U.S. patent application number 17/228596 was filed with the patent office on 2021-08-19 for compositions and methods for controlling insect pests.
The applicant listed for this patent is MONSANTO TECHNOLOGY LLC. Invention is credited to Jodi BEATTIE, Michael John CRAWFORD, Brian Donovan EADS.
Application Number | 20210254094 17/228596 |
Document ID | / |
Family ID | 1000005539122 |
Filed Date | 2021-08-19 |
United States Patent
Application |
20210254094 |
Kind Code |
A1 |
BEATTIE; Jodi ; et
al. |
August 19, 2021 |
COMPOSITIONS AND METHODS FOR CONTROLLING INSECT PESTS
Abstract
Disclosed herein are methods for controlling invertebrate pest
infestations, particularly in plants; compositions and insecticidal
polynucleotides useful in such methods; and plants having improved
resistance to the invertebrate pests. More specifically,
insecticidal polynucleotides and methods of use thereof for
modifying the expression of genes in an insect pest, particularly
through RNA interference are disclosed.
Inventors: |
BEATTIE; Jodi; (Wentzville,
MO) ; CRAWFORD; Michael John; (St. Louis, MO)
; EADS; Brian Donovan; (Kirkwood, MO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MONSANTO TECHNOLOGY LLC |
St. Louis |
MO |
US |
|
|
Family ID: |
1000005539122 |
Appl. No.: |
17/228596 |
Filed: |
April 12, 2021 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
16072824 |
Jul 25, 2018 |
10975387 |
|
|
PCT/US2017/015061 |
Jan 26, 2017 |
|
|
|
17228596 |
|
|
|
|
62287080 |
Jan 26, 2016 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/8286 20130101;
C07K 14/195 20130101; Y02A 40/146 20180101; C12N 15/8218
20130101 |
International
Class: |
C12N 15/82 20060101
C12N015/82; C07K 14/195 20060101 C07K014/195 |
Claims
1. A method of causing mortality or stunting in an insect,
comprising providing in the diet of an insect at least one
insecticidal polynucleotide comprising at least one silencing
element essentially identical or essentially complementary to a
fragment of a target gene sequence of said insect, wherein said
target gene sequence is selected from the group consisting of SEQ
ID NOs:1-859, and wherein ingestion of said insecticidal
polynucleotide by said insect results in mortality or stunting in
said insect.
2. The method of claim 1, wherein said silencing element comprises:
(a) at least one segment of 18 or more contiguous nucleotides
comprising a sequence of 100% complementarity with a fragment of
said target gene of said insect, wherein said target gene has a DNA
sequence selected from the group consisting of SEQ ID NOs:1-859;
(b) at least one segment of 18 or more contiguous nucleotides of a
sequence selected from the group consisting of SEQ ID NOs:860-1718
and 1722-1730 and 1771-1975; (c) a sequence selected from the group
consisting of SEQ ID NOs:860-1718 and 1722-1730 and 1771-1975.
3. The method of claim 1, wherein said insecticidal polynucleotide
is provided in a microbial or plant cell that expresses said
insecticidal polynucleotide, or in a microbial fermentation
product, or is chemically synthesized.
4. The method of claim 1, wherein said at least one insecticidal
polynucleotide is provided in a composition comprising said at
least one insecticidal polynucleotide, wherein said composition is
applied to a surface of said insect or to a surface of a seed or
plant in need of protection from infestation by said insect.
5. The method of claim 4, wherein said composition: (a) comprises a
solid, liquid, powder, suspension, emulsion, spray, encapsulation,
microbeads, carrier particulates, film, matrix, soil drench, or
seed treatment; (b) further comprises one or more components
selected from the group consisting of a carrier agent, a
surfactant, an organosilicone, a polynucleotide herbicidal
molecule, a non-polynucleotide herbicidal molecule, a
non-polynucleotide pesticide, a fungicide, a safener, a fertilizer,
a micronutrient, an insect attractant, and an insect growth
regulator; (c) further comprises at least one pesticidal agent
selected from the group consisting of a patatin, a plant lectin, a
phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a
Xenorhabdus insecticidal protein, a Photorhabdus insecticidal
protein, a Bacillus laterosporous insecticidal protein, a Bacillus
sphaericus insecticidal protein, a bacterium that produces an
insecticidal protein, an entomicidal bacterial species,
Lysinibacillus sphaericus (Bacillus sphaericus), Brevibacillus
laterosporus (Bacillus laterosporus), Chromobacterium species,
Chromobacterium subtsugae, Paenibacillus species, Paenibacillus
lentimorbus, and Paenibacillus popilliae; or (d) is ingested by
said insect.
6. A method for controlling an insect infestation of a plant
comprising contacting, with a dsRNA, an insect that infests a
plant, wherein said dsRNA comprises at least one segment of 18 or
more contiguous nucleotides of a sequence of about 95% to about
100% complementarity with a fragment of a target gene of said
insect, and wherein said target gene has a DNA sequence selected
from the group consisting of SEQ ID NOs:1-859.
7. The method of claim 6, wherein said contacting comprises: (a)
oral delivery to said insect, non-oral delivery to said insect, or
a combination of oral and non-oral delivery to said insect; (b)
application of a composition comprising said dsRNA to a surface of
said insect or to a surface of said plant infested by said insect;
(c) providing said dsRNA in a composition that further comprises
one or more components selected from the group consisting of a
carrier agent, a surfactant, an organosilicone, a polynucleotide
herbicidal molecule, a non-polynucleotide herbicidal molecule, a
non-polynucleotide pesticide, a fungicide, a safener, a fertilizer,
a micronutrient, an insect attractant, and an insect growth
regulator; (d) providing said dsRNA in a composition that further
comprises at least one pesticidal agent selected from the group
consisting of a patatin, a plant lectin, a phytoecdysteroid, a
Bacillus thuringiensis insecticidal protein, a Xenorhabdus
insecticidal protein, a Photorhabdus insecticidal protein, a
Bacillus laterosporous insecticidal protein, a Bacillus sphaericus
insecticidal protein, a bacterium that produces an insecticidal
protein, an entomicidal bacterial species, Lysinibacillus
sphaericus (Bacillus sphaericus), Brevibacillus laterosporus
(Bacillus laterosporus), Chromobacterium species, Chromobacterium
subtsugae, Paenibacillus species, Paenibacillus lentimorbus, and
Paenibacillus popilliae; or (e) providing said dsRNA in a
composition that is ingested by said insect.
8. An insecticidal composition comprising an insecticidally
effective amount of a insecticidal polynucleotide, wherein said
insecticidal polynucleotide comprises at least one segment of 18 or
more contiguous nucleotides comprising a sequence of about 95% to
about 100% complementarity with a fragment of a target gene of an
insect that infests a plant, and wherein said target gene has a DNA
sequence selected from the group consisting of SEQ ID
NOs:1-859.
9. The insecticidal composition of claim 8, wherein said
insecticidal composition: (a) further comprises one or more
components selected from the group consisting of a carrier agent, a
surfactant, an organosilicone, a polynucleotide herbicidal
molecule, a non-polynucleotide herbicidal molecule, a
non-polynucleotide pesticide, a fungicide, a safener, a fertilizer,
a micronutrient, an insect attractant, and an insect growth
regulator; (b) further comprises at least one pesticidal agent
selected from the group consisting of a patatin, a plant lectin, a
phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a
Xenorhabdus insecticidal protein, a Photorhabdus insecticidal
protein, a Bacillus laterosporous insecticidal protein, a Bacillus
sphaericus insecticidal protein, a bacterium that produces an
insecticidal protein, an entomicidal bacterial species,
Lysinibacillus sphaericus (Bacillus sphaericus), Brevibacillus
laterosporus (Bacillus laterosporus), Chromobacterium species,
Chromobacterium subtsugae, Paenibacillus species, Paenibacillus
lentimorbus, and Paenibacillus popilliae; (c) is in a form selected
from the group consisting of a solid, liquid, powder, suspension,
emulsion, spray, encapsulation, microbeads, carrier particulates,
film, matrix, soil drench, insect diet or insect bait, and seed
treatment; or (d) is provided in a microbial or plant cell that
expresses said recombinant RNA, or in a microbial fermentation
product.
10. A plant treated with the insecticidal composition of claim 8,
or a plant grown from seed treated with the insecticidal
composition of claim 8, wherein said plant exhibits improved
resistance to said insect.
11. A method of providing a plant having improved resistance to an
insect, comprising expressing in said plant a recombinant DNA
construct, wherein said recombinant DNA construct comprises DNA
encoding an RNA having a sequence essentially identical or
essentially complementary to at least 18 or more contiguous
nucleotides of said target gene, wherein said target gene has a DNA
sequence selected from the group consisting of SEQ ID NOs:1-859,
and wherein ingestion of said RNA by said insect results in
mortality or stunting in said insect.
12. The method of claim 1 or 11, or the insecticidal composition of
claim 8, wherein said RNA: (a) comprises a strand comprising at
least 21 contiguous nucleotides comprising a sequence of about 95%
to about 100% identity or complementarity with a sequence selected
from the group consisting of SEQ ID NOs:860-1718 and 1722-1975; or
(b) comprises dsRNA.
13. The method or insecticidal composition of claim 12, wherein:
(a) said dsRNA is blunt-ended, or has an overhang at at least one
terminus, or comprises at least one stem-loop; (b) said dsRNA is
chemically synthesized, produced by expression in a microorganism,
produced by expression in a plant cell, or produced by microbial
fermentation; (c) said dsRNA is chemically modified; or (d) said
dsRNA comprises at least one RNA strand comprising a sequence of
about 95% to about 100% identity or complementarity with a sequence
selected from the group consisting of SEQ ID NOs:860-1718 and
1722-1730 and 1731-1975.
14. The method of claim 6 or 12, wherein said dsRNA comprises: (a)
a sequence selected from the group consisting of SEQ ID
NOs:860-1718 and 1722-1730 and 1731-1975, or the complement
thereof, (b) at least one segment of 18 or more contiguous
nucleotides with a sequence of 100% complementarity with a fragment
of said target gene of said insect, wherein said target gene has a
DNA sequence selected from the group consisting of SEQ ID
NOs:1-859; or (c) at least one segment of 18 or more contiguous
nucleotides of an RNA sequence selected from the group consisting
of SEQ ID NOs:860-1718 and 1722-1730 and 1731-1975.
15. The method of any one of claims 1, 6, or 11, or the
insecticidal composition of claim 8, wherein: (a) said insect is a
flea beetle; (b) said insect is a species of a genus selected from
the group consisting of the genera Altica, Anthobiodes, Aphthona,
Aphthonaltica, Aphthonoides, Apteopeda, Argopistes, Argopus,
Arrhenocoela, Batophila, Blepharida, Chaetocnema, Clitea,
Crepidodera, Derocrepis, Dibolia, Disonycha, Epitrix, Hermipyxis,
Hermaeophaga, Hespera, Hippuriphila, Horaia, Hyphasis, Lipromima,
Liprus, Longitarsus, Luperomorpha, Lythraria, Manobia, Mantura,
Meishania, Minota, Mniophila, Neicrepidodera, Nonarthra,
Novofoudrasia, Ochrosis, Oedionychis, Oglobinia, Omeisphaera,
Ophrida, Orestia, Paragopus, Pentamesa, Philopona, Phygasia,
Phyllotreta, Podagrica, Podagricomela, Podontia, Pseudodera,
Psylliodes, Sangariola, Sinaltica, Sphaeroderma, Systena,
Trachyaphthona, Xuthea, and Zipangia; (c) said insect is a species
selected from the group consisting of Altica ambiens (alder flea
beetle), Altica canadensis (prairie flea beetle), Altica chalybaea
(grape flea beetle), Altica prasina (poplar flea beetle), Altica
rosae (rose flea beetle), Altica sylvia (blueberry flea beetle),
Altica ulmi (elm flea beetle), Chaetocnema pulicaria (corn flea
beele), Chaetocnema conofinis (sweet potato flea beetle), Epitrix
cucumeris (potato flea beetle), Systena blanda (palestripped
fleabeetle), and Systena frontalis (redheaded flea beetle); (d)
said insect is a species selected from the group consisting of
Phyllotreta armoraciae (horseradish flea beetle), Phyllotreta
cruciferae (canola flea beetle), Phyllotreta pusilla (western black
flea beetle), Phyllotreta nemorum (striped turnip flea beetle),
Phyllotreta atra (turnip flea beetle), Phyllotreta robusta (garden
flea beetle), Phyllotreta striolata (striped flea beetle),
Phyllotreta undulata, Psylliodes chrysocephala, and Psylliodes
punctulata (hop flea beetle); (e) said insect is an insect pest of
a Brassica species; (f) said insect is a Phyllotreta species and
said target gene has a DNA sequence selected from the group
consisting of SEQ ID NOs:1-551; (g) said insect is Phyllotreta atra
(turnip flea beetle) and said target gene has a DNA sequence
selected from the group consisting of SEQ ID NOs:1-296; (b) said
insect is Phyllotreta cruciferae (canola flea beetle) and said
target gene has a DNA sequence selected from the group consisting
of SEQ ID NOs:297-532; (i) said insect is Phyllotreta striolata
(striped flea beetle) and said target gene has a DNA sequence
selected from the group consisting of SEQ ID NOs:533-551; (j) said
insect is a Psylliodes species and said target gene has a DNA
sequence selected from the group consisting of SEQ ID NOs:552-859;
(k) said insect is Psylliodes chrysocephala and said target gene
has a DNA sequence selected from the group consisting of SEQ ID
NOs:552-859; (l) said insect is a Phyllotreta species and said
silencing element comprises an RNA strand having a sequence
selected from the group consisting of SEQ ID NOs:860-1410; (m) said
insect is Phyllotreta atra (turnip flea beetle), said recombinant
RNA molecule comprises dsRNA, and said dsRNA comprises at least one
RNA sequence selected from the group consisting of SEQ ID
NOs:860-1155; (n) said insect is Phyllotreta cruciferae (canola
flea beetle), said recombinant RNA molecule comprises dsRNA, and
said dsRNA comprises at least one RNA sequence selected from the
group consisting of SEQ ID NOs:1156-1391, 1731-1972, and 1974; (o)
said insect is Phyllotreta striolata (striped flea beetle), said
recombinant RNA molecule comprises dsRNA, and said dsRNA comprises
at least one RNA sequence selected from the group consisting of SEQ
ID NOs:1392-1410, 1973, and 1975; (p) said insect is a Psylliodes
species and said silencing element comprises an RNA strand having a
sequence selected from the group consisting of SEQ ID
NOs:1411-1718; or (q) said insect is Psylliodes chrysocephala, said
recombinant RNA molecule comprises dsRNA, and said dsRNA comprises
at least one RNA sequence selected from the group consisting of SEQ
ID NOs:1411-1718.
16. The method of claim 6 or 11, or the insecticidal composition of
claim 8, wherein: (a) said plant is selected from an ornamental
plant or a crop plant; (b) said plant is a plant in the family
Brassicaceae; (c) said plant is a Brassica species selected from
the group consisting of B. napus, B. juncea, B. carinata, B. rapa,
B. oleracea, B. rupestris, B. septiceps, B. nigra, B. narinosa, B.
perviridus, B. tournefortii, and B. fructiculosa; (d) said plant is
a Brassica plant selected from the group consisting canola,
rapeseed, turnips, and field mustard or turnip rape; (e) said plant
is selected from the group consisting of Glycine max, Linum
usitatissimum, Zea mays, Carthamus tinctorius, Helianthus annuus,
Nicotiana tabacum, Arabidopsis thaliana, Betholettia excelsa,
Ricinus communis, Cocos nucifera, Coriandrum sativum, Gossypium
spp., Arachis hypogaea, Simmondsia chinensis, Solanum tuberosum,
Elaeis guineensis, Olea europaea, Oryza sativa, Cucurbita maxim,
Hordeum vulgare, and Triticum aestivum; or (f) said plant is a
potato plant and said insect is Epitrix cucumeris (potato flea
beetle).
17. The method of claim 11, wherein: (a) said method further
comprises expression in said plant of at least one pesticidal agent
selected from the group consisting of a patatin, a plant lectin, a
phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a
Xenorhabdus insecticidal protein, a Photorhabdus insecticidal
protein, a Bacillus laterosporous insecticidal protein, a Bacillus
sphaericus insecticidal protein, a bacterium that produces an
insecticidal protein, an entomicidal bacterial species,
Lysinibacillus sphaericus (Bacillus sphaericus), Brevibacillus
laterosporus (Bacillus laterosporus), Chromobacterium species,
Chromobacterium subtsugae, Paenibacillus species, Paenibacillus
lentimorbus, and Paenibacillus popilliae; or (b) said method
further comprises expression in said plant of at least one protein
conferring tolerance to an herbicide.
18. The plant having improved resistance to an insect, provided by
the method of claim 11, or fruit, seed, or propagatable parts of
said plant.
19. A recombinant DNA construct comprising a heterologous promoter
operably linked to DNA encoding an RNA transcript comprising at
least 21 contiguous nucleotides of a sequence of about 95% to about
100% identity or complementarity with a sequence selected from the
group consisting of SEQ ID NOs:860-1718 and 1722-1730.
20. The recombinant DNA construct of claim 19, wherein said
heterologous promoter is functional for expression of said RNA
transcript in a bacterium or in a plant cell.
21. A recombinant vector, plant chromosome or plastid, or
transgenic plant cell comprising the recombinant DNA construct of
claim 19.
Description
CROSS-REFERENCE TO RELATED APPLICATION AND INCORPORATION OF
SEQUENCE LISTING
[0001] This application claims priority to U.S. Provisional Patent
Application No. 62/287,080, entitled "Compositions and Methods for
Controlling Insect Pests," filed Jan. 26, 2016, which is
incorporated in its entirety. The sequence listing contained in the
file "P34170WO01_SL.TXT" (2,289,028 bytes, created on Jan. 26,
2017) is filed herewith and is incorporated by reference in its
entirety herein.
FIELD
[0002] Disclosed herein are methods for controlling invertebrate
pest infestations, particularly in plants, compositions and
insecticidal polynucleotides useful in such methods, and plants
having improved resistance to the invertebrate pests. More
specifically, insecticidal polynucleotides and methods of use
thereof for modifying the expression of genes in an insect pest,
particularly through RNA interference are disclosed. Pest species
of interest include flea beetles, such as Phyllotreta spp. and
Psylliodes spp.
BACKGROUND
[0003] Commercial crops are often the targets of attack by
invertebrate pests such as insects. RNA interference (RNAi,
RNA-mediated gene suppression) is an approach that shows promise
for use in environmentally friendly pest control. In invertebrates,
RNAi-based gene suppression was first demonstrated in nematodes
(Fire et al., (1998) Nature, 391:806-811; Timmons & Fire (1998)
Nature, 395:854). Subsequently, RNAi-based suppression of
invertebrate genes using recombinant nucleic acid techniques has
been reported in a number of species, including agriculturally or
economically important pests from various insect and nematode taxa,
such as: root-knot nematodes (Meloidogyne spp.), see Huang et al.
(2006) Proc. Natl. Acad. Sci. USA, 103:14302-14306,
doi:10.1073/pnas.0604698103); cotton bollworm (Helicoverpa
armigera), see Mao et al. (2007) Nature Biotechnol., 25:1307-1313,
doi:10.1038/nbt1352; Western corn rootworm (Diabrotica virgifera
LeConte), see Baum et al. (2007) Nature Biotechnol., 25:1322-1326,
doi:10.1038/nbt1359; sugar beet cyst nematode (Heterodera
schachtii), see Sindhu et al. (2008) J. Exp. Botany, 60:315-324,
doi:10.1093/jxb/em289; mosquito (Aedes aegypti), see Pridgeon et
al. (2008) J. Med. Entomol., 45:414-420, doi:
full/10.1603/0022-2585%282008%2945%5B414%3ATAADRK %5D2.0.CO %3B2;
fruit flies (Drosophila melanogaster), flour beetles (Tribolium
castaneum), pea aphids (Acyrthosiphon pisum), and tobacco hornworms
(Manduca sexta), see Whyard et al. (2009) Insect Biochem. Mol.
Biol., 39:824-832, doi:10.1016/j.ibmb. 2009.09.00; diamondback moth
(Plutella xylostella), see Gong et al. (2011) Pest Manag. Sci., 67:
514-520, doi:10.1002/ps. 2086; green peach aphid (Myzus persicae),
see Pitino et al. (2011) PLoS ONE, 6:e25709,
doi:10.1371/journal.pone.0025709; brown planthopper (Nilaparvata
lugens), see Li et al. (2011) Pest Manag. Sci., 67:852-859,
doi:10.1002/ps.2124; and whitefly (Bemisia tabaci), see Upadhyay et
al. (2011) J Biosci., 36:153-161,
doi:10.1007/s12038-011-9009-1.
SUMMARY
[0004] The present embodiments relate to control of insect species,
especially flea beetle species that are economically or
agriculturally important pests. The compositions and methods
disclosed herein comprise insecticidal polynucleotide molecules
that are useful for controlling or preventing infestation by that
insect species. Several embodiments described herein relate to a
polynucleotide-containing composition (e. g., a composition
containing a dsRNA for suppressing a target gene) that is topically
applied to an insect species or to a plant, plant part, or seed to
be protected from infestation by an insect species. Other
embodiments relate to methods for selecting insect target genes
that are effective targets for RNAi-mediated control of an insect
species.
[0005] Several embodiments relate to a method for controlling an
insect infestation of a plant comprising contacting the insect with
a insecticidal polynucleotide comprising at least one segment of 18
or more contiguous nucleotides with a sequence of about 95% to
about 100% complementarity with a fragment of a target gene of the
insect. In some embodiments, the insecticidal polynucleotide
comprises at least one segment of 21 contiguous nucleotides with a
sequence of 100% complementarity with a fragment of a target gene
of the insect. In some embodiments, the target gene is selected
from the group consisting of Act5C, arginine kinase, COPI (coatomer
subunit) alpha, COPI (coatomer subunit) beta, COPI (coatomer
subunit) betaPrime, COPI (coatomer subunit) delta, COPI (coatomer
subunit) epsilon, COPI (coatomer subunit) gamma, COPI (coatomer
subunit) zeta, RpL07, RpL19, RpL3, RpL40, RpS21, RpS4, Rpn2, Rpn3,
Rpt6, Rpn8, Rpn9, Rpn6-PB-like protein, Sar1, sec6, sec23, sec23A,
shrb (snf7), Tubulin gamma chain, ProsAlpha2, ProsBeta5, Proteasome
alpha 2, Proteasome beta 5, VATPase E, VATPase A, VATPase B,
VATPase D, Vps2, Vps4, Vps16A, Vps20, Vps24, Vps27, Vps28, Vha26
(V-ATPase A), Vha68-2 (V-ATPase D/E), 40S ribosomal protein S14,
and 60S ribosomal protein L13. In some embodiments, the target gene
has a DNA sequence selected from the group consisting of SEQ ID
NOs:1-859. In some embodiments, the insecticidal polynucleotide
comprises a sequence of about 95% to about 100% identity or
complementarity with a sequence selected from the group consisting
of SEQ ID NOs:860-1718 and 1722-1975 or a fragment thereof. In some
embodiments, the insecticidal polynucleotide comprises at least one
segment of 21 contiguous nucleotides of a sequence selected from
the group consisting of SEQ ID NOs:860-1718 and 1722-1975. In some
embodiments, the insecticidal polynucleotide is a dsRNA comprising
an RNA strand with a sequence selected from the group consisting of
SEQ ID NOs:860-1718 and 1722-1975. In various embodiments, the
contacting of the insect is by oral delivery, or by non-oral
contact, e. g., by absorption through the cuticle, or through a
combination of oral and non-oral delivery. In some embodiments, the
insecticidal polynucleotide suppresses a target gene in the insect
and stunts growth, development or reproduction by the insect, or
kills the insect.
[0006] Several embodiments relate to a method of causing mortality
or stunting in an insect, comprising providing in the diet of an
insect at least one insecticidal polynucleotide comprising at least
one silencing element, wherein the at least one silencing element
is essentially identical or essentially complementary to a fragment
of a target gene sequence of the insect, and wherein ingestion of
the insecticidal polynucleotide by the insect results in mortality
or stunting in the insect. In some embodiments, the target gene is
selected from the group consisting of actin, Act5C, arginine
kinase, COPI (coatomer subunit) alpha, COPI (coatomer subunit)
beta, COPI (coatomer subunit) betaPrime, COPI (coatomer subunit)
delta, COPI (coatomer subunit) epsilon, COPI (coatomer subunit)
gamma, COPI (coatomer subunit) zeta, RpL07, RpL19, RpL3, RpL40,
RpL13, RpL14, RpS21, RpS4, RpS14, Rpn2, Rpn3, Rpn7, Rpt6, Rpn8,
Rpn9, Rpn6-PB-like protein, Sar1, sec6, sec23, sec23A, shrb (snf7),
Tubulin gamma chain, ProsAlpha2, ProsBeta5, Proteasome alpha 2,
Proteasome beta 5, VATPase E, VATPase A, VATPase B, VATPase D,
Vps2, Vps4, Vps16A, Vps20, Vps24, Vps27, Vps28, Vha26 (V-ATPase A),
Vha68-2 (V-ATPase DE), 40S ribosomal protein S14, and 60S ribosomal
protein L13. In some embodiments, the target gene sequence is
selected from the group consisting of SEQ ID NOs:1-859. In some
embodiments, the insecticidal polynucleotide comprises a sequence
of about 95% to about 100% identity or complementarity to at least
21 contiguous nucleotides of a sequence selected from the group
consisting of SEQ ID NOs:860-1718 and 1722-1975 or a fragment
thereof. In some embodiments, the insecticidal polynucleotide
comprises a sequence selected from the group consisting of SEQ ID
NOs:860-1718 and 1722-1975. In some embodiments, the insecticidal
polynucleotide is provided in the diet of the insect in the form of
a plant or bacterial cell containing the polynucleotide, or as a
synthetic polynucleotide molecule, or as a fermentation product (e.
g., a hairpin form of a dsRNA, produced in a bacterium). In some
embodiments, the insecticidal polynucleotide is a single-stranded
RNA molecule. In some embodiments, the insecticidal polynucleotide
is a double-stranded RNA molecule. In some embodiments, the
insecticidal polynucleotide is a single-stranded DNA molecule. In
some embodiments, the insecticidal polynucleotide is a
double-stranded DNA molecule. In some embodiments, the insecticidal
polynucleotide is a RNA/DNA hybrid molecule.
[0007] Several embodiments relate to an insecticidal composition
comprising an insecticidally effective amount of a polynucleotide,
wherein the polynucleotide comprises 18 or more contiguous
nucleotides with about 95% to about 100% complementarity with a
corresponding portion of a target gene of an insect that infests a
plant. In some embodiments, the polynucleotide comprises 21
contiguous nucleotides with a sequence of 100% complementarity with
a corresponding portion of the target gene. In some embodiments,
the target gene is selected from the group consisting of actin,
Act5C, arginine kinase, COPI (coatomer subunit) alpha, COPI
(coatomer subunit) beta, COPI (coatomer subunit) betaPrime, COPI
(coatomer subunit) delta, COPI (coatomer subunit) epsilon, COPI
(coatomer subunit) gamma, COPI (coatomer subunit) zeta, RpL07,
RpL19, RpL3, RpL40, RpL13, RpL14, RpS21, RpS4, RpS14, Rpn2, Rpn3,
Rpn7, Rpt6, Rpn8, Rpn9, Rpn6-PB-like protein, Sar1, sec6, sec23,
sec23A, shrb (snf7), Tubulin gamma chain, ProsAlpha2, ProsBeta5,
Proteasome alpha 2, Proteasome beta 5, VATPase E, VATPase A,
VATPase B, VATPase D, Vps2, Vps4, Vps16A, Vps20, Vps24, Vps27,
Vps28, Vha26 (V-ATPase A), Vha68-2 (V-ATPase DE), 40S ribosomal
protein S14, and 60S ribosomal protein L13. In some embodiments,
the polynucleotide comprises 18 or more contiguous nucleotides with
about 95% to about 100% complementarity with a DNA sequence
selected from the group consisting of SEQ ID NOs:1-859 or a
fragment thereof. In some embodiments, the polynucleotide comprises
21 contiguous nucleotides with a sequence of 100% complementarity
with a DNA sequence selected from the group consisting of SEQ ID
NOs:1-859 or a fragment thereof. In some embodiments, the
polynucleotide is a single-stranded RNA molecule. In some
embodiments, the polynucleotide is molecule is a dsRNA molecule. In
some embodiments, the polynucleotide is a single-stranded DNA
molecule. In some embodiments, the polynucleotide is a
double-stranded DNA molecule. In some embodiments, the
polynucleotide is a RNA/DNA hybrid molecule. In some embodiments,
the polynucleotide comprises at least one segment (e. g., an RNA
strand or segment of an RNA strand) with a sequence of about 95% to
about 100% identity or complementarity with a sequence selected
from the group consisting of SEQ ID NOs:860-1718 and 1722-1975 or a
fragment thereof. In some embodiments, polynucleotide comprises at
least one segment (e. g., an RNA strand or segment of an RNA
strand) with a sequence selected from the group consisting of SEQ
ID NOs:860-1718 and 1722-1975 or a fragment thereof. In some
embodiments, polynucleotide comprises at least one segment of 21
contiguous nucleotides (e. g., an RNA strand or segment of an RNA
strand) that is complementary or identical to a sequence selected
from the group consisting of SEQ ID NOs:860-1718 and 1722-1975 or a
fragment thereof. In some embodiments, the insecticidal composition
further comprises one or more of a carrier agent, a surfactant, an
organosilicone, a cationic lipid, a polynucleotide herbicidal
molecule, a non-polynucleotide herbicidal molecule, a
non-polynucleotide pesticide, a fungicide, a safener, an insect
attractant, and an insect growth regulator. Embodiments of the
insecticidal compositions comprise non-polynucleotide insecticides,
e. g., a bacterially produced insecticidal protein.
[0008] Several embodiments relate to a method of providing a plant
having improved resistance to an insect, comprising expressing in
the plant a recombinant DNA construct, wherein the recombinant DNA
construct encodes an insecticidal polynucleotide comprising a
sequence that is essentially identical or essentially complementary
to a fragment of at least one target gene of the insect, and
wherein ingestion of the insecticidal polynucleotide by the insect
results in mortality or stunting in the insect. In some
embodiments, the target gene is selected from the group consisting
of actin, Act5C, arginine kinase, COPI (coatomer subunit) alpha,
COPI (coatomer subunit) beta, COPI (coatomer subunit) betaPrime,
COPI (coatomer subunit) delta, COPI (coatomer subunit) epsilon,
COPI (coatomer subunit) gamma, COPI (coatomer subunit) zeta, RpL07,
RpL19, RpL3, RpL40, RpL13, RpL14, RpS21, RpS4, RpS14, Rpn2, Rpn3,
Rpn7, Rpt6, Rpn8, Rpn9, Rpn6-PB-like protein, Sar1, sec6, sec23,
sec23A, shrb (snf7), Tubulin gamma chain, ProsAlpha2, ProsBeta5,
Proteasome alpha 2, Proteasome beta 5, VATPase E, VATPase A,
VATPase B, VATPase D, Vps2, Vps4, Vps16A, Vps20, Vps24, Vps27,
Vps28, Vha26 (V-ATPase A), Vha68-2 (V-ATPase D/E), 40S ribosomal
protein S14, and 60S ribosomal protein L13. In some embodiments,
the target gene has a DNA sequence selected from the group
consisting of SEQ ID NOs:1-859. In some embodiments, the
insecticidal polynucleotide is single-stranded RNA (ssRNA). In
other embodiments, the insecticidal polynucleotide is
double-stranded RNA (dsRNA), which may comprise single-stranded
portions, such as a loop in a stem-loop structure. In some
embodiments, the insecticidal polynucleotide is a single-stranded
DNA molecule. In some embodiments, the insecticidal polynucleotide
is a double-stranded DNA molecule. In some embodiments, the
insecticidal polynucleotide is a RNA/DNA hybrid molecule. In some
embodiments, the insecticidal polynucleotide comprises a sequence
of about 95% to about 100% identity or complementarity with a
sequence selected from the group consisting of SEQ ID NOs:860-1718
and 1722-1975 or a fragment thereof. In some embodiments, the
insecticidal polynucleotide comprises a sequence of at least 21
contiguous nucleotides that are complementarity or identical to a
sequence selected from the group consisting of SEQ ID NOs:860-1718
and 1722-1975. In some embodiments, the insecticidal polynucleotide
is an RNA (e. g., an RNA strand or segment of an RNA strand)
comprising at least 21 contiguous nucleotides of a sequence
selected from the group consisting of SEQ ID NOs:860-1718 and
1722-1975 or a fragment thereof.
[0009] Several embodiments relate to a recombinant DNA construct
comprising a heterologous promoter operably linked to DNA encoding
an insecticidal polynucleotide comprising a sequence of about 95%
to about 100% identity or complementarity with at least 21
contiguous nucleotides of a sequence selected from the group
consisting of SEQ ID NOs:860-1718 and 1722-1975 or a fragment
thereof. Several embodiments relate to a commercial unit of seed,
such as a bag, in which all or substantially all of the seeds
comprise a recombinant DNA construct comprising a heterologous
promoter operably linked to DNA encoding an insecticidal
polynucleotide comprising a sequence of about 95% to about 100%
identity or complementarity with at least 21 contiguous nucleotides
of a sequence selected from the group consisting of SEQ ID
NOs:860-1718 and 1722-1975 or a fragment thereof. In some
embodiments, the insecticidal polynucleotide is single-stranded RNA
(ssRNA). In other embodiments, the insecticidal polynucleotide is
double-stranded RNA (dsRNA), which may comprise single-stranded
portions, such as a loop in a stem-loop structure. In some
embodiments, the insecticidal polynucleotide is single-stranded DNA
(ssDNA). In other embodiments, the insecticidal polynucleotide is
double-stranded DNA (dsDNA), which may comprise single-stranded
portions, such as a loop in a stem-loop structure. In some
embodiments, the insecticidal polynucleotide is a hybrid RNA/DNA
molecule. In some embodiments, the insecticidal polynucleotide
comprises an RNA strand or segment of an RNA strand comprising a
sequence of about 95% to about 100% identity or complementarity
with at least 21 contiguous nucleotides of a sequence selected from
the group consisting of SEQ ID NOs:860-1718 and 1722-1975 or a
fragment thereof. In some embodiments, the insecticidal
polynucleotide comprises an RNA strand or segment of an RNA strand
comprising a sequence selected from the group consisting of SEQ ID
NOs:860-1718 and 1722-1975 or a fragment thereof.
