Field of the invention

[001] The present invention relates to the field of plant pest control, particularly insect control. Provided is a recombinant DNA sequence encoding a protein designated as BIP1 , which is useful to protect plants from insect damage, as well as insecticidal variant! thereof. Further provided are plants comprising a nucleic acid molecule encoding a BIP1 protein, as well as methods and means for using these nucleic acid sequences for reducing insect damage to plants, particularly cotton plants, especially against Helicoverpa zea and Heliothis virescens.

[002] Background art

[003] Insect pests cause huge economic losses worldwide in crop production, and every year farmers face the threat of yield losses due to insect infestation. Genetic engineering of insect resistance in agricultural crops has been an attractive approach to reduce costs associated with crop-management and chemical control practices. The first generation of insect-resistant crops was introduced into the market in 1996, based on the expression in plants of proteins isolated from the gram-positive soil bacterium Bacillus thuringiensis (abbreviated herein as "Bt").

[004] There is a risk that susceptible insect species may develop resistance against Bt Cry toxins. Consequently, active efforts have been made to identify novel insecticidal proteins. Despite the isolation and characterization of a relatively large number of different insecticidal proteins to date, there remains a need for identification, isolation and characterization of new insecticidal proteins. The reasons for this are manifold. Firstly, due to the specificity of insecticidal proteins towards particular groups of target pests, there is a need to clone genes encoding proteins with different spectra of activity, so that for different crops and different geographic regions suitable proteins

for combating insect pests are available. The specificity of Bt Cry proteins, for example, is mostly limited. Identification of toxins with specificity towards different target insects remains desirable. Second, after prolonged use in one geographic region, insects are known to have the capacity to develop resistance towards chemical insecticides and microbial sprays (for example based on Bt spore-crystal mixtures), and are believed to have the capacity to develop resistance towards plants expressing insecticidal proteins. The development of resistance within insect populations could render existing insecticidal proteins ineffective, creating a need for novel genes and proteins. Third, for health and environmental reasons it is desirable to identify proteins with high, specific insecticidal potency and acute bioactivity towards target insect species.

[005] Khandelwal and Banerjee-Bhatnagar (2003) and Khandelwal et al. (2004a) describe the isolation of a protein from Xenorhabdus nematophila with in vitro cytotoxicity to Helicoverpa armigera hemocytes, and Khandelwal et al. (2004b) describe that such protein is toxic to Helicoverpa armigera larvae when incorporating it into their diet.

[006] He et al. (2004) describe the sequence of a fimbrial major subunit of Xenorhabdus nematophila, which is part of the mannose-resistant fimbrial operon. [007] The current invention describes new forms and new applications of bacterial insecticidal proteins such as those derived from Xenorhabdus nematophilus strains, and variants thereof, particularly towards the engineering of transgenic plants with improved resistance to insect pests.

[008] Summary of the Invention

The invention is summarized in the following numbered paragraphs:

1. An isolated DNA comprising the nucleotide sequence of SEQ ID NO:1 from nucleotide position 6 to nucleotide position 473.

2. An isolated DNA comprising the nucleotide sequence of SEQ ID NO:3 from nucleotide position 6 to nucleotide position 473.

3. An isolated DNA encoding an insecticidal protein comprising the amino acid sequence of SEQ ID NO: 2.

4. An isolated DNA encoding an insecticidal protein comprising the amino acid sequence of SEQ ID NO: 4.

5. An isolated DNA encoding an insecticidal protein comprising the amino acid sequence of SEQ ID NO: 7.

6. A chimeric gene comprising as coding sequence the DNA of paragraph 1 and a promoter which allows expression in plant cells.

7. A chimeric gene comprising as coding sequence the DNA of paragraph 2 and a promoter which allows expression in plant cells.

8. A chimeric gene comprising as coding sequence the DNA of any one of paragraphs 3 to 5 and a promoter which allows expression in plant cells.

9. A plant, seed or plant cell comprising the chimeric gene of paragraph 6.

10. A plant, seed or plant cell comprising the chimeric gene of paragraph 7.

11. A plant, seed or plant cell comprising the chimeric gene of paragraph 8.

12. The plant, seed or cell of paragraph 9, wherein said plant, seed or cell is selected from the group consisting of: corn, cotton, soybean, rice, oilseed rape, cauliflower, and cabbage plants, seeds, or cells.

13. The plant, seed or cell of paragraph 10, wherein said plant, seed or cell is selected from the group consisting of: corn, cotton, soybean, rice, oilseed rape, cauliflower, and cabbage plants, seeds, or cells.

14. The plant, seed or cell of paragraph 11, wherein said plant, seed or cell is selected from the group consisting of: corn, cotton, soybean, rice, oilseed rape, cauliflower, and cabbage plants, seeds, or cells.

15. A method of protecting a plant against damage caused by insects, comprising the step of expressing the chimeric gene of paragraph 6 in cells of said plant.

16. A method of protecting a plant against damage caused by insects, comprising the step of expressing the chimeric gene of paragraph 7 in cells of said plant.

17. A method of protecting a plant against damage caused by insects, comprising the step of expressing the chimeric gene of paragraph 8 in cells of said plant.

18. An isolated insecticidal protein comprising the amino acid sequence of SEQ ID No. 2.

19. An isolated insecticidal protein comprising the amino acid sequence of SEQ ID No. 4.

20. An isolated insecticidal protein comprising the amino acid sequence of SEQ ID No. 7.

21. An isolated insecticidal protein comprising the amino acid sequence of SEQ ID No. 2 from amino acid position 2 to amino acid position 175, fused at its N-terminal end to a plant transit peptide.

22. An isolated insecticidal protein comprising the amino acid sequence of SEQ ID No. 4 from amino acid position 2 to amino acid position 175, fused at its N-terminal end to a plant transit peptide.

23. A method for killing larvae of Helicoverpa zea or Heliothis virescens, comprising contacting said larvae with a protein comprising the amino acid sequence of SEQ ID NO: 2 from amino acid position 2 to amino acid position 157.

24. A method for killing larvae of Helicoverpa zea or Heliothis virescens, comprising contacting said larvae with a protein comprising the amino acid sequence of SEQ ID NO: 4 from amino acid position 2 to amino acid position 157.

[009] Detailed Description of Embodiments of the Invention [010] The present invention provides methods and means for reducing damage to plants caused by pests, particularly insect pests, such as lepidopteran insect pests. The present invention further provides novel nucleic acid sequences and proteins that are distinct from previously described nucleic acid sequences and proteins. These nucleic acids and proteins can be used for controlling insect pests, either by integration and expression of at least one of these new nucleotide sequences in plants or plant cells, or by external treatment of plants or plant parts with compositions comprising the toxins encoded by these nucleic acid molecules.

[011] The present invention provides novel pesticidal toxins derived from bacterial strains, and certain novel uses of known toxins. The pesticidal toxins of the present invention include the proteins designated as "BIP1" or "BIP1 protein", e.g., BIPIa and BIPIb.

[012] In accordance with this invention, a "nucleic acid sequence" refers to a DNA or RNA molecule, in single- or double-stranded form, that encodes any of the BIP1 proteins of this invention. The term "isolated nucleic acid sequence", as used herein, is not limited to a nucleic acid sequence in isolation, but also encompasses a nucleic acid sequence that is no longer in the natural environment where it was isolated from. Thus, an "isolated BIP1 nucleic acid sequence" or an "isolated BIP1 protein sequence", in accordance with this invention, includes the nucleic acid or protein sequence in another bacterial host or in a plant nuclear genome, compared to the original bacterial organism. As used herein, an "isolated BIP1 nucleic acid sequence" or "an isolated

BIP1 protein" does not include a nucleic acid sequence or protein of similar or identical sequence described in one of the databases, publications or patent applications describing a bacterial genomic sequence, to the extent such sequences were not isolated from the organism or were not tested for their utility, such as their insecticidal activity (of the encoded protein). As such, the mere mentioning of a DNA or protein sequence with a mere showing of some sequence homology with known genes, without any isolation of the specific gene, or without reference to a utility as (a DNA encoding) an insecticidal protein, is considered not to form part of the relevant state of the art for this invention. To the extent any such sequence is deemed to form part of the relevant state of the art for this invention, Applicant retains the right to disclaim such sequences from the scope of the invention by reference to the specific sequence and the relevant document reference without this being any added subject matter under existing patent laws, thereby leaving unaffected the patentability of any novel uses of these DNA or protein sequences or novel variants thereof which are specified herein as being useful for insect control.

