Methods and compositions for reducing undesirable phenotypic traits of insecticidal proteins in plants and / or increasing their persistence in plants

Abstract

This disclosure relates to the field of molecular biology. Novel compositions and methods are provided for mitigating undesirable phenotypic characteristics of certain transgenic proteins, including insecticidal proteins, when expressed in transgenic plants. Novel compositions and methods for increasing protein expression levels in transgenic plants are also provided. Engineered peptides and chimeric fusion peptides, as well as methods for producing and using them, are also contemplated.

Description

[0001] Cross-referencing This application claims the benefit of U.S. Provisional Application No. 63 / 550,748, filed February 7, 2024, the disclosure of which is incorporated herein by reference in its entirety.

[0002] References to sequence lists submitted electronically An official copy of this sequence list, in XML format, was submitted electronically via the Patent Centre under the filename "118742-WO-SEC-1 Sequence Listing.XML" and filed concurrently with this specification. The sequence list contained in this XML file is part of this specification and is incorporated herein by reference in its entirety. Technical Field

[0003] This disclosure relates to the fields of plant genetics and molecular biology. Novel compositions and methods are provided for mitigating undesirable phenotypic traits caused by the presence of certain transgenic proteins, including insecticidal proteins, during expression in transgenic plants. Novel compositions and methods are also provided for increasing protein persistence and expression levels in transgenic plants. Novel compositions and methods are also provided for regulating the activity of engineered proteins. Novel engineered peptides, polypeptides, and chimeric polypeptides, as well as methods for producing and using them, are also contemplated. Background Technology

[0004] The use of microbial agents (such as fungi, bacteria, or other insect species) for the biological control of agriculturally significant insect pests offers an environmentally friendly and commercially attractive alternative to synthetic chemical pesticides. Generally, the use of biopesticides carries a lower risk of pollution and environmental harm, and they offer greater target specificity than traditional broad-spectrum chemical insecticides. Furthermore, biopesticides are often less expensive to produce, thus increasing the economic yield of various crops.

[0005] Certain species of Bacillus microorganisms are known to possess pest-killing activity against a range of insect pests, including those in the orders Lepidoptera, Diptera, Coleoptera, and Hemiptera. Bacillus thuringiensis (Bt) and Bacillus popilliae are among the most successful biocontrol agents discovered to date. Insect pathogenicity is also attributed to strains of Bacillus larvae, Bacillus lentimorbus, Bacillus sphaericus, and Bacillus cereus. Microbial insecticides, particularly those derived from Bacillus strains, play an important role in agriculture as an alternative to chemical pest control. In addition, insecticidal proteins have been discovered in other microorganisms, fungi, and plants, showing promise as agricultural controls for transgenic insect traits.

[0006] Insect-enhanced crops have been developed by genetically engineering them to produce pest-killing proteins from Bacillus, ferns, and other sources. For example, maize and cotton plants have been genetically engineered to produce pest-killing proteins isolated from Bacillus thuringiensis strains. These genetically engineered crops are now widely used in agriculture, providing farmers with an environmentally friendly alternative to traditional insect control methods.

[0007] Nevertheless, some transgenic plants expressing insecticidal proteins may exhibit undesirable phenotypic responses at different developmental stages or under different conditions. For example, Milan et al., in U.S. Patent Application Publication No. 2011 / 0023194, noted that expression of Vip2 in plant cells led to severe developmental pathology and phenotypic alterations in the plant itself. One approach is to engineer constructs and identify transgenic plants that strike a balance between efficacy and agronomy. However, for some insecticidal proteins, this may be difficult to achieve, where even very low levels of expression have undesirable phenotypic effects.

[0008] Therefore, there remains a need for new compositions and methods that involve mitigating undesirable phenotypic traits or undesirable agronomic phenotypes in certain transgenic plants, enhancing the expression of insecticidal peptides and other target transgenic peptides in transgenic plants, and increasing the efficacy and persistence of insecticidal proteins in transgenic plants. Summary of the Invention

[0009] In one aspect, compositions and methods are provided for mitigating undesirable phenotypic characteristics caused by the presence of one or more targeted transgenic peptides (e.g., targeted insecticidal peptides) in transgenic plants. The compositions include peptides and nucleic acid molecules encoding multimerizing domains, chimeric fusion peptides, expression constructs comprising nucleic acid molecules, and host cells and plants comprising the chimeric fusion peptide or expression construct. The compositions also include multimerizing domain peptide sequences, including chimeric peptides comprising multimerizing domain peptides, one or more cleavable linkers, and one or more targeted insecticidal peptides. The compositions further comprise transformed bacteria, plants, plant cells, tissues, and seeds.

[0010] On the other hand, a chimeric polypeptide comprising an insecticidal peptide and a heteropolymerization domain is provided. In one embodiment, a chimeric polypeptide comprising an insecticidal peptide and a trimerization domain is provided. In another embodiment, the insecticidal peptide of the chimeric polypeptide exhibits altered activity compared to an insecticidal peptide lacking a heteropolymerization domain. In a non-limiting embodiment, the altered activity is selected from: reducing phytotoxicity in plants, reducing activity in non-target organisms, increasing activity in target organisms, and increasing expression in host plants.

[0011] On the other hand, a chimeric fusion polypeptide is provided, wherein an insecticidal polypeptide and a heteropolymerized 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, the protease cleavage site is specific to proteases present in Lepidoptera or Coleoptera gastrointestinal fluids.

[0012] Isolated or recombinant nucleic acid molecules capable of encoding chimeric polypeptides comprising a polymerized domain, wherein the polymerized domain has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more sequences selected from SEQ ID NO: 1, 2, or 8-16, and amino acid substitutions, deletions, insertions, fragments thereof, and combinations thereof are provided. Nucleic acid sequences complementary to or hybridized with the nucleic acid sequences of the embodiments are also covered. In another aspect, chimeric fusion polypeptides comprising a polymerized domain having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more sequences selected from SEQ ID NO: 1, 2, or 8-16 are provided.

[0013] On the other hand, DNA constructs are also included. DNA constructs comprising a nucleic acid molecule encoding a chimeric polypeptide comprising a polymerizing domain and an insecticidal polypeptide, and optionally one or more adapters are also provided. In a non-limiting embodiment, a DNA construct comprising a nucleic acid molecule encoding a fusion polypeptide comprising: a polymerizing domain having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more sequences selected from SEQ ID NO: 1, 2, or 8-16; and optionally one or more adapters having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more sequences selected from any one of SEQ ID NO: 3-7 or 17; and one or more targeted insecticidal polypeptides. Plants, plant cells, and microorganisms comprising the DNA constructs described herein are also provided. In another embodiment, plants, plant cells, or microorganisms comprising the DNA constructs described herein are provided, wherein these plants, plant cells, or microorganisms further comprise additional polynucleotide sequences encoding different insecticidal proteins. In another embodiment, the additional polynucleotide sequence encodes a chimeric polypeptide comprising an insecticidal polypeptide and a heteropolymerized domain.

[0014] On the other hand, methods for modifying the activity of insecticidal peptides are provided, wherein these methods include engineering the insecticidal peptide to include a heteropolymerization domain, thereby modifying the activity of the insecticidal peptide. In one non-limiting embodiment, the modified activity of the insecticidal peptide is selected from: reducing phytotoxicity in plants, reducing activity in non-target organisms, increasing activity in target organisms, and increasing expression in host plants. In another non-limiting embodiment, the heteropolymerization domain and the insecticidal peptide are joined or linked by a linker sequence. In one non-limiting embodiment, the linker sequence includes at least one protease cleavage site. In another embodiment, the protease cleavage site is specific to proteases present in the gastrointestinal fluid of Lepidoptera or Coleoptera. In one embodiment, the target organism is selected from Coleoptera, Lepidoptera, and Hemiptera, and the non-target organism is selected from non-harmful insects of row crops, maize, soybean, and cotton.

[0015] In one aspect, a method for modifying the activity of a two-component insecticidal peptide system is provided, the method comprising linking a heteropolymerization domain comprising a first component insecticidal peptide of the two-component system to an N-terminus of the heteropolymerization domain, the method further comprising linking a second component insecticidal peptide of the two-component system to a C-terminus of the heteropolymerization domain, thereby modifying the activity of the two-component insecticidal peptide system. In one non-limiting embodiment, the activity of the modified two-component insecticidal peptide system is selected from: reducing phytotoxicity in plants, reducing activity in non-target organisms, increasing activity in target organisms, or increasing expression in a host plant. In another non-limiting embodiment, each of the first and second components of the two-component insecticidal peptide comprises an intermediate heteropolymerization domain linked 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 for proteases present in Lepidoptera or Coleoptera gastrointestinal fluids.

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

[0017] On the other hand, a method for increasing the persistence of insecticidal peptides is provided, the method comprising expressing in plants an engineered chimeric insecticidal peptide comprising a heteropolymerized domain, wherein the expression level of the engineered chimeric insecticidal peptide is increased compared with that of an insecticidal peptide lacking the heteropolymerized domain, and wherein the increased expression level of the engineered chimeric insecticidal peptide increases the persistence of the insecticidal peptide against the target pest.

[0018] On the other hand, a method is provided for designing trimerizing domains or engineering trimerizing domains, the method comprising altering the distribution of amino acid residues in a polypeptide sequence that promote trimerization, wherein (i) the inward-facing amino acid residues are hydrophobic, thereby enabling the formation of a hydrophobic core; and (ii) the outward-facing amino acid residues are capable of electrostatic interactions with adjacent subunits of the trimer.

[0019] In non-limiting embodiments, compositions and methods for designing / engineering chimeric peptides are provided, the chimeric peptides comprising a trimerizing domain containing a heptapeptide repeat comprising an amino acid sequence in the order 'ABCDEF', wherein each 'A' and 'D' amino acid residue comprises a hydrophobic side chain, and wherein each 'E' and 'G' amino acid residue comprises a charged side chain. In another embodiment, the 'A' and 'D' amino acid residues comprise a hydrophobic amino acid selected from valine or leucine. In yet another embodiment, the trimerizing domain of the foregoing embodiments comprises hydrophobic amino acids at positions 1, 8, and 20, wherein each 'E' amino acid is a positively charged amino acid and each 'G' amino acid is a negatively charged amino acid, wherein this pattern may also be reversed, wherein the 'E' position is a negatively charged amino acid and each 'G' is a positively charged amino acid.

[0020] On the other hand, the compositions and methods of the embodiments can be used to produce organisms with enhanced resistance or tolerance to harmful organisms. These organisms, and compositions comprising them, are desirable for agricultural purposes. Attached Figure Description

[0021] Figure 1 The structure of a trimeric complex is shown, wherein each monomeric subunit comprises a trimerized (or multimerized) domain fused to toxin A via a linker, and optionally a proteolytic cleavage site that can serve as an activation switch. The chimeric fusion protein mediates trimer formation via the helical trimerized domain. The trimeric structure prevents the toxin from forming an active complex and inactivates the toxin. Cleavage at the proteolytic cleavage site releases the toxin from the trimerized domain, thereby forming the active complex. In some embodiments, the proteolytic cleavage site may be endogenously present in toxin A.

[0022] Figure 2 The structure of a trimeric complex is shown, where each monomeric subunit contains a bipolar fusion protein comprising two distinct toxins located at either end of a central trimerized (or multimerized) domain. Toxin B is located at the N-terminus of the fusion protein, and toxin A is located at the C-terminus. Two distinct cleavable linkers may be present, indicated by solid and dashed lines.

[0023] Figure 3 The structure of the trimer complex is shown, wherein each monomeric subunit contains a chimeric fusion protein linked by linkers that prevent unfavorable steric interactions between toxin molecules within the trimer complex. The linkers may further contain proteolytic cleavage sequences, shown by solid lines.

[0024] Figure 4(A) The structure of a dimer complex is shown, wherein each monomer subunit includes a dimerized domain fused to a toxin via a linker, and optionally a proteolytic cleavage site that can be used as an activation switch. (B) The structure of a tetramer complex is shown, wherein each monomer subunit includes a tetramerized domain fused to a toxin via a linker, and optionally a proteolytic cleavage site that can be used as an activation switch.

[0025] Figure 5 The structure of the dimer complex is shown, wherein each monomeric subunit contains a bipolar fusion protein with a dimerizing domain. At one end of the fusion protein, a monomeric toxin (toxin A) is present. At the other end of the protein, a dimer protein (toxin B) is present, wherein each toxin A monomer is fused to a dimerizing domain, which binds to each other in solution to form a dimer. Each end of the dimerizing domain has a cleavable linker. The ability to use the dimerizing domain that forms a dimer in solution can mitigate potential aggregation that might occur from fusing a trimerizing domain with a dimerizing toxin. The stoichiometry of the dimer toxin is maintained by the dimerizing domain. It also allows for the delivery of two different toxins within the same reading frame. The efficacy of each toxin can be enhanced by increasing the local concentration of both toxins near the site of action or cell membrane. Bringing the toxins closer together also reduces the LC50 or IC50 value of the insecticidal protein compared to the unfused WT insecticidal protein. IC50 measurement affects the growth-inhibiting concentration in 50% of the test larvae. LC50 measurement affects the lethal concentration (LC50) of 50% of larvae.

[0026] Figure 6 The structure of a heterotrimer is shown, wherein one polymerizing domain forming the trimer in solution has an amino acid containing a large side chain (e.g., tryptophan), while the other two helices have smaller amino acid residues (e.g., alanine) at corresponding positions to accommodate the large amino acid from the first polymerizing domain, thereby forming the trimer. This can be used as a lock-and-key mechanism, allowing two different toxins to bind at the same end of the trimer in a 2:1 ratio. Heterotrimers or heteropolymers containing at least three different toxins are further considered. Detailed Implementation

[0027] It should be understood that this disclosure is not limited to the specific methods, protocols, cell lines, genera, and reagents described, and therefore variations are possible. It should also 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 this disclosure.

[0028] As used herein, the singular forms “a / an” and “the” include a plural indicator unless the context clearly indicates otherwise. Thus, for example, reference to “a / an cell” includes multiple such cells, and reference to “the protein” includes reference to one or more proteins and their equivalents, and so on. All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains, unless otherwise expressly stated.

[0029] As used herein, the terms “undesired phenotypic traits,” “undesired phenotypes” in plants, or “undesired plant phenotypes” include, but are not limited to, phytotoxicity, low protein production in plants, poor plant growth, undesirable developmental pathology, and undesired phenotypic changes.

[0030] As used herein, the terms "fusion peptide," "fusion polynucleotide," "chimeric peptide," or "chimeric fusion peptide" include, but are not limited to, one or more engineered peptides or one or more polynucleotides encoding one or more engineered peptides, wherein the engineered peptides comprise at least one polymerizing domain and at least one target peptide. In certain embodiments, the target peptide may include a peptide having insecticidal activity. In some embodiments, at least one polymerizing domain and at least one target peptide may be linked by an intermediate linker sequence. In yet other embodiments, the linker sequence may further comprise a proteolytic cleavage (splicing) 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 and expressing them in a translationally fused manner, wherein the two polypeptide sequences are directly translated into a fused form, or joined or linked via an intermediate linker peptide sequence. In one embodiment, the linker peptide sequence may be a linker peptide and / or a polymerized domain peptide. In another embodiment, the linker peptide sequence may contain at least one proteolytic cleavage site. In yet another embodiment, three or more polypeptide sequences may be expressed in a translationally fused manner.

[0032] As used herein, the term "multimerizing domain" refers to a multimerizing domain derived from an organism and which may be modified for use as a component of the chimeric fusion protein considered herein. As used herein, the term "engineered multimerizing domain" refers to a multimerizing domain derived from an organism and engineered or modified (by substituting, adding, or deleting one or more amino acid residues to alter its multimerizing activity) for use as a component of the chimeric fusion protein considered herein. The term "heterogeneous multimerizing domain" refers to a multimerizing domain heterologous to the target polypeptide in the chimeric fusion protein.

[0033] Some insecticidal proteins that exhibit high efficacy in certain insects when expressed in plants under certain conditions may induce undesirable phenotypic effects. The mechanisms underlying the undesirable phenotypic traits triggered by recombinant expression of insecticidal proteins in plants are unclear and likely diverse. Possible mechanisms may include negative epitopes driving undesirable plant phenotypic responses (e.g., by blocking essential plant factors, pore formation, and protein aggregation) and may be specific to each insecticidal protein. In one embodiment, reducing undesirable phenotypic traits in plants (including, but not limited to, phytotoxicity) would enable the delivery of certain insecticidal proteins at high doses 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 mimics, and multivalent inhibitors), which have been reviewed in the literature (Omersa et al. (2019) Toxins [Toxins] (Basel) 11(9):545). These strategies may be difficult to apply to insecticidal traits because they must retain insecticidal activity while mitigating undesirable phenotypic traits and be able to be recombinantly expressed in plants.

[0034] It is desirable to create a universal platform for mitigating undesirable phenotypic characteristics caused by the presence of certain target peptides (e.g., insecticidal proteins), for example, by using a universal multimerization strategy, i.e., by fusing multimerization domains that are independent of the mechanism of the undesirable phenotypic characteristic. This document describes compositions and methods for reducing undesirable phenotypic characteristics, compositions and methods for increasing expression, and compositions and methods for increasing the persistence of insecticidal proteins by fusing the insecticidal protein with at least one multimerization domain.

[0035] This disclosure relates to compositions and methods for mitigating undesirable phenotypic characteristics caused by the presence of one or more target transgenic peptides (e.g., target insecticidal peptides) in transgenic plants. These methods involve transforming organisms with a nucleic acid sequence encoding one or more polymerized domains (fused with one or more target peptides (e.g., target insecticidal peptides)) and optionally one or more adapters. In some embodiments, the adapters may further include proteolytic cleavage sites. Specifically, the nucleic acid sequences of the embodiments can be used to prepare plants and microorganisms with pest-killing activity. Thus, transformed bacteria, plants, plant cells, plant tissues, and seeds are provided. The compositions include nucleic acid molecules encoding engineered chimeric fusion peptides, expression constructs containing the nucleic acid molecules, and host cells containing the constructs. The compositions also include engineered polymerized peptide sequences, including chimeric fusion peptides containing one or more polymerized domains, one or more adapters, and one or more target peptides (e.g., target insecticidal peptides). The compositions also include transformed bacteria, plants, plant cells, tissues, and seeds. Nucleic acid sequences can be used to construct expression vectors that are subsequently transformed into target organisms, and to generate the multi-merging domains or chimeric fusion peptides described herein by utilizing various aspects of certain methods known in the art, such as site-directed mutagenesis, domain exchange, or DNA shuffling.

[0036] Chimeric peptides containing at least one polymerizing domain and at least one targeted insecticidal peptide can be used to control or kill harmful organism populations of Lepidoptera, Coleoptera, Diptera, fungi, Hemiptera and nematodes, and can be used to produce compositions with insecticidal activity. The target insect pests include, but are not limited to: Lepidoptera species, including but not limited to: corn ear moth (CEW, Helicoverpa zea), European corn borer (ECB, Ostrinia nubialis), fall armyworm (FAW, Spodoptera frugiperda), southern armyworm (SAW, Spodoptera eridania), soybean looper (SBL, Pseudoplusia includens), diamondback moth (e.g., corn ear moth (Helicoverpa zea Boddie)), and pear bean looper (VBC, Anticarsia gemmatalis Hübner)); and Coleoptera species, including but not limited to: western corn rootworm (Diabrotica virgifera) - WCRW, southern corn rootworm (Diabrotica undecimpunctata howardi) - SCRW and Northern Corn Rootworm (Diabrotica barberi) - NCRW.

[0037] This disclosure relates to compositions and methods for controlling pests. In one embodiment, these methods involve transforming an organism with a nucleic acid sequence encoding a toxin F polypeptide. Specifically, the nucleic acid sequences of the embodiments can be used to prepare plants and microorganisms with pest-killing activity. Thus, transformed bacteria, plants, plant cells, plant tissues, and seeds are provided. The compositions comprise pest-killing nucleic acids and proteins of plant or bacterial species. In one embodiment, the nucleic acid sequence can be used to construct an expression vector subsequently transformed into a target organism, as a probe for isolating other homologous (or partially homologous) genes, and for generating a modified toxin F polypeptide by utilizing multiple aspects of certain methods known in the art, such as directed mutagenesis, domain exchange, or DNA shuffling.

[0038] In one embodiment, the toxin F polypeptide can be used to control or kill populations of Lepidoptera, Coleoptera, Diptera, fungi, Hemiptera, and nematodes, and can be used to produce compositions with pest-killing activity. Target insect pests include, but are not limited to: Lepidoptera species, including but not limited to: the corn ear moth (CEW), the European corn borer (ECB), the fall armyworm (FAW), the soybean looper (SBL), and the diamondback moth (e.g., the corn ear moth); and the pear bean looper, such as the pear bean looper; and Coleoptera species, including but not limited to: the western corn rootworm (WCRW), the southern corn rootworm (SCRW), and the northern corn rootworm (NCRW).

[0039] The terms “insecticide polypeptide” or “insecticide protein” or “toxin” are used herein to refer to a toxin that is toxic to one or more pests. For example, pests may include members of the orders Lepidoptera, Coleoptera, Diptera, Hemiptera, or Nematoda, or proteins that are homologous to such proteins. Target insecticidal proteins have been isolated from organisms and plant species, including but not limited to species of the genera *Bacillus*, *Bacillus thuringiensis* (“Bt”), *Pseudomonas*, *Photorhabdus*, *Xenorhabdus*, *Clostridium bifermentans*, and *Paenibacillus popilliae*. Plant species include but are not limited to species of the genera *Selaginella*, *Polystichum*, *Adiantum*, *Coniogramme*, *Davallia*, *Didymochlaena*, *Humata*, *Onoclea*, and *Tectaria*.

[0040] Methods for utilizing polymerized domains and chimeric polypeptides containing said polymerized domains in transgenic plants The multimerizing domains (MMDs) and chimeric peptides containing them described herein can be used to reduce undesirable plant phenotypes in transgenic plants expressing target peptides (e.g., target insecticidal proteins). The multimerizing domains and chimeric fusion peptides containing them described herein can also be used to improve the expression and efficacy of target peptides, and further to increase the persistence of certain target insecticidal proteins in plants.

[0041] In one embodiment of the non-limiting hypothesis, the mechanisms by which plants respond to certain target peptides (e.g., insecticidal proteins) to produce undesirable phenotypic traits (e.g., but not limited to phytotoxicity) are complex and may include, but are not limited to, biochemical changes (e.g., lipid peroxidation, enzyme inactivation, cell damage or death, and disruption of membrane and ion homeostasis); molecular changes (e.g., chromosomal aberrations, DNA damage, altered cell division, and gene regulation); and physiological changes (e.g., membrane disturbances, altered stomatal aperture, reduced photosynthesis, chlorosis, necrosis, and effects on reproductive growth).

[0042] In one embodiment, the presentation of undesirable plant phenotypes resulting from the expression of one or more target peptides can be reduced by associating one or more polymerizing domains with one or more target peptides (e.g., target insecticidal peptides), and can be used in conjunction with one or more adapter sequences. The one or more polymerizing domains can be the same or different polymerizing domains, and can be used in conjunction with one or more adapter sequences, and may further include cleavable sites.

[0043] In one embodiment, the first chimeric fusion polypeptide can be expressed or co-expressed in a plant with different target chimeric polypeptides, wherein the first fusion polypeptide comprises a polymerizing domain and an insecticidal polypeptide, and wherein the second fusion polypeptide comprises a polymerizing domain and an insecticidal polypeptide.

[0044] In another embodiment, the polymerized domain can be expressed in plants as a fusion protein with a target transgenic polypeptide (e.g., a target insecticidal polypeptide). In some embodiments, fusion proteins are provided comprising one or more target polypeptides fused to a peptide of the polymerized domain, represented by a formula selected from the following: R 1 -LM 1 M 1 -LR 1 R 1 -M 1 M 1 -R 1 R 1 -LM 1 -LR 2 R 2 -LM 1 -LR 1 R 1 -M 1 -R 2 R 2 -M 1 -R 1 M 2 -R 1 -M 1 Wherein R1 is the target transgenic peptide (e.g., the target insecticidal peptide), and R2 is the second target transgenic peptide (e.g., the target insecticidal peptide), and M1 is a multi-domain peptide, and M2 is the same multi-domain peptide as M1 or alternatively, a second multi-domain peptide. The R1 peptide is fused to the M1 peptide directly or via a linker (L) segment. The term "directly" defines the fusion of peptides in the absence of a peptide linker. Therefore, "L" represents the chemically bound or peptide segment to which R1 and M1 are fused within the frame. In an exemplary embodiment, L is a linear peptide to which R1 and M1 are bound via amide bonds, wherein the amide bonds connect the carboxyl terminus of R1 to the amino terminus of L, and the carboxyl terminus of L to the amino terminus of M1, respectively.

[0045] "Intra-frame fusion" means there is no translation termination or interruption between the reading frames of R1 and M1. The linker group (L) is typically a polypeptide of 1 to 500 amino acids in length. The linker connecting the two molecules is preferably designed to (1) allow the two molecules to fold and function independently of each other, (2) not have a tendency to develop ordered secondary structures that could interfere with the functional domains of the two polypeptides, (3) have minimal hydrophobic or charged features to interact with the functional polypeptide domains, and (4) provide spatial separation of R1 and M1. The linker typically consists of a flexible loop or flexible residue or ring at the ends and a rigid core region in the middle for separating R1 from M1. Typically, the surface amino acids in the flexible protein region include Gly, Asn, and Ser. In fact, any arrangement of amino acid sequences containing Gly, Asn, and Ser is expected to satisfy the above criteria for a flexible loop. In some cases, other neutral amino acids (e.g., Ala) may also be used for flexible loop linker sequences. The rigid core region in the linker typically has a helical content that can be designed using modern computational tools. Residues that tend to form helices include Met, Ala, Leu, Glu, and Lys. Additional amino acids may also be included in the linker because a unique restriction site is added to the linker sequence to facilitate the construction of the fusion complex. In one embodiment, the linker has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with a sequence selected from SEQ ID NO: 3-7. In another embodiment, the linker is selected from SEQ ID NO: 3-7.

[0046] In some embodiments, the linker or a portion thereof comprises a sequence selected from the group consisting of (Gly3Ser)n, (Gly4Ser)n, (Gly5Ser)n, (GlynSer)n, or (AlaGlySer)n, where n is an integer. An example of a highly flexible linker is a (GlySer)-rich spacer region present in the pIII protein of filamentous bacteriophages (e.g., phage M13 or fd) (Schaller et al., 1975). Linkers also include those containing endopeptidase recognition sequences (e.g., plasmin, enterokinase, kallikrein, urokinase, tissue plasminogen activator, clostridium protease, rennet, collagenase, Russell's viper venom protease, postproline cleavage enzyme, V8 protease, thrombin, and factor Xa). In some embodiments, the linker comprises amino acids from a multigene expression vector (MGEV) (see International Patent Application Publication No. WO 2007 / 137329), which is cleaved by a vacuole protease. In other embodiments, peptide linker segments from the hinge regions of heavy chain immunoglobulins IgG, IgA, IgM, IgD, or IgE provide angular relationships between the attached polypeptides. Hinge regions where cysteine ​​residues are replaced with serine residues are particularly useful. Linkers of this disclosure include endogenous recognition sequences and derivatives of digestive endopeptidases obtained from crop pests, including the insects disclosed herein. Linkers of this disclosure may include sequences derived from the hinge region of mouse IgG γ2b, wherein cysteine ​​residues have been replaced with serine residues. Fusion proteins are not limited by the form, size, or number of linker sequences used, and the only requirement for the linkers is that they do not adversely interfere with the folding and function of the fused molecules.

[0047] Methods for evaluating the role of multi-merging domains Various assays can be used to assess the mitigation of undesirable plant phenotypes by binding one or more polymerized domains to one or more target peptides (e.g., one or more insecticidal peptides), or by expressing a chimeric fusion peptide comprising a polymerized domain peptide, one or more linkers, and one or more target insecticidal peptides.

[0048] For example, the reduction of undesirable plant phenotypes induced by transgenic or insecticidal activity (e.g., but not limited to phytotoxicity) can be assessed visually or spectrophotometrically by growing transgenic plants or transgenic plant cells or tissues containing a polymerized domain peptide (fused to one or more transgenic or insecticidal proteins) and comparing growth parameters at certain time points with those of appropriate control plants (expressing the same transgene but without the polymerized domain peptide) and non-transgenic blank controls. Similarly, a decline in plant health can be assessed visually or spectrophotometrically in a substantially similar manner. Screening options capable of quantifying expression, phytotoxicity, and insecticidal efficacy include, but are not limited to: maize protoplast assays, dwarf bean transient assays, and crop transient and stability assays.

[0049] Transgenic expression can be assessed using similar experimental methods, such as Western blotting, ELISA, mass spectrometry, Octet, SPR, FRET, and other protein quantification methods to determine the level of transgenic expression.

[0050] Conversion efficiency can be assessed, for example, using one or more rapid maize determination methods disclosed in, such as, U.S. Patent Application Publication No. 2017 / 0121722 (which is incorporated herein by reference in its entirety).

[0051] Methods for generating multipolymerized domains and connectors A variety of multimerization domains and linkers were considered. Multimerization domain peptides can be directly selected from the target peptide-protein fusion library. These multimerization domain peptides can enhance the expression of the target transgenic polypeptide, reduce undesirable phenotypic responses (such as phytotoxicity phenotypes), or alter (i.e. enhance) protein accumulation levels in transgenic plants or appropriate alternatives. As used herein, a “multiplexed domain” or “multiplexed domain peptide” refers to a peptide that, when expressed in a plant as an N-terminal and / or C-terminal fusion partner (linked to a target transgenic polypeptide (e.g., a target insecticidal protein)), has at least one of the following effects: mitigation of one or more undesirable phenotypic responses (e.g., but not limited to phytotoxic phenotypic responses) induced by the expression of the target transgenic polypeptide (e.g., a target insecticidal protein); improvement of the agronomic phenotype of transgenic plants expressing the target polypeptide (e.g., a target insecticidal protein); increase in the expression of one or more target heterologous polypeptides (e.g., a target insecticidal protein) in plants; increase in the accumulation of target heterologous polypeptides (e.g., a target insecticidal protein) in plants; increase in the persistence of one or more target heterologous polypeptides (e.g., a target insecticidal protein) in plants; and / or alter the accumulation level of target heterologous polypeptides (e.g., a target insecticidal protein) in plants. The target protein may be, for example, a protein (e.g., an insecticidal protein) transgenically expressed in plant cells. In a particular embodiment, the multimerization domain may be a dimerization domain, a trimerization domain, or a tetramerization domain.

