Cell-based assay for determining pesticidal protein toxicity

Abstract

The present invention pertains to the field of pest control. In particular, the invention pertains to bioassays to measure the response of insect cells to toxinsm. Furthermore, the invention pertains to methods for identifying receptors which play a role in the interaction of insecticidal toxins and insect cells. The invention thus also pertains to the identification of toxins having an activity against an insect cell, and to toxins that interact with receptor molecules.

Description

[0001] 202227W001

[0002] Cell-Based Assay for Determining Pesticidal Protein Toxicity

[0003] Technical Field

[0004] Methods for assaying the effect of toxin on insect cell lines are provided.

[0005] Background

[0006] Insects, particularly insects within the order Lepidoptera and Hemiptera, are considered a major cause of damage to field crops, resulting in decreased crop yields over infested areas. Lepidopteran pest species which negatively impact agriculture include, but are not limited to, black armyworm, cotton bollworm, tomato fruitworm, and variegated cutworm. Key among hemipteran pests of agricultural importance are stink bugs, whitefly, aphids and psyllids. Biological pest control agents such as Bacillus thuringiensis (Bt) strains expressing pesticidal toxins have been applied to crop plants with satisfactory results against insect pests. The 6- endotoxins (delta endotoxins, also called crystal toxins or Cry proteins) are often used in this respect. The 6-endotoxins are proteins held within a crystalline matrix that are known to possess insecticidal activity when ingested by certain insects. Bt toxins, ICPs, d-endotoxins or crystalline proteins can be solubilized and activated by proteinases in the midgut region of an insect. The toxin binds to a specific receptor, creating a pore in the epithelial cells of the insect gut and finally kills the insect pest (Whalon and Wingerd 2003). Therefore, the toxic effects depend on a complex biological process of activation and receptor recognition in a specific tissue. Usage of transgenic plants expressing Cry proteins has been shown to be effective against biological pests. Accordingly, there is a need to identify rapidly which Cry protein is effective against a given insect pest. It is also important to be able to identify the mode of action of insect control products in order to determine the most efficient way of mitigating resistance.

[0007] The specificity of the Bt endotoxins is dependent on both the activation of the toxin in the insect gut and its ability to bind specific receptors present on the insect’s midgut epithelial cells. The correlation between binding and toxicity was first demonstrated using brush border membrane vesicles (BBMV) prepared from microvilli by use of a technique developed by Wolfersberger (Wolfersberger, M. G. 1990. The toxicity of two Bacillus thuringiensis b-endotoxins to gypsy moth larvae is inversely related to the affinity of binding sites on midgut brush border membranes for the toxins (Experientia 46:475-477. [PubMed] [Google Scholar]). Since this time, efforts to identify and clone toxin receptors were intensified. Many putative Cry toxin receptors have since been reported, of which the best characterized are the aminopeptidase N (APN) receptors and the cadherin-like receptors identified in lepidopterans. In nematodes, glycolipids are believed to be an important class of Cry toxin receptors. A summary of the various receptors is provided in Craig R. Pigott and David J. Ellar, Microbiol Mol Biol Rev. 2007 Jun; 71 (2): 255- 281 . Role of Receptors in Bacillus thuringiensis Crystal Toxin Activity. 202227W001

[0008] 2

[0009] In addition to identifying receptors, it is important to understand the contribution of a domain of the toxins to insect specificity. Using domain information derived from the crystal structures of active CrylAa, Cry2Aa, and Cry3Aa, de Maagd et al. (de Maagd, R. A., A. Bravo, and N. Crickmore. 2001 . How Bacillus thuringiensis has evolved specific toxins to colonize the insect world. Trends Genet. 17:193-199. [PubMed] [Google Scholar]) aligned each of three toxin domains separately and created phylogenetic trees to assess the individual contribution of each domain to insect specificity. The different trees showed that in general, there was a correlation between sequence similarity and specificity, but that relatively unrelated clusters could sometimes have similar activities.

[0010] Once a means of controlling pest is identified, it is also important to consider dietary exposure. Dietary exposure is the major route by which humans can be exposed to insecticidal proteins expressed in transgenic plants. Members of certain groups of Cry proteins are likely to be rapidly degraded following ingestion by humans. It is important to minimize the potential for allergic reactions. Proteins that degrade rapidly are likely to cause less of an allergic reaction, and thus, it is important to determine which proteins degrade rapidly. In this regard, it may be possible to introduce sites into the protein such as protease cleavage sites so that the site is recognized by a different protease. Protease cleavage sites for chymotrypsin, trypsin and pepsin are well known in the art. Chymotrypsin preferentially cleaves peptide amide bonds. Methods for measuring pesticidal activity using live insects are well known in the art. See, for example, Czapla and Lang (1990) J. Econ. Entomol. 83:2480-2485; Andrews et al. (1988) Biochem. J. 252:199-206; Marrone et al. (1985) J. of Economic Entomology 78:290-293; and U.S. Pat. No. 5,743,477. However, in vitro diagnosis of the effect of protein toxins remains a challenge. Although some insect cell lines have been developed for in vitro detection of these toxins, they have to be developed individually for each insect, and they are often difficult to develop. Currently, commercial kits are used to measure cell death for the purpose of this in vitro assay. Disadvantages of these in vivo and in vitro assays include that the assays are not done in real time, the assays use larvae of live insects which are difficult to score and manage, the assays are not high throughput and the results are not always quantitative.