[013] In accordance with the present invention, the terms "protein" or "polypeptide" are used interchangeably to refer to a molecule consisting of a chain of amino acids, without reference to any specific mode of action, size, three-dimensional structure or origin. Hence, a fragment or portion of a BIP1 protein of the invention is still referred to herein as a "protein". The phrase "isolated protein", as used herein, is not limited to a protein in isolation, but also encompasses a protein that is no longer in its natural environment. The natural environment of the protein refers to the environment in which the protein could be found when the nucleotide sequence encoding it was expressed and translated in its natural environment, i.e., in the environment from which the nucleotide sequence was isolated. For example, an isolated protein can be present in

vitro, or in another bacterial host or in a plant cell, or it can be secreted from another bacterial host or from a plant cell.

[014] In accordance with this invention, nucleic acid sequences, including DNA sequences, encoding new BIP1 proteins are isolated and characterized, and novel forms have been artificially made by DNA synthesis. Two specific genes described herein were designated bipia and bipib and their encoded proteins BIPIa and BIPI b, respectively.

[015] In accordance with this invention, a "BIP" protein is an insecticidal protein of about 16 to 20 kDa, particularly between 15 and 40 kDa, or between 16 and 24 kDa, especially a protein smaller then 40 kDa, isolated or derived from Xenorhabdus spp. bacteria, particularly Xenorhabdus nematophila, with at least 50 %, preferably at least 60, 75 or 85 % sequence identity to any one of the proteins of SEQ ID NO: 2, 4, 5, 6 or 7, and any insecticidal fragments or variants thereof such as fusions to signal peptides, selectable marker proteins, or other insecticidal proteins. Such BIP proteins are typically isolatable from the bacterial culture supernatant in the original strain. "kDa", as used herein, refers to the size in kiloDalton of the theoretical molecular weight of a protein, the sequence of which is known, or of the molecular weight observed in standard SDS-PAGE/Western blotting of a protein, whose sequence is not entirely known or certain. For SDS-PAGE/Western blotting, a deviation of up to 5-15 % is to be expected from the theoretical value.

[016] In accordance with this invention, a "BIP1 protein" refers to any protein comprising the smallest fragment of the amino acid sequence of SEQ ID NO: 2 or 4 that retains insecticidal activity (hereinafter referred to as the "smallest toxic fragment"), particularly any insecticidal protein comprising the amino acid sequence of SEQ ID NO:2 from amino acid position 2 to amino acid position 157, or any insecticidal protein comprising the amino acid sequence of SEQ ID NO:4 from amino acid position 2 to

amino acid position 157, preferably a protein with the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4. This includes hybrid or chimeric proteins comprising the smallest toxic fragment of the amino acid sequence of SEQ ID NO:2 or 4. Also included in this definition are variants of the amino acid sequence in SEQ ID NO: 2 or 4, such as amino acid sequences essentially similar to SEQ ID NO: 2 or 4, having a sequence identity of at least 91%, or at least 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% at the amino acid sequence level. In the context of the present invention, "sequence identity" may be determined using pairwise alignments using the GAP program of the Wisconsin package of GCG (Madison, Wisconsin, USA, version 10.2). The GAP program is used with the following parameters for the amino acid sequence comparisons: the 'blosum62' scoring matrix, a 'gap creation penalty ¹ (or 'gap weight') of 8 and a 'gap extension penalty' (or 'length weight') of 2. lnsecticidal proteins according to the present invention may have some, for example 5-10, or less than 5 amino acids added, replaced or deleted without significantly changing, or without changing the insecticidal activity of the protein. Changes in the amino acid sequence that do not change the insecticidal activity of the protein in a negative way are also included in this definition.

[017] In accordance with this invention "BIPIb protein" refers to any protein comprising the smallest fragment of the amino acid sequence of SEQ ID NO: 4 that retains insecticidal activity (hereinafter referred to as "smallest toxic fragment"), preferably the protein comprising or consisting of the amino acid sequence of SEQ ID NO:4. This includes hybrid- or chimeric proteins comprising the smallest toxic fragment. Also included in this definition are variants of the amino acid sequence in SEQ ID NO: 4. Such variants may include essentially similar amino acid sequences, having a sequence identity of at least 91%, or at least 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% at the amino acid sequence level, as determined using pairwise

alignments using the GAP program of the Wisconsin package of GCG (Madison, Wisconsin, USA, version 10.2). The GAP program is used with the following parameters for the amino acid sequence comparisons: the 'blosum62' scoring matrix, a 'gap creation penalty' (or 'gap weight') of 8 and a 'gap extension penalty' (or 'length weight') of 2. Preferably, proteins having some, e.g. 5-10, or less than 5, amino acids added, replaced or deleted without significantly changing the insecticidal activity of the protein, or at least without changing the insecticidal activity of the protein in a negative way, are included in this definition.

[018] As used herein, the term "comprising" is to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps or components, or groups thereof. Thus, reference herein to DNA or protein "comprising the sequence or region X" refers to a DNA or protein including or containing at least the sequence or region X, so that other nucleotide or amino acid sequences can be included at the 5' (or N-terminal) and/or 3' (or C-terminal) end. For example, a nucleotide sequence may comprise the nucleotide sequence encoding a transit peptide, and/or a 5' or 3' leader sequence.

[019] The "smallest toxic fragment" of a BIP1 protein of the invention, as used herein, refers to the smallest fragment or portion of a BIP1 protein retaining insecticidal activity that can be obtained, e.g., by cleavage of a signal peptide or by enzymatic digestion of the full length BIP1 protein. "Smallest toxic fragment" also encompasses the smallest fragment or portion of a BIP1 protein retaining insecticidal activity that can be obtained by making nucleotide deletions in the DNA encoding a BIP1 protein. DNA encoding shorter toxic BIP1 fragments may also be synthesized chemically; thus, the smallest toxic fragment obtainable from transcription and translation of synthetic DNA is included in the definition of smallest toxic fragment.

[020] Suitable Lepidopteran target insects for the BIP1 proteins of the invention include, but are not limited to, corn earworm (Helicoverpa zea), cotton bollworm (Helicoverpa armigera), native budworm (Helicoverpa punctigera), tobacco budworm (Heliothis virescens), european corn borer (Ostrinia nubilalis), fall armyworm (Spodoptera frugiperda), black cutworm (Agrotis ipsilon), pink bollworm (Pectinophora gossypiella), yellow stem borer (Scirphophaga incertulas), leaffolder {Cnaphalocrocis medinalis), pink stem borer (Sesamia inferens), corn spotted stem borer (Chilo partellus), velvet caterpillar (Anticarsia gemmatalis), soybean looper [Pseudoplusia includens), pod borer (Epinotia aporema), and Rachiplusia nu. [021] Other target insects for the BIP proteins of the invention are selected from the _: ϋjst consisting of: Plathypena scabra, Spodoptera exigua, Spodoptera ornithogalli, Chilo suppressalis, Hereitogramma licarisalis, Naranga aenescens, Mycalesis gotama, Marasmia patnalis, Marasmia exigua, Marasmia ruralis, Nymphula depunctalis, Scirpophaga innotata, Spodoptera litura, Chilo polychrysus, Rupela albinella, Diatraea saccharalis, Spodoptera frugiperda, Mythimna unipuncta, Chilo zacconius and Parnara guttata, Agelastica alni, Hypera postica, Hypera brunneipennis, Haltica tombacina, Anthonomus grandis, Tenebrio molitor, Triboleum castaneum, Dicladispa armigera, Trichispa serica, Oulema oryzae, Colaspis brunnea, Lissorhorptrus oryzophilus, Phyllotreta cruciferae, Phyllotreta striolata, Psylliodes punctulata, Entomoscelis americana, Meligethes aeneus, Ceutorynchus sp., Psylliodes chrysocephala, Phyllotreta undulata, Leptinotarsa decemlineata, Diabrotica undecimpunctata undecimpunctata, Diabrotica undecimpunctata howardi, Diabrotica barberi, and Diabrotica virgifera.

