U.S. patent application number 15/348706 was filed with the patent office on 2017-04-27 for methods for genetic control of insect infestations in plants and compositions thereof.
The applicant listed for this patent is Monsanto Technology LLC. Invention is credited to James A. Baum, Claire A. CaJacob, Pascale Feldmann, Gregory R. Heck, Wendy T. Maddelein, Irene Maria Antonia Nooren, Geert Plaetinck, Ty T. Vaughn.
Application Number | 20170114362 15/348706 |
Document ID | / |
Family ID | 37497032 |
Filed Date | 2017-04-27 |
United States Patent
Application |
20170114362 |
Kind Code |
A1 |
Baum; James A. ; et
al. |
April 27, 2017 |
METHODS FOR GENETIC CONTROL OF INSECT INFESTATIONS IN PLANTS AND
COMPOSITIONS THEREOF
Abstract
The present invention relates to control of pest infestation by
inhibiting one or more biological functions. The invention provides
methods and compositions for such control, By feeding one or more
recombinant double stranded RNA molecules provided by the invention
to the pest, a reduction in pest infestation is obtained through
suppression of gene expression. The invention is also directed to
methods for making transgenic plants that express the double
stranded RNA molecules, and to particular combinations of
transgenic pesticidal agents for use in protecting plants from pest
infestation.
Inventors: |
Baum; James A.; (Webster
Groves, MO) ; CaJacob; Claire A.; (Chesterfield,
MO) ; Feldmann; Pascale; (Gent, BE) ; Heck;
Gregory R.; (Crystal Lake Park, MO) ; Nooren; Irene
Maria Antonia; (XH Utrecht, NL) ; Plaetinck;
Geert; (Merelbeke-Bottelare, BE) ; Maddelein; Wendy
T.; (Gijzenzele, BE) ; Vaughn; Ty T.;
(Clayton, MO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Monsanto Technology LLC |
St. Louis |
MO |
US |
|
|
Family ID: |
37497032 |
Appl. No.: |
15/348706 |
Filed: |
November 10, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13855328 |
Apr 2, 2013 |
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15348706 |
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12973783 |
Dec 20, 2010 |
8759611 |
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13855328 |
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11522307 |
Sep 15, 2006 |
7943819 |
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12973783 |
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60718034 |
Sep 16, 2005 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
Y02A 40/164 20180101;
Y02A 40/162 20180101; C12N 15/8285 20130101; C07H 21/04 20130101;
C12N 15/8286 20130101; C12N 15/8218 20130101; C07K 14/43563
20130101; C12N 15/113 20130101; C12N 15/8282 20130101; Y02A 40/146
20180101; C07K 14/43536 20130101 |
International
Class: |
C12N 15/82 20060101
C12N015/82; C07K 14/435 20060101 C07K014/435 |
Claims
1. A recombinant polynucleotide comprising a nucleic acid sequence
selected from the group consisting of: (a) a polynucleotide
comprising a nucleic acid sequence of SEQ ID NO: 819; (b) a
polynucleotide that hybridizes to a nucleic acid sequence of SEQ ID
NO: 819 under wash conditions of 5.times.SSC, 50% formamide and
42.degree. C. for 10 minutes; (c) a polynucleotide comprising at
least 70% sequence identity to a nucleic acid sequence of SEQ ID
NO: 819; (d) a fragment of at least 21 contiguous nucleotides of a
nucleic acid sequence of SEQ ID NO: 819, wherein ingestion by a
coleopteran plant pest of a double stranded ribonucleotide sequence
comprising at least one strand that is complementary to said
fragment inhibits the growth of said pest; and (e) a complement of
the sequence of (a), (b), (c) or (d); wherein the nucleic acid
sequence is operably linked to a heterologous promoter.
2-3. (canceled)
4. The recombinant polynucleotide of claim 1, defined as comprised
on a plant transformation vector.
5. A double stranded ribonucleotide sequence produced from the
expression of a polynucleotide according to claim 1, wherein
ingestion of said ribonucleotide sequence by a coleopteran plant
pest inhibits the growth of said pest.
6. The double stranded ribonucleotide sequence of claim 5, defined
as produced by preparing a recombinant polynucleotide sequence
comprising a first, a second and a third polynucleotide sequence,
wherein the first polynucleotide sequence comprises the recombinant
polynucleotide of claim 1, wherein the third polynucleotide
sequence is linked to the first polynucleotide sequence by the
second polynucleotide sequence, and wherein the third
polynucleotide sequence is substantially the reverse complement of
the first polynucleotide sequence such that the first and the third
polynucleotide sequences hybridize when transcribed into a
ribonucleic acid to form the double stranded ribonucleotide
molecule stabilized by the linked second ribonucleotide
sequence.
7. The double stranded ribonucleotide sequence of claim 5, wherein
ingestion of the polynucleotide sequence by the pest inhibits the
expression of a nucleotide sequence substantially complementary to
said polynucleotide sequence.
8. A cell transformed with the polynucleotide of claim 1.
9. The cell of claim 8, defined as a prokaryotic cell.
10. The cell of claim 8, defined as a eukaryotic cell.
11. The cell of claim 8, defined as a plant or bacterial cell.
12. A plant transformed with the polynucleotide of claim 1.
13. A seed of the plant of claim 12, wherein the seed comprises the
polynucleotide.
14. (canceled)
15. The plant of claim 12, wherein said polynucleotide is expressed
in a cell of the plant as a double stranded ribonucleotide sequence
and ingestion of an insect pest inhibitory amount of said double
stranded ribonucleotide sequence in a diet inhibits the pest from
further feeding on said diet.
16. The plant of claim 15, wherein the insect pest is selected from
the group consisting of Diabrotica virgifera, Diabrotica virgifera
virgifera, Diabrotica virgifera zea, Diabrotica balteata,
Diabrotica barberi, Diabrotica viridula, Diabrotica speciosa, and
Diabrotica undecimpunctata.
17. The plant of claim 15, wherein ingestion of the insect pest
inhibitory amount of the double stranded ribonucleotide sequence
stunts the growth of the pest.
18. A commodity product produced from a plant according to claim
12, wherein said commodity product comprises a detectable amount of
the polynucleotide of claim 1 or a ribonucleotide expressed
therefrom.
19-34. (canceled)
35. The plant of claim 12, wherein said plant is a corn plant.
36. The seed of claim 13, wherein said seed is corn seed.
37. The cell according to claim 11, wherein said plant cell is a
corn plant cell.
38. A cell of the plant of claim 35, wherein said cell comprises
the polynucleotide.
Description
PRIORITY CLAIM
[0001] This application claims the priority of U.S. Provisional
Application Ser. No. 60/718,034, filed Sep. 16, 2005, which
application is incorporated herein by reference in its
entirety.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING SUBMITTED ON A
COMPACT DISC
[0002] The Sequence Listing is submitted on one compact disc (Copy
1), together with a duplicate thereof (Copy 2), each created on
Sep. 15, 2006, and each containing one 669 kb file entitled
"MNDE002.APP.TXT." The material contained on the compact disc is
specifically incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates generally to genetic control
of pest infestations. More specifically, the present invention
relates to recombinant DNA technologies for post-transcriptionally
repressing or inhibiting expression of target coding sequences in
the cell of a pest to provide a pest-protective effect.
[0005] 2. Description of Related Art
[0006] The environment in which humans live is replete with pest
infestation. Pests including insects, arachnids, crustaceans,
fungi, bacteria, viruses, nematodes, flatworms, roundworms,
pinworms, hookworms, tapeworms, trypanosomes, schistosomes,
botflies, fleas, ticks, mites, and lice and the like are pervasive
in the human environment. A multitude of means have been utilized
for attempting to control infestations by these pests. Compositions
for controlling infestations by microscopic pests such as bacteria,
fungi, and viruses have been provided in the form of antibiotic
compositions, antiviral compositions, and antifungal compositions.
Compositions for controlling infestations by larger pests such as
nematodes, flatworm, roundworms, pinworms, heartworms, tapeworms,
trypanosomes, schistosomes, and the like have typically been in the
form of chemical compositions that can be applied to surfaces on
which pests are present or administered to infested animals in the
form of pellets, powders, tablets, pastes, or capsules and the
like. There is a great need in the art for improvement of these
methods and particularly for methods that would benefit the
environment relative to the prior techniques.
[0007] 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 pesticidal
agents. Chemical pesticidal agents are not selective. Applications
of chemical pesticides intended to control invertebrate pests, such
as coleopteran insects including corn rootworm species that are
harmful to various crops and other plants, exert their effects on
non-target fauna as well, often effectively sterilizing a field for
a period of time over which the pesticidal agents have been
applied. Chemical pesticidal agents 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.
Accumulations of these chemical pesticidal agents results in the
development of resistance to the agents and in species higher up
the evolutionary ladder, can act as mutagens and/or carcinogens to
cause irreversible and deleterious genetic modifications. Thus
there has been a particularly long felt need for environmentally
friendly methods for controlling or eradicating insect infestation
on or in plants, i.e., methods that are selective, environmentally
inert, non-persistent, and biodegradable, and that fit well into
pest resistance management schemes.
[0008] Compositions that include Bacillus thuringiensis (Bt)
bacteria have been commercially available and used as
environmentally safe and acceptable insecticides for more than
thirty years. The insecticidal effect of Bt bacteria do not persist
in the environment, are highly selective as to the target species
affected, exert their effects only upon ingestion by a target pest,
and have been shown to be harmless to plants and other non-targeted
organisms, including humans. Transgenic plants containing one or
more genes encoding insecticidal Bt protein are also available in
the art and are remarkably efficient in controlling insect pest
infestation. A substantial result of the use of recombinant plants
expressing Bt insecticidal proteins is a marked decrease in the
amount of chemical pesticidal agents that are applied to the
environment to control pest infestation in crop fields in areas in
which such transgenic crops are used. The decrease in application
of chemical pesticidal agents has resulted in cleaner soils and
cleaner waters running off of the soils into the surrounding
streams, rivers, ponds and lakes. In addition to these
environmental benefits, there has been a noticeable increase in the
numbers of beneficial insects in crop fields in which transgenic
insect resistant crops are grown because of the decrease in the use
of chemical insecticidal agents.
[0009] Antisense methods and compositions have been reported in the
art and are believed to exert their effects through the synthesis
of a single-stranded RNA molecule that in theory hybridizes in vivo
to a substantially complementary sense strand RNA molecule.
Antisense technology has been difficult to employ in many systems
for three principle reasons. First, the antisense sequence
expressed in the transformed cell is unstable. Second, the
instability of the antisense sequence expressed in the transformed
cell concomitantly creates difficulty in delivery of the sequence
to a host, cell type, or biological system remote from the
transgenic cell. Third, the difficulties encountered with
instability and delivery of the antisense sequence create
difficulties in attempting to provide a dose within the recombinant
cell expressing the antisense sequence that can effectively
modulate the level of expression of the target sense nucleotide
sequence.
[0010] There have been few improvements in technologies for
modulating the level of gene expression within a cell, tissue, or
organism, and in particular, a lack of developed technologies for
delaying, repressing or otherwise reducing the expression of
specific genes using recombinant DNA technology. Furthermore, as a
consequence of the unpredictability of these approaches, no
commercially viable means for modulating the level of expression of
a specific gene in a eukaryotic or prokaryotic organism is
available.
[0011] Double stranded RNA mediated inhibition of specific genes in
various pests has been previously demonstrated. dsRNA mediated
approaches to genetic control have been tested in the fruit fly
Drosophila melanogaster (Kennerdell and Carthew, 1998; Kennerdell
and Carthew, 2000). Kennerdell and Carthew (1998) describe a method
for delivery of dsRNA involved generating transgenic insects that
express double stranded RNA molecules or injecting dsRNA solutions
into the insect body or within the egg sac prior to or during
embryonic development.
[0012] Research investigators have previously demonstrated that
double stranded RNA mediated gene suppression can be achieved in
nematodes either by feeding or by soaking the nematodes in
solutions containing double stranded or small interfering RNA
molecules and by injection of the dsRNA molecules. Rajagopal et.
al. (2002) described failed attempts to suppress an endogenous gene
in larvae of the insect pest Spodoptera litura by feeding or by
soaking neonate larvae in 25690899.1-4-solutions containing dsRNA
specific for the target gene, but were successful in suppression
after larvae were injected with dsRNA into the hemolymph of
5.sup.th instar larvae using a microapplicator. Recently, Yadav et
al. (2006) reported that host-generated dsRNA produced in a plant
can protect such plants from infection by nematodes. Similarly,
U.S. Patent App. Pub. No. 2003/0150017 prophetically described a
preferred locus for inhibition of the lepidopteran larvae
Helicoverpa armigera using dsRNA delivered to the larvae by
ingestion of a plant transformed to produce the dsRNA. WO
2005/110068 teaches providing, in the diet of corn rootworm (CRW),
CRW-specific dsRNA directed to essential CRW genes. The dsRNA is
provided in the CRW diet in-vitro and in-planta, with the result
that CRW larvae are stunted or killed after feeding on the diet,
and this effect was demonstrated for several different genes.
[0013] Therefore, there has existed a need for identifying
efficacious nucleotide sequences for use in improved methods of
modulating gene expression by repressing, delaying or otherwise
reducing gene expression within a particular coleopteran pest for
the purpose of controlling pest infestation or to introduce novel
phenotypic traits.
SUMMARY OF THE INVENTION
[0014] In one aspect, the invention provides a method of inhibiting
expression of a target gene in a coleopteran pest. In certain
embodiments, the method comprises modulating or inhibiting
expression of one or more target genes in a coleopteran pest that
causes cessation of feeding, growth, development, reproduction
and/or infectivity and eventually result in the death of the
insect. The method comprises introduction of partial or fully,
stabilized double-stranded RNA (dsRNA), including its modified
forms such as small interfering RNA (siRNA) sequences, into the
cells or into the extracellular environment, such as the midgut,
within a coleopteran pest body wherein the dsRNA enters the cells
and inhibits expression of at least one or more target genes and
wherein the inhibition exerts a deleterious effect upon the
coleopteran pest. The methods and associated compositions may be
used for limiting or eliminating coleopteran pest infestation in or
on any pest host, pest symbiont, or environment in which a pest is
present by providing one or more compositions comprising the dsRNA
molecules described herein in the diet of the pest. The method will
find particular benefit for protecting plants from insect attack.
In one embodiment, the pest is defined as comprising a digestive
system pH within the range of from about 4.5 to about 9.5, from
about 5 to about 9, from about 6 to about 8, and from about pH
7.0.
[0015] In another aspect, the present invention provides exemplary
nucleic acid compositions that are homologous to at least a portion
of one or more native nucleic acid sequences in a target pest. In
certain embodiments, the pest is selected from among Diabrotica sp.
including Western Corn Rootworm (WCR, Diabrotica virgifera or
Diabrotica virgifera virgifera), Southern Corn Rootworm (SCR,
Diabrotica undecimpunctata howardi), Mexican Corn Rootworm (MCR,
Diabrotica virgifera zea), Brazilian Corn Rootworm (BZR, Diabrotica
balteata, Diabrotica viridula, Diabrotica speciosa), Northern Corn
rootworm (NCR, Diabrotica barberi), Diabrotica undecimpunctata; as
well as Colorado Potato Beetle (CPB, Leptinotarsa decemlineata),
Red Flour Beetle (RFB, Tribolium castaneum), and Mexican Bean
Beetle (Epilachna varivestis). In other embodiments the pest is
selected from among Lepidopteran insects including European Corn
Borer (ECB, Ostrinia nubilalis), Black Cutworm (BCW, Agrotis
ipsilon), Corn Earworm (CEW, Helicoverpa zea), Fall Armyworm (FAW,
Spodoptera frugiperda), Cotton Ball Weevil (BWV, Anthonomus
grandis), silkworms (Bombyx mori) and Manduca sexta, and from
Dipteran insects including Drosophila melanogaster, Anopheles
gambiae, and Aedes aegypti. Specific examples of such nucleic acids
provided by the invention are given in the attached sequence
listing as SEQ ID NO:1 through SEQ ID NO:906.
[0016] In yet another aspect, the invention provides a method for
suppression of gene expression in a coleopteran pest such as a corn
rootworm or related species that comprises the step of providing in
the diet of the pest a gene suppressive amount of at least one
dsRNA molecule transcribed from a nucleotide sequence as described
herein, at least one segment of which is complementary to an mRNA
sequence within the cells of the pest. The method may further
comprise observing the death, inhibition, stunting, or cessation of
feeding of the pest. A dsRNA molecule, including its modified form
such as an siRNA molecule, fed to a pest in accordance with the
invention may be at least from about 80, 81, 82, 83, 84, 85, 86,
87, 88 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or about 100%
identical to a RNA molecule transcribed from a nucleotide sequence
selected from the group consisting of SEQ ID NO:1 through SEQ ID
NO:906. In particular embodiments, the nucleotide sequence may be
selected from the group consisting of SEQ ID NO:697, SEQ ID
NOs:813-819, SEQ ID NO:841, and SEQ ID NO:874.
[0017] Accordingly, in another aspect of the present invention, a
set of isolated and purified nucleotide sequences as set forth in
SEQ ID NO:1 through SEQ ID NO:906 is provided. The present
invention provides a stabilized dsRNA molecule or the expression of
one or more miRNAs for inhibition of expression of a target gene in
a coleopteran pest expressed from these sequences and fragments
thereof. A stabilized dsRNA, including a miRNA or siRNA molecule
can comprise at least two coding sequences that are arranged in a
sense and an antisense orientation relative to at least one
promoter, wherein the nucleotide sequence that comprises a sense
strand and an antisense strand are linked or connected by a spacer
sequence of at least from about five to about one thousand
nucleotides, wherein the sense strand and the antisense strand may
be a different length, and wherein each of the two coding sequences
shares at least 80% sequence identity, at least 90%, at least 95%,
at least 98%, or 100% sequence identity, to any one or more
nucleotide sequence(s) set forth in set forth in SEQ ID NO:1
through SEQ ID NO:906.
[0018] Further provided by the invention is a fragment or
concatemer of a nucleic acid sequence selected from the group
consisting of SEQ ID NO:1 through SEQ ID NO:906. In particular
embodiments, the nucleotide sequence may comprise a fragment or
concatemer of a sequence selected from the group consisting of SEQ
ID NO:697, SEQ ID NOs:813-819, SEQ ID NO:841, and SEQ ID
NO:874.
[0019] The fragment may be defined as causing a the death,
inhibition, stunting, or cessation of feeding of a pest when
expressed as a dsRNA and provided to the pest. The fragment may,
for example, comprise at least about 19, 21, 23, 25, 40, 60, 80,
100, 125 or more contiguous nucleotides of any one or more of the
sequences in SEQ ID NO:1 through SEQ ID NO:906, or a complement
thereof. One beneficial DNA segment for use in the present
invention is at least from about 19 to about 23, or about 23 to
about 100 nucleotides up to about 2000 nucleotides or more in
length. Particularly useful will be dsRNA sequences including about
23 to about 300 nucleotides homologous to a pest target sequence.
The invention also provides a ribonucleic acid expressed from any
of such sequences including a dsRNA. A sequence selected for use in
expression of a gene suppression agent can be constructed from a
single sequence derived from one or more target pests and intended
for use in expression of an RNA that functions in the suppression
of a single gene or gene family in the one or more target pests, or
that the DNA sequence can be constructed as a chimera from a
plurality of DNA sequences.
[0020] In yet another aspect, the invention provides recombinant
DNA constructs comprising a nucleic acid molecule encoding a dsRNA
molecule described herein. The dsRNA may be formed by transcription
of one strand of the dsRNA molecule from a nucleotide sequence
which is at least from about 80% to about 100% identical to a
nucleotide sequence selected from the group consisting of SEQ ID
NO:1 through SEQ ID NO:906. Such recombinant DNA constructs may be
defined as producing dsRNA molecules capable of inhibiting the
expression of endogenous target gene(s) in a pest cell upon
ingestion. The construct may comprise a nucleotide sequence of the
invention operably linked to a promoter sequence that functions in
the host cell. Such a promoter may be tissue-specific and may, for
example, be specific to a tissue type which is the subject of pest
attack. In the case of rootworms, for example, it may be desired to
use a promoter providing root-preferred expression.
[0021] Nucleic acid constructs in accordance with the invention may
comprise at least one non-naturally occurring nucleotide sequence
that can be transcribed into a single stranded RNA capable of
forming a dsRNA molecule in vivo through hybridization. Such dsRNA
sequences self assemble and can be provided in the diet of a
coleopteran pest to achieve the desired inhibition.
[0022] A recombinant DNA construct may comprise two different
non-naturally occurring sequences which, when expressed in vivo as
dsRNA sequences and provided in the diet of a coleopteran pest,
inhibit the expression of at least two different target genes in
the cell of the coleopteran pest. In certain embodiments, at least
3, 4, 5, 6, 8 or 10 or more different dsRNAs are produced in a cell
or plant comprising the cell that have a pest-inhibitory effect.
The dsRNAs may expressed from multiple constructs introduced in
different transformation events or could be introduced on a single
nucleic acid molecule. The dsRNAs may be expressed using a single
promoter or multiple promoters. In one embodiments of the
invention, single dsRNAs are produced that comprise nucleic acids
homologous to multiple loci within a pest.
[0023] In still yet another aspect, the invention provides a
recombinant host cell having in its genome at least one recombinant
DNA sequence that is transcribed to produce at least one dsRNA
molecule that functions when ingested by a coleopteran pest to
inhibit the expression of a target gene in the pest. The dsRNA
molecule may be encoded by any of the nucleic acids described
herein and as set forth in the sequence listing. The present
invention also provides a transformed plant cell having in its
genome at least one recombinant DNA sequence described herein.
Transgenic plants comprising such a transformed plant cell are also
provided, including progeny plants of any generation, seeds, and
plant products, each comprising the recombinant DNA.
[0024] The methods and compositions of the present invention may be
applied to any monocot and dicot plant, depending on the
coleopteran pest control desired. Specifically, the plants are
intended to include, without limitation, alfalfa, aneth, apple,
apricot, artichoke, arugula, asparagus, avocado, banana, barley,
beans, beet, blackberry, blueberry, broccoli, brussel sprouts,
cabbage, canola, cantaloupe, carrot, cassava, cauliflower, celery,
cherry, cilantro, citrus, clementine, coffee, corn, cotton,
cucumber, Douglas fir, eggplant, endive, escarole, eucalyptus,
fennel, figs, gourd, grape, grapefruit, honey dew, jicama,
kiwifruit, lettuce, leeks, lemon, lime, Loblolly pine, mango,
melon, mushroom, nut, oat, okra, onion, orange, an ornamental
plant, papaya, parsley, pea, peach, peanut, pear, pepper,
persimmon, pine, pineapple, plantain, plum, pomegranate, poplar,
potato, pumpkin, quince, radiata pine, radicchio, radish,
raspberry, rice, rye, sorghum, Southern pine, soybean, spinach,
squash, strawberry, sugarbeet, sugarcane, sunflower, sweet potato,
sweetgum, tangerine, tea, tobacco, tomato, turf, a vine,
watermelon, wheat, yams, and zucchini plants. Thus, a plant
transformed with a recombinant DNA sequence as set forth in SEQ ID
NO:1 through SEQ ID NO:906, or concatemer, fragment, or complement
thereof, that is transcribed to produce at least one dsRNA molecule
that functions when ingested by a coleopteran pest to inhibit the
expression of a target gene in the pest is also provided by the
invention. In particular embodiments, the recombinant DNA sequence
may be selected from the group consisting of SEQ ID NO:697, SEQ ID
NOs:813-819, SEQ ID NO:841, and SEQ ID NO:874, or fragment,
complement, or concatemer thereof.
