Novel insecticidal proteins and methods of use

Patent metadata

Field	Value
Publication number	US-2016376607-A1
Application number	US-201615197161-A
Country	US
Kind code	A1
Filing date	Jun 29, 2016
Priority date	Mar 14, 2013
Publication date	Dec 29, 2016
Grant date	—

Abstract

Compositions and methods for controlling pests are provided. The methods involve transforming organisms with a nucleic acid sequence encoding an insecticidal protein. In particular, the nucleic acid sequences are useful for preparing plants and microorganisms that possess insecticidal activity. Thus, transformed bacteria, plants, plant cells, plant tissues and seeds are provided. Compositions are isolated insecticidal proteins and nucleic acids. The sequences find use in the construction of expression vectors for subsequent transformation into organisms of interest, as probes for the isolation of other homologous (or partially homologous) genes. The insecticidal proteins find use in controlling or killing lepidopteran, coleopteran, dipteran, fungal, hemipteran, and nematode pest populations and for producing compositions with insecticidal activity.

First claim

That which is claimed:

1 . An isolated insecticidal protein having a structure comprising:
a) a Domain I, comprising a surface hydrophobic patch and a type 1′ β-turn; b) a Domain II; and c) a Domain III, wherein the surface of Domain Hand Domain III comprise a stripe of solvent exposed serine and threonine residues.

2 . The isolated insecticidal protein of claim 1 , wherein Domain I comprises an anti-parallel β-sheet with four short strands and four α-helices designated as helix 1, helix 2, helix 3, and helix 4.

3 . The isolated insecticidal protein of claim 2 , wherein the anti-parallel β-sheet of Domain I comprises the C-terminal end of strand 11, strand 12, and the type 1′ β-turn joins strand 11 and strand 12.

4 . The isolated insecticidal protein of claim 2 , wherein Domain I further comprises a β-hair-pin between helix 2 and helix 3.

5 . The isolated insecticidal protein of claim 1 , wherein the surface hydrophobic patch comprises residues from strand 12, helix 2, helix 3, and the type 1′ β-turn between strand 11 and strand 12.

6 . The isolated insecticidal protein of claim 5 , wherein the hydrophobic patch is about 180-220 Å 2 .

7 . The isolated insecticidal protein of claim 6 , wherein the hydrophobic patch comprises residues corresponding to Phe26, Va148, Pro50, 11e52, Tyr56, Met193, Va1202, His205, Tyr206, Phe207, Trp208, Phe209, and Leu210 of SEQ ID NO: 2.

8 . The isolated insecticidal protein of claim 1 , wherein Domain 1 comprises residue 1 to about residue 65 and about residue 187 to about residue 223 corresponding to the residues of SEQ ID NO: 2.

9 . The isolated insecticidal protein of claim 1 , wherein Domain II comprises five-stranded anti-parallel β-sheet (β5/6-β11-β13-β7-β10) patched on one side by an amphipathic β-hairpin stemmed from β2 and β10.

10 . The isolated insecticidal protein of claim 1 , wherein Domain II comprises about residue 66 to about residue 79; about residue 104 to about residue 154; about residue 175 to about residue 186; and about residue 224 to about residue 234 corresponding to the residues of SEQ ID NO: 2.

11 . The isolated insecticidal protein of claim 1 , wherein Domain III comprises the same five β-strands of Domain II which extend and refold into domain III with a beta-sandwich structure, wherein the three strands β5/6, β11, and β13 make a180° twist in the middle forming a new 3 stranded β-sheet as one side of a β-sandwich and β7 and β10 spray from the central sheet, twist in middle, and hydrophobically pack against strands β5/6, β11, and β13.

12 . The isolated insecticidal protein of claim 1 , wherein Domain III comprises from about residue 80 to about residue 103; about residue 155 to about residue 174; and about residue 235 to about residue 246 corresponding to the residues of SEQ ID NO: 2.

13 . The isolated insecticidal protein of claim 1 , wherein the stripe of exposed serine and threonine residues on the surface of Domain II and Domain III comprises: 3 serine and 4 threonine residues in an alternating motif on strand 11, and 3 serine and 2 threonine residues on strand 7.

