicarb, Chlorpyrifos, Cypermethrin, Deltamethrin, Malathion, Monocrotophos, Abamectin, Acetamiprid, Emamectin Benzoate, Imidacloprid, Indoxacarb, Lambda-Cyhalothrin, Spinosad, Thiodicarb, Gamma-Cyhalothrin, Spiromesifen, Pyridalyl, Flonicamid, Flubendiamide, Triflumuron, Rynaxypyr, Beta-Cyfluthrin, Spirotetramat, Clothianidin, Thiamethoxam, Thiacloprid, Dinetofuran, Flubendiamide, Cyazypyr, Spinosad, Spinotoram, gamma Cyhalothrin, 4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on, Thiodicarb, Avermectin, Flonicamid, Pyridalyl, Spiromesifen, Sulfoxaflor, Profenophos, Thriazophos, Endosulfan; Cotton Fungicides: Etridiazole, Metalaxyl, Quintozene; Soybean Herbicides: Alachlor, Bentazone, Trifluralin, Chlorimuron-Ethyl, Cloransulam-Methyl, Fenoxaprop, Fomesafen, Fluazifop, Glyphosate, Imazamox, Imazaquin, Imazethapyr, (S- )Metolachlor, Metribuzin, Pendimethalin, Tepraloxydim, Glufosinate; Soybean Insecticides: Lambda-cyhalothrin, Methomyl, Parathion, Thiocarb, Imidacloprid, Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid, Dinetofuran, Flubendiamide, Rynaxypyr, Cyazypyr, Spinosad, Spinotoram, Emamectin-Benzoate, Fipronil, Ethiprole, Deltamethrin, P-Cyfluthrin, gamma and lambda Cyhalothrin, 4-[[(6-Chlorpyridin-3-yl)methyl](2,2- difluorethyl)amino]furan-2(5H)-on, Spirotetramat, Spinodiclofen, Triflumuron, Flonicamid, Thiodicarb, beta-Cyfluthrin; Soybean Fungicides: Azoxy strobin, Cy proconazole, Epoxiconazole, Flutriafol, Pyraclostrobin, Tebuconazole, Trifloxystrobin, Prothioconazole, Tetraconazole; Canola Herbicides: Clopyralid, Diclofop, Fluazifop, Glufosinate, Glyphosate, Metazachlor, Trifluralin Ethametsulfuron, Quinmerac, Quizalofop, Clethodim, Tepraloxydim;Canola Fungicides: Azoxystrobin, Carbendazim, Fludioxonil, Iprodione, Prochloraz, Vinclozolin; Canola Insecticides: Carbofuran organophosphates, Pyrethroids, Thiacloprid, Deltamethrin, Imidacloprid, Clothianidin, Thiamethoxam, Acetamiprid, Dinetofuran, (1- Cyfluthrin, gamma and lambda Cyhalothrin, tau-Fluvaleriate, Ethiprole, Spinosad, Spinotoram, Flubendiamide, Rynaxypyr, Cyazypyr, 4-[[(6-Chlorpyridin-3-yl)methyl](2,2- difluorethyl)amino]furan-2(5H)-on.

[0185] In some embodiments the herbicide is Atrazine, Bromacil, Diuron, Chlorsulfuron, Metsulfuron, Thifensulfuron Methyl, Tribenuron, Acetochlor, Dicamba, Isoxaflutole, Nicosulfuron, Rimsulfuron, Pyrithiobac-sodium, Flumioxazin, Chlorimuron-Ethyl, Metribuzin, Quizalofop, S-metolachlor, Hexazinne or combinations thereof.

[0186] In some embodiments the insecticide is Esfenval erate, Chlorantraniliprole, Methomyl, Indoxacarb, Oxamyl or combinations thereof.Pesticidal and Insecticidal Activity

[0187] “Pest” includes but is not limited to, insects, fungi, bacteria, nematodes, mites, ticks and the like. Insect pests include insects selected from the orders Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularly Lepidoptera.

[0188] Those skilled in the art will recognize that not all compounds are equally effective against all pests. Compounds of the embodiments display activity against insect pests, which may include economically important agronomic, forest, greenhouse, nursery ornamentals, food and fiber, public and animal health, domestic and commercial structure, household and stored product pests.

[0189] Larvae of the order Lepidoptera include, but are not limited to, army worms, cutworms, loopers and heliothines in the family Noctuidae Spodoptera frugiperda JE Smith (fall army worm); S. exigua Hubner (beet army worm); S. litura Fabricius (tobacco cutworm, cluster caterpillar); Mamestra configurata Walker (bertha army worm); M. brassicae Linnaeus (cabbage moth); Agrotis ipsilon Hufnagel (black cutworm); A. orthogonia Morrison (western cutworm); A. subterranea Fabricius (granulate cutworm); Alabama argillacea Hubner (cotton leaf worm); Trichoplusia ni Hubner (cabbage looper); Pseudoplusia includens Walker (soybean looper);Anticarsia gemmatalis Hubner (velvetbean caterpillar); Hypena scabra Fabricius (green cloverworm); Heliothis virescens Fabricius (tobacco budworm); Pseudaletia unipuncta Haworth (armyworm); Athetis mindara Barnes and Mcdunnough (rough skinned cutworm); Euxoa messoria Harris (darksided cutworm); Earias insulana Boisduval (spiny bollworm); E. vittella Fabricius (spotted bollworm); Helicoverpa armigera Hubner (American bollworm); H. zea Boddie (corn earworm or cotton bollworm); Melanchra picta Harris (zebra caterpillar); Egira (Xylomyges) curialis Grote (citrus cutworm); borers, casebearers, webworms, coneworms, and skeletonizers from the family Pyralidae Ostrinia nubilalis Hubner (European corn borer); Amyelois transitella Walker (naval orangeworm); Anagasta kuehniella Zeller (Mediterranean flour moth); Cadra cautella Walker (almond moth); Chilo suppressalis Walker (rice stem borer); C. partellus, (sorghum borer); Corcyra cephalonica Stainton (rice moth); Crambus caliginosellus Clemens (corn root webworm); C. teterrellus Zincken (bluegrass webworm); Cnaphalocrocis medinalis Guenee (rice leaf roller); Desmia funeralis Hubner (grape leaffolder); Diaphania hyalinata Linnaeus (melon worm); D. nitidalis Stoll (pickleworm); Diatraea grandiosella Dyar (southwestern corn borer), D. saccharalis Fabricius (surgarcane borer); Eoreuma loftini Dyar (Mexican rice borer); Ephestia elutella Hubner (tobacco (cacao) moth); Galleria mellonella Linnaeus (greater wax moth); Herpetogramma licarsisalis Walker (sod webworm); Homoeosoma electellum Hulst (sunflower moth); Elasmopalpus lignosellus Zeller (lesser cornstalk borer); Achroia grisella Fabricius (lesser wax moth); Loxostege sticticalis Linnaeus (beet webworm); Orthaga thyrisalis Walker (tea tree web moth); Maruca testulalis Geyer (bean pod borer); Plodia interpunctella Hubner (Indian meal moth); Scirpophaga incertulas Walker (yellow stem borer); Udea rubigalis Guenee (celery leaftier); and leafrollers, budworms, seed worms and fruit worms in the family Tortricidae Acleris gloverana Walsingham (Western blackheaded budworm); A. variana Fernaid (Eastern blackheaded budworm); Archips argyrospila Walker (fruit tree leaf roller); A. rosana Linnaeus (European leaf roller); and other Archips species, Adoxophyes orana Fischer von Rbsslerstamm (summer fruit tortrix moth); Cochylis hospes Walsingham (banded sunflower moth); Cydia latiferreana Walsingham (filbertworm); C. pomonella Linnaeus (coding moth); Platynota flavedana Clemens (variegated leafroller); P. stultana Walsingham (omnivorous leafroller); Lobesia botrana Denis & Schiffermuller (European grape vine moth); Spilonota ocellana Denis & Schiffermuller (ey espotted bud moth); Endopiza viteana Clemens (grape berrymoth); Eupoecilia ambiguella Hubner (vine moth); Bonagota salubricola Meyrick (Brazilian apple leafroller); Grapholita molesta Busck (oriental fruit moth); Suleima helianthana Riley (sunflower bud moth); Argyrotaenia spp.; Choristoneura spp..

[0190] Selected other agronomic pests in the order Lepidoptera include, but are not limited to, Alsophila pometaria Harris (fall cankerworm); Anarsia lineatella Zeller (peach twig borer); Anisota senatoria J.E. Smith (orange striped oakworm); Antheraea pernyi Guerin-Meneville (Chinese Oak Tussah Moth); Bombyx mori Linnaeus (Silkworm); Bucculatrix thurberiella Busck (cotton leaf perforator); Colias eurytheme Boisduval (alfalfa caterpillar); Datana integerrima Grote & Robinson (walnut caterpillar); Dendrolimus sibiricus Tschetwerikov (Siberian silk moth), Ennomos subsignaria Hubner (elm spanworm); Erannis tiliaria Harris (linden looper); Euproctis chrysorrhoea Linnaeus (browntail moth); Harrisina americana Guerin-Meneville (grapeleaf skeletonizer); Hemileuca oliviae Cockrell (range caterpillar); Hyphantria cunea Drury (fall webworm); Keiferia lycopersicella Walsingham (tomato pinworm); Lambdina fiscellaria fiscellaria Hulst (Eastern hemlock looper); L. fiscellaria lugubrosa Hulst (Western hemlock looper); Leucoma salicis Linnaeus (satin moth); Lymantria dispar Linnaeus (gypsy moth); Manduca quinquemaculata Haworth (five spotted hawk moth, tomato homworm); M. sexta Haworth (tomato hornworm, tobacco homworm); Operophtera brumata Linnaeus (winter moth); Paleacrita vemata Peck (spring cankerworm); Papilio cresphontes Cramer (giant swallowtail orange dog); Phryganidia californica Packard (California oakworm); Phyllocnistis citrella Stainton (citrus leafminer); Phyllonorycter blancardella Fabricius (spotted tentiform leafminer); Pieris brassicae Linnaeus (large white butterfly); P. rapae Linnaeus (small white butterfly); P. napi Linnaeus (green veined white butterfly); Platyptilia carduidactyla Riley (artichoke plume moth); Plutella xylostella Linnaeus (diamondback moth); Pectinophora gossypiella Saunders (pink bollworm); Pontia protodice Boisduval and Leconte (Southern cabbageworm); Sabulodes aegrotata Guenee (omnivorous looper); Schizura concinna J.E. Smith (red humped caterpillar); Sitotroga cerealella Olivier (Angoumois grain moth); Thaumetopoea pityocampa Schiffermuller (pine processionary caterpillar); Tineola bisselliella Hummel (webbing clothesmoth); Tuta absoluta Meyrick (tomato leafminer); Yponomeuta padella Linnaeus (ermine moth); Heliothis subflexa Guenee; Malacosoma spp. and Orgyia spp.

[0191] Adults and immatures of the order Diptera are of interest, including leafminers Agromyza parvicornis Loew (corn blotch leafminer); midges (including, but not limited to: Contarinia sorghicola Coquillett (sorghum midge); Mayetiola destructor Say (Hessian fly); Sitodiplosis mosellana Gehin (wheat midge); Neolasioptera murtfeldti ana Felt, (sunflower seed midge)); fruit flies (Tephritidae), Oscinella frit Linnaeus (fruit flies); maggots (including, but not limited to: Delia platura Meigen (seedcorn maggot).

[0192] Included as insects of interest are adults and nymphs of the orders Hemiptera and Homoptera such as, but not limited to, adelgids from the family Adelgidae, plant bugs from the family Miridae, cicadas from the family Cicadidae, leafhoppers, Empoasca spp.; from the family Cicadellidae, planthoppers from the families Cixiidae, Flatidae, Fulgoroidea, Issidae and Delphacidae, treehoppers from the family Membracidae, psyllids from the family Psyllidae, whiteflies from the family Aleyrodidae, aphids from the family Aphididae, phylloxera from the family Phylloxeridae, mealybugs from the family Pseudococcidae, scales from the families Asterolecanidae, Coccidae, Dactylopiidae, Diaspididae, Eriococcidae Ortheziidae, Phoenicococcidae and Margarodidae, lace bugs from the family Tingidae, stink bugs from the family Pentatomidae, cinch bugs, Blissus spp.; and other seed bugs from the family Lygaeidae, spittlebugs from the family Cercopidae squash bugs from the family Coreidae and red bugs and cotton stainers from the family Pyrrhocoridae.

