Plants transformed with a DNA sequence from bacillus thuringiensis lethal to lepidoptera

Abstract: A plant cell transformed with all or part of a Bacillus thuringiensis gene coding for either a 130 or 132 kDa protoxin against Lepidoptera, preferably with a part of the gene coding just for the respective 62 or 60 kDa trypsin-digestion product of the protoxin. A plant, regenerated from the transformed cell, is resistant to Lepidoptera.

Description: Background of the Invention This invention relates to a new DNA sequence ("the bt4 gene") from the genome of the strain Bacillus thuringiensis var. aizawai HD-68 (the "Bt HD-68 strain") which is publicly available from the Agricultural Research Culture Collection, Northern Region Research Center, 1815 North University Street, Peoria, Illinois 61604, U.S.A. The bt4 gene encodes a 132 kDa protein (the "Bt4 protoxin") This invention also relates to a 60 kDa protein (the "Bt4 toxin") which can be obtained by trypsin digestion of the Bt4 protoxin The Bt4 toxin is the active ingredient in the crystallized protoxin produced by the Bt HD-68 strain, with a high activity against Lepidoptera species, such as Manduca sexta and Spodoptera species. As is the case for other B. thuringiensis ("Bt") crystal proteins (Höfte et al, 1988), when the crystalline Bt4 protoxin is ingested by insect larvae, it is solubilized and processed in the insect's midgut, releasing the Bt4 toxin . In this regard, Höfte et al (1988) has generally described three types of toxin-producing Lepidoptera-specific B. thuringiensis having the following characteristics: Type A (consisting of 3 subtypes) producing a protoxin of 130 kDa and a toxin of 60 kDa which is toxic against Manduca sexta and Pieris brassicae. Type B producing a protoxin of 133 kDa and a 55 kDa toxin which is toxic against Pieris brassicae. Type C producing a 135 kDa protoxin and a 63 kDa trypsin-activated toxin, showing insecticidal activity against Spodoptera littoralis and Mamestra brassicae. This invention further relates to a chimaeric gene that can be used to transform a plant cell and that contains the following, operably linked, DNA sequences: 1) all or an insecticidally effective part of the bt4 gene encoding all or an insecticidally effective part of the respective Bt4 protoxin, preferably a truncated part of the bt4 gene ("the truncated bt4 gene") encoding just the respective Bt4 toxin; 2) a promoter suitable to direct transcription of all or part of the bt4 gene in the plant cell; and 3) suitable 3' transcription regulation signals for expressing all or part of the bt4 gene in the plant cell. This chimaeric gene is hereinafter referred to as the "bt4 chimaeric gene". Preferably, the plant cell is transformed with a bt4 chimaeric gene comprising the truncated bt4 gene, together with a selectable marker gene, such as the neo gene encoding neomycin phosphotransferase II or NPTII (Reiss et al, 1984), fused with the truncated bt4 gene as a bt4-neo hybrid gene encoding a Bt4 toxin - NPTII fusion protein. This invention still further relates to a plant that is regenerated from the transformed cell and that is resistant to Lepidoptera, particularly Sphingidae such as Manduca sexta and Noctuidae such as Spodoptera species which are major pests of economically important crops such as cotton, corn, soybean, alfalfa, tomato, tobacco, sugarbeet and other vegetables. This invention yet further relates to: a method of locating the C-terminal end of the minimum toxic part or core of a B. thuringiensis protoxin which is like the Bt4 protoxin and is hereinafter referred to as a "Bt4-like protoxin"; a plant cell and plant transformed with a DNA sequence encoding a toxic part of a Bt4-like protoxin having a minimum-length C-terminal end; and a probe for identifying such a DNA sequence. Summary of the Invention In accordance with this invention, a plant cell genome is transformed with the bt4 chimaeric gene containing all or part of the bt4 gene, preferably containing the truncated bt4 gene, whereby the resulting plant cell