FR2838750A1 - New nucleic acid from Photorhabdus luminescens, useful for producing insecticidal polypeptides and insect resistant transgenic plants - Google Patents

Abstract

Isolated nucleic acid (I) sequences of 402 bp (2) and 1260 bp (4), fully defined in the specification that encode at least one polypeptide (II) active against insects, are new. Independent claims are also included for: (1) a nucleic acid sequence (Ia) that: (a) is at least 70% identical with (I); (b) hybridizes under highly stringent conditions with (I); (c) is complementary to (a) or (b); (d) is a fragment of (2) or (4), or their complements, of at least 10 bp; (e) comprises (a)-(d); or (f) is a modified form of (a)-(e), provided they encode a (II); (2) a polypeptide (II) that: (a) is encoded by (I) or (Ia), or has (a) sequence (3) or (5) of 133 and 419 amino acids (aa), respectively; or (b) has at least 80% identity with (a), or (c) is a fragment of (a) with at least 10 consecutive aa; (3) a nucleic acid (Ib) that encodes (II); (4) an expression cassette (EC) containing a promoter linked to (I), (Ia) or (Ib); (5) a cloning and/or expression vector containing EC, (I), (Ia) or (Ib); (6) a viral vector containing (I), (Ia) or (Ib) in its genome, optionally linked to an inducible or constitutive promoter; (7) host cells that contain EC; (8) producing plants that express an insecticidal toxin by introducing EC into at least one cell, then regenerating the cell to a plant; (9) plants or their parts that contain EC; (10) controlling insects by applying a composition containing (II), the cells of (7) or the vector of (5) to infected plants; (11) producing (II) by culturing cells of (7); (12) producing insect-resistant plants by introduction of (I), (Ia) or (Ib); (13) isolated nucleic acid (Ic) comprising about 60 kb from Photorhabdus luminescens, designated plBAC1A2 (CNCM I-2699), present in Escherichia coli DH10B; and (14) an isolated nucleic acid having a 60131 bp sequence (1), reproduced; and (15) strain containing the vector of (5) deposited as plbac1a2.

Description

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The invention particularly relates to isolated nucleotide sequences of Photorhabdus luminescens, toxins encoded by said sequences, processes for obtaining such toxins, compositions comprising such toxins for the control of crop pests or pests (car vectors diseases) for humans and animals, transformed plants expressing such toxins.

There is a very large number of insecticidal chemical agents for the control of insects. However, some of the existing products pose problems of selectivity against a particular category of insects and resistance against these substances.

Very many types of insects are causing serious damage to field crops, including cereals, flowers, fruit. Various alternative techniques have been developed, in particular biological control by other insects or by biological agents such as bacteria capable of producing insecticidal toxins. Transgenic plants expressing such insecticidal toxins are also known.

For example, WO 99/54472 discloses insecticidal toxins isolated from Xenorhabdus nematophilus, and nucleotide sequences encoding such toxins.

US 6,281,413 discloses insecticidal toxins isolated from Photorhabdus luminescens. Photorhabdus luminescens is an entomopathogenic, commensal intestinal bacterium of a nematode of the genus Heterorhabditis. These nematodes colonize insect larvae that they destroy and develop.

There is still a great need to find toxins that are effective against pests, especially against Diptera (especially mosquitoes) and Lepidoptera. The invention aims to obtain plants and / or compositions containing such toxins capable of inactivating or destroying insect pests of cultivated plants or harmful insects vectors of pathogens for humans, animals or plants. The invention also aims at the use of

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 such toxins, for treatment-preventive or curative- crops against pests.

For this purpose, the inventors have succeeded in identifying, using a library of BAC (artificial bacterial chromosome), prepared from Photorhabdus luminescens, nucleotide sequences coding for insecticidal toxins particularly effective against dipterans, especially the species. mosquitoes Aedes aegypti, Culex pipiens, Anopheles gambiae and Anopheles stephensie, and Lepidoptera including Plutella genus.

For this purpose, the subject of the invention is, according to a first aspect, a nucleotide sequence isolated from Photorhabdus luminescens comprising a nucleotide sequence chosen from SEQ ID No. 2 and SEQ ID No. 4, said nucleotide sequence coding for at least one active polypeptide against insects.

The sequence SEQ ID No. 2 corresponds to the nucleotides nt 8113 to nt 8514 of the sequence SEQ ID N1 which is the nucleotide sequence of a BAC, designated BAC 1A2, deposited on July 12, 2001 at the CNCM under the number 1-2699. BAC1A2 is a genomic DNA fragment of Photorhabdus luminescens subsp.laumondii strain TT01 (Fischer-Le Saux M. et al., 1998. Appl., Environ Microbiol., Vol 64, 4246), cloned into the vector pBeloBAC11 (Kim VJ et al., 1996. Genomics, vol 34, 213) in E. coli DH10B TH (Calvin NM and Hanawalt P. C, 1998, J.

Bacteriol., 170, 2796).

The sequence SEQ ID No. 4 corresponds to nucleotides No. 8544 to No. 9803 of the sequence SEQ ID No. 1 of this BAC 1 A2.

The invention also relates to the nucleotide sequences chosen from: a) a nucleotide sequence comprising at least 70% identity with
SEQ ID N 2 or SEQ ID N 4; b) a nucleotide sequence hybridizing under conditions of high stringency with SEQ ID N 2 or SEQ ID N 4; c) a nucleotide sequence complementary to a sequence defined in a) or b); d) a fragment representative of a sequence defined in a), b) or c), of at least 10, preferably at least 20 base pairs;

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 e) a nucleotide sequence comprising a sequence as defined in a), b), c) or d); f) a modified nucleotide sequence of a nucleotide sequence as defined in a), b), c), d) or e); said sequence encoding at least one polypeptide active against insects.

In the present document, the expression nucleotide sequence with insecticidal expression will be used to designate these nucleotide sequences SEQ ID N 2, SEQ ID N 4, and the variant sequences defined in a) to f).

The sequence SEQ ID No. 2 corresponds to an open reading frame designated ORF1 and codes for the 133 amino acid protein of sequence SEQ ID No. 3.

The sequence SEQ ID No. 4 corresponds to an open reading frame designated ORF2 and codes for the 419 amino acid protein of sequence SEQ ID No. 5.

The sequences SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 are clearly distinguishable from the prior art. No significant similarity has been identified, for example using the BESTFIT program known to those skilled in the art, between these sequences and the sequences SEQ ID N 12,13,14 of US 6,281,413.

Only a low similarity (always less than 50%) between the sequence SEQ ID No. 3, and the proteins encoded by the ORF 1 of the document WO99 / 54472 (SEQ ID No. 2, SEQ ID No. 5, SEQ ID No. 9, SEQ ID N 13) was noted.

Similarly, only a low similarity (always less than 50%) between the sequence SEQ ID No. 5 and the proteins encoded by ORF 2 of document WO99 / 54472 (SEQ ID No. 3, SEQ ID No. 7, SEQ ID No. 11, SEQ ID N 14) was noted.

By the term insecticidal activity or active compound against insects, it is meant that the toxins are capable of controlling insects, by killing them or by inactivating them for example by a decrease or a loss of appetite with respect to insects. cultivated plants of interest. The result is typically the destruction, or decrease in growth and / or reproduction of the target pest. By insecticide is also meant biopesticide.

The list of insects above (Diptera, in particular the mosquito species belonging to the genera Aedes, Culex, Anopheles, and Lepidoptera including Plutella) is not exhaustive: the insecticidal efficacy of the sequences according to the invention on other insects can be screened without undue effort by those skilled in the art,

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 now that the BAC 1 A2 and the biological activity test protocol are known, notably using the appropriate techniques presented in the detailed description, in particular examples 2 and 3. Similar tests, using for example another species target insect, also measure said insecticidal activity.

By nucleic acid, nucleic or nucleic acid sequence, polynucleotide, oligonucleotide, polynucleotide sequence, nucleotide sequence, terms which will be used indifferently in the present description, is meant to designate a precise sequence of nucleotides, modified or otherwise, to define a fragment or a region of a nucleic acid, with or without unnatural nucleotides, and which may correspond to both a double-stranded DNA, a single-stranded DNA and transcripts of said DNA. These nucleic acids are isolated from their natural environment, and are natural or artificial.

By "homologous nucleotide sequence" is meant any nucleotide sequence which differs from the nucleotide sequences comprising SEQ ID No. 2 or SEQ ID No. 4 (in particular which differs from the sequences SEQ ID No. 2 and SEQ ID No. 4) by substitution, deletion, and or insertion of a nucleotide or a reduced number of nucleotides, at positions such that these homologous nucleotide sequences possess an insecticidal activity as described above. Preferably, such a homologous nucleotide sequence is identical to at least 70.75% of the nucleotide sequences SEQ ID N 2 and SEQ ID N 4, preferably at least 80, 85%, more preferably at least 90.92, 95, 98.99%.

The percentage of identity between two nucleic acid sequences is intended to denote a percentage of identical nucleotides between the two sequences to be compared, obtained after their better alignment, this percentage being purely statistical and the differences between the two sequences being randomly distributed. and over their entire length. Optimal sequence alignment for comparison can be achieved using mathematical algorithms. In a preferred, nonlimiting manner, mention may be made of the various algorithms mentioned in the document Fundamentals of Database Search (review) Stephen F., Altschul, Bioinformatics: A Trends Guide, 1998: 5: 7-9, in particular Karlin's algorithms and Altschul (1990) Proc. Natl. Acad. Sci. USA 872264, modified in Karlin and Altschul (1993)

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 Proc. Natl. Acad. Sci. USA 90: 5873-5877. In particular, the programs may be used: -BLAST, in particular BLASTN, BLAST2 (Tatusova et al., 1999, "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett., 174: 247-250), gapped BLAST (Altschul et al., 1997, Nucleic Acids Res.

