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Orally active androctonus amoreuxi pesticidal biopeptides

Orally active androctonus amoreuxi pesticidal biopeptides description/claims

This application is a divisional of U.S. application Ser. No. 10/617,978, filed Jul. 11, 2003, which claims the benefit of U.S. Provisional Application No. 60/395,428, filed Jul. 12, 2002, each of which is hereby incorporated in its entirety by reference herein.

The official copy of the sequence listing is submitted concurrently with the specification as a text file via EFS-Web, in compliance with the American Standard Code for Information Interchange (ASCII), with a file name of 346403SequenceListing.txt, a creation date of Jul. 11, 2008, and a size of 28,403 bytes. The sequence listing filed via EFS-Web is part of the specification and is hereby incorporated in its entirety by reference herein.

The present invention relates to naturally occurring pesticides, in particular, to polypeptides isolated from arthropods. The invention further relates to methods of impacting pests, particularly insect pests, involving the pesticidal polypeptides and the corresponding nucleic acid molecules that encode the pesticidal polypeptides.

Chemical insecticides are an integral component of modern agriculture, and are an effective means for reducing crop damage by controlling insect pests. However, chemical agents are under continuous scrutiny due to the potential for environmental contamination, selection of resistant populations of agronomic pests, and toxicity to non-target organisms such as beneficial insects, aquatic organisms, animals and humans. As a result, alternative strategies for insect control are being sought that are effective and yet benign to non-target populations and the environment. One of these strategies is to utilize the mechanisms of naturally occurring pathogens of target pest populations.

Examples of this type of strategy include the use of various forms of the 6-endotoxin produced by the soil dwelling microorganism Bacillus thuringiensis (Bt) as pesticidal agents. These polypeptides have been found to be specifically toxic to particular insects, and microbial formulations have been used commercially for many years as foliarly applied insecticides. It has also recently been found that various forms of the Bt toxin can be toxic to insect pests when expressed inside the tissues of plants on which the insects feed.

Many arthropods express polypeptides capable of killing or incapacitating various pests. Therefore arthropods have been identified as a group of organisms producing polypeptides possessing pesticidal properties. In fact, scorpion venom contains insect-selective toxins affecting ion channels (Zlotkin et al. (1985) Arch. Biochem. Biophys. 240:877-87). The toxicity of the different polypeptides operates through multiple pharmacological and biochemical pathways (Zlotkin et al. (1971) Biochimie (Paris), 53:1073-1078).

Transgenic dicot plants expressing a toxin obtained from Androctonus australis scorpions have been created (U.S. Pat. No. 5,177,308). The AaIT expressing plants have been crossbred to transgenic plants carrying Bt δ-endotoxin yielding plants with two independent insect-specific toxin traits (U.S. Pat. No. 5,177,308).

Insects predominate as rice pests throughout Asia and are most serious in tropical regions where over 60 species are pests. The most serious rice insect pests are commonly categorized as Homoptera (sucking pests such as leafhoppers and planthoppers) and Lepidoptera (stem borers and defoliators).

The identification of new pesticidal polypeptides is desirable for use in pest-management strategies. It is of particular importance to identify pesticidal toxins that are active against insect pests from the orders Homoptera and Lepidoptera, and against insects that have developed resistance to Bt toxins.

Compositions and methods for enhancing plant pest resistance, particularly insect pests are provided. Compositions of the invention include isolated polypeptides, peptides, and amino acid sequences and nucleic acid molecules having a nucleotide sequence encoding the polypeptides, peptides, and amino acid sequences of the invention. The polypeptides of the invention were isolated from arthropod venom and telsons and possess pesticidal activity. In an embodiment, the polypeptides of the invention are orally active and affect pests upon ingestion. The invention provides expression cassettes, host cells, baculovirus expression vectors, transformed plant cells, and plants comprising nucleic acid molecules encoding the polypeptides of the invention. In an embodiment, a nucleotide sequence encoding a pesticidal polypeptide of the invention is inserted into the genome of a baculovirus to produce a pesticidal recombinant baculovirus. In this embodiment the presence of the pesticidal polypeptide in the baculovirus increases the efficiency with which the virus acts to kill or incapacitate the pest, thus, enhancing the effectiveness of the baculovirus as an adjunct or replacement for chemical pest control agents.

In an embodiment of the invention, the promoter to which a nucleotide sequence of the invention or a nucleotide sequence encoding a polypeptide of the invention is operably linked is constitutive, inducible, or tissue-preferred. An embodiment of the invention comprises a vascular tissue preferred promoter operably linked to a nucleotide sequence encoding a polypeptide of the invention. The operably linked promoter is an insect-inducible promoter in one embodiment of the invention.

Methods for altering the pest resistance of a plant are provided. The plant's resistance to pests such as, but not limited to, insects and the pathogens transmitted by insects, may be altered by the methods of the invention. A method of the invention comprises stably transforming into a plant cell a nucleic acid molecule encoding a polypeptide of the invention. The nucleic acid molecule is operably linked to a promoter capable of driving transcription in the plant cell. The transformed plant cell is used to generate a transformed plant. The transformed plant cell and transformed plant are capable of expressing a polypeptide of the invention in the plant cell or plant cells. The polypeptide of the invention possesses pesticidal activity. In an embodiment the pesticidal activity of the polypeptide of the invention is orally active upon ingestion by an insect such as, but not limited to, an insect of the Homopteran, Lepidopteran, and Hymenopteran orders. In an embodiment, the insect infected by the pesticidal activity of the invention exhibits resistance to a Bt toxin.

Methods for identifying polypeptides possessing oral pesticidal activity are provided. In an embodiment, polypeptides are isolated from arthropod venom. The venom is obtained from isolated venom glands. The isolated venom glands have been surgically removed from the organism. In another embodiment, the venom is obtained by harvesting venom from the organism through a process such as, but not limited to, milking the organism. The polypeptides are combined with at least one nutrient to generate a polypeptide solution. The polypeptide solution is fed to insects. Insecticidal activity is assayed. Insecticidal activity includes, but is not limited to, mortality, weight loss, attraction, and repellency.

Some pesticidal polypeptides modulate sodium channels and are sodium channel neurotoxins. In another embodiment of the invention, nucleic acid sequences encoding potential sodium channel neurotoxins are identified from amino acid and nucleotide sequence data derived from a cDNA library of arthropod telsons. The open reading frames encoding potential sodium channels neurotoxins are amplified from the cDNA and cloned into appropriate expression cassettes. The potentially pesticidal amino acids are tested for oral pesticidal activity.

Compositions of the invention provide antibodies that selectively bind to a polypeptide of the invention.

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