RNAI-MEDIATED PEST CONTROL

Abstract

Provided are double stranded polyribonucleotides, expression systems, host cells and methods for controlling animal pests, in paricular insect pests, via RNAi-mediated gene silencing. The animal pest is contacted with a double-stranded RNA from outside the cell(s) of the animal pest and the double-stranded

RNA is taken up by the animal pest. In particular, the methods of the invention are used to alleviate plants from insect pests. Suitable insect target genes for RNAi-mediated gene silencing are disclosed.

Claims

1. A double stranded polyribonucleotide comprising annealed complementary strands, wherein at least one of said strands comprises a polyribonucleotide selected from the group consisting of: (i) polyribonucleotides complementary to at least 21 contiguous nucleotides of a target gene represented by any of SEQ ID NOs 1 to 68, and (ii) polyribonucleotides having at least 80% sequence identity with the polyribonucleotides of (i) over its entire length as determined using the BLASTN alignment tool, wherein ingestion of said polyribonucleotide by an animal pest or said polyribonucleotide being in surface contact with the animal pest controls said animal pest.

2. The double stranded polyribonucleotide according to claim 1, wherein the polyribonucleotide according to (i) is complementary to at least 21 but not more than 2000 contiguous nucleotides of the target gene.

3. The double stranded polyribonucleotide according to claim 1, wherein the polyribonucleotide of (ii) has at least 90% sequence identity with the polyribonucleotides of (i) over its entire length.

4. The double stranded polyribonucleotide according to claim 1, wherein the polyribonucleotide of (ii) has at least 95% sequence identity with the polyribonucleotides of (i) over its entire length.

5. The double stranded polyribonucleotide according to claim 1, wherein the animal pest is selected from insect pest, nematode pest and acarid pest.

6. The double stranded polyribonucleotide according to claim 1, wherein the animal pest is an insect pest.

7. The double stranded polyribonucleotide according to claim 1, wherein the animal pest is controlled by inhibiting the viability, growth, development, movement or reproduction of said animal pest, or by decreasing the pathogenicity or infectivity of said animal pest.

8. An expression cassette system suitable for expression of a polyribonucleotide according to claim 1, comprising (a) a sense template, wherein the sense template is selected from the group consisting of (a1) sense template of a target gene represented by any of SEQ ID NOs 1 to 68, wherein said sense template comprises at least 21 contiguous nucleotides of said target gene, and (a2) sense template having at least 80% sequence identity with the sense template of (a1) over its entire length as determined using the BLASTN alignment tool; (b) a first promotor operably linked to the sense template according to (a); (c) an antisense template, wherein the antisense template is selected from the group consisting of (c1) antisense template of a target gene represented by any of SEQ ID NOs 1 to 68, wherein said sense template comprises at least 21 contiguous nucleotides of said target gene, and (c2) antisense template having at least 80% sequence identity with the antisense template of (c1) over its entire length as determined using the BLASTN alignment tool; (d) a second promotor operably linked to the antisense template according to (c); wherein the polyribonucleotides transcribed from the sense template according to (a) and the antisense template according to (c) are complementary.

9. A non-human host cell comprising a double stranded polyribonucleotide according to claim 1 and/or an expression cassette system suitable for expression of said polyribonucleotide according to claim 8.

10. A method for down-regulating expression of a target gene in an animal pest, comprising contacting said animal pest with a double stranded polyribonucleotide according to claim 1, whereby the double-stranded polyribonucleotide is taken up into the animal pest and down-regulates the expression of the animal pest target gene.

11. The method according to claim 10, wherein the animal pest is selected from insect pest, nematode pest and acarid pest.

12. The method according to claim 10, wherein the animal pest target gene is represented by any of SEQ ID NOs 1 to 68.

13. A formulation, optionally an agrochemical formulation, comprising at least one double stranded polyribonucleotide according claim 1.

14. The formulation according to claim 13 further comprising at least one extender and/or at least one surface-active substance.

15. The formulation according to claim 13, wherein the double stranded polyribonucleotide is in a mixture with at least one further active compound.

16. A method for controlling an animal pest, said method comprising allowing a double stranded polyribonucleotide according to claim 1 or a formulation thereof. is allowed to act on the pest and/or their habitat thereof.

