Method for identifying and evaluating insect control genes

Abstract

Methods and compositions for identifying pesticidal genes are provided. In particular, the methods of the invention comprise systematically designing brush marginal membrane vesicles (BBMVs) from a particular target pest, where the BBMVs are filled with a detectable substance, and then contacting the BBMVs with a candidate pesticidal protein.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 433,569, filed on December 19, 2022, the entire contents of which are hereby incorporated by reference. Background Art

[0003] Insect pests are a major factor in world crop losses. For example, feeding damage by corn rootworms and boll weevil damage can be economically devastating to agricultural producers. Insect - pest - related crop losses caused by corn rootworms alone reach $1 billion per year.

[0004] Certain microbial species of the genus Bacillus are known to have pesticidal activity against a wide range of insect pests, including Lepidoptera, Diptera, Coleoptera, Hemiptera, etc. Bacillus thuringiensis and Bacillus popilliae are among the most successful biocontrol agents discovered to date. Insect pathogenicity has been attributed to the following strains: Bacillus larvae, Bacillus lentimorbus, Bacillus popilliae, Bacillus sphaericus, Bacillus thuringiensis (Harwook, editor (1989) Bacillus [Genus Bacillus] (Plenum Press), page 306), and Bacillus cereus (International Publication No. WO 96 / 10083). Although pesticidal proteins have also been isolated from the vegetative growth stage of the genus Bacillus, pesticidal activity seems to be concentrated in the parasporal crystal protein inclusions. Several genes encoding these pesticidal proteins have been isolated and characterized (see, for example, U.S. Patent Nos. 5,366,892 and 5,840,868).

[0005] Microbial biopesticides, particularly those obtained from Bacillus strains, play an important role in agriculture as an alternative to chemical pest control. Pest-killing proteins (referred to as δ-endotoxins or Cry toxins) isolated from Bacillus thuringiensis strains are initially produced in an inactive protoxin form. These protoxins are cleaved and transformed into active toxins through the action of proteases in the insect gut. See, Rukmini et al. (2000) Biochimie 82:109-116; Oppert (1999) Arch. Insect Biochem. Phys. 42:1-12; and Carroll et al. (1997) J. Invertebrate Pathology 70:41-49. Proteolytic activation of the toxin can include removal of N- and C-terminal peptides from the protein, as well as internal cleavage of the protein. Once activated, the Cry toxin binds with high affinity to receptors on the insect gut epithelial cells, thereby creating leakage channels in the cell membrane, leading to lysis of the insect gut and subsequent death of the insect by starvation and septicemia. See, for example, Li et al. (1991) Nature 353:815-821.

[0006] Genetically engineering crop plants with pest-killing genes to produce pest-killing proteins from Bacillus (and other sources) is a key solution to the growing problem of feeding an increasing world population. Insect pressure, expanding pest distribution areas, and insect resistance to control are all key obstacles to ensuring the world's future food supply. To this end, scientists have made great efforts to discover new genes and modes of action to overcome these obstacles. Nature seems to provide a large number of tools that can be used for such purposes. However, the discovery cost of such genes is high, and candidate genes need to be sorted to identify safe and best-performing genes for use in agriculture. Therefore, there is a need in the art for new methods for effectively identifying novel pest-killing genes and rapidly predicting their performance in plants. Summary of the Invention

[0007] Embodiments of the present invention provide methods and compositions for identifying novel pesticidal genes and predicting the performance of such pesticidal genes in plants against corresponding plant pests. The methods disclosed herein allow for the rapid and efficient screening of a large number of pesticidal genes (and their gene products) to identify potential pesticidal genes that can be used in transgenic crops to control insects. The method for identifying novel pesticidal genes includes systematically designing and constructing brush border membrane vesicles (referred to herein as "BBMV"), wherein the brush border membrane used to prepare the BBMV is derived from a specific target plant pest (e.g., Spodoptera frugiperda, Helicoverpa zea, etc.). A dye, reporter gene, or any means that can be visually observed or detected in solution when the outer membrane of the BBMV begins to leak or is punctured (referred to herein as a "detectable substance") is inserted into the lumen of the BBMV. The BBMV can be used to rapidly screen potential pesticidal gene candidates in a high-throughput manner, wherein various gene candidates are applied to the BBMV, and if leakage is detected, it can be inferred that the candidate gene is active against the corresponding insect from which the brush border membrane is derived. This method does not require a large number of insect bioassays or studies in plants. The degree of leakage can also be correlated with the degree of effectiveness of the gene candidate against a given crop pest. The methods provided herein help to rapidly eliminate pesticidal genes that are ineffective against a given pest from the candidate gene pool in a pesticidal gene discovery program. Pesticidal genes that cause BBMV leakage can be identified and further studied to further characterize and transform into the corresponding crop plants relative to the target pest. The methods of the embodiments are further suitable for automated and high-throughput screening. Detailed Description

