Methods and compositions for mitigating undesirable phenotypic characteristics in plants

Abstract

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Description

METHODS AND COMPOSITIONS FOR MITIGATING UNDESIRABLEPHENOTYPIC CHARACTERISTICS IN PLANTSCROSS REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 550,732, filed on February 7, 2024, the disclosure of which is incorporated herein by reference in its entirety.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The official copy of the sequence listing is submitted electronically via Patent Center as an XML formatted sequence listing with a file named “211786_SequenceListing.xml” created on February 3, 2025, and having a size of 72,181 bytes and is filed in computer readable form concurrently with the specification. The sequence listing contained in this XML formatted document is part of the specification and is herein incorporated by reference in its entirety.FIELD

[0003] This disclosure relates to the field of plant genetics and molecular biology. Provided are novel compositions and methods for mitigating undesirable phenotypic characteristics attributable to the presence of certain transgenic proteins, including insecticidal proteins, when expressed in transgenic plants. Also provided are novel methods for increasing protein durability and expression levels in transgenic plants. Further provided are novel genes that encode pesticidal proteins. These pesticidal proteins and the nucleic acid sequences that encode them are useful in preparing pesticidal formulations and in the production of transgenic pest-resistant plants. Novel engineered peptides, polypeptides, and chimeric polypeptides, and methods of producing and using the same, are also contemplated.BACKGROUND

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

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

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

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

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

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

[0010] In another aspect, isolated or recombinant nucleic acid molecules are provided encoding engineered scaffold proteins, including amino acid substitutions, deletions, insertions, and fragments thereof. Provided are isolated or recombinant nucleic acid molecules capable of encoding an engineered scaffold polypeptide of SEQ ID NO: 1, as well as amino acid substitutions, deletions, insertions, fragments thereof, and combinations thereof. In certain embodiments, polynucleotides are provided that encode engineered scaffold proteins, wherein the polynucleotides comprise a nucleic acid sequence as set forth in SEQ ID NO: 31, as well as nucleic acid substitutions, deletions, insertions, fragments thereof, and combinations thereof. Nucleic acid sequences that are complementary to a nucleic acid sequence of the embodiments or that hybridize to a sequence of the embodiments are also encompassed. In another aspect, an engineered scaffold polypeptide is provided comprising, consisting essentially of, or alternatively consisting of, SEQ ID NO: 1.

[0011] In another aspect, isolated or recombinant nucleic acid molecules are provided encoding engineered scaffold binding peptides, including amino acid substitutions, deletions, insertions, and fragments thereof. Provided are isolated or recombinant nucleic acid molecules capable of encoding scaffold binding peptides of SEQ ID NOs: 2-6, as well as amino acid substitutions, deletions, insertions, fragments thereof, and combinations thereof. In certain embodiments, polynucleotides are provided that encode scaffold binding peptides, wherein the polynucleotidescomprise a nucleic acid sequence as set forth in any one of SEQ ID NOs: 32-36. Nucleic acid sequences that are complementary to a nucleic acid sequence of the embodiments or that hybridize to a sequence of the embodiments are also encompassed. The nucleic acid sequences can be used in DNA constructs or expression cassettes for transformation and expression in organisms, including microorganisms and plants. The nucleotide or amino acid sequences may be synthetic sequences that have been designed for expression in an organism including, but not limited to, a microorganism or a plant.

[0012] In another aspect scaffold binding peptides are encompassed. Also provided are isolated or recombinant scaffold binding peptides, wherein the scaffold binding peptides have at least 90% sequence identity to a sequence selected from SEQ ID NOs: 2-6, as well as amino acid substitutions, deletions, insertions, fragments thereof and combinations thereof.

[0013] In one aspect, the scaffold binding peptide has binding affinity for a polypeptide of interest, for example an insecticidal polypeptide of interest. In another aspect, a scaffold binding peptide having binding affinity for a polypeptide of interest, for example an insecticidal polypeptide of interest, reduces undesirable phenotypic characteristics attributable to the presence of the polypeptide of interest (e.g., insecticidal protein) in plants compared to the polypeptide of interest (e.g., insecticidal protein) lacking the presence of the scaffold binding peptide. In another aspect, a scaffold binding peptide having binding affinity for a polypeptide of interest, for example an insecticidal polypeptide of interest, reduces phytotoxic effects attributable to the presence of the polypeptide of interest (e.g., insecticidal protein) in plants compared to the polypeptide of interest (e g., insecticidal protein) lacking the presence of the scaffold binding peptide.

[0014] In another aspect, a scaffold binding peptide having binding affinity for a polypeptide of interest, for example an insecticidal protein of interest, increases expression of the polypeptide of interest (e.g., insecticidal protein of interest) in plants compared to the polypeptide of interest (e.g., insecticidal protein) lacking the presence of the scaffold binding peptide.

[0015] In another aspect, a scaffold binding peptide having binding affinity for a polypeptide of interest, for example an insecticidal protein of interest, increases durability of the insecticidal protein against a target pest(s) in plants over time compared to the insecticidal protein lacking the presence of the scaffold binding peptide. In yet another aspect, a scaffold binding peptide having binding affinity for an insecticidal protein of interest increases efficacy of the insecticidal proteinin plants compared to the insecticidal protein lacking the presence of the scaffold binding peptide.

[0016] Also provided are isolated or recombinant linkers. In one aspect, the core structure of the linker peptide forms a rigid helical structure. In another aspect, the linker is cleavable and contains an insect specific protease cleavage site. In another aspect, the linker contains a serine protease cleavage site, such as but not limited to a trypsin cleavage site and / or a chymotrypsin cleavage site. In another aspect, the linker contains a cathepsin cleavage site. In another aspect, the linker contains an alkaline proteinase and / or an acidic proteinase cleavage site. In yet another aspect, the linker contains a cleavage site that upon contact with insect gastrointestinal fluid through digestion releases the insecticidal toxin protein when linked to a scaffold binding peptide. In another aspect, linkers are provided comprising, consisting essentially of, or alternatively consisting of, SEQ ID NOs: 7-27, as well as amino acid substitutions, deletions, insertions, fragments thereof and combinations thereof. In certain embodiments, polynucleotides are provided that encode linkers, wherein the polynucleotides comprise a nucleic acid sequence as set forth in any one of SEQ ID NOs: 37-57, as well as nucleic acid substitutions, deletions, insertions, fragments thereof and combinations thereof.

[0017] In one embodiment, provided are expression constructs comprising a polynucleotide which encodes one or more scaffold binding peptides of SEQ ID NOs: 2-6, one or more linker peptides of SEQ ID NOs: 7-27, and one or more insecticidal polypeptides of interest.

[0018] In another aspect, DNA constructs are encompassed. Also provided are DNA constructs comprising nucleic acid molecules encoding one or more scaffold binding peptides of SEQ ID NOs: 2-6, one or more linker peptides of SEQ ID NOs: 7-27, one or more insecticidal polypeptides of interest and combinations thereof.

[0019] In another aspect, chimeric fusion polypeptides are encompassed. Provided are chimeric fusion polypeptides comprising one or more scaffold binding peptides, one or more linker peptides, and one or more insecticidal polypeptides of interest. Also provided are chimeric fusion polypeptides comprising one or more scaffold binding peptides of SEQ ID NOs: 2-6, one or more linker peptides of SEQ ID NOs: 7-27, and one or more insecticidal polypeptides of interest. In a further aspect, the one or more scaffold binding peptides are linked to the insecticidal polypeptide of interest at the N-terminus of the insecticidal polypeptide. In another aspect, the one or more scaffold binding peptides are linked to the insecticidal polypeptide of interest at the C-terminus ofthe insecticidal polypeptide. In one aspect, the one or more scaffold binding peptides are linked to the insecticidal polypeptide of interest at both the N-terminus of the insecticidal polypeptide and the C-terminus of the insecticidal polypeptide.

[0020] In one embodiment, chimeric fusion polypeptides comprising one or more scaffold binding peptides, one or more linker peptides, and one or more insecticidal polypeptides of interest contain an insect specific protease cleavage site located anywhere in the fusion polypeptide, such as but not limited to an insect specific protease cleavage site located in the one or more scaffold binding peptides, an insect specific protease cleavage site located in the one or more linker peptides, or an insect specific protease cleavage site located in the one or more insecticidal polypeptides of interest. In another embodiment, provided are insecticidal polypeptides fused to a cleavable cisacting heterologous inactivation domain, wherein cleavage of the inactivation domain activates the insecticidal protein, and wherein the inactivation domain is not a crystal forming domain of a three-domain insecticidal toxin or an antibody fragment.

[0021] Also provided are chimeric fusion polypeptides comprising (i) a scaffold binding peptide having at least 90% sequence identity to a scaffold binding peptide sequence selected from SEQ ID NOs: 2-6, (ii) a linker having at least 90% sequence identity to a linker sequence selected from SEQ ID NOs: 1-T1 , and (iii) a polypeptide sequence comprising an insecticidal polypeptide of interest.

[0022] In another aspect, the scaffold binding peptide comprises one or more variable domains that bind to one or more target epitopes in an insecticidal protein of interest to reduce or prevent undesirable phenotypic characteristics in plants. In one aspect of a non-limiting hypothesis, the binding of a scaffold binding peptide to an insecticidal protein of interest prevents pore formation by the insecticidal protein, resulting in reduction of the insecticidal protein’s undesirable phenotypic characteristics in plants.

[0023] In another aspect, the insecticidal polypeptide of interest comprises an insecticidal protein having a 3-domain (3d) Cry delta endotoxin architecture or an insecticidal protein having the domain architecture of one or more of domain I, domain II, or domain III of a 3-domain delta endotoxin. In one embodiment, the scaffold binding peptide comprises one or more epitopebinding portions that bind to residues in domain I of an insecticidal protein having a 3-domain delta endotoxin architecture or an insecticidal protein having the domain architecture of domain Iof a 3-domain delta endotoxin. In one embodiment, the scaffold binding peptide comprises one or more epitope-binding portions that bind to residues in helix a-4 in domain I of an insecticidal protein having a 3-domain delta endotoxin architecture or an insecticidal protein having the domain architecture of domain I of a 3-domain delta endotoxin. In another embodiment, the scaffold binding peptide comprises one or more epitope-binding portions that bind to residues in helix a-4 and to residues in helix a-5 in domain I of an insecticidal protein having a 3-domain delta endotoxin architecture or an insecticidal protein having the domain architecture of domain I of a 3-domain delta endotoxin. In a non-limiting hypothesis, binding of the scaffold binding peptide to residues in helix a-4 and to residues in helix a-5 in domain I of an insecticidal protein having a 3-domain delta endotoxin architecture or an insecticidal protein having the domain architecture of domain I of a 3-domain delta endotoxin prevents the insecticidal protein from forming a pore by blocking movement of helix a-4 and / or a-5. In another embodiment, the scaffold binding peptide comprises one or more epitope-binding portions that bind to residues in domain II of an insecticidal protein having a 3-domain delta endotoxin architecture or an insecticidal protein having the domain architecture of domain II of a 3-domain delta endotoxin. In another embodiment, the scaffold binding peptide comprises one or more epitope-binding portions that bind to residues in domain III of an insecticidal protein having a 3-domain delta endotoxin architecture or an insecticidal protein having the domain architecture of domain III of a 3-domain delta endotoxin. In one embodiment, the scaffold binding peptide comprises one or more epitope-binding portions that bind to residues in domain I and domain II of an insecticidal protein having a 3-domain delta endotoxin architecture or an insecticidal protein having the domain architecture of domain I and domain II of a 3-domain delta endotoxin. In another embodiment, the scaffold binding peptide comprises one or more epitope-binding portions that bind to one or more residues in helix a-6 and helix a-7 in domain I and to residues in helix a-8 in domain II of an insecticidal protein having a 3-domain delta endotoxin architecture or an insecticidal protein having the domain architecture of domain I and domain II of a 3-domain delta endotoxin. In another embodiment, the scaffold binding peptide comprises one or more epitope-binding portions that bind to one or more residues in at least one of the following in domain I and / or domain II of an insecticidal protein having a 3- domain delta endotoxin architecture or an insecticidal protein having the domain architecture of one or more of domain I or domain II of a 3-domain delta endotoxin: helix a-4, helix a-5, helix a-6, helix a-7, and helix a-8. In a further embodiment, the scaffold binding peptide comprises one or more epitope-binding portions that bind to one or more arginine and threonine residues in domain I and / or domain II of an insecticidal protein having a 3 -domain delta endotoxin architecture or an insecticidal protein having the domain architecture of one or more of domain I or domain II of a 3-domain delta endotoxin. In a non-limiting hypothesis, binding of the scaffold binding peptide to one or more residues in any one or more of helix a-4, helix a-5, helix a-6, helix a-7, and helix a-8 of an insecticidal protein having a domain architecture of one or more of domain I or domain II of a 3-domain delta endotoxin prevents the insecticidal protein from forming a pore by blocking movement of the one or more of helix a-4, helix a-5, helix a-6, helix a-7, or helix a- 8. In a further embodiment, a scaffold binding peptide identified as a binder for a particular insecticidal protein may be used as a scaffold binding peptide for different, similar class insecticidal protein.

[0024] In another aspect, methods are provided for using an engineered scaffold to identify and generate scaffold binding peptides. In one aspect, methods are provided for using an engineered scaffold to generate libraries of scaffold binding peptides. In another aspect, methods are provided for using an engineered scaffold to generate high diversity phage display scaffold binding peptide loop libraries. In a further aspect, libraries of scaffold binding peptides are generated using oligonucl eotide-directed mutagenesi s .

[0025] In another aspect, methods are contemplated for identifying epitope(s) responsible for phytotoxicity in an insecticidal protein of interest. In one aspect, methods are contemplated for generating scaffold binding peptides that bind over a region of an insecticidal protein that include an epitope responsible for an undesirable phenotypic characteristic, which when bound reduces the undesirable phenotypic characteristic such as phytotoxicity attributable to the insecticidal protein. In a non-limiting hypothesis, the causes for such beneficial effect include but are not limited to locking up protein structure so that conformational changes required for pore formation cannot proceed, blocking unintended plant receptor binding by steric hindrance, and a combination of both.

[0026] In one aspect, methods are provided for reducing undesirable phenotypic characteristics attributable to the presence of certain transgenic proteins, including insecticidal proteins, when expressed in transgenic plants, by co-expressing in a plant one or more scaffold binding peptidesand one or more polypeptides of interest, for example insecticidal polypeptides of interest. In another aspect, methods are provided for reducing phytotoxicity of certain transgenic proteins, including insecticidal proteins, when expressed in transgenic plants, by co-expressing in a plant one or more scaffold binding peptides and one or more polypeptides of interest, for example insecticidal polypeptides of interest. In another aspect, methods are provided for increasing durability of certain insecticidal proteins against target pests when expressed in transgenic plants by co-expressing in a plant one or more scaffold binding peptides and one or more insecticidal polypeptides of interest. In another aspect, methods are provided for increasing expression of certain transgenic proteins, including insecticidal proteins, when expressed in transgenic plants, by co-expressing in a plant one or more scaffold binding peptides and one or more polypeptides of interest, for example insecticidal polypeptides of interest. In yet another aspect, methods are provided for increasing efficacy of certain transgenic proteins, including insecticidal proteins, when expressed in transgenic plants, by co-expressing in a plant one or more scaffold binding peptides and one or more polypeptides of interest, for example insecticidal polypeptides of interest.

[0027] In one aspect, methods are provided for producing chimeric fusion polypeptides comprising one or more scaffold binding peptides, one or more linkers, and one or more insecticidal polypeptides of interest, and for using those chimeric fusion polypeptides for controlling or killing a Lepidopteran, Coleopteran, Hemiptera, nematode, fungi and / or Dipteran pest.

[0028] In one aspect, provided are methods for modifying activity of a polypeptide of interest in a plant, such as an insecticidal polypeptide of interest, wherein the method comprises expressing one or more chimeric fusion polypeptides comprising one or more scaffold binding peptides, one or more linkers, and one or more insecticidal polypeptides of interest in a plant, thereby modifying the activity of the insecticidal polypeptide. In one non-limiting embodiment, the modifying activity of an insecticidal polypeptide is selected from the group consisting of: reduced phytotoxicity in plants, reduced activity in a non-target organism, increased activity in a target organism, and increased expression in a host including a host plant. In one aspect, the target organism is selected from the group consisting of: Coleopteran, Lepidopteran, Hemipteran and nematode pests, and the non-target organism is selected from the group consisting of: non-pest insects of row crops, corn, soybean, and cotton.

[0029] In another aspect, methods are provided for reducing undesirable phenotypic characteristics attributable to certain transgenic proteins, including insecticidal proteins, when expressed in transgenic plants, by expressing one or more of said chimeric fusion polypeptides in a plant. In another aspect, methods are provided for reducing phytotoxicity of certain transgenic proteins, including insecticidal proteins, when expressed in transgenic plants, by expressing one or more of said chimeric fusion polypeptides in a plant. In another aspect, methods are provided for increasing expression of an insecticidal protein in a plant by expressing one or more of said chimeric fusion polypeptides in a plant. In another aspect, methods are provided for increasing efficacy of an insecticidal protein in a plant by expressing one or more of said chimeric fusion polypeptides in a plant. In one aspect, methods are provided for increasing durability of an insecticidal protein in a plant by expressing one or more chimeric polypeptides comprising one or more scaffold binding peptides, one or more linkers, and one or more insecticidal polypeptides of interest in a plant.

[0030] In one aspect, provided are host cells comprising a polypeptide comprising one or more scaffold binding peptides of SEQ ID NOs: 2-6 and a polypeptide comprising one or more insecticidal polypeptides of interest. Also provided are host cells comprising a chimeric fusion polypeptide comprising one or more scaffold binding peptides of SEQ ID NOs: 2-6, one or more linkers of SEQ ID NOs: 7-27, and one or more insecticidal polypeptides of interest.

[0031] In another aspect, transgenic plants and transgenic plant cells comprising one or more scaffold binding peptides of SEQ ID NOs: 2-6 and one or more insecticidal polypeptides of interest are provided herein. In another aspect, transgenic plants and transgenic plant cells are provided comprising one or more of the chimeric fusion polypeptides described herein. In various embodiments, the transgenic plant and transgenic plant cell further comprises one or more additional genes for insect resistance, for example, one or more additional genes for controlling Coleopteran, Lepidopteran, Hemipteran or nematode pests. In one embodiment, transgenic plants and transgenic plant cells are provided comprising more than one insecticidal polypeptide, each of which is expressed or co-expressed with a scaffold binding peptide. In further embodiments, a single scaffold binding peptide is capable of binding to more than one insecticidal polypeptide. It will be understood by one of skill in the art that the transgenic plant may comprise any gene imparting an agronomic trait of interest.

[0032] In another aspect the compositions and methods of the embodiments are useful for the production of organisms with enhanced pest resistance or tolerance. These organisms and compositions comprising the organisms are desirable for agricultural purposes.

[0033] In one aspect compositions and methods for conferring pesticidal activity to bacteria, plants, plant cells, tissues and seeds are provided. In one embodiment, compositions include nucleic acid molecules encoding sequences for pesticidal and insecticidal polypeptides, vectors comprising those nucleic acid molecules, and host cells comprising the vectors. In another embodiment, compositions also include the pesticidal polypeptide sequences and antibodies to those polypeptides. Compositions also comprise transformed bacteria, plants, plant cells, tissues, and seeds.

[0034] In one aspect isolated or recombinant nucleic acid molecules are provided encoding Insecticidal protein B and Insecticidal protein C polypeptides including amino acid substitutions, deletions, insertions, and fragments thereof. Provided are isolated or recombinant nucleic acid molecules capable of encoding Insecticidal protein B and Insecticidal protein C polypeptides of SEQ ID NOs: 58 and 59, respectively, as well as amino acid substitutions, deletions, insertions, fragments thereof, and combinations thereof. In certain embodiments, polynucleotides are provided that encode insecticidal polypeptides, wherein the polynucleotides comprise a nucleic acid sequence as set forth in any one of SEQ ID NOs: 60 and 61 (coding sequences for Insecticidal protein B), and 62 (coding sequence for Insecticidal protein C). Nucleic acid sequences that are complementary to a nucleic acid sequence of the embodiments or that hybridize to a sequence of the embodiments are also encompassed. The nucleic acid sequences can be used in DNA constructs or expression cassettes for transformation and expression in organisms, including microorganisms and plants. The nucleotide or amino acid sequences may be synthetic sequences that have been designed for expression in an organism including, but not limited to, a microorganism or a plant.

[0035] In another aspect Insecticidal protein B and Insecticidal protein C polypeptides are encompassed. Also provided are isolated or recombinant Insecticidal protein B and Insecticidal protein C polypeptides of SEQ ID NOs: 58 and 59, as well as amino acid substitutions, deletions, insertions, fragments thereof and combinations thereof.

[0036] In another aspect methods are provided for producing the polypeptides and for using those polypeptides for controlling or killing a Lepidopteran, Coleopteran, Hemipteran, nematode, fungi, and / or Dipteran pests. In one embodiment, the transgenic plants of the embodiments express one or more of the pesticidal sequences disclosed herein. In various embodiments, the transgenic plant further comprises one or more additional genes for insect resistance, for example, one or more additional genes for controlling Coleopteran, Lepidopteran, Hemipteran or nematode pests. It will be understood by one of skill in the art that the transgenic plant may comprise any gene imparting an agronomic trait of interest.

[0037] In another aspect methods for detecting the nucleic acids and polypeptides of the embodiments in a sample are also included. A kit for detecting the presence of an Insecticidal protein B and Insecticidal protein C polypeptide or detecting the presence of a polynucleotide encoding an Insecticidal protein B or Insecticidal protein C polypeptide in a sample is provided. The kit may be provided along with all reagents and control samples necessary for carrying out a method for detecting the intended agent, as well as instructions for use.

[0038] In another aspect the compositions and methods of the embodiments are useful for the production of organisms with enhanced pest resistance or tolerance. These organisms and compositions comprising the organisms are desirable for agricultural purposes. In another embodiment, the compositions of the embodiments are also useful for generating altered or improved proteins that have pesticidal activity or for detecting the presence of Insecticidal protein B and / or Insecticidal protein C polypeptides.BRIEF DESCRIPTION OF THE FIGURES

[0039] FIG. 1 shows an illustration of the engineered maize phytocystatin scaffold created using AlphaFold with mutagenic loops highlighted.

[0040] FIG. 2 illustrates the binding interface mapped by crosslinking mass spectrometry of insecticidal protein Insecticidal protein A with scaffold binding peptide PMSB10. The four crosslinked residues are in domain I of Insecticidal protein A: T137, R141, R194 and Y198. Regions T137-R141 and R194-Y198 are colored in white marking the epitope while the rest of domain I is shown in black.

[0041] FIG. 3 illustrates the structural similarity between Insecticidal protein A domain I (in darkgrey) and CrylFa domain I (in light grey). On the right is a zoomed-in view of helices a-4, a-5, and a-6 of Insecticidal protein A domain I. The epitope defined by T137-R141 and R194-Y198 based on PMSB10 is shown. Naming of helices follows traditional 3d-Cry toxin helix naming convention as Insecticidal protein A has an extra helix at the N-terminal end. Structure of Insecticidal protein A is modeled by AlphaFold 2.0 and structure of CrylFa is determined by X- ray crystallography.

[0042] FIG. 4 illustrates a structural model of PMSB 10-linkerl8-Insecticidal protein A fusion protein by AlphaLink with distance restraint information from crosslinking Mass Spectroscopy incorporated.

[0043] FIG. 5 illustrates the binding interface mapped by crosslinking mass spectrometry of Insecticidal protein B with scaffold binding peptide PMSB19. The seven crosslinked residues are in domain I and domain II of Insecticidal protein B: T211, R215, R218, T219, S282, S285, and R292. Crosslinked residues are colored in white marking the epitope while the rest of domain I and domain II are shown in black and grey.

[0044] FIG. 6 illustrates a structural model of Insecticidal protein B-linker9-PMSB19 fusion protein by AlphaLink with distance restraint information from crosslinking Mass Spectroscopy incorporated.

[0045] FIG. 7 illustrates the binding interface mapped by crosslinking mass spectrometry of Insecticidal protein B with scaffold binding peptide PMSB23 (not shown). The four crosslinked residues are in domain I of Insecticidal protein B: T120, K125, R129, and S137. Crosslinked residues are colored in white marking the epitope while the rest of domain I are shown in black.

[0046] FIG. 8 illustrates a structural model of Insecticidal protein B-linker9-PMSB23 fusion protein by AlphaLink with distance restraint information from crosslinking Mass Spectroscopy incorporated.DETAILED DESCRIPTION

[0047] It is to be understood that this disclosure is not limited to the particular methodology, protocols, cell lines, genera, and reagents described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure.

[0048] As used herein the singular forms "a", "and", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the protein" includes reference to one or more proteins and equivalents thereof, and so forth. All technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs unless clearly indicated otherwise.

[0049] As used herein, the terms “undesirable phenotypic characteristic(s)”, “undesirable phenotype(s)” in a plant, or “undesirable plant phenotype(s)” include but are not limited to, plant phytotoxicity, low protein production in planta, poor plant growth, developmental pathology, and phenotypic alterations.

[0050] Some insecticidal proteins showing high efficacy in certain insects may induce undesirable phenotypic effects when expressed in plants under certain conditions. The mechanism of action underpinning the undesirable phenotypic characteristics elicited when insecticidal proteins are recombinantly expressed in planta is unknown and is likely very diverse. Possible mechanisms may include negative epitopes that drive undesirable phenotypic plant responses, such as by blocking an essential plant factor, pore formation, and protein aggregation, and may be unique to each insecticidal protein. In one embodiment, reducing undesirable phenotypic characteristics, including but not limited to phytotoxicity, in plants will allow high-dose delivery of certain insecticidal proteins without significantly sacrificing yield and durability. Many strategies have been successfully used to inhibit pore-forming toxins including small molecules, synthetic nanoparticles, antibodies, antibody mimetics, and polyvalent inhibitors that have been reviewed in the literature (Omersa, et al. (2019) Toxins (Basel) 11(9):545). These strategies can be difficult to employ with insecticidal traits as they must preserve insecticidal activity while mitigating undesirable phenotypic characteristics and be capable of being recombinantly expressed in planta.

[0051] Creating a universal platform for mitigating undesirable phenotypic characteristics attributable to the presence of certain polypeptides of interest (e.g., insecticidal proteins) is desirable, for example by using a generic protein binding strategy that is independent of the undesirable phenotypic characteristic mechanism. Described herein are compositions and methods for reducing undesirable phenotypic characteristics, compositions and methods for increasing expression of polypeptides of interest (e.g., insecticidal proteins), and compositions andmethods for increasing durability of polypeptides of interest (e.g., insecticidal proteins), by use of scaffold binding peptides, which are proteins with two variable domains, similar to an antibody, that bind to target epitopes of polypeptides of interest (e g., insecticidal proteins) to prevent undesirable phenotypic characteristics.

[0052] The present disclosure is drawn to compositions and methods for mitigating undesirable phenotypic characteristics attributable to the presence of one or more transgenic polypeptides of interest, such as for example insecticidal polypeptides of interest, in a transgenic plant. In one embodiment, the methods involve transforming organisms with nucleic acid sequences encoding one or more scaffold binding peptides, one or more polypeptides of interest (e.g., insecticidal polypeptides of interest), and optionally one or more linkers. In particular, the nucleic acid sequences of the embodiments are useful for preparing plants and microorganisms that possess pesticidal activity. Thus, transformed bacteria, plants, plant cells, plant tissues and seeds are provided. In some embodiments, the compositions include nucleic acid molecules encoding an engineered scaffold polypeptide, scaffold binding peptides, chimeric polypeptides, expression constructs comprising the nucleic acid molecules, and host cells comprising the constructs. Compositions also include scaffold binding peptide sequences, including chimeric fusion polypeptides optionally comprising one or more scaffold binding peptides, one or more linkers, and one or more polypeptides of interest (e.g., insecticidal polypeptides of interest). Compositions also comprise transformed bacteria, plants, plant cells, tissues and seeds. The nucleic acid sequences find use in the construction of expression vectors for subsequent transformation into organisms of interest and for the generation of altered scaffold, scaffold binding peptide, or chimeric fusion polypeptides described herein by utilizing aspects of certain methods known in the art, such as site directed mutagenesis, domain swapping, or DNA shuffling.

[0053] The scaffold binding peptides expressed or co-expressed with insecticidal polypeptides of interest find use in controlling or killing Lepidopteran, Coleopteran, Dipteran, fungal, Hemipteran and nematode pest populations and for producing compositions with pesticidal activity. Insect pests of interest include, but are not limited to, Lepidoptera species including but not limited to: Com Earworm (CEW, Helicoverpa zed), European Com Borer (ECB, Ostrinia nubialis), Fall Armyworm (FAW, Spodoptera frugiperdd), Southern Armyworm (SAW, Spodoptera eridanid), Soybean looper (SBL, Pseudoplusia includens), diamond-back moth, e.g., Helicoverpa zeaBoddie, and velvet bean caterpillar (VBC, Anticar sia gemmatalis Hubner) and Coleoptera species including but not limited to Western com rootworm (Diabrotica virgifera) - WCRW, Southern corn rootworm (Diabrotica undecimpunctata howardi) - SCRW, and Northern corn rootworm (Diabrotica barberi) - NCRW.

[0054] The present disclosure is also drawn to compositions and methods for controlling pests. In one embodiment, the methods involve transforming organisms with nucleic acid sequences encoding Insecticidal protein B and / or Insecticidal protein C polypeptides. In particular, the nucleic acid sequences of the embodiments are useful for preparing plants and microorganisms that possess pesticidal activity. Thus, transformed bacteria, plants, plant cells, plant tissues and seeds are provided. The compositions include pesticidal nucleic acids and proteins of plant or bacterial species. The nucleic acid sequences find use in the construction of expression vectors for subsequent transformation into organisms of interest, as probes for the isolation of other homologous (or partially homologous) genes, and for the generation of altered Insecticidal protein B and Insecticidal protein C polypeptides by utilizing aspects of certain methods known in the art, such as site directed mutagenesis, domain swapping, or DNA shuffling.

[0055] The Insecticidal protein B and / or Insecticidal protein C polypeptides find use in controlling or killing Lepidopteran, Coleopteran, Dipteran, fungal, Hemipteran and nematode pest populations and for producing compositions with pesticidal activity. Insect pests of interest include, but are not limited to, Lepidoptera species including but not limited to: Corn Earworm (CEW; Helicoverpa zea), European Corn Borer (ECB; Ostrinia nubialis), Fall Armyworm (FAW; Spodoptera frugiperda), Soybean looper (SBL; Pseudoplusia inchidens), diamond-back moth, e.g., Helicoverpa zea Boddie; and velvet bean caterpillar e.g., Anticar sia gemmatalis Hubner and Coleoptera species including but not limited to Western corn rootworm (WCRW; Diabrotica virgifera), Southern com rootworm (SCRW; Diabrotica undecimpunctata howardi) , and Northern corn rootworm (NCRW; Diabrotica barberi).

[0056] By “pesticidal toxin” or “pesticidal protein” is used herein to refer to a toxin that has toxic activity against one or more pests. For example, pests may include members of the Lepidoptera, Diptera, Hemiptera and Coleoptera orders or the Nematoda phylum or a protein that has homology to such a protein. Pesticidal proteins have been isolated from organisms including, for example, Bacillus sp., Bacillus thurengiensis (fB ), Pseudomonas sp., Photorhabdus sp., Xenorhabdus sp.,Clostridium bifermentans and Paenibacillus popilliae, as well as from plant species including but not limited to Selaginella, Polystichum, Adiantum, Coniogramme, Davallia, Didymochlaena, Humata, Onoclea, and Tectaria species.

[0057] In some embodiments an Insecticidal protein B and / or Insecticidal protein C polypeptide includes an amino acid sequence deduced from the full-length nucleic acid sequence disclosed herein and amino acid sequences that are shorter than the full-length sequences, either due to the use of an alternate downstream start site or due to processing that produces a shorter protein having pesticidal activity. Processing may occur in the organism in which the protein is expressed in or in the pest after ingestion of the protein.

[0058] In some embodiments, the Insecticidal protein B and / or Insecticidal protein C polypeptide has an altered spectrum of activity. In another embodiment, the Insecticidal protein B and / or Insecticidal protein C polypeptide has an altered amount of pesticidal activity. In some embodiments, the Insecticidal protein B and / or Insecticidal protein C polypeptide has an altered mode of action or site of action. In some embodiments, the Insecticidal protein B and / or Insecticidal protein C polypeptide has an altered solubility.