[0193] Agronomically important members from the order Homoptera further include, but are not limited to: Acyrthisiphon pisum Harris (pea aphid); Aphis craccivora Koch (cowpea aphid); A. fabae Scopoli (black bean aphid); A. gossypii Glover (cotton aphid, melon aphid); A. maidiradicis Forbes (corn root aphid); A. pomi De Geer (apple aphid); A. spiraecolaPatch (spirea aphid); Aulacorthum solani Kaltenbach (foxglove aphid); Chaetosiphon fragaefolii Cockerell (strawberry aphid); Diuraphis noxia Kurdjumov / Mordvilko (Russian wheat aphid); Dysaphis plantaginea Paaserini (rosy apple aphid); Eriosoma lanigerum Hausmann (woolly apple aphid); Brevicoryne brassicae Linnaeus (cabbage aphid); Hyalopterus pruni Geoffroy (mealy plum aphid); Lipaphis erysimi Kaltenbach (turnip aphid); Metopolophium dirrhodum Walker (cereal aphid); Macrosiphum euphorbiae Thomas (potato aphid); Myzus persicae Sulzer (peach-potato aphid, green peach aphid); Nasonovia ribisnigri Mosley (lettuce aphid); Pemphigus spp. (root aphids and gall aphids); Rhopalosiphum maidis Fitch (corn leaf aphid); R. padi Linnaeus (birdcherry-oat aphid); Schizaphis graminum Rondani (greenbug); Sipha flava Forbes (yellow sugarcane aphid); Sitobion avenae Fabricius (English grain aphid); Therioaphis maculata Buckton (spotted alfalfa aphid); Toxoptera aurantii Boyer de Fonscolombe (black citrus aphid) and T. citricida Kirkaldy (brown citrus aphid); Adelges spp. (adelgids); Phylloxera devastatrix Pergande (pecan phylloxera); Bemisia tabaci Gennadius (tobacco whitefly, sweetpotato whitefly); B. argentifolii Bellows & Perring (silverleaf whitefly); Dialeurodes citri Ashmead (citrus whitefly); Trialeurodes abutiloneus (bandedwinged whitefly) and T. vaporariorum Westwood (greenhouse whitefly); Empoasca fabae Harris (potato leafhopper); Laodelphax striatellus Fallen (smaller brown planthopper); Macrolestes quadrilineatus Forbes (aster leafhopper); Nephotettix cinticeps Uhler (green leafhopper); N. nigropictus Stal (rice leafhopper); Nilaparvata lugens Stal (brown planthopper); Peregrinus maidis Ashmead (corn planthopper); Sogatella furcifera Horvath (white-backed planthopper); Sogatodes orizicola Muir (rice delphacid); Typhlocyba pomaria McAtee (white apple leafhopper); Erythroneoura spp. (grape leafhoppers); Magicicada septendecim Linnaeus (periodical cicada); Icerya purchasi Maskell (cottony cushion scale); Quadraspidiotus perniciosus Comstock (San Jose scale); Pianococcus citri Risso (citrus mealybug); Pseudococcus spp. (other mealybug complex); Cacopsylla pyricola Foerster (pear psylla); Trioza diospyri Ashmead (persimmon psylla).

[0194] Agronomically important species of interest from the order Hemiptera include, but are not limited to: Acrosternum hilare Say (green stink bug); Anasa tristis De Geer (squash bug); Blissus leucopterus leucopterus Say (chinch bug); Corythuca gossypii Fabricius (cotton lace bug); Cyrtopeltis modesta Distant (tomato bug); Dysdercus suturellus Herrich-Schaffer (cotton stainer); Euschistus servus Say (brown stink bug); E. variolarius Palisot de Beauvois (one-spotted stink bug); Graptostethus spp. (complex of seed bugs); Leptoglossus corculus Say (leaf-footed pine seed bug); Lygus lineolaris Palisot de Beauvois (tarnished plant bug); L. Hesperus Knight (Western tarnished plant bug); L. pratensis Linnaeus (common meadow bug); L. rugulipennis Poppius (European tarnished plant bug); Lygocoris pabulinus Linnaeus (common green capsid); Nezara viridula Linnaeus (southern green stink bug); Oebalus pugnax Fabricius (rice stink bug); Oncopeltus fasciatus Dallas (large milkweed bug); Pseudatomoscelis seriatus Reuter (cotton fleahopper).

[0195] Furthermore, embodiments may be effective against Hemiptera such as, Calocoris norvegicus Gmelin (strawberry bug); Orthops campestris Linnaeus; Plesiocoris rugicollis Fallen (apple capsid); Cyrtopeltis modestus Distant (tomato bug); Cyrtopeltis notatus Distant (suckfly); Spanagonicus albofasciatus Reuter (whitemarked fleahopper); Diaphnocoris chlorionis Say (honeylocust plant bug); Labopidicola allii Knight (onion plant bug); Pseudatomoscelis seriatus Reuter (cotton fleahopper); Adelphocoris rapidus Say (rapid plant bug); Poecilocapsus lineatus Fabricius (four-lined plant bug); Nysius ericae Schilling (false chinch bug); Nysius raphanus Howard (false chinch bug); Nezara viridula Linnaeus (Southern green stink bug); Eurygaster spp.; Coreidae spp.; Pyrrhocoridae spp.; Tinidae spp.; Blostomatidae spp.; Reduviidae spp. and Cimicidae spp.

[0196] Also included are adults and larvae of the order Acari (mites) such as Aceria tosichella Keifer (wheat curl mite); Petrobia latens Muller (brown wheat mite); spider mites and red mites in the family Tetranychidae, Panonychus ulmi Koch (European red mite); Tetranychus urticae Koch (two spotted spider mite); (T. mcdanieli McGregor (McDaniel mite); T. cinnabarinus Boisduval (carmine spider mite); T. turkestani Ugarov & Nikolski (strawberry spider mite); flat mites in the family Tenuipalpidae, Brevipalpus lewisi McGregor (citrus flat mite); rust and bud mites in the family Eriophyidae and other foliar feeding mites and mites important in human and animal health.

[0197] Insect pest of interest include the superfamily of stink bugs and other related insects including but not limited to species belonging to the family Pentatomidae (Nezara viridula, Halyomorpha halys, Piezodorus guildini, Euschistus servus, Acrosternum hilare, Euschistus heros, Euschistus tristigmus, Acrosternum hilare, Dichelops furcatus, Dichelops melacanthus, and Bagrada hilaris (Bagrada Bug)), the family Plataspidae (Megacopta cribraria - Bean plataspid) and the family Cydnidae (Scaptocoris castanea - Root stink bug) and Lepidoptera species including but not limited to: diamond-back moth, e.g., Helicoverpa zea Boddie; soybean looper, e.g., Pseudoplusia includens Walker and velvet bean caterpillar e.g., Anticarsia gemmatalis Hiibner.

[0198] Methods for measuring pesticidal activity are well known in the art. See, for example, Czapla and Lang, (1990) J. Econ. Entomol. 83:2480-2485; Andrews, et al., (1988) Biochem. J. 252:199-206; Marrone, et al., (1985) J. of Economic Entomology 78:290-293 and US PatentNumber 5,743,477. Generally, the protein is mixed and used in feeding assays. See, for example Marrone, et al., (1985) J. of Economic Entomology 78:290-293. Such assays can include contacting plants with one or more pests and determining the plant's ability to survive and / or cause the death of the pests.

[0199] Nematodes include parasitic nematodes such as root -knot, cyst and lesion nematodes, including Heterodera spp., Meloidogyne spp. and Globodera spp.; particularly members of the cyst nematodes, including, but not limited to, Heterodera glycines (soybean cyst nematode); Heterodera schachtii (beet cyst nematode); Heterodera avenae (cereal cyst nematode) and Globodera rostochiensis and Globodera pailida (potato cyst nematodes). Lesion nematodes include Pratylenchus spp.Seed Treatment

[0200] To protect and to enhance yield production and trait technologies, seed treatment options can provide additional crop plan flexibility and cost-effective control against insects, weeds and diseases. Seed material can be treated, typically surface treated, with a composition comprising combinations of chemical or biological herbicides, herbicide safeners, insecticides, fungicides, germination inhibitors and enhancers, nutrients, plant growth regulators and activators, bactericides, nematocides, avicides and / or molluscicides. These compounds are typically formulated together with further carriers, surfactants or application-promoting adjuvants customarily employed in the art of formulation. The coatings may be applied by impregnating propagation material with a liquid formulation or by coating with a combined wet or dry formulation. Examples of the various types of compounds that may be used as seed treatments are provided in The Pesticide Manual: A World Compendium, C.D.S. Tomlin Ed., Published by the British Crop Production Council.

[0201] Some seed treatments that may be used on crop seed include, but are not limited to, one or more of abscisic acid, acibenzolar-S-methyl, avermectin, amitrol, azaconazole, azospirillum, azadirachtin, azoxystrobin, Bacillus spp. (including one or more of cereus, firmus, megaterium, pumilis, sphaericus, subtilis and / or thuringiensis species), bradyrhizobium spp. (including one or more of betae, canariense, elkanii, iriomotense, japonicum, liaonigense, pachyrhizi and / or yuanmingense), captan, carboxin, chitosan, clothianidin, copper, cyazypyr, difenoconazole,etidiazole, fipronil, fludioxonil, fluoxastrobin, fluquinconazole, flurazole, fluxofenim, harpin protein, imazalil, imidacloprid, ipconazole, isoflavenoids, lipo-chitooligosaccharide, mancozeb, manganese, maneb, mefenoxam, metalaxyl, metconazole, myclobutanil, PCNB, penflufen, penicillium, penthiopyrad, permethrine, picoxystrobin, prothioconazole, pyraclostrobin, rynaxypyr, S-metolachlor, saponin, sedaxane, TCMTB, tebuconazole, thiabendazole, thiamethoxam, thiocarb, thiram, tolclofos-methyl, triadimenol, trichoderma, trifloxystrobin, triticonazole and / or zinc. PCNB seed coat refers to EPA Registration Number 00293500419, containing quintozen and terrazole. TCMTB refers to 2-(thiocyanomethylthio) benzothiazole.

[0202] Seed varieties and seeds with specific transgenic traits may be tested to determine which seed treatment options and application rates may complement such varieties and transgenic traits in order to enhance yield. For example, a variety with good yield potential but head smut susceptibility may benefit from the use of a seed treatment that provides protection against head smut, a variety with good yield potential but cyst nematode susceptibility may benefit from the use of a seed treatment that provides protection against cyst nematode, and so on. Likewise, a variety encompassing a transgenic trait conferring insect resistance may benefit from the second mode of action conferred by the seed treatment, a variety encompassing a transgenic trait conferring herbicide resistance may benefit from a seed treatment with a safener that enhances the plants resistance to that herbicide, etc. Further, the good root establishment and early emergence that results from the proper use of a seed treatment may result in more efficient nitrogen use, a better ability to withstand drought and an overall increase in yield potential of a variety or varieties containing a certain trait when combined with a seed treatment.Methods for killing an insect pest and controlling an insect population

[0203] In some embodiments methods are provided for killing an insect pest, comprising contacting the insect pest, either simultaneously or sequentially, with an insecticidally-effective amount of a recombinant insecticidal polypeptide of interest in conjunction with a multimerization domain polypeptide of the disclosure. In some embodiments methods are provided for killing an insect pest, comprising contacting the insect pest with an insecticidally- effective amount of one or more of a recombinant insecticidal protein of interest in conjunction with a multimerization domain polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity to one or more of SEQ ID NOs: 1, 2, or 8-16, or a variant or active fragment thereof. In other embodiments, methods are provided for killing an insect pest, comprising contacting the insect pest with an insecticidally-effective amount of a recombinant chimeric fusion polypeptide comprising one of more multimerization domain polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more of SEQ ID NOs: 1, 2, or 8-16, one or more linker peptides having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more of SEQ ID NOs: 3-7 or 17, and one or more insecticidal polypeptides of interest, or a variant or active fragment thereof.

[0204] In some embodiments methods are provided for controlling an insect pest population, comprising contacting the insect pest population, either simultaneously or sequentially, with an insecticidally-effective amount of an insecticidal composition comprising one or more multimerization domain polypeptide and an insecticidal polypeptide of interest. In some embodiments, methods are provided for controlling an insect pest population, comprising contacting the insect pest population with an insecticidally-effective amount of an insecticidal composition comprising one or more multimerization domain polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more of SEQ ID NOs: 1, 2, or 8-16, or a variant or active fragment thereof and an insecticidal polypeptide of interest. In other embodiments, methods are provided for controlling an insect pest population, comprising contacting the insect pest population with an insecticidally-effective amount of an insecticidal composition comprising a recombinant chimeric polypeptide comprising one of more multimerization domain polypeptides having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more of SEQ ID NOs: 1, 2, or 8-16, one or more linker peptides having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more of SEQ ID NOs: 3-7 or 17, and one or more insecticidal polypeptides of interest. As used herein, “controlling a pest population” or “controls a pest” refers to any effect on a pest that results in limiting the damage that the pest causes. Controlling a pest includes, but is not limited to, killing the pest, inhibiting development of the pest, altering fertility or growth of the pest in such a manner that the pest provides less damage to the plant, decreasing the number of offspring produced, producing less fit pests, producing pests more susceptible to predator attack or deterring the pests from eating the plant.

[0205] In some embodiments methods are provided for controlling an insect pest population resistant to a pesticidal protein, comprising contacting the insect pest population, either simultaneously or sequentially, with an insecticidally-effective amount of one or more insecticidal polypeptides of interest in conjunction with one or more multimerization domain polypeptide of the disclosure, and optionally one or more of the linker peptides of the disclosure. In some embodiments, methods are provided for controlling an insect pest population resistant to a pesticidal protein, comprising contacting the insect pest population with an insecticidally- effective amount of one or more insecticidal polypeptides of interest in conjunction with one or more multimerization domain polypeptides having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more of SEQ ID NOs: 1, 2, or 8-16, or a variant or active fragment thereof, and optionally one or more of the linker peptides having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more of SEQ ID NOs: 3-7 or 17.

[0206] In some embodiments methods are provided for controlling an insect pest population resistant to a pesticidal protein, comprising contacting the insect pest population, either simultaneously or sequentially, with an insecticidally-effective amount of a recombinant Toxin F polypeptide of the disclosure. In some embodiments, methods are provided for controlling an insect pest population resistant to a pesticidal protein, comprising contacting the insect pest population with an insecticidally-effective amount of a recombinant Toxin F polypeptide of SEQ ID NOs: 67, respectively, or a variant or insecticidally active fragment thereof.

