U.S. patent application number 14/485103 was filed with the patent office on 2018-05-24 for methods for controlling pests using rnai.
This patent application is currently assigned to DEVGEN NV. The applicant listed for this patent is Thierry BOGAERT, Nicole DAMME, Lies DEGRAVE, Pascale FELDMANN, Laurent KUBLER, Irene NOOREN, Frederic PECQUEUR, Geert PLAETINCK, Romaan RAEMAEKERS, Isabel REMORY, Els VAN BLEU. Invention is credited to Thierry BOGAERT, Nicole DAMME, Lies DEGRAVE, Pascale FELDMANN, Laurent KUBLER, Irene NOOREN, Frederic PECQUEUR, Geert PLAETINCK, Romaan RAEMAEKERS, Isabel REMORY, Els VAN BLEU.
Application Number | 20180142237 14/485103 |
Document ID | / |
Family ID | 38287985 |
Filed Date | 2018-05-24 |
United States Patent
Application |
20180142237 |
Kind Code |
A9 |
RAEMAEKERS; Romaan ; et
al. |
May 24, 2018 |
METHODS FOR CONTROLLING PESTS USING RNAi
Abstract
The present invention relates to methods for controlling pest
infestation using double stranded RNA molecules. The invention
provides methods for producing transgenic cells expressing the
double stranded RNA molecules, as well as compositions and
commodity products containing or treated with such molecules.
Inventors: |
RAEMAEKERS; Romaan; (De
Pinte, BE) ; FELDMANN; Pascale; (Gent, BE) ;
PLAETINCK; Geert; (Bottelare, BE) ; NOOREN;
Irene; (Oegstgeest, NL) ; VAN BLEU; Els;
(Berlare, BE) ; PECQUEUR; Frederic; (Sequedin,
FR) ; KUBLER; Laurent; (Beynost, FR) ; DAMME;
Nicole; (Kruishoutem, BE) ; DEGRAVE; Lies;
(Tieet, BE) ; REMORY; Isabel; (Ressegem, BE)
; BOGAERT; Thierry; (Kortrijk, BE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
RAEMAEKERS; Romaan
FELDMANN; Pascale
PLAETINCK; Geert
NOOREN; Irene
VAN BLEU; Els
PECQUEUR; Frederic
KUBLER; Laurent
DAMME; Nicole
DEGRAVE; Lies
REMORY; Isabel
BOGAERT; Thierry |
De Pinte
Gent
Bottelare
Oegstgeest
Berlare
Sequedin
Beynost
Kruishoutem
Tieet
Ressegem
Kortrijk |
|
BE
BE
BE
NL
BE
FR
FR
BE
BE
BE
BE |
|
|
Assignee: |
DEVGEN NV
Zwijnaarde
BE
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20150065557 A1 |
March 5, 2015 |
|
|
Family ID: |
38287985 |
Appl. No.: |
14/485103 |
Filed: |
September 12, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11992091 |
May 8, 2008 |
8933042 |
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PCT/IB2006/004008 |
Sep 18, 2006 |
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14485103 |
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PCT/IB2006/003446 |
Sep 15, 2006 |
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11992091 |
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60718034 |
Sep 16, 2005 |
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60758191 |
Jan 12, 2006 |
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60771160 |
Feb 7, 2006 |
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60837910 |
Aug 16, 2006 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A01N 63/10 20200101;
A61P 31/04 20180101; C12N 15/8286 20130101; C12N 15/113 20130101;
C12N 2310/14 20130101; Y02A 40/146 20180101; Y02A 40/162
20180101 |
International
Class: |
C12N 15/113 20060101
C12N015/113; A01N 63/02 20060101 A01N063/02 |
Claims
1. An isolated polynucleotide sequence selected from the group
consisting of a polynucleotide sequence comprising a nucleic acid
sequence set forth in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17,
19, 21, 23, 49-158, 159, 160, 163, 168, 173, 178, 183, 188, 193,
198, 203, 208, 215, 220, 225, 230, 247, 249, 251, 253, 255, 257,
259, 275-472, 473, 478, 483, 488, 493, 498, 503, 513, 515, 517,
519, 521, 533-575, 576, 581, 586, 591, 596, 601, 603, 605, 607,
609, 621-767, 768, 773, 778, 783, 788, 793, 795, 797, 799, 801,
813-862, 863, 868, 873, 878, 883, 888, 890, 892, 894, 896,908-1040,
1041, 1046, 1051, 1056, 1061, 1071, 1073, 1075, 1077, 1079, 1081,
1083, 1085, 1087, 1089, 1091, 1093, 1095, 1097, 1099, 1101, 1103,
1105, 1107, 1109, 1111, 1113, 1161-1571, 1572, 1577, 1582, 1587,
1592, 1597, 1602, 1607, 1612, 1617, 1622, 1627, 1632, 1637, 1642,
1647, 1652, 1657, 1662, 1667, 1672, 1677, 1682, 1684, 1686, 1688,
1690, 1692, 1694, 1696, 1698, 1700, 1702, 1704, 1730-2039, 2040,
2045, 2050, 2055, 2060, 2065, 2070, 2075, 2080, 2085, 2090, 2095,
2100, 2102, 2104, 2106, 2108, 2120-2338, 2339, 2344, 2349, 2354,
2359, 2364, 2366, 2368, 2370, 2372, 2384-2460, 2461, 2466, 2471,
2476 and 2481; a polynucleotide sequence having at least 70%
sequence identity to a nucleic acid sequence set forth in SEQ ID
NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 49-158, 159, 160,
163, 168, 173, 178, 183, 188, 193, 198, 203, 208, 215, 220, 225,
230, 24 7' 249, 251, 253, 255, 257, 259, 275-472, 473, 478, 483,
488, 493, 498, 503, 513, 515, 517, 519, 521, 533-575, 576, 581,
586, 591, 596, 601, 603, 605, 607, 609, 621-767, 768, 773, 778,
783, 788, 793, 795, 797, 799, 801, 813-862, 863, 868, 873, 878,
883, 888, 890, 892, 894, 896, 908-1040, 1041, 1046, 1051, 1056,
1061, 1071, 1073, 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089,
1091, 1093, 1095, 1097, 1099, 1101, 1103, 1105, 1107, 1109, 1111,
1113, 1161-1571, 1572, 1577, 1582, 1587, 1592, 1597, 1602, 1607,
1612, 1617, 1622, 1627, 1632, 1637, 1642, 1647, 1652, 1657, 1662,
1667, 1672, 1677, 1682, 1684, 1686, 1688, 1690, 1692, 1694, 1696,
1698, 1700, 1702, 1704, 1730-2039, 2040, 2045, 2050, 2055, 2060,
2065, 2070, 2075, 2080, 2085, 2090, 2095, 2100, 2102, 2104, 2106,
2108, 2120-2338, 2339, 2344, 2349, 2354, 2359, 2364, 2366, 2368,
2370, 2372, 2384-2460, 2461, 2466, 2471, 2476 and 2481; and a
double stranded ribonucleotide sequence produced from the
expression of a polynucleotide sequence, wherein contact of said
ribonucleotide sequence by a pest inhibits the growth of said
pest.
2. The ribonucleotide sequence of claim 1, wherein contact of said
sequence inhibits expression of a nucleotide sequence substantially
complementary to said sequence.
3. A cell transformed with a polynucleotide of any of claim 1.
4. The cell of claim 3, wherein said cell is a bacterial, yeast, or
algal cell.
5. A food product comprising the cell of claim 4.
6. The food product of claim 5, wherein said food product is
selected from the group consisting of stored grains, pet food, and
powdered chocolate.
7. A composition comprising the polynucleotide of claim 1.
8. The composition of claim 7, wherein said composition is selected
from the group consisting of a spray, powder, pellet, gel, capsule,
food product, garment bag, and book.
9. A method for controlling pest infestation, comprising exposing a
pest to a composition comprising a polynucleotide sequence that
inhibits a pest biological activity.
10. The method of claim 9, wherein said polynucleotide sequence is
set forth in any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,
21, 23, 49-158, 159, 160, 163, 168, 173, 178, 183, 188, 193, 198,
203, 208, 215, 220, 225, 230, 247, 249, 251, 253, 255, 257, 259,
275-472, 473, 478, 483, 488, 493, 498, 503, 513, 515, 517, 519,
521, 533-575, 576, 581, 586, 591, 596, 601, 603, 605, 607,
609,621-767, 768, 773, 778, 783, 788, 793, 795, 797, 799, 801,
813-862, 863, 868, 873, 878, 883, 888, 890, 892, 894, 896,
908-1040, 1041, 1046, 1051, 1056, 1061, 1071, 1073, 1075, 1077,
1079, 1081, 1083, 1085, 1087, 1089, 1091, 1093, 1095, 1097, 1099,
1101, 1103, 1105, 1107, 1109, 1111, 1113, 1161 to 1571, 1572, 1577,
1582, 1587, 1592, 1597, 1602, 1607, 1612, 1617, 1622, 1627, 1632,
1637, 1642, 1647, 1652, 1657, 1662, 1667, 1672, 1677, 1682, 1684,
1686, 1688, 1690, 1692, 1694, 1696, 1698, 1700, 1702, 1704,
1730-2039, 2040, 2045, 2050, 2055, 2060, 2065, 2070, 2075, 2080,
2085, 2090, 2095, 2100, 2102, 2104, 2106, 2108, 2120-2338, 2339,
2344, 2349, 2354, 2359, 2364, 2366, 2368, 2370, 2372, 2384-2460,
2461, 2466, 2471, 2476 and 2481.
11. A pesticide comprising a polynucleotide of claim 1.
12. A method for protecting an object from pest infestation,
comprising treating the surface of said object with a composition
comprising a polynucleotide of claim 1.
13. The method of claim 12, wherein said object is selected from
the group consisting of wood, tree, book binding, cloth, and a food
storage container.
14. A method for preventing or treating an insect infestation
comprising administering a composition according to claim 7.
15. A method for preventing or treating a nematode infestation
comprising administering a composition according to claim 7.
16. A method for preventing or treating a fungal infection
comprising administering a composition according to claim 7.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of, and claims
priority to U.S. patent application Ser. No. 11/992,091 filed on
May 8, 2008 which is a national stage filing under 35 U.S.C.
.sctn.371 of International Application No. PCT/IB2006/004008, filed
on Sep. 18, 2006, which claims benefit of 60/837,910, filed on Aug.
16, 2006, and claims benefit of 60/771,160, filed on Feb. 7, 2006,
and claims benefit of 60/758,191 filed on Jan. 12, 2006, and claims
benefit of 60/718,034, filed on Sep. 16, 2005, the contents of each
of which are herein incorporated by reference in their
entireties.
SEQUENCE LISTING
[0002] A Sequence Listing in ASCII text format, submitted under 37
CFR .sctn.1.821, entitled "80386_SEQLIST_ST25.txt", 774 kilobytes
in size, generated on Sep. 11, 2014 and filed via EFS-Web is
provided in lieu of a paper copy. This sequence listing is hereby
incorporated by reference into the specification for its
disclosures.
FIELD OF THE INVENTION
[0003] The present invention relates generally to genetic control
of pest infestations. More specifically, the present invention
relates to recombinant technologies for repressing or inhibiting
expression of target coding sequences in a pest.
INTRODUCTION
[0004] Insect and other pests can cause injury and even death by
their bites or stings. Additionally, many pests transmit bacteria
and other pathogens that cause diseases. For example, mosquitoes
transmit pathogens that cause malaria, yellow fever, encephalitis,
and other diseases. The bubonic plague, or black death, is caused
by bacteria that infect rats and other rodents. Compositions for
controlling microscopic pest infestations have been provided in the
form of antibiotic, antiviral, and antifungal compositions. Methods
for controlling infestations by pests, such as nematodes and
insects, have typically been in the form of chemical compositions
that are applied to surfaces on which pests reside, or administered
to infested animals in the form of pellets, powders, tablets,
pastes, or capsules.
[0005] Commercial crops are often the targets of insect attack.
Substantial progress has been made in the last a few decades
towards developing more efficient methods and compositions for
controlling insect infestations in plants. Chemical pesticides have
been very effective in eradicating pest infestations. However,
there are several disadvantages to using chemical pesticides. Not
only are they potentially detrimental to the environment, but
chemical pesticides are not selective and can pose harm to
non-target flora and fauna. Chemical pesticides persist in the
environment and generally are slow to be metabolized, if at all.
They accumulate in the food chain, and particularly in the higher
predator species. Accumulation of chemical pesticides results in
the development of resistance to the agents and in species higher
up the evolutionary ladder, they can act as mutagens and/or
carcinogens and cause irreversible and deleterious genetic
modifications.
[0006] Because of the dangers associated with chemical pesticides,
biological approaches have been developed for controlling pest
infestations. For example, biological control using protein Cry3A
from Bacillus thuringiensis have effectively controlled Colorado
potato beetle larvae either as formulations sprayed onto the
foliage or expressed in the leaves of potatoes. An alternative
biological agent is double stranded RNA (dsRNA). Over the last few
years, downregulation of genes (also referred to as "gene
silencing") in multicellular organisms by means of RNA interference
has become a well-established technique.
[0007] RNA Interference (RNAi) provides a potentially powerful tool
for controlling gene expression because of its specificity of
target selection and remarkably high efficiency in target mRNA
suppression. RNAi refers to the process of sequence-specific
post-transcriptional gene silencing mediated by short interfering
RNAs (siRNAs) (Zamore, P. et al., Cell 101:25-33 (2000); Fire, A.
et al., Nature 391:806 (1998); Hamilton et al., Science 286,
950-951 (1999); Lin et al., Nature 402:128-129 (1999)). While the
mechanics underlying RNAi are not fully characterized, it is
thought that the presence of dsRNA in cells triggers RNAi by
activating the ribonuclease III enzyme Dicer (Zamore, P. et al.,
(2000); Hammond et al., Nature 404, 293 (2000)). Dicer processes
the dsRNA into short pieces called short interfering RNAs (siRNAs),
which are about 21 to about 23 nucleotides long and comprise about
19 base pair duplexes (Zamore et al., (2000); Elbashir et al.,
Genes Dev., 15, 188 (2001)). Following delivery into cells, the
siRNA molecules associate with an endonuclease complex, commonly
referred to as an RNA-induced silencing complex (RISC), which
brings together the antisense strand of the siRNA and the cellular
mRNA gene target. RISC cleaves the mRNA, which is then released and
degraded. Importantly, RISC is then capable of degrading additional
copies of the target mRNA.
[0008] Accordingly, the present invention provides methods and
compositions for controlling pest infestation by repressing,
delaying, or otherwise reducing gene expression within a particular
pest.
SUMMARY OF THE INVENTION
[0009] In one aspect, the invention provides an isolated
polynucleotide sequence comprising a nucleic acid sequence set
forth in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,
49-158, 159, 160, 163, 168, 173, 178, 183, 188, 193, 198, 203, 208,
215, 220, 225, 230, 247, 249, 251, 253, 255, 257, 259, 275-472,
473, 478, 483, 488, 493, 498, 503, 513, 515, 517, 519, 521,
533-575, 576, 581, 586, 591, 596, 601, 603, 605, 607, 609, 621-767,
768, 773, 778, 783, 788, 793, 795, 797, 799, 801, 813-862, 863,
868, 873, 878, 883, 888, 890, 892, 894, 896, 908-1040, 1041, 1046,
1051, 1056, 1061, 1071, 1073, 1075, 1077, 1079, 1081, 1083, 1085,
1087, 1089, 1091, 1093, 1095, 1097, 1099, 1101, 1103, 1105, 1107,
1109, 1111, 1113, 1161-1571, 1572, 1577, 1582, 1587, 1592, 1597,
1602, 1607, 1612, 1617, 1622, 1627, 1632, 1637, 1642, 1647, 1652,
1657, 1662, 1667, 1672, 1677, 1682, 1684, 1686, 1688, 1690, 1692,
1694, 1696, 1698, 1700, 1702, 1704, 1730-2039, 2040, 2045, 2050,
2055, 2060, 2065, 2070, 2075, 2080, 2085, 2090, 2095, 2100, 2102,
2104, 2106, 2108, 2120-2338, 2339, 2344, 2349, 2354, 2359, 2364,
2366, 2368, 2370, 2372, 2384-2460, 2461, 2466, 2471, 2476 and 2481.
In one embodiment, a double stranded ribonucleotide sequence is
produced from the expression of a polynucleotide sequence, wherein
contact of said ribonucleotide sequence by a pest inhibits the
growth of said pest. In a further embodiment, contact of the
sequence inhibits expression of a nucleotide sequence substantially
complementary to said sequence. In another embodiment, a cell is
transformed with the polynucleotide. In a further embodiment, the
cell is a bacterial, yeast, or algal cell. In a still further
embodiment, a food product, such as stored grains, pet food, or
powdered chocolate, comprises the cell transformed with the
polynucleotide. In yet another embodiment, a composition, such as a
spray, powder, pellet, gel, capsule, food product, garment bag, and
book, comprising the polynucleotide. In yet another embodiment, the
invention provides a pesticide comprising the polynucleotide. In
another embodiment, the invention provides a method for protecting
an object, such as wood, tree, book binding, cloth, and a food
storage container, from pest infestation, comprising treating said
surface with a composition comprising the polynucleotide.
[0010] In another aspect, the invention provides a polynucleotide
sequence having at least 70% sequence identity to a nucleic acid
sequence set forth in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17,
19, 21, 23, 49-158, 159, 160, 163, 168, 173, 178, 183, 188, 193,
198, 203, 208, 215, 220, 225, 230, 247, 249, 251, 253, 255, 257,
259, 275-472, 473, 478, 483, 488, 493, 498, 503, 513, 515, 517,
519, 521, 533-575, 576, 581, 586, 591, 596, 601, 603, 605, 607,
609, 621-767, 768, 773, 778, 783, 788, 793, 795, 797, 799, 801,
813-862, 863, 868, 873, 878, 883, 888, 890, 892, 894, 896,
908-1040, 1041, 1046, 1051, 1056, 1061, 1071, 1073, 1075, 1077,
1079, 1081, 1083, 1085, 1087, 1089, 1091, 1093, 1095, 1097, 1099,
1101, 1103, 1105, 1107, 1109, 1111, 1113, 1161-1571, 1572, 1577,
1582, 1587, 1592, 1597, 1602, 1607, 1612, 1617, 1622, 1627, 1632,
1637, 1642, 1647, 1652, 1657, 1662, 1667, 1672, 1677, 1682, 1684,
1686, 1688, 1690, 1692, 1694, 1696, 1698, 1700, 1702, 1704,
1730-2039, 2040, 2045, 2050, 2055, 2060, 2065, 2070, 2075, 2080,
2085, 2090, 2095, 2100, 2102, 2104, 2106, 2108, 2120-2338, 2339,
2344, 2349, 2354, 2359, 2364, 2366, 2368, 2370, 2372, 2384-2460,
2461, 2466, 2471, 2476 and 2481. In one embodiment, a double
stranded ribonucleotide sequence is produced from the expression of
a polynucleotide sequence, wherein contact of said ribonucleotide
sequence by a pest inhibits the growth of said pest. In a further
embodiment, contact of the sequence inhibits expression of a
nucleotide sequence substantially complementary to said sequence.
In another embodiment, a cell is transformed with the
polynucleotide. In a further embodiment, the cell is a bacterial,
yeast, or algal cell. In a still further embodiment, a food
product, such as stored grains, pet food, or powdered chocolate,
comprises the cell transformed with the polynucleotide. In yet
another embodiment, a composition, such as a spray, powder, pellet,
gel, capsule, food product, garment bag, and book, comprising the
polynucleotide. In yet another embodiment, the invention provides a
pesticide comprising the polynucleotide. In another embodiment, the
invention provides a method for protecting an object, such as wood,
tree, book binding, cloth, and a food storage container, from pest
infestation, comprising treating said surface with a composition
comprising the polynucleotide.
[0011] In another aspect, the invention provides a method for
controlling pest infestation, comprising exposing a pest to a
composition comprising a polynucleotide sequence that inhibits a
pest biological activity. In one embodiment, the polynucleotide
sequence is set forth in any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13,
15, 17, 19, 21, 23, 49-158, 159, 160, 163, 168, 173, 178, 183, 188,
193, 198, 203, 208, 215, 220, 225, 230, 247, 249, 251, 253, 255,
257, 259, 275-472, 473, 478, 483, 488, 493, 498, 503, 513, 515,
517, 519, 521, 533-575, 576, 581, 586, 591, 596, 601, 603, 605,
607, 609, 621-767, 768, 773, 778, 783, 788, 793, 795, 797, 799,
801, 813-862, 863, 868, 873, 878, 883, 888, 890, 892, 894, 896,
908-1040, 1041, 1046, 1051, 1056, 1061, 1071, 1073, 1075, 1077,
1079, 1081, 1083, 1085, 1087, 1089, 1091, 1093, 1095, 1097, 1099,
1101, 1103, 1105, 1107, 1109, 1111, 1113, 1161-1571, 1572, 1577,
1582, 1587, 1592, 1597, 1602, 1607, 1612, 1617, 1622, 1627, 1632,
1637, 1642, 1647, 1652, 1657, 1662, 1667, 1672, 1677, 1682, 1684,
1686, 1688, 1690, 1692, 1694, 1696, 1698, 1700, 1702, 1704,
1730-2039, 2040, 2045, 2050, 2055, 2060, 2065, 2070, 2075, 2080,
2085, 2090, 2095, 2100, 2102, 2104, 2106, 2108, 2120-2338, 2339,
2344, 2349, 2354, 2359, 2364, 2366, 2368, 2370, 2372, 2384-2460,
2461, 2466, 2471, 2476 and 2481.
[0012] In other embodiments, the invention provides for the use of
the isolated nucleotide sequence, the double stranded
ribonucleotide sequence, the cell, the composition, or the
pesticide for preventing or treating an infestation, such as
insect, nematode, or fungal infestation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1-LD: Survival of L. decemlineata on artificial diet
treated with dsRNA. Insects of the second larval stage were fed
diet treated with 50 .mu.l of topically-applied solution of dsRNA
(targets or gfp control). Diet was replaced with fresh diet
containing topically-applied dsRNA after 7 days. The number of
surviving insects were assessed at days 2, 5, 7, 8, 9, & 13.
The percentage of surviving larvae was calculated relative to day 0
(start of assay). Target LD006: (SEQ ID NO: 178); Target LD007 (SEQ
ID NO: 183); Target LD010 (SEQ ID NO: 188); Target LD011 (SEQ ID
NO: 193); Target LD014 (SEQ ID NO: 198); gfp dsRNA (SEQ ID NO:
235).
[0014] FIG. 2-LD: Survival of L. decemlineata on artificial diet
treated with dsRNA. Insects of the second larval stage were fed
diet treated with 50 .mu.l of topically-applied solution of dsRNA
(targets or gfp control). Diet was replaced with fresh diet only
after 7 days. The number of surviving insects was assessed at days
2, 5, 6, 7, 8, 9, 12, & 14. The percentage of surviving larvae
was calculated relative to day 0 (start of assay). Target LD001
(SEQ ID NO: 163); Target LD002 (SEQ ID NO: 168); Target LD003 (SEQ
ID NO: 173); Target LD015 (SEQ ID NO: 215); Target LD016 (SEQ ID
NO: 220); gfp dsRNA (SEQ ID NO: 235).
[0015] FIG. 3-LD: Average weight of L. decemlineata larvae on
potato leaf discs treated with dsRNA. Insects of the second larval
stage were fed leaf discs treated with 20 .mu.l of a
topically-applied solution (10 ng/.mu.l) of dsRNA (target LD002 or
gfp). After two days the insects were transferred on to untreated
leaves every day.
[0016] FIG. 4-LD: Survival of L. decemlineata on artificial diet
treated with shorter versions of target LD014 dsRNA and concatemer
dsRNA. Insects of the second larval stage were fed diet treated
with 50 .mu.l of topically-applied solution of dsRNA (gfp or
targets). The number of surviving insects were assessed at days 3,
4, 5, 6, & 7. The percentage of surviving larvae were
calculated relative to day 0 (start of assay).
[0017] FIG. 5-LD: Survival of L. decemlineata larvae on artificial
diet treated with different concentrations of dsRNA of target LD002
(a), target LD007 (b), target LD010 (c), target LD011 (d), target
LD014 (e), target LD015 (f), LD016 (g) and target LD027 (h).
Insects of the second larval stage were fed diet treated with 50
.mu.l of topically-applied solution of dsRNA. Diet was replaced
with fresh diet containing topically-applied dsRNA after 7 days.
The number of surviving insects were assessed at regular intervals.
The percentage of surviving larvae were calculated relative to day
0 (start of assay).
[0018] FIG. 6-LD. Effects of E. coli strains expressing dsRNA
target LD010 on survival of larvae of the Colorado potato beetle,
Leptinotarsa decemlineata, over time. The two bacterial strains
were tested in separate artificial diet-based bioassays: (a)
AB309-105; data points for pGBNJ003 and pGN29 represent average
mortality values from 5 different bacterial clones, (b) BL21(DE3);
data points for pGBNJ003 and pGN29 represent average mortality
values from 5 different and one single bacterial clones,
respectively. Error bars represent standard deviations.
[0019] FIG. 7-LD. Effects of different clones of E. coli strains
(a) AB309-105 and (b) BL21(DE3) expressing dsRNA target LD010 on
survival of larvae of the Colorado potato beetle, Leptinotarsa
decemlineata, 12 days post infestation. Data points are average
mortality values for each clone for pGN29 and pGBNJ003. Clone 1 of
AB309-105 harbouring plasmid pGBNJ003 showed 100% mortality towards
CPB at this timepoint. Error bars represent standard
deviations.
[0020] FIG. 8-LD. Effects of different clones of E. coli strains
(a) AB309-105 and (b) BL21(DE3) expressing dsRNA target LD010 on
growth and development of larval survivors of the Colorado potato
beetle, Leptinotarsa decemlineata, 7 days post infestation. Data
points are % average larval weight values for each clone (one clone
for pGN29 and five clones for pGBNJ003) based on the data of Table
10. Diet only treatment represents 100% normal larval weight.
[0021] FIG. 9-LD. Survival of larvae of the Colorado potato beetle,
Leptinotarsa decemlineata, on potato plants sprayed by
double-stranded RNA-producing bacteria 7 days post infestation.
Number of larval survivors were counted and expressed in terms of %
mortality. The bacterial host strain used was the
RNaseIII-deficient strain AB309-105. Insect gene target was
LD010.
[0022] FIG. 10-LD. Growth/developmental delay of larval survivors
of the Colorado potato beetle, Leptinotarsa decemlineata, fed on
potato plants sprayed with dsRNA-producing bacteria 11 days post
infestation. The bacterial host strain used was the
RNaseIII-deficient strain AB309-105. Data figures represented as
percentage of normal larval weight; 100% of normal larval weight
given for diet only treatment. Insect gene target was LD010. Error
bars represent standard deviations.
[0023] FIG. 11-LD. Resistance to potato damage caused by larvae of
the Colorado potato beetle, Leptinotarsa decemlineata, by
double-stranded RNA-producing bacteria 7 days post infestation.
Left, plant sprayed with 7 units of bacteria AB309-105 containing
the pGN29 plasmid; right, plant sprayed with 7 units of bacteria
Ab309-105 containing the pGBNJ003 plasmid. One unit is defined as
the equivalent of 1 ml of a bacterial suspension at OD value of 1
at 600 nm. Insect gene target was LD010.
[0024] FIG. 12-LD. Survival of L. decemlineata adults on potato
leaf discs treated with dsRNA. Young adult insects were fed
double-stranded-RNA-treated leaf discs for the first two days and
were then placed on untreated potato foliage. The number of
surviving insects were assessed regularly; mobile insects were
recorded as insects which were alive and appeared to move normally;
moribund insects were recorded as insects which were alive but
appeared sick and slow moving--these insects were not able to right
themselves once placed on their backs. Target LD002 (SEQ ID NO:
168); Target LD010 (SEQ ID NO: 188); Target LD014 (SEQ ID NO: 198);
Target LD016 (SEQ ID NO: 220); gfp dsRNA (SEQ ID NO: 235).
[0025] FIG. 13-LD. Effects of bacterial produced target
double-stranded RNA against larvae of L. decemlineata. Fifty .mu.l
of an OD 1 suspension of heat-treated bacteria expressing dsRNA
(SEQ ID NO: 188) was applied topically onto the solid artificial
diet in each well of a 48-well plate. CPB larvae at L2 stage were
placed in each well. At day 7, a picture was taken of the CPB
larvae in a plate containing (a) diet with bacteria expressing
target 10 double-stranded RNA, (b) diet with bacteria harbouring
the empty vector pGN29, and, (c) diet only.
[0026] FIG. 14-LD Effects on CPB larval survival and growth of
different amounts of inactivated E. coli AB309-105 strain
harbouring plasmid pGBNJ003 topically applied to potato foliage
prior to insect infestation. Ten L1 larvae were fed treated potato
for 7 days. Amount of bacterial suspension sprayed on plants: 0.25
U, 0.08 U, 0.025 U, 0.008 U of target 10 and 0.25 U of pGN29
(negative control; also included is Milli-Q water). One unit (U) is
defined as the equivalent bacterial amount present in 1 ml of
culture with an optical density value of 1 at 600 nm. A total
volume of 1.6 ml was sprayed on to each plant. Insect gene target
was LD010.
[0027] FIG. 15-LD Resistance to potato damage caused by CPB larvae
by inactivated E. coli AB309-105 strain harbouring plasmid pGBNJ003
seven days post infestation. (a) water, (b) 0.25 U E. coli
AB309-105 harbouring pGN29, (c) 0.025 U E. coli AB309-105
harbouring pGBNJ003, (d) 0.008 U E. coli AB309-105 harbouring
pGBNJ003. One unit (U) is defined as the equivalent bacterial
amount present in 1 ml of culture with an optical density value of
1 at 600 nm. A total volume of 1.6 ml was sprayed on to each plant.
Insect gene target was LD010.
[0028] FIG. 1-PC: Effects of ingested target dsRNAs on survival and
growth of P. cochleariae larvae. Neonate larvae were fed oilseed
rape leaf discs treated with 25 .mu.l of topically-applied solution
of 0.1 .mu.g/.mu.l dsRNA (targets or gfp control). After 2 days,
the insects were transferred onto fresh dsRNA-treated leaf discs.
At day 4, larvae from one replicate for every treatment were
collected and placed in a Petri dish containing fresh untreated
oilseed rape foliage. The insects were assessed at days 2, 4, 7, 9
& 11. (a) Survival of E. varivestis larvae on oilseed rape leaf
discs treated with dsRNA. The percentage of surviving larvae was
calculated relative to day 0 (start of assay). (b) Average weights
of P. cochleariae larvae on oilseed rape leaf discs treated with
dsRNA. Insects from each replicate were weighed together and the
average weight per larva determined. Error bars represent standard
deviations. Target 1: SEQ ID NO: 473; target 3: SEQ ID NO: 478;
target 5: SEQ ID NO: 483-; target 10: SEQ ID NO: 488; target 14:
SEQ ID NO: 493; target 16: SEQ ID NO: 498; target 27: SEQ ID NO:
503; gfp dsRNA: SEQ ID NO: 235.
[0029] FIG. 2-PC: Survival of P. cochleariae on oilseed rape leaf
discs treated with different concentrations of dsRNA of (a) target
PC010 and (b) target PC027. Neonate larvae were placed on leaf
discs treated with 25 .mu.l of topically-applied solution of dsRNA.
Insects were transferred to fresh treated leaf discs at day 2. At
day 4 for target PC010 and day 5 for target PC027, the insects were
transferred to untreated leaves. The number of surviving insects
were assessed at days 2, 4, 7, 8, 9 & 11 for PC010 and 2, 5, 8,
9 & 12 for PC027. The percentage of surviving larvae was
calculated relative to day 0 (start of assay).
[0030] FIG. 3-PC: Effects of E. coli strain AB309-105 expressing
dsRNA target PC010 on survival of larvae of the mustard leaf
beetle, P. cochleariae, over time. Data points for each treatment
represent average mortality values from 3 different replicates.
Error bars represent standard deviations. Target 10: SEQ ID NO:
488
[0031] FIG. 1-EV: Survival of E. varivestis larvae on bean leaf
discs treated with dsRNA. Neonate larvae were fed bean leaf discs
treated with 25 .mu.l of topically-applied solution of 1
.mu.g/.mu.l dsRNA (targets or gfp control). After 2 days, the
insects were transferred onto fresh dsRNA-treated leaf discs. At
day 4, larvae from one treatment were collected and placed in a
plastic box containing fresh untreated bean foliage. The insects
were assessed for mortality at days 2, 4, 6, 8 & 10. The
percentage of surviving larvae was calculated relative to day 0
(start of assay). Target 5: SEQ ID NO: 576; target 10: SEQ ID NO:
586; target 15: SEQ ID NO: 591; target 16: SEQ ID NO: 596; gfp
dsRNA: SEQ ID NO: 235.
[0032] FIG. 2-EV: Effects of ingested target dsRNAs on survival of
E. varivestis adults and resistance to snap bean foliar insect
damage. (a) Survival of E. varivestis adults on bean leaf treated
with dsRNA. Adults were fed bean leaf discs treated with 75 .mu.l
of topically-applied solution of 0.1 .mu.g/.mu.l dsRNA (targets or
gfp control). After 24 hours, the insects were transferred onto
fresh dsRNA-treated leaf discs. After a further 24 hours, adults
from one treatment were collected and placed in a plastic box
containing potted fresh untreated whole bean plants. The insects
were assessed for mortality at days 4, 5, 6, 7, 8, & 11. The
percentage of surviving adults was calculated relative to day 0
(start of assay). Target 10: SEQ ID NO: 586; target 15: SEQ ID NO:
591; target 16: SEQ ID NO: 596; gfp dsRNA: SEQ ID NO: 235. (b)
Resistance to bean foliar damage caused by adults of the E.
varivestis by dsRNA. Whole plants containing insects from one
treatment (see (a)) were checked visually for foliar damage on day
9. (i) target 10; (ii) target 15; (iii) target 16; (iv) gfp dsRNA;
(v) untreated.
[0033] FIG. 1-TC: Survival of T. castaneum larvae on artificial
diet treated with dsRNA of target 14. Neonate larvae were fed diet
based on a flour/milk mix with 1 mg dsRNA target 14. Control was
water (without dsRNA) in diet. Four replicates of 10 first instar
larvae per replicate were performed for each treatment. The insects
were assessed for survival as average percentage means at days 6,
17, 31, 45 and 60. The percentage of surviving larvae was
calculated relative to day 0 (start of assay). Error bars represent
standard deviations. Target TC014: SEQ ID NO: 878.
[0034] FIG. 1-MP: Effect of ingested target 27 dsRNA on the
survival of Myzus persicae nymphs. First instars were placed in
feeding chambers containing 50 .mu.l of liquid diet with 2
.mu.g/.mu.l dsRNA (target 27 or gfp dsRNA control). Per treatment,
5 feeding chambers were set up with 10 instars in each feeding
chamber. Number of survivors were assessed at 8 days post start of
bioassay. Error bars represent standard deviations. Target MP027:
SEQ ID NO: 1061; gfp dsRNA: SEQ ID NO: 235.
[0035] FIG. 1-NL: Survival of Nilaparvata lugens on liquid
artificial diet treated with dsRNA. Nymphs of the first to second
larval stage were fed diet supplemented with 2 mg/ml solution of
dsRNA targets in separate bioassays: (a) NL002, NL003, NL005,
NL010; (b) NL009, NL016; (c) NL014, NL018; (d) NL013, NL015, NL021.
Insect survival on targets were compared to diet only and diet with
gfp dsRNA control at same concentration. Diet was replaced with
fresh diet containing dsRNA every two days. The number of surviving
insects were assessed every day
[0036] FIG. 2-NL: Survival of Nilaparvata lugens on liquid
artificial diet treated with different concentrations of target
dsRNA NL002. Nymphs of the first to second larval stage were fed
diet supplemented with 1, 0.2, 0.08, and 0.04 mg/ml (final
concentration) of NL002. Diet was replaced with fresh diet
containing dsRNA every two days. The numbers of surviving insects
were assessed every day.
DETAILED DESCRIPTION OF THE INVENTION
[0037] The present invention provides a means for controlling pest
infestations by exposing a pest to a target coding sequence that
post-transcriptionally represses or inhibits a requisite biological
function in the pest. Following exposure to a target sequence, the
target forms a the dsRNA corresponding to part or whole of an
essential pest gene and causes down regulation of the pest target
via RNA interference (RNAi). As a result of the down regulation of
mRNA, the dsRNA prevents expression of the target pest protein and
hence causes death, growth arrest, or sterility of the pest.
[0038] The present invention finds application in any area where it
is desirable to inhibit viability, growth, development or
reproduction of a pest, or to decrease pathogenicity or infectivity
of a pest. Practical applications include, but are not limited to,
(1) protecting plants against pest infestation; (2) pharmaceutical
or veterinary use in humans and animals (for example to control,
treat, or prevent insect infections in humans and animals); (3)
protecting materials against damage caused by pests; and (4)
protecting perishable materials (such as foodstuffs, seed, etc.)
against damage caused by pests.
[0039] Administering or exposing a double stranded ribonucleic acid
molecule to a pest results in one or more of the following
attributes: reduction in feeding by the pest, reduction in
viability of the pest, death of the pest, inhibition of
differentiation and development of the pest, absence of or reduced
capacity for sexual reproduction by the pest, muscle formation,
juvenile hormone formation, juvenile hormone regulation, ion
regulation and transport, maintenance of cell membrane potential,
amino acid biosynthesis, amino acid degradation, sperm formation,
pheromone synthesis, pheromone sensing, antennae formation, wing
formation, leg formation, development and differentiation, egg
formation, larval maturation, digestive enzyme formation,
haemolymph synthesis, haemolymph maintenance, neurotransmission,
cell division, energy metabolism, respiration, apoptosis, and any
component of a eukaryotic cells' cytoskeletal structure, such as,
for example, actins and tubulins. Any one or any combination of
these attributes can result in an effective inhibition of pest
infestation.
[0040] All technical terms employed in this specification are
commonly used in biochemistry, molecular biology and agriculture;
hence, they are understood by those skilled in the field to which
this invention belongs. Those technical terms can be found, for
example in: MOLECULAR CLONING: A LABORATORY MANUAL, 3rd ed., vol.
1-3, ed. Sambrook and Russel, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., 2001; CURRENT PROTOCOLS IN MOLECULAR
BIOLOGY, ed. Ausubel et al., Greene Publishing Associates and
Wiley-Interscience, New York, 1988 (with periodic updates); SHORT
PROTOCOLS IN MOLECULAR BIOLOGY: A COMPENDIUM OF METHODS FROM
CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, 5.sup.th ed., vol. 1-2, ed.
Ausubel et al., John Wiley & Sons, Inc., 2002; GENOME ANALYSIS:
A LABORATORY MANUAL, vol. 1-2, ed. Green et al., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1997.
[0041] Various techniques using PCR are described, for example, in
Innis et al., PCR PROTOCOLS: A GUIDE TO METHODS AND APPLICATIONS,
Academic Press, San Diego, 1990 and in Dieffenbach and Dveksler,
PCR PRIMER: A LABORATORY MANUAL, 2.sup.nd ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 2003. PCR-primer pairs
can be derived from known sequences by known techniques such as
using computer programs intended for that purpose, e.g., Primer,
Version 0.5, 1991, Whitehead Institute for Biomedical Research,
Cambridge, Mass. Methods for chemical synthesis of nucleic acids
are discussed, for example, in Beaucage & Caruthers, Tetra.
Letts. 22: 1859-62 (1981), and Matteucci & Caruthers, J. Am.
Chem. Soc. 103: 3185 (1981).
[0042] Restriction enzyme digestions, phosphorylations, ligations,
and transformations were done as described in Sambrook et al.,
MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed. (1989), Cold Spring
Harbor Laboratory Press. All reagents and materials used for the
growth and maintenance of bacterial cells were obtained from
Aldrich Chemicals (Milwaukee, Wis.), DIFCO Laboratories (Detroit,
Mich.), Invitrogen (Gaithersburg, Md.), or Sigma Chemical Company
(St. Louis, Mo.) unless otherwise specified.
[0043] Biological activity refers to the biological behavior and
effects of a protein or peptide and its manifestations on a pest.
For example, an inventive RNAi may prevent translation of a
particular mRNA, thereby inhibiting the biological activity of the
protein encoded by the mRNA or other biological activity of the
pest.
[0044] In the present description, an RNAi molecule may inhibit a
biological activity in a pest, resulting in one or more of the
following attributes: reduction in feeding by the pest, reduction
in viability of the pest, death of the pest, inhibition of
differentiation and development of the pest, absence of or reduced
capacity for sexual reproduction by the pest, muscle formation,
juvenile hormone formation, juvenile hormone regulation, ion
regulation and transport, maintenance of cell membrane potential,
amino acid biosynthesis, amino acid degradation, sperm formation,
pheromone synthesis, pheromone sensing, antennae formation, wing
formation, leg formation, development and differentiation, egg
formation, larval maturation, digestive enzyme formation,
haemolymph synthesis, haemolymph maintenance, neurotransmission,
cell division, energy metabolism, respiration, apoptosis, and any
component of a eukaryotic cells' cytoskeletal structure, such as,
for example, actins and tubulins.
[0045] Complementary DNA (cDNA) refers to single-stranded DNA
synthesized from a mature mRNA template. Though there are several
methods, cDNA is most often synthesized from mature (fully spliced)
mRNA using the enzyme reverse transcriptase. This enzyme operates
on a single strand of mRNA, generating its complementary DNA based
on the pairing of RNA base pairs (A, U, G, C) to their DNA
complements (T, A, C, G). Two nucleic acid strands are
substantially complementary when at least 85% of their bases
pair.
[0046] Desired Polynucleotide: a desired polynucleotide of the
present invention is a genetic element, such as a promoter,
enhancer, or terminator, or gene or polynucleotide that is to be
transcribed and/or translated in a transformed cell that comprises
the desired polynucleotide in its genome. If the desired
polynucleotide comprises a sequence encoding a protein product, the
coding region may be operably linked to regulatory elements, such
as to a promoter and a terminator, that bring about expression of
an associated messenger RNA transcript and/or a protein product
encoded by the desired polynucleotide. Thus, a "desired
polynucleotide" may comprise a gene that is operably linked in the
5'- to 3'-orientation, a promoter, a gene that encodes a protein,
and a terminator. Alternatively, the desired polynucleotide may
comprise a gene or fragment thereof, in a "sense" or "antisense"
orientation, the transcription of which produces nucleic acids that
may affect expression of an endogenous gene in the host cell. A
desired polynucleotide may also yield upon transcription a
double-stranded RNA product upon that initiates RNA interference of
a gene to which the desired polynucleotide is associated. A desired
polynucleotide of the present invention may be positioned within a
vector, such that the left and right border sequences flank or are
on either side of the desired polynucleotide. The present invention
envisions the stable integration of one or more desired
polynucleotides into the genome of at least one host cell. A
desired polynucleotide may be mutated or a variant of its wild-type
sequence. It is understood that all or part of the desired
polynucleotide can be integrated into the genome of a host. It also
is understood that the term "desired polynucleotide" encompasses
one or more of such polynucleotides. Thus, a vector of the present
invention may comprise one, two, three, four, five, six, seven,
eight, nine, ten, or more desired polynucleotides.