[0059] Thus, provided herein are isolated or recombinant nucleic acid sequences encoding Insecticidal protein B and Insecticidal protein C polypeptides conferring pesticidal activity. Also provided are the amino acid sequences of Insecticidal protein B and Insecticidal protein C polypeptides. The polypeptides resulting from translation of these Insecticidal protein B and Insecticidal protein C genes allow cells to control or kill pests that ingest it.

[0060] By “insecticidal polypeptide of interest” or “insecticidal protein of interest” is used herein to refer to a protein that has toxic activity against one or more pests. For example, pests may include members of the Lepidoptera, Coleoptera, Diptera, Hemiptera orders or the Nematoda phylum or a protein that has homology to such a protein. Insecticidal proteins of interest have been isolated from organisms including but not limited to, for example, Bacillus sp., Bacillus thurengiensis (fBC), Pseudomonas sp., Photorhabdus sp., Xenorhabdus sp., Clostridium bifermentans and Paenibacillus popilliae, as well as from plant species including but not limited to Selaginella, Polystichum, Davallia, Didymochlaena, Humata, Onoclea, and Tectaria species.

[0061] Members of B. thuringiensis insecticidal protein classes are known to one skilled in the art (see, Crickmore, et al., Microbiology and Molecular Biology Reviews (1998) Vol 62: 807-813;and Crickmore, etal., "Bacillus thuringiensis toxin nomenclature" (2016), at btnomenclature.info / which can be accessed on the world-wide web using the "www" prefix). As used herein, a “B / Cry toxin,” or a “Cry toxin” refers to a parasporal inclusion (crystal) protein from B. thuringiensis that exhibits some experimentally verifiable toxic effect to a target organism, or any protein that has obvious sequence similarity to a known Cry protein (Crickmore, et al., Microbiology and Molecular Biology Reviews (1998) Vol 62: 807).Methods of Utilizing Scaffold Binding Peptides and Chimeric Polypeptides in Transgenic Plants

[0062] The scaffold binding peptides and chimeric fusion polypeptides comprising them as described herein are useful for reducing undesirable plant phenotypes of transgenic plants expressing polypeptides of interest such as insecticidal proteins of interest. The scaffold binding peptides and chimeric fusion polypeptides comprising them as described herein are also useful for improving the expression and efficacy of polypeptides of interest, and also for increasing the durability of certain insecticidal proteins of interest in a plant.

[0063] In one embodiment of a non-limiting hypothesis, the mechanisms causing undesirable phenotypic characteristics, such as but not limited to phytotoxicity, in plants in response to certain polypeptides of interest (e.g., insecticidal proteins) are complicated and may include, but are not limited to biochemical changes such as lipid peroxidation, enzyme inactivation, cell injury or death, and disruption of membrane and ion homeostasis; molecular changes such as chromosomal aberrations, DNA damage, altered cell division, and gene regulation; and physiological changes such as disturbed membrane, altered stomatai opening, reduced photosynthesis, chlorosis, necrosis, and impacts on reproductive growth.

[0064] In one embodiment, the presentation of undesirable plant phenotypes in response to the expression of one or more polypeptides of interest may be reduced by the association of one or more scaffold binding peptides with the one or more polypeptides of interest, such as insecticidal polypeptides of interest. The one or more scaffold binding peptides may be the same scaffold binding peptides, or different scaffold binding peptides, and may be used in conjunction with one or more linker sequences. In another embodiment, the scaffold binding peptide has binding affinity for the one or more polypeptides of interest, such as an insecticidal polypeptide of interest.

[0065] In one embodiment, the one or more scaffold binding peptides may be expressed or coexpressed in a plant with the one or more polypeptide of interest, such as one or more insecticidal polypeptide(s) of interest. The one or more scaffold binding peptides and the one or more transgenic polypeptides of interest may be expressed or co-expressed in the plant using independent plant promoters, wherein the independent plant promoters may be the same promoter or different promoters. Alternatively, the one or more transgenic or insecticidal polypeptide(s) of interest and scaffold binding peptides may be expressed or co-expressed in the plant using the same plant promoter. In one embodiment, the expressed or co-expressed scaffold binding peptides may bind to or associate with the transgenic polypeptide of interest, such as an insecticidal polypeptide of interest, and block the region responsible for generating the undesirable plant phenotype.

[0066] In one embodiment, scaffold binding peptides may be expressed in plants as a chimeric fusion protein to the transgenic polypeptide of interest, such as an insecticidal polypeptide of interest. In some embodiments, chimeric fusion proteins are provided comprising a polypeptide of interest fused to one or more scaffold binding peptides, represented by a formula selected from:R'-L-R2, R2-L- R1, R1- R2or R2- R1wherein R1is a transgenic polypeptide of interest, such as an insecticidal polypeptide of interest, and R2is a scaffold binding peptide. The R1polypeptide is fused either directly or through a linker (L) segment to the R2polypeptide. The term "directly" defines fusions in which the polypeptides are joined without a peptide linker. Thus “L” represents a chemical bound or polypeptide segment to which both R1and R2are fused in frame, most commonly L is a linear peptide to which R1and R2are bound by amide bonds linking the carboxy terminus of R1to the amino terminus of L and carboxy terminus of L to the amino terminus of R2.

[0067] In some embodiments, the linker sequence between the scaffold binding peptide and polypeptide of interest contains an insect specific protease cleavage site. Serine proteases are the primary enzymes for activating toxins for Lepidopteran and Coleopteran insects. For Lepidopteran insects, the protease cleavage site is usually a serine protease site, for example trypsin, and chymotrypsin may also be used. For Coleopteran insects, the protease cleavage site can also be a cathepsin site. For Hemipteran insects, the protease cleavage site can be an alkaline protease and / or an acidic proteinase site. In one embodiment, the core structure of the linker forms a rigidhelical structure. The length of the linker is target protein dependent and needs to reach anywhere on the target protein, which can be small or large. In one embodiment, the length of the linker is between 25 amino acids to 45 amino acids in length. In other embodiments, the length of the linker is between 25 amino acids to 70 amino acids in length. In certain embodiments, the linker has a low bias for amino acids Gin, Ala, and Pro. In another embodiment, the linker can be two subhelices connected by a loop. In one embodiment, if a scaffold binding peptide is fused to the C- terminus of an insecticidal protein and if that insecticidal protein naturally ends in a protease sensitive residue such as arginine or lysine (for example the C-terminus of tryptic core of 3d-Cry toxin), then an additional protease cleavage site may be optional. In one embodiment, the linker has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence selected from SEQ ID NOs: 7-27. In another embodiment, the linker is selected from SEQ ID NOs: 7-27.

[0068] In other embodiments, fusion proteins are provided comprising an Insecticidal protein B or Insecticidal protein C polypeptide or chimeric Insecticidal protein B or Insecticidal protein C polypeptide of the disclosure represented by a formula selected from the group consisting of:R’-L-R2, R2-L- R1, R1- R2or R2- R1

[0069] wherein R1is an Insecticidal protein B or Insecticidal protein C polypeptide or chimeric Insecticidal protein B or Insecticidal protein C of the disclosure and R2is a polypeptide of interest. In some embodiments R1and R2are an Insecticidal protein B or Insecticidal protein C polypeptide or chimeric Insecticidal protein B or Insecticidal protein C of the disclosure. The R1polypeptide is fused either directly or through a linker (L) segment to the R2polypeptide. The term "directly" defines fusions in which the polypeptides are joined without a peptide linker. Thus “L” represents a chemical bound or polypeptide segment to which both R1and R2are fused in frame, most commonly L is a linear peptide to which R1and R2are bound by amide bonds linking the carboxy terminus of R1to the amino terminus of L and carboxy terminus of L to the amino terminus of R2.

[0070] By "fused in frame" is meant that there is no translation termination or disruption between the reading frames of R1and R2. The linking group (L) is generally a polypeptide of between 1 and 500 amino acids in length. The linkers joining the two molecules are preferably designed to (1) allow the two molecules to fold and act independently of each other, (2) not have a propensity for developing an ordered secondary structure which could interfere with the functional domains of the two polypeptides, (3) have minimal hydrophobic or charged characteristic which couldinteract with the functional polypeptide domains and (4) provide steric separation of R1and R2. A linker usually consists of either a flexible loop or flexible residues or loops at the ends and a rigid core region in the middle for separating R1away from R2. Typically surface amino acids in flexible protein regions include Gly, Asn and Ser. Virtually any permutation of amino acid sequences containing Gly, Asn and Ser would be expected to satisfy the above criteria for flexible loop. Other neutral amino acids, such as Ala may also be used in flexible loop linker sequence under certain circumstances. The rigid core region in a linker is typically of helical content that can be designed by modern computational tools. Residues that are favored to form a helix include Met, Ala, Leu, Glu and Lys. Additional amino acids may also be included in the linkers due to the addition of unique restriction sites in the linker sequence to facilitate construction of the fusions.

[0071] In some embodiments, the linkers or portions of the linkers comprise sequences selected from the group of formulas: (Gly.3Ser)n, (Gly4Ser)n, (GlysSer)n, (GlynSer)nor (AlaGlySer)nwhere n is an integer. One example of a highly-flexible linker is the (GlySer)-rich spacer region present within the pill protein of the filamentous bacteriophages, e g. bacteriophages M13 or fd (Schaller, et al., 1975). Also included are linkers in which an endopeptidase recognition sequence is included, such as for example, Plasmin, Enterokinase, Kallikerin, Urokinase, Tissue Plasminogen activator, clostripain, Chymosin, Collagenase, Russell's Viper Venom Protease, Postproline cleavage enzyme, V8 protease, Thrombin and factor Xa. In some embodiments the linker comprises the amino acids from the multi-gene expression vehicle (MGEV; See International Patent Application Publication No. WO2007 / 137329), which is cleaved by vacuolar proteases. In other embodiments, peptide linker segments from the hinge region of heavy chain immunoglobulins IgG, IgA, IgM, IgD or IgE provide an angular relationship between the attached polypeptides. Especially useful are those hinge regions where the cysteines are replaced with serines. In some embodiments, linkers of the present disclosure include the endogenous recognition sequences of digestive endopeptidases obtained from crop pests, including those insects disclosed herein, and derivatives thereof. In other embodiments, linkers of the present disclosure may include sequences derived from murine IgG gamma 2b hinge region in which the cysteines have been changed to serines. The fusion proteins are not limited by the form, size or number of linker sequences employed and the only requirement of the linker is that functionally it does not interfere adversely with the folding and function of the individual molecules of the fusion.Methods of Evaluating the Effects of Scaffold Binding Peptides

[0072] Various assays are available for evaluating the mitigation of undesirable plant phenotypes by the inclusion of one or more scaffold binding peptides with the one or more polypeptides of interest, such as one or more insecticidal polypeptides, whether the scaffold binding peptide is coexpressed with the insecticidal polypeptide in a plant or expressed as a chimeric fusion polypeptide comprising one or more scaffold binding peptides, one or more linkers, and one or more insecticidal polypeptides of interest.

[0073] For example, the mitigation of transgenic or insecticidal active-induced undesirable plant phenotypes, such as but not limited to phytotoxicity, may be visually or spectrophotometrically evaluated by growing transgenic plants or transgenic plant cells or tissues that include a scaffold binding peptide with the transgenic or insecticidal protein and comparing the growth parameters at certain times with appropriate control plants expressing the same transgene without said scaffold binding peptide, and also non-transgenic null controls. Likewise, decreased plant health may be visually or spectrophotometrically evaluated in a substantially similar manner. Options for screening with the ability to quantify expression, phytotoxicity, and insect efficacy include but are not limited to maize protoplast assays, bush bean transient assays, and crop transient and stable assays.

[0074] Transgene expression may be evaluated in a similar experimental format, utilizing tools such as Western blot, ELISA, mass spectrometry, Octet, SPR, FRET and other protein quantitation methods to determine the levels of transgene expression.

[0075] Transformation efficiency may be evaluated, for example, using the Quick corn assay method(s) as disclosed in U.S. Patent No. 11,330,776, which is herein incorporated by reference in its entirety.Methods of Generating Scaffold Binding Peptides

[0076] Many protein scaffolds can be used to initiate the generation of scaffold binding partner peptides consistent with the teachings herein. Desirable properties of a suitable scaffold include a highly stable framework in which randomized surface regions are created and screened for binding to a target protein or other substrate. They are typically small (<100 amino acids), monomeric, and are easily produced and purified from E. coli. The core fold is highly resistant to denaturation byheat and chemical agents such as guanidine HC1 or urea. These may include, but are not limited to, antibody derivatives, single chain antibody, domain antibody, nanobody, fibronectin type III domain (e.g. AdNectin and Centyrin), Anticalins, Affibodies, Alphabody, knottin, peptide aptamer (e.g., thioredoxin scaffold), Avimer, Affilins, Affitin, and ADAPT (albumin-derived affinity protein), nanoCLAMPs, families of protease inhibitor proteins, such as cystatins (e.g., Affimer), phytocystatins, including but not limited to Maize phytocystatin 1 (Zm00001d043175; SEQ ID NO: 65), subtilisin-chymotrypsin inhibitors, subtilisin inhibitor eglin C, potato chymotrypsin inhibitor I family of serine protease inhibitors, Kunitz domains, Atrimers, DARPins, dArmRPs (Designed Armadillo Repeat Proteins), and Repebodies. In addition, sources to initiate the generation of scaffold binding partner peptides further include the small protein SH3 domains (e.g., Fynomers), Protein GB1 domains of proteins, or by synthetic design including any of SEQ ID NOs: 1-6 and 74-79 of U.S. patent application publication number 2016 / 0032278, incorporated herein by reference.

[0077] In one embodiment, scaffold binding peptides are generated using one or multiple rounds of phage display. One method for selection of binding peptides using phage display is disclosed in Sidhu, S.S., etal., Methods in Enzymology, 328:333-363 (2000).

[0078] A variety of scaffold binding peptides and linkers are contemplated. Scaffold binding peptides may be directly selected from peptide-protein of interest fusion libraries for those enhancing expression of transgenic polypeptides of interest, reducing undesirable plant phenotypes, such as phytotoxicity, and for altering (i.e., enhancing) protein accumulation levels in transgenic plants or appropriate surrogate. “Scaffold binding peptide” or “scaffold binding partner peptide” as used herein refers to a peptide that when expressed as an N- and / or C-terminal fusion linked to a transgenic polypeptide of interest, or co-expressed with a transgenic polypeptide of interest, such as for example an insecticidal protein of interest, in planta has at least one of: a mitigating effect on one or more undesirable phenotypic responses of the plant, such as but not limited to phytotoxic phenotypic responses, to the expression of the transgenic polypeptide of interest, such as for example an insecticidal protein of interest; improves the phenotype of transgenic plants expressing polypeptides of interest such as insecticidal proteins of interest; increases the expression of one or more heterologous polypeptides of interest in a plant, such as for example an insecticidal protein of interest; increases accumulation of the heterologouspolypeptide of interest, such as for example an insecticidal protein of interest in the plant; increases the durability of one or more heterologous polypeptides of interest in a plant, such as for example an insecticidal protein of interest; and / or, alters the accumulation levels of the heterologous polypeptide of interest, such as for example an insecticidal protein of interest, in the plant (each of which is considered “scaffold binding peptide activity”). The protein of interest can be, for example, a transgenically expressed protein in a plant cell such as an insecticidal protein. A scaffold binding peptide is engineered to bind to a heterologous polypeptide that is not its naturally occurring binding partner.

[0079] Directly screening for reduced undesirable phenotypic characteristics and / or improved expression may occur in plants, plant cells, plant tissues, yeast or other surrogate assays such as those disclosed herein. Peptides of different sequence, position, length, and sequence of linker may be selected or designed using structural modeling information for testing in appropriate assays. Peptides that increase or decrease the stability of specific protein conformations may be isolated. Many insecticidal proteins are known to undergo significant conformational changes upon activation in the target pest digestive tract. Limiting these conformations to those commensurate with a healthy plant phenotype could be desirable.

[0080] Peptide sequences obtained via phage display may be screened and selected based on their binding affinity in the high micromolar range. In one embodiment, to determine binding kinetics, scaffold binding peptides are subcloned into a modified pET21b(+)_10xCHIS vector and recombinantly expressed in an E. coli expression system. Only scaffold binding peptides that are soluble and stable, as determined by size exclusion chromatography, have binding kinetic metrics measured for their interaction with polypeptides of interest such as insecticidal proteins of interest (including on-rate, off-rate, and Kd values). Higher affinity peptides may be obtained through further mutation and screening in serial rounds of phage biopanning in order to obtain scaffold binding peptides that bind the one or more transgenic polypeptides of interest, such as for example insecticidal proteins of interest, with a binding affinity in the low micromolar to nanomolar range. In one embodiment, a scaffold binding peptide that binds to one or more transgenic polypeptides of interest, such as for example an insecticidal protein of interest, has a binding affinity of less than about 5 pM. In one embodiment, a scaffold binding peptide that binds to one or more transgenic polypeptides of interest, such as for example an insecticidal protein of interest, has a bindingaffinity of less than about 500 nM. In another embodiment, a scaffold binding peptide that binds to one or more transgenic polypeptides of interest, such as for example an insecticidal protein of interest, has a binding affinity of less than about 50 nM. In another embodiment, a scaffold binding peptide that binds to one or more transgenic polypeptides of interest, such as for example an insecticidal protein of interest, has a binding affinity of less than about 5 nM. In another embodiment, the scaffold binding peptides or fusion partner polypeptides may be generated using random peptide generation techniques.

[0081] In one embodiment, "NNK" mutagenesis may be performed where random mutations are desired at certain amino acid residues of the candidate scaffold binding peptide. Single mutation and multiple mutation candidate scaffold binding peptides may be made by a similar manner using mutagenesis oligonucleotides designed to create the selected substitutions at the desired residues of the candidate scaffold binding peptide. Positions with multiple desired mutations are made with degenerate forward primers whereas positions with only one desired mutation are made with nondegenerate primers. Candidate colonies may be amplified by colony PCR, and the PCR product sequenced first with a single primer to confirm the presence of the desired mutation or mutations, and subsequently sequenced fully with multiple primers to identify clones with no additional mutations. Loop regions of the source polypeptides may be particularly targeted for mutagenesis and screening during the generation of scaffold binding peptides. For example, in addition to generating random mutations in the loop regions, the size of the loop region may be expanded by mutagenesis. In one embodiment, one or more of the loop regions of the candidate scaffold binding peptide may be expanded by about 5-10 amino acids.

[0082] Libraries of scaffolds with random loop diversity may be constructed using oligonucleotide-directed mutagenesis of uracil containing templates of the phage display vectors as described in Tonikian et al. (2007) Nat Protoc. 2(6): 1368-86; Kunkel, (1985) PNAS USA 82:488-492; and Kunkel, et al., (1987) Methods Enzymol 154:367-382. In brief, uracil-containing ssDNA of the phage display vector is purified from an E. coli dut- / ung- strain such as CJ236. In one embodiment, oligos are designed containing various lengths of random codons of NNK, where N is any base and K is G or T, with flanking sequences that anneal to the codons encoding amino acids at the bases of the loops. NNK codons encode all 20 amino acids and one stop codon. In another embodiment, oligos are designed containing various lengths of random codons of VVC,where V is A, C or G, to bias the library for hydrophilic amino acids. VVC codons encode nine amino acids (A, D, G, H, N, P, R, S, T) and no stop codon. It is possible to use other degenerative codons strategies to create more diversity including: NNC, NWW, RVK, DVT, NVT, NNT, NTT, RST, and TDK.

[0083] These oligos are annealed to the template and then enzymatically extended and ligated to form covalently closed circular DNA that is electroporated into E. coli. The uracil containing strand is preferentially destroyed and the in vitro synthesized strand is replicated. In this way, DNA mismatches (i.e., loop variations) encoded by the synthetic oligonucleotide are incorporated into the phagemid. Efficient mutagenesis (>80%) is readily achieved, provided the template DNA is highly pure. In this way, random amino acids encoded by the synthetic oligonucleotide are incorporated into the phage display vector. Large libraries with over lOelO independent sequences can be readily obtained using this technique. Other methods for generating diversity libraries includes synthetic DNA approaches and alternative forms of cloning and mutagenesis are known in the art.

[0084] In addition to binders obtained through directed evolution exemplified by biopanning of a scaffold protein-based library as described herein, emerging technology such as Al designed miniproteins may also be utilized to obtain novel binders that mitigate undesirable phenotypic characteristics. Recent examples have seen de novo designed miniproteins neutralizing snake venom toxins (Torres, et al., Nature (2025)) and minibinder agonists of Toll-like Receptor 3(TLR3) (Adams, et al., Nature Communications (2025) 16: 1234). In those cases, the designed miniproteins have advantages of among others, short design time, high stability and tunable affinity. In combination with knowledge of effective epitopes, Al designed miniproteins binding to defined epitopes on insecticidal proteins could provide equal or better binders than those obtained via a library biopanning approach to mitigate undesirable phenotypic characteristics attributable to the presence of insecticidal proteins under certain conditions.Engineered Scaffold Polypeptides, Scaffold Binding Peptides, Linker Peptides, Insecticidal protein B and Insecticidal protein C Polypeptides, and Variants and Fragments Thereof

[0085] In one embodiment, certain engineered scaffold polypeptides are encompassed by the disclosure. In some embodiments, an engineered scaffold comprises, consists essentially of, oralternatively consists of, an amino acid sequence of SEQ ID NO: 1, as well as variants thereof and engineered scaffold polypeptides having sufficient homology to SEQ ID NO: 1. In one embodiment, certain scaffold binding peptides are encompassed by the disclosure. In some embodiments, a scaffold binding peptide comprises, consists essentially of, or alternatively consists of, an amino acid sequence of any one or more of SEQ ID NOs: 2-6, as well as variants thereof and scaffold binding peptides having sufficient homology to any one or more of SEQ ID NOs: 2-6. In another embodiment, certain cleavable linker peptides are encompassed by the disclosure. In some embodiments, a linker peptide comprises, consists essentially of, or alternatively consists of, an amino acid sequence of any one or more of SEQ ID NOs: 7-27, as well as variants thereof and linker peptides having sufficient homology to any one or more of SEQ ID NOs: 7-27.

[0086] In another embodiment, Insecticidal protein B and Insecticidal protein C polypeptides are encompassed by the disclosure. In some embodiments, an Insecticidal protein B and Insecticidal protein C polypeptide comprises, consists essentially of, or alternatively consists of, an amino acid sequence of any one or more of SEQ ID NOs: 58 and 59, as well as variants thereof and Insecticidal protein B and Insecticidal protein C polypeptides having sufficient homology to any one or more of SEQ ID NOs: 58 and 59.

[0087] “Sufficiently identical” or “sufficiently homologous” are used herein to refer to an amino acid sequence that has at least about 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence homology compared to a reference sequence using one of the alignment programs described herein using standard parameters. In some embodiments the sequence homology is against the full-length sequence of an engineered scaffold polypeptide, a scaffold binding peptide, or a linker peptide. In some embodiments, the engineered scaffold polypeptide, scaffold binding 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 greater sequence identity compared to any one of SEQ ID NOs: 1, 2-6,and 7-27, respectively.

[0088] In some embodiments the sequence homology is against the full-length sequence of an Insecticidal protein B or Insecticidal protein C polypeptide. In some embodiments, the Insecticidal protein B or Insecticidal protein C polypeptide 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 greater sequence identity compared to any one of SEQ ID NOs: 58 or 59, respectively. The term “about” when used herein in context with percent sequence identity means + / - 0.5%. One of skill in the art will recognize that these values can be appropriately adjusted to determine corresponding homology of proteins taking into account amino acid similarity and the like. In some embodiments the sequence identity is calculated using ClustalW algorithm in the ALIGNX® module of the Vector NTI® Program Suite (Invitrogen Corporation, Carlsbad, CA) with all default parameters. In some embodiments the sequence identity is across the entire length of polypeptide calculated using ClustalW algorithm in the ALIGNX® module of the Vector NTI® Program Suite (Invitrogen Corporation, Carlsbad, CA) with all default parameters.

[0089] In one embodiment, a chimeric fusion polypeptide including a scaffold binding peptide, a linker peptide, and an insecticidal polypeptide, 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 greater sequence identity compared to any one of the linked, chimeric fusion, or tethered polypeptides described in any of Tables 1, 2, 3, 4, 5, 6, or 7 in the Examples herein.

[0090] As used herein, the terms “protein,” “peptide,” “peptide molecule,” or “polypeptide” includes any molecule that comprises five or more amino acids. It is well known in the art that protein, peptide or polypeptide molecules may undergo modification, including post-translational modifications, such as, but not limited to, disulfide bond formation, glycosylation, phosphorylation or oligomerization. Thus, as used herein, the terms “protein,” “peptide,” “peptide molecule” or “polypeptide” includes any protein that is modified by any biological or non-biological process. The terms “amino acid” and “amino acids” refer to all naturally occurring L-amino acids.

[0091] A “recombinant protein” is used herein to refer to a protein that is no longer in its natural environment, for example in vitro or in a recombinant bacterial or plant host cell.

[0092] “Substantially free of cellular material” as used herein refers to a polypeptide including preparations of protein having less than about 30%, 20%, 10% or 5% (by dry weight) of non- pesticidal protein (also referred to herein as a “contaminating protein”).

[0093] “Fragments” or “biologically active portions” include polypeptide fragments comprising amino acid sequences sufficiently identical to an engineered scaffold, scaffold binding peptide or a linker peptide and that exhibit scaffold binding peptide activity or linker peptide activity as described above. “Fragments” or “biologically active portions” of scaffold binding peptides, or linker peptides include fragments comprising amino acid sequences sufficiently identical to the amino acid sequence set forth in any one of SEQ ID NOs: 2-6 and 7-27, wherein the scaffold binding peptide or linker peptide has scaffold binding peptide activity or linker peptide activity, respectively. Such biologically active portions can be prepared by recombinant techniques and evaluated for scaffold binding peptide activity or linker peptide activity. In some embodiments, the engineered scaffold, scaffold binding peptide, or linker peptide fragment is an N-terminal and / or a C-terminal truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or more amino acids from theN-terminus and / or C-terminus relative to any one of SEQ ID NOs: 1, 2-6, and 7-27, respectively: , e.g., by proteolysis, by insertion of a start codon, by deletion of the codons encoding the deleted amino acids and concomitant insertion of a start codon. In some embodiments, the engineered scaffold, scaffold binding peptide, or linker peptide fragment is an N-terminal truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, amino acids from the N-terminus of any one of SEQ ID NOs: 1, 2-6, and 7-27, respectively . In some embodiments, the engineered scaffold, scaffold binding peptide, or linker peptide fragment is an N-terminal and / or a C-terminal truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or more amino acids from the N-terminus and / or C-terminus relative to any one of SEQ ID NOs: 1, 2-6, and 7-27, respectively.

[0094] “Fragments” or “biologically active portions” include polypeptide fragments comprising amino acid sequences sufficiently identical to an Insecticidal protein B or Insecticidal protein C polypeptide and that exhibit insecticidal activity. “Fragments” or “biologically active portions” ofInsecticidal protein B or Insecticidal protein C polypeptides include fragments comprising amino acid sequences sufficiently identical to the amino acid sequence set forth in any one of SEQ ID NOs: 58 or 59, respectively, wherein the Insecticidal protein B or Insecticidal protein C polypeptide has insecticidal activity. Such biologically active portions can be prepared by recombinant techniques and evaluated for insecticidal activity. In some embodiments, the Insecticidal protein B or Insecticidal protein C polypeptide fragment is an N-terminal and / or a C- terminal truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or more amino acids from the N-terminus and / or C-terminus relative to any one of SEQ ID NOs: 58 and 59, respectively, e.g., by proteolysis, by insertion of a start codon, by deletion of the codons encoding the deleted amino acids and concomitant insertion of a start codon, and / or insertion of a stop codon. In some embodiments, the Insecticidal protein B or Insecticidal protein C 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 any one of SEQ ID NOs: 58 and 59, respectively. In some embodiments, the Insecticidal protein B or Insecticidal protein C polypeptide fragment is an N-terminal and / or a C- terminal truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or more amino acids from the N-terminus and / or C-terminus relative to any one of SEQ ID NOs: 58 and 59, respectively.

[0095] “Variants” as used herein refers to proteins or polypeptides having an amino acid sequence that is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identical to the parental amino acid sequence.

[0096] In some embodiments a scaffold binding 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 greater sequence identity to the full length or a fragment of the amino acid sequence of any one of SEQ ID NOs: 2-6, wherein the scaffold binding peptide has scaffold binding peptide activity.

[0097] In some embodiments an Insecticidal protein B or Insecticidal protein C polypeptidecomprises 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 greater sequence identity to the full length or a fragment of the amino acid sequence of any one of SEQ ID NOs: 58 and 59 respectively, wherein the Insecticidal protein B or Insecticidal protein C polypeptide has insecticidal activity.

[0098] In some embodiments an engineered scaffold, scaffold binding peptide, or linker peptide comprises an amino acid sequence of any one or more of SEQ ID NOs: 1, 2-6 and 7-27, respectively, having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 or more amino acid substitutions compared to the amino acid at the corresponding position of any one or more of the respective SEQ ID NOs: 1, 2-6, and 7-27.

[0099] In some embodiments an Insecticidal protein B or Insecticidal protein C polypeptide comprises an amino acid sequence of any one or more of SEQ ID NOs: 58 and 59, respectively, having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 or more amino acid substitutions compared to the amino acid at the corresponding position of any one or more of the respective SEQ ID NOs: 58 and 59, respectively.

[0100] In some embodiments the sequence identity is across the entire length of the polypeptide calculated using ClustalW algorithm in the ALIGNX® module of the Vector NTI® Program Suite (Invitrogen Corporation, Carlsbad, CA) with all default parameters.

[0101] Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of an engineered scaffold, scaffold binding peptide, linker peptide, or Insecticidal protein B or Insecticidal protein C polypeptide can be prepared by mutations in the DNA. This may also be accomplished by one of several forms of mutagenesis, such as for examplesite-specific double strand break technology, and / or in directed evolution. In some aspects, the changes encoded in the amino acid sequence will not substantially affect the function of the protein. Such variants will possess the desired scaffold binding peptide or linker activity. However, it is understood that the ability of a scaffold binding peptide to confer scaffold binding peptide activity or other polypeptide physical property may be improved or altered by the use of such techniques upon the compositions of this disclosure.

[0102] Conservative amino acid substitutions may be made at one or more predicted nonessential amino acid residues. A “nonessential” amino acid residue is a residue that can be altered without altering the biological activity. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include: amino acids with basic side chains (e.g., lysine, arginine, histidine); acidic side chains (e.g., aspartic acid, glutamic acid); polar, negatively charged residues and their amides (e.g., aspartic acid, asparagine, glutamic, acid, glutamine; uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine); small aliphatic, nonpolar or slightly polar residues (e.g., Alanine, serine, threonine, proline, glycine); nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); large aliphatic, nonpolar residues (e.g., methionine, leucine, isoleucine, valine, cystine); beta-branched side chains (e.g., threonine, valine, isoleucine); aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine); large aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan).

[0103] Amino acid substitutions may be made in nonconserved regions that retain function. In general, such substitutions would not be made for conserved amino acid residues or for amino acid residues residing within a conserved motif, where such residues are essential for protein activity. Examples of residues that are conserved and that may be essential for protein activity include, for example, residues that are identical between all proteins contained in an alignment of similar or related polypeptides to the sequences of the embodiments (e.g., residues that are identical in an alignment of homologous proteins). Examples of residues that are conserved but that may allow conservative amino acid substitutions and still retain activity include, for example, residues that have only conservative substitutions between all proteins contained in an alignment of similar or related polypeptides to the sequences of the embodiments (e.g., residues that have onlyconservative substitutions between all proteins contained in the alignment homologous proteins). However, one of skill in the art would understand that functional variants may have minor conserved or nonconserved alterations in the conserved residues.

[0104] In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, (1982) J Mol Biol. 157(1): 105-32). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.

[0105] It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, ibid). These are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (_3.5); aspartate (_3.5); asparagine (-3.5); lysine (_3.9) and arginine (_4.5). In making such changes, the substitution of amino acids whose hydropathic indices are within +2 is preferred, those which are within +1 are particularly preferred, and those within +0.5 are even more particularly preferred.

[0106] It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. US Patent Number 4,554,101, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein.

[0107] As detailed in US Patent Number 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 +0.1); glutamate (+3.0 +0.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5. +0.1); alanine (—0.5); histidine (—0.5); cysteine (—1.0); methionine (—1.3); valine (-1.5); leucine (—1.8); isoleucine (—1.8); tyrosine (—2.3); phenylalanine (-2.5); tryptophan (—3.4).