(17): 3389-3402), -FASTA (Altschul SF et al., 1990, J. Mol Biol., 403-410), -Clustal W (Thompson, JD et al., 1994, Nucleic Acids Res. 22, 4673-80), -BESTFIT.

The BLAST algorithm is described in detail on the NCBI site http: ww.ncbi.nih.gov.

Preferably, such a homologous nucleotide sequence hybridizes specifically to the sequence complementary to a sequence coding for an insecticidal toxin, under stringent conditions. For example, the following conditions will be used: (1) prehybridization at 42 ° C. for 3 hours in phosphate buffer (20 mM, pH 7.5) containing 5 × SSC (IX SSC corresponds to a solution of 0.15 M NaCl + 0. 015 M citrate sodium), 50% formamide, 7% sodium dodecyl sulfate (SDS), 10X Denhardt's solution, 5% dextran sulfate and 1% salmon sperm DNA; (2) actual hybridization for 20 hours at a temperature dependent on the size of the probe (for example 42 C for a probe larger than 100 nucleotides) followed by 2 washes for 20 minutes at 20 C in 2X SSC + 2% SDS, 1 wash for 20 minutes at 20 C in 0.1X SSC + 0.1% SDS. The last wash is carried out in 0.1X SSC + 0.1% SDS for 30 minutes at 60 ° C. for a probe of size greater than 100 nucleotides.

The hybridization conditions of high stringency described above can be adapted by those skilled in the art for polynucleotides of larger or smaller size, according to appropriate teachings known to those skilled in the art, in particular described in Sambrook et al. (1989, Molecular cloning, A laboratory Manual, Cold Spring Harbor), Maniatis et al., 1982 (Molecular cloning, A laboratory Manual, Cold Spring Harbor Lab., CSH, NY USA or one of its recent reissues and in Ausubel et al., Eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience, NY)

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 By nucleotide fragment, we mean on the one hand any fragment of a nucleotide sequence comprising SEQ ID N 2 or SEQ ID N 4 (in particular any fragment of SEQ ID N 2 or SEQ ID N 4), or any fragment of homologous sequences to these sequences, said fragments coding for a peptide with insecticidal activity as defined above. These nucleotide fragments have at least 10 nucleotides, preferably at least 10, 15, 30, 45, 75, 150, 300 consecutive nucleotides of the sequence from which they are derived. These nucleotide fragments encode peptides having an insecticidal activity preferably of at least 10, 20, 30, 50, 80.90% or even more than 100%, of the insecticidal activity of the sequences SEQ ID N 3 and SEQ ID N 5 encoded by the insecticidal expression sequences SEQ ID N 2 and SEQ ID N 4. To test this insecticidal activity, the skilled person has in particular the method presented in the detailed examples described below. The present document thus teaches those skilled in the art to also use the fragments or homologues defined above, in particular to use the functional elements of these sequences which determine the insecticidal expression, and therefore not to necessarily use the non-essential elements. essential to this expression.

The invention thus relates in one aspect to nucleotide sequences which comprise at least one such fragment and have insecticidal activity.

Representative fragments according to the invention also include probes or primers, which can be used in methods for detecting, identifying, assaying or amplifying nucleic sequences. These methods may involve amplification techniques of the PCR type (described for example in US Pat. No. 4,683,202) or PCR like, for example techniques known to those skilled in the art (Strand Displacement Amplification), TAS (Transcription- Based Amplification System), LCR (Ligase Chain Reaction), NASBA (Nucleic Acid Sequence Based Amplification). For the purposes of the invention, a probe or primer is defined as a single-stranded nucleic acid fragment or a denatured double-stranded fragment comprising at least 8 bases, preferably at least 10, 15, 30 bases, and having a hybridization specificity under specified conditions to form a hybridization complex with a target nucleic acid.

Such probes will in particular be usable in marker-assisted selection programs, for example to monitor the integration of the toxin gene.

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 in the transformed plant. For this at least one of these probes is labeled, for example by a radioactive isotope, and then placed in contact with genomic DNA of the plant, previously digested with restriction enzymes, under conditions allowing the specific hybridization of the labeled probe to the DNA in question.

By modified nucleotide sequence is meant any nucleotide sequence typically obtained by mutagenesis according to appropriate techniques, and comprising modifications with respect to the nucleotide sequences SEQ ID N 2 and SEQ ID N 4, or the variant sequences (nucleotide sequences defined a) to e ) previously). These modifications may result, according to a preferred variant, in an increase in the level of insecticidal expression. For example, site-specific site-specific mutagenesis, described in Upender et al., 1995, Biotechniques 18 (1): 29-30, or DNA shuffling mutagenesis techniques described in US 5,605,793 may be used. for example obtain mutated sequences of SEQ ID N 2 and SEQ ID N 4, by deletions, and screen for active mutants to identify regions all particularly associated with the level of insecticidal activity.

According to another aspect, the invention relates to an insecticidal polypeptide encoded by a nucleotide sequence with insecticidal expression as described above.

The invention also relates to the polypeptides chosen from: a) a polypeptide of sequence SEQ ID No. 3 or SEQ ID No. 5; b) a polypeptide homologous to the polypeptide defined in a) comprising at least
80%, preferably at least 85%, 87%, 90%, 95%, 97% 98%, 99% identity with said polypeptide of a); c) a fragment of at least 10 consecutive amino acids, preferably at least 15,20, 23,25, 30,40, 50,100 consecutive amino acids of a polypeptide defined in a), or b); d) a polypeptide variant polypeptide of amino acid sequences defined in a), b), or c); said polypeptide having insecticidal activity as previously described.

In this text, the term "insecticide polypeptide" or "insecticidal toxin" is used interchangeably to denote a polypeptide according to the invention having an insecticidal activity as described above. The term polypeptide is also used to refer to a protein or a peptide.

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Preferably a polypeptide according to the invention is a polypeptide consisting of the sequence SEQ ID No. 3 or SEQ ID No. 5 or of a sequence having at least 80% identity, preferably at least 85%, 90%, 95% , 98% and 99% identity with SEQ ID N 3 or SEQ ID N 5 after optimal alignment. A polypeptide whose amino acid sequence has an identity percentage of at least 80%, preferably at least 85%, 90%, 95%, 98% and 99% after optimal alignment with a reference sequence is meant polypeptides having certain modifications relative to the reference polypeptide, such as in particular one or more deletions, truncations, an elongation, a chimeric fusion, and / or one or more substitutions.

By polypeptide fragment is meant a polypeptide comprising at least 10 consecutive amino acids, preferably at least 15,20, 25,30, 40,50, 100 consecutive amino acids. The polypeptide fragments according to the invention obtained by cleavage of said polypeptide by a proteolytic enzyme, by a chemical reagent, or by placing said polypeptide in a highly acidic environment also form part of the invention.

In the case of a substitution, one or more consecutive or non-consecutive amino acids may be replaced by equivalent amino acids.

The expression "equivalent amino acid" is intended here to designate any amino acid that may be substituted for one of the amino acids of the basic structure without, however, modifying the essential characteristics or essential functional properties desired, in this case not entailing a loss of insecticidal activity. These equivalent amino acids can be determined either by relying on their structural homology with the amino acids to which they are substituted, or on the results of cross-biological activity tests to which the various polypeptides are likely to give rise. By way of example, mention may be made of the possibilities of substitutions that may be carried out without resulting in a thorough modification of the biological activities of the corresponding modified polypeptides, the replacements, for example, of leucine by valine or isoleucine. , aspartic acid with glutamic acid, glutamine with asparagine, arginine with lysine, etc., the reverse substitutions being naturally conceivable under the same conditions. By variant polypeptide is meant all the polypeptides

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 mutations which may exist naturally, and which correspond in particular to truncations, substitutions, deletions and / or additions of amino acid residues. A variant polypeptide, a homologous polypeptide or a polypeptide fragment according to the invention has at least 10%, preferably at least 20.30, 50, 80, 90% or even more than 100.120, 150% of the insecticidal activity. sequences SEQ ID N 3 and SEQ ID N 5.

The invention also relates to the nucleotide sequences encoding the polypeptides as described above.

In this document, isolated and purified expressions refer to a level of purity achievable using current knowledge of those skilled in the art.

The molecules according to the invention need not be absolutely pure (that is to say containing absolutely no molecules other than cellular macromolecules), but should be sufficiently pure so that those skilled in the art will recognize that they are no longer present in the environment in which they were initially found (the cellular environment).

According to another aspect, the invention relates to an expression cassette comprising a promoter sequence operably linked in the transformed plant to a nucleotide sequence according to the invention coding for an insecticidal toxin, and to a transcription termination region. The preparation of a DNA expression cassette can be performed in a variety of appropriate ways by those skilled in the art.

For example, at least one nucleotide sequence with insecticidal expression as defined above, can be cloned downstream of the promoter using restriction enzymes to ensure its insertion in a suitable orientation to the promoter so that it is expressed. Once this sequence of interest is operatively linked to the promoter, the expression cassette thus formed can be cloned into a plasmid or other vector. The promoter sequence controls transcription into a functional messenger RNA encoding an insecticidal protein.

The expression cassette may be chimeric or natural, but typically it is heterologous to the host, which involves introduction into the host by transformation. A large number of plant promoters are known, mentioned in particular in document W001 / 70778. A constitutive promoter can be used

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 or inducible, a specific promoter of a tissue, an organ, a stage of development, for example specific to the phloem in order to fight against insects that take the elaborated sap.

