Mycogen Plant Science, Inc.

University of Georgia Research Foundation Inc

Dow Agrosciences LLC

Stine Seed Farm, Inc.

Michael J Adang

Elizabeth E Murray

Gang Hua

Donald J Merlo

John D Kemp

Thomas A Rocheleau

Mohd Amir Fursan Abdullah

Juan Luis Jurat Fuentes

Rebecca McNall

Ke Luo

Ebrahim Firoozabady

Richard F Barker

Thomas J McLoughlin

Janet A Klein

Rui Zhang

Carolyn A Stock

Jiang Chen

Mohd Amir-Fursan Abdullah

Jian (John) Chen

The subject invention relates in part to a combination of a cadherin peptide derived fromand a Cry protein capable of inhibiting an insect. In certain embodiments, the combination is capable of overcoming Cry protein resistance in insects that are resistant to the Cry protein alone.

Cadherin fragments for managing insect resistance to cry proteins

The subject invention relates in part to fragments of non-cadherin Cry toxin binding proteins, wherein the fragments potentiate, or act as synergists with, the insecticidal activity of Cry proteins. In some preferred embodiments, the binding protein (a Cry protein receptor on insect midgut cells) is an aminopeptidase. In preferred embodiments, the fragment comprises a Cry protein binding region.

Non-cadherin polypeptide potentitators of cry proteins

This invention relates in part to modifying BtBooster (BtB) peptides, in part to increase their stability in insect midgut digestive juices. Some preferred embodiments of BtB have removed proteinase cleavage sites resulting in increased stability of the modified BtB in the insect gut, while retaining the ability to enhance B.t. proteins for improved insect control. In some preferred embodiments, the protease-stable BtB is used in combination with B.t. spores and/or crystals comprising a Cry protein. Also reported herein is the significant and increased enhancement of Bt toxins against relatively Bt-tolerant insects (and), when used with BtBs. We also describe increased toxin enhancement with cadherin fragments that are stabilized against over-digestion by insect midgut proteinases. We also report enhancement of Bt Cry1F toxin by cadherin fragments.

Modified peptides having toxin-enhancing effects

The subject invention relates in part to the surprising and unexpected discovery that insects that are resistant toCry toxins have measurably altered alkaline phosphatase (ALP) activity as compared to insects that are susceptible to Cry toxins. This and other surprising discoveries reported herein have broad implications in areas such as managing and monitoring the development of insect resistance to B.t. toxins. For example, the subject invention provides a simple and fast assay (enzymatic or otherwise) for detecting ALP activity levels and thereby monitoring the development of resistance by insects to crystal protein insect toxins. There was no prior motivation or suggestion to go about resistance monitoring using this simple and easy approach.

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The subject invention relates in part to the discovery that a fragment from a cadherin of the western corn rootworm enhances Cry3 toxicity to larvae of naturally susceptible species. The subject invention also relates in part to the discovery that a cadherin fragment from a beetle enhances Cry3Aa and Cry3Bb toxicity to coleopteran larvae, particularly those in the family Chrysomelidae. Such cadherin fragments are referred to as Bt Boosters (BtBs). The subject invention can be extended to the use of BtBs with other coleopteran-toxic Cry proteins for controlling a wide range of coleopterans.

The subject invention pertains to the use of peptide fragments of cadherins (including cadherin-like proteins). The subject invention includes a cell (and use thereof) comprising a polynucleotide that expresses the peptide fragment. The subject invention includes methods of feeding the peptides to insects. In preferred embodiments, the peptides are fed to target insects together with one or more insecticidal proteins, preferably (but not limited to) B.t. Cry proteins. When used in this manner, the peptide fragment can not only enhance the apparent toxin activity of the Cry protein against the insect species that was the source of the receptor but also against other insect species. Preferably, the cadherin is a(B.t.) insecticidal crystal protein (Cry) toxin receptor. Preferably, the peptide fragment is a binding domain of the receptor. In some preferred embodiments, the peptide is the binding domain nearest to the membrane proximal ectodomain. Corresponding domains are identifiable in a variety of B.t. toxin receptors.