[0008] One or more embodiments relate to compositions and methods for identifying and predicting the performance of novel pesticidal proteins that exhibit resistance to a given pest. "Resistance" means that a pest (e.g., an insect) is killed upon ingestion or otherwise contacting one or more embodiments of the polypeptide. "Tolerance" is intended to mean impairment or reduction of the movement, feeding, reproduction, or other functions of a pest. These methods include using BBMV, into the lumen of which a detectable substance is inserted. The BBMV contains the brush border membrane of the target pest (referred to herein as the "target pest"). In one embodiment, the target pests consist of Lepidoptera, Diptera, Hemiptera, and Coleoptera, as well as the phylum Nematoda. The BBMV is then contacted with a candidate pesticidal gene (referred to herein as the "candidate gene"). If the candidate gene is active against the BBMV, it will attach to the BBMV and cause lysis of the outer membrane of the BBMV. This lysis will then allow the detectable substance to leak from the BBMV into the solution, which can then be detected and analyzed. The activity and effectiveness of gene candidates against the target pest can be rapidly evaluated by this method.

[0009] "Pesticidal toxin" or "pesticidal protein" is intended to mean a toxin that is toxicologically active against one or more pests, including but not limited to members of the Lepidoptera, Diptera, Hemiptera, and Coleoptera orders, or the phylum Nematoda, or a protein that is homologous to such a protein. Pesticidal proteins include amino acid sequences deduced from the full-length nucleotide sequences disclosed herein, as well as amino acid sequences that are shorter than the full-length sequence (due to the use of alternative downstream start sites, or due to processing that results in a shorter protein with pesticidal activity). Processing may occur within the organism expressing the protein, or within the pest after ingestion of the protein.

[0010] In another aspect, the BBMV concept as described herein can be used to evaluate chemicals or other active ingredients against a target pest. For example, novel chemicals can be screened and tested against a panel of BBMVs in a microtiter plate, and BBMV leakage can be tested, which will indicate the activity or effectiveness of the chemical against the brush border membrane of the target pest.

[0011] In another aspect, one of ordinary skill in the art can use the methods described herein to evaluate and characterize pesticidal proteins. For example, fragments of known or novel pesticidal proteins can be generated to evaluate which portions of the pesticidal protein bind to and cleave the brush border membrane of the target pest.

[0012] In another aspect, one of ordinary skill in the art can rapidly evaluate the effectiveness of pesticidal protein variants against a given target pest. For example, one of ordinary skill in the art can generate multiple variants of a given pesticidal gene, with the aim of expanding the breadth of target pests that the pesticidal gene can control and / or the effectiveness of the pesticidal gene. In this example, the methods described herein can be used to rapidly screen such variants against BBMVs derived from one or more target pests. Variants include polypeptides that differ in amino acid sequence due to mutagenesis.

[0013] In one aspect, one or more embodiments are antibodies that can be used for quantitative or qualitative detection of a protein or peptide molecule of interest, or for detection of post-translational modifications of a protein. As used herein, an antibody or peptide is said to "specifically bind" to a protein or peptide molecule if the binding of the antibody or peptide to the protein or peptide molecule of the embodiment is not competitively inhibited by the presence of non-related molecules. In some aspects, the BBMVs of the embodiments can be used to detect a pesticidal protein of interest by using an antibody. Methods for detecting a protein or peptide of interest using an antibody are known in the art.

[0014] In another aspect, the BBMV of the embodiments can be used to evaluate the effectiveness of a given expression cassette. For example, a given pesticidal gene known to be active against a target pest can be used to evaluate the effectiveness of various post-translational strategies, thereby indicating the best way to express and / or target the pesticidal gene in a plant to control the target pest. As another example, one skilled in the art can utilize the methods described herein to evaluate the effectiveness of gene promoters (including inducible promoters), where the pesticidal gene is operably linked to a candidate inducible promoter and various screens can be performed by adding various inducers. If BBMV formation is leaky, the inducible promoter can be associated with its corresponding inducer and then used for gene expression in transgenic plants.

[0015] "Plant" is intended to mean an entire plant, a plant organ (e.g., leaf, stem, root, etc.), a seed, a plant cell, a propagule, an embryo, and its progeny. A plant cell can be differentiated or undifferentiated (e.g., callus, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells, pollen).