[0047] "Exposing" encompasses any method by which a pest may come
into contact with a dsRNA, wherein the dsRNA comprises annealed
complementary strands, one of which has a nucleotide sequence which
is complementary to at least part of the nucleotide sequence of a
pest target gene to be down-regulated. A pest may be exposed to the
dsRNA by direct uptake (e.g. by feeding), which does not require
expression of dsRNA within the pest. Alternatively, a pest may come
into direct contact with a composition comprising the dsRNA. For
example, a pest may come into contact with a surface or material
treated with a composition comprising a dsRNA. A dsRNA may be
expressed by a prokaryotic (for instance, but not limited to, a
bacterial) or eukaryotic (for instance, but not limited to, a
yeast) host cell or host organism.
[0048] Foreign: "foreign," with respect to a nucleic acid, means
that that nucleic acid is derived from non-host organisms.
According to the present invention, foreign DNA or RNA represents
nucleic acids that are naturally occurring in the genetic makeup of
viruses, mammals, fish or birds, but are not naturally occurring in
the host that is to be transformed. Thus, a foreign nucleic acid is
one that encodes, for instance, a polypeptide that is not naturally
produced by the transformed host. A foreign nucleic acid does not
have to encode a protein product.
[0049] Gene: refers to a polynucleotide sequence that comprises
control and coding sequences necessary for the production of a
polypeptide or precursor. The polypeptide can be encoded by a full
length coding sequence or by any portion of the coding sequence. A
gene may constitute an uninterrupted coding sequence or it may
include one or more introns, bound by the appropriate splice
junctions. Moreover, a gene may contain one or more modifications
in either the coding or the untranslated regions that could affect
the biological activity or the chemical structure of the expression
product, the rate of expression, or the manner of expression
control. Such modifications include, but are not limited to,
mutations, insertions, deletions, and substitutions of one or more
nucleotides. In this regard, such modified genes may be referred to
as "variants" of the "native" gene.
[0050] Genetic element: a "genetic element" is any discreet
nucleotide sequence such as, but not limited to, a promoter, gene,
terminator, intron, enhancer, spacer, 5'-untranslated region,
3'-untranslated region, or recombinase recognition site.
[0051] Genetic modification: stable introduction of a nucleic acid
into the genome of certain organisms by applying methods in
molecular and cell biology.
[0052] "Gene suppression" or "down-regulation of gene expression"
or "inhibition of gene expression" are used interchangeably and
refer to a measurable or observable reduction in gene expression or
a complete abolition of detectable gene expression, at the level of
protein product and/or mRNA product from the target gene.
Down-regulation or inhibition of gene expression is "specific" when
down-regulation or inhibition of the target gene occurs without
manifest effects on other genes of the pest.
[0053] Depending on the nature of the target gene, down-regulation
or inhibition of gene expression in cells of a pest can be
confirmed by phenotypic analysis of the cell or the whole pest or
by measurement of mRNA or protein expression using molecular
techniques such as RNA solution hybridization, nuclease protection,
Northern hybridization, reverse transcription, gene expression
monitoring with a microarray, antibody binding, enzyme-linked
immunosorbent assay (ELISA), Western blotting, radioimmunoassay
(RIA), other immunoassays, or fluorescence-activated cell analysis
(FACS).
[0054] Gymnosperm, as used herein, refers to a seed plant that
bears seed without ovaries. Examples of gymnosperms include
conifers, cycads, ginkgos, and ephedras.
[0055] Homology, as used herein relates to sequences; Protein, or
nucleotide sequences are likely to be homologous if they show a
"significant" level of sequence similarity or more preferably
sequence identity. Truely homologous sequences are related by
divergence from a common ancestor gene. Sequence homologs can be of
two types: (i) where homologs exist in different species they are
known as orthologs. e.g. the .alpha.-globin genes in mouse and
human are orthologs; (ii) paralogues are homologous genes in within
a single species. e.g. the .alpha.- and .beta.-globin genes in
mouse are paralogs.
[0056] Host cell: refers to a microorganism, a prokaryotic cell, a
eukaryotic cell, or cell line cultured as a unicellular entity that
may be, or has been, used as a recipient for a recombinant vector
or other transfer of polynucleotides, and includes the progeny of
the original cell that has been transfected. The progeny of a
single cell may not necessarily be completely identical in
morphology or in genomic or total DNA complement as the original
parent due to natural, accidental, or deliberate mutation.
[0057] Introduction: as used herein, refers to the insertion of a
nucleic acid sequence into a cell, by methods including infection,
transfection, transformation, or transduction.
[0058] Insect pests as used herein pests are include but are not
limited to: from the order Lepidoptera, for example, Acleris spp.,
Adoxophyes spp., Aegeria spp., Agrotis spp., Alabama argillaceae,
Amylois spp., Anticarsia gemmatalis, Archips spp, Argyrotaenia
spp., Autographa spp., Busseola fusca, Cadra cautella, Carposina
nipponensis, Chilo spp., Choristoneura spp., Clysia ambiguella,
Cnaphalocrocis spp., Cnephasia spp., Cochylis spp., Coleophora
spp., Crocidolomia binotalis, Cryptophlebia leucotreta, Cydia spp.,
Diatraea spp., Diparopsis castanea, Earias spp., Ephestia spp.,
Eucosma spp., Eupoecilia ambiguella, Euproctis spp., Euxoa spp.,
Grapholita spp., Hedya nubiferana, Heliothis spp., Hellula undalis,
Hyphantria cunea, Keiferia lycopersicella, Leucoptera scitella,
Lithocollethis spp., Lobesia botrana, Lymantria spp., Lyonetia
spp., Malacosoma spp., Mamestra brassicae, Manduca sexta,
Operophtera spp., Ostrinia Nubilalis, Pammene spp., Pandemis spp.,
Panolis flammea, Pectinophora gossypiella, Phthorimaea operculella,
Pieris rapae, Pieris spp., Plutella xylostella, Prays spp.,
Scirpophaga spp., Sesamia spp., Sparganothis spp., Spodoptera spp.,
Synanthedon spp., Thaumetopoea spp., Tortrix spp., Trichoplusia ni
and Yponomeuta spp.;
[0059] from the order Coleoptera, for example, Agriotes spp.,
Anthonomus spp., Atomaria linearis, Chaetocnema tibialis,
Cosmopolites spp., Curculio spp., Dermestes spp., Epilachna spp.,
Eremnus spp., Leptinotarsa decemlineata, Lissorhoptrus spp.,
Melolontha spp., Orycaephilus spp., Otiorhynchus spp., Phlyctinus
spp., Popillia spp., Psylliodes spp., Rhizopertha spp.,
Scarabeidae, Sitophilus spp., Sitotroga spp., Tenebrio spp.,
Tribolium spp. and Trogoderma spp.;
[0060] from the order Orthoptera, for example, Blatta spp.,
Blattella spp., Gryllotalpa spp., Leucophaea maderae, Locusta spp.,
Periplaneta ssp., and Schistocerca spp.;
[0061] from the order Isoptera, for example, Reticulitemes ssp;
from the order Psocoptera, for example, Liposcelis spp.; from the
order Anoplura, for example, Haematopinus spp., Linognathus spp.,
Pediculus spp., Pemphigus spp. and Phylloxera spp.;
[0062] from the order Mallophaga, for example, Damalinea spp. and
Trichodectes spp.; from the order Thysanoptera, for example,
Franklinella spp., Hercinothrips spp., Taeniothrips spp., Thrips
palmi, Thrips tabaci and Scirtothrips aurantii;
[0063] from the order Heteroptera, for example, Cimex spp.,
Distantiella theobroma, Dysdercus spp., Euchistus spp., Eurygaster
spp., Leptocorisa spp., Nezara spp., Piesma spp., Rhodnius spp.,
Sahlbergella singularis, Scotinophara spp., Triatoma spp., Miridae
family spp. such as Lygus hesperus and Lygus lineoloris, Lygaeidae
family spp. such as Blissus leucopterus, and Pentatomidae family
spp.;
[0064] from the order Homoptera, for example, Aleurothrixus
floccosus, Aleyrodes brassicae, Aonidiella spp., Aphididae, Aphis
spp., Aspidiotus spp., Bemisia tabaci, Ceroplaster spp.,
Chrysomphalus aonidium, Chrysomphalus dictyospermi, Coccus
hesperidum, Empoasca spp., Eriosoma larigerum, Erythroneura spp.,
Gascardia spp., Laodelphax spp., Lacanium corni, Lepidosaphes spp.,
Macrosiphus spp., Myzus spp., Nehotettix spp., Nilaparvata spp.,
Paratoria spp., Pemphigus spp., Planococcus spp., Pseudaulacaspis
spp., Pseudococcus spp., Psylla ssp., Pulvinaria aethiopica,
Quadraspidiotus spp., Rhopalosiphum spp., Saissetia spp.,
Scaphoideus spp., Schizaphis spp., Sitobion spp., Trialeurodes
vaporariorum, Trioza erytreae and Unaspis citri;
[0065] from the order Hymenoptera, for example, Acromyrmex, Atta
spp., Cephus spp., Diprion spp., Diprionidae, Gilpinia polytoma,
Hoplocampa spp., Lasius sppp., Monomorium pharaonis, Neodiprion
spp, Solenopsis spp. and Vespa ssp.;
[0066] from the order Diptera, for example, Aedes spp., Antherigona
soccata, Bibio hortulanus, Calliphora erythrocephala, Ceratitis
spp., Chrysomyia spp., Culex spp., Cuterebra spp., Dacus spp.,
Drosophila melanogaster, Fannia spp., Gastrophilus spp., Glossina
spp., Hypoderma spp., Hyppobosca spp., Liriomysa spp., Lucilia
spp., Melanagromyza spp., Musca ssp., Oestrus spp., Orseolia spp.,
Oscinella fit, Pegomyia hyoscyami, Phorbia spp., Rhagoletis
pomonella, Sciara spp., Stomoxys spp., Tabanus spp., Tannia spp.
and Tipula spp.,
[0067] from the order Siphonaptera, for example, Ceratophyllus spp.
and Xenopsylla cheopis and
[0068] from the order Thysanura, for example, Lepisma
saccharina.
[0069] Monocotyledonous plant (monocot) is a flowering plant having
embryos with one cotyledon or seed leaf, parallel leaf veins, and
flower parts in multiples of three. Examples of monocots include,
but are not limited to turfgrass, maize, rice, oat, wheat, barley,
sorghum, orchid, iris, lily, onion, and palm.
[0070] Pest or target pest refers to insects, arachnids,
crustaceans, fungi, bacteria, viruses, nematodes, flatworms,
roundworms, pinworms, hookworms, tapeworms, trypanosomes,
schistosomes, botflies, fleas, ticks, mites, and lice and the like
that are pervasive in the human environment. A pest may ingest or
contact one or more cells, tissues, or products produced by an
organism transformed with a double stranded gene suppression agent,
as well as a material or surface treated with a double stranded
gene suppression agent.
[0071] Nematodes, or roundworms, are one of the most common phyla
of animals, with over 20,000 different described species (over
15,000 are parasitic). They are ubiquitous in freshwater, marine,
and terrestrial environments, where they often outnumber other
animals in both individual and species counts, and are found in
locations as diverse as Antarctica and oceanic trenches. Further,
there are a great many parasitic forms, including pathogens in most
plants and animals.
[0072] Nematode pests of a particular interest include, for
example, A. caninum, A. ceylancium, H. contortus, O. ostertagi, C.
elegans, C. briggsae, P. pacificus, S. stercoralis, S. ratti, P.
trichosuri, M. arenaria, M. chitwoodi, M. hapla, M. incognita, M.
javanica, M. paraensis, G. rostochiensis, G. pallida, H. glycines,
H. schattii, P. penetrans, P. vulnus, R. similis, Z. punctata, A.
suum, T. canis, B. malayi, D. immitis, O. volvulus, T. vulpis, T.
spiralis, X. index. A. duodenale, A. lumbricoides, as well as
species from the following genera: Aphelenchoides, Nacobbus,
Ditylenchus, Longidorus, Trichodorus, and Bursaphelenchus.
[0073] Normal cell refers to a cell of an untransformed phenotype
or exhibiting a morphology of a non-transformed cell of the tissue
type being examined.
[0074] Operably linked: combining two or more molecules in such a
fashion that in combination they function properly in a cell. For
instance, a promoter is operably linked to a structural gene when
the promoter controls transcription of the structural gene.
[0075] Orthologs are genes that are related by vertical descent
from a common ancestor and encode proteins with the same function
in different species. Due to their separation following a
speciation event, orthologs may diverge, but usually have
similarity at the seqence and structure levels. Two genes that are
derived from a common ancestor and encode proteins with similar
function are referred to as orthologous. Identification of
orthologs is critical for reliable predictions of gene function in
newly sequenced genomes.
[0076] "Pest control agent", or "gene suppression agent" refers to
a particular RNA molecule comprising a first RNA segment and a
second RNA segment, wherein the complementarity between the first
and the second RNA segments results in the ability of the two
segments to hybridize in vivo and in vitro to form a double
stranded molecule. It may generally be preferable to include a
third RNA segment linking and stabilizing the first and second
sequences such that a stem can be formed linked together at one end
of each of the first and second segments by the third segment to
forms a loop, so that the entire structure forms into a stem and
loop structure, or even more tightly hybridizing structures may
form into a stem-loop knotted structure. Alternatively, a
symmetrical hairpin could be formed without a third segment in
which there is no designed loop, but for steric reasons a hairpin
would create its own loop when the stem is long enough to stabilize
itself. The first and the second RNA segments will generally lie
within the length of the RNA molecule and be substantially inverted
repeats of each other and linked together by the third RNA segment.
The first and the second segments correspond invariably and not
respectively to a sense and an antisense sequence with respect to
the target RNA transcribed from the target gene in the target
insect pest that is suppressed by the ingestion of the dsRNA
molecule.
[0077] The pest control agent can also be a substantially purified
(or isolated) nucleic acid molecule and more specifically nucleic
acid molecules or nucleic acid fragment molecules thereof from a
genomic DNA (gDNA) or cDNA library. Alternatively, the fragments
may comprise smaller oligonucleotides having from about 15 to about
250 nucleotide residues, and more preferably, about 15 to about 30
nucleotide residues.
[0078] Pesticide refers to any substance or mixture of substances
intended for preventing, destroying, repelling, or mitigating any
pest. A pesticide may be a chemical substance or biological agent
used against pests including insects, pathogens, weeds, nematodes,
and microbes that compete with humans for food, destroy property,
spread disease, or are a nuisance.
[0079] Phenotype is a distinguishing feature or characteristic of
an organism, which may be altered according to the present
invention by integrating one or more "desired polynucleotides"
and/or screenable/selectable markers into the genome of at least
one cell of a transformed organism. The "desired polynucleotide(s)"
and/or markers may confer a change in the phenotype of a
transformed organism, by modifying any one of a number of genetic,
molecular, biochemical, physiological, or morphological
characteristics or properties of the transformed cell or organism
as a whole.
[0080] Plant and plant tissue: a "plant" is any of various
photosynthetic, eukaryotic, multicellular organisms of the kingdom
Plantae characteristically producing embryos, containing
chloroplasts, and having cellulose cell walls. A part of a plant,
i.e., a "plant tissue" may be treated according to the methods of
the present invention to prevent pest infestation on the plant or
on the part of the plant. Many suitable plant tissues can be
treated according to the present invention and include, but are not
limited to, somatic embryos, pollen, leaves, stems, calli, stolons,
microtubers, and shoots. Thus, the present invention envisions the
treatment of angiosperm and gymnosperm plants such as acacia,
alfalfa, apple, apricot, artichoke, ash tree, asparagus, avocado,
banana, barley, beans, beet, birch, beech, blackberry, blueberry,
broccoli, brussels sprouts, cabbage, canola, cantaloupe, carrot,
cassaya, cauliflower, cedar, a cereal, celery, chestnut, cherry,
chinese cabbage, citrus, clemintine, clover, coffee, corn, cotton,
cowpea, cucumber, cypress, eggplant, elm, endive, eucalyptus,
fennel, figes, fir, geranium, grape, grapefruit, groundnuts, ground
cherry, gum hemlock, hickory, kale, kiwifruit, kohlrabi, larch,
lettuce, leek, lemon, lime, locust, pine, maidenhair, maize, mango,
maple, melon, millet, mushroom, mustard, nuts, oak, oats, okra,
onion, orange, an ornamental plant or flower or tree, papaya, palm,
parsley, parsnip, pea, peach, peanut, pear, peat, pepper,
persimmon, pigeon pea, pine, pineapple, plantain, plum,
pomegranate, potato, pumpkin, radicchio, radish, rapeseed,
raspberry, rice, rye, sorghum, soybean, spinach, spruce, squash,
strawberry, sugarbeet, sugarcane, sunflower, sweet potato, sweet
corn, tangerine, tea, tobacco, tomato, trees, triticale, turf
grasses, turnips, a vine, walnut, watercress, watermelon, wheat,
yams, yew, and zucchini.
[0081] According to the present invention "plant tissue" also
encompasses plant cells. Plant cells include suspension cultures,
callus, embryos, meristematic regions, callus tissue, leaves,
roots, shoots, gametophytes, sporophytes, pollen, seeds and
microspores. Plant tissues may be at various stages of maturity and
may be grown in liquid or solid culture, or in soil or suitable
media in pots, greenhouses or fields. A plant tissue also refers to
any clone of such a plant, seed, progeny, propagule whether
generated sexually or asexually, and descendents of any of these,
such as cuttings or seed.
[0082] Promoter is intended to mean a nucleic acid, preferably DNA
that binds RNA polymerase and/or other transcription regulatory
elements. As with any promoter, the promoters of the current
invention will facilitate or control the transcription of DNA or
RNA to generate an mRNA molecule from a nucleic acid molecule that
is operably linked to the promoter. As stated earlier, the RNA
generated may code for a protein or polypeptide or may code for an
RNA interfering, or antisense molecule.
[0083] Polynucleotide is a nucleotide sequence, comprising a gene
coding sequence or a fragment thereof, a promoter, an intron, an
enhancer region, a polyadenylation site, a translation initiation
site, 5' or 3' untranslated regions, a reporter gene, a selectable
marker or the like. The polynucleotide may comprise single stranded
or double stranded DNA or RNA. The polynucleotide may comprise
modified bases or a modified backbone. The polynucleotide may be
genomic, an RNA transcript (such as an mRNA) or a processed
nucleotide sequence (such as a cDNA). The polynucleotide may
comprise a sequence in either sense or antisense orientations.
[0084] An isolated polynucleotide is a polynucleotide sequence that
is not in its native state, e.g., the polynucleotide is comprised
of a nucleotide sequence not found in nature or the polynucleotide
is separated from nucleotide sequences with which it typically is
in proximity or is next to nucleotide sequences with which it
typically is not in proximity.
[0085] Recombinant nucleotide sequence refers to a nucleic acid
molecule that contains a genetically engineered modification
through manipulation via mutagenesis, restriction enzymes, and the
like.
[0086] RNA interference (RNAi) refers to sequence-specific or
gene-specific suppression of gene expression (protein synthesis)
that is mediated by short interfering RNA (siRNA).
[0087] Sequence identity: as used herein, "sequence identity" or
"identity" in the context of two nucleic acid sequences includes
reference to the residues in the two sequences which are the same
when aligned for maximum correspondence over a specified
region.
[0088] As used herein, percentage of sequence identity means the
value determined by comparing two optimally aligned sequences over
a comparison window, wherein the portion of the polynucleotide
sequence in the comparison window may comprise additions or
deletions (i.e., gaps) as compared to the reference sequence (which
does not comprise additions or deletions) for optimal alignment of
the two sequences. The percentage is calculated by determining the
number of positions at which the identical nucleic acid base occurs
in both sequences to yield the number of matched positions,
dividing the number of matched positions by the total number of
positions in the window of comparison and multiplying the result by
100 to yield the percentage of sequence identity.
[0089] "Sequence identity" has an art-recognized meaning and can be
calculated using published techniques. See COMPUTATIONAL MOLECULAR
BIOLOGY, Lesk, ed. (Oxford University Press, 1988), BIOCOMPUTING:
INFORMATICS AND GENOME PROJECTS, Smith, ed. (Academic Press, 1993),
COMPUTER ANALYSIS OF SEQUENCE DATA, PART I, Griffin & Griffin,
eds., (Humana Press, 1994), SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY,
Von Heinje ed., Academic Press (1987), SEQUENCE ANALYSIS PRIMER,
Gribskov & Devereux, eds. (Macmillan Stockton Press, 1991), and
Carillo & Lipton, SIAM J. Applied Math. 48: 1073 (1988).
Methods commonly employed to determine identity or similarity
between two sequences include but are not limited to those
disclosed in GUIDE To HUGE COMPUTERS, Bishop, ed., (Academic Press,
1994) and Carillo & Lipton, supra. Methods to determine
identity and similarity are codified in computer programs.
Preferred computer program methods to determine identity and
similarity between two sequences include but are not limited to the
GCG program package (Devereux et al., Nucleic Acids Research 12:
387 (1984)), BLASTP, BLASTN, FASTA (Atschul et al., J. Mol. Biol.
215: 403 (1990)), and FASTDB (Brutlag et al., Comp. App. Biosci. 6:
237 (1990)).
[0090] Short hairpin RNA (shRNA) are short single-stranded RNAs
having a high degree of secondary structure such that a portion of
the RNA strand forms a hairpin loop.
[0091] Short interfering RNA (siRNA) refers to double-stranded RNA
molecules from about 10 to about 30 nucleotides long that are named
for their ability to specifically interfere with gene protein
expression.
[0092] Target sequence refers to a nucleotide sequence in a pest
that is selected for suppression or inhibition by double stranded
RNA technology. A target sequence encodes an essential feature or
biological activity within a pest.
[0093] Transcriptional terminators: The expression DNA constructs
of the present invention typically have a transcriptional
termination region at the opposite end from the transcription
initiation regulatory region. The transcriptional termination
region may be selected, for stability of the mRNA to enhance
expression and/or for the addition of polyadenylation tails added
to the gene transcription product. Translation of a nascent
polypeptide undergoes termination when any of the three
chain-termination codons enters the A site on the ribosome.
Translation termination codons are UAA, UAG, and UGA.
[0094] Transformation: A process by which a nucleic acid is stably
inserted into the genome of an organism. Transformation may occur
under natural or artificial conditions using various methods well
known in the art. Transformation may rely on any known method for
the insertion of nucleic acid sequences into a prokaryotic or
eukaryotic host cell, including microorganism-mediated
transformation, viral infection, whiskers, electroporation,
microinjection, polyethylene glycol-treatment, heat shock,
lipofection, and particle bombardment.
[0095] Transgenic organism comprises at least one cell in which an
exogenous nucleic acid has been stably integrated. A transgenic
organism according to the invention is for instance a bacterial, or
eukaryotic, such as a yeast, host cell or host organism. The
bacterium can be chosen from the group comprising Gram-negative and
Gram-positive bacteria, such as, but not limited to, Escherichia
spp. (e.g. E. coli), Bacillus spp. (e.g. B. thuringiensis),
Rhizobium spp., Lactobacilllus spp., Lactococcus spp., etc. The
yeast can be chosen from the group comprising Saccharomyces spp.,
etc.
[0096] Variant: a "variant," as used herein, is understood to mean
a nucleotide sequence that deviates from the standard, or given,
nucleotide or amino acid sequence of a particular gene or protein.
The terms, "isoform," "isotype," and "analog" also refer to
"variant" forms of a nucleotide sequence. "Variant" may also refer
to a "shuffled gene" such as those described in Maxygen-assigned
patents.
[0097] It is understood that the present invention is not limited
to the particular methodology, protocols, vectors, and reagents,
etc., described herein, as these may vary. It is also to be
understood that the terminology used herein is used for the purpose
of describing particular embodiments only, and is not intended to
limit the scope of the present invention. It must be noted that as
used herein and in the appended claims, the singular forms "a,"
"an," and "the" include plural reference unless the context clearly
dictates otherwise. Thus, for example, a reference to "a gene" is a
reference to one or more genes and includes equivalents thereof
known to those skilled in the art and so forth.
[0098] I. Target Pests
[0099] The present invention provides methodology and constructs
for controlling pest infestations by administering, or otherwise
exposing, to a pest a target coding sequence that
post-transcriptionally represses or inhibits a requisite biological
function in the pest. As used herein, the term "pest" refers to
insects, arachnids, crustaceans, fungi, bacteria, viruses,
nematodes, flatworms, roundworms, pinworms, hookworms, tapeworms,
trypanosomes, schistosomes, botflies, fleas, ticks, mites, and lice
and the like that are pervasive in the human environment. A pest
may ingest or contact one or more cells, tissues, or products
produced by an organism transformed with a double stranded gene
suppression agent, as well as a surface or material treated with a
double stranded gene suppression agent.
[0100] A "pest resistance" trait is a characteristic of a
transgenic host that causes the host to be resistant to attack from
a pest that typically inflicts damage to the host. Such pest
resistance can arise from a natural mutation or more typically from
incorporation of recombinant DNA that confers pest resistance. To
impart pest resistance to a transgenic host, a recombinant DNA can,
for example, be transcribed into a RNA molecule that forms a dsRNA
molecule within the tissues or fluids of the recombinant host. The
dsRNA molecule is comprised in part of a segment of RNA that is
identical to a corresponding RNA segment encoded from a DNA
sequence within a pest that prefers to feed on the recombinant
host. Expression of the gene within the target pest is suppressed
by the dsRNA, and the suppression of expression of the gene in the
target pest results in the host being pest resistant.
[0101] Suitable pests include any organism that causes damage to
another organism. The invention contemplates insect, nematode, and
fungal pests in particular.
[0102] Insect as used herein can be any insect, meaning any
organism belonging to the Kingdom Animals, more specific to the
Phylum Arthropoda, and to the Class Insecta or the Class Arachnida.
The methods of the invention are applicable to all insects and that
are susceptible to gene silencing by RNA interference and that are
capable of internalising double-stranded RNA from their immediate
environment.
[0103] In one embodiment of the invention, the insect may belong to
the following orders: Acari, Araneae, Anoplura, Coleoptera,
Collembola, Dermaptera, Dictyoptera, Diplura, Diptera, Embioptera,
Ephemeroptera, Grylloblatodea, Hemiptera, Homoptera, Hymenoptera,
Isoptera, Lepidoptera, Mallophaga, Mecoptera, Neuroptera, Odonata,
Orthoptera, Phasmida, Plecoptera, Protura, Psocoptera,
Siphonaptera, Siphunculata, Thysanura, Strepsiptera, Thysanoptera,
Trichoptera, and Zoraptera.
[0104] In preferred, but non-limiting, embodiments and methods of
the invention the insect is chosen from the group consisting
of:
[0105] (1) an insect which is a plant pest, such as but not limited
to Nilaparvata spp. (e.g. N. lugens (brown planthopper));
Laodelphax spp. (e.g. L. striatellus (small brown planthopper));
Nephotettix spp. (e.g. N. virescens or N. cincticeps (green
leafhopper), or N. nigropictus (rice leafhopper)); Sogatella spp.
(e.g. S. furcifera (white-backed planthopper)); Blissus spp. (e.g.
B. leucopterus leucopterus (chinch bug)); Scotinophora spp. (e.g.
S. vermidulate (rice blackbug)); Acrosternum spp. (e.g. A. hilare
(green stink bug)); Parnara spp. (e.g. P. guttata (rice skipper));
Chilo spp. (e.g. C. suppressalis (rice striped stem borer), C.
auricilius (gold-fringed stem borer), or C. polychrysus
(dark-headed stem borer)); Chilotraea spp. (e.g. C. polychrysa
(rice stalk borer)); Sesamia spp. (e.g. S. inferens (pink rice
borer)); Tryporyza spp. (e.g. T. innotata (white rice borer), or T.
incertulas (yellow rice borer)); Cnaphalocrocis spp. (e.g. C.
medinalis (rice leafroller)); Agromyza spp. (e.g. A. oryzae
(leafminer), or A. parvicornis (corn blot leafminer)); Diatraea
spp. (e.g. D. saccharalis (sugarcane borer), or D. grandiosella
(southwestern corn borer)); Narnaga spp. (e.g. N. aenescens (green
rice caterpillar)); Xanthodes spp. (e.g. X. transversa (green
caterpillar)); Spodoptera spp. (e.g. S. frugiperda (fall armyworm),
S. exigua (beet armyworm), S. littoralis (climbing cutworm) or S.
praefica (western yellowstriped armyworm)); Mythimna spp. (e.g.
Mythmna (Pseudaletia) seperata (armyworm)); Helicoverpa spp. (e.g.
H. zea (corn earworm)); Colaspis spp. (e.g. C. brunnea (grape
colaspis)); Lissorhoptrus spp. (e.g. L. oryzophilus (rice water
weevil)); Echinocnemus spp. (e.g. E. squamos (rice plant weevil));
Diclodispa spp. (e.g. D. armigera (rice hispa)); Oulema spp. (e.g.
O. oryzae (leaf beetle); Sitophilus spp. (e.g. S. oryzae (rice
weevil)); Pachydiplosis spp. (e.g. P. oryzae (rice gall midge));
Hydrellia spp. (e.g. H. griseola (small rice leafminer), or H.
sasakii (rice stem maggot)); Chlorops spp. (e.g. C. oryzae (stem
maggot)); Ostrinia spp. (e.g. O. nubilalis (European corn borer));
Agrotis spp. (e.g. A. ipsilon (black cutworm)); Elasmopalpus spp.
(e.g. E. lignosellus (lesser cornstalk borer)); Melanotus spp.
(wireworms); Cyclocephala spp. (e.g. C. borealis (northern masked
chafer), or C. immaculata (southern masked chafer)); Popillia spp.
(e.g. P. japonica (Japanese beetle)); Chaetocnema spp. (e.g. C.
pulicaria (corn flea beetle)); Sphenophorus spp. (e.g. S. maidis
(maize billbug)); Rhopalosiphum spp. (e.g. R. maidis (corn leaf
aphid)); Anuraphis spp. (e.g. A. maidiradicis (corn root aphid));
Melanoplus spp. (e.g. M. femurrubrum (redlegged grasshopper) M.
differentialis (differential grasshopper) or M. sanguinipes
(migratory grasshopper)); Hylemya spp. (e.g. H. platura (seedcorn
maggot)); Anaphothrips spp. (e.g. A. obscrurus (grass thrips));
Solenopsis spp. (e.g. S. milesta (thief ant)); or spp. (e.g. T.
urticae (twospotted spider mite), T. cinnabarinus (carmine spider
mite); Helicoverpa spp. (e.g. H. zea (cotton bollworm), or H.
armigera (American bollworm)); Pectinophora spp. (e.g. P.
gossypiella (pink bollworm)); Earias spp. (e.g. E. vittella
(spotted bollworm)); Heliothis spp. (e.g. H. virescens (tobacco
budworm)); Anthonomus spp. (e.g. A. grandis (boll weevil));
Pseudatomoscelis spp. (e.g. P. seriatus (cotton fleahopper));
Trialeurodes spp. (e.g. T. abutiloneus (banded-winged whitefly) T.
vaporariorum (greenhouse whitefly)); Bemisia spp. (e.g. B.
argentifolii (silverleaf whitefly)); Aphis spp. (e.g. A. gossypii
(cotton aphid), A. mellifera); Lygus spp. (e.g. L. lineolaris
(tarnished plant bug) or L. hesperus (western tarnished plant
bug)); Euschistus spp. (e.g. E. conspersus (consperse stink bug));
Chlorochroa spp. (e.g. C. sayi (Say stinkbug)); Nezara spp. (e.g.
N. viridula (southern green stinkbug)); Thrips spp. (e.g. T. tabaci
(onion thrips)); Frankliniella spp. (e.g. F. fusca (tobacco
thrips), or F. occidentalis (western flower thrips)); Leptinotarsa
spp. (e.g. L. decemlineata (Colorado potato beetle), L. juncta
(false potato beetle), or L. texana (Texan false potato beetle));
Lema spp. (e.g. L. trilineata (three-lined potato beetle)); Epitrix
spp. (e.g. E. cucumeris (potato flea beetle), E. hirtipennis (flea
beetle), or E. tuberis (tuber flea beetle)); Epicauta spp. (e.g. E.
vittata (striped blister beetle)); Empoasca spp. (e.g. E. fabae
(potato leafhopper)); Myzus spp. (e.g. M. persicae (green peach
aphid)); Paratrioza spp. (e.g. P. cockerelli (psyllid)); Conoderus
spp. (e.g. C. falli (southern potato wireworm), or C. vespertinus
(tobacco wireworm)); Phthorimaea spp. (e.g. P. operculella (potato
tuberworm)); Macrosiphum spp. (e.g. M. euphorbiae (potato aphid));
Thyanta spp. (e.g. T. pallidovirens (redshouldered stinkbug));
Phthorimaea spp. (e.g. P. operculella (potato tuberworm));
Helicoverpa spp. (e.g. H. zea (tomato fruitworm); Keiferia spp.
(e.g. K. lycopersicella (tomato pinworm)); Limonius spp.
(wireworms); Manduca spp. (e.g. M. sexta (tobacco hornworm), or M.
quinquemaculata (tomato hornworm)); Liriomyza spp. (e.g. L.
sativae, L. trifolli or L. huidobrensis (leafminer)); Drosophilla
spp. (e.g. D. melanogaster, D. yakuba, D. pseudoobscura or D.
simulans); Carabus spp. (e.g. C. granulatus); Chironomus spp. (e.g.
C. tentanus); Ctenocephalides spp. (e.g. C. felis (cat flea));
Diaprepes spp. (e.g. D. abbreviatus (root weevil)); Ips spp. (e.g.
I. pini (pine engraver)); Tribolium spp. (e.g. T. castaneum (red
floor beetle)); Glossina spp. (e.g. G. morsitans (tsetse fly));
Anopheles spp. (e.g. A. gambiae (malaria mosquito)); Helicoverpa
spp. (e.g. H. armigera (African Bollworm)); Acyrthosiphon spp.
(e.g. A. pisum (pea aphid)); Apis spp. (e.g. A. melifera (honey
bee)); Homalodisca spp. (e.g. H. coagulate (glassy-winged
sharpshooter)); Aedes spp. (e.g. Ae. aegypti (yellow fever
mosquito)); Bombyx spp. (e.g. B. mori (silkworm), B. mandarina);
Locusta spp. (e.g. L. migratoria (migratory locust)); Boophilus
spp. (e.g. B. microplus (cattle tick)); Acanthoscurria spp. (e.g.
A. gomesiana (red-haired chololate bird eater)); Diploptera spp.
(e.g. D. punctata (pacific beetle cockroach)); Heliconius spp.
(e.g. H. erato (red passion flower butterfly) or H. melpomene
(postman butterfly)); Curculio spp. (e.g. C. glandium (acorn
weevil)); Plutella spp. (e.g. P. xylostella (diamontback moth));
Amblyomma spp. (e.g. A. variegatum (cattle tick)); Anteraea spp.
(e.g. A. yamamai (silkmoth)); Belgica spp. (e.g. B. antartica),
Bemisa spp. (e.g. B. tabaci), Bicyclus spp., Biphillus spp.,
Callosobruchus spp., Choristoneura spp., Cicindela spp., Culex
spp., Culicoides spp., Diaphorina spp., Diaprepes spp., Euclidia
spp., Glossina spp., Gryllus spp., Hydropsyche spp., Julodis spp.,
Lonomia spp., Lutzomyia spp., Lysiphebus spp, Meladema spp,
Mycetophagus spp., Nasonia spp., Oncometopia spp., Papilio spp.,
Pediculus spp., Plodia spp., Rhynchosciara spp., Sphaerius spp.,
Toxoptera spp., Trichoplusa spp., and Armigeres spp. (e.g. A.
subalbatus);
[0106] (2) an insect capable of infesting or injuring humans and/or
animals such as, but not limited to those with piercing-sucking
mouthparts, as found in Hemiptera and some Hymenoptera and Diptera
such as mosquitos, bees, wasps, lice, fleas and ants, as well as
members of the Arachnidae such as ticks and mitesorder, class or
familiy of Acarina (ticks and mites) e.g. representatives of the
families Argasidae, Dermanyssidae, Ixodidae, Psoroptidae or
Sarcoptidae and representatives of the species Amblyomma spp.,
Anocentor spp., Argas spp., Boophilus spp., Cheyletiella spp.,
Chorioptes spp., Demodex spp., Dermacentor spp., Dermanyssus spp.,
Haemophysalis spp., Hyalomma spp., Ixodes spp., Lynxacarus spp.,
Mesostigmata spp., Notoedres spp., Ornithodoros spp., Ornithonyssus
spp., Otobius spp., otodectes spp., Pneumonyssus spp., Psoroptes
spp., Rhipicephalus spp., Sarcoptes spp., or Trombicula spp.;
Anoplura (sucking and biting lice) e.g. representatives of the
species Bovicola spp., Haematopinus spp., Linognathus spp., Menopon
spp., Pediculus spp., Pemphigus spp., Phylloxera spp., or
Solenopotes spp.; Diptera (flies) e.g. representatives of the
species Aedes spp., Anopheles spp., Calliphora spp., Chrysomyia
spp., Chrysops spp., Cochliomyia spp., Culex spp., Culicoides spp.,
Cuterebra spp., Dermatobia spp., Gastrophilus spp., Glossina spp.,
Haematobia spp., Haematopota spp., Hippobosca spp., Hypoderma spp.,
Lucilia spp., Lyperosia spp., Melophagus spp., Oestrus spp.,
Phaenicia spp., Phlebotomus spp., Phormia spp., Sarcophaga spp.,
Simulium spp., Stomoxys spp., Tabanus spp., Tannia spp. or Tipula
spp.; Mallophaga (biting lice) e.g. representatives of the species
Damalina spp., Felicola spp., Heterodoxus spp. or Trichodectes
spp.; or Siphonaptera (wingless insects) e.g. representatives of
the species Ceratophyllus spp., spp., Pulex spp., or Xenopsylla
spp; Cimicidae (true bugs) e.g. representatives of the species
Cimex spp., Tritominae spp., Rhodinius spp., or Triatoma spp.
[0107] and
[0108] (3) an insect that causes unwanted damage to substrates or
materials, such as insects that attack foodstuffs, seeds, wood,
paint, plastic, clothing etc.
[0109] The methods of the invention are applicable to all nematodes
and that are susceptible to gene silencing by RNA interference and
that are capable of internalising double-stranded RNA from their
immediate environment.
[0110] In one embodiment of the invention, the nematode may belong
to the family of the Heteroderidae, encompassing the genera
Heterodera and Globodera.
[0111] In preferred, but non-limiting, embodiments and methods of
the invention the insect is chosen from the group coprising but not
limited to:
[0112] (1) a nematode which is a plant pathogenic nematode, such as
but not limited to: Meloidogyne spp. (e.g. M. incognita, M.
javanica, M. graminicola, M. arenaria, M. chitwoodi, M. hapla or M.
paranaensis); Heterodera spp. (e.g. H. oryzae, H. glycines, H. zeae
or H. schachtii); Globodera spp. (e.g. G. pallida or G.
rostochiensis); Rotylenchulus spp. (e.g. R. reniformis);
Pratylenchus spp. (e.g. P. coffeae, P. Zeae or P. goodeyi);
Radopholus spp. (e.g. R. similis); Hirschmaniella spp. (e.g. H.
oryzae); Ancylostoma spp. (e.g. A. caninum, A. ceylanicum, A.
duodenale or A. tubaeforme); Anisakid; Aphelenchoides spp. (e.g. A.
Besseyi); Ascarids; Ascaris spp., (e.g. A. suum or A.
lumbridoides); Belonolaimus spp.; Brugia spp. (e.g. B. malayi or B.
pahangi); Bursaphelenchus spp.; Caenorhabditis spp. (e.g. C.
elegans, C. briggsae or C. remanei); Clostridium spp. (e.g. C.
acetobutylicum); Cooperia spp. (e.g. C. oncophora); Criconemoides
spp.; Cyathostomum spp. (e.g. C. catinatum, C. coronatum or C.
pateratum); Cylicocyclus spp. (e.g. C. insigne, C. nassatus or C.
radiatus); Cylicostephanus spp. (e.g. C. goldi or C.
longibursatus); Diphyllobothrium; Dirofilaria spp. (e.g. D.
immitis); Ditylenchus spp. (e.g. D. dipsaci, D. destructor or D.
Angustus); Enterobius spp. (e.g. E. vermicularis); Haemonchus spp.
(e.g. H. contortus); Helicotylenchus spp.; Hoplolaimus spp.;
Litomosoides spp. (e.g. L. sigmodontis); Longidorus spp. (e.g. L.
macrosoma); Necator spp. (e.g. N. americanus); Nippostrongylus spp.
(e.g. N. brasiliensis); Onchocerca spp. (e.g. O. volvulus);
Ostertagia spp. (e.g. O. ostertagi); Parastrongyloides spp. (e.g.
P. trichosuri); Paratrichodorus spp. (e.g. P. minor or P. teres);
Parelaphostrongylus spp. (e.g. P. tenuis); Radophulus spp.;
Scutellonerna. spp.; Strongyloides spp. (e.g. S. Ratti or S.
stercoralis); Teladorsagia spp. (e.g. T. circumcincta); Toxascaris
spp. (e.g. T. leonina); Toxocara spp. (e.g. T. canis or T. cati);
Trichinella spp. (e.g. T. britovi, T. spiralis or T. spirae);
Trichodorus spp. (e.g. T. similis); Trichuris spp. (e.g. T. muris,
T. vulpis or T. trichiura); Tylenchulus spp.; Tylenchorhynchus
spp.; Uncinaria spp. (e.g. U. stenocephala); Wuchereria spp. (e.g.
W. bancrofti); Xiphinema spp. (e.g. X. Index or X. americanum).
[0113] (2) a nematode capable of infesting humans such as, but not
limited to: Enterobius vermicularis, the pinworm that causes
enterobiasis; Ascaris lumbridoides, the large intestinal roundworm
that causes ascariasis; Necator and Ancylostoma, two types of
hookworms that cause ancylostomiasis; Trichuris trichiura, the
whipworm that causes trichuriasis; Strongyloides stercoralis that
causes strongyloidiasis; and Trichonella spirae that causes
trichinosis; Brugia malayi and Wuchereria bancrofti, the filarial
nematodes associated with the worm infections known as lymphatic
filariasis and its gross manifestation, elephantiasis, and
Onchocerca volvulus that causes river blindness. Transfer of
nematodes to humans may also occur through blood-feeding mosquitoes
which have fed upon infected animals or humans;
[0114] (3) a nematode capable of infesting animals such as, but not
limited to: dogs (Hookworms e.g. Ancylostoma caninum or Uncinaria
stenocephala, Ascarids e.g. Toxocara canis or Toxascaris leonina,
or Whipworms e.g. Trichuris vulpis), cats (Hookworms e.g.
Ancylostoma tubaeforme, Ascarids e.g. Toxocara cati), fish (herring
worms or cod worms e.g. Anisakid, or tapeworm e.g.