[0207] In some embodiments methods are provided for protecting a plant from an insect pest, comprising expressing in the plant or cell thereof at least one recombinant polynucleotide encoding an insecticidally effective composition comprising one or more multimerization domain polypeptides of the disclosure, optionally a linker peptide of the disclosure, and an insecticidal polypeptide of interest. In some embodiments methods are provided for protecting a plant from an insect pest, comprising expressing in the plant or cell thereof a recombinant polynucleotide encoding an insecticidally effective composition comprising one or more multimerization domain polypeptides having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more of SEQ ID NOs: 1, 2, or 8-16, or variants, optionally one or more linker peptides having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more of SEQ ID NOs: 3-7 or 17, and an insecticidal polypeptide of interest.Insect Resistance Management (IRM) Strategies

[0208] Expression of B. thuringiensis 8-endotoxins in transgenic corn plants has proven to be an effective means of controlling agriculturally important insect pests (Perlak, et al., 1990; 1993). However, in certain instances insects have evolved that are resistant to B. thuringiensis 8- endotoxins expressed in transgenic plants. Such resistance, should it become widespread, would clearly limit the commercial value of germplasm containing genes encoding suchB. thuringiensis 8-endotoxins.

[0209] One way of increasing the effectiveness of the transgenic insecticides against target pests and contemporaneously reducing the development of insecticide-resistant pests is to use non- transgenic (i.e., non-insecticidal protein) refuges (a section of non-insecticidal crops / com) with transgenic crops producing a single insecticidal protein active against target pests. The United States Environmental Protection Agency(epa.gov / oppbppdl / biopesticides / pips / bt_corn_refuge_2006.htm, which can be accessed using the www prefix) publishes the requirements for use with transgenic crops producing a single Bt protein active against target pests. In addition, the National Corn Growers Association, on their website: (ncga.com / insect-resistance-management-fact-sheet-bt-corn, which can be accessed using the www prefix) also provides similar guidance regarding refuge requirements. Due to losses to insects within the refuge area, larger refuges may reduce overall yield.

[0210] Expression of transgenic insecticidal proteins at a high dose, that which kills 99.99% of susceptible insects, will result in greater durability of such insecticidal traits (Tabashnik and Carrier. Nature Biotechnology 35:926. 2017). In one embodiment, chimeric polypeptide compositions comprising a multimerization domain peptide and at least one insecticidal polypeptide of interest that eliminate negative host effects such as phytotoxicity and produce more protein make it possible to produce high dose levels of active ingredient and mitigate insect resistance development.

[0211] Another way of increasing the effectiveness of the transgenic insecticides against target pests and contemporaneously reducing the development of insecticide-resistant pests would be to have a repository of insecticidal genes that are effective against groups of insect pests and which manifest their effects through different modes of action.

[0212] Expression in a plant of two or more insecticidal compositions toxic to the same insect species, each insecticide being expressed at efficacious levels would be another way to achieve control of the development of resistance. This is based on the principle that evolution of resistance against two separate modes of action is far more unlikely than only one. Roush, for example, outlines two-toxin strategies, also called "pyramiding" or "stacking," for management of insecticidal transgenic crops. (The Royal Society. Phil. Trans. R. Soc. Lond. B. (1998) 353: 1777-1786). Stacking or pyramiding of two different proteins each effective against the target pests and with little or no cross-resistance can allow for use of a smaller refuge. The US Environmental Protection Agency requires significantly less (generally 5%) structured refuge of non-Bt corn be planted than for single trait products (generally 20%). There are various ways of providing the IRM effects of a refuge, including various geometric planting patterns in the fields and in-bag seed mixtures, as discussed further by Roush.

[0213] In some embodiments the chimeric polypeptides comprising multimerization domain peptide having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more of SEQ ID NOs: 1, 2, or 8-16, one or more linker peptides having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more of SEQ ID NOs: 3-7 or 17, and one or more insecticidal polypeptides of interest of the disclosure are useful as an insect resistance management strategy in combination (i.e., pyramided) with other pesticidal proteins or other transgenes (i.e., an RNAi trait) including but not limited to Bt toxins, Xenorhabdus sp. or Photorhabdus sp. insecticidal proteins, other insecticidally active proteins, and the like.

[0214] Provided are methods of controlling Lepidoptera and / or Coleoptera and / or Hemiptera insect infestation(s) in a transgenic plant that promote insect resistance management, comprising expressing or co-expressing in the plant at least one of the multimerization domain peptide in conjunction with one or more insecticidal polypeptides of interest to insects in the order Lepidoptera and / or Coleoptera and / or Hemiptera, and optionally at least one of the cleavable linker peptides.

[0215] In some embodiments the methods of controlling Lepidoptera and / or Coleoptera and / or Hemiptera insect infestation in a transgenic plant and promoting insect resistance management comprises the presentation of at least one of the one of the multimerization domain peptides in conjunction with one or more insecticidal polypeptides of interest to insects in the orderLepidoptera and / or Coleoptera and / or Hemiptera, and optionally at least one of the cleavable linker peptides.

[0216] In some embodiments the methods of controlling Lepidoptera and / or Coleoptera and / or Hemiptera insect infestation in a transgenic plant and promoting insect resistance management comprises the presentation of at least one of the multimerization domain peptides having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more of SEQ ID NOs: 1, 2, or 8-16, or variants or active fragments thereof, in conjunction with one or more insecticidal polypeptides of interest to insects in the order Lepidoptera and / or Coleoptera and / or Hemiptera, and optionally at least one of the cleavable linker peptides.

[0217] In some embodiments the methods of controlling Lepidoptera and / or Coleoptera and / or Hemiptera insect infestation in a transgenic plant and promoting insect resistance management comprise expression in the transgenic plant of a multimerization domain peptide having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more of SEQ ID NOs: 1, 2, or 8-16 fused with said insecticidal polypeptide of interest, or variants or insecticidally active fragments thereof and a Cry protein or other insecticidal protein to insects in the order Lepidoptera and / or Coleoptera and / or Hemiptera, where the insecticidal polypeptide of interest and Cry protein have different modes of action.

[0218] Also provided are methods of reducing likelihood of emergence of Lepidoptera and / or Coleoptera and / or Hemiptera insect resistance to transgenic plants expressing in the plants insecticidal proteins to control the insect species, comprising expression of at least one of a multimerization domain peptide having multimerization activity in conjunction with said insecticidal polypeptide of interest to the insect species in combination with a second insecticidal protein to the insect species having different modes of action.

[0219] Also provided are means for effective Lepidoptera and / or Coleoptera and / or Hemiptera insect resistance management of transgenic plants, comprising expressing or co-expressing at high levels in the plants two or more insecticidal proteins or other insecticidal transgenes (e.g., an RNAi trait) toxic to Lepidoptera and / or Coleoptera and / or Hemiptera insects but each exhibiting a different mode of effectuating its killing activity, wherein the two or more insecticidal proteins or other insecticidal transgenes comprise a a multimerization domain peptide and a Cry protein. Also provided are means for effective Lepidoptera and / or Coleoptera and / orHemiptera insect resistance management of transgenic plants, comprising expressing or coexpressing at high levels in the plants two or more insecticidal proteins or other insecticidal transgenes (e.g., an RNAi trait) toxic to Lepidoptera and / or Coleoptera and / or Hemiptera insects but each exhibiting a different mode of effectuating its killing activity, wherein the two or more insecticidal proteins or other insecticidal transgenes comprise a multimerization domain peptide having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more of SEQ ID NOs: 1, 2, or 8-16 fused with said insecticidal polypeptide of interest, or variants or insecticidally active fragments thereof and a Cry protein or other insecticidally active protein.

[0220] In addition, methods are provided for obtaining regulatory approval for planting or commercialization of plants expressing a multimerization domain fused with said insecticidal polypeptide of interest to insects in the order Lepidoptera and / or Coleoptera and / or Hemiptera, comprising the step of referring to, submitting or relying on insect assay binding data showing that multimerization domain peptides having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more of SEQ ID NOs: 1, 2, or 8-16 fused with said insecticidal polypeptide of interest, or variant or insecticidally active fragment thereof does not compete with binding sites for Cry proteins in such insects.Methods for Increasing Plant Yield

[0221] Methods for increasing plant yield are provided. The methods comprise providing a plant or plant cell expressing a polynucleotide encoding multimerization domain peptide fused with said insecticidal polypeptide of interest disclosed herein and growing the plant or a seed thereof in a field infested with a pest against which the insecticidal polypeptide of interest has pesticidal activity. In some embodiments, the multimerization domain fused with said insecticidal polypeptide of interest has pesticidal activity against a Lepidopteran, Dipteran, Hemipteran or nematode pest, and the field is infested with a Lepidopteran, Hemipteran, Dipteran or nematode pest.

[0222] As defined herein, the “yield” of the plant refers to the quality and / or quantity of biomass produced by the plant. “Biomass” as used herein refers to any measured plant product. An increase in biomass production is any improvement in the yield of the measured plant product. Increasing plant yield has several commercial applications. For example, increasing plant leafbiomass may increase the yield of leafy vegetables for human or animal consumption. Additionally, increasing leaf biomass can be used to increase production of plant-derived pharmaceutical or industrial products. An increase in yield can comprise any statistically significant increase including, but not limited to, at least a 1% increase, at least a 3% increase, at least a 5% increase, at least a 10% increase, at least a 20% increase, at least a 30%, at least a 50%, at least a 70%, at least a 100% or a greater increase in yield compared to a plant not expressing the pesticidal sequence.

[0223] In specific methods, plant yield is increased as a result of improved pest resistance of a plant expressing multimerization domain peptide disclosed herein fused with said insecticidal polypeptide of interest. Expression of the multimerization domain peptide disclosed herein fused with said insecticidal polypeptide of interest results in a reduced ability of a pest to infest or feed on the plant, thus improving plant yield.Methods of Processing

[0224] Further provided are methods of processing a plant, plant part or seed to obtain a food or feed product from a plant, plant part or seed comprising multimerization domain peptide. The plants, plant parts or seeds provided herein, can be processed to yield oil, protein products and / or by-products that are derivatives obtained by processing that have commercial value. Nonlimiting examples include transgenic seeds comprising a nucleic acid molecule encoding multimerization domain peptides which can be processed to yield soy oil, soy products and / or soy by-products.

[0225] "Processing" refers to any physical and chemical methods used to obtain any soy product and includes, but is not limited to, heat conditioning, flaking and grinding, extrusion, solvent extraction or aqueous soaking and extraction of whole or partial seeds.

[0226] Embodiments include compositions and methods for mitigating undesirable phenotypic characteristics attributable to the presence of certain transgenic proteins, including insecticidal proteins, when expressed in transgenic plants. Embodiments also include novel methods for increasing protein durability, expression levels, and efficacy in transgenic plants. Embodiments further include novel engineered peptides, polypeptides, and chimeric polypeptides, and methods of producing and using the same.X6

[0227] One embodiment includes a polypeptide comprising an engineered chimeric polypeptide comprising a multimerization domain and an insecticidal polypeptide of interest, wherein the multimerization domain mitigates an undesirable plant phenotype exhibited by the insecticidal polypeptide of interest.

[0228] Another embodiment includes a polypeptide comprising an engineered chimeric polypeptide comprising a multimerization domain and an insecticidal polypeptide of interest, wherein the multimerization domain mitigates an undesirable plant phenotype exhibited by the insecticidal polypeptide of interest, wherein multimerization domain comprises a peptide having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a peptide selected from SEQ ID NOs: 1, 2, or 8-16.

[0229] Another embodiment includes a polypeptide comprising an engineered chimeric polypeptide comprising a multimerization domain and an insecticidal polypeptide of interest, wherein the multimerization domain mitigates an undesirable plant phenotype exhibited by the insecticidal polypeptide of interest, wherein multimerization domain comprises a peptide selected from SEQ ID NOs: 1, 2, or 8-16.

[0230] One embodiment includes a chimeric fusion polypeptide comprising a multimerization domain and an insecticidal polypeptide of interest, wherein the multimerization domain and the insecticidal polypeptide of interest are joined by a cleavable linker sequence.

[0231] Another embodiment includes a chimeric fusion polypeptide comprising a multimerization domain and an insecticidal polypeptide of interest, wherein the multimerization domain and the insecticidal polypeptide of interest are joined by a cleavable linker sequence of any prior embodiment, wherein the chimeric fusion polypeptide exhibits reduced undesirable phenotypic characteristics in a plant when compared to the insecticidal polypeptide without the multimerization domain.

[0232] Another embodiment includes a chimeric fusion polypeptide comprising a multimerization domain and an insecticidal polypeptide of interest, wherein the multimerization domain and the insecticidal polypeptide of interest are joined by a cleavable linker sequence of any prior embodiment, wherein the chimeric fusion polypeptide exhibits increased expression in a plant when compared to the insecticidal polypeptide without the multimerization domain.

[0233] Another embodiment includes a chimeric fusion polypeptide comprising a multimerization domain peptide and an insecticidal polypeptide of interest, wherein the multimerization domain peptide and the insecticidal polypeptide of interest are joined by a cleavable linker sequence, wherein the multimerization domain is capable of interacting with other multimerization domains to form a tetramer, trimer, or a dimer complex.

[0234] Another embodiment includes a chimeric fusion polypeptide comprising a multimerization domain peptide and an insecticidal polypeptide of interest, wherein the multimerization domain peptide and the insecticidal polypeptide of interest are joined by a cleavable linker sequence of any prior embodiment, wherein the multimerization domain peptide has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 1, 2, or 8-16.

[0235] Another embodiment includes a chimeric fusion polypeptide comprising a multimerization domain peptide and an insecticidal polypeptide of interest, wherein the multimerization domain peptide and the insecticidal polypeptide of interest are joined by a cleavable linker sequence of any prior embodiment, wherein the linker sequence has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 3-7 or 17.

[0236] Another embodiment includes a chimeric fusion polypeptide comprising a multimerization domain peptide and an insecticidal polypeptide of interest, wherein the multimerization domain peptide and the insecticidal polypeptide of interest are joined by a cleavable linker sequence of any prior embodiment, wherein the multimerization domain peptide is linked to the insecticidal polypeptide at the N-terminus of the insecticidal polypeptide.

