Modified insecticidal proteins with improved toxicity to lepidopteran insects

Abstract

The present invention provides a modified Cry1Ac protein having enhanced toxicity to resistant Pink Bollworm (PBW) species. Insertion of the specific, closely bound intestinal peptides at one or more recognition sites in Cry1Ac increases retention of toxins in the PBW intestine, resulting in dissolution and pore formation, thereby causing sepsis and larva death. The modified Bt toxins show significant toxicity to resistant Pink Bollworm and thus can be used to produce insect resistant transgenic plants.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology. In particular, the present invention is directed to the development of modified insecticidal toxins with improved toxicity for the control of Lepidopteran insect pests. Background Art

[0002] Lepidoptera poses one of the biggest challenges for insect pest management in Indian agriculture and causes severe damage to important crop plants. Bacillus thuringiensis (Bt) toxins are widely accepted insecticidal proteins and are highly effective / efficient in controlling Lepidoptera insects, especially on cotton. By suppressing key insect pests, Bt crops reduce the dependence on conventional insecticides and the increased yields increase farmers' profits. However, over time, resistance evolved by insect pests reduces the benefits of insecticidal proteins from widely used Bt sprays and transgenic crops. Pink bollworm (Pectinophora gossypiella) (Lepidoptera) is a global pest of cotton and is currently a major threat to cotton cultivation in India. Some recent reports (Tabashnik et al., 2013, Wang et al., 2022) have revealed that field-evolved resistance of pink bollworm to Bt cotton Bollguard II could be a major counterattack against Bt cotton technology, the only transgenic technology currently available to combat pink bollworm.

[0003] It is well known that proteolytic activation of Bt toxins and subsequent binding to the insect gut epithelium are two important steps involved in the toxicity mechanism of Bt Cry proteins. However, mutations in the gene encoding cadherin, a receptor for Cry1Ac in the gut of pink bollworm, led to field-evolved resistance of pink bollworm to Cry1Ac toxin (Fabrick et.al., 2009). Therefore, altered binding or non-binding of toxins to gut receptors is the main reason behind the field-evolved resistance of pink bollworm.

[0004] To cope with the field evolved resistance of pink bollworm, there is a need to continuously upgrade Bt cotton technology by introducing new or improved Bt Cry toxins with enhanced insecticidal activity. Since the field evolved Bt resistance of pink bollworm is attributed to the altered binding or non-binding of toxins to gut receptors, increasing the binding of Cry toxins to insect gut receptors by various means will be a prudent approach to address.

[0005] This problem can be addressed by modifying the binding target, specificity and / or affinity of Bt toxins, with the ultimate goal of producing designer toxins that target resistant species and resist evolving resistance in the field. The use of synthetic gut-binding peptides (GBPs) in the engineering of Bt toxins is a relatively new approach that has the potential to restore and / or enhance the toxicity of Bt toxins to pests that have evolved resistance to Bt toxins (Deist et al., 2014).

[0006] The development of powerful combinatorial technologies such as phage display libraries (PDL) has proposed new methods for selecting binding peptides (Deist et al., 2014 and Sijun Liu et al., 2010). The phage display peptide library is a mixture of filamentous phages with exogenous peptides on its surface and the coding sequence of peptides in viral DNA. Surface display is achieved by fusing the peptide coding sequence to the coat protein coding gene in the minor coat protein cpIII or in the major coat protein cpVIII (Scott and Smith 1990). Each phage clone displays a single peptide, but the library as a whole can represent billions of peptides overall. The important advantage of surface exposure is that it allows easy retrieval of phage display libraries with a large number of peptides for clones whose displayed peptides specifically bind to any given molecular target. Many peptides that bind to receptor molecules have been identified using phage display libraries (Natalia et.al., 2002).

[0007] None of the compositions or methods available in the art have the potential to restore and / or enhance the toxicity of Bt toxins to Lepidoptera insect pests that have evolved resistance to Bt toxins, such as Pectinophora gossypiella, by increasing the binding of Cry toxins to insect gut receptors. Therefore, there is a continuing need to upgrade and develop new and improved insecticidal proteins that can specifically target resistant populations of Lepidoptera insect pests. Summary of the invention

[0008] The present invention overcomes the problems of the prior art and involves modifying Cry1Ac protein using a cotton bollworm gut-binding peptide to enhance its gut binding and ultimately enhance activity. More specifically, the present invention provides a modified Cry1Ac protein with enhanced insecticidal toxicity, which has great potential for generating insect-resistant transgenic plants, thereby leading to a reduction in the application of expensive and environmentally damaging insecticides for sustainable and environmentally friendly agricultural production.

[0009] Purpose of the Invention

[0010] An important object of the present invention is to provide modified CrylAc toxins with enhanced toxicity to lepidopteran insect pests, particularly resistant pink bollworm.

[0011] Another important object of the present invention is to provide a modified Cry1Ac protein having improved insecticidal activity / toxicity to Lepidoptera insects compared to the native Cry1Ac protein and comprising one or more peptide sequences inserted at one or more amino acid positions of Cry1Ac.

[0012] Another important object of the present invention is to provide a method for identifying sites in CrylAc where small peptides can be inserted without affecting toxicity.

[0013] Another object of the present invention is to design a library of novel gut binding peptides (GBPs) capable of binding to the gut of resistant pink bollworm.

[0014] Another object of the present invention is to provide an insecticidal composition comprising a mutant of the Cry1Ac protein.

[0015] Another object of the present invention is to provide a method for preparing insect-resistant transgenic plants.

[0016] Another object of the present invention is to provide transgenic plants that can express mutant Cry1Ac protein and are resistant to lepidopteran insects.

[0017] Another object of the present invention is to provide a method for inhibiting the growth of or killing insect pests, pest populations, or resistant pests of the order Lepidoptera.

[0018] Another object of the present invention is to provide a method for suppressing lepidopteran pests, particularly pink bollworm or resistant populations of pink bollworm using the Cry1Ac mutant. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings illustrate some embodiments of the invention and, together with the description, serve to explain the invention. These drawings are provided by way of illustration and not by way of limitation.

[0020] Figure 1 A schematic diagram of the identification of pink bollworm (PBW) gut-binding peptides using phage display libraries is provided.

[0021] Figure 2 The scheme of overlap extension PCR used for peptide conjugation and modification of Cry1Ac protein is shown.