[0108] Alternatively, alterations may be made to the scaffold binding peptide or linker peptidesequence at the amino or carboxy terminus without substantially affecting activity. This can include insertions, deletions, or alterations introduced by modem molecular methods, such as PCR, including PCR amplifications that alter or extend the protein coding sequence by virtue of inclusion of amino acid encoding sequences in the oligonucleotides utilized in the PCR amplification.

[0109] Variant nucleotide and amino acid sequences of the disclosure also encompass sequences derived from mutagenic and recombinogenic procedures such as DNA shuffling. With such a procedure, one or more different engineered scaffold polypeptide, scaffold binding peptide, linker peptide, or Insecticidal protein B or Insecticidal protein C polypeptide coding regions can be used to create new engineered scaffold polypeptides, scaffold binding peptides, linker peptides, or Insecticidal protein B or Insecticidal protein C polypeptides possessing the desired properties. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. For example, using this approach, sequence motifs encoding a domain of interest may be shuffled between a pesticidal gene and other known pesticidal genes to obtain a new gene coding for a protein with an improved property of interest, such as an increased insecticidal activity. Strategies for such DNA shuffling are known in the art. See, for example, Stemmer, (1994) Proc. Natl. Acad. Sci. USA 91 : 10747-10751; Stemmer, (1994) Nature 370:389-391; Crameri, et al., (1997) Nature Biotech. 15:436-438; Moore, et al., (1997) J. Mol. Biol. 272:336-347; Zhang, et al., (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri, etal., (1998) Nature 391 :288-291; and US Patent Numbers 5,605,793 and 5,837,458.

[0110] In some embodiments the engineered scaffold, scaffold binding peptide, linker peptide, or Insecticidal protein B or Insecticidal protein C polypeptide has a modified physical property. As used herein, the term “physical property” refers to any parameter suitable for describing the physical -chemi cal characteristics of a protein. As used herein, “physical property of interest” and “property of interest” are used interchangeably to refer to physical properties of proteins that are being investigated and / or modified. Examples of physical properties include, but are not limited to, net surface charge and charge distribution on the protein surface, net hydrophobicity and hydrophobic residue distribution on the protein surface, surface charge density, surface hydrophobicity density, total count of surface ionizable groups, surface tension, protein size andits distribution in solution, melting temperature, heat capacity, and second virial coefficient. Examples of physical properties also include, scaffold binding peptides or linker peptides having digestibility of proteolytic fragments in an insect gut. Models for digestion by simulated gastric fluids are known to one skilled in the art (Fuchs, R.L. and J.D. Astwood. Food Technology 50: 83- 88, 1996; Astwood, J.D., et al Nature Biotechnology 14: 1269-1273, 1996; Fu TJ et al J. Agric FoodChem. 50: 7154-7160, 2002).

[0111] In some embodiments an engineered scaffold, scaffold binding peptide, or linker peptide comprises the amino acid sequence of any one or more of SEQ ID NOs: 1, 2-6, and 7-27, respectively.

[0112] In some embodiments an Insecticidal protein B or Insecticidal protein C polypeptide comprises the amino acid sequence of any one or more of SEQ ID NOs: 58 and 59, respectively.

[0113] In some embodiments, chimeric scaffold binding polypeptides are provided comprising regions of at least two different scaffold binding peptides of the disclosure.

[0114] In some embodiments, fusion polypeptides are provided comprising regions of at least two different Insecticidal protein B or Insecticidal protein C polypeptides of the disclosure.

[0115] In some embodiments, chimeric fusion polypeptides are provided comprising regions of at least two different scaffold binding peptides and linker peptides selected from any one or more of SEQ ID NOs: 2-6 and 7-27. In other embodiments, chimeric fusion polypeptides are provided comprising at least one scaffold binding peptide selected from any one of SEQ ID NOs: 2-6 and at least one linker peptide selected from any one of SEQ ID NOs: 7-27. In a further embodiment, the chimeric fusion polypeptide comprising at least one scaffold binding peptide selected from any one of SEQ ID NOs: 2-6 and at least one linker peptide selected from any one of SEQ ID NOs: 7- 27 also comprises at least one polypeptide of interest, for example, at least one insecticidal polypeptide of interest.

[0116] In other embodiments, fusion polypeptides are provided comprising regions of at least two different Insecticidal protein B or Insecticidal protein C polypeptides selected from any one or more of SEQ ID NOs: 58 and 59, respectively.

[0117] In some embodiments, chimeric scaffold binding polypeptide(s) are provided comprising an N-terminal Region of a first scaffold binding peptide of the disclosure operably fused to a C- terminal Region of a second scaffold binding peptide of the disclosure.

[0118] In another embodiment fusion proteins are provided that include within its amino acid sequence an amino acid sequence comprising a scaffold binding peptide and linker peptide of the disclosure. In one embodiment, the scaffold binding peptide and linker peptide may comprise an additional fusion to an insecticidal or other polypeptide of interest. Methods for design and construction of fusion proteins (and polynucleotides encoding same) are known to those of skill in the art. Polynucleotides encoding a scaffold binding peptide and optionally a linker peptide may be fused to signal sequences which will direct the localization of the scaffold binding peptide to particular compartments of a prokaryotic or eukaryotic cell and / or direct the secretion of the embodiments from a prokaryotic or eukaryotic cell.

[0119] For example, in E. coli, one may wish to direct the expression of the scaffold binding peptide to the periplasmic space through, but are not limited to, the use of a pelB signal sequence like the pelB pectate lyase signal sequence, the maltose binding protein (MBP) signal sequence, MBP, the ompA signal sequence, the signal sequence of the periplasmic E. coli heat-labile enterotoxin B-subunit and the signal sequence of alkaline phosphatase. See also the commercially available pMAL series of vectors (particularly the pMAL-p series) available from New England Biolabs (Ipswich, MA).

[0120] In another embodiment fusion proteins are provided that include within its amino acid sequence an amino acid sequence comprising an Insecticidal protein B or Insecticidal protein C polypeptide or chimeric Insecticidal protein B or Insecticidal protein C polypeptide of the disclosure. Methods for design and construction of fusion proteins (and polynucleotides encoding same) are known to those of skill in the art. Polynucleotides encoding an Insecticidal protein B or Insecticidal protein C polypeptide may be fused to signal sequences which will direct the localization of the Insecticidal protein B or Insecticidal protein C polypeptide to particular compartments of a prokaryotic or eukaryotic cell and / or direct the secretion of the Insecticidal protein B or Insecticidal protein C polypeptide of the embodiments from a prokaryotic or eukaryotic cell.

[0121] Plant plastid transit peptide / polypeptide fusions are known in the art. Apoplast transit peptides such as rice or barley alpha-amylase secretion signal are also known in the art. The plastid transit peptide is generally fused N-terminally to the polypeptide to be targeted (e.g., the fusion partner). In one embodiment, the fusion protein may comprise, or alternatively consist essentiallyof, the plastid transit peptide, the scaffold binding peptide to be targeted, optionally a linker peptide, and a polypeptide of interest. In other embodiments, the fusion protein may comprise, or alternatively consist essentially of, the plastid transit peptide and the Insecticidal protein B or Insecticidal protein C polypeptide to be targeted. In such embodiments, the plastid transit peptide is preferably at the N-terminus of the fusion protein but may include additional amino acid residues N-terminal to the plastid transit peptide. In a specific embodiment, the plastid transit peptide is in the N-terminal half, N-terminal third or N-terminal quarter of the fusion protein. Most or all of the plastid transit peptide is generally cleaved from the fusion protein upon insertion into the plastid. In one embodiment, the plastid transit peptide cleavage site may be homogenous or alternatively may vary by 1-10 amino acids. In some embodiments the Insecticidal protein B or Insecticidal protein C polypeptide can be recombinantly fused to a heterologous signal peptide or heterologous transit peptide. The plastid transit peptide can be recombinantly fused to a second protein in one of several ways. For example, a restriction endonuclease recognition site can be introduced into the nucleotide sequence of the transit peptide at a position corresponding to its C- terminal end and the same or a compatible site can be engineered into the nucleotide sequence of the protein to be targeted at its N-terminal end. Care must be taken in designing these sites to ensure that the coding sequences of the transit peptide and the second protein are kept "in frame" to allow the synthesis of the desired fusion protein. In some cases, it may be preferable to remove the initiator methionine of the second protein when the new restriction site is introduced. The introduction of restriction endonuclease recognition sites on both parent molecules and their subsequent joining through recombinant DNA techniques may result in the addition of one or more extra amino acids between the transit peptide and the second protein. This generally does not affect targeting activity as long as the transit peptide cleavage site remains accessible and the function of the second protein is not altered by the addition of these extra amino acids at its N- terminus. Alternatively, one skilled in the art can create a precise cleavage site between the transit peptide and the second protein (with or without its initiator methionine) using gene synthesis (Stemmer, etal., (1995) Gene 164:49-53) or similar methods. In addition, the transit peptide fusion can intentionally include amino acids downstream of the cleavage site. The amino acids at the N- terminus of the mature protein can affect the ability of the transit peptide to target proteins to plastids and / or the efficiency of cleavage following protein import. This may be dependent on theprotein to be targeted. See, e.g., Comai, el al., (1988) J. Biol. Chem. 263(29): 15104-9.Nucleic Acid Molecules, and Variants and Fragments Thereof

[0122] Isolated or recombinant nucleic acid molecules comprising nucleic acid sequences encoding engineered scaffold polypeptides, scaffold binding peptides, linker peptides, chimeric fusion polypeptides, Insecticidal protein B or Insecticidal protein C polypeptides, or biologically active portions thereof, as well as nucleic acid molecules sufficient for use as hybridization probes to identify nucleic acid molecules encoding proteins with regions of sequence homology are provided. 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) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.

[0123] An "isolated" nucleic acid molecule (or DNA) is used herein to refer to a nucleic acid sequence (or DNA) that is no longer in its natural environment, for example in an in vitro or in a heterologous recombinant bacterial or plant host cell. In some embodiments, an isolated nucleic acid molecule, or biologically active portion thereof, is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. An isolated nucleic acid is free of sequences (optimally protein encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. A “recombinant” nucleic acid molecule (or DNA) is used herein to refer to a nucleic acid sequence (or DNA) that is in a recombinant bacterial or plant host cell. In some embodiments, an “isolated” or “recombinant” nucleic acid is free of sequences (preferably protein encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For purposes of the disclosure, “isolated” or “recombinant” when used to refer to nucleic acid molecules excludes isolated chromosomes. For example, in various embodiments, the recombinant nucleic acid molecules encoding engineered scaffold polypeptides, scaffold binding peptides, and linker peptides can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleic acid sequences that naturally flank the nucleic acid molecule ingenomic DNA of the cell from which the nucleic acid is derived.

[0124] In some embodiments an isolated nucleic acid molecule encoding an engineered scaffold polypeptide, a scaffold binding peptide, and a linker peptide has one or more change in the nucleic acid sequence compared to the native or genomic nucleic acid sequence. In some embodiments an isolated nucleic acid molecule encoding Insecticidal protein B or Insecticidal protein C polypeptides has one or more change in the nucleic acid sequence compared to the native or genomic nucleic acid sequence. In some embodiments the change in the native or genomic nucleic acid sequence includes but is not limited to: changes in the nucleic acid sequence due to the degeneracy of the genetic code; changes in the nucleic acid sequence due to the amino acid substitution, insertion, deletion and / or addition compared to the native or genomic sequence; removal of one or more intron; deletion of one or more upstream or downstream regulatory regions; and deletion of the 5’ and / or 3’ untranslated region associated with the genomic nucleic acid sequence. In some embodiments the nucleic acid molecule encoding an engineered scaffold or a scaffold binding peptide is a non-genomic sequence.

[0125] A variety of polynucleotides that encode engineered scaffold polypeptides and scaffold binding peptides with or without linker peptides, or related proteins are contemplated. Such polynucleotides are useful for production of engineered scaffolds and scaffold binding peptides, for example as scaffold binding peptide chimeric fusion proteins, in host cells when operably linked to a suitable promoter, transcription termination and / or polyadenylation sequences.

[0126] A variety of polynucleotides that encode Insecticidal protein B or Insecticidal protein C polypeptides or related proteins are contemplated. Such polynucleotides are useful for production of Insecticidal protein B or Insecticidal protein C polypeptides in host cells when operably linked to a suitable promoter, transcription termination and / or polyadenylation sequences. Such polynucleotides are also useful as probes for isolating homologous or substantially homologous polynucleotides that encode or related proteins.Polynucleotides Encoding Engineered Scaffold Polypeptides, Scaffold Binding Peptides, Linker Peptides, or Insecticidal protein B and Insecticidal protein C Polypeptides

[0127] As noted previously, polynucleotide sources for the generation of scaffold binding peptides or related proteins include scaffold proteins having loop domains involved in the function ofcertain protease inhibitor proteins, such as phytocystatins, subtilisin-chymotrypsin inhibitors, and subtilisin inhibitor eglin c. In addition, sources for the generation of scaffold binding peptides or linker peptides further include polynucleotides encoding the small protein SH3 domains and Protein GB1 domains of proteins. In one embodiment, polynucleotides encoding the AL11 linker peptide are available as the source for the generation of scaffold binding peptides (Robinson et al., Proc. Nat. Acad. Sci., 95(11): 5929-34 (1998)).

[0128] One source of polynucleotides that encode scaffold binding peptides, linker peptides, or related proteins is scaffold binding polynucleotides encoding scaffold binding peptides of SEQ ID NOs: 2-6. These polynucleotides can be used to express scaffold binding peptides or fusion partner polypeptides, for example as chimeric polypeptides.

[0129] One source of polynucleotides that encode shuffled Cry toxin polypeptides Insecticidal protein B or Insecticidal protein C polypeptides or related proteins is a Bacillus bacterium which may contain which may contain an Insecticidal protein B or Insecticidal protein C polynucleotide of any one of SEQ ID NOs: 60 or 61, or 62, encoding an Insecticidal protein B or Insecticidal protein C polypeptide of SEQ ID NOs: 58 or 59, respectively. The polynucleotides of any one or more of SEQ ID NOs: 60 or 61, or 62 can be used to express Insecticidal protein B or Insecticidal protein C polypeptides, respectively, in recombinant bacterial hosts that include but are not limited to Agrobacterium, Bacillus, Escherichia, Salmonella, Pseudomonas and Rhizobium bacterial host cells. The polynucleotides are also useful as probes for isolating homologous or substantially homologous polynucleotides that encode Insecticidal protein B or Insecticidal protein C polypeptides or related proteins.

[0130] Polynucleotides encoding engineered scaffold polypeptides, scaffold binding peptides, fusion polypeptides, including chimeric fusion polypeptides as described herein, or Insecticidal protein B or Insecticidal protein C polypeptides, can also be synthesized de novo from an engineered scaffold polypeptide, a scaffold binding peptide, chimeric polypeptide sequence, or Insecticidal protein B or Insecticidal protein C polypeptide. The sequence of the polynucleotide gene can be deduced from an engineered scaffold polypeptide, a scaffold binding peptide, chimeric polypeptide sequence, or Insecticidal protein B or Insecticidal protein C polypeptide through use of the genetic code. Computer programs such as “BackTranslate” (GCG™ Package, Acclerys, Inc. San Diego, Calif.) can be used to convert a peptide sequence to the corresponding nucleotidesequence encoding the peptide. Examples of engineered scaffold polypeptide and scaffold binding peptide sequences that can be used to obtain corresponding nucleotide encoding sequences include, but are not limited to the engineered scaffold polypeptide of SEQ ID NO: 1 and the scaffold binding peptides of SEQ ID NOs: 2-6. Furthermore, synthetic scaffold, scaffold binding, chimeric fusion polynucleotide, or Insecticidal protein B or Insecticidal protein C sequences of the disclosure can be designed so that they will be expressed in plants.

[0131] In some embodiments the nucleic acid molecule encoding an engineered scaffold polypeptide, a scaffold binding peptide, or a linker peptide is a polynucleotide encoding the polypeptide sequence set forth in any one of SEQ ID NOs: 1, 2-6, or 7-27, respectively, and variants, fragments and complements thereof. In some embodiments the nucleic acid molecule encoding an engineered scaffold polypeptide, a scaffold binding peptide, or a linker peptide is a polynucleotide having the sequence set forth in any one of SEQ ID NOs: 31, 32-36, or 37-57, respectively, and variants, fragments and complements thereof. In some embodiments the nucleic acid molecule encoding an Insecticidal protein B or Insecticidal protein C polypeptide is a polynucleotide having the sequence set forth in any one of SEQ ID NOs: 60 or 61, or 62, respectively, and variants, fragments and complements thereof. “Complement” is used herein to refer to a nucleic acid sequence that is sufficiently complementary to a given nucleic acid sequence such that it can hybridize to the given nucleic acid sequence to thereby form a stable duplex. “Polynucleotide sequence variants” is used herein to refer to a nucleic acid sequence that except for the degeneracy of the genetic code encodes the same polypeptide.

[0132] In some embodiments the nucleic acid molecule encodes an engineered scaffold polypeptide or a scaffold binding peptide variant comprising one or more amino acid substitutions to the amino acid sequence of any one of SEQ ID NOs: 1 or 2-6, respectively. In other embodiments the nucleic acid molecule encodes a linker sequence comprising one or more amino acid substitutions to the amino acid sequence of any one of SEQ ID NOs: 7-27.

[0133] Also provided are nucleic acid molecules that encode transcription and / or translation products that are subsequently spliced to ultimately produce functional engineered scaffold polypeptides, scaffold binding peptides, linker peptides, chimeric polypeptides, or Insecticidal protein B or Insecticidal protein C polypeptides. Splicing can be accomplished in vitro or in vivo, and can involve cis- or trans-splicing. The substrate for splicing can be polynucleotides (e.g., RNAtranscripts) or polypeptides. An example of cis-splicing of a polynucleotide is where an intron inserted into a coding sequence is removed and the two flanking exon regions are spliced to generate an engineered scaffold polypeptide, a scaffold binding peptide or fusion polypeptide encoding sequence, for example as a chimeric polypeptide comprising a scaffold binding peptide, linker peptide, and an insecticidal polypeptide of interest. An example of trans-splicing would be where a polynucleotide is encrypted by separating the coding sequence into two or more fragments that can be separately transcribed and then spliced to form the full-length pesticidal encoding sequence. The use of a splicing enhancer sequence, which can be introduced into a construct, can facilitate splicing either in cis or trans-splicing of polypeptides. Thus, in some embodiments the polynucleotides do not directly encode a full-length engineered scaffold polypeptide, scaffold binding peptide, fusion polypeptide, or Insecticidal protein B or Insecticidal protein C polypeptide, but rather encode a fragment or fragments of an engineered scaffold polypeptide, a scaffold binding peptide, chimeric polypeptide, or Insecticidal protein B or Insecticidal protein C polypeptide. These polynucleotides can be used to express a functional engineered scaffold, scaffold binding peptide, chimeric polypeptide, such as for example a fusion with an insecticidal polypeptide of interest and a linker peptide, or Insecticidal protein B or Insecticidal protein C polypeptide through a mechanism involving splicing, where splicing may occur at the level of polynucleotide (e.g., intron / exon) and / or polypeptide (e.g., intein / extein). This may be useful, for example, in controlling expression of pesticidal activity, since a scaffold binding peptide or chimeric polypeptide comprising the scaffold binding peptide will only be expressed if all required fragments are expressed in an environment that permits splicing processes to generate functional product. In another example, introduction of one or more insertion sequences into a polynucleotide can facilitate recombination with a low homology polynucleotide; use of an intron or intein for the insertion sequence facilitates the removal of the intervening sequence, thereby restoring function of the encoded variant.

[0134] Nucleic acid molecules that are fragments of these nucleic acid sequences encoding an engineered scaffold polypeptide, scaffold binding peptide, linker, or Insecticidal protein B or Insecticidal protein C polypeptide are also encompassed by the embodiments. “Nucleotide fragment” as used herein refers to a portion of the nucleic acid sequence encoding an engineered scaffold, a scaffold binding peptide, a linker, or an Insecticidal protein B or Insecticidal protein Cpolypeptide. A nucleotide fragment of a nucleic acid sequence may encode a biologically active portion of an engineered scaffold, a scaffold binding peptide, or an Insecticidal protein B or Insecticidal protein C polypeptide, or it may be a fragment that can be used as a hybridization probe or PCR primer using methods disclosed below. Nucleic acid molecules that are fragments of a nucleic acid sequence encoding an engineered scaffold, a scaffold binding peptide, or an Insecticidal protein B or Insecticidal protein C polypeptide comprise at least about 21, 24, 27, 30, 33, 36, 39, 45, 60, 75, 90, 100, 120, 150, 180, 210, 240, 270, or 300 contiguous nucleotides or up to the number of nucleotides present in a full-length nucleic acid sequence encoding an engineered scaffold, a scaffold binding peptide, or an Insecticidal protein B or Insecticidal protein C polypeptide disclosed herein, depending upon the intended use. “Contiguous nucleotides” is used herein to refer to nucleotide residues that are immediately adjacent to one another. Fragments of the nucleic acid sequences of the embodiments will encode protein fragments that retain the biological activity of the engineered scaffold, scaffold binding peptide, or Insecticidal protein B or Insecticidal protein C polypeptide, and, hence, retain engineered scaffold activity, scaffold binding peptide activity, or insecticidal activity. “Retains scaffold binding peptide activity” is used herein to refer to a polypeptide having at least about 10%, at least about 30%, at least about 50%, at least about 70%, 80%, 90%, 95% or higher of the activity of any one of the full-length scaffold binding peptides set forth in SEQ ID NOs: 2-6. “Retains insecticidal activity” is used herein to refer to a polypeptide having at least about 10%, at least about 30%, at least about 50%, at least about 70%, 80%, 90%, 95% or higher of the insecticidal activity of any one of the full- length Insecticidal protein B or Insecticidal protein C polypeptides set forth in SEQ ID NOs: 58 and 59, respectively. In some embodiments, the insecticidal activity is against a Lepidopteran species. In some embodiments, the insecticidal activity is against one or more insect pests selected from Soybean Looper (SBL; Pseudophisia inchidens), Fall Armyworm (FAW; Spodoptera frugiperdci) , Corn Earworm (CEW; Helicoverpa zea), Velvet Bean Caterpillar (VBC; Anticarsia gemmatalis) and European Corn Borer (ECB; Ostrinia nubialis). In one embodiment, the insecticidal activity is against a Coleopteran species. In some embodiments, the insecticidal activity is against one or more insect pests of the corn rootworm complex: western com rootworm, Diabrotica virgifera northern corn rootworm, 1). bar her i Southern com rootworm or spotted cucumber beetle; Diabrotica undecimpunctata howardi, Diabrotica speciosa, and the Mexicancorn rootworm, D. virgifera zeae. In one embodiment, the insecticidal activity is against a Diabrotica species.

[0135] "Percent (%) sequence identity" with respect to a reference sequence (subject) is determined as the percentage of amino acid residues or nucleotides in a candidate sequence (query) that are identical with the respective amino acid residues or nucleotides in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any amino acid conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (e.g., percent identity of query sequence = number of identical positions between query and subject sequences / total number of positions of query sequence * 100).

[0136] In some embodiments, an engineered scaffold polynucleotide encodes an engineered scaffold 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 greater sequence identity across the entire length of the amino acid sequence of SEQ ID NO: 1. In some embodiments a scaffold binding polynucleotide encodes a scaffold binding 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 greater sequence identity across the entire length of the amino acid sequence of any one of SEQ ID NOs: 2-6. In some embodiments a linker polynucleotide encodes a linker peptide comprising an amino acid sequence having at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity across the entire length of the amino acid sequence of any one of SEQ ID NOs: 7-27. In some embodiments an Insecticidal protein B or Insecticidal protein C polynucleotide encodes an Insecticidal protein B or Insecticidal protein C 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 greater sequence identity across the entire length of the amino acid sequence of any one of SEQ ID NOs: 58 and 59, respectively.

[0137] In one embodiment, a polynucleotide encodes a chimeric fusion polypeptide including a scaffold binding peptide, a linker peptide, and insecticidal polypeptide, 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 greater sequence identity compared to any one of the linked, chimeric fusion, or tethered polypeptides described in any of Tables 1, 2, 3, 4, 5, 6, or 7 in the Examples herein.

[0138] In one embodiment is contemplated a polynucleotide sequence having at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity across the entire length of the polynucleotide sequence of any one of SEQ ID NOs: 31-57, 60-62, and 64.

[0139] In some embodiments polynucleotides are provided encoding chimeric polypeptides comprising regions of at least two different scaffold binding peptides or linker peptides of the disclosure.

[0140] In some embodiments polynucleotides are provided encoding chimeric polypeptides comprising regions of at least two different Insecticidal protein B or Insecticidal protein C polypeptides of the disclosure.

[0141] In some embodiments polynucleotides are provided encoding chimeric polypeptides comprising an N-terminal Region of a first scaffold binding peptide of the disclosure operably fused to a C-terminal Region of a second scaffold binding peptide of the disclosure.

[0142] In some embodiments polynucleotides are provided encoding chimeric polypeptides comprising an N-terminal Region of a first Insecticidal protein B or Insecticidal protein C polypeptide of the disclosure operably fused to a C-terminal Region of a second Insecticidal protein B or Insecticidal protein C polypeptide of the disclosure.

[0143] The embodiments also encompass nucleic acid molecules encoding engineered scaffold polypeptide, scaffold binding peptide, linker peptide, or Insecticidal protein B or Insecticidal protein C polypeptide variants. “Variants” of the engineered scaffold polypeptide, scaffoldbinding peptide, linker peptide, or Insecticidal protein B or Insecticidal protein C polypeptide encoding nucleic acid sequences include those sequences that encode the engineered scaffold polypeptides, scaffold binding peptide, linker peptide, or Insecticidal protein B or Insecticidal protein C polypeptides disclosed herein but that differ conservatively because of the degeneracy of the genetic code as well as those that are sufficiently identical as discussed above. Naturally occurring allelic variants can be identified with the use of well-known molecular biology techniques, such as polymerase chain reaction (PCR) and hybridization techniques as outlined below. Variant nucleic acid sequences also include synthetically derived nucleic acid sequences that have been generated, for example, by using site-directed mutagenesis but which still encode the engineered scaffold polypeptide or scaffold binding peptide disclosed as discussed below.

[0144] The present disclosure provides isolated or recombinant polynucleotides that encode any of the engineered scaffold polypeptides, scaffold binding peptides, linker peptides, or Insecticidal protein B or Insecticidal protein C polypeptides disclosed herein. Those having ordinary skill in the art will readily appreciate that due to the degeneracy of the genetic code, a multitude of nucleotide sequences encoding engineered scaffold polypeptides, scaffold binding peptides, linker peptides, or Insecticidal protein B or Insecticidal protein C polypeptides of the present disclosure exist.

[0145] The skilled artisan will further appreciate that changes can be introduced by mutation of the nucleic acid sequences, such as for example by introducing one or more nucleotide substitutions, additions and / or deletions into the corresponding nucleic acid sequence disclosed herein, thereby leading to changes in the amino acid sequence of the encoded engineered scaffold polypeptide, scaffold binding peptide, linker peptide, or Insecticidal protein B or Insecticidal protein C polypeptide, without altering the biological activity of the proteins. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Such variant nucleic acid sequences are also encompassed by the present disclosure.

[0146] Alternatively, variant nucleic acid sequences can be made by introducing mutations randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant engineered scaffold polypeptide or scaffold binding peptide mutants can be screened for activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be expressed recombinantly, and the activity of the engineered scaffold polypeptide or scaffoldbinding peptide can be determined using assay techniques disclosed herein or known in the art. The polynucleotides of the disclosure and fragments thereof are optionally used as substrates for a variety of recombination and recursive recombination reactions, in addition to standard cloning methods as set forth in, e.g., Ausubel, Berger and Sambrook, i.e., to produce additional engineered scaffold polypeptide or scaffold binding peptide homologues and fragments thereof with desired properties. Libraries of variant polynucleotides so produced, cells comprising said libraries, and any recombinant polynucleotide produced by such methods are also embodiments of the disclosure. Additionally, such methods optionally comprise selecting a variant polynucleotide from such libraries based on engineered scaffold polypeptide or scaffold binding peptide activity, as is wherein such recursive recombination is done in vitro or in vivo.

[0147] A variety of diversity generating protocols, including nucleic acid recursive recombination protocols are available and fully described in the art. The procedures can be used separately and / or in combination to produce one or more variants of a nucleic acid or set of nucleic acids, as well as variants of encoded proteins. Individually and collectively, these procedures provide robust, widely applicable ways of generating diversified nucleic acids and sets of nucleic acids (including, e.g., nucleic acid libraries) useful, e.g., for the engineering or rapid evolution of nucleic acids, proteins, pathways, cells and / or organisms with new and / or improved characteristics.

[0148] Descriptions of a variety of diversity generating procedures for generating modified nucleic acid sequences, e.g., those coding for polypeptides having engineered scaffold or scaffold binding activity, or having pesticidal activity, or fragments thereof, are found in the following publications and the references cited therein: Soong, et al., (2000) Nat Genet 25(4):436-439; Stemmer, et al., (1999) Tumor Targeting 4:1-4; Ness, et al., (1999) Nat Biotechnol 17:893-896; Chang, et al., (1999) Nat Biotechnol 17:792-697; Minshull and Stemmer, (1999) Curr Opin Chem Biol 3:284- 290; Christians, etal., (1999) Nat Biotechnol 17:259-264; Crameri, etal., (1998) Nature 391 :288- 291; Crameri, etal., (1997) Nat Biotechnol 15:436-438; Zhang, etal., 1991) 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” 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) Science270: 1510; Stemmer, (1995) Bio, Technology 13:549-553; Stemmer, (1994) Nature 370:389-391 and Stemmer, (1994) PNAS USA 91 : 10747-10751.

[0149] Mutational methods of generating diversity include, for example, site-directed mutagenesis (Ling, et al., (1997) Anal Biochem 254(2): 157-178; Dale, etal., (1996) Methods Mol Biol 57:369- 374; Smith, (1985) Ann Rev Genet 19:423-462; Botstein and Shortle, (1985) Science 229:1193- 1201; Carter, (1986) Biochem J 237: 1-7 and Kunkel, (1987) “The efficiency of oligonucleotide directed mutagenesis” in Nucleic Acids & Molecular Biology (Eckstein and Lilley, eds., Springer Verlag, Berlin)); mutagenesis using uracil containing templates (Kunkel, (1985) PNAS USA 82:488-492; Kunkel, et al, (1987) Methods Enzymol 154:367-382 and Bass, etal, (1988) Science 242:240-245); oligonucleotide-directed mutagenesis (Zoller and Smith, (1983) Methods Enzymol 100:468-500; Zoller and Smith, (1987) Methods Enzymol 154:329-350 (1987); Zoller and Smith, (1982) Nucleic Acids Res 10:6487-6500), phosphorothioate-modified DNA mutagenesis (Taylor, et al., (1985) Nucl Acids Res 13:8749-8764; Taylor, et al., (1985) Nucl1Acids Res 13:8765-8787 (1985); Nakamaye and Eckstein, (1986) Nucl Acids Res 14:9679-9698; Sayers, et al., (1988) Nucl Acids Res 16:791-802 and Sayers, et al., (1988) Nucl Acids Res 16:803-814); mutagenesis using gapped duplex DNA (Kramer, et al., (1984) Nucl Acids Res 12:9441-9456; Kramer and Fritz, (1987) Methods Enzymol 154:350-367; Kramer, et al., (1988) Nucl Acids Res 16:7207 and Fritz, etal., (1988) Nucl Acids Res 16:6987-6999).

[0150] Additional suitable methods include point mismatch repair (Kramer, et al., (1984) Cell 38:879-887), mutagenesis using repair-deficient host strains (Carter, et al., (1985) Nucl Acids Res 13:4431-4443 and Carter, (1987) Methods in Enzymol 154:382-403), deletion mutagenesis (Eghtedarzadeh and Henikoff, (1986) Nucl Acids Res 14:5115), restriction-selection and restriction-purification (Wells, et al., (1986) Phil Trans R Soc Lond A 317:415-423), mutagenesis by total gene synthesis (Nambiar, et al., (1984) Science 223: 1299-1301; Sakamar and Khorana, (1988) Nucl Acids Res 14:6361-6372; Wells, et al., (1985) Gene 34:315-323 and Grundstrom, et al., (1985) Nucl Acids Res 13:3305-3316), double-strand break repair (Mandecki, (1986) PNAS USA, 83:7177-7181 and Arnold, (1993) Curr Opin Biotech 4:450-455). Additional details on many of the above methods can be found n Methods Enzymol Volume 154, which also describes useful controls for trouble-shooting problems with various mutagenesis methods.