can be used to provide a plant which produces, in some or all of its tissues, all or at least a toxic part of the Bt4 protoxin, preferably the Bt4 toxin, thereby rendering the plant resistant to Lepidoptera. The transformed plant cells of this invention can also be used to produce, for recovery, the Bt toxins expressed by these cells. Also in accordance with this invention, a process is provided for rendering a plant resistant to Lepidoptera by transforming the plant cell genome with the bt4 chimaeric gene containing all or part of the bt4 gene, preferably the truncated bt4 gene. Further in accordance with this invention, DNA sequences of the bt4 gene are provided that encode the Bt4 protoxin and that include the DNA sequences of the truncated bt4 gene which codes for just the respective Bt4 toxin . A plant cell transformed with all or part of either one of these DNA sequences of the bt4 genes is resistant to Lepidoptera. Still further in accordance with this invention, a method is provided to determine the C-terminus of the minimum toxic part of a Bt4-like protoxin against Lepidoptera, comprising the step of locating the following intact twelve-amino acid sequence in the protoxin: V/L Y I D K/R I E X I P/L V/A T/D wherein X is any amino acid. Also in accordance with this invention is provided a plant and plant cell, the genome of which is transformed with a DNA sequence encoding a toxic part of a Bt4-like protoxin having a minimum length, C-terminal end that consists essentially of, and that preferably terminates with, said twelve-amino acid sequence. Further in accordance with this invention, a nucleotide sequence encoding said twelve-amino acid sequence is utilized as a probe for: 1) identifying a nucleotide sequence, preferably a DNA sequence, encoding a toxic part, preferably the toxin, of a Bt4-like protoxin having a minimum length C-terminal end; and 2) for locating a nucleotide sequence encoding said C-terminal end in a larger nucleotide sequence encoding all or part of a Bt4-like protoxin. The invention related to the above-mentioned Bt4 protein and gene is identified in the enclosed claims. Detailed Description of the Invention In accordance with this invention, the bt4 gene can be isolated from the respective Bt HD-68 strain. For example, the bt4 gene can be identified in the respective Bt strain , using the procedure described in European patent application 86300291.1 (which is incorporated herein by reference). The so-identified bt4 gene can then be sequenced in a conventional manner (Maxam and Gilbert, 1980), to obtain the DNA sequences shown in Fig. 1 . The amino acid sequence of the Bt4 protoxin and toxin can be determined from the DNA sequences of the respective bt4 gene and truncated bt4 gene . The insecticidal spectra of the Bt4 protoxin and Bt4 toxin and the nucleotide sequences of the bt4 gene proves that the protoxin and toxin are different from previously described toxins with activity against Sphingidae and Noctuidae (Höfte et al, 1988). All of the bt4 gene (encoding the Bt4 protoxin) or part of the bt4 gene (encoding a toxic part of the Bt4 protoxin), preferably the truncated bt4 gene (encoding the Bt4 toxin), can be stably inserted in a conventional manner into the nuclear genome of a single plant cell, and the so-transformed plant cell be used to produce a transformed plant that is insect-resistant. In this regard, a disarmed Ti-plasmid, containing the bt4 chimaeric gene, in Agrobacterium (e.g., A. tumefaciens ) can be used to transform the plant cell using the procedures described, for example, in European patent publications 116,718 and 270,822, PCT publication 84/02913 and European patent application 87400544.0 (which are also incorporated herein by reference). The genome of the resulting transformed plant cell and plant contains, integrated therein, the bt4 chimaeric gene. By "an insecticidally effective part" or "a part" of the bt4 gene is meant a DNA sequence encoding a polypeptide which has fewer amino acids then the respective Bt4 protoxin but which