Compositions and methods for screening insecticidal proteins

Abstract

Provided herein are methods for screening and identifying pesticidal proteins, along with recombinant nucleic acids, DNA constructs, vectors, fusion proteins and spores useful in the provided methods. The provided methods allow for efficient high-throughput screening of candidate pesticidal proteins, including naturally occurring and engineered synthetic proteins.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 299,567, filed January 14, 2022, the entire contents of which are incorporated by reference for all purposes.

[0002] The present disclosure relates to the field of plant biotechnology. In particular, the present invention relates to methods for screening insecticidal proteins for desired traits.

[0003] Reference to sequence listings submitted as XML files via EFS-WEB This application is accompanied by a Sequence Listing entitled 109098-1360060.xml, created on January 13, 2023, which is approximately 1,03,846 bytes in size. This Sequence Listing is incorporated herein by reference in its entirety. This Sequence Listing has been submitted herewith via EFS-Web and complies with 37 C.FR § 1.824(a)(2)-(6) and (b). [Background technology]

[0004] Insecticidal proteins may include naturally occurring and synthetic proteins. Candidate insecticidal proteins, particularly those of Bacillus thuringiensis (Bt), may be engineered (i.e., artificially modified) by DNA shuffling, block exchange, site-directed, saturation and random mutagenesis. These engineering techniques often generate large numbers of variants that collectively are referred to as libraries. Such libraries may be screened for desired traits or desired traits. This process requires high-throughput protein production and screening of diverse proteins. Such high-throughput screening is time-consuming and expensive. This process typically involves: 1) using protein engineering techniques to generate a library of diverse insecticidal proteins (i.e., naturally occurring and / or engineered proteins); 2) cloning the library genes in a host organism, such as Escherichia coli or Bacillus species, for protein production; 3) individually picking transformants of the host organism containing the library in multi-well plates to generate seed cultures; 4) expressing the library proteins in multi-well plates and isolating them individually; 5) analyzing the isolated proteins for purity and concentration and normalizing the proteins for use (i.e., by adjusting the protein concentration); 6) assaying the insecticidal activity and other traits of the isolated and analyzed proteins in live insects to identify proteins with desired traits; and 7) repeating the above steps to identify additional proteins with desired traits.

[0005] The drawback of the current screening process is that the experimental throughput is limited to a maximum of several thousand samples, without significant investment in manpower and infrastructure. The rate-limiting factor is that the process involves the use of live insect bioassays and the amount of isolated protein required for the bioassay. Therefore, an increase in throughput over the conventional methods described above is highly desirable in the pursuit of finding new receptor specificities. Summary of the Invention

[0006] This summary is a high-level summary of various aspects of the disclosure, introducing some of the concepts described and illustrated in the specification and the accompanying drawings. This summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used alone to determine the scope of the claimed subject matter. The subject matter should be understood by reference to the entire specification, any or all figures, and appropriate portions of each claim. Some embodiments of the disclosure are described below.

[0007] The present disclosure is based in part on the inventors' discovery of a novel method of screening libraries of putative toxin proteins and / or protein variants using cell (particle) sorting techniques to identify insect-active toxin proteins. In some embodiments, as described further below, the insect-active toxins are individually tethered (i.e., tagged) onto carrier particles (e.g., spores) that contain the genetic information for the tethered protein.

[0008] In one aspect, the present specification provides a recombinant nucleic acid that encodes a fusion protein.In some embodiments, the recombinant nucleic acid comprises:a) a first nucleic acid sequence that encodes a spore coat polypeptide;b) a second nucleic acid sequence that encodes a linker;c) a third nucleic acid sequence that encodes a putative insecticidal polypeptide.

[0009] In some embodiments of the recombinant nucleic acids provided herein, the spore coat polypeptide is derived from a Bacillus bacterium. In some embodiments, the spore coat polypeptide is CotC, CotG, CotB, CotA, CotD, CotE, CotX, CotY, or CotZ. In some embodiments, the spore coat polypeptide is CotC or CotG.

[0010] In some embodiments of the recombinant nucleic acids provided herein, the putative insecticidal polypeptide is a Bacillus thuringiensis crystal (Bt Cry) protein. In some embodiments, the Bt Cry protein comprises at least one modification compared to a wild-type protein. In some embodiments, the modification is the result of DNA shuffling, block swapping, site-directed mutagenesis, saturation mutagenesis, random mutagenesis, or a combination thereof. In some embodiments, the Bt Cry protein modification confers resistance to serine protease digestion.

[0011] In some embodiments, the putative insecticidal polypeptide interacts with a cell surface protein of an insect digestive epithelial cell. In some embodiments, the cell surface protein is an ATP-binding cassette (ABC) transporter protein, a cadherin protein, an aminopeptidase N protein, or an alkaline phosphatase protein. In some aspects, the cell surface protein is an ABC transporter protein. In some embodiments, the ABC transporter protein is PxABCC2, Bm-ABCC2, Sf-ABCC2, Sf-ABCC3, Dv-ABCB1, Bm-ABCB1, Sf-ABCB1, Bm-ABCA2, or Tc-ABCC4. In some embodiments, the ABC transporter protein is PxABCC2, Bm-ABCC2, Sf-ABCC2, or Sf-ABCC3.

[0012] In some embodiments of the recombinant nucleic acids provided herein, the spore coat polypeptide and the putative insecticidal polypeptide are structurally isolated by a linker. In some embodiments, the linker comprises at least one amino acid. In some embodiments, the linker is a polypeptide comprising at least two amino acids. In some embodiments, the linker is structurally flexible. In some embodiments, the linker is resistant to protease digestion. In some embodiments, the linker is a polypeptide comprising an amino acid sequence having at least 80% identity to any of SEQ ID NOs: 20, 21, or 38-61.

[0013] Also provided is a DNA construct comprising any of the recombinant nucleic acids described herein.Also provided is a vector comprising any of the recombinant nucleic acids or DNA constructs described herein.

[0014] Also provided herein are fusion proteins encoded by any of the recombinant nucleic acids, DNA constructs, or vectors described herein.

[0015] Also provided herein is a spore comprising any of the recombinant nucleic acids, DNA constructs, vectors, or fusion proteins described herein. In some embodiments, the spore comprises the fusion protein, and the fusion protein is displayed on the surface of the spore. In some embodiments, the spore is a bacterial spore or a fungal spore.

[0016] Also provided herein are compositions comprising a plurality of any of the spores described herein. In some embodiments, each of the plurality of spores comprises a recombinant nucleic acid encoding a different putative insecticidal polypeptide.

[0017] In another aspect, a method for identifying an insecticidal protein is provided herein. In some embodiments, the method comprises a) contacting any of the fusion proteins or spores described herein with an insecticidal protein receptor, and b) detecting an interaction between the fusion protein or spore and the insecticidal protein receptor. In some embodiments, the interaction indicates that the putative insecticidal protein of the fusion protein or spore is an insecticidal protein. In some embodiments, the insecticidal protein receptor is a purified insecticidal protein receptor protein. In some embodiments, the insecticidal protein receptor is part of an SMA lipid particle. In some embodiments, the insecticidal protein receptor is expressed on a cell. In some embodiments, the cell is an insect cell. In some embodiments, the insecticidal protein receptor is an ABC transporter protein.

[0018] In some embodiments of the methods provided herein, detecting the interaction between the fusion protein or spore and the insecticidal protein receptor comprises performing immunomagnetic separation, flow cytometry, cytotoxicity assay, sequencing, or a combination thereof. In some embodiments, detecting the interaction between the fusion protein or spore and the insecticidal protein receptor protein further comprises isolating a recombinant nucleic acid encoding a fusion protein that interacts with the insecticidal protein receptor protein, and performing sequencing to determine the identity of the insecticidal protein.

[0019] This disclosure includes the following figures. The drawings are intended to illustrate certain embodiments and / or features of the compositions and methods, and to supplement any descriptions of the compositions and methods. The drawings do not limit the scope of the compositions and methods unless the written description expressly indicates such. [Brief description of the drawings]

[0020] [Figure 1]1 shows a method of amplifying cotC or cotG (Bacillus subtilis coat protein genes) by PCR using their own promoters, according to an embodiment of the present disclosure, which results in linking the cot gene to the Bacillus thuringiensis cry gene via a linker to form a Cot-Cry fusion protein with a structurally flexible linker. [Diagram 2] 2 shows an exemplary method for constructing a spore display vector carrying the Bacillus subtilis cotC or cotG genes and the Bacillus thuringiensis cry1Aa gene according to an embodiment of the present disclosure. Two Bacillus-E. coli shuttle vectors were used: pSB634, a fusion of Bacillus cereus pBC16.1 with E. coli pBluescript KS and pHY300PLK, a fusion of Streptococcus faecalis pAMα1 with E. coli pACYC177. [Diagram 3] 3 shows that according to an embodiment of the present disclosure, the Bacillus subtilis cotC or cotG gene was fused to the Bacillus thuringiensis cry1Aa or cry1Fa gene using a linker and terminator derived from the Bacillus thuringiensis cry1Ac gene. The Arg 27 / 28 (R27 / 28) amino acid residues of the Cry1 protein were mutated to Leu (L) to prevent serine protease digestion. [Figure 4]FIG. 4 illustrates a method for detecting Bacillus thuringiensis Cry1 protein displayed on Bacillus subtilis spores using an anti-Cry1 antibody (Ab), according to an embodiment of the disclosure. As shown in the schematic (top panel), the antibody labeled with Alexa Fluor 488 fluorescent dye results in spore fluorescence upon binding. The bottom panel is a micrograph of Spore-Cry1Fa (a spore displaying the Cry1Fa protein) that becomes fluorescent upon binding to the anti-Cry1 antibody (bottom right panel). [Diagram 5] 5 shows a feasibility study demonstrating that Bacillus spores displaying Bacillus thuringiensis Cry proteins can be sorted by flow cytometry, according to an embodiment of the present disclosure. Those spores displaying Cry proteins were fluorescentized with a fluorescent anti-Cry antibody (e.g., as shown in FIG. 4). Two successive sorting experiments were performed: the left panel is the first sort, and the spores in the boxed region were used to obtain the right panel. [Figure 6] FIG. 6 shows micrographs of Sf9 cells (Spodoptera frugiperda cell line) expressing the ABCC2 gene of Spodoptera frugiperda (top panel) (Sf9::Sf-ABCC2) and another cell not expressing the ABCC2 gene (bottom panel) according to an embodiment of the present disclosure. These Sf9 cells were mixed with Spore-Cry1Fa Bacillus subtilis spores displaying the Cry1Fa protein and allowed to bind to the spores. The bound Spore-Cry1Fa spores were visualized as bright spots with Alexa-labeled anti-Cry1Fa antibody by fluorescence microscopy (top right panel). The host Sf9 cells also expressed the GFP gene and were therefore visualized by fluorescence microscopy. The lower panel is a negative control showing that Spore-Cry1Fa did not bind to Sf9 cells that do not express the Sf-ABCC2 protein. [Figure 7]FIG. 7 shows microscopic observation of the cytotoxic activity of Spore-Cry1Fa against Sf9 cells expressing Sf-ABCC2 according to an embodiment of the present disclosure. After Spore-Cry1Fa bound to Sf9 cells, Cry1Fa protein on the spores disrupted the cell membrane within 1 hour (upper panel). Spore-Cry1Fa was visible under a fluorescent microscope as a bright spot with Alexa-labeled anti-Cry antibodies bound on the spores. Longer incubation showed complete disruption of Sf9 cells (lower panel). [Figure 8] 8 shows a method of packaging an ABC transporter protein into a SMALP (styrene-maleic acid lipid particle) according to an embodiment of the present disclosure. The transporter protein expressed in the insect cell membrane was extracted with SMA (styrene-maleic acid). In this figure, "extraction" refers to the removal of the ABC transporter protein from the insect cell membrane with SMA and packaging of the transporter protein with its own phospholipid in the SMALP (styrene-maleic acid lipid particle). [Figure 9] FIG. 9 shows affinity column purification of Sf-ABCC2-FLAG in SMALP using anti-FLAG antibody according to an embodiment of the present disclosure. The ABC transporter proteins used in the examples herein were labeled with a C-terminal FLAG tag. SMALP-Sf-ABCC2-FLAG was loaded onto 200 μl of anti-FLAG antibody-immobilized agarose gel packed into a 1 ml Pierce™ Spin Column (Thermo-Fisher). The column was washed with TBS containing 10% glycerol (TBS-glycerol) and Sf-ABCC2 was eluted with 1.2 ml of elution buffer (0.1 mg / ml FLAG peptide in TBS-10% glycerol). The column eluate was fractionated into six 200 μl fractions and analyzed by dot blotting with Alexa-labeled anti-FLAG antibody. All fractions ("Fr"), including the final spin-off (Fr.7) except the first (Fr.1), showed strong fluorescent signals, confirming that Sf-ABCC2 was successfully eluted from the affinity column. [Figure 10]FIG. 10 shows Bacillus subtilis colonies growing on LB-tet-agar plates according to an embodiment of the present disclosure. These colonies were derived from Spore-pSB634 (no Cry proteins displayed) or Spore-Cry1Fa spores and were selected by immunomagnetic sorting. These spores were bound to SMALP-Sf-ABCC2-FLAG (e.g., as purified in FIG. 9) and sorted with anti-FLAG antibody-immobilized magnetic beads designed to capture only ABCC2-bound spores. The sorted spores were plated on LB-tet-agar plates and incubated at 37° C. for 16 hours to germinate and form colonies. Spore-pSB634, sorted under the same protocol as a negative control, showed only a few colonies (left panel), indicating little binding between this spore sample and Sf-ABCC2 in the SMALP. Selection of Spore-Cry1Fa yielded too many colonies to count (right panel). [Figure 11]FIG. 11 shows sorting of Spore-Cry1Aa (Bacillus subtilis spore-displayed Bacillus thuringiensis Cry1Aa protein) by flow cytometry according to an embodiment of the present disclosure. The Spore-Cry1Aa-Bm-ABCC2 complex was made fluorescent by sequentially mixing with anti-FLAG-mouse-antibody and Alexa-labeled anti-mouse IgG-antibody. The population of fluorescent spores for cell cytometry was reduced by treating with 0.2% SDS (described in Example 8 herein) to partially cleave the bond between Cry1Aa and the ABC transporter protein. This is to demonstrate the feasibility of spore separation by flow cytometry. Microscopic observation revealed that only a few spores retained the fluorescent dye. These fluorescent spores were sorted by a flow cytometer, On-chip Sort cell sorter (On-Chip Biotechnologies). The cell sorter pulled out about 1000 spores as fluorescent, likely bound to Bm-ABCC2. The sorted 1000 fluorescent spores (encircled in the cell sorter output) were sampled and plated on LB-agar plates. The LB plates were copied onto LB-tet-agar plates. Over 800 spores were found to germinate and form colonies on tetracycline selection plates, indicating that they contained a plasmid that directs Cry1Aa to the spore surface, conferring tetracycline resistance. This sorting process indicates that the captured spores can be recovered and the sequence of the Cry protein can be deduced by DNA sequencing of the plasmid. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] I. Introduction This disclosure is based in part on the inventors' discovery of an efficient, high-throughput method for screening libraries of putative insecticidal proteins using spore display technology. Although spore display technology has previously been utilized to display enzymes and antigens (e.g., for vaccine development), this disclosure provides the first application of spore display to screening insecticidal proteins for receptor-binding ability.

[0022] Provided herein are methods for identifying insecticidal proteins, along with recombinant nucleic acids, DNA constructs, vectors, fusion proteins, and spores useful in the provided methods. The provided methods allow for efficient high-throughput screening of candidate insecticidal proteins, including naturally occurring and engineered synthetic proteins. As detailed herein, embodiments of the provided methods include displaying a functionally active insecticidal protein (e.g., Bacillus thuringiensis crystal [Bt Cry] protein) on the surface of a microbial spore (e.g., a bacterial spore or a fungal spore). In some embodiments, the spore surface display includes the use of a fusion protein that includes a spore coat protein linked (e.g., by a structurally flexible linker) to a candidate insecticidal protein. In some embodiments, upon expression of the fusion protein in the spore, the fusion protein translocates to the spore surface to display the insecticidal protein positioned toward the spore exterior (e.g., toward the solvent or medium).

[0023] Prior to the experiments described in the Examples herein, it was not known whether an insecticidal protein (e.g., Bt Cry toxin) could be functionally displayed on the spore surface (i.e., where insecticidal protein receptor binding and subsequent insecticidal function are preserved). In some embodiments, the methods provided herein include fusion proteins that utilize a structurally flexible linker to anchor a putative insecticidal protein to a spore coat protein with some or complete freedom, such that the insecticidal protein (e.g., Cry protein) portion can be properly folded and its receptor-binding domain is exposed to the receptor.

[0024] In some embodiments of the methods provided herein, the insecticidal protein remains attached to the spore during screening, and spores containing a gene encoding the insecticidal protein (e.g., in a spore display vector, as described in more detail below) have the sequence tag of the insecticidal protein fixed to their surface. If a spore carrying a desired variant of the insecticidal protein is found during screening, the sequence of the variant can be determined by sequencing the insecticidal protein gene in the spore. This eliminates the need to separate individual clones in multi-well plates. The spore display system provided herein also has the advantage of allowing flow cytometric sorting of spores, although the E. coli phages used in the phage display system cannot be sorted by flow cytometry.