17. The method according to claim 16, wherein the animal pest is selected from insect pest, nematode pest and acarid pest.

18. A product comprising a double stranded polyribonucleotide according to claim 1 or a formulation thereof for controlling animal pests.

19. The product according to claim 18, wherein the animal pest is selected from insect pest, nematode pest and acarid pest.

20. The product of claim 18 in crop protection.

21. The product of claim 18 in the field of animal health.

22. A method for protecting seed or a germinating plant from animal pests, comprising contacting the seed with a double stranded polyribonucleotide according to claim 1 or a formulation thereof.

23. A seed obtained by a method according to claim 22, wherein the seed comprises said double stranded polyribonucleotide.

24. A transgenic plant expressing a double stranded polyribonucleotide according to claim 1 and/or comprising an expression cassette system thereof and/or comprising a host cell thereof.

Description

EXAMPLES

Preparation Example 1

[0447] For template generation, RNA of the Tribolium castaneum (T. castaneum) 5.sup.th instar, Phaedon cochleariae (P. cochleariae) 2.sup.nd instar and Leptinotarsa decemlineata (L. decemlineata) 2.sup.nd instar larvae was extracted using RNeasy Mini Kit (QIAGEN) according to manufacturer's instructions including DNAse digestion. 1 μg total RNA were applied in cDNA synthesis using the SuperScript™ II Reverse Transcriptase Kit (Thermo Fisher Scientific) with oligo-dT primers (Thermo Fisher Scientific) according to manufacturer's instructions. Gene-specific amplification from 1 μl 1:10 diluted cDNA was carried out using Phusion Flash High-Fidelity PCR Master Mix (Thermo Fisher Scientific) in a 50 μl reaction with a final concentration of 0.5 μM forward and reverse primers, respectively (program: 98° C. 3 min, 35× (98° C. 1 s, Ta (=annealing temperature; as calculated by Thermo Fisher Scientific webcalculator according to the respective primer sequences; see https://www.thermofisher.com/de/de/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/tm-calculator.html) 5 s, 72° C. 15 s), 72° C. 1 min, 12° C. on hold). Purification of the PCR product after agarose gel electrophoresis was carried out using the NucleoSpin® Gel and PCR Clean-Up Kit (Macherey-Nagel) according to manufacturer's instructions, and 500 ng PCR product were used as template for dsRNA production with the MEGAscript™ T7 Transcription Kit (Thermo Fisher Scientific). Purification of dsRNA using LiCl solution was modified by the extension of centrifugation steps to 30 min and the addition of two ethanol wash steps. Nuclease-free water was used to resuspend the dsRNA. GFP-based dsRNA was kindly provided by GreenLight Biosciences and purified with LiCl solution as described above. DNA and dsRNA concentrations were measured with a NanoQuant Plate™ on a Plate reader Infinite 200 PRO (Tecan Life Sciences).

[0448] Using the methods as described above, the dsRNA molecules according to the SEQ ID NOs 69-136 have been obtained.

Use Examples

[0449] 1. Tribolium castaneum dsRNA Delivery by Injection

[0450] Fifth instar for T. castaneum larvae were injected with ˜1 μl of 1 μg/μl dsRNA for the respective target gene. Control larvae were water injected (i.e. without dsRNA). 10 larvae per replicate were performed for each treatment and validation was performed in a dose dependent manner. Survival of T. castaneum larvae postinjection of ˜1 μl of 30 ng/μl or 3 ng/μl with dsRNA of the respective target gene (as indicated in column 1 of table 2) was assessed at days 6 and 10 postinjection for 30 ng/μl and 3 ng/μl and at day 11 for 1 μg/μl. In table 2, the SEQ ID NO of the nucleotide sequence which is representative for the target gene is provided in column 1, and the SEQ ID NO of the nucleotide sequence of the coding strand of the dsRNA molecule which targets said target gene, is provided in column 2. Survival rates were analyzed using Excel 2010 (Microsoft) and Prism 5 (GraphPad) softwares, wherein the survival at day 1 after exposure was set to 100%. The percentage of larval survival was calculated relative to the average of the values at day 6 and 10 postinjection for 30 ng/μl and 3 ng/μl and to the values at day 11 for 1 μg/μl. The following survival rates were obtained (table 2):