Diphyllobothrium), sheep (Wire worms e.g. Haemonchus contortus) and
cattle (Gastro-intestinal worms e.g. Ostertagia ostertagi, Cooperia
oncophora);
[0115] (4) a nematode that causes unwanted damage to substrates or
materials, such as nematodes that attack foodstuffs, seeds, wood,
paint, plastic, clothing etc. Examples of such nematodes include
but are not limited to Meloidogyne spp. (e.g. M. incognita, M.
javanica, M. arenaria, M. graminicola, M. chitwoodi or M. hapla);
Heterodera spp. (e.g. H. oryzae, H. glycines, H. zeae or H.
schachtii); Globodera spp. (e.g. G. pallida or G. rostochiensis);
Ditylenchus spp. (e.g. D. dipsaci, D. destructor or D. angustus);
Belonolaimus spp.; Rotylenchulus spp. (e.g. R. reniformis);
Pratylenchus spp. (e.g. P. coffeae, P. goodeyi or P. zeae);
Radopholus spp. (e.g. R. Similis); Hirschmaniella spp. (e.g. H.
oryzae); Aphelenchoides spp. (e.g. A. besseyi); Criconemoides spp.;
Longidorus spp.; Helicotylenchus spp.; Hoplolaimus spp.; Xiphinema
spp.; Paratrichodorus spp. (e.g. P. minor); Tylenchorhynchus
spp;
[0116] (5) virus transmitting nematodes (e.g. Longidorus macrosoma:
transmits prunus necrotic ring spot virus, Xiphinema americanum:
transmits tobacco ring spot virus, Paratrichadorus teres: transmits
pea early browning virus, or Trichodorus similis: transmits tobacco
rattle virus).
[0117] Fungal pests of particular interest include but are not
limited to the following. In one embodiment of the invention, the
fungus may be a mold, or more particularly a filamentous fungus. In
other embodiments of the invention, the fungus may be a yeast.
[0118] In one embodiment the fungus may be an ascomycetes fungus,
i.e. a fungus belonging to the Phylum Ascomycota.
[0119] In preferred, but non-limiting, embodiments of the invention
the fungal cell is chosen from the group consisting of:
[0120] (1) a fungal cell of, or a cell derived from a plant
pathogenic fungus, such as but not limited to Acremoniella spp.,
Alternaria spp. (e.g. Alternaria brassicola or Alternaria solani),
Ascochyta spp. (e.g. Ascochyta pisi), Botrytis spp. (e.g. Botrytis
cinerea or Botryotinia fuckeliana), Cladosporium spp., Cercospora
spp. (e.g. Cercospora kikuchii or Cercospora zaea-maydis),
Cladosporium spp. (e.g. Cladosporium fulvum), Colletotrichum spp.
(e.g. Colletotrichum lindemuthianum), Curvularia spp., Diplodia
spp. (e.g. Diplodia maydis), Erysiphe spp. (e.g. Erysiphe graminis
f. sp. graminis, Erysiphe graminis f. sp. hordei or Erysiphe pisi),
Erwinia armylovora, Fusarium spp. (e.g. Fusarium nivale, Fusarium
sporotrichioides, Fusarium oxysporum, Fusarium graminearum,
Fusarium germinearum, Fusarium culmorum, Fusarium solani, Fusarium
moniliforme or Fusarium roseum), Gaeumanomyces spp. (e.g.
Gaeumanomyces graminis f. sp. tritici), Gibberella spp. (e.g.
Gibberella zeae), Helminthosporium spp. (e.g. Helminthosporium
turcicum, Helminthosporium carbonum, Helminthosporium mavdis or
Helminthosporium sigmoideum), Leptosphaeria salvinii, Macrophomina
spp. (e.g. Macrophomina phaseolina), Magnaportha spp. (e.g.
Magnaporthe oryzae), Mycosphaerella spp., Nectria spp. (e.g.
Nectria heamatococca), Peronospora spp. (e.g. Peronospora
manshurica or Peronospora tabacina), Phoma spp. (e.g. Phoma betae),
Phakopsora spp. (e.g. Phakopsora pachyrhizi), Phymatotrichum spp.
(e.g. Phymatotrichum omnivorum), Phytophthora spp. (e.g.
Phytophthora cinnamomi, Phytophthora cactorum, Phytophthora
phaseoli, Phytophthora parasitica, Phytophthora citrophthora,
Phytophthora megasperma f. sp. soiae or Phytophthora infestans),
Plasmopara spp. (e.g. Plasmopara viticola), Podosphaera spp. (e.g.
Podosphaera leucotricha), Puccinia spp. (e.g. Puccinia sorghi,
Puccinia striiformis, Puccinia graminis f. sp. tritici, Puccinia
asparagi, Puccinia recondite or Puccinia arachidis), Pythium spp.
(e.g. Pythium aphanidermatum), Pyrenophora spp. (e.g. Pyrenophora
tritici-repentens or Pyrenophora teres), Pyricularia spp. (e.g.
Pyricularia oryzae), Pythium spp. (e.g. Pythium ultimum),
Rhincosporium secalis, Rhizoctonia spp. (e.g. Rhizoctonia solani,
Rhizoctonia oryzae or Rhizoctonia cerealis), Rhizopus spp. (e.g.
Rhizopus chinensid), Scerotium spp. (e.g. Scerotium rolfsil),
Sclerotinia spp. (e.g. Sclerotinia sclerotiorum), Septoria spp.
(e.g. Septoria lycopersici, Septoria glycines, Septoria nodorum or
Septoria tritici), Thielaviopsis spp. (e.g. Thielaviopsis
basicola), Tilletia spp., Trichoderma spp. (e.g. Trichoderma
virde), Uncinula spp. (e.g. Uncinula necator), Ustilago maydis
(e.g. corn smut), Venturia spp. (e.g. Venturia inaequalis or
Venturia pirina) or Verticillium spp. (e.g. Verticillium dahliae or
Verticillium albo-atrum);
[0121] (2) a fungal cell of, or a cell derived from a fungus
capable of infesting humans such as, but not limited to, Candida
spp., particularly Candida albicans; Dermatophytes including
Epidermophyton spp., Trichophyton spp, and Microsporum spp.;
Aspergillus spp. (particularly Aspergillus flavus, Aspergillus
fumigatus, Aspergillus nidulans, Aspergillus niger or Aspergillus
terreus); Blastomyces dermatitidis; Paracoccidioides brasiliensis;
Coccidioides immitis; Cryptococcus neoformans; Histoplasma
capsulatum Var. capsulatum or Var. duboisii; Sporothrix schenckii;
Fusarium spp.; Scopulariopsis brevicaulis; Fonsecaea spp.;
Penicillium spp.; or Zygomycetes group of fungi (particularly
Absidia corymbifera, Rhizomucor pusillus or Rhizopus arrhizus);
[0122] (3) a fungal cell of, or a cell derived from a fungus
capable of infesting animals such as, but not limited to Candida
spp., Microsporum spp. (particularly Microsporum canis or
Microsporum gypseum), Trichophyton mentagrophytes, Aspergillus
spp., or Cryptococcus neoforman;
[0123] and
[0124] (4) a fungal cell of, or a cell derived from a fungus that
causes unwanted damage to substrates or materials, such as fungi
that attack foodstuffs, seeds, wood, paint, plastic, clothing etc.
Examples of such fungi are the moulds, including but not limited to
Stachybotrys spp., Aspergillus spp., Alternaria spp., Cladosporium
spp., Penicillium spp. or Phanerochaete chrysosporium.
[0125] II. Identification of Target Sequences
[0126] The present invention provides a method for identifying and
obtaining a nucleic acid comprising a nucleotide sequence for
producing a dsRNA or siRNA. For example, such a method comprises:
(a) probing a cDNA or genomic DNA library with a hybridization
probe comprising all or a portion of a nucleotide sequence or a
homolog thereof from a targeted pest; (b) identifying a DNA clone
that hybridizes with the hybridization probe; (c) isolating the DNA
clone identified in step (b); and (d) sequencing the cDNA or
genomic DNA fragment that comprises the clone isolated in step (c)
wherein the sequenced nucleic acid molecule transcribes all or a
substantial portion of the RNA nucleotide acid sequence or a
homolog thereof.
[0127] Additionally, the present invention contemplates a method
for obtaining a nucleic acid fragment comprising a nucleotide
sequence for producing a substantial portion of a dsRNA or siRNA
comprising: (a) synthesizing first and a second oligonucleotide
primers corresponding to a portion of one of the nucleotide
sequences from a targeted pest; and (b) amplifying a cDNA or
genomic DNA template in a cloning vector using the first and second
oligonucleotide primers of step (a) wherein the amplified nucleic
acid molecule transcribes a substantial portion of a dsRNA or siRNA
of the present invention.
[0128] In practicing the present invention, a target gene may be
derived from any pest that causes damage to another organism.
Several criteria may be employed in the selection of preferred
target genes. The gene is one whose protein product has a rapid
turnover rate, so that dsRNA inhibition will result in a rapid
decrease in protein levels. In certain embodiments it is
advantageous to select a gene for which a small drop in expression
level results in deleterious effects for the recipient pest. If it
is desired to target a broad range of insect species, for example,
a gene is selected that is highly conserved across these species.
Conversely, for the purpose of conferring specificity, in certain
embodiments of the invention, a gene is selected that contains
regions that are poorly conserved between individual insect
species, or between insects and other organisms. In certain
embodiments it may be desirable to select a gene that has no known
homologs in other organisms.
[0129] As used herein, the term "derived from" refers to a
specified nucleotide sequence that may be obtained from a
particular specified source or species, albeit not necessarily
directly from that specified source or species.
[0130] In one embodiment, a gene is selected that is expressed in
the insect gut. Targeting genes expressed in the gut avoids the
requirement for the dsRNA to spread within the insect. Target genes
for use in the present invention may include, for example, those
that share substantial homologies to the nucleotide sequences of
known gut-expressed genes that encode protein components of the
plasma membrane proton V-ATPase (Dow et al., 1997; Dow, 1999). This
protein complex is the sole energizer of epithelial ion transport
and is responsible for alkalinization of the midgut lumen. The
V-ATPase is also expressed in the Malpighian tubule, an outgrowth
of the insect hindgut that functions in fluid balance and
detoxification of foreign compounds in a manner analogous to a
kidney organ of a mammal.
[0131] In another embodiment, a gene is selected that is
essentially involved in the growth, development, and reproduction
of an insect. Exemplary genes include but are not limited to the
structural subunits of ribosomal proteins and a beta-coatamer gene,
CHD3 gene. Ribosomal proteins such as S4 (RpS4) and S9(RpS9) are
structural constituents of the ribosome involved in protein
biosynthesis and which are components of the cytosolic small
ribosomal subunit, the ribosomal proteins such as L9 and L19 are
structural constituent of ribosome involved in protein biosynthesis
which is localised to the ribosome. The beta-coatamer gene in C.
elegans encodes a protein which is a subunit of a multimeric
complex that forms a membrane vesicle coat Similar sequences have
been found in diverse organisms such as Arabidopsis thaliana,
Drosophila melanogaster, and Saccharomyces cerevisiae. Related
sequences are found in diverse organisms such as Leptinotarsa
decemlineata, Phaedon cochleariae, Epilachna varivetis, Anthonomus
grandis, Tribolium castaneum, Myzus persicae, Nilaparvata lugens,
Chilo suppressalis, Plutella xylostella and Acheta domesticus.
Other target genes for use in the present invention may include,
for example, those that play important roles in viability, growth,
development, reproduction, and infectivity. These target genes
include, for example, house keeping genes, transcription factors,
and insect specific genes or lethal knockout mutations in
Caenorhabditis or Drosophila. The target genes for use in the
present invention may also be those that are from other organisms,
e.g., from a nematode (e.g., Meloidogyne spp. or Heterodera spp.),
other insects or arachnidae (e.g. Leptinotarsa spp., Phaedon spp.,
Epilachna spp., Anthonomus spp., Tribolium spp., Myzus spp.,
Nilaparvata spp., Chilo spp., Plutella spp., or Acheta spp.
Additionally, the nucleotide sequences for use as a target sequence
in the present invention may also be derived from viral, bacterial,
fungal, insect or fungal genes whose functions have been
established from literature and the nucleotide sequences of which
share substantial similarity with the target genes in the genome of
an insect.
[0132] For many of the insects that are potential targets for
control by the present invention, there may be limited information
regarding the sequences of most genes or the phenotype resulting
from mutation of particular genes. Therefore, genes may be selected
based on available information available concerning corresponding
genes in a model organism, such as Caenorhabditis or Drosophila, or
in some other insect species. Genes may also be selected based on
available sequence information for other species, such as nematode
or fungal species, in which the genes have been characterized. In
some cases it will be possible to obtain the sequence of a
corresponding gene from a target insect by searching databases,
such as GenBank, using either the name of the gene or the gene
sequence. Once the sequence is obtained, PCR may be used to amplify
an appropriately selected segment of the gene in the insect for use
in the present invention.
[0133] In order to obtain a DNA segment from the corresponding gene
in an insect species, for example, PCR primers may be designed
based on the sequence as found in C. elegans or Drosophila, or an
insect from which the gene has already been cloned. The primers are
designed to amplify a DNA segment of sufficient length for use in
the present invention. Amplification conditions are selected so
that amplification will occur even if the primers do not exactly
match the target sequence. Alternately, the gene, or a portion
thereof, may be cloned from a genomic DNA or cDNA library prepared
from the insect pest species, using a known insect gene as a probe.
Techniques for performing PCR and cloning from libraries are known.
Further details of the process by which DNA segments from target
insect pest species may be isolated based on the sequence of genes
previously cloned from an insect species are provided in the
Examples. One of ordinary skill in the art will recognize that a
variety of techniques may be used to isolate gene segments from
insect pest species that correspond to genes previously isolated
from other species.
[0134] III. Methods for Inhibiting or Suppressing a Target Gene
[0135] The present invention provides methods for inhibiting gene
expression of one or multiple target genes in a target pest using
stabilized dsRNA methods. The invention is particularly useful for
modulating eukaryotic gene expression, in particular modulating the
expression of genes present in pests that exhibit a digestive
system pH level that is from about 4.5 to about 9.5, more
preferably from about 5.0 to about 8.0, and even more preferably
from about 6.5 to about 7.5. For pests with a digestive system that
exhibits pH levels outside of these ranges, delivery methods may be
desired for use that do not require ingestion of dsRNA
molecules.
[0136] The methods of the invention encompass the simultaneous or
sequential provision of two or more different double-stranded RNAs
or RNA constructs to the same insect, so as to achieve
down-regulation or inhibition of multiple target genes or to
achieve a more potent inhibition of a single target gene.
[0137] Alternatively, multiple targets are hit by the provision of
one double-stranded RNA that hits multiple target sequences, and a
single target is more efficiently inhibited by the presence of more
than one copy of the double stranded RNA fragment corresponding to
the target gene. Thus, in one embodiment of the invention, the
double-stranded RNA construct comprises multiple dsRNA regions, at
least one strand of each dsRNA region comprising a nucleotide
sequence that is complementary to at least part of a target
nucleotide sequence of an insect target gene. According to the
invention, the dsRNA regions in the RNA construct may be
complementary to the same or to different target genes and/or the
dsRNA regions may be complementary to targets from the same or from
different insect species. Use of such dsRNA constructs in a plant
host cell, thus establishes a more potent resistance to a single or
to multiple insect species in the plant. In one embodiment, the
double stranded RNA region comprises multiple copies of the
nucleotide sequence that is complementary to the target gene.
Alternatively, the dsRNA hits more than one target sequence of the
same target gene. The invention thus encompasses isolated double
stranded RNA constructs comprising at least two copies of said
nucleotide sequence complementary to at least part of a nucleotide
sequence of an insect target. DsRNA that hits more than one of the
above-mentioned targets, or a combination of different dsRNA
against different of the above mentioned targets are developed and
used in the methods of the present invention. Suitable dsRNA
nucleotides and dsRNA constructs are described in WO2006/046148 by
applicant, which is incorporated herein in its entirity.
[0138] The terms "hit", "hits", and "hitting" are alternative
wordings to indicate that at least one of the strands of the dsRNA
is complementary to, and as such may bind to, the target gene or
nucleotide sequence.
[0139] The modulatory effect of dsRNA is applicable to a variety of
genes expressed in the pests including, for example, endogenous
genes responsible for cellular metabolism or cellular
transformation, including house keeping genes, transcription
factors, and other genes which encode polypeptides involved in
cellular metabolism.
[0140] As used herein, the phrase "inhibition of gene expression"
or "inhibiting expression of a target gene in the cell of an pest"
refers to the absence (or observable decrease) in the level of
protein and/or mRNA product from the target gene. Specificity
refers to the ability to inhibit the target gene without manifest
effects on other genes of the cell and without any effects on any
gene within the cell that is producing the dsRNA molecule. The
inhibition of gene expression of the target gene in the pest may
result in novel phenotypic traits in the pest.
[0141] "Gene suppression" refers to any of the well-known methods
for reducing the levels of gene transcription to mRNA and/or
subsequent translation of the mRNA. Gene suppression is also
intended to mean the reduction of protein expression from a gene or
a coding sequence including posttranscriptional gene suppression
and transcriptional suppression. Posttranscriptional gene
suppression is mediated by the homology between of all or a part of
a mRNA transcribed from a gene or coding sequence targeted for
suppression and the corresponding double stranded RNA used for
suppression, and refers to the substantial and measurable reduction
of the amount of available mRNA available in the cell for binding
by ribosomes. The transcribed RNA can be in the sense orientation
to effect what is called co-suppression, in the anti-sense
orientation to effect what is called anti-sense suppression, or in
both orientations producing a dsRNA to effect what is called RNA
interference (RNAi).
[0142] Transcriptional suppression is mediated by the presence in
the cell of a dsRNA gene suppression agent exhibiting substantial
sequence identity to a promoter DNA sequence or the complement
thereof to effect what is referred to as promoter trans
suppression. Gene suppression may be effective against a native
host gene associated with a trait, e.g., to provide hosts with
reduced levels of a protein encoded by the native gene or with
enhanced or reduced levels of an affected metabolite. Gene
suppression can also be effective against target genes in pests
that may ingest or contact material containing gene suppression
agents, specifically designed to inhibit or suppress the expression
of one or more homologous or complementary sequences in the cells
of the pest.
[0143] A beneficial method of post transcriptional gene suppression
in hosts employs both sense-oriented and anti-sense-oriented,
transcribed RNA which is stabilized, e.g., as a hairpin and stem
and loop structure. A preferred DNA construct for effecting post
transcriptional gene suppression is one in which a first segment
encodes an RNA exhibiting an anti-sense orientation exhibiting
substantial identity to a segment of a gene targeted for
suppression, which is linked to a second segment in sense
orientation encoding an RNA exhibiting substantial complementarity
to the first segment. Such a construct forms a stem and loop
structure by hybridization of the first segment with the second
segment and a loop structure from the nucleotide sequences linking
the two segments (see WO94/01550, WO98/05770, US 2002/0048814, and
US 2003/0018993).
[0144] According to one embodiment of the present invention, there
is provided a nucleotide sequence, for which in vitro expression
results in transcription of a stabilized RNA sequence that is
substantially homologous to an RNA molecule of a targeted gene in a
pest that comprises an RNA sequence encoded by a nucleotide
sequence within the genome of the pest. Thus, after the pest
uptakes the stabilized RNA sequence, or is otherwise exposed to the
dsRNA, a down-regulation of the nucleotide sequence corresponding
to the target gene in the cells of a target pest is affected.
[0145] Inhibition of a target gene using the stabilized dsRNA
technology of the present invention is sequence-specific in that
nucleotide sequences corresponding to the duplex region of the RNA
are targeted for genetic inhibition. RNA containing a nucleotide
sequences identical to a portion of the target gene is preferred
for inhibition. RNA sequences with insertions, deletions, and
single point mutations relative to the target sequence have also
been found to be effective for inhibition. In performance of the
present invention, it is preferred that the inhibitory dsRNA and
the portion of the target gene share at least from about 80%
sequence identity, or from about 85% sequence identity, or from
about 90% sequence identity, or from about 95% sequence identity,
or from about 99% sequence identity, or even about 100% sequence
identity. Alternatively, the duplex region of the RNA may be
defined functionally as a nucleotide sequence that is capable of
hybridizing with a portion of the target gene transcript. A less
than full length sequence exhibiting a greater homology compensates
for a longer less homologous sequence. The length of the identical
nucleotide sequences may be at least about 25, 50, 100, 200, 300,
400, 500 or at least about 1000 bases. Normally, a sequence of
greater than 20-100 nucleotides should be used, though a sequence
of greater than about 200-300 nucleotides would be preferred, and a
sequence of greater than about 500-1000 nucleotides would be
especially preferred depending on the size of the target gene. The
invention has the advantage of being able to tolerate sequence
variations that might be expected due to genetic mutation, strain
polymorphism, or evolutionary divergence. The introduced nucleic
acid molecule may not need to be absolute homology, may not need to
be full length, relative to either the primary transcription
product or fully processed mRNA of the target gene. Therefore,
those skilled in the art need to realize that, as disclosed herein,
100% sequence identity between the RNA and the target gene is not
required to practice the present invention.
[0146] IV. Methods for Preparing dsRNA
[0147] dsRNA molecules may be synthesized either in vivo or in
vitro. The dsRNA may be formed by a single self-complementary RNA
strand or from two complementary RNA strands. Endogenous RNA
polymerase of the cell may mediate transcription in vivo, or cloned
RNA polymerase can be used for transcription in vivo or in vitro
Inhibition may be targeted by specific transcription in an organ,
tissue, or cell type; stimulation of an environmental condition
(e.g., infection, stress, temperature, chemical inducers); and/or
engineering transcription at a developmental stage or age. The RNA
strands may or may not be polyadenylated; the RNA strands may or
may not be capable of being translated into a polypeptide by a
cell's translational apparatus.
[0148] A RNA, dsRNA, siRNA, or miRNA of the present invention may
be produced chemically or enzymatically by one skilled in the art
through manual or automated reactions or in vivo in another
organism. RNA may also be produced by partial or total organic
synthesis; any modified ribonucleotide can be introduced by in
vitro enzymatic or organic synthesis. The RNA may be synthesized by
a cellular RNA polymerase or a bacteriophage RNA polymerase (e.g.,
T3, T7, SP6). The use and production of an expression construct are
known in the art (see, for example, WO 97/32016; U.S. Pat. Nos.
5,593,874, 5,698,425, 5,712,135, 5,789,214, and 5,804,693). If
synthesized chemically or by in vitro enzymatic synthesis, the RNA
may be purified prior to introduction into the cell. For example,
RNA can be purified from a mixture by extraction with a solvent or
resin, precipitation, electrophoresis, chromatography, or a
combination thereof. Alternatively, the RNA may be used with no or
a minimum of purification to avoid losses due to sample processing.
The RNA may be dried for storage or dissolved in an aqueous
solution. The solution may contain buffers or salts to promote
annealing, and/or stabilization of the duplex strands.
[0149] V. Polynucleotide Sequences
[0150] Provided according to the invention are nucleotide
sequences, the expression of which results in an RNA sequence which
is substantially homologous to an RNA molecule of a targeted gene
in a pest that comprises an RNA sequence encoded by a nucleotide
sequence within the genome of the pest. Thus, after ingestion of
the dsRNA sequence down-regulation of the nucleotide sequence of
the target gene in the cells of the pest may be obtained resulting
in a deleterious effect on the maintenance, viability,
proliferation, reproduction, and infestation of the pest.
[0151] Each "nucleotide sequence" set forth herein is presented as
a sequence of deoxyribonucleotides (abbreviated A, G, C and T).
However, by "nucleotide sequence" of a nucleic acid molecule or
polynucleotide is intended, for a DNA molecule or polynucleotide, a
sequence of deoxyribonucleotides, and for an RNA molecule or
polynucleotide, the corresponding sequence of ribonucleotides (A,
G, C and U) where each thymidine deoxynucleotide (T) in the
specified deoxynucleotide sequence in is replaced by the
ribonucleotide uridine (U).
[0152] As used herein, "nucleic acid" refers to a single or
double-stranded polymer of deoxyribonucleotide or ribonucleotide
bases read from the 5' to the 3' end. A nucleic acid may also
optionally contain non-naturally occurring or altered nucleotide
bases that permit correct read through by a polymerase and do not
reduce expression of a polypeptide encoded by that nucleic acid.
"Nucleotide sequence" or "nucleic acid sequence" refers to both the
sense and antisense strands of a nucleic acid as either individual
single strands or in the duplex.
[0153] The term "ribonucleic acid" (RNA) is inclusive of RNAi
(inhibitory RNA), dsRNA (double stranded RNA), siRNA (small
interfering RNA), mRNA (messenger RNA), miRNA (micro-RNA), tRNA
(transfer RNA, whether charged or discharged with a corresponding
acylated amino acid), and cRNA (complementary RNA) and the term
"deoxyribonucleic acid" (DNA) is inclusive of cDNA and genomic DNA
and DNA-RNA hybrids.
[0154] The words "nucleic acid segment", "nucleotide sequence
segment", or more generally "segment" will be understood by those
in the art as a functional term that includes both genomic
sequences, ribosomal RNA sequences, transfer RNA sequences,
messenger RNA sequences, operon sequences and smaller engineered
nucleotide sequences that express or may be adapted to express,
proteins, polypeptides or peptides.
[0155] Accordingly, the present invention relates to an isolated
nucleic molecule comprising a polynucleotide having a sequence
selected from the group consisting of any of the polynucleotide
sequences of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,
49-158, 159, 160, 163, 168, 173, 178, 183, 188, 193, 198, 203, 208,
215, 220, 225, 230, 247, 249, 251, 253, 255, 257, 259, 275-472,
473, 478, 483, 488, 493, 498, 503, 513, 515, 517, 519, 521,
533-575, 576, 581, 586, 591, 596, 601, 603, 605, 607, 609, 621-767,
768, 773, 778, 783, 788, 793, 795, 797, 799, 801, 813-862, 863,
868, 873, 878, 883, 888, 890, 892, 894, 896, 908-1040, 1041, 1046,
1051, 1056, 1061, 1071, 1073, 1075, 1077, 1079, 1081, 1083, 1085,
1087, 1089, 1091, 1093, 1095, 1097, 1099, 1101, 1103, 1105, 1107,
1109, 1111, 1113, 1161-1571, 1572, 1577, 1582, 1587, 1592, 1597,
1602, 1607, 1612, 1617, 1622, 1627, 1632, 1637, 1642, 1647, 1652,
1657, 1662, 1667, 1672, 1677, 1682, 1684, 1686, 1688, 1690, 1692,
1694, 1696, 1698, 1700, 1702, 1704, 1730-2039, 2040, 2045, 2050,
2055, 2060, 2065, 2070, 2075, 2080, 2085, 2090, 2095, 2100, 2102,
2104, 2106, 2108, 2120-2338, 2339, 2344, 2349, 2354, 2359, 2364,
2366, 2368, 2370, 2372, 2384-2460, 2461, 2466, 2471, 2476 and 2481.
The invention also provides functional fragments of the
polynucleotide sequences of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15,
17, 19, 21, 23, 49-158, 159, 160, 163, 168, 173, 178, 183, 188,
193, 198, 203, 208, 215, 220, 225, 230, 247, 249, 251, 253, 255,
257, 259, 275-472, 473, 478, 483, 488, 493, 498, 503, 513, 515,
517, 519, 521, 533-575, 576, 581, 586, 591, 596, 601, 603, 605,
607, 609, 621-767, 768, 773, 778, 783, 788, 793, 795, 797, 799,
801, 813-862, 863, 868, 873, 878, 883, 888, 890, 892, 894, 896,
908-1040, 1041, 1046, 1051, 1056, 1061, 1071, 1073, 1075, 1077,
1079, 1081, 1083, 1085, 1087, 1089, 1091, 1093, 1095, 1097, 1099,
1101, 1103, 1105, 1107, 1109, 1111, 1113, 1161-1571, 1572, 1577,
1582, 1587, 1592, 1597, 1602, 1607, 1612, 1617, 1622, 1627, 1632,
1637, 1642, 1647, 1652, 1657, 1662, 1667, 1672, 1677, 1682, 1684,
1686, 1688, 1690, 1692, 1694, 1696, 1698, 1700, 1702, 1704,
1730-2039, 2040, 2045, 2050, 2055, 2060, 2065, 2070, 2075, 2080,
2085, 2090, 2095, 2100, 2102, 2104, 2106, 2108, 2120-2338, 2339,
2344, 2349, 2354, 2359, 2364, 2366, 2368, 2370, 2372, 2384-2460,
2461, 2466, 2471, 2476 and 2481. The invention further provides
complementary nucleic acids, or fragments thereof, to any of the
polynucleotide sequences of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15,
17, 19, 21, 23, 49-158, 159, 160, 163, 168, 173, 178, 183, 188,
193, 198, 203, 208, 215, 220, 225, 230, 247, 249, 251, 253, 255,
257, 259, 275-472, 473, 478, 483, 488, 493, 498, 503, 513, 515,
517, 519, 521, 533-575, 576, 581, 586, 591, 596, 601, 603, 605,
607, 609, 621-767, 768, 773, 778, 783, 788, 793, 795, 797, 799,
801, 813-862, 863, 868, 873, 878, 883, 888, 890, 892, 894, 896,
908-1040, 1041, 1046, 1051, 1056, 1061, 1071, 1073, 1075, 1077,
1079, 1081, 1083, 1085, 1087, 1089, 1091, 1093, 1095, 1097, 1099,
1101, 1103, 1105, 1107, 1109, 1111, 1113, 1161-1571, 1572, 1577,
1582, 1587, 1592, 1597, 1602, 1607, 1612, 1617, 1622, 1627, 1632,
1637, 1642, 1647, 1652, 1657, 1662, 1667, 1672, 1677, 1682, 1684,
1686, 1688, 1690, 1692, 1694, 1696, 1698, 1700, 1702, 1704,
1730-2039, 2040, 2045, 2050, 2055, 2060, 2065, 2070, 2075, 2080,
2085, 2090, 2095, 2100, 2102, 2104, 2106, 2108, 2120-2338, 2339,
2344, 2349, 2354, 2359, 2364, 2366, 2368, 2370, 2372, 2384-2460,
2461, 2466, 2471, 2476 and 2481, as well as a nucleic acid,
comprising at least 15 contiguous bases, which hybridizes to any of
the polynucleotide sequences of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13,
15, 17, 19, 21, 23, 49-158, 159, 160, 163, 168, 173, 178, 183, 188,
193, 198, 203, 208, 215, 220, 225, 230, 247, 249, 251, 253, 255,
257, 259, 275-472, 473, 478, 483, 488, 493, 498, 503, 513, 515,
517, 519, 521, 533-575, 576, 581, 586, 591, 596, 601, 603, 605,
607, 609, 621-767, 768, 773, 778, 783, 788, 793, 795, 797, 799,
801, 813-862, 863, 868, 873, 878, 883, 888, 890, 892, 894, 896,
908-1040, 1041, 1046, 1051, 1056, 1061, 1071, 1073, 1075, 1077,
1079, 1081, 1083, 1085, 1087, 1089, 1091, 1093, 1095, 1097, 1099,
1101, 1103, 1105, 1107, 1109, 1111, 1113, 1161-1571, 1572, 1577,
1582, 1587, 1592, 1597, 1602, 1607, 1612, 1617, 1622, 1627, 1632,
1637, 1642, 1647, 1652, 1657, 1662, 1667, 1672, 1677, 1682, 1684,
1686, 1688, 1690, 1692, 1694, 1696, 1698, 1700, 1702, 1704,
1730-2039, 2040, 2045, 2050, 2055, 2060, 2065, 2070, 2075, 2080,
2085, 2090, 2095, 2100, 2102, 2104, 2106, 2108, 2120-2338, 2339,
2344, 2349, 2354, 2359, 2364, 2366, 2368, 2370, 2372, 2384-2460,
2461, 2466, 2471, 2476 and 2481.
[0156] The present invention also provides orthologous sequences,
and complements and fragments thereof, of the polynucleotide
sequences of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,
49-158, 159, 160, 163, 168, 173, 178, 183, 188, 193, 198, 203, 208,
215, 220, 225, 230, 247, 249, 251, 253, 255, 257, 259, 275-472,
473, 478, 483, 488, 493, 498, 503, 513, 515, 517, 519, 521,
533-575, 576, 581, 586, 591, 596, 601, 603, 605, 607, 609, 621-767,
768, 773, 778, 783, 788, 793, 795, 797, 799, 801, 813-862, 863,
868, 873, 878, 883, 888, 890, 892, 894, 896, 908-1040, 1041, 1046,
1051, 1056, 1061, 1071, 1073, 1075, 1077, 1079, 1081, 1083, 1085,
1087, 1089, 1091, 1093, 1095, 1097, 1099, 1101, 1103, 1105, 1107,
1109, 1111, 1113, 1161-1571, 1572, 1577, 1582, 1587, 1592, 1597,
1602, 1607, 1612, 1617, 1622, 1627, 1632, 1637, 1642, 1647, 1652,
1657, 1662, 1667, 1672, 1677, 1682, 1684, 1686, 1688, 1690, 1692,
1694, 1696, 1698, 1700, 1702, 1704, 1730-2039, 2040, 2045, 2050,
2055, 2060, 2065, 2070, 2075, 2080, 2085, 2090, 2095, 2100, 2102,
2104, 2106, 2108, 2120-2338, 2339, 2344, 2349, 2354, 2359, 2364,
2366, 2368, 2370, 2372, 2384-2460, 2461, 2466, 2471, 2476 and 2481
of the invention. Accordingly, the invention encompasses target
genes which are insect orthologs of a gene comprising a nucleotide
sequence as represented in any of SEQ ID NOs: 1, 3, 5, 7, 9, 11,
13, 15, 17, 19, 21, 23, 49-158, 159, 160, 163, 168, 173, 178, 183,
188, 193, 198, 203, 208, 215, 220, 225, 230, 247, 249, 251, 253,
255, 257, 259, 275-472, 473, 478, 483, 488, 493, 498, 503, 513,
515, 517, 519, 521, 533-575, 576, 581, 586, 591, 596, 601, 603,
605, 607, 609, 621-767, 768, 773, 778, 783, 788, 793, 795, 797,
799, 801, 813-862, 863, 868, 873, 878, 883, 888, 890, 892, 894,
896, 908-1040, 1041, 1046, 1051, 1056, 1061, 1071, 1073, 1075,
1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, 1093, 1095, 1097,
1099, 1101, 1103, 1105, 1107, 1109, 1111, 1113, 1161-1571, 1572,
1577, 1582, 1587, 1592, 1597, 1602, 1607, 1612, 1617, 1622, 1627,
1632, 1637, 1642, 1647, 1652, 1657, 1662, 1667, 1672, 1677, 1682,
1684, 1686, 1688, 1690, 1692, 1694, 1696, 1698, 1700, 1702, 1704,
1730-2039, 2040, 2045, 2050, 2055, 2060, 2065, 2070, 2075, 2080,
2085, 2090, 2095, 2100, 2102, 2104, 2106, 2108, 2120-2338, 2339,
2344, 2349, 2354, 2359, 2364, 2366, 2368, 2370, 2372, 2384-2460,
2461, 2466, 2471, 2476 and 2481. By way of example, insect
orthologues may comprise a nucleotide sequence as represented in
any of SEQ ID NOs: 49-123, 275-434, 533-562, 621-738, 813-852,
908-1010, 1161-1437, 1730-1987, 2120-2290, 2384-2438, or a fragment
thereof of at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26
or 27 nucleotides. A non-limiting list of insect or arachnida
orthologs genes or sequences comprising at least a fragment of 15,
preferably at least 17 bp of one of the sequences of the invention
is given in Tables 4.
[0157] The invention also encompasses target genes which are
nematode orthologs of a gene comprising a nucleotide sequence as
represented in any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17,
19, 21, 23, 49-158, 159, 160, 163, 168, 173, 178, 183, 188, 193,
198, 203, 208, 215, 220, 225, 230, 247, 249, 251, 253, 255, 257,
259, 275-472, 473, 478, 483, 488, 493, 498, 503, 513, 515, 517,
519, 521, 533-575, 576, 581, 586, 591, 596, 601, 603, 605, 607,
609, 621-767, 768, 773, 778, 783, 788, 793, 795, 797, 799, 801,
813-862, 863, 868, 873, 878, 883, 888, 890, 892, 894, 896,
908-1040, 1041, 1046, 1051, 1056, 1061, 1071, 1073, 1075, 1077,
1079, 1081, 1083, 1085, 1087, 1089, 1091, 1093, 1095, 1097, 1099,
1101, 1103, 1105, 1107, 1109, 1111, 1113, 1161-1571, 1572, 1577,
1582, 1587, 1592, 1597, 1602, 1607, 1612, 1617, 1622, 1627, 1632,
1637, 1642, 1647, 1652, 1657, 1662, 1667, 1672, 1677, 1682, 1684,
1686, 1688, 1690, 1692, 1694, 1696, 1698, 1700, 1702, 1704,
1730-2039, 2040, 2045, 2050, 2055, 2060, 2065, 2070, 2075, 2080,
2085, 2090, 2095, 2100, 2102, 2104, 2106, 2108, 2120-2338, 2339,
2344, 2349, 2354, 2359, 2364, 2366, 2368, 2370, 2372, 2384-2460,
2461, 2466, 2471, 2476, and 2481 of the invention. By way of
example, nematode orthologs may comprise a nucleotide sequence as
represented in any of SEQ ID NOs: 124-135, 435-446, 563, 564,
739-751, 853, 854, 1011-1025, 1438-1473, 1988-2001, 2291-2298,
2439-2440 of the invention, or a fragment of at least 15, 16, 17,
18, 19, 20 or 21 nucleotides thereof. According to another aspect,
the invention thus encompasses any of the methods described herein
for controlling nematode growth in an organism, or for preventing
nematode infestation of an organism susceptible to nemataode
infection, comprising contacting nematode cells with a
double-stranded RNA, wherein the double-stranded RNA comprises
annealed complementary strands, one of which has a nucleotide
sequence which is complementary to at least part of the nucleotide
sequence of a target gene comprising a fragment of at least 17, 18,
19, 20 or 21 nucleotides of any of the sequences as represented in
SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 49-158, 159,
160, 163, 168, 173, 178, 183, 188, 193, 198, 203, 208, 215, 220,
225, 230, 247, 249, 251, 253, 255, 257, 259, 275-472, 473, 478,
483, 488, 493, 498, 503, 513, 515, 517, 519, 521, 533-575, 576,
581, 586, 591, 596, 601, 603, 605, 607, 609, 621-767, 768, 773,
778, 783, 788, 793, 795, 797, 799, 801, 813-862, 863, 868, 873,
878, 883, 888, 890, 892, 894, 896, 908-1040, 1041, 1046, 1051,
1056, 1061, 1071, 1073, 1075, 1077, 1079, 1081, 1083, 1085, 1087,
1089, 1091, 1093, 1095, 1097, 1099, 1101, 1103, 1105, 1107, 1109,
1111, 1113, 1161-1571, 1572, 1577, 1582, 1587, 1592, 1597, 1602,
1607, 1612, 1617, 1622, 1627, 1632, 1637, 1642, 1647, 1652, 1657,
1662, 1667, 1672, 1677, 1682, 1684, 1686, 1688, 1690, 1692, 1694,
1696, 1698, 1700, 1702, 1704, 1730-2039, 2040, 2045, 2050, 2055,
2060, 2065, 2070, 2075, 2080, 2085, 2090, 2095, 2100, 2102, 2104,
2106, 2108, 2120-2338, 2339, 2344, 2349, 2354, 2359, 2364, 2366,
2368, 2370, 2372, 2384-2460, 2461, 2466, 2471, 2476 and 2481,
whereby the double-stranded RNA is taken up by the fungus and
thereby controls growth or prevents infestation. The invention also
relates to nematode-resistant transgenic plants comprising a
fragment of at least 17, 18, 19, 20 or 21 nucleotides of any of the
sequences as represented in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15,
17, 19, 21, 23, 49-158, 159, 160, 163, 168, 173, 178, 183, 188,
193, 198, 203, 208, 215, 220, 225, 230, 247, 249, 251, 253, 255,
257, 259, 275-472, 473, 478, 483, 488, 493, 498, 503, 513, 515,
517, 519, 521, 533-575, 576, 581, 586, 591, 596, 601, 603, 605,
607, 609, 621-767, 768, 773, 778, 783, 788, 793, 795, 797, 799,
801, 813-862, 863, 868, 873, 878, 883, 888, 890, 892, 894, 896,
908-1040, 1041, 1046, 1051, 1056, 1061, 1071, 1073, 1075, 1077,
1079, 1081, 1083, 1085, 1087, 1089, 1091, 1093, 1095, 1097, 1099,
1101, 1103, 1105, 1107, 1109, 1111, 1113, 1161-1571, 1572, 1577,
1582, 1587, 1592, 1597, 1602, 1607, 1612, 1617, 1622, 1627, 1632,
1637, 1642, 1647, 1652, 1657, 1662, 1667, 1672, 1677, 1682, 1684,
1686, 1688, 1690, 1692, 1694, 1696, 1698, 1700, 1702, 1704,
1730-2039, 2040, 2045, 2050, 2055, 2060, 2065, 2070, 2075, 2080,
2085, 2090, 2095, 2100, 2102, 2104, 2106, 2108, 2120-2338, 2339,
2344, 2349, 2354, 2359, 2364, 2366, 2368, 2370, 2372, 2384-2460,
2461, 2466, 2471, 2476 and 2481. A non-limiting list of nematode
orthologs genes or sequences comprising at least a fragment of 15,
preferably at least 17 bp of one of the sequences of the invention
is given in Tables 5.