[0022] Figure 3 shows a schematic diagram of CrylAc modifications No. 1 to 16. Each selected peptide was inserted into the four insertion sites identified in the CrylAc coding sequence.

[0023] Figure 4Schematic representation of CrylAc modifications No. 17 to 19 is shown. The peptide AISPSRYFYDET (hereinafter referred to as AIS) was introduced at two, three and four identified insertion sites in CrylAc.

[0024] Figure 5 Schematic diagrams of Cry1Ac modifications No. 20 to 22 are shown. Various modifications were performed by combining AIS@282 with APTTWFNSDSIT (hereinafter referred to as APT)@368, SANYNVQAGWTH (hereinafter referred to as SAN)@508, and WAMDGQQHSNNY (hereinafter referred to as WAM)@523.

[0025] Figure 6 Shown is a method for collecting Cry1Ac-resistant Pink Bollworm from an infested field (heavily infested bolls and plants), panels (A): PBW-infested cotton plants, (B): PBW-infested cotton balls with bored PBW inside, (C): PBW on cotton balls, (D): Cotton balls arranged to harvest live PBW.

[0026] Figure 7 The four insertion sites of the peptide in CrylAc labeled 282, 368, 523, and 582 are shown.

[0027] Figure 8 Overlap of native Cry1Ac, Cry1Ac+peptide@282, Cry1Ac+peptide@368, Cry1Ac+peptide@508, Cry1Ac+peptide@523 are shown.

[0028] Fig. 9 Results of overlap extension-PCR for peptide insertion are shown. (a) Generation of two different overlapping fragments, (b) ligation of the two fragments using overlap extension PCR, (c) verification of nucleotide insertion using site-specific PCR, (d) HRM (high resolution melting) spectra of native and inserted fragments.

[0029] Fig.10 CrylAc modifications 1, 5, 9 and 13 are shown (as representatives). Four different peptides introduced at the same site (aa282; RG-SAQGIE) are shown along with the sequence alignment of the inserted region (nucleotides followed by amino acids). The inserted nucleotide and peptide sequences are shown in grey background.

[0030] Fig.11It is shown that multiple peptide insertions were confirmed in Cry1Ac. Lane M: molecular weight marker, Lane 1: native Cry1Ac (molecular weight 3537 bp), Lane 2: Cry1Ac inserted with the first peptide (molecular weight 3537+36=3573 bp), Lane 3: Cry1Ac inserted with the second peptide (molecular weight 3537+36+36=3609 bp), Lane 4: Cry1Ac inserted with the third peptide (molecular weight 3537+36+36+36=3645 bp), and Lane 5: Cry1Ac inserted with the fourth peptide (molecular weight 3537+36+36+36+36=3681 bp).

[0031] Fig.12 Recombinant expression and purification of native and modified Cry1Ac are shown. Expression of native Cry1Ac and one modified toxin (SAN@282) ​​is shown as a representative. Lane M: molecular weight marker, Lanes 1, 2 and 3: total protein analysis of bacterial lysates expressing native Cry1Ac, Lanes 4, 5 and 6: total protein analysis of bacterial lysates expressing modified Cry1Ac (SAN@282). Lanes 7 and 8: 2 and 4ug BSA as standards.

[0032] Fig.13 Results of Western blot analysis of recombinant Cry1Ac (native and modified) are shown. Immunoreactivity of native and modified Cry1Ac (SAN@282) ​​is shown as a representative. M: molecular weight marker, B1: untransformed bacterial cell lysate, Nt: cell lysate expressing native Cry1Ac, lanes (D14, D24, D8 and D13) cell lysate of clones expressing SAN@282.

[0033] Fig.14 Recombinant expression of Cry1Ac with multiple peptides is shown. Lane M: molecular weight marker, Lane 1: native Cry1Ac (predicted molecular weight is 133.12 kDa), Lane 2: Cry1Ac with the first peptide inserted (predicted molecular weight 133.12+1.5=134.62 kDa), Lane 3: Cry1Ac with the second peptide inserted (predicted molecular weight 133.12+1.5+1.5=135.12 kDa), Lane 4: Cry1Ac with the third peptide inserted (predicted molecular weight 133.12+1.5+1.5+1.5=136.62 kDa), Lane 5: Cry1Ac with the fourth peptide inserted (predicted molecular weight 133.12+1.5+1.5+1.5=138.12 kDa).

[0034] Fig.15 Shown is the mortality of Cry1Ac-resistant PBW larvae fed with native and modified Cry1Ac.

[0035] Fig.16 Comparison of growth delay of PBW larvae fed native and modified CrylAc is shown. AD: Artificial diet only, native (Native CrylAc).

[0036] Fig.17 A binary vector carrying the modified Cry1Ac gene is depicted.

[0037] Fig.18 Shown is the progression from explant selection, Agrobacterium infection, embryogenic callus formation, somatic embryogenesis and embryo maturation stages, followed by embryo elongation, shoot formation, root formation, primary hardening and final growth of putative transgenic events.

[0038] Fig.19 Convincing results are shown highlighting the effectiveness of the modified toxin in transgenic cotton. (A) shows reduced pink bollworm larval survival on leaf discs, while (B) shows visual observations of leaf disc feeding, highlighting the significantly reduced consumption in transgenic events compared to NBt and Bt cotton. DETAILED DESCRIPTION

[0039] The present invention generally provides modified Cry1Ac proteins with enhanced toxicity to resistant pink bollworm (PBW) insect populations. More specifically, the present invention relates to identifying specific insertion sites in Cry1Ac and engineering Cry1Ac by inserting gut-binding peptides into said sites based on in silico studies and modification methods. Insertion of said specific, tightly binding gut peptides at one or more identified sites in Cry1Ac increases retention of the toxin in the PBW gut, leading to dissolution and pore formation, thereby causing sepsis and larval death.

[0040] Modified Bt toxins show significant toxicity to resistant pink bollworm and thus can be used to generate insect-resistant transgenic plants. More specifically, developing transgenic cotton expressing modified Cry toxins with higher potency can delay field-evolved insect resistance for a period of time and this will help maintain Bt cotton technology for a longer period of time. DETAILED DESCRIPTION OF THE INVENTION

[0042] First, it should be understood that the following description only illustrates a specific form of the present invention. However, this specific form is only an exemplary embodiment and is not intended to imply any limitation to the scope of the present invention. Therefore, the description and examples should be understood as exemplary embodiments for teaching the present invention and are not intended to be considered in a limiting sense.