[0237] Another embodiment includes a chimeric fusion polypeptide comprising a multimerization domain peptide and an insecticidal polypeptide of interest, wherein the multimerization domain peptide and the insecticidal polypeptide of interest are joined by a cleavable linker sequence of any prior embodiment, wherein the multimerization domain peptide is linked to the insecticidal polypeptide at the C-terminus of the insecticidal polypeptide.

[0238] One embodiment includes a polynucleotide encoding the chimeric fusion polypeptide of any prior embodiment, wherein the polynucleotide further comprises a heterologous regulatory sequence.

[0239] Another embodiment includes a DNA construct comprising the polynucleotide of any prior embodiment.

[0240] One embodiment includes a plant or plant cell comprising a chimeric fusion polypeptide comprising a multimerization domain peptide and an insecticidal polypeptide of interest, wherein the multimerization domain peptide and the insecticidal polypeptide of interest are joined by a cleavable linker sequence of any prior embodiment.

[0241] Another embodiment includes the plant or plant cell of any prior embodiment, wherein the plant or plant cell further comprises one or more additional polypeptides for insect resistance.

[0242] One embodiment includes a composition comprising a chimeric fusion polypeptide comprising a multimerization domain peptide and an insecticidal polypeptide of interest, wherein the multimerization domain peptide and the insecticidal polypeptide of interest are joined by a cleavable linker sequence of any prior embodiment.

[0243] One embodiment includes a method for reducing undesirable phenotypic characteristics of an insecticidal polypeptide in a plant, the method comprising expressing in the plant a recombinant fusion polypeptide comprising an engineered multimerization domain peptide and an insecticidal polypeptide of interest, wherein the engineered multimerization domain peptide and the insecticidal polypeptide of interest are joined by a linker sequence, and wherein expression of the recombinant fusion polypeptide reduces undesirable phenotypic characteristics of the insecticidal polypeptide of interest compared to an insecticidal polypeptide lacking the engineered multimerization domain peptide. Another embodiment includes a method for reducing undesirable phenotypic characteristics of an insecticidal polypeptide in a plant of any prior embodiment, wherein the undesirable phenotypic characteristic is phytotoxicity.

[0244] Another embodiment includes a method for increasing expression of an insecticidal polypeptide in a plant, the method comprising expressing in a plant a recombinant fusion polypeptide comprising an engineered multimerization domain peptide and an insecticidal polypeptide of interest, wherein the engineered multimerization domain peptide and the insecticidal polypeptide of interest are joined by a linker sequence, wherein the level of expression of the insecticidal polypeptide of interest is increased compared to an insecticidal polypeptide lacking the engineered multimerization domain peptide.

[0245] One embodiment includes a method of increasing efficacy of an insecticidal polypeptide in a plant, the method comprising expressing in a plant a recombinant fusion polypeptide comprising an engineered multimerization domain peptide linked to an insecticidal polypeptide of interest, wherein the level of expression of the fusion polypeptide comprising the engineered multimerization domain peptide linked to the insecticidal polypeptide of interest is increased compared to an insecticidal polypeptide lacking the engineered multimerization domain peptide, and wherein the increased level of the fusion polypeptide results in increased efficacy against a target insect pest.

[0246] One embodiment includes a method of increasing durability of an insecticidal polypeptide, the method comprising expressing in a plant a recombinant fusion polypeptide comprising an engineered multimerization domain peptide linked to an insecticidal polypeptide of interest, wherein the level of expression of the insecticidal polypeptide of interest is increased compared to an insecticidal polypeptide lacking the engineered multimerization domain peptide, and wherein the increased level of the insecticidal polypeptide of interest results in increased durability of the insecticidal polypeptide against a target insect pest.

[0247] One embodiment includes the method of any prior embodiment, wherein the multimerization domain peptide has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 1, 2, or 8-16.

[0248] One embodiment includes the method of any prior embodiment, wherein the linker sequence has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 3-7 or 17.

[0249] Another embodiment includes the method of determining phytotoxicity of an insecticidal protein of interest of any prior embodiment, wherein the reporter gene encodes a fluorescent protein and fluorescence is used to identify viable cells.

[0250] The following examples are offered by way of illustration and not by way of limitation.EXAMPLES

[0251] Some insecticidal proteins that show high efficacy in certain insects may induce undesirable phenotypic effects, such as but not limited to phytotoxic phenotypic effects, when expressed in plants under certain conditions. There is a need for methods to reduce undesirable phenotypic characteristics such as phytotoxicity of insecticidal proteins under certain conditions when expressed in plants. Reduction of undesirable phenotypic characteristics such as phytotoxicity of insecticidal proteins in plants may also allow increased expression of the insecticidal protein in plants, which may lead to increased efficacy against target insects, and may also lead to increased durability of the insecticidal protein. The examples below describe a method of reducing undesirable phenotypic characteristics such as phytotoxicity of insecticidal proteins by fusing a multimerization domain to an insecticidal protein. Fusing a multimerization domain to the insecticidal protein may mitigate undesirable phenotypic characteristics such as phytotoxicity while retaining this desired efficacy in the insect. This approach can be used for many insecticidal proteins.

[0252] In one embodiment, the multimerization of a fusion protein comprising a multimerization domain and an insecticidal protein may sequester the fusion polypeptide molecule into an inactive state by inhibiting its downstream function.

[0253] In another embodiment, the engineered fusion protein (trimerization domain fused to an insecticidal protein) may contain a specific activation site in between the trimerization domain (TMD) and the insecticidal protein, such that activation of the insecticidal protein only happens in the insect but not in the plant, thus maintaining the engineered fusion protein inactive in plants.

[0254] In addition to decreasing phytotoxicity, the trimerization of the insecticidal protein may also enhance the efficacy of the insecticidal protein, for example by increasing local insecticidal protein concentration in the cell and / or by allowing higher levels of expression of the chimeric insecticidal protein. Thus, the engineered fusion protein may mitigate undesirable phenotypic characteristics such as phytotoxicity in the plant and enhance the activity of the insecticidal protein in the insect.Example 1- Fusing a multimerization domain to an insecticidal protein

[0255] Some insecticidal proteins (or toxins) that have efficacy against insects may exhibit undesirable phenotypic characteristics such as phytotoxicity in plants. To mitigate undesirable phenotypic characteristics such as phytotoxicity, an insecticidal protein may be engineered by including a heterologous multimerization domain to modulate the activity of the insecticidal protein specifically in the plant. By fusing a multimerization domain to the insecticidal protein(s), in a non-limiting hypothesis the resulting chimeric molecule may be sequestered in an inactive state. This may be achieved, in non-limiting hypotheses for example, by the multimerization domain preventing interaction of the insecticidal protein(s) with the cognate receptor and / or preventing assembly of an active complex, or by preventing assembly of a pore-forming complex in plants, for example.

[0256] Many viruses use trimerization domains to form spike proteins, needed for receptor recognition. In humans, collagen contains three monomeric helix subunits that come together to form a stable triple helix structure. These monomeric subunits can be fused to a protein of interest, such as an insecticidal protein, to multimerize the chimeric fusion polypeptide monomers, for example. Multimerization domains may further be engineered from existing multimerization domains or be generated de novo by various protein engineering techniques. Monomers may be engineered that can multimerize into dimeric, trimeric, or tetrameric structures, for example. Such engineered monomeric polypeptides, when fused to a protein of interest, may modulate the activity of the protein of interest, such as by reducing the phytotoxic activity of the protein of interest in a plant and / or increasing the insecticidal activity of the protein of interest in the target pest, for example.Example 2- Fusion of a multimerization domain to different toxin classes.

[0257] One or more engineered multimerization domains, for example multimerization domain MMD1, was fused to different toxin classes to produce engineered fusion proteins. In one embodiment, the engineered fusion proteins may contain a protease cleavage site in the linker sequence between the multimerization domain and the toxin (Figure 1). Such a cleavable engineered fusion protein was designed to remain intact in a plant, and once ingested by a target insect, the engineered fusion protein may be proteolytically cleaved in insect gut, therebyactivating the toxin (insecticidal protein) in the insect gut. This engineered fusion protein design may allow the toxin to remain inactive, or in a state having a greatly diminished activity, in a plant and only be activated in the target insect.Example 3- Protein expression, purification, and quantification

[0258] Chimeric fusion proteins were created using the Gibson assembly method. The method involved synthesizing fragments of the MMD, linker (where applicable), and junction sequences of approximately 200 base pairs that corresponded to the flanking regions of the N or C terminus of the toxin and the vector. These fragments were then assembled with a pET28a expression vector containing either an upstream or downstream 6X His tag followed by the protein. The resulting plasmids were transformed into BL21 (DE3) cells from Invitrogen (Waltham, MA). Protein expression was done by using autoinduction media. Cells were allowed to grow in the presence of antibiotics for 4 days at 16 degrees. Pellets were collected and stored at -80 degrees overnight. Pellets were lysed in lysis solution 300mM NaCl, 20mM TRIS-HC1 pH 8, 20mM Imidazole, Omni cleave, Lysozyme and protease inhibitor cocktail. Once the samples were resuspended, pellets were additionally lysed using the homogenizer. The lysate was centrifuged at 4 °C for 25 mins @ 24,000 xg and the cleared lysate was carefully collected.

[0259] Ni-NTA resin was resuspended and washed with buffer (300mM NaCl, 20mM TRIS-HC1 pH 8, 20mM Imidazole). Cleared lysate was incubated with equilibrated resin for 60 mins @ 4°C samples were kept on a mixer to ensure adequate protein binding. Resin was washed with ~50 CVs of wash buffer over the resin to remove non-specific host proteins. Proteins were eluted with 3 CVs of elution buffer (300mM NaCl, 20mM TRIS-HC1 pH 8, 500mM Imidazole). Proteins were dialyzed into IX PBS buffer and stored at 4C (for short term) or -80C (for long term). Samples were run over size exclusion chromatography for cleanup. Protein concentration was obtained by using A280 in the nanodrop. For insect bioassays, the concentration of the proteins was determined by gel densitometry. This was done to disassociate the trimer into monomeric subunits by denaturing the proteins to measure the number of monomers in all samples in comparison to the WT protein.Example 4- Insect Bioassay for Toxin A, a fern-derived toxin, with multimerization domain fusions

[0260] Coleopteran in-vitro feeding assays were conducted using a modified Western com rootworm (Diabrotica virgifera virgifera) WCRW artificial diet (Southland Products Inc., Lake Village, AR) in a 96 well format. The clarified and desalted sample (25 pL) was incorporated with diet (50 pL) and allowed to dry. Control wells were incorporated with 25 pL of IX PBS buffer. Twenty-four-hour WCRW diet-fed larvae were placed into each well. Larvae were allowed to feed for 11 days at 27°C. Insects were visually scored as dead, severely stunted (>60%reduction in size compared to control larvae), or not affected eleven days after infestation. The total number of dead and severely stunted larvae were used to calculate the growth inhibition concentration affecting 50% of the test larvae (IC50) and the total numbers of dead larvae were used to calculate the lethal concentration affecting 50% of the larvae (LC50).

[0261] Multimerization domain 1 (MMD1) was engineered from EML4 PDB (4CGC) and was used to construct an engineered fusion toxin. The MMD1 sequence used here was TSDVQDRLSALESRVQQQEDETVLKAA (SEQ ID NO: 8). Table 12 shows sequences for MMD2 - MMD7.

[0262] Table 1 shows insect feeding bioassay results for in-vitro expressed protein Toxin A, and exemplary engineered fusion proteins. The two components of the fusion proteins (MMD and Toxin) were linked together by a linker selected from one of L1-L5. The LC-50 and IC-50 values are shown in ppm. The LC-50 is the concentration at which 50% of the population experienced mortality while the IC-50 is the concentration at which 50% of the population was affected by mortality and severely stunted growth and / or development. The UCL (Upper Confidence Limit) and LCL (Lower Confidence Limit) show the range of LC-50 / IC-50 values and reflect the data quality. Overall, the fusion proteins were as efficacious, or in some cases, were more efficacious than the WT (Control) protein. For fusion proteins that were expressed in-vitro and tested in the feeding assay, the terminus at which the multimerization domain was added did not change the efficacy compared to other fusion proteins. Linkers LI -L5 have different lengths, flexibility, and, except for LI, all contain a cathepsin proteolytic cleavage site. As shown in Table 1, even fusion protein comprising Toxin A fused to a multimerization domain by linker LI (which is a flexible linker lacking a proteolytic cleavage site) at the C or N terminal of the protein, exhibitedimproved insecticidal activity compared to WT control, suggesting that the target insect may be proteolytically cleaving the toxin near both N- and C- terminus in the gut of the target insect. When engineering the fusion proteins, the length of the linker and the terminal at which the helical domain is engineered, may play an important role. This may depend on the size and insolution behavior of the toxin used.Table 1: Insect feeding assay: feeding Toxin A and engineered fusion proteins to WCR.

[0263] Table Key:

[0264] Toxin A - Control is a control with Toxin A that has not been fused to a multimerization domain.

[0265] Toxin A C-term MMD LI is a fusion polypeptide comprising Toxin A with the multimerization domain fused to the C-terminus of Toxin A linked by an intervening LI linker, for example.

[0266] Toxin A N-term MMD LI is a fusion polypeptide comprising Toxin A with the multimerization domain fused to the N-terminus of Toxin A linked by an intervening LI linker, for example.

[0267] The following table shows the different linkers used. The cathepsin proteolytic cleavage (cut) site is highlighted in bold.Table 2: Linker sequencesExample 5- Phytotoxicity assays for Toxin A with different multimerization domains.

