Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
2001-05-17
2004-03-16
Carlson, Karen Cochrane (Department: 1638)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C514S002600, C514S012200, C536S023700, C424S405000, C424S093460
Reexamination Certificate
active
06706860
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to new insecticidal secreted proteins (“ISPs”) isolated from a bacterial strain, preferably a Brevibacillus species strain, most preferably a
Brevibacillus laterosporus
species strain which are insecticidal when ingested in combination with an ISP-complimentary protein such as another ISP protein of this invention, and to DNA sequences encoding such proteins. These proteins are useful to prevent or minimize insect damage, particularly of corn rootworms, to plants in a field.
The present invention also relates to plants, particularly corn plants, that are rendered insecticidal, preferably to coleopteran insects, particularly to Diabrotica spp., Leptinotarsa spp. and Anthonomus species insects, by the expression of the ISP proteins of this invention in cells of said plants.
The present invention also relates to a method for controlling damage by Diabrotica spp., Leptinotarsa spp. or Anthonomus species insects, preferably Diabrotica spp. insect pests, particularly corn rootworms, by having the ISP proteins of the invention, particularly the proteins with the amino acid sequence of any one of SEQ ID No. 2, 4, 8 or 10, or insecticidally-effective fragments thereof, ingested by said insects.
2. Description of the Prior Art
Some of the most destructive pests are found among the Diabroticine beetles. In North America, the three important species of corn rootworms,
Diabrotica virgifera
(the Western corn rootworm),
Diabrotica barberi
(the Northern corn rootworm) and
Diabrotica undecimpunctata howardi
(the Southern corn rootworm) are considered to be the most expensive insect pests to control (Metcalf, 1986, Foreword in “Methods for the Study of Pest Diabrotica”, pp. vii-xv, eds. Krysan, J. L. and Miller, T. A., Springer-Verlag, New York).
Diabrotica virgifera
and
Diabrotica barberi
are considered the most serious insect pests of corn in the major corn-producing states of the United States and Canada (Levine and Oloumi-Sadeghi, 1991, Annu. Rev. Entomol. 36, 229-55). The larvae feed on the roots and thus cause direct damage to corn growth and corn yields. Costs for soil insecticides to control larval damage to the root systems of corn and aerial sprays to reduce beetle damage to corn silks, when combined with crop losses, can approach one billion dollars annually (Metcalf, 1986, supra). Recently, in some US states it was discovered that the crop rotation program of planting soybeans after corn lost its effect as corn rootworms have adapted to this situation.
Bacterial strains and/or genes with toxicity to corn rootworm have been described in U.S. Pat. Nos. 6,023,013; 6,015,553; 6,001,637; 5,906,818; and 5,645,831. Also, PCT publications WO 00/09697, WO 99/57282, WO 98/18932, WO 97/40162, and WO 00/26378 relate to toxins and genes obtainable from Bacillus or other bacterial spp., some of which are described to have toxicity to corn rootworm. WO 98/44137, WO 94/21795 and WO 96/10083 relate to pesticidal Bacillus strains, characterized by pesticidal proteins and auxiliary proteins produced during vegetative growth, some of which are described to have toxicity to corn rootworm. U.S. Pat. No. 5,055,293 describes a method to control corn rootworms by inoculating soil with parasporal-inclusion forming species of
Bacillus laterosporus.
Orlova et al. (1998, Applied Environmental Microbiol. 64, 2723) showed insecticidal activity to mosquitoes associated with protein crystals in crystal-forming strains of
Bacillus laterosporus.
OBJECTS AND SUMMARY OF THE INVENTION
An object of the present invention is to provide novel proteins and DNA sequences encoding such proteins with significant toxicity to insects, preferably Diabrotica spp. insects, particularly corn rootworm.