[0043] The details of one or more embodiments of the present invention are set forth in the following description, which includes, by way of example, specific details of the best mode considered by the inventor to implement the present invention. It should be clear to those skilled in the art that the present invention can be implemented without being limited to these specific details.

[0044] abbreviation

[0045] Bacillus thuringiensis

[0046] E.coli-Escherichia coli

[0047] EDTA-ethylenediaminetetraacetic acid

[0048] GBP-Gut Binding Peptide

[0049] OLE-PCR - Overlap Extension PCR

[0050] PBS - Phosphate Buffered Saline

[0051] PBW-Pink Bollworm

[0052] SDS-PAGE - sodium dodecyl sulfate polyacrylamide gel electrophoresis

[0053] Tris-HCl - Tris hydrochloride

[0054] definition:

[0055] Unless stated to the contrary or otherwise, throughout this specification, the terms "a" and "an" mean one or more, and the term "or" means and / or.

[0056] The use of "comprises," "including," "comprising," "include," "containing," "includes," "containing," and "including" are not intended to be limiting. It is to be understood that both the foregoing general description and this detailed description are exemplary and illustrative only and are not restrictive. Regardless of any indefinite article used, the specification is to be understood as contemplating plurality as well as the singular, unless the context requires otherwise.

[0057] Unless otherwise defined, scientific and technical terms used herein shall have the meanings commonly understood by those of ordinary skill in the art. In addition, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0058] As used herein, biotechnology terms have their conventional meanings as illustrated by the following illustrative definitions:

[0059] As used herein, the term "recombinant" relates to or refers to an organism, cell or genetic material produced by combining genetic material from more than one source.

[0060] The term "modified" refers to a modified cry protein. Synonyms such as mutant, recombinant, engineered, etc. can be used interchangeably and are generally understood by those skilled in the art.

[0061] The term "insect" refers to an insect or pest that feeds on Bt cotton plants. Related terms such as pest, pest population or resistant pest are to be interpreted in a similar manner.

[0062] The present invention relates to the modification of Cry1Ac protein using a pink bollworm gut-binding peptide to enhance its gut binding and ultimately insecticidal activity. The inventors of the present invention identified specific sites in the Cry1Ac coding sequence into which gut-binding peptides can be inserted without changing the toxicity of the protein. This was achieved using computer simulation structural analysis of the Cry1Ac protein. In addition, phage display libraries were used to identify specific and gut-binding peptides (GBPs) that bind to the resistant pink bollworm gut. Gut-binding peptides that frequently appear in the selected phage population and show higher phage recovery rates compared to other clones were selected. The identified gut-binding peptides were then inserted into the specific sites of Cry1Ac identified in the first part using OLE-PCR. In addition, the modified toxins with the inserted gut-binding peptides were recombinantly expressed and partially purified. The partially purified toxins (native and modified) were fed to resistant PBW larvae in artificial diets and the mortality was observed.

[0063] In an important embodiment, the present invention involves identifying specific sites in the CrylAc protein that can be modified / small inserted without changing the conformation and toxicity of the protein.

[0064] In another embodiment, the present invention provides novel gut-binding peptides identified using phage display libraries.

[0065] In another important embodiment, the present invention provides modified CrylAc proteins with enhanced toxicity to Bt-resistant PBW populations in the field.

[0066] In one embodiment of the present invention, one or more 12 amino acid residues of the intestinal binding peptide selected from SEQ ID NO: 1 to 82 are inserted into the amino acid positions 189, 223, 282, 334, 368, 469, 508, 523, 666 and 834 ( Figure 1 ) at least one specific site without affecting the activity and toxicity of the protein.

[0067] In another embodiment, the present invention provides modified CrylAc peptides with one or more gut-binding peptides inserted at two, three, and four different sites of CrylAc.

[0068] Embodiments of the present invention are also directed to suppressing lepidopteran pests, particularly pink bollworm (Pectinophoragos sypiella).

[0069] Embodiments of the present invention also relate to extending and applying this method for modifying proteins to Bt technology in other related plants not limited to Bt cotton plants.

[0070] In one embodiment, the present invention provides a modified Cry1Ac protein comprising one or more gut-binding peptide (GBP) sequences selected from SEQ ID NOs: 1 to 82 inserted at at least one of the amino acid positions 189, 223, 282, 334, 368, 469, 508, 523, 666 and 834 of Cry1Ac, wherein the mutant Cry1Ac protein has improved insecticidal activity / toxicity to Lepidoptera insects, particularly Bt-resistant pink bollworm (PBW), compared to the native Cry1Ac protein.

[0071] In another embodiment of the present invention, the insect is a pest, a pest population or a resistant pest species of the order Lepidoptera, in particular Pink Bollworm (PBW).

[0072] In another embodiment, the present invention provides a method for identifying one or more stable target sites in a native Cry1Ac protein for inserting a gut-binding peptide, comprising:

[0073] i. Computer simulation studies, including structural analysis and insertion of known gut-binding peptides to examine the stability of the protein before and after insertion; and i.

[0074] ii. Identification and selection of stabilizing sites based on stability results.

[0075] In another embodiment, the stable target sites are selected from amino acid positions 189, 223, 282, 334, 368, 469, 508, 523, 666 and 834 of CrylAc.

[0076] In another embodiment, the present invention provides a method for preparing a modified Cry1Ac protein, comprising:

[0077] a) Identification of gut-binding peptide (GBP);

[0078] b) computer simulation to identify one or more stable target sites in the Cry1Ac protein for inserting one or more of the intestine-binding peptides; and

[0079] c) modifying the natural Cry1Ac gene by inserting one or more GBPs into at least one or more recognition sites to obtain a modified Cry1Ac protein,

[0080] Compared with the natural Cry1Ac protein, the modified Cry1Ac protein has improved insecticidal activity / toxicity to Lepidoptera insects, especially Bt-resistant pink bollworm (PBW).

[0081] In another embodiment, a method of identifying a gut-binding peptide comprises:

[0082] i. Screening of phage display libraries using a biopanning scheme;

[0083] ii. Feeding insect larvae with phage display library using artificial diet or coated cotton balls;

[0084] iii. Open the larval intestine;

[0085] iv. eluting bound phage;

[0086] v. amplifying and enriching the eluted phage by transfection into host cells for generating a peptide library;

[0087] vi. Analysis and sequencing of individual clones; and

[0088] vii. selecting gut-binding peptides that bind to insect gut, wherein gut-binding peptides that frequently appear in the selected phage population and show higher phage recovery rates compared to other clones are selected.