[0025] The invention also provides combinations of methods and
compositions for controlling coleopteran pest infestations. One
means provides a dsRNA method as described herein for protecting
plants from insect infestation along with one or more insecticidal
agents that exhibit features different from those exhibited by the
dsRNA methods and compositions. For example, one or more Bt
proteins may be provided in the diet of insect pests in combination
with one or more dsRNAs as described herein. A composition
formulated for topical application or derived using a transgenic
approach that combines dsRNA methods and compositions with Bt may
be used to provide synergies that were not known previously in the
art for controlling insect infestation. One synergy is the
reduction in the level of expression required for either the
dsRNA(s) or the Bt protein(s). When combined together, a lower
effective dose of each pest control agent could be used. It is
believed that the Bt insecticidal proteins create entry pores
through which the dsRNA molecules are able to penetrate more
effectively into spaces remote from the gut of the insect pest, or
more efficiently into the cells in the proximity of lesions created
by the Bt proteins, thus requiring less of either the Bt or the
dsRNA to achieve the desired insecticidal result or the desired
inhibition or suppression of a targeted biological function in the
target pest.
[0026] The present invention therefore provides a composition that
contains two or more different pesticidal agents each toxic to the
same pest or insect species, at least one of which comprises a
dsRNA described herein. In certain embodiments, the second agent
can be an agent selected from the group consisting of a patatin, a
Bacillus thuringiensis insecticidal protein, a Xenorhabdus
insecticidal protein, a Photorhabdus insecticidal protein, a
Bacillus laterosporous insecticidal protein, a Bacillus sphaericus
insecticidal protein, and a lignin. A Bacillus thuringiensis
insecticidal protein can be any of a number of insecticidal
proteins including but not limited to a Cry1, a Cry3, a TIC851, a
CryET70, a Cry22, a TIC901, a TIC1201, a TIC407, a TIC417, a binary
insecticidal protein CryET33 and CryET34, a binary insecticidal
protein CryET80 and CryET76, a binary insecticidal protein TIC100
and TIC101, a binary insecticidal protein PS149B1, a VIP
insecticidal protein, a TIC900 or related protein, or combinations
of the insecticidal proteins ET29 or ET37 with insecticidal
proteins TIC810 or TIC812, and insecticidal chimeras of any of the
preceding insecticidal proteins.
[0027] A ribonucleic acid that is provided in a diet can be
provided in an artificial diet formulated to meet particular
nutritional requirements for maintaining a pest on such diet. The
diet may be supplemented with a pest controlling amount of an RNA
that has been purified from a separate expression system to
determine a pest controlling amount of RNA composition or to
determine extent of suppressive activity upon ingestion of the
supplemented diet by the pest. The diet can also be a recombinant
cell transformed with a DNA sequence constructed for expression of
the agent, the RNA, or the gene suppression agent. Upon ingestion
of one or more such transformed cells by the pest, a desired
phenotypic result is observed, indicating that the agent has
functioned to inhibit the expression of a target nucleotide
sequence that is within the cells of the pest.
[0028] A gene targeted for suppression can encode an essential
protein, the predicted function of which is selected from the group
consisting of muscle formation, juvenile hormone formation,
juvenile hormone regulation, ion regulation and transport, protein
synthesis and transport, digestive enzyme synthesis, 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, an unknown function, and apoptosis.
[0029] Another aspect of the present invention also provides
methods for improving the yield of a crop produced from a crop
plant subjected to insect pest infestation, said method comprising
the steps of a) introducing a polynucleotide comprising a sequence
selected from SEQ ID NO:1 through SEQ ID NO:906 or a complement or
concatemer or fragment thereof into said crop plant; and b)
cultivating the crop plant to allow the expression of said
polynucleotide, wherein expression of the polynucleotide inhibits
feeding by insect pests and loss of yield due to pest
infestation.
[0030] In certain embodiments, the expression of the polynucleotide
produces an RNA molecule that suppresses at least a first target
gene in an insect pest that has ingested a portion of said crop
plant, wherein the target gene performs at least one essential
function selected from the group consisting of feeding by the pest,
viability of the pest, pest cell apoptosis, differentiation and
development of the pest or any pest cell, sexual reproduction by
the pest, muscle formation, muscle twitching, muscle contraction,
juvenile hormone formation and/or reduction, 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, egg
formation, larval maturation, digestive enzyme formation,
haemolymph synthesis, haemolymph maintenance, neurotransmission,
larval stage transition, pupation, emergence from pupation, cell
division, energy metabolism, respiration, cytoskeletal structure
synthesis and maintenance, nucleotide metabolism, nitrogen
metabolism, water use, water retention, and sensory perception.
[0031] In other embodiments, the insect pest is a corn rootworm
pest selected from the group consisting of Diabrotica
undecimpunctata howardi (Southern Corn Rootworm (SCR)), Diabrotica
virgifera virgifera (Western Corn Rootworm (WCR)), Diabrotica
barberi (Northern Corn Rootworm (NCR)), Diabrotica virgifera zea
(Mexican Corn Rootworm (MCR)), Diabrotica balteata (Brazilian Corn
Rootworm (BZR)), Diabrotica viridula (Brazilian Corn Rootworm
(BZR)), and Diabrotica speciosa (Brazilian Corn Rootworm
(BZR)).
[0032] Methods for improving the drought tolerance of a crop
produced from a crop plant subjected to insect pest infestation,
said method comprising the steps of a) introducing a polynucleotide
sequence selected from SEQ ID NO:1 through SEQ ID NO:906, or a
fragment thereof, into said crop plant; and b) cultivating the crop
plant to allow the expression of said polynucleotide, wherein
expression of the polynucleotide inhibits feeding by insects pests
and loss of drought tolerance due to pest infestation, are also
provided.
[0033] Yet another aspect of the invention further provides
agronomically and commercially important products and/or
compositions of matter including, but not limited to, animal feed,
commodities, products and by-products that are intended for use as
food for human consumption or for use in compositions and
commodities that are intended for human consumption including but
not limited to corn flour, corn meal, corn syrup, corn oil, corn
starch, popcorn, corn cakes, cereals, and the like. Such
compositions may be defined as containing detectable amounts of a
nucleotide sequence set forth herein, and thus are also diagnostic
for any transgenic event containing such nucleotide sequences.
These products are useful at least because they are likely to be
derived from crops propagated with fewer pesticides and
organophosphates as a result of their incorporation of the
nucleotides of the present invention for controlling the
infestation of coleopteran pests in plants. Such commodities and
commodity products can be produced from seed produced from a
transgenic plant, wherein the transgenic plant expresses RNA from
one or more contiguous nucleotides of the present invention or
nucleotides of one or more coleopteran pests and the complements
thereof. Such commodities and commodity products may also be useful
in controlling coleopteran pests of such commodity and commodity
products, such as for example, control of flour weevils, because of
the presence in the commodity or commodity product of the pest gene
suppressive RNA expressed from a gene sequence as set forth in the
present invention.
[0034] A method of producing such a commodity product comprising
obtaining a plant transformed with a polynucleotide comprising a
sequence selected from the group consisting of SEQ ID NO:1 through
SEQ ID NO:906, or a concatemer or fragment or complement thereof,
and preparing a commodity product from the plant or part thereof is
also provided. Further, a method of producing food or feed,
comprising obtaining a plant transformed with a polynucleotide
selected from the group consisting of SEQ ID NO:1 through SEQ ID
NO:906 or a fragment or complement thereof, and preparing food or
feed from said plant or part thereof is yet another aspect of the
invention.
[0035] The invention also provides a computer readable medium
having recorded thereon one or more of the nucleotide sequences as
set forth in SEQ ID NO:1 through SEQ ID NO:906, or complements
thereof, for use in a number of computer based applications,
including but not limited to DNA identity and similarity searching,
protein identity and similarity searching, transcription profiling
characterizations, comparisons between genomes, and artificial
hybridization analyses.
BRIEF DESCRIPTION OF THE FIGURES
[0036] FIG. 1: Bioassay of F1 corn plant events transformed with
pMON98503 (SEQ ID NO:820) and challenged with Western Corn Rootworm
(WCR).
[0037] FIG. 2: Bioassay of F1 corn plant events transformed with
pMON98504 comprising concatemer C1 (SEQ ID NO:821) and challenged
with Western Corn Rootworm (WCR).
[0038] FIG. 3: Selection of Dv49 and Dv248 fragments and schematic
design of Dv49-Dv248 concatemer C38.
[0039] FIG. 4: dsRNAs F1-F13 synthesized based on concatemer
C38.
[0040] FIG. 5: DV49-DV248 concatemer 38 dose response (Fragments
F1-F6).
[0041] FIG. 6: DV49-DV248 concatemer 38 dose response (Fragments
F7-F10).
[0042] FIG. 7: DV49-DV248 concatemer 38 dose response (Fragments
F11-F13).
DETAILED DESCRIPTION OF THE INVENTION
[0043] The following is a detailed description of the invention
provided to aid those skilled in the art in practicing the present
invention. Those of ordinary skill in the art may make
modifications and variations in the embodiments described herein
without departing from the spirit or scope of the present
invention.
[0044] The present invention provides methods and compositions for
genetic control of pest infestations. For example, the present
invention provides recombinant DNA technologies to
post-transcriptionally repress or inhibit expression of a target
coding sequence in the cell of a pest to provide a pest-protective
effect by feeding to the pest one or more double stranded or small
interfering ribonucleic acid (RNA) molecules transcribed from all
or a portion of a target coding sequence, thereby controlling the
infestation. Therefore, the present invention relates to
sequence-specific inhibition of expression of coding sequences
using double-stranded RNA (dsRNA), including small interfering RNA
(siRNA), to achieve the intended levels of pest control.
[0045] Isolated and substantially purified nucleic acid molecules
including but not limited to non-naturally occurring nucleotide
sequences and recombinant DNA constructs for transcribing dsRNA
molecules of the present invention are provided that suppress or
inhibit the expression of an endogenous coding sequence or a target
coding sequence in the pest when introduced thereto. Transgenic
plants that (a) contain nucleotide sequences encoding the isolated
and substantially purified nucleic acid molecules and the
non-naturally occurring recombinant DNA constructs for transcribing
the dsRNA molecules for controlling plant pest infestations, and
(b) display resistance and/or enhanced tolerance to the insect
infestations, are also provided. Compositions containing the dsRNA
nucleotide sequences of the present invention for use in topical
applications onto plants or onto animals or into the environment of
an animal to achieve the elimination or reduction of pest
infestation are also described.
[0046] The inventors have herein discovered that, contrary to the
teachings in the prior art, feeding a composition containing double
stranded RNA molecules consisting of sequences found within one or
more expressed nucleotide sequences of a coleopteran species to the
species from which the nucleotide sequences were obtained results
in the inhibition of one or more biological functions within the
coleopteran species. Particularly, the inventors have discovered
that feeding the double stranded RNA molecules described herein to
crop pest species such as corn rootworms results in the death or
inhibition of development and differentiation of insect pests that
ingest these compositions.
[0047] The inventors have identified the nucleotide sequences
described herein as providing plant protective effects against
coleopteran pest species. Amino acid sequences encoded by the cDNA
sequences have been deduced and compared to known amino acid
sequences. Many of the sequences are predicted to encode proteins
that have some annotation information associated with them. The
annotation information that is associated with a particular
nucleotide sequence and protein sequence encoded therefrom is based
on homology or similarity between the amino acid sequences deduced
through translation of the coding sequences described herein as set
forth and amino acid sequences that are known in the art in
publicly available databases.
[0048] cDNA sequences encoding proteins or parts of proteins
essential for survival, such as amino acid sequences involved in
various metabolic or catabolic biochemical pathways, cell division,
reproduction, energy metabolism, digestion, neurological function
and the like were selected for use in preparing double stranded RNA
molecules that were provided in the diet of coleopteran pests. As
described herein, ingestion by a target pest of compositions
containing one or more dsRNAs, at least one segment of which
corresponds to at least a substantially identical segment of RNA
produced in the cells of the target pest, resulted in death,
stunting, or other inhibition of the target pest. These results
indicated that a nucleotide sequence, either DNA or RNA, derived
from a coleopteran pest can be used to construct plant cells
resistant to infestation by the pest. The pest host, for example,
can be transformed to contain one or more of the nucleotide
sequences derived from the coleopteran pest. The nucleotide
sequence transformed into the pest host or symbiont may encode one
or more RNAs that form into a dsRNA sequence in the cells or
biological fluids within the transformed host or symbiont, thus
making the dsRNA available in the diet of the pest if/when the pest
feeds upon the transgenic host or symbiont, resulting in the
suppression of expression of one or more genes in the cells of the
pest and ultimately the death, stunting, or other inhibition of the
pest
[0049] The present invention relates generally to genetic control
of coleopteran pest infestations in host organisms. More
particularly, the present invention includes the methods for
delivery of pest control agents to a coleopteran pest. Such pest
control agents cause, directly or indirectly, an impairment in the
ability of the pest to maintain itself, grow or otherwise infest a
target host or symbiont. The present invention provides methods for
employing stabilized dsRNA molecules in the diet of the pest as a
means for suppression of targeted genes in the pest, thus achieving
desired control of pest infestations in, or about the host or
symbiont targeted by the pest.
[0050] In accomplishing the foregoing, the present invention
provides a method of inhibiting expression of a target gene in a
coleopteran pest, including for example, corn rootworms or other
coleopteran insect species, resulting in the cessation of feeding,
growth, development, reproduction, infectivity, and eventually may
result in the death of the pest. The method comprises in one
embodiment introducing partial or fully stabilized double-stranded
RNA (dsRNA) nucleotide molecules into a nutritional composition
that the pest relies on as a food source, and making the
nutritional composition available to the pest for feeding.
Ingestion of the nutritional composition containing the double
stranded or siRNA molecules results in the uptake of the molecules
by the cells of the pest, resulting in the inhibition of expression
of at least one target gene in the cells of the pest. Inhibition of
the target gene exerts a deleterious effect upon the pest.
[0051] In certain embodiments, dsRNA molecules provided by the
invention comprise nucleotide sequences complementary to a sequence
as set forth in any of SEQ ID NO:1 through SEQ ID NO:906, the
inhibition of which in a pest organism results in the reduction or
removal of a protein or nucleotide sequence agent that is essential
for the pests' growth and development or other biological function.
The nucleotide sequence selected may exhibit from about 80% to at
least about 100% sequence identity to one of the nucleotide
sequences as set forth in SEQ ID NO:1 through SEQ ID NO:906, as set
forth in the sequence listing, including the complement thereof.
Such inhibition can be described as specific in that a nucleotide
sequence from a portion of the target gene is chosen from which the
inhibitory dsRNA or siRNA is transcribed. The method is effective
in inhibiting the expression of at least one target gene and can be
used to inhibit many different types of target genes in the pest.
In particular embodiments, the nucleotide sequence may be selected
from the group consisting of SEQ ID NO:697, SEQ ID NOs:813-819, SEQ
ID NO:841, and SEQ ID NO:874.
[0052] The sequences identified as having a pest protective effect
may be readily expressed as dsRNA molecules through the creation of
appropriate expression constructs. For example, such sequences can
be expressed as a hairpin and stem and loop structure by taking a
first segment corresponding to a sequence selected from SEQ ID NO:1
through SEQ ID NO:906 or a fragment thereof, linking this sequence
to a second segment spacer region that is not homologous or
complementary to the first segment, and linking this to a third
segment that transcribes an RNA, wherein at least a portion of the
third segment is substantially complementary to the first segment.
Such a construct forms a stem and loop structure by hybridization
of the first segment with the third segment and a loop structure
forms comprising the second segment (WO94/01550, WO98/05770, US
2002/0048814A1, and US 2003/0018993A1).
[0053] A. Nucleic Acid Compositions and Constructs
[0054] The invention provides recombinant DNA constructs for use in
achieving stable transformation of particular host or symbiont pest
targets. Transformed host or symbiont pest targets may express
pesticidally effective levels of preferred dsRNA or siRNA molecules
from the recombinant DNA constructs, and provide the molecules in
the diet of the pest. Pairs of isolated and purified nucleotide
sequences may be provided from cDNA library and/or genomic library
information. The pairs of nucleotide sequences may be derived from
any preferred coleopteran pest for use as thermal amplification
primers to generate DNA templates for the preparation of dsRNA and
siRNA molecules of the present invention.
[0055] As used herein, the term "nucleic acid" refers to a single
or double-stranded polymer of deoxyribonucleotide or ribonucleotide
bases read from the 5' to the 3' end. The "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.
The term "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. 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. 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.
[0056] 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 an insect that comprises an RNA sequence encoded by a nucleotide
sequence within the genome of the insect. Thus, after ingestion of
the stabilized RNA sequence down-regulation of the nucleotide
sequence of the target gene in the cells of the insect may be
obtained resulting in a deleterious effect on the maintenance,
viability, proliferation, reproduction and infestation of the
insect.
[0057] As used herein, the term "substantially homologous" or
"substantial homology", with reference to a nucleic acid sequence,
includes a nucleotide sequence that hybridizes under stringent
conditions to the coding sequence as set forth in any of SEQ ID
NO:1 through SEQ ID NO:906 as set forth in the sequence listing, or
the complements thereof. Sequences that hybridize under stringent
conditions to any of SEQ ID NO:1 through SEQ ID NO:906 as set forth
in the sequence listing, or the complements thereof, are those that
allow an antiparallel alignment to take place between the two
sequences, and the two sequences are then able, under stringent
conditions, to form hydrogen bonds with corresponding bases on the
opposite strand to form a duplex molecule that is sufficiently
stable under the stringent conditions to be detectable using
methods well known in the art. Substantially homologous sequences
have preferably from about 70% to about 80% sequence identity, or
more preferably from about 80% to about 85% sequence identity, or
most preferable from about 90% to about 95% sequence identity, to
about 99% sequence identity, to the referent nucleotide sequences
as set forth in any of SEQ ID NO:1 through SEQ ID NO:906 as set
forth in the sequence listing, or the complements thereof.
[0058] As used herein, the term "sequence identity", "sequence
similarity" or "homology" is used to describe sequence
relationships between two or more nucleotide sequences. The
percentage of "sequence identity" between two sequences is
determined by comparing two optimally aligned sequences over a
comparison window, wherein the portion of the 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 or amino acid residue
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. A sequence
that is identical at every position in comparison to a reference
sequence is said to be identical to the reference sequence and
vice-versa. A first nucleotide sequence when observed in the 5' to
3' direction is said to be a "complement" of, or complementary to,
a second or reference nucleotide sequence observed in the 3' to 5'
direction if the first nucleotide sequence exhibits complete
complementarity with the second or reference sequence. As used
herein, nucleic acid sequence molecules are said to exhibit
"complete complementarity" when every nucleotide of one of the
sequences read 5' to 3' is complementary to every nucleotide of the
other sequence when read 3' to 5'. A nucleotide sequence that is
complementary to a reference nucleotide sequence will exhibit a
sequence identical to the reverse complement sequence of the
reference nucleotide sequence. These terms and descriptions are
well defined in the art and are easily understood by those of
ordinary skill in the art.
[0059] As used herein, a "comparison window" refers to a conceptual
segment of at least 6 contiguous positions, usually about 50 to
about 100, more usually about 100 to about 150, in which a sequence
is compared to a reference sequence of the same number of
contiguous positions after the two sequences are optimally aligned.
The comparison window may comprise additions or deletions (i.e.
gaps) of about 20% or less as compared to the reference sequence
(which does not comprise additions or deletions) for optimal
alignment of the two sequences Those skilled in the art should
refer to the detailed methods used for sequence alignment in the
Wisconsin Genetics Software Package Release 7.0, Genetics Computer
Group, 575 Science Drive Madison, Wis., USA) or refer to Ausubel et
al. (1998) for a detailed discussion of sequence analysis.
[0060] The present invention provides DNA sequences capable of
being expressed as an RNA in a cell or microorganism to inhibit
target gene expression in a cell, tissue or organ of an insect. The
sequences comprises a DNA molecule coding for one or more different
nucleotide sequences, wherein each of the different nucleotide
sequences comprises a sense nucleotide sequence and an antisense
nucleotide sequence connected by a spacer sequence coding for a
dsRNA molecule of the present invention. The spacer sequence
constitutes part of the sense nucleotide sequence or the antisense
nucleotide sequence and forms within the dsRNA molecule between the
sense and antisense sequences. The sense nucleotide sequence or the
antisense nucleotide sequence is substantially identical to the
nucleotide sequence of the target gene or a derivative thereof or a
complementary sequence thereto. The dsDNA molecule may be placed
operably under the control of a promoter sequence that functions in
the cell, tissue or organ of the host expressing the dsDNA to
produce dsRNA molecules. In one embodiment, the DNA sequence may be
derived from a nucleotide sequence as set forth in SEQ ID NO:1
through SEQ ID NO:906 in the sequence listing.
[0061] The invention also provides a DNA sequence for expression in
a cell of a plant that, upon expression of the DNA to RNA and
ingestion by a target pest achieves suppression of a target gene in
a cell, tissue or organ of an insect pest. The dsRNA at least
comprises one or multiple structural gene sequences, wherein each
of the structural gene sequences comprises a sense nucleotide
sequence and an antisense nucleotide sequence connected by a spacer
sequence that forms a loop within the complementary and antisense
sequences. The sense nucleotide sequence or the antisense
nucleotide sequence is substantially identical to the nucleotide
sequence of the target gene, derivative thereof, or sequence
complementary thereto. The one or more structural gene sequences is
placed operably under the control of one or more promoter
sequences, at least one of which is operable in the cell, tissue or
organ of a prokaryotic or eukaryotic organism, particularly a
plant.
[0062] A gene sequence or fragment for pest control according to
the invention may be cloned between two tissue specific promoters,
such as two root specific promoters which are operable in a
transgenic plant cell and therein expressed to produce mRNA in the
transgenic plant cell that form dsRNA molecules thereto. The dsRNA
molecules contained in plant tissues are ingested by an insect so
that the intended suppression of the target gene expression is
achieved.
[0063] A nucleotide sequence provided by the present invention may
comprise an inverted repeat separated by a "spacer sequence." The
spacer sequence may be a region comprising any sequence of
nucleotides that facilitates secondary structure formation between
each repeat, where this is required. In one embodiment of the
present invention, the spacer sequence is part of the sense or
antisense coding sequence for mRNA. The spacer sequence may
alternatively comprise any combination of nucleotides or homologues
thereof that are capable of being linked covalently to a nucleic
acid molecule. The spacer sequence may comprise a sequence of
nucleotides of at least about 10-100 nucleotides in length, or
alternatively at least about 100-200 nucleotides in length, at
least 200-400 about nucleotides in length, or at least about
400-500 nucleotides in length.
[0064] The nucleic acid molecules or fragment of the nucleic acid
molecules or other nucleic acid molecules in the sequence listing
are capable of specifically hybridizing to other nucleic acid
molecules under certain circumstances. As used herein, two nucleic
acid molecules are said to be capable of specifically hybridizing
to one another if the two molecules are capable of forming an
anti-parallel, double-stranded nucleic acid structure. A nucleic
acid molecule is said to be the complement of another nucleic acid
molecule if they exhibit complete complementarity. Two molecules
are said to be "minimally complementary" if they can hybridize to
one another with sufficient stability to permit them to remain
annealed to one another under at least conventional
"low-stringency" conditions. Similarly, the molecules are said to
be complementary if they can hybridize to one another with
sufficient stability to permit them to remain annealed to one
another under conventional "high-stringency" conditions.
Conventional stringency conditions are described by Sambrook, et
al. (1989), and by Haymes et al. (1985).
[0065] Departures from complete complementarity are therefore
permissible, as long as such departures do not completely preclude
the capacity of the molecules to form a double-stranded structure.
Thus, in order for a nucleic acid molecule or a fragment of the
nucleic acid molecule to serve as a primer or probe it needs only
be sufficiently complementary in sequence to be able to form a
stable double-stranded structure under the particular solvent and
salt concentrations employed.
[0066] Appropriate stringency conditions which promote DNA
hybridization are, for example, 6.0.times. sodium chloride/sodium
citrate (SSC) at about 45.degree. C., followed by a wash of
2.0.times.SSC at 50.degree. C., are known to those skilled in the
art or can be found in Current Protocols in Molecular Biology
(1989). For example, the salt concentration in the wash step can be
selected from a low stringency of about 2.0.times.SSC at 50.degree.