[0052] Direct screening for undesirable phenotypic reductions and / or expression improvements can be performed in plants, plant cells, plant tissues, yeast, or other alternative assays (such as those disclosed herein). Structural modeling information can be used to select or design peptides with different sequences, positions, lengths, and linker sequences for testing in appropriate assays. Peptides that increase or decrease the conformational stability of specific proteins can be isolated. Many insecticidal proteins are known to undergo significant conformational changes upon activation within the digestive tract of target pests. Restricting these conformations to those adapted to the phenotype of healthy plants may be ideal.

[0053] Engineering modification of polymerized domains, linker peptides and their variants In one embodiment, this disclosure covers certain engineered polymerized domain peptides. In some embodiments, the engineered polymerized domain peptide comprises, substantially comprises, or alternatively comprises: an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more sequences selected from SEQ ID NO: 1, 2, or 8-16, and variants thereof, and engineered polymerized domain peptides having sufficient homology with any one or more of SEQ ID NO: 1, 2, or 8-16. In one embodiment, this disclosure covers certain polymerized domains. In some embodiments, the polymerized domain peptide comprises, or alternatively comprises: an amino acid sequence of any one or more of SEQ ID NO: 1, 2, or 8-16, and variants thereof, and polymerized domain peptides having sufficient homology with any one or more of SEQ ID NO: 1, 2, or 8-16. In another embodiment, this disclosure covers certain cleavable linker peptides. In some embodiments, the adaptor peptide comprises, or alternatively consists of, the amino acid sequence of any one or more of SEQ ID NO: 3-7 or 17, and variants thereof, and the adaptor peptide having sufficient homology with any one or more of SEQ ID NO: 3-7 or 17.

[0054] As used herein, “sufficient identity” or “sufficient homology” refers to an amino acid sequence that, using standard parameters and one of the alignment procedures described herein, 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 with respect to a reference sequence. In some embodiments, sequence homology refers to the full-length sequence of an engineered polymerized domain peptide, polymerized domain peptide, or linker peptide. In some embodiments, the engineered polymerized domain polypeptide, polymerized 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 with any of SEQ ID NO: 1, 2, 8-16 or 3-7, 17, or 67. When used herein with respect to percentage of sequence identity, the term “about” means + / - 0.5%. Those skilled in the art will recognize that these values ​​can be appropriately adjusted to determine the corresponding homology of proteins, taking into account amino acid similarity, etc. In some embodiments, sequence identity is calculated using the ClustalW algorithm in the ALIGNX® module of the Vector NTI® program suite (Invitrogen Corporation, Carlsbad, California) with all default parameters. In some embodiments, sequence identity across the full-length polypeptide is calculated using the ClustalW algorithm in the ALIGNX® module of the Vector NTI® program suite (Invitrogen Corporation, Carlsbad, California) with all default parameters.

[0055] As used herein, the terms “protein,” “peptide,” “peptide molecule,” or “polypeptide” include any molecule comprising five or more amino acids. It is well known in the art that protein, peptide, or polypeptide molecules can be modified, including post-translational modifications such as, but not limited to, disulfide bond formation, glycosylation, phosphorylation, or oligomerization. Therefore, as used herein, the terms “protein,” “peptide,” “peptide molecule,” or “polypeptide” include any protein modified by any biological or non-biological process. The terms “amino acid” and “multiple amino acids” refer to all naturally occurring L-amino acids.

[0056] In this article, “recombinant protein” is used to refer to a protein that is no longer in its natural environment (e.g., in vitro or in recombinant bacterial or plant host cells).

[0057] As used herein, “substantially free of cellular material” refers to a polypeptide comprising a protein formulation having less than about 30%, 20%, 10%, or 5% (on a dry weight) of non-toxic biohazard proteins (also referred to herein as “contamination proteins”).

[0058] The term "fragment" or "bioactive moiety" includes polypeptide fragments containing an amino acid sequence substantially identical to that of a polymerizing domain peptide or a linker peptide, and exhibiting polymerizing activity or linker peptide activity as described above. The "fragment" or "bioactive moiety" of a polymerizing domain or linker peptide includes a fragment containing an amino acid sequence substantially identical to one or more sequences selected from SEQ ID NO: 1, 2, 8-16, 3-7, or 17, having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, wherein the polymerizing domain peptide or linker peptide exhibits polymerizing activity or linker peptide activity, respectively. Such bioactive moieties can be prepared using recombinant techniques, and the polymerizing activity or linker peptide activity can be evaluated. In some embodiments, the polymerized domain peptide or linker peptide fragment is a 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 NO: 1, 2, 8-16 or 3-7 or 17, for example by proteolysis, by inserting a start codon, or by deleting a codon encoding the missing amino acid while simultaneously inserting a start codon. In some embodiments, the polymerized 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, or 20 amino acids from the N-terminus of any one of SEQ ID NO: 1, 2, 8-16, 3-7, or 17. In some embodiments, the polymerized domain peptide or linker peptide fragment is a N-terminal and / or 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, or 20 or more amino acids from the N-terminus and / or C-terminus of any one of SEQ ID NO: 1, 2, 8-16, 3-7, or 17.

[0059] In one embodiment, the "fragment" or "bioactive portion" comprises a polypeptide fragment containing an amino acid sequence substantially identical to that of the toxin F polypeptide and exhibiting insecticidal activity. The "fragment" or "bioactive portion" of the toxin F polypeptide comprises a fragment containing an amino acid sequence substantially identical to that of SEQ ID NO: 67, wherein the toxin F polypeptide has insecticidal activity. Such bioactive portions can be prepared using recombinant techniques and their insecticidal activity can be evaluated. In some embodiments, the toxin F polypeptide fragment is a truncated N-terminus and / or C-terminus 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, for example by proteolysis, by inserting a start codon, by deleting a codon encoding the missing amino acid and simultaneously inserting a start codon, and / or inserting 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 a N-terminal and / or 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 of SEQ ID NO: 67.

[0060] As used herein, “variant” refers to a protein or polypeptide 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 that of its parent.

[0061] In some embodiments, the polymerized 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 with the full length or fragment of the amino acid sequence of any one of SEQ ID NO: 1, 2, 8-16, wherein the polymerized domain peptide has polymerizing activity.

[0062] In some embodiments, the polymerized domain peptide or linker peptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more sequences selected from any one or more of SEQ ID NO: 1, 2, 8-16, 3-7, or 17, and having substituted 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids compared to the amino acids at the corresponding positions in any one or more of SEQ ID NO: 1, 2, 8-16, 3-7, or 17.

[0063] In some embodiments, sequence identity across the full-length peptide is calculated using the ClustalW algorithm in the ALIGNX® module of the Vector NTI® program suite (Ingenie, Carlsbad, CA) with all default parameters.

[0064] Methods for such operations are generally known in the art. For example, amino acid sequence variants of polymerizing domain peptides or linker peptides can be prepared by mutations in DNA. This can also be accomplished by one of several forms of mutagenesis, such as site-specific double-strand break techniques and / or directed evolution. In some respects, the changes encoded in the amino acid sequence will have substantially no effect on the function of the protein. Such variants will possess the desired polymerizing or linker activity. However, it should be understood that the ability to confer polymerizing activity or other peptide physical properties by using such techniques on the compositions of this disclosure can be improved or altered.

[0065] Conservative amino acid substitutions can be made at one or more predicted non-essential amino acid residues. A "non-essential" amino acid residue is a residue that can be altered without changing its biological activity. A "conservative amino acid substitution" is a substitution 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); amino acids with acidic side chains (e.g., aspartic acid, glutamic acid); amino acids with polar, negatively charged residues and their amides (e.g., aspartic acid, asparagine, glutamic acid, glutamine); amino acids with non-polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine); and amino acids with small aliphatic, nonpolar, or slightly polar residues (e.g., alanine, serine, threonine, proline). Amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); amino acids with large aliphatic, nonpolar residues (e.g., methionine, leucine, isoleucine, valine, cysteine); amino acids with β-branched side chains (e.g., threonine, valine, isoleucine); amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine); amino acids with large aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan).

[0066] Amino acid substitutions can occur in non-conserved regions that retain function. Typically, such substitutions are not targeted at conserved amino acid residues or amino acid residues within a conserved motif, where such residues are essential for protein activity. Examples of conserved residues that may be essential for protein activity include, for example, residues that are identical across all proteins included in alignments with sequence-similar or related peptides of the examples (e.g., identical residues in homologous protein alignments). Examples of conserved residues that allow for conserved amino acid substitutions and still retain activity include, for example, residues with only conserved substitutions across all proteins included in alignments with sequence-similar or related peptides of the examples (e.g., only conserved substitutions across all proteins in homologous protein alignments). However, those skilled in the art will understand that functional variants may have minor conserved or non-conserved changes in conserved residues.

[0067] When making such changes, the hydrophilicity index of the amino acid can be considered. The importance of the hydrophilic amino acid index in conferring interactive biological functions to proteins is generally understood in the field (Kyte and Doolittle, (1982) JMol Biol. [Journal of Molecular Biology], 157(1):105-32). The acceptance of the relative hydrophilicity of the amino acid contributes to the secondary structure of the resulting protein, which in turn defines the protein's interactions with other molecules, such as enzymes, matrix, receptors, DNA, antibodies, antigens, etc.

[0068] It is known in the art that certain amino acids can be substituted with other amino acids having similar hydrophilicity indices or scores, and still produce proteins with similar biological activities, i.e., proteins that still achieve biological functional equivalence. Each amino acid is assigned a hydrophilicity index (Kyte and Doolittle, ibid.) based on its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine ​​(+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); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). When making such changes, it is preferred to use amino acid substitutions with a hydrophilicity index of +2 or less, particularly preferred to use amino acid substitutions with a hydrophilicity index of +1 or less, and even more particularly preferred to use amino acid substitutions with a hydrophilicity index of +0.5 or less.

[0069] It is also understood in the art that similar amino acid substitutions can be made efficiently based on hydrophilicity. U.S. Patent No. 4,554,101 illustrates that the maximum local average hydrophilicity of a protein (e.g., governed by the hydrophilicity of its adjacent amino acids) is related to the biological properties of the protein.

[0070] As detailed in U.S. Patent No. 4,554,101, the following hydrophilicity values ​​have been assigned to the amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0, +0.1); glutamic acid (+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).

[0071] Alternatively, the amino or carboxyl termini of the polymerized domain peptide or linker peptide sequence can be altered without substantially affecting activity. This can include insertions, deletions, or alterations introduced by modern molecular methods such as PCR, including PCR amplification that alters or elongates the protein-coding sequence due to the presence of amino acid-coding sequences in the oligonucleotides used in the PCR amplification.

[0072] The variant nucleotide and amino acid sequences disclosed herein also encompass sequences derived from mutagenesis and recombination procedures, such as DNA shuffling. In the case of such procedures, one or more different multimerized domain peptide or linker peptide coding regions can be used to generate novel engineered multimerized domains or linker peptides with desired properties. In this manner, recombinant polynucleotide libraries are generated from a population of related sequence polynucleotides containing sequence regions having basic sequence identity and capable of homologous recombination in vitro or in vivo. For example, using this method, sequence motifs encoding a target domain can be shuffled between pest-killing genes and other known pest-killing genes to obtain new genes encoding proteins with improved target properties, such as increased insecticidal activity. Such DNA shuffling strategies are known in the art. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 91:10747-10751; Stemmer (1994) Nature [Nature] 370:389-391; Crameri et al. (1997) Nature Biotech. [Nature Biotechnology] 15:436-438; Moore et al. (1997) J. Mol. Biol. [Journal of Molecular Biology] 272:336-347; Zhang et al., (1997) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 94:4504-4509; Crameri et al., (1998) Nature [Nature] 391:288-291; and U.S. Patent Nos. 5,605,793 and 5,837,458.

[0073] In some embodiments, engineered polymerized domain peptides or linker peptides possess modified physical properties. As used herein, the term "physical property" refers to any parameter suitable for describing the physicochemical characteristics of a protein. As used herein, "target physical property" and "target property" are used interchangeably and refer to the physical properties of the protein being studied 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 distribution of hydrophobic residues on the protein surface, surface charge density, surface hydrophobic density, total count of ionized groups on the surface, surface tension, protein size and its distribution in solution, melting temperature, heat capacity, and second in-situ force coefficient. Examples of physical properties also include the digestibility of polymerized domain peptides or linker peptides with proteolytic fragments in the insect gut. Models simulating gastric digestion are known to those skilled in the art (Fuchs, RL and JD Astwood. Food Technology 50: 83-88, 1996; Astwood, JD et al., Nature Biotechnology 14: 1269-1273, 1996; Fu TJ et al., J. Agric Food Chem. 50:7154-7160, 2002).

[0074] In some embodiments, engineered polymerized domain peptides or adapter peptides are provided. In one embodiment, the polymerized domain peptide or adapter peptide comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more sequences selected from any one or more of SEQ ID NO: 1, 2, 8-16, 3-7, or 17.

[0075] In some embodiments, chimeric multi-domain polypeptides are provided that comprise regions of at least two different multi-domains of the present disclosure.

[0076] In some embodiments, chimeric polypeptides are provided, comprising at least two different insecticidal polypeptides linked to a polymerizing domain. In other embodiments, chimeric fusion polypeptides are provided, comprising: at least one polymerizing domain having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a peptide selected from SEQ ID NO: 1, 2, or 8-16; and at least one adapter peptide having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more sequences selected from SEQ ID NO: 3-7 or 17. In another embodiment, the chimeric fusion polypeptide further comprises at least one target polypeptide, for example, at least one target insecticidal polypeptide, the chimeric fusion polypeptide comprising: at least one polymerized domain peptide having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more sequences selected from SEQ ID NO: 1, 2, or 8-16; and at least one adapter peptide having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more sequences selected from any one of SEQ ID NO: 3-7 or 17.

[0077] In another embodiment, fusion proteins are provided, the amino acid sequences of which include amino acid sequences containing the polymerized domain peptide and adaptor peptide of this disclosure. In one embodiment, the polymerized domain peptide and adaptor peptide may comprise additional fusion with an insecticidal peptide or other target peptide. Methods for designing and constructing fusion proteins (and the polynucleotides encoding them) are known to those skilled in the art. The polynucleotides encoding the polymerized domain peptide and optionally the adaptor peptide may be fused with signal sequences that will guide the fusion peptide to be located in a specific compartment of a prokaryotic or eukaryotic cell, and / or guide the embodiment to be secreted from a prokaryotic or eukaryotic cell.

[0078] For example, in *Escherichia coli*, one might wish to direct the expression of polymerized domain peptides into the periplasmic space using, but not limited to, pelB signal sequences (such as the pelB pectic acid lyase signal sequence), maltose-binding protein (MBP) signal sequences, MBP, ompA signal sequences, signal sequences of the periplasmic *Escherichia coli* heat-labile enterotoxin B subunit, and alkaline phosphatase signal sequences. See also the commercially available pMAL series vectors (particularly the pMAL-p series) from New England Biolabs (Ipswich, MA).

[0079] Plant plasmid transport peptide / peptide fusions are known in the art. Apoplast transport peptides, such as rice or barley α-amylase secretion signals, are also known in the art. Plasmid transport peptides are typically fused to the N-terminus of a target peptide (e.g., a fusion partner). In one embodiment, the fusion protein may comprise, or alternatively consist of, the plasmid transport peptide, the target polymerized domain peptide, optional linker peptide, and the target peptide. In such embodiments, the plasmid transport peptide is preferably located at the N-terminus of the fusion protein, but may include additional amino acid residues at the N-terminus of the plasmid transport peptide. In specific embodiments, the plasmid transport peptide is located at the N-terminal half, N-terminal third, or N-terminal quarter of the fusion protein. When a plasmid is inserted, most or all of the plasmid transport peptide is typically cleaved from the fusion protein. In one embodiment, the plasmid transport peptide cleavage site may be uniform, or alternatively may differ by 1-10 amino acids. The plasmid transport peptide can be recombinantly fused to a second protein in one of several ways. For example, restriction endonuclease recognition sites can be introduced into the nucleotide sequence of the transport peptide corresponding to its C-terminus, and identical or compatible sites can be engineered to the nucleotide sequence of the protein to be targeted at its N-terminus. Care must be taken to design these sites to ensure that the coding sequences of the transport peptide and the second protein remain “in the box” to allow for the synthesis of the desired fusion protein. In some cases, when introducing new restriction sites, it is preferable to remove the initiation factor methionine of the second protein. Introducing restriction endonuclease recognition sites on two parent molecules, and their subsequent ligation via recombinant DNA technology, can result in the addition of one or more additional amino acids between the transport peptide and the second protein. This generally does not affect targeting activity, as long as the transport peptide cleavage site remains accessible, and the addition of these additional amino acids at its N-terminus does not alter the function of the second protein. Alternatively, those skilled in the art can use gene synthesis (Stemmer et al., (1995) Gene [Gene] 164:49-53) or similar methods to generate precise cleavage sites between the transport peptide and the second protein (with or without its initiator methionine). Additionally, transport peptide fusions can intentionally include amino acids downstream of the cleavage site. The amino acids at the N-terminus of a mature protein can affect the ability of a transport peptide to target the protein to the plasmid and / or the cleavage efficiency after protein input. This may depend on the protein to be targeted. See, for example, Comai et al., (1988) J. Biol. Chem. 263(29):15104-9.

[0080] Nucleic acid molecules and their variants and fragments Provided are isolated or recombinant nucleic acid molecules comprising nucleic acid sequences encoding polypeptides or linker peptides or their biologically active portions, as well as nucleic acid molecules sufficient to be used as hybridization probes for identifying nucleic acid molecules encoding proteins having sequence homology regions. As used 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), as well as DNA or RNA analogs produced using nucleotide analogs. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA.

[0081] The term “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, such as in vitro or in heterologous recombinant bacterial or plant host cells. In some embodiments, when produced by recombinant technology, the isolated nucleic acid molecule or its biologically active portion is substantially free of other cell material or culture medium, or when synthesized chemically, it is substantially free of chemical precursors or other chemicals. The isolated nucleic acid does not contain sequences naturally located flanking the nucleic acid in the genomic DNA of the organism from which it is derived (i.e., sequences located at the 5' and 3' ends of the nucleic acid) (preferably protein-coding sequences). The term “recombinant” nucleic acid molecule (or DNA) is used herein to refer to a nucleic acid sequence (or DNA) in recombinant bacterial or plant host cells. In some embodiments, “isolated” or “recombinant” nucleic acids do not contain sequences naturally located flanking the nucleic acid in the genomic DNA of the organism from which it is derived (i.e., sequences located at the 5' and 3' ends of the nucleic acid) (preferably protein-coding sequences). For the purposes of this disclosure, “isolated” or “recombinant” when used to refer to nucleic acid molecules excludes isolated chromosomes. For example, in various embodiments, the recombinant nucleic acid molecule encoding engineered scaffold polymerized domain peptides and linker peptides may contain nucleic acid sequences of 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, which are naturally located on the flanking side of the nucleic acid molecule in the cellular genomic DNA from which the nucleic acid is derived.

[0082] In some embodiments, the isolated nucleic acid molecules encoding polymerized domain peptides and adaptor peptides have one or more variations in their nucleic acid sequences compared to the natural or genomic nucleic acid sequences. In some embodiments, the alterations to the natural or genomic nucleic acid sequences include, but are not limited to: nucleic acid sequence alterations due to the degeneracy of the genetic code; nucleic acid sequence alterations due to amino acid substitutions, insertions, deletions, and / or additions compared to the natural or genomic sequences; removal of one or more introns; deletion of one or more upstream or downstream regulatory regions; and deletion of 5' and / or 3' untranslated regions associated with the genomic nucleic acid sequence. In some embodiments, the nucleic acid molecules encoding polymerized domain peptides are non-genomic sequences.

[0083] Various polynucleotides encoding engineered multi-domain peptides and multi-domain proteins (with or without linker peptides) or related proteins were considered. When operatively linked to suitable promoter sequences, transcription termination sequences, and / or polyadenylation sequences, such polynucleotides can be used to generate engineered multi-domain peptides in host cells, for example, as multi-domain peptide fusion proteins. Such polynucleotides can also be used as probes to isolate homologous or substantially homologous polynucleotides encoding related proteins.

[0084] Polynucleotides encoding multipolymerized domains or linker peptides One source of polynucleotides encoding multi-domain peptides, adaptor peptides, or related proteins is a multi-domain polynucleotide encoding a multi-domain peptide having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more sequences selected from SEQ ID NO: 1, 2, 8-16, 3-7, or 17. These polynucleotides can be used to express multi-domain peptides as chimeric polypeptides.

[0085] Polynucleotides encoding multi-domain polypeptides or fusion polypeptides (including chimeric polypeptides as described herein) can also be synthesized de novo from engineered multi-domain polypeptide or chimeric polypeptide sequences. The sequence of the polynucleotide gene can be deduced from the multi-domain chimeric polypeptide sequence using the genetic code. Computer programs such as BackTranslate (GCG™ package, Aclair, San Diego, CA) can be used to convert peptide sequences into corresponding nucleotide sequences encoding peptides. Examples of multi-domain polypeptide sequences that can be used to obtain corresponding nucleotide-encoding sequences include, but are not limited to, multi-domain polypeptides of SEQ ID NO: 1, 2, or 8-16. Furthermore, the multi-domain fusion polynucleotide sequences of this disclosure can be engineered for expression in plants.

[0086] In some embodiments, the nucleic acid molecule encoding the multimerging domain polypeptide is a polynucleotide, a variant thereof, a fragment thereof, or a complementary sequence thereof encoding the polypeptide sequence shown in any one of SEQ ID NO: 1, 2, or 8-16. In one embodiment, the nucleic acid molecule encoding the multimerging domain polypeptide is selected from any one of SEQ ID NO: 89-97.

[0087] In this paper, "complementary sequence" refers to a nucleic acid sequence that is fully complementary to a given nucleic acid sequence, such that it can hybridize with the given nucleic acid sequence to form a stable double strand. "Polynucleotide sequence variant" refers to a nucleic acid sequence that encodes the same polypeptide except for the degeneracy of the genetic code.

[0088] In some embodiments, the nucleic acid molecule encodes a polymerized domain polypeptide or variant comprising one or more amino acid substitutions for an amino acid sequence of any one of SEQ ID NOs: 1, 2, or 8-16. In other embodiments, the nucleic acid molecule encodes an adapter sequence comprising one or more amino acid substitutions for an amino acid sequence of any one of SEQ ID NOs: 3-7 or 17. In one embodiment, the nucleic acid molecule encoding the polymerized domain polypeptide is selected from any one of SEQ ID NOs: 84-88 and 98.

[0089] Nucleic acid molecules encoding transcription and / or translation products are also provided, which are subsequently spliced ​​to ultimately produce functional multimerging-domain peptides or chimeric peptides. Splicing can be performed in vitro or in vivo and can involve cis or trans splicing. The substrates used for splicing can be polynucleotides (e.g., RNA transcripts) or peptides. An example of cis splicing of polynucleotides is the excision of introns in the inserted coding sequence and the splicing of exon regions on both sides, thereby generating a multimerging-domain peptide or fusion peptide coding sequence, such as a chimeric peptide containing a multimerging domain, a linker peptide, and a target insecticidal peptide. An example of trans splicing is the encryption of polynucleotides by separating the coding sequence into two or more fragments, which can be transcribed individually and then spliced ​​to form a full-length pest-killing coding sequence. The use of splicing enhancer sequences that can be introduced into the construct can facilitate cis or trans splicing of the peptide. Therefore, in some embodiments, the polynucleotide does not directly encode a full-length multimerging-domain polypeptide or fusion polypeptide, but rather encodes one or more fragments of a multimerging-domain polypeptide or chimeric polypeptide. These polynucleotides can be used to express functional multimerging-domain or chimeric polypeptides (e.g., fusions with a target insecticidal polypeptide and a linker peptide) via mechanisms involving splicing, where splicing can occur at the polynucleotide level (e.g., intron / exon) and / or the polypeptide level (e.g., intron / exon). This can be used, for example, to control the expression of pest-killing activity, because if all the necessary fragments are expressed in an environment that allows the splicing process to generate a functional product, only the multimerging domain or the chimeric polypeptide containing the multimerging-domain peptide is expressed. In another example, introducing one or more insert sequences into a polynucleotide can facilitate recombination with low-homology polynucleotides; the use of introns or introns targeting the insert sequences facilitates the removal of the intercalated sequences, thereby restoring the function of the encoded variant.

[0090] Nucleic acid molecules that are fragments of these nucleic acid sequences encoding multi-domain peptides are also covered in the examples. As used herein, a “nucleotide fragment” refers to a portion of a nucleic acid sequence encoding a multi-domain peptide. A nucleotide fragment of a nucleic acid sequence may encode the biologically active portion of the multi-domain peptide, or it may be a fragment that can be used as a hybridization probe or PCR primer using the methods disclosed below. Nucleic acid molecules that are fragments of nucleic acid sequences encoding multi-domain peptides comprise at least about 21, 24, 27, 30, 33, 36, 39, 45, 60, 75, 90, 120, 150, 180, 210, 240, 270, or 300 consecutive nucleotides, or up to the number of nucleotides present in the full-length nucleic acid sequence encoding the engineered multi-domain peptides disclosed herein, depending on the intended use. “Consecutive nucleotides” herein refers to nucleotide residues that are adjacent to each other. The nucleic acid sequence fragments of the examples will encode protein fragments that retain the biological activity of the multi-domain peptide and thus retain the activity of the multi-domain. "Retained polymerization 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 any of the full-length polymerization domain peptides shown in SEQ ID NO: 1, 2 or 8-16.

[0091] The "sequence identity percentage (%)" relative to the reference sequence (topic sequence) is determined as the percentage of amino acid residues or nucleotides in the candidate sequence (query sequence) that are identical to the corresponding amino acid residues or nucleotides in the reference sequence, after alignment and the introduction of gaps (if necessary) to achieve the maximum sequence identity percentage, and without considering any conserved substitutions of amino acids as part of sequence identity. Alignments performed for determining the sequence identity percentage can be performed in various ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment across the full length of the sequences being compared. The identity percentage between two sequences is a function of the number of common positions shared by the sequences (e.g., identity percentage of the query sequence = number of common positions between the query and topic sequences / total number of positions in the query sequence × 100).

[0092] In some embodiments, the polymerized domain polynucleotide encodes a polymerized 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 over the entire length of the amino acid sequence of SEQ ID NO: 1, 2, or 8-16. In some embodiments, the polymerized domain polynucleotide encodes a polymerized 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 over the entire length of the amino acid sequence of any one of SEQ ID NO: 1, 2, or 8-16. In some embodiments, the 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 over the entire length of the amino acid sequence of any one of SEQ ID NO: 3-7 or 17.

[0093] In one embodiment, a polynucleotide sequence is considered that has 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 over the entire length with respect to the polynucleotide sequence of any one of SEQ ID NO: 84-164.

[0094] In another embodiment, a polypeptide sequence is considered that has 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 over its entire length with the polypeptide sequence of any one of SEQ ID NO: 18-83.

[0095] The examples also cover nucleic acid molecules encoding variants of multi-domain peptides. “Variants” of nucleic acid sequences encoding multi-domain polypeptides or peptides include those sequences encoding the multi-domains disclosed herein but which exhibit conserved differences due to the degeneracy of the genetic code, as well as those sequences that are sufficiently identical as discussed above. Naturally occurring allelic variants can be identified using well-known molecular biology techniques, such as polymerase chain reaction (PCR) and hybridization techniques outlined below. Variant nucleic acid sequences also include synthetically derived nucleic acid sequences that have been generated, for example, by site-directed mutagenesis, but still encode the disclosed multi-domain peptides discussed below.

[0096] This disclosure provides isolated or recombinant polynucleotides encoding any of the polymerized domains or linker peptides disclosed herein. Those skilled in the art will readily understand that, due to the degeneracy of the genetic code, there are many nucleotide sequences encoding the polymerized domains or linker peptides disclosed herein.

[0097] Those skilled in the art will further understand that alterations can be introduced through mutations in nucleic acid sequences, for example, by introducing one or more nucleotide substitutions, additions, and / or deletions into the corresponding nucleic acid sequences disclosed herein, thereby resulting in changes to the amino acid sequence of the encoded polymerized domain peptide without altering the protein's biological activity. Mutations can be introduced using standard techniques, such as directed mutagenesis and PCR-mediated mutagenesis. Such variant nucleic acid sequences are also covered by this disclosure.

[0098] Alternatively, variant nucleic acid sequences can be prepared by randomly introducing mutations into all or part of the coding sequence (e.g., through saturation mutagenesis), and the obtained multi-domain peptide mutants can be screened for activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be recombinantly expressed, and the activity of the multi-domain peptide can be determined using assays disclosed herein or known in the art. The polynucleotides and fragments thereof disclosed herein are optionally used as substrates for various recombination and recursive recombination reactions, in addition to standard cloning methods described, such as those by Ausubel, Berger, and Sambrook, i.e., to generate additional multi-domain peptide homologs and fragments thereof with desired properties. Variant polynucleotide libraries thus generated, cells containing said libraries, and any recombinant polynucleotides generated by such methods are also embodiments of this disclosure. Furthermore, such methods optionally include selecting variant polynucleotides from such libraries based on the activity of the multi-domain peptide, as in which such recursive recombination is performed in vitro or in vivo.

[0099] Various diversity generation schemes, including nucleic acid recursive recombination schemes, are available and well described in the art. These procedures can be used alone and / or in combination to generate one or more variants of nucleic acids or sets of nucleic acids, as well as variants of the proteins they encode. Individually and holistically, these procedures provide robust and widely applicable methods for generating diverse nucleic acids and sets of nucleic acids, including, for example, nucleic acid libraries, which can be used for, for example, the engineering or rapid evolution of nucleic acids, proteins, pathways, cells, and / or organisms with novel and / or improved characteristics.