is still toxic to Lepidoptera. Such a part of the bt4 gene can encode a Bt4 protoxin which has been truncated towards at least one trypsin cleavage site of the protoxin (U.S. patent application 821,582, filed January 22, 1986; European patent application 86300291.1) The resulting transformed plant can be used in a conventional breeding scheme to produce more transformed plants with the same Lepidoptera-resistant characteristics or to introduce all or part of the bt4 gene into other varieties of the same or related plant species. Seeds, which are obtained from the transformed plants, contain all or part of the bt4 gene as a stable genomic insert. Cells of the transformed plant can be cultured to produce the Bt4 toxin which can be recovered for use in conventional insecticide compositions against Lepidoptera (U.S. patent application 821,582; European patent application 86300291.1). Preferably, all or part of the bt4 gene is provided in the bt4 chimaeric gene and is inserted in a plant genome downstream (i.e., 3') of, and under the control of, a promoter which can direct the expression of the gene in the plant's cells. Preferred promoters include the strong constitutive 35S promoter (Odell et al, 1985) of the cauliflower mosaic virus. 35S promoters have been obtained from different isolates: CM1841 (Gardner et al, 1981), Cabb-JI (the "35S3 promoter") and CabbB-5 (Hull and Howell, 1987). Other preferred promoters include the TR1' promoter and the TR2' promoter which drive the expression of the 1' and 2' genes, respectively, of the T-DNA (Velten et al, 1984) [the "TR1' promoter" and "TR2' promoter", respectively]. Alternatively, a promoter can be utilized which is not constitutive but rather is specific for one or more tissues or organs of the plant whereby all or a toxic part of the Bt4 protoxin is expressed only in cells of the specific tissue(s) or organ(s). For example, all or part of the bt4 gene could be selectively expressed in the green tissues of a plant by placing the gene under the control of a light-inducible promoter such as the promoter of the ribulose-1,5-bisphosphate carboxylase small unit gene of the plant itself or of another plant such as pea as disclosed in U.S. patent application 821,582 and European patent application 86300291.1 (which are also incorporated herein by reference). Another alternative is to use a promoter whose expression is inducible by temperature or chemical factors. It is also preferred that all or part of the bt4 gene be provided in the bt4 chimaeric gene and be inserted in a plant genome upstream (i.e., 5') of suitable 3' transcription regulation signals (i.e., transcription termination and polyadenylation signals) such as the 3' untranslated end of the octopine synthase ("ocs") gene (Gielen et al, 1984) or T-DNA gene 7 (Velten and Schell, 1985). It is further preferred that all or part of the bt4 gene be provided in the bt4 chimaeric gene and be inserted in a plant genome in the same transcriptional unit as, and under the control of, the same promoter as a selectable marker gene. This hybrid bt4 -marker gene will, thereby, be expressed in a transformed plant as a fusion protein (U.S. patent application 821,582; European patent application 86300291.1; Vaeck et al, 1987). Any conventional marker gene can be utilized, with which it is possible to make N-terminal gene fusions and the expression of which can be used to select transformed plant cells. An example of a suitable selectable marker gene is an antibiotic resistance gene such as the neo gene coding for neomycin phosphotransferase II conferring kanamycin resistance (European patent application 87400544.0; U.S. patent application 821,582). Any DNA or RNA sequence, encoding the above-defined twelve-amino acid sequence, can be used in a conventional manner as a probe for identifying the location, in a gene encoding a Bt4-like protoxin, of the minimum length, C-terminal end of the protoxin. This probe can also be used to identify other homologous sequences present in other Bt. strains by means