[0011] Electronic methods for analyzing cellular compositions have been in use for many years. Particularly, impedance-based assays have been utilized for a variety of different applications, including measuring cell migration, counting cells or measuring cell growth, but very few of these methods have been used in connection with insect lines and very few have been used to study the effect of toxin proteins on target cells. Most of the impedance-based assays to date have been developed to investigate the cytotoxic effect of potential therapeutic agents upon target cells. These assays are based upon the principle of cellular impedance, where adherent cells act as insulators impeding the flow of an alternating microampere electric current between electrodes. A change in measured impedance within such a system (i.e. due to a change in the current flow) between and amongst electrodes can identify a change in the quantity or presence of cells. Thus, target cells which adhere to electrodes may impede the flow of the current and the presence of adherent target cells on the substrate hence results in increased impedance due to their insulating effect. The addition of immune effector cells which specifically bind to the adherent target cells, resulting in target cell death, decreases the impedance since fewer target cells are present to produce an insulating effect. Thus, impedance can be used to measure 202227W001

[0012] 3 target cell death and the number of target cells present on the electrodes. The method is sensitive and can also allow the measurement of real time target cell killing. xCELLigence® technology is based on impedance sensing. The functional unit of the xCELLigence® technology is E-Plates®, which are the gold microelectrodes. For impedance measurement, a very low electric current is applied to the plates, flowing through an electrically conductive solution (cell culture media) from one microelectrode to the other. As soon as cells adhere to these microelectrodes they act as electrical insulators, thereby influencing the impedance signal. The technology has mainly been used to identify potential cytotoxic agents for pharmaceutical purposes. To date, the technology has not been applied extensively to study protein toxins. In addition, there are very few examples of the use of xCELLigence® or other cytotoxic assays on insect cells.

[0013] Garcia-Reina et al. (Insect Science (2017) 24: 358-370) shows the use of xCELLigence® real time analyzer on an insect cell line. The authors characterized the impedance profile of a Tribolium castaneum (rust flour beetle) cell line in response to abiotic stress factors like thermal stress and UV light. There was no study of the effect of protein toxins. Zhou et al. (In Vitro Cellular & Developmental Biology - Animal (2020) 56: 10-14) shows fall armyworm cell lines from larval midguts. Cytotoxicity assays were performed in a 96 well plate by measuring fluorescence. The analysis was not done in real time and the impedance profile of the cell lines was not measured. Bondzio et al. (PLOS ONE 2013 8(7) e67079) studied the effect of CrylAb on porcine intestinal cells in order to screen for toxicity of GM food. The xCELLigence® system was used to evaluate cell survival after CrylAb treatment. Insect cell lines were not used. Zhou et al (Cell Cycle 18:13, 1498-1512) used the xCELLgence® system for real-time monitoring of cell properties of BmGem2 knockout cells in silkworm (Bombyx). No toxins were added to the cell line.

[0014] Thus, there have been no studies directed to a bioassay for measurement of the response of insect cells to toxins perse.

[0015] Summary

[0016] The invention relates to a method of detecting the activity of a protein toxin on an insect cell line, the method comprising: attaching a set of insect cells to a cell plate, measuring cell attachment by electronic sensing of resistance or impedance in real time, adding a protein toxin to the cell line on the cell plate, and measuring the change in cell index over time that results from the addition of the protein toxin. In general, the cell plate is a multi-well culture dish with microelectrodes at the bottom of each well or e-plate.

[0017] In another embodiment, there is a method of determining the activity of a protein toxin on an insect cell line by attaching a set of insect cells to a cell plate, adding a protein toxin to the cell line on the cell plate, and monitoring cell death by measuring an increase in cell detachment from the plate. 202227W001

[0018] 4

[0019] The methods of this invention further include treating the protein toxin with a protease before, concurrent with or following the addition of the protein toxin to the insect cell line. In one aspect, the protease tested is trypsin.

[0020] The insect cells that may be tested according to the methods of the present invention are preferably from hemipteran or lepidopteran cell lines, but other cell lines may be tested by the techniques set forth herein. Preferably are the hemipteran or lepidopteran cells lines being midgut or embryonic cell lines.

[0021] Insect Cell lines that over-express a toxin receptor or that express a mutated version of a toxin receptor may also be used in the methods of the present invention. Hence, the techniques and methods of the present invention may be used to investigate the cytotoxicity of a toxin protein, as well as for identifying and characterizing receptors that interact with the toxin protein.

[0022] In one aspect, the methods herein involve analyzing the response of the insect cells to the protein toxin at various times after toxin addition or toxin activation.

[0023] In another embodiment, there is provided a method of identifying the active regions of a protein toxin, the method comprising creating a series of mutations in a gene encoding a protein toxin, transcribing such mutagenized genes to produce a protein, contacting insect cells with such mutagenized toxin proteins, measuring cell index of the cell lines in response to each mutagenized protein, thereby allowing for the screening of a plurality of mutagenized toxins. The gene may be mutated by way of a single nucleic acid change, substitution, deletion or insertion.

[0024] In related embodiments, the methods involve contacting the insect cell lines with a toxin protein fused with a marker, linker or other variant or tag. In one embodiment the toxin is fused with a protein moiety for simplified purification like the maltose binding protein (MBP).

[0025] In another embodiment, the methods include contacting the insect cells with a supernatant or cell extract from a transformant expressing a mutated toxin and assaying contacted cells for changes in cell index.

[0026] In another embodiment, the methods include contacting the insect cells with a supernatant or cell extract from a bacterium expressing a toxin and assaying contacted cells for changes in cell index.

[0027] In another embodiment, there is a provided a method of screening cellular extracts for the presence of a cytotoxic agent that has activity against a cell line, the method comprising: attaching insect cells to a plate, adding a cellular extract to the insect cells, measuring the cell index following the addition of the cellular extract. The cell index can be measured in real time, using xCELLigence, for example. 202227W001

[0028] 5

[0029] Description of the Figures

[0030] Figure 1 shows data using SF9 cells (Lepidoptera) seeded on e-Plates® treated either with buffer or different toxins at a serial dilution of 1 :5. Fig. 1 A shows data when the cells were exposed to BP005 toxin at a concentration of 0.1 mg / ml and a serial dilution of 1 :5. Fig. 1 B shows the effect when the cells were exposed to Axmi554 toxin at a concentration of 0.5 mg / ml and a serial dilution of 1 :5. Fig. 1C shows a control where pH10 buffer was added to the Sf9 cells seeded on the plates. BP005 shows strong concentration dependent activity on SF9 cells whereas Axmi554 shows only low activity.

[0031] Figure 2 is a set of charts showing squash bug cells (Hemiptera) treated with BSA or BP005, Axmi554 or R1 toxins Fig. 2A shows the effect of BP005 when added to cells at the indicated concentration, 20 hours after the cells are seeded onto the plate. Fig. 2B shows the same as Fig. 2A, but with Axmi554 added to the cells. Fig. 2C shows the same as Fig. 2A, but with R1 added to the cells. Fig. 2D shows a control where BSA was added to the cells. BP005 and Axmi554 show concentration dependent activity on the hemiptera squash bug cells whereas R1 does not.

