Insecticidal proteins

Patent metadata

Field	Value
Publication number	US-9522937-B2
Application number	US-201213623921-A
Country	US
Kind code	B2
Filing date	Sep 21, 2012
Priority date	Mar 28, 2007
Publication date	Dec 20, 2016
Grant date	Dec 20, 2016

Abstract

Compositions and methods for controlling plant pests are disclosed. In particular, novel engineered hybrid insecticidal proteins (eHIPs) having toxicity to at least corn rootworm are provided. By fusing unique combinations of complete or partial variable regions and conserved blocks of at least two different Bacillus thuringiensis (Bt) Cry proteins or a modified Cry proteins an eHIP having activity against corn rootworm is designed. Nucleic acid molecules encoding the novel eHIPs are also provided. Methods of making the eHIPs and methods of using the eHIPs and nucleic acids encoding the eHIPs of the invention, for example in transgenic plants to confer protection from insect damage are also disclosed.

First claim

What is claimed is:

1. An isolated nucleic acid molecule encoding an engineered hybrid insecticidal protein comprising in an N-terminus to C-terminus direction an N-terminal region of a Cry3A protein fused to a C-terminal region of a Cry1Aa or Cry1Ab protein, wherein at least one crossover position between the Cry3A protein and the Cry1Aa or Cry1Ab protein is located in conserved block 2, conserved block 3 or variable region 4, and optionally comprising:
(a) at the C-terminus a protoxin tail region of a Bt Cry protein; or
(b) at the N-terminus a peptidyl fragment; or
(c) both (a) and (b),

wherein the engineered hybrid insecticidal protein has at least 80% identity to SEQ ID NO:64 and has insecticidal activity against western corn rootworm ( Diabrotica virgifera ).

2. The isolated nucleic acid molecule according to claim 1 , wherein the Cry3A is a Cry3Aa or a modified Cry3Aa.

3. The isolated nucleic acid molecule according to claim 2 , wherein (a) the Cry3Aa comprises SEQ ID NO:68 or SEQ ID NO:135, or (b) the modified Cry3Aa comprises SEQ ID NO:70, and wherein the Cry1Ab comprises SEQ ID NO:72.

4. The isolated nucleic acid molecule according to claim 1 , wherein the hybrid insecticidal protein comprises at the C-terminus a protoxin tail region of a Bt Cry Protein.

5. The isolated nucleic acid molecule according to claim 4 , wherein the protoxin tail region is from a Bt Cry protein that is active against a lepidopteran insect.

6. The isolated nucleic acid molecule according to claim 5 , wherein the Bt Cry protein is a Cry1A.

7. The isolated nucleic acid molecule according to claim 6 , wherein the Cry1A is a Cry1Aa or a Cry1Ab.

8. The isolated nucleic acid molecule according to claim 7 , wherein the Cry1Ab protoxin tail region comprises at least 38 amino acids.

9. The isolated nucleic acid molecule according to claim 8 , wherein the Cry1 Ab protoxin tail region comprises an amino acid sequence that corresponds to amino acids 611-648 of SEQ ID NO: 72.

10. The isolated nucleic acid molecule according to claim 9 , wherein the Cry1 Ab protoxin tail region comprises amino acids 611-648 of SEQ ID NO: 72.

11. The isolated nucleic acid molecule according to claim 1 , wherein the hybrid insecticidal protein comprises at the N-terminus a peptidyl fragment comprising at least 9 amino acids.

12. The isolated nucleic acid molecule according to claim 11 , wherein the peptidyl fragment comprises the amino acid sequence YDGRQQHRG (SEQ ID NO: 132) or the amino acid sequence TSNGRQCAGIRP (SEQ ID NO: 133).

13. The isolated nucleic acid molecule according to claim 12 , wherein the peptidyl fragment is selected from the group consisting of SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131 and SEQ ID NO: 132.

14. The isolated nucleic acid molecule according to claim 1 , wherein the crossover position between Cry3A and Cry1 Aa or Cry1 Ab is located in a region between amino acids corresponding to amino acid 6 of conserved block 3 to amino acid 7 of conserved block 4.

15. The isolated isolated nucleic acid molecule according to claim 14 , wherein the crossover position is located in conserved block 3 immediately following an amino acid corresponding to Ser450, Phe454, or Leu468 of SEQ ID NO: 70.