[0010] In related aspects, provided herein are man-made
compositions comprising a insecticidal polynucleotide as described
herein, such as ssRNA, dsRNA, ssDNA, dsDNA or hybrid RNA/DNA
formulations useful for topical application to a plant or substance
in need of protection from an insect infestation; recombinant
constructs and vectors useful for making transgenic plant cells and
transgenic plants; formulations and coatings useful for treating
plants (including plant seeds or propagatable parts such as
tubers); plant seeds or propagatable parts such as tubers treated
with or containing a polynucleotide as described herein as well as
commodity products and foodstuffs produced from such plants; seeds,
or propagatable parts (especially commodity products and foodstuffs
having a detectable amount of a polynucleotide disclosed herein).
Several embodiments relate to polyclonal or monoclonal antibodies
that bind a peptide or protein encoded by a sequence or a fragment
of a sequence selected from the group consisting of SEQ ID
NOs:1-859. Several embodiments relate to polyclonal or monoclonal
antibodies that bind a peptide or protein encoded by a sequence or
a fragment of a sequence selected from the group consisting of SEQ
ID NOs:860-1718 and 1722-1975 or the complement thereof. Such
antibodies are made by routine methods as known to one of ordinary
skill in the art.
DETAILED DESCRIPTION
[0011] The present embodiments relate to methods and compositions
for controlling insect pests, in particular the group of
coleopteran insects commonly known as "flea beetles", of which
there are several genera. Disclosed herein are target genes
identified as useful for designing insecticidal polynucleotide
agents for preventing or treating flea beetle infestations,
especially of commercially important plants. The methods and
compositions are especially useful for preventing or treating flea
beetle infestations of commercially important Brassica species
including species commercially used as oilseed, food, or livestock
feed (e. g., canola, rapeseed, turnips, and field mustard or turnip
rape). Such Brassica species include B. napus, B. juncea, B.
carinata, B. rapa, B. oleracea, B. rupestris, B. septiceps, B.
nigra, B. narinosa, B. perviridus, B. tournefortii, and B.
fructiculosa. Also disclosed are sequences for suppressing one or
more flea beetle target genes. Several embodiments relate to
insecticidal polynucleotide agents that suppress flea beetle target
genes. In some embodiments, insecticidal polynucleotides and
recombinant DNA molecules and constructs useful in methods of
controlling insect pests, especially flea beetles are provided.
Several embodiments relate to insecticidal compositions, as well as
to transgenic plants resistant to infestation by insect pests.
Several embodiments relate to methods of identifying efficacious
insecticidal polynucleotide agents, for example, single-stranded
RNA molecules, double-stranded RNA molecules, single-stranded DNA
molecules, double-stranded DNA molecules, or hybrid RNA/DNA
molecules for controlling insect pests and methods for identifying
target genes that are likely to represent essential functions,
making these genes preferred targets for RNAi-mediated silencing
and control of insect pests.
[0012] Several embodiments relate to methods and compositions for
inhibiting or controlling flea beetle infestation of a plant by
inhibiting in the flea beetle the expression of one or more target
gene selected from the group consisting of actin, Act5C, arginine
kinase, COPI (coatomer subunit) alpha, COPI (coatomer subunit)
beta, COPI (coatomer subunit) betaPrime, COPI (coatomer subunit)
delta, COPI (coatomer subunit) epsilon, COPI (coatomer subunit)
gamma, COPI (coatomer subunit) zeta, RpL07, RpL19, RpL3, RpL40,
RpL13, RpL14, RpS21, RpS4, RpS14, Rpn2, Rpn3, Rpn7, Rpt6, Rpn8,
Rpn9, Rpn6-PB-like protein, Sar1, sec6, sec23, sec23A, shrb (snf7),
Tubulin gamma chain, ProsAlpha2, ProsBeta5, Proteasome alpha 2,
Proteasome beta 5, VATPase E, VATPase A, VATPase B, VATPase D,
Vps2, Vps4, Vps16A, Vps20, Vps24, Vps27, Vps28, Vha26 (V-ATPase A),
Vha68-2 (V-ATPase DE), 40S ribosomal protein S14, and 60S ribosomal
protein L13. In some embodiments, inhibiting the expression of one
or more target gene in the flea beetle results in stunting or
mortality.
[0013] Several embodiments relate to an insecticidal polynucleotide
molecule, such as a ssRNA, a dsRNA, a ssDNA, a dsDNA, or a RNA/DNA
hybrid, which comprises one or more segments comprising 18 or more
contiguous nucleotides, for example 21 or more contiguous
nucleotides, having 95% to about 100% (e. g., about 95%, about 96%,
about 97%, about 98%, about 99%, or about 100%) complementarity
with a fragment of an insect target gene selected from the group
consisting of actin, Act5C, arginine kinase, COPI (coatomer
subunit) alpha, COPI (coatomer subunit) beta, COPI (coatomer
subunit) betaPrime, COPI (coatomer subunit) delta, COPI (coatomer
subunit) epsilon, COPI (coatomer subunit) gamma, COPI (coatomer
subunit) zeta, RpL07, RpL19, RpL3, RpL40, RpL13, RpL14, RpS21,
RpS4, RpS14, Rpn2, Rpn3, Rpn7, Rpt6, Rpn8, Rpn9, Rpn6-PB-like
protein, Sar1, sec6, sec23, sec23A, shrb (snf7), Tubulin gamma
chain, ProsAlpha2, ProsBeta5, Proteasome alpha 2, Proteasome beta
5, VATPase E, VATPase A, VATPase B, VATPase D, Vps2, Vps4, Vps16A,
Vps20, Vps24, Vps27, Vps28, Vha26 (V-ATPase A), Vha68-2 (V-ATPase
D/E), 40S ribosomal protein S14, and 60S ribosomal protein L13. In
some embodiments, the insecticidal polynucleotide comprises
multiple segments each of 18 or more contiguous nucleotides with a
sequence of about 95% to about 100% (e. g., about 95%, about 96%,
about 97%, about 98%, about 99%, or about 100%) complementarity
with a fragment of a DNA sequence selected from the group
consisting of SEQ ID NOs:1-859. In some embodiments, the
insecticidal polynucleotide comprises at least 21 contiguous
nucleotides having 100% complementarity with a fragment of a DNA
sequence selected from the group consisting of SEQ ID NOs:1-859. In
some embodiments, the insecticidal polynucleotide comprises
segments complementary to different regions of a target gene, or
can comprise multiple copies of a segment. In some embodiments, the
insecticidal polynucleotide comprises multiple segments, each of 18
or more contiguous nucleotides with a sequence of about 95% to
about 100% (e. g., about 95%, about 96%, about 97%, about 98%,
about 99%, or about 100%) complementarity with a fragment of a
different target gene; in this way multiple target genes, or
multiple insect species, can be suppressed.
[0014] Several embodiments relate to a insecticidal polynucleotide
(e.g., a ssRNA, a dsRNA, a ssDNA, a dsDNA, or a RNA/DNA hybrid)
molecule which inhibits the expression of one or more insect target
genes selected from the group consisting of actin, Act5C, arginine
kinase, COPI (coatomer subunit) alpha, COPI (coatomer subunit)
beta, COPI (coatomer subunit) betaPrime, COPI (coatomer subunit)
delta, COPI (coatomer subunit) epsilon, COPI (coatomer subunit)
gamma, COPI (coatomer subunit) zeta, RpL07, RpL19, RpL3, RpL40,
RpL13, RpL14, RpS21, RpS4, RpS14, Rpn2, Rpn3, Rpn7, Rpt6, Rpn8,
Rpn9, Rpn6-PB-like protein, Sar1, sec6, sec23, sec23A, shrb (snf7),
Tubulin gamma chain, ProsAlpha2, ProsBeta5, Proteasome alpha 2,
Proteasome beta 5, VATPase E, VATPase A, VATPase B, VATPase D,
Vps2, Vps4, Vps16A, Vps20, Vps24, Vps27, Vps28, Vha26 (V-ATPase A),
Vha68-2 (V-ATPase DE), 40S ribosomal protein S14, and 60S ribosomal
protein L13. Several embodiments relate to an insecticidal
polynucleotide having a length greater than that which is typical
of naturally occurring regulatory small RNAs (such as endogenously
produced siRNAs and mature miRNAs), e. g, the polynucleotide is at
least about 30 contiguous base-pairs in length. In some
embodiments, the insecticidal polynucleotide has a length of
between about 50 to about 500 base-pairs. In some embodiments, the
insecticidal polynucleotide is at least 50 base pairs in length. In
some embodiments, the insecticidal polynucleotide is formed from
two separate, essentially complementary strands (e. g., where each
strand is separately provided, or where each strand is encoded on a
separate DNA molecule, or where the two strands are encoded on
separate sections of a DNA and are separately transcribed or made
separate, for example, by the action of a recombinase or nuclease),
wherein at least one strand comprises a sequence of about 95% to
about 100% (e. g., about 95%, about 96%, about 97%, about 98%,
about 99%, or about 100%) identity or complementarity with at least
21 contiguous nucleotides of a sequence selected from the group
consisting of SEQ ID NOs:860-1718 and 1722-1975 or a fragment
thereof. In some embodiments, the insecticidal polynucleotide is
double stranded and blunt-ended, e. g., two separate, equal-length
strands which form the double-stranded polynucleotide through
intermolecular hybridisation. In some embodiments, the insecticidal
polynucleotide is double stranded and has an overhang at one or
both ends (termini), e. g., two separate, unequal-length strands
which form the double-stranded polynucleotide through
intermolecular hybridisation; the overhang can be a single
nucleotide or 2, 3, 4, 5, 6, or more nucleotides, and can be
located on the 5' end or on the 3' end of a strand. In some
embodiments, the insecticidal polynucleotide comprises at least one
stem-loop, e. g., a single polynucleotide molecule that forms a
double-stranded region through intramolecular hybridization
adjacent to a "hairpin" secondary structure. In some embodiments,
the insecticidal polynucleotide is formed from a single
self-hybridizing hairpin, wherein one "arm" of the hairpin
comprises a sequence of about 95% to about 100% (e. g., about 95%,
about 96%, about 97%, about 98%, about 99%, or about 100%) identity
or complementarity to at least 21 contiguous nucleotides of a
sequence selected from the group consisting of SEQ ID NOs:860-1718
and 1722-1975 or a fragment thereof. In embodiments,
self-hybridizing polynucleotides which form hairpins (or partial
hairpins) include polynucleotide molecules that comprise "spacer"
nucleotides or a single-stranded "loop region" between the
double-strand-forming complementary "arms" of sense sequence and
anti-sense sequence. In embodiments, such spacers or loops include
nucleotides having a sequence unrelated (not complementary or
identical to) the target gene corresponding to the double-stranded
portion of the hairpin. In embodiments, such spacers or loops
include nucleotides having a sequence complementary or identical to
the target gene. Examples of spacers or loops include those encoded
by SEQ ID NOs:1719-1721. In embodiments, the insecticidal
polynucleotide comprises multiple stem-loops, with or without
spacer nucleotides between each stem-loop. In embodiments, the
insecticidal polynucleotide comprises a modified stem-loop such as
a "stabilized anti-sense" loop or a "stabilized sense" loop; see,
e. g., U.S. Pat. Nos. 7,855,323 and 9,006,414, which are
incorporated by reference in their entirety herein.
[0015] The insecticidal polynucleotide can be chemically
synthesized (e. g., by in vitro transcription, such as
transcription using a T7 polymerase or other polymerase), or can be
produced by expression in a microorganism, by expression in a plant
cell, or by microbial fermentation. The insecticidal polynucleotide
can be chemically modified, e. g., to improve stability, ease of
formulation, or efficacy. In some embodiments, the insecticidal
polynucleotide molecule is provided in a microbial or plant cell
that expresses the insecticidal polynucleotide (such as a hairpin
form of a dsRNA), or in a microbial fermentation product.
[0016] A variety of methods for designing and producing a variety
of forms of insecticidal polynucleotide are known in the art and
are useful in the compositions and methods disclosed herein. See,
for example, the following patents which are incorporated by
reference in their entirety herein: (1) U.S. Pat. No. 8,598,332 to
Waterhouse et al., which discloses recombinant DNA constructs
comprising DNA encoding sense RNA and anti-sense RNA sequences in a
single transcript that forms an artificial "hairpin" RNA structure
with a double-stranded RNA stem by base-pairing between the sense
and anti-sense nucleotide sequences; embodiments include hairpins
with spacer nucleotides between the sense and anti-sense nucleotide
sequences; (2) U.S. Pat. No. 8,158,414 to Rommens et al., which
discloses recombinant DNA constructs including convergently
oriented first and second promoters, which produce, e. g., an RNA
duplex that is formed by annealing of two separate RNA transcripts;
and (3) U.S. Pat. Nos. 7,855,323 and 9,006,414 to Huang et al.,
which disclose recombinant DNA constructs including DNA encoding
"stabilized anti-sense" transcripts which form a loop of
anti-sense-oriented RNA for suppressing one or more target genes;
recombinant DNA constructs can be designed to similarly encode
"stabilized sense" transcripts which form a loop of sense-oriented
RNA for suppressing one or more target genes.
[0017] Embodiments of the compositions comprising insecticidal
polynucleotides described herein further comprise one or more
additional components or adjuvants, e. g., a carrier agent, an
encapsulation agent, an emulsifying agent, a surfactant, an
organosilicone, a cationic lipid, a spreading agent, a
photoprotective agent, a rainfastness agent, a polynucleotide
herbicidal molecule, a non-polynucleotide herbicidal molecule, a
non-polynucleotide pesticide, a non-polynucleotide fungicide, a
safener, a bait, an insect attractant, an insect pheromone, and an
insect growth regulator. In embodiments, these additional
components or adjuvants are edible or digestible if ingested by a
flea beetle.
[0018] In embodiments, the insecticidal polynucleotide disclosed
herein are used in combination with a non-nucleotide pesticidal
agent such as a small-molecule pesticidal agent or a proteinaceous
pesticidal agent, either concurrently or sequentially. Examples of
non-nucleotide pesticidal agents include patatins, plant lectins,
phytoecdysteroids, and bacterial insecticidal proteins (e. g.,
insecticidal proteins from Bacillus thuringiensis, Xenorhabdus sp.,
Photorhabdus sp., Brevibacillus laterosporus (previously Bacillus
laterosporus), Lysinibacillus sphaericus (previously Bacillus
sphaericus), Chromobacterium sp., Chromobacterium subtsugae,
Paenibacillus sp., Paenibacillus lentimorbus, and Paenibacillus
popilliae), a bacterium that produces an insecticidal protein, and
an entomicidal bacterial species. In embodiments, the compositions
comprising polynucleotides for flea beetle control such as the
insecticidal polynucleotide described herein can further comprise,
or can be used concurrently or sequentially with, conventional
pesticides such as Spiromesifen, Spirodiclofen, Spirotetramat,
Pyridaben, Tebufenpyrad, Tolfenpyrad, Fenpyroximate, Flufenerim,
Pyrimidifen, Fenazaquin, Rotenone, Cyenopyrafen, Hydramethylnon,
Acequinocyl, Fluacrypyrim, Aluminium phosphide, Calcium phosphide,
Phosphine, Zinc phosphide, Cyanide, Diafenthiuron, Azocyclotin,
Cyhexatin, Fenbutatin oxide, Propargite, Tetradifon, Bensultap,
Thiocyclam, Thiosultap-sodium, Flonicamid, Etoxazole, Clofentezine,
Diflovidazin, Hexythiazox, Chlorfluazuron, Bistrifluron,
Diflubenzuron, Flucycloxuron, Flufenoxuron, Hexaflumuron,
Lufenuron, Novaluron, Noviflumuron, Teflubenzuron, Triflumuron,
Buprofezin, Cyromazine, Hydroprene, Kinoprene, Methoprene,
Fenoxycarb, Pyriproxyfen, Pymetrozine, Pyrifluquinazon,
Chlorfenapyr, Tralopyril, B.t. (Bacillus thuringiensis) var.
aizawai, B.t. var. israelensis, B.t. var. kurstaki, B.t. var.
sphaericus, B.t. var. tenebrionensis, Bacillus thuringiensis crop
proteins including Cry1Ab, Cry1Ac, Cry1Fa, Cry2Ab, mCry3A, Cry3Ab,
Cry3Bb, Cry34/35Ab1, Methyl bromide and other alkyl halides,
Chloropicrin, Sulfuryl fluoride, Benclothiaz, Chinomethionat,
Cryolite, Methylneodecanamide, Benzoximate, Cymiazole,
Fluensulfone, Azadirachtin, Bifenazate, Amidoflumet, Dicofol,
Plifenate, Cyflumetofen, Pyridalyl, Beauveria bassiana GHA,
Sulfoxaflor, Spinetoram, Spinosad, Spinosad, Emamectin benzoate,
Lepimectin, Milbemectin, Abamectin, Methoxyfenozide,
Chromafenozide, Halofenozide, Tebufenozide, Amitraz,
Chlorantraniliprole, Cyantraniliprole, Flubendiamide,
alpha-endosulfan, Chlordane, Endosulfan, Fipronil, Acetoprole,
Ethiprole, Pyrafluprole, Pyriprole, Indoxacarb and Metaflumizone,
Acrinathrin, Allethrin, Allethrin-cis-trans, Allethrin-trans,
beta-Cyfluthrin, beta-Cypermethrin, Bifenthrin, Bioallethrin,
Bioallethrin S-cyclopentenyl, Bioresmethrin, Cycloprothrin,
Cyfluthrin, Cyhalothrin, Cypermethrin, Cyphenothrin
[(1R)-trans-isomers], Dimefluthrin, Empenthrin [(EZ)-(1R)-isomers],
Esfenvalerate, Etofenprox, Fenpropathrin, Fenvalerate,
Flucythrinate, Flumethrin, Gamma-cyhalothryn, lambda-Cyhalothrin,
Meperfluthrin, Metofluthrin, Permethrin, Phenothrin
[(1R)-trans-isomer], Prallethrin, Profluthrin, Protrifenbute,
Resmethrin, Silafluofen, tau-Fluvalinate, Tefluthrin, Tetramethrin,
Tetramethrin [(1R)-isomers], Tetramethylfluthrin,
theta-Cypermethrin, Tralomethrin, Transfluthrin, zeta-Cypermethrin,
alpha-Cypermethrin, Deltamethrin, DDT, and Methoxychlor,
Thiodicarb, Alanycarb, Aldicarb, Bendiocarb, Benfuracarb,
Butoxycarboxim, Carbaryl, Carbofuran, Carbosulfan, Ethiofencarb,
Fenobucarb, Formetanate, Furathiocarb, Isoprocarb, Methiocarb,
Methomyl, Metolcarb, Oxamyl, Pirimicarb, Propoxur, Thiofanox,
Triazamate, Trimethacarb, XMC, Xylylcarb, Chlorpyrifos, Malathion,
Acephate, Azamethiphos, Azinphos-ethyl, Azinphos-methyl, Cadusafos,
Chlorethoxyfos, Chlorfenvinphos, Chlormephos, Chlorpyrifos-methyl,
Coumaphos, Cyanophos, Demeton-S-methyl, Diazinon, Dichlorvos/DDVP,
Dicrotophos, Dimethoate, Dimethylvinphos, Disulfoton, EPN, Ethion,
Ethoprophos, Famphur, Fenamiphos, Fenitrothion, Fenthion, Fonofos,
Fosthiazate, Imicyafos, Isofenphos-methyl, Mecarbam, Methamidophos,
Methidathion, Mevinphos, Monocrotophos, Naled, Omethoate,
Oxydemeton-methyl, Parathion, Parathion-methyl, Phenthoate,
Phorate, Phosalone, Phosmet, Phosphamidon, Phoxim,
Pirimiphos-ethyl, Profenofos, Propaphos, Propetamphos, Prothiofos,
Pyraclofos, Pyridaphenthion, Quinalphos, Sulfotep, Tebupirimfos,
Temephos, Terbufos, Tetrachlorvinphos, Thiometon, Triazophos,
Trichlorfon, Vamidothion Imidacloprid, Thiamethoxam, Acetamiprid,
Clothianidin, Dinotefuran, Nitenpyram, Nithiozine, Nicotine,
Thiacloprid, chlorantraniliprole, and cyantraniliprole. In
embodiments, the compositions comprising insecticidal
polynucleotide for flea beetle control in Brassica species,
including canola, further comprise, or are used concurrently or
sequentially with, foliar sprays including one or more pesticides
selected from the group consisting of Deltamethrin, Cypermethrin,
Lambda-cyhalothrin, Permethrin, Carbaryl, Carbofuran, and
Malathion, or seed treatments comprising one or more pesticides
selected from the group consisting of Thiamethoxam, Imidacloprid,
and Clothianadin.
[0019] In embodiments, the compositions comprising insecticidal
polynucleotides for flea beetle control described herein can
further comprise, or can be used concurrently or sequentially with,
conventional fungicides such as bupirimate, dimethirimol,
ethirimol, cyprodinil, pyrimethanil, mepanipyrim, fenpiclonil,
fludioxonil; phenylamides, benalaxyl, furalaxyl, metalaxyl,
R-metalaxyl, ofurace, oxadixyl, benomyl, carbendazim, debacarb,
fuberidazole, thiabendazole, chlozolinate, dichlozoline, iprodine,
myclozoline, procymidone, vinclozolin, carboxin, fenfuram,
flutolanil, mepronil, oxycarboxin, thifluzamide, guazatine, dodine,
iminoctadine, azoxystrobin, kresoxim-methyl, metominostrobin, or
trifloxystrobin, ferbam, mancozeb, maneb, metiram, propineb,
thiram, zineb, ziram, captafol, captan, dichlofluanid, fluoromide,
folpet, tolyfluanid, copper hydroxide, copper oxychloride, copper
sulfate, cuprous oxide, mancopper, oxine-copper, dinocap,
nitrothal-isopropyl, edifenphos, iprobenphos, isoprothiolane,
phosdiphen, pyrazopho, toclofos-methyl, acibenzolar-S-methyl,
harpin, anilazine, blasticidin-S, chinomethionat, chloroneb,
chlorothalonil, cymoxanil, dichione, diclomezine, dicloran,
diethofencarb, dimethomorph, dithianon, etridiazole, famoxadone,
fenamidone, fentin, fenpyrazamine, ferimzone, fluazinam,
flusulfamide, fosetyl-aluminium, hymexazol, kasugamycin,
methasulfocarb, pencycuron, phthalide, polyoxins, probenazole,
propamocarb, pyroquilon, quinoxyfen, quintozene, sulfur,
triazoxide, tricyclazole, validamycin, azaconazole, bitertanol,
propiconazole, difenoconazole, diniconazole, cyproconazole,
epoxiconazole, fluquinconazole, flusilazole, flutriafol,
hexaconazole, imazalil, imibenconazole, ipconazole, tebuconazole,
tetraconazole, fenbuconazole, metconazole, myclobutanil,
perfurazoate, penconazole, bromuconazole, pyrifenox, prochloraz,
triadimefon, triadimenol, triflumizol, triticonazole, triforine,
ancymidol, fenarimol or nuarimol, dodemorph, fenpropidin,
fenpropimorph, spiroxamine, tridemorph, and fenhexamid. In
embodiments, the compositions comprising insecticidal
polynucleotides for flea beetle control in Brassica species,
including canola, further comprise, or are used concurrently or
sequentially with, foliar sprays including one or more fungicides
selected from the group consisting of Azoxystrobin, Bacillus
subtilis strain QST 2808, Boscalid, Fluxopyroxad, Pyraclostrobin,
Metconazole, Prothioconazole, Penthiopyrad, Picoxystrobin, and
Thiophanate Methyl, or seed treatments including one or more
fungicides selected from the group consisting of Azoxystrobin,
Metalaxyl, Trifloxystrobin, Pyraclostrobin, Sedaxane, Penflufen,
Fludioxonil, and Mefenoxam.
[0020] In embodiments, the compositions comprising insecticidal
polynucleotides for flea beetle described herein can further
comprise, or can be used concurrently or sequentially with,
conventional herbicides such as glyphosate, auxin-like herbicides
such as dicamba, phosphinothricin, glufosinate,
2,2-dichloropropionic acid (Dalapon), acetolactate synthase
inhibitors such as sulfonylurea, imidazolinone, triazolopyrimidine,
pyrimidyloxybenzoates, and phthalide, bromoxynil, cyclohexanedione
(sethoxydim) and aryloxyphenoxypropionate (haloxyfop), sulfonamide
herbicides, triazine herbicides, 5-methyltryptophan, aminoethyl
cysteine, pyridazinone herbicides such as norflurazon,
cyclopropylisoxazole herbicides such as isoxaflutole,
protoporphyrinogen oxidase inhibitors, herbicidea containing an
aryloxyalkanoate moiety, phenoxy auxins such as 2,4-D and
dichlorprop, pyridyloxy auxins such as fluroxypyr and triclopyr,
aryloxyphenoxypropionates (AOPP) acetyl-coenzyme A carboxylase
(ACCase) inhibitors such as haloxyfop, quizalofop, and diclofop,
and 5-substituted phenoxyacetate protoporphyrinogen oxidase IX
inhibitors such as pyraflufen and flumiclorac. In embodiments, the
compositions comprising insecticidal polynucleotides for flea
beetle control in Brassica species, including canola, further
comprise, or are used concurrently or sequentially with, one or
more post-emergence herbicides selected from the group consisting
of Quizalofop, Sethoxydim, Clethodim, and Clopyralid. In
embodiments, the compositions comprising insecticidal
polynucleotides for flea beetle control in herbicide-resistant
Brassica species, including herbicide-resistant canola, further
comprise, or are used concurrently or sequentially with, one or
more herbicides selected from the group consisting of Imazamox,
Glyphosate, and Glufosinate.
[0021] The compositions and methods disclosed are useful for
inhibiting or controlling flea beetle infestation of a plant, such
as a Brassica species. In embodiments, the compositions and methods
are used to treat a growing plant, such as a field of Brassica
plants. Embodiments include compositions comprising insecticidal
polynucleotides disclosed herein in a composition in the form of a
solid, liquid, powder, suspension, emulsion, spray, encapsulation,
microbeads, carrier particulates, film, matrix, soil drench, or
seed treatment composition. In embodiments, such compositions are
applied to a surface of the plant in need of protection from or
treatment for flea beetle infestations, or applied directly to the
flea beetles, or provided in an ingestible form to the flea
beetles. In embodiments, a composition comprising insecticidal
polynucleotides disclosed herein is applied directly to
ungerminated seeds (such as ungerminated Brassica species seeds),
providing plants germinated from the treated seeds increased
resistance to flea beetle infestations; examples of seed treatment
methods are disclosed in U.S. patent application Ser. No.
14/143,836, which is incorporated by reference in its entirety
herein. An embodiment includes a Brassica seed that is treated by
directly contacting the seed with an insecticidal polynucleotide
(such as a ssRNA, dsRNA, ssDNA, dsDNA or RNA/DNA hybrid molecule)
disclosed herein, followed by germination into a Brassica plant
that exhibits increased resistance to flea beetle infestations.
[0022] Unless defined otherwise, all technical and scientific terms
used have the same meaning as commonly understood by one of
ordinary skill in the art. Generally, the nomenclature used and the
manufacturing or laboratory procedures described below are well
known and commonly employed in the art. Conventional methods are
used for these procedures, such as those provided in the art and
various general references. Where a term is provided in the
singular, the inventors also contemplate aspects described by the
plural of that term. Where there are discrepancies in terms and
definitions used in references that are incorporated by reference,
the terms used in this application shall have the definitions
given. Other technical terms used have their ordinary meaning in
the art in which they are used, as exemplified by various
art-specific dictionaries, for example, "The American Heritage.RTM.
Science Dictionary" (Editors of the American Heritage Dictionaries,
2011, Houghton Mifflin Harcourt, Boston and New York), the
"McGraw-Hill Dictionary of Scientific and Technical Terms"
(6.sup.th edition, 2002, McGraw-Hill, New York), or the "Oxford
Dictionary of Biology" (6.sup.th edition, 2008, Oxford University
Press, Oxford and New York). The inventors do not intend to be
limited to a mechanism or mode of action. Reference thereto is
provided for illustrative purposes only.
[0023] Unless otherwise stated, nucleic acid sequences in the text
of this specification are given, when read from left to right, in
the 5' to 3' direction. One of skill in the art would be aware that
a given DNA sequence is understood to define a corresponding RNA
sequence which is identical to the DNA sequence except for
replacement of the thymine (T) nucleotides of the DNA with uracil
(U) nucleotides. Thus, providing a specific DNA sequence is
understood to define the RNA equivalent and vice versa. A given
first polynucleotide sequence, whether DNA or RNA, further defines
the sequence of its exact complement (which can be DNA or RNA), i.
e., a second polynucleotide that hybridizes perfectly to the first
polynucleotide by forming Watson-Crick base-pairs. By "essentially
complementary" is meant that a polynucleotide strand (or at least
one strand of a double-stranded polynucleotide) is designed to
hybridize (generally under physiological conditions such as those
found in a living plant or animal cell) to a target gene or to a
fragment of a target gene or to the transcript of the target gene
or the fragment of a target gene; one of skill in the art would
understand that such hybridization does not necessarily require
100% sequence complementarity. A first nucleic acid sequence is
"operably" connected or "linked" with a second nucleic acid
sequence when the first nucleic acid sequence is placed in a
functional relationship with the second nucleic acid sequence. For
example, a promoter sequence is "operably linked" to DNA if the
promoter provides for transcription or expression of the DNA.
Generally, operably linked DNA sequences are contiguous.
[0024] The term "polynucleotide" commonly refers to a DNA or RNA
molecule containing multiple nucleotides and generally refers both
to "oligonucleotides" (a polynucleotide molecule of 18-25
nucleotides in length) and longer polynucleotides of 26 or more
nucleotides. Polynucleotides also include molecules containing
multiple nucleotides, including non-canonical nucleotides or
chemically modified nucleotides as commonly practiced in the art;
see, e. g., chemical modifications disclosed in the technical
manual "RNA Interference (RNAi) and DsiRNAs", 2011 (Integrated DNA
Technologies Coralville, Iowa).
[0025] Generally, insecticidal polynucleotides as described herein,
whether DNA or RNA or both, and whether single- or double-stranded,
comprise at least one segment of 18 or more contiguous nucleotides
(or, in the case of double-stranded polynucleotides, at least 18
contiguous base-pairs) that are essentially identical or
complementary to a fragment of equivalent size of the DNA of a
target gene or the target gene's RNA transcript. Throughout this
disclosure, "at least 18 contiguous" means "from about 18 to about
10,000, including every whole number point in between". Thus,
embodiments include compositions comprising oligonucleotides having
a length of 18-25 nucleotides (18-mers, 19-mers, 20-mers, 21-mers,
22-mers, 23-mers, 24-mers, or 25-mers), or medium-length
polynucleotides having a length of 26 or more nucleotides
(polynucleotides of 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,
55, 56, 57, 58, 59, 60, about 65, about 70, about 75, about 80,
about 85, about 90, about 95, about 100, about 110, about 120,
about 130, about 140, about 150, about 160, about 170, about 180,
about 190, about 200, about 210, about 220, about 230, about 240,
about 250, about 260, about 270, about 280, about 290, or about 300
nucleotides), or long polynucleotides having a length greater than
about 300 nucleotides (e. g., polynucleotides of between about 300
to about 400 nucleotides, between about 400 to about 500
nucleotides, between about 500 to about 600 nucleotides, between
about 600 to about 700 nucleotides, between about 700 to about 800
nucleotides, between about 800 to about 900 nucleotides, between
about 900 to about 1000 nucleotides, between about 300 to about 500
nucleotides, between about 300 to about 600 nucleotides, between
about 300 to about 700 nucleotides, between about 300 to about 800
nucleotides, between about 300 to about 900 nucleotides, or about
1000 nucleotides in length, or even greater than about 1000
nucleotides in length, for example up to the entire length of a
target gene including coding or non-coding or both coding and
non-coding portions of the target gene). Where an insecticidal
polynucleotide is double-stranded, such as the dsRNAs described in
the working Examples, its length can be similarly described in
terms of base pairs. Double-stranded insecticidal polynucleotides
can further be described in terms of one or more of the
single-stranded components.
[0026] The insecticidal polynucleotides described herein can be
single-stranded (ss) or double-stranded (ds). "Double-stranded"
refers to the base-pairing that occurs between sufficiently
complementary, anti-parallel nucleic acid strands to form a
double-stranded nucleic acid structure, generally under
physiologically relevant conditions. Embodiments include those
wherein the polynucleotide is selected from the group consisting of
sense single-stranded DNA (ssDNA), sense single-stranded RNA
(ssRNA), double-stranded RNA (dsRNA), double-stranded DNA (dsDNA),
a double-stranded DNA/RNA hybrid, anti-sense ssDNA, or anti-sense
ssRNA; a mixture of polynucleotides of any of these types can be
used. In some embodiments, the insecticidal polynucleotide is
double-stranded RNA of a length greater than that which is typical
of naturally occurring regulatory small RNAs (such as endogenously
produced siRNAs and mature miRNAs). In some embodiments, the
insecticidal polynucleotide is double-stranded RNA of at least
about 30 contiguous base-pairs in length. In some embodiments, the
insecticidal polynucleotide is double-stranded RNA with a length of
between about 50 to about 500 base-pairs. In some embodiments, the
insecticidal polynucleotide can comprise components other than
standard ribonucleotides, e. g., an embodiment is an RNA that
comprises terminal deoxyribonucleotides.
[0027] Insecticidal polynucleotides of any size can be used, alone
or in combination, in the various methods and compositions
described herein. In some embodiments, a single insecticidal
polynucleotide is used to make a composition (e. g., a composition
for topical application). In other embodiments, a mixture or pool
of different insecticidal polynucleotides is used; in such cases
the insecticidal polynucleotides can be for a single target gene or
for multiple target genes.
[0028] In various embodiments, an insecticidal polynucleotide as
described herein comprises naturally occurring nucleotides, such as
those which occur in DNA and RNA. In certain embodiments, the
insecticidal polynucleotide is a combination of ribonucleotides and
deoxyribonucleotides, for example, synthetic polynucleotides
consisting mainly of ribonucleotides but with one or more terminal
deoxyribonucleotides or one or more terminal dideoxyribonucleotides
or synthetic polynucleotides consisting mainly of
deoxyribonucleotides but with one or more terminal
dideoxyribonucleotides. In certain embodiments, the insecticidal
polynucleotide comprises non-canonical nucleotides such as inosine,
thiouridine, or pseudouridine. In certain embodiments, the
insecticidal polynucleotide comprises chemically modified
nucleotides. Examples of chemically modified oligonucleotides or
polynucleotides are well known in the art; see, for example, U.S.
Patent Publication 2011/0171287, U.S. Patent Publication
2011/0171176, U.S. Patent Publication 2011/0152353, U.S. Patent
Publication 2011/0152346, and U.S. Patent Publication 2011/0160082,
which are herein incorporated by reference. Illustrative examples
include, but are not limited to, the naturally occurring
phosphodiester backbone of an oligonucleotide or polynucleotide
which can be partially or completely modified with
phosphorothioate, phosphorodithioate, or methylphosphonate
internucleotide linkage modifications, modified nucleoside bases or
modified sugars can be used in oligonucleotide or polynucleotide
synthesis, and oligonucleotides or polynucleotides can be labeled
with a fluorescent moiety (e. g., fluorescein or rhodamine) or
other label (e. g., biotin).