[0100] Descriptions of various diversity-generating procedures for producing modified nucleic acid sequences (e.g., those encoding polypeptides or fragments thereof with engineered polymerizing or cytotoxic activity) can be found in the following publications and references cited therein: Soong et al., (2000) Nat Genet [Nature Genetics] 25(4):436-439; Stemmer et al., (1999) Tumor Targeting [Tumor Targeting] 4:1-4; Ness et al., (1999) Nat Biotechnol [Nature Biotechnology] 17:893-896; Chang et al., (1999) Nat Biotechnol [Nature Biotechnology] 17:792-697; Minshull and Stemmer, (1999) Curr Opin Chem Biol [Contemporary Perspectives on Biochemistry] 3:284-290; Christians et al., (1999) Nat Biotechnol [Nature Biotechnology] 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) J Mol Biol 255:373-386; Stemmer, (1996) "Sexual PCR and Assembly PCR" is published 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.

[0101] Mutation methods that generate diversity include, for example, 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 (edited by Eckstein and Lilley, Springer Verlag) [Springer Publishing Company, Berlin]); mutagenesis using templates containing uracil (Kunkel, (1985) PNAS USA [Proceedings of the National Academy of Sciences of the United States of America] 82:488-492; Kunkel et al., (1987) Methods Enzymol [Enzymological Methods] 154:367-382 and Bass et al., (1988) Science [Science] 242:240-245); oligonucleotide directed mutagenesis (Zoller and Smith, (1983) Methods Enzymol [Enzymological Methods] 100:468-500; Zoller and Smith, (1987) Methods Enzymol [Enzymological Methods] 154:329-350 (1987); Zoller and Smith, (1982) Nucleic Acids Research [Nucleic Acids Research] 10:6487-6500); DNA mutagenesis modified with phosphate thioester (Taylor et al., (1985) Nucl Acids Res [Nucleic Acid Research] 13:8749-8764; Taylor et al., (1985) Nucl Acids Res [Nucleic Acid Research] 13:8765-8787 (1985); Nakamaye and Eckstein, (1986) Nucl Acids Res [Nucleic Acid Research] 14:9679-9698;Sayers et al., (1988) Nucl Acids Res [Nucleic Acid Research] 16:791-802 and Sayers et al., (1988) Nucl Acids Res [Nucleic Acid Research] 16:803-814; mutagenesis using nicked double-stranded DNA (Kramer et al., (1984) Nucl Acids Res [Nucleic Acid Research] 12:9441-9456; Kramer and Fritz, (1987) Methods Enzymol [Enzymatic Methods] 154:350-367; Kramer et al., (1988) Nucl Acids Res [Nucleic Acid Research] 16:7207 and Fritz et al., (1988) Nucl Acids Res [Nucleic Acid Research] 16:6987-6999).

[0102] Other 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 Soc Lond A 317:415-423), and mutagenesis via whole-genome synthesis (Nambiar et al., (1984) Science). 223:1299-1301; Sakamar and Khorana, (1988) Nucl Acids Res [Nucleic Acid Research] 14:6361-6372; Wells et al., (1985) Gene [Gene] 34:315-323 and Grundström et al., (1985) Nucl Acids Res [Nucleic Acid Research] 13:3305-3316), double-strand break repair (Mandecki, (1986) PNAS USA [Proceedings of the National Academy of Sciences of the United States of America], 83:7177-7181 and Arnold, (1993) Curr OpinBiotech [Contemporary Perspectives on Biotechnology] 4:450-455). Further details of many of the above methods can be found in MethodsEnzymol [Enzymological Methods] Volume 154, which also describes useful controls for troubleshooting using various mutagenesis methods.

[0103] Based on the teachings provided herein, the nucleotide sequences of the examples can also be used to isolate corresponding sequences from other sources that serve as sources of polynucleotides capable of generating polypeptides with multipolymerized domains. In this way, such sequences can be identified based on sequence homology with the sequences shown herein using methods such as PCR and hybridization. The examples cover sequences selected based on sequence identity with any of the sequences or fragments shown herein.

[0104] In hybridization methods, all or part of the harmful organism-killing nucleic acid sequence can be used to screen cDNA or genomic libraries. Methods for constructing such cDNA and genomic libraries are generally known in the art and disclosed in Sambrook and Russell, (2001), ibid. Hybridization probes can be genomic DNA fragments, synthetic oligonucleotides, cDNA fragments, RNA fragments, or other oligonucleotides, and can be labeled with a detectable group (such as 32P or any other detectable label, such as other radioisotopes, fluorescent compounds, enzymes, or enzyme cofactors). Degenerate primers may also be used, designed based on conserved nucleotides or amino acid residues in the nucleic acid sequence or the encoded amino acid sequence. Probes typically contain a nucleic acid sequence region that, under stringent conditions, hybridizes with at least about 12, at least about 25, at least about 50, 75, 100, 125, 150, 175, or 200 consecutive nucleotides of a nucleic acid sequence encoding a fragment or variant of the polymerized domain polypeptide disclosed herein. Methods and stringent conditions for preparing probes for hybridization are generally known in the art and are disclosed in Sambrook and Russell, (2001), ibid., which are incorporated herein by reference.

[0105] For example, the complete nucleic acid sequence encoding the polymerized domain polypeptide disclosed herein, or one or more portions thereof, can be used as a probe capable of specifically hybridizing with the corresponding nucleic acid sequence encoding the polymerized domain polypeptide or fusion partner polypeptide-like sequence and messenger RNA. To achieve specific hybridization under different conditions, such probes include sequences that are unique and preferably at least about 10 nucleotides in length, or at least about 20 nucleotides in length. Such probes can be used to amplify the corresponding sequence from a selected sample source. This technique can be used as a diagnostic assay to determine the presence of the coding sequence in a target sample. Hybridization techniques include screening hybridization databases of plated DNA libraries (patches or colonies; see, for example, Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual (2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY)).

[0106] Hybridization of such sequences can be performed under stringent conditions. As used herein, “stringent conditions” or “stringent hybridization conditions” refer to conditions under which the probe hybridizes to its target sequence to a detectably higher degree than it hybridizes to other sequences (e.g., at least 2 times higher than background). Stringent conditions are sequence-dependent and will vary under different conditions. By controlling the stringency of hybridization and / or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous detection). Alternatively, stringent conditions can be adjusted to allow for some mismatches in the sequences in order to detect a lower degree of similarity (heterologous detection). Typically, the probe length is less than about 1000 nucleotides, preferably less than 500 nucleotides.

[0107] AlphaFold In another embodiment, the AlphaFold computational program can be used to identify portions of the polypeptides disclosed herein, including but not limited to domains, structurally critical regions, minimal active core polypeptides, receptor-binding domains, active sites, and protease cleavage sites.

[0108] AlphaFold is a computational method that periodically predicts protein structures with atomic-level precision, even in the absence of known similar structures. The AlphaFold network takes the primary amino acid sequence and homology alignment sequences as input and directly predicts the 3D coordinates of all heavy atoms in a given protein. The AlphaFold method is scalable, capable of handling very long proteins and accurately resolving domains and domain stacking. Furthermore, the model provides accurate, residue-by-residue-based estimates, making its structure predictions reliable. (Jumper, J., Evans, R., Pritzel, A. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583-589 (2021)).

[0109] Antibody The disclosure also covers antibodies against polymerized domain peptides or variants or fragments thereof for use with respect to the embodiments. The antibodies disclosed herein include polyclonal and monoclonal antibodies and fragments thereof that retain their ability to bind to polymerized domain peptides. It is believed that antibodies, monoclonal antibodies, or fragments thereof can bind to a molecule provided that the antibody, monoclonal antibody, or fragment thereof can specifically react with the molecule, thereby binding the molecule to the antibody, monoclonal antibody, or fragment thereof. The terms “antibody” (Ab) or “monoclonal antibody” (Mab) are intended to include complete molecules and fragments thereof or binding regions or domains (e.g., fragments like Fab and F(ab).sub.2) capable of binding haptens. Such fragments are typically produced by proteolytic cleavage (e.g., papain or pepsin). Alternatively, hapten-binding fragments can be produced by applying recombinant DNA technology or by synthetic chemistry.

[0110] The methods for preparing the antibodies disclosed herein are generally known in the art. See, for example, Antibodies, A Laboratory Manual, edited by Harlow and David Lane, Cold Spring Harbor Laboratory, New York (1988), and the references cited therein. Standard references explaining the general principles of immunology include: Klein, J. Immunology: The Science of Cell-Noncell Discrimination, John Wiley & Sons, New York (1982); Dennett et al., Monoclonal Antibodies, Hybridoma: A New Dimension in Biological Analyses, Plenum Press, New York (1980); and Campbell, “Monoclonal Antibody Technology,” in Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 13, Burdon et al. (eds.), Elsevier, Amsterdam (1984). Antibodies targeting or their antigen-binding moieties can be produced using a variety of techniques, including conventional monoclonal antibody methods such as the standard somatic cell hybridization technique described in Kohler and Milstein, (1975) Nature [Nature] 256:495. Other techniques for producing monoclonal antibodies can also be used, such as viral or oncogenic transformation of B lymphocytes. The animal system used for preparing hybridomas is the mouse system. Immunoimmunization protocols and techniques for isolating spleen cells for fusion are known in the art. Fusion couples (e.g., mouse myeloma cells) and fusion procedures are also known. The antibodies and monoclonal antibodies disclosed herein can be prepared using polymerized domain peptides as antigens.

[0111] A kit is provided for detecting the presence of a polymeric domain polypeptide in a sample or for detecting the presence of a nucleotide sequence encoding a polymeric domain polypeptide. In one embodiment, the kit provides an antibody-based reagent for detecting the presence of a polymeric domain polypeptide in a tissue sample. In another embodiment, the kit provides a labeled nucleic acid probe that can be used to detect the presence of one or more polynucleotides encoding a polymeric domain polypeptide. The kit is provided with appropriate reagents and controls for performing the detection method, as well as instructions for use of the kit.

[0112] Nucleotide constructs, expression cassettes, and vectors The use of the term "nucleotide construct" herein is not intended to limit the embodiments to nucleotide constructs containing DNA. Those skilled in the art will recognize that nucleotide constructs, particularly polynucleotides and oligonucleotides composed of ribonucleotides, as well as combinations of ribonucleotides and deoxyribonucleotides, can also be used in the methods disclosed herein. The nucleotide constructs, nucleic acids, and nucleotide sequences of the embodiments further encompass all complementary forms of such constructs, molecules, and sequences. Furthermore, the nucleotide constructs, nucleotide molecules, and nucleotide sequences of the embodiments encompass all nucleotide constructs, molecules, and sequences that can be used in the plant transformation methods of the embodiments, including but not limited to those composed of deoxyribonucleotides, ribonucleotides, and combinations thereof. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogs. 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-loop structures, etc.

[0113] Other embodiments involve transformed organisms, such as those selected from plant and insect cells, bacteria, yeast, baculoviruses, protozoa, nematodes, and algae. The transformed organism contains the DNA molecule of the embodiments, an expression cassette containing the DNA molecule, or a vector containing the expression cassette, which can be stably incorporated into the genome of the transformed organism.

[0114] The DNA construct provides sequences of the embodiments for expression in a target organism. The construct will include regulatory sequences operatively linked to the 5' and 3' of the sequences of the embodiments. As used herein, the term "operatively linked" refers to the association of two or more nucleic acid fragments on a single nucleic acid fragment such that the function of one nucleic acid fragment is influenced by the other. For example, when a promoter is capable of influencing the expression of a coding sequence, it is operatively linked to that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). The coding sequence may be operatively linked to the regulatory sequence in either the sense or antisense direction. Optionally, operatively linked may also mean that the linked nucleic acid sequences are contiguous and, if necessary, two protein-coding regions are linked in the same reading frame. The construct may additionally contain at least one additional gene to be co-transformed into an organism. Alternatively, additional genes may be provided on multiple DNA constructs.

[0115] Such a DNA construct has multiple restriction sites for inserting a polymerized domain polypeptide, adaptor peptide, toxin F or fusion partner, or one or more gene sequences into the present disclosure, placing them under transcriptional regulation of the regulatory region. The DNA construct may additionally contain a selective marker gene.

[0116] Generally, the DNA construct will include, in the transcriptional direction from 5' to 3', a transcription and translation initiation region (i.e., the promoter), the DNA sequence of the example, and a transcription and translation termination region (i.e., the termination region) that functions in the host organism. The transcription initiation region (i.e., the promoter) may be natural, similar, exogenous, or heterologous to the host organism and / or sequence of the example. Additionally, the promoter may be a natural sequence or, alternatively, a synthetic sequence. As used herein, the term "exogenous" means that the promoter is not found in the natural organism in which it is introduced. When referring to a promoter or any other nucleotide or amino acid sequence as "exogenous" or "heterogeneous," it means that the sequence originates from a foreign species; or, if originating from the same species, it means that the sequence has undergone substantial modification at its constituent and / or genomic loci compared to its natural form due to deliberate human intervention. For example, the promoter operatively linked to a heterologous polynucleotide originates from a species different from the species from which the polynucleotide is derived; or, if from the same / similar species, one or both have undergone substantial modifications compared to their original form and / or genomic loci, or the promoter is not a natural promoter of the operatively linked polynucleotide. As used herein, chimeric genes contain coding sequences operatively linked to a transcription start region heterologous to that coding sequence. When the promoter is a native sequence, the expression of the operatively linked sequence is altered compared to wild-type expression, resulting in a phenotypic change.

[0117] In some embodiments, the DNA construct comprises a polynucleotide encoding a polymerized domain polypeptide or one or more chimeric polypeptides of the embodiments, and may optionally include a polynucleotide encoding one or more target genes. In some embodiments, the DNA construct comprises a polynucleotide encoding a chimeric fusion protein comprising a polymerized domain polypeptide of the embodiments, one or more adapter peptides, and one or more target polypeptides (e.g., a target insecticidal polypeptide).

[0118] In some embodiments, the DNA construct may also include a transcription enhancer sequence. As used herein, the term "enhancer" refers to a DNA sequence that can stimulate promoter activity and may be an intrinsic element of the promoter or a heterologous element inserted to enhance promoter level or tissue specificity. Various enhancers are known in the art, including, for example, introns that have gene expression-enhancing properties in plants, ubiquitin introns (i.e., corn ubiquitin intron 1 (see, e.g., NCBI sequence S94464)), ω-enhancers or ω'-enhancers (Gallie et al., (1989) Molecular Biology of RNA, Cech ed. (Liss, New York) 237-256 and Gallie et al., (1987) Gene 60:217-25), CaMV 35S enhancers (see, e.g., Benfey et al., (1990) EMBO J. 9:1685-96), and enhancers of U.S. Patent No. 7,803,992 may also be used. The above list of transcriptional enhancers is not intended to be limiting. Any suitable transcriptional enhancer may be used in the examples.

[0119] The termination region may be natural for the transcription start region, natural for the target DNA sequence to which it is operatively linked, natural for the plant host, or derived from another source (i.e., exogenous or heterologous for the promoter, target sequence, plant host, or any combination thereof).

[0120] Convenient termination regions can be obtained from Ti plasmids of Agrobacterium tumefaciens, such as octopaline synthase and carmine synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. [Molecular Genetics and General Genetics] 262:141-144; Proudfoot, (1991) Cell [Cell] 64:671-674; Sanfacon et al. (1991) Genes Dev. [Genes and Development] 5:141-149; Mogen et al. (1990) Plant Cell [Plant Cell] 2:1261-1272; Munroe et al. (1990) Gene [Genes] 91:151-158; Ballas et al. (1989) Nucleic Acids Res. [Nucleic Acids Research] 17:7891-7903; and Joshi et al. (1987) Nucleic Acids Res. [Nucleic Acids Research] 15:9627-9639.

[0121] Nucleic acids can be optimized to increase expression in the host organism when appropriate. Therefore, in the case of a plant as the host organism, synthetic nucleic acids can be synthesized using plant-preferred codons to improve expression. For a discussion of host-preferred use, see, for example, Campbell and Gowri, (1990) Plant Physiol. [Plant Physiology] 92:1-11. For example, while the nucleic acid sequences of the examples can be expressed in both monocotyledonous and dicotyledonous plant species, the sequences can be modified to account for specific preferences and GC content preferences of monocotyledonous or dicotyledonous plants, as these preferences have shown differences (Murray et al. (1989) Nucleic Acids Res. [Nucleic Acid Research] 17:477-498). Thus, corn preference for specific amino acids can be derived from known gene sequences of corn. Twenty-eight genes from corn plants are listed in Table 4 of Murray et al. (ibid.). Methods for synthesizing plant-preferred genes are available in the art. See, for example, Murray et al., (1989) Nucleic Acids Res. [Nucleic Acids Research] 17:477-498, and Liu H et al. Mol Bio Rep [Molecular Biology Reports] 37:677-684, 2010, which are incorporated herein by reference. Maize (Zea maize) can also be used in tables. The recombinant nucleic acid molecules of chimeric polypeptides have corn-optimized codons.

[0122] Other sequence modifications are known to enhance gene expression in the host cell. These include the removal of sequences encoding pseudopolyadenylation signals, exon-intron splicing site signals, transposon-like repeat sequences, and other well-characterized sequences that may be detrimental to gene expression. The GC content of a sequence can be adjusted to the average level for a given host cell, as calculated by referencing known genes expressed in the host cell. As used herein, the term “host cell” refers to a cell containing a vector and supporting the replication and / or expression of the expression vector. Host cells can be prokaryotic cells such as Escherichia coli, or eukaryotic cells such as yeast, insect, amphibian, or mammalian cells, or monocotyledonous or dicotyledonous plant cells. An example of a monocotyledonous plant host cell is a corn host cell. Where possible, sequences are modified to avoid predicted hairpin-shaped secondary mRNA structures.

[0123] The expression box can also contain a 5' leader sequence. Such a leader sequence can enhance the translation. Translational leader sequences are known in the art and include: piconera virus leader sequences, such as the EMCV leader sequence (5' untranslated region of encephalomyocarditis) (Elroy-Stein et al., (1989) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences], 86:6126-6130); potato virus Y leader sequences, such as the TEV leader sequence (tobacco etch virus) (Gallie et al., (1995) Gene [Gene] 165(2):233-238), the MDMV leader sequence (corn dwarf mosaic virus), and human immunoglobulin heavy chain binding protein (BiP) (Macejak et al., (1991) Nature [Nature] 353:90-94); and untranslated leader sequences of the coat protein mRNA from alfalfa mosaic virus (AMV RNA 4) (Jobling et al., (1987) Nature [Nature]). 325:622-625); Tobacco mosaic virus (TMV) leader sequence (Gallie et al., (1989) Molecular Biology of RNA, edited by Cech (Liss, New York), pp. 237-256) and maize chlorotic mottle virus (MCMV) leader sequence (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 “signal sequences” or “lead sequences” to facilitate the transport of peptides during or after co-translation to certain intracellular structures, such as chloroplasts (or other plastids), endoplasmic reticulum, or Golgi apparatus.

[0124] As used herein, a “signal sequence” refers to a sequence known or suspected of causing co-translational or post-translational transmembrane peptide transport. In eukaryotes, this typically involves secretion into the Golgi apparatus, accompanied by some form of glycosylation. Prototoxins are often synthesized from bacterial insecticidal toxins, which are then activated by proteolysis in the gut of the target pest (Chang, (1987) Methods Enzymol. [Enzymological Methods] 153:507-516). In some embodiments, the signal sequence is located in a native sequence or may be derived from the sequence described in the examples. As used herein, the term “leader sequence” refers to any sequence that, upon translation, produces an amino acid sequence sufficient to trigger co-translational transport of the peptide chain with subcellular organelles. Thus, this includes leader sequences that target transport and / or glycosylation by entering the endoplasmic reticulum, vacuoles, plastids (including chloroplasts, mitochondria), etc. Proteomic studies of chloroplasts in higher plants have identified numerous nuclear-encoded thylakoid lumen proteins (Kieselbach et al., FEBS LETT [Federation of European Biochemical Societies Letters] 480:271-276, 2000; Peltier et al., Plant Cell [Plant Cell] 12:319-341, 2000; Bricker et al., Biochem. Biophys Acta [Acta Biochimica et Biophysica Sinica] 1503:350-356, 2001), and according to this disclosure, lumen-targeting signal peptides of these nuclear-encoded thylakoid lumen proteins may be used. (See Kieselbach et al., Photosynthesis Research [Photosynthesis Research], 78:249-264, 2003, and in particular Table 2 of that publication, which discloses 85 proteins from the chloroplast lumen, which is incorporated herein by reference).

[0125] Suitable chloroplast transport peptides (CTPs) well known to those skilled in the art also include chimeric CTs, including but not limited to N-terminal, central, or C-terminal domains from the following CTPs: rice (Oryzasativa) 1-deoxy-D-xylulose-5-phosphate synthase, rice superoxide dismutase, rice soluble starch synthase, rice NADP-dependent malate enzyme, rice phosphate 2-dehydro-3-deoxyheptanoate aldolase 2, rice L-ascorbic acid peroxidase 5, rice phosphoglucan water-based dual kinase, corn ssRUBISCO, corn β-glucosidase, corn malate dehydrogenase, and corn M-type thioredoxin (see U.S. Patent Application Publication 2012 / 0304336).

[0126] Recombinant nucleic acid molecules encoding polymerized domain peptides or chimeric peptides (to be targeted to chloroplasts) can be optimized for expression in chloroplasts to accommodate differences in usage between the plant cell nucleus and this organelle. In this way, target nucleic acids can be synthesized using chloroplast-preferred sequences.

[0127] In preparing expression cassettes, various DNA fragments can be manipulated to provide DNA sequences that are in the appropriate orientation and at the appropriate time within the appropriate reading frame. This can be achieved by using adapters or linkers to ligate the DNA fragments, or by employing other manipulations to provide convenient restriction sites, remove redundant DNA, and eliminate restriction sites. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, and substitution techniques such as transitions and transversions may be involved.

[0128] Many promoters are available for implementing these embodiments. A promoter can be selected based on the desired results. Nucleic acids can be combined with constitutive, tissue-biased, inducible, or other promoters for expression in a host organism. Suitable constitutive promoters for use in plant host cells include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 1999 / 43838 and U.S. Patent No. 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. [Theoretical and Applied Genetics] 81:581-588); MAS (Velten et al., (1984) EMBO J. [Journal of the European Society for Molecular Biology] 3:2723-2730); ALS promoter (US Patent No. 5,659,026), etc. Other constitutive promoters include, for example, those discussed in US Patent No. 6,177,611.

[0129] Depending on the desired results, gene expression from an inducible promoter may be beneficial. Of particular interest in regulating nucleotide sequences used in this embodiment for expression in plants is the wound-inducible promoter. Such wound-inducible promoters can respond to damage caused by insect feeding and include the potato glycoprotein inhibitor (pin II) gene (Ryan, (1990) Ann. Rev. Phytopath. [Annals of Plant Pathology] 28:425-449; Duan et al., (1996) Nature Biotechnology [Nature Biotechnology] 14:494-498); wun1 and wun2; win1 and win2 (Stanford et al., (1989) Mol. Gen. Genet. [Molecular Genetics and General Genetics] 215:200-208); systemins (McGurl et al., (1992) Science [Science] 225:1570-1573); WIP1 (Rohmeier et al., (1993) Plant Mol. Biol. [Plant Molecular Biology] 22:783-792; Eckelkamp et al., (1993) FEBS Letters [European Federation of Biochemical Societies Letters] 323:73-76); MPI gene (Corderok et al., (1994) Plant J. 6(2):141-150), etc.

[0130] Additionally, pathogen-inducible promoters can be used in the methods and nucleotide constructs of the embodiments. Such pathogen-inducible promoters include those derived from pathogenesis-associated proteins (PR proteins) that are induced upon pathogen infection; for example, PR proteins, SAR proteins, β-1,3-glucanase, chitinase, etc. See, for example, Redolfi et al., (1983) Neth. J. Plant Pathol. [Dutch Journal of Plant Pathology] 89:245-254; Uknes et al., (1992) PlantCell [Plant Cell] 4:645-656; and Van Loon, (1985) Plant Mol. Virol. [Plant Molecular Virology] 4:111-116. See also WO 1999 / 43819.

[0131] Of particular interest are promoters expressed locally at or near the site of pathogen infection. See, for example, Marineau et al., (1987) Plant Mol. Biol. [Plant Molecular Biology] 9:335-342; Matton et al., (1989) Molecular Plant-Microbe Interactions [Molecular Plant-Microbe Interactions] 2:325-331; Somsisch et al., (1986) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 83:2427-2430; Somsisch et al., (1988) Mol. Gen. Genet. [Molecular Genetics and General Genetics] 2:93-98; and Yang, (1996) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 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) Plant J. 3:191-201; Siebertz et al., (1989) Plant Cell 1:961-968. Of particular interest is the inducible promoter of the maize PRms gene, whose expression is induced by the pathogen Fusarium moniliforme (see, for example, Cordero et al., (1992) Physiol. Mol. PlantPath. 41:189-200).

[0132] Chemically regulated promoters can be used to regulate gene expression in plants by applying exogenous chemical regulators. Depending on the target, the promoter can be a chemically inducible promoter, in which a chemical is applied to induce gene expression, or a chemically repressive promoter, in which a chemical is applied to suppress gene expression. Chemically inducible promoters are known in the art and include, but are not limited to, the corn In2-2 promoter activated by a benzenesulfonamide herbicide safener, the corn GST promoter activated by a hydrophobic electrophilic compound used as a pre-emergence herbicide, and the tobacco PR-1a promoter activated by salicylic acid. Other targeted chemical regulatory promoters include steroid-responsive promoters (see, for example, glucocorticoid-inducible promoters in Schena et al., (1991) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 88:10421-10425 and McNellis et al., (1998) Plant J [Plant Journal] 14(2):247-257) as well as tetracycline-inducible and tetracycline-inhibiting promoters (see, for example, Gatz et al., (1991) Mol. Gen. Genet. [Molecular Genetics and General Genetics] 227:229-237).

[0133] Tissue-biased promoters can be used to target the expression of multi-merging domain peptides or fusion peptides (including chimeric peptides containing multi-merging domain peptides, one or more adaptor peptides, and one or more target peptides (e.g., target insecticidal peptides)) in specific plant tissues. Tissue-biased promoters include those described in the following literature: 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. GenGenet. 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. If necessary, such promoters can be modified to achieve weak expression.

[0134] Leaf-preferred 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.

[0135] Root-biased or root-specific promoters are known and can be selected from many promoters obtained from the literature or re-isolated from different compatible species. See, for example, Hire et al., (1992) Plant Mol. Biol. 20(2):207-218 (Root-specific glutamine synthase gene in soybean); Keller and Baumgartner, (1991) Plant Cell 3(10):1051-1061 (Root-specific control element in GRP 1.8 gene of common bean); Sanger et al., (1990) Plant Mol. Biol. 14(3):433-443 (Root-specific promoter of mannan synthase (MAS) gene in Agrobacterium tumefaciens); and Miao et al., (1991) Plant Cell 3(1):11-22 (Full-length cDNA clone encoding cytosolic glutamine synthase (GS) expressed in soybean roots and root nodules). See also Bogusz et al., (1990) Plant Cell 2(7):633-641, which describes two root-specific promoters isolated from hemoglobin genes from the nitrogen-fixing non-leguminous plant Parasponia andersonii and the related non-nitrogen-fixing non-leguminous plant Trema tomentosa. The promoters of these genes are linked to β-glucuronidase reporter genes and have been introduced into both the non-leguminous crop Nicotiana tabacum and the leguminous crop Lotus corniculatus, and root-specific promoter activity has been preserved in both cases. Leach and Aoyagi, (1991) describe their analysis of the promoters of the highly expressed rolC and rolD root-inducing genes from Agrobacterium rhizogenes (see Plant Science (Limerick) 79(1):69-76). They concluded that enhancers and tissue-biased DNA determinants are dissociated in those promoters.Teeri et al. (1989) used gene fusion with lacZ to show that Agrobacterium T-DNA genes encoding octopus alkaloid synthase are active, particularly in the epidermis of root tips, and that the TR2' gene is root-specific in intact plants and stimulated by wounds in leaf tissues, a particularly desirable combination of features when used with insecticidal or larval-killing genes (see EMBO J. [Journal of the European Society for Molecular Biology] 8(2):343-350). The TR1' gene fused with nptII (neomycin phosphotransferase II) showed similar characteristics. Other root-preferred promoters include the VfENOD-GRP3 gene promoter (Kuster et al., (1995) Plant Mol. Biol. [Plant Molecular Biology] 29(4):759-772); and the rolB promoter (Capana et al., (1994) Plant Mol. Biol. [Plant Molecular Biology] 25(4):681-691). See also U.S. Patent No. 5,401,836.

[0136] “Seed-preferred” promoters include “seed-specific” promoters (those that are active during seed development, such as promoters of seed storage proteins) and “seed-germination” promoters (those that are active during seed germination). See Thompson et al., (1989) BioEssays [Biological Analysis] 10:108. Such seed-preferred promoters include, but are not limited to, Cim1 (cytokinin-induced messenger); cZ19B1 (19 kDa zeatin); and milps (inositol-1-phosphate synthase) (see, U.S. Patent No. 6,225,529). γ-zeatin and Glb-1 are endosperm-specific promoters. For dicotyledons, seed-specific promoters include, but are not limited to: Kunitz trypsin inhibitor 3 (KTi3) (Jofuku and Goldberg, (1989) Plant Cell 1:1079-1101), β-coumarin, rapeseed protein, β-conglycinin, glycinin 1, soybean lectin, cruciferous proteins, etc. For monocotyledons, seed-specific promoters include, but are not limited to: 15 kDa zeatin, 22 kDa zeatin, 27 kDa zeatin, g-zeatin, waxy protein, shrunken 1, shrunken 2, globulin 1, etc. See also WO 2000 / 12733, which discloses seed-preferred promoters from the end1 and end2 genes. In dicotyledonous plants, seed-specific promoters include, but are not limited to: the seed coat promoter pBAN from the genus *Arabidopsis*; and early seed promoters from the genus *Arabidopsis*, p26, p63, and p63tr. Promoters exhibiting “tissue-preferred” expression in a specific tissue are expressed at a higher level in that tissue than in at least one other plant tissue. Some tissue-preferred promoters are expressed almost exclusively in a particular tissue.

[0137] Weak promoters can be used when low levels of expression are required. Generally, as used herein, the term "weak promoter" refers to a promoter that drives expression of the coding sequence at low levels. Low-level expression is aimed at levels between approximately 1 / 1000 of transcripts and approximately 1 / 100,000 of transcripts and approximately 1 / 500,000 of transcripts. Alternatively, it should be recognized that the term "weak promoter" also encompasses promoters that drive expression only in a few cells but not in others, thus having low overall expression levels. When a promoter drives expression at unacceptably high levels, portions of the promoter sequence can be deleted or modified to reduce expression levels.