[0268] Chimeric proteins containing different multimerization domains and Toxin A were obtained by gene synthesis. A multimerization domain was fused to the C- or the N-terminus of the toxin protein by a linker selected from L1-L5. The corresponding recombinant polynucleotide encoding the fusion protein were cloned into a transient expression system under the control of the viral promoter dMMV (Dey, et. al., (1999) Plant Mol. Biol. 40:771-782). The Agrobacterium strains containing each of the constructs were infiltrated into leaves as described in Kapila, et. al., (1997) Plant Science 122: 101-108, for example. Briefly, the unifoliate leaves of bush bean (common bean, Phaseolus vulgaris) were agro-infiltrated with normalized bacterial cell cultures of test and control strains. The different transiently expressed constructs comprising Toxin-MMD fusion proteins showed less phytotoxicity than that shown by transiently expressed constructs comprising Toxin A alone under the test conditions (see Table 3).

[0269] Phytotoxicity scoring for all bush bean was assessed based on the following: None-no negative plant phenotype observed, low- some negative plant phenotype observed (bruising / low levels of browning), moderate- significant negative plant phenotype observed (browning tissue on -50% of the leaf), strong- severe negative plant phenotype (tissue death, curling of leaves)

[0270] Transient protein expression of the fusion proteins was confirmed by a mass spectrometry -based protein identification method using extracted protein lysates from infiltrated leaf tissues as described in Patterson et al. (1998) 10(22): 1-24, Current Protocol in Molecular Biology published by John Wiley & Son Inc).Table 3: Phytotoxicity assay in bush bean for chimeric fusion proteins containing Toxin A and multimerization domain.

[0271] Table Key:

[0272] Toxin A - Control is a control with Toxin A that has not been fused to a trimerization domain.

[0273] Toxin A-C-term-MMDl-Ll is a fusion polypeptide containing Toxin A with the trimerization domain fused to the C-terminus of Toxin A linked by an intervening LI linker, for example.

[0274] Toxin A-N-term-MMDl-Ll is a fusion polypeptide containing Toxin A with the trimerization domain fused to the N-terminus of Toxin A linked by an intervening LI linker, for example.

[0275] Table 3 shows the results from a bush bean transient expression experiment and the phytotoxicity observed under test conditions for exemplary fusion constructs comprising a multimerization domain and Toxin A. When Toxin A is fused to a multimerization domain, there is a decrease in the level of phytotoxicity observed under test conditions for most of the fusion constructs in the bush bean transient expression assay. Expression levels for many of the fusion constructs that show a reduction in phytotoxicity is much higher than the expression levels of Toxin A-Control. This indicated that phytotoxicity may be mitigated in the bush bean, which may allow for more protein accumulation in the bush bean. Phytotoxicity caused by Toxin A is greatly reduced from Strong to Low or Moderate, when a helical domain is fused at the C- terminal end of the protein, mediated by a linker, which may further be accompanied by greater levels of fusion protein accumulation, compared to Toxin A -control. The results in Table 3 also show that the linker, and the type of engineered helical domain may also modulate mitigation of phytotoxicity and protein levels in the bush-bean transient expression system.Fusion of a multimerization domain Toxin A reduces the level of phytotoxicity in Soy Platform

[0276] In soy, constructs comprising fusion polypeptides were tested for phytotoxicity mitigation effects. Results shown in Table 4 below. The fusion constructs were tested in two fusion orientations: (i) “Toxin A-N-term-MMDl-L” orientation, which is a fusion polypeptide containing Toxin A with the trimerization domain fused to the N-terminus of Toxin A where the two peptides are linked by an intervening “L” linker selected from one of L1-L5 ; and (ii) “Toxin A-C-term-MMDl-L” orientation, which is a fusion polypeptide containing Toxin A with the trimerization domain fused to the C-terminus of Toxin A where the two peptides are linked by an intervening “L” linker selected from one of L1-L5. As shown in Table 4, all TOXIN-MMDfusion constructs exhibited lower phytotoxicity compared to Toxin A alone under the test conditions.

[0277] Phytotoxicity scoring was assessed based on the following: Low: 1-20% of the disk shows browning / yellowing, Mild / Moderate: 21-60% of the disk shows browning / yellowing, Strong: 61-100% of the disk shows browning / yellowingTable 4: Phytotoxicity assay in soybean for chimeric fusion proteins containing Toxin A and trimerization domain

[0278] Table Key:

[0279] Toxin A - Control is a control with Toxin A that has not been fused to a multimerization domain.

[0280] Toxin A C-term MMD1 LI is a fusion polypeptide containing Toxin A with the multimerization domain (MMD1) fused to the C-terminus of Toxin A linked by an intervening LI linker, for example.

[0281] Toxin A N-term MMD1 LI is a fusion polypeptide containing Toxin A with the multimerization domain (MMD1) fused to the N-terminus of Toxin A linked by an intervening LI linker, for example.

[0282] Table 4 shows the results from the phytotoxicity assay in soy platform. Phytotoxicity score is provided for the phytotoxicity observed under test conditions for fusion constructs comprising a multimerization domain and Toxin A. When Toxin A is fused to the multimerization domain, there is a decrease in the level of phytotoxicity under test conditions observed for most of the fusion constructs that have a multimerization domain fused to Toxin A. For most of the constructs, it is believed that the reduction of phytotoxicity could be attributed to the decrease in protein expression. For the following construct: Toxin A-C-term MMD1 L5 however, phytotoxicity is reduced from strong to low and the level of protein expression is higher than that of the WT control protein. Constructs with the MMD1 multimerization domain and linkers L1-L5 are the same constructs shown in Table 1 but tested in a different assay system. Like in Table 1, the multimerization domain and the linkers were fused to the N- or C- terminus of Toxin A.Fusion of Multimerization domain to Toxin A reduces phytotoxicity and increases insecticidal efficacy in stable corn transformants

[0283] Corn plants were transformed with constructs comprising polynucleotide encoding Toxin A fused to the multimerization domain MMD1 with an intervening linker sequence. As a negative control, com plants were transformed with a construct comprising a polynucleotide encoding Toxin A WT protein (Toxin A - Control). The following linkers were used in this study: LI (SEQ ID NO: 3) - a flexible linker without a cathepsin proteolytic cleavage site;; L5 (SEQ ID NO: 7) - a linker that includes a cathepsin proteolytic cleavage site; and L2 (SEQ ID NO: 4) - a flexible linker with a cathepsin proteolytic cleavage site.Table 5. Shows phytotoxicity and root injury data in stable corn transformants with polynucleotides encoding Toxin A fused to MMD1.

[0284] Protein processing in plants based on western blot analysis indicates the extent of protein processing (proteolytic cleavage of protein) that occurred in root tissue. The results are categorized as follows: (+++): all fusion protein was processed (proteolytically cleaved) to separate MMD1 from Toxin A: (++): Two populations of proteins were present — full-length fusion protein and processed protein; (+): Little to no processing was observed.Table 6. Summarizes the stable com data shown in table 5 above by averaging for each type of fusion polypeptide

[0285] Table 5 and 6 Key:

[0286] Toxin A C-term MMD1-L is a fusion polypeptide containing Toxin A with the MMD1 multimerization domain fused to the C-terminus of Toxin A wherein the two peptides were linked by an intervening linker L (LI, L2, or L5).

[0287] Toxin A-CONTROL is a control expressing only the Toxin A WT gene that has not been fused to a multimerization domain.

[0288] LEAF AREA is a measurement that represents the surface area of a leaf in square centimeters (cmA2) for corn plants. This value is derived by converting the WP SV PIXELAREA, which is a base pixel-counting area. The PIXEL AREA is initially obtained by capturing digital images of the com leaves and using image analysis software to count the number of pixels that correspond to the leaf area. Each pixel represents a small unit of area, and by summing the total number of pixels, we get an initial measurement of the leafs surface area in pixel units. This pixel count is then converted to a standardized unit of measurement, such as square centimeters, using a conversion factor that accounts for the resolution and scale of the images. This conversion allows for standardized comparisons of leaf area across different samples and experiments.CRWNIS (Com RootWorm Nodal Injury Score) is based on the Iowa State 0-3 Node Injury Scale.CRWNISDescription VALUE0.00 No feeding damage (lowest rating that can be given). j 00 One node (circle of roots), or the equivalent of an entire node, eaten back to within approximately V / 2 inches of the stalk (soil line on the 7th node).2.00 Two complete nodes eaten.3.00 Three or more nodes eaten (highest rating that can be given).

[0289] Tables 5 and 6 indicate that the linkers used to link a toxin to a multimerization domain in a chimeric fusion polypeptide may be functionally relevant when engineering a chimeric fusion protein. In the instant test, when no cathepsin proteolytic cleavage site was used in the linker, the fusion protein stayed intact in all tested plant samples. Phytotoxicity caused by Toxin A in these transgenic plants was reduced as indicated by the transgenic plants expressing Toxin A-C-term-MMDl-L chimeric fusion polypeptide having a higher leaf area as compared to the transgenic plants expressing Toxin A without the MMD1 multimerization domain. When a long flexible linker was used that contains a cathepsin proteolytic cleavage site, most of the plants showed proteolytic cleavage of the chimeric fusion polypeptide (processing) resulting in the separation of Toxin A from the multimerization domain. Phytotoxicity in these transgenic plants, in which the chimeric fusion polypeptide was processed, was comparable to the phytotoxicity observed in transgenic plants expressing Toxin A CONTROL. When a short linker comprising a cathepsin proteolytic cleavage site was used, phytotoxicity was reduced as indicated by having a higher leaf area as compared to the transgenic plants expressing Toxin A without the multimerization domain. All fusion constructs and Toxin A - CONTROL showed different levels of protection. It is of note that the transgenic plants expressing a recombinant polynucleotide encoding a chimeric fusion protein (comprising Toxin A and MMD1) showed increased level of the protein in the plant as compared to transgenic plants expressing polynucleotide encoding Toxin A only (Control). The increased chimeric fusion protein levels in the plants likely resulted in better root protection against WCRW compared to control transgenic plants expressing Toxin A only, as indicated by CRWNIS data.Example 6- Insect Bioassay for Toxin B (a non-Bt-derived toxin) with multimerization domain fusions

[0290] Coleopteran in-vitro feeding assays were conducted using a modified Western com rootworm (Diabrotica virgifera virgifera) WCRW artificial diet (Southland Products Inc., Lake Village, AR) in a 96 well format. The clarified and desalted sample (25 pL) was incorporated with diet (50 pL) and allowed to dry. Control wells were incorporated with 25 pL of IX PBS buffer. Twenty-four-hour WCRW diet-fed larvae were placed into each well. Larvae were allowed to feed for 11 days at 27°C. Insects were visually scored as dead, severely stunted (>60%reduction in size compared to control larvae), or not affected eleven days after infestation. The total number of dead and severely stunted larvae were used to calculate the growth inhibition concentration affecting 50% of the test larvae (IC50) and the total numbers of dead larvae were used to calculate the lethal concentration affecting 50% of the larvae (LC50).Table 7: Insect feeding assay: feeding Toxin B and engineered fusion proteins to WCRW

[0291] Table Key:

[0292] Toxin B C-term MMD1 LI is a fusion polypeptide containing Toxin B with the trimerization domain fused to the C-terminus of Toxin B linked by an intervening LI linker, for example.

[0293] Toxin B N-term MMD1 LI is a fusion polypeptide containing Toxin B with the trimerization domain fused to the N-terminus of Toxin B linked by an intervening LI linker, for example.

[0294] Toxin B - Control is a control with Toxin B that was not fused to a trimerization domain.

[0295] Table 7 shows the results for the in-vitro expressed protein Toxin B including the WT and exemplary engineered fusion proteins linked together by a linker, selected from one of LILS, to the MMD1 helical multimerization domain. The LC-50 and IC-50 are shown in ppm. The LC-50 is the concentration at which 50% of the population experienced mortality while the IC- 50 is the concentration at which 50% of the population was affected by mortality and severely stunted growth and / or development. The UCL (Upper Confidence Limit) and LCL (Lower Confidence Limit) show the range of LC-50 / IC-50 values and reflect the data quality. Overall, the fusion proteins exhibited activity in the assay that was similar to the WT proteins. In the case of the fusion protein having linker L3, the fusion protein exhibited increased insecticidal activity compared to that of the WT protein. Table 7 shows the importance of the linker length, flexibility, and the terminus of Toxin B to which the multimerization domain is fused in the fusion protein. When Toxin B is fused to the N-terminal end of the multimerization domain using linker L3, the efficacy is comparable or slightly higher than that of the WT protein; however, when the samemultimerization domain and linker are used to fuse the protein to the C-terminal end, the efficacy is abolished.Example 7- Phytotoxicity assays in bush bean for Toxin B with MMD1 multimerization domain.

[0296] Chimeric proteins containing MMD1 multimerization domains (Helix) and Toxin B were obtained by gene synthesis. The Multimerization domain was added to the C or the N-terminus of the protein fused by linkers mentioned previously (L1-L5) The corresponding genes were cloned into a transient expression system under control of the viral promoter dMMV (Dey, et. al., (1999) Plant Mol. Biol. 40:771-782). The Agrobacterium strains containing each of the constructs were infiltrated into leaves as described in Kapila, et. al., (1997) Plant Science 122: 101-108, for example. Briefly, the unifoliate leaves of bush bean (common bean, Phaseolus vulgaris) were agro-infiltrated with normalized bacterial cell cultures of test and control strains. Phytotoxicity scoring for all bush bean was assessed based on the following: None- no negative plant phenotype observed, low- some negative plant phenotype observed (bruising / low levels of browning), moderate- significant negative plant phenotype observed (browning tissue on -50% of the leaf), strong- severe negative plant phenotype (tissue death, curling of leaves).

[0297] Transient protein expression of the fusion proteins was confirmed by a mass spectrometry -based protein identification method using extracted protein lysates from infiltrated leaf tissues (Patterson, (1998) 10(22): 1-24, Current Protocol in Molecular Biology published by John Wiley & Son Inc).Table 8: Phytotoxicity assay in bush bean for chimeric fusion proteins containing Toxin B and trimerization domain.