[0029] The term "recombinant", as used to refer to a polynucleotide
(such as insecticidal polynucleotide molecules or recombinant DNA
constructs described herein), means that the polynucleotide is not
a naturally occurring molecule, i. e., that human intervention is
required for the polynucleotide to exist. A recombinant
polynucleotide is produced using recombinant nucleic acid
techniques, or by chemical synthesis, and can include combinations
of sequences that do not occur in nature (e. g., combinations of a
heterologous promoter and a DNA encoding an RNA to be expressed, or
an RNA molecule that comprises concatenated segments of a target
gene that do not in nature occur adjacent to one another). A
recombinant polynucleotide can be biologically produced in a cell
(such as a bacterial or plant or animal cell), for example, when
that cell is transfected or transformed with a vector encoding the
recombinant polynucleotide (e. g., a vector encoding a hairpin form
of a dsRNA, produced in a bacterium). A recombinant polynucleotide
can comprise sequences of nucleotides designed in silico using
appropriate algorithms.
[0030] The insecticidal polynucleotides disclosed herein are
generally designed to suppress or silence one or more genes
("target genes"). The term "gene" refers to any portion of a
nucleic acid that provides for expression of a transcript or
encodes a transcript, or that is a hereditable nucleic acid
sequence. A "gene" can include, but is not limited to, a promoter
region, 5' untranslated regions, transcript encoding regions that
can include intronic regions, 3' untranslated regions, or
combinations of these regions. In embodiments, the target genes can
include coding or non-coding sequence or both. In other
embodiments, the target gene has a sequence identical to or
complementary to a messenger RNA, e. g., in embodiments the target
gene is a cDNA.
[0031] As used herein, the term "isolated" refers to separating a
molecule from other molecules normally associated with it in its
native or natural state. The term "isolated" thus may refer to a
DNA molecule that has been separated from other DNA molecule(s)
which normally are associated with it in its native or natural
state. Such a DNA molecule may be present in a recombined state,
such as a recombinant DNA molecule. Thus, DNA molecules fused to
regulatory or coding sequences with which they are not normally
associated, for example as the result of recombinant techniques,
are considered isolated, even when integrated as a transgene into
the chromosome of a cell or present with other DNA molecules.
[0032] By "insecticidally effective" is meant effective in inducing
a physiological or behavioural change in an insect (e. g., adult or
larval flea beetles) that infests a plant such as, but not limited
to, growth stunting, increased mortality, decrease in reproductive
capacity or decreased fecundity, decrease in or cessation of
feeding behavior or movement, or decrease in or cessation of
metamorphosis stage development. In some embodiments, application
of an insecticidally effective amount of the insecticidal
polynucleotide, such as a dsRNA molecule, to a plant improves the
plant's resistance to infestation by the insect. In some
embodiments, application of an insecticidally effective amount of
the insecticidal polynucleotide to a crop plant improves yield (e.
g., increased biomass, increased seed or fruit production, or
increased oil, starch, sugar, or protein content) of that crop
plant, in comparison to a crop plant not treated with the
insecticidal polynucleotide. While there is no upper limit on the
concentrations and dosages of an insecticidal polynucleotide as
described herein that can be useful in the methods and compositions
provided herein, lower effective concentrations and dosages will
generally be sought for efficiency and economy. Non-limiting
embodiments of effective amounts of an insecticidal polynucleotide
include a range from about 10 nanograms per milliliter to about 100
micrograms per milliliter of an insecticidal polynucleotide in a
liquid form sprayed on a plant, or from about 10 milligrams per
acre to about 100 grams per acre of polynucleotide applied to a
field of plants, or from about 0.001 to about 0.1 microgram per
milliliter of polynucleotide in an artificial diet for feeding the
insect. Where compositions as described herein are topically
applied to a plant, the concentrations can be adjusted in
consideration of the volume of spray or treatment applied to plant
leaves or other plant part surfaces, such as flower petals, stems,
tubers, fruit, anthers, pollen, leaves, roots, or seeds. In one
embodiment, a useful treatment for herbaceous plants using
insecticidal polynucleotides as described herein is about 1
nanomole (nmol) of polynucleotides per plant, for example, from
about 0.05 to 1 nmol polynucleotides per plant. Other embodiments
for herbaceous plants include useful ranges of about 0.05 to about
100 nmol, or about 0.1 to about 20 nmol, or about 1 nmol to about
10 nmol of polynucleotides per plant. In certain embodiments, about
40 to about 50 nmol of a single-stranded polynucleotide as
described herein are applied. In certain embodiments, about 0.5
nmol to about 2 nmol of an insecticidal polynucleotide as described
herein is applied. In certain embodiments, a composition containing
about 0.5 to about 2.0 milligrams per milliliter, or about 0.14
milligrams per milliliter of an insecticidal polynucleotide (or a
single-stranded 21-mer) as described herein is applied. In certain
embodiments, a composition of about 0.5 to about 1.5 milligrams per
milliliter of an insecticidal polynucleotide as described herein of
about 50 to about 200 or more nucleotides is applied. In certain
embodiments, about 1 nmol to about 5 nmol of an insecticidal
polynucleotide as described herein is applied to a plant. In
certain embodiments, a polynucleotide composition as topically
applied to the plant comprises at least one polynucleotide as
described herein at a concentration of about 0.01 to about 10
milligrams per milliliter, or about 0.05 to about 2 milligrams per
milliliter, or about 0.1 to about 2 milligrams per milliliter. Very
large plants, trees, or vines can require correspondingly larger
amounts of insecticidal polynucleotides. When using long
insecticidal polynucleotide molecules that can be processed into
multiple oligonucleotides (e. g., multiple oligonucleotides encoded
by a single recombinant DNA molecule as disclosed herein), lower
concentrations can be used. Non-limiting examples of effective
insecticidal polynucleotide treatment regimes include a treatment
of between about 0.1 to about 1 nmol of polynucleotide molecule per
plant, or between about 1 nmol to about 10 nmol of polynucleotide
molecule per plant, or between about 10 nmol to about 100 nmol of
polynucleotide molecule per plant.
Methods of Causing Insect Mortality and of Controlling Insect
Infestations
[0033] Several embodiments relate to a method of causing mortality
or stunting in an insect, comprising providing in the diet of an
insect at least one insecticidal polynucleotide comprising at least
one silencing element essentially identical or essentially
complementary to a fragment of a target gene sequence of the
insect, wherein the target gene sequence is selected from the group
consisting of SEQ ID NOs:1-859, and wherein ingestion of the
insecticidal polynucleotide by the insect results in mortality or
stunting in the insect. These methods are useful for controlling
insect infestations of a plant, for example for prevention or
treatment of a flea beetle infestation of a crop plant,
particularly commercially important Brassica species.
[0034] In embodiments, the at least one silencing element comprises
a sequence having about 95% to about 100% identity or
complementarity with a sequence selected from the group consisting
of SEQ ID NOs:860-1718 and 1722-1975 or a fragment thereof. In
embodiments, the at least one silencing element comprises 18 or
more contiguous nucleotides with a sequence of 100% complementarity
with a fragment of the target gene of the insect, wherein the
target gene has a DNA sequence selected from the group consisting
of SEQ ID NOs:1-859. In embodiments, the at least one silencing
element comprises at least one segment of 18 or more contiguous
nucleotides of a sequence selected from the group consisting of SEQ
ID NOs:860-1718 and 1722-1975 or a fragment thereof. In
embodiments, the at least one silencing element comprises a
sequence selected from the group consisting of SEQ ID NOs:860-1718
and 1722-1975 or a fragment thereof. In embodiments the at least
one silencing element comprises a sequence of about 95% to about
100% identity or complementarity with a sequence selected from the
group consisting of SEQ ID NOs:860-1718 and 1722-1975 or a fragment
thereof.
[0035] In embodiments, the insecticidal polynucleotide is provided
in a microbial or plant cell that expresses the insecticidal
polynucleotide, or in a microbial fermentation product, or is
chemically synthesized. In embodiments, the insecticidal
polynucleotide comprises dsRNA. In embodiments, the dsRNA is
blunt-ended, or has an overhang at at least one terminus, or
comprises at least one stem-loop. In embodiments, the dsRNA is
provided by convenient techniques commonly used. In embodiments,
the dsRNA is chemically synthesized, produced by expression in a
microorganism, produced by expression in a plant cell, or produced
by microbial fermentation. In embodiments, the dsRNA is made from
naturally occurring ribonucleotides; in other embodiments the dsRNA
is chemically modified.
[0036] In embodiments, the method is useful for causing mortality
or stunting in insects that are pests of commercially important
crop plants, such as an insect pest of a Brassica species. In
embodiments, the insect is a flea beetle. In embodiments, the
insect is a species of a genus selected from the group consisting
of the genera Altica, Anthobiodes, Aphthona, Aphthonaltica,
Aphthonoides, Apteopeda, Argopistes, Argopus, Arrhenocoela,
Batophila, Blepharida, Chaetocnema, Clitea, Crepidodera,
Derocrepis, Dibolia, Disonycha, Epitrix, Hermipyxis, Hermaeophaga,
Hespera, Hippuriphila, Horaia, Hyphasis, Lipromima, Liprus,
Longitarsus, Luperomorpha, Lythraria, Manobia, Mantura, Meishania,
Minota, Mniophila, Neicrepidodera, Nonarthra, Novofoudrasia,
Ochrosis, Oedionychis, Oglobinia, Omeisphaera, Ophrida, Orestia,
Paragopus, Pentamesa, Philopona, Phygasia, Phyllotreta, Podagrica,
Podagricomela, Podontia, Pseudodera, Psylliodes, Sangariola,
Sinaltica, Sphaeroderma, Systena, Trachyaphthona, Xuthea, and
Zipangia. In embodiments, the insect is a species selected from the
group consisting of Altica ambiens (alder flea beetle), Altica
canadensis (prairie flea beetle), Altica chalybaea (grape flea
beetle), Altica prasina (poplar flea beetle), Altica rosae (rose
flea beetle), Altica sylvia (blueberry flea beetle), Altica ulmi
(elm flea beetle), Chaetocnema pulicaria (corn flea beele),
Chaetocnema conofinis (sweet potato flea beetle), Epitrix cucumeris
(potato flea beetle), Systena blanda (palestripped fleabeetle), and
Systena frontalis (redheaded flea beetle). In embodiments, the
insect is a species selected from the group consisting of
Phyllotreta armoraciae (horseradish flea beetle), Phyllotreta
cruciferae (canola flea beetle), Phyllotreta pusilla (western black
flea beetle), Phyllotreta nemorum (striped turnip flea beetle),
Phyllotreta atra (turnip flea beetle), Phyllotreta robusta (garden
flea beetle), Phyllotreta striolata (striped flea beetle),
Phyllotreta undulata, Psylliodes chrysocephala, and Psylliodes
punctulata (hop flea beetle).
[0037] Embodiments of the method include those in which the
insecticidal polynucleotide is designed to silence a target gene in
a genus- or species-specific manner, for example, wherein (a) the
insect is a Phyllotreta species and the target gene has a DNA
sequence selected from the group consisting of SEQ ID NOs:1-551;
(b) the insect is Phyllotreta atra (turnip flea beetle) and the
target gene has a DNA sequence selected from the group consisting
of SEQ ID NOs:1-296; (c) the insect is Phyllotreta cruciferae
(canola flea beetle) and the target gene has a DNA sequence
selected from the group consisting of SEQ ID NOs:297-532; (d) the
insect is Phyllotreta striolata (striped flea beetle) and the
target gene has a DNA sequence selected from the group consisting
of SEQ ID NOs:533-551; (e) the insect is a Psylliodes species and
the target gene has a DNA sequence selected from the group
consisting of SEQ ID NOs:552-859; or (f) the insect is Psylliodes
chrysocephala and the target gene has a DNA sequence selected from
the group consisting of SEQ ID NOs:552-859. Embodiments of the
method also include those wherein (a) the insect is a Phyllotreta
species and the insecticidal polynucleotide comprises a sequence
selected from the group consisting of SEQ ID NOs:860-1410 or a
fragment thereof; (b) the insect is Phyllotreta atra (turnip flea
beetle) and the insecticidal polynucleotide comprises a sequence
selected from the group consisting of SEQ ID NOs:860-1155 or a
fragment thereof, (c) the insect is Phyllotreta cruciferae (canola
flea beetle) and insecticidal polynucleotide comprises a sequence
selected from the group consisting of SEQ ID NOs:1156-1391,
1731-1972, and 1974 or a fragment thereof, (d) the insect is
Phyllotreta striolata (striped flea beetle) and the insecticidal
polynucleotide comprises a sequence selected from the group
consisting of SEQ ID NOs:1392-1410, 1973, and 1975 or a fragment
thereof, (e) the insect is a Psylliodes species and the
insecticidal polynucleotide comprises a sequence selected from the
group consisting of SEQ ID NOs:1411-1718 or a fragment thereof, or
(f) the insect is Psylliodes chrysocephala and the insecticidal
polynucleotide comprises a sequence selected from the group
consisting of SEQ ID NOs:1411-1718 or a fragment thereof.
[0038] Embodiments of the method include those wherein at least one
insecticidal polynucleotide is provided in a composition comprising
the insecticidal polynucleotide, wherein the composition is applied
to a surface of the insect or to a surface of a seed or plant in
need of protection from infestation by the insect. Embodiments of
such compositions include those where the composition is a solid,
liquid, powder, suspension, emulsion, spray, encapsulation,
microbeads, carrier particulates, film, matrix, soil drench, or
seed treatment. In many embodiments, the composition is formulated
in a form that is ingested by the insect. In embodiments, the
composition further includes one or more components selected from
the group consisting of a carrier agent, a surfactant, an
organosilicone, a polynucleotide herbicidal molecule, a
non-polynucleotide herbicidal molecule, a non-polynucleotide
pesticide, a fungicide, a safener, a fertilizer, a micronutrient,
an insect attractant, and an insect growth regulator. In
embodiments, the composition further comprises at least one
pesticidal agent selected from the group consisting of a patatin, a
plant lectin, a phytoecdysteroid, a Bacillus thuringiensis
insecticidal protein, a Xenorhabdus insecticidal protein, a
Photorhabdus insecticidal protein, a Bacillus laterosporous
insecticidal protein, a Bacillus sphaericus insecticidal protein, a
bacterium that produces an insecticidal protein, an entomicidal
bacterial species, Lysinibacillus sphaericus (Bacillus sphaericus),
Brevibacillus laterosporus (Bacillus laterosporus), Chromobacterium
species, Chromobacterium subtsugae, Paenibacillus species,
Paenibacillus lentimorbus, and Paenibacillus popilliae.
[0039] Several embodiments relate to a method for controlling an
insect infestation of a plant comprising contacting the plant
and/or an insect that infests a plant with a insecticidal
polynucleotide, wherein the insecticidal polynucleotide comprises
at least one segment of 18 or more contiguous nucleotides with a
sequence of about 95% to about 100% (e. g., about 95%, about 96%,
about 97%, about 98%, about 99%, or about 100%) complementarity
with a fragment of a target gene of the insect selected from the
group consisting of actin, Act5C, arginine kinase, COPI (coatomer
subunit) alpha, COPI (coatomer subunit) beta, COPI (coatomer
subunit) betaPrime, COPI (coatomer subunit) delta, COPI (coatomer
subunit) epsilon, COPI (coatomer subunit) gamma, COPI (coatomer
subunit) zeta, RpL07, RpL19, RpL3, RpL40, RpL13, RpL14, RpS21,
RpS4, RpS14, Rpn2, Rpn3, Rpn7, Rpt6, Rpn8, Rpn9, Rpn6-PB-like
protein, Sar1, sec6, sec23, sec23A, shrb (snf7), Tubulin gamma
chain, ProsAlpha2, ProsBeta5, Proteasome alpha 2, Proteasome beta
5, VATPase E, VATPase A, VATPase B, VATPase D, Vps2, Vps4, Vps16A,
Vps20, Vps24, Vps27, Vps28, Vha26 (V-ATPase A), Vha68-2 (V-ATPase
DE), 40S ribosomal protein S14, and 60S ribosomal protein L13. In
some embodiments, the insecticidal polynucleotide comprises at
least one segment of 18 or more contiguous nucleotides with a
sequence of about 95% to about 100% complementarity with a fragment
of a DNA sequence selected from the group consisting of SEQ ID
NOs:1-859. In some embodiments, the insecticidal polynucleotide
comprises at least one segment of 21 contiguous nucleotides with a
sequence of 100% complementarity with a fragment of a DNA sequence
selected from the group consisting of SEQ ID NOs:1-859. In this
context "controlling" includes inducement of a physiological or
behavioural change in an insect (adult or larvae or nymphs) such
as, but not limited to, growth stunting, increased mortality,
decrease in reproductive capacity, decrease in or cessation of
feeding behavior or movement, or decrease in or cessation of
metamorphosis stage development. In some embodiments, the
insecticidal polynucleotide comprises a sequence selected from the
group consisting of SEQ ID NOs:860-1155 or a fragment thereof, or
the complement thereof.
[0040] In various embodiments, the insect is a flea beetle, e. g.,
a species of a genus selected from the group consisting of the
genera Altica, Anthobiodes, Aphthona, Aphthonaltica, Aphthonoides,
Apteopeda, Argopistes, Argopus, Arrhenocoela, Batophila,
Blepharida, Chaetocnema, Clitea, Crepidodera, Derocrepis, Dibolia,
Disonycha, Epitrix, Hermipyxis, Hermaeophaga, Hespera,
Hippuriphila, Horaia, Hyphasis, Lipromima, Liprus, Longitarsus,
Luperomorpha, Lythraria, Manobia, Mantura, Meishania, Minota,
Mniophila, Neicrepidodera, Nonarthra, Novofoudrasia, Ochrosis,
Oedionychis, Oglobinia, Omeisphaera, Ophrida, Orestia, Paragopus,
Pentamesa, Philopona, Phygasia, Phyllotreta, Podagrica,
Podagricomela, Podontia, Pseudodera, Psylliodes, Sangariola,
Sinaltica, Sphaeroderma, Systena, Trachyaphthona, Xuthea, and
Zipangia. In some embodiments, the insect is selected from the
group consisting of Altica ambiens (alder flea beetle), Altica
canadensis (prairie flea beetle), Altica chalybaea (grape flea
beetle), Altica prasina (poplar flea beetle), Altica rosae (rose
flea beetle), Altica sylvia (blueberry flea beetle), Altica ulmi
(elm flea beetle), Chaetocnema pulicaria (corn flea beele),
Chaetocnema conofinis (sweet potato flea beetle), Epitrix cucumeris
(potato flea beetle), Systena blanda (palestripped fleabeetle), and
Systena frontalis (redheaded flea beetle). In some embodiments, the
insect is selected from the group consisting of Phyllotreta
armoraciae (horseradish flea beetle), Phyllotreta cruciferae
(canola flea beetle), Phyllotreta pusilla (western black flea
beetle), Phyllotreta nemorum (striped turnip flea beetle),
Phyllotreta atra (turnip flea beetle), Phyllotreta robusta (garden
flea beetle), Phyllotreta striolata (striped flea beetle),
Phyllotreta undulata, Psylliodes chrysocephala, and Psylliodes
punctulata (hop flea beetle).
[0041] The plant can be any plant that is subject to infestation by
an insect that can be controlled by the insecticidal
polynucleotides disclosed herein. Plants of particular interest
include commercially important plants, including row crop plants,
vegetables, and fruits, and other plants of agricultural or
decorative use. Examples of suitable plants are provided under the
heading "Plants". The method is especially useful for controlling
an insect infestation of an ornamental plant or a crop plant.
Various embodiments of the method include those wherein the plant
is a plant in the family Brassicaceae, including a Brassica species
selected from the group consisting of B. napus, B. juncea, B.
carinata, B. rapa, B. oleracea, B. rupestris, B. septiceps, B.
nigra, B. narinosa, B. perviridus, B. tournefortii, and B.
fructiculosa. In other embodiments, the plant is selected from the
group consisting of Glycine max, Linum usitatissimum, Zea mays,
Carthamus tinctorius, Helianthus annuus, Nicotiana tabacum,
Arabidopsis thaliana, Betholettia excelsa, Ricinus communis, Cocus
nucifera, Coriandrum sativum, Gossypium spp., Arachis hypogaea,
Simmondsia chinensis, Solanum tuberosum, Elaeis guineensis, Olea
europaea, Oryza sativa, Cucurbita maxim, Hordeum vulgare, and
Triticum aestivum.
[0042] Methods include those developed for specific flea beetle
pests for a given plant, e. g., wherein the plant is a potato plant
and the insect is Epitrix cucumeris (potato flea beetle). In some
embodiments, specific target genes are identified as targets for
RNAi-mediated control in a given insect species. Various
embodiments of the method include those wherein (a) the insect is
Phyllotreta atra (turnip flea beetle) and the target gene has a DNA
sequence selected from the group consisting of SEQ ID NOs:1-296;
(b) the insect is Phyllotreta cruciferae (canola flea beetle) and
the target gene has a DNA sequence selected from the group
consisting of SEQ ID NOs:297-532; (c) the insect is Phyllotreta
striolata (striped flea beetle) and the target gene has a DNA
sequence selected from the group consisting of SEQ ID NOs:533-551;
or (d) the insect is Psylliodes chrysocephala and the target gene
has a DNA sequence selected from the group consisting of SEQ ID
NOs:552-859.
[0043] In some embodiments, specific insecticidal polynucleotides
are developed for specific target genes in a given insect species.
Embodiments of the method include those wherein (a) the insect is
Phyllotreta atra (turnip flea beetle) and the insecticidal
polynucleotide comprises at least one sequence selected from the
group consisting of SEQ ID NOs:860-1155 or a fragment thereof, (b)
the insect is Phyllotreta cruciferae (canola flea beetle) and the
insecticidal polynucleotide comprises at least one sequence
selected from the group consisting of SEQ ID NOs:1156-1391,
1731-1972, and 1974 or a fragment thereof; (c) the insect is
Phyllotreta striolata (striped flea beetle) and the insecticidal
polynucleotide comprises at least one sequence selected from the
group consisting of SEQ ID NOs:1392-1410, 1973, and 1975 or a
fragment thereof, or (d) the insect is Psylliodes chrysocephala and
the insecticidal polynucleotide comprises at least one sequence
selected from the group consisting of SEQ ID NOs:1411-1718 or a
fragment thereof.
[0044] The method includes contacting an insect, such as a flea
beetle, with an insecticidal polynucleotide. Embodiments include
contacting via oral delivery to the insect, or non-oral delivery to
the insect, or a combination of oral and non-oral delivery to the
insect. Embodiments include contacting insects in the adult stage,
or in larval stages, or in the egg stage. In some embodiments,
contacting results in mortality (death) or stunting (growth
stunting or decrease in or cessation of metamorphosis stage
development) of the insect, thereby preventing or treating
infestation of the plant by the insect. In some embodiments,
contacting results in inducement of a physiological or behavioural
change in an insect (adult or larvae or nymphs) that results in a
decreased ability of the insect to infest or damage a plant, for
example, a decrease in reproductive capacity, or a decrease in or
cessation of feeding behavior or movement.
[0045] In some embodiments of the method, the contacting comprises
application of a composition comprising one or more insecticidal
polynucleotide to a surface of the insect or to a surface of the
plant infested by the insect. The composition can comprise or be in
the form of a solid, liquid, powder, suspension, emulsion, spray,
encapsulation, microbeads, carrier particulates, film, matrix, soil
drench, or seed treatment. In some embodiments, the contacting
comprises providing the insecticidal polynucleotide in a
composition that further comprises one or more components selected
from the group consisting of a carrier agent, a surfactant, an
organosilicone, a polynucleotide herbicidal molecule, a
non-polynucleotide herbicidal molecule, a non-polynucleotide
pesticide, a fungicide, a safener, an insect attractant, and an
insect growth regulator. In some embodiments, the contacting
comprises providing the one or more insecticidal polynucleotides in
a composition that further comprises at least one pesticidal agent
selected from the group consisting of a patatin, a plant lectin, a
phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a
Xenorhabdus insecticidal protein, a Photorhabdus insecticidal
protein, a Bacillus laterosporous insecticidal protein, a Bacillus
sphaericus insecticidal protein, a bacterium that produces an
insecticidal protein, an entomicidal bacterial species,
Lysinibacillus sphaericus (Bacillus sphaericus), Brevibacillus
laterosporus (Bacillus laterosporus), Chromobacterium species,
Chromobacterium subtsugae, Paenibacillus species, Paenibacillus
lentimorbus, and Paenibacillus popilliae.
[0046] In some embodiments of the method, the contacting comprises
providing one or more insecticidal polynucleotides in a composition
that is ingested by the insect, such as in a liquid, emulsion, or
powder applied to a plant on which the insect feeds, or in the form
of bait. Such compositions can further comprise one or more
components selected from the group consisting of a carrier agent, a
surfactant, an organosilicone, a polynucleotide herbicidal
molecule, a non-polynucleotide herbicidal molecule, a
non-polynucleotide pesticide, a fungicide, a safener, an insect
attractant, and an insect growth regulator. Such compositions can
further comprise at least one pesticidal agent selected from the
group consisting of a patatin, a plant lectin, a phytoecdysteroid,
a Bacillus thuringiensis insecticidal protein, a Xenorhabdus
insecticidal protein, a Photorhabdus insecticidal protein, a
Bacillus laterosporous insecticidal protein, a Bacillus sphaericus
insecticidal protein, a bacterium that produces an insecticidal
protein, an entomicidal bacterial species, Lysinibacillus
sphaericus (Bacillus sphaericus), Brevibacillus laterosporus
(Bacillus laterosporus), Chromobacterium species, Chromobacterium
subtsugae, Paenibacillus species, Paenibacillus lentimorbus, and
Paenibacillus popilliae. In embodiments, the combination of the
insecticidal polynucleotide and the non-polynucleotide pesticidal
agent provides a level of insect control that is greater than the
sum of the effects of the insecticidal polynucleotide and the
non-polynucleotide pesticidal agent components if tested
separately.
Insecticidal Compositions
[0047] Several embodiments relate to an insecticidal composition
comprising an insecticidally effective amount of an insecticidal
polynucleotide, such as a ssRNA, dsRNA, ssDNA, dsDNA or hybrid
RNA/DNA molecule, wherein the insecticidal polynucleotide comprises
at least 18 or more contiguous nucleotides with a sequence of about
95% to about 100% (e. g., about 95%, about 96%, about 97%, about
98%, about 99%, or about 100%) complementarity with a fragment of
an insect target gene selected from the group consisting of actin,
Act5C, arginine kinase, COPI (coatomer subunit) alpha, COPI
(coatomer subunit) beta, COPI (coatomer subunit) betaPrime, COPI
(coatomer subunit) delta, COPI (coatomer subunit) epsilon, COPI
(coatomer subunit) gamma, COPI (coatomer subunit) zeta, RpL07,
RpL19, RpL3, RpL40, RpL13, RpL14, RpS21, RpS4, RpS14, Rpn2, Rpn3,
Rpn7, Rpt6, Rpn8, Rpn9, Rpn6-PB-like protein, Sar1, sec6, sec23,
sec23A, shrb (snf7), Tubulin gamma chain, ProsAlpha2, ProsBeta5,
Proteasome alpha 2, Proteasome beta 5, VATPase E, VATPase A,
VATPase B, VATPase D, Vps2, Vps4, Vps16A, Vps20, Vps24, Vps27,
Vps28, Vha26 (V-ATPase A), Vha68-2 (V-ATPase DE), 40S ribosomal
protein S14, and 60S ribosomal protein L13. In some embodiments the
insecticidal polynucleotide comprises at least 18 or more
contiguous nucleotides having about 95% to about 100%
complementarity with a fragment of a DNA sequence selected from the
group consisting of SEQ ID NOs:1-859. In some embodiments the
insecticidal polynucleotide comprises at least 21 contiguous
nucleotides having 100% complementarity with a fragment of a DNA
sequence selected from the group consisting of SEQ ID NOs:1-859. In
embodiments, the insecticidal polynucleotide comprises a sequence
selected from the group consisting of SEQ ID NOs:860-1718 and
1722-1975 or a fragment thereof.
[0048] In various embodiments of the insecticidal composition, the
insect is a flea beetle, e. g., a species of a genus selected from
the group consisting of the genera Altica, Anthobiodes, Aphthona,
Aphthonaltica, Aphthonoides, Apteopeda, Argopistes, Argopus,
Arrhenocoela, Batophila, Blepharida, Chaetocnema, Clitea,
Crepidodera, Derocrepis, Dibolia, Disonycha, Epitrix, Hermipyxis,
Hermaeophaga, Hespera, Hippuriphila, Horaia, Hyphasis, Lipromima,
Liprus, Longitarsus, Luperomorpha, Lythraria, Manobia, Mantura,
Meishania, Minota, Mniophila, Neicrepidodera, Nonarthra,
Novofoudrasia, Ochrosis, Oedionychis, Oglobinia, Omeisphaera,
Ophrida, Orestia, Paragopus, Pentamesa, Philopona, Phygasia,
Phyllotreta, Podagrica, Podagricomela, Podontia, Pseudodera,
Psylliodes, Sangariola, Sinaltica, Sphaeroderma, Systena,
Trachyaphthona, Xuthea, and Zipangia. In some embodiments, the
insect is selected from the group consisting of Altica ambiens
(alder flea beetle), Altica canadensis (prairie flea beetle),
Altica chalybaea (grape flea beetle), Altica prasina (poplar flea
beetle), Altica rosae (rose flea beetle), Altica sylvia (blueberry
flea beetle), Altica ulmi (elm flea beetle), Chaetocnema pulicaria
(corn flea beele), Chaetocnema conofinis (sweet potato flea
beetle), Epitrix cucumeris (potato flea beetle), Systena blanda
(palestripped fleabeetle), and Systena frontalis (redheaded flea
beetle). In some embodiments, the insect is selected from the group
consisting of Phyllotreta armoraciae (horseradish flea beetle),
Phyllotreta cruciferae (canola flea beetle), Phyllotreta pusilla
(western black flea beetle), Phyllotreta nemorum (striped turnip
flea beetle), Phyllotreta atra (turnip flea beetle), Phyllotreta
robusta (garden flea beetle), Phyllotreta striolata (striped flea
beetle), Phyllotreta undulata, Psylliodes chrysocephala, and
Psylliodes punctulata (hop flea beetle).
[0049] The insecticidal composition is useful for treating a plant
or area in the vicinity of a plant to provide protection or
treatment from insects, especially flea beetles. A related aspect
is a plant treated with an insecticidal composition as described
herein, or a seed of the treated plant, wherein the plant exhibits
improved resistance to the insect (e. g., improved resistance to
flea beetles). In some embodiments, the plant exhibiting improved
resistance to the insect is characterized by improved yield, when
compared to a plant not treated with the insecticidal composition.
In an embodiment, yield (oilseed biomass or oil content) in canola
or oilseed rape plants is improved by application of an
insecticidally effective amount of a insecticidal polynucleotide
targetting one or more genes identified from Phyllotreta cruciferae
(canola flea beetle); in particular embodiments, the target gene
has a DNA sequence selected from the group consisting of SEQ ID
NOs:297-532. The plant can be any plant that is subject to
infestation by an insect that can be controlled by the insecticidal
composition. Plants of particular interest include commercially
important plants, including row crop plants, vegetables, and
fruits, and other plants of agricultural or decorative use.
Examples of suitable plants are provided under the heading
"Plants". The method is especially useful for controlling an insect
infestation of an ornamental plant or a crop plant. Various
embodiments include those wherein the plant is a plant in the
family Brassicaceae, including a Brassica species selected from the
group consisting of B. napus, B. juncea, B. carinata, B. rapa, B.
oleracea, B. rupestris, B. septiceps, B. nigra, B. narinosa, B.
perviridus, B. tournefortii, and B. fructiculosa. In other
embodiments, the plant is selected from the group consisting of
Glycine max, Linum usitatissimum, Zea mays, Carthamus tinctorius,
Helianthus annuus, Nicotiana tabacum, Arabidopsis thaliana,
Betholettia excelsa, Ricinus communis, Cocus nucifera, Coriandrum
sativum, Gossypium spp., Arachis hypogaea, Simmondsia chinensis,
Solanum tuberosum, Elaeis guineensis, Olea europaea, Oryza sativa,
Cucurbita maxim, Hordeum vulgare, and Triticum aestivum.
[0050] In some embodiments, the insecticidal composition is
developed for specific flea beetle pests for a given plant, e. g.,
where the plant is a potato plant and the insect is Epitrix
cucumeris (potato flea beetle). In some embodiments, the
insecticidal composition is developed for specific target genes in
a given insect species. Specific embodiments of the insecticidal
composition include those wherein (a) the insect is Phyllotreta
atra (turnip flea beetle) and the target gene has a DNA sequence
selected from the group consisting of SEQ ID NOs:1-296; (b) the
insect is Phyllotreta cruciferae (canola flea beetle) and the
target gene has a DNA sequence selected from the group consisting
of SEQ ID NOs:297-532; (c) the insect is Phyllotreta striolata
(striped flea beetle) and the target gene has a DNA sequence
selected from the group consisting of SEQ ID NOs:533-551; or (d)
the insect is Psylliodes chrysocephala and the target gene has a
DNA sequence selected from the group consisting of SEQ ID
NOs:552-859.
[0051] In some embodiments the insecticidal polynucleotide molecule
of use in this method is provided as an isolated insecticidal
polynucleotide molecule (not part of an expression construct, e.
g., lacking additional elements such as a promoter or terminator
sequences). Such insecticidal polynucleotide molecules can be
relatively short, such as single- or double-stranded RNA, DNA or
hybrid RNA/DNA molecules of between about 18 to about 300 or
between about 50 to about 500 nucleotides (for single-stranded
polynucleotides) or between about 18 to about 300 or between about
50 to about 500 base-pairs (for double-stranded polynucleotides).
In embodiments the polynucleotide is a dsRNA comprising a sequence
selected from the group consisting of SEQ ID NOs:860-1718 and
1722-1975 or a fragment thereof.