[0032] Figure 3 shows real time monitoring of the effect of Axmi554 toxins on AC20 (hemiptera) cells. It shows the concentration dependent activity of Axmi554 on AC20 hemiptera cells.

[0033] Figure 4 shows trypsin-dependent potentiation of Axmi554 activity on AC20 cells in real time. Test cells were seeded on a 96-well plate. Fig. 4A shows the effect of Axmi554 when added 20 hours after AC cells were plated. Fig. 4B shows the effects when trypsin is added at 1 ug / mL concentration simultaneously with Axmi554 on AC20 cells.

[0034] Description

[0035] Determining Pesticidal Protein Activity Generally

[0036] The present invention is drawn to a bioassay for determining the effect of a pesticidal toxin on an insect cell line, for determining the receptors that interact with a pesticidal toxin and for determining the regions of the protein sequence that confer activity on a pesticidal toxin. The identification of these features is important for identifying new or improved toxins and for determining how to manage pest resistance or tolerance. It is known that pests may become resistant or tolerant to certain toxins upon prolonged or repeated treatment with the same toxin which makes it important to understand the specificity for such resistance or tolerance. Therefore, it is desirable to identify new or modified toxins to which such tolerant or insensitive pest are still sensitive.

[0037] The methods of this invention involve contacting insect cell lines with toxin proteins or pesticidal proteins. By “toxin protein”, "pesticidal toxin" or "pesticidal protein" is intended a toxin that has toxic activity against one or more pests, including, but not limited to, members of the Lepidoptera, Diptera, Hemiptera, and Coleoptera orders, or the Nematoda phylum, or a protein that has homology to such a protein. Pesticidal proteins have been isolated from organisms including, for example, Bacillus sp., Clostridium bifermentans and Paenibacillus popilliae. 202227W001

[0038] 6

[0039] Pesticidal proteins that may be tested according to the present invention include amino acid sequences deduced from the full-length nucleotide sequences as well as amino acid sequences that are shorter than the full-length sequences, either due to the use of an alternate downstream start site, or due to processing that produces a shorter protein having pesticidal activity. In reality, the shortened sequences may be reflective of processing that may occur in the organism the protein is expressed in, or in the pest after ingestion of the protein.

[0040] In order to test the proteins for activity in the bioassay of the invention, cell lines were treated with purified proteins alone, and / or with cell extract from an organism such as bacteria like E.coli which has been transformed with a vector comprising a nucleotide sequence encoding a pesticidal protein or with an cell extract from an organism which naturally (e.g. without transformation of a respective vector or construct) expresses one or more pesticidal proteins. Various known nucleotide sequences may be used in the construction of expression vectors or cassettes for subsequent transformation into organisms and the generation of pesticidal proteins by methods known in the art. As well, the proteins may be altered such as domain swapping or DNA shuffling. These altered proteins may also be tested according to the methods set forth herein. The wildtype or altered proteins may be tested for activity against lepidopteran, hemipteran, coleopteran, dipteran, and nematode pest populations.

[0041] Use of Cell Index as a Measuring of the Toxicity of a Pesticidal Toxin

[0042] The xCELLigence real-time cell analysis (RTCA) system may be used to monitor alterations in cell adhesion following genetic manipulations, overexpression of a protein of interest or in response to pesticidal treatment. This technology works by measuring electron flow transmitted between gold microelectrodes, fused to the bottom surface of a microtiter plate, in the presence of an electrically conductive solution such as tissue culture medium, a salt solution or a buffered solution. Cells that adhere to the surface disrupt the interaction between the electrodes and the solution and thus impede electron flow. This impedance (resistance to alternating current) is expressed as cell index. Cell index is dependent on cell number, cell morphology, cell size and on the strength of cell attachment to the substrate coating the plate. Thus, when using the xCelligence system it is important to have an appropriate control test.

[0043] The advantage of using xCELLigence are that it provides real time assessment of cell adhesion in an entire population of cells. The results are sensitive and multiple experimental conditions can be measured at the same time and in the same plate.

[0044] The change in impedance in the present invention is correlated with the number of target cells present and thus cell death will affect (decrease) the impedance. A decrease in impedance upon addition or incubation with pesticidal toxins is indicative of the cytotoxic effect of the toxin. Thus, if the toxin or fragment thereof has a cytotoxic effect on the target insect cells (i.e. is capable of inducing and / or causing cell death of the target cells), a decrease in impedance may be detected. Generally, the more cell death induced by the toxin, the greater the decrease in impedance which is detected. The method of the invention may be used not only to determine whether a protein toxin has a cytotoxic effect on a target cell, but may be used to determine the level of the cytotoxic effect in relation to time (i.e. how quickly the cytotoxic effect appeared). 202227W001

[0045] 7

[0046] Determining Domains Responsible for Activity

[0047] The present invention is also drawn to a bioassay for determining which domains of pesticidial proteins like Bt endotoxins have activity, such as the toxin and the stabilizing or crystallization domain. It is also possible to test hybrid toxins having a toxic core domain operably linked to a heterologous stabilizing domain. For example, one could create a hybrid toxin having domains operably linked so that when translated, a functional chimeric protein is produced. Some examples of domains are the “death domain”, EGF-like motifs or repeats. The invention herein provides, in one embodiment, an assay for the effect of 6-endotoxins such as the Cry toxins. Trypsin potentiates the effect of certain toxins on certain cell lines, and the present bioassay provides a means of determining the effect of protease on the activity of a pesticidal toxin. Delta-endotoxins generally have five conserved sequence domains, and three conserved structural domains (see, for example, de Maagd et al. (2001) Trends Genetics 17:193-199). The first conserved structural domain consists of seven alpha helices and is involved in membrane insertion and pore formation. Domain II consists of three beta-sheets arranged in a Greek key configuration, and domain III consists of two antiparallel beta-sheets in “jelly-roll” formation (de Maagd et al., 2001 , supra). Domains II and III are involved in receptor recognition and binding and are therefore considered determinants of toxin specificity.