In one embodiment of the invention, a protein is provided comprising the amino acid sequence of the smallest active toxin of the protein of SEQ ID No. 2, wherein said smallest active toxin is:
a) a fragment of the protein of SEQ ID No. 2, and
b) insecticidal to
Diabrotica virgifera
larvae when ingested by said larvae in combination with the protein of SEQ ID No. 4 from amino acid position 51 to 457. Also provided is a protein comprising the amino acid sequence of the smallest active toxin of the protein of SEQ ID No. 4,
wherein said smallest active toxin is:
a) a fragment of the protein of SEQ ID No. 4, and
b) insecticidal to
Diabrotica virgifera
larvae when ingested by said insect in combination with the protein of SEQ ID No. 2 from amino acid position 38 to 871.
Particularly preferred is a protein characterized by an amino acid sequence comprising the sequence of SEQ ID No. 2 from amino acid position 38 to amino acid position 768 or 781, preferably the protein characterized by the amino acid sequence of SEQ ID No. 2 or SEQ ID No. 10; and a protein characterized by an amino acid sequence comprising the sequence of SEQ ID No. 4 from amino acid position 51 to amino acid position 449 or 457, preferably a protein characterized by the amino acid sequence of SEQ ID No. 4 or SEQ ID No. 8.
A further object of the invention is a protein comprising the amino acid sequence of the protease-digestion fragment of the protein encoded by the isp1A DNA deposited at the BCCM-LMBP under accession number LMBP 4009, which protease-digestion fragment is insecticidal to
Diabrotica virgifera
upon combined application with the protein of SEQ ID. No. 4 from amino acid position 51 to amino acid position 457; and a protein comprising the amino acid sequence of the protease-digestion fragment of the protein encoded by the isp2A DNA deposited at the BCCM-LMBP under accession number LMBP 4009, which protease-digestion fragment is insecticidal to
Diabrotica virgifera
upon combined application with the protein of SEQ ID No. 2 from amino acid position 38 to amino acid position 871; particularly wherein said protease-digestion fragment is obtainable by treatment with coleopteran gut juice.
Also provided in accordance with this invention is a DNA sequence encoding the above proteins, particularly a DNA comprising an artificial DNA sequence having a different codon usage compared to the naturally occurring DNA sequence but encoding the same protein sequence, preferably contained in a chimeric gene operably linked to a plant-expressible promoter region (i.e., a promoter region which is suitable for expression in plant cells, this can be from bacterial, viral or plant origin or can be artificially made); particularly a promoter region which is preferentially active in root tissue.
In one embodiment of the invention, the promoter in said chimeric gene comprises the DNA sequence of SEQ ID No. 5 or 6 or a DNA hybridizing thereto under stringent hybridization conditions.
In a further embodiment of this invention, the chimeric gene further comprises a signal peptide for secretion from the cell or for targeting to a cellular organelle, particularly a chloroplast transit peptide.
Also provided is a plant cell, a plant or a seed, comprising any of these chimeric genes integrated in their cells, particularly a combination of the chimeric gene encoding the ISP1A, or an insecticidally effective fragment thereof, and the chimeric gene encoding the ISP2A protein, or an insecticidally effective fragment thereof; particularly a corn cell, plant or seed.
In another embodiment of this invention, a micro-organism transformed to contain any of the above DNA sequences is provided.
Also provided is a process for controlling insects, particularly a process for rendering a plant resistant to coleopteran insects, comprising expressing any of the ISP proteins of this invention in cells of a plant, and regenerating transformed plants from said cells which are resistant to insects. In such process, the insect is preferably selected from the group consisting of: rootworms, weevils, potato beetles, Diabrotica species, Anthonomus spp., Leptinotarsa spp.,
Agelastica alni, Hypera postica, Hypera brunneipennis, Haltica tombacina, Anthonomus grandis, Tenebrio molitor, Triboleum castaneum, Dicladispa
Arnaut Greta
Boets Annemie
Damme Nicole
Van Rie Jeroen
Bayer BioScience N.V.
Carlson Karen Cochrane
Liu Samuel Wei
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