According to one embodiment, the expression of the gene of interest is regulated, in addition to the promoter sequence, by the use of appropriate sequences capable of enhancing the activity of this promoter sequence, such as introns, for example intron of actin 1 or 2, the DSV intron of the tobacco yellow mosaic (Morris et al.

(1992), Virology, 187: 633), enhancer sequences, e.g. certain elements of the CaMV35S promoter and octopine synthase genes (US 5,290,924), leader sequences (typically sequences located between the site of initiation of transcription and the beginning of the coding sequence, particularly consensus leader sequences that stabilize the mRNA and prevent inappropriate transcription initiation), e.g., the EMCV leader (Leroy-Stein et al., 1989, PNAS USA , 86: 6126-6130), the TMV leader (Gallie et al., 1989, Molecular Biology of RNA, pages 237-256). Among the terminator sequences are the nos terminator (Bevan et al., 1983, Nucleic Acids Res., 11 (2), and the histone gene terminator (EPO 633 317).

According to one variant, the expression cassette contains subcellular addressing sequences, in particular a sequence coding for a signal peptide allowing the secretion of the protein, or a sequence coding for a transit peptide that sends the toxin into the chloroplasts (by way of example). example the optimized transit peptide described in EP 0 508 909).

According to another aspect the invention relates to a cloning and / or expression vector comprising an expression cassette as described above. According to one embodiment, the vector is a virus type and comprises in its genome a polynucleotide sequence according to the invention, operably linked to an appropriate inducible or constitutive promoter.

According to another aspect the invention relates to a host cell transformed with the nucleic sequences described above.

According to another aspect, the invention relates to a host cell comprising such an expression cassette. Typically the host organism is a bacterium, a yeast, a plant cell, a fungus.

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 According to another aspect, the invention relates to a method for preparing a polypeptide using at least one vector or a cell as described above. The invention also relates to a recombinant polypeptide obtainable by this method.

According to another aspect the invention relates to a process for preparing a synthetic polypeptide using an amino acid sequence of an insecticidal polypeptide as defined above.

According to another aspect the invention relates to the use of a nucleotide sequence, a vector, a host cell as described above for the biosynthesis of insecticidal polypeptides. The term, of course, refers to at least one.

According to another aspect the invention relates to the nucleotide sequences according to the invention, recorded on a support, called recording medium, whose shape and nature facilitate the reading, analysis and exploitation of said sequences.

Computer-readable media such as magnetic, optical, electrical, or hybrid media such as floppy disks or disks, CD ROMs, or recording cassettes may be preferred among these media.

According to another aspect, the invention relates to plant cells transformed with a vector as defined above, with the aid of such a cellular host capable of infecting said plant cells by allowing integration into the genome of said cells, nucleotide promoter sequences initially contained in the genome of the vector used.

The transformation of the plants can be obtained by various appropriate techniques known to those skilled in the art, in particular referred to in the references: Ed. DUNOD Plant Physiology, Volume 2 Development Heller, Esnault and Lance, 2000, Methods of Molecular Biology: Plantgene transfer and Expression protocols, Hansen et al., 1999, Recent advances in the transformation of plants, Trends in plant science, Vol 4, No. 6, 226, SBGelvin, The introduction and expression of transgenes in plants, Current opinion in biotechnology, 1998, 227, Hellens et al., 2000, Trends in plant science, Vol 5, No. 10, A guide to Agrobacterium binary Ti Vectors, 446, Franken et al., Recombinant proteins from transgenic plants, Current opinion in biotechnology, 8: 411- 416, et al., 1998, Insect-resistant

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 transgenic plants, Tibtech, vol 16,168, Michelmore. M, 2000, Genomic approaches to plant disease resistance, Current opinion in biotechnology, 3: 125-131.

The term transformation refers to a genetic manipulation of plant cells that can be transformed such as callus cells, embryos, cells suspended in cultures (cultures derived from, for example, calli, embryos, leaves, young inflorescences, anther) plants. The term "transgenic" or "transformed" with reference to a plant cell, a plant part, refers to those elements comprising a DNA segment (preferably a nucleotide sequence or an expression cassette according to the invention purified isolated pre-selected which has been introduced into said element by a method of genetic transformation.

These include: protoplast transformation, biolistic or microprojectile bombardment techniques, transformation using bacteria including Agrobacterium or viral vectors, directly in planta processes.

In the case of protoplast transformation, protoplasts are typically isolated, either mechanically or enzymatically to separate cell walls. Protoplasts are typically obtained from callus cell lines obtained from immature embryos, immature inflorescences, mesocotyls, and anthers. Protoplasts can be transformed by Agrobacterium or by direct insertion of DNA facilitated by polyethylene glycol treatment or electroporation (Lazzeri, Methods Mol Biol., 49: 95-106, 1995). In some species, protoplasts can be isolated from mesophylls, a method that provides results independent of the genotype.

Among the biolistic methods recalled in Finer et al., T. Particle bombardment mediated transformation, Current Topics in Microbiology and Immunology, Vol.

240, can be used in particular the barrel bombardment of particles coated with the plasmid DNA of interest, in particular using a tungsten or gold particle gun.

Biolistic methods may be preferred to agrolistic methods described in Hansen et al. Agrolistic transformation of plant cells: integration of T-strands

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 generated in planta. Proc Natl Acad Sci USA 1996, 93: 14978-14983, in particular in order to integrate a low copy number of the transgene.

Many transformation techniques using Agrobacterium are useful, such as those described in Zupan, J. et al. (2000) The transfer of DNA from Agrobacterium tumefaciens into plants: a feast of fundamental insights. Plant J. 23: 11-28, US 6 037 522, US 6,265,538. The Agrobacterium tumefaciens transformation can be carried out for Dicotyledonous (tobacco, potato) and Monocotyledonous (wheat, barley, sorghum, maize, rice). especially) even if they are not usually their natural host. In particular, known techniques will be used for wheat (McCormac et al., Euphytica, vol 99 (1): 17-25,1998), maize (Ishidia et al., 1996, Nat Biotechnol., 14 (6): 745 -750.1996).

In addition, since gene integration occurs primarily at random in the genome, there is often variability among transgenic plants.

Transformants comprising a single copy of the transgene, segregated as a Mendelian character with a uniform expression from one generation to the next will be sought to obtain. Stable expression of the transgene with a desired level of expression is thus sought.

Those skilled in the art know a large number of plasmids usable in the context of the invention, recalled in particular in the document Trends in plant science oct 2000 vol 5 n 10 p 446, and adapt the transformation conditions to the plasmid used. For example, it is possible to use, in a preferred manner, certain binary Ti plasmids, such as pBIN19, pMON, pGREEN, the accessible sequence of which makes it possible to define precise restriction maps.

The so-called in planta transformation, described in particular in Bechtold et al., 1993, In planta Agrobacterium-mediated gene transfer by infiltration of adult Arabidopsis thaliana plants, C.R. Acad. Sci, Paris, Life Sciences 316, 1194-1199, and Clough et al., 1998, Floral dip: simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana, Plant J. 16,735-743, has the advantage of not requiring any step of tissue culture. The transgene is introduced into intact plants as naked DNA or Agrobacterium, usually at the zygote stage.

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 Techniques and agents for selecting plant cells and / or plant tissues incorporating marker nucleotide sequences associated with the gene of interest are also well known to those skilled in the art, and include, but are not limited to, the use of marker genes such as genes conferring resistance to an antibiotic or to herbicides, or positive selection systems, cited for example in Gelvin 1998, in particular the system based on selection on mannose, in the presence of the gene for selection of the MPI (Mannose-6 Phosphate Isomerase) (Hansen and Wright, 1999), or selection systems coupled to the elimination of marker genes after selection (Ebinuma et al., 1997). Finally, the transformed plants can also be screened by PCR screening in the absence of selection marker genes (McGarvey and Kaper, 1991).

According to another aspect the invention relates to a process for obtaining a plant expressing at least one insecticidal toxin according to the invention in its tissues, comprising the introduction of an expression cassette comprising a nucleotide sequence with an insecticidal expression such as than previously described in at least one plant cell, then the culture of the cell thus transformed so as to regenerate a plant containing in its genome said expression cassette. Said cassette is stably integrated into the genome.

In one embodiment the method further comprises identifying and selecting transformed cells capable of regenerating plants expressing insecticidal toxins relative to an untransformed plant.

The selection of transformation products, i.e. resulting immediately from the transformation process, and the resulting transgenic plants, can be achieved in different ways. Plant culture growth techniques are known to those skilled in the art, for example in Mcormick, 1986, Plant Cell Reports, 5, 80-84. The recombinant DNA comprising at least one nucleotide sequence according to the invention is transmitted during a reproductive cycle of the transgenic plant to its offspring so as to be expressed in the plants of the offspring. The invention therefore also relates to the tissues or parts of plants, plants, or seeds containing the nucleic acid sequences according to the invention described above. The term "plant tissue" refers to any tissue of a plant, plant or culture. This term includes plants

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 whole, plant cells, plant organs, plant seeds, protoplasts, calli, cell cultures and any other plant cells organized as a functional and / or structural unit. Parts of regenerated plants such as flowers, seeds, leaves, stems, fruits, pollen, tubers and the like are also within the scope of the invention.

The tissues, parts of plants, plants or seeds, are resistant to insects.