[022] The N- and C-terminal amino acid sequences of the smallest toxic fragment may be conveniently determined by amino acid sequence determination of the above fragments using routine techniques available in the art.

[023] As used herein, the term "bip1 DNA" or "BIP1 DNA" refers to any DNA sequence encoding a BIP1 protein, such as a DNA encoding the "BIPIa protein" or "BIPIb protein", as defined above, e.g., the DNA of SEQ ID NO: 1 or 3 from nucleotide position 6 to nucleotide position 473. This includes naturally-occurring, artificial, or synthetic DNA sequences encoding the proteins of SEQ ID NO: 2, 4, 5, 6, or 7, preferably encoding the proteins of SEQ ID NO: 2 or 4 from amino acid position 2 to amino acid position 157, or their insecticidal fragments or variants as defined above. Also included herein are DNA sequences encoding insecticidal proteins, which are similar enough to the DNA sequences provided in the sequence listing, or any DNA encoding a BIP protein of the invention, that they can (i.e., have the ability to) hybridize to these DNA sequences under stringent hybridization conditions. [024] "Stringent hybridization conditions", as used herein, refers particularly to the following conditions: immobilizing the relevant DNA on a filter, and prehybridizing the filters for either 1 to 2 hours in 50% formamide, 5% SSPE, 2x Denhardt's reagent and 0.1% SDS at 42° C, or 1 to 2 hours in 6x SSC, 2x Denhardt's reagent and 0.1% SDS at 68 ⁰ C. The denatured (Digoxigenin- or radio-) labeled probe is then added directly to the prehybridization fluid and incubation is carried out for 16 to 24 hours at the appropriate temperature mentioned above. After incubation, the filters are then washed for 30 minutes at room temperature in 2x SSC, 0.1% SDS, followed by 2 washes of 30 minutes each at 68°C in 0.5 x SSC and 0.1% SDS. An autoradiograph is established by exposing the filters for 24 to 48 hours to X-ray film (Kodak XAR-2 or

equivalent) at -70 ⁰ C with an intensifying screen (2Ox SSC = 3M NaCI and 0.3M sodium citrate; 10Ox Denhardt's reagent= 2%(w/v) bovine serum albumin, 2%(w/v) Ficoll™ and 2% (w/v) polyvinylpyrrolidone; SDS = sodium dodecyl sulfate; 2Ox SSPE= 3.6M NaCI, 02M Sodium phosphate and 0.02M EDTA pH7.7). One of ordinary skill in the art will readily be able to modify the particular conditions and parameters specified above while retaining the desired stringent hybridization conditions. [025] There are many approaches known in the art for the isolation of variants of the DNA sequences of the invention. For example, variants can be detected and isolated from bacterial strains, e.g., Xenorhabdus nematophila, by hybridization as described supra, and/or by PCR technology, as known in the art. Specific or degenerate primers can be made to regions of the BIP1 DNA sequences, and used to amplify variants from known or novel bacterial strains.

[026] Variants of the BIP1 DNA of the invention include DNA sequences encoding the insecticidal BIP1 protein variants described above, or a DNA sequence, encoding an insecticidal protein, with at least 93%, at least 94%, at least 95%, 96% or 97%, at least 98 % or at least 99% sequence identity to SEQ ID NO: 1 or 3 from nucleotide position 6 to nucleotide position 473. The sequence identities referred to are calculated using the GAP program of the Wisconsin package of GCG (Madison, Wisconsin, USA) Version 10.2. The GAP program is used with the following parameters for nucleic acids: the "nwsgapdna" scoring matrix, a "gap creation penalty" (or "gap weight") of 50 and a "gap extension penalty'"(or "length weight") of 3. Stringent hybridization conditions are as defined above.

[027] "Insecticidal activity" of a protein, as used herein, means the capacity of a protein to kill insects when such protein is fed to insects, preferably by expression in a recombinant host such as a plant. It is understood that a protein has insecticidal activity

if it has the capacity to kill the insect during at least one of its developmental stages, preferably the larval stage.

[028] "Insect-controlling amounts" of a protein, as used herein, refers to an amount of protein which is sufficient to limit damage on a plant, caused by insects at any stage of development {e.g., insect larvae) feeding on such plant, to commercially acceptable levels. Limiting insect damage to a plant may be the result of, for example, killing the insects or inhibiting insect development, fertility or growth in such a manner that the insect inflicts less damage to a plant and plant yield is not significantly adversely affected.

[029] In accordance with this invention, insects susceptible to the new BIP1 proteins of the invention are contacted with this protein in insect-controlling amounts, preferably insecticidal amounts. Preferred target insects for the proteins of this invention are economically damaging insect pests of corn, cotton, rice or soybean plants, particularly in Northern and Southern American countries, Asia and Australia. The term "plant", as used herein, encompasses whole plants as well as parts of plants, such as leaves, stems, seeds, flowers or roots.

[030] The terms "BIP1 protein", "BIP1 protein of this invention", as used herein, refers to any one of the new proteins or variants thereof in accordance with this invention, e.g., the proteins defined herein as BIPIa or BIPIb protein.

[031] A BIP1 protein, as used herein, can be a protein in the full-length size or can be in a truncated form as long as the insecticidal activity is retained, or can be a combination of several proteins or protein domains in a hybrid or fusion protein. A

"BIP1 toxin" refers to an insecticidal fragment or portion of a BIP1 protein, particularly the smallest toxic fragment thereof.

[032] The nucleic acid sequence, particularly the DNA sequence, encoding the BIP1 protein of this invention can be made synthetically and can be inserted in expression

vectors to produce high amounts of BIP1 proteins. The BIP1 proteins can be used to prepare specific monoclonal or polyclonal antibodies in a conventional manner (Hόfte et al., 1988; Harlow and Lane, 1988).

[033] In one embodiment of the invention, antibodies that specifically bind to the BIP1 protein are provided. In particular, monoclonal or polyclonal antibodies that bind to BIP1 or to fragments or variants thereof are provided. Also included are fragments of monoclonal or polyclonal antibodies, which retain the ability to bind to the BIP1 protein or fragment against which they were raised (e.g., a single-chain antibody). An antibody to a BIP1 protein can be prepared by using the BIP1 protein as an antigen in an animal (such as rabbit or mouse), using methods known in the art. Suitable methods for preparing antibodies include those described in Harlow and Lane "Using Antibodies: A Laboratory Manual" (New York: Cold Spring Harbor Laboratory Press, 1998); and in Liddell and Cryer "A Practical Guide to Monoclonal Antibodies" (Wiley and Sons, 1991). The antibodies can be used to isolate, identify, characterize or purify the BIP1 protein to which it binds. For example, the antibody can be used to detect the BIP1 protein in a sample, by allowing antibody and protein to form an immunocomplex, and detecting the presence of the immunocomplex, for example through ELISA or immunoblots. [034] In a further embodiment of the invention PCR primers and/or probes and kits for detecting the BIP1 DNA sequences are provided. PCR primer pairs (wherein each primer is at least 15 to 25, preferably at least 18 or 20 nucleotides in length) to amplify BIP1 DNA from samples can be synthesized based on SEQ ID NO: 1 or 3, as known in the art (see, e.g., Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press; and McPherson at al. (2000) PCR - Basics: From Background to Bench, First Edition, Springer Verlag, Germany). Likewise, DNA fragments of SEQ ID NO: 1 or 3 can be used as hybridization probes. A BIP1 detection kit may comprise either BIP1 specific primers or BIP1 specific probes,

and an associated protocol to use the primers or probe to detect BIP1 DNA in a sample. For example, such a detection kit may be used to determine, whether a plant has been transformed with a BIP1 gene (or part thereof) of the invention, such as the DNA of SEQ ID NO: 1 or 3.