of conventional methods and by means of computer homology searches. This twelve-amino acid sequence also can be used in a conventional manner (Höfte et al, 1988) to prepare polyclonal and/or monoclonal antibodies which are directed against this sequence and which can be used to identify Bt4-like protoxins, preferably Bt4-like toxins. The following Examples illustrate the invention. The figures, referred to in the Examples, are as follows: Figure 1 shows the nucleotide sequence and deduced amino acid sequence of the ORF of the bt4 gene extending from nucleotide 264 to a TAG termination codon at nucleotide 3758. The truncated bt4 gene, coding for just the Bt4 toxin, extends from nucleotide 264 to nucleotide 2039. The C-terminal twelve-amino acid sequence of the Bt4 toxin extends from nucleotide 2004 to nucleotide 2039 and is underlined in Fig. 1 . Trypsin cleavage sites of the Bt684 protoxin are at amino acids 27 and 603. Figure 2 shows comparisons of the amino acid sequences of the Bt18 protoxin [no. 7] and the Bt4 protoxin [no. 6] of this invention with the Bt2 protoxin (Höfte et al, 1986) [no. 2] and the Bt15 protoxin (Honée et al, 1988) [no. 5] . The amino acids which are homologous are printed in capitals. An arrowhead indicates the end of the amino acid sequence of the Bt18 toxin (at amino acid position 601). Figure 3 shows the alignment of the minimum length, C-terminal ends of the amino acid sequences of the toxic parts (e.g., the toxin) of the Bt4 protoxin and of other homologous Bt4-like protoxins, i.e., the Bt14, Bt15 and Bt18 protoxins. A consensus twelve-amino acid sequence at the C-terminal ends of the toxic parts of these Bt4-like protoxins, the integrity of which sequence is essential for retention of the anti-Lepidoptera activity of such toxic parts, is shown in a box. This consensus amino acid sequence corresponds for the respective protoxins to the following amino acid positions: Bt4 581-592 (this invention) Bt18 590-601 Bt14 625-636 (Brizzard and Whiteley, 1988) Bt15 605-616 (Honée et al, 1988; European patent application 89401499.2). Unless otherwise stated in the Examples, all procedures for making and manipulating recombinant DNA were carried out by the standardized procedures described in Maniatis et al, Molecular Cloning - A Laboratory Manual , Cold Spring Harbor Laboratory (1982). Example 1 : Cloning of the bt4 gene Protoxin crystals were isolated from the Bt HD-68 strain using the procedure described by Mahillon and Delcour (1984). SDS-PAGE (Laemmli, 1970) of the purified crystals showed the presence of one major 132 kDa protein band. Trypsin digestion, followed by SDS-PAGE, showed the presence of a single 60 kDa protein band that showed toxic activity against Spodoptera exigua and M. sexta , and that consequently contained the toxic fragment of the Bt4 protoxin. The toxicity tests of the 60 kDa tryptic fragment (Bt4 toxin) were performed as described by Höfte et al (1988), and the results are summarized in Table II. In Table II, toxicity is expressed as 50% lethal concentration in ng/cm<2> (which is the concentration required to kill 50% of insects tested). M. sexta Heliothis virescens Mamestra brassica 5.0 >256 >1350 S. littoralis Spodoptera exigua > 256 52.85 In order to clone the bt4 gene, total DNA was prepared from the Bt HD-68 strain, and this genomic library was partially digested with Sau 3a and size-fractioned on a sucrose gradient. Fractions containing DNA between 4kb and 6kb were ligated to the Bgl II-digested and BAP-treated cloning vector pEcoR251 (deposited under accession no. 4711 at the Deutsche Sammlung von Mikroorganismen und Zellkulturen ("DSM"), Braunschweig, Federal Republic of Germany). Recombinant E. coli clones were screened with an internal 1.1 Kb Hind III fragment from the bt2 gene as a probe (Höfte et al, 1986; European patent publication 0,193,259). One positive clone contained a plasmid carrying a DNA fragment hybridizing to the probe. DNA sequencing of the fragment, according to Maxam and Gilbert (1980), revealed the presence of