[0151] The nucleotide sequences of the embodiments can also be used to isolate correspondingsequences from other sources that serve as sources of polynucleotides from which engineered scaffold polypeptides or scaffold binding peptides may be generated, in light of the teachings provided herein. In this manner, methods such as PCR, hybridization, and the like can be used to identify such sequences based on their sequence homology to the sequences set forth herein. Sequences that are selected based on their sequence identity to any of the entire sequences set forth herein or to fragments thereof are encompassed by the embodiments.

[0152] In hybridization methods, all or part of the pesticidal nucleic acid sequence can be used to screen cDNA or genomic libraries. Methods for construction of such cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook and Russell, (2001), supra. The hybridization probes may be genomic DNA fragments, synthetic oligonucleotides, cDNA fragments, RNA fragments or other oligonucleotides and may be labeled with a detectable group such as 32P or any other detectable marker, such as other radioisotopes, a fluorescent compound, an enzyme or an enzyme co-factor. Degenerate primers designed on the basis of conserved nucleotides or amino acid residues in the nucleic acid sequence or encoded amino acid sequence can additionally be used. The probe typically comprises a region of nucleic acid sequence that hybridizes under stringent conditions to at least about 12, at least about 25, at least about 50, 75, 100, 125, 150, 175 or 200 consecutive nucleotides of nucleic acid sequences encoding engineered scaffold polypeptides, scaffold binding peptides, or Insecticidal protein B or Insecticidal protein C polypeptides of the disclosure or a fragment or variant thereof. Methods for the preparation of probes for hybridization and stringency conditions are generally known in the art and are disclosed in Sambrook and Russell, (2001), supra, herein incorporated by reference.

[0153] For example, an entire nucleic acid sequence, encoding an engineered scaffold polypeptide, a scaffold binding peptide, or an Insecticidal protein B or Insecticidal protein C polypeptide disclosed herein or one or more portions thereof may be used as a probe capable of specifically hybridizing to corresponding nucleic acid sequences encoding an engineered scaffold polypeptide, a scaffold binding peptide, an Insecticidal protein B or Insecticidal protein C polypeptide, or fusion partner polypeptide-like sequences and messenger RNAs. To achieve specific hybridization under a variety of conditions, such probes include sequences that are unique and are preferably at least about 10 nucleotides in length or at least about 20 nucleotides in length. Such probes may be used to amplify corresponding sequences from a chosen sample source. This technique may be used asa diagnostic assay to determine the presence of coding sequences in a sample of interest. Hybridization techniques include hybridization screening of plated DNA libraries (either plaques or colonies; see, for example, Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

[0154] Hybridization of such sequences may be carried out under stringent conditions. “Stringent conditions” or “stringent hybridization conditions” is used herein to refer to conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and / or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, preferably less than 500 nucleotides in length.Polypeptides of Interest

[0155] General categories of polypeptides of interest include, for example, those encoded by genes involved in information, such as zinc fingers, those involved in communication, such as kinases, those involved in biosynthetic pathways, and those involved in housekeeping, such as heat shock proteins. More specific categories of transgenes encoding polypeptides of interest, for example, include sequences encoding important traits for agronomics, insect resistance, disease resistance, herbicide resistance, sterility, grain characteristics, oil, starch, carbohydrate, phytate, protein, nutrient, metabolism, digestability, kernel size, sucrose loading, and commercial products.

[0156] Traits such as oil, starch, and protein content can be genetically altered in addition to using traditional breeding methods. Modifications include increasing content of oleic acid, saturated and unsaturated oils, increasing levels of lysine and sulfur, providing essential amino acids, and also modification of starch. Protein modifications to alter amino acid levels are described in U.S. Patent Nos. 5,703,049, 5,885,801, 5,885,802, and 5,990,389 and WO 98 / 20122, herein incorporated by reference.

[0157] Insect resistance genes encoding insecticidal polypeptides of interest may encode resistance to pests such as rootworm, cutworm, armyworm, European Corn Borer, and the like as listed above. Such genes include, for example, polypeptides of interest such as insecticidal proteins from Pseudomonas sp. such as PSEEN3174 (Monalysin; (2011) PLoS Pathogens 7: 1-13); from Pseudomonas protegens strain CHAO and Pf-5 (previously fluorescens) (Pechy-Tarr, (2008) Environmental Microbiology 10:2368-2386; GenBank Accession No. EU400157); from Pseudomonas taiwanensis (Liu, et al., (2010) J. Agric. Food Chem., 58: 12343-12349) and from Pseudomonas pseudoalcaligenes (Zhang, et al., (2009) Annals of Microbiology 59:45-50 and Li, et al., (2007) Plant Cell Tiss. Organ Cult. 89: 159-168); insecticidal proteins from Photorhabdus sp. and Xenorhabdus sp. (Hinchliffe, et al., (2010) The Open Toxicology Journal, 3: 101-118 and Morgan, et al., (2001) Applied and Envir. Micro. 67:2062-2069); US Patent Number 6,048,838, and US Patent Number 6,379,946; a PIP-1 polypeptide ofUS 9,688,730; an AHP-1A and / or A1TP- 1B polypeptide of US 9,475,847; a PIP -47 polypeptide of US Patent Application Publication Number US20160186204; an IPD045 polypeptide, an IPD064 polypeptide, an IPD074 polypeptide, an IPD075 polypeptide, and an IPD077 polypeptide of International Patent Application Publication Number WO2016 / 114973; an IPD080 polypeptide of International Patent Application Publication Number W02018 / 075350; an IPD078 polypeptide, an IPD084 polypeptide, an IPD085 polypeptide, an IPD086 polypeptide, an IPD087 polypeptide, an IPD088 polypeptide, and an IPD089 polypeptide of International Patent Application Publication Number WO2018 / 084936; PIP-72 polypeptide of US Patent Application Publication Number US20160366891; a PtIP-50 polypeptide and a PtIP-65 polypeptide of US Patent Application Publication NumberUS20170166921; an IPD098 polypeptide, an IPD059 polypeptide, an IPD108 polypeptide, an IPD109 polypeptide of International Patent Application Publication Number WO20 18 / 232072; a PtIP-83 polypeptide of US Patent Application Publication Number US20160347799; a PtIP-96 polypeptide of US Patent Application Publication Number US20170233440; an ZPD079 polypeptide of International Patent Application Publication Number WO2017 / 23486; an IPD082 polypeptide of International Patent Application Publication Number WO2017 / 105987; an IPD090 polypeptide of International Patent Application Publication Number WO2017 / 192560; an IPD093 polypeptide of International Patent Application Publication Number WO2018 / 111551; an IPD103 polypeptide of International Patent Application Publication NumberWO2018 / 005411; an IPD101 polypeptide of International Patent Application Publication Number WO2018 / 118811; an IPD121 polypeptide of International Patent Application Publication Number WO2018 / 208882; and 8-endotoxins including but not limited to a Cryl, Cry2, Cry3, Cry4, Cry5, Cry6, Cry7, Cry8, Cry9, Cry 10, Cryl 1, Cry 12, Cryl3, Cryl4, Cryl5, Cryl6, Cryl7, Cryl8, Cryl9, Cry20, Cry21, Cry 22, Cry23, Cry 24, Cry25, Cry26, Cry27, Cry28, Cry29, Cry30, Cry31, Cry32, Cry33, 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 Cry 72 classes of 6-endotoxin polypeptides and the B. thuringiensis cytolytic cytl and cyt2 genes. Members of these classes of B. thuringiensis insecticidal proteins well known to one skilled in the art (see, Crickmore, et al., "Bacillus thuringiensis toxin nomenclature" (2011), at lifesci.sussex.ac.uk / home / Neil Crickmore / Bt / which can be accessed on the world-wide web using the "www" prefix).

[0158] Examples of insecticidal polypeptides of interest encoding 8-endotoxins also include but are not limited to Cryl A proteins of US Patent Numbers 5,880,275, 7,858,849, and 8,878,007; a CrylAc mutant of US9,512,187; a DIG-3 or DIG-11 toxin (N-terminal deletion of a-helix 1 and / or a-helix 2 variants of cry proteins such as CrylA, Cry3A) of US Patent Numbers 8,304,604, 8.304,605 and 8,476,226; Cry IB of US Patent Application Publication Number US20060112447, US Patent Application Publication Number US20160194364, and US Patent Numbers 9,404,121 and 8,772,577; CrylB variants of PCT Publication Number WO2016 / 61197 and Serial Number PCT / US 17 / 27160; CrylC of US Patent Number 6,033,874; CrylD protein of US20170233759; a CrylE protein of PCT Serial Number PCT / US17 / 53178; a CrylF protein of US Patent Numbers 5,188,960 and 6,218,188; CrylA / F chimeras of US Patent Numbers 7,070,982; 6,962,705 and 6,713,063; a Cryll protein of PCT Publication number WO 2017 / 0233759; a Cryl J variant of US Publication US20170240603; a Cry2 protein such as Cry2Ab protein of US Patent Number 7,064,249 and Cry2A.127 protein of US 7208474; a Cry3 A protein including but not limited to an engineered hybrid insecticidal protein (efflP) created by fusing unique combinations of variable regions and conserved blocks of at least two different Cry proteins (US Patent Application Publication Number 2010 / 0017914); a Cry4 protein; a Cry5 protein; a Cry6 protein; Cry8 proteins of US Patent Numbers 7,329,736, 7,449,552, 7,803,943, 7,476,781, 7,105,332, 7,339,092,7,378,499, 7,462,760, and 9,593,345; a Cry9 protein such as such as members of the Cry9A, Cry9B, Cry9C, Cry9D, Cry9E and Cry9F families including the Cry9 protein of US Patent 9,000,261 and 8,802,933, and US Serial Number WO 2017 / 132188; a Cryl5 protein of Naimov, et al., (2008) Applied and Environmental Microbiology, 74:7145-7151; a Cryl4 protein of US Patent Number US8,933,299; a Cry22, a Cry34Abl protein of US Patent Numbers 6,127,180, 6,624,145 and 6,340,593; a truncated Cry34 protein of US Patent Number US8,816,157; a CryET33 and cryET34 protein of US Patent Numbers 6,248,535, 6,326,351, 6,399,330, 6,949,626, 7,385,107 and 7,504,229; a CryET33 and CryET34 homologs of US Patent Publication Number 2006 / 0191034, 2012 / 0278954, and PCT Publication Number WO 2012 / 139004; a Cry35Abl protein of US Patent Numbers 6,083,499, 6,548,291 and 6,340,593; a Cry46 protein of US Patent Number 9,403,881, a Cry 51 protein, a Cry binary toxin; a TIC901 or related toxin; TIC807 of US Patent Application Publication Number 2008 / 0295207; TIC853 of US Patent US8, 513,493; ET29, ET37, TIC809, TIC810, TIC812, TIC127, TIC128 of PCT US 2006 / 033867; engineered Hemipteran toxic proteins of US Patent Application Publication Number US20160150795; AXMI-027, AXMI-036, and AXMI-038 of US Patent Number 8,236,757; AXMI-031, AXMI- 039, AXMI-040, AXMI-049 of US Patent Number 7,923,602; AXMI-018, AXMI-020 and AXMI-021 of WO 2006 / 083891; AXMI-010 of WO 2005 / 038032; AXMI-003 of WO 2005 / 021585; AXMI-008 ofUS Patent Application Publication Number 2004 / 0250311; AXMI-006 of US Patent Application Publication Number 2004 / 0216186; AXMI-007 of US Patent Application Publication Number 2004 / 0210965; AXMI-009 of US Patent Application Number 2004 / 0210964; AXMI-014 of US Patent Application Publication Number 2004 / 0197917; AXMI-004 of US Patent Application Publication Number 2004 / 0197916; AXMI-028 and AXMI-029 of WO 2006 / 119457; AXMI-007, AXMI-008, AXMI-0080rf2, AXMI-009, AXMI-014 and AXMI-004 of WO 2004 / 074462; AXMI-150 of US Patent Number 8,084,416; AXMI-205 of US Patent Application Publication Number 2011 / 0023184; AXMI-011, AXMI-012, AXMI-013, AXMI-015, AXMI-019,AXMI-044, AXMI-037, AXMI-043, AXMI-033, AXMI-034, AXMI-022, AXMI-023, AXMI- 041, AXMI-063 and AXMI-064 of US Patent Application Publication Number 2011 / 0263488;AXMI046, AXMI048, AXMI050, AXMI051, AXMI052, AXMI053, AXMI054, AXMI055,AXMI056, AXMI057, AXMI058, AXMI059, AXMI060, AXMI061, AXMI067, AXMI069,AXMI071, AXMI072, AXMI073, AXMI074, AXMI075, AXMI087, AXMI088, AXMI093,AXMI070, AXMI080, AXMI081, AXMI082, AXMI091, AXMI092, AXMI096, AXMI097AXMI098, AXMI099, AXMI100, AXMI101, AXMI102, AXMI103, AXMI104, AXMI107AXMI108, AXMI109, AXMI110, AXMI111, AXMI112, AXMI114, AXMI116, AXMI117AXMI118, AXMI119, AXMI120, AXMI121, AXMI122, AXMI123, AXMI124, AXMI125AXMI126, AXMI127, AXMI129, AXMI151, AXMI161, AXMI164, AXMI183, AXMI132AXMI137, AXMI138 of US Patent US8461421 and US8,461,422; AXMI-R1 and related proteins of US Patent Application Publication Number 2010 / 0197592; AXMI221Z, AXMI222z, AXMI223z, AXMI224z and AXMI225z of WO 2011 / 103248; AXMI218, AXMI219, AXMI220,AXMI226, AXMI227, AXMI228, AXMI229, AXMI230 and AXMI231 of WO 2011 / 103247; AXMI-115, AXMI-113, AXMI-005, AXMI-163 and AXMI-184 of US Patent Number 8,334,431; AXMI-001, AXMI-002, AXMI-030, AXMI-035 and AXMI-045 of US Patent Application Publication Number 2010 / 0298211; AXMI-066 and AXMI-076 of US Patent Application Publication Number 2009 / 0144852; AXMI128, AXMI130, AXMI131, AXMI133, AXMI140, AXMI141, AXMI142, AXMI143, AXMI144, AXMI146, AXMI148, AXMI149, AXMI152,AXMI153, AXMI154, AXMI155, AXMI156, AXMI157, AXMI158, AXMI162, AXMI165,AXMI166, AXMI167, AXMI168, AXMI169, AXMI170, AXMI171, AXMI172, AXMI173,AXMI174, AXMI175, AXMI176, AXMI177, AXMI178, AXMI179, AXMI180, AXMI181,AXMI182, AXMI185, AXMI186, AXMI187, AXMI188, AXMI189 of US Patent Number8,318,900; AXMI079, AXMI080, AXMI081, AXMI082, AXMI091, AXMI092, AXMI096,AXMI097, AXMI098, AXMI099, AXMI100, AXMI101, AXMI102, AXMI103, AXMI104,AXMI107, AXMI108, AXMI109, AXMI110, dsAXMIl l l, AXMI112, AXMI114, AXMI116, AXMI117, AXMI118, AXMI119, AXMI120, AXMI121, AXMI122, AXMI123, AXMI124, AXMI1257, AXMI1268, AXMI127, AXMI129, AXMI164, AXMI151, AXMI161, AXMI183, AXMI132, AXMI138, AXMI137 of US Patent US8461421; AXMI192 of US Patent US8,461,415; AXMI281 of US Patent Application Publication Number US20160177332; AXMI422 of US Patent Number US8,252,872; cry proteins such as CrylA and Cry3A having modified proteolytic sites of US Patent Number 8,319,019; a CrylAc, Cry2Aa and CrylCa toxin protein from Bacillus thuringiensis strain VBTS 2528 of US Patent Application Publication Number 2011 / 0064710. The Cry proteins MP032, MP049, MP051, MP066, MP068, MP070, MP091S, MP109S, MP114, MP121, MP134S, MP183S, MP185S, MP186S, MP195S, MP197S,MP208S, MP209S, MP212S, MP214S, MP217S, MP222S, MP234S, MP235S, MP237S,MP242S, MP243, MP248, MP249S, MP251M, MP252S, MP253, MP259S, MP287S, MP288S,MP295S, MP296S, MP297S, MP300S, MP304S, MP306S, MP3 IOS, 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, MP661 S, MP666S, MP672S, MP696S, MP704S, MP724S, MP729S, MP739S,MP755S, MP773S, MP799S, MP800S, MP801S, MP802S, MP803S, MP805S, MP809S,MP815S, MP828S, MP831S, MP844S, MP852, MP865S, MP879S, MP887S, MP891S, MP896S, MP898S, MP935S, MP968, MP989, MP993, MP997, MP1049, MP1066, MP1067, MP1080, MP1081, MP1200, MP1206, MP1233, and MP1311 of US Serial Number 62 / 607372. The insecticidal activity of Cry proteins is well known to one skilled in the art (for review, see, van Frannkenhuyzen, (2009) J. Invert. Path. 101 :1-16). The use of Cry proteins as transgenic plant traits is well known to one skilled in the art and Cry-transgenic plants comprising insecticidal polypeptides of interest including but not limited to plants expressing Cry 1 Ac, CrylAc+Cry2Ab, CrylAb, CrylA.105, CrylF, CrylFa2, CrylF+CrylAc, Cry2Ab, Cry3A, mCry3A, Cry3Bbl, Cry34Abl, Cry35Abl, Vip3A, mCry3A, Cry9c and CBI-Bt have received regulatory approval (see, Sanahuja, (2011) Plant Biotech Journal 9:283-300 and the CERA. (2010) GM Crop Database Center for Environmental Risk Assessment (CERA), ILSI Research Foundation, Washington D.C. at cera-gmc.org / index. php?action=gm_crop_database which can be accessed on the world-wide web using the “www” prefix). More than one pesticidal proteins well known to one skilled in the art can also be expressed in plants as an insecticidal polypeptide of interest such as Vip3Ab & CrylFa (US2012 / 0317682); CrylBE & CrylF (US2012 / 0311746); CrylCA & CrylAB (US2012 / 0311745); CrylF & CryCa (US2012 / 0317681); CrylDA& CrylBE (US2012 / 0331590); CrylDA & CrylFa (US2012 / 0331589); CrylAB & CrylBE (US2012 / 0324606); CrylFa & Cry2Aa and Cryll & CrylE (US2012 / 0324605); Cry34Ab / 35Ab & Cry6Aa (US20130167269); Cry34Ab / VCry35Ab & Cry3Aa (US20130167268); CrylDa & CrylCa (US 9796982); Cry3Aa&Cry6Aa (US 9798963); and Cry3A & Cry 1 Ab or Vip3Aa (US9,045,766). Insecticidal proteins of interest also include insecticidal lipases including lipid acyl hydrolases of US Patent Number 7,491,869, and cholesterol oxidases such as from Streptomyces (Purcell et al. (1993) Biochem Biophys Res Commun 15: 1406-1413). Insecticidal proteins of interst also include VIP (vegetative insecticidal proteins) toxins of US Patent Numbers 5,877,012, 6,107,279 6,137,033, 7,244,820, 7,615,686, and 8,237,020 and the like. Other VIP proteins are well known to one skilled in the art (see, lifesci.sussex.ac.uk / home / Neil_Crickmore / Bt / vip.html which can be accessed on the worldwide web using the "www" prefix). Pesticidal proteins of interest also include Cyt proteins including CytlA variants of PCT Serial Number PCT / US2017 / 000510; Pesticidal proteins of interest also include toxin complex (TC) proteins, obtainable from organisms such as Xenorhabdus, Photorhabdus and Paenibacillus (see, US Patent Numbers 7,491,698 and 8,084,418). Some TC proteins have “stand alone” insecticidal activity and other TC proteins enhance the activity of the stand-alone toxins produced by the same given organism. The toxicity of a “stand-alone” TC protein (from Photorhabdus, Xenorhabdus or Paenibacillus, for example) can be enhanced by one or more TC protein “potentiators” derived from a source organism of a different genus. There are three main types of TC proteins. As referred to herein, Class A proteins (“Protein A”) are stand-alone toxins. Class B proteins (“Protein B”) and Class C proteins (“Protein C”) enhance the toxicity of Class A proteins. Examples of Class A proteins are TcbA, TcdA, XptAl and XptA2. Examples of Class B proteins are TcaC, TcdB, XptBIXb and XptCIWi. Examples of Class C proteins are TccC, XptClXb and XptBIWi. Pesticidal proteins of interest also include spider, snake and scorpion venom proteins. Examples of spider venom peptides include but not limited to lycotoxin-1 peptides and mutants thereof (US Patent Number 8,334,366). The combinations generated can also include multiple copies of any one of the polynucleotides of interest.

[0159] Genes encoding polypeptides of interest for disease resistance traits include detoxification genes, such as against fumonosin (U.S. Patent No. 5,792,931); avirulence (avr) and disease resistance (R) genes (Jones et al. (1994) Science 266:789 Martin et al. (1993) Science 262: 1432; and Mindrinos el al. (1994) Cell 78: 1089); and the like.

[0160] Herbicide resistance traits may include genes encoding polypeptides of interest for resistance to herbicides that act to inhibit the action of acetolactate synthase (ALS), in particularthe sulfonylurea-type herbicides (e.g., the S4 and / or Hra mutations in ALS), genes coding for resistance to herbicides that act to inhibit action of glutamine synthase, such as phosphinothricin or basta (e.g, the bar gene), genes providing resistance to glyphosate, such as GAT (glyphosate / V-acetyltransferase; U.S. Patent 6,395,485), EPSPS (enolpyruvylshikimate-3-phosphate synthase; U.S. Patents 6,867,293, 5,188,642, 5,627,061), or GOX (glyphosate oxidoreductase; U.S. Patent No. 5,463,175), or other such genes known in the art. The nptll gene encodes resistance to the antibiotics kanamycin and geneticin.

[0161] Any of the pesticidal proteins listed above and disclosed herein may be an insecticidal polypeptide of interest according to the compositions and methods of the invention.AlphaFold

[0162] In another embodiment, portions of the polypeptides disclosed herein including, but not limited to, domains, structurally significant regions, minimal active core polypeptide, receptor binding domains, active sites, scaffold binding peptide regions, and protease cleavage sites may be identified using the AlphaFold computational program.

[0163] AlphaFold is a computational method that can regularly predict protein structures with atomic accuracy even in cases in which no similar structure is known. The AlphaFold network directly predicts the 3D coordinates of all heavy atoms for a given protein using the primary amino acid sequence and aligned sequences of homologues as inputs. The AlphaFold methods are scalable to very long proteins with accurate domains and domain-packing, and the model is able to provide precise, per-residue estimates of its reliability that should enable confident use of its structure predictions. (Jumper, J., Evans, R., Pritzel, A. el al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583-589 (2021).Antibodies

[0164] Antibodies to an engineered scaffold polypeptide, a scaffold binding peptide, or an Insecticidal protein B or Insecticidal protein C polypeptide of the embodiments or to variants or fragments thereof are also encompassed. The antibodies of the disclosure include polyclonal and monoclonal antibodies as well as fragments thereof which retain their ability to bind to an engineered scaffold polypeptide, a scaffold binding peptide, or an Insecticidal protein B orInsecticidal protein C polypeptide. An antibody, monoclonal antibody or fragment thereof is said to be capable of binding a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody, monoclonal antibody or fragment thereof. The term "antibody" (Ab) or "monoclonal antibody" (Mab) is meant to include intact molecules as well as fragments or binding regions or domains thereof (such as, for example, Fab and F(ab).sub.2 fragments) which are capable of binding hapten. Such fragments are typically produced by proteolytic cleavage, such as papain or pepsin. Alternatively, hapten-binding fragments can be produced through the application of recombinant DNA technology or through synthetic chemistry.

[0165] Methods for the preparation of the antibodies of the present disclosure are generally known in the art. For example, see, Antibodies, A Laboratory Manual, Ed Harlow and David Lane (eds.) Cold Spring Harbor Laboratory, N.Y. (1988), as well as the references cited therein. Standard reference works setting forth the general principles of immunology include: Klein, J. Immunology: The Science of Cell-Noncell Discrimination, John Wiley & Sons, N.Y. (1982); Dennett, et al., Monoclonal Antibodies, Hybridoma: A New Dimension in Biological Analyses, Plenum Press, N.Y. (1980) and Campbell, "Monoclonal Antibody Technology," In Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 13, Burdon, etal., (eds.), Elsevier, Amsterdam (1984). Antibodies against or antigen-binding portions thereof can be produced by a variety of techniques, including conventional monoclonal antibody methodology, for example the standard somatic cell hybridization technique of Kohler and Milstein, (1975) Nature 256:495. Other techniques for producing monoclonal antibody can also be employed such as viral or oncogenic transformation of B lymphocytes. An animal system for preparing hybridomas is a murine system. Immunization protocols and techniques for isolation of immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known. The antibody and monoclonal antibodies of the disclosure can be prepared by utilizing engineered scaffold polypeptides or scaffold binding peptides as antigens.

[0166] A kit for detecting the presence of a scaffold binding peptide or an Insecticidal protein B or Insecticidal protein C polypeptide or detecting the presence of a nucleotide sequence encoding a scaffold binding peptide or an Insecticidal protein B or Insecticidal protein C polypeptide in a sample is provided. In one embodiment, the kit provides antibody-based reagents for detecting the presence of a scaffold binding peptide or an Insecticidal protein B or Insecticidal protein Cpolypeptide in a tissue sample. In another embodiment, the kit provides labeled nucleic acid probes useful for detecting the presence of one or more polynucleotides encoding a scaffold binding peptide or an Insecticidal protein B or Insecticidal protein C polypeptide. The kit is provided along with appropriate reagents and controls for carrying out a detection method, as well as instructions for use of the kit.Nucleotide Constructs, Expression Cassettes and Vectors

[0167] The use of the term "nucleotide constructs" herein is not intended to limit the embodiments to nucleotide constructs comprising DNA. Those of ordinary skill in the art will recognize that nucleotide constructs, particularly polynucleotides and oligonucleotides composed of ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides, may also be employed in the methods disclosed herein. The nucleotide constructs, nucleic acids, and nucleotide sequences of the embodiments additionally encompass all complementary forms of such constructs, molecules, and sequences. Further, the nucleotide constructs, nucleotide molecules, and nucleotide sequences of the embodiments encompass all nucleotide constructs, molecules, and sequences which can be employed in the methods of the embodiments for transforming plants including, but not limited to, those comprised of deoxyribonucleotides, ribonucleotides, and combinations thereof. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. The nucleotide constructs, nucleic acids, and nucleotide sequences of the embodiments also encompass all forms of nucleotide constructs including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures and the like.

[0168] A further embodiment relates to a transformed organism such as an organism selected from plant and insect cells, bacteria, yeast, baculovirus, protozoa, nematodes and algae. The transformed organism comprises a DNA molecule of the embodiments, an expression cassette comprising the DNA molecule or a vector comprising the expression cassette, which may be stably incorporated into the genome of the transformed organism.

[0169] The sequences of the embodiments are provided in DNA constructs for expression in the organism of interest. The construct will include 5' and 3' regulatory sequences operably linked to a sequence of the embodiments. The term "operably linked" as used herein refers to the associationof two or more nucleic acid fragments on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation. Optionally, operably linked may also mean that the nucleic acid sequences being linked are contiguous and where necessary to join two protein coding regions in the same reading frame. The construct may additionally contain at least one additional gene to be co-transformed into the organism. Alternatively, the additional gene(s) can be provided on multiple DNA constructs.

[0170] Such a DNA construct is provided with a plurality of restriction sites for insertion of the scaffold binding peptide, linker peptide, fusion partner, or Insecticidal protein B or Insecticidal protein C polypeptide gene sequence(s) of the disclosure to be under the transcriptional regulation of the regulatory regions. The DNA construct may additionally contain selectable marker genes.

[0171] The DNA construct will generally include in the 5' to 3' direction of transcription: a transcriptional and translational initiation region (i.e., a promoter), a DNA sequence of the embodiments, and a transcriptional and translational termination region (i.e., termination region) functional in the organism serving as a host. The transcriptional initiation region (i.e., the promoter) may be native, analogous, foreign, or heterologous to the host organism and / or to the sequence of the embodiments. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence. The term "foreign" as used herein indicates that the promoter is not found in the native organism into which the promoter is introduced. Where the promoter or any other nucleotide or amino acid sequence is "foreign" or "heterologous" in reference to a sequence is a sequence that originates from a foreign species or, if from the same species, is substantially modified from its native form in composition and / or genomic locus by deliberate human intervention. For example, a promoter operably linked to a heterologous polynucleotide is from a species different from the species from which the polynucleotide was derived, or, if from the same / analogous species, one or both are substantially modified from their original form and / or genomic locus, or the promoter is not the native promoter for the operably linked polynucleotide. As used herein, a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence. Where the promoter is a native ornatural sequence, the expression of the operably linked sequence is altered from the wild-type expression, which results in an alteration in phenotype.

[0172] In some embodiments the DNA construct comprises a polynucleotide encoding a scaffold binding peptide, chimeric fusion polypeptide(s), or Insecticidal protein B or Insecticidal protein C polypeptide of the embodiments, and may optionally include a polynucleotide encoding one or more genes of interest. In some embodiments the DNA construct comprises a polynucleotide encoding a chimeric fusion protein comprising one or more scaffold binding peptides, one or more linker peptides, and one or more polypeptides of interest, such as an insecticidal polypeptide of interest, of the embodiments.

[0173] In some embodiments the DNA construct comprises a polynucleotide encoding a fusion protein comprising an Insecticidal protein B or Insecticidal protein C polypeptide of the embodiments.

[0174] In some embodiments the DNA construct may also include a transcriptional enhancer sequence. As used herein, the term an “enhancer” refers to a DNA sequence which can stimulate promoter activity, and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. Various enhancers are known in the art including for example, introns with gene expression enhancing properties in plants (, the ubiquitin intron (i.e., the maize ubiquitin intron 1 (see, for example, NCBI sequence S94464)), the omega enhancer or the omega prime enhancer (Gallie, et al., (1989) Molecular Biology of RNA ed. Cech (Liss, New York) 237-256 and Gallie, et al., (1987) Gene 60:217-25), the CaMV 35S enhancer (see, e g., Benfey, et al, (1990) EMBO J. 9: 1685-96) and the enhancers of US Patent Number 7,803,992 may also be used. The above list of transcriptional enhancers is not meant to be limiting. Any appropriate transcriptional enhancer can be used in the embodiments.

[0175] The termination region may be native with the transcriptional initiation region, may be native with the operably linked DNA sequence of interest, may be native with the plant host or may be derived from another source (i.e., foreign or heterologous to the promoter, the sequence of interest, the plant host or any combination thereof).

[0176] Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also, Guerineau, et al., (\99\)Mol. Gen. Genet. 262:141-144; Proudfoot, (1991) Cell 64:671-674; Sanfacon, etal., (1991)Genes Dev. 5:141-149; Mogen, etal., (1990) Plant Cell 2: 1261-1272; Munroe, etal., (1990) Gene 91 : 151-158; Ballas, etal., (1989) Nucleic Acids Res. 17:7891-7903 and Joshi, etal., (1987) Nucleic AcidRes. 15:9627-9639.

[0177] Where appropriate, a nucleic acid may be optimized for increased expression in the host organism. Thus, where the host organism is a plant, the synthetic nucleic acids can be synthesized using plant-preferred codons for improved expression. See, for example, Campbell and Gowri, (1990) Plant Physiol. 92: 1-11 for a discussion of host-preferred usage. For example, although nucleic acid sequences of the embodiments may be expressed in both monocotyledonous and dicotyledonous plant species, sequences can be modified to account for the specific preferences and GC content preferences of monocotyledons or dicotyledons as these preferences have been shown to differ (Murray et al. (1989) Nucleic Acids Res. 17:477-498). Thus, the maize-preferred for a particular amino acid may be derived from known gene sequences from maize. Maize usage for 28 genes from maize plants is listed in Table 4 of Murray, et al., supra. Methods are available in the art for synthesizing plant-preferred genes. See, for example, Murray, et al., (1989) Nucleic Acids Res. 17:477-498, and Liu H et al. Mol Bio Rep 37:677-684, 2010, herein incorporated by reference. A Zea maize usage table can be also found at kazusa.or.jp / / cgi- bin / show.cgi?species=4577, which can be accessed using the www prefix. A Glycine max usage table can be found at kazusa.or.jp / / cgi-bin / show.cgi?species=3847&aa=l&style=N, which can be accessed using the www prefix. In some embodiments the recombinant nucleic acid molecule encoding a scaffold binding peptide, Insecticidal protein B or Insecticidal protein C polypeptide, or chimeric polypeptide has maize optimized codons.