[035] Because of the degeneracy of the genetic code, some amino acid codons can be replaced by others without changing the amino acid sequence of the protein. Furthermore, some amino acids can be substituted by other equivalent amino acids without changing, or without significantly changing the insecticidal activity of the protein, or at least without decreasing the insecticidal activity of the protein. For example, conservative amino acid substitutions include interchanging amino acids within categories: basic (e.g. Arg, His, Lys), acidic (e.g. Asp, GIu), nonpolar (e.g. Ala, VaI, Trp, Leu, lie, Pro, Met, Phe, Trp, GIy), and polar (e.g. Ser, Thr, Tyr, Cys, Asn, GIn). Such substitutions within categories fall within the scope of the invention as long as the insecticidal activity of the BIP1 protein is substantially the same, or not significantly decreased. In addition, non-conservative amino acid substitutions fall within the scope of the invention as long as the insecticidal activity of the BIP1 protein is substantially the same, or not significantly decreased. Variants or equivalents of the DNA sequences of the invention include DNA sequences hybridizing to the BIP1 DNA sequence of SEQ ID NO: 1 under stringent hybridization conditions. Such variants or equivalents should still encode a protein with the same or substantially the same insecticidal characteristics as the protein of this invention. Variants or equivalents as used herein also include DNA sequences having a different codon usage compared to the native BIP1 genes of this invention but which encode a protein with the same insecticidal activity and with the same or substantially the same amino acid sequence. The BIP1 DNA sequences can be codon-optimized by adapting the codon usage to that most preferred in plant genes, particularly to genes native to the plant genus or species of

interest, i.e., into which expression of a BIP protein is desired (Bennetzen & Hall, 1982; ltakura et a!., 1977) using available codon usage tables (e.g., more adapted towards expression in cotton, soybean corn or rice). Codon usage tables for various plant species have been published by, for example, lkemura (1993) and Nakamura et al. (2000).

[036] Long stretches of AT or GC nucleotides may be removed and suitable restriction sites may be introduced.

[037] In addition, the N-terminus of a BIP1 protein can be modified to have an optimum translation initiation context, thereby adding or deleting one or more amino acids at the N-terminal of the protein. In most cases, it is preferred that the proteins of the invention to be expressed in plants cells start with a Met-Asp or Met-Ala dipeptide for optimal translation initiation, hence requiring the insertion in the BIP1 DNA of a codon encoding an Asp or Ala amino acid downstream of the start codon as a new second codon (if such second codon is not already Ala or Asp). [038] The DNA sequences may also be modified to remove illegitimate splice sites or plant transcription termination sequences. As bacterial genes may contain motifs that are recognized in other hosts, especially in eukaryotic host such as plants, as 5' or 3 ¹ splice sites or plant transcription termination sequences, transcription in those other hosts may be ineffective or may be terminated prematurely, resulting in truncated mRNA. Illegitimate splice sites or transcription termination sites, or sequences resembling such sites, can be identified by computer-based analysis of the DNA sequences and/or by PCR analysis as known in the art.

[039] Any DNA sequence differing in its codon usage but encoding the same protein or a similar protein with substantially the same insecticidal activity can be constructed, depending on the particular purpose. It has been described in prokaryotic and eukaryotic expression systems that changing the codon usage to that of the host cell

has benefits for gene expression in foreign hosts (Bennetzen & Hall, 1982; ltakura et al., 1977). Codon usage tables are available in the literature (Wada et al., 1990; Murray et al., 1989) and in the major DNA sequence databases (e.g. EMBL at Heidelberg, Germany) and as described by Nakamura et al (2000). Accordingly, one of ordinary skill in the art can readily construct synthetic DNA sequences so that the same or substantially the same proteins are produced. It is evident that alternate DNA sequences can be made once the amino acid sequence of the BIP1 proteins of this invention is known. Such alternate DNA sequences include synthetic or semi-synthetic DNA sequences that have been changed in order to inactivate certain sites in the gene. This inactivation can be accomplished by, for example, adapting the overall codon usage to that of a more related host organism, such as that of the host organism in which expression is desired. Several techniques for modifying the codon usage to that preferred by the host cells can be found in the patent and scientific literature. The exact method of codon usage modification is not critical for this invention as long as most or all of the cryptic regulatory sequences or processing elements (such as plant polyadenylation signals, or plant splice sites) have been replaced by other sequences, and preferably the AT-content of the coding region approaches that of the coding regions in the host organism.

[040] Small modifications to a DNA sequence such as described above can be routinely made, e.g., by PCR-mediated mutagenesis (Ho et al., 1989, White et al., 1989). More substantial modifications to a DNA sequence can routinely be made by de novo DNA synthesis of a desired coding region using available techniques. [041] The phrase "substantially the same," when used herein in reference to the amino acid sequence of a BIP1 protein, refers to an amino acid sequence that differs no more than 5%, or no more than 2%, from the amino acid sequence of the protein compared to. When referring to toxicity of a BIP1 protein, the phrase "substantially the

same" refers to a protein whose mean LC ₅₀ value differs by no more than a factor of 2 to 5, particularly 2, from the mean LC ₅₀ value obtained for the protein compared to. In this context, "mean LC ₅₀ " is the concentration of protein causing 50% mortality of the test population, calculated from three independent bioassays carried out using the same bioassay conditions. LC ₅₀ values are calculated with Probit analysis, using the program POLO PC (from LeOra Software, 1987, Berkely, California). It is understood, that 95% (or 90%) confidence limits (an associated parameter calculated with Probit analysis) are calculated for the LC ₅₀ values of each of the two proteins to be compared in order to determine whether a statistically significant difference in LC ₅₀ values exists. In one embodiment of this invention, the toxicity of the two proteins is seen to be substantially the same, if the confidence limits overlap and substantially different if the confidence limits do not overlap.

[042] The BIP1 DNA sequences of the invention, prepared from total DNA, can be ligated in suitable expression vectors and transformed in a bacterial strain, such as E. coli or another bacterial strain. In one embodiment of the invention, for expression in bacteria such as E. coli, a DNA encoding the bacterial signal peptide of the BIP protein or a suitable other bacterial signal peptide (e.g., one originating from a secreted protein made by the host cell) is included in the DNA construct. The clones can then be screened by conventional colony immunoprobing methods (French et al., 1986) for expression of the toxin with monoclonal or polyclonal antibodies raised against the BIP1 proteins.

[043] The bacterial clones can be screened for production of BIP1 proteins (cell lysate can be run on SDS-PAGE gels using standard methods and standard western- blotting procedures can be carried out), or the bacteria or purified or semi-purified BIP protein can be tested for their insecticidal activity compared to control bacteria using methods known in the art (see, e.g., Khandelwal et al. 2004b). The clones can also be

analysed for the presence of mRNA encoding BIP1 protein using standard PCR procedures, such as RT-PCR.

[044] The genes encoding the BIP1 proteins of this invention can be sequenced in a conventional manner (Maxam and Gilbert, 1980; Sanger, 1977) to obtain the DNA sequence. Sequence comparisons indicated that the genes are different from previously described genes encoding insecticidal toxins. [045] An insecticidally-effective part of the DNA sequences, encoding an insecticidally-effective portion of the newly identified BIP1 proteins, can be made in a conventional manner after sequence analysis of the gene. The amino acid sequence of the BIP1 proteins can be determined from the DNA sequence of the isolated DNA sequences. The phrase "an insecticidally effective part (or portion or fragment)" of DNA sequences encoding the BIP1 protein, also referred to herein as a "truncated gene" or "truncated DNA," as used herein refers to a DNA sequence encoding a polypeptide that is insecticidal, but has fewer amino acids than the BIP1 full length protein form. [046] In order to express all or an insecticidally-effective part of the DNA sequence encoding a BIP1 protein of this invention in E. coli, in other bacterial strains, or in plants, suitable restriction sites can be introduced, flanking the DNA sequence. This can be done by site-directed mutagenesis, using well-known procedures (see, e.g., Stanssens et a)., 1989; White et al., 1989). In order to obtain improved expression in plants, the codon usage of the BIP1 gene or insecticidally effective BIP1 gene part of this invention can be modified to form an equivalent, modified or artificial gene or gene part in accordance with PCT publications WO 91/16432 and WO 93/09218 and publications EP 0 385 962, EP 0 359 472 and US 5,689,052. Such improved DNA coding sequences are illustrated in SEQ ID NO: 1 and 3. The BIP1 genes or gene parts may also be inserted in the plastid, mitochondrial or chloroplast genome and

expressed there using a suitable promoter (see, e.g., Mc Bride et al., 1995; US 5,693,507).