[0025] Recent attempts to use a related technique, phage display, to screen Bt Cry variants for receptor binding activity have proven unsuccessful (Fujii et al., 2012, Mol. Biotech. 54:888-899). A limitation to phage display is that because phage particles are produced in E. coli, heterogeneous genes from Bacillus species (e.g., Bt Cry proteins and their variants) may not be reliably expressed. This is particularly problematic when the screening library contains a wide variety of protein sequences. Indeed, Fujii et al. (2012, Mol. Biotech. 54:888-899) found that phage display did not result in improved insecticidal activity. Furthermore, results indicate that the structure of the phage-displayed Bt Cry protein may not be the same as that of the native protein. The methods and compositions provided herein overcome this limitation. In some embodiments, the methods herein allow for homogeneous expression of a Bacillus gene (eg, a Bt Cry insecticidal protein gene) in a Bacillus host.

[0026] In summary, the methods provided herein have several advantages over other insecticidal protein screening techniques, including, but not limited to: 1) high-throughput screening of candidate insecticidal protein libraries (e.g., using cell sorting techniques such as immunomagnetic separation and flow cytometry); 2) greater tolerance to expression and screening of candidate insecticidal proteins (e.g., Bt Cry proteins) from Bacillus species compared to phage display, since spores can be produced in Bacillus species; 3) increased flexibility of the receptor for candidate insecticidal proteins.

[0027] II. Terminology All technical and scientific terms used herein are intended to have the same meaning as commonly understood by those skilled in the art, unless otherwise defined below. References to techniques used herein are intended to refer to techniques commonly understood in the art, including variations of those techniques and / or equivalent technical substitutions that would be apparent to those skilled in the art. Although the following terms are believed to be well understood by those skilled in the art, the following definitions are provided to facilitate the description of the subject matter of this disclosure.

[0028] As used herein, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to an "antibody" optionally includes combinations of two or more such molecules, and the like.

[0029] The term "about" as used herein refers to the normal range of error for the respective value, readily known to one of ordinary skill in the art, e.g., ±20%, ±10%, or ±5% within the intended meaning of the recited value.

[0030] As used herein, the terms "comprising" or "comprise" are open ended. When used in reference to a subject nucleic acid (or amino acid sequence), it refers to a nucleic acid sequence (or amino acid sequence) that includes the subject sequence as a part or entire sequence.

[0031] The term "plurality" refers to two or more entities. Thus, a "plurality of individuals" refers to at least two individuals. In some embodiments, the term plurality refers to more than half of a total. For example, in some embodiments, a "plurality of a population" refers to more than half of the members of the population.

[0032] The term "Next Generation Discovery Technology" or "NGDT" is used to refer to certain embodiments of the present disclosure, as described in more detail below.

[0033] The term "Cry" refers to members of the Bacillus thuringiensis crystal protein family, members of which can be insecticidal.

[0034] The term "Cry1Fa" refers to a Cry protein (or, when italicized, [cry1Fa], the gene encoding said protein) that is highly active against Spodoptera frugiperda (Sf, Fall Armyworm) and known to bind to Sf-ABCC2. The amino acid sequence of Cry1Fa is shown in SEQ ID NO:1, and the nucleotide sequence of the protein coding region of cry1Fa is shown in SEQ ID NO:2.

[0035] The term "Cry1Aa" refers to a Cry protein (or, when italicized, [cry1Aa], the gene encoding said protein) that is highly active against Bombyx mori (Bm, Silkworm) and Plutella xylostella (Px, Diamondback Moth) and is known to bind to Bm / Px-ABCC2. The amino acid sequence of Cry1Aa is shown in SEQ ID NO:3, and the nucleotide sequence of the protein coding region of cry1Aa is shown in SEQ ID NO:4.

[0036] The term "ATP-binding cassette transporter" or "ABC transporter" refers to a large family of transporter proteins represented in all extant phyla. The term "ABCC" refers to ABC transporter subfamily C, type 2 (ABCC2) and / or type 3 (ABCC3) proteins (or, when italicized [ABCC], the genes encoding said proteins). The amino acid sequence of Px-ABCC2 is shown in SEQ ID NO:5, and the nucleotide sequence of the protein coding region of Px-ABCC2 is shown in SEQ ID NO:6. The amino acid sequence of Bm-ABCC2 is shown in SEQ ID NO:7, and the nucleotide sequence of the protein coding region of Bm-ABCC2 is shown in SEQ ID NO:8. The amino acid sequence of Sf-ABCC2 is shown in SEQ ID NO:9, and the nucleotide sequence of the protein coding region of Sf-ABCC2 is shown in SEQ ID NO:10. The amino acid sequence of an Sf-ABCC2 mutant derived from a Cry1Fa-resistant Sf strain (herein referred to as the "Sf-ABCC2 mutant") is shown in SEQ ID NO:11, and the nucleotide sequence of the protein coding region of the Sf-ABCC2 mutant is shown in SEQ ID NO:12. The amino acid sequence of Sf-ABCC3 is shown in SEQ ID NO: 13, and the nucleotide sequence of the protein coding region of Sf-ABCC3 is shown in SEQ ID NO: 14. Because there are variations in protein sequences within the same class of ABC transporters (e.g., ABCC2 proteins in different insect species), ABCCs are generally referred to herein by insect species abbreviations, such as Sf (Fall armyworm, Spodoptera frugiperda), Bm (Silkworm, Bombyx mori), Dv (Western corn rootworm (WCR), Diabrotica virgifera virgifera), Tc (Red flour beetle, Tribolium castaneum), etc., to indicate the source.

[0037] As used herein, the term "spore" without modification refers to the spore of any spore-producing microorganism. Examples of microorganisms that produce spores include bacterial and fungal species, as detailed herein below. Spores from a particular source may be referred to herein by a genus or species name, for example, generally B. thuringiensis spores or Bacillus spores. In some embodiments, the present disclosure provides spores that include a protein (e.g., a protein expressed by the spore that may be expressed on the surface of the spore) and / or a vector (e.g., where the protein is not expected to be displayed on the surface of the spore). Such spores are referred to as "Spore-[protein name]" (e.g., Spore-Cry1Fa or Spore-Cry1Aa) or "Spore-[vector name]" (e.g., Spore-pSB634 or Spore-pHY300PLK).

[0038] The term "CotC" refers to the Bacillus subtilis spore coat type C protein (or, when italicized, [cotC], the gene encoding said protein). The amino acid sequence of CotC is shown in SEQ ID NO:15, and the nucleotide sequence of cotC (including its promoter) is shown in SEQ ID NO:16.

[0039] The term "CotG" refers to the Bacillus subtilis spore coat type G protein (or, when italicized, [cotG], the gene encoding said protein). The amino acid sequence of CotG is shown in SEQ ID NO:17, and the nucleotide sequence of cotG (including its promoter) is shown in SEQ ID NO:18.

[0040] The term "cry1Ac terminator" refers to the translation terminator of the Bt cry1Ac gene having a double hairpin RNA structure, the nucleotide sequence of which is shown in SEQ ID NO:19.

[0041] The terms "Cry1Aa leader sequence" and "Cry1Fa leader sequence" refer to a short (about 27 amino acids) naturally occurring amino acid sequence attached to the N-terminus of mature Cry1Aa and Cry1Fa proteins, which is removed by protease digestion in the insect gut, converting the protoxin to a mature toxin. In some embodiments, the leader sequence serves as a flexible linker between the Cot protein and the Cry protein. The Cry1Aa leader sequence is shown in SEQ ID NO:20, and the Cry1Fa leader sequence is shown in SEQ ID NO:21.

[0042] The term "spore display vector" refers to a plasmid vector in which an insecticidal protein is cloned together with a spore coat protein gene to express (or display) the protein (e.g., on the surface of one or more spores). In this disclosure, two Bacillus-E. coli shuttle vectors, pSB634 (SEQ ID NO: 22) containing Bacillus cereus pBC16.1 and E. coli pBluescript-KS, and pHY300PLK (SEQ ID NO: 23) containing Streptococcus faecalis pAM-alpha and E. coli pACYC177, were used as examples.

[0043] The term "tet" refers to the antibiotic compound tetracycline.

[0044] The term "LB" refers to Luria Broth. In some embodiments, LB contains 10 g tryptone, 5 g yeast extract, and 10 g NaCl in 1 L of water. In this disclosure, 1 / 2 LB means 2-fold diluted LB (e.g., diluted with water) and "LB-Agar" refers to solid LB medium made with 1.5% agar.

[0045] The term "Sf9" refers to a clonally isolated insect cell line of the parent cell line Spodoptera frugiperda Sf21 IPLB-Sf21-AE (derived from pupal ovary tissue).

[0046] The terms "nucleic acid" and "polynucleotide" are used interchangeably and, as used herein, refer to both sense and antisense strands of RNA, cDNA, genomic DNA, mitochondrial DNA, and synthetic forms and mixed polymers of the above. In certain embodiments, nucleotides refer to ribonucleotides, deoxynucleotides, or modified forms of any type of nucleotide, and combinations thereof. The term also includes, but is not limited to, single-stranded and double-stranded forms of DNA and / or RNA. Furthermore, the polynucleotides disclosed herein, such as circular DNA templates, nucleic acid concatemers disclosed herein, can contain either or both naturally occurring and modified nucleotides linked together by naturally occurring and / or non-naturally occurring nucleotide bonds. As will be readily understood by those skilled in the art, nucleic acid molecules can be chemically or biochemically modified or contain non-natural or derivatized nucleotide bases. Such modifications include, for example, labels, methylation, substitution of one or more analogs of naturally occurring nucleotides, internucleotide modifications, such as uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendant moieties (e.g., polypeptides), intercalators (e.g., acridines, psoralens, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.). The above terms are also intended to include any topological conformation, including single-stranded, double-stranded, partially duplexed, triplexed, hairpinned, circular, and padlocked conformations. Reference to a nucleic acid sequence includes its complement, unless otherwise specified. Thus, reference to a nucleic acid molecule having a particular sequence should be understood to include its complementary strand, its complementary sequence. Nucleotide sequences are "complementary" if they hybridize specifically (e.g., according to Watson-Crick base-pairing rules) in solution. The term also includes codon-optimized nucleic acids that encode the same polypeptide sequence.It is also understood that the nucleic acids can be unpurified, purified, or bound to synthetic materials such as, for example, beads or column matrices.

[0047] Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants, alleles, orthologs, SNPs and complementary sequences thereof, as well as the sequence explicitly indicated.

[0048] The term "identity" or "substantial identity" as used in the context of polynucleotide or polypeptide sequences described herein refers to a sequence having at least 60% sequence identity with a reference sequence. Alternatively, the percent identity can be any integer between 60% and 100%. Exemplary embodiments include at least the following: 60%, 65%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% compared to a reference sequence using the programs described herein; preferably, BLAST using standard parameters as described below. One of skill in the art will recognize that these values ​​can be appropriately adjusted to determine the corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame alignment, and the like.

[0049] For sequence comparison, typically, one sequence acts as a reference sequence to which test sequence is compared.When using sequence comparison algorithm, test sequence and reference sequence are input into computer, partial sequence coordinates are designated as necessary, and sequence algorithm program parameters are designated.Default program parameters can be used, or alternative parameters can be designated.The sequence comparison algorithm then calculates the percent sequence identity of test sequence to reference sequence based on program parameters.

[0050] As used herein, a "comparison window" includes reference to any one of a number of consecutive positions selected from the group consisting of 20 to 600, usually about 50 to about 200, more usually about 100 to about 150, where a sequence can be compared to a reference sequence of the same number of consecutive positions after the two sequences are optimally aligned. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed by the local homology algorithm by Smith and Waterman Add.APL.Math.2:482 (1981), the homology alignment algorithm by Needleman and Wunsch J.Mol.Biol.48:443 (1970), the similarity search method by Pearson and Lipman Proc.Natl.Acad.Sci.(USA)85:2444 (1988), computer implementations of these algorithms (e.g., BLAST), or by manual alignment and visual inspection.

[0051] Particularly suitable algorithms for determining percent sequence identity and percent sequence similarity are the BLAST and BLAST 2.0 algorithms described in Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1977) Nucleic Acids Res. 25:3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) website. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or meet some positive threshold score T when aligned with words of the same length in the database sequences. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds to initiate searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence as far as the cumulative alignment score can be increased. The cumulative score is calculated using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatched residues; always <0) for nucleotide sequences. For amino acid sequences, a score matrix is ​​used to calculate the cumulative score. The extension of the word hits in each direction is stopped when the cumulative alignment score falls by an amount X from its maximum achieved value; the cumulative score falls below 0 due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=1, N=-2, and a comparison of both strands.For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0052] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, it is preferred that the smallest sum probability in a comparison of a test nucleic acid to a reference nucleic acid is less than about 0.01, more preferably less than about 10 -5 less than about 10 -20 A nucleic acid is considered to be similar to a reference sequence if it is less than

[0053] "Polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. As used herein, the term encompasses amino acid chains of any length, including full-length proteins, in which the amino acid residues are linked by covalent peptide bonds.

[0054] The amino acids in the polypeptides described herein may be any of the 20 natural amino acids, D-stereoisomers of natural amino acids, unnatural amino acids, and chemically modified amino acids. Unnatural amino acids (i.e., those not naturally found in proteins) are also known in the art, as described, for example, in Zhang et al. "Protein engineering with unnatural amino acids" Curr. Opin. Struct. Biol. 23(4):581-587 (2013); Xie et al. "Adding amino acids to the genetic repertoire", 9(6):548-54 (2005)) and all references cited therein. β and γ amino acids are known in the art and are also contemplated herein as unnatural amino acids.

[0055] As used herein, chemically modified amino acids refer to amino acids whose side chains have been chemically modified. For example, the side chains can be modified to include signaling moieties, such as fluorophores or radiolabels. The side chains can also be modified to include new functional groups, such as thiols, carboxylic acids, or amino groups. Post-translationally modified amino acids are also included in the definition of chemically modified amino acids.

[0056] Conservative amino acid substitutions are also contemplated. For example, conservative amino acid substitutions can be made at one or more amino acid residues, for example, at one or more lysine residues of any of the polypeptides provided herein. Those skilled in the art will know that conservative substitution refers to the replacement of an amino acid residue with another amino acid residue that is biologically and / or chemically similar. Each of the following eight groups contains amino acids that are conservative substitutions for each other: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) Cysteine ​​(C), Methionine (M)

[0057] For example, when arginine to serine is referred to, conservative substitutions of the serine (e.g., threonine) are also contemplated. Non-conservative substitutions, such as replacing lysine with asparagine, are also contemplated.

[0058] As used herein, the term "primer" refers to an oligonucleotide that can anneal (in some embodiments, specifically anneal to a nucleic acid target) to allow DNA polymerase and / or reverse transcriptase to bind thereto, thereby serving as a starting point for DNA synthesis when placed under conditions (e.g., in the presence of nucleotides and agents for polymerization, such as DNA polymerase, at a suitable temperature and pH) that induce synthesis of a primer extension product. In some embodiments, one or more primers are used to amplify plant nucleic acids (e.g., using the polymerase chain reaction; PCR).

[0059] III. Recombinant Nucleic Acids, Constructs, Vectors and Spores In one aspect, provided herein is a recombinant nucleic acid encoding a fusion protein, the recombinant nucleic acid comprising: a) a first nucleic acid sequence encoding a spore coat polypeptide; b) a second nucleic acid sequence encoding a linker; and c) a third nucleic acid sequence encoding a putative insecticidal polypeptide.

[0060] A. Spore coat polypeptide In some embodiments of the recombinant nucleic acid, the spore coat polypeptide is from any microorganism that produces spores. In some embodiments, the microorganism is an endospore-producing bacterium. In some embodiments, the bacterium is a species of any of the following genera: Acetonema, Actinomyces, Alkalibacillus, Ammoniphilus, Amphibacillus, Anaerobacter, Anaerospora, Aneurinibacillus, Anoxybacillus, Bacillus, or Bacillus subtilis. Bacillus, Brevibacillus, Caldanaerobacter, Caloramator, Caminicella, Cerasibacillus, Clostridium, Clostridiisalibacter, Cohnella, Coxiella (i.e., Coxiella burnetii), burnetii), Dendrosporobacter, Desulfotomaculum, Desulfosporomusa, Desulfosporosinus, Desulfovirgula, Desulfunispora, Desulfurispora, Filifactor actor), Filobacillus, Gelria, Geobacillus, Geosporobacter, Gracilibacillus, Halobacillus, Halonatronum, Heliobacterium, Heliophilum, Laceyella,Lentibacillus, Lysinibacillus, Mahella, Metabacterium, Moorella, Natroniella, Oceanobacillus, Orenia, Ornithinibacillus, Oxalophagus, Oxobacter, Paenibacillus lus, Paraliobacillus, Pelospora, Pelotomaculum, Piscibacillus, Planifilum, Pontibacillus, Propionispora, Salinibacillus, Salsuginibacillus, Seinonella, Shimazuella ella, Sporacetigenium, Sporoanaerobacter, Sporobacter, Sporobacterium, Sporohalobacter, Sporolactobacillus, Sporomusa, Sporosarcina, Sporotalea, Sporotomac ulum, Syntrophomonas, Syntrophospora, Tenuibacillus, Tepidibacter, Terribacillus, Thalassobacillus, Thermoacetogenium, Thermoactinomyces, Thermoalkalibacillus,Thermoanaerobacter, Thermoanaeromonas, Thermobacillus, Thermoflavimicrobium, Thermovenablum, Tuberibacillus, Virgibacillus, or Vulcanobacillus.