[0178] Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other well-characterized sequences that may be deleterious to gene expression. The GC content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. The term "host cell" as used herein refers to a cell which contains a vector and supports the replication and / or expression of the expression vector is intended. Host cells may be prokaryotic cells such as E. coli or eukaryotic cells such as yeast, insect, amphibian or mammalian cells or monocotyledonous or dicotyledonous plant cells. An example of a monocotyledonous host cell isa maize host cell. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures.

[0179] The expression cassettes may additionally contain 5' leader sequences. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein, et al., (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie, et al., (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf Mosaic Virus), human immunoglobulin heavy-chain binding protein (BiP) (Macejak, et al., (1991) Nature 353:90-94); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling, et al., (1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie, et al., (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256) and maize chlorotic mottle virus leader (MCMV) (Lommel, et al., (1991) Virology 81 :382-385). See also, Della-Cioppa, et al., (1987) Plant Physiol. 84:965-968. Such constructs may also contain a “signal sequence” or “leader sequence” to facilitate co-translational or post-translational transport of the peptide to certain intracellular structures such as the chloroplast (or other plastid), endoplasmic reticulum or Golgi apparatus.

[0180] “Signal sequence” as used herein refers to a sequence that is known or suspected to result in cotranslational or post-translational peptide transport across the cell membrane. In eukaryotes, this typically involves secretion into the Golgi apparatus, with some resulting glycosylation. Insecticidal toxins of bacteria are often synthesized as protoxins, which are proteolytically activated in the gut of the target pest (Chang, (1987) Methods Enzymol. 153:507-516). In some embodiments, the signal sequence is located in the native sequence or may be derived from a sequence of the embodiments. “Leader sequence” as used herein refers to any sequence that when translated, results in an amino acid sequence sufficient to trigger co-translational transport of the peptide chain to a subcellular organelle. Thus, this includes leader sequences targeting transport and / or glycosylation by passage into the endoplasmic reticulum, passage to vacuoles, plastids including chloroplasts, mitochondria, and the like. Research in proteomics of the higher plant chloroplast has identified numerous nuclear-encoded thylakoid lumen proteins (Kieselbach et al. FEBS ££77480:271-276, 2000; Peltier et al. Plant Cell 12:319-341, 2000; Bricker et al. Biochim. Biophys Acta 1503:350-356, 2001), the lumen targeting signal peptide of which can potentially beused in accordance with the present disclosure. (See Kieselbach et al., Photosynthesis Research, 78:249-264, 2003, in particular, Table 2 of this publication disclosing 85 proteins from the chloroplast lumen, which is incorporated herein by reference.

[0181] Suitable chloroplast transit peptides (CTP) are well known to one skilled in the art also include chimeric CT’s comprising but not limited to, an N-terminal domain, a central domain or a C-terminal domain from a CTP from Oryza sativa 1 -decoy -D xylose-5 -Phosphate Synthase Oryza / iw-Superoxide dismutase Oryza sativa- soluble starch synthase Oryza sativa- ADP-dependent Malic acid enzyme Oryza .s / / / -Phospho-2-dehydro-3-deoxyheptonate Aldolase 2 Oryza sativa- L-Ascorbate peroxidase 5 Oryza .sz / Z / va-Phosphoglucan water dikinase, Zea Mays ssRUBISCO, Zea A / ay.s-beta-glucosidase, Zea A / ay.s-Malate dehydrogenase, Zea Mays Thioredoxin M-type (See US Patent Application Publication 2012 / 0304336).

[0182] The recombinant nucleic acid molecule encoding a scaffold binding peptide, chimeric fusion polypeptide, or Insecticidal protein B or Insecticidal protein C polypeptide to be targeted to the chloroplast may be optimized for expression in the chloroplast to account for differences in usage between the plant nucleus and this organelle. In this manner, the nucleic acids of interest may be synthesized using chloroplast-preferred sequences.

[0183] In preparing the expression cassette, the various DNA fragments may be manipulated so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved.

[0184] A number of promoters can be used in the practice of the embodiments. The promoters can be selected based on the desired outcome. The nucleic acids can be combined with constitutive, tissue-preferred, inducible or other promoters for expression in the host organism. Suitable constitutive promoters for use in a plant host cell include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 1999 / 43838 and US Patent Number 6,072,050; the core CaMV 35S promoter (Odell, et al., (1985) Nature 313:810- 812); rice actin (McElroy, et al., (1990) Plant Cell 2: 163-171); ubiquitin (Christensen, et al.,(1989) Plant Mol. Biol. 12:619-632 and Christensen, et al., (1992) Plant Mol. Biol. 18:675-689); pEMU (Last, etal., (1991) Theor. Appl. Genet. 81 :581-588); MAS (Velten, et al., (1984) EMBO J. 3:2723-2730); ALS promoter (US Patent Number 5,659,026) and the like. Other constitutive promoters include, for example, those discussed in US Patent Number 6,177,611.

[0185] Depending on the desired outcome, it may be beneficial to express the gene from an inducible promoter. Of particular interest for regulating the expression of the nucleotide sequences of the embodiments in plants are wound-inducible promoters. Such wound-inducible promoters, may respond to damage caused by insect feeding, and include potato proteinase inhibitor (pin II) gene (Ryan, (1990) Ann. Rev. Phytopath. 28:425-449; Duan, et al., (1996) Nature Biotechnology 14:494-498); wunl and wun2; winl and win2 (Stanford, et al., (19 9 Mol. Gen. Genet. 215:200- 208); systemin (McGurl, et aL, (1992) Science 225: 1570-1573); WIP1 (Rohmeier, et al., (1993) Plant Mol. Biol. 22:782-692; Eckelkamp, et al., (1993) FEBS Letters 323:72-66); MPI gene (Corderok, et al., (1994) Plant J. 6(2): 141 -150) and the like.

[0186] Additionally, pathogen-inducible promoters may be employed in the methods and nucleotide constructs of the embodiments. Such pathogen-inducible promoters include those from pathogenesis-related proteins (PR proteins), which are induced following infection by a pathogen; e.g., PR proteins, SAR proteins, beta-l,3-glucanase, chitinase, etc. See, for example, Redolfi, et al., (1983) Neth. J. Plant Pathol. 89:245-254; Uknes, et al., (1992) Plant Cell 4: 645-656 and Van Loon, (1985) Plant Mol. Virol. 4:111-116. See also, WO 1999 / 43819.

[0187] Of interest are promoters that are expressed locally at or near the site of pathogen infection. See, for example, Marineau, et al., (1987) Plant Mol. Biol. 9:335-342; Matton, et al., (1989) Molecular Plant-Microbe Interactions 2:325-331; Somsisch, et al., (1986) Proc. Natl. Acad. Sci. USA 83:2427-2430; Somsisch, et al., (1988) Mol. Gen. Genet. 2:93-98 and Yang, (1996) Proc. Natl. Acad. Sci. USA 93: 14972-14977. See also, Chen, etal., (1996) Plant J. 10:955-966; Zhang, et al., (1994) Proc. Natl. Acad. Sci. USA 91 :2507-2511; Warner, etal., (1993) Plant J. 3: 191-201; Siebertz, et aL, (1989) Plant Cell 1 :961-968. Of particular interest is the inducible promoter for the maize PRms gene, whose expression is induced by the pathogen Fusarium moniliforme (see, for example, Cordero, et al., (1992) Physiol. Mol. Plant Path. 41 : 189-200).

[0188] Chemical -regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. Depending upon the objective, thepromoter may be a chemical-inducible promoter, where application of the chemical induces gene expression or a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical-inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-la promoter, which is activated by salicylic acid. Other chemi cal -regulated promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena, et al., (1991) Proc. Natl. Acad. Set. USA 88: 10421-10425 and McNellis, et al., (1998) Plant J. 14(2):247-257) and tetracyclineinducible and tetracycline-repressible promoters (see, for example, Gatz, et al., (1991) Mol. Gen. Genet. 227:229-237).

[0189] Tissue-preferred promoters can be utilized to target enhanced scaffold binding peptide, fusion polypeptide, or Insecticidal protein B or Insecticidal protein C polypeptide, including chimeric polypeptides comprising one or more scaffold binding peptides, one or more linker peptides, and one or more polypeptides of interest, such as an insecticidal polypeptide of interest, expression within a particular plant tissue. Tissue-preferred promoters include those discussed in Yamamoto, et al., (1997) Plant J. 12(2)255-265; Kawamata, et al., (1997) Plant Cell Physiol. 38(7):792-803; Hansen, et al., (1997) Mol. Gen Genet. 254(3):337-343; Russell, et al., (1997) Transgenic Res. 6(2):157-168; Rinehart, et al., (1996) Plant Physiol. 112(3): 1331-1341; Van Camp, et al., (1996) Plant Physiol. 112(2):525-535; Canevascini, et al., (1996) Plant Physiol. 112(2):513-524; Yamamoto, etal., (1994) Plant Cell 'Physiol. 35(5):772-678; Lam, (199A Results Probl. CellDiffer. 20: 181-196; Orozco, etal., (1993) Plant Mol Biol. 23(6):1129-1138; Matsuoka, et al., (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590 and Guevara-Garcia, et al., (1993) Plant J. 4(3):495-505. Such promoters can be modified, if necessary, for weak expression.

[0190] 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):772-678; 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.

[0191] Root-preferred or root-specific promoters are known and can be selected from the manyavailable from the literature or isolated de novo from various compatible species. See, for example, Hire, el al., (1992) Plant Mol. Biol. 20(2):207-218 (soybean root-specific glutamine synthetase gene); Keller and Baumgartner, (1991) Plant Cell 3(10): 1051-1061 (root-specific control element in the GRP 1.8 gene of French bean); Sanger, et al., (1990) Plant Mol. Biol. 14(3):433-443 (rootspecific promoter of the mannopine synthase (MAS) gene of Agrobacterhim tumefaciens) and Miao, et al., (1991) Plant Cell 3(1): 11-22 (full-length cDNA clone encoding cytosolic glutamine synthetase (GS), which is expressed in roots and root nodules of soybean). See also, Bogusz, et al., (1990) Plant Cell 2(7):633-641, where two root-specific promoters isolated from hemoglobin genes from the nitrogen-fixing nonlegume Parasponia andersonii and the related non-nitrogen- fixing nonlegume Trema tomentosa are described. The promoters of these genes were linked to a b -glucuronidase reporter gene and introduced into both the nonlegume Nicotiana tabacum and the legume Lotus corniculatus, and in both instances root-specific promoter activity was preserved. Leach and Aoyagi, (1991) describe their analysis of the promoters of the highly expressed rolC and rolD root-inducing genes of Agrobacterium rhizogenes (see, Plant Science (Limerick) 79(l):69-76). They concluded that enhancer and tissue-preferred DNA determinants are dissociated in those promoters. Teeri, et al., (1989) used gene fusion to lacZ to show that the Agrobacterium T-DNA gene encoding octopine synthase is especially active in the epidermis of the root tip and that the TR2' gene is root specific in the intact plant and stimulated by wounding in leaf tissue, an especially desirable combination of characteristics for use with an insecticidal or larvicidal gene (see, EMBO J. 8(2):343-350). The TRI' gene fused to nptll (neomycin phosphotransferase II) showed similar characteristics. Additional root-preferred promoters include the VfENOD-GRP3 gene promoter (Kuster, etal., (1995) Plant Mol. Biol. 29(4): 759-772) and rolB promoter (Capana, et al., (1994) Plant Mol. Biol. 25(4):681-691. See also, US Patent Number 5,401,836.

[0192] "Seed-preferred" promoters include both "seed-specific" promoters (those promoters active during seed development such as promoters of seed storage proteins) as well as "seedgerminating" promoters (those promoters active during seed germination). See, Thompson, et al., (1989) BioEssays 10:108. Such seed-preferred promoters include, but are not limited to, Ciml (cytokinin-induced message); cZ19Bl (maize 19 kDa zein); and milps (myo-inositol-1 -phosphate synthase) (see, US Patent Number 6,225,529). Gamma-zein and Glb-1 are endosperm-specificpromoters. For dicots, seed-specific promoters include, but are not limited to, Kunitz trypsin inhibitor 3 (KTi3) (Jofuku and Goldberg, (1989) Plant Cell 1 : 1079-1101), bean b-phaseolin, napin, b-conglycinin, glycinin 1, soybean lectin, cruciferin, and the like. For monocots, seed-specific promoters include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein, waxy, shrunken 1, shrunken 2, globulin 1, etc. See also, WO 2000 / 12733, where seed-preferred promoters from end I and end2 genes are disclosed. In dicots, seed specific promoters include but are not limited to seed coat promoter from Arabidopsis, pB AN; and the early seed promoters from Arabidopsis, p26, p63, and p63tr. A promoter that has “preferred” expression in a particular tissue is expressed in that tissue to a greater degree than in at least one other plant tissue. Some tissuepreferred promoters show expression almost exclusively in the particular tissue.

[0193] Where low level expression is desired, weak promoters will be used. Generally, the term "weak promoter" as used herein refers to a promoter that drives expression of a coding sequence at a low level. By low level expression at levels of between about 1 / 1000 transcripts to about 1 / 100,000 transcripts to about 1 / 500,000 transcripts is intended. Alternatively, it is recognized that the term “weak promoters” also encompasses promoters that drive expression in only a few cells and not in others to give a total low level of expression. Where a promoter drives expression at unacceptably high levels, portions of the promoter sequence can be deleted or modified to decrease expression levels.

[0194] Such weak constitutive promoters include, for example the core promoter of the Rsyn7 promoter (WO 1999 / 43838 and US Patent Number 6,072,050), the core 35 S CaMV promoter, and the like. Other constitutive promoters include, for example, those disclosed in US Patent Number 6,177,611.

[0195] The above list of promoters is not meant to be limiting. Any appropriate promoter can be used in the embodiments.

[0196] Generally, the expression cassette will comprise a selectable marker gene for the selection of transformed cells. Selectable marker genes are utilized for the selection of transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones and 2,4-dichlorophenoxyacetate (2,4-D). Additional examples of suitable selectable marker genesinclude, but are not limited to, genes encoding resistance to chloramphenicol (Herrera Estrella, et al., (1983) EMBO J. 2:987-992); methotrexate (Herrera Estrella, et al., (1983) Nature 303:209- 213 and Meijer, et al., (1991) Plant Mol. Biol. 16:807-820); streptomycin (Jones, et al., (1987) Mol. Gen. Genet. 210:86-91); spectinomycin (Bretagne-Sagnard, et al., (1996) Transgenic Res. 5: 131-137); bleomycin (Hille, etal., (1990) Plant Mol. Biol. 7: 171-176); sulfonamide (Guerineau, et al., (1990) Plant Mol. Biol. 15: 127-136); bromoxynil (Stalker, et al., (1988) Science 242:419- 423); glyphosate (Shaw, et al., (1986) Science 233:478-481 and US Patent Application Serial Numbers 10 / 004,357 and 10 / 427,692); phosphinothricin (DeBlock, et al., (1987) EMBO J. 6:2513-2518). See generally, Yarranton, (1992) Curr. Opin. Biotech. 3:506-511; Christopherson, et al., (1992) Proc. Natl. Acad. Sci. USA 89:6314-6318; Yao, et al., (1992) Cell 71:62-62; Reznikoff, (1992) Mol. Microbiol. 6:2419-2422; Barkley, et al., (1980) in The Operon, pp. 177-220; Hu, et al., (1987) Cell 48:555-566; Brown, etal., (1987) Cell 49: 603 -612; Figge, etal., (1988) Cell 52:712-622; Deuschle, et al., (1989) Proc. Natl. Acad. Sci. USA 86:5400-5404; Fuerst, et al., (1989) Proc. Natl. Acad. Sci. USA 86:2549-2553; Deuschle, et al., (1990) Science 248:480-483; Gossen, (1993) Ph.D. Thesis, University of Heidelberg; Reines, et al., (1993) Proc. Natl. Acad. Sci. USA 90:1917-1921; Labow, et al., (1990) Mol. Cell. Biol. 10:3343-3356; Zambretti, et al., (1992) Proc. Natl. Acad. Sci. USA 89:3952-3956; Bairn, etal., (1991) Proc. Natl. Acad. Sci. USA 88:5072-5076; Wyborski, etal., (1991) Nucleic Acids Res. 19:4647-4653; Hillenand-Wissman, (1989) Topics Mol. Struc. Biol. 10: 143-162; Degenkolb, et al., (1991) Antimicrob. Agents Chemother. 35:1591-1595; Kleinschnidt, et al., (1988) Biochemistry 27:1094-1104; Bonin, (1993) Ph.D. Thesis, University of Heidelberg; Gossen, et al., (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Oliva, et al., (1992) Antimicrob. Agents Chemother. 36:913-919; Hlavka, et al., (1985) Handbook of Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin) and Gill, et al., (1988) Vo / z / re 334:721-724.

[0197] The above list of selectable marker genes is not meant to be limiting. Any selectable marker gene can be used in the embodiments.Plant Transformation

[0198] The methods of the embodiments involve introducing a polypeptide or polynucleotide into a plant. The methods of the embodiments involve introducing a scaffold binding peptide or fusion polypeptide or polynucleotide, including chimeric fusion polypeptides comprising one or morescaffold binding peptides, one or more linker peptides, and one or more polypeptides of interest, such as an insecticidal polypeptide of interest, into a plant. "Introducing" as used herein means presenting to the plant the polynucleotide or polypeptide in such a manner that the sequence gains access to the interior of a cell of the plant. The methods of the embodiments do not depend on a particular method for introducing a polynucleotide or polypeptide into a plant, only that the polynucleotide(s) or polypeptide(s) gains access to the interior of at least one cell of the plant. Methods for introducing polynucleotide(s) or polypeptide(s) into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.

[0199] "Stable transformation" is a transformation in which the polynucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by the progeny thereof. "Transient transformation" as used herein means that a polynucleotide is introduced into the plant and does not integrate into the genome of the plant. “Plant” as used herein generically includes whole plants, plant organs, plant tissues, seeds, plant cells, seeds and progeny of the same. The plant is a monocot or dicot. Plant cells include, without limitation, cells from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen and microspores. A “plant element" is intended to reference either a whole plant or a plant component, which may comprise differentiated and / or undifferentiated tissues, for example but not limited to plant tissues, parts, and cell types. In one embodiment, a plant element is one of the following: whole plant, seedling, meristematic tissue, ground tissue, vascular tissue, dermal tissue, seed, leaf, root, shoot, stem, flower, fruit, stolon, bulb, tuber, corm, keiki, shoot, bud, tumor tissue, and various forms of cells and culture (e.g., single cells, protoplasts, embryos, callus tissue). It should be noted that a protoplast is not technically "intact" plant cell (as naturally found with all components), as protoplasts lack a cell wall. “Plant organ" refers to plant tissue or a group of tissues that constitute a morphologically and functionally distinct part of a plant. A plant element "is synonymous to a portion" of a plant, and refers to any part of the plant, and can include distinct tissues and / or organs, and may be used interchangeably with tissue" throughout. Similarly, a “plant reproductive element" is intended to generically reference any part of a plant that is able to initiate other plants via either sexual or asexual reproduction of that plant, for example but not limited to: seed, seedling, root, shoot, cutting, scion,graft, stolon, bulb, tuber, corm, keiki, or bud. The plant element may be in plant or in a plant organ, tissue culture, or cell culture.

[0200] Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection (Crossway, et al., (1986) Biotechniques 4:320-334), electroporation (Riggs, et al., (1986) Proc. Natl. Acad. Set. USA 83:5602-5606), Agrobacterium -mediated transformation (US Patent Numbers 5,563,055 and 5,981,840), direct gene transfer (Paszkowski, et al., (1984) EMBO J. 3:2717-2722) and ballistic particle acceleration (see, for example, US Patent Numbers 5,879,918; 5,886,244 and 5,932,782; Tomes, et al., (1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips, (Springer-Verlag, Berlin) and McCabe, et al., (1988) Biotechnology 6:923- 926) and Led transformation (WO 00 / 28058). For potato transformation see, Tu, et al., (1998) Plant Molecular Biology 37:829-838 and Chong, et al., (2000) Transgenic Research 9:71-78. Additional transformation procedures can be found in Weissinger, et al., (1988) Ann. Rev. Genet. 22:421-477; Sanford, etal., (1987) Particulate Science and Technology 5:27-37 (onion); Christou, et al., (1988) Plant Physiol. 87:671-674 (soybean); McCabe, et al., (1988) Bio / Technology 6:923- 926 (soybean); Finer and McMullen, (1991) In Vitro Cell Dev. Biol. 27P: 175-182 (soybean); Singh, etal., (1998) Theor. Appl. Genet. 96:319-324 (soybean); Datta, etal., (1990) Biotechnology 8:736-740 (rice); Klein, et al., (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein, et al., (1988) Biotechnology 6:559-563 (maize); US Patent Numbers 5,240,855; 5,322,783 and 5,324,646; Klein, et al., (1988) Plant Physiol. 91 :440-444 (maize); Fromm, et al., (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren, et al., (1984) Nature (London) 311 :762-664; US Patent Number 5,736,369 (cereals); Bytebier, et al., (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet, et al., (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman, etal., (Longman, New York), pp. 197-209 (pollen); Kaeppler, et al., (1990) Plant Cell 'Reports 9:415-418 and Kaeppler, et al., (1992) Theor. Appl. Genet. 84:560-566 (whisker-mediated transformation); D'Halluin, et al., (1992) Plant Cell 4: 1495-1505 (electroporation); Li, et al., (1993) Plant Cell Reports 12:250-255 and Christou and Ford, (1995) Annals of Botany 75:407-413 (rice); Osjoda, et al., (1996) Nature Biotechnology 14:745-750(maize via Agrobacterium tumefaciens).

[0201] In specific embodiments, the sequences of the embodiments can be provided to a plant using a variety of transient transformation methods. Such transient transformation methods include, but are not limited to, the introduction of the scaffold binding partner or chimeric fusion polynucleotide or variants and fragments thereof directly into the plant or the introduction of the transcript into the plant or the introduction of the scaffold binding partner or chimeric fusion polypeptide transcript into the plant. Such transient transformation methods include, but are not limited to, the introduction of the Insecticidal protein B and / or Insecticidal protein C polynucleotide or variants and fragments thereof directly into the plant or the introduction of the Insecticidal protein B and / or Insecticidal protein C transcript into the plant. Such methods include, for example, microinjection or particle bombardment. See, for example, Crossway, et al., (1986) Mol Gen. Genet. 202:179-185; Nomura, et al, (1986) Plant Sci. 44:53-58; Hepler, et al., (1994) Proc. Natl. Acad. Sci. 91 :2176-2180 and Hush, et al., (1994) The Journal of Cell Science 107:775- 784. Alternatively, the scaffold binding partner or chimeric fusion polypeptide, or Insecticidal protein B and / or Insecticidal protein C transcript can be transiently transformed into the plant using techniques known in the art. Such techniques include viral vector system and the precipitation of the polynucleotide in a manner that precludes subsequent release of the DNA. Thus, transcription from the particle-bound DNA can occur, but the frequency with which it is released to become integrated into the genome is greatly reduced. Such methods include the use of particles coated with polyethylimine (PEI; Sigma #P3143).

[0202] Methods are known in the art for the targeted insertion of a polynucleotide at a specific location in the plant genome. In one embodiment, the insertion of the polynucleotide at a desired genomic location is achieved using a site-specific recombination system. See, for example, WO 1999 / 25821, WO 1999 / 25854, WO 1999 / 25840, WO 1999 / 25855 and WO 1999 / 25853. Briefly, the polynucleotide of the embodiments can be contained in transfer cassette flanked by two nonidentical recombination sites. The transfer cassette is introduced into a plant have stably incorporated into its genome a target site which is flanked by two non-identical recombination sites that correspond to the sites of the transfer cassette. An appropriate recombinase is provided and the transfer cassette is integrated at the target site. The polynucleotide of interest is thereby integrated at a specific chromosomal position in the plant genome.

[0203] Plant transformation vectors may be comprised of one or more DNA vectors needed for achieving plant transformation. For example, it is a common practice in the art to utilize plant transformation vectors that are comprised of more than one contiguous DNA segment. These vectors are often referred to in the art as “binary vectors”. Binary vectors as well as vectors with helper plasmids are most often used for Agrobacterium-mediated transformation, where the size and complexity of DNA segments needed to achieve efficient transformation is quite large, and it is advantageous to separate functions onto separate DNA molecules. Binary vectors typically contain a plasmid vector that contains the cis-acting sequences required for T-DNA transfer (such as left border and right border), a selectable marker that is engineered to be capable of expression in a plant cell, and a “gene of interest” (a gene engineered to be capable of expression in a plant cell for which generation of transgenic plants is desired). Also present on this plasmid vector are sequences required for bacterial replication. The cis-acting sequences are arranged in a fashion to allow efficient transfer into plant cells and expression therein. For example, the selectable marker gene and the pesticidal gene are located between the left and right borders. Often a second plasmid vector contains the trans-acting factors that mediate T-DNA transfer from Agrobacterium to plant cells. This plasmid often contains the virulence functions (Vir genes) that allow infection of plant cells by Agrobacterium, and transfer of DNA by cleavage at border sequences and vir-mediated DNA transfer, as is understood in the art (Hellens and Mullineaux, (2000) Trends in Plant Science 5:446-451). Several types of Agrobacterium strains (e.g. LBA4404, GV3101, EHA101, EHA105, etc.) can be used for plant transformation. The second plasmid vector is not necessary for transforming the plants by other methods such as microprojection, microinjection, electroporation, polyethylene glycol, etc.

[0204] In general, plant transformation methods involve transferring heterologous DNA into target plant cells (e.g., immature or mature embryos, suspension cultures, undifferentiated callus, protoplasts, etc.), followed by applying a maximum threshold level of appropriate selection (depending on the selectable marker gene) to recover the transformed plant cells from a group of untransformed cell mass. Following integration of heterologous foreign DNA into plant cells, one then applies a maximum threshold level of appropriate selection in the medium to kill the untransformed cells and separate and proliferate the putatively transformed cells that survive from this selection treatment by transferring regularly to a fresh medium. By continuous passage andchallenge with appropriate selection, one identifies and proliferates the cells that are transformed with the plasmid vector. Molecular and biochemical methods can then be used to confirm the presence of the integrated heterologous gene of interest into the genome of the transgenic plant.

[0205] Explants are typically transferred to a fresh supply of the same medium and cultured routinely. Subsequently, the transformed cells are differentiated into shoots after placing on regeneration medium supplemented with a maximum threshold level of selecting agent. The shoots are then transferred to a selective rooting medium for recovering rooted shoot or plantlet. The transgenic plantlet then grows into a mature plant and produces fertile seeds (e.g., Hiei, et al., (1994) The Plant Journal 6:271-282; Ishida, et al., (1996) Nature Biotechnology 14:745-750). Explants are typically transferred to a fresh supply of the same medium and cultured routinely. A general description of the techniques and methods for generating transgenic plants are found in Ayres and Park, (1994) Critical Reviews in Plant Science 13:219-239 and Bommineni and Jauhar, (1997) Maydica 42: 107 -120. Since the transformed material contains many cells; both transformed and non-transformed cells are present in any piece of subjected target callus or tissue or group of cells. The ability to kill non-transformed cells and allow transformed cells to proliferate results in transformed plant cultures. Often, the ability to remove non-transformed cells is a limitation to rapid recovery of transformed plant cells and successful generation of transgenic plants.

[0206] The cells that have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick, et al., (1986) Plant Cell Reports 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having constitutive or inducible expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure that expression of the desired phenotypic characteristic has been achieved.

[0207] The nucleotide sequences of the embodiments may be provided to the plant by contacting the plant with a virus or viral nucleic acids. Generally, such methods involve incorporating the nucleotide construct of interest within a viral DNA or RNA molecule. It is recognized that the recombinant proteins of the embodiments may be initially synthesized as part of a viralpolyprotein, which later may be processed by proteolysis in vivo or in vitro to produce the desired scaffold binding peptide or chimeric fusion polypeptide, or Insecticidal protein B or Insecticidal protein C polypeptide. It is also recognized that such a viral polyprotein, comprising at least a portion of the amino acid sequence of a scaffold binding peptide or chimeric fusion polypeptide, or Insecticidal protein B and / or Insecticidal protein C polypeptide of the embodiments, may have the desired activity, such as pesticidal activity. Such viral polyproteins and the nucleotide sequences that encode for them are encompassed by the embodiments. Methods for providing plants with nucleotide constructs and producing the encoded proteins in the plants, which involve viral DNA or RNA molecules, are known in the art.

[0208] Methods for transformation of chloroplasts are known in the art. See, for example, Svab, etal., (1990) roc. Natl. Acad. Sci. USA 87:8526-8530; Svab and Maliga, (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab and Maliga, (1993) EMBO J. 12:601-606. The method relies on particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination. Additionally, plastid transformation can be accomplished by transactivation of a silent plastid-borne transgene by tissue-preferred expression of a nuclear-encoded and plastid-directed RNA polymerase. Such a system has been reported in McBride, et al., (1994) Proc. Natl. Acad. Sci. USA 91 :7301-7305.

[0209] The embodiments further relate to plant-propagating material of a transformed plant of the embodiments including, but not limited to, seeds, tubers, corms, bulbs, leaves and cuttings of roots and shoots.

[0210] The embodiments may be used for transformation of any plant species, including, but not limited to, monocots and dicots. Examples of plants of interest include, but are not limited to, com (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B.Juncea), particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solarium tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava Manihot esculenta), coffee (Coffea spp.), sugarcane (Saccharum spp.), oats, barley, and vegetables.

[0211] Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca saliva), green beans (Phaseohis vulgaris), lima beans (Phaseohis limensis), peas (Lathyrus spp.), and members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo). Plants of the embodiments include crop plants (for example, com, alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, etc.), such as com and soybean plants.

[0212] Turf grasses include, but are not limited to: annual bluegrass (Poa annua), annual ryegrass (Lolium multifloruniy, Canada bluegrass (Poa compressa),' Chewing’s fescue Festuca rubra), colonial bentgrass (Agrostis tenuis), creeping bentgrass (Agrostis palustris), crested wheatgrass (Agropyron desertorum),' fairway wheatgrass (Agropyron cristatum),' hard fescue (Festuca longifolia),' Kentucky bluegrass (Poa pratensis), orchardgrass (Dactylis glomerata),' perennial ryegrass (Lolium perenne),' red fescue (Festuca rubra),' redtop (Agrostis alba), rough bluegrass (Poa trivialis),' sheep fescue (Festuca ovina),' smooth bromegrass (Bromus inermis),' tall fescue (Festuca arundinacea),' timothy (Phleum pratense),' velvet bentgrass (Agrostis canina), weeping alkaligrass (Puccinellia distans),' western wheatgrass (Agropyron smithii),' Bermuda grass (Cynodon spp.); St. Augustine grass (Stenotaphrum secundatum), zoysia grass (Zoysia spp.); Bahia grass (Paspalum notatum), carpet grass Axonopus affinis),' centipede grass (Eremochloa ophiuroides), kikuyu grass (Pennisetum clandesinum),' seashore paspalum (Paspalum vaginatum), blue gramma (Bouteloua gracilis),' buffalo grass Buchloe dactyloids),' sideoats gramma (Bouteloua curtipendula).

[0213] Plants of interest include grain plants that provide seeds of interest, oil-seed plants, and leguminous plants. Seeds of interest include grain seeds, such as corn, wheat, barley, rice, sorghum, rye, millet, etc. Oil-seed plants include cotton, soybean, safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, flax, castor, olive, etc. Leguminous plants include beans and peas. Beans include guar, locust bean, fenugreek, soybean, garden beans, cowpea, mung bean, lima bean, fava bean, lentils, chickpea, etc.

[0214] Following introduction of heterologous foreign DNA into plant cells, the transformation or integration of heterologous gene in the plant genome is confirmed by various methods such as analysis of nucleic acids, proteins and metabolites associated with the integrated gene.

[0215] PCR analysis is a rapid method to screen transformed cells, tissue or shoots for the presenceof incorporated gene at the earlier stage before transplanting into the soil (Sambrook and Russell, (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). PCR is carried out using oligonucleotide primers specific to the gene of interest or Agrobacterium vector background, etc.