[047] For obtaining enhanced expression in monocot plants such as corn or rice, an intron (e.g., a monocot intron) can also be added to the chimeric gene. For example, the insertion of the intron of the maize Adh1 gene into the 5' regulatory region has been shown to enhance expression in maize (Callis et. al., 1987). Likewise, the HSP70 intron, as described in US 5,859,347, may be used to enhance expression. The DNA sequence of the BIP1 gene or its insecticidal part can be further changed in a translationally neutral manner. Such changes may modify possibly inhibiting DNA sequences present in the gene part by means of site-directed intron insertion and/or by introducing changes to the codon usage. Changes in codon usage may be, e.g., to adapt the codon usage to that most preferred by plants, particularly the host plant (Murray et al., 1989), without changing, or without significantly changing, the encoded amino acid sequence.

[048] In accordance with one embodiment of this invention, the proteins may be targeted to intracellular organelles such as plastids, chloroplasts, mitochondria, or are secreted from the cell. For this purpose, in one embodiment of this invention, the chimeric genes of the invention comprise a coding region encoding a signal or target peptide, linked to the BIP1 protein-coding region of the invention. Peptides that may be included in the proteins of this invention are the transit peptides for chloroplast or other plastid targeting, such as duplicated transit peptide regions from plant genes whose gene product is targeted to the plastids, the optimized transit peptide of Capellades et al. (US 5,635,618), the transit peptide of ferredoxin-NADP ⁺ oxidoreductase from spinach (Oelmuller et al., 1993), the transit peptide described in Wong et al. (1992) and the targeting peptides in published PCT patent application WO 00/26371. Alternative peptides include those signalling secretion of a protein linked to such peptide (outside

the cell), such as the secretion signal of the potato proteinase inhibitor Il (Keil et al., 1986), the secretion signal of the alpha-amylase 3 gene of rice (Sutliff et al., 1991) and the secretion signal of tobacco PR1 protein (Cornelissen et al., 1986). [049] Useful signal peptides in accordance with the invention include the chloroplast transit peptide (e.g., Van Den Broeck et al., 1985), or the optimized chloroplast transit peptide of US 5,510,471 and US 5,635,618 causing transport of the protein to the chloroplasts, a secretory signal peptide or a peptide targeting the protein to other plastids, mitochondria, the ER, or another organelle. A BIP1 protein containing an optimized transit peptide is illustrated in SEQ ID NO: 7. Signal sequences for targeting to intracellular organelles or for secretion outside the plant cell or to the cell wall are found in naturally targeted or secreted proteins, such as those described by Klδsgen et al. (1989), Klosgen and Weil (1991), Neuhaus & Rogers (1998), Bih et al. (1999), Morris et al. (1999), Hesse et al. (1989), Tavladoraki et al. (1998), Terashima et al. (1999), Park et al. (1997), Shcherban et al. (1995), all of which are incorporated herein by reference. Alternative signal sequences include the signal peptide sequences from targeted or secreted proteins of corn, cotton, soybean or rice. [050] To allow secretion of the BIP1 proteins to the outside of the transformed host cell, an appropriate secretion signal peptide may be fused to the amino terminal end (N-terminal end) of the BIP1 protein. Also, any putative native bacterial secretion signal peptide can be deleted and replaced by an appropriate signal peptide, such as a eukaryotic secretion signal peptide as described above. Particularly, amino acids 1 to 23 of the BIP1 proteins of the invention comprise a putative bacterial signal peptide. Amino acids 1 to 23 may be removed and replaced by a Methionine amino acid, by a Met-Ala or Met-Asp dipeptide, or by an appropriate signal peptide, such as a plant signal peptide as described above, see, e.g., the proteins of SEQ ID NO: 2, 4, and 7. Putative signal peptides can be detected using computer based analysis, using

programs such as the program Signal Peptide search (SignalP V1.1 or 2.0), using an appropriate matrix (e.g., for prokaryotic gram-positive bacteria) and a threshold score of less than 0.5, a threshold score of 0.25, or less (see, e.g., Von Heijne, Gunnar, 1986 and Nielsen et al., 1996).

[051] Furthermore, the binding properties of the BIP1 proteins of the invention can be evaluated, using methods known in the art (see, e.g., Van Rie et al., 1990), to determine if the BIP1 proteins of the invention bind to sites in the insect gut, such as the midgut, that are not recognized (or competed for) by other bacterial proteins, lnsecticidal bacterial proteins with different binding sites (for which there is no competition for binding) in relevant susceptible insects are very valuable. Such proteins can be used to replace known bacterial proteins to which insects may have developed resistance, or to use in combination with insecticidal bacterial proteins having a different mode of action to prevent or delay the development of insect resistance against bacterial proteins, particularly when expressed in a plant. Because of the characteristics of the BIP1 toxins of the present invention, they are extremely useful for transforming plants, e.g., monocots such as corn and rice and dicots such as cotton, Brassica species (such as cauliflower or cabbage) and soybean, to protect these plants from insect damage. The binding properties of the BIP1 proteins of the current invention are different compared to the known Bt toxins that are currently used in transgenic plants, such as Cry or VlP proteins. Such different binding properties can be measured by routine binding assays as described above or in US patent 6,291 ,156 and US patent 6,137,033.

[052] Especially for insect resistance management purposes for a specific insect pest, it is preferred to combine a BIP1 protein of this invention with another insect control protein, particularly a bacterial Cry protein, such as Cry1 B, Cry1C, Cry1 D, Cry1 E, Cry1 F, Cry2A or Cry1 Ac protein, or a VIP or VIP-like protein, such as VIP3A,

preferably a protein which does not recognise at least one binding site recognised by such BIP1 protein. Suitable insect control proteins to combine with the BIP1 proteins of this invention, particularly for simultaneous expression in plants (such as maize, cotton, rice or soybean plants), include, but are not limited to, the Cry proteins, such as a protein comprising the Cry1 F toxic fragment or hybrids derived from a Cry1 F protein (e.g., the hybrid Cry1A-Cry1 F proteins described in US 6,326,169; US 6,281 ,016; US 6,218,188, or toxic fragments thereof), a protein comprising the Cry1A-type proteins or toxic fragments thereof, preferably the Cry 1Ac protein or hybrids derived from the CryiAc protein (e.g., the hybrid Cry1Ab-Cry1Ac protein described in US 5,880,275) or a protein comprising the CrylAb protein or insecticidal fragments thereof as described in EP451878, a protein comprising the Cry2Ab protein toxic fragment, such as the Cry2Ab protein-transit peptide fusion protein described in US patent 6,489,542; a protein comprising the Cry2Ae, Cry2Af or Cry2Ag toxic fragments as described in WO02/057664, a protein comprising the Cry protein toxic fragments as described in WO01/47952, a protein comprising the VIP3Aa protein or a toxic fragment thereof as described in Estruch et al. (1996) and US 6,291 ,156, insecticidal proteins from Xenorhabdus spp. as described in WO98/50427, insecticidal proteins from Serratia (particularly from S. entomophila) or Photorhabdus species strains, such as Tc-proteins from Photorhabdus as described in WO98/08932 (e.g., Waterfield et al., 2001; Ffrench- Constant and Bowen, 2000). In one embodiment, such co-expression is easily obtained by transforming a plant already expressing an insect control protein with a DNA encoding a BIP1 protein of this invention, or by crossing plants transformed with a known insect control protein with plants transformed with one or more BIP1 proteins of this invention. For maize, rice, cotton or soybean plants, the BIP1 protein may be used as first insect control protein and as second insect control protein the CrylAb, CryiAc, Cry2Ae or VIP3Aa proteins or proteins comprising their toxic fragments, hybrids or

variants thereof can be used. Methods for obtaining expression of different insecticidal proteins in the same plant in an effort to minimize or prevent resistance development to transgenic insect-resistant plants are described in EP 0408 403. The different proteins can be expressed in the same plant, or each can be expressed in a single plant and then combined in the same plant by crossing the single plants with one another. For example, in hybrid seed production, each parent plant can express a single protein. Upon crossing the parent plants to produce hybrids, both proteins are combined in the hybrid plant.