[0048] Amino acid substitutions may be made in non-conserved regions that retain function. In general, such substitutions would not be made for conserved amino acid residues, or for amino acid residues residing within a conserved motif, where such residues are essential for protein activity. Examples of residues that are conserved and that may be essential for protein activity include, for example, residues that are identical between all proteins contained in an alignment of similar or related toxins to the sequences of the invention (e.g., residues that are identical in an alignment of homologous proteins). Examples of residues that are conserved but that may allow conservative amino acid substitutions and still retain activity include, for example, residues that have only conservative substitutions between all proteins contained in an alignment of similar or related toxins to the sequences of the invention (e.g., residues that have only conservative substitutions between all proteins contained in the alignment homologous proteins). However, one of skill in the art would understand that functional variants may have minor conserved or non-conserved alterations in the conserved residues.

[0049] Alternatively, variant nucleotide sequences can be made by introducing mutations randomly along all or part of the target mutation region(s), such as by permutation or saturation mutagenesis, and the resultant mutants can be screened for ability to confer pesticidal activity to identify mutants that retain activity or show improved activity. Following mutagenesis, the encoded protein can be expressed recombinantly, and the activity of the protein can be determined using the methods of the current invention

[0050] “Variants” of the pesticidal proteins may be generated, for example, by using site-directed mutagenesis but which still encode the pesticidal proteins disclosed in the present invention as discussed below. Variant proteins encompassed by the present invention are biologically active, that is they continue to possess the desired biological activity of the reference protein. The skilled artisan will further appreciate that changes can be introduced by mutation of the 202227W001

[0051] 8 nucleotide sequences of the invention thereby leading to changes in the amino acid sequence of the encoded pesticidal proteins, without altering the biological activity of the proteins. Thus, variant isolated nucleic acid molecules can be created by introducing one or more nucleotide substitutions, additions, or deletions into the corresponding nucleotide sequence disclosed herein, such that one or more amino acid substitutions, additions or deletions are introduced into the target mutation region(s) of the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Such variant nucleotide sequences are also encompassed by the present invention. In some cases, the variant may have at least about 30%, at least about 50%, at least about 70%, or at least about 80% of the pesticidal activity of the wildtype protein. Other methods for measuring pesticidal activity are well known in the art. See, for example, Czapla and Lang (1990) J. Econ. Entomol. 83: 2480-2485; Andrews et al. (1988) Biochem. J. 252:199-206; Marrone et al. (1985) J. of Economic Entomology 78:290-293; and U.S. Pat. No. 5,743,477, all of which are herein incorporated by reference in their entirety. The present invention allows for real time and rapid testing of pesticidal proteins and their variants.

[0052] The skilled artisan will further appreciate that changes can be introduced by mutation of the nucleotide sequences of the invention thereby leading to changes in the amino acid sequence of the encoded pesticidal proteins, without altering the biological activity of the proteins. Thus, variant isolated nucleic acid molecules can be created by introducing one or more nucleotide substitutions, additions, or deletions into the corresponding nucleotide sequence disclosed herein, such that one or more amino acid substitutions, additions or deletions are introduced into the target mutation region(s) of the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Such variant nucleotide sequences are also encompassed by the present invention.

[0053] For example, conservative amino acid substitutions may be made at one or more, predicted, nonessential amino acid residues. A “nonessential” amino acid residue is a residue that can be altered from the reference sequence of a pesticidal protein without substantially altering the biological activity, whereas an “essential” amino acid residue is required for biological activity. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0054] Alternatively, alterations may be made to the protein sequence of many proteins at the amino or carboxy terminus without substantially affecting activity. This can include insertions, deletions, or alterations introduced by modern molecular methods, such as PCR, including PCR amplifications that alter or extend the protein coding sequence by virtue of inclusion of amino acid encoding sequences in the oligonucleotides utilized in the PCR amplification. Alternatively, the protein sequences added can include entire protein-coding sequences, such as those used 202227W001

[0055] 9 commonly in the art to generate protein fusions. Such fusion proteins are often used to (1) increase expression or stability of a protein of interest (2) introduce a binding domain, enzymatic activity, or epitope to facilitate either protein purification, protein detection, or other experimental uses known in the art (3) target secretion or translation of a protein to a subcellular organelle, such as the periplasmic space of Gram-negative bacteria, or the endoplasmic reticulum of eukaryotic cells, the latter of which often results in glycosylation of the protein.

[0056] Variant nucleotide and amino acid sequences of the present invention also encompass sequences derived from mutagenic and recombinogenic procedures such as DNA shuffling. With such a procedure, one or more different pesticidal protein coding regions can be used to create a new pesticidal protein possessing the desired properties. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. For example, using this approach, sequence motifs encoding a domain of interest may be shuffled between a pesticidal gene of the invention and other known pesticidal genes to obtain a new gene coding for a protein with an improved property of interest, such as an increased insecticidal activity. Strategies for such DNA shuffling are known in the art. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91 :10747- 10751 ; Stemmer (1994) Nature 370:389-391 ; Crameri et al. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al. (1998) Nature 391 :288-291 ; and U.S. Pat. Nos. 5,605,793 and 5,837,458.

[0057] It is also possible to create hybrid or linked pesticidal toxins and to test their activity. For example, additional amino acid sequences that may be tested in the present invention may include fusion partner amino acid sequences which create a fusion protein. By way of example, fusion partner amino acid sequences may be used in the assay or may assist in detection and / or purification of the isolated pesticidal toxin. Non-limiting examples include metal-binding (e.g. polyhistidine) fusion partners, maltose binding protein (MBP), Protein A, glutathione S- transferase (GST), fluorescent protein sequences (e.g. GFP), epitope tags such as myc, FLAG and haemagglutinin tags. The isolated peptides, variant and / or derivatives of the present invention may be produced by any means known in the art, including but not limited to, chemical synthesis and recombinant DNA technology.