The invention includes the resulting fertile transgenic plants as well as their offspring and the product of this offspring. Transgenic hybrid plants, obtained by crossing at least one plant according to the invention with another, also form part of the invention. The transgenic plants according to the invention comprise in particular a transgenic plant TO or RO, that is to say the first plant generated from transformed plant cells, the transgenic plant T1 or R1, that is to say the first one. generation of offspring, and subsequent offspring plants of subsequent generations that understand and express the recombinant DNA. For example, one will proceed according to the following steps: - obtaining of parent plants of a first line, and a second line (donor line comprising a transgene according to the invention) - pollination of flowers of the first parent by the pollen of the second parent - harvesting seeds produced by the first parent.

Backcrossing can be performed to introgress the insecticidal DNA of interest.

To confirm the presence of the expression cassette, and in particular the toxin gene of interest according to the invention in the regenerated plants, a large number of suitable techniques may be used, for example PCR analysis or assay techniques. Southern blot hybridization, to determine the structure of the recombinant DNA, the detection of RNA transcribed from the DNA of the gene of interest expressed in transformed plant cells, techniques for tracking the production of proteins encoded by the gene of interest, such as gel electrophoresis, Western blot techniques.

The plants transformed according to the methods described may be monocotyledons or dicotyledons, in particular maize, wheat, barley, sorghum, potato, pea, soya, tobacco, rapeseed, tomato, sunflower, cotton,

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 rice, beans, cauliflower, broccoli, lettuce, radish, celery, spinach, onion, garlic, carrot, apple, pear, melon, cherry, peach, apricot, raspberry, strawberry, pineapple, avocado, banana, sugar cane.

Those skilled in the art will further be able to increase the level of production by transformed plants by increasing the GC base content of the coding sequences. In addition, those skilled in the art will use optionally modified sequences to obtain an appropriate expression according to whether they are monocotyledonous or dicotyledonous plants.

According to another aspect, the subject of the invention is a composition for the control of harmful insects as well as processes using such compositions, these compositions comprising at least one insecticidal toxin or insecticidal compound according to the invention as described above. .

By insecticidal composition is meant a phytosanitary or pharmaceutical composition comprising as active ingredient an effective amount of insecticidal toxin according to the invention. In use for the protection of cultures, the compositions will typically be in association with a solid or liquid, agriculturally acceptable carrier and / or an agriculturally acceptable surfactant. In the pharmaceutical field, mosquitoes being disease vectors for humans and animals, the pharmaceutical compositions will comprise the appropriate pharmaceutically acceptable carriers associated with the insecticidal toxin. Such carriers are known to those skilled in the art, for example described in US 5,877,322.

Within the various insecticide compositions for crop protection according to the present invention, the insecticidal compound is used in effective but non-phytotoxic amounts. By effective amount and non-phytotoxic means an amount of active material sufficient to allow control or destruction of insects present or likely to appear on crops, and causing for said crops no significant symptoms of phytotoxicity. Such an amount is likely to vary within wide limits depending on the insect to be combated, the type of culture, the climatic conditions, and the compounds included in the insecticidal composition according to the invention. This quantity can

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 easily be determined by systematic field trials, within the reach of the skilled person.

In one embodiment, an insecticidal composition comprises at least one host cell as described above.

For their use in practice, the different insecticidal compositions according to the invention are typically associated with a support, solid or liquid, which can be used in the agricultural field, and optionally at least one surfactant and / or one or more agents. auxiliary.

In particular, as supports, are used the inert and usual supports; as surfactants, the usual surfactants in the field of the formulation of compositions for use in agriculture, especially for the treatment or protection of crops, can be used.

The insecticidal compositions according to the invention typically comprise between 0.00001 and 100%, preferably between 0.001 and 80%, of insecticidal compound. The proportions and percentages used or described in this text are proportions or percentages by weight.

In the present description, the term "support" denotes an organic or inorganic material, natural or synthetic, with which the active ingredient is associated to facilitate its application, in particular on the plant, or on seeds or on the soil. This support is therefore generally inert and it must be acceptable in agriculture, especially by the treated plant. The support optionally used for the formulation of compositions according to the invention may be solid or liquid.

As examples of solid supports that can be used for the formulation of compositions according to the invention, mention may be made of natural or synthetic silicates, resins, waxes, fine powders or clay granules, in particular of kaolin clay, diatoms, bentonite or acidic clay, synthetic hydrated silicon oxide, talcs, ceramics, other minerals including sericite, quartz, sulfur, activated carbon, calcium carbonate, hydrated silica or industrial fertilizers such as ammonium sulphate, ammonium phosphate, ammonium nitrate, urea or ammonium chloride.

As examples of liquid carriers that can be used for the formulation of compositions according to the invention, mention may be made of water, alcohols and especially the

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 methanol or ethanol, ketones and especially acetone, methyl ethyl ketone or cyclohexanone, petroleum fractions, aromatic hydrocarbons including benzene, toluene, xylene, ethylbenzene or methylnaphthalene, non-aromatic hydrocarbons including hexane, cyclohexane, kerosene or gas oil, liquefied gas, esters including ethyl acetate and butyl acetate, nitriles including acetonitrile and isobutyronitrile, ethers including ether diisopropyl or dioxane, amides including N, N-dimethylformamide or N, N-dimethylacetamide, halogenated hydrocarbons including dichloromethane, trichloroethane or carbon tetrachloride, dimethylsulfoxide, vegetable oils including soybean oil or cotton oil.

The surfactant may be an emulsifying, dispersing or wetting agent of ionic or nonionic type. For example, mention may be made of polyacrylic acid salts, lignosulfonic acid salts, phenolsulphonic or naphthalenesulphonic acid salts, polycondensates of ethylene oxide with fatty alcohols or with fatty acids or with fatty amines, substituted phenols, in particular alkylphenols or arylphenols, salts of sulphosuccinic acid esters, taurine derivatives, in particular alkyltaurates, phosphoric esters of polyoxyethylated alcohols or phenols; mention may be made especially of alkylsulfonate salts, alkylarylsulfonates, alkylaryl ethers, their polyoxyethylene derivatives, polyethylene glycol ethers, polyalcohol esters, sugar derivatives, alcohols and others.

The presence of at least one surfactant is generally appropriate when at least one of the active substances and / or the inert support are not soluble, especially in water, in the case where the application vector agent is 'water.

The insecticidal compositions according to the invention may also contain any kind of other ingredients or agents such as, for example, protective colloids, adhesives, thickeners, thixotropic agents, penetrating agents, stabilizing agents including phosphate isopropyl acid, 2,6-ditert-butyl-4-methylphenol, 2-tert-butyl-4-methoxyphenol and 3-tert-butyl-4-methoxyphenol, vegetable or mineral oils, fatty acids or their esters , sequestering agents, dispersing agents including casein, gelatin,

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 saccharides and especially starch powder, gum arabic, certain cellulose derivatives or alginic acid, lignin derivatives, bentonite, water-soluble synthetic polymers, in particular polyvinyl alcohol, polyvinylpyrrolidone polyacrylic acids, etc., as well as other active ingredients known for their pesticidal properties, especially insecticides or fungicides; or for their properties promoting the growth of plants, especially fertilizers; or for their regulating properties of the growth of plants or insects.

More generally the insecticidal compound can be combined with any solid or liquid additives corresponding to the usual techniques of formulation, particularly the formulation of products or compositions intended for uses or uses in agriculture or public hygiene.

Thus, the compositions according to the invention can take the form of quite a number of formulations among which mention may be made of oily solutions, emulsifiable concentrates, wettable powders, fluid formulations and in particular aqueous suspensions or aqueous emulsions, granules, powders, aerosols, nebulizing formulations, especially misting formulations, very low volume formulations, pastes, emulsions, concentrated suspensions, as well as any mixtures, combinations or combinations of these different forms.

In most cases, for formulations of the dust or dispersion powder type, the content of insecticidal compound may be up to 100%; likewise, for formulations in the form of granules, in particular those obtained by extrusion, by compacting, by impregnation of a granulated support, by granulation from a powder, the content of insecticidal compound in these granules is most often included between 0.5 and 80%.

The insecticidal compositions according to the invention, referred to as concentrated compositions, comprising an insecticidal compound which are in the form of emulsifiable or soluble concentrates, generally comprise from 25 to 100% of active ingredients, the emulsions or solutions ready for application containing, as to them, from 0.00001 to 20% of active ingredients.

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 According to one embodiment, the active ingredient A is combined, where appropriate, with at least one other active ingredient or insecticidal compound derived from Photorhabdus luminescens or other microorganisms.

In addition to the solvent, the emulsifiable concentrates may contain, when necessary, 2 to 20% of suitable additives such as stabilizing agents, surfactants, penetrating agents, corrosion inhibitors, dyes or adhesives previously cited.

The insecticidal compositions according to the invention in the form of concentrated suspensions, also applicable in spraying, are prepared in such a way as to obtain a fluid, stable product which does not settle; they usually contain from 2 to 75% of active ingredient, from 0.5 to 15% of surfactants, from 0.1 to 10% of thixotropic agents, from 0 to 10% of suitable additives, such as foam, corrosion inhibiting agents, stabilizing agents, penetrating agents and adhesives; and, as carrier, water or an organic liquid in which the or active ingredient is little or not soluble, or mixtures of several of these solvents, organic or otherwise.

Some solid organic materials or mineral salts may be dissolved in the support to inhibit or prevent sedimentation; or even such materials can be used as antifreezes for water.

The insecticidal compositions according to the invention which take the form of wettable or sprayable powders are usually prepared so that they contain from 20 to 95% of active ingredients.

Furthermore, they usually contain, in addition to a solid support, from 0 to 5% of a wetting agent, from 3 to 10% of a dispersing agent, and, where appropriate, from 0 to 10% of one or more stabilizing agents and / or other additives, such as penetrating agents, adhesives, anti-caking agents, dyes, etc.