[0089] In another embodiment, the present invention provides a polynucleotide comprising a nucleic acid molecule encoding the modified Cry1Ac protein of the present invention.

[0090] In yet another embodiment, the present invention provides a vector comprising the polynucleotide of the present invention.

[0091] In another embodiment, the present invention provides a host cell transformed with the vector of the present invention, wherein the cell is capable of expressing the modified CrylAc.

[0092] In another embodiment, the host cell is a plant cell.

[0093] In yet another embodiment, the present invention provides a method for preparing an insect-resistant transgenic plant, comprising:

[0094] a. Stably integrate the polynucleotide into the genome of the host / plant cell; and

[0095] b. Obtaining a stably transformed plant from a plant / plant cell capable of expressing the modified CrylAc, wherein the stably transformed plant is resistant to lepidopteran insects.

[0096] In another embodiment, the present invention provides a library of gut binding peptides (GBPs) comprising peptides having 12 amino acid residues and defined by SEQ ID NOs: 1 to 82, wherein the peptides are capable of binding to one or more gut receptors of Lepidoptera insects.

[0097] In another embodiment, the present invention provides an insecticidal formulation comprising an effective amount of a modified Cry1Ac protein.

[0098] In another embodiment, the present invention provides a composition comprising an insecticide formulation and one or more formulating agents selected from but not limited to the group consisting of: adjuvants, surfactants, thickeners, stickers, petroleum oils, crop oil concentrates, stabilizers, solvents, hygroscopic agents, deposition promoters, defoamers, buffers and activators.

[0099] In another embodiment, the present invention provides a method of inhibiting the growth of an insect pest, an insect pest population, or a resistant Lepidopteran insect pest, comprising contacting the insect pest with a modified CrylAc protein.

[0100] In another embodiment, the present invention provides a method of using an insecticide formulation to improve crop yield.

[0101] In another embodiment, the present invention provides the design and construction of a binary vector for expressing Cry1Ac and its modified forms.

[0102] In another embodiment, the present invention provides for the generation of transgenic samples by Agrobacterium-mediated transformation, plant regeneration, and subsequent results of insect feeding bioassays.

[0103] In another embodiment, the present invention provides a transgenic plant cell, plant or plant part carrying the modified CrylAc protein of the present invention.

[0104] Example

[0105] The following examples specifically describe the manner in which the present invention can be implemented. However, the embodiments disclosed herein do not limit the scope of the present invention in any way.

[0106] Example 1: Identification and Preparation of Modified Cry1Ac Protein

[0107] 1.1 Identification of gut-binding peptide (GBP)

[0108] A phage display library containing 12 amino acid residue peptides was screened using a biopanning protocol with resistant pink bollworm larvae (Liu et al., 2010). Larvae were fed with the phage display library using artificial diet or coated cotton balls. The larval gut was then dissected and bound phages were eluted. The eluted phages were amplified and enriched by transfecting host cells to generate a peptide library. The library was screened by screening individual clones or by subsequent Next-Gen sequencing. The overall protocol is shown in Figure 1 Screening of the library generated a list of several hundred peptides containing 12 amino acid residues (36 nucleotides). However, only 84 peptides showing higher abundance were considered.

[0109] 1.2 In silico identification of sites suitable for peptide insertion in Cry1Ac

[0110] In order to identify the site where the intestinal binding peptide can be inserted in Cry1Ac, computer simulation research was carried out. The method is to use molecular docking to randomly insert peptide sequences in different Cry1Ac sites and study the stability of toxins before and after insertion. The selected peptide (ligand) is processed to use LigPrep in Schrodinger to carry out docking research. The selected peptide Cry1Ac model is carried out interaction analysis to obtain a deep understanding of the precise site and intensity of binding. All ionization states are significantly present for pH range 7.0+ / -2.0. Desalination is carried out, and tautomers are subsequently produced. ZRANK docking scores have been produced, and the interface salvation energies (interface salvation energies) (kcal / mol) of Cry1Ac for peptide integration have been studied.

[0111] 1.3 Modification of the Cry1Ac gene by inserting the recognized GBP

[0112] The peptide sequences obtained for bacterial expression were codon optimized, and primers were designed for inserting these intestinal binding nucleotides at the selected sites of Cry1Ac. From the obtained peptide list, four peptides, AIS, APT, SAN and WAM, were initially selected for cloning purposes. OLE-PCR was used to achieve the insertion of 36 nucleotides at specific sites in the Cry1Ac coding sequence. Amplification was performed using specific PCR conditions. The overall scheme of OLE PCR is shown in Figure 2 .

[0113] Each selected peptide was inserted at 4 different insertion sites (i.e. 282, 368, 508 and 523), resulting in 16 different modified toxins (numbered 1 to 16). A schematic diagram of the obtained modifications is shown in Figure 3. The modified toxins were recombinantly expressed, partially purified and subjected to insect feeding bioassays. From the results obtained, the best performing peptides and corresponding sites in Cry1Ac were selected.

[0114] Representative sequence of an exemplary modified CrylAc with a single insertion (the inserted peptide is shown in bold)

[0115] SEQ ID NO:83 (peptide AISPSRYFYDET inserted at 189 / 190)

[0116]

[0117] SEQ ID NO:84 (peptide APTTWFNSDSIT inserted at 223 / 224)

[0118]

[0119] SEQ ID NO:85 (peptide MPLRYPTQVTLD inserted at 282 / 283)

[0120]

[0121] SEQ ID NO:86 (peptide SANYNVQAGWTH inserted at 334 / 335)

[0122]

[0123] SEQ ID NO:87 (peptide DGSMLNRMRGFS inserted at 368 / 369)

[0124]

[0125] SEQ ID NO:88 (peptide WAMDGQQHSNNY inserted at 479 / 480)

[0126]

[0127] SEQ ID NO:89 (peptide EPELDNARVLQI inserted at position 508 / 509)

[0128]

[0129] SEQ ID NO:90 (peptide APTTWFNSDSIT inserted at position 523 / 524)

[0130]

[0131] SEQ ID NO:91 (peptide APTTWFNSDSIT inserted at position 666 / 667)

[0132]

[0133] SEQ ID NO:92 (peptide WAMDGQQHSNNY inserted at 834 / 835)

[0134]

[0135] 1.4 Cry1Ac modification with multiple insertions of peptides

[0136] It is believed that inserting more copies of the peptide can enhance the binding to the intestine, resulting in higher toxicity. The peptide AIS was inserted at two, three and four different sites in Cry1Ac and modified (No.17 to No.19), such as Figure 4 As shown in . As previously described, multiple insertions of peptides were also performed using molecular cloning techniques. Peptides were added one at a time at the desired insertion sites. Successful insertion of the various peptides was confirmed by gel migration plots and DNA sequencing. Modified CrylAcs with two, three and four insertions of the AIS along with the previously used single insertion were recombinantly expressed, partially purified and subjected to insect feeding bioassays.