[0058] Domain swapping or shuffling is another mechanism for generating altered pesticidal proteins. Domains may be swapped between pesticidal proteins, resulting in hybrid or chimeric toxins with improved pesticidal activity or target spectrum. Methods for generating recombinant proteins and testing them for pesticidal activity are generally known in the art (see, for example, Naimov et al. (2001) Appl. Environ. Microbiol. 67:5328-5330; de Maagd et al. (1996) Appl. Environ. Microbiol. 62:1537-1543; Ge et al. (1991) J. Biol. Chem. 266:17954-17958; Schnepf et al. (1990) J. Biol. Chem. 265:20923-20930; Rang et al. 91999) Appl. Environ. Microbiol. 65:2918-2925) but improved methods to test recombinant proteins for pesticidial activity are provided with the methods of this invention. 202227W001

[0059] 10

[0060] By “linked” is intended that a covalent bond is produced between two or more molecules. Methods that may be used for modification and / or linking of polypeptide ligands such as toxins, include mutagenic and recombinogenic approaches including, but not limited to, site-directed mutagenesis, chimeric polypeptide construction, and DNA shuffling. Polypeptide modification methods also include methods for covalent modification of polypeptides. “Operably linked” means that the linked molecules carry out the function intended by the linkage.

[0061] The term “hybrid toxin” is used to indicate a genetic fusion, having domains operably linked so that, when translated, a functional chimeric protein is formed having, in the aggregate, the properties of the individual domains. “Domain” is used to indicate a region or portion of a protein or confers a recognizable function or structure which contributes to the overall functionality of the protein. It is recognized that a DNA sequence which encodes a protein domain is also encompassed by this definition.

[0062] Identifying Receptors that Bind to Pesticidal Toxins

[0063] The insect specificity of a particular pesticidal toxin may be determined by the presence of the receptor in specific insect species. Binding of the toxins may be specific for the receptor of some insect species and variant receptors may display none binding properties (see, for example Hofte et al. (1989) Microbiol Rev 53: 242-255). It has been shown that pesticidal toxins have the ability to bind to the apical membranes of midgut epithelial cells and this binding triggers a process that will end in cell lysis. There are proteins located in the apical membranes that recognize and bind to the toxin, acting as a receptor. The receptor will mediate the response of the insect cell to the toxin.

[0064] Multiple receptor protein classes for Cry proteins have been identified within insects, and multiple examples exist within each receptor class. Resistance to a particular Cry protein may develop, for example, by means of a mutation within the toxin-binding portion of a cadherin domain of a receptor protein (reference: Gomez, I., Sanchez, J., Miranda, R., Bravo, A., Soberon, M. (2002) Cadherin-like receptor binding facilitates proteolytic cleavage of helix alpha- 1 in domain I and oligomer pre-pore formation of Bacillus thuringiensis Cry1 Ab toxin. FEBS Lett. 513:242-246). These structures of several Cry toxins have been studied and have been shown to be comprised of three distinct domains (reviewed in de Maagd et al., 2003 Structure, diversity, and evolution of protein toxins from spore-forming entomopathogenic bacteria. Annu. Rev. Genet. 37:409-433.). Domain II is formed by three anti-parallel beta sheets packed together in a beta prism and is thought to play an important role in binding insect midgut receptors. Similarly, Domain III binds certain classes of receptor proteins.

[0065] The methods disclosed herein can help uncover additional information about the receptor binding site of various pesticidal toxins and may help to predict the risks that insects develop tolerance to pesticidal proteins based on receptor mutations or help to identify new pesticidal toxins to which otherwise tolerant pests are still sensitive.

[0066] In one embodiment, methods for screening receptors that bind to the toxin polypeptides are disclosed herein. Both the polypeptides and fragments thereof (for example, toxin binding 202227W001

[0067] 11 peptides) may be used in screening assays for pesticidal toxins that bind to receptors and exhibit desired binding characteristics. Desired binding characteristics include, but are not limited to binding affinity, binding site specificity, association and dissociation rates, and the like. The screening assays may be conducted in intact cells (i.e. cell line) according to the methods disclosed herein and include exposing a toxin protein or a ligand binding domain to a receptor and measuring the cell index according to the methods disclosed herein. In this regard, “toxicity” means the decreased viability of a cell. “Viability” is intended to mean the ability of a cell to proliferate and / or differentiate and / or maintain its biological characteristics in a manner characteristic of that cell in the absence of a particular cytotoxic agent.

[0068] As a proof of principle, Sf9 cells were transformed with Cry 1 Ac receptor. These Sf9 cells were plated on the xCelligence device and Cry1 Ac was added. The results showed that the cells expressing the receptor swelled and died whereas untransformed Sf9 cells as control did not. Thus, the techniques and methods discussed herein can be used to identify which receptors bind to a specific toxin or the other way around. Further with the quantitative read out, the receptor toxin-interaction can be further analyzed in terms of relevance of individual amino acid positions in the receptor and the toxin which can be mutated as described before.

[0069] Toxins Activation by Proteases

[0070] Insects have proteases in their midgut that digest plant tissue and may proteolytically activate toxins by releasing the so-called crystallization domain. In contrast, certain insect cells may lack in protease. The addition of protease to such cell lines allows for the study of the effect of protease on activation of the pesticidal toxin. The inventors have shown that trypsin potentiates the effect of certain pesticidal proteins such as Axmi554. The amount of potentiation depends on the concentration of trypsin as well as the concentration of the toxin. Trypsin is capable of potentiating both purified pesticidal toxin and cell extract containing the toxin. However, the activating or potentiating effect of the protease depends on whether the toxin indeed contains a crystallization domain and whether it can be removed by the protease through cleavage at an appropriate protease cleavage site.

[0071] Additional Definitions

[0072] “Fragments” or “biologically active portions” include polypeptide fragments that exhibit pesticidal activity. A biologically active portion of a pesticidal protein can be a polypeptide that is, for example, 10, 25, 50, 100, 150, 200, 250 or more amino acids in length. Such biologically active portions can be prepared by recombinant techniques and evaluated for pesticidal activity. Preferred fragments may be designed or produced by the proteolytic or genetic removal of the crystallization domain.