14 . The isolated insecticidal protein of claim 13 , wherein the 3 serine and 4 threonine residues on strand 11 have an average solvent accessibility of about 78.5A 2 and the 3 serine and 2 threonine residues on strand 7 average solvent exposure of about 59.3 A 2 .

15 . The isolated insecticidal protein of claim 1 wherein the protein has an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:2;
(b) a protein that is encoded by the nucleotide sequence of SEQ ID NO:1; and
(c) an amino acid sequence having at least 85% sequence identity to an amino acid sequence of (a) or (b).

16 . An isolated nucleic acid molecule selected from the group consisting of:
(a) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1; (b) a nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2; (c) a nucleotide sequence encoding a protein having insecticidal activity comprising an amino acid sequence characterized by at least 85% sequence identity to an amino acid sequence of (b); and (d) a nucleotide sequence encoding a protein having insecticidal activity comprising an amino acid sequence characterized by at least 85% sequence identity to an amino acid sequence of (b).

17 . The isolated nucleic acid molecule of claim 16 , wherein said nucleic acid molecule is a synthetic molecule that has been designed for expression in a plant.

18 . A DNA construct comprising the nucleic acid molecule of claim 16 .

19 . The DNA construct of claim 18 , further comprising a nucleic acid molecule encoding a heterologous polypeptide.

20 . A host cell that contains the DNA construct of claim 18 .

21 . The host cell of claim 20 that is a bacterial cell.

22 . The host cell of claim 20 that is a plant cell.

23 . A transgenic plant comprising the host cell of claim 22 .

24 . The transgenic plant of claim 23 , wherein said plant is selected from the group consisting of maize, sorghum, wheat, cabbage, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugar beet, sugarcane, tobacco, barley, and oilseed rape.

25 . Transformed seed of the plant of claim 24 , wherein the seed comprise the DNA construct.

26 . A composition comprising the protein of claim 1 .

27 . The composition of claim 26 , wherein said composition is selected from the group consisting of a powder, dust, pellet, granule, spray, emulsion, colloid, and solution.

28 . The composition of claim 27 , wherein said composition is prepared by desiccation, lyophilization, homogenization, extraction, filtration, centrifugation, sedimentation, or concentration of a culture of micor-organisms.

29 . A method for controlling a pest population comprising contacting said population with an insecticidally-effective amount of the protein of claim 26 .

30 . A method for killing a pest comprising contacting said pest with, or feeding to said pest, an insecticidally-effective amount of the protein of claim 26 .

31 . A method for producing a polypeptide with insecticidal activity, comprising culturing the host cell of claim 21 under conditions in which a nucleic acid molecule encoding the polypeptide is expressed, said polypeptide being selected from the group consisting of:
(a) a polypeptide comprising the amino acid sequence of SEQ ID NO:2 or 10;
(b) a polypeptide that is encoded by the nucleotide sequence of SEQ ID NO:1 or 9; and
(c) a polypeptide sequence having at least 85% sequence identity to a polypeptide sequence of (a) or (b).

32 . A plant having stably incorporated into its genome a DNA construct comprising a nucleotide sequence that encodes a protein having insecticidal activity, wherein said nucleotide sequence is selected from the group consisting of:
(a) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 or 9; (b) a nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2 or 10; and (c) a nucleotide sequence encoding a protein having insecticidal activity comprising an amino acid sequence characterized by at least 85% sequence identity to an amino acid sequence of (b); wherei

Assignees

• Pioneer Hi Bred Int

Inventors

• Abad Andre R
• Hou Zhenglin
• Liu Lu
• Poland Brad
• Schellenberger Ute
• Udranszky Ingrid
• Wang Jimei
• Wei Jun-Zhi
• Wolfe Thomas C
• Xie Weiping
• Zhou Lan
• Zhu Genhai

Classifications

• C12N15/8286 Primary

  for insect resistance · CPC title

• C07K14/325

  Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins · CPC title

• A01N37/46

  N-acyl derivatives · CPC title

• C07K14/32

  from Bacillus (G) · CPC title

• A01N63/02

  Human Necessities · mapped topic