[0052] In some embodiments, the insecticidal composition is in a
form selected from the group consisting of a solid, liquid, powder,
suspension, emulsion, spray, encapsulation, microbeads, carrier
particulates, film, matrix, soil drench, insect diet or insect
bait, and seed treatment. In some embodiments, the insecticidal
composition is provided in a form that is ingested by the insect,
such as in a liquid, emulsion, or powder applied to a plant on
which the insect feeds, or in the form of bait. The insecticidal
compositions can further comprise one or more components selected
from the group consisting of a carrier agent, a surfactant, an
organosilicone, a polynucleotide herbicidal molecule, a
non-polynucleotide herbicidal molecule, a non-polynucleotide
pesticide, a fungicide, a safener, an insect attractant, and an
insect growth regulator. The insecticidal compositions can further
comprise at least one pesticidal agent selected from the group
consisting of a patatin, a plant lectin, a phytoecdysteroid, a
Bacillus thuringiensis insecticidal protein, a Xenorhabdus
insecticidal protein, a Photorhabdus insecticidal protein, a
Bacillus laterosporous insecticidal protein, a Bacillus sphaericus
insecticidal protein, a bacterium that produces an insecticidal
protein, an entomicidal bacterial species, Lysinibacillus
sphaericus (Bacillus sphaericus), Brevibacillus laterosporus
(Bacillus laterosporus), Chromobacterium species, Chromobacterium
subtsugae, Paenibacillus species, Paenibacillus lentimorbus, and
Paenibacillus popilliae. In some embodiments, the combination of
the insecticidal polynucleotide molecule and the non-polynucleotide
pesticidal agent provides a level of insect control that is greater
than the sum of the effects of the insecticidal polynucleotide
molecule and the non-polynucleotide pesticidal agent components if
tested separately.
[0053] Embodiments of the compositions optionally comprise the
appropriate stickers and wetters required for efficient foliar
coverage as well as UV protectants to protect insecticidal
polynucleotides, such as dsRNAs, from UV damage. Such additives are
commonly used in the bioinsecticide industry and are known to those
skilled in the art. Compositions for soil application can comprise
granular formulations that serve as bait for insect larvae.
Embodiments include a carrier agent, a surfactant, an
organosilicone, a polynucleotide herbicidal molecule, a
non-polynucleotide herbicidal molecule, a non-polynucleotide
pesticide, a fungicide, a safener, an insect attractant, and an
insect growth regulator.
[0054] Embodiments of compositions may comprise a "transfer agent",
an agent that, when combined with a composition comprising an
insecticidal polynucleotide as disclosed herein that is topically
applied to the surface of an organism, enables the polynucleotide
to enter the cells of that organism. Such transfer agents can be
incorporated as part of the composition comprising a insecticidal
polynucleotide as disclosed herein, or can be applied prior to,
contemporaneously with, or following application of the composition
comprising an insecticidal polynucleotide as described herein. In
some embodiments, a transfer agent is an agent that improves the
uptake of an insecticidal polynucleotide by an insect. In some
embodiments, a transfer agent is an agent that conditions the
surface of plant tissue, e. g., seeds, leaves, stems, roots,
flowers, or fruits, to permeation by an insecticidal polynucleotide
into plant cells. In some embodiments, the transfer agent enables a
pathway for a polynucleotide through cuticle wax barriers, stomata,
and/or cell wall or membrane barriers into plant cells.
[0055] Suitable transfer agents include agents that increase
permeability of the exterior of the organism or that increase
permeability of cells of the organism to polynucleotides. Suitable
transfer agents include a chemical agent, or a physical agent, or
combinations thereof. Chemical agents for conditioning or transfer
include (a) surfactants, (b) an organic solvent or an aqueous
solution or aqueous mixtures of organic solvents, (c) oxidizing
agents, (d) acids, (e) bases, (f) oils, (g) enzymes, or
combinations thereof. In some embodiments, application of an
insecticidal polynucleotide and a transfer agent optionally
includes one or more of an incubation step, a neutralization step
(e. g., to neutralize an acid, base, or oxidizing agent, or to
inactivate an enzyme), a rinsing step, or combinations thereof.
Suitable transfer agents can be in the form of an emulsion, a
reverse emulsion, a liposome, or other micellar-like composition,
or can cause the polynucleotide composition to take the form of an
emulsion, a reverse emulsion, a liposome, or other micellar-like
composition. Embodiments of transfer agents include counter-ions or
other molecules that are known to associate with nucleic acid
molecules, e. g., cationic lipids, inorganic ammonium ions, alkyl
ammonium ions, lithium ions, polyamines such as spermine,
spermidine, or putrescine, and other cations. Embodiments of
transfer agents include organic solvents such as DMSO, DMF,
pyridine, N-pyrrolidine, hexamethylphosphoramide, acetonitrile,
dioxane, polypropylene glycol, or other solvents miscible with
water or that dissolve phosphonucleotides in non-aqueous systems
(such as is used in synthetic reactions). Embodiments of transfer
agents include naturally derived or synthetic oils with or without
surfactants or emulsifiers, e. g., plant-sourced oils, crop oils
(such as those listed in the 9.sup.th Compendium of Herbicide
Adjuvants, publicly available on-line at herbicide.adjuvants.com),
paraffinic oils, polyol fatty acid esters, or oils with short-chain
molecules modified with amides or polyamines such as
polyethyleneimine or N-pyrrolidine.
[0056] Embodiments of transfer agents include organosilicone
preparations. For example, a suitable transfer agent is an
organosilicone preparation that is commercially available as SILWET
L-77.RTM. brand surfactant having CAS Number 27306-78-1 and EPA
Number: CAL.REG.NO. 5905-50073-AA, and currently available from
Momentive Performance Materials, Albany, N.Y. In embodiments where
a SILWET L-77.RTM. brand surfactant organosilicone preparation is
used as transfer agent in the form of a spray treatment (applied
prior to, contemporaneously with, or following application of the
composition comprising an insecticidal polynucleotide as disclosed
herein) of plant leaves or other plant surfaces, freshly made
concentrations in the range of about 0.015 to about 2 percent by
weight (wt percent) (e. g., about 0.01, 0.015, 0.02, 0.025, 0.03,
0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08,
0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,
1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2,
2.3, 2.5 wt percent) are efficacious in preparing a leaf or other
plant surface for transfer of a polynucleotide as disclosed herein
into plant cells from a topical application on the surface. One
embodiment includes a composition that comprises a polynucleotide
and a transfer agent including an organosilicone preparation such
as Silwet L-77 in the range of about 0.015 to about 2 percent by
weight (wt percent) (e. g., about 0.01, 0.015, 0.02, 0.025, 0.03,
0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08,
0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,
1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2,
2.3, 2.5 wt percent). One embodiment includes a composition that
comprises a polynucleotide and a transfer agent including SILWET
L-77.RTM. brand surfactant in the range of about 0.3 to about 1
percent by weight (wt percent) or about 0.5 to about 1%, by weight
(wt percent).
[0057] Organosilicone compounds useful as transfer agents for use
in compositions and methods disclosed herein include, but are not
limited to, compounds that include: (a) a trisiloxane head group
that is covalently linked to, (b) an alkyl linker including, but
not limited to, an n-propyl linker, that is covalently linked to,
(c) a polyglycol chain, that is covalently linked to, (d) a
terminal group. Trisiloxane head groups of such organosilicone
compounds include, but are not limited to, heptamethyltrisiloxane.
Alkyl linkers can include, but are not limited to, an n-propyl
linker. Polyglycol chains include, but are not limited to,
polyethylene glycol or polypropylene glycol. Polyglycol chains can
include a mixture that provides an average chain length "n" of
about "7.5". In certain embodiments, the average chain length "n"
can vary from about 5 to about 14. Terminal groups can include, but
are not limited to, alkyl groups such as a methyl group.
Organosilicone compounds useful as transfer agents for use in
compositions and methods disclosed herein include, but are not
limited to, trisiloxane ethoxylate surfactants or polyalkylene
oxide modified heptamethyl trisiloxane. An example of a transfer
agent for use in compositions and methods disclosed herein is
Compound I:
##STR00001##
[0058] (Compound I: polyalkyleneoxide heptamethyltrisiloxane,
average n=7.5).
[0059] Organosilicone compounds useful as transfer agents for use
in compositions and methods disclosed herein are used, e. g., as
freshly made concentrations in the range of about 0.015 to about 2
percent by weight (wt percent) (e. g., about 0.01, 0.015, 0.02,
0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07,
0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,
0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0,
2.1, 2.2, 2.3, 2.5 wt percent).
[0060] Embodiments of transfer agents include one or more salts
such as ammonium chloride, tetrabutylphosphonium bromide, and
ammonium sulfate, provided in or used with a composition comprising
an insecticidal polynucleotide disclosed herein. In some
embodiments, ammonium chloride, tetrabutylphosphonium bromide,
and/or ammonium sulfate are used at a concentration of about 0.5%
to about 5% (w/v), or about 1% to about 3% (w/v), or about 2%
(w/v). In certain embodiments, the composition comprising an
insecticidal polynucleotide comprises an ammonium salt at a
concentration greater or equal to 300 millimolar. In certain
embodiments, the composition comprising an insecticidal
polynucleotide comprises an organosilicone transfer agent in a
concentration of about 0.015 to about 2 percent by weight (wt
percent) as well as ammonium sulfate at concentrations from about
80 to about 1200 millimolar or about 150 millimolar to about 600
millimolar.
[0061] Embodiments of transfer agents include a phosphate salt.
Phosphate salts useful in a composition comprising an insecticidal
polynucleotide include, but are not limited to, calcium, magnesium,
potassium, or sodium phosphate salts. In certain embodiments, the
composition comprising an insecticidal polynucleotide includes a
phosphate salt at a concentration of at least about 5 millimolar,
at least about 10 millimolar, or at least about 20 millimolar. In
certain embodiments, the composition comprising an insecticidal
polynucleotide includes a phosphate salt in a range of about 1
millimolar to about 25 millimolar or in a range of about 5
millimolar to about 25 millimolar. In certain embodiments, the
composition comprising an insecticidal polynucleotide includes
sodium phosphate at a concentration of at least about 5 millimolar,
at least about 10 millimolar, or at least about 20 millimolar. In
certain embodiments, the composition comprising an insecticidal
polynucleotide includes sodium phosphate at a concentration of
about 5 millimolar, about 10 millimolar, or about 20 millimolar. In
certain embodiments, the composition comprising an insecticidal
polynucleotide includes a sodium phosphate salt in a range of about
1 millimolar to about 25 millimolar or in a range of about 5
millimolar to about 25 millimolar. In certain embodiments, the
composition comprising an insecticidal polynucleotide includes a
sodium phosphate salt in a range of about 10 millimolar to about
160 millimolar or in a range of about 20 millimolar to about 40
millimolar. In certain embodiments, the composition comprising an
insecticidal polynucleotide includes a sodium phosphate buffer at a
pH of about 6.8.
[0062] Embodiments of transfer agents include surfactants and/or
effective molecules contained therein. Surfactants and/or effective
molecules contained therein include, but are not limited to, sodium
or lithium salts of fatty acids (such as tallow or tallowamines or
phospholipids) and organosilicone surfactants. In certain
embodiments, the composition comprising an insecticidal
polynucleotide is formulated with counter-ions or other molecules
that are known to associate with nucleic acid molecules.
Non-limiting examples include, tetraalkyl ammonium ions, trialkyl
ammonium ions, sulfonium ions, lithium ions, and polyamines such as
spermine, spermidine, or putrescine. In certain embodiments, the
composition comprising an insecticidal polynucleotide is formulated
with a non-polynucleotide herbicide e. g., glyphosate, auxin-like
benzoic acid herbicides including dicamba, chloramben, and TBA,
glufosinate, auxin-like herbicides including phenoxy carboxylic
acid herbicide, pyridine carboxylic acid herbicide, quinoline
carboxylic acid herbicide, pyrimidine carboxylic acid herbicide,
and benazolin-ethyl herbicide, sulfonylureas, imidazolinones,
bromoxynil, delapon, cyclohezanedione, protoporphyrinogen oxidase
inhibitors, and 4-hydroxyphenyl-pyruvate-dioxygenase inhibiting
herbicides. In certain embodiments, the composition comprising an
insecticidal polynucleotide is formulated with a non-polynucleotide
pesticide, e. g., a patatin, a plant lectin, a phytoecdysteroid, a
Bacillus thuringiensis insecticidal protein, a Xenorhabdus
insecticidal protein, a Photorhabdus insecticidal protein, a
Bacillus laterosporous insecticidal protein, and a Bacillus
sphaericus insecticidal protein.
Methods of Providing Plants with Improved Insect Resistance
[0063] Several embodiments relate to a method of providing a plant
having improved resistance to an insect, comprising expressing in
the plant a recombinant DNA construct, wherein the recombinant DNA
construct comprises DNA encoding an insecticidal polynucleotide
comprising a sequence of at least 21 contiguous nucleotides that
are essentially identical or essentially complementary to a
fragment of at least one insect target gene selected from the group
consisting of actin, Act5C, arginine kinase, COPI (coatomer
subunit) alpha, COPI (coatomer subunit) beta, COPI (coatomer
subunit) betaPrime, COPI (coatomer subunit) delta, COPI (coatomer
subunit) epsilon, COPI (coatomer subunit) gamma, COPI (coatomer
subunit) zeta, RpL07, RpL19, RpL3, RpL40, RpL13, RpL14, RpS21,
RpS4, RpS14, Rpn2, Rpn3, Rpn7, Rpt6, Rpn8, Rpn9, Rpn6-PB-like
protein, Sar1, sec6, sec23, sec23A, shrb (snf7), Tubulin gamma
chain, ProsAlpha2, ProsBeta5, Proteasome alpha 2, Proteasome beta
5, VATPase E, VATPase A, VATPase B, VATPase D, Vps2, Vps4, Vps16A,
Vps20, Vps24, Vps27, Vps28, Vha26 (V-ATPase A), Vha68-2 (V-ATPase
DE), 40S ribosomal protein S14, and 60S ribosomal protein L13. In
some embodiments, the DNA construct comprises DNA encoding an
insecticidal polynucleotide comprising a sequence essentially
identical or essentially complementary to a fragment of a DNA
sequence selected from the group consisting of SEQ ID NOs:1-859.
Several embodiments relate to a plant produced by such method. In
some embodiments, the DNA construct comprises DNA encoding an
insecticidal polynucleotide having about 95% to about 100% (e. g.,
about 95%, about 96%, about 97%, about 98%, about 99%, or about
100%) identity or complementarity with at least 21 contiguous
nucleotides of a sequence selected from the group consisting of SEQ
ID NOs:860-1718 and 1722-1975 or a fragment thereof.
[0064] In some embodiments, the recombinant DNA construct further
comprises a heterologous promoter operably linked to the DNA
encoding an insecticidal polynucleotide, wherein the heterologous
promoter is functional in a plant cell. "Heterologous" refers to
nucleic acid sequences that are not usually operably linked in a
native or naturally occurring genome; by "heterologous promoter" is
meant that the promoter is not natively operably linked with the
DNA encoding an insecticidal polynucleotide. Promoters functional
in a plant cell include those listed under the heading
"Promoters".
[0065] In some embodiments, the recombinant DNA construct is
expressed in the plant by means of transgenic expression or
transient expression. In some embodiments, the method further
comprises expression in the plant of at least one pesticidal agent
selected from the group consisting of a patatin, a plant lectin, a
phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a
Xenorhabdus insecticidal protein, a Photorhabdus insecticidal
protein, a Bacillus laterosporous insecticidal protein, a Bacillus
sphaericus insecticidal protein, a bacterium that produces an
insecticidal protein, an entomicidal bacterial species,
Lysinibacillus sphaericus (Bacillus sphaericus), Brevibacillus
laterosporus (Bacillus laterosporus), Chromobacterium species,
Chromobacterium subtsugae, Paenibacillus species, Paenibacillus
lentimorbus, and Paenibacillus popilliae. The pesticidal agent can
be expressed from the same recombinant DNA construct that comprises
the DNA encoding an insecticidal polynucleotide, or from a
different recombinant DNA construct.
[0066] A related aspect is a plant having improved resistance to an
insect (e. g., improved resistance to flea beetles), or the seed of
such a plant, wherein the plant is provided by the method
comprising expressing in the plant a recombinant DNA construct,
wherein the recombinant DNA construct comprises DNA encoding an
insecticidal polynucleotide having a sequence essentially identical
or essentially complementary to a fragment of at least one target
gene of the insect, wherein the target gene is selected from the
group consisting of actin, Act5C, arginine kinase, COPI (coatomer
subunit) alpha, COPI (coatomer subunit) beta, COPI (coatomer
subunit) betaPrime, COPI (coatomer subunit) delta, COPI (coatomer
subunit) epsilon, COPI (coatomer subunit) gamma, COPI (coatomer
subunit) zeta, RpL07, RpL19, RpL3, RpL40, RpL13, RpL14, RpS21,
RpS4, RpS14, Rpn2, Rpn3, Rpn7, Rpt6, Rpn8, Rpn9, Rpn6-PB-like
protein, Sar1, sec6, sec23, sec23A, shrb (snf7), Tubulin gamma
chain, ProsAlpha2, ProsBeta5, Proteasome alpha 2, Proteasome beta
5, VATPase E, VATPase A, VATPase B, VATPase D, Vps2, Vps4, Vps16A,
Vps20, Vps24, Vps27, Vps28, Vha26 (V-ATPase A), Vha68-2 (V-ATPase
D/E), 40S ribosomal protein S14, and 60S ribosomal protein L13. In
some embodiments, the recombinant DNA construct comprises DNA
encoding an insecticidal polynucleotide comprising a sequence
essentially identical or essentially complementary to a fragment of
a DNA sequence selected from the group consisting of SEQ ID
NOs:1-859. In some embodiments, the plant exhibiting improved
resistance to the insect is characterized by improved yield, when
compared to a plant not expressing the recombinant DNA construct.
In an embodiment, yield (oilseed biomass or oil content) in canola
or oilseed rape plants is improved by expressing in the canola or
oilseed rape plants an insecticidal polynucleotide, such as a dsRNA
molecule, targetting one or more genes of Phyllotreta cruciferae
(canola flea beetle), e. g., wherein the target gene has a DNA
sequence selected from the group consisting of SEQ ID NOs:297-532.
Several embodiments relate to fruit, seed, or propagatable parts of
the plant provided by this method and exhibiting improved
resistance to the insect. The plant can be any plant that is
subject to infestation by an insect that can be controlled by
expressing in the plant the recombinant DNA construct according to
this method. Plants of particular interest include commercially
important plants, including row crop plants, vegetables, and
fruits, and other plants of agricultural or decorative use.
Examples of suitable plants are provided under the heading
"Plants". The method is especially useful for providing an
ornamental plant or a crop plant with improved resistance to flea
beetles. Various embodiments of the method include those wherein
the plant is a plant in the family Brassicaceae, including a
Brassica species selected from the group consisting of B. napus, B.
juncea, B. carinata, B. rapa, B. oleracea, B. rupestris, B.
septiceps, B. nigra, B. narinosa, B. perviridus, B. tournefortii,
and B. fructiculosa. In other embodiments, the plant is selected
from the group consisting of Glycine max, Linum usitatissimum, Zea
mays, Carthamus tinctorius, Helianthus annuus, Nicotiana tabacum,
Arabidopsis thaliana, Betholettia excelsa, Ricinus communis, Cocus
nucifera, Coriandrum sativum, Gossypium spp., Arachis hypogaea,
Simmondsia chinensis, Solanum tuberosum, Elaeis guineensis, Olea
europaea, Oryza sativa, Cucurbita maxim, Hordeum vulgare, and
Triticum aestivum. In an embodiment, the method provides a potato
plant with improved resistance to Epitrix cucumeris (potato flea
beetle).
[0067] Embodiments of the method provide a plant having improved
resistance to one or more flea beetle species, e. g., a species of
a genus selected from the group consisting of the genera Altica,
Anthobiodes, Aphthona, Aphthonaltica, Aphthonoides, Apteopeda,
Argopistes, Argopus, Arrhenocoela, Batophila, Blepharida,
Chaetocnema, Clitea, Crepidodera, Derocrepis, Dibolia, Disonycha,
Epitrix, Hermipyxis, Hermaeophaga, Hespera, Hippuriphila, Horaia,
Hyphasis, Lipromima, Liprus, Longitarsus, Luperomorpha, Lythraria,
Manobia, Mantura, Meishania, Minota, Mniophila, Neicrepidodera,
Nonarthra, Novofoudrasia, Ochrosis, Oedionychis, Oglobinia,
Omeisphaera, Ophrida, Orestia, Paragopus, Pentamesa, Philopona,
Phygasia, Phyllotreta, Podagrica, Podagricomela, Podontia,
Pseudodera, Psylliodes, Sangariola, Sinaltica, Sphaeroderma,
Systena, Trachyaphthona, Xuthea, and Zipangia. In embodiments, the
insect is selected from the group consisting of Altica ambiens
(alder flea beetle), Altica canadensis (prairie flea beetle),
Altica chalybaea (grape flea beetle), Altica prasina (poplar flea
beetle), Altica rosae (rose flea beetle), Altica sylvia (blueberry
flea beetle), Altica ulmi (elm flea beetle), Chaetocnema pulicaria
(corn flea beele), Chaetocnema conofinis (sweet potato flea
beetle), Epitrix cucumeris (potato flea beetle), Systena blanda
(palestripped fleabeetle), and Systena frontalis (redheaded flea
beetle). In embodiments, the method provides a plant having
improved resistance to an insect selected from the group consisting
of Phyllotreta armoraciae (horseradish flea beetle), Phyllotreta
cruciferae (canola flea beetle), Phyllotreta pusilla (western black
flea beetle), Phyllotreta nemorum (striped turnip flea beetle),
Phyllotreta atra (turnip flea beetle), Phyllotreta robusta (garden
flea beetle), Phyllotreta striolata (striped flea beetle),
Phyllotreta undulata, Psylliodes chrysocephala, and Psylliodes
punctulata (hop flea beetle).
[0068] In some embodiments, the method is developed for specific
target genes in a given insect species. In some embodiments, a
plant having improved resistance to Phyllotreta atra (turnip flea
beetle), wherein the target gene has a DNA sequence selected from
the group consisting of SEQ ID NOs:1-296, is provided. In some
embodiments, a plant having improved resistance to Phyllotreta
cruciferae (canola flea beetle), wherein the target gene has a DNA
sequence selected from the group consisting of SEQ ID NOs:297-532,
is provided. In some embodiments, a plant having improved
resistance to Phyllotreta striolata (striped flea beetle), wherein
the target gene has a DNA sequence selected from the group
consisting of SEQ ID NOs:533-551, is provided. In some embodiments,
a plant having improved resistance to Psylliodes chrysocephala,
wherein the target gene has a DNA sequence selected from the group
consisting of SEQ ID NOs:552-859, is provided. In some embodiments,
a plant having improved resistance to Phyllotreta atra (turnip flea
beetle) that expresses from a recombinant DNA construct a dsRNA
strand comprising a sequence of about 95% to about 100% identity
with a sequence selected from the group consisting of SEQ ID
NOs:860-1155 or a fragment thereof is provided. In some
embodiments, a plant having improved resistance to Phyllotreta
cruciferae (canola flea beetle) that expresses from a recombinant
DNA construct an insecticidal polynucleotide comprising a sequence
of about 95% to about 100% identity with a sequence selected from
the group consisting of SEQ ID NOs:1156-1391, 1731-1972, and 1974
or a fragment thereof is provided. In some embodiments, a plant
having improved resistance to Phyllotreta striolata (striped flea
beetle) that expresses from a recombinant DNA construct an
insecticidal polynucleotide comprising a strand comprising a
sequence of about 95% to about 100% identity with a sequence
selected from the group consisting of SEQ ID NOs:1392-1410, 1973,
and 1975 or a fragment thereof is provided. In some embodiments, a
plant having improved resistance to Psylliodes chrysocephala that
expresses from a recombinant DNA construct an insecticidal
polynucleotide comprising a strand comprising a sequence of about
95% to about 100% identity with a sequence selected from the group
consisting of SEQ ID NOs:1411-1718 or a fragment thereof is
provided.
Recombinant DNA Constructs Encoding Insecticidal Polynucleotides
for Insect Control
[0069] Several embodiments relate to a recombinant DNA construct
comprising a heterologous promoter operably linked to DNA encoding
an insecticidal polynucleotide transcript comprising a sequence of
about 95% to about 100% (e. g., about 95%, about 96%, about 97%,
about 98%, about 99%, or about 100%) identity or complementarity to
at least 21 contiguous nucleotides of a sequence selected from the
group consisting of SEQ ID NOs:860-1718 and 1722-1975 or a fragment
thereof.
[0070] In some embodiments of the recombinant DNA construct, the
insecticidal polynucleotide transcript comprises a sequence of
about 95% to about 100% (e. g., about 95%, about 96%, about 97%,
about 98%, about 99%, or about 100%) identity or complementarity
with at least 21 contiguous nucleotides of a sequence selected from
the group consisting of SEQ ID NOs:860-1718 and 1722-1975 or a
fragment thereof. In some embodiments, the insecticidal
polynucleotide transcript forms dsRNA double-stranded insecticidal
polynucleotide. In some embodiments, the insecticidal
polynucleotide transcript is a dsRNA comprising an RNA strand
comprising at least one segment of 18 or more contiguous
nucleotides of an RNA sequence selected from the group consisting
of SEQ ID NOs:860-1718 and 1722-1975 or a fragment thereof. In some
embodiments, the insecticidal polynucleotide transcript is a dsRNA
comprising an RNA strand comprising at least one segment of 21
contiguous nucleotides of an RNA sequence selected from the group
consisting of SEQ ID NOs:860-1718 and 1722-1975 or a fragment
thereof. In some embodiments, the insecticidal polynucleotide
transcript is a dsRNA comprising at least one RNA strand comprising
a sequence of about 95% to about 100% identity or complementarity
with a sequence selected from the group consisting of SEQ ID
NOs:860-1718 and 1722-1975 or a fragment thereof. In some
embodiments, the insecticidal polynucleotide transcript is a dsRNA
comprising an RNA strand comprising a sequence selected from the
group consisting of SEQ ID NOs:860-1718 and 1722-1975 or a fragment
thereof. In some embodiments, the insecticidal polynucleotide
transcript forms single-stranded insecticidal polynucleotide. In
some embodiments, the insecticidal polynucleotide transcript is a
ssRNA comprising at least one segment of 18 or more contiguous
nucleotides of an RNA sequence selected from the group consisting
of SEQ ID NOs:860-1718 and 1722-1975 or a fragment thereof. In some
embodiments, the insecticidal polynucleotide transcript is a ssRNA
comprising at least one segment of 21 contiguous nucleotides of an
RNA sequence selected from the group consisting of SEQ ID
NOs:860-1718 and 1722-1975 or a fragment thereof. In some
embodiments, the insecticidal polynucleotide transcript is a ssRNA
comprising a sequence of about 95% to about 100% identity or
complementarity with a sequence selected from the group consisting
of SEQ ID NOs:860-1718 and 1722-1975 or a fragment thereof. In some
embodiments, the insecticidal polynucleotide transcript is a ssRNA
comprising a sequence selected from the group consisting of SEQ ID
NOs:860-1718 and 1722-1975 or a fragment thereof.
[0071] In some embodiments of the recombinant DNA construct, the
heterologous promoter is functional for expression of the
insecticidal polynucleotide transcript in a bacterium. In some
embodiments where the recombinant DNA construct is to be expressed
in a bacterium, the bacterium is selected from the group consisting
of Escherichia coli, Bacillus species, Pseudomonas species,
Xenorhabdus species, or Photorhabdus species. In other embodiments,
the recombinant DNA construct comprises a heterologous promoter
that is functional in a plant cell.
[0072] In some embodiments, the recombinant DNA construct is
contained in a recombinant vector, such as a recombinant plant
virus vector or a recombinant baculovirus vector. In embodiments,
the recombinant DNA construct is integrated into a plant chromosome
or plastid, e. g., by stable transformation.
[0073] Related aspects include a transgenic plant cell comprising
in its genome the recombinant DNA construct, and a transgenic plant
comprising such a transgenic plant cell. Transgenic plant cells and
plants are made by methods known in the art, such as those
described under the heading "Making and Using Transgenic Plant
Cells and Transgenic Plants". Further aspects include a commodity
product produced from such a transgenic plant, and transgenic
progeny seed or propagatable plant part of the transgenic
plant.
Related Information and Techniques
Plants
[0074] The methods and compositions described herein for treating
and protecting plants from insect infestations are useful across a
broad range of plants. Suitable plants in which the methods and
compositions disclosed herein can be used include, but are not
limited to, cereals and forage grasses (rice, maize, wheat, barley,
oat, sorghum, pearl millet, finger millet, cool-season forage
grasses, and bahiagrass), oilseed crops (soybean, oilseed brassicas
including canola and oilseed rape, sunflower, peanut, flax, sesame,
and safflower), legume grains and forages (common bean, cowpea,
pea, fava bean, lentil, tepary bean, Asiatic beans, pigeonpea,
vetch, chickpea, lupine, alfalfa, and clovers), temperate fruits
and nuts (apple, pear, peach, plums, berry crops, cherries, grapes,
olive, almond, and Persian walnut), tropical and subtropical fruits
and nuts (citrus including limes, oranges, and grapefruit; banana
and plantain, pineapple, papaya, mango, avocado, kiwifruit,
passionfruit, and persimmon), vegetable crops (solanaceous plants
including tomato, eggplant, and peppers; vegetable brassicas;
radish, carrot, cucurbits, alliums, asparagus, and leafy
vegetables), sugar, tuber, and fiber crops (sugarcane, sugar beet,
stevia, potato, sweet potato, cassava, and cotton), plantation
crops, ornamentals, and turf grasses (tobacco, coffee, cocoa, tea,
rubber tree, medicinal plants, ornamentals, and turf grasses), and
forest tree species. Specific plant species of interest are plants
in the family Brassicaceae, including the Brassica species B.
napus, B. juncea, B. carinata, B. rapa, B. oleracea, B. rupestris,
B. septiceps, B. nigra, B. narinosa, B. perviridus, B.
tournefortii, and B. fructiculosa. Additional plant species of
interest are Glycine max, Linum usitatissimum, Zea mays, Carthamus
tinctorius, Helianthus annuus, Nicotiana tabacum, Arabidopsis
thaliana, Betholettia excelsa, Ricinus communis, Cocos nucifera,
Coriandrum sativum, Gossypium spp., Arachis hypogaea, Simmondsia
chinensis, Solanum tuberosum, Elaeis guineensis, Olea europaea,
Oryza sativa, Cucurbita maxim, Hordeum vulgare, and Triticum
aestivum.
Additional Construct Elements
[0075] Embodiments of the polynucleotides and nucleic acid
molecules disclosed herein can comprise additional elements, such
as promoters, small RNA recognition sites, aptamers or ribozymes,
additional and additional expression cassettes for expressing
coding sequences (e. g., to express a transgene such as an
insecticidal protein or selectable marker) or non-coding sequences
(e. g., to express additional suppression elements). For example,
an aspect provides a recombinant DNA construct comprising a
heterologous promoter operably linked to DNA encoding an RNA
transcript comprising a sequence of about 95% to about 100%
identity or complementarity with a sequence selected from the group
consisting of SEQ ID NOs:860-1718 and 1722-1975 or a fragment
thereof. In another embodiment, a recombinant DNA construct
comprising a promoter operably linked to DNA encoding: (a) an RNA
transcript comprising a sequence of about 95% to about 100%
identity or complementarity with a sequence selected from the group
consisting of SEQ ID NOs:860-1718 and 1722-1975 or a fragment
thereof, and (b) an aptamer, is stably integrated into the plant's
genome from where RNA transcripts comprising the RNA aptamer and
the RNA silencing element are expressed in cells of the plant; the
aptamer serves to guide the RNA silencing element to a desired
location in the cell. In another embodiment, inclusion of one or
more recognition sites for binding and cleavage by a small RNA (e.
g., by a miRNA or an siRNA that is expressed only in a particular
cell or tissue) allows for more precise expression patterns in a
plant, wherein the expression of the recombinant DNA construct is
suppressed where the small RNA is expressed. Such additional
elements are described below.
Promoters
[0076] Promoters of use in the compositions and methods disclosed
herein are functional in the cell in which the construct is
intended to be transcribed. Generally these promoters are
heterologous promoters, as used in recombinant constructs, i. e.,
they are not in nature found to be operably linked to the other
nucleic elements used in the constructs. In various embodiments,
the promoter is selected from the group consisting of a
constitutive promoter, a spatially specific promoter, a temporally
specific promoter, a developmentally specific promoter, and an
inducible promoter. In many embodiments the promoter is a promoter
functional in a plant, for example, a pol II promoter, a pol III
promoter, a pol IV promoter, or a pol V promoter.
[0077] Non-constitutive promoters suitable for use with the
recombinant DNA constructs disclosed herein include spatially
specific promoters, temporally specific promoters, and inducible
promoters. Spatially specific promoters can include organelle-,
cell-, tissue-, or organ-specific promoters (e. g., a
plastid-specific, a root-specific, a pollen-specific, or a
seed-specific promoter for expression in plastids, roots, pollen,
or seeds, respectively). In many cases a seed-specific,
embryo-specific, aleurone-specific, or endosperm-specific promoter
is especially useful. Temporally specific promoters can include
promoters that tend to promote expression during certain
developmental stages in a plant's growth cycle, or during different
times of day or night, or at different seasons in a year. Inducible
promoters include promoters induced by chemicals or by
environmental conditions such as, but not limited to, biotic or
abiotic stress (e. g., water deficit or drought, heat, cold, high
or low nutrient or salt levels, high or low light levels, or pest
or pathogen infection). MicroRNA promoters are useful, especially
those having a temporally specific, spatially specific, or
inducible expression pattern; examples of miRNA promoters, as well
as methods for identifying miRNA promoters having specific
expression patterns, are provided in U. S. Patent Application
Publications 2006/0200878, 2007/0199095, and 2007/0300329, which
are specifically incorporated herein by reference. An
expression-specific promoter can also include promoters that are
generally constitutively expressed but at differing degrees or
"strengths" of expression, including promoters commonly regarded as
"strong promoters" or as "weak promoters".
[0078] Promoters include the following examples: an opaline
synthase promoter isolated from T-DNA of Agrobacterium; a
cauliflower mosaic virus 35S promoter; enhanced promoter elements
or chimeric promoter elements such as an enhanced cauliflower
mosaic virus (CaMV) 35S promoter linked to an enhancer element (an
intron from heat shock protein 70 of Zea mays); root specific
promoters such as those disclosed in U.S. Pat. Nos. 5,837,848;
6,437,217 and 6,426,446; a maize L3 oleosin promoter disclosed in
U.S. Pat. No. 6,433,252; a promoter for a plant nuclear gene
encoding a plastid-localized aldolase disclosed in U. S. Patent
Application Publication 2004/0216189; cold-inducible promoters
disclosed in U.S. Pat. No. 6,084,089; salt-inducible promoters
disclosed in U.S. Pat. No. 6,140,078; light-inducible promoters
disclosed in U.S. Pat. No. 6,294,714; pathogen-inducible promoters
disclosed in U.S. Pat. No. 6,252,138; and water deficit-inducible
promoters disclosed in U.S. Patent Application Publication
2004/0123347 A1. All of the above-described patents and patent
publications disclosing promoters and their use, especially in
recombinant DNA constructs functional in plants are incorporated
herein by reference.