a large ORF of 3495 bp as shown in Fig. 1 . The ORF encodes the Bt4 protoxin of 132 kDa and the trypsin-digested Bt4 toxin of 60 kDa. The amino acid sequence of the Bt4 protoxin was deduced from the DNA sequence of the bt4 gene. Comparison of the amino acid sequence of the Bt4 protoxin with the Bt2 protoxin (Höfte et al, 1986), the Bt14 protoxin (Brizzard and Whitely, 1988), and the Bt15 protoxin (Höfte, 1988) and the Bt18 protoxin showed clearly that the C-terminal part of the protoxin is highly conserved (Fig. 2 ). The N-terminal part of the Bt4 protoxin revealed local stretches of homology with the Bt2 and Bt15 protoxins. The N-terminal sequence of the 60 kDa Bt4 toxin was determined. To this end, the toxic fragment of the trypsin-digested total crystal Bt4 proteins is blotted on polybrene-coated glass fiber (European patent application 86401,933.6), and an approximately 60 kDa fragment is extracted for amino acid sequencing as described in Example 1. The determined N-terminal amino acid sequence corresponds with the amino acid sequence deduced from the nucleotide sequence in Figure 1 . Example 2 :Expression of the bt4 gene in E. coli In order to express the bt4 gene in E. coli and in plants, different gene cassettes were made. a)Construction of a bt4 gene cassette A Nco I site is introduced at the ATG-initiation codon of the bt4 gene. A 25-mer oligonucleotide with the following sequence: is synthesized using an Applied Biosystems device according to the procedure of Itakura et al (1984). This allows the change of the TT nucleotides ahead of the ATG (Fig. 1 ) into CC (underlined above in the oligonucleotide sequence) by site directed mutagenesis (Stanssens et al, 1987), yielding a NcoI site. A 1.2 kb NdeI-EcoRI fragment containing the N-terminal sequence of the bt4 gene is cloned in the pMa5-8 plasmid, yielding the plasmid "pJB300". Single stranded DNA of this plasmid is made, and the oligonucleotide is inserted by a gapped duplex procedure (Stanssens et al, 1987). This yields the plasmid "pJB301" which has a Nco I site at the ATG initiation codon of the bt4 gene. In a second step, an Nco I- Eco RI 1150 bp fragment, encoding the 5' end nucleotide sequence of the bt4 gene of pJB301, is cloned in pJB65 (Botterman and Zabeau, 1987) digested with Nco I and Eco RI, yielding plasmid "pJB302". Subsequently, a polylinker is introduced in pJB302 downstream of the EcoRI site by exchanging an appropriate restriction fragment from pLK37 (Botterman and Zabeau, 1987), yielding plasmid "pJB303". The presence of a polylinker region downstream of the N-terminal fragment of the bt4 gene allows the reconstruction of the intact bt4 gene in pJB303 with an engineered NcoI site at the ATG initiation codon to produce plasmid "pJB304" containing the bt4 gene under the control of the lambda Pr promoter. b ) Construction of a C-terminal truncated bt4 gene cassette and a bt4-neo hybrid gene cassette The active toxic part of the Bt4 protoxin comprises a 60 kDa trypsin digestion product. Instead of expressing the whole gene, it is also possible to express a Bt4 toxin-encoding gene fragment or a derivative thereof in plants. To this end, a truncated bt4 gene fragment is constructed. In order to be able to select easily transgenic plants producing sufficient Bt4 toxin to kill Lepidoptera, a bt4-neo hybrid gene is also constructed. A Bcl I site is localized 1887 bp downstream of the ATG initiation codon (Fig. 1 ) and downstream of the coding sequence for the Bt4 toxin. To construct a bt4-neo hybrid gene and a bt4 C-terminal truncated gene fragment, the filled Bcl I end is ligated to the filled EcoRI site of pLKM91 and the filled Hind III site of pLK94 (Botterman and Zabeau, 1987), yielding plasmids "pJB305" and "pJB306" respectively. In pJB305, the neo gene is fused in frame to the bt4 gene, and in pJB306, a translation codon is positioned immediately downstream of the bt4 N-terminal gene fragment. c) Expression of the bt4 gene and derived gene fragments in E. coli Plasmid pJB304 from Example 3a is transformed into