[053] It is well known that Bt Cry proteins are expressed as protoxins, which are converted into the toxic core by proteolysis in the insect gut. When combining the BIP1 proteins of the invention with Bt Cry proteins, it is understood that Cry genes encoding either the full protoxin or the toxic core or any intermediate form may be used. [054] For selection purposes, and for increasing the weed control options, the transgenic plants of the invention may also be transformed with a DNA encoding a protein conferring resistance to a broad-spectrum herbicide, e.g., herbicides based on glufosinate or glyphosate.

[055] The insecticidally effective BIP1 gene part or its equivalent, preferably the BIP1 chimeric gene, encoding an insecticidally effective portion of the BIP1 protein, can be stably inserted in a conventional manner into the nuclear genome of a single plant cell, and the so-transformed plant cell can be used in a conventional manner to produce a transformed plant that is insect-resistant. In this regard, a T-DNA vector, containing the insecticidally effective BIP1 gene part, in Agrobacterium tumefaciens can be used to transform the plant cell. Thereafter, a transformed plant can be regenerated from the transformed plant cell using the procedures described, for example, in EP 0 116 718, EP 0 270 822, PCT publication WO 84/02913 and published European Patent application EPO 242 246 and in Gould et al. (1991). The construction of a T-DNA

vector for Agrobacterium-mediated plant transformation is well known in the art. The T- DNA vector may be either a binary vector as described in EP 0 120 561 and EP 0 120 515 or a co-integrate vector which can integrate into the Agrobacterium Ti-plasmid by homologous recombination, as described in EP 0 116 718. Preferred T-DNA vectors each contain a promoter operably linked to the insecticidally effective BIP1 gene part between T-DNA border sequences, or at least located to the left of the right border sequence. Border sequences are described in Gielen et al. (1984). Other types of vectors can be used to transform the plant cell, using procedures such as direct gene transfer (as described, for example in EP 0 223 247), pollen mediated transformation (as described, for example in EP 0 270 356 and WO 85/01856), protoplast transformation as, for example, described in US 4,684,611 , plant RNA virus-mediated transformation (as described, for example in EP 0 067 553 and US 4,407,956), liposome-mediated transformation (as described, for example in US 4,536,475), and other methods, such as the recently described methods for transforming certain lines of corn (e.g., US 6,140,553; Fromm et al., 1990; Gordon-Kamm et al., 1990) and rice (Shimamoto et al., 1989; Datta et al. 1990) and the method for transforming monocots generally (PCT publication WO 92/09696). A suitable method for cotton transformation is described in PCT patent publication WO 00/71733. For rice transformation, reference is made to the methods described in WO92/09696, WO94/00977 and WO95/06722. [056] The terms "maize" and "corn" are used herein synonymously, referring to Zea mays. Cotton as used herein refers to Gossypium spp., particularly G. hirsutum and G. barbadense. The term "rice" refers to Oryza spp., particularly O. sativa. "Soybean" refers to Glycine spp, particularly G. max.

[057] Besides transformation of the nuclear genome, also transformation of the plastid genome (e.g., the chloroplast genome) is included in the invention. Kota et al.

(1999) have described a method to over-express a Cry2Aa protein in tobacco chloroplasts.

[058] The resulting transformed plant can be used in a conventional plant breeding scheme to produce more transformed plants with the same characteristics or to introduce the insecticidally effective BIP1 gene part into other varieties of the same or related plant species. Seeds, which are obtained from the transformed plants, contain the insecticidally effective BIP1 gene part as a stable genomic insert. Cells of the transformed plant can be cultured in a conventional manner to produce the insecticidally effective portion of the BIP1 toxin or protein, which can be recovered for use in conventional insecticide compositions, particularly insecticide compositions against Lepidoptera (see, e.g., US 5,254,799).

[059] The insecticidally effective BIP1 gene part is inserted in a plant cell genome so that the inserted gene is downstream (i.e., 3') of, and under the control of, a promoter which can direct the expression of the gene part in the plant cell. This may be accomplished by inserting the BIP1 chimeric gene in the plant cell genome, for example in the nuclear or plastid (e.g., chloroplast) genome. [060] Suitable promoters include, but are not limited to: the strong constitutive 35S promoters (the "35S promoters") of the cauliflower mosaic virus (CaMV) of isolates CM 1841 (Gardner et al., 1981), CabbB-S (Franck et al., 1980) and CabbB-JI (Hull and Howell, 1987); the 35S promoter described by Odell et al. (1985), promoters from the ubiquitin family (e.g., the maize ubiquitin promoter of Christensen et al., 1992, EP 0 342 926, see also Cornejo et al., 1993), the gos2 promoter (de Pater et al., 1992), the emu promoter (Last et al., 1990), Arabidopsis actin promoters such as the promoter described by An et al. (1996), rice actin promoters such as the promoter described by Zhang et al. (1991) and the promoter described in US 5,641 ,876; promoters of the Cassava vein mosaic virus (WO 97/48819, Verdaguer et al. (1998)) , the pPLEX series

of promoters from Subterranean Clover Stunt Virus (WO 96/06932, particularly the S7 promoter), a alcohol dehydrogenase promoter, e.g., pAdhiS (GenBank accession numbers X04049, X00581), and the TR1' promoter and the TR2' promoter (the "TR1 ¹ promoter" and "TR2' promoter", respectively) which drive the expression of the 1' and 2' genes, respectively, of the T-DNA (Velten et al., 1984). Alternatively, a promoter can be utilized which is not constitutive but rather is specific for one or more tissues or organs of the plant (e.g., leaves and/or roots) whereby the inserted BIP1 gene part is expressed only in cells of the specific tissue(s) or organ(s). For example, the insecticidally effective BIP1 gene part could be selectively expressed in the leaves of a plant (e.g., corn, cotton, rice, soybean) by placing the insecticidally effective gene part under the control of a light-inducible promoter such as the promoter of the ribulose-1 ,5-bisphosphate carboxylase small subunit gene of the plant itself or of another plant, such as pea, as disclosed in US 5,254,799. The promoter can, for example, be chosen so that the BIP1 gene of the invention is only expressed in those tissues or cells on which the target insect pest feeds so that feeding by the susceptible target insect will result in reduced insect damage to the host plant, compared to plants which do not express the BIP1 gene. An insect pest mainly damaging the roots can thus effectively be controlled by expressing a BIP1 gene under a root specific promoter. A promoter preferentially active in roots is described in WOOO/29566. A suitable promoter for root preferential expression is the ZRP promoter (and modifications thereof) as described in US 5,633,363. Another alternative is to use a promoter whose expression is inducible, for example a wound-inducible promoter such as, e.g., the MPI promoter described by Cordera et al. (1994), which is induced by wounding (such as caused by insect feeding), or a promoter inducible by a chemical, such as dexamethasone as described by Aoyama and Chua (1997) or a promoter inducible by temperature, such as the heat shock promoter described in US 5,447,858,

or a promoter inducible by other external stimuli. In monocot plants, such as corn and rice, the Agrobacterium TR2' promoter, or variants thereof, are a preferred wound- induced promoter to drive transcription of a chimeric BIP1 gene of the invention, see WO 03/093483.