[0158] According to another embodiment, the invention encompasses
target genes which are fungal orthologs of a gene comprising a
nucleotide sequence as represented in any of SEQ ID Nos: 1, 3, 5,
7, 9, 11, 13, 15, 17, 19, 21, 23, 49-158, 159, 160, 163, 168, 173,
178, 183, 188, 193, 198, 203, 208, 215, 220, 225, 230, 247, 249,
251, 253, 255, 257, 259, 275-472, 473, 478, 483, 488, 493, 498,
503, 513, 515, 517, 519, 521, 533-575, 576, 581, 586, 591, 596,
601, 603, 605, 607, 609, 621-767, 768, 773, 778, 783, 788, 793,
795, 797, 799, 801, 813-862, 863, 868, 873, 878, 883, 888, 890,
892, 894, 896, 908-1040, 1041, 1046, 1051, 1056, 1061, 1071, 1073,
1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, 1093, 1095,
1097, 1099, 1101, 1103, 1105, 1107, 1109, 1111, 1113, 1161-1571,
1572, 1577, 1582, 1587, 1592, 1597, 1602, 1607, 1612, 1617, 1622,
1627, 1632, 1637, 1642, 1647, 1652, 1657, 1662, 1667, 1672, 1677,
1682, 1684, 1686, 1688, 1690, 1692, 1694, 1696, 1698, 1700, 1702,
1704, 1730-2039, 2040, 2045, 2050, 2055, 2060, 2065, 2070, 2075,
2080, 2085, 2090, 2095, 2100, 2102, 2104, 2106, 2108, 2120-2338,
2339, 2344, 2349, 2354, 2359, 2364, 2366, 2368, 2370, 2372,
2384-2460, 2461, 2466, 2471, 2476 and 2481 of the invention. By way
of example, fungal orthologs may comprise a nucleotide sequence as
represented in any of SEQ ID NOs:136-158, 447-472, 565-575,
752-767, 855-862, 1026-1040, 1474-1571, 2002-2039, 2299-2338,
2441-2460, or a fragment of at least 17, 18, 19, 20, 21, 22, 23,
24, 25, 26 or 27 nucleotides thereof. According to another aspect,
the invention thus encompasses any of the methods described herein
for controlling fungal growth on a cell or an organism, or for
preventing fungal infestation of a cell or an organism susceptible
to fungal infection, comprising contacting fungal cells with a
double-stranded RNA, wherein the double-stranded RNA comprises
annealed complementary strands, one of which has a nucleotide
sequence which is complementary to at least part of the nucleotide
sequence of a target gene comprising a fragment of at least 17, 18,
19, 20 or 21 nucleotides of any of the sequences as represented in
SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 49-158, 159,
160, 163, 168, 173, 178, 183, 188, 193, 198, 203, 208, 215, 220,
225, 230, 247, 249, 251, 253, 255, 257, 259, 275-472, 473, 478,
483, 488, 493, 498, 503, 513, 515, 517, 519, 521, 533-575, 576,
581, 586, 591, 596, 601, 603, 605, 607, 609, 621-767, 768, 773,
778, 783, 788, 793, 795, 797, 799, 801, 813-862, 863, 868, 873,
878, 883, 888, 890, 892, 894, 896, 908-1040, 1041, 1046, 1051,
1056, 1061, 1071, 1073, 1075, 1077, 1079, 1081, 1083, 1085, 1087,
1089, 1091, 1093, 1095, 1097, 1099, 1101, 1103, 1105, 1107, 1109,
1111, 1113, 1161-1571, 1572, 1577, 1582, 1587, 1592, 1597, 1602,
1607, 1612, 1617, 1622, 1627, 1632, 1637, 1642, 1647, 1652, 1657,
1662, 1667, 1672, 1677, 1682, 1684, 1686, 1688, 1690, 1692, 1694,
1696, 1698, 1700, 1702, 1704, 1730-2039, 2040, 2045, 2050, 2055,
2060, 2065, 2070, 2075, 2080, 2085, 2090, 2095, 2100, 2102, 2104,
2106, 2108, 2120-2338, 2339, 2344, 2349, 2354, 2359, 2364, 2366,
2368, 2370, 2372, 2384-2460, 2461, 2466, 2471, 2476 and 2481,
whereby the double-stranded RNA is taken up by the fungus and
thereby controls growth or prevents infestation. The invention also
relates to fungal-resistant transgenic plants comprising a fragment
of at least 17, 18, 19, 20 or 21 of any of the sequences as
represented in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,
23, 49-158, 159, 160, 163, 168, 173, 178, 183, 188, 193, 198, 203,
208, 215, 220, 225, 230, 247, 249, 251, 253, 255, 257, 259,
275-472, 473, 478, 483, 488, 493, 498, 503, 513, 515, 517, 519,
521, 533-575, 576, 581, 586, 591, 596, 601, 603, 605, 607, 609,
621-767, 768, 773, 778, 783, 788, 793, 795, 797, 799, 801, 813-862,
863, 868, 873, 878, 883, 888, 890, 892, 894, 896, 908-1040, 1041,
1046, 1051, 1056, 1061, 1071, 1073, 1075, 1077, 1079, 1081, 1083,
1085, 1087, 1089, 1091, 1093, 1095, 1097, 1099, 1101, 1103, 1105,
1107, 1109, 1111, 1113, 1161-1571, 1572, 1577, 1582, 1587, 1592,
1597, 1602, 1607, 1612, 1617, 1622, 1627, 1632, 1637, 1642, 1647,
1652, 1657, 1662, 1667, 1672, 1677, 1682, 1684, 1686, 1688, 1690,
1692, 1694, 1696, 1698, 1700, 1702, 1704, 1730-2039, 2040, 2045,
2050, 2055, 2060, 2065, 2070, 2075, 2080, 2085, 2090, 2095, 2100,
2102, 2104, 2106, 2108, 2120-2338, 2339, 2344, 2349, 2354, 2359,
2364, 2366, 2368, 2370, 2372, 2384-2460, 2461, 2466, 2471, 2476 and
2481. A non-limiting list of fungal orthologs genes or sequences
comprising at least a fragment of 15, preferably at least 17 bp of
one of the sequences of the invention is given in Tables 6.
[0159] In a further embodiment, a dsRNA molecule of the invention
comprises any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,
23, 49-158, 159, 160, 163, 168, 173, 178, 183, 188, 193, 198, 203,
208, 215, 220, 225, 230, 247, 249, 251, 253, 255, 257, 259,
275-472, 473, 478, 483, 488, 493, 498, 503, 513, 515, 517, 519,
521, 533-575, 576, 581, 586, 591, 596, 601, 603, 605, 607, 609,
621-767, 768, 773, 778, 783, 788, 793, 795, 797, 799, 801, 813-862,
863, 868, 873, 878, 883, 888, 890, 892, 894, 896, 908-1040, 1041,
1046, 1051, 1056, 1061, 1071, 1073, 1075, 1077, 1079, 1081, 1083,
1085, 1087, 1089, 1091, 1093, 1095, 1097, 1099, 1101, 1103, 1105,
1107, 1109, 1111, 1113, 1161-1571, 1572, 1577, 1582, 1587, 1592,
1597, 1602, 1607, 1612, 1617, 1622, 1627, 1632, 1637, 1642, 1647,
1652, 1657, 1662, 1667, 1672, 1677, 1682, 1684, 1686, 1688, 1690,
1692, 1694, 1696, 1698, 1700, 1702, 1704, 1730-2039, 2040, 2045,
2050, 2055, 2060, 2065, 2070, 2075, 2080, 2085, 2090, 2095, 2100,
2102, 2104, 2106, 2108, 2120-2338, 2339, 2344, 2349, 2354, 2359,
2364, 2366, 2368, 2370, 2372, 2384-2460, 2461, 2466, 2471, 2476 and
2481, though the sequences set forth in SEQ ID NOs: 1, 3, 5, 7, 9,
11, 13, 15, 17, 19, 21, 23, 49-158, 159, 160, 163, 168, 173, 178,
183, 188, 193, 198, 203, 208, 215, 220, 225, 230, 247, 249, 251,
253, 255, 257, 259, 275-472, 473, 478, 483, 488, 493, 498, 503,
513, 515, 517, 519, 521, 533-575, 576, 581, 586, 591, 596, 601,
603, 605, 607, 609, 621-767, 768, 773, 778, 783, 788, 793, 795,
797, 799, 801, 813-862, 863, 868, 873, 878, 883, 888, 890, 892,
894, 896, 908-1040, 1041, 1046, 1051, 1056, 1061, 1071, 1073, 1075,
1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, 1093, 1095, 1097,
1099, 1101, 1103, 1105, 1107, 1109, 1111, 1113, 1161-1571, 1572,
1577, 1582, 1587, 1592, 1597, 1602, 1607, 1612, 1617, 1622, 1627,
1632, 1637, 1642, 1647, 1652, 1657, 1662, 1667, 1672, 1677, 1682,
1684, 1686, 1688, 1690, 1692, 1694, 1696, 1698, 1700, 1702, 1704,
1730-2039, 2040, 2045, 2050, 2055, 2060, 2065, 2070, 2075, 2080,
2085, 2090, 2095, 2100, 2102, 2104, 2106, 2108, 2120-2338, 2339,
2344, 2349, 2354, 2359, 2364, 2366, 2368, 2370, 2372, 2384-2460,
2461, 2466, 2471, 2476 and 2481 are not limiting. A dsRNA molecule
of the invention can comprise any contiguous target gene from a
pest species (e.g., about 15 to about 25 or more, or about 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, or 25 or more contiguous
nucleotides).
[0160] By "isolated" nucleic acid molecule(s) is intended a nucleic
acid molecule, DNA or RNA, which has been removed from its native
environment. For example, recombinant DNA molecules contained in a
DNA construct are considered isolated for the purposes of the
present invention. Further examples of isolated DNA molecules
include recombinant DNA molecules maintained in heterologous host
cells or purified (partially or substantially) DNA molecules in
solution. Isolated RNA molecules include in vitro RNA transcripts
of the DNA molecules of the present invention. Isolated nucleic
acid molecules, according to the present invention, further include
such molecules produced synthetically.
[0161] Nucleic acid molecules of the present invention may be in
the form of RNA, such as mRNA, or in the form of DNA, including,
for instance, cDNA and genomic DNA obtained by cloning or produced
synthetically. The DNA or RNA may be double-stranded or
single-stranded. Single-stranded DNA may be the coding strand, also
known as the sense strand, or it may be the non-coding strand, also
referred to as the anti-sense strand.
[0162] VI. Sequence Analysis
[0163] Unless otherwise indicated, all nucleotide sequences
determined by sequencing a DNA molecule herein were determined
using an automated DNA sequencer (such as the Model 373 from
Applied Biosystems, Inc.). Therefore, as is known in the art for
any DNA sequence determined by this automated approach, any
nucleotide sequence determined herein may contain some errors.
Nucleotide sequences determined by automation are typically at
least about 95% identical, more typically at least about 96% to at
least about 99.9% identical to the actual nucleotide sequence of
the sequenced DNA molecule. The actual sequence can be more
precisely determined by other approaches including manual DNA
sequencing methods well known in the art. As is also known in the
art, a single insertion or deletion in a determined nucleotide
sequence compared to the actual sequence will cause a frame shift
in translation of the nucleotide sequence such that the predicted
amino acid sequence encoded by a determined nucleotide sequence may
be completely different from the amino acid sequence actually
encoded by the sequenced DNA molecule, beginning at the point of
such an insertion or deletion.
[0164] In another aspect, the invention provides an isolated
nucleic acid molecule comprising a polynucleotide which hybridizes
under stringent hybridization conditions to a portion of the
polynucleotide in a nucleic acid molecule of the invention
described above. By a polynucleotide which hybridizes to a
"portion" of a polynucleotide is intended a polynucleotide (either
DNA or RNA) hybridizing to at least about 15 nucleotides, and more
preferably at least about 20 nucleotides, and still more preferably
at least about 30 nucleotides, and even more preferably more than
30 nucleotides of the reference polynucleotide. These fragments
that hybridize to the reference fragments are useful as diagnostic
probes and primers. For the purpose of the invention, two sequences
hybridize when they form a double-stranded complex in a
hybridization solution of 6.times.SSC, 0.5% SDS, 5.times.Denhardt's
solution and 100 .mu.g of non-specific carrier DNA. See Ausubel et
al., section 2.9, supplement 27 (1994). Sequences may hybridize at
"moderate stringency," which is defined as a temperature of
60.degree. C. in a hybridization solution of 6.times.SSC, 0.5% SDS,
5.times.Denhardt's solution and 100 .mu.g of non-specific carrier
DNA. For "high stringency" hybridization, the temperature is
increased to 68.degree. C. Following the moderate stringency
hybridization reaction, the nucleotides are washed in a solution of
2.times.SSC plus 0.05% SDS for five times at room temperature, with
subsequent washes with 0.1.times.SSC plus 0.1% SDS at 60.degree. C.
for 1 h. For high stringency, the wash temperature is increased to
68.degree. C. For the purpose of the invention, hybridized
nucleotides are those that are detected using 1 ng of a
radiolabeled probe having a specific radioactivity of 10,000
cpm/ng, where the hybridized nucleotides are clearly visible
following exposure to X-ray film at -70.degree. C. for no more than
72 hours.
[0165] The present application is directed to such nucleic acid
molecules which are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 96%, 97%, 98%, 99% or 100% identical to a nucleic acid
sequence described in any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15,
17, 19, 21, 23, 49-158, 159, 160, 163, 168, 173, 178, 183, 188,
193, 198, 203, 208, 215, 220, 225, 230, 247, 249, 251, 253, 255,
257, 259, 275-472, 473, 478, 483, 488, 493, 498, 503, 513, 515,
517, 519, 521, 533-575, 576, 581, 586, 591, 596, 601, 603, 605,
607, 609, 621-767, 768, 773, 778, 783, 788, 793, 795, 797, 799,
801, 813-862, 863, 868, 873, 878, 883, 888, 890, 892, 894, 896,
908-1040, 1041, 1046, 1051, 1056, 1061, 1071, 1073, 1075, 1077,
1079, 1081, 1083, 1085, 1087, 1089, 1091, 1093, 1095, 1097, 1099,
1101, 1103, 1105, 1107, 1109, 1111, 1113, 1161-1571, 1572, 1577,
1582, 1587, 1592, 1597, 1602, 1607, 1612, 1617, 1622, 1627, 1632,
1637, 1642, 1647, 1652, 1657, 1662, 1667, 1672, 1677, 1682, 1684,
1686, 1688, 1690, 1692, 1694, 1696, 1698, 1700, 1702, 1704,
1730-2039, 2040, 2045, 2050, 2055, 2060, 2065, 2070, 2075, 2080,
2085, 2090, 2095, 2100, 2102, 2104, 2106, 2108, 2120-2338, 2339,
2344, 2349, 2354, 2359, 2364, 2366, 2368, 2370, 2372, 2384-2460,
2461, 2466, 2471, 2476 and 2481. Preferred, however, are nucleic
acid molecules which are at least 95%, 96%, 97%, 98%, 99% or 100%
identical to the nucleic acid sequence shown in of SEQ ID NOs: 1,
3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 49-158, 159, 160, 163, 168,
173, 178, 183, 188, 193, 198, 203, 208, 215, 220, 225, 230, 247,
249, 251, 253, 255, 257, 259, 275-472, 473, 478, 483, 488, 493,
498, 503, 513, 515, 517, 519, 521, 533-575, 576, 581, 586, 591,
596, 601, 603, 605, 607, 609, 621-767, 768, 773, 778, 783, 788,
793, 795, 797, 799, 801, 813-862, 863, 868, 873, 878, 883, 888,
890, 892, 894, 896, 908-1040, 1041, 1046, 1051, 1056, 1061, 1071,
1073, 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, 1093,
1095, 1097, 1099, 1101, 1103, 1105, 1107, 1109, 1111, 1113,
1161-1571, 1572, 1577, 1582, 1587, 1592, 1597, 1602, 1607, 1612,
1617, 1622, 1627, 1632, 1637, 1642, 1647, 1652, 1657, 1662, 1667,
1672, 1677, 1682, 1684, 1686, 1688, 1690, 1692, 1694, 1696, 1698,
1700, 1702, 1704, 1730-2039, 2040, 2045, 2050, 2055, 2060, 2065,
2070, 2075, 2080, 2085, 2090, 2095, 2100, 2102, 2104, 2106, 2108,
2120-2338, 2339, 2344, 2349, 2354, 2359, 2364, 2366, 2368, 2370,
2372, 2384-2460, 2461, 2466, 2471, 2476 and 2481. Differences
between two nucleic acid sequences may occur at the 5' or 3'
terminal positions of the reference nucleotide sequence or anywhere
between those terminal positions, interspersed either individually
among nucleotides in the reference sequence or in one or more
contiguous groups within the reference sequence.
[0166] As a practical matter, whether any particular nucleic acid
molecule is at least 95%, 96%, 97%, 98% or 99% identical to a
reference nucleotide sequence refers to a comparison made between
two molecules using standard algorithms well known in the art and
can be determined conventionally using publicly available computer
programs such as the BLASTN algorithm. See Altschul et al., Nucleic
Acids Res. 25:3389-3402 (1997).
[0167] In one embodiment of the invention, a nucleic acid comprises
an antisense strand having about 15 to about 30 (e.g., about 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)
nucleotides, wherein the antisense strand is complementary to a RNA
sequence or a portion thereof encoding a protein that controls cell
cycle or homologous recombination, and wherein said siNA further
comprises a sense strand having about 15 to about 30 (e.g., about
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)
nucleotides, and wherein said sense strand and said antisense
strand are distinct nucleotide sequences where at least about 15
nucleotides in each strand are complementary to the other
strand.
[0168] In one embodiment, the present invention provides
double-stranded nucleic acid molecules of that mediate RNA
interference gene silencing. In another embodiment, the siNA
molecules of the invention consist of duplex nucleic acid molecules
containing about 15 to about 30 base pairs between oligonucleotides
comprising about 15 to about 30 (e.g., about 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides. In yet
another embodiment, siNA molecules of the invention comprise duplex
nucleic acid molecules with overhanging ends of about 1 to about 32
(e.g., about 1, 2, or 3) nucleotides, for example, about
21-nucleotide duplexes with about 19 base pairs and 3'-terminal
mononucleotide, dinucleotide, or trinucleotide overhangs. In yet
another embodiment, siNA molecules of the invention comprise duplex
nucleic acid molecules with blunt ends, where both ends are blunt,
or alternatively, where one of the ends is blunt.
[0169] An siNA molecule of the present invention may comprise
modified nucleotides while maintaining the ability to mediate RNAi.
The modified nucleotides can be used to improve in vitro or in vivo
characteristics such as stability, activity, and/or
bioavailability. For example, a siNA molecule of the invention can
comprise modified nucleotides as a percentage of the total number
of nucleotides present in the siNA molecule. As such, a siNA
molecule of the invention can generally comprise about 5% to about
100% modified nucleotides (e.g., about 5%, 10%, 15%, 20%, 25%, 30%,
35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or
100% modified nucleotides). The actual percentage of modified
nucleotides present in a given siNA molecule will depend on the
total number of nucleotides present in the siNA. If the siNA
molecule is single stranded, the percent modification can be based
upon the total number of nucleotides present in the single stranded
siNA molecules. Likewise, if the siNA molecule is double stranded,
the percent modification can be based upon the total number of
nucleotides present in the sense strand, antisense strand, or both
the sense and antisense strands.
[0170] VII. Nucleic Acid Constructs
[0171] A recombinant nucleic acid vector may, for example, be a
linear or a closed circular plasmid. The vector system may be a
single vector or plasmid or two or more vectors or plasmids that
together contain the total nucleic acid to be introduced into the
genome of the bacterial host. In addition, a bacterial vector may
be an expression vector. Nucleic acid molecules as set forth in SEQ
ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 49-158, 159,
160, 161, 162, 163, 168, 173, 178, 183, 188, 193, 198, 203, 208,
215, 220, 225, 230, 240-246, 247, 249, 251, 253, 255, 257, 259,
275-472, 473, 478, 483, 488, 493, 498, 503, 508-512, 513, 515, 517,
519, 521, 533-575, 576, 581, 586, 591, 596, 601, 603, 605, 607,
609, 621-767, 768, 773, 778, 783, 788, 793, 795, 797, 799, 801,
813-862, 863, 868, 873, 878, 883, 888, 890, 892, 894, 896,
908-1040, 1041, 1046, 1051, 1056, 1061, 1066-1070, 1071, 1073,
1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, 1093, 1095,
1097, 1099, 1101, 1103, 1105, 1107, 1109, 1111, 1113, 1161-1571,
1572, 1577, 1582, 1587, 1592, 1597, 1602, 1607, 1612, 1617, 1622,
1627, 1632, 1637, 1642, 1647, 1652, 1657, 1662, 1667, 1672, 1677,
1682, 1684, 1686, 1688, 1690, 1692, 1694, 1696, 1698, 1700, 1702,
1704, 1730-2039, 2040, 2045, 2050, 2055, 2060, 2065, 2070, 2075,
2080, 2085, 2090, 2095, 2100, 2102, 2104, 2106, 2108, 2120-2338,
2339, 2344, 2349, 2354, 2359, 2364, 2366, 2368, 2370, 2372,
2384-2460, 2461, 2466, 2471, 2476 and 2481, or fragments thereof
can, for example, be suitably inserted into a vector under the
control of a suitable promoter that functions in one or more
microbial hosts to drive expression of a linked coding sequence or
other DNA sequence. Many vectors are available for this purpose,
and selection of the appropriate vector will depend mainly on the
size of the nucleic acid to be inserted into the vector and the
particular host cell to be transformed with the vector. Each vector
contains various components depending on its function
(amplification of DNA or expression of DNA) and the particular host
cell with which it is compatible. The vector components for
bacterial transformation generally include, but are not limited to,
one or more of the following: a signal sequence, an origin of
replication, one or more selectable marker genes, and an inducible
promoter allowing the expression of exogenous DNA.
[0172] Promoters
[0173] "Operably linked", as used in reference to a regulatory
sequence and a structural nucleotide sequence, means that the
regulatory sequence causes regulated expression of the linked
structural nucleotide sequence. "Regulatory sequences" or "control
elements" refer to nucleotide sequences located upstream (5'
noncoding sequences), within, or downstream (3' non-translated
sequences) of a structural nucleotide sequence, and which influence
the timing and level or amount of transcription, RNA processing or
stability, or translation of the associated structural nucleotide
sequence. Regulatory sequences may include promoters, translation
leader sequences, introns, enhancers, stem-loop structures,
repressor binding sequences, and polyadenylation recognition
sequences and the like.
[0174] An expression vector for producing a mRNA can also contain
an inducible promoter that is recognized by the host bacterial
organism and is operably linked to the nucleic acid encoding, for
example, the nucleic acid molecule coding the D. v. virgifera mRNA
or fragment thereof of interest. Inducible promoters suitable for
use with bacterial hosts include .beta.-lactamase promoter, E. coli
.lamda. phage PL and PR promoters, and E. coli galactose promoter,
arabinose promoter, alkaline phosphatase promoter, tryptophan (trp)
promoter, and the lactose operon promoter and variations thereof
and hybrid promoters such as the tac promoter. However, other known
bacterial inducible promoters are suitable.
[0175] In certain embodiments, the genes can be derived from
different insects in order to broaden the range of insects against
which the agent is effective. When multiple genes are targeted for
suppression or a combination of expression and suppression, a
polycistronic DNA element can be fabricated as illustrated and
disclosed in Fillatti, Application Publication No. US
2004-0029283.
[0176] Selectable Marker Genes
[0177] A recombinant DNA vector or construct of the present
invention will typically comprise a selectable marker that confers
a selectable phenotype on transformed cells. Selectable markers may
also be used to select for cells that contain the exogenous nucleic
acids encoding polypeptides or proteins of the present invention.
The marker may encode biocide resistance, such as antibiotic
resistance (e.g., kanamycin, G418 bleomycin, hygromycin, etc.).
Examples of selectable markers include, but are not limited to, a
neo gene which codes for kanamycin resistance and can be selected
for using kanamycin, G418, etc., a bar gene which codes for
bialaphos resistance; a nitrilase gene which confers resistance to
bromoxynil, and a methotrexate resistant DHFR gene. Examples of
such selectable markers are illustrated in U.S. Pat. Nos.
5,550,318; 5,633,435; 5,780,708 and 6,118,047.
[0178] A recombinant vector or construct of the present invention
may also include a screenable marker. Screenable markers may be
used to monitor expression. Exemplary screenable markers include a
.beta.-glucuronidase or uidA gene (GUS) which encodes an enzyme for
which various chromogenic substrates are known (Jefferson, 1987;
Jefferson et al., 1987); a .beta.-lactamase gene (Sutcliffe et al.,
1978), a gene which encodes an enzyme for which various chromogenic
substrates are known (e.g., PADAC, a chromogenic cephalosporin); a
luciferase gene (Ow et al., 1986) a xylE gene (Zukowsky et al.,
1983) which encodes a catechol dioxygenase that can convert
chromogenic catechols; an .alpha.-amylase gene (Ikatu et al.,
1990); a tyrosinase gene (Katz et al., 1983) which encodes an
enzyme capable of oxidizing tyrosine to DOPA and dopaquinone which
in turn condenses to melanin; an .alpha.-galactosidase, which
catalyzes a chromogenic .alpha.-galactose substrate.
[0179] A transformation vector can be readily prepared using
methods available in the art. The transformation vector comprises
one or more nucleotide sequences that is/are capable of being
transcribed to an RNA molecule and that is/are substantially
homologous and/or complementary to one or more nucleotide sequences
encoded by the genome of the insect, such that upon uptake of the
RNA there is down-regulation of expression of at least one of the
respective nucleotide sequences of the genome of the pest.
[0180] VIII. Methods for Genetic Engineering
[0181] The present invention contemplates introduction of a
nucleotide sequence into a organism to achieve pest inhibitory
levels of expression of one or more dsRNA molecules. The inventive
polynucleotides and polypeptides may be introduced into a host
cell, such as bacterial or yeast cell, by standard procedures known
in the art for introducing recombinant sequences into a target host
cell. Such procedures include, but are not limited to,
transfection, infection, transformation, natural uptake, calcium
phosphate, electroporation, microinjection biolistics and
microorganism-mediated transformation protocols. The methods chosen
vary with the host organism.
[0182] A transgenic organism of the present invention is one that
comprises at least one cell it its genome in which an exogenous
nucleic acid has been stably integrated. Thus, a transgenic
organism may contain only genetically modified cells in certain
parts of its structure.
[0183] Accordingly, the present invention also encompasses a
transgenic cell or organism comprising any of the nucleotide
sequences or recombinant DNA constructs described herein. The
invention further encompasses prokaryotic cells (such as, but not
limited to, gram-positive and gram-negative bacterial cells) and
eukaryotic cells (such as, but not limited to, yeast cells or plant
cells).
[0184] For example, the present invention contemplates introducing
a target gene into a bacterium, such as Lactobacillus. The nucleic
acid constructs can be integrated into a bacterial genome with an
integrating vector. Integrating vectors typically contain at least
one sequence homologous to the bacterial chromosome that allows the
vector to integrate. Integrations appear to result from
recombinations between homologous DNA in the vector and the
bacterial chromosome. For example, integrating vectors constructed
with DNA from various Bacillus strains integrate into the Bacillus
chromosome (EP 0 127,328). Integrating vectors may also be
comprised of bacteriophage or transposon sequences. Suicide vectors
are also known in the art.
[0185] Construction of suitable vectors containing one or more of
the above-listed components employs standard recombinant DNA
techniques. Isolated plasmids or DNA fragments are cleaved,
tailored, and re-ligated in the form desired to generate the
plasmids required. Examples of available bacterial expression
vectors include, but are not limited to, the multifunctional E.
coli cloning and expression vectors such as Bluescript.TM.
(Stratagene, La Jolla, Calif.), in which, for example, a D. v.
virgifera protein or fragment thereof, may be ligated into the
vector in frame with sequences for the amino-terminal Met and the
subsequent 7 residues of .beta.-galactosidase so that a hybrid
protein is produced; pIN vectors (Van Heeke and Schuster, 1989);
and the like.
[0186] The invention also contemplates introducing a target gene
into a yeast cell. A yeast recombinant construct can typically
include one or more of the following: a promoter sequence, fusion
partner sequence, leader sequence, transcription termination
sequence, a selectable marker. These elements can be combined into
an expression cassette, which may be maintained in a replicon, such
as an extrachromosomal element (e.g., plasmids) capable of stable
maintenance in a host, such as yeast or bacteria. The replicon may
have two replication systems, thus allowing it to be maintained,
for example, in yeast for expression and in a prokaryotic host for
cloning and amplification. Examples of such yeast-bacteria shuttle
vectors include YEp24 (Botstein et al., 1979), pCl/1 (Brake et al.,
1984), and YRp17 (Stinchcomb et al., 1982). In addition, a replicon
may be either a high or low copy number plasmid. A high copy number
plasmid will generally have a copy number ranging from about 5 to
about 200, and typically about 10 to about 150. A host containing a
high copy number plasmid will preferably have at least about 10,
and more preferably at least about 20.
[0187] Useful yeast promoter sequences can be derived from genes
encoding enzymes in the metabolic pathway. Examples of such genes
include alcohol dehydrogenase (ADH) (EP 0 284044), enolase,
glucokinase, glucose-6-phosphate isomerase,
glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH),
hexokinase, phosphofructokinase, 3-phosphoglycerate mutase, and
pyruvate kinase (PyK) (EP 0 3215447). The yeast PHOS gene, encoding
acid phosphatase, also provides useful promoter sequences
(Myanohara et al., 1983). In addition, synthetic promoters that do
not occur in nature also function as yeast promoters. Examples of
such hybrid promoters include the ADH regulatory sequence linked to
the GAP transcription activation region (U.S. Pat. Nos. 4,876,197
and 4,880,734). Examples of transcription terminator sequences and
other yeast-recognized termination sequences, such as those coding
for glycolytic enzymes, are known to those of skill in the art.
[0188] Alternatively, the expression constructs can be integrated
into the yeast genome with an integrating vector. Integrating
vectors typically contain at least one sequence homologous to a
yeast chromosome that allows the vector to integrate, and
preferably contain two homologous sequences flanking the expression
construct. Integrations appear to result from recombinations
between homologous DNA in the vector and the yeast chromosome
(Orr-Weaver et al., 1983). An integrating vector may be directed to
a specific locus in yeast by selecting the appropriate homologous
sequence for inclusion in the vector. See Orr-Weaver et al., supra.
One or more expression constructs may integrate, possibly affecting
levels of recombinant protein produced (Rine et al., 1983).
[0189] IX. Quantifying Inhibition of Target Gene Expression
[0190] Inhibition of target gene expression may be quantified by
measuring either the endogenous target RNA or the protein produced
by translation of the target RNA and the consequences of inhibition
can be confirmed by examination of the outward properties of the
cell or organism. Techniques for quantifying RNA and proteins are
well known to one of ordinary skill in the art. Multiple selectable
markers are available that confer resistance to ampicillin,
bleomycin, chloramphenicol, gentamycin, hygromycin, kanamycin,
lincomycin, methotrexate, phosphinothricin, puromycin,
spectinomycin, rifampicin, and tetracyclin, and the like.
[0191] In certain embodiments gene expression is inhibited by at
least 10%, preferably by at least 33%, more preferably by at least
50%, and yet more preferably by at least 80%. In particularly
preferred embodiments of the invention gene expression is inhibited
by at least 80%, more preferably by at least 90%, more preferably
by at least 95%, or by at least 99% within cells in the pest so a
significant inhibition takes place. Significant inhibition is
intended to refer to sufficient inhibition that results in a
detectable phenotype (e.g., cessation of larval growth, paralysis
or mortality, etc.) or a detectable decrease in RNA and/or protein
corresponding to the target gene being inhibited. Although in
certain embodiments of the invention inhibition occurs in
substantially all cells of the pest, in other preferred embodiments
inhibition occurs in only a subset of cells expressing the gene.
For example, if the target gene plays an essential role in cells in
an insect alimentary tract, inhibition of the gene within these
cells is sufficient to exert a deleterious effect on the
insect.
[0192] X. Exposing Pest to dsRNA
[0193] A pest can be exposed to a dsRNA in any suitable manner that
permits administering the dsRNA to the pest. For example, the pest
can be contacted with the dsRNA in pure or substantially pure form,
for example an aqueous solution containing the dsRNA. In one
embodiment, the insect may be simply "soaked" or "sprayed" with an
aqueous solution comprising the dsRNA. Alternatively, the pest may
be "sprayed" with a solution comprising a dsRNA.
[0194] Alternatively, the dsRNA may be linked to a food component
of the pest, such as a food component for a mammalian pathogenic
pest, in order to increase uptake of the dsRNA by the insect.
Ingestion by a pest permits delivery of the pest control agents to
the pest and results in down-regulation of a target gene in the
host. Methods for oral introduction may include, for example,
directly mixing dsRNA with a, pest's food, as well as engineered
approaches in which a species that is used as food is engineered to
express the dsRNA or siRNA, then fed to the pest to be affected.
For example, a bacteria, such as Lactobacillus, may be transformed
with a target sequence and then fed to a pest. In one embodiment,
for example, the dsRNA or siRNA molecules may be incorporated into,
or overlaid on the top of, the insect's diet.
[0195] In other embodiments the pest may be contacted with a
composition containing the inventive dsRNA. The composition may, in
addition to the dsRNA, contain further excipients, diluents, or
carriers.
[0196] The dsRNA may also be incorporated in the medium in which
the pest grows or infests. For example, a dsRNA may be incorporated
into a food container or protective wrapping as a means for
inhibiting pest infestation. Wood, for example, may be treated with
a solution comprising a dsRNA to prevent pest infestation.
[0197] In other embodiments, the dsRNA is expressed in a bacterial
or fungal cell and the bacterial or fungal cell is taken up or
eaten by the insect species.
[0198] As illustrated in the examples, bacteria can be engineered
to produce any of the dsRNA or dsRNA constructs of the invention.
These bacteria can be eaten by the insect species. When taken up,
the dsRNA can initiate an RNAi response, leading to the degradation
of the target mRNA and weakening or killing of the feeding insect.
Alternatively, dsRNA producing bacteria or yeast cells can be
sprayed directly onto the crops.
[0199] Some bacteria have a very close interaction with the host
plant, such as, but not limited to, symbiotic Rhizobium with the
Legminosea (for example Soy). Such recombinant bacteria could be
mixed with the seeds (for instance as a coating) and used as soil
improvers.
[0200] A virus such as a baculovirus which specifically infects
insects may be also be used. This ensures safety for mammals,
especially humans, since the virus will not infect the mammal, so
no unwanted RNAi effect will occur.
[0201] Possible applications include intensive greenhouse cultures,
for instance crops that are less interesting from a GMO point of
view, as well as broader field crops such as soy.
[0202] This approach has several advantages, eg: since the problem
of possible dicing by a plant host is not present, it allows the
delivery of large dsRNA fragments into the gut lumen of the feeding
pest; the use of bacteria as insecticides does not involve the
generation of transgenic crops, especially for certain crops where
transgenic variants are difficult to obtain; there is a broad and
flexible application in that different crops can be simultaneously
treated on the same field and/or different pests can be
simultaneously targeted, for instance by combining different
bacteria producing distinct dsRNAs.
[0203] XI. Products
[0204] The present invention provides numerous products that can
encompass a dsRNA for use in controlling pests. For example, the
invention provides pharmaceutical or veterinary compositions for
treating or preventing a pest disease or infection of humans or
animals, respectively. Such compositions comprise at least one
dsRNA or RNA construct, or nucleotide sequence or recombinant DNA
construct encoding the dsRNA or RNA construct, or wherein the RNA
comprises annealed complementary strands, one of which has a
nucleotide sequence which corresponds to a target nucleotide
sequence of an pest target gene that causes the disease or
infection, and at least one carrier, excipient, or diluent suitable
for pharmaceutical use.
[0205] Alternatively, a pharmaceutical or veterinary composition
may be used as a composition suitable for topical use, such as
application on the skin of an animal or human. For example, a dsRNA
may be used in a liquid composition to be applied to the skin as
drops, gel, aerosol, cream, ointment, etc. Additionally, a dsRNA
may be integrated into a transdermal patch or other medical device
for treating or preventing a disease or condition. Other
conventional pharmaceutical dosage forms may also be produced,
including tablets, capsules, pessaries, transdermal patches,
suppositories, etc. The chosen form will depend upon the nature of
the target pest and hence the nature of the disease it is desired
to treat.
[0206] Oral vaccines, for example, can be produced using the
inventive constructs and methods. For example, a vaccine can be
constructed by producing a dsRNA in bacteria (e.g. lactobacillus)
which can be included in food and functions as an oral vaccine
against insect infection. Accordingly, the invention provides
constructs and methods for treating and/or preventing a pest
disease or condition, comprising administering to a subject in need
of such treatment and/or prevention, any of the compositions as
herein described, said composition comprising at least one
double-stranded RNA or double stranded RNA construct comprising
annealed complementary strands, one of which has a nucleotide
sequence which is complementary to at least part of a nucleotide
sequence of a pest target gene that causes the disease or
condition.
[0207] While the inventive compositions may be used for treating a
disease or condition in a subject patient, the compositions and
methods may also be used as a means for protecting a substrate or
material from pest infestation. The nature of the excipients
included in the composition and the physical form of the
composition may vary depending upon the nature of the substrate
that it is desired to treat.
[0208] For example, such a composition may be a coating or a powder
that can be applied to a substrate as a means for protecting the
substrate from infestation by an insect and thereby preventing
pest-induced damage to the substrate or material. Thus, in one
embodiment, the composition is in the form of a coating on a
suitable surface which adheres to, and is eventually ingested by an
insect which comes into contact with the coating. Such a
composition can be used to protect any substrate or material that
is susceptible to infestation by or damage caused by a pest, for
example foodstuffs and other perishable materials, and substrates
such as wood.
[0209] For example, the composition may be a liquid that is brushed
or sprayed onto or imprinted into the material or substrate to be
treated. Thus, a human user can spray the insect or the substrate
directly with the composition
[0210] For example, houses and other wood products can be destroyed
by termites, powder post beetles, and carpenter ants. By treating
wood or house siding with a composition comprising a dsRNA, it may
be possible to reduce pest infestation. Likewise, a tree trunk may
be treated with a composition comprising a dsRNA.
[0211] Flour beetles, grain weevils, meal moths, and other pests
feed on stored grain, cereals, pet food, powdered chocolate, and
almost everything else in the kitchen pantry that is not protected.
Accordingly, the present invention provides a means for treating
cereal boxes and other food storage containers and wrapping with a
composition comprising a target dsRNA.
[0212] Larvae of clothes moths eat clothes made from animal
products, such as fur, silk and wool. Thus, it may be desirable to
treat hangers, closet organizers, and garment bags with the
inventive dsRNA. Book lice and silverfish are pests of libraries
because they eat the starchy glue in the bindings of books.
Accordingly, the present invention provides compositions for
treating books from pest infestation and destruction.
[0213] In one embodiment, the composition is in the form of a bait.
The bait is designed to lure the insect to come into contact with
the composition. Upon coming into contact therewith, the
composition is then internalized by the insect, by ingestion for
example and mediates RNAi to thus kill the insect. The bait may
depend on the species being targeted. An attractant may also be
used. The attractant may be a pheromone, such as a male or female
pheromone for example. The attractant acts to lure the insect to
the bait, and may be targeted for a particular insect or may
attract a whole range of insects. The bait may be in any suitable
form, such as a solid, paste, pellet or powdered form.
[0214] The bait may also be carried away by the insect back to the
colony. The bait may then act as a food source for other members of
the colony, thus providing an effective control of a large number
of insects and potentially an entire insect pest colony. This is an
advantage associated with use of the double stranded RNA or
bacteria expressing the dsRNA of the invention, because the delayed
action of the RNAi mediated effects on the pests allows the bait to
be carried back to the colony, thus delivering maximal impact in
terms of exposure to the insects.
[0215] The baits may be provided in a suitable "housing" or "trap".
Such housings and traps are commercially available and existing
traps may be adapted to include the compositions of the invention.
The housing or trap may be box-shaped for example, and may be
provided in pre-formed condition or may be formed of foldable
cardboard for example. Suitable materials for a housing or trap
include plastics and cardboard, particularly corrugated cardboard.
The inside surfaces of the traps may be lined with a sticky
substance in order to restrict movement of the insect once inside
the trap. The housing or trap may contain a suitable trough inside
which can hold the bait in place. A trap is distinguished from a
housing because the insect can not readily leave a trap following
entry, whereas a housing acts as a "feeding station" which provides
the insect arachnid with a preferred environment in which they can
feed and feel safe from predators.
[0216] It is clear that numerous products and substrates can be
treated with the inventive compositions for reducing pest
infestation. Of course, the nature of the excipients and the
physical form of the composition may vary depending upon the nature
of the substrate that is desired to treat. For example, the
composition may be a liquid that is brushed or sprayed onto or
imprinted into the material or substrate to be treated, or a
coating that is applied to the material or substrate to be
treated.
[0217] Specific examples are presented below of methods for
identifying target sequences and introducing the sequences into
various cells and compositions. They are meant to be exemplary and
not as limitations on the present invention.
Example 1
Silencing C. elegans Target Genes in C. elegans in High Throughput
Screening
[0218] A C. elegans genome wide library was prepared in the pGN9A
vector (WO 01/88121) between two identical T7-promoters and
terminators, driving its expression in the sense and antisense
direction upon expression of the T7 polymerase, which was induced
by IPTG.
[0219] This library was transformed into the bacterial strain
AB301-105 (DE3) in 96 well plate format. For the genome wide
screening, these bacterial cells were fed to the nuclease deficient
C. elegans nuc-1 (e1392) strain.
[0220] Feeding the dsRNA produced in the bacterial strain AB301-105
(DE3), to C. elegans nuc-1 (e1392) worms, was performed in a 96
well plate format as follows: nuc-1 eggs were transferred to a
separate plate and allowed to hatch simultaneously at 20.degree. C.
for synchronization of the L1 generation. 96 well plates were
filled with 100 .mu.L liquid growth medium comprising IPTG and with
10 .mu.L bacterial cell culture of OD.sub.6001 AB301-105 (DE3) of
the C. elegans dsRNA library carrying each a vector with a C.
elegans genomic fragment for expression of the dsRNA. To each well,
4 of the synchronized L1 worms were added and were incubated at
25.degree. C. for at least 4 to 5 days. These experiments were
performed in quadruplicate. In the screen 6 controls were used:
[0221] pGN29=negative control, wild type [0222]
pGZ1=unc-22=twitcher phenotype [0223] pGZ18=chitin
synthase=embryonic lethal [0224] pGZ25=pos-1=embryonic lethal
[0225] pGZ59=bli-4D=acute lethal [0226] ACC=acetyl co-enzym A
carboxylase=acute lethal
[0227] After 5 days, the phenotype of the C. elegans nuc-1 (e1392)
worms fed with the bacteria producing dsRNA were compared to the
phenotype of worms fed with the empty vector (pGN29) and the other
controls. The worms that were fed with the dsRNA were screened for
lethality (acute or larval) lethality for the parent (Po)
generation, (embryonic) lethality for the first filial (F1)
generation, or for growth retardation of Po as follows: (i) Acute
lethality of Po: L1's have not developed and are dead, this
phenotype never gives progeny and the well looks quite empty; (ii)
(Larval) lethality of Po: Po died in a later stage than L1, this
phenotype also never gives progeny. Dead larvae or dead adult worms
are found in the wells; (iii) Lethality for F1: L1's have developed
until adult stage and are still alive. This phenotype has no
progeny. This can be due to sterility, embryonic lethality (dead
eggs on the bottom of well), embryonic arrest or larval arrest
(eventually ends up being lethal): (iv) Arrested in growth and
growth retardation/delay: Compared to a well with normal
development and normal # of progeny.
[0228] For the target sequences presented in Table 1A, it was
concluded that dsRNA mediated silencing of the C. elegans target
gene in nematodes, such as C. elegans, had a fatal effect on the
growth and viability of the worm.
[0229] Subsequent to the above dsRNA silencing experiment, a more
detailed phenotyping experiment was conducted in C. elegans in a
high throughput format on 24 well plates. The dsRNA library
produced in bacterial strain AB301-105 (DE3), as described above,
was fed to C. elegans nuc-1 (e1392) worms on 24 well plates as
follows: nuc-1 eggs were transferred to a separate plate and
allowed to hatch simultaneously at 20 C for synchronization of the
L1 generation. Subsequently 100 of the synchronized L1 worms were
soaked in a mixture of 500 .mu.A S-complete fed medium, comprising
5 .mu.g/mL cholesterol, 4 .mu.L/mL PEG and 1 mM IPTG, and 500 .mu.A
of bacterial cell culture of OD.sub.6001 AB301-105 (DE3) of the C.
elegans dsRNA library carrying each a vector with a C. elegans
genomic fragment for expression of the dsRNA. The soaked L1 worms
were rolled for 2 hours at 25 C.
[0230] After centrifugation and removal of 950 .mu.L of the
supernatant, 5 .mu.L of the remaining and resuspended pellet
(comprising about 10 to 15 worms) was transferred in the middle of
each well of a 24 well plate, filled with a layer of agar LB broth.
The inoculated plate was incubated at 25.degree. C. for 2 days. At
the adult stage, 1 adult worm was singled and incubated at
25.degree. C. for 2 days for inspection of its progeny. The other
adult worms are inspected in situ on the original 24 well plate.
These experiments were performed in quadruplicate.
[0231] This detailed phenotypic screen was repeated with a second
batch of worms, the only difference being that the worms of the
second batch were incubated at 20 C for 3 days.
[0232] The phenotype of the worms fed with C. elegans dsRNA was
compared to the phenotype of C. elegans nuc-1 (e1392) worms fed
with the empty vector.
[0233] Based on this experiment, it was concluded that silencing
the C. elegans target genes as represented in Table 1A had a fatal
effect on the growth and viability of the worm and that the target
gene is essential to the viability of nematodes. Therefore these
genes are good target genes to control (kill or prevent from
growing) nematodes via dsRNA mediated gene silencing. Accordingly,
the present invention encompasses the use of nematode orthologs of
the above C. elegans target gene to control nematode infestation in
a variety of organisms and materials.