[0061] In some embodiments, the spore coat polypeptide is derived from a Bacillus species. In some embodiments, the spore coat polypeptide is derived from a Bacillus species (e.g., Bacillus subtilis) spore coat (Cot) protein. In some embodiments, the Cot protein is CotC, CotG, CotB, CotA, CotD, CotE, CotX, CotY, or CotZ. In some embodiments, the Cot protein is CotC or CotG.

[0062] In some embodiments of the recombinant nucleic acids provided herein, a first nucleic acid sequence encoding a spore coat polypeptide comprises a nucleic acid sequence having at least 60% identity (e.g., at least 65% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity) to SEQ ID NO: 16 or SEQ ID NO: 18. In some embodiments, the first nucleic acid sequence encodes a spore coat polypeptide comprising an amino acid sequence having at least 60% identity to SEQ ID NO: 15 or SEQ ID NO: 17.

[0063] In some embodiments of the recombinant nucleic acids provided herein, the spore coat polypeptide is derived from a spore-producing fungus, hi some embodiments, the fungus is from any of the following phyla: Zygemycota, Ascomycota, Basidiomycota, or Oomycota.

[0064] B. Putative insecticidal polypeptides The recombinant nucleic acids provided herein can encode a putative insecticidal polypeptide of any of the following structural classes: App, Cry, Cyt, Gpp, Mcf, Mpf, Mpp, Mtx, Pra, Prb, Spp, Tpp, Vip, Vpa, or Vpb. See, e.g., Crickmore, N., et al. "A structure-based nomenclature for Bacillus thuringiensis and other bacteria-derived pesticidal proteins." Journal of Invertebrate Pathology, 107438 (2020); doi.org / 10.1016 / j.jip.2020.107438.

[0065] In some embodiments, the insecticidal polypeptide is a Bacillus thuringiensis insecticidal protein (including, but not limited to, a Cry protein, a vegetative insecticidal protein (VIP), and an insecticidal chimera of any of the foregoing insecticidal proteins). In some embodiments, the putative insecticidal polypeptide is a Bacillus thuringiensis crystal (Bt Cry) protein.

[0066] Examples of Cry proteins include Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ad, Cry1Ae, Cry1Af, Cry1Ag, Cry1Ah, Cry1Ai, Cry1Aj, Cry1Ba, Cry1Bb, Cry1Bc, Cry1Bd, Cry1Be, Cry1Bf, Cry1Bg, Cry1Bh, Cry1Bi, Cry1Ca, Cry1Cb, Cry1Da, Cry1Db, Cry1Dc, Cry1Dd, Cry1Ea, Cry1Eb, Cry1Fa, Cry1Fb, Cry1Ga, Cry1Gb, Cry1Gc, Cry1Ha, Cr y1Hb, Cry1Hc, Cry1Ia, Cry1Ib, Cry1Ic, Cry1Id, Cry1Ie, Cry1If, Cry1Ig, Cry1Ja, Cry1Jb, Cry1Jc, Cry1Jd, Cry1Ka, Cry1La, Cry1Ma, Cry1Na, Cry1Nb, Cry2Aa, Cry2Ab, Cry2Ac, Cry2Ad, Cry2Ae, Cry2Af, Cry2Ag, Cry2Ah, Cry2Ai, Cry2Aj, Cry2Ak, Cry2Al, Cry2Ba, Cry3Aa, Cry3Ba, Cry3Bb, Cry3Ca, Cry4Aa , Cry4Ba, Cry4Ca, Cry4Cb, Cry4Cc, Cry5Aa, Cry5Ab, Cry5Ac, Cry5Ad, Cry5Ba, Cry5Ca, Cry5Da, Cry5Ea, Cry6Aa, Cry6Ba, Cry7Aa, Cry7Ab, Cry7Ac, Cry7 Ba, Cry7Bb, Cry7Ca, Cry7Cb, Cry7Da, Cry7Ea, Cry7Fa, Cry7Fb, Cry7Ga, Cry7Gb, Cry7Gc, Cry7Gd, Cry7Ha, Cry7Ia, Cry7Ja, Cry7Ka, Cry7Kb, Cry7La, Cry 8Aa, Cry8Ab, Cry8Ac, Cry8Ad, Cry8Ba, Cry8Bb, Cry8Bc, Cry8Ca, Cry8Da, Cry8Db, Cry8Ea, Cry8Fa, Cry8Ga, Cry8Ha, Cry8Ia, Cry8Ib, Cry8Ja, Cry8Ka, C ry8Kb, Cry8La, Cry8Ma, Cry8Na, Cry8Pa, Cry8Qa, Cry8Ra, Cry8Sa, Cry8Ta, Cry9Aa, Cry9Ba, Cry9Bb, Cry9Ca, Cry9Da, Cry9Db, Cry9Dc, Cry9Ea, Cry9Eb,Cry9Ec、Cry9Ed、Cry9Ee、Cry9Fa、Cry9Ga、Cry10Aa、Cry11Aa、Cry11Ba、Cry11Bb、Cry12Aa,Cry13Aa、Cry14Aa、Cry14Ab、Cry15Aa、Cry16Aa、Cry17Aa、Cry18Aa、Cry18Ba、Cry18Ca、Cry19Aa、Cry19Ba、Cry19Ca、Cry20Aa、Cry20Ba、Cry21Aa、Cry21Ba、Cry21Ca、Cry21Da、Cry21Ea、Cry21Fa、Cry21Ga、Cry21Ha、Cry22Aa、Cry22Ab、Cry22Ba、Cry22Bb、Cry23Aa、Cry24Aa、Cry24Ba、Cry24Ca、Cry25Aa、Cry26Aa、Cry27Aa、Cry28Aa、Cry29Aa、Cry29Ba、Cry30Aa、Cry30Ba、Cry30Ca、Cry30Da、Cry30Db、Cry30Ea、Cry30Fa、Cry30Ga,Cry31Aa、Cry31Ab、Cry31Ac、Cry31Ad、Cry32Aa、Cry32Ab、Cry32Ba、Cry32Ca、Cry32Cb、Cry32Da、Cry32Ea、Cry32Eb、Cry32Fa、Cry32Ga、Cry32Ha、Cry32Hb、Cry32Ia、Cry32Ja、Cry32Ka、Cry32La、Cry32Ma、Cry32Mb、Cry32Na、Cry32Oa、Cry32Pa、Cry32Qa、Cry32Ra、Cry32Sa、Cry32Ta、Cry32Ua、Cry33Aa、Cry34Aa、Cry34Ab、Cry34Ac、Cry34Ba、Cry35Aa、Cry35Ab、Cry35Ac、Cry35Ba、Cry36Aa、Cry37Aa、Cry38Aa、Cry39Aa、Cry40Aa、Cry40Ba、Cry40Ca、Cry40Da、Cry41Aa、Cry41Ab、Cry41Ba、Cry42Aa、Cry43Aa、Cry43Ba、Cry43Ca、Cry43Cb、Cry43Cc、Cry44Aa、Cry45Aa、Cry46Aa Cry46Ab、Cry47Aa、Cry48Aa、Cry48Ab、Cry49Aa、Cry49Ab、Cry50Aa、Cry50Ba、Cry51Aa、Cry52Aa、Cry52Ba、Cry53Aa、Examples include, but are not limited to, Cry53Ab, Cry54Aa, Cry54Ab, Cry54Ba, Cry55Aa, Cry56Aa, Cry57Aa, Cry57Ab, Cry58Aa, Cry59Aa, Cry59Ba, Cry60Aa, Cry60Ba, Cry61Aa, Cry62Aa, Cry63Aa, Cry64Aa, Cry65Aa, Cry66Aa, Cry67Aa, Cry68Aa, Cry69Aa, Cry69Ab, Cry70Aa, Cry70Ba, Cry70Bb, Cry71Aa, Cry72Aa, Cry73Aa, or any combination of the foregoing. A list of such Cry proteins and related information can be found, for example, at the Bacterial Pesticidal Protein Resource Center (BPPRC) (Crickmore, N., Berry, C., Panneerselvam, S., Mishra, R., Connor, T. R. and Bonning, B. C. (2020) Bacterial Pesticidal Protein Resource Center, bpprc.org). Sequences are available through the BPPRC and NCBI databases (ncbi.nlm.nih.gov).

[0067] Non-limiting examples of members of the Vip3 class and their respective GenBank accession numbers, U.S. patent or patent publication numbers include Vip3Aa1 (AAC37036), Vip3Aa2 (AAC37037), Vip3Aa3 (see U.S. Pat. No. 6,137,033), Vip3Aa4 (AAR81079), Vip3Aa5 (AAR81080), Vip3Aa6 (AAR81081), Vip3Aa7 (AAK95 326), Vip3Aa8 (AAK97481), Vip3Aa9 (CAA76665), Vip3Aa10 (AAN60738), Vip3Aa11 (AAR36859), Vip3Aa12 (AAM22456), Vip3Aa13 (AAL69542), Vip3Aa14 (AAQ12340), Vip3Aa15 (AAP51131), Vip3Aa16 (AAW65132), Vip3Aa17 (U.S. Patent No. 6,603,063 specification), Vip3Aa18 (AAX49395), Vip3Aa19 (DQ241674), Vip3Aa19 (DQ539887), Vip3Aa20 (DQ539888), Vip3Aa21 (ABD84410), Vip3Aa22 (AAY41) 427), Vip3Aa23(AAY41428), Vip3Aa24(BI880913), Vip3Aa25(EF608501), Vip3Aa26(EU294496), Vip3Aa27(EU332167), Vip3Aa28(FJ494817), Vi p3Aa29(FJ626674), Vip3Aa30(FJ626675), Vip3Aa31(FJ626676), Vip3Aa32(FJ626677), Vip3Aa33(GU073128), Vip3Aa34(GU073129), Vip3Aa35( GU733921), Vip3Aa36(GU951510), Vip3Aa37(HM132041), Vip3Aa38(HM117632), Vip3Aa39(HM117631), Vip3Aa40(HM132042), Vip3Aa41(HM13204 3), Vip3Aa42 (HQ587048), Vip3Aa43 (HQ594534), Vip3Aa44 (HQ650163), Vip3Ab1 (AAR40284), Vip3Ab2 (AAY88247), Vip3Ac1 (see U.S. Patent Application Publication No. 20040128716), Vip3Ad1 (see U.S. Patent Application Publication No. 20040128716), Vip3Ad2 (CAI43276), Vip3Ae1 (CAI43277), Vip3Af1 (see U.S. Patent Application Publication No. 7,378,493), Vip3Af2 ( ADN08753), Vip3Af3 (HM117634), Vip3Ag1 (ADN08758), Vip3Ag2 (FJ556803), Vip3Ag3 (HM117633), Vip3Ag4 (HQ414237), Vip3Ag5 (HQ542193), Vip3Ah1 (DQ832323), Vip3Ba1 (AAV70653), Vip3Ba2 (HM117635), Vip3Bb1 (see U.S. Pat. No. 7,378,493), Vip3Bb2 (AB030520) and Vip3Bb3 (ADI48120).

[0068] In some embodiments, the insecticidal polypeptide can be a protein other than a B. thuringiensis protein. For example, the insecticidal polypeptide can be an α-amylase, a peroxidase, a cholesterol oxidase, a patatin, a protease, a protease inhibitor, an urease, an α-amylase inhibitor, a pore forming protein, a chitinase, a lectin, an engineered antibody or antibody fragment, a Bacillus cereus protein, a B. thuringiensis ... cereus insecticidal proteins, Xenorhabdus genus (e.g., X. nematophila or X. bovienii) insecticidal proteins, Photorhabdus genus (e.g., P. luminescens or P. asymobiotica) insecticidal proteins, Brevibacillus genus (e.g., B. laterosporous) insecticidal proteins, Lysinibacillus genus (e.g., L. sphearicus) insecticidal proteins, Chromobacterium genus (e.g., C. subtsugae or C. piscinae) insecticidal proteins, The insecticidal protein may be a Yersinia (e.g., Y. entomophaga) insecticidal protein, a Paenibacillus (e.g., P. propylaea) insecticidal protein, a Clostridium (e.g., C. bifermentans) insecticidal protein, a Pseudomonas (e.g., P. fluorescens) insecticidal protein, and lignin. In other embodiments, the insecticidal polypeptide may be at least one insecticidal protein from Photorhabdus, Xenorhabdus, Serratia, or an insecticidal toxin complex (Tc) from Yersinia.In some embodiments, the insecticidal polypeptide can be an ADP-ribosyltransferase from an insecticidal bacterium, such as Photorhabdus. In some embodiments, the insecticidal polypeptide can be a VIP protein, such as VIP1 and / or VIP2 from B. cereus. In some embodiments, the insecticidal polypeptide can be a bicomponent toxin from an insecticidal bacterium, such as ISP1A and ISP2A from B. laterosporous, or BinA and BinB from L. sphaericus. In some embodiments, the insecticidal polypeptide can be an engineered version or a hybrid or chimera of any of the aforementioned insecticidal polypeptides.

[0069] Other non-limiting examples of insecticidal polypeptides include DIG-657 (see U.S. Patent Application Publication No. 2015366211); PtIP-96 (see U.S. Patent Application Publication No. 2017233440); PIP-72 (see U.S. Patent Application Publication No. 2016366891); PIP-83 (see U.S. Patent Application Publication No. 2016347799); PIP-50 (see U.S. Patent Application Publication No. 2017166921); IPD73 (see U.S. Patent Application Publication No. 2019119334); IPD090 (see U.S. Patent Application Publication No. 201 9136258;IPD80 (see U.S. Patent Application Publication No. 2019256563);IPD078, IPD084, IPD086, IPD087, IPD089 (see U.S. Patent Application Publication No. 2020055906);IPD093 (see WO 2018111551);IPD059 (see WO 2018232072);IPD113 (see WO 2019178042);IPD121 (see WO 2018208882);IPD11 0 (see WO 2019178038); IPD103 (see WO 2019125717); IPD092; IPD095; IPD097; IPD099; IPD100, IPD105; IPD106; IPD107; IPD111; IPD112 (see WO 2020055885); IPD102 (see WO 2020076958); Cry1B.868 and Cry1Da_7 (see U.S. Patent Application Publication No. 2020-032289); TIC107 (U.S. Patent Application Publication No. 2020-032289); No. 8049071; Cry2Ab and Cry1A.105 (see U.S. Pat. No. 10,584,391); Cry1F, Cry34Ab1, Cry35Ab1 (see U.S. Pat. No. 10,407,688); TIC6757, TIC7472, TIC7473, TIC6757 (see U.S. Pat. App. Pub. No. 2017058294); TIC3668, TIC3669, TIC3670, TIC4076, TIC4078, TIC4260, TIC4346, TIC4826, TIC4861, TIC4862, TIC4863,TIC-3668 (see U.S. Patent Application Publication No. 2016319302); TIC7040, TIC7042, TIC7381, TIC7382, TIC7383, TIC7386, TIC7388, TIC7389 (see U.S. Patent Application Publication No. 2018291395); TIC7941 (see U.S. Patent Application Publication No. 2020229445); TIC836, TIC860, TIC867, TIC868, TIC869, and TIC1100 (see International Publication No. 2016061391); TIC2160 (International Publication No. U.S. Patent Application Publication No. 2016061392), ET66, TIC400, TIC800, TIC834, TIC1415, AXMI-001, AXMI-002, AXMI-030, AXMI-035, and AXMI-045 (see U.S. Patent Application Publication No. 20130117884), AXMI-52, AXMI-58, AXMI-88, AXMI-97, AXMI-102, AXMI-112, AXMI-117, AXMI-100 (see U.S. Patent Application Publication No. 201-0310543), AXMI-115, AXMI-1 13, AXMI-005 (see U.S. Patent Application Publication No. 20130104259), AXMI-134 (see U.S. Patent Application Publication No. 20130167264), AXMI-150 (see U.S. Patent Application Publication No. 20100160231), AXMI-184 (see U.S. Patent Application Publication No. 20100004176), AXMI-196, AXMI-204, AXMI-207, AXMI-209 (see U.S. Patent Application Publication No. 2011-0030096), AXMI-218, AXMI-220 (see U.S. Patent Application Publication No. 20140245491), AXMI-221z, AXMI-222z, AXMI-223z, AXMI-224z, AXMI-225z (see U.S. Patent Application Publication No. 20140196175), AXMI-238 (see U.S. Patent Application Publication No. 20140033363), AXMI-270 (see U.S. Patent Application Publication No. 20140223598), AXMI-345 (see U.S. Patent Application Publication No. 20140373195), AXMI-335 (see International Publication No. 2013134523),DIG-3 (see U.S. Patent Application Publication No. 20130219570), DIG-5 (see U.S. Patent Application Publication No. 20100317569), DIG-11 (see U.S. Patent Application Publication No. 20100319093), AfIP-1A (see U.S. Patent Application Publication No. 20140033361), AfIP-1B (see U.S. Patent Application Publication No. 20140033361), PIP-1A PIP-1B (see U.S. Patent Application Publication No. 20140007292), PSEEN3174 (see U.S. Patent Application Publication No. 20140007292), AECFG-592740 (see U.S. Patent Application Publication No. 20140007292), P put_1063 (see US Patent Application Publication No. 20140007292), DIG-657 (see International Publication No. 2015195594), Pput_1064 (see US Patent Application Publication No. 20140007292), GS-135 (see US Patent Application Publication No. 20120233726), GS153 (see US Patent Application Publication No. 20120192310), GS154 (see US Patent Application Publication No. 20120192310), GS155 (see US Patent Application Publication No. 20120192310), DIG-911 and DIG-180 (see US Patent Application Publication No. 20150264940); and the like.