In order to obtain these spraying powders or wettable powders, the active ingredient (s) are intimately mixed in appropriate mixers with the additional substances, and they are milled with suitable mills or other grinders. Spray powders are then obtained, the wettability and suspension of which are particularly advantageous.

They can be suspended with water at any desired concentration.

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Rather than wettable powders, it is possible to produce insecticidal compositions according to the invention which are in the form of pastes.

The conditions and methods for producing and using these pastes are similar to those of the wettable or sprayable powders.

As has already been said, aqueous dispersions and emulsions, for example the insecticidal compositions obtained by diluting with water a wettable powder or an emulsifiable concentrate according to the invention, are included in the general scope of the present invention. .

The emulsions may be of the water-in-oil or oil-in-water type and may have a thick or fairly thick consistency.

More generally, the compositions according to the invention can take many forms of formulations; thus, these compositions comprising an insecticidal compound can be used as an aerosol generator; bait (ready to use); concentrate for bait preparation; bait in stoc; suspension of capsules; product for cold fogging; powder for dusting; emulsifiable concentrate; emulsion of aqueous / aqueous type; oily / inverse type emulsion; encapsulated pellet; fine granulated; concentrated for seed treatment; compressed gas; gas generator product; on grain; granulated bait; granulated; product for hot nebulization; macrogranulated; microgranulated; powder dispersed in oil; concentrated suspension dilutable in oil; liquid miscible in oil; dough ; rod for agropharmaceutical use; plate bait; powder for dry seed treatment; bait on broken pieces; treated or coated seed; smoke candle; smoke cartridge; smoke; smoke granulate; smoke stick; smoke tablet; smoke box; soluble concentrate; soluble powder; seed treatment liquid; concentrated suspension (= flowable concentrate); track; liquid for very low volume application; suspension for very low volume application; vapor diffuser product; granules or tablets to be dispersed in water; wettable powder for wet treatment; granules or tablets soluble in water; soluble powder for seed treatment; wettable powder.

Numerous examples (in particular A to E) of insecticidal compositions described in document FR 2 805 971 can be used with the toxins according to the invention.

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 Furthermore, the invention relates to a method of controlling insects comprising applying to plants infected with said insects an insecticidally effective amount of an insecticidal composition as described above or recombinant vectors as described. previously.

The invention also relates to a process for obtaining insecticidal toxins comprising: - obtaining at least one host cell as described above, - culturing under conditions suitable for the expression of a nucleotide sequence according to the invention. invention leading to the production of at least one active toxin against insects; - collection of toxins produced.

The invention also relates to a strain comprising a vector as described above, deposited at the CNCM under the reference plbac 1 a2 CNCM 1-2699.

Other advantages of the invention will become apparent in the detailed description which follows.

The global approach used to obtain a BAC library then tested for their entomotoxic activity is described, followed by the procedures in detail.

Initially, the entire genome sequence of the entomopathogenic bacterium Photorhabdus luminescens strain TT01 was identified (with the exception of two regions of ambiguous sequences). Several libraries of genomic DNA fragments were constructed according to methods described (L. Frangeul et al., 1999. Microbiology 145, 2625-2634) and introduced by transformation into Escherichia coli DH10B: a small library (1 to 2 kb), a medium size (5 to 20 kb) and a BAC library containing large fragments (50 kb on average). The chosen "shotgun" approach involves the sequencing of small random fragments. These fragments were then assembled using Phred and Phrap software (developed by P. Green, University of Washington) to obtain contiguous sequences (contigs). The sequenced ends of medium sized library fragments and those of the BAC library, and the sequencing of combinatorial PCR products, made it possible to link these

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 contiguous sequences and obtain the genomic sequence, in the form of a contig of 5.7 Mb. To identify BACs with insecticidal activity, the inventors proceeded in parallel to two methods.

According to a first method, the inventors have identified by research of similarity "in silico", from the genomic sequences, genes potentially responsible for an entomotoxic activity.

In a second method, the BAC library containing Photorhabdus DNA fragments was completely screened to identify Escherichia coli clones with insecticidal activity. This library, constructed using the vector pBeloBACl 1, was deposited (CNCM No. 1-2478) on July 12, 2001.

The inventors have thus succeeded in identifying a clone, designated BAC1A2, having an entomotoxic activity.

The procedures used for the sequencing and the analysis of the genome of Photorhabdus luminescens, in particular BAC1A2 (EXAMPLE 1), and the entomopathogenic activity tests on larvae, in particular those of Aedes, are described in detail (EXAMPLE 2), Culex (EXAMPLE 3), Plutella (EXAMPLE 4).

EXAMPLE 1: Sequencing and analysis of the genome of Photorhabdus luminescens The inventors used the DNA of strain TT01 of Photorhabdus luminescens to begin sequencing its genome whose size is 5.7 million base pairs. Different genomic DNA libraries of Photorhabdus luminescens have been constructed for these purposes. From clones of these different libraries 65,000 reads of sequences of about 500 base pairs of quality were performed - corresponding to a coverage of 7 times the length of the genome - thus covering about 98-99% of this genome with non-redundant sequences.

Assembling these sequences using computer tools (Ewing B. et al., 1998. Genome Res., 8, 175-185; Ewing B. & Green P. 1998. Genome Res., 8, 186-194; et al., 1998. Genome Res.8, 195-202, Contigs-Tool-Box, L. Frangeul et al., Unpublished Results, Genomic Laboratory for Pathogenic Microorganisms, Institut Pasteur, Paris) yielded about 450 contigs (contiguous sequences), the ultimate goal being to connect these contigs to obtain a contig

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 of 5,681,324 base pairs corresponding to the total genomic sequence of Photorhabdus luminescens.

The contigs obtained were connected using various techniques briefly summarized below: - Using low copy number vectors was constructed a "scaffold" covering the entire genome. In order to achieve this, the inventors have used genomic DNA libraries of Photorhabdus luminescens, in particular a library constructed using BAC vectors (one copy per cell), the average sizes of the inserted DNA fragments being about 50 kb. Sequencing the ends of these fragments allows the prediction of links between adjacent contigs.

The comparison of the ends of the contigs obtained with the "control" genome of a phylogenetically close bacterium (Escherichia coli), the sequence of which is known, was used to predict links between adjacent contigs in the studied genome (Photorhabdus).

- Different PCR techniques (combinatorial PCR, reverse PCR, the "walk on the chromosome" technique) were then used to fill the gaps between adjacent contigs.

The genomic DNA library was obtained in the bacterial vector (pBeloBAC11, California Institute of Technology) according to the following steps: Step 1: bacterial culture and plugging - Inoculation of 10 ml of peptone water in the evening with a single colony Photorhabdus luminescens strain TT01; - Incubate overnight with agitation at 37 C; - Measurement of optical density at 600 nm; - Take a culture volume containing 2,109 bacteria knowing that one unit of OD equals 5,110 bacteria / ml; - Equilibrate each tube by adding sterile peptone water to obtain a final volume of 10 ml; - Centrifuge the bacteria at 4 C for 20 min at 4000 rpm; - Remove the supernatant and resuspend the pellet in 5 ml of washing buffer (1 M NaCl, 1 OmM Tris-HCl pH = 7.6);

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Centrifuge the bacteria at 4 C for 20 min at 4000 rpm; - Discard the supernatant and take the pellet in 1 ml of TE IX; - Melt the low melting point agarose and store at 55 ° C; - Prepare the molds for making the caps by closing one side with parafilm and putting them on ice; - Mix in a 2 ml eppendorf tube, 1 ml of the pellet taken up in 1 ml of TE IX and 1 ml of the agarose low melting point at 55 C; - Well homogenize the mixture and deposit 100 [il per mold. Leave the caps 10 minutes on the ice; - Prepare the Proteinase K solution. For a cap strip, prepare 5 ml of 0.5M EDTA / 0.5% N-lauryl sarcosine solution with 2 mg / ml Proteinase K in a 30 ml falcon tube (Proteinase K should be added just before use); Overnight incubation at 55 ° C without agitation in a dry bath or a water bath; - Remove the tubes containing the corks from the oven and place them for 15 minutes in the ice; - Open the tube and put the drip tray to remove Proteinase K solution without losing the caps; - Prepare the PMSF (Phenyl Methyl Sulfonyl Fluoride) solution by adding 10 l PMSF / Isopropanol (thawed for 30 min at 55 ° C.) in 10 ml TE IX (prepared just before use because the PMSF rapidly loses its activity in aqueous solution ); - Add 5 ml of the PMSF / TE IX solution per tube and incubate for 30 min at 55 ° C .; - Place the caps for 15 minutes on the ice; - Open the tube and put the drip tray to remove the PMSF / TE IX solution; - Wash the caps by adding 10 ml of TE IX for 20 min at room temperature; - Open the tube and put the drip tray to eliminate the TE IX without losing the plugs; - Wash the caps again 2 times in the same way with TE IX; - To preserve the caps, empty the tubes and add 10 ml of ETDA 0.5 M pH 8; conservation at 4 C for several months;

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 If the caps are used immediately for the restriction, they are stored in the TE IX solution at 4 ° C. overnight.

Step 2: partial digestion of the DNA in pieces of agarose - the agarose pieces are washed three times for 15 minutes at room temperature in a solution of TE (1x) with moderate stirring; the agarose pieces are equilibrated twice in 300 μl of a Hind III digestion buffer (1x) (Boehringer or Biolabs) for 30 minutes at room temperature; the digestion buffer is removed and an ice-cold Hind III digestion buffer (1 ml per piece of agarose) containing 20 U of Hind III (Boehringer or Biolabs) is added; - incubate for two hours in ice; the pieces of agarose are transferred into a 37 C waterbath and incubated for 10 minutes to 30 minutes (depending on the DNA contained in the agarose pieces); and the digestion is stopped by the addition of 100 μl of a 250 mM EDTA solution (pH 8).