Example 2
Identification of D. melanogaster Ortholos
[0234] As described above in Example 1, numerous C. elegans lethal
sequenes were identified and can be used for identifying orthologs
in other species and genera. For example, the C. elegans lethal
sequences can be used to identify orthologous D. melanogasters
sequences. That is, each C. elegans sequence can be querried
against a public database, such as GenBank, for orthologous
sequences in D. melanogaster. Potential D. melanogaster orthologs
were selected that share a high degree of sequence homology (E
value preferably less than or equal to 1E-30) and the sequences are
blast reciprocal best hits, the latter means that the sequences
from different organisms (e.g. C. elegans and D. melanogaster) are
each other's top blast hits. For example, sequence C from C.
elegans is compared against sequences in D. melanogaster using
BLAST. If sequence C has the D. melanogaster sequence D as best hit
and when D is compared to all the sequences of C. elegans, also
turns out to be sequence C, then D and C are reciprocal best hits.
This criterium is often used to define orthology, meaning similar
sequences of different species, having similar function. The D.
melanogaster sequence identifiers are represented in Table 1A.
Example 3
Leptinotarsa decemlineata (Colorado Potato Beetle)
A. Cloning Partial Gene Sequences from Leptinotarsa
decemlineata
[0235] High quality, intact RNA was isolated from 4 different
larval stages of Leptinotarsa decemlineata (Colorado potato beetle;
source: Jeroen van Schaik, Entocare CV Biologische
Gewasbescherming, Postbus 162, 6700 AD Wageningen, the Netherlands)
using TRIzol Reagent (Cat. Nr. 15596-026/15596-018, Invitrogen,
Rockville, Md., USA) following the manufacturer's instructions.
Genomic DNA present in the RNA preparation was removed by DNase
treatment following the manufacturer's instructions (Cat. Nr. 1700,
Promega). cDNA was generated using a commercially available kit
(SuperScript.TM. III Reverse Transcriptase, Cat. Nr. 18080044,
Invitrogen, Rockville, Md., USA) following the manufacturer's
instructions.
[0236] To isolate cDNA sequences comprising a portion of the LD001,
LD002, LD003, LD006, LD007, LD010, LD011, LD014, LD015, LD016 and
LD018 genes, a series of PCR reactions with degenerate primers were
performed using Amplitaq Gold (Cat. Nr. N8080240, Applied
Biosystems) following the manufacturer's instructions.
[0237] The sequences of the degenerate primers used for
amplification of each of the genes are given in Table 2-LD, which
displays Leptintarsa decemlineata target genes including primer
sequences and cDNA sequences obtained. These primers were used in
respective PCR reactions with the following conditions: 10 minutes
at 95.degree. C., followed by 40 cycles of 30 seconds at 95.degree.
C., 1 minute at 55.degree. C. and 1 minute at 72.degree. C.,
followed by 10 minutes at 72.degree. C. The resulting PCR fragments
were analyzed on agarose gel, purified (QIAquick Gel Extraction
kit, Cat. Nr. 28706, Qiagen), cloned into the pCR8/GW/topo vector
(Cat. Nr. K2500 20, Invitrogen), and sequenced. The sequences of
the resulting PCR products are represented by the respectiveSEQ ID
NOs as given in Table 2-LD and are referred to as the partial
sequences. The corresponding partial amino acid sequence are
represented by the respective SEQ ID NOs as given in Table 3-LD,
where the start of the reading frame is indicated in brackets.
B. dsRNA Production of the Leptinotarsa decemlineata Genes
[0238] dsRNA was synthesized in milligram amounts using the
commercially available kit T7 Ribomax.TM. Express RNAi System (Cat.
Nr. P1700, Promega). First two separate single 5' T7 RNA polymerase
promoter templates were generated in two separate PCR reactions,
each reaction containing the target sequence in a different
orientation relative to the T7 promoter.
[0239] For each of the target genes, the sense T7 template was
generated using specific T7 forward and specific reverse primers.
The sequences of the respective primers for amplifying the sense
template for each of the target genes are given in Table 8-LD. The
conditions in the PCR reactions were as follows: 4 minutes at
95.degree. C., followed by 35 cycles of 30 seconds at 95.degree.
C., 30 seconds at 55.degree. C. and 1 minute at 72.degree. C.,
followed by 10 minutes at 72.degree. C. The anti-sense T7 template
was generated using specific forward and specific T7 reverse
primers in a PCR reaction with the same conditions as described
above. The sequences of the respective primers for amplifying the
anti-sense template for each of the target genes are given in Table
8-LD. The resulting PCR products were analyzed on agarose gel and
purified by PCR purification kit (Qiaquick PCR Purification Kit,
Cat. Nr. 28106, Qiagen) and NaClO.sub.4 precipitation. The
generated T7 forward and reverse templates were mixed to be
transcribed and the resulting RNA strands were annealed, DNase and
RNase treated, and purified by sodium acetate, following the
manufacturer's instructions. The sense strand of the resulting
dsRNA for each of the target genes is given in Table 8-LD. Table
8-LD displays sequences for preparing ds RNA fragments of
Leptinotarsa decemlineata target sequences and concatemer
sequences, including primer sequences.
C. Screening dsRNA Targets Using Artificial Diet for Activity
Against Leptinotarsa decemlineata
[0240] Artificial diet for the Colorado potato beetle was prepared
as follows (adapted from Gelman et al., 2001, J. Ins. Sc., vol. 1,
no. 7, 1-10): water and agar were autoclaved, and the remaining
ingredients (shown in Table 2 below) were added when the
temperature dropped to 55.degree. C. At this temperature, the
ingredients were mixed well before the diet was aliquoted into
24-well plates (Nunc) with a quantity of 1 ml of diet per well. The
artificial diet was allowed to solidify by cooling at room
temperature. Diet was stored at 4.degree. C. for up to three
weeks.
TABLE-US-00001 TABLE 2 Ingredients for Artificial diet Ingredients
Volume for 1 L water 768 ml agar 14 g rolled oats 40 g Torula yeast
60 g lactalbumin 30 g hydrolysate casein 10 g fructose 20 g Wesson
salt mixture 4 g tomato fruit powder 12.5 g potato leaf powder 25 g
b-sitosterol 1 g sorbic acid 0.8 g methyl paraben 0.8 g Vanderzant
vitamin 12 g mix neomycin sulfate 0.2 g aureomycin 0.130 g
rifampicin 0.130 g chloramphenicol 0.130 g nystatin 0.050 g soybean
oil 2 ml wheat germ oil 2 ml
[0241] Fifty .mu.l of a solution of dsRNA at a concentration of 1
mg/ml was applied topically onto the solid artificial diet in the
wells of the multiwell plate. The diet was dried in a laminair flow
cabin. Per treatment, twenty-four Colorado potato beetle larvae
(2.sup.nd stage), with two insects per well, were tested. The
plates were stored in the insect rearing chamber at 25.+-.2.degree.
C., 60% relative humidity, with a 16:8 hours light:dark
photoperiod. The beetles were assessed as live or dead every 1, 2
or 3 days. After seven days, for targets LD006, LD007, LD010,
LD011, and LD014, the diet was replaced with fresh diet with
topically applied dsRNA at the same concentration (1 mg/ml); for
targets LD001, LD002, LD003, LD015, and LD016, the diet was
replaced with fresh diet only. The dsRNA targets were compared to
diet only or diet with topically applied dsRNA corresponding to a
fragment of the GFP (green fluorescent protein) coding sequence
(SEQ ID NO: 235).
[0242] Feeding artificial diet containing intact naked dsRNAs to L.
decemlineata larvae resulted in significant increases in larval
mortalities as indicated in two separate bioassays (FIGS.
1LD-2LD).
[0243] All dsRNAs tested resulted ultimately in 100% mortality
after 7 to 14 days. Diet with or without GFP dsRNA sustained the
insects throughout the bioassays with very little or no
mortality.
[0244] Typically, in all assays observed, CPB second-stage larvae
fed normally on diet with or without dsRNA for 2 days and molted to
the third larval stage. At this new larval stage the CPB were
observed to reduce significantly or stop altogether their feeding,
with an increase in mortality as a result.
D. Bioassay of dsRNA Targets Using Potato Leaf Discs for Activity
Against the Leptinotarsa decemlineata
[0245] An alternative bioassay method was employed using potato
leaf material rather than artificial diet as food source for CPB.
Discs of approximately 1.1 cm in diameter (or 0.95 cm.sup.2) were
cut out off leaves of 2 to 3-week old potato plants using a
suitably-sized cork borer. Treated leaf discs were prepared by
applying 20 .mu.A of a 10 ng/.mu.l solution of target LD002 dsRNA
or control gfp dsRNA on the adaxial leaf surface. The leaf discs
were allowed to dry and placed individually in 24 wells of a
24-well multiplate (Nunc). A single second-larval stage CPB was
placed into each well, which was then covered with tissue paper and
a multiwell plastic lid. The plate containing the insects and leaf
discs were kept in an insect chamber at 28.degree. C. with a
photoperiod of 16 h light/8 h dark. The insects were allowed to
feed on the leaf discs for 2 days after which the insects were
transferred to a new plate containing fresh treated leaf discs.
Thereafter, the insects were transferred to a plate containing
untreated leaf discs every day until day 7. Insect mortality and
weight scores were recorded.
[0246] Feeding potato leaf discs with surface-applied intact naked
dsRNA of target LD002 to L. decemlineata larvae resulted in a
significant increase in larval mortalities (i.e. at day 7 all
insects were dead; 100% mortality) whereas control gfp dsRNA had no
effect on CPB survival. Target LD002 dsRNA severely affected the
growth of the larvae after 2 to 3 days whereas the larvae fed with
gfp dsRNA at the same concentration developed as normal (FIG.
3-LD).
D. Screening Shorter Versions of dsRNAs Using Artificial Diet for
Activity Against Leptinotarsa decemlineata
[0247] This example exemplifies the finding that shorter (60 or 100
bp) dsRNA fragments on their own or as concatemer constructs are
sufficient in causing toxicity towards the Colorado potato
beetle.
[0248] LD014, a target known to induce lethality in Colorado potato
beetle, was selected for this example. This gene encodes a V-ATPase
subunit E (SEQ ID NO: 15).
[0249] A 100 base pair fragment, LD014_F1, at position 195-294 on
SEQ ID NO: 15 (SEQ ID NO: 159) and a 60 base pair fragment,
LD014_F2, at position 235-294 on SEQ ID NO: 15 (SEQ ID NO: 160)
were further selected. See also Table 7-LD.
[0250] Two concatemers of 300 base pairs, LD014_C1 and LD014_C2,
were designed (SEQ ID NO: 161 and SEQ ID NO: 162). LD014_C1
contained 3 repeats of the 100 base pair fragment described above
(SEQ ID NO: 159) and LD014_C2 contained 5 repeats of the 60 base
pair fragment described above (SEQ ID NO: 160). See also Table
7-LD.
[0251] The fragments LD014_F1 and LD014_F2 were synthesized as
sense and antisense primers. These primers were annealed to create
the double strands DNA molecules prior to cloning. XbaI and XmaI
restrictions sites were included at the 5' and 3' ends of the
primers, respectively, to facilitate the cloning.
[0252] The concatemers were made as 300 base pairs synthetic genes.
XbaI and XmaI restrictions sites were included at the 5' and 3'
ends of the synthetic DNA fragments, respectively, to facilite the
cloning.
[0253] The 4 DNA molecules, i.e. the 2 single units (LD014_F1 &
LD014_F2) and the 2 concatemers (LD014_C1 & LD014_C2), were
digested with XbaI and XmaI and subcloned in pBluescriptII SK+
linearised by XbaI and XmaI digests, resulting in recombinant
plasmids p1, p2, p3, & p4, respectively.
[0254] Double-stranded RNA production: dsRNA was synthesized using
the commercially available kit T7 Ribomax.TM. Express RNAi System
(Cat. Nr. P1700, Promega). First two separate single 5' T7 RNA
polymerase promoter templates were generated in two separate PCR
reactions, each reaction containing the target sequence in a
different orientation relative to the T7 promoter. For LD014_F1,
the sense T7 template was generated using the specific T7 forward
primer oGBM159 and the specific reverse primer oGBM164 (represented
herein as SEQ ID NO: 204 and SEQ ID NO: 205, respectively) in a PCR
reaction with the following conditions: 4 minutes at 95.degree. C.,
followed by 35 cycles of 30 seconds at 95.degree. C., 30 seconds at
55.degree. C. and 1 minute at 72.degree. C., followed by 10 minutes
at 72.degree. C. The anti-sense T7 template was generated using the
specific forward primer oGBM163 and the specific T7 reverse primer
oGBM160 (represented herein as SEQ ID NO: 206 and SEQ ID NO: 207,
respectively) in a PCR reaction with the same conditions as
described above. The resulting PCR products were analyzed on
agarose gel and purified by PCR purification kit (Qiaquick PCR
Purification Kit, Cat. Nr. 28106, Qiagen) and NaClO.sub.4
precipitation. The generated T7 forward and reverse templates were
mixed to be transcribed and the resulting RNA strands were
annealed, Dnase and Rnase treated, and purified by sodium acetate,
following the manufacturer's instructions. The sense strand of the
resulting dsRNA is herein represented by SEQ ID NO: 203.
[0255] For LD014_F2, the sense T7 template was generated using the
specific T7 forward primer oGBM161 and the specific reverse primer
oGBM166 (represented herein as SEQ ID NO: 209 and SEQ ID NO: 210,
respectively) in a PCR reaction with the following conditions: 4
minutes at 95.degree. C., followed by 35 cycles of 30 seconds at
95.degree. C., 30 seconds at 55.degree. C. and 1 minute at
72.degree. C., followed by 10 minutes at 72.degree. C. The
anti-sense T7 template was generated using the specific forward
primer oGBM165 and the specific T7 reverse primer oGBM162
(represented herein as SEQ ID NO: 211 and SEQ ID NO: 212,
respectively) in a PCR reaction with the same conditions as
described above. The resulting PCR products were analyzed on
agarose gel and purified by PCR purification kit (Qiaquick PCR
Purification Kit, Cat. Nr. 28106, Qiagen) and NaClO.sub.4
precipitation. The generated T7 forward and reverse templates were
mixed to be transcribed and the resulting RNA strands were
annealed, Dnase and Rnase treated, and purified by sodium acetate,
following the manufacturer's instructions. The sense strand of the
resulting dsRNA is herein represented by SEQ ID NO: 208.
[0256] Also for the concatemers, separate single 5' T7 RNA
polymerase promoter templates were generated in two separate PCR
reactions, each reaction containing the target sequence in a
different orientation relative to the T7 promoter. The recombinant
plasmids p3 and p4 containing LD014_C1 & LD014_C2 were
linearised with XbaI or XmaI, the two linear fragments for each
construct purified and used as template for the in vitro
transcription assay, using the T7 promoters flanking the cloning
sites. Double-stranded RNA was prepared by in vitro transcription
using the T7 RiboMAX.TM. Express RNAi System (Promega). The sense
strands of the resulting dsRNA for LD014_C1 and LD014_C2 are herein
represented by SEQ ID NO: 213 and 2114, respectively.
[0257] Shorter sequences of target LD014 and concatemers were able
to induce lethality in Leptinotarsa decemlineata, as shown in FIG.
4-LD.
G. Screening dsRNAs at Different Concentrations Using Artificial
Diet for Activity Against Leptinotarsa decemlineata
[0258] Fifty .mu.l of a solution of dsRNA at serial ten-fold
concentrations from 1 .mu.g/.mu.l (for target LD027 from 0.1
.mu.g/.mu.l) down to 0.01 ng/.mu.l was applied topically onto the
solid artificial diet in the wells of a 24-well plate (Nunc). The
diet was dried in a laminair flow cabin. Per treatment, twenty-four
Colorado potato beetle larvae (2.sup.nd stage), with two insects
per well, were tested. The plates were stored in the insect rearing
chamber at 25.+-.2.degree. C., 60% relative humidity, with a 16:8
hours light:dark photoperiod. The beetles were assessed as live or
dead at regular intervals up to day 14. After seven days, the diet
was replaced with fresh diet with topically applied dsRNA at the
same concentrations. The dsRNA targets were compared to diet
only.
[0259] Feeding artificial diet containing intact naked dsRNAs of
different targets to L. decemlineata larvae resulted in high larval
mortalities at concentrations as low as between 0.1 and 10 ng
dsRNA/.mu.l as shown in FIG. 5-LD.
H. Cloning of a CPB Gene Fragment in a Vector Suitable for
Bacterial Production of Insect-Active Double-Stranded RNA
[0260] While any efficient bacterial promoter may be used, a DNA
fragment corresponding to an MLB gene target was cloned in a vector
for the expression of double-stranded RNA in a bacterial host (See
WO 00/01846).
[0261] The sequences of the specific primers used for the
amplification of target genes are provided in Table 8. The template
used is the pCR8/GW/topo vector containing any of target sequences.
The primers are used in a PCR reaction with the following
conditions: 5 minutes at 98.degree. C., followed by 30 cycles of 10
seconds at 98.degree. C., 30 seconds at 55.degree. C. and 2 minutes
at 72.degree. C., followed by 10 minutes at 72.degree. C. The
resulting PCR fragment is analyzed on agarose gel, purified
(QIAquick Gel Extraction kit, Cat. Nr. 28706, Qiagen), blunt-end
cloned into Srf I-linearized pGNA49A vector (reference to
WO00188121A1), and sequenced. The sequence of the resulting PCR
product corresponds to the respective sequence as given in Table 8.
The recombinant vector harboring this sequence is named
pGBNJ003.
[0262] The sequences of the specific primers used for the
amplification of target gene fragment LD010 are provided in Table 8
(forward primer SEQ ID NO: 191 and reverse primer SEQ ID NO: 190).
The template used was the pCR8/GW/topo vector containing the LD010
sequence (SEQ ID NO: 11). The primers were used in a PCR reaction
with the following conditions: 5 minutes at 98.degree. C., followed
by 30 cycles of 10 seconds at 98.degree. C., 30 seconds at
55.degree. C. and 2 minutes at 72.degree. C., followed by 10
minutes at 72.degree. C. The resulting PCR fragment was analyzed on
agarose gel, purified (QIAquick Gel Extraction kit, Cat. Nr. 28706,
Qiagen), blunt-end cloned into Srf I-linearized pGNA49A vector
(reference to WO 00/188121A1), and sequenced. The sequence of the
resulting PCR product corresponds to SEQ ID NO: 188 as given in
Table 8. The recombinant vector harboring this sequence was named
pGBNJ003.
I. Expression and Production of a Double-Stranded RNA Target in Two
Strains of Escherichia coli: (1) AB309-105, and, (2) BL21(DE3)
[0263] The procedures described below were followed in order to
express suitable levels of insect-active double-stranded RNA of
target LD010 in bacteria. An RNaseIII-deficient strain, AB309-105,
was used in comparison to wild-type RNaseIII-containing bacteria,
BL21(DE3).
[0264] Transformation of AB309-105 and BL21(DE3)
[0265] Three hundred ng of the plasmid was added to and gently
mixed in a 50 .mu.l aliquot of ice-chilled chemically competent E.
coli strain AB309-105 or BL21(DE3). The cells were incubated on ice
for 20 minutes before subjecting them to a heat shock treatment of
37.degree. C. for 5 minutes, after which the cells were placed back
on ice for a further 5 minutes. Four hundred and fifty .mu.l of
room temperature SOC medium was added to the cells and the
suspension incubated on a shaker (250 rpm) at 37.degree. C. for 1
hour. One hundred .mu.l of the bacterial cell suspension was
transferred to a 500 ml conical flask containing 150 ml of liquid
Luria-Bertani (LB) broth supplemented with 100 .mu.g/ml
carbenicillin antibiotic. The culture was incubated on an Innova
4430 shaker (250 rpm) at 37.degree. C. overnight (16 to 18
hours).
[0266] Chemical Induction of Double-Stranded RNA Expression in
AB309-105 and BL21(DE3)
[0267] Expression of double-stranded RNA from the recombinant
vector, pGBNJ003, in the bacterial strain AB309-105 or BL21(DE3)
was made possible since all the genetic components for controlled
expression are present. In the presence of the chemical inducer
isopropylthiogalactoside, or IPTG, the T7 polymerase will drive the
transcription of the target sequence in both antisense and sense
directions since these are flanked by oppositely oriented T7
promoters.
[0268] The optical density at 600 nm of the overnight bacterial
culture was measured using an appropriate spectrophotometer and
adjusted to a value of 1 by the addition of fresh LB broth. Fifty
ml of this culture was transferred to a 50 ml Falcon tube and the
culture then centrifuged at 3000 g at 15.degree. C. for 10 minutes.
The supernatant was removed and the bacterial pellet resuspended in
50 ml of fresh S complete medium (SNC medium plus 5 .mu.g/ml
cholesterol) supplemented with 100 .mu.g/ml carbenicillin and 1 mM
IPTG. The bacteria were induced for 2 to 4 hours at room
temperature.
[0269] Heat Treatment of Bacteria
[0270] Bacteria were killed by heat treatment in order to minimize
the risk of contamination of the artificial diet in the test
plates. However, heat treatment of bacteria expressing
double-stranded RNA is not a prerequisite for inducing toxicity
towards the insects due to RNA interference. The induced bacterial
culture was centrifuged at 3000 g at room temperature for 10
minutes, the supernatant discarded and the pellet subjected to
80.degree. C. for 20 minutes in a water bath. After heat treatment,
the bacterial pellet was resuspended in 1.5 ml MilliQ water and the
suspension transferred to a microfuge tube. Several tubes were
prepared and used in the bioassays for each refreshment. The tubes
were stored at -20.degree. C. until further use.
J. Laboratory Trials to Test Escherichia coli Expressing dsRNA
Target LD010 Against Leptinotarsa decemlineata
[0271] Two bioassay methods were employed to test double-stranded
RNA produced in Escherichia coli against larvae of the Colorado
potato beetle: (1) artificial diet-based bioassay, and, (2)
plant-based bioassay.
[0272] Artificial Diet-Based Bioassays
[0273] Artificial diet for the Colorado potato beetle was prepared
as described previously in Example 4. A half milliliter of diet was
dispensed into each of the wells of a 48-well multiwell test plate
(Nunc). For every treatment, fifty .mu.l of an OD 1 suspension of
heat-treated bacteria (which is equivalent to approximately
5.times.10.sup.7 bacterial cells) expressing dsRNA was applied
topically onto the solid diet in the wells and the plates were
allowed to dry in a laminair flow cabin. Per treatment, forty-eight
2.sup.nd stage Colorado potato beetle larvae, one in each well
containing diet and bacteria, were tested. Each row of a plate
(i.e. 8 wells) was considered as one replicate. The plates were
kept in the insect rearing chamber at 25.+-.2.degree. C., 60.+-.5%
relative humidity, with a 16:8 hours light:dark photoperiod. After
every 4 days, the beetles were transferred to fresh diet containing
topically-applied bacteria. The beetles were assessed as alive or
dead every one or three days post infestation. For the survivors,
growth and development in terms of larval weight was recorded on
day 7 post infestation.
[0274] For RNaseIII-deficient E. coli strain AB309-105, bacteria
containing plasmid pGBNJ003 and those containing the empty vector
pGN29 (reference to WO 00/188121A1) were tested in bioassays for
CPB toxicity. Bacteria harboring the pGBNJ003 plasmid showed a
clear increase in insect mortality with time, whereas little or no
mortality was observed for pGN29 and diet only control (FIGS. 6a-LD
& 7a-LD). The growth and development of Colorado potato beetle
larval survivors, 7 days after feeding on artificial diet
containing bacteria expressing dsRNA target LD010, was severely
impeded (Table 10-LD, FIG. 8a-LD).
[0275] For E. coli strain BL21(DE3), bacteria containing plasmid
pGBNJ003 and those containing the empty vector pGN29 were tested
against the Colorado potato beetle larvae. Similar detrimental
effects were observed on larvae fed diet supplemented with
BL21(DE3) bacteria as for the RNAseIII-deficient strain, AB309-105
(FIGS. 6b-LD & 7b-LD). However, the number of survivors for the
five clones were higher for BL21(DE3) than for AB309-105; at day
12, average mortality values were approximately 25% lower for this
strain compared to the RNase III deficient strain. Also, the
average weights of survivors fed on diet containing BL21(DE3)
expressing dsRNA corresponding to target LD010 was severely reduced
(Table 10-LD, FIG. 8b-LD).
[0276] The delay in growth and development of the CPB larvae fed on
diet containing either of the two bacterial strains harboring
plasmid pGBNJ003 was directly correlated to feeding inhibition
since no frass was visible in the wells of refreshed plates from
day 4 onwards when compared to bacteria harboring the empty vector
pGN29 or the diet only plate. This observation was similar to that
where CPB was fed on in vitro transcribed double-stranded RNA
topically applied to artificial diet (see Example 3D); here,
cessation of feeding occurred from day 2 onwards on treated
diet.
[0277] Plant-Based Bioassays
[0278] Whole potato plants were sprayed with suspensions of
chemically induced bacteria expressing dsRNA prior to feeding the
plants to CPB larvae. The potato plants of variety `line 5` were
grown from tubers to the 8-12 unfolded leaf stage in a plant growth
room chamber with the following conditions: 25.+-.2.degree. C., 60%
relative humidity, 16:8 hour light:dark photoperiod. The plants
were caged by placing a 500 ml plastic bottle upside down over the
plant with the neck of the bottle firmly placed in the soil in a
pot and the base cut open and covered with a fine nylon mesh to
permit aeration, reduce condensation inside and prevent larval
escape. Fifteen Colorado potato beetle larvae at the L1 stage were
placed on each treated plant in the cage. Plants were treated with
a suspension of E. coli AB309-105 harboring the pGBNJ003 plasmids
(clone 1; FIG. 7a-LD) or pGN29 plasmid (clone 1; see FIG. 7a-LD).
Different quantities of bacteria were applied to the plants: 66,
22, and 7 units, where one unit is defined as 10.sup.9 bacterial
cells in 1 ml of a bacterial suspension at optical density value of
1 at 600 nm wavelength. In each case, a total volume of 1.6 ml was
sprayed on the plant with the aid of a vaporizer. One plant was
used per treatment in this trial. The number of survivors were
counted and the weight of each survivor recorded.
[0279] Spraying plants with a suspension of E. coli bacterial
strain AB309-105 expressing target dsRNA from pGBNJ003 led to a
dramatic increase in insect mortality when compared to pGN29
control. The mortality count was maintained when the amount of
bacteria cell suspension was diluted 9-fold (FIG. 9-LD). The
average weights of the larval survivors at day 11 on plants sprayed
with bacteria harboring the pGBNJ003 vector were approximately
10-fold less than that of pGN29 (FIG. 10-LD). Feeding damage by CPB
larvae of the potato plant sprayed with bacteria containing the
pGBNJ003 plasmid was much reduced when compared to the damage
incurred on a potato plant sprayed with bacteria containing the
empty vector pGN29 (FIG. 11-LD).
[0280] These experiments showed that double-stranded RNA
corresponding to an insect gene target sequence produced in either
wild-type or RNaseIII-deficient bacterial expression systems is
toxic towards the insect in terms of substantial increases in
insect mortality and growth/development delay for larval survivors.
It is also clear from these experiments that an exemplification was
provided for the effective protection of plants/crops from insect
damage by the use of a spray of a formulation consisting of
bacteria expressing double-stranded RNA corresponding to an insect
gene target.
K. Testing Various Culture Suspension Densities of Escherichia coli
Expressing dsRNA Target LD010 Against Leptinotarsa decemlineata
[0281] Preparation and treatment of bacterial cultures are
described in Example 3J. Three-fold serial dilutions of cultures
(starting from 0.25 unit equivalents) of Escherichia coli
RNAseIII-deficient strain AB309-105 expressing double-stranded RNA
of target LD010 were applied to foliages of the potato plant of
variety `Bintje` at the 8-12 unfolded leaf stage. Ten L1 larvae of
the L. decemlineata were placed on the treated plants with one
plant per treatment. Scoring for insect mortality and growth
impediment was done on day 7 (i.e., 7 days post infestation).
[0282] As shown in FIG. 14-LD, high CPB larval mortality (90 to
100%) was recorded after 1 week when insects were fed potato plants
treated with a topical application by fine spray of
heat-inactivated cultures of E. coli harboring plasmid pGBNJ003
(for target 10 dsRNA expression) at densities 0.25, 0.08 and 0.025
bacterial units. At 0.008 units, about a third of the insects were
dead, however, the surviving insects were significantly smaller
than those in the control groups (E. coli harbouring the empty
vector pGN29 and water only). Feeding damage by CPB larvae of the
potato plant sprayed with bacteria containing the pGBNJ003 plasmid
at concentrations 0.025 or 0.008 units was much reduced when
compared to the damage incurred on a potato plant sprayed with
bacteria containing the empty vector pGN29 (FIG. 15-LD).
L. Adults are Extremely Susceptible to Orally Ingested dsRNA
Corresponding to Target Genes
[0283] The example provided below highlights the finding that adult
insects (and not only insects of the larval stage) are extremely
susceptible to orally ingested dsRNA corresponding to target
genes.
[0284] Four targets were chosen for this experiment: targets 2, 10,
14 and 16 (SEQ ID NO: 168, 188, 198 and 220, respectively). GFP
fragment dsRNA (SEQ ID NO: 235) was used as a control. Young adults
(2 to 3 days old) were picked at random from our laboratory-reared
culture with no bias towards insect gender. Ten adults were chosen
per treatment. The adults were prestarved for at least 6 hours
before the onset of the treatment. On the first day of treatment,
each adult was fed four potato leaf discs (diameter 1.5 cm.sup.2)
which were pretreated with a topical application of 25 .mu.l of 0.1
.mu.g/.mu.l target dsRNA (synthesized as described in Example 3A;
topical application as described in Example 3E) per disc. Each
adult was confined to a small petridish (diameter 3 cm) in order to
make sure that all insects have ingested equal amounts of food and
thus received equal doses of dsRNA. The following day, each adult
was again fed four treated leaf discs as described above. On the
third day, all ten adults per treatment were collected and placed
together in a cage consisting of a plastic box (dimensions 30
cm.times.20 cm.times.15 cm) with a fine nylon mesh built into the
lid to provide good aeration. Inside the box, some moistened filter
paper was placed in the base. Some (untreated) potato foliage was
placed on top of the paper to maintain the adults during the
experiment. From day 5, regular assessments were carried out to
count the number of dead, alive (mobile) and moribund insects. For
insect moribundity, adults were laid on their backs to check
whether they could right themselves within several minutes; an
insect was considered moribund only if it was not able to turn onto
its front.
[0285] Clear specific toxic effects of double-stranded RNA
correpsonding to different targets towards adults of the Colorado
potato beetle, Leptinotarsa decemlineata, were demonstrated in this
experiment (FIG. 12-LD). Double-stranded RNA corresponding to a gfp
fragment showed no toxicity towards CPB adults on the day of the
final assessment (day 19). This experiment clearly showed that the
survival of CPB adults was severely reduced only after a few days
of exposure to dsRNA when delivered orally. For example, for target
10, on day 5, 5 out of 10 adults were moribund (sick and slow
moving); on day 6, 4 out of 10 adults were dead with three of the
survivors moribund; on day 9 all adults were observed dead.
[0286] As a consequence of this experiment, the application of
target double-stranded RNAs against insect pests may be broadened
to include the two life stages of an insect pest (i.e. larvae and
adults) which could cause extensive crop damage, as is the case
with the Colorado potato beetle.
Example 4
Phaedon cochleariae (Mustard Leaf Beetle)
A. Cloning of a Partial Sequence of the Phaedon cochleariae
(Mustard Leaf Beetle) PC001, PC003, PC005, PC010, PC014, PC016 and
PC027 Genes Via Family PCR
[0287] High quality, intact RNA was isolated from the third larval
stage of Phaedon cochleariae (mustard leaf beetle; source: Dr.
Caroline Muller, Julius-von-Sachs-Institute for Biosciences,
Chemical Ecology Group, University of Wuerzburg,
Julius-von-Sachs-Platz 3, D-97082 Wuerzburg, Germany) using TRIzol
Reagent (Cat. Nr. 15596-026/15596-018, Invitrogen, Rockville, Md.,
USA) following the manufacturer's instructions. Genomic DNA present
in the RNA preparation was removed by DNase (Cat. Nr. 1700,
Promega) treatment following the manufacturer's instructions. cDNA
was generated using a commercially available kit (SuperScript.TM.
III Reverse Transcriptase, Cat. Nr. 18080044, Invitrogen,
Rockville, Md., USA) following the manufacturer's instructions.
[0288] To isolate cDNA sequences comprising a portion of the PC001,
PC003, PC005, PC010, PC014, PC016 and PC027 genes, a series of PCR
reactions with degenerate primers were performed using Amplitaq
Gold (Cat. Nr. N8080240, Applied Biosystems) following the
manafacturer's instructions.
[0289] The sequences of the degenerate primers used for
amplification of each of the genes are given in Table 2-PC. Table
2-PC displays Phaedon cochleariae target genes including primer
sequences and cDNA sequences obtained. These primers were used in
respective PCR reactions with the following conditions: 10 minutes
at 95.degree. C., followed by 40 cycles of 30 seconds at 95.degree.
C., 1 minute at 55.degree. C. and 1 minute at 72.degree. C.,
followed by 10 minutes at 72.degree. C. The resulting PCR fragments
were analyzed on agarose gel, purified (QIAquick Gel Extraction
kit, Cat. Nr. 28706, Qiagen), cloned into the pCR4/TOPO vector
(Cat. Nr. K4530-20, Invitrogen) and sequenced. The sequences of the
resulting PCR products are represented by the respective SEQ ID
NO:s as given in Table 2-PC and are referred to as the partial
sequences.
[0290] The corresponding partial amino acid sequence are
represented by the respective SEQ ID NO:s as given in Table 3-PC.
Table 3-PC provides amino acid sequences of cDNA clones, and the
start of the reading frame is indicated in brackets.
B. dsRNA Production of the Phaedon cochleariae Genes
[0291] dsRNA was synthesized in milligram amounts using the
commercially available kit T7 Ribomax.TM. Express RNAi System (Cat.
Nr. P1700, Promega). First two separate single 5' T7 RNA polymerase
promoter templates were generated in two separate PCR reactions,
each reaction containing the target sequence in a different
orientation relative to the T7 promoter.
[0292] For each of the target genes, the sense T7 template was
generated using specific T7 forward and specific reverse primers.
The sequences of the respective primers for amplifying the sense
template for each of the target genes are given in Table 8-PC.
Table 8-PC provides details for preparing ds RNA fragments of
Phaedon cochleariae target sequences, including primer
sequences.
[0293] The conditions in the PCR reactions were as follows: 1
minute at 95.degree. C., followed by 20 cycles of 30 seconds at
95.degree. C., 30 seconds at 60.degree. C. and 1 minute at
72.degree. C., followed by 15 cycles of 30 seconds at 95.degree.
C., 30 seconds at 50.degree. C. and 1 minute at 72.degree. C.
followed by 10 minutes at 72.degree. C. The anti-sense T7 template
was generated using specific forward and specific T7 reverse
primers in a PCR reaction with the same conditions as described
above. The sequences of the respective primers for amplifying the
anti-sense template for each of the target genes are given in Table
8-PC. The resulting PCR products were analyzed on agarose gel and
purified by PCR purification kit (Qiaquick PCR Purification Kit,
Cat. Nr. 28106, Qiagen) and NaClO.sub.4 precipitation. The
generated T7 forward and reverse templates were mixed to be
transcribed and the resulting RNA strands were annealed, DNase and
RNase treated, and purified by sodium acetate, following the
manufacturer's instructions. The sense strand of the resulting
dsRNA for each of the target genes is given in Table 8-PC.
C. Laboratory Trials to Test dsRNA Targets, Using Oilseed Rape Leaf
Discs for Activity Against Phaedon cochleariae Larvae
[0294] The example provided below is an exemplification of the
finding that the mustard leaf beetle (MLB) larvae are susceptible
to orally ingested dsRNA corresponding to own target genes.
[0295] To test the different double-stranded RNA samples against
MLB larvae, a leaf disc assay was employed using oilseed rape
(Brassica napus variety SW Oban; source: Nick Balaam, Sw Seed Ltd.,
49 North Road, Abington, Cambridge, CB1 6AS, UK) leaf material as
food source. The insect cultures were maintained on the same
variety of oilseed rape in the insect chamber at 25.+-.2.degree. C.
and 60.+-.5% relative humidity with a photoperiod of 16 h light/8 h
dark. Discs of approximately 1.1 cm in diameter (or 0.95 cm.sup.2)
were cut out off leaves of 4- to 6-week old rape plants using a
suitably-sized cork borer. Double-stranded RNA samples were diluted
to 0.1 .mu.g/.mu.l in Milli-Q water containing 0.05% Triton X-100.
Treated leaf discs were prepared by applying 25 .mu.l of the
diluted solution of target PC001, PC003, PC005, PC010, PC014,
PC016, PC027 dsRNA and control gfp dsRNA or 0.05% Triton X-100 on
the adaxial leaf surface. The leaf discs were left to dry and
placed individually in each of the 24 wells of a 24-well multiplate
containing 1 ml of gellified 2% agar which helps to prevent the
leaf disc from drying out. Two neonate MLB larvae were placed into
each well of the plate, which was then covered with a multiwell
plastic lid. The plate (one treatment containing 48 insects) was
divided into 4 replicates of 12 insects per replicate (each row).
The plate containing the insects and leaf discs were kept in an
insect chamber at 25.+-.2.degree. C. and 60.+-.5% relative humidity
with a photoperiod of 16 h light/8 h dark. The insects were fed
leaf discs for 2 days after which they were transferred to a new
plate containing freshly treated leaf discs. Thereafter, 4 days
after the start of the bioassay, the insects from each replicate
were collected and transferred to a Petri dish containing untreated
fresh oilseed rape leaves. Larval mortality and average weight were
recorded at days 2, 4 7, 9 and 11.
[0296] P. cochleariae larvae fed on intact naked target
dsRNA-treated oilseed rape leaves resulted in significant increases
in larval mortalities for all targets tested, as indicated in FIG.
1(a). Tested double-stranded RNA for target PC010 led to 100%
larval mortality at day 9 and for target PC027 at day 11. For all
other targets, significantly high mortality values were reached at
day 11 when compared to control gfp dsRNA, 0.05% Trition X-100
alone or untreated leaf only: (average value in
percentage.+-.confidence interval with alpha 0.05) PC001
(94.4.+-.8.2); PC003 (86.1.+-.4.1); PC005 (83.3.+-.7.8); PC014
(63.9.+-.20.6); PC016 (75.0.+-.16.8); gfp dsRNA (11.1.+-.8.2);
0.05% Triton X-100 (19.4.+-.10.5); leaf only (8.3.+-.10.5).
[0297] Larval survivors were assessed based on their average
weight. For all targets tested, the mustard leaf beetle larvae had
significantly reduced average weights after day 4 of the bioassay;
insects fed control gfp dsRNA or 0.05% Triton X-100 alone developed
normally, as for the larvae on leaf only (FIG. 1(b)--PC).
D. Laboratory Trials to Screen dsRNAs at Different Concentrations
Using Oilseed Rape Leaf Discs for Activity Against Phaedon
cochleariae Larvae
[0298] Twenty-five .mu.l of a solution of dsRNA from target PC010
or PC027 at serial ten-fold concentrations from 0.1 .mu.g/.mu.l
down to 0.1 ng/.mu.l was applied topically onto the oilseed rape
leaf disc, as described in Example 4D above. As a negative control,
0.05% Triton X-100 only was administered to the leaf disc. Per
treatment, twenty-four mustard leaf beetle neonate larvae, with two
insects per well, were tested. The plates were stored in the insect
rearing chamber at 25.+-.2.degree. C., 60.+-.5% relative humidity,
with a 16:8 hours light:dark photoperiod. At day 2, the larvae were
transferred on to a new plate containing fresh dsRNA-treated leaf
discs. At day 4 for target PC010 and day 5 for target PC027,
insects from each replicate were transferred to a Petri dish
containing abundant untreated leaf material. The beetles were
assessed as live or dead on days 2, 4, 7, 8, 9, and 11 for target
PC010, and 2, 5, 8, 9 and 12 for target PC027.
[0299] Feeding oilseed rape leaf discs containing intact naked
dsRNAs of the two different targets, PC010 and PC027, to P.
cochleariae larvae resulted in high mortalities at concentrations
down to as low as 1 ng dsRNA/.mu.1 solution, as shown in FIGS. 2
(a) and (b). Average mortality values in percentage.+-.confidence
interval with alpha 0.05 for different concentrations of dsRNA for
target PC010 at day 11, 0 .mu.g/.mu.l: 8.3.+-.9.4; 0.1 .mu.g/.mu.l:
100; 0.01 .mu.g/.mu.l: 79.2.+-.20.6; 0.001 .mu.g/.mu.l:
58.3.+-.9.4; 0.0001 .mu.g/.mu.l: 12.5.+-.15.6; and for target PC027
at day 12, 0 .mu.g/.mu.l: 8.3.+-.9.4; 0.1 .mu.g/.mu.l: 95.8.+-.8.2;
0.01 .mu.g/.mu.l: 95.8.+-.8.2; 0.001 .mu.g/.mu.l: 83.3.+-.13.3;
0.0001 .mu.g/.mu.l: 12.5.+-.8.2.
E. Cloning of a MLB Gene Fragment in a Vector Suitable for
Bacterial Production of Insect-Active Double-Stranded RNA
[0300] What follows is an example of cloning a DNA fragment
corresponding to an MLB gene target in a vector for the expression
of double-stranded RNA in a bacterial host, although any vector
comprising a T7 promoter or any other promoter for efficient
transcription in bacteria, may be used (reference to
WO0001846).
[0301] The sequences of the specific primers used for the
amplification of target genes are provided in Table 8. The template
used is the pCR8/GW/topo vector containing any of target sequences.
The primers are used in a PCR reaction with the following
conditions: 5 minutes at 98.degree. C., followed by 30 cycles of 10
seconds at 98.degree. C., 30 seconds at 55.degree. C. and 2 minutes
at 72.degree. C., followed by 10 minutes at 72.degree. C. The
resulting PCR fragment is analyzed on agarose gel, purified
(QIAquick Gel Extraction kit, Cat. Nr. 28706, Qiagen), blunt-end
cloned into Srf I-linearized pGNA49A vector (reference to
WO00188121A1), and sequenced. The sequence of the resulting PCR
product corresponds to the respective sequence as given in Table 8.
The recombinant vector harbouring this sequence is named pGBNJ00
(to be completed).
[0302] The sequences of the specific primers used for the
amplification of target gene fragment PC010 are provided in Table
8-PC. The template used was the pCR8/GW/topo vector containing the
PC010 sequence (SEQ ID NO: 253). The primers were used in a
touch-down PCR reaction with the following conditions: 1 minute at
95.degree. C., followed by 20 cycles of 30 seconds at 95.degree.
C., 30 seconds at 60.degree. C. with temperature decrease of
-0.5.degree. C. per cycle and 1 minute at 72.degree. C., followed
by 15 cycles of 30 seconds at 95.degree. C., 30 seconds at
50.degree. C. and 1 minute at 72.degree. C., followed by 10 minutes
at 72.degree. C. The resulting PCR fragment was analyzed on agarose
gel, purified (QIAquick Gel Extraction kit, Cat. Nr. 28706,
Qiagen), blunt-end cloned into Srf I-linearized pGNA49A vector
(reference to WO00188121A1), and sequenced. The sequence of the
resulting PCR product corresponds to SEQ ID NO: 488 as given in
Table 8-PC. The recombinant vector harbouring this sequence was
named pGCDJ001.