[0137] Because the introduction of the same peptide in more than one site did not show any further enhancement of activity or toxicity, the inventors explored the best performing peptide-insertion site combinations (from the initial 16 modifications). The best modifications with combinations of peptides and their corresponding sites were selected from the feeding bioassay data, namely APT at position 368, SAN at position 508, and WAM at position 523. Combining these modifications with the first successful modification with AIS at position 282 resulted in modifications No. 20 to 22, as Figure 5 shown.

[0138] Representative sequence of an exemplary modified CrylAc with multiple peptide insertions (the inserted peptides are shown in bold)

[0139] SEQ ID NO:93 (peptide APTTWFNSDSIT inserted at positions 282 / 283 and 368 / 369)

[0140]

[0141] SEQ ID NO:94 (peptide APTTWFNSDSIT inserted at 282 / 283, 368 / 369 and 508 / 509)

[0142]

[0143] SEQ ID NO:95 (same peptide inserted at 282 / 283, 368 / 369, 508 / 509 and 523 / 524)

[0144]

[0145] SEQ ID NO:96 (different peptides inserted at 282 / 283 and 368 / 369)

[0146]

[0147] SEQ ID NO:97 (three different peptides inserted at 282 / 283, 368 / 369 and 508 / 509)

[0148]

[0149] SEQ ID NO:98 (four different peptide insertions at 282 / 283, 368 / 369, 508 / 509 and 523 / 524)

[0150]

[0151] The above exemplary modified CrylAc peptide sequences according to the claimed invention are provided for illustrative purposes and should not be construed as limiting.

[0152] Example 2: Recombinant Expression and Partial Purification of Native and Modified Cry1Ac Proteins

[0153] 2.1 Confirmation of protein expression

[0154] The peptides of all modified Cry1Ac clones and the insertion of the correct open reading frame were confirmed by sequencing. For primary culture, Escherichia coli cells (DH5-α) with recombinant plasmid (pKK223-3) were inoculated into 6ml LB medium containing ampicillin (50 μg / ml) and grown overnight at 37°C under continuous shaking at 180rpm. The secondary culture of 50ml LB (ampicillin @ 50 μg / ml) was inoculated with 1% volume of primary culture and incubated at 37°C / 200rpm. The culture was grown until the OD at 600nm reached 0.6. Then, the flask was transferred to 28°C / 200rpm for 24h. After 24 hours, the culture medium was centrifuged at 5000g for 10 minutes. The particles (cell agglomerates, pellets, pellets) were stored at -20°C until further use.

[0155] From 50ml culture medium, 1ml is processed to check protein expression. The remaining culture medium is centrifuged at 8,000g for 10min. The supernatant is discarded and the particles are stored at -20°C until further use. The particles of 1ml culture are resuspended in 0.9% NaCl. Centrifuge again at 8,000g for 10 minutes and discard the supernatant. The particles are resuspended in 1mL PBS by vortexing. The sample is then sonicated for 5 seconds (1 pulse @ 30% amplitude) to lyse the cells. Cell lysate (80 μL) is mixed with 20 μL loading dye and heated at 98°C for 10min. The sample is then cooled at room temperature for 5min and centrifuged at 8,000g for 10min. The supernatant (40 μL) is loaded onto an SDS (12% resolution) gel. Electrophoresis is performed at a constant current of 20mA. After electrophoresis, the gel is stained with Coomassie Brilliant Blue R-250 and decolorized with methanol and acetic acid. After confirmation of protein expression in pilot cultures, pellets obtained from bulk cultures were processed for purification of inclusion bodies (IBs).

[0156] 2.2 Purification of Cry1Ac inclusion bodies

[0157] The frozen cell pellets are thawed, and 1 g of the pellets are resuspended in 50 ml of lysis buffer (50 mM Tris-HCL, 50 mM EDTA, 15% sucrose, pH 8.0) containing lysozyme (2 mg / ml). The suspension is fully mixed by vortexing, and incubated for 1 hour at 37 ° C under constant oscillation. The suspension is then homogenized for 15 minutes using a tissue dissolver (@ frequency level -25). The ultrasonic treatment is then performed for 10 minutes (10 second pulses @ 30% amplitude). The resulting lysate is centrifuged for 15 min at 10,000 x g at 4 ° C. The supernatant is discarded, and the pellet is washed vigorously with TritonX-100 buffer (2% triton in 0.5 m NaCl). The lysate is centrifuged for 15 minutes at 10,000 x g at 4 ° C, and washed three times with TritonX-100.

[0158] The obtained particles were washed with 25 mL of 0.5 M NaCl solution and fully mixed by pipetting. The suspension was vortexed vigorously for 5 min and centrifuged at 15000 x g for 15 min at 4 ° C. This step (washed with 0.5 M NaCl) was repeated 5 times. All wash fractions (supernatant of each wash) were retained for SDS-PAGE analysis.

[0159] Distilled water 25mL is added to the particles and mixed by vigorous vortex for 5 minutes, and then centrifuged at 15000xg for 15 minutes at 4°C. Repeat this step once. By vigorous mixing or by vortexing for 5 minutes, the particles containing the purified IB obtained after the final wash are dissolved in 5mL of solubilization buffer (50mM sodium carbonate, pH 8.0). The mixture is incubated at 37°C under 200rpm continuous shaking for 2-3h, and then centrifuged at 15000xg for 15min at room temperature. Collect the supernatant, and analyze the dissolved IB on SDS-PAGE and store at -20°C for future use.

[0160] 2.3 Collection and artificial rearing of Cry1Ac-resistant populations of PBW

[0161] For testing and validation purposes, pink bollworm larvae were collected from Bt cotton fields from a historically known hotspot (Akola, Maharashtra) for PBW challenge. Bt cotton plants were confirmed by using CrylAc lateral flow strips (LFS). Approximately 16,000 mature Bt cotton balls showing signs of larval bores, holes or feeding larvae were collected and brought back to the insect rearing room (heavily infested plants and cotton balls are shown in Figure 6 Design a special tray and arrange the infested cotton balls to ensure that the larvae will fall into the tray below (such as Figure 6 shown in panel D).