[0070] In some embodiments, the putative insecticidal polypeptide comprises at least one modification relative to the wild-type protein (i.e., the protein from which it is derived). In some embodiments, the modification is the result of DNA shuffling, block swapping, site-directed mutagenesis, saturation mutagenesis, random mutagenesis, or a combination thereof (e.g., as described in Cong et al., ed. Akhurst et al., 2002, Proceedings of the 4th Pacific Rim Conferences on Biotechnology of Bacillus thuringiensis and its environmental impact: 118-123, U.S. Pat. No. 8,530,411, Hou et al., 2019, Toxins 11(3): 162, and U.S. Patent Publication No. 20210147492). In some embodiments, the modification is a deletion of one or more nucleotides compared to the wild-type protein. In some embodiments, the modification is an insertion of one or more nucleotides relative to the wild-type protein. In some embodiments, the modification is a truncation, i.e., deletion, of one or more nucleotides from the 5' and / or 3' ends of the wild-type protein. In some embodiments, the putative insecticidal polypeptide is derived from a Bt Cry protein and the modification to the wild-type protein confers resistance to serine protease digestion (i.e., inhibition of serine protease activity). In some embodiments, serine protease inhibition enhances the insecticidal activity of the Bt Cry protein (see, e.g., Pardo-Lopez, et al., 2009, Peptides 30(3):589-595).

[0071] In some embodiments of the recombinant nucleic acids provided herein, the nucleic acid sequence encoding the putative insecticidal polypeptide comprises a nucleic acid sequence having at least 60% identity (e.g., at least 65% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity) to SEQ ID NO:2 or SEQ ID NO:4. In some embodiments, the nucleic acid sequence encodes a putative insecticidal polypeptide comprising an amino acid sequence having at least 60% identity to SEQ ID NO:1 or SEQ ID NO:3.

[0072] One family of Cry proteins is known as the three-domain Cry family and includes proteins with three domains in the active toxin protein. These domains, called domain I, domain II, and domain III, are described, for example, in Bravo et al., 2008, Toxicon 49(4):423-435 and Palma et al., 2014, Toxins 6(12):3296-3325. Such Cry proteins can be expressed as protoxins that contain additional domains IV-VII, which are generally not required for toxicity. In some embodiments, the putative insecticidal polypeptides provided herein include domains I, II, and III. In some embodiments, the putative insecticidal polypeptides include domains I, II, III, and at least one of domains IV, V, VI, and VII.

[0073] C. Insecticidal protein receptor In some embodiments, the putative insecticidal polypeptides of the present disclosure can interact with cell surface proteins of pest cells (e.g., insect cells). Pests that may be targeted by the putative insecticidal proteins may include any of those listed below. In some embodiments, the pest cells are epithelial cells. In some embodiments, the pest cells are part of a pest digestive system. The cell surface proteins of the present disclosure may include any known receptor or any potential receptor for an insecticidal protein. In some embodiments, the cell surface protein is a receptor for any of the putative insecticidal proteins listed above. In some embodiments, the cell surface protein is a known Bt Cry protein receptor. In some embodiments, the cell surface protein is an ATP-binding cassette (ABC) transporter protein, a cadherin protein, an aminopeptidase N protein, or an alkaline phosphatase protein. In some embodiments, the cell surface protein is a VIP receptor, such as, for example, VPAC1 and / or VPAC2. In some embodiments, the cell surface protein is a scavenger receptor (e.g., SR-C) (see, e.g., Wang et al., 2019, Appl. Environ. Microbiol. 85(16):e00579-19).

[0074] In some embodiments, the putative insecticidal polypeptide interacts with an ABC transporter protein. Recently, ABC transporters have been identified as receptors for Bacillus thuringiensis (Bt) insecticidal proteins (Sato et al., 2019, Toxins 11:124 and Endo et al., 2018, J. Biol. Chem. 293:8569-8577). For example, a Bt insecticidal toxin called Cry1Fa (Bt Cry insecticidal toxin) utilizes ABCC2 of Spodoptera frugiperda (Fall Armyworm) as its receptor to kill insects. Cry1Aa toxin binds to two of the six extracellular loops (ECLs) of Bm-ABCC2, ECL1 and ECL4, as described by Sato et al. (2019, Toxins 11:124). Cry1Fa is also predicted to bind to ECL1 and ECL4 of Sf-ABCC2 based on structural similarity (FIG. 8). In some embodiments, the ABC transporter protein has two transmembrane α-helical bundles, each consisting of six helices, as shown in FIG. 8. In some embodiments, between the two bundles is a first ATPase (nucleotide-binding) domain. In some embodiments, there is a second ATPase domain that is bound to the C'-terminus of the last α-helix of the second α-helical bundle. In some embodiments, both ATPase domains are in the cytoplasm (i.e., inside the cell) (FIG. 8). In some embodiments, ABC transporter proteins that do not contain a second ATPase retain the same conformation of the α-helical bundle and ECL (i.e., compared to ABC transporter proteins that have both ATPase domains). In some embodiments, the first ATPase domain (i.e., the ATPase domain between the two alpha-helical bundles described above) is necessary to help identify ECL1 and ECL4. In some embodiments, the ABC transporter protein does not comprise a second ATPase domain.

[0075] In some embodiments, the ABC transporter may include a protein from any of the ABC transporter subfamilies, including ABCA, ABCB, ABCC, ABCD, ABCE, ABCF, ABCG, and ABCH. The ABC transporter protein may be from any of the pests listed below. In some embodiments, the ABC transporter protein is PxABCC2, Bm-ABCC2, Sf-ABCC2, Sf-ABCC3, Dv-ABCB1, Bm-ABCB1, Sf-ABCB1, Bm-ABCA2, or Tc-ABCC4. In some embodiments, the ABC transporter protein is PxABCC2, Bm-ABCC2, Sf-ABCC2, or Sf-ABCC3. In some embodiments, the ABC transporter protein retains ABC transporter function. In some embodiments, the ABC transporter protein does not retain ABC transporter function (e.g., an ABC transporter without a second ATPase domain, as described above). In some embodiments, the ABC transporter protein comprises an amino acid sequence having at least 60% identity (e.g., at least 65% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity) to any of SEQ ID NOs: 5, 7, 9, 11, or 13. In some embodiments, the ABC transporter protein is encoded by a nucleic acid sequence having at least 60% identity to any of SEQ ID NOs: 6, 8, 10, 12, or 14.

[0076] In some embodiments, the cell surface protein may be from a pest known to be resistant to one or more insecticides. In some embodiments, the cell surface protein is a mutant cell surface protein known to have reduced interaction with one or more insecticidal proteins of interest. For example, Cry1Fa-resistant colonies of S. frugiperda were found to have a mutation in its ABCC2 that Cry1Fa was unable to bind (Banerjee et al., 2017, Sci. Rep. 7:10877). These reports indicate that insecticidal proteins can be engineered to bind to new receptors or mutants of existing receptors to overcome resistance. For example, new engineered proteins that bind to mutants of ABCC2 of Cry1Fa-resistant S. frugiperda may be able to overcome resistance. Similarly, engineered proteins that bind to S. frugiperda ABCC3 may be active against Cry1Fa-resistant S. frugiperda. In some embodiments, the cell surface protein comprises a mutant form of an insecticidal protein receptor. In some embodiments, the cell surface protein comprises a mutant of ABCC2 from Cry1Fa-resistant S. frugiperda. In some embodiments, the Sf ABCC2 mutant comprises an amino acid sequence having at least 60% identity to SEQ ID NO:11. In some embodiments, the Sf ABCC2 mutant is encoded by a nucleic acid sequence having at least 60% identity to SEQ ID NO:12.

[0077] In some embodiments, the cell surface protein comprises one or more modifications relative to the wild-type protein from which it is derived. In some embodiments, the modifications are the result of DNA shuffling, block swapping, site-directed mutagenesis, saturation mutagenesis, random mutagenesis, or a combination thereof (e.g., as described in Cong et al., ed. Akhurst et al., 2002, Proceedings of the 4th Pacific Rim Conferences on Biotechnology of Bacillus thuringiensis and its environmental impact: 118-123, U.S. Pat. No. 8,530,411, Hou et al., 2019, Toxins 11: 162, and U.S. Patent Publication No. 20210147492). In some embodiments, the modifications are deletions of one or more nucleotides compared to the wild-type protein. In some embodiments, the modifications are insertions of one or more nucleotides relative to the wild-type protein. In some embodiments, the modification is a truncation, ie, a deletion, of one or more nucleotides from the 5' and / or 3' end of the wild-type protein.

[0078] D. Target pest The insecticidal proteins provided herein can be active against a variety of target pests, including lepidopteran pests, coleopteran pests, hemipteran pests, dipteran pests, Lygus pests, and nematode pests.

[0079] In some embodiments, the insecticidal protein may have activity against one or more of the following non-limiting examples of lepidopteran pests: Spodoptera genus, such as S. frugiperda (fall armyworm), S. littoralis (Egyptian cotton leafworm), S. ornithogalli (yellowstriped armyworm), S. praefica (western yellowstriped armyworm), S. eridania (southern armyworm), S. litura (Common cutworm / Oriental cutworm), S. scutellari (Spodoptera spp. ... leafworm) and / or S. exigua (beet armyworm); the genus Ostrinia, for example O. nubilalis (European corn borer) and / or O. furnacalis (Asian corn borer); the genus Plutella, for example P. xylostella (diamondback moth); the genus Agrotis, for example A. ipsilon (black cutworm), A. segetum (common cutworm), A. gladiaria (claybacked cutworm), cutworm), and / or A. orthogonia (light-coloured western cutworm); the genus Striacosta, e.g. S. albicosta (western bean cutworm); the genus Helicoverpa, e.g. H.zea (corn earworm), H. punctigera (native budworm) and / or H. armigera (cotton bollworm); the genus Heliothis, such as H. virescens (tobacco budworm); the genus Diatraea, such as D. grandiosella (southwestern corn borer) and / or D. saccharalis (sugarcane borer); the genus Trichoplusia, such as T. ni (cabbage looper), looper); the genus Sesamia, for example S. nonagroides (Mediterranean corn borer), S. inferens (pink stem borer) and / or S. calamistis (pink stem borer); the genus Pectinophora, for example P. gossypiella (pink bollworm); the genus Cochylis, for example C. hospes (banded sunflower moth); the genus Manduca, for example M. sexta (tobacco hornworm), hornworm) and / or M. quinquemaculata (tomato hornworm); the genus Elasmopalpus, e.g. E. lignosellus (lesser cornstalk borer); the genus Pseudoplusia, e.g. P. includens (P.includens (soybean looper); spp. Anticarsia, e.g. A. gemmatalis (velvetbean caterpillar); spp. Plathypena, e.g. P. scabra (green cloverworm); spp. Pieris, e.g. P. brassicae (cabbage butterfly); spp. Papaipema, e.g. P. nebris (stalk borer); spp. Pseudaletia, e.g. P. unipuncta (common armworm); armyworm); the genus Peridroma, for example P. sausia (variegated cutworm); the genus Keiferia, for example K. lycopersicella (tomato pinworm); the genus Artogeia, for example A. rapae (imported cabbageworm); the genus Phthorimaea, for example P. operculella (potato tuber moth); the genus Chrysodeixis, for example C. includens (soybean looper); the genus Feltia, for example F. ducens (dingy cutworm); cutworm); the genus Chilo, e.g. C. suppressalis (striped stem borer), the genus Cnaphalocrocis, e.g. C. medinalis (rice leaffolder), the genus Conogethes, e.g. C. punctiferalis (C.punctiferalis (Yellow peach moth), Mythimna spp. such as M. separata (Oriental armyworm), Atetis spp. such as A. lepigone (Two-spotted armyworm), or any combination thereof.

[0080] In some embodiments, the insecticidal protein can have activity against coleopteran pests. In embodiments, the disclosed proteins have activity against the genus Diabrotica. Diabrotica is a genus of coleopteran beetles commonly referred to as "corn rootworms" or "cucumber beetles." Exemplary Diabrotica species include, but are not limited to, Diabrotica barberi (northern corn rootworm), D. virgifera virgifera (western corn rootworm), D. undecimpunctata howardii (southern corn rootworm), D. balteata (banded cucumber beetle), D. undecimpunctata undecimpunctata (western spotted cucumber beetle), D. scutellari (striped ... beetle), D. significata (3-spotted leaf beetle), D. speciosa (chrysanthemum beetle), D. virgifera zeae (Mexican corn rootworm), rootworm), D. beniensis, D. cristata, D. curviplustalata, D. dissimilis, D. elegantula, D. emorsitans, D. graminea, D. hispanloe, D. lemniscata, D.including D. linsleyi, D. milleri, D. nummularis, D. occlusal, D. porrecea, D. scutellata, D. tibialis, D. trifasciata and D. viridula; and any combination thereof. Other non-limiting examples of coleopteran pests according to the present invention include the genus Leptinotarsa, such as L. decemlineata (Colorado potato beetle); the genus Chrysomela, such as C. scripta (Cottonwood leaf beetle); the genus Hypothenemus, such as H. hampei (coffee berry borer); the genus Sitophilus, such as S. zeamais (maize weevil); the genus Epitrix, such as E. hirtipennis (tobacco flea beetle); beetle), E. cucumeris (potato leaf beetle); the genus Phyllotreta, for example P. cruciferae (crucifer flea beetle), and P. pusilla (western black flea beetle); the genus Anthonomus, for example A. eugenii (pepper weevil); the genus Hemicrepidus, for example H. memnonius (wireworm); the genus Melanotus, for example M. communis (wireworm);communis (wireworm); the genus Ceutorhychus, for example C. assimilis (cabbage seedpod weevil); the genus Phyllotreta, for example P. cruciferae (crucifer flea beetle); the genus Aeolus, for example A. mellillus (wireworm); the genus Aeolus, for example A. mancus (wheat wireworm); the genus Horistonotus, for example H. uhlerii (sand wireworm); the genus Sphenophorus such as S. maidis (maize billbug), S. zeae (timothy billbug), S. parvulus (bluegrass billbug), and S. callosus (southern corn billbug); the genus Phyllophaga (White grubs); the genus Chaetocnema such as C. pulicaria (corn flea beetle); the genus Popillia such as P. japonica (Japanese beetle); beetle); the genus Epilachna, such as E. varivestis (Mexican bean beetle); the genus Cerotoma, such as C. trifurcate (bean leaf beetle); the genus Epicauta, such as E. pestifera and E. lemniscata (E.lemniscata (Blister beetle); and any combination of the above.

[0081] The disclosed insecticidal proteins can be active against Hemiptera, Diptera, Lygus, and / or other borer and sucker insects of the orders Orthoptera or Thysanoptera. Dipteran insects include, but are not limited to, the genus Liriomyza, such as L. trifolii (leafminer) and L. sativae (vegetable leafminer); the genus Scrobipalpula, such as S. absoluta (tomato leafminer); the genus Delia, such as D. platura (seedcorn maggot), D. brassicae (cabbage maggot) and D. radicum (cabbage root fly). fly); Psilia genus, such as P. rosae (carrot rust fly); Tetanops genus, such as T. myopaeformis (sugarbeet root maggot); and any combination of the above. Orthoptera insects include, but are not limited to, the genus Melanoplus, such as M. differential grasshopper, M. femurrubrum (Redlegged grasshopper), M. bivittatu (Two striped grasshopper), M. schizophyllum (Siberian ... Insects of the order Orthoptera include, but are not limited to, any now known or later identified Orthoptera insects, including, but not limited to, any insects of the order Orthoptera ...Thysanoptera insects include any now known or later identified Thysanoptera insects, including, but not limited to, the genus Frankliniella, e.g., F. occidentalis (western flower thrips) and F. fusca (tobacco thrips); and the genus Thrips, e.g., T. tabaci (onion thrips), T. palmi (melon thrips); and any combination of the above.