[0073] “Pest” includes but is not limited to, insects, fungi, bacteria, nematodes, mites, ticks, and the like. Insect pests include insects selected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularly Coleoptera, Lepidoptera, and Diptera. 202227W001

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[0075] The order Lepidoptera includes the families Papilionidae, Pieridae, Lycaenidae, Nymphalidae, Danaidae, Satyridae, Hesperiidae, Sphingidae, Saturniidae, Geometridae, Arctiidae, Noctuidae, Lymantriidae, Sesiidae, and Tineidae. Insects in the order Lepidoptera include without limitation any insect now known or later identified that is classified as a Lepidopteran, including those insect species within suborders Zeugloptera, Glossata, and Heterobathmiina, and any combination thereof. Exemplary Lepidopteran insects include, but are not limited to, Ostrinia spp. such as O. nubilalis (European corn borer); Plutella spp. such as P. xylostella (diamondback moth); Spodoptera spp. such as S. frugiperda (fall armyworm), S. omithogalli (yellowstriped armyworm), S. praefica (western yellowstriped armyworm), S. eridania (southern armyworm) and S. exigua (beet armyworm); Agrotis spp. such as A. ipsilon (black cutworm), A. segetum (common cutworm), A. gladiaria (claybacked cutworm), and A. orthogonia (pale western cutworm); Striacosta spp. such as S. albicosta (western bean cutworm); Helicoverpa spp. such as H. zea (corn earworm), H. punctigera (native budworm), S. Uttoralis (Egyptian cotton leafworm) and H. armigera (cotton bollworm); Heliothis spp. such as H. virescens (tobacco budworm); Diatraea spp. such as D. grandiosella (southwestern corn borer) and D.saccharalis (sugarcane borer); Trichoplusia spp. such as 7. ni (cabbage looper); Sesamia spp. such as S. nonagroides (Mediterranean corn borer); Pectinophora spp. such as P. gossypiella (pink bollworm); Cochylis spp. such as C. hospes (banded sunflower moth); Manduca spp. such as M. sexta (tobacco hornworm) and M. quinquemaculata (tomato hornworm); Elasmopalpus spp. such as E. lignosellus (lesser cornstalk borer); Pseudoplusia spp. such as P. includens (soybean looper); Anticarsia spp. such as A. gemmatalis (velvetbean caterpillar); Plathypena spp. such as P. scabra (green cloverworm); Pieris spp. such as P. brassicae (cabbage butterfly), Papaipema spp. such as P. nebris (stalk borer); Pseudaletia spp. such as P. unipuncta (common armyworm); Peridroma spp. such as P. saucia (variegated cutworm); Keiferia spp. such as K. lycopersicella (tomato pin worm); Artogeia spp. such as A. rapae (imported cabbageworm); Phthorimaea spp. such as P. operculella (potato tuberworm); Crymodes spp. such as C. devastator (glassy cutworm); Feltia spp. such as F. ducens (dingy cutworm); and any combination of the foregoing. In one aspect of this embodiment, the insecticidal proteins of the invention are active against black cutworm, sugar cane borer, and / or southwestern corn borer.

[0076] The order Coleoptera includes the suborders Adephaga and Polyphaga. Suborder Adephaga includes the superfamilies Caraboidea and Gyrinoidea, while suborder Polyphaga includes the superfamilies Hydrophiloidea, Staphylinoidea, Cantharoidea, Cleroidea, Elateroidea, Dascilloidea, Dryopoidea, Byrrhoidea, Cucujoidea, Meloidea, Mordelloidea, Tenebrionoidea, Bostrichoidea, Scarabaeoidea, Cerambycoidea, Chrysomeloidea, and Curculionoidea. Superfamily Caraboidea includes the families Cicindelidae, Carabidae, and Dytiscidae. Superfamily Gyrinoidea includes the family Gyrinidae. Superfamily Hydrophiloidea includes the family Hydrophilidae. Superfamily Staphylinoidea includes the families Silphidae and Staphylinidae. Superfamily Cantharoidea includes the families Cantharidae and Lampyridae. Superfamily Cleroidea includes the families Cleridae and Dermestidae. Superfamily Elateroidea includes the families Elateridae and Buprestidae. Superfamily Cucujoidea includes the family Coccinellidae. Superfamily Meloidea includes the family Meloidae. Superfamily Tenebrionoidea includes the family Tenebrionidae. Superfamily Scarabaeoidea includes the families Passalidae and Scarabaeidae. Superfamily Cerambycoidea includes the family Cerambycidae. Superfamily 202227W001

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[0078] Chrysomeloidea includes the family Chrysomelidae. Superfamily Curculionoidea includes the families Curculionidae and Scolytidae.

[0079] The order Diptera includes the Suborders Nematocera, Brachycera, and Cyclorrhapha. Suborder Nematocera includes the families Tipulidae, Psychodidae, Culicidae, Ceratopogonidae, Chironomidae, Simuliidae, Bibionidae, and Cecidomyiidae. Suborder Brachycera includes the families Stratiomyidae, Tabanidae, Therevidae, Asilidae, Mydidae, Bombyliidae, and Dolichopodidae. Suborder Cyclorrhapha includes the Divisions Aschiza and Aschiza. Division Aschiza includes the families Phoridae, Syrphidae, and Conopidae. Division Aschiza includes the Sections Acalyptratae and Calyptratae. Section Acalyptratae includes the families Otitidae, Tephritidae, Agromyzidae, and Drosophilidae. Section Calyptratae includes the families Hippoboscidae, Oestridae, Tachinidae, Anthomyiidae, Muscidae, Calliphoridae, and Sarcophagidae.