Step 3: Size
Separation of partially digested DNA made by PFGE using CHEF DRIII (Bio-Rad) apparatus: - 1% LMP agarose gel in Tris-acetate-EDTA buffer (1x) at 13 C; - reverse the current every 3 to 15 seconds at 6 V / cm for 16 hours; - load at least two pieces of agarose and the marker; - color the marker and a line or part of the line to check the partial digestion; - excise strips (non-colored part) of different sizes (ie 50 to 100 kb and 150 to 250 kb); the agarose strips can be stored at 4 ° C. in a solution of TE.

Step 4: Preparation of the vector

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 isolation of pBeloBACl 11 by the cesium chloride method; digestion with Hind III; - dephosphorylation with CIP (Calf Intestine Phosphatase).

Step 5: Ligation and transformation - the agarose libraries containing the DNA are melted at 60 ° C for 10 minutes; the molten agarose / DNA solution is equilibrated for 15 minutes at 45 ° C .; gelase (Epicenter Technologies) is added at the rate of 1 U per 100 l of gel strip (do not add a digestion buffer which causes certain problems at the time of ligation); the digestion is carried out for 1 hour at 45 ° C .; The ligation is carried out in 50 l of a solution containing pBeloBACII (2 l), T4 ligase (1 l at 1:10), a 10x T4 buffer (5 ul), and the DNA / agarose solution (42). 1), incubation for 20 hours at 16 ° C .; the ligation medium is heated for 15 minutes at 65 ° C .; the ligation medium was dialysed against a Tris-EDTA buffer using Millipore membranes of 0.025 mM pore size VS; - 1 or 2 l of the ligation solution are introduced by electroporation into E. coli DH10B cells (Gibco BRL) using 2 mm wide electroporation cuvettes with the following settings 2.5 kV, 25 F and 200 Q ; after electroporation, the cells are resuspended in 600 μl of SOC or NYZ medium and then incubated for 45 minutes at 37 ° C. with shaking; 10 and 100 μl of each cell suspension are spread in LB agar medium containing chloramphenicol (12.5 μg / ml), X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside; 50 μg / ml) and IPTG (isopropyl-β-D-thiogalactopyranoside, 25 g / ml); and the boxes thus obtained are incubated overnight.

 the size of the average insert is of the order of 50 kb.

 Step 6: Plasmid DNA and sequencing inserts.

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Extraction of the plasmid DNA was performed by the alkaline lysis technique in 24-well plates.

Sequencing of the inserts of the clones was carried out by the PE Big Dye KIT according to the conditions recommended by the manufacturer using the oligonucleotides specific for each vector.

The reactions are then introduced into the thermocycler in order to undergo 35 cycles composed of the following three stages: Denaturation for 10 seconds at 96 ° C., hybridization for 10 seconds at 50 ° C., elongation for 4 minutes at 56 ° C.

Then precipitation is carried out with 76% ethanol for 20 minutes at room temperature.

The plates are then centrifuged for 35 minutes at 2200 g and then drained on absorbent paper and then centrifuged and returned to absorbent paper for 1 minute at 500 g in order to leave no trace of ethanol.

The DNA pellets are then: either taken up in a formamide-EDTA-dextran blue solution and then denatured for 2 minutes at 96 ° C .; the plates are then immediately placed in the ice, an aliquot then being deposited on acrylamide gel of an automatic sequencer PE-377; - is taken up in a 0.3mM EDTA solution; an aliquot is then automatically deposited in a PE-3700 automatic sequencer.

Step 7: Mapping BAC clones on the genome of Photorhabdus luminescens Knowing almost the entire genome sequence of the bacterium Photorhabdus luminescens, as well as the sequences of the ends of the inserts of the clones of the various libraries, the inventors carried out a mapping allowing position all of these clones on the genome.

EXAMPLE 2: entomopathogenic activity tests on Aedes larvae, conducted from the BAC library described above.

 1) Protocol used

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 For the screening of the library of BACs, the test comprises first two steps: Step 1: The BACs are cultured in plates comprising 96 wells closed with a parafilm in LB medium (Luria-Bertani) / chloramphenicol at 12 μg / ml overnight at 37 C, with stirring at 250 rpm. Volume: 200 μl / well.

2nd step :
The next day the plates are centrifuged at 1100 g for 10 minutes and the LB (which is toxic to the Aedes larvae) is aspirated. 200 l of sterile water are then added.

The bacteria can then bind and a measurement is made of OD 595.

Similarly for the screening of individual BACs, the test comprises two first steps.

Step 1: In this case, the culture of BACs in vial is performed on LB / chloramphenicol overnight until the log of OD 595 is at least 1.

Step 2: The bacteria are then centrifuged for 10 minutes at 5000 rpm and the pellet is washed with distilled water and centrifuged again. The bacteria are then resuspended in water and sampled in triplicate (or more) in 96-well plates.

A dilution series in water can be performed to evaluate the potential activity of these samples. The maximum concentration can result in a D0595 of 1 (go to step 3).

Activity evaluation can also be performed on samples of dry frozen bacteria. In this case, the bacteria are cultured, washed with sterile water, resuspended in a small volume of water and frozen.

Step 3: Following these steps 1 and 2, varying according to the type of screening, proceed to step 3.

The eggs of Aedes aegypti are stirred in water for two minutes, which causes them to hatch. About 45 minutes after the newly hatched larvae are

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 distributed in each well. Each well contains 10 l of this larval solution containing 5 to 10 larvae. The structure of the pipette is adapted accordingly.

Variations in the number of larvae per well is not critical since if the BAC is toxic, the larvae are killed and the bacteria are not destroyed.

Step 4: Leave the plates at room temperature and measure the OD 595 at different times after the addition of Aedes larvae, especially at 24 hours and 48 hours. We can see with the naked eye if the larvae feed on bacteria or not. Generally, Aedes larvae are observed at about 20 hours, possibly at 24 to 48 hours.

Step 5: Measurement of results As a positive control, wild strains of Photorhabdus are used. As negative controls, an E. coli strain DH10B (the strain used for the BACs) and a control BAC, that is to say the strain DH10B containing the BAC vector without inserted fragment, is used.

2) Results In these tests on Aedes larvae, in which the Aedes were fed with E. coli expressing each BAC clone, the complete library of BAC was screened. The inventors have identified a positive BAC. The results have been repeated with this BAC many times and are reproducible. This BAC, plate 1 line A column 2, designated BAC1A2, causes paralysis and / or death of Aedes larvae. Controls used in these assays include: a random BAC clone that does not exert an effect against Aedes larvae, E. coli strain DH10B (the strain in which the BAC library was cloned), a wild-type strain type Photorhabdus luminescensTTO1.

The inventors have observed larval alteration in the case of BAC 1A2 about 20 hours after the plates are infected with the larvae. The differences are particularly noticeable after 48 hours after infection. The larvae begin to slow down their movements and then no longer seem to feed on the medium containing the bacteria as well as in the wells containing the random clone

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 BAC and the DH10B. The larvae then lie down in the bottom of the well, no longer swim to the surface to collect air. The activity observed with BAC 1A2 is similar to that observed with a sample of wild-type Photorhabdus, whereas the strain of E. coli DH10B is inactive.

In the 96-well plates using Photorhabdus luminescens, larvae were killed with a D0595 for the bacteria of 0.1 before the addition of Aedes larvae. Death is also observed with an OD of 0.5 for BAC 1A2.

In mosquito trials, the percentage of mortality was 100% with wild-type Photorhabdus, and in the range of 90-95% for BAC 1A2.

In addition, heat sensitivity tests were performed. Photorhabdus, BAC1A2 and Ecoli DH10B were heated at different temperatures to determine the remaining insecticidal activity. For this, a night Photorhabdus culture was collected and then suspended in sterile water. 1 ml samples were heated at 30 ° C., 40 ° C., 50 ° C., 60 ° C., 70 ° C., 80 ° C. or at 100 ° C. for 15 minutes. The results showed that the larvae died when Photorhabdus was heated to 70 C. The larvae survived and absorbed the bacteria when the temperature was 80 C or higher. All the tests carried out show that the insecticidal toxin secreted by Photorhabdus is stable up to about 60 C. Tests on BAC 1A2 also show a stability up to 60 C, the larvae die in wells containing BAC 1A2 which were heated to 60 C.

For the DH10B and random BAC control samples, larvae were able to feed on heated bacteria at all tested temperatures. In contrast, the toxins obtained by the inventors retain their insecticidal activity typically several weeks stored at about 4 C.

The clone BAC 1 A2 (deposit CNCM 1-2699), the size of the inserted fragment is 60131 bps, comprises: a set of genes similar to genes isolated from Escherichia coli (housekeeping genes); the gene of sequence SEQ ID No. 4, coding for the protein of sequence SEQ ID
N 5, which has homology with a juvenile hormone esterase gene

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beetle, flanked by the SEQ ID N 2 sequence gene coding for the protein of sequence SEQ ID No. 3. In addition, a gene coding for a transcriptional regulator adjoins this putative operon. Since these genes (SEQ ID No. 4 and SEQ ID No. 2) were probably responsible for the entomotoxic activity observed, the inventors first restricted the sequence of BAC 1 A2 to a region containing mainly the SEQ ID N 4 genes and
SEQ ID N 2.