TABLE-US-00002 TABLE 2 SEQ ID SEQ ID dsRNA Survival at Survival at Survival at target gene molecule 1 μg/μl [%] 30 μg/μl [%] 3 μg/μl [%] 1 69 0 10 50 2 70 0 0 0 3 71 0 0 0 4 72 0 0 0 5 73 0 10 50 6 74 0 0 50 7 75 0 0 0 8 76 0 0 50 9 77 0 20 55 10 78 0 0 0 11 79 0 0 50 12 80 0 0 5 13 81 0 0 10 14 82 0 0 50 15 83 0 0 50 16 84 0 0 0 17 85 0 0 50 18 86 0 0 50 19 87 0 0 0 20 88 90 NA NA 21 89 0 0 55 22 90 0 25 65 23 91 0 0 0 24 92 0 5 10 25 93 0 10 5 26 94 0 5 5

[0451] 2. P. cochleariae dsRNA Oral Application

[0452] Feeding bioassays were adopted to suit an industrial screening situation. Application of target dsRNA diluted in 0.1% emulsifier W (Poly(oxy-1,2-ethanediyl), alpha-(1,1′-biphenyl)-4-yl-omega-hydroxy-, benzylated; CAS 104376-72-9; Saltigo GmbH) to leaf discs placed on 0.75% agar in a 12-well plate system was performed by a custom-built spraying device in rates ranging from 0.3 μg, 1 μg, 3 μg to 10 μg dsRNA per leaf disc. Spraying of 0.1% emulsifier W alone and 3 μg/leaf disc of dsGFP served as surfactant and dsRNA negative controls, respectively. Per plate, always one of the two controls as well as all three concentrations of one of the dsRNAs were included and repeated three times. Two young 2.sup.nd instar P. cochleariae larvae were placed in each well and allowed to feed for three days. Afterwards, one larva was frozen in liquid nitrogen for usage in expression analysis while the other larva was monitored over 10 days with untreated diet exchanged on days 3, 5, 6 and 7. In tables 3 and 4, the SEQ ID NO of the nucleotide sequence which is representative for the target gene is provided in column 1, and the SEQ ID NO of the nucleotide sequence of the coding strand of the dsRNA molecule which targets said target gene, is provided in column 2. Survival rates were analyzed using Excel 2010 (Microsoft) and Prism 5 (GraphPad) softwares, wherein the survival at day 3 after exposure was set to 100%. The following survival rates were obtained (tables 3 and 4):

[0453] Table 3:

TABLE-US-00003 TABLE 3 Survival Survival Survival SEQ at at at Survival at ID SEQ ID 10 μg/leaf 3 μg/leaf 1 μg/leaf 0.3 μg/leaf target dsRNA disc [%] disc [%] at disc [%] disc [%] gene molecule at 7 days 7 days at 7 days at 7 days 27 95 0 11 11 0 28 96 33 11 22 22 29 97 0 NA NA NA 30 98 0 22 0 0 31 99 44 0 0 11 32 100 NA 78 100 89 33 101 11 11 0 33 34 102 89 22 44 78 35 103 22 0 0 0 36 104 33 0 0 0 37 105 100 NA NA NA 38 106 22 NA NA NA 39 107 44 NA NA NA 40 108 78 67 89 100 41 109 0 NA NA NA 42 110 78 22 33 44 43 111 NA NA NA 0

[0454] SEQ ID SEQ ID Survival at 10 Survival at 3 Survival at 1 .sub.Kg/leaf Survival at 0.3

[0455] target gene dsRNA Kg/leaf disc Kg/leaf disc disc [%] at 7 days Kg/leaf disc [%] [0456] molecule [%] at 7 days [%] at 7 days at 7 days