F. Expression and Production of a Double-Stranded RNA Target in Two
Strains of Escherichia coli AB309-105
[0303] The procedures described below are followed in order to
express suitable levels of insect-active double-stranded RNA of
insect target in bacteria. In this experiment, an
RNaseIII-deficient strain, AB309-105 is used.
Transformation of AB309-105
[0304] Three hundred ng of the plasmid were added to and gently
mixed in a 50 .mu.l aliquot of ice-chilled chemically competent E.
coli strain AB309-105. The cells were incubated on ice for 20
minutes before subjecting them to a heat shock treatment of
37.degree. C. for 5 minutes, after which the cells were placed back
on ice for a further 5 minutes. Four hundred and fifty .mu.l of
room temperature SOC medium was added to the cells and the
suspension incubated on a shaker (250 rpm) at 37.degree. C. for 1
hour. One hundred .mu.l of the bacterial cell suspension was
transferred to a 500 ml conical flask containing 150 ml of liquid
Luria-Bertani (LB) broth supplemented with 100 .mu.g/ml
carbenicillin antibiotic. The culture was incubated on an Innova
4430 shaker (250 rpm) at 37.degree. C. overnight (16 to 18
hours).
Chemical Induction of Double-Stranded RNA Expression in
AB309-105
[0305] Expression of double-stranded RNA from the recombinant
vector, pGBNJ003, in the bacterial strain AB309-105 was made
possible since all the genetic components for controlled expression
are present. In the presence of the chemical inducer
isopropylthiogalactoside, or IPTG, the T7 polymerase will drive the
transcription of the target sequence in both antisense and sense
directions since these are flanked by oppositely oriented T7
promoters.
[0306] The optical density at 600 nm of the overnight bacterial
culture was measured using an appropriate spectrophotometer and
adjusted to a value of 1 by the addition of fresh LB broth. Fifty
ml of this culture was transferred to a 50 ml Falcon tube and the
culture then centrifuged at 3000 g at 15.degree. C. for 10 minutes.
The supernatant was removed and the bacterial pellet resuspended in
50 ml of fresh S complete medium (SNC medium plus 5 .mu.g/ml
cholesterol) supplemented with 100 .mu.g/ml carbenicillin and 1 mM
IPTG. The bacteria were induced for 2 to 4 hours at room
temperature.
Heat Treatment of Bacteria
[0307] Bacteria were killed by heat treatment in order to minimize
the risk of contamination of the artificial diet in the test
plates. However, heat treatment of bacteria expressing
double-stranded RNA is not a prerequisite for inducing toxicity
towards the insects due to RNA interference. The induced bacterial
culture was centrifuged at 3000 g at room temperature for 10
minutes, the supernatant discarded and the pellet subjected to
80.degree. C. for 20 minutes in a water bath. After heat treatment,
the bacterial pellet was resuspended in a total volume of 50 ml of
0.05% Triton X-100 solution. The tube was stored at 4.degree. C.
until further use
G. Laboratory Trials to Test Escherichia coli Expressing dsRNA
Targets Against Phaedon cochleariae
Leaf Disc Bioassays
[0308] The leaf-disc bioassay method was employed to test
double-stranded RNA from target PC010 produced in Escherichia coli
(from plasmid pGCDJ001) against larvae of the mustard leaf beetle.
Leaf discs were prepared from oilseed rape foliage, as described in
Example 4. Twenty .mu.l of a bacterial suspension, with an optical
density measurement of 1 at 600 nm wavelength, was pipetted onto
each disc. The leaf disc was placed in a well of a 24-multiwell
plate containing 1 ml gellified agar. On each leaf disc were added
two neonate larvae. For each treatment, 3 replicates of 16 neonate
larvae per replicate were prepared. The plates were kept in the
insect rearing chamber at 25.+-.2.degree. C. and 60.+-.5% relative
humidity, with a 16:8 hours light:dark photoperiod. At day 3 (i.e.
3 days post start of bioassay), larvae were transferred to a new
plate containing fresh treated (same dosage) leaf discs. The leaf
material was refreshed every other day from day 5 onwards. The
bioassay was scored on mortality and average weight. Negative
controls were leaf discs treated with bacteria harbouring plasmid
pGN29 (empty vector) and leaf only.
A clear increase in mortality of P. cochleariae larvae with time
was shown after the insects were fed on oilseed rape leaves treated
with a suspension of RNaseIII-deficient E. coli strain AB309-105
containing plasmid pGCDJ001, whereas very little or no insect
mortality was observed in the case of bacteria with plasmid pGN29
or leaf only control (FIG. 3-PC).
Plant-Based Bioassays
[0309] Whole plants are sprayed with suspensions of chemically
induced bacteria expressing dsRNA prior to feeding the plants to
MLB. The are grown from in a plant growth room chamber. The plants
are caged by placing a 500 ml plastic bottle upside down over the
plant with the neck of the bottle firmly placed in the soil in a
pot and the base cut open and covered with a fine nylon mesh to
permit aeration, reduce condensation inside and prevent insect
escape. MLB are placed on each treated plant in the cage. Plants
are treated with a suspension of E. coli AB309-105 harbouring the
pGBNJ001 plasmids or pGN29 plasmid. Different quantities of
bacteria are applied to the plants: for instance 66, 22, and 7
units, where one unit is defined as 10.sup.9 bacterial cells in 1
ml of a bacterial suspension at optical density value of 1 at 600
nm wavelength. In each case, a total volume of between 1 and 10 ml
s sprayed on the plant with the aid of a vaporizer. One plant is
used per treatment in this trial. The number of survivors are
counted and the weight of each survivor recorded.
[0310] Spraying plants with a suspension of E. coli bacterial
strain AB309-105 expressing target dsRNA from pGBNJ003 leed to a
dramatic increase in insect mortality when compared to pGN29
control. These experiments show that double-stranded RNA
corresponding to an insect gene target sequence produced in either
wild-type or RNaseIII-deficient bacterial expression systems is
toxic towards the insect in terms of substantial increases in
insect mortality and growth/development delay for larval survivors.
It is also clear from these experiments that an exemplification is
provided for the effective protection of plants/crops from insect
damage by the use of a spray of a formulation consisting of
bacteria expressing double-stranded RNA corresponding to an insect
gene target.
Example 5
Epilachna varivetis (Mexican Bean Beetle)
A. Cloning Epilachna varivetis Partial Gene Sequences
[0311] High quality, intact RNA was isolated from 4 different
larval stages of Epilachna varivetis (Mexican bean beetle; source:
Thomas Dorsey, Supervising Entomologist, New Jersey Department of
Agriculture, Division of Plant Industry, Bureau of Biological Pest
Control, Phillip Alampi Beneficial Insect Laboratory, PO Box 330,
Trenton, N.J. 08625-0330, USA) using TRIzol Reagent (Cat. Nr.
15596-026/15596-018, Invitrogen, Rockville, Md., USA) following the
manufacturer's instructions. Genomic DNA present in the RNA
preparation was removed by DNase treatment following the
manafacturer's instructions (Cat. Nr. 1700, Promega). cDNA was
generated using a commercially available kit (SuperScript.TM. III
Reverse Transcriptase, Cat. Nr. 18080044, Invitrogen, Rockville,
Md., USA) following the manufacturer's instructions.
[0312] To isolate cDNA sequences comprising a portion of the EV005,
EV009, EV010, EV015 and EV016 genes, a series of PCR reactions with
degenerate primers were performed using Amplitaq Gold (Cat. Nr.
N8080240, Applied Biosystems) following the manufacturer's
instructions.
[0313] The sequences of the degenerate primers used for
amplification of each of the genes are given in Table 2-EV, which
displays Epilachna varivetis target genes including primer
sequences and cDNA sequences obtained. These primers were used in
respective PCR reactions with the following conditions: for EV005
and EV009, 10 minutes at 95.degree. C., followed by 40 cycles of 30
seconds at 95.degree. C., 1 minute at 50.degree. C. and 1 minute 30
seconds at 72.degree. C., followed by 7 minutes at 72.degree. C.;
for EV014, 10 minutes at 95.degree. C., followed by 40 cycles of 30
seconds at 95.degree. C., 1 minute at 53.degree. C. and 1 minute at
72.degree. C., followed by 7 minutes at 72.degree. C.; for EV010
and EV016, 10 minutes at 95.degree. C., followed by 40 cycles of 30
seconds at 95.degree. C., 1 minute at 54.degree. C. and 1 minute 40
seconds at 72.degree. C., followed by 7 minutes at 72.degree. C.
The resulting PCR fragments were analyzed on agarose gel, purified
(QIAquick Gel Extraction kit, Cat. Nr. 28706, Qiagen), cloned into
the pCR4/TOPO vector (Cat. Nr. K.sub.4530-20, Invitrogen), and
sequenced. The sequences of the resulting PCR products are
represented by the respective SEQ ID NO:s as given in Table 2-EV
and are referred to as the partial sequences. The corresponding
partial amino acid sequences are represented by the respective SEQ
ID NO:s as given in Table 3-EV, where the start of the reading
frame is indicated in brackets.
B. dsRNA Production of the Epilachna varivetis Genes
[0314] dsRNA was synthesized in milligram amounts using the
commercially available kit T7 Ribomax.TM. Express RNAi System (Cat.
Nr. P1700, Promega). First two separate single 5' T7 RNA polymerase
promoter templates were generated in two separate PCR reactions,
each reaction containing the target sequence in a different
orientation relative to the T7 promoter.
[0315] For each of the target genes, the sense T7 template was
generated using specific T7 forward and specific reverse primers.
The sequences of the respective primers for amplifying the sense
template for each of the target genes are given in Table 8-EV.
[0316] The conditions in the PCR reactions were as follows: 1
minute at 95.degree. C., followed by 20 cycles of 30 seconds at
95.degree. C., 30 seconds at 60.degree. C. and 1 minute at
72.degree. C., followed by 15 cycles of 30 seconds at 95.degree.
C., 30 seconds at 50.degree. C. and 1 minute at 72.degree. C.
followed by 10 minutes at 72.degree. C. The anti-sense T7 template
was generated using specific forward and specific T7 reverse
primers in a PCR reaction with the same conditions as described
above. The sequences of the respective primers for amplifying the
anti-sense template for each of the target genes are given in Table
8-EV. The resulting PCR products were analyzed on agarose gel and
purified by PCR purification kit (Qiaquick PCR Purification Kit,
Cat. Nr. 28106, Qiagen) and NaClO.sub.4 precipitation. The
generated T7 forward and reverse templates were mixed to be
transcribed and the resulting RNA strands were annealed, DNase and
RNase treated, and purified by sodium acetate, following the
manufacturer's instructions. The sense strand of the resulting
dsRNA for each of the target genes is given in Table 8-EV.
C. Laboratory Trials to Test dsRNA Targets Using Bean Leaf Discs
for Activity Against Epilachna varivetis Larvae
[0317] The example provided below is an exemplification of the
finding that the Mexican bean beetle (MBB) larvae are susceptible
to orally ingested dsRNA corresponding to own target genes.
[0318] To test the different double-stranded RNA samples against
MBB larvae, a leaf disc assay was employed using snap bean
(Phaseolus vulgaris variety Montano; source: Aveve NV, Belgium)
leaf material as food source. The same variety of beans was used to
maintain insect cultures in the insect chamber at 25.+-.2.degree.
C. and 60.+-.5% relative humidity with a photoperiod of 16 h
light/8 h dark. Discs of approximately 1.1 cm in diameter (or 0.95
cm.sup.2) were cut out off leaves of 1- to 2-week old bean plants
using a suitably-sized cork borer. Double-stranded RNA samples were
diluted to 1 .mu.g/.mu.l in Milli-Q water containing 0.05% Triton
X-100. Treated leaf discs were prepared by applying 25 .mu.l of the
diluted solution of target Ev005, Ev010, Ev015, Ev016 dsRNA and
control gfp dsRNA or 0.05% Triton X-100 on the adaxial leaf
surface. The leaf discs were left to dry and placed individually in
each of the 24 wells of a 24-well multiplate containing 1 ml of
gellified 2% agar which helps to prevent the leaf disc from drying
out. A single neonate MBB larva was placed into each well of a
plate, which was then covered with a multiwell plastic lid. The
plate was divided into 3 replicates of 8 insects per replicate
(row). The plate containing the insects and leaf discs were kept in
an insect chamber at 25.+-.2.degree. C. and 60.+-.5% relative
humidity with a photoperiod of 16 h light/8 h dark. The insects
were fed on the leaf discs for 2 days after which the insects were
transferred to a new plate containing freshly treated leaf discs.
Thereafter, 4 days after the start of the bioassay, the insects
were transferred to a petriplate containing untreated fresh bean
leaves every day until day 10. Insect mortality was recorded at day
2 and every other day thereafter.
[0319] Feeding snap bean leaves containing surface-applied intact
naked target dsRNAs to E. varivestis larvae resulted in significant
increases in larval mortalities, as indicated in FIG. 1. Tested
double-stranded RNAs of targets Ev010, Ev015, & Ev016 led to
100% mortality after 8 days, whereas dsRNA of target Ev005 took 10
days to kill all larvae. The majority of the insects fed on treated
leaf discs containing control gfp dsRNA or only the surfactant
Triton X-100 were sustained throughout the bioassay (FIG.
1-EV).
D. Laboratory Trials to Test dsRNA Targets Using Bean Leaf Discs
for Activity Against Epilachna varivestis Adults
[0320] The example provided below is an exemplification of the
finding that the Mexican bean beetle adults are susceptible to
orally ingested dsRNA corresponding to own target genes.
[0321] In a similar bioassay set-up as for Mexican bean beetle
larvae, adult MBBs were tested against double-stranded RNAs
topically-applied to bean leaf discs. Test dsRNA from each target
Ev010, Ev015 and Ev016 was diluted in 0.05% Triton X-100 to a final
concentration of 0.1 .mu.g/.mu.l. Bean leaf discs were treated by
topical application of 30 .mu.l of the test solution onto each
disc. The discs were allowed to dry completely before placing each
on a slice of gellified 2% agar in each well of a 24-well multiwell
plate. Three-day-old adults were collected from the culture cages
and fed nothing for 7-8 hours prior to placing one adult to each
well of the bioassay plate (thus 24 adults per treatment). The
plates were kept in the insect rearing chamber (under the same
conditions as for MBB larvae for 24 hours) after which the adults
were transferred to a new plate containing fresh dsRNA-treated leaf
discs. After a further 24 hours, the adults from each treatment
were collected and placed in a plastic box with dimensions 30
cm.times.15 cm.times.10 cm containing two potted and untreated
3-week-old bean plants. Insect mortality was assessed from day 4
until day 11.
[0322] All three target dsRNAs (Ev010, Ev015 and Ev016) ingested by
adults of Epilachna varivestis resulted in significant increases in
mortality from day 4 (4 days post bioassay start), as shown in FIG.
2(a)-EV. From day 5, dramatic changes in feeding patterns were
observed between insects fed initially with target-dsRNA-treated
bean leaf discs and those that were fed discs containing control
gfp dsRNA or surfactant Triton X-100. Reductions in foliar damage
by MBB adults of untreated bean plants were clearly visible for all
three targets when compared to gfp dsRNA and surfactant only
controls, albeit at varying levels; insects fed target 15 caused
the least damage to bean foliage (FIG. 2(b)-EV).
E. Cloning of a MBB Gene Fragment in a Vector Suitable for
Bacterial Production of Insect-Active Double-Stranded RNA
[0323] What follows is an example of cloning a DNA fragment
corresponding to an MLB gene target in a vector for the expression
of double-stranded RNA in a bacterial host, although any vector
comprising a T7 promoter or any other promoter for efficient
transcription in bacteria, may be used (reference to
WO0001846).
[0324] The sequences of the specific primers used for the
amplification of target genes are provided in Table 8-EV. The
template used is the pCR8/GW/topo vector containing any of target
sequences. The primers are used in a PCR reaction with the
following conditions: 5 minutes at 98.degree. C., followed by 30
cycles of 10 seconds at 98.degree. C., 30 seconds at 55.degree. C.
and 2 minutes at 72.degree. C., followed by 10 minutes at
72.degree. C. The resulting PCR fragment is analyzed on agarose
gel, purified (QIAquick Gel Extraction kit, Cat. Nr. 28706,
Qiagen), blunt-end cloned into Srf I-linearized pGNA49A vector
(reference to WO00188121A1), and sequenced. The sequence of the
resulting PCR product corresponds to the respective sequence as
given in Table 8-EV. The recombinant vector harbouring this
sequence is named pGBNJ00XX.
F. Expression and Production of a Double-Stranded RNA Target in Two
Strains of Escherichia coli: (1) AB309-105, and, (2) BL21(DE3)
[0325] The procedures described below are followed in order to
express suitable levels of insect-active double-stranded RNA of
insect target in bacteria. An RNaseIII-deficient strain, AB309-105,
is used in comparison to wild-type RNaseIII-containing bacteria,
BL21(DE3).
Transformation of AB309-105 and BL21(DE3)
[0326] Three hundred ng of the plasmid are added to and gently
mixed in a 50 .mu.l aliquot of ice-chilled chemically competent E.
coli strain AB309-105 or BL21(DE3). The cells are incubated on ice
for 20 minutes before subjecting them to a heat shock treatment of
37.degree. C. for 5 minutes, after which the cells are placed back
on ice for a further 5 minutes. Four hundred and fifty .mu.l of
room temperature SOC medium is added to the cells and the
suspension incubated on a shaker (250 rpm) at 37.degree. C. for 1
hour. One hundred .mu.l of the bacterial cell suspension is
transferred to a 500 ml conical flask containing 150 ml of liquid
Luria-Bertani (LB) broth supplemented with 100 .mu.g/ml
carbenicillin antibiotic. The culture is incubated on an Innova
4430 shaker (250 rpm) at 37.degree. C. overnight (16 to 18
hours).
Chemical Induction of Double-Stranded RNA Expression in AB309-105
and BL21(DE3)
[0327] Expression of double-stranded RNA from the recombinant
vector, pGBNJ003, in the bacterial strain AB309-105 or BL21(DE3) is
made possible since all the genetic components for controlled
expression are present. In the presence of the chemical inducer
isopropylthiogalactoside, or IPTG, the T7 polymerase will drive the
transcription of the target sequence in both antisense and sense
directions since these are flanked by oppositely oriented T7
promoters.
[0328] The optical density at 600 nm of the overnight bacterial
culture is measured using an appropriate spectrophotometer and
adjusted to a value of 1 by the addition of fresh LB broth. Fifty
ml of this culture is transferred to a 50 ml Falcon tube and the
culture then centrifuged at 3000 g at 15.degree. C. for 10 minutes.
The supernatant is removed and the bacterial pellet resuspended in
50 ml of fresh S complete medium (SNC medium plus 5 .mu.g/ml
cholesterol) supplemented with 100 .mu.g/ml carbenicillin and 1 mM
IPTG. The bacteria are induced for 2 to 4 hours at room
temperature.
Heat Treatment of Bacteria
[0329] Bacteria are killed by heat treatment in order to minimize
the risk of contamination of the artificial diet in the test
plates. However, heat treatment of bacteria expressing
double-stranded RNA is not a prerequisite for inducing toxicity
towards the insects due to RNA interference. The induced bacterial
culture is centrifuged at 3000 g at room temperature for 10
minutes, the supernatant discarded and the pellet subjected to
80.degree. C. for 20 minutes in a water bath. After heat treatment,
the bacterial pellet is resuspended in 1.5 ml MilliQ water and the
suspension transferred to a microfuge tube. Several tubes are
prepared and used in the bioassays for each refreshment. The tubes
are stored at -20.degree. C. until further use.
G. Laboratory Trials to Test Escherichia coli Expressing dsRNA
Targets Against Epilachna varivetis
Plant-Based Bioassays
[0330] Whole plants are sprayed with suspensions of chemically
induced bacteria expressing dsRNA prior to feeding the plants to
MBB. The are grown from in a plant growth room chamber. The plants
are caged by placing a 500 ml plastic bottle upside down over the
plant with the neck of the bottle firmly placed in the soil in a
pot and the base cut open and covered with a fine nylon mesh to
permit aeration, reduce condensation inside and prevent insect
escape. MMB are placed on each treated plant in the cage. Plants
are treated with a suspension of E. coli AB309-105 harbouring the
pGBNJ001 plasmids or pGN29 plasmid. Different quantities of
bacteria are applied to the plants: for instance 66, 22, and 7
units, where one unit is defined as 10.sup.9 bacterial cells in 1
ml of a bacterial suspension at optical density value of 1 at 600
nm wavelength. In each case, a total volume of between 1 and 10 ml
sprayed on the plant with the aid of a vaporizer. One plant is used
per treatment in this trial. The number of survivors are counted
and the weight of each survivor recorded.
[0331] Spraying plants with a suspension of E. coli bacterial
strain AB309-105 expressing target dsRNA from pGBNJ003 lead to a
dramatic increase in insect mortality when compared to pGN29
control. These experiments show that double-stranded RNA
corresponding to an insect gene target sequence produced in either
wild-type or RNaseIII-deficient bacterial expression systems is
toxic towards the insect in terms of substantial increases in
insect mortality and growth/development delay for larval survivors.
It is also clear from these experiments that an exemplification is
provided for the effective protection of plants/crops from insect
damage by the use of a spray of a formulation consisting of
bacteria expressing double-stranded RNA corresponding to an insect
gene target.
Example 6
Anthonomus grandis (Cotton Boll Weevil)
A. Cloning Anthonomus grandis Partial Sequences
[0332] High quality, intact RNA was isolated from the 3 instars of
Anthonomus grandis (cotton boll weevil; source: Dr. Gary Benzon,
Benzon Research Inc., 7 Kuhn Drive, Carlisle, Pa. 17013, USA) using
TRIzol Reagent (Cat. Nr. 15596-026/15596-018, Invitrogen,
Rockville, Md., USA) following the manufacturer's instructions.
Genomic DNA present in the RNA preparation was removed by DNase
treatment following the manafacturer's instructions (Cat. Nr. 1700,
Promega). cDNA was generated using a commercially available kit
(SuperScript.TM. III Reverse Transcriptase, Cat. Nr. 18080044,
Invitrogen, Rockville, Md., USA) following the manufacturer's
instructions.
[0333] To isolate cDNA sequences comprising a portion of the AG001,
AG005, AG010, AG014 and AG016 genes, a series of PCR reactions with
degenerate primers were performed using Amplitaq Gold (Cat. Nr.
N8080240, Applied Biosystems) following the manafacturer's
instructions.
[0334] The sequences of the degenerate primers used for
amplification of each of the genes are given in Table 2-AG. These
primers were used in respective PCR reactions with the following
conditions: for AG001, AG005 and AG016, 10 minutes at 95.degree.
C., followed by 40 cycles of 30 seconds at 95.degree. C., 1 minute
at 50.degree. C. and 1 minute and 30 seconds at 72.degree. C.,
followed by 7 minutes at 72.degree. C.; for AG010, 10 minutes at
95.degree. C., followed by 40 cycles of 30 seconds at 95.degree.
C., 1 minute at 54.degree. C. and 2 minutes and 30 seconds at
72.degree. C., followed by 7 minutes at 72.degree. C.; for AG014,
10 minutes at 95.degree. C., followed by 40 cycles of 30 seconds at
95.degree. C., 1 minute at 55.degree. C. and 1 minute at 72.degree.
C., followed by 7 minutes at 72.degree. C. The resulting PCR
fragments were analyzed on agarose gel, purified (QIAquick Gel
Extraction kit, Cat. Nr. 28706, Qiagen), cloned into the
pCR8/GW/TOPO vector (Cat. Nr. K2500-20, Invitrogen) and sequenced.
The sequences of the resulting PCR products are represented by the
respective SEQ ID NO:s as given in Table 2-AG and are referred to
as the partial sequences. The corresponding partial amino acid
sequence are represented by the respective SEQ ID NO:s as given in
Table 3-AG.
B. dsRNA Production of the Anthonomus grandis (Cotton Boll Weevil)
Genes
[0335] dsRNA was synthesized in milligram amounts using the
commercially available kit T7 Ribomax.TM. Express RNAi System (Cat.
Nr. P1700, Promega). First two separate single 5' T7 RNA polymerase
promoter templates were generated in two separate PCR reactions,
each reaction containing the target sequence in a different
orientation relative to the T7 promoter.
[0336] For each of the target genes, the sense T7 template was
generated using specific T7 forward and specific reverse primers.
The sequences of the respective primers for amplifying the sense
template for each of the target genes are given in Table 8-AG. A
touchdown PCR was performed as follows: 1 minute at 95.degree. C.,
followed by 20 cycles of 30 seconds at 95.degree. C., 30 seconds at
60.degree. C. with a decrease in temperature of 0.5.degree. C. per
cycle and 1 minute at 72.degree. C., followed by 15 cycles of 30
seconds at 95.degree. C., 30 seconds at 50.degree. C. and 1 minute
at 72.degree. C., followed by 10 minutes at 72.degree. C. The
anti-sense T7 template was generated using specific forward and
specific T7 reverse primers in a PCR reaction with the same
conditions as described above. The sequences of the respective
primers for amplifying the anti-sense template for each of the
target genes are given in Table 8-AG. The resulting PCR products
were analyzed on agarose gel and purified by PCR purification kit
(Qiaquick PCR Purification Kit, Cat. Nr. 28106, Qiagen) and
NaClO.sub.4 precipitation. The generated T7 forward and reverse
templates were mixed to be transcribed and the resulting RNA
strands were annealed, DNase and RNase treated, and purified by
sodium acetate, following the manufacturer's instructions. The
sense strand of the resulting dsRNA for each of the target genes is
given in Table 8-AG.
C. Laboratory Trials to Test dsRNA Targets, Using Artificial Diet
for Activity Against the Larvae of the House Cricket, Acheta
domesticus
[0337] House crickets, Acheta domesticus, were maintained at Insect
Investigations Ltd. (origin: Blades Biological Ltd., Kent, UK). The
insects were reared on bran pellets and cabbage leaves. Mixed sex
nymphs of equal size and no more than 5 days old were selected for
use in the trial. Double-stranded RNA was mixed with a wheat-based
pelleted rodent diet (rat and mouse standard diet, B & K
Universal Ltd., Grimston, Aldbrough, Hull, UK). The diet, BK001P,
contains the following ingredients in descending order by weight:
wheat, soya, wheatfeed, barley, pellet binder, rodent 5 vit min,
fat blend, dicalcium phosphate, mould carb. The pelleted rodent
diet was finely ground and heat-treated in a microwave oven prior
to mixing, in order to inactivate any enzyme components. All rodent
diet was taken from the same batch in order to ensure consistency.
The ground diet and dsRNA were mixed thoroughly and formed into
small pellets of equal weight, which were allowed to dry overnight
at room temperature.
[0338] Double-stranded RNA samples from targets and gfp control at
concentrations 10 .mu.g/.mu.l are applied in the ratio 1 g ground
diet plus 1 ml dsRNA solution, thereby resulting in an application
rate of 10 mg dsRNA per g pellet. Pellets are replaced weekly. The
insects are provided with treated pellets for the first three weeks
of the trial. Thereafter untreated pellets are provided. Insects
are maintained within lidded plastic containers (9 cm diameter, 4.5
cm deep), ten per container. Each arena contains one treated bait
pellet and one water source (damp cotton wool ball), each placed in
a separate small weigh boat. The water is replenished ad lib
throughout the experiment.
[0339] Assessments are made at twice weekly intervals, with no more
than four days between assessments, until all the control insects
had either died or moulted to the adult stage (84 days). At each
assessment the insects are assessed as live or dead, and examined
for abnormalities. From day 46 onwards, once moulting to adult
commences, all insects (live and dead) are assessed as nyumph or
adult. Surviving insects are weighed on day 55 of the trial. Four
replicates are performed for each of the treatments. During the
trial the test conditions are 25 to 33.degree. C. and 20 to 25%
relative humidity, with a 12:12 hour light:dark photoperiod.
D. Cloning of a MLB Gene Fragment in a Vector Suitable for
Bacterial Production of Insect-Active Double-Stranded RNA
[0340] What follows is an example of cloning a DNA fragment
corresponding to an MLB gene target in a vector for the expression
of double-stranded RNA in a bacterial host, although any vector
comprising a T7 promoter or any other promoter for efficient
transcription in bacteria, may be used (reference to
WO0001846).
[0341] The sequences of the specific primers used for the
amplification of target genes are provided in Table 8. The template
used is the pCR8/GW/topo vector containing any of target sequences.
The primers are used in a PCR reaction with the following
conditions: 5 minutes at 98.degree. C., followed by 30 cycles of 10
seconds at 98.degree. C., 30 seconds at 55.degree. C. and 2 minutes
at 72.degree. C., followed by 10 minutes at 72.degree. C. The
resulting PCR fragment is analyzed on agarose gel, purified
(QIAquick Gel Extraction kit, Cat. Nr. 28706, Qiagen), blunt-end
cloned into Srf I-linearized pGNA49A vector (reference to
WO00188121A1), and sequenced. The sequence of the resulting PCR
product corresponds to the respective sequence as given in Table 8.
The recombinant vector harbouring this sequence is named
pGBNJ00XX.
E. Expression and Production of a Double-Stranded RNA Target in Two
Strains of Escherichia coli: (1) AB309-105, and, (2) BL21(DE3)
[0342] The procedures described below are followed in order to
express suitable levels of insect-active double-stranded RNA of
insect target in bacteria. An RNaseIII-deficient strain, AB309-105,
is used in comparison to wild-type RNaseIII-containing bacteria,
BL21(DE3).
Transformation of AB309-105 and BL21(DE3)
[0343] Three hundred ng of the plasmid are added to and gently
mixed in a 50 .mu.l aliquot of ice-chilled chemically competent E.
coli strain AB309-105 or BL21(DE3). The cells are incubated on ice
for 20 minutes before subjecting them to a heat shock treatment of
37.degree. C. for 5 minutes, after which the cells are placed back
on ice for a further 5 minutes. Four hundred and fifty .mu.l of
room temperature SOC medium is added to the cells and the
suspension incubated on a shaker (250 rpm) at 37.degree. C. for 1
hour. One hundred .mu.l of the bacterial cell suspension is
transferred to a 500 ml conical flask containing 150 ml of liquid
Luria-Bertani (LB) broth supplemented with 100 .mu.g/ml
carbenicillin antibiotic. The culture is incubated on an Innova
4430 shaker (250 rpm) at 37.degree. C. overnight (16 to 18
hours).
Chemical Induction of Double-Stranded RNA Expression in AB309-105
and BL21(DE3)
[0344] Expression of double-stranded RNA from the recombinant
vector, pGBNJ003, in the bacterial strain AB309-105 or BL21(DE3) is
made possible since all the genetic components for controlled
expression are present. In the presence of the chemical inducer
isopropylthiogalactoside, or IPTG, the T7 polymerase will drive the
transcription of the target sequence in both antisense and sense
directions since these are flanked by oppositely oriented T7
promoters.
[0345] The optical density at 600 nm of the overnight bacterial
culture is measured using an appropriate spectrophotometer and
adjusted to a value of 1 by the addition of fresh LB broth. Fifty
ml of this culture is transferred to a 50 ml Falcon tube and the
culture then centrifuged at 3000 g at 15.degree. C. for 10 minutes.
The supernatant is removed and the bacterial pellet resuspended in
50 ml of fresh S complete medium (SNC medium plus 5 .mu.g/ml
cholesterol) supplemented with 100 .mu.g/ml carbenicillin and 1 mM
IPTG. The bacteria are induced for 2 to 4 hours at room
temperature.
Heat Treatment of Bacteria
[0346] Bacteria are killed by heat treatment in order to minimise
the risk of contamination of the artificial diet in the test
plates. However, heat treatment of bacteria expressing
double-stranded RNA is not a prerequisite for inducing toxicity
towards the insects due to RNA interference. The induced bacterial
culture is centrifuged at 3000 g at room temperature for 10
minutes, the supernatant discarded and the pellet subjected to
80.degree. C. for 20 minutes in a water bath. After heat treatment,
the bacterial pellet is resuspended in 1.5 ml MilliQ water and the
suspension transferred to a microfuge tube. Several tubes are
prepared and used in the bioassays for each refreshment. The tubes
are stored at -20.degree. C. until further use.
F. Laboratory Trials to Test Escherichia coli Expressing dsRNA
Targets Against Anthonomus grandis
Plant-Based Bioassays
[0347] Whole plants are sprayed with suspensions of chemically
induced bacteria expressing dsRNA prior to feeding the plants to
CBW. The are grown from in a plant growth room chamber. The plants
are caged by placing a 500 ml plastic bottle upside down over the
plant with the neck of the bottle firmly placed in the soil in a
pot and the base cut open and covered with a fine nylon mesh to
permit aeration, reduce condensation inside and prevent insect
escape. CBW are placed on each treated plant in the cage. Plants
are treated with a suspension of E. coli AB309-105 harbouring the
pGBNJ001 plasmids or pGN29 plasmid. Different quantities of
bacteria are applied to the plants: for instance 66, 22, and 7
units, where one unit is defined as 10.sup.9 bacterial cells in 1
ml of a bacterial suspension at optical density value of 1 at 600
nm wavelength. In each case, a total volume of between 1 and 10 ml
sprayed on the plant with the aid of a vaporizer. One plant is used
per treatment in this trial. The number of survivors are counted
and the weight of each survivor recorded.
[0348] Spraying plants with a suspension of E. coli bacterial
strain AB309-105 expressing target dsRNA from pGBNJ003 lead to a
dramatic increase in insect mortality when compared to pGN29
control. These experiments show that double-stranded RNA
corresponding to an insect gene target sequence produced in either
wild-type or RNaseIII-deficient bacterial expression systems is
toxic towards the insect in terms of substantial increases in
insect mortality and growth/development delay for larval survivors.
It is also clear from these experiments that an exemplification is
provided for the effective protection of plants/crops from insect
damage by the use of a spray of a formulation consisting of
bacteria expressing double-stranded RNA corresponding to an insect
gene target.
Example 7
Tribolium castaneum (Red Flour Beetle)
A. Cloning Tribolium castaneum Partial Sequences
[0349] High quality, intact RNA was isolated from all the different
insect stages of Tribolium castaneum (red flour beetle; source: Dr.
Lara Senior, Insect Investigations Ltd., Capital Business Park,
Wentloog, Cardiff, CF3 2PX, Wales, UK) using TRIzol Reagent (Cat.
Nr. 15596-026/15596-018, Invitrogen, Rockville, Md., USA) following
the manufacturer's instructions. Genomic DNA present in the RNA
preparation was removed by DNase treatment following the
manafacturer's instructions (Cat. Nr. 1700, Promega). cDNA was
generated using a commercially available kit (SuperScript.TM. III
Reverse Transcriptase, Cat. Nr. 18080044, Invitrogen, Rockville,
Md., USA) following the manufacturer's instructions.
[0350] To isolate cDNA sequences comprising a portion of the TC001,
TC002, TC010, TC014 and TC015 genes, a series of PCR reactions with
degenerate primers were performed using Amplitaq Gold (Cat. Nr.
N8080240, Applied Biosystems) following the manafacturer's
instructions.
[0351] The sequences of the degenerate primers used for
amplification of each of the genes are given in Table 2-TC. These
primers were used in respective PCR reactions with the following
conditions: 10 minutes at 95.degree. C., followed by 40 cycles of
30 seconds at 95.degree. C., 1 minute at 50.degree. C. and 1 minute
and 30 seconds at 72.degree. C., followed by 7 minutes at
72.degree. C. (TC001, TC014, TC015); 10 minutes at 95.degree. C.,
followed by 40 cycles of 30 seconds at 95.degree. C., 1 minute at
54.degree. C. and 2 minutes and 30 seconds at 72.degree. C.,
followed by 7 minutes at 72.degree. C. (TC010); 10 minutes at
95.degree. C., followed by 40 cycles of 30 seconds at 95.degree.
C., 1 minute at 53.degree. C. and 1 minute at 72.degree. C.,
followed by 7 minutes at 72.degree. C. (TC002). The resulting PCR
fragments were analyzed on agarose gel, purified (QIAquick Gel
Extraction kit, Cat. Nr. 28706, Qiagen), cloned into the
pCR8/GW/TOPO vector (Cat. Nr. K2500-20, Invitrogen), and sequenced.
The sequences of the resulting PCR products are represented by the
respective SEQ ID NO:s as given in Table 2-TC and are referred to
as the partial sequences. The corresponding partial amino acid
sequences are represented by the respective SEQ ID NO:s as given in
Table 3-TC.
B. dsRNA Production of the Tribolium castaneum Genes
[0352] dsRNA was synthesized in milligram amounts using the
commercially available kit T7 Ribomax.TM. Express RNAi System (Cat.
Nr. P1700, Promega). First two separate single 5' T7 RNA polymerase
promoter templates were generated in two separate PCR reactions,
each reaction containing the target sequence in a different
orientation relative to the T7 promoter.
[0353] For each of the target genes, the sense T7 template was
generated using specific T7 forward and specific reverse primers.
The sequences of the respective primers for amplifying the sense
template for each of the target genes are given in Table 8-TC. The
conditions in the PCR reactions were as follows: 1 minute at
95.degree. C., followed by 20 cycles of 30 seconds at 95.degree.
C., 30 seconds at 60.degree. C. (-0.5.degree. C./cycle) and 1
minute at 72.degree. C., followed by 15 cycles of 30 seconds at
95.degree. C., 30 seconds at 50.degree. C. and 1 minute at
72.degree. C., followed by 10 minutes at 72.degree. C. The
anti-sense T7 template was generated using specific forward and
specific T7 reverse primers in a PCR reaction with the same
conditions as described above. The sequences of the respective
primers for amplifying the anti-sense template for each of the
target genes are given in Table 8-TC. The resulting PCR products
were analyzed on agarose gel and purified by PCR purification kit
(Qiaquick PCR Purification Kit, Cat. Nr. 28106, Qiagen) and
NaClO.sub.4 precipitation. The generated T7 forward and reverse
templates were mixed to be transcribed and the resulting RNA
strands were annealed, DNase and RNase treated, and purified by
sodium acetate, following the manufacturer's instructions. The
sense strand of the resulting dsRNA for each of the target genes is
given in Table 8-TC.
C. Laboratory Trials to Test dsRNA Targets, Using Artificial Diet
for Activity Against Tribolium castaneum Larvae
[0354] The example provided below is an exemplification of the
finding that the red flour beetle (RFB) larvae are susceptible to
orally ingested dsRNA corresponding to own target genes.
[0355] Red flour beetles, Tribolium castaneum, were maintained at
Insect Investigations Ltd. (origin: Imperial College of Science,
Technology and Medicine, Silwood Park, Berkshire, UK). Insects were
cultured according to company SOP/251/01. Briefly, the beetles were
housed in plastic jars or tanks. These have an open top to allow
ventilation. A piece of netting was fitted over the top and secured
with an elastic band to prevent escape. The larval rearing medium
(flour) was placed in the container where the beetles can breed.
The stored product beetle colonies were maintained in a controlled
temperature room at 25.+-.3.degree. C. with a 16:8 hour light:dark
cycle.
[0356] Double-stranded RNA from target TC014 (with sequence
corresponding to SEQ ID NO: -799) was incorporated into a mixture
of flour and milk powder (wholemeal flour: powdered milk in the
ratio 4:1) and left to dry overnight. Each replicate was prepared
separately: 100 .mu.A of a 10 .mu.g/.mu.1 dsRNA solution (1 mg
dsRNA) was added to 0.1 g flour/milk mixture. The dried mixture was
ground to a fine powder. Insects were maintained within Petri
dishes (55 mm diameter), lined with a double layer of filter paper.
The treated diet was placed between the two filter paper layers.
Ten first instar, mixed sex larvae were placed in each dish
(replicate). Four replicates were performed for each treatment.
Control was Milli-Q water. Assessments (number of survivors) were
made on a regular basis. During the trial, the test conditions were
25-33.degree. C. and 20-25% relative humidity, with a 12:12 hour
light:dark photoperiod.
[0357] Survival of larvae of T. castaneum over time on artificial
diet treated with target TC014 dsRNA was significantly reduced when
compared to diet only control, as shown in FIG. 1.
D. Cloning of a RFB Gene Fragment in a Vector Suitable for
Bacterial Production of Insect-Active Double-Stranded RNA
[0358] What follows is an example of cloning a DNA fragment
corresponding to an RFB gene target in a vector for the expression
of double-stranded RNA in a bacterial host, although any vector
comprising a T7 promoter or any other promoter for efficient
transcription in bacteria, may be used (reference to
WO0001846).
[0359] The sequences of the specific primers used for the
amplification of target genes are provided in Table 8-TC. The
template used is the pCR8/GW/topo vector containing any of target
sequences. The primers are used in a PCR reaction with the
following conditions: 5 minutes at 98.degree. C., followed by 30
cycles of 10 seconds at 98.degree. C., 30 seconds at 55.degree. C.
and 2 minutes at 72.degree. C., followed by 10 minutes at
72.degree. C. The resulting PCR fragment is analyzed on agarose
gel, purified (QIAquick Gel Extraction kit, Cat. Nr. 28706,
Qiagen), blunt-end cloned into Srf I-linearized pGNA49A vector
(reference to WO00188121A1), and sequenced. The sequence of the
resulting PCR product corresponds to the respective sequence as
given in Table 8-TC. The recombinant vector harbouring this
sequence is named pGBNJ00 XX.
E. Expression and Production of a Double-Stranded RNA Target in Two
Strains of Escherichia coli: (1) AB309-105, and, (2) BL21(DE3)
[0360] The procedures described below are followed in order to
express suitable levels of insect-active double-stranded RNA of
insect target in bacteria. An RNaseIII-deficient strain, AB309-105,
is used in comparison to wild-type RNaseIII-containing bacteria,
BL21(DE3).
Transformation of AB309-105 and BL21(DE3)
[0361] Three hundred ng of the plasmid are added to and gently
mixed in a 50 .mu.l aliquot of ice-chilled chemically competent E.
coli strain AB309-105 or BL21(DE3). The cells are incubated on ice
for 20 minutes before subjecting them to a heat shock treatment of
37.degree. C. for 5 minutes, after which the cells are placed back
on ice for a further 5 minutes. Four hundred and fifty .mu.l of
room temperature SOC medium is added to the cells and the
suspension incubated on a shaker (250 rpm) at 37.degree. C. for 1
hour. One hundred .mu.l of the bacterial cell suspension is
transferred to a 500 ml conical flask containing 150 ml of liquid
Luria-Bertani (LB) broth supplemented with 100 .mu.g/ml
carbenicillin antibiotic. The culture is incubated on an Innova
4430 shaker (250 rpm) at 37.degree. C. overnight (16 to 18
hours).
Chemical Induction of Double-Stranded RNA Expression in AB309-105
and BL21(DE3)
[0362] Expression of double-stranded RNA from the recombinant
vector, pGBNJ003, in the bacterial strain AB309-105 or BL21(DE3) is
made possible since all the genetic components for controlled
expression are present. In the presence of the chemical inducer
isopropylthiogalactoside, or IPTG, the T7 polymerase will drive the
transcription of the target sequence in both antisense and sense
directions since these are flanked by oppositely oriented T7
promoters.