C. to a high stringency of about 0.2.times.SSC at 50.degree. C. In
addition, the temperature in the wash step can be increased from
low stringency conditions at room temperature, about 22.degree. C.,
to high stringency conditions at about 65.degree. C. Both
temperature and salt may be varied, or either the temperature or
the salt concentration may be held constant while the other
variable is changed. A nucleic acid for use in the present
invention may specifically hybridize to one or more of nucleic acid
molecules from WCR or complements thereof under such conditions.
Preferably, a nucleic acid for use in the present invention will
exhibit at least from about 80%, or at least from about 90%, or at
least from about 95%, or at least from about 98% or even about 100%
sequence identity with one or more nucleic acid molecules as set
forth in SEQ ID NO:1 through SEQ ID NO:906 as set forth in the
sequence listing.
[0067] Nucleic acids of the present invention may also be
synthesized, either completely or in part, especially where it is
desirable to provide plant-preferred sequences, by methods known in
the art. Thus, all or a portion of the nucleic acids of the present
invention may be synthesized using codons preferred by a selected
host. Species-preferred codons may be determined, for example, from
the codons used most frequently in the proteins expressed in a
particular host species. Other modifications of the nucleotide
sequences may result in mutants having slightly altered
activity.
[0068] dsRNA or siRNA nucleotide sequences comprise double strands
of polymerized ribonucleotide and may include modifications to
either the phosphate-sugar backbone or the nucleoside.
Modifications in RNA structure may be tailored to allow specific
genetic inhibition.
[0069] In one embodiment, the dsRNA molecules may be modified
through an enzymatic process so that siRNA molecules may be
generated. The siRNA can efficiently mediate the down-regulation
effect for some target genes in some insects. This enzymatic
process may be accomplished by utilizing an RNAse III enzyme or a
DICER enzyme, present in the cells of an insect, a vertebrate
animal, a fungus or a plant in the eukaryotic RNAi pathway
(Elbashir et al., 2002; Hamilton and Baulcombe, 1999). This process
may also utilize a recombinant DICER or RNAse III introduced into
the cells of a target insect through recombinant DNA techniques
that are readily known to the skilled in the art. Both the DICER
enzyme and RNAse III, being naturally occurring in an insect or
being made through recombinant DNA techniques, cleave larger dsRNA
strands into smaller oligonucleotides. The DICER enzymes
specifically cut the dsRNA molecules into siRNA pieces each of
which is about 19-25 nucleotides in length while the RNAse III
enzymes normally cleave the dsRNA molecules into 12-15 base-pair
siRNA. The siRNA molecules produced by the either of the enzymes
have 2 to 3 nucleotide 3' overhangs, and 5' phosphate and 3'
hydroxyl termini. The siRNA molecules generated by RNAse III enzyme
are the same as those produced by Dicer enzymes in the eukaryotic
RNAi pathway and are hence then targeted and degraded by an
inherent cellular RNA-degrading mechanism after they are
subsequently unwound, separated into single-stranded RNA and
hybridize with the RNA sequences transcribed by the target gene.
This process results in the effective degradation or removal of the
RNA sequence encoded by the nucleotide sequence of the target gene
in the insect. The outcome is the silencing of a particularly
targeted nucleotide sequence within the insect. Detailed
descriptions of enzymatic processes can be found in Hannon
(2002).
[0070] A nucleotide sequence of the present invention can be
recorded on computer readable media. As used herein, "computer
readable media" refers to any tangible medium of expression that
can be read and accessed directly by a computer. Such media
include, but are not limited to: magnetic storage media, such as
floppy discs, hard disc, storage medium, and magnetic tape: optical
storage media such as CD-ROM; electrical storage media such as RAM
and ROM; optical character recognition formatted computer files,
and hybrids of these categories such as magnetic/optical storage
media. A skilled artisan can readily appreciate that any of the
presently known computer readable mediums can be used to create a
manufacture comprising computer readable medium having recorded
thereon a nucleotide sequence of the present invention.
[0071] As used herein, "recorded" refers to a process for storing
information on computer readable medium. A skilled artisan can
readily adopt any of the presently known methods for recording
information on computer readable medium to generate media
comprising the nucleotide sequence information of the present
invention. A variety of data storage structures are available to a
skilled artisan for creating a computer readable medium having
recorded thereon a nucleotide sequence of the present invention.
The choice of the data storage structure will generally be based on
the means chosen to access the stored information. In addition, a
variety of data processor programs and formats can be used to store
the nucleotide sequence information of the present invention on
computer readable medium. The sequence information can be
represented in a word processing text file, formatted in
commercially-available software such as WordPerfect and Microsoft
Word, or represented in the form of an ASCII text file, stored in a
database application, such as DB2, Sybase, Oracle, or the like. The
skilled artisan can readily adapt any number of data processor
structuring formats (e.g. text file or database) in order to obtain
computer readable medium having recorded thereon the nucleotide
sequence information of the present invention.
[0072] Computer software is publicly available which allows a
skilled artisan to access sequence information provided in a
computer readable medium. Software that implements the BLAST
(Altschul et al., 1990) and BLAZE (Brutlag, et al., 1993) search
algorithms on a Sybase system can be used to identify open reading
frames (ORFs) within sequences such as the Unigenes and EST's that
are provided herein and that contain homology to ORFs or proteins
from other organisms. Such ORFs are protein-encoding fragments
within the sequences of the present invention and are useful in
producing commercially important proteins such as enzymes used in
amino acid biosynthesis, metabolism, transcription, translation,
RNA processing, nucleic acid and a protein degradation, protein
modification, and DNA replication, restriction, modification,
recombination, and repair.
[0073] The present invention further provides systems, particularly
computer-based systems, which contain the sequence information
described herein. Such systems are designed to identify
commercially important fragments of the nucleic acid molecule of
the present invention. As used herein, "a computer-based system"
refers to the hardware means, software means, and data storage
means used to analyze the nucleotide sequence information of the
present invention. The minimum hardware means of the computer-based
systems of the present invention comprises a central processing
unit (CPU), input means, output means, and data storage means. A
skilled artisan can readily appreciate that any one of the
currently available computer-based system are suitable for use in
the present invention.
[0074] As used herein, "a target structural motif," or "target
motif," refers to any rationally selected sequence or combination
of sequences in which the sequences or sequence(s) are chosen based
on a three-dimensional configuration that is formed upon the
folding of the target motif. There are a variety of target motifs
known in the art. Protein target motifs include, but are not
limited to, enzymatic active sites and signal sequences. Nucleic
acid target motifs include, but are not limited to, promoter
sequences, cis elements, hairpin structures and inducible
expression elements (protein binding sequences).
[0075] B. Recombinant Vectors and Host Cell Transformation
[0076] A recombinant DNA 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 DNA 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 NO:1 through SEQ ID
NO:906 or fragments or complements 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.
[0077] Expression and cloning vectors generally contain a selection
gene, also referred to as a selectable marker. This gene encodes a
protein necessary for the survival or growth of transformed host
cells grown in a selective culture medium. Typical selection genes
encode proteins that (a) confer resistance to antibiotics or other
toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline,
(b) complement auxotrophic deficiencies, or (c) supply critical
nutrients not available from complex media, e.g., the gene encoding
D-alanine racemase for Bacilli. Those cells that are successfully
transformed with a heterologous protein or fragment thereof produce
a protein conferring drug resistance and thus survive the selection
regimen.
[0078] 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.
[0079] The term "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.
[0080] Alternatively, the expression constructs can be integrated
into the 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.
[0081] 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.
[0082] 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), pC1/1 (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.
[0083] 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.
[0084] 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
(On-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).
[0085] The present invention also contemplates transformation of a
nucleotide sequence of the present invention into a plant to
achieve pest inhibitory levels of expression of one or more dsRNA
molecules. 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 insect.
[0086] The transformation vector may be termed a dsDNA construct
and may also be defined as a recombinant molecule, an insect
control agent, a genetic molecule or a chimeric genetic construct.
A chimeric genetic construct of the present invention may comprise,
for example, nucleotide sequences encoding one or more antisense
transcripts, one or more sense transcripts, one or more of each of
the aforementioned, wherein all or part of a transcript therefrom
is homologous to all or part of an RNA molecule comprising an RNA
sequence encoded by a nucleotide sequence within the genome of an
insect.
[0087] In one embodiment the plant transformation vector comprises
an isolated and purified DNA molecule comprising a promoter
operatively linked to one or more nucleotide sequences of the
present invention. The nucleotide sequence is selected from the
group consisting of SEQ ID NO:1 through SEQ ID NO:906 as set forth
in the sequence listing. The nucleotide sequence includes a segment
coding all or part of an RNA present within a targeted pest RNA
transcript and may comprise inverted repeats of all or a part of a
targeted pest RNA. The DNA molecule comprising the expression
vector may also contain a functional intron sequence positioned
either upstream of the coding sequence or even within the coding
sequence, and may also contain a five prime (5') untranslated
leader sequence (i.e., a UTR or 5'-UTR) positioned between the
promoter and the point of translation initiation.
[0088] A plant transformation vector may contain sequences from
more than one gene, thus allowing production of more than one dsRNA
for inhibiting expression of two or more genes in cells of a target
pest. One skilled in the art will readily appreciate that segments
of DNA whose sequence corresponds to that present in different
genes can be combined into a single composite DNA segment for
expression in a transgenic plant. Alternatively, a plasmid of the
present invention already containing at least one DNA segment can
be modified by the sequential insertion of additional DNA segments
between the enhancer and promoter and terminator sequences. In the
insect control agent of the present invention designed for the
inhibition of multiple genes, the genes to be inhibited can be
obtained from the same insect species in order to enhance the
effectiveness of the insect control agent. 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.
[0089] Promoters that function in different plant species are also
well known in the art. Promoters useful for expression of
polypeptides in plants include those that are inducible, viral,
synthetic, or constitutive as described in Odell et al. (1985),
and/or promoters that are temporally regulated, spatially
regulated, and spatio-temporally regulated. Preferred promoters
include the enhanced CaMV35S promoters, and the FMV35S promoter.
For the purpose of the present invention, e.g., for optimum control
of species that feed on roots, it may be preferable to achieve the
highest levels of expression of these genes within the roots of
plants. A number of root-enhanced promoters have been identified
and are known in the art (Lu et al., 2000; U.S. Pat. Nos. 5,837,848
and 6,489,542).
[0090] A recombinant DNA vector or construct of the present
invention will typically comprise a selectable marker that confers
a selectable phenotype on plant cells. Selectable markers may also
be used to select for plants or plant cells that contain the
exogenous nucleic acids encoding polypeptides or proteins of the
present invention. The marker may encode biocide resistance,
antibiotic resistance (e.g., kanamycin, G418 bleomycin, hygromycin,
etc.), or herbicide resistance (e.g., glyphosate, 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 mutant EPSP synthase gene which encodes glyphosate
resistance; a nitrilase gene which confers resistance to
bromoxynil; a mutant acetolactate synthase gene (ALS) which confers
imidazolinone or sulfonylurea resistance; 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.
[0091] 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); an R-locus gene, which encodes a product
that regulates the production of anthocyanin pigments (red color)
in plant tissues (Dellaporta et al., 1988); 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.
[0092] Preferred plant transformation vectors include those derived
from a Ti plasmid of Agrobacterium tumefaciens (e.g. U.S. Pat. Nos.
4,536,475, 4,693,977, 4,886,937, 5,501,967 and EP 0 122 791).
Agrobacterium rhizogenes plasmids (or "Ri") are also useful and
known in the art. Other preferred plant transformation vectors
include those disclosed, e.g., by Herrera-Estrella (1983); Bevan
(1983), Klee (1985) and EP 0 120 516.
[0093] In general it is preferred to introduce a functional
recombinant DNA at a non-specific location in a plant genome. In
special cases it may be useful to insert a recombinant DNA
construct by site-specific integration. Several site-specific
recombination systems exist which are known to function implants
include cre-lox as disclosed in U.S. Pat. No. 4,959,317 and FLP-FRT
as disclosed in U.S. Pat. No. 5,527,695.
[0094] Suitable methods for transformation of host cells for use
with the current invention are believed to include virtually any
method by which DNA can be introduced into a cell, such as by
direct delivery of DNA such as by PEG-mediated transformation of
protoplasts (Omirulleh et al., 1993), by
desiccation/inhibition-mediated DNA uptake (Potrykus et al., 1985),
by electroporation (U.S. Pat. No. 5,384,253), by agitation with
silicon carbide fibers (Kaeppler et al., 1990; U.S. Pat. No.
5,302,523; and U.S. Pat. No. 5,464,765), by Agrobacterium-mediated
transformation (U.S. Pat. No. 5,591,616 and U.S. Pat. No.
5,563,055) and by acceleration of DNA coated particles (U.S. Pat.
No. 5,550,318; U.S. Pat. No. 5,538,877; and U.S. Pat. No.
5,538,880), etc. Through the application of techniques such as
these, the cells of virtually any species may be stably
transformed. In the case of multicellular species, the transgenic
cells may be regenerated into transgenic organisms.
[0095] Methods for the creation of transgenic plants and expression
of heterologous nucleic acids in plants in particular are known and
may be used with the nucleic acids provided herein to prepare
transgenic plants that exhibit reduced susceptibility to feeding by
a target pest organism such as corn rootworms. Plant transformation
vectors can be prepared, for example, by inserting the dsRNA
producing nucleic acids disclosed herein into plant transformation
vectors and introducing these into plants. One known vector system
has been derived by modifying the natural gene transfer system of
Agrobacterium tumefaciens. The natural system comprises large Ti
(tumor-inducing)-plasmids containing a large segment, known as
T-DNA, which is transferred to transformed plants. Another segment
of the Ti plasmid, the vir region, is responsible for T-DNA
transfer. The T-DNA region is bordered by terminal repeats. In the
modified binary vectors the tumor-inducing genes have been deleted
and the functions of the vir region are utilized to transfer
foreign DNA bordered by the T-DNA border sequences. The T-region
may also contain a selectable marker for efficient recovery of
transgenic plants and cells, and a multiple cloning site for
inserting sequences for transfer such as a dsRNA encoding nucleic
acid.
[0096] A transgenic plant formed using Agrobacterium transformation
methods typically contains a single simple recombinant DNA sequence
inserted into one chromosome and is referred to as a transgenic
event. Such transgenic plants can be referred to as being
heterozygous for the inserted exogenous sequence. A transgenic
plant homozygous with respect to a transgene can be obtained by
selfing an independent segregant transgenic plant to produce F1
seed. One fourth of the F1 seed produced will be homozygous with
respect to the transgene. Germinating F1 seed results in plants
that can be tested for heterozygosity or homozygosity, typically
using a SNP assay or a thermal amplification assay that allows for
the distinction between heterozygotes and homozygotes (i.e., a
zygosity assay).
[0097] C. Nucleic Acid Expression and Target Gene Suppression
[0098] The present invention provides, as an example, a transformed
host or symbiont pest target organism, transformed plant cells and
transformed plants and their progeny. The transformed plant cells
and transformed plants may be engineered to express one or more of
the dsRNA or siRNA sequences described herein to provide a
pest-protective effect. These sequences may be used for gene
suppression in a pest organism, thereby reducing the predation by
the pest on a protected transformed host or symbiont organism. As
used herein the words "gene suppression" are intended to refer to
any of the well-known methods for reducing the levels of gene
transcription to mRNA and/or subsequent translation of the
mRNA.
[0099] 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).
[0100] 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
plant gene associated with a trait, e.g., to provide plants 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 plant
pests that may ingest or contact plant 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. Post-transcriptional gene suppression by
anti-sense or sense oriented RNA to regulate gene expression in
plant cells is disclosed in U.S. Pat. Nos. 5,107,065, 5,759,829,
5,283,184, and 5,231,020. The use of dsRNA to suppress genes in
plants is disclosed in WO 99/53050, WO 99/49029, U.S. Patent
Application Publication No. 2003/0175965, and 2003/0061626, U.S.
patent application Ser. No. 10/465,800, and U.S. Pat. Nos.
6,506,559, and 6,326,193.
[0101] A beneficial method of post transcriptional gene suppression
in plants 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).
[0102] 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
an insect that comprises an RNA sequence encoded by a nucleotide
sequence within the genome of the insect. Thus, after the insect
ingests the stabilized RNA sequence incorporated in a diet or
sprayed on a plant surface, a down-regulation of the nucleotide
sequence corresponding to the target gene in the cells of a target
insect is affected.
[0103] 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 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.
[0104] 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.
[0105] 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 insect 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 insect, in other preferred
embodiments inhibition occurs in only a subset of cells expressing
the gene. For example, if the gene to be inhibited plays an
essential role in cells in the insect alimentary tract, inhibition
of the gene within these cells is sufficient to exert a deleterious
effect on the insect.
[0106] 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.
[0107] 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. No's.
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.
[0108] For transcription from a transgene in vivo or an expression
construct, a regulatory region (e.g., promoter, enhancer, silencer,
and polyadenylation) may be used to transcribe the RNA strand (or
strands). Therefore, in one embodiment, the nucleotide sequences
for use in producing RNA molecules may be operably linked to one or
more promoter sequences functional in a microorganism, a fungus or
a plant host cell. Ideally, the nucleotide sequences are placed
under the control of an endogenous promoter, normally resident in
the host genome. The nucleotide sequence of the present invention,
under the control of an operably linked promoter sequence, may
further be flanked by additional sequences that advantageously
affect its transcription and/or the stability of a resulting
transcript. Such sequences are generally located upstream of the
operably linked promoter and/or downstream of the 3' end of the
expression construct and may occur both upstream of the promoter
and downstream of the 3' end of the expression construct, although
such an upstream sequence only is also contemplated.
[0109] As used herein, the term "insect 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 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. The insect 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.
[0110] As used herein, the term "genome" as it applies to cells of
an insect or a host encompasses not only chromosomal DNA found
within the nucleus, but organelle DNA found within subcellular
components of the cell. The DNA's of the present invention
introduced into plant cells can therefore be either chromosomally
integrated or organelle-localized. The term "genome" as it applies
to bacteria encompasses both the chromosome and plasmids within a
bacterial host cell. The DNA's of the present invention introduced
into bacterial host cells can therefore be either chromosomally
integrated or plasmid-localized.
[0111] 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 and that
may ingest or contact one or more cells, tissues, or fluids
produced by a pest host or symbiont transformed to express or
coated with a double stranded gene suppression agent or that may
ingest plant material containing the gene suppression agent. As
used herein, a "pest resistance" trait is a characteristic of a
transgenic plant, transgenic animal, transgenic host or transgenic
symbiont that causes the plant, animal, host, or symbiont to be
resistant to attack from a pest that typically is capable of
inflicting damage or loss to the plant, animal, host or symbiont.
Such pest resistance can arise from a natural mutation or more
typically from incorporation of recombinant DNA that confers pest
resistance. To impart insect resistance to a transgenic plant 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 plant. 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 an insect pest that
prefers to feed on the recombinant plant. Expression of the gene
within the target insect pest is suppressed by the dsRNA, and the
suppression of expression of the gene in the target insect pest
results in the plant being insect resistant. Fire et al. (U.S. Pat.
No. 6,506,599) generically described inhibition of pest
infestation, providing specifics only about several nucleotide
sequences that were effective for inhibition of gene function in
the nematode species Caenorhabditis elegans. Similarly, Plaetinck
et al. (US 2003/0061626) describe the use of dsRNA for inhibiting
gene function in a variety of nematode pests. Mesa et al. (US
2003/0150017) describe using dsDNA sequences to transform host
cells to express corresponding dsRNA sequences that are
substantially identical to target sequences in specific pathogens,
and particularly describe constructing recombinant plants
expressing such dsRNA sequences for ingestion by various plant
pests, facilitating down-regulation of a gene in the genome of the
pest and improving the resistance of the plant to the pest
infestation.
[0112] The present invention provides for inhibiting gene
expression of one or multiple target genes in a target pest using
stabilized dsRNA methods. The invention is particularly useful in
the modulation of eukaryotic gene expression, in particular the
modulation of 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 plant 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.
[0113] 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.
[0114] As used herein, the phrase "inhibition of gene expression"
or "inhibiting expression of a target gene in the cell of an
insect" 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 insect pest
may result in novel phenotypic traits in the insect pest.
[0115] The present invention provides in part a delivery system for
the delivery of the insect control agents to insects through their
exposure to a diet containing the insect control agents of the
present invention. In accordance with one of the embodiments, the
stabilized dsRNA or siRNA molecules may be incorporated in the
insect diet or may be overlaid on the top of the diet for
consumption by an insect. The present invention also provides in
part a delivery system for the delivery of the insect control
agents to insects through their exposure to a microorganism or host
such as a plant containing the insect control agents of the present
invention by ingestion of the microorganism or the host cells or
the contents of the cells. In accordance with another embodiment,
the present invention involves generating a transgenic plant cell
or a plant that contains a recombinant DNA construct transcribing
the stabilized dsRNA molecules of the present invention. As used
herein, the phrase "generating a transgenic plant cell or a plant"
refers to the methods of employing the recombinant DNA technologies
readily available in the art (e.g., by Sambrook, et al., 1989) to
construct a plant transformation vector transcribing the stabilized
dsRNA molecules of the present invention, to transform the plant
cell or the plant and to generate the transgenic plant cell or the
transgenic plant that contain the transcribed, stabilized dsRNA
molecules.
[0116] In still another embodiment, non-pathogenic, attenuated
strains of microorganisms may be used as a carrier for the insect
control agents and, in this perspective, the microorganisms
carrying such agents are also referred to as insect control agents.
The microorganisms may be engineered to express a nucleotide
sequence of a target gene to produce RNA molecules comprising RNA
sequences homologous or complementary to RNA sequences typically
found within the cells of an insect. Exposure of the insects to the
microorganisms result in ingestion of the microorganisms and
down-regulation of expression of target genes mediated directly or
indirectly by the RNA molecules or fragments or derivatives
thereof.
[0117] The present invention alternatively provides exposure of an
insect to the insect control. agents of the present invention
incorporated in a spray mixer and applied to the surface of a host,
such as a host plant. In an exemplary embodiment, ingestion of the
insect control agents by an insect delivers the insect control
agents to the gut of the insect and subsequently to the cells
within the body of the insect. In another embodiment, infection of
the insect by the insect control agents through other means such as
by injection or other physical methods also permits delivery of the
insect control agents. In yet another embodiment, the RNA molecules
themselves are encapsulated in a synthetic matrix such as a polymer
and applied to the surface of a host such as a plant. Ingestion of
the host cells by an insect permits delivery of the insect control
agents to the insect and results in down-regulation of a target
gene in the host.
[0118] It is envisioned that the compositions of the present
invention can be incorporated within the seeds of a plant species
either as a product of expression from a recombinant gene
incorporated into a genome of the plant cells, or incorporated into
a coating or seed treatment that is applied to the seed before
planting. The plant cell containing a recombinant gene is
considered herein to be a transgenic event.
[0119] It is believed that a pesticidal seed treatment can provide
significant advantages when combined with a transgenic event that
provides protection from coleopteran pest infestation that is
within the preferred effectiveness range against a target pest. In
addition, it is believed that there are situations that are well
known to those having skill in the art, where it is advantageous to
have such transgenic events within the preferred range of
effectiveness.
[0120] The present invention provides in part a delivery system for
the delivery of insect control agents to insects. The stabilized
dsRNA or siRNA molecules of the present invention may be directly
introduced into the cells of an insect, or introduced into an
extracellular cavity, interstitial space, lymph system, digestive
system, into the circulation of the insect through oral ingestion
or other means that one skilled in the art may employ. Methods for
oral introduction may include direct mixing of RNA with food of the
insect, 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 insect to be affected. In one embodiment, for example, the
dsRNA or siRNA molecules may be incorporated into, or overlaid on
the top of, the insect's diet. In another embodiment, the RNA may
be sprayed onto a plant surface. In still another embodiment, the
dsRNA or siRNA may be expressed by microorganisms and the
microorganisms may be applied onto a plant surface or introduced
into a root, stem by a physical means such as an injection. In
still another embodiment, a plant may be genetically engineered to
express the dsRNA or siRNA in an amount sufficient to kill the
insects known to infect the plant.