the NF1 strain. SDS-PAGE reveals that this NF1 strain (pJB304) then produces a 132 kDa protein upon temperature induction of the Pr-promoter at 42 DEG C. This 132 kDa protoxin is not present in a control NF1 strain. The expressed Bt4 protein shows a similar toxic activity against M. sexta and S. exigua as the trypsin-treated crystals of the Bt HD-68 strain. This 132 kDa protein is purified from the E. coli clone using the procedure described for the Bt2 protoxin (Höfte et al, 1986) and contains the same 60 kDa tryptic fragment. Furthermore, plasmids pJB305 and pJB306 from Example 3 b are transformed into the NF1 strain. Temperature induction of these strains at 42 DEG C shows that pJB305 directs the expression of a Bt4-NPTII fusion protein which exhibits NPTII activity and which shows toxic activity against M. sexta and S. exigua. pJB306 directs the expression of a 60 kDa protein, the migration on SDS-PAGE of which corresponds to that of the Bt4 toxin and which shows in vitro toxic activity against M. sexta and S. exigua. Example 3 : Introducing the bt4 chimaeric gene into plant expression vectors The different bt4 gene cassettes of Example 3a- b are placed under the control of promoters active in plant cells in intermediate plant expression vectors as described below. The Nco I- BgI II fragment from pJB304 (Example 3 a ) which encodes the bt4 gene, the Nco I- Xba I fragment from pJB306 (Example 3 b ) which encodes the truncated bt4 gene, and the NcoI-BglII fragment from pJB305 (Example 3 b ), which encodes the bt4 -neo hybrid gene, are also isolated and are cloned in the intermediate T-DNA vectors, described above, under the control of the 35S3 and TR2' promoters of the vectors. The respective bt4 gene fragments are cloned between the ClaI and BamHI sites localized immediately downstream of the TR2' promoter in pGSH160, and the bt4 - neo hybrid gene is cloned between the Cla l and Bam HI sites of pGSH150. This yields the vectors "pGSTX64" "pGSTX65" and "pGSTX53", respectively. In a similar way, these bt4 gene fragments are cloned in vectors pDE160 and pDE150, yielding the vectors "pGSTX66", pGSTX67" and "pGSTX54", respectively. Example 4: Identification of the bt4 gene fragment encoding the toxic part of the Bt4 protoxin based on the presence of a consensus sequence localized at the C-terminus of the toxic part A bt4 gene cassette has been constructed in the following way. A Nco I site is introduced at the ATG initiation codon by site directed mutagenesis using a synthetic oligonucleotide and the pMa/c vector system (Stanssens et al, 1987). The sequence TTTATGG at the initiation codon is converted to TCCATGG containing a Nco I site overlapping with the ATG initiation codon as described in Example 3. The amino acid sequence at the C-terminus of the 62kDa Bt18 toxin is highly homologous to the corresponding sequence in the 60 kDa Bt4 toxin. A DNA sequence, encoding the consensus sequence of Fig. 3 , is identified at nucleotide positions 2004 to 2039 of the bt4 gene in Fig 1 . A suitable Bcl I site for producing a C-terminal truncation is localized at position 2151 in Fig. 1 , 112 nucleotides downstream of the consensus sequence. This truncated bt4 gene directs the synthesis of a 70 kDa protein showing insect toxicity. T he consensus sequence of the bt4 gene identifies the bt4 gene fragment encoding the minimal C-terminal truncated toxic part of the Bt4 protoxin, as shown by expression of the gene fragment in E. coli. This amino acid sequence at the C-terminus is also highly homologous to the corresponding sequence in the Bt14 and Bt15 protoxins (Fig. 3 ). Example 5 : Transformation of tobacco and tomato plants Using standard procedures (Deblaere et al, 1988), the intermediate plant expression vectors (pGSTX50, pGSTX52-54 and pGSTX60-67) of Example 3 are transferred to the Agrobacterium strain C58C1Rif R (U.S. patent application 821,582; European patent application 86300291.1) carrying the disarmed Ti-plasmid pGV2260 (Deblaere et al, 1988). Transconjugants with cointegrate plasmids, consisting