[0363] The optical density at 600 nm of the overnight bacterial
culture is measured using an appropriate spectrophotometer and
adjusted to a value of 1 by the addition of fresh LB broth. Fifty
ml of this culture is transferred to a 50 ml Falcon tube and the
culture then centrifuged at 3000 g at 15.degree. C. for 10 minutes.
The supernatant is removed and the bacterial pellet resuspended in
50 ml of fresh S complete medium (SNC medium plus 5 .mu.g/ml
cholesterol) supplemented with 100 .mu.g/ml carbenicillin and 1 mM
IPTG. The bacteria are induced for 2 to 4 hours at room
temperature.
Heat Treatment of Bacteria
[0364] Bacteria are killed by heat treatment in order to minimise
the risk of contamination of the artificial diet in the test
plates. However, heat treatment of bacteria expressing
double-stranded RNA is not a prerequisite for inducing toxicity
towards the insects due to RNA interference. The induced bacterial
culture is centrifuged at 3000 g at room temperature for 10
minutes, the supernatant discarded and the pellet subjected to
80.degree. C. for 20 minutes in a water bath. After heat treatment,
the bacterial pellet is resuspended in 1.5 ml MilliQ water and the
suspension transferred to a microfuge tube. Several tubes are
prepared and used in the bioassays for each refreshment. The tubes
are stored at -20.degree. C. until further use.
F. Laboratory Trials to Test Escherichia coli Expressing dsRNA
Targets Against Tribolium castaneum
Plant-Based Bioassays
[0365] Whole plants are sprayed with suspensions of chemically
induced bacteria expressing dsRNA prior to feeding the plants to
RFB. The are grown from in a plant growth room chamber. The plants
are caged by placing a 500 ml plastic bottle upside down over the
plant with the neck of the bottle firmly placed in the soil in a
pot and the base cut open and covered with a fine nylon mesh to
permit aeration, reduce condensation inside and prevent insect
escape. RFB are placed on each treated plant in the cage. Plants
are treated with a suspension of E. coli AB309-105 harbouring the
pGBNJ001 plasmids or pGN29 plasmid. Different quantities of
bacteria are applied to the plants: for instance 66, 22, and 7
units, where one unit is defined as 10.sup.9 bacterial cells in 1
ml of a bacterial suspension at optical density value of 1 at 600
nm wavelength. In each case, a total volume of between 1 and 10 ml
s sprayed on the plant with the aid of a vaporizer. One plant is
used per treatment in this trial. The number of survivors are
counted and the weight of each survivor recorded.
[0366] Spraying plants with a suspension of E. coli bacterial
strain AB309-105 expressing target dsRNA from pGBNJ003 leed to a
dramatic increase in insect mortality when compared to pGN29
control. These experiments show that double-stranded RNA
corresponding to an insect gene target sequence produced in either
wild-type or RNaseIII-deficient bacterial expression systems is
toxic towards the insect in terms of substantial increases in
insect mortality and growth/development delay for larval survivors.
It is also clear from these experiments that an exemplification is
provided for the effective protection of plants/crops from insect
damage by the use of a spray of a formulation consisting of
bacteria expressing double-stranded RNA corresponding to an insect
gene target.
Example 10
Myzus persicae (Green Peach Aphid)
a. Cloning Myzus persicae Partial Sequences
[0367] High quality, intact RNA was isolated from nymphs of Myzus
persicae (green peach aphid; source: Dr. Rachel Down, Insect &
Pathogen Interactions, Central Science Laboratory, Sand Hutton,
York, YO41 1LZ, UK) using TRIzol Reagent (Cat. Nr.
15596-026/15596-018, Invitrogen, Rockville, Md., USA) following the
manufacturer's instructions. Genomic DNA present in the RNA
preparation was removed by DNase treatment following the
manafacturer's instructions (Cat. Nr. 1700, Promega). cDNA was
generated using a commercially available kit (SuperScript.TM. III
Reverse Transcriptase, Cat. Nr. 18080044, Invitrogen, Rockville,
Md., USA) following the manufacturer's instructions.
[0368] To isolate cDNA sequences comprising a portion of the MP001,
MP002, MP010, MP016 and MP027 genes, a series of PCR reactions with
degenerate primers were performed using Amplitaq Gold (Cat. Nr.
N8080240, Applied Biosystems) following the manafacturer's
instructions.
[0369] The sequences of the degenerate primers used for
amplification of each of the genes are given in Table 2-MP. These
primers were used in respective PCR reactions with the following
conditions: for MP001, MP002 and MP016, 10 minutes at 95.degree.
C., followed by 40 cycles of 30 seconds at 95.degree. C., 1 minute
at 50.degree. C. and 1 minute 30 seconds at 72.degree. C., followed
by 7 minutes at 72.degree. C.; for MP027, a touchdown program was
used: 10 minutes at 95.degree. C., followed by 10 cycles of 30
seconds at 95.degree. C., 40 seconds at 60.degree. C. with a
decrease in temperature of 1.degree. C. per cycle and 1 minute 10
seconds at 72.degree. C., followed by 30 cycles of 30 seconds at
95.degree. C., 40 seconds at 50.degree. C. and 1 minute 10 seconds
at 72.degree. C., followed by 7 minutes at 72.degree. C.; for
MP010, 10 minutes at 95.degree. C., followed by 40 cycles of 30
seconds at 95.degree. C., 1 minute at 54.degree. C. and 3 minutes
at 72.degree. C., followed by 7 minutes at 72.degree. C. The
resulting PCR fragments were analyzed on agarose gel, purified
(QIAquick Gel Extraction kit, Cat. Nr. 28706, Qiagen), cloned into
the pCR8/GW/TOPO vector (Cat. Nr. K2500-20, Invitrogen), and
sequenced. The sequences of the resulting PCR products are
represented by the respective SEQ ID NO:s as given in Table 2-MP
and are referred to as the partial sequences. The corresponding
partial amino acid sequences are represented by the respective SEQ
ID NO:s as given in Table 3-MP.
B. dsRNA Production of Myzus persicae Genes
[0370] dsRNA was synthesized in milligram amounts using the
commercially available kit T7 Ribomax.TM. Express RNAi System (Cat.
Nr. P1700, Promega). First two separate single 5' T7 RNA polymerase
promoter templates were generated in two separate PCR reactions,
each reaction containing the target sequence in a different
orientation relative to the T7 promoter.
[0371] For each of the target genes, the sense T7 template was
generated using specific T7 forward and specific reverse primers.
The sequences of the respective primers for amplifying the sense
template for each of the target genes are given in Table 8-MP. A
touchdown PCR was performed as follows: 1 minute at 95.degree. C.,
followed by 20 cycles of 30 seconds at 95.degree. C., 30 seconds at
55.degree. C. (for MP001, MP002, MP016, MP027 and gfp) or 30
seconds at 50.degree. C. (for MP010) with a decrease in temperature
of 0.5.degree. C. per cycle and 1 minute at 72.degree. C., followed
by 15 cycles of 30 seconds at 95.degree. C., 30 seconds at
45.degree. C. and 1 minute at 72.degree. C. followed by 10 minutes
at 72.degree. C. The anti-sense T7 template was generated using
specific forward and specific T7 reverse primers in a PCR reaction
with the same conditions as described above. The sequences of the
respective primers for amplifying the anti-sense template for each
of the target genes are given in Table 8-MP. The resulting PCR
products were analyzed on agarose gel and purified by PCR
purification kit (Qiaquick PCR Purification Kit, Cat. Nr. 28106,
Qiagen) and NaClO.sub.4 precipitation. The generated T7 forward and
reverse templates were mixed to be transcribed and the resulting
RNA strands were annealed, DNase and RNase treated, and purified by
sodium acetate, following the manufacturer's instructions. The
sense strand of the resulting dsRNA for each of the target genes is
given in Table 8-MP.
C. Laboratory Trials to Test dsRNA Targets Using Liquid Artificial
Diet for Activity Against Myzus persicae
[0372] Liquid artificial diet for the green peach aphid, Myzus
persicae, was prepared based on the diet suitable for pea aphids
(Acyrthosiphon pisum), as described by Febvay et al. (1988)
[Influence of the amino acid balance on the improvement of an
artificial diet for a biotype of Acyrthosiphon pisum (Homoptera:
Aphididae). Can. J. Zool. 66: 2449-2453], but with some
modifications. The amino acids component of the diet was prepared
as follows: in mg/100 ml, alanine 178.71, beta-alanine 6.22,
arginine 244.9, asparagine 298.55, aspartic acid 88.25, cysteine
29.59, glutamic acid 149.36, glutamine 445.61, glycine 166.56,
histidine 136.02, isoleucine 164.75, leucine 231.56, lysine
hydrochloride 351.09, methionine 72.35, ornithine (HCl) 9.41,
phenylalanine 293, proline 129.33, serine 124.28, threonine 127.16,
tryptophane 42.75, tyrosine 38.63, L-valine 190.85. The amino acids
were dissolved in 30 ml Milli-Q H.sub.2O except for tyrosine which
was first dissolved in a few drops of 1 M HCl before adding to the
amino acid mix. The vitamin mix component of the diet was prepared
as a 5.times. concentrate stock as follows: in mg/L, amino benzoic
acid 100, ascorbic acid 1000, biotin 1, calcium panthothenate 50,
choline chloride 500, folic acid 10, myoinositol 420, nicotinic
acid 100, pyridoxine hydrochloride 25, riboflavin 5, thiamine
hydrochloride 25. The riboflavin was dissolved in 1 ml H2O at
50.degree. C. and then added to the vitamin mix stock. The vitamin
mix was aliquoted in 20 ml per aliquot and stored at -20.degree. C.
One aliquot of vitamin mix was added to the amino acid solution.
Sucrose and MgSO.sub.4.7H.sub.2O was added with the following
amounts to the mix: 20 g and 242 mg, respectively. Trace metal
stock solution was prepared as follows: in mg/100 ml,
CuSO.sub.4.5H.sub.2O 4.7, FeCl.sub.3.6H.sub.2O 44.5,
MnCl.sub.2.4H2O 6.5, NaCl 25.4, ZnCl.sub.2 8.3. Ten ml of the trace
metal solution and 250 mg KH.sub.2PO.sub.4 was added to the diet
and Milli-Q water was added to a final liquid diet volume of 100
ml. The pH of the diet was adjusted to 7 with 1 M KOH solution. The
liquid diet was filter-sterilised through an 0.22 .mu.m filter disc
(Millipore).
[0373] Green peach aphids (Myzus persicae; source: Dr. Rachel Down,
Insect & Pathogen Interactions, Central Science Laboratory,
Sand Hutton, York, YO41 1LZ, UK) were reared on 4- to 6-week-old
oilseed rape (Brassica napus variety SW Oban; source: Nick Balaam,
Sw Seed Ltd., 49 North Road, Abington, Cambridge, CB1 6AS, UK) in
aluminium-framed cages containing 70 .mu.m mesh in a controlled
environment chamber with the following conditions: 23.+-.2.degree.
C. and 60.+-.5% relative humidity, with a 16:8 hours light:dark
photoperiod.
[0374] One day prior to the start of the bioassay, adults were
collected from the rearing cages and placed on fresh detached
oilseed rape leaves in a Petri dish and left overnight in the
insect chamber. The following day, first-instar nymphs were picked
and transferred to feeding chambers. A feeding chamber comprised of
10 first instar nymphs placed in a small Petri dish (with diameter
3 cm) covered with a single layer of thinly stretched parafilm M
onto which 50 .mu.l of diet was added. The chamber was sealed with
a second layer of parafilm and incubated under the same conditions
as the adult cultures. Diet with dsRNA was refreshed every other
day and the insects' survival assessed on day 8 i.e. 8.sup.th day
post bioassay start. Per treatment, 5 bioassay feeding chambers
(replicates) were set up simultaneously. Test and control (gfp)
dsRNA solutions were incorporated into the diet to a final
concentration of 2 .mu.g411. The feeding chambers were kept at
23.+-.2.degree. C. and 60.+-.5% relative humidity, with a 16:8
hours light:dark photoperiod. A Mann-Whitney test was determined by
GraphPad Prism version 4 to establish whether the medians do differ
significantly between target 27 (MP027) and gfp dsRNA.
[0375] In the bioassay, feeding liquid artificial diet supplemented
with intact naked dsRNA from target 27 (SEQ ID NO: 1061) to nymphs
of Myzus persicae using a feeding chamber, resulted in a
significant increase in mortality, as shown in FIG. 1. Average
percentage survivors for target 27, gfp dsRNA and diet only
treatment were 2, 34 and 82, respectively. Comparison of target 027
with gfp dsRNA groups using the Mann-Whitney test resulted in an
one-tailed P-value of 0.004 which indicates that the median of
target 027 is significantly different (P<0.05) from the expected
larger median of gfp dsRNA. The green peach aphids on the liquid
diet with incorporated target 27 dsRNA were noticeably smaller than
those that were fed on diet only or with gfp dsRNA control (data
not presented).
D. Cloning of a GPA Gene Fragment in a Vector Suitable for
Bacterial Production of Insect-Active Double-Stranded RNA
[0376] What follows is an example of cloning a DNA fragment
corresponding to a GPA gene target in a vector for the expression
of double-stranded RNA in a bacterial host, although any vector
comprising a T7 promoter or any other promoter for efficient
transcription in bacteria, may be used (reference to
WO0001846).
[0377] The sequences of the specific primers used for the
amplification of target genes are provided in Table 8-MP. The
template used is the pCR8/GW/topo vector containing any of target
sequences. The primers are used in a PCR reaction with the
following conditions: 5 minutes at 98.degree. C., followed by 30
cycles of 10 seconds at 98.degree. C., 30 seconds at 55.degree. C.
and 2 minutes at 72.degree. C., followed by 10 minutes at
72.degree. C. The resulting PCR fragment is analyzed on agarose
gel, purified (QIAquick Gel Extraction kit, Cat. Nr. 28706,
Qiagen), blunt-end cloned into Srf I-linearized pGNA49A vector
(reference to WO00188121A1), and sequenced. The sequence of the
resulting PCR product corresponds to the respective sequence as
given in Table 8-MP. The recombinant vector harbouring this
sequence is named pGBNJ00XX.
E. Expression and Production of a Double-Stranded RNA Target in Two
Strains of Escherichia coli: (1) AB309-105, and, (2) BL21(DE3)
[0378] The procedures described below are followed in order to
express suitable levels of insect-active double-stranded RNA of
insect target in bacteria. An RNaseIII-deficient strain, AB309-105,
is used in comparison to wild-type RNaseIII-containing bacteria,
BL21(DE3).
Transformation of AB309-105 and BL21(DE3)
[0379] Three hundred ng of the plasmid are added to and gently
mixed in a 50 .mu.A aliquot of ice-chilled chemically competent E.
coli strain AB309-105 or BL21(DE3). The cells are incubated on ice
for 20 minutes before subjecting them to a heat shock treatment of
37.degree. C. for 5 minutes, after which the cells are placed back
on ice for a further 5 minutes. Four hundred and fifty .mu.l of
room temperature SOC medium is added to the cells and the
suspension incubated on a shaker (250 rpm) at 37.degree. C. for 1
hour. One hundred .mu.l of the bacterial cell suspension is
transferred to a 500 ml conical flask containing 150 ml of liquid
Luria-Bertani (LB) broth supplemented with 100 .mu.g/ml
carbenicillin antibiotic. The culture is incubated on an Innova
4430 shaker (250 rpm) at 37.degree. C. overnight (16 to 18
hours).
Chemical Induction of Double-Stranded RNA Expression in AB309-105
and BL21(DE3)
[0380] Expression of double-stranded RNA from the recombinant
vector, pGBNJ003, in the bacterial strain AB309-105 or BL21(DE3) is
made possible since all the genetic components for controlled
expression are present. In the presence of the chemical inducer
isopropylthiogalactoside, or IPTG, the T7 polymerase will drive the
transcription of the target sequence in both antisense and sense
directions since these are flanked by oppositely oriented T7
promoters.
[0381] The optical density at 600 nm of the overnight bacterial
culture is measured using an appropriate spectrophotometer and
adjusted to a value of 1 by the addition of fresh LB broth. Fifty
ml of this culture is transferred to a 50 ml Falcon tube and the
culture then centrifuged at 3000 g at 15.degree. C. for 10 minutes.
The supernatant is removed and the bacterial pellet resuspended in
50 ml of fresh S complete medium (SNC medium plus 5 .mu.g/ml
cholesterol) supplemented with 100 .mu.g/ml carbenicillin and 1 mM
IPTG. The bacteria are induced for 2 to 4 hours at room
temperature.
Heat Treatment of Bacteria
[0382] Bacteria are killed by heat treatment in order to minimise
the risk of contamination of the artificial diet in the test
plates. However, heat treatment of bacteria expressing
double-stranded RNA is not a prerequisite for inducing toxicity
towards the insects due to RNA interference. The induced bacterial
culture is centrifuged at 3000 g at room temperature for 10
minutes, the supernatant discarded and the pellet subjected to
80.degree. C. for 20 minutes in a water bath. After heat treatment,
the bacterial pellet is resuspended in 1.5 ml MilliQ water and the
suspension transferred to a microfuge tube. Several tubes are
prepared and used in the bioassays for each refreshment. The tubes
are stored at -20.degree. C. until further use.
F. Laboratory Trials to Test Escherichia coli Expressing dsRNA
Targets Against Myzus persicae
Plant-Based Bioassays
[0383] Whole plants are sprayed with suspensions of chemically
induced bacteria expressing dsRNA prior to feeding the plants to
GPA. The are grown from in a plant growth room chamber. The plants
are caged by placing a 500 ml plastic bottle upside down over the
plant with the neck of the bottle firmly placed in the soil in a
pot and the base cut open and covered with a fine nylon mesh to
permit aeration, reduce condensation inside and prevent insect
escape. GPA are placed on each treated plant in the cage. Plants
are treated with a suspension of E. coli AB309-105 harbouring the
pGBNJ001 plasmids or pGN29 plasmid. Different quantities of
bacteria are applied to the plants: for instance 66, 22, and 7
units, where one unit is defined as 10.sup.9 bacterial cells in 1
ml of a bacterial suspension at optical density value of 1 at 600
nm wavelength. In each case, a total volume of between 1 and 10 ml
s sprayed on the plant with the aid of a vaporizer. One plant is
used per treatment in this trial. The number of survivors are
counted and the weight of each survivor recorded.
[0384] Spraying plants with a suspension of E. coli bacterial
strain AB309-105 expressing target dsRNA from pGBNJ003 lead to a
dramatic increase in insect mortality when compared to pGN29
control. These experiments show that double-stranded RNA
corresponding to an insect gene target sequence produced in either
wild-type or RNaseIII-deficient bacterial expression systems is
toxic towards the insect in terms of substantial increases in
insect mortality and growth/development delay for larval survivors.
It is also clear from these experiments that an exemplification is
provided for the effective protection of plants/crops from insect
damage by the use of a spray of a formulation consisting of
bacteria expressing double-stranded RNA corresponding to an insect
gene target.
Example 11
Nilaparvata lugens (Brown Plant Hopper)
A. Cloning Nilaparvata lugens Partial Sequences
[0385] From high quality total RNA of Nilaparvata lugens (source:
Dr. J. A. Gatehouse, Dept. Biological Sciences, Durham University,
UK) cDNA was generated using a commercially available kit
(SuperScript.TM. III Reverse Transcriptase, Cat N.sup.o. 18080044,
Invitrogen, Rockville, Md., USA) following the manufacturer's
protocol.
[0386] To isolate cDNA sequences comprising a portion of the
Nilaparvata lugens NL001, NL002, NL003, NL004, NL005, NL006, NL007,
NL008, NL009, NL010, NL011, NL012, NL013, NL014, NL015, NL016,
NL018, NL019, NL021, NL022, and NL027 genes, a series of PCR
reactions with degenerate primers were performed using Amplitaq
Gold (Cat N.sup.o. N8080240; Applied Biosystems) following the
manufacturer's protocol.
[0387] The sequences of the degenerate primers used for
amplification of each of the genes are given in Table 2-NL. These
primers were used in respective PCR reactions with the following
conditions: for NL001: 5 minutes at 95.degree. C., followed by 40
cycles of 30 seconds at 95.degree. C., 1 minute at 55.degree. C.
and 1 minute at 72.degree. C., followed by 10 minutes at 72.degree.
C.: for NL002: 3 minutes at 95.degree. C., followed by 40 cycles of
30 seconds at 95.degree. C., 1 minute at 55.degree. C. and 1 minute
at 72.degree. C., followed by 10 minutes at 72.degree. C.; for
NL003: 3 minutes at 95.degree. C., followed by 40 cycles of 30
seconds at 95.degree. C., 1 minute at 61.degree. C. and 1 minute at
72.degree. C., followed by 10 minutes at 72.degree. C.; for NL004:
10 minutes at 95.degree. C., followed by 40 cycles of 30 seconds at
95.degree. C., 1 minute at 51.degree. C. and 1 minute at 72.degree.
C.; for NL005: 10 minutes at 95.degree. C., followed by 40 cycles
of 30 seconds at 95.degree. C., 1 minute at 54.degree. C. and 1
minute at 72.degree. C., followed by 10 minutes at 72.degree. C.;
for NL006: 10 minutes at 95.degree. C., followed by 40 cycles of 30
seconds at 95.degree. C., 1 minute at 55.degree. C. and 3 minute 30
seconds at 72.degree. C., followed by 10 minutes at 72.degree. C.;
for NL007: 10 minutes at 95.degree. C., followed by 40 cycles of 30
seconds at 95.degree. C., 1 minute at 54.degree. C. and 1 minute 15
seconds at 72.degree. C., followed by 10 minutes at 72.degree. C.;
for NL008: 10 minutes at 95.degree. C., followed by 40 cycles of 30
seconds at 95.degree. C., 1 minute at 53.degree. C. and 1 minute at
72.degree. C., followed by 10 minutes at 72.degree. C.; for NL009:
10 minutes at 95.degree. C., followed by 40 cycles of 30 seconds at
95.degree. C., 1 minute at 55.degree. C. and 1 minute at 72.degree.
C., followed by 10 minutes at 72.degree. C.; for NL010: 10 minutes
at 95.degree. C., followed by 40 cycles of 30 seconds at 95.degree.
C., 1 minute at 54.degree. C. and 2 minute 30 seconds at 72.degree.
C., followed by 10 minutes at 72.degree. C.; for NL011: 10 minutes
at 95.degree. C., followed by 40 cycles of 30 seconds at 95.degree.
C., 1 minute at 55.degree. C. and 1 minute at 72.degree. C.; for
NL012: 10 minutes at 95.degree. C., followed by 40 cycles of 30
seconds at 95.degree. C., 1 minute at 55.degree. C. and 1 minute at
72.degree. C.; for NL013: 10 minutes at 95.degree. C., followed by
40 cycles of 30 seconds at 95.degree. C., 1 minute at 54.degree. C.
and 1 minute 10 seconds at 72.degree. C., followed by 10 minutes at
72.degree. C.; for NL014: 10 minutes at 95.degree. C., followed by
40 cycles of 30 seconds at 95.degree. C., 1 minute at 53.degree. C.
and 1 minute at 72.degree. C., followed by 10 minutes at 72.degree.
C.; for NL015: 10 minutes at 95.degree. C., followed by 40 cycles
of 30 seconds at 95.degree. C., 1 minute at 54.degree. C. and 1
minute 40 seconds at 72.degree. C., followed by 10 minutes at
72.degree. C.; for NL016: 10 minutes at 95.degree. C., followed by
40 cycles of 30 seconds at 95.degree. C., 1 minute at 54.degree. C.
and 1 minute 40 seconds at 72.degree. C., followed by 10 minutes at
72.degree. C.; for NL018: 10 minutes at 95.degree. C., followed by
40 cycles of 30 seconds at 95.degree. C., 1 minute at 54.degree. C.
and 1 minute 35 seconds at 72.degree. C., followed by 10 minutes at
72.degree. C.; for NL019: 10 minutes at 95.degree. C., followed by
40 cycles of 30 seconds at 95.degree. C., 1 minute at 55.degree. C.
and 1 minute at 72.degree. C., followed by 10 minutes at 72.degree.
C.; for NL021: 10 minutes at 95.degree. C., followed by 40 cycles
of 30 seconds at 95.degree. C., 1 minute at 54.degree. C. and 1
minute 45 seconds at 72.degree. C., followed by 10 minutes at
72.degree. C.: for NL022: 10 minutes at 95.degree. C., followed by
40 cycles of 30 seconds at 95.degree. C., 1 minute at 54.degree. C.
and 1 minute 45 seconds at 72.degree. C., followed by 10 minutes at
72.degree. C.; and for NL027: 10 minutes at 95.degree. C., followed
by 40 cycles of 30 seconds at 95.degree. C., 1 minute at 54.degree.
C. and 1 minute 45 seconds at 72.degree. C., followed by 10 minutes
at 72.degree. C. The resulting PCR fragments were analyzed on
agarose gel, purified (QIAquick Gel Extraction kit, Cat. Nr. 28706,
Qiagen), cloned into the pCR8/GW/topo vector (Cat. Nr. K2500 20,
Invitrogen), and sequenced. The sequences of the resulting PCR
products are represented by the respective SEQ ID NO:s as given in
Table 2-NL and are referred to as the partial sequences. The
corresponding partial amino acid sequences are represented by the
respective SEQ ID NO:s as given in Table 3-NL.
B. Cloning of a Partial Sequence of the Nilaparvata lugens NL023
Gene Via EST Sequence
[0388] From high quality total RNA of Nilaparvata lugens (source:
Dr. J. A. Gatehouse, Dept. Biological Sciences, Durham University,
UK) cDNA was generated using a commercially available kit
(SuperScript.TM. III Reverse Transcriptase, Cat N.sup.o. 18080044,
Invitrogen, Rockville, Md., USA) following the manufacturer's
protocol.
[0389] A partial cDNA sequence, NL023, was amplified from
Nilaparvata lugens cDNA which corresponded to a Nilaparvata lugens
EST sequence in the public database Genbank with accession number
CAH65679.2. To isolate cDNA sequences comprising a portion of the
NL023 gene, a series of PCR reactions with EST based specific
primers were performed using PerfectShot.TM. ExTaq (Cat N.sup.o.
RROO5A, Takara Bio Inc.) following the manafacturer's protocol.
[0390] For NL023, the specific primers oGBKWO02 and oGBKWO03
(represented herein as SEQ ID NO: 1157 and SEQ ID NO: 1158,
respectively) were used in two independent PCR reactions with the
following conditions: 3 minutes at 95.degree. C., followed by 30
cycles of 30 seconds at 95.degree. C., 30 seconds at 56.degree. C.
and 2 minutes at 72.degree. C., followed by 10 minutes at
72.degree. C. The resulting PCR products were analyzed on agarose
gel, purified (QIAquick.RTM. Gel Extraction Kit; Cat. N.sup.o.
28706, Qiagen), cloned into the pCR4-TOPO vector (Cat N.sup.o.
K4575-40, Invitrogen) and sequenced. The consensus sequence
resulting from the sequencing of both PCR products is herein
represented by SEQ ID NO: 1111 and is referred to as the partial
sequence of the NL023 gene. The corresponding partial amino acid
sequence is herein reperesented as SEQ ID NO: 1112.
C. dsRNA Production of Nilaparvata lugens Genes
[0391] dsRNA was synthesized in milligram amounts using the
commercially available kit T7 Ribomax.TM. Express RNAi System (Cat.
Nr. P1700, Promega). First two separate single 5' T7 RNA polymerase
promoter templates were generated in two separate PCR reactions,
each reaction containing the target sequence in a different
orientation relative to the T7 promoter.
[0392] For each of the target genes, the sense T7 template was
generated using specific T7 forward and specific reverse primers.
The sequences of the respective primers for amplifying the sense
template for each of the target genes are given in Table 4. The
conditions in the PCR reactions were as follows: for NL001: 4
minutes at 94.degree. C., followed by 35 cycles of 30 seconds at
94.degree. C., 30 seconds at 60.degree. C. and 1 minute at
72.degree. C., followed by 10 minutes at 72.degree. C.; for NL002:
4 minutes at 94.degree. C., followed by 35 cycles of 30 seconds at
94.degree. C., 30 seconds at 60.degree. C. and 1 minute at
72.degree. C., followed by 10 minutes at 72.degree. C.; for NL003:
4 minutes at 94.degree. C., followed by 35 cycles of 30 seconds at
94.degree. C., 30 seconds at 66.degree. C. and 1 minute at
72.degree. C., followed by 10 minutes at 72.degree. C.; for NL004:
4 minutes at 95.degree. C., followed by 35 cycles of 30 seconds at
95.degree. C., 30 seconds at 54.degree. C. and 1 minute at
72.degree. C., followed by 10 minutes at 72.degree. C.; for NL005:
4 minutes at 95.degree. C., followed by 35 cycles of 30 seconds at
95.degree. C., 30 seconds at 57.degree. C. and 1 minute at
72.degree. C., followed by 10 minutes at 72.degree. C.; for NL006:
4 minutes at 95.degree. C., followed by 35 cycles of 30 seconds at
95.degree. C., 30 seconds at 54.degree. C. and 1 minute at
72.degree. C., followed by 10 minutes at 72.degree. C.; for NL007:
4 minutes at 95.degree. C., followed by 35 cycles of 30 seconds at
95.degree. C., 30 seconds at 51.degree. C. and 1 minute at
72.degree. C., followed by 10 minutes at 72.degree. C.; for NL008:
4 minutes at 95.degree. C., followed by 35 cycles of 30 seconds at
95.degree. C., 30 seconds at 54.degree. C. and 1 minute at
72.degree. C., followed by 10 minutes at 72.degree. C.; for NL009:
4 minutes at 95.degree. C., followed by 35 cycles of 30 seconds at
95.degree. C., 30 seconds at 54.degree. C. and 1 minute at
72.degree. C., followed by 10 minutes at 72.degree. C.; for NL010:
4 minutes at 95.degree. C., followed by 35 cycles of 30 seconds at
95.degree. C., 30 seconds at 54.degree. C. and 1 minute at
72.degree. C., followed by 10 minutes at 72.degree. C.; for NL011:
4 minutes at 95.degree. C., followed by 35 cycles of 30 seconds at
95.degree. C., 30 seconds at 53.degree. C. and 1 minute at
72.degree. C., followed by 10 minutes at 72.degree. C.; for NL012:
4 minutes at 95.degree. C., followed by 35 cycles of 30 secondes at
95.degree. C., 30 seconds at 53.degree. C. and 1 minute at
72.degree. C., followed by 10 minutes at 72.degree. C.; for NL013:
4 minutes at 95.degree. C., followed by 35 cycles of 30 seconds at
95.degree. C., 30 seconds at 55.degree. C. and 1 minute at
72.degree. C., followed by 10 minutes at 72.degree. C.; for NL014:
4 minutes at 95.degree. C., followed by 35 cycles of 30 seconds at
95.degree. C., 30 seconds at 51.degree. C. and 1 minute at
72.degree. C., followed by 10 minutes at 72.degree. C.; for NL015:
4 minutes at 95.degree. C., followed by 35 cycles of 30 seconds at
95.degree. C., 30 seconds at 55.degree. C. and 1 minute at
72.degree. C., followed by 10 minutes at 72.degree. C.; for NL016:
4 minutes at 95.degree. C., followed by 35 cycles of 30 seconds at
95.degree. C., 30 seconds at 57.degree. C. and 1 minute at
72.degree. C., followed by 10 minutes at 72.degree. C.; for NL018:
4 minutes at 95.degree. C., followed by 35 cycles of 30 seconds at
95.degree. C., 30 seconds at 55.degree. C. and 1 minute at
72.degree. C., followed by 10 minutes at 72.degree. C.; for NL019:
4 minutes at 95.degree. C., followed by 35 cycles of 30 seconds at
95.degree. C., 30 seconds at 54.degree. C. and 1 minute at
72.degree. C., followed by 10 minutes at 72.degree. C.; for NL021:
4 minutes at 95.degree. C., followed by 35 cycles of 30 seconds at
95.degree. C., 30 seconds at 55.degree. C. and 1 minute at
72.degree. C., followed by 10 minutes at 72.degree. C.; for NL022:
4 minutes at 95.degree. C., followed by 35 cycles of 30 seconds at
95.degree. C., 30 seconds at 53.degree. C. and 1 minute at
72.degree. C., followed by 10 minutes at 72.degree. C.; for NL023:
4 minutes at 95.degree. C., followed by 35 cycles of 30 seconds at
95.degree. C., 30 seconds at 52.degree. C. and 1 minute at
72.degree. C., followed by 10 minutes at 72.degree. C.; and for
NL027: 4 minutes at 95.degree. C., followed by 35 cycles of 30
secondes at 95.degree. C., 30 seconds at 52.degree. C. and 1 minute
at 72.degree. C., followed by 10 minutes at 72.degree. C. The
anti-sense T7 template was generated using specific forward and
specific T7 reverse primers in a PCR reaction with the same
conditions as described above. The sequences of the respective
primers for amplifying the anti-sense template for each of the
target genes are given in Table 4-NL. The resulting PCR products
were analyzed on agarose gel and purified by PCR purification kit
(Qiaquick PCR Purification Kit, Cat. Nr. 28106, Qiagen). The
generated T7 forward and reverse templates were mixed to be
transcribed and the resulting RNA strands were annealed, DNase and
RNase treated, and purified by sodium acetate, following the
manufacturer's instructions, but with the following modification:
RNA peppet is washed twice in 70% ethanol. The sense strand of the
resulting dsRNA for each of the target genes is given in Table
8-NL.
[0393] The template DNA used for the PCR reactions with T7 primers
on the green fluorescent protein (gfp) control was the plasmid
pPD96.12 (the Fire Lab,
http://genome-www.stanford.edu/group/fire/), which contains the
wild-type gfp coding sequence interspersed by 3 synthetic introns.
Double-stranded RNA was synthesized using the commercially
available kit T7 RiboMAX.TM. Express RNAi System (Cat. N.sup.o.
P1700, Promega). First two separate single 5' T7 RNA polymerase
promoter templates were generated in two separate PCR reactions,
each reaction containing the target sequence in a different
orientation relative to the T7 promoter. For gfp, the sense T7
template was generated using the specific T7 FW primer oGAU183 and
the specific RV primer oGAU182 (represented herein as SEQ ID NO:
236 and SEQ ID NO: 237, respectively) in a PCR reaction with the
following conditions: 4 minutes at 95.degree. C., followed by 35
cycles of 30 seconds at 95.degree. C., 30 seconds at 55.degree. C.
and 1 minute at 72.degree. C., followed by 10 minutes at 72.degree.
C. The anti-sense T7 template was generated using the specific FW
primer oGAU181 and the specific T7 RV primer oGAU184 (represented
herein as SEQ ID NO: 238 and SEQ ID NO: 239, respectively) in a PCR
reaction with the same conditions as described above. The resulting
PCR products were analyzed on agarose gel and purified
(QIAquick.RTM. PCR Purification Kit; Cat. N.sup.o. 28106, Qiagen).
The generated T7 FW and RV templates were mixed to be transcribed
and the resulting RNA strands were annealed, DNase and RNase
treated, and purified by precipitation with sodium acetate and
isopropanol, following the manufacturer's protocol, but with the
following modification: RNA peppet is washed twice in 70% ethanol.
The sense strands of the resulting dsRNA is herein represented by
SEQ ID NO: 235.
D. Laboratory Trials to Screen dsRNA Targets Using Liquid
Artificial Diet for Activity Against Nilaparvata lugens
[0394] Liquid artificial diet (MMD-1) for the rice brown
planthopper, Nilaparvata lugens, was prepared as described by
Koyama (1988) [Artificial rearing and nutritional physiology of the
planthoppers and leafhoppers (Homoptera: Delphacidae and
Deltocephalidae) on a holidic diet. JARQ 22: 20-27], but with a
modification in final concentration of diet component sucrose:
14.4% (weight over volume) was used. Diet components were prepared
as separate concentrates: 10.times. mineral stock (stored at
4.degree. C.), 2.times. amino acid stock (stored at -20.degree. C.)
and 10.times. vitamin stock (stored at -20.degree. C.). The stock
components were mixed immediately prior to the start of a bioassay
to 4/3.times. concentration to allow dilution with the test dsRNA
solution (4.times. concentration), pH adjusted to 6.5, and
filter-sterilised into approximately 500 .mu.A aliquots.
[0395] Rice brown planthopper (Nilaparvata lugens) was reared on
two-to-three month old rice (Oryza sativa cv Taichung Native 1)
plants in a controlled environment chamber: 27.+-.2.degree. C., 80%
relative humidity, with a 16:8 hours light:dark photoperiod. A
feeding chamber comprised 10 first or second instar nymphs placed
in a small petri dish (with diameter 3 cm) covered with a single
layer of thinly stretched parafilm M onto which 50 .mu.A of diet
was added. The chamber was sealed with a second layer of parafilm
and incubated under the same conditions as the adult cultures but
with no direct light exposure. Diet with dsRNA was refreshed every
other day and the insects' survival assessed daily. Per treatment,
5 bioassay feeding chambers (replicates) were set up
simultaneously. Test and control (gfp) dsRNA solutions were
incorporated into the diet to a final concentration of 2 mg/ml. The
feeding chambers were kept at 27.+-.2.degree. C., 80% relative
humidity, with a 16:8 hours light:dark photoperiod. Insect survival
data were analysed using the Kaplan-Meier survival curve model and
the survival between groups were compared using the logrank test
(Prism version 4.0).
[0396] Feeding liquid artificial diet supplemented with intact
naked dsRNAs to Nilaparvata lugens in vitro using a feeding chamber
resulted in significant increases in nymphal mortalities as shown
in four separate bioassays (FIGS. 1(a)-(d)-NL; Tables 1a-d-NL).
These results demonstrate that dsRNAs corresponding to different
essential BPH genes showed significant toxicity towards the rice
brown planthopper.
[0397] Effect of gfp dsRNA on BPH survival in these bioassays is
not significantly different to survival on diet only
[0398] Tables 10a-d-NL show a summary of the survival of
Nilaparvata lugens on artificial diet supplemented with 2 mg/ml
(final concentration) of the following targets; in Table 10(a)-NL:
NL002, NL003, NL005, NL010; in Table 10(b)-NL NL009, NL016; in
Table 10(c)-NL NL014, NL018; and in Table 10(d)-NL NL013, NL015,
NL021. In the survival analysis column, the effect of RNAi is
indicated as follows: +=significantly decreased survival compared
to gfp dsRNA control (alpha <0.05); -=no significant difference
in survival compared to gfp dsRNA control. Survival curves were
compared (between diet only and diet supplemented with test dsRNA,
gfp dsRNA and test dsRNA, and diet only and gfp dsRNA) using the
logrank test.
E. Laboratory Trials to Screen dsRNAs at Different Concentrations
Using Artificial Diet for Activity Against Nilaparvata lugens
[0399] Fifty .mu.l of liquid artificial diet supplemented with
different concentrations of target NL002 dsRNA, namely 1, 0.2,
0.08, and 0.04 mg/ml (final concentration), was applied to the
brown planthopper feeding chambers. Diet with dsRNA was refreshed
every other day and the insects' survival assessed daily. Per
treatment, 5 bioassay feeding chambers (replicates) were set up
simultaneously. The feeding chambers were kept at 27.+-.2.degree.
C., 80% relative humidity, with a 16:8 hours light:dark
photoperiod. Insect survival data were analysed using the
Kaplan-Meier survival curve model and the survival between groups
were compared using the logrank test (Prism version 4.0).
Feeding liquid artificial diet supplemented with intact naked
dsRNAs of target NL002 at different concentrations resulted in
significantly higher BPH mortalities at final concentrations of as
low as 0.04 mg dsRNA per ml diet when compared with survival on
diet only, as shown in FIG. 2-NL and Table 9-NL. Table 9-NL
summarizes the survival of Nilaparvata lugens artificial diet
feeding trial supplemented with 1, 0.2, 0.08, & 0.04 mg/ml
(final concentration) of target NL002. In the survival analysis
column the effect of RNAi is indicated as follows: +=significantly
decreases survival compared to diet only control (alpha <0.05);
-=no significant differences in survival compared to diet only
control. Survival curves were compared using the logrank test.
F. Cloning of a BPH Gene Fragment in a Vector Suitable for
Bacterial Production of Insect-Active Double-Stranded RNA
[0400] What follows is an example of cloning a DNA fragment
corresponding to a BPH gene target in a vector for the expression
of double-stranded RNA in a bacterial host, although any vector
comprising a T7 promoter or any other promoter for efficient
transcription in bacteria, may be used (reference to
WO0001846).
[0401] The sequences of the specific primers used for the
amplification of target genes are provided in Table 8. The template
used is the pCR8/GW/topo vector containing any of target sequences.
The primers are used in a PCR reaction with the following
conditions: 5 minutes at 98.degree. C., followed by 30 cycles of 10
seconds at 98.degree. C., 30 seconds at 55.degree. C. and 2 minutes
at 72.degree. C., followed by 10 minutes at 72.degree. C. The
resulting PCR fragment is analyzed on agarose gel, purified
(QIAquick Gel Extraction kit, Cat. Nr. 28706, Qiagen), blunt-end
cloned into Srf I-linearized pGNA49A vector (reference to
WO00188121A1), and sequenced. The sequence of the resulting PCR
product corresponds to the respective sequence as given in Table
8-NL. The recombinant vector harbouring this sequence is named
pGBNJ00.
G. Expression and Production of a Double-Stranded RNA Target in Two
Strains of Escherichia coli: (1) AB309-105, and, (2) BL21(DE3)
[0402] The procedures described below are followed in order to
express suitable levels of insect-active double-stranded RNA of
insect target in bacteria. An RNaseIII-deficient strain, AB309-105,
is used in comparison to wild-type RNaseIII-containing bacteria,
BL21(DE3).
Transformation of AB309-105 and BL21(DE3)
[0403] Three hundred ng of the plasmid are added to and gently
mixed in a 50 .mu.l aliquot of ice-chilled chemically competent E.
coli strain AB309-105 or BL21(DE3). The cells are incubated on ice
for 20 minutes before subjecting them to a heat shock treatment of
37.degree. C. for 5 minutes, after which the cells are placed back
on ice for a further 5 minutes. Four hundred and fifty .mu.l of
room temperature SOC medium is added to the cells and the
suspension incubated on a shaker (250 rpm) at 37.degree. C. for 1
hour. One hundred .mu.l of the bacterial cell suspension is
transferred to a 500 ml conical flask containing 150 ml of liquid
Luria-Bertani (LB) broth supplemented with 100 .mu.g/ml
carbenicillin antibiotic. The culture is incubated on an Innova
4430 shaker (250 rpm) at 37.degree. C. overnight (16 to 18
hours).
Chemical Induction of Double-Stranded RNA Expression in AB309-105
and BL21(DE3)
[0404] Expression of double-stranded RNA from the recombinant
vector, pGBNJ003, in the bacterial strain AB309-105 or BL21(DE3) is
made possible since all the genetic components for controlled
expression are present. In the presence of the chemical inducer
isopropylthiogalactoside, or IPTG, the T7 polymerase will drive the
transcription of the target sequence in both antisense and sense
directions since these are flanked by oppositely oriented T7
promoters.
[0405] The optical density at 600 nm of the overnight bacterial
culture is measured using an appropriate spectrophotometer and
adjusted to a value of 1 by the addition of fresh LB broth. Fifty
ml of this culture is transferred to a 50 ml Falcon tube and the
culture then centrifuged at 3000 g at 15.degree. C. for 10 minutes.
The supernatant is removed and the bacterial pellet resuspended in
50 ml of fresh S complete medium (SNC medium plus 5 .mu.g/ml
cholesterol) supplemented with 100 .mu.g/ml carbenicillin and 1 mM
IPTG. The bacteria are induced for 2 to 4 hours at room
temperature.