[0121] Specifically, in practicing the present invention in WCR,
the stabilized dsRNA or siRNA may be introduced in the midgut
inside the insect and achieve the desired inhibition of the
targeted genes. The dsRNA or siRNA molecules may be incorporated
into a diet or be overlaid on the diet as discussed above and may
be ingested by the insects. In any event, the dsRNA's of the
present invention are provided in the diet of the target pest. The
target pest of the present invention will exhibit a digestive tract
pH from about 4.5 to about 9.5, or from about 5 to about 8.5, or
from about 6 to about 8, or from about 6.5 to about 7.7, or about
7.0. The digestive tract of a target pest is defined herein as the
location within the pest that food that is ingested by the target
pest is exposed to an environment that is favorable for the uptake
of the dsRNA molecules of the present invention without suffering a
pH so extreme that the hydrogen bonding between the double-strands
of the dsRNA are caused to dissociate and form single stranded
molecules.
[0122] It is also anticipated that dsRNA's produced by chemical or
enzymatic synthesis may be formulated in a manner consistent with
common agricultural practices and used as spray-on products for
controlling insect infestations. The formulations may include the
appropriate stickers and wetters required for efficient foliar
coverage as well as UV protectants to protect dsRNAs from UV
damage. Such additives are commonly used in the bioinsecticide
industry and are well known to those skilled in the art. Such
applications could be combined with other spray-on insecticide
applications, biologically based or not, to enhance plant
protection from insect feeding damage.
[0123] The present inventors contemplate that bacterial strains
producing insecticidal proteins may be used to produce dsRNAs for
insect control purposes. These strains may exhibit improved insect
control properties. A variety of different bacterial hosts may be
used to produce insect control dsRNAs. Exemplary bacteria may
include E. coli, B. thuringiensis, Pseudomonas sp., Photorhabdus
sp., Xenorhabdus sp., Serratia entomophila and related Serratia
sp., B. sphaericus, B. cereus, B. laterosporus, B. popilliae,
Clostridium bifermentans and other Clostridium species, or other
spore-forming gram-positive bacteria. In certain embodiments,
bacteria may be engineered for control of pests such as
mosquitoes.
[0124] The present invention also relates to recombinant DNA
constructs for expression in a microorganism. Exogenous nucleic
acids from which an RNA of interest is transcribed can be
introduced into a microbial host cell, such as a bacterial cell or
a fungal cell, using methods known in the art.
[0125] The nucleotide sequences of the present invention may be
introduced into a wide variety of prokaryotic and eukaryotic
microorganism hosts to produce the stabilized dsRNA or siRNA
molecules. The term "microorganism" includes prokaryotic and
eukaryotic microbial species such as bacteria, fungi and algae.
Fungi include yeasts and filamentous fungi, among others.
[0126] Illustrative prokaryotes, both Gram-negative and
Gram-positive, include Enterobacteriaceae, such as Escherichia,
Erwinia, Shigella, Salmonella, and Proteus; Bacillaceae;
Rhizobiceae, such as Rhizobium; Spirillaceae, such as
photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio,
Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such
as Pseudomonas and Acetobacter; Azotobacteraceae, Actinomycetales,
and Nitrobacteraceae. Among eukaryotes are fungi, such as
Phycomycetes and Ascomycetes, which includes yeast, such as
Saccharomyces and Schizosaccharomyces; and Basidiomycetes, such as
Rhodotorula, Aureobasidium, Sporobolomyces, and the like.
[0127] For the purpose of plant protection against insects, a large
number of microorganisms known to inhabit the phylloplane (the
surface of the plant leaves) and/or the rhizosphere (the soil
surrounding plant roots) of a wide variety of important crops may
also be desirable host cells for manipulation, propagation,
storage, delivery and/or mutagenesis of the disclosed recombinant
constructs. These microorganisms include bacteria, algae, and
fungi. Of particular interest are microorganisms, such as bacteria,
e.g., genera Bacillus (including the species and subspecies B.
thuringiensis kurstaki HD-1, B. thuringiensis kurstaki HD-73, B.
thuringiensis sotto, B. thuringiensis berliner, B. thuringiensis
thuringiensis, B. thuringiensis tolworthi, B. thuringiensis
dendrolimus, B. thuringiensis alesti, B. thuringiensis galleriae,
B. thuringiensis aizawai, B. thuringiensis subtoxicus, B.
thuringiensis entomocidus, B. thuringiensis tenebrionis and B.
thuringiensis san diego); Pseudomonas, Erwinia, Serratia,
Klebsiella, Zanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas,
Methylophilius, Agrobacterium, Acetobacter, Lactobacillus,
Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes; fungi,
particularly yeast, e.g., genera Saccharomyces, Cryptococcus,
Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Of
particular interest are such phytosphere bacterial species as
Pseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens,
Acetobacter xylinum, Agrobacterium tumefaciens, Rhodobacter
sphaeroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenes
eutrophus, and Azotobacter vinlandii; and phytosphere yeast species
such as Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca,
Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces
rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S.
odorus, Kluyveromyces veronae, and Aureobasidium pollulans.
[0128] D. Transgenic Plants
[0129] The present invention provides seeds and plants having one
or more transgenic event. Combinations of events are referred to as
"stacked" transgenic events. These stacked transgenic events can be
events that are directed at the same target pest, or they can be
directed at different target pests. In one embodiment, a seed
having the ability to express a nucleic acid provided herein also
has the ability to express at least one other insecticidal agent,
including, but not limited to, an RNA molecule the sequence of
which is derived from the sequence of an RNA expressed in a target
pest and that forms a double stranded RNA structure upon expressing
in the seed or cells of a plant grown from the seed, wherein the
ingestion of one or more cells of the plant by the target pest
results in the suppression of expression of the RNA in the cells of
the target pest.
[0130] In certain embodiments, a seed having the ability to express
a dsRNA the sequence of which is derived from a target pest also
has a transgenic event that provides herbicide tolerance. One
beneficial example of a herbicide tolerance gene provides
resistance to glyphosate, N-(phosphonomethyl) glycine, including
the isopropylamine salt form of such herbicide.
[0131] In the present method, combination of expression of an
insecticidal amount of a dsRNA within the cells of a transgenic
seed or plant grown from the seed coupled with treatment of the
seed or plant with certain chemical or protein pesticides may be
used to provide unexpected synergistic advantages, including
unexpectedly superior efficacy for protection against damage to the
resulting transgenic plant by the target pest. In particular
embodiments, treatment of a transgenic seed that is capable of
expressing certain constructs that form dsRNA molecules, the
sequence of which are derived from one or more sequences expressed
in a corn rootworm, with from about 100 gm to about 400 gm of
pesticide per 100 kg of seed provides unexpectedly superior
protection against corn rootworm. In addition, it is believed that
such combinations are also effective to protect the emergent plants
against predation by other pests. The seeds of the present
invention may also be used to decrease the cost of pesticide use,
because less pesticide can be used to obtain a required amount of
protection than when such methods are not used. Moreover, because
less pesticide is used and because it is applied prior to planting
and without a separate field application, it is believed that the
subject method is therefore safer to the operator and to the
environment, and is potentially less expensive than conventional
methods.
[0132] By "synergistic" it is meant to include the synergistic
effects of the combination on the pesticidal activity (or efficacy)
of the combination of the transgenic event and the pesticide.
However, it is not intended that such synergistic effects be
limited to the pesticidal activity, but that they should also
include such unexpected advantages as increased scope of activity,
advantageous activity profile as related to type and amount of
damage reduction, decreased cost of pesticide and application,
decreased pesticide distribution in the environment, decreased
pesticide exposure of personnel who produce, handle and plant corn
seeds, and other advantages known to those skilled in the art.
[0133] Pesticides and insecticides that are useful in compositions
in combination with the methods and compositions of the present
invention, including as seed treatments and coatings as well as
methods for using such compositions can be found, for example, in
U.S. Pat. No. 6,551,962, the entirety of which is incorporated
herein by reference.
[0134] Although it is believed that the seed treatments can be
applied to a transgenic seed in any physiological state, it may be
preferred that the seed be in a sufficiently durable state that it
incurs no damage during the treatment process. Typically, the seed
would be a seed that had been harvested from the field; removed
from the transgenic plant; and separated from any other non-seed
plant material. The seed would preferably also be biologically
stable to the extent that the treatment-would cause no biological
damage to the seed. In one embodiment, for example, the treatment
can be applied to seed corn that has been harvested, cleaned and
dried to a moisture content below about 15% by weight. In an
alternative embodiment, the seed can be one that has been dried and
then primed with water and/or another material and then re-dried
before or during the treatment with the pesticide. Within the
limitations described, it is believed that the treatment can be
applied to the seed at any time between harvest of the seed and
sowing of the seed. As used herein, the term "unsown seed" is meant
to include seed at any period between the harvest of the seed and
the sowing of the seed in the ground for the purpose of germination
and growth of the plant. When it is said that unsown seed is
"treated" with the pesticide, such treatment is not meant to
include those practices in which the pesticide is applied to the
soil, rather than to the seed. For example, such treatments as the
application of the pesticide in bands, "T"-bands, or in-furrow, at
the same time as the seed is sowed are not considered to be
included in the present invention.
[0135] The pesticide, or combination of pesticides, can be applied
"neat", that is, without any diluting or additional components
present. However, the pesticide is typically applied to the seeds
in the form of a pesticide formulation. This formulation may
contain one or more other desirable components including but not
limited to liquid diluents, binders to serve as a matrix for the
pesticide, fillers for protecting the seeds during stress
conditions, and plasticizers to improve flexibility, adhesion
and/or spreadability of the coating.
[0136] The subject pesticides can be applied to a seed as a
component of a seed coating. Seed coating methods and compositions
that are known in the art are useful when they are modified by the
addition of one of the embodiments of the combination of pesticides
of the present invention. Such coating methods and apparatus for
their application are disclosed in, for example, U.S. Pat. Nos.
5,918,413, 5,891,246, 5,554,445, 5,389,399, 5,107,787, 5,080,925,
4,759,945 and 4,465,017. Seed coating compositions are disclosed,
for example, in U.S. Pat. Nos. 5,939,356, 5,882,713, 5,876,739,
5,849,320, 5,834,447, 5,791,084, 5,661,103, 5,622,003, 5,580,544,
5,328,942, 5,300,127, 4,735,015, 4,634,587, 4,383,391, 4,372,080,
4,339,456, 4,272,417 and 4,245,432, among others.
[0137] The pesticides that are useful in the coating are those
pesticides that are described herein. The amount of pesticide that
is used for the treatment of the seed will vary depending upon the
type of seed and the type of active ingredients, but the treatment
will comprise contacting the seeds with an amount of the
combination of pesticides that is pesticidally effective. When
insects are the target pest, that amount will be an amount of the
insecticide that is insecticidally effective. As used herein, an
insecticidally effective amount means that amount of insecticide
that will kill insect pests in the larvae or pupal state of growth,
or will consistently reduce or retard the amount of damage produced
by insect pests.
[0138] In general, the amount of pesticide that is applied to the
seed in the treatment will range from about 10 gm to about 2000 gm
of the active ingredient of the pesticide per 100 kg of the weight
of the seed. Preferably, the amount of pesticide will be within the
range of about 50 gm to about 1000 gm active per 100 kg of seed,
more preferably within the range of about 100 gm to about 600 gm
active per 100 kg of seed, and even more preferably within the
range of about 200 gm to about 500 gm of active per 100 kg of seed
weight. Alternatively, it has been found to be preferred that the
amount of the pesticide be over about 60 gm of the active
ingredient of the pesticide per 100 kg of the seed, and more
preferably over about 80 gm per 100 kg of seed.
[0139] The pesticides that are used in the treatment must not
inhibit germination of the seed and should be efficacious in
protecting the seed and/or the plant during that time in the target
insect's life cycle in which it causes injury to the seed or plant.
In general, the coating will be efficacious for approximately 0 to
120 days after sowing. The pesticides of the subject invention can
be applied to the seed in the form of a coating.
[0140] Benefits provided by the present invention may include, but
are not limited to: the ease of introducing dsRNA into the insect
cells, the low concentration of dsRNA which can be used, the
stability of dsRNA, and the effectiveness of the inhibition. The
ability to use a low concentration of a stabilized dsRNA avoids
several disadvantages of anti-sense interference. The present
invention is not limited to in vitro use or to specific sequence
compositions, to a particular set of target genes, a particular
portion of the target gene's nucleotide sequence, or a particular
transgene or to a particular delivery method, as opposed to the
some of the available techniques known in the art, such as
antisense and co-suppression. Furthermore, genetic manipulation
becomes possible in organisms that are not classical genetic
models.
[0141] In practicing the present invention, selections can be
carried out to ensure that the presence of the nucleotide sequences
that are transcribed from the recombinant construct are not harmful
to non-pest cells. This can be achieved by targeting genes that
exhibit a low degree of sequence identity with corresponding genes
in a plant or a vertebrate animal. Preferably the degree of the
sequence identity is less than approximately 80%. More preferably
the degree of the sequence identity is less than approximately 70%.
Most preferably the degree of the sequence identity is less than
approximately 60%.
[0142] In addition to direct transformation of a plant with a
recombinant DNA construct, transgenic plants can be prepared by
crossing a first plant having a recombinant DNA construct with a
second plant lacking the construct. For example, recombinant DNA
for gene suppression can be introduced into first plant line that
is amenable to transformation to produce a transgenic plant that
can be crossed with a second plant line to introgress the
recombinant DNA for gene suppression into the second plant
line.
[0143] The present invention can be, in practice, combined with
other insect control traits in a plant to achieve desired traits
for enhanced control of insect infestation. Combining insect
control traits that employ distinct modes-of-action can provide
insect-protected transgenic plants with superior durability over
plants harboring a single insect control trait because of the
reduced probability that resistance will develop in the field.
[0144] The mechanism of insecticidal activity of B. thuringiensis
crystal proteins has been studied extensively in the past decade.
It has been shown that the crystal proteins are toxic to the larval
form of the insect only after ingestion of the protein. In
lepidopteran larvae, an alkaline pH and proteolytic enzymes in the
insect mid-gut solubilize the proteins, thereby allowing the
release of components that are toxic to the insect. These toxic
components disrupt the mid-gut cells, cause the insect to cease
feeding, and, eventually, bring about insect death. For this
reason, B. thuringiensis toxins have proven themselves to be
effective and environmentally safe insecticides in dealing with
various insect pests. Coleopteran and hemipteran insects, and
likely dipteran, lygus and other piercing and sucking insects
exhibit a gut pH that is slightly acidic, and so the Bt toxins that
are effective against lepidopteran larvae are ineffective against
these pests. The slightly acidic pH of the gut of these insects is
also believed to be more hospitable to the compositions of the
present invention, and without intending to be limited to a
particular theory, it is likely that the alkaline pH of the gut of
lepidopteran larvae is a contributing reason that prior attempts to
exhibit dsRNA efficacy has failed (Fire et al. U.S. Pat. No.
6,506,559; Mesa et al. Patent Publication No. US2003/0150017;
Rajagopal et al., 2002; Tabara et al., 1998). It is believed
therefore that the dsRNA methods disclosed herein should be
preferentially used in compositions and in plants to control
coleopteran, dipteran, hemipteran, lygus, and piercing and sucking
insects. The methods and compositions set forth herein are
particularly useful for targeting genes for suppression in insects
exhibiting a gut pH of from about 4.5 to about 9.5, or from about
5.0 to about 9.0, or from about 5.5 to about 8.5, or from about 6.0
to about 8.0, or from about 6.5 to about 7.7, or from about 6.8 to
about 7.6, or about 7.0. However, insects and other pest species
that exhibit a gut pH of from about 7.5 to about 11.5, or from
about 8.0 to about 11.0, or from about 9.0 to about 10.0, such as
lepidopteran insect larvae, are also intended to be within the
scope of the present invention. This is particularly true when a
dsRNA specific for inhibiting a gene in a lepidopteran larvae is
provided in the diet of the larvae along with one or more Bt
proteins, that, with respect to the Bt protein would ordinarily be
toxic to that lepidopteran larvae when provided at or above a
threshold level. The presence of one or more Bt toxins toxic to the
same insect species would effectively reduce the gut pH, providing
a stable environment for the double stranded RNA molecules to exert
their effects in suppressing a target gene in the insect pest.
[0145] It is anticipated that the combination of certain stabilized
dsRNA constructs with one or more insect control protein genes will
result in synergies that enhance the insect control phenotype of a
transgenic plant. Insect bioassays employing artificial diet- or
whole plant tissue can be used to define dose-responses for larval
mortality or growth inhibition using both dsRNAs and insect control
proteins. One skilled in the art can test mixtures of dsRNA
molecules and insect control proteins in bioassay to identify
combinations of actives that are synergistic and desirable for
deployment in insect-protected plants (Tabashnik, 1992). Synergy in
killing insect pests has been reported between different insect
control proteins (for review, see Schnepf et al., 1998). It is
anticipated that synergies will exist between certain dsRNAs and
between certain dsRNAs and certain insect control proteins.
[0146] The invention also relates to commodity products containing
one or more of the sequences of the present invention, and produced
from a recombinant plant or seed containing one or more of the
nucleotide sequences of the present invention are specifically
contemplated as embodiments of the present invention. A commodity
product containing one or more of the sequences of the present
invention is intended to include, but not be limited to, meals,
oils, crushed or whole grains or seeds of a plant, or any food
product comprising any meal, oil, or crushed or whole grain of a
recombinant plant or seed containing one or more of the sequences
of the present invention. The detection of one or more of the
sequences of the present invention in one or more commodity or
commodity products contemplated herein is defacto evidence that the
commodity or commodity product is composed of a transgenic plant
designed to express one or more of the nucleotides sequences of the
present invention for the purpose of controlling insect infestation
using dsRNA mediated gene suppression methods.
[0147] D. Obtaining Nucleic Acids
[0148] The present invention provides a method for obtaining a
nucleic acid comprising a nucleotide sequence for producing a dsRNA
or siRNA. In one embodiment, such a method comprises: (a) probing a
cDNA or gDNA library with a hybridization probe comprising all or a
portion of a nucleotide sequence or a homolog thereof from a
targeted insect; (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 gDNA 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.
[0149] In another embodiment, a method of the present invention for
obtaining a nucleic acid fragment comprising a nucleotide sequence
for producing a substantial portion of a dsRNA or siRNA comprises:
(a) synthesizing first and a second oligonucleotide primers
corresponding to a portion of one of the nucleotide sequences from
a targeted insect; and (b) amplifying a cDNA or gDNA 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.
[0150] In practicing the present invention, a target gene may be
derived from a corn rootworm (CRW), such as a WCR or a SCR, or any
insect species that causes damage to the crop plants and subsequent
yield losses. It is contemplated that 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 insect. If it is desired to target a broad range of insect
species 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.
[0151] 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.
[0152] 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
vacuolar and 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. In another embodiment, the
V-ATPase may be Vha68-2, or a homolog or ortholog thereof (e.g. as
found in SEQ ID NO:821).
[0153] 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 a CHD3
gene, a .beta.-tubulin gene, and a gene encoding a protein
predicted to be involved in transport. The CHD3 gene in Drosophila
melanogaster encodes a protein with ATP-dependent DNA helicase
activity that is involved in chromatin assembly/disassembly in the
nucleus. Similar sequences have been found in diverse organisms
such as Arabidopsis thaliana, Caenorhabditis elegans, and
Saccharomyces cerevisiae. The beta-tubulin gene family encodes
microtubule-associated proteins that are a constituent of the
cellular cytoskeleton. Related sequences are found in such diverse
organisms as C. elegans, and Manduca sexta. Proteins predicted to
be subunits of the endosomal sorting complex required for transport
(ESCRT)-III (Babst et al., 2002), e.g. Dv49, are found in diverse
organisms including mammals, yeast, and insects such as D.
virgifera. Another transport-related protein is the
.beta.'-coatomer protein, abbreviated as .beta.'Cop, that encodes a
product involved in retrograde (Golgi to ER) transport. Similar or
predicted sequences have been identified in C. elegans and D.
virgifera, e.g. Dv248 (SEQ ID NO:.
[0154] Other target genes for use in the present invention may
include, for example, those that play important roles in the
viability, growth, development, reproduction and infectivity. These
target genes may be one of the house keeping genes, transcription
factors and insect specific genes or lethal knockout mutations in
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., C. elegans). Additionally, the nucleotide sequences for use
in the present invention may also be derived from plant, viral,
bacterial 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. According to one aspect of the present invention for WCR
control, the target sequences may essentially be derived from the
targeted WCR insect. Some of the exemplary target sequences from
cDNA library from WCR that encode D. virgifera proteins or
fragments thereof which are homologues of known proteins may be
found in the Sequence Listing. Nucleic acid molecules from D.
virgifera encoding homologs of known proteins are known (Andersen
et al., U.S. patent application Ser. No. 10/205,189).
[0155] For the purpose of the present invention, the dsRNA or siRNA
molecules may be obtained from the CRW by polymerase chain
(PCR.TM.) amplification of a target CRW gene sequences derived from
a corn rootworm gDNA or cDNA library or portions thereof. The WCR
larvae may be prepared using methods known to the ordinary skilled
in the art and DNA/RNA may be extracted. Larvae with various sizes
ranging from 1st instars to fully-grown CRWs may be used for the
purpose of the present invention for DNA/RNA extraction. Genomic
DNA or cDNA libraries generated from WCR may be used for PCR.TM.
amplification for production of the dsRNA or siRNA.
[0156] The target genes may be then be PCR.TM. amplified and
sequenced using the methods readily available in the art. One
skilled in the art may be able to modify the PCR.TM. conditions to
ensure optimal PCR.TM. product formation. The confirmed PCR.TM.
product may be used as a template for in vitro transcription to
generate sense and antisense RNA with the included minimal
promoters.
[0157] The present inventors contemplate that nucleic acid
sequences identified and isolated from any insect species in the
insect kingdom may be used in the present invention for control of
WCR and another targeted insects. In one aspect of the present
invention, the nucleic acid may be derived from a coleopteran
species. Specifically, the nucleic acid may be derived from leaf
beetles belonging to the genus Diabrotica (Coleoptera,
Chrysomelidae) and more specifically the nucleic acid molecules of
the present invention may be derived from species in the virgifera
group. Most specifically, the nucleic acid molecules of the present
invention may be derived from Diabrotica virgifera virgifera
LeConte that is normally referred to as WCR. The isolated nucleic
acids may be useful, for example, in identifying a target gene and
in constructing a recombinant vector that produce stabilized dsRNAs
or siRNAs of the present invention for protecting plants from WCR
insect infestations.
[0158] Therefore, in one embodiment, the present invention
comprises isolated and purified nucleotide sequences from WCR or
Lygus that may be used as the insect control agents. The isolated
and purified nucleotide sequences may comprise those as set forth
in the sequence listing.
[0159] The nucleic acids from WCR or other insects that may be used
in the present invention may also comprise isolated and
substantially purified Unigenes and EST nucleic acid molecules or
nucleic acid fragment molecules thereof. EST nucleic acid molecules
may encode significant portions of, or indeed most of, the
polypeptides. 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. Alternatively, the nucleic acid molecules for use in the
present invention may be from cDNA libraries from WCR, or from any
other coleopteran pest species.
[0160] Nucleic acid molecules and fragments thereof from WCR, or
other coleopteran pest species may be employed to obtain other
nucleic acid molecules from other species for use in the present
invention to produce desired dsRNA and siRNA molecules. Such
nucleic acid molecules include the nucleic acid molecules that
encode the complete coding sequence of a protein and promoters and
flanking sequences of such molecules. In addition, such nucleic
acid molecules include nucleic acid molecules that encode for gene
family members. Such molecules can be readily obtained by using the
above-described nucleic acid molecules or fragments thereof to
screen, for instance, cDNA or gDNA libraries obtained from D. v.
virgifera or other coleopterans, or from Lygus hesperus. Methods
for forming such libraries are well known in the art.