[0162] The larvae are carefully removed from the tray and transferred to artificial diet (AD). The first generation is maintained on AD containing 0.1ppm partially purified Cry1Ac protein. For each future generation, the concentration of Cry1Ac is gradually increased. The development of larvae to pupae, adult mating, egg collection and hatching are carried out according to the standard practice of PBW artificial breeding. As the concentration of Cry1Ac increases, larval growth and molting are affected. Sometimes, the larval duration is longer than normal. Pupariation and eclosion (adult emergence) are also seriously affected (especially at higher concentrations). However, despite the conditions, the PBW population slowly increases. The generations of the Cry1Ac-resistant PBW population, the details of the toxin concentration, the number of larvae per generation, and the number of larvae obtained are shown in Table 1 below.

[0163] Table 1

[0164]

[0165] Once stabilized at 10 ppm Cry1Ac, larvae were constantly maintained at the same concentration for further passages and used in feeding bioassays as needed.

[0166] 2.4 Efficacy of modified Cry1Ac protein against resistant PBW larvae

[0167] The modified toxins were incorporated into artificial diets and feeding bioassays were performed for susceptible and resistant PBW larvae. The native Cry1Ac protein was used as a control. PBW larvae were fed as follows: artificial diet (AD), and AD combined with native Cry1Ac protein (5 ppm) or modified Cry1Ac protein (5 ppm). Larval growth and mortality were monitored daily.

[0168] 3. Results

[0169] 3.1 Identification of midgut binding peptide (GBP) in resistant PBW larvae

[0170] Using phage display libraries, a list of various gut-binding peptides was obtained. A list of all abundant peptides along with codon-optimized nucleotide sequences is listed in Table 2 below. Four peptides (AIS, APT, SAN and WAM) were randomly selected from them for insertion and further study.

[0171] Table 2

[0172]

[0173]

[0174]

[0175] 3.2 Identification of peptide insertion sites in Cry1Ac

[0176] The coding sequence of Cry1Ac comprises 1179 amino acid residues. Based on the data obtained in the molecular docking study, 10 potential amino acid residues (189, 223, 282, 334, 368, 469, 508, 523, 666, 834) in which peptide insertion can be completed were identified. Table 3 below lists the residues identified for peptide binding based on the ZRANK score, wherein the residues with ZRANK value <-67.00 are selected (ZRANK score is -104.00 for natural Cry1Ac).

[0177] Table 3

[0178]

[0179]

[0180] Among the identified sites, four sites, namely 282, 368, 523 and 582, showed the most stable protein conformation after peptide insertion in the computer simulation study. Some representative insertion sites (amino acid residues) used during the computer simulation study with inserted peptides (i.e., 282, 368, 523 and 582) are shown in Figure 7 and the resulting sequence of the peptide after insertion is shown in Table 4.

[0181] Table 4

[0182]

[0183] The four selected peptides were inserted at the sites identified in CrylAc, one and / or more each time and multiple toxin models were constructed. These models were further docked with wild-type and mutant cadherin models and examined for energy.

[0184] Superposition of the predicted structures indicated that the core Cry1Ac structure was not affected by binding of the peptide in these loops. Figure 8 Overlays of modified toxins with peptides inserted at different positions are shown.

[0185] From these studies, it was concluded that there are potential sites in the coding sequence of Cry1Ac where a 12 amino acid residue (36 nucleotide) gut-binding peptide could be introduced without altering the structure and conformation of the toxin.

[0186] 3.3 Modification of Cry1Ac by insertion of a single peptide

[0187] Various modifications were achieved by inserting GBP at specific sites in Cry1Ac using the overlap extension PCR method (Bryksin and Matsumara, 2018). Fig. 9 The steps involved in PCR, agarose gel electrophoresis, high resolution melting analysis and the results obtained are shown in FIG. Fig. 9 middle:

[0188] Representative figure (a) shows the generation of two different overlapping fragments

[0189] Representative figure (b) shows the joining of two fragments using overlap extension PCR

[0190] Representative figure (c) shows the verification of mutations using mutation-specific PCR

[0191] · Representative figure (d) shows the HRM spectrum of the mutant fragment.

[0192] Successful insertion of the peptide into Cry1Ac was confirmed by sequencing the amplified PCR product. The DNA sequence of the modified Cry1Ac toxin showed 36 nucleotides inserted at the designated insertion site. As a representative, the sequence alignment of the native toxin and the modified toxin for the insertion region used for modification (AIS@282, APT@282, SAN@282, and WAM@523) is shown in Fig.10 middle.

[0193] 3.4 Modification of Cry1Ac by Insertion of Multiple Peptides

[0194] Modified Cry1Ac with multiple peptide insertions (No. 17 to No. 22) were also generated using OLE PCR technology. Successful insertions were initially confirmed by agarose gel electrophoresis or by gel shift assay ( Fig.11 ). Restriction digestion and agarose gel electrophoresis analysis showed the expected increase in DNA molecular weight.

[0195] 3.5 Recombinant expression and partial purification of native and modified Cry1Ac proteins

[0196] All Cry1Ac modified by binding peptides were cloned into vector pKK223-3 and expressed in E. coli. Partially purified native and modified Cry1Ac proteins were analyzed by SDS-PAGE followed by Coomassie staining. The results are shown in Fig.12 The predicted molecular weight of Cry1Ac is about 133.12 kDa. For native Cry1Ac, a band with a predicted molecular weight is visible. The results of a modified recombinant Cry1Ac with a single peptide insertion are shown here. The predicted molecular weight of the modified (peptide-inserted) Cry1Ac is about 134.66 kDa (the addition of the peptide increases the molecular weight by about 1.5 kDa). This can also be confirmed by visual observation of the mobility change of the modified Cry1Ac. Use BSA of known concentration as a standard to visually measure the partially purified protein from IB and use it for insect feeding bioassay.

[0197] Total proteins from bacterial lysates were separated by SDS-PAGE on 12% acrylamide gels; sample preparation, gel running, staining, and transfer to nitrocellulose membranes were performed as previously described (Fitches et al., 2004). The primary (anti-Cry1Ac) antibody used in Western blotting was diluted to 1:5000 for incubation with the blot. Detection of immunoreactive bands was performed using a chemiluminescent detection kit. Western blotting results showed immunoreactive bands at predicted molecular weights for native (133.12 kDa) and modified Cry1Ac (134.65 kDa).