Heat Treatment of Bacteria
[0406] Bacteria are killed by heat treatment in order to minimise
the risk of contamination of the artificial diet in the test
plates. However, heat treatment of bacteria expressing
double-stranded RNA is not a prerequisite for inducing toxicity
towards the insects due to RNA interference. The induced bacterial
culture is centrifuged at 3000 g at room temperature for 10
minutes, the supernatant discarded and the pellet subjected to
80.degree. C. for 20 minutes in a water bath. After heat treatment,
the bacterial pellet is resuspended in 1.5 ml MilliQ water and the
suspension transferred to a microfuge tube. Several tubes are
prepared and used in the bioassays for each refreshment. The tubes
are stored at -20.degree. C. until further use.
H. Laboratory Trials to Test Escherichia coli Expressing dsRNA
Targets Against Nilaparvata lugens
Plant-Based Bioassays
[0407] Whole plants are sprayed with suspensions of chemically
induced bacteria expressing dsRNA prior to feeding the plants to
BPH. The are grown from in a plant growth room chamber. The plants
are caged by placing a 500 ml plastic bottle upside down over the
plant with the neck of the bottle firmly placed in the soil in a
pot and the base cut open and covered with a fine nylon mesh to
permit aeration, reduce condensation inside and prevent insect
escape. BPH are placed on each treated plant in the cage. Plants
are treated with a suspension of E. coli AB309-105 harbouring the
pGBNJ001 plasmids or pGN29 plasmid. Different quantities of
bacteria are applied to the plants: for instance 66, 22, and 7
units, where one unit is defined as 10.sup.9 bacterial cells in 1
ml of a bacterial suspension at optical density value of 1 at 600
nm wavelength. In each case, a total volume of between 1 and 10 ml
s sprayed on the plant with the aid of a vaporizer. One plant is
used per treatment in this trial. The number of survivors are
counted and the weight of each survivor recorded.
[0408] Spraying plants with a suspension of E. coli bacterial
strain AB309-105 expressing target dsRNA from pGBNJ003 leed to a
dramatic increase in insect mortality when compared to pGN29
control. These experiments show that double-stranded RNA
corresponding to an insect gene target sequence produced in either
wild-type or RNaseIII-deficient bacterial expression systems is
toxic towards the insect in terms of substantial increases in
insect mortality and growth/development delay for larval survivors.
It is also clear from these experiments that an exemplification is
provided for the effective protection of plants/crops from insect
damage by the use of a spray of a formulation consisting of
bacteria expressing double-stranded RNA corresponding to an insect
gene target.
Example 10
Chilo suppressalis (Rice Striped Stem Borer)
A. Cloning of Partial Sequence of the Chilo suppressalis Genes Via
Family PCR
[0409] High quality, intact RNA was isolated from the 4 different
larval stages of Chilo suppressalis (rice striped stem borer) using
TRIzol Reagent (Cat. Nr. 15596-026/15596-018, Invitrogen,
Rockville, Md., USA) following the manufacturer's instructions.
Genomic DNA present in the RNA preparation was removed by DNase
treatment following the manafacturer's instructions (Cat. Nr. 1700,
Promega). cDNA was generated using a commercially available kit
(SuperScript.TM. III Reverse Transcriptase, Cat. Nr. 18080044,
Invitrogen, Rockville, Md., USA) following the manufacturer's
instructions.
[0410] To isolate cDNA sequences comprising a portion of the CS001,
CS002, CS003, CS006, CS007, CS009, CS011, CS013, CS014, CS015,
CS016 and CS018 genes, a series of PCR reactions with degenerate
primers were performed using Amplitaq Gold (Cat. Nr. N8080240,
Applied Biosystems) following the manafacturer's instructions.
[0411] The sequences of the degenerate primers used for
amplification of each of the genes are given in Table 2-CS. These
primers were used in respective PCR reactions with the following
conditions: 10 minutes at 95.degree. C., followed by 40 cycles of
30 seconds at 95.degree. C., 1 minute at 55.degree. C. and 1 minute
at 72.degree. C., followed by 10 minutes at 72.degree. C. The
resulting PCR fragments were analyzed on agarose gel, purified
(QIAquick Gel Extraction kit, Cat. Nr. 28706, Qiagen), cloned into
the pCR4/TOPO vector (Cat. Nr. K2500-20, Invitrogen), and
sequenced. The sequences of the resulting PCR products are
represented by the respective SEQ ID NO:s as given in Table 2-CS
and are referred to as the partial sequences. The corresponding
partial amino acid sequences are represented by the respective SEQ
ID NO:s as given in Table 3-CS.
B. dsRNA Production of the Chilo suppressalis Genes
[0412] dsRNA was synthesized in milligram amounts using the
commercially available kit T7 Ribomax.TM. Express RNAi System (Cat.
Nr. P1700, Promega). First two separate single 5' T7 RNA polymerase
promoter templates were generated in two separate PCR reactions,
each reaction containing the target sequence in a different
orientation relative to the T7 promoter.
[0413] For each of the target genes, the sense T7 template was
generated using specific T7 forward and specific reverse primers.
The sequences of the respective primers for amplifying the sense
template for each of the target genes are given in Table 8-CS. The
conditions in the PCR reactions were as follows: 4 minutes at
95.degree. C., followed by 35 cycles of 30 seconds at 95.degree.
C., 30 seconds at 55.degree. C. and 1 minute at 72.degree. C.,
followed by 10 minutes at 72.degree. C. The anti-sense T7 template
was generated using specific forward and specific T7 reverse
primers in a PCR reaction with the same conditions as described
above. The sequences of the respective primers for amplifying the
anti-sense template for each of the target genes are given in Table
8-CS. The resulting PCR products were analyzed on agarose gel and
purified by PCR purification kit (Qiaquick PCR Purification Kit,
Cat. Nr. 28106, Qiagen) and NaClO.sub.4 precipitation. The
generated T7 forward and reverse templates were mixed to be
transcribed and the resulting RNA strands were annealed, DNase and
RNase treated, and purified by sodium acetate, following the
manufacturer's instructions. The sense strand of the resulting
dsRNA for each of the target genes is given in Table 8-CS.
C. Laboratory Trials to Test dsRNA Targets, Using Artificial Diet
for Activity Against Chilo suppressalis Larvae
[0414] Rice striped stem borers, Chilo suppressalis, (origin:
Syngenta, Stein, Switzerland) were maintained on a modified
artificial diet based on that described by Kamano and Sato, 1985
(in: Handbook of Insect Rearing. Volumes I & II. P Singh and RF
Moore, eds., Elsevier Science Publishers, Amsterdam and New York,
1985, pp 448). Briefly, a litre diet was made up as follows: 20 g
of agar added to 980 ml of Milli-Q water and autoclaved; the agar
solution was cooled down to approximately 55.degree. C. and the
remaining ingredients were added and mixed thoroughly: 40 g corn
flour (Polenta), 20 g cellulose, 30 g sucrose, 30 g casein, 20 g
wheat germ (toasted), 8 g Wesson salt mixture, 12 g Vanderzant
vitamin mix, 1.8 g sorbic acid, 1.6 g nipagin (methylparaben), 0.3
g aureomycin, 0.4 g cholesterol and 0.6 g L-cysteine. The diet was
cooled down to approx. 45.degree. C. and poured into rearing trays
or cups. The diet was left to set in a horizontal laminair flow
cabin. Rice leaf sections with oviposited eggs were removed from a
cage housing adult moths and pinned to the solid diet in the
rearing cup or tray. Eggs were left to hatch and neonate larvae
were available for bioassays and the maintenance of the insect
cultures. During the trials and rearings, the conditions were
28.+-.2.degree. C. and 80.+-.5% relative humidity, with a 16:8 hour
light:dark photoperiod.
[0415] The same artificial diet is used for the bioassays but in
this case the diet is poured equally in 24 multiwell plates, with
each well containing 1 ml diet. Once the diet is set, the test
formulations are applied to the diet's surface (2 cm.sup.2), at the
rate of 50 .mu.l of 1 .mu.g/.mu.l dsRNA of target. The dsRNA
solutions are left to dry and two first instar moth larvae are
placed in each well. After 7 days, the larvae are transferred to
fresh treated diet in multiwell plates. At day 14 (i.e. 14 days
post bioassay start) the number of live and dead insects is
recorded and examined for abnormalities. Twenty-four larvae in
total are tested per treatment.
[0416] An alternative bioassay is performed in which treated rice
leaves are fed to neonate larvae of the rice striped stem borer.
Small leaf sections of Indica rice variety Taichung native 1 are
dipped in 0.05% Triton X-100 solution containing 1 .mu.g/.mu.l of
target dsRNA, left to dry and each section placed in a well of a 24
multiwell plate containing gellified 2% agar. Two neonates are
transferred from the rearing tray to each dsRNA treated leaf
section (24 larvae per treatment). After 4 and 8 days, the larvae
are transferred to fresh treated rice leaf sections. The number of
live and dead larvae are assessed on days 4, 8 and 12; any
abnormalities are also recorded.
D. Cloning of a SSB Gene Fragment in a Vector Suitable for
Bacterial Production of Insect-Active Double-Stranded RNA
[0417] What follows is an example of cloning a DNA fragment
corresponding to an SSB gene target in a vector for the expression
of double-stranded RNA in a bacterial host, although any vector
comprising a T7 promoter or any other promoter for efficient
transcription in bacteria, may be used (reference to
WO0001846).
[0418] The sequences of the specific primers used for the
amplification of target genes are provided in Table 8. The template
used is the pCR8/GW/topo vector containing any of target sequences.
The primers are used in a PCR reaction with the following
conditions: 5 minutes at 98.degree. C., followed by 30 cycles of 10
seconds at 98.degree. C., 30 seconds at 55.degree. C. and 2 minutes
at 72.degree. C., followed by 10 minutes at 72.degree. C. The
resulting PCR fragment is analyzed on agarose gel, purified
(QIAquick Gel Extraction kit, Cat. Nr. 28706, Qiagen), blunt-end
cloned into Srf I-linearized pGNA49A vector (reference to
WO00188121A1), and sequenced. The sequence of the resulting PCR
product corresponds to the respective sequence as given in Table
8-CS. The recombinant vector harbouring this sequence is named
pGBNJ00XX.
E. Expression and Production of a Double-Stranded RNA Target in Two
Strains of Escherichia coli: (1) AB309-105, and, (2) BL21(DE3)
[0419] The procedures described below are followed in order to
express suitable levels of insect-active double-stranded RNA of
insect target in bacteria. An RNaseIII-deficient strain, AB309-105,
is used in comparison to wild-type RNaseIII-containing bacteria,
BL21(DE3).
Transformation of AB309-105 and BL21(DE3)
[0420] Three hundred ng of the plasmid are added to and gently
mixed in a 50 .mu.l aliquot of ice-chilled chemically competent E.
coli strain AB309-105 or BL21(DE3). The cells are incubated on ice
for 20 minutes before subjecting them to a heat shock treatment of
37.degree. C. for 5 minutes, after which the cells are placed back
on ice for a further 5 minutes. Four hundred and fifty .mu.l of
room temperature SOC medium is added to the cells and the
suspension incubated on a shaker (250 rpm) at 37.degree. C. for 1
hour. One hundred .mu.l of the bacterial cell suspension is
transferred to a 500 ml conical flask containing 150 ml of liquid
Luria-Bertani (LB) broth supplemented with 100 .mu.g/ml
carbenicillin antibiotic. The culture is incubated on an Innova
4430 shaker (250 rpm) at 37.degree. C. overnight (16 to 18
hours).
Chemical Induction of Double-Stranded RNA Expression in AB309-105
and BL21(DE3)
[0421] Expression of double-stranded RNA from the recombinant
vector, pGBNJ003, in the bacterial strain AB309-105 or BL21(DE3) is
made possible since all the genetic components for controlled
expression are present. In the presence of the chemical inducer
isopropylthiogalactoside, or IPTG, the T7 polymerase will drive the
transcription of the target sequence in both antisense and sense
directions since these are flanked by oppositely oriented T7
promoters.
[0422] The optical density at 600 nm of the overnight bacterial
culture is measured using an appropriate spectrophotometer and
adjusted to a value of 1 by the addition of fresh LB broth. Fifty
ml of this culture is transferred to a 50 ml Falcon tube and the
culture then centrifuged at 3000 g at 15.degree. C. for 10 minutes.
The supernatant is removed and the bacterial pellet resuspended in
50 ml of fresh S complete medium (SNC medium plus 5 .mu.g/ml
cholesterol) supplemented with 100 .mu.g/ml carbenicillin and 1 mM
IPTG. The bacteria are induced for 2 to 4 hours at room
temperature.
[0423] Heat Treatment of Bacteria
[0424] Bacteria are killed by heat treatment in order to minimise
the risk of contamination of the artificial diet in the test
plates. However, heat treatment of bacteria expressing
double-stranded RNA is not a prerequisite for inducing toxicity
towards the insects due to RNA interference. The induced bacterial
culture is centrifuged at 3000 g at room temperature for 10
minutes, the supernatant discarded and the pellet subjected to
80.degree. C. for 20 minutes in a water bath. After heat treatment,
the bacterial pellet is resuspended in 1.5 ml MilliQ water and the
suspension transferred to a microfuge tube. Several tubes are
prepared and used in the bioassays for each refreshment. The tubes
are stored at -20.degree. C. until further use.
F. Laboratory Trials to Test Escherichia coli Expressing dsRNA
Targets Against Chilo suppressalis
Plant-Based Bioassays
[0425] Whole plants are sprayed with suspensions of chemically
induced bacteria expressing dsRNA prior to feeding the plants to
SSB. The are grown from in a plant growth room chamber. The plants
are caged by placing a 500 ml plastic bottle upside down over the
plant with the neck of the bottle firmly placed in the soil in a
pot and the base cut open and covered with a fine nylon mesh to
permit aeration, reduce condensation inside and prevent insect
escape. SSB are placed on each treated plant in the cage. Plants
are treated with a suspension of E. coli AB309-105 harbouring the
pGBNJ001 plasmids or pGN29 plasmid. Different quantities of
bacteria are applied to the plants: for instance 66, 22, and 7
units, where one unit is defined as 10.sup.9 bacterial cells in 1
ml of a bacterial suspension at optical density value of 1 at 600
nm wavelength. In each case, a total volume of between 1 and 10 ml
s sprayed on the plant with the aid of a vaporizer. One plant is
used per treatment in this trial. The number of survivors are
counted and the weight of each survivor recorded.
[0426] Spraying plants with a suspension of E. coli bacterial
strain AB309-105 expressing target dsRNA from pGBNJ003 leed to a
dramatic increase in insect mortality when compared to pGN29
control. These experiments show that double-stranded RNA
corresponding to an insect gene target sequence produced in either
wild-type or RNaseIII-deficient bacterial expression systems is
toxic towards the insect in terms of substantial increases in
insect mortality and growth/development delay for larval survivors.
It is also clear from these experiments that an exemplification is
provided for the effective protection of plants/crops from insect
damage by the use of a spray of a formulation consisting of
bacteria expressing double-stranded RNA corresponding to an insect
gene target.
Example 9
Plutella xylostella (Diamondback Moth)
A. Cloning of a Partial Sequence of the Plutella xylostella
[0427] High quality, intact RNA was isolated from all the different
larval stages of Plutella xylostella (Diamondback moth; source: Dr.
Lara Senior, Insect Investigations Ltd., Capital Business Park,
Wentloog, Cardiff, CF3 2PX, Wales, UK) using TRIzol Reagent (Cat.
Nr. 15596-026/15596-018, Invitrogen, Rockville, Md., USA) following
the manufacturer's instructions. Genomic DNA present in the RNA
preparation was removed by DNase treatment following the
manufacturer's instructions (Cat. Nr. 1700, Promega). cDNA was
generated using a commercially available kit (SuperScript.TM. III
Reverse Transcriptase, Cat. Nr. 18080044, Invitrogen, Rockville,
Md., USA) following the manufacturer's instructions.
[0428] To isolate cDNA sequences comprising a portion of the PX001,
PX009, PX010, PX015, PX016 genes, a series of PCR reactions with
degenerate primers were performed using Amplitaq Gold (Cat. Nr.
N8080240, Applied Biosystems) following the manufacturer's
instructions.
[0429] The sequences of the degenerate primers used for
amplification of each of the genes are given in Table 2-PX. These
primers were used in respective PCR reactions with the following
conditions: 10 minutes at 95.degree. C., followed by 40 cycles of
30 seconds at 95.degree. C., 1 minute at 50.degree. C. and 1 minute
and 30 seconds at 72.degree. C., followed by 7 minutes at
72.degree. C. (for PX001, PX009, PX015, PX016); 10 minutes at
95.degree. C., followed by 40 cycles of 30 seconds at 95.degree.
C., 1 minute at 54.degree. C. and 2 minute and 30 seconds at
72.degree. C., followed by 7 minutes at 72.degree. C. (for PX010).
The resulting PCR fragments were analyzed on agarose gel, purified
(QIAquick Gel Extraction kit, Cat. Nr. 28706, Qiagen), cloned into
the pCR8/GW/TOPO vector (Cat. Nr. K2500-20, Invitrogen) and
sequenced. The sequences of the resulting PCR products are
represented by the respective SEQ ID NO:s as given in Table 2-PX
and are referred to as the partial sequences. The corresponding
partial amino acid sequence are represented by the respective SEQ
ID NO:s as given in Table 3-PX.
B. dsRNA Production of the Plutella xylostella Genes
[0430] dsRNA was synthesized in milligram amounts using the
commercially available kit T7 Ribomax.TM. Express RNAi System (Cat.
Nr. P1700, Promega). First two separate single 5' T7 RNA polymerase
promoter templates were generated in two separate PCR reactions,
each reaction containing the target sequence in a different
orientation relative to the T7 promoter.
[0431] For each of the target genes, the sense T7 template was
generated using specific T7 forward and specific reverse primers.
The sequences of the respective primers for amplifying the sense
template for each of the target genes are given in Table 8-PX. The
conditions in the PCR reactions were as follows: 1 minute at
95.degree. C., followed by 20 cycles of 30 seconds at 95.degree.
C., 30 seconds at 60.degree. C. (-0.5.degree. C./cycle) and 1
minute at 72.degree. C., followed by 15 cycles of 30 seconds at
95.degree. C., 30 seconds at 50.degree. C. and 1 minute at
72.degree. C., followed by 10 minutes at 72.degree. C. The
anti-sense T7 template was generated using specific forward and
specific T7 reverse primers in a PCR reaction with the same
conditions as described above. The sequences of the respective
primers for amplifying the anti-sense template for each of the
target genes are given in Table 8-PX. The resulting PCR products
were analyzed on agarose gel and purified by PCR purification kit
(Qiaquick PCR Purification Kit, Cat. Nr. 28106, Qiagen) and
NaClO.sub.4 precipitation. The generated T7 forward and reverse
templates were mixed to be transcribed and the resulting RNA
strands were annealed, DNase and RNase treated, and purified by
sodium acetate, following the manufacturer's instructions. The
sense strand of the resulting dsRNA for each of the target genes is
given in Table 8-PX.
C. Laboratory Trials to Test dsRNA Targets, Using Artificial Diet
for Activity Against Plutella xylostella Larvae
[0432] Diamond-back moths, Plutella xylostella, were maintained at
Insect Investigations Ltd. (origin: Newcastle University,
Newcastle-upon-Tyne, UK). The insects were reared on cabbage
leaves. First instar, mixed sex larvae (approximately 1 day old)
were selected for use in the trial. Insects were maintained in
Eppendorf tubes (1.5 ml capacity). Commercially available
Diamond-back moth diet (Bio-Serv, NJ, USA), prepared following the
manafacturer's instructions, was placed in the lid of each tube
(0.25 ml capacity, 8 mm diameter). While still liquid, the diet was
smoother over to remove excess and produce an even surface.
[0433] Once the diet has set the test formulations are applied to
the diet's surface, at the rate of 25 .mu.l undiluted formulation
(1 .mu.g/.mu.l dsRNA of targets) per replicate. The test
formulations are allowed to dry and one first instar moth larva is
placed in each tube. The larva is placed on the surface of the diet
in the lid and the tube carefully closed. The tubes are stored
upside down, on their lids such that each larva remains on the
surface of the diet. Twice weekly the larvae are transferred to new
Eppendorf tubes with fresh diet. The insects are provided with
treated diet for the first two weeks of the trial and thereafter
with untreated diet.
[0434] Assessments are made twice weekly for a total of 38 days at
which point all larvae are dead. At each assessment the insects are
assessed as live or dead and examined for abnormalities. Forty
single larva replicates are performed for each of the treatments.
During the trial the test conditions are 23 to 26.degree. C. and 50
to 65% relative humidity, with a 16:8 hour light:dark
photoperiod.
D. Cloning of a DBM Gene Fragment in a Vector Suitable for
Bacterial Production of Insect-Active Double-Stranded RNA
[0435] What follows is an example of cloning a DNA fragment
corresponding to a DBM gene target in a vector for the expression
of double-stranded RNA in a bacterial host, although any vector
comprising a T7 promoter or any other promoter for efficient
transcription in bacteria, may be used (reference to
WO0001846).
[0436] The sequences of the specific primers used for the
amplification of target genes are provided in Table 8-PX. The
template used is the pCR8/GW/topo vector containing any of target
sequences. The primers are used in a PCR reaction with the
following conditions: 5 minutes at 98.degree. C., followed by 30
cycles of 10 seconds at 98.degree. C., 30 seconds at 55.degree. C.
and 2 minutes at 72.degree. C., followed by 10 minutes at
72.degree. C. The resulting PCR fragment is analyzed on agarose
gel, purified (QIAquick Gel Extraction kit, Cat. Nr. 28706,
Qiagen), blunt-end cloned into Srf I-linearized pGNA49A vector
(reference to WO00188121A1), and sequenced. The sequence of the
resulting PCR product corresponds to the respective sequence as
given in Table 8-PX. The recombinant vector harbouring this
sequence is named pGBNJ00XX.
E. Expression and Production of a Double-Stranded RNA Target in Two
Strains of Escherichia coli: (1) AB309-105, and, (2) BL21(DE3)
[0437] The procedures described below are followed in order to
express suitable levels of insect-active double-stranded RNA of
insect target in bacteria. An RNaseIII-deficient strain, AB309-105,
is used in comparison to wild-type RNaseIII-containing bacteria,
BL21(DE3).
Transformation of AB309-105 and BL21(DE3)
[0438] Three hundred ng of the plasmid are added to and gently
mixed in a 50 .mu.A aliquot of ice-chilled chemically competent E.
coli strain AB309-105 or BL21(DE3). The cells are incubated on ice
for 20 minutes before subjecting them to a heat shock treatment of
37.degree. C. for 5 minutes, after which the cells are placed back
on ice for a further 5 minutes. Four hundred and fifty .mu.l of
room temperature SOC medium is added to the cells and the
suspension incubated on a shaker (250 rpm) at 37.degree. C. for 1
hour. One hundred .mu.l of the bacterial cell suspension is
transferred to a 500 ml conical flask containing 150 ml of liquid
Luria-Bertani (LB) broth supplemented with 100 .mu.g/ml
carbenicillin antibiotic. The culture is incubated on an Innova
4430 shaker (250 rpm) at 37.degree. C. overnight (16 to 18
hours).
Chemical Induction of Double-Stranded RNA Expression in AB309-105
and BL21(DE3)
[0439] Expression of double-stranded RNA from the recombinant
vector, pGBNJ003, in the bacterial strain AB309-105 or BL21(DE3) is
made possible since all the genetic components for controlled
expression are present. In the presence of the chemical inducer
isopropylthiogalactoside, or IPTG, the T7 polymerase will drive the
transcription of the target sequence in both antisense and sense
directions since these are flanked by oppositely oriented T7
promoters.
[0440] The optical density at 600 nm of the overnight bacterial
culture is measured using an appropriate spectrophotometer and
adjusted to a value of 1 by the addition of fresh LB broth. Fifty
ml of this culture is transferred to a 50 ml Falcon tube and the
culture then centrifuged at 3000 g at 15.degree. C. for 10 minutes.
The supernatant is removed and the bacterial pellet resuspended in
50 ml of fresh S complete medium (SNC medium plus 5 .mu.g/ml
cholesterol) supplemented with 100 .mu.g/ml carbenicillin and 1 mM
IPTG. The bacteria are induced for 2 to 4 hours at room
temperature.
Heat Treatment of Bacteria
[0441] Bacteria are killed by heat treatment in order to minimise
the risk of contamination of the artificial diet in the test
plates. However, heat treatment of bacteria expressing
double-stranded RNA is not a prerequisite for inducing toxicity
towards the insects due to RNA interference. The induced bacterial
culture is centrifuged at 3000 g at room temperature for 10
minutes, the supernatant discarded and the pellet subjected to
80.degree. C. for 20 minutes in a water bath. After heat treatment,
the bacterial pellet is resuspended in 1.5 ml MilliQ water and the
suspension transferred to a microfuge tube. Several tubes are
prepared and used in the bioassays for each refreshment. The tubes
are stored at -20.degree. C. until further use.
F. Laboratory Trials to Test Escherichia coli Expressing dsRNA
Targets Against Plutella xylostella
Plant-Based Bioassays
[0442] Whole plants are sprayed with suspensions of chemically
induced bacteria expressing dsRNA prior to feeding the plants to
DBM. The are grown from in a plant growth room chamber. The plants
are caged by placing a 500 ml plastic bottle upside down over the
plant with the neck of the bottle firmly placed in the soil in a
pot and the base cut open and covered with a fine nylon mesh to
permit aeration, reduce condensation inside and prevent insect
escape. DBM are placed on each treated plant in the cage. Plants
are treated with a suspension of E. coli AB309-105 harbouring the
pGBNJ001 plasmids or pGN29 plasmid. Different quantities of
bacteria are applied to the plants: for instance 66, 22, and 7
units, where one unit is defined as 10.sup.9 bacterial cells in 1
ml of a bacterial suspension at optical density value of 1 at 600
nm wavelength. In each case, a total volume of between 1 and 10 ml
sprayed on the plant with the aid of a vaporizer. One plant is used
per treatment in this trial. The number of survivors are counted
and the weight of each survivor recorded.
[0443] Spraying plants with a suspension of E. coli bacterial
strain AB309-105 expressing target dsRNA from pGBNJ003 leed to a
dramatic increase in insect mortality when compared to pGN29
control. These experiments show that double-stranded RNA
corresponding to an insect gene target sequence produced in either
wild-type or RNaseIII-deficient bacterial expression systems is
toxic towards the insect in terms of substantial increases in
insect mortality and growth/development delay for larval survivors.
It is also clear from these experiments that an exemplification is
provided for the effective protection of plants/crops from insect
damage by the use of a spray of a formulation consisting of
bacteria expressing double-stranded RNA corresponding to an insect
gene target.
Example 12
Acheta domesticus (House Cricket)
A. Cloning Acheta domesticus Partial Sequences
[0444] High quality, intact RNA was isolated from all the different
insect stages of Acheta domesticus (house cricket; source: Dr. Lara
Senior, Insect Investigations Ltd., Capital Business Park,
Wentloog, Cardiff, CF3 2PX, Wales, UK) using TRIzol Reagent (Cat.
Nr. 15596-026/15596-018, Invitrogen, Rockville, Md., USA) following
the manufacturer's instructions. Genomic DNA present in the RNA
preparation was removed by DNase treatment following the
manafacturer's instructions (Cat. Nr. 1700, Promega). cDNA was
generated using a commercially available kit (SuperScript.TM. III
Reverse Transcriptase, Cat. Nr. 18080044, Invitrogen, Rockville,
Md., USA) following the manufacturer's instructions.
[0445] To isolate cDNA sequences comprising a portion of the AD001,
AD002, AD009, AD015 and AD016 genes, a series of PCR reactions with
degenerate primers were performed using Amplitaq Gold (Cat. Nr.
N8080240, Applied Biosystems) following the manafacturer's
instructions.
[0446] The sequences of the degenerate primers used for
amplification of each of the genes are given in Table 2-AD. These
primers were used in respective PCR reactions with the following
conditions: 10 minutes at 95.degree. C., followed by 40 cycles of
30 seconds at 95.degree. C., 1 minute at 50.degree. C. and 1 minute
and 30 seconds at 72.degree. C., followed by 7 minutes at
72.degree. C. The resulting PCR fragments were analyzed on agarose
gel, purified (QIAquick Gel Extraction kit, Cat. Nr. 28706,
Qiagen), cloned into the pCR8/GW/topo vector (Cat. Nr. K2500 20,
Invitrogen) and sequenced. The sequences of the resulting PCR
products are represented by the respective SEQ ID NO:s as given in
Table 2-AD and are referred to as the partial sequences. The
corresponding partial amino acid sequence are represented by the
respective SEQ ID NO:s as given in Table 3-AD.
B. dsRNA Production of the Acheta domesticus Genes
[0447] dsRNA was synthesized in milligram amounts using the
commercially available kit T7 Ribomax.TM. Express RNAi System (Cat.
Nr. P1700, Promega). First two separate single 5' T7 RNA polymerase
promoter templates were generated in two separate PCR reactions,
each reaction containing the target sequence in a different
orientation relative to the T7 promoter.
[0448] For each of the target genes, the sense T7 template was
generated using specific T7 forward and specific reverse primers.
The sequences of the respective primers for amplifying the sense
template for each of the target genes are given in Table 8-AD. The
conditions in the PCR reactions were as follows: 1 minute at
95.degree. C., followed by 20 cycles of 30 seconds at 95.degree.
C., 30 seconds at 60.degree. C. (-0.5.degree. C./cycle) and 1
minute at 72.degree. C., followed by 15 cycles of 30 seconds at
95.degree. C., 30 seconds at 50.degree. C. and 1 minute at
72.degree. C., followed by 10 minutes at 72.degree. C. The
anti-sense T7 template was generated using specific forward and
specific T7 reverse primers in a PCR reaction with the same
conditions as described above. The sequences of the respective
primers for amplifying the anti-sense template for each of the
target genes are given in Table 8-AD. The resulting PCR products
were analyzed on agarose gel and purified by PCR purification kit
(Qiaquick PCR Purification Kit, Cat. Nr. 28106, Qiagen) and
NaClO.sub.4 precipitation. The generated T7 forward and reverse
templates were mixed to be transcribed and the resulting RNA
strands were annealed, DNase and RNase treated, and purified by
sodium acetate, following the manufacturer's instructions. The
sense strand of the resulting dsRNA for each of the target genes is
given in Table 8-AD.
C. Laboratory Trials to Test dsRNA Targets, Using Artificial Diet
for Activity Against Acheta domesticus Larvae
[0449] House crickets, Acheta domesticus, were maintained at Insect
Investigations Ltd. (origin: Blades Biological Ltd., Kent, UK). The
insects were reared on bran pellets and cabbage leaves. Mixed sex
nymphs of equal size and no more than 5 days old were selected for
use in the trial. Double-stranded RNA is mixed with a wheat-based
pelleted rodent diet (rat and mouse standard diet, B & K
Universal Ltd., Grimston, Aldbrough, Hull, UK). The diet, BK001P,
contains the following ingredients in descending order by weight:
wheat, soya, wheatfeed, barley, pellet binder, rodent 5 vit min,
fat blend, dicalcium phosphate, mould carb. The pelleted rodent
diet is finely ground and heat-treated in a microwave oven prior to
mixing, in order to inactivate any enzyme components. All rodent
diet is taken from the same batch in order to ensure consistency.
The ground diet and dsRNA are mixed thoroughly and formed into
small pellets of equal weight, which are allowed to dry overnight
at room temperature.
[0450] Double-stranded RNA samples from targets and gfp control at
concentrations 10 .mu.g/.mu.l were applied in the ratio 1 g ground
diet plus 1 ml dsRNA solution, thereby resulting in an application
rate of 10 mg dsRNA per g pellet. Pellets are replaced weekly. The
insects are provided with treated pellets for the first three weeks
of the trial. Thereafter untreated pellets are provided. Insects
are maintained within lidded plastic containers (9 cm diameter, 4.5
cm deep), ten per container. Each arena contains one treated bait
pellet and one water source (damp cotton wool ball), each placed in
a separate small weigh boat. The water is replenished ad lib
throughout the experiment.
[0451] Assessments are made at twice weekly intervals, with no more
than four days between assessments, until all the control insects
had either died or moulted to the adult stage (84 days). At each
assessment the insects are assessed as live or dead, and examined
for abnormalities. From day 46 onwards, once moulting to adult has
commenced, all insects (live and dead) are assessed as nymph or
adult. Surviving insects are weighed on day 55 of the trial. Four
replicates are performed for each of the treatments. During the
trial the test conditions are 25 to 33.degree. C. and 20 to 25%
relative humidity, with a 12:12 hour light:dark photoperiod.
D. Cloning of a HC Gene Fragment in a Vector Suitable for Bacterial
Production of Insect-Active Double-Stranded RNA
[0452] What follows is an example of cloning a DNA fragment
corresponding to a HC gene target in a vector for the expression of
double-stranded RNA in a bacterial host, although any vector
comprising a T7 promoter or any other promoter for efficient
transcription in bacteria, may be used (reference to
WO0001846).
[0453] The sequences of the specific primers used for the
amplification of target genes are provided in Table 8. The template
used is the pCR8/GW/topo vector containing any of target sequences.
The primers are used in a PCR reaction with the following
conditions: 5 minutes at 98.degree. C., followed by 30 cycles of 10
seconds at 98.degree. C., 30 seconds at 55.degree. C. and 2 minutes
at 72.degree. C., followed by 10 minutes at 72.degree. C. The
resulting PCR fragment is analyzed on agarose gel, purified
(QIAquick Gel Extraction kit, Cat. Nr. 28706, Qiagen), blunt-end
cloned into Srf I-linearized pGNA49A vector (reference to
WO00188121A1), and sequenced. The sequence of the resulting PCR
product corresponds to the respective sequence as given in Table
8-AD. The recombinant vector harbouring this sequence is named
pGBNJ00XX.
E. Expression and Production of a Double-Stranded RNA Target in Two
Strains of Escherichia coli: (1) AB309-105, and, (2) BL21(DE3)
[0454] The procedures described below are followed in order to
express suitable levels of insect-active double-stranded RNA of
insect target in bacteria. An RNaseIII-deficient strain, AB309-105,
is used in comparison to wild-type RNaseIII-containing bacteria,
BL21(DE3).
Transformation of AB309-105 and BL21(DE3)
[0455] Three hundred ng of the plasmid are added to and gently
mixed in a 50 .mu.A aliquot of ice-chilled chemically competent E.
coli strain AB309-105 or BL21(DE3). The cells are incubated on ice
for 20 minutes before subjecting them to a heat shock treatment of
37.degree. C. for 5 minutes, after which the cells are placed back
on ice for a further 5 minutes. Four hundred and fifty .mu.l of
room temperature SOC medium is added to the cells and the
suspension incubated on a shaker (250 rpm) at 37.degree. C. for 1
hour. One hundred .mu.l of the bacterial cell suspension is
transferred to a 500 ml conical flask containing 150 ml of liquid
Luria-Bertani (LB) broth supplemented with 100 .mu.g/ml
carbenicillin antibiotic. The culture is incubated on an Innova
4430 shaker (250 rpm) at 37.degree. C. overnight (16 to 18
hours).
Chemical Induction of Double-Stranded RNA Expression in AB309-105
and BL21(DE3)
[0456] Expression of double-stranded RNA from the recombinant
vector, pGBNJ003, in the bacterial strain AB309-105 or BL21(DE3) is
made possible since all the genetic components for controlled
expression are present. In the presence of the chemical inducer
isopropylthiogalactoside, or IPTG, the T7 polymerase will drive the
transcription of the target sequence in both antisense and sense
directions since these are flanked by oppositely oriented T7
promoters.
[0457] The optical density at 600 nm of the overnight bacterial
culture is measured using an appropriate spectrophotometer and
adjusted to a value of 1 by the addition of fresh LB broth. Fifty
ml of this culture is transferred to a 50 ml Falcon tube and the
culture then centrifuged at 3000 g at 15.degree. C. for 10 minutes.
The supernatant is removed and the bacterial pellet resuspended in
50 ml of fresh S complete medium (SNC medium plus 5 .mu.g/ml
cholesterol) supplemented with 100 .mu.g/ml carbenicillin and 1 mM
IPTG. The bacteria are induced for 2 to 4 hours at room
temperature.
Heat Treatment of Bacteria
[0458] Bacteria are killed by heat treatment in order to minimise
the risk of contamination of the artificial diet in the test
plates. However, heat treatment of bacteria expressing
double-stranded RNA is not a prerequisite for inducing toxicity
towards the insects due to RNA interference. The induced bacterial
culture is centrifuged at 3000 g at room temperature for 10
minutes, the supernatant discarded and the pellet subjected to
80.degree. C. for 20 minutes in a water bath. After heat treatment,
the bacterial pellet is resuspended in 1.5 ml MilliQ water and the
suspension transferred to a microfuge tube. Several tubes are
prepared and used in the bioassays for each refreshment. The tubes
are stored at -20.degree. C. until further use.
F. Laboratory Trials to Test Escherichia coli Expressing dsRNA
Targets Against Acheta domesticus
Plant-Based Bioassays
[0459] Whole plants are sprayed with suspensions of chemically
induced bacteria expressing dsRNA prior to feeding the plants to
HC. The are grown from in a plant growth room chamber. The plants
are caged by placing a 500 ml plastic bottle upside down over the
plant with the neck of the bottle firmly placed in the soil in a
pot and the base cut open and covered with a fine nylon mesh to
permit aeration, reduce condensation inside and prevent insect
escape. HC are placed on each treated plant in the cage. Plants are
treated with a suspension of E. coli AB309-105 harbouring the
pGBNJ001 plasmids or pGN29 plasmid. Different quantities of
bacteria are applied to the plants: for instance 66, 22, and 7
units, where one unit is defined as 10.sup.9 bacterial cells in 1
ml of a bacterial suspension at optical density value of 1 at 600
nm wavelength. In each case, a total volume of between 1 and 10 ml
s sprayed on the plant with the aid of a vaporizer. One plant is
used per treatment in this trial. The number of survivors are
counted and the weight of each survivor recorded.
[0460] Spraying plants with a suspension of E. coli bacterial
strain AB309-105 expressing target dsRNA from pGBNJ003 leed to a
dramatic increase in insect mortality when compared to pGN29
control. These experiments show that double-stranded RNA
corresponding to an insect gene target sequence produced in either
wild-type or RNaseIII-deficient bacterial expression systems is
toxic towards the insect in terms of substantial increases in
insect mortality and growth/development delay for larval survivors.
It is also clear from these experiments that an exemplification is
provided for the effective protection of plants/crops from insect
damage by the use of a spray of a formulation consisting of
bacteria expressing double-stranded RNA corresponding to an insect
gene target.
Example 13
Pyricularia grisea (Rice Blast)
A. Cloning P. grisea Partial Sequences
[0461] High quality, intact RNA is isolated from different growth
stages of P. grisea using TRIzol Reagent (Cat. Nr.
15596-026/15596-018, Invitrogen, Rockville, Md., USA) following the
manufacturer's instructions. Genomic DNA present in the RNA
preparation is removed by DNase treatment following the
manafacturer's instructions (Cat. Nr. 1700, Promega). cDNA is
generated using a commercially available kit (SuperScript.TM. III
Reverse Transcriptase, Cat. Nr. 18080044, Invitrogen, Rockville,
Md., USA) following the manufacturer's instructions.
[0462] To isolate cDNA sequences comprising a portion of a target
gene, PCR is performed with degenerate primers using Amplitaq Gold
(Cat. Nr. N8080240, Applied Biosystems) following the
manafacturer's instructions. The resultant PCR products are
fractionated and sequenced.
B. dsRNA Production of P. grisea Genes
[0463] dsRNA is synthesized in milligram amounts using a
commercially available kit, such as T7 Ribomax.TM. Express RNAi
System (Cat. Nr. P1700, Promega), following the manufacturer's
instructions. The resulting PCR products are analyzed on an agarose
gel and purified by a PCR purification kit (e.g. Qiaquick PCR
Purification Kit, Cat. Nr. 28106, Qiagen) and NaClO.sub.4
precipitation. The producr T7 forward and reverse templates are
mixed and the resulting RNA strands are annealed, then DNase and
RNase treated, and purified by sodium acetate, following the
manufacturer's instructions.
C. Expression and Production of a Double-Stranded RNA Target in Two
Strains of Escherichia coli: (1) AB309-105, and, (2) BL21(DE3)
[0464] The procedures described below are followed in order to
express suitable levels of fungal double-stranded RNA of fungal
target in bacteria. An RNaseIII-deficient strain, AB309-105, is
used in comparison to wild-type RNaseIII-containing bacteria,
BL21(DE3).
Transformation of AB309-105 and BL21(DE3)
[0465] Three hundred ng of the plasmid are added to and gently
mixed in a 50 .mu.l aliquot of ice-chilled chemically competent E.
coli strain AB309-105 or BL21(DE3). The cells are incubated on ice
for 20 minutes before subjecting them to a heat shock treatment of
37.degree. C. for 5 minutes, after which the cells are placed back
on ice for a further 5 minutes. Four hundred and fifty .mu.l of
room temperature SOC medium is added to the cells and the
suspension incubated on a shaker (250 rpm) at 37.degree. C. for 1
hour. One hundred .mu.l of the bacterial cell suspension is
transferred to a 500 ml conical flask containing 150 ml of liquid
Luria-Bertani (LB) broth supplemented with 100 .mu.g/ml
carbenicillin antibiotic. The culture is incubated on an Innova
4430 shaker (250 rpm) at 37.degree. C. overnight (16 to 18
hours).
Chemical Induction of Double-Stranded RNA Expression in AB309-105
and BL21(DE3)
[0466] Expression of double-stranded RNA from the recombinant
vector, pGBNJ003, in the bacterial strain AB309-105 or BL21(DE3) is
made possible since all the genetic components for controlled
expression are present. In the presence of the chemical inducer
isopropylthiogalactoside, or IPTG, the T7 polymerase will drive the
transcription of the target sequence in both antisense and sense
directions since these are flanked by oppositely oriented T7
promoters.
[0467] The optical density at 600 nm of the overnight bacterial
culture is measured using an appropriate spectrophotometer and
adjusted to a value of 1 by the addition of fresh LB broth. Fifty
ml of this culture is transferred to a 50 ml Falcon tube and the
culture then centrifuged at 3000 g at 15.degree. C. for 10 minutes.
The supernatant is removed and the bacterial pellet resuspended in
50 ml of fresh S complete medium (SNC medium plus 5 .mu.g/ml
cholesterol) supplemented with 100 .mu.g/ml carbenicillin and 1 mM
IPTG. The bacteria are induced for 2 to 4 hours at room
temperature.
Heat Treatment of Bacteria
[0468] Bacteria are killed by heat treatment in order to minimise
the risk of contamination of the artificial diet in the test
plates. However, heat treatment of bacteria expressing
double-stranded RNA is not a prerequisite for inducing toxicity
towards the insects due to RNA interference. The induced bacterial
culture is centrifuged at 3000 g at room temperature for 10
minutes, the supernatant discarded and the pellet subjected to
80.degree. C. for 20 minutes in a water bath. After heat treatment,
the bacterial pellet is resuspended in 1.5 ml MilliQ water and the
suspension transferred to a microfuge tube. Several tubes are
prepared and used in the bioassays for each refreshment. The tubes
are stored at -20.degree. C. until further use.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20180142237A9).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20180142237A9).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References