[0161] As used herein, the phrase "coding sequence", "structural
nucleotide sequence" or "structural nucleic acid molecule" refers
to a nucleotide sequence that is translated into a polypeptide,
usually via mRNA, when placed under the control of appropriate
regulatory sequences. The boundaries of the coding sequence are
determined by a translation start codon at the 5'-terminus and a
translation stop codon at the 3'-terminus. A coding sequence can
include, but is not limited to, genomic DNA, cDNA, EST and
recombinant nucleotide sequences.
[0162] The term "recombinant DNA" or "recombinant nucleotide
sequence" refers to DNA that contains a genetically engineered
modification through manipulation via mutagenesis, restriction
enzymes, and the like.
[0163] 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, the present inventors
contemplate that selection of appropriate genes from insect pests
for use in the present invention may be accomplished through use of
information available from study of the corresponding genes in a
model organism such in Drosophila, in some other insect species, or
even in a nematode species, in a fungal species, in a plant
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 sequence from, for example,
Drosophila, another insect, a nematode, a fungus, or a plant from
which the gene has been cloned. Once the sequence is obtained,
PCR.TM. may be used to amplify an appropriately selected segment of
the gene in the insect for use in the present invention.
[0164] In order to obtain a DNA segment from the corresponding gene
in an insect species, PCR.TM. primers may be designed based on the
sequence as found in WCR or other insects from which the gene has
been cloned. The primers are designed to amplify a DNA segment of
sufficient length for use in the present invention. DNA (either
genomic DNA or cDNA) is prepared from the insect species, and the
PCR.TM. primers are used to amplify the DNA segment. 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 gDNA or cDNA
library prepared from the insect pest species, using the WCR gene
or another known insect gene as a probe. Techniques for performing
PCR.TM. 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 WCR or other 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.
[0165] When an insect is the target pest for the present invention,
such pests include but are not limited to: from the order
Lepidoptera, for example,
[0166] 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.;
[0167] from the order Coleoptera, for example,
[0168] Agriotes spp., Anthonomus spp., Atomaria linearis,
Chaetocnema tibialis, Cosmopolites spp., Curculio spp., Dermestes
spp., Diabrotica 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.;
[0169] from the order Orthoptera, for example,
[0170] Blatta spp., Blattella spp., Gryllotalpa spp., Leucophaea
maderae, Locusta spp., Periplaneta ssp., and Schistocerca spp.;
[0171] from the order Isoptera, for example,
[0172] Reticulitemes ssp;
[0173] from the order Psocoptera, for example,
[0174] Liposcelis spp.;
[0175] from the order Anoplura, for example,
[0176] Haematopinus spp., Linognathus spp., Pediculus spp.,
Pemphigus spp. and Phylloxera Spp.;
[0177] from the order Mallophaga, for example,
[0178] Damalinea spp. and Trichodectes spp.;
[0179] from the order Thysanoptera, for example,
[0180] Franklinella spp., Hercinothrips spp., Taeniothrips spp.,
Thrips palmi, Thrips tabaci and Scirtothrips aurantii;
[0181] from the order Heteroptera, for example,
[0182] 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.;
[0183] from the order Homoptera, for example,
[0184] 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;
[0185] from the order Hymenoptera, for example,
[0186] Acromyrmex, Atta spp., Cephus spp., Diprion spp.,
Diprionidae, Gilpinia polytoma, Hoplocampa spp., Lasius sppp.,
Monomorium pharaonis, Neodiprion spp, Solenopsis spp. and Vespa
ssp.;
[0187] from the order Diptera, for example,
[0188] 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 frit, Pegomyia hyoscyami,
Phorbia spp., Rhagoletis pomonella, Sciara spp., Stomoxys spp.,
Tabanus spp., Tannia spp. and Tipula spp.,
[0189] from the order Siphonaptera, for example,
[0190] Ceratophyllus spp. and Xenopsylla cheopis and
[0191] from the order Thysanura, for example,
[0192] Lepisma saccharina.
[0193] It has been found that the present invention is particularly
effective when the insect pest is a Diabrotica spp., and especially
when the pest is Diabrotica virgifera virgifera (Western Corn
Rootworm, WCR), Diabrotica barberi (Northern Corn Rootworm, NCR),
Diabrotica virgifera zea (Mexican Corn Rootworm, MCR), Diabrotica
balteata (Brazilian Corn Rootworm (BZR) or Brazilian Corn Rootworm
complex (BCR) consisting of Diabrotica viridula and Diabrotica
speciosa), or Diabrotica undecimpunctata howardi (Southern Corn
Rootworm, SCR).
EXAMPLES
[0194] The inventors herein have identified means for controlling
coleopteran pest infestation by providing a double stranded
ribonucleic acid molecules in the diet of pests. Surprisingly, the
inventors have discovered double stranded ribonucleic acid
molecules that function upon ingestion by the pest to inhibit a
biological function in the 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. Any one or any combination of
these attributes can result in an effective inhibition of pest
infestation, and in the case of a plant pest, inhibition of plant
infestation. For example, when used as a diet composition
containing a pest inhibitory sufficient amount of one or more
double stranded ribonucleic acid molecules provided topically to a
plant, as a seed treatment, as a soil application around a plant,
or when produced by a plant from a recombinant DNA molecule present
within the cells of a plant, plant pest infestation is unexpectedly
dramatically reduced. The Examples set forth herein below are
illustrative of the invention when applied to a single pest.
However, the skilled artisan will recognize that the methods,
formulae, and ideas presented in the Examples are not intended to
be limiting, and are applicable to all coleopteran pest species
that can consume food sources that can be formulated to contain a
sufficient amount of a pest inhibitory agent consisting at least of
one or more double stranded RNA molecules exemplified herein
intended to suppress some essential feature about or function
within the pest.
Example 1
Identification of Target Nucleotide Sequences for Preparation of
dsRNA Useful for Controlling Corn Rootworms
[0195] Corn rootworm cDNA libraries (LIB149, LIB 150, LIB3027,
LIB3373) were constructed from whole larvae, pupae and from
dissected midgut sections, and nucleotide sequence information was
obtained (see Andersen et al., U.S. patent application Ser. No.
10/205,189 filed Jul. 24, 2002, incorporated herein specifically by
reference in its entirety). In addition, cDNA libraries were
constructed from whole larvae at different developmental stages and
at different times within each developmental stage in order to
maximize the number of different EST sequences from the Diabrotica
species. Libraries LIB5444 and LIB5462 were constructed
respectively from mRNA pools obtained from first (1 gram) and third
(2.9 grams) instar Western Corn Rootworm larvae. Harvested insects
were rapidly frozen by insertion into liquid nitrogen. The insects
were ground in a mortar and pestle maintained at or below
-20.degree. C. by chilling on dry ice and/or with the addition of
liquid nitrogen to the mortar until the tissue was ground into a
fine powder. RNA was extracted using TRIzol.RTM. reagent
(Invitrogen) according to the manufacturer's instructions. Poly A+
RNA was isolated from the total RNA prep using DYNABEADS Oligo dT
(Invitrogen) following the manufacturer's instructions. A cDNA
library was constructed from the Poly A+ RNA using the
SuperScript.TM. Plasmid System (Invitrogen). cDNA was size
fractionated using chromatography. The fourth and fifth fractions
were collected and ligated into the pSPORT1 vector (Life
Technologies Inc., Gaithersburg Md.) between the Sal1 and Not1
restriction endonucleases recognition sites, and transformed into
E. coli DH10B electro-competent cells by electroporation. The first
instar larvae library yielded about 420,000 colony-forming units.
The third instar larvae library yielded about 2.78.times.10.sup.6
colony forming units. Colonies from LIB149, LIB150 were washed from
the plates, mixed to uniformity by vortexing briefly, and pooled
into Tris-EDTA buffer. Half of the wash was brought to 10%
glycerol, aliquoted into cryovials, and stored at -70.degree. C.
The other half was used to produce plasmid DNA using a Quiagen
midi-prep purification column, or its equivalent. Purified plasmid
DNA was aliquoted to microcentrifuge tubes and stored at
-20.degree. C.
[0196] Colonies from the Diabrotica virgifera cDNA libraries
LIB5444 and LIB5462 were amplified individually in a high viscosity
medium. Approximately 200,000 colony-forming units from LIB5444 and
600,000 colony-forming units from LIB5462 were mixed on a stir
plate separately in 500 ml LB medium containing 0.3% SeaPrep
Agarose.RTM. and 50 mg/l carbenecillin at 37.degree. C. and then
rapidly cooled in a water/ice bath for 1 hour allowing uniform
suspension of the bacterial colonies. The inoculated libraries were
then grown at 30.degree. C. for 42 hours. After incubation, the
cells were mixed for 5 minutes on a stir plate. The medium was then
transferred to two 250 ml centrifuge bottles. The bacterial cells
were pelleted at 10,000.times.g for 10 minutes. The medium was
removed from the bottles and the cells were resuspended in a total
of 20 ml of LB medium with 50 mg/l carbenecillin. Dimethyl
sulfoxide was added to 10% to preserve the cells in freezing. Both
libraries were amplified to a final titer of 10.sup.8
colony-forming units per milliliter. Samples of the Diabrotica
virgifera cDNA libraries LIB5444 and LIB5462 were combined and
adjusted to a DNA concentration of about 1.25 micrograms per
microliter in sterile distilled and deionized water and aliquoted
into twenty five cryovials, each cryovial containing about 8.75
micrograms of DNA. These samples were deposited by the
applicant(s)/inventors with the American Type Culture Collection
(ATCC) located at 10801 University Boulevard, Manassas, Va., USA
ZIP 20110-2209 on Jun. 10, 2004 and referred to as LIB5444/62. The
ATCC provided the Applicant with a deposit receipt, assigning the
ATCC Deposit Accession No. PTA-6072.
[0197] Corn rootworm high molecular weight cDNA libraries, i.e.,
LIB5496 and LIB5498, were prepared essentially as described above
for the production of corn rootworm cDNA libraries. Libraries
LIB5496 and LIB5498 were constructed respectively from mRNA pools
obtained from first (1 gram) and second and third (1 gram) instar
Western Corn Rootworm larvae. Briefly, insects were quickly frozen
in liquid nitrogen. The frozen insects were reduced to a fine
powder by grinding in a mortar and pestle. RNA was extracted using
TRIzol.RTM. reagent (Invitrogen) following the manufacturer's
instructions. Poly A+ RNA was isolated from the total RNA prep
using DYNABEADS Oligo dT (Invitrogen). A high molecular weight cDNA
library was made from 20 micrograms of Poly A+ RNA using the
SuperScript.TM. Plasmid System (Invitrogen). The cDNA was size
fractionated on a 1% agarose gel in TAE, and cDNA between the range
of 1 Kb to 10 Kb was collected and ligated into the pSPORT1 vector
in between the Sal1 and Not1 restriction sites and transformed into
E. coli DH10B electro-competent cells by electroporation. LIB5496
yielded a total titer of about 3.5.times.10.sup.6 colony forming
units. LIB5498 yielded a total titer of about 1.0.times.10.sup.6
colony forming units. Colonies from the corn rootworm high
molecular weight cDNA libraries LIB5496 and LIB5498 were amplified
individually in a high viscosity medium. Approximately 600,000
colony-forming units from LIB5496 and LIB5498 were mixed on a stir
plate separately in 500 ml LB medium containing 0.3% SeaPrep
Agarose.RTM. and 50 mg/l carbenecillin at 37.degree. C. and then
rapidly cooled in a water/ice bath for 1 hour allowing uniform
suspension of the bacterial colonies. The libraries were then grown
at 30.degree. C. for 42 hours. After incubation, the cells were
mixed for 5 minutes on a stir plate. The medium was then
transferred to two 250 mL centrifuge bottles. The bacterial cells
were pelleted at 10,000.times.g for 10 minutes. The medium was
removed from the bottles and the cells were resuspended in a total
of 20 mL of LB medium with 50 mg/L carbenecillin. Dimethyl
sulfoxide was added to 10% to preserve the cells in freezing. Both
libraries were amplified to a final titer of 10.sup.8
colony-forming units per milliliter. Inserted cDNA sequence
information was obtained from the corn rootworm species-specific
plasmid libraries.
[0198] The Andersen et al. rootworm libraries together with
additional sequences from the libraries LIB5444 and LIB5462
initially produced about 18,415 individual EST sequences consisting
of approximately 1.0.times.10.sup.7 nucleotide residues. The
average length of an EST sequence was about 586 nucleotide
residues. These EST sequences were subjected to bioinformatics
algorithms that resulted in the assembly of contig sequences
referred to herein as UNIGENE sequences, and individual EST
sequences that could not be compiled by overlap identity with other
EST sequences, referred to herein as singletons. The LIB5444 and
LIB5462 libraries were then sequenced much deeper, resulting in
additional individual EST sequences. EST sequences obtained from
libraries, i.e., LIB149, LIB150, LIB3027, LIB3373, LIB5444,
LIB5462, LIB5496 and LIB5503 were selected for further
investigation in feeding bioassays as set forth below and the
corresponding sequences are given in the sequence listing.
[0199] The EST sequences isolated from CRW cDNA libraries were
assembled, where possible, into UNIGENE sets and these assembled
Unigene sequences are included in the sequence listing. A UNIGENE
is a gene-oriented cluster formed from the overlap of individual
EST sequences within regions of sequence identity to form a larger
sequence. Pontius et al. (2003). Each nucleotide sequence as set
forth in the sequence listing was analyzed to identify the presence
of open reading frames. Amino acid sequence information deduced
from open reading frames was compared to known amino acid sequence
information available in public databases in order to deduce the
extent of amino acid sequence identity or similarity to those known
amino acid sequences. Biological function, if any, associated with
known amino acid sequences in public databases was annotated to the
amino acid sequences deduced from the cDNA library nucleotide
sequence information. Annotations provided information that was
suggestive of the function of a protein that may be expressed from
a particular gene that gave rise to a particular cDNA sequence, but
was not outcome determinative. Based on the suggestive annotation
information, certain cDNA sequences were characterized as those
that encoded a protein that was likely involved in some biological
function within corn rootworm cells that was either essential to
life, or that was necessary for ensuring health and vitality to a
cell, or were likely to be involved in cellular integrity, cell
maintenance, reproductive capacity, and the like.
[0200] Sequences selected for further investigation were used in
the construction of double stranded RNA molecules for incorporation
into CRW diet. Thermal amplification primer pairs were designed
based on cDNA and EST starting sequences to obtain sequences used
in feeding assays. Primer pairs were constructed either as a pair
of nucleotide sequences, each member of a primer pair exhibiting
perfect complementarity either to a sense or to an antisense
sequence. Some primer pair sequences were constructed so that each
member of the pair exhibited a sequence containing a T7 phage RNA
polymerase promoter at it's 5' end. Preferably a higher fidelity
first amplification reaction was carried out using a first primer
pair lacking a T7 promoter to generate a first amplicon using CRW
genomic DNA as template. Preferably, a cDNA or a mRNA sequence is
used as the template for the synthesis of a dsRNA molecule for use
in the present invention because eukaryotic genome sequences are
recognized in the art to contain sequences that are not present
within the mature RNA molecule. A sample of the first amplicon
generated from the higher fidelity first amplification reaction was
then used as template in a second thermal amplification reaction
with a second primer pair containing the T7 promoter sequence to
produce a second amplicon that contained a T7 promoter at or
embedded within the 5' end of each strand of the second amplicon.
The complete nucleotide sequence of the second amplicon was
obtained in both directions and compared to the nucleotide sequence
as reported for the cDNA, and discrepancies between the two
sequences, if any, were noted. Generally, sequences prepared using
genome DNA as template were inconsistent with further use as dsRNA
molecules for use in achieving significant levels of suppression
because of variations within the genome sequences that were not
present within the mRNA or cDNA sequence.
[0201] An in vitro transcription reaction typically contained from
about 1 to about 2 micrograms of linearized DNA template, T7
polymerase reaction buffer from a 10.times. concentrate,
ribonucleotides ATP, CTP, GTP, and UTP at a final concentration of
from between 50 and 100 mM each, and 1 unit of T7 RNA polymerase
enzyme. The RNA polymerase reaction was incubated at about
37.degree. C., depending on the optimal temperature of the RNA
polymerase used according to the manufacturers' instructions, for a
period of time ranging from several minutes to several hours.
Generally, reactions were carried out for from about 2 to about 6
hours for transcription of template sequences up to about 400
nucleotides in length, and for up to 20 hours for transcription of
template sequences greater than about 400 nucleotides in length.
Heating the reaction to 65.degree. C. for fifteen minutes
terminates RNA transcription. RNA transcription products were
precipitated in ethanol, washed, air dried and resuspended in RNAse
free water to a concentration of about 1 microgram per microliter.
Most transcripts which took advantage of the opposing T7 promoter
strategy outlined above produced double stranded RNA in the in
vitro transcription reaction, however, a higher yield of double
stranded RNA was obtained by heating the purified RNA to 65.degree.
C. and then slowly cooling to room temperature to ensure proper
annealing of sense and antisense RNA segments. Double stranded RNA
products were then incubated with DNAse I and RNAse at 37.degree.
C. for one hour to remove any DNA or single stranded RNA present in
the mixture. Double stranded RNA products were purified over a
column according to the manufacturers' instructions (AMBION
MEGAscript.RTM. RNAi KIT) and resuspended in 10 mM Tris-HCl buffer
(pH 7.5) or RNAse free water to a concentration of between 0.1 and
1.0 microgram per microliter.
[0202] The following nucleotide sequences were derived first as
cDNA sequences identified in a corn rootworm mid-gut cDNA library
(Andersen et al., ibid), and were adapted for use in constructing
double stranded RNA molecules for use in testing the efficacy of
inhibiting a biological function in a pest by feeding double
stranded RNA molecules in the diet of the pest.
[0203] A. Chd3 Homologous Sequences
[0204] CHD genes have been identified in numerous eukaryotes, and
the corresponding proteins are proposed to function as
chromatin-remodeling factors. The term CHD is derived from the
three domains of sequence homology found in CHD proteins: a chromo
(chromatin organization modifier) domain, a SNF2-related
helicase/ATPase domain, and a DNA-binding domain, each of which is
believed to confer a distinct chromatin-related activity. CHD
proteins are separated into two categories based on the presence or
absence of another domain of sequence homology, a PHD zinc finger
domain, typically associated with chromatin related activity. CHD3
related proteins possess a PHD zinc finger domain, but CHD1 related
proteins do not. Experimental observations have suggested a role
for CHD3 proteins in repression of transcription, and in some
species have been shown to be a component of a complex that
contains histone deacetylase as a subunit. Deacetylation of
histones is correlated with transcriptional inactivation, and so
CHD3 proteins have been implicated to function as repressors of
transcription by virtue of being a component of a histone
deacetylase complex (Ogas et al., 1999). Thus, suppression of CHD3
protein synthesis may be a useful target for double stranded RNA
mediated inhibition of coleopteran pests.
[0205] B. Beta-Tubulin Homologous Sequences
[0206] Tubulin proteins are important structural components of many
cellular structures in all eukaryote cells and principally in the
formation of microtubules. Inhibition of microtubule formation in
cells results in catastrophic effects including interference with
the formation of mitotic spindles, blockage of cell division, and
the like. Therefore, suppression of tubulin protein formation may
be a useful target for double stranded RNA mediated inhibition.
[0207] C. 40 kDa V-ATPase Homologous Sequences
[0208] Energy metabolism within subcellular organelles in
eukaryotic systems is an essential function. Vacuolar ATP synthases
are involved in maintaining sufficient levels of ATP within
vacuoles. Therefore, vacuolar ATP synthases may be a useful target
for double stranded RNA mediated inhibition.
[0209] D. EF1.alpha. Homologous Sequences
[0210] Transcription elongation and transcription termination
factors are essential to metabolism and may be advantageous targets
for double stranded RNA mediated inhibition.
[0211] E. 26S Proteasome Subunit p28 Homologous Sequences
[0212] The 26S proteasome is a large, ATP-dependent, multi-subunit
protease that is highly conserved in all eukaryotes. It has a
general function in the selective removal of various short-lived
proteins that are first covalently linked to ubiquitin and then
subsequently degraded by the 26S proteasome complex. The ubiquitin
pathway plays an important role in the control of the cell cycle by
the specific degradation of a number of regulatory proteins
including mitotic cyclins and inhibitors of cyclin-dependent
kinases such as p27 of mammalian cells. Thus, the suppression of
26S proteasome synthesis and suppression of synthesis of its
component subunits may be preferred targets for double stranded RNA
mediated inhibition. (Smith et al., 1997).
[0213] F. Juvenile Hormone Epoxide Hydrolase Homologous
Sequences
[0214] Insect juvenile hormone controls and regulates a variety of
necessary biological processes within the insect life cycle
including but not necessarily limited to metamorphosis,
reproduction, and diapause. Juvenile hormone (JH) concentrations
are required to peak at appropriate times within the haemolymph of
the larval form of an insect pest, in particular lepidopteran and
coleopteran larvae, and then must be degraded in order to terminate
the effects of the hormone response. Enzymes involved in decreasing
the concentration of juvenile hormone are effective through two
primary pathways of metabolic degradation. One pathway involves
juvenile hormone esterase (JHE), which hydrolyzes the methyl ester
providing the corresponding acid. The second pathway utilizes
juvenile hormone epoxide hydrolase (JHEH) to achieve hydrolysis of
the epoxide, resulting in formation of the diol. The contribution
of JHE in the degradation of JH is well understood and has been
found to be invariate between the lepidoptera and coleoptera
species. Inhibition of JH esterase has been associated with severe
morphological changes including but not limited to larval
wandering, deferred pupation, and development of malformed
intermediates. In contrast, the contribution of JHEH in JH
metabolism is less well understood and had been shown to vary
between the species, but recent studies point to evidence that
suggests that JHEH may be the primary route of metabolism of JH
(Brandon J. Fetterolf, Doctoral Dissertation, North Carolina State
University (Feb. 10, 2002) Synthesis and Analysis of Mechanism
Based Inhibitors of Juvenile Hormone Epoxide Hydrolase from Insect
Trichoplusia ni). In any event, disruption of either JH degradation
pathway using gene suppression technology could be an effective
target for double stranded RNA mediated pest inhibition.
[0215] G. Swelling Dependent Chloride Channel Protein Homologous
Sequences
[0216] Swelling dependent chloride channel proteins have been
postulated to play a critical role in osmoregulation in eukaryotic
animal cell systems. Therefore, a nucleotide sequence exhibiting
the ability to express an amino acid sequence that exhibits
homology to previously identified swelling dependent chloride
channel proteins may be a useful target for RNA inhibition in a
pest.
[0217] H. Glucose-6-Phosphate 1-Dehydrogenase Protein Homologous
Sequences
[0218] Glucose-6-phosphate 1-dehydrogenase protein (G6PD) catalyzes
the oxidation of glucose-6-phosphate to 6-phosphogluconate while
concomitantly reducing the oxidized form of nicotinamide adenine
dinucleotide phosphate (NADP+) to NADPH. NADPH is known in the art
as a required cofactor in many eukaryotic biosynthetic reactions,
and is known to maintain glutathione in its reduced form. Reduced
glutathione acts as a scavenger for dangerous oxidative metabolites
in eukaryotic cells, and with the assistance of the enzyme
glutathione peroxidase, convert harmful hydrogen peroxide to water
(Beutler et al., 1991). Therefore, G6PD may be a preferable target
for double stranded RNA mediated inhibition in a coleopteran
pest.
[0219] I. Act42A Protein Homologous Sequences
[0220] Actin is a ubiquitous and highly conserved eukaryotic
protein required for cell motility and locomotion (Lovato et al.,
2001). A number of CRW cDNA sequences were identified that were
predicted to likely encode actin or proteins exhibiting amino acid
sequence structure related to actin proteins. Therefore, genes
encoding actin homologues in a pest cell may be useful targets for
double stranded RNA mediated inhibition.
[0221] J. ADP-Ribosylation Factor 1 Homologous Sequences
[0222] ADP ribosylation factors have been demonstrated to be
essential in cell function in that they play integral roles in the
processes of DNA damage repair, carcinogenesis, cell death, and
genomic stability. Thus, it would be useful to be able to
selectively disrupt transcription of ADP-ribosylation factors in
coleopteran pest species using double stranded RNA mediated
inhibition.