The inventors have further tested subclones containing the two ORFs of BAC 1A2 cited above, namely ORF1 encoding SEQ ID No. 3 and ORF2 encoding SEQ ID No. 5.

Specifically, two subclones, plg16g1 and plgl6el2, were transformed into E. coli strain DH10B. The clone plgl6gl, deposited at the CNCM under the number 1-2698, is a genomic DNA fragment of Photorhabdus luminescens, strain TT01 (Fischer-Le Saux M. et al., (1999) Int.J. System Bacteriol. 49, 1645-56), cloned into the vector pSYX34 (Xu S. and Fomenkov A. (1994) - Biotechniques, vol 15,57) in E. coli XL10-Gold Kanr (Stratagene).

From frozen samples, Photorhabdus, BAC1A2, DH10B, plgl6gl and plg16e12 bacteria were cultured. 200 l of each culture were pipetted into 12 wells of a 96 well plate. Plates were shaken for 10 minutes at 1000 rpm. The supernatant was removed. 200 l of sterile water were added to each well and the bacteria were resuspended. OD 595 was measured, with each well containing about 10 Aedes aegypti larvae. Both clones, plgl6gl and plgl6el2 have an insecticidal effect on larvae, which is even faster and stronger than the effect of Photorhabdus and BAC 1A2. This result is likely related to the higher copy number (5 copies per cell) of the pSYX34 vector compared to the pBELOBACll vector (1 copy per cell). Control larvae (DH10B and water) are unaffected and remain very active. Larvae in contact with Photorhabdus or BAC 1A2 show slow motion and remain in the bottom of the well. Larvae in contact with clones plg16g1 and plg16e12 are immobile and appear to be dead.

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 EXAMPLE 3: entomopathogenic activity on Culex pipiens larvae, conducted from the BAC library described above.

The larvae were prepared analogously to those of Aedes.

The inventors have prepared: a subclone (construction deposited at the CNCM under the N 1-2752) containing the genes of sequence SEQ ID No. 2 (ORF 1) and SEQ ID No. 4 (ORF 2): these genes have been amplified by PCR and cloned at the EcoRV site of pBluescript SK. This plasmid is named pDIA700 clones n 10 (sequence verified).

a subclone (construction deposited at the CNCM under N 1-2753) containing the genes of SEQ ID No. 2 (ORF 1) and SEQ ID No. 4 (ORF 2), with deletion of the 3 'part of the gene sequence SEQ ID N 4; plasmid pDIA700 (# 10) was restricted with EcoRI to remove the last third of SEQ ID N 4. This plasmid is named pDIA701.

The results obtained were as follows, demonstrating that ORF 2 is essential for insecticidal activity against Culex pipiens.

Results on Culex pipiens larvae

<Tb>
<tb> Locus <SEP> 1
<tb> pDIA700 / 10 <SEP> pDIA701 <SEP> Control
<tb> ORF1 <SEP> + <SEP> ORF2 <SEP> ORF1 + ORF2 <SEP> tr.3 '<SEP> water
<tb> 24 <SEP> h <SEP> DO <SEP> 2 <SEP> DO <SEP> 2
<tb> Replicat <SEP> 1 <SEP> Dead <SEP> 10 <SEP> 0 <SEP> 0
<tb> V <SEP> ivantes <SEP> 0 <SEP> 11 <SEP> 11
<tb> Replicat <SEP> 2 <SEP> Dead <SEP> 10 <SEP> 0 <SEP> 0
<tb> Live <SEP> 0 <SEP> 12 <SEP> 12
<tb> Total <SEP> Dead <SEP> 20 <SEP> 0 <SEP> 0
<tb> Live <SEP> 0 <SEP> 23 <SEP> 23
<tb>% <SEP> Mortality <SEP> 100% <SEP> 0% <SEP> 0%
<tb> 48 <SEP> h
<tb> Replicat <SEP> 1 <SEP> Dead <SEP> 10 <SEP> 0 <SEP> 0
<tb> V <SEP> ivantes <SEP> 0 <SEP> 11 <SEP> 11
<tb> Replicat <SEP> 2 <SEP> Dead <SEP> 10 <SEP> 0 <SEP> 0
<tb> Live <SEP> 0 <SEP> 12 <SEP> 12
<tb> Total <SEP> Dead <SEP> 20 <SEP> 0 <SEP> 0
<tb> Live <SEP> 0 <SEP> 23 <SEP> 23
<tb>% <SEP> Mortality <SEP> 100% <SEP> 0% <SEP> 0%
<Tb>

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 Other series of tests on different species of mosquitoes have demonstrated the effectiveness especially in Culex pipiens, Anopheles gambiae, Anopheles stephensie, summarized in Table 1.

Table 1

<Tb>
<tb> STAGE <SEP> LI <SEP> LI-L2 <SEP> L1 <SEP> L1
<tb> Culex <SEP> pipiens <SEP> Aedes <SEP> aegypti <SEP> Anopheles <SEP> gambiae <SEP> Anopheles <SEP> stephensia
<tb> 48H <SEP> 48H <SEP> 48H <SEP> 48H
<tb> Photorhabdus <SEP> 16 <SEP> alive <SEP> 40 <SEP> alive <SEP> 6 <SEP> alive <SEP> 15 <SEP> alive
<tb> 59 <SEP> dead <SEP> / 75 <SEP> 35 <SEP> dead <SEP> / 75 <SEP> 69 <SEP> dead / 75 <SEP> 60 <SEP> dead <SEP> / 75
<tb> 79% <SEP> 47% <SEP> 92% <SEP> 80%
<tb> Bac <SEP> 1 <SEP> A2 <SEP> 5 <SEP> alive <SEP> 44 <SEP> alive <SEP> 7 <SEP> alive <SEP> 6 <SEP> alive <SEP>
<tb> 70 <SEP> dead <SEP> / 75 <SEP> 31 <SEP> dead <SEP> / 75 <SEP> 68 <SEP> dead / 75 <SEP> 69 <SEP> dead <SEP> / <SEP > 75 <SEP>
<tb> 93% <SEP> 41% <SEP> 91% <SEP> 92%
<tb> P18F1 <SEP> 44 <SEP> alive <SEP> 62 <SEP> alive <SEP> 39 <SEP> alive <SEP> 41 <SEP> alive
<tb> control <SEP> 31 <SEP> dead <SEP> / 75 <SEP> 13 <SEP> dead <SEP> / <SEP> 75 <SEP> 36 <SEP> dead <SEP> / <SEP> 75 <SEP> 34 <SEP> dead <SEP> / <SEP> 75 <SEP>
<tb> negative <SEP> (without <SEP> 41% <SEP> 17% <SEP> 48 <SEP>% <SEP> 45%
<tb> insert)
<Tb>
EXAMPLE 4: Entomopathogenic activity tests on Plutella xylostella larvae, conducted from the BAC library described above.

(i) A series of tests was performed according to the following protocol. Bacteria (strain E. coli DH10B, containing plasmid pBeloBAC11) were cultured overnight in 5 ml LB + chloramphenicol at 28 ° C. with shaking at 200 rpm. The D054o of each crop has been systematically taken. Each culture is diluted with LB to a concentration of 5x107 bacteria / ml. Six leaves (3 cm in diameter) are incubated in a diluted culture for one hour with 0.05% Tween 20 (Sigma). The treated leaves are put in 6-well plates and 5 larvae are placed in each well, ie 30 larvae tested per clone. Each well contains an agar medium containing 15 g / l agar (Difco) and 1.5 g / l antifungal (nipagin, Sigma).

Preferably, the larvae used for this test are late-stage larvae of the L2 stage (2nd instar larvae), selected just before their moulting to the L3 stage. In

<Desc / Clms Page number 35>

 Indeed, the inventors have observed that the larvae were significantly more sensitive to the bacterium at this stage L2 and that the tests there were much more reproducible.

The tests were carried out at 28 C with a photoperiod of 11h: 13h (night: day).

After 2 days, the treated leaves were replaced by untreated leaves. The larval mortality level was observed at 48h, 72h and 144h after larval contact with treated leaves. Controls were used for screening tests, including: Photorhabdus TT01, and / or BAC8d10 (containing the locus te ) as positive controls. Negative controls corresponding to non-insert constructs were adequately used (pBeloBACll in E. coli DH10B for BAC clones, pSYX34 in E.coli XL10 for plgl6gl subclone, E.coli XLIBlue for pDIA subclones) . All these bioassays were made with a strain of Plutella xylostella native to Martinique.

The mortality rate of the larvae was corrected against the negative control using the Abbott equation (Abbott W. S., J. Econ Entomol., 18: 265-267).

The inventors have demonstrated, according to a first series of tests, the insecticidal activity of BAC1A2 against lepidopterans, in particular Plutella xylostella, when the larvae were in the late larval L2 stage.

<Tb>
<Tb>

Clones <SEP>% <SEP> of <SEP> mortality <SEP> ABBOTT
<tb> 48h <SEP> 72h <SEP> 144h
<tb> BAC <SEP> 1a02 <SEP> 30% <SEP> 50% <SEP> 58%
<tb> 26% <SEP> 26% <SEP> 26%
<tb> Plg <SEP> 16g1 <SEP> 43% <SEP> 58% pDIA700 <SEP>#<SEP> 96% <SEP> 100% <SEP> 100%
<tb> pDIA701 <SEP>#<SEP> 12% <SEP> 12% <SEP> 23%
<Tb>
* a% of Abbott mortality greater than 20% is considered significant # the concentration of antifungal (nipagin) used in the agar medium (for the support of the leaves) was decreased by 5 times during the bioassays carried out with the sub -clones pDIA.

<Desc / Clms Page number 36>

 (ii) Another series of tests on Plutella demonstrated the insecticidal effect, using the following protocol.