[0082] The disclosed insecticidal proteins may be active against nematodes. As used herein, the term "nematodes" includes any now known or later identified organism classified in the kingdom Animalia, phylum Nematoda, including, but not limited to, the orders Diplopoda (e.g., Enoplida, Isolaimida, Mononchida, Dorylaimida, Trichocephalida, Mermithida, Muspiceida, Araeolaimida, Chromadorida, and the like). dorida, Desmoscolecida, Desmodorida, and Monhysterida) and / or nematodes (e.g., Rhabdita, Strongylida, Ascaridida, Spirurida, Camallanida, Diplogasterida, Tylenchida, and Aphelenchida). Nematodes include, but are not limited to, parasitic nematodes such as root-knot nematodes, cyst nematodes, and / or lesion nematodes. Exemplary genera of nematodes according to the present invention include, but are not limited to, Meloidogyne (root-knot nematodes), Heterodera (cyst nematodes), Globodera (cyst nematodes), Radopholus (burrowing nematodes), Rotylenchulus (reniform nematodes), Pratylenchus (lesion nematodes), Aphelenchoides (foliar nematodes), Helicotylenchus (spiral nematodes), and Helicotylenchus (spiral nematodes).nematodes), Hoplolaimus (lance nematodes), Paratrichodorus (stubby-root nematodes), Longidorus, Nacobus (false root-knot nematodes), Subanguina, Belonlaimus (sting nematodes), Criconemella, Criconemoides (sting nematodes), Ditylenchus, Dolichodorus, Hemicriconemoides, Hemicycliophora, Hirschmaniella, Hypsoperine, Macroposthonia, Melinius, Punctodera, Quinisulcius, Scutellonema, Xiphinema (dagger nematodes), Tylenchorhynchus (stunt nematodes), Tylenchulus, Bursaphelenchus (round worms), and any combination thereof. Exemplary plant parasitic nematodes according to the present disclosure include, but are not limited to, Belonolaimus gracilis, Belonolaimus longicaudatus, Bursaphelenchus xylophilus (pine wood nematode), Criconemoides ornata, Ditylenchus destructor (potato rot nematode), and the like.nematode), Ditylenchus dipsaci (stem and bulb nematode), Globodera pallida (potato cyst nematode), Globodera rostochiensis (golden nematode), Heterodera glycines (soybean cyst nematode), Heterodera schachtii (sugar beet cyst nematode); Heterodera zeae (corn cyst nematode), Heterodera avenae avenae (cereal cyst nematodes), Heterodera carotae, Heterodera trifolii, Hoplolaimus columbus, Hoplolaimus galeatus, Hoplolaimus magnistylus, Longidorus breviannulatus, Meloidogyne arenaria, Meloidogyne chitwoodi, Meloidogyne hapla, Meloidogyne incognita, Meloidogyne javanica javanica, Mesocriconema xenoplax, Nacobbus aberrans, Naccobus dorsalis, Paratrichodorus christiei, Paratrichodorus minorminor, Pratylenchus brachyurus, Pratylenchus crenatus, Pratylenchus hexincisus, Pratylenchus neglectus, Pratylenchus penetrans, Pratylenchus projectus, Pratylenchus scribneri, Pratylenchus tenuicaudatus, Pratylenchus thornei, Pratylenchus zeae, Punctodera chacoensis chaccoensis, Quinisulcius acutus, Radopholus similis, Rotylenchulus reniformis, Tylenchorhynchus dubius, Tylenchulus semipenetrans (citrus nematode), Siphinema americanum, X. mediterraneanum, and any combination thereof.

[0083] E. Linker In some embodiments of the recombinant nucleic acid provided herein, the nucleic acid sequence encodes a linker. In some embodiments, the linker links the spore coat polypeptide to the putative insecticidal polypeptide. In some embodiments, the spore coat polypeptide and the putative insecticidal polypeptide are structurally isolated by the linker. The "structurally isolated" polypeptides in the fusion proteins described herein can fold into their proper form and thus maintain functionality. For example, an insecticidal polypeptide structurally isolated from a spore coat polypeptide can fold into its proper shape and maintain 1) the ability to bind to a target protein (e.g., an insect cell surface protein receptor) and 2) its insecticidal activity. Similarly, a spore coat polypeptide structurally isolated from a putative insecticidal protein can fold into its proper shape and maintain the ability to position the fusion protein in the spore coat. An important feature of the linkers described herein is the ability to keep the putative insecticidal polypeptide attached to the spore in which it is expressed. The linkers of the present disclosure can be optimized to produce a desired effect (e.g., structural separation) in the fusion protein encoded by the recombinant nucleic acid. Linker design aspects and considerations are described, for example, in Klein et al., 2014, Protein Eng. Des. Sel. 27(10):325-330. In some embodiments, the linker is resistant to protease digestion. In some embodiments, the protease digestion resistance of the linker keeps the putative insecticidal polypeptide linked to the spore coat polypeptide (i.e., keeps the putative insecticidal protein attached to the surface of the spore in which it is expressed). In some embodiments, the linker is resistant to protease digestion in a protease-rich environment (e.g., sporulation and / or lysed Bacillus culture medium).

[0084] In some embodiments, the linker described herein comprises at least one amino acid. In some embodiments, the linker is a polypeptide comprising at least two amino acids. The linker sequence can increase the range of orientations that the polypeptide of the fusion protein can adopt. The peptide linker can be, for example, 1 to 100 or more amino acids long (e.g., 1 aa, 2 aa, 3 aa, 4 aa, 5 aa, 10 aa, 15 aa, 20 aa, 25 aa, 30 aa, 35 aa, 40 aa, 45 aa, 50 aa, 55 aa, 60 aa, 65 aa, 70 aa, 75 aa, 80 aa, 85 aa, 90 aa, 95 aa, 100 aa or more). Depending on the length, the linker sequence can have various conformations in the secondary structure, such as helix, beta strand, coil / bend, and turn. In some examples, the linker sequence can have an extended conformation and function as an independent domain that does not interact with adjacent protein domains. Linker sequences can be structurally flexible or structurally rigid. Flexible linkers provide some degree of movement or interaction between polypeptide domains and are generally rich in small or polar amino acids such as Gly and Ser. Rigid linkers can be used to help maintain a certain distance between domains and maintain their independent functions.

[0085] In some embodiments, the recombinant nucleic acids described herein encode a linker having at least 60% identity (e.g., at least 65% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity) to any of the linkers in Table 1 or any of the linkers described in Klein et al., 2014, Protein Eng. Des. Sel. 27(10):325-330. In some embodiments, the linker is a polypeptide comprising an amino acid sequence having at least 60% identity (e.g., at least 65% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity) to any of SEQ ID NOs: 20, 21, or 38-61. In some embodiments, the linker comprises a sequence derived from a hydrophilic protein (e.g., maltose binding protein). In some embodiments, the linker comprises one or more repeats of the amino acid sequence GGGGS (SEQ ID NO:38) followed by an alanine residue and a serine residue. The number of GGGGS repeats can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more. In some embodiments, the linker comprises a Bt Cry protein leader sequence (e.g., the amino acid sequence of SEQ ID NO:20 or SEQ ID NO:21).

[0086] [Table 1]

[0087] [Table 2]

[0088] [Table 3]

[0089] F. DNA Constructs and Vectors Also provided herein is a DNA construct comprising a promoter operably linked to any of the recombinant nucleic acids described herein. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. A number of promoters can be used in the constructs described herein. A promoter is a region or sequence located upstream and / or downstream of the transcription start that is involved in the recognition and binding of RNA polymerase and other proteins to initiate transcription. A promoter can be a eukaryotic promoter or a prokaryotic promoter. In some embodiments, the promoter is an inducible promoter. In some aspects, the promoter is a constitutive promoter. In some embodiments, the promoter is an endogenous promoter for a spore coat polypeptide gene (i.e., the promoter drives expression of the gene in its natural genomic context). In some embodiments, the promoter is an endogenous promoter for the particular spore coat polypeptide gene encoded by the recombinant nucleic acid. In some aspects, the promoter is an endogenous CotC or CotG promoter. In some embodiments, the promoter is a CotA, CotB, CotD, CotE, CotX, CotY, or CotZ promoter. In some embodiments, the promoter is a Bacillus species promoter. In some embodiments, the promoter is under sigma K factor control in Bacillus species.

[0090] In some embodiments, the promoter is a sporulation-specific promoter. Genes involved in spore synthesis and structure have been identified and cloned, and promoter sequences from such genes have been isolated and characterized. Those skilled in the art will understand that by selecting among these promoters and regulatory sequences, it is possible to control the physical location of expression of a polypeptide of interest in the spore or vegetative cell, as well as the timing of expression in the life cycle of the spore and / or vegetative cell. See, for example, Hill et al., Soc. Appl. Bacteriol. Symp. Ser. 23:129S-134S (1998), which demonstrates that model proteins such as luciferase and β-galactosidase can be directed to endospores during the sporulation process by operably linking the nucleic acid sequences encoding these proteins to a sporulation-specific promoter. Hill et al. further demonstrate that these model enzymes are effectively conserved and retain their activity. See also U.S. Patent Application Publication No. 20030165538.

[0091] The recombinant nucleic acids provided herein can be included in an expression cassette for expression in a host cell or organism of interest. The cassette can include 5' and 3' regulatory sequences operably linked to the recombinant nucleic acids provided herein that allow for expression of a fusion protein. The cassette can further include at least one additional gene or genetic element that is co-transformed into the organism. When additional genes or elements are included, the components are operably linked. Alternatively, the additional genes or elements can be provided on multiple expression cassettes. Such expression cassettes comprise multiple restriction and / or recombination sites for insertion of polynucleotides under the transcriptional control of the regulatory regions. The expression cassette can include, in the 5' to 3' direction of transcription: a transcriptional and translational initiation region (i.e., a promoter) that functions in the cell or organism of interest, a recombinant nucleic acid disclosed herein, and a transcriptional and translational termination region (i.e., a termination region). The promoters described herein can direct or drive expression of a coding sequence in a host cell. The regulatory regions (i.e., promoters, transcriptional regulatory regions, and translational termination regions) can be endogenous or heterologous to the host cell or each other. As used herein, "heterologous" with respect to a sequence is a sequence that originates from a foreign species or, if from the same species, is substantially altered in composition and / or genomic locus from its native form by deliberate human intervention.

[0092] Additional regulatory signals include, but are not limited to, transcription initiation sites, operators, activators, enhancers, other regulatory elements, ribosome binding sites, start codons, termination signals, etc. See Sambrook et al. (1992) Molecular Cloning: A Laboratory Manual, ed. Maniatis et al. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY); Davis et al., eds. (1980) Advanced Bacterial Genetics (Cold Spring Harbor Laboratory Press), Cold Spring Harbor, NY, and references cited therein.

[0093] The expression cassette may also contain a selectable marker gene for selecting transformed cells. Marker genes include genes that confer antibiotic resistance, such as those that confer hygromycin resistance, ampicillin resistance, gentamicin resistance, neomycin resistance, to name a few. Additional selectable markers are known and any may be used.

[0094] In preparing an expression cassette, various DNA fragments can be manipulated to provide the DNA sequences in the proper orientation and, if necessary, in the proper reading frame. To this end, DNA fragments can be joined using adapters or linkers, or other manipulations can be involved to provide convenient restriction sites, removal of excess DNA, removal of restriction sites, etc. To this end, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, such as transitions and transversions, can be involved.

[0095] Further provided herein is a vector comprising a recombinant nucleic acid or expression cassette as described herein. It is contemplated that the vector has the necessary functional elements to direct and regulate the transcription of the inserted nucleic acid. These functional elements include, but are not limited to, a promoter, a region upstream or downstream of the promoter, such as an enhancer that can regulate the transcriptional activity of the promoter, an origin of replication, a suitable restriction site to facilitate cloning of an insert adjacent to the promoter, an antibiotic resistance gene or other marker that can be useful for selecting cells containing the vector or a vector containing an insert, an RNA splice junction, a transcription termination region, or any other region that can be useful for facilitating the expression of the inserted gene or hybrid gene (see generally Sambrook et al. Molecular Cloning: A Laboratory Manual, 4). th (Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2012). The vector can be, for example, a plasmid.

[0096] Also provided are host cells comprising the recombinant nucleic acids, DNA constructs, or vectors described herein. In some embodiments, the host cells are bacterial or fungal cells. The host cells can be any spore-producing microbial cell, including, for example, cells derived from any of the bacterial or fungal species listed above. The host cells can be in vitro, ex vivo, or in vivo host cells. Populations of any of the host cells described herein are also provided. Also provided are cell cultures comprising one or more host cells described herein. Methods for the culture and production of many cells, including cells of bacterial (e.g., E. coli and other bacterial strains) origin, are available in the art. See, e.g., Sambrook (supra), Ausubel et al., (2010) Current protocols in molecular biology, John Wiley and Sons, New York, and Berger and Kimmel (1987) Guide to Molecular Cloning Techniques, Methods in Enzymology 152:3-812, and Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, 3 rd Ed., Wiley-Liss, NY and references cited therein; Doyle and Griffiths (1997) Mammalian Cell Culture: Essential Techniques John Wiley and Sons, NY; Humason (1979) Animal Tissue Techniques, 4 th See Ed. WH Freeman and Company; and Ricciardelli, et al., (1989) In vitro Cell Dev. Biol. 25:1016-1024.

[0097] As used herein, the phrase "introducing" in the context of introducing a nucleic acid into a cell refers to the translocation of a nucleic acid sequence from outside the cell to inside the cell. Various methods of such transfer are contemplated, including, but not limited to, electroporation, nanoparticle delivery, viral delivery, contact with nanowires or nanotubes, receptor-mediated internalization, cell-penetrating peptide-mediated transfer, liposome-mediated transfer, DEAE-dextran, lipofectamine, calcium phosphate, or any method now known or to be identified in the future for introducing a nucleic acid into a prokaryotic or eukaryotic host. Targeted nuclease systems (e.g., RNA-guided nucleases (CRISPR-Cas9), transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), or megaTALs (MTs) (Li et al. Signal Transduction and Targeted Therapy 5, Article No. 1 (2020)) can also be used to introduce nucleic acids, such as nucleic acids encoding recombinant proteins described herein, into a host cell.

[0098] G. Fusion Proteins Also provided herein are fusion proteins encoded by any of the recombinant nucleic acids, DNA constructs, or vectors described herein.

[0099] Any of the polypeptides or proteins described herein can further comprise a detectable moiety, such as a fluorescent protein or a fragment thereof. Examples of fluorescent proteins include, but are not limited to, yellow fluorescent protein (YFP, e.g., Venus), green fluorescent protein (GFP), and red fluorescent protein (RFP), as well as derivatives of these proteins, such as mutant derivatives. See, for example, Chudakov et al. "Fluorescent Proteins and Their Applications in Imaging Living Cells and Tissues", Physiological Reviews 90(3):1103-1163 (2010); and Specht et al., "A Critical and Comparative Review of Fluorescent Tools for Live-Cell Imaging", Annual Review of Physiology 79:93-117 (2017).

[0100] Any of the polypeptides or proteins described herein can further comprise a domain or sequence useful for protein isolation. In some embodiments, the polypeptide comprises an affinity tag, such as a FLAG tag (SEQ ID NO:24), a Myc tag (EQKLISEEDL (SEQ ID NO:26)), a polyhistidine tag (e.g., an 8XHis tag (SEQ ID NO:27)), an albumin binding protein, alkaline phosphatase, an AU1 epitope, an AU5 epitope, a biotin-carboxy carrier protein (BCCP), to name a few. In some embodiments, the affinity tag is useful for protein isolation. See Kimple et al. "Overview of Affinity Tags for Protein Purification", Curr. Protoc. Protein Sci. 73:Unit-9.9 (2013). In some embodiments, the polypeptides or proteins described herein comprise a signal sequence useful for protein isolation. See, e.g., Low et al. "Optimisation of signal peptide for recombinant protein secretion in bacterial host" Applied Microbiology and Biotechnology 97:3811-3826 (2013). In some embodiments, the polypeptides or proteins described herein comprise a protease recognition site. Such protease recognition sites can be useful, inter alia, to allow removal of a signal peptide or affinity purification tag following protein isolation.

[0101] H. spores Also provided herein is a microbial spore comprising any of the recombinant nucleic acids, DNA constructs, vectors, or fusion proteins described herein. In some embodiments, the fusion protein is expressed as a component of the spore. In some embodiments, the fusion protein is displayed on the surface of the spore.

[0102] In some embodiments, the spores are bacterial or fungal spores. In some embodiments, the spores are from any of the spore-producing microorganisms listed in Section A of this disclosure. In some embodiments, the spores are from a Bacillus species that is not known to produce insecticidal proteins in its spores.