[0016] "Transgenic plant" or "transformed plant" or "stably transformed" plant or cell or tissue refers to a plant in which an exogenous nucleic acid sequence or DNA fragment has been incorporated or integrated into a plant cell. These nucleic acid sequences include those that are exogenous or not present in an untransformed plant cell, as well as those that can be endogenous or present in an untransformed plant cell. "Heterologous" generally means that a nucleic acid sequence is not endogenous to the cell or a part of the native genome in which it is located and has been added to the cell by infection, transfection, microinjection, electroporation, microprojection, etc.

[0017] Among such DNA sequences encoding proteins having various pesticidal resistances or insect tolerances, including: Cry1F protein or hybrids derived from Cry1F protein (e.g., hybrid Cry1A-Cry1F proteins or their toxic fragments described in U.S. 6,326,169, US 6,281,016, U.S. 6,218,188), Cry1A type proteins or their toxic fragments, Cry1Ac protein or hybrids derived from this Cry1Ac protein (e.g., hybrid Cry1Ab-Cry1Ac protein described in US 5,880,275), or Cry1Ab or Bt2 protein or their insecticidal fragments as described in EP451878, Cry2Ae, Cry2Af or Cry2Ag proteins or their toxic fragments as described in WO2002 / 057664, Cry1A.105 protein (SEQ ID No.7) or its toxic fragment described in WO 2007 / 140256, VIP3Aa19 protein with NCBI accession number ABG20428, VIP3Aa20 protein with NCBI accession number ABG20429 (SEQ ID No.2 in WO 2007 / 142840), VIP3A proteins produced in COT202 or COT203 cotton events (WO2005 / 054479 and WO2005 / 054480 respectively), Cry proteins as described in WO2001 / 47952, VIP3Aa proteins or their toxic fragments as described in Estruch et al. (1996), Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 28:93(11):5389-94 and U.S. 6,291,156, insecticidal proteins from strains of the genus Xenorhabdus (as described in WO 98 / 50427), Serratia (particularly from S. entomophila) or Photorhabdus species, Tc protein from Photorhabdus as described in WO98 / 08932 (e.g., Waterfield et al., 2001, Appl. Environ. Microbiol. [Applied and Environmental Microbiology] 67(11):5017-24; French-Constant and Bowen, 2000, Cell Mol Life Sci. [Cell and Molecular Life Sciences]; 57(5):828-33). This article includes any variant or mutant of any of these proteins that differs from any of the above-mentioned proteins (which can effectively combat the target) in some (1-10 or 1-5) amino acids, or any variant or mutant fused with a transit peptide (such as a plastid transit peptide) or another protein or peptide.

[0018] In various embodiments, the methods described herein can be used to identify an optimal combination of pesticidal proteins against one or more target pests. These methods can also be used to measure any negative impacts when pesticidal genes are combined with other genes or agents, which may indicate unfavorable combinations that could affect the effectiveness of the pesticidal genes. For example, such combinations can include genes for traits such as herbicide tolerance, insect tolerance, drought tolerance, nematode control, water use efficiency, nitrogen use efficiency, improved nutritional value, disease resistance, improved photosynthesis, improved fiber quality, stress tolerance, improved reproduction, etc., or combinations with certain chemicals, hormones, or other agents. It is anticipated that the effects of environmental variables (such as heat, alkalinity / acidity) on one or more pesticidal genes and their respective performance can be evaluated.

[0019] Typically, the “plant expression cassette” is inserted into a “plant transformation vector”. The plant transformation vector can consist of one or more DNA vectors required to effect plant transformation. For example, plant transformation vectors composed of more than one contiguous DNA segment are commonly utilized in the art. These vectors are commonly referred to in the art as “binary vectors”. Binary vectors, as well as vectors with helper plasmids, are most commonly used for Agrobacterium-mediated transformation, where the size and complexity of the DNA segments required for efficient transformation are quite large, and it is advantageous to separate the functions onto different DNA molecules. Binary vectors typically contain a plasmid vector that contains cis-acting sequences required for T-DNA transfer (such as left and right borders), a selectable marker engineered to be expressed in plant cells, and a “gene of interest” (a gene engineered to be expressed in plant cells, and by which transgenic plants are desired to be generated). Sequences required for bacterial replication are also present on this plasmid vector. The cis-acting sequences are arranged in a manner that permits efficient transfer into and expression in plant cells. For example, the selectable marker gene and the pesticidal gene are located between the left and right borders. Typically, a second plasmid vector contains the trans-acting factors that mediate the transfer of T-DNA from Agrobacterium to plant cells. This plasmid typically contains virulence functions (Vir genes) that allow Agrobacterium to infect plant cells and transfer DNA by cleavage at the border sequences and vir-mediated DNA transfer, as understood in the art (Hellens and Mullineaux (2000) Trends in Plant Science 5:446-451). Several types of Agrobacterium strains (such as LBA4404, GV3101, EHA101, EHA105, etc.) can be used for plant transformation. The second plasmid vector is not required for transforming plants by other methods such as microprojectile bombardment, microinjection, electroporation, polyethylene glycol, etc.