[061] The insecticidally effective BIP1 gene part may be inserted into the plant genome so that the inserted gene part is upstream (Ae., 5 ¹ ) of suitable 3' end transcription regulation signals (i.e., transcript formation and polyadenylation signals). This is preferably accomplished by inserting the BIP1 chimeric gene in the plant cell genome. Suitable polyadenylation and transcript formation signals include those of the CaMV 35S gene, the nopaline synthase gene (Depicker et al., 1982), the octopine synthase gene (Gielen et al., 1984) and the T-DNA gene 7 (Velten and Schell, 1985), which act as 3'-untranslated DNA sequences in transformed plant cells. [062] Introduction of the T-DNA vector into Agrobacterium can be carried out using known methods, such as electroporation or triparental mating. [063] The insecticidally-effective BIP1 gene part can optionally be inserted in the plant genome as a hybrid gene (US 5,254,799; Vaeck et al., 1987) under the control of the same promoter as a selectable or scorable marker gene, such as the neo gene (EP 0 242 236) encoding kanamycin resistance, so that the plant expresses a fusion protein that is easily detectable.

[064] Transformation of plant cells can also be used to produce the proteins of the invention in large amounts in plant cell cultures, e.g., to produce a BIP1 protein that can then be applied onto crops after proper formulation. When reference to a transgenic plant cell is made herein, this refers to a plant cell (or also a plant protoplast) as such in isolation or in tissue culture, or to a plant cell (or protoplast) contained in a plant or in a differentiated organ or tissue, and both possibilities are specifically included herein. Hence, a reference to a plant cell in the description or

claims is meant to refer not only to isolated cells in culture, but also to any plant cell, wherever it may be located or in whatever type of plant tissue or organ it may be present.

[065] All or part of the BIP1 gene, encoding an insecticidal, particularly anti- Lepidopteran, protein, can also be used to transform other microorganisms, including bacteria, such as a B. thuringiensis, which may already have insecticidal activity against Lepidoptera or Coleoptera. Thereby, a transformed Bt strain can be produced which is useful for combating a wide spectrum of Lepidopteran and/or Coleopteran insect pests or for combating additional Lepidopteran insect pests. Transformation of bacteria, such as bacteria of the genus Pseudomonas, Agrobacterium, Bacillus or Escherichia, with all or part of the BIP1 gene of this invention, incorporated in a suitable cloning vehicle, can be carried out in a conventional manner, using, e.g., conventional electroporation techniques as described in Mahillon et al. (1989) and in PCT Patent publication WO 90/06999.

[066] Transformed Bacillus species strains containing the BIP1 gene of this invention can be fermented by conventional methods (Dulmage, 1981; Bemhard and Utz, 1993) to provide high yields of cells. Under appropriate growth conditions, these strains can produce BIP1 protein in high yields.

[067] Alternative suitable host microorganisms in which the BIP1 genes can be expressed are fungi, algae, or viruses, particularly species which are plant colonizing (e.g., (endo)symbiontic) species or insect pathogens.

[068] An insecticidal, particularly anti-Lepidopteran, composition of this invention can be formulated in a conventional manner using the microorganisms transformed with the BIP1 gene, or a BIP1 protein, or an insecticidally effective BIP1 portion as an active ingredient, together with suitable carriers, diluents, emulsifiers and/or dispersants (e.g., as described by Bernhard and Utz, 1993). This insecticide composition can be

formulated as a wettable powder, pellets, granules or dust or as a liquid formulation with aqueous or non-aqueous solvents as a foam, gel, suspension, concentrate, etc.. Examples of compositions comprising insecticidal bacterial spores are described in WO96/10083.

[069] A method for controlling insects, particularly Lepidoptera, in accordance with this invention can comprise applying (e.g., spraying), to a locus (area) to be protected, an insecticidal amount of the BIP1 proteins or compositions comprising the BIP1 proteins or comprising host cells transformed with the BIP1 genes of this invention. The locus to be protected can include, for example, the habitat of the insect pests or growing vegetation (e.g. application to the foliage) or an area where vegetation is to be grown (e.g. application to soil or water). In one embodiment, a composition according to the present invention comprises an insecticidal amount of at least one of the BIP1 proteins of the invention, which may be produced by a bacterial host. Such a composition may be applied to leaves, soil, or seed coating. [070] The term "contacting" is used herein to mean, "to bring into physical contact with." Contacting a plant with an insecticidal protein means that the insecticidal protein is brought into contact with cells of the plant, either internally (for example by expression in the plant) or externally (for example by applying compositions comprising the insecticidal protein externally to the plant). It is understood that the term does not indicate the length of time of contact, but comprises any period of contact. When referring to a method of protecting a plant against insect damage comprising contacting said plant (or cells or tissues thereof) with an insecticidal protein of the invention, the contact may be long enough and extensive enough (with a high enough amount of protein contacting a large enough number of cells) to prevent or reduce insect damage. [071] This invention further relates to a method for controlling Lepidopteran cotton pests, such as bollworms, budworms, and earworms. Specific Lepidopteran cotton

pests that may be controlled by the methods of the present invention include, but are not limited to, those selected from the group of Helicoverpa zea (Corn Earworm), Helicoverpa armigera (Cotton Bollworm), Helicoverpa punctigera (Native Bollworm), Heliothis virescens (Tobacco Budworm), Spodoptera frugiperda (Fall Armyworm) and Pectinophora gossypiella (Pink Bollworm). The method of controlling Lepidopteran cotton pests comprises applying to an area or plant to be protected, a BIP1 protein as defined herein, such as a BIPIa or BIPI b protein or the toxic portions thereof, all as defined herein. This may be accomplished by contacting a cotton plant with a BIP1 protein of this invention, for example by planting a cotton plant transformed with a BIP1 gene of this invention, or spraying a composition containing a BIP1 protein of this invention. The invention also relates to the use of the BIP1 proteins of this invention, against Lepidopteran cotton insect pests to minimize damage to cotton plants. [072] This invention further relates to a method for controlling Lepidopteran maize pests, such as earworms, armyworms, and com borers. Specific maize pests that may be controlled by the methods of the present invention may be selected from the group of Helicoverpa zea (Corn Earworm), Agrotis ipsilon (Black Cutworm), Ostrinia nubilalis (European Corn Borer) and Spodoptera frugiperda (Fall Armyworm). The method comprises applying to an area or plant to be protected, a BIP1 protein as defined herein, such as a BIPIa or BIPIb protein, all as defined herein. This may be accomplished by contacting a maize plant with a BIP1 protein of this invention, for example by planting a maize plant transformed with a BIP1 gene of this invention, or spraying a composition containing a BIP1 protein of this invention. The invention also relates to the use of the BIP1 proteins of this invention, against Lepidopteran maize insect pests to minimize damage to maize plants.

[073] This invention further relates to a method for controlling Lepidopteran rice pests, such as rice stemborers, rice skippers, rice cutworms, rice armyworms, rice

caseworms, and rice leaffolders. Specific rice pests that may be controlled by the methods of the present invention may be selected from the group of Yellow Stem Borer (Scirphophaga incertulas), Leaffolder (Cnaphalocrocis medinalis), Pink Stem Borer (Sesamia inferens) and Corn Spotted Stem Borer (Chilo partellus). The method comprises applying to an area or plant to be protected, a BIP1 protein as defined herein, such as a BIPIa or BIP1 B protein, all as defined herein. This may be accomplished by contacting a rice plant with a BIP1 protein of this invention, for example by planting a rice plant transformed with a BIP1 gene of this invention, or spraying a composition containing a BIP1 protein of this invention. The invention also relates to the use of the BIP1 proteins of this invention, against Lepidopteran rice insect pests to minimize damage to rice plants.

[074] This invention further relates to a method for controlling Lepidopteran soybean pests. Specific soybean pests that may be controlled by the methods of the present invention may be selected from the group of Velvet Bean Caterpillar {Anticarsia gemmatalis), Soybean Looper (Pseudoplusia includens), Beet Armyworm (Spodoptera exigua), Yellowstriped Armyworm (Spodoptera ornithogalli), Corn Earworm (Helicoverpa zea), Pod Borer (Epinotia aporema) and Rachiplusia nu. This method comprises applying to an area or plant to be protected, a BIP1 protein as defined herein, such as a B!P1a or BIPIb protein, all as defined herein. This may be accomplished by contacting a soybean plant with a BIP1 protein of this invention, for example by planting a soybean plant transformed with a BIP1 gene of this invention, or spraying a composition containing a BIP1 protein of this invention. The invention also relates to the use of the BIP1 proteins of this invention, against Lepidopteran soybean insect pests to minimize damage to soybean plants.

