RNAI FOR THE CONTROL OF PHYTOPATHOGENIC FUNGI AND OOMYCETES BY INHIBITING THE EXPRESSION OF CYP51 GENES

• US 20160215290A1
• Filed: 07/09/2014
• Published: 07/28/2016
• Est. Priority Date: 07/10/2013
• Status: Active Grant

• Current Assignee: BASF SE
• Original Assignee: BASF SE
• Inventors: Kogel, Karl-Heinz
• Granted Patent: US 11,441,147 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A01N 57/16 containing heterocyclic radicals
• CPC Class Codes: C12N 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• CPC Class Codes: C12N 15/8218 Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
• CPC Class Codes: C12N 15/8282 for fungal resistance
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/51 in polymeric form, e.g. multimers, concatemers
• CPC Class Codes: C12N 2310/531 Stem-loop; Hairpin
• CPC Class Codes: C12Y 114/1307 Sterol 14-demethylase (1.14.13.70)

Abstract

The present invention relates to dsRNA molecules for inhibiting the expression of CYP51 gene(s) from a fungal or oomycete phytopathogen and the use of such inhibitory dsRNA molecules for controlling phytopathogenic fungi and/or oomycetes. The present invention further relates to DNA sequences providing a transcriptional template for inhibitory dsRNA molecules or antisense RNA; and phytopathogen-tolerant transgenic plants with such DNA sequences.

Claims

1. A dsRNA molecule capable of inhibiting the expression of the CYP51A gene, the CYP51B gene and the CYP51C gene from a phytopathogen of the genus Fusarium, the dsRNA comprising:

i) a sense sequence being at least 70% identical to at least 20 contiguous nucleotides of the coding sequence of the CYP51A gene;

ii) a sense sequence being at least 70% identical to at least 20 contiguous nucleotides of the coding sequence of the CYP51B gene;

iii) a sense sequence being at least 70% identical to at least 20 contiguous nucleotides of the coding sequence of the CYP51C gene; and

iv) antisense sequences being substantially complementary to the sense sequences (i), (ii), and (iii);

wherein the arrangement of the sequences within the dsRNA molecule allows hybridization of each sense sequence with its substantially complementary antisense sequence.

2. The dsRNA molecule of claim 1, wherein the phytopathogen is Fusarium graminearum, and

the sense sequence (i) is at least 70% identical to at least 20 contiguous nucleotides of SEQ ID NO:

1;

the sense sequence (ii) is at least 70% identical to at least 20 contiguous nucleotides of SEQ ID NO;

2; and

the sense sequence (iii) is at least 70% identical to at least 20 contiguous nucleotides of SEQ ID NO;

3.

3. The dsRNA molecule of claim 2, wherein

the sense sequence (i) is at least 70% identical to at least 20 contiguous nucleotides of SEQ ID NO:

4;

the sense sequence (ii) is at least 70% identical to at least 20 contiguous nucleotides of SEQ ID NO;

5; and

the sense sequence (iii) is at least 70% identical to at least 20 contiguous nucleotides of SEQ ID NO;

6.

4. The dsRNA molecule of claim 1, wherein the phytopathogen is Fusarium oxysporum, and

the sense sequence (i) is at least 70% identical to at least 20 contiguous nucleotides of SEQ ID NO:

8;

the sense sequence (ii) is at least 70% identical to at least 20 contiguous nucleotides of SEQ ID NO;

9; and

the sense sequence (iii) is at least 70% identical to at least 20 contiguous nucleotides of SEQ ID NO;

10.

5. The dsRNA molecule of claim 1, wherein the phytopathogen is Fusarium verticilliodes, and

the sense sequence (i), is at least 70% identical to at least 20 contiguous nucleotides of SEQ ID NO:

11;

the sense sequence (ii) is at least 70% identical to at least 20 contiguous nucleotides of SEQ ID NO;

12; and

the sense sequence (iii) is at least 70% identical to at least 20 contiguous nucleotides of SEQ ID NO;

13.

6. The dsRNA molecule of claim 1 comprising a sense sequence that is at least 70% identical to SEQ ID NO:

7, and an antisense sequences being substantially complementary to said sense sequences, wherein the arrangement of the sequences within the dsRNA molecule allows hybridization of said sense sequence with said antisense sequence.

8. The dsRNA molecule of claim 5, capable of inhibiting the expression of a first CYP51 gene and a second CYP51 gene which are different CYP51 genes from the phytopathogen, the dsRNA comprising:

a first sense sequence being at least 70% identical to at least 20 contiguous nucleotides of the coding sequence of the first CYP51 gene,

a second sense sequence being at least 70% identical to at least 20 contiguous nucleotides of the coding sequence of the second CYP51 gene, and

antisense sequences being substantially complementary to the sense sequences;

wherein the arrangement of the sequences within the dsRNA molecule allows hybridization of each sense sequence with its substantially complementary antisense sequence.