[0103] Also provided herein is a composition comprising a mixture of spores, wherein at least two of the spores in the mixture comprise different putative insecticidal polypeptides. In some embodiments, the composition comprises a plurality of spores described herein, wherein the plurality of spores comprises spores comprising recombinant nucleic acids encoding different putative insecticidal polypeptides. For example, the mixture or plurality of spores may comprise a spore comprising a first recombinant nucleic acid encoding a first putative insecticidal polypeptide and a spore comprising a second recombinant nucleic acid encoding a second putative insecticidal polypeptide. The mixture or plurality of spores may comprise spores encoding a wide range of putative insecticidal polypeptides. In some embodiments, the mixture or plurality of spores comprises spores comprising up to 10x10^8 different putative insecticidal polypeptides (e.g., up to 10x10^7, up to 10x10^6, or up to 10x10^5). In some embodiments, the composition comprises a plurality of spores described herein, wherein each of the plurality of spores comprises a recombinant nucleic acid encoding a different putative insecticidal polypeptide.

[0104] IV. METHODS FOR IDENTIFYING INSECTICID PROTEINS AND INSECTICID PROTEIN RECEPTORS In another aspect, a method for screening and identifying insecticidal proteins is provided herein. In some embodiments, the method comprises displaying an insecticidal protein library on bacterial or fungal spores (e.g., spores of Bacillus species) and screening against one or more receptor proteins. In some embodiments, the method comprises contacting the fusion protein or a spore containing the fusion protein with the insecticidal protein receptor. In some embodiments, the insecticidal protein receptor protein is an ABC transporter protein. In some embodiments, the method comprises detecting an interaction between the fusion protein or the spore and the insecticidal protein receptor. In some embodiments, such an interaction indicates that the putative insecticidal protein of the fusion protein or the spore is an insecticidal protein.

[0105] In some embodiments, the fusion protein or spores are contacted with a purified insecticidal protein receptor protein. In some embodiments, the fusion protein or spores are contacted with a crude extract containing the insecticidal protein receptor protein. In some embodiments, the fusion protein or spores are contacted with the insecticidal protein receptor in an environment that mimics the relevant biological environment. For example, the simulated environment can be an insect midgut environment. The environment can be a neutral or alkaline pH (i.e., pH level of 7.0 or higher) environment, such as a buffer solution. In some embodiments, the pH is maintained at 7.5 pH using Tris-buffered saline (TBS). In some embodiments, the pH is maintained at 8.0 pH to 11.0 pH. For example, the pH can be maintained up to 11.0 pH (e.g., 11 pH, 10.5 pH, 10 pH, 9.5 pH, 9 pH, 8.5 pH, or 8 pH). An appropriate alkaline buffer (e.g., pK of 8.2 or higher) can be used. aBuffers having a pH of about 10 are known in the art and include, but are not limited to, CAPS, CABS, cholamine chloride, MOPS, BES, TES, HEPES, DIPSO, MOBS, acetamidoglycine, TAPSO, TEA, POPSO, HEPPSO, EPS, HEPPS, tricine, tris, glycinamide, glycylglycine, HEPBS, bicine, TAPS, AMPB, CHES, CAPSO, and AMP. In some embodiments, a CAPS buffer or a CABS buffer is used to maintain the pH at about 10.

[0106] In some embodiments, the insecticidal protein receptor is a membrane protein. Specific production and / or isolation methods can be used to purify the membrane protein (i.e., to maintain the structure and / or function of the membrane protein). In some embodiments, the protein is purified as part of a copolymer. For example, in some embodiments, the insecticidal protein receptor is purified as part of a styrene maleic acid (SMA) lipid particle (SMALP) (e.g., by the method described in Example 7 herein). In some embodiments, the receptor protein is purified as part of an SMA copolymer having a specific S;M ratio (i.e., the ratio of styrene to maleic anhydride in the copolymer). This ratio can be determined by experimentation by one of skill in the art. In some embodiments, the S:M ratio for the purification of ABC transporter proteins is 2:1 to 3:1 (e.g., 2:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, or 3:1). By preparing insecticidal protein receptor proteins (e.g., ABC transporter proteins) in SMALPs, the structure and function of the receptor protein can be preserved. The use of SMALP-ABC transporter proteins in Cry binding assays has not been described before. Previously, detergents such as DDM have been used to purify ABC transporters for Cry binding assays. Without being bound to any particular theory, the structure and function of ABC transporters may be adversely affected by detergents that displace or remove insect phospholipids that support the structure of the ABC transporter.

[0107] The insecticidal protein receptor protein of the present disclosure may be expressed in and / or purified from any suitable cell (e.g., insect cell, prokaryotic cell, eukaryotic cell, etc.). In some embodiments, the receptor protein is expressed in an insect cell. In some aspects, the receptor protein is expressed from a baculovirus expression vector. Recombinant proteins produced in insect cells using a baculovirus vector undergo post-translational modification. Suitable vectors are known to those of skill in the art. Insect cells useful for expressing the receptor protein herein include Sf9 cells, Sf21 cells, silkworm pupa cells, or other insect cell lines known to those of skill in the art. Other cells that may be useful for expressing the receptor protein herein include Expi293 cells and HEK293 cells. See Willcoxon et al., 2016, J.Biotech 217:72-81 and Niu et al., 2020, Sci.Reports 2020(10):15830.

[0108] In some embodiments, the receptor protein is expressed in and purified from prokaryotic cells. There are numerous E. coli expression vectors known to those skilled in the art that are useful for expressing and purifying receptor proteins. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis, and other Enterobacteriaceae, such as Salmonella, Senatia, and various Pseudomonas species. In these prokaryotic hosts, expression vectors can also be made that typically contain expression control sequences (e.g., origins of replication) compatible with the host cell. Additionally, a promoter is operably linked to the sequence encoding the receptor protein. Exemplary well-known promoters include the lactose promoter system, the tryptophan (Trp) promoter system, the beta-lactamase promoter system, and the promoter system from phage lambda. Additionally, yeast expression can be used, for example, the receptor protein can be expressed by Pichia pastoris or S. cerevisiae.

[0109] Mammalian cells also allow for the expression and purification of proteins. Vectors useful for the expression of active proteins in mammalian cells are known in the art and can include genes that confer hygromycin resistance, geneticin or G418 resistance, or other genes or phenotypes suitable for use as selection markers, or methotrexate resistance for gene amplification. Several suitable host cell lines capable of secreting intact proteins have been developed in the art, including CHO cells, HeLa cells, HEK-293 cells, HEK-293T cells, U2OS cells, or any other primary or transformed cell line. Other suitable host cell lines include COS-7 cells, myeloma cell lines, Jurkat cells, and the like. Expression vectors for these cells can include expression control sequences such as origins of replication, promoters, enhancers, and necessary information processing sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences.

[0110] The expression vector for the expression of the proteins described herein may also include a nucleic acid encoding the proteins described herein under the control of an inducible promoter, such as a tetracycline-inducible promoter or a glucocorticoid-inducible promoter. The nucleic acid may also be under the control of a tissue-specific promoter to promote expression of the nucleic acid in a particular cell, tissue or organ. Any regulatable promoter is also contemplated, such as metallothionein promoters, heat shock promoters, and other regulatable promoters, of which many examples are known in the art. In addition, the Cre-loxP inducible system, as well as the Flp recombinase inducible promoter system, both of which are known in the art, may also be used.

[0111] In some embodiments of the methods provided herein, the fusion protein or spores are contacted with an insecticidal protein receptor on the surface of a cell (i.e., not a purified protein or a protein present in a cell lysate). In some embodiments, the cell is an insect cell (e.g., derived from any of the insects listed in Section D: Target Pests above). In some embodiments, the cell is part of a cell culture. In some embodiments, the cell is present in a live insect.

[0112] In some embodiments, detecting the interaction between the fusion protein or spore and the insecticidal protein receptor comprises performing immunomagnetic separation, flow cytometry, cytotoxicity assay, sequencing, or a combination thereof. In some embodiments, an epitope on the insecticidal protein receptor protein (e.g., FLAG epitope) is used with an antibody against the epitope to purify the insecticidal protein receptor (e.g., via immunomagnetic separation). If the fusion protein or spore interacts with the receptor, it is purified together with the receptor protein. In some embodiments, the spore that interacts with the receptor protein can be identified by flow cytometry. In some embodiments, the spore that interacts with the receptor protein can be identified by cytotoxicity assay (e.g., as described in Example 5 herein).

[0113] In some embodiments of the methods provided herein, the putative insecticidal polypeptide is tethered to the spore (e.g., via fusion to a spore coat polypeptide), allowing the putative insecticidal polypeptide to be identified after sorting of bound spores (i.e., spores that have interacted with the receptor protein). This aspect allows for high-throughput screening and distinguishes the methods herein from previous insect-active toxin screening methods in which putative insecticidal proteins had to be individually produced in a host microorganism (e.g., in a 96-well plate with each well having a host cell expressing a different putative insecticidal protein) and tested separately against the receptor protein.

[0114] In some embodiments, detecting an interaction between the fusion protein or spore and the insecticidal protein receptor further comprises isolating a recombinant nucleic acid encoding the fusion protein that interacts with the insecticidal protein receptor protein and sequencing to determine the identity of the insecticidal protein.

[0115] In some embodiments, the methods provided herein include the following steps (e.g., as demonstrated in the Examples herein), which, although numbered, certain steps may be performed in a different order and / or simultaneously than those listed. 1. A library of diversified insecticidal proteins is generated by using various protein engineering techniques and isolating naturally occurring insecticidal proteins; 2. Cloning the library from step 1 into a spore display vector to display the proteins on the surface of spores; 3. A protein sample of the desired receptor (e.g., an ABCC2 mutant from a Cry1Fa-resistant S. frugiperda colony to overcome resistance) is produced in a receptor expression system (e.g., an insect cell line such as Sf9 using a baculovirus expression system or a live insect such as a silkworm); 4. The receptor protein is extracted from the cell membrane so that its native protein structure is preserved and purified for the next step; 5. The spores produced from step 2 are screened for their ability to bind to the receptor produced in step 4 using cell sorting techniques such as immunomagnetic separation and flow cytometry; 6. Repeat steps 1-5 above until one or more insecticidal proteins are obtained.

[0116] In some embodiments, a method for identifying insecticidal proteins capable of binding to a particular insecticidal protein receptor is provided, the method comprising: (a) displaying a library of putative insecticidal proteins on the surface of microbial endospores from a spore display vector cloned into a host microorganism in an orientation such that the receptor-binding portion of the putative insecticidal protein is exposed to solvent and capable of binding to the insect cell receptor protein; (b) identifying the insecticidal protein receptor protein (i.e., toxin receptor) in a form that retains the native receptor protein structure and is capable of binding to the putative insecticidal protein; (c) using cell (particle) sorting techniques (e.g., flow cytometry or magnetic sorting) to sort the putative insecticidal spores based on the binding ability of the putative insecticidal protein to the insect cell membrane protein prepared in (b); (d) optionally determining the genetic sequence of the putative insecticidal protein tethered to the spores that bind to the receptor protein; and, optionally, (e) determining the cytotoxicity of the toxin protein against the spores sorted in (c) using cultured cells such as Sf9 and HEK293 in which the toxin receptor is cloned and expressed.

[0117] Methods for identifying insecticidal protein receptors for a particular insecticidal protein are also contemplated herein. In some embodiments, a fusion protein or spore containing a known or putative insecticidal polypeptide can be contacted with a library of receptor proteins (e.g., as described herein). In some embodiments, the library includes known receptors or engineered synthetic receptors (e.g., produced by DNA shuffling, block swapping, site-directed mutagenesis, saturation mutagenesis, random mutagenesis, or combinations thereof). In some embodiments, an interaction between the fusion protein or spore is detected as described above (e.g., using an epitope on the fusion protein in an immunomagnetic separation). In some embodiments, such an interaction indicates that the receptor protein is a receptor for the insecticidal polypeptide of the fusion protein or spore.

[0118] V. Pest Control Methods Also contemplated herein is a method of using the fusion protein or spore described herein as a biological agent for pest control. As used herein, "pest control" refers to the reduction of the adverse effect of a pest on a plant. A pest or plant pest is a species, strain, or biotype of a plant, animal, or pathogen that directly or indirectly damages a plant or plant product, or that causes damage to or disease in a plant or plant product. In the context of this disclosure, a "pest" is an insect pest. In some embodiments, the pest is any of the pests listed in Section III.D above. In some embodiments, pest control is achieved by biocidal activity (i.e., by killing the pest). With respect to pests, biocidal activity can be referred to as insecticidal activity. In some embodiments, pest control is achieved by reducing the impact of a pest on a plant.

[0119] In some embodiments, a composition comprising a fusion protein or spores as described herein is applied to a plant, plant seed or plant propagation material (e.g., a seed, a leaf, a stem, a root, or any other plant tissue). In some embodiments, the composition comprises a bacterium producing a spore as described herein. In some embodiments, the composition comprises other pesticides known in the art. In some embodiments, the composition comprises additional auxiliary ingredients conventionally used in the art, such as excipients, stabilizers, carriers, dispersants, fertilizers, nutrients, growth additives, stabilizers, "slow release" aids, colorants, and where appropriate, surface active substances (surfactants). Suitable auxiliary ingredients include all substances conventionally used in crop protection products.

[0120] In some embodiments, the composition comprises one or more excipients. The one or more excipients can be one or more stabilizers, one or more additives, one or more carriers, one or more dispersants, one or more fertilizers, or any combination thereof. In one example, the one or more excipients comprises acetone.

[0121] In some embodiments, the composition comprises one or more stabilizers and / or other additives. The stabilizers and / or additives include, but are not limited to, penetrants, adhesives, anti-caking agents, dyes, dispersants, wetting agents, emulsifiers, defoamers, antibacterial agents, antifreeze, pigments, colorants, buffers and carriers. The composition may further comprise surfactants and / or adjuvants.

[0122] In some embodiments, the compositions disclosed herein may further comprise one or more carriers. Examples of carriers include, but are not limited to, solid carriers, sponges, fabrics, and synthetic materials. The synthetic materials may be porous synthetic materials. Additional carriers may include organic carriers such as wax, linoleum, paraffin, dextrose granules, sucrose granules, and maltose-dextrose granules. Alternatively, the carrier may be an inorganic carrier such as natural clay, kaolin, pyrophyllite, bentonite, alumina, montmorillonite, diatomaceous earth, chalk, diatomaceous earth, calcium phosphate, calcium and magnesium carbonate, sulfur, lime, flour, or talc. The composition may be adsorbed onto the carrier.

[0123] In some embodiments, the composition includes one or more dispersants. The dispersants may be negatively charged anionic dispersants. The dispersants may be non-ionic dispersants.

[0124] In some embodiments, the composition comprises a fertilizer, nutrient, or other growth additive. The fertilizer may be a chemical fertilizer. The fertilizer may be an organic fertilizer. The fertilizer may be an inorganic fertilizer. The fertilizer may be a granular or powdered fertilizer. The fertilizer may be a liquid fertilizer. The fertilizer may be a slow release fertilizer.

[0125] In some embodiments, the pest control methods provided herein include treating a plant, plant seed, or plant propagation material with a pesticidal amount of the fusion protein or spores described herein (or a composition comprising the fusion protein or spores described herein). As used herein, a "pesticidal amount" refers to an amount of the fusion protein, spore, or composition that can kill, control, or infect a target pest, slow the growth or reproduction of a target pest, reduce the population of a target pest, and / or reduce damage to the plant caused by a target pest. The pesticidal amount of a given fusion protein, spore, or composition will vary depending on factors including, but not limited to, the plant species, surface area of ​​the plant, plant seed, or plant propagation material, type of carrier, the presence or absence of other active ingredients, method of formulation, route of delivery, the particular fusion protein or spore used, the source of the fusion protein or spore (i.e., the spore-producing microorganism that produces the fusion protein or spore), the target pest species, and the severity of the pest infection or damage to the plant.

[0126] The pest control methods provided herein can be carried out in any suitable manner known to those skilled in the art, depending on the intended purpose and the prevailing circumstances, i.e., by spraying, wetting, injecting, misting, dusting, brushing, seed dressing, dusting or injecting a composition comprising the fusion protein or spores described herein. In some embodiments, the composition is applied directly to the plant, plant seed or plant propagation material. In some embodiments, the composition is applied indirectly to the plant, plant seed or plant propagation material (e.g., by adding the composition to irrigation water, plant habitat, soil, plant growth container, etc.). For spore-forming bacteria and fungi, the application rate for plant propagation material (e.g., seed treatment) is about 1×10 4 ~1×10 12 (or more) spores / seed. In a further embodiment, the spore concentration is about 1×10 6 pieces~approx. 1×10 11 1 x 10 spores / seed 6 pieces~approx. 1×10 10 1 x 10 spores / seed 6 pieces~approx. 1×109 1 x 10 spores / seed 6 pieces~approx. 1×10 8 spores / seed, or 1 x 10 6 pieces~approx. 1×10 7 In a further embodiment, the spore concentration may range from about 1×10 7 pieces~approx. 1×10 11 1 x 10 spores / seed 7 pieces~approx. 1×10 10 1 x 10 spores / seed 7 pieces~approx. 1×10 9 spores / seed or 1 x 10 7 pieces~approx. 1×10 8 In a further embodiment, the spore concentration may range from about 1×10 7 The spores / seeds may be

[0127] The propagation material can be treated with the composition before using the propagation material for plant propagation, for example, seeds are dressed before sowing. The active ingredient can also be applied to the seed kernel by immersing the kernel in a liquid composition or by coating the kernel with a solid composition (coating). Examples of formulations of the compositions comprising the fusion proteins or spores described herein that can be used in the methods provided herein include, but are not limited to, solutions, granules, pellets, beads, dusts, sprayable powders, emulsions, coated granules, and suspension concentrates. EXAMPLES

[0128] The following examples are offered to illustrate, but not to limit the claimed invention.