[0138] Such weakly constitutive promoters include, for example, the core promoter of the Rsyn7 promoter (WO 1999 / 43838 and U.S. Patent No. 6,072,050), the core 35S CaMV promoter, etc. Other constitutive promoters include, for example, those disclosed in U.S. Patent No. 6,177,611.

[0139] The list of promoters above is not intended to be limiting. Any suitable promoter can be used in the embodiments.

[0140] Typically, expression cassettes contain selective marker genes for selecting transformed cells. Selective marker genes are used to select transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), and genes conferring resistance to herbicide compounds such as glufosinate, bromosulfuron, imidazolinone, and 2,4-dichlorophenoxyacetic acid (2,4-D). Other examples of suitable selective marker genes include, but are not limited to, genes encoding resistance to: chloramphenicol (Herrera Estrella et al., (1983) EMBO J. [Journal of the European Society for Molecular Biology] 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); sulfonamides (Guerineau et al., (1990) Plant Mol. Biol. [Plant Molecular Biology] 15:127-136); bromonazine (Stalker et al., (1988) Science [Science] 242:419-423); glyphosate (Shaw et al., (1986) Science [Science] 233:478-481 and US patent application serial numbers 10 / 004,357 and 10 / 427,692); glufosinate (DeBlock et al., (1987) EMBO J. [Journal of the European Society for Molecular Biology] 6:2513-2518). See also 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.[Molecular Microbiology] 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 [Proceedings of the National Academy of Sciences of the United States of America] 86:5400-5404; Fuerst et al., (1989) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 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; Baim 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. [Hot Topics in Molecular Structural Biology] 10:143-162; Degenkolb et al., (1991) Antimicrob. Agents Chemother. [Antibacterial Agents and Chemotherapy] 35:1591-1595; Kleinschnidt et al., (1988) Biochemistry 27:1094-1104; Bonin, (1993) Ph. D.Thesis, University of Heidelberg, Germany; 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.

[0141] The list of selective marker genes above is not intended to be limiting. Any selective marker gene may be used in the examples.

[0142] Plant transformation The methods of the embodiments involve introducing polymerized domain peptides or fusion peptides or polynucleotides (including chimeric fusion peptides comprising polymerized domain peptides, one or more adaptor peptides, and one or more target peptides (e.g., target insecticidal peptides) into plants. As used herein, “introduction” means presenting a polynucleotide or peptide to a plant in such a manner that the sequence can enter the interior of a plant cell. The methods of the embodiments do not depend on a specific method of introducing a polynucleotide or peptide into a plant, as long as the polynucleotide or peptide can enter the interior of at least one cell of the plant. Methods for introducing polynucleotides or peptides into plants are known in the art and include, but are not limited to, stable transformation, transient transformation, and virus-mediated transformation.

[0143] A “stable transformation” is a transformation in which a polynucleotide construct introduced into a plant integrates into the plant’s genome and can be inherited by its offspring. As used herein, a “transient transformation” means the introduction of a polynucleotide into a plant that does not integrate into the plant’s genome. As used herein, “plant” generally includes the whole plant, plant organs, plant tissues, seeds, plant cells, and their offspring. Plants are monocotyledonous or dicotyledonous. Plant cells include, but are not limited to, cells derived from: seeds, suspension cultures, embryos, meristematic regions, callus, leaves, roots, buds, gametophytes, sporophytes, pollen, and microspores. “Plant element” is intended to refer to the whole plant or plant component, which may include differentiated and / or undifferentiated tissues, such as, but not limited to, plant tissues, parts, and cell types. In one embodiment, a plant element is one of the following: whole plant, seedling, meristem, ground tissue, vascular tissue, cortical tissue, seed, leaf, root, bud, stem, flower, fruit, stolon, bulb, tuber, corm, asexual terminal shoot, bud, young shoot, tumor tissue, and various forms of cells and cultures (e.g., single cells, protoplasts, embryos, and callus). It should be noted that a protoplast is not a technically “complete” plant cell (as all components are naturally found) because protoplasts lack cell walls. “Plant organ” refers to a plant tissue or a group of tissues that constitute different morphological and functional parts of a plant. “Plant element” is synonymous with “part” of a plant and refers to any part of a plant, and may include different tissues and / or organs, and may be used interchangeably with “tissue” throughout the text. Similarly, "plant propagation element" is intended in a general sense to refer to any part of a plant that can be used to create another plant through sexual or asexual reproduction of that plant, such as, but not limited to: seeds, seedlings, roots, buds, cuttings, scions, grafted seedlings, stolons, bulbs, tubers, corms, asexual terminal shoots, or young buds. Plant elements can be present in plants or in plant organs, tissue cultures, or cell cultures.

[0144] The transformation scheme and the scheme for introducing nucleotide sequences into plants can vary depending on the type of plant or plant cell to be targeted for transformation (i.e., monocots or dicots). Suitable methods for introducing nucleotide sequences into plant cells and subsequently inserting them into the plant genome include microinjection (Crossway et al., (1986) Biotechniques [Biotechnology] 4:320-334), electroporation (Riggs et al., (1986) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 83:5602-5606), Agrobacterium-mediated transformation (US Patent Nos. 5,563,055 and 5,981,840), direct gene transfer (Paszkowski et al., (1984) EMBO J. [Journal of the European Society for Molecular Biology] 3:2717-2722), and ballistic particle acceleration (see, for example, US Patent Nos. 5,879,918; 5,886,244 and 5,932,782; Tomes et al., (1995) in: Plant Cell, Tissue, and Organ Culture: Fundamental Methods). In *Plant Cell, Tissue and Organ Culture: Basic Methods*, edited by Gamborg and Phillips (Springer-Verlag, Berlin); and McCabe et al., (1988) *Biotechnology* 6:923-926; and the Lecl transformation method (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. Other transformation procedures can be found in the following literature: Weissinger et al., (1988) Ann. Rev. Genet. [Annals of Genetics] 22:421-477; Sanford et al., (1987) Particulate Science and Technology [Particulate Science and Technology] 5:27-37 (Onion); Christou et al., (1988) Plant Physiol.[Plant Physiology] 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 (Corn); Klein et al., (1988) Biotechnology 6:559-563 (Corn); US Patent Nos. 5,240,855; 5,322,783 and 5,324,646; Klein et al., (1988) Plant Physiol. 91:440-444 (Corn); Fromm et al., (1990) Biotechnology 8:833-839 (Corn); Hooykaas-Van Slogteren et al., (1984) Nature (London) 311:763-764; US Patent No. 5,736,369 (Cereals); Bytebier et al., (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al., (1985) Published in: The Experimental Manipulation of Ovule Tissues, edited by 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.[Theoretical and Applied Genetics] 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).

[0145] In certain embodiments, various transient conversion methods can be used to provide the sequence of the embodiment to the plant. Such transient conversion methods include, but are not limited to, the direct introduction of fusion polynucleotides or variants and fragments thereof into the plant, the introduction of transcripts into the plant, or the introduction of fusion polypeptide transcripts into the plant. Such methods include, for example, microinjection or particle bombardment. See, for example, Crossway et al., (1986) Mol Gen. Genet. [Molecular Genetics and General Genetics] 202:179-185; Nomura et al., (1986) Plant Sci. [Plant Science] 44:53-58; Hepler et al., (1994) Proc. Natl. Acad. Sci. [Proceedings of the National Academy of Sciences of the United States of America] 91:2176-2180; and Hush et al., (1994) The Journal of Cell Science [Journal of Cell Science] 107:775-784. Alternatively, one or more polynucleotides can be transiently converted into the plant using techniques known in the art. Such techniques include viral vector systems and polynucleotide precipitation that prevents the subsequent release of DNA. Therefore, transcription can proceed from the particle-bound DNA, but the frequency with which it is released to integrate into the genome is greatly reduced. Such methods involve the use of particles coated with polyethyleneimine (PEI; Sigma #P3143).

[0146] Methods for targeted insertion of polynucleotides at specific locations in a plant genome are known in the art. In one embodiment, the insertion of the polynucleotide at the 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 embodiment may be contained within a transfer cassette flanked by two distinct recombination sites. The transfer cassette is introduced into a plant that has stably incorporated the target site into its genome, flanked by two distinct recombination sites corresponding to the site on the transfer cassette. A suitable recombinase is provided, and the transfer cassette is integrated into the target site. Thus, the target polynucleotide is integrated at a specific chromosomal location in the plant genome.

[0147] Plant transformation vectors can consist of one or more DNA vectors required to achieve plant transformation. For example, it is common practice in the art to use plant transformation vectors composed of more than one continuous DNA fragment. These vectors are generally referred to in the art as “binary vectors.” Binary vectors, as well as vectors with helper plasmids, are most commonly used for Agrobacterium-mediated transformation, where the size and complexity of the DNA fragments required for efficient transformation are considerable, and it is advantageous to isolate function onto individual DNA molecules. Binary vectors typically contain a plasmid vector containing cis-acting sequences required for T-DNA transfer (e.g., left and right boundaries), a selective marker engineered to be expressed in plant cells, and a “target gene” (a gene engineered to be expressed in plant cells where transgenic plants are desired). The sequences required for bacterial replication are also present on this plasmid vector. The cis-acting sequences are arranged in a manner that allows efficient transfer to and expression in plant cells. For example, a selective marker gene and a pest-killing gene are located between the left and right boundaries. Typically, the second plasmid vector contains trans-acting factors that mediate T-DNA transformation from Agrobacterium to plant cells. As understood in the art, this plasmid typically contains a virulence function (Vir gene) that allows infection of plant cells by Agrobacterium, as well as DNA transfer via boundary sequence cleavage and vir-mediated DNA transfer (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. Transformation of plants by other methods such as microscopy, microinjection, electroporation, and polyethylene glycol does not require a second plasmid vector.

[0148] Typically, plant transformation involves transferring heterologous DNA into target plant cells (e.g., immature or mature embryos, suspension cultures, undifferentiated callus, protoplasts, etc.), followed by applying an appropriate selection pressure (depending on the selectable marker gene) at a maximum threshold level to recover the transformed plant cells from a population of untransformed cells. After the heterologous DNA is integrated into the plant cells, an appropriate selection pressure at a maximum threshold level is then applied in the culture medium to kill the untransformed cells, and the presumed transformed cells that survived the selection treatment are isolated and proliferated by periodically transferring them to fresh culture medium. Cells transformed with plasmid vectors can be identified and proliferated through continuous passage and application of appropriate selection pressure. The presence of the target heterologous gene integrated into the genome of the transgenic plant can then be confirmed using molecular and biochemical methods.

[0149] Typically, explants are transferred to freshly supplied, identical media and cultured routinely. Subsequently, after being placed on regeneration medium supplemented with a maximum threshold level of selectant, the transformed cells differentiate into shoots. The shoots are then transferred to a selective rooting medium for the recovery of rooted shoots or plantlets. The transgenic plantlets then grow into mature plants and produce fertile seeds (e.g., Hiei et al., (1994) The Plant Journal 6:271-282; Ishida et al., (1996) Nature Biotechnology 14:745-750). Typically, explants are transferred to freshly supplied, identical media and cultured routinely. A general description of the techniques and methods used to generate transgenic plants is found in the following literature: Ayres and Park, (1994) Critical Reviews in Plant Science 13:219-239 and Bommineni and Jauhar, (1997) Maydica 42:107-120. Because transformed material contains many cells, both transformed and untransformed cells are present simultaneously in any part of the target callus or tissue or cell population. The ability to kill untransformed cells and allow transformed cells to proliferate produces transformed plant cultures. Generally, the ability to remove untransformed cells limits the rapid recovery of transformed plant cells and the successful generation of transgenic plants.

[0150] Transformed cells can be cultured into plants using conventional methods. See, for example, McCormick et al., (1986) Plant Cell Reports 5:81-84. These plants can then be cultured and pollinated with the same or different transformed lines, and hybrids with constitutive or inducible expression of the desired phenotypic trait can be identified. Two or more generations can be cultured to ensure that the expression of the desired phenotypic trait is stably maintained and inherited, and then seeds are harvested to ensure that the expression of the desired phenotypic trait has been achieved.

[0151] The nucleotide sequences of the embodiments can be provided to plants by contacting them with viruses or viral nucleic acids. Typically, such methods involve incorporating the target nucleotide construct into viral DNA or RNA molecules. It should be understood that the recombinant proteins of the embodiments can initially be synthesized as part of a viral polypeptide and then processed in vivo or in vitro by proteolysis to produce the desired polymerized domain polypeptides or fusion chimeric polypeptides. It should also be understood that such viral polyps containing at least a portion of the amino acid sequence of the polymerized domain polypeptides or chimeric polypeptides of the embodiments can possess the desired activity, such as necrotic bioactivity. Such viral polyps and the nucleotide sequences encoding them are covered in the embodiments. Methods involving viral DNA or RNA molecules for providing nucleotide constructs to plants and producing the encoded proteins in plants are known in the art.

[0152] Methods for transforming chloroplasts are known in the art. See, for example, Svab et al., (1990) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 87:8526-8530; Svab and Maliga, (1993) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 90:913-917; Svab and Maliga, (1993) EMBO J. [Journal of the European Society for Molecular Biology] 12:601-606. This method relies on particle gun delivery of DNA containing selectively labeled markers and targeting the DNA to the plastid genome via homologous recombination. Alternatively, plastid transformation is achieved by transactivating silent plastid-carried transgenes through tissue-biased expression of nuclear-encoded and plastid-directed RNA polymerases. Such a system has been reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 91:7301-7305.

[0153] These embodiments further relate to plant propagation materials of the transformed plants of the embodiments, including but not limited to seeds, tubers, bulbs, leaves, and cuttings of roots and buds.

[0154] These embodiments can be used to transform any plant species, including but not limited to monocots and dicots. Examples of target plants include, but are not limited to, maize (corn), Brassica species (e.g., Brassica napus, Brassica rapa, Brassica juncea) (especially those Brassica species that can be used as seed oil sources), alfalfa (Medicago sativa), rice, rye (Secalecereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., Pennisetum glaucum, Panicum miliaceum, Setariaitalica, Eleusine coracana), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), and wheat (Triticum). The following are listed: Soybean (Glycine max), Tobacco (Bureto), Potato (Solanum tuberosum), Peanut (Arachis hypogaea), Cotton (Gossypium barbadense, Gossypium hirsutum), Sweet potato (Ipomoeabatatus), Cassava (Manihot esculenta), Coffee (Coffea spp.), Sugarcane (Saccharum spp.), Oats, Barley, and Vegetables.

[0155] Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactucasativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (species of the genus Lathyrus), and members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and melon (C. melo). Plants in the examples include crop plants (e.g., corn, alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, etc.), such as corn and soybean plants.

[0156] Turfgrasses include, but are not limited to: annual bluegrass (Poa annua); annual ryegrass (Lolium multiflorum); Canada bluegrass (Poa compressa); Chewing's fescue (Festuca rubra); Colonia lbentgrass (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 foxgrass (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 (Puccinelliadistans); western wheatgrass (Agropyron smithii); bermudagrass (Cynodon species); St. Augustine grass (St.Augustine grass (Stenotaphrum secundatum); Zoysia grass (a species of the genus Zoysia); Bahia grass (Paspalum notatum); Carpetgrass (Axonopus affinis); Centipede grass (Eremochloa ophiuroides); Kikuyu grass (Pennisetum clandesinum); Seashore paspalum (Paspalumvaginatum); Blue gramma (Bouteloua gracilis); Buffalo grass (Buchloe dactyloids); Sideoats gramma (Bouteloua curtipendula).

[0157] Target plants include cereal plants, oilseed plants, and legumes that provide the target seeds. Target seeds include cereal seeds such as corn, wheat, barley, rice, sorghum, rye, and millet. Oilseed plants include cotton, soybean, safflower, sunflower, brassica, maize, alfalfa, palm, coconut, flax, castor bean, and olive. Legumes include beans and peas. Beans include guar beans, locust beans, fenugreek, soybeans, green beans, cowpeas, mung beans, lima beans, broad beans, sea peas, chickpeas, and so on.

[0158] After introducing heterologous DNA into plant cells, the transformation or integration of the heterologous gene into the plant genome is confirmed by various methods (such as analyzing nucleic acids, proteins and metabolites associated with the integrated gene).

[0159] PCR analysis is a rapid method for screening transformed cells, tissues, or shoots for incorporated genes at an early stage before transplantation into soil (Sambrook and Russell, (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York). PCR is performed using oligonucleotide primers specific to the target gene or Agrobacterium vector background.

[0160] Plant transformation can be confirmed by gene DNA blot analysis (Sambrook and Russell, (2001), ibid.). In RNA blot analysis, RNA is isolated from specific tissues of the transformant, fractionated in formaldehyde agar gel, and blotted onto a nylon filter according to standard procedures conventionally used in the art (Sambrook and Russell, (2001), ibid.). The expression of RNA encoded by the fusion polynucleotide or pest-killing gene is then tested by hybridizing the filter with a radioactive probe derived from the fusion polynucleotide using methods known in the art (Sambrook and Russell, (2001), ibid.). Protein blotting, biochemical assays, etc., can be performed on transgenic plants to confirm the presence of proteins encoded by pest-killing genes using standard procedures (Sambrook and Russell, 2001, ibid.) using antibodies that bind to one or more epitopes present on the polymerized domain polypeptide or fusion polypeptide.

[0161] Methods of introducing genome editing technology into plants In some embodiments, genome editing techniques can be used to introduce the disclosed fusion polynucleotide composition into the plant genome, or genome editing techniques can be used to edit previously introduced fusion polynucleotides in the plant genome. For example, the disclosed polynucleotide can be introduced into the desired location in the plant genome using double-strand break techniques (such as TALEN, large-scale nucleases, zinc finger nucleases, CRISPR-Cas, etc.). For example, for site-specific insertion purposes, the CRISPR-Cas system can be used to introduce the disclosed polynucleotide into the desired location in the genome. The desired location in the plant genome can be any target site required for insertion, such as a genomic region suitable for breeding, or it can be a target site located within a genomic window with an existing target trait. The existing target trait may be an endogenous trait or a previously introduced trait.

[0162] In some embodiments, where the disclosed fusion polynucleotide has been previously introduced into the genome, genome editing techniques can be used to alter or modify the introduced polynucleotide sequence. Site-specific modifications that can be introduced into the disclosed fusion polynucleotide composition include those produced using any method for introducing site-specific modifications, including but not limited to the use of gene repair oligonucleotides or the use of double-strand break techniques such as TALEN, large-scale nucleases, zinc finger nucleases, CRISPR-Cas, etc. Such techniques can be used to modify previously introduced polynucleotides by inserting, deleting, or substituting nucleotides within the introduced polynucleotide. Alternatively, double-strand break techniques can be used to add additional nucleotide sequences to the introduced polynucleotide. Additional sequences that can be added include additional expression elements (such as enhancer sequences and promoter sequences). In another embodiment, genome editing techniques can be used to localize additional insecticidal active proteins near the disclosed fusion polynucleotide composition within the plant genome to generate molecular stacks of target proteins (e.g., insecticidal active proteins).

[0163] The terms “altered target site,” “altered target sequence,” “modified target site,” and “modified target sequence” are used interchangeably herein and mean a target sequence as disclosed herein that contains at least one alteration when compared to an unaltered target sequence. Such an “alteration” includes, for example: (i) substitution of at least one nucleotide, (ii) deletion of at least one nucleotide, (iii) insertion of at least one nucleotide, or (iv) any combination of (i)-(iii).

[0164] Stacking of traits in transgenic plants Transgenic plants may contain stacks of one or more fusion polynucleotides disclosed herein with one or more additional polynucleotides, resulting in the production or repression of multiple polypeptide sequences. Transgenic plants containing polynucleotide sequence stacks can be obtained by one or both of conventional breeding methods or genetic engineering methods. These methods include, but are not limited to, breeding single lines each containing the target polynucleotide, transforming transgenic plants containing the gene disclosed herein along with subsequent genes, and co-transforming genes into a single plant cell. As used herein, the term "stack" includes the presence of multiple traits in the same plant (i.e., incorporating both traits into the nuclear genome, incorporating one trait into the nuclear genome and one trait into the plastid genome, or incorporating both traits into the plastid genome). In a non-limiting example, a "stacked trait" includes a molecular stack in which sequences are physically adjacent to each other. As used herein, a trait refers to a phenotype derived from a particular sequence or set of sequences. Co-transformation of genes can be performed using a single transformation vector containing multiple genes or genes carried separately on multiple vectors. If sequences are stacked by genetically transforming plants, the target polynucleotide sequences can be combined at any time and in any order. Co-transformation schemes can be used to introduce the desired polynucleotide along with any combination of transformation cassettes. For example, if two sequences are introduced, they can be contained in separate transformation cassettes (trans) or in the same transformation cassette (cis). Expression of the sequences can be driven by the same promoter or by different promoters. In some cases, it may be necessary to introduce a transformation cassette that will inhibit the expression of the desired polynucleotide. This can be combined with any combination of other repressor or overexpression cassettes to produce the desired combination of traits in the plant. It should be further appreciated that site-specific recombination systems can be used to stack polynucleotide sequences at desired genomic locations. See, for example, WO 1999 / 25821, WO 1999 / 25854, WO 1999 / 25840, WO 1999 / 25855 and WO 1999 / 25853, all of which are incorporated herein by reference.

[0165] In some embodiments, one or more polynucleotides encoding the polymerized domain polypeptides disclosed herein, alone or stacked with one or more other insect resistance traits, or one or more fusion polypeptides (including fusion polynucleotides encoding chimeric polypeptides comprising one or more polymerized domain polypeptides, one or more linker peptides, and one or more target polypeptides (e.g., target insecticidal polypeptides)), may be stacked with one or more other input traits (e.g., herbicide resistance, fungal resistance, viral resistance, stress tolerance, disease resistance, male sterility, stem strength, etc.) or output traits (e.g., increased yield, modified starch, improved oil properties, balanced amino acids, high lysine or methionine, increased digestibility, improved fiber quality, drought resistance, etc.). Therefore, the polynucleotide embodiments can be used to provide complete agronomic programs with the ability to flexibly and cost-effectively control any number of agronomic pests, resulting in improved crop quality.

[0166] Genetically modified organisms (GMOs) that can be stacked include, but are not limited to: GMOs that confer herbicide resistance; GMOs that confer or contribute to altered cereal characteristics; genes that control male sterility; genes that create sites for site-specific DNA integration; genes that affect resistance to abiotic stresses; genes that confer increased yield; genes that confer digestibility in plants; and GMOs that confer resistance to insects or diseases.

[0167] Examples of transgenic organisms conferring insect resistance include genes encoding Bacillus thuringiensis proteins, their derivatives, or synthetic polypeptides modeled thereon. See, for example, Geiser et al., (1986) Gene [Gene] 48:109, which discloses the clone and nucleotide sequence of the Bt δ-endotoxin gene. Furthermore, DNA molecules encoding the δ-endotoxin gene are available from the American Type Culture Collection (Rockville, Md., MD), for example, ATCC® Registry numbers 40098, 67136, 31995, and 31998. Other non-limiting examples of genetically engineered Bacillus thuringiensis transgenic strains are given in the following patents and patent applications: U.S. Patent Nos. 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, 7,1 79,965, 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.

[0168] Genes encoding pest-killing proteins can also be stacked, including but not limited to: insecticidal proteins from *Pseudomonas* species, such as PSEEN3174 (Monalysin, (2011) PLoS Pathogens, 7:1-13); insecticidal proteins from *Pseudomonas protegens* strains CHA0 and Pf-5 (formerly *fluorescens*) (Pechy-Tarr, (2008) Environmental Microbiology, 10:2368-2386: GenBank accession number EU400157); insecticidal proteins from *Pseudomonas taiwanensis* (Liu et al., (2010) J. Agric. Food Chem., 58:12343-12349); and from *Pseudomonas pseudoalkaloidosa* (Zhang et al., (2009) Annals of Microbiology 59:45-50 and Li et al., (2007) Plant Cell Tiss. OrganCult. 89:159-168) insecticidal proteins; insecticidal proteins from species of the genera *Luminobacterium* and *Bacillus* (Hinchliffe et al., (2010) The Open Toxinology Journal 3:101-118 and Morgan et al., (2001) Applied and Envir. Micro. [Applied and Environmental Microbiology] 67:2062-2069), insecticidal proteins from US Patent Nos. 6,048,838 and 6,379,946; PIP-1 peptide from US Patent No. 9,688,730; AfIP-1A and / or AfIP-1B peptides from US Patent No. 9,475,847; PIP-47 peptide from US Patent No. 10,006,045; IPD045, IPD064, IPD074, IPD075, and IPD077 peptides from PCT Publication No. WO 2016 / 114973; IPD080 peptide from International Patent Application Publication No. WO 2018 / 075350; International Patent Application Publication No. WO IPD078 peptide, IPD084 peptide, IPD085 peptide, IPD086 peptide, IPD087 peptide, IPD088 peptide, and IPD089 peptide of 2018 / 084936;PIP-72 peptide in US Patent Publication No. US 20160366891; PtIP-50 and PtIP-65 peptides in US Patent Application Publication No. US 20170166921; IPD098, IPD059, IPD108, and IPD109 peptides in International Patent Application Publication No. WO 2018 / 232072; PtIP-83 peptide in US Publication No. US 20160347799; PtIP-96 peptide in US Publication No. US 20170233440; IPD079 peptide in PCT Publication No. WO 2017 / 23486; International Patent Application Publication No. WO The following peptides are listed: IPD082 peptide (2017 / 105987), IPD090 peptide (International Patent Application Publication No. WO2017 / 192560), IPD093 peptide (International Patent Application Publication No. WO2018 / 111551), IPD103 peptide (International Patent Application Publication No. WO2018 / 005411), and IPD101 peptide (International Patent Application Publication No. WO2018 / 118811).The IPD121 polypeptide and δ-endotoxins, including but not limited to Cry1, Cry2, Cry3, Cry4, Cry5, Cry6, Cry7, Cry8, Cry9, Cry10, Cry11, Cry12, Cry13, Cry14, Cry15, Cry16, Cry17, Cry18, Cry19, Cry20, Cry21, Cry22, Cry23, Cry24, Cry25, Cry26, Cry27, Cry28, Cry29, Cry30, Cry31, Cry32, and Cr, as described in International Patent Application Publication No. WO2018 / 208882, are also mentioned. The δ-endotoxin genes of the classes y33, Cry34, Cry35, Cry36, Cry37, Cry38, Cry39, Cry40, Cry41, Cry42, Cry43, Cry44, Cry45, Cry46, Cry47, Cry49, Cry50, Cry51, Cry52, Cry53, Cry54, Cry55, Cry56, Cry57, Cry58, Cry59, Cry60, Cry61, Cry62, Cry63, Cry64, Cry65, Cry66, Cry67, Cry68, Cry69, Cry70, Cry71, and Cry72, as well as the cytolytic Cyt1 and Cyt2 genes of Bacillus thuringiensis. Members of these classes of Bacillus thuringiensis insecticidal proteins are well known to those skilled in the art (see, Crickmore et al., “Bacillus thuringiensis toxin nomenclature” (2011), lifesci.sussex.ac.uk / home / Neil_Crickmore / Bt / , which can be accessed on the World Wide Web with the “www” prefix).