[0198] 3.6 Recombinant Expression of Cry1Ac with Multiple Peptide Insertions

[0199] In SDS-PAGE analysis, Cry1Ac modified with multiple peptide insertions (No.17 to No.22) showed successful recombinant expression and the expected migration shift ( Fig.14 ).

[0200] 3.7 Efficacy of modified Cry1Ac protein against Cry1Ac-resistant PBW larvae

[0201] Susceptible larvae fed with native and modified Cry1Ac proteins showed 100% mortality. The results of the insect feeding bioassay were shown in Fig.15 middle.

[0202] All modified toxins (No. 1 to No. 7) showed increased toxicity to resistant PBW compared to native Cry1Ac. On the 10th day of the bioassay, larvae Mod. No. 5 fed with modified Cry1Ac showed about 50% mortality, while 13% larval mortality was recorded for native Cry1Ac. These results show that the peptide insertion does not affect protein toxicity, but it significantly increases protein toxicity to Cry1Ac-resistant PBW larvae.

[0203] In addition, the surviving larvae showed significant growth retardation, e.g. Fig.16 On day 13 of the assay, larvae were carefully removed from the feed blocks and growth data were recorded.

[0204] Cry1Ac with multiple peptides inserted showed similar toxicity levels to single inserted Cry1Ac for resistant PBW. Combinations of different gut-binding peptides and their insertion at different sites in Cry1Ac can be further used to enhance the toxicity of Cry1Ac. Therefore, the present invention has great potential to solve the problem of PBW resistance to natural Cry1Ac. When expressed in cotton plants, the modified toxin can essentially treat multiple geographically resistant populations.

[0205] Example 4: Production of transgenics

[0206] 4.1 Design and construction of binary vectors expressing Cry1Ac and its modified forms

[0207] The Cry1Ac gene sequence from Bacillus thuringiensis and its modified forms were codon optimized using codons used for cotton. Constructs containing codon-optimized gene sequences encoding Cry1Ac and its modified forms were synthesized from GenScript, USA. The Cry1Ac gene was expressed under the figwort mosaic virus (FMV) 35s promoter, and the selectable marker neomycin phosphotransferase II (nptII) gene was expressed under the nopaline synthase (NOS) promoter ( Fig.17 The fully synthesized construct sequence was cloned into an empty vector using the PmeI-XhoI restriction sites. The presence of the inserted gene and the stability of the final construct were verified by restriction digestion and sequencing. The resulting binary vector was named pASPL818 and moved into Agrobacterium strain LBA4404.

[0208] 4.2 Agrobacterium-mediated transformation and plant regeneration

[0209] Agrobacterium tumefaciens (Agrobacterium tumefaciens) strain LBA4404 is used for transformation.By carrying the bacterial strain of binary vector (pASPL818 carrying Cry1Ac gene sequence) at 28 ℃ in Luria-Bertani (LB) medium supplemented with 50mg / L kanamycin, 50mg / L rifampicin and 250mg / L streptomycin and incubate overnight.Then by centrifugal collection bacterial cells, wash twice with 10mM magnesium chloride, and be resuspended in the liquid MS medium supplemented with 200 μM acetosyringone, and realize the final optical density (OD600) of 0.8.The seed of cotton cultivar Coker 312 is carried out surface sterilization by 70% ethanol treatment in 1 minute, immersed in 1% sodium hypochlorite 10 minutes subsequently, and washed with sterile distilled water.Seed is germinated in the culture bottle containing the MS medium supplemented with 30g / L sucrose and 8g / L agar. Hypocotyls from 8-9 day old seedlings were used as explants. At 25°C, on MS medium supplemented with 100 μM acetosyringone, 0.5 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.1 mg / L zeatin, explants were co-cultivated with Agrobacterium suspension under a photoperiod of 16 hours of illumination and 8 hours of darkness for 72 hours. After co-cultivation, explants were washed with cefotaxime (300 mg / L) and placed on a selection medium consisting of MS medium supplemented with 30 g / L sucrose, 8 g / L agar, 0.5 mg / L 2,4-D, 0.1 mg / L zeatin, 50 mg / L kanamycin and 400 mg / L carbenicillin. Explants were then incubated at 28°C for 4 weeks with a 16 hour photoperiod, wherein the selection medium was changed every 2 weeks. The process was continued for 3-4 cycles (each 4 weeks) of progeny culture. Observe kanamycin resistant callus and transfer to the embryo induction medium containing the MS medium supplemented with 30g / L sucrose, 8g / L agar, 0.1mg / L 2,4-D, 0.1mg / L zeatin and 50mg / L kanamycin.The culture was further incubated for 4 weeks at 28 ℃ with 16 hours photoperiod.Then embryogenic callus was selectively placed on the embryo proliferation medium containing the MS medium supplemented with 30g / L sucrose, 8g / L agar, 0.1mg / L zeatin, 0.1mg / L indoleacetic acid (IAA) and 50mg / L kanamycin, and incubated at 28 ℃ until different embryo development stages were observed.Harvest cotyledon embryos and be placed on the embryo elongation medium comprising the half-strength MS medium supplemented with 30g / L sucrose, 8g / L agar and 50mg / L kanamycin.The elongated embryo with fully developed bud and root was taken for primary hardening. The primary hardened plants were then transferred to soil in pots and acclimatized in a growth chamber at 28°C with a 16 h photoperiod for further growth and development.

[0210] 4.3 Pink bollworm feeding bioassay

[0211] Leaf disc bioassays were performed using a Bt-resistant pink bollworm population. Leaf discs were obtained from transgenic cotton plants engineered to express a modified Cry1Ac toxin. Each leaflet (2 cm 2 ) were placed in separate containers for insect feeding, with one first-instar larva of pink bollworm on each disc. Visual observations of larval consumption of leaf discs were made regularly, and larval mortality was recorded daily.