[0223] K. Transcription Factor IIB Protein Homologous Sequences
[0224] Transcription elongation and transcription termination
factors, as indicated above, are essential to metabolism and may be
advantageous targets for double stranded RNA mediated inhibition to
control or eliminate coleopteran pest infestation.
[0225] L. Chitinase Homologous Sequences
[0226] Chitin is a .beta.(1.fwdarw.4)homopolymer of
N-acetylglucosamine and is found in insect exoskeletons. Chitin is
formed from UDP-N-acetlglucosamine in a reaction catalyzed by
chitin synthase. Chitin is a structural homopolymer polysaccharide,
and there are many enzymatic steps involved in the construction of
this highly branched and cross-linked structure. Chitin gives
shape, rigidity and support to insects and provides a scaffolding
to which internal organs such as muscles are attached. Chitin must
also be degraded to some extent to mediate the steps involved in
the insect molting process. Therefore, it is believed that double
stranded RNA mediated inhibition of proteins in these pathways
would be useful as a means for controlling coleopteran pest
infestation.
[0227] M. Ubiquitin Conjugating Enzyme Homologous Sequences
[0228] The ubiquitin pathway plays an important role in the control
of the cell cycle by the specific degradation of a number of
regulatory proteins including mitotic cyclins and inhibitors of
cyclin-dependent kinases such as p27 of mammalian cells. Thus,
genes encoding ubiquitin and associated components may be a
preferred target for double stranded RNA mediated inhibition.
(Smith et al., 1997). The ubiquitin-dependent proteolytic pathway
is one of the major routes by which intracellular proteins are
selectively destroyed in eukaryotes. Conjugation of ubiquitin to
substrate proteins is mediated by a remarkably diverse array of
enzymes. Proteolytic targeting may also be regulated at steps
between ubiquitination of the substrate and its degradation to
peptides by the multi-subunit 26S protease. The complexity of the
ubiquitin system suggests a central role for protein turnover in
eukaryotic cell regulation, and implicates other proteins in the
pathway including ubiquitin-activating enzyme,
ubiquitin-conjugating enzyme, ubiquitin-protein ligase, and 26S
proteasome subunit components. Therefore, it is believed that
double stranded RNA mediated inhibition of proteins in this pathway
would be useful as a means for controlling coleopteran pest
infestation.
[0229] N. Glyceraldehyde-3-Phosphate Dehydrogenase Homologous
Sequences
[0230] The glycolytic pathway is an essential pathway in most
organisms and is involved in the production of metabolic energy
from the degradation of glucose. One important enzyme in the second
stage of the glycolytic pathway is glyceraldehyde-3-phosphate
dehydrogenase (G3PDH), which, in the presence of NAD+ and inorganic
phosphate, catalyzes the oxidation of 3-phospho-glyceraldehyde to
3-phosphoglyceroyl-phosphate along with the formation of NADH. The
important component of this reaction is the storage of energy
through the formation of NADH. Genes encoding enzymes associated
with the glycolytic pathway, and particularly genes: encoding
enzymes involved in the steps useful in formation of energy
reserves may be particularly useful targets for double stranded RNA
mediated inhibition in coleopteran pest species.
[0231] O. Ubiquitin B Homologous Sequences
[0232] As described above, the ubiquitin protein degradation
pathway plays an important role in the control of the cell cycle by
the specific degradation of a number of regulatory proteins
including mitotic cyclins and inhibitors of cyclin-dependent
kinases such as p27 of mammalian cells. Thus, genes encoding
ubiquitin and associated components may be a preferred target for
double stranded RNA mediated inhibition. (Smith et al., 1997).
[0233] P. Juvenile Hormone Esterase Homologs
[0234] As indicated above, insect juvenile hormone controls and
regulates a variety of necessary biological processes within the
insect life cycle including but not necessarily limited to
metamorphosis, reproduction, and diapause. Disruption of JH
synthesis or degradation pathways using gene suppression technology
could be an effective target for double stranded RNA mediated pest
inhibition.
[0235] Q. Alpha Tubulin Homologous Sequences
[0236] Eukaryotic cells generally utilize cytoskeletal structural
elements that are important, no t only as a mechanical scaffold,
but also in sustaining the shape of the cell. Semiflexible
microfilaments make cells mobile, help them to divide in mitosis
(cytokinesis) and, in vertebrate and invertebrate animals, are
responsible for muscular contraction. The relatively stiff
microtubules which are made up of alpha and beta tubulin proteins
play an important role in acting as a sort of highway for transport
of vesicles and organelles and in the separation of chromosomes
during mitosis (karyokinesis). The flexible intermediate filaments
provide at least additional strength to the overall cellular
structure. The cytoskeleton is also known to be involved in
signaling across the cell cytoplasm. Taking these functions into
account, it is believed that any disruption of the cytoskeleton or
even subtle changes of its integrity may cause pathological
consequences to a cell.
[0237] R. Transport Related Sequences
[0238] As indicated above, sorting and transport of various
molecules within a cell, including to appropriate organelles, as
well as their secretion is an important physiological function.
Such sorting pathways could include those relying on the endosomal
sorting complex required for transport (ESCRT), complexes I-III,
among others. Thus, functions related to transport of polypeptides
and other molecules may also be a preferred target for
dsRNA-mediated inhibition.
Example 2
Insect Feeding Bioassays
[0239] Samples of double stranded RNA (dsRNA) were subjected to
bioassay with a selected number of target pests. The dsRNA was
prepared from sequences identified according to Example 1 using
either a full contig sequence in the case of SEQ ID Nos: 1-6, or a
sequence amplified from the assembled contig using the primer pairs
as set forth in the sequence listing. Varying does of dsRNA were
applied as an overlay to corn rootworm artificial diet according to
the following procedure. Diabrotica virgifera virgifera (WCR) eggs
were obtained from Crop Characteristics, Inc., Farmington, Minn.
The non-diapausing WCR eggs were incubated in soil for about 13
days at 24 C, 60% relative humidity, in complete darkness. On day
13 the soil containing WCR eggs was placed between #30 and #60 mesh
sieves and the eggs were washed out of the soil using a high
pressure garden hose. The eggs were surface disinfested by soaking
in LYSOL for three minutes, rinsed three times with sterile water,
washed one time with a 10% formalin solution and then rinsed three
additional times in sterile water. Eggs treated in this way were
dispensed onto sterile coffee filters and hatched overnight at
27.degree. C., 60% relative humidity, in complete darkness.
[0240] To prepare dsRNA, amplicons of selected sequences were
cloned into a plasmid vector capable of replication in E. coli and
sufficient amounts of plasmid DNA was recovered to allow for in
vitro T7 RNA polymerase transcription from the embedded convergent
T7 promoters at either end of the cloned fragment. Double stranded
RNA was produced and subjected to bioassay; one RNA segment
comprising the sequence as set forth in the sequence listing, the
other RNA segment being substantially the reverse complement of the
nucleotide sequence, uridines appropriately positioned in place of
thymidines. A sample of double stranded RNA (dsRNA) was treated
with DICER or with RNAse III to produce sufficient quantities of
small interfering RNA's (siRNA). Samples containing siRNA or dsRNA
were overlayed onto CRW diet bioassay as described above and larvae
were allowed to feed as set forth below.
[0241] A sample of double stranded RNA was either added directly to
each well containing insect diet as indicated above, or was
modified prior to being added to insect diet. Modification of
double stranded RNA followed the instructions for RNAse III (AMBION
CORPORATION, Austin, Tex.) or DICER (STRATAGENE, La Jolla, Calif.)
provided by the manufacturer. RNAse III digestion of double
stranded RNA produced twenty-one and twenty-two nucleotide duplexes
containing 5' phosphorylated ends and 3' hydroxyl ends with 2-3
base overhangs, similar to the .about.21-26 base pair duplexed
short interfering RNA (siRNA) fragments produced by the dicer
enzyme in the eukaryotic pathway identified by Hamilton et. al.
(1999) and Elbashir et. al. (2001a). This collection of short
interfering RNA duplexes was further purified and a sample
characterized by polyacrylamide gel electrophoresis to determine
the integrity and efficiency of duplex formation. The purity and
quantity of the sample was then determined by spectrophotometry at
a wavelength of 250 nanometers, and unused sample retained for
further use by storage at -20.degree. C.
[0242] Insect diet was prepared essentially according to Pleau et
al. (2002), with the following modifications. 9.4 grams of SERVA
agar was dispensed into 540 milliliters of purified water and
agitated until the agar was thoroughly distributed. The water/agar
mixture was heated to boiling to completely dissolve the agar, and
then poured into a WARING blender. The blender was maintained at
low speed while 62.7 grams of BIO-SERV DIET mix (F9757), 3.75 grams
lyophilized corn root, 1.25 milliliters of green food coloring, and
0.6 milliliters of formalin was added to the hot agar mixture. The
mixture was then adjusted to pH 9.0 with the addition of a 10%
potassium hydroxide stock solution. The approximately 600
milliliter volume of liquid diet was continually mixed at high
speed and maintained at from about 48.degree. C. to about
60.degree. C. using a sterilized NALGENE coated magnetic stir bar
on a magnetic stirring hot plate while being dispensed in aliquots
of 200 microliters into each well of FALCON 96-well round bottom
microtiter plates. The diet in the plates was allowed to solidify
and air dry in a sterile biohood for about ten minutes.
[0243] Thirty (30) microliter volumes of test samples containing
either control reagents or double stranded RNA in varying
quantities was overlayed onto the surface of the insect diet in
each well using a micro-pipettor repeater. Insect diet was allowed
to stand in a sterile biohood for up to one half hour after
application of test samples to allow the reagents to diffuse into
the diet and to allow the surface of the diet to dry. One WCR
neonate larva was deposited to each well with a fine paintbrush.
Plates were then sealed with MYLAR and ventilated using an insect
pin. 12-72 insect larvae were tested per dose depending on the
design of the assay. The bioassay plates were incubated at
27.degree. C., 60% relative humidity in complete darkness for 12-14
days. The number of surviving larvae per dose was recorded at the
12-14 day time point. Larval mass was determined using a suitable
microbalance for each surviving larva. Data was analyzed using
JMP.COPYRGT.4 statistical software (SAS Institute, 1995) and a full
factorial ANOVA was conducted with a Dunnet's test to look for
treatment effects compared to the untreated control (P<0.05). A
Tukey-Kramer post hoc test was performed to compare all pairs of
the treatments (P<0.05). The results of the CRW larvae feeding
assays exhibited significant growth inhibition and mortality
compared to controls as explained below.
Example 3
Results of Insect Feeding Bioassays
[0244] Artificial diet sufficient for rearing corn rootworm larvae
was prepared by applying samples of double stranded RNA sequences
identified as described in Example 1 using bioassays carried out as
described in Example 2. Corn rootworm larvae were typically allowed
to feed on the diet for twelve days and mortality and stunting
monitored in comparison to rootworms allowed to feed only on
negative and positive control diets. The results of the studies
confirmed significant levels (p<0.05) of larval stunting and/or
mortality using dsRNAs containing portions sequences homologous to
a variety of different gene classes. The sequences and vectors
yielding significant stunting and/or mortality and the
corresponding SEQ ID NO for the sequence expressed as a dsRNA are
given in Tables 1-5 below.
TABLE-US-00001 TABLE 1 dsRNA Constructs Demonstrating Significant
Stunting and/or Mortality in Effect in Insect Feeding Bioassays
with Southern Corn Rootworm or Western Corn Rootworm Vector
Sequence Expressed as dsRNA SEQ ID NO RNAi-pIC17553:001 Apple 697
RNAi-pIC17504:049 EST Full Length V-ATPase 695 RNAi-pIC17554:001
Rpl 9 698 RNAi-pIC17555:001 Rpl 19 699 RNAi-pIC17504:050 Section
6.0 V-ATPase 711 RNAi-pIC19514:001 WCR mRNA capping enzyme LIB5496-
696 028-A1-M1-A7 RNAi-pIC17552:003 LIB5462-042-A1-M1-H10 700
Dv.6_CG9355 DUSKY STRUCTURAL CONSTITUENT OF CUTICLE
RNAi-pIC17546:003 LIB5462-091-A1-M1-G3 701 Dv.1_CG6217 KNICKKOPF
UNK RNAi-pIC17546:001 Dv.1_CG6217 1 RNAi-pIC17549:001 Dv.4_CG1435 4
RNAi-pIC17550:001 LIB5444-065-A1-M1-D5 5 Dv.5_cg1915_1
RNAi-pIC17551:001 LIB5462-012-A1-M2-B2 703 Dv.5_cg1915_2
RNAi-pIC17552:001 Dv.6_CG9355 6 RNAi-pMON78412:002 Dv.7_CG3416;
putative Mov34:CG3416 10 ortholog RNAi-pMON96172:002 Dv.8_CG1088;
putative vacuolar 14 H+ ATPase E subunit: CG1088 ortholog
RNAi-pMON96168:002 Dv.9_CG2331; putative ATPase activity: 18 CG2331
ortholog RNAi-pMON78424:002 Dv.10_CG6141; putative ribosomal
protein 22 L9: CG6141 ortholog RNAi-pMON78425:001 Dv.11_CG2746 26
RNAi-pMON78444:002 Dv.12_CG1341; putative proteasome 30 regulatory
particle, rpt1: CG1341 ortholog RNAi-pMON78416:002 Dv.13_CG11276;
putative ribosomal 34 protein S4: CG11276 ortholog
RNAi-pMON78434:001 Dv.14_CG17927_2; putative myosin heavy 38 chain:
CG17927 ortholog RNAi-pMON78439:001 Dv.16_CG5394; putative
glutamyl-prolyl- 46 tRNA synthetase: CG5394 ortholog
RNAi-pMON78438:001 Dv.17_CG10149; putative proteasome 50 p44.5
subunit, rpn6: CG10149 ortholog RNAi-pMON78435:002 Dv.18_CG1404;
putative RAN small 54 monomeric GTPase: CG1404 ortholog
RNAi-pMON78449:002 Dv.19_CG18174; putative proteasome 58 regulatory
particle, lid subcomplex, rpn11: CG18174 ortholog
RNAi-pMON78419:001 Dv.20_CG3180_1; putative DNA-directed 62 RNA
polymerase II: CG3180 ortholog RNAi-pMON78440:002 Dv20_CG3180_2;
putative DNA-directed 706 RNA polymerase II: CG3180 ortholog
RNAi-pMON78420:001 Dv.21_CG3320; putative Rab1: CG3320 70 ortholog
RNAi-pMON78410:002 Dv.22_CG3395; putative Ribosomal 74 protein S9:
CG3395 ortholog RNAi-pMON78422:001 Dv.23_CG7269; putative helicase:
78 CG7269 ortholog RNAi-pMON78423:001 Dv.25_CG9012; putative
Clathrin heavy 86 chain: CG9012 ortholog RNAi-pMON78414:006 DV.26
sec1~5' half of EST 710 RNAi-pMON78414:001 Dv.26_CG9261; putative
sodium/ 90 potassium-exchanging ATPase: CG9261 ortholog
RNAi-pMON78413:001 Dv.27_CG12052; putative RNA 94 polymerase II
transcription factor: CG12052 ortholog. RNAi-pMON78427:001
Dv.35_CG3762; putative Vha68-2: 126 CG3762 ortholog.
RNAi-pMON97122:001 C1_Dv.35; putative Vha68-2: CG3762 713 ortholog;
concatamer RNAi-pMON97127:001 C2_Dv.35; putative Vha68-2: CG3762
714 ortholog; concatamer RNAi-pMON97125:001 C3_Dv.35; putative
Vha68-2: CG3762 715 ortholog; concatamer RNAi-pMON78441:001
Dv.39_CG9078; putative sphingolipid 142 delta-4 desaturase;
stearoyl-CoA 9- desaturase: CG9078 ortholog RNAi-pMON97114:001
Dv.41_CG2637; Female sterile Ketel; 150 involved in protein-nucleus
import: CG2637 ortholog RNAi-pMON97140:001 Dv.44_CG1244; Putative
nucleic acid 162 binding activity: CG1244 ortholog
RNAi-pMON78429:001 Dv.46_CG10689; putative RNA helicase: 170
CG10689 ortholog. RNAi-pMON78432:001 Dv.48_CG33196; putative
transmembrane 178 receptor protein tyrosine kinase: CG33196
ortholog RNAi-pMON78428:001 Dv.49_CG8055_1; putative binding, 182
carrier activity: CG8055_1 ortholog RNAi-pMON78428:003
Dv.49_CG8055_1; putative binding, 704 carrier activity: CG8055_1
ortholog Free region selected from DV.49 RNAi-pMON78426:001
Dv.50_CG10110_1; putative Cleavage and 186 polyadenylation
specificity factor: CG10110_1 ortholog RNAi-pMON96185:001
Dv.55_CG5931; putative splicing factor 202 activity, RNA helicase
activity: CG5931 ortholog RNAi-pMON78442:001 Dv.57_CG2968; putative
hydrogen- 206 exporting ATPase: CG2968 ortholog RNAi-pMON78431:001
Dv.58_CG1751; putative signal peptidase: 210 CG1751 ortholog
RNAi-pMON96177:001 Dv.61_CG3725_1; putative Calcium 222 ATPase:
CG3725 ortholog RNAi-pMON96183:001 Dv.62_CG3612; putative
bellwether: 230 CG3612 ortholog RNAi-pMON96180:002 Dv.65_CG7033;
putative chaperone 242 activity: CG7033 ortholog RNAi-pMON96176:001
Dv.66_CG32019; putative bent: CG32019 246 ortholog
RNAi-pMON96170:002 Dv.67_CG16916; putative endopeptidase 250
activity: CG16916 ortholog RNAi-pMON96166:001 Dv.70_CG5771;
putative Rab-protein 11: 258 CG5771 ortholog RNAi-pMON96179:001
Dv.72_CG6831; putative rhea: CG6831 266 ortholog RNAi-pMON96186:001
Dv.73_CG10119; putative Lamin C: 270 CG10119 ortholog
RNAi-pMON96160:002 Dv.74_CG6375; putative pitchoune: 274 CG6375
ortholog RNAi-pMON97137:001 Dv.77_CG4214; putative Syntaxin 5; 286
involved in intracellular protein transport: CG4214 ortholog
RNAi-pMON96167:001 Dv.82_CG8264; putative Bx42: CG8264 302 ortholog
RNAi-pMON96171:001 Dv.83_CG11397; putative gluon: 306 CG11397
ortholog RNAi-pMON96187:003 Dv.85_CG4494; putative protein binding
314 activity: CG4494 ortholog RNAi-pMON96174:001 Dv.86_CG5055;
putative bazooka: 318 CG5055 ortholog RNAi-pMON97126:001
Dv.88_CG8756; function unknown; 326 contains chitin binding domain:
CG8756 ortholog RNAi-pMON97130:001 Dv.93_CG8515; putative
structural 342 constituent of cuticle; contains chitin binding
domain: CG8515 ortholog RNAi-pMON97109:001 Dv.99_CG2446; Unknown;
lethal in 366 Drosophila & low homology with human: CG2446
ortholog RNAi-pMON97111:001 Dv.105_CG1250_1; GTPase activator, 390
involved in intracellular protein transport: CG1250 ortholog
RNAi-pMON97112:001 Dv.105_CG1250_2; GTPase activator, 394 involved
in intracellular protein transport: CG1250 ortholog
RNAi-pMON97107:001 Dv.107_CG14813; COPI vesicle coat; 398 involved
in Golgi to ER intracellular protein transport: CG14813 ortholog
RNAi-pMON97115:001 Dv.108_CG17248; n-synaptobrevin; 402 involved in
intracellular protein transport: CG17248 ortholog
RNAi-pMON97133:001 Dv.113; function unknown; WCR unique 422
sequence RNAi-pMON97121:001 Dv.122_CG3164; putative ATP-binding 454
cassette transporter activity: CG3164 ortholog RNAi-pMON97134:001
Dv.127; function unknown; no homology 470 with human
RNAi-pMON97171:001 Dv.146; function unknown, WCR unique 514
sequence RNAi-pMON97166:001 Dv.147; function unknown, WCR unique
518 sequence RNAi-pMON97167:001 Dv.149; function unknown, WCR
unique 526 sequence RNAi-pMON97169:001 Dv.155; function unknown,
WCR unique 550 sequence RNAi-pMON97173:001 Dv.162; function
unknown, WCR unique 578 sequence RNAi-pMON97170:001 Dv.170;
function unknown, WCR unique 610 sequence
TABLE-US-00002 TABLE 2 dsRNA Constructs Causing Significant
Stunting Levels in Feeding Bioassays with Western Corn Rootworm
(WCR) Larvae Vector Sequence Expressed as dsRNA RNAi-pIC17553:001
Apple RNAi-pIC17504:049 EST Full Length V-ATPase RNAi-pIC17554:001
Rpl 9 RNAi-pIC17555:001 Rpl 19 RNAi-pIC17504:050 Section 6.0
V-ATPase RNAi-pIC16005:001 V-ATPase D subunit 1 RNAi-pIC19514:001
WCR mRNA capping enzyme LIB5496-028-A1-M1-A7 RNAi-pIC17546:001
Dv.1_CG6217 RNAi-pIC17549:001 Dv.4_CG1435 RNAi-pIC17551:001
LIB5462-012-A1-M2-B2 Dv.5_cg1915_2 RNAi-pIC17552:001 Dv.6_CG9355
RNAi-pMON78412:001 Dv.7_CG3416 RNAi-pMON78412:002 Dv.7_CG3416;
putative Mov34:CG3416 ortholog RNAi-pMON96172:002 Dv.8_CG1088;
putative vacuolar H+ ATPase E subunit: CG1088 ortholog
RNAi-pMON96168:002 Dv.9_CG2331; putative ATPase activity: CG2331
ortholog RNAi-pMON78424:002 Dv.10_CG6141; putative ribosomal
protein L9: CG6141 ortholog RNAi-pMON78425:001 Dv.11_CG2746
RNAi-pMON78444:002 Dv.12_CG1341; putative proteasome regulatory
particle, rpt1: CG1341 ortholog RNAi-pMON78416:002 Dv.13_CG11276;
putative ribosomal protein S4: CG11276 ortholog RNAi-pMON78434:001
Dv.14_CG17927_2; putative myosin heavy chain: CG17927 ortholog
RNAi-pMON78439:001 Dv.16_CG5394; putative glutamyl-prolyl-tRNA
synthetase: CG5394 ortholog RNAi-pMON78435:002 Dv.18_CG1404;
putative RAN small monomeric GTPase: CG1404 ortholog
RNAi-pMON78449:002 Dv.19_CG18174; putative proteasome regulatory
particle, lid subcomplex, rpn11: CG18174 ortholog
RNAi-pMON78440:002 Dv.20_CG3180_2; putative DNA-directed RNA
polymerase II: CG3180 ortholog RNAi-pMON78420:002 Dv.21_CG3320;
putative Rab1: CG3320 ortholog RNAi-pMON78410:001 Dv.22_CG3395;
putative Ribosomal protein S9: CG3395 ortholog RNAi-pMON78422:001
Dv.23_CG7269; putative helicase: CG7269 ortholog RNAi-pMON78423:001
Dv.25_CG9012; putative Clathrin heavy chain: CG9012 ortholog
RNAi-pMON78414:001 Dv.26_CG9261; putative
sodium/potassium-exchanging ATPase: CG9261 ortholog
RNAi-pMON78413:001 Dv.27_CG12052; putative RNA polymerase II
transcription factor: CG12052 ortholog. RNAi-pMON97122:001
C1_Dv.35; putative Vha68-2: CG3762 ortholog; concatamer
RNAi-pMON97127:001 C2_Dv.35; putative Vha68-2: CG3762 ortholog;