[0169] Examples of delta-endotoxins include, but are not limited to, the Cry1A protein of U.S. Patent Nos. 5,880,275 and 7,858,849; the DIG-3 or DIG-11 toxins (N-terminal deletion of α-helix 1 and / or α-helix 2 variants of Cry proteins (e.g., Cry1A) of U.S. Patent Application Serial No. 10 / 525,318, U.S. Patent Application Publication No. US20160194364, and Cry1B of U.S. Patent Nos. 9,404,121 and 8,772,577; and PCT Publication No. WO Variants of Cry1B (serial number PCT / US17 / 27160, 2016 / 61197); Cry1C (US Patent No. 6,033,874); Cry1D protein (US20170233759); Cry1E protein (PCT Publication No. WO2018 / 075197); Cry1F (US Patent Nos. 5,188,960, 6,218,188); Cry1A / F chimeras (US Patent Nos. 7,070,982, 6,962,705, and 6,713,063); and US Patent Publication No. Cry1J variant of 20170240603; Cry2 protein such as Cry2Ab protein of US Patent No. 7,064,249; Cry3A protein, including but not limited to engineered hybrid insecticidal protein (eHIP) produced by a unique combination of variable and conserved regions of at least two different Cry proteins (US Patent Application Publication No. 2010 / 0017914); Cry4 protein; Cry5 protein; Cry6 protein; and more. Cry8 proteins, including 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; Cry9 proteins, such as members of the Cry9A, Cry9B, Cry9C, Cry9D, Cry9E, and Cry9F families; and Cry15 proteins, described in Naimov et al. (2008). Applied and Environmental Microbiology 74:7145-7151; Cry22 and Cry34Ab1 proteins in US patents 6,127,180, 6,624,145, and 6,340,593; CryET33 and CryET34 proteins in US patents 6,248,535, 6,326,351, 6,399,330, 6,949,626, 7,385,107, and 7,504,229; US patent publication number 2006 / 0191034,CryET33 and CryET34 homologs as published in PCT Publication No. WO 2012 / 0278954 and PCT Publication No. WO 2012 / 139004; Cry35Ab1 protein as published in US Patent Nos. 6,083,499, 6,548,291 and 6,340,593; Cry46 protein, Cry51 protein, and Cry binary toxin as published in US Patent No. 9,403,881; TIC901 or related toxins; TIC807 as published in US Patent No. 2008 / 0295207; ET29, ET37, TIC809, TIC810, TIC812, TIC127, and TIC128 as published in PCTUS 2006 / 033867; and US Patent Application Publication No. US The engineered hemipteran toxin protein of 20160150795; AXMI-027, AXMI-036, and AXMI-038 of US Patent No. 8,236,757; AXMI-031, AXMI-039, AXMI-040, and AXMI-049 of US Patent No. 7,923,602; AXMI-018, AXMI-020, and AXMI-021 of WO 2006 / 083891; AXMI-010 of WO 2005 / 038032; WO AXMI-003 of 2005 / 021585; AXMI-008 of U.S. Patent Application Publication No. 2004 / 0250311; AXMI-006 of U.S. Patent Application Publication No. 2004 / 0216186; AXMI-007 of U.S. Patent Application Publication No. 2004 / 0210965; AXMI-009 of U.S. Patent Application Publication No. 2004 / 0210964; AXMI-014 of U.S. Patent Application Publication No. 2004 / 0197917; AXMI-004 of U.S. Patent Application Publication No. 2004 / 0197916; AXMI-028 and AXMI-029 of WO 2006 / 119457; WO AXMI-007, AXMI-008, AXMI-0080rf2, AXMI-009, AXMI-014, and AXMI-004 of U.S. Patent No. 2004 / 074462; AXMI-150 of U.S. Patent No. 8,084,416; AXMI-205 of U.S. Patent Application Publication No. 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 U.S. Patent Application Publication No. 2011 / 0263488; U.S. Patent No. 8461421 and U.S. Patent No. 8461421. AXMI046 of 8,461,422AXMI048, AXMI050, AXMI051, AXMI052, AXMI053, AXMI054, AXMI055, AXMI056, AXMI057, AXMI058, AXMI059, AXMI060, AXMI061, AXMI067, AXMI069, AXMI071, AXMI072, AXM I073, AXMI074, AXMI075, AXMI087, AXMI088, AXMI093, AXMI070, AXMI080, AXMI081, AXMI082, AXMI091, AXMI092, AXMI096, AXMI097, AXMI098, AXMI099, AXMI100, AXMI10 1. 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; AXMI-R1 and related proteins of U.S. Patent Application Publication No. 2010 / 0197592; WO 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 U.S. Patent No. 8,334,431; and AXMI-001, AXMI-002, and AXMI-002 of U.S. Patent Application Publication No. 2010 / 0298211. AXMI-030, AXMI-035, and AXMI-045; AXMI-066 and AXMI-076 of U.S. Patent Application Publication No. 2009 / 0144852; AXMI128, AXMI130, AXMI131, AXMI133, AXMI140, AXMI141, AXMI142, AXMI143, AXMI144, AXMI146, AXMI148, AXMI149, AXMI152, and AXMI153 of U.S. Patent No. 8,318,900.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; U.S. patent US AXMI079, AXMI080, AXMI081, AXMI082, AXMI091, AXMI092, AXMI096, AXMI097, AXMI098, AXMI099, AXMI100, AXMI101, AXMI102, AXMI103, AXMI104, AXMI107, AXMI108, AXMI109, AXMI110, dsAXMI111, AXMI112 of 8461421 , AXMI114, AXMI116, AXMI117, AXMI118, AXMI119, AXMI120, AXMI121, AXMI122, AXMI123, AXMI124, AXMI125 7. AXMI1268, AXMI127, AXMI129, AXMI164, AXMI151, AXMI161, AXMI183, AXMI132, AXMI138, AXMI137; U.S. patent US AXMI192 of U.S. Patent Application Publication No. 8,461,415; AXMI281 of U.S. Patent Application Publication No. 20160177332; AXMI422 of U.S. Patent No. 8,252,872; and Cry proteins with modified proteolytic sites, such as Cry1A and Cry3A, of U.S. Patent No. 8,319,019; and Cry1Ac, Cry2Aa, and Cry1Ca toxin proteins from Bacillus thuringiensis strain VBTS 2528 of U.S. Patent Application Publication No. 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, and MP249S as described in US Patent 11,492,639.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. Other Cry proteins are well known to those 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), btnomenclature.info / , which can be accessed on the World Wide Web with the “www” prefix). The insecticidal activity of Cry proteins is well known to those skilled in the art (see van Frannkenhuyzen, (2009) J. Invert. Path. 101:1-16 for a review). The use of Cry proteins as traits in transgenic plants is well known to those skilled in the art, and Cry transgenic plants (including but not limited to Cry1Ac, Cry1Ac+Cry2Ab, Cry1Ab, Cry1A.105, Cry1F, Cry1Fa2, Cry1F+Cry1Ac, Cry2Ab, Cry3A, mCry3A, Cry3Bb1, Cry34Ab1, Cry35Ab1, Vip3A, mCry3A, Cry9c, and CBI-Bt) have been approved by regulatory authorities (see Sanahuja, (2011) PlantBiotech Journal 9:283-300 and CERA (2010) Transgenic Crop Database Center for Environmental Risk Assessment (CERA), ILSI Research Foundation, Washington, D.C. Vip3Ab and Cry1Fa (US 2012 / 0317682); Cry1BE and Cry1F (US 2012 / 0311746); Cry1CA and Cry1AB (US 2012 / 0311745); Cry1F and CryCa (US 2012 / 0317681); Cry1DA and Cry1BE (US 2012 / 0331590); Cry1DA and Cry1Fa (US 2012 / 0331589); Cry1AB and Cry1BE (US 2012 / 0324606); Cry1Fa and Cry2Aa, and Cry1I and Cry1E (US 2012 / 0317682); Cry1BE and Cry1F ... 2012 / 0324605); Cry34Ab / 35Ab and Cry6Aa (US20130167269); Cry34Ab / VCry35Ab and Cry3Aa (US 20130167268); Cry1Da and Cry1Ca (US9796982); Cry3Aa and Cry6Aa (US 9798963); and Cry3A and Cry1Ab or Vip3Aa (US 9,045,766). Harmful biocides also include insecticidal lipases, including the lipid acyl hydrolase of US Patent No. 7,491,869, and cholesterol oxidases, such as those from Streptomyces (Purcell et al., (1993) BiochemBiophys Res Commun [Biochemistry and Biophysics Research Communications] 15:1406-1413). Pest-killing proteins also include VIP (nutritional insecticidal protein) toxins, as described in U.S. Patent Nos. 5,877,012, 6,107,279, 6,137,033, 7,244,820, 7,615,686, and 8,237,020. Other VIP proteins are well known to those 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). Pest-killing proteins also include MP467 complex (TC) proteins, which are available from organisms such as pathogenic bacteria, luminescent bacteria, and spore-forming bacteria (see U.S. Patent Nos. 7,491,698 and 8,084,418). Some TC proteins have "independent" insecticidal activity, and others enhance the activity of independent toxins produced by the same given organism. The toxicity of "independent" TC proteins (such as those from the genera *Luminobacterium*, *Pathobacterium*, or *Bacillus*) can be enhanced by one or more "synergists" derived from organisms of different genera. There are three main types of TC proteins. As mentioned in this article, class A proteins ("protein A") are independent 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, XptA1, and XptA2. Examples of Class B proteins are TcaC, TcdB, XptB1Xb, and XptC1Wi. Examples of Class C proteins are TccC, XptC1Xb, and XptB1Wi. Pesticide-killing proteins also include spider, snake, and scorpion venom proteins. Examples of spider venom peptides include, but are not limited to, lycotoxin-1 peptide and its mutants (US Patent No. 8,334,366).

[0170] According to the compositions and methods of the present invention, any of the pest-killing proteins listed above and disclosed herein can be targeted insecticidal peptides.

[0171] Gene silencing In some embodiments, the stacked traits may be in the form of silencing one or more targeted polynucleotides, resulting in inhibition of one or more target harmful biological peptides. In some embodiments, this silencing is achieved using an inhibitory DNA construct.

[0172] In some embodiments, one or more polynucleotides of a polypeptide or fragment or variant thereof encoding a polymerized domain polypeptide or chimeric polypeptide (comprising one or more polymerized domain polypeptides, one or more adaptor peptides, and one or more target polypeptides (e.g., target insecticidal polypeptides)) may be stacked with one or more polynucleotides encoding one or more polypeptides (as shown above) having insecticidal activity or agronomic traits, and may optionally further include one or more polynucleotides that provide gene silencing to one or more target polynucleotides discussed below.

[0173] Further transgenic conferring resistance in insects may involve using interfering RNA molecules to downregulate the expression of target genes in insect pest species. RNA interference refers to the process of sequence-specific posttranscriptional gene silencing in animals mediated by short interfering RNA (siRNA) (Fire et al., (1998) Nature [Nature] 391:806). RNAi transgenics can include, but are not limited to, dsRNA, siRNA, miRNA, iRNA, antisense RNA, or sense RNA molecules that downregulate the expression of target genes in insect pests. PCT Publication WO 2007 / 074405 describes methods for inhibiting the expression of target genes in invertebrate pests, including the Colorado potato beetle. PCT Publication WO 2005 / 110068 describes methods for inhibiting the expression of target genes in invertebrate pests, particularly the western maize rootworm, as a means of controlling insect infestation. In addition, PCT Publication WO 2009 / 091864 describes compositions and methods for inhibiting target genes from insect pest species, including pests from the genus *Hemiberlesia lataniae*.

[0174] RNAi transgenes targeting the H subunit of vacuole ATPase can be used to control populations and infections of harmful Coleoptera, as described in U.S. Patent Application Publication 2012 / 0198586. PCT Publication WO 2012 / 055982 describes ribonucleic acid (RNA or double-stranded RNA) encoding the following target genes that inhibit or downregulate expression: insect ribosomal proteins, such as ribosomal protein L19, ribosomal protein L40, or ribosomal protein S27A; insect proteasome subunits, such as Rpn6, Pros 25, Rpn2, proteasome β1 subunit protein, or Pros β2 protein; insect β-exoclase of COPI vesicles, γ-exoclase of COPI vesicles, β'-exoclase protein of COPI vesicles, or ζ-exoclase; insect tetraspanin 2. Protein A (proposed transmembrane domain protein); insect proteins belonging to the actin family, such as actin 5C; insect ubiquitin-5E protein; insect Sec23 protein, which is a GTPase activator involved in intracellular protein transport; insect wrinkling proteins that are unconventional myosin involved in locomotion; insect neck-bending proteins involved in the regulation of nuclear alternative mRNA splicing; insect vesicle H+-ATPase G subunit proteins; and insect Tbp-1 such as Tat-binding proteins. PCT Publication WO 2007 / 035650 describes ribonucleic acid (RNA or double-stranded RNA) that inhibits or downregulates the expression of target genes encoding Snf7. U.S. Patent Application Publication 2011 / 0054007 describes a polynucleotide silencing element targeting RPS10. PCT Publication WO 2016 / 205445 describes polynucleotide silencing elements and target polynucleotides that reduce fertility, including NCLB, MAEL, BOULE, and VgR. U.S. Patent Application Publications 2014 / 0275208 and US2015 / 0257389 describe polynucleotide silencing elements targeting RyanR (DvSSJ1) 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 exosome subunit nucleic acid molecules that confer resistance to Coleoptera and Hemiptera pests.U.S. Patent Application Publications 2012 / 029750, US 20120297501, and 2012 / 0322660 describe interfering RNAs (RNA or double-stranded RNA) that, when taken up by insect pest species, function to downregulate the expression of target genes in the insect pests, wherein the RNA contains at least one silencing element, wherein the silencing element is a double-stranded RNA region comprising an annealed complementary strand, one strand of the double-stranded RNA region comprising or consisting of a nucleotide sequence that is at least partially complementary to a target nucleotide sequence in the target gene. U.S. Patent Application Publication 2012 / 0164205 describes potential targets for interfering with double-stranded RNA to inhibit harmful invertebrate organisms, including: Chd3 homologous sequences, β-tubulin homologous sequences, 40 kDa V-ATPase homologous sequences, EF1α homologous sequences, 26S protein subunit p28 homologous sequences, juvenile hormone epoxide hydrolase homologous sequences, swelling-dependent chloride channel protein homologous sequences, glucose-6-phosphate 1-dehydrogenase protein homologous sequences, Act42A protein homologous sequences, ADP-ribosome factor 1 homologous sequences, transcription factor IIB protein homologous sequences, chitinase homologous sequences, ubiquitin conjugation enzyme homologous sequences, glyceraldehyde-3-phosphate dehydrogenase homologous sequences, ubiquitin B homologous sequences, juvenile hormone esterase homologs, and α-tubulin homologous sequences.

[0175] Applications in pest control The general methods for using strains containing nucleic acid sequences or variants thereof from the embodiments as pest control agents or for the engineering of other organisms are known in the art.

[0176] Microbial hosts known to occupy the "phytosphere" (leaf surface, leaf margin, rhizosphere, and / or root surface) of one or more target crops can be selected. These microorganisms are selected to be able to successfully compete with wild-type microorganisms in a specific environment, provide stable maintenance and expression of one or more genes expressing one or more polymerized domain peptides or chimeric fusion peptides (containing one or more polymerized domain peptides, one or more adaptor peptides, and one or more target peptides (e.g., target insecticidal peptides)), and ideally, increase protection against pests from environmental degradation and inactivation.

[0177] Alternatively, multi-domain peptides or chimeric peptides (comprising one or more multi-domain peptides, one or more adaptor peptides, and one or more target peptides (e.g., target insecticidal peptides)) can be produced by introducing a heterologous gene into a cell host. Expression of the heterologous gene directly or indirectly leads to the production and maintenance of the pest-killing agent within the cell. The cells are then treated under conditions that prolong the activity of the toxin produced in the cell when the cells are applied to the environment of the target pest. The resulting product retains the toxicity of the toxin. These naturally encapsulated multi-domain peptides or chimeric peptides (comprising one or more multi-domain peptides, one or more adaptor peptides, and one or more target peptides (e.g., target insecticidal peptides)) can then be formulated using conventional techniques for application to the environment inhabited by the target pest (e.g., soil, water, and plant leaves). See, for example, EPA 0192319 and the references cited therein.

[0178] Pesticide Combination In some embodiments, the active ingredient (e.g., an insecticidal peptide) can be applied in the form of a composition and can be applied simultaneously or sequentially with other compounds to the crop area or plant to be treated. These compounds can be fertilizers, herbicides, cryoprotectants, surfactants, detergents, pest-killing soaps, dormant oils, polymers, and / or time-release or biodegradable carrier formulations that allow for long-term administration to the target area after a single application. They can also be selective herbicides, chemical insecticides, viricides, microbial agents, amoebics, pest control agents, fungicides, bactericides, nematicides, molluscicides, or mixtures of several of these products, if desired, along with other agriculturally acceptable carriers, surfactants, or adjuvants commonly used in the field of formulation. Suitable carriers and adjuvants can be solid or liquid and correspond to substances commonly used in formulation technology, such as natural or recycled minerals, solvents, dispersants, wetting agents, thickeners, binders, or fertilizers. Similarly, the formulation can be prepared as an edible "bait" or molded into a pest "trap" to allow the target pest to feed on or ingest the pest-killing agent.

[0179] Methods of applying active ingredients or agrochemical compositions (containing chimeric polypeptides (comprising one or more polymerized domains, one or more linker peptides, and one or more target polypeptides (e.g., target insecticidal polypeptides)) and / or one or more toxin F polypeptides) include foliar application, seed coating, and soil application. The number of applications and the rate of application depend on the intensity of the corresponding pest infestation.

[0180] The composition can be formulated into powders, dusts, pills, granules, sprays, emulsions, colloids, solutions, etc., and can be prepared by conventional methods such as drying, lyophilization, homogenization, extraction, filtration, centrifugation, sedimentation, or concentration of cell cultures containing peptides. In all such compositions containing at least one such biocidal peptide, the peptide can be present at a concentration of about 1 to about 99% by weight.

[0181] The methods disclosed herein can be used to kill or reduce the number of Lepidoptera, Diptera, Heteroptera, Nematodes, Hemiptera, or Coleoptera pests in a given area, or can be used preventively in environmental areas to prevent the invasion of susceptible pests. Preferably, the pest ingests or comes into contact with a biocidally effective amount of the polypeptide. As used herein, "biocidally effective amount" means an amount of pest capable of causing death or significantly reducing the growth, feeding, or normal physiological development of at least one pest. This amount will vary depending on factors such as, for example, the specific target pest to be controlled, the specific environment, location, plant, crop, or agricultural site to be treated, environmental conditions, and the method, rate, concentration, stability, and quantity of application of the biocidally effective polypeptide composition. The formulation may also vary depending on climatic conditions, environmental factors, and / or application frequency and / or the severity of pest infestation.

[0182] The desired biocidal composition can be prepared by formulating a suspension of bacterial cells, crystals, and / or spores or isolated protein components using a desired agriculturally acceptable carrier. The composition can be formulated prior to application in an appropriate manner (e.g., lyophilization, freeze-drying, drying) or in an aqueous carrier, culture medium, or suitable diluent (e.g., saline or other buffer). The formulated composition can be in the form of a dust or granular material, a suspension in oil (plant or mineral) or water or oil / water emulsion, or as a wettable powder, or in combination with any other carrier material suitable for agricultural applications. Suitable agricultural carriers can be solid or liquid and are well known in the art. The term "agriculturally acceptable carrier" encompasses all adjuvants, inert components, dispersants, surfactants, thickeners, binders, etc., commonly used in biocidal formulation techniques; these are well known to those skilled in the art. Formulations can be mixed with one or more solid or liquid adjuvants and prepared by various methods (e.g., homogenizing, blending, and / or grinding the biocidal composition with a suitable adjuvant using conventional formulation techniques). Suitable formulations and application methods are known. Plants can also be treated with one or more chemical compositions, including one or more herbicides, insecticides, or fungicides. Exemplary chemical compositions include: cereal herbicides: isoproturon, bromobenzonitrile, iodobenzonitrile, phenoxy compounds, chlorsulfuron, clodinafop-methyl, quizalofop-p-ethyl, pyrifluquinazon, oxazolyl quizalofop-p-ethyl, diflubenzuron, flusulfanilamide, mesosulfuron-methyl, fensulfuron-methyl, flusulfanilamide, iodosulfuron-methyl, propanilamide, flupyrflupyrazole, mesosulfuron-methyl, flubutyrazole, cyclopyrazole, sulfonylurea, sulfonylurea, methoxysulfuron, fluthiamethoxam, oxadiazon, pyrrolidinone; cereal fungicides: carbendazim, chlorothalonil, azoxystrobin, cyclopyrrolidone, pyrimethanil, butylmorpholine, flutriafol, azoxystrobin, quinoxaline, tebuconazole, azoxystrobin, siloxystrobin, azoxystrobin, pyraclostrobin, azoxystrobin, propiconazole, thiophanate-methyl Acetazole, flupyraclostrobin; cereal insecticides: dimethoate, lambda-cyhalothrin, deltamethrin, α-cyhalothrin, β-flupyrrothrin, bifenthrin, imidacloprid, thiamethoxam, thiamethoxam, acetamiprid, dinotefuran, chlorpyrifos, methamidophos, acephate, pirimicarb, thiophanate-methyl; corn herbicides: atrazine, metolachlor, bromobenzonitrile, acetochlor, dicamba, dichloropyridine acid, (S-)-dimethalin, glufosinate, glyphosate, isoxaflutole, (S-)-metolachlor, mesosulfuron, nicosulfuron, flusulfuron, sulfadiazine, sulfadiazine, formamide sulfadiazine, pyrimethanil, tembotrione, pyrimimethoxam, ketofosulfuron, fluthiamethoxam, pyrrolizofuron;Insecticides for corn: Carbofuran, Chlorpyrifos, Bifenthrin, Fipronil, Imidacloprid, Lambda-cyhalothrin, Heptamethrin, Terbufos, Thiamethoxam, Thiamethoxam, Spirodiclofen, Chlorantraniliprole, Chlorfenapyr, Deltamethrin, Thiamethoxam, β-Cypermethrin, Cypermethrin, Bifenthrin, Lufenuron, Chlorantraniliprole, Heptamethrin, Pyrimethanil, Ethylbutazone, Cypermethrin, Thiamethoxam, Acetamiprid, Dinotefuran, Abamectin, Methionil, Spirodiclofen, Spirotetramethrin; Fungicides for corn: Seed dressing ester, Thiram, Prothioconazole, Tebuconazole, Azoxystrobin; Herbicides for cotton: Diuron, Furfluralin, MSMA, Oxyfluorfen, Prochloraz, Trifluralin, Pyrazosulfuron, Clethodim, Pyrazosulfuron-butyl, Glyphosate, Datura Herbicides: pendimethalin, pyrimethanil sodium, trifluralin, dexamethasone, glufosinate, propyzamide, sepium; Cotton insecticides: acephate, aldicarb, chlorpyrifos, cypermethrin, deltamethrin, malathion, phorate, abamectin, acetamiprid, indoxacarb, lambda-cyhalothrin, spinosad, thiamethoxam, γ-cyhalothrin, spirodiclofen, acetamiprid, flufenoxuron, chlorantraniliprole, chlorfenapyr, β-cyhalothrin, spirotetramat, thiamethoxam, thiamethoxam, fipronil, chlorantraniliprole, cyantraniliprole, spinosad, ethyl spinosad, γ-cyhalothrin, 4-[[(6-chloropyridin-3-yl)methyl](2,2-) [-difluoroethyl)amino]furan-2(5H)-one, thiamethoxam, abamectin, flupyradifurone, acetamiprid, spirodiclofen, flupyradifurone, profenofos, triazophos, endosulfan; Cotton fungicides: terbufos, metalaxyl, quinalphos; Soybean herbicides: metolachlor, bentazon, trifluralin, chlorpyrifos-ethyl, chlorpyrifos-methyl, oxadiazon, flusulfanilamide, pyrifluquinazon, glyphosate, methoxyfenozide, metribuzin, imidacloprid, (S-)-metolachlor, cypermethrin, pendimethalin, doxycycline, glufosinate; Soybean insecticides: lambda-cyhalothrin, methomyl, parathion, thiocarb, imidacloprid, thiamethoxam, thiamethoxam, acetamiprid, dinotefuran, chlorantraniliprole Chlorantraniliprole, cypermethrin, spinosad, ethyl spinosad, inmethrin-benzoate, fipronil, acetamiprid, deltamethrin, β-cyhalothrin, γ and λ cyhalothrin, 4-[[(6-chloropyridin-3-yl)methyl](2,2-difluoroethyl)amino]furan-2(5H)-one, spirotetramat, spirodiclofen, chlorfenapyr, flufenoxuron, thiamethoxam, β-cyhalothrin; Soybean fungicides: azoxystrobin, cyclohexane, flutriafol, fenpyroxenol, pyraclostrobin, tebuconazole, azoxystrobin, prothioconazole, tetraflufenozide; Canola herbicides: dichloropyridinic acid, quizalofop-p-ethyl, pyrifluquinazon, glufosinate, glyphosate, pyrimethanil, trifluralin, sulfanilamide, quinclorac, quizalofop-p-ethyl, clethodim, doxycycline;Canola rapeseed fungicides: azoxystrobin, carbendazim, fludioxonil, iprodione, propiconazole, tebuconazole; Canola rapeseed insecticides: carbofuran, organophosphates, pyrethroids, thiamethoxam, deltamethrin, imidacloprid, thiamethoxam, acetamiprid, dinotefuran, β-cyhalothrin, γ and λ cyhalothrin, τ-cyhalothrin, acetamiprid, spinosad, ethyl spinosad, chlorantraniliprole, chlorantraniliprole, cyantraniliprole, 4-[[(6-chloropyridin-3-yl)methyl](2,2-difluoroethyl)amino]furan-2(5H)-one.

[0183] In some embodiments, the herbicide is atrazine, chlorpyrifos, diuron, chlorsulfuron, metsulfuron-methyl, thifensulfuron-methyl, bensulfuron-methyl, acetochlor, dicamba, isoxaflutole, nicosulfuron, sulfadiazine, pyrimethanil sodium, propyzoxystrobin, chlorsulfuron-ethyl, metribuzin, quizalofop-P-ethyl, S-metolachlor, hexazinne, or a combination thereof.

[0184] In some embodiments, the insecticide is cypermethrin, chlorantraniliprole, methomyl, indoxacarb, glufosinate, or a combination thereof.

[0185] Pesticide and insecticide activity "Pests" include, but are not limited to, insects, fungi, bacteria, nematodes, mites, ticks, etc. Insect pests include insects selected from the following orders: Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., especially Lepidoptera.

[0186] Those skilled in the art will recognize that not all compounds are equally effective against all pests. The compounds of the examples show activity against insect pests, which may include economically important agronomic, forestry, greenhouse, nursery ornamental plant, food and fiber, public and animal health, domestic and commercial structures, and household and stored product pests.

[0187] Lepidoptera larvae include, but are not limited to: noctuid moths, cutworms, inchworms, and subfamily Noctuidae (Noctuidae family); fall armyworm (Spodoptera frugiperda JE Smith); beet armyworm (S. exigua Hübner); tobacco cutworm (S. litura Fabricius); bertha armyworm (Mamestra configurata Walker); cabbage moth (M. brassicae Linnaeus); black cutworm (Agrotis ipsilon Hufnagel); western cutworm (A. orthogonia Morrison); and granulate cutworm (A. subterranea Fabricius). Cutworm); Cotton leaf worm (Alabama argillacea Hübner); Cabbage looper (Trichoplusia ni Hübner); Soybean looper (Pseudoplusia includens Walker); Green cloverworm (Hypena scabra Fabricius); Tobacco budworm (Heliothis virescens Fabricius); Armyworm (Pseudaletia unipuncta Haworth); Rough skinned cutworm (Athetis mindara Barnes and Mcdunnough); Darksided cutworm (Euxoa messoria Harris). (cutworm); cotton bollworm (Earias insulana Boisduval); spiny bollworm; green-striped borer moth (E.)* *Vitella Fabricius* (spotted bollworm); *Helicoverpa armigera Hübner* (American bollworm); *Cornus earworm* (corn earworm or cotton bollworm); *Melanchra picta Harris* (zebra caterpillar); *Egira (Xylomyges) curialis Grote* (citrus cutworm); borers, sheath moths, web-forming insects, coneworms, and skeletonizers from the family Pyralidae (*Ostrinia nubilalis* Hübner, European corn borer); *Amyelois transitella Walker* (naval orange borer). Orange worm); Mediterranean flour moth (Anagasta kuehniella Zeller); Almond moth (Cadra cautella Walker); Rice stem borer (Chilo suppressalis Walker); Sorghum borer (C. partellus); Rice moth (Corcyracephalonica Stainton); Corn root webworm (Crambus caliginosellus Clemens); Bluegrass webworm (Crambus teterrellus Zincken); Rice leaf roller (Cnaphalocrocis) medinalis Guenée (rice leaf roller); Desmia funeralis Hübner (grape leaffolder); Diaphania hyalinata Linnaeus (melonworm); D. cucumber hyalinata (Cucumber hyalinata).llidis Stoll (pickleworm); Diatraea grandiosella Dyar (southwestern corn borer); sugarcane borer (D.Sugarcane borer (Saccharalis Fabricius); Mexican rice borer (Eoreuma loftiniDyar); Tobacco moth (Ephestia elutella Hübner); Great wax moth (Galleria mellonella Linnaeus); Sod webworm (Herpetogramma licarsisalis Walker); Sunflower moth (Homoeosoma electellum Hulst); Lesser corn stalk borer (Elasmopalpus lignosellus Zeller); Small wax moth (Achroia grisella) Fabricius (lesser waxmoth); Loxostege sticticalis Linnaeus (beet webworm); Orthaga thyrisalis Walker (tea tree web moth); Marucatestulalis Geyer (bean pod borer); Plodia interpunctella Hübner (Indian meal moth); Scirpophaga incertulas Walker (yellow stem borer); Udea rubigalis Guenée (celery leafroller); and leafrollers, aphids, seed borers, and fruit borers of the Tortricidae family, including the western black-headed longwinged leafroller (Acleris gloverana). Walsingham (Western blackheaded budworm); Eastern blackheaded longwinged roller (A.* *Variana Fernald* (Eastern blackheaded budworm); *Archips argyrospila Walker* (fruittree leaf roller); *A. rosana Linnaeus* (European leafroller); and other species of the genus *Variana*, including *Adoxophyes orana Fischer von Rösslerstamm* (summer fruit tortrix moth); *Cochylishospes Walsingham* (banded sunflower moth); *Cydialatiferreana Walsingham* (filbertworm); *C. pomonella Linnaeus* (codling moth); and *Platynota flavedana Clemens* (variegated rice leaf roller). leafroller); Dutch carnation leafroller (P. )stultana Walsingham (omnivorous leafroller); Lobesia botrana Denis & Schiffermüller (European grape vine moth); Spilonota ocellana Denis & Schiffermüller (eyespotted bud moth); Endopiza viteana Clemens (grape berry moth); Eupoecilia ambiguella Hübner (grape vine moth); Bonagotasalubricola Meyrick (Brazilian apple leafroller); Grapholita molesta Busck (oriental fruit moth); Suleima helianthana Riley (sunflower bud moth); species of the genus *Argyrotaenia*; species of the genus *Choristoneura*.

[0188] Other agronomical pests selected from the Lepidoptera include, but are not limited to, the fall cankerworm (Alsophilapometaria Harris); the peach twig borer (Anarsia lineatella Zeller); and the orange-striped rhinoceros moth (Anisota senatoria JE).Smith (orange striped oakworm); Antheraea pernyi Guérin-Méneville (Chinese oak tussah moth); Bombyx mori Linnaeus (silkworm); Bucculatrix thurberiella Busck (cotton leaf perforator); Colias eurytheme Boisduval (alfalfa caterpillar); Datanaintegerrima Grote & Robinson (walnut caterpillar); Dendrolimus sibiricus Tschetwerikov (Siberian silk moth); Ennomos subsignaria Hübner (elm looper moth) spanworm); linden looper (Erannistiliaria Harris); browntail moth (Euproctis chrysorrhoea Linnaeus); wild cotton skeletonizer (Harrisina americana Guérin-Méneville); range caterpillar (Hemileuca oliviae Cockrell); fall webworm (Hyphantria cunea Drury); tomato pinworm (Keiferia lycopersicella Walsingham); Eastern hemlock looper (Lambdina fiscellaria fiscellaria Hulst); western hemlock looper (L.fiscellaria lugubrosa Hulst (Western hemlock looper); Leucoma salicis Linnaeus (Satin moth); Lymantria dispar Linnaeus (Gypsy moth); Manduca quinquemaculata Haworth (Five-spotted hawkmoth, tomato hornworm); M. sexta Haworth (Tomato hornworm, tobacco hornworm); Operaphtera brumata Linnaeus (Winter moth); Paleacrita vernata Peck (Spring cankerworm); Papilio cresphontes Cramer (giantswallowtail, orange dog); California oakworm (Phryganidia californica Packard); Citrus leafminer (Phyllocnistis citrella Stainton); spotted tentiform leafminer (Phyllonorycterblancardella Fabricius); large white butterfly (Pieris brassicae Linnaeus); small white butterfly (P. rapae Linnaeus); dark-veined cabbage 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 Guenée (omnivorous looper); Schizura concinna JESmith (redhumped caterpillar); Sitotroga cerealella Olivier (Angoumois) The following moths are listed: * ...