[0079] Plant vascular- or phloem-specific promoters of interest
include a rolC or rolA promoter of Agrobacterium rhizogenes, a
promoter of a Agrobacterium tumefaciens T-DNA gene 5, the rice
sucrose synthase RSs1 gene promoter, a Commelina yellow mottle
badnavirus promoter, a coconut foliar decay virus promoter, a rice
tungro bacilliform virus promoter, the promoter of a pea glutamine
synthase GS3A gene, a invCD111 and invCD141 promoters of a potato
invertase genes, a promoter isolated from Arabidopsis shown to have
phloem-specific expression in tobacco by Kertbundit et al. (1991)
Proc. Nat. Acad. Sci. USA., 88:5212-5216, a VAHOX1 promoter region,
a pea cell wall invertase gene promoter, an acid invertase gene
promoter from carrot, a promoter of a sulfate transporter gene
Sultr1;3, a promoter of a plant sucrose synthase gene, and a
promoter of a plant sucrose transporter gene.
[0080] Promoters suitable for use with a recombinant DNA construct
or polynucleotide disclosed herein include polymerase II ("pol II")
promoters and polymerase III ("pol III") promoters. RNA polymerase
II transcribes structural or catalytic RNAs that are usually
shorter than 400 nucleotides in length, and recognizes a simple run
of T residues as a termination signal; it has been used to
transcribe siRNA duplexes (see, e. g., Lu et al. (2004) Nucleic
Acids Res., 32:e171). Pol II promoters are therefore preferred in
certain embodiments where a short RNA transcript is to be produced
from a recombinant DNA construct. In one embodiment, the
recombinant DNA construct includes a pol II promoter to express an
RNA transcript flanked by self-cleaving ribozyme sequences (e. g.,
self-cleaving hammerhead ribozymes), resulting in a processed RNA,
such as a single-stranded RNA that binds to the transcript of the
flea beetle target gene, with defined 5' and 3' ends, free of
potentially interfering flanking sequences. An alternative approach
uses pol III promoters to generate transcripts with relatively
defined 5' and 3' ends, i. e., to transcribe an RNA with minimal 5'
and 3' flanking sequences. In some embodiments, Pol III promoters
(e. g., U6 or H1 promoters) are preferred for adding a short
AT-rich transcription termination site that results in 2 base-pair
overhangs (UU) in the transcribed RNA; this is useful, e. g., for
expression of siRNA-type constructs. Use of pol III promoters for
driving expression of siRNA constructs has been reported; see van
de Wetering et al. (2003) EMBO Rep., 4: 609-615, and Tuschl (2002)
Nature Biotechnol., 20: 446-448. Baculovirus promoters such as
baculovirus polyhedrin and p10 promoters are known in the art and
commercially available; see, e. g., Invitrogen's "Guide to
Baculovirus Expression Vector Systems (BEVS) and Insect Cell
Culture Techniques", 2002 (Life Technologies, Carlsbad, Calif.) and
F. J. Haines et al. "Baculovirus Expression Vectors", undated
(Oxford Expression Technologies, Oxford, UK).
[0081] The promoter element can include nucleic acid sequences that
are not naturally occurring promoters or promoter elements or
homologues thereof but that can regulate expression of a gene.
Examples of such "gene independent" regulatory sequences include
naturally occurring or artificially designed RNA sequences that
comprise a ligand-binding region or aptamer (see "Aptamers", below)
and a regulatory region (which can be cis-acting). See, for
example, Isaacs et al. (2004) Nat. Biotechnol., 22:841-847, Bayer
and Smolke (2005) Nature Biotechnol., 23:337-343, Mandal and
Breaker (2004) Nature Rev. Mol. Cell Biol., 5:451-463, Davidson and
Ellington (2005) Trends Biotechnol., 23:109-112, Winkler et al.
(2002) Nature, 419:952-956, Sudarsan et al. (2003) RNA, 9:644-647,
and Mandal and Breaker (2004) Nature Struct. Mol. Biol., 11:29-35.
Such "riboregulators" could be selected or designed for specific
spatial or temporal specificity, for example, to regulate
translation of DNA that encodes a silencing element for suppressing
a target gene only in the presence (or absence) of a given
concentration of the appropriate ligand. One example is a
riboregulator that is responsive to an endogenous ligand (e. g.,
jasmonic acid or salicylic acid) produced by the plant when under
stress (e. g., abiotic stress such as water, temperature, or
nutrient stress, or biotic stress such as attach by pests or
pathogens); under stress, the level of endogenous ligand increases
to a level sufficient for the riboregulator to begin transcription
of the DNA that encodes a silencing element for suppressing a
target gene.
Transgene Transcription Units
[0082] In some embodiments, the recombinant DNA construct or
polynucleotide disclosed herein comprises a transgene transcription
unit. A transgene transcription unit comprises DNA sequence
encoding a gene of interest, e. g., a natural protein or a
heterologous protein. A gene of interest can be any coding or
non-coding sequence from any species (including, but not limited
to, non-eukaryotes such as bacteria, and viruses; fungi, protists,
plants, invertebrates, and vertebrates. Particular genes of
interest are genes encoding one or more proteins conferring
resistance to an herbicide and genes encoding at least one
pesticidal agent selected from the group consisting of a patatin, a
plant lectin, a phytoecdysteroid, a Bacillus thuringiensis
insecticidal protein, a Xenorhabdus insecticidal protein, a
Photorhabdus insecticidal protein, a Bacillus laterosporous
insecticidal protein, a Bacillus sphaericus insecticidal protein,
and an insecticidal protein produced by any of Lysinibacillus
sphaericus (Bacillus sphaericus), Brevibacillus laterosporus
(Bacillus laterosporus), Chromobacterium species, Chromobacterium
subtsugae, Paenibacillus species, Paenibacillus lentimorbus, and
Paenibacillus popilliae. The transgene transcription unit can
further comprise 5' or 3' sequence or both as required for
transcription of the transgene.
Introns
[0083] In some embodiments, the recombinant DNA construct or
polynucleotide comprises DNA encoding a spliceable intron. By
"intron" is generally meant a segment of DNA (or the RNA
transcribed from such a segment) that is located between exons
(protein-encoding segments of the DNA or corresponding transcribed
RNA), wherein, during maturation of the messenger RNA, the intron
present is enzymatically "spliced out" or removed from the RNA
strand by a cleavage/ligation process that occurs in the nucleus in
eukaryotes. The term "intron" is also applied to non-coding DNA
sequences that are transcribed to RNA segments that can be spliced
out of a maturing RNA transcript, but are not introns found between
protein-coding exons. One example of these are spliceable sequences
that that have the ability to enhance expression in plants (in some
cases, especially in monocots) of a downstream coding sequence;
these spliceable sequences are naturally located in the 5'
untranslated region of some plant genes, as well as in some viral
genes (e. g., the tobacco mosaic virus 5' leader sequence or
"omega" leader described as enhancing expression in plant genes by
Gallie and Walbot (1992) Nucleic Acids Res., 20:4631-4638). These
spliceable sequences or "expression-enhancing introns" can be
artificially inserted in the 5' untranslated region of a plant gene
between the promoter but before any protein-coding exons. Examples
of such expression-enhancing introns include, but are not limited
to, a maize alcohol dehydrogenase (Zm-Adh1), a maize Bronze-1
expression-enhancing intron, a rice actin 1 (Os-Act1) intron, a
Shrunken-1 (Sh-1) intron, a maize sucrose synthase intron, a heat
shock protein 18 (hsp18) intron, and an 82 kilodalton heat shock
protein (hsp82) intron. U.S. Pat. Nos. 5,593,874 and 5,859,347,
specifically incorporated by reference herein, describe methods of
improving recombinant DNA constructs for use in plants by inclusion
of an expression-enhancing intron derived from the 70 kilodalton
maize heat shock protein (hsp70) in the non-translated leader
positioned 3' from the gene promoter and 5' from the first
protein-coding exon.
Gene Suppression Elements
[0084] In some embodiments, the recombinant DNA construct or
polynucleotide comprises DNA encoding additional gene suppression
element for suppressing a target gene other than a flea beetle
target gene. The target gene to be suppressed can include coding or
non-coding sequence or both.
[0085] Suitable gene suppression elements are described in detail
in U. S. Patent Application Publication 2006/0200878, which
disclosure is specifically incorporated herein by reference, and
include one or more of: [0086] (a) DNA that comprises at least one
anti-sense DNA segment that is anti-sense to at least one segment
of the gene to be suppressed; [0087] (b) DNA that comprises
multiple copies of at least one anti-sense DNA segment that is
anti-sense to at least one segment of the gene to be suppressed;
[0088] (c) DNA that comprises at least one sense DNA segment that
is at least one segment of the gene to be suppressed; [0089] (d)
DNA that comprises multiple copies of at least one sense DNA
segment that is at least one segment of the gene to be suppressed;
[0090] (e) DNA that transcribes to RNA for suppressing the gene to
be suppressed by forming double-stranded RNA and comprises at least
one anti-sense DNA segment that is anti-sense to at least one
segment of the gene to be suppressed and at least one sense DNA
segment that is at least one segment of the gene to be suppressed;
[0091] (f) DNA that transcribes to RNA for suppressing the gene to
be suppressed by forming a single double-stranded RNA and comprises
multiple serial anti-sense DNA segments that are anti-sense to at
least one segment of the gene to be suppressed and multiple serial
sense DNA segments that are at least one segment of the gene to be
suppressed; [0092] (g) DNA that transcribes to RNA for suppressing
the gene to be suppressed by forming multiple double strands of RNA
and comprises multiple anti-sense DNA segments that are anti-sense
to at least one segment of the gene to be suppressed and multiple
sense DNA segments that are at least one segment of the gene to be
suppressed, and wherein the multiple anti-sense DNA segments and
the multiple sense DNA segments are arranged in a series of
inverted repeats; [0093] (h) DNA that comprises nucleotides derived
from a plant miRNA; [0094] (i) DNA that comprises nucleotides of a
siRNA; [0095] (j) DNA that transcribes to an RNA aptamer capable of
binding to a ligand; and [0096] (k) DNA that transcribes to an RNA
aptamer capable of binding to a ligand, and DNA that transcribes to
regulatory RNA capable of regulating expression of the gene to be
suppressed, wherein the regulation is dependent on the conformation
of the regulatory RNA, and the conformation of the regulatory RNA
is allosterically affected by the binding state of the RNA
aptamer.
[0097] In some embodiments, an intron is used to deliver a gene
suppression element in the absence of any protein-coding exons
(coding sequence). In one example, an intron, such as an
expression-enhancing intron (preferred in certain embodiments), is
interrupted by embedding within the intron a gene suppression
element, wherein, upon transcription, the gene suppression element
is excised from the intron. Thus, protein-coding exons are not
required to provide the gene suppressing function of the
recombinant DNA constructs disclosed herein.
Transcription Regulatory Elements
[0098] In some embodiments, the recombinant DNA construct or
polynucleotide comprises DNA encoding a transcription regulatory
element. Transcription regulatory elements include elements that
regulate the expression level of the recombinant DNA construct
(relative to its expression in the absence of such regulatory
elements). Examples of suitable transcription regulatory elements
include riboswitches (cis- or trans-acting), transcript stabilizing
sequences, and miRNA recognition sites, as described in detail in
U. S. Patent Application Publication 2006/0200878, specifically
incorporated herein by reference.
Transgenic Plant Cells and Transgenic Plants
[0099] The recombinant DNA constructs disclosed herein can be
stacked with other recombinant DNA for imparting additional traits
(e. g., in the case of transformed plants, traits including
herbicide resistance, pest resistance, cold germination tolerance,
water deficit tolerance, and the like) for example, by expressing
or suppressing other genes. Constructs for coordinated decrease and
increase of gene expression are disclosed in U.S. Patent
Application Publication 2004/0126845 A1, specifically incorporated
by reference.
[0100] In certain transgenic plant cells and transgenic plants, it
is sometimes desirable to concurrently express a gene of interest
while also modulating expression of a flea beetle target gene.
Thus, in some embodiments, the transgenic plant contains
recombinant DNA further comprising a gene expression element for
expressing at least one gene of interest, and transcription of the
recombinant DNA construct for flea beetle control is preferably
effected with concurrent transcription of the gene expression
element. In embodiments, the transgenic plant expresses DNA
encoding a insecticidal polynucleotide transcript as disclosed
herein for suppression of a flea beetle target gene, and also
expresses DNA encoding a non-nucleotide pesticidal agent such as a
small-molecule pesticidal agent or a proteinaceous pesticidal
agent; such DNAs can be stacked in a single recombinant construct
or expression cassette, or alternatively can be expressed from
discrete recombinant constructs or expression cassettes. Examples
of non-nucleotide pesticidal agents include patatins, plant
lectins, phytoecdysteroids, and bacterial insecticidal proteins (e.
g., insecticidal proteins from Bacillus thuringiensis, Xenorhabdus
sp., Photorhabdus sp., Brevibacillus laterosporus (Bacillus
laterosporus), Lysinibacillus sphaericus (Bacillus sphaericus),
Chromobacterium sp., Chromobacterium subtsugae, Paenibacillus sp.,
Paenibacillus lentimorbus, and Paenibacillus popilliae). In
embodiments, the transgenic plant expresses DNA encoding a
recombinant RNA transcript as disclosed herein for suppression of a
flea beetle target gene, and also expresses DNA encoding one or
more proteins conferring tolerance to an herbicide. Examples of
proteins conferring tolerance to an herbicide include
5-enolpyruvylshikimate-3-phosphate synthases (EPSPS; see, e. g.,
U.S. Pat. Nos. 5,627,061, 5,633,435 RE39247, 6,040,497, and
5,094,945, and PCT International Application Publications
WO04074443 and WO04009761), glyphosate oxidoreductase (GOX; U.S.
Pat. No. 5,463,175), glyphosate decarboxylase (PCT International
Application Publication WO05003362, U.S. Pat. No. 7,405,347, and U.
S. Patent Application Publication 2004/0177399),
glyphosate-N-acetyl transferase (GAT; U.S. Pat. No. 7,714,188)
conferring tolerance to glyphosate; dicamba monooxygenase
conferring tolerance to auxin-like herbicides such as dicamba (U.S.
Pat. No. 7,105,724); phosphinothricin acetyltransferase (pat or
bar) conferring tolerance to phosphinothricin or glufosinate (U.S.
Pat. No. 5,646,024); 2,2-dichloropropionic acid dehalogenase
conferring tolerance to 2,2-dichloropropionic acid (Dalapon) (PCT
International Application Publication WO9927116); acetohydroxy acid
synthase or acetolactate synthase conferring tolerance to
acetolactate synthase inhibitors such as sulfonylurea,
imidazolinone, triazolopyrimidine, pyrimidyloxybenzoates and
phthalide (U.S. Pat. No. 6,225,105); haloarylnitrilase (Bxn) for
conferring tolerance to bromoxynil (U.S. Pat. No. 4,810,648);
modified acetyl-coenzyme A carboxylase for conferring tolerance to
cyclohexanedione (sethoxydim) and aryloxyphenoxypropionate
(haloxyfop) (U.S. Pat. No. 6,414,222); dihydropteroate synthase
(sul 1) for conferring tolerance to sulfonamide herbicides (U.S.
Pat. No. 5,719,046); 32 kDa photosystem II polypeptide (psbA) for
conferring tolerance to triazine herbicides (Hirschberg et al.,
1983, Science, 222:1346-1349); anthranilate synthase for conferring
tolerance to 5-methyltryptophan (U.S. Pat. No. 4,581,847);
dihydrodipicolinic acid synthase (dap A) for conferring to
tolerance to aminoethyl cysteine (PCT International Application
Publication WO8911789); phytoene desaturase (crt1) for conferring
tolerance to pyridazinone herbicides such as norflurazon (Japan
Patent JP06343473); hydroxyphenylpyruvate dioxygenase, a
4-hydroxyphenylacetic acid oxidase and a 4-hydroxyphenylacetic
1-hydrolase (U.S. Pat. No. 7,304,209) for conferring tolerance to
cyclopropylisoxazole herbicides such as isoxaflutole (U.S. Pat. No.
6,268,549); modified protoporphyrinogen oxidase I (protox) for
conferring tolerance to protoporphyrinogen oxidase inhibitors (U.S.
Pat. No. 5,939,602); aryloxyalkanoate dioxygenase (AAD-1) for
conferring tolerance to an herbicide containing an aryloxyalkanoate
moiety (WO05107437); a serine hydroxymethyltransferase (US Patent
Application Publication 2008/0155716), a glufosinate-tolerant
glutamine synthase (US Patent Application Publication
2009/0018016). Examples of such herbicides include phenoxy auxins
(such as 2,4-D and dichloroprop), pyridyloxy auxins (such as
fluroxypyr and triclopyr), aryloxyphenoxypropionates (AOPP)
acetylcoenzyme A carboxylase (ACCase) inhibitors (such as
haloxyfop, quizalofop, and diclofop), and 5-substituted
phenoxyacetate protoporphyrinogen oxidase IX inhibitors (such as
pyraflufen and flumiclorac). The nucleotide sequences of the
nucleic acids encoding herbicide-tolerance proteins and the
sequences of the herbicide-tolerance proteins, as disclosed in the
U. S. patent and patent application publications cited in this
paragraph are incorporated herein by reference.
[0101] In some embodiments, the recombinant DNA constructs
disclosed herein can be transcribed in any plant cell or tissue or
in a whole plant of any developmental stage. Transgenic plants can
be derived from any monocot or dicot plant, such as, but not
limited to, plants of commercial or agricultural interest, such as
crop plants (especially crop plants used for human food or animal
feed), wood- or pulp-producing trees, vegetable plants, fruit
plants, and ornamental plants. Examples of plants of interest
include grain crop plants (such as wheat, oat, barley, maize, rye,
triticale, rice, millet, sorghum, quinoa, amaranth, and buckwheat);
forage crop plants (such as forage grasses and forage dicots
including alfalfa, vetch, clover, and the like); oilseed crop
plants (such as cotton, safflower, sunflower, soybean, canola,
rapeseed, flax, peanuts, and oil palm); tree nuts (such as walnut,
cashew, hazelnut, pecan, almond, and the like); sugarcane, coconut,
date palm, olive, sugarbeet, tea, and coffee; wood- or
pulp-producing trees; vegetable crop plants such as legumes (for
example, beans, peas, lentils, alfalfa, peanut), lettuce,
asparagus, artichoke, celery, carrot, radish, the brassicas (for
example, cabbages, kales, mustards, and other leafy brassicas,
broccoli, cauliflower, Brussels sprouts, turnip, kohlrabi), edible
cucurbits (for example, cucumbers, melons, summer squashes, winter
squashes), edible alliums (for example, onions, garlic, leeks,
shallots, chives), edible members of the Solanaceae (for example,
tomatoes, eggplants, potatoes, peppers, ground cherries), and
edible members of the Chenopodiaceae (for example, beet, chard,
spinach, quinoa, amaranth); fruit crop plants such as apple, pear,
citrus fruits (for example, orange, lime, lemon, grapefruit, and
others), stone fruits (for example, apricot, peach, plum,
nectarine), banana, pineapple, grape, kiwifruit, papaya, avocado,
and berries; plants grown for biomass or biofuel (for example,
Miscanthus grasses, switchgrass, jatropha, oil palm, eukaryotic
microalgae such as Botryococcus braunii, Chlorella spp., and
Dunaliella spp., and eukaryotic macroalgae such as Gracilaria spp.,
and Sargassum spp.); and ornamental plants including ornamental
flowering plants, ornamental trees and shrubs, ornamental
groundcovers, and ornamental grasses. Specific plant species of
interest in which a recombinant DNA construct is transcribed to
provide resistance to flea beetles are plants in the family
Brassicaceae, including the Brassica species B. napus, B. juncea,
B. carinata, B. rapa, B. oleracea, B. rupestris, B. septiceps, B.
nigra, B. narinosa, B. perviridus, B. tournefortii, and B.
fructiculosa. Additional plant species of interest in which a
recombinant DNA construct is transcribed to provide resistance to
flea beetles are Glycine max, Linum usitatissimum, Zea mays,
Carthamus tinctorius, Helianthus annuus, Nicotiana tabacum,
Arabidopsis thaliana, Betholettia excelsa, Ricinus communis, Cocos
nucifera, Coriandrum sativum, Gossypium spp., Arachis hypogaea,
Simmondsia chinensis, Solanum tuberosum, Elaeis guineensis, Olea
europaea, Oryza sativa, Cucurbita maxim, Hordeum vulgare, and
Triticum aestivum.
[0102] Also disclosed herein are commodity products produced from a
transgenic plant cell, plant, or seed expressing a recombinant DNA
construct imparting improved resistance to flea beetles as
disclosed herein, including, but not limited to, harvested leaves,
roots, shoots, tubers, stems, fruits, seeds, or other parts of a
plant, meals, oils, extracts, fermentation or digestion products,
crushed or whole grains or seeds of a plant, or any food or
non-food product including such commodity products produced from a
transgenic plant cell, plant, or seed as disclosed herein. The
detection of one or more of nucleic acid sequences of the
recombinant DNA constructs for flea beetle control as disclosed
herein in one or more commodity or commodity products contemplated
herein is defacto evidence that the commodity or commodity product
contains or is derived from a transgenic plant cell, plant, or seed
expressing such a recombinant DNA construct.
[0103] Generally a transgenic plant having in its genome a
recombinant DNA construct as disclosed herein exhibits increased
resistance to an insect infestation, specifically increased
resistance to a flea beetle infestation. In various embodiments,
for example, where the transgenic plant expresses a recombinant DNA
construct for flea beetle control that is stacked with other
recombinant DNA for imparting additional traits, the transgenic
plant has at least one additional altered trait, relative to a
plant lacking the recombinant DNA construct, selected from the
group of traits consisting of: [0104] (a) improved abiotic stress
tolerance; [0105] (b) improved biotic stress tolerance; [0106] (c)
modified primary metabolite composition; [0107] (d) modified
secondary metabolite composition; [0108] (e) modified trace
element, carotenoid, or vitamin composition; [0109] (f) improved
yield; [0110] (g) improved ability to use nitrogen, phosphate, or
other nutrients; [0111] (h) modified agronomic characteristics;
[0112] (i) modified growth or reproductive characteristics; and
[0113] (j) improved harvest, storage, or processing quality.
[0114] In some embodiments, the transgenic plant is characterized
by: improved tolerance of abiotic stress (e. g., tolerance of water
deficit or drought, heat, cold, non-optimal nutrient or salt
levels, non-optimal light levels) or of biotic stress (e. g.,
crowding, allelopathy, or wounding); by a modified primary
metabolite (e. g., fatty acid, oil, amino acid, protein, sugar, or
carbohydrate) composition; a modified secondary metabolite (e. g.,
alkaloids, terpenoids, polyketides, non-ribosomal peptides, and
secondary metabolites of mixed biosynthetic origin) composition; a
modified trace element (e. g., iron, zinc), carotenoid (e. g.,
beta-carotene, lycopene, lutein, zeaxanthin, or other carotenoids
and xanthophylls), or vitamin (e. g., tocopherols) composition;
improved yield (e. g., improved yield under non-stress conditions
or improved yield under biotic or abiotic stress); improved ability
to use nitrogen, phosphate, or other nutrients; modified agronomic
characteristics (e. g., delayed ripening; delayed senescence;
earlier or later maturity; improved shade tolerance; improved
resistance to root or stalk lodging; improved resistance to "green
snap" of stems; modified photoperiod response); modified growth or
reproductive characteristics (e. g., intentional dwarfing;
intentional male sterility, useful, e. g., in improved
hybridization procedures; improved vegetative growth rate; improved
germination; improved male or female fertility); improved harvest,
storage, or processing quality (e. g., improved resistance to pests
during storage, improved resistance to breakage, improved appeal to
consumers); or any combination of these traits.
[0115] In another embodiment, transgenic seed, or seed produced by
the transgenic plant, has modified primary metabolite (e. g., fatty
acid, oil, amino acid, protein, sugar, or carbohydrate)
composition, a modified secondary metabolite composition, a
modified trace element, carotenoid, or vitamin composition, an
improved harvest, storage, or processing quality, or a combination
of these. In another embodiment, it can be desirable to change
levels of native components of the transgenic plant or seed of a
transgenic plant, for example, to decrease levels of an allergenic
protein or glycoprotein or of a toxic metabolite.
EXAMPLES
Example 1
[0116] This example illustrates non-limiting embodiments of coding
DNA sequences useful as target genes for controlling insect species
and for making compositions for controlling insects and
insect-resistant transgenic plants, and identifies insecticidal
polynucleotide sequences useful for controlling insect species.
More specifically, embodiments of target genes identified by name
(annotation) and sequence identifier (SEQ ID NO.) for controlling
flea beetles are provided in SEQ ID NOs:1-859, and embodiments of
dsRNA sequences ranging in size from 135 to 352 base pairs and
designed to suppress these target genes are provided in SEQ ID
NOs.:860-1718.
TABLE-US-00001 TABLE 1 Target Gene dsRNA SEQ ID SEQ ID NO. NO.* 1
860 2 861 3 862 4 863 5 864 6 865 7 866 8 867 9 868 10 869 11 870
12 871 13 872 14 873 15 874 16 875 17 876 18 877 19 878 20 879 21
880 22 881 23 882 24 883 25 884 26 885 27 886 28 887 29 888 30 889
31 890 32 891 33 892 34 893 35 894 36 895 37 896 38 897 39 898 40
899 41 900 42 901 43 902 44 903 45 904 46 905 47 906 48 907 49 908
50 909 51 910 52 911 53 912 54 913 55 914 56 915 57 916 58 917 59
918 60 919 61 920 62 921 63 922 64 923 65 924 66 925 67 926 68 927
69 928 70 929 71 930 72 931 73 932 74 933 75 934 76 935 77 936 78
937 79 938 80 939 81 940 82 941 83 942 84 943 85 944 86 945 87 946
88 947 89 948 90 949 91 950 92 951 93 952 94 953 95 954 96 955 97
956 98 957 99 958 100 959 101 960 102 961 103 962 104 963 105 964
106 965 107 966 108 967 109 968 110 969 111 970 112 971 113 972 114
973 115 974 116 975 117 976 118 977 119 978 120 979 121 980 122 981
123 982 124 983 125 984 126 985 127 986 128 987 129 988 130 989 131
990 132 991 133 992 134 993 135 994 136 995 137 996 138 997 139 998
140 999 141 1000 142 1001 143 1002 144 1003 145 1004 146 1005 147
1006 148 1007 149 1008 150 1009 151 1010 152 1011 153 1012 154 1013
155 1014 156 1015 157 1016 158 1017 159 1018 160 1019 161 1020 162
1021 163 1022 164 1023 165 1024 166 1025 167 1026 168 1027 169 1028
170 1029 171 1030 172 1031 173 1032 174 1033 175 1034 176 1035 177
1036 178 1037 179 1038 180 1039 181 1040 182 1041 183 1042 184 1043
185 1044 186 1045 187 1046 188 1047 189 1048 190 1049 191 1050 192
1051 193 1052 194 1053 195 1054 196 1055 197 1056 198 1057 199 1058
200 1059 201 1060 202 1061 203 1062 204 1063 205 1064 206 1065 207
1066 208 1067 209 1068 210 1069 211 1070 212 1071 213 1072 214 1073
215 1074 216 1075 217 1076 218 1077 219 1078 220 1079 221 1080 222
1081 223 1082 224 1083 225 1084 226 1085 227 1086 228 1087 229 1088
230 1089 231 1090 232 1091 233 1092 234 1093 235 1094 236 1095 237
1096 238 1097 239 1098 240 1099 241 1100 242 1101
243 1102 244 1103 245 1104 246 1105 247 1106 248 1107 249 1108 250
1109 251 1110 252 1111 253 1112 254 1113 255 1114 256 1115 257 1116
258 1117 259 1118 260 1119 261 1120 262 1121 263 1122 264 1123 265
1124 266 1125 267 1126 268 1127 269 1128 270 1129 271 1130 272 1131
273 1132 274 1133 275 1134 276 1135 277 1136 278 1137 279 1138 280
1139 281 1140 282 1141 283 1142 284 1143 285 1144 286 1145 287 1146
288 1147 289 1148 290 1149 291 1150 292 1151 293 1152 294 1153 295
1154 296 1155 297 1156 298 1157 299 1158 300 1159 301 1160 302 1161
303 1162 304 1163 305 1164 306 1165 307 1166 308 1167 309 1168 310
1169 311 1170 312 1171 313 1172 314 1173 315 1174 316 1175 317 1176
318 1177 319 1178 320 1179 321 1180 322 1181 323 1182 324 1183 325
1184 326 1185 327 1186 328 1187 329 1188 330 1189 331 1190 332 1191
333 1192 334 1193 335 1194 336 1195 337 1196 338 1197 339 1198 340
1199 341 1200 342 1201 343 1202 344 1203 345 1204 346 1205 347 1206
348 1207 349 1208 350 1209 351 1210 352 1211 353 1212 354 1213 355
1214 356 1215 357 1216 358 1217 359 1218 360 1219 361 1220 362 1221
363 1222 364 1223 365 1224 366 1225 367 1226 368 1227 369 1228 370
1229 371 1230 372 1231 373 1232 374 1233 375 1234 376 1235 377 1236
378 1237 379 1238 380 1239 381 1240 382 1241 383 1242 384 1243 385
1244 386 1245 387 1246 388 1247 389 1248 390 1249 391 1250 392 1251
393 1252 394 1253 395 1254 396 1255 397 1256 398 1257 399 1258 400
1259 401 1260 402 1261 403 1262 404 1263 405 1264 406 1265 407 1266
408 1267 409 1268 410 1269 411 1270 412 1271 413 1272 414 1273 415
1274 416 1275 417 1276 418 1277 419 1278 420 1279 421 1280 422 1281
423 1282 424 1283 425 1284 426 1285 427 1286 428 1287 429 1288 430
1289 431 1290 432 1291 433 1292 434 1293 435 1294 436 1295 437 1296
438 1297 439 1298 440 1299 441 1300 442 1301 443 1302 444 1303 445
1304 446 1305 447 1306 448 1307 449 1308 450 1309 451 1310 452 1311
453 1312 454 1313 455 1314 456 1315 457 1316 458 1317 459 1318 460
1319 461 1320 462 1321 463 1322 464 1323 465 1324 466 1325 467 1326
468 1327 469 1328 470 1329 471 1330 472 1331 473 1332 474 1333 475
1334 476 1335 477 1336 478 1337 479 1338 480 1339 481 1340 482 1341
483 1342 484 1343 485 1344 486 1345 487 1346 488 1347 489 1348 490
1349 491 1350 492 1351 493 1352
494 1353 495 1354 496 1355 497 1356 498 1357 499 1358 500 1359 501
1360 502 1361 503 1362 504 1363 505 1364 506 1365 507 1366 508 1367
509 1368 510 1369 511 1370 512 1371 513 1372 514 1373 515 1374 516
1375 517 1376 518 1377 519 1378 520 1379 521 1380 522 1381 523 1382
524 1383 525 1384 526 1385 527 1386 528 1387 529 1388 530 1389 531
1390 532 1391 533 1392 534 1393 535 1394 536 1395 537 1396 538 1397
539 1398 540 1399 541 1400 542 1401 543 1402 544 1403 545 1404 546
1405 547 1406 548 1407 549 1408 550 1409 551 1410 552 1411 553 1412
554 1413 555 1414 556 1415 557 1416 558 1417 559 1418 560 1419 561
1420 562 1421 563 1422 564 1423 565 1424 566 1425 567 1426 568 1427
569 1428 570 1429 571 1430 572 1431 573 1432 574 1433 575 1434 576
1435 577 1436 578 1437 579 1438 580 1439 581 1440 582 1441 583 1442
584 1443 585 1444 586 1445 587 1446 588 1447 589 1448 590 1449 591
1450 592 1451 593 1452 594 1453 595 1454 596 1455 597 1456 598 1457
599 1458 600 1459 601 1460 602 1461 603 1462 604 1463 605 1464 606
1465 607 1466 608 1467 609 1468 610 1469 611 1470 612 1471 613 1472
614 1473 615 1474 616 1475 617 1476 618 1477 619 1478 620 1479 621
1480 622 1481 623 1482 624 1483 625 1484 626 1485 627 1486 628 1487
629 1488 630 1489 631 1490 632 1491 633 1492 634 1493 635 1494 636
1495 637 1496 638 1497 639 1498 640 1499 641 1500 642 1501 643 1502
644 1503 645 1504 646 1505 647 1506 648 1507 649 1508 650 1509 651
1510 652 1511 653 1512 654 1513 655 1514 656 1515 657 1516 658 1517
659 1518 660 1519 661 1520 662 1521 663 1522 664 1523 665 1524 666
1525 667 1526 668 1527 669 1528 670 1529 671 1530 672 1531 673 1532
674 1533 675 1534 676 1535 677 1536 678 1537 679 1538 680 1539 681
1540 682 1541 683 1542 684 1543 685 1544 686 1545 687 1546 688 1547
689 1548 690 1549 691 1550 692 1551 693 1552 694 1553 695 1554 696
1555 697 1556 698 1557 699 1558 700 1559 701 1560 702 1561 703 1562
704 1563 705 1564 706 1565 707 1566 708 1567 709 1568 710 1569 711
1570 712 1571 713 1572 714 1573 715 1574 716 1575 717 1576 718 1577
719 1578 720 1579 721 1580 722 1581 723 1582 724 1583 725 1584 726
1585 727 1586 728 1587 729 1588 730 1589 731 1590 732 1591 733 1592
734 1593 735 1594 736 1595 737 1596 738 1597 739 1598 740 1599 741
1600 742 1601 743 1602 744 1603
745 1604 746 1605 747 1606 748 1607 749 1608 750 1609 751 1610 752
1611 753 1612 754 1613 755 1614 756 1615 757 1616 758 1617 759 1618
760 1619 761 1620 762 1621 763 1622 764 1623 765 1624 766 1625 767
1626 768 1627 769 1628 770 1629 771 1630 772 1631 773 1632 774 1633
775 1634 776 1635 777 1636 778 1637 779 1638 780 1639 781 1640 782
1641 783 1642 784 1643 785 1644 786 1645 787 1646 788 1647 789 1648
790 1649 791 1650 792 1651 793 1652 794 1653 795 1654 796 1655 797
1656 798 1657 799 1658 800 1659 801 1660 802 1661 803 1662 804 1663
805 1664 806 1665 807 1666 808 1667 809 1668 810 1669 811 1670 812
1671 813 1672 814 1673 815 1674 816 1675 817 1676 818 1677 819 1678
820 1679 821 1680 822 1681 823 1682 824 1683 825 1684 826 1685 827
1686 828 1687 829 1688 830 1689 831 1690 832 1691 833 1692 834 1693
835 1694 836 1695 837 1696 838 1697 839 1698 840 1699 841 1700 842
1701 843 1702 844 1703 845 1704 846 1705 847 1706 848 1707 849 1708
850 1709 851 1710 852 1711 853 1712 854 1713 855 1714 856 1715 857
1716 858 1717 859 1718 *RNA sequences are provided for the
anti-sense strand of the dsRNA in 5' to 3' direction. **T44966 and
T44967 are positive controls based on a Phyllotreta striolata
arginine kinase mRNA disclosed in Zhao et al. (2008), Fur. I
Entomol., 5:815.