[0216] Plant transformation may be confirmed by Southern blot analysis of genomic DNA (Sambrook and Russell, (2001) supra). In Northern blot analysis, RNA is isolated from specific tissues of transformant, fractionated in a formaldehyde agarose gel, and blotted onto a nylon filter according to standard procedures that are routinely used in the art (Sambrook and Russell, (2001) supra). Expression of RNA encoded by the scaffold binding peptide or fusion polynucleotide or pesticidal gene is then tested by hybridizing the filter to a radioactive probe derived from a scaffold binding peptide or fusion polynucleotide, by methods known in the art (Sambrook and Russell, (2001) supra). Western blot, biochemical assays and the like may be carried out on the transgenic plants to confirm the presence of protein encoded by the pesticidal gene by standard procedures (Sambrook and Russell, 2001, supra) using antibodies that bind to one or more epitopes present on the scaffold binding peptide or fusion polypeptides.Methods to Introduce Genome Editing Technologies into Plants

[0217] In some embodiments, the disclosed scaffold binding partner, chimeric fusion polynucleotide, or polynucleotide compositions encoding any transgenic protein that causes or is suspected of causing undesirable phenotypic characteristics, such as an insecticidal polypeptide of interest, including pre-existing transgenic events can be introduced into the genome of a plant using genome editing technologies, or previously introduced scaffold binding partner, chimeric fusion polynucleotides, or polynucleotide encoding any transgenic protein that causes or is suspected of causing undesirable phenotypic characteristics, such as an insecticidal polypeptide of interest, including pre-existing transgenic events in the genome of a plant may be edited using genome editing technologies. For example, the disclosed polynucleotides can be introduced into a desired location in the genome of a plant through the use of double-stranded break technologies such as TALENs, meganucleases, zinc finger nucleases, CRISPR-Cas, and the like. For example, the disclosed polynucleotides can be introduced into a desired location in a genome using a CRISPR-Cas system, for the purpose of site-specific insertion. The desired location in a plantgenome can be any desired target site for insertion, such as a genomic region amenable for breeding or may be a target site located in a genomic window with an existing trait of interest. Existing traits of interest could be either an endogenous trait or a previously introduced trait.

[0218] In some embodiments, where the disclosed scaffold binding partner or chimeric fusion polynucleotide, or polynucleotide encoding any transgenic protein that causes or is suspected of causing undesirable phenotypic characteristics, such as an insecticidal polypeptide of interest, including pre-existing transgenic events has previously been introduced into a genome, genome editing technologies may be used to alter or modify the introduced polynucleotide sequence. Site specific modifications that can be introduced into the disclosed scaffold binding partner or chimeric fusion polynucleotide, or polynucleotide encoding any transgenic protein that causes or is suspected of causing undesirable phenotypic characteristics, such as an insecticidal polypeptide of interest, including pre-existing transgenic events compositions include those produced using any method for introducing site specific modification, including, but not limited to, through the use of gene repair oligonucleotides, or through the use of double-stranded break technologies such as TALENs, meganucleases, zinc finger nucleases, CRISPR-Cas, and the like. Such technologies can be used to modify the previously introduced polynucleotide through the insertion, deletion or substitution of nucleotides within the introduced polynucleotide. Alternatively, double-stranded break technologies can be used to add additional nucleotide sequences to the introduced polynucleotide. Additional sequences that may be added include, additional expression elements, such as enhancer and promoter sequences. In another embodiment, genome editing technologies may be used to position additional insecticidally -active proteins in close proximity to the disclosed scaffold binding partner, chimeric fusion polynucleotide, or polynucleotide encoding any transgenic protein that causes or is suspected of causing undesirable phenotypic characteristics, such as an insecticidal polypeptide of interest, including pre-existing transgenic events compositions disclosed herein within the genome of a plant, in order to generate molecular stacks of proteins of interest such as insecticidally-active proteins.

[0219] An “altered target site,” “altered target sequence,” “modified target site,” and “modified target sequence” are used interchangeably herein and refer to a target sequence as disclosed herein that comprises at least one alteration when compared to non-altered target sequence. Such "alterations" include, for example: (i) replacement of at least one nucleotide, (ii) a deletion of atleast one nucleotide, (iii) an insertion of at least one nucleotide, or (iv) any combination of (i) - (iii).Stacking of Traits in Transgenic Plants

[0220] Transgenic plants may comprise a stack of one or more insecticidal polynucleotides disclosed herein, such as those encoding polypeptides of interest, including insecticidal polypeptides of interest, with one or more additional polynucleotides resulting in the production or suppression of multiple polypeptide sequences. In one embodiment, transgenic plants may comprise a stack of one or more polynucleotides encoding scaffold binding peptides or chimeric fusion polynucleotides or polynucleotides encoding insecticidal polypeptides disclosed herein with one or more additional polynucleotides resulting in the production or suppression of multiple polypeptide sequences. In another embodiment, transgenic plants may comprise a stack of one or more polynucleotides encoding insecticidal polypeptides of interest with one or more additional polynucleotides encoding polypeptides of interest and do not comprise a scaffold binding peptide. Transgenic plants comprising stacks of polynucleotide sequences can be obtained by either or both of traditional breeding methods or through genetic engineering methods. These methods include, but are not limited to, breeding individual lines each comprising a polynucleotide of interest, transforming a transgenic plant comprising a gene disclosed herein with a subsequent gene and co- transformation of genes into a single plant cell. As used herein, the term “stacked” includes having the multiple traits present in the same plant (i.e., both traits are incorporated into the nuclear genome, one trait is incorporated into the nuclear genome and one trait is incorporated into the genome of a plastid or both traits are incorporated into the genome of a plastid). In one nonlimiting example, “stacked traits” comprise a molecular stack where the sequences are physically adjacent to each other. A trait, as used herein, refers to the phenotype derived from a particular sequence or groups of sequences. Co-transformation of genes can be carried out using single transformation vectors comprising multiple genes or genes carried separately on multiple vectors. If the sequences are stacked by genetically transforming the plants, the polynucleotide sequences of interest can be combined at any time and in any order. The traits can be introduced simultaneously in a co-transformation protocol with the polynucleotides of interest provided by any combination of transformation cassettes. For example, if two sequences will be introduced,the two sequences can be contained in separate transformation cassettes (trans) or contained on the same transformation cassette (cis). Expression of the sequences can be driven by the same promoter or by different promoters. In certain cases, it may be desirable to introduce a transformation cassette that will suppress the expression of the polynucleotide of interest. This may be combined with any combination of other suppression cassettes or overexpression cassettes to generate the desired combination of traits in the plant. It is further recognized that polynucleotide sequences can be stacked at a desired genomic location using a site-specific recombination system. See, for example, WO 1999 / 25821, WO 1999 / 25854, WO 1999 / 25840, WO 1999 / 25855 and WO 1999 / 25853, all of which are herein incorporated by reference.

[0221] In some embodiments, one or more of the polynucleotides encoding the scaffold binding peptides, fusion polypeptide(s) disclosed herein, including fusion polynucleotides encoding chimeric polypeptides comprising one or more scaffold binding peptides, one or more linker peptides, and one or more polypeptides of interest, such as an insecticidal polypeptide of interest, or Insecticidal protein B or Insecticidal protein C polypeptides, alone or stacked with one or more additional insect resistance traits can be stacked with one or more additional input traits (e.g., herbicide resistance, fungal resistance, virus resistance, stress tolerance, disease resistance, male sterility, stalk strength, and the like) or output traits (e.g., increased yield, modified starches, improved oil profile, balanced amino acids, high lysine or methionine, increased digestibility, improved fiber quality, drought resistance, and the like). Thus, the polynucleotide embodiments can be used to provide a complete agronomic package of improved crop quality with the ability to flexibly and cost effectively control any number of agronomic pests.

[0222] Transgenes useful for stacking include but are not limited to: transgenes that confer resistance to an herbicide; transgenes that confer or contribute to an altered grain characteristic; genes that control male-sterility; genes that create a site for site specific DNA integration; genes that affect abiotic stress resistance; genes that confer increased yield, genes that confer plant digestibility; and transgenes that confer resistance to insects or disease.

[0223] Examples of transgenes encoding insecticidal polypeptides of interest that confer resistance to insects include those disclosed herein.Gene Silencing

[0224] In some embodiments, the stacked trait may be in the form of silencing of one or more polynucleotides of interest resulting in suppression of one or more target pest polypeptides. In some embodiments, the silencing is achieved using a suppression DNA construct.

[0225] In some embodiments, one or more polynucleotide encoding the polypeptides of the scaffold binding peptides or chimeric fusion polypeptides comprising one or more scaffold binding peptides, one or more linker peptides, and one or more polypeptides of interest, such as an insecticidal polypeptide of interest, or fragments or variants thereof may be stacked with one or more polynucleotides encoding one or more polypeptides having insecticidal activity or agronomic traits as set forth supra and optionally may further include one or more polynucleotides providing for gene silencing of one or more target polynucleotides as discussed infra. In some embodiments, one or more polynucleotide encoding the polypeptides of the Insecticidal protein B or Insecticidal protein C polypeptide or fragments or variants thereof may be stacked with one or more polynucleotides encoding one or more polypeptides having insecticidal activity or agronomic traits as set forth supra and optionally may further include one or more polynucleotides providing for gene silencing of one or more target polynucleotides as discussed infra.

[0226] Further transgenes that confer resistance to insects may relate to down-regulation of expression of target genes in insect pest species by interfering ribonucleic acid (RNA) molecules through RNA interference. RNA interference refers to the process of sequence-specific post- transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs) (Fire, et al., (1998) Nature 391 :806). RNAi transgenes may include but are not limited to expression of dsRNA, siRNA, miRNA, iRNA, antisense RNA, or sense RNA molecules that down-regulate expression of target genes in insect pests. PCT Publication WO 2007 / 074405 describes methods of inhibiting expression of target genes in invertebrate pests including Colorado potato beetle. PCT Publication WO 2005 / 110068 describes methods of inhibiting expression of target genes in invertebrate pests including in particular Western corn rootworm as a means to control insect infestation. Furthermore, PCT Publication WO 2009 / 091864 describes compositions and methods for the suppression of target genes from insect pest species including pests from the Lygus genus.

[0227] RNAi transgenes are provided for targeting the vacuolar ATPase H subunit, useful for controlling a coleopteran pest population and infestation as described in US Patent Application Publication 2012 / 0198586. PCT Publication WO 2012 / 055982 describes ribonucleic acid (RNAor double stranded RNA) that inhibits or down regulates the expression of a target gene that encodes: an insect ribosomal protein such as the ribosomal protein LI 9, the ribosomal protein L40 or the ribosomal protein S27A; an insect proteasome subunit such as the Rpn6 protein, the Pros 25, the Rpn2 protein, the proteasome beta 1 subunit protein or the Pros beta 2 protein; an insect P- coatomer of the COPI vesicle, the y-coatomer of the COPI vesicle, the '- coatomer protein or the (^-coatomer of the COPI vesicle; an insect Tetraspanine 2 A protein which is a putative transmembrane domain protein; an insect protein belonging to the actin family such as Actin 5C; an insect ubiquitin-5E protein; an insect Sec23 protein which is a GTPase activator involved in intracellular protein transport; an insect crinkled protein which is an unconventional myosin which is involved in motor activity; an insect crooked neck protein which is involved in the regulation of nuclear alternative mRNA splicing; an insect vacuolar H+-ATPase G-subunit protein and an insect Tbp-1 such as Tat-binding protein. PCT publication WO 2007 / 035650 describes ribonucleic acid (RNA or double stranded RNA) that inhibits or down regulates the expression of a target gene that encodes Snf7. US Patent Application publication 2011 / 0054007 describes polynucleotide silencing elements targeting RPS10. PCT publication WO 2016 / 205445 describes polynucleotide silencing elements that reduce fecundity, with target polynucleotides, including NCLB, MAEL, BOULE, and VgR. US Patent Application publication 2014 / 0275208 and US2015 / 0257389 describes polynucleotide silencing elements targeting RyanR (DvSSJl) and PAT3. PCT publications WO / 2016 / 138106, WO 2016 / 060911, WO 2016 / 060912, WO 2016 / 060913, and WO 2016 / 060914 describe polynucleotide silencing elements targeting COPI coatomer subunit nucleic acid molecules that confer resistance to Coleopteran and Hemipteran pests. US Patent Application Publications 2012 / 029750, US 20120297501, and 2012 / 0322660 describe interfering ribonucleic acids (RNA or double stranded RNA) that functions upon uptake by an insect pest species to down- regulate expression of a target gene in said insect pest, wherein the RNA comprises at least one silencing element wherein the silencing element is a region of double-stranded RNA comprising annealed complementary strands, one strand of which comprises or consists of a sequence of nucleotides which is at least partially complementary to a target nucleotide sequence within the target gene. US Patent Application Publication 2012 / 0164205 describe potential targets for interfering double stranded ribonucleic acids for inhibiting invertebrate pests including: a Chd3 Homologous Sequence, a Beta-Tubulin Homologous Sequence, a 40 kDa V-ATPase HomologousSequence, a EFla Homologous Sequence, a 26S Proteosome Subunit p28 Homologous Sequence, a Juvenile Hormone Epoxide Hydrolase Homologous Sequence, a Swelling Dependent Chloride Channel Protein Homologous Sequence, a Glucose-6-Phosphate 1 -Dehydrogenase Protein Homologous Sequence, an Act42A Protein Homologous Sequence, a ADP-Ribosylation Factor 1 Homologous Sequence, a Transcription Factor IIB Protein Homologous Sequence, a Chitinase Homologous Sequences, a Ubiquitin Conjugating Enzyme Homologous Sequence, a Glyceraldehyde-3 -Phosphate Dehydrogenase Homologous Sequence, an Ubiquitin B Homologous Sequence, a Juvenile Hormone Esterase Homolog, and an Alpha Tubuliln Homologous Sequence.Use in Pesticidal Control

[0228] General methods for employing strains comprising a nucleic acid sequence of the embodiments or a variant thereof, in pesticide control or in engineering other organisms as pesticidal agents are known in the art.

[0229] Microorganism hosts that are known to occupy the "phytosphere" (phylloplane, phyllosphere, rhizosphere, and / or rhizoplana) of one or more crops of interest may be selected. These microorganisms are selected so as to be capable of successfully competing in the particular environment with the wild-type microorganisms, provide for stable maintenance and expression of the gene(s) expressing one or more of the scaffold binding peptides or chimeric fusion polypeptides comprising one or more scaffold binding peptides, one or more linker peptides, and one or more polypeptides of interest, such as an insecticidal polypeptide of interest and desirably provide for improved protection of the pesticide from environmental degradation and inactivation.

[0230] Alternatively, the scaffold binding peptide or chimeric polypeptides comprising one or more scaffold binding peptides, one or more linker peptides, and one or more polypeptides of interest, such as an insecticidal polypeptide of interest are produced by introducing a heterologous gene into a cellular host. Alternatively, the Insecticidal protein B or Insecticidal protein C polypeptide is produced by introducing a heterologous gene into a cellular host. Expression of the heterologous gene results, directly or indirectly, in the intracellular production and maintenance of the pesticide. These cells are then treated under conditions that prolong the activity of the toxin produced in the cell when the cell is applied to the environment of target pest(s). The resulting product retains the toxicity of the toxin. These naturally encapsulated scaffold binding peptide orchimeric polypeptides comprising one or more scaffold binding peptides, one or more linker peptides, and one or more polypeptides of interest, such as an insecticidal polypeptide of interest may then be formulated in accordance with conventional techniques for application to the environment hosting a target pest, e.g., soil, water, and foliage of plants. Further, these naturally encapsulated Insecticidal protein B or Insecticidal protein C polypeptides may then be formulated in accordance with conventional techniques for application to the environment hosting a target pest, e.g., soil, water, and foliage of plants. See, for example EPA 0192319, and the references cited therein.Pesticidal Compositions

[0231] In some embodiments the active ingredients such as insecticidal polypeptides can be applied in the form of compositions and can be applied to the crop area or plant to be treated, simultaneously or in succession, with other compounds. These compounds can be fertilizers, weed killers, Cryoprotectants, surfactants, detergents, pesticidal soaps, dormant oils, polymers, and / or time-release or biodegradable carrier formulations that permit long-term dosing of a target area following a single application of the formulation. They can also be selective herbicides, chemical insecticides, virucides, microbicides, amoebicides, pesticides, fungicides, bacteriocides, nematocides, molluscicides or mixtures of several of these preparations, if desired, together with further agriculturally acceptable carriers, surfactants or application-promoting adjuvants customarily employed in the art of formulation. Suitable carriers and adjuvants can be solid or liquid and correspond to the substances ordinarily employed in formulation technology, e.g. natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, binders or fertilizers. Likewise, the formulations may be prepared into edible “baits” or fashioned into pest “traps” to permit feeding or ingestion by a target pest of the pesticidal formulation.

[0232] Methods of applying an active ingredient or an agrochemical composition that contains at least one of the scaffold binding peptide or chimeric fusion polypeptides comprising one or more scaffold binding peptides, one or more linker peptides, and one or more polypeptides of interest, such as an insecticidal polypeptide of interest and / or of the Insecticidal protein B or Insecticidal protein C polypeptide(s) include leaf application, seed coating and soil application. The number of applications and the rate of application depend on the intensity of infestation by thecorresponding pest.

[0233] The composition may be formulated as a powder, dust, pellet, granule, spray, emulsion, colloid, solution, or such like, and may be prepared by such conventional means as desiccation, lyophilization, homogenation, extraction, fdtration, centrifugation, sedimentation or concentration of a culture of cells comprising the polypeptide. In all such compositions that contain at least one such pesticidal polypeptide, the polypeptide may be present in a concentration of from about 1% to about 99% by weight.

[0234] Lepidopteran, Dipteran, Heteropteran, nematode, Hemiptera or Coleopteran pests may be killed or reduced in numbers in a given area by the methods of the disclosure or may be prophylactically applied to an environmental area to prevent infestation by a susceptible pest. Preferably the pest ingests or is contacted with, a pesticidally-effective amount of the polypeptide. “Pesticidally-effective amount” as used herein refers to an amount of the pesticide that is able to bring about death to at least one pest or to noticeably reduce pest growth, feeding or normal physiological development. This amount will vary depending on such factors as, for example, the specific target pests to be controlled, the specific environment, location, plant, crop or agricultural site to be treated, the environmental conditions and the method, rate, concentration, stability, and quantity of application of the pesticidally-effective polypeptide composition. The formulations may also vary with respect to climatic conditions, environmental considerations, and / or frequency of application and / or severity of pest infestation.

[0235] The pesticide compositions described may be made by formulating either the bacterial cell, Crystal and / or spore suspension or isolated protein component with the desired agri cultural ly- acceptable carrier. The compositions may be formulated prior to administration in an appropriate means such as lyophilized, freeze-dried, desiccated or in an aqueous carrier, medium or suitable diluent, such as saline or other buffer. The formulated compositions may be in the form of a dust or granular material or a suspension in oil (vegetable or mineral) or water or oil / water emulsions or as a wettable powder or in combination with any other carrier material suitable for agricultural application. Suitable agricultural carriers can be solid or liquid and are well known in the art. The term “agriculturally-acceptable carrier” covers all adjuvants, inert components, dispersants, surfactants, tackifiers, binders, etc. that are ordinarily used in pesticide formulation technology; these are well known to those skilled in pesticide formulation. The formulations may be mixedwith one or more solid or liquid adjuvants and prepared by various means, e.g., by homogeneously mixing, blending and / or grinding the pesticidal composition with suitable adjuvants using conventional formulation techniques. Suitable formulations and application methods are known. The plants can also be treated with one or more chemical compositions, including one or more herbicide, insecticides or fungicides. Exemplary chemical compositions include: Cereals Herbicides: Isoproturon, Bromoxynil, Ioxynil, Phenoxies, Chlorsulfuron, Clodinafop, Diclofop, Diflufenican, Fenoxaprop, Florasulam, Fluoroxypyr, Metsulfuron, Triasulfuron, Flucarbazone, lodosulfuron, Propoxycarbazone, Picolinafen, Mesosulfuron, Beflubutamid, Pinoxaden, Amidosulfuron, Thifensulfuron Methyl, Tribenuron, Flupyrsulfuron, Sulfosulfuron, Pyrasulfotole, Pyroxsulam, Flufenacet, Tralkoxydim, Pyroxasulfon; Cereals Fungicides: Carbendazim, Chlorothalonil, Azoxystrobin, Cyproconazole, Cyprodinil, Fenpropimorph, Epoxiconazole, Kresoxim-methyl, Quinoxyfen, Tebuconazole, Trifloxystrobin, Simeconazole, Picoxystrobin, Pyraclostrobin, Dimoxystrobin, Prothioconazole, Fluoxastrobin; Cereals Insecticides: Dimethoate, Lambda-cyhalthrin, Deltamethrin, alpha-Cypermethrin, P-cyfluthrin, Bifenthrin, Imidacloprid, Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid, Dinetofuran, Clorphyriphos, Metamidophos, Oxidemethon-methyl, Pirimicarb, Methiocarb; Maize Herbicides: Atrazine, Alachlor, Bromoxynil, Acetochlor, Dicamba, Clopyralid, (S-) Dimethenamid, Glufosinate, Glyphosate, Isoxaflutole, (S-)Metolachlor, Mesotrione, Nicosulfuron, Primisulfuron, Rimsulfuron, Sulcotrione, Foramsulfuron, Topramezone, Tembotrione, Saflufenacil, Thiencarb azone, Flufenacet, Pyroxasulfon; Maize Insecticides: Carbofuran, Chlorpyrifos, Bifenthrin, Fipronil, Imidacloprid, Lambda-Cyhalothrin, Tefluthrin, Terbufos, Thiamethoxam, Clothianidin, Spiromesifen, Flubendiamide, Triflumuron, Rynaxypyr, Deltamethrin, Thiodicarb, P-Cyfluthrin, Cypermethrin, Bifenthrin, Lufenuron, Triflumoron, Tefluthrin, Tebupirimphos, Ethiprole, Cyazypyr, Thiacloprid, Acetamiprid, Dinetofuran, Avermectin, Methiocarb, Spirodiclofen, Spirotetramat; Maize Fungicides: Fenitropan, Thiram, Prothioconazole, Tebuconazole, Trifloxystrobin; Cotton Herbicides: Diuron, Fluometuron, MSMA, Oxyfluorfen, Prometryn, Trifluralin, Carfentrazone, Clethodim, Fluazifop-butyl, Glyphosate, Norflurazon, Pendimethalin, Pyrithiobac-sodium, Trifloxysulfuron, Tepraloxydim, Glufosinate, Flumioxazin, Thidiazuron; Cotton Insecticides: Acephate, Aldicarb, Chlorpyrifos, Cypermethrin, Deltamethrin, Malathion, Monocrotophos, Abamectin, Acetamiprid, Emamectin Benzoate, Imidacloprid,Indoxacarb, Lambda-Cyhalothrin, Spinosad, Thiodicarb, Gamma-Cyhalothrin, Spiromesifen, Pyridalyl, Flonicamid, Flubendiamide, Triflumuron, Rynaxypyr, Beta-Cyfluthrin, Spirotetramat, Clothianidin, Thiamethoxam, Thiacloprid, Dinetofuran, Flubendiamide, Cyazypyr, Spinosad, Spinotoram, gamma Cyhalothrin, 4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan- 2(5H)-on, Thiodicarb, Avermectin, Flonicamid, Pyridalyl, Spiromesifen, Sulfoxaflor, Profenophos, Thriazophos, Endosulfan; Cotton Fungicides: Etridiazole, Metalaxyl, Quintozene; Soybean Herbicides: Alachlor, Bentazone, Trifluralin, Chlorimuron-Ethyl, Cloransulam-Methyl, Fenoxaprop, Fomesafen, Fluazifop, Glyphosate, Imazamox, Imazaquin, Imazethapyr, (S- )Metolachlor, Metribuzin, Pendimethalin, Tepraloxydim, Glufosinate; Soybean Insecticides: Lambda-cyhalothrin, Methomyl, Parathion, Thiocarb, Imidacloprid, Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid, Dinetofuran, Flubendiamide, Rynaxypyr, Cyazypyr, Spinosad, Spinotoram, Emamectin-Benzoate, Fipronil, Ethiprole, Deltamethrin, P-Cyfluthrin, gamma and lambda Cyhalothrin, 4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on, Spirotetramat, Spinodiclofen, Triflumuron, Flonicamid, Thiodicarb, beta-Cyfluthrin; Soybean Fungicides: Azoxystrobin, Cyproconazole, Epoxiconazole, Flutriafol, Pyraclostrobin, Tebuconazole, Trifloxystrobin, Prothioconazole, Tetraconazole; Canola Herbicides: Clopyralid, Diclofop, Fluazifop, Glufosinate, Glyphosate, Metazachlor, Trifluralin Ethametsulfuron, Quinmerac, Quizalofop, Clethodim, Tepraloxydim; Canola Fungicides: Azoxystrobin, Carbendazim, Fludioxonil, Iprodione, Prochloraz, Vinclozolin; Canola Insecticides: Carbofuran organophosphates, Pyrethroids, Thiacloprid, Deltamethrin, Imidacloprid, Clothianidin, Thiamethoxam, Acetamiprid, Dinetofuran, P-Cyfluthrin, gamma and lambda Cyhalothrin, tau- Fluvaleriate, Ethiprole, Spinosad, Spinotoram, Flubendiamide, Rynaxypyr, Cyazypyr, 4-[[(6- Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on.

[0236] In some embodiments the herbicide is Atrazine, Bromacil, Diuron, Chlorsulfuron, Metsulfuron, Thifensulfuron Methyl, Tribenuron, Acetochlor, Dicamba, Isoxaflutole, Nicosulfuron, Rimsulfuron, Pyrithiobac-sodium, Flumioxazin, Chlorimuron-Ethyl, Metribuzin, Quizalofop, S-metolachlor, Hexazinne or combinations thereof.

[0237] In some embodiments the insecticide is Esfenval erate, Chlorantraniliprole, Methomyl, Indoxacarb, Oxamyl or combinations thereof.Pesticidal and Insecticidal Activity

[0238] “Pest” includes but is not limited to, insects, fungi, bacteria, nematodes, mites, ticks and the like. Insect pests include insects selected from the orders Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularly Lepidoptera.

[0239] Those skilled in the art will recognize that not all compounds are equally effective against all pests. Compounds of the embodiments display activity against insect pests, which may include economically important agronomic, forest, greenhouse, nursery ornamentals, food and fiber, public and animal health, domestic and commercial structure, household and stored product pests.

[0240] Larvae of the order Lepidoptera include, but are not limited to, armyworms, cutworms, loopers and heliothines in the family Noctuidae Spodoptera frugiperda JE Smith (fall armyworm); S. exigua Hiibner (beet armyworm); 5. litura Fabricius (tobacco cutworm, cluster caterpillar); Mamesira configurata Walker (bertha armyworm); M. brassicas Linnaeus (cabbage moth); Agrotis ipsilon Hufnagel (black cutworm); A. orthogonia Morrison (western cutworm); A. subterranea Fabricius (granulate cutworm); Alabama argillacea Hiibner (cotton leaf worm); Trichoplusia Hi Hiibner (cabbage looper); Pseudoplusia includens Walker (soybean looper); Anticarsia gemmatalis Hiibner (velvetbean caterpillar); Hypena scabra Fabricius (green cloverworm); Heliothis virescens Fabricius (tobacco budworm); Psendaletia unipuncta Haworth (armyworm); Athetis mindara Barnes and Mcdunnough (rough skinned cutworm); Euxoa messoria Harris (darksided cutworm); Earias insulana Boisduval (spiny bollworm); E. vittella Fabricius (spotted bollworm); Elelicoverpa armigera Hiibner (American bollworm); H. zea Boddie (com earworm or cotton bollworm); Melanchra picta Harris (zebra caterpillar); Egira (Xylomyges) curialis Grote (citrus cutworm); borers, casebearers, webworms, coneworms, and skeletonizers from the family Pyralidae Ostrinia nubilalis Hiibner (European corn borer); Amyelois transitella Walker (naval orangeworm); Anagasta kuehniella Zeller (Mediterranean flour moth); Cadra cautella Walker (almond moth); Chilo suppressalis Walker (rice stem borer); C. partellus, (sorghum borer); Corcyra cephalonica Stainton (rice moth); Crambus caliginosellus Clemens (com root webworm); C. teterrelhis Zincken (bluegrass webworm); Cnaphalocrocis medinalis Guenee (rice leaf roller); Desmia funeralis Hiibner (grape leaffolder); Diaphania hyalinata Linnaeus (melon worm); D. nitidalis Stoll (pickleworm); Diatraea grandiosella Dyar(southwestern corn borer), D. saccharalis Fabricius (surgarcane borer); Eoreuma loftini Dyar (Mexican rice borer); Ephestia elutella Hiibner (tobacco (cacao) moth); Galleria mellonella Linnaeus (greater wax moth); Herpetogramma licarsisalis Walker (sod webworm); Homoeosoma electellum Hulst (sunflower moth); Elasmopalpus lignosellus Zeller (lesser cornstalk borer); Achroia grisella Fabricius (lesser wax moth); Loxostege sticticalis Linnaeus (beet webworm); Orthaga thyrisalis Walker (tea tree web moth); Maruca testulalis Geyer (bean pod borer); Plodia interpunctella Hiibner (Indian meal moth); Scirpophaga incertulas Walker (yellow stem borer); Udea rubigalis Guenee (celery leaftier); and leafrollers, budworms, seed worms and fruit worms in the family Tortricidae Acleris gloverana Walsingham (Western blackheaded budworm); A. variana Femald (Eastern blackheaded budworm); Archips argyrospila Walker (fruit tree leaf roller); A. rosana Linnaeus (European leaf roller); and other Archips species, Adoxophyes orana Fischer von Rbsslerstamm (summer fruit tortrix moth); Cochylis hospes Walsingham (banded sunflower moth); Cydia latiferreana Walsingham (filbertworm); C. pomonella Linnaeus (coding moth); Platynota flavedana Clemens (variegated leafroller); P. stultana Walsingham (omnivorous leafroller); Lobesia botrana Denis & Schiffermiiller (European grape vine moth); Spilonota ocellana Denis & Schiffermiiller (eyespotted bud moth); Endopiza viteana Clemens (grape berry moth); Eupoecilia ambiguella Hiibner (vine moth); Bonagota salubricola Meyrick (Brazilian apple leafroller); Grapholita molesta Busck (oriental fruit moth); Suleima helianthana Riley (sunflower bud moth); Argyrotaenia spp. Chori stoneura spp..

[0241] Selected other agronomic pests in the order Lepidoptera include, but are not limited to, Alsophila pomeiaria Harris (fall cankerworm); Anarsia lineatella Zeller (peach twig borer); Anisota senatoria J.E. Smith (orange striped oakworm); Antheraea pernyi Guerin-Meneville (Chinese Oak Tussah Moth); Bombyx mori Linnaeus (Silkworm); Bucculatrix thurberiella Busck (cotton leaf perforator); Colias eurytheme Boisduval (alfalfa caterpillar); Datana integerrima Grote & Robinson (walnut caterpillar); Dendrolimus sibiricus Tschetwerikov (Siberian silk moth), Ennomos subsignaria Hiibner (elm spanworm); Erannis tiliaria Harris (linden looper); Euproctis chrysorrhoea Linnaeus (browntail moth); Harrisina americana Guerin-Meneville (grapeleaf skeletonizer); Heniileuca oliviae Cockrell (range caterpillar); Hyphantria cunea Drury (fall webworm); Keiferia lycopersicella Walsingham (tomato pinworm); Lambdina fiscellaria fiscellaria Hulst (Eastern hemlock looper); L. fiscellaria lugubrosa Hulst (Western hemlocklooper); Leucoma salicis Linnaeus (satin moth); Lymantria dispar Linnaeus (gypsy moth); Manduca quinquemaculata Haworth (five spotted hawk moth, tomato homworm); M. sexta Haworth (tomato hornworm, tobacco homworm); Operophtera brumata Linnaeus (winter moth); Paleacrita vernata Peck (spring cankerworm); Papilio cresphontes Cramer (giant swallowtail orange dog); Phryganidia californica Packard (California oakworm); Phyllocnistis citrella Stainton (citrus leafminer); Phyllonorycter blancardella Fabricius (spotted tentiform leafminer); Pieris brassicae Linnaeus (large white butterfly); P. rapae Linnaeus (small white butterfly); P. napi Linnaeus (green veined white butterfly); Platyptilia carduidactyla Riley (artichoke plume moth); Plutella xylostella Linnaeus (diamondback moth); Pectinophora gossypiella Saunders (pink bollworm); Pontia protodice Boisduval and Leconte (Southern cabbageworm); Sabulodes aegrotata Guenee (omnivorous looper); Schizura concinna J.E. Smith (red humped caterpillar); Sitotroga cerealella Olivier (Angoumois grain moth); Thaumetopoea pityocampa Schiffermuller (pine processionary caterpillar); Tineola bisselliella Hummel (webbing clothesmoth); Tula absoluta Meyrick (tomato leafminer); Yponomeuta padella Linnaeus (ermine moth); Heliothis subflexa Guenee; Malacosoma spp. and Orgyia spp.

