Novel insecticidal toxin receptors and methods of use

Patent metadata

Field	Value
Publication number	US-2022380418-A1
Application number	US-201917266318-A
Country	US
Kind code	A1
Filing date	Aug 2, 2019
Priority date	Aug 13, 2018
Publication date	Dec 1, 2022
Grant date	—

Abstract

The disclosure relates to insecticidal toxin resistance management and screening of novel insecticidal toxins. One embodiment relates to the isolation, characterization, compositions, and methods of use relating to polynucleotides encoding novel insecticidal toxin receptors and the polypeptides encoded thereby. The polynucleotides and polypeptides are useful in identifying or designing novel insecticidal toxin receptor ligands including novel insecticidal toxins.

First claim

That which is claimed:

1 . A heterologous polynucleotide comprising:
a. the nucleotide sequence set forth in any one of SEQ ID NOs: 45-91; b. a nucleotide sequence encoding the amino acid sequence set forth in any one of SEQ ID NOs: 180-226; or c. a nucleotide sequence having at least about 90% sequence identity to the nucleotide sequence set forth in any one of SEQ ID NOs: 45-91, wherein said nucleotide sequence having at least about 90% sequence identity to the nucleotide sequence set forth in any one of SEQ ID NOs: 45-91 encodes a polypeptide having toxin binding activity.

2 . The heterologous polynucleotide of claim 1 , wherein said heterologous polynucleotide comprises a nucleotide sequence encoding a polypeptide having insecticidal toxin binding activity.

3 . An expression cassette comprising the nucleotide sequence of claim 1 operably linked to a promoter.

4 . A host cell comprising the expression cassette of claim 3 .

5 . The host cell of claim 4 , wherein said cell is a microorganism.

6 . The host cell of claim 5 , wherein said microorganism is selected from the group consisting of yeast and bacteria.

7 . The host cell of claim 6 , wherein said cell is an insect cell.

8 . The host cell of claim 7 , wherein said cell is a mammalian cell.

9 . A heterologous polypeptide having the amino acid sequence comprising:
a. the amino acid sequence set forth in any one of SEQ ID NOs: 180-226; or b. a variant of the amino acid sequence set forth in any one of SEQ ID NOs: 180-226, wherein said sequence variant has insecticidal toxin binding activity and shares at least about 90% sequence identity with the amino acid sequence set forth in SEQ ID NO: 180-226.

10 . A polypeptide complex comprising a heterologous polypeptide comprising the heterologous polypeptide of claim 9 , wherein said heterologous polypeptide is bound to an insecticidal toxin.

11 . An antibody preparation specific for the polypeptide of claim 9 .

12 . A transformed cell of interest having stably incorporated within its genome a heterologous nucleotide sequence comprising:
a. the nucleotide sequence set forth in any one of SEQ ID NOs: 45-91; b. a nucleotide sequence encoding the amino acid sequence set forth in any one of SEQ ID NOs: 180-226; or c. a nucleotide sequence having at least about 90% sequence identity to the nucleotide sequence set forth in any one of SEQ ID NOs: 45-91, wherein said nucleotide sequence having at least about 90% sequence identity to the nucleotide sequence set forth in any one of SEQ ID NOs: 45-91 encodes a polypeptide having toxin binding activity.

13 . The transformed cell of claim 12 , wherein said cell is a plant cell.

14 . The transformed cell of claim 12 , wherein said cell is an insect cell.

15 . A method for identifying compounds that bind to the polypeptide of claim 4 , said method comprising:
a. contacting the polypeptide of claim 4 with one or more test compounds; and b. determining whether the test compound binds to the polypeptide.

16 . A method for generating a variant of any one of SEQ ID NOs: 45-314 in an insect comprising modifying the endogenous sequence of any one of SEQ ID NOs: 45-179.

17 . The method of claim 16 , wherein the insect expresses a variant polypeptide of any one of SEQ ID NOs: 180-314.

18 . The method of claim 16 , wherein the variant does not express the corresponding polypeptide as set forth in SEQ ID NO: 180-314.

19 . A method for selecting altered susceptibility of an insect to an insecticidal toxin, said method comprising:
a. identifying in an insect:
i. alterations of the nucleotide sequence of any one of SEQ ID NOs: 45-179;
ii. alterations of the amino acid sequence of any one of SEQ ID NOs: 180-314; or
iii. changes in expression of any one of SEQ ID NOs: 45-314; and
b. selecting for altered susceptibility of said insect.

20 . The method of claim 19 , wherein the insect is a genetically edited insect.

21 . An insect cell comprising at least one edited genomic sequence, wherein the edited genomic sequence is different from the native genomic sequence encoding a polypeptide having at least 95% sequence identity to a sequence set forth in any one of SEQ ID NOs: 180-314.

22 . The insect cell of claim 21 , wherein the edited sequence is no longer functional.

23 . The insect cell of claim 21 , wherein the edited sequence is a toxin receptor.

24 . The insect cell of claim 23 , wherein the toxin receptor comprises a polypeptide encoded by a polynucleotide having at least 95% sequence identity to any one of SEQ ID NOs: 45-179.

25 . The insect cell of claim 23 , wherein the toxin receptor no longer binds the toxin.

26 . The insect cell of claim 21 , wherein the insect cell is resistant to an insecticidal toxin.

27 . An insect comprising the insect cell of claim 21 .

28 . An insect colony comprising the insect of claim 26 .

29 . The insect cell of claim 21 , wherein a regulatory element is edited.

30 . The insect cell of claim 21 , wherein the expression of the polypeptide has reduced expression in the insect cell.

31 . The insect cell of claim 29 , wherein the regulatory element is a promoter.

32 . A kit for screening populations of insects, wherein said kit comprises a means for assaying an altered level of expression or an altered genomic sequence of a sequence as set forth in any one of SEQ ID NOs: 45-314, or variants or fragments thereof, wherein the alteration indicates an insect resistant to an insecticidal toxin.

Assignees

• Pioneer Hi Bred Int

Inventors

• Bazyk Claire Marie
• Clark Catherine J
• Cong Ruth
• Hasler James M
• Hou Jingtong
• Kakani Naga Kishore
• Kapka-Kitzman Deirdre
• Leng Song
• Mathis John P
• Nelson Mark Edward
• Yamamoto Takashi

Classifications

• Y02A40/146

  Genetically Modified [GMO] plants, e.g. transgenic plants · CPC title

• C12Q1/6888

  for detection or identification of organisms · CPC title

• C07K14/43563 Primary

  from insects · CPC title

• C12Q2600/158

  Expression markers · CPC title

• C12Q2600/156

  Polymorphic or mutational markers · CPC title