[0129] Example 1. Cloning of Bt cry1Aa and cry1Fa genes in a spore display vector The Bt Cry insecticidal protein was displayed on the surface of Bacillus subtilis spores. To enable the displayed Cry protein to bind to an ABC transporter receptor, a structurally flexible linker containing the leader sequence of the Cry protein itself was inserted between the spore coat (CotC / G) and the Cry protein.

[0130] The cry1Aa (SEQ ID NO: 4) and cry1Fa (SEQ ID NO: 2) genes, including leader sequences, were synthesized by GenScript Japan (Tokyo, Japan). To make Cry1Aa and Cry1Fa resistant to serine proteases, the R28L or R27L mutation was included in the synthesized cry1Aa or cry1Fa genes. The cotC (SEQ ID NO: 16) and cotG (SEQ ID NO: 18) genes, including the promoter (5' upstream region), were amplified from a genomic DNA preparation of Bacillus subtilis ISW1214 using primers 1F (SEQ ID NO: 28 and 29) and 2R (SEQ ID NO: 30 and 31) in Table 2. These primers were designed to link cotC and cotG to the nucleotides encoding L1 (short: GGGGGSAS [SEQ ID NO: 54]) and L2 (long: GGGGSGGGGSAS [SEQ ID NO: 55]) linker peptides according to the scheme shown in Figure 1.

[0131] As shown in Figure 1, a two-step amplification was performed to obtain the promoter-cotC / G-linker (L1 or L2). The first amplification with primer 3R L1 (SEQ ID NO: 32) added part of the linker, and the second amplification with primer 3R L2 (SEQ ID NO: 33) (Table 2) was directed to the complete linker sequence and NcoI site. The product of the second amplification was cloned into pGEM-T easy (Promega, Madison, WI, US).

[0132] The cry1Aa gene was amplified from the synthetic cry1Aa gene using primer 4F (SEQ ID NO: 34) and primer 5R (SEQ ID NO: 35) in Table 2, adding NcoI and BamHI restriction sites. The PCR product with an NcoI site at the 5' end and a BamHI site at the 3' end of the protein coding sequence was cloned into pGEM-T-easy. These restriction enzyme sites were used to clone other cry genes such as cry1Fa. These two pGEM-T clones, one containing the cotC / G-L1 / L2 genes, was digested with XhoI and NcoI, and the other containing the cry1Aa was digested with NcoI and BamHI. These two DNA fragments, XhoI-cotC / G-linker-NcoI and NcoI-cry1Aa-BamHI, were cloned by three-way ligation into pSB634 (SEQ ID NO: 22) digested with XhoI and BamHI. The pSB634 plasmid was described by Sasaki et al. (1996, Curr. Microbiol. 32:195-200). The cot-cry fragment was cloned into pHY300PLK (SEQ ID NO: 23) into which a synthetic cry1Ac terminator and XhoI site were cloned. The cloning scheme is shown in FIG. 2.

[0133] Because pHY300PLK does not have an XhoI site in its multiple cloning site (MCS), an XhoI site was inserted between the XbaI and BamHI sites of the MCS by cloning a small piece of DNA consisting of linker F and linker R oligonucleotides (SEQ ID NOs: 36 and 37), as shown in Table 2.

[0134] The final spore display plasmid is shown in Figure 3. In this figure, "Shuttle Vector" refers to pSB634 or pHY300PLK. To clone other cry genes, the cry1Aa gene in the final constructs of pSB634 and pHY300PLK carrying cotC / G-L1 / 2-cry1Aa was digested, and new genes such as cry1Fa were cloned using the NcoI and BamHI sites.

[0135] [Table 4]

[0136] Example 2. Preparation of Spore-pSB634, Spore-Cry1Aa and Spore-Cry1Fa A method was established to isolate Bacillus subtilis spores expressing Cry proteins on their surface. To prevent contamination of vegetative cells, Bacillus cultures were grown on the surface of 1 / 2 LB agar plates for complete and synchronous sporulation.

[0137] As shown in Figure 3, after cloning the cot-cry gene into pSB634 and pHY300PLK, Bacillus subtilis ISW1214 (host strain) was transformed with these plasmid constructs by electroporation. Preparation of electrocompetent Bacillus cells and electroporation were performed according to the protocols of TaKaRa Bio Inc., Shiga, Japan, from which pHY300PLK and Bacillus subtilis host strains were purchased. The transformed Bacillus cells were spread onto 1 / 2 LB-tet agar plates to confluent growth and incubated at 37 °C. After complete sporulation, the spores were collected in 1 ml of TBS (pH 7.5 containing 20 mM Tris-HCl, 150 mM NaCl) per plate and transferred to a 15 ml centrifuge tube. The tube was filled to 14 ml with TBS and the spores were suspended. The spores were centrifuged at 14,000xg for 30 minutes and the pellet was saved. The spores in the pellet were gently but completely suspended in 14 ml of TBS without forming bubbles and centrifuged again at the same speed and time. This procedure of suspension and centrifugation is called "washing" and this term is used in other examples to mean the same or similar procedure. The final pellet was suspended in 1 ml of TBS and kept frozen at -20°C if long-term storage is required. This spore suspension was examined under a microscope to determine the spore count. There were no differences in the preparation of Spore-pSB634 except that the cot-cry gene was not cloned into pSB634. Wild-type spores of B. subtilis ISW1214 were prepared in the same way except that 1 / 2 LB only (no tet) plates were used.

[0138] Example 3. Confirmation of expression of Cry1Aa and Cry1Fa on the spore surface The expression of Bt Cry protein on the surface of B. subtilis spores was confirmed by microscopic observation and flow cytometry using fluorescent anti-Cry antibodies. B. subtilis spores expressing Cry protein could be selected by flow cytometry.

[0139] Soluble, asporogenic Cry1Aa and Cry1Fa proteins were produced by cloning these genes in pSB634 under the cry1Ca promoter as described by Sasaki et al. (1996, Curr. Microbiol. 32:195-200), and Cry proteins were purified according to the method reported by Yamamoto et al. (1989, ACS Symposium Series 432:46-60). Using these purified Cry proteins, antibodies against Cry1Aa and Cry1Fa were produced in guinea pigs and labeled with Alexa Fluor 488 ("Alexa") using a labeling kit (Molecular Probe Inc., Eugene, OR, USA). A few μl of 10 9 Spores / ml Spore-Cry1Aa / Cry1Fa suspension were mixed with an equal volume of antiserum and incubated at 25° C. for 1 h. Spores were then washed in TBS by centrifugation as described in Example 2 and observed under a fluorescent microscope. The model shown in the top panel of FIG. 4 shows how Cry proteins on Bacillus spores were detected by fluorescent antibodies. The model shows only one Cry protein molecule on the spore surface, although multiple molecules are likely present. Fluorescence microscopy (FIG. 4, bottom) showed that most, if not all, spores were fluorescent, confirming that Cry proteins were expressed and localized to the spore surface.

[0140] The localization of Cry1Fa on the spore surface was confirmed by flow cytometry using an On-chip Sort microchip cell sorter from On-Chip Biotechnologies Co. Ltd. (Tokyo, Japan). In this experiment, 10% Spore-Cry1Fa conjugated with Alexa-labeled anti-Cry1Fa antibody was mixed with Spore-Cry1Fa without antibody. As shown in Figure 5 (left panel), the sorter isolated 9.17% fluorescent spores as a small peak. The remaining 90.8% spores (major peak) were not fluorescent. These 9.17% fluorescent spores were resorbed (Figure 5, right panel), revealing their purity to be 74.5%. Note that the spore number scales are different between the two graphs. The results confirmed the microscopic observation that Bacillus spores indeed express Cry proteins. It was also confirmed that Bacillus spores could be isolated by flow cytometry.

[0141] Example 4. Binding between Sf9 cells expressing ABC transporters and Bacillus spores displaying Cry proteins Bt Cry proteins displayed on the surface of B. subtilis spores were able to bind to their respective receptors functionally expressed on the surface of Sf9 insect cells.

[0142] To express ABC transporter proteins in Sf9 cells, genes encoding Bm-ABCC2 (SEQ ID NO: 8), Sf-ABCC2 (SEQ ID NO: 10), Sf-ABCC2 mutant (SEQ ID NO: 12) and Sf-ABCC3 (SEQ ID NO: 14) were synthesized by GenScript Japan (Tokyo, Japan). They were labeled with a FLAG tag at the C-terminus as shown in the SEQ ID NOs. These genes were cloned into the Bac-to-Bac™ baculovirus expression system from Thermo-Fisher Scientific (Tokyo, Japan) according to their published protocols. Briefly, the transporter genes were cloned into pFastBac™ using the AcMNPV polyhedrin promoter. The EGFP (Enhanced Green Fluorescent Protein) gene, which serves as a marker, was also cloned into the same vector between the NheI and SphI sites. The GFP gene was amplified from pEGFP-1 (Clontech, Mountain View, CA, USA). After the pFastBac expression plasmid was transformed into DH10Bac™ cells, transposition occurred between the mini-Tn7 element on the pFastBac vector and the mini-attTn7 target site on the bacmid, generating a recombinant bacmid. After the transposition reaction was completed, the high molecular weight recombinant bacmid DNA was isolated and the bacmid DNA was transfected into Sf9 cells using ExpiFectamine™ Transfection Reagent to generate recombinant baculovirus. After 72 hours, the medium covering the cells was collected and clarified by centrifugation at 500xg for 5 minutes. The supernatant was used as the virus solution. Nakaishi et al. (2018, Journal of Insect Biotechnology and Sericology 87(2):45-51) cloned Px-ABCC2 with a FLAG tag (SEQ ID NO:6) into a baculovirus to provide the virus. Sf9 cells expressing this transporter protein were generated in the same manner as for other transporter genes and used to test the cytotoxicity of Spore-Cry1Aa.Sf9 cells were maintained in Grace's supplemented insect medium with 10% heat-inactivated fetal bovine serum (Thermo-Fisher) at 26 °C. For Spore-Cry binding and cell viability assays, Sf9 cells with >80% confluency were infected with virus suspensions. Infected Sf9 cells were incubated at 26 °C for 72 h. To examine whether Spore-Cry binds to Sf9 cells expressing ABC transporters, Spore-Cry suspended in PBS (8.1 mM disodium phosphate-1.5 mM monopotassium phosphate buffer, pH 7.4, 2.7 mM potassium chloride, 137 mM NaCl) was mixed with Sf9 cell suspensions in insect medium and observed under a microscope. To confirm that the particles found on the Sf9 cell surface were spores expressing Cry proteins, Alexa-labeled anti-Cry antibodies were added to the spores and observed under a fluorescent microscope. Figure 6 shows an example of Spore-Cry1Fa bound to Sf9 expressing Sf-ABCC2. Bound spores were seen as grey spots on the cells by bright field microscopy (Figure 6, top left panel) and as white spots by fluorescence microscopy (Figure 6, top right panel). Alexa-labeled anti-Cry1Fa antibody bound to Cry1Fa on the spores bound to the Sf9 cell surface. Spore-Cry1Fa binding was not observed in Sf9 cells without Sf-ABCC2 (Figure 6, bottom left and right panels) or in Sf9 cells expressing Sf-ABCC2 mutants. Sf9 cells were visible under a fluorescence microscope because EGFP was cocloned with the cry genes.

[0143] Example 5. Cytotoxicity of Bacillus spores expressing Cry proteins Bt Cry proteins displayed on the surface of Bacillus spores were toxic to Sf9 insect cells only if the cells expressed a receptor for the Cry protein.

[0144] After the cells were mixed with Spore-Cry1Fa, the mixture was incubated at 26° C. for 60 minutes. As shown in FIG 7, Sf9 cells expressing Sf-ABCC2 swelled (FIG 7, upper left and upper right panels) and then completely collapsed (FIG 7, lower left and lower right panels). The lower right panel of FIG 7 shows GFP leaking out of the cells, indicating that the cell wall had completely collapsed.

[0145] Cytotoxicity of Spore-Cry1Fa was not observed in Sf9 cells expressing no ABC transporters or Sf9 cells expressing Sf-ABCC2 mutants. Similar observations were made with the combination of Spore-Cry1Aa and Sf9-expressing Bm-ABCC2. Cytotoxicity assays of Spore-Cry are summarized as follows: Spore-Cry1Aa was cytotoxic to Px-ABCC2, Bm-ABCC2, and Sf-ABCC3; Spore-Cry1Fa was cytotoxic to Sf-ABCC2 mutants derived from Cry1Fa-resistant S. frugiperda colonies, but not to Sf-ABCC2 mutants.

[0146] Example 6. Production of ABC transporter proteins in Silkworm Pupae The receptor for the Bt Cry insecticidal protein was expressed in silkworm pupae using the baculovirus expression system to produce large amounts of the receptor protein.

[0147] The synthesized Bm-ABCC2, Sf-ABCC2 and Sf-ABCC3 genes were sent to Sysmex Corporation (Kobe, Japan) to produce the proteins using ProCube™ technology. Sysmex's ProCube Technology is described on the website (Sysmex Corporation ProCube Technology, procube.sysmex.co.jp / eng / p / e_silkworm_outline / ). Briefly, the ABC transporter genes were cloned into a baculovirus expression system to generate viruses. The recombinant baculoviruses were propagated in B. mori (silkworm) cells and injected into 10 silkworm pupae. The virus-infected silkworm pupae were homogenized by sonication in TBS containing a protease inhibitor cocktail and a melanization inhibitor. The cell membrane fragments generated by sonication were collected by differential centrifugation as follows. The homogenized pupal tissue was centrifuged at 1,000xg to remove large-sized material. The pellet was further sonicated and centrifuged two more times. The supernatants from the low speed centrifugations were combined and centrifuged at 100,000xg for 1 hour to collect the pellet. The pellet was then suspended in 10 ml of TBS, divided into ten 1 ml aliquots (1 ml per pupa) and kept frozen at -80°C. This sample, called the "membrane fragment", was used to extract and purify ABC transporter proteins.

[0148] Example 7. Extraction and purification of ABC transporter proteins ABC transporters produced in silkworm pupal cell membranes were extracted, packaged into SMALPs (Styrene Malecic Acid Lipid Particles), and purified by affinity chromatography utilizing a FLAG tag bound to the C-terminus of the second ATPase of the transporter protein. The FLAG tag bound to the anti-FLAG antibody means that the tag is exposed to the solvent. The results support the predicted structure of the ABC transporter protein in SMALPs.

[0149] ABC transporter proteins were extracted with SMA (styrene maleic acid). In this example, "solubilized with SMA" or "extracted with SMA" means that ABC transporter proteins were extracted from insect cell membranes and packaged into SMALPs (Styrene Maleic Acid Lipid Particles) as shown in FIG. 8. Ready-to-use powder SMA reagent was purchased from Cube Biotech (Monheim, Germany) and dissolved in TBS at 5%. Three different S:M ratios of SMA reagents were used to extract Sf-ABCC2. The S:M ratio is the ratio of styrene (S) to maleic anhydride (M) in the SMA copolymer. For example, S:M=3:1 is a copolymer [styrene] with n=3, m=1. n -[Maleic anhydride] m The membrane fragment suspension prepared in Example 6 was mixed with an equal volume of 5% SMA in TBS and incubated at 25°C for 2 hours. The mixture was centrifuged at 100,000xg for 30 minutes at 4°C. The supernatant and precipitate were analyzed by SDS-PAGE and Western blotting using anti-FLAG antibody to confirm Sf-ABCC2 in the supernatant. Only SMA with S:M ratios of 2.3:1 and 3:1 extracted Sf-ABCC2, while SMA with S:M=1.4:1 did not extract Sf-ABCC2. SMA is thought to punch ABC transporters out of the cell membrane as small membrane fragments with the phospholipid bilayer of the cell, as shown in Figure 8. The fragments are SMALPs. In this model (Figure 8, right panel), the ATPase region of the transporter (nucleotide binding region) together with the ECL (extracellular loop) that binds to the domain II loop region of the Cry protein and the FLAG tag that is attached to the second ATPase are exposed to the solvent allowing binding to both the Cry protein and anti-FLAG antibodies. All ABC transporters used in this invention have a FLAG tag attached to the C-terminus of the second ATPase.