[0298] Table Key:

[0299] Toxin B - WT is a control with Toxin B that has not been fused to a trimerization domain.

[0300] Toxin B C-term EML4MMD1 LI is a fusion polypeptide containing Toxin B with the trimerization domain fused to the C-terminus of Toxin B linked by an intervening LI linker, for example.

[0301] Toxin B N-term EML4MMD1 Llis a fusion polypeptide containing Toxin B with the trimerization domain fused to the N-terminus of Toxin B linked by an intervening LI linker, for example.

[0302] When Toxin B is fused to a multimerization domain (MMD1), phytotoxicity of the fusion protein is reduced. However, the reduction of phytotoxicity could be partly caused by the lower fusion protein expression compared to the unfused Toxin B (WT) level in control Bush Bean leaves. Toxin B MMD1 fusion protein levels are greatly reduced when compared to the WT- control protein (Toxin B without MMD1 fusion). Based on the results obtained in Table 3, it is possible that fusing Toxin B to other MMDs may result in higher fusion protein expression in Bush Bean compared to WT Toxin B, which may result in even lower phytotoxicity in bush bean assays.Example 8- Phytotoxicity assay in bush bean for Toxin C (a fern-derived toxin) fusion construct

[0303] Toxin C was fused to MMD1 multimerization domain by a linker L6 (LCAS) containing a CASPASE (CAS) proteolytic site linking Toxin C and MMD1 sequences. Polynucoeotides encoding Toxin C, unfused or fused to MMD1, were cloned into a transient expression system under control of the viral promoter DMMV (Dey, et. al., (1999) Plant Mol. Biol. 40:771-782). The Agrobacterium strains containing each of the constructs were infiltrated into leaves. The agro-infiltration method of introducing an Agrobacterium cell suspension to plant cells of intact tissues so that reproducible infection and subsequent plant derived transgene expression may be measured or studied is well known in the art (Kapila, et. al., (1997) Plant Science 122: 101 -108). Briefly, the unifoliate leaves of bush bean (common bean, Phaseolus vulgaris) were agroinfiltrated with normalized bacterial cell cultures of test and control strains. Leaf discs were excised from each plantlet and infested with four neonates of Com Earworm (CEW; Helicoverpa zea), European Corn Borer (ECB; Ostrinia nubialis), Fall Armyworm (FAW; Spodoptera frugiperda), Southern Armyworm (SAW), and Soybean looper (SBL; Pseudoplusia includens). Leaf discs from a control were generated with Agrobacterium containing only empty expression vector. The consumption of the leaf tissue was scored three days after infestation. Plants were also scored at 4 fays post infiltration for visual negative plant health effects from the expressed protein using a panel of infiltrated control plants with known phenotypes. The scores for leaf damage and phenotype can be seen below.

[0304] The scores for leaf damage and phenotype can be seen below.Table 9: Phytotoxicity and Efficacy in bush bean for chimeric fusion proteins containing Toxin C and MMD1 multimerization domain.

[0305] Table Key:

[0306] Toxin C (core) C-term MMD1 LCAS is a fusion polypeptide containing Toxin C with the trimerization domain fused to the C-terminus of Toxin C where the two peptides are linked by an intervening L linker containing as CASPASE (CAS) cut (cleavage) site.

[0307] Toxin C-(core) is a WT control without a fusion partner.

[0308] Toxin C-(core)-C-term-MMDl-LCAS fusion protein shows higher expression and increased efficacy against FAW, and an overall decrease in leaf damage caused by FAW when compared to the Toxin C-(core) WT control.

[0309] As shown in Table 9, under the testing conditions of this bush bean assay, fusing MMD1 multimerization domain to Toxin C resulted in an increased average concentration of the fusion protein in the bush-bean expression assay and exhibited a reduced level of leaf damage from FAW, compared to expressing Toxin C WT (Toxin C without MMD1). In addition, under the assay conditions, a very mild phytotoxicity was observed when Toxin C was expressed in the plant. When the fusion protein was expressed, no phytotoxicity was observed under the assay conditions. Both phytotoxicity score and efficacy assays were carried out from the same bush bean plant.Example 9 - Phytotoxicity assay in bush bean for TOXIN D (a bacterial-derived toxin)

[0310] Toxin D was tested in the bush-bean platform to examine phytotoxicity. Two different multimerization domains (MMD1 or MMD2) were fused to either the N-terminus or C-terminus of Toxin D with the L5 linker (see Table 7 below). The gene of interest was fused to a multimerization domain by a linker containing a proteolytic site linking the sequences. The corresponding polynucleotides encoding the gene of interest, either unfused or fused to the multimerization domain, were cloned into a transient expression system under the control of the viral promoter DMMV (Dey et al., 1999, Plant Mol. Biol. 40:771-782). Agrobacterium strains containing each of the constructs were infiltrated into leaves using the agro-infiltration method, which introduces an Agrobacterium cell suspension to plant cells of intact tissues to achieve reproducible infection and subsequent plant-derived transgene expression (Kapila et al., 1997, Plant Science 122: 101-108).

[0311] Briefly, the unifoliate leaves of bush bean (Phaseolus vulgaris) were agro-infiltrated with normalized bacterial cell cultures of test and control strains. Leaf discs were excised from eachplantlet and infested with four neonates of various insect pests, including Com Earworm (CEW; Helicoverpa zea), European Corn Borer (ECB; Ostrinia nubialis), Fall Armyworm (FAW; Spodoptera frugiperda), Southern Armyworm (SAW), and Soybean Looper (SBL; Pseudoplusia includens). Leaf discs from a control were generated with Agrobacterium containing only the empty expression vector. The consumption of the leaf tissue was scored three days after infestation. Plants were also scored at four days post-infiltration for visual negative plant health effects from the expressed protein, using a panel of infiltrated control plants with known phenotypes. Table 10 shows the results for average concentration of Toxin D and phytotoxicity measured by assessing leaf damage.Table 10: Phytotoxicity assay in bush bean for chimeric fusion proteins containing Toxin D and trimerization domain.

[0312] Table Key:

[0313] Toxin D C-term MMD1 or MMD2 L5 is a fusion polypeptide comprising Toxin D with the MMD fused to the C-terminus of Toxin D where the two peptides are linked by an intervening L5 linker.

[0314] Toxin D N-term MMD1 or MMD2 L5 is a fusion polypeptide comprising Toxin D with the MMD fused to the N-terminus of Toxin D where the two peptides are linked by an intervening L5 linker.

[0315] Toxin D - WT is a control with Toxin D that has not been fused to a trimerization domain

[0316] Even though phytotoxicity was strong for Toxin D -WT and all fusion proteins under test conditions, the accumulation of this toxin was higher when a fusion protein was expressed illcompared to expression of the unfused Toxin D-WT. It was noted that the accumulation of protein was higher when the multimerization domain was fused to the N-terminal end of Toxin D in comparison to the constructs that had the multimerization domain at the C-terminal end of Toxin D. In one non-limiting interpretation of the results, the presence of the multimerization domain appears to provide lower phytotoxicity from the Toxin D insecticidal protein; this happens perhaps early during plant transfection of the bush bean assay. This may allow the cells to express higher levels of the fusion protein before phytotoxicity was observed under test conditions. Thus, fusing a trimerization domain to the N-terminal end of Toxin D may reduce phytotoxicity of the Toxin D insecticidal protein when compared to the unfused Toxin D insecticidal protein based on total protein accumulation under test conditions. In this example, the phytotoxicity may be reduced further if the level of Toxin D-MMD fusion can be reduced to levels comparable to the Toxin D WT control levels in the plant, perhaps by fusing Toxin D to other MMDs.Example 10- Engineering of new trimerization domains for use in constructing engineered fusion toxins.

[0317] Sequence searches were performed with SEQ ID NO: 8 as the query sequence and using BLASTP tool from the NCBI BLAST+ package (Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, Madden TL. BLAST+: architecture and applications. BMC Bioinformatics, 2009, Dec 15; 10:421. d. An e-value cutoff of 0.001 was used for search strategies. All the remaining parameters were defaults. From the queries of the above- mentioned databases multiple homologs of SEQ ID NO: 8 was identified. The homologs and the organisms they were identified from are shown in Table 11 below. illTable 11: Sequence search results using SEQ ID NO: 8 as the query sequence

[0318] In one non-limiting embodiment, the main factors for engineering a stable trimerization domain are believed to be a distribution of amino acid residues in a polypeptide sequence that allows in a trimer formation where (i) the inward facing amino acid residues are hydrophobic, allowing formation of a hydrophobic core; and (ii) the outward facing amino acid residues allow for electrostatic interactions with adjacent subunits of the trimer. Based on the sequence of SEQ ID NO: 1, different repeats of the following pattern were added within the primary sequence to facilitate trimer formation and stability: every 1st and 4th amino acid residue have a hydrophobic side-chain; every 5th amino acid was replaced with an arginine; and every 7th amino acid was replaced to glutamic acid. The sequences of the engineered MMDs are shown in Table 12 below.Table 12: Engineered multimerization domains (MMDs).Methods for Identifying Cisgenic Sequences for use in Crop Improvement.

[0319] The following is a description of an exemplary workflow for how to identify a nucleotide (or peptide) sequence, or set of sequences, of interest within a plant genome, particularly for the crop plant species or variety intended for agricultural improvement. This method is distinct from conventional sequence search matching, such as typical gene or coding region BLAST searches for homologous genes sequences.

[0320] The methods here envision a situation where one desires to find nucleotide or peptide sequences (or even protein structural predictions) that match closely to that of the query sequence, but for which there is no obvious good or singular sequence match in a genome (such as a direct end-to-end perfect match). These methods envision searching for patches of comparatively short subsequences (i.e. subsequences of the query) in the target (plant, crop) genomic sequences of interest, and regardless of whether they are in recognized gene boundaries or intergenic regions.

[0321] These native crop plant subsequences so found by these methods may then be assembled into the singular unified sequence of interest resembling or even matching the original query. This sequence may be made into a physical nucleotide through various molecular biology techniques. Subsequently this physical polynucleotide may be re-introduced into the (crop) plant, by various means, to achieve in the end a fully functioning reproductively competent crop plant containing in its genome that sequence, most often especially in a manner capable of expression as mRNA and thence protein product.

[0322] Step_l. Target Sequence Identification. The Target Sequence is selected. Usually this may be a particular amino acid sequence. It could also be a specific nucleotide sequence that is desired. Where protein sequences are the desired subject, the nucleotide sequence matches itself may matter less, given the recognized redundant ambiguity of some amino acids’ codons, provided that upon virtual or in-vivo translation of the nucleotide sequence would contribute some portion or up to all of the Target Sequence. This ideotype query Target Sequence, regardless of its origin, will be used to search for related native sequences in the plant and crop species, as described below in steps 3, 4, 5, 6.

[0323] Importantly, the Target Sequence need not be an exact nucleotide match to a sourced sequence, because synonymous codons within may encode for the same peptide. Secondly, the ulTarget Sequence need not encode an exact peptide match either, due to the ability of differing amino acid sequences to encode for peptides having the same or similar physical peptide structure, and like same or similar function. These differing sequences may be referred to as Target Sequence Analogs.

[0324] Accordingly, to various degrees the search criteria, such as those in the examples below, may judiciously proceed in such manner as to allow ambiguities in the search parameters, and in the interpretation of the matching sequences as being useful to arrive at the Target Sequence or Target Sequence Analogs.

[0325] Step_2. Source Plant Species Genomic Sequences. This method requires a Source Organism and its genomics sequences to be known, or at least in part. Source Organism means a Plant Species, which may nor may not presently be recognized as a crop. These methods, however, could be applied to any living organism, single celled or multi-celled, and not restricted to plants. Plant examples are specifically discussed below.

[0326] “Plant Species” is defined to mean a set of plants that are linked by reproductive compatibility with the crop plant species anchoring this set of plant organisms. In one embodiment, each species member of the set is from the same genera, but not necessarily strictly a single recognized species. There may be occasions of inter-generic members in a set. Sexual compatibility among plant species is common, for plant species classifications were not necessarily guided by reproductive isolation, or reproductive compatibility was not known when they were classified, and / or some species were defined at different time and geographies whereof such information was not known. All intra-specific genomic variations (races, lines, inbreds, hybrids, mutants, ecotypes, varieties, subspecies, etc.) are considered conspecific here, part of the same species.

[0327] Genomic Sequences (inclusive of transcriptome or mRNA or CDS sequences) are needed to perform the search. Genomics sequences are now widely available or can be readily produced for a species if presently lacking.

[0328] Step_3. BLAST and related Searches. The BLAST suite of tools is a widely recognized method to search for related nucleotide and peptide tools. Many BLAST parameters can be adjusted to achieve sensitive searches of large genomics and peptide databases.

[0329] Example: For the query sequences “TSDVQDRLSALESRVQQQEDETVLKAA” (SEQ ID NO: 8), using BLAST (at settings Matrix: BLOSUM62 Gap Penalties: Existence: 11, Extension: 1) to search the public rice genome assembly Osa7 (374,471,240 nucleotides), a partial match was identified:Query= “T SD VQDRL S ALESRVQQQEDETVLKAA”Length=27Score ESequences producing significant alignments: (Bits) ValueChr2 26.6 2.1>Chr2Length=35937250Score = 26.6 bits (57), Expect = 2.1, Method: Composition-based stats.Identities = 11 / 17 (65%), Positives = 15 / 17 (88%), Gaps = 0 / 17 (0%)Frame = +2Query 5 QDRLSALESRVQQQEDE 21+DRLSAL+SR ++QE ESbjct 20802773 EDRLSALKSRDKEQEAE 20802823Lambda K H0.301 0.115 0.267GappedLambda K H0.267 0.0410 0.140Effective search space used: 3120592950Database: osalv7_all_conPosted date: Nov 28, 2020 10:46 PMNumber of letters in database: 374,471,240Number of sequences in database: 14Neighboring words threshold: 13

[0330] In this way the sub-sequence of exact match “DRLSAL” (SEQ ID NO: 165) was identified. The sequences here is derived from a location on rice chromosome 2. Similar searches may uncover more sequence relationships across this rice genome or other plant genomes.