[0020] The active ingredients that may be used or evaluated in the embodiments of the present invention are generally applied in the form of a composition and can be applied to a solution simultaneously or sequentially with other compounds and contacted with the BBMV of the embodiment. These compounds can be fertilizers, herbicides, cryoprotectants, surfactants, detergents, pesticidal soaps, dormant oils, polymers, and / or timed-release or biodegradable carrier formulations that allow for long-term dosing of the target area after a single application of the formulation. They can also be selective herbicides, chemical insecticides, virucides, microbicides, amoebicides, pesticides, fungicides, bactericides, nematicides, molluscicides, or a mixture of several of these formulations, if desired, together with additional agriculturally acceptable carriers, surfactants, or application-promoting adjuvants commonly used in the formulation art. Suitable carriers and adjuvants can be solid or liquid and correspond to substances commonly employed in formulation technology, such as natural or regenerated minerals, solvents, dispersants, wetting agents, thickeners, binders, or fertilizers. Similarly, the formulation can be prepared as an edible "bait" or made into a pest "trap" to allow the target pest to ingest or take in the pesticidal formulation.

[0021] "Pest" includes, but is not limited to, insects, fungi, bacteria, nematodes, mites, ticks, etc. Insect pests include insects selected from the following orders: Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., especially Coleoptera, Lepidoptera, and Diptera.

[0022] Coleoptera includes Adephaga and Polyphaga. Adephaga includes Caraboidea and Gyrinoidea, while Polyphaga includes Hydrophiloidea, Staphylinoidea, Cantharoidea, Cleroidea, Elateroidea, Dascilloidea, Dryopoidea, Byrrhoidea, Cucujoidea, Meloidea, Mordelloidea, Tenebrionoidea, Bostrichoidea, Scarabaeoidea, Cerambycidae, Chrysomeloidea and Curculionoidea. Caraboidea includes Cicindelidae, Carabidae and Dytiscidae. Gyrinoidea includes Gyrinidae. Hydrophiloidea includes Hydrophilidae. Staphylinoidea includes Silphidae and Staphylinidae. Cantharoidea includes Cantharidae and Lampyridae. Cleroidea includes Cleridae and Dermestidae. Elateroidea includes Elateridae and Buprestidae. Cucujoidea includes Coccinellidae. Meloidea includes Meloidae. Tenebrionoidea includes Tenebrionidae. Scarabaeoidea includes Passalidae and Scarabaeidae. Cerambycidae includes Cerambycidae. Chrysomeloidea includes Chrysomelidae. Curculionoidea includes Curculionidae and Scolytidae.

[0023] Diptera includes Nematocera, Brachycera, and Cyclorrhapha. Nematocera includes Tipulidae, Psychodidae, Culicidae, Ceratopogonidae, Chironomidae, Simuliidae, Bibionidae, and Cecidomyiidae. Brachycera includes Stratiomyidae, Tabanidae, Therevidae, Asilidae, Mydidae, Bombyliidae, and Dolichopodidae. Cyclorrhapha includes Aschiza and Schizophora. Aschiza includes Phoridae, Syrphidae, and Conopidae. Schizophora includes Acalyptratae and Calyptratae. Acalyptratae includes Otitidae, Tephritidae, Agromyzidae, and Drosophilidae. Calyptratae includes Hippoboscidae, Oestridae, Tachinidae, Anthomyiidae, Muscidae, Calliphoridae, and Sarcophagidae.

[0024] Lepidoptera includes Papilionidae, Pieridae, Lycaenidae, Nymphalidae, Danaidae, Satyridae, Hesperiidae, Sphingidae, Saturniidae, Geometridae, Arctiidae, Noctuidae, Lymantriidae, Sesiidae, and Tineidae.

[0025] Nematodes include parasitic nematodes such as root-knot nematodes, cyst nematodes, and root-lesion nematodes, including species of Heterodera spp., Meloidogyne spp., and Globodera spp.; also including members of cyst nematodes, including but not limited to Heterodera glycines (soybean cyst nematode), Heterodera schachtii (beet cyst nematode), Heterodera avenae (cereal cyst nematode); and Globodera rostochiensis and Globodera pallida (potato cyst nematode). Root-lesion nematodes include species of Pratylenchus spp.