Initially three doses of frozen bacteria were tested on Plutella: 0.014 g / 5ml, 0.007g / 5ml and 0.035g / 5ml of water. 150/1 of bacteria were added to the surface of a Plutella dish. The cup was placed in a hood for drying.

Once the cups are dried, each is infested with 10 Plutella larvae (4 days old). We observe: - 100% mortality in all Photorhabdus treatments; - 95% mortality for treatment with 0.0014g / 5ml of BAC 1A2; - 90% mortality for treatment with 0.007g / 5ml of BAC 1 A2; - some mortality for DH10B control with 42% for treatment
0,014g / 5ml.

Thus, the wild strain of Photorhabdus has a strong insecticidal effect on Plutella larvae. The effect obtained with the BAC 1A2 is significantly higher compared to the control.

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 BIBLIOGRAPHIC REFERENCES Fischer-Le Saux M. et al. 1998. Appl. About. Microbiol., Vol 64, 4246; Kim V.J. et al. 1996. Genomics, vol 34, 213; Calvin N.M. and Hanawalt P. C, 1998, J. Bacteriol., 170, 2796; Stephen F., Altschul, Bioinformatics: A Trends Guide, 1998: 5: 7-9; Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 872264, modified in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877; Tatusova et al., 1999, "Blast 2 sequences - a new tool for comparative protein and nucleotide sequences"; FEMS Microbiol Lett. 174: 247-250; Altschul et al., 1997, Nucleic Acids Res. 25 (17): 3389-3402; Altschul S.F. et al., 1990, J. Mol. Biol., 403-410; Thompson, J. D. et al., 1994, Nucleic Acids Res. 22, 4673-80; Sambrook et al., (1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor); Maniatis et al., 1982 (Molecular cloning, A laboratory Manual, Cold Spring Harbor Lab, CSH, NY USA or one of his recent reissues, Ausubel et al., Eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience, NY) Upender et al., 1995, Biotechniques 18 (1): 29-30 Morris et al (1992), Virology, 187: 633, Leroy-Stein et al., 1989, PNAS USA, 86: 6126-6130, Gallie et al., 1989, Molecular Biology of RNA, pp. 237-256, Ed DUNOD Plant Physiology, Volume 2 Development Heller, Esnault and Lance, 2000, Methods of Molecular Biology: Plantgene transfer and Expression protocols, Hansen et al., 1999, Recent advances in the transformation of plants, Trends in plant science, Vol 4, No. 6, 226, SBGelvin, The introduction and expression of transgenes in plants, Current opinion in biotechnology, 1998, 227;

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Hellens et al., 2000, Trends in Plant Science, Vol 5, No. 10, A Guide to Agrobacterium binary Ti Vectors, 446;
Franken et al., Recombinant proteins from transgenic plants, Current opinion in biotechnology, 8: 411-416; Schuler et al., 1998, Insect-resistant transgenic plants, Tibtech, vol 16,168, Michelmore. M, 2000, Genomic Approaches to Plant Disease Resistance, Current Opinion in Biotechnology, 3: 125-131; Lazzeri, Methods Mol Biol., 49: 95-106, 1995; Finer et al., T. Particle Bombardment-mediated Transformation, Current Topics in Microbiology and Immunology, Vol. 240; Zupan, J. et al. (2000) The transfer of DNA from Agrobacterium tumefaciens into plants: a feast of fundamental insights. Plant J. 23: 11-28, US 6,037,522, US 6,265,538; Hansen et al. Agrolistic transformation of plant cells: integration of T-strands generated in planta. Proc Natl Acad Sci USA 1996, 93: 14978-14983; McCormac et al., Euphytica, vol 99 (1): 17-25,1998; Ishidia et al., 1996, Nat Biotechnol., 14 (6): 745-750, 1996; Trends in plant science Oct 2000 Vol 5 n 10 p 446; Bechtold et al., 1993, In planta Agrobacterium-mediated gene transfer by infiltration of adult Arabidopsis thaliana plants, CR Acad. Sci, Paris, Life Sciences 316, 1194-1199; Clough et al., 1998, Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana, Plant J. 16,735-743; Gelvin 1998; Hansen and Wright, 1999; Ebinuma et al., 1997; McGarvey and Kaper, 1991; Mcormick, 1986, Plant Cell Reports, 5, 80-84; L. Frangeul et al., 1999. Microbiology 145, 2625-2634; Ewing B. et al., 1998. Genome Res. 8, 175-185; Ewing B. & Green P. 1998. Genome Res. 8, 186-194; Gordon et al. 1998. Genome Res. 8, 195-202;

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 Contigs-Tool-Box, L. Frangeul et al., Unpublished Results, Genomic Laboratory for Pathogenic Microorganisms, Institut Pasteur, Paris; Fischer - The Saux M. et al. (1999) Int. J. Syst. Bacteriol., 49, 1645-56); Xu S. and Fomenkov A. (1994) - Biotechniques, Vol 15, 57;

Claims (24)

1. isolated nucleotide sequence characterized in that it comprises a nucleotide sequence selected from SEQ ID N 2 or SEQ ID N 4, said nucleotide sequence encoding at least one polypeptide active against insects.  2. A nucleotide sequence characterized in that it is chosen from: a) a nucleotide sequence comprising at least 70% identity with a nucleotide sequence according to claim 1; b) a nucleotide sequence hybridizing under conditions of high stringency with a nucleotide sequence according to claim 1; c) a nucleotide sequence complementary to a sequence defined in a) or b); d) a fragment representative of a sequence defined in a), b) or c), of at least 10 base pairs; e) a nucleotide sequence comprising a sequence as defined in a), b), c) or d); f) a modified nucleotide sequence of a nucleotide sequence as defined in a), b), c), d) or e); said sequence encoding at least one polypeptide active against insects.  3. Insecticidal polypeptide characterized in that it is encoded by a nucleotide sequence according to any one of claims 1 or 2.  An insecticidal polypeptide characterized in that it comprises a polypeptide sequence chosen from: a) a polypeptide of sequence SEQ ID No. 3 or SEQ ID No. 5; b) a polypeptide homologous to the polypeptide defined in a) having at least 80% identity with said polypeptide of a); <Desc / Clms Page number 41>  c) a fragment of at least 10 consecutive amino acids of a polypeptide defined in a), or b); d) a variant polypeptide of polypeptides of amino acid sequences defined in a), b), or c). 5. A nucleotide sequence characterized in that it encodes a polypeptide according to claim 3 or 4. 6. Nucleotide sequence according to any one of claims 1, 2,5 characterized in that the toxin is active against Diptera, including mosquito species Aedes aegypti, Culex pipiens, Anopheles gambiae and Anopheles stephensia, and / or Lepidoptera including Plutella xylostella. An expression cassette comprising a promoter sequence operably linked to a nucleotide sequence according to any one of claims 1, 2, 5 and 6. Cloning and / or expression vector characterized in that it comprises an expression cassette according to claim 7 or a nucleotide sequence according to any one of claims 1, 2, 5 and 6. 9. Virus vector characterized in that it comprises in its genome a polynucleotide sequence according to any one of claims 1, 2, 5 and 6 operably linked to an inducible promoter or appropriate constitutive. 10. Host cell comprising an expression cassette according to claim 7. 11. Host cell according to claim 10 characterized in that it is a bacterium, a yeast, a plant cell, a fungus. <Desc / Clms Page number 42>  12. Insecticidal composition characterized in that it comprises a polypeptide according to one of claims 3 or 4, or a host cell according to claim 10 or 11 accompanied by a suitable carrier. 13. Process for obtaining a plant expressing at least one toxin against insects, characterized in that it comprises the introduction of an expression cassette according to claim 7 in at least one plant cell, and then the culture of the cell thus transformed so as to regenerate a plant containing in its genome said expression cassette. Plant or part of a plant, comprising an expression cassette according to claim 7, wherein the plants or parts of plants are transformed and resistant to insects. 15. A plant or plant part according to claim 14 having an increase in the expression of a toxin against insects compared to an unprocessed plant. 16. Plant or plant part according to any one of claims 14 to 15 characterized in that it is a mono or dicotyledonous, including wheat or corn. 17. A method of controlling insects characterized in that it comprises the application to plants infected with said insects of an insecticidally effective amount of an insecticidal composition according to claim 12 or vectors according to claim 8 . 18. Process for obtaining insecticidal toxins characterized in that it comprises: - obtaining at least one host cell according to claim 11; culturing under conditions suitable for the expression of a nucleotide sequence according to any one of claims 1, 2, 5 and 6 and leading to the production of at least one active toxin against insects; <Desc / Clms Page number 43>  - collection of toxins produced.  19. Process for obtaining an insect-resistant plant, characterized in that it comprises the introduction of a nucleotide sequence according to any one of claims 1, 2, 5 and 6, the expression of said sequence. nucleotide being at a level sufficient to act against insects. 20. Isolated nucleotide sequence characterized in that it comprises the DNA fragment of Photorhabdus luminescens of about 60 kb designated plBACIA2 deposited under the number CNCM-2699, present in the strain E. coli DH10B. 21. Isolated nucleotide sequence characterized in that it corresponds to SEQ IDN 1. 22. A strain comprising a vector according to claim 8, deposited at the CNCM under the reference plbac 1 a2 CNCM 1-2699. 23. Use of a nucleotide sequence according to one of claims 1, 2, 5 and 6, of a vector according to claim 8, of a cell according to claim 10 or 11, for the preparation of an insecticidal polypeptide. 24. A pharmaceutical composition comprising an insecticidal polypeptide according to claim 3 or 4.