of pGV2260 and the respective intermediate plant expression vectors, are selected. These Agrobacterium strains are then used to transform tobacco plants (Nicotiana tabacum var. Petit Havana SR-1) and tomato plants ( Licopersicon esculentum) as described in European patent application 87400141.5. Example 6: Lepidoptera Resistance of the transformed tobacco and tomato plants of Example 5 The insecticidal activity, expressed in leaves of the transformed tomato and tobacco plants of Example 5, is evaluated by recording the growth rate and mortality of Spodoptera sp and Manduca sexta larvae fed on these leaves. These results are compared with the growth rate of larvae fed on untransformed leaves. Toxicity assays are performed as described in U.S. patent application 821,582 and European patent application 86300291.1. The mortality rate of Spodoptera sp. and M. sexta is significantly higher among those feeding on the transformed tomato and tobacco leaves. Needless to say, this invention is not limited to tobacco or tomato plants transformed with the bt4 gene or truncated bt4 gene. It also relates to any plant transformed with all or part of the bt4 gene, such as rapeseed, alfalfa, sunflowers, cotton, corn, soybeans, potato, brassicas, sugar beets and other vegetables. Also, DNA sequences other than those shown in Fig. 1 for the bt4 and truncated bt4 genes can be used for transforming plants. In this regard, the DNA sequences of Fig. 1 can be modified: 1) by replacing some codons with others that code either for the same amino acids or for other amino acids; and/or 2) by deleting or adding some codons; provided that such modifications do not substantially alter the insecticidal properties of the encoded proteins. Furthermore, other DNA recombinants containing the aforementioned DNA sequences in association with other foreign DNA sequences, particularly the DNA of vectors suitable for transforming plants, are encompassed by this invention. In this regard, this invention is not limited to the specific plasmids containing the bt4, truncated bt4, heretofore described, but rather encompasses any DNA recombinants containing a DNA sequence that is their equivalent. For example, the bt4 chimaeric gene of this invention can be provided between the T-DNA border repeats of a binary vector as described in European patent publication 270,822 to produce binary vectors containing all or part of the bt4 gene under the control of the TR2' promoter. Further, the invention relates to all DNA recombinants that include the respective bt4 or truncated bt4 gene and that are suitable for transforming microorganisms (e.g., plant-associated bacteria such as Pseudomonas, Xanthomonas or yeasts such as Streptomyces cerevisiae) under conditions which enable the bt4 or truncated bt4 gene to be expressed and to be recoverable from said microorganisms or to be transferred to a plant cell. Moreover, the minimum length, C-terminal end of a toxic part of a Bt4-like protoxin of this invention can include up to about 100, preferably no more than about 25, particularly no more than about 10, additional amino acids after the intact twelve-amino acid sequence of Figure 3 . In this regard, the twelve-amino acid sequence need not be the last C-terminal amino acids of such a toxic part of a Bt4-like protoxin, provided that any additional C-terminal amino acids do not substantially alter the insecticidal properties of the toxic part of the Bt4-like protoxin. REFERENCES Botterman J. and Zabeau M. (1987), DNA 6, 583-591. Brizzard B. and Whiteley H. (1988), Nucleic Acids Research 16 , 4168-4169. Deblaere R., Reynaerts A., Höfte H., Hernalsteens J.-P., Leemans J. and Van Montagu M. (1988), Methods in Enzymology 153, 277-292. Gardner, Howarth, Hahn, Brown-Luedi, Shepard and Messing (1981), Nucleic Acids Research 9 , 2871-2887. Gielen J, De Beukeleer M., Seurinck J., Deboeck F., De Greve H., Lemmer M., Van Montagu M. and Schell J. (1984), EMBO J. 3 , 835-845. Höfte H., Dissertation Thesis, State University of Ghent, Belgium, 1988. Höfte H., De Greve H., Seurinck J., Janssens S., Mahillon J., Ampe C., Vandekerckhove J., Vanderbruggen H., Van Montagu M., Zabeau M. and Vaeck M, (1986), Eur. J. Bioch 161 , 273-280. Höfte H., Van Rie J., Janssens S., Van Houtven A., Verbruggen H. and Valck M. (1988), Appl. Environ. Microbiol. 