[0026] Hemipteran pests (including species designated as Hemiptera, Homoptera, or Heteroptera) include but are not limited to: species of Lygus spp., such as Lygus hesperus (western tarnished plant bug), Lygus lineolaris, and Lygus elisus; aphids, such as Myzus persicae, Aphis gossypii, Myzus cerasi, Aphis glycines Matsumura; Nilaparvata lugens and Nephotettix spp. (green leafhoppers of rice); and stink bugs, such as Acrosternum hilare, Halyomorpha halys, Nezara viridula, Oebalus pugnax, Pentatoma rufipes, Rhaphigaster nebulosa, and Troilus luridus.

[0027] For embodiments of major crops, insect pests include: Corn ( Maize):European corn borer (Ostrinia nubilalis); black cutworm (Agrotis ipsilon); corn earworm (Helicoverpa zea); fall armyworm (Spodoptera frugiperda); southwestern corn borer (Diatraea grandiosella); lesser cornstalk borer (Elasmopalpus lignosellus); sugarcane borer (Diatraea saccharalis); Western corn rootworm (Diabrotica virgifera); northern corn rootworm (Diabrotica longicornis barberi); southern corn rootworm (Diabrotica undecimpunctata howardi); Melanotus spp.) wireworms; northern masked chafer (Cyclocephala borealis); southern masked chafer (Cyclocephala immaculata); Japanese beetle (Popillia japonica); corn flea beetle (Chaetocnema pulicaria); maize billbug (Sphenophorus maidis); corn leaf aphid (Rhopalosiphum maidis); corn root aphid (Anuraphis maidiradicis); chinch bug (Blissus leucopterus leucopterus); redlegged grasshopper (Melanoplus femurrubrum); migratory grasshopper (Melanoplus sanguinipes); seedcorn maggot (Hylemya platura); corn blot leafminer (Agromyza parvicornis); grass thrips (Anaphothrips obscrurus); thief ant (Solenopsis milesta); twospotted spider mite (Tetranychus urticae);. Sorghum: Chilo partellus (sorghum borer); Spodoptera frugiperda (fall armyworm); Spodoptera cosmioides; Spodoptera eridania; Helicoverpa zea (corn earworm); Elasmopalpus lignosellus (lesser cornstalk borer); Feltia subterranea (granulate cutworm); Phyllophaga crinita (white grub); Eleodes, Conoderus, and Aeolus spp. (wireworms); Oulema melanopus (cereal leaf beetle); Chaetocnema pulicaria (corn flea beetle); Sphenophorus maidis (maize billbug); Rhopalosiphum maidis (corn leaf aphid); Sipha flava (yellow sugarcane aphid); Blissus leucopterus leucopterus (chinch bug); Contarinia sorghicola (sorghum midge); Tetranychus cinnabarinus (carmine spider mite); Tetranychus urticae (twospotted spider mite); Wheat: Mythimna separata (army worm); Spodoptera frugiperda (fall armyworm); Elasmopalpus lignosellus (lesser cornstalk borer); Agrotis orthogonia (western cutworm); Elasmopalpus lignosellus (lesser cornstalk borer); Oulema melanopus (cereal leaf beetle); Hypera punctata (clover leaf weevil); Diabrotica undecimpunctata howardi (southern corn rootworm); Russian wheat aphid; Schizaphis graminum (greenbug); Macrosiphum avenae (English grain aphid); Melanoplus femurrubrum (redlegged grasshopper); Melanoplus differentialis (differential grasshopper); Melanoplus sanguinipes (migratory grasshopper); Mayetiola destructor (Hessian fly); Sitodiplosis mosellana (wheat midge); Meromyza americana (wheat stem maggot); Hylemya coarctata (wheat bulb fly); Frankliniella fusca (tobacco thrips); Cephus cinctus (wheat stem sawfly); Aceria tulipae (wheat curl mite); Sunflower; Sunflower bud moth (Suleima helianthana); Sunflower moth (Homoeosoma electellum); Sunflower beetle (zygogramma exclamationis); Carrot beetle (Bothyrus gibbosus); Sunflower seed midge (Neolasioptera murtfeldtiana); Cotton: Green cotton bollworm (Heliothis virescens, cotton budworm); Corn earworm (Helicoverpa zea) (cotton bollworm); Beet armyworm (Spodoptera exigua); Pink bollworm (Pectinophora gossypiella); Boll weevil (Anthonomus grandis); Cotton aphid (Aphis gossypii); Cotton fleahopper (Pseudatomoscelis seriatus); Bandedwinged whitefly (Trialeurodes abutilonea); Tarnished plant bug (Lygus lineolaris); Redlegged grasshopper (Melanoplus femurrubrum); Differential grasshopper (Melanoplus differentialis); Onion thrips (Thrips tabaci); Tobacco thrips (Franklinkiella fusca); Carmine spider mite (Tetranychus cinnabarinus); Two-spotted spider mite (Tetranychus urticae); Rice: Diatraea saccharalis (sugarcane borer); Spodoptera frugiperda (fall armyworm); Spodoptera cosmioides; Spodoptera eridania; Helicoverpa zea (corn earworm); Colaspis brunnea (grape colaspis); Lissorhoptrus oryzophilus (rice waterweevil); Sitophilus oryzae (rice weevil); Nephotettix nigropictus (rice leafhopper); Blissus leucopterus leucopterus (chinch bug); Acrosternum hilare (green stink bug); Chilo suppressalis (Asiatic rice borer); Soybean: Pseudoplusia includens, soybean looper; Anticarsia gemmatalis, velvetbean caterpillar; Plathypena scabra, green cloverworm; Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black cutworm; Spodoptera exigua, beet armyworm; Spodoptera cosmioides; Spodoptera eridania; Heliothis virescens, cotton budworm; Helicoverpa zea (bollworm); Epilachna varivestis, Mexican bean beetle; Myzus persicae, green peach aphid; Empoasca fabae, potato leafhopper; Acrosternum hilare, green stink bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplus differentialis, differential grasshopper; Hylemya platura, seedcorn maggot; Sericothrips variabilis, soybean thrips; Thrips tabaci, onion thrips; Tetranychus turkestani, strawberry spider mite; Tetranychus urticae, two-spotted spider mite; Barley: European corn borer (Ostrinia nubilalis); black cutworm (Agrotis ipsilon); greenbug (Schizaphis graminum); chinch bug (Blissus leucopterus leucopterus); green stink bug (Acrosternum hilare); brown stink bug (Euschistus servus); neotropical brown stink bug (Euschistus heros); seedcorn maggot (Delia platura); Hessian fly (Mayetiola destructor); brown wheat mite (Petrobia latens); Rapeseed : cabbage aphid (Brevicoryne brassicae); Flea beetle (Phyllotretacruciferae); Bertha armyworm (Mamestra configurata); Diamond-back moth (Plutella xylostella); Root maggots (Delia ssp.).