[0117] The embodiments of dsRNA sequences provided in Table 1 are
generally useful for RNA-mediated suppression of the corresponding
target gene identified in Table 1. These dsRNAs are useful for
controlling insects, especially flea beetles, including the source
species from which the target genes in Table 1 were identified.
RNA-mediated suppression of one or more of the target genes
provided in Table 1, or use of one or more of the dsRNAs provided
in Table 1, is useful for causing mortality or stunting, or
otherwise controlling, target insect species in the following
genera: Altica, Anthobiodes, Aphthona, Aphthonaltica, Aphthonoides,
Apteopeda, Argopistes, Argopus, Arrhenocoela, Batophila,
Blepharida, Chaetocnema, Clitea, Crepidodera, Derocrepis, Dibolia,
Disonycha, Epitrix, Hermipyxis, Hermaeophaga, Hespera,
Hippuriphila, Horaia, Hyphasis, Lipromima, Liprus, Longitarsus,
Luperomorpha, Lythraria, Manobia, Mantura, Meishania, Minota,
Mniophila, Neicrepidodera, Nonarthra, Novofoudrasia, Ochrosis,
Oedionychis, Oglobinia, Omeisphaera, Ophrida, Orestia, Paragopus,
Pentamesa, Philopona, Phygasia, Phyllotreta, Podagrica,
Podagricomela, Podontia, Pseudodera, Psylliodes, Sangariola,
Sinaltica, Sphaeroderma, Systena, Trachyaphthona, Xuthea, and
Zipangia. In embodiments, compositions comprising adsRNA for
suppression of one or more of the target genes provided in Table 1
(e. g., a composition comprising an effective amount of one or more
of the dsRNAs provided in Table 1) are useful for controlling at
least one of Altica ambiens (alder flea beetle), Altica canadensis
(prairie flea beetle), Altica chalybaea (grape flea beetle), Altica
prasina (poplar flea beetle), Altica rosae (rose flea beetle),
Altica sylvia (blueberry flea beetle), Altica ulmi (elm flea
beetle). Chaetocnema pulicaria (corn flea beele), Chaetocnema
conoinis (sweet potato flea beetle), Epitrix cucumeris (potato flea
beetle), Systena blanda (palestripped fleabeetle), and Systena
frontalis (redheaded flea beetle), thus preventing or treating
plant infestation by these species. For example, a composition
comprising an effective amount of one or more of the dsRNAs
provided in Table 1 is useful for preventing or treating
infestation of potato plants by Epitrix cucumeris (potato flea
beetle).
[0118] In embodiments, RNA-mediated suppression of one or more of
the target genes provided in Table 1, or use of one or more of the
dsRNAs provided in Table 1, is useful for causing mortality or
stunting in flea beetle species in the genera Phyllotreta and
Psylliodes, thus preventing or treating plant infestation by these
species. In specific embodiments, RNA-mediated suppression of one
or more of the target genes provided in Table 1, or use of one or
more of the dsRNAs provided in Table 1, is useful for causing
mortality or stunting in at least one flea beetle species selected
from the group consisting of Phyllotreta armoraciae (horseradish
flea beetle), Phyllotreta cruciferae (canola flea beetle),
Phyllotreta pusilla (western black flea beetle), Phyllotreta
nemorum (striped turnip flea beetle), Phyllotreta atra (turnip flea
beetle), Phyllotreta robusta (garden flea beetle), Phyllotreta
striolata (striped flea beetle), Phyllotreta undulata, Psylliodes
chrysocephala, and Psylliodes punctulata (hop flea beetle). In
embodiments, RNA-mediated suppression of one or more of the target
genes having a sequence selected from the group consisting of SEQ
ID NOs:1-296 is used to cause mortality or stunting in Phyllotreta
atra (turnip flea beetle) adults or larvae, for example, by
contacting Phyllotreta atra adults, larvae, or eggs with an
effective amount of a dsRNA comprising a sequence selected from the
group consisting of SEQ ID NOs:860-1155. In embodiments,
RNA-mediated suppression of one or more of the target genes having
a sequence selected from the group consisting of SEQ ID NOs:297-532
is used to cause mortality or stunting in Phyllotreta cruciferae
(canola flea beetle) adults or larvae, for example, by contacting
Phyllotreta cruciferae adults, larvae, or eggs with an effective
amount of a dsRNA comprising a sequence selected from the group
consisting of SEQ ID NOs:1156-1391, 1731-1972, and 1974. In
embodiments, RNA-mediated suppression of one or more of the target
genes having a sequence selected from the group consisting of SEQ
ID NOs:533-551 is used to cause mortality or stunting in
Phyllotreta striolata (striped flea beetle) adults or larvae, for
example, by contacting Phyllotreta striolata adults, larvae, or
eggs with an effective amount of a dsRNA comprising a sequence
selected from the group consisting of SEQ ID NOs:1392-1410, 1973,
and 1975. In embodiments, RNA-mediated suppression of one or more
of the target genes having a sequence selected from the group
consisting of SEQ ID NOs:552-859 is used to cause mortality or
stunting in Psylliodes chrysocephala adults or larvae, for example,
by contacting Psylliodes chrysocephala adults, larvae, or eggs with
an effective amount of a dsRNA comprising a sequence selected from
the group consisting of SEQ ID NOs:1411-1718.
[0119] Plants which can be protected by such infestation by
transgenic expression or topical application of one or more of the
dsRNAs provided in Table 1 include any plant species or variety
that is subject to infestation by flea beetles, especially plants
of economic importance, including ornamental plants and crop
plants. Embodiments of such plants include plants in the family
Brassicaceae (mustard family), such as a plant in the genus
Brassica including, for example, one of the following: B. napus
(rapeseed, including cultivars such as canola and rutabaga), B.
juncea (Indian mustard), B. carinata (Abyssinian mustard), B. rapa
(turnip), B. oleracea (wild cabbage, including domesticated
cultivars such as, kale, cabbage, broccoli, cauliflower, brussels
sprouts, etc.)B. rupestris (brown mustard), B. septiceps (seventop
mustard), B. nigra (black mustard), B. narinosa (broadbeaked
mustard), B. perviridus (mustard spinach), B. tournefortii (asian
mustard), and B. fructiculosa (Mediterranean cabbage). In
additional embodiments, the target plants may include, but not
limited to, one of the following: Glycine max (soybean), Linum
usitatissimum (linseed/flax), Zea mays (maize), Carthamus
tinctorius (safflower), Helianthus annuus (sunflower), Nicotiana
tabacum (tobacco), Arabidopsis thaliana, Betholettia excelsa
(Brazil nut), Ricinus communis (castor bean), Cocos nucifera
(coconut), Coriandrum sativum (coriander), Gossypium spp. (cotton),
Arachis hypogaea (groundnut or peanut), Simmondsia chinensis
(jojoba), Solanum tuberosum (potato) Elaeis guineensis (oil palm),
Olea europaea (olive), Oryza sativa (rice), Cucurbita maxima
(squash), Hordeum vulgare (barley), and Triticum aestivum
(wheat).
[0120] An aspect includes compositions comprising an effective
amount of one or more of the dsRNAs provided in Table 1 for topical
treatment of a plant to be treated for, or be protected from, flea
beetle infestation. Another aspect includes a recombinant DNA
construct encoding at least one strand of at least one the dsRNAs
provided in Table 1 for transgenic expression in a plant that has
improved resistance to flea beetle infestation, in comparison to a
plant not expressing such a construct.
Example 2
[0121] This example illustrates non-limiting embodiments of testing
the efficacy of dsRNA sequences and validating the dsRNA's utility
for suppressing expression of target genes for controlling insect
species. More specifically this example illustrates a method
comprising contacting an insect, such as a flea beetle adult or
larva, with one or more dsRNAs designed to cause stunting or
mortality in the insect. Other embodiments include methods where
the dsRNA is delivered to the insect by oral delivery (e. g., on or
in a food material ingested by the insect), or through non-oral
delivery (e. g., delivery through the insect's cuticle, or delivery
by contacting an egg of the insect).
[0122] In one embodiment, a feeding assay is used to determine
efficacy of a dsRNA in causing stunting or mortality in insects,
such as flea beetles. To test the efficacy of the dsRNA to kill or
stunt flea beetles, a single discriminating dose (for example, 100
nanograms/milliliter) is used to identify dsRNAs with measureable
ability to kill or stunt flea beetles at that dose. A negative
control dsRNA, such as a dsRNA targetting green fluorescent protein
(GFP), is also included in the assay. Each dsRNA is coated evenly
onto 1.0 centimeter diameter canola leaf discs and placed in
multiwell trays, with 2 male and 2 female adult flea beetles or 4
flea beetle larvae per well. Every 24 hours for a set period (e.
g., 2 weeks), new, freshly-coated leaves are provided. Stunting and
mortality are scored periodically (e. g., daily, or every 2 or 3
days).
[0123] The dsRNAs that show efficacy in this single-dose assay are
tested further. Using a similar protocol, varying doses of dsRNAs
are tested, as described above, to determine the LC50 dose for each
of the active dsRNAs. Bioassays include 12-24 insects per dose,
performed in triplicate. Stunting and mortality is assessed over a
2 week period, scored on every third day.
[0124] The dsRNA sequences that are confirmed to be effective in
suppressing a target gene in a sequence-specific manner are useful
for identifying efficacious RNA delivery agents and formulations.
The insecticidal activity of formulations containing dsRNA can be
optimized by various techniques, such as modifying the chemical
entities in the formulation or modifying the ratio of the chemical
components in the formulation. Non-limiting examples of delivery
agents and formulations are provided in Example 5.
Example 3
[0125] This example illustrates non-limiting embodiments of methods
for validating dsRNA efficacy for suppressing or silencing a target
gene in an insect cell or causing stunting or mortality in an
insect. More specifically this example illustrates methods for
testing dsRNA for efficacy in preventing or treating flea beetle
infestations in whole plants.
[0126] Polynucleotides (such as the dsRNA sequences described in
Examples 1 and 2) that have been confirmed to be effective in
suppressing a target gene in a sequence-specific manner are further
evaluated in whole plant assays. In one method, the polynucleotides
(e. g., anti-sense RNA, dsRNA) are applied directly to the insect
surface (e. g. by spraying or dusting). In another method, the
polynucleotides are provided to the insect in an insect diet (e.
g., in a bacterial or plant cell expressing a polynucleotide such
as a hairpin form of a dsRNA, or in an artificial bait containing a
polynucleotide). Stunting and mortality are scored periodically, as
described in Example 2.
[0127] In various methods that are also appropriate for large-scale
application (e. g., to fields of crop plants), the polynucleotide
is applied in a foliar application through aerial or terrestrial
spraying or dusting or chemigation on the leaf surface to control
early season damage from the adult stage of the life cycle, or
applied as a seed treatment to control larval or adult stages of
the insect life cycle, or applied as a soil in-furrow or drench
application to control larval or adult stages of the insect life
cycle. An example of a foliar testing regime includes treating the
plant immediately after emergence from the ground and evaluating
foliar damage caused by adult flea beetles 1-2 weeks after plant
emergence. For in-furrow or seed treatment similar timing for
damage evaluation is followed.
Example 4
[0128] The polynucleotides are generally designed to modulate
expression by inducing regulation or suppression of an insect
target gene and are designed to have a nucleotide sequence
essentially identical or essentially complementary to the
nucleotide sequence an insect target gene or cDNA (e. g., SEQ ID
NOs:1-859) or to the sequence of RNA transcribed from an insect
target gene, which can be coding sequence or non-coding sequence.
This example describes non-limiting techniques useful in the design
and selection of polynucleotides to modulate expression of an
insect target gene.
Selection of Effective Polynucleotides by "Tiling"
[0129] Polynucleotides of use in the compositions and methods
disclosed herein need not be of the full length of a target gene,
and in many embodiments are of much shorter length in comparison to
the target gene. An example of a technique that is useful for
selecting effective polynucleotides for insect control is "tiling",
or evaluation of polynucleotides corresponding to adjacent or
partially overlapping segments of a target gene.
[0130] Effective polynucleotides can be identified by "tiling" gene
targets in selected length fragments, e. g., fragments of 200-300
nucleotides in length, with partially overlapping regions, e. g.,
of about 25 nucleotides, along the length of the target gene. To
suppress a single gene, polynucleotides are designed to correspond
to (have a nucleotide identity or complementarity with) regions
that are unique to the target gene; the selected region of the
target gene can include coding sequence or non-coding sequence (e.
g., promoter regions, 3' untranslated regions, introns and the
like) or a combination of both.
[0131] Where it is of interest to design a polynucleotide effective
in suppressing multiple target genes, the multiple target gene
sequences are aligned and polynucleotides designed to correspond to
regions with high sequence homology in common among the multiple
targets. Conversely, where it is of interest to design a
polynucleotide effective in selectively suppressing one among
multiple target sequences, the multiple target gene sequences are
aligned and polynucleotides designed to correspond to regions with
no or low sequence homology in common among the multiple
targets.
[0132] In a non-limiting example, polynucleotides (e.g, anti-sense
single-stranded RNAs, dsRNAs, anti-sense single-stranded DNAs,
dsDNAs) capable of inducing suppression are designed for each of
the target genes listed in Table 1 as follows. Multiple
polynucleotides, each of 200-300 nucleotides in length and
comprising a sequence complementary to a fragment of a target gene
having a sequence selected from SEQ ID NOs:1-859 are designed so
that each polynucleotide's sequence overlaps about 25 nucleotides
of the next adjacent polynucleotide's sequence, in such a way that
the multiple polynucleotides in combination cover the full length
of the target gene. (Similarly, double-stranded polynucleotides can
be designed by providing pairs of sense and anti-sense
polynucleotides, each pair of polynucleotides overlapping the next
adjacent pair of polynucleotides.)
[0133] The polynucleotides are tested by any convenient means for
efficacy in silencing the insect target gene. Examples of a
suitable test include the bioassays described herein in the working
Examples. Another test involves the topical application of the
polynucleotides either directly to individual insects or to the
surface of a plant to be protected from an insect infestation. One
desired result of treatment with a polynucleotide as disclosed
herein is prevention or control of an insect infestation, e. g., by
inducing in an insect a physiological or behavioural change such
as, but not limited to, growth stunting, increased mortality,
decrease in reproductive capacity, decrease in or cessation of
feeding behavior or movement, or decrease in or cessation of
metamorphosis stage development. Another desired result of
treatment with a polynucleotide as disclosed herein is provision of
a plant that exhibits improved resistance to an insect
infestation.
[0134] The tiling procedure can be repeated, if desired. A
polynucleotide found to provide desired activity can itself be
subjected to a tiling procedure. For example, multiple overlapping
polynucleotides are designed, each of 50-60 nucleotides in length
and comprising a sequence complementary to the fragment of a target
gene having a sequence selected from SEQ ID NOs:1-859 for which a
single polynucleotide of 300 nucleotides was found to be effective.
Additional rounds of tiling analysis can be carried out, where
polynucleotides as short as 18, 19, 20, or 21 nucleotides are
tested.
[0135] Effective polynucleotides of any size can be used, alone or
in combination, in the various methods disclosed herein. In some
embodiments, a single polynucleotide is used to make a composition
(e. g., a composition for topical application, or a recombinant DNA
construct useful for making a transgenic plant). In other
embodiments, a mixture or pool of different polynucleotides is
used; in such cases the polynucleotides can be for a single target
gene or for multiple target genes. In some embodiments, a
polynucleotide is designed to target different regions of the
target gene, e. g., an insecticidal polynucleotide can comprise
multiple segments that correspond to different exon regions of the
target gene, and "spacer" nucleotides which do not correspond to a
target gene can optionally be used in between or adjacent to the
segments.
Thermodynamic Considerations in Selecting Insecticidal
Polynucleotides
[0136] Polynucleotides can be designed or their sequence optimised
using thermodynamic considerations. For example, insecticidal
polynucleotides can be selected based on the thermodynamics
controlling hybridization between one nucleic acid strand (e. g., a
polynucleotide or an individual siRNA) and another (e. g., a target
gene transcript)
[0137] Methods and algorithms to predict nucleotide sequences that
are likely to be effective at RNAi-mediated silencing of a target
gene are known in the art. Non-limiting examples of such methods
and algorithms include "i-score", described by Ichihara et al.
(2007) Nucleic Acids Res., 35(18): 123e; "Oligowalk", publicly
available at ma.urmc.rochester.edu/servers/oligowalk and described
by Lu et al. (2008) Nucleic Acids Res., 36:W104-108; and "Reynolds
score", described by Khovorova et al. (2004) Nature Biotechnol.,
22:326-330.
Permitted Mismatches
[0138] By "essentially identical" or "essentially complementary" is
meant that the polynucleotide (or at least one strand of a
double-stranded polynucleotide) has sufficient identity or
complementarity to the target gene or to the RNA transcribed from a
target gene (e. g., the transcript) to suppress expression of a
target gene (e. g., to effect a reduction in levels or activity of
the target gene transcript and/or encoded protein). Polynucleotides
need not have 100 percent identity or complementarity to a target
gene or to the RNA transcribed from a target gene to suppress
expression of the target gene (e. g., to effect a reduction in
levels or activity of the target gene transcript or encoded
protein, or to provide control of an insect species). In some
embodiments, the polynucleotide or a portion thereof is designed to
be essentially identical to, or essentially complementary to, a
sequence of at least 18 or 19 contiguous nucleotides in either the
target gene or the RNA transcribed from the target gene. In some
embodiments, the polynucleotide or a portion thereof is designed to
be exactly identical to, or exactly complementary to, a sequence of
21 contiguous nucleotides in either the target gene or the RNA
transcribed from the target gene. In certain embodiments, an
"essentially identical" polynucleotide has 100 percent sequence
identity or at least about 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,
93, 94, 95, 96, 97, 98, or 99 percent sequence identity when
compared to the sequence of 18 or more contiguous nucleotides of
the target gene or an RNA transcribed from the target gene. In
certain embodiments, an "essentially complementary" polynucleotide
has 100 percent sequence complementarity or at least about 83, 84,
85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99
percent sequence complementarity to 18 or more contiguous
nucleotides of the target gene or RNA transcribed from the target
gene.
[0139] Polynucleotides comprising mismatches to the target gene or
transcript can be used in certain embodiments of the compositions
and methods disclosed herein. In some embodiments, the
polynucleotide comprises at least 18 or at least 19 contiguous
nucleotides that are essentially identical or essentially
complementary to a segment of equivalent length in the target gene
or target gene's transcript. In certain embodiments, a
polynucleotide of 19 contiguous nucleotides that is essentially
identical or essentially complementary to a segment of equivalent
length in the target gene or target gene's transcript can have 1 or
2 mismatches to the target gene or transcript. In certain
embodiments, a polynucleotide of 20 or more nucleotides that
comprises a contiguous 19 nucleotide span of identity or
complementarity to a segment of equivalent length in the target
gene or target gene's transcript can have 1 or 2 mismatches to the
target gene or transcript. In certain embodiments, a polynucleotide
of 21 continuous nucleotides that is essentially identical or
essentially complementary to a segment of equivalent length in the
target gene or target gene's transcript can have 1, 2, or 3
mismatches to the target gene or transcript. In certain
embodiments, a polynucleotide of 22 or more nucleotides that
contains a contiguous 21 nucleotide span of identity or
complementarity to a segment of equivalent length in the target
gene or target gene's transcript can have 1, 2, or 3 mismatches to
the target gene or transcript.
[0140] In designing polynucleotides with mismatches to a target
gene or to an RNA transcribed from the target gene, mismatches of
certain types and at certain positions that are more likely to be
tolerated can be used. In certain embodiments, mismatches formed
between adenine and cytosine or guanosine and uracil residues are
used as described by Du et al. (2005) Nucleic Acids Res.,
33:1671-1677. In some embodiments, mismatches in 19 base-pair
overlap regions are located at the low tolerance positions 5, 7, 8
or 11 (from the 5' end of a 19-nucleotide target), at medium
tolerance positions 3, 4, and 12-17 (from the 5' end of a
19-nucleotide target), and/or at the high tolerance positions at
either end of the region of complementarity, e. g., positions 1, 2,
18, and 19 (from the 5' end of a 19-nucleotide target) as described
by Du et al. (2005) Nucleic Acids Res., 33:1671-1677. Tolerated
mismatches can be empirically determined in routine assays such as
those described herein in the working Examples.
[0141] In some embodiments, the polynucleotides comprise additional
nucleotides (e.g., for reasons of stability or for convenience in
cloning or synthesis). In one embodiment, the polynucleotide is a
single-stranded RNA comprising an RNA strand with a segment of at
least 21 contiguous nucleotides of a sequence selected from the
group consisting of SEQ ID NOs:860-1718 and 1722-1975 and further
comprising an additional 5' G or an additional 3' C or both,
adjacent to the segment. In one embodiment, the polynucleotide is a
dsRNA comprising an RNA strand with a segment of at least 21
contiguous nucleotides of a sequence selected from the group
consisting of SEQ ID NOs:860-1718 and 1722-1975 and further
comprising an additional 5' G or an additional 3' C or both,
adjacent to the segment. In another embodiment, the polynucleotide
is a double-stranded RNA comprising additional nucleotides to form
an overhang, for example, a dsRNA comprising 2 deoxyribonucleotides
to form a 3' overhang.
Embedding Active Insecticidal Polynucleotides in Neutral
Sequence
[0142] In an embodiment, a polynucleotide with a sequence
complementary to the target gene and which is responsible for an
observed suppression of the target gene is embedded in "neutral"
sequence, e. g., inserted into additional nucleotides that have no
sequence identity or complementarity to the target gene. Neutral
sequence can be desirable, e. g., to increase the overall length of
a polynucleotide. For example, it can be desirable for a
polynucleotide to be of a particular size for reasons of stability,
cost-effectiveness in manufacturing, or biological activity.
[0143] It has been reported that in another coleopteran species,
Diabrotica virgifera, dsRNAs greater than or equal to approximately
60 base-pairs (bp) are required for biological activity in
artificial diet bioassays; see Bolognesi et al. (2012) PLoS ONE
7(10): e47534. doi:10.1371/journal.pone.0047534. Thus, in one
embodiment, a 21-base-pair dsRNA corresponding to a target gene in
Table 1 and found to provide control of an insect infestation is
embedded in neutral sequence of an additional 39 base pairs, thus
forming a polynucleotide of about 60 base pairs. In another
embodiment, a single 21-base-pair polynucleotide is found to be
efficacious when embedded in larger sections of neutral sequence,
e. g., where the total polynucleotide length is from about 60 to
about 300 base pairs. In another embodiment, at least one segment
of at least 21 contiguous nucleotides of a sequence selected from
the group consisting of SEQ ID NOs:860-1718 and 1722-1975 is
embedded in larger sections of neutral sequence to provide an
efficacious insecticidal polynucleotide. In another embodiment,
segments from multiple sequences selected from the group consisting
of SEQ ID NOs:860-1718 and 1722-1975 are embedded in larger
sections of neutral sequence to provide an efficacious insecticidal
polynucleotide.
[0144] It is anticipated that the combination of certain
recombinant polynucleotides disclosed herein (e. g., single-strand
RNA, dsRNA, single-strand DNA, or dsDNA comprising a sequence
selected from SEQ ID NOs:860-1718 and 1722-1975, or active
fragments thereof) with one or more non-polynucleotide pesticidal
agents will result in an improvement in prevention or control of
insect infestations, when compared to the effect obtained with the
polynucleotide alone or the non-polynucleotide pesticidal agent
alone. Routine insect bioassays such as the bioassays described
herein in the working Examples are useful for defining
dose-responses for larval mortality or growth inhibition using
combinations of the polynucleotides disclosed herein and one or
more non-polynucleotide pesticidal agents (e. g., a patatin, a
plant lectin, a phytoecdysteroid, a Bacillus thuringiensis
insecticidal protein, a Xenorhabdus insecticidal protein, a
Photorhabdus insecticidal protein, a Bacillus laterosporous
insecticidal protein, and a Bacillus sphaericus insecticidal
protein). One of skill in the art can test combinations of
polynucleotides and non-polynucleotide pesticidal agents in routine
bioassays to identify combinations of bioactives that are effective
for use in protecting plants from insect infestations.
Example 5
[0145] This example illustrates non-limiting embodiments of methods
of testing the efficacy of insecticidal polynucleotides in flea
beetles. More specifically this example illustrates a method
including oral delivery of polynucleotides to flea beetles,
resulting in stunting or mortality in the flea beetles.
[0146] P. cruciferae were collected from a canola field where no
pesticides had been applied in the previous 3 months. Three dsRNAs
(SEQ ID NOs:1169, 1193, and 1392) targeting Phyllotreta genes and
one negative control dsRNA targeting GFP were tested on groups of
30 P. cruciferae. The dsRNAs were resuspended in water and applied
to 6 millimeter leaf discs (55.+-.6 milligrams each) at a
discriminating dose of 50 nanograms dsRNA/milligram leaf tissue,
which were fed to groups of 5 flea beetles. Leaf discs with freshly
applied dsRNA were replaced every other day, and the number of
surviving individuals was recorded over a 2-week period. A low
non-specific mortality rate was observed in the negative-control
insect groups (3 out of 30 insects dying over 2 weeks, or 10%
non-specific mortality). Mortality was observed beginning at day 4
and continuing through the 2 week period. Specific mortality was
observed for all dsRNA treatments (Table 2). Correcting for
non-specific mortality (subtracting non-specific mortality rate of
3 insects per group for corrected N=27), the percent mortality
observed at the end of the 2-week period was 85% (SEQ ID NO:1169),
0.70% (SEQ ID NO:1193), and 0.63% (SEQ ID NO:1392). These results
demonstrated the efficacy of the dsRNAs in causing mortality in
flea beetles when provided in the flea beetles' diet.
TABLE-US-00002 TABLE 2 Cumulative number of dead P. cruciferae (N =
30) Negative SEQ ID SEQ ID SEQ ID Day control NO: 1169 NO: 1193 NO:
1392 0 0 0 0 0 2 1 2 1 1 4 2 6 2 2 6 2 9 3 3 8 2 13 8 9 10 3 18 16
15 12 3 23 19 18 14 3 26 22 20
[0147] In a second series of experiments carried out in a similar
manner, several dsRNAs were tested at a discriminating dose of 50
nanograms dsRNA/milligram leaf tissue on P. cruciferae; two lower
doses (15 nanograms dsRNA/milligram leaf tissue and 2 nanograms
dsRNA/milligram leaf tissue) were also tested. Ten beetles were
tested at each dose. The negative control (five replicates) used
was a dsRNA targetting the bacterial gene uidA encoding
beta-glucuronidase (NCBI accession number NC_000913.3). Leaf discs
with freshly applied dsRNA were replaced every other day, and
mortality was recorded over a 12 day period. The overall mortality
rate for the negative controls was 4% (likely due to handling
injuries) over the 12-day observation period. The observed
cumulative mortality (N=10) following 12 days exposure to the
dsRNAs are provided in Table 3; the negative control mortality
values are given as an average (N=5). Eight of the dsRNAs
(indicated by a mortality rating of +++) caused 90-100% mortality
at the highest dose and were still highly effective (80% or higher
mortality) at the lowest dose tested. Some dsRNAs (indicated by a
mortality rating of ++) induced mortality in a proportion of the
insects at the highest dose, but were less effective at lower doses
(<20% mortality).
TABLE-US-00003 TABLE 3 dsRNA cumulative mortality (N = 10 ) SEQ ID
50 15 2 Mortality NO**: ng/mg ng/mg ng/mg Rating 870 3 1 0 - 876 3
1 0 - 1156 2 0 0 - 1157 10 3 0 - 1158 2 0 0 - 1159 5 2 0 - 1160 2 0
0 - 1161 10 3 1 - 1163 4 4 2 - 1164 10 8 4 ++ 1165 8 7 5 ++ 1166 5
2 2 - 1167 6 2 0 - 1168 8 5 0 - 1169 10 10 8 +++ 1170 9 5 3 - 1171
10 10 6 ++ 1392 3 2 0 - 1393 6 1 0 - 1186 9 7 5 - 1394 9 9 5 - 1187
9 9 8 +++ 1193 10 10 9 +++ 1210 6 3 1 - 1219 8 3 2 - 1224 10 9 8
+++ 1234 10 7 6 ++ 1243 5 2 0 - 1258 9 4 2 - 1396 9 6 5 ++ 1397 6 3
0 - 1398 8 7 7 ++ 1399 10 10 8 +++ 1400 2 1 1 - 1403 9 6 6 - 1404
10 7 4 - 1405 6 6 2 - 1406 9 9 7 +++ 1407 10 9 8 +++ 1408 9 9 9 +++
negative control 0.6 0.6 0.4 - (GFP) negative control 0.4 0.6 0.2 -
(beta-glucuronidase) negative control 0.2 0.4 0.6 - (water only)
*+++rating indicates high (>80%) mortalities for all three
doses; ++rating indicates high mortalities for the highest dose,
and within 40 to 70% mortality with the lower two doses. **
sequences are provided for the anti-sense strand of the dsRNA in 5`
to 3` direction.
[0148] Other techniques for delivering these or similar
insecticidal polynucleotides are contemplated and include applying
the polynucleotides directly to the insect surface (e. g. by
spraying or dusting), or providing the polynucleotidesto the insect
in a diet or bait (e. g., in a bacterial or plant cell expressing a
dsRNA, or in an artificial bait containing the dsRNA). In an
embodiment, a hairpin version of the Phyllotreta insecticidal
polynucleotide with the sequence SEQ ID NO: 1169 is designed; this
hairpin version is encoded by the DNA sequence SEQ ID NO:1722,
which contains, in 5' to 3' order, anti-sense sequence (nucleotide
positions 1-267), loop sequence (nucleotide positions 268-373)
which does not contain matches to Phyllotreta sequences, and sense
sequence (nucleotide positions 374-640). This DNA sequence is
expressed as a single-stranded RNA transcript, where the anti-sense
and sense regions anneal to form the double-stranded "stem" region
of the hairpin. The construct is expressed in a bacterium, such as
E. coli; the resulting dsRNA hairpin produced in the bacterium is
provided to flea beetles as a crude or purified fermentation
product, or in the form of the bacterial cells. Similar constructs
are designed encoding dsRNAs having modified stem-loops, such as
"stabilized anti-sense" or "stabilized sense" versions, which
contain stabilized loops formed by an extended anti-sense or sense
sequence, respectively, of sequence corresponding to the intended
target gene.
Example 6
[0149] This example discloses embodiments related to polynucleotide
molecules having a nucleotide sequence containing specific
modifications such as nucleotide substitutions. Embodiments of such
modifications include modified polynucleotides that provide
improved sequence discrimination between the intended target gene
of the insect pest of interest, and genetic sequences of other,
non-target species.
[0150] Selected dsRNAs identified in Table 1 were screened for
unintended sequence matches to a sequence of at least 19 contiguous
nucleotides identified in a non-target gene or a non-target
organism (NTO, e. g., Apis mellifera, Bombus impatiens and B.
terrestris; Coleomegilla spp.; Danaus plexippus; Homo sapiens;
Megachile rotundata; Mus musculus; and Brassica rapa). Nucleotide
changes are made in an original polynucleotide sequence to
eliminate contiguous sequence matches to a non-target gene or
non-target organism. Examples of such modified polynucleotide
sequences are provided by SEQ ID NO:1723, which corresponds to SEQ
ID NO:1393 (which targets the same flea beetle gene as does the
insecticidal polynucleotide of SEQ ID NO:1392), and SEQ ID NO:1724,
which corresponds to SEQ ID NO:1169.
Example 7
[0151] This example discloses embodiments related to polynucleotide
molecules having a nucleotide sequence for silencing a target gene
in more than one species. Embodiments include dsRNA sequences of at
least 21 contiguous nucleotides identified as having 100%
complementarity or identity to more than one ortholog of a target
gene.
[0152] Table 4 provides a list of sequences, each at least 21
contiguous nucleotides in length and identified by the sequence's
coordinates in a dsRNA for one flea beetle species, wherein the
identical sequence is also found in a dsRNA for a different flea
beetle species. These sequences are useful to design insecticidal
polynucleotides against multiple species in which the target
sequence co-occurs. For example, SEQ ID NO:1186 (targetting a
Phyllotreta cruciferae COPI alpha target gene, SEQ ID NO:327)
contains five sequences of at least 21 contiguous nucleotides at
positions 1-71, 88-116, 136-209, 238-266, and 274-296, all of which
match a sequence in SEQ ID NOs:882 and 888 (targetting a
Phyllotreta atra COPI alpha target genes, SEQ ID NOs:23 and 29,
respectively); these five sequences are therefore useful in
targeting a gene in the two Phyllotreta species.