[0150] The size of SMALPs is determined by the S:M ratio and must be large enough to contain lipid particles with ABC transporter proteins. SMA with an S:M ratio of 1.4:1 is thought to generate lipid particles of an inappropriate size for ABC transporters. SMALPs containing Sf-ABCC2 were purified by affinity chromatography using anti-FLAG antibody-immobilized agarose gel obtained from MBL International Corp., Woburn, MA, USA. One aliquot of 1 ml of cell membrane fragment sample was divided into two 500 μl aliquots. One 500 ul aliquot was estimated to contain approximately 150 μg of ABC transporter protein by SDS-PAGE. For affinity chromatography, one 500 μl aliquot was mixed with 500 μl of 5% SMA, incubated, and centrifuged to obtain approximately 1 ml of SMA-extracted Sf-ABCC2 (i.e., SMALP-Sf-ABCC2). SDS-PAGE showed that approximately 50% of the transporter protein was extracted. SMALP-Sf-ABCC2 was loaded onto 200 μl of anti-FLAG antibody-immobilized agarose gel packed into a 1 ml Pierce™ Spin Column (Thermo-Fisher). The column was washed with TBS containing 10% glycerol (TBS-glycerol), and SMALP-Sf-ABCC2 was eluted with 1.2 ml of elution buffer (0.1 mg / ml FLAG peptide in TBS with 10% glycerol). The column eluate was fractionated into six 200 μl fractions and analyzed by dot blotting as shown in FIG. 9. In FIG. 9, all 200 μl fractions ("Fr." including the last spin-off (Fr.7) except the first (Fr.1)) showed strong fluorescent signals, confirming that Sf-ABCC2 was successfully eluted from the affinity column.

[0151] All fractions except Fr.1 were combined and concentrated to 300 ul with an Amicon Ultra Centrifugal Filter (purchased from MilliporeSigma, Saint Louis, MO, USA, 50 kDa cutoff). The concentrated column eluate was then desalted in a 2 ml Zeba Spin Desalting Column (Thermo-Fisher) to remove the FLAG peptide. Approximately 300 μl of this affinity purified ABC transporter, called SMALP-Sf-ABCC2, was obtained. SDS-PAGE and Western blotting with anti-FLAG antibody showed a single band at approximately 150 kDa. This was divided into six 50 μl aliquots, flash frozen in liquid nitrogen, and stored at -80°C. The successful purification of SMALP-Sf-ABCC2 by anti-FLAG-antibody affinity chromatography indicates that the FLAG tag is exposed to solvent. This supports the SMALP model shown in Figure 8. It is likely based on the SMALP model that the extracellular loops (ECLs) of ABCC2 are also solvent exposed and capable of binding Cry proteins.

[0152] Example 8. Selection of Bacillus spores expressing Cry proteins based on the interaction between the Cry protein and its specific receptor Bacillus subtilis spores expressing Cry proteins and specifically binding their receptors were isolated by immunomagnetic separation using the FLAG tag bound to the receptor and anti-FLAG antibodies immobilized on magnetic beads, and it was demonstrated that the receptor-bound spores could be sorted by flow cytometry.

[0153] Spore sorting experiments were performed by immunomagnetic separation and flow cytometry. The purpose of the sorting is to extract Bacillus spores expressing Cry proteins by utilizing the affinity between Cry proteins on spores and specific ABC transporter proteins. To demonstrate the feasibility of spore sorting, Spore-Cry1Fa was sorted with Sf-ABCC2, and Spore-Cry1Aa was sorted with Bm-ABCC2. Immunomagnetic separation was performed using Spore-Cry1Fa bound to SMALP-Sf-ABCC2. Spores bound to the transporter were extracted using anti-FLAG antibody-immobilized magnetic beads as follows. Approximately 10 spores suspended in 100 μl of TBS were sorted by immunomagnetic separation. 6 Spore-Cry1Fa spores were mixed with 50 μl of affinity purified SMALP-Sf-ABCC2 from Example 7, incubated at 25° C. for 2 hours, and washed twice with 1.5 ml of TBS by centrifugation at 14,000×g for 30 minutes. The washed spores were suspended in 100 μl of TBS and mixed with 5 μl of anti-FLAG antibody immobilized magnetic beads (MilliporeSigma catalog number M8823). The spores bound to the magnetic beads were held on a magnet and washed five times with TBS. The washed spores were suspended in 100 μl of TBS. Spore-Cry1Fa bound to SMALP-Sf-ABCC2 was separated from the anti-FLAG antibody immobilized magnetic beads containing 1.5 mg / ml FLAG peptide in 10 mM TBS. This final spore suspension was plated on LB-tet plates. The exact same experiment was performed using Spore-pSB634, which does not express Cry proteins, and the results are shown in Figure 10.

[0154] As shown in Figure 10, the magnetic beads were incubated with 10% of Spore-pSB634, which does not express Cry proteins. 6 Only 9 spores (colonies) were pulled from 10 spores (Figure 10, left panel). The number of spores was much lower than expected as background levels of non-specific binding. On the other hand, the beads captured a large number of spores (i.e., too many confluent growths) in the Spore-Cry1Fa starting sample (Figure 10, right panel).

[0155] To examine whether magnetic separation can selectively isolate Spore-Cry1Fa, a similar experiment was carried out using a mixture of Spore-Cry1Fa and wild-type spores. 6 (approximately 10%) of Spore-Cry1Fa spores and 10 7 wild-type spores were present. Mixture-2 contained 2 x 10 3 (0.1%) Spore-Cry1Fa and 2 × 10 6 Mixture-3 consisted of 2 x 10 wild-type spores. 3 (0.01%) Spore-Cry1Fa and 2 × 10 7 Mixture-4 contained 2 x 10 wild-type spores. 3 (0.001%) Spore-Cry1Fa and 2 × 10 8 wild-type spores. These samples were for separating spores between tet-resistant Spore-Cry1Fa and tet-sensitive wild-type spores, so the sorted spores could be identified by tet selection. The spores separated with immunomagnetic beads were first plated on LB plates (without tet) and then transferred to LB-tet plates. Mix-1 produced 359 spores (colonies), Mix-2 produced 180 spores (colonies), Mix-3 produced 1500 spores (colonies), and Mix-4 produced 1700 spores (colonies) on the LB-tet plate. To increase the recovery of 0.01% and 0.001% Spore-Cry1Fa in Mix-3 and 4, 50 μl of anti-FLAG antibody-immobilized magnetic beads was used instead of 5 μl. The number of these spores captured by the magnetic beads indicated that the separation was selective. It was found that 70% of the spores from Mixture-1, 61% of the spores from Mixture-2, 60% of the spores from Mixture-3, and 70% of the spores from Mixture-4 were tetracycline-resistant Spore-Cry1Fa, strongly indicating that SMALP-Sf-ABCC2 selectively bound to Spore-Cry1Fa, and confirmed the SMALP-ABC transporter model shown in Figure 8.

[0156] Sorting of Bacillus spores expressing Cry proteins based on receptor affinity was performed by flow cytometry. In this case, Spore-Cry1Aa was used together with Bm-ABCC2 solubilized in 1% Triton X100, followed by affinity purification similar to SMALP-Sf-ABCC2. Spore-Cry1Aa suspended in PBS was labeled with 5 μl of Bm-ABCC2 (approximately 0.3 mg / ml) in PBS containing 1% Triton X100 by simply mixing and incubating at 25°C for 2 hours. Unbound Bm-ABCC was removed by washing the spores with 10 mM sodium phosphate buffer (pH 7.4) containing 0.2% SDS (sodium dodecyl sulfate) and 500 mM NaCl. The Spore-Cry1Aa-Bm-ABCC2 complex was then made fluorescent by sequentially mixing with anti-FLAG-mouse-antibody and Alexa-labeled anti-mouse IgG-antibody. The spore complexes were washed in PBS containing 0.02% Triton X100 and observed by fluorescence microscopy. As a result of observation, only a few spores retained the fluorescent dye, so they were sorted by flow cytometer using On-chip Sort cell sorter (On-Chip Biotechnologies). The cell sorter pulled out about 1000 spores that were probably bound to Bm-ABCC2 as fluorescence. The use of 0.2% SDS in the washing solution separated Bm-ABCC2 from many spores, but was done intentionally to examine whether the cell sorter could select the few remaining fluorescent spores. As shown in Figure 11, 1000 fluorescent spores were separated and plated on LB plates. The LB plates were copied onto LB-tet plates. More than 800 spores were found to germinate and form colonies on the tet selection plate. Some of these colonies were picked, and the plasmid in those spores was confirmed to be the cry1Aa gene by PCR and sequencing (Example 9). This experiment with Spore-Cry1Aa confirmed that Bacillus spores expressing Cry proteins can be sorted by flow cytometry on the basis of receptor affinity.

[0157] Example 9. Identification of cry genes in selected spores To identify the Cry proteins displayed on the spore surface, plasmids in spores sorted by flow cytometry were isolated and sequenced.

[0158] To identify cry genes in the sorted spores by flow cytometry, approximately 10 colonies on the LB-tet plate shown in Figure 10 were picked and the cell density was OD 600nm The cells were grown in 1 ml of LB-tet broth for several hours at 37 °C in a shaker until the β-terminal end of the colonies reached 0.2–0.3. The cells were collected by centrifugation and plasmids were isolated from their colonies following the method described by Hou et al. (2019, Toxins 11:162). This plasmid isolation method by Hou et al. is for Bacillus thuringiensis but worked well with B. subtilis. The isolated plasmids were sequenced to determine the cry genes in the spores.

[0159] All patents, patent publications, patent applications, journal articles, books, technical references, and the like, discussed in this disclosure are hereby incorporated by reference in their entirety for all purposes.

[0160] It should be understood that the figures and descriptions of the present disclosure are simplified to show elements relevant to a clear understanding of the present disclosure. It should be understood that the drawings are presented for illustrative purposes and are not presented as structural diagrams. Omitted details and modifications or alternative embodiments are within the understanding of those skilled in the art.

[0161] It will be understood that in certain aspects of the disclosure, multiple components may be substituted for single components, and multiple components may be substituted for single components, to provide an element or structure or to perform a given function or functions, and such substitutions are deemed to be within the scope of the disclosure, except where such substitutions would not be operative to practice a particular embodiment of the disclosure.

[0162] The examples presented herein are intended to illustrate potential and specific implementations of the present disclosure. It will be understood that the examples are primarily intended to illustrate the present disclosure for those skilled in the art. There may be variations in these diagrams or the operations described herein without departing from the spirit of the present disclosure. For example, in certain cases, method steps or operations may be performed or executed in a different order, or operations may be added, deleted, or modified.

[0163] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range is also specifically disclosed, to the smallest fraction of the unit of the lower limit, unless the context clearly dictates otherwise. Any narrower range between any stated or unstated intervening value in a stated range and any other stated or intervening value in that stated range is encompassed. The upper and lower limits of these smaller ranges may be independently included or excluded in the range, and each range in which either, either or both limits are included in the smaller range is also encompassed within the technology, subject to any specifically excluded limits in the stated range. Where a stated range includes one or both of the limits, ranges excluding one or both of those included limits are also included.

[0164] In the foregoing description, numerous specific details are set forth to provide a more complete understanding of the present invention. However, it will be apparent to one skilled in the art that the invention described in this disclosure may be practiced without one or more of these specific details. In other instances, well-known features and procedures well known to those skilled in the art have not been described to avoid obscuring the invention. The embodiments of the present disclosure have been described for purposes of illustration and not limitation. Although the present invention has been described primarily with reference to certain embodiments, it is anticipated that other embodiments will become apparent to those skilled in the art upon reading this disclosure, and such embodiments are intended to be included within the scope of the method of the present invention. Thus, the present disclosure is not limited to the embodiments described above or shown in the drawings, and various embodiments and modifications may be made without departing from the scope of the following claims.

[0165] [Table 5]

[0166] [Table 6]

[0167] [Table 7]

[0168] [Table 8]

[0169] [Table 9]

[0170] [Table 10]

[0171]

Table 11

[0172]

Table 12

[0173]

Table 13

[0174]

Table 14

[0175]

Table 15

[0176]

Table 16

[0177]

Table 17

[0178]

Table 18

[0179]

Table 19

Claims

1. A recombinant nucleic acid encoding a fusion protein, comprising: a) a first nucleic acid sequence encoding a spore coat polypeptide; b) a second nucleic acid sequence encoding a linker; and c) a third nucleic acid sequence encoding a putative insecticidal polypeptide. A recombinant nucleic acid comprising:

2. 2. The recombinant nucleic acid of claim 1, wherein the spore coat polypeptide is derived from a Bacillus bacterium.

3. 3. The recombinant nucleic acid of claim 1 or 2, wherein the spore coat polypeptide is CotC, CotG, CotB, CotA, CotD, CotE, CotX, CotY, or CotZ.

4. 3. The recombinant nucleic acid of claim 1, wherein the spore coat polypeptide is CotC or CotG.

5. 3. The recombinant nucleic acid of claim 1 or 2, wherein the putative insecticidal polypeptide is a Bacillus thuringiensis crystal (Bt Cry) protein.

6. 6. The recombinant nucleic acid of claim 5, wherein the Bt Cry protein comprises at least one modification compared to the wild-type protein.

7. 7. The recombinant nucleic acid of claim 6, wherein the modifications are the result of DNA shuffling, block swapping, site-directed mutagenesis, saturation mutagenesis, random mutagenesis, or a combination thereof.

8. 7. The recombinant nucleic acid of claim 6, wherein the Bt Cry protein modification confers resistance to serine protease digestion.

9. 3. The recombinant nucleic acid of claim 1 or 2, wherein the putative insecticidal polypeptide interacts with a cell surface protein of an insect digestive system epithelial cell.

10. 10. The recombinant nucleic acid of claim 9, wherein the cell surface protein is an ATP-binding cassette (ABC) transporter protein, a cadherin protein, an aminopeptidase N protein, or an alkaline phosphatase protein.

11. 10. The recombinant nucleic acid of claim 9, wherein the cell surface protein is an ABC transporter protein.

12. 11. The recombinant nucleic acid of claim 10, wherein the ABC transporter protein is PxABCC2, Bm-ABCC2, Sf-ABCC2, Sf-ABCC3, Dv-ABCB1, Bm-ABCB1, Sf-ABCB1, Bm-ABCA2, or Tc-ABCC4.

13. 11. The recombinant nucleic acid of claim 10, wherein the ABC transporter protein is PxABCC2, Bm-ABCC2, Sf-ABCC2, or Sf-ABCC3.

14. 3. The recombinant nucleic acid of claim 1, wherein the spore coat polypeptide and the putative insecticidal polypeptide are structurally separated by the linker.

15. The recombinant nucleic acid of claim 1 or 2, wherein the linker comprises at least one amino acid.

16. 3. The recombinant nucleic acid of claim 1, wherein the linker is a polypeptide comprising at least two amino acids.

17. The recombinant nucleic acid of claim 1 or 2, wherein the linker is structurally flexible.

18. The recombinant nucleic acid of claim 1 or 2, wherein the linker is resistant to protease digestion.

19. 3. The recombinant nucleic acid of claim 1, wherein the linker is a polypeptide comprising an amino acid sequence having at least 80% identity to any of SEQ ID NOs: 20, 21, or 38-61.

20. A DNA construct comprising a promoter operably linked to the recombinant nucleic acid of claim 1 or 2.

21. A vector comprising the recombinant nucleic acid of claim 1 or 2.

22. A fusion protein encoded by the recombinant nucleic acid of claim 1 or 2.

23. A spore comprising a recombinant nucleic acid described in claim 1 or 2, or a fusion protein encoded by the recombinant nucleic acid described in claim 1 or 2.

24. 24. The spore of claim 23, wherein the fusion protein is displayed on the surface of the spore.

25. The spore described in claim 23, which is a bacterial spore or a fungal spore.

26. A composition comprising a plurality of spores according to claim 23, wherein each of the plurality of spores comprises a recombinant nucleic acid encoding a different putative insecticidal polypeptide.

27. 1. A method for identifying an insecticidal protein, comprising: a) contacting (i) a fusion protein encoded by a recombinant nucleic acid of claim 1 or 2 or (ii) a spore containing the recombinant nucleic acid of claim 1 or 2 or the fusion protein encoded by a recombinant nucleic acid of claim 1 or 2 with an insecticidal protein receptor; and b) detecting an interaction between the fusion protein or spores and the insecticidal protein receptor; wherein said interaction indicates that the putative insecticidal protein of said fusion protein or spore is an insecticidal protein.

28. 28. The method of claim 27, wherein the insecticidal protein receptor is a purified insecticidal protein receptor protein.

29. 28. The method of claim 27, wherein the insecticidal protein receptor is part of an SMA lipid particle.

30. 28. The method of claim 27, wherein the insecticidal protein receptor is expressed on a cell.

31. 31. The method of claim 30, wherein the cell is an insect cell.

32. 28. The method of claim 27, wherein the insecticidal protein receptor is an ABC transporter protein.

33. 28. The method of claim 27, wherein detecting the interaction between the fusion protein or spore and the insecticidal protein receptor comprises performing immunomagnetic separation, flow cytometry, cytotoxicity assay, sequencing, or a combination thereof.

34. 34. The method of claim 33, wherein detecting the interaction between the fusion protein or spore and the insecticidal protein receptor protein further comprises isolating and sequencing the recombinant nucleic acid encoding the fusion protein that interacts with the insecticidal protein receptor protein to determine the identity of the insecticidal protein.

35. The method described in claim 27, wherein the spore contains a fusion protein encoded by the recombinant nucleic acid described in claim 1 or 2, and the fusion protein is displayed on the surface of the spore.

36. The method of claim 35, wherein the spores are bacterial spores or fungal spores.

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