Peptides for inhibiting insects

The subject invention pertains to the use of peptide fragments of cadherins (including cadherin-like proteins). The subject invention includes a cell (and use thereof) comprising a polynucleotide that expresses the peptide fragment. The subject invention includes methods of feeding the peptides to insects. In preferred embodiments, the peptides are fed to target insects together with one or more insecticidal proteins, preferably (but not limited to). Cry proteins. When used in this manner, the peptide fragment can not only enhance the apparent toxin activity of the Cry protein against the insect species that was the source of the receptor but also against other insect species. Preferably, the cadherin is a() insecticidal crystal protein (Cry) toxin receptor. Preferably, the peptide fragment is a binding domain of the receptor. In some preferred embodiments, the peptide is the binding domain nearest to the membrane proximal ectodomain. Corresponding domains are identifiable in a variety of. toxin receptors.

Method for inhibiting insects with an insect cadherin ectodomain

A method for expressing insecticidal protein structural genes in cotton plant genomes is provided. In the preferred embodiments this invention comprises placing a structural gene for thecrystal protein under control of a plant or a T-DNA promoter and ahead of a polyadenylation site followed by insertion of said promoter/structural gene combination into a plant genome by utilizing anTi plasmid-based transformation system. The modified Ti plasmid is then used to transform recipient plant cells. Also provided are the plants and tissues produced by this method and bacterial strains, plasmids, and vectors useful for execution of this invention.

Insect resistant cotton plants

Materials and methods for identifying novel pesticide agents are disclosed herein. Specifically exemplified is a full length aminopeptidase N isolated from, insect cells expressing APN, and methods of screening pesticide agents using the same. Also disclosed are methods of identifying novel APN inhibitors.

Methods and materials for identifying novel pesticide agents

A method for expressing insecticidal protein structural genes in plant genomes is provided. In the preferred embodiments this invention comprises placing a structural gene for thecrystal protein under control of a plant or a T-DNA promoter and ahead of a polyadenylation site followed by insertion of said promoter/structural gene combination into a plant genome by utilizing anTi plasmid-based transformation system. The modified Ti plasmid is then used to transform recipient plant cells. Also provided are the plants and tissues produced by this method and bacterial strains, plasmids, and vectors useful for execution of this invention.

Insect resistant plants

Materials and methods for identifying novel pesticide agents are disclosed herein. Specifically exemplified is a full length aminopeptidase N isolated from , insect cells expressing APN, and methods of screening pesticide agents using the same. Also disclosed are methods of identifying novel APN inhibitors.

The,var. kurstaki HD-73 crystal protein gene was cloned into pBR322.,cells harboring this recombinant plasmid produced a 130 kD protoxin that was toxic to,(tobacco hornworm) larvae. Plasmids having the 3&prime;-end of the protoxin gene deleted where also constructed.,cells harboring these deleted plasmids produced an active, soluble 68 kD toxin, provided that the 3&prime;-deletion had not removed sequences encoding the 68 kD toxin. The invention provides methods to produce 68 kD toxin protein by constructing partial protoxin genes encoding the toxin followed by expression of the genes in living cells. Useful plasmids and cells are also provided.

Insecticidal protein fragments

The,HD-73 crystal protein gene was cloned into pBR322.,cells harboring this recombinant plasmid produced a 130 kD protoxin that was toxic to,(tobacco hornworm) larvae. Plasmids having the 3&prime;-end of the protoxin gene deleted where also constructed.,cells harboring these deleted plasmids produced an active, soluble 68 kD toxin, provided that the 3&prime;-deletion had not removed sequences encoding the 68 kD toxin. The invention provides methods to produce 68 kD toxin protein by constructing partial protoxin genes encoding the toxin followed by expression of the genes in living cells. Useful plasmids and cells are also provided.

The Bacillus thuringiensis var. kurstaki HD-73 crystal protein gene was cloned into pBR322. E. coli cells harboring this recombinant plasmid produced a 130 kD protoxin that was toxic to Manduca sexta (tobacco hornworm) larvae. Plasmids having the 3'-end of the protoxin gene deleted where also constructed. E. coli cells harboring these deleted plasmids produced an active, soluble 68 kD toxin, provided that the 3'-deletion had not removed sequences encoding the 68 kD toxin. The invention provides methods to produce 68 kD toxin protein by constructing partial protoxin genes encoding the toxin followed by expression of the genes in living cells. Useful plasmids and cells are also provided.

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Athens, GA, United States of America

Michael J Adang