[0189] The target species include adult and immature Diptera, including the corn leafminer (Agromyza parvicornis Loew); chironomids (including but not limited to: sorghum midge (Contarinia sorghicola Coquillett); Hessian fly (Mayetiola destructor Say); wheat midge (Sitodiplosismosellana Géhin); sunflower seed midge (Neolasiopteramurtfeldtiana Felt); fruit flies (Tephritidae) and Swedish straw flies (Oscinella frit Linnaeus); and maggots, including but not limited to: seed fly (Delia platura Meigen). maggot).

[0190] The target insects include adults and nymphs of the orders Hemiptera and Homoptera, such as, but not limited to: ball aphids from the family Adelgidae, mirid bugs from the family Miridae, cicadas from the family Cicadidae, leafhoppers from the genus *Empoasca spp.* of the family Cicadidae, 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, and mealybugs from the family Pseudococcidae. Mealybugs include scale insects from the families Asterolecanidae, Coccidae, Dactylopiidae, Diaspididae, Eriococcidae, Ortheziidae, Phoenicococcidae, and Margarodidae; lace bugs from the family Ticetridae; stink bugs from the family Pentatomidae; cinch bugs and other seed bugs from the genus Lygaeidae; foamhoppers from the family Cercopidae; pumpkin bugs from the family Coreidae; and autumn chiggers and cotton bugs from the family Pyrrhocoridae.

[0191] Important agricultural members from the order Hemiptera include, but are not limited to: pea aphid (Acyrthisiphonpisum Harris); cowpea aphid (Aphis craccivora Koch); black bean aphid (A. fabae Scopoli); cotton aphid (A. gossypii Glover); corn root aphid (A. maidiradicis Forbes); apple aphid (A. pomi De Geer); and spirea aphid (A.Spiraecola Patch, Spiraea 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); Rice-wheat aphid (R.*Padi Linnaeus* (bird cherry-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 root phylloxera). phylloxera); tobacco whitefly (Bemisia tabaci Gennadius) (tobacco whitefly, sweet potato whitefly); silver leaf whitefly (B. argentifolii Bellows & Perring); citrus whitefly (Dialeurodes citri Ashmead, citrus whitefly); banded winged whitefly (Trialeurodes abutiloneus) (banded winged whitefly and greenhouse whitefly (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 Stål (rice leafhopper); Nilaparvata lugens Stål (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 leafhopper); *Magicicadaseptendecim Linnaeus* (periodical cicada); *Icerya purchasi Maskell* (cottony cushion scale); *Quadraspidiotus perniciosus Comstock* (San Jose scale); *Planococcus citri Risso* (citrus mealybug); *Pseudococcus spp.*(Other mealybug groups); pear psyllid (Cacopsylla pyricola Foerster, pear psylla); persimmon psyllid (Trioza diospyri Ashmead, persimmon psylla).

[0192] Important agricultural species from the order Hemiptera include, but are not limited to: *Acrosternumhilare 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-Schäffer* (cotton stainer); *Euschistus servus Say* (brownstink bug); and *E. variolarius Palisot de*. Beauvois (one-spottedstink 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. pratensis Linnaeus (L. pratensis).* *Russula 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); *Pseudatomoscelisseriatus* Reuter (cotton fleahopper).

[0193] In addition, the examples are effective against Hemiptera, such as the strawberry bug (Calocoris norvegicus Gmelin); the wild okra bug (Orthops campestris Linnaeus); the apple capsid bug (Plesiocoris rugicollis Fallen); the tomato bug (Tomato worm); the black-spotted smoke bug (Cyrtopeltis notatus Distant); the white-marked fleahopper bug (Spanagonicus albofasciatus Reuter); the honeylocust plant bug (Diaphnocoris chlorionis Say); the onion plant bug (Labopidicola allii Knight); and the cotton bug (Pseudatomoscelis seriatus Reuter). fleahopper); Adelphocoris rapidus Say (rapidplant bug); Poecilocapsus lineatus Fabricius (four-linedplant bug); Nysius ericae Schilling (false chinch bug); Nysius raphanus Howard (false chinch bug); Nezaraviridula Linnaeus (southern green stink bug); Eurygaster spp.; Coreidae spp.; Pyrrhocoridae spp.; Tinidae spp.; Blostomatidae spp.); species of the genus *Reduviidaes* and species of the genus *Cimicidae*.

[0194] This also includes adult and larval mites of the order Acari (mites), such as the wheat curl mite (Aceria tosichella Keifer); the brown wheat mite (Petrobia latens Müller); spider mites and red mites from the family Tetranychus; the European red mite (Panonychus ulmi Koch); the two-spotted spider mite (Tetranychusurticae Koch); the McDaniel mite (T. mcdanieli McGregor); the carmine spider mite (T. cinnabarinus Boisduval); and the strawberry spider mite (T. turkestani Ugarov & Nikolski). (mite); flat mites in the family Ceratomyidae, short-haired mites (Brevipalpus lewisi McGregor) (citrus flat mite); rust mites and bud gall mites in the family Eriophyidae, as well as other leaf-feeding mites and mites important to human and animal health.

[0195] The target insect pests include stink bugs and other related insect superfamilies, including but not limited to species belonging to the following families: Stink Bugs (including *Halyomorpha halys*, *Piezodorus guildini*, *Euschistus heros*, *Euschistus tristigmus*, *Dichelops furcatus*, *Dichelops melacanthus*, and *Bagrada hilaris*), Plataspidae (including *Megacoptacribraria* and *Bean plataspid*), and Scaptocoris castanea (including *Scaptocoris castanea*). Root stink bugs, and lepidopteran species including but not limited to: diamondback moths, such as corn earworms; soybean cutworms, such as soybean looper moths; and pea cutworms (such as pear bean cutworms).

[0196] Methods for measuring pest-killing activity are well known in the art. See, for example, Czapla and Lang, (1990) J. Econ. Entomol. [Journal of Economic Entomology] 83:2480-2485; Andrews et al., (1988) Biochem. J. [Journal of Biochemistry] 252:199-206; Marrone et al., (1985) J. of Economic Entomology [Journal of Economic Entomology] 78:290-293; and U.S. Patent No. 5,743,477. Typically, proteins are mixed and used in feeding assays. See, for example, Marrone et al., (1985), J. of Economic Entomology [Journal of Economic Entomology] 78:290-293. Such assays may include exposing a plant to one or more pests and determining the plant's ability to survive and / or cause the pest's death.

[0197] Nematodes include parasitic nematodes such as root-knot nematodes, cyst nematodes, and saprophytic nematodes, including species of the genera *Heterodera*, *Meloidogyne*, and *Globodera*; particularly members of the cyst nematode genera, including but not limited to: *Heteroderaglycines* (soybean cyst nematode); *Heteroderaschachtii* (beet cyst nematode); *Heterodera avenae* (cereal cyst nematode); and *Globodera rostochiensis* and *Globodera pailida* (potato cyst nematodes). Nematodes include species of the genus *Pratylenchus* spp.

[0198] Seed treatment To protect and enhance yield production and trait technology, seed treatment programs can provide additional crop planning flexibility and cost-effective control against insects, weeds, and diseases. Seed material can be treated with compositions containing combinations of chemical or biological herbicides, herbicide safeners, insecticides, fungicides, germination inhibitors and enhancers, nutrients, plant growth regulators and activators, bactericides, nematicides, birdicides, and / or molluscicides, typically as a surface treatment. These compounds are typically formulated with additional carriers, surfactants, or adjuvants commonly used in the formulation field. Coatings can be applied by impregnating the propagating material with a liquid formulation or by coating with a combination of wet or dry formulations. Examples of various types of compounds that can be used as seed treatments are provided below: The Pesticide Manual: A World Compendium, edited by CDSTomlin, published by the British Crop Production Council.

[0199] Seed treatments that can be used on crop seeds include, but are not limited to, one or more of the following: abscisic acid, acibenzolar-S-methyl, abamectin, glyphosate, azaconazole, *Azospirillum*, azadirachtin, pyraclostrobin, *Bacillus* species (including one or more of *Bacillus cereus*, *Bacillus firmus*, *Bacillus megaterium*, *Bacillus pumilis*, *Bacillus sphaericus*, *Bacillus subtilis*, and / or *Bacillus thuringiensis* species), and *bradyrhizobium* species (including *bradyrhizobium betae*, *bradyrhizobium canariense*, and *bradyrhizobium espp.*). *Iriomote elkanii*, *bradyrhizobium iriomotense*, *bradyrhizobium japonicum*, *bradyrhizobium liaonigense*, *bradyrhizobium pachyrhizi*, and / or *bradyrhizobium yunmingensis* One or more of the following (yuanmingense): captan, carbendazim, chitosan, thiamethoxam, copper, cyantraniliprole, difenoconazole, chlorpyrifos, fipronil, fludioxonil, fluazinam, fluquinazole, chlorpyrifos, fluazinam, chlorpyrifos oxime, hypersensitive protein, imidacloprid, imidacloprid, tebuconazole, isoflavones, lipid chitosan oligosaccharides, mancozeb, manganese, mancozeb, metalaxyl, metalaxyl, tebuconazole, cyproconazole, PCNB, fluazinam, penicillin, pyraclostrobin, permethrin, azoxystrobin, prothioconazole, pyraclostrobin, chlorantraniliprole, methyl metolachlor, saponins, fluazinam, TCMTB, tebuconazole, thiabendazole, thiamethoxam, thiocarb, thiram, methyl thiophanate, triazole, Trichoderma, azoxystrobin, tebuconazole and / or zinc. PCNB seed coat refers to EPA registration number 00293500419, which contains quinalphos and chlorpyrifos. TCMTB refers to 2-(thiocyanomethylthio)benzothiazole.

[0200] Seed varieties and seeds with specific transgenic traits can be tested to determine which seed treatments and application rates can complement such varieties and transgenic traits, thereby increasing yield. For example, varieties with good yield potential but susceptibility to head smut can benefit from seed treatments that provide protection against head smut, and varieties with good yield potential but susceptibility to cyst nematodes can benefit from seed treatments that provide protection against cyst nematodes, etc. Similarly, varieties that include transgenic traits conferring insect resistance can benefit from a second mode of action conferred by seed treatments, and varieties that include transgenic traits conferring herbicide resistance can benefit from seed treatments that use safeners (which enhance the plant's resistance to that herbicide, etc.). Furthermore, when combined with seed treatments, the good root establishment and early emergence resulting from the proper use of seed treatments can lead to more efficient nitrogen use, better drought resistance, and an overall increase in the yield potential of one or more varieties containing a particular trait.

[0201] Methods for killing insect pests and controlling insect populations In some embodiments, methods for killing insect pests are provided, the methods comprising contacting the insect pest simultaneously or sequentially with an insecticidally effective amount of a target recombinant insecticidal polypeptide and a polymerized domain polypeptide of the present disclosure. In some embodiments, methods for killing insect pests are provided, the methods comprising contacting the insect pest with an insecticidally effective amount of one or more target recombinant insecticidal proteins and a polymerized domain polypeptide (having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more of SEQ ID NO: 1, 2, or 8-16) or a variant thereof or an active fragment thereof. In other embodiments, methods for killing insect pests are provided, comprising contacting the insect pest with an insecticidal amount of a recombinant chimeric fusion polypeptide (comprising one or more polymerized domain polypeptides (having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more of SEQ ID NO: 1, 2, or 8-16), one or more linker peptides (having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more of SEQ ID NO: 3-7 or 17), and one or more targeted insecticidal polypeptides) or variants thereof or active fragments thereof.

[0202] In some embodiments, methods for controlling insect pest populations are provided, the methods comprising contacting the insect pest population simultaneously or sequentially with an insecticidally effective amount of an insecticidal composition comprising one or more polymerized domain peptides (having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more of SEQ ID NO: 1, 2, or 8-16) or variants or active fragments thereof and a target insecticidal peptide. In other embodiments, methods for controlling insect pest populations are provided, comprising contacting the insect pest population with an insecticidally effective amount of an insecticidal composition comprising a recombinant chimeric polypeptide having one or more polymerized domain polypeptides (having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more of SEQ ID NO: 1, 2, or 8-16), one or more linker peptides (having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more of SEQ ID NO: 3-7 or 17), and one or more targeted insecticidal polypeptides. As used herein, “controlling a pest population” or “controlling a pest” refers to any measures taken against a pest that limit the damage caused by the pest. Controlling pests includes, but is not limited to, killing pests in a certain way, inhibiting the development of pests, altering the fertility or growth of pests, so that pests cause less damage to plants, reduce the number of offspring produced, produce pests with weaker adaptability, produce pests that are vulnerable to predators, or prevent pests from eating plants.

[0203] In some embodiments, methods are provided for controlling insect pest populations resistant to cytotoxic proteins, the methods comprising contacting the insect pest population simultaneously or sequentially with an insecticidally effective amount of one or more target insecticidal polypeptides, one or more polymerized domain polypeptides of the present disclosure, and optionally one or more adaptor peptides of the present disclosure. In some embodiments, methods are provided for controlling insect pest populations resistant to cytotoxic proteins, the methods comprising contacting the insect pest population with an insecticidally effective amount of one or more target insecticidal polypeptides, one or more polymerized domain polypeptides (having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more of SEQ ID NO: 1, 2, or 8-16) or variants thereof or active fragments thereof, and optionally one or more adaptor peptides (having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more of SEQ ID NO: 3-7 or 17).

[0204] In some embodiments, methods are provided for controlling insect pest populations resistant to cytotoxic proteins, the methods comprising contacting the insect pest population simultaneously or sequentially with an insecticidally effective amount of the recombinant toxin F polypeptide of the present disclosure. In some embodiments, methods are provided for controlling insect pest populations resistant to cytotoxic proteins, the methods comprising contacting the insect pest population, respectively, with an insecticidally effective amount of the recombinant toxin F polypeptide of SEQ ID NO: 67 or a variant thereof or an insecticidal active fragment.

[0205] In some embodiments, methods for protecting plants from insect pests are provided, the methods comprising expressing at least one recombinant polynucleotide encoding an insecticidal effective composition in the plant or its cells, the insecticidal effective composition comprising one or more polymerized domain polypeptides of the present disclosure, optionally a linker peptide of the present disclosure, and a target insecticidal polypeptide. In some embodiments, methods for protecting plants from insect pests are provided, the methods comprising expressing a recombinant polynucleotide encoding an insecticidal effective composition in the plant or its cells, the insecticidal effective composition comprising one or more polymerized domain polypeptides (having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more of SEQ ID NO: 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 with one or more of SEQ ID NO: 3-7 or 17), and a target insecticidal polypeptide.

[0206] Insect Control Management (IRM) Strategies Expression of Bacillus thuringiensis δ-endotoxin in transgenic maize plants has proven to be an effective means of controlling agriculturally important insect pests (Perlak et al., 1990; 1993). However, in some cases, insects have evolved resistance to Bacillus thuringiensis δ-endotoxin expressed in transgenic plants. If such resistance is widespread, it will obviously limit the commercial value of germplasm containing genes encoding such Bacillus thuringiensis δ-endotoxin.

[0207] One approach to increasing the effectiveness of genetically modified insecticides against target pests while simultaneously reducing the development of insecticide-resistant pests is to use non-GMO (i.e., non-insecticide protein) sanctuaries (a portion of the non-insecticide crop / corn) alongside GMO crops that produce a single insecticide protein active against the target pest. The United States Environmental Protection Agency (epa.gov / oppbppdl / biopesticides / pips / bt_corn_refuge_2006.htm, accessible with the www prefix) has published requirements for use with GMO crops producing a single Bt protein active against the target pest. Additionally, the National Corn Growers Association provides similar guidance on sanctuary requirements on its website (ncga.com / insect-resistance-management-fact-sheet-bt-corn, accessible with the www prefix). Larger sanctuaries may reduce overall yield due to insect damage within the sanctuary.

[0208] Expressing transgenic insecticidal proteins at high doses (killing 99.99% of susceptible insects) would result in greater persistence of such insecticidal traits (Tabashnik and Carrier. Nature Biotechnology 35:926. 2017). In one embodiment, a chimeric peptide composition comprising a polymerized domain peptide and at least one targeted insecticidal polypeptide eliminates negative host effects (e.g., phytotoxicity) and produces more protein, enabling the production of high-dose levels of the active ingredient and mitigating insect resistance development.

[0209] Another approach to increase the effectiveness of genetically modified insecticides against target pests while simultaneously reducing the development of insecticide-resistant pests is to have a reservoir of insecticidal genes that can effectively combat insect pests and exert their effects through different modes of action.

[0210] Expressing two or more insecticidal compositions toxic to the same insect species in plants, each insecticide expressed at an effective level, is another method for controlling the development of resistance. This is based on the principle that resistance evolution against two different modes of action is far less likely than against only one. For example, Roush outlines a dual-toxin strategy for managing insecticidal transgenic crops, also known as a “pyramid structure” or “stack” (The Royal Society. Phil. Trans. R.Soc. Lond. B. [Philosophical Transactions of the Royal Society, London, Series B], (1998) 353:1777-1786). A stack or pyramid structure of two different proteins, each effectively resistant to the target pest and with little or no cross-resistance, can allow for the use of smaller shelters. The U.S. Environmental Protection Agency requires significantly fewer structured shelters (typically 5%) for non-Bt maize grown compared to single-trait products (typically 20%). Various methods exist for providing IRM effects of shelter, including various geometric planting patterns in the field and in-bag seed mixes, as further discussed by Roush.

[0211] In some embodiments, chimeric peptides comprising polymerized domain peptides (having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more of SEQ ID NO: 1, 2, or 8-16), one or more adapter peptides (having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more of SEQ ID NO: 3-7 or 17), and one or more insecticidal peptides of the present disclosure may be combined with other pest-killing proteins or other transgenic (i.e., RNAi traits) (including but not limited to Bt toxins, insecticidal proteins of pathogenic Bacillus species or luminescent Bacillus species, other insecticidal active proteins, etc.) (i.e., pyramidalization) as an insect control strategy.

[0212] Methods for promoting insect-resistant management in transgenic plants for controlling Lepidoptera and / or Coleoptera and / or Hemiptera insect infestation are provided, comprising expressing or co-expressing in the plant at least one polymerized domain peptide, and one or more targeted insecticidal peptides (targeting Lepidoptera and / or Coleoptera and / or Hemiptera insects), and optionally at least one cleavable linker peptide.

[0213] In some embodiments, methods for controlling Lepidoptera and / or Coleoptera and / or Hemiptera insect infestation and promoting insect resistance management in transgenic plants include presenting at least one polymerized domain peptide, and one or more targeted insecticidal peptides (targeting Lepidoptera and / or Coleoptera and / or Hemiptera insects), and optionally at least one cleavable linker peptide.

[0214] In some embodiments, the method of controlling Lepidoptera and / or Coleoptera and / or Hemiptera insect infestation and promoting insect resistance management in transgenic plants includes presenting at least one polymerized domain peptide (having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more of SEQ ID NO: 1, 2, or 8-16) or a variant or active fragment thereof, and one or more targeted insecticidal polypeptides (targeting Lepidoptera and / or Coleoptera and / or Hemiptera insects), and optionally at least one cleavable linker peptide.

[0215] In some embodiments, a method for controlling Lepidoptera and / or Coleoptera and / or Hemiptera insect infestation and promoting insect resistance management in transgenic plants includes expressing in the transgenic plant a polymerized domain peptide fused with the target insecticidal polypeptide (having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more of SEQ ID NO: 1, 2, or 8-16) or a variant thereof or an insecticidal active fragment, as well as Cry protein or other insecticidal proteins (targeting Lepidoptera and / or Coleoptera and / or Hemiptera insects), wherein the target insecticidal polypeptide and Cry protein have different modes of action.

[0216] Methods for reducing the likelihood of Lepidoptera and / or Coleoptera and / or Hemiptera insects developing resistance to transgenic plants (in which insecticidal proteins are expressed to control insect species) are also provided, including expressing at least one polymerizable domain peptide with polymerizing activity and a combination of the target insecticidal polypeptide (targeting these insect species) with a second insecticidal protein having a different mode of action against these insect species.

[0217] It also provides means for the effective management of Lepidoptera and / or Coleoptera and / or Hemiptera insect resistance in transgenic plants, which include the high-level expression or co-expression in plants of two or more insecticidal proteins or other insecticidal transgenes (e.g., RNAi traits) that are toxic to Lepidoptera and / or Coleoptera and / or Hemiptera insects, but each exhibits a different pattern of exercising its killing activity, wherein the two or more insecticidal proteins or other insecticidal transgenes comprise polymerized domain peptides and Cry proteins. Means for effectively managing Lepidoptera and / or Coleoptera and / or Hemiptera insect resistance in transgenic plants are also provided, including expressing or co-expressing in plants two or more insecticidal proteins or other insecticidal transgenes (e.g., RNAi traits) that are toxic to Lepidoptera and / or Coleoptera and / or Hemiptera, but each exhibits a different pattern of exercising its killing activity, at high levels, wherein the two or more insecticidal proteins or other insecticidal transgenes comprise a polymerized domain peptide fused to the said target insecticidal polypeptide (having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more of SEQ ID NO: 1, 2, or 8-16) or a variant thereof or an insecticidal active fragment, as well as Cry protein or other insecticidal active proteins.

[0218] Additionally, methods are provided for obtaining regulatory approval for the cultivation or commercialization of plants expressing a multipolymerized domain fused with the target insecticidal peptide against Lepidoptera and / or Coleoptera and / or Hemiptera insects. These methods include the steps of: referencing, submitting, or relying on insect assay binding data showing that the multipolymerized domain peptide fused with the target insecticidal peptide (having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more of SEQ ID NO: 1, 2, or 8-16) or a variant thereof or an insecticidal active fragment does not compete for binding sites with Cry proteins in such insects.

[0219] Methods to increase plant yield Methods for increasing plant yields are provided. These methods include providing a plant or plant cell that expresses a polynucleotide encoding a polymerized domain peptide (fused with the target insecticidal polypeptide) disclosed herein, and growing the plant or its seeds in a field infested with a pest to which the target insecticidal polypeptide has pest-killing activity. In some embodiments, the polymerized domain fused with the target insecticidal polypeptide has pest-killing activity against Lepidoptera, Hemiptera, Diptera, or Nematode pests, and the field is infested with Lepidoptera, Hemiptera, Diptera, or Nematode pests.

[0220] As defined herein, “yield” of a plant refers to the quality and / or quantity of biomass produced by the plant. As used herein, “biomass” means any measured plant product. An increase in biomass yield is any improvement in the yield of the measured plant product. Increased plant yield has several commercial applications. For example, increasing plant leaf biomass can increase the yield of leafy vegetables for human or animal consumption. Additionally, increasing leaf biomass can be used to increase the yield of plant-derived pharmaceuticals or industrial products. Increases in yield can include any statistically significant increase, including but not limited to increases of at least 1%, at least 3%, at least 5%, at least 10%, at least 20%, at least 30%, at least 50%, at least 70%, at least 100%, or greater compared to plants that do not express pest-killing sequences.

[0221] In certain methods, plant yield is increased due to improved pest resistance in plants expressing the disclosed polymerized domain peptide fused with the target insecticidal polypeptide. Expression of the disclosed polymerized domain peptide fused with the target insecticidal polypeptide reduces the ability of pests to infect or consume the plant, thereby improving plant yield.

[0222] Processing methods Further, methods are provided for processing plants, plant parts, or seeds to obtain food or feed products from plants, plant parts, or seeds containing polymerized domain peptides. The plants, plant parts, or seeds provided herein can be processed to produce oils, protein products, and / or byproducts derived from plants, plant parts, or seeds that have commercial value through processing. Non-limiting examples include transgenic seeds containing nucleic acid molecules encoding polymerized domain peptides, which can be processed to produce soybean oil, soybean products, and / or soybean byproducts.

[0223] "Processing" means any physical and chemical method used to obtain any soybean product, and includes, but is not limited to, heat conditioning, exfoliation and milling, extrusion, solvent extraction or aqueous soaking, and whole or partial seed extraction.

[0224] Examples include compositions and methods for mitigating undesirable phenotypic traits caused by the presence of certain transgenic proteins, including insecticidal proteins, during expression in transgenic plants. Examples also include novel methods for increasing the persistence, expression levels, and efficacy of proteins in transgenic plants. Examples further include novel engineered peptides, polypeptides, and chimeric polypeptides, and methods for producing and using them.

[0225] One embodiment includes a polypeptide comprising an engineered chimeric polypeptide comprising a polymerized domain and a target insecticidal polypeptide, wherein the polymerized domain mitigates undesirable plant phenotypes exhibited by the target insecticidal polypeptide.

[0226] Another embodiment includes a polypeptide comprising an engineered chimeric polypeptide comprising a polymerizing domain and a target insecticidal polypeptide, wherein the polymerizing domain mitigates undesirable plant phenotypes exhibited by the target insecticidal polypeptide, wherein the polymerizing domain comprises a peptide having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with a peptide selected from SEQ ID NO: 1, 2, or 8-16.

[0227] Another embodiment includes a polypeptide comprising an engineered chimeric polypeptide comprising a polymerizing domain and a target insecticidal polypeptide, wherein the polymerizing domain mitigates undesirable plant phenotypes exhibited by the target insecticidal polypeptide, wherein the polymerizing domain comprises a peptide selected from SEQ ID NO: 1, 2 or 8-16.

[0228] One embodiment includes a chimeric fusion polypeptide comprising a polymerized domain and a target insecticidal polypeptide, wherein the polymerized domain and the target insecticidal polypeptide are linked by a cleavable linker sequence.

[0229] Another embodiment includes a chimeric fusion polypeptide comprising a polymerized domain and a target insecticidal polypeptide, wherein the polymerized domain and the target insecticidal polypeptide are linked by a cleavable linker sequence of any of the foregoing embodiments, wherein the chimeric fusion polypeptide exhibits reduced undesirable phenotypic characteristics in plants compared to an insecticidal polypeptide without the polymerized domain.

[0230] Another embodiment includes a chimeric fusion polypeptide comprising a polymerized domain and a target insecticidal polypeptide, wherein the polymerized domain and the target insecticidal polypeptide are linked by a cleavable linker sequence of any of the foregoing embodiments, wherein the chimeric fusion polypeptide exhibits increased expression in plants compared to an insecticidal polypeptide without the polymerized domain.

[0231] Another embodiment includes a chimeric fusion polypeptide comprising a polymerized domain peptide and a target insecticidal polypeptide, wherein the polymerized domain peptide and the target insecticidal polypeptide are linked by a cleavable linker sequence, wherein the polymerized domain is capable of interacting with other polymerized domains to form a tetramer, trimer, or dimer complex.

[0232] Another embodiment includes a chimeric fusion polypeptide comprising a polymerized domain peptide and a target insecticidal polypeptide, wherein the polymerized domain peptide and the target insecticidal polypeptide are linked by a cleavable linker sequence of any of the foregoing embodiments, wherein the polymerized domain peptide has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 1, 2, or 8-16.

[0233] Another embodiment includes a chimeric fusion polypeptide comprising a polymerized domain peptide and a target insecticidal polypeptide, wherein the polymerized domain peptide and the target insecticidal polypeptide are linked by a cleavable adapter sequence of any of the foregoing embodiments, wherein the adapter sequence has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 3-7 or 17.

[0234] Another embodiment includes a chimeric fusion polypeptide comprising a polymerized domain peptide and a target insecticidal polypeptide, wherein the polymerized domain peptide and the target insecticidal polypeptide are linked by a cleavable linker sequence of any of the foregoing embodiments, wherein the polymerized domain peptide is linked to the insecticidal polypeptide at the N-terminus of the insecticidal polypeptide.

[0235] Another embodiment includes a chimeric fusion polypeptide comprising a polymerized domain peptide and a target insecticidal polypeptide, wherein the polymerized domain peptide and the target insecticidal polypeptide are linked by a cleavable linker sequence of any of the foregoing embodiments, wherein the polymerized domain peptide is linked to the insecticidal polypeptide at the C-terminus of the insecticidal polypeptide.

[0236] One embodiment includes a polynucleotide encoding any of the chimeric fusion polypeptides of the foregoing embodiments, wherein the polynucleotide further comprises a heterologous regulatory sequence.

[0237] Another embodiment includes a DNA construct comprising any of the polynucleotides described in the foregoing embodiments.

[0238] One embodiment includes a plant or plant cell containing a chimeric fusion polypeptide comprising a polymerized domain peptide and a target insecticidal polypeptide, wherein the polymerized domain peptide and the target insecticidal polypeptide are linked by a cleavable linker sequence of any of the foregoing embodiments.

[0239] Another embodiment includes any of the plants or plant cells of the foregoing embodiments, wherein the plant or plant cells further contain one or more additional insect-resistant polypeptides.

[0240] One embodiment includes a composition comprising a chimeric fusion polypeptide containing a polymerized domain peptide and a target insecticidal polypeptide, wherein the polymerized domain peptide and the target insecticidal polypeptide are linked by a cleavable linker sequence of any of the foregoing embodiments.

[0241] One embodiment includes a method for reducing undesirable phenotypic characteristics of an insecticidal peptide in a plant, the method comprising expressing in the plant a recombinant fusion peptide comprising an engineered polymerized domain peptide and a target insecticidal peptide, wherein the engineered polymerized domain peptide and the target insecticidal peptide are linked by a linker sequence, and wherein expression of the recombinant fusion peptide reduces the undesirable phenotypic characteristics of the target insecticidal peptide compared to an insecticidal peptide lacking the engineered polymerized domain peptide. Another embodiment includes any of the foregoing embodiments of the method for reducing undesirable phenotypic characteristics of an insecticidal peptide in a plant, wherein the undesirable phenotypic characteristic is phytotoxicity.

[0242] Another embodiment includes a method for increasing the expression of an insecticidal peptide in a plant, the method comprising expressing in the plant a recombinant fusion peptide comprising an engineered polymerized domain peptide and a target insecticidal peptide, wherein the engineered polymerized domain peptide and the target insecticidal peptide are linked by a linker sequence, wherein the expression level of the target insecticidal peptide is increased compared to an insecticidal peptide lacking the engineered polymerized domain peptide.