[0242] Adults and immatures of the order Diptera are of interest, including leafminers Agromyza parvicornis Loew (corn blotch leafminer); midges (including, but not limited to: Contarinia sorghicola Coquillett (sorghum midge); Mayetiola destructor Say (Hessian fly); Sitodiplosis mosellana Gehin (wheat midge); NeoIasi opter a murtfeldtiana Felt, (sunflower seed midge)); fruit flies (Tephritidae), Oscinella frit Linnaeus (fruit flies); maggots (including, but not limited to: Delia platura Meigen (seedcorn maggot).

[0243] Included as insects of interest are adults and nymphs of the orders Hemiptera and Homoptera such as, but not limited to, adelgids from the family Adelgidae, plant bugs from the family Miridae, cicadas from the family Cicadidae, leafhoppers, Empoasca spp.; from the family Cicadellidae, planthoppers from the families Cixiidae, Flatidae, Fulgoroidea, Issidae and Delphacidae, treehoppers from the family Membracidae, psyllids from the family Psyllidae, whiteflies from the family Aleyrodidae, aphids from the family Aphididae, phylloxera from the family Phylloxeridae, mealybugs from the family Pseudococcidae, scales from the families Asterolecanidae, Coccidae, Dactylopiidae, Diaspididae, Eriococcidae Ortheziidae, Phoenicococcidae and Margarodidae, lace bugs from the family Tingidae, stink bugs from thefamily Pentatomidae, cinch bugs, Blissus spp.; and other seed bugs from the family Lygaeidae, spittlebugs from the family Cercopidae squash bugs from the family Coreidae and red bugs and cotton stainers from the family Pyrrhocoridae.

[0244] Agronomically important members from the order Homoptera further include, but are not limited to: Acyrthisiphon pisum Harris (pea aphid); Aphis craccivora Koch (cowpea aphid); A. fabae Scopoli (black bean aphid); A. gossypii Glover (cotton aphid, melon aphid); A. maidiradicis Forbes (corn root aphid); A. pomi De Geer (apple aphid); A. spiraecola Patch (spirea aphid); Aulacorthum solani Kaltenbach (foxglove aphid); Chcietosiphon 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 Geoffrey (mealy plum aphid); Lipaphis erysimi Kaltenbach (turnip aphid); Metopolophium dirrhodum Walker (cereal aphid); Macrosiphum euphorbiae Thomas (potato aphid); Myzus persicae Sulzer (peach-potato aphid, green peach aphid); Nasonovia ribisnigri Mosley (lettuce aphid); Pemphigus spp. (root aphids and gall aphids); Rhopalosiphum maidis Fitch (corn leaf aphid); R. padi Linnaeus (bird cherry-oat aphid); Schizaphis gramimim Rondani (greenbug); Sipha jlava Forbes (yellow sugarcane aphid); Sitobion avenae Fabricius (English grain aphid); Therioaphis maculata Buckton (spotted alfalfa aphid); Toxoptera aurantii Boyer de Fonscolombe (black citrus aphid) and T. citricida Kirkaldy (brown citrus aphid); Adelges spp. (adelgids); Phylloxera devastatrix Pergande (pecan phylloxera); Bemisia tabaci Gennadius (tobacco whitefly, sweetpotato whitefly); B. argentifolii Bellows & Perring (silverleaf whitefly); Dialeurodes ciiri Ashmead (citrus whitefly); Trialeurodes abutiloneus (bandedwinged whitefly) and T. vaporariorum Westwood (greenhouse whitefly); Empoasca fabae Harris (potato leafhopper); Laodelphax striatellus Fallen (smaller brown planthopper); Macrolestes quadri lineatus Forbes (aster leafhopper); Nephotettix cinticeps Uhler (green leafhopper); N. nigropictus Stal (rice leafhopper); Nilaparvata lugens Stal (brown planthopper); Peregrinus maidis Ashmead (corn planthopper); Sogatella furcifera Horvath (white- backed planthopper); Sogatodes orizicola Muir (rice delphacid); Typhlocyba pomaria McAtee (white apple leafhopper); Erythroneoura spp. (grape leafhoppers); Magicicada septendecim Linnaeus (periodical cicada); Icerya purchasi Maskell (cottony cushion scale); Quadraspidiotus perniciosus Comstock (San Jose scale); Pianococcus citri Risso (citrus mealybug); Pseudococcusspp. (other mealybug complex); Cacopsylla pyricola Foerster (pear psylla); Trioza diospyri Ashmead (persimmon psylla).

[0245] Agronomically important species of interest from the order Hemiptera include, but are not limited to: Acrosternum hilare Say (green stink bug); Anasa tristis De Geer (squash bug); Blissus leucopterus leucopterus Say (chinch bug); Corythuca gossypii Fabricius (cotton lace bug); Cyrtopeltis modesta Distant (tomato bug); Dysdercus suturellus Herri ch- Schaffer (cotton stainer); Euschistus servus Say (brown stink bug); E. variolarius Palisot de Beauvois (one-spotted stink bug); Graptostethus spp. (complex of seed bugs); Leptoglossus corculus Say (leaf-footed pine seed bug); Lygus lineolaris Palisot de Beauvois (tarnished plant bug); L. Hesperus Knight (Western tarnished plant bug); L. pratensis Linnaeus (common meadow bug); L. rugulipennis Poppius (European tarnished plant bug); Lygocoris pabulinus Linnaeus (common green capsid); Nezara viridula Linnaeus (southern green stink bug); Oebalus pugnax Fabricius (rice stink bug); Oncopeltus fasciatus Dallas (large milkweed bug); Pseudatomoscelis seriatus Reuter (cotton fleahopper).

[0246] Furthermore, embodiments may be effective against Hemiptera such as, Calocoris norvegicus Gmelin (strawberry bug); Orthops campestris Linnaeus; Plesiocoris rugicollis Fallen (apple capsid); Cyrtopeltis modestus Distant (tomato bug); Cyrtopeltis notatus Distant (suckfly); Spanagonicus albofasciatus Reuter (whitemarked fleahopper); Diaphnocoris chlorionis Say (honeylocust plant bug); Labopidicola allii Knight (onion plant bug); Pseudatomoscelis seriatus Reuter (cotton fleahopper); Adelphocoris rapidus Say (rapid plant bug); Poecilocapsus lineatus Fabricius (four-lined plant bug); Nysius ericae Schilling (false chinch bug); Nysius raphanus Howard (false chinch bug); Nezara viridula Linnaeus (Southern green stink bug); Eurygaster spp.; Coreidae spp.; Pyrrhocoridae spp.; Tinidae spp.; Blostomatidae spp.; Reduviidae spp. and Cimicidae spp.

[0247] Also included are adults and larvae of the order Acari (mites) such as Aceria tosichella Keifer (wheat curl mite); Petrobia lalens Muller (brown wheat mite); spider mites and red mites in the family Tetranychidae, Panonychus ulmi Koch (European red mite); Tetranychus urticae Koch (two spotted spider mite); (T. mcdanieli McGregor (McDaniel mite); T. cinnabarinus Boisduval (carmine spider mite); T. turkestani Ugarov & Nikolski (strawberry spider mite); flat mites in the family Tenuipalpidae, Brevipalpus lewisi McGregor (citrus flat mite); rust and budmites in the family Eriophyidae and other foliar feeding mites and mites important in human and animal health.

[0248] Insect pest of interest include the superfamily of stink bugs and other related insects including but not limited to species belonging to the family Pentatomidae (Nezara viridula, Halyomorpha halys, Piezodorus guildini, Euschistus servus, Acrosternum hilar e, Euschistus heros, Euschistus tristignius, Acrosternum hilare, Dichelops furcatus, Dichelops melacanthus, and Bagrada hilaris (Bagrada Bug)), the family Plataspidae (Megacopta cribraria - Bean plataspid) and the family Cydnidae (Scaptocoris castanea - Root stink bug) and Lepidoptera species including but not limited to: diamond-back moth, e.g., Helicoverpa zea Boddie; soybean looper, e.g., Pseudoplusia includens Walker and velvet bean caterpillar e.g., Anticarsia gemmatalis Htibner.

[0249] Methods for measuring pesticidal activity are well known in the art. See, for example, Czapla and Lang, (1990) J. Econ. Entomol. 83:2480-2485; Andrews, et al., (1988) Biochem. J. 252: 199-206; Marrone, et al., (1985) J. of Economic Entomology 78:290-293 and US Patent Number 5,743,477. Generally, the protein is mixed and used in feeding assays. See, for example Marrone, et al., (1985) J. of Economic Entomology 78:290-293. Such assays can include contacting plants with one or more pests and determining the plant's ability to survive and / or cause the death of the pests.

[0250] Nematodes include parasitic nematodes such as root-knot, cyst and lesion nematodes, including Heterodera spp., Meloidogyne spp. and Globodera spp.; particularly members of the cyst nematodes, including, but not limited to, Heterodera glycines (soybean cyst nematode); Heterodera schachtii (beet cyst nematode); Heterodera avenae (cereal cyst nematode) and Globodera rostochiensis and Globodera pailida (potato cyst nematodes). Lesion nematodes include Pratylenchus spp.Seed Treatment

[0251] To protect and to enhance yield production and trait technologies, seed treatment options can provide additional crop plan flexibility and cost-effective control against insects, weeds and diseases. Seed material can be treated, typically surface treated, with a composition comprising combinations of chemical or biological herbicides, herbicide safeners, insecticides, fungicides,germination inhibitors and enhancers, nutrients, plant growth regulators and activators, bactericides, nematocides, avicides and / or molluscicides. These compounds are typically formulated together with further carriers, surfactants or application-promoting adjuvants customarily employed in the art of formulation. The coatings may be applied by impregnating propagation material with a liquid formulation or by coating with a combined wet or dry formulation. Examples of the various types of compounds that may be used as seed treatments are provided in The Pesticide Manual: A World Compendium, C.D.S. Tomlin Ed., Published by the British Crop Production Council.

[0252] Some seed treatments that may be used on crop seed include, but are not limited to, one or more of abscisic acid, acibenzolar-S-methyl, avermectin, amitrol, azaconazole, azospirillum, azadirachtin, azoxystrobin, Bacillus spp. (including one or more of cereus, firmus, megaterium, pumilis, sphaericus, subtilis and / or thuringiensis species), bradyrhizobium spp. (including one or more of betae, canariense, elkanii, iriomotense, japonicum, liaonigense, pachyrhizi and / or yuanmingense), captan, carboxin, chitosan, clothianidin, copper, cyazypyr, difenoconazole, etidiazole, fipronil, fludioxonil, fluoxastrobin, fluquinconazole, flurazole, fluxofenim, harpin protein, imazalil, imidacloprid, ipconazole, isoflavenoids, lipo-chitooligosaccharide, mancozeb, manganese, maneb, mefenoxam, metalaxyl, metconazole, myclobutanil, PCNB, penflufen, penicillium, penthiopyrad, permethrine, picoxystrobin, prothioconazole, pyraclostrobin, rynaxypyr, S-metolachlor, saponin, sedaxane, TCMTB, tebuconazole, thiabendazole, thiamethoxam, thiocarb, thiram, tolclofos-methyl, triadimenol, trichoderma, trifloxystrobin, triticonazole and / or zinc. PCNB seed coat refers to EPA Registration Number 00293500419, containing quintozen and terrazole. TCMTB refers to 2-(thiocyanomethylthio) benzothiazole.

[0253] Seed varieties and seeds with specific transgenic traits may be tested to determine which seed treatment options and application rates may complement such varieties and transgenic traits in order to enhance yield. For example, a variety with good yield potential but head smut susceptibility may benefit from the use of a seed treatment that provides protection against head smut, a variety with good yield potential but cyst nematode susceptibility may benefit from the use of a seed treatment that provides protection against cyst nematode, and so on. Likewise, a variety encompassing a transgenic trait conferring insect resistance may benefit from the second mode of action conferred by the seed treatment, a variety encompassing a transgenic trait conferringherbicide resistance may benefit from a seed treatment with a safener that enhances the plants resistance to that herbicide, etc. Further, the good root establishment and early emergence that results from the proper use of a seed treatment may result in more efficient nitrogen use, a better ability to withstand drought and an overall increase in yield potential of a variety or varieties containing a certain trait when combined with a seed treatment.Methods for Killing an Insect Pest and Controlling an Insect Population

[0254] In some embodiments methods are provided for killing an insect pest, comprising contacting the insect pest, either simultaneously or sequentially, with an insecticidally-effective amount of an insecticidal polypeptide of interest in conjunction with a scaffold binding peptide of the disclosure, either as a chimeric fusion polypeptide or expressed or co-expressed polypeptides. In one embodiment methods are provided for killing an insect pest, comprising contacting the insect pest with an insecticidally-effective amount of one or more of an insecticidal protein of interest in conjunction with a scaffold binding peptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more of SEQ ID NOs: 2-6, or a variant or active fragment thereof. In another embodiment, methods are provided for killing an insect pest, comprising contacting the insect pest with an insecticidally-effective amount of a chimeric fusion polypeptide comprising one or more scaffold binding peptides of SEQ ID NOs: 2-6, one or more linker peptides of SEQ ID NOs: 7-27, and one or more insecticidal polypeptides of interest, or a variant or active fragment thereof. In other embodiments, methods are provided for killing an insect pest, comprising contacting the insect pest with an insecticidally-effective amount of a chimeric fusion polypeptide comprising one or more scaffold binding peptides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more of SEQ ID NOs: 2-6, one or more linker peptides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more of SEQ ID NOs: 1- 1 , and one or more insecticidal polypeptides of interest, or a variant or active fragment thereof.

[0255] In some embodiments methods are provided for killing an insect pest or insect pest population, comprising contacting the insect pest, either simultaneously or sequentially, with an insecticidally-effective amount of an Insecticidal protein B or Insecticidal protein C polypeptideof the disclosure. In some embodiments methods are provided for killing an insect pest or insect pest population, comprising contacting the insect pest with an insecticidally-effective amount of one or more of a pesticidal protein having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more of SEQ ID NOs: 58 or 59, or a variant or insecticidally active fragment thereof.

[0256] In some embodiments methods are provided for controlling an insect pest population, comprising contacting the insect pest population, either simultaneously or sequentially, with an insecticidally-effective amount of an insecticidal composition comprising one or more scaffold binding peptides and an insecticidal polypeptide of interest. In some embodiments, methods are provided for controlling an insect pest population, comprising contacting the insect pest population with an insecticidally-effective amount of an insecticidal composition comprising one or more scaffold binding peptides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more of SEQ ID NOs: 2-6, or a variant or active fragment thereof and an insecticidal polypeptide of interest, or a variant or active fragment thereof. In other embodiments, methods are provided for controlling an insect pest population, comprising contacting the insect pest population with an insecticidally-effective amount of an insecticidal composition comprising a chimeric fusion polypeptide comprising one of more scaffold binding peptides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more of SEQ ID NOs: 2-6, one or more linker peptides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more of SEQ ID NOs: 7-27, and one or more insecticidal polypeptides of interest, or a variant or active fragment thereof. As used herein, “controlling a pest population” or “controls a pest” refers to any effect on a pest that results in limiting the damage that the pest causes. Controlling a pest includes, but is not limited to, killing the pest, inhibiting development of the pest, altering fertility or growth of the pest in such a manner that the pest provides less damage to the plant, decreasing the number of offspring produced, producing less fit pests, producing pests more susceptible to predator attack or deterring the pests from eating the plant.

[0257] In some embodiments methods are provided for controlling an insect pest population, comprising contacting the insect pest population, either simultaneously or sequentially, with an insecticidally-effective amount of one or more of an Insecticidal protein B or Insecticidal proteinC polypeptide of the disclosure. In some embodiments, methods are provided for controlling an insect pest population, comprising contacting the insect pest population with an insecticidally- effective amount of one or more of an Insecticidal protein B or Insecticidal protein C polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more of SEQ ID NOs: 58 or 59, respectively, or a variant or insecticidally active fragment thereof.

[0258] In some embodiments methods are provided for controlling an insect pest population resistant to a pesticidal protein, comprising contacting the insect pest population, either simultaneously or sequentially, with an insecticidally-effective amount of one or more insecticidal polypeptides of interest in conjunction with one or more scaffold binding peptides of the disclosure, and optionally one or more of the linker peptides of the disclosure. In some embodiments, methods are provided for controlling an insect pest population resistant to a pesticidal protein, comprising contacting the insect pest population with an insecticidally-effective amount of one or more insecticidal polypeptides of interest in conjunction with one or more scaffold binding peptides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more of SEQ ID NOs: 2-6, or a variant or active fragment thereof, and optionally one or more of the linker peptides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more of SEQ ID NOs: 7- 27, or a variant or active fragment thereof.

[0259] In some embodiments methods are provided for controlling an insect pest population resistant to a pesticidal protein, comprising contacting the insect pest population, either simultaneously or sequentially, with an insecticidally-effective amount of one or more of an Insecticidal protein B or Insecticidal protein C polypeptide of the disclosure. In some embodiments, methods are provided for controlling an insect pest population resistant to a pesticidal protein, comprising contacting the insect pest population with an insecticidally-effective amount of one or more of an Insecticidal protein B or Insecticidal protein C polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more of SEQ ID NOs: 58 or 59, respectively, or a variant or insecticidally active fragment thereof.

[0260] In some embodiments methods are provided for protecting a plant from an insect pest,comprising expressing in the plant or cell thereof at least one polynucleotide encoding an insecticidally effective composition comprising one or more scaffold binding peptides of the disclosure, optionally a linker peptide of the disclosure, and an insecticidal polypeptide of interest. In some embodiments methods are provided for protecting a plant from an insect pest, comprising expressing in the plant or cell thereof a polynucleotide encoding an insecticidally effective composition comprising one or more scaffold binding peptides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more of SEQ ID NOs: 2-6, or variants or active fragment thereof, optionally one or more linker peptides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more of SEQ ID NOs: 7-27, or a variant or active fragment thereof, and an insecticidal polypeptide of interest.

[0261] In some embodiments methods are provided for protecting a plant from an insect pest, comprising expressing in the plant or cell thereof at least one polynucleotide encoding an Insecticidal protein B or Insecticidal protein C polypeptide of the disclosure. In some embodiments methods are provided for protecting a plant from an insect pest, comprising expressing in the plant or cell thereof a polynucleotide encoding one or more Insecticidal protein B or Insecticidal protein C polypeptides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more of SEQ ID NOs: 58 or 59, respectively, or variants or insecticidally active fragments thereof.Insect Resistance Management (IRM) Strategies

[0262] Expression of B. thuringiensis 8-endotoxins in transgenic corn plants has proven to be an effective means of controlling agriculturally important insect pests (Perlak, et al., 1990; 1993). However, in certain instances insects have evolved that are resistant to B. thuringiensis 8- endotoxins expressed in transgenic plants. Such resistance, should it become widespread, would clearly limit the commercial value of germplasm containing genes encoding such B. thuringiensis 6-endotoxins.

[0263] One way of increasing the effectiveness of the transgenic insecticides against target pests and contemporaneously reducing the development of insecticide-resistant pests is to use non-transgenic (i.e., non-insecticidal protein) refuges (a section of non-insecticidal crops / corn) with transgenic crops producing a single insecticidal protein active against target pests. The United States Environmental Protection Agency(epa.gov / oppbppdl / biopesticides / pips / bt_corn_refuge_2006.htm, which can be accessed using the www prefix) publishes the requirements for use with transgenic crops producing a single Bt protein active against target pests. In addition, the National Com Growers Association, on their website: (ncga.com / insect-resistance-management-fact-sheet-bt-corn, which can be accessed using the www prefix) also provides similar guidance regarding refuge requirements. Due to losses to insects within the refuge area, larger refuges may reduce overall yield.

[0264] Expression of transgenic insecticidal proteins at a high dose, that which kills 99.99% of susceptible insects, will result in greater durability of such insecticidal traits (Tabashnik and Carrier. Nature Biotechnology 35:926. 2017). In one embodiment, scaffold binding peptides or chimeric fusion polypeptide compositions comprising an insecticidal polypeptide of interest that eliminate undesirable host effects such as phytotoxicity and produce more protein make it possible to produce high dose levels of active ingredient (such as expression level of an insecticidal protein in a host plant) and mitigate insect resistance development.

[0265] Another way of increasing the effectiveness of the transgenic insecticides against target pests and contemporaneously reducing the development of insecticide-resistant pests would be to have a repository of insecticidal genes that are effective against groups of insect pests and which manifest their effects through different modes of action.

[0266] Expression in a plant of two or more insecticidal compositions toxic to the same insect species, each insecticide being expressed at efficacious levels would be another way to achieve control of the development of resistance. This is based on the principle that evolution of resistance against two separate modes of action is far more unlikely than only one. Roush, for example, outlines two-toxin strategies, also called "pyramiding" or "stacking," for management of insecticidal transgenic crops. (The Royal Society. Phil. Trans. R. Soc. Lond. B. (1998) 353: 1777- 1786). Stacking or pyramiding of two different proteins each effective against the target pests and with little or no cross-resistance can allow for use of a smaller refuge. The US Environmental Protection Agency requires significantly less (generally 5%) structured refuge of non- / ? / corn be planted than for single trait products (generally 20%). There are various ways of providing theIRM effects of a refuge, including various geometric planting patterns in the fields and in-bag seed mixtures, as discussed further by Roush.

[0267] In some embodiments the scaffold binding peptides or chimeric fusion polypeptides comprising one of more scaffold binding peptides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more of SEQ ID NOs: 2-6, or variants or active fragment thereof, one or more linker peptides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more of SEQ ID NOs: 7-27, or variants or active fragment thereof, and one or more insecticidal polypeptides of interest of the disclosure are useful as an insect resistance management strategy in combination (i.e., pyramided) with other pesticidal proteins or other transgenes (i.e., an RNAi trait) including but not limited to Bt toxins, Xenorhabdus sp. or Photorhabdus sp. insecticidal proteins, other insecticidally active proteins, and the like.

[0268] In some embodiments the Insecticidal protein B or Insecticidal protein C polypeptides of the disclosure are useful as an insect resistance management strategy in combination (i.e., pyramided) with other pesticidal proteins or other transgenes (i.e., an RNAi trait) including but not limited to Bt toxins, Xenorhabdus sp. or Photorhabdus sp. insecticidal proteins, other insecticidally active proteins, and the like.

[0269] Provided are methods of controlling Lepidoptera and / or Coleoptera and / or Hemiptera insect infestation(s) in a transgenic plant that promote insect resistance management, comprising expressing or co-expressing in the plant at least one of the scaffold binding peptides in conjunction with one or more insecticidal polypeptides of interest to insects in the order Lepidoptera and / or Coleoptera and / or Hemiptera, and optionally at least one of the cleavable linker peptides. Provided are methods of controlling Lepidoptera and / or Coleoptera insect infestation(s) in a transgenic plant that promote insect resistance management, comprising expressing in the plant at least two different insecticidal proteins having different modes of action.

[0270] In some embodiments the methods of controlling Lepidoptera and / or Coleoptera and / or Hemiptera insect infestation in a transgenic plant and promoting insect resistance management comprises the presentation of at least one of the scaffold binding peptides in conjunction with one or more insecticidal polypeptides of interest to insects in the order Lepidoptera and / or Coleoptera and / or Hemiptera, and optionally at least one of the cleavable linker peptides.

[0271] In some embodiments the methods of controlling Lepidoptera and / or Coleoptera insect infestation in a transgenic plant and promoting insect resistance management comprises the presentation of at least one of the Insecticidal protein B or Insecticidal protein C polypeptide insecticidal proteins to insects in the order Lepidoptera and / or Coleoptera.

[0272] In some embodiments the methods of controlling Lepidoptera and / or Coleoptera and / or Hemiptera insect infestation in a transgenic plant and promoting insect resistance management comprises the presentation of at least one of the scaffold binding peptides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more of SEQ ID NOs: 2-6, or variants or active fragment thereof, optionally one or more linker peptides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more of SEQ ID NOs: 7-27, or a variant or active fragment thereof, in conjunction with one or more insecticidal polypeptides of interest to insects in the order Lepidoptera and / or Coleoptera and / or Hemiptera, and optionally at least one of the cleavable linker peptides.

[0273] In some embodiments the methods of controlling Lepidoptera and / or Coleoptera insect infestation in a transgenic plant and promoting insect resistance management comprises the presentation of at least one of the Insecticidal protein B or Insecticidal protein C insecticidal proteins to insects in the order Lepidoptera and / or Coleoptera in combination with an insecticidal polypeptide of interest.

[0274] In some embodiments the methods of controlling Lepidoptera and / or Coleoptera and / or Hemiptera insect infestation in a transgenic plant and promoting insect resistance management comprise expression in the transgenic plant of a scaffold binding peptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more of SEQ ID NOs: 2-6, or variants or active fragment thereof, having binding affinity for an insecticidal polypeptide of interest, optionally one or more linker peptides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more of SEQ ID NOs: 7-27, or a variant or active fragment thereof, in conjunction with said insecticidal polypeptide of interest, or variants or insecticidally active fragments thereof and a Cry protein or other insecticidal protein to insects in the order Lepidoptera and / or Coleoptera and / or Hemiptera, where the insecticidal polypeptide of interest and Cry protein have different modes of action.

[0275] Also provided are methods of reducing likelihood of emergence of Lepidoptera and / orColeoptera and / or Hemiptera insect resistance to transgenic plants expressing in the plants insecticidal proteins to control the insect species, comprising expression of at least one of a scaffold binding peptide having affinity for an insecticidal polypeptide of interest in conjunction with said insecticidal polypeptide of interest to the insect species in combination with a second insecticidal protein to the insect species having different modes of action.

[0276] Also provided are methods of reducing likelihood of emergence of Lepidoptera and / or Coleoptera insect resistance to transgenic plants expressing in the plants insecticidal proteins to control the insect species, comprising expression of at least one of an Insecticidal protein B or Insecticidal protein C polypeptide insecticidal to the insect species in combination with a second insecticidal protein to the insect species having different modes of action.

[0277] Also provided are means for effective Lepidoptera and / or Coleoptera and / or Hemiptera insect resistance management of transgenic plants, comprising expressing or co-expressing at high levels in the plants two or more insecticidal proteins or other insecticidal transgenes (e.g., an RNAi trait) toxic to Lepidoptera and / or Coleoptera and / or Hemiptera insects but each exhibiting a different mode of effectuating its killing activity, wherein the two or more insecticidal proteins or other insecticidal transgenes comprise a scaffold binding peptide having affinity for an insecticidal polypeptide of interest in conjunction with said insecticidal polypeptide of interest and a Cry protein. Also provided are means for effective Lepidoptera and / or Coleoptera and / or Hemiptera insect resistance management of transgenic plants, comprising expressing or co-expressing at high levels in the plants two or more insecticidal proteins or other insecticidal transgenes (e.g., an RNAi trait) toxic to Lepidoptera and / or Coleoptera and / or Hemiptera insects but each exhibiting a different mode of effectuating its killing activity, wherein the two or more insecticidal proteins or other insecticidal transgenes comprise at least one of a scaffold binding peptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more of SEQ ID NOs: 2-6, or variants or active fragment thereof, having binding affinity for an insecticidal polypeptide of interest, optionally one or more linker peptides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more of SEQ ID NOs: 7-27, or a variant or active fragment thereof, in conjunction with said insecticidal polypeptide of interest, or variants or insecticidally active fragments thereof and a Cry protein or other insecticidally active protein.

[0278] Also provided are means for effective Lepidoptera and / or Coleoptera insect resistance management of transgenic plants, comprising co-expressing at high levels in the plants two or more insecticidal proteins toxic to Lepidoptera and / or Coleoptera insects but each exhibiting a different mode of effectuating its killing activity, wherein the two or more insecticidal proteins comprise an Insecticidal protein B or Insecticidal protein C polypeptide and a Cry protein. Also provided are means for effective Lepidoptera and / or Coleoptera insect resistance management of transgenic plants, comprising co-expressing at high levels in the plants two or more insecticidal proteins toxic to Lepidoptera and / or Coleoptera insects but each exhibiting a different mode of effectuating its killing activity, wherein the two or more insecticidal proteins comprise an Insecticidal protein B or Insecticidal protein C polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more of SEQ ID NOs: 58 or 59, or variants thereof and a Cry protein or other insecticidally active protein.

[0279] In addition, methods are provided for obtaining regulatory approval for planting or commercialization of plants expressing at least one scaffold binding peptide having affinity for an insecticidal polypeptide of interest in conjunction with said insecticidal polypeptide of interest to insects in the order Lepidoptera and / or Coleoptera and / or Hemiptera, comprising the step of referring to, submitting or relying on insect assay binding data showing that one or more of the scaffold binding peptides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more of SEQ ID NOs: 2-6, or variants or active fragment thereof, having binding affinity for an insecticidal polypeptide of interest, optionally one or more linker peptides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more of SEQ ID NOs: 7-27, or a variant or active fragment thereof, in conjunction with said insecticidal polypeptide of interest, or variant or insecticidally active fragment thereof does not compete with binding sites for Cry proteins in such insects.

[0280] In addition, methods are provided for obtaining regulatory approval for planting or commercialization of plants expressing proteins insecticidal to insects in the order Lepidoptera and / or Coleoptera, comprising the step of referring to, submitting or relying on insect assay binding data showing that the Insecticidal protein B or Insecticidal protein C polypeptide does not compete with binding sites for Cry proteins in such insects. In addition, methods are provided forobtaining regulatory approval for planting or commercialization of plants expressing proteins insecticidal to insects in the order Lepidoptera and / or Coleoptera, comprising the step of referring to, submitting or relying on insect assay binding data showing that the Insecticidal protein B or Insecticidal protein C polypeptide of SEQ ID NOs: 58 or 59, or variant thereof does not compete with binding sites for Cry proteins in such insects.Methods for Increasing Plant Yield

[0281] Methods for increasing plant yield are provided. The methods comprise providing a plant or plant cell expressing a polynucleotide encoding one or more scaffold binding peptides having affinity for an insecticidal polypeptide of interest in conjunction with said insecticidal polypeptide of interest disclosed herein and growing the plant or a seed thereof in a field infested with a pest against which the insecticidal polypeptide of interest has pesticidal activity. In some embodiments, the scaffold binding peptide having affinity for an insecticidal polypeptide of interest in conjunction with said insecticidal polypeptide of interest has pesticidal activity against a Lepidopteran, Dipteran, Hemipteran or nematode pest, and the field is infested with a Lepidopteran, Hemipteran, Dipteran or nematode pest.

[0282] As defined herein, the “yield” of the plant refers to the quality and / or quantity of biomass produced by the plant. “Biomass” as used herein refers to any measured plant product. An increase in biomass production is any improvement in the yield of the measured plant product. Increasing plant yield has several commercial applications. For example, increasing plant leaf biomass may increase the yield of leafy vegetables for human or animal consumption. Additionally, increasing leaf biomass can be used to increase production of plant-derived pharmaceutical or industrial products. An increase in yield can comprise any statistically significant increase including, but not limited to, at least a 1% increase, at least a 3% increase, at least a 5% increase, at least a 10% increase, at least a 20% increase, at least a 30%, at least a 50%, at least a 70%, at least a 100% or a greater increase in yield compared to a plant not expressing the pesticidal sequence.

[0283] In specific methods, plant yield is increased as a result of improved pest resistance of a plant expressing at least one scaffold binding peptide disclosed herein having affinity for an insecticidal polypeptide of interest in conjunction with said insecticidal polypeptide of interest.Expression of the at least one scaffold binding peptide having affinity for an insecticidal polypeptide of interest in conjunction with said insecticidal polypeptide of interest results in a reduced ability of a pest to infest or feed on the plant, thus improving plant yield.

[0284] In specific methods, plant yield is increased as a result of improved pest resistance of a plant expressing at least one Insecticidal protein B or Insecticidal protein C polypeptide disclosed herein having insecticidal activity in conjunction with another insecticidal polypeptide of interest. Expression of the at least one Insecticidal protein B or Insecticidal protein C polypeptide in conjunction with another insecticidal polypeptide of interest results in a reduced ability of a pest to infest or feed on the plant, thus improving plant yield.Methods of Processing

[0285] Further provided are methods of processing a plant, plant part or seed to obtain a food or feed product from a plant, plant part or seed comprising at least one scaffold binding peptide, chimeric fusion polypeptide, and / or at least one Insecticidal protein B or Insecticidal protein C polynucleotide. The plants, plant parts or seeds provided herein, can be processed to yield oil, protein products and / or by-products that are derivatives obtained by processing that have commercial value. Non-limiting examples include transgenic seeds comprising a nucleic acid molecule encoding one or more scaffold binding peptides which can be processed to yield soy oil, soy products and / or soy by-products.