[075] To obtain the BIP1 toxin or protein, cells of the recombinant hosts expressing the BIP1 protein can be grown in a conventional manner on a suitable culture medium.

The produced protein can be separated and purified from the lysed cells, or when secreted, from the growth medium. If the proteins are not secreted, the cells can be lysed using conventional means such as enzyme degradation, by sonication or by using detergents or the like. The BIP protein can then be separated and purified by standard techniques such as chromatography, extraction, electrophoresis, or the like. [076] The term "gene" as used herein means any DNA or RNA fragment comprising a region (the "transcribed region") which may be transcribed into an RNA molecule (e.g., an mRNA) in a cell, operably linked to suitable regulatory regions, e.g., a plant- expressible promoter. A gene may thus comprise several operably linked fragments such as a promoter, a 5' leader sequence, a coding region, and a 3' nontranslated sequence, comprising a polyadenylation site. A gene endogenous to a particular organism (such as a plant species or a bacterial strain) is a gene, which is naturally found in that organism in nature. A "chimeric gene," when referring to a BIP1 DNA of this invention, refers to a BIP1 DNA sequence having 5' and/or 3 ¹ regulatory sequences different from the naturally-occurring bacterial 5' and/or 3' regulatory sequences, which drive the expression of the BIP1 gene in its native host cell. [077] The term "expression of a gene" when referring to the BIP1 genes of the invention, refers to the process wherein a DNA coding region which is operably linked to appropriate regulatory regions, such as to a promoter, is transcribed and translated into a protein.

[078] For the purpose of this invention the "sequence identity" of two related nucleotide or amino acid sequences, expressed as a percentage, refers to the number of positions in the two optimally aligned sequences which have identical residues (x100) divided by the number of positions compared. A gap, i.e., a position in an alignment where a residue is present in one sequence but not in the other is regarded as a position with non-identical residues. To calculate sequence identity between two

sequences for the purpose of this invention, the GAP program, which uses the

Needleman and Wunsch algorithm (1970) and which is provided by the Wisconsin

Package, Version 10.2, Genetics Computer Group (GCG), 575 Science Drive,

Madison, Wisconsin 53711 , USA, may be used. The GAP parameters used are a gap creation penalty = 50 (nucleotides) / 8 (amino acids), a gap extension penalty = 3

(nucleotides) / 2 (amino acids), and a scoring matrix "nwsgapdna" (nucleotides) or

"blosum62" (amino acids).

[079] GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimizes the number of gaps. The default parameters are a gap creation penalty = 50

(nucleotides) / 8 (proteins) and gap extension penalty = 3 (nucleotides) / 2 (proteins).

For nucleotides, the default scoring matrix used is "nwsgapdna" and for proteins the default scoring matrix is "blosum62" (Henikoff & Henikoff, 1992).

[080] These and/or other embodiments of this invention are reflected in the claims, which form part of the description of the invention.

[081] The following Examples illustrate the invention, and are not provided to limit the invention or the protection sought. The sequence listing referred to in the Examples, the Claims and the Description is as follows:

[082] SEQ ID NO: 1 : DNA coding sequence and amino acid sequence of the BIPIa gene optimized for expression in plants

[083] SEQ ID NO: 2: amino acid sequence of the BIPIa protein

[084] SEQ ID NO: 3: DNA coding sequence and amino acid sequence of the BIPIb gene optimized for expression in plants

[085] SEQ ID NO: 4: amino acid sequence of the BIPI b protein

[086] SEQ ID NO: 5: amino acid sequence of BIPIa protein with bacterial signal sequence

[087] SEQ ID NO: 6: amino acid sequence of BIPIb protein with bacterial signal sequence

[088] SEQ ID NO: 7: amino acid sequence of the OTP transit peptide fused to the BIP1A protein

[089] Unless stated otherwise in the Examples, all recombinant DNA techniques are carried out according to standard protocols as described in Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY, in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA and in Volumes I and Il of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK). Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfax (1993) by R.D.D. Croy, jointly published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications, UK. Standard materials and methods for polymerase chain reactions can be found in Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, and in McPherson at al. (2000) PCR - Basics: From Background to Bench, First Edition, Springer Verlag, Germany.

[090] It should be understood that the preceding is merely a detailed description of particular embodiments of this invention and that numerous changes to the disclosed embodiments can be made in accordance with the disclosure herein without departing from the spirit or scope of the invention. The preceding description, therefore, is not meant to limit the scope of the invention. Rather, the scope of the invention is to be determined only by the appended claims and their equivalents.

[091] EXAMPLES

[092] Example 1: production of DNA sequences encoding BIPIa and BIPIb. [093] Synthetic DNA sequences encoding the BIPIa and BIPI b proteins of the invention are made using standard DNA synthesis techniques. Codon usage is optimized for expression in cotton and other plants. The resulting sequence is shown in SEQ ID NO: 1 and 3, the encoded proteins in SEQ ID NO: 3 and 4. For optimal expression in cotton, a plant codon-optimized DNA encoding the transit peptide-BIP fusion protein of SEQ ID NO: 7 is also constructed.

[094] Using standard recombinant DNA methodology (see, e.g., Khandelwal et al., 2004b) expression of the BIP1 proteins is secured in bacterial hosts such as BL21 or WK6 cells. For expression in these bacterial hosts, the BIP1 forms of SEQ ID NO: 5 and 6 (with bacterial signal peptides) are used. SDS-PAGE analysis of transformed cell lysates shows that proteins of the expected molecular weight are produced. As negative controls cell lysate of non-transformed E. coli cells are used. [095] Protein purified from cell lysate of recombinant E. coli cultures, expressing BIPIa or BIPI b, is used in standard insect bioassays (surface contamination assays on Heliothis artificial food, which are scored after 7 days) which are repeated twice. The results on insect toxicity are summarized in Table 2 below. Plus symbols indicate significant insect toxicity over the negative control. [096] Table 2:

[097] Hz: Helicoverpa zea, Hz: Heliothis virescens,"+":significant toxicity above control -, "-": no significant (background) toxicity

[098] For the BIPIa protein and BIPIb protein, significant insect toxicity is found in surface contamination assays with Helicoverpa zea and Heliothis virescens larvae. In addition, undiluted cell lysate of recombinant E. coli cells expressing BIPIa show significant toxicity against Ostrinia nubilalis compared to the control.

[099] Similar significant toxicity results are obtained with an BIPIa and BIPIb protein substantially purified by DEAE column chromatography.

[0100] Example 2: Production of BIP1 protein in transformed plants [0101] Plant expression vectors are constructed comprising a plant-expressible promoter, operably linked to the DNA sequence encoding a BIP1 protein of SEQ ID NO: 1 or 3, and a 3' polyadenylation signal. A leader sequence, such as that from the chlorophyll a/b binding protein gene from Petunia (Harpster et al. 1988), is inserted 5' of the BIP1 DNA. For expression in cotton, the optimized sequences of SEQ ID NO: 1 and 3 are used. In other constructs, the transit peptide fusion protein of SEQ ID NO: 7 is used in the chimeric gene.

[0102] The promoters used to drive the BIP1 gene are selected from the constitutive promoters CaMV 35S (Hull and Howell, 1987), maize ubiquitin promoter, rice actin promoter and Cassava Vein Mosaic Virus promoter. As 3' transcript termination and polyadenylation signal the 3'35S, 3'nos (Depicker et al. 1982), 3'ocs or 3'gene7 are used. For Agrobacterium mediated transformation, the expression cassette is inserted into a T-DNA vector, between the right and left border sequence. [0103] Transformed cotton plants, proven to have the BIP1 chimeric gene stably inserted in their genome, show enhanced resistance to cotton insect pests such as H. zea, H. virescens and H. armigera.

[0104] This invention is not limited to the above cotton plants, transformation methods, vector constructs (promoters, 3'ends, etc.) or the particular BIP1 proteins or DNA sequences used. The invention includes variants or equivalents of the BIP1 proteins retaining insecticidal activity.

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