[0243] One embodiment includes a method for increasing the efficacy of an insecticidal peptide in a plant, the method comprising expressing in the plant a recombinant fusion peptide comprising an engineered multi-merging domain peptide linked to the target insecticidal peptide, wherein the expression level of the fusion peptide comprising the engineered multi-merging domain peptide linked to the target insecticidal peptide is increased compared to an insecticidal peptide lacking the engineered multi-merging domain peptide, and wherein the increased level of the fusion peptide results in increased efficacy against the target insect pest.

[0244] One embodiment includes a method for increasing the persistence of an insecticidal peptide, the method comprising expressing in a plant a recombinant fusion peptide comprising an engineered polymerized domain peptide linked to a target insecticidal peptide, wherein the expression level of the target insecticidal peptide is increased compared to an insecticidal peptide lacking the engineered polymerized domain peptide, and wherein the increased expression level of the target insecticidal peptide results in increased persistence of the insecticidal peptide against the target insect pest.

[0245] One embodiment includes the method of any of the foregoing embodiments, wherein the polymerized domain peptide has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NO: 1, 2, or 8-16.

[0246] One embodiment includes the method of any of the foregoing embodiments, wherein the connector sequence has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NO: 3-7 or 17.

[0247] Another embodiment includes a method for determining the phytotoxicity of any of the target insecticidal proteins of the foregoing embodiments, wherein the reporter gene encodes a fluorescent protein, and the fluorescence is used to identify viable cells.

[0248] The following examples are provided in an illustrative manner, but not in a restrictive manner.

[0249] Example Some insecticidal proteins, when expressed in plants under certain conditions, may induce undesirable phenotypic effects in certain insects, such as, but not limited to, phytotoxicity. Methods are needed to reduce the undesirable phenotypic characteristics (e.g., phytotoxicity) of insecticidal proteins when expressed in plants under certain conditions. Reducing the undesirable phenotypic characteristics (e.g., phytotoxicity) of insecticidal proteins in plants can also increase the expression of insecticidal proteins in plants, which can lead to increased efficacy against target insects and can also lead to increased persistence of the insecticidal proteins. The following examples describe a method for reducing the undesirable phenotypic characteristics (e.g., phytotoxicity) of insecticidal proteins by fusing a polymerizing domain with the insecticidal protein. Fusing a polymerizing domain with the insecticidal protein can mitigate undesirable phenotypic characteristics (e.g., phytotoxicity) while retaining the desired efficacy in insects. This method can be used for many insecticidal proteins.

[0250] In one embodiment, the fusion protein containing a multipolymerized domain and an insecticidal protein undergoes multipolymerization, which can isolate the fusion peptide molecule in an inactive state by inhibiting its downstream functions.

[0251] In another embodiment, the engineered fusion protein (a trimerized domain fused with an insecticidal protein) may contain a specific activation site between the trimerized domain (TMD) and the insecticidal protein, such that the insecticidal protein is activated only in insects and not in plants, thereby keeping the engineered fusion protein inactive in plants.

[0252] In addition to reducing phytotoxicity, trimerization of insecticidal proteins can enhance their efficacy, for example, by increasing local concentrations in cells and / or by enabling chimeric insecticidal proteins to be expressed at higher levels. Therefore, engineered fusion proteins can mitigate undesirable phenotypic traits in plants (such as phytotoxicity) and enhance the activity of insecticidal proteins in insects.

[0253] Example 1 - Fusion of multipolymerized domains with insecticidal proteins Some insecticidal proteins (or toxins) effective against insects can exhibit undesirable phenotypic traits (e.g., phytotoxicity) in plants. To mitigate these undesirable phenotypic traits (e.g., phytotoxicity), insecticidal proteins can be engineered to specifically modulate their activity in plants by including heteropolymerization domains. By fusing the polymerization domain with one or more insecticidal proteins, the resulting chimeric molecule can, in a non-limiting hypothesis, be isolated in an inactive state. For example, in a non-limiting hypothesis, this can be achieved by the polymerization domain preventing the interaction of one or more insecticidal proteins with their homologous receptors and / or preventing the assembly of the active complex, or preventing the assembly of the pore-forming complex in the plant.

[0254] Many viruses use trimerizing domains to form spike proteins required for receptor recognition. In humans, collagen contains three monomeric helical subunits that bind together to form a stable triple helix structure. For example, these monomeric subunits can fuse with target proteins (such as insecticidal proteins), thereby causing the chimeric fusion polypeptide monomer to polymerize. The polymerizing domain can be further engineered from existing polymerizing domains or generated de novo using various protein engineering techniques. For example, monomers can be engineered to polymerize into dimer, trimer, or tetramer structures. When such engineered monomeric polypeptides fuse with target proteins, the activity of the target protein can be modulated, for example, by reducing the phytotoxic activity of the target protein in plants and / or increasing the insecticidal activity of the target protein in target pests.

[0255] Example 2 - Fusion of multipolymerized domains with different types of toxins.

[0256] One or more engineered multimerizing domains (e.g., multimerizing domain MMD1) are fused with different classes of toxins to prepare engineered fusion proteins. In one embodiment, the engineered fusion protein may contain a protease cleavage site in the linker sequence between the multimerizing domain and the toxin. Figure 1 Such cleavable engineered fusion proteins are designed to remain intact in plants, and once ingested by target insects, they can be proteolytically cleaved in the insect's gut, thereby activating the toxins (insecticides) present there. This engineered fusion protein design allows the toxins to remain inactive or significantly reduced in activity in plants, and to be activated only within the target insect.

[0257] Example 3 - Protein Expression, Purification, and Quantification Chimeric fusion proteins were generated using the Gibson assembly method. This method involves synthesizing fragments of the MMD, adapter (where applicable), and linker sequence, each approximately 200 base pairs long, corresponding to the N or C terminus of the toxin and the flanking region of the vector. These fragments were then assembled with the pET28a expression vector, which contains an upstream or downstream 6X His tag, followed by the protein. The resulting plasmid was transformed into BL21(DE3) cells from Ingenium (Waltham, MA). Protein expression was performed using a self-inducing medium. Cells were grown at 16°C for 4 days in the presence of antibiotics. The precipitate was collected and stored overnight at -80°C. The precipitate was lysed in a lysis solution (300 mM NaCl, 20 mM TRIS-HCl pH 8, 20 mM imidazole, Omnicleave, lysozyme, and a mixture of protease inhibitors). Once the sample was resuspended, the precipitate was further lysed using a homogenizer. The lysate was centrifuged at 24,000 xg for 25 min at 4°C, and the clarified lysate was carefully collected.

[0258] Resuspend the Ni-NTA resin and wash with buffer (300 mM NaCl, 20 mM TRIS-HCl pH 8, 20 mM imidazole). Incubate the clarified lysate with equilibrated resin at 4°C for 60 min, placing the sample on a mixer to ensure adequate protein binding. Wash the resin with approximately 50 CV of wash buffer to remove non-specific host proteins. Elute the protein with 3 CV of elution buffer (300 mM NaCl, 20 mM TRIS-HCl pH 8, 500 mM imidazole). Dialyze the protein to 1X PBS buffer and store at 4°C (short-term) or -80°C (long-term). Purify the sample by size exclusion chromatography. Protein concentration was obtained using an A280 nanodrop. For insect bioassays, protein concentration was determined by gel density assay. This was done to measure the number of monomers in all samples compared to WT protein by denaturing the protein and dissociating the trimer into monomeric subunits.

[0259] Example 4 - Insect bioassay of toxin A (a fern-derived toxin) with multimerized domain fusion structures In vitro feeding assays of Coleoptera were performed using a modified Western Corn Rootworm (Diabrotica virgifera virgifera) WCRW artificial feed (Southland Products Inc., Lake Village, Arkansas) in a 96-well format. A clarified and desalted sample (25 µL) was added to the feed (50 µL) and allowed to dry. 25 µL of 1X PBS buffer was added to the control wells. Larvae fed the WCRW feed for 24 hours were placed into each well. Larvae were allowed to feed at 27°C for 11 days. At day 11 post-infection, insects were visually scored as dead, severely stunted (size reduction >60% compared to control larvae), or unaffected. The total number of dead and severely stunted larvae was used to calculate the growth-inhibiting concentration (IC50) affecting 50% of the test larvae, and the total number of dead larvae was used to calculate the lethal concentration (LC50) affecting 50% of the larvae.

[0260] Multimerized domain 1 (MMD1) is engineered based on EML4 PDB (4CGC) and used to construct engineered fusion toxins. The MMD1 sequence used here is TSDVQDRLSALESRVQQQEDETVLKAA (SEQ ID NO: 8). Table 12 shows the sequences of MMD2-MMD7.

[0261] Table 1 shows the results of insect feeding bioassays for the in vitro expressed toxin A protein and the exemplary engineered fusion protein. The two components of the fusion protein (MMD and toxin) are linked together by a linker selected from one of L1-L5. LC-50 and IC-50 values ​​are shown in ppm. LC-50 is the concentration at which 50% mortality occurs, while IC-50 is the concentration at which 50% mortality occurs along with severe growth and / or developmental retardation. UCL (upper confidence limit) and LCL (lower confidence limit) show the range of LC-50 / IC-50 values ​​and reflect data quality. Overall, the fusion protein was as effective as the WT (control) protein, or more effective in some cases. For the fusion protein expressed in vitro and tested in feeding assays, the addition of the multimerizing domain terminus did not alter the efficacy compared to other fusion proteins. Linkers L1-L5 have different lengths and flexibility, and all except L1 contain cathepsin proteolytic cleavage sites. As shown in Table 1, compared to the WT control, even fusion proteins containing toxin A fused to a polymerized domain via linker L1 (a flexible linker lacking proteolytic cleavage sites) at the C or N-terminus of the protein exhibited improved insecticidal activity. This suggests that target insects can proteolytically cleave the toxin near both the N- and C-termini in the target insect gut. When engineering fusion proteins, the length of the linker and the engineered ends of the helical domains may play a crucial role. This may depend on the size of the toxin used and its behavior in solution.

[0262] Table 1: Insect feeding assay: WCRs were fed toxin A and engineered fusion protein.

[0263] Table Notes: The toxin A control is a control of toxin A that is not fused with the multimerizing domain.

[0264] Toxin A C-terminal MMD L1 is a fusion polypeptide containing toxin A, wherein a polymerized domain is fused to the C-terminus of toxin A via, for example, an intermediate L1 linker.

[0265] Toxin A N-terminal MMD L1 is a fusion polypeptide containing toxin A, wherein a polymerized domain is fused to the N-terminus of toxin A via, for example, an intermediate L1 linker.

[0266] The table below shows the different adapters used. Cathepsin proteolytic cleavage (shearing) sites are highlighted in bold.

[0267] Table 2: Connector Sequence Example 5 - Determination of phytotoxicity of toxin A with different polymerization domains.

[0268] Chimeric proteins containing different polymerization domains and toxin A were obtained through gene synthesis. The polymerization domains were fused to the C- or N-terminus of the toxin protein via linkers selected from L1-L5. The corresponding recombinant polynucleotides 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). For example, Agrobacterium strains containing each of these constructs were infiltrated into leaves, as described in Kapila et al., (1997) Plant Science 122:101-108. In short, Agrobacterium infiltration was performed on single leaves of common bean (Phaseolus vulgaris) using standardized bacterial cell cultures of test and control strains. Under the test conditions, constructs containing different transient expressions of the toxin-MMD fusion protein exhibited less phytotoxicity than constructs containing transient expression of toxin A alone (see Table 3).

[0269] All dwarf bean phytotoxicity scores were assessed based on the following: None - no negative plant phenotypes observed, Low - some negative plant phenotypes observed (bruising or slight browning), Moderate - significant negative plant phenotypes observed (browning of approximately 50% of leaf tissue), Strong - severe negative plant phenotypes (tissue death, leaf curling).

[0270] The transient protein expression of the fusion protein was confirmed using a mass spectrometry-based protein identification method with protein lysates extracted from infiltrated leaf tissues, as described in Patterson, (1998) 10(22):1-24, Current Protocol in Molecular Biology (published by John Wiley & Son Inc).

[0271] Table 3: Phytotoxicity determination of chimeric fusion proteins containing toxin A and polymerized domains in dwarf bean.

[0272] Table Notes: The toxin A control is a control containing toxin A that is not fused with the trimerizing domain.

[0273] Toxin AC-terminus-MMD1-L1 is a fusion polypeptide containing toxin A and a trimerized domain fused to the C-terminus of toxin A (via, for example, the intermediate L1 linker).

[0274] Toxin AN-terminal-MMD1-L1 is a fusion polypeptide containing toxin A and a trimerized domain fused to the N-terminus of toxin A (via, for example, the intermediate L1 linker).

[0275] Table 3 shows the results of exemplary fusion constructs containing a polymerizing domain and toxin A in transient expression experiments in dwarf bean, along with the phytotoxicity observed under the test conditions. When toxin A was fused with the polymerizing domain, most fusion constructs showed reduced phytotoxicity levels observed under the test conditions in the dwarf bean transient expression assay. The expression levels of many fusion constructs showing reduced phytotoxicity were significantly higher than those of the toxin A-control. This suggests that phytotoxicity in dwarf bean can be mitigated, potentially allowing for greater protein accumulation. Compared to the toxin A-control, phytotoxicity induced by toxin A was significantly reduced (from strong to low or moderate) when the helical domain was fused to the C-terminus of the protein via a linker-mediated process, which may further be accompanied by higher levels of fusion protein accumulation. The results in Table 3 also show that the type of linker and the type of engineered helical domain can modulate the mitigation of phytotoxicity and protein levels in the dwarf bean transient expression system.

[0276] The fusion of multimerized domain toxin A reduced phytotoxicity levels in soybean platforms. In soybean, constructs containing fusion peptides were tested for their ability to reduce phytotoxicity. The results are shown in Table 4 below. Fusion constructs were tested in two fusion orientations: (i) the "toxin AN-terminal-MMD1-L" orientation, which is a fusion peptide containing toxin A and a trimerized domain fused to the N-terminus of toxin A, wherein the two peptides are linked by an intermediate "L" linker selected from one of L1-L5; and (ii) the "toxin AC-terminal-MMD1-L" orientation, which is a fusion peptide containing toxin A and a trimerized domain fused to the C-terminus of toxin A, wherein the two peptides are linked by an intermediate "L" linker selected from one of L1-L5. As shown in Table 4, under the test conditions, all toxin-MMD fusion constructs exhibited lower phytotoxicity compared to toxin A alone.

[0277] Phytotoxicity scores are assessed based on the following: Low: 1%-20% of leaves show browning / yellowing; Mild / Moderate: 21%-60% of leaves show browning / yellowing; Strong: 61%-100% of leaves show browning / yellowing. Table 4: Phytotoxicity assay of chimeric fusion proteins containing toxin A and trimeric domains in soybeans Table Notes: The toxin A control is a control of toxin A that is not fused with the multimerizing domain.

[0278] Toxin A C-terminal MMD1 L1 is a fusion polypeptide containing toxin A and a multipolymerized domain (MMD1) fused to the C-terminus of toxin A (connected via, for example, the intermediate L1 linker).

[0279] Toxin A N-terminal MMD1 L1 is a fusion polypeptide containing toxin A and a multipolymerized domain (MMD1) fused to the N-terminus of toxin A (connected via, for example, the intermediate L1 linker).

[0280] Table 4 shows the results of phytotoxicity assays in the soybean platform. Phytotoxicity scores are provided for the phytotoxicity observed under test conditions for fusion constructs containing a polymerizing domain and toxin A. When toxin A is fused with the polymerizing domain, a decrease in phytotoxicity was observed for most fusion constructs with the polymerizing domain fused with toxin A under test conditions. For most constructs, the decrease in phytotoxicity is believed to be attributable to reduced protein expression. However, for the construct with toxin AC-terminated MMD1 L5, phytotoxicity decreased from strong to low, and protein expression levels were higher than those of the WT control protein. Constructs with the MMD1 polymerizing domain and linker L1–L5 are identical to those shown in Table 1 but were tested in different assay systems. As shown in Table 1, the polymerizing domain and linker were fused to the N- or C-terminus of toxin A.

[0281] In stable maize transformants, the fusion of polymeric domains with toxin A reduces phytotoxicity and increases insecticidal efficacy. Maize plants were transformed with a construct containing a polynucleotide encoding toxin A (fused to the multimerizing domain MMD1 via an intermediate linker sequence). As a negative control, maize plants were transformed with a construct containing a polynucleotide encoding the toxin A WT protein (toxin A - control). The following linkers were used in this study: L1 (SEQ ID NO: 3) - a flexible linker without a cathepsin cleavage site; L5 (SEQ ID NO: 7) - a linker including a cathepsin cleavage site; and L2 (SEQ ID NO: 4) - a flexible linker containing a cathepsin cleavage site.

[0282] Table 5 shows data on phytotoxicity and root damage in stable maize transformants containing a polynucleotide encoding toxin A fused with MMD1.

[0283] Protein processing in plants based on Western blot analysis indicates the extent of protein processing (proteolytic cleavage) occurring in root tissues. Results are categorized as follows: (+++): All fusion proteins were processed (proteolytically cleaved) to separate MMD1 from toxin A; (++): Two protein populations were present—full-length fusion proteins and processed proteins; (+): Minimal or no processing was observed.

[0284] Table 6. The stable maize data shown in Table 5 above are summarized by averaging for each type of fusion peptide. Notes to Tables 5 and 6: Toxin A C-terminal MMD1-L is a fusion polypeptide containing toxin A and an MMD1 polymerized domain fused to the C-terminus of toxin A, wherein the two peptides are linked by an intermediate linker L (L1, L2 or L5).

[0285] The toxin A-control is a control that expresses only the toxin A WT gene that is not fused with the multimerization domain.

[0286] Leaf area is a measurement of the surface area of ​​maize plant leaves, measured in square centimeters (cm²). This value is derived by converting WP_SV_PIXELAREA (area based on basic pixel counts). Pixel area is initially obtained by capturing digital images of maize leaves and counting the number of pixels corresponding to the leaf area using image analysis software. Each pixel represents a small unit of area; by summing the total number of pixels, we obtain an initial measurement of the leaf surface area in pixels. This pixel count is then converted to a standardized unit of measurement, such as square centimeters, using a conversion factor that takes into account the image resolution and scale. This conversion allows for standardized comparisons of leaf areas from different samples and experiments.

[0287] The CRWNIS (Corn Rootworm Intersegmental Damage Score) is based on the Iowa State University's 0-3 segmental damage scale.

[0288] Tables 5 and 6 indicate that the linker used to connect the toxin to the polymerizing domain in the chimeric fusion polypeptide may have functional relevance when the chimeric fusion protein is engineered. In the tests of this invention, the fusion protein remained intact in all tested plant samples when no cathepsin proteolytic cleavage site was used in the linker. The phytotoxicity induced by toxin A was reduced in these transgenic plants, as indicated by the following: transgenic plants expressing the toxin AC-terminal-MMD1-L chimeric fusion polypeptide had a larger leaf area compared to transgenic plants expressing toxin A without the MMD1 polymerizing domain. When a long, flexible linker containing a cathepsin proteolytic cleavage site was used, most plants showed proteolytic cleavage (processing) of the chimeric fusion polypeptide, resulting in the separation of toxin A from the polymerizing domain. The phytotoxicity in these transgenic plants (where the chimeric fusion polypeptide was processed) was comparable to that observed in transgenic plants expressing the toxin A control. When short linkers containing cathepsin proteolytic cleavage sites were used, phytotoxicity was reduced, as indicated by larger leaf area compared to transgenic plants expressing toxin A without polymerizing domains. All fusion constructs and toxin A-controls showed varying degrees of protection. Notably, transgenic plants expressing recombinant polynucleotides encoding chimeric fusion proteins (containing toxin A and MMD1) showed increased protein levels in the plants compared to transgenic plants expressing only the polynucleotide encoding toxin A (control). As suggested by CRWNIS data, the increased levels of chimeric fusion proteins in the plants compared to control transgenic plants expressing only toxin A may have provided better protection against WCRW in the roots.

[0289] Example 6 - Insect bioassay of toxin B (a non-Bt-derived toxin) with multimerized domain fusions In vitro feeding assays of Coleoptera were performed using a modified Western Corn Rootworm (corn root leaf beetle) WCRW artificial diet (Southland Products, Lake Village, Arkansas) in a 96-well format. A clarified and desalted sample (25 µL) was added to the diet (50 µL) and allowed to dry. 25 µL of 1X PBS buffer was added to the control wells. Larvae fed the WCRW diet for 24 hours were placed into each well. Larvae were allowed to feed at 27°C for 11 days. At day 11 post-infection, insects were visually scored as dead, severely stunted (size reduction > 60% compared to control larvae), or unaffected. The total number of dead and severely stunted larvae was used to calculate the growth-inhibiting concentration (IC50) affecting 50% of the test larvae, and the total number of dead larvae was used to calculate the lethal concentration (LC50) affecting 50% of the larvae.

[0290] Table 7: Insect feeding assays: WCRWs were fed toxin B and engineered fusion protein. Table Notes: Toxin B C-terminal MMD1 L1 is a fusion polypeptide containing toxin B and a trimerized domain fused to the C-terminus of toxin B (via, for example, the intermediate L1 linker).

[0291] Toxin B N-terminal MMD1 L1 is a fusion polypeptide containing toxin B and a trimerized domain fused to the N-terminus of toxin B (via, for example, the intermediate L1 linker).

[0292] The toxin B control is a control of toxin B that is not fused with the trimerizing domain.

[0293] Table 7 shows the results for in vitro expression of toxin B proteins (including WT and engineered fusion proteins linked to the MMD1 helical polymerization domain via a linker selected from L1-L5). LC-50 and IC-50 are shown in ppm. LC-50 is the concentration at which 50% mortality occurs, while IC-50 is the concentration at which 50% mortality occurs along with severe growth and / or developmental retardation. UCL (upper confidence limit) and LCL (lower confidence limit) show the range of LC-50 / IC-50 values ​​and reflect data quality. Overall, the fusion proteins exhibited similar activity to the WT protein in the assays. In the case of fusion proteins with linker L3, the fusion proteins showed increased insecticidal activity compared to the WT protein. Table 7 shows the importance of linker length, flexibility, and the terminator of toxin B fused to the polymerization domain in the fusion protein. When the toxin B is fused to the N-terminus of the polymerizing domain using the linker L3, the efficacy is comparable to or slightly higher than that of the WT protein; however, when the protein is fused to the C-terminus using the same polymerizing domain and linker, the efficacy is lost.

[0294] Example 7 - Determination of phytotoxicity of toxin B with MMD1 polymerized domain in dwarf bean.

[0295] Chimeric proteins containing the MMD1 multimerizing domain (helix) and toxin B are obtained through gene synthesis. The multimerizing domain is added to the C or N-terminus of the protein fused via the aforementioned linkers (L1-L5). The corresponding gene is cloned into a transient expression system under the control of the viral promoter dMMV (Dey et al., (1999) Plant Mol. Biol. [Plant Molecular Biology] 40:771-782). For example, Agrobacterium strains containing each of these constructs are infiltrated into leaves, as described in Kapila et al., (1997) Plant Science [Plant Science] 122:101-108. Briefly, single leaves of dwarf bean (common bean) are infiltrated with Agrobacterium using standardized bacterial cell cultures of test and control strains. All dwarf bean phytotoxicity scores were assessed based on the following: None - no negative plant phenotypes observed, Low - some negative plant phenotypes observed (bruising or slight browning), Moderate - significant negative plant phenotypes observed (browning of approximately 50% of leaf tissue), Strong - severe negative plant phenotypes (tissue death, leaf curling).

[0296] The transient protein expression of the fusion protein was confirmed using protein lysates extracted from infiltrated leaf tissues via a mass spectrometry-based protein identification method (Patterson, (1998) 10(22):1-24, Current Protocol in Molecular Biology, published by John Wiley & Son Inc).

[0297] Table 8: Phytotoxicity assay of chimeric fusion proteins containing toxin B and trimerization domains in dwarf bean.

[0298] Table Notes: Toxin B - WT is a control of toxin B that is not fused with the trimerized domain.

[0299] Toxin B C-terminal EML4MMD1 L1 is a fusion polypeptide containing toxin B and a trimerized domain fused to the C-terminus of toxin B (via, for example, the intermediate L1 linker).

[0300] Toxin B N-terminal EML4MMD1 L1 is a fusion polypeptide containing toxin B and a trimerized domain fused to the N-terminus of toxin B (via, for example, the intermediate L1 linker).

[0301] When toxin B is fused with the multimerizing domain (MMD1), the phytotoxicity of the fusion protein is reduced. However, this reduction in phytotoxicity may be partly due to lower expression of the fusion protein compared to the unfused toxin B (WT) level in control dwarf bean leaves. The level of the toxin B MMD1 fusion protein was significantly lower than that of the WT-control protein (toxin B without MMD1 fusion). Based on the results obtained in Table 3, fusing toxin B with other MMDs may result in higher fusion protein expression in dwarf beans compared to WT toxin B, which could lead to even lower phytotoxicity in dwarf bean assays.

[0302] Example 8 - Phytotoxicity assay of toxin C (fern-derived toxin) fusion construct in dwarf bean Toxin C is fused to the MMD1 polymerization domain via a linker L6 (LCAS) containing a CASPASE (CAS) proteolytic site, which connects the toxin C and MMD1 sequences. Under the control of the viral promoter DMMV, polynucleotides encoding toxin C (unfused or fused with MMD1) are cloned into transient expression systems (Dey et al., (1999) Plant Mol. Biol. [Plant Molecular Biology] 40:771-782). Agrobacterium strains containing each of these constructs are infiltrated into leaves. Agrobacterium infiltration is a well-known technique in the art, involving the introduction of Agrobacterium cell suspensions into intact plant cells to allow for the measurement or study of reproducible infection and subsequent plant-derived transgenic expression (Kapila et al., (1997) Plant Science [Plant Science] 122:101-108). Briefly, single leaves of dwarf bean (common bean) are infiltrated with Agrobacterium using standardized bacterial cell cultures of test and control strains. Leaf discs were excised from each small plant and infected with four newborn larvae of the corn ear borer (CEW), European corn borer (ECB), fall armyworm (FAW), southern armyworm (SAW), and soybean looper (SBL). Leaf discs from the control were produced using Agrobacterium containing only the empty expression vector. Leaf tissue consumption was scored three days post-infection. Four days post-infection, a group of infiltrated control plants with known phenotypes were also used to score the visually negative plant health effects of the expressed protein. Leaf damage and phenotype scoring are described below.

[0303] The scoring of leaf damage and phenotype can be found below.

[0304] Table 9: Phytotoxicity and efficacy of chimeric fusion proteins containing toxin C and MMD1 polymerized domains in dwarf bean.

[0305] Table Notes: Toxin C (core) C-terminal MMD1 LCAS is a fusion polypeptide containing toxin C and a trimerized domain fused to the C-terminus of toxin C, wherein the two peptides are linked by an intermediate L-linker containing a CASPASE (CAS) cleavage site.

[0306] Toxin C- (core) is a WT control without fusion partner.

[0307] Compared with the toxin C-(core) WT control, the toxin C-(core)-C-terminal-MMD1-LCAS fusion protein showed higher expression and increased anti-FAW efficacy, and reduced leaf damage caused by FAW overall.

[0308] As shown in Table 9, under the test conditions of this dwarf bean assay, compared with toxin C WT (toxin C without MMD1), fusing the MMD1 polymerizing domain with toxin C resulted in an increase in the average concentration of the fusion protein in the dwarf bean expression assay and showed a decrease in the level of leaf damage caused by FAW. Furthermore, under the assay conditions, very slight phytotoxicity was observed when toxin C was expressed in the plant. No phytotoxicity was observed under the assay conditions when the fusion protein was expressed. Phytotoxicity scoring and efficacy assays were performed on the same dwarf bean plant.

[0309] Example 9 - Phytotoxicity determination of toxin D (bacterial-derived toxin) in dwarf beans Toxin D was tested on a dwarf bean platform to examine its phytotoxicity. Two different polymerization domains (MMD1 or MMD2) were fused to the N-terminus or C-terminus of toxin D via an L5 linker (see Table 7 below). The target gene was fused to the polymerization domain via a linker containing a proteolytic site, which linked the sequence. Under the control of the viral promoter DMMV, the corresponding polynucleotide encoding the target gene (unfused or fused with the polymerization domain) was cloned into a transient expression system (Dey et al., 1999, Plant Mol. Biol. [Plant Molecular Biology] 40:771-782). Using the Agrobacterium infiltration method, Agrobacterium strains containing each construct were infiltrated into leaves, which introduced Agrobacterium cell suspensions into intact plant cells, thereby enabling reproducible infection and subsequent plant-derived transgene expression (Kapila et al., (1997) Plant Science [Plant Science] 122:101-108).

[0310] In summary, single leaves of dwarf bean (common bean) were infiltrated with Agrobacterium using standardized bacterial cell cultures of test and control strains. Leaf discs were cut from each plantlet and infected with newborns of four different insect pests, including the corn ear moth (CEW), European corn borer (ECB), fall armyworm (FAW), southern armyworm (SAW), and soybean looper (SBL). Leaf discs from the control were produced using Agrobacterium containing only the empty expression vector. The amount of leaf tissue consumed was scored three days after infiltration. Four days after infiltration, a group of infiltrated control plants with known phenotypes were also used to score the visually negative plant health effects of the expressed protein. Table 10 shows the mean concentration of toxin D and the results of phytotoxicity as measured by leaf damage assessment.

[0311] Table 10: Phytotoxicity assay of chimeric fusion proteins containing toxin D and trimerizing domains in dwarf bean.

[0312] Table Notes: Toxin D C-terminal MMD1 or MMD2 L5 is a fusion polypeptide containing toxin D and an MMD fused to the C-terminus of toxin D, wherein the two peptides are linked by a central L5 linker.

[0313] Toxin D N-terminal MMD1 or MMD2 L5 is a fusion polypeptide containing toxin D and an MMD fused to the N-terminus of toxin D, wherein the two peptides are linked by a central L5 linker.

[0314] Toxin D - WT is a control of toxin D that is not fused with the trimerized domain.