[0028] The following examples are provided by way of illustration and not limitation.

[0029] Examples

[0030] Materials and methods: BBMV (brush border membrane vesicles)

[0031] The BBMV material was prepared from dissected third or fourth instar fall armyworm (Spodoptera frugiperda) (referred to herein as "FAW"), corn earworm (Helicoverpa zea) (referred to herein as "Hz"), or tobacco budworm (Heliothis virescens) (referred to herein as Hv), as described by Wolfersberger (1987) and modified.

[0032] FAW BBMV insect material was also prepared from whole insects (whole body, WB). The same Wolfersberger protocol was followed until after the first MgCl2 precipitation step. The pellet was resuspended in half-strength MET containing 12 mM MgCl2. The BBMV was loaded onto a 30% / 40% / 45% sucrose gradient containing 12 mM MgCl2. The gradient was centrifuged at 27,000 rpm for 1 hour at 4°C. The bands with sucrose above 45% were removed and diluted 1:10 with half-strength MET containing 12 mM MgCl2. The material was centrifuged again at 30,000 xg for 30 min at 4°C. The supernatant was removed, and each pellet was resuspended in half-strength MET, homogenized, aliquoted, and flash-frozen in liquid nitrogen. The BCA assay was used to determine the concentration of the vesicles.

[0033] Protein sample preparation

[0034] ARP166 and its variants were transformed into BL21 Gold cells. The cultures were grown in LB at 37°C until the OD600nm was approximately 0.6 - 0.8. Protein expression was induced with 1 mM IPTG, and the temperature was lowered to 18°C overnight. The cells were harvested and frozen until needed. The cells were lysed with BugBuster, and the soluble fusion protein was separated using an MBP affinity column. The buffer was 50 mM Hepes pH 8, 200 mM NaCl, 10 mM maltose. The protein concentration was determined using gel densitometry.

[0035] ARP540 and its variants were transformed into T7 cells. The cultures were grown in Instant TB in 48-well blocks at 37°C for 24 hours. 100 ul of 0.1 mm glass beads and 250 ul of 50 mM Hepes (pH 8), 200 mM NaCl, 5 mM EDTA were added to the pelleted cells. The samples were bead-beaten twice for 3 min each, with a 1-minute break in between. The cell debris was pelleted at 4000 rpm for 10 min. The soluble material was used for the bbmv leakage assay. Protein expression was examined by SDS PAGE.