concatamer RNAi-pMON97125:001 C3_Dv.35; putative Vha68-2: CG3762
ortholog; concatamer RNAi-pMON78427:007 Dv.35_CG3762; putative
Vha68-2: CG3762 ortholog. RNAi-pMON78441:001 Dv.39_CG9078; putative
sphingolipid delta-4 desaturase; stearoyl- CoA 9-desaturase: CG9078
ortholog RNAi-pMON97114:001 Dv.41_CG2637; Female sterile Ketel;
involved in protein-nucleus import: CG2637 ortholog
RNAi-pMON78429:001 Dv.46_CG10689; putative RNA helicase: CG10689
ortholog. RNAi-pMON78432:001 Dv.48_CG33196; putative transmembrane
receptor protein tyrosine kinase: CG33196 ortholog
RNAi-pMON78428:001 Dv.49_CG8055_1; putative binding, carrier
activity: CG8055_1 ortholog RNAi-pMON78428:003 Dv.49_CG8055_1;
putative binding, carrier activity: CG8055_1 ortholog Free region
selected from DV.49 RNAi-pMON78426:001 Dv.50_CG10110_1; putative
Cleavage and polyadenylation specificity factor: CG10110_1 ortholog
RNAi-pMON96185:001 Dv.55_CG5931; putative splicing factor activity,
RNA helicase activity: CG5931 ortholog RNAi-pMON78442:001
Dv.57_CG2968; putative hydrogen-exporting ATPase: CG2968 ortholog
RNAi-pMON78431:001 Dv.58_CG1751; putative signal peptidase: CG1751
ortholog RNAi-pMON96177:001 Dv.61_CG3725_1; putative Calcium
ATPase: CG3725 ortholog RNAi-pMON96182:001 Dv.61_CG3725_2; putative
Calcium ATPase: CG3725 ortholog RNAi-pMON96183:001 Dv.62_CG3612;
putative bellwether: CG3612 ortholog RNAi-pMON96180:002
Dv.65_CG7033; putative chaperone activity: CG7033 ortholog
RNAi-pMON96180:001 Dv.65_CG7033; putative chaperone activity:
CG7033 ortholog RNAi-pMON96176:001 Dv.66_CG32019; putative bent:
CG32019 ortholog RNAi-pMON96170:002 Dv.67_CG16916; putative
endopeptidase activity: CG16916 ortholog RNAi-pMON96166:001
Dv.70_CG5771; putative Rab-protein 11: CG5771 ortholog
RNAi-pMON96179:001 Dv.72_CG6831; putative rhea: CG6831 ortholog
RNAi-pMON96186:001 Dv.73_CG10119; putative Lamin C: CG10119
ortholog RNAi-pMON96160:002 Dv.74_CG6375; putative pitchoune:
CG6375 ortholog RNAi-pMON96160:001 Dv.74_CG6375; putative
pitchoune: CG6375 ortholog RNAi-pMON97137:002 Dv.77_CG4214;
putative Syntaxin 5; involved in intracellular protein transport:
CG4214 ortholog RNAi-pMON96167:001 Dv.82_CG8264; putative Bx42:
CG8264 ortholog RNAi-pMON96171:001 Dv.83_CG11397; putative gluon:
CG11397 ortholog RNAi-pMON96187:003 Dv.85_CG4494; putative protein
binding activity: CG4494 ortholog RNAi-pMON97126:001 Dv.88_CG8756;
function unknown; contains chitin binding domain: CG8756 ortholog
RNAi-pMON97109:001 Dv.99_CG2446; Unknown; lethal in Drosophila
& low homology with human: CG2446 ortholog RNAi-pMON97111:001
Dv.105_CG1250_1; GTPase activator, involved in intracellular
protein transport: CG1250 ortholog RNAi-pMON97107:001
Dv.107_CG14813; COPI vesicle coat; involved in Golgi to ER
intracellular protein transport: CG14813 ortholog
RNAi-pMON97115:001 Dv.108_CG17248; n-synaptobrevin; involved in
intracellular protein transport: CG17248 ortholog
RNAi-pMON97121:001 Dv.122_CG3164; putative ATP-binding cassette
transporter activity: CG3164 ortholog RNAi-pMON97171:001 Dv.146;
function unknown, WCR unique sequence RNAi-pMON97166:001 Dv.147;
function unknown, WCR unique sequence RNAi-pMON97167:001 Dv.149;
function unknown, WCR unique sequence RNAi-pMON97169:001 Dv.155;
function unknown, WCR unique sequence RNAi-pMON97173:001 Dv.162;
function unknown, WCR unique sequence RNAi-pMON97170:001 Dv.170;
function unknown, WCR unique sequence
TABLE-US-00003 TABLE 3 dsRNA Constructs Causing Significant
Mortality Levels in Feeding Bioassays with Western Corn Rootworm
(WCR) Larvae Vector Sequence Expressed as dsRNA RNAi-pIC17553:001
Apple RNAi-pIC17504:049 EST Full Length V-ATPase RNAi-pIC17555:001
Rpl 19 RNAi-pIC17554:001 Rpl 9 RNAi-pIC17504:050 Section 6.0
V-ATPase RNAi-pIC17504:054 V-ATPase subunit 2 sequence from
Diabrotica virgifera virgifera Full EST sequence serving as
positive control RNAi-pIC19514:001 WCR mRNA capping enzyme
LIB5496-028-A1-M1-A7 RNAi-pIC17546:001 Dv.1_CG6217
RNAi-pIC17549:001 Dv.4_CG1435 RNAi-pIC17550:001
LIB5444-065-A1-M1-D5 Dv.5_cg1915_1 RNAi-pMON78412:002 Dv.7_CG3416;
putative Mov34:CG3416 ortholog RNAi-pMON96172:001 Dv.8_CG1088;
putative vacuolar H+ ATPase E subunit: CG1088 ortholog
RNAi-pMON96168:001 Dv.9_CG2331; putative ATPase activity: CG2331
ortholog RNAi-pMON78424:002 Dv.10_CG6141; putative ribosomal
protein L9: CG6141 ortholog RNAi-pMON78425:001 Dv.11_CG2746
RNAi-pMON78444:001 Dv.12_CG1341; putative proteasome regulatory
particle, rpt1: CG1341 ortholog RNAi-pMON78416:002 Dv.13_CG11276;
putative ribosomal protein S4: CG11276 ortholog RNAi-pMON78434:001
Dv.14_CG17927_2; putative myosin heavy chain: CG17927 ortholog
RNAi-pMON78435:001 Dv.18_CG1404; putative RAN small monomeric
GTPase: CG1404 ortholog RNAi-pMON78449:001 Dv.19_CG18174; putative
proteasome regulatory particle, lid subcomplex, rpn11: CG18174
ortholog RNAi-pMON78440:001 Dv.20_CG3180; putative DNA-directed RNA
polymerase II: CG3180 ortholog RNAi-pMON78420:001 Dv.21_CG3320;
putative Rab1: CG3320 ortholog RNAi-pMON78410:001 Dv.22_CG3395;
putative Ribosomal protein S9: CG3395 ortholog RNAi-pMON78422:001
Dv.23_CG7269; putative helicase: CG7269 ortholog RNAi-pMON78414:006
DV.26 sec1~5' half of EST RNAi-pMON78414:001 Dv.26_CG9261; putative
sodium/potassium-exchanging ATPase: CG9261 ortholog
RNAi-pMON97122:001 C1_Dv.35; putative Vha68-2: CG3762 ortholog;
concatamer RNAi-pMON97127:001 C2_Dv.35; putative Vha68-2: CG3762
ortholog; concatamer RNAi-pMON97125:001 C3_Dv.35; putative Vha68-2:
CG3762 ortholog; concatamer RNAi-pMON78427:001 Dv.35_CG3762;
putative Vha68-2: CG3762 ortholog. RNAi-pMON97114:001 Dv.41_CG2637;
Female sterile Ketel; involved in protein-nucleus import: CG2637
ortholog RNAi-pMON97140:001 Dv.44_CG1244; Putative nucleic acid
binding activity: CG1244 ortholog RNAi-pMON78429:001 Dv.46_CG10689;
putative RNA helicase: CG10689 ortholog. RNAi-pMON78428:001
Dv.49_CG8055_1; putative binding, carrier activity: CG8055_1
ortholog RNAi-pMON78428:003 Dv.49_CG8055_1; putative binding,
carrier activity: CG8055_1 ortholog Free region selected from DV.49
RNAi-pMON78426:001 Dv.50_CG10110_1; putative Cleavage and
polyadenylation specificity factor: CG10110_1 ortholog
RNAi-pMON96185:001 Dv.55_CG5931; putative splicing factor activity,
RNA helicase activity: CG5931 ortholog RNAi-pMON78431:001
Dv.58_CG1751; putative signal peptidase: CG1751 ortholog
RNAi-pMON96177:001 Dv.61_CG3725_1; putative Calcium ATPase: CG3725
ortholog RNAi-pMON96182:001 Dv.61_CG3725_2; putative Calcium
ATPase: CG3725 ortholog RNAi-pMON96180:001 Dv.65_CG7033; putative
chaperone activity: CG7033 ortholog RNAi-pMON96176:001
Dv.66_CG32019; putative bent: CG32019 ortholog RNAi-pMON96170:001
Dv.67_CG16916; putative endopeptidase activity: CG16916 ortholog
RNAi-pMON96166:001 Dv.70_CG5771; putative Rab-protein 11: CG5771
ortholog RNAi-pMON96160:001 Dv.74_CG6375; putative pitchoune:
CG6375 ortholog RNAi-pMON97137:001 Dv.77_CG4214; putative Syntaxin
5; involved in intracellular protein transport: CG4214 ortholog
RNAi-pMON96167:001 Dv.82_CG8264; putative Bx42: CG8264 ortholog
RNAi-pMON96187:002 Dv.85_CG4494; putative protein binding activity:
CG4494 ortholog RNAi-pMON97126:001 Dv.88_CG8756; function unknown;
contains chitin binding domain: CG8756 ortholog RNAi-pMON97130:001
Dv.93_CG8515; putative structural constituent of cuticle; contains
chitin binding domain: CG8515 ortholog RNAi-pMON97111:001
Dv.105_CG1250_1; GTPase activator, involved in intracellular
protein transport: CG1250 ortholog RNAi-pMON97107:001
Dv.107_CG14813; COPI vesicle coat; involved in Golgi to ER
intracellular protein transport: CG14813 ortholog
RNAi-pMON97115:001 Dv.108_CG17248; n-synaptobrevin; involved in
intracellular protein transport: CG17248 ortholog
RNAi-pMON97133:001 Dv.113; function unknown; WCR unique sequence
RNAi-pMON97121:001 Dv.122_CG3164; putative ATP-binding cassette
transporter activity: CG3164 ortholog RNAi-pMON97134:001 Dv.127;
function unknown; no homology with human
TABLE-US-00004 TABLE 4 dsRNA Constructs Causing Significant
Stunting Levels in Feeding Bioassays with Southern Corn Rootworm
(SCR) Larvae Vector Sequence Expressed as dsRNA RNAi-pMON96172:001
Dv8_CG1088; putative vacuolar H+ ATPase E subunit: CG1088 ortholog
RNAi-pMON96168:001 Dv9_CG2331; putative ATPase activity: CG2331
ortholog RNAi-pMON78424:001 Dv.10_CG6141 RNAi-pMON96155:002
Dv.10_CG6141; putative ribosomal protein L9: CG6141 ortholog.
RNAi-pMON78425:001 Dv.11_CG2746 RNAi-pMON96158:002 Dv.11_CG2746;
putative Ribosomal protein L19: CG2746 ortholog. RNAi-pMON78416:001
Dv.13_CG11276 RNAi-pMON78434:001 Dv.14_CG17927_2; putative myosin
heavy chain: CG17927 ortholog RNAi-pMON78435:001 Dv.18_CG1404;
putative RAN small monomeric GTPase: CG1404 ortholog
RNAi-pMON78449:001 Dv.19_CG18174; putative proteasome regulatory
particle, lid subcomplex, rpn11: CG18174 ortholog
RNAi-pMON78419:001 Dv.20_CG3180 RNAi-pMON78420:001 Dv.21_CG3320;
putative Rab1: CG3320 ortholog RNAi-pMON78414:001 Dv.26_CG9261;
putative sodium/potassium-exchanging ATPase: CG9261 ortholog
RNAi-pMON97122:001 C1_Dv35; putative Vha68-2: CG3762 ortholog;
concatamer RNAi-pMON97125:001 C3_Dv35; putative Vha68-2: CG3762
ortholog; concatamer RNAi-pMON78427:008 Dv.35_CG3762; putative
Vha68-2: CG3762 ortholog. RNAi-pMON78428:001 Dv.49_CG8055_1;
putative binding, carrier activity: CG8055_1 ortholog
RNAi-pMON78442:001 Dv.57_CG2968; putative hydrogen-exporting
ATPase: CG2968 ortholog RNAi-pMON96177:001 Dv61_CG3725_1; putative
Calcium ATPase: CG3725 ortholog RNAi-pMON96166:001 Dv70_CG5771;
putative Rab-protein 11: CG5771 ortholog RNAi-pMON97126:001
Dv88_CG8756; function unknown; contains chitin binding domain:
CG8756 ortholog RNAi-pMON97111:001 Dv105_CG1250_1; GTPase
activator, involved in intracellular protein transport: CG1250
ortholog RNAi-pMON97112:001 Dv105_CG1250_2; GTPase activator,
involved in intracellular protein transport: CG1250 ortholog
RNAi-pMON97107:001 Dv107_CG14813; COPI vesicle coat; involved in
Golgi to ER intracellular protein transport: CG14813 ortholog
RNAi-pMON97121:001 Dv122_CG3164; putative ATP-binding cassette
transporter activity: CG3164 ortholog
TABLE-US-00005 TABLE 5 dsRNA Constructs Causing Significant
Mortality Levels in Feeding Bioassays with Southern Corn Rootworm
(SCR) Larvae Vector Sequence Expressed as dsRNA RNAi-pIC17546:003
LIB5462-091-A1-M1-G3 Dv1_CG6217 KNICKKOPF UNK RNAi-pIC17504:055
V-ATPase subunit 2 sequence from Diabrotica virgifera virgifera
Full EST sequence serving as positive control RNAi-pMON96155:001
Dv.10_CG6141; putative ribosomal protein L9: CG6141 ortholog
RNAi-pMON96158:001 Dv.11_CG2746; putative Ribosomal protein L19:
CG2746 ortholog RNAi-pMON78416:001 Dv.13_CG11276 RNAi-pMON96154:003
Dv.14_CG17927; putative myosin heavy chain: CG17927 ortholog; cells
grown in S Complete medium. RNAi-pMON78438:001 Dv.17_CG10149;
putative proteasome p44.5 subunit, rpn6: CG10149 ortholog
RNAi-pMON78449:001 Dv.19_CG18174; putative proteasome regulatory
particle, lid subcomplex, rpn11: CG18174 ortholog
RNAi-pMON78440:001 Dv.20_CG3180; putative DNA-directed RNA
polymerase II: CG3180 ortholog RNAi-pMON96156:001 Dv.20_CG3180;
putative RNA polymerase II 140 kD subunit: CG3180 ortholog
RNAi-pMON78420:001 Dv.21_CG3320; putative Rab1: CG3320 ortholog
RNAi-pMON78427:006 Dv.35_CG3762; putative Vha68-2: CG3762 ortholog.
RNAi-pMON78428:001 Dv.49_CG8055_1; putative binding, carrier
activity: CG8055_1 ortholog RNAi-pMON96177:001 Dv61_CG3725_1;
putative Calcium ATPase: CG3725 ortholog RNAi-pMON96166:001
Dv70_CG5771; putative Rab-protein 11: CG5771 ortholog
RNAi-pMON96174:001 Dv86_CG5055; putative bazooka: CG5055 ortholog
RNAi-pMON97111:001 Dv105_CG1250_1; GTPase activator, involved in
intracellular protein transport: CG1250 ortholog RNAi-pMON97107:001
Dv107_CG14813; COPI vesicle coat; involved in Golgi to ER
intracellular protein transport: CG14813 ortholog
Example 4
Transgenic Plant Transformation and Bioassays
[0245] Briefly, the sequence encoding a dsRNA construct as
described above is linked at the 5' end to a sequence consisting of
a 35S promoter operably linked to a maize hsp70 intron and at the
3' end to a Nos3' transcription termination and polyadenylation
sequence. This expression cassette is placed downstream of a
glyphosate selection cassette. These linked cassettes are then
placed into an Agrobacterium tumefaciens plant transformation
functional vector, used to transform maize tissue to glyphosate
tolerance, and events selected and transferred to soil. R.sub.0
plant roots are fed to western corn rootworm larvae (WCR,
Diabrotica virgifera). Transgenic corn roots are handed-off in
Petri dishes with MS0D medium containing antibiotics and glyphosate
for in vitro selection. Two WCR larvae are infested per root in
each dish with a fine tip paintbrush. The dishes are sealed with
Parafilm to prevent the larvae from escaping. The assays are placed
into a 27.degree. C., 60% RH Percival incubator in complete
darkness. Contamination and larval quality are monitored. After six
days of feeding on root tissue, the larvae are transferred to WCR
diet in a 96 well plate. The larvae are allowed to feed on the diet
for eight days making the full assay fourteen days long. Larval
mass and survivorship are recorded for analysis. A one-way ANOVA
analysis and a Dunnett's test is performed on the larval mass data
to look for statistical significance compared to an untransformed
negative control. WCR larvae stunting is measured after feeding on
two events and compared to growth of larvae fed on negative control
plants.
[0246] Transgenic corn plants (R.sub.0) generated are planted into
10-inch pots containing Metromix soil after reaching an appropriate
size. When plants reach the V4 growth stage, approximately 1000
Western corn rootworm (WCR, Diabrotica virgifera) eggs are infested
into the root zone. Non-transgenic corn of the same genotype is
infested at a similar growth stage to serve as a negative control.
Eggs are pre-incubated so hatch occurs within 24 hours of
infestation. Larvae are allowed to feed on the root systems for 3
weeks. Plants are removed from the soil and washed so that the
roots can be evaluated for larval feeding. Root damage is rated
using a Node Injury Scale (NIS) to score the level of damage where
a 0 indicates no damage, a 1 indicates that one node of roots is
pruned to within 1.5 inches, a 2 indicates that 2 nodes are pruned,
while a 3 indicates that 3 nodes are pruned. Because the plants
being used for evaluation are directly out of tissue culture after
transformation and because transformation events are unique, only a
single plant is evaluated per event at this time. The plants in the
assay that present signs or symptoms of larval feeding indicate
that a successful infestation is obtained. Negative control plant
roots are moderately to severely damaged averaging whereas roots of
the transgenic plants provide substantial control of larval
feeding, with about 0.2 or less on the Node Injury Scale.
Example 5
Implementing Insect Pest Gene Suppression Using A ta-siRNA Mediated
Silencing Method
[0247] An alternative method to silence genes in a plant pest uses
the recently discovered class of trans-acting small interfering RNA
(ta-siRNA) (Dalmay et al., 2000; Mourrain et al., 2000; Peragine et
al, 2004; Vazquez et al, 2004). ta-siRNA are derived from single
strand RNA transcripts that are targeted by naturally occurring
miRNA within the cell. Methods for using microRNA to trigger
ta-siRNA for gene silencing in plants are described in U.S.
Provisional Patent Application Ser. No. 60/643,136 (Carrington et
al. 2004), incorporated herein by reference in its entirety. At
least one pest specific miRNA expressed in gut epithelial cells of
corn rootworm larvae is identified. This pest specific miRNA is
then used to identify at least one target RNA transcript sequence
complementary to the miRNA that is expressed in the cell. The
corresponding target sequence is a short sequence of no more than
21 contiguous nucleotides that, when part of a RNA transcript and
contacted by its corresponding miRNA in a cell type with a
functional RNAi pathway, leads to slicer-mediated cleavage of said
transcript. Once miRNA target sequences are identified, at least
one miRNA target sequence is fused to a second sequence that
corresponds to part of a pest gene that is to be silenced using
this method. For example, the miRNA target sequence(s) is fused to
any of SEQ ID NO:1 through SEQ ID NO:906, or a fragment thereof,
such as a sequence of the corn rootworm vacuolar ATPase (V-ATPase)
gene. The miRNA target sequence can be placed at the 5' end, the 3'
end, or embedded in the middle of the target sequence. It may be
preferable to use multiple miRNA target sequences corresponding to
multiple miRNA genes, or use the same miRNA target sequence
multiple times in the chimera of the miRNA target sequence and the
target gene sequence. The target gene sequence can be of any
length, with a minimum of 21 bp.
[0248] The chimera of the miRNA target sequence(s) and the target
gene sequence is expressed in plant cells using any of a number of
appropriate promoter and other transcription regulatory elements,
as long as the transcription occurs in cell types subject to being
provided in the diet of the pest, e.g. corn roots for control of
corn rootworm.
[0249] This method may have the additional advantage of delivering
longer RNA molecules to the target pest. Typically, dsRNAs produced
in plants are rapidly processed by Dicer into short RNA's that may
not be effective when fed exogenously to some pests. In this
method, a single strand transcript is produced in the plant cell,
taken up by the pest, and converted into a dsRNA in the pest cell
where it is then processed into ta-siRNA capable of
post-transcriptionally silencing one or more genes in one or more
target pests.
Example 6
Method for Providing a DNA Sequence for dsRNA-Mediated Gene
Silencing
[0250] This example illustrates a method for providing a DNA
sequence for dsRNA-mediated gene silencing. More specifically, this
example describes selection of an improved DNA useful in
dsRNA-mediated gene silencing by (a) selecting from a target gene
an initial DNA sequence including more than 21 contiguous
nucleotides; (b) identifying at least one shorter DNA sequence
derived from regions of the initial DNA sequence consisting of
regions predicted to not generate undesirable polypeptides and not
exhibiting identity with known sequences such as
homologs/orthologs, and (c) selecting a DNA sequence for
dsRNA-mediated gene silencing that includes the at least one
shorter DNA sequence. Undesirable polypeptides include, but are not
limited to, polypeptides homologous to allergenic polypeptides and
polypeptides homologous to known polypeptide toxins.
[0251] WCR V-ATPase has been demonstrated to function in corn
rootworm feeding assays to test dsRNA mediated silencing as a means
of controlling larval growth. A cDNA sequence from a target gene,
such as vacuolar ATPase gene (V-ATPase) from Western corn rootworm
(WCR) (Diabrotica virgifera virgifera LeConte), is selected for use
as an initial DNA sequence. This initial DNA sequence can be
screened to identify regions within which every contiguous fragment
including at least 21 nucleotides matches fewer than 21 out of 21
contiguous nucleotides of known vertebrate sequences. Sequence
segments that are greater than about 100 contiguous nucleotides
free of such 21/21 hits are identified. Thus criteria including
segment length, GC content, sequence, predicted function based on
sequence or function of a corresponding gene in a model organism,
and predicted secondary structure (e.g. Elbashir, et al., 2001b)
may be used to select and design sequence(s) for use. Different
combinations of these sequence segments are combined to construct
chimeric DNA sequences for expression as dsRNA and use in insect
feeding bioassays as described above.
Example 7
Additional Results of Insect Feeding Bioassays with Sequences
Selected from EST Database
[0252] This example illustrates additional sequences found to be
effective in causing larval stunting and/or mortality when ingested
by rootworm larvae as double stranded RNA sequences. Methods for
rearing corn rootworm larvae, application of dsRNA, and insect
bioassays are as described in Examples 1-3. The results of the
studies confirmed significant levels (p<0.05) of larval stunting
and/or mortality using dsRNAs containing portions of sequences
homologous to a variety of different gene classes. The sequences
and vectors yielding significant stunting and/or mortality and the
corresponding SEQ ID NO for the sequence expressed as a dsRNA are
given in Table 6 below. pMON98503, an exemplary binary vector used
in corn transformation, contains the following elements between the
right and left T-DNA borders for transfer into a plant cell:
e35S-HSP70-DV49 (antisense orientation)-universal spacer-DV49
(sense orientation)-hsp17; ACT (promoter and intron)-CTP2 transit
signal-CP4-NOS.: pMON98504, another exemplary binary vector used in
corn transformation, contains the following elements between the
right and left borders: e35S-HSP70-C1 (antisense
orientation)-universal spacer-C1 (sense orientation)-hsp17; ACT
(promoter and intron)-CTP2 transit signal-CP4-NOS.