[0457] 27 95 0 11 11 0

[0458] 28 96 33 11 22 22

[0459] 29 97 0 NA NA NA

[0460] 30 98 0 22 0 0

[0461] 31 99 44 0 0 11

[0462] 32 100 NA 78 100 89

[0463] 33 101 11 11 0 33

[0464] 34 102 89 22 44 78

[0465] 35 103 22 0 0 0

[0466] 36 104 33 0 0 0

[0467] 37 105 100 NA NA NA

[0468] 38 106 22 NA NA NA

[0469] 39 107 44 NA NA NA

[0470] 40 108 78 67 89 100

[0471] 41 109 0 NA NA NA

[0472] 42 110 78 22 33 44

[0473] 43 111 NA NA NA 0

TABLE-US-00004 TABLE 4 Survival at Survival at Survival at Survival at SEQ ID 10 μg/leaf 3 μg/leaf 1 μg/leaf 0.3 μg/leaf SEQ ID dsRNA disc [%] disc [%] disc [%] disc [%] target gene molecule at 10 days at 10 days at 10 days at 10 days 27 95 0  0 0  0 28 96 0  0 11  11 29 97 0 NA NA NA 30 98 0 22 0  0 31 99 0  0 0 11 32 100 NA 56 67  56 33 101 0 11 0 22 34 102 67 22 44  67 35 103 0  0 0  0 36 104 0  0 0  0 37 105 89 NA NA NA 38 106 11 NA NA NA 39 107 22 NA NA NA 40 108 78 56 78  78 41 109 0 NA NA NA 42 110 33 22 22  22 43 111 NA NA NA NA

[0474] 3. P. cochleariae dsRNA Delivery by Injection

[0475] Approximately 150 ng dsRNA was injected into the lateral abdomen of nine ice-sedated 2.sup.nd instar larvae of P. cochleariae per replicate with a Microinjector FemtoJet® (Eppendorf) with pulled borosilicate glass capillaries (Hilgenberg) and repeated three times. GFP dsRNA served as negative control. After a short recovery time, larvae were placed into 9 cm Petri dishes containing leaves on moistened filter paper. On day one, larvae that died of disruptive injection were replaced by spare injected larvae. For expression analysis, larvae were frozen in liquid nitrogen on the third day. Leaves were exchanged every one or two days and mortality was monitored over 10 days. Similar results as described in table 4 were obtained.

[0476] 4. L. decemlineata dsRNA Oral Application

[0477] Feeding bioassays were adopted to suit an industrial screening situation. Application of target dsRNA diluted in 0.1% emulsifier W to leaf discs placed on 1.2% agar in a 12-well plate system was performed by a custom-built spraying device in rates ranging from 1 μg to 100 ng dsRNA per leaf disc. Spraying of 0.1% emulsifier W alone and 1 μg/leaf disc of dsGFP served as surfactant and dsRNA negative controls, respectively. Per plate, always one of the two controls as well as all three concentrations of one of the dsRNAs were included and repeated three times. One young 2.sup.nd instar L. decemlineata larvae was placed in each well and allowed to feed for three days and monitored over 7 days with untreated diet exchanged on days 3, 4, 5 and 6. In table 5, the SEQ ID NO of the nucleotide sequence which is representative for the target gene is provided in column 1, and the SEQ ID NO of the nucleotide sequence of the coding strand of the dsRNA molecule which targets said target gene, is provided in column 2. Survival rates were analyzed using Excel 2010 (Microsoft) and Prism 5 (GraphPad) softwares, wherein the survival at day 1 after exposure was set to 100%. The following survival rates were obtained (table 5):

TABLE-US-00005 TABLE 5 Survival at Survival at Survival at SEQ ID SEQ ID 1 μg/leaf 0.1 μg/leaf 0.01 μg/leaf target dsRNA disc [%] disc [%] disc [%] gene molecule at 7 days at 7 days at 7 days 44 112 0 0 22 45 113 0 0 67 46 114 33 67 100 47 115 0 0 44 48 116 0 0 22 49 117 0 0 33 50 118 0 0 0 51 119 0 0 78 52 120 0 0 0 53 121 0 0 100 54 122 11 0 0 55 123 0 0 78 56 124 11 0 100 57 125 0 0 0 58 126 0 11 22 59 127 33 89 100 60 128 0 0 0 61 129 22 56 100 62 130 0 0 33 63 131 0 0 22 64 132 0 0 78 65 133 22 78 100 66 134 0 11 100 67 135 0 0 100 68 136 0 0 0