[0331] Step_4. Direct Text Pattern Searches. Another method for sequence searching is a direct text string match. This can be enabled by simple scripting languages such as Perl among others.For this method the target sequence can be ‘chopped’ into various overlapping subsequences to initiate the search.

[0332] For example, if one is looking for hexamer matches, one might array out the starting sequence “TSDVQDRLSALESRVQQQEDETVLKAA” (SEQ ID NO: 8), as a series beginning with “TSDVQD” (SEQ ID NO: 166), “SDVQDR” (SEQ ID NO: 167), “DVQDRL” (SEQ ID NO: 168), and so on to the last hexamer “TVLKAA” (SEQ ID NO: 169) in the string. These hexamers subqueries can be searched against the genome in both DNA strands direction of the genomic assembly, and an array of matches will be found. Depending upon the length of the subsequences, one may get a complete net coverage of the original complete starting sequence, in this case “TSDVQDRLSALESRVQQQEDETVLKAA” (SEQ ID NO: 8). Like for the BLAST search, one can create a table of subsequences and their address locations where they were found in the genome assemblies. This can be useful to document the origin of the sequence from the target plant species.

[0333] The advantage of the direct string search is that it can detect very short sequences and exact matches thereof, something BLAST was not designed to chiefly do. (BLAST was rather designed to enable searches of long(er) sequences with some ambiguities or mismatches, something direct string matches is not well suited).Table 13: Cisgenic Sequences identified as additional multimerization domains (MMDs).Example 11- Toxin Fusion Examples

[0334] Various toxin fusion concepts are contemplated herein.Bipolar toxin fusion

[0335] Contemplated herein is a composition and method of modulating activity of a toxin by fusing a first toxin to one end of a multimerization domain, for example the N-terminal end, of amultimerization domain and fusing a second toxin to another end, for example the C-terminal end, of the multimerization domain (see Figure 2).

[0336] In one non-limiting embodiment, the first and the second toxin are the same toxin. In another non-limiting embodiment, the first and the second toxin may be different toxins. For example, the activity of one or more toxins may be modulated by fusing the one or more toxins to a trimerization domain. A bipolar fusion polypeptide may be constructed, for example, by fusing Toxin 1 to the N-terminal end of the trimerization domain, and by fusing Toxin 2 to the C-terminal end of the trimerization domain. In an exemplary embodiment, fusing multiple phytotoxic toxins to one single multimeric domain can provide phytotoxicity mitigation of the multiple toxins by forcing formation of an inactive multimer. Adding a cleavable linker between the toxin and the multimerization domain may provide a mechanism for separating the one or more toxin(s) from the multimerization domain and freeing the one or more toxin(s) to form an active form in a target organism, for example in an insect pest. The cleavage linkers can be different for each of the toxins to alter the rate of activation of each toxin.Bipolar fusion of Toxin E (a fern-derived toxin) and Toxin F (a Cry protein having a 3d-delta endotoxin architecture) in a Bush-Bean assay

[0337] A bipolar fusion construct was created using two insecticidal proteins: Toxin E (SEQ ID NO: 66) and Toxin F (SEQ ID NO: 67). The two insecticidal proteins were linked by two different linkers: LCAS-linker which has a linker and a caspase cleavable site or (No cut) linker, which has just a linker with no caspase cleavable site, and a multimerization domain (MMD4 or MMD7). The corresponding polynucleotides encoding the gene of interest, either unfused or fused to the multimerization domain, were cloned into a transient expression system under the control of the viral promoter DMMV (Dey et al., 1999, Plant Mol. Biol. 40:771-782). Agrobacterium strains containing each of the constructs were infiltrated into leaves using the agro-infiltration method, which introduces an Agrobacterium cell suspension to plant cells of intact tissues to achieve reproducible infection and subsequent plant-derived transgene expression (Kapila et al., 1997, Plant Science 122: 101-108).

[0338] Briefly, the unifoliate leaves of bush bean (Phaseolus vulgaris) were agro-infiltrated with normalized bacterial cell cultures of test and control strains. Leaf discs were excised from eachplantlet and infested with four neonates of various insect pests, including Com Earworm (CEW; Helicoverpa zea), European Corn Borer (ECB; Ostrinia nubialis), Fall Armyworm (FAW; Spodoptera frugiperda), Southern Armyworm (SAW), and Soybean Looper (SBL; Pseudoplusia includens). Leaf discs from a control were generated with Agrobacterium containing only the empty expression vector. The consumption of the leaf tissue was scored three days after infestation. Plants were also scored at four days post-infiltration for visual negative plant health effects from the expressed protein, using a panel of infiltrated control plants with known phenotypes.

[0339] Table 14 shows results of the assay. In summary, the results indicated that under the instant assay conditions, a fusion construct linking Toxin E to Toxin F by means of a multimerization domain in between the two toxins reduces phytotoxicity and increases efficacy against leaf damage caused by FAW and CEW compared to the corresponding unfused Toxin E or Toxin F (WT) controls. The tested fusion construct exhibited efficacy against both FAW and CEW insects, whereas the individual toxins only exhibited efficacy against FAW or CEW. The scores for leaf damage and phenotype for the bipolar toxins can be seen below.Table 14: Phytotoxicity and Efficacy in bush bean for chimeric fusion proteins using the Bipolar fusion concept.

[0340] Table Key:

[0341] Both phytotoxicity score and efficacy test were done from the same transfected Bush Bean plant.

[0342] Toxin E was fused to the N-terminal end of a multimerization domain, and Toxin F was fused to the C-terminal end of the multimerization domain. Two separate fusion constructs were made using two different multimerization domains, namely MMD4 and MMD7.

[0343] A cleavable linker containing a CASPASE L(CAS) cut (cleavage) site between the toxin and the multimerization domain was engineered.

[0344] LCAS is a small linker added at the ends of the CAS sequence: GGSVDVADGGS (SEQ ID No. 17). Constructs lacking a cut (cleavage) site were also created and labeled herein as ‘No cut’. ‘No cut’ indicates that there is no CASPASE cut (cleavage) site. The toxin is fused to the multimerization domain by a linker.

[0345] Toxin F and Toxin E - Control are controls with toxins that were not fused to a multimerization domain.

[0346] In one embodiment, if the toxin protein is processed by the insect in the insect gut, such as by proteolytic cleavage of an endogenous proteolytic cleavage site, then the inclusion of a protease activation cut (cleavage) site in the fusion protein may not be needed. In such case, the toxin can be linked directly by a linker to the multimerization domain without including a protease cleavage site. Further, the placement of the multimerization domain may depend on the location of the endogenous proteolytic cleavage site in the toxin protein. For example, if the endogenous proteolytic cleavage site is located near the N-terminus of the toxin protein, then the multimerization may be fused proximal to the N-terminus of the toxin protein such that the proteolytic cleavage of the endogenous proteolytic cleavage site results in separation of the multimerization domain from the toxin protein.

[0347] Table 14 shows the results of all fusion proteins tested where Toxin E was fused to Toxin F through various linkers. As shown in the table, Toxin E has strong efficacy against CEW, providing good leaf damage protection against CEW but not against FAW, under the instant test conditions. Conversely, Toxin F protects against FAW but not CEW, under the instant test conditions. Both toxins exhibit strong phytotoxicity in bush bean, under the instant test conditions.

[0348] When the toxins are fused through a MMD and a linker, the fusion proteins have insecticidal activity against both CEW and FAW insect pests, demonstrating that the toxins have insecticidal activity against their respective target insects. Additionally, some of the fusion toxins show a reduction in phytotoxicity, decreasing from strong phytotoxicity in unfused Toxin control, to low phytotoxicity when the Toxin is fused to an MMD. Despite reduced protein expression levels for Toxin F, the efficacy and lower phytotoxicity are greater in the fusion proteins for both insects compared to the individual toxins.

[0349] The table also highlights the important role of the engineered helical domain in reducing phytotoxicity. For example, when the LCAS linker, which contains a CASPASE cleavable site, is used, phytotoxicity is alleviated more effectively with the MMD7 multimerization domain compared to the MMD4 multimerization domain. However, when the MMD4 multimerization domain is used without a cleavable linker between Toxin E and the multimerization domain, phytotoxicity is further reduced. This may be due to Toxin E being proteolytically processed further in the insect gut but not in the plant.

[0350] This example underscores the importance of linker length and cleavability when engineering bipolar fusion toxins. The length of the linkers must provide enough flexibility to prevent the proteins from misfolding while retaining some rigidity between the protein and the MMDs such that the cleavable site is not easily accessible to avoid premature processing in the plant.Increasing efficacy of a toxin by increasing local concentration of the toxin by fusing a multimerization domain

[0351] In one embodiment, fusing one or more toxin proteins to a multimerization domain may increase the efficacy of the toxins by increasing their local concentration in the target insect. This can facilitate the formation of an active toxin in the insect gut by increasing the local concentration of the toxin molecules and thereby enhancing the likelihood that the toxin molecules interact with each other to form an active complex, such as by increasing the rate of pore formation. Toxin G (a fern-derived toxin) or Toxin H (a fern-derived toxin) were fused to different MMDs. The two components of the fusion proteins (MMD and Toxin) were linked together by a linker LCAS. The LC-50 and IC-50 values are shown in ppm. The LC-50 is theconcentration at which 50% of the population experienced mortality while the IC-50 is the concentration at which 50% of the population was affected by mortality and severely stunted growth and / or development. The UCL (Upper Confidence Limit) and LCL (Lower Confidence Limit) show the range of LC-50 / IC-50 values and reflect the data quality.Table 15: Insect feeding assay: feeding Toxin G and engineered fusion proteins to FAWTable 16: Insect feeding assay: feeding Toxin H and engineered fusion proteins to FAW

[0352] Table Key:

[0353] Toxin G or Toxin H N-term MMD LCAS is a fusion polypeptide containing Toxin G or Toxin H with different multimerization domains fused to the N-terminus of the toxin where the two polypeptides are linked by an intervening LCAS linker.

[0354] Toxin G - Control is a control with Toxin G that has not been fused to a multimerization domain.

[0355] Toxin H - Control is a control with Toxin H that has not been fused to a multimerization domain.

[0356] In vitro, under certain test conditions, the wildtype (unfused to MMD) versions of Toxin G and Toxin H exhibit low efficacy, as neither toxin in their WT form exhibited a measurable LC50. However, by adding a multimerization domain to the N-terminus of Toxin G or Toxin H, increased efficacy against insects (measured as lower IC50 values) was observed for some of the fusion proteins compared to the WT protein. Under the test conditions, weak LC50 values were observed for many of the fusion constructs, which was an improvement over the WT control. Thus, the overall efficacy increased with the fusion of a multimerization domain to the Toxin G or Toxin H polypeptides tested under these conditions.

[0357] For insect bioassays, the concentration of the proteins was determined by gel densitometry. This involved denaturing the proteins to disassociate the trimer into monomeric subunits, allowing for the measurement of the number of monomers in all samples in comparison to the WT protein.Decreasing activity of a toxin by fusing to a multimerization domain.

[0358] In one embodiment, the modulating activity of a toxin may comprise mitigating phytotoxicity of the toxin, such that the toxin has reduced phytotoxicity when fused to a multimerization domain.Two-component system

[0359] In one embodiment, insecticidal protein Al and insecticidal protein Bl may be part of a two-component system, wherein both components are necessary to form an active complex. Suchtwo-component bipolar fusion construct can provide phytotoxicity mitigation for two different toxins in one fusion construct, where such two-component system may comprise of Toxin A and Toxin B that interact with each other to form an active complex, wherein the two-component insecticidal protein Al and insecticidal protein Bl and the intervening multimerization domain brings the two components in close proximity to each other. Thus, allowing complex formation after at least one of the two toxins is released, for example by proteolytic cleavage of the linker sequence.

[0360] In one non-limiting embodiment, insecticidal protein Al is fused to a MMD at one end of the multimeric domain, and insecticidal protein Bl is fused to the other end of the MMD domain. This allows expression of the two insecticidal proteins in one open reading frame as a fusion polypeptide. The presence of the MMD in between the two toxins may hold the two different toxins trapped in an inactive state modulating phytotoxicity of both toxins in the uncleaved fusion protein, and cleaving of one or both toxins from the fusion protein releasing the component(s) to form an active complex with its counterpart.Binary Toxins.

[0361] This approach can also be used on binary toxins. One component can be fused to one end of a multimerization domain and the other component fused to the opposite end of the multimerization domain. The components may remain separated by the multimerization domain and may only encounter each other after cleavage of a protease cleavage site in the intervening linker sequence, wherein the specific protease is present in the target insect gut but is not present in a plant cell. In this way, toxins can be activated in the target insect gut by a specific cleavable protease site engineered into the linker sequence. In one embodiment, multiple cleavage sites, each specific to a specific enzyme may be engineered into the linker sequence on either side of the multimerization domain to effect different rates of proteolytic cleavage and release of a toxin from an inactive multimeric complex, so that the freed toxin can be activated as an insecticidal toxin. iBreaking a toxin into two components separated by a multimerization domain.

[0362] In another embodiment, a bivalent toxin can also be separated into the two components and fused to either end of a multimerization domain, such that the bivalent toxin may remain separated by the trimer and may only encounter each other after protease cleavage of the linker sequence, release of the two components to interact with each other leading to activation in the insect gut.Dimerization domains.