[0315] Although toxin D-WT and all fusion proteins exhibited strong phytotoxicity under the test conditions, the accumulation of this toxin was higher when the fusion proteins were expressed compared to the expression of the unfused toxin D-WT. Notably, protein accumulation was higher when the polymerizing domain was fused to the N-terminus of toxin D compared to the construct with a polymerizing domain at the C-terminus of toxin D. In a non-limiting interpretation of the results, the presence of the polymerizing domain appears to reduce the phytotoxicity of the toxin D insecticidal protein; this may occur early in the transfection of plants as determined in dwarf bean. This could allow cells to express higher levels of the fusion protein before phytotoxicity is observed under the test conditions. Therefore, fusion of the trimerizing domain to the N-terminus of toxin D can reduce the phytotoxicity of the toxin D insecticidal protein based on total protein accumulation under the test conditions compared to the unfused toxin D insecticidal protein. In this example, further reduction in phytotoxicity could be achieved if the level of the toxin D-MMD fusion could be reduced to a level comparable to the toxin D WT control in plants, which could potentially be achieved by fusing toxin D with other MMDs.

[0316] Example 10 - Engineering for constructing new trimeric domains of engineered toxins.

[0317] Sequence ID NO:8 was used as the query sequence and a sequence search was performed using the 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 the search strategy. All other parameters were default values. Multiple homologs of SEQ ID NO:8 were identified by querying the database described above. The homologs and the organisms from which they were identified are shown in Table 11 below.

[0318] Table 11: Sequence search results using SEQ ID NO:8 as the query sequence In a non-limiting embodiment, the primary factor in engineering the stable trimerizing domain is considered to be the distribution of amino acid residues in the polypeptide sequence that allows trimer formation, wherein (i) the inward-facing amino acid residues are hydrophobic, thereby enabling the formation of a hydrophobic core; and (ii) the outward-facing amino acid residues are capable of electrostatic interactions with adjacent subunits of the trimer. Based on the sequence of SEQ ID NO: 1, different repeats in the following pattern were added to the primary sequence to promote trimer formation and stability: each of the 1st and 4th amino acid residues has a hydrophobic side chain; each of the 5th amino acid is replaced with arginine; and each of the 7th amino acids is replaced with glutamic acid. The sequences of the engineered MMD are shown in Table 12 below.

[0319] Table 12: Engineering Modification of Multimerized Structural Domains (MMD).

[0320] Methods for identifying cis-gene sequences used in crop improvement.

[0321] The following describes an exemplary workflow for identifying target nucleotide (or peptide) sequences or sequences in plant genomes, particularly for crop plant species or varieties intended for agricultural improvement. This method differs from conventional sequence search matching, such as BLAST searches targeting typical genes or coding regions of homologous gene sequences.

[0322] The methods described in this paper assume a scenario where researchers want to find nucleotide or peptide sequences (or even protein structure predictions) that closely match a query sequence, but no obvious high-quality or unique sequence match (e.g., a direct end-to-end perfect match) exists in the genome. These methods envision searching for relatively short subsequence fragments (i.e., subsequences of the query sequence) within the genome sequence of the target (plant, crop) genome, regardless of whether they are within identified gene boundaries or in intergenetic regions.

[0323] These natural crop plant subsequences found through these methods can then be assembled into a single, unified target sequence that is similar to or even matches the original query sequence. This sequence can then be prepared into physical nucleotides using various molecular biology techniques. Subsequently, this physical polynucleotide can be reintroduced into the (crop) plant in various ways to ultimately obtain a fully functional, reproductive crop plant containing the sequence in its genome, most commonly in a manner that allows it to be expressed as mRNA and subsequently produce a protein product.

[0324] Step 1. Target Sequence Identification. Select a target sequence. Typically, this can be a specific amino acid sequence. It could also be a specific nucleotide sequence. When the desired subject is a protein sequence, given the well-established redundancy and degeneracy of some amino acid codons, nucleotide sequence matching itself may be less important, as long as the nucleotide sequence, after virtual or in vivo translation, produces part or at most all of the target sequence. This specific query target sequence (regardless of its origin) will be used to search for relevant natural sequences in plant and crop species, as described below in steps 3, 4, 5, and 6.

[0325] Importantly, the target sequence does not need to be a perfect nucleotide match to the source sequence, as synonymous codons can encode the same peptide. Secondly, the target sequence does not need to encode a perfectly matching peptide, because different amino acid sequences can encode peptides with the same or similar physical peptide structures and substantially the same or similar functions. These different sequences can be referred to as target sequence analogs.

[0326] Therefore, to varying degrees, search criteria (such as those in the following examples) can be wisely implemented in a manner that allows for ambiguity in search parameters and interpretation of matching sequences, and that ambiguity is used to obtain target sequences or target sequence analogues.

[0327] Step 2. Source plant species genome sequence. This method requires that the source organism and its genome sequence be known, or at least partially known. A source organism refers to a plant species, which may or may not currently be considered a crop. However, these methods can be applied to any living organism (single-celled or multicellular), and are not limited to plants. Examples of plants are discussed specifically below.

[0328] “Plant species” is defined as a group of plants that are reproductively compatible with the crop plant species that anchor the group of plant organisms. In one embodiment, each species member of the group belongs to the same genus, but is not necessarily a single, strictly recognized species. Intergeneric members may exist within the group. Sexual compatibility between plant species is common because plant species classification is not necessarily guided by reproductive isolation, or reproductive compatibility is unknown when they are classified, and / or some species are defined at different times and places when such information was unknown. All intraspecific genomic variations (races, lines, inbred lines, heterozygotes, mutants, ecotypes, varieties, subspecies, etc.) are considered to be of the same species (part of the same species) in this document.

[0329] A genome sequence (including transcriptome, mRNA, or CDS sequence) is required for the search. Genome sequences are now widely available, or can be easily generated if the genome sequence of a certain species is currently unavailable.

[0330] Step 3. BLAST and Related Searches. The BLAST toolkit is a widely recognized method for searching related nucleotides and peptides. Many BLAST parameters can be tuned for sensitive searches of large genomics and peptide databases.

[0331] Example: For the query sequence “TSDVQDRLSALESRVQQQEDETVLKAA” (SEQ ID NO: 8), a BLAST search was performed using a matrix (BLOSUM62; gap penalty: presence: 11, extension: 1) on the public rice genome assembly Osa7 (374,471,240 nucleotides), identifying partial matches: The exact matching subsequence “DRLSAL” (SEQ ID NO: 165) was identified in this way. This sequence originates from a location on rice chromosome 2. Similar searches may reveal more sequence relationships in this rice genome or other plant genomes.

[0332] Step 4. Direct Text Pattern Search. Another method for sequence searching is direct text string matching. This can be implemented using a simple scripting language such as Perl. For this method, the target sequence can be "splittered" into various overlapping subsequences to initiate the search.

[0333] For example, if searching for hexamer matches, the starting sequence “TSDVQDRLSALESRVQQQEDETVLKAA” (SEQ ID NO: 8) can be arranged into a series of fragments, starting with “TSDVQD” (SEQ ID NO: 166), “SDVQDR” (SEQ ID NO: 167), “DVQDRL” (SEQ ID NO: 168), and so on, up to the last hexamer in the string, “TVLKAA” (SEQ ID NO: 169). These hexamer subqueries can be searched against the genome in both DNA strand directions during genome assembly, thus finding a series of matches. Depending on the length of the subsequences, a complete net coverage of the original full starting sequence (“TSDVQDRLSALESRVQQQEDETVLKAA” (SEQ ID NO: 8) in this example) may be obtained. Similar to BLAST searches, a table of subsequences and their positions in genome assembly can be created. This can be used to record the origin of the sequences in target plant species.

[0334] The advantage of direct string search is that it can detect very short sequences and their exact matches, which is not the primary design purpose of BLAST. (BLAST was originally designed for searching longer sequences with some ambiguity or mismatches, which is not suitable for direct string matching).

[0335] Table 13: Cisgene sequences identified as additional multimerization domains (MMDs).

[0336] Example 11 - Toxin Fusion Example This article considers multiple toxin fusion concepts.

[0337] Bipolar toxin fusion This article considers compositions and methods for modulating the activity of toxins, which are carried out by fusing a first toxin to one end of a polymerized domain (e.g., the N-terminus of the polymerized domain) and fusing a second toxin to the other end of the polymerized domain (e.g., the C-terminus of the polymerized domain) (see [link to article]). Figure 2 ).

[0338] In one non-limiting embodiment, the first toxin and the second toxin are the same toxin. In another non-limiting embodiment, the first toxin and the second toxin can be different toxins. For example, the activity of one or more toxins can be modulated by fusing one or more toxins with a trimerizing domain. A bipolar fusion polypeptide can be constructed, for example, by fusing toxin 1 with the N-terminus of the trimerizing domain and toxin 2 with the C-terminus of the trimerizing domain. In an exemplary embodiment, fusing multiple phytotoxic toxins with a single trimerizing domain can mitigate the phytotoxicity of multiple toxins by promoting the formation of an inactive polymer. Adding a cleavable linker between the toxin and the trimerizing domain can provide a mechanism to separate one or more toxins from the trimerizing domain and release one or more toxins to form an active form in a target organism (e.g., an insect pest). For each toxin, the cleavage linker can be different, thereby altering the activation rate of each toxin.

[0339] The bipolar fusion of toxin E (a fern-derived toxin) and toxin F (a Cry protein with a 3d-δ endotoxin architecture) in dwarf beans was determined. The 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 are linked by two distinct linkers and a polymerizing domain (MMD4 or MMD7): an LCAS-linker (with both a linker and a caspase-cleavable site) or a (non-cleavable) linker (with only a linker and no caspase-cleavable site). Under the control of the viral promoter DMMV, the corresponding polynucleotide encoding the target gene (unfused or fused with the polymerizing domain) was cloned into a transient expression system (Dey et al., 1999, Plant Mol. Biol. [Plant Molecular Biology] 40:771-782). Using the Agrobacterium infiltration method, Agrobacterium strains containing each construct were infiltrated into leaves. This method introduces Agrobacterium cell suspensions into plant cells of intact tissues, thereby enabling reproducible infection and subsequent plant-derived transgene expression (Kapila et al., (1997) Plant Science 122:101-108).

[0340] In summary, single leaves of dwarf bean (common bean) were infiltrated with Agrobacterium using standardized bacterial cell cultures of test and control strains. Leaf discs were excised from each plantlet and infected with newborns of four different insect pests, including the corn ear moth (CEW), European corn borer (ECB), fall armyworm (FAW), southern armyworm (SAW), and soybean looper (SBL). Leaf discs from the control were produced using Agrobacterium containing only the empty expression vector. The amount of leaf tissue consumed was scored three days after infiltration. Four days after infiltration, a group of infiltrated control plants with known phenotypes were also used to score the negative visual effects on plant health caused by the expressed protein.

[0341] Table 14 shows the results of the assays. In summary, the results indicate that, under the assay conditions of this invention, the fusion construct linking toxin E and toxin F via a polymerizing domain between the two toxins reduces phytotoxicity and increases efficacy against leaf damage induced by FAW and CEW, compared to the corresponding unfused toxin E or toxin F (WT) controls. The tested fusion construct showed efficacy against both FAW and CEW insects, while the individual toxins showed efficacy against only FAW or CEW. Leaf damage and phenotypic scores for the bipolar toxins are provided below.

[0342] Table 14: Phytotoxicity and efficacy of chimeric fusion proteins constructed using the bipolar fusion concept in dwarf bean.

[0343] Table Notes: Phytotoxicity scores and efficacy tests were conducted on the same transfected dwarf bean plant.

[0344] Toxin E was fused to the N-terminus of the polymerized domain, and toxin F was fused to the C-terminus of the polymerized domain. Two separate fusion constructs were prepared using two different polymerized domains (i.e., MMD4 and MMD7).

[0345] The cuttable connector was engineered to contain CASPASE L (CAS) cleavage (cutting) sites between the toxin and the polymerized structural domain.

[0346] LCAS is a small adapter added to the end of the CAS sequence: GGSVDVADGGS (SEQ ID No. 17). Constructs lacking cleavage (cutting) sites were also created and are labeled "non-cleavage" herein. 'Non-cleavage' indicates the absence of a CASPASE cleavage (cutting) site. The toxin was fused to the polymerized domain via the adapter.

[0347] Toxin F and Toxin E are controls of toxins that are not fused with multimerizing domains.

[0348] In one embodiment, if the toxin protein is processed by the insect within its gut, for example, through proteolytic cleavage at an endogenous proteolytic cleavage site, it may not be necessary to include a protease-activated cleavage (splitting) site in the fusion protein. In such a case, the toxin can directly connect to the polymerization domain via a linker without including a protease cleavage site. Furthermore, the placement of the polymerization domain can depend on the location of the endogenous proteolytic cleavage site within the toxin protein. For example, if the endogenous proteolytic cleavage site is located near the N-terminus of the toxin protein, the polymerization can fuse near the N-terminus, causing proteolytic cleavage at the endogenous proteolytic cleavage site to lead to the separation of the polymerization domain from the toxin protein.

[0349] Table 14 shows the results of all tested fusion proteins, where toxin E fuses with toxin F via various linkers. As shown in the table, under the test conditions of this invention, toxin E is highly effective against CEW, providing good protection against leaf damage, but ineffective against FAW. Conversely, under the test conditions of this invention, toxin F is resistant to FAW but not to CEW. Under the test conditions of this invention, both toxins exhibit strong phytotoxicity in dwarf beans.

[0350] When the toxins were fused via MMD and the linker, the fusion proteins exhibited insecticidal activity against both CEW and FAW insect pests, demonstrating that the toxins possessed insecticidal activity against their respective target insects. Furthermore, some fusion toxins showed reduced phytotoxicity, decreasing from the strong phytotoxicity of the unfused toxin control to the low phytotoxicity observed when the toxins were fused with MMD. Despite reduced protein expression levels in toxin F, the fusion proteins against both insects demonstrated higher efficacy and lower phytotoxicity compared to the individual toxins.

[0351] The table also highlights the important role of engineered helical domains in reducing phytotoxicity. For example, when using LCAS linkers containing CASPASE cleavable sites, the MMD7 polymerizing domain is more effective at mitigating phytotoxicity than the MMD4 polymerizing domain. However, when the MMD4 polymerizing domain is used without a cleavable linker between toxin E and the polymerizing domain, phytotoxicity is further reduced. This may be because toxin E is further proteolytically processed in the insect gut rather than in the plant.

[0352] This example highlights the importance of connector length and cutability when engineering bipolar fusion toxins. The connector length must provide sufficient flexibility to prevent protein misfolding, while maintaining a certain rigidity between the protein and the MMD (mitochondrial metaplasia of the protein and the microdiode) to make the cutable site less accessible, thus avoiding premature processing in the plant.

[0353] The efficacy of toxins can be enhanced by increasing the local concentration of toxins through the fusion of multi-merging domains. In one embodiment, fusing one or more toxin proteins with a polymerizing domain can enhance the efficacy of the toxin by increasing the local concentration of the toxin in the target insect. This can promote the formation of active toxins in the insect gut by increasing the local concentration of toxin molecules, thereby increasing the likelihood that toxin molecules will interact with each other to form active complexes, for example by increasing the rate of pore formation. Toxin G (a fern-derived toxin) or toxin H (a fern-derived toxin) is fused with different MMDs. The two components of the fusion protein (MMD and toxin) are linked together via a linker LCAS. LC-50 and IC-50 values ​​are expressed in ppm. LC-50 is the concentration at which 50% of the population shows mortality, while IC-50 is the concentration at which 50% of the population shows mortality and severe growth and / or developmental retardation. UCL (upper confidence limit) and LCL (lower confidence limit) show the range of LC-50 / IC-50 values ​​and reflect data quality.

[0354] Table 15: Insect feeding assays: FAW were fed toxin G and engineered fusion protein Table 16: Insect feeding assays: FAW were fed toxin H and engineered fusion protein Table Notes: Toxin G or toxin H N-terminal MMD LCAS is a fusion polypeptide containing toxin G or toxin H and different polymerized domains fused to the N-terminus of the toxin, wherein the two polypeptides are linked by a central LCAS linker.

[0355] The toxin G-control is a control of toxin G that is not fused with the multimerizing domain.

[0356] The toxin H-control is a control containing toxin H that is not fused with the multimerizing domain.

[0357] In vitro, under certain test conditions, the wild-type (non-fused with MMD) versions of toxins G and H exhibited low efficacy because neither of the WT forms of these toxins showed a measurable LC50. However, by adding a polymerizing domain to the N-terminus of toxin G or toxin H, some fusion proteins showed increased efficacy against insects (measured as lower IC50 values) compared to the WT proteins. Under the test conditions, many fusion constructs showed lower LC50 values, an improvement over the WT control. Therefore, overall efficacy increased with the fusion of the polymerizing domain with the toxin G or toxin H peptides tested under these conditions.

[0358] For insect bioassays, protein concentration is determined by gel density assay. This involves denaturing the protein to dissociate the trimer into monomeric subunits, thereby enabling the determination of the number of monomers in all samples compared to WT protein.

[0359] The activity of toxins can be reduced by fusing with multimerized structural domains.

[0360] In one embodiment, modulating the activity of a toxin may include reducing the phytotoxicity of the toxin, such that the toxin has reduced phytotoxicity when fused with a polymerized structural domain.

[0361] Two-component system.

[0362] In one embodiment, insecticidal protein A1 and insecticidal protein B1 may be part of a two-component system, where both components are essential for the formation of the active complex. Such a two-component bipolar fusion construct can provide phytotoxicity reduction for two different toxins in a single fusion construct, wherein such a two-component system may comprise toxin A and toxin B, which interact with each other to form an active complex, wherein the two-component insecticidal proteins A1 and B1, along with an intermediate polymerizing domain, bring the two components into close proximity. This allows for the formation of the complex after at least one of the two toxins has been released (e.g., via proteolytic cleavage of the linker sequence).

[0363] In one non-limiting embodiment, insecticidal protein A1 is fused to the MMD at one end of the polymerized domain, and insecticidal protein B1 is fused to the other end of the MMD domain. This allows the two insecticidal proteins to be expressed as a fusion polypeptide within a single reading frame. The presence of the MMD between the two toxins can lock the two distinct toxins into an inactive state, thereby modulating the phytotoxicity of the two toxins in the uncleaved fusion protein, and cleaving one or both toxins from the fusion protein to release one or both components, thereby forming an active complex with their corresponding components.

[0364] Binary poison.

[0365] This method can also be used for binary toxins. One component can fuse to one end of a multimerizing domain, while the other component fuses to the other end. The components can be kept separate by the multimerizing domain and can come into contact with each other only after being cleaved by a protease cleavage site in the intermediate linker sequence, where the specific protease is present in the target insect gut but not in the plant cell. In this way, the toxin can be activated in the target insect gut by engineering specific cleavable protease sites into the linker sequence. In one embodiment, multiple cleavage sites (each specific to a particular enzyme) can be engineered into the linker sequence on either side of the multimerizing domain to achieve different rates of proteolytic cleavage and release of the toxin from the inactive multimer complex, such that the released toxin can be activated as an insecticidal toxin.

[0366] The toxin is broken down into two components separated by polymerized structural domains.

[0367] In another embodiment, the divalent toxin can also be separated into two components and fused to either end of the multimerized domain, such that the divalent toxin can remain separated by the trimer and can only come into contact with each other after protease cleavage of the linker sequence, releasing the two components to interact with each other, resulting in activation in the insect gut.

[0368] Dimerization domains.

[0369] In one embodiment, the multimerizing domain may include a dimerizing domain ( Figure 4 A). For example, a dimerizing domain can be used to isolate monomeric toxins in an inactive dimer conformation. In one non-limiting embodiment, the leucine zipper forming the dimer consists of a characteristic motif containing leucine repeats. They dimerize in solution due to internal hydrophobic modes and external electrostatic interactions. This dimerizing domain can be fused with phytotoxic toxins, thereby isolating them in an inactive dimer conformation. It can also be used to isolate dimeric toxins and prevent them from being flexible in solution. Fusing the leucine zipper with the toxin can prevent the toxin from binding to the receptor and / or forming a higher-order oligomeric structure for pore formation. The fusion protein can further include an intermediate linker sequence and a cleavable site. The dimerizing domain can also be engineered to produce bipolar fusions similar to the concepts discussed above.

[0370] In another embodiment, the dimerizing domain can be used to aggregate two different proteins together, wherein at least one protein is a dimer (see [link to documentation]). Figure 5 This could allow for the delivery of two different mate toxins within a single reading frame. It could also allow for alteration of the phytotoxic properties of the two toxins in plants, while simultaneously allowing for the maintenance of high local concentrations of both toxins in the intestinal cells of insects; this could potentially alter the efficacy of the mate toxins.

[0371] Tetrameric and multimeric protein bundles.

[0372] Any protein that forms a helix or multimeric bundle can be used as a fusion partner protein. Figure 4 B). Polymeric proteins can fuse with toxins to insulate them in an inactive state and prevent the phytotoxicity of polymeric toxins.

[0373] In one embodiment, toxin D can be fused to one end of the polymerizing domain, and toxin E can be fused to the other end of the polymerizing domain to reduce the phytotoxicity of both toxins. In another embodiment, a linker containing a protease-specific sequence can be used to fuse the toxins, thereby allowing the toxin fusion construct to remain inactive in the plant, while the cleavage of the protease-specific sequence and the release of the toxin molecule from the fusion construct ensures that the toxin protein is activated only in the insect gut.

[0374] One example of a multimerizing domain forming a tetramer originates from the C-terminal helical bundle of a prokaryotic sodium channel, which forms a stable tetramer structure in solution. In this structure, Arg243 forms hydrogen bonds with Thr239 of the adjacent helix and interacts with Tyr242 via CH / π stacking, as explained in (Katsumasa et al. 2012 Nat Commun [Nature Communications] 3: 793-793). Helical domains forming multimer bundles can be used as fusion proteins to mitigate phytotoxicity. For example, fusing multimerizing domains with phytotoxic toxins can isolate the toxin in an inactive state by bringing multiple units of the toxin close to each other in a manner that inhibits pore formation.

[0375] The formation rate of active complexes can be increased by multiplying structural domains.

[0376] Fusion proteins comprising polymerizing domains and insecticidal proteins can also be used to facilitate faster formation of active complexes. This can be achieved by bringing two or more toxin subunits closer together to each other to obtain a faster complex formation rate and by increasing local protein concentration. For example, insecticidal proteins function by forming pore-forming macromolecular complexes, which can be enhanced by fusing subunits with polymerizing domains, resulting in more than one toxin subunit being close to each other and / or near the cell surface, which can increase the rate of pore formation. In this non-limiting embodiment, engineered fusion proteins can not only reduce phytotoxicity but also enhance toxin activity, as shown in Tables 15 and 16.

[0377] Lock-key trimerization.

[0378] This article considers compositions and methods for engineering trimerization domains to promote heterotrimer formation. In one embodiment, heterotrimerization can be achieved, for example, by adding an amino acid residue with a large hydrophobic side chain (like tryptophan) to one of the monomer subunit helices and adding a small amino acid (like alanine) to the corresponding positions of the other two monomer subunit helices, wherein the three amino acid residues are close to each other in the trimer, wherein the presence of tryptophan and alanine in the other two subunits promotes the formation of heterotrimers and inhibits the formation of homotrimers due to steric hindrance. In this way, a "lock-and-key" mechanism for selective heterotrimer formation can be achieved. Thus, each trimerization domain can be fused with different insecticidal peptides, thereby enabling the close proximity of two or more different insecticidal peptides to each other using heterotrimerization.

[0379] Cleavage site: The trimer is activated using insect-specific protein hydrolysis cleavage sites.

[0380] To render a toxin inactive in plants but active in the insect gut, a specific protease recognition / cleavage site can be engineered between the polymerizing domain and the target toxin. The toxin is fused to the trimeric domain or any other polymerizing domain via a linker. Within the linker, a cleavable sequence specific to the target pest is introduced. In a non-limiting embodiment, a cathepsin site (LSQSLSQS; SEQ ID NO: 170) is used for underground insects, and a caspase site (VDVAD; SEQ ID NO: 171) is used for aboveground insects. Any cleavable linker specific to the target pest can be used to make the fusion protein active only in the target pest. Insect gut-specific proteases (including, but not limited to, serine proteases), including trypsin, chymotrypsin, and related proteases, can also be used.

[0381] Cleavage sites can be selected to target only target insect pests and not non-target insects; therefore, this strategy can be used to retarget toxins. In one embodiment, such specific targeting / retargeting can be achieved by selecting a protease recognition site that is recognized only by a protease (present only in target insects but not in non-target insects). The toxin may be inactive in non-target pests but can be activated in target insect pests. Using this method, phytotoxicity can be mitigated and toxins can be targeted at target insect pests.

[0382] Connector and connector length For trimeric or multimeric fusion proteins to mitigate phytotoxicity and kill target insects, the toxin should possess sufficient flexibility. In a non-limiting assumption, the fusion protein may aggregate if the components of the fusion product are too close to each other. The flexibility of the linker can also play a role in the function of the fusion protein. In a non-limiting embodiment, the linker should preferably be structurally restrictive (rigid) to prevent excessive flexibility that would prevent the linker from being cut in the plant. Linker length may also be important for the stability of the fusion protein. The length and flexibility of the linker can depend on the multimeric domain and the toxin fused to it. The linker can be as simple as a GGS, or it can have some structural features that separate the toxin from the multimeric bundle and guide it away (see [link to GGS]). Figure 3 This can be particularly helpful when fusing large toxins.

[0383] Methods for engineering multimeric subunits: Korendovych IV and DeGrado WF reviewed the design of multiple helical bundles based on geometric structural properties. (QRev Biophys. [Quarterly Review of Biophysics] 2020 Feb 11;53:e3). Alpha helices that form trimers in solution can be engineered using known helical amino acid geometries. The classic left-handed helix has a geometric repeat of seven residues, denoted as “abcdefg”. For alpha helices that form trimers in solution, each a' and d' amino acid is a hydrophobic amino acid, preferably valine or leucine. The pattern does not need to be consistent. For example, positions 1, 8, and 20 can be hydrophobic amino acids. This can create a strongly hydrophobic core that keeps the trimer together in solution. To facilitate interactions with adjacent helices within the trimer, charged amino acids forming salt bridges alternate within the helical structure; where each e' amino acid is a positively charged amino acid and each g' amino acid is a negatively charged amino acid. This pattern can also be reversed, where each e' position is a negatively charged amino acid and each g' position is a positively charged amino acid.

[0384] An α-helical structure that forms a dimer in solution can be obtained, much like the classic leucine zipper, where leucine residues can be placed at every 7th (g') position to form a heptapeptide repeat. This facilitates dimerization and maintains a hydrophobic core within the dimer. The formation of the dimer structure also benefits from the selectivity of electrostatic interactions with adjacent α-helices.

[0385] To obtain a tetrameric helical structure in solution, simply replacing the amino acid valine with isoleucine at the hydrophobic core produces selectivity for tetrameric helical bundles. The structure of the tetrameric bundles is further enhanced by adding tryptophan in the middle of the helix, which forms a CH / π stack and extends towards the hydrophobic core. This allows the correct stoichiometric geometry to selectively form a tetramer rather than a trimer structure.

[0386] Optimization of fusion protein design: This paper considers the use of plant tissue extracts as in vitro diagnostics for predicting the stability of fusion proteins in vivo. This may require using recombinant fusion proteins generated as described herein and incubating them with plant tissue from a target crop. Construct design optimization can be performed on (but is not limited to): the toxin sequence itself, the length and sequence of the linker, proteolytic cleavage sites, multimerization domains, and the orientation of all components within the construct. Diagnostic assays can be used to optimize the fusion design so that the fusion retains its multimerized structure and is not easily degraded or pre-activated in plants. This will ensure that stable multimerized fusion proteins confer a reduced plant response but still maintain equivalent or increased insecticidal efficacy. ...

Claims

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

2. The chimeric fusion polypeptide of claim 1, wherein the multimerizing domain is a trimerizing domain.

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

4. The polypeptide of claim 3, wherein the altered activity is selected from: reducing phytotoxicity in plants, reducing activity in non-target organisms, increasing activity in target organisms, and increasing expression in host plants.

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

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

7. The polypeptide of claim 6, wherein the protease cleavage site is specific to proteases present in the gastrointestinal fluid of Lepidoptera.

8. The polypeptide of claim 6, wherein the protease cleavage site is specific to proteases present in the gastrointestinal fluid of Coleoptera.

9. The polypeptide of claim 1, wherein the polymerized domain has at least 90% sequence identity with a sequence selected from the group consisting of SEQ ID NO 1, 2 and 8-16.

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

11. A DNA construct comprising the polynucleotide as described in claim 10.

12. A plant or plant cell comprising the polynucleotide as described in claim 10.

13. The plant or plant cell of claim 12, wherein the plant or plant cell further comprises additional polynucleotide sequences encoding different insecticidal proteins.

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

15. A composition comprising the chimeric fusion polypeptide as described in claim 1.

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

17. The method of claim 16, wherein the modified activity of the insecticidal polypeptide is selected from the group consisting of: reducing phytotoxicity in plants, reducing activity in non-target organisms, increasing activity in target organisms, and increasing expression in host plants.

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

19. The method of claim 18, wherein the adapter 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 proteases present in the gastrointestinal fluid of Lepidoptera.

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

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-harmful insects of corn, soybean, and cotton.

24. A method for modifying the activity of a two-component insecticidal peptide system, the method comprising connecting a heteropolymerization domain comprising a first component of the two-component system to one end of the heteropolymerization domain, and connecting a second component of the two-component system to the other end of the heteropolymerization domain, thereby modifying the activity of the two-component insecticidal peptide system.

25. The method of claim 24, wherein the modified activity of the two-component insecticidal peptide system is selected from: reducing phytotoxicity in plants, reducing activity in non-target organisms, increasing activity in target organisms, and increasing expression in host plants, or any combination thereof.

26. The method of claim 24, wherein each of the first and second components of the two-component insecticidal polypeptide is linked to the intermediate heteropolymerized domain via a linker sequence.

27. The method of claim 26, wherein the adapter 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 proteases present in the gastrointestinal fluid of Lepidoptera.

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

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

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

32. A method for designing trimerizing domains, the method comprising altering the distribution of amino acid residues in a polypeptide sequence that promote trimerization, wherein (i) the inward-facing amino acid residues are hydrophobic, thereby enabling the formation of a hydrophobic core; and (ii) the outward-facing amino acid residues are capable of electrostatic interactions with adjacent subunits of the trimer.

33. The chimeric polypeptide of claim 2, wherein the trimerizing domain comprises a heptapeptide repeat comprising an amino acid sequence in the order 'ABCDEFG', wherein each 'A' and 'D' amino acid residue comprises a hydrophobic side chain, and wherein each 'E' and 'G' amino acid residue comprises a charged side chain.

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