Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
2000-01-18
2003-10-07
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C800S279000, C800S288000, C536S023700, C435S468000, C435S418000, C435S419000, C435S252300, C435S255100, C435S258100, C435S071100
Reexamination Certificate
active
06630619
ABSTRACT:
FIELD OF THE INVENTION
The present invention concerns the identification and isolation of a new class of protein toxins with specificity for insects, which are produced by bacteria from the genera Xenorhabdus and Photorhabdus. In addition, the present invention relates to the incorporation of genes encoding this class of toxin into, for example, insect-specific viruses (including entomopox and nuclear polyhedrosis viruses), bacteria (including Gracilicutes, Firmicutes, Tenericutes and Mendosicutes), yeast and plants for control of insect pests.
BACKGROUND OF THE INVENTION
Insect pathogenic nematodes of the families Steinernematidae and Heterorhabditidae are known to be symbiotically associated with bacteria of the genera Xenorhabdus and Photorhabdus respectively. It has been observed that these bacteria have the ability to kill a wide range of different insects without the aid of their nematode partners. The present inventors have isolated polynucleotide molecules encoding a new class of protein insecticidal toxins from
Xenorhabdus nematophilus
strain A24 and
Photorhabdus luminescens
strain V16/1.
DISCLOSURE OF THE INVENTION
In a first aspect, the present invention provides an isolated polynucleotide molecule encoding an insecticidal toxin, said polynucleotide molecule comprising a nucleotide sequence which substantially corresponds to the nucleotide sequence shown as SEQ ID NO: 1 or SEQ ID NO: 2.
In a second aspect, the present invention provides an isolated polynucleotide molecule encoding an insecticidal toxin, said polynucleotide molecule comprising a nucleotide sequence having at least 85%, more preferably at least 95%, sequence identity to the nucleotide sequence shown as SEQ ID NO: 2.
In a third aspect, the present invention provides an insecticidal toxin, in a substantially pure form, which toxin comprises an amino acid sequence having at least 95% sequence identity to that shown as SEQ ID NO: 3.
In a fourth aspect, the present invention provides an insecticidal toxin, in a substantially pure form, which toxin comprises an amino acid sequence having at least 85%, more preferably at least 95%, sequence identity to that shown as SEQ ID NO: 4.
Most preferably, the insecticidal toxin of the third or fourth aspect comprises an amino acid sequence substantially corresponding to that shown as SEQ ID NO: 3 or SEQ ID NO: 4 respectively.
In a fifth aspect the present invention provides a recombinant microorganism, the recombinant microorganism being characterised in that it is transformed with and expresses the polynucleotide molecule of the first or second aspects of the present invention.
The microorganisms which may be usefully transformed with the polynucleotide molecule of the first or second aspects of the present invention include bacteria, such as Escherichia, Gracilicutes, Firmicutes, Tenericutes and Mendosicutes; protozoa and yeast. The microorganism can be transformed by routine methods using expression vectors comprising the toxin-encoding polynucleotide molecule operably linked to a suitable inducible or constitutive promoter sequence.
In a sixth aspect, the present invention provides a method of producing an insecticidal toxin, said method comprising:
(i) culturing a microorganism according to the fourth aspect under conditions suitable for the expression of the toxin-encoding polynucleotide molecule, and
(ii) optionally recovering the expressed insecticidal toxin.
In a seventh aspect, the present invention provides a recombinant insect-specific virus, the recombinant insect-specific virus being characterised in that it includes within a non-essential region of its genome the polynucleotide molecule of the first or second aspects of the present invention operably linked to a suitable inducible or constitutive promoter sequence.
The recombinant insect-specific virus of the seventh aspect is preferably selected from entomopox and nuclear polyhedrosis viruses. The recombinant virus can be produced by routine methods such as homologous recombination.
In an eighth aspect, the present invention provides a method for killing pest insects, said method comprising applying to an area infested with said insects an effective amount of a recombinant microorganism according to the fourth aspect and/or a recombinant virus according to the seventh aspect, optionally in admixture with an acceptable agricultural carrier.
In a ninth aspect, the present invention provides a plant transformed with, and capable of expressing, the polynucleotide molecule of the first or second aspects of the present invention.
The plant according to the ninth aspect may be any plant of agricultural, arboricultural, horticultural or ornamental value that is susceptible to damage by feeding pest insects. However, preferably, the plant is selected from plants of agricultural value such as cereals (e.g.; wheat and barley), vegetable plants (e.g.; tomato and potato) and fruit trees (e.g., citrus trees and apples). Other preferred plants include tobacco and cotton.
The plant can be transformed by routine methods including Agrobacterium transformation and electroporation. Preferably, the toxin-encoding polynucleotide molecule is operably linked to a suitable inducible or constitutive promoter sequence. Particularly preferred promoter sequences include the cauliflower mosaic virus (CaMV 35 S) promoter element and promoter elements from the sub-clover stunt virus (SCSV).
The term “substantially corresponds” as used herein in relation to the nucleotide sequence is intended to encompass minor variations in the nucleotide sequence which due to degeneracy do not result in a change in the encoded protein. Further this term is intended to encompass other minor variations in the sequence which may be required to enhance expression in a particular system but in which the variations do not result in a decrease in biological activity of the encoded protein.
The term “substantially corresponding” as used herein in relation to the amino acid sequence is intended to encompass minor variations in the amino acid sequence which do not result in a decrease in biological activity of the insecticidal toxin. These variations may include conservative amino acid substitutions. The substitutions envisaged are:
G, A, V, I, L, M; D, E; N, Q; S, T; K, R, H; F, Y, W, H; and P. N&agr;-alkalamino acids.
The term “comprise”, “comprises” and “comprising” as used throughout the specification are intended to refer to the inclusion of a stated step, component or feature or group of steps, components or features with or without the inclusion of a further step, component or feature or group of steps, components or features.
The invention will hereinafter be further described by way of reference to the following, non-limiting example and accompanying figures.
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Jouanin et al. Transgenic plants for insect resistance Plant Science 131 1998 1-11.*
Smigocki et al. cytokinin-mediated insect resistance in Nicotiana plants transformed with the ipt gene 23: 325-335 1993.*
Pang et al. Expression of a gene encoding a scorpion insectotoxin peptide in yeast, bacteria and plants Gene 116 1992 165-172.*
Broun et al. Catalytic plasticity of fatty acid modification enzymes underlying chemical diversityy of plant lipids Science vol. 282 Nov. 13, 1998.*
Lazar et al. Transforming growth factor a: mutation of aspartic acid 47 and leucine 48 resultd in different biological activites Mar. 1988 p. 1247-1252 vol. 8, No. 3.*
Bowie et al. Deciphering the message in protein sequences: tolerance to amino acid substitutions Science, vol. 247.*
Hongsthong et al: “Optimum conditions for insecticidal toxin production by Photorhabdus luminescens” Abstracts of the General Meeting of the American Society for Microbiology, The Society, Washington, DC, US, No. 95, May 1, 1995, pp. 408-AbstrQ-48, XP002076055 ISSN: 1060-2011.
Bowen DJ et al
Commonwealth Scientific and Industrial Organisation
Fox David T.
Kubelik Anne
McDermott & Will & Emery
LandOfFree
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