[0080] The methods provided herein may be effective against or involve Hemiptera such as Lygus hesperus, Lygus lineolaris, Lygus pratensis, Lygus rugulipennis Popp, Lygus pabulinus, Calocoris norvegicus, Orthops compestris, Plesiocoris rugicollis, Cyrtopeltis modestus, Cyrtopeltis notatus, Spanagonicus albofasciatus, Diaphnocoris chlorinonis, Labopidicola allii, Pseudatomoscelis seriatus, Adelphocoris rapidus, Poecilocapsus lineatus, Blissus leucopterus, Nysius ericae, Nysius raphanus, Euschistus servus, Nezara viridula, Eurygaster, Coreidae, Pyrrhocoridae, Tinidae, Blostomatidae, Reduviidae, and Cimicidae. Pests of interest also include Araecerus fasciculatus, coffee bean weevil; Acanthoscelides obtectus, bean weevil; Bruchus rufmanus, broadbean weevil; Bruchus pisorum, pea weevil; Zabrotes subfasciatus, Mexican bean weevil; Diabrotica balteata, banded cucumber beetle; Cerotoma trifurcata, bean leaf beetle; Diabrotica virgifera, Mexican corn rootworm; Epitrix cucumeris, potato flea beetle; Chaetocnema confinis, sweet potato flea beetle; Hypera postica, alfalfa weevil; Anthonomus quadrigibbus, apple curculio; Sternechus paludatus, bean stalk weevil; Hypera brunnipennis, Egyptian alfalfa weevil; Sitophilus granaries, granary weevil; Craponius inaequalis, grape curculio; Sitophilus zeamais, maize weevil; Conotrachelus nenuphar, plum curculio; Euscepes postfaciatus, West Indian sweet potato weevil; Maladera castanea, Asiatic garden beetle; Rhizotrogus majalis, European chafer; Macrodactylus subspinosus, rose chafer; Tribolium confusum, confused flour beetle; Tenebrio obscurus, dark mealworm; Tribolium castaneum, red flour beetle; Tenebrio molitor, yellow mealworm.

[0081] Other insect lines may be tested in the methods disclosed herein, the only limitation being whether a change in cell index can be determined using an instrument such as xCelligence.

[0082] Example 1 Cell Lines

[0083] Exemplary cell lines used were Sf9 cells, AC20 cells and squash bug (Hemipteran) cells. Sf9 cells are Lepidoptera cells, and more particularly, the Sf9 insect cell line is a clonal isolate derived from the parental Spodoptera frugiperda cell line IPLB-Sf-21-AE). Sf9 cells can be maintained in suspension culture. Sf9 cells attach firmly to surfaces, and their small, regular size makes them exceptional for the formation of monolayers and plaques. https: / / www.thermofisher.com / de / de / home / references / qibco-cell-culture-basics / cell- morpholoqvZmorpholoqv-of-sf9-cells.html 202227W001

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[0085] AC20 is an established insect cell line (AC20) from embryos of Constricted leaf hopper, Agallia constricta Hempitera). It was obtained from USDA Lab, ARS, BCIRL, Columbia, MO. AC20 cells are maintained as adherent cultures.

[0086] Squash bug cells lines have been described for example in Goodman, C.L., Ringbauer, J.A., Li, YF. et al. Cell lines derived from the squash bug, Anasa tristis (Coreidae: Hemiptera). In Vitro Cell. Dev.Biol. -Animal 53, 417-420 (2017). https: / / doi.org / 10.1007 / s11626-017-0134-5 Insect cell line freezing was performed as follows: Cells were grown to confluency in T25 flasks. Spend medium was removed from the flasks. For freezing 8 ampules, 4 mL (90% PBS / 10% medium) solution into each T25 flask. The cells are detached by pipetting medium up and down over attached cells or by using trypsin-EDTA. The cells are transferred from flasks into sterile 15 mL tubes and are gently mixed by inverting the tube a few times. The cells are then placed on ice. Approximately 0.1 mL of cells are removed for counting. The minimum density of the cells should be 3-4 X 106cells / mL to proceed. If the concentration is lower, the cells can be centrifuged and medium removed until this concentration is reached. The cells are diluted 1 :1 with 10% DMSO in medium as follows: while on ice, 4 mL of 10% DMSO solution is added to the cell solution, the tube is gently inverted after addition is complete. This is done dropwise to give cells time to adjust to the addition of DMSO. For freezing, the goal is to decrease the temperature approximately 1 °C per minute. This can be achieved using cell freezing containers (e.g. CoolCell® Cell Freezing Container) or by a styrofoam holder that is placed in a liquid nitrogen tank set at a height above the surface of the liquid nitrogen. The cells are frozen overnight in a -80 °C freezer. For long term storage, cells are best kept in liquid nitrogen.

[0087] Insect cell line thawing was performed as follows: A water bath was warmed to 30° C. Medium was warmed. The ampules are placed in the water bath until they are completely thawed. The contents of the ampules are transferred to centrifuge tubes and the ampules are centrifuged for 5 minutes at 4 °C, 1 ,000 g. The supernatant is discarded. 5 mL of medium was added and the cells was pipetted up and down until completely in suspension. The cell suspension is transferred into a T25flask. The flask is placed in a 25 C incubator and growth is monitored.

[0088] Example 2 Real Time Monitoring Using xCelligence

[0089] For real time cell viability monitoring xCELLigence instrument from ACEA (Model: MP, s / n: 28-1- 180401764-2) was employed to monitor the dynamic response of the insect cell lines to toxin stimulation via measurement of cell index (Cl). Cl is a parameter to describe electronic impedance, which corresponds to the number of cells attaching to the bottom of a microelectrode embedded (E-plate) wells as an index of cell viability. Cry toxins disrupt cell attachment and cause cell rounding (i.e. reduce cell spreading), thus lowering the Cl values. An xCelligence well was seeded with 50,000 cells before being placed in an incubator that has the same temperature set up as the cell line culture incubator. Cells were allowed to attach and grow until a certain cell index before reagents were applied. For Sf9 cells, it takes about 2 hours, and for AC20 cells, it takes about 20 hours. Once the cells are grown, toxins or other control compounds can be added, either directly to cell plates or toxins may be mixed with other substances before being added to cell plates. As described below, to potentiate toxin activity, trypsin may be added. 202227W001

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[0091] Example 3 Purification of BP005, Cry and Axmi Toxins

[0092] BP005 was synthesized and cloned as described in U.S. Patent Application No. 16 / 478,236. which is incorporated by reference. Purified BP005 was submitted to bioassay vs. hemipteran pests according to standard protocol and such purified BP005 was used in the bioassays described herein.

[0093] Axmi554 was synthesized and purified as described in U.S. Patent No. 10,435,707, which is incorporated by reference.