16. The isolated nucleic acid molecule according to claim 15 , wherein the crossover position is located in conserved block 3 immediately following Ser450, Phe454, or Leu468 of SEQ ID NO: 70.

17. An isolated nucleic acid molecule encoding a hybrid insecticidal protein comprising at least two crossover positions between an amino acid sequence from a Cry3A protein and an amino acid sequence from a Cry1 Aa or a Cry1 Ab protein.

18. The isolated nucleic acid molecule according to claim 17 , wherein (a) the first crossover position is located in conserved block 2 and the second crossover position is located in conserved block 3; or (b) the first crossover position is located in conserved block 3 and the second crossover position is located in conserved block 4.

19. The isolated nucleic acid molecule according to claim 18 , wherein the Cry3A is a Cry3Aa or a modified Cry3Aa.

20. The isolated nucleic acid molecule according to claim 19 , wherein (a) the first crossover position between Cry3Aa and Cry1Aa or Cry1Ab or modified Cry3Aa and Cry1Aa or Cry1Ab is located in conserved block 2 immediately following an amino acid corresponding to Asp232 of SEQ ID NO: 70 and a second crossover position between Cry1Aa or Cry1Ab and Cry3Aa or Cry1Aa or Cry1Ab and modified Cry3Aa is located in conserved block 3 immediately following an amino acid corresponding to Leu476 of SEQ ID NO: 72; or (b) the first crossover position between Cry3Aa and Cry1Aa or Cry1Ab or modified Cry3Aa and Cry1Aa or Cry1Ab is located in conserved block 3 immediately following an amino acid corresponding to Leu468 of SEQ ID NO: 70 and the second crossover position between Cry1Aa or Cry1Ab and Cry3Aa or Cry1Aa or Cry1Ab and modified Cry3Aa is located in conserved block 4 immediately following an amino acid corresponding to Ile527 of SEQ ID NO: 72.

21. The isolated nucleic acid molecule according to claim 20 , wherein (a) the first crossover position between Cry3Aa and Cry1Aa or Cry1Ab or modified Cry3Aa and Cry1Aa or Cry1Ab is located in conserved block 2 immediately following Asp232 of SEQ ID NO: 70 and the second crossover position between Cry1Aa or Cry1Ab and Cry3Aa or Cry1Aa or Cry1Ab and modified Cry3Aa is located in conserved block 3 immediately following Leu476 of SEQ ID NO: 72; or (b) the first crossover position between Cry3Aa and Cry1Aa or Cry1Ab or modified Cry3Aa and Cry1Aa or Cry1Ab is located in conserved block 3 immediately following Leu468 of SEQ ID NO: 70 and the second crossover position between Cry1Aa or Cry1Ab and Cry3Aa or Cry1Aa or Cry1Ab and modified Cry3Aa is located in conserved block 4 immediately following Ile527 of SEQ ID NO: 72.

22. The isolated nucleic acid molecule according to claim 1 , wherein the insecticidal protein is additionally active against northern corn rootworm ( Diabrotica undecimpunctata ) or Mexican corn rootworm ( D. zea ).

23. The isolated nucleic acid molecule according to claim 1 , wherein the hybrid insecticidal protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 62; SEQ ID NO: 64, SEQ ID NO: 147, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 159 and SEQ ID NO: 160.

24. The isolated nucleic acid molecule according to claim 23 comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 61; SEQ ID NO: 63, SEQ ID NO: 146, SEQ ID NO: 152, SEQ ID NO: 154 and SEQ ID NO: 158.

25. An expression cassette comprising a heterologous promoter sequence operatively linked to the nucleic acid molecule according to any one of claims 1 - 24 .

26. A recombinant vector comprising the expression cassette of claim 25 .

27. A transgenic host cell comprising the expression cassette of claim 25 .

28. The transgenic host

Assignees

• Syngenta Participations Ag

Inventors

• Hart Hope
• Chen Jeng Shong
• Defontes Cheryl M
• Walters Frederick

Classifications

• C12N15/8286

  for insect resistance · CPC title

• C07K2319/55

  containing a fusion with a toxin, e.g. diphteria toxin · CPC title

• C07K14/325 Primary

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

• A01N63/50

  Isolated enzymes; Isolated proteins (peptides A01N37/46) · CPC title

• Y02A40/146

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