[0363] In one embodiment, the multimerization domain may comprise a dimerization domain (Figure 4A). For example, a dimerization domain can be used to sequester monomeric toxins in an inactive dimeric conformation. In one non-limiting embodiment, dimer-forming Leucine zippers are comprised of a signature motif containing leucine repeats. They dimerize in solution due to an inner hydrophobic pattern and outer electrostatic interactions. This dimerization domain can be fused to phytotoxic toxins, thus sequestering them in an inactive dimeric conformation. It can also be used to sequester toxins that are dimeric and prevent them from having flexibility in solution. Fusing a leucine zipper to a toxin can prevent the toxin from binding the receptor and / or forming higher oligomeric structure for pore formation. The fusion protein may further comprise an intervening linker sequence and a cleavable site. A dimerization domain may also be engineered to produce a bi-polar fusion similar to the concept discussed above.

[0364] In another embodiment, the dimerization domain can be used to bring together two different proteins where at least one of them is a dimer (see Figure. 5). This may allow two different partner toxins to be delivered within one reading frame. This may also allow the modification of the phytotoxicity profile for the two toxins in plants while allowing the local cell concentration of the two toxins to be higher in the insect midgut cells; perhaps altering the efficacy of the partner toxins.Tetrameric and multimeric protein bundles.

[0365] Helices or any proteins that form multimeric bundles can be used as fusion partner proteins (Figure 4B). The multimeric proteins can be fused to toxins to sequester them in an inactive state and prevent phytotoxicity of multimeric toxins.

[0366] In one embodiment, Toxin D can be fused to the multimerization domain at one end, and Toxin E can be fused at the opposite end of the multimerization domain to reduce phytotoxicity of both toxins. In another embodiment, the toxin may be fused using a linker comprising protease specific sequence, allowing the toxin fusion construct to remain inactive in a plant, whereas cleavage of the protease specific sequence and release of the toxin molecule from the fusion construct, leads to activation of the toxin protein only in the insect gut.

[0367] One example of a tetramer-forming multimerization domain is from the C-terminal helical bundle of a prokaryotic sodium channel that forms a stable tetrameric structure in solution where Arg243 forms a hydrogen bond with Thr239 of the adjacent helix and interacts with Tyr242 by CH / 71 stacking as explained in (Katsumasa et al. 2012 Nat Commun 3: 793-793). Helical domains that form multimeric bundles can be used as fusion proteins to mitigate phytotoxicity. Fusing multimeric domains to phytotoxic toxins may sequester the toxin in an inactive state by bringing multiple units of the toxin close to one another in a manner that inhibits pore formation, for example.Increasing the rate of active complex formation by multimerization domain.

[0368] A fusion protein comprising a multimerization domain and an insecticidal protein can also be used to aid a faster active complex formation. This can be accomplished by bringing two or more subunits of a toxin in proximity to each other for a faster rate of complex formation and by increasing the local protein concentration. For example, an insecticidal protein that functions by forming a pore-forming macromolecular complex may be helped by fusing the subunit to a multimerization domain, resulting in more than one toxin subunit in close proximity to each other, and / or close to the cell surface, which may increase the rate of pore formation. In this nonlimiting embodiment, the engineered fusion protein may not only mitigate phytotoxicity but also enhance the activity of the toxin as shown in Tables 15 and 16. liiLock and key trimerization.

[0369] Contemplated herein are composition and method of engineering a trimerization domain to force hetero-trimer formation. In one embodiment, hetero-trimerization may be achieved, for example, by adding an amino acid residue like tryptophan with a bulky hydrophobic side-chain to one of the monomeric subunit helixes, and adding a small amino acid, like alanine, to the corresponding position of the other two monomeric subunit helixes, wherein the three amino acid residues come in proximity to each other in a trimer, wherein the presence of the tryptophan and the presence of alanine in the other two subunits encourages a hetero-trimer formation, and discourages a homo-trimer formation due to steric clash. In this way, a “lock-and-key" mechanism may be achieved for selective hetero-trimer formation. Consequently, each of the trimerization domains may be fused to a different insecticidal polypeptide, making it possible to bring together two or more different insecticidal polypeptides in close proximity to each other by virtue of hetero-trimerization.Cleavage sites: Trimer activation using insect specific proteolytic cleavage sites.

[0370] To keep the toxin in the inactive state in plants but active in the insect gut, a specific protease recognition / cleavage site can be engineered between the multimerization domain and the toxin of interest. The toxin is fused to the trimeric domain or any other multimeric domain by a linker. Within the linker, a cleavable sequence is introduced specific to the target pest. In one non-limiting embodiment, a cathepsin site (LSQSLSQS; SEQ IDNO: 170) is used for belowground insects, a caspase site (VDVAD; SEQ ID NO: 171) is used for above ground insects. Any cleavable linker specific to the target pest can be used to make the fusion protein activatable only in the target pest. Insect gut specific proteases including but not limited to serine proteases can also be used, which includes trypsin, chymotrypsin and related proteases.

[0371] The cleavage site can be selected to target only target insect pests but not non-target insects; thus, this strategy can be used to re-target a toxin. In one embodiment, such specific targeting / retargeting can be achieved by electing a protease recognition site that is only recognized by a protease that is present only in the target insect but is absent in non-target insects.12SThe toxin may be inactive in the non-target pest and activated in the target insect pest. Using this approach, phytotoxicity may be mitigated and target toxins directed to target insect pests.Linkers and linker length

[0372] For the trimer or multimeric fusion protein to mitigate phytotoxicity and kill the target insect, the toxin should have enough flexibility. In a non-limiting hypothesis, if the components of the fusion product are too close to each other the fusion proteins may aggregate. Flexibility of the linker may also play a role in the functioning of the fusion protein. In one non-limiting embodiment, the linker should preferably be structurally restrictive (rigid) to prevent too much flexibility so that the linker is not cleaved in plants. The linker length may also be important for the stability of the fusion protein. The length and flexibility of the linker may be dependent on the multimerization domain and the toxin fused to it. The linker may be as simple as GGS or it may have some structural characteristics separating and guiding the toxin away from the multimeric bundle (see figure 3). This can be particularly helpful when fusing large toxins.Method for engineering multimeric subunits:

[0373] A design of multiple helix bundles based on geometric structural properties is reviewed by Korendovych IV, DeGrado WF. (Q Rev Biophys. 2020 Feb 11 ;53 :e3). An alpha helix may be engineered that forms trimers in solution by using a known helical amino acid geometrical order. The classical left-handed helix has a seven-residue geometric repeat labeled, ‘abcdefg’. For an alpha helix to form a trimer in solution, every a’ and d’ amino acid is hydrophobic, preferentially: valine or leucine. The pattern does not need to be consistent. For example, there can be a hydrophobic amino acid in the 1st position, in the 8th and 20th position. This may yield a strong hydrophobic core that holds the trimer together in solution. To aid the interactions with neighboring helixes within the trimer, charged amino acids forming salt bridges are alternated within the helical structure; where every e’ amino acid is a positively charged and every g’ amino acid is a negatively charged amino acid. This pattern can also be reversed where every e’ position is a negatively charged amino acid and every g’ is a positively charged amino acid.

[0374] Alpha helical structure may be obtained that form dimers in solution just like the classical leucine zippers, a leucine can be placed in every 7th (g’) position to form a heptad repeat. Thisfacilitates dimerization and maintains a hydrophobic core in the dimer. The mediation of the dimeric structure is also aided by the selectivity of electrostatic interactions with the neighboring alpha helix.

[0375] To obtain a tetrameric helical structure in solution, a simple change of the amino acid valine to isoleucine at the hydrophobic core can yield a selectivity for a tetrameric helical bundle. The structure of the tetrameric bundle is also fortified by the addition of a tryptophan at the middle of the helix that forms CH / K stacking and projects towards the hydrophobic core. This allows the correct stochiometric geometry to select for the formation of a tetramer and not a trimeric structure.Optimization of Fusion Protein Design:

[0376] Contemplated herein is the use of plant tissue extracts as an in vitro diagnostic prediction of fusion protein stability in vivo. This could entail using recombinant fusion proteins generated as mentioned herein and subjected to incubation with plant tissue from the target crop. Construct design optimizations may be made to the following, but not limited to, the toxin se...

Claims

THAT WHICH IS CLAIMED IS:

1. A chimeric fusion polypeptide comprising an insecticidal polypeptide and a heterologous multimerization domain.

2. The chimeric fusion polypeptide of claim 1, wherein the multimerization domain is a trimerization domain.

3. The polypeptide of claim 1 or 2, wherein the chimeric fusion polypeptide exhibits an altered activity compared to an insecticidal polypeptide lacking the heterologous multimerization domain.

4. The polypeptide of claim 3, wherein altered activity is selected from: reduced phytotoxicity in plants, reduced activity in a non-target organism, increased activity in a target organism, and increased expression in a host plant.

5. The polypeptide of claim 1, wherein the insecticidal polypeptide and the heterologous multimerization domain are linked by a linker sequence.

6. The polypeptide of claim 5, wherein the linker sequence comprises at least one protease cleavage site.

7. The polypeptide of claim 6, protease cleavage site is specific to a protease present in Lepidopteran gastrointestinal fluid.

8. The polypeptide of claim 6, protease cleavage site is specific to a protease present in Coleopteran gastrointestinal fluid.

9. The polypeptide of claim 1, wherein the multimerization domain has at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NOs 1, 2, and 8-16.

10. A polynucleotide encoding the chimeric fusion polypeptide of claim 1, wherein the polynucleotide further comprises a heterologous regulatory sequence.

11. A DNA construct comprising the polynucleotide of claim 10.

12. A plant or plant cell comprising the polynucleotide of claim 10.

13. A plant or plant cell of claim 12, wherein the plant or plant cell further comprises an additional polynucleotide sequence encoding a different insecticidal protein.

14. A plant or plant cell of claim 13, wherein the additional polynucleotide sequence encodes a chimeric fusion polypeptide comprising an insecticidal polypeptide and a heterologous multimerization domain.

15. A composition comprising the chimeric fusion polypeptide of claim 1.

16. A method for modifying activity of an insecticidal polypeptide, wherein the method comprises engineering the insecticidal polypeptide to include a heterologous multimerization domain, thereby modifying the activity of the insecticidal polypeptide.

17. The method of claim 16, wherein the modified activity of an insecticidal polypeptide is selected from the group consisting of: reduced phytotoxicity in plants, reduced activity in a nontarget organism, increased activity in a target organism, and increased expression in a host plant.

18. The method of claim 16, wherein the heterologous multimerization domain and the insecticidal polypeptide are linked by a linker sequence.

19. The method of claim 18, wherein the linker sequence comprises at least one protease cleavage site.

20. The method of claim 19, wherein the at least one protease cleavage site is specific to a protease present in Lepidopteran gastrointestinal fluid.

21. The polypeptide of claim 19, wherein the at least one protease cleavage site is specific to a protease present in Coleopteran gastrointestinal fluid.

22. The method of claim 17, wherein the target organism is selected from the group consisting of coleoptera, lepidoptera, and hemiptera.

23. The method of claim 17, wherein the non-target organism is selected from the group consisting of: non-pest insects of corn, soybean, and cotton.

24. A method for modifying an activity of a two-component insecticidal polypeptide system, the method comprising linking a heterologous multimerization domain comprising a first component of the two-component system to one end of the heterologous multimerization domain and linking a second component of the two-component system to the other end of the heterologous multimerization domain, thereby modifying the activity of the two-component insecticidal polypeptide system.

25. The method of claim 24, wherein modified activity of a two-component insecticidal polypeptide system is selected from: reduced phytotoxicity in plants, reduced activity in a nontarget organism, increased activity in a target organism, and increased expression in a host plant, or any combination thereof.

26. The method of claim 24, wherein each one of the first component and second 4component of the two-component insecticidal polypeptide is linked to the intervening heterologous multimerization domain by a linker sequence.

27. The method of claim 26, wherein the linker sequence further comprises at least one protease cleavage site.

28. The method of claim 27, wherein the at least one protease cleavage site is specific to a protease present in Lepidopteran gastrointestinal fluid.

29. The polypeptide of claim 27, wherein the at least one protease cleavage site is specific to a protease present in Coleopteran gastrointestinal fluid.

30. A method of increasing efficacy of an insecticidal polypeptide, the method comprising expressing in a plant an engineered chimeric insecticidal polypeptide linked to a heterologous multimerization domain, wherein the level of expression of the engineered chimeric insecticidal polypeptide is increased compared to an insecticidal polypeptide lacking the heterologous multimerization domain, and wherein the increased level of the engineered chimeric insecticidal polypeptide results in increased efficacy against a target pest.

31. A method of increasing durability of an insecticidal polypeptide, the method comprising expressing in a plant an engineered chimeric insecticidal polypeptide linked to a heterologous multimerization domain, wherein the level of expression of the engineered chimeric insecticidal polypeptide is increased compared to an insecticidal polypeptide lacking the heterologous multimerization domain, and wherein the increased level of the engineered chimeric insecticidal polypeptide increases durability of the insecticidal polypeptide against a target pest.

32. A method of designing a trimerization domain, the method comprising changing the distribution of amino acid residues in a polypeptide sequence that promote trimerization, wherein (i) the inward facing amino acid residues are hydrophobic, allowing formation of a hydrophobic core; and (ii) the outward facing amino acid residues allow for electrostatic interactions with adjacent subunits of the trimer.

33. The chimeric polypeptide of claim 2, wherein the trimerization domain comprises a heptad repeat comprising an amino acid sequence in the order ‘ABCDEFG’, wherein every ‘A’ and ‘D’ amino acid residue comprises a hydrophobic side-chain, and wherein every ‘E’ and ‘G’ amino acid residue comprises a charged side-chain. i