54 , 2010-2017. Hull and Howell (1987), Virology 86 , 482-483. Itakura K., Rossi J. and Wallace R. (1984), Annual Review of Biochemistry 53 , 323-356. Honée G., Van der Salm J. and Visser B. (1988), Nucleic Acids Research 16 , 6240. Laemmli U. (1970), Nature 227 , 680-685. Mahillon J. and Delcour J. (1984), J. Microbiol. Methods 3 , 69-76. Maxam A.M. and Gilbert W. (1980), Methods in Enzymol. 65 , 499-560. Odell J., Nagy F. and Chua N. H. (1985), Nature 313 , 810-812. Reiss B., Sprengel R. and Schaller H. (1984), EMBO J. 3 , 3317-3322. Stanssens P., McKeown Y., Friedrich K. and Fritz H.J (198 7 ), "Oligonucleotide-directed construction of mutations by the gapped duplex DNA method using the pMa/c plasmid vectors", published in the collection of additional experimental procedures distributed at the EMBO laboratory course on "Directed mutagenesis and protein engineering" in July 1987 at the Max Planck Institute für Biochimie, Martinsried, Federal Republic of Germany. Vaeck M., Reynaerts A., Höfte H., Jansens S., De Beukeleer M., Dean C., Zabeau M., Van Montagu M. and Leemans J. (1987), Nature 327 , 33-37. Velten J., Velten L., Hain R. and Schell J. (1984) EMBO J. 3 , 2723-2730. Velten J. and Schell J. (1985), Nucleic Acids Research 13, 6981-6998.

Claims: A DNA comprising the nucleotide sequence of Figure 1 from nucleotide position 345 to nucleotide position 2039. A DNA comprising the nucleotide sequence of Figure 1 from nucleotide position 264 to nucleotide position 3758. A DNA sequence encoding the protein of Figure 1 from amino acid position 28 to amino acid position 592. A DNA sequence encoding the protein of Figure 1 from amino acid position 1 to amino acid position 592. A DNA sequence encoding the protein of Figure 1 from amino acid position 1 to amino acid position 1165. A protein comprising the amino acid sequence of Figure 1 from amino acid position 28 to amino acid position 592. A protein comprising the amino acid sequence of Figure 1 from amino acid position 1 to amino acid position 592. A protein comprising the amino acid sequence of Figure 1 from amino acid position 1 to amino acid position 1165. A chimaeric gene, characterized by the following, operably linked, DNA sequences: (a) the DNA of any one of Claims 1 to 5, (b) a promoter capable of directing transcription of said DNA in a plant cell; and (c) suitable 3' transcription regulation signals for expressing said DNA in a plant cell. A transformed plant cell, comprising the chimaeric gene of Claim 9. A plant comprising the chimaeric gene of Claim 9. A seed of the plant of Claim 11, comprising the chimaeric gene of Claim 9. A process for rendering a plant resistant to an insect, characterized by the step of: transforming the plant genome with the chimaeric gene of Claim 9. The process of claim 13 wherein said insect is Manduca sexta or a Spodoptera species insect. The process of claim 14 wherein said Spodoptera insect is Spodoptera exigua. An insecticide composition against Lepidopteran insects, comprising the protein of any one of claims 6 to 8. The composition of Claim 16, wherein said Lepidopteran insects are Manduca sexta or a Spodoptera species insect. The composition of Claim 17, wherein said Spodoptera species insect is Spodoptera exigua. A microorganism transformed to contain the DNA of any one of Claims 1 to 5. The microorganism of Claim 19 which is E. coli, a plant-associated bacterium such as Pseudomonas or Xanthomonas, or a yeast such as Streptomyces cerevisiae.

Inventors: BOTTERMAN JOHAN, HOFTE HERMAN, JOOS HENK, PEFEROEN MARNIX, BOTTERMAN, JOHAN, HOFTE, HERMAN, JOOS, HENK, PEFEROEN, MARNIX
Applicants: BAYER BIOSCIENCE NV, BAYER BIOSCIENCE N.V.
Application year: 1989
Main classification: C12N15
IPC codes: C07K14, C12N15