[0094] Example 4: BP005 and Axmi554 on SF9 cells

[0095] Example 5: Squash bug cells (Hemiptera) - BP005, Axmi554, R1 , BSA, medium, and PBS Example 6: AC20 - Intact 554 show activity on AC2

[0096] Example 7: Trypsin potentiates Axmi554 activity on AC20 cells to about 25 folds

[0097] Example 4 Effect of Addition of BP005, Axmi554 on Sf9 Cells

[0098] Sf9 cells were plated on xCelligence E-plates. The cells were kept on the plates for 2 hours before addition of media (as control), BP005 (0.5 mg / mL), Axmi554 (1 mg / mL) and pH 10 buffer as control. As shown in Figure 1 , BP005 showed strong activity against Sf9 cells on E- plates, as evidenced by the decrease in cell index. The time for the cell index to reach zero (meaning all cells were killed) varied from between 5 hours to 15 hours, depending on the ratio of BP005 that was added to the cells. Axmi554 did not show activity against Sf9 activity as assessed on e-Plates.

[0099] Example5: Squash bug cells (Hemiptera) - BP005, Axmi554, R1 , BSA, medium, and PBS Figure 2 is a set of charts showing BP005, Axmi554 and R1 (R1=AmxiR1 described in WQ2010 / 091230) on squash bug cells (Hemiptera). Fig. 2A shows the effect of BP005 when added to the cells at the indicated concentrations, 20 hours after the cells are seeded onto the plate. Fig. 2B shows the same as Fig. 2A, but with Axmi554 added to the cells. Fig. 2c shows the same as Fig. 2A, but with R1 added to the cells. Fig. 2D shows a control where BSA and PBS were each added to the cells.

[0100] Example 6 Effect of Axmi554 on AC20 Cells

[0101] Figure 3 shows real time monitoring of the effect of Axmi554 toxins on AC20 (hemiptera) cells. It shows the concentration dependent activity of Axmi554 on AC20 hemiptera cells

[0102] Example 7 Effect of Trypsin on Axmi554 on AC20 Cells

[0103] Fig.4 shows that Trypsin potentiates Axmi554 activity on AC20 cells

[0104] Axmi554 at 1 .4 mg / mL was treated with 1 ug / ml trypsin at 4C for overnight. The sample mixture was then applied to the AC20 cells that were seeded on the E-plate and cultured for 20 hrs. Serial dilution of the trypsin-treated Axmi554 was also applied to AC20 cells to identify the minimal Axmi554 concentration needed to kill the cells. The results show that trypsin potentiates activity of Axmi554 several fold when applied at a concentration of 1 ug / mL to Axmi554 extract. Trypsin activated the activity of purified Axmi554 more than 125-fold. Even when applied at concentrations as low as 0.5 ug / mL, trypsin was shown to potentiate activity of Axmi554 on AC20 cells. 202227W001

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[0106] A skilled person will recognize that many suitable variations of the method may be substituted for or used in addition to those described above and in the claims. It should be understood that the implementation of other variations and modifications of the embodiments of the invention and its various aspects will be apparent to one skilled in the art and the invention is not limited by the specific embodiments described herein and in the claims. Therefore, it is contemplated to cover the present embodiments of the invention and any and all modifications, variations and equivalents that fall within the true spirit and scope of the basic underlying principles described and claimed herein.

Claims

202227W00117Claims1 . A method of testing the effect of a pesticidal toxin in an insect cell line, the method comprising:- attaching a set of insect cells to a surface,- contacting the cells with an aliquot of the toxin, measuring cell death by a decrease in cell attachment to a surface, as determined by real time electronic sensing of resistance or impedance.

2. The method of claim 1 , wherein the cells are placed on multi-cell culture plate and the real time electronic sensing is performed in the multi-cell plate.

3. The method of claim 1 to 2, wherein the cells are contacted with a bacterial cell extract, wherein the bacterial cells were transformed with a vector for expression of the pesticidal toxin, prior to contacting the cells.

4. The method of claim 1 to 3, wherein the pesticidal toxin is a Lepidopteran or Hemipteran toxin5. The method of claim 4, wherein the Lepidopteran or Hemipteran toxin is selected from the group consisting of a MTX, monalysin and a 6-endotoxin.

6. The method of claim 5, wherein the insect cell line is a hemipteran or lepidopteran cell line.

7. The method of claim 6, wherein the cells are from Squash bug or AC20 cell lines8. The method of claim 1 to 7, further comprising treating the toxin with a protease prior to, concurrent with, or following the addition of the cells.

9. The method of claim 8, wherein the protease is trypsin.

10. A method of testing the effect of a domain of a pesticidal protein for its effect on toxicity, the method comprising:- mutating a protein by changing one or more amino acids;- contacting a cell line with the mutated pesticidal protein;- measuring cell death by a decrease in cell attachment to a surface by electronic sensing of resistance or impedance; and- contacting a cell line with wildtype pesticidal protein and measuring the resulting cell death for comparison.11 . The method of claim 10, where the cell line is AC20 cell line.202227W0011812. The method of claim 10, where the cell line is a Squash bug cell line.

13. A method for identifying a toxin with activity against an insect cell, the method comprising:- plating a population of insect cells on a plate to which the insect cells adhere;- adding a compound to the insect cells:- measuring the cell index following the addition of the compound;- comparing the results with the cell index of the same cells when no or inactivated compound is added.

14. A method of identifying receptor molecules that interact with a pesticidal toxin comprising:- transfecting an insect cell line with the vector so as to over-express the putative receptor molecule in the cell line;- plating the insect cell line on a device that is capable of measuring the cell index of the cell line by way of electronic sensing of resistance;- adding a pesticidal toxin to the insect cell line that overexpresses the putative receptor; and- determining the effect of cell index caused by the addition of the toxin.

15. A method of identifying a pesticidal toxin that interact with a receptor molecule comprising:- transfecting an insect cell line with the vector so as to over-express the receptor molecule in the cell line;- plating the insect cell line on a device that is capable of measuring the cell index of the cell line by way of electronic sensing of resistance;- adding a pesticidal toxin to the insect cell line that overexpresses the receptor- determining the effect of cell index caused by the addition of the toxin.

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