# US 2014/0194351 A1 · Hemipteran-and Coleopteran Active Toxin Proteins from Bacillus Thuringiensis

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- Number: US 2014/0194351 A1
- Document ID: US20140194351A1
- Status: Published application
- Kind: A1
- Application number: 14/107,453
- Filed: 2013-12-16
- Published: 2014-07-10
- Earliest claimed: 2007-04-27
- Assignees: Monsanto Technology LLC
- Inventors: James A. Baum; Stanislaw Flasinski; Gregory R. Heck; Stephen R. Penn; Uma Rao Sukuru; Xiaohong Shi
- Web page: https://patentgrep.com/patent/US20140194351A1

## CPC

- C12N 15/8286: {for insect resistance}
- A01N 63/50: Isolated enzymes; Isolated proteins (peptides A01N37/46)
- C07K 14/325: Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins
- Y02A 40/146: Genetically Modified [GMO] plants, e.g. transgenic plants
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A01N: PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF (preservation of food or foodstuff A23); BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES (preparations for medical, dental or toiletry purposes which kill or prevent the growth or proliferation of unwanted organisms A61K); PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

A novel Bacillus thuringiensis crystal protein exhibiting insect inhibitory activity is disclosed. Growth of Lygus insects is significantly inhibited by providing the novel crystal protein in Lygus insect diet. Polynucleotides encoding the crystal protein, transgenic plants and microorganisms that contain the polynucleotides, isolated peptides derived from the crystal protein, and antibodies directed against the crystal protein are also provided. Methods of using the crystal protein and polynucleotides encoding the crystal protein to control Hemipteran insects are also disclosed.

## Claims

### Claim at position 1

1-20. (canceled)

### Claim 21

A DNA construct comprising a heterologous promoter operably linked to a polynucleotide sequence encoding an insect inhibitory protein exhibiting at least 99% identity to the amino acid sequence as set forth in SEQ ID NO:5.

### Claim 22 (depends on 21)

The DNA construct of claim 21, wherein said insect inhibitory protein exhibits 100% sequence identity to SEQ ID NO:5.

### Claim 23 (depends on 21)

The DNA construct of claim 21, wherein said insect inhibitory protein inhibits a Hemipteran insect, a Heteropteran insect, a Leptinotarsa sp. insect or a Homopteran insect.

### Claim 24 (depends on 23)

The DNA construct of claim 23, wherein said Hemipteran insect is Lygus.

### Claim 25 (depends on 23)

The DNA construct of claim 23, wherein said Homopteran insect is an aphid, a hopper, or a whitefly.

### Claim 26 (depends on 21)

The DNA construct of claim 21, wherein said polynucleotide sequence is selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:44, SEQ ID NO: 45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53.

### Claim 27 (depends on 21)

A transgenic plant, or a plant part derived therefrom, comprising the DNA construct of claim 21.

### Claim 28 (depends on 21)

A transformed host cell comprising the DNA construct of claim 21.

### Claim 29 (depends on 28)

The transformed host cell of claim 28, wherein said host cell is a bacterial cell or a plant cell.

### Claim 30 (depends on 29)

A plant derived from the transformed host cell of claim 29, said plant comprising said DNA construct.

### Claim 31 (depends on 30)

A seed produced from the plant of claim 30, said seed comprising said DNA construct.

### Claim 32 (depends on 31)

A progeny plant from the seed of claim 31, said progeny plant comprising said DNA construct.

### Claim 33

A method for controlling a Lygus insect, said method comprising:
(a) providing a Lygus inhibitory amount of a TIC807 insect inhibitory protein or an insect inhibitory protein fragment derived therefrom, wherein said TIC807 insect inhibitory protein exhibits at least 99% sequence identity to SEQ ID NO:5; and
(b) contacting said Lygus insect with said inhibitory amount of said TIC807 insect inhibitory protein or said insect inhibitory protein fragment derived therefrom, thereby controlling said Lygus insect.

### Claim 34 (depends on 33)

The method of claim 33, wherein said Lygus inhibitory amount of said TIC807 insect inhibitory protein or said insect inhibitory protein fragment derived therefrom is provided in a Lygus diet in step (a) and said Lygus is contacted in step (b) by permitting said Lygus to feed on said diet.

### Claim 35 (depends on 34)

The method of claim 34, wherein said Lygus diet is a transgenic plant, said transgenic plant comprising a DNA construct comprising a heterologous promoter operably linked to a polynucleotide sequence encoding an insect inhibitory protein exhibiting at least 99% sequence identity to SEQ ID NO:5.

### Claim 36 (depends on 33)

The method of claim 33, wherein said Lygus inhibitory amount of said TIC807 insect inhibitory protein or said insect inhibitory protein fragment derived therefrom is provided in step (a) by spraying on a plant a composition comprising a DNA construct comprising a heterologous promoter operably linked to a polynucleotide sequence encoding an insect inhibitory protein exhibiting at least 99% sequence identity to SEQ ID NO:5.

### Claim 37

An insect inhibitory composition comprising a TIC807 insect inhibitory protein that exhibits at least 99% sequence identity to SEQ ID NO:5.

### Claim 38 (depends on 37)

The insect inhibitory composition of claim 37, further comprising at least one other insect inhibitory agent that is different from said TIC807 insect inhibitory protein, wherein said insect inhibitory agent is selected from the group consisting of ET29, ET37, TIC809, TIC810, TIC812, TIC127, TIC128, AXMI-027, AXMI-036, AXMI-038, AXMI-018, AXMI-020, and AXMI-021, AXMI-010, AXMI-003, AXMI-008, AXMI-006, AXMI-007, AXMI-009, AXMI-014, AXMI-004, AXMI-028, AXMI-029, and AXMI-0080rf2.

### Claim 39 (depends on 37)

A method for controlling at least one insect pest, said method comprising:
(a) providing an insect inhibitory amount of the insect inhibitory composition of claim 37; and
(b) contacting said insect pest with said insect inhibitory composition.

### Claim 40 (depends on 39)

The method of claim 39, wherein said at least one insect pest is a Lygus insect.