[0036] ARP793 and its variants were transformed into BL21 Star. The cultures were grown in LB at 37°C until the OD600nm was approximately 0.6 - 0.7. Protein expression was induced with 0.1 mM IPTG, and the temperature was lowered to 18°C overnight. The cells were harvested and frozen until needed. The cells were resuspended in 50 mM sodium carbonate (pH 10.5) and lysed using a microfluidic device. The soluble fusion protein was separated using an MBP affinity column. The buffer was 50 mM sodium carbonate (pH 10.5), 10 mM maltose. The protein concentration was determined by BCA assay and gel densitometry.

[0037] FAW leakage assay

[0038] Add an appropriate amount of dye solution (80 mM 5(6)-carboxyfluorescein, 1 μM CHAPS, 100 mM Hepes (pH 7.3), 200 mM NaCl, 5 mM EDTA, 0.26 N NaOH) to the thawed and precipitated FAW bbmv. Entrap the dye by vortexing and sonication alternately three times for 30 seconds. Separate the entrapped dye from the free dye by SEC (PD10 column, Superdex 75 10 / 300GL or HiLoad Superdex 75 pg 16 / 600). Determine the amount of BBMV by BCA assay. Combine the positive fractions and dilute to 0.05 mg / ml BBMV. If needed, add trypsin at 0.1 mg / ml to the entrapped bbmv. Mix the protein and the entrapped BBMV and monitor the fluorescence signal (excitation 480 nm, emission 520 nm) for 3 hours. After the run is complete, add 1% triton to obtain the total leakage signal.

[0039] Hz leakage assay

[0040] Add an appropriate amount of dye solution (80 mM calcein, 1 μM CHAPS, 50 mM Hepes (pH 8), 200 mM NaCl, 5 mM EDTA, 0.33 N NaOH) to the thawed and precipitated Hz bbmv. Entrap the dye by vortexing and icing alternately three times for 30 seconds. Separate the entrapped dye from the free dye by SEC (G50 column, PD10 column, Superdex 75 10 / 300GL or HiLoad Superdex 75 pg 16 / 600). Determine the amount of bbmv by BCA assay. Combine the positive fractions and dilute to 0.05 mg / ml bbmv. Equilibrate the bbmv on ice for at least 1 hour. If needed, add trypsin to increase the reaction rate (0.1 mg / ml for ARP540 or 0.02 mg / ml for ARP793). Mix the protein and the bbmv and monitor the fluorescence signal (excitation 480 nm, emission 520 nm) for 5 hours. After the run is complete, add 1% triton to obtain the total leakage signal.

[0041] Hv leakage assay

[0042] An appropriate amount of dye solution (80 mM calcein, 1 μM CHAPS, 50 mM Hepes (pH 8), 250 mM trehalose, 1x HALT, 1 mM PMSF, 0.33 N NaOH) was added to the thawed, pelleted Hv bbmv. The dye was encapsulated by vortexing and ice treatment alternately 3 times for 30 seconds each. The encapsulated dye was separated from the free dye by SEC (G50 column). The amount of bbmv was determined by BCA assay. The positive fractions were combined and diluted to 0.05 mg / ml. The bbmv was equilibrated on ice for at least 30 min. The protein and bbmv were mixed, and the fluorescence signal (excitation 480 nm, emission 520 nm) was monitored for 1 hour.

[0043] Results

[0044] FAW leakage assay with ARP166 improved variant detection

[0045] The purified fusion ARP166 and five variants were tested in FAW gut and WB leakage assays as well as FAW insect bioassays. The EC50s of each variant and the wild-type protein were collected (Table 1A). The variants were then ranked / grouped based on the magnitude of improvement relative to the wild-type protein. These same variants were tested in both FAW gut (Table 1B and Figure 1B ) and WB (Table 1C and Figure 1C ) leakage assays. The figure shows the percentage of dye release from bbmv at 30 min. Error bars are the standard deviation of 3 biological replicates. These values are also shown in the table and ranked based on the change in improvement relative to the wild-type protein. Table 1D shows the comparison of all 3 FAW assays.

[0046] Table 1A: FAW EC50 data for wild-type and variants.