9. The dsRNA molecule of claim 5, wherein

the phytopathogen is Fusarium graminearum and the coding sequence(s) of the CYP51 gene(s) is/are selected from SEQ ID NOs:

1-3;

or

the phytopathogen is Fusarium oxysporum and the coding sequence(s) of the CYP51 gene(s) is/are selected from SEQ ID NOs;

8-10;

or

the phytopathogen is Fusarium verticillioides and the coding sequence(s) of the CYP51 gene(s) is/are selected from SEQ ID NOs;

11-13;

or

the phytopathogen is Blumeria graminis and the coding sequence of the CYP51 gene is selected from SEQ ID NO;

14;

or

the phytopathogen is Magnaporthe grisea and the coding sequence(s) of the CYP51 gene(s) is/are selected from SEQ ID NOs;

15 and 16;

or

the phytopathogen is Sclerotinia sclerotinium and the coding sequence of the CYP51 gene is selected from SEQ ID NO;

17;

or

the phytopathogen is Phakopsora pachyrhizi and the coding sequence of the CYP51 gene is selected from SEQ ID NO;

18;

or

the phytopathogen is Botrytis cinerea and the coding sequence of the CYP51 gene is selected from SEQ ID NO;

19;

or

the phytopathogen is Puccinia triticina and the coding sequence of the CYP51 gene is selected from SEQ ID NO;

20;

or

the phytopathogen is Puccinia graminis and the coding sequence of the CYP51 gene is selected from SEQ ID NO;

21;

or

the phytopathogen is Puccinia recondita and the coding sequence of the CYP51 gene is selected from SEQ ID NO;

22;

or

the phytopathogen is Pyrenophora teres and the coding sequence of the CYP51 gene is selected from SEQ ID NO;

23;

or

the phytopathogen is Pyrenophora tritici and the coding sequence(s) of the CYP51 gene(s) is/are selected from SEQ ID NOs;

24 and 25;

or

the phytopathogen is Phaeosphaeria nodorum and the coding sequence of the CYP51 gene is selected from SEQ ID NO;

26;

or

the phytopathogen is Septoria tritici and the coding sequence of the CYP51 gene is selected from SEQ ID NO;

27;

or

the phytopathogen is Cochliobolus sativus and the coding sequence of the CYP51 gene is selected from SEQ ID NO;

28;

or

the phytopathogen is Ustilago maydis and the coding sequence of the CYP51 gene is selected from SEQ ID NO;

29;

or

the phytopathogen is Rhynchosporium secalis and the coding sequence of the CYP51 gene is selected from SEQ ID NO;

30;

or

the phytopathogen is Venturia inaequalis and the coding sequence of the CYP51 gene is selected from SEQ ID NO;

31.

10. The dsRNA molecule of claim 1, wherein the sequence identity of each sense sequence pertains to at least 25.

11. The dsRNA molecule of claim 1, wherein the sequence identity of each sense sequence to its respective reference sequence is at least 75%.

12. The dsRNA molecule of claim 1, wherein each sense sequence is located on a different RNA strand of the dsRNA molecule than its corresponding antisense sequence.

13. The dsRNA molecule of claim 1, wherein the sense sequences and the antisense sequences are located on a single RNA strand that loops back on itself so as to form a hairpin structure.

14. A DNA sequence or multitude of DNA sequences providing a transcriptional template of at least the antisense sequence(s) of the dsRNA molecule of claim 1.

15. The DNA sequence(s) of claim 14 providing a transcriptional template of said dsRNA molecule.

16. The DNA sequence(s) of claim 15, wherein the transcriptional template is operably linked to at least one promoter.

17. The DNA sequence(s) of claim 16, wherein the at least one promoter is functional in a plant cell.

18. A transgenic plant comprising the DNA sequence(s) of claim 14.

19. The transgenic plant of claim 18, wherein the plant is capable of generating at least the antisense sequence(s) of said dsRNA molecule.

20. The transgenic plant of claim 19, wherein the plant is capable of generating said dsRNA molecule.

21. The transgenic plant of claim 18, wherein the transgenic plant is a cereal crop and the phytopathogen is from the genus Fusarium.

22. A method for controlling a fungal or oomycete phytopathogen, wherein a transgenic plant according to claim 18 is cultivated to allow the generation of RNA comprising the antisense sequence(s) of said dsRNA molecule, and said RNA inhibits the growth and/or propagation of said phytopathogen.

23. A composition for controlling a fungal or oomycete phytopathogen comprising the dsRNA molecule of claim 1, and a plant-compatible carrier.

24. A method for controlling a fungal or oomycete phytopathogen, wherein a plant infested by or at risk of being infested by said phytopathogen and/or the vicinity of said plant is contacted with an effective amount of the composition of claim 23.

7. (canceled)

25. A dsRNA molecule capable of inhibiting the expression of at least one CYP51 gene from a fungal or oomycete phytopathogen selected from Table 1, the dsRNA comprising:

a sense sequence being at least 70% identical to at least 20 contiguous nucleotides of the coding sequence of said at least one CYP51 gene, and

an antisense sequence being substantially complementary to said sense sequence,

wherein the arrangement of the sequences within the dsRNA molecule allows hybridization of said sense sequence and said corresponding antisense sequence.