TABLE-US-00004 TABLE 4 QUERY Polynucleotide SEQ SUBJECT ID
Polynucleotide NO: start end SEQ ID NO: 1186 1 71 882,888 1186 88
116 882,888 1186 136 209 882,888 1186 238 266 882,888 1186 274 296
882,888 1191 51 96 900-908, 910 1191 137 177 900-908, 910 1191 179
216 900-908, 910 1191 218 255 900-908, 910 1191 257 342 900-908,
910 1192 2 25 900-908, 910 1192 66 106 900-908, 910 1192 108 145
900-908, 910 1192 147 184 900-908, 910 1192 186 271 900-908, 910
1192 273 298 900-908, 910 1192 300 330 900-908, 910 1191 58 96
900-908, 910 1192 300 336 900-908, 910 1191 44 96 900-908, 910 1191
179 200 900-908, 910 1192 108 129 900-908, 910 1191 218 242
900-908, 910 1192 147 171 900-908, 910 1191 257 277 900-908, 910
1192 186 206 900-908, 910 1191 257 334 900-908, 910 1192 186 263
900-908, 910 1191 257 320 900-908, 910 1192 186 249 900-908, 910
1193 15 101 911, 915, 916, 919 1194 6 92 911, 915, 916, 919 1195 2
107 911, 915, 916, 919 1195 121 207 911, 915, 916, 919 1196 2 122
911, 915, 916, 919 1196 136 222 911, 915, 916, 919 1197 2 20 911,
915, 916, 919 1197 22 42 911, 915, 916, 919 1197 44 180 911, 915,
916, 919 1197 194 280 911, 915, 916, 919 1198 18 38 911, 915, 916,
919 1198 40 176 911, 915, 916, 919 1198 190 276 911, 915, 916, 919
1199 5 25 911, 915, 916, 919 1199 27 163 911, 915, 916, 919 1199
177 263 911, 915, 916, 919 1200 16 152 911, 915, 916, 919 1200 166
252 911, 915, 916, 919 1201 19 105 911, 915, 916, 919 1202 2 119
911, 915, 916, 919 1202 133 219 911, 915, 916, 919 1203 14 100 911,
915, 916, 919 1193 123 189 911, 915, 916, 919 1194 114 180 911,
915, 916, 919 1195 229 295 911, 915, 916, 919 1196 244 310 911,
915, 916, 919 1197 302 350 911, 915, 916, 919 1198 298 350 911,
915, 916, 919 1199 285 352 911, 915, 916, 919 1200 274 340 911,
915, 916, 919 1201 127 193 911, 915, 916, 919 1202 241 307 911,
915, 916, 919 1203 122 188 911, 915, 916, 919 1193 123 196 911,
915, 916, 919 1193 198 230 911, 915, 916, 919 1194 114 187 911,
915, 916, 919 1194 189 221 911, 915, 916, 919 1195 229 302 911,
915, 916, 919 1195 304 336 911, 915, 916, 919 1196 1 122 911, 915,
916, 919 1196 244 317 911, 915, 916, 919 1196 319 351 911, 915,
916, 919 1197 60 180 911, 915, 916, 919 1198 56 176 911, 915, 916,
919 1199 43 163 911, 915, 916, 919 1199 285 351 911, 915, 916, 919
1200 32 152 911, 915, 916, 919 1200 274 347 911, 915, 916, 919 1201
127 200 911, 915, 916, 919 1201 202 234 911, 915, 916, 919 1202 241
314 911, 915, 916, 919 1202 316 348 911, 915, 916, 919 1203 122 195
911, 915, 916, 919 1203 197 229 911, 915, 916, 919 1204 6 25 920,
921, 922, 923, 924 1204 27 49 920, 921, 922, 923, 924 1204 51 118
920, 921, 922, 923, 924 1204 186 214 920, 921, 922, 923, 924 1204
219 268 920, 921, 922, 923, 924 1204 270 295 920, 921, 922, 923,
924 1204 306 351 920, 921, 922, 923, 924 1205 180 199 920, 921,
922, 923, 924 1205 201 223 920, 921, 922, 923, 924 1205 225 292
920, 921, 922, 923, 924 1206 10 29 920, 921, 922, 923, 924 1206 31
53 920, 921, 922, 923, 924 1206 55 122 920, 921, 922, 923, 924 1206
190 218 920, 921, 922, 923, 924 1206 223 272 920, 921, 922, 923,
924 1206 274 299 920, 921, 922, 923, 924 1206 310 351 920, 921,
922, 923, 924 1207 11 33 920, 921, 922, 923, 924 1207 35 102 920,
921, 922, 923, 924 1207 170 198 920, 921, 922, 923, 924 1207 203
252 920, 921, 922, 923, 924 1207 254 279 920, 921, 922, 923, 924
1207 290 335 920, 921, 922, 923, 924 1208 18 40 920, 921, 922, 923,
924 1208 42 109 920, 921, 922, 923, 924 1208 177 205 920, 921, 922,
923, 924 1208 210 259 920, 921, 922, 923, 924 1208 261 286 920,
921, 922, 923, 924 1208 297 342 920, 921, 922, 923, 924 1209 101
120 920, 921, 922, 923, 924 1209 122 144 920, 921, 922, 923, 924
1209 146 213 920, 921, 922, 923, 924 1209 281 309 920, 921, 922,
923, 924 1209 314 351 920, 921, 922, 923, 924 1205 2 91 920, 921,
922, 923, 924 1205 114 142 920, 921, 922, 923, 924 1205 144 178
920, 921, 922, 923, 924 1209 35 63 920, 921, 922, 923, 924 1209 65
99 920, 921, 922, 923, 924 1204 306 347 920, 921, 922, 923, 924
1207 290 331 920, 921, 922, 923, 924 1208 297 338 920, 921, 922,
923, 924 1207 290 352 920, 921, 922, 923, 924 1208 297 351 920,
921, 922, 923, 924 1205 18 91 920, 921, 922, 923, 924 1224 1 43
947-949, 951-956 1224 57 95 947-949, 951-956 1224 97 145 947-949,
951-956 1224 147 175 947-949, 951-956 1224 177 208 947-949, 951-956
1224 210 262 947-949, 951-956 1224 285 350 947-949, 951-956 1225
150 192 947, 949, 951-956 1225 206 244 947, 949, 951-956 1225 246
294 947, 949, 951-956 1225 296 324 947, 949, 951-956 1225 326 351
947, 949, 951-956 1226 258 300 947, 949, 951-955 1226 314 351 947,
949, 951-955 1227 253 295 947, 949, 951-955 1227 309 347 947, 949,
951-955 1228 206 248 947, 949, 951-956 1228 262 300 947, 949,
951-956 1228 302 350 947, 949, 951-956 1229 171 213 947, 949,
951-956 1229 227 265 947, 949, 951-956 1229 267 315 947, 949,
951-956 1229 317 345 947, 949, 951-956 1230 252 294 947, 949,
951-955 1230 308 346 947, 949, 951-955 1231 191 233 947, 949,
951-956 1231 247 285 947, 949, 951-956 1231 287 335 947, 949,
951-956 1232 196 238 947, 949, 951-956 1232 252 290 947, 949,
951-956 1232 292 340 947, 949, 951-956 1233 209 251 947, 949,
951-956 1233 265 303 947, 949, 951-956 1233 305 351 947, 949,
951-956 1224 177 202 947-949, 951-956 1225 1 72 947, 949, 951-956
1225 74 135 947, 949, 951-956 1225 137 192 947, 949, 951-956 1226
110 180 947, 949, 951-955 1226 182 243 947, 949, 951-955 1226 245
300 947, 949, 951-955 1227 105 175 947, 949, 951-955 1227 177 238
947, 949, 951-955 1227 240 295 947, 949, 951-955 1228 58 128 947,
949, 951-956 1228 130 191 947, 949, 951-956 1228 193 248 947, 949,
951-956 1229 23 93 947, 949, 951-956 1229 95 156 947, 949, 951-956
1229 158 213 947, 949, 951-956 1230 104 174 947, 949, 951-955 1230
176 237 947, 949, 951-955 1230 239 294 947, 949, 951-955 1231 43
113 947, 949, 951-956 1231 115 176 947, 949, 951-956 1231 178 233
947, 949, 951-956 1232 48 118 947, 949, 951-956 1232 120 181 947,
949, 951-956 1232 183 238 947, 949, 951-956 1233 61 131 947, 949,
951-956 1233 133 194 947, 949, 951-956 1233 196 251 947, 949,
951-956 1224 227 262 947-949, 951-956 1224 57 94 947-949, 951-956
1225 2 72 947, 949, 951-956 1225 206 243 947, 949, 951-956 1226 1
96 947, 949, 951-955 1226 98 180 947, 949, 951-955 1227 2 91 947,
949, 951-955 1227 93 175 947, 949, 951-955 1227 309 346 947, 949,
951-955 1228 2 44 947, 949, 951-956 1228 46 128 947, 949, 951-956
1228 262 299 947, 949, 951-956 1229 11 93 947, 949, 951-956 1229
227 264 947, 949, 951-956 1230 2 90 947, 949, 951-955 1230 92 174
947, 949, 951-955 1230 308 345 947, 949, 951-955 1231 2 29 947,
949, 951-956 1231 31 113 947, 949, 951-956 1231 247 284 947, 949,
951-956 1232 2 34 947, 949, 951-956 1232 36 118 947, 949, 951-956
1232 252 289 947, 949, 951-956 1233 2 47 947, 949, 951-956 1233 49
131 947, 949, 951-956 1233 265 302 947, 949, 951-956 1226 7 96 947,
949, 951-955 1227 1 91 947, 949, 951-955 1226 8 96 947, 949,
951-955 1227 3 91 947, 949, 951-955 1230 1 90 947, 949, 951-955
1226 54 96 947, 949, 951-955 1227 49 91 947, 949, 951-955 1228 1 44
947, 949, 951-956 1230 48 90 947, 949, 951-955 1233 5 47 947, 949,
951-956 1224 125 145 947-949, 951-956 1225 274 294 947, 949,
951-956 1228 330 350 947, 949, 951-956 1229 295 315 947, 949,
951-956 1231 315 335 947, 949, 951-956 1232 320 340 947, 949,
951-956 1233 333 351 947, 949, 951-956 1234 50 135 960, 964 1234
149 168 960, 964 1234 170 350 960, 964 1235 175 260 960, 963, 964
1235 274 293 960, 963, 964 1235 295 351 960, 963, 964 1236 190 275
960, 963, 964 1236 289 308 960, 963, 964
1236 310 351 960, 963, 964 1237 269 350 960, 963, 964 1238 51 136
960, 964 1238 150 169 960, 964 1238 171 350 960, 964 1239 50 135
960, 964 1239 149 168 960, 964 1239 170 351 960, 964 1240 103 188
960, 963, 964 1240 202 221 960, 963, 964 1240 223 351 960, 963, 964
1241 269 351 960, 963, 964 1242 87 172 960, 963, 964 1242 186 205
960, 963, 964 1242 207 351 960, 963, 964 1235 2 23 960, 963, 964
1235 25 114 960, 963, 964 1236 2 38 960, 963, 964 1236 40 129 960,
963, 964 1237 2 39 960, 963, 964 1237 41 117 960, 963, 964 1237 119
208 960, 963, 964 1240 2 42 960, 963, 964 1241 2 39 960, 963, 964
1241 41 117 960, 963, 964 1241 119 208 960, 963, 964 1242 2 26 960,
963, 964 1234 38 135 960, 964 1234 170 211 960, 964 1235 25 136
960, 963, 964 1235 163 260 960, 963, 964 1235 295 336 960, 963, 964
1236 1 38 960, 963, 964 1236 40 151 960, 963, 964 1236 178 275 960,
963, 964 1237 81 117 960, 963, 964 1237 119 230 960, 963, 964 1237
257 350 960, 963, 964 1238 39 136 960, 964 1238 171 212 960, 964
1239 38 135 960, 964 1239 170 211 960, 964 1240 2 64 960, 963, 964
1240 91 188 960, 963, 964 1240 223 264 960, 963, 964 1241 81 117
960, 963, 964 1241 119 230 960, 963, 964 1241 257 351 960, 963, 964
1242 2 48 960, 963, 964 1242 75 172 960, 963, 964 1242 207 248 960,
963, 964 1243 118 158 965, 966, 968, 969 1243 283 304 965, 966,
968, 969 1243 310 348 965, 966, 968, 969 1244 165 205 965, 966,
968, 969 1244 330 351 965, 966, 968, 969 1246 205 245 965, 966,
967, 968, 969 1247 206 246 965, 966, 967, 968, 969 1248 163 203
965, 966, 968, 969 1248 328 349 965, 966, 968, 969 1245 1 20 967
1245 22 42 967 1245 104 135 967 1245 137 155 967 1245 187 209 967
1246 36 58 965, 966, 967, 968, 969 1247 37 59 965, 966, 967, 968,
969 1244 330 352 965, 966, 968, 969 1249 2 20 970, 973, 974, 978,
979 1250 5 84 969-971, 973-979 1250 86 270 969-971, 973-979 1250
278 306 969-971, 973-979 1250 326 351 969-971, 973-979 1251 2 52
969-971, 973-979 1251 54 238 969-971, 973-979 1251 246 274 969-971,
973-979 1251 294 322 969-971, 973-979 1253 115 194 969-971, 973-979
1253 196 351 969-971, 973-979 1254 3 31 970, 971, 973, 974, 977-979
1254 51 79 970, 971, 973, 974, 977-979 1255 282 352 970, 971,
973-978, 1626, 1629 1256 1 53 970, 971, 973-978 1256 55 239 970,
971, 973-978 1256 247 275 970, 971, 973-978 1256 295 323 970, 971,
973-978 1257 6 85 970, 971, 973-978 1257 87 271 970, 971, 973-978
1257 279 307 970, 971, 973-978 1257 327 351 970, 971, 973-978 1250
1 84 969-971, 973-979 1250 326 352 969-971, 973-979 1251 294 319
969-971, 973-979 1253 112 194 969-971, 973-979 1254 51 76 970, 971,
973, 974, 977-979 1255 279 352 970, 971, 973-978, 1626, 1629 1256
295 320 970, 971, 973-978 1257 3 85 970, 971, 973-978 1252 31 89
972 1252 91 236 972 1252 238 266 972 1252 277 337 972 1249 2 23
970, 973, 974, 978, 979 1249 25 44 970, 973, 974, 978, 979 1250 34
84 969-971, 973-979 1251 1 52 969-971, 973-979 1251 294 325
969-971, 973-979 1251 327 346 969-971, 973-979 1253 144 194
969-971, 973-979 1254 51 82 970, 971, 973, 974, 977-979 1254 84 103
970, 971, 973, 974, 977-979 1255 311 352 970, 971, 973-978, 1626,
1629 1256 3 53 970, 971, 973-978 1256 295 326 970, 971, 973-978
1256 328 347 970, 971, 973-978 1257 35 85 970, 971, 973-978 1250 47
84 969-971, 973-979 1251 15 52 969-971, 973-979 1253 157 194
969-971, 973-979 1255 324 352 970, 971, 973-978, 1626, 1629 1256 16
53 970, 971, 973-978 1257 48 85 970, 971, 973-978 1250 2 84
969-971, 973-979 1250 86 241 969-971, 973-979 1251 54 209 969-971,
973-979 1253 1 68 969-971, 973-979 1253 91 194 969-971, 973-979
1253 196 352 969-971, 973-979 1255 169 235 970, 971, 973-978, 1626,
1629 1255 258 352 970, 971, 973-978, 1626, 1629 1256 55 210 970,
971, 973-978 1257 1 85 970, 971, 973-978 1257 87 242 970, 971,
973-978 1250 86 110 969-971, 973-979 1251 54 78 969-971, 973-979
1253 2 68 969-971, 973-979 1253 196 220 969-971, 973-979 1255 39
130 970, 971, 973-978, 1626, 1629 1255 132 235 970, 971, 973-978,
1626, 1629 1256 55 79 970, 971, 973-978 1257 87 111 970, 971,
973-978 1250 326 350 969-971, 973-979 1251 294 318 969-971, 973-979
1253 110 194 969-971, 973-979 1254 51 75 970, 971, 973, 974,
977-979 1255 277 352 970, 971, 973-978, 1626, 1629 1256 295 319
970, 971, 973-978 1257 327 352 970, 971, 973-978 1250 32 84
969-971, 973-979 1253 142 194 969-971, 973-979 1255 309 352 970,
971, 973-978, 1626, 1629 1257 33 85 970, 971, 973-978 1249 46 68
970, 973, 974, 978, 979 1250 61 84 969-971, 973-979 1251 29 52
969-971, 973-979 1253 171 194 969-971, 973-979 1254 105 127 970,
971, 973, 974, 977-979 1256 30 53 970, 971, 973-978 1257 62 85 970,
971, 973-978 1259 192 226 999-1004 1259 249 298 999-1004 1259 321
352 999-1004 1260 190 224 999-1004 1260 247 296 999-1004 1260 319
351 999-1004 1261 99 133 999-1004 1261 156 205 999-1004 1261 228
289 999-1004 1261 300 334 999-1004 1262 82 116 999-1004 1262 139
188 999-1004 1262 211 272 999-1004 1262 283 317 999-1004 1262 319
350 999-1004 1263 324 351 999-1004 1264 191 225 999-1004 1264 248
297 999-1004 1264 320 351 999-1004 1265 98 132 999-1004 1265 155
204 999-1004 1265 227 288 999-1004 1265 299 333 999-1004 1259 95
145 999-1004 1259 165 226 999-1004 1260 93 143 999-1004 1260 163
224 999-1004 1261 1 52 999-1004 1261 72 133 999-1004 1262 1 35
999-1004 1262 55 116 999-1004 1263 227 277 999-1004 1263 297 351
999-1004 1264 94 144 999-1004 1264 164 225 999-1004 1265 2 51
999-1004 1265 71 132 999-1004 1259 15 40 999-1004 1259 42 64
999-1004 1259 66 88 999-1004 1259 90 145 999-1004 1259 165 219
999-1004 1260 13 38 999-1004 1260 40 62 999-1004 1260 64 86
999-1004 1260 88 143 999-1004 1260 163 217 999-1004 1261 2 52
999-1004 1261 72 126 999-1004 1262 55 109 999-1004 1263 1 60
999-1004 1263 105 127 999-1004 1263 147 172 999-1004 1263 174 196
999-1004 1263 198 220 999-1004 1263 222 277 999-1004 1263 297 352
999-1004 1264 14 39 999-1004 1264 41 63 999-1004 1264 65 87
999-1004 1264 89 144 999-1004 1264 164 218 999-1004 1265 71 125
999-1004 1259 190 226 999-1004 1260 188 224 999-1004 1261 97 133
999-1004 1262 80 116 999-1004 1263 322 351 999-1004 1264 189 225
999-1004 1265 96 132 999-1004 1259 96 145 999-1004 1260 94 143
999-1004 1261 3 52 999-1004 1263 228 277 999-1004 1264 95 144
999-1004 1265 1 51 999-1004 1259 165 218 999-1004 1260 163 216
999-1004 1261 72 125 999-1004 1262 55 108 999-1004 1263 2 60
999-1004 1263 297 350 999-1004 1264 164 217 999-1004 1265 71 124
999-1004 1266 2 39 1016-1023 1266 41 87 1016-1023 1266 89 129
1016-1023 1267 2 74 1016-1023 1267 76 122 1016-1023 1267 124 164
1016-1023 1268 2 53 1016-1023 1268 55 101 1016-1023
1268 103 143 1016-1023 1269 2 72 1016-1023 1269 74 120 1016-1023
1269 122 162 1016-1023 1270 2 107 1016-1023 1270 109 155 1016-1023
1270 157 197 1016-1023 1271 2 62 1016-1023 1271 64 110 1016-1023
1271 112 152 1016-1023 1266 131 177 1016-1023 1266 179 207
1016-1023 1266 209 243 1016-1023 1267 166 212 1016-1023 1267 214
242 1016-1023 1267 244 278 1016-1023 1268 145 191 1016-1023 1268
193 221 1016-1023 1268 223 257 1016-1023 1269 164 210 1016-1023
1269 212 240 1016-1023 1269 242 276 1016-1023 1270 199 245
1016-1023 1270 247 275 1016-1023 1270 277 311 1016-1023 1271 154
200 1016-1023 1271 202 230 1016-1023 1271 232 266 1016-1023 1266
209 294 1016-1023 1266 296 316 1016-1023 1267 1 74 1016-1023 1267
244 329 1016-1023 1267 331 351 1016-1023 1268 223 308 1016-1023
1268 310 330 1016-1023 1269 242 327 1016-1023 1269 329 349
1016-1023 1270 35 107 1016-1023 1270 277 351 1016-1023 1271 232 317
1016-1023 1271 319 339 1016-1023 1266 296 318 1016-1023 1267 4 74
1016-1023 1268 310 332 1016-1023 1269 1 72 1016-1023 1269 329 351
1016-1023 1270 37 107 1016-1023 1271 319 341 1016-1023 1266 131 157
1016-1023 1267 166 192 1016-1023 1268 145 171 1016-1023 1269 164
190 1016-1023 1270 199 225 1016-1023 1271 154 180 1016-1023 1295
113 168 1026, 1027, 1024 1295 170 201 1026, 1027, 1024 1295 203 233
1026, 1027, 1024 1295 236 258 1026, 1027, 1024 1297 128 183 1026,
1027, 1024 1297 185 216 1026, 1027, 1024 1297 218 248 1026, 1027,
1024 1296 1 44 1025, 1028, 1029, 1515-1518, 1520 1298 7 57 1025,
1028, 1029 1298 59 96 1025, 1028, 1029 1298 110 132 1025, 1028,
1029 1298 143 210 1025, 1028, 1029 1295 17 87 1026, 1027, 1024 1297
32 102 1026, 1027, 1024 1294 127 153 1027 1295 17 46 1026, 1027,
1024 1297 32 61 1026, 1027, 1024 1298 5 57 1025, 1028, 1029 1320
249 268 1033, 1035, 1076 1324 2 23 1036, 1037, 1038 1326 14 38
1036, 1037, 1038 1326 95 117 1036, 1037, 1038 1326 152 174 1036,
1037, 1038 1326 227 248 1036, 1037, 1038 1327 44 65 1036, 1037,
1038 1321 65 86 1037 1321 88 132 1037 1322 2 34 1037 1322 36 67
1037 1322 135 156 1037 1322 158 202 1037 1323 1 35 1037 1323 37 68
1037 1323 136 157 1037 1323 159 203 1037 1324 2 51 1036, 1037, 1038
1324 56 93 1036, 1037, 1038 1324 95 126 1036, 1037, 1038 1324 194
215 1036, 1037, 1038 1324 217 261 1036, 1037, 1038 1325 2 31 1037
1325 33 64 1037 1325 132 153 1037 1325 155 199 1037 1326 227 276
1036, 1037, 1038 1326 281 318 1036, 1037, 1038 1326 320 350 1036,
1037, 1038 1327 44 93 1036, 1037, 1038 1327 98 135 1036, 1037, 1038
1327 137 168 1036, 1037, 1038 1327 236 257 1036, 1037, 1038 1327
259 303 1036, 1037, 1038 1324 2 24 1036, 1037, 1038 1326 227 249
1036, 1037, 1038 1327 44 66 1036, 1037, 1038 1328 3 59 1039-1043,
1045, 1594, 1597, 1598 1328 70 146 1039-1043, 1045, 1594, 1597,
1598 1328 148 215 1039-1043, 1045, 1594, 1597, 1598 1328 232 263
1039-1043, 1045, 1594, 1597, 1598 1328 328 350 1039-1043, 1045,
1594, 1597, 1598 1329 22 53 1039-1043 1045 1329 118 140 1039-1043
1045 1330 31 62 1039-1043 1045 1330 127 149 1039-1043 1045 1331 28
84 1039-1043, 1045, 1594, 1597, 1598 1331 95 171 1039-1043, 1045,
1594, 1597, 1598 1331 173 240 1039-1043, 1045, 1594, 1597, 1598
1331 257 288 1039-1043, 1045, 1594, 1597, 1598 1332 2 26 1039-1043,
1045, 1594, 1597, 1598 1332 37 113 1039-1043, 1045, 1594, 1597,
1598 1332 115 182 1039-1043, 1045, 1594, 1597, 1598 1332 199 230
1039-1043, 1045, 1594, 1597, 1598 1332 295 317 1039-1043, 1045,
1594, 1597, 1598 1333 4 60 1039-1043, 1045, 1594, 1597, 1598 1333
71 147 1039-1043, 1045, 1594, 1597, 1598 1333 149 216 1039-1043,
1045, 1594, 1597, 1598 1333 233 264 1039-1043, 1045, 1594, 1597,
1598 1333 329 351 1039-1043, 1045, 1594, 1597, 1598 1334 42 98
1039-1043, 1045, 1594, 1597, 1598 1334 109 185 1039-1043, 1045,
1594, 1597, 1598 1334 187 254 1039-1043, 1045, 1594, 1597, 1598
1334 271 302 1039-1043, 1045, 1594, 1597, 1598 1335 2 47 1039-1043,
1045, 1594, 1597, 1598 1335 49 116 1039-1043, 1045, 1594, 1597,
1598 1335 133 164 1039-1043, 1045, 1594, 1597, 1598 1335 229 251
1039-1043, 1045, 1594, 1597, 1598 1336 34 90 1039-1043, 1045, 1594,
1597, 1598 1336 101 177 1039-1043, 1045, 1594, 1597, 1598 1336 179
246 1039-1043, 1045, 1594, 1597, 1598 1336 263 294 1039-1043, 1045,
1594, 1597, 1598 1338 40 96 1039-1043, 1045, 1594, 1597, 1598 1338
107 183 1039-1043, 1045, 1594, 1597, 1598 1338 185 252 1039-1043,
1045, 1594, 1597, 1598 1338 269 300 1039-1043, 1045, 1594,
1597,1598 1328 35 59 1039-1043, 1045, 1594, 1597, 1598 1331 60 84
1039-1043, 1045, 1594, 1597, 1598 1332 1 26 1039-1043, 1045, 1594,
1597, 1598 1333 36 60 1039-1043, 1045, 1594, 1597, 1598 1334 74 98
1039-1043, 1045, 1594, 1597, 1598 1336 66 90 1039-1043, 1045, 1594,
1597, 1598 1338 72 96 1039-1043, 1045, 1594, 1597, 1598 1331 1 26
1039-1043, 1045, 1594, 1597, 1598 1334 2 40 1039-1043, 1045, 1594,
1597, 1598 1336 2 32 1039-1043, 1045, 1594, 1597, 1598 1338 2 38
1039-1043, 1045, 1594, 1597, 1598 1328 36 59 1039-1043, 1045, 1594,
1597, 1598 1331 61 84 1039-1043, 1045, 1594, 1597, 1598 1332 3 26
1039-1043, 1045, 1594, 1597, 1598 1333 37 60 1039-1043, 1045, 1594,
1597, 1598 1334 75 98 1039-1043, 1045, 1594, 1597, 1598 1336 67 90
1039-1043, 1045, 1594, 1597, 1598 1338 73 96 1039-1043, 1045, 1594,
1597, 1598 1329 172 191 1039-1043 1045 1329 265 296 1039-1043 1045
1329 298 317 1039-1043 1045 1329 319 343 1039-1043 1045 1330 181
200 1039-1043 1045 1330 274 305 1039-1043 1045 1330 307 326
1039-1043 1045 1330 328 352 1039-1043 1045 1335 283 302 1039-1043,
1045, 1594, 1597, 1598 1337 1 19 1043, 1045 1337 93 124 1043, 1045
1337 126 145 1043, 1045 1337 147 171 1043, 1045 1329 319 352
1039-1043 1045 1337 147 180 1043, 1045 1386 2 38 1054-1057, 1060,
1496, 1498 1386 40 59 1054- 1057, 1060, 1496, 1498 1386 70 95
1054-1057, 1060, 1496, 1498 1386 103 161 1054-1057, 1060, 1496,
1498 1386 196 262 1054-1057, 1060, 1496, 1498 1388 2 51 1054-1057,
1060, 1496, 1498 1388 53 72 1054-1057, 1060, 1496, 1498 1388 83 108
1054-1057, 1060, 1496, 1498 1388 116 174 1054-1057, 1060, 1496,
1498 1388 209 275 1054-1057, 1060, 1496, 1498 1390 88 119
1054-1057, 1060, 1492, 1496-1498 1390 145 194 1054-1057, 1060,
1492, 1496-1498 1390 196 215 1054-1057, 1060, 1492, 1496-1498 1390
226 251 1054-1057, 1060, 1492, 1496-1498 1390 259 317 1054-1057,
1060, 1492, 1496-1498 1391 19 50 1054-1057, 1060, 1492, 1496-1498
1391 76 125 1054-1057, 1060, 1492, 1496-1498 1391 127 146
1054-1057, 1060, 1492, 1496-1498 1391 157 182 1054-1057, 1060,
1492, 1496-1498 1391 190 248 1054-1057, 1060, 1492, 1496-1498 1391
283 349 1054-1057, 1060, 1492, 1496-1498 1384 20 39 1055, 1058,
1059, 1061-1063, 1500 1384 62 84 1055, 1058, 1059, 1061-1063, 1500
1384 134 204 1055, 1058, 1059, 1061-1063, 1500 1384 206 249 1055,
1058, 1059, 1061-1063, 1500 1384 251 270 1055, 1058, 1059,
1061-1063, 1500 1384 284 305 1055, 1058, 1059, 1061-1063, 1500 1385
21 40 1055, 1058, 1059, 1061-1063, 1500 1385 63 85 1055, 1058,
1059, 1061-1063, 1500 1385 135 205 1055, 1058, 1059, 1061-1063,
1500 1385 207 250 1055, 1058, 1059, 1061-1063, 1500 1385 252 271
1055, 1058, 1059, 1061-1063, 1500 1385 285 306 1055, 1058, 1059,
1061-1063, 1500 1387 2 21 1055, 1058, 1059, 1061-1063, 1500, 1057
1387 23 42 1055, 1058, 1059, 1061-1063, 1500, 1057 1387 56 77 1055,
1058, 1059, 1061-1063, 1500, 1057 1389 1 55 1055, 1058, 1059,
1061-1063, 1500 1389 57 100 1055, 1058, 1059, 1061-1063, 1500 1389
102 121 1055, 1058, 1059, 1061-1063, 1500 1389 135 156 1055, 1058,
1059, 1061-1063, 1500 1386 196 255 1054-1057, 1060, 1496, 1498 1388
209 268 1054-1057, 1060, 1496, 1498 1391 283 342 1054-1057, 1060,
1492, 1496-1498 1387 257 321 1055, 1058, 1059, 1061-1063, 1500,
1057 1387 332 351 1055, 1058, 1059, 1061-1063, 1500, 1057 1390 25
56 1054-1057, 1060, 1492, 1496-1498 1384 284 321 1055, 1058, 1059,
1061-1063, 1500 1385 285 322 1055, 1058, 1059, 1061-1063, 1500 1387
56 93 1055, 1058, 1059, 1061-1063, 1500, 1057 1389 135 172 1055,
1058, 1059, 1061-1063, 1500 1386 196 332 1054-1057, 1060, 1496,
1498 1388 209 345 1054-1057, 1060, 1496, 1498 1391 283 352
1054-1057, 1060, 1492, 1496-1498 1384 323 348 1055, 1058, 1059,
1061-1063, 1500 1385 324 349 1055, 1058, 1059, 1061-1063, 1500 1387
95 120 1055, 1058, 1059, 1061-1063, 1500, 1057 1387 128 153 1055,
1058, 1059, 1061-1063, 1500, 1057 1387 161 186 1055, 1058, 1059,
1061-1063, 1500, 1057 1387 191 210 1055, 1058, 1059, 1061-1063,
1500, 1057 1389 174 199 1055, 1058, 1059, 1061-1063, 1500 1389 207
232 1055, 1058, 1059, 1061-1063, 1500 1389 240 265 1055, 1058,
1059, 1061-1063, 1500 1389 270 289 1055, 1058, 1059, 1061-1063,
1500 1283 177 208 1064-1070 1284 130 161 1064-1070 1284 301 351
1064-1070 1286 210 241 1064-1070 1287 126 157 1064-1070 1287 297
351 1064-1070 1288 326 351 1064-1070, 1071
1289 41 72 1064-1070 1289 212 288 1064-1070 1289 320 351 1064-1070
1290 178 209 1064-1070 1291 228 259 1064-1070, 1071 1292 43 74
1064-1070 1292 214 290 1064-1070 1292 322 351 1064-1070 1293 330
351 1064-1070, 1071 1377 37 68 1064-1070 1377 208 284 1064-1070
1377 316 348 1064-1070 1289 212 274 1064-1070 1292 214 276
1064-1070 1377 208 270 1064-1070 1285 123 148 1067, 1069, 1071 1288
107 132 1064-1070, 1071 1291 9 34 1064-1070, 1071 1293 111 136
1064-1070, 1071 1287 297 347 1064-1070 1289 212 262 1064-1070 1292
214 264 1064-1070 1377 208 258 1064-1070 1287 297 352 1064-1070
1289 212 266 1064-1070 1292 214 268 1064-1070 1377 208 262
1064-1070 1285 18 61 1067, 1069, 1071 1288 2 45 1064-1070, 1071
1293 6 49 1064-1070, 1071 1357 1 40 1079-1082, 1152-1154, 1455,
1457, 1458 1357 63 85 1079-1082, 1152-1154, 1455, 1457, 1458 1357
102 157 1079-1082, 1152-1154, 1455, 1457, 1458 1357 159 189
1079-1082, 1152-1154,1455, 1457, 1458 1357 191 322 1079-1082,
1152-1154, 1455, 1457, 1458 1358 25 47 1079-1082, 1152-1154, 1455,
1457, 1458 1358 64 119 1079-1082, 1152-1154, 1455, 1457, 1458 1358
121 151 1079-1082, 1152-1154, 1455, 1457, 1458 1358 153 284
1079-1082, 1152-1154, 1455, 1457, 1458 1359 31 53 1079-1082,
1152-1154, 1455, 1457, 1458 1359 70 125 1079-1082, 1152-1154, 1455,
1457, 1458 1359 127 157 1079-1082, 1152-1154, 1455, 1457, 1458 1359
159 290 1079-1082, 1152-1154, 1455, 1457, 1458 1360 38 60
1079-1082, 1152-1154, 1455, 1457, 1458 1360 77 132 1079-1082,
1152-1154, 1455, 1457, 1458 1360 134 164 1079-1082, 1152-1154,
1455, 1457, 1458 1360 166 297 1079-1082, 1152-1154, 1455, 1457,
1458 1361 1 41 1079-1082, 1152-1154, 1455, 1457, 1458 1361 64 86
1079-1082, 1152-1154, 1455, 1457, 1458 1361 103 158 1079-1082,
1152-1154, 1455, 1457, 1458 1361 160 190 1079-1082, 1152-1154,
1455, 1457, 1458 1361 192 323 1079-1082, 1152-1154, 1455, 1457,
1458 1362 31 53 1079-1082, 1152-1154, 1455, 1457, 1458 1362 70 125
1079-1082, 1152-1154, 1455, 1457, 1458 1362 127 157 1079-1082,
1152-1154, 1455, 1457, 1458 1362 159 290 1079-1082, 1152-1154,
1455, 1457, 1458 1363 33 55 1079-1082, 1152-1154, 1455, 1457, 1458
1363 72 127 1079-1082, 1152-1154, 1455, 1457, 1458 1363 129 159
1079-1082, 1152-1154, 1455, 1457, 1458 1363 161 292 1079-1082,
1152-1154, 1455, 1457, 1458 1364 4 59 1079-1082, 1152-1154, 1455,
1457, 1458 1364 61 91 1079-1082, 1152-1154, 1455, 1457, 1458 1364
93 224 1079-1082, 1152-1154, 1455, 1457, 1458 1365 35 57 1079-1082,
1152-1154, 1455, 1457, 1458 1365 74 129 1079-1082, 1152-1154, 1455,
1457, 1458 1365 131 161 1079-1082, 1152-1154, 1455, 1457, 1458 1365
163 294 1079-1082, 1152-1154, 1455, 1457, 1458 1366 2 26 1079-1082,
1152-1154, 1455, 1457, 1458 1366 28 58 1079-1082, 1152-1154, 1455,
1457, 1458 1366 60 191 1079-1082, 1152-1154, 1455, 1457, 1458 1367
2 116 1079, 1080, 1081, 1082, 1152, 1153, 1154 1358 322 345
1079-1082, 1152-1154, 1455, 1457, 1458 1359 328 350 1079-1082,
1152-1154, 1455, 1457, 1458 1362 328 351 1079-1082, 1152-1154,
1455, 1457, 1458 1363 330 352 1079-1082, 1152-1154, 1455, 1457,
1458 1364 262 285 1079-1082, 1152-1154, 1455, 1457, 1458 1365 332
351 1079-1082, 1152-1154, 1455, 1457, 1458 1366 229 252 1079-1082,
1152-1154, 1455, 1457, 1458 1367 154 177 1079-1082, 1152-1154 1358
322 344 1079-1082, 1152-1154, 1455, 1457, 1458 1362 328 350
1079-1082, 1152-1154, 1455, 1457, 1458 1364 262 284 1079-1082,
1152-1154, 1455, 1457, 1458 1366 229 251 1079-1082, 1152-1154,