TABLE-US-00006 TABLE 6 Additional dsRNA Constructs Demonstrating
Significant Stunting and/or Mortality Effect in Insect Feeding
Bioassays with Southern Corn Rootworm or Western Corn Rootworm
Vector Sequence Expressed as dsRNA SEQ ID NO pMON98356 Dv164;
function unknown, WCR unique sequence 726 pMON98354 Dv172; function
unknown, WCR unique sequence 727 pMON97191 Dv189; function unknown,
WCR unique sequence 728 pMON98359 Dv200; function unknown, WCR
unique sequence 729 pMON38880 Dv207_F39H11.5; putative pbs-7,
endopeptidase: F39H11.5 ortholog 730 pMON101054 Dv208_F58F12_1;
putative Mitochondrial F1F0-ATP synthase, subunit 731 delta/ATP16:
F58F12_1 ortholog pMON98437 Dv210_K11H12_2; putative rpl-15,
structural constituent of ribosome: 732 K11H12_2 ortholog pMON98435
Dv211_R12E2_3; putative rpn-8, translation initiation factor, 26S
733 proteasome regulatory complex, subunit RPN8: R12E2_3 ortholog
pMON98447 Dv212_C17H12_14; putative vha-8, hydrogen-exporting
ATPase: 734 C17H12_14 ortholog pMON98448 Dv213_B0464_1; putative
drs-1, tRNA ligase: B0464_1 ortholog 735 pMON101059
Dv214_F53G12_10; putative rpl-7, structural constituent of
ribosome: 736 F53G12_10 ortholog pMON98442 Dv216_C52E4_4; putative
rpt-1, ATPase subunit of the 19S regulatory 737 complex of the
proteasome: C52E4_4 ortholog pMON98441 Dv218_K01G5_4; putative
ran-1, small monomeric GTPase: K01G5_4 738 ortholog pMON98440
Dv219_C15H11_7; putative pas-1, endopeptidase: C15H11_7 ortholog
739 pMON101081 Dv223_R10E11.1; putative cbp-1, homolog of
transcriptional cofactors 740 CBP and p300: R10E11.1 ortholog
pMON101050 Dv224_F11C3_3; putative unc-54, ATP binding; motor
activity: 741 F11C3_3 ortholog pMON101051 Dv225_C37H5_8; putative
hsp-6, heat shock protein 6: C37H5_8 742 ortholog pMON38888
Dv226_C47E12.5; putative uba-1, ubiquitin activating enzyme: 743
C47E12.5 ortholog pMON38887 Dv227_F54A3.3; putative Chaperonin
complex component, TCP-1 744 gamma subunit: F54A3.3 ortholog
pMON101110 Dv229_D1081.8; putative Myb-like DNA binding: D1081.8
ortholog 745 pMON101052 Dv230_F55A11_2; putative syn-3, protein
transporter; syntaxin: 746 F55A11_2 ortholog pMON101107
Dv231_C30C11.1; putative mitochondrial ribosomal protein L32: 747
C30C11.1 ortholog pMON101055 Dv232_B0250_1; putative rpl-2,
structural constituent of ribosome: 748 B0250_1 ortholog pMON98446
Dv233_F54C9_5; putative rpl-5, 5S rRNA binding, structural 749
constituent of ribosome: F54C9_5 ortholog pMON101138
Dv235_C04F12.4; putative rpl-14, large ribosomal subunit L14
protein: 750 C04F12.4 ortholog pMON98449 Dv236_C01G8_5; putative
erm-1, Ezrin/Radixin/Moesin (ERM) family 751 of cytoskeletal
linkers: C01G8_5 ortholog pMON98439 Dv237_F57B9_10; putative rpn-6,
proteasome Regulatory Particle, 752 Non-ATPase-like: F57B9_10
ortholog pMON98436 Dv240_F53A3_3; putative rps-22, structural
constituent of ribosome: 753 F53A3_3 ortholog pMON101078
Dv241_F32H2.5; putative alcohol dehydrogenase, zinc-dependent: 754
F32H2.5 ortholog pMON101058 Dv242_B0336_2; putative arf-1, small
monomeric GTPase: B0336_2 755 ortholog pMON101057 Dv244_C14B9_7;
putative rpl-21, structural constituent of ribosome: 756 C14B9_7
ortholog pMON98444 Dv245_C26F1_4; putative rps-30, Ribosomal
Protein, Small subunit: 757 C26F1_4 ortholog pMON98434
Dv247_C13B9_3; putative delta subunit of the coatomer (COPI) 758
complex: C13B9_3 ortholog pMON101053 Dv248_F38E11_5; putative
vesicle coat complex COPI, beta' subunit: 759 F38E11_5 ortholog
pMON98445 Dv249_F37C12_9; putative rps-14, structural constituent
of ribosome: 760 F37C12_9 ortholog pMON101056 Dv250_CD4_6; putative
pas-6, endopeptidase: CD4_6 ortholog 761 pMON101104 Dv251_D1007.12;
putative rpl-24.1, structural constituent of ribosome: 762 D1007.12
ortholog pMON101088 Dv252_C49H3.11; putative rps-2, structural
constituent of ribosome: 763 C49H3.11 ortholog pMON101079
Dv253_C26D10.2; putative hel-1, ATP-dependent RNA helicase: 764
C26D10.2 ortholog pMON101085 Dv254_B0336.10; putative rpl-23,
structural constituent of ribosome: 765 B0336.10 ortholog pMON38879
Dv255_C36A4.2; putative member of Cytochrome P450 family: 766
C36A4.2 ortholog pMON101087 Dv256_K05C4.1; putative pbs-5,
proteasome beta subunit: K05C4.1 767 ortholog pMON101082
Dv257_F29G9.5; putative rpt-2, 26S proteasome regulatory complex:
768 F29G9.5 ortholog pMON101084 Dv258_F40F8.10; putative rps-9,
structural constituent of ribosome: 769 F40F8.10 ortholog
pMON101083 Dv259_K07D4.3; putative rpn-11, 26S proteasome
regulatory complex, 770 subunit RPN11: K07D4.3 ortholog pMON101080
Dv260_F49C12.8; putative rpn-7, proteasome Regulatory Particle,
Non- 771 ATPase-like: F49C12.8 ortholog pMON101115 Dv261_D1054.2;
putative pas-2, endopeptidase: D1054.2 ortholog 772 pMON101141
Dv263_F55A3.3; putative metalloexopeptidase: F55A3.3 ortholog 773
pMON101126 Dv264_F56F3.5; putative rps-1, structural constituent of
ribosome: 774 F56F3.5 ortholog pMON101133 Dv266_C09D4.5; putative
rpl-19, structural constituent of ribosome: 775 C09D4.5 ortholog
pMON38881 Dv268_R06A4.9; putative Polyadenylation factor I complex,
subunit 776 PFS2: R06A4.9 ortholog pMON101135 Dv271_F37C12.4;
putative rpl-36, structural constituent of 777 ribosome:F37C12.4
ortholog pMON101132 Dv273_F54E7.2; putative rps-12, structural
constituent of ribosome: 778 F54E7.2 ortholog pMON101139
Dv274_C23G10.4; putative rpn-2, proteasome Regulatory Particle, 779
Non-ATPase-like: C23G10.4 ortholog pMON101130 Dv275_C03D6.8;
putative rpl-24.2, structural constituent of ribosome: 780 C03D6.8
ortholog pMON101119 Dv276_C26E6.4; putative DNA-directed RNA
polymerase: C26E6.4 781 ortholog pMON101134 Dv277_R13A5.8; putative
rpl-9, structural constituent of ribosome: 782 R13A5.8 ortholog
pMON101127 Dv279_F42C5.8; putative rps-8, structural constituent of
ribosome: 783 F42C5.8 ortholog pMON101122 Dv280_F13B10.2; putative
rpl-3, large ribosomal subunit L3: F13B10.2 784 ortholog pMON101116
Dv281_T05C12.7; putative cct-1, Chaperonin complex component, 785
TCP-1 alpha subunit: T05C12.7 ortholog pMON101125 Dv282_F07D10.1;
putative rpl-11.2, structural constituent of ribosome: 786 F07D10.1
ortholog pMON38883 Dv283_T05H4.6; putative Peptide chain release
factor 1 (eRF1): 787 Dv283_T05H4.6 ortholog pMON101124
Dv284_C47E8.5; putative daf-21, Heat shock 90 protein, chaperone
788 activity: C47E8.5 ortholog pMON101120 Dv285_M03F4.2; putative
act-4, actin: M03F4.2 ortholog 789 pMON101137 Dv286_F25H5.4;
putative eft-2, translation elongation factor: F25H5.4 790 ortholog
pMON101140 Dv287_F26D10.3; putative hsp-1, Heat shock protein:
F26D10.3 791 ortholog pMON101117 Dv288_F28D1.7; putative rps-23,
structural constituent of ribosome: 792 F28D1.7 ortholog pMON38886
Dv290_CG11979; putative H-exporting ATPase: CG11979 ortholog 793
pMON38885 Dv291_CG13628; putative H-exporting ATPase: CG13628
ortholog 794 pMON101103 Dv293_CG31237; putative DNA-directed RNA
polymerase II: CG31237 795 ortholog pMON101096 Dv294_CG8669;
putative cryptocephal; transcription factor, involved in 796
molting cycle, pupariation and metamorphosis: CG8669 ortholog
pMON101095 Dv295_CG8048; putative Vacuolar H+ ATPase 44 kD C
subunit: 797 CG8048 ortholog pMON101100 Dv298_CG9032; putative
H-exporting ATPase: CG9032 ortholog 798 pMON101111 Dv299_CG17369;
putative H-exporting ATPase: CG17369 ortholog 799 pMON101129
Dv303_CG4152; putative ATP-dependent RNA helicase: CG4152 800
ortholog pMON101136 Dv305_CG4916; putative ATP-dependent RNA
helicase: CG4919 801 ortholog pMON101131 Dv315_CG9160; putative
NADH dehydrogenase: CG9160 ortholog 802 pMON101123 Dv316_CG8764;
putative ubiquinol-cytochrome-c reductase: CG8764 803 ortholog
pMON98364 C4_Dv49_CG8055 concatemer; putative binding, carrier
activity: 804 CG8055 ortholog pMON98365 C5_Dv49_CG8055 concatemer;
putative binding, carrier activity: 805 CG8055 ortholog. pMON98368
C6 concatemer of highly effective WCR targets, ~50% GC criterion,
consisting 806 of segments in order 5'-3' from Dv26, Dv49, Dv23,
Dv20, Dv13, Dv22, Dv18 pMON98369 C7 concatemer of insect-specific
targets, ~50% GC criterion, 807 consisting of segments in order
5'-3' from Dv6, Dv1, Dv88, Dv93, Dv4, Dv113, Dv127, Dv99 pMON98372
C8 concatemer; putative sodium/potassium-exchanging ATPase: 808
CG9261 ortholog pMON98373 C9 concatemer; putative
sodium/potassium-exchanging ATPase: 809 CG9261 ortholog pMON98366
C10 concatemer of genes with putative same/different mode of
action, ~50% 810 GC criterion pMON98367 C12 concatemer of highly
effective WCR targets, ~50% GC criterion, 811 consisting of
segments in order 5'-3' from Dv23-Dv13-Dv26-Dv18- Dv49-Dv22-Dv20.
pMON98371 C14 concatemer of gene targets active in several
different organisms, ~50% 812 GC criterion pMON98503 Comprising
DV49 putative ESCRT-III (endosomal sorting complex 820 required for
transport III) complex subunit from Diabrotica virgifera pMON98504
Comprising putative Vha68-2: CG3762 ortholog; 250 bp concatamer C1;
821 pMON102862 Dv319_CG14750; putative ESCRTII, Vps25: CG14750
ortholog 835 pMON102863 Dv320_CG9712; putative ESCRTI, Vps23:
CG9712 ortholog 836 pMON102861 Dv321_CG12770; putative ESCRTI,
Vps28: CG12770 ortholog 837 pMON102865 Dv322_CG14542; putative
ESCRT III, Vps2: CG14542 ortholog 838 pMON102866 Dv323_CG4071;
putative ESCRT III, Vsp20: CG4071 ortholog 839 pMON102871
Dv326_CG3564; putative protein carrier, component of the COPI
vesicle 840
coat: CG3564 ortholog pMON102873 Dv327_CG6223; putative coatomer,
component of the COPI vesicle 841 coat: CG6223 ortholog pMON102877
Dv328_CG6948; putative Clathrin light chain, coat of coated pit:
842 CG6948 ortholog pMON102872 Dv330_CG9543; putative COPI vesicle
coat: CG9543 ortholog 843 pMON102879 Dv331_CG5183; putative KDEL
sequence binding: CG5183 ortholog 844 pMON102867 Dv335_F11C1.6;
putative nhr-25, DNA binding: F11C1.6 ortholog 845 pMON102870
Dv337_CG18734; putative furin 2, serine-type endopeptidase: CG18734
846 ortholog pMON102875 Dv329_CG7961; putative coatomer, component
of the COPI vesicle 874 coat: CG7961 ortholog
[0253] Efficacy tests were conducted as follows, utilizing progeny
of corn plants transformed with selected insect control
constructs:
[0254] 1. Seven days post planting: incubate 10,000 WCR eggs per
event (10 plants per event) at 25.degree. C., 60% RH in complete
darkness for seven days.
[0255] 2. Fourteen days post infestation: plants are transplanted
from 4''peat pots to 8'' pots; v4 root tip samples may be taken for
gene expression studies.
[0256] 3. Fourteen days post planting: wash the WCR eggs out of the
soil. Place the eggs and soil into a 60-mesh screen and place a
30-mesh screen on top of the 60-mesh screen to protect the eggs
from the water stream. Rinse thoroughly with warm water using a
spray nozzle until the soil is removed.
[0257] 4. Suspend the eggs in a 2% (w/v) Difco agar solution, 25 mL
of solution per 1 mL of eggs. The eggs are infested into the soil
in about 3 or 4 aliquots using, for example, an Eppendorf repeater
pipette, about 1000 eggs per plant. Holes are made into the soil
using a spatula prior to infestation and covered after
infestation.
[0258] 5. Twenty-eight days post planting v8 root tip samples may
be taken for gene expression studies.
[0259] 6. Thirty-five days post planting; the assay is evaluated.
Plants are cut down using pruning sheers, leaving about 6'' of
stalk. Plant stakes containing the event information are
hole-punched and zip tied to the stalk. As much soil as possible is
removed from the root system. The remainder of the soil is washed
off using a spray hose.
[0260] 7. The roots are examined and given a root damage rating by
using the Olsen (0-3) NIS scale for WCR larvae damage.
[0261] FIG. 1 and FIG. 2 illustrate insect control results obtained
following challenge of F1 corn plants (derived from plants
transformed with either pMON98503 or pMON98504) with WCR. In growth
chamber efficacy tests performed essentially as described above,
NIS scores at or below the economic injury threshold were seen in
progeny of events derived by transformation with pMON98503 or
pMON98504.
[0262] Full length EST DNA sequences were assembled for selected
genes described in Examples 3 and 7 displaying significant activity
versus Western Corn Rootworm. These EST sequences are listed in
Table 7:
TABLE-US-00007 TABLE 7 Assembled EST Sequences for WCR Targets
Assembled Sequence SEQ ID NO Dv9 full length (aka Apple); putative
ortholog of CG2331 813 Dv10 full length (Rp19); putative ortholog
of CG6141 814 Dv11 full length (Rpl19); putative ortholog of CG2746
815 Dv13 full length (Rps4); putative ortholog of CG11276 816 Dv35
full length v-ATPase A; ortholog of CG3762 817 Dv49 full length
ortholog of CG8055 818 Dv248 full length putative ortholog of
CG6699 819
Example 8
Creation and Efficacy Results for Dv49 and Dv248 Sequences
[0263] Portions of the assembled EST and adjacent sequences of Dv49
(SEQ ID NO:818) and Dv248 (SEQ ID NO:819) were selected for further
bioactivity assays based on criteria including predicted function,
phenotype of knockout mutants of corresponding coding regions in
other organisms, segment length, GC content, similarity to known
sequences, and predicted secondary structure (e.g. Elbashir, et
al., 2001b). The respective Dv49 and Dv248 sequences were
synthesized in vitro based on their predicted activity against WCR
individually, as well as grouped as shown in FIGS. 3-4, and Table
8, and applied to WCR larvae.
TABLE-US-00008 TABLE 8 Dv49 and Dv248 Fragments Assessed for
Efficacy Against WCR Fragment SEQ ID NO F1 822 F2 823 F3 824 F4 825
F5 826 F6 827 F7 828 F8 829 F9 830 F10 831 F11 832 F12 833 F13
834
[0264] Fragments F1-F3 correspond to portions of the full length
Dv49 transcript. Fragments F4-F6 correspond to portions of the
Dv248 transcript or flanking region. Fragments F7-F13 are
concatemers of two or more of fragments F1-F6, as shown in FIG. 4.
Fragment F13 (SEQ ID NO:834) represents the C38 (Dv49-Dv248)
concatemer.
[0265] Dose Response data for F1-F13 is shown in FIGS. 5-7. As
shown in FIG. 5, activity (larval % mortality) of fragments F4-F6,
comprising Dv248-derived sequences (FIG. 4), was significantly
better than the control when fed to WCR larvae at 0.1 ppm. Fragment
F6 displayed significant activity when fed at 0.02 ppm as well.
Activity of fragment F5 at the lowest dose is likely an artifact,
since surviving larvae displayed no stunting.
[0266] As shown in FIG. 6, each of fragments F7-F10 displays
statistically significant activity when fed to WCR larvae at 0.1
ppm. Fragments F9 and F10 also displayed statistically significant
activity when fed to WCR larvae at 0.02 ppm, and fragment F10
displayed statistically significant activity when fed to WCR larvae
at 0.01 ppm as well. Activity of F8 at 0.01 ppm may be an
artifact.
[0267] As shown in FIG. 7, fragments F1 i-F13 display statistically
significant activity when fed to WCR larvae at 0.02 ppm or higher.
Additionally, the largest fragments (F12 and F13) show activity at
0.005 ppm and higher.
Example 9
Additional Active Concatemer Sequences Derived from Concatemer
C6
[0268] As shown in Table 9, portions of active concatemer C6 (SEQ
ID NO:806), derived from pMON98368, were identified in diet overlay
bioassays (performed as described e.g. in Example 2) as inhibiting
the growth and/or survival of corn rootworm (WCR). The C6 full
length concatemer contains 7 target subfragments of 70 bp each, as
noted in Table 6 and Table 9.
TABLE-US-00009 TABLE 9 Efficacy of full length C6 concatemer and
selected sub-portions in rootworm diet bioassay (SEQ ID NOs: 806,
847-873). % Mortality SEQ Concatemer Segments at 1 ppm ID NO
C6_full Dv26-Dv49-Dv23-Dv20-Dv13- 100 806 length Dv22-Dv18 C6.1
Dv26-Dv49 35.3 847 C6.2 Dv26-Dv49-Dv23 57.1 848 C6.3
Dv26-Dv49-Dv23-Dv20 84.7 849 C6.4 Dv26-Dv49-Dv23-Dv20-Dv13 51 850
C6.5 Dv26-Dv49-Dv23-Dv20-Dv13- 41.3 851 Dv22 C6.6 Dv49-Dv23 94.6
852 C6.7 Dv49-Dv23-Dv20 75.6 853 C6.8 Dv49-Dv23-Dv20-Dv13 50 854
C6.9 Dv49-Dv23-Dv20-Dv13-Dv22 56.4 855 C6.10
Dv49-Dv23-Dv20-Dv13-Dv22- 52.5 856 Dv18 C6.11 Dv23-Dv20 74.6 857
C6.12 Dv23-Dv20-Dv13 63.4 858 C6.13 Dv23-Dv20-Dv13-Dv22 58.5 859
C6.14 Dv23-Dv20-Dv13-Dv22-Dv18 60.8 860 C6.15 Dv20-Dv13 57.5 861
C6.16 Dv20-Dv13-Dv22 20 862 C6.17 Dv20-Dv13-Dv22-Dv18 44.8 863
C6.18 Dv13-Dv22 71.1 864 C6.19 Dv13-Dv22-Dv18 52.5 865 C6.20
Dv22-Dv18 44.6 866 C6.28 Dv26 58.9 867 C6.29 Dv49 72.3 868 C6.30
Dv23 71.9 869 C6.31 Dv20 62.6 870 C6.32 Dv13 54.2 871 C6.33 Dv22
56.7 872 C6.34 Dv18 44.6 873
[0269] Full length concatemer C7 contains 8 target sub-fragments of
70 bp each, as noted in Table 6. Similarly, full length concatemer
C12 contains 7 target sub-fragments from
Dv23-Dv13-Dv26-Dv18-Dv49-Dv22-Dv20, ranging in length between 53-80
bp, as noted in Table 6 and Table 10.
TABLE-US-00010 TABLE 10 Composition of full length C12 concatemer
and selected sub-portions (SEQ ID NOs: 811, 887-892). Included
sequence (SEQ ID NO) in Concatemer Segments 5'-3' direction
C12_full Dv23-Dv13-Dv26-Dv18-Dv49-Dv22- 811 length Dv20 C12.1
Dv23-Dv13 887 C12.2 Dv23-Dv13-Dv26 888 C12.3 Dv23-Dv13-Dv26-Dv18
889 C12.4 Dv23-Dv13-Dv26-Dv18-Dv49 890 C12.5
Dv23-Dv13-Dv26-Dv18-Dv49-Dv22 891 C12.6 Dv13-Dv26 892 C12.7
Dv13-Dv26-Dv18 893 C12.8 Dv13-Dv26-Dv18-Dv49 894 C12.9
Dv13-Dv26-Dv18-Dv49-Dv22 895 C12.10 Dv13-Dv26-Dv18-Dv49-Dv22-Dv20
896 C12.11 Dv26-Dv18 897 C12.12 Dv26-Dv18-Dv49 898 C12.13
Dv26-Dv18-Dv49-Dv22 899 C12.14 Dv26-Dv18-Dv49-Dv22-Dv20 900 C12.15
Dv18-Dv49 901 C12.16 Dv18-Dv49-Dv22 902 C12.17 Dv18-Dv49-Dv22-Dv20
903 C12.18 Dv49-Dv22 904 C12.19 Dv49-Dv22-Dv20 905 C12.20 Dv22-Dv20
906
[0270] Table 11 lists additional sequences for use in targeting
other coleopteran pests, including Diabrotica sp. and other
Coccinellidae and Chrysomelidae.
TABLE-US-00011 TABLE 11 Additional Coleopteran Target Sequences
Sequence and source SEQ ID NO Dbar248_CG6699 (D. barberi) 875
Dbal248_CG6699 (D. balteata) 876 Du248_CG6699 (D. undecimpunctata
howardi) 877 Dz248_CG6699 (D. virgifera zea) 878 Dv248_CG6699 (D.
virgifera virgifera) 879 Ev248_CG6699 (Epilachna varivestis) 880
Ld248_CG6699 (Leptinotarsa decemlineata) 881 Dbal49_CG8055_2 (D.
balteata) 882 Db49_CG8055_2 (D. barberi) 883 Du49_CG8055_2 (D.
undecimpunctata howardi) 884 Dz49_CG8055_2 (D. virgifera zea) 885
Dv49_CG8055_2 (D. virgifera virgifera) 886
[0271] All of the compositions and methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of the foregoing
illustrative embodiments, it will be apparent to those of skill in
the art that variations, changes, modifications, and alterations
may be applied to the composition, methods, and in the steps or in
the sequence of steps of the methods described herein, without
departing from the true concept, spirit, and scope of the
invention. More specifically, it will be apparent that certain
agents that are both chemically and physiologically related may be
substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope, and concept of the invention as defined
by the appended claims.
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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=US20170114362A1).
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=US20170114362A1).
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