[0212] Example 5: Results and analysis of evaluation conclusions

[0213] 5.1 Transformation of modified CryAc genes in cotton

[0214] To evaluate the effectiveness of the modified Cry1Ac, a binary vector carrying the modified gene was constructed and introduced into cotton plants via Agrobacterium-mediated transformation ( Fig.17 ). Highly fragile callus is derived from the hypocotyl of Coker 312 and subsequently screened for resistance to kanamycin. After 3-4 cycles (4 weeks) of subculture, somatic embryogenesis was observed. Within 3-5 weeks on the selective medium, spherical embryo clusters formed by 2-3g of embryogenic callus undergoing transformation. Each cluster represents an independent change event. These embryos enter the cotyledon stage and usually germinate within a few weeks. Most of the buds effectively take root without the need for additional treatment. When transferred to greenhouse soil conditions, all plants show regular morphology and undergo flowering ( Fig.18 ). Gene insertion was confirmed by PCR, while copy number was determined using Western blot analysis.

[0215] 5.2 Bioassay analysis

[0216] Leaf disc feeding assays were performed on four different transgenic events to evaluate the enhanced effectiveness of the modified toxins compared to native Cry1Ac expressed in conventional Bt cotton. Transgenic cotton events expressing the modified Cry1Ac toxin showed enhanced toxicity to resistant pink bollworm. On the third day of the bioassay, larvae showed approximately 50% mortality on all events, while 18% larval mortality was observed on cotton expressing native Cry1Ac ( Fig.19 A). These findings indicate that the peptide insertion did not impair protein toxicity; in fact, it substantially amplified protein toxicity against Cry1Ac-resistant PBW larvae. On day 5, Cry1AcModE2 and Cry1AcModE3 events showed 85% larval mortality, while Cry1AcModE1 and Cry1AcModE4 recorded 71% and 69% mortality, respectively ( Fig.19A). Meanwhile, larval mortality on non-Bt cotton (NBt) and conventional Bt cotton was 5% and 19%, respectively. Visual observation of leaf disc feeding highlighted the significantly reduced consumption in the transgenic events compared to NBt and Bt cotton ( Fig.19 B). In summary, these results highlight the efficacy of modified toxin-expressing transgenic cotton events, providing a valuable tool for combating Bt-resistant pink bollworm. Thus, the present invention has substantial potential in addressing the challenge of PBW resistance to Bt cotton.

[0217] advantage

[0218] 1. A unique approach to mitigate / solve the problem of Bt resistant lepidopteran pests, especially pink bollworm.

[0219] 2. The present invention can be applied to all cotton growing geographical areas facing the above mentioned problems.

[0220] 3. The present invention has high efficacy in delaying the evolution of insect resistance in the field and maintaining Bt cotton technology for a longer duration.

[0221] 4. The present invention has improved toxicity to pests, especially resistant pink bollworm.

Claims

1. A modified Cry1Ac protein comprising one or more gut-binding peptide (GBP) sequences selected from SEQ ID NOs: 1 to 82 inserted at at least one of the amino acid positions 189, 223, 282, 334, 368, 469, 508, 523, 666 and 834 of Cry1Ac, wherein the mutant Cry1Ac protein has improved insecticidal activity / toxicity to Lepidoptera insects, particularly Bt-resistant pink bollworm (PBW), compared to a native Cry1Ac protein.

2. The modified Cry1Ac protein according to claim 1, wherein the insect is a pest, a pest group, or a resistant pest species of Lepidoptera, in particular pink bollworm (PBW).

3. A method for identifying one or more stable target sites in a native Cry1Ac protein for inserting a gut-binding peptide as defined in claim 1, comprising: i. Computer simulation studies, including structural analysis and insertion of known gut-binding peptides to examine the stability of the protein before and after insertion; as well as ii. Identification and selection of stabilizing sites based on stability results.

4. The method of claim 3, wherein the stabilizing target site is selected from amino acid positions 189, 223, 282, 334, 368, 469, 508, 523, 666 and 834 of CrylAc.

5. A method for preparing the modified Cry1Ac protein according to claim 1, comprising: a. Identify the gut-binding peptide (GBP) as defined in claim 1; b. computer simulation to identify one or more stable target sites in the Cry1Ac protein for inserting one or more of the intestinal binding peptides; as well as c. modifying the natural Cry1Ac gene by inserting one or more GBPs at at least one or more identified sites to obtain the modified Cry1Ac protein, Compared with the natural Cry1Ac protein, the modified Cry1Ac protein has improved insecticidal activity / toxicity to Lepidoptera insects, especially Bt-resistant pink bollworm (PBW).

6. The method according to claim 5, comprising: i. Screening of phage display libraries using a biopanning scheme; ii. Feeding insect larvae with phage display library using artificial diet or coated cotton bolls; iii. Open the larval intestine; iv. eluting bound phage; v. amplifying and enriching the eluted phage by transfection into host cells for generating a peptide library; vi. Analysis and sequencing of individual clones; as well as vii. selecting gut-binding peptides that bind to insect gut, wherein gut-binding peptides that frequently appear in the selected phage population and show higher phage recovery rates compared to other clones are selected. 7 . A polynucleotide comprising a nucleic acid molecule encoding the modified Cry1Ac protein of claim 1 . A vector comprising the polynucleotide according to claim 7.

9. A host cell transformed with the vector of claim 8, wherein the cell is capable of expressing the modified CrylAc of claim 1.

10. The host cell according to claim 9, wherein the host cell is a plant cell.

11. A method for preparing an insect-resistant transgenic plant, comprising: a. stably integrating the polynucleotide according to claim 7 into the genome of a host / plant cell; as well as b. Obtaining a stably transformed plant from a plant / plant cell capable of expressing the modified CrylAc, wherein the stably transformed plant is resistant to lepidopteran insects.

12. A library of gut binding peptides (GBPs) comprising peptides having 12 amino acid residues and defined by SEQ ID NOs: 1 to 82, wherein the peptides are capable of binding to one or more gut receptors of Lepidoptera insects.

13. An insecticide formulation comprising an effective amount of the modified Cry1Ac protein according to claim 1.

14. A composition comprising the insecticide formulation of claim 15 and one or more formulating agents selected from but not limited to the group consisting of: adjuvants, surfactants, thickeners, stickers, petroleum oils, crop oil concentrates, stabilizers, solvents, hygroscopic agents, precipitation promoters, defoamers, buffers and activators.

15. A method for inhibiting the growth of an insect pest, an insect pest population, or a resistant Lepidoptera insect pest, comprising contacting the insect pest with the modified Cry1Ac protein of claim 1.

16. A method for improving crop yield using the insecticide formulation of claim 15 or the composition of claim 16.

17. A transgenic plant cell, plant or plant part carrying the modified Cry1Ac protein of claim 1.

Images