[0047]

[0048]

[0049] Table 1B: FAW gut leakage assay data for wild-type and variants at 30 min

[0050]

[0051] Table 1C: FAW WB leakage assay data for wild-type and variants at 30 min

[0052]

[0053] Table 1D: Ranking of wild-type and variants for each assay

[0054]

[0055] The ranking of each detection method is very similar, which shows the effectiveness of the bbmv leakage assay. It is also important that the WB bbmv preparation method produces results similar to those of the intestinal bbmv preparation method. This result would not occur without additional treatment of WB bbmv with a sucrose gradient.

[0056] The insecticidal activity of the variants was scored by eye and by comparison with the size of untreated insects in the bioassay plate. The degree of stunting was scored from 0, 1, 2, 3 or 4. A score of 0 indicates no stunting, a score of 1 indicates 1%-25% stunting, a score of 2 indicates 26%-50% stunting, a score of 3 indicates 51%-75% stunting, and a score of 4 indicates 76%-100% stunting. Hz leakage assay of variants improved with ARP540

[0057] ARP540 and five variant lysates were tested in the Hz leakage assay and the Hz insect bioassay. The EC50 of each variant and the wild-type protein was collected (Table 2A). The variants were ranked / grouped based on the improvement relative to the wild-type protein. These same variants were tested in the Hz leakage assay (Table 2B and Figure 2B ). The figure shows the percentage of dye released from bbmv at 3 hours. The error bars are the standard deviation of 3 biological replicates. These values are also shown in the table and are ranked based on the change in improvement relative to the wild-type protein. Table 2C shows a comparison of the Hz insect bioassay and the Hz leakage assay.

[0058] Similarly, the ranking between the insect bioassay and the bbmv leakage assay is similar, indicating that the leakage assay can be used to screen for improved variants.

[0059] The insecticidal activity of the variants was scored by eye and by comparison with the size of untreated insects in the bioassay plate. The degree of stunting was scored from 0, 1, 2, 3 or 4. A score of 0 indicates no stunting, a score of 1 indicates 1%-25% stunting, a score of 2 indicates 26%-50% stunting, a score of 3 indicates 51%-75% stunting, and a score of 4 indicates 76%-100% stunting.

[0060] Table 2A: Hz EC50 data for wild-type and variants

[0061]

[0062] Table 2B: Hz intestinal leakage data for wild-type and variants at 3 hours

[0063]

[0064]

[0065] Table 2C: Ranking of Each Detection Method for Wild-Type and Variants

[0066]

[0067] Hz leakage assay of variants improved with ARP793

[0068] Purified fusion ARP793 and 5 variants were tested in the Hz leakage assay and the Hz insect bioassay. The EC50s of each variant and the wild-type protein were collected (Table 3A). The variants were ranked / grouped based on the magnitude of improvement relative to the wild-type protein. These same variants were tested in the Hz leakage assay (Table 3B and Figure 3B ). The figure shows the percentage of dye release from bbmv at 4 hours. These values are also shown in the table and ranked based on the percentage change in improvement relative to the wild-type protein. Table 3C shows a comparison of the Hz insect bioassay and the Hz leakage assay.

[0069] Similarly, the ranking between the insect bioassay and the bbmv leakage assay was similar, indicating that the leakage assay can be used to screen for improved variants.

[0070] The insecticidal activity of the variants was accomplished via eye scoring and by comparing with the size of untreated insects in the bioassay plate. The degree of stunting was scored from 0, 1, 2, 3, or 4. A score of 0 indicates no stunting, 1 indicates 1%-25% stunting, 2 indicates 26%-50% stunting, 3 indicates 51%-75% stunting, and 4 indicates 76%-100% stunting.

[0071] Table 3A: Hz EC50 Data for Wild-Type and Variants

[0072]

[0073]

[0074] Table 3B: Hz Gut Leakage Data for Wild-Type and Variants at 3 Hours

[0075]

[0076] Table 3C: Ranking of Each Detection Method for Wild-Type and Variants

[0077]

[0078] All publications and patent applications mentioned in the specification indicate the state of the art of those skilled in the art to which embodiments of the present invention pertain. All publications and patent applications are hereby incorporated by reference in their entirety as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0079] Although the foregoing embodiments have been described in detail for purposes of clarity of understanding, it will be apparent that certain changes and modifications may be made within the scope of the appended claims.

Claims

1. A method for determining whether a gene is active against a target pest, the method comprising the following steps: a. generating BBMV from the brush border membrane of the target pest, wherein the BBMV contains a detectable substance in its lumen; b. contacting the candidate gene with the BBMV of step (a); c. evaluating whether the candidate gene causes leakage of the BBMV; and d. thereby determining whether the gene is active against the target pest.

2. The method according to claim 1, wherein the BBMV is produced by a target pest comprising Lepidoptera, Diptera, Hemiptera, Coleoptera, and Nematoda.

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