[0286] "Processing" refers to any physical and chemical methods used to obtain any soy product and includes, but is not limited to, heat conditioning, flaking and grinding, extrusion, solvent extraction or aqueous soaking and extraction of whole or partial seeds.

[0287] Embodiments include compositions and methods for mitigating undesirable phenotypic characteristics attributable to the presence of certain transgenic proteins, including insecticidal proteins, when expressed in transgenic plants. Embodiments also include novel methods for increasing protein durability, expression levels, and efficacy in transgenic plants. Embodiments further include novel engineered peptides, polypeptides, and chimeric fusion polypeptides, and methods of producing and using the same.

[0288] One embodiment includes a polypeptide comprising a scaffold binding peptide havingbinding affinity for an insecticidal polypeptide of interest, wherein the insecticidal polypeptide of interest comprises an insecticidal protein having the domain architecture of a 3 -domain delta endotoxin, wherein the scaffold binding peptide mitigates an undesirable plant phenotype exhibited by the insecticidal polypeptide of interest.

[0289] Another embodiment includes the polypeptide comprising a scaffold binding peptide having binding affinity for an insecticidal polypeptide of interest, wherein the insecticidal polypeptide of interest comprises an insecticidal protein having the domain architecture of a 3- domain delta endotoxin, wherein the scaffold binding peptide mitigates an undesirable plant phenotype exhibited by the insecticidal polypeptide of interest of any prior embodiment, wherein the scaffold binding peptide has a binding affinity of between less than 5 nM to less than 5 pM.

[0290] Another embodiment includes the polypeptide comprising a scaffold binding peptide having binding affinity for an insecticidal polypeptide of interest, wherein the insecticidal polypeptide of interest comprises an insecticidal protein having the domain architecture of a 3- domain delta endotoxin, wherein the scaffold binding peptide mitigates an undesirable plant phenotype exhibited by the insecticidal polypeptide of interest of any prior embodiment, wherein the scaffold binding peptide comprises a peptide having at least 90% sequence identity to a peptide selected from SEQ ID NOs: 2-6.

[0291] Another embodiment includes the polypeptide comprising a scaffold binding peptide having binding affinity for an insecticidal polypeptide of interest, wherein the insecticidal polypeptide of interest comprises an insecticidal protein having the domain architecture of a 3- domain delta endotoxin, wherein the scaffold binding peptide mitigates an undesirable plant phenotype exhibited by the insecticidal polypeptide of interest of any prior embodiment, wherein the scaffold binding peptide comprises a peptide selected from SEQ ID NOs: 2-6.

[0292] Another embodiment includes a plant or plant cell comprising the polypeptide comprising a scaffold binding peptide having binding affinity for an insecticidal polypeptide of interest, wherein the insecticidal polypeptide of interest comprises an insecticidal protein having the domain architecture of a 3-domain delta endotoxin, wherein the scaffold binding peptide mitigates an undesirable plant phenotype exhibited by the insecticidal polypeptide of interest of any prior embodiment.

[0293] Another embodiment includes the polypeptide comprising a scaffold binding peptidehaving binding affinity for an insecticidal polypeptide of interest, wherein the insecticidal polypeptide of interest comprises an insecticidal protein having the domain architecture of a 3- domain delta endotoxin, wherein the scaffold binding peptide mitigates an undesirable plant phenotype exhibited by the insecticidal polypeptide of interest of any prior embodiment, wherein the scaffold binding peptide is not an antibody or an antibody fragment.

[0294] One embodiment includes a polypeptide comprising a scaffold binding peptide having binding affinity for an insecticidal polypeptide of interest, wherein the scaffold binding peptide mitigates an undesirable plant phenotype exhibited by the insecticidal polypeptide of interest.

[0295] Another embodiment includes the polypeptide comprising a scaffold binding peptide having binding affinity for an insecticidal polypeptide of interest, wherein the scaffold binding peptide mitigates an undesirable plant phenotype exhibited by the insecticidal polypeptide of interest of any prior embodiment, wherein the scaffold binding peptide has a binding affinity of between less than 5 nM to less than 5 pM.

[0296] Another embodiment includes the polypeptide comprising a scaffold binding peptide having binding affinity for an insecticidal polypeptide of interest, wherein the scaffold binding peptide mitigates an undesirable plant phenotype exhibited by the insecticidal polypeptide of interest of any prior embodiment, wherein the scaffold binding peptide comprises a peptide having at least 90% sequence identity to a peptide selected from SEQ ID NOs: 2-6.

[0297] Another embodiment includes the polypeptide comprising a scaffold binding peptide having binding affinity for an insecticidal polypeptide of interest, wherein the scaffold binding peptide mitigates an undesirable plant phenotype exhibited by the insecticidal polypeptide of interest of any prior embodiment, wherein the scaffold binding peptide comprises a peptide selected from SEQ ID NOs: 2-6.

[0298] Another embodiment includes the polypeptide comprising a scaffold binding peptide having binding affinity for an insecticidal polypeptide of interest, wherein the scaffold binding peptide mitigates an undesirable plant phenotype exhibited by the insecticidal polypeptide of interest of any prior embodiment, wherein the insecticidal polypeptide of interest comprises an insecticidal protein having the domain architecture of one or more of domain I, domain II, or domain III of a 3-domain delta endotoxin.

[0299] Another embodiment includes a plant or plant cell comprising the polypeptide comprisinga scaffold binding peptide having binding affinity for an insecticidal polypeptide of interest, wherein the scaffold binding peptide mitigates an undesirable plant phenotype exhibited by the insecticidal polypeptide of interest of any prior embodiment.

[0300] Another embodiment includes the polypeptide comprising a scaffold binding peptide having binding affinity for an insecticidal polypeptide of interest, wherein the scaffold binding peptide mitigates an undesirable plant phenotype exhibited by the insecticidal polypeptide of interest of any prior embodiment, wherein the scaffold binding peptide is not an antibody or an antibody fragment.

[0301] One embodiment includes a chimeric fusion polypeptide comprising a scaffold binding peptide and an insecticidal polypeptide of interest, wherein the insecticidal polypeptide of interest comprises an insecticidal protein having the domain architecture of a 3-domain delta endotoxin, wherein the scaffold binding peptide and the insecticidal polypeptide of interest are linked by at least one cleavable linker sequence.

[0302] Another embodiment includes the chimeric fusion polypeptide comprising a scaffold binding peptide and an insecticidal polypeptide of interest, wherein the insecticidal polypeptide of interest comprises an insecticidal protein having the domain architecture of a 3-domain delta endotoxin, wherein the scaffold binding peptide and the insecticidal polypeptide of interest are linked by at least one cleavable linker sequence of any prior embodiment, wherein the insecticidal polypeptide exhibits reduced undesirable phenotypic characteristics in a plant when compared to the insecticidal polypeptide without the scaffold binding peptide.

[0303] Another embodiment includes the chimeric fusion polypeptide comprising a scaffold binding peptide and an insecticidal polypeptide of interest, wherein the insecticidal polypeptide of interest comprises an insecticidal protein having the domain architecture of a 3-domain delta endotoxin, wherein the scaffold binding peptide and the insecticidal polypeptide of interest are linked by at least one cleavable linker sequence of any prior embodiment, wherein the insecticidal polypeptide exhibits increased expression in a plant when compared to the insecticidal polypeptide without the scaffold binding peptide.

[0304] Another embodiment includes the chimeric fusion polypeptide comprising a scaffold binding peptide and an insecticidal polypeptide of interest, wherein the insecticidal polypeptide of interest comprises an insecticidal protein having the domain architecture of a 3-domain deltaendotoxin, and wherein the scaffold binding peptide and the insecticidal polypeptide of interest are linked by at least one cleavable linker sequence of any prior embodiment, wherein the scaffold binding peptide has at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 2-6.

[0305] Another embodiment includes the chimeric fusion polypeptide comprising a scaffold binding peptide and an insecticidal polypeptide of interest, wherein the insecticidal polypeptide of interest comprises an insecticidal protein having the domain architecture of a 3-domain delta endotoxin, and wherein the scaffold binding peptide and the insecticidal polypeptide of interest are linked by at least one cleavable linker sequence of any prior embodiment, wherein the cleavable linker sequence has a length of between 25 to 70 amino acids, a strong core helical bias, and optionally a low bias for the amino acids glutamine, alanine, and proline.

[0306] Another embodiment includes the chimeric fusion polypeptide comprising a scaffold binding peptide and an insecticidal polypeptide of interest, wherein the insecticidal polypeptide of interest comprises an insecticidal protein having the domain architecture of a 3-domain delta endotoxin, and wherein the scaffold binding peptide and the insecticidal polypeptide of interest are linked by at least one cleavable linker sequence of any prior embodiment, wherein the cleavable linker sequence comprises a protease cleavage site selected from a serine protease site, a trypsin site, a chymotrypsin site, a cathepsin site, an alkaline protease site, and an acidic proteinase site.

[0307] Another embodiment includes the chimeric fusion polypeptide comprising a scaffold binding peptide and an insecticidal polypeptide of interest, wherein the insecticidal polypeptide of interest comprises an insecticidal protein having the domain architecture of a 3-domain delta endotoxin, and wherein the scaffold binding peptide and the insecticidal polypeptide of interest are linked by at least one cleavable linker sequence, wherein the cleavable linker sequence comprises a protease cleavage site selected from a serine protease site, a trypsin site, a chymotrypsin site, a cathepsin site, an alkaline protease site, and an acidic proteinase site of any prior embodiment, wherein the cleavable linker sequence comprises two, three, or more protease cleavage sites.

[0308] Another embodiment includes the chimeric fusion polypeptide comprising a scaffold binding peptide and an insecticidal polypeptide of interest, wherein the insecticidal polypeptide of interest comprises an insecticidal protein having the domain architecture of a 3-domain deltaendotoxin, and wherein the scaffold binding peptide and the insecticidal polypeptide of interest are linked by at least one cleavable linker sequence of any prior embodiment, wherein the cleavable linker sequence has at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 7-27.

[0309] Another embodiment includes the chimeric fusion polypeptide comprising a scaffold binding peptide and an insecticidal polypeptide of interest, wherein the insecticidal polypeptide of interest comprises an insecticidal protein having the domain architecture of a 3-domain delta endotoxin, and wherein the scaffold binding peptide and the insecticidal polypeptide of interest are linked by at least one cleavable linker sequence of any prior embodiment, wherein the scaffold binding peptide is linked to the insecticidal polypeptide at the N-terminus of the insecticidal polypeptide.

[0310] Another embodiment includes the chimeric fusion polypeptide comprising a scaffold binding peptide and an insecticidal polypeptide of interest, wherein the insecticidal polypeptide of interest comprises an insecticidal protein having the domain architecture of a 3-domain delta endotoxin, and wherein the scaffold binding peptide and the insecticidal polypeptide of interest are linked by at least one cleavable linker sequence of any prior embodiment, wherein the scaffold binding peptide is linked to the insecticidal polypeptide at the C-terminus of the insecticidal polypeptide.

[0311] Another embodiment includes a polynucleotide encoding the chimeric fusion polypeptide comprising a scaffold binding peptide and an insecticidal polypeptide of interest, wherein the insecticidal polypeptide of interest comprises an insecticidal protein having the domain architecture of a 3-domain delta endotoxin, and wherein the scaffold binding peptide and the insecticidal polypeptide of interest are linked by at least one cleavable linker sequence of any prior embodiment.

[0312] Another embodiment includes a DNA construct comprising the polynucleotide encoding the chimeric fusion polypeptide comprising a scaffold binding peptide and an insecticidal polypeptide of interest, wherein the insecticidal polypeptide of interest comprises an insecticidal protein having the domain architecture of a 3-domain delta endotoxin, wherein the scaffold binding peptide and the insecticidal polypeptide of interest are linked by at least one cleavable linker sequence of any prior embodiment.

[0313] Another embodiment includes a plant or plant cell comprising the chimeric fusion polypeptide comprising a scaffold binding peptide and an insecticidal polypeptide of interest, wherein the insecticidal polypeptide of interest comprises an insecticidal protein having the domain architecture of a 3 -domain delta endotoxin, and wherein the scaffold binding peptide and the insecticidal polypeptide of interest are linked by at least one cleavable linker sequence of any prior embodiment.

[0314] Another embodiment includes the plant or plant cell comprising the chimeric fusion polypeptide comprising a scaffold binding peptide and an insecticidal polypeptide of interest, wherein the insecticidal polypeptide of interest comprises an insecticidal protein having the domain architecture of a 3 -domain delta endotoxin, wherein the scaffold binding peptide and the insecticidal polypeptide of interest are linked by at least one cleavable linker sequence of any prior embodiment, wherein the plant or plant cell further comprises one or more additional polypeptides for insect resistance.

[0315] Another embodiment includes a composition comprising the chimeric fusion polypeptide comprising a scaffold binding peptide and an insecticidal polypeptide of interest, wherein the insecticidal polypeptide of interest comprises an insecticidal protein having the domain architecture of a 3 -domain delta endotoxin, and wherein the scaffold binding peptide and the insecticidal polypeptide of interest are linked by at least one cleavable linker sequence of any prior embodiment.

[0316] One embodiment includes a chimeric fusion polypeptide comprising a scaffold binding peptide and an insecticidal polypeptide of interest, wherein the scaffold binding peptide and the insecticidal polypeptide of interest are linked by at least one cleavable linker sequence.

[0317] Another embodiment includes the chimeric fusion polypeptide comprising a scaffold binding peptide and an insecticidal polypeptide of interest, wherein the scaffold binding peptide and the insecticidal polypeptide of interest are linked by at least one cleavable linker sequence of any prior embodiment, wherein the insecticidal polypeptide exhibits reduced undesirable phenotypic characteristics in a plant when compared to the insecticidal polypeptide without the scaffold binding peptide.

[0318] Another embodiment includes the chimeric fusion polypeptide comprising a scaffold binding peptide and an insecticidal polypeptide of interest, wherein the scaffold binding peptideI l land the insecticidal polypeptide of interest are linked by at least one cleavable linker sequence of any prior embodiment, wherein the insecticidal polypeptide exhibits increased expression in a plant when compared to the insecticidal polypeptide without the scaffold binding peptide.

[0319] Another embodiment includes the chimeric fusion polypeptide comprising a scaffold binding peptide and an insecticidal polypeptide of interest, wherein the scaffold binding peptide and the insecticidal polypeptide of interest are linked by at least one cleavable linker sequence of any prior embodiment, wherein the insecticidal polypeptide of interest comprises an insecticidal protein having the domain architecture of one or more of domain I, domain II, or domain III of a 3 -domain delta endotoxin.

[0320] Another embodiment includes the chimeric fusion polypeptide comprising a scaffold binding peptide and an insecticidal polypeptide of interest, wherein the scaffold binding peptide and the insecticidal polypeptide of interest are linked by at least one cleavable linker sequence, wherein the insecticidal polypeptide of interest comprises an insecticidal protein having the domain architecture of one or more of domain I, domain II, or domain III of a 3 -domain delta endotoxin of any prior embodiment, wherein the scaffold binding peptide comprises a binding site that binds to one or more residues in domain I, domain II, or domain III of the insecticidal protein having the domain architecture of one or more of domain I, domain II, or domain III of a 3 -domain delta endotoxin.

[0321] Another embodiment includes the chimeric fusion polypeptide comprising a scaffold binding peptide and an insecticidal polypeptide of interest, wherein the scaffold binding peptide and the insecticidal polypeptide of interest are linked by at least one cleavable linker sequence, wherein the insecticidal polypeptide of interest comprises an insecticidal protein having the domain architecture of one or more of domain I, domain II, or domain III of a 3 -domain delta endotoxin of any prior embodiment, wherein the scaffold binding peptide comprises a binding site that binds to one or more residues in helix a-4, helix a-5, helix a-6, helix a-7, or helix a-8 of an insecticidal protein having the domain architecture of one or more of domain I or domain II of a 3-domain delta endotoxin.

[0322] Another embodiment includes the chimeric fusion polypeptide comprising a scaffold binding peptide and an insecticidal polypeptide of interest, wherein the scaffold binding peptide and the insecticidal polypeptide of interest are linked by at least one cleavable linker sequence ofany prior embodiment, wherein the scaffold binding peptide has at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 2-6.

[0323] Another embodiment includes the chimeric fusion polypeptide comprising a scaffold binding peptide and an insecticidal polypeptide of interest, wherein the scaffold binding peptide and the insecticidal polypeptide of interest are linked by at least one cleavable linker sequence of any prior embodiment, wherein the cleavable linker sequence has a length of between 25 to 70 amino acids, a strong core helical bias, and optionally a low bias for the amino acids glutamine, alanine, and proline.

[0324] Another embodiment includes the chimeric fusion polypeptide comprising a scaffold binding peptide and an insecticidal polypeptide of interest, wherein the scaffold binding peptide and the insecticidal polypeptide of interest are linked by at least one cleavable linker sequence of any prior embodiment, wherein the cleavable linker sequence comprises a protease cleavage site selected from a serine protease site, a trypsin site, a chymotrypsin site, a cathepsin site, an alkaline protease site, and an acidic proteinase site.

[0325] Another embodiment includes the chimeric fusion polypeptide comprising a scaffold binding peptide and an insecticidal polypeptide of interest, wherein the scaffold binding peptide and the insecticidal polypeptide of interest are linked by at least one cleavable linker sequence, wherein the cleavable linker sequence comprises a protease cleavage site selected from a serine protease site, a trypsin site, a chymotrypsin site, a cathepsin site, an alkaline proteas...

Claims

CLAIMSTHAT WHICH IS CLAIMED IS:

1. A polypeptide comprising a scaffold binding peptide having binding affinity for an insecticidal polypeptide of interest, wherein the insecticidal polypeptide of interest comprises an insecticidal protein having the domain architecture of a 3-domain delta endotoxin, and wherein the scaffold binding peptide mitigates an undesirable plant phenotype exhibited by the insecticidal polypeptide of interest.

2. The polypeptide of claim 1, wherein the scaffold binding peptide has a binding affinity of between less than 5 nM to less than 5 pM.

3. The polypeptide of claim 1, wherein the scaffold binding peptide comprises a peptide having at least 90% sequence identity to a peptide selected from SEQ ID NOs: 2-6.

4. The polypeptide of claim 3, wherein the scaffold binding peptide comprises a peptide selected from SEQ ID NOs: 2-6.

5. A polypeptide comprising a scaffold binding peptide having binding affinity for an insecticidal polypeptide of interest, wherein the scaffold binding peptide mitigates an undesirable plant phenotype exhibited by the insecticidal polypeptide of interest.

6. The polypeptide of claim 5, wherein the scaffold binding peptide has a binding affinity of between less than 5 nM to less than 5 pM.

7. The polypeptide of claim 5, wherein the scaffold binding peptide comprises a peptide having at least 90% sequence identity to a peptide selected from SEQ ID NOs: 2-6.

8. The polypeptide of claim 7, wherein the scaffold binding peptide comprises a peptide selected from SEQ ID NOs: 2-6.

9. The polypeptide of claim 5, wherein the insecticidal polypeptide of interest comprises an insecticidal protein having the domain architecture of one or more of domain I, domain II, or domain III of a 3-domain delta endotoxin.

10. A plant or plant cell comprising the polypeptide of claim 1 or 5.

11. The polypeptide of claim 1 or 5, wherein the scaffold binding peptide is not an antibody or an antibody fragment.

12. A chimeric fusion polypeptide comprising a scaffold binding peptide and an insecticidal polypeptide of interest, wherein the insecticidal polypeptide of interest comprises an insecticidal protein having the domain architecture of a 3-domain delta endotoxin, and wherein the scaffold binding peptide and the insecticidal polypeptide of interest are linked by at least one cleavable linker sequence.

13. The chimeric fusion polypeptide of claim 12, wherein the insecticidal polypeptide exhibits reduced undesirable phenotypic characteristics in a plant when compared to the insecticidal polypeptide without the scaffold binding peptide.

14. The chimeric fusion polypeptide of claim 12, wherein the insecticidal polypeptide exhibits increased expression in a plant when compared to the insecticidal polypeptide without the scaffold binding peptide.

15. A chimeric fusion polypeptide comprising a scaffold binding peptide and an insecticidal polypeptide of interest, wherein the scaffold binding peptide and the insecticidal polypeptide of interest are linked by at least one cleavable linker sequence.

16. The chimeric fusion polypeptide of claim 15, wherein the insecticidal polypeptide exhibits reduced undesirable phenotypic characteristics in a plant when compared to the insecticidal polypeptide without the scaffold binding peptide.

17. The chimeric fusion polypeptide of claim 15, wherein the insecticidal polypeptide exhibits increased expression in a plant when compared to the insecticidal polypeptide without the scaffold binding peptide.

18. The chimeric fusion polypeptide of claim 15, wherein the insecticidal polypeptide of interest comprises an insecticidal protein having the domain architecture of one or more of domain I, domain II, or domain III of a 3-domain delta endotoxin.

19. The chimeric fusion polypeptide of claim 18, wherein the scaffold binding peptide comprises a binding site that binds to one or more residues in domain I , domain II, or domain III of the insecticidal protein having the domain architecture of one or more of domain I, domain II, or domain III of a 3 -domain delta endotoxin.

20. The chimeric fusion polypeptide of claim 18, wherein the scaffold binding peptide comprises a binding site that binds to one or more residues in helix a-4, helix a-5, helix a-6, helix a-7, or helix a-8 of an insecticidal protein having the domain architecture of one or more of domain I or domain II of a 3 -domain delta endotoxin.

21. The chimeric fusion polypeptide of claim 12 or 15, wherein the scaffold binding peptide has at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 2-6.

22. The chimeric fusion polypeptide of claim 12 or 15, wherein the cleavable linker sequence has a length of between 25 to 70 amino acids, a strong core helical bias, and optionally a low bias for the amino acids glutamine, alanine, and proline.

23. The chimeric fusion polypeptide of claim 12 or 15, wherein the cleavable linker sequence comprises a protease cleavage site selected from a serine protease site, a trypsin site, a chymotrypsin site, a cathepsin site, an alkaline protease site, and an acidic proteinase site.

24. The chimeric fusion polypeptide of claim 23, wherein the cleavable linker sequence comprises two, three, or more protease cleavage sites.

25. The chimeric fusion polypeptide of claim 12 or 15, wherein the cleavable linker sequence has at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 7-27.

26. The chimeric fusion polypeptide of claim 12 or 15, wherein the scaffold binding peptide is linked to the insecticidal polypeptide at the N-terminus of the insecticidal polypeptide.

27. The chimeric fusion polypeptide of claim 12 or 15, wherein the scaffold binding peptide is linked to the insecticidal polypeptide at the C-terminus of the insecticidal polypeptide.

28. A polynucleotide encoding the chimeric fusion polypeptide of claim 12 or 15.

29. A DNA construct comprising the polynucleotide of claim 28.

30. A plant or plant cell comprising the chimeric fusion polypeptide of claim 12 or 15.

31. The plant or plant cell of claim 30, wherein the plant or plant cell further comprises one or more additional polypeptides for insect resistance.

32. A composition comprising the chimeric fusion polypeptide of claim 12 or 15.

33. A stacked insecticidal polypeptide composition comprising two or more distinct insecticidal polypeptides of interest, each insecticidal polypeptide comprising a scaffold binding peptide and linker.

34. The stacked insecticidal polypeptide composition of claim 33, wherein the scaffold binding peptides for the two or more distinct insecticidal polypeptides of interest are the same.

35. A method for reducing undesirable phenotypic characteristics of an insecticidal polypeptide in a plant, the method comprising co-expressing in a plant a polypeptide comprising an scaffold binding peptide and a polypeptide comprising an insecticidal polypeptide of interest, wherein the insecticidal polypeptide of interest comprises an insecticidal protein having the domain architecture of a 3 -domain delta endotoxin, and wherein co-expression of the scaffold binding peptide with the insecticidal polypeptide of interest reduces undesirable phenotypic characteristics in the plant compared to the insecticidal polypeptide without the scaffold binding peptide.

36. A method for reducing undesirable phenotypic characteristics of an insecticidal polypeptide in a plant, the method comprising expressing in the plant a chimeric fusion polypeptide comprising a scaffold binding peptide and an insecticidal polypeptide of interest, wherein the insecticidal polypeptide of interest comprises an insecticidal protein having the domain architecture of a 3 -domain delta endotoxin, wherein the scaffold binding peptide and the insecticidal polypeptide of interest are linked by at least one cleavable linker sequence, and wherein expression of the chimeric fusion polypeptide reduces undesirable phenotypiccharacteristics of the insecticidal polypeptide of interest compared to an insecticidal polypeptide lacking the scaffold binding peptide.

37. A method for reducing undesirable phenotypic characteristics of an insecticidal polypeptide in a plant, the method comprising co-expressing in a plant a polypeptide comprising a scaffold binding peptide and a polypeptide comprising an insecticidal polypeptide of interest, wherein co-expression of the scaffold binding peptide with the insecticidal polypeptide of interest reduces undesirable phenotypic characteristics in the plant compared to the insecticidal polypeptide without the scaffold binding peptide.

38. A method for reducing undesirable phenotypic characteristics of an insecticidal polypeptide in a plant, the method comprising expressing in the plant a chimeric fusion polypeptide comprising a scaffold binding peptide and an insecticidal polypeptide of interest, wherein the scaffold binding peptide and the insecticidal polypeptide of interest are linked by at least one cleavable linker sequence, and wherein expression of the chimeric fusion polypeptide reduces undesirable phenotypic characteristics of the insecticidal polypeptide of interest compared to an insecticidal polypeptide lacking the scaffold binding peptide.

39. The method of any one of claims 35, 36, 37, or 38, wherein the undesirable phenotypic characteristic is phytotoxicity.

40. A method for increasing expression of an insecticidal polypeptide in a plant, the method comprising expressing in a plant a chimeric fusion polypeptide comprising a scaffold binding peptide and an insecticidal polypeptide of interest, wherein the scaffold binding peptide and the insecticidal polypeptide of interest are linked by at least one cleavable linker sequence, wherein the level of expression of the insecticidal polypeptide of interest is increased compared to an insecticidal polypeptide lacking the scaffold binding peptide.

41. A method of increasing efficacy of an insecticidal polypeptide in a plant, the method comprising expressing in a plant a chimeric fusion polypeptide comprising a scaffold binding peptide and an insecticidal polypeptide of interest, wherein the scaffold binding peptide and the insecticidal polypeptide of interest are linked by at least one cleavable linker sequence, wherein the level of expression of the insecticidal polypeptide of interest isincreased compared to an insecticidal polypeptide lacking the scaffold binding peptide, and wherein the increased level of the insecticidal polypeptide of interest results in increased efficacy against a target insect pest.

42. A method of increasing durability of an insecticidal polypeptide, the method comprising expressing in a plant a chimeric fusion polypeptide comprising a scaffold binding peptide and an insecticidal polypeptide of interest, wherein the scaffold binding peptide and the insecticidal polypeptide of interest are linked by at least one cleavable linker sequence, wherein the level of expression of the insecticidal polypeptide of interest is increased compared to an insecticidal polypeptide lacking the scaffold binding peptide, and wherein the increased level of the insecticidal polypeptide of interest results in increased durability of the insecticidal polypeptide against a target insect pest.

43. The method of any of claims 37, 38, 39, 40, 41, or 42 wherein the insecticidal protein has the domain architecture of one or more of domain I, domain II, or domain III of a 3 -domain delta endotoxin.

44. The method of any one of claims 35, 36, 37, 38, 39, 40, 41, 42, or 43, wherein the scaffold binding peptide has at least 90% sequence identity to a sequence selected from SEQ ID NOs: 2-6.

45. The method of any one of claims 36, 38, 40, 41, 42, 43, or 44, wherein the cleavable linker sequence has a length of between 25 to 70 amino acids, a strong core helical bias, and optionally a low bias for the amino acids glutamine, alanine, and proline.

46. The method of any one of claims 36, 38, 40, 41, 42, 43, 44, or 45, wherein the cleavable linker sequence comprises a protease cleavage site selected from a serine protease site, a trypsin site, a chymotrypsin site, a cathepsin site, an alkaline protease site, and an acidic proteinase site.

47. The method of any one of claims 36, 38, 40, 41, 42, 43, 44, 45, or 46, wherein the cleavable linker sequence has at least 90% sequence identity to a sequence selected from SEQ IDNOs: 7-27.

48. An engineered scaffold protein having two peptide loops that can be mutagenized to function as Complementarity-Determining Regions that bind to an insecticidal protein comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1.

49. The engineered scaffold protein of claim 48, wherein the 80% sequence identity is directed to the portion of the protein that excludes the two peptide loops.

50. A method for generating a scaffold binding peptide having binding affinity for an insecticidal polypeptide of interest comprising: a. Generating scaffold binding peptide libraries with random loop diversity using oligonucleotide-directed mutagenesis; b. Biopanning said libraries against the insecticidal protein of interest; and c. Selecting binding scaffold binding partner peptides having binding affinity for the insecticidal polypeptide of interest using phage display of scaffold binding partner libraries.

51. A method for determining phytotoxicity of an insecticidal protein of interest comprising: a. Transfecting maize leaf mesophyll protoplasts with a plasmid DNA construct containing two expression cassettes, wherein the first expression cassette comprises a reporter gene and the second expression cassette comprises an insecticidal gene of interest driven by a different promoter than the first expression cassette; b. Counting the viable transfected cells over a time course from an initial time point to a final time point; and c. Calculating the percent reduction in cell count from the initial time point to the final time point where a larger percent reduction in cell count is indicative of phytotoxicity of the insecticidal protein.

52. The method of claim 51, wherein the reporter gene encodes a fluorescent protein and fluorescence is used to identify viable cells.

53. A computer-implemented method for use in generating linkers for fusion of insecticidalpolypeptides and scaffold binding peptides, wherein the linkers form rigid helical structures, the computer-implemented method comprising using a sequence space diffusion model that generates protein sequences and structures simultaneously to generate linkers having a length of between 25 to 70 amino acids, a strong core helical bias, and optionally a low bias for amino acids glutamine, alanine, and proline.

54. An insecticidal polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 58, or a fragment thereof having insecticidal activity.

55. The insecticidal polypeptide of claim 54, wherein the amino acid sequence has at least 95% sequence identity to SEQ ID NO: 58.

56. The insecticidal polypeptide of claim 54 or 55, wherein the polypeptide has insecticidal activity against a Lepidopteran, Coleopteran, or Hemipteran agricultural pest.

57. A chimeric insecticidal protein comprising portions of at least two different insecticidal polypeptides of claim 54 or 55.

58. A fusion protein comprising an insecticidal polypeptide of claim 54 or 55.

59. An agricultural composition comprising at least one insecticidal polypeptide of claim 54 or 55.

60. A transgenic plant or plant cell comprising the insecticidal polypeptide of claim 54 or 55.

61. A method of inhibiting growth or killing an insect pest or pest population, comprising contacting the insect pest with the insecticidal polypeptide of claim 54 or 55.

62. A method of controlling insect pest damage to plants comprising providing the insecticidal polypeptide of claim 54 or 55 to an insect pest or pest population for ingestion, wherein said insecticidal polypeptide is produced by a transgenic plant and is present in at least one of said plants.

63. A method for controlling pest infestation comprising providing in the diet of the pest the transgenic plant of claim 60, or a part thereof.

64. A method for improving the yield of a crop comprising growing the transgenic plant of claim 60, wherein the yield of the crop is increased in the presence of the insect pest relative to the crop not comprising said transgenic plant.

65. The method of claim of any one of claims 61-64, wherein the insect pest or pest population is resistant to at least one Cry insecticidal protein.

66. The method of any one of claims 62-65, wherein the transgenic plant is selected from corn, soybean, wheat, rice, sorghum, sunflower, canola, barley, sugarcane, potatoes, tomatoes, cotton, rape seed, peanut, and alfalfa.

67. The method of any one of claims 61-66, wherein the insect pest or insect pest population is an agriculturally important species in the Order Lepidoptera.

68. The method of claim 67, wherein the insect pest or insect pest population is selected from corn earworm, European corn borer, fall armyworm, soybean looper, velvet bean caterpillar, and diamondback moth.