1455, 1457, 1458 1367 154 176 1079-1082, 1152-1154 1368 126 145
1083, 1087 1368 153 177 1083, 1087 1368 186 208 1083, 1087 1368 224
253 1083, 1087 1369 161 180 1083, 1084, 1089 1370 124 143 1083,
1087 1370 151 175 1083, 1087 1370 184 206 1083, 1087 1370 222 251
1083, 1087 1369 278 303 1083, 1084, 1089 1369 182 201 1083, 1084,
1089 1371 1 71 1093-1095, 1098, 1100-1103 1371 82 122 1093-1095,
1098, 1100-1103 1371 124 180 1093-1095, 1098, 1100-1103 1371 182
244 1093-1095, 1098, 1100-1103 1372 16 56 1093-1095, 1098,
1100-1103 1372 58 114 1093-1095, 1098, 1100-1103 1372 116 178
1093-1095, 1098, 1100-1103 1373 2 61 1093-1095, 1098, 1100-1103
1373 72 112 1093-1095, 1098, 1100-1103 1373 114 170 1093-1095,
1098, 1100-1103 1373 172 234 1093-1095, 1098, 1100-1103 1374 1 68
1093-1095, 1098, 1100-1103 1374 79 119 1093-1095, 1098, 1100-1103
1374 121 177 1093-1095, 1098, 1100-1103 1374 179 241 1093-1095,
1098, 1100-1103 1375 2 41 1093-1095, 1098, 1100-1103 1375 52 92
1093-1095, 1098, 1100-1103 1375 94 150 1093-1095, 1098, 1100-1103
1375 152 214 1093-1095, 1098, 1100-1103 1376 7 47 1093-1095, 1098,
1100-1103 1376 49 105 1093-1095, 1098, 1100-1103 1376 107 169
1093-1095, 1098, 1100-1103 1371 124 148 1093-1095, 1098, 1100-1103
1372 58 82 1093-1095, 1098, 1100-1103 1373 114 138 1093-1095, 1098,
1100-1103 1374 121 145 1093-1095, 1098, 1100-1103 1375 94 118
1093-1095, 1098, 1100-1103 1376 49 73 1093-1095, 1098, 1100-1103
1371 182 276 1093-1095, 1098, 1100-1103 1371 278 333 1093-1095,
1098, 1100-1103 1372 116 210 1093-1095, 1098, 1100-1103 1372 212
267 1093-1095, 1098, 1100-1103 1372 269 309 1093-1095, 1098,
1100-1103 1372 311 352 1093-1095, 1098, 1100-1103 1373 172 266
1093-1095, 1098, 1100-1103 1373 268 323 1093-1095, 1098, 1100-1103
1373 325 351 1093-1095, 1098, 1100-1103 1374 179 273 1093-1095,
1098, 1100-1103 1374 275 330 1093-1095, 1098, 1100-1103 1374 332
351 1093-1095, 1098, 1100-1103 1375 152 246 1093-1095, 1098,
1100-1103 1375 248 303 1093-1095, 1098, 1100-1103 1375 305 345
1093-1095, 1098, 1100-1103 1376 107 201 1093-1095, 1098, 1100-1103
1376 203 258 1093-1095, 1098, 1100-1103 1376 260 300 1093-1095,
1098, 1100-1103 1376 302 342 1093-1095, 1098, 1100-1103 1371 278
303 1093-1095, 1098, 1100-1103 1372 212 237 1093-1095, 1098,
1100-1103 1373 268 293 1093-1095, 1098, 1100-1103 1374 275 300
1093-1095, 1098, 1100-1103 1375 248 273 1093-1095, 1098, 1100-1103
1376 203 228 1093-1095, 1098, 1100-1103 1371 124 155 1093-1095,
1098, 1100-1103 1372 58 89 1093-1095, 1098, 1100-1103 1373 114 145
1093-1095, 1098, 1100-1103 1374 121 152 1093-1095, 1098, 1100-1103
1375 94 125 1093-1095, 1098, 1100-1103 1376 49 80 1093-1095, 1098,
1100-1103 1371 182 241 1093-1095, 1098, 1100-1103 1372 116 175
1093-1095, 1098, 1100-1103 1373 172 231 1093-1095, 1098, 1100-1103
1374 179 238 1093-1095, 1098, 1100-1103 1375 152 211 1093-1095,
1098, 1100-1103 1376 107 166 1093-1095, 1098, 1100-1103 1371 278
325 1093-1095, 1098, 1100-1103 1372 212 259 1093-1095, 1098,
1100-1103 1373 268 315 1093-1095, 1098, 1100-1103 1374 275 322
1093-1095, 1098, 1100-1103 1375 248 295 1093-1095, 1098, 1100-1103
1376 203 250 1093-1095, 1098, 1100-1103 1309 1 69 1114, 1115 1309
71 96 1114, 1115 1309 98 144 1114, 1115 1309 152 195 1114, 1115
1309 197 237 1114, 1115 1309 254 352 1114, 1115 1310 25 92 1114,
1115 1310 94 119 1114, 1115 1310 121 167 1114, 1115 1310 175 218
1114, 1115 1310 220 260 1114, 1115 1310 277 351 1114, 1115 1311 7
74 1114, 1115 1311 76 101 1114, 1115 1311 103 149 1114, 1115 1311
157 200 1114, 1115 1311 202 242 1114, 1115 1311 259 351 1114, 1115
1312 9 76 1114, 1115 1312 78 103 1114, 1115 1312 105 151 1114, 1115
1312 159 202 1114, 1115 1312 204 244 1114, 1115 1312 261 352 1114,
1115 1309 2 69 1114, 1115 1309 254 327 1114, 1115 1310 22 92 1114,
1115 1310 277 350 1114, 1115 1311 4 74 1114, 1115 1311 259 332
1114, 1115 1312 6 76 1114, 1115 1312 261 334 1114, 1115 1315 56 87
1117, 1119-1125 1315 91 114 1117, 1119-1125 1315 116 144 1117,
1119-1125 1315 146 171 1117, 1119-1125 1315 185 225 1117, 1119-1125
1315 275 294 1117, 1119-1125 1315 317 342 1117, 1119-1125 1316 22
45 1117, 1119-1125 1316 47 75 1117, 1119-1125 1316 77 102 1117,
1119-1125 1316 116 156 1117, 1119-1125 1316 206 225 1117, 1119-1125
1316 248 273 1117, 1119-1125 1316 275 317 1117, 1119-1125 1318 144
175 1117, 1119-1125 1318 179 202 1117, 1119-1125 1318 204 232 1117,
1119-1125 1318 234 259 1117, 1119-1125 1318 273 313 1117, 1119-1125
1319 2 20 1117, 1119-1125 1319 24 47 1117, 1119-1125 1319 49 77
1117, 1119-1125 1319 79 104 1117, 1119-1125 1319 118 158 1117,
1119-1125 1319 208 227 1117, 1119-1125 1319 250 275 1117, 1119-1125
1319 277 319 1117, 1119-1125 1313 17 81 1118, 1125 1313 92 117
1118, 1125 1313 137 204 1118, 1125 1313 215 240 1118, 1125 1313 242
291 1118, 1125 1313 293 329 1118, 1125 1314 22 89 1118-1125 1314
100 125 1118-1125 1314 127 176 1118-1125 1314 178 214 1118-1125
1317 25 92 1118, 1120-1122, 1124, 1125 1317 103 128 1118,
1120-1122, 1124, 1125 1317 130 179 1118, 1120-1122, 1124, 1125 1317
181 217 1118, 1120-1122, 1124, 1125 1314 332 351 1118-1125 1315 27
51 1117, 1119-1125 1316 275 306 1117, 1119-1125 1318 115 139 1117,
1119-1125 1319 277 308 1117, 1119-1125 1314 330 351 1118-1125 1315
25 51 1117, 1119-1125 1316 275 303 1117, 1119-1125 1317 333 351
1118, 1120-1122, 1124, 1125 1318 113 139 1117, 1119-1125 1319 277
305 1117, 1119-1125 1314 262 293 1118-1125
1314 295 314 1118-1125 1314 316 351 1118-1125 1315 11 51 1117,
1119-1125 1317 265 296 1118, 1120-1122, 1124, 1125 1317 298 317
1118, 1120-1122, 1124, 1125 1317 319 351 1118, 1120-1122, 1124,
1125 1318 45 76 1117, 1119-1125 1318 78 97 1117, 1119-1125 1318 99
139 1117, 1119-1125 1316 275 324 1117, 1119-1125 1319 277 326 1117,
1119-1125 1299 50 96 1126-1131, 1651-1653 1300 2 43 1126-1131,
1651-1653 1300 66 112 1126-1131, 1651-1653 1300 123 154 1126-1131,
1651-1653 1300 183 211 1126-1131, 1651-1653 1300 213 232 1126-1131,
1651-1653 1300 264 310 1126-1131, 1651-1653 1301 1 35 1126-1131
1301 37 84 1126-1131 1301 107 153 1126-1131 1301 164 195 1126-1131
1301 224 252 1126-1131 1301 254 273 1126-1131 1301 305 351
1126-1131 1302 26 54 1126-1131, 1651-1653 1302 56 75 1126-1131,
1651-1653 1302 107 153 1126-1131, 1651-1653 1303 11 39 1126-1131,
1651-1653 1303 41 60 1126-1131, 1651-1653 1303 92 138 1126-1131,
1651-1653 1299 50 114 1126-1131, 1651-1653 1299 131 162 1126-1131,
1651-1653 1299 215 236 1126-1131, 1651-1653 1299 248 270 1126-1131,
1651-1653 1299 272 309 1126-1131, 1651-1653 1300 264 328 1126-1131,
1651-1653 1302 107 171 1126-1131, 1651-1653 1302 188 219 1126-1131,
1651-1653 1302 272 293 1126-1131, 1651-1653 1302 305 327 1126-1131,
1651-1653 1302 329 351 1126-1131, 1651-1653 1303 92 156 1126-1131,
1651-1653 1303 173 204 1126-1131, 1651-1653 1303 257 278 1126-1131,
1651-1653 1303 290 312 1126-1131, 1651-1653 1303 314 351 1126-1131,
1651-1653 1299 272 352 1126-1131, 1651-1653 1299 272 344 1126-1131,
1651-1653 1299 272 318 1126-1131, 1651-1653 1299 272 310 1126-1131,
1651-1653 1304 2 25 1132, 1134, 1135, 1136 1304 36 169 1132, 1134,
1135, 1136 1304 186 214 1132, 1134, 1135, 1136 1304 231 250 1132,
1134, 1135, 1136 1304 264 307 1132, 1134, 1135, 1136 1306 2 70
1132, 1134, 1135, 1136 1306 87 115 1132, 1134, 1135, 1136 1306 132
151 1132, 1134, 1135, 1136 1306 165 208 1132, 1134, 1135, 1136 1307
1 27 1132, 1134, 1135, 1136 1307 29 69 1132, 1134, 1135, 1136 1307
80 213 1132, 1134, 1135, 1136 1307 230 258 1132, 1134, 1135, 1136
1307 275 294 1132, 1134, 1135, 1136 1307 308 351 1132, 1134, 1135,
1136 1308 2 33 1132, 1134, 1135, 1136 1308 44 177 1132, 1134, 1135,
1136 1308 194 222 1132, 1134, 1135, 1136 1308 239 258 1132, 1134,
1135, 1136 1308 272 315 1132, 1134, 1135, 1136 1347 2 28 1132,
1134, 1135, 1136 1347 39 172 1132, 1134, 1135, 1136 1347 189 217
1132, 1134, 1135, 1136 1347 234 253 1132, 1134, 1135, 1136 1347 267
310 1132, 1134, 1135, 1136 1305 1 133 1133, 1137, 1138, 1580, 1581,
1583 1305 135 154 1133, 1137, 1138, 1580, 1581, 1583 1305 156 178
1133, 1137, 1138, 1580, 1581, 1583 1305 195 280 1133, 1137, 1138,
1580, 1581, 1583 1305 330 351 1133, 1137, 1138, 1580, 1581, 1583
1344 13 35 1133, 1137, 1138, 1580, 1581, 1583 1344 52 137 1133,
1137, 1138, 1580, 1581, 1583 1344 187 208 1133, 1137, 1138, 1580,
1581, 1583 1345 50 181 1133, 1137, 1138, 1580, 1581, 1583 1345 183
202 1133, 1137, 1138, 1580, 1581, 1583 1345 204 226 1133, 1137,
1138, 1580, 1581, 1583 1345 243 328 1133, 1137, 1138, 1580, 1581,
1583 1346 2 81 1133, 1137, 1138 1346 131 152 1133, 1137, 1138 1304
1 25 1132, 1134, 1135, 1136 1304 264 334 1132, 1134, 1135, 1136
1306 165 235 1132, 1134, 1135, 1136 1307 46 69 1132, 1134, 1135,
1136 1308 10 33 1132, 1134, 1135, 1136 1308 272 342 1132, 1134,
1135, 1136 1347 5 28 1132, 1134, 1135, 1136 1347 267 337 1132,
1134, 1135, 1136 1304 101 169 1132, 1134, 1135, 1136 1306 1 70
1132, 1134, 1135, 1136 1306 237 310 1132, 1134, 1135, 1136 1306 312
352 1132, 1134, 1135, 1136 1307 145 213 1132, 1134, 1135, 1136 1308
109 177 1132, 1134, 1135, 1136 1347 104 172 1132, 1134, 1135, 1136
1307 38 69 1132, 1134, 1135, 1136 1308 1 33 1132, 1134, 1135, 1136
1344 187 239 1133, 1137, 1138, 1580, 1581, 1583 1344 241 272 1133,
1137, 1138, 1580, 1581, 1583 1344 286 308 1133, 1137, 1138, 1580,
1581, 1583 1344 310 329 1133, 1137, 1138, 1580, 1581, 1583 1346 131
183 1133, 1137, 1138 1346 185 216 1133, 1137, 1138 1346 230 252
1133, 1137, 1138 1346 254 273 1133, 1137, 1138 1305 227 280 1133,
1137, 1138, 1580, 1581, 1583 1344 84 137 1133, 1137, 1138, 1580,
1581, 1583 1345 275 328 1133, 1137, 1138, 1580, 1581, 1583 1346 28
81 1133, 1137, 1138 1346 290 351 1133, 1137, 1138 1348 44 66 1139,
1140, 1141, 1142 1348 104 129 1139, 1140, 1141, 1142 1348 131 156
1139, 1140, 1141, 1142 1348 158 195 1139, 1140, 1141, 1142 1348 167
189 1139, 1140, 1141, 1142 1348 197 237 1139, 1140, 1141, 1142 1348
215 234 1139, 1140, 1141, 1142 1348 4 49 1139, 1140, 1141, 1142
1348 4 74 1139, 1140, 1141, 1142 1348 4 85 1139, 1140, 1141, 1142
1349 19 90 1143 1349 146 207 1143 1349 209 256 1143 1350 32 51
1144-1151, 1703 1350 71 90 1144-1151, 1703 1351 118 137 1144-1151
1351 157 176 1144-1151 1353 81 100 1144-1151 1353 120 139 1144-1151
1354 1 42 1144-1151 1354 218 237 1144-1151 1354 257 276 1144-1151
1356 43 62 1144-1151 1356 82 101 1144-1151 1350 176 201 1144-1151,
1703 1350 209 231 1144-1151, 1703 1350 236 267 1144-1151, 1703 1351
262 287 1144-1151 1351 295 317 1144-1151 1351 322 351 1144-1151
1353 225 250 1144-1151 1353 258 280 1144-1151 1353 285 316
1144-1151 1356 187 212 1144-1151 1356 220 242 1144-1151 1356 247
278 1144-1151 1350 236 265 1144-1151, 1703 1351 322 352 1144-1151
1353 285 314 1144-1151 1356 247 276 1144-1151 1350 287 306
1144-1151, 1703 1350 323 348 1144-1151, 1703 1352 2 21 1147, 1150,
1151, 1703 1352 38 63 1147, 1150, 1151, 1703 1355 5 24 1147, 1150,
1151, 1703 1355 41 66 1147, 1150, 1151, 1703 1356 298 317 1144-1151
1358 322 351 1079-1082, 1152-1154, 1455, 1457, 1458 1364 262 291
1079-1082, 1152-1154, 1455, 1457, 1458 1366 229 258 1079-1082,
1152-1154, 1455, 1457, 1458 1367 154 183 1079-1082, 1152-1154 1361
2 41 1079-1082, 1152-1154, 1455, 1457, 1458 1210 1 35 1431 1211 30
63 1431 1213 15 48 1431 1217 19 52 1431 1357 162 200 1079-1082,
1152-1154, 1455, 1457, 1458 1358 124 162 1079-1082, 1152-1154,
1455, 1457, 1458 1359 130 168 1079-1082, 1152-1154, 1455, 1457,
1458 1360 137 175 1079-1082, 1152-1154, 1455, 1457, 1458 1361 163
201 1079-1082, 1152-1154, 1455, 1457, 1458 1362 130 168 1079-1082,
1152-1154, 1455, 1457, 1458 1363 132 170 1079-1082, 1152-1154,
1455, 1457, 1458 1364 64 102 1079-1082, 1152-1154, 1455, 1457, 1458
1365 134 172 1079-1082, 1152-1154, 1455, 1457, 1458 1366 31 69
1079-1082, 1152-1154, 1455, 1457, 1458 1054 198 244 1492, 1496,
1497, 1498 1054 285 319 1492, 1496, 1497, 1498 1056 191 237 1492,
1496, 1497, 1498 1056 278 312 1492, 1496, 1497, 1498 1057 14 60
1492, 1496, 1497, 1498 1057 101 135 1492, 1496, 1497, 1498 1057 233
264 1492, 1496, 1497, 1498 1060 269 315 1492, 1496, 1497, 1498 1390
10 50 1054-1057, 1060, 1492, 1496-1498 1390 142 173 1054-1057,
1060, 1492, 1496-1498 1391 73 104 1054-1057, 1060, 1492, 1496-1498
1055 85 131 1493, 1494, 1495, 1499, 1500 1058 117 163 1493, 1494,
1495, 1499, 1500 1059 118 164 1493, 1494, 1495, 1499, 1500 1061 109
155 1493, 1494, 1495, 1499, 1500 1062 222 268 1493, 1494, 1495,
1499, 1500 1063 262 308 1493, 1494, 1495, 1499, 1500 1054 15 64
1492, 1496, 1497, 1498 1054 165 196 1492, 1496, 1497, 1498 1056 8
57 1492, 1496, 1497, 1498 1056 158 189 1492, 1496, 1497, 1498 1060
86 135 1492, 1496, 1497, 1498 1060 236 267 1492, 1496, 1497, 1498
1386 199 248 1054-1057, 1060, 1496, 1498 1388 212 261 1054- 1057,
1060, 1496, 1498 1391 286 335 1054-1057, 1060, 1492, 1496-1498 1384
311 351 1055, 1058, 1059, 1061-1063, 1500 1385 312 351 1055, 1058,
1059, 1061-1063, 1500 1387 83 123 1055, 1058, 1059, 1061-1063,
1500, 1057 1389 162 202 1055, 1058, 1059, 1061-1063, 1500 1296 69
101 1025, 1028, 1029, 1515, 1516, 1517, 1518, 1520 909 305 340 1531
960 70 101 1543, 1551 964 259 290 1543, 1551 1305 180 226 1133,
1137, 1138, 1580, 1581, 1583 1133 195 226 1580, 1581 1137 52 83
1580, 1581 1344 37 83 1133, 1137, 1138, 1580, 1581, 1583 1345 228
274 1133, 1137, 1138, 1580, 1581, 1583 1305 180 217 1133, 1137,
1138, 1580, 1581, 1583 1344 37 74 1133, 1137, 1138, 1580, 1581,
1583 1345 228 265 1133, 1137, 1138, 1580, 1581, 1583 1039 176 210
1590, 1594, 1597, 1598 1040 142 176 1590, 1594, 1597, 1598 1041 248
282 1590, 1594, 1597, 1598 1042 141 175 1590, 1594, 1597, 1598 1328
175 209 1039-1043, 1045, 1594, 1597, 1598 1331 2 54 1039-1043,
1045, 1594, 1597, 1598 1331 200 234 1039-1043, 1045, 1594, 1597,
1598 1332 142 176 1039-1043, 1045, 1594, 1597, 1598 1333 176 210
1039-1043, 1045, 1594, 1597, 1598 1334 16 68 1039-1043, 1045, 1594,
1597, 1598 1334 214 248 1039-1043, 1045, 1594, 1597, 1598 1335 76
110 1039-1043, 1045, 1594, 1597, 1598 1336 8 60 1039-1043, 1045,
1594, 1597, 1598 1336 206 240 1039-1043, 1045, 1594, 1597, 1598
1338 14 66 1039-1043, 1045, 1594, 1597, 1598 1338 212 246
1039-1043, 1045, 1594, 1597, 1598 1255 165 199 970, 971, 973-978,
1626, 1629 976 129 163 1626, 1629 1299 98 129 1126-1131, 1651-1653
1300 312 343 1126-1131, 1651-1653 1302 155 186 1126-1131, 1651-1653
1303 140 171 1126-1131, 1651-1653 1350 314 345 1144-1151, 1703
1352 29 60 1147, 1150, 1151, 1703 1355 32 63 1147, 1150, 1151,
1703
Example 8
[0153] This example discloses embodiments related to polynucleotide
molecules having a nucleotide sequence containing specific
modifications such as nucleotide substitutions. Embodiments of such
modifications include modified polynucleotides that provide
improved sequence discrimination between the intended target gene
of the insect pest of interest, and genetic sequences of other,
non-target species.
[0154] Double-stranded RNAs identified in Table 1 were screened for
sequence matches to a sequence of at least 19 contiguous
nucleotides identified in a non-target gene or a non-target
organism (NTO, e. g., Apis mellifera, Bombus impatiens and B.
terrestris; Bombyx mori; Bos taurus; Canis lupus familiaris;
Coleomegilla maculata; Danio rerio; Danaus plexippus; Daphnia
pulex; Equus caballus; Gallus gallus; Homo sapiens; Megachile
rotundata; Mus musculus; Sus scrofa; Brassica napus; Brassica
oleracea; Brassica rapa; Glycine max; Gossypium raimondii; Solanum
tuberosum; Solanum lycopersicum; and Zea mays). Nucleotide changes
are made to eliminate contiguous sequence matches over 19
nucleotides to a non-target gene or non-target organism. Examples
of such modified polynucleotide sequences are provided by SEQ ID
NO:1725, which corresponds to SEQ ID NO:870 (which targets the
target gene having SEQ ID NO:11), SEQ ID NO:1726, which corresponds
to SEQ ID NO:1224 (which targets the target gene having SEQ ID
NO:365), SEQ ID NO:1727, which corresponds to SEQ ID NO:875 (which
targets the target gene having SEQ ID NO:16), SEQ ID NO:1728, which
corresponds to SEQ ID NO:1193 (which targets the target gene having
SEQ ID NO:334), SEQ ID NO:1729, which corresponds to SEQ ID NO:871
(which targets the target gene having SEQ ID NO:12), and SEQ ID
NO:1730, which corresponds to SEQ ID NO:1187 (which targets the
target gene having SEQ ID NO:328).
[0155] These modified polynucleotide sequences SEQ ID NOs:1725-1730
are useful for designing polynucleotides for providing improved
discrimination between the intended target species and non-target
organisms. For example, embodiments of insecticidal compositions
useful for controlling or preventing flea beetle infestations
comprise an effective amount of a dsRNA comprising one RNA strand
having a sequence selected from the group consisting of SEQ ID
NOs:1725-1730, or a fragment thereof. In embodiments, insecticidal
compositions for causing stunting or mortality in flea beetles
comprise an effective amount of a dsRNA comprising one RNA strand
having at least one segment of at least 21 contiguous nucleotides
of a sequence selected from the group consisting of SEQ ID
NOs:1725-1730. In embodiments, insecticidal compositions for
causing stunting or mortality in flea beetles comprise an effective
amount of an RNA molecule comprising at least one segment of at
least 21 contiguous nucleotides of a sequence selected from the
group consisting of SEQ ID NOs:1725-1730 wherein the at least one
segment is embedded in larger sections of neutral sequence to
provide an efficacious insecticidal polynucleotide. In another
embodiment, segments of at least 21 contiguous nucleotides from
multiple sequences selected from the group consisting of SEQ ID
NOs:1725-1730 are embedded in larger sections of neutral sequence
to provide an efficacious insecticidal polynucleotide for
controlling flea beetles. Methods for selecting efficacious
insecticidal polynucleotides are described elsewhere in this
application, e. g., the method including oral delivery of
insecticidal polynucleotides to flea beetles, resulting in stunting
or mortality in the flea beetles, described in Example 5. These
modified polynucleotide sequences SEQ ID NOs:1725-1730 are useful
in a method of causing mortality or stunting in an insect,
comprising providing in the diet of an insect, such as a flea
beetle, at least one polynucleotide that comprises at least one
segment of at least 21 contiguous nucleotides of a sequence
selected from the group consisting of SEQ ID NOs:1725-1730, wherein
ingestion of the polynucleotide by the insect results in mortality
or stunting in the insect; in embodiments, the polynucleotide
comprises a sequence selected from the group consisting of SEQ ID
NOs:1725-1730. Polynucleotides comprising a sequence of SEQ ID
NOs:1725-1730 are useful in a method for controlling an insect
infestation of a plant comprising contacting, with a
polynucleotide, an insect that infests a plant, wherein the
polynucleotide comprises at least one segment of at least 21
contiguous nucleotides of a sequence selected from the group
consisting of SEQ ID NOs:1725-1730, whereby the insect infestation
is controlled; in embodiments, the polynucleotide comprises a
sequence selected from the group consisting of SEQ ID
NOs:1725-1730. Polynucleotides as described herein are useful in a
method of providing a plant having improved resistance to an
insect, comprising expressing in the plant a recombinant DNA
construct, wherein the recombinant DNA encodes a polynucleotide
that comprises at least one segment of at least 21 contiguous
nucleotides of a sequence selected from the group consisting of SEQ
ID NOs:1725-1730, wherein ingestion of the polynucleotide by the
insect results in mortality or stunting in the insect; in some
embodiments, the polynucleotide comprises a sequence selected from
the group consisting of SEQ ID NOs:1725-1730.
Example 9
[0156] This example discloses additional insecticidal
polynucleotide molecules with complementarity to both Phyllotreta
cruciferae and Psylloides striolata. A total of two hundred-forty
(240) polynucleotide sequences were engineered (SEQ ID NOs
1731-1971). These polynucleotides target gene transcripts for
calmodulin, casein kinase 1a and elongation factori 1-alpha across
both flea beetle species. Fifty-one polynucleotides are tiling
polynucleotides that cover the transcript length of these genes.
The following Table 5 summarizes these fifty-one tiling
polynucleotides and the transcripts targeted.
TABLE-US-00005 TABLE 5 Insecticidal Polynucleotides and target
transcripts Insecticidal Polynucleotide SEQ ID NO Target transcript
1731 Rpn7_PHYST-05JAN16-TRPT0002041:1 1732
Rpn7_PHYST-05JAN16-TRPT0002041:3 1733
TubulinG_PHYST-05JAN16-TRPT0002886:1 1734
TubulinG_PHYST-05JAN16-TRPT0002886:2 1735
TubulinG_PHYST-05JAN16-TRPT0002886:3 1736
TubulinG_PHYST-05JAN16-TRPT0002886:4 1737
TubulinG_PHYST-05JAN16-TRPT0002886:5 1738
COPI_Delta_PHYST-05JAN16-TRPT0009108:2 1739
COPI_Delta_PHYST-05JAN16-TRPT0009108:5 1740
COPI_Delta_PHYST-05JAN16-TRPT0009108:6 1741
COPI_Delta_PHYST-05JAN16-TRPT0009108:11 1742
COPI_Delta_PHYST-05JAN16-TRPT0009108:12 1743
sec23_PHYST-05JAN16-TRPT0011013:1 1744
sec23_PHYST-05JAN16-TRPT0011013:2 1745
sec23_PHYST-05JAN16-TRPT0011013:3 1746
sec23_PHYST-05JAN16-TRPT0011013:4 1747
COPI_Gamma_PHYST-05JAN16-TRPT0018736:1 1748
COPI_Gamma_PHYST-05JAN16-TRPT0018736:2 1749
COPI_Gamma_PHYST-05JAN16-TRPT0018736:3 1750
COPI_Gamma_PHYST-05JAN16-TRPT0018736:4 1751
COPI_Gamma_PHYST-05JAN16-TRPT0018736:5 1752
Rpt6_PHYST-05JAN16-TRPT0036466:1 1753
Rpt6_PHYST-05JAN16-TRPT0036466:2 1754
Rpt6_PHYST-05JAN16-TRPT0036466:3 1755
Rpt6_PHYST-05JAN16-TRPT0036466:4 1756
Rpt6_PHYST-05JAN16-TRPT0036466:5 1757
actin_PHYST-05JAN16-TRPT0037252:1 1758
actin_PHYST-05JAN16-TRPT0037252:3 1759
actin_PHYST-05JAN16-TRPT0037252:5 1760
vATPaseA_PHYST-05JAN16-TRPT0041323:1 1761
RpL13_PHYST-05JAN16-TRPT0045203:1 1762
RpL13_PHYST-05JAN16-TRPT0045203:2 1763
RpS14_PHYST-05JAN16-TRPT0045884:1 1764
RpS14_PHYST-05JAN16-TRPT0045884:2 1765
actin_PHYCR-05JAN16-TRPT0000169:2 1766
actin_PHYCR-05JAN16-TRPT0000169:3 1767
vATPase-A_PHYCR-05JAN16-TRPT0001064:1 1768
vATPase-A_PHYCR-05JAN16-TRPT0001064:3 1769
vATPase-A_PHYCR-05JAN16-TRPT0001064:4 1770
vATPase-A_PHYCR-05JAN16-TRPT0001064:5 1771
vATPase-A_PHYCR-05JAN16-TRPT0001064:6 1772
vATPase-A_PHYCR-05JAN16-TRPT0001064:7 1773
vATPase-A_PHYCR-05JAN16-TRPT0001064:8 1774
tubulinG_PHYCR-05JAN16-TRPT0001496:1 1775
tubulinG_PHYCR-05JAN16-TRPT0001496:2 1776
tubulinG_PHYCR-05JAN16-TRPT0001496:3 1777
tubulinG_PHYCR-05JAN16-TRPT0001496:4 1778
tubulinG_PHYCR-05JAN16-TRPT0001496:5 1779
tubulinG_PHYCR-05JAN16-TRPT0001496:6 1780
RpS14_PHYCR-05JAN16-TRPT0002697:1 1782
RpS14_PHYCR-05JAN16-TRPT0002697:2
Example 10
[0157] This example illustrates non-limiting embodiments of methods
of testing the efficacy of insecticidal polynucleotides in flea
beetles. More specifically this example illustrates a method
comprising oral delivery of dsRNAs to flea beetles, resulting in
stunting or mortality in the flea beetles.
[0158] P. cruciferae and P. striolata were collected from a canola
field where no pesticides had been applied in the previous 3
months. Two dsRNAs (SEQ ID NOs:1972 and 1974) targeting Phyllotreta
cruciferae genes (COPI_delta and Rpt6, respectively) and two dsRNAs
(SEQ ID Nos:1973 and 1975) targeting Phyllotreta striolata genes
(COPI_delta and Rpt6, respectively) and one negative control dsRNA
targeting GFP were tested on groups of 30 P. cruciferae or P.
striolata by using 10 insects in three separate replications. The
dsRNAs were resuspended in water and applied to 9 millimeter leaf
discs (55.+-.6 milligrams each) at a dose of 50 nanograms
dsRNA/milligram leaf tissue, which were fed to groups of 5 flea
beetles. Leaf discs with freshly applied dsRNA were replaced every
other day, and the number of surviving individuals was recorded
over a 12-day period.
[0159] A low non-specific mortality rate was observed in the
negative-control insect groups (4 out of 30 insects dying over 12
days for P. cruciferae, or 13% non-specific mortality and 8 out of
30 insects dying over 12 days for P. striolata, or 26% non-specific
mortality). Mortality was observed beginning at day 2 and
continuing through the 12 day period. Specific mortality was
observed for all dsRNA treatments (Table 6). Correcting for
non-specific mortality (subtracting non-specific mortality rate of
4 or 8 insects per group for corrected N=26 or N=22), the percent
mortality observed at the end of the 12 day period ranged from
36-38% for the dsRNAs tested. These results demonstrated the
efficacy of the dsRNAs in causing mortality in flea beetles when
provided in the flea beetles' diet and their cross species
effect.
TABLE-US-00006 TABLE 6 Mortality in flea beetles fed an artificial
diet containing insecticidal polynucleotide SEQ Target Species
Cumulative mortality ID gene tested (days after treatment, N = 30)
NO species against d0 d2 d5 d7 d9 d12 crucifer 0 0 1 1 2 4 NA
crucifer 0 1 2 2 3 5 1972 crucifer crucifer 0 0 4 8 10 10 1973
striped crucifer 0 0 2 6 6 9 1974 crucifer crucifer 0 1 3 7 8 9
1975 striped crucifer 0 0 0 2 6 8 striped 0 0 1 4 7 8 NA striped 0
0 1 2 6 8 1972 crucifer striped 0 0 3 9 10 10 1973 striped striped
0 0 3 9 9 10 1974 crucifer striped 0 0 3 7 7 8 1975 striped striped
0 1 3 5 8 9
[0160] All of the materials and methods disclosed and claimed
herein can be made and used without undue experimentation as
instructed by the above disclosure. Although the materials and
methods disclosed herein have been described in terms of preferred
embodiments and illustrative examples, it will be apparent to those
of skill in the art that variations can be applied to the materials
and methods described herein without departing from the concept,
spirit and scope of this invention. All such similar substitutes
and modifications apparent to those skilled in the art are deemed
to be within the spirit, scope and concept of this invention as
defined by the appended claims.
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
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20210254094A1).
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
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20210254094A1).
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